Acarbose, orlistat, α-glucosidase (catalog no. G5003), pancreatic lipase (catalog no. L3126), paranitrophenyl-glucopyranoside (p-NPG), p-nitro phenyl butyrate (p-NPB), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and Oil Red O (ORO) staining solution were procured from Sigma Aldrich. Nonidet P­40, morpholine propane sulphonic acid (MOPS), isopropyl alcohol, Insulin, Dulbecco’s modified Eagle’s medium (DMEM), fetal Bovine serum (FBS), 3­isobutyl­1­methyl­xanthine (IBMX), penicillin and streptomycin were procured from Thermo Scientifics. Other chemicals, solvents and reagents used were of Analytical Grade.

Fresh whole plants of T. purpurea were obtained from Chinthalapatteda area near Nagari and Tirupati, Andhra Pradesh, India. Their identity was authenticated by a taxonomist (Voucher Specimen Accession Number-1259) and deposited in the herbarium of Department of Botany, S.V. University, Tirupati. T. purpurea plants were shade dried, crushed to crude powder and extracted with chloroform following cold extraction method. The chloroform extract was then fractionated using hexane, ethyl acetate, chloroform and methanol based on their polarity in a column chromatography using silica gel as the column material. All the filtrates were concentrated under reduced pressure in Heidolph rotary-evaporator. Based on phytochemical analysis, chloroform fraction of T. purpurea (CFTp) was used for further studies.

Briefly, a 0.3 mM solution of DPPH was prepared in methanol and 500 µL of this solution was added to 1 mL of CFTp at different concentrations (100 - 500 µg/mL) [16] . These solutions were mixed and incubated in the dark for 30 min at room temperature. The absorbance was measured at 517 nm against a blank lacking scavenger. Vitamin C was used as a standard. The antioxidant or free radical inhibitory activity was calculated according to the following formula

%inhibition = ((Ac – As)/Ac) × 100

where, Ac—Absorbance of control, As—Absorbance of sample.

A 2.5 mL aliquot of CFTp was mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide [17] . The mixture was incubated at 50˚C for 20 min, followed by addition of 2.5 mL of 10% trichloro acetic acid and centrifuged at 3000 rpm for 10 min. Then, 2.5 mL of the upper layer of the solution was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride, after 10 min, absorbance was measured at 700 nm. An increase in the absorbance of the reaction mixture indicated increased reducing power of CFTp. The experiment was carried out in triplicate, using vitamin C as a positive control.

Briefly, 500 µL of CFTp and/or standard inhibitor (acarbose) at a concentrations of 100 - 500 µg/mL were incubated with 54 µL (1.0 U/mL) of α-glucosidase solution (in 100 mM phosphate buffer pH 6.8) and 446 µL of phosphate buffer for 15 min at 37˚C [18] . To this, 250 µL of p-nitrophenyl D-glucoside solution (5 mM) in 100 mM phosphate buffer (pH 6.8) was added and incubated for 20 min at 37˚C. Absorbance of liberated yellow colour p-nitrophenol was read at 405 nm using UV-visible spectrophotometer. All the readings were measured in triplicate and the average was considered. The percentage of enzyme inhibition was calculated using the formula specified below and the inhibitory activity was expressed as percentage of the control without inhibitor

%inhibition = ((Ac – As)/Ac) × 100

where, Ac—Absorbance of control, As—Absorbance of sample.

Briefly, an enzyme-buffer was prepared by the addition of 30 µL of lipase solution (2.5 mg/mL) in 10 mM morpholine propane sulphonic acid (MOPS) and 1 mM EDTA, (pH 6.8) to 850 µL of Tris buffer (100 mM Tris-HCl and 5 mM CaCl₂, pH 7.0) [19] . Then 100 µL of CFTp (100 - 500 µg/mL) was mixed with 880 µL of enzyme buffer and incubated for 15 min at 37˚C. To this, 20 µL substrate solution (10 mM p-nitro phenyl butyrate in dimethyl formamide) was added and incubated for 15 min at 37˚C. The lipase activity was determined spectrophotometrically by measuring the hydrolysis of p-nitrophenyl butyrate to p-nitrophenol at 400 nm. All assays were carried out in triplicate and the calculation was done according to the following formula. Orlistat was used as standard drug.

%inhibition = ((Ac – As)/Ac) × 100

where, Ac—Absorbance of control, As—Absorbance of sample.

Pre-confluent pre-adipocytes (3T3-L1 cells, from NCCS Pune), 2500 cells/well or mature adipocytes 5000 cells/well were seeded in 96 well culture plates using DMEM medium supplemented with 10% FBS and 1% antibiotic and incubated at 37˚C with 5% CO₂ for 48 or 72 h [20] . Then, cells were treated with CFTp. After overnight incubation, cytotoxicity/cell viability were determined by adding 10 µL of MTT [3-(4,5-dimethylthizol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt] (0.5 mg/mL in PBS), incubated at 37˚C for 4 h. At the end of incubation, culture media was discarded and the wells were washed with PBS. Later, 150 µL of dimethyl sulfoxide (DMSO) was added to all the wells, and incubated for 30 min at room temperature with constant shaking. Absorbance was read at 540 nm using Microplate Reader and subsequently percentage (%) of cell viability will be calculated using following equation to determine the formazan concentration, which is proportional to the number of live cells.

%Inhibition of proliferation = %untreated cell viability (100) − %drug treated cell viability.

3T3-L1 cells were cultured ingrowth media (GM) consisting of DMEM supplemented with 10% fetal Bovine serum (FBS) and 2mM glutamine. The cells were grown according to a well-established protocol described previously. Briefly, for differentiation, 3T3-1 cells were cultured in GM to full confluence. Two days after confluence (referred to as day 0), the cells were switched to differentiation media (DM) consisting of DMEM supplemented with 10% FBS, 10 mg/mL insulin, 1 M dexamethasone and 0.5 mM IBMX (isobutylmethylxanthine) and cultured for three days. Next, the cells were maintained in DM but containing only insulin (10 mg/mL) and the medium was changed every 2 - 3 days. The cells normally differentiate into mature adipocytes in a week. The 3T3-L1 preadipocytes were differentiated as described above in the presence of CFTp or vehicle (PBS).

For Oil Red O staining, at the end of incubation period, cell monolayers were washed twice with PBS (pH 7.4) and, fixed in 10% buffered formalin solution in PBS for 1 h, washed twice with DW and then stained with 0.5% Oil Red O stain for 30 min at room temperature. Excess Oil Red O dye was washed with DW and photographs were taken in inverted microscope using digital camera system. In another set of experiment, the stained adipocytes were treated with 60% isopropanol (to extract intracellular Oil Red O stain) and the absorbance (Optical density, OD) was read at 520 nm [21] .

%Adipogenesis was calculated as OD of treated cells/OD of untreated cells × 100.

Glycerol release was measured to assess the lipolytic effect from adipocytes and examined according to the Millipore kit procedure. Briefly, differentiated adipocytes were incubated at 37˚C in 5% CO₂ atmosphere with the CFTp in sterile Hank’s balanced salt solution containing 2% Bovine serum albumin (BSA). At the interval of 12 h and 24 h, the 10 µL supernatant from the 96 well plates were withdrawn and mixed with the 80 µL of glycerol assay reagent in a separate 96 well plate. After incubation of 1 h, the absorbance of the solution was measured at 540 nm using a microplate reader [22] . The amount of glycerol released was calculated by the equation of glycerol standard curve. Nor epinephrine, quercetin and forskolin were used as positive standards.

Total RNA was isolated from 3T3-L1 cells/adipose tissue by using tri-reagent (Sigma Aldrich, USA) according to manufacturer’s protocol and reverse transcribed to obtain cDNA using DNA synthesis kit (Applied Bio Systems, Foster City, USA) [23] . Two nanograms of cDNA were used for semi-quantitative RT-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 the specific the primers. The primer sequences used for PCR analysis were as follows:

PPAR-γ F: 5’-GACCGAGTGTGACGACAAG-3’;

R: 5’-CGTGATTTCTCAGCCGCGT-3’

Glut4 F: 5’-AAAAGTGCCTGAAACCAGAG-3’;

R: 5’-TCACCTCCTGCTCTAAAAGG-3’

FAS F: 5’-ATGTGGTACGGAAGGTGGAG-3’;

R: 5’-TGGCTACCTTCGTCTGTGTG-3’

ACC2 F: 5’-ACCTTGTTGGGGAGAAGTGC-3’;

R: 5’-AGGGCCAAGGTGTCATAAGC-3’

β-Actin F: 5’-ACCTTCCAGCAGATGTGGAT-3’;

R: 5’-AGAAGCACTTGCGGTGCACGA-3’.

Male WNIN rats and diets were obtained from National Institute of Nutrition (NIN), Hyderabad, India. After one-week quarantine period for safe health of experimental rats, they were fed with either normal diet or freshly prepared HFD (15 g/rat/day) for 16 weeks as mentioned in experimental design and water ad libitum and standard laboratory conditions (temperature: 22˚C ± 2˚C; humidity: 40% - 60%) were maintained. Normal diet contained all the recommended macro and micronutrients (carbohydrate-56%, protein-18.5%, fat-8%, fiber-12% and adequate levels of minerals and vitamins). High fat diet contained starch-42%, casein-23%, lard oil-23%, cholesterol-2%. Cellulose-5%, mineral mixture (AIN-93G)-3.5%, vitamin mixture (AIN-93VX)-1%, L-cystine-0.3%, choline bitartrate-0.2%. Rats initially weighing 180 - 200 g were randomly divided into six groups of six each (n = 6). To test the activity of CFTp, 100 or 200 mg/kg b.wt. of CFTp was suspended in 0.5% carboxy methyl cellulose (CMC), and orally administered for 42 days from 10^th week onwards using an intra-gastric tube. All experimental protocols were followed as per institutional animal ethical committee guidelines (No: 55/2012/(i)/a/CPCSEA/IAEC/SVU/MBJ, Dt: 08.07.2012).

Group 1: Normal control (normal diet control)

Group 2: High fat diet (HFD) control

Group 3: HFD + orlistat 5 mg/kg b.wt.

Group 4: HFD + CFTp 100 mg/kg b.wt.

Group 5: HFD + CFTp 200 mg/kg b.wt.

The body composition, body weight, fat percent of each rat was measured by Total Body Electrical Conductivity (TOBEC) using small animal body composition analysis system (EM-SCAN, Model SA-3000 Multi detector, Springfield, USA). At the end of the experiment, blood was collected from overnight fasted animals under inhalation of anesthesia by heart puncture method; plasma was separated by centrifugation at 2500 rpm for 15 min.

Plasma leptin and adiponectin are important adipokines and their levels were measured in experimental rats by using enzyme-linked immunosorbent assay kits (Crystal Chem, Downers Grove, IL, USA), performed in duplicate, as per the manufacturer’s guidelines and were expressed in ng/mL.

At the end of the experiment OGTT was performed [24] . After overnight fasting of experimental rats, glucose was administered orogastrically at a dose of 2.0 g/kg b.wt. and blood samples were collected from supraorbital sinus at 0, 30, 60, 90 and 120 min. Glucose levels were estimated at all intervals.

Statistical analysis was performed using Turkey’s-HSD multiple range post hoc test p < 0.05 IBM SPSS version 23. Data were expressed as the mean ± standard deviation (SD).

The antioxidant capacity of the chloroform fraction of T. purpurea (CFTp) was measured by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric Reducing Antioxidant Power (FRAP) assays. In-vitro antioxidant activity of CFTp on DPPH free radicals had shown significant scavenging activity in a dose dependent manner and its activity was close to vitamin C at 500 µg/mL of CFTp (Figure 1(A)). Similarly, the dose-dependent ferric reducing power of CFTp which was found to be about 75 % of Ascorbic acid, indicating its potential free radical scavenging activity (Figure 1(B)).

In the present study as shown in Figure 2(A) and Figure 2(B), a significant and dose-dependent inhibition of both α-glucosidase and pancreatic lipase was noticed. At 500 µg/mL of CFTp, the maximum inhibition of 81% and 75% was observed for α-glucosidase and pancreatic lipase respectively.

The cytotoxic effects of CFTp on the viability of 3T3-L1 cells was analysed at 48

h using MTT assay. No cytotoxic effect was observed up to a dose of 250 µg/mL of CFTp, but thereafter, about 10% - 15% of cell death was noticed (Figure 3(A)).

The microscopic observation of Oil Red O stained 3T3-L1 cells indicates that, groups treated with CFTp shows decreasing number of adipocytes and reduced lipid accumulation in adipocytes (in a dose dependent manner) when compared to untreated cells (Figure 3(D)). In addition, measurement of absorbance of Oil Red O stain, extracted (using isopropanol) from lipid droplets of 3T3-L1 cells, indicates the extent of adipocytes differentiation. Our results showed that, CFTp (250 µg/mL) could considerably inhibit adipocyte differentiation when compared to the untreated cells (Figure 3(B)). To understand the effect of CFTp on lipolysis of 3T3-L1 cells, glycerol release into the surrounding medium was estimated spectrophotometrically. A significant increase in glycerol content was observed in groups treated with CFTp when compared to untreated cells and the maximum lipolytic activity was noticed at a concentration of 250 µg/mL (Figure 3(C)).

The mRNA expression of FAS, GLUT4, ACC-2 and PPAR-γ in 3T3-L1 cells in the presence and absence of CFTp (Figure 4). The expression of FAS, PPAR-γ and ACC-2 were down-regulated, while GLUT4 was up-regulated with increasing concentration of CFTp. Their expression was compared to that of house-keeping gene β-actin.

Table 1 depicts change in body weight and body composition of experimental rats. Consumption of HFD for 16 weeks resulted in significant increase in body weights (458 ± 3.45 g) and total fat levels (53.245 ± 2.75 g) in HFD control group, compared to normal control group of rats whose body weight and total fat were 302 ± 7.11 g and 29.22 ± 3.04 g, respectively. Oral administration of CFTp (100 and 200 mg/kg b.wt.) for 42 days (from 12 to 16 weeks) considerably reduced body weight and body composition in a dose dependent manner. At a dose of 200 mg/kg b.wt., CFTp could substantially limit the body weight gain and total fat to 312 ± 8.16 g and 96.265 ± 4.78 g respectively.

Figure 5 depicts the levels of leptin and adiponectin in control and experimental obese rats. There was a marked increase in leptin levels, while decrease in adiponectin levels found in HFD-fed obese rats compared to the normal rats. Interestingly, treatment with CFTp has significantly (p < 0.05) decreased the levels of leptin, while the levels of adiponectin increased in HFD-fed obese rats.

All data in the table are shown as mean ±SD (n = 6). * indicates significant difference between normal control and HFD control groups. # indicates significant difference between HFD control and other groups. CFTp1 = 100 µg/mL; CFTp2 = 200 µg/mL.

Figure 6 depicts the results of oral glucose tolerance test performed on control and experimental obese rats. In the normal control group of rats, blood glucose level reached its maximum value at 60 min after glucose load and declined to near basal level at 120 min, whereas, in HFD-induced obese rats, the peak increase in blood glucose level was noticed even after 60 min and remained high over the next 60 min. Administration of CFTp (100 and 200 mg/kg b.wt) or orlistat to obese rats elicited a significant decrease in blood glucose level at 60 min and beyond when compared with HFD control rats.