Antiobesity activity of Zingiber officinale

Context: Zingiber officinale Roscoe (Zingiberaceae) rhizome, known commonly as ginger is extensively used in Indian traditional system of medicine for treatment of various disorders. The ethanolic Z. officinale extract is reported to have various activity such as antidiabetic, antihyperlipidemic and antioxidant activity in experimental animals. Objective: To evaluate anti-obesity effect of aqueous Z. officinale extract in murine model of high fat diet (HFD)induced obesity. Materials and Methods: Male Wistar rats fed with HFD (20 g/day/rat, p.o) for a period of 42 days were used to induce obesity. Aqueous Z. officinale extract (20 mg/kg b.w.) administered orally to HFD fed rats from day 8 to 50 days for a period of 42 days. Body weight gain, serum lipids, insulin and leptin parameters were measured. Results: Oral feeding of the aqueous Z. officinale extract (20 mg/kg) to HFD-induced obese rats for a period of 42 days resulted in significant reduction in body weight gain, insulin, leptin, lipids as compared to rats fed HFD alone. Further, the extract also showed significant increase in high density lipoprotein (HDL-C) levels. Discussion and Conclusion: These results show that aqueous Z. officinale extract possess significant anti-obesity potential.


INTRODUCTION
Obesity generally described as a condition of abnormal body weight resulting from an accumulation of extra adipose tissue, basically in response to a state of positive energy balance that occurs when intake exceeds energy expenditure.It is a growing global health problem in the present era.2] Obesity is a disorder not a disease, but, it has been recognized as a chronic disease by National Institute of health consensus conference in 1985 due to the increasing prevalence and associated risks. 2 This situation currently prevailing widely, anti-obesity drugs expanding rapidly in the market, but drugs are sparse in relation to need.Hence, novel anti-obesity drug is required to handle the problem.Plants and natural products are long been recognized as an important sources for the therapeutically effective medicines.Approximately 80% of the world's population relies on plants for the natural products and medication.Of the 520 new drugs approved between 1983 and 1994, 39% were of natural origin and 60-80% of antibacterial and anticancer drugs were derived from natural products.Infect several Indian medicinal plants or extracts have been used as anti-obesity plant. 3.officinale, commonly known as Ginger, (family Zingiberaceae).Z. officinale rhizome contains 1-4% essential oil and an oleoresin.The essential oil varies as a function of geographical origin, but the chief constituent is sesquiterpene hydrocarbons.These compounds include (-)-zingiberene, (+)-ar-curcumene, (-)-β-sesquiphellandrene, and β-bisabolene.][6] However, aqueous extract of Z. officinale has not been investigated for its anti-obesity potential so far.Hence, this study was carried out to test the hypothesis that aqueous extract of Z. officinale may have anti-obesity activity in high fat diet (HFD)-induced obesity in Wistar rats.

Preparation of aqueous Z. officinale extract
All plant materials were washed; shade dried and powdered, then extracted with water in a soxhlet apparatus for 72 hours.The solvent was removed under reduced pressure to give a dry extract, 5% yield w/w with respect to the crude material and stored at -20°C till experiment was not started.The weighed amount of aqueous extract was suspended in 1% gum acacia in normal saline for pharmacological activity.This extract was used for phytochemical analysis for standardization.

Macroscopical studies
The material of Z. officinale were subjected to macroscopical studies which comprised of organoleptic characteristics of the drug viz., size, colour, odour, taste, smell, texture, fracture etc.

Microscopical studies
Qualitative microscopic evaluation was carried out by taking powder.5 g of the drug sample was taken, powdered.The powdered material was taken on an 85 mesh sieve and allowed in slow running water for washing away the minerals.The materials were cleared in chloral hydrate, wash with distilled water and mounted in glycerin, then observed characters. 7,8

Animals and Diets
The female Wistar rats (100-150 g) were procured from central animal house facility of Hamdard University, New Delhi, India.The animals were housed in standard polypropylene cages and maintained under controlled room temperature (22 ± 2°C) and humidity (55 ± 5%) with 12 h light and 12 h dark cycle.All the rats in Normal group were provided with commercially available rodent chow diet (Amrut rat feed, Nav Maharastra Chakan Oil Mills Ltd, Delhi, India) and tap water ad libitum.The guidelines of committee for the purpose of control and supervision of experiments on animals (CPCSEA), Government of India were followed and prior permission was sought from the Institutional Animal Ethics Committee, Jamia Hamdard, New Delhi for conducting the study.

HFD-induced obesity
Rats were fed a HFD (20 g/rat/day) in a pellet form and water ad libitum for the period of 6 weeks.HFD was purchased from National Centre for Laboratory Animal Science (NCLAS), National Institute of Nutrition (NIN), Hyderabad, India.Composition of HFD is shown in (Table 1).

Experimental Design
In this study, a total of 50 rats were used and divided into five groups of 6 rats each.Group 0020I: normal healthy control rats fed with rodent chow diet and administered 1% gum acacia (1 ml/kg, body weight (bw)) for the period of 42 days (NC); Group II: obesity control rats fed with HFD (20g/rat/day) for the period of 42 days (HFD); Group III: rats fed with HFD (20g/rat/day) for the period of 42 days , treated with aqueous Z. officinale extract (20 mg/kg, bw) for period of 42 days (HFD+ZO); Group IV: rats fed with HFD (20g/rat/day) for the period of 42 days, treated with Orlistate (5 mg/kg, bw) for period of 42 days (HFD+ ORL); Group V: rats fed with rodent chow diet for the period of 42 days + treated with aqueous Z. officinale extract (20 mg/kg, bw) for period of 42 days (ZO per se).All the drugs were administered orally via a standard orogastric cannula (Table 2).Food and water intakes were measured daily for the period of 42 days at the same time.Food intake and water intake were measured on per cage basis (6 rats per cage) and the average food and water consumed were calculated.At the end of the experimental period (on 43 th day), the animals were anesthetized with ether, following overnight fasting.Blood was drawn by retro-orbital method into a tube and the serum was obtained by centrifugation.After collection of blood, rats were sacrificed; heart and perirenal, epididymal, mesenteric fat pads were excised immediately, rinsed with phosphate buffer saline and weighed.The serum and heart samples were stored at -70° C until analysis.

Anthropometric measurements
The body weights were determined once a week.Body mass index (BMI) was calculated from formula: BMI = Body weight (g)/ length 2 (cm 2 )

Biochemical serum analyses
Serum glucose, triglyceride (TG) and total cholesterol (TC) levels were determined by enzymatic methods using commercial assay kits (SPAN Diagnostics Ltd , Surat, India) according to the manufacturer's protocols.Serum High-density lipoprotein cholesterol (HDL-C) levels were determined using HDL Cholesterol Test Kit (Reckon Diagnostics P. Ltd, Baroda, India).Serum leptin and insulin levels were measured by immunoassays using a commercially available ELISA assay kit from Ray Biotech, Inc (Norcrcoss, US) and Crystal Chem Inc (Downers Grove, IL 60515, USA) respectively.Low-density lipoprotein cholesterol (LDL-C) levels were calculated using Friedewald formula. 9Serum very low density lipoprotein cholesterol fraction (VLDL-C) concentration was calculated by deduction of the sum of HDL-C and LDL-C concentrations from that of TC.

Statistical analysis
Data are expressed as the mean ± standard error of the mean (SEM).
The statistical significance of difference between the mean values for the treatment groups was analysed by ANOVA (analysis of variance) followed by Dunnett's t-test using Graph pad InStat® version 3.06 (Graph Pad Software, San Diego, CA, USA).Values of p < 0.05 were considered significant.

Macroscopical Studies
The Z. officinale are light brown colored irregular shaped rhizome, odour pleasant, Pungent taste and rough textured shown in (Figure .1).The organoleptic evaluations of Z. officinale were showed in (Table 3).

Microscopic study
For microscopic study, 5 g of the drug sample was taken, powdered.The powdered material was taken on a 85 mesh sieve and allowed in slow running water for washing away the minerals.The materials were cleared in chloral hydrate, wash with distilled water and mounted in glycerin, then observed charcters 8 (Figure 2).

Effect on Food and Water intake
Negligible (P>0.05)change in food and water intake was observed in HFD fed group as compared to the NC group.Administration of aqueous Z. officinale extract (20 mg/kg) along with HFD (HFD + ZO group) did not produce significant change in the food and water intake as compared to HFD group (Table 4).

Effect on anthropometric Parameters
In the HFD group body weight gain of rats was greater than the values for the NC.HFD supplemented with 20 mg/kg aqueous Z. officinale extract reduced body weight gain of the rats.However, there was no signi ficant difference in body weight gain among the NC and ZO per se treated groups (Table 4).

Effect on organ weight and fat pad analysis
No significant changes in weights of heart, kidney, liver and spleen in drug treated albino rats as compared to control and high fat diet fed group except weight of kidney was reduced significantly (P<0.05).However, drug treatment has variably restricted increase in fat pad weights compared to high fat diet group significantly (P<0.01)(Table 5 & 6 ).

Effect on serum lipid levels
Serum TG levels were significantly higher in HFD fed group compared to that of NC group, HFD+ ZO group (p<0.01).Levels of serum TC, LDL-C, VLDL-C were similar to tendency of serum triglyceride level (p < 0.01).However, TC, LDL-C and HDL-C were significantly (p < 0.05) affected in HFD+ ZO treated group Serum levels of HDL-C were higher in HFD+ ORL treated group compared to HFD fed and NC group, significant difference was recognized (p<0.001)(Table 7).

Effect on serum insulin, lipase and leptin levels
It was observed after 42 days of treatment, HFD + ZO group showed serum lipase levels in HFD fed group was significantly higher than that of normal control but significant reduction observed in HFD + ZO group (p < 0.05).Result of HFD + ORL group, HFD + ZO group were mentioned in Table 8.

Histopathological studies
Results reveal that drug significantly reduced the fat accumulation in liver, heat and kidney (Figure 3, 4 & 5 respectively).

DISCUSSION
Present study was carried out to investigate the anti-obesity effects of aqueous Z. officinale extract in HFD-induced obesity in Wistar rats.Obesity, which affects up to 30% of the adult population in developed countries.Obesity is associated with serious mortalities including high incidence of type 2 diabetes, hyperlipidemia, hypercholesterolemia, cardiovascular disease and many more.HFD is thought to be one of the main factors for development of obesity.[12][13] In the present study, we found that the body weight was markedly reduced in the animal groups treated by Z. officinale to vehicle-treated control.The inhibition of body weight gain did not depend upon a decreased food or energy intake, because there was no significant correlation in diet intake between the control and experimental groups, but was caused by preventing and/or delaying of fat absorption.Similarly, we found that the feeding of Z. officinale for 6 weeks suppressed the increases in organ weights of the heart kidney, liver , spleen and adipose tissue weights of mesentric , perirenal and uterine fat pad weights induced by a HFD.On the other hand, it is reported that feeding with a high fat diet in wistar rats leads to increase in weight of body organs such as liver, heart, spleen and both kidneys.It is also reported that high fat diet induces substantial increase in deposition of fat in the mesenteric, perirenal and uterine region in wistar rats.The present study results showed that compared to that of NC, Serum TG, TC, LDL-C, VLDL-C levels were significantly elevated in HFD fed rats (HFD group) decreased in HFD+ ZO treated group more than that of HFD fed group; indicating potential lipid lowering activity of the test drug.Among various fat deposits, visceral fat mass correlated to insulin resistance in animal model. 14Individuals with visceral by obesity have impaired insulin action, 15 and prevents insulin resistance by removing visceral fat mass 16 From adipose tissue, two groups of inflammatory proteins are produced (i) IL-6 and TNF-α [inflammatory mediators], (ii) leptin, adiponectin and resistin [adipocytokines]. 17Human obesity is characterized by variable degrees of hyperleptinemia and leptin resistance. 18The result of the present study indicates insulin sensitivity improved in rat fed with the drug.The drug induced melioration of insulin resistance which is supported by down regulation of leptin.Thus, visceral fat accumulation and improved insulin resistance in rat model with HFD induced obesity may possibly be through down regulation of leptin, TNF-α, SREBP1γ, and PPARγ2 gene expression.Uygun et al reported that leptin might contribute to hepatic steatosis by promoting insulin resistance and also by altering insulin signaling in hepatocytes, which consequently promote increased intracellular fatty acid. 19Hence, drug treatment to rats with HFD prevents increase of these levels due to decrease of the body fat content.The Z. officinale administration also showed significant activity against lipase activity.Conclusively, observed reduction in body weight gain, serum lipids, insulin, leptin levels suggests that aqueous extract of Z. officinale possess significant anti-obesity potential.

ABBREVIATIONS USED
Dried Z. officinale fruits were purchased from Local market Kharibaoli, Delhi and authenticated by Dr. H. B. Singh, expert taxonomist, National Institute of Science Communication and Information Resources (NISCAIR), New Delhi, India.

Figure 3 :
Figure 3: Changes observed in liver of different groups.

Figure 4 :
Figure 4: Changes observed in heart of different groups

Figure 5 :
Figure 5: Changes observed in kidney of different groups

Table 6 : Effect of aqueous Z. officinale extract (20 mg/kg) on Fat pad weight profile in albino rats after 42 days of treatment Fat pad weights (g/100g of animal weight)
All values were expressed as Mean ± SEM, (n=6); ##P<0.01,###P<0.001,#P<0.05 as compared to the NC group; one way ANOVA followed by Dunnet's t-test.