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.