Journal of Ayurveda and Integrated Medical Sciences

Introduction

Hyperlipidemia is a secondary metabolic dysregulation associated with diabetes. Besides the cause-effect relationship with diabetes, elevated serum levels of triglycerides, cholesterol and LDL are major risk factors for the premature development of cardiovascular diseases like arthrosclerosis, hypertension, coronary heart disease etc.^[1]

Hyperlipidemia is one of the greatest risk factors contributing to the prevalence and severity of atherosclerosis and subsequent coronary heart disease.^[2] Treatment of hyperlipidemia involves diet control, exercise, and the use of lipid-lowering diets and drugs.^[3] The most commonly employed drugs for treatment of hyperlipidemia include hydroxyl methyl glutarate coenzyme A (HMG-CoA) reductase inhibitors, also called as statins. Other drugs employed for treatment of hyperlipidemia include bile acid sequestrants (anion-exchange resins) such as cholestyramine and colestipol; fibrates such as clofibrate, gemfibrozil, fenofibrate, ciprofibrate, and bezafibrate; niacin; cholesterol absorption inhibitors such as ezetimibe; and omega-3-fatty acids.^[4]

Liver synthesizes two-third of the total cholesterol made in the body. Endogenous cholesterol biosynthesis in the liver is mainly controlled by rate limiting enzyme, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, which catalyzes the conversion of HMG-CoA to mevalonic acid.^[5] Drugs that lower cholesterol level mainly work by inhibiting the HMG-CoA reductase activity.^[6,7] Despite the significant clinical benefits provided by statins^[8], many patients do not achieve the recommended low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol target goals.^[9]

Moreover, the use of statins is not preferred in more than 40% of patients, mostly due to the occurrence of several side effects including myalgia, myopathy, liver disease, and rhabdomyolysis in more severe cases.^[10,11] This limits the use of statins and incites a search for new natural drugs to combat hypercholesterolemia as well as cholesterol induced oxidative stress and atherosclerosis.

Elevated lipid levels result from increased absorption through the gut or enhanced

i.e., late autumn season (November to January) from their natural habitat, Dhanvantari Vana Bangalore University Campus and Reserve Forest, Jaraka Bande Kaval, Bangalore North. The specimens were authenticated by Dr. Ravikumar K. Taxonomist and Senior Botanist’s from FRLHT TDU, Yelahanka Bangalore – 560064. Quantity sufficient matured barks of Panchavalkalas were cut in to small pieces separately and were dried under mild sunlight covering thin cloth for 2 days and later shade dried for 10 days. Then the dried barks were powdered using pulverizer at Sanjeevini Pharma Kengeri, Bangalore. The phytochemical analysis, quantification of marker compounds and in vitro study is carried out at Skanda Life Sciences Pvt. Ltd., Bangalore-560091.

Formulation details - Preparation of aqueous and alcoholic extract

20 g of dried sample powder-PVK weighted and dissolved in 100 ml water or 100 ml of ethanol for aqueous and alcoholic extract in 500ml beaker with Aluminium foil covered on it. Then the beaker was kept on hot water bath at 50°C for 4 hours. After incubation period the extract was filtered with Whatman filter paper, and the filtrate was collected in 50ml beaker. Residue present over the filtrate was taken for further use. Then the filtrate was kept at 70°C for few hours until the extract got completely dried and turned into semisolid form. This semi solid sample was weighted and used for dosing.

Phytochemical Analysis

Various Chemical tests were carried out for detection of organic chemical constituents Phyto chemical examinations were carried out for all the extracts as per the standard methods.^[18,19]

Study of marker compounds Caffeic acid and Gallic acid through HPLC analysis

A. Caffeic acid standard (Sigma Aldrich, 99% w/v) stock solution (1mg/ml) was prepared in MEOH. Caffeic acid was quantified using the peak area after HPLC analysis. Chromatography analysis for the quantification of the chemical marker Caffeic acid in Ficus virens, Ficus religiosa L., Ficus benghalensis L. and Thespasia populnea L. was conducted using an instrument (Shimadzu LC-MS Prominence 20AT) at ambient temperature (40°C) on an C18 column (250 × 4.6 mm, 5.0 µm particle size). The mobile

Justification for the selection of species: Literature survey suggests that rabbit will be used to evaluate the anti-atherosclerosis activity.

No. of groups: 15

No. of groups: 6

Body weight: 170-200gm

Identification: By cage card, crystal picric acid color body marking.

Acclimatization: 5 days

Treatment protocol and group allocation:

The animals were divided into fifteen groups of six animals each and individually housed in cages. The normal control group continued to be fed a laboratory pellet chow ad libitum. The cafeteria diet-control group received the cafeteria diet in addition to the normal pellet diet (NPD). The Standard control group received Atorvastatin along with HFD and NPD. The remaining 12 groups were fed with the cafeteria diet and NPD along with the test drug 200mg/kg body weight p.o. Treatment was continued for 30 days. The animals were weighed at the start of the experiment and on 30^th day thereafter.

Husbandry Conditions:

Animals were housed under standard air-conditioned laboratory conditions.

• Temperature: Maximum: 24°C and minimum 23°C
• Relative humidity: Maximum: 63% and minimum 48%
• 12 h light and 12 h dark cycle.

The maximum and minimum temperature and relative humidity in the experimental room was recorded once daily.

Equipment Details and Chemicals:

• Weighing balance: Make: Weigh well, model: WWTT
• Biochemical analyzer: Make: Robonik, Model: Prietest- COMPACT
• Glucose Kit: Make: Robonik, Lot no: 191431
• TG kit: Make: Robonik, Lot no: 192902
• Total Cholesterol: Make: Asritha (CH-10/1019)
• HDL: Make: Robonik: Lot no: 191705

of Plasma and change in body weight were calculated and analysis done as per the protocol.

Atherogenic index of Plasma is calculated using formula

AIP=Log (serum triglyceride/serum HDLc)^[25]

Estimation of Lipid ratios:

Lipid ratios can provide information on risk factors difficult to quantify by routine analyses and could be a better mirror of the metabolic and clinical interactions between lipid fractions. These ratios could be potential indicators for risk stratification of ICAS (Intracranial atherosclerotic stenosis) and provide guidance in lipid-lowering therapy.

Histopathology: The liver tissue was stored at −80°C for further histopathology analysis.

Slide preparation procedure: Tissues were washed for formalin clearing over-night in running water. Tissues were prepared for staining after serial dilution of alcohol by rehydration and dehydration method. The tissues were embedded in paraffin blocks for sectioning in microtome. The sections were made at 4 µ thickness suitable for staining.

Staining procedure: The tissue fixed slides were deparaffinized using xylene and further rehydrated using serially diluted alcohol. Further sections were stained using Eosin-hematoxylin. The slides were dehydrated by serially diluted alcohol. Microscopical examinations of H&E (hematoxylin and eosin) stained slides were conducted by research microscope at 40x resolution for better visibility and detailed examination. The changes are recorded based on circulatory changes, infiltrative changes and proliferative changes.

Data Compilation and Statistical Analysis

Average of all the data were compiled and SEM were calculated. All the data was analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. Values <0.05 were considered as statistically significant.^[26]

Results

Phytochemical screening:

Preliminary phytochemical screening of the aqueous and Ethanolic extracts

Table 3: Caffeic acid content of test sample

Table 4: Gallic acid content of test sample

Table 5. Effect of PVK bark extracts on Lipid profile of rats fed with trial drug for 4 weeks

Figure 1: Histopathology of Liver Tissue - Control, HFD Control, Standard Control, AE PVK, EE PVK and AE Vata

Figure 2: Histopathology of Liver tissue - EE Vata, AE Udumbara, EE Udumbura, AE Aswatta, EE Aswatta and AE Plaksha

Figure 3: Histopathology of Liver Tissue- EE Plaksha, AE Pareesha and EE of Pareesha

when compared to PVK Aqueous Extract and Ethanolic, aqueous extracts of individual drugs separately.^[33,34] Polyphenols represent the major class for the pancreatic lipase inhibitor. They bind to the enzyme by polyvalent sites present in them. When compared to PVK Aqueous Extract, PVK Ethanolic Extract has better extraction and solubility of Phenolic compounds the key phyto-constituents responsible for anti-hyperlipidemic action.

It is well accepted that enzymes are the major regulators of the lipid metabolism; wherein HMG-CoA reductase is one of the most clinically important enzymes involved in the cholesterol biosynthetic pathway. Changes in the reductase activity are closely related to changes in the overall rate of cholesterol synthesis. This suggests that the inhibition of HMG-CoA reductase would be an effective mean to lower plasma cholesterol.^[35] Thus, this enzyme is the target of the widely available cholesterol lowering drugs known, collectively, as statins.^[36] Although, most of the HMG-CoA reductase inhibitors have some adverse side effects, PVK Ethanolic Extract having rich bioactive substances such as Flavonoid’s, Polyphenols, Tannins, etc. inhibits HMG-CoA reductase activity, and produces antihyperlipidemic action. Our results are in concordance with earlier reports demonstrating that the reduction in HMG-CoA reductase activity is responsible for the hypolipidemic property of natural agents.^[37] This lipid lowering activity might be due to the pleiotropic effect that the compound exhibits through reduced HMG-CoA reductase activity, with significant antioxidant property and increased lipoprotein lipase activity of PVK responsible for hydrolysis of plasma lipids. PVK Ethanolic Extract showed marked anti-hyperlipidemic activity properties and might be a drug candidate.

The reduction of total cholesterol by the PVK extract was associated with a decrease of its LDL fraction (LDL-C) (Table-5), which is the target of several hypolipidemic drugs. This result suggests that the cholesterol-lowering effect of the extract may result from the rapid catabolism of LDL-C through its hepatic receptors for final elimination in the form of bile acids as demonstrated by Khanna et al.^[38]

Elevated serum TG is considered as an independent risk factor for cardiovascular diseases.^[39] In the triglyceride metabolism di-aceyl-glycerol transferase (DAGT) and lipoprotein lipase plays an important

two third of plasma cholesterol is found in the LDL. And the plasma clearance of LDL particle is mediated primarily by LDL receptors located in the liver, which removes more than 75% of LDL from the plasma. In the present study the test drug PVK together and individually both EE and AE have shown significant reduction in the LDL-C level (Table-5). It might due to its capacity to induce a greater number of LDL receptors in the liver and thereby decreasing the plasma LDL- C level. Hence the hypolipidemic effect could be due to an increased catabolism of cholesterol in to bile acids in liver. Another possible mechanism is inhibition of HMG CoA reductase a rate limiting enzyme in the biosynthesis of cholesterol.^[42] Thus, the test drug might have either inhibition of absorption of dietary cholesterol or has inhibitory action on HMG CoA reductase enzyme. Furthermore, the fraction of Ficus virens which was identified as Octadecanyl-O-αD-glucopyranosyl (6′ → 1″)-O-α-D-glucopyranoside a new and completely different structure than the other inhibitors have been studied for in silico molecular interaction and inhibitory action on HMG CoA reductase enzyme and confirms the uncompetitive mode of inhibition observed in vitro study.^[43]

HDL is considered as protective lipoproteins that decrease the risk of coronary heart disease. This protective effect may result from participation of HDL in reverse cholesterol transport, the process by which the excess cholesterol is acquired from cells and transferred to the liver for excretion.^[44] In the present study the Atorvastatin has shown significantly increase in the HDL- C level and test drug-PVK has showed a moderate elevation (Table-5). Thus, test drug has shown moderate protective activity.

In the same way, oxidative stress also plays a pivotal role in atherosclerosis and subsequent CVD. Therefore, finding an HMG-CoA reductase inhibitor with antioxidant property is of great potential in the treatment and management of hypercholesterolemia and cholesterol induced oxidative stress.

Dyslipidemia is very common in type 2 diabetes (T2DM) mellitus affecting around 72%-85% patients.^[45] Lipid abnormalities in patients with diabetes, often termed “diabetic dyslipidemia”, are typically characterized by high total cholesterol (T-Chol), high triglycerides (Tg),

with Standard drug Atorvastatin, Test drug PVK Ethanolic Extract, Ashwata Aqueous Extract and Pareesha Aqueous Extract when compared with HFD Control (P<0.01). Decrease in liver weight and body weight is due to loss of cholesterol, due to anti-hyperlipidemic and antioxidant activity of the PVK test drug. However, the test drug did not show any significant changes in the relative body weight and organ weight such as kidneys, heart and lungs (Table-6).

HFD produced significant increase in Liver weight when compared to the control group where in the weight gain of kidneys, heart and lungs were not significant.

Histopathological study of Liver tissue is carried out in all groups to demonstrate effects of PVK extracts on liver tissue. In the control group which represents Hematoxylin eosin staining of normal liver with glomerulus. It shows normal histological tissue architecture of regular classical hepatic lobules with central vein and the peripheral portal area. Each hepatic lobule consisted of normal hepatocytes cords from the center to the periphery (Fig.1) HFD fed animals revealed numerous histopathological changes indicating severe hepatic steatosis (accumulation of lipid droplets in hepatic tissue), disrupted liver architecture marked central vein dilation, congestion of portal veins and blood sinusoids (Fig.1). The hepatic parenchyma of the standard treatment received group showed features of moderate hepatic steatosis and hydropic degeneration (Fig.1). The hepatic architecture was normal in the group treated with Aqueous extract of PVK and the cell borders were visible and the size of sinusoids was normal (Fig.1). Similarly, animals treated with Alcoholic extract of PVK group showed improvement of the hepatic architecture. For instance, most hepatocytes had regular nuclei, the amount and size of vacuoles decreased considerably, the cell borders were visible, and the size of sinusoids was normal (Fig.1). Similarly in other individual groups treated with single drugs of PVK both in aqueous and Ethanolic extracts are showing significant changes when compared to HFD control group (Fig.2&3). Earlier reports also demonstrates that Lipid drops are usually accumulated in hepatic tissue, called hepatic steatosis under the progress of atherosclerosis especially hyperlipidemia stage. However, enzymes such as Superoxide dismutase

Conclusion

The present study showed PVK Ethanolic Extract has significant antihyperlipidemic activity due to presence of Flavonoids, Tannins, and polyphenols. PVK have all such properties like Antidiabetic, Antioxidant and Pancreatic lipase enzyme inhibition activity. When it is compared with Atorvastatin, in vivo study has proven it has significantly reduced the lipids and brought changes in liver architecture. The present study has proven Lekhana and Medhogna Karma / Antihyperlipidemic activity of PVK which demonstrate the interference in the whole set of the pathophysiology involved in metabolic disease like diabetes, dyslipidemia and hyperlipidemia in particular. Hence it can be exploited as an antihyperlipidemic therapeutic agent or adjuvant in existing therapy for the treatment of Medorogas which includes Hyperlipidemias.

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