2009 The Authors Doi: 10.1111/j.1742-7843.2008.00362.x Journal compilation 2009 Nordic Pharmacological Society.

. Basic & Clinical Pharmacology & Toxicology, 104, 366373

Blackwell Publishing Ltd

Protective Effects of Ethanolic Extract of Zingiber ofcinale Rhizome on the Development of Metabolic Syndrome in High-Fat Diet-Fed Rats
Srinivas Nammi1, Satyanarayana Sreemantula2 and Basil D. Roufogalis1
2 1 Herbal Medicines Research and Education Centre, Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia, and Department of Pharmacology, College of Pharmaceutical Sciences, Andhra University, Visakhapatnam 530003, Andhra Pradesh, India

(Received 4 August 2008; Accepted 2 October 2008) Abstract: Metabolic syndrome, including obesity, dyslipidaemia, hyperglycaemia and insulin resistance that predisposes type 2 diabetes is a major disease problem around the world and a plethora of herbal medicines are claimed to be effective in controlling these disorders. The rhizome of Zingiber ofcinale (Zingiberaceae) is commonly used as a spice in various foods and beverages. Apart from its other traditional medical uses, Z. ofcinale has been used to control diabetes and dyslipidaemia. In the present study, the protective effects of an ethanolic extract of Z. ofcinale on the development of metabolic syndrome were investigated in a high-fat diet-fed rat model at doses of 100, 200 and 400 mg/kg body weight. The marked rise in body weights, glucose, insulin, total cholesterol, LDL cholesterol, triglycerides, free fatty acids and phospholipids in serum of the rats that followed 6 weeks of high-fat diet treatment were signicantly reduced by Z. ofcinale treatment. However, no signicant change in serum HDL cholesterol was observed either with high-fat diet or Z. ofcinale compared to both control groups. The present results provide scientic evidence to substantiate the traditional use of Z. ofcinale in preventing metabolic disorders.

Metabolic syndrome including the presence of obesity, insulin resistance and dyslipidaemia that predisposes type 2 diabetes is becoming more prevalent in recent years [1,2]. According to recent estimates, approximately 215 million people worldwide suffer from diabetes and 8090% of them from type 2 diabetes [3]. The modern lifestyle of increased intake of high-calorie cafeteria fast food associated with decreased energy expenditure also contributes to the current rising prevalence of obesity and type 2 diabetes [4]. Recent epidemiological studies also revealed that 90% of all patients with type 2 diabetes are or have been overweight, and indicated that obesity is a strong risk factor and cause of type 2 diabetes and associated metabolic disturbances [5,6]. The events of hyperglycaemia and hyperlipidaemia, and their association present major risk factors for the development of diabetic and cardiovascular complications [7]. To reduce these serious complications and negative outcome of the metabolic syndrome, the control not only of blood glucose but also of lipids is necessary [8]. Therefore, new medicinal agents with dual properties on controlling both blood glucose and lipids are in great demand. The currently available therapeutic options such as dietary modication or a combination of synthetic antidiabetic, hypolipidaemic drugs have their own limitations and undesirable side-effects [7]. Hence, there is an increased demand to search and evaluate traditional approaches for the treatment of metabolic disorders, particularly the use of herbal medicines.

Author for correspondence: Srinivas Nammi, Herbal Medicines Research and Education Centre, Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia (fax +61 2 9351 4391, e-mail snammi@pharm.usyd.edu.au).

Zingiber ofcinale Roscoe (Zingiberaceae) commonly known as ginger, a well-known food spice, has been used traditionally in a wide variety of ailments [9,10]. The major chemical constituents of ginger rhizome are essential volatile oils and non-volatile pungent compounds [11,12]. The volatile oil components mainly consist of various terpenoids. The non-volatile compounds include the gingerols, shogaols, paradols and zingerone. Among them, the gingerols and shogaols were identied as the major gingerderived bioactive constituents that are found in fresh and dried ginger, respectively [13]. In laboratory experiments, ethanolic extract of Z. ofcinale has been shown to reduce plasma lipids in cholesterol-fed hyperlipidaemic rabbits [14,15] and in streptozotocininduced diabetic rats [16] and was also found to inhibit LDL oxidation in atherosclerotic mice [17]. Besides, the aqueous extract of Z. ofcinale has also been shown to reduce serum cholesterol and triglycerides in normal rats [18]. However, no change in plasma lipids was observed in normal rats treated with the whole powder [19,20] or fresh juice [21] of Z. ofcinale. In contrary, the hypoglycaemic and antidiabetic potential of Z. ofcinale were investigated and reported to be variable. From low to moderate, but signicant blood glucose lowering effect of the juice of Z. ofcinale was observed in both normal and diabetic animals [19,22]. The ethanolic extract has also been shown to lower blood glucose in normal rabbits [23] and rats [24] and in alloxan [25] and streptozotocin-induced diabetic rats [24,26]. However, the ethanolic extract of ginger was reported to elicit no effect on blood glucose in normal rats [27]. In addition, Singhal and Johsi [28] reported an elevation of blood glucose in normal rats treated with Z. ofcinale

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rhizome powder. The anti-obesity actions of ginger extracts were also noticed recently in chemical- [29] and in high-fat diet-induced obese mice [30]. Although consumption of high-fat containing diets is known to be a major cause of developing metabolic syndrome in human beings, a large body of the above reported work on Z. ofcinale has been carried out in either normal- or chemical-induced disease models. Hence, in the present study, we investigated the in vivo effects of the ethanolic extract of Z. ofcinale for its glucose and lipid regulating activities in high-fat diet-fed rat model of metabolic syndrome.

Experimental design and treatments. The rats were weight-matched and divided into six groups, each consisting of six rats. The normal- and the high-fat diet control groups received the standard diet and the high-fat diet, respectively, and treated with vehicle (1% sodium CMC) by oral gavage once daily for 6 weeks. The Z. ofcinale 100, 200 and 400 groups received the high-fat diet and treated, respectively, with 100, 200 and 400 mg/kg of Z. ofcinale extract by oral gavage once daily for 6 weeks, while the positive control group (rosiglitazone) received the high-fat diet and treated with 3 mg/kg of rosiglitazone by oral gavage once daily for 6 weeks. Blood collection and experimental parameters. Following an overnight fasting for 12 hr, blood sampling was done in rats under conscious condition from the lateral saphenous vein before (baseline) and at 6-week after onset of the treatments. The sample tubes were allowed to clot for 30 min. before centrifuging at 850 g for 15 min. Serum was separated and stored at 20 until analysis for biochemical parameters. Body weight data. The daily body weights were recorded in all the groups of rats every day between 4 and 4.30 p.m. and continued up to 6 weeks. Determination of serum glucose. Serum glucose was measured using an enzymatic colorimetric assay based on glucose oxidaseperoxidase (GOD-POD) method following manufacturers instructions with absorbance measured at 505 nm using a Shimadzu UV-1201 UV/VIS spectrophotometer. Determination of serum insulin. Serum insulin was measured using an enzyme-immuno assay (EIA) method following the manufacturers instructions with uorescence measured at an excitation of 405 nm using a POLARstar OPTIMA Fluorimeter (BMG Labtech, Germany). Homeostatic model assessment of insulin resistance (HOMA-IR). HOMA-IR, a measure of insulin resistance index [31] was calculated from the real-time fasting serum glucose and fasting insulin concentrations of different groups of rats using the mathematical HOMA-IR formula: HOMA-IR = (fasting serum insulin in U/ml fasting serum glucose in mg/dl)/405. Determination of serum total cholesterol. Total cholesterol in serum was estimated using an enzymatic colorimetric assay based on cholesterol oxidase method following the manufacturers instructions with absorbance measured at 600 nm using a Shimadzu UV-1201 UV/VIS spectrophotometer. Determination of serum HDL cholesterol. Serum HDL cholesterol was estimated using an enzymatic colorimetric assay based on the cholesterol oxidase method after removal of other lipoproteins by precipitation with phosphotungstate-magnesium, following the manufacturers instructions with absorbance measured at 600 nm using a Shimadzu UV-1201 UV/VIS spectrophotometer. Determination of serum LDL cholesterol. Serum LDL cholesterol was calculated indirectly by the Friedewalds equation [32] LDL = Total cholesterol [HDL + (TGL/5)]. Determination of serum triglycerides. Serum triglycerides were measured using an enzymatic colorimetric assay based on GPO-DAOS method following the manufacturers instructions with absorbance measured at 600 nm using a Shimadzu UV-1201 UV/VIS spectrophotometer.

Materials and Methods

Chemicals used. Rosiglitazone was obtained as generous gift sample from Dr. Reddys Laboratories, India. Rat insulin enzyme immunoassay kit was obtained from SPI-Bio, France whereas the other diagnostic kits used in the studies were purchased from Wako Diagnostics, Japan. All other chemicals used were of analytical grade. Plant extract and standardization. The 95% ethanolic standardized extract of Z. ofcinale rhizome used in the studies was obtained from Lipa Pharmaceuticals, Australia. Briey, fresh rhizomes of Z. ofcinale were vacuum dried at low temperature, shredded and triple reuxed with 95% (v/v) ethanol/water at low temperature and pressure. The ltrate was then concentrated at low temperature and pressure and vacuum dried to yield the nal extract (20 : 1). The extract was standardized for the content of gingerols using 6gingerol as a reference standard using a colorimetric method of analysis and found to contain not <10% of gingerols. Briey, 1 g of the extract was mixed with 70 ml of absolute ethanol and placed in water bath at 65 for 1 hr under constant shaking. The volume was then made up to 100 ml with absolute ethanol, mixed well and ltered. To 1 ml of the ltrate in a volumetric ask, 0.5 ml of potassium ferricyanide was added and left in dark place for 5 min. The volume was then made up to 50 ml with 0.1 mM HCl and allowed for 15 min. for complete colour development. The absorbance of the sample was measured at 660 nm and the content of 6-gingerol was calculated using the standard curve. Animals and diets. Adult male Wistar rats (140150 g) obtained from Mahaveera Enterprises (Hyderabad, India) were used in the studies. They were weight-matched and divided into groups of six animals each and housed three per cage in custom modied polypropylene cages having stainless steel vertical mesh that divides the cage space into three compartments. Each animal was housed in each compartment so that the animals were able to see each other, thus isolation stress if any, was minimized. The animal facility was well-ventilated and maintained at an ambient temperature of 24 2 having 5060% relative humidity with 12-hr light and dark cycle. Rats were acclimatized to the laboratory conditions for 1 week before experimentation and provided with standard diet and water ad libitum. Both the standard and high-fat diets were supplied by Snam Vijaya Feeds (Hyderabad, India) following the recommendations of the National Institute of Nutrition, Hyderabad. The standard diet contained (in weight percent) approximately 60% carbohydrate, 25% protein, 5% fat, 7% crude bre and the high-fat diet contained 15% carbohydrate, 17.5% protein, 60% fat, 5% crude bre and 2% cholesterol. The use and care of the animals in the experimental protocol has been approved by the local Institutional Animal Ethics Committee (Regd. No. 516/01/A/CPCSEA) following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

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Fig. 1. Effect of ethanolic extract of Zingiber ofcinale (ZO) on high-fat diet (HFD)-induced body weight gain in rats. (A) Daily recordings of the mean body weight changes of the experimental groups of rats and (B) comparison of the mean body weights of rats prior to (open columns) and after treatment for 6 weeks (closed columns) either with the HFD alone or with Zingiber ofcinale or rosiglitazone (RSG). Each bar indicates the mean S.E.M. of 56 rats. Signicant difference from normal control at identical times: ###P < 0.001. Signicant difference from HFD control at identical times: ***P < 0.001. Signicant difference from the respective pre-treated value: aP < 0.001.

Determination of serum free fatty acids. Serum free fatty acid levels were measured using an enzymatic colorimetric assay based on acyl-CoA synthetase-acyl-CoA oxidase (ACS-ACOD) method following the manufacturers instructions with absorbance measured at 550 nm using a Shimadzu UV-1201 UV/VIS spectrophotometer. Determination of serum phospholipids. Serum phospholipids were measured using an enzymatic colorimetric assay based on CODDAOS method following the manufacturers instructions with absorbance measured at 600 nm using a Shimadzu UV-1201 UV/VIS spectrophotometer. Data and statistical analysis. All the results are expressed as means S.E.M. To analyse the quantitative differences among the experimental groups before or after treatments, the respective data were subjected to analysis of variance (anova) using Graphpad Instat (version 3.0) statistical programme. Post hoc comparisons were made using StudentNewmanKeuls multiple comparisons test. Statistical differences in individual groups before and after treatments were detected using Students paired t-test. In all tests, P < 0.05 value was used as the criterion for statistical signicance.

Results Body weight data. The changes in the mean body weight of the experimental groups of rats over the 6-weeks treatment period are shown in g. 1A. There was no signicant difference in the initial body weights (from 133.7 6.8 g to 144.2 2.0 g; n = 6) and body weights after 2 weeks in different groups. But by the end of 34 weeks, Z. ofcinale extract treatment signicantly suppressed the body weight gain by 1723% compared with the animals fed with high-fat diet alone and this difference was more pronounced by 2734% and gained higher signicance after 6 weeks. High-fat diet alone-treated animals exhibited signicant increase in body weight compared with their pre-treated values (211.3 4.5 g versus 142.9 2.3 g; P < 0.001) or with the control group received standard diet alone (211.3 4.5 g versus 156.9 6.2 g; P < 0.001) at the end of 6 weeks (g. 1B). The groups

2009 The Authors Journal compilation 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 366373

ZINGIBER OFFICINALE ON HIGH-FAT DIET-INDUCED METABOLIC SYNDROME Table 1. Effect of ethanolic extract of Zingiber ofcinale on high-fat diet (HFD)-induced metabolic derangements of glucose and insulin in rats. Serum parameters studied Glucose (mg/dl) Group Normal control HFD control ZO (100 mg/kg) ZO (200 mg/kg) ZO (400 mg/kg) RSG (3 mg/kg) Before 113.4 6.9 121.0 10.8 107.5 7.5 79.2 7.4 73.9 8.4 75.9 8.7 After 89.6 6.1 208.8 12.4###,a 126.8 9.1*** 126.4 7.2***,b 93.1 11.6*** 118.8 7.7*** Before 5.7 0.3 7.0 0.6 5.7 0.2 5.9 0.3 6.1 0.3 5.9 0.2 Insulin (U/ml) After 5.8 0.2 14.7 1.7###,a 10.4 0.5***,##,a 9.2 0.7***,b 6.9 0.5*** 6.1 0.4*** Insulin resistance index Before 1.6 0.1 2.1 0.3 1.6 0.1 1.2 0.1 1.1 0.1 1.1 0.1 After

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1.3 0.1 7.4 0.7###,a 3.2 0.2***,##,a 2.8 0.2***,#,a 1.6 0.3*** 1.8 0.2***

Values represent the mean S.E.M. of 56 rats. ZO, Zingiber ofcinale; RSG, rosiglitazone. Signicant difference from normal control at identical times: ###P < 0.001; ##P < 0.01; #P < 0.05. Signicant difference from HFD control at identical times: ***P < 0.001. Signicant difference from the respective pre-treated value: aP < 0.001; bP < 0.01.

treated with the various doses of Z. ofcinale or rosiglitazone along with high-fat diet showed signicant (P < 0.001) reduction in body weight compared with high-fat diettreated control, whereas the body weights of Z. ofcinale treated rats were not signicantly different from their initial body weights or from the normal control rats after 6 weeks. Serum glucose. As shown in table 1, the serum glucose concentration prior to the treatments among the experimental groups varied within the normal physiological range although the levels are signicantly different among some of the groups. Rats fed high-fat diet alone showed signicant (P < 0.001; n = 6) elevation of fasting blood glucose compared with their pretreated glucose level or with the normal controls (n = 5) fed a standard diet at the end of 6 weeks. Zingiber ofcinale extract treatment at doses of 100, 200 and 400 mg/kg body weight along with high-fat diet produced signicant reduction (P < 0.001) in fasting blood glucose compared with the high-fat diet alone-treated controls in a dose-dependent manner and the results are comparable with the values observed with the positive control, rosiglitazone (P < 0.001). Moreover, the serum glucose levels in the Z. ofcinaletreated groups were not signicantly different from their pre-treated glucose levels (except Z. ofcinale 200 group) or from the normal control group at the end of the treatment. Serum insulin. Prior to the treatments, serum insulin concentration is not signicantly different among the experimental groups. However, serum insulin levels were found to be signicantly (P < 0.001) higher in the high-fat diet-treated control group (n = 6) compared with their pre-treated insulin level or with the normal control rats (n = 5) at the end of 6 weeks (table 1). Zingiber ofcinale treatment along with high-fat diet produced signicant (P < 0.001) and dose-dependent reduction in serum insulin compared with the high-fat diet control and comparable with the insulin levels found in the rosiglitazone-treated group. No signicant difference in serum insulin was observed in rats treated with the 400 mg/kg dose of Z. ofcinale compared with their pre-treated levels.

Homeostatic model assessment of insulin resistance (HOMA-IR). The development of insulin resistance as assessed by the mathematical HOMA-IR model in different groups of rats is shown in table 1. Prior to the treatment, all the groups exhibited similar insulin sensitivity. Nevertheless, rats fed high-fat diet alone developed insulin resistance, an effect which is statistically signicant (P < 0.001) compared with their pre-treated HOMA-IR index or with that of the rats fed standard diet alone at the end of 6 weeks. The groups of rats treated with Z. ofcinale extract at all doses showed signicant (P < 0.001) and dose-dependent reduction in insulin resistance (increased insulin sensitivity) compared with the high-fat diet control. The positive control rosiglitazone also produced signicant (P < 0.001) increase in insulin sensitivity compared with high-fat diet only-fed animals. Serum total cholesterol. Table 2 shows the total cholesterol levels among the various experimental groups. Prior to the treatment, serum total cholesterol concentrations were not statistically different among the groups. Rats fed with high-fat diet alone showed signicant increase (P < 0.01) in total cholesterol level (n = 6) when compared with the normal control group (n = 5) at the end of 6 weeks treatment or with their pretreated level (P < 0.05). Zingiber ofcinale extract produced signicant and dose-dependent reduction in total cholesterol level at doses of 100 (P < 0.01), 200 (P < 0.01) and 400 mg/kg (P < 0.001) compared with the high-fat diet-fed control. Furthermore, the serum total cholesterol concentrations in all the Z. ofcinale-treated groups were not signicantly different from their respective pre-treated total cholesterol levels or from the normal control group at the end of the treatment. Serum HDL cholesterol. The levels of HDL cholesterol observed in different groups of rats are shown in table 2. The serum HDL concentrations before the treatments were not signicantly different among the different groups. Also interestingly, no signicant change in HDL cholesterol was observed in high-fat diet

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SRINIVAS NAMMI ET AL. Table 2.

Effect of ethanolic extract of Zingiber ofcinale on high-fat diet (HFD)-induced metabolic changes of cholesterol in rats. Serum parameters studied Total cholesterol (mg/dl) Group Normal control HFD control ZO (100 mg/kg) ZO (200 mg/kg) ZO (400 mg/kg) RSG (3 mg/kg) Before 67.7 5.0 73.1 7.1 63.2 7.0 64.4 7.7 53.4 3.6 52.2 4.4 After 79.4 7.1 106.7 10.4#,c 62.2 11.7** 59.9 8.8** 45.0 2.9*** 48.6 3.8*** HDL cholesterol (mg/dl) Before 53.2 4.0 52.9 6.7 42.3 4.0 44.8 7.7 37.0 3.4 36.1 4.9 After 57.5 3.9 48.7 8.7 45.1 10.7 45.8 6.9 31.7 2.5 36.3 4.0 LDL cholesterol (mg/dl) Before 8.5 1.6 12.1 1.8 10.0 1.6 8.5 1.5 8.1 2.1 7.7 2.8 After 15.5 4.7 34.0 8.5##,c 7.7 2.2*** 5.6 2.1*** 5.8 1.7*** 5.0 1.7***

Values represent the mean S.E.M. of 56 rats. ZO, Zingiber ofcinale; RSG, rosiglitazone. Signicant difference from normal control at identical times: ##P < 0.01; #P < 0.05. Signicant difference from HFD control at identical times: ***P < 0.001; **P < 0.01. Signicant difference from the respective pre-treated value: cP < 0.05.

control rats (n = 6) when compared with their pre-treated HDL cholesterol or with the normal control group (n = 5) at the end of the treatment. Zingiber ofcinale treatment also however, did not signicantly alter the serum HDL cholesterol compared with either of the two control groups. Serum LDL cholesterol. The levels of LDL cholesterol as calculated by Friedewalds equation in various groups of experimental rats are shown in table 2. The LDL concentrations, before the treatments, were not statistically different among the groups. Rats fed high-fat diet alone showed signicant elevation of serum LDL cholesterol (n = 6) compared with their pre-treated (P < 0.05) serum LDL concentration or with the serum LDL of normal control rats (P < 0.01; n = 5) at the end of 6 weeks treatment. In the contrary, the groups treated with Z. ofcinale showed signicant (P < 0.001) reduction in LDL cholesterol in a dose-effective manner compared with the high-fat diet-fed control. Furthermore, the serum LDL cholesterol concentrations, in all the Z. ofcinale-treated groups, were not signicantly different from their respective pre-treated LDL cholesterol levels or from the normal control group at the end of the treatment.

Serum triglycerides. The levels of triglycerides in the different groups are shown in table 3. Serum triglyceride concentrations were not statistically different among the groups before the treatments. The high-fat diet-fed group showed signicant (P < 0.001) increase in serum triglycerides (n = 6) when compared with their pre-treated serum triglyceride concentration or with the serum triglycerides level of the control group (n = 5) at the end of the treatment. Zingiber ofcinale at all the three dose levels showed statistically signicant (P < 0.001) and dose-dependent reduction in serum triglycerides compared to the high-fat diet-fed control group and this difference is comparable with that observed with rosiglitazone (P < 0.001). Furthermore, the serum triglyceride concentrations, in all the Z. ofcinale-treated groups, were not signicantly different from their respective pre-treated serum triglyceride levels or from the normal control group at the end of the treatment. Serum free fatty acids. As given in table 3, serum free fatty acids were not signicantly different among the groups before the treatments. However, serum free fatty acid levels were found to be

Table 3. Effect of ethanolic extract of Zingiber ofcinale on high-fat diet (HFD)-induced metabolic changes of lipids in rats. Serum parameters studied Triglycerides (mg/dl) Group Normal control HFD control ZO (100 mg/kg) ZO (200 mg/kg) ZO (400 mg/kg) RSG (3 mg/kg) Before 30.3 4.5 40.5 9.1 54.5 8.4 55.5 6.6 41.5 7.2 42.2 12.0 After 32.2 3.3 119.8 8.8###,a 47.2 7.3*** 42.3 9.1*** 37.5 5.6*** 36.5 5.6*** Free fatty acids (mg/dl) Before 587.9 79.7 528.3 68.1 565.0 82.5 498.3 79.3 512.9 81.9 623.8 76.1 After 400.5 56.7 1296.7 103.6###,a 973.3 74.4*,##,c 865.4 133.4*,# 695.5 127.9** 727.5 114.8** Phospholipids (mg/dl) Before 168.0 13.1 186.8 12.4 157.4 4.7 166.4 13.7 162.6 8.8 172.0 10.5 After 166.8 17.2 256.8 16.3##,c 221.6 15.8b 181.1 15.0* 174.5 20.3* 192.1 12.9*

Values represent the mean S.E.M. of 56 rats. ZO, Zingiber ofcinale; RSG, rosiglitazone. Signicant difference from normal control at identical times: ###P < 0.001; ##P < 0.01; #P < 0.05. Signicant difference from HFD control at identical times: ***P < 0.001; **P < 0.01; *P < 0.05. Signicant difference from the respective pre-treated value: aP < 0.001; bP < 0.01; cP < 0.05.
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signicantly (P < 0.001) higher in the high-fat diet-treated control group (n = 6) compared with their pre-treated serum free fatty acid level or with the serum free fatty acid concentration of normal control rats (n = 5) at the end of 6 weeks. Zingiber ofcinale extract produced signicant and dosedependent reduction in serum free fatty acid levels at doses of 100 (P < 0.05), 200 (P < 0.05) and 400 mg/kg (P < 0.01) compared with the high-fat diet-fed control, in spite of signicant increase in serum free fatty acid with the 100 mg/kg of Z. ofcinale compared with their respective pre-treated levels or with the normal control at 6 weeks. Serum phospholipids. As shown in table 3, serum phospholipids concentrations were not statistically different among the groups before the treatments. Rats fed high-fat diet alone showed signicant (P < 0.05) increase in serum phospholipids level (n = 6) when compared with their pre-treated level or with the normal control group (P < 0.01; n = 5) at the end of 6 weeks treatment. Zingiber ofcinale extract produced dosedependent reduction in serum phospholipids, an effect reaching statistical signicance at doses of 200 (P < 0.05) and 400 mg/kg (P < 0.05) compared with the high-fat dietfed control. Furthermore, the serum phospholipid concentrations, in the Z. ofcinale-treated groups, except the lowest dose, were not signicantly different from their respective pre-treated serum phospholipid levels or from the normal control group at the end of the treatment. Discussion In the present study, we examined the protective effects of Z. ofcinale in high-fat diet-fed rats, a metabolic model of obesity and type 2 diabetes that is similar to human metabolic syndrome [33]. Since rats have similar metabolic patterns as that of human beings, it is rational to use this disease model to examine the prophylactic effects of chronic treatment of Z. ofcinale. To our knowledge, this is the rst study to use high-fat diet-induced rat model in investigating the protective effects of Z. ofcinale on in vivo glucose and lipid homeostasis. Metabolic syndrome is a complex polygenic disorder resulting in part from the contribution of impaired insulin secretion and/or impaired insulin action on its receptors [34]. When carbohydrates are in low supply, or their breakdown is incomplete, fats become the preferred source of energy [35]. As a result, fatty acids are mobilized into the general circulation leading to secondary triglyceridaemia in which the total serum lipids, including triglycerides, cholesterol and phospholipids increase, leading to life-threatening lipid disorders [36]. The development of metabolic syndrome is inuenced by a combination of genetic and environmental factors. Among the environmental factors, long-term high-fat intake is most intensively studied because of its contribution to the development of metabolic syndrome in human beings and rodents [37]. Apparently, a high proportion of daily energy derived from fat component is a

common situation in current day lifestyle in most societies of the world. The high prevalence of metabolic disorders is probably related to abnormal blood lipid proles that may be due to long-term effects of high-fat intake [38]. High-fat diet-fed rats showed signicant increase in body weight, serum glucose, and insulin and reduced insulin sensitivity. The present study reveals that the ethanolic extract of Z. ofcinale effectively suppresses the body weight gain, lowers serum glucose and insulin and increases insulin sensitivity upon concomitant administration along with the high-fat diet. Mascolo et al. [23] demonstrated that the ethanolic extract of Z. ofcinale treatment to normal rabbits showed potential hypoglycaemic activity. In contrast, isolated studies in normal rats [27] and in non-diabetic patients with coronary artery disease [39] have shown no effect on blood glucose. However, a large body of compelling evidence from various studies has shown signicant blood glucose lowering activity in experimental animals [19,2426]. Zingiber ofcinale has also been shown to improve insulin sensitivity by reducing elevated serum glucose and insulin in chemical-induced obese mice [29]. Clearly, the results of these earlier observations substantiate our ndings in the high-fat diet-fed rats. Most recently, Isa et al. [40] reported an up-regulation of adiponectin by 6-shogaol and 6-gingerol, the major components of Z. ofcinale. They also reported that 6-shogaol but not 6-gingerol possesses signicant PPAR- agonistic activity. It is clear that plasma adiponectin concentration and its mRNA expression level decrease in the obese and insulinresistant states [41] and it is believed that exogenous administration or up-regulation of endogenous adiponectin by pharmacological intervention improves insulin sensitivity followed by an increase in fatty acid oxidation and a decrease in serum triglycerides levels [42]. Thus, these latter ndings [40] strongly lend support to the results of our present study in the high-fat diet-treated rats by suggesting that the glucose-regulating and insulin-sensitizing effects of the active component(s) of Z. ofcinale could be at least in-part attributed to PPAR- agonistic activity and/or upregulation of adiponectin. Dyslipidaemia is the most important modiable risk factor contributing to the development of atherosclerosis in type 2 diabetes [43]. Thus the importance of blood levels of triglycerides, free fatty acids, cholesterol and phospholipids in the pathogenesis of lipid disorders have been extensively reviewed [44]. In the present study, high-fat diet treatment markedly increased the serum triglycerides, free fatty acids, phospholipids, total- and LDL- but not HDL-cholesterol. Presumably, these changes may be in part due to enhanced cholesterol biosynthesis during high intake of fatty diet [45]. The increase in LDL-cholesterol may be due to the reduced expression or activity of the LDL-receptor sites in response to high-fat diet treatment [46]. Therefore, lowering the LDL-cholesterol level may be an important factor in lowering the serum total cholesterol level in rats fed a high-fat diet. Zingiber ofcinale administration along with the high-fat diet effectively reduced the serum total cholesterol and

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SRINIVAS NAMMI ET AL. 4 Aude YW, Mego P, Mehta JL. Metabolic syndrome: dietary interventions. Curr Opin Cardiol 2004;19:473 9. 5 Bray GA, Bellanger T. Epidemiology, trends, and morbidities of obesity and the metabolic syndrome. Endocrine 2006;29:109 17. 6 Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin resistance and type 2 diabetes. Nature 2006;444:840 6. 7 Lender D, Sysko SK. The metabolic syndrome and cardiometabolic risk: scope of the problem and current standard of care. Pharmacotherapy 2006;26:3S 12S. 8 Moller DE. New drug targets for type 2 diabetes and the metabolic syndrome. Nature 2001;414:821 7. 9 Afzal M, Al-Haddi D, Menon M, Pesek M, Dharmi MSI. Ginger: an ethnomedical, chemical and pharmacological review. Drug Metab Drug Interact 2001;18:159 90. 10 Chrubasik S, Pittler MH, Roufogalis BD. Zingiberis rhizoma: a comprehensive review on the ginger effect and efcacy proles. Phytomedicine 2005;12:684 701. 11 Govindarajan V. Ginger-chemistry technology and quality evaluation: Part-1. Crit Rev Food Sci Nutr 1982;17:1 96. 12 Govindarajan V. Ginger-chemistry technology and quality evaluation: Part-2. Crit Rev Food Sci Nutr 1982;17:189258. 13 Connell D, Sutherland M. A re-examination of gingerol, shogaol and zingerone, the pungent principles of ginger (Zingiber ofcinale Roscoe). Aust J Chem 1969;22:1033 43. 14 Bhandari U, Sharma JN, Zafar R. The protective action of ethanolic ginger extract in cholesterol-fed rabbits. J Ethnopharmacol 1998;61:167 71. 15 Verma SK, Singh M, Jain P, Bordia A. Protective effect of ginger, Zingiber ofcinale Rosc on experimental atherosclerosis in rabbits. Ind J Exp Biol 2004;42:736 8. 16 Bhandari U, Kanojia R, Pillai KK. Effect of ethanolic extract of Zingiber ofcinale on dyslipidaemia in diabetic rats. J Ethnopharmacol 2005;97:227 30. 17 Fuhrman B, Rosenblat M, Hayek T, Coleman R, Aviram M. Ginger extract consumption reduces plasma cholesterol, inhibits LDL oxidation and attenuates development of atherosclerosis in atherosclerotic, apolipoprotein E-decient mice. J Nutr 2000;130:1124 31. 18 Thomson M, Al-Qattan KK, Al-Sawan SM, Alnaqeeb MA, Khan I, Ali M. The use of ginger (Zingiber ofcinale Rosc.) as a potential anti-inammatory and antithrombotic agent. Prostaglandins Leukot Essent Fatty Acids 2002;67:475 8. 19 Akhani SP, Viswarkarma SL, Goyal RK. Antidiabetic activity of Zingiber ofcinale in streptozotocin-induced type I diabetic rats. J Pharm Pharmacol 2004;56:101 5. 20 Sivakumar V, Sivakumar S. Effect of indigenous herbal compound preparation Trikatu on the lipid proles of atherogenic diet and standard diet fed Rattus norvegicus. Phytother Res 2004;18:976 81. 21 Ahmed RS, Sharma SB. Biochemical studies on combined effects of garlic (Allium sativum Linn) and ginger (Zingiber ofcinale Rosc) in albino rats. Ind J Exp Biol 1997;35:8413. 22 Sharma M, Shukla S. Hypoglycemic effect of ginger. J Res Ind Yoga Homeop 1977;12:127 30. 23 Mascolo N, Jain R, Jain SC, Capasso F. Ethnopharmacologic investigation of ginger (Zingiber ofcinale). J Ethnopharmacol 1989;27:129 40. 24 Ojewole JAO. Analgesic, anti-inammatory and hypoglycemic effects of ethanol extract of Zingiber ofcinale (Roscoe) Rhizomes (Zingiberaceae) in mice and rats. Phytother Res 2006;20:76472. 25 Kar A, Choudary BK, Bandyopadhyay NG. Comparative evaluation of hypoglycemic activity of some Indian medicinal plants in alloxan diabetic rats. J Ethnopharmacol 2003;84:1058. 26 Al-Amin ZM, Thomson M, Al-Qattan KK, Peltonen-Shalaby R, Ali M. Anti-diabetic and hypolipidaemic properties of ginger

LDL-cholesterol in addition to marked reduction in serum triglycerides, free fatty acids and phospholipids in concordance with earlier reports [1417]. The reduction of LDLcholesterol by Z. ofcinale could be due to prevention of the suppressive action of high-fat diet on the LDL-receptor site. In contrast to increased serum HDL-cholesterol observed by Bhandari et al. [16], the serum HDL-cholesterol concentration is not altered in our studies either by the high-fat diet or by Z. ofcinale treatment. The reason for this variability could be due to the differences in the kind of experimental disease models used for the studies. Earlier studies have demonstrated that Z. ofcinale through its activity on hepatic cholesterol-7-hydroxylase, stimulates the conversion of hepatic cholesterol to bile acids [21,47]. More recently, Han et al. [30] found that Z. ofcinale increased the faecal excretion of cholesterol, suggesting that ginger may block absorption of cholesterol in the gut. Studies in apolipoprotein E-decient mice have also disclosed that the lipid lowering effects of Z. ofcinale could have possibly resulted, at least in part, from the inhibition of cellular cholesterol biosynthesis [17] which also supports an earlier observation by Tanabe et al. [48] revealing the presence of an active component in Z. ofcinale that acts on the liver to reduce cholesterol biosynthesis. Thus, the molecular mechanisms responsible for the observed lipid lowering effects of Z. ofcinale could be due a single or multiple effects of its active components on potential sites of action leading to decreased intestinal fat absorption and/or decreased lipid biosynthesis and/or enhanced cholesterol elimination. In conclusion, the ethanolic extract of Z. ofcinale showed remarkable protection from the high-fat dietinduced metabolic disturbances by strongly suppressing the body weight gain, protection from hyperglycaemic, hyperlipidaemic and insulin-resistant conditions. Thus the present ndings emphasize that the rhizome of Z. ofcinale possesses potential medicinal value and hence its traditional consumption in foods as a spice is benecial in the prevention of metabolic disorders caused by high-fat diet. Further studies are being undertaken to explain fully the mechanism(s) of the glucose and lipid metabolism-regulating activities of Z. ofcinale. Acknowledgement This research study was supported under the SESQUI postdoctoral research grant provided to S.N. by the University of Sydney, Australia.

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2009 The Authors Journal compilation 2009 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 104, 366373