Dietary fat is not directly absorbed from the small intestine unless it has been subjected to the action of pancreatic lipase⁴. The obesity epidemic in the world today, is an unintended consequence of the economic, social and technological advances realized during the past several decades. With recognition to the epidemic, obesity has an increasing awareness of the need to improve the quality and effectiveness of available treatments. The current core of treatments for obesity includes behavior therapy aimed at modifying eating related activities, exercise to increase caloric expenditure and diets to lower calorie and fat intake. Pharmacological treatments are generally considered as an adjunct to this core therapy⁵.

The use of herbs as medicine, for various medical ailments, dates back to the Aryan period. With onset of scientific research in herbals, it is becoming clearer that the medicinal herbs have a potential in today’s synthetic era, as numbers of medicines are becoming resistant. According to one estimate only 20% of the plant flora has been studied and 60% of synthetic medicines owe their origin to plants. Ancient knowledge coupled with scientific principles can come to the forefront and provide us with powerful remedies to eradicate the diseases. All over the world, today we are looking for a natural system of healing that is comprehensive and complete that is not merely some curious form of folk healing but a real and rational system of medicine that is sensitive to both nature and the Earth. The benefits of herbs are many and varied. Various herbs can be used to treat obesity depending on the physical constitution and food habits of an individual. The main advantage of herbs is that these can be taken on a long-term basis without a risk of serious side-effects⁶.

The herbs used in the treatment of obesity are: Guggul, Shilajit, Zinziber officinalis, Piper nigrum, Emblica officinalis, and Terminalia bellericz^7. Moringa oleifera Lam (synonym: Moringa pterygosperma), a small tree (7–12 m high) with thick grey bark, fragrant white flowers, and long green pods, is commonly referred to as the ‘drumstick tree’ or ‘horseradish tree’⁸. It is a member of the Moringaceae family that grows throughout most of the tropics, including Pakistan, Bangladesh, Afghanistan, and northwest India⁹. Moringa oleifera seeds are used in Gujarat for anti obesity purpose (personal communication).To prove the same scientifically, the present study we are interested to screen anti-obesity activity of Moringa oleifera seeds.

MATERIALS AND METHODS:

MATERIALS:

List of equipments:

Semi automatic analyzer (Glaxosmithkline Pharmaceutical Ltd., Bombay), centrifuge and homogenizer (Remi motors, Bombay).

METHODS:

Procurement of plant material:

Moringa oleifera Lam. seeds were collected during May 2009, from Byranahalli ranges, in Nelamangala taluk of Bangalore district, Karnataka. The collected seeds were authentified by Dr. Siddappa, Head of the department of Botany, Sree Siddaganga College of Arts Science and Commerce for Boys, B.H Road, Tumkur. The specimen was kept in herbarium of Sree Siddaganga College of Pharmacy and the voucher number is SSCP/Pcol 07/08-09.

Extraction procedure:

Moringa oleifera Lam. seeds were shade dried and coarse powdered in mixer grinder. About 100 g of the powder was extracted with 300 ml of 95% v/v ethanol by maceration process for 24 h. The solvent was removed completely by evaporating the extract at 40^°C and reddish brown residue (yield 10.20% w/w with respect to starting dry material) was obtained. The extract was stored in airtight container in a cool place and used throughout the study.

Preparation of suspension:

A weighed amount of dried ethanol extract of the seeds of Moringa oleifera Lam. was suspended in 1% aqueous Tween-80 solution and used for the present study.

Chemicals required:

Beef tallow (Purchased from the local slaughter house, Tumkur), normal feed (Amrut laboratory animal feed, Pranav Agro Industries Ltd. Sangli, Maharastra), casein (Himedia Laboratories Pvt. Ltd., Mumbai), corn starch (LOBA Chemicals, Mumbai ), vitamin and mineral mix (Abbott Healthcare Pvt. Ltd., Mumbai), corn oil (S.d.fine Chem. Ltd., Bombay), cholic acid (LOBA Chemicals, Mumbai), cholesterol (S.d.fine Chem. Ltd., Bombay), oleic acid (S.d.fine Chem. Ltd., Bombay), biochemical kits like Triglyceride E-Test kit, Total cholesterol E-Test kit, HDL-cholesterol E-Test kit (ERBA diagnostics Mannheim GmbH, Germany) and orlistat (Meyer’s Pharmaceutical Ltd., Bangalore). All solvents and other chemicals used were of analytical grade.

Experimental Animals:

Female albino Swiss mice (3 weeks old) and male Wistar rats (6 weeks old) were obtained from the animal house of Sree Siddaganga College of Pharmacy, Tumkur. They were housed in groups of six under standard laboratory conditions of temperature (25 ± 2^°C) and 12/12 h light/dark cycle. Animals had free access to standard pellet diet and water ad libitum.

The distribution of animals in the groups, the sequence of trials and the treatment allotted to each group were randomized, throughout the experiment. Laboratory animal handling and experimental procedures were performed in accordance with the guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and experimental protocol was approved by Institutional Animal Ethical Committee (IAEC/ SSCPT /60/2008-09).

Preliminary Phytochemical Evaluation:

The MOE was tested for the presence of phytochemical constituents like alkaloids, glycosides, saponins, tannins, steroids and flavonoids.

Screening methods for the evaluation of anti-obesity activity:

a) High-fat diet (HFD) induced obesity in mice fed for nine weeks¹⁰.

Composition and preparation of high fat diets:

Compositions of different experimental high fat diets for 100 g are as shown in Table 1. For the preparation of diets, all the ingredients except beef tallow were weighed and mixed in the ascending order of their weight. Finally the beef tallow was weighed, melted at 60^°C and pellets were made after mixing with the whole quantity of powder.

Table 1: Composition of experimental high-fat diets

Ingredients	HFD	g/100 g diet
Ingredients	HFD	HFD + 1% MOE	HFD + 2% MOE	HFD+0.012% Orlistat
Beef tallow	40	40	40	40
Casein	26	25	24	26
Corn starch	10	10	10	10
Powdered NPD*	10	10	10	10
Sugar	9	9	9	9
Mineral mix	4	4	4	4
Vitamin mix	1	1	1	1
MOE	0	1	2	0
Orlistat	0	0	0	0.012

*NPD – Normal pellet diet

Experimental study design:

Female albino Swiss mice of (3 weeks old) were housed for 1 week under a 12 h/12 h light/dark cycle in a temperature and humidity controlled room. The animals were given free access to food and water. After adaptation to the above conditions for 1 week, the healthy animals were randomized depending on body weight, grouped as Group I- NPD fed control; Group II- Powdered NPD + 2% MOE; Group III- HFD fed control; Group IV- HFD + 1% MOE; Group V- HFD + 2% MOE; Group VI- HFD + 0.012% orlistat and used in these experiments. Mice were allocated into two dietary regimens and were fed on either normal pellet diet (NPD) or high-fat diet (HFD) ad libitum, for a period of nine weeks. The parameters like body weight, food consumption, serum glucose, wet weight of feces, TG in feces, lipid parameters likeTG, TC, HDL-c, LDL-c, VLDL-c, TC/HDL-c , LDL-c/HDL-c, parametrial adipose tissue (PAT) weight, organ weights like heart, kidney and liver, liver TG.

Measurement of body weight and total food consumption¹¹:

Initial body weight of each mouse in all six groups was measured and they were allocated into two dietary regimens and fed either NPD or HFD as explained in Table 8 for the period of nine weeks. At the end of each week, weight of mice in each group was measured using a standard weighing machine till the end of nine weeks of study. The net weight gain was calculated as:

Also percentage change in the body weight of each group was calculated as:

Insulin tolerance test (ITT)¹²:

Insulin tolerance of various groups was estimated by ITT. At the end of 8 weeks of treatment, insulin (5 IU/kg, i.p.) was administered to 12 h fasted mice and blood samples were collected at 0 (before insulin administration), 15, 30 and 45 min after insulin administration by tail cut method. Sodium citrate was added as anti-coagulant to the collected samples and centrifuged at 5000 rpm for 10 min. The plasma was separated and plasma glucose level was estimated by using glucose kit (DIALAB). The results were expressed as integrated area under curve for glucose (AUC _glucose), which was calculated by trapezoid rule:

Fat excretion in feces of mice¹³:

Mice which consumed the NPD and HFD for nine weeks, feces wet weight was measured and triglyceride content in feces obtained during the last 24 h were measured using ERBA Triglyceride E-Test kit.

Estimation of biochemical parameters in blood serum:

After the completion of nine weeks of treatment, blood samples were collected from retro-orbital plexus of mice under anesthesia with diethyl ether. Blood samples were centrifuged at 5000 rpm for 10 min and serum was obtained. The biochemical parameters like TG, TC, HDL-c were estimated in the serum. LDL-c and VLDL-c in serum were calculated as per Friedewald’s equation¹⁴.

The markers of dyslipidemia such as TC/HDL-c ratio and LDL-c/HDL-c ratio were also calculated as:

Estimation of organ and tissue weights:

After nine weeks of treatment, the female mice were sacrificed with overdose of diethyl ether and the adipose tissue surrounding uterus¹⁵ was dissected and weighed. The organs liver, heart and kidney were also dissected and weighed¹⁶. The ratios of weight of liver, heart and kidney to the weight of animal were calculated as follows:

Estimation of liver triglyceride:

A portion (0.5 g) of the liver was homogenized in Krebs Ringer phosphate buffer (7.4, 4.5 ml), the homogenate (0.2 ml) was extracted with chloroform/methanol (2:1, v/v, 4 ml) and the extract was concentrated. The residue was analyzed using ERBA Triglyceride E-Test kit¹⁵.

b) Lipid emulsion tolerance test (LETT) model in rats

Composition and preparation of lipid emulsion (LE)¹³:

The composition of lipid emulsion is given in the Table 2. The lipid emulsion was prepared as follows; corn oil was triturated with cholic acid in a mortar. In a separate beaker cholesterol was dissolved in oleic acid, added to the contents in mortar and triturated until crackling sound was obtained. Finally saline was mixed to the contents in the mortar to obtain lipid emulsion.

Table 2: Composition of lipid emulsion

Sl. No	Ingredients	Quantity
1	Corn oil	3 ml
2	Cholic acid	40 mg
3	Cholesterol	800mg
4	Oleic acid	2 ml
5	Saline	3 ml

Experimental study design:

Male Wistar rats of (six weeks old) were housed for 1 week under a 12 h/12 h light/dark cycle in a temperature and humidity controlled room. The animals were given free access to food and water. After adaptation to the above conditions for one week, the healthy animals were grouped as shown in Table 3 and used in these experiments.

Table 3: Experimental study design for lipid emulsion tolerance test (LETT) in rats

Groups (n=6)	Treatments	Parameter
I	Normal saline control	Plasma triglyceride level
II	Lipid emulsion control
III	Lipid emulsion + Moringa oleifera seed extract 500 mg/kg
IV	Lipid emulsion + Moringa oleifera seed extract 1000 mg/kg
V	Lipid emulsion + (standard) Orlistat 45 mg/kg

After rats had been deprived of food overnight, they were orally administered 1 ml of lipid emulsion. Group I was treated with saline, Group II with lipid emulsion and Groups III, IV and V were treated with 500 mg/kg MOE, 1000 mg/kg MOE and 45 mg/kg olistat, 30 min before giving LE. Blood samples were collected from tail vein by cutting tip of the tail at 0, 0.5, 1, 2, 3, 4 and 5 h after the administration of LE. Sodium citrate (3.8%) is used as anti coagulant and blood samples were centrifuged at 5500 rpm for 10 min and plasma was obtained. The plasma triacylglycerol concentration was determined using ERBA Triglyceride E-Test kit.

Statistical analysis:

The data were expressed as Mean ± S.E.M. Statistical comparisons were performed by one way ANOVA followed by Tukey’s post- test using Graph Pad Prism version 4.0, U.S.A. Differences were considered significant at P< 0.05.

RESULTS:

Preliminary phytochemical evaluation.

The phytochemical constituents present in MOE was given in Table 4.

Table 4: Preliminary phytochemical evaluation of MOE.

Sl No.	Chemical tests	Observation	Inference
1	Dragendroff’s test	+	Alkaloids present
2	Mayer’s test	+	Alkaloids present
3	Hager’s test	+	Alkaloids present
4	Wagner’s test	+	Alkaloids present
5	Keller-killiani test	+	Glycosides present
6	Foam test	+	Saponins present
7	Haemolytic test	+	Saponins present
8	Salkowski reaction	+	Steroids present
9	Liebermann-Burchard reaction	+	Steroids present
10	Lead acetate solution	-	Tannins absent
11	5% FeCl₃ solution	-	Tannins absent
12	Shinoda test	+	Flavonoids present

Effect of nine weeks treatment with different doses of MOE and orlistat on various parameters of HFD fed mice.

Body weight:

Consumption of HFD for nine weeks has showed increase in the body weight of mice compared to NPD fed control, whereas the results are not statistically significant. Furthermore, HFD fed mice have showed significant (^aP<0.05) percentage change in body weight (53.36%) compared to NPD fed group (32.10%).

Chronic treatment with higher dose of MOE (2%) in HFD fed mice exhibited significant (^aP<0.05; ^bP<0.01; ^cP<0.001) reduction in the body weight on 1^st, 8^th and 9^th week compared to HFD fed control (Fig. 1) and exhibited significantly (^aP<0.05) decreased percentage change in body weight (32.62%). Treatment of orlistat (0.012%) to HFD fed mice exhibited significant (^aP<0.05; ^bP<0.01; ^cP<0.001) reduction in body weight at 1^st, 6^th and 9^th week and also showed significant (^cP<0.001) decrease in percentage change in body weight (24.78%) compared to HFD fed control. Treatment with lower dose of MOE (1%) however showed significant (^cP<0.001) reduction in body weight only at 1^st week, whereas further treatment failed to reduce body weight significantly and there is no significant percentage change in body weight (38.91%). Treatment of NPD fed mice containing 2% MOE exhibited significant (^dP<0.05; ^eP<0.01; ^fP<0.001) reduction in body weight at the end of 1^st, 4^th, 5^th, 8^th and 9^th week compared to NPD fed control. We observed that there is no significant percentage change in body weight of NPD fed mice treated with 2% MOE (42.04%) compared to NPD fed control (Fig. 2).

Fig.1. Effect of nine weeks treatment of different doses of MOE and orlistat on body weight of HFD fed mice.

Data represents the Mean ± S.E.M. for 6 mice. ^aP<0.05; ^bP<0.01; ^cP<0.001 compared to HFD fed control; ^dP<0.05; ^eP<0.01; ^fP<0.001 compared to NPD fed control (one way ANOVA followed by Tukey’s post-test).

Food consumption, wet weight of feces, TG in feces, parametrial adipose tissue weight and liver TG

The average food consumption was found to be increased significantly (^cP<0.001) in HFD fed control (581.62 ± 0.59) as compared to NFD fed control (532.78 ± 0.51). The average food intake in the HFD fed mice containing MOE (1% and 2%) and orlistat (0.012%) was found to be different and significant (^cP<0.001) compared to HFD fed control. Oral administration of NPD fed mice containing MOE (2%) has showed significantly (^fP<0.001) increased in food intake compared to NPD fed control (Table 5).

Fig.2. Effect of nine weeks treatment of different doses of MOE and orlistat on percentage change in body weight of HFD fed mice.

(G1) NPD fed control; (G2) NPD+2% MOE; (G3) HFD fed control; (G4) HFD+1% MOE; (G5) HFD+2% MOE; (G6) HFD+0.012% orlistat. Data represents the Mean ± S.E.M. for 6 mice. ^aP<0.05; ^cP<0.001 compared to HFD fed control (one way ANOVA followed by Tukey’s post-test).

Consumption of HFD for nine weeks has shown significant (^cP<0.001) decrease in the wet weight of feces compared to NPD fed control. Treatment with MOE (1 and 2%) and orlistat (0.012%) to HFD fed mice has significantly (^cP<0.001) increased the feces weight at the end of nine weeks, compared to HFD fed control. Feces weight also significantly (^fP<0.001) increased in NPD fed mice treated with MOE 2% compared to NPD fed control.

Mice fed with HFD for nine weeks exhibited significant decreased levels of TG in feces compared to NPD fed control. Treatment with different doses of MOE (1 and 2%) and orlistat (0.012%) for nine weeks had shown significantly higher TG in feces compared to HFD fed control. TG levels in feces also significantly (^fP<0.001) increased in NPD fed mice treated with MOE 2% compared to NPD fed control. The results were summarized in Table 5.

Feeding HFD for nine weeks has significantly (^bP<0.01) increased parametrial adipose tissue weight compared to NPD fed control. HFD fed mice treated with different doses of MOE (1 and 2%) and orlistat (0.012%) had not significantly reduced the parametrial adipose tissue weight compared to HFD fed control. Also there was no significant reduction in parametrial adipose tissue weight of NPD mice treated with 2% MOE compared to NPD fed control (Table 5).

Feeding the HFD for nine weeks resulted in fatty liver with accumulation of triglyceride. The accumulation of hepatic triglyceride by the high fat diet was significantly (^cP<0.001) higher compared to NPD fed control. Treatment with MOE (1 and 2%) and orlistat (0.012%) to HFD fed mice exhibited significant decrease in the liver TG as compared to HFD fed control. Moreover, no significant reduction was observed in MOE (2%) treated NPD fed mice compared to NPD fed control (Table 5).

Insulin tolerance test (ITT)

From the insulin tolerance test, it is possible to know the extent of peripheral utilization of glucose. At the end of eight weeks of respective treatment, administration of insulin (5 IU/kg) has produced significant (^aP<0.05; ^cP<0.001) reduction in the plasma glucose levels of NPD fed mice over the period of 0 to 45 min. Further, HFD fed mice subjected to insulin challenge did not exhibit a marked fall in plasma glucose levels suggested that, these HFD fed mice were not able utilize the exogenously administered insulin to reduce the glucose levels. This observation may be due to the loss of insulin sensitivity resulted from chronic administration of high fat diet. However, treatment with MOE (2%) and orlistat (0.012%) showed significant (^cP<0.001) reduction in plasma glucose level at 0 to 45 min compared to HFD fed control (Fig.3A).

Fig.3. Effects of nine weeks treatment of different doses of MOE on insulin Tolerance Test (ITT) in HFD fed mice.

[A] Plasma glucose levels were measured prior to, and after s.c. administration of insulin alone (5 U/kg), or in combination with MOE or orlistat. [B] Area under curve for glucose (AUC_glucose) values for 0-45 min post insulin injection. Data represents the Mean ± S.E.M. for 6 mice^aP<0.05; ^cP<0.001 compared to HFD fed control (one way ANOVA followed by Tukey’s post-test).

Integrated area under the glucose curve over 45 min (AUC_glucose) of HFD fed control was significantly (^cP<0.001) higher compared to NPD-fed. Treatment with MOE/orlistat produced a significantly (P<0.001) decreased AUC _glucose compared to HFD fed control (Fig. 3B).

Further estimation of AUC values indicated that, there is a 70.04% reduction in the plasma glucose level of NPD fed control compared to HFD fed control. Treatment of MOE and orlistat to HFD fed mice showed 35.05% and 39.07% reduction in plasma glucose levels respectively compared to HFD fed control (Fig. 3B).

Estimation of lipid parameters:

Consumption of HFD for nine weeks had shown significant (^bP<0.01; ^cP<0.001) increase in the STG, STC, VLDL-c and LDL-c levels and decreased serum HDL-c levels, as compared to NPD fed control. Treatment with MOE (1 and 2%) and orlistat (0.012%) to HFD fed mice exhibited significant (^cP<0.001) reduction in STG, STC, VLDL-c and LDL-c levels and increased serum HDL-c levels, as compared to HFD fed control (Fig. 4and 5). Whereas administration of higher dose of MOE to NPD fed mice failed to reduce levels of STG, STC, VLDL-c and LDL-c levels and increase serum HDL- c , as compared to NPD fed control.

Mice fed with HFD for nine weeks had shown higher level of dyslipidemic markers such as TC/HDL-c and LDL-c/HDL-c ratios as compared to NPD fed control and results were found to be significant (^cP<0.001). Treatment with different doses of MOE and orlistat has significantly (^cP<0.001) reduced markers of dyslipidemia. Whereas there is no significant reduction in markers of dyslipidemia of NPD fed mice treated with higher dose MOE compared to NPD fed control (Fig. 5).

Fig.4. Effect of nine weeks treatment of different doses of MOE and orlistat on [A] Serum TG [B] Serum TC [C] Serum HDL-c of HFD fed mice.(G1) NPD fed control; (G2) NPD+2% MOE; (G3) HFD fed control; (G4) HFD+1% MOE; (G5) HFD+2% MOE; (G6) HFD+0.012% orlistat. Data represent the Mean ± S.E.M. for 6 mice. ^bP<0.01; ^cP<0.001 compared to HFD fed control (one way ANOVA followed by Tukey’s post-test).

Fig.5. Effect of nine weeks treatment of different doses of MOE and orlistat on [A] LDL-c [B] VLDL-c [C] TC/HDL-c ratio [D] LDL-c/HDL-c ratio of HFD fed mice.

(G1) NPD fed control; (G2) NPD+2% MOE; (G3) HFD fed control; (G4) HFD+1% MOE; (G5) HFD+2% MOE; (G6) HFD+0.012% orlistat. Data represent the Mean ± S.E.M. for 6 mice. ^bP<0.01; ^cP<0.001 compared to HFD fed control (one way ANOVA followed by Tukey’s post-test).

Estimation of organ weight and the ratios of weight of liver, heart and kidney to the weight of animal

Consumption of high fat diet for nine weeks exhibited no significant increase in the heart and liver weight and no significant decrease in kidney weight compared to NPD fed control. Treatment with MOE (2%) and orlistat (0.012%) to HFD fed mice showed, significant (^aP<0.05; ^cP<0.001) reduction in liver weight and increase in kidney weight whereas failed to reduce significantly in heart weight compared to HFD fed control. Treatment with lower dose of MOE (1%) has no significant effect on heart, kidney and liver weight compared to HFD fed control. Treatment of NPD fed mice containing MOE 2% also not shown significantly altered weight of heart, kidney and liver compared to NPD fed control (Table 6).

Consumption of high fat diet for nine weeks has not shown significant increase in the heart and liver ratio and no significant decrease in kidney weight compared to NPD fed control. Treatment with higher dose of MOE (2%) and orlistat (0.012%) to HFD fed mice exhibited significant (^aP<0.05; ^cP<0.001) reduction in liver ratio and increase on kidney ratio but no significant reduction in heart ratio compared to HFD fed control. Treatment with MOE 2% to NPD fed mice also not shown significant effect on heart, kidney and liver ratios compared to NPD fed control. The values were summarized in Table 6.

Effect of different doses of MOE and orlistat on rat plasma triglyceride level in Lipid emulsion tolerance test (LETT).

All the rats were administered with 1 ml of lipid emulsion orally and plasma TG level was measured at different time intervals from 0 to 5 h using diagnostic kit. In the treated groups, rats were administered with the fixed dose of MOE/orlistat 30 min prior to administration of lipid emulsion and then TG was measured.

The plasma TG level in to lipid emulsion control group was found to be significantly (^bP<0.01; ^cP<0.001) higher from 2 to 4 h compared normal saline control. Treatment with higher dose of MOE (1000 mg/kg) has significantly (^cP<0.001) reduced Plasma TG from 0.5 to 5 h compared to lipid emulsion control. Whereas lower dose of MOE (500 mg/kg) failed to reduce TG level at initial tested time points. Moreover, these treatments showed significantly reduced plasma TG from 2 to 5 h compared to lipid emulsion control (Fig. 6A).

Integrated area under the TG curve over 5 h (AUC _TG) of lipid emulsion control was significantly (^cP<0.001) higher compared to normal saline control. Treatment with MOE/orlistat produced a significantly (^cP<0.001) reduced AUC _TGcompared to lipid emulsion control.

Further estimation of AUC values indicated that, there is a 50.59% reduction in the plasma TG level of normal saline control group compared to lipid emulsion control group. Treatment of rats with MOE (500 and 1000 mg/kg) and orlistat (45 mg/kg) showed 45.59%, 67.04% and 34.16% significant (^cP<0.001) reduction in plasma TG levels respectively compared to lipid emulsion control (Fig. 6B).

Fig.6. Effect of MOE and orlistat on lipid emulsion tolerance test in rats.

[A] Plasma TG was measured before and after the oral administration of 1 ml lipid emulsion, or in combination with MOE and orlistat. [B] Area Under Curve for TG (AUC _Plasma _TG) values for 0 to 5 h. Data represents the Mean ± S.E.M. for 6 rats ^bP<0.01; ^cP<0.001 compared to lipid emulsion control(one way ANOVA followed by Tukey’s post-test).

DISCUSSION:

Recently, obesity is increasing in developed countries including Europe, United States and Japan. Obesity is closely associated with life-style-related diseases such as hyperlipidemia, hypertension, arteriosclerosis and non-insulin-dependent diabetes mellitus and with increased risk of coronary heart disease¹⁴. Dietary fat is mainly responsible for increasing body weight and adiposity in humans and animals more effectively compared to dietary carbohydrate¹⁰. There are several cellular and molecular targets are available to treat obesity such as:

Affecting fat absorption: lipase inhibitor e.g. Orlistat Reducing food intake:

a. Combined norepinephrine and serotonin re-uptake inhibitor e.g. Sibutramine

b. sympathomimetic amine e.g. Phentermine

c. Selective serotonin re-uptake inhibitor e.g. Fluoxetine

Inhibiting the synthesis and release of appetite stimulating factors such as hypothalamic neuropeptide Y e.g. Leptin¹⁷.

There are a number of reports showing that Moringa oleifera seeds are pre-clinically anti-hypertensive, hypoglycemic and anti-atherosclerotic^18,19. Therefore in the present study we evaluated the effect of MOE in two obesity models such as high-fat diet-induced obesity in mice and lipid emulsion tolerance test in normal rats.

In the present study, we examined effect of MOE on high-fat diet-induced obesity in mice. Obesity was induced by feeding high-fat diet containing 40% fat for nine weeks. We found that the consumption of HFD by mice for nine weeks increased the body weight and PAT weight. The increased body weight in HFD might be due to the consumption of an energy-rich diet of saturated fats (beef tallow) and its deposition in body pads coupled with decreased energy expenditure when compared to NPD fed mice. Treatment with MOE to HFD fed mice reduced the increase in body weight and PAT weight whereas; orlistat was successful only in reducing body weight rather than decreasing PAT weight. The decrease in the body weight may be attributed to the reduction in food and water intake caused by constituents that affect brain centers involved in satiety and hunger or could have inhibited digestive enzymes or decreased bioavailability of nutrient caused by factors like saponins present in plant extract.

At the end of eight weeks of treatment, we performed insulin tolerance test in HFD induced obesity mice model. High-fat diet has been shown to induce insulin resistance by different mechanisms. One possible mechanism for insulin resistance is through Randle or glucose-fatty acid cycle²⁰. Briefly, high triglyceride levels resulting from HFD could lead to increased fatty acid availability and oxidation. The preferential use of increased fatty acids over glucose for oxidation by different tissue could result in the insulin-mediated reduction of hepatic glucose output and reduce the glucose uptake/utilization in skeletal muscle leading to compensatory hyperinsulinemia, a common feature of insulin resistance²¹. Mice treated with MOE and orlistat showed the significant reduction in plasma glucose level over 45 min whereas, in HFD fed mice there is no decrease in the plasma glucose levels.

At the end of nine weeks of treatment the feces was collected from all the six groups and feces TG was measured. We observed that MOE and orlistat might inhibit pancreatic lipase or bind TG to increase fecal excretion. So the feces weight and feces TG was increased in the treated groups compared to HFD fed mice.

The hypertriglyceridemia observed in HFD fed mice may be due to increased absorption and formation of triglycerides in the form of chylomicrons from exogenous fat-rich diet or through combination of increased endogenous production of TG-enriched hepatic VLDL and decreased TG-uptake in peripheral tissues²². Hypercholesterolemia may be attributed to increased absorption of dietary cholesterol from HFD²³. We observed increased level of liver TG and serum lipids such as STG, STC, LDL-c and VLDL-c in HFD fed mice. Further, hyperlipidemia might result either from the inhibition of TG synthesis in liver or increased peripheral clearance of TG by stimulating LPL and/or inhibition of dietary cholesterol absorption from the intestine. The mechanism by which liver and serum lipids are lowered by dietary or other agents is of interest. The calories in excess of the requirement of the normal animal or man are known to be stored in the adipose tissue. The LPL (lipoprotein lipase) and HSL (hormone sensitive lipase) of the adipose tissue responsible for the uptake of triglycerides and mobilization in the fed and starved states respectively and skeletal muscle LPL seem to determine the level of serum triglycerides. A related aspect is the role of substrate cycle between TG and FFA between adipose tissue and liver in determining TG levels in liver, serum and adipose tissue²⁴. TG secretion from liver to serum is reported to be reduced in mice fed high fat diets²⁵. It is of significance that MOE stimulates TG secretion and also elevates skeletal muscle LPL both of which lead to lowering of liver and serum TG²⁶. Another possible mechanism that explains the low serum levels of TG in the MOE/orlistat treated groups was that MOE/orlistat might inhibit pancreatic lipase or bind TG to increase fecal excretion. The administration of MOE and orlistat for nine weeks resulted in a significant reduction in STG, STC, LDL-c, VLDL-c and other dyslipidemic markers, indicating their potent hypolipidemic activity.

In humans and most animal models, the development of obesity leads not only to increased fat depots in classical adipose tissue locations, but also to significant lipid deposits within and around other tissues and organs, a phenomenon known as ectopic fat storage. The possible locations of ectopic fat in key target-organs of cardiovascular control (heart, blood vessels, liver and kidneys) and to propose how ectopic fat storage can play a role in the pathogenesis of cardiovascular diseases associated with obesity. In animals fed a high-fat diet, cardiac fat depots within and around the heart impair both systolic and diastolic functions, and may in the long term promote heart failure. Accumulation of fat around blood vessels (perivascular fat) may affect vascular function in a paracrine manner, as perivascular fat cells secrete vascular relaxing factors, pro-atherogenic cytokines and smooth muscle cell growth factors. Furthermore, high amounts of perivascular fat could mechanically contribute to the increased vascular stiffness seen in obesity. Accumulation of fat in the liver may cause fatty liver and increases liver TG. Finally, renal sinus may limit the outflow of blood and lymph from the kidney, which would alter intra renal physical forces and promote sodium re absorption and arterial hypertension. Taken together, ectopic fat storage in key target-organs of cardiovascular control may impair their functions, contributing to the increased prevalence of cardiovascular diseases in obese subjects²⁷. Feeding high-fat diet for nine weeks considerably resulted in deposition of fat in heart, liver and kidney. Whereas, there is no significant increase in heart weight of HFD fed mice compared to NPD fed control. Furthermore, there is a significant reduction in the liver weight and increase in kidney weight of MOE and orlistat treated groups compared to HFD fed control.

In the second model, lipid emulsion tolerance test there was an increase in the plasma TG of lipid emulsion group after oral administration of lipid emulsion in rats. We examined the effects of MOE and orlistat on plasma TG concentration and found that both reduced the elevation of plasma TG levels at different time intervals. These results suggested that the MOE and orlistat reduces the small intestinal absorption of dietary fat by inhibiting the pancreatic lipase activity.

CONCLUSION:

The present study provides clear evidence that, ethanol extract of Moringa oleifera Lam. seeds were helpful in treating the HFD induced obesity. Anti-obesity actions of ethanol extract of Moringa oleifera Lam. seeds in experimental animals may be partly mediated through delaying the intestinal absorption of dietary fat by inhibiting pancreatic lipase activity. The present study clearly indicated that the extract of Moringa oleifera Lam. seeds exhibited a potent anti-obesity action and supports the traditional usage. Moreover it might help in preventing obesity complications and serve as good adjuvant in the present armamentarium of anti-obesity drugs.

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Received on 25.08.2011

Accepted on 08.09.2011

Research J. Pharmacology and Pharmacodynamics. 3(6): Nov.-Dec., 2011, 318-328

Treatment	Average food intake (g/week)	Wet weight of feces (g)	TG in feces (mg/g of feces)	PAT weight (g)	Liver TG (mg/0.5 g of liver)
NPD fed control	532.78 ± 0.51^c	1.02 ± 0.01^c	264.65 ± 0.33^c	0.28 ± 0.02^b	90.40 ± 9.06^c
NPD + 2% MOE	565.78 ± 0.54^f	0.38 ± 0.00^f	415.39 ± 0.13^f	0.33 ± 0.08	81.42 ± 5.23
HFD fed control	581.62 ± 0.59	0.21 ± 0.00	151.53 ± 0.00	0.98 ± 0.14	135.07 ± 4.86
HFD + 1% MOE	543.01 ± 0.01^c	0.42 ± 0.00^c	406.00 ± 0.02^c	0.94 ± 0.09	96.21 ± 3.50^c
HFD + 2% MOE	551.15 ± 0.05^c	0.48 ± 0.00^c	500.01 ± 0.01^c	0.84 ± 0.06	96.14 ± 5.03^c
HFD + 0.012% orlistat	563.58 ± 0.56^c	0.35 ± 0.00^c	417.26 ± 0.18^c	0.93 ± 0.21	101.84 ± 1.89^b

Treatment	Weight of organ (g/100 g)				Weight of organ Animal weight
Treatment	Heart	Kidney	Liver	Heart	Kidney	Liver
NPD fed control	0.54 ± 0.03	0.70 ± 0.07	4.86 ± 0.33	5.40 ± 0.69	6.96 ± 0.69	48.60 ± 3.60
NPD + 2% MOE	0.65 ± 0.04	0.83 ± 0.07	5.02 ± 0.20	6.46 ± 0.44	8.30 ± 0.71	50.16 ± 2.02
HFD fed control	0.49 ± 0.03	0.56 ± 0.03	6.12 ± 0.40	4.89 ± 0.27	5.57 ± 0.27	61.22 ± 3.97
HFD + 1% MOE	0.51 ± 0.03	0.61 ± 0.01	4.78 ± 0.16	5.14 ± 0.34	6.08 ± 0.12	47.81 ± 1.63
HFD + 2% MOE	0.58 ± 0.03	0.99 ± 0.08^c	4.60 ± 0.53^a	5.79 ± 0.32	9.88 ± 0.78^c	45.97 ± 5.25^a
HFD +0.012% orlistat	0.49 ± 0.05	0.64 ± 0.03	4.57 ± 0.36^a	4.94 ± 0.45	6.37 ± 0.29	45.71 ± 3.59^a