Research Grape Seed Extract Abstracts

The cellular and molecular basis of health benefits of grape seed extract Red grape seed extract containing and other antioxidants are being used as nutritional supplements by many health conscious individuals. The beneficial effects of grape seed proanthocyanidins (GSPE) have been reported, however, little is known about their mechanism(s) of action. One of the beneficial effects of GSPE is chemoprevention of cellular damage. The precise mechanism by which GSPE mediates, chemoprevention is not yet understood. This report addresses this issue. We investigated the mechanisms of actions of GSPE, which ameliorates chemotherapy-induced toxic effects of Idarubicin (Ida) and 4,-hydroxyperoxycyclophosphamide (4-HC) in normal human Chang liver cells. Exposure to GSPE resulted in a significant reduction in apoptosis in response to the cytotoxicity of chemotherapeutic agents. RT-PCR analysis showed a significant increase in the anti-apoptotic gene Bcl-2 and a decrease in the cell cycle associated and proapoptotic genes, c-myc and p53 in cells treated with GSPE. These results suggest that some of the chemopreventive effects of GSPE are mediated by upregulating Bcl-2 and down regulating c-myc and p53 genes. Joshi SS, Kuszynski CA, Bagchi D Curr Pharm Biotechnol 2001 Jun;2(2):187-200. Free radicals scavenging action and anti-enzyme activities of procyanidines from Vitis vinifera. A mechanism for their capillary protective action The scavenging by procyanidines (polyphenol oligomers from Vitis vinifera seeds, CAS 85594-37-2) of reactive oxygen species (ROS) involved in the onset (HO degrees) and the maintenance of microvascular injury (lipid radicals R degrees, RO degrees, ROO degrees) has been studied in phosphatidylcholine liposomes (PCL), using two different models of free radical generation: a) iron-promoted and b) ultrasound-induced lipid peroxidation. In a) lipid peroxidation was assessed by determination of thiobarbituric acid-reactive substances (TBARS); in b) by determination of conjugated dienes, formation of breakdown carbonyl products (as 2,4-dinitrophenylhydrazones) and loss of native phosphatidylcholine. In the iron-promoted (Fenton-driven) model, procyanidines had a remarkable, dose-dependent antilipoperoxidant activity (IC50 = 2.5 mumol/l), more than one order of magnitude greater than that of the monomeric unit (IC50 = 50 mumol/l), activity which is due, at least in part, to their metal-chelating properties. In the more specific model b), which discriminates between the initiator (hydroxyl radical from water sonolysis) and the propagator species of lipid peroxidation (the peroxyl radical, from autooxidation of C-centered radicals), procyanidines are highly effective in preventing conjugated diene formation in both the induction (IC50 = 0.1 mumol/l) and propagation (IC50 = 0.05 mumol/l) phases (the scavenging effect of alpha-tocopherol was weaker, with IC50 of 1.5 and 1.25 mumol/l). In addition, procyanidines at 0.5 mumol/l markedly delayed the onset of the breakdown phase (48 h), totally inhibiting during this time the formation of degradation products (the lag-time induced by alpha-tocopherol was only of 24 h at 10 mumol/l concentration). The HO degrees entrapping capacity of these compounds was further confirmed by UV studies and by electron spin resonance (ESR) spectroscopy, using DMPO as spin trapper: procyanidines markedly reduced, in a dose-dependent fashion, the signal intensity of the DMPO-OH radical spin adduct (100% inhibition at 40 mumol/l). The results of the second part of this study show that procyanidines, in addition to free radical scavenging action, strongly and non-competitively, inhibit xanthine oxidase activity, the enzyme which triggers the oxy radical cascade (IC50 = 2.4 mumol/l). In addition procyanidines non-competitively inhibit the activities of the proteolytic enzymes collagenase (IC50 = 38 mumol/l) and elastase (IC50 = 4.24 mumol/l) and of the glycosidases hyaluronidase and beta-glucuronidase (IC50 = 80 mumol/l and 1.1 mumol/l), involved in the turnover of the main structural components of the extravascular matrix collagen, elastin and hyaluronic acid. Maffei, Facino R, Carini M, Aldini G, Bombardelli E, Morazzoni P, Morelli R. Arzneimittelforschung 1994 May;44(5):592-601. Regulation of inducible adhesion molecule expression in human endothelial cells by grape seed proanthocyanidin extract

Altered expression of cell adhesion molecule expression has been implicated in a variety of chronic inflammatory conditions. Regulation of adhesion molecule expression by specific redox sensitive mechanisms has been reported. Grape seed proanthocyanidins have been reported to have potent antioxidant properties. We evaluated the effects of grape seed proanthocyanidin extract (GSPE) on the expression of TNFalpha-induced ICAM-1 and VCAM-1 expression in primary human umbilical vein endothelial cells (HUVEC). GSPE at low concentrations (1-5 micrograms/ml), down-regulated TNFalpha- 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 VCAM-1 was also effective in down-regulating actual endothelial cell/leukocyte interaction. GSPE treatment significantly decreased TNFalpha-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 as detected by a NF-kappaB reporter assay. The potent inhibitory effect of low concentrations of GSPE on agonist- induced VCAM-1 expression suggests therapeutic potential of this extract in inflammatory conditions and other pathologies involving altered expression of VCAM- 1. Sen CK, Bagchi D. Mol Cell Biochem 2001 Jan;216(1-2):1-7.

Selective cytotoxic activity of grape peel and seed extracts against oral tumor cell lines Grape seed extracts were more cytotoxic than grape peel extracts. Methanol and 70% methanol extracts of grape seed selectively killed two human oral tumor cell lines, more efficiently than human gingival fibroblasts. ESR spectroscopy revealed that these extracts produced radicals under alkaline conditions and enhanced the radical intensity of sodium ascorbate at higher concentrations. On the other hand, lower concentration of these extracts slightly reduced the radical intensity of sodium ascorbate, and scavenged superoxide anion, generated by hypoxanthine and xanthine oxidase reaction. These properties of grape seed extracts suggest their possible application for cancer prevention. Shirataki Y et al. Anticancer Res 2000 Jan-Feb;20(1A):423-6.

Oxygen free radical scavenging abilities of vitamins C and E, and a grape seed proanthocyanidin extract in vitro Proanthocyanidins, a group of polyphenolic bioflavonoids, have been reported to exhibit a wide range of biological, pharmacological and chemoprotective properties against oxygen free radicals. We have assessed the concentration-dependent oxygen free radical scavenging abilities of a grape seed proanthocyanidin extract (GSPE), vitamin C and vitamin E succinate (VES) as well as superoxide dismutase, catalase and mannitol against biochemically generated superoxide anion and hydroxyl radical using a chemiluminescence assay and cytochrome c reduction. A concentration-dependent inhibition was demonstrated by GSPE. At a 100 mg/l concentration, GSPE exhibited 78-81% inhibition of superoxide anion and hydroxyl radical. Under similar conditions, vitamin C inhibited these two oxygen free radicals by approximately 12-19%, while VES inhibited the two radicals by 36-44%. The combination of superoxide dismutase and catalase inhibited superoxide anion by approximately 83%, while mannitol resulted in an 87% inhibition of hydroxyl radical. The results demonstrate that GSPE is a more potent scavenger of oxygen free radicals as compared to vitamin C and VES. Bagchi D et al. Res Commun Mol Pathol Pharmacol 1997 Feb;95(2):179-89. Grape seed proanthocyanidin reduces cardiomyocyte apoptosis by inhibiting ischemia/ reperfusion-induced activation of JNK-1 and C-JUN The mechanism of cardioprotection with red wine consumption was studied by examining the antideath signaling cascade of one of the principle components of red wine, proanthocyanidins. Grape seed proanthocyanidin extract (GSPE) was administered orally (100 mg/kg/d) supplemented with regular diet for 3 weeks to a group of rats while the other group was given the regular diet only for the same period of time. After 3 weeks, rats were sacrificed, hearts excised, and perfused via Langendorff mode. After stabilization, hearts were perfused in the working mode for baseline measurement of contractile function. Hearts were then made globally ischemic for 30 min followed by 2 h of reperfusion. Contratile function was continuously monitored during reperfusion, and free radical production was examined by electron spin resonance (ESR) technique. Cardiomyocyte apoptosis was examined by TUNEL staining in conjunction with an antibody against myocin heavy chain to specifically detect myocytes. Induction of JNK-1 and c-fos proteins was studied by Western blot analysis using respective antibodies followed by densitometric scanning. The results indicated significant induction of JNK-1 and c-fos proteins in the ischemic/reperfused myocardium, which was inhibited by the proanthocyanidin extract. In concert, GSPE significantly reduced the appearance of apoptotic cardiomyocytes in the ischemic/reperfused hearts. GSPE also significantly reduced the appearance of the reactive oxygen species in the hearts. Improved postischemic contractile recovery was achieved with GSPE suggesting its cardioprotective action. The results of this study indicated that GSPE functioned as an in vivo antioxidant, and its cardioprotective properties may be at least partially attributed to its ability to block antideath signal through the inhibition of proapoptotic transcription factor and gene, JNK-1 and c-Jun. Sato M, Bagchi D, Tosaki A, Das DK. Free Radic Biol Med 2001 Sep 15;31(6):729-37.

Anti-tumor-promoting activity of a polyphenolic fraction isolated from grape seeds in the mouse skin two-stage initiation-promotion protocol and identification of B5- 3'-gallate as the most effective antioxidant constituent present in grape seeds are known to exert anti-inflammatory, anti-arthritic and anti-allergic activities, prevent skin aging, scavenge oxygen free radicals and inhibit UV radiation-induced peroxidation activity. Since most of these events are associated with the tumor promotion stage of carcinogenesis, these studies suggest that grape seed polyphenols and the procyanidins present therein could be anticarcinogenic and/or anti-tumor-promoting agents. Therefore, we assessed the anti-tumor-promoting effect of a polyphenolic fraction isolated from grape seeds (GSP) employing the 7,12- dimethylbenz[a]anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol 13-acetate (TPA)-promoted SENCAR mouse skin two-stage carcinogenesis protocol as a model system. Following tumor initiation with DMBA, topical application of GSP at doses of 0.5 and 1.5 mg/mouse/application to the dorsal initiated mouse skin resulted in a highly significant inhibition of TPA tumor promotion. The observed anti-tumor-promoting effects of GSP were dose dependent and were evident in terms of a reduction in tumor incidence (35 and 60% inhibition), tumor multiplicity (61 and 83% inhibition) and tumor volume (67 and 87% inhibition) at both 0.5 and 1.5 mg GSP, respectively. Based on these results, we directed our efforts to separate and identify the individual polyphenols present in GSP and assess their antioxidant activity in terms of inhibition of epidermal lipid peroxidation. Employing HPLC followed by comparison with authentic standards for retention times in HPLC profiles, physiochemical properties and spectral analysis, nine individual polyphenols were identified as catechin, epicatechin, procyanidins B1-B5 and C1 and -3'-gallate. Five of these individual polyphenols with evident structural differences, namely catechin, , procyanidin B5, and procyanidin B5-3'-gallate, were assessed for antioxidant activity. All of them significantly inhibited epidermal lipid peroxidation, albeit to different levels. A structure- activity relationship study showed that with an increase in the degree of polymerization in polyphenol structure, the inhibitory potential towards lipid peroxidation increased. In addition, the position of linkage between inter-flavan units also influences lipid peroxidation activity; procyanidin isomers with a 4-6 linkage showed stronger inhibitory activity than isomers with a 4-8 linkage. A sharp increase in the inhibition of epidermal lipid peroxidation was also evident when a gallate group was linked at the 3'-hydroxy position of a procyanidin dimer. Procyanidin B5-3'-gallate showed the most potent antioxidant activity with an IC(50) of 20 microM in an epidermal lipid peroxidation assay. Taken together, for the first time these results show that grape seed polyphenols possess high anti-tumor-promoting activity due to the strong antioxidant effect of procyanidins present therein. In summary, grape seed polyphenols in general, and procyanidin B5-3'-gallate in particular, should be studied in more detail to be developed as cancer chemopreventive and/or anticarcinogenic agents. Zhao J, Wang J, Chen Y, Agarwal R. Carcinogenesis 1999 Sep;20(9):1737-45.

The preceding abstracts were obtained from the Medline service maintained by the National Institutes of Health.