Sulforaphane vs Cancer & Neurodegeneration -- Articles & Patents

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Sulforaphane vs Cancer

http://www.sciencealert.com/broccoli-pills-could-help-fight-existing-cancers-health-experts-say
2 December 2015

Broccoli pills could help fight existing cancers, health experts say
Always eat your veggies.
by Peter Dockrill

A diet high in green vegetables is known to reduce the incidence of cancer, and now scientists have found evidence that they could help fight cancer already present in tumours.

Researchers in the US have found that a compound in dark-green vegetables (aka cruciferous vegetables) called sulforaphane may be able to treat cancer and help existing cancer drugs work more effectively.

Sulforaphane is found in the highest concentrations in young sprouts of broccoli, but it's also present in Brussels sprouts, kale, cauliflower, and cabbage. In addition to natural produce, you can also buy sulforaphane in the dietary supplement broccoli sprout extract (BSE).

In a new study conducted by researchers at the Texas A&M Health Science Centre, 28 human volunteers aged 50+ who were already undergoing routine colonoscopies were surveyed on their cruciferous vegetable-eating habits.

When the researchers examined the volunteers' colon biopsies, they found that those who ate more servings of dark-green vegetables had higher levels of expression of a tumour suppressor gene (called p16) than those who ate few or no cruciferous vegetables.

What surprised the researchers was finding that the p16 benefits from vegetable intake persisted even when volunteers indicated that they didn’t eat vegetables every day. That's strange, because suloraphane usually clears out of the body in less than 24 hours after being consumed.

"This hints at the possibility that epigenetic mechanisms are initially triggered by sulforaphane and its metabolites, and downstream mechanisms could be sustained, at least in the short-term, even after compounds are eliminated from the body," said one of the researchers, Praveen Rajendran.

What this means is eating cruciferous vegetables or taking sulforaphane in a supplement form – effectively, a broccoli pill – may end up changing your genes, helping your body to get better at preventing tumour growth. The findings are published in Clinical Epigenetics.

Previous research by the same team had found that sulforaphane could inhibit colon and prostate cancer cells in laboratory tests, but discovering that the benefits extend to humans is significant.

"Our work provides comprehensive proof-of-principle using cell-based, animal and human studies that dietary compounds like sulforaphane can be chemopreventive," said Rajendran. "However, we’re not quite ready to recommend everyone take a BSE supplement, and it’s certainly worth reiterating what nutritionists have said for years: eat your vegetables."

http://news.tamhsc.edu/?post=our-mothers-told-us-to-eat-our-vegetables-now-we-know-why
November 30, 2015

Our mothers told us to eat our vegetables: Now we know why

by

Christina Sumners

A compound in broccoli and other cruciferous vegetables may be able to not only help prevent cancer but also help to treat it — a new approach researchers at Texas A&M Health Science Center are calling “from the table to the bedside.” Although no one is suggesting giving up traditional chemotherapy and radiation treatments for cancer, compounds from food may actually help cancer drugs work more effectively.

This cancer-fighting compound is called sulforaphane, and it is found in vegetables like Brussels sprouts, kale, cauliflower and cabbage, but its highest concentrations are in the young sprouts of broccoli. Sulforaphane can also be found in a dietary supplement called broccoli sprout extract, or BSE.

Researchers at the Texas A&M Health Science Center Institute of Biosciences and Technology (IBT) in Houston, along with collaborators in Oregon, had previously found that sulforaphane could inhibit colon and prostate cancer cells in the laboratory. They’ve now shown that it seems to help humans as well.

Roderick H. Dashwood, professor and director of the Center for Epigenetics & Disease Prevention at the Texas A&M IBT, takes a “field-to-clinic” approach to cancer prevention. He and a collaborator, Praveen Rajendran, Ph.D., assistant professor at the center, published a new study in the journal Clinical Epigenetics that indicates a BSE supplement may help prevent or even treat colon cancer and hints at the biological pathways involved.

The BSE supplement seems to be generally safe. “We have not seen any serious adverse events in healthy volunteers who consumed BSE pills for seven days,” Rajendran said, although some people did experience mild abdominal discomfort. He cautions, however, that not all broccoli supplements are necessarily as effective as the one tested. “We have used a standardized broccoli extract in our study provided by Johns Hopkins University,” Rajendran said. “This BSE supplement is being evaluated in several other clinical trials around the country, but I’m not sure other, similar supplements available to the public have the same level of active ingredients, including sulforaphane.”

In a separate clinical study, 28 human volunteers over the age of 50, who were undergoing routine colonoscopies, were surveyed for their cruciferous vegetable-eating habits. When their colon biopsies were examined, those who ate more servings were found to have higher levels of expression of the tumor suppressor gene p16 than those who ate few or no cruciferous vegetables. This effect on p16 held even for people who didn’t eat these vegetables every single day, which may seem strange, as a single serving of sulforaphane is generally cleared from the body in less than 24 hours. “This hints at the possibility that epigenetic mechanisms are initially triggered by sulforaphane and its metabolites, and downstream mechanisms could be sustained, at least in the short-term, even after compounds are eliminated from the body.” In other words, eating vegetables containing sulforaphane can actually change your genes to make your body better able to prevent tumor growth.

However, it’s not all good news. In animal models, sulforaphane was shown to generally inhibit the development of colon cancer, but it’s a bit of a two-edged sword. Sulforaphane induces a protein called Nrf2, which has beneficial antioxidant and detoxifying effects — and is obviously good for fighting cancer. Later in the development of cancer, though, Nrf2 can also have a role in tumor growth and can even enhance the buildup of plaque in the arteries. “Because of all this, we believe that Nrf2 status is worthy of further investigation,” Rajendran said, “not just for cancer treatment but for its role in modulating cardiovascular disease.”

“Our work provides comprehensive proof-of-principle using cell-based, animal and human studies that dietary compounds like sulforaphane can be chemopreventive,” or able to help prevent cancer, Rajendran said. “However, we’re not quite ready to recommend everyone take a BSE supplement, and it’s certainly worth reiterating what nutritionists have said for years: eat your vegetables.”

http://www.clinicalepigeneticsjournal.com/content/7/1/102
Clinical Epigenetics 2015, 7:102
doi:10.1186/s13148-015-0132-y
18 September 2015

Nrf2 status affects tumor growth, HDAC3 gene promoter associations, and the response to sulforaphane in the colon

Praveen Rajendran, Wan-Mohaiza Dashwood, Li Li, Yuki Kang, Eunah Kim, Gavin Johnson, Kay A. Fischer, Christiane V. Löhr, David E. Williams, Emily Ho, Masayuki Yamamoto, David A. Lieberman and Roderick H. Dashwood

Abstract

Background

The dietary agent sulforaphane (SFN) has been reported to induce nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2)-dependent pathways as well as inhibiting histone deacetylase (HDAC) activity. The current investigation sought to examine the relationships between Nrf2 status and HDAC expression in preclinical and translational studies.

Results

Wild type (WT) and Nrf2-deficient (Nrf2 −/+ ) mice were treated with the colon carcinogen 1,2-dimethylhydrazine (DMH) followed by 400 ppm SFN in the diet (n = 35 mice/group). WT mice were more susceptible than Nrf2 −/+ mice to tumor induction in the colon. Tumors from WT mice had higher HDAC levels globally and locally on genes such as cyclin-dependant kinase inhibitor 2a (Cdkn2a/p16) that were dysregulated during tumor development. The average tumor burden was reduced by SFN from 62.7 to 26.0 mm 3 in WT mice and from 14.6 to 11.7 mm 3 in Nrf2 −/+ mice. The decreased antitumor activity of SFN in Nrf2 −/+ mice coincided with attenuated Cdkn2a promoter interactions involving HDAC3. HDAC3 knockdown in human colon cancer cells recapitulated the effects of SFN on p16 induction. Human subjects given a broccoli sprout extract supplement (200 μmol SFN equivalents), or reporting more than five cruciferous vegetable servings per week, had increased p16 expression that was inversely associated with HDAC3 in circulating peripheral blood mononuclear cells (PBMCs) and in biopsies obtained during screening colonoscopy.

Conclusions

Nrf2 expression varies widely in both normal human colon and human colon cancers and likely contributes to the overall rate of tumor growth in the large intestine. It remains to be determined whether this influences global HDAC protein expression levels, as well as local HDAC interactions on genes dysregulated during human colon tumor development. If corroborated in future studies, Nrf2 status might serve as a biomarker of HDAC inhibitor efficacy in clinical trials using single agent or combination modalities to slow, halt, or regress the progression to later stages of solid tumors and hematological malignancies.

https://en.wikipedia.org/wiki/Sulforaphane

Sulforaphane

Names
IUPAC name : 1-Isothiocyanato-4-methylsulfinylbutane
Identifiers
CAS Number 4478-93-7 Yes
ChEBI     CHEBI:47807
ChEMBL     ChEMBL48802
ChemSpider 5157
PubChem 5350

Properties

Chemical formula : C6H11NOS2
Molar mass     177.29 g/mol

Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

Sulforaphane is a compound within the isothiocyanate group of organosulfur compounds. It is obtained from cruciferous vegetables such as broccoli, Brussels sprouts or cabbages. It is produced when the enzyme myrosinase transforms glucoraphanin, a glucosinolate, into sulforaphane upon damage to the plant (such as from chewing), which allows the two compounds to mix and react. Young sprouts of broccoli and cauliflower are particularly rich in glucoraphanin.

Occurrence and isolation

Sulforaphane was identified in broccoli sprouts, which, of the cruciferous vegetables, have the highest concentration of sulforaphane.[1] It is also found in Brussels sprouts, cabbage, cauliflower, bok choy, kale, collards, Chinese broccoli, broccoli raab, kohlrabi, mustard, turnip, radish, arugula, and watercress.
Research

Basic research on sulforaphane includes its potential effect on mechanisms of neurodegenerative disorders and cancer; however, results to date are contradictory.[2][3] Sulforaphane is under study for a potential neuroprotective effect on recovery from spinal cord injury[4] and as a possible factor in Helicobacter pylori-associated gastric diseases.[5][6]

References

Zhang Y, Talalay P, Cho CG, Posner GH (March 1992). "A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure". Proc. Natl. Acad. Sci. U.S.A. 89 (6): 2399–2403. doi:10.1073/pnas.89.6.2399. PMC 48665. PMID 1549603.
http://www.pnas.org/cgi/pmidlookup?view=long&pmid=1549603

Tarozzi A, Angeloni C, Malaguti M, Morroni F, Hrelia S, Hrelia P (2013). "Sulforaphane as a potential protective phytochemical against neurodegenerative diseases". Oxid Med Cell Longev (Review) 2013: 415078. doi:10.1155/2013/415078. PMC 3745957. PMID 23983898.
http://www.pnas.org/cgi/pmidlookup?view=long&pmid=1549603

Grabacka MM, Gawin M, Pierzchalska M (2014). "Phytochemical modulators of mitochondria: the search for chemopreventive agents and supportive therapeutics". Pharmaceuticals (Basel) (Review) 7 (9): 913–42. doi:10.3390/ph7090913. PMC 4190497. PMID 25192192.

Koushki D, Latifi S, Javidan AN, Matin M (June 2014). "Efficacy of some non-conventional herbal medications (sulforaphane, tanshinone IIA, and tetramethylpyrazine) in inducing neuroprotection in comparison with interleukin-10 after spinal cord injury: A meta-analysis". J Spinal Cord Med (Meta-analysis) 38: 13–22. doi:10.1179/2045772314Y.0000000215. PMID 24969510.

Moon JK, Kim JR, Ahn YJ, Shibamoto T (2010). "Analysis and anti-Helicobacter activity of sulforaphane and related compounds present in broccoli ( Brassica oleracea L.) sprouts". J. Agric. Food Chem. 58 (11): 6672–7. doi:10.1021/jf1003573. PMID 20459098.

Fahey JW, Haristoy X, Dolan PM, Kensler TW, Scholtus I, Stephenson KK, Talalay P, Lozniewski A (2002). "Sulforaphane inhibits extracellular, intracellular, and antibiotic-resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors". Proc. Natl. Acad. Sci. U.S.A. 99 (11): 7610–5. doi:10.1073/pnas.112203099. PMC 124299. PMID 12032331.

http://www.pnas.org/cgi/pmidlookup?view=long&pmid=1549603
Oxid Med Cell Longev. 2013; 2013: 415078.
25 July 2013
doi: 10.1155/2013/415078
PMCID: PMC3745957

Sulforaphane as a Potential Protective Phytochemical against Neurodegenerative Diseases

Andrea Tarozzi, Cristina Angeloni, Marco Malaguti, Fabiana Morroni, Silvana Hrelia, and Patrizia Hrelia

Abstract

A wide variety of acute and chronic neurodegenerative diseases, including ischemic/traumatic brain injury, Alzheimer's disease, and Parkinson's disease, share common characteristics such as oxidative stress, misfolded proteins, excitotoxicity, inflammation, and neuronal loss. As no drugs are available to prevent the progression of these neurological disorders, intervention strategies using phytochemicals have been proposed as an alternative form of treatment. Among phytochemicals, isothiocyanate sulforaphane, derived from the hydrolysis of the glucosinolate glucoraphanin mainly present in Brassica vegetables, has demonstrated neuroprotective effects in several in vitro and in vivo studies. In particular, evidence suggests that sulforaphane beneficial effects could be mainly ascribed to its peculiar ability to activate the Nrf2/ARE pathway. Therefore, sulforaphane appears to be a promising compound with neuroprotective properties that may play an important role in preventing neurodegeneration.

1. Introduction

Acute and chronic neurodegenerative diseases, including stroke, traumatic brain injury (TBI), Alzheimer's disease (AD), and Parkinson's disease (PD), are illnesses associated with high morbidity and mortality, and few or no effective options are available for their treatment [1, 2]. These diseases result in acute, as well as gradual and progressive neurodegeneration, leading to brain dysfunction and neuronal death. Although molecular mechanisms involved in the pathogenesis of acute and chronic neurodegenerative diseases remain elusive, oxidative stress, misfolding, aggregation, accumulation of proteins, perturbed Ca2+ homeostasis, excitotoxicity, inflammation, and apoptosis have been implicated as possible causes of neurodegeneration in the previously mentioned neurological disorders [3, 4]. In addition, recent studies demonstrated that acute brain injuries are also environmental risk factors associated with chronic neurodegenerative diseases [5–7].

In the last few years, there has been a growing interest in a number of pharmacological approaches aimed at preventing and counteracting the neuronal dysfunction and death associated with neurodegenerative diseases. However, while enormous efforts have been made to identify agents that could be used to alleviate debilitating neurodegenerative disorders, a source of potentially beneficial agents, namely, phytochemicals, would appear to have significant benefits in counteracting neurodegenerative diseases. Phytochemicals have long been recognized as exerting different biological effects, including antioxidant, antiallergic, antiinflammatory, antiviral, antiproliferative, and anticarcinogenic effects [8–10]. Considering that these age-related neurological disorders are multifactorial and that no drugs are available to stop their progression, intervention strategies using phytochemicals have been proposed as an alternative form of treatment for their prevention. Among phytochemicals, sulforaphane (isothiocyanato-4-(methylsulfinyl)-butane) (SF) has been demonstrated to have neuroprotective effects in several experimental paradigms. Reports in the literature have shown a pleiotropic role of this natural compound, thanks to its ability to address different targets and to modulate different pathways in neuronal/glial cells.

In this review, we will discuss the most recent experimental evidence on the role of SF in counteracting brain oxidative stress in both acute and chronic neurodegenerative diseases. SF bioavailability is also considered, since it is a fundamental aspect in the evaluation of the “in vivo” bioactivity of a nutritional compound.

2. Sulforaphane Bioavailability

Various Brassica vegetables and especially broccoli contain glucoraphanin. Following cutting or chewing, it is hydrolyzed into the corresponding isothiocyanate SF either by the plant thioglucosidase myrosinase or by bacterial thioglucosidases in the colon [11].

Because of its lipophilicity [12] and molecular size, SF is likely to passively diffuse into the enterocytes [13]. After absorption, SF is conjugated with glutathione (SF-GSH) by glutathione-S-transferase (GST) leading to maintenance of a concentration gradient and facilitating a fast passive absorption into the cell [14]. It is metabolized via the mercapturic acid pathway, producing predominantly cysteinylglycine (SF-CG), cysteine (SF-Cys), and N-acetyl-cysteine (SF-NAC) conjugates that are excreted in the urine [15].

Pharmacokinetic studies in both humans and animals showed that the plasma concentration of SF and its metabolites increased rapidly, reaching a maximum between 1 and 3 h after administration of either SF, glucosinolate, or broccoli [16–21]. In particular, Veeranki and colleagues [21] reported the ability of SF and its metabolites to reach different tissues in the gastrointestinal and genitourinary tracts and other organs such as liver, pancreas, lung, and heart, in vastly different concentrations and that bioactivity, in terms of induction of cytoprotective phase II enzymes, may differ significantly among organs. Both plasma and tissue levels of these SF metabolites are rapidly eliminated through urinary excretion within 12–24 h reflecting the rapid elimination of SF. The in vivo bioactivity of each SF metabolite is still unclear, although many in vitro studies have shown the ability of SF-Cys, and SF-NAC metabolites to exert some bioactivity [22–24]. These data suggest the hypothesis that repeated consumption of SF or cruciferous vegetables is required to maintain the SF metabolite concentration in tissues.

Interestingly, more recent SF bioavailability studies in human subjects consuming broccoli showed its bioconversion into isothiocyanate erucin (isothiocyanato-4-(methylthio)-butane) (ER), a sulfide analog [25, 26]. Whether this conversion from SF to ER is important for the health promoting effects of glucosinolate still remains to be determined although some reports provide a glimpse into the possibility of differing activities between these two isothiocyanates [27–29].

In order to exert protective effects towards neurodegenerative disorders or improve brain function, SF must traverse the blood-brain barrier (BBB) and accumulate in the central nervous system (CNS). As reported in the following sections of this review, various studies in animal models of neurodegeneration suggest the ability of SF to reach CNS and to display protective effects at this level. In this context, Jazwa et al. [30] demonstrated in mice that after SF gavage, SF is able to cross the BBB and to accumulate in cerebral tissues such as the ventral midbrain and striatum, with a maximum increase and disappearance after 15 min and 2 h, respectively. Interestingly, Clarke et al. [19] also detected SF-GSH, SF-Cys and SF-NAC metabolites, but not SF alone, in the CNS in a similar experimental in vivo model after 2 h and 6 h. However, the authors suggest that low levels of the various SF metabolites recorded in the CNS indicate their poor ability to cross the BBB. These results show the ability of SF to quickly reach the CNS and the potential contribution of SF metabolites to prolong the presence of SF at this level because they are unstable under physiological conditions and readily dissociate back to SF [21, 30].

3. Protective Effects of Sulforaphane against Oxidative Stress

Oxidative stress results from an imbalance of pro-oxidant/antioxidant homeostasis that leads to an abnormal production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). The main ROS/RNS involved in neurodegeneration are superoxide anion radical (O2•−), hydrogen peroxide (H2O2), the highly reactive hydroxyl radical (•OH), and nitric oxide (NO) that can react with superoxide anion to produce peroxynitrite [31]. At high levels, ROS can react with different cell molecules, causing damage to DNA, lipids, and proteins and modulate intracellular signaling pathways, leading to cellular degeneration and apoptosis. ROS can also initiate proinflammatory pathways, further exacerbating the deleterious oxidized environment. The brain is particularly vulnerable to oxidative stress because of its high oxygen consumption, high content of oxidizable polyunsatured fatty acids, and low antioxidant defense capacities especially in aging brains [32–34]. Oxidative stress is involved in many neurodegenerative diseases and is a proposed mechanism for age-related degenerative processes as a whole [35, 36]. Numerous studies have provided compelling evidence that oxidative stress is an important causative factor in PD [2, 37–40], AD [41–43], amyotrophic lateral sclerosis (ALS) [44, 45], and multiple sclerosis (MS) [46, 47].

Cells possess a complex network of nonenzymatic and enzymatic components to counteract oxidative stress. GSH is the major nonenzymatic regulator of intracellular redox homeostasis. On the other hand, enzymatic antioxidants include glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), NAD(P)H-quinone oxidoreductase 1 (NQO1), thioredoxin reductase (TR), heme oxygenase 1 (HO1), peroxiredoxins, and many others. These enzymes are now recognized as primary defense mechanisms against many degenerative and chronic disease conditions [48]. These antioxidants and cytoprotective enzymes are regulated by a common mechanism that involves two proteins: nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like-ECH-associated protein 1 (Keap1) [49, 50]. Under basal conditions, Nrf2 is sequestered in the cytoplasm by its repressor protein Keap1 [51]. Keap1 contains several reactive cysteine residues that serve as sensors of the intracellular redox state. Nrf2 is released from Keap1 upon oxidative or covalent modification of thiols in some of these cysteine residues. Nrf2 translocates to the nucleus where it heterodimerizes with small Maf proteins before binding to the antioxidant responsive element (ARE) [35, 52] within the promoter regions of many cytoprotective genes [36]. In addition, Nrf2 has a key role against inflammation thanks to its ability to antagonize the transcription factor nuclear factor-κB (NF-κB) which regulates the expression of inflammatory genes [37].

ARE induction by chemical activators has been shown to protect neuronal cell lines against various oxidative damages induced by dopamine, hydrogen peroxide (H2O2), and glutamate [38–40]. SF has been demonstrated to increase many ARE-dependent antioxidant enzymes in different cell systems [41–43], such as GR, GPx, glutaredoxin (GLRX), thioredoxin (TX), TR, HO1, and NQO1. It has been shown that SF directly interacts with Keap1 by covalent binding to its thiol groups [44].

Negi et al. [45] demonstrated that SF increased the expression of Nrf2 and of downstream targets HO-1 and NQO-1 in Neuro2a cells and the sciatic nerve of diabetic animals. SF was also effective in counteracting oxidative stress induced by antipsychotic drugs in human neuroblastoma SK-N-SH cells, increasing GSH levels and inducing NQO1 activity [46].

Sulforaphane prevented oxidative stress-induced cytotoxicity in rat striatal cultures by raising the intracellular GSH content via an increase in γ-GCS expression induced by the activation of the Nrf2-antioxidant responsive element pathway [47].

It has also been observed that oxidative stress can inactivate peroxiredoxins, an important family of cysteine-based antioxidant enzymes that exert neuroprotective effects in several models of neurodegeneration [48, 53–55]. Interestingly, in both neurons and glia, SF treatment upregulates sulfiredoxin, an enzyme responsible for reducing hyperoxidized peroxiredoxins [56]. SF pretreatment also leads to attenuation of the tetrahydrobiopterin (BH4) induced ROS production thanks to the increase in mRNA levels and enzymatic activity of NQO1 in DAergic cell lines CATH.a and SK-N-BE(2)C [57].

Kraft et al. [58] demonstrated the importance of ARE activation in astrocytes of a mixed primary culture system. They observed that SF induced an ARE-mediated genetic response that is highly selective for astrocytes over neurons and conveys neuroprotection from oxidative insults initiated by H2O2 or nonexcitotoxic glutamate toxicity. Innamorato et al. [59] observed a direct association between the protective effect of SF against oxidative stress induced by lipopolysaccharide with HO-1 induction in BV2 microglial cells.

Oxidative stress induces Ca2+-dependent opening of the mitochondrial inner membrane permeability transition pore (PTP), causing bioenergetic failure and subsequent death in different cell models, including those related to acute brain injury [60–62]. Intraperitoneal injection of rats with a nontoxic level of SF resulted in resistance of isolated nonsynaptic brain mitochondria to peroxide-induced PTP opening [63], and this could contribute to the neuroprotection observed with SF.

BBB damage following oxidative stress has been extensively investigated [64]. Postinjury induction of Nrf2-driven genes by SF treatment attenuated the loss of endothelial cells and tight junction proteins and reduced BBB permeability and cerebral edema [65]. Another study demonstrated that SF administration reduced BBB permeability in a rat subarachnoid hemorrhage model likely through the antioxidative effects of the activated Nrf2-ARE pathway [66].

Less attention has been focused on oxidative damage at the blood-cerebrospinal fluid (CSF) barrier (BCSFB) located at the choroid plexus (CP) epithelium. Even modest changes in the CPs may have a marked impact on the brain. For example, changes in CP function have been implicated in Alzheimer's disease [67]. A study by Xiang et al. [68] demonstrated that SF can protect the BCSFB in vitro from damage caused by H2O2 and reduced H2O2-induced cell death in primary CP epithelial cells and a CP cell line Z310.

Summarizing, the observed protective effects of SF against brain oxidative stress are mainly associated with Nrf2 activation and the resulting upregulation of antioxidant cytoprotective proteins and elevation of GSH (Figure 1).

Figure 1
Proposed mechanism of neuroprotective effects provided by SF through Keap1/Nrf2 transcriptional activation of the antioxidant system. Adapted from [124].

4. Protective Effects of Sulforaphane against Acute Neurodegeneration
4.1. Ischemic Brain Injury

The pathophysiology of ischemic brain injury involves various biochemical mechanisms, such as glutamate-mediated excitotoxicity, the generation of ROS, apoptosis, and inflammation [69]. In adults, brain ischemic insults typically result from stroke or cardiac arrest, while in infants, cerebral ischemia is mediated by complications during labor and delivery, resulting in neonatal hypoxic-ischemic encephalopathy. In both groups, restoring blood flow to the ischemic brain is essential to salvage neurons. However, reperfusion itself causes additional and substantial brain damage referred to as “reperfusion injury.”

In a neonatal hypoxia/ischemia brain injury model, Ping et al. [70] observed that SF significantly increased Nrf2 and HO-1 expression which was accompanied by reduced infarct volume. In particular, SF treatment reduced the number of apoptotic neurons, activated macroglia, and some oxidative parameters such as the amount of 8-hydroxy-2-deoxyguanosine and MDA level. In a similar model of ischemia/reperfusion induced by either oxygen and glucose deprivation or hemin in immature mouse hippocampal neurons, SF treatment activated the ARE/Nrf2 pathway of antioxidant defenses and protected immature neurons from delayed cell death [71]. Zhao et al. [69] demonstrated that delayed administration of a single dose of SF significantly decreased cerebral infarct volume in rats following focal ischemia. Moreover, in rat cortical astrocytes, SF treatment before or after oxygen and glucose deprivation significantly reduced cell death, stimulating the Nrf2 pathway of antioxidant gene expression [72]. In contrast to these data, Porritt et al. [73] showed that SF treatment initiated after photothrombosis-induced permanent cerebral ischemia in mice did not interfere with key cellular mechanisms involved in tissue damage. The authors suggest that the small volume of infarcted cortical tissue resulting from the photothrombosis injury might result in the generation of relatively smaller amounts of ROS and may explain why they did not observe any neuroprotection after SF administration. In addition, Srivastava et al. [74] recorded that the pretreatment of rats with SF decreased the nuclear accumulation of Nrf2 following cerebral ischemia/reperfusion injury. On this topic, the authors speculate that rapid accumulation of SF in the brain and subsequent upregulation of Nrf2 and antioxidant enzymes may reduce the need for the later adaptive increase in Nrf2 expression following stroke.

These lines of evidence indicate that SF may counteract ischemia/reperfusion due to its ability to modulate Nrf2 and intracellular redox signaling.

4.2. Traumatic Brain Injury

Traumatic brain injury (TBI) is defined as damage to the brain caused by external mechanical force [75]. Survivors of TBI are left with long-term disabilities, and even a mild TBI can leave people with cognitive impairments, difficulty in concentrating, headaches, and fatigue [76]. TBI is a complex disease process [77] that results in early phase of mechanical damage of brain tissue and a secondary phase of cellular and molecular events that cause oxidative damage and brain cell death [78, 79]. Despite advances in prevention measures, surgical, and diagnostic techniques, no pharmacological treatment has so far been found to confer neuroprotection by targeting secondary injury mechanisms [76].

Recent studies in a rat model of TBI showed that postinjury administration of SF reduces the BBB impairment and cerebral edema after TBI [65, 80]. In particular, Zhao et al. [80] showed that SF attenuated aquaporin-4 (AQP4) channel loss in the injury core and further increased AQP4 protein levels in the penumbra region at 24 h and 3 days following TBI. In contrast to the early increase of AQP4 levels, the decrease in cerebral edema was observed only at 3 days, confirming the important role of AQP4 channels to clear the water in excess and to maintain the brain water homeostasis [81]. However, the authors suggest that the observed SF neuroprotective effect may be due to a combination of mechanisms that include decreased BBB permeability, enhanced cell survival, and/or increased AQP4 channel levels. In particular, the restoration of AQP4 channel activity prevented the impaired clearance of extracellular potassium with neuronal depolarization and glutamate release. It should be noted that the glutamate release is involved in an important sequel of CNS injury [80]. In the same rat model of TBI, Zhao et al. [65] demonstrated that postinjury administration of SF preserved BBB function through the reduction of endothelial cell markers and tight junction protein loss. These protective effects were mediated by the activity of Nrf2. In particular, SF increased the expression of Nrf2-driven cytoprotective genes such as GSTα3, GPx, and HO-1 in the parietal cortex and brain microvessels. More recent papers confirmed these findings in both rat and mice models of TBI [82]. Interestingly, Dash et al. [83] showed that in addition to vascular protection of SF, postinjury SF treatment preserved neurological function in injured animals. This improvement was demonstrated by enhanced learning and memory and by improved performance in a working memory task. The authors propose that the ability of SF to improve the hippocampal- and prefrontal cortex-dependent cognitive function could be ascribed to its ability to protect the neurons and other cell types of the neurovascular unit from the oxidative damage elicited by TBI. Taken together, these findings suggest that SF may protect against the various pathophysiological consequences of TBI and other neurological traumatic injuries. On this topic, a recent study demonstrated that SF provides neuroprotective effects in the spinal cord after contusive injury [84].

5. Protective Effects of Sulforaphane against Chronic Neurodegeneration
5.1. Alzheimer's Disease

Alzheimer's disease (AD) is the most common neurodegenerative disease that accounts for most cases of dementia experienced by older people and is characterized by a progressive decline in memory and impairment of at least one other cognitive function [85].

This neurodegenerative disease is characterized by the accumulation of amyloid beta (Aβ) peptides that result in oxidative damage, inflammation and increased intracellular calcium levels [86, 87]. Two major hallmarks of AD are the extracellular aggregation of Aβ peptides and the intracellular precipitation/aggregation of hyperphosphorylated Tau (forming neurofibrillary tangles) protein [87]. In particular, Aβ 1–40 and Aβ 1–42 peptides, produced by the cleavage of the precursor protein, can exist in multiple aggregation forms, including soluble oligomers or protofibrils, and insoluble fibrils, which are responsible for various pathological effects [88, 89].

Several studies showed that increased oxidative stress, the impaired protein-folding function of the endoplasmatic reticulum, and deficient proteasome- and autophagic-mediated clearance of damaged proteins accelerated the accumulation of Aβ peptides and Tau protein in AD [90, 91].

In this context, Kwak et al. [92] demonstrated that the neuroprotective effects of SF against oxidative stress, in terms of protein carbonyl formation and cytotoxicity elicited by hydrogen peroxide, could be ascribed to its ability to induce proteasome expression in murine neuroblastoma Neuro2A cells. In similar cellular models, Park et al. [93] confirmed the ability of SF to enhance the proteasome activities and to protect the neuronal cells from Aβ1–42-mediated cytoxicity. More recent studies reported that SF induced the expression of heat shock protein 27, demonstrating that SF-stimulated proteasome activity may contribute to cytoprotection [94]. These data suggest that induction of proteasome by SF may facilitate the clearance of the Aβ1–42 peptides and lead to the improvement of protein misfolding in AD. Kim et al. [95] investigated the potential neuroprotective effects of SF in an Aβ1–40 peptide-induced AD acute mouse model. In particular, they recorded the ability of SF to ameliorate the cognitive function impairment although it did not directly interact with Aβ. These findings reinforce the indirect neuroprotective effects of SF against Aβ toxicity.

5.2. Parkinson's Disease

Parkinson's disease (PD) is an age-related neurodegenerative disease with progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta and with accumulation of neuronal inclusions known as Lewy bodies [96]. The exact etiology of PD remains to be fully elucidated, but the most reliable theories propose either an environmental [97, 98] or a genetic [99] origin, or a combination of both. Genetic studies have demonstrated that α-synuclein protein, a principal component of Lewy body inclusions [100], is a key participant in the pathogenesis of this disorder [101–103]. The exact biological function of α-synuclein and the mechanism by which mutations in this gene lead to neuron loss are still not clear, although it has been observed that an excess of α-synuclein protein can cause DA neuron loss [104].

Overwhelming evidence indicates that oxidative damage induced by ROS participates in the progression of DA neurons. In particular, the metabolism of dopamine (DA) might be responsible for the high basal levels of oxidative stress in the SN. Autooxidation of dopamine leads to the formation of neurotoxic species such as electrophilic DA quinone and ROS including superoxide anion (O2•) and H2O2 [105]. DA quinone is also thought to cause mitochondrial dysfunction [106] and to mediate α-synuclein-associated neurotoxicity in PD by covalently modifying α-synuclein monomer [107] and by stabilizing the toxic protofibrillar α-synuclein [108].

Using a Drosophila model of α-synucleinopathy, Trinh et al. [109] observed that the neuronal death accompanying α-synuclein expression is enhanced by loss-of-function mutations in genes involved in the phase II detoxification pathway, specifically, glutathione metabolism. This neuronal loss can be overcome by pharmacological inducers, including SF, that increase glutathione synthesis or glutathione conjugation activity. They also observed similar neuroprotective effects of SF in Drosophila parkin mutants, another loss-of-function model of PD.

Several in vitro studies showed that SF was able to significantly reduce DA quinone levels in dopaminergic cell lines, such as CATH.a and SK-N-BE(2)C, as well as in mesencephalic dopaminergic neurons, evoked by 6-hydroxydopamine (6-OHDA) and BH4 [110]. In particular, Han et al. [57] demonstrated that SF can protect dopaminergic cells from the cytotoxicity of 6-OHDA and BH4 by removal of intracellular DA quinone, because NQO1 enzyme activity and mRNA level are increased by SF treatment and quinone-modified proteins are decreased.

In addition, DA quinone may yield neurotoxic species following its reaction with cellular thiols to form the 5-S-cysteinyl-dopamine (CysDA) [111–113]. CysDA adducts have been reported in human brain tissue and are elevated in the brains of patients suffering from PD [114]. We have demonstrated that SF is able to protect primary cortical neurons against CysDA-induced injury. In particular, we found that the protection exerted by SF against this neurotoxin is linked to the activation of ERK1/2, to the associated release of Nrf2 from Keap1, and to a subsequent increase in the expression and activity of specific detoxifying phase II enzymes [115]. Moreover, we demonstrated that SF prevented the dopaminergic-like neuroblastoma SH-SY5Y cell death, in terms of apoptosis and necrosis, induced by oxidant compounds, such as H2O2 and 6-OHDA, by its abilities to increase endogenous GSH, enzymes involved in GSH metabolism including GST and GR, and to normalize the intracellular redox status (Figure 2) [116]. Interestingly, we recorded similar in vitro neuroprotective effects also with the erucin generated by bioconversion of the SF suggesting a neuroprotective role of SF metabolites in PD [117].

Figure 2

SF prevents 6-OHDA-induced ROS formation in SH-SY5Y cells. Representative images of SH-SY5Y cells incubated with SF for 24 h and then treated with 6-OHDA for 3 h. At the end of incubation, ROS formation was determined by fluorescence probe, ...

Deng et al. [118] observed that SF inhibited 6-OHDA-induced cytotoxicity in SH-SY5Y cells through increasing Nrf2 nuclear translocation and HO-1 expression in a PI3 K/Akt-dependent manner. Further, other authors confirmed that Nrf2 activation by SF may play an important role in DA neuron protection against 6-OHDA-induced toxicity in rat organotypical nigrostriatal cocultures [119]. As regards in vivo neurodegeneration models, Jazwa et al. [30] demonstrated that SF induced an Nrf2-dependent phase II response in the basal ganglia and protected against nigral dopaminergic cell death, astrogliosis, and microgliosis in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of PD. Further, we reported the ability of SF to exert neuroprotective effects on DA neurons in 6-OHDA-lesioned mice. In particular, these effects may be attributed to SF ability to enhance GSH levels and its dependent enzymes, including GST and GR, and to modulate neuronal survival pathways, such as ERK1/2 [120].

6. Conclusions

Several in vitro and in vivo studies have demonstrated the ability of SF to prevent various neurodegenerative processes that underlie stroke, traumatic brain injury, AD, and PD. The ability of SF to exert neuroprotective effects in different acute and chronic neurodegenerative diseases could be ascribed to its peculiar ability to activate the Nrf2/ARE pathway. Nrf2 is a recent therapeutic target in neurodegenerative diseases because it regulates several genes that have been implicated in protection against neurodegenerative conditions [121, 122]. In this context, SF presents many advantages, such as good pharmacokinetics and safety after oral administration as well as the potential ability to penetrate the BBB and deliver its neuroprotective effects in the central nervous system [123]. Based on these considerations, SF appears to be a promising compound with neuroprotective properties that may play an important role in preventing neurodegenerative diseases.

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Patents : Sulforaphane

Method for extracting multifunctional sulforaphane from broccoli sprouting vegetable
CN101514174

The invention discloses a method for extracting multifunctional sulforaphane from broccoli sprouting vegetable. The method comprises: fresh broccoli sprouting vegetable which grows for 6-10 days is taken as raw material, and is then crushed to be 100 meshes of sieve after being frozen and dried; after that, 100 parts by weight of the processed material is taken to be added with de-ionized water, methylene dichloride, 0.001-0.003 parts by weight of Vc and 0.1-0.3 parts by weight of Na2S, the pH value is adjusted to be 4-6, and the mixed solution is hydrolyzed for 6-10h at 15-35 DEG C; finally, the sulforaphane is obtained after being filtered, washed and purified. As the broccoli sprouting vegetable which grows for 6-10 days is taken as raw material, the method can effectively increase the production rate of the sulforaphane and plays the role of activation for using myrosase to hydrolyze sulpho-glucoside by adding the Vc; meanwhile, sodium sulfide is added for overcoming the interference of Fe in the material, thus improving the production rate of the sulforaphane. The obtained sulforaphane has multiple functions such as cancer resistance, oxidation resistance and the like as well as good application prospect.

The present invention discloses a method for extracting from broccoli sprouts in the multifunction sulforaphane. This method takes growth of 6 to 10 days of fresh broccoli sprouts for raw materials, freeze-dried crushed a 100-mesh sieve; then called 100 parts quality raw learn treated, deionized water, methylene chloride, 0.001 ~ 0.003 parts by mass of Vc, 0.1 ~ 0.3 parts by weight of NaS, adjusted to pH 4-6, in 15 ~ 35 ° C temperature of the hydrolysis of 6 to 10 hours; and finally filtered, washed, purified sulforaphane have. The present invention is selected growth of 6 to 10 days of broccoli sprouts for raw materials can effectively improve the extraction yield of sulforaphane, and by adding Vc myrosinase hydrolysis of glucosinolates activation plays a role, while adding sodium sulfide to overcome raw material Fe interference and improve the yield of sulforaphane, resulting sulforaphane has multiple functions of anti-cancer, anti-oxidation, has good prospects.
A method to extract from broccoli sprouts of multifunction sulforaphane

TECHNICAL FIELD

The present invention relates to a method to extract sulforaphane, and more particularly to a multifunction sulforaphane extracted from broccoli sprouts in Alkyl methods.

Background technique

Sulforaphane also called sulforaphane, formula C6H11NOS2, iso-thiocyanate derivative, relative molecular mass The amount of 177.29, is a potent anti-cancer substances vegetables, which are more present in cruciferous vegetables, and cruciferous to Broccoli with higher levels, it turned broccoli broccoli seed and seedling period were higher than those of mature vegetables. US Johns Paul Talalay Hopkins University School of Medicine published proof, broccoli sprouts (broccoli sprouts) in anti-cancer compounds The content of sulforaphane than mature broccoli (broccoli) 20-30 times more. British scientists in early 1997 once The results show that the anti-cancer vegetables, broccoli and Brussels sprouts contain very rich in glucose iso cyanate salts Sulphur The auxiliary compounds. Gluconeogenesis from Sulphur cyanate decomposes a sulforaphane, and separating from broccoli with Sulforaphane growth gene DNA material, this material implanted in a variety of cabbage and radish, will help people to cancer cells Resistance cells and reduce the risk of cancer. Brassica Johns Hopkins University School of Medicine Chemooprotection Lab found Broccoli contains a lot of iso cyanate Sulphur and Sulphur these iso cyanate excitable Live body's own anti-cancer substances "Phase Two Enzymes". This enzyme can neutralize suspected carcinogen, prevent carcinogens Mass destruction of healthy genes within cells. Japan's Agriculture Research Institute also said isothiocyanate can prevent melanoma cancer Growth. Among them, the most dynamic sulforaphane is a class of isothiocyanates, which have anti-cancer effects of breast cancer in rats Obtained sufficient proof.

The prior art, the preparation method sulforaphane mainly chemical and enzymatic. Chemical method is through chemical synthesis Methods. Stereochemistry in synthesis of chiral synthesis method, this method from the perspective of stereochemistry to produce sulforaphane, Process is simple, but difficult to control, it is seldom used. Sulforaphane, existing research enzymatic production are from the cruciferous vegetables Before sulforaphane extracted body, obtained by hydrolysis mixture containing sulforaphane. The mixture was separated and purified hydrolyzed prepared Sulforaphane; separation and purification commonly used solvent extraction, followed by high performance liquid chromatography (HPLC), gas chromatography and mass spectrometry Binding or reverse HPLC purity identification.
Application No. 200510030467.9 of Chinese invention patent application discloses a brassica vegetable raw material preparation Levin Turnip sulfane approach. This method after brassica seeds, flowers, stems, leaves crushed sulfur mustard plant's own use Nucleotide enzyme hydrolysis under specific pH value, extracted with ethyl acetate, silica gel adsorption, ethyl acetate impurity, isopropanol, ethyl Alcohol, isopropyl alcohol and petroleum ether, or a mixed solution of ethanol and petroleum ether elution sulforaphane, thus effectively improving the sulforaphane Alkane content. This method does not add sulfur mustard exogenous nucleotide enzyme, can reduce production costs, simplify the extraction and purification process. Application No. 200810026202.8 of Chinese invention patent application discloses a broccoli seed extract and its preparing France, the preparation method is hydrolyzed broccoli seeds, degreasing, inactivated and concentrated to give broccoli seed extract, Tim Plus accessories can be obtained health products. The main component of broccoli extract is Isothiocyanates compound, wherein the highest activity and The highest concentrations of sulforaphane. However, these two methods are not versatile sulforaphane extracted for broccoli sprouts, And its extraction methods do not consider the impact of the growth cycle of broccoli sprouts sulforaphane, nor does it consider broccoli sprouts Dish itself affect the mineral content of the extraction yield, therefore, the extraction yield is not high, sulforaphane loss is large.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the disadvantages of the prior art and to provide a less loss of sulforaphane, extraction yield, mention Take a simple extraction procedure versatile method of sulforaphane from broccoli sprouts in.
The purpose of the present invention is realized by the following technical solutions:
A method to extract from broccoli sprouts multifunction sulforaphane, characterized in that it comprises the following steps and processes condition:
(1)

(1) raw material selection and pre-treatment: Take the growth of 6 to 10 days of fresh broccoli sprouts for raw materials, freeze-dried Sifted through a 100 mesh sieve;
(2)

(2) biphasic hydrolysis: Weigh learn step (1) raw materials processed 100 parts by mass, deionized water, methylene Alkoxy, 0.001 to 0.003 parts by mass of Vc, 0.1 ~ 0.3 parts by mass of Na2S, adjusted to pH 4-6, at a temperature of 15 ~ 35 ° C Hydrolysis of 6 to 10 hours; the resulting hydrolysis sulforaphane directly extracted with methylene chloride; the deionized water per 100g Raw materials added 100 ~ 500ml; the methylene chloride is added per 100g of raw materials 100 ~ 200ml;
(3)

(3) filtering, washing and purification: the step (2) the resulting solution was filtered with filter paper, the filtrate was layered, take the methylene Alkoxy alternate layers, the aqueous layer was 2 to 4 times with methylene chloride washings were washed with methylene chloride, the methylene chloride layer with two alternate The combined methylene chloride solution was washed with methylene chloride extracts; After the dichloromethane extracts were concentrated by rotary evaporation over Sephadex LH-20 column chromatography, eluting with acetone, concentrating the eluate lyophilized to give after sulforaphane. Purity by HPLC 75 to 85%.
Said step (1) the growth of broccoli sprouts preferably 7 to 8 days of fresh broccoli sprouts.
Said step (2) of the pH is adjusted phosphate buffer pH.
Hydrolysis of the step (2) is at a speed of 150 ~ 300rpm hydrolysis under magnetic stirring.
Said step (3) obtained after lyophilization sulforaphane also include methanol scheduled after using 0.22μm membrane filtration water system, the Sulforaphane was high purity product storage stand at -20 ° C freezer.
Compared with the prior art, the invention has the following advantages:
(1)

(1) The present inventors have found creative with broccoli sprouts growing increase in the number of days for which the body content of sulforaphane first accumulator Volume increases and then decreases, while the content of sulforaphane to grow 6 to 10 days of broccoli sprouts is maintained at a high level, Especially in growth to a maximum of sulforaphane content on the 7th day of broccoli sprouts. Therefore, the growth of 6 to 10 days to select Zealand flower bud Seedling vegetables as raw materials can effectively improve the extraction yield of sulforaphane.
(2)

(2) different non-polar organic solvent has a great influence on the extraction rate of sulforaphane from the chemical structure sulforaphane Constructions can be seen by the sulforaphane glucosinolate, sulfonic acid oxime and a side chain, whose choice of solvent extraction Can be used ether, methylene chloride, ethyl acetate and chloroform extraction rate of sulforaphane organic solvent with a dielectric constant Increases increases, dichloromethane as extraction solvent extraction rate greater degree higher than the ether, ethyl acetate and chloroform, because This present invention extraction agent sulforaphane choice dichloromethane. For dichloromethane toxicity, since the compound has a very Good volatility, and the rate of photolysis quickly, the initial degradation product of phosgene and carbon monoxide, and then reconverted to carbon Dioxide and hydrochloric acid. So that it can be completely removed in the process of follow-up treatment will not cause accumulation in the product. And the presence of oxygen, It is readily biodegradable, and thus will not cause bioaccumulation.
(3)

(3) of the present invention is the generation of sulforaphane mainly from the hydrolysis of glucosinolates substance precursor generating itself, hydrolyzed The journey takes itself involved in myrosinase, the present invention, by adding an appropriate amount of Vc myrosinase hydrolysis of glucosinolates played a shock Live action, at the same time, the presence of Fe ions in the feedstock will inhibit the enzyme activity mustard, tested, 100g broccoli sprouts Dish iron ion of about 2.3mg, the present invention is removed by Na2S added broccoli sprouts in the Fe ions; it is Consider broccoli sprouts contain myrosinase own characteristics and Fe ions, the present invention is added Vc and Na2S during hydrolysis, Greatly improving the yield of sulforaphane. If the sodium sulfide was added the extract would cause excessive emulsification, resulting in points From difficulties, loss of a small amount of sulforaphane, so experiment sulforaphane yield declined slightly.
(4)

(4) The present invention takes the control of the external environment hydrolysis sulforaphane precursor while solvent extraction methods, namely bipolar Acid Hydrolysis prepared sulforaphane. Preparation by double hydrolysis step sulforaphane, simplifying the process, the maximum To retain the integrity of sulforaphane, and saves energy. This method is easy to realize industrialization, for the realization of sulforaphane Industrial production is significant.
(5)

(5) The present invention has multiple functions of anti-cancer, anti-oxidation from broccoli sprout extract sulforaphane, with good Application prospects.

Detailed description

Below in conjunction with embodiments of the present invention will be further described, it should be noted that the embodiments of the present invention does not constitute Ming requirements to limit the scope of protection.

Example 1

A versatile sulforaphane extracted from broccoli sprouts method, including the steps and process conditions:
(1)

(1) raw material selection and pre-treatment: Take grow seven days fresh broccoli sprouts for raw materials, freeze-dried and then pulverized 100-mesh sieve;
(2)

(2) biphasic hydrolysis: learn said step (1) was treated feedstock 100g, was added 100mL of deionized water, 100mL Methylene chloride, 0.003 Vc, 0.2 of Na2S, with phosphate buffer pH adjusted to pH 5, at a temperature of 28 ° C, Magnetic stirring speed of 150rpm hydrolysis eight hours; pH phosphate buffer is disodium hydrogen phosphate and sodium dihydrogen phosphate mixed Thereof.
(3)

(3) filtering, washing and purification: the step (2) the resulting solution was filtered with filter paper, the filtrate was layered, take the methylene Alkoxy alternate layers, the aqueous layer was washed with methylene chloride three times was washed with methylene chloride, the methylene chloride layer with dichloromethane spare Washings were combined dichloromethane extracts were obtained; the dichloromethane extract was concentrated by rotary evaporation after over Sephadex LH-20 column chromatography Analysis, acetone eluate was concentrated and lyophilized resulting sulforaphane 0.1g, store stand at -20 ° C freezer. A certain amount Sulforaphane after constant volume of methanol, water system with 0.22μm membrane filter, determined by HPLC, sulforaphane purity of 85%.

Example 2

A versatile sulforaphane extracted from broccoli sprouts method, including the steps and process conditions:
(1)

(1) raw material selection and pre-treatment: Take 6 days grow fresh broccoli sprouts for raw materials, freeze-dried and then pulverized 100-mesh sieve;
(2)

(2) biphasic hydrolysis: learn said step (1) was treated feedstock 100g, was added 500mL of deionized water, 200mL Methylene chloride, 0.003g of Vc, 0.1g of Na2S, phosphate buffer with pH adjusted to pH 4, at 35 ° C temperature 6 hours of reflux extraction.
(3)

(3) filtering, washing and purification: the step (2) the resulting solution was filtered with filter paper, the filtrate was layered, take the methylene Alkoxy alternate layers, the aqueous layer was washed 4 times with dichloromethane, the dichloromethane layer was washed with dichloromethane to give the combined alternate Dichloromethane extracts; The dichloromethane extracts were concentrated by rotary evaporation over Sephadex LH-20 column chromatography, eluting with acetone, The eluate was concentrated and freeze-drying the resulting alkyl sulforaphane after lyophilization was sulforaphane. Purity by HPLC was 75%.

Example 3

A versatile sulforaphane extracted from broccoli sprouts method, including the steps and process conditions:
(1)

(1) raw material selection and pre-treatment: Take grow 10 days fresh broccoli sprouts for raw materials, freeze-dried powder Broken through the 100 mesh sieve;
(2)

(2) biphasic hydrolysis: learn said step (1) was treated feedstock 100g, was added 300mL of deionized water, 150mL Methylene chloride, 0.002g of Vc, 0.1g of Na2S, with pH phosphate buffer (disodium hydrogen phosphate and sodium dihydrogen phosphate) Adjusting pH to 6 at a temperature of 15 ° C, under magnetic stirring speed of 300rpm hydrolysis 10 hours;
(3)

(3) filtering, washing and purification: the step (2) the resulting solution was filtered with filter paper, the filtrate was layered, take the methylene Alkoxy alternate layers, the aqueous layer was washed with methylene chloride and the methylene chloride layer was washed twice with dichloromethane, the combined spare obtained Dichloromethane extracts; The dichloromethane extracts were concentrated by rotary evaporation over Sephadex LH-20 column chromatography, eluting with acetone, The eluate was concentrated and lyophilized resulting sulforaphane, Purity by HPLC 82.3%.
Example 4
A versatile sulforaphane extracted from broccoli sprouts method, including the steps and process conditions:
(1)

(1) raw material selection and pre-treatment: take eight days grow fresh broccoli sprouts for raw materials, freeze-dried and then pulverized 100-mesh sieve;
(2)

(2) biphasic hydrolysis: learn said step (1) was treated feedstock 100g, was added 200mL of deionized water, 200mL Methylene chloride, 0.001g of Vc, 0.2g of Na2S ,, with pH phosphate buffer (disodium hydrogen phosphate and sodium dihydrogen phosphate) Adjusting pH to 5.5, at a temperature of 25 ° C, under magnetic stirring speed of 200rpm hydrolysis 9 hours;
(3)

(3) filtering, washing and purification: the step (2) the resulting solution was filtered with filter paper, the filtrate was layered, take the methylene Alkoxy alternate layers, the aqueous layer was washed 3 times with dichloromethane, the dichloromethane layer was washed with dichloromethane to give the combined alternate Dichloromethane extracts; The dichloromethane extracts were concentrated by rotary evaporation over Sephadex LH-20 column chromatography, eluting with acetone, The eluate was concentrated and lyophilized resulting sulforaphane, Purity by HPLC 85.6%.

Example 5

A versatile sulforaphane extracted from broccoli sprouts method, including the steps and process conditions:
(1)

(1) raw material selection and pre-treatment: take nine days grow fresh broccoli sprouts for raw materials, freeze-dried and then pulverized 100-mesh sieve;
(2)

(2) biphasic hydrolysis: learn said step (1) was treated feedstock 100g, was added 400mL of deionized water, 200mL Methylene chloride, 0.002g of Vc, 0.2g of Na2S, with pH phosphate buffer (disodium hydrogen phosphate and sodium dihydrogen phosphate) Adjusting pH to 5.5, at a temperature of 26 ° C, under magnetic stirring speed of 150rpm hydrolysis 10 hours;
(3)

(3) filtering, washing and purification: the step (2) the resulting solution was filtered with filter paper, the filtrate was layered, take the methylene Alkoxy alternate layers, the aqueous layer was washed with methylene chloride and the methylene chloride layer was washed twice with dichloromethane, the combined spare obtained Dichloromethane extracts; The dichloromethane extracts were concentrated by rotary evaporation over Sephadex LH-20 column chromatography, eluting with acetone, The eluate was concentrated and lyophilized resulting sulforaphane, Purity by HPLC 84.8%.
Sulforaphane anticancer effect: 6 Example
Sulforaphane Example 1 was prepared. Effect of sulforaphane to detect prostate cancer cell growth by MTT assay. Its original Li is present in the mitochondria of living cells succinate dehydrogenase, can be yellow MTT is reduced to insoluble blue-violet crystals A Month for (Formazan) and deposited in cells, cell death does not exist in the mitochondrial succinate dehydrogenase having activity, MTT Is not reduced, there is no blue-violet crystals produced. After dissolved in DMSO Formazan, detection on its optical density microplate reader Value, thereby indirectly reflect the number of viable cells. Specific process is as follows:
1, the cells were passaged: after cell culture flasks were passaged covered wall posts. The old culture medium was decanted, added 3-4mL Washed twice in Hanks. Join EDTA- trypsin 2-3mL, at room temperature to digest 5-10min, after joining 3-4mL Fresh medium, digestion was terminated. Centrifuged (1000r / min, 10min), the supernatant was decanted. Join 10-12mL of fresh medium Nutrient solution, mix, spread the culture bottle, typically a transfer II.
2, cryopreservation: cells covered the bottom wall, the frozen cells. The old culture medium was decanted, added 3 ~ 4mL of Hanks Washed twice. EDTA- trypsin was added 2-3mL, at room temperature to digest 5 ~ 10min, then fresh medium was added 3 ~ 4mL Liquid, terminate digestion. Mix, suck out a small part of the cell count. Frozen cells at a concentration of 1 ~ 2 × 106 个 / mL. from Heart (1000r / min, 10min), the supernatant was decanted. Join cryopreservation solution (culture medium: serum :DMSO = 7:2:1), mixing, Dispensed into vials. -20 ° C overnight, then placed in liquid nitrogen at Guankou 30min, then placed in liquid nitrogen tank.
3, cell recovery: recovery of cells needed quickly removed from liquid nitrogen tanks, shaking water bath at 37 ° C 1 ~ 2min, it Completely thawed. The thawed cells into the centrifuge tube, supplemented with fresh medium 4-5mL of. Centrifuged (1000r / min, 10min), The supernatant was discarded. Adding fresh culture medium 6-7mL, placed in the incubator.
Detected by MTT sulforaphane impact on human prostate cancer cell growth.
4, digestion and vaccination
With 0.02% EDTA digestion logarithmic phase of prostate cancer, and containing 10% fetal bovine serum RPMI-1640 medium into a single cell suspension using a hemocytometer counts per well 3 × 10 <3> cells were seeded at 96 Orifice plates in a volume of 200μL.
5, culture: in CO2 incubator 37 ° C, under 5% CO2 and saturated humidity conditions, culture 24h.
6, sulforaphane added: observe the state of cells, to be adherent cells, the cell culture medium was discarded, each well separately Sulforaphane was added to a final concentration of 2μg / mL, 4μg / mL, 8μg / mL, 16μg / mL, the culture was 24μg / mL in 200μL, The control group with the same volume containing 0.5% of anhydrous methanol was treated culture, continuous culture 5 days, the medium was changed every two days.
7, the measurement result: the end of culture, MTT solution was added to each well 20μL in the experimental group and the control group, at 37 ° C followed Continued incubation 4h, termination of culture. Carefully draw hole supernatant was discarded and the supernatant was added DMSO 200μL, oscillation 10min, so purple crystals dissolve, the absorbance of each well (OD) in a microplate reader at 490nm wavelength, record the results. Cancer inhibition rate = (1 OD values in the experimental group / control group OD value) × 100%. Respectively solvent control group and 16μg / mL The sulforaphane sample cell for five days, the fifth day under an inverted microscope when the medium was changed the next day, the cells form the basic growth State of change and change shape, with a digital camera to take pictures. Seen from the MTT assay results, sulforaphane for prostate cancer Has a lot of growing cells inhibited cell growth with different concentrations of sulforaphane treated sample were subject to different processes Rejection of sulforaphane higher the dose, the more obvious inhibition. In the 24μg / mL concentrations, fine human prostate cancer Cell growth in the third day will be subject to more significant inhibition, inhibition rate was 45.48%.
7, antioxidant in broccoli sprouts Sulforaphane Example
Sulforaphane Example 1 was prepared. In Escherichia coli, Staphylococcus aureus, Staphylococcus white, Bacillus subtilis Bacteria are indicator bacteria, bacteriostatic diameter measured using the Oxford Cup six times and averaged.
Sterilization: The test required dish, Oxford cup, metal forceps placed 160 ° C oven dry heat sterilization 90min. Medium, pipette tip into the high-pressure steam autoclave 121 ° C heat sterilization 20min.
Preparation of the test bacteria suspension: picked for the test inoculated in a test tube filled with a nutrient solution in 5mLLB placed in an incubator Bacteria cultured in 37 ° C 24h. As for the 4 ° C freezer after use.
Colony counts: diluted broth to 10 <-1>, 10 <-2>, 10 <-3> 10 <-4> four concentrations, take 0.1mL coated plates at 37 ° C culture 24h. Observe the situation colonies colony count, the number of colonies concentration select alternate 10 <6> cfu / mL in.
Antibacterial: were taken with a pipette various test bacteria 0.1mL, uniformly coated on the plate, with no visible water droplets Accurate, bacteriostatic test immediately. Oxford with sterile forceps into the cup medium were injected with different concentrations 100uL Levin Turnip sulfane preparation liquid, sterile water to make a blank and a control antibiotics, the plate upside down on tissue culture incubator 37 ° C Yang 24h, remove the inhibition zone diameters were measured and photographed.
Results: were prepared at a concentration of sulforaphane 4ug / mL, 8ug / mL, 16ug / mL and 24ug / mL were gold grape Cocci, white aureus, Escherichia coli, a common bacterium Bacillus subtilis antimicrobial effect, the results shown in Table 1.

Table 1 inhibitory effect of sulforaphane
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As can be seen from Table 1, the inhibitory effect of sulforaphane staphylococcus aureus, Staphylococcus white is very obvious, for hay Bacillus antibacterial better, E. coli has some inhibitory effect.

Natural drug extract
CN103385989

The present invention relates to a natural medicinal extracts, the natural medicinal extracts concentrated decoction made Alismataceae radish seed extract, alisol B acetate and sulforaphane by 2-5: 3-7: 5-8 The composition ratio of the composition by the cancer chemotherapy related diarrhea have excellent efficacy and can also play a synergistic effect of cancer chemotherapy.

TECHNICAL FIELD

The present invention relates to a natural medicinal extracts, and more particularly relates to a concentrated decoction Alismataceae radish seed extract, alisol B acetate and sulforaphane composed of natural medicinal extracts, the extraction of natural medicine preparation method thereof and extract natural medicine for the treatment of cancer and synergies from the manufacture of a medicament to reduce cancer chemotherapy-associated diarrhea in use.

Background technique

Cancer is currently endanger human life, health, one of the major diseases, which treatment including surgery, chemotherapy, radiation therapy, medical treatment, and the effect of chemotherapy in the treatment of cancer is particularly prominent. Chemotherapeutic drugs alone or in combination with a patient will cause diarrhea and other side effects, diarrhea after chemotherapy known as chemotherapy-associated diarrhea. In recent years, along with chemotherapy drugs such as 5-fluorouracil, taxanes, hydroxycamptothecin, irinotecan, capecitabine, etc. The advent and popularization and application of chemotherapy-associated diarrhea incidence increased year by year, and if not active treatment, patients easily lead to dehydration, malnutrition, serum electrolyte imbalance, acid-base balance, not only for the rehabilitation of the body's tremendous disadvantage, but also to bring some difficulties periodic chemotherapy, so the chemotherapy-associated diarrhea effective therapy has become an important part of the overall treatment of patients with cancer.

Chemotherapy-associated diarrhea on their clinical performance, undoubtedly medicine "diarrhea" category, in medicine for research Diarrhea, impressive, but there is no fight cancer chemotherapy can relieve diarrhea and synergy of medicine . Therefore, to develop a high efficiency and low toxicity, low cost, easy to spread can be used to alleviate cancer chemotherapy-associated diarrhea and act natural synergy with chemotherapy in clinical medicine to become a serious problem.
Alisma only contained in the "Shen Nong's Herbal Classic" as a top grade, stating that his "sweet, warm, tonic win, in addition to cold and heat evil, deficiency of power, long muscles, eyes and ears Jiufu smart. Indications spleen diarrhea. Radish Seed is a cruciferous plant radish Raphanus sativus L. Dry mature seeds can be used for the treatment of diet stagnation, abdominal pain, bloating Indigestion Xie Li, in recent years, Alisma and radish seed anticancer effects are confirmed, the present inventors have plenty of each formulation proportion of research and experiment, finally won the best pharmaceutical compositions of the present invention, the ratio is innovative composition, and proved its excellent efficacy of cancer chemotherapy-associated diarrhea and can simultaneously chemotherapeutic drug, thereby completing the present invention.

SUMMARY OF THE INVENTION

A natural medicines of the present invention provides an extract from Alisma radish seed extract concentrated decoction, alisol B acetate and sulforaphane weight by 2-5: 3-7: 5-8 composition ratio.

It said natural medicinal extracts, which concentrated decoction Zexie radish seed extract, alisol B acetate and the amount of sulforaphane ratio is 5: 7: 8.

Among them, Alisma radish seed extract concentrated decoction, alisol B acetate obtained can be purified by conventional methods known to a person skilled in the art, the use of these ingredients obtained by conventional methods or the use of commercially available chemical composition natural medicines of the present invention can achieve the same effect.

Preferably, the above production method to extract natural medicine alisma radish seed extract was concentrated decoction is:
According to the ratio of compatible 1-4:1 take Alisma, radish seed, soaked in distilled water, heating boiling, filtering, plus dregs distilled water, soak the same method, boiling, filtered, and the filtrate was mixed twice, heating concentrated, super membrane filtration, dried under reduced pressure, that is.

Said natural medicine extracts alisol B acetate methods of preparation are:

The Alisma meal percolation with 80% ethanol, 10% ethanol recovery, the oily extract obtained by a silica gel column, with a volume ratio of 10: 1 petroleum ether - ethyl acetate after, then the solvent ratio of 10 : 3 petroleum ether - ethyl acetate, thin layer chromatography, fractions were collected to give crystals with petroleum ether - ethyl acetate to obtain alisol B acetate.

It said natural medicine extract sulforaphane preparation method is:

Grated radish seed was added an excess of hexane, and digested with phosphate buffer pH 7.0, were added methylene chloride and extracted three times, the combined solvent, the organic solvent was removed under reduced pressure, the residue was dissolved in a volume fraction of 10% ethanol solution , and extracted three times with n-hexane to remove the oil, then the ethanol phase was extracted three times with dichloromethane, the combined solvents, i.e., methylene chloride was removed by rotary evaporation.

The present invention also provides the natural medicinal extracts of preferred methods of preparation, including:
（1）

(1) Preparation of radish seed extract concentrated decoction Alismataceae
According to the ratio of compatible 1-4:1 take Alisma, radish seed, soaked in distilled water, heating boiling, filtering, plus dregs distilled water, soak the same method, boiling, filtered, and the filtrate was mixed twice, heating concentrated, super membrane filtration, dried under reduced pressure, that is.
（2）

(2) Preparation of B acetate Orientalol alcohol
The Alisma meal percolation with 80% ethanol, 10% ethanol recovery, the oily extract obtained by a silica gel column, with a volume ratio of 10: 1 petroleum ether - ethyl acetate after, then the solvent ratio of 10 : 3 petroleum ether - ethyl acetate, thin layer chromatography, fractions were collected to give crystals with petroleum ether - ethyl acetate to obtain alisol B acetate.
（3）

(3) Preparation of sulforaphane
Grated radish seed was added an excess of hexane, and digested with phosphate buffer pH 7.0, were added methylene chloride and extracted three times, the combined solvent, the organic solvent was removed under reduced pressure, the residue was dissolved in a volume fraction of 10% ethanol solution , and extracted three times with n-hexane to remove the oil, then the ethanol phase was extracted three times with dichloromethane, the combined solvents, i.e., methylene chloride was removed by rotary evaporation.
（4）

(4) pro rata to select Alismataceae radish seed extract concentrated decoction, alisol B acetate and sulforaphane, uniformly mixed, that is, too.
Preparation method of the natural medicinal extracts, further comprising: the step (4) the product obtained by conventional pharmaceutical means further contains natural medicinal extracts made from 100mg or 200mg size capsules or tablets.

The present invention is a natural medicinal extracts anti chemotherapy-associated diarrhea and suppression of tumors beneficial effects as follows:

1, the present invention is a natural medicinal extracts, in irinotecan cause diarrhea model study in mice showed that: for irinotecan-induced diarrhea in mice has obvious antagonism response.

2, prove that this natural medicinal extracts can enhance the inhibition of tumor chemotherapy drugs, using a mouse tumor model to observe the natural medicinal extracts and cyclophosphamide in vivo inhibition of tumor-bearing mice. Studies have shown that: the natural medicinal extracts can significantly increase cyclophosphamide for inhibition of tumor.

3, the present inventors to the original alisma radish seed extract concentrated decoction, alisol B acetate in combination with sulforaphane, and through a lot of experiments to determine the amount of the ratio between these components, so that the The composition play a good synergy, get unexpected results. After repeated experiments we study, the proportion of the components of natural medicine extract of the present invention is between 2-5: 5-8 produced a synergistic effect when unexpected, synergistic obvious side effects play: 3-7 very good results, especially in the proportion of the amount of each component is 5: 7: 8:00 particularly evident, this is the present inventors have paid a lot of creative work and obtained by those of ordinary skill in just simply can not get this through disclosure seed ratio ranges and optimal dosage ratio.

4, the natural medicinal extracts concentrated decoction Alismataceae radish seed extract, alisol B acetate and sulforaphane each component can be obtained by conventional means, including components such as acetic acid alisol B ester sulforaphane also commercially available, such a composition itself get convenient, low-cost results. This is also the inventor by both the excellent efficacy and a higher industrial applicability of the product have acquired a lot of creative work.
detailed description
The following examples and test examples further illustrate the present invention, natural medicine extract preparation, and their use in anti-diarrhea and anti-tumor benefits.

implementation plan

Preparing various extracts of: a first step

(1) Preparation of radish seed extract concentrated decoction Alismataceae
According to the ratio of compatible 1-4:1 take Alisma, radish seed, soaked in distilled water, heating boiling, filtering, plus dregs distilled water, soak the same method, boiling, filtered, and the filtrate was mixed twice, heating concentrated, super membrane filtration, dried under reduced pressure, that is.
（2）

(2) Preparation of B acetate Orientalol alcohol
The Alisma meal percolation with 80% ethanol, 10% ethanol recovery, the oily extract obtained by a silica gel column, with a volume ratio of 10: 1 petroleum ether - ethyl acetate after, then the solvent ratio of 10 : 3 petroleum ether - ethyl acetate, thin layer chromatography, fractions were collected to give crystals with petroleum ether - ethyl acetate to obtain alisol B acetate.
（3）

(3) Preparation of sulforaphane
Grated radish seed was added an excess of hexane, and digested with phosphate buffer pH 7.0, were added methylene chloride and extracted three times, the combined solvent, the organic solvent was removed under reduced pressure, the residue was dissolved in a volume fraction of 10% ethanol solution , and extracted three times with n-hexane to remove the oil, then the ethanol phase was extracted three times with dichloromethane, the combined solvents, i.e., methylene chloride was removed by rotary evaporation.

Step two: Formulation of

（1）Preparation of the present invention, natural medicine extract capsules (1)
Prepared as described above were weighed steps Alismataceae radish seed decoction concentrated extract 25mg, alisol B acetate 35mg and sulforaphane 40mg, while weighed starch 395mg, magnesium stearate 5mg, uniformly mixed, filled into capsules.

(2) Preparation of the present invention, natural medicine extract tablets

Prepared as described above were weighed steps Alismataceae radish seed decoction concentrated extract 25mg, alisol B acetate 35mg and sulforaphane 40mg, while weighed starch 295mg, lactose 100mg, uniformly mixed with wet granulation, dried, granulated and 5mg of magnesium stearate, compressed into tablets.

Efficacy and pharmacological profile:

In the following test examples composition: Alismataceae radish seed extract concentrated decoction, alisol proportion of the amount of sulforaphane B acetate of 5: 7: 8. The inventors of the above-mentioned components used in a proportion of 2-5: 3-7: ratio between the portfolio composition of a variety of 5-8 were obtained when the same experiment, were found in such amount in the range of natural medicinal extracts can get a good synergistic effect on cancer chemotherapy-associated diarrhea can play a very good effect and a synergistic effect of cancer treatment. The following is a pharmacodynamic data obtained in the preferred embodiment:

Test Example 1: Pharmacodynamic study of natural medicinal extracts therapeutic effects of chemotherapy-associated diarrhea
1, the test material
（1）

(1) Alisma, radish seed
（2）

(2) before the mice were male, the experiment were fed ad libitum water.
2, test methods:
The mice were randomly divided into five groups, each group 5, A group of model group, group B Alismataceae radish seed decoction concentrated extract 10g.kg <-1> (containing the equivalent amount of crude drug), group C alisol B acetate 10g.kg <-1> (containing the equivalent amount of crude drug), D group sulforaphane 10g.kg <-1> (containing the equivalent amount of crude drug), E group of the present invention natural medicinal extracts 10g.kg <-1> (containing the equivalent amount of crude drug), irinotecan was administered intraperitoneally (75mg / kg), 1 once a day for four days, to copy the model of chemotherapy-associated diarrhea. B, C, D, E group fed continuously with the corresponding extract eight days, the mice were placed in metabolic cages, cage bottom spread a white paper to observe the stool, and the mice were observed perianal conditions and fecal contamination of the tail, combined with a cotton swab to stimulate defecation were observed twice daily record. The degree of diarrhea standard rates as follows: 0: normal stool or no; 1 point: mild diarrhea, stool visible light squishy; 2 points: moderate diarrhea, stool wetter without molding. And mild perianal coloring; 3 points, severe diarrhea, watery and accompanied by severe perianal coloring.
3, the experimental results
Compared with the model group, natural medicines of the present invention extract significantly reduced the incidence of diarrhea. The results are shown in Table 1.
Table associated diarrhea incidence and impact of a score of mice treated compositions of this invention
[Image]
Note: a crude drug containing the equivalent amount
Pharmacodynamic study of natural medicinal extracts of the present invention the anti-tumor effect of: Test Example 2
1, the experimental material
（1）

(1) Kunming mice, weighing 20 ± 2g, free access to food before the experiment drinking, 24h natural light.
（2）

(2) cyclophosphamide, Jiangsu Hengrui Medicine Co., Ltd. pharmaceutical products.
2, test methods:
S 180 sarcoma in mice growth inhibition test
Using passaging 5-7 days, the growth of good milky ascites diluted with an appropriate amount of sterile saline tumor cell suspension, cell number of 10 <7> / mL, n = 5, right armpit of each mouse inoculated subcutaneously 0.2mL ( Tumor cells containing 2 × 10 <6> a). Inoculation next day randomized experimental group of natural medicines of the present invention will extract into (200,100mg / mL) both dose groups, each mouse gavage 0.5 ml, while giving cyclophosphamide 10g · kg <-1> · d <-1>; the negative control group was given 0.5 mL of distilled water; positive drug group were given cyclophosphamide 10g · kg <-1> · d <-1>, administered continuously for 10 days. Regular feeding, drinking water, food limitation. Withdrawal next day, all the mice were killed off the cervical spine, stripping subcutaneous tumor mass of solid tumors and weighed according to the following formula to calculate the tumor inhibition rate (%):
Tumor inhibition rate = [control group mean tumor weight (C) - the experimental group mean tumor weight (T)] / average tumor weight in the control group (C)
3, the test results
The present invention is a natural medicinal extracts the low and high dose groups can enhance the effect of cyclophosphamide agent and the degree of inhibition of S 180, in particular the role of the strongest in the high-dose group. (Table 2)
Table natural medicinal extracts two invention for inhibition of tumor S 180 mice

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