INTRODUCTION:

Obesity is almost invariable in developed countries and all most all people accumulate some fat as they get older. The WHO acknowledged that obesity (BMI>30kg/m²) is worldwide problem which is also affects many developing country. The present obesity epidemic is mainly due to changes in lifestyle behaviour (although genetic may be involved in some individuals). There has been a trebling in the prevalence of obesity in the UK over the last three decades as well as a vast increase in developing countries the growing obesity problem in humans has affected children, adults and older people (Praveen Kumar and Michael Clark, 2012).

Ficus religiosa (Moraceae) is a large perennial tree, glabrous when young, found throughout the plains of India upto 170m altitude in the Himalayas, largely planted as an avenue and roadside tree especially near temples.

Received on 10.04.2015 Modified on 02.05.2015

Research J. Pharm. and Tech. 8(6): June, 2015; Page 679-682

DOI: 10.5958/0974-360X.2015.00107.9

The plants have been used in traditional Indian medicine for various range of ailments. Traditionally the bark is used as an antibacterial, antiprotozoal, antiviral, astringent, antidiarrhoeal, in the treatment of gonorrhea, ulcers, and the leaves used for skin diseases. The leaves reported antivenom activity and regulates the menstrual cycle (Anonymous, 2001; Hemaiswarya et al. ,2009; Khan et al., 2011).

The following medicinal plants have been reported to possess anti-obesity activity: Aegle marmelos, Allium sativum, Aloe vera, Alpina galanga, bauhinia variegate, Camellia sinensis, Cannabis sativa, Carum carvi, Cinnamomum zeylanicum, Cissus quadrangularis, Crocus sativus, Curcuma longa, Cyperus rotundus, Embeliaribes, Ferula asafoetida, Garcinia cambogia, Glycyrrhiza glabra, Gymnema sylvestre, Hibiscus sabdariffa, Mimosa pudica, Momordica charantia, Morinda citrifolia, Nelumbo nucifera, Punica granatum, Stellaria media,, Syzygium aromaticum, Tamarindus indica, Trigonella foenum-graecum and Ziziphus jujube (Marta González-Castejón, 2011)

MATERIALS AND METHODS:

Preparation of ethanolic extract Ficus religiosa:

The shade dried and coarsely powdered leaves of Ficus religiosa was defatted with petroleum ether (60-80˚c) for three days by triple maceration. The defatted marc was extracted with ethanol by triple maceration and filtered. The filtrate was concentrated under reduced pressure to obtain a solid residue which was dark green in colour.

Drugs and chemicals required:

Ethanolic extract of Ficus religiosa, Orlistat (Reshape-Mayer organics, Mumbai), Tris Hcl buffer, tricholoroacetic acid, 5’5 dithiobis (2- nitrobenzoic acid) and n-butanol (AR grade).

Animals:

Male Wistar albino rats, weighing 200-225 g, were employed in the present study. The rats were exposed to 12 h light-dark cycles. The experimental protocol was duly approved by the Institutional Animal Ethics Committee.

Experimental protocol:

Five groups, each comprising six Wistar rats were employed in the present study. The control group of animals was given normal diet and water ad libitum. Except the control group of animals, experimental rats were fed with high fat diet for 28 consecutive days. Extract and the standard drug were administered to the experimental rats from day 8 to 28^th day for 21 consecutive days. Body weight (initial and final), Thoracic circumference, Abdominal circumference were determined for these animals. The blood samples were withdrawn from retro orbital plexus on the 29^th day of the experiment, from the overnight fasted rats for the estimation of the biochemical parameters such as Aspartate transaminase (AST), Alanine transaminase (ALT), Total cholesterol (TC), Triglycerides (TG), High Density Lipoprotein (HDL), Low Density Lipoprotein (LDL), Very Low Density Lipo Protein (VLDL), and glucose. Then, these rats were sacrificed by cervical dislocation under anaesthesia (Thiopental sodium 40mg/kg i.p) as per CPCSEA guidelines. The epididymal fat tissue and peritoneal fat tissue were removed from the experimental rats by means of dissection and weighed. The liver, kidney, heart, epididymal and peritoneal fat were isolated from these animals. These organs were washed with normal saline and blotted with sterile filter paper and the weight of these organs were noted. A portion of the liver tissue was used for the estimation of Thiobarbituric acid reactive substances (TBARS) and Reduced glutathione (GSH).

Group I (Normal control group):

These group of animals were given normal diet and water ad libitum. Standard diet comprises of Corn starch 50 g/kg, Fish flour- 160 g/kg, Bean flour-140 g/kg, Wheat flour - 410 g/kg and vegetable oil-40 g/kg (Agarwal ,1988).

Group II (High fat diet group):

Rats were administered high fat diet for 28 consecutive days (ad libitum). High fat diet comprises of Corn starch 250 g/kg, Fish flour- 160 g/kg, Bean flour-140 g/kg, Wheat flour -130 g/kg and Fat- 320 g/kg. The ethanolic extract of Ficus religiosa and standard drug (Orlistat) were prepared as suspension using 1% CMC.

Group III (EEFR 100 mg/kg treated group):

HFD administered rats were treated with 100 mg/kg; p.o of ethanolic extract of Ficus religiosa from day 8 to 28^th day for 21 days.

Group IV (EEFR 200 mg/kg treated group):

HFD administered rats were treated with 200 mg/kg; p.o of ethanolic extract of Ficus religiosa from day 8 to 28^th day for 21 days.

Group V (Standard Orlistat 10 mg/kg treated group):

HFD administered rats were treated with Orlistat 10 mg/kg; p.o. from day 8 to 28^th day for 21 days.

Biochemical estimation of serum markers:

Commercial assay kits (Span Diagnostics Ltd., Surat, India) were used for the estimation of blood glucose, serum triglycerides (TGs), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), aspartate amino transferase (AST), and alanine amino transferase (ALT) (Parveen Kumar et al., 2014). All the groups of animals were sacrificed on 29^th day by cervical dislocation and the liver was isolated immediately and used for the biochemical estimation. Freshly excised liver homogenate (10 %) was prepared with 0.1 M Tris HCl buffer (P^H-7.4) and the homogenate was kept in ice water for 30 min and centrifuged at 4˚C (2000 g,10 min).The supernatant of homogenate was separated and which was used to estimate following biochemical markers.

Estimation of reduced glutathione:

Equal quantity of liver homogenate was mixed with 10 % tricholoro acetic acid and the mixture was centrifuged to separate proteins. To 0.01 mL of this supernatant, 2 mL of phosphate buffer (PH-8.4). 0.5 mL of 5’5 dithiobis (2- nitrobenzoic acid) and 0.4 mL of distilled water were added. Mixture was vortexed and the absorbance was taken at 415 nm with 15 mins. The concentration of reduced glutathione was expressed as nmol/mg of proteins (Beutler et al.,1963).

Estimation of TBARS

The thiobarbituric acid reactive substance (TBARS) level was estimated as per the standard procedure. To each test tube,0.5 mL of supernatant,0.5 mL normal saline, 1 mL of 20 % tricholoroacetic acid (TCA) and 0.25 mL of TBA reagent (200 mg of thiobarbituric acid in 30 mL distilled water and 30 mL of acetic acid) were added. The test tubes were kept for boiling at 95˚ c for one hour. To each test tube, 3 mL of n-butanol was added and mixed well. These test tubes were centrifuged at 3000 rpm for 10 minutes. The separated butanol layer was collected and read in a spectrophotometer against blank at 535 nm. Concentration of thiobarbituric reactive substance was expressed in terms of malondialdehyde nmol/mg of protein (Okhawaet al., 1979).

Statistical analysis:

All the results were expressed as mean ± standard error of means (SEM graph pad prism v.5.0 software. The body weight, abdominal circumference and thoracic circumference of the rats and serum biochemical parameters and liver tissue homogenate TBARS and GSH were statistically analysed by one way ANOVA followed by Tukey’s multiple range tests. P< 0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION:

TABLE: 1 EFFECT OF EEFR ON BODY WEIGHT OF THE RATS

Groups	Body weight(g)		Weight gain(g)
Groups	Initial weight	Final weight	Weight gain(g)
Control	208.35±3.60	251.83±1.85	43.48
High fat diet(HFD)	219±3.72	366.16±4.81^a	147.16
EFR100	205.3±5.41	314.33±3.95^b	109.03
EFR200	223±4.09	287.83±3.68^b	64.83
Orlistat	215±4.35	276.83±1.60^b	61.83

*mean of six readings ± SEM

Digits in parenthesis indicate dose in mg/kg. Data were expressed as mean ± SEM, n=6 rats per group. a- indicate statistical significance (p<0.05) difference when compared to normal control group. b- indicate statistical significance (p<0.05) difference when compared to HFD.

The weight gain of the normal rat was found to be 43.48 g, whereas the body weight of the HFD induced rats were found to be 147.16 g. Treatment of EEFR at the dose level of 100 and 200mg/kg b.w significantly decreases the body weight(109.09 g and 64.83 g respectively) in a dose dependent manner (p< 0.05). The standard drug orlistat (10mg/kg b.w) also reduce the body weight (61.83 g) significantly (p< 0.05). The results are presented in Table 1.

TABLE: 2 EFFECT OF EEFR ON ABDOMINAL AND THORACIC CIRCUMFERENCE OF RAT

Groups	Abdominal circumference in cms	Thoracic circumference in cms
Control	16.98±0.17	15.6±0.12
High fat diet(HFD)	22.15±0.28^a	17.53±0.15^a
EFR100	20.44±0.15^b	16.9±0.16^b
EFR200	18.6±0.13^b	16.64±0.28^b
Orlistat	17.13±0.41^b	16.26±0.18^b

* mean of three readings ±SEM

Digits in parenthesis indicate dose in mg/kg. Data were expressed as mean ± SEM, n=6 rats per group. a- indicate statistical significance (p<0.05) difference when compared to normal control group. b- indicate statistical significance (p<0.05) difference when compared to HFD.

Treatment of EEFR at the dose level of 100 and 200 mg/kg b.w significantly decreases the abdominal (20.44 cms and 18.6 cms respectively) and thoracic circumference (16.9 cms and 16.64 cms respectively) in a dose dependent manner (p< 0.05). The standard drug orlistat (10 mg/kg b.w) also reduce the abdominal (17.23 cms) and thoracic circumference (16.26 cms) significantly (p< 0.05). The results are presented in table 2.

The increase in organ weight (heart, liver, right and left kidney) and White Adipose Tissue (Epididymal and peritoneal fat) weight were noted with the HFD induced rats. Treatment of EEFR 100,200 and orlistat significantly decreases the organ weights as well as WAT weight. The results are presented in Table 3.

TABLE: 3 EFFECT OF EEFR ON ORGAN WEIGHT AND WAT

Groups	Organ weight (g)				WAT(g)
Groups	Heart	Liver	Right kidney	Left kidney	Epididymal	Peritoneal
Control	0.695±0.006	6.295±0.171	0.591±0.03	0.63±0.03	2.7±0.14	1.45±0.107
High fat diet (HFD)	1.231±0.07^a	11.18±0.259^a	1.57±0.01^a	1.69±0.02^a	5.91±0.12^a	3.53±0.199^a
EFR100	0.906±0.014^b	9.31±0.102^b	0.835±0.05^b	0.818±0.01^b	4.83±0.04^b	2.75±0.04^b
EFR200	0.838±0.01^b	7.65±0.201^b	0.736±0.01^b	0.923±0.02^b	3.67±0.08^b	2.39±0.06^b
Orlistat	0.765±0.008^b	6.78±0.07^b	0.67±0.02^b	0.751±0.04^b	3.23±0.05^b	1.91±0.03^b

* mean of three readings ±SEM

TABLE: 4 EFFECT OF EEFR ON SERUM BIOCHEMICAL PARAMETERS

Groups

(mg/dL)

HDL

(mg/dL)

LDL

(mg/dL)

VLDL

(mg/dL)

Glucose

(mg/dL)

AST

(IU/L)

ALT

(IU/L)

Control

90±

1.591

62.2±

2.8

34.06±

0.43

55.56±

1.03

14.16±

0.35

106.2±

1.46

52.87±

0.56

23.17±

0.48

High fat diet (HFD)

153±

1.61^a

157.2±

1.64^a

17.87±

0.14^a

99.82±

1.30^a

25.1±

1.20^a

151.10±

1.93^a

98.03±

0.78^a

64.23±

0.36^a

Orlistat

111.66±

2.37^b

73.11±

0.46^b

32.4±

0.36^b

64.18±

1.62^b

17.23±

0.15^b

123.59±

1.03^b

58.58±

0.90^b

29.42±

0.41^b

EFR100

124.33±

3.54^b

102.6±

2.90^b

24.99±

0.40^b

82.77±

2.86^b

21.84±

0.23^b

138.09±

1.36^b

73.45±

0.74^b

44.71±

0.27^b

EFR200

114±

2.40^b

85±

1.22^b

30.23±

0.30^b

73.99±

1.21^b

18.33±

0.44^b

129.99±

0.74^b

62.59±

0.69^b

34.42±

0.62^b

* mean of three readings ±SEM

TABLE: 5 EFFECT OF EEFR ON HEPATIC TBARS AND GSH

Groups	TBARS (nmole/mg)	GSH (nmole/mg)
Control	111.73±1.26	105.53±2.14
High fat diet(HFD)	371.37±5.99^a	80.06±1.11^a
EFR100	277.26±4.12^b	87.27±1.23^b
EFR200	209.52±2.42^b	92.2±1.59^b
Orlistat	180.59±3.16^b	98.18±1.43^b

*mean of three readings ±SEM

The elevated levels of TC, TG, HDL, LDL, VLDL, glucose, AST, ALT and TBARS and decrease in the level of GSH were recorded in the HFD induced rats. Treatment of EEFR 100 and 200 mg/kg b.w, p.o significantly attenuated the elevated serum parameters and augmented GSH level in a dose dependent manner. The standard drug orlistat also significantly altered and normalizes the above mentioned biochemical parameters. The results are presented in Table 4 and 5.

CONCLUSION:

Treatment of EEFR at the dose level of 100 and 200mg/kg b.w significantly decreases the body weight, organ weight(heart, liver and kidney), thoracic circumference and abdominal circumference. It significantly attenuated the elevated serum parameters such as TBARS, TG, TC, AST, ALT, glucose, LDL and VLDL and augmented GSH and HDL level in a dose dependent manner. The reduction of TBARS and elevation of GSH by the extract treatment revealed the inhibition of oxidative stress by virtue of its antioxidant activity (Gupta, 2011; Kirana, 2009)

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