GRAPE EXTRACT


By Biomedical Engineer Mark Lubin

13 Patents issued or pending with USPTO



Grapes have a long and abundant history in human use.  The cultivation and domestication of the plant appears to have started 4,000-6,000 BC in what is now called the Middle East.  Use of grapes as a food and in winemaking then rapidly spread to the rest of the Mediterranean, Europe, and the near East.  People often enjoy the various grape products, such as fruit, raisins, juice and wine. Grape skin, flesh, and seed contain various (and often different) nutrient elements, such as vitamins, minerals, carbohydrates, edible fibers and phytochemicals. Polyphenols are the most important phytochemicals in grape because they possess many biological activities and health-promoting benefits.1-3  The phenolic compounds mainly include anthocyanins, flavanols, flavonols, stilbenes (resveratrol) and phenolic acids.4-6   The dietary consumption of grape and its products is associated with a lower incidence of degenerative diseases such as cardiovascular disease, diabetes and its aftermath, certain types of cancers, inflammatory disease such as arthritis, microbial processes, and degeneration of cognitive and vision functions.  Most recent interest has focused on the bioactive phenolic compounds in grape.  Anthocyanins, flavanols, flavonols, and resveratrol are the most important grape polyphenols.  The anthocyanins are pigments, and mainly exist in grape skins while the others are distributed in the flesh, seeds and stems.    


Anthocyanins are pigments, and mainly exist in grape skins. Flavonoids are widely distributed in grapes, especially in seeds and stems, and principally contain (+)-catechins, (−)-epicatechin and procyanidin polymers. Anthocyanins are the main polyphenolics in red grapes, while flavan-3-ols are more abundant in white varieties.7-9  Polyphenolics from grapes and red wines attracted the attention of scientists to define their chemical composition and their properties for human health.10  The reported evidences of beneficial health effects of phenolic compounds include inhibiting some degenerative diseases, such as cardiovascular diseases,11-14 certain types of cancers,15-17 reducing plasma oxidation stress, and slowing aging.18-19  Phenolic compounds are also regarded as preservatives against microbes and oxidation for food.20-21   What’s more, in vivo assays showed that phenolic compounds are bioavailable.10,22  Therefore, besides wine and juice, grape diet supplements would be promising functional foods worthy of popularization.  However, some reports have also shown that at higher concentrations the effect of phenolic compounds on health was negative and some structures in particular promoted the negative effects.23   In addition, some high molecular weight phenolics could not be absorbed.24,25


Grape – Research


Recently, growing interests on phenolic compounds from grapes have focused on their biological activities linked to human health benefits.  These benefits, and the metabolic pathways by which they are realized, have been explored in over 13,000 published research papers available from NLM (the National Library of Medicine).  Only the research pertinent to antioxidant and anti-inflammatory activities have been referenced in this report.


Anti-Oxidant Activity


Being most the notable bioactivity of phenolic compounds from grapes, the antioxidative characteristics have been widely studied, including scavenging of free radicals, inhibition of lipid oxidation, reduction of hydroperoxide formation, and so on.18,19 Several methods were employed to evaluate the antioxidant capacities of phenolic compounds extracted from various grapes or different parts of grapes and wide differences in antioxidant capacities were revealed.55-61  


Many researchers have tried to discover which phenolic compounds and chemical structure(s) are mainly responsible for the antioxidant activities of grape extracts. For same phenolic compounds, 50% and 25% (v/v) concentrations showed the same antioxidant activities, both being better than that of the 10% (v/v) concentration. The result suggested that perhaps the antioxidant capacity of phenolics has a concentration saturation limit, and above this limit, the activity could not increase further with the concentration.66  However, the relationship between phenolic compounds and antioxidant capacity was inconsistent among the results from different studies, which indicated that, besides the concentration, the antioxidant capacities of phenolic compounds were affected by other factors.49,67  In a study, malvidin-3-glucoside showed the highest antioxidant capacity in wine anthocyanins.35  Although total phenolic index was lower in grape flesh than in grape skin because anthocyanins were absent in the flesh, they possessed equal amounts of reactivity to hydroxyl radicals.13  In another study, the results also showed that the anti-radical activity was due to the flavanols, rather than anthocyanins.68  The results showed that procyanidin polymers with higher degrees of polymerization had higher antioxidant activities.46  However, Faria et al.74 showed that in five fractions of different degrees of procyanidins polymers, the second degree fraction displayed the highest antioxidant capacity (scavenging peroxyl radicals). A similar result was obtained by Soobratteea et al.75, who showed that the most antioxidative compound in various phenolics was procyanidin dimer, and the decrease in antioxidant capacity was in order of procyanidin dimer, flavanol, flavonol, hydroxycinnamic acids and simple phenolic acids. Diphenols are more effectively antioxidant than simpler phenols due to stabilization of the phenoxy-radical through hydrogen bonding.50 The high molecule weight compounds might be as important as the monomer flavanols such as catechin, which have been demonstrated high antioxidant potential in phenolic compounds.76 Furthermore, the antioxidant activity of a sample could be synergic effect among several compositions, rather than a single compound.47,77


Pinelo et al. studied the impact of solvent on the antioxidant activity of catechin, resveratrol and grape extracts dissolved in ethanol, methanol and water. The maximum antiradical activity was in ethanol, then in methanol, and the minimum was in water.45 By in vitro physiological procedure such as digestive enzymatic extraction, phenolic compounds from grape seed displayed a higher phenolic content and antioxidant capacity than by chemical procedure, which could be employed for the aim of getting dietary supplements from grapes.78


The antioxidative characteristics of phenolic compounds are mainly ascribed to their free radical scavenging and metal chelating properties, as well as their effects on cell signaling pathways and on gene expression.75,79 Arora et al 80 found that flavonoids displayed higher antioxidant capacity against metal-ion-induced peroxidation than peroxyl-radical-induced peroxidation. The mechanism was mainly speculated to react directly to generate phenoxyl radicals81, which was stable and cuts off the reaction chains. The chemical functional group and structure is OH for antioxidant capacity of phenolic compounds. The number of OH group and its position on the ring of molecule determined the antioxidant capacity of flavonols.80 When the OH added onto the flavonoid nucleus, the activity enhanced, while substituted by the OCH3 groups, the activity diminished. The results were proved by Majo et al.67,82 The o-diphenoxyl groups in resveratrol were determined to exhibit higher antioxidant activity than other compositions.83


Cardio-protective properties


Postprandial hyperlipemia and oxidative stress, a well-defined risk factor for atherosclerosis, could be reduced by grape seed extracts or phenolic-rich grape juice. These oxidative stress factors refer to plasma lipid hydroperoxides, serum lipid peroxidation products, malondialdehyde-modified-LDL (MDA-LDL). The lipid-bound polyphenols increasing in serum were found even two hours after intake of phenolics, and MDA-LDL was detected after six weeks.48,69,84   Grape seed extracts protected the rat liver against oxidative damage induced by irradiation in vivo, and remained the activities of superoxide dismutase and catalase at normal level.85  


Grape seed extracts (5–50 μg/mL) rich in polyphenols displayed reduction of platelet adhesion and aggregation and generation of superoxide anion, and were more effective than pure resveratrol.12   Shanmuganayagam et al.11 Auger et al employed rabbits to investigate the potential of phenolic compounds to defend the hypercholesterolemic-induced platelet aggregation. After intake of the grape juice (225 mL/day), which was rich in polyphenolics, with hypercholesterolemic diet for 96 days, platelet aggregation in rabbits was significantly ameliorated and the development of atheroma was near 30% lower than that of the control group. Aortic fatty streak areas of hamster also showed significant reduction in the groups receiving catechin (84%) or quercetin (80%) or resveratrol (76%) in comparison to the controls.37,86 Dell Agli et al 79 showed anthocyanins from wine and grape skin inhibited phosphodiesterase-5 activity, which reduced the risk of cardiovascular diseases by vasorelaxation. Falchi et al. [13] made ischemic to isolated heart of rats for 30 min followed by two hours of reperfusion, and found that the ischemic reperfusion injury was significantly inhibited in the rats after 30days consumption of the extracts of flesh and skin of grapes, and flesh and skin of grapes exhibited equal effect of cardioprotection.


Castilla et al.87 found that phenolic compounds significantly ameliorated plasma lipid levels. After drinking 100 mL red grape juice/day for 14 days, the concentration of cholesterol-standardized-tocopherol and antioxidant capacity of plasma were significantly increased, and oxidized LDL and LDL were significantly reduced. The plasma level of HDL and apolipoprotein A-I were also elevated.  In addition, consumption of red wine resulted to high concentrations of HDL cholesterol14 which linked to control of the risk of coronary heart diseases.  Ardevol et al.88 reported that treatment of differentiated 3T3-L1 cells with procyanidin extracts reduced HSL in the mRNA levels, and inhibited triacylglycerol synthesis and boost its hydrolysis. After feeding to hamsters at a moderate dose of grape extracts, the plasma cholesterol was reduced 11% on average.86 Moreover, plasma apolipoprotein A1 concentration was increased 26%, 22%, and 19%, induced by catechin, quercetin, and resveratrol, respectively.37 For hemodialysis patients, phenolics of grapes are offered to prevent from inflammation.  Red grape juice significantly reduced plasma monocyte chemoattractant protein-1, an inflammatory factor involved with cardiovascular disease risk, after three weeks’ consumption.87 Tsang et al 14 showed that after two weeks of daily red wine consumption (375 mL), the maximum concentrations of conjugated dienes and TBAES in Cu-oxidized LDL were reduced. It was reported that red wine consumption reduced oxidative stress induced by Cu-oxidized LDL and increased HDL cholesterol concentrations. Grape juices showed complete inhibition of copper-induced oxidation of human LDL at the concentration of 0.01%.89   Phenolic compounds in grapes have showed effective power to regulate the plasma lipid and oxidative stress.


Anticancer Activity


Many evidences have shown that the extracts from grapes and its products had anticancer activity. Hudson et al.90 reported that the grape skin extract induced prostate tumor cell lines apoptosis with high rates. The extract from pomace remaining after wine production inhibited activities of matrix metalloproteinases-2 and -9, and expressed a significant antiproliferative effect on human colon adenocarcinoma cells (Caco-2), which implied by-product of wine would help to fight against carcinogenesis.15,91  Phenolics of grape juice also significantly inhibited carcinogen-induced DNA adduct formation in rat model17 and inhibited DNA synthesis in breast cancer cells.16  Phenolic compounds had dual effects on cells, and modulated cell proliferation was notably dose-dependent.92  At high concentration, they were attributed to direct toxic effect and induced cells to death.93


Anti-inflammatory Activity


Phenolic compounds in grapes, especially in grape seeds, have showed significant anti-inflammation effects on rats, mice and human, and the contributive molecules may be flavonols, flavanols and procyanidins (oligomeric flavonoids) 7,36,102 Bralley et al.103 found that extracts from grape skins and seeds inhibited mouse ear inflammation, edema, and polymorphonuclear leukocyte infiltration induced by 12-O-tetradecanoylphorbol 13-acetate, after treated with the extracts for 30 minutes. Moreover, the effect of the combination of grape seeds and skins almost paralleled to that of indomethacin, a common drug against degenerative diseases of joint. These findings indicated that phenolic compounds in grapes possessed obviously anti-inflammatory activity.


The mechanism of anti-inflammation of procyanidins was investigated, and the results showed that it might inhibit releasing proinflammation factors.  Immunomodulation was the main pathway, and antioxidative action was another pathway for the anti-inflammation effect of grape phenolics.7,36,104 Panico et al.36 employed human chondrocytes assays to prove this. After treatment with a combination of extract of grape wine and IL-1b, a notable decrease was detected in the concentration of nitric oxide, prostaglandins E2 and reactive oxygen species in human chondrocytes culture, compared to control groups, and the effects were equal or super to that of indomethacin. Li et al.104 demonstrated that proanthocyanidins could prevent the increase of MDA in rat paws with arthritis induced by carrageenan at the concentration of 10 mg/kg by injection. Nitric oxide synthase activity and N-acetyl-β-d- glucosaminidase were also successfully inhibited by proanthocyanidins.  Inhibition or reduction of the cytokine gene expression may be a basic pathway to anti-inflammation for grape phenolics.7,102,104   After pre-treated with extracts of grape seed procyanidins, human adipocytes and macrophage-like cell lines produced less IL-6 and MCP-1 induced by inflammatory stimulus, and increase in anti-inflammatory adipokine and adiponectin appears.  The results demonstrated that grape seeds procyanidins might modulate adipokine and cytokine gene expression related to anti-inflammation.7 Terra et al.102 reported that grape seed procyanidins inhibited the increase of C-reaction protein in rat plasma induced by high fat feed, and the same trend in IL-6 and TNF-α was detected in the mesenteric white adipose tissue (WAT).  Further research demonstrated that CRP mRNA expression was decreased in the liver and mesenteric WAT, while adiponectin mRNA expression was increased in the mesenteric WAT. Then, lipid metabolic disorder and inflammation were availably inhibited. The results indicated that procyanidins in grapes inhibited inflammation at mRNA levels, and major health benefits brought by them involved in decreasing the risk of diseases link to high fat diets and obesity, such as cardiovascular and metabolic disorders.


Anti-aging Effects


It was found that polyphenolics presented in foods might be beneficial in reversing the course of neuronal and behavioral aging. Due to their notable antioxidant activity, such as scavenging free radical, they could prevent organs and tissues from oxidative damage, and modify the body negative mechanism of redox status. The evidences were obtained by observing the behaviors of rats, from age 19 to 21 months. After drinking the 10% grape juice, improvements were detected on release of dopamine from striatal slices, as well as cognitive performance in the Morris water maze, while the 50% grape juice improved action capacity.105  Further research discovered that supplement with grape seed extracts (100 mg/kg b.wt.) for 30 days, phenolic compounds from the extracts inhibited the accumulation of age-related oxidative DNA damages in neural tissue.106  Balu et al.107 reported the decreased incidence of free radical-induced lipid peroxidation in the central nervous system of aged rats.


Antimicrobial Activity


Plant polyphenols have been demonstrated potential antibacterial48,107,108, antifungal28,110, and antiviral111,112 activities.  Rodriguez-Vaquero et al 113 have showed that grape wine inhibited microbial, especially Escherichia coli growth, and the inhibition increased as the polyphenol concentration increased, and clarified wines were inactive against all bacteria tested.  The extracts of alcohol-free red and white wine exhibited antimicrobial activity to some pathogens such as Staphylococcus aureus, Escherichia coli and Candida albicans.114 The results suggested that polyphenolic compounds contained in red wines were responsible for the antimicrobial effects.  Some studies reported phenolic compounds inhibited other food-borne species such as Salmonella typhimurium115 and Listerial monocytogenes.62 Various bacterial species exhibit different sensitivities towards phenolic compounds. Papadopoulou et al 114 demonstrated Staphylococcus aureus were most sensitive to wine extracts, followed by Escherichia coli and the least effect of inhibition was detected in Candida albicans.  Research to define and quantify the antimicrobial effects of polyphenols is a very broad field resulting in thousands of publications.21,54,62,111,112,115,117,118,122  


The bioactivities of phenolic compounds from grapes are summarized below.


Phenolic compound Bioactivity References

resveratrol free radical scavenging [76,81]

 antiproliferation [83,100]

 enhancing plasma NO level [123]

 regulating lipid metabolism [37]

 protection against membrane oxidation [124]

quercetin antibacterial [20]

 enhancing plasma NO level [123]

catechin anticancer [74]

 free radical scavenging [13,68,83]

 antibacterial [119]

 anti-inflammation [36]

 protection against membrane oxidation [124]

flavone antiproliferation [99]

flavonol free radical scavenging [75,81]

procyanidin anticancer [74,94]

 free radical scavenging [75]

 anti-inflammation [8,102,125]

 antioxidant [89]

anthocyanin vasorelaxation [79]

 free radical scavenger [97]

 antibacterial [118,119]

 antioxidant [89]

 inducing apoptosis [126]

gallic acid free radical scavenger [76]

epicatechin antibacterial [76,118]

 


Several studies showed rapid absorption of the polyphenolics, such as procyanidins, quercetin and flavanols from grapes into plasma, with plasma concentrations peaking at two or three hours after ingestion.31,48,87,127-129  The increase of lipid-bound polyphenolics in serum could be detected, and as a result of the bioactivity of polyphenolics, significant decrease was detected on lipid peroxidation in serum.48  Moreover, after two weeks of daily red wine consumption (375 mL), plasma levels of total phenolic concentrations increased significantly, and trace levels of metabolites, mainly glucuronides and methyl glucuronides of (+)-catechin and (−)-epicatechin, were detected in plasma which could not be found in a control group, indicating that phenolic compounds could be absorbed by human digestion system and enter the blood successfully.14  Despite these detected results the absorption mechanisms remain unclear.


The potential toxicity of some polyphenols from grape has been investigated. Negative toxicity effects were noted after exposing the two cell lines to epicatechin for 24 hours or more time, and when the concentration was 3–7 fold higher than that of expressing positively antioxidant activity.23,98   Moreover, the compounds with a gallate group exhibited more potential toxicity than those without the gallate group.23  In addition, noticeable DNA damage was induced in mice spleen cells by incubating with higher concentration (150 μmol/L) of catechin.98  Grape extracts was also found to promote mitomycin C inducing sister chromatid exchange at concentration from 75 to 300 μg/mL in human peripheral blood lymphocytes.139  The compounds with polyphenols, caffeic acid, gallic acid, and rutin hydrate enhanced MMC-induced clastogenicity at accordant concentrations, suggesting that the negative effects of phenolic compounds were related to the synergistic effect of some molecules, and the concentration was not always a crucial factor.


Grape and products from grape have been consumed for a long time. The studies have demonstrated an inverse association between intake of grape and products from grape and mortality from age-related diseases such as coronary heart diseases. The health benefits of grapes are thought to arise mainly from bioactivities of their polyphenols. Anthocyanins, flavonoids and resveratrol are the major functional components that are responsible for most of biological activities of grape. Tremendous progress has been obtained for the extraction, analysis and biological activities of polyphenols in grape. The bioactive compounds were usually extracted from grape using the liquid-liquid extraction, and high-performance liquid chromatography with UV or MS detection could be applied to analysis of active components in grape. The grape and its main components anthocyanins, flavonoids and resveratrol have a variety of bioactivities, such as antioxidant, cardio-protective, anticancer, anti-inflammation, antiaging and antimicrobial activities, which are closely related to the prevention against disease and promotion of health, making greater potential for grape in the field of food and pharmaceutical application. The structure-activity relationships of some polyphenols have been studied, and the results obtained could be used to modify structure of polyphenol as well as to design and synthesize novel polyphenols with special function. Most of phenolic compounds were bioavailable, but some high molecular weight phenolics could not be absorbed. In addition, the effect of some phenolic compounds was negative on health at higher concentration, and some structures promoted the negative effect.


In the future, the extraction methods of polyphenols from grape should be improved, and the by-products of wine industry should be utilized effectively. The crude extracts from grape could be used as diet supplements for health-protection after defining the levels or limits to make sure the dose is safe for health, but bioactive components at high purity should be used instead of crude extracts in medicinal preparations from grape. In order to explore more effective functional food or pharmaceutical products based on grape, more wide pharmacological studies should be carried out to determine new pharmacodynamic effects, such as anti-influenza, anti-obesity and antidiabetic activities. The relationship of structure-activity should be studied further, and the key mechanisms of bioactivities should be understood clearly. In addition, more attention should be paid to minor components in grape because special pharmacodynamic effects could be found from minor components. The structural diversities and pronounced biological activities of compounds in grape indicate that grape are worthy of further studies that may lead to the identification of new functional constituents. The polyphenols from grape will widely be employed to prevent and treat these diseases in association with reactive oxygen species, such as atherosclerosis, coronary heart diseases and cancer.














































































































































Grape – Safety



Because of the long, historically pervasive, and ubiquitous use of all varieties of grape in every conceivable manner of food and herbal medicine use worldwide, no appropriate specific safety statement can be made other than care should be exercised when a known or suspected allergy to grapes or grape byproducts is present in any given individual.