Sushma Devi,Randhir Singh

Department of Pharmaceutical Sciences,Maharishi Markandeshwar University,Mullana,Ambala,India

Abstract

Keywords:Vitis vinifera;Hypercholesterolemia;Cholesterol diet;Total phenolic content

1.Introduction

In food and pharmaceutical science,functional food is a growing field for the betterment of health.Functional foods are well-known to have positive effects on human health and helpful in prevent various disorders such as CVD,cancer,diabetes,cholesterol risk or other metabolic disorders.In addition,commercial and research activities on functional food/or their ingredients are in great stipulate.Grapes seed,fruit and their extract are already used as functional food[1].It is well known thatVitis viniferaleaves are edible and used for cooking.Young leaves wrapped around other foods and then baked,they impart a pleasant flavor and taste. Leaves consumed in traditional foods(Dolmathes)and used for diarrhoea,vomiting and varicose treatment[2].The leaves(especially red lecccaves)have antiinflammatory and astringent properties.Leaves decoction used as a wash for mouth ulcers and as douche for treating vaginal discharge.Red grape leaves are also helpful in treatment of varicose veins,haemorrhoids and capillary fragility[3].Various pharmacological activities like antioxidant,antidiabetic[4],hepatoprotective(leaves extract)[5],anti-atherosclerotic,antitumor,antioxidant,collagen stabilization,cytoprotective,hair growth,anti-ischemic(seed extract)were reported in experimental animals[6–8].Functional ingredients of grape include several flavonoids with a phenolic nature such as monomeric flavanols,dimeric,trimeric and polymeric procyanidins,and phenolic acids[1].Various reports indicated that extract ofVitis viniferaleaves have strong antioxidant activity due to presence of active phytoconstituents such as,phenolic,flavonoids,tannins etc[4].According to literature,in medicinal plants presences of flavonoids are responsible for antihyperlipidemic,tannins and saponins cause the inhibition of lipid absorption.So,the study was designed to evaluate the anti-hypercholesterolemic potential ofVitis viniferared leaves extract.Further,positive results can promote uses of grape leaves as functional food.

2.Material and methods

2.1.Chemical used

Gallic acid,Methanol(Nice chemicals),Cholesterol(Hi Media),Cholic acid(Hi Media),Simvastatin(sample from Beta Drugs Pvt.Ltd)and spectrophotmetric analysis was carried out by using UV spectrophotometer(Shimadzu).Erba diagnostic kits(Transasia,Mumbai)were used for estimation of total cholesterol,total triglyceride and HDL levels in serum.

2.2.Plant material

Fresh red leaves ofVitis viniferawas collected on April 2013 from the Tau Devilal National herbal park,Khizrabad,Haryana,India and authenticated by Dr.Shiddamallayya N.,National Ayurveda Dietetics Research Institute,Banglore,India(specimen number RRCBI-MUS-125).

2.3.Preparation of extract

Leaves ofVitis viniferawas washed in water,shade dried and grinded into coarse powder.Then,weighed plant material was packed into soxhlet and extraction was carried for 72h using methanol and water.The extract was concentrated using vacuum rotary evaporator at 40℃,dried,weighed and stored in a refrigerator at 4℃ throughout the duration of study.The% extraction yields of both extracts were calculated using formula[9]:

2.4.Qualitative estimation of phytoconstituents

The qualitative phytochemical screening of VVME and VVAE was carried out to determine phytoconstituents present by using standard test[10,11].

2.5.Quantitative estimation of phytoconstituents

The quantitative amount of total phenolic content(mg/g of gallic acid equivalent dry weight)[12],total flavonoids content(rutin equivalents(mg/g))[13],total tannin content(rutin equivalents(mg/g))[14],total alkaloids content(mg of atropine/g of extract)[15],total saponin content(diosgenin equivalents(mg/g))[16],total steroid content(cycloartenol equivalents(mg/g))[16]and total terpenoids content[15]were estimated using standard procedure.

2.6.Animals

Wistar male rats were used in the study and experimental protocol was duly approved by Institutional Animal Ethics Committee(MMCP/IAEC/13/36).Animals were kept as per the guidelines of committee for the purpose of control and supervision of experiments on animals(CPCSEA)in Department of Pharmaceutical Sciences,Maharishi Markandeshwar University,Ambala,India.Animals were fed normal chow diet andad libitumunder controlled environmental condition of temperature(24–28℃),relative humidity 60%–70% and natural light/dark cycle(12:12).

2.7.Induction of hypercholesterolemia with cholesterol diet

Wistar male rats(220–250g)were procured under controlled environmental conditions.Atherosclerosis was induced by administration of cholesterol diet(cholesterol 2% w/w and cholic acid 0.5% w/w along with basal diet)for 21days.Simvastatin(dose 10mg/kg),VVME and VVAE(dose 100mg/kg,200mg/kg and 400mg/kg)were administered for 21days[17,18].

2.7.1.Changes in body weight

The change in the body weights was recorded weekly and % change in body weights was calculated using formula[19]:

2.7.2.Biochemical estimations

Blood was collectedviaretro-orbital plexus on 21st day and centrifuged at 3000rpm for 10min and serum was separated.The serum glucose,triglycerides,total cholesterol and HDL level was measured using enzymatic kits.The levels of LDL and VLDL were calculated using Friedewald equation[17].

2.7.3.Liver functions test

The levels of SGOT and SGPT were also analysed in the blood samples at the end of study using enzymatic kits.

2.7.4.Histopathological studies

Then,animals were sacrificed and heart was isolated for histopathology.A portion of heart tissue was dissected out and fixed in 10% formalin solution and histopathological studies were carried out.

2.7.5.Atherogenic index

Atherogenic index and % protection was also calculated at the end of study using formulas[20]:

2.8.Statistical analysis

All the data were shown mean values and represented as mean± SEM.Statistical analysis was done with Dunnett’s multiple comparison tests using Graph pad Instat Demo software(version 3.10).In statistical analysis,p＜0.05 was considered to be significant;b=vscholesterol control;c=vs100mg/kg dose;d=vs200mg/kg dose;p＜0.05=*;p＜0.01=?;p＜0.001=#.

3.Results and discussion

3.1.Qualitative and quantitative estimation of phytoconstituents

The % extraction yield of VVME and VVAE was found as 9.2% and 11.5% w/w,respectively.VVME and VVAE revealed that flavonoids,phenolic,tannins,saponins,steroids and terpenoids present.Moreover,alkaloids present in VVAE only.In VVME,total tannins content,total flavonoids content and total phenolic content was found in major amount(Table 1).

Table 1Quantitative estimation of phytoconstituents in VVME and VVAE.

3.2.Change in body weight of experimental animals

The weight gain in the rats was found significant with administration of cholesterol diet during the experimentation.The % change in body weight of VVME 100mg/kg,200mg/kg and 400mg//kg group animals were found 13.5%,11.8% and11.7%,respectively.The% change in body weight of VVAE100mg/kg,200mg/kg and 400mg/kg groups were found 22.2%,23.6% and 17.6% respectively.Excessive cholesterol feeding leads to susceptibility to hypercholesterolemia and arteriosclerosis and further promotes the development of obesity and dyslipidemia in both humans and rodents by altering the plasma cholesterol and triglyceride levels[15].According to literature,hypercholesterolemic animals are used to study the cholesterol homeostasis as convenient models. As well as, this model is obliging to understand the association between cholesterol metabolism disorders,atherogenesis or possible treatments to reduce lipid levels in drugtrials[21].

3.3.Effect of VVME and VVAE on glucose level

Different doses of VVME and VVAE statistically lowered the glucose level as compared to cholesterol control.(Table 2)

Table 2Effect of VVME and VVAE on glucose and lipid levels in cholesterol induced hypercholesterolemia.

3.4.Effect of VVME and VVAE on lipids level

The lipids level except HDL level was found to be significantly elevated in experimental animals and different doses of VVME and VVAE produced a significant attenuation in lipid level.Whereas,HDL level was found to be decreased in cholesterol control animals and administration of VVME and VVAE significantly elevated(p＜0.05)the HDL level after 21days treatment.(Table 2)

High cholesterol diets acts as extrinsic inducer and significantly increase the cholesterol,triglyceride,LDL levels and decrease HDL level.Increase in LDL has been indicated one of the risk factors in development of atherosclerosis and other related cardiovascular disorders[22].High triglyceride levels also a marker and important risk factor that influences lipid deposition and clotting mechanisms. Numerous experimental reportsshowed that cholesterol and high dietary fat induce hypercholesterolemia in animal models [23]. Similar results were alsoobserved with the high cholesterol diet animals have increaselipid status and increased lipid level act as indicator of establishment of hypercholesterolemia in animal models [24]. Moreover,HDL is inversely associated with total cholesterol and severalevidences are accessible regarding this fact. A reduction in HDLlevel may impair the clearance of cholesterol from the arterialwall and speed up the development of atherosclerosis that furtherlead to ischemic heart diseases[25].

3.5.Effect of VVME and VVAE on SGOT and SGPT level

Administration of cholesterol diet resulted in elevation of SGOT and SGPT levels in cholesterol control animals as compared to normal control after 21 days.Different doses of extracts significantly attenuated the elevated SGOT and SGPT levels.(Fig.1)The elevated level in cholesterol control animals may be due to leakage of the enzymes into the serum and damage the integrity of the heart and liver.Also,increased level of these enzymes is reported as indicators of deliberate risk of cardiovascular disease [26]. In case of severe hepatocellular injury, SGOTand SGPT are released into serum. In the absence of viral hepatitis and alcoholism, increased SGPT level can lead to a higher riskof cardiovascular disease with more risk in women. Also a highSGOT content is found in heart which becomes more elevated inmyocardial infarction case[27].Also,increased level of SGOT and SGPT level in cholesterol control animals were supported by histology results that showed the disruption of endothelial lining in aorta,presence of foamy macrophage,increase thickness of lining and cardiovascular distress in cholesterol control animals.

3.6.Histopathology of blood vessels

The histology of blood vessels(tunica intima+media thickness)in cholesterol control showed disruption of the endothelial lining.Within the tunica intima and media were seen lipids containing elongated smooth muscle cells in single and aggregates of foamy macrophages and thickness was found to be 53.2μm.In VVAE 400mg/kg section,layers of artery appeared intact except for disruption of the endothelial lining.Within tunica intima and media were seen lipids containing smooth muscle cells in single and aggregates of foamy macrophages.The thickness of tunica intima+media was found 45.25μm.In VVME 400mg/kg section,the layers of artery appeared intact and few areas appeared disrupted.There were seen few scattered lipid containing spindle cells between the tunica intima and tunica media and thickness was found to be 45.0μm(Fig.2).

Fig.2.Histology of blood vessels(Tunica intima+media thickness)of different groups.

3.7.Effect on atherogenic index and % protection

The cholesterol control have significant atherogenic index as compared to normal control(0.422?high risk).In VVME 400mg/kg found lowest atherogenic index(0.120)and maximum % protection as compared to other experimental groups.(Table 3)In VVME 400mg/kg group,atherogenic index and lipid profile were significantly improved with an improvement in the thickening of aortic walls.Also,it can be state that decreased lipid levels might be an experimental tool to determine antiatherogenicity of plant extract or other metabolites[28].

Table 3Effect on atherogenic index and% protection in cholesterol induced hypercholesterolemia.

4.Conclusion

Vitis viniferaextracts were found effective in reducing cholesterol level and improved HDL level in experimentally induced atherosclerosis in rats.Also,the administration of extract decreases the disruption of endothelial lining and thickness of blood vessel lining in experimental animals.The effectiveness ofVitis viniferaextract as anti-hypercholesterolemic agent due to presence of active phytoconstituents and antioxidative constituents.Further,active phytoconstituents Resveratrol isolated from VVME and its anti-atherosclerosis and anti-dyslipidemic activities in process.

Conflict of interest

Authors have no conflict of interest.

Acknowledgements

Authors convey their gratitude to Navdeep Bharti,Humed Life Sciences Pvt.Ltd.and Maharishi Markandeshwar University for support of this project.