Zincovit tablets (grape seed extract and zinc containing multivitamin-mineral nutritional food supplement) were supplied by Apex Laboratories Private Ltd., Chennai, India. Atorvastatin (Atocor 10 mg tablets; Dr. Reddy’s Laboratory Ltd., Hyderabad, India) was purchased from pharmacy of Kasturba hospital, Manipal, Karnataka (India). The diagnostic kits for estimation of triglyceride (TG), total cholesterol (T-CHO) and high-density lipoprotein cholesterol (HDL-C) were procured from Aspen Laboratories, New Delhi (India). The reagents were equilibrated at room temperature for 30 min before use, either at the start of analysis or when reagent containers were refilled.

Preparation of aqueous solution of Zincovit tablets for oral administration: Zincovit tablet is a single combined formulation of vitamins, minerals and grape seed extract. Each tablet of Zincovit weighs 850 mg. Ten tablets of Zincovit were crushed and fine powder form was dissolved in 100 ml of distilled water containing 2 g gum acacia (2% gum acacia). The aqueous solution of Zincovit tablets was stored in an amber coloured bottle at 4°C in refrigerator.

Adult male Wistar rats aged 8-12 weeks weighing 150-250 g were used. They were housed in individual cages, maintained under the temperature (22–24°C), 12-h light/12-h dark cycle and relative humidity 40–60%. The animals were fed with a normal pellet diet obtained from Amrit Feeds Ltd., Pune, India and they had free access to water. Animals described as fasted were deprived of food for 12-h but had allowed free access to water. The experimental protocol was approved by the Institutional Animal Ethics Committee (IAEC/KMC/86/2012) and experiments were conducted according to the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines.

The hypercholesterolemic diet included hydrogenated vegetable oil (Vanaspati Ghee) and cholesterol extra pure powder made in to 20% suspension in coconut oil. The suspension was administered at the dose of 5 ml/kg/day at evening hours for 30 consecutive days [15].

Experimental procedure: In the experiment, 54 adult male Wistar rats were divided into nine groups (n= 6). The corresponding doses of drugs were administered orally till 30 days as follow-

Group I: Normal control animals received 2% gum acacia (1 ml/kg/day)

Group II: Hypercholesterolemic control animals received cholesterol in oil (5 ml/kg/day) + 2% gum acacia (1 ml/kg/day)

Group III: Hypercholesterolemic animals received cholesterol in oil (5 ml/kg/day) + Atorvastatin (0.9 mg/kg/day)

Group IV: Normal animals received Zincovit tablets with grape seed extract (40 mg/kg/day)

Group V: Normal animals received Zincovit tablets with grape seed extract (80 mg/kg/day)

Group VI: Normal animals received Zincovit tablets with grape seed extract (160 mg/kg/day)

Group VII: Hypercholesterolemic animals received cholesterol in oil (5 ml/kg/day) + Zincovit tablets with grape seed extract (40 mg/kg/day)

Group VIII: Hypercholesterolemic animals received cholesterol in oil (5ml/kg/day) + Zincovit tablets with grape seed extract (80 mg/kg/day)

Group IX: Hypercholesterolemic animals received cholesterol in oil (5ml/kg/day) + Zincovit tablets with grape seed extract (160 mg/kg/day)

At the end of the experimental period, the animals were anesthetized with ketamine (80 mg/kg; i.p.) following a 12 h fast. Serum was obtained by centrifugation of blood at 3,000 rpm for 20 min at 4°C using a refrigerated centrifuge (MIKRO 22R, Andreas Hettich GmbH & Co. KG, Germany). The resulting supernatant (serum) was stored at-20°C.

Serum was analysed further for assay of triglyceride (TG), total cholesterol (T-CHO) and high-density lipoprotein cholesterol (HDL-C) according to the standard protocols given along with respective kits (Aspen Laboratories, New Delhi, India). Low-density lipoprotein cholesterol (LDL-C) and Very low-density lipoprotein cholesterol (VLDL-C) was calculated by using Friedewald’s equation:

VLDL-C = Triglycerides (TG)/5

LDL-C = Total cholesterol – (HDL-C+VLDL-C)

Using Statistical Package for the Social Sciences (SPSS version 16.0; SPSS Inc., Chicago, USA), data were expressed as mean ± standard error of mean and analysed by one-way analysis of variance (ANOVA) followed by post-hoc Tukey test. A level for p ≤ 0.05 was considered to be statistically significant.

There was no significant impact of combined formulation of grape seed extract and Zincovit tablets on lipid profile among normal animals in comparison with normal control group.

In hypercholesterolemic control animals, there was significant increase in serum triglyceride, total cholesterol, low-density lipoprotein cholesterol and very low-density lipoprotein cholesterol level when compared with normal control animals.

Both atorvastatin and combined formulation of grape seed extract and Zincovit tablets significantly attenuated triglyceride, total cholesterol, low-density lipoprotein cholesterol, very low-density lipoprotein cholesterol (p < 0.001) and improved high-density lipoprotein cholesterol level (p < 0.001) among hypercholesterolemic treated rats in comparison with hypercholesterolemic untreated animals [Table/Fig-1a–e].

[Table/Fig-1]:

Effect of combined formulation of grape seed extract and Zincovit tablets among experimental animal groups on lipid profile: (a) triglyceride level (b) total cholesterol level; (c) High-density lipoprotein cholesterol level (d) Low-density lipoprotein cholesterol level; (e) Very low-density lipoprotein cholesterol level, n = 6, number of rats in each group; HC, hypercholesterolemic; ZVT, zincovit tablets with grape seed extract. Values are mentioned as mean. Error bars, +/- 2 standard error of the mean ****p<0.001, *p<0.01, a and b- compared to normal control and hypercholesterolemic control group.

In this study, we investigated the influence of combined formulation of grape seed extract and Zincovit tablets (nutritional food supplement) on lipid profile of Wistar rats. Increased levels of different types of lipids have been implicated in the production of atherosclerosis. Reactive oxygen species induced depletion of antioxidants is a key factor for the initiation of atherosclerosis and the development of cardiovascular disease [16]. Oxidized low-density lipoprotein (LDL) is present in atherosclerotic plaques and is involved in the transition of stable atherosclerotic lesions into active lesions [4]. The precise mechanisms involved have not been fully explained, but oxidation of LDL is believed to play a role in the initiation of atherosclerosis, leading to LDL uptake by macrophages and foam-cell formation, but becoming less important in the later stages of the condition [17,18]. Current knowledge includes effective percutaneous and/or medical management of coronary disease and heart failure [19]. The beneficial effects of medical treatments may be related to their known antioxidant properties [20]. This study demonstrated that, all the three different doses (40, 80 and 160 mg/kg) of combined formulation of grape seed extract and Zincovit tablets treated animals exhibited drastic decrease in serum triglycerides, total cholesterol, LDL-C, VLDL-C and rise of HDL-C in comparison to hypercholesterolemic control group animals. The anti-hyperlipidemic effect of single combined formulation of grape seed extract and Zincovit tablet was comparable with the standard drug atorvastatin (0.9 mg/kg) treated animals and the variations were statistically non-significant. All statins including atorvastatin are well known for the risk of myopathy, rhabdomyolysis and hepatotoxicity in addition to wide range of drug interactions in a dose dependent fashion. Thus, the combined formulation of grape seed extract and Zincovit tablets (nutritional food supplement) can be a better add-on/substitute drug for statins in hyperlipidemic conditions.

Earlier, we had reported combined formulation of grape seed extract and Zincovit tablets for its strong in vitro, in vivo antioxidant, anti-hyperglycemic and anti-cataractogenic potential [10–14]. A recent study has shown that a combination of IH636 grape seed proanthocyanidin extract and niacin-bound chromium can decrease total cholesterol, LDL and oxidized LDL level in hypercholesterolemic human subjects [9]. In one of the study, it has been suggested that proanthocyanidins, might trap reactive oxygen species in plasma and interstitial fluid of the arterial wall, thereby inhibit the oxidation of LDL and show an anti-atherosclerotic activity [21]. In the current study, the anti-hyperlipidemic property of combined formulation of grape seed extract and Zincovit tablets might be attributed to the synergistic interplay of constituents of Zincovit tablets, such as-grape seed extract proanthocyanidins which comprise only procyanidins [subunits constituted of (+) catechin (C) and (–)-epicatechin (EC)], Vitamins A, B, C, D, E, folic acid, biotin and minerals like zinc, copper, selenium, magnesium, manganese, chromium and molybdenum mainly, which are promoters of antioxidant activity and act against oxidative stress.

Before moving from bench to bedside, it is essential to further investigate the influence of long term therapy of the combined formulation of grape seed extract and zinc containing multivitamin-mineral nutritional food supplement (Zincovit tablet) on lipid profile in other relevant models. The therapeutic effect seen in animal studies cannot always be entirely extrapolated to humans. Hence, clinical evaluation should be performed to precisely define the cardio-protective role of Zincovit tablets with grape seed extract in humans

Thus, the present study demonstrates that the single combined formulation of grape seed extract and Zincovit tablet is the potential functional nutritional food supplements that could offer a novel therapeutic opportunity against diet-induced hypercholesterolemia in Wistar rats. Our study opens the perspective to clinical studies which could improve the clinical outcome of patients subjected to percutaneous angioplasty/statin therapy, a novel view likely to give rise to the performance of clinical trials devised to demonstrate the validity of this paradigm as nutritional food supplement. “This information would eventually complement our findings, opening the way to sustain cardiovascular disease progression in human population”.