Quantitative Mitochondrial Proteomics Study on Protective Mechanism of Grape Seed Proanthocyanidin Extracts Against Ischemia/Reperfusion Heart Injury in Rat

Cardiac ischemia/reperfusion（I/R） injury is a critical condition,often associated with high morbidity and mortality.The cardioprotective effect of grape seed proanthocyanidin extracts（GSPE） against oxidant injury during I/R has been described in previous studies.However,the underlying molecular mechanisms have not been fully elucidated.This study investigated the effect of GSPE on reperfusion arrhythmias especially ventricular tachycardia（VT） and ventricular fibrillation（VF）,the lactic acid accumulation and the ultrastructure of ischemic cardiomyocytes as well as the global changes of mitochondria proteins in in vivo rat heart model against I/R injury.GSPE significantly reduced the incidence of VF and VT,lessened the lactic acid accumulation and attenuated the ultrastructure damage.Twenty differential proteins related to cardiac protection were revealed by isobaric tag for relative and absolute quantitation（iTRAQ） profiling.These proteins were mainly involved in energy metabolism.Besides,monoamine oxidase A（MAOA） was also identified.The differential expression of several proteins was validated by Western blot.Our study offered important information on the mechanism of GSPE treatment in ischemic heart disease.

Grape Seed Proanthocyanidin Extract Alleviates Arsenic-induced Oxidative Reproductive Toxicity in Male Mice

Objective To determine the ability of grape seed proanthocyanidin extract （GSPE） in alleviating arsenic-induced reproductive toxicity. Methods Sixty male Kunming mice received the following treatments by gavage： normal saline solution （control）; arsenic trioxide （ATO; 4 mg/kg）; GSPE （400 mg/kg）; ATO＋GSPE （100 mg/kg）; ATO＋GSPE （200 mg/kg） and ATO＋GSPE （400 mg/kg）. Thereafter, the mice were sacrificed and weighed, and the testis was examined for pathological changes. Nuclear factor （erythroid-derived 2）-like 2 （Nrf2）, heme oxygenase 1 （HO1）, glutathione S-transferase （GST）, NAD（P）H dehydrogenase, and quinone 1 [NQO1） expression in the testis was detected by real-time PCR. Superoxide dismutase （SOD）, glutathione （GSH）, total antioxidative capability （T-AOC）, malondialdehyde （MDA）, 8-hydroxydeoxyguanosine （8-OHdG）, and reproductive indexes were analyzed. Results ATO-treated mice showed a significantly decreased sperm count and testis somatic index and activity levels of SOD, GSH, and T-AOC than control group. Compared to the ATO-treated group, ATO ＋GSPE group showed recovery of the measured parameters. Mice treated with ATO＋high-dose GSPE showed the highest level of mRNA expression of Nrf2, HO, NO.O1, and GST. Conclusion GSPE alleviates oxidative stress damage in mouse testis by activating Nrf2 signaling, thus counteracting arsenic-induced reproductive toxicity.

Inhibitory Effects of Grape Seed Proanthocyanidin Extract on Selenite-induced Cataract Formation and Possible Mechanism

This study investigated the inhibitory effect of grape seed proanthocyanidin extract(GSPE) on selenite-induced cataract formation in rats and the possible mechanism.Eighty 8-day-old Sprague-Dawley rats were divided randomly into 5 groups:control group,model group,three GSPE groups(low dose,medium dose and high dose).Control group received subcutaneous injection of physiological saline.Model group was given subcutaneous injection of sodium selenite(20 μmol/kg body weight) on the postpartum day 10,and once every other day for consecutive three times thereafter.GSPE treated groups were respectively administered GSPE at doses of 50,100,and 200 mg/kg body weight intragastrically 2 days prior to the selenite injection(that was,on the postpartum day 8),and once daily for fourteen consecutive days thereafter.The opacity of lenses was observed,graded and photographed under the slit lamp microscopy and the maximal diameter of the nuclear cataract plaques was measured.The lenses were analyzed for superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GSH-PX),malondialdehyde(MDA),calcium(Ca 2+),nitric oxide(NO) and anti-hydroxyl radical ability(anti-OH).The histomorphology of lenses was observed with HE staining under a light microscope.The levels of calpainⅡ,and iNOS protein and mRNA expression in lenses were detected by using immunohistochemistry and real-time quantitative RT-PCR.The results showed subcutaneous injection of sodium selenite led to severe nuclear cataract in model group,and the achievement ratio of model group was 100%.As compared with model group,the degree of lenses opacity and the maximal diameter of nuclear cataract plaques were significantly reduced in GSPE-treated groups.Moreover,we observed selenite treatment caused a significant decrease in the activities of antioxidative enzymes(SOD,CAT,GSH-PX) and anti-OH ability,accompanied by a significant increase in the levels of MDA,NO,Ca 2+ as well as iNOS,and calpainⅡ protein and mRNA expression.Administration of GSPE could dose-dependently preserve the activities of these antioxidative enzymes and anti-OH ability,accompanied by a significant reduction in the levels of MDA,NO,Ca 2+ as well as iNOS,and calpainⅡ protein and mRNA expression.These results suggested that GSPE markedly prevented selenite-induced cataract formation probably by suppressing the generation of lipid peroxidation and free radicals as well as the activation of iNOS,and calpainⅡ in the lenses.

Neuroprotective Effects of Grape Seed Procyanidin Extracton Ischemia-Reperfusion Brain Injury

Objective Oxidative stress （OS） plays a crucial role in ischemic stroke. Grape seed procyanidin extract （GSPE） was reported to be a critical regulator of OS. We hypothesized that GSPE might also be protective in ischemia-reperfusion brain injury. This study aimed to explore whether GSPE administration can protect mice from ischemia-reperfusion brain injury. Methods Transient middle cerebral artery occlusion （MCAO） was conducted followed by reperfusion for 24 hours to make ischemia-reperfusion brain injury in mice that received GSPE （MCAOG, n=60） or normal saline （MCAONS, n=60）. Sham-operated mice （GSPE group and normal saline group） were set as controls. The neurological severity score （NSS） was used to evaluate neural function impairment 1 hour, 24 hour, 3 days and 7 days after MCAO. Mice underwent brain T2WI imaging with a 3T animal MRI scanner 24 hours after reperfusion, and the stroke volume of brains were calculated according to abnormal signal intensity. Immunohistopathological analysis of brain tissues at 24 h after reperfusion was performed for neuronal nuclear antigen （NeuN）, CD34, Bcl-2, and Bax. Glutathione peroxidation （GSH-Px） activity and the level of malonaldehyde （MDA） of brain tissue were also examined. The above indexes were compared among the groups statistically.Results Significant functional improvement was observed 24 hours after MCAO in MCAOG group compared to MCAONS group （P〈0.05）. MCAOG group had smaller cerebral stroke volume （22.46 ± 11.45 mm3 vs. 47.84±9.06 mm3, P〈0.05） than MCAONS group 24 hours after MCAO. More mature NeuN-immunoreactive neurons and more CD34-positive cells in peri-infarct zones were observed in brain tissue of MCAOG mice 24 h after MCAO than that of MCAONS mice （both P〈0.05）. MCAONS mice had significantly higher number of Bax-positive cells in brain tissue than MCAOG （P〈0.05）. The mean MDA level was significantly lower （P〈0.05） and the GSH-Px activity was significantly higher （P〈0.05） in brains of MCAOG mice compared to those of MCAONS mice. Conclusion GSPE administration protects mice from ischemia-reperfusion brain injury through attenuating oxidative stress and apoptosis, promoting angiogenesis, and activating antioxidant enzyme GSH-Px. GSPE may represent a new therapeutical direction for the treatment of ischemia-reperfusion brain injury.

Grape Seed Proanthocyanidin Extract Ameliorates Streptozotocin-induced Cognitive and Synaptic Plasticity Deficits by Inhibiting Oxidative Stress and Preserving AKT and ERK Activities

Progressive memory loss and cognitive impairment are the main clinical manifestations of Alzheimer’s disease(AD).Currently,there is no effective drug available for the treatment of AD.Previous studies have demonstrated that the cognitive impairment of AD is associated with oxidative stress and the inhibition of AKT and ERK phosphorylation.Grape seed proanthocyanidin extract(GSPE)has been shown to have strong antioxidant effect and can protect the nervous system from oxidative stress damage.This study aimed to investigate the protective effect of GSPE on the cognitive and synaptic impairments of AD using a sporadic AD rat model induced by intracerebroventricular(ICV)injection of streptozotocin(STZ)(ICV-STZ).Rats were treated with GSPE(50,100,or 200 mg/kg every day)by intragastrical(ig.)administration for continuous 7 weeks,and ICV-STZ(3 mg/kg)was performed on the first day and third day of week 5.Learning and memory abilities were assessed by the Morris water maze(MWM)test at week 8.After behavioral test,hippocampal long-term potentiation(LTP)was recorded,and the levels of malondialdehyde(MDA),superoxide dismutases(SOD),glutathione(GSH)and the protein expression of AKT and ERK were measured in the hippocampus and cerebral cortex of rats.Our study revealed that ICV-STZ significantly impaired the working learning ability and hippocampal LTP of rats,significantly increased the levels of MDA,and decreased the activity of SOD and GSH in the hippocampus and cerebral cortex.In contrast,GSPE treatment prevented the impairment of cognitive function and hippocampal LTP induced by ICV-STZ,decreased the level of MDA,and increased the level of SOD and GSH.Furthermore,Western blot results showed that GSPE treatment could prevent the loss of AKT and ERK activities in the hippocampus and cerebral cortex induced by ICV-STZ.Our findings demonstrate that GSPE treatment could ameliorate the impairment of cognitive ability and hippocampal synaptic plasticity in a rat model of sporadic AD by inhibiting oxidative stress and preserving AKT and ERK activities.Therefore,GSPE may be an effective agent for the treatment of cognitive deficits associated with sporadic AD.

Effects of grape seed extract on meat color and premature browning of meat patties in high-oxygen packaging

This study investigated the effects of grape seed extract(GSE)on fresh and cooked meat color and premature browning(PMB)in ground meat patties(85% beef and 15% pork back fat)packaged under high-oxygen modified atmospheres(HiOx-MAP).The GSE was added to patties at concentrations of 0,0.10,0.25,0.50 and 0.75 g kg^(-1).This study evaluated the surface color,pH,lipid oxidation,and total viable counts(TVC)of raw patties,and the internal color and pH of patties cooked to a temperature of 66 or 71℃ over 10-day storage at 4℃.Compared with the control(0 g kg^(-1) GSE),GSE improved the color stability(P<0.05)and significantly inhibited the lipid and myoglobin oxidation of raw patties from day 5 to 10,but GSE had no effect(P>0.05)on TVC.Patties containing 0.50 and 0.75 g kg^(-1) GSE cooked to 66℃ exhibited greater(P<0.05)interior redness than the control and reduced the PMB of cooked patties in the late storage stage.These results suggested that 0.50 and 0.75 g kg^(-1) GSE can improve fresh meat color and minimize PMB of HiOx-MAP patties.

Beneficial clinical effects of grape seed proanthocyanidin extract on the progression of carotid atherosclerotic plaques

Background Atherosclerotic plaques indicate the occurrence of ischemia events and it is a difficult task for clinical physicians. Grape seed proanthocyanidin extract （GSPE） has been reported to exert an antiatherogenic effect by inducing regression of atherosclerotic plaques in animal experimental studies. In this study, the antiatherogenic effect of GSPE has been investigated in clinical use. Methods Consecu- tive 287 patients diagnosed with asymptomatic carotid plaques or abnormal plaque free carotid intima-media thickness （CIMT） were ran- domly assigned to the GSPE group （n = 146） or control group （n = 141）. The patients in the GSPE group received GSPE 200 mg per day orally, while patients in the control group were only enrolled in a lifestyle intervention program. Carotid ultrasound examination was per- formed at baseline and 6, 12, 24 months during follow-up. Mean maximum CIMT （MMCIMT）, plaque score, echogenicity of plaques and ischemic vascular events were recorded. Results As anticipated, after treatment, GSPE resulted in significant reduction in MMCIMT pro- gression （4.2% decrease after six months, 4.9% decrease after 12 months and 5.8% decrease after 24 months） and plaque score （10.9% de- crease after six months, 24.1% decrease after 12 months and 33.1% decrease after 24 months） for the primary outcome, while MMCIMT and plaque score were stable and even increased with the time going on in control group. The number of plaques and unstable plaques also de- creased after treatment of GSPE. Furthermore, the carotid plaque can disappear after treatment with GSPE. The incidence rate for transitory ischemic attack （TIA）, arterial revascularization procedure, and hospital readmission for unstable angina in GSPE group were statistically significant lower （P = 0.02, 0.08, 0.002, respectively） compared with the control group. Conclusions GSPE inhibited the progression of MMCIMT and reduced carotid plaque size in GSPE treated patients, and with extended treatment, the superior efficacy on MMCIMT and carotid plaque occurred. Furthermore, the GSPE group showed lower rates of clinical vascular events.

Grape Seed Procyanidin Extract Reduces Arsenic- Induced Renal Inflammatory Injury in Male Mice

The aim of the present study is to evaluate the ability and mechanism by which grape seed procyanidin extract （GSPE） relieves arsenic trioxide （As2O3）-induced renal inflammatory injury. Therefore, male Kunming mice were treated with As2O3 and/or GSPE by gavage for 5 weeks. Mice were then sacrificed and inflammatory cytokines of kidneys were examined by ELISA, whereas the expression levels of molecules involved in the nuclear factor （NF）-KB signaling pathway were evaluated by both qRT-PCR and Western blot. Our results indicate that GSPE prevents As2O3-mediated renal inflammatory injury by inhibiting activation of the NF-KB signaling pathway and inflammatory cytokine production, while promoting expression of anti-inflammatory cytokines.

In vitro Evaluating the Influence of Grape Seed Polyphenol Extract on the Digestibility of Macronutrients

Interaction between dietary polyphenol and proteins including digestive enzymes may result in reduced digestibility of food macronutrients, thus lowering absorption of nutrients that contributing to high energy accumulation in human body. The objective of this study was to investigate the effect of grape seed polyphenol extract （GSPE） on the digestibility of starch, food lipid and food protein by digestive enzymes such as u-amylase, lipase, pepsin and trypsin. The digestion of each substrate was conducted at the optimal pH and temperature of specific enzyme. Bread containing different amount of grape pomace was used as a real food model and its digestion was conducted under simulated digestion condition. Concentrations of reducing sugar, fatty acid and amino acids in enzyme digested mixtures were determined as indicators of starch, lipid and protein digestions, respectively. Results indicate that GSPE significantly inhibited the digestion of starch, cooking oil and casein, but did not inhibit digestion of whey protein and egg white protein. Instead, the digestion of egg white and whey protein by trypsin was moderately enhanced in the presence of GSPE. However, under simulated human digestion condition, the grape pomace in the bread significantly reduced the digestibility of bread starch and protein.

Role of survivin in apoptosis induced by grape seed procyanidin extract in human bladder cancer BIU87 cells

Objects： The aim of this study was to research the effect of grape seed procyanidin extract （GSPE） on cell apotosis in human bladder cancer BIU87 cells and investigate its molecular mechanism. Methods： BIU87 cells were treated with different concentrations of GSPE and cultured for 24 h in vitro while untreated group as control, MTT[3- （4,5-dimethylthiazole- 2-yl） -2, 5-diphenyltetrazolium bromide] assay, Hoechst 33258 stainning, flow cytometry, RT-PCR and Western blot were used to detect the apoptotic induction effect of GSPE on BIU87 cells. Results： We found that GSPE induced cell apoptosis in BIU87 cells by a dose-dependent manner. Semiquantitated RT-PCR and Western blot analyses indicated that GSPE increased the expression of caspase-3 and decreased the expression of survivin （P 〈 0.01）. Conclusion： GSPE induces apoptosis in BIU87 cells in vitro, and the effect maybe related with its down-regulation of survivin.

Vitamin C, Grape Seed Extract and Citrus Bioflavonoids Protect the Skin against Photoaging: A Review

The skin is a major protective organ of the body. It is constantly exposed to the environment and is very resilient. But exposure to ultraviolet (UV) rays from the sun results in the production of reactive oxygen species (ROS) and subsequent inflammatory responses that can overwhelm the innate protective mechanisms of the skin. This results in damage and premature aging. Strategies to mitigate this premature photoaging might include avoidance of sunlight. However, some sunlight exposure is beneficial to health. One notable example of this is the production of vitamin D. A more practical approach to preventing adverse effects of UV light in the skin is antioxidant supplementation. Dietary antioxidants may help control ROS propagation following UV light exposure. To further evaluate the utility of antioxidants in protecting the skin, in vitro, in vivo and human studies of three well known dietary antioxidants are reviewed and discussed. The data clearly demonstrate that vitamin C, grape seed extract and citrus bioflavonoids have the potential to reduce the damaging effects of excess sun exposure via antioxidant, anti-inflammatory and immunomodulating mechanisms. As such, regular ingestion of dietary antioxidants appears to be a useful strategy for protecting the skin against photoaging.

Grape Seed Extract and the Fetal Ductus Arteriosus: A Potential Danger of a Common Herbal Supplement

A female at 28 weeks gestation was referred to pediatric cardiology for a fetal arrhythmia. The echocardiogram revealed premature constriction of the fetal ductus arteriosus. Her work up was unremarkable except for her use of an herbal supplement, grape seed extract, which is advertised as a potent anti-inflammatory medication, and has biochemical properties similar to other medications that have been shown to cause premature ductal constriction. The use of herbal remedies increases each year. Although the public is inundated with radio, television, and internet advertisements for these products, little unbiased information regarding the possible dangers of toxicity or adverse reactions exists. As physicians, we need to be aware of these products, and counsel our patients accordingly.

A novel approach of proteomics to study the mechanism of action of grape seed proanthocyanidin extracts on diabetic retinopathy in rats

Background Diabetic retinopathy （DR） is a leading cause of visual impairment and blindness among the people of occupational age. To prevent the progress of retina injury, effective therapies directed toward the key molecular target are required. Grape seed proanthocyanidin extracts （GSPE） have been reported to be effective in treating diabetic complications, while little is discussed about the functional protein changes. Methods We used streptozotocin （STZ） to induce diabetes in rats. GSPE （250 mg/kg body weight per day） were administrated to diabetic rats for 24 weeks. Serum glucose, glycated hemoglobin and advanced glycation end products （AGEs） were determined. Consequently, 2-D difference gel electrophoresis and mass spectrometry were used to investigate retina protein profiles among control, STZ-induced diabetic rats, and GSPE treated diabetic rats. Results GSPE significantly reduced the AGEs of diabetic rats （P 〈0.05）. Moreover, GSPE significantly suppressed the vascular lesions of central regions, decreased capillary enlargements and neovascularization, similar to those of the control rats under light microscope. Eighteen proteins were found either up-regulated or down-regulated in the retina of STZ-induced diabetic rats. And seven proteins in the retina of diabetic rats were found to be back-regulated to normal levels after GSPE therapy. These back-regulated proteins are involved in many important biological processes such as heat shock, ubiquitin-proteasome system, cell proliferation, cell growth and glucose metabolism. Conclusions These findings might promote a better understanding for the mechanism of DR, and provide novel targets for evaluating the effects of GSPE therapy.

Grape Seed Extract Attenuates Demyelination in Experimental Autoimmune Encephalomyelitis Mice by Inhibiting Inflammatory Response of Immune Cells

Objective: To examine the anti-inflammatory effect of grape seed extract(GSE) in animal and cellular models and explore its mechanism of action. Methods: This study determined the inhibitory effect of GSE on macrophage inflammation and Th1 and Th17 polarization in vitro. Based on the in vitro results, the effects and mechanisms of GSE on multiple sclerosis(MS)-experimental autoimmune encephalomyelitis(EAE) mice model were further explored. The C57BL/6 mice were intragastrically administered with 50 mg/kg of GSE once a day from the 3rd day to the 27th day after immunization. The activation of microglia, the polarization of Th1 and Th17and the inflammatory factors such as tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), IL-6, IL-12, IL-17and interferon-γ(IFN-γ) secreted by them were detected in vitro and in vivo by flow cytometry, enzyme linked immunosorbent assay(ELISA), immunofluorescence staining and Western blot, respectively. Results: GSE reduced the secretion of TNF-α, IL-1β and IL-6 in bone marrow-derived macrophages stimulated by lipopolysaccharide(P<0.01), inhibited the secretion of TNF-α, IL-1β, IL-6, IL-12, IL-17 and IFN-γ in spleen cells of EAE mice immunized for 9 days(P<0.05 or P<0.01), and reduced the differentiation of Th1 and Th17 mediated by CD3 and CD28 factors(P<0.01). GSE significantly improved the clinical symptoms of EAE mice, and inhibited spinal cord demyelination and inflammatory cell infiltration. Peripherally, GSE downregulated the expression of toll-like-receptor 4(TLR4) and Rho-associated kinase(ROCKⅡ, P<0.05 or P<0.01), and inhibited the secretion of inflammatory factors(P<0.01 or P<0.05). In the central nervous system, GSE inhibited the infiltration of CD45+CD11b+and CD45+CD4+cells, and weakened the differentiation of Th1 and Th17(P<0.05). Moreover, it reduced the secretion of inflammatory factors(P<0.01), and prevented the activation of microglia(P<0.05). Conclusion: GSE had a beneficial effect on the pathogenesis and progression of EAE by inhibiting inflammatory response as a potential drug and strategy for the treatment of MS.

Buffalo stirred yoghurt fortified with grape seed extract:New insights into its functional properties

Grape seed extract(GSE)is considered one of the major byproducts,which are rich in phenolic content and possess several health-promoting activities including antibacterial,anticancer,and antioxidant.The current research was devoted to improve the bioactive properties of stirred yoghurt through supplementation with GSE at three concentrations(0.1,0.25,and 0.5 g/100 g yoghurt).The obtained stirred yoghurts were analyzed for determining their composition,pH,syneresis,viscosity,and sensory evaluation.Furthermore,some functional properties including antioxidant,antibacterial,anticancer activities were estimated.Significant increments(p≤0.05)were observed in total solids,ash contents,pH,water holding capacity,and viscosity values,especially when 0.5%GSE was added.Moreover,adding 0.5%GSE showed higher(p≤0.05)in vitro antioxidant,antibacterial,and anticancer activities.Interestingly,the improved bioactive properties resulting from adding GSE,even at the higher concentration,were not accompanied by any change in sensory properties.In particular,the color descriptor was mostly preferred by panelists when 0.5%GSE was added.The assessors did not observe any off-flavors in any of the tested stirred yoghurt samples.Therefore,the functional properties of stirred yoghurt could be improved by adding GSE preparation without affecting its organoleptic properties.