|Year : 2013 | Volume
| Issue : 3 | Page : 229-236
Anti-obesity activity of chloroform-methanol extract of Premna integrifolia in mice fed with cafeteria diet
Prashant Y Mali1, Papiya Bigoniya1, Shital S Panchal2, Irrappa S Muchhandi3
1 Department of Pharmacology, Radharaman College of Pharmacy, Ratibad, Bhopal, Madhya Pradesh, India
2 Department of Pharmacology, Institute of Pharmacy, Nirma University, Ahmedabad, Gujarat, India
3 Department of Pharmacology, H.S.K. College of Pharmacy, Bagalkot, Karnataka, India
|Date of Submission||27-Sep-2012|
|Date of Decision||01-Feb-2013|
|Date of Acceptance||11-Mar-2013|
|Date of Web Publication||23-Aug-2013|
Prashant Y Mali
Department of Pharmacology, Radharaman College of Pharmacy, Ratibad, Bhopal, Madhya Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aim of the study: Aim of the present study was to evaluate the anti-obesity activity of chloroform:methanol extract of P. integrifolia (CMPI) in mice fed with cafeteria diet. Materials and Methods: Female Swiss Albino mice were divided into six groups, which received normal and cafeteria diet, standard drug simvastatin (10 mg/kg) and CMPI (50, 100 and 200 mg/kg) daily for 40 days. Parameters such as body weight, body mass index (BMI), Lee index of obesity (LIO), food consumption, locomotor behavior, serum glucose, triglyceride, total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL), atherogenic index, organ weight and organ fat pad weight were studied for evaluating the anti-obesity activity of P. integrifolia. High performance liquid chromatography (HPLC) fingerprint profile of chloroform-methanol extract was also studied using quercetin as the reference standard. Results: There was a significant increase in body weight, BMI, LIO, food consumption, organ weight (liver and small intestine), organ fat pad weight (mesenteric and peri-renal fat pad) and in the levels of serum glucose, triglyceride, total cholesterol, LDL and VLDL with a significant decrease in locomotor behavior (ambulation, rearing, grooming) and HDL level in cafeteria diet group. Animals treated with CMPI showed dose dependent activity. P. integrifolia (200 mg/kg) supplementation attenuated all the above alterations, which indicates the anti-obesity activity. HPLC fingerprint profile of CMPI showed two peaks in the solvent system of 50 mm potassium diphosphate (pH-3 with ortho phosphoric acid): Methanol (30:70 v/v) at 360 nm. Conclusion: Present findings suggest that, CMPI possessed anti-obesity activity that substantiated its ethno-medicinal use in the treatment of obesity.
Keywords: Cafeteria diet, chloroform-methanol extract (1:1), obesity, Premna integrifolia
|How to cite this article:|
Mali PY, Bigoniya P, Panchal SS, Muchhandi IS. Anti-obesity activity of chloroform-methanol extract of Premna integrifolia in mice fed with cafeteria diet. J Pharm Bioall Sci 2013;5:229-36
|How to cite this URL:|
Mali PY, Bigoniya P, Panchal SS, Muchhandi IS. Anti-obesity activity of chloroform-methanol extract of Premna integrifolia in mice fed with cafeteria diet. J Pharm Bioall Sci [serial online] 2013 [cited 2020 Jan 25];5:229-36. Available from: http://www.jpbsonline.org/text.asp?2013/5/3/229/116825
| Introduction|| |
Obesity is a serious illness that can lead to many medical complications. It results from an imbalance between food intake and energy expenditure, culminating in excessive accumulation of fat in adipose tissue, liver, muscle, pancreatic islets, and other organs involved in metabolism. , Its prevalence is on a continuous rise in all age groups of many of the developed countries in the world.  Obesity is rapidly turning into an epidemic afflicting much of the industrialized world, resulting in a prohibitive health and economic burden on society. ,, Obesity is a multifactorial, chronic disorder that has reached a pandemic proportion world-wide. , Nearly one third of the world's adult population (1.3 billion people) was overweight or obese in 2005 and if recent trends continue, by 2030 nearly two third of the world's adult population (3.3 billion people) could be either overweight or obese.  Moreover, obese and overweight patients are at higher risk from coronary artery disease, hypertension, hyperlipidemia, diabetes mellitus, cancers, gall bladder disorders, cerebrovascular accidents, osteoarthritis, restrictive pulmonary disease and sleep apnea. , Recently, much attention has been focused on some food factors/natural compounds that may be beneficial in preventing high fat diet induced body fat accumulation and possibly reduce the risk of diabetes and heart disease. ,
P. integrifolia Linn. (Verbenaceae), commonly known as Arni or Agnimantha , have been widely used for obesity and other obesity associated disorders.  P. integrifolia has been reported for its potential actions such as antidiabetic and hypoglycemic, , anti-inflammatory, , immunomodulatory  cardiac stimulant,  analgesic and antibacterial,  anti-arthritic, , antioxidant, , hepatoprotective and in-vitro cytotoxic, ,, antihyperglycaemic,  antiparasitic,  and hypolipidemic. , The plant mainly contains p-methoxy cinnamic acid and linalool, ,, linoleic acid, β-sitosterol and flavone luteolin,  iridoid glycoside,  premnine, ganiarine and ganikarine, premnazole, aphelandrine, pentacyclic terpene betulin, caryophellen, premnenol, premnaspirodiene, clerodendrin-A, ,,,,, three diterpenoids namely 1 β,3 α,8 β-trihydroxy-pimara-15-ene, 6 α, 11, 12, 16-tetrahydroxy-7-oxo-abieta-8, 11, 13-triene and 2 α,19-dihydroxy-pimara-7,15-diene. , Moreover, alkaloids, proteins, carbohydrates, amino acids, steroids, flavonoids, glycosides, tannins and phenolic compounds were found in preliminary phytochemical screening of (CMPI).  Therefore, the objective of the present study was to evaluate the anti-obesity activity of CMPI in mice fed with cafeteria diet in preclinical experimental animal model.
| Materials and Methods|| |
Chemicals and reagents
Glucose, triglyceride, total cholesterol, high density lipoprotein (HDL) kits were obtained from ERBA Diagnostics, Mannheim GmbH, Germany. Cafeteria diet was purchased from local market of Bhopal, Madhya Pradesh. Simvastatin was obtained from USV Ltd., Baddi, India. Petroleum ether, chloroform and methanol were obtained from SD Fine-Chem Ltd, Mumbai, India. All other chemicals and reagents used for experimental work were of analytical grade.
Plant material and preparation of extract
Fresh, well developed P. integrifolia plants and their roots were collected from the region of North Karnataka, India in the month of September 2008 and it was authenticated by a taxonomist, Department of Botany, Basaveshwar Science College, B.V.V.S. Campus, Bagalkot, Karnataka, India. Voucher specimen (No. B.Sc/Bot/13/08) was deposited in the same. The fresh roots were cleaned in 10% KMNO 4 , dried in the shade and powdered through sieve #44 for uniform size. The root powder (4 kg) was extracted with petroleum ether (40-60°C) for defating and subsequently with 1:1 ratio of chloroform-methanol (55-60°C) for 24 h by using soxhlet apparatus. After the residue extraction, the excess solvent was completely removed by using a rotatory flash evaporator to get concentrated, then completely dried in freeze drier and preserved in an airtight container under refrigeration. Percentage yield of CMPI was 1.29% and then it was used for evaluation of anti-obesity activity.
High performance liquid chromatography fingerprint profile
High performance liquid chromatography (HPLC) fingerprint profile of CMPI was studied using quercetin as the reference standard followed by following chromatographic conditions.
- Mobile phase: 50 Mm potassium diphosphate (pH-3 with ortho phosphoric acid): Methanol (30:70 V/v) at 360 nm.
- Wave length: 360 nm.
- Flow rate: 1 ml/min.
- Column: C18 (250 × 4.6 mm, 5u).
- HPLC system: Refurbished water isocratic reverse phase-HPLC system.
- Pump: Waters.
- Detector: Waters 486 U.V-Visible.
- Injector: Rehodyne.
- Reference standard: Quercetin.
- Software: Chromatography software.
Female Swiss albino mice (22-26 gm) were used for the study. Inbreed animals were obtained from the central animal house of Radharaman College of Pharmacy, Bhopal. The animals were housed at room temperature (25 ± 1°C) with 50 ± 5% relative humidity and given standard laboratory feed (Hindustan Lever, India) and water ad libitum throughout the experimental period. The study was approved by Institutional Animal Ethics Committee, Radharaman College of Pharmacy, Bhopal.
Acute toxicity study
Acute toxicity study of CMPI was performed as per the Organization for Economic Co-operation and Development (OECD) guideline No. 425 and 420 followed by up and down and Fixed dose method. Based on these agreements, a limit test was performed to categorize the toxicity class of the compound and then main test was performed to estimate the exact 50% of lethal dose (LD 50 ). , The dose range of 50, 100 and 200 mg/kg was selected for CMPI. The doses selected for the study starts from 1/10, 1/20 and 1/30 of the LD 50 .
Experimental protocol for anti-obesity activity
Female Swiss Albino mice (22-26 gm) were randomly divided into six groups of six mice in each and treated are as follows:
Group I: Received standard laboratory feed, i.e., the normal diet.
Group II: Received cafeteria diet in pellets forms.
Group III: Simvastatin (10 mg/kg, orally) was administered daily.
Group IV: CMPI (50 mg/kg, orally) was administered daily.
Group V: CMPI (100 mg/kg, orally) was administered daily.
Group VI: CMPI (200 mg/kg, orally) was administered daily.
Preparation of cafeteria diet for induction of obesity
The method described by Harris and Kulkarni was followed with some modifications. , Cafeteria diet (highly palatable, energy rich animal diet that includes a variety of human snack foods) consists of 3 diets, which includes (condensed milk 48 g + bread 48 g), (chocolate 18 g + biscuits 36 g + dried coconut 36 g), (cheese 48 g + boiled potatoes 60 g). Cafeteria diet was presented in the form of pellets to 5 groups of 6 mice in each for 40 days.
Body weights of mice (g) were recorded on 1, 10, 20, 30 and 40 day in each group. 
Body mass index and lee index of obesity
Body mass index (BMI) and lee index of obesity (LIO) of mice were recorded on 1 and 40 day of study (i.e., initial and final body weight and body height) and was measured by using formulas, 
BMI = Body weight in gm/(Height in cm) 2
LIO = Body weight in gm (1/3)/Nasoanal length in cm
Food consumption study was carried out on 1, 10, 20, 30 and 40 day and recorded at 1 h, 2 h and 3 h of time intervals. The food consumption was estimated by subtracting the amount of food left on the grid from initial food weight. 
Locomotor behavior study was performed on 40 day by using open field behavior test after 30 min of drug administration. Open field test was performed by placing mice in the center of apparatus and recording the ambulation (by counting the number of horizontal and vertical compartments traversed by animal), frequency of rearing and grooming for a 5 min test period was recorded. ,,
On 41 day, the blood samples were taken by penetrating the retro-orbital plexus with a fine glass capillary. The blood samples were centrifuged at 2500 rpm for 15 min to separate the serum and preserved (−20°C) for analysis of glucose (Trinder's Method), triglycerides (Glycerol phosphate oxidase (GPO)-Trinder Method, End Point), total cholesterol (Modified Roeschau's Method), high density lipoprotein-c (HDL-c) (Phosphotungstic Acid Method), low density lipoprotein-c (LDL-c) (Calculated using Friedewald's equation, LDL-c = total cholesterol- very low density lipoprotein (VLDL-c - HDL-c), VLDL-c (VLDL-c = Triglyceride/5) and atherogenic index (AI = LDL-c + VLDL-c/HDL-c) were estimated. ,,
Mice were sacrificed under diethyl ether anesthesia on 41 day. Organs such as brain, liver, stomach, heart, small intestine, spleen, lungs, kidneys, and adrenal glands were removed and weighed. 
Organ fat pad weight
Mesenteric fat pad
Mice were sacrificed under diethyl ether anesthesia on 41 day. The fat deposited on mesentery, i.e., duodenum to the large intestine were removed and weighed.
Peri-renal fat pad
Mice were sacrificed under diethyl ether anesthesia on 41 day. The fat deposited on left and right kidneys were removed and weighed. 
All experimental data were statistically analyzed and expressed as means ± SEM. The significance of difference between control and treated animals for different parameters were determined by using one-way analysis of variance followed by multiple comparisons Dunnett's test. P < 0.05 value was considered as statistically significant.
| Results|| |
Effect of CMPI on body weight
[Table 1] shows the effect of CMPI on body weight in normal and experimental group of mice. Cafeteria diet group showed significant (P0 < 0.001) increase in body weight on 10, 20, 30, and 40 day as compared to a normal diet group. Cafeteria diet fed mice treated with simvastatin showed significant ( P < 0.05- P < 0.001) decrease in body weight on 10, 20, 30 and 40 day as compared to cafeteria diet group. Oral administration of CMPI group (200 mg/kg) showed significant ( P < 0.05- P < 0.001) reduction in body weight at 20, 30 and 40 day as compared to cafeteria diet group.
|Table 1: Effect of CMPI on body weight in normal and experimental group of mice |
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Effect of CMPI on BMI and LIO
Effect of CMPI on BMI and LIO in normal and experimental group of mice is shown in [Table 2]. Feeding the cafeteria diet to mice was found to significantly ( P < 0.001) increase the final BMI and LIO when compared to mice fed with normal diet. Cafeteria diet fed mice treated with simvastatin, CMPI (200 mg/kg) groups showed a significant ( P < 0.001) decrease in the final BMI and LIO when compared with cafeteria diet group.
|Table 2: Effect of CMPI on BMI and LIO in normal and experimental group of mice |
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Effect of CMPI on food consumption
[Table 3] shows the effect of CMPI on food consumption in normal and experimental group of mice. Food consumption was found to be significantly ( P < 0.001 ) increased on 1, 10, 20, 30 and 40 day as compared to a normal diet group. Simvastatin administered mice showed a significant ( P < 0.01, P < 0.001) decrease in food consumption on 30 and 40 day as compared to cafeteria diet group. The CMPI (200 mg/kg) treated mice showed significantly ( P < 0.05, P < 0.01) decreased food consumption on 30 and 40 day as compared to cafeteria diet group.
|Table 3: Effect of CMPI on food consumption in normal and experimental group of mice |
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Effect of CMPI on locomotor behavior
[Table 4] shows the effect of CMPI on locomotor behavior in normal and experimental group of mice. Cafeteria diet group showed significant ( P < 0.001) decrease in ambulation, rearing and grooming as compared to a normal diet group. Cafeteria diet fed mice treated with simvastatin, CMPI (200 mg/kg) groups showed a significant increase in ambulation ( P < 0.001), rearing ( P < 0.05) and grooming ( P < 0.05) as compared to cafeteria diet group.
|Table 4: Effect of CMPI on locomotor behavior in normal and experimental group of mice |
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Effect of CMPI on biochemical profile
Effect of CMPI on biochemical profile in normal and experimental group of mice is shown in [Table 5]. Feeding the cafeteria diet to mice was found to significantly ( P < 0.001) increase the levels of serum glucose, triglycerides, total cholesterol, LDL, VLDL, atherogenic index and significantly ( P < 0.001) decrease the level of HDL when compared to mice fed with normal diet. Cafeteria diet fed mice treated with simvastatin, CMPI (200 mg/kg) groups showed a significant ( P < 0.001) decrease in the levels of serum glucose, triglycerides, total cholesterol, LDL, VLDL, AI and significant ( P < 0.001) increase in the levels of HDL when compared with cafeteria diet group.
|Table 5: Effect of CMPI on biochemical profile in normal and experimental group of mice |
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Effect of CMPI on organ weight
[Table 6] shows the effect of CMPI on organ weight in normal and experimental group of mice. The weight of organs such as liver and small intestine in cafeteria diet group was found to be significantly ( P < 0.001) increased as compared to a normal diet group. Simvastatin administered mice showed a significant decrease in organ weight such as liver ( P < 0.01) and small intestine ( P < 0.001) as compared to cafeteria diet group. The CMPI (200 mg/kg) treated mice showed significantly ( P < 0.05) decreased weight of liver and small intestine as compared to cafeteria diet group. There was no significant difference between the organ weights of brain, stomach, heart, spleen, lungs, kidneys and adrenal glands of cafeteria diet group and other experimental groups.
|Table 6: Effect of CMPI on organ weight in normal and experimental group of mice |
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Effect of CMPI on organ fat pad weight
Effect of CMPI on organ fat pad weight in normal and experimental group of mice is shown in [Table 7]. Feeding the cafeteria diet to mice was found to significantly ( P < 0.01, P < 0.001) increase the weights of mesenteric and peri-renal fat pads when compared to mice fed with normal diet. Cafeteria diet fed mice treated with simvastatin, CMPI (200 mg/kg) groups showed a significant (P < 0.01, P < 0.001) decrease in the weights of mesenteric and peri-renal fat pads compared with cafeteria diet group.
|Table 7: Effect of CMPI on organ fat pad weight in normal and experimental group of mice |
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| Discussion|| |
Obesity is arguably biggest challenge among the epidemics facing world-wide.  A cafeteria diet induced obesity model is the simplest obesity induction model and possibly the one that most closely resembles the reality of obesity in humans.  Cafeteria diet has been previously reported to increase energy intake and cause obesity in humans  as well as in animals.  The cafeteria diet has been reported to induce hyperphagia in rats,  which results in higher fats stores resulting in increased body weight and organ weight.  The results of our present study was in line with the above findings as we have observed a significant increase in body weight, BMI, LIO, food consumption, organ weight (liver and small intestine), and organ fat pad weight (mesenteric and peri-renal fat pad) in cafeteria diet induced group. The elevated consumption of foods rich in calories such as high fat food is associated with the low physical activity. Moreover, studies have shown that mice fed a high fat diet displayed lower frequencies of ambulation, rearing and grooming as compared to mice fed with a balanced diet. , These results were in agreement with our present studies as we have noticed decreased ambulation, rearing and grooming in cafeteria diet group as compared to a normal diet group. Treatment with CMPI groups showed a significant increase in ambulation, rearing and grooming suggesting its beneficial role in maintaining the physical activity and locomotor behavior.
High fat diet induced obesity can lead to insulin resistance. Obesity is associated with a decreased capacity of insulin to regulate glucose metabolism in the peripheral tissues.  Reports have strongly suggested that obesity is strong associated with imbalance in glucose and insulin homeostasis.  In our present study, cafeteria diet fed mice showed abnormally increased blood glucose levels. Treatment with P. integrifolia markedly brought down the levels of blood glucose suggesting its anti-hyperglycemic effect. Obesity is associated with an unfavorable lipid profile. Lipid abnormalities related to obesity include an elevated serum concentration of triglycerides, cholesterol, LDL and VLDL as well as a reduction in serum HDL.  It is shown that cafeteria diet elevates serum triglyceride levels essentially by preventing its uptake and clearance by inhibiting catabolizing enzymes like lipoprotein lipase (LPL) and lecithin cholesterol acetyl transferase.  Cafeteria diet induced hypercholesterolemia has been related to its ability to alter the physico-chemical properties of lipoproteins and thereby prevent their uptake by the liver for clearance.  High fat diet increases both LDL-cholesterol and oxidative stress that results in increased oxidized LDL levels leading to atherosclerotic plaque formation.  Moreover, cafeteria diet decreases the levels of HDL, which is considered to be the good cholesterol that is anti-atherogenic in natur.  The above findings were in supportive with our present results as we have observed increased serum levels of triglycerides, total cholesterol, LDL, VLDL, and decreased levels of serum HDL in cafeteria diet induced animals. Animals treated with P. integrifolia showed markedly reduced serum levels of triglycerides, total cholesterol, LDL, VLDL, and increased levels of serum HDL. The reduction in triglycerides level is due to an increase in activity of endothelium bound LPL which hydrolyzes the triglyceride into fatty acids or may be due to an inhibition of peripheral lipolysis so that fatty acids are not released and get converted into triglyceride.  The decline in VLDL levels in treated groups could be directly correlated to a decline in triglyceride levels as it is well established that VLDL particles are the main transporters of triglycerides in plasma.  A decrease in serum cholesterol and LDL level is due to decreased cholesterol absorption from the intestine by binding with bile acids within the intestine and increasing fecal bile acids excretion.  Tannins present in P. integrifolia have been reported to increase fecal bile acid excretion, thereby leading to reduction in cholesterol levels.  Elevated levels of HDL helps to carry cholesterol back to the liver, where it gets excreted as bile salts.  In our present study, mice fed with P. integrifolia were found to significantly increase serum HDL levels suggesting its cardioprotective nature. The increased level of HDL observed in CMPI compare to the cafeteria diet group may be due to an increased activity of lecithin cholesterol acyl transferase (an enzyme which incorporates free cholesterol from LDL into HDL and transfer it back to VLDL and intermediate density lipoprotein).  Atherogenic index of plasma has recently been proposed as a marker of plasma atherogenicity because it is increased in people at higher risk for coronary heart diseases and is inversely correlated with LDL particle size. , In our present study, treatment with P. integrifolia showed a reduction in atherogenic index, which could be due to the presence of alkaloids (premnazole), flavonoids (luteolin), sterols (β-sitosterol), amino acids (linoleic acid-fatty acid), carbohydrates, tannins and polyphenolic compounds. ,,,,,,,, However, HPLC fingerprint profile of CMPI [Figure 1] showed two peaks in the solvent system of 50 Mm potassium diphosphate (pH-3 with Ortho Phosphoric acid): Methanol (30:70 v/v) at 360 nm. The reference standard was used as quercetin [Figure 2]. Thus, it is suggested that assessment of active constituents and clinical evaluation of P. integrifolia would give a positive lead in the successful treatment of obesity. However, further series of studies are required to prove its clinical reliability, safety and efficacy.
|Figure 1: Chromatogram of chloroform-methanol extract of Premna integrifolia|
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| Acknowledgments|| |
Authors are thankful to the Principal and Management of Radharaman College of Pharmacy, Ratibad, Bhopal, Madhya Pradesh, India for encouragement and availing of the laboratory facilities during the course of the investigation.
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[Table 6], [Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 7]