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ActiVin®

(IH636) RESEARCH OVERVIEW

ActiVin®, Dry Creek Nutritions' novel IH636 grape seed proanthocyanidin extract, is a nutritional ingredient used in a wide variety of dietary supplements and nutraceutical food, beverage and cosmetic products. It is also the most actively researched grape seed extract on the market today. Over the past two years, more than 60 research presentations, publications and abstracts have shown that ActiVin possesses a wide range of antioxidant, chemoprotective and biological properties, including studies showing that ActiVin: Following is a comprehensive overview of the independent university research performed exclusively on ActiVin, including a complete list of references.

STRESS RELATED RESEARCH
ANTIOXIDANT RESEARCH
TOBACCO RESEARCH
CANCER RESEARCH
CHEMOTHERAPY RESEARCH
CARDIOVASCULAR RESEARCH
HYPERTENSION RESEARCH
OTHER RESEARCH

STRESS RELATED RESEARCH

Activin Against Chronic Pancreatitis in Humans

This study reports two cases in which patients suffering from chronic pancreatitis symptoms were ameliorated with grape seed proanthocyanidin extract (ActiVin) after traditional therapy had failed. ActiVin was found to reduce both pain index and incidence of vomiting in these patients.

—Banerjee B, et al., Beneficial Effects of Grape Seed Proanthocyanidin Extract in the Treatment of Chronic Pancreatitis, American Journal of Gastroenterology, 93: Abstract 175, 1653, 1998; Banerjee B, et al., Grape Seed Proanthocyanidin Extract for the Treatment of Chronic Pancreatitis, Free Radical Biology & Medicine, 25: Abstract 264, S93, 1998; Banerjee B and Bagchi D, Beneficial Effects of a Novel IH636 Grape Seed Proanthocyanidin Extract in the Treatment of Chronic Pancreatitis. Digestion (submitted).

Acute and Chronic Stress Study

The protective ability of ActiVin against acute and chronic stress-induced gastric and intestinal mucosal lipid peroxidation, DNA fragmentation and membrane microviscosity were determined in rats, and correlated with increased production of oxygen free radicals. ActiVin provided significant protection against acute and chronic stress-induced gastrointestinal oxidative injury as demonstrated by reduced lipid peroxidation, DNA damage and membrane microviscosity.

—Bagchi M, et al., Acute and Chronic Stress-Induced Gastrointestinal Injury in Rats, and Protection by a novel IH636 Grape Seed Proanthocyanidin Extract (GSPE), Free Radical Biology & Medicine, 25: Abstract 235, S83, 1998; Bagchi M, et al., Acute and Chronic Stress-Induced Oxidative Gastrointestinal Injury in Rats, and the Protective Ability of a Novel Grape Seed Proanthocyanidin Extract, Nutrition Research, 19:1189-1199, 1999.

ANTIOXIDANT RESEARCH

• A potent free radical scavenger and inhibitor of free radical-induced cellular damage, including lipid peroxidation and DNA fragmentation (in vitro & in vivo).
• Bioavailable and a far more powerful antioxidant than vitamins E, C and beta-carotene, as well as a combination of vitamins E and C (in vivo).
• Protects against H2O2-induced oxidative injury to macrophage (white blood) and brain cells (in vitro).
• Enhances vitamin C protection against LDL and VLDL oxidation (in vitro).

Grape seed proanthocyanidins are natural antioxidants, which are known to possess a broad spectrum of biological, pharmacological and chemoprotective properties against free radicals and oxidative stress. We have assessed the concentration- or dose-dependant free radical scavenging abilities of ActiVin®, a novel IH636 grape seed proanthocyanidin extract, in both in vitro and in vivo models, and compared these data with vitamins C, E and b -carotene. ActiVin demonstrated excellent concentration/dose-dependent inhibitions in both in vitro and in vivo experiments.

ActiVin In Vitro Study

The free radical scavenging abilities (RSA) of ActiVin, vitamin E and vitamin C against biochemically generated superoxide anion and hydroxyl radical were assessed in vitro at varying concentrations via cytochrome c reduction and chemiluminescence response. Chemiluminescence is a general assay for the production of reactive oxygen species, while cytochrome c reduction is a specific assay for superoxide anion. At 50 mg/l, ActiVin demonstrated 84% and 98% greater RSA against superoxide anion and hydroxyl radical, respectively, as compared to natural vitamin E, and at 100 mg/l, ActiVin demonstrated 439% and 575% greater RSA against superoxide anion and hydroxyl radical, respectively, as compared to vitamin C.

—Bagchi D, et al., Oxygen Free Radical Scavenging Abilities of Vitamins C and E, and a Grape Seed Proanthocyanidin Extract In Vitro, Research Communications in Molecular Pathology and Pharmacology, 95:170-189, 1997.

ActiVin In Vivo Study

The protective abilities of ActiVin, vitamin E, vitamin C, beta-carotene and a combination of vitamins E and C against TPA-induced lipid peroxidation and DNA fragmentation in the brain and liver tissues of mice, as well as against free radical production in the peritoneal macrophages of mice, were assessed in vivo. TPA (12-O-tetradecanoylphorbol-13-acetate) is a well-known inducer of free radicals and tumor promotion in living organisms. Pretreatment of mice with ActiVin (100 mg/kg), vitamin E (100 mg/kg), vitamin C (100 mg/kg), beta-carotene (50 mg/kg) and a combination of vitamins E and C (100 mg/kg each) decreased TPA-induced free radical production in peritoneal macrophage cells by 71%, 43%, 16%, 17% and 51%, respectively, via chemiluminescence response, and 69%, 32%, 15%, 18% and 47%, respectively, via cytochrome c reduction as compared to controls. Pretreatment of mice with the same dosages of ActiVin, vitamin E, vitamin C, beta-carotene and a combination of vitamins E and C decreased TPA-induced DNA fragmentation by 50%, 31%, 14%, 11% and 40%, respectively, in brain tissue, and 47%, 30%, 10%, 11% and 38%, respectively, in liver tissue, while lipid peroxidation was reduced by 61%, 45%, 13%, 8% and 48%, respectively, in brain tissue, 46%, 36%, 12%, 7% and 39%, respectively, in liver mitochondria, and 59%, 47%, 14%, 12% and 53%, respectively, in liver microsomes compared to controls. Pretreatment of mice with ActiVin (25, 50 and 100 mg/kg) resulted in a significant dose-dependent inhibition of TPA-induced free radical production in peritoneal macrophage cells, and lipid peroxidation and DNA fragmentation in brain and liver tissues compared to controls.

—Bagchi D, et al., Protective Effects of Grape Seed Proanthocyanidins and Selected Antioxidants Against TPA-Induced Hepatic and Brain Lipid Peroxidation and DNA Fragmentation, and Peritoneal Macrophage Activation in Mice, General Pharmacology, 30:771-776, 1998.

The results of these in vitro and in vivo experiments demonstrate that ActiVin is bioavailable to these vital organs and tissues, and provides significantly greater protection against oxygen free radicals and free radical-induced lipid peroxidation and DNA damage than vitamins E, C and beta-carotene, as well as a combination of vitamins E and C.

Laser Scanning Confocal Microscopy Study

Laser Scanning Confocal Microscopy (LSCM) is a novel technique used to assess free radical-induced modulation of intracellular oxidized states as an index of oxidative damage to cultured cells at a single cell level, and to determine the protective effect of a given antioxidant. This LSCM study was designed to assess the protective ability of ActiVin against hydrogen peroxide (H2O2)-induced oxidative stress in cultured macrophage J774A.1 and neuroactive PC-12 adrenal pheochromocytoma cells. The overall intracellular oxidized states of these cells were measured following incubation with H2O2 and/or ActiVin at an excitation wavelength of 513 nm by LSCM using 2,7-dichlorofluorescein diacetate as the probe. Signals were quantitated by integrating fluorescence over a user defined cell number. Approximately 5.8- and 4.5-fold increases in fluorescence intensity were observed following incubation of macrophage J774A.1 and neuroactive PC-12 cells with 0.5 mM H2O2 for 24 hr, respectively. Pretreatment of the macrophage J774A.1 cells with 50 mg/l and 100 mg/l ActiVin decreased H2O2–induced fluorescence intensity by 36% and 70%, respectively, while under these same conditions 50% and 70% decreases, respectively, in fluorescence intensities were observed in neuroactive PC-12 cells. These results indicate that ActiVin can significantly protect against hydrogen peroxide-induced oxidative stress in cultured macrophage J774A.1 and neuroactive PC-12 cells.

—Bagchi D, et al., Hydrogen Peroxide-Induced Modulation of Intracellular Oxidized States in Cultured Macrophage J774A.1 and Neuroactive PC-12 Cells, and Protection by a Novel Grape Seed Proanthocyanidin Extract, Phytotherapy Research, 12:568-571, 1998.

Biological Role on Diverse Multicellular Organisms

Naturally occurring proanthocyanidin antioxidants have been reported to possess a broad spectrum of biological, pharmacological and medicinal properties against free radicals and oxidative stress. In our previous studies, IH636 proanthocyanidin extract (GSPE, commercially known as ActiVin) demonstrated excellent concentration- and dose- dependent free radical scavenging abilities both in vitro and in vivo models, and provided significantly better free radical scavenging ability compared to vitamins C, E, and b-carotene. GSPE demonstrated significant cytotoxicity towards human breast, lung and gastric cancer cells, while enhancing the growth and viability of normal cells. Additionally, GSPE protected against tobacco-induced apoptotic cell death in human oral keratinocytes by modulating the expression of p53 genes, and also protected human Chang epithelial cells against chemotherapeutic drugs idarubicin (Ida) or 4-hydroxyperoxycyclophosphamide (4-HC) induced cytotoxicity. Ida or 4-HC-induced apoptotic cell death was significantly reduced in the presence of GSPE. The expression of Bcl-2 gene was significantly increased, while the expressions of p53 and c-myc genes were significantly decreased in GSPE-treated cells. These results suggest that the chemoprotective effects of GSPE are mediated probably by modulation of bcl-2 and/or p53 gene expression. Recently, we demonstrated that GSPE pre-exposure protects acetaminophen-induced hepatotoxicity and nephrotoxicity in mice. These anti-toxic properties may be due to its inhibitory effects on drug metabolizing enzymes. Along this line, we found that 100 and 250 mg/ml of GSPE inhibited aniline hydroxylation 40% and 60%, respectively, in uninduced microsomes, and 25% and 50%, respectively, in induced microsomes, indicating GSPE’s ability to influence cytochrome P450 2E1 activity. In the present study, the cytoprotective ability of GSPE pre-exposure was examined on doxorubicin-induced cardiotoxicity, amiodarone-induced pulmonary toxicity, dimethylnitrosamine-induced splenotoxicity, CdCl2-inducd nephrotoxicity and MOCAP-induced neurotoxicity. In each experiment, half of the test animals (mice) were orally fed GSPE for seven days prior to toxicant exposure, while the other half received no GSPE. Parameters of analysis included changes in serum chemistry (ALT, BUN, and CK), histopathology and integrity of genomic DNA. Results indicate that 7-day GSPE pre-exposure prior to the drugs/toxicants such as doxorubicin, amiodarone, dimethylnitrosamine and CdCl2 provided near complete protection in terms of serum chemistry changes (ALT, BUN, and CK), and abolished both forms of cell death, e.g., apoptosis and necrosis. In addition, DNA damage in various tissues triggered by these agents was significantly reduced. Histopathological examination of multiple organs reflected the pattern of serum chemistry and DNA changes. Surprisingly, MOCAP exposure showed symptoms of severe neurotoxicity coupled with serum chemistry changes in the absence of any significant genomic change or brain pathology, where GSPE provided only partial protection. Taken together, these results suggest that GSPE may be beneficial and protect multiple organs from drug and chemical-induced toxic assaults.

–Bagchi D, et al., Recent Advances in Grape Seed Proanthocyanidin Research: Its Biological Role in Diverse Multicellular Organisms. 1999 SFRR Europe Winter Meeting on Bio-Flavonoids & Polyphenols in Health & Disease, Dinard, France, Abstract OP25, 1999.

Oxygen Free Radical Scavenging Properties of Proanthocyanidins

Proanthocyanidins are a group of phenolic bioflavonoids found in fruits, vegetables, seeds and bark. We compared oxygen free radical (OFR) scavenging properties of seven widely used proanthocyanidin products: ActiVin, Berkem Grape Seed, Bio Vin Plus, Leucoselect, OptiPure, Polyphenolics and Pycnogenol. OFR scavenging ability was examined by adding the same amount (10 mg/ml) of various proanthocyanidins to the OFR generating systems. Superoxide radicals (O2-) were generated by the action of xanthine on xanthine oxidase. Irradiating coumarin-3-carboxylic acid (CCA) with 137Cs–ray produced hydroxyl radicals HO) and peroxyl radicals were generated by dissolving 2,2k-azobis (2-amidinopropane) dihydrochloride (AAPH) in PBS buffer. O2- scavenging activity was studied by determining lag period to completely exhaust the antioxidant ability of the compounds. OH scavenging activity was tested with their abilities to reduce CCA while peroxyl radical scavenging ability was determined by their ability to remove peroxyl radical. The O2-, OH and peroxyl radical scavenging activities were compared against ascorbate (20 mM), dimethyl sulfoxide (1 mM) and Trolox (100mM), respectively. All the proanthocyanidins tested scavenged O2-, OH and peroxyl radicals. The potency of O2- scavenging activity was in the order of ActiVin> Berkem Grape Seed> Leucoselect> OptiPure/ Polyphenolics> Pycnogenol> Bio Vin Plus while those for OH and peroxyl radicals were ActiVin/Pycnogenol> Polyphenolics> OptiPure>Berkem/Bio Vin Plus> Leucoselect, and Polyphenolics/Leucoselect/Berkem Grape Seed/Bio Vin Plus> ActiVin/OptiPure> Pycnogenol, respectively. The results indicate that proanthocyanidins, irrespective of source, are potent scavengers of OFR.

–Maulik G, et al., Oxygen Free Radical Scavenging Properties of Proanthocyanidins. Free Radical Biology & Medicine, 27(Suppl. 1): Abstract 90, S39, 1999.

TOBACCO RESEARCH

Tobacco Apoptosis Study

• Inhibits tobacco-induced oxidative damage and cell death significantly greater than vitamins E and C, as well as a combination of vitamins E and C (in vitro).

• Protects against acetaminophen-induced liver and kidney toxicity (in vivo).

• Provides protection against multiple organ toxicities induced by Amiodarone (lung), dimethylnitrosamine (spleen), cadmium chloride (kidney) and MOCAP (brain) (in vivo).

• Enhances detoxification in vivo by inhibiting microsomal cytochrome P450 2E1 isozymes.

Free radicals that appear during physiological processes may lead to programmed cell death (apoptosis) in some pathological conditions, such as when free radical production exceeds the antioxidant levels in the tissue. In other words, antioxidants can suppress apoptosis induced by excess free radical production. Previous studies have demonstrated that tobacco induces dramatic apoptopic cell death in cultured human oral keratinocyte (mucosal) cells. Approximately 9%, 29% and 35% apoptopic cell death were observed in human oral keratinocyte cells following treatment with 100, 200 and 300 m g/ml of tobacco extract, respectively. Recently, the protective ability of ActiVin, a novel IH636 grape seed extract, was assessed against tobacco-induced apoptosis in human oral keratinocyte cells using Flow Cytometry. Pretreatment of the 300 m g/ml tobacco-treated cells with 100 m g ActiVin/ml reduced tobacco-induced apoptopic cell death by approximately 85% in human oral keratinocyte cells, while pretreatment of the 300 m g/ml tobacco-treated cells with a combination of vitamins E and C (75 m M each) reduced tobacco-induced apoptopic cell death by only 46%. —Bagchi M, et al., Smokeless Tobacco, Oxidative Stress, Apoptosis and Antioxidants in Human Oral Keratinocytes, Free Radical Biology & Medicine, 26:992-1000, 1999.

Modulation of p53 Gene

The p53 gene is a tumor suppressor and cell cycle regulatory gene. Mutations of the p53 gene occur in high frequency in most forms of cancer. Modulation of p53 gene expression and apoptopic cell death of human oral keratinocytes increases with increasing concentrations of smokeless tobacco extract (STE). In the present study, human oral keratinocytes were treated with STE (0-300 mg/ml) for 24 hr, and changes in the expression of p53 gene were assessed by RT-PCR (reverse transcriptase-polymerase chain reaction) (b-tubulin was used as the housekeeping gene). Approximately a 2.0-fold increase in p53 gene expression was observed following incubation of the cells with 100 mg/ml of STE, beyond which the expression of p53 dramatically decreased with higher concentrations, confirming increased apoptopic cell death with a higher concentrations of STE. Furthermore, the comparative abilities of vitamin C (75 mM), vitamin E (75 mM), a combination of vitamins C plus E (75 mM each) and ActiVin (100 mg/ml) were determined. The results demonstrate that STE significantly modulates p53 gene expression, while these antioxidants reduced STE-induced changes by 22-46%. ActiVin demonstrated significantly better protection as compared to vitamins C and E, individually and in combination.

—Bagchi M, et al., Smokeless Tobacco Induced Modulation of p53 Gene and Protective Effect of Antioxidants, 38th Annual Meeting of the Society of Toxicology, 48(Suppl. 1): Abstract 307, 65, 1999.

Protective Effects of Vitamins C and E and Activin on Tobacco Induced Oxidative Stress and Modulation of bcl-2, p53 and cmyc Genes

The oral use of moist tobacco products chewing tobacco has greatly increased in recent years. The use of smokeless tobacco is associated with the cancers of the mouth, lip, nasal cavities, esophagus and gut. Oral cancer accounts for 3% of all the cancers in the U.S.A. and is the seventh most common cancer. The p53 gene is defined as a tumor suppressor gene. Indeed, mutations in p53 were found to occur in high frequency in most of the common types of human cancer. Stabilization and activation of p53 following variety of genotoxic insults result in the execution of its biological activities. Antioxidants have been shown to demonstrate a broad spectrum of biological, pharmacological, and chemopreventive properties against free radicals and oxidative stress, and may delay an onset of carcinogenesis. Previous studies in our laboratories have demonstrated that ActiVin, a novel grape seed proanthocyanidin extract (GSPE), exhibits excellent free radical scavenging ability both in vitro and in vivo models, and potentiates selective cytotoxicity toward human breast, lung, and gastric cancer cells. Previously, we have reported the oxidative stress and DNA damage in human oral cells following treatment with different concentrations of STE. Apoptotic cell death of human oral keratinocytes was also increased with increasing concentrations of STE, and 51-85% decreases in apoptotic cell death were observed with vitamins C, E, and GSPE, respectively. In the present study, keratinocytes were isolated and cultured from human oral tissues and treated with (0-30mg/ml) of STE for 24 hours and change in the expression of a p53 gene was measured by reverse transcriptase polymerase chain reaction (RT-PCR). Thirty-five cycles were performed for five minutes at 94° C and b-tubulin was used as the housekeeping gene. Approximately a 2.0 fold increase in p53 gene expression was observed following incubation of the oral keratinocytes with 100 mg/ml of STE, beyond which the expression of p53 decreased. Thus, p53 expression dramatically reduced with higher concentration of STE, confirming increased apoptotic cell death with a higher concentration of STE as reported by us earlier. Also, the expression of antiapoptotic Bcl-2 gene also increased with GSPE treatment when measured similarly using RT-PCR. In addition, the expression level of transcription factor c-myc gene responsible for cell cycle growth also altered following treatment with GSPE. Furthermore, the comparative abilities of vitamin C (75mm), vitamin E (75mm), a combination of vitamins C plus E (75mm each), and GSPE (100mg/ml) were determined. Our results demonstrate that STE significantly modulates Bcl-2, p53 and c-myc gene expression, and approximately 22-46% protection was observed with antioxidants. GSPE demonstrated significantly better protection as compared to vitamins C and E, singly and in combination. - Bagchi M, et al., Protective Effects of Vitamins C and E and a Novel Grape Seed Proanthocyanidin Extract on Smokeless Tobacco Induced Oxidative Stress and Modulation of Bcl-2, p53 and c-myc Genes, 1999 SFRR Europe Winter Meeting on Bio-Flavonoids & Polyphenols in Health & Disease, Dinard, France, Abstract P44, 1999.

CANCER RESEARCH

Human Cancer Cell Study

• Inhibits and even kills human cancer cells, including breast, lung and stomach cancer cells, while enhancing the growth and viability of normal cells (in vitro).
• Protects normal human liver cells from the toxic effects of chemotherapy drugs (in vitro).
• Inhibits the initiation, promotion and progression of DMN-induced liver carcinogenesis (in vivo).

In a recent study, researchers assessed ActiVin-induced cytotoxicity towards selected human cancer cells, including cultured MCF-7 human breast cancer cells, CRL 1739 human gastric adenocarcinoma cells and A-427 human lung cancer cells, by phase contrast microscopy and MTT cytotoxicity assay, and compared these effects with two normal cultured cells, including normal human gastric mucosal cells and J774A.1 murine macrophage cells. The results demonstrated that ActiVin exhibits selective cytotoxicity towards cultured MCF-7 human breast cancer cells, CRL 1739 human gastric adenocarcinoma cells and A-427 human lung cancer cells at 25 mg/l and 50 mg/l concentrations, while ActiVin enhances the growth and viability of the normal cultured cells at these concentrations.

—Ye X, et al., Cytotoxic Effects of a Novel IH636 Grape Seed Proanthocyanidin Extract (GSPE) on Cultured Human Cancer Cells, Molecular and Cellular Biochemistry, 196:99-108, 1999.

In Vivo Ih636 Grape Seed Proanthocyanidin Extract (GSPE) Exposure Inhibits Mouse Liver Microsomal Cyp4502E1-Dependent Aniline Hydroxlation in Vitro

Previous studies in our laboratories have demonstrated the protective abilities of a novel IH636 GSPE against oxygen free radicals, tobacco-included programmed cell death in human oral keratinocytes, myocardial ischemia-reperfusion injury and infarction, and drug and chemically-induced multi-organ toxicity. This study compared the in vitro aniline hydroxylation patterns of in vivo GSPE pre-exposed hepatic microsomes with induced (1% acetone in drinking water for 3 days) and uninduced rat liver microsomes. Male B6C3F1 mice were fed GSPE containing diet (ADI: 100 mg/kg body wt.) for 4 weeks. Liver microsomes were isolated from both control and GSPE-exposed mice, and aniline hydroxylation was assessed as specific marker of CYP4502E1 activity. Interestingly, GSPE supplementation for 4 weeks inhibited aniline hydroxylation by 40%, as compared to the control animals. Similar to in vivo GSPE preexposed mouse liver microsomes, rat liver microsomes when incubated with various concentrations of GSPE preexposed mouse liver microsomes, rat liver microsomes when incubated with various concentrations of GSPE (100 and 250 mg/ml), inhibited aniline hydroxylation to various degrees (Uninduced: 40% and 60% and Induced: 25% and 50%, respectively). These data were compared with hydroxylation pattern of another hepatoprotective agent, such as 4-aminobenzamide (4-AB), which shows that 4-AB did not alter aniline hydroxylation at all. Collectively, these results may suggest that the ability of GSPE to inhibit CYP4502E1 is an additional cytoprotective attribute, in conjunction with its novel antioxidant efficacy. The above research shows that ActiVin can act as a very good detoxifying agent against acetone-induced cytochrome P4502E1 induction by reducing aniline hydroxylation, a marker of cytochrome P4502E1 activity.

–Bagchi D, et al., In Vivo IH636 Grape Seed Proanthocyanidin Extract (GSPE) Exposure Inhibits Mouse Liver Microsomal CYP4502E1-Dependent Aniline Hydroxylation, 39th Annual Meeting of the Society of Toxicology Meeting, 54(Suppl. 1): Abstract 468, 100, 2000; Ray SD, et al., Differential Effects of IH636 Grape Seed Proanthocyanidin Extract and a DNA Repair Modulator 4-aminobenzamide on Liver Microsomal Cytochrome P450-2E1-Dependent Aniline Hydroxylation. Toxicology Letters (submitted).

IH636 Grape Seed Proanthocyanidin Extract (GSPE) Exposure Significantly Attenuates Dimethylnitrosamine (DMN)-Induced Liver Cancer and Mortality in ICR Mice.

Although carcinogenesis is a genetically regulated multistep process, our knowledge to intervene its initiation, promotion and progression stages are very limited. The goal of this study was to determine whether pre-, post, and coexposure of GSPE followed by DMN prevents/reduces/delays onset of liver tumor formation in male B6C3F1 mice and, if so, whether GSPE interferes with any of the stages of cancer development. Animals in this study were divided into six groups: I-Control, II-GSPE alone, III-DMN alone, IV-GSPE +DMN, V-3 months DMN to 6 months GSPE, and VI-3 months GSPE to 9 months DMN. DMN was administered ip (10 mg/kg once a week), and with drinking water (1 mg/ml DMN) for the first 3 months, whereas GSPE was mixed with a lab chow (approx. ADI: 100 mg/kg/b.w.). Results show that DMN induced liver tumor formation and animal lethality (Gr. III-tumors: 85%; deaths: 29%) were powerfully antagonized by co-administration of GSPE diet + DMN (Gr. IV-tumors: 72%; death: 11%). More than 66% of the DMN alone animals had numerous tumors (5+), which was reduced to 48% in the GSPE + DMN group. GSPE’s influence on the stage between promotion and progression was also remarkable (Gr. VI-only 28% had 5 or more tumors). Similarly, GSPE also effectively interfered with the tumor initiation process (Gr. V). Serum chemistries did not change significantly during the entire study, and 9 month oral exposure of GSPE alone had no adverse effects. This study proposes that long-term GSPE exposure may negatively influence and interfere with all the three stages of DMN-induced liver carcinogenesis.

–Ray SD, et al., IH636 Grape Seed Proanthocyanidin Extract (GSPE) Exposure Significantly Attenuates Dimethylnitrosamine (DMN)-Induced Liver Cancer and Mortality in ICR Mice. 91st Annual Meeting of the American Association for Cancer Research, 41: Abstract 2928, 460, 2000.

CHEMOTHERAPY RESEARCH

Chemotherapy in Vitro Study

Although anticancer chemotherapeutic agents can be very effective at inhibiting the growth of cancer cells in vitro and in vivo, toxicity to normal cells is a major problem. Normal human liver cells were treated with two widely used chemotherapy drugs, Idarubicin (30 nM) and 4-hydroxyperoxy-cyclophosphamide (4-HC) (1 mg/ml), with or without ActiVin (25 mg/ml), and the growth rate of the cells were determined using MTT assay. This study demonstrated that ActiVin significantly decreased the growth inhibitory effects of Idarubicin and 4-HC on normal human liver cells in vitro. ActiVin was also shown to enhance the expression of bcl-2 gene in normal human liver cells. It is worthwhile to mention that bcl-2 stimulates an antioxidant pathway at sites of oxygen free radical generation, and thus potentiates antioxidant functions of cellular systems. These results indicate that ActiVin is a potential candidate to ameliorate the toxic effects associated with these chemotherapeutic agents towards normal healthy cells.

—Joshi SS, et al., Amelioration of Cytotoxic Effects of Idarubicin and 4HC on Chang Liver Cells by a Novel Grape Seed Proanthocyanidin Extract, The FASEB Journal, 12: Abstract 4484, A774, 1998.

Modulation of Gene Expression: Protective Mechanisms Against Chemotherapy - Induced Cell Damage

The molecular mechanisms of action of ActiVin towards chemotherapy drug-induced toxicity towards normal human cells were investigated. Chang liver cells, isolated from normal human liver samples, and human peripheral blood mononuclear cells were treated with the chemotherapeutic agents 4-hydroxy-peroxycyclophosphamide (4-HC) (1 mg/ml) or Idarubicin (1 mM/ml) with or without ActiVin (25 mg/ml) for 48 hr. Apoptosis (programmed cell death) was measured by flow cytometry. Apoptosis was significantly decreased in the presence of ActiVin compared to administration of the drugs alone. Similar effects of ActiVin were also seen with antisense oligonucleotide-induced apoptosis in human liver cells. Subsequently, ActiVin treated liver cells and blood mononuclear cells were analyzed for the expression of key cell cycle/apoptosis regulatory genes such as bcl-2, p53 and c-myc by RT-PCR and Western blot techniques. ActiVin significantly increased expression of bcl-2 antioxidant gene and decreased expression of tumor inducing c-myc gene and apoptosis regulatory p53 gene. The results suggest that the chemopreventive effects of ActiVin are mediated by alterations in bcl-2, p53 and c-myc gene expression, and that ActiVin can be used to ameliorate chemotherapy-induced toxicity to normal cells and tissues.

—Joshi SS, et al., Chemopreventive Effects of a Novel Grape Seed Proanthocyanidin Extract Via Alterations in Functions of bcl-2 and p53 genes, The FASEB Journal, 13: Abstract LB139, 1999; Joshi SS, et al., Amelioration of the Cytotoxic Effects of Chemotherapeutic Agents by Grape Seed Proanthocyanidin Extract. Antioxidants & Redox Signaling, 1(4): 563-570, 1999.

Renal Protection in Vivo Study

This study focused on the protective effects of ActiVin on acetaminophen-induced nephrotoxicity and genomic DNA damage in kidneys. Male ICR mice (3 months old) were fed 100 mg ActiVin/kg orally for 7 days followed by intraperitoneal administration of acetaminophen 500 mg/kg for 24 hr. Blood was collected for determination of BUN (blood urea nitrogen), and the kidneys examined for histopathology and DNA damage. Exposure to acetaminophen alone caused greater than 3-fold increase in BUN compared to the controls (acetaminophen – 67 mg BUN/dl, control – 21 mg BUN/dl). While ActiVin alone didn’t cause any damage to the kidneys (19 mg BUN/dl), its pre-exposure to acetaminophen-treated animals significantly reduced damage to the kidneys (32 mg BUN/dl). Histopathological evaluation of kidney sections mirrored the serum chemistry findings. Acetaminophen-induced genomic DNA damage and its modulation by ActiVin were also demonstrated. In conclusion, these findings suggest that ActiVin may protect kidney and renal function in mice from acetaminophen-induced toxicity.

—Ray SD, et al., Effect of a Novel IH636 Grape Seed Proanthocyanidin Extract on Acetaminophen-Induced Nephrotoxicity, Journal of the American College of Nutrition, 17: Abstract 49, 508, 1998.

Doxorubicin-Induced Cardiotoxicity

Cardiotoxicity of Doxorubicin is a major limiting factor for clinical application of this important anticancer drug. Since DNA is the primary target of Doxorubicin in vivo, this study was designed to investigate whether ActiVin via its antioxidant properties can provide protection against Doxorubicin-induced DNA damage and prevents cardiotoxicity. Male ICR mice (30-40 gms) were pretreated with ActiVin (100 mg/kg/day for 7 days p.o.) followed by Doxorubicin (20 mg/kg i.p.), or Doxorubicin alone. The animals were then sacrificed and their CPK activities were determined. Hearts were collected for histopathology and DNA analysis. Results indicate that Doxorubicin alone caused significant heart injury, whereas pre-exposure to GSPE significantly reduced Doxorubicin-induced cardiotoxicity and prevented DNA damage. Histopathological evaluation of PAS and H&E stained heart sections indicated that ActiVin successfully intervened Doxorubicin-induced cardiotoxic events and protected cells from apoptopic and necrotic deaths. ActiVin’s protection was fully equivalent to that of 3-aminobenzamide (3-AB), a promising new anti-cancer drug known to significantly inhibit DNA damage.

—Wong V, et al., Antioxidant Grape Seed Proanthocyanidin Extract (GSPE) and a DNA Repair Modulator 3-Aminobenzamide (3-AB) Protect Doxorubicin (DOX)-Induced Cardiotoxicity in Antioxidant Grape Seed Proanthocyanidin Extract (GSPE) and a DNA Repair Modulator 3-Aminobenzamide (3-AB) Protect Doxorubicin (DOX)-Induced Cardiotoxicity In Vivo, 38th Annual Meeting of the Society of Toxicology, 48(Suppl. 1): Abstract 731, 156, 1999.

Protection Against Multiple Organ Drug and Chemical-Induced Toxicity

Drug- and/or chemical-induced degradation of DNA in cells has serious biological consequences such as apoptopic and necrotic cell death, mutation and/or carcinogenic transformation. Recently, we demonstrated that ActiVin provides significant cellular protection against acetaminophen-induced liver and kidney toxicity, and several other acetaminophen-sensitive intracellular targets. This study examined the protective effects of ActiVin against Amiodarone-induced lung toxicity, dimethylnitrosamine-induced spleen toxicity, cadmium chloride-induced kidney toxicity and MOCAP-induced neurotoxicity. Parameters of analysis included changes in serum chemistry, histopathology and integrity of genomic DNA. Results indicate that animals (mice) fed ActiVin for 7-days prior to exposure to toxic levels of Amiodarone, cadmium chloride and dimethylnitrosamine received near complete protection in terms of serum chemistry changes (ALT, BUN and CK), and totally eliminated both apoptopic and necrotic cell death, compared to animals exposed to the toxicants in the absence of ActiVin. In addition, ActiVin also provided significant protection against DNA damage triggered by these toxicants to various degrees. Evaluation of H&E stained lung, kidney and spleen tissues mirrored the serum chemistry and DNA changes. Surprisingly, MOCAP-exposed animals showed symptoms of severe neurotoxicity coupled with serum chemistry changes with minimal DNA damage and brain pathology, of which ActiVin provided partial protection. Taken together, this study suggests that ActiVin-provides significant protection against multiple organ toxicity from a variety of drug and chemical-induced toxic assaults in vivo.

—Ray SD, et al., A Novel Grape Seed Proanthocyanidin Extract (GSPE) Protects Multiple Target Organ Toxicities Induced by Amiodarone (Lung), Dimethylnitrosamine (Spleen), CdCl2 (Kidney), and MOCAP (Brain), The FASEB Journal, 13: Abstract 175.2, A187, 1999.

Cytochrome P450-2E1 Dependent Aniline Hydroxylation

Grape seed proanthocyanidin extract (IH636 GSPE) prevents acetaminophen (AAP)-induced liver injury in mice (Ray et al., 1999, Arch. Biochem. Biophys.): this anti-AAP action may be due to its antioxidant effect or, in part, due to its ability to inhibit microsomal drug metabolizing enzyme(s), such as CYP4502E1. Since CYP4502E1 primarily metabolizes AAP in mice and rats, this study was undertaken to investigate whether various concentrations of GSPE inhibit CYP4502E1 activity in vitro. Microsomes were insolated from induced (1% acetone in drinking water for three days) and uninduced male SD rats (three months old). Microsomes (both induced and uninduced) were incubated with or without various concentrations of GSPE. Their abilities to hydroxylate aniline were then determined. Aniline hydroxylation is used as a specific marker of CYP4502E1 activity. Results show that 100 and 250mg/ml of GSPE inhibited aniline hydroxylation 40% and 60%, respectively, in uninduced microsomes, and 25% and 50%, respectively, in induced microsomes. We also compared the effect of GSPE with another anti-AAP agent 4-aminobenzamide (4-AB). In comparison to GSPE, 4-AB did not alter aniline hydroxylation at all. In summary, these findings suggest that the anti-AAP activity of GSPE may be due, in part, to its interference with drug metabolizing activities, such as CYP4502E1.

—Ray SD, et al., IH636 Grape Seed Proanthocyanidin Extract Inhibits Cytochrome P450-IIE1 Dependent Aniline Hydroxylation in Induced and Uninduced Rat Liver Microsomes, Journal of the American College of Nutrition 18: Abstract 50, 533, 1999.

CARDIOVASCULAR RESEARCH

Cardioprotection In Vivo Study

• Lowers blood pressure and decreases glycosylated hemoglobin levels in rats.
• Provides cardioprotection by improving left ventricular function, decreasing myocardial infarction and reducing oxidative damage caused by ischemia-reperfusion injury (in vivo).
• Protects against Doxorubicin-induced cardiotoxicity (in vivo).
• Effect On Atherosclerosis Models

Grape seed proanthocyanidins extracts (GSPE) are known to exhibit a broad spectrum of chemoprotective and cardioprotective properties against oxidative stress. In this study, we have assessed the comparative protective effects of vitamin C and vitamin E, and GSPE (ActiVin), singly, and in combination, in an in vitro cupric –ion mediated LDL + VLDL oxidation model which stimulates the initiating step in atherosclerosis. Incubation of this model with a combination of GSPE and vitamin C in ratios of 0:5:100 and 54:100, respectively, exhibited 264% and 49% synergism, respectively, while a combination of GSPE and vitamin E in ratios of 13:100 and 66:100, respectively, demonstrated 21% and 133% synergism, respectively. A combination of GSPE, vitamin C, and vitamin E in ratios of 9:26:65 and 49:15:36, respectively, exhibited 65% and 59% synergism, respectively. These results suggest that GSPE can significantly potentiates antioxidant synergism. Furthermore, we assessed the efficacy of GSPE supplementation in hamsters since these animals have a similar lipid profile to hypercholesterolemic humans when fed a hypercholesterolemic diet of 0.2% cholesterol and 10% coconut oil (HD). After 10 weeks of feeding a HD, these animals developed foam cells, a biomarker of early stage of atherosclerosis. Atherosclerosis (% of aorta covered with foam cells) was reduced by approximately 50% and 63% following supplementation of these animals with 50 mg/kg and 100 mg/kg of GSPE, respectively, in conjunction with HD. These data demonstrate that GSPE may provide significant health benefits by potentiating antioxidant synergism and dramatically ameliorating the incidence of atherosclerosis as demonstrated by reducing the formation of foam cells.

–Vinson JA, et al., Beneficial Effect of a Novel Grape Seed Proanthocyanidin Extract in Atherosclerosis Models. Free Radical Biology & Medicine, 27(Suppl. 1): Abstract 90, S45, 1999.

Amelioration of Cardiomyocyte Apoptosis Through Inhibition Cardiomyocyte Apoptosis Through Inhibition of JNK1 and cJUN

Grape seed proanthocyanidins (GSPE) are natural antioxidants, which are known to possess a broad spectrum of biological, pharmacological and chemoprotective properties against free radicals and oxidative stress. Oxygen free radicals play a crucial role in the pathogenesis of a variety of cardiovascular diseases including congestive heart failure, cardiomyopathy, atherosclerosis, hypertrophy, hypertension, arrhythmias, and ischemic heart disease. In the present study, the cardioprotective effects of GSPE (ActiVin) were evaluated using an isolated perfused rat heart, which was subjected to ischemia and reperfusion. Rats were fed GSPE (100 mg/kg/day, p.o.) for three weeks. After 21 days, the rats were anesthetized with sodium pentobarbital (70 mg/kg) (i.p) and heparinized with 500 IU (i.v.). The hearts were rapidly excised, placed in ice-cold buffer, and both aorta and pulmonary vein were cannulated. The hearts were placed in the working mode and then subjected to 30 min of global ischemia followed by 120 min of reperfusion. Systolic aortic pressure, heart rate, the first derivatives of positive pressure (dp/dtmax) and developed pressure (DP) were recorded at baseline, and during 30, 60 and 120 min of reperfusion using pressure transducer connected with the data capture system. The coronary flow (CF) was measured by timed collections of coronary effluent. At the end of each experiment, the hearts were incubated for 10 min in a 1% solution of buffered triphenyltetrazolium chloride (TTC), preheated at 37°C. The atriums were excised and frozen. The ventricles were sliced transversely approximately 1mm in thickness and incubated with TTC for 10 min. Each slice was weighed and scanned with computer-assisted scanner. The risk area, which consisted of whole ventricle, was stained in red by TTC and the infarct area remaining unstained was measured using computerized planimetry software. These areas were multiplied by the weight of each slice. And the results were added to obtain the weight of the risk zone and the infarct zone. Infarct size was expressed as the ratio of the infarct zone to the risk zone. Cardiomyocytes were isolated from each heart and the degree of apoptosis was evaluated by TUNEL method. The extent of DNA fragmentation was assessed by subjecting the DNAs into electrophoresis. Western blot analysis was performed to estimate the abundance of c-Jun and JNK-1 proteins. Gas exerted cardioprotective effects as evidenced by the improved post-ischemic ventricular recovery (DP, dp/dtmax, CF, AF, AP) and its ability to reduce myocardial infarct size. GSPE also reduced apoptotic cardiomyocyte death and DNA laddering and attenuated ischemia/reperfused-mediated increased abundance of c-JUN and JNK-1 proteins. In vitro study using paniranic acid fluorescence assay was undertaken to examine the antioxidative property of these compounds. Our results demonstrated that GSPE possesses potent peroxyl radical and hydroxyl radical scavenging properties. The results of this study demonstrated that GSPE possesses potent cardioprotective properties as evidenced by its ability to reduce both necrosis and apoptosis. Reperfusion of the ischemic myocardium induces apoptosis in concert with the enhancement of proapoptotic factor, c-JUN and JNK-1. Our results suggest that GSPE reduces apoptotic cell death probably by attenuating ischemia/reperfusion-induced increased abundance of c-JUN and JNK-1 proteins. The cardioprotective ability of GSPE, may at least in part, be attributed to its ability to scavenge peroxyl and hydroxyl radicals, which are generated in the heart during ischemia and reperfusion. This suggests that GSPE may be used as a potential therapeutic agent to cure ischemic heart disease.

–Das DK, et al., Amelioration of Cardiomyocyte Apoptosis by IH636 Grape Seed Proanthocyanidin Extract Through the Inhibition of JNK1 and cJUN, 1999 SFRR Europe Winter Meeting on Bio-Flavonoids & Polyphenols in Health & Disease, Dinard, France, Abstract OP26, 1999.

Effects of Chromium and Grape Seed Extraction the Lipid Profile of Hypercholesterolemic Patients

Hypercholesterolemia, a significant cardiovascular risk factor, is prevalent in the American population. Although many pharmaceuticals provide adequate therapeutic benefits, the majority carries significant adverse reactions such as liver problems with the "statins". Accordingly, natural means to lower cholesterol levels via safe means would be welcomed. We examined hypercholesterolemic patients (220-300 mg/dl) in a randomized, double-blind, placebo-controlled study. Three groups of 10 patients each received placebo bid, chromium polynicotinate (Cr) (ChromeMate) 200 mg bid, or Cr 200 mg bid plus Grape Seed Extract (GSE) (ActiVin) 100 mg bid. Over 2 months, the change in total cholesterol from baseline among groups was: placebo- 3.5% ± 4 (SEM), Cr- 10% ± 5, and the combination of Cr + GSE- 16.5% ± 3. The decrease in the last group was significantly different from placebo (p < 0.01). The major change was in the LDL levels: placebo- 3% ± 4, Cr- 14% ± 4, and the combination- 20% ± 6. Again, the combination significantly decreased LDL when compared to placebo. HDL levels did not essentially change among groups. Also, there was no significant difference for the triglyceride concentrations among the groups. In this basically, normotensive population, the blood pressure changes were not significantly different. Currently, we are examining serum oxidized LDL concentrations in these patients. We conclude that a combination of natural products, specifically Cr and GSE, can decrease LDL levels significantly. Our results demonstrate that when GSE and ChromeMate taken in combination significantly reduced elevated cholesterol levels in hypercholesterolemic patients.

–Talpur NA, et al., Effects of Chromium and Grape Seed Extract on the Lipid Profile of Hypercholesterolemic Patients. The FASEB Journal, 14: Abstract 503.8, A727, 2000.

Regulation of Inducible VCAM-1 Expression by Grape Seed Proanthocyanidin Extract

Altered expression of cell adhesion molecules has been implicated in a variety of chronic inflammatory conditions. Regulation of adhesion molecule expression via specific redox sensitive mechanisms has been reported. Grape seed proanthocyanidins have potent antioxidant properties. We evaluated the effects of grape seed proanthocyanidin extract (GSPE) on TNF-alpha-induced ICAM-1 and VCAM-1 expression in primary human umbilical vein endothelial cells (HUVEC). At low concentrations (1-5 microgram/ml), GSPE down-regulated TNF-alpha-induced VCAM-1 expression but not ICAM-1 expression in HUVEC. Such regulation of inducible VCAM-1 by GSPE was also observed at the mRNA expression level. A cell-cell co-culture assay was performed to verify whether the inhibitory effect of GSPE on the expression of inducible VCAM-1 was also effective in down regulating actual endothelial cell/leukocyte interaction. GSPE treatment significantly decreased TNF-alpha-induced adherence of T-cells to HUVEC. Although several studies have postulated NF-kappaB as the molecular site where redox active substances act to regulate agonist-induced ICAM-1 and VCAM-1 gene expression, inhibition of inducible VCAM-1 gene expression by GSPE was not through a NF-kappaB dependent pathway. The inhibitory effect of GSPE on agonist-induced VCAM-1 expression suggests therapeutic potential of this extract in pathologies involving elevated levels of inducible VCAM-1 expression. Our results show that GSPE may help significantly in the treatment of skin rashes, dermatitis, psoriasis, acne, and arthritis, cardiovascular and other inflammatory disorders.

—Sen CK and Bagchi D, Regulation of Inducible VCAM-1 Expression by Grape Seed Proanthocyanidin Extract. The FASEB Journal, 14 Abstract 484.20, A695, 2000; Sen CK and Bagchi D, Regulation of Inducible Adhesion Molecule Expression in Human Endothelial Cells by Grape Seed Proanthocyanidin Extract. Molecular & Cellular Biochemistry (submitted).

HYPERTENSION RESEARCH

Age-Related Hypertension and Glyosylated Hemoglobin Study

The pathogenesis of the aging phenomenon and chronic diseases associated with aging is attributed, in part, to glycosylation of proteins and nucleic acids, and augmented free radical formation causing increased tissue damage. In normotensive rats, it was demonstrated that chronic supplementation of ActiVin (250 ppm) in conjunction with ChromeMate® niacin-bound chromium (5 ppm chromium) and OptiZinc® methionine-bound zinc (18 ppm zinc) significantly reduced systolic blood pressure and glycosylated hemoglobin (HbA1C), as well as decreased lipid peroxidation and free radical formation. These results demonstrate potential long-term health benefits of ActiVin used in combination with ChromeMate and OptiZinc.

—Preuss HG, et al., Chromium, Zinc, and Grape seed Extract (Flavonoids) Can Overcome Age-Related Increases in SBP of Normotensive Rats, Journal of the American College of Nutrition, 16: Abstract 43, 481, 1997; Tyson DA, et al., Acute Effects of Grape Seed Extract on the Systolic Blood Pressure of Normotensive and Hypertensive Rats, Journal of American College of Nutrition, (submitted).

Effect on Blood Pressure IN Spontaneously Hypertensive Rats

Elevated blood pressure has been attributed in some situations to perturbations in the glucose/insulin, NO and/or renin-angiotensin systems. Various natural products have been shown to influence these systems favorably. Among these are antioxidants such as chromium (Cr), grape seed extracts (GSE) and zinc (Zn). Accordingly, we wished to examine simultaneously these elements, alone and in combination, for comparison. We examined six dietary groups containing eight spontaneously hypertensive rats (SHR). In addition to a control group ingesting the basic diet, the other groups ate the basic diet with the addition of Cr, or GSE or Cr + GSE, or Cr + GSE + Zn. At the end of three weeks, there are no significant differences in average body weights. Systolic BP (SBP) was measured by tail plethysmography. Over three weeks, the changes compared to baseline in the respective SBP of SHR were Con + 25.7± 5.5, Cr + 7.9± 2.1, GSE – 2.1 ± 4.9, Zn + 12.9 ± 4.2, C-GSE + 0.7 ± 4.3 and Cr- GSE- Zn –2.9 ± 5.9 mm Hg ± SEM. All other values were statistically lower than control, with Cr and Zn alone showing the lesser changes from control. Adding Cr and Zn to GSE produced no more difference than GSE alone. Results from Losarten challenge indicate that lessened activity if the renin-angiotensin system was involved, at least in part, in lowering SBP with each constituent. HbA1c and serum glucose, insulin, transaminase, alkaline phosphatase, cholesterol and triglycerides were not different among groups, but creatine was statistically lower in all groups compared to control. We conclude that oral Cr, GSE, and Zn all lower SBP via alterations in the renin-angiotensin system, that GSE has the greatest effect and that combining these ingredients with GSE works no better than GSE alone at the concentrations examined.

—Talpur N, et al., Effects of Chromium, Grape Seed Extract, and Zinc Alone and in Combination on Blood Pressure of Spontaneously Hypertensive Rats, Journal of the American College of Nutrition, 18: Abstract 35, 527, 1999.

OTHER RESEARCH:

• Reduces chronic pancreatitis in human subjects (human).
• Inhibits acute and chronic stress-induced gastrointestinal injury (in vivo).
• Topical application enhances sun protection factor (SPF) in human subjects (human).
• ActiVin and ChromeMate taken in combination significantly reduces elevated cholesterol levels in hypercholesterolemic patients.
• Significantly helps in the treatment of skin rashes, dermatitis, psoriasis, acne, and arthritis, cardiovascular and other inflammatory disorders.

CONCLUSION

The results of these experiments demonstrate that ActiVin:

• Is a potent free radical scavenger and inhibitor of free radical-induced cellular damage, including lipid peroxidation and DNA fragmentation (in vivo),
• Is bioavailable and a more powerful antioxidant than vitamins E, C and beta-carotene (in vivo),
• Inhibits tobacco-induced oxidative damage and cell death (in vitro),
• Inhibits and even kills human cancer cells, while enhancing the growth of normal cells (in vitro),
• Helps protect cells from the toxic effects of chemotherapy drugs by enhancing the antioxidant regulatory gene bcl-2, down regulating the cancer-inducing gene c-myc, and modulating the apoptosis (programmed cell death) regulatory gene p53 (in vitro),
• Improves ventricular heart function, decreases myocardial infarction and reduces the tissue damage caused by cardiac ischemia-reperfusion injury (in vivo),
• Lowers blood pressure and decreases glycosylated hemoglobin in hypertensive models (in vivo),
• Protects against acetaminophen-induced liver toxicity (in vivo),
• Reduces chronic pancreatitis in human subjects,
• Inhibits acute and chronic stress-induced gastrointestinal injury (in vivo),
• Protects against doxorubicin-induced cardiotoxicity (in vivo),
• Protects multiple target organs against drug and chemical-induced toxicity including amiodarone (lung toxicity), dimethylnitrosamine (spleen toxicity), cadmium chloride (kidney toxicity) and MOCAP (brain toxicity) (in vivo),
• Reduces apoptotic cell death probably by attenuating ischemia/reperfusion-induced increased abundance of c-Jun and JNK-1 proteins,
• Inhibits microsomal CYP4502E1-dependent aniline hydroxylation in vitro,
• Provides long-term protection and inhibits the initiation, promotion and progression of cancer resulting from exposure to certain environmental pollutants such as dimethylnitrosamine (DMN),
• Significantly helps in the treatment of skin rashes, dermatitis, psoriasis, acne, arthritis, cardiovascular and other inflammatory disorders,
• ActiVin in conjunction with ChromeMate significantly reduces elevated cholesterol levels in hypercholesterolemic patients.