Anti obesity activity of Piper

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Mitigating efcacy of piperine in the physiological derangements of highfat 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 da Department of Biochemistry, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, Indiab Department of Botany, Sri Venkateswara University, Tirupati 517502, Andhra Pradesh, Indiac National Center for Laboratory Animal Sciences, National Institute of Nutrition (Indian Council of Medical Research), Hyderabad 500007, Andhra Pradesh, Indiad Department of Biotechnology, Sreenidhi Institute of Science and Technology, Hyderabad, Andhra Pradesh,India

a r t i c l e i n f o

Article history:Received 17 May 2014Received in revised form 13 July 2014Accepted 17 July 2014Available online 31 July 2014

Keywords:PiperineHigh fat dietBody compositionLipid prolesAntioxidants

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 hyper-tension, diabetes, osteoarthritis, heart diseases, liver steatosis etc. Pharmacological intervention withnatural product-based drugs is considered a healthier alternative to treat obesity. This study was aimedto evaluate anti-obesity effects of piperine on high fat diet (HFD) induced obesity in rats. Piperine wasisolated from methanolic extract of Piper nigrum by using column chromatography and conrmed byLCMS 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 for42 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, plasmaand tissue lipid proles, liver antioxidants were explained. The activities of lipase, amylase and lipidmetabolic marker enzymes such as HMG-CoA reductase, carnitine palmitoyl transferase (CPT), fatty acidsynthase (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.wthas signicantly ( p < 0.05) reversed the HFD-induced alterations in experimental rats in a dose depen-dant manner, the maximum therapeutic effect being noted at a dose of 40 mg/kg b.wt. Our study con-cludes that piperine can be well considered as an effective bioactive molecule to suppress of bodyweight, 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 orunwarranted fat accumulation and growth of adipose tissue lead-ing to obesity [1] . Because of its rising prevalence and its associa-tion with chronic health disorders such as insulin resistance,dyslipidemia, hypertension, cardio vascular diseases, non alcoholicfatty liver and osteoarthritis, obesity has become a major healthconcern in developed and developing countries [24] .

Different kinds of therapies are available to help control obesityincluding appetite regulation, lipid digestion and absorption, pro-motion of lipolysis, inhibition of adipogenesis and behavior modi-cation [5] . Among these, diet management, physical exercise andbehavior modication are indispensable to control obesity. Despite

potential global market for anti-obesity drugs, presently there are afew FDA approved drugs to treat obesity. Although the currentlyavailable drugs such as orlistat, sibutramine and rimonabant havemodest clinical efcacy, their use is often restricted due to associ-ated gastrointestinal or cardiovascular or central nervous systemside effects. Among these drugs orlistat, the pancreatic lipaseinhibitor is clinically approved for obesity treatment [6] . Eventhough the occurrence of obesity continues to increase in modernsociety; there are no satisfactory pharmacological therapies for itstreatment. 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 deriveddirectly or indirectly from them. These phytoconstituents remain afocus of attention in our quest for the development of novelemphasizes the need for anti-obesity drugs. According to World

http://dx.doi.org/10.1016/j.cbi.2014.07.008

0009-2797/ 2014 Elsevier Ireland Ltd. All rights reserved.

Corresponding author. Tel.: +91 984 9086856; fax: +91 877 2289414.E-mail address: [email protected] (B. Meriga).

Chemico-Biological Interactions 221 (2014) 4251

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Health Organization (WHO) report [7] , continuous exploration andscreening of medicinal plants with satisfactory weight manage-ment efciency. A growing body of facts has clearly establishedthat medicinal plants and their constituents play a fundamentalrole in appetite regulation and energy homeostasis [8] .

Since prehistoric age, traditional formulations derived frommedicinal plants have been used for therapeutic purposes. To date,there is growing attention on therapeutic efcacy of spices includ-ing pepper because their intake appears to be connected withtreatment of certain chronic diseases as reported by severalresearchers [9,10] . Piper nigrum Linn commonly known as blackpepper has been traditionally used to treat cholera, dyspepsia, gas-tric ailments, and diarrhea [3,4,10] . Previous studies have shownthe presence of phytochemicals like piperine, piperidine and pellit-orine oil in P. nigrum Linn [911] . Moreover, to our best knowledge,data that deliberated the anti-obesity effects of piperine on diet-induced obesity in rats that dealt with pathophysiological factorsare limited. Therefore, this study was intended to investigate themitigating efcacy of piperine on pathophysiological deragmentsin HFD-induced obese rats.

2. Materials and methods

2.1. Animals

All experiments related to diet induced obesity were carried outwith male SD rats. Experiments were conducted at National Centerfor Laboratory Animal Sciences, National Institute of Nutrition,Hyderabad, India (Regd. No. 154/1999/CPCSEA). Animals werehoused individually in standard polycarbonate cages with top grillhaving facilities for holding pelleted diet and drinking water inpolycarbonate bottles with stainless steel sipper tubes (Technip-last, Italy) at 22 2 C, with 1416 air changes per hour with a rel-ative humidity 5060% with a 12 h light/dark cycle. After initialacclimatization, the animals were grouped based on their body

weights. All procedures involving laboratory animals were inaccordance with the Institute Animal Ethical Committee (IAECNo.: P11F/IAEC/2013/MB/SVU/SD Rats/M48).

2.2. Chemicals

To measure plasma glucose and insulin, kits were procuredfrom Stanbio Laboratory USA, Bio-Merieux, RCS, Lyon, France,respectively. Plasma was analyzed for total cholesterol, triglycer-ides, phospholipids, free fatty acids, VLDL, HDL by colorimetricmethods using kits (Nicholas Piramal India Ltd, Mumbai). Orlistat(Cat No. 04139) was obtained from SigmaAldrich. All otherreagents 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, itsidentity 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 thedepartmental herbarium. Seeds were shade dried and pulverizedto a coarse powder and sequentially extracted with hexane, ethylacetate, methanol and water. The respective ltrates obtainedwere evaporated to dryness in a rotary evaporator to obtain theirdried extracts. Based on preliminary phytochemical analysis,methanolic extract was found to possess important phytochemi-cals. Therefore, we proceeded further with methanolic extract

and isolated piperine by column chromatography. The purityand structure of piperine was conrmed by HPLC and mass

spectroscopy respectively at Indian Institute of Chemical Technol-ogy, Hyderabad, India [12] .

2.4. Induction of obesity

SD rats weighing 180200 g were randomly divided into sixgroups of six each. Normal control rats (Group-I) were fed with

normal pellet chow of standard composition containing all therecommended macro and micronutrients prepared according toAIN-93 guidelines with water ad libitum . High fat diet was pre-pared with a composition of beef tallow 29.5%, casein 22.0%, starch23.0%, cellulose 17.9%, L -cystine 4.0%, choline chloride 0.3%, vita-min mixture 1.8%, salt mixture 1.5% prepared at National Instituteof Nutrition, as per nutrition guidelines. Group-II to Group-VI ratswere initially fed with HFD for 15 weeks to induce obesity (20 gdaily) and from 16th week on wards, different doses of piperine(20, 30 and 40 mg/kg b.wt) were supplemented for 42 days alongwith 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 HFDeach, and 20 g of diet/day/rat was given aiming for a daily doseof 20, 30 and 40 mg/kg b.wt as mentioned in experimental design.The exact dose of piperine consumed was calculated from dailyfood intake.

2.6. Acute toxicity studies

The acute oral toxicity studies were performed in overnightfasted animals as per Organization for Economic Co-operationand Development (OECD) guidelines. The animals did not showany abnormal behavior or mortality at a dose range of 200400 mg/kg b.wt of piperine. Hence its 1/10th concentrations wereused as the therapeutic dose in the present study.

2.7. Experimental protocol

Group I Normal control (normal diet control)Group II High fat diet (HFD) controlGroup III HFD + orlistat5 mg/kg b.wtGroup IV HFD + piperine20 mg/kg b.wtGroup V HFD + piperine30 mg/kg b.wtGroup 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. Atthe end of the experiment, blood was collected from overnight

fasted animals under inhalation of anesthesia by retro orbitalpuncture method, plasma was separated by centrifugation at2500 rpmfor 15 min. For the measurement of nutrient metaboliteslike food intake, water intake, urine volume and fecal weight, bothcontrol and experimental rats were placed in the metabolic cagesfor 72 h (Techniplast, Italy).

2.9. Body composition

Body composition of experimental animals was assessed at theend 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 wereused to compare levels of body adiposity between the control and

experimental groups. Lean body mass, fat free mass and total bodyfat percent were calculated as described previously [3] .

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2.10. Heart rate, systolic and diastolic blood pressure

Heart rate, systolic and diastolic blood pressure of all experi-mental rats were measured at the end of the experiment by using12 channel BP apparatus (IITC Life sciences, Cas No. 91387) by tailcup non invasive method according to manufacturers protocol.

2.11. Determination of adiposity index

Adiposity index was measuredby using the formula: Sum of thetotal 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 over-night fasted rats under inhalation of anaesthesia by retro-orbitalpuncture method. Plasma glucose was estimated using kits (CatNo. 1060-500, Stanbio laboratory, USA). Plasma level of insulinwas determined using kits from Bio-Merieux, RCS, Lyon, France.Insulin resistance was calculated using the homeostasis modelassessment of Matthews et al., 1985 [14] .

2.13. Estimation of leptin and adiponectin

Plasma leptin and adiponectin activities were measured byusing enzyme-linked immunosorbent assay kits (Crystal Chem,Downers Grove, IL, USA), performed in duplicate, as per the man-ufacturers guidelines and expressed in ng mL 1 .

2.14. Oral glucose tolerance test (OGTT)

OGTT was performed at the end of the experiment, afterovernight fasting, glucose was administered orogastrically at adose of 2.0 g/kg b.wt and blood samples were collected from supraorbital sinus at 0,30,60,90 and 120 min. Glucose levels were esti-mated at all intervals [15] .

2.15. Assay of plasma amylase and lipase

Plasma amylase and lipase activities were determined bykinetic 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 centrifugedat 4000 rpm/min for 10 min to separate plasma which was thenstored at 80 C for further biochemical analysis. Total cholesterol,HDL, LDL and triacylglyceride levels were estimated by CHOD-PAPmethod and GPO-PAP method.

2.17. Tissue lipid analysis

Tissue lipids were extracted from experimental animals by themethod of Floch et al. (1957) [16] using a chloroformmethanolmixture (2:1, v/v). The liver tissues were rinsed with ice cold phys-iological saline, dried, homogenized in cold chloroformmethanol(2:1, v/v) and contents were extracted after 24 h. The extractionwas repeated four times. The combined ltrate was washed with0.7% KCl and the aqueous layer was discarded. The organic layer

was made up to a known volume with chloroform and used for tis-sue lipid analysis.

2.18. Assay of lipid metabolic marker enzymes

Enzyme activities were analyzed as per the proceduresdescribed 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) wasassayed 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 SigmaAldrich, USA) were measured by kit methods.

2.19. Liver antioxidants analysis

After the completion of experimental period rats we anesthe-tized and sacriced. Then the liver was excised, rinsed in ice coldnormal saline followed by ice-cold 10% KCl solution, blotted, driedand weighed. A 10% w/v homogenate was prepared in ice-cold KClsolution and centrifuged at 2000 rpm for 10 min at 4 C. The super-natants thus obtained were used for the estimation of thiobarbitu-

ric 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 ineach group. All the data were statistically evaluated with SPSS n10.0software. 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 indi-cate 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 analy-sis of piperine showed molecular formula C 17 H22 NO 3 . The LCMSspectrum, library search and the structure of obtained piperineshown in Fig. 1 .

The changes in body weight, food and water intake in differentgroups of animals during the experimental period are summarizedin Table 1 and Fig. 2 (AE). There was signicant change in food andwater intake in HFD-fed group when compared to normal controlgroup. Feeding on HFD increased substantially body weight andBMI in experimental rats when compared to normal group. HFD-

induced increase in body weight, BMI, total fat, fat percentageand fat free mass were signicantly curtailed by piperine supple-mentation in HFD-fed rats, in a dose dependent manner (20, 30and 40 mg/kg b.wt). The maximum therapeutic effect of piperinewas found at a dose of 40 mg/kg b.wt.

We recorded heart rate, systolic and diastolic blood pressure innormal diet fed and HFD-fed groups of rats. When compared tonormal control group, a signicant increase ( p < 0.05) in heart rate,systolic and diastolic blood pressure was recorded in HFD-fedgroup which was signicantly ( p < 0.05) decreased by piperinesupplementation in a dose dependent manner as shown in Table 2 .

Experimental animals fed with HFD for 21 weeks exhibited anincreased 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 organweight, fat pad weight and adiposity index in a dose dependant

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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, insulinarea under curve (AUC), leptin and adiponectin in control andexperimental 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 anddecrease in adiponectin in HFD induced obese rats over their nor-mal control rats. Supplementation with piperine tended to restorethe 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 per-formed in control and experimental obese rats. In normal controlrats, maximum elevation in blood glucose level was observed at60 min after glucose load and declined to near basal level at120 min, whereas, in HFD-induced obese rats, the peak increasein blood glucose level was noticed even after 60 min and remainedhigh over the next 60 min. Interestingly, supplementation withpiperine or orlistat to obese rats resulted in a signicant decrease

in blood glucose level at 60 min and beyond when compared withHFD control rats.

The enzyme activities of pancreatic lipase and amylase of nor-mal and experimental obese rats are represented in Fig. 6 . Wefound 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 obeserats signicantly reduced the activity of amylase and pancreatic

lipase.Fig. 7 (A and B) represents plasma and liver lipid proles of con-

trol and experimental obese rats. The concentration of plasma TGs,total cholesterol, PLs, free fatty acids, VLDL, LDL and liver lipidswere signicantly increased, except HDL, in HFD induced obeserats when compared to normal rats. Nonetheless, treatment withpiperine (40 mg/kg b.wt) signicantly ( p < 0.05) reduced the con-centrations of plasma and liver lipids, in obese rats to near normallevel, while HDL was elevated.

The activities of important lipid metabolism enzymes like(acetyl CoA carboxylase) ACC, (fatty acid synthase) FAS, (carnitinepalmitoyl transferase) CPT, HMG-CoA reductase, LPL and LCAT incontrol and experimental groups of rats are displayed in Fig. 8 . Asignicant reduction ( p < 0.05) in the level of CPT, LPL and LCAT

and a concomitant increase in the level of ACC, FAS and HMG-CoA reductase was observed in HFD control rats. Supplementation

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 362 13.9 14.2 0.85 21.8 2.6HFD control 527 8.2 a , 15.08 0.6 25.1 3.6HFD + orlistat 5 mg/kg b.wt 344.3 30.5 b , 14.7 0.7 24.2 2.8

HFD + piperine 20 mg/kg b.wt 409.2 8.77b ,

14.4 0.4 22.7 1.4HFD + piperine 30 mg/kg b.wt 391.2 12.3 b , 14.3 0.6 22.9 1.07HFD + piperine 40 mg/kg b.wt 369.2 13.4 b , 14.1 0.4 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.

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of piperine, (40 mg/kg b.wt) showed a signicant increase in theactivities of CPT, LPL and LCAT and decrease of ACC, FAS andHMG-CoA reductase in experimental animals, as that of orlistat.

Fig. 9 summarizes the levels of TBARS, GSH, SOD, CAT and GPxin the liver of control and experimental groups of rats. The activi-ties 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 tobe increased. Treatment with piperine or orlistat showed a signif-icant increase in the activities of SOD, CAT, GSH and GPx and con-comitant decrease in the level of TBARS in liver of experimentalobese rats, proving the potent antioxidant activity of piperine.

4. Discussion

The quest for novel and safe therapeutic molecules continues tocombat different aliments. Plants and herbs serve as potential

perennial source of to explore such novel bioactive factors. In thepresent work we reported that high fat diet induces obesity andassociated pathophysiological changes in the experimental ani-mals which are signicantly reverted by the treatment with piper-ine in a dose dependent manner. Obesity which was marked byincreased body weight, BMI, blood pressure, total fat, fat percent-age, fat free mass and organ weights in SD rat [22] . There is a goodrapport between body weights relative to organ weight and therisk of metabolic syndrome is successive and graded [23] . It isthe energy intake that matters in relation to the development of overweight and the energy intake is often high when HFD isconsumed in large amounts. This involved two growth mecha-nisms: 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 pressureand BMI are widely used as determining factors of fatness in

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 354.2 7.3 131.4 5.2 101.3 2.2HFD control 387 7.09 a , 157.1 12.1 a , 123.6 2.3 a ,

HFD + orlistat 5 mg/kg b.wt 367.1 4.16 b , 132.2 8.7 b , 109.4 6.8 b ,

HFD + piperine 20 m g/kg b .wt 383.4 6.5 NS 155.5 7.9 NS 122.2 5.2 NS

HFD + piperine 30 m g/kg b .wt 375.4 5.59 b , 149.9 2.4 b , 110.6 5.6 b ,

HFD + piperine 40 m g/kg b .wt 366.5 7.6 b , 141.9 9.6 b , 100.1 5.4 b ,



NS Non signicant.



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epidemiological studies, because they are highly interconnectedwith body fat [25] . Our present studies depicted a signicantincrease in blood pressure and BMI in HFD supplementation inaccordance with Akiyama et al. [25] . Increased BMI may occurdue to the increase of caloric intake resulting in more adipose tis-sue deposition. The high levels of BMI are associated with substan-

tial increases in fat mass and this index is most useful as a measureof 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 Williamset al. [27] . In our present study, there was a signicant increase insystolic, diastolic and mean arterial blood pressures as well asincrease in heart rate in HFD-fed rats when compared to normalrats. This might be due to intake of HFD in short periodwhich increase Ca 2+ channel dysregulation, leading to elevatedtransmembrane Ca 2+ inux. Up regulation in Ca 2+ current densityis connected with signicant elevation of blood pressure [28] . Sup-plementation of piperine or orlistat caused signicant attenuationin these changes in HFD induced obese rats.

Insulin resistance and hyperglycemia are hallmarks of meta-bolic syndrome [29] . In the present study, plasma glucose andinsulin levels were analyzed as indices of glycemic control andmoreover HFD-induced animals developed a hyperglycemic stateassociated with insulin resistance and/or glucose intolerance. Thisis in line with previous reports [3,30] . Under physiological condi-tions, insulin enhances lipogenesis and inhibits lipolysis whichleads to increased circulating levels of glucose and lipids thusresulting in impaired insulin secretion [31] . Supplementation of

piperine signicantly lowered plasma glucose, insulin level andinsulin 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, isa key appetite regulator and plays a pivotal role in food intakeand energy expenditure. It has been suggested that obese personsare insensitive to endogenous production of leptin and its secretionlevels are found to be positively correlated with the degree of tri-glyceride stores in adipose tissue [4951] . In our study supplemen-tation of piperine decreased leptin and increased adiponectin inplasma of HFD induced obese rats. These observations indicate thatreduction in plasma leptin and increase in adiponectin levels aftertreatment with piperine may be due to decreased lipid accumula-tion in adipocytes. Many reports have shown that consumption of

foods rich in phenolic compounds and minerals increase adiponec-tin and decrease leptin levels in obese individuals [3234] .

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 10.04 0.3 6 2.45 0.2 7 1.02 0.1 2.03 0.04 1.09 0.07 1.96 0.1 0.64 0.06HFD control 14.9 0.5 a , 3.21 0.2 a , 1.38 0.08 a, 2.86 0.51 a , 1.81 0.07 a , 3.65 0.17 a , 0.99 0.12 a ,

HFD + orlistat 5 m g/kg b .wt 12.89 0.3 b , 2.77 0.19 b , 1.16 0.13 b , 2.09 0.07 b , 1.57 0.08 b , 2.79 0.2 b , 0.77 0.08 b ,

HFD+ piperine 20 mg/kgb.wt 13.57 0.75 b , 3.02 0.16 b , 1.22 0.07 b , 2.49 0.43 b , 1.61 0.19 b , 3.03 0.05 b , 0.89 0.1 b ,

HFD+ piperine 30 mg/kgb.wt 12.82 0.64 b ,

2.64 0.33 b ,

1.16 0.09 b ,

2.27 0.155 b ,

1.5 0.14 b ,

2.77 0.3 b ,

0.83 0.09 b ,

HFD+ piperine 40 mg/kgb.wt 11.12 0.45 b , 2.55 0.46 b , 1.09 0.04 b , 2.14 0.06 b , 1.43 0.15 b , 2.19 0.12 b , 0.8 0.06 b ,



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;aSignicantly 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 dl 1) Insulin ( l U ml 1) Insulin resistance

Control 80.33 10.8 2.2 0.6 3.6 0.09HFD control 144 13.9 a , 7.1 0.8 a , 5.5 1.07 a ,

HFD + orlistat 5 mg/kg b.wt 96.3 8.7 b , 5.4 0.6 b , 4.1 0.01 b ,

HFD+ piperine 20 mg/kgb.wt 125.1 6.06 b , 6.2 0.67 b , 4.7 0.05 b ,





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Amylase and lipase provide fascinating targets in the develop-ment of anti-obesity and anti-diabetic compounds. a -amylase,one of the key digestive enzymes secreted from the pancreas andsalivary glands, is involved in hydrolytic process of starch [34] .Many synthetic and crude drugs are reported to inhibit a -amylaseactivity [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 ananti-obesity effect. Dietary lipids, which are major source of unde-sirable calories, are not directly absorbed from the intestine unlessthey are subjected to the action of pancreatic lipase. Pancreaticlipase hydrolyzes triglycerides into glycerol and fatty acids [37] .Therefore, drugs that can target pancreatic lipase are consideredto be valuable therapeutic agents for treating diet induced obesityin humans. In our study, supplementation of piperine or orlistatnormalized the elevated levels of amylase and lipase. It is possiblethat the reduced levels of pancreatic amylase and lipase might rep-resent a mechanism that would obstruct the digestion and absorp-tion of carbohydrates and lipids leading to lesser weight reduction

in piperine treated HFD-fed obese rats. Our results are in line withprevious reports [3638] .

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)

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 statisticallysignicant at p < 0.05; a Signicantly different from control; b Signicantly different from HFD control.

Fig. 5. Effectof piperine on glucose tolerancein control andexperimental obeserats.Values aremean SD, n = 6; Valuesare statistically signicant at p < 0.05; aSignicantlydifferent from control; b Signicantly different from HFD control.

Fig. 6. Effect of piperine on amylase and pancreatic lipase activities in normal andexperimental obese rats. Values are mean SD, n = 6; Values are statisticallysignicant at p < 0.05; a Signicantly different from control; b Signicantly differentfrom HFD control.



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and LDL and a decrease in serum level of HDL [39] . During fedstate, increment of chylomicrons synthesis and absorption is acommon factor which leads to increase in TC, TGs level and endog-enous VLDL production [40] . In the present study, supplementationof HFD produced experimental obesity as evidenced by increasedlipid proles in experimental rats. TGs elevation was due to dietarycholesterol which is reported to reduce fatty acid oxidation, whichin turn increases the levels of hepatic and plasma TGs. The exces-sive accumulation of TGs in the lipid stores is associated with anumber of metabolic complications [39] . Elevated TC and LDL lev-els amplify the risk of developing hypertension and cardio vasculardiseases (CVDs). On the other hand, high HDL is helpful in trans-porting excess cholesterol to the liver for excretion through bile[40] . Phospholipids play important role in the transport of triglyc-erides [41] . In our study, in HFD fed rats, the elevated level of PLsmay be due to the elevated levels of FFAs and total cholesterolwhich 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 whichpiperine contributes in improving lipid prole is by decreasingcholesterol absorption and secretion from the intestine whichleads to lowered availability of FFAs to the liver.

The liver is regarded as one of the central metabolic organs inthe body, regulating and maintaining lipid homeostasis throughkey enzymes such as ACC, FAS, CPT, HMG-CoA reductase, LPL andLCAT. ACC is the rate-limiting enzyme in de novo lipogenesis. Ithas been reported that ACC is a physiological inhibitor of CPT byproducing malonyl-CoA in the liver [43] . Fatty acids are trans-formed to acetyl-CoA by means of mitochondrial oxidation. CPTis the rate-controlling enzyme in this process of fatty acid oxida-tion. Endogenous FFA synthesis is inhibited through the suppres-sion of ACC and FAS [44] .

Supplementation of HFD enhances fat deposition in adiposetissue and it can be loweredby reducing lipid uptake by adipocytesvia suppressing lipoprotein lipase or reducing lipid synthesisthrough inhibiting FASor ACC. Piperine or orlistat reduced the lipidcontent in tissues of experimental animals by inhibiting the saidenzymes. FAS inhibitors have been reported to provide a potentialpathway to target obesity therapy [45] . Conversion of HMG-CoA tomevalonate, an NADPH-dependent reduction reaction, is the rstcommitted step in cholesterol biosynthesis catalyzed by HMG-CoA reductase [46] . The decrease in cholesterol content may beattributed to increased level of plasma HDL, increase in the activityof lipoprotein lipase and plasma LCAT, which are known to be

Fig. 7. (A) and(B) Effect of piperine onplasma and liver lipid proles in normal and experimental obeserats.Values are mean SD, n = 6; Values are statisticallysignicantat p < 0.05; a Signicantly different from control; b Signicantly 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; a Signicantly different from control; b Signicantly different from HFD control.

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involved in transport of tissue cholesterol to liver for its excretion.HFD induced hyperlipidemia down regulates the LCAT activity inplasma, which impairs the capability of HDL to remove cholesteroland leads to cholesterol accumulation in the blood. Supplementa-tion of piperine could revert these enzymes activities and inhibitcholesterol biosynthesis.

It is well established that the biomarkers of oxidative stressare much elevated in liver at an early stage in many metabolicdiseases, 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 antioxidantenzymes and TBARS level in liver. Intracellular antioxidantenzymes and TBARS are the determining factors of oxidativedamage at cellular level. HFD induced hyperglycemia observed inthis study might be an imperative aspect to increase lipidperoxidation causing reduction of antioxidant defense status andindicating the development of oxidative stress in HFD-fed ratswhich are interconnectedwith other studies [47,48] . In the presentstudy, increasedactivities of SOD, CAT, GPx and GSH anddecreasedlevel of TBARS, in signicant levels were found in HFD-fed rats,however, piper supplementation decreased hepatic SOD, CAT andGPx activities in them. This indicates that piperine has a major rolein inhibiting HFD induced intracellular oxidative stress.

5. Conclusions

To conclude, supplementation of piperine caused signicantattenuation in the physiological changes produced by HFD in rats.This might be due to the deterrence of pathological mechanismsresponsible for lipid storage and weight gain, possibly by revertingleptin and adiponectin activity and increasing energy expenditure.Together, these observations strongly suggest that piperine, amajor phytoconstituent of black pepper serves as an effective ther-apeutic 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-NewDelhi, India (Grant No.: BT/PR7799/PBD/17/849/2013) for provid-ing Junior Research Fellowship and Financial Assistance to carryout this research work and also thankful to Dr Rama Rao (IndianInstitute of Chemical Technology-India), Dr P Suresh (Director-NCLAS, NIN), Dr R Ravindar Naik (Technical Ofcer-A), NationalInstitute of Nutrition-India, for their constant encouragement and

their valuable suggestions.

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Anti obesity activity of Piper