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Japanese Knotweed



The invasive Japanese Knotweed contains the highest known concentration of resveratrol.
The leaves are edible, and the plant has many applications in traditional Chinese medicine.









Green Deane : Japanese Knotweed: Dreadable Edible

Peter G. Becker : Bionic Knotweed Control

Patents : Extraction of Resveratrol &c from Knotweed

CN103396295 : Extraction method for resveratrol from giant knotweed rhizome

CN102838456 : Technology for preparing resveratrol by using traditional Chinese medicine giant knotweed rhizome

CN102379930 : Method for extracting total anthraquinone aglycones from giant knotweed

CN102320934 : New method for extracting resveratrol from giant knotweed rhizome

CN102070410 : Microwave-combined enzymatic method for extracting resveratrol from giant knotweed rhizome

CN101993354 : Method for extracting resveratrol

CN101870640 : Method for preparing resveratrol extract from giant knotweed and product

CN101811939 : Process for dynamical countercurrent extraction of resveratrol in giant knotweed

CN101735999 : Method for improving content of resveratrol...

CN101643754 : New technique for extracting and preparing high-purity resveratrol from giant knotweed

CN101397242 : Technique for extracting and purifying resveratrol from fresh giant knotweed rhizome

CN101204428 : Substance extracted from giant knotweed for diabetes mellitus and complication thereof

CN101167975 : Method for preparing polygonum paleaceum dry extract by hot water extraction...

CN1978473 : Resveratrol imprinted polymer preparation and extraction method for resveratrol

CN1800122 : Giant knotweed rhizome active ingredient emodin extraction method

CN1546503 : Method for preparing polygonin and resveratrol

CN1621401 : Method for extracting high purity resveratrol from giant knotweed rhizome


CN1251361 : Process for extracting resveratrol from Chinese medicine giant knotweed root

CN1513823 : Technology of super critical caron dioxide extraction of resveratrol from polygonum cuspidatum

CN1384088 : Extraction process of resveratrol from giant knotweed

CN1341587 : Method for preparing resveratrol from giant knotweed rhizome

Some "Traditional Chinese Medicine" Patents incorporating Knotweed



http://www.eatheweeds.com

Japanese Knotweed: Dreadable Edible

by

Green Deane

...Japanese Knotweed is listed by the World Conservation Union as one of the world’s worst invasive species. Perhaps it should be planted in countries where starvation is annual. Introduced into Great Britain by 1825 Japanese Knotweed has been on the decimation list for more than 30 years and has to be disposed at landfills licensed to handle the dreaded edible. In fact they spend some two billion pounds to combat it annually, which as of this writing is about three billion dollars a year. It increased the construction cost of the 2012 Olympic stadium by some 70 million pounds. Japanese Knotweed is also “invading” New Zealand, Australia, and Tasmania. It arrived in North America in the late 180os and is officially found in 39 of the 50 United States, probably more, and six provinces of Canada. It’s an invasive weed in Ohio, Vermont, West Virginia, New York, Alaska, Pennsylvania, Oregon and Washington. About the only place where they are not upset with the plant is where it’s native, southeast Asia. What do they know the rest of the world doesn’t? It is said that Japanese Knotweed out lives the gardener and the garden.

Knotweed, in the Buckwheat family, is not liked in western nations because it grows around three feet a month, sends roots down some 10 feet, grows through concrete, damaging roads, dams, buildings and just about anything made by man. It’s a pain in the asphalt. Forages take advantage of it eating — raw or cooked — young shoots, growing tips of larger plants and unfurled leaves on the stalk and branches. Many folks say it tastes like rhubarb but I think a lemony green is more accurate, crunchy and tender. For the health conscious it is a major source of resveratrol and Vitamin C … a noxious weed AND very healthy. Tsk…Tsk… The California Department of Food and Agriculture and the book Cornucopia II both say the rhizomes are edible. No references are given as to how to cook them nor have I tried. Usually the roots are used medicinally. Giant Knotweed, Polygonum sachalinense (Fallopia sachalinensis) is similarly consume except its fruit is eaten as well, or stored in oil..

Green Deane’s “Itemized” Plant Profile: Japanese Knotweed

IDENTIFICATION: Polygonum cuspidatum: A semi-woody perennial, fast growing, hollow, bamboo-like stems forming dense, leafy thickets, woody with age. Young shoots are red. Leaves simple, toothless, hairless, alternating, broadly ovate with a pointed tip, 3 to 6 inches long, 2 to 4½ inches wide, on a long leaf stem. Flowers branching in spike-like clusters, individual flowers are 1/8 inch across, white to greenish or pinkish, with 5 petals, 8 stamens. Male and female flowers separate (dioecious.) Female flowers can produce small 3-angled black-brown fruit. Seed production is uncommon.

TIME OF YEAR: Purple shoots appear in spring, flowers late summer, early autumn.

ENVIRONMENT: Riverbanks, roadsides, moist, disturbed areas.

METHOD OF PREPARATION: Young shoots, growing tips, young leaves boiled or steamed and eaten like asparagus, or chilled and served with a dressing. Can be used in pies. soups, aspics, sauces, jams, chutneys even wines. The roots, actually rhizomes, are sometimes eaten. It is good fodder for grazing animals, including cattle, sheep, goats, horses and donkeys. Old stems have been used to make matches. It is high in oxalic acid so if you avoid spinach or rhubarb you should avoid knotweed….




 



http://www.newtritionink.de/shop/pdf/english.pdf

Bionic Knotweed Control

by

Peter G. Becker

Japanese Knotweed; the most pernicious weed in the northern hemisphere has been rated as a biological catastrophe, for it`s monstrous tendency of overrunning the sensitive wetland areas of our nature reserves with monopolizing thickets due to which endangered native plant societies are often permanently displaced and their last occurrences extinguished.

So Japanese Knotweed is the most invasive plant on the northern globe and often treated with massive amounts of highly toxic herbicides. My goal is to teach folks how to take a bite out of their knotweed invasion, because the costs for other means of controlling polygonum cuspidatum and the damage done to construction sites and real estate are horrendous, while this project would not only save taxpayers their due, but yield a fantastic new foodsource, supply employment to any willing sponsor and protect our native environment; by turning this botanic pest into an economical blessing.

Not until 2002 did I realize that Japanese Knotweed was not just another edible wildplant, that I introduced to the attendance of my herbwalks. I would bring a jar of Knotweed jam and buttered breadslices along any excursion and whenever we passed a knotweed stand I would pass forward my knowledge of it`s fascinating culinary attributes as I was taught by Monsieur Dumaine and Euell Gibbons.

In order to expand my monologue I started surfing the internet and learned that Fallopia japonica found mention mostly on gardening websites where it`s victims exchanged their experiences about the plants eradication or vice versa their failure to control it`s growth.

Knotweed populations everywhere. A futuristic prediction would be that this plant may change the face of our landscape. Why let this precious regional produce go to waste ?
While counting hundreds of populations in the Wiesbaden Area I learned that several nearby wetlands were infested even worse and what damage common ignorance causes. Every m2 of Knotweed growth produces an average 12-15 Kg of biomass.

Up to a hundred dry hollow stalks persist the elements for years to come, withdraw and conserve the soils nutriments.

While I found numerous larger monopolizing patches where the native habitat has been completely displaced,

I discovered several invasive species websites dedicated to Knotweed management all collectively ignorant to the fact that Knotweed is first and foremost a fantastic foodsource; which`s became an invasive weed due to a lack of natural enemies.

Although it`s been predicted that knotweed`s in the process of a culinary boom as Wild Garlic has been going through in the last fifteen years and proven by the fact that ever more often the plant finds mention in cookbooks, the idea to connect it`s edibility against it`s invasive attributes, has not been really recognized yet. Mainly because specialists und consumers generally harvest the sprouts only over a span of ten days before it get`s to fibrous. Here, my semi-cultivation makes all the difference.

In 2003 I had my booklet My Appetite; Your Herbicide completed, eager to offer advice and avoid further damage of our ecosystem by unnecessary, losses to native plant societies and waste of potential capital.

But to volunteer with local control programs turned out to be the hardest part; of establishing my Bionic Knotweed Control.

Specialists all over the world agree that a manual control is virtually impossible and that successful management usually requires herbicides.

Due to it`s vigorous growth of, especially in watersheds, up to 30 cm a day, harvest by wildfoodists has traditionally been limited to a span of probably 10 days; before the stalks become to fibrous. That`s why knotweed has never become a popular produce item of any culinary or agricultural importance. Here`s the significant difference between plain destructive mowing and disposal of the biomass, compared to semi-cultivating designated populations to reap an agricultural value and renature it`s native inhabitants.

While the manual control is hard labour, the bionic treatment has an instant symptomatic impact on the plants aggressive tendency because once your action is a harvest and your supplied with it ́s beneficial profit; the entire measure gains a constructive and very motivation aspect which no other mean of control can match.

In order to handle the advance of the knotweed invasion the most sustainable way, I suggest to semi-cultivate any population that suitable for human consumption in terms of the “Bio” standard. That way a good percentage of general knotweed control may be financially covered and communities would be able to save a lot of tax money presently squandered by temporary means.

Each designated population is thoroughly cleaned, all stalks removed, burned and the ashes, after Phase 1, returned to the soil before the process of renaturing begins.
To attempt the control of a full grown population is useless because the organism has already began it`s secondary metabolism and all sprouts are too fibrous for Knotty Foods

The tolerable leaf development during harvest (Phase 2). This can only be accomplished by daily operation throughout the entire 10 weeks of the plants primary growth period.
After the harvest, the populations have to be weeded from all the tiny sprout bushels representing a secondary sort of growth that develop their leafs immediately upon surfacing. These sprouts are collected for the isolation of resveratrol and cosmetic application.

The sprouts reach a length of 30-40 cm a day and the average yield is 70 g per day and m2..

…In 2004 four populations were threatening to form a huge thicket to monopolize the meadow and displace the entire fauna and flora.

In 2005 the Bionic Control began and throughout the season all leaf development was inhibited to weaken the plant, while 7 kg of knotweed- sprouts per m2 were the average harvest.

In 2006 the combined effect of semi-cultivation and renaturing of the native plant- society has proven that this sustainable method is faster than all other current management approaches and yields a profit of 140 Euros per m2.



Patents : Extraction of Resveratrol &c from Knotweed


Method for extracting resveratrol from giant knotweed by enzymolysis
CN104087623

The invention relates to a method for extracting resveratrol from a natural plant giant knotweed, particularly a method for extracting resveratrol from giant knotweed by enzymolysis. The method comprises the following steps: by using commercially available giant knotweed rhizome as the raw material, carrying out enzymolysis reaction, alcohol deep extraction and separation concentration, and drying to obtain the resveratrol crystal powder product. The method has the characteristics of sufficient enzymolysis, short time, low operating cost, high operation safety and the like, fully utilizes the raw materials and organic solvent, is beneficial to environmental protection since the waste liquid is mainly water, and can easily implement industrialized popularization and application. The resveratrol crystal powder product prepared by the method has high purity, and the maximum purity is 98.31%. The overall yield is high, and the maximum overall yield is 1.40%. The method can be widely used for producing the resveratrol crystal product. The product prepared by the method can be widely used in fine chemical engineering production of medical drugs for preventing cardiovascular diseases, protecting the liver and resisting cancers and skin-care beauty-treatment products.

An enzymatic method of extraction from Polygonum cuspidatum resveratrol, relates to a method for preparing natural plants from Polygonum cuspidatum resveratrol. The present invention is a commercially available Polygonum cuspidatum rhizome as raw material, enzymatic reactions, the depth of extraction and separation of the alcohol concentration, the dry process is simple and prepared resveratrol crystalline powder products. The method of the present invention has fully digested and the time is short, low operating costs, operating safety, make full use of raw materials and organic solvents, mainly for waste water, are environmentally friendly, easy industrial application and so on. Prepared by the method of the present invention, resveratrol crystalline powder products with high purity, up 98.31 percent; total yield, up 1.40% and so on. The present invention can be widely used in the production of resveratrol crystal products; prepared by the method of the present invention can be widely used in the prevention of cardiovascular disease and liver cancer drugs for medical and skin care cosmetics fine chemical production.

I. Technical Field

The present invention belongs to the field of resveratrol extracted from natural plants, and more particularly from natural plant Resveratrol Polygonum cuspidatum preparation method.

BACKGROUND

Technical

Resveratrol is a polyphenolic compound containing a non-flavonoid stilbene structure, chemical name 3,4 ', 5-hydroxyl Stilbene group, white or pale yellow crystalline powder. Resveratrol not only prevent cancer cancer disease, but also pre Preventing cardiovascular and cerebrovascular diseases, with liver, antioxidant, regulating blood lipids balance, anti-aging and other health effects, is On human health have a significant positive effect of the active substance, and therefore it is widely used in medicine and health aspects. Since Resveratrol is a stilbene polyphenols substances, phenols and alkenes having properties when exposed to air Or lighting conditions, resveratrol may be oxidized or isomerization occurs irreversible transformation generated substance, physiologically active Destruction loss, so in order to improve the yield of resveratrol and extend storage time, production of resveratrol and Paul Deposit process more stringent requirements. Resveratrol exists in giant knotweed, peanuts, mulberries, grapes and other natural plant Branch And mainly relatively stable molecular form Polydatin inside the object. Especially in C. quinoa Polygonum cuspidatum Resveratrol and resveratrol glucoside total content higher than other natural plants, industrial production huge potential benefits. therefore, Resveratrol from Polygonum cuspidatum extract is the focus of today's study.

Existing extracted from Polygonum cuspidatum resveratrol, such as April 17, 2013 published Publication No. CN 103044210A of "one kind of Resveratrol from Polygonum cuspidatum preparation of the production process" patent, a method is disclosed: In Polygonum cuspidatum as raw material, enzyme dry, continuous countercurrent extraction tank acetone reflux extraction, filtered and concentrated, ethyl acetate Ester extraction, concentration, chromatography on silica gel, and concentrated to obtain crystals of resveratrol crystal products. The main disadvantage of this method Is: ① constant enzymatic reaction time is long, long time high temperature is not conducive to the stability of the chemical structure of resveratrol; ② Since the exposure of resveratrol in the light conditions to reduce the content of volatile leaving live, so after drying operation is not digested Conducive to the stability of the chemical structure of resveratrol; ③ due to the low boiling point of acetone, volatile, with a certain toxicity, Mixed prone to the risk of explosion with air, so its a countercurrent extraction solvent, the insulation and the corresponding device Sealing relatively higher; ④ using ethyl acetate - petroleum ether as the eluting solvent separation, wherein toxic petroleum ether Large and flammable solvent recovery process involves a big security risk.

III. SUMMARY OF THE INVENTION

The present invention is directed to an existing shortage extracted from Polygonum cuspidatum resveratrol method provided a tiger from The method of enzyme extract resveratrol rod having fully digested, and time is short, simple equipment, into operation The low, safe operation, environmental protection, easy to promote industrial production and so on.

The main principles of the present invention are: Resveratrol from Polygonum mainly in the form of the presence of resveratrol glucoside, and its molecular Formula C 20 H 22 O 8, the chemical name 3,4 ', 5-trihydroxy-stilbene -3-β-D- glucoside, which in the hot water bath It has good solubility, soluble in organic solvent such as methanol, ethanol and the like. Suitable pH values ​​at a certain temperature and stirred Under the conditions, Piceid gradually dissolved from Polygonum cuspidatum, while the action of the enzyme solution in the β-D polydatin Glycosidic bond to hydrolysis resveratrol (C 14 H 12 O 3) and glucose. In addition, the enzyme can also damage the plant Polygonum cuspidatum Cell wall, is conducive polydatin dissolution and transformation.
Achieve the object of the present invention is technical solution: A method enzyme extracted from Polygonum cuspidatum resveratrol to Commercially available Polygonum cuspidatum rhizome as raw material, enzymatic reactions, the depth of extraction and separation of the alcohol concentration, drying the simple process Resveratrol prepared crystalline powder products. Specific steps of the method are as follows:

(1) enzymatic reaction

The first commercially available feedstock is placed in the roots of Polygonum cuspidatum mill pulverized, and then by 240 ~ 320 mesh sieve into Line sub-screen, to collect the powder sieved, spare, unscreened particles return mill pulverized. then, Polygonum cuspidatum powder mass (g): mass cellulase (mg): acetic acid - sodium acetate buffer solution volume (mL) of Ratio of 1 (1 to 10): (10 to 50), the ratio, first with concentrated hydrochloric acid or sodium hydroxide solution to adjust the buffer solution pH value was 4-6 after the Polygonum cuspidatum powder and adjusting pH value of buffer solution, followed by placement in a batch Reactor, at 30 ~ 60 ° C and the thermostat 80 ~ 200r / min speed, after stirring for 15 ~ 30min, plus The cellulase, continued constant stirring, enzymatic reaction carried out 15 ~ 30h, to obtain enzymatic reaction after the solid-liquid mixture Compound.

(2) an alcohol depth extraction

Paragraph (1) after the step is complete, the first paragraph (1) a solid-liquid mixture obtained in step enzymatic reaction after the filter is placed in The solid-liquid separation, the filtrate was collected separately (i.e. the filtrate extracted after hydrolysis) and the solid residues. After following The solid residue mass (g): the volume fraction of the volume of a methanol solution of 50% to 75% (mL) of the ratio of 1 (5-20) The proportion of the solid residue in methanol and placed in the extraction tank, at a temperature of 35 ~ 65 ° C, For the first stirring speed is 80 ~ 200r / min in a condition after the alcohol extract 15 ~ 60mm, and the first Filtered, collected the first alcohol extract filtrate and solid residues, respectively. Then, take the first alcohol extract equal time Volume, equal concentrations of methanol solution and the solid residue was collected for the first time, and then back into the extraction tank at a temperature To 35 ~ 65 ° C, stirring speed of 80 ~ 200r / min under conditions of a second alcohol extract after 15 ~ 60min, And a second filter, and the solid residue was collected by filtration second alcohol extract respectively, so repeat 1-3 times. The solid residue was used to extract the depth of alcohol. Finally, each sub-alcohol extract combined filtrate collected, placed in evaporator Concentrator, evaporative concentration to no alcohol taste so far collected concentrate and solvent evaporation, respectively. Collected Solvent (ie methanol), can be reused. The combined filtrate and enzyme extracted various times alcohol extract collected concentrate To obtain resveratrol crude liquid separation and purification for the next step.

(3) Preparation of Resveratrol crystalline powder products

Initial separation and elution macroporous resin

Paragraph (2) after completion of step, according to the macroporous resin mass (g): Volume (2) obtained in step resveratrol crude liquid (ML) of the ratio of 1 (6-10) ratio by a conventional macroporous resin filling method, macroporous resin filled in The separation column, with constant flow pump, a volume of 1 ~ 3BV (that is, 1 to 3 times the bed volume of the filler), the volume Fraction of 10 to 20% ethanol, a flow rate of 1 ~ 3BV / h is passed into the interior of the separation column, were rinsed Run and collect effluent wash, recovery of ethanol solvent by evaporation, can be reused. Then, said resveratrol Crude alcohol solution at a flow rate 1 ~ 3BV / h is passed into the separation column to adsorb and collect effluent solution. Emissions after solution treatment collection compliance. Then, a volume of 2 ~ 5BV distilled water to 1 ~ 3BV / h Within the separation column flow was passed through the first elution for the unadsorbed component (mainly sugar and Vegetable protein substances) elution, collecting the first time out of the eluent, the treated discharge standards. Then again, 3 ~ 5BV first volume, the volume fraction of 20 to 30% ethanol, a flow rate of 1 ~ 3BV / h through The separation column into the second elution portion for the pigment elution, the second outflow Eluent, recovering ethanol solvent by evaporation, can be reused. After 3 ~ 5BV volume, the volume fraction of 30 to 50% ethanol, a flow rate of 1 ~ 3BV / h through the separation column into the third elution, The substance is used Polydatin elution, collecting the third out of the eluent and concentrated by evaporation is carried out Concentrated by evaporation, collected evaporative liquid (ie, ethanol solvent) and concentrate (ie Polydatin enriched concentrate), respectively. Polydatin enrichment of concentrate collected reserved for other uses. Then the volume was 4 ~ 10BV volume fraction 60 to 80% ethanol, and then at a flow rate 1 ~ 3BV / h were passed into the fourth eluting said separation column, Resveratrol for eluting material, the fourth effluent was collected eluate was concentrated by evaporation by means of evaporation of the concentrated Shrink, were collected and evaporated liquid (ie, ethanol solvent) and concentrate (ie resveratrol-rich concentrate). Collected The evaporative liquid (ie, ethanol solvent) can be reused; to concentrate collected (ie resveratrol-rich concentrate) The next step for the preparation of resveratrol crystal products. Macroporous resin within the separation column by conventional hydrogen Sodium hydroxide - Hydrochloric acid regeneration process can be recycled. Said macroporous resin NKA-9 type or NKA-II-type or AB-8 type or HPD950 type or HPD400 type.

Preparation of Resveratrol crystalline powder products

Article (3) -① step after completion, according to the quality of neutral alumina (g): Article (3) -① step collected concentrate (i.e. Resveratrol concentrate enriched) has a volume (mL) of the ratio of 1 (10 to 20), the ratio, by a conventional method to fill Neutral alumina packed into a chromatography column, the first with constant flow pump, a volume of 1 ~ 3BV of volume fraction 60 to 80% ethanol, the flow rate of 1 ~ 3BV / h is passed into the rinse chromatography on a column, collecting the flow Run out of the wash, and concentrated by evaporation, evaporator recovering ethanol solvent recycling. Then the respective concentration Condensing fluid (i.e. resveratrol enriched concentrate) 1 ~ 3BV and volume, the volume fraction of 60 to 80% B Alcohol solution, the flow rate of 1 ~ 3BV / h is passed into the column decolorized effluent collected stainer (i.e. Resveratrol solution after bleaching). Then, the collected destaining solution (ie resveratrol solution after bleaching), Concentrated by evaporation of the evaporator concentrate collected evaporative liquid (ie, ethanol solvent), respectively, and a concentrated liquid (ie resveratrol Alcohol concentrate). Evaporation of liquid (ie, ethanol solvent) collected reusable; to concentrate collected (ie, Resveratrol concentrate) is obtained first solution purified resveratrol. Finally, according to the activated carbon Mass (g): the first volume of the solution was purified resveratrol (mL) of the ratio of 1 (50 to 100) ratio, the first A purified resveratrol solution and activated carbon, placed in the bleaching tank, the mixture was stirred at room temperature for the first 1 ~ 5h Secondary purification (purified through active carbon), the mixture of the second purified subjected to suction filtration points Do not collect smoke residue (ie activated charcoal solid) and the filtrate pump (second solution is resveratrol purified), The collected smoke residue for recycling, reuse. The filtrate was pumped (ie, the second purified resveratrol Solution), concentrated by evaporation, a concentrated solution to be concentrated by evaporation before the concentration by volume of the volume of 1/5 ~ 1/30 So far were collected evaporative liquid (ie, ethanol solvent) and concentrate (ie, the second purified resveratrol concentration Condensing fluid). Evaporative liquid (i.e., an ethanol solvent) collected reusable; of concentrate collected (i.e., the second Resveratrol concentrate purified), placed in a freezer oven, first at -10 ~ -18 ° C pre-freezing 2 ~ 3h, And then at a pressure of 20 ~ 60Pa, at the temperature of -40 ~ -60 ° C conditions, freeze-dried 24 ~ 30h, to obtain Quality purity of 98.07% ~ 98.31%, a yield of 1.12% to 1.40% of the crystalline powder resveratrol products.

After adopting the technical scheme of the present invention, there are the following effects:

1. The present invention is commercially available Polygonum cuspidatum rhizome as raw material, after grinding to a certain mesh, cellulase and Under certain conditions the role of a buffer solution of pH value, enzyme converted to resveratrol, and then into a solution of methanol Line depth extraction, and therefore the total yield of resveratrol product, the total yield up to 1.40%;

2. Macroporous resin separation process of the present invention, a different volume fraction (ie, 20 to 30% 30 to 50%, 60 to 80%) in ethanol solution were adsorbed resveratrol crude resin washed punch Off, and finally has the further use of alumina and activated carbon decolorization impurity, so the quality resveratrol products High purity, the highest purity of 98.31%.

3. Cellulase of the present invention uses less, at least the amount of enzyme converted into dry powder Polygonum cuspidatum count about 1 ‰, Fully digested and the time is short, about 15 to 30 hours, greatly shorten the production cycle, reduce production cost, easy push Wide industrial production;

4. Methanol extract of the present invention take the first solid-liquid separation section extracting the way, a significant reduction in methanol solution The amount of solvents to improve extraction efficiency and yield of Polygonum cuspidatum resveratrol raw materials;

5. The present invention is mainly used in the organic solvent is methanol and ethanol, low toxicity solvent, the boiling point of methanol and ethanol Moderate, easy recycling, make full use of organic solvents and saving energy consumption, mainly waste water, in line with environmental Production requirements and safe operation;

6. The present invention, since the colored impurities are mainly flavonoids and easy to combine with Al 2 O 3, so a Al 2 O 3, and with the use of activated carbon has a method for purification of resveratrol, resveratrol efficient removal of enrichment Parts of residual colored impurities; Furthermore, Al 2 O 3 directly at 500 ~ 700 ° C was calcined 1 ~ 2h, on renewable Recycling, emissions-free, easy to operate. After Al 2 O 3 to remove most of the pigment and impurities, residual color Prime only a small amount of activated carbon can be removed, reducing the amount of active carbon used, further reducing production costs.

The present invention can be widely used in the production of resveratrol crystal products, the method of the present invention, products can be Widely used in the prevention of cardiovascular disease and liver cancer drugs for medical and skin care and beauty fine chemical products.

detailed description

Below in conjunction with specific embodiments, the present invention further.

Example 1

An enzymatic method specific steps to extract from Polygonum cuspidatum resveratrol, the method is as follows:

(1) enzymatic reaction

The first commercially available feedstock is placed in the roots of Polygonum cuspidatum mill pulverized, and then dividing by 280 mesh sieve Sieve powder, spare, unscreened screened to collect particles and then return mill pulverized. Then, Quality Polygonum cuspidatum powder (g): mass cellulase (mg): acetic acid - sodium acetate buffer solution volume (mL) ratio 1:2:20 proportion of, first with concentrated hydrochloric acid or sodium hydroxide solution to adjust the pH of buffer solution 4.7 After the powder Polygonum cuspidatum buffer solution to adjust the pH value and, in turn placed in a batch reactor at 45 ° C At constant and 200r / min speed, stirring 20min, and then added cellulase, continued constant stirring, into Line enzymatic reaction 20h, the solid-liquid mixture to obtain enzymatic reaction after.

(2) an alcohol depth extraction

Paragraph (1) after the step is complete, the first paragraph (1) a solid-liquid mixture obtained in step enzymatic reaction after the filter is placed in The solid-liquid separation, the filtrate is collected (i.e. the filtrate extracted enzyme), respectively, and the solid residue (i.e. enzymes Polygonum cuspidatum powder solution after the reaction). After a mass of solid residue (g): a volume fraction of 65% methanol solution of Volume (mL) ratio of 1:10 ratio, to the solid residue in methanol and placed in the extraction tank At a temperature of 45 ° C, stirring speed after the first alcohol extract 30min under 200r / min conditions, and Filtration for the first time, collected the first alcohol extract filtrate and solid residues, respectively. Then, take the first alcohol Equal volumes picked up on time, and equal concentrations of methanol solution and the solid residue was collected for the first time, and then back into the extraction tank At a temperature of 45 ° C, stirring speed after the second alcohol extraction 30min under 200r / min conditions, and A second filter for collecting filtered liquid and solid residues second alcohol extraction respectively, repeat once with In the depth of the solid residue of alcohol extract. Finally, each sub-alcohol extract combined filtrate collected, concentrated by evaporation in place Reduction reactor, evaporative concentration to no alcohol taste so far collected concentrate and solvent evaporation, respectively. The collected Solvent (ie methanol), can be reused. The combined filtrate concentrated liquid extract after enzymatic hydrolysis and each sub-alcohol extract collected on Resveratrol obtained crude liquid separation and purification for the next step.

(3) Preparation of Resveratrol crystalline powder products

initial separation and elution macroporous resin

Paragraph (2) after completion of step, according to the macroporous resin mass (g): Volume (2) obtained in step resveratrol crude liquid (ML) of the ratio of 1:6, the first conventional macroporous resin filling method, the macroporous resin is filled into the separation column The constant current pump, a volume of 2BV, the volume fraction of 20% ethanol, to 2BV / h of flow rate Inside passage into said separation column for rinse, wash and collect effluent run, recovery of ethanol solvent by evaporation, Reusable. Then, the resveratrol crude liquid to flow 1BV / h is passed into the separation column Adsorption and collect effluent solution. Emissions after solution treatment collection compliance. Then, the volume of 3BV of distilled water, the flow rate 1BV / h is passed into the separation column was first eluted, not for the Adsorption component (mainly sugar and vegetable protein substances) elution, collecting the first time out of the eluant Treatment discharge standards. Then again, the first volume of 4BV volume fraction of 25% ethanol, to 2BV / h flow through into the inside of the second separation column eluting for the pigment portion of elution, collecting Second outflow eluent recovering ethanol solvent by evaporation, can be reused. After 4BV volume of body Fraction of 45% ethanol, the flow rate of 2BV / h is passed into the third separation column eluted, The substance is used Polydatin elution, collecting the third out of the eluent and concentrated by evaporation is carried out Concentrated by evaporation, collected evaporative liquid (ie, ethanol solvent) and concentrate (ie Polydatin enriched concentrate), respectively. Polydatin enrichment of concentrate collected reserved for other uses. Then 8BV volume, the volume fraction of 70% Of ethanol, and then to the 2BV / h flow into said separation column was eluted fourth for the White Resveratrol substance elution, collecting the fourth flowing eluent and concentrated by evaporation of the evaporator concentrate, respectively Collected evaporative liquid (ie, ethanol solvent) and concentrate (ie resveratrol-rich concentrate). Evaporation of liquid collected (Ie, ethanol solvent) can be reused; the collected concentrate (ie resveratrol-rich concentrate) for the next Step preparation of resveratrol crystal products. Macroporous resin within the separation column by conventional sodium hydroxide - Hydrochloric acid regeneration process, can be recycled. Said macroporous resin NKA-9 type.

Preparation of Resveratrol crystalline powder products

Article (3) -① step after completion, according to the quality of neutral alumina (g): Article (3) -① step collected concentrate (ie, white Resveratrol-rich concentrate) volume (mL) of the ratio of 1:15, using conventional filling method neutral oxygen After the aluminum is filled into a chromatography column, first with a constant flow pump, a volume of 2BV, the volume fraction of 70% ethanol Solution, the flow rate of 2BV / h is passed into the column were rinsed out of the collected washings were run, and through Concentrated by evaporation, evaporative recovery of ethanol solvent recycling. Then the respective concentrate (ie resveratrol Concentrate Enrichment) and 2BV volume, the volume fraction of 70% ethanol at a flow rate 2BV / h through Into the chromatography column within decolorization, discoloration collected effluent liquid (ie resveratrol solution after bleaching). However After decoloration was collected (ie resveratrol after bleaching solution), concentrated by evaporation of the evaporator concentrate, divided Do not collect evaporative liquid (ie, ethanol solvent) and concentrate (ie resveratrol concentrate). Evaporation of liquid collected (ie, Ethanol solvent), reusable; the collected concentrate (ie resveratrol concentrate) is first prepared A solution of purified resveratrol. Finally, according to the activated carbon mass (g): first purified resveratrol Volume (mL) ratio is the ratio of 1:75 in a solution, the first solution resveratrol purified and activated carbon, Placed in the bleaching tank, and at room temperature for 3h second purification (purified through active carbon) After Effects Second purified mixture subjected to suction filtration to obtain a solution of resveratrol second purified. And then the second Times the volume of the resveratrol was purified, concentrated by evaporation, a concentrated solution to be concentrated by evaporation with concentrated After shrinking the volume of 1/5 up front, were collected and evaporated liquid (ie, ethanol solvent) and concentrate (ie, a second purification Resveratrol concentrate). Evaporative liquid (i.e., an ethanol solvent) collected reusable; the collected and concentrated Liquid (ie resveratrol concentrates second purified), placed in a freezer oven, first in pre-freezing -18 ° C 2h, and then at a pressure of 20Pa, temperature conditions at -50 ° C, lyophilized 27h, to obtain the quality of purity 98.31%, a yield of 1.40% resveratrol crystalline powder products.

Example 2

An enzyme extracted from Polygonum cuspidatum resveratrol method, the same as in Example 1, wherein:

Paragraph (1) step, the sub-240 mesh sieve, quality Polygonum cuspidatum powder (g): mass cellulase (mg): acetic acid - Sodium acetate buffer solution volume (mL) ratio of 1:1:10, adjusting the pH of the buffer solution to 4.0, in At 60 ° C constant temperature, stirring speed 150r / min rotation speed conditions, the enzymatic reaction 30h.

(2) step, the mass of the solid residue (g): the volume of methanol solution of 50% volume fraction (mL) ratio of 1:20, a methanol solution of 50% volume fraction, at a temperature of 35 ° C, the stirring speed is 150r / min speed Under the conditions, for the first time alcohol extraction 60min. At a temperature of 35 ° C, the stirring speed is 150r / min speed of strip Next member, a second alcohol extraction 60min, so repeated twice.

Article (3) -① step, macroporous resin mass (g): Volume (2) obtained in step resveratrol crude liquid (mL) Ratio of 1:8, when rinsed ethanol content was 10% by volume of ethanol 1BV, rinse flow Is 1BV / h; on column flow resveratrol crude liquid was 2BV / h; first elution, the elution volume of distilled water Is 2BV, flow 2BV / h; eluted second, the volume fraction of ethanol solution of 20% ethanol body 3BV volume of the elution flow rate 1BV / h; the third elution, the volume fraction of ethanol solution of 30% acetic Alcoholic liquid is 5BV, elution flow rate 1BV / h; in the fourth elution, ethanol solution of 60% volume fraction, Ethanol volume of 10BV of the elution flow 3BV / h; on the macroporous resin AB-8 type.

Paragraph (3) -② step quality neutral alumina (g): Article (3) -① step collected concentrate (ie resveratrol Enriched concentrate) has a volume (mL) ratio of 1:10; when rinsed ethanol volume fraction of 60% ethanol Volume of solution 3BV, rinse flow rate 1BV / h; decolorization, ethanol volume fraction of 60% ethanol Resveratrol solution after the first purification: activated carbon mass (g); volume of solution 1BV, flow rate 1BV / h The volume (mL) ratio of 1:50, was stirred at room temperature 1h, evaporated concentrate volume was concentrated to a volume of 1/15 the front; When freeze-drying, to pre-freezing at -14 ° C 2.5h, and then at a pressure of 40Pa, temperature conditions at -60 ° C, frozen Drying 30h, to obtain a purity of 98.07% mass yield of 1.12% resveratrol crystalline powder products.

Example 3

An enzyme extracted from Polygonum cuspidatum resveratrol method, the same as in Example 1, wherein:

Paragraph (1) step, the sub-320 mesh sieve, quality Polygonum cuspidatum powder (g): mass cellulase (mg): acetic acid - Sodium acetate buffer solution volume (mL) ratio of 1:10:50, adjusting the pH of the buffer solution to 6.0, in At 30 ° C constant temperature, stirring speed is 80r / min rotation speed conditions, the enzymatic reaction 15h.

(2) step, the mass of the solid residue (g): the volume of methanol solution of 50% volume fraction (mL) ratio of 1:5, a methanol solution of 75% volume fraction, at a temperature of 65 ° C, the stirring speed is 80r / min bar speed Under parts, for the first time an alcohol extract 15min. At a temperature of 65 ° C, the stirring speed is 80r / min rotation speed conditions, A second alcohol extraction 15min, so repeated 3 times.

Article (3) -① step, macroporous resin mass (g): Volume (2) obtained in step resveratrol crude liquid (mL) Ratio of 1:10, when rinsing, ethanol volume fraction of 15% ethanol solution volume 3BV, rinse stream Amount 3BV / h; the column flow resveratrol crude liquid was 3BV / h; first elution, eluted with distilled water body Volume of 5BV, flow 3BV / h; eluted second, the volume fraction of ethanol solution of 30% ethanol solution 5BV volume of elution flow 3BV / h; in the third elution, ethanol solution of 50% volume fraction, Ethanol body is 3BV, elution flow 3BV / h; in the fourth elution, the volume fraction of the ethanol solution 80% ethanol solution volume 4BV the elution flow 1BV / h; on the macroporous resin NKA- II type.

Paragraph (3) -② step quality neutral alumina (g): Article (3) -① step collected concentrate (ie resveratrol Enriched concentrate) has a volume (mL) ratio of 1:20; when rinsed ethanol volume fraction of 80% ethanol Volume of solution 1BV, rinse flow 3BV / h; decolorization, ethanol volume fraction of 80% ethanol Resveratrol first purified solution: activated carbon mass (g); volume of solution 3BV, flow 3BV / h The volume (mL) ratio of 100, with stirring at room temperature 5h, concentrated by evaporation of the liquid volume before concentration of 1/30 volume; When freeze-drying, the first pre-freezing in -10 ° C 3h, then at a pressure of 60Pa, a temperature of -40 ° C conditions, freeze dry Dry 24h, to obtain a purity of 98.15% mass yield of 1.12% resveratrol crystalline powder products.



Novel method for extracting resveratrol from giant knotweed
CN104263763

The invention relates to a novel method for extracting resveratrol from giant knotweed, which comprises the following steps: a. pulverizing giant knotweed, and passing through a 40-60-mesh screen; b. carrying out enzymolysis with composite enzymes; c. carrying out continuous reflux ultrasonic assisted extraction; d. passing through a macroporous resin column; and e. crystallizing. By using the giant knotweed as the raw material, the method fully utilizes enzymolysis to convert resveratrol glycoside in the raw material into resveratrol, and uses a continuous reflux ultrasonic process to extract the resveratrol, thereby achieving the goal of enhancing the resveratrol yield. The method has the advantages of simple technique, short production cycle, high yield and the like, and is suitable for large-scale production.


The present invention relates to a new method for extracting resveratrol from Polygonum cuspidatum, which process comprises the steps: a. Polygonum cuspidatum sifted through 40 to 60 mesh sieve; b. Enzymatic hydrolysis; c. Continuous countercurrent ultrasonic assisted extraction; d. Macroporous resin column; e. crystallization. The present invention uses as raw material Polygonum cuspidatum, take full advantage of the raw material in the enzymatic conversion of resveratrol resveratrol glucoside, and then taken out in a continuous countercurrent ultrasonic Bo Fati resveratrol, in order to improve the yield of resveratrol purpose. The invention has simple process, short production cycle, yield advantages for large-scale production.

TECHNICAL FIELD

The present invention is in the field of natural plant extraction and separation, a new method of extracting resveratrol from Polygonum cuspidatum relates, in particular, relates to an enzymatic hydrolysis - continuous countercurrent method of ultrasonic extraction of resveratrol.

Background technique

Polygonum cuspidatum Polygonum roots and root dry perennial herb. Bitter, slightly cold, antibacterial, anti-viral and anti-tumor efficacy, extensive clinical application. In recent years, effective ingredients in Polygonum has conducted in-depth studies, resveratrol, anthraquinone compounds and flavonoids as its main active ingredient, with the highest content of resveratrol. Modern pharmacological experiments show that resveratrol has anti-bacterial anti-inflammatory, lowering blood pressure, prevention of cardiovascular disease, anti-tumor activity of inhibiting platelet physiology.

Currently, the extraction method of Resveratrol from Polygonum mainly ethanol extraction, alkaline extraction and acid precipitation, supercritical CO 2 extraction method, there is a lack of these technologies, such as long extraction, extraction time, solvent large amount of low extraction; alkali extraction and acid precipitation, extraction rate, complex composition; supercritical extraction require special equipment, high pressure, high operating costs, and polar extracts have certain requirements.

Resveratrol Polygonum mainly in the form of glycosides. In recent years, cellulase abroad widely used in various fields in the country will be applied to herbal extract still in its infancy. Most of the active ingredients of Chinese herbal medicines have been wrapped in the plant cell wall, the cell wall is cellulose, and cellulose to glucose is from the β- 1,4-β-D- glucoside linkage of a glucose-β-D- composed of cellulose enzymatic hydrolysis will generate glucose degradation polydatin lose Polydatin yuan to improve the extraction rate of resveratrol. Based on the above principles, the present invention is to extract resveratrol provides a new approach and provide the basis for the application of cellulase in the extraction of active ingredients of traditional Chinese medicine.

SUMMARY

Object of the present invention to overcome the deficiencies of the prior art and to provide a hydrolysis - continuous countercurrent ultrasonic extraction method resveratrol, resveratrol improves dissolution rate and shorten the extraction time, the process is simple, suitable for large-scale produce.

The present invention comprises the following steps:

a. Take raw material Polygonum cuspidatum, crushed, over 40 to 60 mesh sieve;

b. The pulverized to better Polygonum cuspidatum adding 2 to 4 times as much water, sodium acetate / acetic acid buffer solution adjust PH 4.0 to 5.0, then add the weight of the raw material 3 ~ 5 ‰ of cellulase and pectinase (ratio of 0.8: 0.2), temperature at 45 ~ 55 ° C, stirring, reaction time of 2 ~ 3h;

c. The enzymatic conversion of materials added after 3 to 5 times of 80 to 90% ethanol into a countercurrent extraction apparatus extracts the ultrasound, ultrasound power 150 ~ 250W, extraction time is 60 ~ 90min;

d. The extract was centrifuged in step c, the supernatant was removed, pretreated macroporous resin adsorption column, first with distilled water and cleaning, with 50 to 70% 5 ~ 7BV ethanol eluate, concentrated under reduced pressure to obtain a crude product;

e. The crude product obtained in step d was repeated with crystallized twice from ethanol to obtain resveratrol.

Pretreatment step d macroporous adsorption of the present invention, resin is first with ethanol wet packed column, the column flow continues with ethanol cleaning, always check the outflow of ethanol solution, when out of the water and ethanol solution opaque white color mixing phenomenon can not, then wash away with plenty of distilled ethanol, to no alcohol taste.

The present invention is described in step d macroporous resin model NKA-9, H1020, NKAⅡ, D-101.

In the invention, the cellulase full destruction of plant cell wall structure, the active ingredient intracellular easy dissolution, while Polygonum cuspidatum resveratrol glucoside in the role of cellulase, converted into resveratrol, make resveratrol greatly improve the yield of alcohol; and pectinase can periwinkle and other insoluble pectin was dissolved. Recycling countercurrent extraction technology and ultrasonic assisted extraction technology to accelerate the dissolution of active ingredients from Polygonum powder, facilitated diffusion and mass transfer of the solvent to improve the extraction rate of resveratrol from Polygonum cuspidatum, shorten the extraction time and reduce solvent usage, simple process, high yield.

It will be further described with reference to specific embodiments of the present invention, but the scope of protection of the present invention is not limited to the following embodiments.

detailed description:

Example 1:

Polygonum cuspidatum material crushed through a 40 mesh sieve, taking powder 300g, was added 2 times water, sodium acetate / acetic acid buffer solutions adjust PH 4.0, then add 3 ‰ weight of the material of cellulase and pectinase (ratio of 0.8: 0.2), temperature at 45 ° C, stirring, hydrolysis time 3h, then enzymatic conversion material after adding 4 times 90% ethanol into the countercurrent extraction ultrasonic extraction device, ultrasonic power 200W, extraction time to 80min, liquid centrifugal extraction, the supernatant was removed, NKAⅡ pretreated macroporous resin adsorption column, first with distilled water and cleaning, with 50 percent of 5BV ethanol eluate, concentrated under reduced pressure to give the crude product, crystallized with ethanol was repeated twice to obtain resveratrol 2.55g, yield of 0.85%, a purity of 98.1%.

Example 2:

Polygonum cuspidatum material crushed through a 40 mesh sieve, taking powder 300g, was added 4 times as much water, sodium acetate / acetic acid buffer solutions adjust PH 5.0, then add 4 ‰ weight of the material of cellulase and pectinase (ratio of 0.8: 0.2), temperature at 50 ° C, stirring, hydrolysis time 2.5h, then the material is added after the enzymatic conversion of 80% ethanol and 5 times into the countercurrent extraction ultrasonic extraction device, ultrasonic power 150W, extraction time is 90min, liquid centrifugal extraction, the supernatant was removed, NKA-9 macroporous resin adsorption on the pretreated column, first with distilled water and removing impurities, and then 70% ethanol 6BV the eluate under reduced pressure. concentrated to give crude repeated crystallization with ethanol twice, to give resveratrol 2.76g, yield 0.92%, a purity of 98.6%.

Example 3:

Polygonum cuspidatum material crushed through a 40 mesh sieve, taking powder 300g, water was added 3 times, sodium acetate / acetic acid buffer solutions adjust PH 4.5, then add the weight of the material 5 ‰ cellulase and pectinase (ratio of 0.8: 0.2), temperature at 55 ° C, stirring, hydrolysis time 2h, then enzymatic conversion material after adding 3 times 85% ethanol into the countercurrent extraction ultrasonic extraction device, ultrasonic power 250W, extraction time to 60min, liquid centrifugal extraction, the supernatant was removed, D-101 macroporous resin adsorption on the pretreated column, first with distilled water and removing impurities, and then 60% ethanol 7BV eluate concentrated under reduced pressure , crude, crystallized with ethanol was repeated twice to give resveratrol 2.88g, yield 0.96%, a purity of 97.9%. 



Extraction method for resveratrol from giant knotweed rhizome
CN103396295

The invention provides an extraction method for resveratrol from giant knotweed rhizome. Plants like giant knotweed rhizome, peanut rhizome, branches and leaves of grape and mulberry leaves having undergone enzymatic hydrolysis and fermentation can be used as raw materials. The extraction method comprises the following steps: adding superoxide dismutase according to the proportion of the raw materials and then adding an ethanol solvent with a concentration of 80% for extraction; combining obtained extracts, carrying out vacuum concentration and complete recovery of ethanol, adding a saturated chitosan solution into an obtained concentrate, and allowing an extract to be precipitated in a deposition manner; adding an ethanol solvent weighing 20 times of an obtained precipitate and having a concentration of 40% to dissolve the precipitate, then carrying out filtering, concentrating a filtrate, adding a chitosan solution into the concentrated filtrate and carrying out standing and centrifugation at normal temperature so as to obtain a solid precipitate; and adding an ethanol solvent weighing 20 times of the solid precipitate and having a concentration of 40% to dissolve the solid precipitate, then carrying out filtering, concentrating a filtrate, adding a chitosan solution into the concentrated filtrate, carrying out standing and centrifugation at normal temperature so as to obtain a solid precipitate and washing the solid precipitate with purified water until a washing liquid does not contain a pigment;; and successively carrying out vacuum drying and crushing so as to obtain a product with purity of 50 to 60%. Compared with a conventional production method, the extraction method provided by the invention enables yield of resveratrol to be increased by more than 30%.
 
The present invention provides a composition of resveratrol from Polygonum cuspidatum extract method used by the enzymatic fermentation good feed, for Polygonum cuspidatum, peanut roots, grape leaves, mulberry and other plants as a raw material. After the material is added in accordance with the proportion of added superoxide dismutase concentration of 80% ethanol solvent extraction, extracts were combined and concentrated in vacuo and complete closing of ethanol, the concentrate was added a saturated solution of chitosan to precipitate precipitate manner extract was added 20 times the weight of the precipitate was 40% ethanol solvent and dissolving the precipitate filtered, the filtrate was concentrated chitosan solution was allowed to stand at room temperature, centrifuged to give a solid precipitate. The precipitate was again added 20 times by weight of 40% ethanol solvent and dissolving the precipitate filtered, the filtrate was concentrated chitosan solution was allowed to stand at room temperature to give a precipitate solid was isolated by centrifugation, washed with purified water until the washing liquid amelanotic under component state, after vacuum drying pulverized acquired 50% -65% of the product; compared to conventional production methods, the yield can be increased by more than 30%.

An extract from Polygonum cuspidatum resveratrol component method

I. Technical Field

The present invention relates to a method for extracting resveratrol from Polygonum cuspidatum ingredients. Also applies to the peanut roots, Grape leaves, mulberry and other plants as raw material to extract resveratrol. Belonging to the field of biotechnology.

BACKGROUND

Resveratrol is a natural antioxidant, can reduce blood viscosity, non-platelet inhibition Normal clotting, prevention impatient cram, cerebral embolism, liver damage, ischemic heart protective effect and move Vein vasodilatation maintain blood flow and microcirculation, it can occur and prevent the development of cancer, has anti Atherosclerosis and coronary heart disease, ischemic heart disease, Alzheimer's disease, viral hepatitis, gastric ulcer and Enhance wound healing immune system activity, and inhibit tumor.

Preparation of resveratrol currently divided into chemical synthesis method, plant cell culture, microwave extraction, ultrasonic Extraction and solvent extraction method five categories; chemical synthesis and plant cell culture conditions harsh, complex Miscellaneous, low yield, difficult to implement industrialization; ultrasonic method and microwave method has also increased the role of yield but in This process, due to the special nature of the product easily oxidized raw product, not suitable, it is difficult to implement, use The most widely used method or plant extracts.

Now used in production methods can be divided into two processes; one is the alcohol extraction and then extracted with ethyl acetate Take, then the anti-alcohol extracted, filtered and concentrated and dried to give the product. The other is directly extracted with ethyl acetate, Then the anti-alcohol extracted, filtered and concentrated and dried to give the product. These existing technologies are present long working hours, the More equipment, solvent loss, the yield is not high, production consumption, high cost common drawback; more It is essential that the above method can not be excluded (blocked) due to the heat extraction process fully, so that raw materials remain in Among the enzymes accelerate the decomposition of low yield losses caused.

III. SUMMARY OF THE INVENTION

Object of the present invention is to provide an extract from Polygonum cuspidatum resveratrol component method, which uses a single Solvent extraction process is simple, efficient, low cost production process is short, high yield production methods.

To achieve the object of the present invention, the present invention employs the following technical implementation of the program:

(1), the enzymatic treatment by adding 1 ‰ good raw material weight of superoxide dismutase, 5 80% times the weight concentrations of ethanol solvent extraction, the temperature control at 45-65 ° C degrees, every 2 hours Total extracted four times;

(2) After the extract obtained above were combined, the temperature controlled at 55--60 ° C or less, and subjected to vacuum Concentrated and complete recovery of ethanol, the concentrate was added a saturated solution of chitosan in accordance with the raw material weight ratio of 5%, standing 8--12 hours at room temperature the extract to precipitate a precipitate manner, until complete precipitation was filtered, the solid interception Precipitated fraction;

(3), the solid precipitate, the precipitate was added 20 times the weight of the alcohol solvent is 40--45% ethanol, To precipitate dissolved and filtered, the insoluble matter was discarded, and the filtered liquid temperature control in real 55--60 ° C Empty state, concentrated to ethanol-free ingredients, add a saturated solution of chitosan material by weight proportion of 5%, in 8--12 hours standing at room temperature, centrifuged to obtain a precipitate of a solid;

(4), the precipitate was again added 20 times the weight of the alcohol degree of 40--45% ethanol solvent, the precipitate of Dissolved and filtered, the insoluble matter was discarded, and the filtered liquid temperature control heating 55--60 ° C in a vacuum state State and concentrated to ethanol-free ingredients, add a saturated solution of chitosan material weight ratio of 5%, at room temperature 8--12 hours standing, a precipitate obtained by centrifugation of the solid, to give a solid precipitate with pure water Washing liquid to wash the non-pigmented component status;

(5), after washing the precipitate by vacuum drying and crushing acquired 50% -65% of the product.

The method of extracting resveratrol from Polygonum cuspidatum ingredients, add the raw material weight of 1 ‰ superoxide Dismutase prevents the production process present in the feed plant for decomposing enzyme inactivated polydatin No longer occur over enzymatic decomposition; thereby protecting the losses during extraction of resveratrol ingredient

The method of extracting a component from Polygonum cuspidatum resveratrol, with 5% of the saturated solution of chitosan Quick guide precipitated liquid concentrate product.

The present invention is a method of extracting ingredients from Polygonum cuspidatum resveratrol, which measured by the process of amplification; its First, the extraction process continues to inhibit the enzyme added to the reaction decarboxylase agents 'superoxide dismutase' in Present in the raw material extraction process plant exploded polydatin enzyme activity for lose not occur over Enzymes break down; thereby protecting the losses during extraction of resveratrol ingredient than the existing industry (companies) may mention High yield of more than 30%; while reducing the content of the product produced by enzymatic hydrolysis of harmful free radicals; second, mining With 40% of the content of ethanol solvent with solvent from the chitosan solution to accelerate the entry into aqueous solutions of resveratrol Alcohol precipitation quickly and fully precipitated by the turbid liquid to solid form. Change the traditional way of the process 2-4 The day is part of the natural product precipitated within 12 hours to complete; Third, lack of emodin products 0.7% Far below the industry standard of 2.8% of the enterprise; anthraquinone impurity content is less than 1/10 of the traditional process.

The present invention is weight of raw materials added 1 ‰ of superoxide dismutase to prevent and protect during extraction over points Losses caused by the solution and reduce the content of the product due to hydrolysis to produce harmful free radicals; by adding 5% A saturated solution of chitosan fast boot concentrate precipitated. Superoxide dismutase and chitosan are non-toxic Add ingredients in the process does not cause unnecessary harm.

The present invention refers to components containing resveratrol Polygonum cuspidatum, peanut roots, grape leaves, mulberry leaves, etc. Plant as a raw material, especially Polygonum cuspidatum; weight ratio of the raw material of the present invention is added superoxide dismutase is insoluble Form solution state, chitosan is dissolved in water refers to a saturated solution. Due to the purity of industry products Typically 50% of the content of the specification is not very high, the product contains half of the accounting process using soluble impurities so Tim Plus chitosan solution as a guide to speed up the process of precipitating agent production process and accordingly reduced working hours.

The present invention provides a method for extracting ingredients from Polygonum cuspidatum resveratrol, its existing production side Compared method, the yield can be increased by 30%.

V. DETAILED DESCRIPTION

The following examples further combining said fine description of the invention.

The process steps of the present invention is obtained by using parallel test optimization, and real vertical enlarged manner Shi;

Example 1:

After digestion by --- after taking resveratrol content of 1.4% Polygonum cuspidatum Ingredients 1 kg (including exclusion Water ratio) added 1 g of superoxide dismutase while adding 5 liters of 80% ethanol concentration of the solvent, the heating temperature control After extraction 50 ° C and 2 hours at reflux, the extract was filtered, then added to 5 liters of the ethanol extract 2 hours, e.g. This 4 times, the extract batches were combined to control the temperature at 60 ° C recovering the ethanol was concentrated in vacuo, The extract was concentrated under the weight of the raw material ratio of 5% chitosan was added a saturated solution was allowed to stand for 10 hours at room temperature Precipitation, to be completely precipitated after filtration;

Interception solid precipitate portion was added 20 times the mass of the precipitate concentration of 40% ethanol solvent to give The precipitates were dissolved and filtration was repeated, insolubles were discarded, filtrate control the heating temperature 60 ° C in vacuo shape State added to the concentrate to ethanol-saturated solution of chitosan raw ingredient weight ratio of 2%, and allowed to stand at room temperature 10 hours precipitation. Centrifuged to give a solid precipitate;

The precipitate was again added 20 times the weight of the alcohol of 40% ethanol solvent to dissolve the precipitate and had Filtered and insolubles were discarded, and then the filtrate was concentrated to a temperature control without ethanol was heated at 60 ° C under vacuum After adding a saturated solution of chitosan material composition weight ratio of 5% and allowed to stand for 10 hours at room temperature, centrifuged The extract obtained solid precipitated. Washed with pure water until the washing liquid under no pigment component state.

The washed precipitate was dried in vacuo to give 57% of the ground product 23 g, yield 93.6%.

Example 2:

After fermentation by taking resveratrol content of 1.4% Polygonum cuspidatum 10 kg of raw materials (excluding water content) Add 10 grams of superoxide dismutase while adding 50 liters of 80% ethanol solvent concentration, heating temperature control 50 ° C reflux extraction for 2 hours after the extract was filtered off, then 5 liters of an ethanol extract of two hours, so 4 Times, the extracts were obtained by controlling the temperature of each batch in 60 ° C ethanol recovery was concentrated in vacuo, and concentrated After extract the raw material weight ratio of 5% was added a saturated solution of chitosan, stand for 12 hours at room temperature, precipitation, After completion of the precipitation to be filtered.

Interception solid precipitate portion was added 20 times the mass of the precipitate concentration of 40% ethanol solvent to give The precipitates were dissolved and filtration was repeated, insolubles were discarded, filtrate control the heating temperature 60 ° C in vacuo shape State added to the concentrate to ethanol-saturated solution of chitosan raw ingredient weight ratio of 2%, and allowed to stand at room temperature 12 hours precipitation. Centrifuged to give a solid precipitate.

The precipitate was again added 20 times the weight of the alcohol of 40% ethanol solvent to dissolve the precipitate and had Filtered and insolubles were discarded, and then the filtrate was concentrated to a temperature control without ethanol was heated at 60 ° C under vacuum After adding a saturated solution of chitosan material composition weight ratio of 5% and allowed to stand 12 hours at room temperature, centrifuged The extract obtained solid precipitated. Washed with pure water until the washing liquid under no pigment component state.

The washed precipitate was dried in vacuo to give 55% of the pulverized product 238 g, yield 93.5%.

Example 3:

After fermentation by taking resveratrol content of 1.4% Polygonum cuspidatum 100 kg of raw materials (excluding water content) Add 100 grams of superoxide dismutase while adding 500 liters of 80% ethanol concentration of the solvent, the heating temperature control 50 ° C reflux extraction for 2 hours after the extract was filtered off, then 5 liters of an ethanol extract of two hours, so 4 Times, the extracts were obtained by controlling the temperature of each batch in 60 ° C ethanol recovery was concentrated in vacuo, and concentrated After extract the raw material weight ratio of 5% was added a saturated solution of chitosan, stand for 12 hours at room temperature, precipitation, After completion of the precipitation to be filtered.

Interception solid precipitate portion was added 20 times the mass of the precipitate concentration of 40% ethanol solvent to give The precipitates were dissolved and filtration was repeated, insolubles were discarded, filtrate control the heating temperature 60 ° C in vacuo shape State added to the concentrate to ethanol-free composition of raw materials after a 2% weight ratio of chitosan saturated sugar solution and allowed to stand at room temperature 12 hours precipitation. Centrifuged to give a solid precipitate.

The precipitate was again added 20 times the weight of the alcohol of 40% ethanol solvent to dissolve the precipitate and had Filtered and insolubles were discarded, and then the filtrate was concentrated to a temperature control without ethanol was heated at 60 ° C under vacuum After adding a saturated solution of chitosan material composition weight ratio of 5% and allowed to stand 12 hours at room temperature, centrifuged The extract obtained solid precipitated. Washed with pure water until the washing liquid under no pigment component state.

The washed precipitate was dried in vacuo to give 54% of the pulverized product 2.4 kg, yield 92.5%.

Example 4:

After fermentation by taking resveratrol content of 1.4% Polygonum cuspidatum 500 kg of raw materials (excluding water content) Add 0.5 kg of superoxide dismutase while adding 2500 l of 80% ethanol concentration of the solvent, the heating temperature control System 50 ° C and extracted under reflux for 2 hours, filtered off after the extract was added 5 liters of ethanol extract 2 hours, So 4 times, the extract batches were combined to control the temperature at 60 ° C recovering the ethanol was concentrated in vacuo, The extract was concentrated under the weight of the raw material ratio of 5% chitosan was added a saturated solution was allowed to stand for 12 hours at room temperature Precipitation, to be precipitated completely filtered.

Interception solid precipitate portion was added 20 times the mass of the precipitate concentration of 40% ethanol solvent to give The precipitates were dissolved and filtration was repeated, insolubles were discarded, filtrate control the heating temperature 60 ° C in vacuo shape State no ethanol was added to the concentrated feed ingredient after weight ratio of 2% chitosan solution was allowed to stand at room temperature for 12 hours When precipitated. Centrifuged to give a solid precipitate.

The precipitate was again added 20 times the weight of the alcohol of 40% ethanol solvent to dissolve the precipitate and had Filtered and insolubles were discarded, and then the filtrate was concentrated to a temperature control without ethanol was heated at 60 ° C under vacuum After adding a saturated solution of chitosan material composition weight ratio of 5% and allowed to stand 12 hours at room temperature, centrifuged The extract obtained solid precipitated. Washed with pure water until the washing liquid under no pigment component state.

The washed precipitate was dried in vacuo to give 55% of the pulverized product 11.5 kg, 90.4% yield.



Technology for preparing resveratrol by using traditional Chinese medicine giant knotweed rhizome
CN102838456

The invention provides a technology for preparing resveratrol by using traditional Chinese medicine giant knotweed rhizomes, which comprises the following steps of: crushing dried giant knotweed rhizomes, passing the crushed giant knotweed rhizomes through a screen to obtain power, adding water for soaking, adding compound enzymes and fermenting the mixture for 24-72h at constant temperature being 35-60DEG C; and conducting extraction by using ethanol, conducting elution, collecting the eluent, concentrating the eluent and drying the concentrate to obtain the resveratrol. The technology provided by the invention has the advantages that the process is safe and economic, the operation is simple and convenient, and the large-scale production and preparation of high-purity resveratrol by using plants and/or extract containing polydatin and/or resveratrol are easy to realize.

The present invention provides a process for preparing medicine from Polygonum cuspidatum resveratrol process which comprises the dried herbs Polygonum cuspidatum crushed and sieved, added enzyme complex after water infiltration at 35 ° C ~ 60 ° C at a constant temperature of fermentation 24 ~ 72h; then ethanol extracted eluate, concentration, drying derived resveratrol. The process of the invention safe, simple and economical, operation, and easy to implement from the group consisting of Polydatin and / or resveratrol plants and / or extract resveratrol prepared large-scale production of high purity.

TECHNICAL FIELD

The present invention relates to the field of active ingredients from plants, particularly from a Chinese medicine Polygonum cuspidatum resveratrol preparation process.  

Background technique

Resveratrol (also known as three-stilbene phenol), a chemical called 3,4,5, - non-flavonoid polyphenols trihydroxy stilbene (3,4,5, -trihydrolystilbene), containing a stilbene structure . Resveratrol is a plant, when subjected to stress in itself a synthesis of antitoxin, an important success activity, inhibition of cancer, lower blood lipids, prevention of cardiovascular diseases, anti-oxidation, anti-aging and other aspects have a more significant role, following Taxol has been hailed as another new green anticancer drugs. Mainly in Polygonum cuspidatum, grapes, peanuts and other plants, research shows that the highest concentration of resveratrol in Polygonum cuspidatum.  

New investigation found that on "Extract from Polygonum cuspidatum resveratrol" aspects of literature and patents it has been reported. China patent "preparation of high purity resveratrol from Polygonum cuspidatum process" (ZL201110125337) disclosed the use of an organic solvent extraction, alumina column chromatography purification of resveratrol, Chinese patent "a purified extract from Polygonum cuspidatum white a new method of resveratrol "(ZL200510019788.1) disclosed is the use of acid catalyzed hydrolysis, macroporous resin separation and purification of resveratrol, Chinese patent" new Preparation method "polydatin and resveratrol (ZL200310112538.3 ) disclosed is the use of polyamide chromatography separation and purification of resveratrol, Chinese patent "resveratrol extract from Polygonum cuspidatum purification process more than 98%" (ZL200810032067.8) after the first Polygonum cuspidatum extract fermentation using dedicated flocculant precipitate alumina chromatographic separation and purification of resveratrol, resveratrol currently using alkaline elution literature has not been reported.  

SUMMARY

Object of the present invention is to provide a simple, low cost, rapid method for preparing resveratrol, the process steps are as follows:

1) The Polygonum cuspidatum herbs dried and pulverized to obtain Polygonum cuspidatum powder;

2) adding water infiltration Polygonum cuspidatum powder enzyme was added after the invasion of Polygonum cuspidatum powder, temperature of fermentation, so that resveratrol analogs polydatin transformed into resveratrol;

3) with ethanol reflux extraction;

4) The extracts were combined, filtered, and concentrated; the precipitate with purified water, centrifuged, the supernatant;

5) The supernatant macroporous resin, eluted with purified water began to colorless, then eluted with alkaline, alkaline eluent was collected, adjusted pH = 2.5-3.5, centrifuged and the supernatant;

6) The supernatant continue on macroporous resin, eluting first to colorless with pure water, the second with 10% ethanol, is discarded, and the third with 50% to 95% ethanol, The eluent was collected;

7) The collected eluate was concentrated to dryness.  

A preferred embodiment of the present invention is:

1) The Polygonum cuspidatum herbs dried and pulverized to obtain Polygonum cuspidatum powder;

2) adding water infiltration Polygonum cuspidatum powder enzyme was added after the invasion of Polygonum cuspidatum powder, at a temperature controlled within 35 ° C ~ 60 ° C container, thermostat 24 to 72 hours of fermentation, so Polygonum cuspidatum resveratrol analogs glycosides converted into resveratrol;

3) P. cuspidatum added weight of 6 to 12 times 40 to 95% ethanol reflux extraction;

4) The extracts were combined, filtered and concentrated recycling ethanol to no alcohol taste, add 1 to 3 times the water sedimentation, centrifugation, and the supernatant;

5) the supernatant macroporous resin, eluted with purified water began to colorless, and then with 0.4% to 1% of alkaline elution, collecting the eluent alkaline with dilute hydrochloric pH = 2.5-3.5 , centrifuged and the supernatant;

6) The supernatant continue on macroporous resin, eluting first to colorless with pure water, the second with 10% to 35% ethanol 2 ~ 3BV, discarded, and the third with 50% 95% ethanol elution, collecting 3 ~ 6BV eluent;

7) The collected eluent was concentrated to dryness.  

More preferred aspect of the present invention to provide for:

(1) crushed dried herbs to Polygonum cuspidatum by ≥90% 80 mesh sieve;

(2) adding water infiltration Polygonum cuspidatum powder; adding water, the enzyme complex enzyme with water in the proportion of 1 (g): 150-250 (ml), adjusted pH = 3-5, then added to the infiltration after Polygonum cuspidatum powder , at a temperature controlled within 50 ° C ~ 60 ° C container, thermostat 50 to 60 hours of fermentation, so that resveratrol analogs polydatin transformed into resveratrol;

(3) P. cuspidatum added 8 to 12 times the weight of 80% ethanol, reflux extraction twice, each 1 to 2 hours;

(4) The two Polygonum cuspidatum extracts were combined, filtered and concentrated ethanol recovery to almost no alcohol taste, add 1 to 3 times the water sedimentation, centrifugation, and the supernatant;

(5) The supernatant macroporous resin, begin with purified water to elute substantially colorless, and then with 0.4% to 0.5% of alkaline elution, collecting alkaline eluent and 0.5mol / L dilute hydrochloric acid adjust pH = 3, centrifuged and the supernatant;

(6) The supernatant continue on macroporous resin, elute to substantially colorless first with pure water, the second with 20% to 25% ethanol 2 ~ 3BV, discarded, and the third with 50 % ~ 60% ethanol, the eluate was collected 3 ~ 6BV;

(7) The collected eluate was concentrated to dryness.  

More preferred aspect of the present invention provides, step (2) of the complex enzyme with water use ratio of enzyme: water = 1 (g): 200 (ml), PH value is adjusted to 5; amount of enzyme Polygonum cuspidatum powder weight ratio: enzyme: Polygonum cuspidatum powder = 1-3: 80-100. The most preferred: with the addition of Polygonum cuspidatum powder weight ratio of enzyme: enzyme: Polygonum cuspidatum powder = 1: 100.  

The present invention is preferably a strong base in the base. Such as sodium hydroxide, potassium hydroxide and the like.  

The present invention has the following advantages:

1, Document only alkaline extraction, the present invention is the first time alkaline elution, and at a certain concentration, the target component targeted relatively strong, less impurities interference.  

2, the resin may be AB-8, D101 and other resveratrol with a conventional resin adsorption using a broader scope;

3, the whole process cycle is short, simple equipment, low cost, in addition to ethanol, other organic reagents no.  

4, the product purity is over 65%.  

BRIEF DESCRIPTION

FIG. 1 is a product of high performance liquid chromatogram.  

FIG. 1 is a product of high-performance liquid chromatogram

Example 1:

The dried herbs Polygonum cuspidatum Polygonum cuspidatum crushed into powder, over 80 mesh sieve, taking Polygonum cuspidatum powder 100g, add water infiltration; 1g composite enzyme was added 200ml water, adjusted to pH = 5, then stir in flour Polygonum cuspidatum, fermentation 48h on 50 ° C water bath. 80% ethanol was extracted twice, first 12 times, 1.5h, 8 times the amount of the second, 1h, filtered and the combined extracts were concentrated to 500ml, 500ml of water was added, centrifuged and the supernatant, the D101 resin (Tianjin sea Chemical Co., Ltd.), pure water rinse to substantially colorless, then eluted with 0.5% NaOH solution, collect 3BV, 0.5mol / L dilute hydrochloric pH = 3, centrifuged and the supernatant, and then on D101 resin, eluting first with pure water to a substantially colorless, the second eluted with 25% ethanol, 3BV, the third time with 60% ethanol, collected 5BV60% ethanol eluate, the eluate was concentrated, and dried , HPLC assay 60.2% purity resveratrol.  

Example 2:

The dried herbs Polygonum cuspidatum Polygonum cuspidatum crushed into powder, over 80 mesh sieve, taking Polygonum cuspidatum powder 100g, add water infiltration; 1g composite enzyme was added 200ml water, adjusted to pH = 5, then stir in flour Polygonum cuspidatum, fermentation 52h on 50 ° C water bath. 80% ethanol was extracted twice, first 12 times, 1.5h, 10 times a second, 1h, filtered and the combined extracts were concentrated to 500ml, 600ml of water was added, centrifuged and the supernatant, the AB -8 resin (Xi'an Xiao Lan Technology Co., Ltd.), pure water rinse to substantially colorless, then eluted with 0.6% NaOH solution, collect 3BV, 0.5mol / L dilute hydrochloric pH = 3, centrifuged and the supernatant again on AB-8 resin, eluting first with pure water to a substantially colorless, the second with 30% ethanol 3BV, the third time with 75% ethanol, collected 5BV75% ethanol eluate, eluted It was concentrated, dried, HPLC assay 61.1% purity resveratrol.  

Example 3:

The dried herbs Polygonum cuspidatum Polygonum cuspidatum crushed into powder, over 80 mesh sieve, taking Polygonum cuspidatum powder 100g, add water infiltration; 1g composite enzyme was added 200ml water, adjusted to pH = 5, then stir in flour Polygonum cuspidatum, fermentation 60h on 50 ° C water bath. 80% ethanol was extracted twice, first 12 times, 1.5h, 10 times a second, 1h, filtered and the combined extracts were concentrated to 500ml, 600ml of water was added, centrifuged and the supernatant, the AB -8 resin, pure water rinse to essentially colorless, then eluted with 0.5% NaOH aqueous solution, was collected 3BV, 0.5mol / L dilute hydrochloric pH = 3, centrifuged and the supernatant, and then the resin AB-8, a first eluted with pure water to a substantially colorless, the second eluted with 25% ethanol, 3BV, the third time with 60% ethanol, collected 5BV60% ethanol eluate, the eluate was concentrated, dried, HPLC determination, 65.1% purity resveratrol.  

Example 4:

The dried herbs Polygonum cuspidatum Polygonum cuspidatum crushed into powder, over 80 mesh sieve, taking Polygonum cuspidatum powder 80g, water infiltration; 1g composite enzyme was added 150ml water, adjusted to pH = 3, then stir in flour Polygonum cuspidatum, fermentation 50h on 60 ° C water bath. 80% ethanol was extracted twice, first 8 times the amount, 1.5h, 12 times a second, 2h, filtered and the combined extracts were concentrated to 500ml, 500ml of water was added, centrifuged and the supernatant, the AB -8 resin, pure water rinse to essentially colorless, then eluted with 0.4% NaOH aqueous solution, was collected 5BV, 0.5mol / L dilute hydrochloric pH = 3, centrifuged and the supernatant, and then the resin AB-8, a first eluted with pure water to a substantially colorless, the second time with 20% ethanol 2BV, the third time with 50% ethanol, collected 6BV50% ethanol eluate, the eluate was concentrated, dried, HPLC determination, 62.5% purity resveratrol.  

Example 5:

The dried herbs Polygonum cuspidatum Polygonum cuspidatum crushed into powder, over 80 mesh sieve, taking Polygonum cuspidatum powder 100g, add water infiltration; 3g of compound enzymes added 250ml water, adjusted to pH = 4, then stir in flour Polygonum cuspidatum, fermentation 24h on 35 ° C water bath. 40% ethanol was extracted twice, first 6 times, 1.5h, 12 times a second, 2h, filtered, and the combined extracts were concentrated to 500ml, 1500ml of water was added, centrifuged and the supernatant, the AB -8 resin, pure water rinse to essentially colorless, then eluted with 1% NaOH aqueous solution, was collected 4BV, 0.5mol / L dilute hydrochloric pH = 2.5, centrifuged and the supernatant, and then the resin AB-8, a first eluted with pure water to a substantially colorless, the second with 10% ethanol 3BV, the third time with 95% ethanol, collected 3BV95% ethanol eluate, the eluate was concentrated, dried, HPLC determination, 61.5% purity resveratrol.  

Example 6:

The dried herbs Polygonum cuspidatum Polygonum cuspidatum crushed into powder, over 80 mesh sieve, taking Polygonum cuspidatum powder 80g, water infiltration; 3g of compound enzymes added 220ml water, adjusted to pH = 3.5, then stir in flour Polygonum cuspidatum, fermentation 48h on 45 ° C water bath. 60% ethanol reflux extraction twice, first 8 times the amount, 1h, 10 times a second, 2h, filtered, and the combined extracts were concentrated to 500ml, 800ml of water was added, centrifuged and the supernatant, the AB- 8 resin, pure water rinse to essentially colorless, then eluted with 0.5% NaOH aqueous solution, was collected 5BV, 0.5mol / L dilute hydrochloric pH = 3.5, centrifuged and the supernatant, and then the resin AB-8, the first elution with purified water to a substantially colorless, the second with 30% ethanol 2BV, the third with 80% ethanol, collected 6BV80% ethanol eluent, concentrating the eluate and drying, HPLC assay yield 62.5% purity resveratrol.  

Obviously, the above-described embodiments of the present invention is merely to clearly illustrate the present invention made by way of example, and not limitative of the embodiments of the present invention. Those of ordinary skill in the art that, based on the above description may be made other changes or changes in different forms. It could not be here without exhaustive of all embodiments. Changes or changes in these apparent spirit of the present invention belong to the corollary still in the midst of the scope of the present invention. 



Method for extracting total anthraquinone aglycones from giant knotweed
CN102379930

The invention discloses a method for extracting total anthraquinone aglycones from giant knotweed, and the method comprises the following steps: smashing giant knotweed medicinal material, then adding ethanol in a certain concentration, performing heating reflux, filtering, diluting the filtrate, degreasing through a low-polarity solvent, and then adding n-butanol for extraction, evaporating extract liquor to obtain an n-butanol extract, adding a low-polarity organic solvent and inorganic acid into water phase after extraction for hydrolysis, washing organic phase after hydrolysis with water till reaching neutrality, evaporating to obtain a hydrolysis product, merging the hydrolysis product with a n-butanol layer, performing macroporous resin chromatography, and recrystallizing to obtain a total anthraquinone aglycone pure product above 99%.; The method is low in cost, simple in process and high in purity of the extract.

The present invention discloses a method of extracting total anthraquinone aglycone from Polygonum cuspidatum, the process by adding crushed herbs Polygonum cuspidatum after certain concentrations of ethanol, heated to reflux, filtered, and the filtrate was diluted by the low polar solvent degreasing added n-butyl alcohol extract was evaporated to dryness to give n-butanol extract, the aqueous phase was extracted after adding a low polar organic solvent and an inorganic acid hydrolysis, roar and the organic phase washed with water until neutral and evaporated to dryness to give the hydrolysis product, the hydrolysis product butanol layers were combined, via a macroporous resin chromatography, recrystallization, to obtain more than 99% of the total pure anthraquinone aglycone, the method is low cost, simple process, extraction of high purity.

TECHNICAL FIELD

The present invention relates to a method of extracting total anthraquinone aglycone method, particularly relates to a extract from Polygonum cuspidatum anthraquinone aglycone approach.

Background technique

Polygonum cuspidatum for the plants, surface brown to gray-brown, with a clear vertical wrinkles, spots and fibrous roots fibrous scar, and branches on the top section and the sheath-like scar bud scales. Long section between 2-3cm. Quality hard and difficult to break, fracture surface brown, fibrous, and the sheath Kibe easily separated, the cortex is thinner, accounted for most of the wood portion, radially, the central marrow cavity shape or form, a longitudinal section of the diaphragm, gas micro, taste bitter, astringent.

Knotweed rhizomes can be for medicinal purposes, there is blood scattered sputum, chills and detoxification, anti-inflammatory pain, jaundice and other effects to heat, knotweed rhizome containing free anthraquinone and anthraquinone glycosides, such as emodin, rhein, , physcion, physcion -8-OD- glucoside and emodin -8-OD- glucoside and so on. Said anthraquinone substance has antibacterial, diarrhea, anti-inflammatory role of detoxification, can be used in medical, health and household chemicals, and good market prospects, but the current extraction methods exist to extract total anthraquinone glycosides yuan is not high purity, high extraction costs, complex extraction process and other issues.

SUMMARY

Object of the present invention is to provide a total anthraquinone aglycone high purity extract from Polygonum cuspidatum anthraquinone aglycone method of extracting low cost, simple extraction process, the extracted.

The above object of the present invention is achieved by the following technical solution to achieve: a method of extracting total anthraquinone aglycone from Polygonum cuspidatum, comprising the following steps:

(1) Polygonum added extraction solvent by heating reflux extraction, the residue was removed by filtration, the extract was concentrated to give a crude extract concentrate;

(2) The crude extract concentrate is diluted with water and then with a low polar organic solvent degreasing, degreasing was after dilution;

(3) the addition of n-butanol in the dilution after degreasing, the n-butanol extract was concentrated and n-butanol extract;

(4) to the water n-butanol phase after adding a low polar organic solvents and inorganic acids, heating hydrolysis reaction, after hydrolysis, remove the upper organic phase was washed with water until aqueous phase was neutral, organic evaporated The solvent to obtain a hydrolyzate;

Hydrolyzate of step (5) in step (4) obtained in (3) obtained in n-butanol extracts were combined, dried macroporous resin chromatography, alcohol-water system gradient to give the total anthraquinone aglycone crude;

(6) The total anthraquinone aglycone crude product was recrystallized to obtain high-purity total anthraquinone aglycone products.

In the above steps:

Extraction step of the present invention (1) the solvent is aqueous ethanol, wherein the volume percent of ethanol is preferably 70 to 90% aqueous ethanol used is preferably 3 to 5 times the mass of Polygonum cuspidatum. Wherein preferably from the place of origin of P. cuspidatum Polygonum cuspidatum tubers, dried and then pulverized by the extraction solvent extraction.

The present invention, in step (1) was heated under reflux time is preferably 5 to 8 hours, the heating temperature is 75 ° C-80 ° C, the extraction mixture is preferably 3 to 4 times.

The present invention, in step (1) is preferably in the extract was concentrated to the original volume of 1/15 ~ 1/10.

The present invention in step (2) in the crude extract and concentrate volume ratio of water mass 1g: 10 ~ 15mL, the low polarity organic solvent is methylene chloride or cyclohexane, preferably methylene chloride, in an amount dilutions of the crude extract representing a total volume of 0.3 to 0.5 times, 2-3 times the number of fat, skim combined extracts were obtained after dilution.
Inventive step (3) of the n-butanol is preferably added in an amount accounting for 0.5 to 1 times the crude extract dilutions of the total volume of extraction times and preferably 1 to 2 times.

Step of the present invention (4) in the low polar organic solvent is preferably methylene chloride, preferably in an amount accounting for dilution of the crude extract of a total volume of 1/5 to 1/4.
pH value of the inventive step (4) of the n-butanol in the aqueous phase after addition of an inorganic acid solution is preferably adjusted to 4-5, heating hydrolysis reaction is preferably 5 ~ 6h, the heating temperature is 60 ° C-70 ° C wherein the mineral acid is preferably sulfuric acid or hydrochloric acid, preferably sulfuric acid.

Inventive step (5) in the low polar macroporous resin macroporous resin, the low polarity is preferably a macroporous resin AB-8 or HP-20, preferably HP-20, the alcohol-water system comprise a volume percentage of 10%, respectively, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% ethanol solution, after macroporous resin chromatography, collecting ethanol percentage of 40-80% as eluent part segment, and evaporated to dryness to obtain a total of anthraquinone aglycone crude.

Step of the present invention (6) in ethanol recrystallized volume percent ethanol content of 90 to 98% of the total amount of ethanol anthraquinone aglycone from 8 to 10 times the total mass of the crude product, recrystallization when the temperature is - 5 ~ -10 ° C, allowed to stand for 5 to 8 hours, and then crystallization purity higher than 99% of total anthraquinone aglycone products.
The present invention has the following advantages: the extraction process of the invention is simple, low cost, the total extract anthraquinone aglycone product purity of over 99%, high purity, can be widely used in medical, health care and household chemicals, and good market prospects .

Below in conjunction with embodiments of the present invention will be further described, but they are not limited to the present invention, wherein the raw materials used in the absence of exceptional indicated, are commercially available.

Example 1

The dried pulverized rhizome of Polygonum cuspidatum, take 500 g was added to 2,500 g of 75% volume percent of ethanol was heated under reflux in a water bath to extract eight hours, filtered, and then after the filtration was repeated 2 times, the combined extracts. The extract was concentrated to 200 g, diluted 3000mL water, stir well. To the dilution was added 1200 ml of dichloromethane and extracted, twice repeated and the combined extracts recovered dichloromethane. Down the layers the aqueous layer was added 1700 mL of n-butanol, stirred extraction was repeated twice, and the combined extracts were concentrated and recovering a n-butanol, to give 10.74 g of solid. To the aqueous layer was extracted by n-butanol was added after the mass percentage of 98% sulfuric acid to adjust the pH value to 5, and then 650 ml of methylene chloride was added, stirred, the hydrolysis reaction was heated in an oil bath for 6 hours and then cooled to room temperature, separation of the upper The organic layer was washed with water until the aqueous layer became neutral basis, to recover the solvent was concentrated to give 9.45 g solid. The 10.74 grams of n-butanol extract was combined with 9.45 g hydrolyzate with 1000mL volume percentage of 20% aqueous ethanol and the dissolution liquid by ultrasound in addition to gas, filtered, and then the sample through the current pump has been installed to the column and by 500 g pretreated macroporous resin HP-20 chromatography column. After loading is complete, eluted with 3 column volumes of pure water in addition to sugar, desalting, and then by a volume percent content of 10%, respectively, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 100% (V ethanol: V water) of the alcohol-water system gradient eluate 40% -80% of the segment, combined and concentrated to give 6.32 g ethanol recovery pale yellow solid crude product. The crude product was dissolved in 60 g of 95% by volume percentage of alcohol, heated to reflux for 30 minutes, filtered, and the filtrate was cooled to room temperature and then stored at -8 ° C still 8 hours to precipitate 2.12 g orange solid pure content tests showed a purity of 99.31%.

Example 2

The dried pulverized rhizome of Polygonum cuspidatum, take 700 g was added 3500 g of 80% volume percent of ethanol was heated under reflux in a water bath to extract seven hours, filtered, and then after the filtration was repeated 2 times, the combined extracts. The extract was concentrated to 250 grams, diluted 3000mL water, stir well. To the dilution was added 1400 ml of dichloromethane and extracted, twice repeated and the combined extracts recovered dichloromethane. Down the layers the aqueous layer was added 1800 mL of n-butanol, stirred extraction was repeated twice, and the combined extracts were concentrated and recovering a n-butanol, to give 12.86 g of solid. To the aqueous layer was extracted by n-butanol was added after the mass content of 98% sulfuric acid to adjust the pH value to 4.5, then 650 ml of methylene chloride was added, stirred, the hydrolysis reaction was heated in an oil bath for 5 hours and then cooled to room temperature, divided the upper organic layer was washed with water until the aqueous layer became neutral basis, to recover the solvent was concentrated to give 13.43 g of solid. The 12.86 grams of n-butanol extract was combined with 13.43 g hydrolyzate with 1000mL volume percentage of 20% aqueous ethanol and the dissolution liquid by ultrasound in addition to gas, filtered, and then the sample through the current pump has been installed to the column and by 500 g pretreated macroporous resin HP-20 chromatography column. After loading is complete, eluted with 3 column volumes of pure water in addition to sugar, desalting, and then were treated with the following volume percent of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 100% (V ethanol: V water) of the alcohol-water system gradient eluate 40% -80% of the segment, combined and concentrated to give 8.12 g of alcohol recovered crude pale yellow solid. The crude product was dissolved in 80 g of 98% by volume percentage of ethanol was heated to reflux for 40 minutes, filtered, and the filtrate was cooled to room temperature and then stored at -5 ° C still seven hours to precipitate 3.31 g orange solid pure, determination It showed a purity of 99.25%.

Example 3

The dried roots of Polygonum cuspidatum powdered, was added 4500 g 1000 g take volume percent of 85% ethanol was heated under reflux in a water bath to extract 6 hours, filtered, and then filtered after extraction was repeated 3 times, the combined extracts. The extract was concentrated to 400 g, diluted 4500mL water, stir well. To the dilution was added 1700 ml of dichloromethane and extracted, twice repeated and the combined extracts recovered dichloromethane. Down the layers the aqueous layer was added 3000mL of n-butanol, stirred extraction was repeated twice, and the combined extracts were concentrated and recovering a n-butanol, to give 17.24 g of solid. To the aqueous layer was extracted by n-butanol was added after the mass content of 98% sulfuric acid to adjust the pH value to 4.7, then 1000 ml of methylene chloride was added, stirred, the hydrolysis reaction was heated in an oil bath for 5 hours and then cooled to room temperature, divided the upper organic layer was washed with water until the aqueous layer became neutral basis, to recover the solvent was concentrated to give 15.53 g of solid. The 17.24 grams of n-butanol extract was combined with 15.53 g hydrolyzate with 1000mL volume percentage of 20% aqueous ethanol and the dissolution liquid by ultrasound in addition to gas, filtered, and then the sample through the current pump has been installed to the column and by 500 g pretreated macroporous resin HP-20 chromatography column. After loading is complete, eluted with 3 column volumes of pure water in addition to sugar, desalting, and then were treated with a volume percent content of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 100% (V ethanol: V water) of the alcohol-water system gradient eluate 40% -80% of the segment, combined and concentrated to give 10.13 g of alcohol recovered crude pale yellow solid. The crude product was dissolved in 90 g of 95% ethanol was heated at reflux for 50 minutes, filtered, and the filtrate was cooled to room temperature and then to -7 ° C still save six hours to precipitate 4.86 g orange solid pure, purity determination indicates that 99.11% .

Example 4

The dried pulverized rhizome of Polygonum cuspidatum, take 600 g was added 1800 g of 90% volume percent of ethanol was heated under reflux in a water bath to extract five hours, filtered, and then filtered after extraction was repeated 3 times, the combined extracts. The extract was concentrated to 500 g was added 5000mL dilution water, stir well. To the dilution was added 1650 ml of cyclohexane extraction was repeated 3 times, extracts were combined, recovered cyclohexane. Down the layers the aqueous layer was added 2750 ml of n-butanol, stirred extraction was repeated once, and the combined extracts were concentrated and recovering a n-butanol, n-butanol extract obtained, to the aqueous layer was extracted by n-butanol was added after the mass percent adjusting the pH of 98% sulfuric acid to 4, and then 1375 ml of methylene chloride was added, stirred, the hydrolysis reaction was heated in an oil bath for 6 hours and then cooled to room temperature, separated and the upper organic layer was washed with water until the aqueous layer became neutral basis, and concentrated recovered solvent to give a hydrolyzate. The n-butanol extract and the hydrolyzate combined with 1000mL volume percentage of 20% aqueous ethanol and the dissolution liquid by ultrasound in addition to gas, filtered, and then by the current pump has been installed to sample column and pretreated 500 g of macroporous resin HP-20 chromatography column. After loading is complete, eluted with 3 column volumes of pure water in addition to sugar, desalting, and then by a volume percent content of 10%, respectively, 20%, 30%, 40%, 50%, 60%, 70%, 80 %, 100% (V ethanol: V water) of the alcohol-water gradient solvent system, from 40% to 80% of eluent segment, combined, and concentrated to give a pale yellow solid was recovered crude ethanol, the crude product was dissolved in percent volume 95% alcohol, heated to reflux for 30 minutes, filtered, and the filtrate was cooled to room temperature and then stored at -8 ° C still 8 hours to precipitate an orange solid pure, purity determination showed 99.15%.
 
The above embodiments of the present invention are illustrative only, and the scope of the present invention is not limited to the above embodiments. Ordinary skill in the art based on the above parameters and content of the present invention disclosed taken range, can achieve the object of the present invention.



New method for extracting resveratrol from giant knotweed rhizome
CN102320934

he invention relates to a new method for extracting resveratrol from giant knotweed rhizomes. The method comprises the following steps of pulverization, extraction, concentration, hydrolyzation, crystallization, filtration, drying, and the like. The method does not require enzymatic hydrolysis or column chromatography, extracts polydatin and resveratrol from giant knotweed directly by using methanol (ethanol), decomposes glycosidic bonds on the polydatin with acids, neutralizes redundant acids with alkalis, and obtains resveratrol with a content of above 50%. The invention does not require column chromatography, has less solvent consumption, simple operations, a short production period, low cost, and high yield, and is applicable to industrial mass production.

The present invention is a new method for extracting resveratrol from Polygonum cuspidatum rhizome. The method includes crushing, extraction, concentration, hydrolysis, crystallization, filtration, drying and other steps. This method does not require enzymatic hydrolysis, column chromatography, extracted from Polygonum cuspidatum resveratrol in polydatin and direct methanol (ethanol) out, and then the acid glycoside bond Polydatin on decomposition, plus soda and excess acid, obtained by the method of resveratrol content of greater than 50%. The present invention is not subjected to column chromatography, solvent consumption of small, simple operation, short production cycle, low cost, high yield and is suitable for industrial mass production.

TECHNICAL FIELD

The present invention relates to a new method for extracting resveratrol from Polygonum cuspidatum rhizome, belonging to the technical field of natural products chemistry.

Background technique

Polydatin (polydatin) aka Polydatin its structural formula is as follows:
[Image]

Resveratrol (resveratrol), the formula is as follows:
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Resveratrol is extracted from the roots and rhizomes of grape skins and grape branch knotweed Polygonum plants out of a flavonoids. Resveratrol Polygonum medicine mainly in the form of glycosides, a small amount of resveratrol in free coexistence, herbs Polygonum cuspidatum is the main active ingredient. Polydatin resveratrol and a variety of biological activity. Such as anti-oxidation, anti-tumor, improve microcirculation, blood fat, lowering blood pressure.

Currently, the method for extracting resveratrol from Polygonum cuspidatum now include the use of herbs itself after enzymatic hydrolysis extract plus extract or the like to extract plus enzymatic hydrolysis such as patent CN1251361A, CN101698634A, CN101338327, CN1896255A, etc. after enzymatic hydrolysis, most of the extraction solvent is an organic solvent, these methods are subject to hydrolysis polydatin into resveratrol. But need a constant temperature digestion process, so as to ensure adequate enzymatic and enzymatic production occupies a larger space, a long reaction time, and fermented herbs if not promptly treated, it can lead to the fermentation of resveratrol decreased, which takes a large human and material resources, will inevitably lead to lower productivity, higher costs.

Patent CN1513823 A discloses a supercritical CO2 extraction process of resveratrol, which uses supercritical CO2 extraction process, and mixed with ethanol and 2-propanol as a solvent modifier. This process is simple, short production cycle, reduce solvent consumption, low cost, high yield, but supercritical CO2 extraction technology is currently used in large-scale production there is a certain degree of difficulty, can only stay in the experimental stage.

Patent CN1546503A discloses a polydatin resveratrol and new preparation methods and Patent CN101397242A discloses an extract from fresh Polygonum cuspidatum The process of purification of resveratrol, since the content of resveratrol in Polygonum cuspidatum containing low , the vast majority is in the form of glycosides to Polygonum cuspidatum. Because there is no such preparation to glycosides transforming the resulting low yield large losses. And the former polyamide chromatography, but fine-grained polyamide, easy to plug the column.

Patent CN1724495A discloses a process for the preparation of resveratrol from Polygonum cuspidatum herbal plants, through ethanol extraction, concentration, and extracted with ethyl acetate, cellulose as stationary phase column chromatography, as required during operation ultrasound to ultrasound dissolved extract can be extracted, and the ethyl acetate extraction process emulsification obviously, big loss. Although cellulose as the stationary phase for the first time, but still difficult to promote use.

Patent CN1962592A discloses an isolated and purified from traditional Chinese medicine Polygonum cuspidatum resveratrol glucoside and resveratrol approach. Extracted with ethanol, macroporous resin column pre-separation, then the high-speed countercurrent chromatography, the large amount of an organic solvent, time-consuming and costly.

Patent CN1513822A discloses an extract resveratrol from Polygonum cuspidatum process, the first use of special microorganisms added to improve the bioconversion of Polygonum cuspidatum resveratrol content, the use of 40% ethanol and 4-methyl-2-pentanol mixture as the extraction solvent, microwave extraction. Although the present invention is to simplify the process and operation process and shorten the production cycle, reduce costs, but the microwave extraction technology is limited to laboratory operations.

Patent CN101348805A discloses a new process to extract resveratrol. Alkaline extraction, and the extract after adjusting the pH, flocculant clarifying agent treatment, enzymatic hydrolysis, filtration, the precipitate was dried to obtain resveratrol content of 50%; which was further dilute alkali wash, then washed to neutral, then dissolved in a lot of hot water, the water was allowed to stand at room temperature, precipitation sedimentation, filtration and drying to obtain resveratrol content of 98% of the products. Alkaline extraction solution viscosity, filtration long time, a large filtrate loss, high energy consumption, long period, complicated operation.

Patent CN1621401A discloses a high purity extract from Polygonum cuspidatum resveratrol way is through different polar solvent extract derived after multiple reflow, this method complex process, the amount of solvent consumption, long production cycle, solvent recovery difficult.

Patent CN1384088A discloses a method for extracting resveratrol from Polygonum cuspidatum plant through the organic solvent extract of Polygonum cuspidatum Polygonum cuspidatum glycosides, organic synthesis by means of hydrolyzing enriched it with their composite filler ratio after pressurizing the high pressure column in a dedicated apparatus, the high pressure principle by HPLC column chromatography, eluting with chloroform and ethyl acetate gradient. This method is cumbersome, long production cycle, and elution process using highly toxic chloroform.

Patent CN1760166A discloses a new method of purifying resveratrol from Polygonum cuspidatum extract, the extraction method using water, and then extract enriched polydatin out by acid catalyzed hydrolysis reaction, but after a certain enrichment product the proportion of pre-treatment macroporous adsorption resin column to separate and purify water and ethanol. Polydatin slightly soluble in water, resveratrol is insoluble in water, with water as solvent extraction, extraction polydatin incomplete and raw resveratrol could not be extracted.

In recent years, patents have been reported, extracted with an alkaline solution, the drawback is that alkaline extract viscosity, filtration speed is very slow, easy to plug the filtration time consuming.

Patent CN101811939A discloses a tank resveratrol group dynamic countercurrent extraction process by alkali extraction and acid hydrolysis and then ethanol precipitation, activated carbon decolorization out some impurities in through the resin column and a silica gel column. As a result of the present invention, the tank extraction group, large equipment footprint, the extraction solvent is alkaline, alkaline extract high viscosity, filtered difficult, and large after hydrolysis, the subsequent processing equipment pressure, high energy consumption. CN101805755A discloses a purified extract from Polygonum cuspidatum resveratrol process, the method is to use its own enzyme hydrolysis knotweed, fermentation product by alkali extraction and acid precipitation, passed through the column. There is a long hydrolysis time, hydrolysis is not sufficient, an alkali extract difficult to filter its shortcomings process.

In order to solve the above problems, the present invention provides a novel method for extracting the roots from the plant Polygonum cuspidatum resveratrol, without the enzymatic method, column chromatography, to obtain direct polydatin alcohol extract and resveratrol then add the acid to the glycoside bond polydatin exploded, plus excess acid and alkali, placed crystallization. This method is simple, less solvent consumption, short production cycle, low cost, high yield and is suitable for industrial mass production.

SUMMARY

The method of the present invention is based on the plant Polygonum cuspidatum rhizome as raw material, extraction, concentration, hydrolysis, crystallization, filtration, drying and prepared.
Follow these steps:

a. The Polygonum cuspidatum rhizome crushed.

b. Press 1: 8 ~ 1: 20 (m / v) ratio of Polygonum cuspidatum meal and 60 to 90% methanol (ethanol) of Polygonum cuspidatum extract reflux.

c. Concentrated to no alcohol taste.

d. Concentrate and acid hydrolysis stabilizers stirring.

e. Adjusting the pH to neutral hydrolyzate.

f. Standing crystallization, filtration, drying crystals.

Wherein step a crushing granularity 10 mesh sieve to obtain a coarse powder.

Wherein step b reflux extraction time was 1 ~ 3h, 0.5 ~ 2h, 0.5 ~ 1h, extracted three times, extraction temperature control between 50 ° C ~ 85 ° C.

Wherein the acid used in step d of sulfuric acid, hydrochloric acid, acetic acid, phosphoric any one stabilizer is sodium citrate, hydrolysis temperature is between 50 ° C ~ 80 ° C, hydrolyzing time of 0.5 to 3 hours. Concentrate: Acid: stabilizing agent = 100: 0.5 to 5: 1

Wherein the pH adjusting step e the reagent used is sodium hydroxide, sodium carbonate and sodium bicarbonate, calcium oxide, calcium hydroxide or a solid solution.

The following examples further illustrate the invention, but the present invention is not limited to the contents contained in the following examples.

Example A:

Take Polygonum cuspidatum meal 500g, was added 65% methanol 5000ml, extracted three hours at 80 ° C ~ 85 ° C under reflux, filtered extract was collected for the first time. The residue was added 65% methanol 5000ml, extracted 80 ° C ~ 85 ° C under reflux for 2 hours, filtered, and collected by a second extraction liquid. The residue was added 65% methanol 5000ml, extracted 80 ° C ~ 85 ° C under reflux for 1 hour, collected by filtration third extract. Three combined extracts were concentrated under reduced pressure to no alcohol taste. Press concentrate volume of hydrochloric acid and sodium citrate ratio of 100: 3: 1 ratio by adding hydrochloric acid and sodium citrate, hydrolyzed by stirring for 1 hour at 75 ~ 80 ° C water bath, let cool, add 10% sodium hydroxide solution to adjust to neutral pH, crystallized on standing overnight, filtered, and the crystals were collected and dried under reduced pressure to obtain resveratrol 8.7g. HPLC Determination of 51.18%.

Example Two:

Take Polygonum cuspidatum meal 500g, with 70% methanol 4000ml, extracted for 2 hours at 80 ° C ~ 85 ° C reflux, filtered, and collected the first extract. Residue added 70% methanol 4000ml, extracted 80 ° C ~ 85 ° C under reflux for 2 hours, filtered, collected the second extract. Residue added 70% methanol 4000ml, extracted for 1 hour at 80 ° C ~ 85 ° C reflux, filtered, and collected the third extract. Three combined extracts were concentrated under reduced pressure to no alcohol taste. Concentrate by volume of sulfuric acid and sodium citrate ratio 100: 2: 1 ratio of sulfuric acid and sodium citrate was added, followed by stirring at 70 ~ 80 ° C water bath for 2 hours hydrolysis, allowed to cool, adjusted with 5% sodium hydroxide solution to neutral pH, crystallized on standing overnight, filtered, and the crystals were collected and dried under reduced pressure to obtain resveratrol 8.1g, HPLC content of 51.34% was measured.

Example Three:

Take Polygonum cuspidatum meal 500g, added 80% methanol 9000ml, extracted for 2 hours at 80 ° C ~ 85 ° C reflux, filtered, and collected the first extract. The residue was added 80% methanol 9000ml, extracted 80 ° C ~ 85 ° C under reflux for 2 hours, filtered, collected the second extract. The residue was added 80% methanol 9000ml, extracted 80 ° C ~ 85 ° C under reflux for 1 hour, filtered, collecting the third extract. Three combined extracts were concentrated under reduced pressure to no alcohol taste. , Based on concentrated solution of hydrochloric acid and sodium citrate volume ratio of 100: 3: 1 ratio of hydrochloric acid and sodium citrate was added, followed by stirring at 70 ~ 80 ° C water bath for 2 hours hydrolysis, let cool, add solid sodium hydroxide to adjust the pH neutral, crystallized on standing overnight, filtered, and the crystals were collected and dried under reduced pressure to obtain resveratrol 8.5g, HPLC content of 51.75% was measured.

Example Four:

Polygonum cuspidatum meal 500g, 85% methanol was added 7000ml, extracted for 2 hours at 80 ° C ~ 85 ° C reflux, filtered, and collected the first extract. The residue was added 85% methanol 7000ml, extracted 80 ° C ~ 85 ° C under reflux for 2 hours, filtered, collected the second extract. The residue was added 85% methanol 7000ml, extracted 80 ° C ~ 85 ° C under reflux for 1 hour, collected by filtration third extract. Three combined extracts were concentrated under reduced pressure to no alcohol taste. Press concentrate volume ratio of phosphoric acid and sodium citrate 100: 3: 1 ratio of phosphoric acid and sodium citrate was added, followed by stirring at 70 ~ 80 ° C water bath for 2 hours hydrolysis, allowed to cool, the solution was adjusted with 5% sodium bicarbonate pH to neutral stand overnight crystallization, filtration, crystals were collected and dried under reduced pressure to give resveratrol 8.7g, determined by HPLC content of 50.46%.

Example five:

Polygonum cuspidatum meal 5kg, 85% methanol was added 50L, extracted for 2 hours at 80 ° C ~ 85 ° C reflux, filtered, and collected the first extract. The residue was added 85% methanol 50L, extracted 80 ° C ~ 85 ° C under reflux for 2 hours, filtered, and collected by a second extraction liquid. The residue was added 85% methanol 50L, extracted 80 ° C ~ 85 ° C under reflux for 1 hour, collected by filtration third extract. Three combined extracts were concentrated under reduced pressure to no alcohol taste. Press concentrate volume of hydrochloric acid and sodium citrate ratio of 100: 4: 1 ratio by adding hydrochloric acid and sodium citrate ,, stirred at 70 ~ 80 ° C hydrolysis for 2 hours and let cool, add 5% sodium hydroxide solution to adjust pH to neutral, crystallized on standing overnight, filtered, and the crystals were collected and dried under reduced pressure to obtain resveratrol 91g, determined by HPLC content of 51.27%.

Example VI:

Polygonum cuspidatum meal 500kg, added 80% methanol 5000L, extracted for 2 hours at 80 ° C ~ 85 ° C reflux, filtered, and collected the first extract. The residue was added 80% methanol 5000L, extracted 80 ° C ~ 85 ° C under reflux for 2 hours, filtered, collected the second extract. The residue was added 80% methanol 5000L, extracted 80 ° C ~ 85 ° C under reflux for 1 hour, filtered, collecting the third extract. Three combined extracts were concentrated under reduced pressure to no alcohol taste. Hydrochloric acid was added to the concentrate according 100L 3L, sodium citrate was added 1kg, at 70 ~ 80 ° C was stirred for 2 hours hydrolysis, allowed to cool, with 5% sodium hydroxide solution to adjust the pH to neutral, crystallized on standing overnight, filtration, crystals were collected by dried under reduced pressure to give resveratrol 8.8kg, HPLC determination of 52.34%.

Example VII:

Polygonum cuspidatum meal 500kg, 85% methanol was added 4000L, extracted for 3 hours at 80 ° C ~ 85 ° C reflux, filtered, and collected the first extract. The residue was added 85% methanol 4000L, extracted 80 ° C ~ 85 ° C under reflux for 2 hours, filtered, collected the second extract. The residue was added 85% methanol 4000L, extracted 80 ° C ~ 85 ° C under reflux for 1 hour, collected by filtration third extract. Three combined extracts were concentrated under reduced pressure to no alcohol taste. Press concentrate volume of hydrochloric acid and sodium citrate ratio of 100: 3: 1 ratio of hydrochloric acid and sodium citrate was added, followed by stirring at 70 ~ 75 ° C hydrolysis for 2 hours and let cool, add solid sodium hydroxide to adjust the pH to neutral place overnight crystallization, filtration, crystals were collected and dried under reduced pressure to give resveratrol 8.5kg, determined by HPLC content of 51.92%.

Example VII:

Polygonum cuspidatum meal 500kg, 85% methanol was added 5500L, extracted for 2 hours at 80 ° C ~ 85 ° C reflux, filtered, and collected the first extract. The residue was added 85% methanol 5500L, extracted 80 ° C ~ 85 ° C under reflux for 2 hours, filtered, collected the second extract. The residue was added 85% methanol 5500L, extracted 80 ° C ~ 85 ° C under reflux for 1 hour, filtered, collected the third extract. Three combined extracts were concentrated under reduced pressure to no alcohol taste. Press concentrate, sulfuric acid and sodium citrate volume ratio of 100: 3: 1 ratio of sulfuric acid and sodium citrate was added, followed by stirring at 70 ~ 75 ° C water bath for hydrolysis of 2 hours, allowed to cool, add solid sodium hydroxide to adjust pH neutral, crystallized on standing overnight, filtered, and the crystals were collected and dried under reduced pressure to obtain resveratrol 8.7kg, determined by HPLC content of 51.51%.



Microwave-combined enzymatic method for extracting resveratrol from giant knotweed rhizome
CN102070410  

The invention provides a microwave-combined enzymatic method for extracting resveratrol from giant knotweed rhizome, which comprises the following steps of: weighing a plurality of parts of 5.000g of giant knotweed rhizome, preparing into mixed solution in a liquid-to-material mass ratio of 20:1 by using 80 volume percent ethanol solution as an extracting agent, and adding 10mg of cellulase for enzymolysis under the optimal enzymatic extraction conditions, namely at the temperature of 50 DEG C and the pH value of 5.0 for 30 minutes, wherein a microwave-assisted extraction single factor experiment proves that microwave power has slight influence on the yield of the resveratrol, so only the influence of microwave time is considered; and performing microwave heating on the mixed solution subjected to enzymolysis under the power of 510W, performing suction-filtering on extracting solution under reduced pressure, concentrating, and drying at a low temperature to obtain a resveratrol sample. The resveratrol is extracted by the microwave-combined enzymatic method, so that on the one hand, the extraction ratio is greatly improved, and on the other hand, compared with a simple enzymatic extraction method, the microwave-combined enzymatic method greatly shortens extraction time; a simple microwave extraction method is high in speed but low in the extraction ratio; and the microwave-combined enzymatic method fully combines the advantages of the simple enzymatic extraction method and the simple microwave extraction method, so that production cost is reduced, and the method is more suitable for industrial production.

TECHNICAL FIELD

The present invention provides a combination of microwave Enzymatic extraction of Resveratrol from Polygonum method involves enzymatic extraction and microwave extraction technology.

BACKGROUND

Polygonum cuspidatum, also known as big-leaf tongue mains, spotted purple dragon root, its roots and stems contain anthraquinones and stilbene compounds effective active substances in its class component stilbene resveratrol (resveratrol) is an important phytoalexins, can enhance plant resistance to pathogens, and have antioxidant, free radical scavenging, anti-aging, anti-cancer, cardiovascular protection and plant estrogen and hepatoprotective effect, is a great value of natural substance.

Currently, there are more than ten countries and regions in the development of the world's raw materials and preparations resveratrol, another new green anticancer drug Taxol has been hailed after being widespread concern. Major domestic extracted from Polygonum cuspidatum resveratrol, extracted with an organic solvent and more, and with a solvent such as chloroform as eluent, separated by a silica gel column, the time required is longer, larger toxic solvents such as chloroform, solvent residue problems difficult to solve, Polygonum cuspidatum only single component extraction and separation, and can not make full use of the resources of Polygonum cuspidatum, low economic value. Most of the active ingredients of Chinese herbal medicines have been wrapped in the plant cell wall and extracted with an organic solvent used in general, difficult to destroy the cell wall, a lower extraction rate, foreign-1980s has been treated by the enzymatic extraction of natural products, researchers reported It showed that enzymatic action can make the cell wall loose, broken, reducing the mass transfer resistance, accelerate the release of the active ingredient, thereby increasing extraction rate, enhance the value of raw materials. The microwave heating method has a short time, uniform heating, producing good quality products, easy to automate control and so on. Microwave can be directly compared to the role of molecules, the thermal motion of molecules increased, while raising the temperature from this thermal effect can quickly destroy the cell walls, making Chinese medicinal herbs have active ingredients faster separations extracted. The present invention uses the extraction of the enzyme, microwave technology combined effect of resveratrol, enzymes and microwave and combination of both methods give full play to their respective advantages, the extraction time is short, high extraction rate, product quality, easy to automate Available in factory production.

SUMMARY

A microwave combination Enzymatic extraction of Resveratrol from Polygonum method, the main problems to be solved is the most suitable conditions for enzymatic extraction, microwave extraction conditions, and how to combine the two extraction problems, the present invention is determined by a series of experiments the microwave digestion combined with resveratrol extract optimum extraction conditions.

Example:

Weigh 5.000g Polygonum cuspidatum powder several parts, with the volume fraction of 80% ethanol as extracting solvent, dubbed liquid feed mixture 20:1 mass ratio, the optimum extraction conditions enzyme that is 50 ° C temperature, ph value of 5.0 under the conditions of cellulase enzyme added 10mg 30min. Because microwave assisted extraction single-factor test showed that resveratrol has little effect on the yield of microwave power, so only consider the impact of microwave time. By the hydrolysis mixture over 510W power microwave heating, the extract reduced pressure suction filtration, dried and concentrated to low temperature, the sample was resveratrol.

Precautions: It was found that by increasing the microwave power can increase the extraction yield, but little effect, likely to cause excessive power within the container vigorously boiling liquid, boiling medicine will produce large amounts of foam, rapid discharge extraction vessel, causing the active ingredient loss microwave should be used and intermittent, to control the temperature, to prevent violent boiling liquid.

The present invention in combination with microwave Enzymatic extraction of resveratrol on the one hand dramatically increase the extraction rate, on the other hand, and simple enzymatic extraction compared to shorten the extraction time, simply microwave extraction although time is fast but the extraction rate, the present method combines the full advantages of both methods, reduce production costs, more suitable for industrial production.



Method for extracting resveratrol
CN101993354

The invention discloses a method for extracting resveratrol, belongs to a method for preparing a compound by extracting and distilling. The method comprises the following steps of: soaking traditional Chinese medicine material of giant knotweed in water, drying and crushing into giant knotweed powder; adding water, which is 10 times as much as the giant knotweed powder in volume ratio, processing with 300w ultrasonic for 10-15min; adding 1wt%-5wt% of cellulase and carrying out enzymolysis reaction at constant temperature of 40-60 DEG C to obtain giant knotweed powder subjected to enzymolysis; adding a mixed solution of absolute ethyl alcohol and 2-propanol, which is 10 times as much as the giant knotweed powder and used as a modifier; supercritically extracting with CO2 and collecting extracts; carrying out vacuum decompressed concentration to obtain a concentrated filtrate containing resveratrol; and finally chromatographing and refining by using macroporous adsorption resin. By using the method, the consumption of an extraction solvent can be greatly reduced, the cost can be reduced, the process is easy to operate, the extraction solvent CO2 can be recycled, little pollution to environment can be caused.

DESCRIPTION

The present invention discloses a method for extracting resveratrol, belongs with extractive distillation method for the preparation of compounds. The present invention is a medicine raw material Polygonum cuspidatum flooding after drying and pulverizing a powdery raw material Polygonum cuspidatum, a volume ratio of 10 times the amount of water added, the 300w ultrasonic treatment for 10-15 minutes, then the weight percentage of 1% to 5% cellulase, at 40 ~ 60 ° C constant temperature obtained after enzymatic hydrolysis reaction Polygonum cuspidatum powder; adding 10 times the amount of ethanol and a mixture of 2-propanol as modifier, supercritical CO extraction, collection the extract was vacuum concentrated to give the filtrate was concentrated under reduced pressure to contain resveratrol, macroporous resin chromatography refining. This method can greatly reduce the amount of solvent extraction, reduce costs, the process is simple and easy to operate, CO extraction solvent can be recycled, environmental pollution and so on.

TECHNICAL FIELD

The present invention relates to a method for preparing compounds of extractive distillation, in particular enzyme - supercritical fluid extraction process of extracting resveratrol from Polygonum method.

Background technique

Giant knotweed (Polygonum Cuspidatum Sieb.et Zucc) Polygonum Polygonum small shrubs, as China's traditional Chinese medicine, mainly in the East, South, Liaoning, Shaanxi, Gansu, Sichuan, Guizhou and Yunnan and other places. With blood and pain, heat and dampness, the role of cough and phlegm. Resveratrol (Resveratrol) is one of the active ingredients of traditional Chinese medicine Polygonum cuspidatum, and its chemical name is 3,4 ', 5-trihydroxy - trans - stilbene (3,4', 5-trihydroxystilbene), molecular formula C14H12O3, molecular weight 228, which is structured as follows:

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Resveratrol is a human health has a significant role in the efficacy of natural active substances, has caused widespread concern in the international community. By scientific research and clinical applications show that resveratrol has anti-cancer, anti-oxidation and prevent thrombosis, liver protection and free radical scavenging and other effects, the elderly degenerative diseases such as Parkinson's disease, dementia, Alzheimer's , rheumatic disease have a better prevention and treatment. In cosmetics, it has to get rid of melasma and whitening effect; it solidified lipid metabolism and platelet always make an impact, can prevent coronary heart disease and atherosclerosis and other diseases. Thus, resveratrol has become scientists attach great importance to natural active ingredients, it has great medicinal value and market prospects.

China Patent CN1621401A, discloses "a high-purity extract from Polygonum cuspidatum resveratrol method" which is extracted from Polygonum dried after pretreatment of coarse roots added a certain amount of organic solvent reflux recovered solvent, using different pole the operation of the reflux solvent, the solvent recovery section, centrifugation, sedimentation and water after treatment with an adsorbent, to obtain a high-purity crystalline resveratrol.

This method uses large amounts of organic solvents, serious harm to the environment, low product yield, high cost, is not suitable for industrial production.

China Patent CN100340536C, disclosed "supercritical CO2 extraction process of resveratrol from Polygonum" system using supercritical CO2 extraction process, and with absolute ethanol and 2-propanol mixed solvent as modifier, can greatly reduce the amount of solvent extraction, reduce cost, simple process, high yield, good product quality, CO2 extraction solvent can be recycled, for environmental pollution.

Chinese patent CN1090603C disclosed "an extract from traditional Chinese medicine in battle tiger resveratrol", the extraction process is: Polygonum cuspidatum powdered material is added cellulase enzyme reaction carried out at constant temperature for 48-72 hours to obtain enzyme raw materials; coupled with solvent extraction was concentrated to obtain semi-finished products containing resveratrol, and then refined to obtain. HSBC has a source of raw materials, simple process, high yield advantage, the end of the cost.

Supercritical CO2 fluid extraction is the high-tech field of modern separation occurs, it can effectively reduce the amount of organic solvent extraction efficiency; extraction solvent has selected volatile extract more clean, less pollution to the environment and other characteristics .

However, in the above technique, since just selected Polygonum cuspidatum a simple pre-treatment process, thus affecting the yield of the product.

SUMMARY

The present invention addresses the above drawbacks of the prior art, there is provided a method for enzymatic treatment to extract resveratrol tiger battle.

The present invention is achieved by the following technical solutions:

A method for extracting resveratrol, Polygonum cuspidatum flooding after Chinese medicine raw materials dried pulverized into powder Polygonum cuspidatum materials by volume than 10 times the amount of added water, treated by ultrasonic 300w for 10-15 minutes, then weight percent 1% to 5% cellulase at 40 ~ 60 ° C constant temperature obtained after enzymatic hydrolysis reaction Polygonum cuspidatum powder; Polygonum cuspidatum powder was added to 10 times the amount of ethanol and 2-propanol as a mixture of modifiers, supercritical CO2 extraction, the extract was collected, concentrated under reduced pressure to give the filtrate was concentrated under vacuum containing resveratrol; macroporous resin chromatography refining.

The method of extraction of resveratrol, which is a powdery raw material medicine Polygonum cuspidatum Polygonum cuspidatum soaked in water 48 to 72 hours, remove and dry, pulverized and sieved through a 40 mesh sieve charged.

The method of extraction of resveratrol, its enzymatic hydrolysis reaction to adjust the pH value of 4.5 to 5.5, hydrolysis 60 ~ 120min, take precipitate.

The method of extraction of resveratrol, the supercritical CO2 extraction from Polygonum powder is transferred after enzymatic extraction kettle, adjust the extraction vessel pressure of 20 ~ 35Mpa, extraction temperature is 40 ~ 55 ° C, parsing kettle pressure 7 ~ 5Mpa, resolve temperature 40 ~ 55 ° C, to start the cycle, while adding ethanol and 2-propanol to 8:2 volume ratio of 7/3 of the mixture as a modifier, supercritical CO2 60 ~ 120min after extraction, the autoclave was adjusted parsed 5 ~ 6.5Mpa, analytical temperature 40 ~ 50 ° C, collecting the extract.

The method of the resveratrol extract, the extract is at 40 ~ 50 ° C, under a vacuum of -40cmHg ~ -60cmHg conditions, vacuum-concentrated under reduced pressure to recover the modifier collected containing resveratrol The filtrate was concentrated.

The method of extraction of resveratrol, the purification is large holes containing filtrate was concentrated resveratrol has washed adsorption resin column chromatography, concentrated under reduced pressure, freeze drying, purity ≥98 % of resveratrol.

Advantage of the present invention is that: 1, the use of traditional Chinese medicine Polygonum cuspidatum enzymatic methods and sonication, the raw material can be effectively separated, greatly improving the utilization of resources; 2, supercritical CO2 extraction technology, process is simple, yield high, high purity, solvent consumption, no organic solvent residue, the process of health hazards and environmental pollution is small; 3, macroporous isolated and purified resveratrol, ideal separation process is simple; resin recycling reusable, low cost, simple equipment, low investment, better prospects for industrialization.

BRIEF DESCRIPTION

Figure is a process flow diagram of the present invention.

The accompanying drawings and the following embodiments of the present invention will be described in detail.

Example 1:

Weigh herbs Polygonum cuspidatum 10g, 20ml water for 48 hours, remove and dry, crushed through a 40 mesh sieve; 100mL of distilled water was added, treated by 300W ultrasound for 15 minutes and further pulverization, adjusting the pH value at 4.5 to 4.8 by adding cellulase 0.1g, maintaining the temperature at 45 ~ 48 ° C, after hydrolysis 60min, fractionated precipitation, drying into powder Polygonum cuspidatum after enzymatic hydrolysis; the powder into the supercritical CO2 extraction kettle, kettle regulating extraction pressure 20Mpa, extraction temperature 40 ° C, pressure was resolved 5Mpa, a temperature of 40 ° C, extracted, while adding a mixture of 70ml ethanol and 30ml of 2-propanol from the modifying agent, after extraction 60min, the autoclave was adjusted resolves 5Mpa analytical temperature of 40 ° C, collect the extract, resveratrol was crude; recovering modifier, at 40 ° C, vacuum degree of vacuum -40cmHg filtrate was concentrated under reduced pressure and concentrated to give; and finally, macroporous resin chromatography, and further purified purity of 97.8% resveratrol product 0.056g.

Example 2:

Take herbs Polygonum cuspidatum 10g, 20ml water for 48 hours, remove dry, sifted through a 40 mesh screen, added 100mL of distilled water, treated by 300W ultrasound for 15 minutes and further pulverized to adjust pH 4.8 to 5.0, was added cellulase 0.2g , maintaining the temperature at 45 ~ 50 ° C, hydrolysis 90min after dispensing precipitate was dried to a powder Polygonum cuspidatum digestion; the powder into the supercritical CO2 extraction kettle, kettle regulating extraction pressure 30Mpa, extraction temperature of 50 ° C analytical autoclave pressure 6Mpa, a temperature of 48 ° C, extracted, while adding 80ml of ethanol and 20ml of 2-propanol as modifier, extraction 90min after adjusting resolve autoclave pressure 5.8Mpa, resolve temperature 45 ° C, collect the extract, resveratrol was crude; finally recovered modifier, at 45 ° C, vacuum degree of vacuum -50cmHg filtrate was concentrated under reduced pressure and concentrated to give; and finally, macroporous resin chromatography, further refined purity of 98.5% resveratrol product 0.083g.

Example 3:

Take herbs Polygonum cuspidatum 10g, 20ml water for 48 hours, remove dry, sifted through a 40 mesh screen, added 100mL of distilled water, treated by 300W ultrasound for 15 minutes and further pulverized to make tiger battle fibers become loose, it is conducive to C. quinoa release resveratrol effective materials; adjust the pH from 4.6 to 5.2, was added cellulase 0.3g, maintaining the temperature at 47 ~ 52 ° C, after hydrolysis 120min, fractionated precipitation, drying into powder Polygonum cuspidatum after hydrolysis, the powder loading the supercritical CO2 extraction kettle, kettle regulating extraction pressure 35Mpa, extraction temperature of 55 ° C, the autoclave pressure to resolve 7Mpa, a temperature of 53 ° C, extracted, while adding 80ml ethanol and 20ml of 2-propanol as modifiers, after extraction 120min, adjust resolve autoclave pressure 6.5Mpa, resolve temperature 48 ° C. The extract was collected to give crude resveratrol; and finally, recovery modifier, at 50 ° C, vacuum degree of vacuum -60cmHg filtrate was concentrated under reduced pressure and concentrated to give; and finally, by macroporous resin adsorption chromatography have been washed further refined to 98 percent purity resveratrol product 0.079g.



Method for preparing resveratrol extract from giant knotweed and product
CN101870640

The invention discloses a method for preparing a resveratrol extract from giant knotweed and a product. The method comprises the following steps of: pulverizing giant knotweed, screening, adding yeast accounting for 0.5-3 wt% of medicinal powder, evenly stirring, and fermenting for 4-10 days; adding an organic solvent which is 6-12 times of the medicinal powder by weight to reflux in the water bath 2-4 times and 1-3 hours each time, concentrating the extracting solution, and drying to obtain the crude extract; and refluxing in the water bath of 20-50% ethanol (which is 3-6 times of the crude extract by weight) for 0.5-3 hours, vacuum-filtering, concentrating the filtrate, filtering, and drying the filter residues to obtain the resveratrol extract.; Compared with the prior art, the invention has the advantages of simple extraction process, low equipment investment, simple operation, low production cost, higher conversion rate and extraction yield of resveratrol, high resource utilization ratio and favorable industrial production potential.

DESCRIPTION

The present invention discloses a method for extraction and product preparation Resveratrol extract from Polygonum cuspidatum herbs, the method of P. cuspidatum is crushed, sieved powder was added 0.5-3% by weight of yeast, stir, ferment 4- 10 days; adding 6-12 times the weight of the organic solvent in a water bath at reflux for 2-4 times, 1-3 hours each time, the extract was concentrated and dried to obtain a crude extract; crude extract with 3-6 times the weight of 20- 50% ethanol-water bath under reflux for 0.5-3 hours, filtration, and the filtrate was concentrated and then filtered, the filter residue was dried to obtain resveratrol extract. Compared with the prior art, the present invention is used in the extraction process is simple, small investment and easy operation, low production cost, high conversion rate and resveratrol extract, resource utilization, good prospects for industrial production .

TECHNICAL FIELD

The present invention relates to a method for extracting resveratrol from Polygonum cuspidatum herbs, belonging to the field of purification techniques pharmaceutically active ingredient.

Background technique:

The current technical solution, resveratrol is mainly prepared by chemical synthesis methods, but because there is a large investment in equipment, process complexity, high cost and low yield less than satisfactory, in recent years, people attempt to extract polydatin (aka polydatin) and resveratrol from natural plant Polygonum cuspidatum, resveratrol is a very beneficial to human health, a variety of diseases have a significant role in the efficacy of natural active substances , it has caused widespread concern in the international community. By scientific research and clinical applications show that resveratrol has anti-cancer, anti-oxidation and prevent thrombosis, liver protection and free radical scavenging and other effects, the elderly degenerative diseases such as Parkinson's disease, dementia, Alzheimer's , rheumatic disease have a better prevention and treatment; in cosmetics, it has to get rid of melasma and whitening effect; it solidified lipid metabolism and platelet always make an impact, can prevent coronary heart disease and atherosclerosis and other diseases. With the continuous development of biological science and technology, medicinal value and the amount of resveratrol in the increasingly also surge. While inside it contains a lot of natural plant Polygonum cuspidatum polydatin and resveratrol, but because of complex components, difficult to extract, combined with the current extraction process is not perfect, so the extraction rate and purity are low.

Publication No. CN1724495A, entitled "prepared from herbal plants Polygonum cuspidatum resveratrol," the invention patent application discloses P. cuspidatum with an organic solvent extraction, extraction, concentration, silica gel column chromatography to obtain resveratrol process ;

Publication No. CN1385535A, entitled "Chinese medicine to improve the content of resveratrol in Polygonum cuspidatum approach" invention patent application discloses the use of the water infiltration natural fermentation, organic solvent extraction to obtain a crude extract of Polygonum cuspidatum method concentrates;

Publication No. as CN1896255A, entitled "microbial conversion material Polygonum cuspidatum resveratrol extract high purity process," the invention patent application discloses the use of special materials microbial bioconversion of Polygonum cuspidatum, Polygonum cuspidatum extract preparation process;

Publication No. CN1384088A, name "a method for preparing extracting resveratrol from Polygonum cuspidatum plant," the invention patent application discloses the use of P. cuspidatum organic solvent extraction, concentration, extraction, concentration, hydrolysis, column chromatography, to obtain resveratrol methods. Resveratrol above patent literature is the result of the extraction process are extraction, enzymatic reactions or acid hydrolysis, column chromatography steps, and the process steps used in the present invention is completely different. Moreover, existing technical solutions are present complex process, complicated operation, high extraction costs, resveratrol low conversion rate and other shortcomings, so the degree of industrialization and its yield is still not satisfactory yield.

SUMMARY:

Technical problem to be solved by the present invention are: to overcome the deficiencies of the prior art, to provide a prepared medicine extracted from Polygonum cuspidatum resveratrol content of more than 50% of the extraction methods and products thereof. The method of extraction rate used in the present invention, product purity, and can be reached kilogram quantities of industrial production targets.

Aspect of the present invention: Preparation of resveratrol extract extracted from Polygonum cuspidatum herbs as: the herbs Polygonum cuspidatum crushing, screening, adding 0.5-3% by weight of powder yeast, stir, ferment 4-10 days; then was added 6-12 times the weight of the organic solvent in a water bath at reflux for 2-4 times, 1-3 hours each time, the extract was concentrated and dried to obtain a crude extract; crude extract 20-50% by weight of ethanol 3-6 times with water bath reflux for 0.5-3 hours, filtration, and the filtrate was concentrated and then filtered, the filter residue dried to obtain resveratrol extract.

Specifically, the method comprising the steps of:

(1) The Polygonum cuspidatum crushed herbs, over 20-40 mesh sieve;

(2) Fermentation: take 0.5 to 3 parts by weight of yeast dissolved in warm water after 26-32 ° C with 2.5 to 4.5 times the weight, 100 parts by weight of P. cuspidatum powder, stir, ferment 4-10 days;

(3) Extraction: Add 6-12 times the weight of the organic solvent to the fermented medicinal herbs, in a water bath at reflux for 2-4 times, 1-3 hours each time, the combined extracts were dried and concentrated under reduced pressure to obtain a crude extract;

(4) Purification: The crude extract of crushed 3-6 times added 20 to 50% by weight of ethanol, water bath under reflux for 0.5 to 3 hours, vacuum filtration, the filtrate was concentrated to no alcohol taste, filtration, the filter residue dried under reduced pressure , that was resveratrol extract.

The above yeast wine yeast, baker's yeast or wine yeast.

Said organic solvent is ethyl acetate, ethyl acetate and petroleum ether in any proportion or a mixed solvent system of ethyl acetate and diethyl ether mixed solvent system in any proportion.
More specifically, the method of preparation of Resveratrol from Polygonum cuspidatum extract herbs extract comprising the steps of:

(1) P. cuspidatum and crushed, over 20 mesh sieve;

(2) Fermentation: Take 1 part by weight of wine yeast, after 28 ~ 30 ° C with 2.5 wt. Dissolved in warm water, 100 parts by weight of P. cuspidatum powder, stir, maintaining the temperature at 28 ~ 30 ° C, fermentation 6 days;

(3) Extraction: herbs fermented to 8 times by weight was added ethyl acetate, 60-80 ° C water bath under reflux for 2 times, the first two hours, one hour a second time, the combined extracts were concentrated under reduced pressure after dried to obtain a crude extract;

(4) Purification: The crude extract was pulverized, was added 6 times by weight of 40% ethanol, 60-80 ° C water bath under reflux for 1 hour, vacuum filtration, the filtrate was concentrated until no smell of alcohol, filtration, residues 40-70 ° C under reduced pressure and dried to obtain resveratrol extract.

Using resveratrol extract preparation extracted from herbs Polygonum cuspidatum resveratrol extract preparation obtained above. The resulting extract resveratrol content of more than 50%.

Here are the inventors experimental research in the process of completion of the present invention carried out:

A crude extract preparation method thereof

1, yeast species of choice accurately weighed Polygonum cuspidatum medicinal powder 20g, the following were added to the yeast 0.2g (dissolved in 18mL of water was added), stir, sealed at room temperature for 7 days, 90% ethanol was added 120mL After fermentation, 60 ° C water bath reflux for 2 times, each time 1 hour, vacuum filtration, the combined extracts were concentrated to recover the solvent, the crude extract was dried under reduced pressure, accurately weighed 10mg, dissolved in methanol and set the volume to 25mL, filtered through 0.45μm pore membrane filtration, precision drawing continued filtrate 20μL injected into the chromatograph to measure the content of resveratrol, the results are as follows:

According to the results in Table 1, the selection of wines Angel active dry yeast (average extraction rate of 0.88%).

2, fermentation time accurately weighed Polygonum cuspidatum medicinal powder 20g, wine yeast were added 0.2g (dissolved in 18mL of water was added), stir, sealed room temperature for 1 to 12 days, 90% ethanol was added 120mL After fermentation, 60 ° C water bath reflux for 2 times, each time 1 hour, vacuum filtration, the combined extracts were concentrated to recover the solvent, the crude extract was dried under reduced pressure, accurately weighed 10mg, dissolved in methanol and set the volume to 25mL, filtered through 0.45μm pore membrane filtration, precision drawing continued filtrate 20μL injected into the chromatograph to measure the content of resveratrol, the results are as follows:

Due to the effect of the influence of temperature fermentation and processing water that, considering, select appropriate fermentation time to six days, the temperature is 28 ~ 30 ° C, 2.5 to 3 times the amount of water.

3, add water hydrolysis of the test precision that (after adding the amount of water dissolved in the table) take Polygonum cuspidatum medicinal powder 20g, were added to wine yeast 0.20g, stir, sealed room temperature for 7 days, and ethyl acetate was added 60mL after fermentation, 70 ° C water bath under reflux for 2 times, each time 1 hour, vacuum filtration, the combined extracts were concentrated to recover the solvent, the crude extract was dried under reduced pressure, accurately weighed 10mg, dissolved in methanol and set the volume to 25mL, 0.45 μm filter membrane, precision drawing continued filtrate 20μL injected into the chromatograph to measure the content of resveratrol, the results are as follows:

4, the amount of yeast test accurately weighed Polygonum cuspidatum medicinal powder 20g, the amount added to the table wine yeast (dissolved in 50mL of water was added), stir, sealed room temperature for 6 days, 120mL ethyl acetate was added after fermentation, 70 ° C water bath reflux for 2 times, each time 1 hour, vacuum filtration, the combined extracts were concentrated to recover the solvent, the crude extract was dried under reduced pressure, accurately weighed 10mg, dissolved in methanol and set the volume to 25mL, filtered through 0.45μm pore membrane filtration, precision drawing continued filtrate 20μL injected into the chromatograph to measure the content of resveratrol, the results are as follows:

5, the choice of solvent extraction from Polygonum accurately weighed medicinal powder 20g, were added 0.3g wine yeast (dissolved in 50mL of water was added), stir, sealed room temperature for 6 days, the following solvents were added 120mL After fermentation, 70 ° C water bath reflux for 2 times, each time 1 hour, vacuum filtration, the combined extracts were concentrated to recover the solvent, the crude extract was dried under reduced pressure, accurately weighed 10mg, dissolved in methanol and set the volume to 25mL, filtered through 0.45μm pore membrane filtration, precision drawing continued filtrate 20μL injected into the chromatograph to measure the content of resveratrol, the results are as follows:

6, the extraction temperature is selected accurately weighed Polygonum cuspidatum medicinal powder 20g, were added 0.3g wine yeast (dissolved in 50mL of water was added), stir, sealed room temperature for 6 days, respectively 120mL ethyl acetate was added after fermentation, the following temperature water bath reflux for 2 times, each time 1 hour, vacuum filtration, the combined extracts were concentrated to recover the solvent, the crude extract was dried under reduced pressure, accurately weighed 10mg, dissolved in methanol and set the volume to 25mL, filtered through 0.45μm pore membrane filtration, precision drawing continued filtrate 20μL injected into the chromatograph to measure the content of resveratrol, the results are as follows:

7, the amount of solvent test accurately weighed Polygonum cuspidatum medicinal powder 20g, were added 0.3g wine yeast (dissolved in 50mL of water was added), stir, sealed room temperature for 6 days, respectively, following the amount of ethyl acetate was added after fermentation, 70 ° C water bath under reflux for 2 times, each time 1 hour, vacuum filtration, the combined extracts were concentrated to recover the solvent, the crude extract was dried under reduced pressure, accurately weighed 10mg, dissolved in methanol and set the volume to 25mL, micro through 0.45μm hole membrane filter, precision drawing continued filtrate 20μL injected into the chromatograph to measure the content of resveratrol, the results are as follows:

8, extraction time test: Precision Weigh Polygonum cuspidatum medicinal powder 20g, were added 0.3g wine yeast (dissolved in 50mL of water was added), stir, sealed room temperature for 6 days, respectively 160mL ethyl acetate was added after fermentation, the following temperature water bath under reflux for 2 times at the following table, after reflow vacuum filtration, the combined extracts were concentrated to recover the solvent, the crude extract was dried under reduced pressure, accurately weighed 10mg, dissolved in methanol and set the volume to 25mL, 0.45 μm filter membrane, precision drawing continued filtrate 20μL injected into the chromatograph to measure the content of resveratrol, the results are as follows:

According to Table 8 and FIG. 8, FIG. 9, select reflux extraction for 3 hours (i.e.: the first two hours, the second time 1 hour).

9, when the yeast fermentation whether sugar: Precision Weigh Polygonum cuspidatum medicinal powder 20g, respectively, in the following table wine yeast was added 0.3g (dissolved in water or sugar was added 75mL), stir, sealed room temperature for 6 days, respectively, after the addition of acetic acid fermentation ethyl 160mL, 70 ° C water bath under reflux for 2 times, the first two hours, the second one hour after reflow, vacuum filtration, the combined extracts were concentrated to recover the solvent, the crude extract was dried under reduced pressure, accurately weighed 10mg, dissolved in methanol and set the volume to 25mL, through 0.45μm filter membrane, precision drawing continued filtrate 20μL injected into the chromatograph to measure the content of resveratrol, the results are as follows:

Second, the crude extract was purified

1, accurately weighed Polygonum cuspidatum crude extract 0.31g, 40% ethanol was added 15mL sonication and filtered. The filtrate was evaporated done Sample No. 1, the transfer of the remaining precipitation after adding 15mL dissolved in ethyl acetate, petroleum ether was added 15mL, precipitation, did play No. 2 sample, determination of resveratrol, respectively. The results showed that the higher the content of the sample 1.

Conclusion: 40% ethanol, high content of resveratrol, the election of 40% ethanol as crude extract was purified extraction solvent.

2, accurately weighed Polygonum cuspidatum crude extract 0.2g, add 40% ethanol 10mL, 70 ° C water bath reflux for 1 hour, and filtered. Residue of Sample No. 1, the filtrate evaporated to dryness sample 2, weighed amount of sample 2 were added 3mL, 5mL dissolved in ethyl acetate, filtered, and the filtrate evaporated to doing sample No. 3 and No. 4 were measured white Resveratrol content. The results are as follows:

3, accurately weighed Polygonum cuspidatum crude extract (resveratrol content of 20 ~ 23%) 1.1206g, adding 40% ethanol 56ml, 70 ° C water bath under reflux for 1 hour, filtered, and the filtrate was evaporated to dryness, weighed 0.7270g calculate the transfer rate of resveratrol, and all were from the upper crystalline extract Determination of resveratrol content. The results are as follows:
Purification of Resveratrol Transfer rate: 98.72%; all of resveratrol extract content: 35%; upper crystallization of resveratrol content: 72%.

4, accurately weighed Polygonum cuspidatum crude extract (resveratrol content of 20 ~ 23%) 0.2030g, adding 40% ethanol 10mL, 70 ° C water bath reflux for 1 hour, allowed to cool filtered and the filtrate evaporated to no alcohol taste ( approximately 2mL), filtered, and the residue evaporated to dryness. Determination of resveratrol. The results are as follows:
The filtrate was evaporated to 1/5 volume left resveratrol content: 56.06%.

5, the crude extract was purified verification taking Polygonum cuspidatum extract crude (resveratrol content of about 23%) 0.5g, accurately weighed, add 40% ethanol 25mL, 70 ° C water bath reflux for 1 hour, allowed to cool filtered, the filtrate was evaporated to no alcohol taste (about 5mL), filtered, and the residue evaporated to dryness. Determination of resveratrol. The results are as follows:

6, accurately weighed crude extract from Polygonum 4g, add 40% ethanol 200mL, 70 ° C water bath reflux for 1 hour, allowed to cool filtered and the filtrate evaporated to no alcohol taste (about 50mL), filtered, and the residue evaporated to dryness. Determination of resveratrol is 64%.

7. Extraction - Purification of the whole process of verification

Both said powder P. cuspidatum, each 100g, were added cellulase and yeast (300mL water was added after dissolution), placed in 30 ° C water bath, and 6 days of fermentation, the fermentation to the sample after the addition of ethyl acetate 800ml, 70 ° C water bath under reflux for 2 times, the first two hours, the second time 1 hour. The extract was concentrated to recover the solvent and dried under reduced pressure, pulverized, 1:50 was added 40% ethanol was refluxed for 70 ° C water bath for one hour, filtered, and concentrated to no alcohol odor (about 160 ~ 200mL), allowed to cool after filtration the residue dried under reduced pressure. Determination of resveratrol.

6, accurately weighed crude extract from Polygonum 4g, add 40% ethanol 200mL, 70 ° C water bath reflux for 1 hour, allowed to cool filtered and the filtrate evaporated to no alcohol taste (about 50mL), filtered, and the residue evaporated to dryness. Determination of resveratrol is 64%.

7. Extraction - Purification of the whole process of verification

Both said powder P. cuspidatum, each 100g, were added cellulase and yeast (300mL water was added after dissolution), placed in 30 ° C water bath, and 6 days of fermentation, the fermentation to the sample after the addition of ethyl acetate 800mL, 70 ° C water bath under reflux for 2 times, the first two hours, the second time 1 hour. The extract was concentrated to recover the solvent and dried under reduced pressure, pulverized, 1:50 was added 40% ethanol was refluxed for 70 ° C water bath for one hour, filtered, and concentrated to no alcohol odor (about 160 ~ 200mL), allowed to cool after filtration the residue dried under reduced pressure. Determination of resveratrol.

8, plus a large number of verification

The said P. cuspidatum powder 1kg, added cellulase and yeast (dissolved water 2.5L added), is placed in a water bath at 30 ° C, fermentation for 7 days, to the sample after fermentation ethyl acetate was added 8L, 70 ° C water bath reflux 2 times, the first two hours, the second time for 1 hour. The extract was concentrated to recover the solvent and dried under reduced pressure, pulverized, 1:50 was added 40% ethanol was refluxed for 70 ° C water bath for one hour, filtered, and concentrated until no odor of alcohol (about a 1/5 volume), allowed to cool after filtration, the residue was dried under reduced pressure. Determination of resveratrol content:

Results: The rate was 3.11% cream, the average content of crude extract resveratrol was 23.68% and the purification was average content of 61.81%.

Compared with the prior art, the present invention is used in the extraction process is simple, small investment and easy operation, low production cost, high conversion rate and resveratrol extract, resource utilization, good prospects for industrial production .

Brief Description:

FIG. 1 is a diagram fermentation time study results;

Figure 2 is the amount of water results in Fig study;

Figure 3 is the amount of yeast examine the results of Fig;

Figure 4 is a kind of solvent examine the results of FIG;

Figure 5 is a diagram bath temperature results of the examinations;

Figure 6 is a diagram solvent usage results of the examinations;

Figure 7 is a diagram solvent usage results of the examinations;

Figure 8 is resveratrol extraction rate index reflux time study results shown in Fig;

FIG. 9 is the content of resveratrol as an indicator of reflux time study results in Fig.

Example 1:

The Polygonum cuspidatum herbs crushed, over 20 mesh sieve spare; take Polygonum cuspidatum powder and wine yeast (weight ratio of 100: 1), 2.5 times the amount of yeast in warm water (28 ~ 30 ° C) was dissolved, add herbs Polygonum cuspidatum powder, stir , maintain 28 ~ 30 ° C, 6 days fermentation; 8-fold amount of ethyl acetate was added to the solvent the fermented medicinal herbs, 60 ° C water bath under reflux for 2 times, 2 hours each time, the combined extracts were dried and concentrated under reduced pressure Polygonum cuspidatum extract was crude. The crude extract of Polygonum cuspidatum pulverized, add 4 times the amount of 30% ethanol, 60 ° C water bath under reflux for 0.5 hours, vacuum filtration, the filtrate was concentrated to no alcohol taste, filtration, residue 40 ° C under reduced pressure and dried to obtain white Resveratrol extract. After testing, the resulting extract resveratrol content of 61.32%.

Example 2:

The Polygonum cuspidatum herbs crushed, over 20 mesh sieve spare; take Polygonum cuspidatum powder and white wine yeast (weight ratio 200: 1), 3.0 times the amount of yeast with warm water (26 ~ 28 ° C) was dissolved, add herbs Polygonum cuspidatum powder, stir , maintain 26 ~ 28 ° C, 8 days of fermentation; to the fermented medicinal 10-fold amount of ethyl acetate was added - diethyl ether (arbitrary ratio) mixed solvent, 70 ° C water bath under reflux 3 times, 1 hour each time, and the combined extracts solution, dried and concentrated under reduced pressure to obtain a crude extract of Polygonum cuspidatum. The crude extract of Polygonum cuspidatum crushed 3 times the amount of 40% ethanol, 70 ° C water bath reflux for 1 hour, vacuum filtration, the filtrate was concentrated to no alcohol taste, filtration, residue 50 ° C under reduced pressure and dried to obtain white Resveratrol extract. After testing, the resulting extract resveratrol content of 61.78%.

Example 3:

The Polygonum cuspidatum herbs crushed, over 30 mesh sieve spare; take Polygonum cuspidatum powder and baker's yeast (weight ratio of 100: 2), 3.0 times the amount of yeast with warm water (28 ~ 32 ° C) was dissolved, add herbs Polygonum cuspidatum powder, stir , maintain 28 ~ 32 ° C, 6 days of fermentation; to the fermented medicinal herbs is added 12-fold amount of ethyl acetate - petroleum ether (arbitrary ratio) mixed solvent, 70 ° C water bath under reflux for 2 times, the first two hours, second 1 hour combined extracts were dried and concentrated under reduced pressure to obtain a crude extract of Polygonum cuspidatum. The crude extract of Polygonum cuspidatum crushed 3 times the amount of 50% ethanol, 70 ° C water bath under reflux for 1.5 hours and vacuum filtration, the filtrate was concentrated to no alcohol taste, filtration, residue 60 ° C under reduced pressure and dried to obtain white Resveratrol extract. After testing, the resulting extract resveratrol content of 62.05%.

Example 4:

The Polygonum cuspidatum herbs crushed, over 40 mesh sieve spare; take Polygonum cuspidatum powder and wine yeast (weight ratio of 100: 3), 4.5 times the amount of yeast with warm water (28 ~ 32 ° C) was dissolved, add herbs Polygonum cuspidatum powder, stir maintain 28 ~ 32 ° C, fermentation seven days; adding 6 times the amount of ethyl acetate solvent to the fermentation of herbs, 75 ° C water bath under reflux for 2 times, the first one hour, the second two hours, and the combined extracts solution, dried and concentrated under reduced pressure to obtain a crude extract of Polygonum cuspidatum. The crude extract of Polygonum cuspidatum pulverized, adding 6 times the amount of 40% ethanol, 65 ° C water bath reflux for 2 hours, vacuum filtration, the filtrate was concentrated to no alcohol taste, filtration, residue 70 ° C under reduced pressure and dried to obtain white Resveratrol extract. After testing, the resulting extract resveratrol content of 61.67%.



Process for dynamical countercurrent extraction of resveratrol in giant knotweed
CN101811939

The invention relates to a process for dynamical countercurrent extraction of resveratrol in giant knotweed, which comprises the steps of: pulverizing giant knotweed drier; adding a certain alkaline aqueous solvent; extracting in a tank group type dynamic countercurrent mode; hydrolyzing extracted liquid acid; converting resveratrol glycoside into the resveratrol; removing partial impurities of the hydrolyzed product once by the methods of alcohol precipitation and active carbon decoloration; purifying by using macroporous absorbent resin; and finally obtaining 98% of resveratrol product by silica gel column chromatography and recrystallization refining. The yield of the obtained resveratrol is 0.92%, the utilization quantity of the extracting solvent can be reduced by 45% at least, thermal energy is correspondingly saved by 60%, and the extraction rate is enhanced by 20%.

TECHNICAL FIELD

The present invention relates to a dynamic countercurrent extraction process Resveratrol from Polygonum.

BACKGROUND

Giant knotweed (Polygonum cuspidatum Sieb.et Zucc. ) Polygonum quality of the grass like a small shrub, its roots contain a lot of stilbene compound resveratrol (Resveratrol) and polydatin (aka Polydatin), the chemical name is 3,4 resveratrol ', 5-trihydroxy stilbene, natural resveratrol often aglycone and glycoside exists in two forms. Polydatin activity differs from resveratrol, but the human body contains glucosidase in the digestive process can be hydrolyzed to polydatin resveratrol, which play resveratrol on human physiology Features. The study shows that resveratrol has anti-cancer, treatment of cardiovascular disease, lowering blood pressure, anti-platelet aggregation, anti-oxidation and anti-free radical, anti-bacterial, beauty and other effects, is a raw material widely used drugs, and health care products, beauty added products and health food ingredients.

Currently, most of the resveratrol from Polygonum cuspidatum purified using conventional solvent extraction methods, including dipping method, percolation method, boiling water extraction method, reflux extraction method, etc., with the development of technology continues to improve and new extraction device, Some modern extraction methods for extracting resveratrol, a common main enzymatic extraction, microwave assisted extraction, ultrasonic extraction, supercritical CO2 fluid extraction method.

Pot groups dynamic countercurrent extraction process, is a series of two or more dynamic extraction tank unit, an extraction solvent solute concentration gradient of each spice jar reverse transported through the tanks from low to high in turn along the inner tank group, and with the drug material to maintain a certain extraction time and times apply. The main advantage of this technology to solid-liquid (solvent and herbs) active ingredient concentration in the two phases gradient difference, gradual diffusion of the active ingredients in the herbs to lower the initial concentration of solvent extraction, to maximize the transfer of active ingredients from herbs purpose. While using pot groups countercurrent extraction process, can be utilized effectively a concentration gradient of the solid-liquid two-phase, increasing the concentration of poor extraction rate, liquid extract concentration increased, the extraction cycle is short, the use of herbs to extract the body as the filter layer, may improve clarity.

SUMMARY

Object of the present invention is to provide a low power consumption, time is short, solvent extraction process of Resveratrol from Polygonum with less dynamic countercurrent high yield.

Dynamic countercurrent extraction of the invention Resveratrol from Polygonum process comprising the steps of:

1, the dynamic countercurrent extraction: Take dried cuspidatum, crushed, over 40 mesh sieve, weighed aliquot 3-7, 3-7 to extract only the tank as a group, aliquots of Polygonum cuspidatum extract only cans were placed 3-7 each jar at room temperature followed by extraction cycle three times, each extraction times are 0.5 to 2 hours, stirring frequency dynamic extraction of 20 to 200 rev / min, the first extraction the first extraction tank is added 6-10 times the amount of pH 8-12 alkaline water extraction liquid, second and third extract were added 3-6 times the amount of water, after the first extraction tank first extraction liquid into the filter tank reserve, the first liquid extraction tank as a second first second extraction solvent extraction, the extraction liquid filter back after the third extraction liquid extraction tank as a second second extraction solvent, the resulting liquid extract as first third extraction solvent extraction tank, extracting tank that is N times per extraction solvent are added on a first extraction tank N + 1 times extract, the second extraction tank, a third from cans, the first N extracted in the first extraction tank is added on a tank of liquid extract were added on the basis of 1-3 times the amount of the alkaline aqueous extract, collect cans of each extract of the first extraction liquid;

2, and the resulting extract was at pH 2-5,50 ° C-100 ° C conditions, hydrolysis for 1-5 hours, the reaction was cooled, the standby;

3, the hydrolyzate was dispersed in 5 to 10 times by weight of 50% -100% ethanol, add 0.5-1 times the weight of the activated carbon was heated under reflux with stirring for 1-2 hours, allowed to stand for cooling, the supernatant was concentrated to dip paste;

Extract water

4, the above steps the resulting dispersion, filtered and the filtrate, according to a flow rate of 1-4BV / h on the pretreated macroporous resin adsorption, 1-6 times the bed volume of distilled water elution the flow rate was 1-4BV / h, discard the water eluent, then 1-6 times the bed volume of 60% -100% ethanol by 1-4BV / h speed eluate, and recovering ethanol concentrated to a thick paste, drying, dry extract, resveratrol crude;

5, the upper and collected to resveratrol crude product was separated by silica gel column chromatography, eluting with a gradient mixed solvent, and then recrystallized twice from ethanol to give white crystals of pure resveratrol.

The present invention is dynamic countercurrent extraction Resveratrol from Polygonum process has the following advantages:

1, pot groups dynamic countercurrent extraction instead of the traditional single tank multiple extractions, ensure inter-tank groups larger concentration of active ingredient is poor, has greatly increased the extraction impetus to accelerate the extraction rate, shorten the extraction cycle, tank group extractor through repeated use of the solvent, the solvent concentration in the final to improve the active ingredients, reduce the subsequent concentrated energy, compared with conventional extraction process, the extraction solvent consumption by at least 45%, and the corresponding energy savings of about 60%, extraction rate increased by 20%, and increases as the number of pots set, this advantage is more obvious.

2, stirred dynamically extract instead of the traditional static extraction, can effectively avoid Polygonum cuspidatum powder stick pan pasting;

3, by acid-catalyzed hydrolysis reaction can be greatly improved Polygonum cuspidatum resveratrol content, improve the utilization of resources.

4, heavy alcohol use, activated carbon bleaching, macroporous resin separation and purification technology of resveratrol, ideal separation effect, simple process.

5, using normal phase chromatography on silica gel purified resveratrol crude obtain content up to 98% pure resveratrol.

Specific implementation methods

Example 1:


Polygonum cuspidatum of dried herbs, crushed, over 40 mesh sieve, weighed three, each 100g, respectively, set within the extraction tank 1,2,3. Add 10 times the amount in the 1st tank pH = 10 of aqueous sodium hydroxide solution at room temperature, stirring means to open, so that up to 100 rev / min speed, one hour to extract, the extract was filtered, stored as a stock solution; 1 No. canister added 8 times the amount of sodium hydroxide solution at room temperature pH = 10, using the same conditions as the last extracted a second extraction, after 1 hour, turn off the power, the extract was transferred to a 2 tank, and 2 No. 3 times the amount of supplementary tank pH = 10 at room temperature aqueous sodium hydroxide solution, seal, open stirring means, so as to extract the extraction conditions according to the 1st tank 1 hour, filtered extract, stored as stock solution 2; No. 1, then after the second extraction liquor tank was transferred to the 2nd tank, the tank is added to the 1st 8 times the amount of sodium hydroxide solution pH = 10, according to the previous extraction conditions to extract the last 1 hour extraction is completed, the extract was transferred to a tank 2, and 1 tank were slagging operations, in accordance with the conditions of the last 2 cans a second extraction, extract the resulting liquid quickly transferred to the tank 3, and 3 cans supplement 3 times the amount of sodium hydroxide solution pH = 10, according to the above conditions were extracted liquid was 3; and so on, 2 cans of the third extract as 3 cans a second extraction solvent extraction, stock solution was 4; 3 tank was added to 8 times the amount of sodium hydroxide solution pH = 10, and a third extraction liquid was 5; the above-mentioned liquid were combined to give a total extract, Polydatin resveratrol extract a total of 20.4mg / g. The crude extract with 50% sulfuric acid to adjust pH = 2,70 ° C hydrolysis was stirred for 1 hour and cooled for 24 hours, filtered and washed with water to give a neutral extract containing resveratrol. After the hydrolysis of resveratrol extract dispersed in 70% ethanol, activated carbon, with a small amount of 70% ethanol soaked and heated to reflux 6min, cooling, to join together the material, 70% ethanol added to the total amount of material the 8-fold (W / V). Stirred for 1.5 hours, 24 hours, and an upper suction filtered supernatant was washed 6 times the 70% ethanol precipitation, centrifugation or suction filtration, combined supernatant, recovering ethanol under reduced pressure to obtain extract. The extract obtained above water dispersion to the 3BV / h pretreated macroporous resin adsorption separation column, eluting first with water, then water - ethanol system gradient elution, TLC tracking and detection, collection resveratrol segment eluent, concentrated to remove ethanol under reduced pressure, to give crude resveratrol content of 50.5%. Take resveratrol crude product was dissolved with a small amount of ethyl acetate, the sample volume silica 1/40 (W / W). Petroleum ether: ethyl acetate (5:1) as an eluent, 10mL / min gradient flow rate, the resveratrol-containing fractions were combined, concentrated and recrystallized twice from ethanol to give white crystals, i.e., white Resveratrol, as determined by HPLC purity of 98% extraction rate 0.92%.

Example 2:

Polygonum cuspidatum of dried herbs, crushed, over 40 mesh sieve, weighed five copies, each 100g, respectively, set within the extraction tank 1,2,3,4,5. Was added in the 1st tank 8 pH = 8 times the amount of sodium hydroxide aqueous solution at room temperature, stirring means to open, so that up to 150 rev / min speed, 1.5 hours extraction, the extract was filtered, stored as a stock solution; 1 No. 6 times the amount of the tank was added aqueous sodium hydroxide solution at room temperature pH = 8, using the same conditions as the last extracted a second extraction, after 1.5 hours, turn off the power, the extract was transferred to a 2 tank, and 2 No. 2 times the amount of supplementary tank at room temperature sodium hydroxide aqueous solution pH = 8, sealed, open stirring means, so that the extraction conditions according to the 1st tank were extracted for 1.5 hours. The extract was filtered, stored as a liquid 2; then after 1 second extraction liquor tank was transferred to the 2nd tank to tank 1 was added 6 times the amount of sodium hydroxide solution pH = 8, according to the former secondary extraction conditions last extraction 1.5 extraction childhood ended, the extract was transferred to a tank 2, and 1 tank were slagging operations, in accordance with the conditions of the last 2 cans a second extraction, extract the resulting liquid quickly transferred to 3 tanks, 2 and 3 times the amount of supplementary tank of sodium hydroxide solution pH = 8, according to the above conditions, the extraction yield reservoir 3; the third and so on, 4 cans extract cans as 5 second extraction solvent extraction to give the accumulator 6; 5 to 6 times the amount of the tank was added aqueous sodium hydroxide solution pH = 8, a third extraction liquid was 7; the said liquid were combined to give a total extract, resveratrol glucoside and resveratrol extract a total of 18.6mg / g. The crude extract with 50% sulfuric acid to adjust pH = 4,60 ° C hydrolysis was stirred for 2 hours and cooled for 24 hours, filtered and washed with water to give a neutral extract containing resveratrol. The hydrolysis of resveratrol extract dispersed in 80% ethanol, activated carbon, with a small amount of 80% ethanol and heated to reflux immersion 5min, after cooling, the material was added together with 80% ethanol added to the total amount of material 6 times (W / V). Stirred for 2 hours, 24 hours, and an upper suction filtered supernatant was then washed 4 times with 80% ethanol precipitation, suction filtration or centrifugation, the supernatant combined, recovering ethanol under reduced pressure to obtain extract. The extract obtained above water dispersion to the 2BV / h pretreated macroporous resin adsorption separation column, eluting first with water, then water - ethanol system gradient elution, TLC tracking and detection, collection resveratrol segment eluent, concentrated to remove ethanol under reduced pressure, to give crude resveratrol in an amount of 51.2%. Take resveratrol crude product was dissolved with a small amount of ethyl acetate, the sample volume silica 1/40 (W / W). Petroleum ether: ethyl acetate (6:1) as an eluent, 10mL / min gradient flow rate, the resveratrol-containing fractions were combined, concentrated and recrystallized twice from ethanol to give white crystals, i.e., white Resveratrol, as determined by HPLC purity of 98% extraction rate 0.89%.



Method for improving content of resveratrol by converting giant knotweed materials through immobilized enzyme method
CN101735999

The invention discloses a method for improving the content of resveratrol by converting giant knotweed materials through an immobilized enzyme method. The method comprises the steps of biologically converting a crude extract of the giant knotweed materials with a specific immobilized enzyme to convert resveratrol analogues therein into the resveratrol in a short time and then obtaining the resveratrol with a high purity by extraction and separation technologies.; The method can increase the resveratrol content of the giant knotweed materials by 10 to 20 times, solves the problems of overlong conversion time, difficult control on catalytic time as well as increased cost caused by the fact that the enzyme cannot be recovered after the reaction in the similar techniques, and simultaneously obtains over 95 percent of resveratrol and a side-product archen with a certain purity by adopting the separation and refining techniques such as chromatography, crystallization and the like.

The present invention provides a method of transforming immobilized enzyme Polygonum cuspidatum extract resveratrol to improve the method, which uses a special material immobilized enzyme crude extract of Polygonum cuspidatum biotransformation short period of time in which resveratrol is converted to analog resveratrol, then use extraction technology to obtain high purity resveratrol. The present invention can improve the material Polygonum cuspidatum resveratrol 10-20 times, to avoid similar technology conversion time is too long, time is not easy to control, and the catalytic enzyme caused an increase in cost can not be recovered after the completion of the reaction problem, while using chromatography, crystallization and other separation and purification technology, access to more than 95% of the by-products of a certain purity resveratrol and emodin two products.

It was transformed into an immobilized enzyme material to improve the content of resveratrol in Polygonum cuspidatum method

FIELD:

The present invention relates to a biological conversion technologies in natural plant extract the active substance in the field and in particular to provide a method of using immobilized enzyme conversion material purified Resveratrol from Polygonum process.

Background technique:

Giant knotweed (Polygonum cuspidatum Sie bet Zucc) Polygonum Polygonum small shrubs, as China's traditional Chinese medicine, which is rich in resveratrol (3,4 ', 5-trimethyl-hydroxy-trans-bis styrene, Resvertrol). Resveratrol as an active non-flavonoid polyphenols. Plant widely found in grapes, peanuts and other natural foods or drugs, is a kind of clear physiological functions of physiologically active substances, with the efficacy of anti-cancer and treatment of cardiovascular diseases.

New investigation found that, at home and abroad for the "extraction biotransformation resveratrol" aspects of literature and patents have been reported.

As Chinese patent CN101255449 process: select specific bacteria, enzyme production in the culture medium, 25 ° C-50 ° C under the culture supernatant was collected as a crude enzyme solution. Then samples containing polydatin crushed to 60 mesh, by a certain percentage added to the crude enzyme solution was allowed to stand after conversion 10-24h, extracted by ethanol was heated to reflux way to get resveratrol crude.

China Patent CN101338327A provided an extract from Polygonum cuspidatum resveratrol purity of more than 98% of the process is first crushed Polygonum cuspidatum extract ethanol, and concentrated using an enzyme recovered in the 45 ° C-50 ° C constant temperature hydrolysis 4-5 days and then inactivate the enzyme was filtered, purified by chromatography using alumina residue after filtration through flocculation, and finally crystallized and dried to obtain the product.

"Traditional Chinese Medicine" (2009.09) Li Yi non-published papers, et al, describes a enzymatic extraction of Resveratrol from Polygonum process, "China Modern Medicine" (2008.02) silver Sheng et al., Published papers, introduces a cellulase extraction studies of resveratrol from Polygonum.

Documents of the new investigation showed that the current use of biological conversion process to produce high purity resveratrol has been a lot of methods. However, the prevalence of long reaction time, reaction time is difficult to control, process complexity, low conversion rate problem, the most important after the other methods can not be transformed enzyme recycled again, indirectly raises the production of resveratrol cost.

SUMMARY

Object of the present invention is that in order to overcome the above drawbacks, providing an immobilized enzyme technology to produce high purity resveratrol from Polygonum cuspidatum of the process that increases the material Polygonum cuspidatum resveratrol 10-20 times, to avoid similar art conversion time is too long, time is not easy to control, and the catalytic enzyme caused an increase in cost can not be recovered after the reaction was complete problem.

To achieve the above object, the process steps of the present invention comprises:

a) selecting a viability of greater than or equal to 5000U / g of β- glucosidase, an enzyme preparation concentration of 20% solution;

b) sodium alginate in distilled water at room temperature and stir, distilled water and sodium alginate weight ratio of 2 to 5:100, standing swelling 20min, then water heating at 70 ~ 100 ° C, the so its fully dissolved, cooled to 45 ° C after the completion of the following;

c) The enzyme solution was poured into a sodium alginate gel, stir, uniform dropped 2-5% concentration of CaCl2 solution to form gel particles, adjusting the flow rate of the particles formed in diameter between 2 ~ 4mm, complete after particulate filtration, washed with distilled water again after use was added to a concentration of 2 to 5% CaCl2 solution at 4 ~ 10 ° C was allowed to stand at 4 ~ 6h, then filtered particles, rinsed thoroughly with distilled water, using the water separator paper gauze the particle surface water drain, that was immobilized enzyme, sealed and stored at 4 ~ 10 ° C conditions;

d) Select cuspidatum, fully dried, crushed by the mill, over 40 to 60 mesh sieve;

e) The Polygonum cuspidatum pulverized added at a concentration of 80 to 95% ethanol, at 30 ~ 35 ° C under a mixed system for ultrasonic treatment, solid-liquid ratio of Polygonum cuspidatum: ethanol = 1g:3ml, power 80 ~ 150W, extraction 3 times, each time 1 ~ 2h, after the completion of filtration and the combined filtrate;

f) The filtrate was evaporated to 6-9% of the original volume, then add water thereto to adjust pH to 4.0 to 6.0, alternate;

g) The resulting extract was added to step up the immobilized enzyme extract: immobilized enzyme = 1ml:0.03 ~ 0.1g, at 45 ~ 55 ° C under low speed agitation 15 ~ 24h, after completion of immobilized enzyme catalysis was filtered off;

h) after the crude extract is catalyzed by volume of body fluids than the rough: ethyl acetate = extracted three times the proportion of 1:3 to 5, were extracted with ethyl acetate to give crude extract resveratrol;

i) and column chromatography separation, concentration, crystallization, resveratrol products.

Step d), to optimize water heating temperature 80 ~ 90 ° C.

In step c), the most suitable concentration of CaCl2 solution is 2.5 to 3%.

Step g), the most suitable temperature of the catalytic reaction is 49 ~ 50 ° C.

Utilizing enzyme immobilization technology to improve the stability of the converting enzyme, but after the conversion can be quickly separated from the immobilized enzyme reaction was to avoid the impact of the conversion of residual enzyme after subsequent separation and purification process and improve material Polygonum cuspidatum resveratrol content is more than 10 times.
And enzyme can be reused several times, supplemented by microwave low temperature, rapid extraction technology, to simplify the production process and operation, shorten the production cycle, dramatically reduce production costs, improve product quality, combined with chromatography and crystallization separation and purification technology, you can get 95 % purity resveratrol and certain byproducts emodin and other two products.

Example 1:

1, the immobilized enzyme production:

a) acquisition of β- glucosidase, viability is greater than or equal to 5000U / g, and an enzyme preparation concentration of 20% was reserved.

b) the ratio by weight of distilled water and sodium alginate is from 2 to 5:100 distilled water and weighed alginate, sodium alginate in distilled water at room temperature, stir and let stand swell 20min, then at 80 ~ 90 ° C under water heating to fully dissolve, uniform color, non-condensing particulate standard, cooled to 45 ° C or less to complete.

c) the prepared enzyme solution was poured into a sodium alginate gel, stir until uniform, then using a syringe or a cross-flow pump, a 7-gauge needle uniform dropped 2.5% to 3% concentration of CaCl2 solution to form gel particles, flow rate was adjusted so that the particle diameter is formed between 2 ~ 4mm, filtered off after completion of the particles, the use of distilled water was added again after 2.5 to 3% strength CaCl2 solution at 4 ~ 10 ° C was allowed to stand at 4 ~ 6h, then filtered particles, thoroughly rinsed with distilled water, using a water separator paper gauze particle surface water drain, that was immobilized enzyme, sealed and stored at 4 ~ 10 ° C conditions.

2. Preparation of crude extract of Polygonum cuspidatum:

d) Select cuspidatum, fully dried, crushed by the mill, over 40 to 60 mesh sieve.

e) ultrasonic extraction: The Polygonum cuspidatum crushed, using 80 to 95% ethanol at a ratio of 1g:3mL (Polygonum cuspidatum Dry weight: ethanol, Polygonum cuspidatum units of g, methanol unit mL) were mixed in 30 ~ 35 ° C under the hybrid system using ultrasonic treatment, power control in 80 ~ 150W, extracted three times, each time 1 ~ 2h, after the completion of filtration and the combined filtrate.

f) The filtrate was evaporated to 6-9% of the original volume, to which was then added the ethanol used in the step of 5 to 10% water, pH adjusted to between 4.0 to 6.0, the standby.
3, crude extract of Polygonum cuspidatum extract and transformation

j) step up the extract was added to the immobilized enzyme extract: immobilized enzyme = 1ml:0.03 ~ 0.1g, at 49 ~ 50 ° C under low speed agitation 15 ~ 24h. After completion of the immobilized enzyme catalytic filtration, washed with distilled water saving, can be reused.

h) after the crude extract is catalyzed by volume of body fluids than the rough: ethyl acetate = 1:5 proportion extracted three times, the combined ethyl acetate, acquired resveratrol crude extract.

i) purified resveratrol: by column chromatography, concentration, crystallization, resveratrol Gifts

Example 2:

1, the immobilized enzyme production: Example 1

2. Preparation of crude extract of Polygonum cuspidatum:

a) Select cuspidatum, fully dried, crushed by the mill, over 40 to 60 mesh sieve.

b) ultrasonic extraction: Use 80 to 95% methanol ratio of 1/3 (Polygonum cuspidatum Dry weight: volume methanol, Polygonum cuspidatum units of g, methanol unit mL) at 30 ~ 35 ° C under ultrasonic treatment on a mixed system, power control in 80 ~ 150W, extracted three times, each time 1 ~ 1.5h, after the completion of filtration and the combined filtrate.

c) The filtrate was evaporated to 6-9% of the original volume, and added thereto ethanol of the original 5 to 10% of water, adjusting the pH to between 4.0 to 6.0, the standby.
3, Polygonum cuspidatum extract crude and refined conversion mention:

d) in the above Step extract added 5% by weight of the immobilized enzyme extract, the extract according to the above crude extract: ethyl acetate = 1:3 proportion of ethyl acetate was added, at 45 ~ 52 ° C using a stirrer at low speed for 15 ~ 24h.

e) catalytic processes, replacement every 5 ~ 8h 1 ethyl acetate three times and the combined ethyl acetate layers alternate.

f) after completion of ethyl completely removed, in order to prevent contamination of the immobilized enzyme after the ethyl acetate layer was completely removed, the immobilized enzyme was filtered off, washed with distilled water after preservation, can be reused.

4. Resveratrol refined

Using conventional methods column chromatography separation, concentration, crystallization, resveratrol products.



New technique for extracting and preparing high-purity resveratrol from giant knotweed
CN101643754

The invention provides a new technique for extracting and preparing high-purity resveratrol from giant knotweed, belonging to the technical field of extracting resveratrol from the plant and solving the problems that the extraction ratio of extracting resveratrol from giant knotweed in the prior art, the purity of the extracted product is not high, the extraction cost is high and the like. The production technique comprises the following steps: crushing the materials of giant knotweed, extracting the crushed the materials with a solvent by adopting a vacuum blasting means, combining the extracted solution, and carrying out enzymolysis fermentation the extracted solution after concentrating, adjusting the pH value of resveratrol liquid after enzymolysis by dilute acid, absorbing by macroporous resin, washing, crystallizing to obtain the resveratrol with the purity of more than 98 percent. The technique can lead the yield of resveratrol to be more than 90 percent, greatly reduces production cost and is suitable for industrial promotion.

The present invention provides a new process for preparing high purity extract resveratrol from Polygonum cuspidatum, belonging to the technical field to extract resveratrol from plants to solve the prior art resveratrol extracted from the extraction rate is not in battle tiger high purity extraction is not high, high cost of extraction. Production process: After grinding the raw material Polygonum cuspidatum, vacuum blasting means, followed by a solvent extraction, the combined extracts were concentrated and then enzymatic fermentation, enzymes break down the resveratrol solution pH was adjusted with dilute acid, macroporous resin adsorption, washing, crystallization, to obtain more than 98% content of resveratrol. The process can yield more than 90% of resveratrol, greatly reduce production costs, suitable for industrial promotion.

TECHNICAL FIELD

The present invention belongs to the extraction process of resveratrol from plants, and more particularly relates to the preparation of a high purity extract from Polygonum cuspidatum C. quinoa

Background technique

Polygonum cuspidatum (Rhizoma Polygoni Cuspidati) Polygonaceae Polygonaceae plant Polygonum cuspidatum Polygonum cuspidatum Sieb.et Zucc., dried rhizome and roots. Polygonum cuspidatum Polygonum containing glycosides, organic acids, glucoside, a polysaccharide Class, containing stilbene compounds: resveratrol (resveratrol) that is 3,4 ', 5-trihydroxy stilbene (3,4', 5-tri -hydroxystilbene), polydatin (Polydatin) ie resveratrol 3-O-β-D- glucoside ( rerveratrol-3-O-β-D-glucoside), detoxification, cool Solutions Department, stomach and clear food effect. Resveratrol is Considered a phytoalexin produced in the plant pathogenic attack deteriorates and the environment. Its pharmacological effects: antibacterial , Anti-oxidation, prevent heart disease, cancer, anti-platelet aggregation, protect the liver, the role of estrogen, radiation, immune regulation Anti-AIDS activity and the like. Resveratrol has been hailed as paclitaxel after another new green anti-cancer drugs, it is food, medicine Medicine and other aspects of the application more widely. Polygonum cuspidatum extract resveratrol from the prior art have enzymatic extraction, solvent extraction, microwave extraction And so on, but because of a single extraction methods and extraction conditions employed and so there is the extraction rate is not high, the product purity is not extracted High extraction cost disadvantages.

SUMMARY

Object of the present invention is to provide a new extraction process for preparing high purity resveratrol from Polygonum cuspidatum, the solution from the prior art Tiger battle to extract resveratrol extraction rate is not high, the product purity extraction is not high, high cost of extraction, this extraction process Rate, high product purity, low extraction cost, simple process steps.
The method of preparation of high-purity extract resveratrol from Polygonum cuspidatum new process of the invention: After Polygonum cuspidatum raw material crushing and vacuum Blasting (ie, first with extreme pressure, then relief, momentary vacuum, cell wall material damage), followed by a solvent extraction The combined extracts were concentrated and then enzymatic fermentation decomposition, enzymatic decomposition of resveratrol with a dilute acid solution after pH adjustment, macroporous Adsorption resin, washed, and then concentrated to crystallization to obtain high-purity resveratrol.

Significant advantage of the present invention:

(1) of the present invention employs the Polygonum cuspidatum crushed during vacuum blasting, Polygonum cuspidatum so thoroughly pulverized, for subsequent extraction of reach Better results, making extraction more fully.

Enzymolysis (2) of the present invention, the extract was concentrated and Polygonum cuspidatum resveratrol glycosides and other substances off under enzymatic hydrolysis To be converted to glucose-based resveratrol significantly improve the extraction rate of resveratrol.

(3) In the present invention, after digestion macroporous resin adsorption, washing, crystallization, purification of resveratrol from Polygonum make better Before and after each step with each other, forming an integral role, making the tiger battle extraction rate of resveratrol in more than 95% white Resveratrol purity of over 98% and shorten the extraction time, greatly reduce production costs, greatly improve product quality, with There are significant economic benefits for large-scale industrial application.

Ingredients: Polygonum cuspidatum: dried rhizome and root of Polygonum cuspidatum Polygonum plants.

Extraction steps are as follows:

(1) raw material crushing Polygonum cuspidatum: Polygonum cuspidatum grinding particle size of 10-100 mesh.

(2) vacuum blasting (vacuum instant expansion, which is to use extreme pressure, then relief, momentary vacuum, so that the material Cell wall damage. )

(3) solvent extraction: vacuum blasting solvent extraction step, the mass ratio of solvent knotweed is 5:1-20:1, mention Take a temperature 10-50 ° C, 1-5 times extraction times, extraction time 2-5 hours; the solvent is water or an alcohol or alcohols And any water mixture ratio. Wherein the alcohol is methanol or ethanol in one. (4) The combined extracts were concentrated to: control The extract is concentrated at a temperature below 80 ° C, and concentrated to a density of 1.03-1.08g / cm <3>.

Conduct (5) after enzymatic decomposition fermentation: the enzyme is glucoamylase, dosage concentrate 0.1% to 1.0% by mass, fermentation At a temperature of 25-50 ° C, the reaction time is 24-96 hours.

(6) resveratrol liquid enzyme decomposed with dilute acid to adjust the pH value, the adjusted hydrolyzate as dilute hydrochloric acid or dilute sulfuric Acid, sulfuric acid or dilute hydrochloric acid at a concentration of 0.5 to 10% by weight, a pH adjusted hydrolyzate 3-5.

(7) by macroporous resin, washed: macroporous resin after washing with water to neutral, first with the volume concentration After 10-40% ethanol impurity, and then the volume concentration of 80% ethanol to elute resveratrol, and then concentrated eluant Shrink: control the temperature of the eluent was concentrated below 80 ° C, and concentrated to a density of 1.03-1.08g / cm <3>.

(8) Crystallization: concentrate resveratrol elution crystallization solution was added, the crystallization solution is chloroform or acetone, or two By a mixture in any proportion, freeze crystallization temperature of -30-0 ° C, the crystallization time is 5-12 hours, one or more times Recrystallization; crystallization solution and the amount of resveratrol concentrate was eluted in a volume ratio of 2-10:1.

The above steps to get resveratrol content of about 98%, more than 90% yield of resveratrol,

The following are several embodiments of the present invention, further illustrate the invention, but the present invention is not limited thereto.

Example 1

The Polygonum cuspidatum material crushed to 30 mesh after blasting vacuum at room temperature, and extracted three times with methanol, methanol and Polygonum cuspidatum quality ratio Is 5:1, extraction temperature at 10 ° C, extraction time 5 hours; the combined extracts were concentrated to control the temperature of the extract in 80 ° C or less, and concentrated to a density of 1.03g / cm <3>. The concentrate was added 0.3% by mass glucoamylase decomposition, temperature controlled at 40 ° C, Fermentation time 72 hours resveratrol was decomposed with enzyme concentration of 0.5 wt% dilute hydrochloric PH value to 4.0, macroporous tree Fat absorption, macroporous resin after washing with water until neutral and then with the volume concentration of 30% ethanol wash impurity concentration by volume Of 80% ethanol to resveratrol. The eluate was concentrated (control the temperature of the eluant was concentrated below 80 ° C, and concentrated to an airtight Degree of 1.03g / cm <3>) After elution the concentrated solution was added 2-fold amount (by volume) of a solution of crystalline (Anti-chloro-: acetone = 3 mixture of Co-solution) were dissolved, freezing the crystallization temperature of -30 ° C, the crystallization time is 12 hours to give 98.3% pure product, resveratrol Alcohol yield of 93.2% can be achieved.

Example 2

The Polygonum cuspidatum material crushed to 10 mesh after blasting vacuum at room temperature, extracted five times with ethanol, ethanol and Polygonum cuspidatum quality ratio 20:1 is extracted at a temperature of 50 ° C, 2 hours extraction time; combined extracts control the temperature of the extract was concentrated Below 80 ° C, and concentrated to a density of 1.08g / cm <3>. The concentrate was added 1.0% by mass glucoamylase decomposition, temperature controlled at 25 ° C Fermentation time 96 hours, resveratrol was decomposed with enzyme concentration of 10% by weight sulfuric acid to adjust PH 5, macroporous resin Adsorption, macroporous resin after washing with water until neutral and then with the volume concentration of 10% ethanol wash impurity concentration of 80% by volume The resveratrol in ethanol. The eluate was concentrated (control the temperature of the eluant was concentrated below 80 ° C, and concentrated to a density of 1.03g / cm <3>) After elution the concentrated solution was added 10 times (volume ratio) was dissolved in chloroform, freeze crystallization temperature of -0 ° C, Crystallization time of 12 hours, the product purity of 98.0%, the yield of resveratrol can reach 92.4%.

Example 3

The Polygonum cuspidatum material crushed to 100 mesh after blasting vacuum at room temperature, extracted three times with ethanol, ethanol and the quality of Polygonum cuspidatum 15:1 ratio, extraction temperature at 20 ° C, 3 hours extraction time; combined extracts control the temperature of the extract was concentrated Below 80 ° C, and concentrated to a density of 1.05g / cm <3>. The concentrate was added 0.1% by mass glucoamylase decomposition, temperature controlled at 40 ° C Fermentation time of 24 hours, resveratrol was decomposed with enzyme concentration of 5% by weight sulfuric acid to adjust PH 3, macroporous resin Adsorption, macroporous resin after washing with water until neutral, after concentration by volume of 40% ethanol wash impurity concentration by volume Of 80% ethanol to resveratrol. The eluate was concentrated (control the temperature of the eluant was concentrated below 80 ° C, and concentrated to an airtight Degree of 1.05g / cm <3>), the solution was added 5 volumes of eluate concentrate (volume ratio) was dissolved in acetone, freeze crystallization, a temperature of -20 ° C, the crystallization time of 10 hours to obtain a product with a purity of 97.5%, the yield of resveratrol can reach 91.4%.

Example 4

The Polygonum cuspidatum material crushed to 80 mesh after blasting vacuum at room temperature, extracted four times with methanol, methanol and Polygonum cuspidatum quality ratio 10:1 is extracted at a temperature of 30 ° C, 4 hours extraction time; combined extracts control the temperature of the extract was concentrated Below 80 ° C, and concentrated to a density of 1.07g / cm <3>. The concentrate was added 0.8% by mass of glucoamylase decomposition, temperature controlled at 45 ° C Fermentation time 80 hours, resveratrol was decomposed with enzyme concentration of 6% by weight of dilute hydrochloric acid to adjust PH 3, macroporous resin Adsorption, macroporous resin after washing with water until neutral and then with the volume concentration of 30% ethanol wash impurity concentration of 80% by volume The resveratrol in ethanol. The eluate was concentrated (control the temperature of the eluant was concentrated below 80 ° C, and concentrated to a density of 1.07g / cm <3>) After elution the concentrated solution was added 8 times (by volume) of chloroform and acetone, a mixed solution by dissolving any ratio, Frozen crystallization temperature of -25 ° C, the crystallization time is 8 hours, product purity of 98.7%, the yield of resveratrol can be reached To 93.8%.

Example 5

The Polygonum cuspidatum material crushed to 30 mesh after blasting vacuum at room temperature, extracted twice with methanol, methanol and Polygonum cuspidatum quality ratio 8:1, extraction temperature at 25 ° C, extraction time 4 hours; the combined extracts were concentrated to control the temperature of the extract in 80 ° C or less, and concentrated to a density of 1.06g / cm <3>. The concentrate was added 0.6% by mass glucoamylase decomposition, temperature controlled at 45 ° C, Fermentation time 65 hours, resveratrol was decomposed with enzyme concentration of 2% by weight sulfuric acid to adjust PH 4.5, macroporous resin Adsorption, macroporous resin after washing with water until neutral and then with the volume concentration of 25% ethanol wash impurity concentration by volume Of 80% ethanol to resveratrol. The eluate was concentrated (control the temperature of the eluant was concentrated below 80 ° C, and concentrated to an airtight Degree of 1.06g / cm <3>), the solution was added 5 volumes of eluate concentrate (volume ratio) mixed solution of chloroform and acetone 4 to 1 by dissolving , Freeze crystallization temperature of -15 ° C, the crystallization time was 6 hours, the product purity of 99.2%, the yield of resveratrol can 93.7%.



Technique for extracting and purifying resveratrol from fresh giant knotweed rhizome
CN101397242

The invention discloses a process method for extracting and purifying resveratrol from fresh giant knotweed rhizome, the process method is characterized in that the process method comprises the following steps: the collected fresh giant knotweed rhizome is cleaned, air-dried, cut into slices and weighed; ethanol with the weight which is 4 to 8 times of the weight of the giant knotweed rhizome and the concentration of 60 percent to 85 percent is added, the soaking and the extraction are carried out for 24 hours at 30 DEG C to 60 DEG C, extract liquid is collected and filtered; the secondary extraction is carried out by using the same method, filtrate is combined, the pressure is reduced as far as possible and a thick paste is obtained by evaporation, 4 times of inactivated silica gel with 70 to 140 meshes is added and evenly stirred with the thick paste, the blow drying by cold air is carried out, the chromatography is carried out by 6 times of the sample amount of the silica gel with 70 to 140 meshes, ethanol-ethyl acetate-petroleum ether is used for elution at the temperature of 60 to 90 DEG C, the part containing the resveratrol is guided to merge by using the thin layer chromatography result, the pressure is reduced, the solvent is recycled, a crude solid is obtained, and a pure resveratrol needle-like crystal is obtained by recrystallization in ethanol. The process method has simple manufacturing process, easy realization, low production cost and high product purity.

The present invention discloses a method for extracting from fresh Polygonum cuspidatum The process of purification of resveratrol, comprising the steps of: collecting fresh Polygonum cuspidatum washed, dried, cut into thin slices, weighed; Polygonum cuspidatum added considerable weight 4-8 times of ethanol, 60% -85%, in the 30 ° C-60 ° C at 24 hours soaking extraction, the extract was collected, and filtered; the above-described method to extract the same time, the combined filtrate again evaporated under reduced pressure to a thick possible paste, add 4 times 70-140 mesh silica gel and inactivated a thick paste mix, cold dry, with six times the sample size 70-140 mesh silica gel using ethanol - ethyl acetate - petroleum ether 60-90 elution temperature ° C, combined with a thin layer analysis guide section contains resveratrol, the recovered solvent under reduced pressure to give a crude solid recrystallized from ethanol to obtain pure resveratrol needles. The present invention is a manufacturing process is simple, easy to implement, low production cost, high product purity is obtained.

TECHNICAL FIELD

Extraction and purification process of the present invention relates to a traditional Chinese medicine chemical composition, in particular a new Fresh Polygonum cuspidatum extract, process for purification of resveratrol.

Background technique

Resveratrol can reduce blood lipids, inhibit platelet aggregation, anti-tumor, anti-aging, protection Cardiovascular system, effective against cancer, its medical value and health value has been full World recognized.

Practice has proved that traditional Chinese medicine Polygonum cuspidatum resveratrol in the form of multi-glycosides exist, and a small amount Free coexistence of resveratrol is Polygonum cuspidatum important active ingredient, however, to extract from Polygonum cuspidatum high Purity resveratrol need to go through a certain process method. The investigation found that the new, Chinese patent number: 200610031850,3 discloses a "microbial conversion material Polygonum cuspidatum extract high purity resveratrol work Art ", which is characterized by: its first use of yeast and other microorganisms Polygonum material biotransformation, so Polygonum cuspidatum resveratrol resveratrol analog conversion, then add a certain percentage of ethanol and hydrocarbon alcohol, The use of microwave low temperature extraction, filtered and concentrated to give resveratrol.

Patent disclosed above is dried using as raw material Polygonum cuspidatum, Polygonum cuspidatum material needed for microbes Material biotransformation, and ultrasonic vibration, microwave-assisted extraction methods, such party Method process more complicated operation, long cycle, the extracted resveratrol purity is not high.

SUMMARY

Object of the present invention is to solve the deficiencies of the prior art, to provide a low cost of production in the new In fresh Polygonum cuspidatum extract, process for purification of high purity resveratrol.

Aspect of the present invention is implemented as follows: A fresh extract from Polygonum cuspidatum, Purified Resveratrol process comprising the steps of:

(1) to collect fresh Polygonum cuspidatum washed, dried, cut into thin slices, weighing;

(2), 4-8 times added considerable weight Polygonum cuspidatum 60% -85% ethanol at 30 ° C-60 ° C under immersion Soak 24 hours extraction, the extract was collected, and filtered;

(3), extracted once with the same method as above, and the combined filtrate again evaporated under reduced pressure to a thick possible paste;

(4), the resulting paste subjected to silica gel column chromatography, to isolate and purify Resveratrol: 4 with Times 70-140 mesh silica gel and inactivated a thick paste mix, cold dry, with six times the sample size of 70-140 Mesh silica gel chromatography, eluting with ethanol - ethyl acetate - petroleum ether yielded at a temperature of 60-90 ° C, with TLC analysis combined guide section contains resveratrol, the recovered solvent under reduced pressure to give a crude solid, Recrystallized from ethanol to obtain pure resveratrol needles.
Step of the present invention, (2) the optimal concentration of ethanol was 80% and the extraction 5h at 60 ° C under.

Advantage of the present invention is that: 1, as a raw material in fresh Polygonum cuspidatum, Polygonum cuspidatum can free Resveratrol and glycosides state is not destroyed, improve the extraction rate of resveratrol; 2, due to the new Adding fresh Polygonum cuspidatum ethanol solution, which can effectively isolate resveratrol and resveratrol analogues, The process is simple, the cost of extracting the bottom; 3, the use of silica gel column chromatography using ethanol - ethyl acetate - Petroleum ether as eluent to obtain greater than 95% purity resveratrol product, its isolation, purification treatment effect miss you.

Specific implementation methods

The collection of fresh Polygonum cuspidatum washed, dried, cut into thin slices, weighed 500g, was added 3000g Concentration of 80% ethanol, at a temperature of 60 ° C soak for 5 hours extraction, the extract was collected, too Filtered, and then extracted once again in the same manner, and the combined filtrate, the combined filtrate was evaporated to a thick Cream, with 4 70-140 mesh silica gel deactivated with a thick paste mix, cold dry, with six times the amount of sample The 70-140 mesh silica gel using ethanol - ethyl acetate - petroleum ether at a temperature of 75 ° C Eluted, precipitated white needle crystal was filtered to obtain crystals with a thin layer containing the results of the analysis to guide the merger Resveratrol part, solvent is recovered under reduced pressure to give a crude solid recrystallized from ethanol to obtain pure resveratrol Needle-like crystals, that was greater than 95% purity resveratrol products.



Substance extracted from giant knotweed for diabetes mellitus and complication thereof
CN101204428

The invention relates to a substance which is extracted from giant knotweed for curing diabetes mellitus and complicating diseases. By using the substance extracted from the giant knotweed, the medicine for curing diabetes mellitus and the complicating diseases can be produced effectively, and the treatment problems of diabetes mellitus and the complicating diseases are solved. The substance is coarse powder of the giant knotweed and can be prepared through the following steps: putting the giant knotweed in a round flask, adding 6 times quantity of grain alcohol to soak the giant knotweed; refluxing and extracting and filtering the grain alcohol, and the reflux extraction and the filtering are carried out to medical slag which is added with the grain alcohol which is 6 times of the medical slag. The filtrate water are combined for two times, the filtrate water is decompressed for recycling the grain alcohol, and the filtrate water is added in the pretreatment and reborn macro pore adsorption resin column; the resin column is elutriated with distilled water until the eluate is colorless, and the resin column is elutriated with the grain alcohol, the mass concentration of which is 70 percent, the eluate is collected till the eluate does not contain any component of anthraquinone, and the grain alcohol is recycled, and the total anthraquinone which is the extracted substance of the giant knotweed can be gained when the eluate is volatilized on the water bath. The invention has the advantages that the preparation method of the product is scientific, the operation is easy, the quality of the total anthraquinone which is the preparation substance is good, the purity quotient is high and the effect is good. The substance can be used for preparing the medicine which is used for curing diabetes mellitus and the complicating diseases, and the substance which benefits mankind is a creative of the development and the application of the giant knotweed medicine.

The present invention relates to a method of treating diabetes and complications of substance extracted from Polygonum cuspidatum, the drug may be effective for treating diabetes and its complications prepared to solve the problem of the treatment of diabetes and its complications, the meal was the medicinal substance, set round flask, add 6 times the amount of ethanol soaking, reflux extraction, filtration, dregs and then six times the amount of ethanol reflux extraction, filtration, twice combined filtrate decompression recovery of ethanol, concentrated pretreatment and regeneration macroporous resin column and eluted with distilled water to the eluent a colorless, then the mass concentration of 70% ethanol eluate, the eluate to contain anthraquinones far, the recovery of ethanol, water bath Polygonum cuspidatum extract evaporated to dryness to give the total anthraquinone substance, its products of scientific preparation, easy operation, good preparation material total anthraquinone quality, high purity, good effect, for the preparation of effective drugs for treating diabetes and its complications, the drug is Polygonum cuspidatum development and application on a major innovation, making benefit of mankind.

A method of treating diabetes and complications of substance extracted from Polygonum cuspidatum

I. Technical Field

The present invention relates to the field of medicine, in particular extracted from Polygonum cuspidatum and treating diabetes and Complications substances.

BACKGROUND Technical

Diabetes and its complications (diabetes mellitus, DM) is a Chinese medicine "Diabetes" category, based on more Drink, eat, polyuria, body weight loss, or turbid urine, urinary disorders characterized by sweet. Modern medicine recognized Diabetes is a by a variety of causes chronic metabolic diseases, due to the lack of insulin Or antagonize the hormone insulin or the insulin does not increase the normal physiological role to play in target cells and cause Glucose, protein and lipid metabolism disorder syndrome. Divided into insulin-dependent diabetes clinic Disease (IDDM) that is type I and non-insulin dependent diabetes mellitus (NIDDM) that is type II of two types, the current Approximately 157 million diabetics in the world, China has more than 20 million people, and the prevalence in the year Growth, has become a threat to human health in the top three killers, which occurs in addition to genetic and environmental factors Vegetarian and diet related, insulin resistance has become one of the important reasons for diabetes, the relevant Data show that during the transition from developing to developed countries significantly higher incidence. Lifestyle Application and adjustment of antidiabetic drugs is the main means to control type 2 DM and complications, hypoglycemic drugs currently still In western medicine, there is hypoglycemia, gastrointestinal reactions and other side effects and poor efficacy of the symptoms of diabetes And other issues, and with the extension of treatment time, also need to increase the dose, and therefore need further research and development of long-term, Efficient, low toxicity hypoglycemic agents, especially the control of drug complications and improve insulin resistance is still medicine Experts goal.

Chinese medicine in the prevention and treatment of DM has great advantages, Chinese medicine, its occurrence and spleen, kidney, gas Yin deficiency, blood stasis, and clinical take qi and yin, kidney and spleen, traditional Chinese medicine treatment Diabetes have a good effect, and the small toxic side effects of traditional Chinese medicine, the role of mild-lasting, with combined therapy Effect, can delay complications advantage, after all, to find new drugs hypoglycemic an important way. First use Advanced science and technology, a good grasp of Chinese multi-target, multi-link, the combined effects of regulation and functional characteristics, To focus on the prevention of complications of DM, DM prevention are the advantages of traditional Chinese medicine, but also the majority of diabetes Common needs of patients and society as a whole.

Motherland medicine: basic pathogenesis of diabetes is Yin-chun, loss, hot side wins. Jin homologous blood each other Zisheng conversion, Yin deficiency blood will be insufficient, and heat dissipation can be gravel body fluid, Hao Shang Yin, Yin make more deficiency, Yin deficiency is not sufficient pulse Road may cause poor blood, bleeding within the stop, stasis and qi stagnation. Moreover, those who long Diabetes, The PVI inevitable injuries, signs of deficiency Diego now, if Qi is weak transport blood, blood deficiency is incomprehensible, from chronic illness Into the network, into the network of long virtual blood stasis syndrome, stasis Serve, then Chen who is not when to go, when the new students who can not Health, more blood and more virtual stasis, stasis and the more the more virtual, reinforce each other, cross-phase infestation. Leading to bad blood yin and yang, Blood stasis. So there must be Diabetes pathological bleeding. Since most diabetics have blood stasis syndrome Like. Clinical blood circulation method can improve patients' sugar, fat metabolism and blood viscosity state and Neurovascular complications symptoms.

Polygonum cuspidatum, also known as "Kowloon root", "Yin Yang Lotus" tepid, bitter, liver, gall bladder, lung. Polygonum Polygonum cuspidatum plants (Polygonum cuspidatum Sieb.et Zucc) dried rhizomes and roots. main Function is expelling wind and dampness, stasis and pain, cough and phlegm. Polygonum cuspidatum as hypoglycemic blood stasis in folk medicine Already applied in recent years, there are scattered reports of hypoglycemic Polygonum cuspidatum, but not in-depth study more, for lack of hypoglycemic Discussion on the mechanism. Polygonum cuspidatum as antidiabetic medicine used more often in addition to the compound, the single application and about its taste drop Modern research sugar component and hypoglycemic effect is small.

Modern pharmacological studies have shown that Polygonum cuspidatum hypoglycemic, anti-oxidation, inotropic, anti-shock, improve microcirculation Ring, inhibiting platelet aggregation, lowering blood pressure, cough, asthma, improve immunity, pathogenic microorganisms, Ming Significant intestinal muscle relaxation, anti-tumor, increasing the role of white blood cells and platelets and anti-oxidation. For the treatment of Diabetes, hyperlipidemia, upper gastrointestinal bleeding, hepatitis, neonatal jaundice, leukopenia, lung Inflammation, arthritis, burns, vaginitis, and vitreous hemorrhage embolism. Polygonum cuspidatum containing mainly free anthraquinone and anthracene Quinone glycosides, tannins, flavonoids, polysaccharides and a small amount of amino acids and other ingredients. Experimental studies have shown that the existing Polygonum cuspidatum Aqueous extract of a- glucosidase strongly inhibited, and has been reported that Polygonum cuspidatum anthraquinones compounds It was a hypoglycemic active ingredient; rabbits intravenously has been reported to be extracted from Polygonum cuspidatum oxalic acid, can cause low blood sugar shock. Another report from Polygonum tannin in alloxan diabetic mice have a good hypoglycemic effect. But its Study on hypoglycemic effect also limited water decoction, hypoglycemic active site and the active ingredient is not clear, it Study on hypoglycemic mechanism is also limited to the overall level, how to filter Polygonum cuspidatum extract containing the active moiety The main chemical components - anthraquinone and for the treatment of diabetes and its complications, there are so far no reports.

III. SUMMARY OF THE INVENTION

For the above, an object of the invention is an active part of the screening of Polygonum cuspidatum extract from Polygonum cuspidatum treatment Substance diabetes and its complications, drug therapy may be an effective preparation of diabetes and its complications, resolve Problems treatment of diabetes and its complications, its technical solutions to address is the substance from the dried root of Polygonum cuspidatum Slice the stems and roots made taking Polygonum cuspidatum slices, coarsely ground, set a round-bottom flask, plus 6 times the amount of ethanol (70%) After soaking 30min, extracted reflux 2h, filtered, residue 6 times the amount of ethanol (70%) and then reflux extraction 1 Times, extracting 2h, filtered, and the filtrate combined twice, vacuum recovery of ethanol, and then concentrated to pretreatment Students macroporous resin column and eluted with distilled water to the eluent a colorless, then the mass concentration of 70% Ethanol eluate, eluent free to Anthraquinones far, the recovery of ethanol, water bath On evaporated to dryness to give the Polygonum cuspidatum total anthraquinone substance, yield 2-2.5% purity 64-70.7%. Said preparatory Macroporous resin adsorption process and regeneration column macroporous resin used for adsorption and regeneration to be pretreated, Wherein the pretreatment is to take the D301 macroporous resin with a concentration of 95% ethanol for 24h, constantly Stirring, after removal of air bubbles in a column, with a concentration of 95% ethanol elution, the eluate to thoroughly wash After the Ming and evaporated without residue, then washed successively with 1mol / LNaOH 3 volumes of water, 2 volumes Distilled water, 1mol / LHCL 3 volumes of eluting solution, and finally eluted with distilled water to neutral pH, Thereafter, the macroporous resin regeneration, that is eluted with 1mol / LNaOH aqueous wash eluent to transparent, Then eluted with 2 volumes of distilled water, and then 1mol / LHCL aqueous elution wash eluent to transparent, Finally eluted with distilled water to neutral pH (reusable), the process flow process shown in Figure 1.

Present Description Product Preparation science, easy operation, good preparation material total anthraquinone quality, high purity, using the results Well, for the preparation of effective drugs for treating diabetes and its complications, it is the development and application of drugs Polygonum cuspidatum A major innovation, making benefit of mankind.

IV BRIEF DESCRIPTION

Figure 1 is a process flow diagram for preparing the product of the invention.

Figure 2 is a product of the invention the determination of the standard curve.

Figure 3 is a graph showing the different volumes of product invention to α- glucosidase Vigor.

V. DETAILED DESCRIPTION

According to the aforementioned aspect, the present invention is made in the implementation of the following specific ways.

The knotweed rhizome and roots to its soil, wash static, crushed into coarse powder (50-80 mesh), take 1000 G meal, add 6000ml mass concentration of 70% ethanol soaked after 30min, reflux extraction 2h, Filtration, liquid reserve, then add 6000ml dregs mass concentration of 70% ethanol reflux extraction After 2h, filtered, twice combined filtrates under reduced pressure to recover ethanol, and concentrated to a volume of 50%, was added pretreatment Management and regeneration of macroporous adsorption resin column (called macroporous resin column and pretreated with regeneration Before), eluted with distilled water until the eluate is colorless, then is eluted with 70% ethanol concentration, collecting Eluent eluent to contain Anthraquinones far, the recovery of ethanol, evaporated on a water bath did Polygonum cuspidatum extract Extract total anthraquinone 21.25 g, 70.7% purity, it can be used to prepare an effective treatment of diabetes and its complications Disease Drugs, the extract by the test for the treatment of diabetes and its complications have a prominent role in the disease, than the original Polygonum cuspidatum The original drug is much better, the effect did not expect for the development before the relevant experimental data as follows.

(A) total anthraquinone Polygonum cuspidatum chromogenic reaction

1. Feigl reaction

Take a little of Polygonum cuspidatum total anthraquinone in a test tube, dissolved in water, 25% aqueous sodium carbonate, 4% A 5% solution of formaldehyde and benzene o-nitrobenzene, a little different, a mixture heated on a water bath for 2 minutes, resulting in significant The purple.

2. The reaction with an alkaline agent

Take polygonum anthraquinone 5mg, add water about 5ml, stir, take the supernatant, add chloroform 10ml, Shaking extraction, get a chloroform solution, evaporated and 2 drops of 5% sodium hydroxide was dropped, was cherry red.

3. Magnesium acetate color reaction

Take a little polygonum anthraquinone, dissolved in ethanol, drops on filter paper, drying, spray 0.5% magnesium acetate A Alcohol solution, heated to 90 degrees minutes was cherry red.

4 .. TLC

Take polygonum anthraquinone 15mg, plus 2.5mol L sulfuric acid 5ml / solution, heating hydrolysis for 30 minutes, put Cold, extracted with chloroform 2 times, each 5ml, combined chloroform solution, evaporated and the residue dissolved in chloroform 1ml make Solution, as the test solution. Another emodin reference substance 2mg, add methanol made per 1ml containing 1mg Solution as the reference solution. Learn the test, reference solution 2ul, respectively, point to the same silicon Gel G plate with ethyl acetate volume: methanol: water = 100:16.5:13.5 as the agent, exhibition Open out, drying, UV light (365nm), under review. Test products for chromatography, in that the reference The corresponding position chromatography was the same orange fluorescent spots; smoked rear ammonia vapor, fluorescent under inspection Visual, spot turns red.

(B) Determination of Polygonum cuspidatum total anthraquinone

Colorimetric determination of total content of anthraquinone, emodin as standard, smoked spectrophotometry Received degrees.

1. Preparation of the reference solution

Emodin reference substance 10mg, accurately weighed, placed in 100ml volumetric flask, dissolved in methanol and diluted To the mark (100ug / ml), shake, that was (per 1ml containing emodin 0.1mg).

2. Preparation of standard curve

Precision drawing reference solution 4.0,6.0,8.0,10.0,12.0,14.0ml respectively set 25ml capacity Bottle, recovering methanol on a water bath, add 5% to 2% sodium hydroxide lye mix, shake, Dissolve with a sintered funnel III filtration, the filtrate, the reagent blank, according to spectrophotometry (2005 Edition Appendix VB), 30min later, in a wavelength of 520nm absorbance was measured (A), respectively. In absorbance The vertical axis, the concentration of abscissa, the absorbance (A) - concentration (C) of the curve, the regression equation It is: A = 5.26 × C + 0.021, correlation coefficient r = 0.9996. Linear range: 0.016 ~ 0.056mg / ml, Table 1:

Table 1 Determination Polygonum cuspidatum total anthraquinone standard curve data

Standard curve shown in Figure 2.

3. Determination of content of the sample:

Accurately weighed dried to constant weight polygonum anthraquinone 7.5mg, a 100ml round-bottomed flask, add 2.5mol / L sulfuric acid solution, 20ml, was heated under reflux for 1 hour, coolish chloroform of about 30ml, reflux was continued 2 hours, cooled, displacing the separating funnel, washed with a small amount of chloroform container, lotion into the separating funnel In, get a chloroform layer set 50ml volumetric flask, acid and extracted with chloroform 2 times, each 8ml, and the chloroform solution Into 50ml volumetric flask, add chloroform to the mark, shake, that was the precise amount of the solution 10ml, evaporated to dryness; precision Add equal amount of 2% ammonia solution and 5% sodium hydroxide solution, a mixed solution of 20ml to dissolve, with weeping III Melt funnel filtration, the filtrate, the reagent blank, at the wavelength of 520nm absorbance was measured separately, Calculated by linear regression equation polygonum anthraquinone emodin (C15H10O5) is the reference against its total anthracene Quinone content.

Measured according to the above extraction method of total anthraquinone in Polygonum cuspidatum total anthraquinone content was measured 6 times and the results Table 2.

Table 2 polygonum anthraquinone assay results

Above table can be seen that the average content of this experiment extracted from Polygonum total anthraquinone anthraquinone is 67.33%.

Influence (c) polygonum anthraquinone large model of diabetes in mice

1 Materials

1.1 Drugs

Polygonum anthraquinone, press extraction process to extract; metformin hydrochloride, Shanghai Pharmaceutical (Group) Co., Company Xinyi Pharmaceutical Factory, batch number: 060310,0.25g / piece, 48 / box.

1.2 Reagents

Streptozotocin, sigma company; saline, Zhengzhou Yonghe Pharmaceutical Co., Ltd., batch number: 060 810 012, specifications 500ml / bottle; citric acid (AR), Hubei Pharmaceutical company Bose station, batch No: 20020623; sodium citrate (AR), Suzhou Chemical Reagent Factory, batch number: 20010624; conc. Sulfuric acid, Luoyang City Chemical Reagent Factory, batch number: 051 102; glucose kit, Zhejiang Dongou Biotechnology Ltd., batch number: 2006060350; glycated serum protein (GSP) kit of Nanjing Jiancheng Institute of Biotechnology of the first branch, batch number: 20061007; glycogen kit, Nanjing Jiancheng Bioengineering Cheng Institute, batch number: 20060907; glacial acetic acid (AR), China Laiyang both Chemical Co. Division Lot: 20050321; SOD kit (measuring total), Nanjing Jiancheng Institute of Biotechnology, batch number: 20,070,131; malondialdehyde (MDA) test kit, Nanjing Jiancheng Institute of Biotechnology, batch number: 20070206; LDL cholesterol assay kit (LDL-C), Zhejiang East Ou Biological Engineering Co., batch Number: 2006080265; high-density lipoprotein cholesterol assay kit (HDL-C), Zhejiang East Ou biological Engineering Co., Ltd., batch number: 2006120002; triglyceride assay kit (TG), Zhejiang East Ou Health Engineering Co., Ltd., batch number: 2006110001; total cholesterol assay kit (TCH), Zhejiang East Ou Biological Engineering Co., Ltd., batch number: 2006090002; insulin (INS) radioimmunoassay kit Beijing Chemclin Biotechnology Co., Ltd., batch number: 20063082; Leptin (Leptin) Radiation Immunoassay kit, People's Liberation Army General Hospital, RIA Technology Development Center, Lot 070126; C- Peptide (C-P) radioimmunoassay kit (RIA), Beijing Chemclin Biotechnology Co., Ltd., batch number: 20,070,125; insulin antibodies (INS-Ab) radioimmunoassay kit, Beijing Chemclin biotechnology Surgery Ltd., batch number: 20070125.

1.3 Instrument

UV-2000 UV-Vis spectrophotometer: UNICO (Shanghai) Co., Ltd. Instrument; heated water Bath, Beijing Guangming Instrument Factory; centrifuge, Beijing medical equipment repair shop; FA (N) / JA (N) series Electronic balance, Shanghai People Bridge Precision Instrument Co., Ltd.; adjustable pipette, the Hai Leibo analytical instruments Limited; OLYMPUS optical microscope; Hitachi H-7500 transmission electron microscope.

1.4 Animals

Mice: Kunming, male, weight: 18 ~ 20g; provided by the Experimental Animal Center of Hebei Province, together Grid No: 611 040. Rat: Wistar, male, body weight: 180 ~ 200g; Hebei Province experimental animals Was center, Certificate of Conformity: 701,022.

1.5 test solution preparation

Before according to kit instructions, is added a certain amount of concentrated sulfuric acid, stirring to dissolve, the use of: the reagent formulation New feature.
Formulated citrate buffer pH: 4.2: citric acid 0.1mol / L + sodium citrate 0.1mol / L 12.3 + 7.7

Weigh 1292.4mg citric acid and sodium citrate 1132.3mg dubbed 100ml solution, and the solution that is A citrate buffer Ph 4.2, in order to ensure its accuracy available Ph instrument calibration deployment.

(Streptozotocin is not very stable, and greater stimulation of blood vessels with buffer formulated streptozotocin Reduce these side effects that): prepared before use.
10% formalin fixative formulation: 36% formaldehyde solution 10ml, paired with distilled water 100ml.

4% glutaraldehyde fixative formulation of: disodium hydrogen phosphate 35.61g, paired with double distilled water 1000ml; Sodium dihydrogen phosphate 27.60g, paired with double distilled water 1000ml. Take disodium hydrogen phosphate solution and 40.5ml Sodium dihydrogen after mixing 9.5ml solution of 25% glutaraldehyde was added 16.0ml, coupled with double-distilled water to 100ml.

2 Statistical

Data was analyzed by SPSS 10.0for windows statistical software, measurement data between groups were compared using ANOVA, using Ridit level data analysis.

3 Experimental methods and results

3.1 Effect of polygonum anthraquinone mouse model of diabetes

Take male mice, normal feeding for 3 days after fasting 12h, intravenous injection of streptozotocin 80 (Mg / kg, 0.02ml / 10g), injection after the first 11 days of fasting 12h, trailing blood test fasting blood glucose level, Select the blood glucose> 11.1mmol / L and has significantly more drink, more food, more urinary symptoms in mice 50, press Blood glucose levels were randomly divided into five groups, namely large, medium and small dose Polygonum cuspidatum, positive control group and model group, Are fed large, medium and small dose polygonum anthraquinone (400mg / kg, 200mg / kg, 100mg / kg, 0.2ml / 10g), metformin (0.5g / kg, 25mg / ml, 0.2ml / 10g) and the same volume of saline, Another 10 mice served as the control group, fed with normal saline. Administered once daily, continuous Administered for 30 days. The first day of administration 10,20,30 tail blood test blood glucose level. The first 30 days of fasting 12h After the last administration 1h, blood, blood sugar (according to kit instructions), glycosylated serum protein (press agent Kit instructions), the mice were sacrificed, the liver glycogen measured (measured according to kit instructions); pancreas, 10% formalin Lin was fixed, for biopsy. The results are shown in Table 3,4,5.

Table on streptozotocin-induced blood glucose in mice 3 polygonum anthraquinone <Img class = "EMIRef" id = "099981881-if0001" />

**: Indicates Compared with model group P <0.01 *: represents compared with the model group P <0.05

As can be seen from the above chart, compared with the control group, the model group blood glucose levels at the start of the first 10, 20, 30 days were significantly higher than the control group (p <0.01), described streptozotocin diabetic model made successful. versus Model group than in the administration of the first 20 days, large, medium dose polygonum anthraquinone group and metformin group can significantly Lowering blood glucose levels (p <0.01), low-dose polygonum anthraquinone can significantly lower blood sugar levels (p <0.05), In the first 30 days of administration, large, medium and small doses of polygonum anthraquinone group and metformin group can significantly reduce blood Sugar levels (p <0.01).

Table 4 polygonum anthraquinone streptozotocin-induced diabetic mice glycated serum protein And the impact of glycogen <Img class = "EMIRef" id = "099981881-if0002" />
**: Indicates Compared with model group P <0.01 *: represents compared with the model group P <0.05

As can be seen from the table, compared with the control group, the model group made glycogen levels were significantly reduced, glycosylated blood Albumin levels were significantly higher (p <0.01). Compared with model group, large dose of polygonum anthraquinone and Second Metformin could significantly increase glycogen levels (p <0.01), low-dose polygonum anthraquinone significantly elevated liver Glycogen levels (p <0.05). Large and small doses of polygonum anthraquinone and metformin significantly decreased serum glycated Serum protein levels (p <0.01), the dose polygonum anthraquinone can significantly reduce serum glycated serum protein levels Level (p <0.05).

Table 5 polygonum anthraquinone streptozotocin-induced diabetic mice pancreas tissue

"-" For the islet cell cytoplasm rich, atrophy, edema and vacuolar degeneration were normal does not appear; "+" Islet cells showed atrophy without edema and vacuolar degeneration; "++" Most of the islet cells Shrink, a small number of cells without degeneration of edema. "+++" Islet cells shrink, Meanwhile edema and degeneration obvious.

As can be seen from the table, all rich white mice islet cell cytoplasm, does not appear to shrink, edema And vacuolar degeneration were normal, compared with the control group, model group, the vast majority of islet cells shrink, with When edema and vacuolar degeneration obvious by Ridit test P <0.01, illustrate successful model. And model Group, the large, medium and small doses of polygonum anthraquinone group and metformin group most abundant islet cell cytoplasm, Islet volume increases, a small cell atrophy, decreased cell cytoplasm and nucleus presented crowding, By Ridit test P <0.01. Metformin group than in large, medium dose polygonum anthraquinone islets Abundant cytoplasm, shrink less, edema and vacuolar degeneration lighter by Ridit test P <0.01. The results suggest that: large, medium and small doses of polygonum anthraquinone streptozotocin-induced islet injury protection Effect, obviously stronger than metformin.

3.2 Effect of polygonum anthraquinone diabetic rats

Take 100 male rats, fasting 12h, in which 90 intravenous injection of streptozotocin 60mg / kg (with a pH 4.2 citrate buffer preparation), another 10 to completely blank control group, Intravenous injection of an equal volume of citrate buffer. Day 10 tail blood test blood glucose level, blood glucose level select > 11.1mmol / L and has significantly more drink, more food, 50 urinary symptoms in rats, according to a random blood glucose level Were divided into five groups fed large, medium and small doses of polygonum anthraquinone (400mg / kg, 200mg / kg, 100mg / kg, formulated at a concentration of 20mg / ml, 10mg / ml, 5mg / ml, the volume of gavage was 2ml / 100g) Solution, metformin 208mg / kg (20.8mg / ml, 2ml / 100g), model group and control group Were given the same volume of saline, administered once a day for consecutive 30 days, respectively, in the first 10 administration, 20, 30 days fasting blood glucose level, in the last day of fasting blood sugar before 12; drench 2h after taking Blood serum was separated, blood glucose (FBG), insulin (Ins), insulin antibodies, C peptide, leptin, Triglyceride (TG), cholesterol (TC), low density lipoprotein (LDL-C), high density lipoprotein (HDL-C) Level, superoxide dismutase (SOD), lipid peroxidation product malondialdehyde (MDA) level. Big killed After the rat pancreas and kidney for routine pathology and pancreas as transmission electron microscope to observe the pathological changes. Results Table 6,7,8,9,10.

Table 6 Effect of polygonum anthraquinone diabetic rats glucose <Img class = "EMIRef" id = "099981881-if0003" />

Compared with model group, ** indicates P <0.01, * indicates P <0.05

As can be seen from the table: compared with the control group, model group, blood glucose levels at the start of the first 10,20,30 Day was significantly higher than the control group (P <0.01), described streptozotocin diabetic model made successful. With the model group Than the first 20, 30 days metformin significantly reduced the blood glucose level after administration (P <0.01), on the 10th day of blood No significant effect on glucose levels; high dose polygonum anthraquinone can significantly reduce the blood glucose level on the 20th day (P <0.05), may Significantly reduced blood glucose levels on day 30 (P <0.01); dose polygonum anthraquinone can significantly reduce the first 20, 30 Day blood glucose level (P <0.01), lowering blood glucose level trends in the first 10 days; a small dose of polygonum anthraquinone have lower first 30 days blood glucose level trend.

Table 7 polygonum anthraquinone streptozotocin-induced diabetic rats serum insulin, Effect of leptin, insulin antibody, C- peptide <Img class = "EMIRef" id = "099981881-if0004" />
n = 6 **: compared with the control group indicates P <0.01 *: representation than in the control group P <0.05

As can be seen from the table, compared with the control group, the model group insulin, C-peptide levels were significantly lower (P <0.01), Leptin levels were significantly higher (P <0.01), insulin antibody levels did not change significantly. Compared with model group, Metformin, significantly higher dose of insulin polygonum anthraquinone group (P <0.01) insulin dose group Significantly increased (P <0.05), low-dose group had elevated insulin trend; middle dose group and low dose group C Peptide was significantly higher (P <0.01) levels, metformin group had elevated levels of C-peptide trend, but large doses Obvious; large, medium and small and metformin group can significantly reduce leptin levels (P <0.01); metformin Guanidine had significantly lower insulin antibody levels (P <0.01), and a large dose of insulin antibodies was significantly higher in the water Level (P <0.05).

Table 8 polygonum anthraquinone streptozotocin-induced diabetic rats serum malondialdehyde (MDA) And superoxide dismutase (SOD) Activity in Rats <Img class = "EMIRef" id = "099981881-
if0005" />

**: Indicates compared with model group P <0.01 *: represents compared with model group P <0.05

As can be seen from the table, compared with the control group, the model group of lipid peroxidation (LPO) metabolites prop The amount of the dialdehyde (MDA) was significantly higher than the control group, and SOD activity was significantly lower than the control group (P <0.01), Description lipid peroxidation injury and SOD decreased streptozotocin induced diabetic model exists. And the mold Type group, the medium and small doses of polygonum anthraquinone and metformin significantly decreased serum levels of MDA (P <0.01), high-dose polygonum anthraquinone can significantly reduce the serum levels of MDA (P <0.05), large, Dose Polygonum cuspidatum total anthraquinone can and metformin significantly increased activity of SOD after making model (P <0.01), Small doses of polygonum anthraquinone elevated SOD activity trend (P> 0.05).

Table 9 polygonum anthraquinone induced diabetic rat model lipid metabolism in streptozotocin

**: Indicates compared with model group P <0.01 *: represents compared with model group P <0.05

As can be seen from the table, compared with the control group, made streptozotocin diabetic rats model TG, TC, LDL-C levels were significantly higher than the control group (P <0.01), HDL-C levels were significantly lower than the control group (P <0.01), It shows that the model exists lipid metabolism disorders. Compared with model group, each group could improve lipid perfusion to varying degrees Metabolic disorders. Large doses of polygonum anthraquinone can significantly reduce TG, TC and LDL-C levels (P <0.01), Significantly increased HDL-C levels (P <0.01); dose polygonum anthraquinone can significantly reduce TC, TG, LDL-C levels (P <0.01), significantly increased HDL-C levels (P> 0.01); low-dose polygonum anthraquinone Can significantly reduce LDL-C levels (P <0.01), significantly increased HDL-C levels (P> 0.01), Ariake Significantly reduce TC and TG levels (P <0.05); metformin significantly reduced TC, TG, LDL-C water Level (P <0.01) and significantly increased HDL-C levels (P <0.01).

Table 10 Effect of polygonum anthraquinone kidney and pancreas of diabetic rats

For the islet cell cytoplasm rich, atrophy, edema and vacuolar degeneration were normal does not appear; "+" It appears to shrink as islet cells, without degenerative change and edema; "++" islet cells found mostly wilt Shrink, few cells degeneration edema. "+++" Islet cells shrink, and edema and Vacuolation.

As can be seen from the table, blank islets rich cytoplasm, cell in the body, also showed the nucleus Loose state. Compared with the control group, the model group significant portion of the vast majority of islet cell atrophy, cell Pulp significantly reduced, the cell body to narrow, dense nucleus, some cells appear edema and vacuolation, by Ridit Test, P <0.01, illustrate successful model.
Compared with model group, high dose group polygonum anthraquinone most pancreatic Cytoplasm rich island, islet volume increases, a small number of cell atrophy, degeneration Edema by Ridit test, P <0.01. Dose polygonum anthraquinone islets most cells atrophy, cell Significantly reduced cytoplasm and nucleus presented crowding, and a small part of cell degeneration edema, dried Ridit test, P <0.05; metformin improved islet injury trend. The results suggest that Polygonum cuspidatum Total anthraquinone of streptozotocin-induced islet injury in rats.

(Iv) Effect of Polygonum cuspidatum total anthraquinone α- glucosidase Activity

1 Materials

1.1 Reagents

α- glucosidase, sigma Company, Lot 081K7415; PNPG (4- nitrophenol -α-D - Glucopyranoside), sigma company; glutathione, sigma company; polygonum anthraquinone, double-distilled Water, 67mmol / L phosphate buffer (pH6.8), 0.1mol / L Na2CO3.

1.2 Instrument

752 spectrophotometer, Shanghai Third Analytical Instrument Factory; FA (N) / JA (N) series of electronic balance, on Precision Instrument Co., Ltd. China Sea Bridge; TGL-16G high-speed refrigerated centrifuge, Shanghai Anting Scientific Instrument Factory; DZKW-4 electron temperature water bath, Beijing Medical Instrument Factory Wing bright; adjustable pipette, Shanghai Leibo Analytical Instruments Limited.

2 Experimental Methods

By detecting α- glucosidase activity decreased, indirect determination polygonum anthraquinone glucose α- Glucosidase inhibitory activity.

Control tubes α- glucosidase enzyme activity assay: the PNPG (4- nitrophenol -α-D--gluco Glucoside) as substrate. Reaction system: 67mmol / L phosphate buffer (pH6.8) 1.7ml, 1mg / ml Glutathione 50μl, 20mg / mlα- glucosidase 5μl, 37 ° C for 10min after, Added 0.010mol / L PNPG50μl, after the reaction at 37 ° C 10min, was added 0.1mol / L Na2CO3 Stop Solution 10ml. Measured under the action of enzymes released amount (A2 value) nitrophenol at 400nm place.

Polygonum cuspidatum total anthraquinone inhibition assay activity: Weigh Polygonum cuspidatum total anthraquinone 100mg, treated with activated carbon removal Color, dry, add distilled water to 5ml, formulated at a concentration of 20mg / ml solution of polygonum anthraquinone. Minute Do not take 25,50,100,200,400μl was added to the enzyme activity assay system (system buffer A corresponding reduction in volume), followed by incubation with the enzyme is first reacted (37 ° C) 10min, plus substrate 10min, Na2CO3 The reaction was stopped, absorbance was measured at 400nm (A3 values). Chinese medicine extract liquid plus buffer is empty Tube reaction step above, measured at 400nm absorbance (A1 value). Are buffer plus glutathione plus PNPG stop solution to zeroing tube.

Statistical analysis

Data was analyzed by SPSS 10.0 for windows statistical software using statistical curve fitting analysis.

Define α- glucosidase activity unit is: pH6.8,37 ° C per minute when released 1μmolPNP A dynamic unit. The inhibitory activity was defined units: pH6.8,37 ° C within the same time make a Inactivation of enzyme activity unit as a suppression unit. = The percent inhibition The inhibitory activity / enzyme activity test × 100% The same determination into account in this experiment enzymes and inhibitors vitality, consistent main reagent consumption, it Percentage inhibition is calculated as simplified: percent inhibition = A / B × 100%. Note: A = control tube A2 Value - (the value of the test tube A3 - blank tube A1 value) B = control tube A2 value. The results are shown in Table 11.

Table 11 Effect of different volume polygonum anthraquinone α- glucosidase Activity

Figure 3 shows that within a certain range of different amounts of polygonum anthraquinone α- glucosidase inhibitory Activity curve fitting analysis showed different amounts of polygonum anthraquinone with α- glucosidase activity inhibition rate deposit Model relationships in S-shaped curve. Equation lny = 0.004141 + (- 31.426988 / x) (F = 758.43959, P value (Signif F) = 0.0001 <0.0005, at 0.05 significance level, reject the null Suppose, the equation can be considered significant. Wherein the value of the independent variable T = -27.540, P = 0.0001, according to inspection 0.05 Inspection standards, reject the null hypothesis can be considered as independent variables X regression coefficient is not zero. ) Was calculated by adding 45.07μl (20mg / ml) polygonum anthraquinone solution can generate more than 50% of the inhibitory activity, suggesting that the α- glucosidase has a good combination of power and a strong inhibitory effect.

(V) Conclusion

Anthraquinone is one of the main active ingredient knotweed, based on a lot of relevant literature, laboratory The preliminary research data and experimental results of this study, we believe that the main active portion anthraquinone its hypoglycemic Bit.

Polygonum anthraquinone significantly reduced by the adrenaline, had made alloxan hyperglycemia model mice blood sugar Level and streptozotocin had made big mouse model of diabetes blood glucose and glycated serum protein levels, there are pro Trends into insulin secretion and hepatic glycogen formation, can reduce diabetic rats triglycerides, cholesterol Alcohol, low-density lipoprotein levels, increased HDL levels, reduce the lipid peroxidation in the blood Between MDA level product, enhancement of SOD activity. Pathological observation by light microscopy analysis showed that: total anthraquinone Polygonum cuspidatum Protect modeling large, islet cells in mice against drugs damage modeling islet cells to make Model rats kidney has a protective effect, reduce the distal convoluted tubule cells edema state; Observation points Analysis showed that: Polygonum cuspidatum total anthraquinone can increase the number of mitochondria in rat renal tubular epithelial cells, reducing mitochondrial iso Chromatin quantity, reduce the mitochondria is dissolved and disappeared; by improving the organizational structure of certain microscopic Extent, help slow the disease process in diabetic rat kidney. Also polygonum anthraquinone can be reduced in vitro Low α- glucosidase activity. Hypoglycemic effect polygonum anthraquinone and its promotion of insulin secretion, promoting glucose Original synthesis, metabolism of free radicals in a certain relationship. At the same time, it can also reduce glycated serum protein levels Flat, lower blood lipid levels, thereby delaying the development of diabetic complications, effectively applied to the preparation Diabetes and its complications drugs, is a major innovation on medicinal Polygonum cuspidatum, a prominent economic and social efficiency Benefits for Chinese medicine has made creative contributions.



Method for preparing polygonum paleaceum dry extract by hot water extraction and ethanol precipitation and/or its purification part
CN101167975

The invention discloses a method for preparing a paleaceous knotweed extract. Paleaceous knotweed and pure water are put into the container, and bathed in the 100 DEG C water and reflowed to lixiviate for 30-90min; alcohol is charged into the filter liquor; the mixture is placed under the temperature of 0-8 DEG C till the filter liquor and the alcohol are fully mixed, and then is filtered to obtain spirituous filter liquor; the spirituous filter liquor is decompressed and condensed into paleaceous knotweed extract acquired by hot water extraction and ethanol precipitation under the temperature of 70-85 DEG C, and the paleaceous knotweed extract acquired by hot water extraction and ethanol precipitation is condensed into 0.1-0.4 times of original volume and braised to eliminate the alcohol and obtain the residue; the residue is dissolved by distilled water and filtered the insoluble matter toobtain the filter liquor; through strong-acid cationic ion resin columns, the filter liquor is firstly washed into achromatic color by water and then eluted by 3N of NH4OH; the ammonia eluent is collected and condensed into ointment; and the ointment is put on the silica gel columns and eluted by acetone-carbinol, the acetone-carbinol eluent is kept on concentrating till the white powder is deposited.

The present invention discloses a grass dragon's blood boiled dry extract alcohol precipitation and / or preparation of purified fraction. The grass dragon's blood into a container with water, water at 100 ° C water bath reflux extraction 30 ~ 90min, filtration to obtain filtrate was added ethanol, placed to the filtrate mixed with alcohol in 0 ~ 8 ° C under then filtration was alcoholic filtrate was concentrated under reduced pressure and then placed in boiling alcohol precipitation grass dragon's blood extract, then concentrated to the original volume of 0.1 to 0.4 times at 70 ~ 85 ° C after the residue was distilled ethanol , the residue was dissolved with distilled water, insoluble matter was filtered off and the filtrate, the filtrate was passed through a strong acid cation resin column, washed with water until colorless, and then eluted with 3N NHOH, ammonia eluate was collected, concentrated to give paste, the paste on a silica gel column with acetone - methanol to give the grass dragon's blood boiled dry extract alcohol precipitation. The grass dragon's blood alcohol precipitation boiled dry extract was concentrated to precipitate a white powder precipitated partially purified.

TECHNICAL FIELD

The present invention relates to a grass dragon's blood boiled dry extract alcohol precipitation and / or preparation of purified fractions, which belongs Pharmaceutical fields.

Background technique

At present, many due to the antiviral drug resistance and side effects of long-term and limit its clinical application Use, such as commonly used in clinical treatment of influenza amantadine and rimantadine methyl derivative structure on the existence of God After toxic, easy to produce resistant strains and influenza B viruses invalid defects; currently approved by the FDA for Prevention and treatment of respiratory syncytial virus infection drug ribavirin only, but there are bone marrow cell toxicity when its intravenous administration And the presence of a small amount when administered by aerosol through the respiratory tract neutropenia, excessive doses can cause headache, can not Against anemia and other side effects; anti-herpes virus drugs acyclovir herpes simplex virus can lead to drug resistance, resulting in Respiratory viral infection caused by the incidence of continued unabated.

Grass dragon's blood is more commonly used herbs, Polygonaceae, perennial herb, the origin of Yunnan, Sichuan. Of bitter, acrid, Slightly astringent, slightly warm. At present, experimental studies with anti-inflammatory and anti-tumor herbs in the dragon's blood applied research The results show that it has anti-inflammatory and anti-tumor effect. And it has been through modern analytical chemistry methods of dragon's blood on the grass Isolated chemical composition, purification, structural analysis and identification of pharmacological activity, antioxidant activity and anti-inflammatory analgesic, Dragon's blood drawn grass may contain plant sterols or steroid saponins, glycosides and anthraquinone or sugars, phenols and tannins Quality and other substances, initially revealed the pharmacological foundation grass dragon's blood detoxification role, but will be made of dragon's blood grass Methods related to drugs has become a new topic in the field of medical research.

SUMMARY

Object of the present invention is to provide a grass dragon's blood boiled dry extract alcohol precipitation and / or preparing purified fraction Law, the purpose of using heat detoxification grass of dragon's blood.

Purposes of technical solution of the present invention is: a grass dragon's blood boiled dry extract alcohol precipitation and / or purification section Preparation, prepared as follows:

(1) The grass dragon's blood and water mass ratio 1:8 to 15 placed in a container, dip in boiling water bath for reflux Mention 30 ~ 90min, and then suction filtered and the filtrate residue were obtained;

(2) repeating steps (1) 2 to 3 times, and the resulting filtrate was mixed, then add 95% to the mixing volume of the filtrate 98% alcohol, placed in 0 ~ 8 ° C down to the filtrate mixed with alcohol, then was suction filtered alcoholic liquid;

(3) the alcoholic filtrate under reduced pressure at 70 ~ 85 ° C under grass dragon's blood boiled and concentrated to give alcohol precipitation extract the grass Dragon's blood boiling alcohol precipitation extract was concentrated to the original volume of 0.1 to 0.4 times the residue obtained after distilling off the ethanol, distilled water The residue was dissolved, insoluble material was filtered off and the filtrate, the filtrate was passed through a strong acid cation resin column, washed with water to Colorless, and then eluted with 3N NH4OH, ammonia eluate was collected and concentrated to give a paste, the paste on silica gel Column with acetone - methanol to give the grass dragon's blood boiled dry extract alcohol precipitation.

The above-prepared grass dragon's blood alcohol precipitation boiled dry extract was concentrated to precipitate a white powder that prepared the purification unit Minute.
Wherein with acetone - methanol substance of ratio of 1:1.

The present invention provides a method of making simple, raw materials required for a wide range of sources. The present invention provides a method of manufacture Take the grass dragon's blood boiled dry extract alcohol precipitation and / or purified fractions yield, good quality. The experiment proved Preparation Dragon's blood boiled grass dry extract alcohol precipitation and / or purification section has a good anti-virus prevalent role

Example 1

First, take the following steps to make the grass dragon's blood alcohol precipitation boiled dry extract:

(1) will 130g grass dragon's blood and placed in a round bottom flask 1850ml water, placed in boiling water bath for reflux 90min, and then suction filtered and the filtrate residue were obtained;

(2) repeating steps (1) 2 to 3 times, and the resulting filtrate was mixed, and then mixed with the volume of filtrate was added 98 % Alcohol, place overnight at 8 ° C under suction, the filtrate was alcoholic;

(3) the alcoholic filtrate to 85 ° C and concentrated under reduced pressure to give the grass's Blood 15ml boiling alcohol precipitation extract.
Repeat steps (1) to (3) Preparation of 50ml grass dragon's blood boiling alcohol precipitation extract;

(4) Preparation of 50ml grass dragon's blood boiling alcohol precipitation extract was concentrated to 0.4 times the volume of the residue obtained after distilling off the ethanol Residue, the residue was dissolved with distilled water, insoluble matter was filtered off and the filtrate, the filtrate was passed through a strong acid cation resin column, Washed with water until colorless, and then eluted with 3N NH4OH collected ammonia eluent and concentrated to give a paste, the paste It was on a silica gel column, using a molar ratio of 1:1 acetone - methanol to give the grass dragon's blood alcohol precipitation boiled dry dip paste;

Dragon's blood boiled grass can dry extract concentrated alcohol precipitation needed to precipitate a white powder, which obtained purified fractions.

Example 2

First, take the following steps to make the grass dragon's blood alcohol precipitation boiled dry extract:

(1) will 130g grass dragon's blood and 1300ml water placed in a round bottom flask in a boiling water bath for reflux 60min, Filtration, the filtrate and the residue were obtained;

(2) repeating steps (1) 2 to 3 times, and the resulting filtrate was mixed, and then added to the filtrate mixed with an equal volume of 97 % Alcohol, after standing overnight at 4 ° C under suction, the filtrate was alcoholic;

(3) the alcoholic filtrate to 75 ° C and concentrated under reduced pressure to give the grass's Blood 15ml boiling alcohol precipitation extract. Repeat steps (1) to (3) Preparation of 50ml grass dragon's blood boiling alcohol precipitation extract;

(4) Preparation of 50ml grass dragon's blood boiling alcohol precipitation extract was concentrated to 0.3 times the volume of the residue obtained after distilling off the ethanol Residue, the residue was dissolved with distilled water, insoluble matter was filtered off and the filtrate, the filtrate was passed through a strong acid cation resin column, Washed with water until colorless, and then eluted with 3N NH4OH collected ammonia eluent and concentrated to give a paste, the paste It was on a silica gel column, using a molar ratio of 1:1 acetone - methanol to give the grass dragon's blood alcohol precipitation boiled dry dip paste.

The grass needed dragon's blood alcohol precipitation boiled dry extract was concentrated to precipitate a white powder, which obtained purified fractions.

Example 3

First, take the following steps to make the grass dragon's blood alcohol precipitation boiled dry extract:

(1) will 130g grass dragon's blood and 1040ml water placed in a round bottom flask in a boiling water bath for reflux 30min, Filtration, the filtrate and the residue obtained respectively; repeat 2 or 3 times.

(2) repeating steps (1) 2 to 3 times, and the resulting filtrate was mixed, and then added to the filtrate mixed with an equal volume of 95 % Alcohol, after standing overnight at 0 ° C under suction, the filtrate was alcoholic;

(3) the alcoholic filtrate to 70 ° C and concentrated under reduced pressure to give the grass's Blood 15ml boiling alcohol precipitation extract. Repeat steps (1) to (3) Preparation of 50ml grass dragon's blood boiling alcohol precipitation extract;

(4) Preparation of 50ml grass dragon's blood boiling alcohol precipitation extract was concentrated to 0.1 times the volume of the residue obtained after distilling off the ethanol Residue, the residue was dissolved with distilled water, insoluble matter was filtered off and the filtrate, the filtrate was passed through a strong acid cation resin column, Washed with water until colorless, and then eluted with 3N NH4OH collected ammonia eluent and concentrated to give a paste, the paste He was on a silica gel column, using a molar ratio of 1:1 acetone - methanol to give the grass dragon's blood boiled dry extract alcohol precipitation.

The grass needed dragon's blood alcohol precipitation boiled dry extract was concentrated to precipitate a white powder, which obtained purified fractions.



Resveratrol imprinted polymer preparation and extraction method for resveratrol
CN1978473

This invention relates to resveratrol imprinted polymers and the method for the extraction of resveratrol. Imprinted polymer preparation include: take trans-resveratrol as a template elements, and functional monomers: 4-vinyl pyridine, crosslinker: glycol dimethacrylate, initiator: azobisisobutyronitrile all dissolved in acetone, water bath polymerization; Polymer crushing, screening, using acetic acid-methanol to cleared out the template molecule and un-reacted reagents, to product resveratrol imprinted polymer particles. Extracting resveratrol from include: giant knotweed rhizome extracted with methanol, concentration and add in molecular imprinting column, take methanol-water as the mobile phase to separate; collect outflow component, get resveratrol (80% purity) and emodin (90% purity).; The invention of the molecular imprinting stationary phase with high selectivity, and can separate resveratrol cis-isomer, can also separate other structural analogues (including the glycoside of giant knotweed rhizome etc.).

Resveratrol Imprinted Polymer preparation and extraction methods for resveratrol. Preparation of imprinted polymers include: trans-resveratrol molecule as a template, and functional monomer: 4-vinylpyridine, a crosslinking agent: ethylene glycol dimethacrylate, initiator: azobisisobutyronitrile nitrile was dissolved in acetone, the polymerization bath; polymer was ground and sieved with acetic acid - methanol and unreacted reagents template molecules elute to obtain resveratrol imprinted polymer particles. The method of extracting resveratrol from Polygonum cuspidatum include: Polygonum cuspidatum extract was concentrated in methanol was added molecular imprinting the column with methanol - water as the mobile phase; eluting component was collected to give resveratrol (80% purity) and emodin (purity 90%). The present invention has a high molecular imprinting stationary phase selectivity, separable cis and trans isomers of resveratrol, but also to separate other structural analogs (including Polygonum cuspidatum Polygonum cuspidatum glycosides, etc.). The present invention is a highly efficient separation and purification results.

TECHNICAL FIELD:

The present invention pertains to the synthesis of new chemical separation material, separation and application of resveratrol in Polygonum cuspidatum.

BACKGROUND OF THE INVENTION:

Resveratrol (Resveratrol) is a natural active substance, studies suggest that resveratrol has significant anti-cancer, A wide range of activity against high cholesterol, anti-bacterial, anti-inflammatory, cardiovascular protection and prevention of osteoporosis. Resveratrol exists naturally and trans Both cis configuration, wherein the biologically active trans-isomer higher than the cis isomer.

Polygonum cuspidatum is a traditional Chinese herbal medicine, Polygonum Polygonum cuspidatum plant roots. Because it contains trans-resveratrol, it can be used as resveratrol The main sources. At present, there are several ways to extract from Polygonum cuspidatum resveratrol.

Polygonum cuspidatum containing a variety of ingredients, including Resveratrol glycosides; anthraquinones (emodin, rhubarb phenol, etc.); phenols; HUANG Ketones and glycosides; also contains polysaccharides, simple sugars, organic acids, trace elements and tannin. Since the composition of complex, using pass Conventional methods (such as a liquid - liquid extraction, solid-phase bonded silica gel adsorption method) of Polygonum cuspidatum extract resveratrol, steps still needed to compare Cumbersome, resulting in increased time and cost of the solvent.

A technique of molecular imprinting polymer technology is based on the target molecule as a template, prepare a molecular recognition, molecular imprinting Polymer in clinical drug analysis, separation of enantiomers and isomers, solid phase extraction, membrane separation technology and other aspects have to be good With prospects. Application of molecularly imprinted polymer separation and extraction of the active ingredients of traditional Chinese medicine, with high selectivity and efficient characteristics, can Reducing the separation step, to reduce costs, the technology has value and development prospects.

SUMMARY OF THE INVENTION:

The purpose of the present invention is to solve using traditional methods of Resveratrol from Polygonum cuspidatum extract more complicated steps, leading to Solvent and increase the time cost, there is provided a resveratrol molecularly imprinted polymer (hereinafter referred to as Resveratrol Imprinted Polymer) Extraction Method for the Preparation and resveratrol.

Preparation of resveratrol imprinted polymers of the present invention to provide, comprising the steps of:

1), the resveratrol molecule as a template, the functional monomer 4-vinylpyridine, ethylene glycol dimethacrylate cross-linking agent Ester, initiator azobisisobutyronitrile was dissolved in acetone; wherein the molar ratio of template molecule and functional monomer is between 1:4 to 1:6, The molar ratio of functional monomer and the crosslinking agent is between 1:5 to 1:6; the total amount of the crosslinking agent and initiator functionality to monomer molar ratio of about 1: 100; by function and the total amount of crosslinking agent per gram of monomer dissolved in about 1mL of acetone (in imprinted polymer preparation, the template molecule, functional Application of the monomer with the crosslinking agent more 1:4:20 molar ratio); the solution after oxygen, sealed, polymerized in a water bath at 60 ° C 24h;

2), obtained in the previous step polymer was ground and sieved to 10% acetic acid - methanol solution and methanol elution to remove the polymer The template molecule and unreacted reagents, having a particle diameter of about 36μm resveratrol imprinted polymer particles.

In the course of the study, compared with different functional monomers: α- methacrylic acid (MAA), acrylamide (AM) and 4- Vinylpyridine (4-VP), and different solvent: ethyl acetate, acetone conditions imprinted polymer obtained, of which 4-vinyl Pyridine monomer, acetone as a solvent to give a polymer with optimal selectivity. The imprinted polymers with non-imprinted polymer (to remove Without imprinted molecule, other synthesis conditions with MIPs synthesized polymers) proved relatively imprinted polymer having class Similar to the recognition site immunoaffinity column, for resveratrol good selectivity.

A method of resveratrol imprinted polymer prepared as described above is separated from Polygonum cuspidatum extract resveratrol, with the method Body as follows:

1), after the crushing of Polygonum cuspidatum, extracted with methanol;

2) After the extract was concentrated, prepared as described above containing resveratrol imprinted polymer particles of 150mm × 4.6mm Molecular imprinting the column (can also be used for semi-preparative column), a volume ratio of 80/20 methanol - water as the mobile phase was separated;

3), in the separation step, in addition to resveratrol and emodin, other components out of the column first, followed by resveratrol, Finally emodin components sequentially collected effluent, respectively resveratrol (80% purity) and emodin (purity 90%).

Advantages and effects of the invention:

The present invention provides a method for synthesizing new separation materials and highly efficient separation method resveratrol. The method in trans C. quinoa Resveratrol (trans-resveratrol) as template molecule, using molecular imprinting imprinted polymer prepared resveratrol. And C. quinoa Resveratrol imprinted polymer is a chromatographic stationary phase, the establishment of Resveratrol from Polygonum extraction methods, this method can be separated in step For complete Polygonum cuspidatum extract resveratrol separation and extraction, recovery rate above 80%, while the separation of Emodin Polygonum cuspidatum (Emodin), recovery rate of 98%.

The method of the present invention, the molecularly imprinted stationary phase separation has good selectivity, it can be separated trans and cis resveratrol.

Molecularly imprinted polymers of the present invention also provides the ability to further structural analogs (including Polygonum cuspidatum contained polydatin etc.) Isolation and resveratrol. Trans-resveratrol and other structural analogues in different methanol - water mobile phase under conditions have very different The retention factor (k), can therefore be separated. Resveratrol imprinted polymer can be used for complex systems (such as plants) Belarus Efficient Extraction of Resveratrol separation.

The present invention is applied to extract Polygonum cuspidatum resveratrol, a highly efficient separation and purification results.

BRIEF DESCRIPTION:

FIG. 1 is a cis and trans resveratrol in MIPs (with 4-vinyl pyridine as functional monomer, ethylene glycol dimethacrylate Ester as crosslinking agent, prepared in acetone as solvent) Chromatographic Resolution Figure stationary phase, wherein, cis- represents cis-resveratrol, Trans- It represents trans-resveratrol;

Figure 2 is a mobile phase of water content on resveratrol and its analogs retained molecularly imprinted column (retention factor k to represent) Impact schematic diagram, ■, represents trans-resveratrol; represents bisphenol A; ●, represents polydatin; △, table It shows cis-resveratrol; represents catechol;

Figure 3 is a MIPs for three functional monomers resveratrol adsorption curve. Figure, P1, P2, P3, respectively In α- methacrylic acid, acrylamide and 4-vinylpyridine as functional monomer preparation resveratrol imprinted polymer are acetate Ester solvent;

Figure 4 (a), (b), (c) is imprinted polymer prepared from the monomer for three functions of cis and trans resveratrol chromatographic separation FIG. Figure, P1, P2, P3 respectively in order to resveratrol methacrylic acid, acrylamide and 4-vinyl pyridine as functional monomer prepared α- Alcohol imprinted polymers, are ethyl acetate as solvent;

Figure 5 is Polygonum cuspidatum extract resveratrol and emodin separation chromatograms;

Figure 6 (a), (b) is a reversed-phase liquid chromatography column by MIP after the separation of pure resveratrol and emodin Chromatogram; FIG, B resveratrol after fractionation (peak a resveratrol) chromatogram, C is separated emodin Component (peak b emodin) chromatogram.

Example 1:

Preparation of molecularly imprinted polymer:

The trans-resveratrol (template molecules), 4-vinyl pyridine (functional monomer), ethylene glycol dimethacrylate (crosslinking agent), Azobisisobutyronitrile (initiator) was dissolved in acetone, weighing 0.45g (2.0mmol) of trans-resveratrol, dissolved in about 9mL Acetone, were added successively 4-vinyl pyridine 0.84g (8.0mmol), ethylene glycol dimethacrylate 7.9g (40mmol), even Azobisisobutyronitrile 0.094g (0.57mmol), was dissolved ultrasound 15min, nitrogen-oxygen 10min, and then the mixture was transferred into the ammeter Bottle, sealed, 60 ° C water bath for polymerization 24h. The polymer was pulverized and sieved to obtain particles having a particle size of amorphous. Has 10% acetic acid - methanol solution and methanol elution, to remove the template molecule polymer and unreacted reagents, the particles are charged Chromatography Column (150mm × 4.6mm), and for chromatographic separation. Molecularly imprinted stationary phase separation has good selectivity, can be separated from the anti Cis-resveratrol, see chromatograms (Figure 1, wherein, cis- represents cis-resveratrol, Trans- represents trans-resveratrol).

Studies have shown that molecular imprinted polymers can be separated other structural analogs (including polydatin contained Polygonum cuspidatum). Trans White Veratrum alcohol and other structural analogues in different methanol - water mobile phase under conditions have very different retention factor (k), that is able to Enough is separated (see Fig. 2, Fig., ■, represents trans-resveratrol; represents bisphenol A; ●, represents polydatin; △, It represents cis-resveratrol; represents catechol).

Example 2:

Ethyl acetate instead of acetone as the solvent, the other conditions are the same, the preparation of molecularly imprinted polymers were obtained for C. quinoa Isolated resveratrol imprinted polymer.

MIPs compare different functional monomers (α- methacrylic acid, acrylamide and 4-vinyl pyridine) obtained solid The experimental results show that the acrylamide monomer the resulting polymer has a certain selectivity, but lower than in 4-vinyl pyridine monomer The resulting polymer adsorption properties of three polymers comparison shown in Figure 3 (figure, P1, P2, P3 respectively, methacrylic acid with α-, Resveratrol Imprinted Polymers of acrylamide and 4-vinyl pyridine as functional monomers and solvents are ethyl acetate). Three kinds of poly For chromatograms compound cis and trans resveratrol isolated Figure 4 (Fig., P1, P2, P3 respectively in α- methacrylic acid, Resveratrol Imprinted Polymers of acrylamide and 4-vinyl pyridine as functional monomers and solvents are ethyl acetate).

Example 3

Polygonum cuspidatum extract and chromatographic separation:

After Polygonum cuspidatum crushed, over 60 mesh sieve, add methanol ultrasonic after extraction 24h. After 20min then filtered ultrasound to obtain an extract . After the extract was concentrated, molecularly imprinted column using methanol - water (volume ratio of 80/20) as the mobile phase separation. Collected stream The components, the solvent was evaporated to give resveratrol (80% purity) and emodin (purity 90%). Polygonum cuspidatum extract color separation Spectrum shown in Figure 5. In separation, in addition to resveratrol and emodin, other components out of the column first, followed by resveratrol And, finally, emodin, efficient separation and purification results.

It was determined to prove that resveratrol and emodin recovery this extract obtained were approximately 82% and 98%, five times the separation Results (parallel experiments) obtained in the table below.

Table 1 component recovery and purity molecularly imprinted polymer Polygonum cuspidatum extract resveratrol and emodin separation (Average of five)

By pure resveratrol imprinted polymer after extraction and separation, reversed-phase liquid chromatography method for the determination to get resveratrol and emodin Degree (column: particle size 5μm containing the stationary phase Inertsil ODS-2C18, a size of 150 × 4.6mm stainless Analysis Column) chromatography shown in Figure 6 (wherein, B in FIG resveratrol after fractionation (peak a resveratrol) chromatogram, Figure is C After fractionation emodin (emodin peak b) chromatograms are shown in Table 1 calculated purity.



Giant knotweed rhizome active ingredient emodin extraction method
CN1800122

The invention provides a method for extracting the effective component archin of the giant knotweed rhizome, which puts the drugs into five extracting tanks after shattering them to in turn extract the E, D, C, B, A tank with the extracting solution: 30%-70% alcohol whose amount is 8-12 volume of the drug's quality, the temperature: 35-65 deg. and the time: 20-50 minutes, so that the effect component of the extracting solution of the five tanks descendent, it then discharges opening the tank which has the highest effect component and discharges the dross of the tank which has been five solution extracted, it dose interval removal to the other extracting solution and dose four cycles and then amalgamates the extracting solutions.

TECHNICAL FIELD

The present invention belongs to the field of natural medicine extract the active ingredients, the active ingredients from herbs relates Emodin Polygonum cuspidatum Extracting methods, in particular to take a group of tank countercurrent extraction process Emodin from Polygonum cuspidatum active ingredient party law.

Background technique

Shujin wine rheumatism is a mainly Polygonum cuspidatum suberectstem water Korea and other herbs as raw materials, add white wine dip Bubble extracted from the wine. It has rheumatism, Shujin network functions. Mainly used for rheumatic arthralgia, Bruises, aching pain, acid waist paralysis embolism rheumatism and joint pain, bruises, aching pain, waist Pain embolism. The study, whose main ingredient was the medicinal efficacy emodin. Emodin on Staphylococcus aureus Bacteria, Escherichia coli, Shigella flexneri significantly inhibited vascular smooth muscle cell proliferation Inhibition, can inhibit mesangial cell proliferation, inhibiting mesangial cell autocrine or paracrine white fine Intracellular interleukin-1, interleukin-6, tumor necrosis factor brought glomerular local inflammation effect on people Lung cancer A-549 significantly inhibited cell division and so on.

Currently, industrial production Shujin wine rheumatism mainly hot liquor reflux and percolation method. Heat recovery Flow percolation extraction of traditional Chinese medicine industry is the most widely used extraction techniques, but both techniques exist solution Dosage, long extraction time and low efficiency. Group dynamic tank countercurrent extraction technology (Multi-stage countercurrent extraction, MCCE) is a dynamic extraction and countercurrent extraction Technology combined <[1]> Chinese medicine extraction technology. It made full use of the active ingredient between the solvent and herbs Concentration gradient, gradual diffusion of the active ingredients in the herbs to the starting concentration is relatively low solvent sets mention that Active ingredients to maximize dissolution, with low temperature extraction, extraction efficiency, less solvent, and concentrated Process energy consumption is low. At present, the application of technology tends MCCE wide, as has been reported from motherwort Leonurine extracted, extract alcohol from Millettia Millettia in.

SUMMARY

Object of the present invention is insufficient for getting rheumatoid wine traditional production process, from getting an offer The main active ingredient emodin way wine rheumatism medicine Polygonum cuspidatum extract.

Through the following technical solutions: (1) first reverse gradient extraction: crush the herbs Polygonum cuspidatum, Were weighed into the same amount of A, B, C, D, E five extraction tank, tank E Xianxiang extraction solvent is added, Solvent extraction using a concentration of 35 to 70% ethanol, the solvent is used in an amount of 8 to 12 times the volume of the quality of medicine, mention Take the temperature of 35 ~ 65 ° C, phase extraction time is 20 ~ 50min, sequentially E, D, C, B, A Extracting tank, so that A, active ingredient content B, C, D, E five tank descending extract; (2) Countercurrent extraction: Collect five tanks highest active ingredient content of the extract, the next step was concentrated phase, Abandoned dregs of medicine where the extraction solvent extraction has five tanks were slagging and add herbs, other extraction Liquid separator in accordance with a reverse cell migration (C to A, E to C, B to E, A to D), after the migration Tank without a solvent supplemented with fresh solvent, four cycles, combined extract.

The method of the present invention, the use of an extraction solvent concentration of 35 to 70% ethanol; extraction temperature is 35 ~ 65 ° C; The solvent is used in an amount of 8 to 12 times the volume of crude drug; extraction time is 20 ~ 50min.

The present invention ensures that each unit of P. cuspidatum extract the solvent remains large concentrated active ingredients Poor, greatly increasing the driving force to extract and accelerate the rate of extraction; extraction rate has the advantage that the active ingredient, and Increasing the concentration of active ingredient of the final solvent; the solvent extraction unit for each participation of all tank herbs Extracting, by recycling, greatly reducing the absolute amount of the solvent, and concentrated to reduce the subsequent consumption. Soxhlet back Flow extraction process compared to the present invention uses a dynamic group tank countercurrent extraction (MCCE) extraction rate slightly There are dropped, but medicines reduce solvent consumption per unit of 37.5%, extraction time is reduced by 92.5%; Compared to percolation extraction process, the method of the present invention in a unit medicinal solvent consumption under the same circumstances, Emodin extraction rate MCCE technology improved 26.6%, unit herbs extraction time savings of 88%; Compared with the hot reflux extraction process, the extraction rate of the present invention is a method of emodin improved by 12.5%, unit herbs Solvent consumption reduced by 50%, unit herbs extraction time savings of 85%. Accordingly, the present invention party Pot method for getting a group of rheumatoid wine countercurrent production, with high extraction efficiency, low temperature, solvent, and other significant savings Feature.

BRIEF DESCRIPTION

Figure 1 is a group of tank countercurrent extraction gradient flow chart.

Figure 2 is a flowchart of a method of operating a countercurrent extraction, wherein P1, P2, P3, P4, P5 represents one cycle Ring five extraction stage, A, B, C, D, E represents five from cans, circles represent herbs, square Graphical representation of the solvent, and the black dots represent the content of the active substance.

Embodiments of the present invention in conjunction with the accompanying drawings for further instructions.

Embodiment 1

1. Reverse Gradient Extraction

Referring to Figure 1, the pulverized herbs Polygonum cuspidatum, a certain amount weighed into five extraction tank, the tank was added to the E The extraction solvent, the choice of 35% to 70% alcohol solution of 8 to 12 times the volume, the temperature was raised to 35 ~ 65 ° C, Extract 20 ~ 50min. After completion of the extraction, the extract E tank was transferred to the tank D, E was added to the tank Into fresh solvent, the temperature was raised to 35 ~ 65 ° C, extracted 20 ~ 50min. After the extraction is completed, D tank The extract was transferred to a C tank, the tank extract E is transferred to tank D, E to the addition of fresh solvent tank Addition, the temperature was raised to 35 ~ 65 ° C, extracted 20 ~ 50min. After completion of the extraction, the extract tank C switch Move tank B, the tank D extract was transferred to a C tank, the tank extract E is transferred to the tank D The tank E is added to fresh solvent, the temperature was raised to 35 ~ 65 ° C, extracted 20 ~ 50min. Extraction End After the extraction tank B was transferred to the tank A, the tank C is transferred to the extract tank B, the D Extracts tank C is transferred to a tank, the tank extract E is transferred to tank D, E was added to the tank Fresh solvent, the temperature was raised to 35 ~ 65 ° C, extracted 20 ~ 50min. (Fig. 1 P1 stage).

2. Countercurrent extraction

Referring to Figure 2, after stage P1, the A tank of the extract was transferred to a storage tank, the tank C Extraction A tank was transferred to the E tank extract was transferred to a C tank, the tank has been discharged E extract five tiger Stick herbs, add fresh herbs. Then, the B tank extract was transferred to a tank E, the D tank mention Take was transferred to a tank B, adding fresh solvent to the D tank. The temperature was raised to 35 ~ 65 ° C, 20 ~ 50min extraction (Fig. 2 P2 stage).

After phase P2, the tank E extract was transferred to a storage tank, the tank B transferred to the extract E Tank, the tank D extract was transferred to a tank B, D tank discharge have been extracted five times Polygonum cuspidatum herbs, plus The fresh herbs. Then, the A tank of extract was transferred to the tank D, the tank C is transferred to the extract A tank to tank C by adding fresh solvent. The temperature was raised to 35 ~ 65 ° C, extracted 20 ~ 50min (Figure 2 The P3 stage).

After phase P3, the tank D extract was transferred to a storage tank, the tank A is transferred to extract D Tank, the tank C extract was transferred to the A tank, tank discharge C have been extracted five times Polygonum cuspidatum herbs, plus The fresh herbs. Then, the extract E tank C is transferred to the tank, the tank B is transferred to the extract E tank, tank B was added to the fresh solvent. The temperature was raised to 35 ~ 65 ° C, extracted 20 ~ 50min (Figure 2 The P4 stage).

After the P4 phase, the tank C is transferred to the extract tank, the tank extract E is transferred to C Tank, the B tank extract was transferred to E tank, tank discharge B have been extracted five times Polygonum cuspidatum herbs, plus The fresh herbs. Then, the tank D extract was transferred to a tank B, the tank A is transferred to the extract D tank to tank A fresh solvent was added. The temperature was raised to 35 ~ 65 ° C, extracted 20 ~ 50min (Figure 2 The P5 stage).

According to the above solvent migration patterns, continuous cycle extraction. After completion of the extraction, collection A, B, C, D, E five tank extract was concentrated to the next stage, abandoned dregs. The method of the present invention, the extraction solvent Using the concentration of agent 35 to 70% ethanol; extraction temperature is 35 ~ 65 ° C;) solvent in an amount of 8 to 12 times Volume medicinal quality; phase extraction time is 20 ~ 50min.

Second Embodiment

The pulverized herbs Polygonum cuspidatum, each weighing 10g into A, B, C, D, E five extraction tank to tank E Adding 8 to 12 times the volume, 35 to 70% ethanol solution, the temperature was raised to 35 ~ 65 ° C, the extraction stage 20 ~ 50min. The orthogonal design factor level in Table 1, according to "reverse gradient extraction" and "countercurrent extraction" Operation points extracted. After completion of the extraction, the combined extracts, the amount of volume, sampling and analysis emodin content. The results in Table 2.

Table 1 factor level table

Table 2 orthogonal experiment

Third Embodiment

(1) Best tank countercurrent extraction process group

The pulverized herbs Polygonum cuspidatum, each weighing 10g into A, B, C, D, E five extraction tank to tank E Added 10 volumes of 70% ethanol solution, the temperature was raised to 65 ° C, the extraction stage 35min. Press the "reverse Gradient to extract "and" countercurrent extraction "operation points extracted. After completion of the extraction, the combined extracts were The amount of volume, sampling and analysis of the content of emodin.

(2) heat reflux extraction

Weigh 10g of P. cuspidatum within 250mL round bottom flask was added 100mL 70% medicinal alcohol, Fitted with a condenser, 90 ° C under reflux after 2h, rapid cooling, herbs and extracts were separated by filtration, in After the residue was added 100mL 70% of a pharmaceutically acceptable alcohol, refluxing was continued for 2h, the combined extracts were extracted Determination Liquid emodin.
(3)

(3) percolation extraction

Weigh 10g of P. cuspidatum, doubling the amount of solvent is added, sealed 2h. Herbs installed infiltration Luk barrel, Since the upper portion percolator Slowly add 10 times the amount of 70% alcohol impregnated. Open the percolator after impregnation is completed Under the mouth, collecting percolate. Percolation process is time-consuming 3h, spent 100mL solvent.
(4)

(4) Soxhlet extraction

P. cuspidatum particles weighed 10g, placed Soxhlet extractor, add 70% ethanol 160mL at 90 ° C extraction 8h.

The Polygonum cuspidatum heat reflux, percolation, soxhlet extraction technology and process MCCE comparing results shown in Table 3. Compared with Soxhlet reflux extraction technology, extraction rate MCCE process declined slightly, but the unit was dissolved herbs Agent consumption by 37.5%, extraction time is reduced by 92.5%; percolation extraction process compared to the Units under the same circumstances herbs solvent consumption, emodin extraction rate MCCE technology improved 26.6% Herbs extraction unit time savings of 88%; and heat reflux extraction process compared, MCCE process large Flavin extraction rate of 12.5%, solvent consumption per unit reduced by 50% herbs, medicinal materials extraction unit Time savings of 85%. Visible, tank group countercurrent extraction process, both to ensure a high extraction efficiency, reduce Extraction temperature, the biggest advantage is saving extraction solvent, reducing energy consumption in the subsequent concentrated by evaporation.

Table 3 Comparison of Different Extraction Methods

Without further elaboration, we believe that the use of the foregoing disclosure, those skilled in the art can most Maximize application of the present invention. Accordingly, the foregoing preferred embodiment be construed as merely illustrative, And not in any way limit the scope of the present invention.



Method for preparing polygonin and resveratrol
CN1546503

The invention relates to a method for preparing polygonin and resveratrol which comprises, using the conventional technological process for extraction from plants containing giant knotweed rhizome glycosides and / or chenopodium album alcohols and / or their extracts, separation by chromatography, and purifying.

The present invention relates to a self-contained polydatin and resveratrol substance was isolated and prepared polydatin Resveratrol A method, particularly to a method for separating and preparing polydatin and resveratrol from plant extracts. The method of the present invention can be Preparation and industrialization polydatin resveratrol implementation.

BACKGROUND

polydatin (/ glycosides) (aka Polydatin polydatin, spruce glycosides peicin). The chemical structure of 3,4 ', 5-trimethyl -3-Β- hydroxy stilbene single -D- glucoside (3,4 ', 5-trihydroxy-stilbene-3-β-D-glucoside). Its structure is as follows:

[Image]

Resveratrol (resveratrol), chemical structure of 3,4 ', 5-trihydroxy stilbene, trans-stilbene (ie, stilbene, stilbene) Compound. Its structure is as follows:

[Image]

Polydatin and resveratrol have diverse biological activities such as anti-oxidation, inhibit tumor formation and development, Microcirculation and the role of shock therapy.

Separation and purification method polydatin and resveratrol are set out in the literature include solvents (such as ethyl acetate, diethyl ether) extraction France, silica gel column chromatography (for example, Yang Yun, Feng Weisheng, the chemical composition of traditional Chinese medicine extraction and separation Manual, Chinese medicine Press, 1998: 205-207; Sun Wenji, natural medicine ingredient extraction and separation and preparation, Chinese Medical Science and Technology Press, second edition, 1999: 315-317). In recent years, also appeared in a number of patents related to the preparation of documents and Polydatin its aglycone. For example, "resveratrol Preparation of alcohol polydatin "(Chinese Patent Application No. 01118461.2), the method is the column chromatography and high-speed Countercurrent chromatography together, namely a first step towards separation of resveratrol and resveratrol glucoside by silica gel column chromatography (Mobile phase chromatography halogenated hydrocarbons (e.g. chloroform, methylene chloride, carbon tetrachloride), fatty alcohols or ketones (e.g., methanol, ethanol, propanol, propoxy Ketone)), then the first step of this second separated product was isolated by countercurrent chromatography to give the target product. In another Patent "resveratrol and resveratrol glucoside Separation and Its Application" (Application No. 00121100.5), the method involves First mention was subjected to extraction with ethyl acetate, and then using chloroform - methanol (or ethanol) or ethyl acetate - ethanol as elution chromatography Was subjected to silica gel column chromatography, and then with methanol - chloroform as a solvent was recrystallized to give crystals resveratrol glucoside, and through the secondary Chromatography (eluent: chloroform - ethyl acetate) and recrystallization (crystallization solvent: acetone - chloroform) to obtain resveratrol.
Obviously, The method also involves a large number of organic solvents, repeated use. Extraction of resveratrol and resveratrol glucoside from Polygonum cuspidatum reports Also found in Japanese Patent Publication 60-9455 (1985). The patented method is the use of ether as the extraction solvent to obtain the desired product, A process which is more complex, volatile and ether, flammable, toxic than ethyl acetate, methanol and other solvents stronger.

These methods have been disclosed above, more suitable for the preparation of a small laboratory scale under the large-scale industrial production, It limited its application. First, from the separation process, the above-described methods are to varying degrees by silica gel column chromatography as an essential separation of the hand segment. Silica gel column there is a small amount of sample separation, the amount of filler big disadvantage, and silica filler is difficult to recycle, the production Limited scale, the higher cost of industrial production. The high-speed countercurrent chromatography applications, such as the Chinese Patent 01118461.2, Is obtained by silica gel column chromatography purity of more than 70% and 90% of the product was chromatographed twice counterflow, over the entire process Part of a multi-step process involving a variety of equipment. In general, as compared with silica gel column chromatography, countercurrent chromatography sample amount is more Little harder to meet industrial production requirements.

In addition, the solvent used, the solvent system used in the above document are to varying degrees using a second class solvent such as halogenated hydrocarbons (Chloroform, etc.) or methanol, to ensure production safety conditions at the scale of production is relatively difficult and high cost solvent, The final product will inevitably involve harmful solvents residue problems.

The present invention provides a method comprising polydatin and resveratrol from plants or plant extracts early separation and purification of Polygonum cuspidatum Glycosides and methods of resveratrol. The method is simple, easy to implement large-scale industrial production.

The main feature of the present invention is that the first time the combination of polyamide chromatographic separation applied Polydatin and / or resveratrol Purified preparation.

Extraction and purification method of the present invention polydatin and resveratrol feed comprises one or more polydatin containing and / Or whole plant or plant part of a plant of resveratrol chemical composition, such as roots and / or stems and / or leaves and / or flowers and / or fruit. example Such as Polygonum cuspidatum (Polygonum cuspidatum), Polygonum (Polygonum multiflorum), library spruce (Picea glehnii), Maple Ivy (Cissus assamica), grapes (Vitis Vinifera), peanuts (Arachis hypogaea), Rheum (Rheum palmatum), tanguticum rhubarb (Rheum tenguticum), whole plant medicinal rhubarb (Rheum officinale) and other plants or Its a part of the base of the original plant.

Extraction and purification method of the present invention polydatin and resveratrol material may also be part of the base of the original plant or above Extracts from the beginning, these early extract include extracts of the above plants, for example, water extract, an alcohol extract, ethyl acetate extract Etc., it can also be extracted, extracts, etc. The resulting extract and the like after treatment.

When the base of the original plant or part thereof as a raw material in the polyamide before chromatography, using aqueous or non-aqueous organic Solvents such as ethanol and the like, in order to conventional techniques known in the art of flow at a certain temperature extraction, to obtain containing Polydatin And / or resveratrol extract. These extracts may be appropriate with a polyamide chromatography Before Li, for example, clarification, extraction, concentration, drying and the like.

Polyamide chromatography process of the present invention comprises:

(1) containing Polydatin and / or the resveratrol extracts from the beginning of the adsorption sample: the sample method can be used in an appropriate The solvent extract was dissolved at the beginning, such aqueous or non-aqueous solvents include methanol, ethanol, acetone or the like solvent, which preferably contains Aqueous ethanol as solvent; the sample can be loaded Polydatin / or resveratrol-containing solution and can also be the solution was poly Amide mixed solid sample after sample. Which contains polydatin / or resveratrol solution using conventional techniques and may contain from methods Polydatin containing and / or resveratrol extract polydatin and / or resveratrol plant material obtained, it can be extracted The solution was appropriate pretreatment (such as clarification, concentration, extraction, etc.) after the resulting solution.

(2) Chromatography eluant: After the sample was extracted on an aqueous ethanol system as a mobile phase chromatographic elution. The elution system So the concentration of a particular aqueous ethanol, a concentration gradient may be aqueous ethanol. Elution effluent was collected to afford containing Polydatin and / or resveratrol fractions.

(3) collect or contain Polydatin were collected and eluted fractions / or resveratrol.

(4) Treatment of fractions Polydatin and / or resveratrol: polydatin collected containing and / or elution fractions resveratrol, Further use one or more of the following treatment processes: (1) The collected fractions were concentrated and crystallization; or (2) The collected fractions were chromatographed twice; or (3) using decolorizing charcoal to fractions were collected, concentrated and recrystallization. thus To obtain the target composition Polydatin and / or resveratrol.

The positive effect of the present invention is that: the present invention will first be applied to the polyamide chromatography polydatin (and resveratrol) sub From purification, through the combination of the nature of the object, give full play to the unique advantages of this technology in Polydatin scale preparation of Potential. The salient features of the present invention is embodied in a method: polyamide column can recycling, no need to replace fill between batches Replace column materials; control of industrial production conditions easy to implement, required equipment is simple, easy operation, safety, production Low cost, easy to implement process conditions under different production scale conversion and transfer. On the basis of the polyamide chromatography, In water - to complete all of the production process of ethanol system, the solvent system is more secure and significantly reduce the solvent used in the production process Production staff as well as the potential impact on the environment, more suitable for large-scale industrial production.

DETAILED DESCRIPTION

The following are specific examples of the process of the present invention will be described, the scope of the present invention does not pose any restrictions.

Example 1.1

polydatin new preparation methods and implementation of resveratrol

1. Polygonum cuspidatum extract plant dry rhizome (Chinese Herbal Medicine) 100g, at a temperature of 77-85 ° C to 800ml of 50% Aqueous ethanol reflux extraction 2 times, each extraction for 2 hours. The combined extracts were concentrated under reduced pressure (60 ° C, 0.07-0.1Mpa) to total Volume 300ml, 2N sodium hydroxide solution concentrate to adjust the pH to 9-10, allowed to stand for about 2 hours at room temperature, centrifuged (4000r / min × 5min), to the residue, the supernatant was used as loading backup solution.

2. Separating the sample to an alternate polyamide column (column volume of about 500ml), flow rate of about 100ml / hr on the sample solution, the sample Adsorption to first move the ribbon near the bottom of the column 1/2 stop loading. Successively with 300ml 20% aqueous ethanol, 1000ml 60% ethanol Water, 1000ml 90% aqueous ethanol, atmospheric or superatmospheric pressure chromatography gradient elution rate of about 300ml / hr. Collect 60%, respectively, Water fraction eluted with 90% ethanol, and concentrated under reduced pressure to a volume of the original 1/20 (60 ° C, 0.07-0.1Mpa), filtration, respectively, to give the product I, product II. (Suction filtrate is incorporated on the next sample solution).

The product I about 1.6g, add water to dissolve 95-100% ethanol solution to a final concentration of 30% alcohol, loaded onto an alternate polyamide Column (column volume 100ml) were chromatographed twice, with 100ml 25% aqueous ethanol, 400ml 60% aqueous ethanol gradient elution, respectively, Off, elution rate of about 50ml / min. 60% aqueous ethanol eluate was collected, concentrated under reduced pressure (55 ° C, 0.07-0.1Mpa) to 30ml, 4 ° C crystallization conditions standing for 1 hour, filtered, and the insoluble matter was dried under vacuum 12-72 hours (40 ° C, 0.08-0.1Mpa, pentoxide Phosphorus desiccant) to give the final product Polydatin 1.2g. By HPLC containing Polydatin 99.1%.

The product I (Polydatin) Detection: <1> H-NMR (actone-d6) δ: 8.35 (2H, s, C3,4'-OH), 7.43 (2H, d, C2 ', 6'-H), 7.08 (1H, d, J = 16, aH), 6.89 (1H, d, J = 16, β-H), 6.83 (2H, d, J = 8, C3 ', 5'- H), 6.76 (2H, d, C2 ', 6'-H), 6.47 (1H, t, C4-H), 4.93 (1H, d, 1 "-H), 4.46-3.91 (4H, C2", 3 ", 4", 6 "-OH), 3.3-3.73 (6H, C2 ", 3", 4 ", 5", 6 "-H).

II product about 0.5g, after 95-100% ethanol to dissolve, add water to an alcohol solution containing a final concentration of 50%, and loaded onto an alternate polyamide Amine column (column volume 50ml) were chromatographed twice, respectively 100ml 60% aqueous ethanol, 150ml 95% aqueous ethanol gradient Elution rate of about 20ml / min. Of 95% aqueous ethanol eluate was collected, concentrated under reduced pressure (50 ° C, 0.07-0.1Mpa) to 20ml, 4 ° C crystallization conditions stand for 1 hour, filtered, the insoluble was vacuum dried for 24 hours (40 ° C, 0.08-0.1Mpa, dry phosphorus pentoxide Drying agent) to give the final product resveratrol 0.2g. Containing resveratrol by HPLC 97.5%.

The product II (resveratrol) Detection: EI-MS (m / z): 228 (100%). <1> HNMR (Acetone-d6): 8.45 (1H, s, C4-OH), 8.18 (2H, s, C3 ', 5'-OH), 7.40 (2H, d, J = 8.7, C2,6-H), 7.00 (1H, d, J = 16.2, a-H), 6.86 (1H, d, J = 16.2, β-H), 6.81 (2H, d, J = 8.7, C3,5-H), 6.52 (2H, d, J = 2.1, C2 ', 6'-H) , 6.25 (1H, t, J = 2.1, C4'-H).

After the column with 90-95% ethanol followed by water, eluting regeneration aside.

Example 1.2 polydatin new preparation methods and implementation of resveratrol

Embodiment, atmospheric or superatmospheric pressure gradient elution chromatography. Collected respectively 60% and 90% ethanol aqueous fractions under reduced pressure and Is concentrated to a volume of 1/3 (60 ° C, 0.07-0.1Mpa), other steps from Example 1.1.

The new preparation method of Example 1.3 polydatin and resveratrol implementation

Embodiment, atmospheric or superatmospheric pressure gradient elution chromatography. Collected respectively 60% and 90% ethanol aqueous fractions and Save Pressure concentrated to the original volume of 1/50 (60 ° C, 0.07-0.1Mpa), other steps same as in Example 1.1.

Preparation
Example 2
embodiment Polydatin

1. Polygonum cuspidatum rhizome 100g, appropriate ground to 800ml ethanol percolation extraction, filtration, concentrated under reduced pressure (50 ° C, 0.07 -0.1Mpa) To a final volume of 50ml, 100ml of water was added, and extracted three times with 200ml of ethyl acetate, the extract was concentrated under reduced pressure (50 ° C, 0.07-0.1Mpa) to 150ml, polyamide powder mixed sample, the solvent evaporated under reduced pressure, as a backup sample loading.

2. The samples were loaded on a dry sample in the alternate polyamide column (column volume of about 600ml), followed by water to 1000ml, 3000ml 30% ethanol and water as the mobile phase chromatography with a flow rate 600ml / hr. 30% ethanol was collected and eluted with water effluent, and The stream which contains Polydatin were combined, concentrated under reduced pressure to 100ml (60 ° C, 0.07-0.1Mpa), filtration to give the crude product 2g, Wherein polydatin content of 84%.

3. First product was 95-100% ethanol solution, filtered and the filtrate was added to water containing a final concentration of 30% alcohol, added 1% (ml / ml) medicine Boil 3 minutes with charcoal powder, hot filtered and the filtrate concentrated under reduced pressure to 50ml, 4 ° C under crystallization was allowed to stand 0.5 hours, filtered, The resulting crystals were vacuum-dried four hours (100 ° C, 0.08-0.1Mpa, phosphorus pentoxide drying agent) to give the final product Polydatin 1.5g. By HPLC containing Polydatin 99.82%.

Example 3.1
polydatin implement large-scale industrial production method

1. P. cuspidatum Pieces 500kg, by countercurrent extraction, concentrated under reduced pressure (60 ° C, 0.07-0.1Mpa) have concentrated liquid extract 1200L, with 2N sodium hydroxide solution to pH 10 solution was concentrated and allowed to stand at room temperature for about 2 hours, to the residue was centrifuged, the supernatant was As loading backup solution.

2. The polyamide powder (100-200 mesh), to conventional techniques known in the art of flow means a column (column volume of about 800L), total 2 Genzhu installed, installed after backup.

3. The sample solutions were loaded to the alternate adsorption of two polyamide column, the sample flow rate of about 6L / hr., When the sample solution is sucked The first ribbon is attached to the bottom of the column moved nearly 4/5 stops on the sample. Sequentially 800L 30% aqueous ethanol, 2000L 60% ethanol plus water Pressure chromatography elution pressure 10Bar., 2 60% ethanol was collected column fractions of water, the combined and concentrated under reduced pressure to 100L (60 ° C, 0.07-0.1Mpa), i.e. 1/20 of the original volume, allowed to stand for 0.5 hours at room temperature, centrifuged to obtain a yellowish white powder 7300g, By HPLC containing Polydatin 82.2%. Concentrated under reduced pressure were recovered ethanol as an extraction solution or a low concentration of elution Backup solution.

4. Column with 5% sodium hydroxide solution were soaked, replace solvent once a day for 3 days after soaking in water Off until the effluent pH of 8-9 with 10% acetic acid and then eluted 2000L, and finally washed with water until neutral, the backup and recovery column state.

5. Recrystallization purification: The above product was chromatographed on 7300g, 80L 95% ethanol to dissolve, filtered, and the filtrate add water to Alcohol containing a final concentration of 30%, adding 0.3% (ml / ml) medicinal powder activated carbon boil for 3 minutes, filtered hot and the filtrate was concentrated under reduced pressure to 50L, allowed to stand at room temperature for 3 hours and centrifuged to give nearly white crystalline powder, vacuum, vacuum dried for 12 hours (60 ° C, 0.08-0.1Mpa, Phosphorus pentoxide drying agent) to give the final product Polydatin 5500g.

Example 3.2

polydatin method of preparation

In embodiments may be used sequentially with 800L 30% aqueous ethanol, 2000L 60% aqueous ethanol at atmospheric pressure Chromatography eluant. collect 2 60% ethanol water column fractions were combined and concentrated in vacuo to a rear 1/5 of its original volume, at room temperature for 0.5 hours, which He steps from Example 3.1.
3.3 Preparation Example embodiments Polydatin

The column was collected in two embodiments fraction eluted with 60% aqueous ethanol, the combined and concentrated under reduced pressure to 1/10 of the original volume, Ethanol and water may be pressurized Chromatography eluant to 800L 30% aqueous ethanol, 2000L 60% ethanol and water pressure chromatography eluting standing at room temperature 4 hours. Other steps from

Example 3.1.

In the specific implementation process, increasing concentrations of aqueous ethanol gradient also optionally water, 10 to 30% aqueous ethanol, 30 to 60% Aqueous ethanol; water 0 to 20% ethanol, 95% ethanol, 20% water; 30 to 60% aqueous ethanol, 60 to 95% aqueous ethanol.

Polydatin detection:

Content: This product is detected by HPLC, containing polydatin 99.93%.

Residue on ignition: 0.1% (under the relevant provisions of the Chinese Pharmacopoeia 2000 edition of Appendix matching).

Heavy Metals: compliance with the relevant provisions under the Chinese Pharmacopoeia 2000 edition of Appendix items:( Not use a residual solvent, solvent II).

Microbial Limit Tests: According to Chinese Pharmacopoeia 2000 edition of Appendix checks compliance.
IR (KBr, cm <-1>): 3373 (a phenolic hydroxyl group υo-H), 3026 (benzene υC-H), 1605,1591,1514,1448 (benzene bone Frame vibration), 1341 (a phenolic -OH, υC-OH), 1263 (unsaturated ether C-O-C, υC-O-C), 1172 (methylene saturated ring and a C-H Stretching vibration, υC-H), 1075 (six-membered ring stretching vibration of secondary alcohols, υC-OH), 1019,996,961 (C-H bending on the benzene ring Vibration, δC-H), 839 (para-substituted on the phenyl ring C-H bending vibration, δC-H), 680 (m-substituted on the phenyl ring C-H bending vibration, δ C-H).
UV: UV absorption of the sample and resolution (Table 1).

Table 1 Sample UV absorption and resolve

Between the measured value (M + H) <+> = 391.1378, theoretical value (M + H) <+> = 391.1387, measured and theoretical values: high resolution mass spectrometry The error is in the range of measurement requirements, consistent with the sample formula: C20H22O8. MS database error range of the formula It is C20H23O8 (M + H) <+>, molecular formula C20H22O8 consistent with the sample.

Example Results Discussion and Analysis of implementation

Based on chromatographic separation method of choice First, the nature of the object Polydatin see: it is soluble in methanol, ethanol Alcohol, hot water, can be dissolved in ethyl acetate, sodium bicarbonate and aqueous sodium hydroxide solution, slightly soluble in water, insoluble in ether. Secondly, From the need to produce the feasibility and cost accounting to see: First, the production process should have the feasibility, routing the more pure and simple, No cross, the greater the feasibility of large-scale production; secondly with the operability and safety; taking into account the low consumption, low cost in principle.

The present invention is applied to the polyamide chromatography polydatin (and resveratrol) is isolated by the nature of the target Organic combination, give full play to the unique advantages in technology Polydatin scale preparation of.

two. Basis and purpose of chromatographic separation techniques to determine embodiment employs clarification step of loading the appropriate samples Pretreatment, aims to reduce the interference of impurities, because herbs Polygonum cuspidatum extract, containing not only the active ingredient, but also contains a lot of Before invalid ingredients such as anthraquinone, tannins, polysaccharides and flavonoids and other ingredients, so the use of column chromatography, can be achieved by The column appropriate sample pretreatment to simplify the separation of work, but also reduce the pollution of the column, the column to improve utilization. Sample pre-treatment sample may also be realized by other technical methods, such as extraction liquid extraction methods.

In the method of the present invention, the primary extract containing resveratrol polydatin and can be used on wet or dry samples. Chromatography eluant Preferably ethanol - water as the eluting solvent, three kinds of organic solvent is ethanol, low production cost, safe, and chromatographed eluting Using a gradient of concentration series of ethanol - water as solvent gradient system, 0-30% ethanol by water → 30 ~ 60% aqueous ethanol, Enrichment achieved is the active ingredient (Polydatin), the purpose of removing the interference of impurities, 30 to 60% ethanol water column after elution, can Higher concentrations of ethanol water continues to give eluted fractions containing resveratrol. The method of the present invention is realized in the same root Chromatography column, by changing the solvent leaving a gradient elution system of active ingredient isolated and enrichment purposes, chromatography and used Small elution solvent, a small amount of post-processing. Column in the elution component resveratrol has been completed, while preliminary column again Students, after washing with water and then make the appropriate change elution, the column can basically reach the standby state. The process is simple, economic security, Preparation of a large quantity of products.

Third, the outstanding feature of this embodiment is reflected in:

1. Solvent system: the method of the present invention may be implemented in the water - complete extract and resveratrol polydatin under ethanol system, divided From purified. Has been disclosed in resveratrol polydatin and separation techniques, all involving the use of a different degree, there are two types of Solvent such as diethyl ether, chloroform, methanol and ethyl acetate as the extraction solvent or solvent elution chromatography. Obviously, Experimental and production operations in the preparation process, the solvent system of the present invention, the method more secure, significantly reduces the solvent used in the production process Production staff as well as the potential impact on the environment.

2. Industrialization, large-scale production: separation in the preparation of documents disclosed polydatin and resveratrol, the use of a silica gel column Chromatography, organic solvent extraction, countercurrent chromatography and other methods. Obviously, in these methods, their preparation and preparative scale amounts Enlarge necessarily limited by its own technical conditions. For example, when the silica gel column chromatography to achieve the industrial production, since (1) as Regeneration step chromatography on silica filler is too complex and generally difficult to regenerate, and packed conditions and conditions of a silica gel column chromatography Very strict requirements; (2) The silica gel column chromatography elution solvent is generally an organic solvent not containing water, such as chloroform, acetic acid Ethyl, not only the high cost of these solvents, and the use of more stringent process conditions; therefore, to achieve a silica gel column chromatography Industrial production there must be a substantial increase in production cost constraints and the technical difficulty. The use of a solvent such as ethyl ether extraction When taking, use and disposal of the solvent there are similar problems. As high-speed countercurrent chromatography, under the existing technical conditions, Its preparation batch volume generally only milligrams, industrial production is difficult to achieve under the technical conditions. In contrast, in the present Out method, since (1) may be water - to complete all of the production process of ethanol system; (2) a polyamide column can be regenerated Use, no need to replace the filler between the batch and re-packed; control (3) industrial production conditions easy to achieve, it Required equipment is simple, easy operation, safety, low production cost, easy to implement conversion process conditions under different production scale and Handover, so the applications, highlighting the advantages of the present invention is a method of the prior art methods can not be compared.



Method for extracting high purity resveratrol from giant knotweed rhizome
CN1621401

The present invention is method of extracting high purity resveratrol from giant knotweed. Pre-treated dry coarse giant knotweed powder is added with certain amount of organic solvent for reflux extraction, and after the organic solvent is recovered, it is extracted with different kinds of polar solvent. Through further recovering partial solvent, centrifugation, and precipitate treatment with water and adsorbent, high purity resveratrol crystal is obtained. The present invention has the advantages of flexible technological process, simple operation, simple apparatus, less solvent consumption and high product yield.

[ No Description ]



Process for extracting resveratrol from Chinese medicine giant knotweed root
CN1251361

The extraction process of medicinal raw material resveratrol includes the following steps: adding complex enzyme to powdered bushy knotweed root raw material; making zymolysis reaction for 48-72 hr. under the condition of constant temp. to obtain the enzymolyzed raw material; adopting solvent extraction and concentration processes to obtain the intermediate product containing resversatrol, refining to obtain the invented product. It possesses the advantages of rich raw material source, simple process, high yield and low cost.

DESCRIPTION

Resveratrol (Resveratrol, 3,5,4 'trihydroxy trans - stilbene), also known as Qi pyrogallol, because Pharmacological activity of anti-cancer, anti-bacterial, anti-inflammatory, anti-allergic, anti-oxidation, blood fat and other aspects have been widely Pan used in food, health products, cosmetics and other fields. Currently the main source of resveratrol from grape skins And some beans to extract content is extremely limited. The content of resveratrol in grape skin is considered the most High, it is only up to 0.5 to 1 / million. China Sichuan origin in the form of traditional Chinese medicine Polygonum cuspidatum glycosides present in C. quinoa Resveratrol content, the theoretical value of the skins 200 times! And Polygonum cuspidatum has always been to use only as
medicines, with A very small, resulting in a serious slow-moving giant knotweed, a waste of valuable resources.
Object of the present invention is to develop sources of resveratrol, there is provided a medicine extract resveratrol from Polygonum cuspidatum Alcohol-forming process.

Resveratrol extracted from traditional Chinese medicine Polygonum cuspidatum process of the present invention, wherein said extraction process To: Add enzyme in Polygonum cuspidatum powdered material is carried out at a constant temperature digestion for 48 to 72 hours to give Enzyme raw materials; followed by a solvent extraction, concentrated to give semi-finished products containing resveratrol, and then refined to obtain. this Extraction of the invention containing from polydatin very rich raw materials for traditional Chinese medicine Polygonum cuspidatum, resveratrol hydrolysis Glycosides to extract resveratrol, has the following advantages:

1) Chinese medicine Polygonum cuspidatum easy planting, cuttings, seeds can be broadcast, high yield, low price, convenient source, Follow-up resource security.

2) the process is simple, attention is good, perfect glycoside hydrolysis, high yield, good quality and low cost. Experimental results show that after the expansion of three batches of resveratrol from Polygonum cuspidatum extract yield of 85%. Products by the China Medical ASTRI drugs test, the purity reached 99.63%, 99.68%, 99.78%. Higher than the international index 1.7 percentage points to 99.5% of the sigma standard purity requirements.

3) less investment in equipment, low cost, no pollution, safety.

The following is an embodiment of the present invention.

FIG. 1 is a flow chart of the present invention.

Referring to Figure 1, taking traditional Chinese medicine Polygonum cuspidatum milled into powder form, adding complex enzyme hydrolysis reaction at a constant temperature 70 hours, material obtained after hydrolysis by solvent extraction and concentrated to obtain a mixture containing about 30% of resveratrol semifinished Product, and then refined to obtain resveratrol.



Technology of super critical caron dioxide extraction of resveratrol from polygonum cuspidatum
CN1513823

A supercritical CO2 process for extracting resveratrol from giant knotweed rhizome features that supercritical CO2 extraction is used, and the mixture of absolute alcohol and 2-propanol is used as the modifier. Its advantages are less consumption of extracting solvent, low cost, high output rate and quality of product and no environmental pollution.
 
BACKGROUND

Giant knotweed (Polygonum cuspidatum Sie bet Zucc) Polygonaceae Polygonum small shrubs, as China's traditional Chinese medicine and modern pharmaceutical research to prove that resveratrol (Resveratrol) (3,4 ', 5-trihydroxy-stilbene) having a variety of pharmacological effects, mainly Performance in terms of anti-cancer and treatment of cardiovascular diseases.
New investigation found that at home and abroad "Extract Resveratrol from Polygonum cuspidatum" aspects of literature and expertise Lee has been reported. As Chinese patent CN1251361 French patent FR2795964 extract process To: Add powdered enzyme in raw material Polygonum cuspidatum enzymatic reaction is carried out at a constant temperature, 48-72 Hours to get digested material, solvent extraction and then concentrated to obtain semi-finished products containing resveratrol, set 24--120 hours, with stirring, and then extracted with ethyl acetate, ethyl acetate, recovered, and then by Further purification process to obtain resveratrol and resveratrol glucoside. "Tianjin Pharmacy" (1999-11 -07) Published "knotweed chemical composition, pharmacological effects and extraction and separation" is the root of Polygonum cuspidatum meal With ethanol extraction, recovery of ethanol to extract form, dissolved in water, filtered, and concentrated aqueous solution, concentrated Polyamide shrink liquid chromatography, eluted with 20% ethanol polydatin. China Patent CN1239141 discloses a process for the extraction of resveratrol from grape stems roots and leaves. but In general, such methods complex process operation, long cycle, low quality products.

At home and abroad are using conventional solvent extraction method, there is no mention by supercritical CO2 extraction Take reports. Supercritical CO2 fluid extraction is the high-tech field of modern separation occurs, you can Reduce the amount of organic solvent is used, the extraction efficiency and high selectivity, time-saving; volatile solvent extraction, Extract a cleaner, less environmental pollution, easy to change operating conditions.

It is an object of the present invention is to provide a supercritical CO2 extraction technology from Polygonum cuspidatum extract resveratrol technology, which overcomes the conventional solvent extraction long time and solvent consumption A large amount of residual solvent quantity, the extraction rate is relatively low, product quality is not high shortcomings. Ultra CO2 extraction process is not only simple, high yield, and less solvent consumption, product quality it is good.

For the purposes of the process steps of the present invention comprises

(1) First Polygonum cuspidatum rhizome crushed, over 60 mesh sieve;

(2) charged supercritical CO2 extraction kettle, adjust extraction conditions;

(3) collecting the extract was concentrated;

(4) chromatographic separation, concentration, crystallization, and finally ≥95% purity resveratrol.

Supercritical CO2 extraction conditions have a significant impact on the effect of Polygonum cuspidatum extract resveratrol, the test Inspection, extraction conditions of the invention as follows: extraction pressure was: 35-25Mpa, temperature 60 ° C-40 ° C, the autoclave pressure to resolve 8-5Mpa, temperature 60 ° C-40 ° C;

Hydrocarbon mixture with an alcohol such as ethanol and 2-propanol as modifier

When the temperature has concentrated a great influence on the stability of resveratrol, the test to determine the present invention Concentrated optimum temperature 45 ° C-60 ° C, vacuum degree ≤-65cmHg.

The present invention uses supercritical CO2 technology can overcome the conventional solvent extraction solvent Large, long extraction time, solvent residue and more complex procedure and other issues, can greatly reduce the mention Take solvent consumption, reduce costs, environmental pollution, mainly CO2 extraction solvent, can be recycled use. High product yield and quality, first extract can be obtained more than 18% purity of Polygonum cuspidatum C. quinoa Resveratrol extract, and then by chromatography, concentration, crystallization, freeze-drying and other refined the process can be prepared

Preparation of high purity (≥95%) of resveratrol.

The accompanying drawings, the present invention is a process flow diagram 1 Polygonum cuspidatum cleaning materials, dry, Crushed 2 supercritical CO2 extraction filter 3, 4 and concentrated by column chromatography eluting 5 6 Resveratrol Principal component was collected and concentrated 7 8 9 High-purity crystalline resveratrol 10 drying, storage 11 large flavin

DETAILED DESCRIPTION

concrete steps in this process are:

(1) The dried roots of Polygonum cuspidatum crushed, over 60 mesh sieve, called Polygonum cuspidatum 50g powder;

(2) the powder into the extraction vessel, the extraction pressure was adjusted, adjust the temperature set value Start the cycle, while adding the modifier ethanol 2-propanol = 80:20 (ratio by volume). extraction Conditions: extraction pressure was 25Mpa, extraction temperature 50 ° C, the autoclave pressure to resolve: 6.0Mpa, Solutions Analysis of temperature 46 ° C;

(3) and extracted for 1.5 hours, the autoclave was adjusted resolves 5.7Mpa, analytical temperature 45 ° C. Receive Set extract, after a one-time extraction, extract resveratrol content of more than 18%, the extraction rate of 75. 8%.

(4) and concentrated to recover the modifier. Concentration temperature is 45 ° C, the degree of vacuum -50cmHg.

(5) by column chromatography, concentrated under reduced pressure, crystallization, freeze drying, to obtain a purity of 95.1% Resveratrol 0.25lg.



Extraction process of resveratrol from giant knotweed
CN1384088

During the extraction of resveratrol from giant knotwood, giant knotwood glycoside is hydrolyzed and enriched through organic synthesis. The extraction process includes mixing giant knotwood raw material and composite stuffing, high pressure chromatography in a high-pressure chromatographic column apparatus, gradient elution with chloroform and ethyl acetate and thin chromatographic tracking detection. The said process can reach a kilogram level yield and high product purity. The present invention may be used in extracting resveratrol and other similar product from giant knotwood material.

DESCRIPTION

A method for preparing extracting resveratrol from Polygonum cuspidatum plant

FIELD:

The present invention relates to a method for extracting resveratrol, especially C. quinoa is an extract from Polygonum cuspidatum plant Preparation of resveratrol.

Background technique:

In the current technical solution, resveratrol mainly through chemical synthesis to complete, but Large investment in equipment, process complexity, high cost and low yield and other failings of its existence. In recent years, People are trying to extract from natural plant Polygonum cuspidatum resveratrol and has made encouraging progress. Polygonum cuspidatum is our pass Chinese herbal medicine system, which contains a large number of internal polydatin (aka polydatin) and resveratrol, and C. quinoa Resveratrol is a kind of human health has a significant role in the efficacy of natural active substances, the international community has caused Widespread concern. By scientific research and clinical applications show that resveratrol has anti-cancer, anti-oxidant and anti
Only the formation of thrombus, protect the liver and free radical scavenging and other effects in the elderly degenerative diseases such as Parkinson's , Dementia, Alzheimer's disease, rheumatic diseases have better preventive and therapeutic effect; in the dressing Produced in the country, it has to get rid of melasma and whitening effect; it lipid metabolism and platelet coagulation always make Influential, can prevent coronary heart disease and atherosclerosis and other diseases. With biological science and technology continue to send Exhibition medicinal value, the amount of resveratrol and also in the increasingly dramatically. Natural plant Polygonum cuspidatum, although its contents There are a lot of polydatin and resveratrol, but because of complex components, difficult to extract, combined with the current extraction work Art is not perfect, so the extraction rate and purity are low. Applicants Beijing Fu Man biotechnology research Which, in its Publication No. CN1277954A, entitled "Separation of resveratrol and resveratrol glucoside and Its application "discloses a technical solution Polygonum cuspidatum roots were extracted with an organic solvent, extraction, concentration, silicon Rubber plug chromatography, crystallization, recrystallization and get resveratrol process; patent applicants Beijing-dimensional days of pure Tong Biotechnology Co., Ltd., in its Publication No. CN1323776A, entitled "Resveratrol and C. quinoa Preparation resveratrol glucoside "technical solution is characterized by: extraction with an organic solvent containing resveratrol and Polydatin plants, animals, microorganisms obtaining the extract, and then were subjected to column chromatography and high-speed Countercurrent chromatography is obtained. The above-described Patent Document resveratrol extraction process is mainly through the extraction enzyme Silicic acid hydrolysis reaction or plug chromatography, were introduced with the claims of the invention prepared in the same method. Summing up Above, the existing technical solutions and patent literature, are separating resveratrol and polydatin as separate projects Further consideration mentioned standard, not the first by synthetic means making Polygonum cuspidatum resveratrol enriched to higher levels when Pure, so the degree of industrialization and its yield, yield is still unsatisfactory.

SUMMARY:

Object of the present invention to overcome deficiencies of the prior art, by providing a Polygonum cuspidatum Polydatin plants by means of hydrolyzing organic synthesis enriched it with their appropriate composite filling material Ratio, after pressing a dedicated high pressure column apparatus using a high performance liquid chromatography column pressure principle Chromatography, eluted with chloroform and ethyl acetate gradient, tracking and detection by thin layer chromatography, thus achieving production Product recovery, good purity and up to kilogram quantities of industrial production of an extract from Polygonum cuspidatum plant Preparation of resveratrol.

The basic process of the present invention are as follows:

1. extract:

The dried material Polygonum cuspidatum pulverized with an organic solvent heated to reflux and extracted three times its amount of organic solvent Volume ratio of the raw materials were 3-5 times, 2-4 times, 1-3 times, extraction time were small 3:2:1 When combined extracts all times and recovering the organic solvent under reduced pressure, distillation and concentrated to a thick paste;

2. Water-soluble:

After the obtained paste is added to 10 to 20 times its volume ratio of the amount of water, heated to the boiling point was allowed to stand 1-3 Hours, at a temperature of not less than 50 ° C the upper part of the supernatant was decanted, and concentrated to 30-40B ° C (Baume);

3. extraction:

The resulting Baume degree 30-40B ° C dilute solution, according to volume ratio, respectively (3-5): (2 -4): (2-4) times the amount of ethyl acetate was added, extracted three times after various times extracts were combined, adjusted with its base PH value of 8.0-10.0, allowed to stand, filtered, and concentrated under reduced pressure to a volume of extract 1 / 15-1 / 20, junction Polydatin crystallized crude extract;

4. Hydrolysis enrichment:

The resulting polydatin crude extract, according to volume ratio plus 2-10% 2-10 times the amount of acid hydrolysis After 4-8 hours hydrolysis enrichment, cooling, for 24 hours, filtered, washed acid eligible resveratrol crude;

5. Isolation and Purification:

The resulting crude was dissolved in ethyl acetate resveratrol, pressure pump and its proportion to the preparation of compound Special filler into a high-pressure chromatography column apparatus, pressurized by HPLC column chromatography principle Separation, eluting with chloroform and ethyl acetate gradient, tracking and detection by thin layer chromatography, collecting resveratrol Eluent segment, again concentrated under reduced pressure to a small volume crystallization, high purity resveratrol eligible for crude;

6. product:

The high purity resveratrol obtained crude repeated recrystallization from ethanol, is eligible purity greater than 99% Resveratrol white crystalline solid product.

The basic process of the present invention, the extraction with an organic solvent under reflux heating may be: methanol, ethanol, The ratio of any combination propanol, acetone, ethyl acetate or these solvents.

The basic process of the present invention, the PH value thereof was adjusted extract bases may be used: ammonia, carbonate Sodium bicarbonate, calcium hydroxide, sodium hydroxide, potassium hydroxide, etc., and all inorganic and organic bases.

The basic process of the present invention, the use of dilute acid hydrolysis enrichment for: all except hydrochloric acid acid.

The basic process of the present invention, in proportion to the preparation of the composite filler may be: silica gel and trioxide Aluminum by weight 6:1 formulated.

The basic process of the present invention, the gradient of chloroform and ethyl acetate of a volume ratio may be sequentially It is: 6:1,3:1,1:1.

The basic process of the present invention, the dedicated high pressure column pressure may range device: 2- 15MPa.

The basic process of the present invention, the pressurized column chromatography mobile phase can be used: halogenated hydrocarbons and carboxylic acid esters Volume ratio, or any mixture of fatty alcohols or aliphatic ketones with water in any ratio, respectively, constitute a group 2-8 Minute. The halogenated hydrocarbon is chloroform, methylene chloride, carbon tetrachloride; carboxylic ester is ethyl acetate, ethyl formate; Fatty alcohols or aliphatic ketones as methanol, ethanol, propanol, acetone.

Specific implementation methods:

Below in conjunction with embodiments of the present invention will be further described.

Example 1:

The dried material Polygonum cuspidatum pulverized with ethanol and ethyl acetate their volume ratio of 1:1 combination heating back Stream extracted three times its volume of the extraction solvent volume ratio of the raw material was 3 times, 2 times, 1 times, mention Take time respectively 3:2:1 hours combined each time and extract the organic solvent recovered under reduced pressure, distillation and concentration to Thick paste; paste is obtained according to the ratio of the volume of 10-fold amount of water was added, heated to boiling point for 1 hour standing When, at a temperature of not less than 50 ° C the upper part of the supernatant was decanted, and concentrated to 30B ° C (Baume); The resulting Baume dilute solution 30B ° C, according to their volume ratio was 3:2:2 times the amount of ethyl acetate was added, After extraction three times each combined extracts were washed with concentrated aqueous ammonia, and its PH value of 8.0, allowed to stand, filtered and concentrated under reduced pressure The extract was reduced to 1/15 of the volume, crystallized polydatin crude extract; the resulting crude extract resveratrol glycosides, After its volume ratio of 2% dilute sulfuric acid hydrolysis of 2 times the amount of 4 hours, cooled, allowed to stand for 24 hours, after Filter, wash acid eligible resveratrol crude; the obtained crude product was dissolved in ethyl acetate resveratrol, pressure pump Its silica and aluminum oxide with a weight ratio of 6:1 formulated by a composite filler, into a dedicated high pressure layer

Analytical column pressurizing equipment pressurizing 5MPa column chromatography, followed by volume ratio 6:1,3:1,1:1 A mixture of chloroform and ethyl acetate gradient elution times, tracking and detection by thin layer chromatography, collecting C. quinoa Resveratrol eluent segment, again concentrated under reduced pressure to a small volume crystallization, high purity resveratrol eligible for crude; the The obtained high purity resveratrol crude repeated recrystallization from ethanol, is eligible purity greater than 99% as white Resveratrol crystalline solid product.

Example 2:

The dried material Polygonum cuspidatum pulverized with ethanol and ethyl acetate their volume ratio of 1:1 combination heating back Stream extracted three times the amount of their extraction solvent volume ratio was 4 times of raw materials, 3 times, 2 times, to mention Take time respectively 3:2:1 hours combined each time and extract the organic solvent recovered under reduced pressure, distillation and concentration to Thick paste; The resulting paste is added 15 times its volume ratio of the amount of water, heated to boiling point for 2 hours standing When, at a temperature of not less than 50 ° C the upper part of the supernatant was decanted, and concentrated to 35B ° C (Baume); The resulting Baume dilute solution 35B ° C, according to their volume ratio was 4:3:3 times the amount of ethyl acetate was added, After extraction three times each combined extracts were washed with concentrated aqueous ammonia, and its PH value of 9.0, allowed to stand, filtered and concentrated under reduced pressure The extract was reduced to a volume of 1/18, crystallized polydatin crude extract; the resulting crude extract resveratrol glycosides, After its volume ratio of 6% plus 5 times the amount of dilute sulfuric acid hydrolysis of 6 hours, cooled, allowed to stand for 24 hours, after Filter, wash acid eligible resveratrol crude; obtained as described in Example 1.

Example 3:

The dried material Polygonum cuspidatum pulverized with ethanol and ethyl acetate their volume ratio of 1:1 combination heating back Stream extracted three times its volume of the extraction solvent volume ratio of 5 times the raw material, 4-fold, 3-fold, mention Take time respectively 3:2:1 hours combined each time and extract the organic solvent recovered under reduced pressure, distillation and concentration to Thick paste; The resulting paste is added 20 times its volume ratio of the amount of water, heated to boiling point for 3 hours standing When, at a temperature of not less than 50 ° C the upper part of the supernatant was decanted, and concentrated to 40B ° C (Baume); The resulting Baume dilute solution 40B ° C, according to their volume ratio was 5:4:4 times the amount of ethyl acetate was added, Extracted three times each time after the extracts were combined, neutralized with concentrated aqueous ammonia, and its PH value of 10.0, it was allowed to stand, filtered and the filtrate The extract was concentrated to a volume of 1/20 crystallized polydatin crude extract; the resulting crude extract polydatin Thereof, the ratio of its 10% volume of 10 times the amount of dilute sulfuric acid hydrolysis for 8 hours, cooled, allowed to stand for 24 hours, Filtered, washed acid eligible resveratrol crude; obtained as described in Example 1.

Pressure column layer was separated according to embodiments of the invention, the use of a variety can be formulated according to the proportion of complex Combined filler being fed special high-pressure chromatography apparatus therein, thus achieving up to kilogram quantities of industrial Production needs.

As can be seen with the above embodiments, the present invention has the following advantages over current technology:

1. Polydatin by using the means of hydrolyzing organic synthesis enrichment after the line separation and extraction, reduced Consumption of raw materials, improved product yield;

2. Using a dedicated high-voltage equipment for high pressure column chromatography separation layer, changing the existing atmospheric column The layers were separated mode, low cost, short cycle, high degree of industrialization of production;

3. Technology played tight, easy operation, high product purity.

The present invention can be used in the extraction of resveratrol and other similar products from Polygonum cuspidatum plant industrialization Production, can also replace the current method of producing various types of resveratrol.



Method for preparing resveratrol from giant knotweed rhizome
CN1341587

The method for preparing resveratrol by using bushy knotweed root includes the following steps: using fresh bushy knotweed root and pulping, placing the obtained pulp material in a container, storing for 24-120 hr. at 10-50 deg.C, and stirring, then using ethyl acetate to make extraction, recovering ethyl acetate, further making purification treatment so as to obtain resveratrol. Because the resveratrol glycoside being in the bushy knotweed root can be converted into resveratrol under the condition without foreign enzyme added. so it has the advantages of simple operation, high yield and low cost.

TECHNICAL FIELD

The present invention relates to a method of separating from Polygonum cuspidatum extract resveratrol.

Background technique

Polygonum cuspidatum resveratrol (resveratrol) and its glycosides, resveratrol with lowering blood pressure, Anti-oxidation, improve microcirculation, lower blood pressure and inhibition of staphylococcus, streptococcus and so on.

The method of extracting resveratrol mainly two categories firstly by adding combined Enzymes Resveratrol The glycosides were digested by solvent extraction, separation and purification, high production cost. Another direct By solvent extraction, separation and resveratrol glycosides and other ingredients, the product yield is low.

SUMMARY

The object of the present invention is to overcome the above drawbacks of the prior art and to provide a simple process Single, low-cost method for preparing resveratrol from Polygonum cuspidatum collected high rate.

Object of the present invention can be achieved by the following technical solution: A method of making resveratrol from Polygonum cuspidatum Alcohols, characterized in that the process comprises the following steps. First, fresh Polygonum beating placed Container, placed in 10 ~ 50 ° C for 24 to 120 hours, with stirring, and then extracted with ethyl acetate, Recycling ethyl acetate, and further purification treatment to obtain resveratrol.

The same with the present invention, respectively Polygonum cuspidatum comparison test, test and existing direct extraction The results showed that the yield obtained by the present invention, resveratrol than direct extraction can be increased more than 2 times. Since the present invention Resveratrol from Polygonum cuspidatum glycosides converted under conditions without adding exogenous enzymes to resveratrol Alcohol, which has a simple, high yielding, low cost.

BRIEF DESCRIPTION

Figure 1 is a flow chart of the invention.

Results

The following specific embodiments and the accompanying drawings, the present invention will be further described.

Example 1

Take fresh Polygonum cuspidatum I, was determined to contain resveratrol 100g (not including glycosides) is 0.16g, weighed 500g, Crushed into pulp, placed in 30 ± 2 ° C for 96 hours, and placed five minutes per hour during stirring with acetic acid Extraction with ethyl acetate, solvent recovery, and then purified to give white crystals namely resveratrol 1.89g. If the tiger Cane syrup directly extracted with ethyl acetate, and purified in the same manner to obtain resveratrol 0.61g.

Example 2

Take fresh Polygonum cuspidatum I, was determined to contain resveratrol 100g (not including glycosides) is 0.16g, weighed 500g, Pulverized slurried and placed in 40 ± 2 ° C for 72 hours, every two hours during placement stirred for 5 minutes, with ethyl Ethyl extraction, solvent recovery, and then purified to give white crystals namely resveratrol 1.80g.

Example 3

Take fresh Polygonum cuspidatum II, was determined 100g contains resveratrol (not including glycosides) is 0.19g, weighed 500g, Pulverized slurried in 96 hours at 20 ± 2 ° C, during placement stirred for 10 minutes every three hours, with Ethyl acetate solvent recovery, and then purified to give white crystals namely resveratrol 2.02g. If the The Polygonum cuspidatum slurry directly extracted with ethyl acetate, and purified in the same manner to obtain resveratrol 0.65g.

Example 4

Take fresh Polygonum cuspidatum II, was determined 100g contains resveratrol (not including glycosides) is 0.19g, weighed 500g, Pulverized slurried in 96 hours at 20 ± 2 ° C, during placement stirred for 10 minutes every three hours, with Ethyl acetate solvent recovery, and then purified to give white crystals namely resveratrol 2.01g.

Example 5

Take fresh Polygonum cuspidatum III, was determined 100g contains resveratrol (not including glycosides) is 0.15g, weighed 500g, Pulverized slurried and placed in 35 ± 2 ° C at 96 hours, every two hours during placement stirred for 5 minutes, with ethyl Ethyl extraction, solvent recovery, and then purified to give white crystals namely resveratrol 1.75g. If this Polygonum cuspidatum slurry directly extracted with ethyl acetate, and purified in the same manner to obtain resveratrol 0.55g.

Example 6

Take fresh Polygonum cuspidatum III, was determined 100g contains resveratrol (not including glycosides) is 0.15g, weighed 500g, Pulverized slurry, placed in 25 ± 2 ° C at 120 hours, every two hours during placement was stirred for 5 minutes, Ethyl acetate solvent recovery, and then purified to give white crystals namely resveratrol 1.81g.

Example 7

Take fresh Polygonum cuspidatum IV, was determined 100g contains resveratrol (not including glycosides) is 0.12g, weighed 500g, Pulverized slurried and placed in 35 ± 2 ° C at 96 hours, every two hours during placement stirred for 5 minutes, with ethyl Ethyl extraction, solvent recovery, and then purified to give white crystals namely resveratrol 1.40g. If this Polygonum cuspidatum slurry directly extracted with ethyl acetate, and purified in the same manner to obtain resveratrol 0.45g.

Example 8

Take fresh Polygonum cuspidatum IV, was determined 100g contains resveratrol (not including glycosides) is 0.12g, weighed 500g, Pulverized slurry, placed in 25 ± 2 ° C at 120 hours, every two hours during placement was stirred for 5 minutes, Ethyl acetate solvent recovery, and then purified to give white crystals namely resveratrol 1.45g.



Some "Traditional Chinese Medicine" Patents incorporating Knotweed
 
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Spleen-warming and cold-dispelling preparation for chronic diarrhea and preparation method thereof
CN104383009

Biscuit with capabilities of clearing away heat and toxic materials and preparation method of biscuit
CN104381410

Purple sweet potato cake capable of nourishing Yin and protecting liver and preparation method thereof
CN104381381

Botanical fungicide
CN104381348

Traditional Chinese medicine with functions of invigorating spleen, supplementing qi, stimulating appetite and preserving health
CN104367958

Traditional Chinese medicine composition for treating anti-tuberculosis medicament induced liver injury
CN104367956

Traditional Chinese medicine composition for treating pains in necks, shoulders, waists and legs
CN104367921

Pharmaceutical composition for treating qi deficiency and blood stasis-type apoplexy and preparation method thereof
CN104367896

Herbal medication for treating seborrheic dermatitis
CN104367894

Traditional Chinese medicine ointment for treating tinea of feet and hands and preparing method thereof
CN104367878

Chinese medicinal composition for treating primary hypertension
CN104367851

Traditional Chinese medicine decoction for treating thrombocytopenia
CN104367803

Traditional Chinese medicine preparation for treating gingivitis
CN104367792

Traditional Chinese medicine preparation for treating optic atrophy caused by spleen-kidney yang deficiency
CN104367762

Traditional Chinese medicine anesthetic for tooth pulling surgery and preparation method thereof
CN104367733

Traditional Chinese and western medicine composite drops for treating onychomycosis and preparation method thereof
CN104367681

Blood circulation-promoting and dysentery-stopping preparation for chronic dysentery as well as preparation method thereof
CN104367650

Chinese medicinal decoction capable of clearing heat and freeing strangury
CN104353034

Ciliatenerve knotweed root-containing traditional Chinese medicine composition for treating diabetes
CN104352974

Traditional Chinese medicine for treating duck viral hepatitis and preparation method of traditional Chinese medicine
CN104352959

Traditional Chinese medicine composition for treating diabetes
CN104352942

Traditional Chinese medicine preparation for treatment of whooping cough
CN104352868

Traditional Chinese medicine decoction piece combination preparation of herbal tea of Guangdong, preparation method and combined package
CN104352838

Preparation method of traditional Chinese medicine composition for treating chicken leucocytozoonosis
CN104352837

Traditional Chinese medicine composition for treating chronic dysentery and preparation method thereof
CN104352802

Traditional Chinese medicine preparation for treating infertility caused by hypomenorrhea and preparation method thereof
CN104352776

Traditional Chinese medicine composition for treating dysentery and preparation method thereof
CN104352701

External traditional Chinese medicine paste for treating acutesuperficial lymphangitis
CN104352666

Oral administration medicine for diminishing inflammation and arresting bleeding for department of gynecology and obstetrics
CN104352651

Traditional Chinese medicine composition with functions of clearing heat and promoting diuresis for acute bacillary dysentery and preparation method
CN104352584

Medicine for treating burn, traumatic injury, trauma hemorrhage and ulcer disease of lower limb
CN104337944

Chinese medicinal pills for treating ankylosing spondylitis
CN104337929

Traditional Chinese medicine preparation for treating mania and preparation method of traditional Chinese medicine preparation
CN104324327

Pharmaceutical composition for treating hepatitis B and preparation method of pharmaceutical composition
CN104324259

Traditional Chinese medicine composition for treating kidney deficiency type lumbar disc herniation and preparation method of traditional Chinese medicine composition
CN104324162

External-use traditional Chinese medicine for treating cervical spondylosis
CN104324086

Traditional Chinese medicine facial mask for removing acne and whitening and preparation method of traditional Chinese medicine facial mask
CN104323960

Health-preserving sesame paste and preparation method thereof
CN104323346

Cactus faint-scent honeysuckle tea and preparation method thereof
CN104322781

Culture medium capable of increasing resveratrol content of gentrin knotweed and preparation method thereof
CN104313061

Heart-nourishing and nerve-soothing health persimmon vinegar and preparation method thereof
CN104312891

Preparation method of perfoliate knotweed herb red pigment
CN104312199

Culture medium for white needle mushroom and preparation method thereof
CN104311301

Nutrient flammulina velutipes culture medium and preparation method thereof
CN104311297

Traditional Chinese medicine combination for curing hyperlipidemia
CN104306943

Capsule for treating prostatitis
CN104306900

Traditional Chinese medicine for treating hemophilia and preparation method thereof
CN104306783

Traditional Chinese medicine composition for treating primary hypotension
CN104306769

Traditional Chinese medicine for treating oral ulcer
CN104306711

Drug for curing hyperlipidemia
CN104306703

Tongmai Decoction
CN104306700

Traditional Chinese medicine composition for treating cough and preparation method and application
CN104306652

Traditional Chinese medicine composition for treating acute diarrhea with capability of clearing away heat and toxic materials and preparation method of traditional Chinese medicine composition
CN104306624

Traditional Chinese medicine dispersible tablet capable of nourishing blood and tranquilization
CN104306541  

Chinese chestnut and apricot blood replenishing honey and preparation method thereof
CN104304888

Lactating sow feed containing edible fungi residue and preparation method thereof
CN104304735

Traditional Chinese medicine composition for treating acute icteric hepatitis
CN104288710

Chinese patent medicinal preparation for treating angina pectoris
CN104288709

Traditional Chinese medicine composition for treating bacterial pneumonia as well as preparation method and application thereof
CN104288642

Pharmaceutical preparation for treating tinea pedis
CN104288632

Traditional Chinese medicine composition used for treating whitecomb of chicken
CN104288561

Medicament for treating chronic obstructive pulmonary disease
CN104288544

Externally-used traditional Chinese medicine preparation for treating burn and scald and preparation method thereof
CN104288520

Traditional Chinese medicine composition for treating acute bacillary dysentery and for clearing heat and promoting diuresis and preparation method thereof
CN104288416

Traditional Chinese medicine for treating rheumatoid arthritis
CN104288387

Traditional Chinese medicine composition for resisting superbacteria NDM-1 drug resistance gene bacteria
CN104288315

Morning tea powder with effect of refreshing, and preparation method thereof
CN104286319

Traditional Chinese medicinal preparation for treating damp-heat type uterine prolapse
CN104274612

Traditional Chinese medicine composition for treating fatty liver
CN104274610

Nerves-calming and qi-benefiting capsule and preparation method thereof
CN104274557

Nutritious rice capable of promoting health of digestive function and preparation method of nutritious rice
CN104273454

Summer sunstroke-prevention goose feed
CN104273363

Nutrient milky dry sauce and preparation method thereof
CN104273232

Interior decoration leveling putty with mosquito and insect expelling function
CN104231710

Production method of fabric affinitive with skin
CN104228308

Traditional Chinese medicine preparation for treating hand-foot-and-mouth disease and production method thereof
CN104225568

Traditional Chinese medicine for treating stomach ulcer and duodenal ulcer
CN104225536

Traditional Chinese medicine for treating cholecystitis
CN104225490

Traditional Chinese medicine for treating chronic cholecystitis
CN104225486

Traditional Chinese medicine for treating upper respiratory infection
CN104225412

Traditional Chinese medicine for treating clustered acne around eye sockets
CN104225325

Traditional Chinese medicine composition for curing sciatica and preparation method and application method thereof
CN104225261

Traditional Chinese medicine for treating tinnitus cerebri caused by syndrome of qi depression transforming into fire
CN104225197

Medicine for recovering plasma ablation operation wound
CN104225136

Traditional Chinese medicine for treating liver Qi stagnation type globus hysteriocus
CN104225107

Pharmaceutical preparation for treating prosopalgia
CN104225105

Traditional Chinese medicine pills for treating appendicitis
CN104225095

Traditional Chinese medicine decoction for treating infantile pneumonia
CN104225070

Fish medicine for treating bacterial rotten gill disease
CN104224970

Herbal medicine for treating dysentery
CN104224944

Novel functional food for synchronously reducing hyperglycaemia, hypertension and hyperlipidemia
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Medicated food preparation for treating neurasthenia and preparation method thereof
CN104222862

Use method of moult-preventing feed for ducks
CN104222613

Feed additive for enhancing immunity of piglets
CN104222520

Medical tea formula used for treating acute infectious hepatitis
CN104222364

Cultivation method of polygonaceae perennial herbaceous plants Japanese knotweed
CN104221653

Traditional Chinese medicine composition having fat-reducing efficacy
CN104248728

Traditional Chinese medicine for treatment of bacillary dysentery
CN104248698

Growing method for multiflower knotweed root
CN104247617

Novel method for extracting resveratrol from giant knotweed
CN104263763

Traditional Chinese medicine for treating hepatitis B
CN104258353

Medicated wine for meridian unblocking, wind dispelling, collaterals activating and pain relieving
CN104258304

Traditional Chinese medicine for treating mumps
CN104258299

Bone-strengthening powder
CN104258294

Traditional Chinese medicinal preparation for treating arteriosclerosis and preparation method thereof
CN104258280

Traditional Chinese medicine for treating gastric ulcer
CN104258262

Traditional Chinese medicine composition for nursing vaginas of women in puerperium
CN104258236

Traditional Chinese medicine for treating impetigo
CN104258233

Traditional Chinese medicine composition for treating hematuria
CN104258217

Traditional Chinese medicine composition for treating dysuria
CN104258216

Traditional Chinese medicine for treating chronic laryngitis with low recurrence rate
CN104258213

Traditional Chinese medicine composition for treating hypertension due to vital energy and blood deficiency and preparation method of composition
CN104258202

Preparation method of traditional Chinese medicine composition for treating acne
CN104258190

Traditional Chinese medicine composition for treating tubercular peritonitis as well as preparation method and application of composition
CN104258084

Traditional Chinese medicine composition for preventing and treating porcine reproductive and respiratory syndrome
CN104258047

Medicine for treating waist and neck osteoproliferation
CN104257951

Traditional Chinese medicine preparation for treating blood-stasis obstruction type gastroparesis syndrome
CN104257950

Externally applied liniment for treating otitis media
CN104257945

Chinese medicinal preparation for lowering blood fat
CN104257915

Traditional Chinese medicine preparation for treating constipation
CN104257766

Preparation method of natural anti-dandruff and caring shampoo
CN104257562

Beverage for young peacock
CN104256176

Fish feed for preventing pancreatic necrosis disease of salmon and trout
CN104256112

Externally applied ointment for treating eczema
CN104208535

Traditional Chinese medicine composition used for treating rheumatism
CN104208417

Traditional Chinese medicine for treating damp-heat stagnation type abdominal mass
CN104208332

Medicated bath powder for dispelling wind, eliminating dampness and warming and activating meridians
CN104208216

Blood lipid and blood glucose reducing Chinese wolfberry root-bark health care tea and preparation method thereof
CN104206595

Herba Taxilli health-care tea capable of lowering blood pressure and blood lipid and preparation method thereof
CN104206594

Distiller's yeast and preparation method thereof as well as method for making wine by virtue of distiller's yeast
CN104194999

Water purification drinking water bottle capable of being carried with users
CN104193047

Traditional Chinese medicine composition for treating acute infectious jaundice type hepatitis
CN104189789

Traditional Chinese medicine for treating damp-heat type 2 diabetes mellitus and preparation method thereof
CN104189646

Traditional Chinese medicine powder for inhibiting viral nervous necrosis of sea fish
CN104189480

Traditional Chinese medicine compound for treating osteoporosis, and preparation method of traditional Chinese medicine compound
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Anti-inflammatory tincture and preparing method thereof
CN104189397  




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