Abstract
Hyperlipidemia can lead to atherosclerosis by lipoprotein deposition inside the vessel wall and oxidative stress induction that leads to the formation of atherosclerotic plaque. Oxidized low-density lipoprotein particles (Ox-LDL) have a key role in the pathogenesis of atherosclerosis. The lipid-lowering properties and antioxidants of the grape seed can be beneficial in atherosclerosis prevention. We conducted a randomized double-blind placebo-controlled crossover clinical trial. Fifty-two mildly hyperlipidemic individuals were divided into two groups that received either 200 mg/day of the red grape seed extract (RGSE) or placebo for 8 weeks. After an 8-week washout period, the groups were crossed over for another 8 weeks. Lipid profiles and Ox-LDL were measured at the beginning and the end of each phase. RGSE consumption reduced total cholesterol (−10.68±26.76 mg/dL, P=.015), LDL cholesterol (−9.66±23.92 mg/dL, P=.014), and Ox-LDL (−5.47±12.12 mg/dL, P=.008). While triglyceride and very low–density lipoprotein cholesterol were decreased and high-density lipoprotein cholesterol was increased by RGSE, the changes were not statistically significant. RGSE consumption decreases Ox-LDL and has beneficial effects on lipid profile—consequently decreasing the risk of atherosclerosis and cardiovascular disorders—in mild hyperlipidemic individuals.
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Lipid-lowering properties of grape products have been investigated in many animal and some human studies, some with promising results, and some investigators have debated their effect on lipid profiles. 5 –9 Antioxidants contained in the red grape seed extract (RGSE) are able to inactivate superoxide anions and prevent lipid peroxidation. 10 RGSE has cardioprotective effects against reperfusion-induced injury by free radicals after ischemia. 2,11 In this study, we evaluated the lipid-lowering properties of RGSE and its effect on Ox-LDL in mild hyperlipidemic individuals.
A randomized double-blind placebo-controlled clinical trial was carried out at Shahid Modarres Hospital (Tehran, Iran). The target population were adults aged 21–64 years, with triglyceride (TG) >150 mg/dL and total cholesterol >200 mg/dL. Individuals with severe hyperlipidemia (TG >300 mg/dL, total cholesterol >250 mg/dL), body mass index (BMI) >30 kg/m2, heart failure, chronic renal failure, chronic hepatic disease, malignancy, any lipid-lowering drug usage, vegetarian diet, alcohol, and cigarette use were excluded from the study. During a nonrandom sequential sampling of subjects participating in the screening program for cardiovascular disease at the hospital's clinics, 52 subjects were randomly assigned into two groups (RGSE and placebo) using a computerized random numbers table. Individuals responsible for grouping the subjects and the executive committee were unaware of group assignments. All participants signed the informed consent and the study was registered in
The grape seed capsule used in this study was prepared at the Drug Applied Research Center (Tabriz, Iran). 12 Capsule ingredients were as follows: dicalcium phosphate, gelatin, microcrystalline cellulose, and 100 mg of RGSE. Each capsule contained the equivalent to 5–8 grape seeds. In an analysis performed on this capsule, it contained at least 95% of proanthocyanidins and 80% polyphenolic compounds.
Recruited individuals participated in a series of meetings to get acquainted with the aim and significance of the study. Participants were advised not to change their lifestyle, general nutritional habits, and daily physical activity during the study period. Patients were encouraged to continue consuming any dietary supplementation or medication they were using before the study, but were asked to stop consumption of any grape product during the active phases.
The study design is illustrated in Figure 1. Initial blood samples were taken after overnight fasting at the beginning of the study. Specimens were centrifuged and the extracted serum was stored at −94°C. The RGSE group received RGSE capsules (containing 100 mg RGSE) twice a day for 8 weeks, and the placebo group received similar-looking capsules (filled with starch) for the same amount of time. A second blood sample was collected at the end of the first round. After an 8-week washout period, the groups were crossed over. Two more blood samples were collected at the beginning and end of the second round. Blood pressure, BMI, and physical activity (number and length of activities per week) were recorded at the time of sample collection. Daily nutritional intake was recorded using a food frequency questionnaire at the beginning and end of each round. The questionnaires enabled the interviewer to calculate the amount of each dietary intake category (protein, carbohydrate, fat, and fiber) as grams/day and categorize it on a scale of 0 to 10.
Schematic representation of the study design.
Serum total cholesterol, TG, and high-density lipoprotein cholesterol (HDL-C) were measured by an enzymatic colorimetric method with an automated chemical analyzer (Abbott analyzer; Abbott Laboratories, Abbott Park, North Chicago, IL, USA). The very low–density lipoprotein cholesterol (VLDL-C) level was calculated by dividing TG by five (TG/5), and low-density lipoprotein cholesterol (LDL-C) was calculated with the Friedewald formula. Ox-LDL was measured using an ELISA test (Mercodia Oxidized LDL ELISA kit; Mercodia, Inc., Uppsala, Sweden).
Changes in dietary intake were analyzed by the Wilcoxon signed ranks test. The paired samples t-test was employed to compare before and after values in each group. Between-group comparisons were carried out using the independent samples t-test. Data were analyzed with SPSS software (SPSS, Inc., Chicago, IL, USA) and P<.05 was considered statistically significant.
Fifty-two patients were initially recruited. Four patients failed to complete the first round because of lack of cooperation. From 48 patients (20 males, 28 females) finishing the first round, 6 patients were lost to follow-up in the washout period. All 42 remaining patients (18 males and 24 females) completed the second round, and analysis was performed on a total of 90 cases. Values that were different from the median by more than 1.5 interquartile ranges were considered outliers, resulting in the exclusion of 15 cases (Fig. 1).Ten subjects had unusual (very low or very high) laboratory values due to improper sample handling or a laboratory error. Lipid profile of the other five cases clearly placed the subject in the severe hyperlipidemic range, both before and after treatment.
The mean age of the patients was 48.22 (±9.07 SD) years. In spite of all precautions, the daily protein (P<.001) and carbohydrate (P<.001) intake had increased significantly during active phases, but mean daily fat and fiber ingestion had no meaningful change.
HDL-C, LDL-C, VLDL-C, Ox-LDL, TG, total cholesterol, weight, and systolic and diastolic blood pressure data are shown in the Table 1.
Comparing mean change of variables between RGSE and placebo groups.
Statistically significant.
TG, triglyceride; RGSE, red grape seed extract; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; VLDL-C, very low–density lipoprotein cholesterol; Ox-LDL, oxidized low-density lipoprotein cholesterol.
The placebo and RGSE groups had no significant difference in any variable mean at the beginning of the study. The mean weight was significantly increased after 8 weeks in both RGSE (P=.007) and placebo (P=.024) groups. The RGSE group exhibited a significant decrease in total cholesterol (−10.68±26.76 mg/dL, P=.015), LDL-C (−9.66±23.92 mg/dL, P=.014), and Ox-LDL (−5.47±12.12 mg/dL, P=.008) (Table 1).
Comparing the mean change of variables after treatment between groups, the RGSE group exhibited a greater decrease in Ox-LDL (−11.23±16.21 mg/dL, P=.004) and total cholesterol (−16.31±30.75 mg/dL, P=.043) compared to the placebo group (Table 1).
Our results show that RGSE can effectively reduce serum total cholesterol, LDL-C, and Ox-LDL in individuals with mild hyperlipidemia, but has no significant impact on TG, HDL-C, and VLDL-C. Animal and in vitro studies have found no toxic effect and few possible mutagenic effects of the grape seed extract at very high doses (4–5 g/kg). 13,14 We administered relatively smaller doses to our study subjects and excluded patients with severe hyperlipidemia or other underlying disease, further minimizing any risk of RGSE consumption. We observed a meaningful increase in the carbohydrate and protein consumption and weight in both groups suggesting a possible confounding effect on serum lipid profiles. Regression analysis did not find any of these variables to be a predictor of lipid profiles or Ox-LDL.
French people consume more fat, exercise less, and smoke more than other western societies, still their death from cardiovascular disease is much lower; a phenomenon known as the French paradox. 15 It is verified that polyphenolic derivatives in red wine (e.g., oligomeric proanthocyanidins) are the origin of this chemoprotective effect. 15 –17
Current data regarding the effect of RGSE and other grape products on plasma lipid profiles are inconsistent. Many studies have found RGSE to improve plasma lipid profiles in animal models. 5,6 The proanthocyanidin-rich grape seed extract had no effect on plasma lipid profiles in cholesterol-fed rabbits, whereas it decreased the number of Ox-LDL-positive foam cells in atherosclerotic lesions in the aorta. 7 A few studies have investigated the lipid-lowering and antioxidant activity of RGSE in humans. In a study conducted on 24 heavy smoker subjects, Vigna et al. found no significant change in plasma lipid profiles after 4 weeks consumption of 75 mg RGSE twice daily; however, RGSE decreased the susceptibility of LDL-C oxidation. 8 In another study of 40 hypercholesterolemic individuals divided into four groups, combined administration of niacin-bound chromium and the grape seed proanthocyanidin extract decreased total cholesterol and LDL-C after 2 months. 9 With previous studies in mind, we included individuals with mild hyperlipidemia and only used RGSE as an intervention against placebo to avoid any confounding factor in both the population selection and in the treatment module. We also increased the sample size and RGSE dose and extended the treatment duration. The results of our study confirm the lipid-lowering and antioxidant effects of RGSE.
Many studies have investigated the effects of RGSE on LDL-C oxidation. In an in vitro model, the grape seed showed a greater dose-dependent scavenging ability against the superoxide anion and the hydroxyl radical compared to vitamins C and E. 18 The effect of RGSE, red wine, and grape juice on curtailing the oxidation of LDL-C has been verified by many studies. 2,8,19 Our results confirm RGSE as a potent antioxidant that can reduce serum Ox-LDL.
Despite promising results, there are some limitations in our study. The sample size and treatment time were limited. We used RGSE as a whole and did not distinguish between different contents of the grape seed extract and their possibly different impacts on lipid profiles. Food frequency questionnaires were accurately designed, but further details on the constituents of each food category, which could have helped in additional analysis of confounding effects, were not recorded.
Considering the cardioprotective effect of grape seed found in previous studies and with regard to our results, it could be suggested that RGSE consumption has favorable effects on serum lipid profile and decreases Ox-LDL, ultimately decreasing the risk of atherosclerosis and cardiovascular disorders in mild hyperlipidemic individuals. Long-term effects of RGSE on hyperlipidemic patients have yet to be investigated.
Footnotes
Acknowledgments
The authors appreciate the assistance of the physicians and staff of Shahid Modarres Hospital cardiology clinics. They also wish to thank all participating patients for their cooperation.
Author Disclosure Statement
No competing financial interests exist.