Tephrosia purpurea Fraction Attenuates Lipid Accumulation and Adipogenesis in 3T3-L1 Adipocytes and Reduces Body Weight in High Fat Diet Induced Obese Rats

The anti-adipogenic and anti-obesity activity of 
chloroform fraction of Tephrosia purpurea (CFTp) on 3T3-L1 adipocytes and high fat diet 
(HFD)-fed obese rats was evaluated in this study. A substantial and dose 
dependent inhibition of α-glucosidase 
(81%) and lipase (75%) activities by CFTp was noticed. Treatment with CFTp (250 
μg/mL) significantly inhibited 3T3-L1 adipocytes differentiation and lipid 
accumulation. A semi-quantitative 
RT-PCR analysis of 3T3-L1 cells revealed down regulation of mRNA expression of 
peroxisome proliferator-activated receptor-γ (PPAR-γ), fatty acid synthase (FAS) 
and acetyl CoA carboxylase-2 (ACC-2), while 
glucose transporter type-4 (GLUT-4) expression was up-regulated in a 
dose dependent manner with CFTp. Further, oral administration of CFTp (200 
mg/kg.b.wt.) significantly reduced body weight gain, fat mass, blood glucose and 
leptin levels in high fat diet (HFD)-induced obese rats. Taken together, these 
findings demonstrate that CFTp possesses potent anti-obesity activities.

Advances in Bioscience and Biotechnology overweight of which 650 million are obese in the world [1] [2]. A sea change in food habits, work culture, increased snaking frequency, reduced physical activity and sedentary life styles have been the prime causes for enhanced MetS cases world over, especially in developing countries. The situation of childhood obesity is more alarming than adults [3].
Although a few FDA approved drugs are available to treat obesity or diabetes, drugs that can target both diabetes and obesity are lacking. In fact, some of the anti-obesity drugs have been withdrawn from the market due to their side effects [4] [5] [6]. In view of the high demand for safe, effective anti-obesity and antidiabetic drugs and considering the side effects associated with existing synthetic drugs, there is a growing necessity to explore natural product based therapeutic alternatives. Targeting key carbohydrate and lipid metabolizing enzymes or molecules that reduce adipogenesis or/and insulin resistance have been considered as potential means to develop effective drugs to attenuate obesity or/and diabetes [7].
Compounds that interfere in the transcriptional regulation of key genes associated with lipid metabolism, insulin resistance and adipogenesis like peroxisome proliferator-activated receptor-γ, fatty acid synthase, acetyl CoA carboxylase-2, glucose transporter type-4 etc., have been found to be useful in developing effective therapeutics [8] [9] [10] [11]. Adipokines like leptin and adiponectin have been reported to play decisive roles in the regulation of obesity and insulin resistance and hence could be targeted as therapeutic molecules [12]. Leptin is produced from white adipose tissue and plays a negative feedback role in the regulation of energy expenditure through controlling specific neuronal groups of hypothalamus. In overweight/obese subjects a condition called leptin resistance occurs that leads to addicted food intake and eventually to more obesity. The expression of adiponectin is inversely related to obesity and exerts its action through modulating PPAR-γ and AMPK pathways [13].
Tephrosia purpurea (L.) Pers. is a perennial herb belonging to the family Fabaceae, distributed in Asian countries. It is commonly known as "Sarapunkha" in classical Ayurvedic texts and is traditionally used to treat cough, cold, cirrhosis, splenomegaly, abdominal swelling and also as an antidote in folklore medicine. Previous studies have identified several active components including flavonoids and other phytochemicals such as pongamol, semiglabrin, lanceolatins A and B, lupeol, rutin and β-sitosterol in T. purpurea extract [14]. Pharmacological studies on T. purpurea extracts showed hepatoprotective, anti-inflammatory, anti-allergic, antioxidant and antimicrobial activity [15]. In the present work, we evaluated anti-adipogenic and anti-obesity activity of T. purpurea extract, CFTp in 3T3-L1 adipocytes and HFD-fed obese rat model.

DPPH Antioxidant Assay
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.

Ferric-Reducing Antioxidant Power (FRAP) Assay
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.

Assay of α-Glucosidase Activity
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.

Assay of Pancreatic Lipase Activity
Briefly where, Ac-Absorbance of control, As-Absorbance of sample.

Cell Viability by MTT Assay
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% CO 2 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 %Inhibition of proliferation = %untreated cell viability (100) − %drug treated cell viability.

Adipocyte Differentiation-Measurement of Cellular Lipid Contents by Oil Red O Staining
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.

Lipolysis: Measurement of Glycerol Content
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% CO 2 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.

RT-PCR-mRNA Expression
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

Animals and Diets
Male WNIN rats and diets were obtained from National Institute of Nutrition

Measurement of Body Weight and Body Composition Parameters
The body composition, body weight, fat percent of each rat was measured by

Estimation of Leptin and Adiponectin Levels
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.

Oral Glucose Tolerance Test (OGTT)
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
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).

Antioxidant Activity of CFTp by DPPH and FRAP Assays
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)).

Inhibitory Effect of CFTp on α-Glucosidase and Pancreatic Lipase Activities
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.

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

Effect of CFTp on Adipocyte Differentiation, Lipid Content and Glycerol Release in 3T3-L1 Cells
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)).

Effect of CFTp on mRNA Expression of FAS, GLUT4, ACC-2 and PPAR-γ
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.     Values are mean ± SD, n = 6. Values are statistically significant at *p < 0.05. a* significantly different from normal control and b* significantly different from HFD control. 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.

Discussion
The cases of obesity, diabetes, hypertension and CVDs have tremendously in- Overall, these findings support pharmacological effects of CFTp, which can be treated as natural product therapy in patients with obesity-related compilations.
Inhibiting the activity of key enzymes of carbohydrate and lipid metabolism has been considered as potential therapeutic target to contain obesity and diabetes [25]. In the present study CFTp substantially inhibited the activity of α-glucosidase and pancreatic lipase ( Figure 2). Previous studies on phytochemicals of Oncoba spinosa and Ficus carica have shown inhibition of α-amylase, α-glucosidase and pancreatic lipase activity leading to anti-obesity and anti-diabetic activity [26] [27]. Piper and Capsicum extracts have brought about weight reduction in diet induced obese rat models through inhibition of key lipid metabolizing enzymes [28] [29].
Our studies on mRNA expression level showed down-regulation of PPAR-γ, FAS and ACC-2 but up-regulation of Glut-4 in the presence of CFTp. This indicates that, CFTp exerts anti-adipogenic activity through modulation of master transcriptional regulator PPAR-γ as well as ACC-2 and FAS [10] [11].
Down-regulation of ACC-2 by CFTp might lead to increased oxidation of fatty acids and favours glucose uptake (as evident from increased expression of Glut-4) leading to its oxidation and might contribute to decrease insulin resistance [9]. leading to reduced inflammation, insulin resistance and obesity [30] [31]. Our results suggest that, CFTp has beneficial effects in the management of adipogenesis, hyperlipidemia and insulin resistance.

Conclusion
These findings suggest that, CFTp attenuates insulin resistance and obesity through inhibition of key enzymes of carbohydrate and lipid metabolism, modulation of leptin and adiponectin levels in HFD-induced obese rats and through transcriptional regulation of mRNA expression of PPAR-γ, FAS, ACC-2 and Glut-4 levels in 3T3-L1 adipocytes. Thus, CFTp could be considered as an effective therapeutic agent to treat obesity and insulin resistance.