Collection and extraction of plant material
The bark of Terminalia paniculata was collected from Seshachalam forest spread around Tirupati, Andhra Pradesh, India. Its identity was authenticated by a Taxonomist, Department of Botany, at S.V. University, Tirupati, voucher number 136, and a specimen has been preserved at the departmental herbarium. The bark of T. paniculata was dried under shade, pulverized to coarse powder and extracted with ethanol. The filtrate obtained was evaporated to dryness at 50-65°C in a rotary vacuum evaporator to obtain a dark colored molten mass.
For this study, male Sprague–Dawley rats (n = 36), weighing 150-160 g were obtained from National Institute of Nutrition, Hyderabad, India. All rats were housed under 22 ± 2°C temperature, humidity (40-50%) and light–dark cycle (12-12 ± 1 h) and allowed food and water ad libetum, for ten weeks. Experimental protocols were followed as per Institutional animal ethics committee guidelines (IAEC) (Regd No: 438/01a/CPCSEA, Date: 17-07-2001). The Committee granted permission to authors and passed resolution to carryout animal work (Resolution No: 36/2012-2013/ (i)/a/CPCSEA/IAEC/SVU/MB-MRG).
Composition of high fat diet
High fat diet (HFD) was obtained from National centre for laboratory animal sciences (NCLAS), National Institute of Nutrition (NIN), Hyderabad, India. Diet composition is as follows, Casein (342 g), Cystine (30 g), Starch (172 g), Sucrose (50 g), Cellulose (50 g), G.N. Oil (25 g), Thallow (190 g), Mineral mixture (35 g), Vit. Mixture (10 g).
Rats were randomly divided in to six groups of six each. Group 1: Normal control group (NC), Group 2: High fat diet group (HFD), Group 3: HFD + Orlistat (30 mg/kg b.wt), Group 4: HFD + TPEE (100 mg/kg b.wt), Group 5: HFD + TPEE (150 mg/kg b.wt) and Group 6: HFD + TPEE (200 mg/kg b.wt).
Measurement of body and organ weights
At the end of the experimental period, rats were weighted and their weights noted. Later, they were fasted overnight, anaesthetized, sacrificed, organs (liver, spleen, kidney and testis were) were surgically removed, wet weights were measured with experimental electrical balance (Shimadzu) and stored at -80°C for further studies.
Estimation of liver lipid profiles
Liver tissues were washed in ice-cold saline (0.9%), blotted on absorbent paper and weighed. Hepatic lipids were extracted from 1 g liver with chloroform and methanol (2:1 v/v) according to the procedure of Folch . Hepatic cholesterol, triglycerides and free fatty acids were estimated by commercially available kits (Randox Laboratories).
Assay of Liver antioxidant enzymes and lipid peroxidation
Superoxide dismutase (SOD) activity was determined spectrophotometrically according to a modified method of Beyer and Fridovich . Liver tissue (100 mg) was lysed in isotonic buffer (10 mM Tric-HCl (pH 7.4), containing 200 mM mannitol, 50 mM sucrose and 1 mM EDTA) and centrifuged at 8000 rpm for 10 min. The supernatant was added to a reaction mixture containg PBS (pH 7.8), 0.1 mM EDTA, 12 mM L-methionine, 75 μM nitroblue tetrazolium (NBT) and 2 μM riboflavin to a total volume of 3 ml. The reaction mixture was kept under a fluorescent light for 15 min at 20-25°C and then measured spectrophotometrically at 240 nm. One unit of SOD represents the amount of enzyme required to inhibit the rate of NBT oxidation by 50%. The activity was expressed as units/mg protein.
Catalase (CAT) activity was measured by a modified method of Aebi . Hydrogen peroxide (H2O2) decomposition by CAT was measured spectrophotometrically at 240 nm. The molar extinction coefficient of 0.043 mM-1 cm-1 was used to determine CAT activity. One unit of enzyme activity is equal to the micromole of H2O2 degraded per minute per milligram of protein (min-1 mg-1).
Lipid peroxidation was estimated by the modified method of Buege and Aust . Briefly the MDA levels were estimated by measuring thiobarbituric acid reactive substances (TBARS) and expressed in terms of malodialdehyde (MDA) content. Before the assay, liver tissues were washed in 0.9% ice-cold saline, blotted on absorbent paper and weighed. Each sample was minced in a Tri-HCl buffer (pH 7.4) and homogenized. After centrifugation at 3000 g for 10 min at 4°C, the clear homogenate was used for biochemical assay. 125 μl of supernatant (S1) was mixed with 50 μl of PBS (pH7.4), 125 μl of 20% trichloroacetic acid containing 1% butylhydoxytoluene and centrifuged (1000 g, 10 min,4°C). Then 200 μl of supernatant (S2) was mixed with 40 μl of HCl (0.6 M) and 160 μl of Tris-Thiobarbituric acid (120 mM) and the mixture was heated at 80-85°C for 10-12 min. The absorbance was measured spectrophotometrically at 530 nm. The MDA levels were expressed as μmol/L/mg protein/mg tissue.
RNA extraction and semiquantitative RT-PCR
Total RNA was isolated from the liver tissue by using tri-reagent (Sigma-Aldrich, USA) according to manufacturer’s protocol and reverse transcribed to obtain cDNA using DNA synthesis kit (Applied Biosystems, Foster City, USA). 20 ng of cDNA was used for semi-quantitative PCR. The PCR amplification was performed for 38 cycles using the following cycling conditions: 30 sec of denaturation at 94°C, 30 sec of annealing at 59°C and 1 min of extension at 72°C, with following primers: FAS (F:ATGTGGTACGGAAGGTGGAG; R: TGGCTACCTTCGTCTGTGTG), AMPK-1α (F: GGTCCTGGTGGTTTCTGTTG; R: ATGATGTCAGATGGTGAATT) and RPL-19 (F: CGTCCTCCGCTGTGGTAAA; R: AGTACCCTTCCTCTTCCCTAT).
Western blot analysis
Liver tissue protein was extracted with lysis buffer (Sigma Aldrich, USA) and quatified using Bradford method. Equal amount of proteins were resolved on 10% SDS-PAGE gel and transferred to nitrocellulose membrane. To block nonspecific binding sites, blots were incubated at 4°C with 5% (v/v) skimmed milk for 1 hr followed by overnight incubation in primary antibodies of rabbit anti-FAS and mouse anti- β actin (Santa Cruz Biotechnology, USA) at 1:1000 dilution. The immunoreactive antigen was then recognized by incubation with Horse radish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology). Immunoreactive bands were visualized with chemiluminescence detection system (Thermo Fisher Scientific, USA).
Liver morphology and histopathology
Liver tissues from all groups of rats were collected and kept in 10% formalin solution. A small piece of tissue was sectioned with microtome, fixed on slides and stained using haematoxilin and eosin (H&E) staining procedures and observed under optical microscope.
Results are expressed as mean ± S.D (standard deviation). The statistical analysis of results was done by using t-test and one- way analysis (ANOVA) followed by Ducan’s. Values with p < 0.05 were considered to be statistically significant.