INTRODUCTION:

Obesity, a condition in which an abnormally large amount of fat is stored in the adipose tissue, resulting in an increase in body weight, is one of the major public health problems in developed and developing countries. In general, it is accepted that obesity results from disequilibrium between energy intake and expenditure¹.

Obesity may be defined as excess weight with a body mass index (BMI) greater than 30kg/m². Morbid obesity is more serious condition, which is correlated with a weight 170% greater than the ideal weight, or overweight or else a BMI greater than 40kg/m².²BMI is 20-25 (Health Person), BMI is 25-30 (Over Weight), BMI is ≥ 35 (OBESE)³.

Most obesity can be effectively treated for compliant patients, using a focused lifestyle intervention based on a whole-foods, low-glycemic- load, phytonutrient-rich diet combined with exercise and stress management, there are patients who do not respond predictably to normally successful interventions⁴. In addition to the key role in the screening of novel compounds for effects on food intake and/or body weight, animal models have utility in the identification of the physiological and genetic basis of obesity, which can result in the discovery and validation of new therapeutic targets [e.g. through the generation and characterization of transgenic animals]⁵. Attempts have been made to reduce body weight with such pharmacological intervention that possesses minimal side effects.

Plants have been used as traditional natural medicines for healing many diseases. In particular, various oriental medicinal plants are reported to have biological activity⁶. There are many such plants considered as medicinally useful because of array of bioactive compounds. One such plant Citrus maxima (Pomelo) belong to family Ruteceae known to contain many bioactive constituents which claims to posses anti-obesity activity. Citrus maxima also known to possess anti-inflammatory, anti-tumor, anti-diabetic, anti-microbial, anti-depressant, hypoglycemic and hypocholesterolemic activities⁷.Since no official reports are available on anti-obesity activity of Citrus maxima leaves. Thus present study was carried out with the aim to evaluate anti-obesity activity of ethanolic extract of Citrus maxima leaves on cafeteria diet and drug induced obese rats.

MATERIALS AND METHODS

All chemicals and reagents will be of pure analytical grade and obtained from local suppliers. The leaves were washed with double distilled water, shade dried and powered. 500 g of the powdered material of C. maxima was extracted with 80% ethanol for 72h in a cold percolation method. After that the solvent was evaporated under pressure to get the crude extract i.e. Ethanolic Extract of Citrus Maxima (EECM)⁸. Wistar Albino rats (150 to 200 g) and albino mice (20-25 g) of either sex were used for this study. They were maintained under standard conditions (temperature 22 ± 2°C, relative humidity 60±5% and 12 h light/dark cycle) and had free access to standard pellet diet and water ad libitum. The animals were housed in sanitized polypropylene cages containing sterile paddy husk as bedding. The Institutional Animal Ethics Committee reviewed and approved the experimental protocol. All the procedures were performed in accordance with Institutional Animal ethics committee constituted as per the direction of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

General Procedure:

Acute toxicity evaluation:

Acute toxicity study of the Citrus Maxima leaves extract were performed as per the OECD guidelines 425 at a limit dose of 2000 mg/kg or 5000 mg/kg. The doses were administered by oral route in albino mice (20-25g). Animals were observed individually at least once during the first 30 minutes after dosing, periodically during the first 24 hours (with special attention given during the first 4 hours), and daily thereafter, for total 14 days for sign of toxicity and/or mortality⁹ . As per OECD guidelines for toxicity studies, 1/10^th& 1/5^thLD₅₀ cut off value of the extract were selected as screening dose.

Cafetaria Diet Induced Obesity in Rats

Experimental Design:

The Wistar albino rats (150-200g) of either sex were randomly distributed into 5 groups (n=6) and were treated as given below.

Group I: Normal control

Group II: Obese control (Cafeteria diet)

Group III: Standard control (Cafeteria diet + Orlistat 10mg/kg, p.o. daily)

Group IV: Treatment group (Cafeteria diet + EECM low dose, p.o.daily )

Group V: Treatment group (Cafeteria diet + EECM high dose, p.o. daily)

The Cafeteria diet consist of three diets

a) Condensed milk (8g) + bread (8g)

b) Chocolate (3g) + biscuits (6g) + dried coconut (6g)

c) Cheese (8g) + boiled potato (10g)

These diet were fed along with normal diet in rotation for a total of 6 weeks¹⁰.

Treatment:

All the animals except in Group I, obesity were induced by feeding cafeteria diet along with normal diet for six weeks and continued along with the treatment for next four weeks. All the treatments were given post orally daily once. Before and after the treatment the animals were fasted for 2 h to improve the absorption rate.

Olanzapine Induced Obesity in Rats

Experimental Design:

The Wistar albino rats (150-200g) of female rats were selected and the obesity was induced by drug Olanzapine (2mg/kg) for 28 days¹¹.

The animals were divided into following groups containing 6 animals each.

Group I: Normal control

Group II: Obese control (Olanzapine 2mg/kg )

Group III: Standard control (Olanzapine 2mg/kg + Orlistat 10mg/kg, p.o. daily)

Group IV: Treatment group (Olanzapine 2mg/kg + EECM low dose, p.o daily )

Group V: Treatment group (Olanzapinie 2mg/kg + EECM high dose, p.o daily)

Treatment:

All the treatments were carried out for 28 days. Before and after the treatment the animals were fasted for 2 h to improve the absorption rate.

In both the models, the weekly body weight analysis was carried out for entire duration of the study. At end of the study, the blood samples were collected from all the groups of the animals through the orbital sinus and the lipid proﬁle was estimated.

Evaluation:

Body Weight and Food Intake:

The body weight (g) was recorded on day one and then weekly using a weighing balance. In addition to this, the daily food intake for each group was measured.

Body Temperature:

The body temperature was noted using a rectal thermometer before administration of the drug and after 30 and 60 min of drug administration, with a contact time of 1 minute weekly once.

Biochemical Estimations:

After the completion of the experiment, all the animals were anesthetized and blood samples were collected by carotid bleeding separately into sterilized dry centrifugation tubes and allowed to stand for 30 minutes at 20–25°C. The clear serum was separated at 2500rpm for 10min using a centrifuge. The levels of serum glucose, total TC, TG, HDL-C, LDL-C, VLDL-C with commercial kits.

Estimation of liver weight and liver triglyceride content:

After completion of the experiment, animals were sacrificed with an overdose of diethyl ether. The livers were quickly removed, weighed and used for estimation of triglyceride¹².

Statistical analysis:

The Statistical analysis will be carried out using analysis variation (ANOVA) test.

RESULTS:

Table 1: Percentage yield of crude extract of C. maxima leaves.

Solvent	Colour and consistency	Method	Percentage yield
Ethanolic	Dark green and waxy	Continuous hot percolation	15.93%

Preliminary Phytochemical Screening:

Table 2: Preliminary phytochemical screening of ethanolic extract of Citrus maxima leaves.

Sl. No.	Test	Result
1.	Alkaloids	+ve
2.	Carbohydrates	+ve
3.	Flavonoids	+ve
4.	Glycosides	+ve
5.	Saponins	+ve
6.	Steroids	-ve
7.	Tannins	+ve
8.	Proteins	-ve

Determination of Antiobesity Activity:

1. Cafeteria Diet Induced Obesity:

1.1 Effect of Citrus maxima on change in body weight and food intake:

There was significant increase in the body weight in obese control group compared to normal control group. Animal were treated with orlistat and citrus maxima both low dose (200mg/kg) and high dose (400mg/kg) showed significant reduced body weight compared to obese control group.

1.2 Effect of Citrus maxima on body temperature:

The body temperature was noted using a rectal thermometer before drug administration and after 30 min, and 60 min of drug administration respectively, with a contact time of 1 minute weekly once and compared.

There was decrease in the body temperature of rats with obese control when compared to normal control group. Animal was treated with orlistat and Citrus maxima both low dose (200mg/kg) and high dose (400mg/kg) showed increased body temperature compared to cafeteria diet induced obesity in rats.

1.3 Effect of Citrus maxima on serum lipid profile on cafeteria diet induced obesity

Serum lipid profile (TC, TG, LDL-C, VLDL-C, SGOT, SGPT) showed significant increase in the level of serum of cafeteria induced rats when compared to normal control group. Treated animals with EECM showed significant decrease in serum lipid level compared to obese control. On the other hand the serum HDL cholesterol level significantly increased in treated groups as compared to obese control group. Significant increase in serum HDL cholesterol level was seen

Table 3: Effect of C. maxima extract on Body weight and food intake in cafeteria diet induced obesity.

Parameters	Normal control	Obese control	Standard Orlistat	Low dose 200 mg/kg	High dose 400 mg/kg
Initial weight (g)	160.50±1.25	162.36±0.98	160.50±0.85	160.75±68	162.25±1.13
Final weight (g)	173.7±1.174	243.3±1.304***	179.5±0.62***	207.5±0.67***	186.2±0.65***
Food intake per week	140.2±0.98	163.4±1.58***	157.7±1.23**	160.25±0.98*	160.70±1.25*

All the values are Mean± SEM, n=6 ns P>0.05, *P<0.05, **P<0.01, ***P<0.001 One- way ANOVA followed by Dunnette’s test compared to p ^#toxic control

Table 4: Effect of C. maxima extract on Body temperature in cafeteria diet induced obesity.

Parameters	Normal control	Obese control	Standard Orlistat	Low dose 200mg/kg	High dose 400 mg/kg
Initial temp. before drug administration	36.10±0.03	36.10±0.04	36.10±0.07	36.10±0.09	36.10±0.07
30 min after drug administration	36.11±0.04	36.20±0.07	36.43±0.10	36.37±0.08	36.42±0.12
60 min after drug administration	36.10±0.03	36.20±0.07	36.44±0.08	36.37±0.07	36.40±0.06

All the values are Mean± SEM, n=6 ns P>0.05, *P<0.05, **P<0.01, ***P<0.001 One- way ANOVA followed by Dunnette’s test compared to p # toxic control.

Table 5: Antiobesity effect of C. maxima extract on serum parameters in cafeteria diet induced obesity in rats.

Parameters	Normal	Obese control	Standard	Low dose	High dose
Treatment	Saline	Cafeteria diet	Orlistat(10mg/kg)	Drug (200mg/kg)	Drug (400mg/kg)
TC	68.0±0.22	88.75±0.32**	69.84±0.14***	79.76±0.97***	75.77±0.23***
TG	82.54±0.56	130.0±0.47***	93.38±0.51***	104.3±0.41***	98.05±0.12***
HDL	44.49±0.29	35.11±0.32***	44.96±0.25***	40.68±0.52***	43.22±0.25***
LDL	51.71±0.29	74.71±0.34***	49.57±0.48***	54.31±0.48***	51.75±0.30***
VLDL	16.51±0.11	26.01±0.09***	18.58±0.15***	20.87±0.09***	19.58±0.04***
SGOT	26.13±0.27	50.19±0.20***	31.23±0.20***	43.12±0.11***	34.83±0.21***
SGPT	28.12±0.61	56.91±0.20***	34.44±0.16***	40.88±0.17***	35.88±0.19***

All the values are Mean± SEM, n=6 ns P>0.05, *P<0.05, **P<0.01, ***P<0.001 One- way ANOVA followed by Dunnette’s test compared to p ^#toxic control.

1.4 Effect on serum Glucose level:

Table 6: Effect of C. maxima extract on Glucose level in cafeteria diet induced.

Parameters	Normal	Obese control	Standard	Low dose	High dose
Treatment	Saline	Cafeteria diet	Orlistat (10mg/kg)	Drug (200mg/kg)	Drug (400mg/kg)
Glucose level	64.73±0.51	108.40±0.48***	97.04±0.33***	101.30±0.53***	98.21±0.29***
All the values are Mean± SEM, n=6 ns P>0.05, P<0.05, P<0.01, **P<0.001 One- way ANOVA followed by Dunnette’s test compared to p ^#toxic control.

Table 10: Antiobesity effect of C. maxima extract on Serum parameters in Olanzapine induced.

Parameters	Normal control	Obese control	Standard	Low dose	High dose
Treatment	Saline	Olanzapine	Orlistat(10mg/kg)	Drug (200 mg/kg)	Drug (400 mg/kg)
TC	67.96±0.27	98.32±1.78***	70.77±0.61***	88.48±1.28*	86.08±1.004**
TG	81.27±09	128.5±1.04***	110.3±0.47***	125.4±0.90*	114.9±1.42***
HDL	43.04±0.32	33.87±0.31^ns	42.50±0.32***	37.32±0.09**	41.67±0.38*
LDL	50.17±0.47	63.44±0.17***	53.64±0.08***	61.42±0.57**	56.80±0.33***
VLDL	16.34±0.15	23.90±0.07***	18.25±0.10***	23.03±0.08***	21.83±0.28***
SGOT	28.32±0.25	50.87±0.23***	35.61±0.18***	43.02±0.18***	39.64±0.15***
SGPT	31.60±0.27	150.5±0.44***	72.07±0.61***	112.9±0.30***	98.73±0.20***

All the values are Mean± SEM, n=6 ns P>0.05, *P<0.05, **P<0.01, ***P<0.001 One- way ANOVA followed by Dunnette’s test compared to p ^#toxic control.

2.4 Effect on serum Glucose level:

Table 11: Effect of C. maxima extract on Glucose level in olanzapine induced obesity.

Parameters	Normal	Obese control	Standard	Low dose	High dose
Treatment	Saline	Olanzapine	Orlistat (10mg/kg)	Drug (200mg/kg)	Drug (400mg/kg)
Glucose level	71.52±0.34	94.78±0.22***	80.21±0.25***	90.13±0.34***	85.59±0.48***

All the values are Mean± SEM, n=6 ns P>0.05, *P<0.05, **P<0.01, ***P<0.001 One- way ANOVA followed by Dunnette’s test compared to p ^#toxic control.

2.5 Effect of C. maxima extract on Liver organ weight and Liver TG

Table 12: Effect on Liver organ weight and Liver TG level .

Parameter	Normal Control	Obese Control	Standard	Low dose	High dose
Weight (g)	5.25±0.05	8.50±0.06***	6.12±0.07***	7.49±0.03***	6.65±0.05***
Liver TG level	87.60±0.46	153.1±0.32***	103.3±0.45***	128.9±0.32***	111.0±0.41***
All the values are Mean± SEM, n=6 ns P>0.05, P<0.05, P<0.01, **P<0.001 One- way ANOVA followed by Dunnette’s test compared to p ^#toxic control.

DISCUSSION:

Obesity is characterized by increased adipose tissue mass that results from both increased fat cell number and increased fat cell size. This result demonstrated that the rats in the cafeteria diet (CD) and olanzapine induced both causes gain in weight throughout the experimental period, when compared to the initial body weight than the rats in the normal control group. There was a significant increase of food intake in both models that was improved significantly in all treated groups. Effect of orlistat (standard) alone on weight loss and other serum lipid parameter was more potent than the two other treated groups with EECM. The effect on lipid profile was found to be in a dose dependent manner; that is, EECM at a dose of 400 mg/kg shows a better effect in comparison to 200mg/kg. The higher values of serum lipid profile (TG, TC, LDL, VLDL, SGOT, SGPT) were observed in obese group when compared to the normal group, that was decreased significantly in EECM treated group. The effect of EECM caused to increase the level of HDL-cholesterol was also significant. The effect of EECM on liver weight and liver TG level was also seen

to be significant, which showed decrease in weight compared to obese control. The results showed that obesity causes elevation in body weight and reduces lipid metabolism as clearly seen by the marked elevation of liver enzymes and lipid level. However, EECM reverses all the parameters thus suggesting its weight reducing potential.

The above results indicate that the high dose (400 mg/kg) of Citrus maxima extract showed more significant effect in reducing obesity compared to the low dose (200 mg/kg) and showed relatively same effect as that of standard orlistat. Thus, from the present study it can be concluded that the ethanolic extract of Citrus maxima leaves is beneficial to the weight management. Further, studies are required in order to determine the active principles of this plant, followed by identification of the mechanistic approach of EECM that helps in weight management.

ACKNOWLEDGEMENTS:

The authors are thankful to the authorities of A Shama Rao Foundation Mangalore, Karnataka, India for the facilities.

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Received on 14.05.2016 Modified on 28.05.2016

Research J. Pharm. and Tech. 2016; 9(7):907-912.

DOI: 10.5958/0974-360X.2016.00173.6

Parameter	Normal Control	Obese Control	Standard	Low dose	High dose
Weight (g)	5.73±0.088	8.845±0.98***	6.158±0.89***	7.573±0.78***	6.95±1.75***
Liver TG level	87.63±0.15	142.1±0.71***	97.40±0.43***	108.4±1.67**	103.10±0.32***

Parameters	Normal control	Obese control	Standard	Low dose	High dose
Intial weight (g)	159.9±0.61	161.1±0.36	161.2±0.48	160.9±0.24	160.3±0.60
Final weight (g)	173.9±0.24	231.1±0.84***	208.7±0.65***	222.3±0.49***	217.4±0.49***
Food in taken per week	140.5±0.48	172.5±0.27***	165.3±0.77***	162.7±1.2**	163.5±0.91*

Parameters	Normal control	Obese control	Standard Orlistat	Low dose 200mg/kg	High dose 400mg/kg
Initial temp. before drug administration	36.8±0.04	36.5±0.07	36.7±0.06	36.5±0.07	36.7±0.04
30 min after drug administration	37.0±0.07	37.4±0.12	38.0±0.08	37.6±0.03	37.8±0.09
60 min after drug administration	37.2±0.03	37.6±0.08	38.6±0.07	37.9±0.08	38.3±0.05