Effect of Macrotyloma uniflorum on antiobesity in rats fed with a high fat diet

Abstract

1 Introduction Obesity is a universal health burden develops from an inequity between food consumption and energy disbursement which causes excessive deposition of fat in adipose tissue, liver tissue, muscle, pancreatic islets and other organs involved in metabolism results in dyslipidaemia, glucose intolerance, coronary heart disease, diabetes, hypertension, non-alcoholic fatty liver disease and cancer (Isabelle et al., 2017). Globally, 600 million people are obese and 19 billion adults are overweight (Jian Bing et al., 2016). Fat absorption process is mediated by pancreatic lipase (PL) and militarization of fat stored in adipose tissues is mediated by triglyceride lipase (TGL) (Rudolf et al., 2012). Lifestyle modification and high energy diet have increase the incidence of obesity (Hasani et al., 2013) There are several antiobesity drugs are available, however, they have perilous side effects and hence medicinal plant including crude extracts and isolated compound from plant can be used to induce weight loss and prevent diet induced obesity (Matson and Fallon, 2012). The potential of natural products against obesity is still largely unexplored and can be an excellent alternative for the safe and effective antiobesity drugs from natural origin. Macrotyloma uniflorum traditionally used as an antiobesity natural food supplement’s in India. It belongs to the family Fabaceae and has been used in ethnomedicine for treating haemorrhoids, tumours, bronchitis, cardiopathy, nephrolithiasis, urolithiasis, splenomegaly, strangury, hiccough, ophthalmopathy, verminosis, kidney stones, inflammation, and liver-related abnormalities (Bigoniya et al., 2014). As an effort to evaluate scientifically on the ethanolic extract of M. uniflorum leaves (EEMUL), ethanolic extract of M. uniflorum seeds (EEMUS), and ethanolic extract of M. uniflorum seeds and leaves combination (EESLC) against obesity. The preliminary investigation was carried out on In vitro inhibitory activity of fractions against PL. The potent PL inhibitor fractions was further characterised by In vivo anti-obesity including food intake, body weight, blood serum lipid profile and hepatoprotective potential on high fat diet (HFD) induced male albino Wistar rats. 2 Materials and methods 2.1 Plant collection and extraction M. uniflorum leaves and seeds were collected from organic cultivation field and authenticated by Rapinat Herbarium, Trichy, Tamil Nadu, India (voucher number VB001) for future reference. The extraction was performed by standard procedure (Sasidharan et al., 2011) using coarse powder of M. uniflorum seed, leaves and mixture. 100 g of each coarse powder suspended separately in 300 mL of ethanol. The extract was filter through 420-μm stainless steel filter and excess solvent was removed by rotary evaporator and recovered yields were 22.4% ± 1.25 (leaves), 19.5% ± 2.07 (seed) and 56.8% ± 1.65 (leaf and seed) and all the filtrate was stored at 4 °C until further use. 2.2 In vitro pancreatic lipase inhibition assay An assay of PL activity was performed by standard method, described by Moreno et al. (2003), with some modification. Briefly, different concentrations (250, 500, 1000, and 2000 mg/mL) of EEMUS, EEMUL and EESLC added separately to test tubes contain Tris-HCl buffer and incubated for 3 min at 37 °C and then 0.5 mL aliquot of porcine PL (250 mg/mL, type II, Sigma Chemical Co.) was added to each test tube to initiate the reactions. After 30 min of incubation, all the test tube was immersed in boiling water for 2 min to stop the reaction and then cooled. The free fatty acid concentration was determined according to manufacture protocol. using commercial kit (NEFA kit, Randox, China). 2.3 Animals Adult healthy male albino Wistar rats were, bred and reared in the Central Animal House, Department of Animal Science, SASTRA University. Weight matched animals (180–200 g) were selected and kept in natural conditions (12 h light/12 h dark). The animals were allowed free access to water and a standard pellet diet (Amrut Animal Feed Laboratory, Pranav Agro Industries Ltd., Bangalore, India). The standard pellet diet contained 21.1% protein, 5.1% fat, 60.0% carbohydrate, 3.9% fibre, 7.9% minerals, and 2.0% vitamins and HFD consist 34.1% of beef tallow along with standard pellet diet. Handling and experimental protocol ethical committee approval was obtained from SASTRA University Institutional Animal Ethics Committee (Reg. No. 376/SASTRA/IAEC/RPP). 2.3.1 Experimental design EESLC showed potent PL inhibitor when compared to EEMUL and EEMUS. Therefore, EESLC further analysed on In vivo studies. Initially, before the study, all test animals were fed a standard diet. The animals were then divided into six groupings with six rats in each group as follows. Group 1 fed the standard pellet diet for 12 weeks; Group 2 fed with the HFD for 12 weeks; Group 3 fed with the HFD for the first 8 weeks, and then received EESLC (200 mg/kg BW) along with the HFD for the next 4 weeks; Group 4 fed with HFD for the first 8 weeks and then received EESLC (400 mg/kg BW) along with the HFD for next 4 weeks; Group 5 fed with HFD for the first 8 weeks and then received EESLC (600 mg/kg BW) along with the HFD for the next 4 weeks. Group 6 fed with HFD for the first 8 weeks and then received orlistat (10 mg/kg BW) along with the HFD for the next 4 weeks. After the experimental period, all the rats were anesthetized intramuscularly with ketamine. Blood samples were collected from both control and experimental groups for biochemical analysis. 2.3.2 Biochemical estimations Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and Alkaline phosphatase (ALP) Gamma-glutamyl transpeptidase (GGT), Triacylglycerol (TG) and total cholesterol (TC) estimation kits were purchased from Sigma-Aldrich, USA. The biochemical parameters were analysed according to manufacturer instruction using auto-analyser (Indiko model-Thermo Fisher scientific, USA). Low-density lipoprotein cholesterol (LDL-C) and very low-density lipoprotein cholesterol (VLDL-C) levels were calculated as prescribed previously (So Young et al., 2015, Mauro et al., 2006). 2.4 Statistical analysis Results are expressed as mean ± S.D. Statistically, all the data were compared using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT) by the SPSS software version 11.5 and p-values <0.05 considered as statistically significant. 2.5 High performance thin layer chromatography (HPTLC) HPTLC was carried out according to a previous report (Misra et al., 2008). Briefly, 2 µl of EESLC was loaded on 5 mm band length in the 3 × 10 Silica gel 60F254 TLC plate using a Hamilton syringe and CAMAG LINOMAT 5 instrument (Mumbai, India). After the saturation of solvent vapour the sample loaded plate was kept in the TLC twin with mobile phase (ethyl acetate-methanol-water (10:1.35:1)) up to 90 mm. The developed plate was dried in hot air in order to evaporate the solvents and then kept in a photo-documentation chamber (CAMAG REPROSTAR 3) to capture the images in white light, UV 254 nm, and UV 366 nm. After derivatization, the plate was fixed in the scanner stage (CAMAG TLC SCANNER 3) and was scanned at 500 nm. The peak table, peak display, and peak densitogram was recorded. Furthermore, the identified bioactive compounds in EESLC were characterised by In silico docking, in order to understand the inhibitory mechanisms. 2.6 Molecular docking analysis The structure of rat pancreatic lipase – RPL (PDB code – 1BU8) and Human Pancreatic lipase – HPL (PDB code – 2PPL) were retrieved from the protein data bank (www.rcsb.org) and adipose triglyceride lipase (ATGL) was predicted by homology modelling MODELLER (Natalie et al., 2010) using ATGL sequence retrieved from NCBI. The refined protein and its possible conformation sites were obtained using a procheck analysis with Ramachandran plot (Laskowsky et al., 1993). The structure of identified compound from HPTLC analysis of study subjects were retrieved from PubChem (www.pubchem.ncbi.nlm.nih.gov). Docking analysis was performed using Discovery Studio 2.5 (Accelrys Inc., 2010). Docking calculations were performed with program LibDock16 implemented in Discovery studio 2.5. The binding affinities are expressed as docking scores by logarithm of the dissociation constant (lgKd). 3 Results 3.1 Pancreatic lipase inhibition The efficacy of EEMUS, EEMUL, and EESLC on PL activity was depicted in Fig. 1. EESLC showed dose dependent activity on PL inhibition. The standard drug, orlistat, showed 61.1% inhibition and EESLC showed 58.9% inhibition at the concentration of 50 µg/ml. EFMUS and EEMUL showed 47.8% and 46.2% inhibition, respectively. The activity of EESLC was analogous to that of the positive control orlistat and EESLC showed the significant inhibitory activity with an IC50 value of 30 µg/ml. This indicated that EESLC had prominent PL inhibitory potential compared to EEMUS and EEMUL. Hence EESLC were further investigated In-vivo anti-obesity effect in animal model using HFD-induced rats. Fig. 1 Pancreatic lipase Inhibition assay – Orlistat, EEMUS, EEMUL and EESLC. 3.2 Effects of EESLC on reduction of body weight and obesity The optimized dose was 400 mg/kg BW was finalized as the optimal dose for the present study. Changes in water intake, food intake and final body weight in both control and experimental rats were shown in Table 1. HFD-induced obesity rat final body weight (297.72 ± 17.15 g), food intake (29.91 ± 1.14 g), and water intake (27.63 ± 2.35 mL) were significantly elevated when compared to normal control (NC) rats (213.70 ± 8.87 g, 20.96 ± 1.11 g and 24.91 ± 2.11 mL, respectively). Supplementation of EESLC at 400 mg/kg BW for 4 weeks caused a substantial reduction in final BW (216.45 ± 8.15 g), whereas, EESLC fed group food intake slightly decreased (22.12 ± 1.28), when compared to HFD control (29.91 ± 1.14 g), but significantly increased in food intake on normal control group (20.96 ± 1.11 g). The obese status was recorded in HFD (BMI – 7.38 ± 0.31) control group rat. Interestingly, our results showed similar BMI on Orlistat treated (BMI 4.35 ± 0.143) and EESLC (400 mg/kg) treated (BMI 4.36 ± 0.136) rats. Table 1 Effect of ethanolic extract of M. uniflorum seed and leaf combination (EESLC) on body weight, food intake, and water intake on high-fat diet (HFD) fed obese rats. Groups Body weight (g) Average food intake (g) Average water intake (mL) Initial Final Control 183.81 ± 10.17 213.70 ± 8.87a 20.96 ± 1.11a 24.91 ± 2.11a HFD 185.34 ± 9.13 297.72 ± 17.15b 29.91 ± 1.14b 27.63 ± 2.35b HFD + EESLC (200 mg/kg, bw) 184.34 ± 10.21 273.54 ± 14.23c 28.65 ± 1.31c 26.12 ± 2.12c HFD + EESLC (400 mg/kg, bw) 188.13 ± 10.91 216.45 ± 8.15d,a 22.12 ± 1.28d 25.32 ± 2.34d,a HFD + EESLC (600 mg/kg, bw) 187.32 ± 9.02 249.45 ± 17.65e 24.01 ± 1.21e 27.12 ± 2.15b HFD + Orlistat 180.64 ± 11.01 203.39 ± 11.06a,d 26.66 ± 1.08d 25.73 ± 1.97d,c Values are means ± SD of six rats in each group. Values not sharing a common superscript differ significantly at p < 0.05. Duncan’s Multiple Range Test (DMRT). 3.3 Effects of EESLC on high fat-diet induced obesity As mentioned in Table 2, it has been observed that rat fed on high fat with EESLC Supplement consecutively for 28 days, resulted decreased level of serum lipid profile. The significant (p < 0.01) increased level of serum TG, TC, LDL-C and VLDL-C were observed in HFD-fed group and a significant (p < 0.01) decrease in serum HDL-C levels when compared to the NC group rats receiving normal fed. The HFD group that was administered EESLC showed a significant (p < 0.01) decrease in lipid metabolic parameters including TC, TG, LDL-C, and VLDL-C compared to HFD control group. Oral administration of orlistat for 4 weeks along with a HFD caused a noteworthy decrease in TG, TC, LDL, and VLDL levels and an upsurge in HDL compared to the HFD control group. However, no significant (p > 0.05) variance in serum lipid profile between the NC group and EESLC group was observed. Table 2 Effect of ethanolic extract of M. uniflorum seed and leaf combination (EESLC) on lipid profiles of high-fat diet (HFD) fed obese rats. Groups TC (mg/dL) TG (mg/dL) HDL-C (mg/dL) VLDL-C (mg/dL) LDL-C (mg/dL) Control 78.16 ± 3.00a 61.14 ± 2.43a 48.81 ± 3.16a 12.22 ± 0.73a 17.15 ± 1.24a HFD 77.87 ± 2.98a 60.09 ± 2.73a 49.97 ± 3.21a 12.01 ± 0.48a 15.85 ± 0.82a HFD + EESLC (200 mg/kg, bw) 150.21 ± 5.94b 145.09 ± 5.98b 29.14 ± 2.65b 29.01 ± 2.32b 92.03 ± 6.64b HFD + EESLC (400 mg/kg, bw) 139.67 ± 4.35c 129.23 ± 4.23c 35.78 ± 2.90c 25.84 ± 1.90c 78.05 ± 4.56c HFD + EESLC (600 mg/kg, bw) 89.82 ± 5.04d 70.09 ± 2.76d 43.38 ± 3.18d 14.02 ± 1.42d 32.42 ± 2.17d HFD + Orlistat 118.34 ± 4.90e 110.43 ± 2.43e 40.67 ± 3.09e 22.08 ± 1.67e 55.59 ± 3.54e Values are means ± SD for six rats. Values not sharing a common superscript differ significantly at p < 0.05. Duncan’s Multiple Range Test (DMRT). 3.4 Effects of EESLC on liver enzymes As shown in Table 3, the EESLC with fed exhibits a hepatoprotective effects indicated by the decreased level of liver enzymes AST, ALT, ALP, and GGT. The level of AST, ALT, ALP, and GGT were significantly increased (96.72 ± 2.06, 42.90 ± 3.79, 104.45 ± 6.47 and 34.48 ± 0.29 IU/L) in the HFD control group compared to NC group. The liver enzyme level significantly decreased AST (82.69 ± 5.32 IU/L), ALT (35.74 ± 1.78 IU/L), ALP (90.31 ± 4.02 IU/L) and GGT (23.86 ± 0.24 IU/L) in all EESLC treated HFD rats. Table 3 Effect of ethanolic extract of M. uniflorum seed and leaf combination (EESLC) on liver enzymes of high fat diet (HFD) fed obese rats. Groups AST (IU/L) ALT (IU/L) ALP (IU/L) GGT (IU/L) Control 75.83 ± 2.43a 28.46 ± 2.47a 85.34 ± 2.14a 21.88 ± 0.23a HFD 96.72 ± 2.06b 42.90 ± 3.79b 104.45 ± 6.47b 34.48 ± 0.29b HFD + EESLC (200 mg/kg, bw) 84.33 ± 5.50c 37.66 ± 3.72c 94.33 ± 5.92c 25.53 ± 1.64c HFD + EESLC (400 mg/kg, bw) 82.69 ± 5.32d 35.74 ± 1.78d 90.31 ± 4.02d 23.86 ± 0.24d HFD + EESLC (600 mg/kg, bw) 86.0 ± 3.76e 36.99 ± 4.91e 92.66 ± 6.10e 24.0 ± 5.21e HFD + Orlistat 79.15 ± 2.24d 32.89 ± 1.45d 87.42 ± 3.68d 22.05 ± 0.27d Values are means ± SD for six rats. Values not sharing a common superscript differ significantly at p < 0.05. Duncan’s Multiple Range Test (DMRT). 3.5 HPTLC analysis of EESLC The EESLC loaded TLC plate was visualized under UV light at 254 nm and visible light, without derivatization. Fig. 2 represents the presence of bioactive compounds gallic acid and kaempferol in EESLC on TLC plate photograph. The qualitative and quantitative presence of gallic acid and kaempferol was confirmed by HPTLC co-chromatography and overlain absorption spectra with quantitative reference standard, when scanned at 365 nm (Misra et al., 2008). The densitometric chromatogram of HPTLC fingerprint of the EESLC is shown in Fig. 3a–c. The peaks resolving at Rf 0.13 and 0.72 in test solution were found to be superimposing with those of respective standards. On the basis of calibration curve linear range of gallic acid and kaempferol in EESLC were found to be 1.17–300 µg/ml and 1.95–500 µg/ml respectively. The Regression equation (Y = 757,131X − 17,232 and Y = 351,194X + 15,589) and correlation coefficient was found not less than 0.999, which revealed a good linearity response of bioactive principles in EESLC. Fig. 2 TLC chromatogram under UV-254 nm and Visible light 1. GAS – Galic acid standard 2. KS – Kaempferol standard 3.CS – p-Coumaric acid standard 4. EESLC. Fig. 3 HPTLC densitometric chromatogram a. Kaempferol standard, b. GAS – Galic acid standard, and c. EESLC. 3.6 Molecular docking analysis To elucidate the affinity and mode of binding of Orlistat, kaempferol and gallic acid (ligands) with the PL enzymes (RPL -1BU8 and HPL-2PPL) and adipose triglyceride lipase (ATGL protein), docking analysis were performed. The details of libdoc scores and energy values of ligand poses with high binding energy are summarized in Table 4. Structural interaction differences in ligands with nucleotide binding sites are shown in Fig. 4a–h. The Orlistat showed high binding energy and libdoc scores with RPL and HPL (−72.384 and −76.713 kcal/mol), when compared to kaempferol and gallic acid interactions. In RPL and HPL β-sheet side chain of ILE536 (RPL), ASP767 and MET655 (HPL) residues are interact with Orlistat (Fig. 4a & b) and none of the residues of ATGL showed interactions. Kaempferol and galic acid showed higher binding efficiency than that of orilstat. RPL active sites – LYS232, LYS329, ASN240 and LEU213, HPL active sites – PRO813, GLN814, MET534, ILE536 and ATGL active sites – GLY803 and GLN766 are interact with Kaempferol and showed binding energy −40.387, −15.477 and −40.387 respectively. Galic acid interact with active sites residues of RPL – VAL805, MET655, GLY803, GLN766, HPL – GLN814, PRO813, ILE536, MET534 and ATGL – VAL805, GLY803 and MET655 with hydrogen bond and showed binding energy −15.477, −40.387 and −15.477 respectively. Table 4 Ligand and protein docking complex with binding energy (kcal/mol) and LibDock score. Ligand Protein Residues Poses Absolute energy (kcal/mol) Libdock score H-bond Length (Å) Orlistat RPL -1BU8 ILE 536 98 −72.384 149.593 1 1.643 HPL ASP 767MET 655 91 −76.713 146.018 3 1.9042.144 ATGL NIR NIR NIR NIR NIR NIR 

 Kaempferol RPL -1BU8 LYS 232LYS 329ASN 240LEU 213 5 −40.387 101.973 4 1.9382.0801.8122.156 HPL PRO 813GLN 814MET 534ILE 536 25 −15.477 64.604 5 2.3892.2792.0672.287 ATGL GLY 803GLN 766 30 −40.387 108.824 2 2.2482.428 

 Galic acid RPL -1BU8 VAL 805MET 655GLY 803GLN 766 23 −15.477 85.759 5 1.8792.4702.1512.349 HPL GLN 814PRO 813 ILE 536MET 534 29 −40.387 81.546 5 2.3892.2782.2052.0512.118 ATGL VAL 805GLY 803MET 655 20 −15.477 84.912 3 1.8581.9352.004 Fig. 4 Docking conformation of a. Orlistat with binding pocket of RPL, b. Kaempferol with binding pocket of RPL, c. Kaempferol with binding pocket of HPL, d. Kaempferol with binding pocket of ATL e. Galic acid with binding pocket of RPL f. Galic acid with binding pocket of HPL. 4 Discussion In this study we found that combination of M. uniflorum leaves and seeds decrease the obesity of rats fed with high fat diet. PL is an important enzyme for triacylglycerol absorption and hydrolyses of triacylglycerol to form a monoacylglycerol and free fatty acid. Substrates for the lipase enzyme are long-chain triacylglycerols (Kim et al., 2016). In fact, currently two anti-obesity drugs such as orlistat, as PL inhibitor and sibutramine, as appetite suppressant were approved by US FDA (Bujjirao and Kumar, 2013). However, administration of these drugs can cause adversative side effects, like steatorrhea, faecal incontinence and flatulence, hence its limited in use (Shin et al., 2003, Shi and Burn, 2004, Zhang et al., 2008). HFD-fed cause the development of obesity by acceleration of body weight with expanded adipose mass (Buettner et al., 2006). Many previous studies describing the obesity with the consumption of HFD in albino rats (Schrauwen and Westerterp, 2000, Hariri and Thibault, 2010). Current findings are coinciding with those of earlier experiments, where rats fed with HFD gained additional weight than those fed a normal diet (Kumar et al., 2013). In HFD and NC groups Body weight and daily food intake showed no statistical correlation. The HFD-fed groups constantly up taking the analogous amounts of food which providing higher calorie value diet. As a result, the calorie content of the diet increases the total body weight, resulting obese state in HFD groups. Administration of EESLC to rats fed a HFD resulted in a remarkable reduction in body weight compared to the HFD-group rats (Mukherjee, 2003). The hypercholesterolaemic effect may be ascribed to an increased dietary cholesterol intake (John Griffin and Alice Lichtenstein, 2013) and subsequently increased rate of intestinal cholesterol absorption (García Otín et al., 2007). Saturated fatty acids and monounsaturated fatty acids are rich in animal fat; hence the serum LDL cholesterol and serum triacylglycerols level are increased (Lopez Alvarenga et al., 2010). Therefore, the lowering of serum lipid profiles can be used as additional key factor for antiobesity. Current study revealed that the administration of a EESLC cause the elevated level of lipid profile in plasma of HFD fed rats. Furthermore, instigation of intestinal fat absorption, lipolysis, and gastric lipases cause the modification of the lipid profile in HFD-fed group. Increasing TC and LDL-C level in blood plasma are prone to developing coronary heart disease (Walkowiak et al., 2013) whereas increase HDL-C in blood plasma can cause transportation of additional cholesterol to the liver for the elimination of bile (Yoshizaki et al., 2014). The bioactive principles of EESLC may cause decreasing the absorption of fat and cholesterol through inhibition of PL activity. Our finding agreed with a previous report by Han et al. (2003). Liver markers such as AST, ALT, ALP, and GGT were found in elevated concentrations in the hepatic cells of EESLC treated rats. In the present study, HFD rats showed significant decreases level of hepatic enzymes, which exhibit hepatoprotective and consistent with the observations of previous report (Abbas and Hussein, 2013). The potential outcome of the present study due to the presence of quantitative bioactive principles such as kaempferol and galic acid in the EESLC. The standard drug, orlistat, binds to the serine amino acid on active sites of PL. Therefore, the substrate lipids are unable to bind with the enzyme and are not metabolized into simpler forms monoglycerols and free fatty acids. In addition, there is no absorption of fat, which results in the reduction of obesity (Zhi et al., 1995, Guerciolini, 1997). Molecular docking assessment reveals high binding potential of kaempferol and galic acid towards the active sites of PL and ATGL enzymes. Kaempferol and galic acid in HPL of C-terminal domain active sites predicted with hydrophobic interactions by hydrogen bond at inner core residues PRO813, GLN814, MET534 and ILE536. PL activity requires co-lipase as co factor to which C-terminal domain binds (Ganesh Kumar et al., 2015). Kaempferol and galic acid interacts and inhibit the docking lid from accomplishing PL protein optimal conformation. The compound binding to theses catalytic interaction with other nearby charged residues (LYS232 and LYS329) are expected to alter the stability and PL inhibition by docking simulations (Benarous and Abderrahman, 2011). Cumulatively, the computational and experimental data presented here propose that these high content of kaempferol and galic acid in EESLC have definite effect on lipid metabolism by inhibition of PL through reducing the intestinal absorption of dietary fats. 5 Conclusion Current study, exhibit the ameliorative potential of EESLC on HFD-fed obesity in albino Wistar rats. Through animal model and docking analysis, we confirmed that EESLC was an appropriate and potential anti-obesity nutraceutical candidate.