Chemico-Biological Interactions 221 (2014) 4251

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Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint

Mitigating efcacy of piperine in the physiological derangements of high

fat diet induced obesity in Sprague Dawley rats
Parim BrahmaNaidu a, Harishankar Nemani c, Balaji Meriga a,, Santosh Kumar Mehar b, Sailaja Potana c,
Sajjalaguddam Ramgopalrao d
a

Department of Biochemistry, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India
Department of Botany, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, India
National Center for Laboratory Animal Sciences, National Institute of Nutrition (Indian Council of Medical Research), Hyderabad 500007, Andhra Pradesh, India
d
Department of Biotechnology, Sreenidhi Institute of Science and Technology, Hyderabad, Andhra Pradesh,India
b
c

a r t i c l e

i n f o

Article history:
Received 17 May 2014
Received in revised form 13 July 2014
Accepted 17 July 2014
Available online 31 July 2014
Keywords:
Piperine
High fat diet
Body composition
Lipid proles
Antioxidants

a b s t r a c t
An increased risk of obesity has become a common public health concern as it is associated with hypertension, diabetes, osteoarthritis, heart diseases, liver steatosis etc. Pharmacological intervention with
natural product-based drugs is considered a healthier alternative to treat obesity. This study was aimed
to evaluate anti-obesity effects of piperine on high fat diet (HFD) induced obesity in rats. Piperine was
isolated from methanolic extract of Piper nigrum by using column chromatography and conrmed by
LCMS analysis. Male SD rats were fed HFD initially for 15 weeks to induce obesity. After induction of
obesity, piperine was supplemented in different doses (20, 30 and 40 mg/kg b.wt) through HFD for
42 days to experimental rats. HFD induced changes in body weight, body composition, fat percentage,
adiposity index, blood pressure, plasma levels of glucose, insulin resistance, leptin, adiponectin, plasma
and tissue lipid proles, liver antioxidants were explained. The activities of lipase, amylase and lipid
metabolic marker enzymes such as HMG-CoA reductase, carnitine palmitoyl transferase (CPT), fatty acid
synthase (FAS), acetyl-CoA carboxylase (ACC), lecithin-cholesterol acyl transferase (LCAT) and lipoprotein
lipase (LPL) were assessed in experimental rats. Supplementation of piperine at a dose of 40 mg/kg b.wt
has signicantly (p < 0.05) reversed the HFD-induced alterations in experimental rats in a dose dependant manner, the maximum therapeutic effect being noted at a dose of 40 mg/kg b.wt. Our study concludes that piperine can be well considered as an effective bioactive molecule to suppress of body
weight, improve insulin and leptin sensitivity, ultimately leading to regulate obesity.
2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Obesity, a nutritional disorder, is dened as nonstandard or
unwarranted fat accumulation and growth of adipose tissue leading to obesity [1]. Because of its rising prevalence and its association with chronic health disorders such as insulin resistance,
dyslipidemia, hypertension, cardio vascular diseases, non alcoholic
fatty liver and osteoarthritis, obesity has become a major health
concern in developed and developing countries [24].
Different kinds of therapies are available to help control obesity
including appetite regulation, lipid digestion and absorption, promotion of lipolysis, inhibition of adipogenesis and behavior modication [5]. Among these, diet management, physical exercise and
behavior modication are indispensable to control obesity. Despite
Corresponding author. Tel.: +91 984 9086856; fax: +91 877 2289414.
E-mail address: balaji.meriga@gmail.com (B. Meriga).
http://dx.doi.org/10.1016/j.cbi.2014.07.008
0009-2797/ 2014 Elsevier Ireland Ltd. All rights reserved.

potential global market for anti-obesity drugs, presently there are a

few FDA approved drugs to treat obesity. Although the currently
available drugs such as orlistat, sibutramine and rimonabant have
modest clinical efcacy, their use is often restricted due to associated gastrointestinal or cardiovascular or central nervous system
side effects. Among these drugs orlistat, the pancreatic lipase
inhibitor is clinically approved for obesity treatment [6]. Even
though the occurrence of obesity continues to increase in modern
society; there are no satisfactory pharmacological therapies for its
treatment. Supplementation of drugs that target lipid mobilization,
utilization and reduction of nutrient absorption comprises one of
the most important therapeutic approaches.
Phytoconstituents have always been a commendable source of
drugs and many of the currently available drugs have been derived
directly or indirectly from them. These phytoconstituents remain a
focus of attention in our quest for the development of novel
emphasizes the need for anti-obesity drugs. According to World

P. BrahmaNaidu et al. / Chemico-Biological Interactions 221 (2014) 4251

Health Organization (WHO) report [7], continuous exploration and

screening of medicinal plants with satisfactory weight management efciency. A growing body of facts has clearly established
that medicinal plants and their constituents play a fundamental
role in appetite regulation and energy homeostasis [8].
Since prehistoric age, traditional formulations derived from
medicinal plants have been used for therapeutic purposes. To date,
there is growing attention on therapeutic efcacy of spices including pepper because their intake appears to be connected with
treatment of certain chronic diseases as reported by several
researchers [9,10]. Piper nigrum Linn commonly known as black
pepper has been traditionally used to treat cholera, dyspepsia, gastric ailments, and diarrhea [3,4,10]. Previous studies have shown
the presence of phytochemicals like piperine, piperidine and pellitorine oil in P. nigrum Linn [911]. Moreover, to our best knowledge,
data that deliberated the anti-obesity effects of piperine on dietinduced obesity in rats that dealt with pathophysiological factors
are limited. Therefore, this study was intended to investigate the
mitigating efcacy of piperine on pathophysiological deragments
in HFD-induced obese rats.
2. Materials and methods
2.1. Animals
All experiments related to diet induced obesity were carried out
with male SD rats. Experiments were conducted at National Center
for Laboratory Animal Sciences, National Institute of Nutrition,
Hyderabad, India (Regd. No. 154/1999/CPCSEA). Animals were
housed individually in standard polycarbonate cages with top grill
having facilities for holding pelleted diet and drinking water in
polycarbonate bottles with stainless steel sipper tubes (Techniplast, Italy) at 22 2 C, with 1416 air changes per hour with a relative humidity 5060% with a 12 h light/dark cycle. After initial
acclimatization, the animals were grouped based on their body
weights. All procedures involving laboratory animals were in
accordance with the Institute Animal Ethical Committee (IAEC
No.: P11F/IAEC/2013/MB/SVU/SD Rats/M48).
2.2. Chemicals
To measure plasma glucose and insulin, kits were procured
from Stanbio Laboratory USA, Bio-Merieux, RCS, Lyon, France,
respectively. Plasma was analyzed for total cholesterol, triglycerides, phospholipids, free fatty acids, VLDL, HDL by colorimetric
methods using kits (Nicholas Piramal India Ltd, Mumbai). Orlistat
(Cat No. 04139) was obtained from SigmaAldrich. All other
reagents used in the experiments were of analytical grade and of
high purity.
2.3. Preparation of extracts
Dried P. nigrum seeds were obtained from the local market, its
identity was authenticated by Taxonomist (Dr. K Madhavachetty),
Department of Botany, at Sri Venkateswara University, India,
voucher number 2271, and a specimen has been preserved at the
departmental herbarium. Seeds were shade dried and pulverized
to a coarse powder and sequentially extracted with hexane, ethyl
acetate, methanol and water. The respective ltrates obtained
were evaporated to dryness in a rotary evaporator to obtain their
dried extracts. Based on preliminary phytochemical analysis,
methanolic extract was found to possess important phytochemicals. Therefore, we proceeded further with methanolic extract
and isolated piperine by column chromatography. The purity
and structure of piperine was conrmed by HPLC and mass

43

spectroscopy respectively at Indian Institute of Chemical Technology, Hyderabad, India [12].

2.4. Induction of obesity
SD rats weighing 180200 g were randomly divided into six
groups of six each. Normal control rats (Group-I) were fed with
normal pellet chow of standard composition containing all the
recommended macro and micronutrients prepared according to
AIN-93 guidelines with water ad libitum. High fat diet was prepared with a composition of beef tallow 29.5%, casein 22.0%, starch
23.0%, cellulose 17.9%, L-cystine 4.0%, choline chloride 0.3%, vitamin mixture 1.8%, salt mixture 1.5% prepared at National Institute
of Nutrition, as per nutrition guidelines. Group-II to Group-VI rats
were initially fed with HFD for 15 weeks to induce obesity (20 g
daily) and from 16th week on wards, different doses of piperine
(20, 30 and 40 mg/kg b.wt) were supplemented for 42 days along
with HFD as mentioned below in experimental design.
2.5. Drug preparation
Piperine, 1.6 g, 1.8 g and 2.4 g were mixed with 4 kg of HFD
each, and 20 g of diet/day/rat was given aiming for a daily dose
of 20, 30 and 40 mg/kg b.wt as mentioned in experimental design.
The exact dose of piperine consumed was calculated from daily
food intake.
2.6. Acute toxicity studies
The acute oral toxicity studies were performed in overnight
fasted animals as per Organization for Economic Co-operation
and Development (OECD) guidelines. The animals did not show
any abnormal behavior or mortality at a dose range of 200
400 mg/kg b.wt of piperine. Hence its 1/10th concentrations were
used as the therapeutic dose in the present study.
2.7. Experimental protocol
Group
Group
Group
Group
Group
Group

I Normal control (normal diet control)

II High fat diet (HFD) control
III HFD + orlistat5 mg/kg b.wt
IV HFD + piperine20 mg/kg b.wt
V HFD + piperine30 mg/kg b.wt
VI HFD + piperine40 mg/kg b.wt

2.8. Measurement of body weight

The body weight of each rat was measured once in a week. At
the end of the experiment, blood was collected from overnight
fasted animals under inhalation of anesthesia by retro orbital
puncture method, plasma was separated by centrifugation at
2500 rpm for 15 min. For the measurement of nutrient metabolites
like food intake, water intake, urine volume and fecal weight, both
control and experimental rats were placed in the metabolic cages
for 72 h (Techniplast, Italy).
2.9. Body composition
Body composition of experimental animals was assessed at the
end of experiment by Total Body Electrical Conductivity (TOBEC)
using small animal body composition analysis system (EM-SCAN,
Model SA-3000 Multi detector, Springeld, USA). TOBEC data were
used to compare levels of body adiposity between the control and
experimental groups. Lean body mass, fat free mass and total body
fat percent were calculated as described previously [3].

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2.10. Heart rate, systolic and diastolic blood pressure

2.18. Assay of lipid metabolic marker enzymes

Heart rate, systolic and diastolic blood pressure of all experimental rats were measured at the end of the experiment by using
12 channel BP apparatus (IITC Life sciences, Cas No. 91387) by tail
cup non invasive method according to manufacturers protocol.

Enzyme activities were analyzed as per the procedures

described in respective kits. carnitine palmitoyl transferase (CPT)
was estimated by ELISA Kit (Cat No. MBS705997 Mybiosource),
total acetyl-CoA carboxylase (ACC) was measured by ELISA Kit
(Cat No. 7996 Path Scan). Total fatty acid synthase (FAS) was
assayed by ELISA Kit (Cat No. 7689 MAK107 Sigma Aldrich, USA).
Lecithin-cholesterol acyltransferase (LCAT) (Roar Biomedical,
Inc.), lipoprotein lipase (LPL) (Cat No. STA-610 Cell bio labs INC,
USA) and HMG-CoA reductase activity (Cat No. CS1090 Sigma
Aldrich, USA) were measured by kit methods.

2.11. Determination of adiposity index

Adiposity index was measured by using the formula: Sum of the
total body fat weights/bodyweights  100 [13].
2.12. Estimation of glucose, insulin and insulin resistance
At the end of the experiment, blood was collected from overnight fasted rats under inhalation of anaesthesia by retro-orbital
puncture method. Plasma glucose was estimated using kits (Cat
No. 1060-500, Stanbio laboratory, USA). Plasma level of insulin
was determined using kits from Bio-Merieux, RCS, Lyon, France.
Insulin resistance was calculated using the homeostasis model
assessment of Matthews et al., 1985 [14].
2.13. Estimation of leptin and adiponectin
Plasma leptin and adiponectin activities were measured by
using enzyme-linked immunosorbent assay kits (Crystal Chem,
Downers Grove, IL, USA), performed in duplicate, as per the manufacturers guidelines and expressed in ng mL1.
2.14. Oral glucose tolerance test (OGTT)
OGTT was performed at the end of the experiment, after
overnight fasting, glucose was administered orogastrically at a
dose of 2.0 g/kg b.wt and blood samples were collected from supra
orbital sinus at 0,30,60,90 and 120 min. Glucose levels were estimated at all intervals [15].
2.15. Assay of plasma amylase and lipase
Plasma amylase and lipase activities were determined by
kinetic method using the commercial kits available at Labtest,
Minas Gerais, Brazil and Bioclin, Minas Gerais, Brazil, respectively.
2.16. Plasma lipid analysis
For estimation of lipid proles, blood samples were centrifuged
at 4000 rpm/min for 10 min to separate plasma which was then
stored at 80 C for further biochemical analysis. Total cholesterol,
HDL, LDL and triacylglyceride levels were estimated by CHOD-PAP
method and GPO-PAP method.
2.17. Tissue lipid analysis
Tissue lipids were extracted from experimental animals by the
method of Floch et al. (1957) [16] using a chloroformmethanol
mixture (2:1, v/v). The liver tissues were rinsed with ice cold physiological saline, dried, homogenized in cold chloroformmethanol
(2:1, v/v) and contents were extracted after 24 h. The extraction
was repeated four times. The combined ltrate was washed with
0.7% KCl and the aqueous layer was discarded. The organic layer
was made up to a known volume with chloroform and used for tissue lipid analysis.

2.19. Liver antioxidants analysis

After the completion of experimental period rats we anesthetized and sacriced. Then the liver was excised, rinsed in ice cold
normal saline followed by ice-cold 10% KCl solution, blotted, dried
and weighed. A 10% w/v homogenate was prepared in ice-cold KCl
solution and centrifuged at 2000 rpm for 10 min at 4 C. The supernatants thus obtained were used for the estimation of thiobarbituric acid substances (TBARS) (Fraga et al. 1988) [17], assay of GSH
(Ellman, 1959) [18], SOD (Kakkar et al. 1984) [19], CAT (Aebi,
1984) [20] and GpX by Paglia and Valentine (1967) [21].
2.20. Statistical analysis
All the results are expressed as the Mean SD for six animals in
each group. All the data were statistically evaluated with SPSSn10.0
software. Hypothesis testing methods included one way analysis of
variance (ANOVA) followed by Least Signicant Difference (LSD)
test. Signicance level at p < 0.05, 0.001 were considered to indicate statistical signicance.

3. Results
Piperine, (1-[5-(1,3-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]
piperidine) is a naturally occurring alkaloid found rich in P. nigrum,
a widely used important spice across the world. The LCMS analysis of piperine showed molecular formula C17H22NO3. The LCMS
spectrum, library search and the structure of obtained piperine
shown in Fig. 1.
The changes in body weight, food and water intake in different
groups of animals during the experimental period are summarized
in Table 1 and Fig. 2(AE). There was signicant change in food and
water intake in HFD-fed group when compared to normal control
group. Feeding on HFD increased substantially body weight and
BMI in experimental rats when compared to normal group. HFDinduced increase in body weight, BMI, total fat, fat percentage
and fat free mass were signicantly curtailed by piperine supplementation in HFD-fed rats, in a dose dependent manner (20, 30
and 40 mg/kg b.wt). The maximum therapeutic effect of piperine
was found at a dose of 40 mg/kg b.wt.
We recorded heart rate, systolic and diastolic blood pressure in
normal diet fed and HFD-fed groups of rats. When compared to
normal control group, a signicant increase (p < 0.05) in heart rate,
systolic and diastolic blood pressure was recorded in HFD-fed
group which was signicantly (p < 0.05) decreased by piperine
supplementation in a dose dependent manner as shown in Table 2.
Experimental animals fed with HFD for 21 weeks exhibited an
increased organ and fat pad weights (Table 3) and adiposity index
(Fig. 3). However, supplementation of piperine through HFD for
42 days (16th week to 21st week) resulted in decreased organ
weight, fat pad weight and adiposity index in a dose dependant

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P. BrahmaNaidu et al. / Chemico-Biological Interactions 221 (2014) 4251

Fig. 1. LCMS spectrum and structure of piperine.

Table 1
Effect of piperine on body weight, food and water intake in normal and experimental obese rats.
Groups

Body weight (g)

Food intake (g/rat/day)

Water intake (ml/rat/day)

Control
HFD control
HFD + orlistat 5 mg/kg b.wt
HFD + piperine 20 mg/kg b.wt
HFD + piperine 30 mg/kg b.wt
HFD + piperine 40 mg/kg b.wt

362 13.9
527 8.2a,
344.3 30.5b,
409.2 8.77b,
391.2 12.3b,
369.2 13.4b,

14.2 0.85
15.08 0.6
14.7 0.7
14.4 0.4
14.3 0.6
14.1 0.4

21.8 2.6
25.1 3.6
24.2 2.8
22.7 1.4
22.9 1.07
22.8 2.1

Values are mean SD, n = 6.

Values are statistically signicant at p < 0.05.
a
Signicantly different from control.
b
Signicantly different from HFD control.

manner. The maximum mitigating activity was noted at 40 mg/

kg b.wt which is similar to that of orlistat.
The result of plasma glucose, insulin, insulin resistance, insulin
area under curve (AUC), leptin and adiponectin in control and
experimental obese rats are depicted in Fig. 4(A and B) and Table 4.
There was a signicant (p < 0.001, p < 0.05) elevation in plasma
glucose, plasma insulin, insulin resistance, leptin levels and
decrease in adiponectin in HFD induced obese rats over their normal control rats. Supplementation with piperine tended to restore
the changes in the above parameters in a dose dependant manner,
the maximum activity being observed with 40 mg/kg b.wt of
piperine.
Fig. 5 summarizes the results of oral glucose tolerance test performed in control and experimental obese rats. In normal control
rats, maximum elevation in blood glucose level was observed 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. Interestingly, supplementation with
piperine or orlistat to obese rats resulted in a signicant decrease
in blood glucose level at 60 min and beyond when compared with
HFD control rats.

The enzyme activities of pancreatic lipase and amylase of normal and experimental obese rats are represented in Fig. 6. We
found that HFD-fed obese rats showed signicant elevation
(p < 0.05) in amylase and pancreatic lipase activity. Nevertheless,
supplementation of piperine (40 mg/kg b.wt) or orlistat to obese
rats signicantly reduced the activity of amylase and pancreatic
lipase.
Fig. 7(A and B) represents plasma and liver lipid proles of control and experimental obese rats. The concentration of plasma TGs,
total cholesterol, PLs, free fatty acids, VLDL, LDL and liver lipids
were signicantly increased, except HDL, in HFD induced obese
rats when compared to normal rats. Nonetheless, treatment with
piperine (40 mg/kg b.wt) signicantly (p < 0.05) reduced the concentrations of plasma and liver lipids, in obese rats to near normal
level, while HDL was elevated.
The activities of important lipid metabolism enzymes like
(acetyl CoA carboxylase) ACC, (fatty acid synthase) FAS, (carnitine
palmitoyl transferase) CPT, HMG-CoA reductase, LPL and LCAT in
control and experimental groups of rats are displayed in Fig. 8. A
signicant reduction (p < 0.05) in the level of CPT, LPL and LCAT
and a concomitant increase in the level of ACC, FAS and HMGCoA reductase was observed in HFD control rats. Supplementation

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P. BrahmaNaidu et al. / Chemico-Biological Interactions 221 (2014) 4251

Fig. 2. Effect of piperine on lean mass (A), total fat (B), fat% (C) and fat free mass (D) and BMI (E) in normal and experimental obese rats.

Table 2
Effect of piperine on heart rate, systolic and diastolic blood pressure in normal and experimental obese rats.
Groups

Heart rate (beats/min)

Systolic blood pressure (mm Hg)

Diastolic blood pressure (mm Hg)

Control
HFD control
HFD + orlistat 5 mg/kg b.wt
HFD + piperine 20 mg/kg b.wt
HFD + piperine 30 mg/kg b.wt
HFD + piperine 40 mg/kg b.wt

354.2 7.3
387 7.09a,
367.1 4.16b,
383.4 6.5NS
375.4 5.59b,
366.5 7.6b,

131.4 5.2
157.1 12.1a,
132.2 8.7b,
155.5 7.9NS
149.9 2.4b,
141.9 9.6b,

101.3 2.2
123.6 2.3a,
109.4 6.8b,
122.2 5.2NS
110.6 5.6b,
100.1 5.4b,

Values are mean SD, n = 6.

Values are statistically signicant at p < 0.05.
a
Signicantly different from control.
b
Signicantly different from HFD control.
NS
Non signicant.

of piperine, (40 mg/kg b.wt) showed a signicant increase in the

activities of CPT, LPL and LCAT and decrease of ACC, FAS and
HMG-CoA reductase in experimental animals, as that of orlistat.
Fig. 9 summarizes the levels of TBARS, GSH, SOD, CAT and GPx
in the liver of control and experimental groups of rats. The activities of SOD, CAT, GSH and GPx in liver were signicantly (p < 0.05)
lower in obese control rats while the level of TBARS was found to
be increased. Treatment with piperine or orlistat showed a significant increase in the activities of SOD, CAT, GSH and GPx and concomitant decrease in the level of TBARS in liver of experimental
obese rats, proving the potent antioxidant activity of piperine.
4. Discussion
The quest for novel and safe therapeutic molecules continues to
combat different aliments. Plants and herbs serve as potential

perennial source of to explore such novel bioactive factors. In the

present work we reported that high fat diet induces obesity and
associated pathophysiological changes in the experimental animals which are signicantly reverted by the treatment with piperine in a dose dependent manner. Obesity which was marked by
increased body weight, BMI, blood pressure, total fat, fat percentage, fat free mass and organ weights in SD rat [22]. There is a good
rapport between body weights relative to organ weight and the
risk of metabolic syndrome is successive and graded [23]. It is
the energy intake that matters in relation to the development of
overweight and the energy intake is often high when HFD is
consumed in large amounts. This involved two growth mechanisms: hyperplasia and hypertrophy of adipocytes [24]. However,
on treatment with piperine there was a signicant decrease in
body weight, which proved its anti-obese action. Blood pressure
and BMI are widely used as determining factors of fatness in

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Table 3
Effect of piperine on organ and fat pad weights in normal and experimental obese rats.
Groups

Liver
(g)

Kidney
(g)

Heart
(g)

Lung
(g)

Epididymal
(g/100 g b.wt fat)

Retroperitoneal
(g/100 g b.wt fat)

Mesentric
(g/100 g b.wt fat)

Control
HFD control
HFD + orlistat 5 mg/kg b.wt
HFD + piperine 20 mg/kg b.wt
HFD + piperine 30 mg/kg b.wt
HFD + piperine 40 mg/kg b.wt

10.04 0.36
14.9 0.5a,
12.89 0.3b,
13.57 0.75b,
12.82 0.64b,
11.12 0.45b,

2.45 0.27
3.21 0.2a,
2.77 0.19b,
3.02 0.16b,
2.64 0.33b,
2.55 0.46b,

1.02 0.1
1.38 0.08a,
1.16 0.13b,
1.22 0.07b,
1.16 0.09b,
1.09 0.04 b,

2.03 0.04
2.86 0.51a,
2.09 0.07b,
2.49 0.43b,
2.27 0.155b,
2.14 0.06b,

1.09 0.07
1.81 0.07a,
1.57 0.08b,
1.61 0.19b,
1.5 0.14b,
1.43 0.15b,

1.96 0.1
3.65 0.17a,
2.79 0.2b,
3.03 0.05b,
2.77 0.3b,
2.19 0.12b,

0.64 0.06
0.99 0.12a,
0.77 0.08b,
0.89 0.1b,
0.83 0.09b,
0.8 0.06b,

Values are mean SD, n = 6.

Values are statistically signicant at p < 0.05.
a
Signicantly different from control.
b
Signicantly different from HFD control.

Table 4
Effect of piperine on plasma glucose, insulin and insulin resistance in normal and experimental obese rats.
Groups

Glucose (mg dl1)

Insulin (lU ml1)

Insulin resistance

Control
HFD control
HFD + orlistat 5 mg/kg b.wt
HFD + piperine 20 mg/kg b.wt
HFD + piperine 30 mg/kg b.wt
HFD + piperine 40 mg/kg b.wt

80.33 10.8
144 13.9a,
96.3 8.7b,
125.1 6.06b,
109.2 4.7b,
100.3 6.04b,

2.2 0.6
7.1 0.8a,
5.4 0.6b,
6.2 0.67b,
6.8 0.69 b,
6.2 0.31b,

3.6 0.09
5.5 1.07a,
4.1 0.01b,
4.7 0.05b,
4.5 0.07 b,
4.8 0.09b,

Values are mean SD, n = 6.

Values are statistically signicant at p < 0.001.
a
Signicantly different from control.
b
Signicantly different from HFD control.

Fig. 3. Effect of piperine on adiposity index in control and experimental obese rats.
Values are mean SD, n = 6; Values are statistically signicant at p < 0.05;
a
Signicantly different from control; bSignicantly different from HFD control.

epidemiological studies, because they are highly interconnected

with body fat [25]. Our present studies depicted a signicant
increase in blood pressure and BMI in HFD supplementation in
accordance with Akiyama et al. [25]. Increased BMI may occur
due to the increase of caloric intake resulting in more adipose tissue deposition. The high levels of BMI are associated with substantial increases in fat mass and this index is most useful as a measure
of obesity [26]. Oral treatment with piperine decreased BMI. This is

due the antioxidant nature of piperine which is involved in mood,

sleep and appetite regulation. Our results are in line with Williams
et al. [27]. In our present study, there was a signicant increase in
systolic, diastolic and mean arterial blood pressures as well as
increase in heart rate in HFD-fed rats when compared to normal
rats. This might be due to intake of HFD in short period
which increase Ca2+ channel dysregulation, leading to elevated
transmembrane Ca2+ inux. Up regulation in Ca2+ current density
is connected with signicant elevation of blood pressure [28]. Supplementation of piperine or orlistat caused signicant attenuation
in these changes in HFD induced obese rats.
Insulin resistance and hyperglycemia are hallmarks of metabolic syndrome [29]. In the present study, plasma glucose and
insulin levels were analyzed as indices of glycemic control and
moreover HFD-induced animals developed a hyperglycemic state
associated with insulin resistance and/or glucose intolerance. This
is in line with previous reports [3,30]. Under physiological conditions, insulin enhances lipogenesis and inhibits lipolysis which
leads to increased circulating levels of glucose and lipids thus
resulting in impaired insulin secretion [31]. Supplementation of
piperine signicantly lowered plasma glucose, insulin level and
insulin resistance in treated rats compared with HFD control rats.
Adipocytokines such as leptin, adiponectin, resistin, visfatin,
IL-6 and TNF-a play major role in energy homeostasis. Leptin, is
a key appetite regulator and plays a pivotal role in food intake
and energy expenditure. It has been suggested that obese persons
are insensitive to endogenous production of leptin and its secretion
levels are found to be positively correlated with the degree of triglyceride stores in adipose tissue [4951]. In our study supplementation of piperine decreased leptin and increased adiponectin in
plasma of HFD induced obese rats. These observations indicate that
reduction in plasma leptin and increase in adiponectin levels after
treatment with piperine may be due to decreased lipid accumulation in adipocytes. Many reports have shown that consumption of
foods rich in phenolic compounds and minerals increase adiponectin and decrease leptin levels in obese individuals [3234].

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P. BrahmaNaidu et al. / Chemico-Biological Interactions 221 (2014) 4251

Fig. 4. (A) and (B) Effect of piperine on AUC, leptin and adiponectin in plasma of normal and experimental obese rats. Values are mean SD, n = 6; Values are statistically
signicant at p < 0.05; aSignicantly different from control; bSignicantly different from HFD control.

Fig. 5. Effect of piperine on glucose tolerance in control and experimental obese rats. Values are mean SD, n = 6; Values are statistically signicant at p < 0.05; aSignicantly
different from control; bSignicantly different from HFD control.

Amylase and lipase provide fascinating targets in the development of anti-obesity and anti-diabetic compounds. a-amylase,
one of the key digestive enzymes secreted from the pancreas and
salivary glands, is involved in hydrolytic process of starch [34].
Many synthetic and crude drugs are reported to inhibit a-amylase
activity [35]. These classical a-amylase inhibitors act by reducing
post-prandial hyperglycemia by slowing down the digestion of
carbohydrates and, thus, leading to reduced energy intake [36].
Activating lipase or inhibiting pancreatic lipase would have an
anti-obesity effect. Dietary lipids, which are major source of undesirable calories, are not directly absorbed from the intestine unless
they are subjected to the action of pancreatic lipase. Pancreatic
lipase hydrolyzes triglycerides into glycerol and fatty acids [37].
Therefore, drugs that can target pancreatic lipase are considered
to be valuable therapeutic agents for treating diet induced obesity
in humans. In our study, supplementation of piperine or orlistat
normalized the elevated levels of amylase and lipase. It is possible
that the reduced levels of pancreatic amylase and lipase might represent a mechanism that would obstruct the digestion and absorption of carbohydrates and lipids leading to lesser weight reduction
in piperine treated HFD-fed obese rats. Our results are in line with
previous reports [3638].

Fig. 6. Effect of piperine on amylase and pancreatic lipase activities in normal and
experimental obese rats. Values are mean SD, n = 6; Values are statistically
signicant at p < 0.05; aSignicantly different from control; bSignicantly different
from HFD control.

The fundamental mechanism underlying dyslipidemic changes

in HFD-induced obesity involves elevating the levels of TG, VLDL,
Total cholesterol (TC), Phospholipids (PLs), Free fatty acids (FFAs)

P. BrahmaNaidu et al. / Chemico-Biological Interactions 221 (2014) 4251

and LDL and a decrease in serum level of HDL [39]. During fed
state, increment of chylomicrons synthesis and absorption is a
common factor which leads to increase in TC, TGs level and endogenous VLDL production [40]. In the present study, supplementation
of HFD produced experimental obesity as evidenced by increased
lipid proles in experimental rats. TGs elevation was due to dietary
cholesterol which is reported to reduce fatty acid oxidation, which
in turn increases the levels of hepatic and plasma TGs. The excessive accumulation of TGs in the lipid stores is associated with a
number of metabolic complications [39]. Elevated TC and LDL levels amplify the risk of developing hypertension and cardio vascular
diseases (CVDs). On the other hand, high HDL is helpful in transporting excess cholesterol to the liver for excretion through bile
[40]. Phospholipids play important role in the transport of triglycerides [41]. In our study, in HFD fed rats, the elevated level of PLs
may be due to the elevated levels of FFAs and total cholesterol
which can promote the synthesis of PLs [42]. Supplementation of
piperine signicantly lowered plasma and liver lipid proles of
HFD-induced obese rats. The possible mechanism through which
piperine contributes in improving lipid prole is by decreasing
cholesterol absorption and secretion from the intestine which
leads to lowered availability of FFAs to the liver.

49

The liver is regarded as one of the central metabolic organs in

the body, regulating and maintaining lipid homeostasis through
key enzymes such as ACC, FAS, CPT, HMG-CoA reductase, LPL and
LCAT. ACC is the rate-limiting enzyme in de novo lipogenesis. It
has been reported that ACC is a physiological inhibitor of CPT by
producing malonyl-CoA in the liver [43]. Fatty acids are transformed to acetyl-CoA by means of mitochondrial oxidation. CPT
is the rate-controlling enzyme in this process of fatty acid oxidation. Endogenous FFA synthesis is inhibited through the suppression of ACC and FAS [44].
Supplementation of HFD enhances fat deposition in adipose
tissue and it can be lowered by reducing lipid uptake by adipocytes
via suppressing lipoprotein lipase or reducing lipid synthesis
through inhibiting FAS or ACC. Piperine or orlistat reduced the lipid
content in tissues of experimental animals by inhibiting the said
enzymes. FAS inhibitors have been reported to provide a potential
pathway to target obesity therapy [45]. Conversion of HMG-CoA to
mevalonate, an NADPH-dependent reduction reaction, is the rst
committed step in cholesterol biosynthesis catalyzed by HMGCoA reductase [46]. The decrease in cholesterol content may be
attributed to increased level of plasma HDL, increase in the activity
of lipoprotein lipase and plasma LCAT, which are known to be

Fig. 7. (A) and (B) Effect of piperine on plasma and liver lipid proles in normal and experimental obese rats. Values are mean SD, n = 6; Values are statistically signicant at

p < 0.05; aSignicantly different from control; bSignicantly different from HFD control.

Fig. 8. Effect of piperine on lipid metabolic marker enzymes of normal and experimental obese rats. Values are mean SD, n = 6; Values are statistically signicant at

p < 0.05; aSignicantly different from control; bSignicantly different from HFD control.

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P. BrahmaNaidu et al. / Chemico-Biological Interactions 221 (2014) 4251

Fig. 9. Effect of piperine on liver antioxidants in normal and experimental obese rats. Values are mean SD, n = 6; Values are statistically signicant at p < 0.05; aSignicantly
different from control; bSignicantly different from HFD control. Activity is expressed as 50% of inhibition of epinephrine auto oxidation per min for SOD; lmoles of hydrogen
peroxide decomposed per min per mg of protein for catalase; lmoles of glutathione oxidized per min per mg of protein for GPx; lg/mg of protein for GSH; mM/100 g of tissue
for TBARS.

involved in transport of tissue cholesterol to liver for its excretion.

HFD induced hyperlipidemia down regulates the LCAT activity in
plasma, which impairs the capability of HDL to remove cholesterol
and leads to cholesterol accumulation in the blood. Supplementation of piperine could revert these enzymes activities and inhibit
cholesterol biosynthesis.
It is well established that the biomarkers of oxidative stress
are much elevated in liver at an early stage in many metabolic
diseases, including obesity. High fat diet consumption contributes
to unwarranted formation of ROS which leads to oxidative stress
[47]. In the present study, we assessed the activity of antioxidant
enzymes and TBARS level in liver. Intracellular antioxidant
enzymes and TBARS are the determining factors of oxidative
damage at cellular level. HFD induced hyperglycemia observed in
this study might be an imperative aspect to increase lipid
peroxidation causing reduction of antioxidant defense status and
indicating the development of oxidative stress in HFD-fed rats
which are interconnected with other studies [47,48]. In the present
study, increased activities of SOD, CAT, GPx and GSH and decreased
level of TBARS, in signicant levels were found in HFD-fed rats,
however, piper supplementation decreased hepatic SOD, CAT and
GPx activities in them. This indicates that piperine has a major role
in inhibiting HFD induced intracellular oxidative stress.
5. Conclusions
To conclude, supplementation of piperine caused signicant
attenuation in the physiological changes produced by HFD in rats.
This might be due to the deterrence of pathological mechanisms
responsible for lipid storage and weight gain, possibly by reverting
leptin and adiponectin activity and increasing energy expenditure.
Together, these observations strongly suggest that piperine, a
major phytoconstituent of black pepper serves as an effective therapeutic agent for the management of obesity and hypertension.
Conict of Interest
The authors declare that there are no potential conicts of
interest.

Acknowledgements
Authors are thankful to Department of Bio Technology-New
Delhi, India (Grant No.: BT/PR7799/PBD/17/849/2013) for providing Junior Research Fellowship and Financial Assistance to carry
out this research work and also thankful to Dr Rama Rao (Indian
Institute of Chemical Technology-India), Dr P Suresh (DirectorNCLAS, NIN), Dr R Ravindar Naik (Technical Ofcer-A), National
Institute of Nutrition-India, for their constant encouragement and
their valuable suggestions.
References
[1] A.E. Decle, A.V. Mathew, R. Cunard, AMPK mediates the initiation of kidney
disease induced by a high-fat diet, J. Am. Nephrol. 22 (2011) 18461855.
[2] D.B.F. Carla, F.B. Fernanda, S.A.F. Glaura, Diet-induced obesity in rats leads to a
decrease in sperm motility, Rep. Biol. Endocrinol. 9 (2011) 3242.
[3] I.J.N. Padmavathi, Y.D. Kishore, L. Venu, M. Ganeshan, N. Harishankar, N.V.
Giridharan, M. Raghunath, Prenatal and perinatal zinc restriction: effects on
body composition, glucose tolerance and insulin response in rat offspring, Exp.
Physiol. 94 (2009) 761769.
[4] Z. Min, Z. Baocai, C. Mengjie, Differential responses of hepatic endoplasmic
reticulum stress and inammation in diet-induced obese rats with high-fat
diet rich in lard oil or soybean oil, PLoS One 28 (2013) e78620e78632.
[5] D.T. Villareal, S. Chode, N. Parimi, D.R. Sinacore, T. Hilton, V.R. Armamento, N.
Napoli, Q. Clifford, S. Krupa, Weight loss, exercise, or both and physical
function in obese older adults, New Eng. J. Med. 364 (2011) 12181229.
[6] G.A. Agbor, J.A. Vinson, J.E. Oben, J.Y. Ngogang, Antioxidant effect of herbs and
spices on copper mediated oxidation of lower and very low density
lipoprotein, Chin. J. Nat. Med. 8 (2010) 114120.
[7] WHO, 2013. Facts on obesity. http://www.who.int/features/factles/obesity/
en/ (accessed on 24.2.2014).
[8] N. Ravirajsingh, C. Menaka, V.R. Umed, Anti-obesity potential of Clerodendron
glandulosum. Coleb leaf aqueous extract, J. Ethanopharmocol. 135 (2011) 338
343.
[9] A. Agbor, A. Luli, S. Julianne, Piper species protect cardiac, hepatic and renal
antioxidant status of atherogenic diet fed hamsters, Food Chem. 134 (2012)
13541359.
[10] K. Srinivasan, P.S. Patole, C.L. Kaul, P. Ramarao, Reversal of glucose intolerance
by pioglitazone in high-fat diet fed rats, Exp. Clin. Pharmacol. 26 (2004) 327
333.
[11] S.B. Masood, P. Imran, T.S. Muhammad, A.R. Muhammad, S. Farhan, A. Waqas,
Black pepper and health claims: a comprehensive treatise, Crit. Rev. Food Sci.
Nutr. 53 (2013) 875886.
[12] V.R. Rao, S.S. Raju, V.U. Sarma, F. Sabine, K.H. Babu, J.M. Rao, Simultaneous
determination of bioactive compounds in Piper nigrum L. and a species
comparison study using HPLC-PDA, Nat. Prod. Res. 13 (2011) 12881294.

P. BrahmaNaidu et al. / Chemico-Biological Interactions 221 (2014) 4251

[13] S.M. Jeyakumar, A. Vajreswari, N.V. Giridharan, Chronic dietary vitamin A
supplementation regulates obesity in an obese mutant WNIN/Ob rat model,
Obesity 14 (2006) 5259.
[14] D.R. Matthews, J.P. Hosker, A.S. Rudenski, B.A. Naylor, D.F. Treacher, R.C.
Turner, Homeostasis model assessment: insulin resistance and beta-cell
function from fasting plasma glucose and insulin concentrations in man,
Diabetologia 28 (1985) 412429.
[15] S.S.S. Prasad, M. Sukapaka, V.K. Prathipati, H. Nemani, U.K. Putcha, S. Pothana,
Carbenoxolone treatment ameliorated metabolic syndrome in WNIN/Ob obese
rats, but induced severe fat loss and glucose intolerance in lean rats, PLoS One
12 (2012) e50216.
[16] J. Floch, M. Lee, G.H.S. Stanley, A simple method for the isolation and
purication of total lipids from animal tissues, J. Biol. Chem. 226 (1957) 497
509.
[17] C.G. Fraga, B.E. Leibouitz, A.L. Toppel, Lipid peroxidation measured as TBARS in
tissue slices: characterization and comparison with homogenates and
microsomes, Free Radic. Biol. Med. 4 (1988) 155161.
[18] G.L. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophys. 82 (1959) 70
77.
[19] B. Kakkar, P. Das, N. Viswanathan, A modied spectrophotometric assay of
SOD, Ind. J. Biochem. Biophys. 21 (1984) 130132.
[20] H. Aebi, Catalase in vitro, Methods Enzymol. 105 (1984) 121126.
[21] D. Paglia, W. Valentine, Studies on the quantitative and qualitative
characterization of erythrocyte glutathione peroxidase, J. Lab. Clin. Med. 70
(1967) 158169.
[22] B. Uma, S.C. Hemantkumar, N.B. Ajay, Anti-obesity effect of standardized
ethanol extract of Embelia ribes in murine model of high fat diet-induced
obesity, Pharma Nutr. 1 (2013) 5057.
[23] G.A. Colditz, W.C. Willett, M.J. Stampfer, J.E. Manson, C.H. Hennekens, R.A.
Arky, F.E. Speizer, Weight as a risk factor for clinical diabetes in women, Am. J.
Epidemiol. 132 (1990) 501513.
[24] F.P. Mancini, A. Lanni, L. Sabatino, M. Moreno, A. Giannino, F. Contaldo, V.
Colantuoni, F. Goglia, Fenobrate prevents and reduces body weight gain and
adiposity in diet induced obese rats, FEBS Lett. 491 (2001) 154158.
[25] T. Akiyama, I. Tachibana, H. Shirohara, High-fat hypercaloric diet induces
obesity, glucose intolerance and hyperlipidemia in normal adult male Wistar
rat, Diab. Clin. Pract. 31 (1996) 2735.
[26] B.M. Yalcin, E.M. Sahin, E. Yalcin, Which anthropometric measurements is
most closely related to elevated blood pressure, Fam. Pract. 22 (2005) 541
547.
[27] G. Williams, K. Lyttle, T. Clarke, Supplementation with pumpkin seed oil
improves plasma lipid prole and cardiovascular outcomes of female nonovariectomized and ovariectomized Sprague Dawley rats, Phytother. Res. 22
(2008) 873877.
[28] D.W. Wilde, K.D. Massey, G.K. Walker, A. Vollmer, R.J. Grekin, High-fat diet
elevates blood pressure and cerebrovascular muscle Ca2+ current,
Hypertension 35 (2000) 832837.
[29] M.F. Gregor, G.S. Hotamisligil, Inammatory mechanisms in obesity, Ann. Rev.
Immunol. 29 (2011) (2011) 415445.
[30] S.C. Woods, R.J. Seeley, P.A.A. Rushing, Controlled high fat diet induces an
obese syndrome in rats, J. Nutr. 133 (2003) 10811087.
[31] A.R. Saltiel, C.R. Kahn, Insulin signalling and the regulation of glucose and lipid
metabolism, Nature 414 (2001) 799806.
[32] N.M. Wedick, A.M. Brennan, Q. Sun, Effects of caffeinated and decaffeinated
coffee in biological risk factors for type 2 diabetes: a randomized controlled
trial, Nutr. J. 10 (2011) 93.

51

[33] R. Landerberg, Q. Sun, E.B. Rimm, A. Cassidy, A. Scalbert, C.S. Mantzoros, F.B.
Hu, R.M. van Dam, Selected dietary avonoids are associated with markers of
inammation and endothelial dysfunction in U.S. women, J. Nutr. 141 (2011)
618625.
[34] N. Kei, M. Toshitaka, M. Hiromi, K. Masafumi, Revisiting the cardiometabolic
relevance of serum amylase, BMC Res. Notes 4 (2011) 419426.
[35] K. Kobayashi, Y. Saito, I. Nakazawa, F. Yoshizaki, Screening of crude drugs for
inuence on amylase activity and postprandial blood glucose in mouse
plasma, Biol. Pharm. Bull. 23 (2000) 12501253.
[36] J. Maury, T. Issad, D. Perdereau, B. Gouhot, P. Ferre, J. Girard, Effect of acarbose
on glucose homeostasis, lipogenesis and lipogenic enzyme gene expression in
adipose tissue of weaned rats, Diabetologia 36 (1993) 503509.
[37] L.F.G. Van, I.L. Mertens, C.E. De Block, Mechanisms linking obesity with
cardiovascular disease, Nature 444 (2006) 875880.
[38] J.K. Kyung, L. Myoung-Su, J. Keunae, Piperidine alkaloids from Piper
retrofractum Vahl. Protect against high-fat diet-induced obesity by regulating
lipid metabolism and activating AMP-activated protein kinase, Biochem.
Biophys. Res. Commun., 411 (2011) 219225.
[39] Y. Park, J.M. Storkson, W. Liu, J. Albright, M.E. Cook, M.W. Pariza, Structureactivity relationship of conjugated linoleic acid and its cognates in inhibiting
heparin-releasable lipoprotein lipase and glycerol release from fully
differentiated 3T3-L1 adipocytes, J. Nutr. Biochem. 15 (2004) 561569.
[40] A. Garjani, F. Fathiazad, A. Zakheri, N.A. Akbari, Y. Azarmie, A. Fakhrjoo, S.
Andalib, N. Maleki-Dizaji, The effect of total extract of Securigera securidaca L.
seeds on serum lipid proles, antioxidant status, and vascular function in
hypercholesterolemic rats, J. Ethonopharmocol. 126 (2009) 525532.
[41] Y. Shi, P. Burn, Lipid metabolic enzymes: emerging drug targets for the
treatment of obesity, Nat. Rev. Drug Dis. 3 (2004) 695710.
[42] R.M. Cohn, K.S. Roth, Lipid and Lipoprotein Metabolism, Biochemistry and
Disease, Williams and Wilkins Publishers, Baltimore, 1996, p. 280.
[43] M.R. Munday, Regulation of mammalian acetyl-CoA carboxylase, Biochem.
Soc. Trans. 30 (2002) 10591064.
[44] K. Browning, S.R. Fortna, A. Hajnal, Y. Roux-en, Gastric bypass reverses the
effects of diet-induced obesity to inhibit the responsiveness of central vagal
motoneurones, J. Phys. 9 (2013) 23572372.
[45] C.H. Peng, L.K. Liu, C.M. Chuang, C.C. Chyau, C.N. Huang, C.J. Wang, Mulberry
water extracts possess an anti-obesity effect and ability to inhibit hepatic
lipogenesis and promote lipolysis, J. Agric. Food Chem. 59 (2011) 26632671.
[46] D. Rozman, K. Monostory, Perspectives of the non-statin hypolipidemic agents,
Pharmacol. Ther. 127 (2010) 1940.
[47] S.S. Reddy, P. Ramatholisamma, R. Karuna, D. Saralakumari, Preventive effect
of Tinospora cordifolia against high-fructose diet-induced insulin resistance
and oxidative stress in male wistar rats, Food Chem. Toxicol. 47 (2009) 2224
2229.
[48] A.K. Sharma, S. Bharti, J. Bhatia, Sesamol alleviates diet-induced
cardiometabolic syndrome in rats via up-regulating PPARc, PPARa and eNOS, J. Nutr. Biochem. 23 (2012) 14821489.
[49] K. Suresh, S. Sunil, V. Neeru, Screening of antidiabetic and antihyperlipidemic
potential of oil from Piper longum and piperine with their possible mechanism,
Exp. Opin. Pharmacother. 14 (2013) 17231735.
[50] S.I. Taqvi, A.J. Shah, A.H. Gilani, Blood pressure lowering and vasomodulator
effects of piperine, J. Cardiovasc. Pharm. 52 (2008) 452458.
[51] G. Saravanan, P. Ponmurugan, M.A. Deepa, B. Senthilkumar, Antiobesity action
of gingerol: effect on lipid prole, insulin, leptin, amylase and lipase on male
obese rats induced by a high-fat diet, (2014) doi: 10.1002/jsfa.6642.