STUDY OF ANTIATHEROSCLEROTIC ACTIVITY OF POLYHERBAL PREPARATION USING RAT AS AN EXPERIMENTAL ANIMAL MODEL

STUDY OF ANTIATHEROSCLEROTIC ACTIVITY OF POLYHERBAL PREPARATION USING RAT AS AN EXPERIMENTAL ANIMAL MODEL

J.P. Machhi* and N.N. Shah

Department of Pharmacology, A.R. College of Pharmacy & G.H. Patel Institute of Pharmacy, Vallabh Vidyanagar- 388 120, Gujarat, India

ABSTRACT

Atherosclerosis is one of the risk factors for coronary artery disease. The present study highlights the antiatherosclerotic activity of Methanolic extract of polyherbal preparation in experimentally induced atherosclerotic rats. Atherosclerosis was developed in male Albino Wistar rats, which were randomly divided into five groups of six animals each; by feeding with high cholesterol diet for 21 days. Group 1 received normal diet. Group 2 received high cholesterol diet (HCD) which served as control. Group 5 served as standard, administered with Atorvastatin (10 mg/kg) along with HCD and Group 3 and Group 4 were administered with methanolic extract of polyherbal preparation (300 mg/kg and 600 mg/kg) along with HCD. Methanolic extract of polyherbal preparation reduced the raised serum level of total cholesterol,  triglyceride , LDL ,VLDL and increased the serum  HDL level as compared to the control group(High cholesterol group).There was increased HMG CoA/Mevalonate ratio in liver. There was also depletion of GSH content and increased level of SOD and MDA in liver. Methanolic extract of polyherbal preparation treated groups (300 mg/kg and 600 mg/kg) exhibited less damage to endothelial lining of aorta as compared to control group (High Cholesterol Diet),which may be attributed to hypo-cholesterolemic activity of polyherbal preparation, which may be attributed to hypocholesterolemic activity of polyherbal preparation.

High cholesterol diet

INTRODUCTION: Atherosclerosis  is  a  disease  of  blood  vessels  and  known  colloquially  as  “hardening  of  the  arteries”.  It  is  characterized  by  the  accumulation  of  fatty  substance,  cholesterol,  cellular  waste  products,  calcium  and  other  substances  in  the  inner  lining  of  an  artery.  Major  complications  of  atherosclerosis  include  angina  pectoris,  myocardial  infarction  and  stroke,  which  are  recognized  as  leading  causes  of  morbidity  and  mortality  in  Western  countries.  The  World  Health  Organization  (WHO)  predicted  that  heart  diseases  and  stroke  are  becoming  more  deadly,  with  a  projected  combined  death  of  24  million  by  2030 ¹.

Due  to  accumulation  of  fat, cholesterol  and  other  substances, plaque  builds up inside the arteries.  Arteries  are  blood vessels  that  carry  oxygen-rich  blood  to  the  heart  and  other  parts  of  the  body.

Over  time,  plaque  hardens  and  narrows  the  arteries.  This  limits  the  flow  of  oxygen-rich  blood  to  the  organs  and  other  parts  of  the  body. Atherosclerosis  can  lead  to  serious  problems,  including  heart  attack,  stroke,  or  even  death ². The  development  of  atherosclerosis  is  a  complex  and  multistep  process.

There  are  a  number  of  genetic,  metabolic,  and  environmental  factors  involved  in  the  formation  and evolution of the atherosclerotic plaque.  Lipoprotein oxidation and oxidative processes in  general  play  an  important  role  in  the  pathogenesis  of  atherosclerosis ³.

Disorders  of  lipid  metabolism  are  menifested  by  elevation  of  the  plasma  concentration  of  the  various  lipid  and  lipoprotein  fractions  (total  and  LDL  cholesterol, VLDL  cholesterol, triglycerides  and  chylomicrons) they  result,  predominantly  in  cardiovascular  diseases. So  hyperlipidemia  is  one  of  the  major  cause  of  atherosclerosis  and   associated  conditions ⁴.

Synthetic  antilipidemic  drugs  clearly  reduce  cardiovascular  mortality  but  are  expensive  and  sometimes  highly  prone  to  side  effects. However they  are  not  covered  by  most  health  care  insurers  when  used  primarily  for  prophylactic  purpose ⁵. The plant kingdom represents a rich storehouse of organic compounds,many of which have been used for medicinal and other purpose. There exists a plethora of knowledge, information and benefits of herbal drugs in our ancient literature of Ayurvedic and Unani medicines. Ethanomedicines are replete with description of plant medicines and the grandma’s pouch that has been called from years of medical wisdom is excellent proof of efficacy of the experimental medicines ⁶.

Acorus  calamus    has  been  indicated  in  the  Indian  System  of  Medicine  (ISM)  to  be  useful  in  the  aggravation  of  ‘medas’,  its  hypolipidemic  activity  has  been  explored ⁷. Its antiatherosclerotic effect may be due to presence of saponin which forms complex with cholesterol by binding plasma lipid and thereby alters cholesterol metabolism and absorption ⁸. Curcuma longa shows hypocholesterolemic effect due to curcumin which enhance excretion of cholesterol in bile. It also protect LDL from oxidation ⁹.

Cyperus rotundus having antioxidant and anti-atherosclerotic effect due to presence of flavanoids, polyphenols and terpenes which reduce absorption of total cholesterol and triglycerides ¹⁰. Picrirhiza kurroa  having ayurvedic properties of tikta rasa, laguruksha guna, and katuvipaka. Based on these properties it is having antioxidant and antiatherosclerotic effect ¹¹. Plumbago zeylanica, due to presence of plumbagin, prevents accumulation of cholesterol and triglyceride in liver and aorta and regress atheromatous plaque in thoracic and abdominal aortas ¹². 

Herbal  medicinal  preparations  play  an  especially  important  role  in  prevention  of  atherosclerosis. Their  therapeutic  action  is  directed  against  important  mechanisms  involved  in  the  development  of  atherosclerosis. The  herbal  alternative  have  a  very  low  incident  of  side  effects  and  can  be  recommended  for  medically  supervised  self-treatment ⁵.

The  aim  of  the  current  investigation  is  to  evaluate  the  antioxidant  and antiatherosclerotic  activity  of  the  polyherbal  formulation  containing  following  six  herbal  drugs  using  rat as experimental  model.

• Acorus calamus
• Curcuma longa
• Cyperus rotundus
• Picrorrhiza kurroa
• Plumbago zeylanica

MATERIAL AND METHODS:

Plant Material: Methanolic extracts  of all six plants were procured from AMSAR PVT. LTD., Indore.

Composition of Polyherbal Preparation:

Dried powdered methanolic extracts of all five plants were mixed according to the above praportation. Freshly prepared aqueous solution of this mixed extract (PHE) was used for experimental study.

Drugs and Chemicals: Greetish sample of Atorvastatin was obtained from Astron Pharmaceutical Contract Research Organization,Ahmedabad. All other reagents and chemicals were of analytical grade.

Animals: Healthy male albino rats (250-350g) of Wistar strain were used for the study with the approval of Institute’s animal ethics committee. The animals were purchased from Jay Research Foundation,Surat. The animals were housed in a large spacious cage, bedded with husk and were given food and water. The animal house was ventilated with a 12hr light/dark cycle, throughout the experimental period. Animal experimentation was conducted according to the current institutional regulations. The animals were maintained on a commercial rat feed manufactured by M/s. Pranav Agro Industries Ltd., India, under the trade name ‘Amrut rat feed’. The feed contains 5% fat, 21% protein, 55% nitrogen free extract, 4% fiber (wt/wt) with adequate vitamin and mineral content.

After the completion of duration of study (21 days), the animals were fasted overnight and sacrificed by cervical decapitation. Their livers were dissected out on a petri dish kept in an ice-bath to keep the sample fresh. Sufficient care was tsken to complete the entire procedure within 5-7 min to prevent loss of the enzyme activity in liver samples. The aorta was excised immediately, washed with cold saline. The aorta was fixed by 10% formalin for histopathological studies

Acute Toxicity Studies: The methanolic extract of polyherbal preparation showed no mortality in Albino Wistar rats up to the dose 3000 mg/kg for duration of 24 hours.Hence,as per OECD guidelines 1/10^th and 1/5^th of 3000mg/kg i.e. 300mg/kg and 600mg/kg were selected as doses for the study.

Methods:

Preparation  of  High  Cholesterol  Diet (HCD) ¹³: The  HCD  consist  of  95%  commercial rat feed, 4%  cholesterol  and  1%  cholic acid.

Experimental  design: Rats (n=30)  were  randomized  into  following  groups:

After  the  duration  of  treatment  blood  was  collected  from  the retro-orbital sinus  and  tests  were  done  for  listed  biochemical  parameters.

Measurement of various Parameters:

Physical Parameters ¹⁵: The body weight was recorded on the first day and then last day of the study period in each group.

Biochemical Estimations ¹⁶: Lipid parameters were determined in blood serum. At the end of 21 days,animals were fasted overnight and blood was collected from retro orbital plexus under light ether anaesthesia, centrifuged at 2500  rpm  for  20  minutes.  The  serum  obtained  will  be  kept  at  4^oC  until  used. The quantitative estimation of lipid profile was carried out using Infinite triglycerides liquid for triglycerides, Infinite cholesterol liquid for total cholesterol and Autozyme for HDL-C, ACCUREX in Brambhatt laboratory, Anand.

Estimation  of  VLDL-C  and  LDL-C  will  be  done  by  using  the  Friedward’s  formula.

VLDL-C = Triglycerides/5

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

Measurement of Coronary Disease Risk Factor ¹⁷: Atherogenic Index (AI), which is a measure of the atherogenic potential of an agent, was calculated using the following formula and the results were tabulated.

Atherogenic Index =   Total serum triglyceride

Total serum HDL-C

% Protection =  AI of control - AI of treated group  ×100

AI of control

Assay of HMG CoA Reductase Activity ¹⁸: The  method  described  by  Rao  and  Ramkrishan  will  be  used  for  the  estimation  of  HMG-CoA  reductase  activity

The  tissue  homogenate  will be  prepared  by  homogenizing  1  gm  of  tissue  (liver)  with 10  ml  of  saline  arsenate  solution.  Mix  equal  volumes  of  the  fresh  10%  tissue  homogenate  and  diluted  perchloric  acid.  Allow  to  stand  for  5    minute  and  centrifuge  (2000  rpm, 10  minutes). Treat  1.0  ml  of  filtrate  with 0.5ml  of  freshly  prepared  hydroxylamine  reagent  (alkaline  hydroxylamine  reagent  in  the  case  of  HMG-CoA),  mix,  after  5  minute  add  1.5  ml  of  ferric  chloride  reagent  to  the  same  tube  and  shake  well.  Take  readings  after  10  minutes  at  540  nm  vs. a  similarly  treated  saline/arsenate  blank.

Detrrmination of Antioxidant Parameters: For estimation of antioxidant parameters,the livers were homogenized (10%w/v) in ice cold phosphate buffer and centrifuged at 2000 rpm for 5 min at 4 ˚C in a cooling centrifuge (Remi Instruments,Mumbai).After centrifugation,the supernatant was separated and stored for further analysis.

Assay for Thiobarbituric acid reactive substance (Maleic dialdehyde) ¹⁹: Lipid peroxidation was estimated colorimetrically in the liver by quantifying TBARS according to the method of Ohkawa and Ohishi. For the estimation,0.5 ml of supernatant was treated with 0.2 ml of of 8.1% sodium dodecyl sulphate, 1.5 ml of 20% acetic acid adjusted to pH 3.5 with NaOH and 1.5 ml of 0.8% solution of thiobarbituric acid.The volume of the mixture was adjusted to 4.0 ml with distelled water and heated in a water bath at 85˚C for 60 min. Light pink color was developed.After cooling with tap water, 1.0 ml of distilled water and 5.0 ml of mixture of n-butenol and pyridine (15:1,V/V) was added.

The mixture was shaken vigorously and them centrifuged at 4000 rpm for 5 min. After centrifugation, the organic layer was separated and its absorbance was read at 532 nm using a UV-visible spectrophotometer (UV-1700, Schimadzu, Japan)  against a reagent blank. The amount of TBARS was calculated by using 1.56×10⁵M^-1cm^-1 as molar extinction co-efficient and the level of lipid peroxidation was expressed as nmol of maleic dialdehyde/mg of protein (MDA).

Assay for Reduced Glutathion Content ²⁰: Reduced glutathione (GSH) was determined colorimetrically by the method of Ellman. The Ellman’s reagent was prepared by dissolving 39.6 mg DTNB in 10 ml phosphate buffer (ph=7). For estimation of GSH, 0.5 ml of supernatant was treated with 3.0 mt of ethanol and 0.5 ml of Ellman’s reagent. The absorbance of the developed yellow color was read at 412 nm using a UV-visible spectrophotometer (UV-1700, Schimadzu, Japan) against a reagent blank. The reduced glutathione content was calculated by using 13,600M^-1 cm^-1  as the molar extinction co-efficient as nmol GSH formed/mg protein.

Determination of Superoxide Dismutase Activity ²¹: Superoxide dismutase activity was assayed in terms of its ability to inhibit the radical-mediated autoxidation of epinephrine; using the method described by Misra and Fridovich.

The reaction mixture consisted of 0.5 ml of carbonate buffer (pH=9.7), 0.5 ml of supernatant, 0.1 mi of EDTA solution (1×10^-4 M) and 0.1 ml of Epinephrine solution (3×10^-3 M).The changes in the absorbance of this solution was read at 480 nm for 3 min at 30 sec intervals using a UV-visible spectrophotometer (UV-1700, Schimadzu, Japan) against reagent blank. The activity of superoxide dismutase was calculated by using    4020 M^-1 cm^-1 as molar extinction co-efficient and expressed as Units/min/mg of protein.

Histopathology of Aorta: For histopathology, the rats were sacrificed by cervical decapitation and their aortas were dissected out. During the procedure,ice was used to keep the aorta samples fresh and avoid any degradation. The aortas were stored in 10% formaline solution and sent to a local pathological laboratory for hematoxyline and eosine staining.

Statistical Analysis: The results are expressed as mean ± standard error of mean (SEM).The data were analyzed using one-way analysis of variance (one-way ANOVA) followed by Tukey’s test for comparison between groups. The criterion for statistical significance was p<0.05.

RESULTS:

TABLE 1: EFFECT OF METHANOLIC EXTRACT OF POLYHERBAL  PREPARATION ON BODY WEIGHT (GM) IN  ALBINO WISTAR RATS

TABLE 2: EFFECT OF METHANOLIC EXTRACT OF POLYHERBAL  PREPARATION ON SERUM LIPID PROFILE IN  ALBINO WISTAR RATS

Expressed as mean ± SEM, (n=6) for each group. Data were analyzed by One way ANOVA followed by Tukey test; * p<0.001, represent significant difference  when compared with normal group; # p<0.001, represent significant difference  when compared with control group(High cholesterol diet).

TABLE 3: EFFECT OF METHANOLIC EXTRACT OF POLYHERBAL  PREPARATION ON CORONARY RISK FACTORS

Expressed as mean ± SEM, (n=6) for each group. * p<0.001, represent significant difference when compared with normal group; # p<0.001,represent significant difference when compared with control group(High cholesterol diet).

TABLE 4: EFFECT OF METHANOLIC EXTRACT OF POLYHERBAL  PREPARATION ON HMGCoA/MEVALONATE RATIO AND ANTIOXIDANT ENZYME LEVELS IN LIVER

Expressed as mean ± SEM, (n=6) for each group. * p<0.001, represent significant difference  when compared with normal group; # p<0.001, ## p<0.05, represent significant difference  when compared with control group (High cholesterol diet).

Histopathology of Aorta: In histopathology study, normal group showed no pathological changes in endothelial lining. Control group (High cholesterol diet) showed severe damage to endothelial lining. Methanolic extract of polyherbal preparation treated groups (300 mg/kg and 600 mg/kg) showed less damage to the endothelial lining as compared to control group. Atorvastatin treated group showed mild damage to the endothelial lining.

DISCUSSION: In present study,the antiatherosclerotic activity of methanolic extract of polyherbal preparation was evaluated in rats receiving high cholesterol diet (HCD). Determination of body weight in experimentally induced atherosclerosis is considered to be a positive factor to find out the prognosis of disease ²². Results indicated increase in body weight of animals from the beginning to the end of the experiment in all five groups, but at the end, increase in body weight is low in methanolic extract of polyherbal preparation treated groups as compared to high cholesterol diet treated group.

Cholesterol is an essential structural element of the biological membranes.In addition, it is the precursor of many compounds such as synthesis of bile acids,steroid hormones and vitamin D. Despite this, high concentration of serum cholesterol increases the risk of coronary heart dieses (CHD) ²³.

Our study demonstrated that rats fed with a high cholesterol diet (control group) exhibited a higher level of total cholesterol in serum as compared to rats fed with a standard laboratory diet (Normal group); while oral administration of methanolic extract of polyherbal preparation dose (300 mg/kg, 600 mg/kg, p.o) reduced the raised level of total cholesterol in serum.

In our study, the administration of  methanolic extract of polyherbal preparation dose (300 mg/kg,600 mg/kg, p.o) significantly lowered triglyceride and VLDL level in serum. It is widely accepted that the elevation of plasma LDL-C level is major risk factor for CHD ²⁴. Direct correlation between LDL-C level and atherosclerosis as well as the reversibility of the related pathological events by lowering the serum level of LDL-C has already been reported ²⁵.

The results indicated that, the high concentration of LDL-C in serum was significantly reduced by oral administration of methanolic extract of polyherbal preparation; which might constitute a good candidate for the treatment of atherosclerosis by lowering serum LDL-C level.

Another risk factor for developing atherosclerosis is the reduced serum level of HDL-C. This effect which is largely attributed to its central role to reserve cholesterol transport, a process whereby excess cell cholesterol is up taken and which is subsequently processed by HDL-C particles for further delivery to the liver for metabolism ²⁶. Therefore, it is logical that an increase in HDL-C level can contribute to lower the risk of atherosclerosis ²⁷. The results clearly indicated that methanolic extract of polyherbal preparation was capable of increasing level of  HDL-C in serum.

High atherogenic index (A.I.) is believed to be an important for atherosclerosis. Our data clearly indicated that methanolic extract of polyherbal preparation is capable of potentially decreasing this risk factor. Similar results were reported by chloroform extract of Mimosa pudica Leaves ²⁸.

HMG CoA reductase is the rate limiting enzyme in the cholesterol biosynthetic pathway. It converts HMG CoA to mevalonate. In the present study, HMG CoA reductase activity was indirectly measured in terms of the ratio between HMG CoA and mevalonate ¹⁸. The ratio was found to be inversely prapotional to HMG CoA reductase activity, indicated that an increase in the ratio inferred a decrease in the enzyme activity.The methanolic extract of polyherbal preparation produced a significant and dose dependant (300 mg/kg and 600 mg/kg,p.o.) increase in HMG CoA/mevalonate ratio in liver as compared to the normal group.Similar results were also reported with ethanolic extract of Ficus religiosa Linn leaves ²⁹.

Many reports have demonstrated that high Cholesterol diet-induced atherosclerosis has tight relations with vascular damage and oxidative stress, which is involved in the pathogenesis of a great number of diseases ³⁰. Atherosclerosis leads to tissue oxidant stress, due to reduction in antioxidant capacity and free radical load generated by high cholesterol diet.

Recently, some clinical studies suggest dyslipidaemia as one of the major risk factors for coronary disease, while preclinical observations demonstrate that atherosclerosis promotes accumulation of oxidised low-density lipoprotein (Ox-LDL) in the arterial wall, which plays a major role in the initiation and progression of the cardiovascular dysfunction associated with atherosclerosis ³¹. Since oxidation of LDL plays a significant role in atherogenesis, amelioration of oxidative stress is equally important as controlling or decreasing dyslipidaemia. In this work, we investigated activities of three major defensive antioxidant agents.

SOD is the first line of defence against free radicals, while GSH-Px is responsible for most of the decomposition of lipid peroxides in cells and may thus protect the cell from the deleterious effects of peroxides. In agreement with previous reports ³², it was observed a significant decrease in the accumulation of SOD and GSH-Px in atherosclerotic rats, compared to those in the normal group. In addition, the content of MDA, the product of lipid peroxidation, was elevated in the atherosclerotic rats.

Taken together with the above results, the imbalance between oxidative stress generation and antioxidants formation could occur after feeding a high cholesterol diet. Nevertheless, Methanolic extract of polyherbal preparation (300 mg/kg,p.o. and 600 mg/kg,p.o.) could prevent this pathological process, which indicated its therapeutic and preventive effect on hepatosteatosis induced by high cholesterol diet.

It was derived from present study that curcuma longa extract has a hypocholesterolemic effect on rats fed on a high cholesterol diet to induce experimental atherosclerosis. This effect could be explained on the basis of an enhanced excretion of cholesterol in bile by curcumin with a concomitant reduction in bile cholesterol saturation and elevated faecal fat excretion ³³. It means that the excess cholesterol from diet is removed by excretion. However, in the control group cholesterol is accumulated in plasma and tissue. Furthermore, it has been suggested that Curcuma longa extract produces a decrease in triglyceride levels in human ³⁴.

It is generally assumed that some antioxidants can prevent atherosclerosis by protecting LDL from oxidation ³⁵ and are also associated with an antihypercholesterolemic effect. For the above reason, It was investigated whether curcuma longa extract could influence the development of atherosclerotic lesions after its antioxidant activity and a hypocholesterolemic effect was found. In present study the same positive effect  conformed its antiatherosclerotic activity.

Picrirhiza kurroa contains iridoid glycosides (including picroside I, II, III, pikuroside, kutkoside and 6-feruloyl catalpol), cucurbitacin glycosides, androsin, apocynin, and other organic acids such as vanillic and cinnamic acids. It is having ayurvedic properties of tikta rasa, laguruksha guna, and katuvipaka. Based on these properties, one may anticipate its pharmacodynamic activity on lipids specifically related to lipid disorders ³⁶. Hypolipidemic, free radical scavenger, antioxidant and anti-inflammatory effect of Picrorhiza kurroa has been investigated ³⁷. The current study further confirmed its antioxidant and hypolipidemic activity.

It has been demonstrated that saponins which are present in Acorus calamus, are known to form complexes with cholesterol by binding plasma lipids, there by altering cholesterol metabolism and lipid absorption ³⁸. The presence of tannins and saponin in Capparis deciduas causes the inhibition of lipid absorption ³⁹. So here, antiatherosclerotic effect of Acorus calamus might be due to presence of saponins.

It also has been reported that treatment with  Plumbagin, an active constituent of Plumbago zeylanica, prevented the accumulation of cholesterol and triglyceride in liver and aorta and regressed atheromatous plaque of the thoracic as well as abdominal aortas ^{40, 41}.

Plumbagin also having antiplatelet activity which is useful in many coronary heart diseses ⁴². In present study, it was found that treatment group showed reduced cholesterol and triglyceride level as compare to normal group.

Cyperus rotundus significantly reduces total cholesterol as compared to the normal control. The activity may be due to the presence of flavanoid compounds.

In addition, Cyperus rotundus containing tannins, polyphenols, flavanolols, terpenes, alkaloids, and saponins. Its antioxidant property may be attributed to  these phytochemicals ⁴³.

CONCLUSION: It can be concluded from the present study that methanolic extract of polyherbal preparation at the dose of 300 mg/kg and 600 mg/kg,p.o. shows antiatherosclerotic activity in high cholesterol diet model in rats. Methanolic extract of polyherbal preparation may have antiatherosclerotic activity due to inhibition of the HMG CoA enzyme pathway  and  its antioxidant effect.

ACKNOWLEDGEMENT: Special thanks to Nirzarini N. Shah, Dilip K. Jani for their continuous encouragement, support and freedom to do research work.

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    How to cite this article:

    Machhi JP and Shah NN: Study of Antiatherosclerotic Activity of Polyherbal Preparation using rat as an Experimental Animal Model. Int J Pharm Sci Res. 3(10); 4010-4018.

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