Collection of plant material and preparation of plant extracts

T. cordifolia stem was obtained from the local area of Pune District; Maharashtra, India. The stems were collected in winter season (November), stems were shade dried and pulverized with the help of grinder. The powdered stem was then preserved in an airtight container. The dried powder material (600 gm) was soaked in 1500 ml of 90% ethanol for ten days and was shaken occasionally. The whole mixture was filtered by a piece of clean, white cotton followed by Whatman filter paper. The filtrate was dried using a vacuum rotary evaporator at an optimum temperature of 40 °C to prevent loss of important plant constituents and to obtain the crude ethanolic extract of T.cordifolia (yield-20 gm). The concentrated aqueous ethanol extract was partitioned by the ethyl acetate fraction (Kupchan method) [8].

Drugs and chemicals

T. cordifolia stem was purchased from Manikarnika aushdhalaya Pune, MS (India). Chemicals for high-fat diet (cholesterol, cholic acid, and vitamin D3) were purchased from Uttam Chemicals Pune, MS (India). All other chemicals were purchased from local sources and were of analytical grade.

Experimental animal

Healthy male Wistar rats (120–170 g) were procured from D-Global building, Sai Park, Shewalwadi road, Uruli Devachi Pune, MS (India). Animals were housed in a group of 6 per cage in standard polypropylene cages (32.5 × 21 × 14) cm lined with raw husk. The animal house was maintained on 12 light/dark cycle approximately 22 ± 2 °C, relative humidity 60–70%. All animals were provided with a standard laboratory diet (Nutrivet Lifescience, Maharashtra, India) and water ad libitum. All experimental procedures were carried out by the guidelines prescribed by CPCSEA and study was approved by the IAEC. (Approval No. MCP/IAEC/003/2019).

Induction of atherosclerosis

High-fat diet with vitamin D3 induces atherosclerosis in rats. Rats were fed esophageal injection with vitamin D3 (700,000 IU/kg) in the first four days followed by feeding with high lipid emulsion (4% of cholesterol, 1% cholic acid, 0.5% PTU and salad oil) [17]. The success of the model was determined by gross anatomy and histology at 80 days after the beginning of the diet. The standard group received Atorvastatin and the test groups rats were received EATC (100 mg/kg & 200 mg/kg p.o.) in the last 20 days. The normal control group received distilled water and the disease control group as well as diseased group both received high-fat diet with regular chow to all groups.

Animal grouping and dosing methods

Grouping was done by simple allocation of the animal.

The rats were divided into five groups, six animals in each group.

Group I: Normal control rats receiving vehicle i.e. distilled water.

Group II: Disease control rats administered with a high-fat diet for 80 days.

Group III: Standard group (HFD 60 days followed by 20 days 10 mg/kg standard (Atorvastatin).

Group IV: Test group 1 (HFD 60 days followed by 20 days 100 mg/kg EATC).

Group V: Test group 2 (HFD 60 days followed by 20 days 200 mg/kg EATC).

Biochemical analysis

The blood samples were withdrawn on the 60th and 80th day from the retro-orbital venous plexus of rats without any coagulant for the separation of serum. After collecting the blood into microcentrifuge tubes, it was kept aside for 1 h at room temperature and then serum was isolated by centrifugation at 2000 rpm for 15 min and stored until analyzed for various biochemical parameters. The biochemical analysis was carried out by auto-analyser with ERBA kit which is present in our institute’s laboratory.

Histopathology

Aorta and liver were isolated and fixed in 10% neutral buffer formalin and then dehydrated by successively passing through a gradient of a mixture of ethyl alcohol and water. The samples were rinsed by xylene and embedded in paraffin. Tissue sections (5 µm thickness) were cut stained with hematoxylin and eosin dye (H and E) and histological examination were done using light microscopy.

Statistical analysis

All the data were expressed as mean ± standard error of mean using one-way analysis of variance followed by the turkey comparison test using computerized GraphPad prism (version 5.0, trial version, GraphPad software, USA) software at a level of significance of p < 0.05.