*3.4. In Vivo Assessment of CLL Extract in an HFD Rat Anti-Obesity Activity* 3.4.1. Experimental Animals

Male albino rats of Sprague Dawley strain weighing 100–134 g (115.7 ± 7.6 g as mean ± SD) obtained from Animal House of National Research Centre, Cairo, Egypt, were kept on standard chow diet (8% fat) or high-fat diet (HFD) (30% saturated fat), provided with water ad libitum. Animals were kept individually in stainless steel metabolic cages at 25 ◦C; water and food were given ad libitum. All experiments were carried out according to the research protocols established by Research Ethics Committee in Faculty of Pharmacy, Cairo University, and by Medical Research Ethics Committee (MREC) in NRC, which follow the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals Ethical Approval Certificate No. MP (1959).

#### 3.4.2. In Vivo Assay Experimental Design

Twenty-four rats were randomized into two groups and received either standard chow diet (8% fat, *n* = 6), as a normal control, or an HFD (30% saturated fat, *n* = 18) to induce obesity for 8 weeks [47]. Body weight and food intake were recorded every week. After induction of obesity, rats were divided into three subgroups and still fed on HFD. For subgroup 1, rats were fed on HFD and given the vehicle as obese control. For subgroup 2, rats were fed on HFD and given oral dose of Orly (10 mg/kg RBW/day) for 4 weeks as anti-obesity drug group. Rats in subgroup 3 were fed on HFD and given oral administration of crude methanol extract of *A. carambola* leaf (CLL), prepared as described in Section 3.1 (300 mg/kg RBW/day) for four weeks. Normal control rats were continued to be fed on standard chow diet for four weeks. Body weight and food intake were recorded every week. At the end of the experiment, blood samples were collected for determination of plasma total cholesterol (T-Ch) [48], high-density lipoprotein cholesterol (HDL-Ch) [49], low-density lipoprotein cholesterol (LDL-Ch) [50], and triglycerides (TG) [51]. T-Ch/HDL-Ch ratio was calculated as an indicator of cardiovascular risk. Plasma butyrylcholinesterase (BChE) [52], plasma α-amylase activity [53] was assessed. Plasma malondialdehyde (MDA) [54] and plasma catalase activity (CAT) [55] were estimated as indicators of lipid peroxidation and oxidative stress, respectively. For assessment of liver functions, the activity of plasma transaminases aspartate transaminase (AST) and alanine transaminase (ALT) were estimated, according to the method of [56]. Plasma level of creatinine [57], urea [58], and uric acid [59] were determined to assess changes in kidney functions. Plasma insulin [60] and blood glucose levels [61] were determined. Insulin resistance was calculated based on homeostasis model assessment of insulin resistance (HOMA-IR), according to [62]: (fasting plasma glucose (FPG) (mmoL/L) × fasting plasma insulin (FPI) (μU/mL))/22.5.

The animal experiment has been carried out according to Ethics Committee, National Research Centre, Cairo, Egypt, following the recommendations of the National Institutes of Health Guide for Care and Use of Laboratory Animals (Publication No. 85-23, revised 1985).
