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EVALUATION OF ANTI-OBESITY EFFECT OF AQUEOUS EXTRACT OF MUCUNA PRURIENS SEEDS ON RATS

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Objective: Evaluation of the anti-obesity effect of aqueous extract of Mucuna pruriens seeds on rats.Methods: Male Sprague-Dawley (SD) rats were subjected to high-fat diet (HFD) for 12 wk. L-DOPA (12.5 mg/kg, p. o.) as standard drug and aqueous extract of Mucuna pruriens (AEMP) seeds (200 mg/kg, p. o. and 400 mg/kg, p. o.) as test drugs were administered in last 4 wk along with HFD. Body weight, food intake, body mass index (BMI), serum total cholesterol (TC), triglyceride (TG) and high-density lipoprotein (HDL) levels were measured at the end of fourth, eighth and twelfth wk, while white adipose tissue (WAT) mass and brain dopamine levels were measured at the end of the twelfth wk.Results: AEMP (200 mg/kg, p. o.) and (400 mg/kg, p. o.) treated groups showed a significant decrease in food intake and weight gain without altering BMI. Moreover, TG levels were lower in treated groups as compared to the HFD group, but no significant changes were observed in TC and HDL levels. L-DOPA-treated group showed a significant decrease in body weight, food intake, BMI and WAT. Both AEMP and L-DOPA-treated groups showed an increase in brain dopamine levels as compared to disease control group (p<0.05).Conclusion: L-DOPA and AEMP showed anti-obesity activity by reducing body weight gains, food intake and WAT weights; modulating TG with increased brain dopamine level which correlates to the inhibitory action of dopamine on reward mechanism.
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Original Article
EVALUATION OF ANTI-OBESITY EFFECT OF AQUEOUS EXTRACT OF MUCUNA PRURIENS
SEEDS ON RATS
JAVID MANSURI
a
, ARCHANA PARANJAPE
b
a
Parul Institute of Pharmacy, Parul University, Waghodia, Gujarat, India,
b
Edutech Learning Solutions Pvt. Ltd., Vadodara, India
Email: javid.mansuri@gmail.com
Received: 20 Oct 2016 Revised and Accepted: 17 Jan 2017
ABSTRACT
Objective: Evaluation of the anti-obesity effect of aqueous extract of Mucuna pruriens seeds on rats.
Methods: Male Sprague-Dawley (SD) rats were subjected to high-fat diet (HFD) for 12 wk. L-DOPA (12.5 mg/kg, p. o.) as standard drug and aqueous
extract of Mucuna pruriens (AEMP) seeds (200 mg/kg, p. o. and 400 mg/kg, p. o.) as test drugs were administered in last 4 wk along with HFD. Body
weight, food intake, body mass index (BMI), serum total cholesterol (TC), triglyceride (TG) and high-density lipoprotein (HDL) levels were
measured at the end of fourth, eighth and twelfth wk, while white adipose tissue (WAT) mass and brain dopamine levels were measured at the end
of the twelfth wk.
Results: AEMP (200 mg/kg, p. o.) and (400 mg/kg, p. o.) treated groups showed a significant decrease in food intake and weight gain without
altering BMI. Moreover, TG levels were lower in treated groups as compared to the HFD group, but no significant changes were observed in TC and
HDL levels. L-DOPA-treated group showed a significant decrease in body weight, food intake, BMI and WAT. Both AEMP and L-DOPA-treated groups
showed an increase in brain dopamine levels as compared to disease control group (p<0.05).
Conclusion: L-DOPA and AEMP showed anti-obesity activity by reducing body weight gains, food intake and WAT weights; modulating TG with
increased brain dopamine level which correlates to the inhibitory action of dopamine on reward mechanism.
Keywords: Mucuna pruriens, Dopamine, High fat diet, Body weight, Food intake
© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijpps.2 017v9i3.15781
INTRODUCTION
Obesity is a medical condition in which life is hindered by excess
body fat. The generally accepted benchmark is the BMI. The world
health organisation (WHO) classifies population with a BMI of<18.5
kg/m
2
as underweight, and those with a BMI of 18.5-24.9 kg/m
2
as
normal weight. A BMI in the range of 25.0-29.9 kg/m
2
as grade 1
overweight. If it is between 30.0-39.9 kg/m
2
, the patient is said to be
obese or grade 2 overweight, while those with a BMI of 40 kg/m
2
are
deemed to be grade 3 overweight or morbidly obese. The prevalence
of obesity is increasing not only in adults but also among children
and adolescents. The prevalence of obesity has increased steadily
over the past five decades and may have a significant impact on the
quality adjusted life years [1]. The causes of obesity may include
dietary, exercise, social, cultural and financial factors. Sibutramine
was widely marketed and prescribed until 2010 when it was
withdrawn after a large study showed that it increased the risk of
cardiovascular events and strokes and had minimal efficacy. An
endocannabinoid receptor antagonist, rimonabant was withdrawn
from the market due to concerns about its safety, including the risk
of seizures and suicidal tendencies. At present only one drug orlistat
has been approved for long-term use in the treatment of obesity.
Orlistat promotes 5 to 10% loss of body weight and has their own
limitations and side effects. This currently licensed drug is best
when used in combination with diet, exercise, and behaviour change
regimens. However, they do not cure obesity and weight rebound
when discontinued. Some drugs are employed to treat clinical
obesity is associated with adverse effects such as nausea, insomnia,
constipation, gastrointestinal problems, and potential adverse
cardiovascular effects. Thus, there is a great demand for the search
of new and safer anti-obesity medicines [2].
Several neurotransmitters (dopamine, norepinephrine and
serotonin), as well as peptides and hormones like ghrelin, are
involved in the regulation of food intake [3, 4]. Of particular interest
is dopamine, since this neurotransmitter seems to regulate food
intake [5] by modulating food reward via the meso-limbic circuitry
of the brain [6]. In fact, drugs that block dopamine D
2
receptors
increase appetite and result in significant weight gain [7, 8] whereas
drugs that increase brain dopamine are anorexigenic [9-11].
Additionally, an increase in body weight is a side effect of many
commonly used drugs. Particularly, anti-dopaminergically acting
neuroleptics, tricyclic antidepressants, lithium, and some
anticonvulsants contribute to weight gain. Similarly, in the obesity
body mass index is negatively correlated with D
2
receptor density in
the striatum [12, 13], which might reflect neuroadaptation
secondary to over stimulation with palatable food [14, 15]. Thus,
increased food intake may be a compensatory behaviour for low
dopaminergic drive [16, 17]. Recently it is reported that lower
striatal activation in response to food intake was associated with
obesity. Furthermore, this relation was modulated by genetically
determined D
2
receptor availability [18, 19].
Plants have been the basis for traditional medicine systems.
Numerous preclinical and clinical studies, with various herbal
medicines, have reported significant improvement in controlling
body weight, without any noticeable adverse effects.
Mucuna pruriens Linn. DC. (Leguminosae) known as ‘‘velvet bean”
and ‘‘atmagupta’’ is a climbing legume, endemic in India and in other
parts of the tropics including Central and South America. In the
Ayurvedic system of medicine, Mucuna pruriens was used for the
management of male infertility [20], nervous disorders [21] and also
as an aphrodisiac [22]. Mucuna pruriens seed powder contains a
large amount of L-DOPA (1.5%), which is a dopamine precursor and
effective remedy for the relief in Parkinson's disease [23]. Mucuna
pruriens seeds in addition to L-DOPA contain 5-hydroxytryptamine
(5-HT), tryptamine, mucunine and mucunadine. Ethanolic extract of
Mucuna pruriens shows protection against haloperidol-induced
tardive dyskinesia in rats [24]. Mucuna pruriens has been reported
to inhibit chlorpromazine-induced hyperprolactinaemia in man [25].
Mucuna pruriens has proven to be more effective than L-DOPA in
parkinson’s disease in an animal model [26]. It also shows anti-
diabetic [27], anticancer [28], anti-oxidant [29], anti-hyperlipidemic
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 9, Issue 3, 2017
Mansuri et al.
Int J Pharm Pharm Sci, Vol 9, Issue 3, 111-115
112
[30] and protective effect against nephrotoxicity [31]. These reports
led us to hypothesise that high content of dopamine in Mucuna
pruriens seed could serve as a potent therapeutic agent for the
obesity. However, their efficacy needs to be scientifically evaluated
in vivo experiments. In light of above observations, current
investigation was carried out to study the effect of dopamine-
containing plant Mucuna pruriens (L.) DC. var. utilis in HFD induced
obesity on rats.
MATERIALS AND METHODS
Chemicals and reagents
L-DOPA was obtained from Torrent Research Centre, Bhat, India; all
other reagents used in this study were of analytical grade.
Preparation of extracts
Seeds of Mucuna pruriens (L.) DC. var. utilis were purchased from the
authorised dealer and authenticated by National Institute of Science
Communication and Information Resources (voucher specimen no.:
NISCAIR/RHMD/consult/2016/3001-28-1), India. For the extraction,
freshly collected seeds of Mucuna pruriens were dried in the shade
and pulverized to get a coarse powder. One kg seed powder of
Mucuna pruriens was initially defatted with 750 ml of petroleum
ether and then aqueous extract was prepared by cold maceration
method. After 24 h, the extract was filtered using whatman filter
paper (No. 1) and then concentrated under reduced pressure (bath
temp. 50 °C) and finally dried in a vacuum desiccator [31].
Experimental animals
Male SD rats weighing 250-300 g were used in the study. The
animals were housed in a group of 6 rats per cage under well-
controlled conditions of temperature (22±2 °C), humidity (55±5%)
and light-dark cycles (12:12h). They were maintained under
standard environmental conditions and were fed a standard rat
chow diet with water given ad libitum. The study was approved by
Institutional Animal Ethical Committee, Parul institute of pharmacy,
Parul University, Vadodara, Gujarat, India (PIPH 19/12).
Experimental design
Male SD rats were acclimatised for 1 wk. Obesity in the rat was
induced by a HFD. The composition of HFD is mentioned in table 1
[32, 33]. The rats were divided into five groups consisting of six rats
in each as follows:
Group I: Normal protein diet (NPD)
Group II: HFD
Group III: HFD+AEMP (200 mg/kg, p. o.)
Group IV: HFD+AEMP (400 mg/kg, p. o.)
Group V: HFD+L-DOPA (12.5 mg/kg, p. o.)
Group, I was fed NPD, while groups II, III, IV and V were fed HFD for 12
wk that is throughout the study. At the end of 8 wk, group III, IV and V
were treated with test extract or standard drug for 4 wk along with
HFD. Body weight, food intake, BMI and serum TC, TG and HDL levels
were measured at the end of 4, 8 and 12 wk. The epididymal WAT
mass and brain dopamine level were measured at the end of 12 wk.
Table 1: Composition of HFD
Ingredients
W
eigh
t (g)
Energy (Kcal
)
50
0.2
1
Animal fat
20
0.18
Sucrose
29.8
0.11
Sodium chloride
0.2
-
100 g
0.5
Kcal/100 g
Collection of blood samples
At the end of fourth, eighth and twelfth wk, blood was collected
under inhalation anaesthesia by a retro-orbital puncture from
overnight fasted animals. Blood was allowed to clot for 30 min at
room temperature. Serum was separated by centrifugation at 4000-
5000 rpm for 15 min and analysed for serum TC, TG and HDL levels.
Estimation of TC, TG and HDL levels
TC, TG and HDL were estimated by using a Bayer diagnostic kit
(Bayer Diagnostic India Ltd.)
Fat pad analysis
At the end of the 12 wk, animals were decapitated between 09:00
and 12:00 h. After sacrificing by decapitation, the epididymal WAT
was dissected out. The collected fat was weighed immediately and
compared with the other groups [34].
Brain dopamine levels
Preparation of tissue extract
On the last day of the experiment, rats were sacrificed, and the
whole brain was dissected out, weighed and was homogenised in 3
ml HCl butanol in a cool environment. The sample was subsequently
centrifuged for 10 min at 2000 rpm. 0.8 ml of the supernatant phase
was removed and added to an eppendorf reagent tube containing 2
ml of heptane and 0.25 ml 0.1 M HCl. After 10 min, the tube was
shaken and centrifuged under the same conditions to separate two
phases. Upper organic phase was discarded and the aqueous phase
was used for dopamine assay.
Dopamine assay
0.02 ml of the HCL phase+0.005 ml 0.4 M HCL+0.01 ml EDTA
0.01 ml iodine solution was added for oxidation
After 2 min, 1 ml sodium thiosulphate in 5 M sodium hydroxide was
added to stop the reaction
10 M acetic acid was added 1.5 min later
Solution was then heated to 100 °C for 6 min
Excitation and emission spectra were read (330 to 375 nm) in a
spectrofluorophotometer
(Shimadzu RF-5301 PC) when the samples again reached room
temperature
Tissue values (fluorescence of tissue extract minus fluorescence of
tissue blank) were compared with an internal reagent standard
(fluorescence of internal reagent standard minus fluorescence of
internal reagent blank). Tissue blanks for the assay were prepared
by adding the reagents of the oxidation step in reversed order
(sodium thiosulphate before iodine). Internal reagent standards
were obtained by adding 0.005 ml bidistilled water and 0.1 ml HCl
butanol to 20 ng of dopamine standard [35, 36].
Statistical analysis
All data were presented as mean±standard error of means (SEM). One-
way analysis of variance (ANOVA) followed by Tukey’s test was used for
statistical analysis to compare more than two groups, while two-way
ANOVA was used to compare values of the different time period of the
same group. P values of less than 0.05 were considered significant.
RESULTS
Body weight
Body weight was measured every wk till twelve wk. The body
weights of all groups (II, III, IV and V) were significantly increased
Mansuri et al.
Int J Pharm Pharm Sci, Vol 9, Issue 3, 111-115
113
compared to the control group (group I) for first 8 wk. After 12 wk
the L-DOPA and AEMP (400 mg/kg) groups had significantly
(p<0.05) lower mean body weights than HFD group. The mean body
weight in the HFD group increased by 46.67 g after 12 wk of the
experimental period, whereas L-DOPA and AEMP (400 mg/kg)
group lost 23 g and 30 g respectively. In normal and AEMP (200
mg/kg) treated groups mean body weight gain was found to be 9.66
g and 6.66 g respectively (table 2).
Table 2: Body weights at different time interval
Normal
HFD
L
-
DOPA
AEMP (200 mg/kg)
AEMP (400 mg/kg)
Body weight
(g)
Initial
321.67±11.67
268.34±14.24
270.00±15.00
303.34±3.33
303.34±8.81
8
th
wk
369.66±15.275(55)
420.00±35.11
(152.3)
*
390.00±30.00
(120.67)
460.00±25.16
(147.3)
443.33±12.01(140.0)
12
th
wk
379.33±14.53
(9.66)
466.67±38.44
(46.67)
×
366.66±33.33(
-
23.33)
+
466.67±33.33(6.66)
+
413.33±6.67 (
-
30.00)
+
Values are expressed as means±SEM, n=6 in each g roup. Values are significantly diffe rent (p<0.05) by one way ANO VA followed by Tukey’s
test,
*,
g of bo dy weight gaine d d uring the first 8 wk of the experimental period,
×,+
g of bo dy weight gaine d a lter ed after 12 wk experimental
perio d.
Food intake
There was a significant increase in food intake per wk among the
HFD treated rats as compared to the normal diet-fed rats up to 8 wk.
The rats treated with AEMP (200 mg/kg, p. o. and 400 mg/kg, p. o.)
showed a significant decrease in food intake as compared to the HFD
group. L-DOPA-treated group also exhibited a significant reduction
in food intake as compared to the HFD group (fig. 1).
1 d
2 wk
4 wk
6 wk
8 wk
10 wk
12 wk
0
10
20
30
40
50
60
70
80
HFD+ 400 mg/kg AEMP
Normal
HFD
HFD+L- DOPA
HFD+ 200 mg/kg AEMP
*
Time
Food intake (g)
Fig. 1: Effect of L-DOPA and AEMP on food intake, Each line represents the mean±SEM, n=6;
*
p<0.05 compared with normal group after 12
wk of induction of HFD;
p<0.05 compared to Group II after 12 wk (analysed by two-way ANOVA followed by Bonferroni test)
Body mass index
HFD treated rats showed a significant increase in BMI every wk till
12 wk as compared to control group. L-DOPA-treated group showed
significant lower BMI whereas AEMP (200 mg/kg and 400 mg/kg)
treated group showed no significant change in the BMI.
AEMP treated groups showed preventive effect (fig. 2).
Normal
HFD
HFD+L-DOPA
HFD+ 200 mg/kg AEMP
HFD+ 400 mg/kg AEMP
0.0
0.2
0.4
0.6
0.8
1.0
1 day
After 8 weeks
After 12 weeks
*** ** ***
*
BMI (g/cm
2)
Fig. 2: Effect of L-DOPA and AEMP on BMI, Each Bar represents the mean±SEM, n=6;
***
p<0.001 compared with the normal group after 8
wk of induction of HFD;
p<0.05 compared to Group II after 12 wk (analysed by two-way ANOVA followed by Bonferroni test).
Mansuri et al.
Int J Pharm Pharm Sci, Vol 9, Issue 3, 111-115
114
Serum lipid profile
Feeding of HFD caused a significant (p<0.05) increase in serum levels
of TG as compared to normal diet fed rats. L-DOPA and AEMP (200
mg/kg and 400 mg/kg) treated groups showed significantly lower
serum TG levels than in HFD group. However, no alteration was found
in TC and HDL levels by treatment with either drug (table 3).
Brain dopamine levels
Brain dopamine levels of HFD group were significantly (p<0.05)
reduced compared with the control group. However, groups
administered with L–DOPA and AEMP (200 mg/kg and 400 mg/kg)
showed significant (p<0.05) increase in brain dopamine levels as
compared to the HFD group (fig. 3).
Table 3: Serum levels of TG, TC and HDL of rat (after the 12 wk experimental period)
Serum level (mg/dl)
Groups
Normal
HFD
L
-
DOPA
AEMP (200 mg/kg)
AEMP (400 mg/kg)
TG
75.33±7.86
155.0±5.29
*
118.3±4.41
95.33±4.84
94.33±21.96
TC
58.67±10.37
70.67±6.489
85.67±3.33
6
3.00±9.07
67.33±2.02
HDL
37.93±1.67
40.33±4.48
48.70±3.2
37.47±5.23
42.23±1.39
Results are presented as means±SEM, n=6.,
*
, Means with different letters in the same row are significantly different (p<0.05) by one-way ANOVA
followed by Tukey’s test.
Normal
HFD
HFD+L-DOPA
HFD+ AEMP 200 mg/kg
HFD+AEMP 400 mg/kg
0.0
0.5
1.0
1.5
2.0
*
••
••
Brain dopamine level (pg/mg)
Fig. 3: Effect of L-DOPA and AEMP on brain dopamine level, Each
bar represents the mean±SEM, n=6;
*
, p<0.05 compared to
group I,
●●
, p<0.01 compared to Group II (analysed by one-way
ANOVA followed by Tukey’s test)
White adipose tissue
Feeding a high-fat diet for 12 wk produced a significant (p<0.05)
increase in epididymal WAT weight of HFD treated the group as
compared to normal diet fed rats.
Normal
HFD
HFD+L-DOPA
HFD+ AEMP 200 mg/kg
HFD+AEMP 400 mg/kg
0
2
4
6
8
*
WAT (g)
Fig. 4: Effect of L-DOPA and AEMP on white adipose tissue, Each
bar represents the mean±SEM, n=6;
*
, p<0.05 compared to
group I,
, p<0.05 compared to Group II (analysed by one-way
ANOVA followed by Tukey’s test)
There were a significant reduction in the epididymal fat mass in the
L-DOPA and AEMP (400 mg/kg) treated groups while no significant
alteration was observed in AEMP (200 mg/kg) group as compared
to HFD (fig. 4).
DISCUSSION
In the present study, the anti-obesity activity of dopamine and
dopamine-containing plant Mucuna pruriens was investigated on
HFD induced rat model for obesity.
During the first 8 wk of the experimental period, administration of
HFD was found to increase the body weight in all groups. Treatment
with L-DOPA and AEMP (200 mg/kg and 400 mg/kg) for subsequent
4 wk attenuated the increase in body weight as compared to non-
treatment HFD group. Further, in L-DOPA group and in AEMP (400
mg/kg) group there was a significant reduction in body weight as
compared to pre-treatment weights in rats in these groups. The
reduction in body weight in treated groups was found to be
convergent with a reduction in food intake observed in all treated
groups as compared to the HFD group. In L-DOPA group, this was
supported further by significant reduction of BMI. Lowering of BMI
by AEMP (200 mg/kg and 400 mg/kg) although not statistically
significant also supports the anti-obesity activity of Mucuna pruriens.
The results of present study correlate with the previous studies
showing the role of brain dopamine and its receptors in reward
mechanism and weight gain. In the present study, brain dopamine
levels were found to be increased in all treatment groups as
compared to HFD groups which confirm the body weight reducing
the effect of dopamine.
White adipose tissue mass was significantly reduced by L-DOPA and
AEMP (400 mg/kg) but not significantly altered in AEMP (200
mg/kg) treated groups. Although AEMP (200 mg/kg) treated group
showed prevention of accumulation of adipose tissue and so
adiposity. Serum TG levels were significantly decreased in all treated
groups with no effect on TC and HDL levels. This shows that the anti-
obesity effect seen with L-DOPA and Mucuna pruriens in the present
study is not because of their effect on lipid levels and it is mainly
governed centrally by altering brain dopamine levels.
CONCLUSION
The present study revealed that Mucuna pruriens seeds had weight
lowering agent. The brain dopamine assay indicated that the
presence of L-DOPA (dopamine precursor) in the seed extracts
might be the constituents responsible for these activities.
CONFLICT OF INTERESTS
The authors declare that there is no conflict of interest
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How to cite this article
Javid Mansuri, Archana Paranjape. Evaluation of the anti-
obesity effect of aqueous extract of Mucuna pruriens seeds on
rats. Int J Pharm Pharm Sci 2017;9(3):111-115.
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