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Oleocanthal vs Cancer








http://news.rutgers.edu/research-news/ingredient-olive-oil-looks-promising-fight-against-cancer/20150211#.VnZtsqTR1FV
February 12, 2015

Ingredient in Olive Oil Looks Promising in the Fight Against Cancer

Oleocanthal kills cancer cells with their own enzymes

by

Ken Branson

Extra-virgin olive oil contains an ingredient, oleocanthal, that kills cancer cells without harming healthy cells, researchers have found.

A Rutgers nutritional scientist and two cancer biologists at New York City’s Hunter College have found that an ingredient in extra-virgin olive oil kills a variety of human cancer cells without harming healthy cells.

The ingredient is oleocanthal, a compound that ruptures a part of the cancerous cell, releasing enzymes that cause cell death.

Paul Breslin, professor of nutritional sciences in the School of Environmental and Biological Sciences, and David Foster and Onica LeGendre of Hunter College, report that oleocanthal kills cancerous cells in the laboratory by rupturing vesicles that store the cell’s waste. LeGendre, the first author, Foster, the senior author, and Breslin have published their findings in Molecular and Cellular Oncology.

According to the World Health Organization’s World Cancer Report 2014, there were more than 14 million new cases of cancer in 2012 and more than 8 million deaths.

Scientists knew that oleocanthal killed some cancer cells, but no one really understood how this occurred. Breslin believed that oleocanthal might be targeting a key protein in cancer cells that triggers a programmed cell death, known as apoptosis, and worked with Foster and Legendre to test his hypothesis after meeting David Foster at a seminar he gave at Rutgers.

“We needed to determine if oleocanthal was targeting that protein and causing the cells to die,” Breslin said.  

After applying oleocanthal to the cancer cells, Foster and LeGendre discovered that the cancer cells were dying very quickly – within 30 minutes to an hour. Since programmed cell death takes between 16 and 24 hours, the scientists realized that something else had to be causing the cancer cells to break down and die.

LeGendre, a chemist, provided the answer: The cancer cells were being killed by their own enzymes. The oleocanthal was puncturing the vesicles inside the cancer cells that store the cell’s waste – the cell’s “dumpster,” as Breslin called it, or “recycling center,” as Foster refers to it. These vesicles, known as lysosomes are larger in cancer cells than in healthy cells, and they contain a lot of waste. “Once you open one of those things, all hell breaks loose,” Breslin said.

But oleocanthal didn’t harm healthy cells, the researchers found. It merely stopped their life cycles temporarily – “put them to sleep,” Breslin said. After a day, the healthy cells resumed their cycles.  

The researchers say the logical next step is to go beyond laboratory conditions and show that oleocanthal can kill cancer cells and shrink tumors in living animals. “We also need to understand why it is that cancerous cells are more sensitive to oleocanthal than non-cancerous cells,” Foster said.







Patents

FR2911872
Extraction of oleocanthal from virgin olive oil


Process for enriching an extract containing oleocanthal, comprises forming a bisulfite compound by mixing the extract with a bisulfite of alkaline or alkaline earth metal or quaternary ammonium and eliminating the derivatives that are not reacting with the bisulfite, and deprotecting the bisulfite compound. An independent claim is included for a process for extracting oleocanthal from virgin olive oil, comprising preparing an extract containing oleocanthal, and enriching the extract by implementing the above process.

The present invention relates to a novel process for enriching an extract containing oleocanthal likely to provide extracts high in oleocanthal, and a new method for extracting oleocanthal from olive oil. Maintaining the health and comfort of life has become a major concern of developed country populations. This results in growing demand for medicines, medical care and quality controlled food products. In particular, in the field of food, much research has been devoted to food supplements and supplements, leading the development of areas of "nutraceuticals" and "functional foods" for the development of food or supplements food intended to promote biological functions affected by aging. In addition, consumers are looking for today increasingly natural products or natural origin that has been treated as little denaturing as possible.

Epidemiological studies have shown the beneficial health effects of the Mediterranean diet plans that include a high intake of fiber, fruits, vegetables, and olive oil is the main source of fat. Olive oil in particular contain mono-unsaturated fatty acids (oleic acid) as well as phenolic compounds which have the effect of delaying the oxidation of LDL cholesterol. Among these phenolic compounds, there are phenolic alcohols, such as tyrosol and hydroxytyrosol, benzoic acids, caffeic acid and esterified with elenolic tyrosol and hydroxytyrosol and flavonoids such as flavonols and flavones. Patent FR 2825022 describes a method for recovery of aqueous phase in processes for the preparation of olive oil by extraction of polyphenols contained therein for use in dietetic and cosmetic compositions. P. Andrews et al., J. Agric. Food Chem. 51: 5, 1415-1420 (2003) have shown that olive oil contains a phenolic derivative B1704FR 2 particular, namely deacetoxy ligstroside aglycone, which is responsible for its typical taste. G. K. Beauchamp et al. Nature, Vol. 437, 45-46 (Sept. 2005), showed that the aglycone deacetoxy ligstroside or oleocanthal contained in virgin olive oil obtained by cold pressed, this inhibitory properties COX1 and COX2 enzymes, c ' is to say, neighboring antiinflammatory properties than ibuprofen molecule used for many years as anti-inflammatory drug non-stéroidieri. These oleocanthal the properties are also disclosed in WO 2006122128. The oleocanthal or deacetoxy ligstroside-aglycone, was detected in olive oils from various sources, particularly in virgin olive oil obtained by cold pressing at very low concentrations generally vary from 200 ppm (mass) or 0.002 to 0.02% by weight, depending on the origin of the olive oil.

It can be represented by the general formula (I): HO 0 (I) A pharmaceutical or nutraceutical use of this compound requires the acquisition of more concentrated fractions. Moreover, oleocanthal is a very fragile product, easily destroyed by heat and oxidation, as are also the most other polyphenolic compounds naturally present in vegetable oils. Various methods for extracting phenolic compounds in olive oil were compared by Montedoro G. et al., J. Agric. Food Chem., 1992, 40, 9, 1571- 1576. According to the authors, to be optimal, extraction should be made with a methanol / water mixture 80/20. 3 B1704FR the purification of the extract is then performed by washing with hexane to extract solubilized in acetonitrile. However, the authors have optimized this method compared to total phenolic compounds in olive oil, and their study is not directly transposable to obtain extracts rich in oleocanthal.
In addition, a significant portion of the triglyceride compounds is still present even after purification. Other purifications oleocanthal are described in the literature. They use systematically purifications by chromatography on a glass plate, on open column, or by preparative chromatography, and are for the purpose of obtaining the product purified for characterization.

Thus, the amount of oleocanthal in olive oil is very low (20 to 20C) is 0.002 ppm to 0.02% of oleocanthal), it is difficult to obtain an extract enriched enough oleocanthal without using the chromatographic methods.

It is therefore desirable to develop a process for obtaining extracts high in oleocanthal content. Studies conducted by the applicant showed that it is possible to effectively enrich an extract containing oleocanthal including through the formation of intermediate compounds bisulfite. The present invention relates to a process for enriching a particular extract containing oleocanthal may provide extracts which oleocanthal content is greater than or equal to about 30%. The present invention also provides a process for enriching a sample containing oleocanthal, providing an extract titrated oleocanthal greater than 90% free of triglyceride compounds and pheno-lic impurities, in particular of tyrosol and hydroxytyrosol. The present invention also relates to a method of extraction and enrichment of oleocanthal from virgin olive oil, preferably virgin olive oil B1704FR prepared by first cold pressing, providing a titrated extract oleocanthal in greater than 10% and can go beyond 90%.

The enrichment process a sample containing oleocanthal according to the present invention comprises the following successive steps. a) forming a bisulfite compound by mixing said extract with an alkali metal sulfite or alkaline earth metal or quaternary ammonium, e.g., sodium bisulfite, and elimination derivatives unreacted with said bisulfite; b) deprotection of the bisulfite compound. Step a) comprises the formation of a bisulfite compound intermediate. In general, the term bisulfite compound a compound obtained by alkali metal bisulfite addition or alkaline earth metal or quaternary ammonium on aldehydes or ketones. Removal of the derivatives that do not react with the bisulfite is preferably carried out by liquid / liquid extraction. Step b) of the process according to the invention consists of deprotection of the bisulfite compound. This deprotection may be carried out in acidic medium, in a basic medium or in the presence of a silylating agent, in particular chlorotrimethylsilane.

In a preferred embodiment, the oleocanthal used initially containing extract is in the form of a yellowish oil, and mixing said extract with bisulfite is carried out by solubilization of the extract in an aqueous solution of bisulfite, possible - 30 LEMENT mixed with a solvent or mixture of solvents miscible with water. These solvents may be selected from alkyl acetates, alcohols or acetonitrile. It can be a sodium bisulfite solution composed of a mixture of water and ethanol. 35 According to this embodiment, the amount of bisulfite, preferably sodium bisulfite, may be calculated so as to selectively form the bisulphite salts oleocanthal (I) of ligstroside aglycone B1704FR derivative represented by the formula (II) -dessous, and derivatives of oleuropein aglycone represented by the formulas (III) and (IV) below. 5 HO o // o ~~ H3CO2C (II) CHO CH3 HO HO HO H3CO2C YY1 derivatives which do not have aldehyde (e.g., 1 - acétoxypinorésinol and pinoresinol) or which have not reacted with bisulfite of sodium are then removed by liquid / liquid extraction using a solvent immiscible with water, in particular using dichloro-methane 15 or ethyl acetate.

The bisulfite compound is then obtained by evaporating the aqueous phase. After deprotection, a mixture is obtained wherein oleocanthal prevails and the compounds of formula (I: I), (III) and (IV) are present in substantially equivalent 20. The process just described can achieve an extract having a high content of oleocanthal, on the order of at least 30%, and without B1704FR (IV) 6 intervene organic chemical reagents whose use produces adverse effects on the environment. According to an advantageous variant of the process according to the invention may be provided in addition, between steps a) and b), a selective precipitation step of the bisulfite compound of oleocanthal. This selective precipitation step which occurs before the deprotection step, aims to obtain selectively oleocanthal thereby increasing significantly its content in the final extract compared to the original extract.

The implementation of this step may allow in fact to obtain an extract of oleocanthal purity greater than 90%. In one embodiment, the selective precipitation step is carried out by solubilization of the bisulfite compound in water or in an alcohol, for example methanol, and then selective precipitation of the bisulfite compound of oleocanthal by a non-solvent preferably selected from the group consisting of alkyl acetates, ethers, ketones and acetonitrile, ethyl ether being particularly preferred. In this variant, bisulfite compound deprotection step is then performed as described above. In another aspect, the invention relates to a process for extracting oleocanthal from virgin olive oil, which consists of preparing an extract containing oleocanthal, said extract and enrich implementing the enrichment process as described above.

In a preferred embodiment, the preparation of said extract comprises the following successive steps: - addition of a hydrocarbon in olive oil; extraction with a mixture of binary solvent water / alcohol or acetone, or ternary water / alcohol or acetone / solvent immiscible with water; removal of organic phase and total or partial concentration of the aqueous phase 35. The step of adding the hydrocarbon in olive oil helps to facilitate separation between the organic phase and the aqueous phase. The hydrocarbon used may include 5 B1704FR 7 to 10 carbon atoms and is preferably selected from the group consisting of hexane, cyclohexane and heptane. For the implementation of the following extraction step, in the case of a binary mixture of water / alcohol solvents, the alcohol is selected preferably from the group consisting of methanol, ethanol, n- propanol, isopropanol.

When using a ternary mixture of solvents water / alcohol or acetone / solvent immiscible with water, it may be for example a chlorinated solvent, a ketone or an alkyl acetate. The chlorinated solvent generally has 1 to 8 carbon atoms and may be selected preferably from the group consisting of dichloromethane, chloroform and chlorocyclohexane, the ketone preferably has from 4 to 8 carbon atoms and can be selected from the group consisting of methyl and pentanones, and alkyl acetate generally contains from 3 to 8 carbon atoms and is preferably methyl acetate or ethyl. Solvent proportions used may vary depending on operating conditions, but they are general-ment between 80/20 and 40/60 in the case of a binary mixture of water / alcohol or acetone, and is advantageously used for example a mixture of water / methanol 60/40. The next step of partially purified extract and effectively remove components entrained during extraction whose physicochemical properties are close to those of oleocanthal.

This step is crucial to obtain an extract with a high concentration oleocanthal.

The concentration of the extract may be complete by performing evaporation to dryness so as to obtain a dry extract, which is then used in the enrichment step. In case of partial concentration, it is advantageous to then carry out a centrifugation step before proceeding to the enrichment step. Partial concentration of the rate must be at least 50%. The enrichment process described in particular allows to isolate oleocanthal almost all other B1704FR 8 phenolic compounds originally present in olive oil. These include phenolic alcohols such as tyrosol and hydroxytyrosol; free acid of the benzoic acid series such as protocatechuic acid, gallic, vanillic, syringic, or cinnamic series such as pcoumarique acids, caffeic, sinapic and; esterified derivatives of caffeic acid (verbascoside) or elenolic acid (oleuropein glycosylated or not, responsible for much of the bitterness of olive oil), these two acids being esterified or tyrosol hydroxytyrosol; and finally flavonoids such as flavones (luteolin) and flavonols (quercetin and kaemférol glycosylated or not).

The oleocanthal is also isolated from other components in olive oil such as hydrocarbons (eg squalene), sterols (eg R-sitosterol), triterpene alcohols, and tocopherol (in the form a, p and y). It is particularly interesting to note that the extract obtained contains oleocanthal strongly predominant phenolic compound as well as small amounts of aglycone derivatives of oleuropein and ligstroside aglycone, whereas tyrosol and hydroxytyrosol and their derivatives in particular acetates, are completely eliminated, as well as triglyceride compounds. As mentioned above, according to the variant used, the method of the invention allows to obtain an extract containing not only oleocanthal with a content of at least 30% by weight, but ausssi derivatives aglycone oleuropein and aglycone ligstroside in significant amounts. The proportions of these four compounds may be substantially equivalent.

Such an extract containing them may be of economic interest. The following examples illustrate the invention in more detail without limiting its scope. Unless otherwise indicated, ratios and percentages are by weight. B1704FR 9 Example 1 Preparation of an extract containing oleocanthal from olive oil This example describes the extraction of oleocanthal May 1 mixture of binary solvents. From a containing 56 mg olive oil oleocanthal per kilogram firstly an extraction as described below. In a 50 liter reactor was charged with 10 kg of olive oil 10 and 20 liters of cyclohexane. Leer for liquid / liquid extraction is added 20 liters of methanol / water 40/60 v / v and stirred medium at room temperature for 5 minutes and then allowed to settle the two phases. The extraction is repeated a 15 second time under the same conditions.

Is added to the first aqueous phase 20 liters of cyclohexane. The medium is stirred at room temperature for 5 minutes then allowed to settle the two phases. Is carried out a second washing of the aqueous phase 20 with the same cyclohexane phase. The two aqueous phases are pooled and concentrated by evaporation to dryness by not exceeding 40 C in the bath (an oil). This gives about 4.7 g of extract 12% to 25 oleocanthal. Example 2 Formation of the bisulfite compound and deprotection in acid medium is prepared fresh solution of sodium bisulfite in 30 dissolving 0.817 g in 100 ml of water (molecular weight: 104.06 g / mol, 7,85.10-2 mole per liter) . 20 ml of dilute aqueous sodium bisulfite (1,57.10-3 mole bisulfite) to the appropriate amount of extract containing approximately 400 mg of oleocanthal 35 (molecular weight: 304.13 g / mol, 1,31.10 -3 mol), obtained according to the procedure described in Example 1.

If the medium is too heterogeneous, a small amount of ethanol is added 10 B1704FR. The solution is stirred at room temperature for 1 hour. If ethanol is added, it is evaporated in a rotary evaporator.

Then directs liquid / liquid water / dichloromethane the resulting aqueous phase to remove the derivatives having no aldehyde functions or unreacted with bisulfite. The aqueous phase is evaporated in a rotary evaporator, and obtained a residue A. The obtained R residue is taken up the minimum volume of dilute HC1 solution (pH 3 to 5) for a total solubility of the product. Is allowed to react the reaction mixture stirred 15 for 30 minutes at room temperature. The solution is extracted 3 times with an equal volume of dichloromethane. Then dried over Na2SO4, the dichloromethane layers combined, then the solution is filtered and 20 dichloromethane phase is concentrated in a rotary evaporator in order to recover purified oleocanthal. Give about 1.0 g of product containing a mixture of phenolic compounds (majority oleocanthal, ligstroside aglycone derivative (II) and derivatives of oleuropein aglycone 25 (III) and (IV) in equal proportions), free from compounds pinorésinols (1-acetoxypinoresinol and pinoresinol for example).

The title oleocanthal in the mixture is of the order of 30%. 30 Example 3 This example describes a variation of Example 2 wherein the bisulfite compound is oleocanthal precipitate prior to deprotection. For this, the R residue is prepared as described in Example 2, and then taken up in a minimum of methanol. 35 The bisulfite compound of oleocanthal is selectively precipitated by addition of ethyl ether. The white precipitate is recovered by simple filtration. B1? 04FR 11 The deprotection in acid medium is then carried out on the precipitate as described in Example 2. Give about 280 mg of oleocanthal purity greater than 90%.
Example 4 Formation of the bisulfite compound and deprotection in basic medium The bisulfite compound is prepared according to the conditions described in Example 2.

To perform its deprotection in basic medium, the R residue obtained in Example 2 is taken up in the minimum volume of NaOH solution (pH between 8 and 9) for a total solubility of the product. Is allowed to react the reaction mixture stirred 15 for 30 minutes at room temperature. The solution is extracted 3 times with an equal volume of dichloromethane. Then dried over Na2SO4, the dichloromethane layers combined, then the solution is filtered and 20 dichloromethane phase is concentrated on a rotary evaporator to recover oleocanthal purified. Give about 0.9 g of product containing a mixture of phenolic compounds (majority oleocanthal, ligstroside aglycone derivative (II) and derivatives of oleuropein aglycone 25 (III) and (IV) in equal proportions), free from compounds pinoresinols (1-acetoxypinoresinol and pinoresinol for example). The title oleocanthal in the mixture is of the order of 30%. 30 Example 5 This example describes a variation of Example 4, wherein the bisulfite compound is oleocanthal precipitate prior to deprotection.

For this, the R residue is prepared as described in Example 2, and then taken up in a minimum of methanol and 35 of the bisulfite compound oleocanthal is selectively precipitated by adding ethyl ether. The white precipitate is recovered by simple filtration. B1704FR 12 basic medium deprotection is then carried out on the precipitate as described in Example 4. Give about 250 mg of oleocanthal purity greater than 90%.

Example 6 Formation of the bisulfite compound and deprotection in the presence of chlorotrimethylsilane The bisulfite compound is prepared according to the conditions described in Example 2.

To perform its deprotection in the presence of chlorotrimethylsilane, taken up R residue obtained in Example 2 in 5 ml of acetonitrile. Then is added 0.4 q of chlorotrimethylsilane (Molecular weight: 108.64 g / mol, 3,68.10-3 mole) and left to react the reaction mixture stirred for 2 hours at 400C. Allowed to cool to room temperature and the reaction medium is evaporated to dryness. The residue thus obtained is taken up in 20 ml of water and 20 the solution was extracted 3 times with an equal volume of dichloromethane. Then dried over Na2SO4, the dichloromethane layers combined, and the solution is filtered and the dichloromethane phase is concentrated on a rotary evaporator to recover 25 oleocanthal purified. Give about 0.8 g containing a mixture of phenolic compounds (oleocanthal (I) majority, ligstroside aglycone derivative (II) and derivatives of oleuropein aglycone (III) and (IV) in equal proportions), 30 free of compounds pinoresinols (1-acetoxypinoresinol and pinoresinol for example).

The title oleocanthal in the mixture is of the order of 30%. Example 7 This example describes a variation of Example 6, in 35 which the bisulfite compound is oleocanthal precipitate prior to deprotection. B1704FR 13 For this, the R residue is prepared as described in Example 2, and then taken up in a minimum of methanol. The bisulfite compound of oleocanthal is selectively precipitated by addition of ethyl ether. The white precipitate is recovered by simple filtration. Deprotecting in the presence of chlorotrimethylsilane is then carried out on the precipitate as described in Example 6. Give about 230 mg of 10 oleocanthal purity greater than 90%. B1704FR



Method for extracting oleocanthal from olive oil
CN103483196

The invention discloses a method for extracting oleocanthal from olive oil. The method comprises the steps as follows: water is added to the olive oil and modified starch for mixing, and then the mixture is subjected to spray drying; subcritical extraction is performed twice with a fluid formed by propane and dimethyl ether in a weight ratio of (40-60):(40-60) serving as an extracting agent; the subcritical extraction is performed again with absolute ethyl alcohol serving as an extracting agent; and spray drying is performed after an extraction liquid is concentrated,, so that a finished product is prepared.; According to the method, the extraction process is simple, the usage amount of an organic solvent is small, the residual quantity is low, the final product yield is about 2%, the content can reach 30%-40%, and the oleocanthal with higher quality is effectively obtained from the olive oil which is difficult to extract.

The present invention discloses a kind of method that olive stimulates aldehyde of extracting from sweet oil, first sweet oil and modified starch water adding to be mixed to rear spraying drying, then take fluid that propane and dme form by the weight ratio of 40~60:40~60 as twice of extraction agent sub critical extraction, take dehydrated alcohol as extraction agent sub critical extraction again again, after extracting solution is concentrated, spraying drying makes finished product.Extraction process of the present invention is simple, and the organic solvent usage quantity is few, residual quantity is low, and the yield of the finished product is about 2%, and content can reach 30%~40%, and the olive that effectively from the sweet oil that is difficult for being extracted, obtains higher quality stimulates aldehyde.



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