Abstract

INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease and its prevalence has been reported among 20-30% of the population of Western countries (Paschos, Paletas, 2009Paschos P, Paletas K. Non alcoholic fatty liver disease and metabolic syndrome. Hippokratia. 2009;13(1):9-19.). NAFLD is a term used to describe a range of related diseases: the earliest stage of the disease is liver steatosis, which is described by triglyceride deposition in the form of lipid droplets in the cytoplasm of hepatocytes associated with enlargement of the liver (hepatomegaly). In steatosis, triglyceride drops contain more than 5% of cytoplasm of hepatocytes (Anderson, Borlak, 2008Anderson N, Borlak J. Molecular mechanisms and therapeutic targets in steatosis and steatohepatitis. Pharmacological reviews. 2008;60(3):311-357.; Cohen, Horton, Hobbs, 2011Cohen JC, Horton JD, Hobbs HH. Human fatty liver disease: old questions and new insights. Science. 2011;332(6037):1519-1523. ). Liver steatosis can be extended to non-alcoholic steatohepatitis (NASH), which is distinguished from simple steatosis by hepatocyte injuries, infiltration of inflammatory factors, and/or collagen deposition (fibrosis). NASH can be eventually developed into cirrhosis. About 10-29% of people with NASH would suffer from cirrhosis within 10 years (Argo, Caldwell, 2009Argo CK, Caldwell SH. Epidemiology and natural history of non-alcoholic steatohepatitis. Clinics in liver disease. 2009;13(4):511-531.). In cirrhosis, hepatocytes are replaced by scar tissue (cicatrix), which is mainly composed of type1 collagen. Ultimately, cirrhosis can be developed into liver cancer. Studies have shown that 27% of people with cirrhosis, induced by NASH, have been diagnosed with liver cancer (Starley, Calcagno, Harrison, 2010Starley BQ, Calcagno CJ, Harrison SA. Nonalcoholic fatty liver disease and hepatocellular carcinoma: a weighty connection. Hepatology . 2010;51(5):1820-1832.). NAFLD has also been associated with cardiovascular diseases, metabolic syndrome, and insulin resistance (Kim, et al., 2012aKim D, Choi SY, Park EH, Lee W, Kang JH, Kim W, Kim YJ, Yoon JH, Jeong SH, Lee DH. Nonalcoholic fatty liver disease is associated with coronary artery calcification. Hepatology. 2012a;56(2):605-613.; Parker et al., 2012Parker HM, Johnson NA, Burdon CA, Cohn JS, O’Connor HT, George J. Omega-3 supplementation and non-alcoholic fatty liver disease: a systematic review and meta-analysis. Journal of hepatology. 2012;56(4):944-951.; Paschos, Paletas, 2009). Elevation of a few factors in the liver, including free fatty acids supply from diet, de novo lipogenesis, insulin resistance, oxidative stress, and inflammatory markers play roles in inducing liver steatosis (Chen, Varghese, Ruan, 2014Chen Y, Varghese Z, Ruan XZ. The molecular pathogenic role of inflammatory stress in dysregulation of lipid homeostasis and hepatic steatosis. Genes & Diseases. 2014;1(1):106-112.; Cohen, et al., 2011; Sumida et al., 2013Sumida Y, Niki E, Naito Y, Yoshikawa T. Involvement of free radicals and oxidative stress in NAFLD/NASH. Free radical research. 2013;47(11):869-880.), and controlling of any of these factors can be effective in the improvement of the fatty liver.

Ginger is from the root of Zingiber officinale and is one of the most used spices. In the traditional Chinese medicine, ginger is used to treat inflammation (Rahimlou et al., 2016Rahimlou M, Yari Z, Hekmatdoost A, Alavian SM, Keshavarz SA. Ginger supplementation in nonalcoholic fatty liver disease: a randomized, double-blind, placebo-controlled pilot study. Hepatitis monthly. 2016;16(1).), and its active ingredients have shown anti-diabetic, anti-cancer, anti-pain, and anti-inflammatory properties (Rahimlou, et al., 2016; Young, et al., 2005Young H-Y, Luo Y-L, Cheng H-Y, Hsieh W-C, Liao J-C, Peng W-H. Analgesic and anti-inflammatory activities of [6]-gingerol. Journal of ethnopharmacology . 2005;96(1):207-210.). Moreover, ginger extract has demonstrated antioxidant activity and can decrease blood sugar, insulin, and blood triglyceride (Rahimlou, et al., 2016). Ginger retains nutritional value due to having a variety of bioactive compounds, including gingerols, zingerone, and shogaols (Butt, Sultan, 2011Butt MS, Sultan MT. Ginger and its health claims: molecular aspects. Critical reviews in food science and nutrition. 2011;51(5):383-93.). The spicy taste of fresh ginger rhizome is attributed to the presence of gingerols, a group of volatile phenolic compounds, among which 6-gingerol is the major compound that is responsible for the plant’s sharpness and spice. Other gingerols, such as 4, 8, 10, and 12-gingerols are also present in lower concentrations which are sensitive to heat and will turn into shogaols at high temperatures creating bitter, spices and sweets aromas (Wohlmuth, et al., 2005Wohlmuth H, Leach DN, Smith MK, Myers SP. Gingerol content of diploid and tetraploid clones of ginger (Zingiber officinale Roscoe). Journal of agricultural and food chemistry. 2005;53(14):5772-5778.). The compound 6-gingerol was first isolated from the rhizome of ginger as a volatile yellow oil at room temperature in 1879. After the discovery of 6-gingerol, numerous studies focused on determining its structure (Thresh, 1879Thresh J. Proximate analysis of the rhizome of of Zingiber officinale and comparitive examination of typical specimens of commercial gingers. Pharm J. 1879;10:171.). A number of pre-clinical studies have shown the effects of gingerols in the treatment of diabetes, obesity, diarrhea, allergies, pain, fever, rheumatoid arthritis, inflammation, and various forms of cancer. Moreover, ginger and its metabolites, as strong antioxidants, have been known for their abilities to inhibit free radical oxidation and nitric oxide production. Also, numerous studies have shown that gingerol plays protective role in liver, kidney, and cardiopulmonary, and central nervous systems by providing antioxidant, anti-nausea, anti-gastric acid, anti-angiogenesis, and antimicrobial effects (Semwal, et al., 2015Semwal RB, Semwal DK, Combrinck S, Viljoen AM. Gingerols and shogaols: important nutraceutical principles from ginger. Phytochemistry. 2015;117:554-568.).

Knowing the proven properties of gingerol holding antioxidant as well as other remedial effects (Dugasani, et al., 2010Dugasani S, Pichika MR, Nadarajah VD, Balijepalli MK, Tandra S, Korlakunta JN. Comparative antioxidant and anti-inflammatory effects of [6]-gingerol,[8]-gingerol,[10]-gingerol and [6]-shogaol. Journal of ethnopharmacology . 2010;127(2):515-520.; Semwal, et al., 2015Semwal RB, Semwal DK, Combrinck S, Viljoen AM. Gingerols and shogaols: important nutraceutical principles from ginger. Phytochemistry. 2015;117:554-568.; Tripathi, et al., 2007Tripathi S, Maier KG, Bruch D, Kittur DS. Effect of 6-gingerol on pro-inflammatory cytokine production and costimulatory molecule expression in murine peritoneal macrophages. Journal of Surgical Research. 2007;138(2):209-213.), the therapeutic properties of 6-gingerol in improving and treating non-alcoholic fatty liver and the relevant tissue damages were investigated in the present study in mice models.

MARERIAL AND METHODS

Compounds

6-Gingerol was purchased from Sigma Aldrich Company. Diagnostic kits were purchased from Iran’s Biochemistry Co. for the measurement of cholesterol, triglyceride, glucose, HDL-C (high-density lipoprotein cholesterol), LDL-C (low-density lipoprotein cholesterol), VLDL-C (very low-density lipoprotein cholesterol), Aspartate transaminase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP), SOD (superoxide dismutase), and catalase. The YaK052 rat Elisa kit (Japan’s Yanaihara Company) was used to measure leptin. Rat Elisa kit (Otsuka Pharmaceutical of Japan) was used to measure adiponectin, and American ALPCO Diagnostics ultra-sensitive Rat Elisa kit was used to measure insulin.

Animals

A total of 30 male NMRI mice weighing 25 ± 4 grams were purchased from the Pasteur Institute (Iran) and they were kept at Razi Laboratory in Tehran, Science and Research Unit, under standard conditions with 12 hr of light/dark cycle and a relative humidity of 50-70% at 21 ± 2 ºC. After one week, the animals received high-fat diet for induction of fatty liver, except for the control group which received regular water and food, for a total of 30 days. After confirming the induction of fatty liver in the animals by biochemical and histological assessments, the treatment started and continued for another 30 days. All experiments were performed in accordance with the international guidelines set in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996) and approved by the Research and Ethics Committee of Science and Research Branch, Azad University.

Animals were first weighed and were randomly divided into the 5 following groups (n = 6 per group):

Control group: receiving regular water and food

Positive control group: fatty liver-induced mice receiving silymarin (90 mg/kg) for 30 days once every other day via oral gavage

Negative control group: fatty liver-induced mice receiving the solvent of 6-gingerol (in distilled water) for 30 days once every other day via oral gavage

Group I: fatty liver-induced mice receiving 6-gingerol (400 mg/kg) for 30 days once every other day via oral gavage

Group II: fatty liver-induced mice receiving 6-gingerol (800 mg/kg) for 30 days once every other day via oral gavage

Fatty liver induction method

A high-fat diet consisting of two parts was provided in order to induce fatty liver: the first part contained the compounds that were mixed with the animals’ fed pellets providing full-fat food, and the second part contained the compounds that were fed to mice via gavage for 30 days (Table I). Animals were weighed every week. The induction of fatty liver in animals was confirmed at the end of 30 days by biochemical and histological examinations.

Histological and biochemical evaluations

At the end of the treatment, the animals’ body weights were measured. Animals were then anesthetized by inhalation of diethyl ether, after which blood sample was taken from the cardiac ventricles using 2.5-mL syringes. The blood serum was isolated and stored at -20 ºC until use for biochemical tests. The liver tissue was immediately removed and divided into two parts: one part was subjected to liquid nitrogen for further biochemical tests and the other part was fixed in 10% formalin buffer solution for histopathological evaluation. Fixed tissues were cross-sectioned into 5-mm sections according to routine protocols and were further stained with hematoxylin and eosin (H&E). The slides were examined by light microscopy.

In order to use liver tissue for the level determination of hepatic lipid profiles, hormones, and antioxidant enzymes, liver (10%, w/v) was homogenized in 50 mM phosphate buffer (pH 7.0) and was then centrifuged at 9000 rpm at 4 ºC for 20 min, after which the supernatants were separated and stored in Eppendorf tubes at -70 ºC until analysis. The level of these biochemical parameters were determined by the previously mentioned assay kits according to the manufacturers’ protocols.

The level of other biochemical parameters, including insulin, glucose, adiponectin, leptin, HDL, VLDL, LDL, AST, ALT, and ALP were measured in the blood serum using commercially available kits mentioned previously. In order to separate blood serum, blood samples were allowed to clot for 30 min at room temperature (RT) and were then centrifuged at 2500 rpm at RT for 10 min.

The insulin resistance index was evaluated using homeostasis model assessment (HOMA) formula: HOMA = fasting serum insulin (mU/L) x fasting plasma glucose (mM)/22.5 (Kim, et al., 2012aKim D, Choi SY, Park EH, Lee W, Kang JH, Kim W, Kim YJ, Yoon JH, Jeong SH, Lee DH. Nonalcoholic fatty liver disease is associated with coronary artery calcification. Hepatology. 2012a;56(2):605-613.). The liver weight index (%) was also obtained by the following formula (Kim, et al., 2012b): Liver Weight Index (%) = liver weight/body weight × 100.

Statistical analyses of data

One-way ANOVA and Tukey test were used for statistical examination of data. The results were reported as Mean ± SD. The level of statistical significance was set at p<0.001, p<0.01, and p<0.05. Statistical analyzes were performed by SPSS software and the graphs were drawn using Excel software.

RESULTS

In the present study, non-alcoholic fatty liver was induced in NMRI male mice using a high-fat diet. After 30 days of using a high-fat and high-cholesterol diet, fatty liver induction was confirmed by the biochemical and histological assessments of the liver. Subsequently, animals were treated with 6-gingerol at 400 and 800 mg/kg doses for 30 days. After treatment, evaluation of biochemical parameters, including lipid profiles, antioxidant enzymes, adiponectin and leptin hormones as well as histopathologic examinations were performed on the liver.

Effect of 6-gingerol on liver lipid profile

The levels of triglyceride, cholesterol, LDL-C and VLDL-C in the negative control group showed a considerable increase (p<0.001) compared with the control group after 30 days of receiving high-fat diet, whereas the level of HDL-C decreased significantly (p<0.001) in the negative control group compared with the control group (Table II). The positive control group as well as groups I and II showed substantial reductions in the levels of triglyceride, cholesterol, LDL-C, and VLDL-C compared with the negative control group after 30 days of treatment. In contrast, HDL-C level increased significantly in the positive control group and in both groups I and II compared with the negative control group (Table II).

Effect of 6-gingerol on fasting blood glucose, insulin and insulin resistance levels

After receiving 30 days of high-fat diet, the levels of fasting blood glucose, insulin, and insulin resistance (HOMA) showed a significant increase (p<0.001) in the negative control group compared with the control group. In contrast, administration of silymarin (positive control group) and both doses of 6-gingerol for 30 days decreased the level of these parameters considerably in the treated groups in comparison with the negative control group (Figure 1A-C).

FIGURE 1
Changes in the levels of fasting blood glucose (A), insulin (B), and insulin resistance (HOMA) (C) affected by 6-gingerol in fatty liver-induced mice. Values are reported as mean ± SD. +++ p<0.001 as compared with the control group; ** p<0.01 and *** p<0.001 as compared with the negative control group.

Effect of 6-gingerol on leptin and adiponectin levels

As shown in Figure 2A, the level of leptin increased in the negative control group compared with the control group (p<0.001). Meanwhile, after 30 days of treatment, the leptin level was notably improved in the positive control as well as in groups I and II compared with the negative control group (p<0.001). On the other hand, the level of adiponectin was significantly lowered in the negative control group compared with the control group (p<0.001) while it was elevated in the treated groups I and II compared with the negative control group (p<0.001) (Figure 2B).

FIGURE 2
Changes in the levels of leptin (A) and adiponectin (B) affected by 6-gingerol in fatty liver-induced mice. Values are reported as mean ± SD. ++ p<0.01 and +++ p<0.001 as compared with the control group. *** p<0.001 as compared with the negative control group.

Effect of 6-gingerol on the hepatic and antioxidant enzymes

Before treatment with 6-gingerol, the amount of hepatic enzymes, including AST, ALT, and ALP were considerably high in the negative control group compared with the control group (p<0.001). In the cases of treating with 90 mg/kg silymarin (positive control group) and both doses of 6-gingerol, the levels of these enzymes decreased notably in the treated groups in comparison with the negative group (p<0.001) (Table III).

The levels of antioxidant enzymes, including SOD and catalase were also measured before and after treatment with 6-gingerol. Before treatment, the amount of these enzymes were much lower in the negative control group than the control group (p<0.001). After treating with silymarin and 6-gingerol, the level of SOD increased significantly in the positive control group and in group II compared with the negative control group (p<0.001). However, the increase in the level of SOD in group I was not as notable as group II. Regarding the catalase enzyme, its level was higher in the positive control group as well as in groups I and II compared with the negative control group (p<0.001) (Table III).

Effect of 6-gingerol on body weight changes and liver weight index

The body weights of mice were monitored in all groups throughout the period of the study. The initial weights, measured at day 30, and the final weights of all groups are shown in Table IV. At the beginning of the experiment, the body weights were not significantly different between the groups. Also, after receiving a high-fat diet (the 30^th day of the experiment) and also at the end of the study period (after receiving treatment), no significant difference was observed in the body weights between different groups.

After receiving high-fat diet, the liver weight and the index of liver weight were significantly increased in the negative control group compared with the control group (p<0.001). However, after being treated with silymarin and 6-gingerol, the values of these two parameters were notably reduced in the positive control group and groups I and II, respectively, compared with the negative control group (p<0.001) (Table IV).

Histological examinations

As it is demonstrated in Figure 3, the liver section of the control group was healthy with no lesion, and the hepatocytes, sinusoidal spaces, and central and portal veins could be seen in intact forms (Figure 3A). After receiving a high-fat diet for 30 days, the liver tissue of the negative control group was fattened and the deposited lipid droplets could be seen in the liver tissue. Also, the accumulation of the inflammatory cells was clearly visible in the tissue sections of the negative control group (Figure 3B). Liver tissue examination of the positive control group showed that receiving silymarin at dose of 90 mg/kg for 30 days could well recover the tissue lesions in this group as neither lipid droplets nor accumulation of the inflammatory cells were observed (Figure 3C). Microscopic observations of the liver tissue of group I (receiving 6-gingerol at 400 mg/kg) showed partial improvement as there was a minor accumulation of both inflammatory cells and fat droplets inside the hepatocytes (Figure 3D). Histological examination of the liver tissue of group II (receiving 6-gingerol at 800 mg/kg) indicated an improvement in the status of the liver tissue compared with the negative control group as there was a significant reduction in the accumulation of both inflammatory cells and lipid droplets in hepatocytes. Meanwhile, the tissue images of group II were similar to the control group (Figure 3E).

FIGURE 3
H&E-stained sections of liver from the following groups: control (A), negative control (fatty liver + solvent) (B), and positive control (fatty liver + silymarin) (C), group I (fatty liver + 6-gingerol 400 mg/kg) (D), group II (fatty liver + 6-gingerol 800 mg/kg) (E).

DISCUSSION

In this study, a high-fat diet was used to create fatty liver in mice. After 30 days of receiving high-fat diet, the results demonstrated that the fatty liver-related parameters, including triglyceride, cholesterol, LDL, leptin, and liver enzymes were increased while adiponectin, HDL, and antioxidant enzymes were decreased. Meanwhile, histological examinations of the fatty liver confirmed the development of steatosis. These changes confirmed the creation of fatty liver in the animals.

Triglycerides and cholesterol are among important lipids whose over-received amounts lead to hypertriglyceridemia and hypercholesterolemia. Non-alcoholic fatty liver is specified by the accumulation of triglycerides in hepatocytes formed by the esterification of free fatty acids and glycerol (Magesh, et al., 2006Magesh V, Singh JPV, Selvendiran K, Ekambaram G, Sakthisekaran D. Antitumour activity of crocetin in accordance to tumor incidence, antioxidant status, drug metabolizing enzymes and histopathological studies. Molecular and cellular biochemistry. 2006;287(1):127-135.; Shen, Qian, 2006Shen X-C, Qian Z-Y. Effects of crocetin on antioxidant enzymatic activities in cardiac hypertrophy induced by norepinephrine in rats. Die Pharmazie-An International Journal of Pharmaceutical Science s. 2006;61(4):348-352.; Xiang, et al., 2006Xiang M, Qian Z-Y, Zhou C-H, Liu J, Li W-N. Crocetin inhibits leukocyte adherence to vascular endothelial cells induced by AGEs. Journal of ethnopharmacology . 2006;107(1):25-31.). In numerous studies, researchers have been able to induce fatty liver in animals using high-cholesterol and high-calorie diets. A group of researchers showed that fatty liver can be induced in animals in a short period of time by using high-calorie diet containing fructose, cholesterol and fat (Clapper, et al., 2013Clapper JR, Hendricks MD, Gu G, Wittmer C, Dolman CS, Herich J, Athanacio J, Villescaz C, Ghosh SS, Heilig JS. Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment. American Journal of Physiology-Gastrointestinal and Liver Physiology. 2013;305(7):G483-G495.). In another study, researchers evaluated the effect of high-fat diet on the induction of fatty liver over various times. They found that changes related to the fatty liver appeared only within two weeks after receiving a high-fat diet in animals (Gauthier, Favier, Lavoie, 2006Gauthier M-S, Favier R, Lavoie J-M. Time course of the development of non-alcoholic hepatic steatosis in response to high-fat diet-induced obesity in rats. British journal of nutrition. 2006;95(02):273-281.). The fatty diet used in the present study could effectively induce fatty liver in mice after 30 days. Consumption of the present high-fat diet caused changes of dyslipidemia, which was identified by increasing the serum levels of total cholesterol, triglyceride, and LDL and also by the reduction in the HDL level. These changes were also previously reported in non-alcoholic fatty liver (Noeman, Hamooda, Baalash, 2011Noeman SA, Hamooda HE, Baalash AA. Biochemical study of oxidative stress markers in the liver, kidney and heart of high fat diet induced obesity in rats. Diabetology & metabolic syndrome. 2011;3(1):1.).

According to various studies, high-fat diet could increase the size, the weight as well as the percentage of lipid accumulation in the liver (VanSaun, et al., 2009VanSaun MN, Lee IK, Washington MK, Matrisian L, Gorden DL. High fat diet induced hepatic steatosis establishes a permissive microenvironment for colorectal metastases and promotes primary dysplasia in a murine model. The American journal of pathology. 2009;175(1):355-364.; Zou, et al., 2006Zou Y, Li J, Lu C, Wang J, Ge J, Huang Y, Zhang L, Wang Y. High-fat emulsion-induced rat model of nonalcoholic steatohepatitis. Life sciences. 2006;79(11):1100-1107.). However, the diet used in the present study, despite the induction of non-alcoholic fatty liver, did not significantly increase the body weights in mice. Furthermore, the body weight in the group receiving 6-gingerol showed no significant changes, whereas the liver weight index (%) was significantly reduced. Accordingly, gingerol has shown to cause reduction in the body weight of obese rats (Saravanan, et al., 2014Saravanan G, Ponmurugan P, Deepa MA, Senthilkumar B. Anti-obesity action of gingerol: effect on lipid profile, insulin, leptin, amylase and lipase in male obese rats induced by a high-fat diet. Journal of the Science of Food and Agriculture. 2014;94(14):2972-2977.).

In the present study, treatment with 6-gingerol, in a dose-dependent mode, significantly decreased TG, LDL-C and VLDL levels and improved HDL-C level in the treated groups. In accordance with our study, it has been shown that gingerol could reduce the levels of the lipid profiles in obese rats and also decrease the absorption of lipids, fat, and cholesterol by inhibiting pancreatic lipase activity (Saravanan, et al., 2014Saravanan G, Ponmurugan P, Deepa MA, Senthilkumar B. Anti-obesity action of gingerol: effect on lipid profile, insulin, leptin, amylase and lipase in male obese rats induced by a high-fat diet. Journal of the Science of Food and Agriculture. 2014;94(14):2972-2977.). Another study has demonstrated that 6-gingerol, by regulating key genes associated with inflammation and lipid metabolism, could offer its protective effect against non-alcoholic steatohepatitis (Tzeng, et al., 2015Tzeng T-F, Liou S-S, Chang CJ, Liu I-M. 6-gingerol protects against nutritional steatohepatitis by regulating key genes related to inflammation and lipid metabolism. Nutrients. 2015;7(2):999-1020.). 6-gingerol is the active constituent of fresh ginger, which has shown to improve lipid profiles efficiently (Mazidi, et al., 2016Mazidi M, Gao H-K, Rezaie P, Ferns GA. The effect of ginger supplementation on serum C-reactive protein, lipid profile and glycaemia: a systematic review and meta-analysis. Food & nutrition research. 2016;60(1):32613.). Another study has shown that ginger extract had hypoglycemic effects and could lower cholesterol and LDL in diabetic rats (Al-Noory, Amreen, Hymoor, 2013Al-Noory AS, Amreen A-N, Hymoor S. Antihyperlipidemic effects of ginger extracts in alloxan-induced diabetes and propylthiouracil-induced hypothyroidism in (rats). Pharmacognosy research. 2013;5(3):157.).

In the present case, induction of fatty liver in mice caused a significant increase in the levels of glucose, insulin, and insulin resistance (HOMA). Mazidi et al. have reported that ginger could effectively reduce the levels of glucose, insulin, and insulin resistance in obese rats (Mazidi, et al., 2016). This hypoglycemic effects of ginger could be attributed to its contents of phenols, polyphenols, and flavonoids (Shanmugam, et al., 2011Shanmugam KR, Mallikarjuna K, Kesireddy N, Reddy KS. Neuroprotective effect of ginger on anti-oxidant enzymes in streptozotocin-induced diabetic rats. Food and Chemical Toxicology . 2011;49(4):893-897.). As one of the active phenolic compounds of ginger, 6-gingerol has shown to increase the cellular glucose adsorption by increasing the expression of type 4 glucose carrier gene (Li, et al., 2012Li Y, Tran VH, Duke CC, Roufogalis BD. Gingerols of Zingiber officinale enhance glucose uptake by increasing cell surface GLUT4 in cultured L6 myotubes. Planta medica. 2012;78(14):1549-1555.). Meanwhile, gingerol has been reported to decrease the pancreatic amylase secretion, resulting in the reduction of the intestinal absorption of carbohydrates, and also increase insulin sensitivity (Ali, Amreen, Hymoor, 2006Ali H, Houghton P, Soumyanath A. α-Amylase inhibitory activity of some Malaysian plants used to treat diabetes; with particular reference to Phyllanthus amarus. Journal of ethnopharmacology. 2006;107(3):449-455.). These phenomena could be associated with the anti-obesity and anti-hyperglycemic effects of gingerol (Saravanan, et al., 2014Saravanan G, Ponmurugan P, Deepa MA, Senthilkumar B. Anti-obesity action of gingerol: effect on lipid profile, insulin, leptin, amylase and lipase in male obese rats induced by a high-fat diet. Journal of the Science of Food and Agriculture. 2014;94(14):2972-2977.). Also, a group of researchers examined the therapeutic potential of 6-gingerol in improving hyperglycemia in diabetic rats. According to their observations, 6-gingerol increased the insulin secretion in response to glucose and improved glucose tolerance. They suggested that 6-gingerol could increase insulin secretion by facilitating the exocytosis of insulin-containing vesicles (Samad, et al., 2017Samad MB, Mohsin MNAB, Razu BA, Hossain MT, Mahzabeen S, Unnoor N, Muna IA, Akhter F, Kabir AU, Hannan J. [6]-Gingerol, from Zingiber officinale, potentiates GLP-1 mediated glucose-stimulated insulin secretion pathway in pancreatic β-cells and increases RAB8/RAB10-regulated membrane presentation of GLUT4 transporters in skeletal muscle to improve hyperglycemia in Lepr db/db type 2 diabetic mice. BMC complementary and alternative medicine. 2017;17(1):395.). Accordingly, in the present study, insulin level was also higher in the group receiving 800 mg/kg of 6-gingerol.

In 2015, it was reported that 6-gingerol regulated glucose metabolism through the AMPK pathway (Lee, et al., 2015Lee JO, Kim N, Lee HJ, Moon JW, Lee SK, Kim SJ, Kim JK, Park SH, Kim HS. [6]-Gingerol Affects Glucose Metabolism by Dual Regulation via the AMPKα2-Mediated AS160-Rab5 Pathway and AMPK-Mediated Insulin Sensitizing Effects. Journal of cellular biochemistry. 2015;116(7):1401-1410.). It was also shown that 6-gingerol could offer anti-hyperglycemic effects by increasing muscle glycogen sediments via adjusting the synthesis and the activity of glycogen (Samad, et al., 2017Samad MB, Mohsin MNAB, Razu BA, Hossain MT, Mahzabeen S, Unnoor N, Muna IA, Akhter F, Kabir AU, Hannan J. [6]-Gingerol, from Zingiber officinale, potentiates GLP-1 mediated glucose-stimulated insulin secretion pathway in pancreatic β-cells and increases RAB8/RAB10-regulated membrane presentation of GLUT4 transporters in skeletal muscle to improve hyperglycemia in Lepr db/db type 2 diabetic mice. BMC complementary and alternative medicine. 2017;17(1):395.). Consequently, the plasma glucose level in the present study was lower in the group treated with 800 mg/kg of 6-gingerol than the group receiving 400 mg/kg of this compound, indicating higher uptake of glucose.

In the present study, the levels of leptin and adiponectin showed significant increase in the negative control group compared with the control group. Treatment with 6-gingerol and silymarin resulted in a significant decrease in leptin level and a significant increase in adiponectin level. Accordingly, researchers have demonstrated that 6-gingerol improved insulin sensitivity following an increase in adiponectin levels (Isa, et al., 2008Isa Y, Miyakawa Y, Yanagisawa M, Goto T, Kang M-S, Kawada T, Morimitsu Y, Kubota K, Tsuda T. 6-Shogaol and 6-gingerol, the pungent of ginger, inhibit TNF-α mediated downregulation of adiponectin expression via different mechanisms in 3T3-L1 adipocytes. Biochemical and biophysical research communications. 2008;373(3):429-434.). The hypoglycemic and hypolipidemic effects of ginger extract have been shown in rats fed with high-fat diet by improving the levels of lipid profiles and decreasing the insulin and glucose levels. It was demonstrated that the relationship between the expression of leptin, adiponectin, PPARα and PPARγ in the liver could be the key mechanism for providing hypolipidemic effects by ginger (de las Heras, et al., 2016de las Heras N, Valero-Muñoz M, Martín-Fernández B, Ballesteros S, López-Farré A, Ruiz-Roso B, Lahera V. Molecular factors involved in the hypolipidemic-and insulin-sensitizing effects of a ginger (Zingiber officinale Roscoe) extract in rats fed a high-fat diet. Applied Physiology, Nutrition, and Metabolism. 2016;42(2):209-215.). The researchers have stated that 6-gingerol can reverse the regulation of adiponectin expression in adipocytes with its anti-inflammatory effects (Isa, et al., 2008). Several studies have also confirmed the effects of ginger and 6-gingerol on the reduction of serum leptin levels in treated animals (Saravanan, et al., 2014Saravanan G, Ponmurugan P, Deepa MA, Senthilkumar B. Anti-obesity action of gingerol: effect on lipid profile, insulin, leptin, amylase and lipase in male obese rats induced by a high-fat diet. Journal of the Science of Food and Agriculture. 2014;94(14):2972-2977.; Wadikar, Premavalli, 2011Wadikar D, Premavalli K. Appetizer administration stimulates food consumption, weight gain and leptin levels in male Wistar rats. Appetite. 2011;57(1):131-133.).

In the present study, high-fat diet significantly increased the levels of AST, ALT and ALP enzymes in the negative control group compared with the control group, and prescription of 6-gingerol significantly reduced the levels of these enzymes. Accordingly, it has been shown that 6-gingerol could significantly reduce the levels of AST and ALT in non-alcoholic steatohepatitis (Tzeng, et al., 2015Tzeng T-F, Liou S-S, Chang CJ, Liu I-M. 6-gingerol protects against nutritional steatohepatitis by regulating key genes related to inflammation and lipid metabolism. Nutrients. 2015;7(2):999-1020.). Several studies have also shown that hydroalcoholic, aqueous, and ethanolic extracts of ginger could significantly decrease the level of all the mentioned liver enzymes (Al-Naqeeb, et al., 2003Al-Naqeeb MA, Thomson M, Al-Qattan K, Kamel F, Mustafa T, Ali M. Biochemical and histopathological toxicity of an aqueous extract of ginger in female rats. Kuwait journal of science and engineering. 2003;30(2):35-48.; Bhandari, et al., 2003Bhandari U, Shamsher AA, Pillai K, Khan M. Antihepatotoxic activity of ginger ethanol extract in rats. Pharmaceutical biology. 2003;41(1):68-71.; Poorrostami, Farokhi, Heidari, 2014Poorrostami A, Farokhi F, Heidari R. Effect of hydroalcoholic extract of ginger on the liver of epileptic female rats treated with lamotrigine. Avicenna Journal of Phytomedicine. 2014;4(4):276.). Interestingly, another study has shown that ginger extract was capable of reducing the activity of these enzymes in the rats poisoned with carbon tetrachloride and acetaminophen (Yemitan, Izegbu, 2006Yemitan OK, Izegbu MC. Protective effects of Zingiber officinale (Zingiberaceae) against carbon tetrachloride and acetaminophen-induced hepatotoxicity in rats. Phytotherapy Research. 2006;20(11):997-1002.).

Previous investigations have shown that high-fat diets increased the production of free radicals and induced oxidative stress. In fact, hypercholesterolemia was associated with lipid peroxidation and it decreased the activity of antioxidant enzymes, ultimately leading to cellular damage (Amirkhizi, et al., 2010Amirkhizi F, Siassi F, Minaie S, Djalali M, Rahimi A, Chamari M. Is obesity associated with increased plasma lipid peroxidation and oxidative stress in women? ARYA Atheroscler. 2010;2(4).). More studies have demonstrated that increasing the oxidative stress and production of free radicals, due to fat accumulation in the liver, would lead to the development of liver steatosis, fibrosis, and cirrhosis (Mohajeri, 2013Mohajeri D. Effects of Solanum lycopersicum L. on serum lipid profile and oxidative stress in liver tissue of high fat fed diet rats. 2013.). Several studies have also shown that the use of high-fat diet in animals caused a significant reduction in the serum level of antioxidants, including SOD and catalase (Liu, Lloyd, 2013Liu J, Lloyd SG. High-fat, low-carbohydrate diet alters myocardial oxidative stress and impairs recovery of cardiac function after ischemia and reperfusion in obese rats. Nutrition research. 2013;33(4):311-321.; Lu, Chiang, 2001Lu Y-F, Chiang C-F. Effect of dietary cholesterol and fat levels on lipid peroxidation and the activities of antioxidant enzymes in rats. International journal for vitamin and nutrition research. 2001;71(6):339-346.; Zou, et al., 2006Zou Y, Li J, Lu C, Wang J, Ge J, Huang Y, Zhang L, Wang Y. High-fat emulsion-induced rat model of nonalcoholic steatohepatitis. Life sciences. 2006;79(11):1100-1107.). Herein, SOD and catalase levels were decreased significantly by consumption of high-fat diet, whereas prescribing 6-gingerol significantly increased the amount of these enzymes. Ginger has shown to present high antioxidant effects due to containing active compounds, in particular gingerol (Chang, et al., 1994Chang W, Chang Y, Lu F, Chiang H-C. Inhibitory effects of phenolics on xanthine oxidase. Anticancer research. 1994;14(2A):501-506.). Increasing the level of antioxidant enzymes, such as SOD and catalase, by ginger extract has been furthermore confirmed by other researches (Amin, Hamza, 2006Amin A, Hamza AA. Effects of Roselle and Ginger on cisplatin-induced reproductive toxicity in rats. Asian journal of andrology. 2006;8(5):607-612.; Motawi, et al., 2011Motawi TK, Hamed MA, Shabana MH, Hashem RM, Naser AFA. Zingiber officinale acts as a nutraceutical agent against liver fibrosis. Nutrition & metabolism. 2011;8(1):40.; Poorrostami, Farokhi, Heidari, 2014Poorrostami A, Farokhi F, Heidari R. Effect of hydroalcoholic extract of ginger on the liver of epileptic female rats treated with lamotrigine. Avicenna Journal of Phytomedicine. 2014;4(4):276.). It has been reported that phenolic compounds in ginger, including 6-gingerol inhibit free radicals and lipid peroxidation, protect liver, and increase the antioxidants (Siddaraju, Dharmesh, 2007Siddaraju MN, Dharmesh SM. Inhibition of gastric H+, K+-ATPase and Helicobacter pylori growth by phenolic antioxidants of Zingiber officinale. Molecular nutrition & food research. 2007;51(3):324-332.; Aeschbach, et al., 1994Aeschbach R, Löliger J, Scott B, Murcia A, Butler J, Halliwell B, Aruoma O. Antioxidant actions of thymol, carvacrol, 6-gingerol, zingerone and hydroxytyrosol. Food and Chemical Toxicology. 1994;32(1):31-36.; Chung, Yow, Benzie, 2003Chung WY, Yow CM, Benzie IF. Assessment of membrane protection by traditional Chinese medicines using a flow cytometric technique: preliminary findings. Redox report. 2003;8(1):31-33.).

The most important characteristic of steatosis is fat accumulation in the liver cells, resulting in both insulin resistance and inflammation in the liver. (Janczyk, Socha, 2012Janczyk W, Socha P. Non-alcoholic fatty liver disease in children. Clinics and research in hepatology and gastroenterology. 2012;36(3):297-300.). In the development of steatosis, cellular ballooning and inflammation are visible (Nalbantoglu, Blunt, 2014Nalbantoglu I, Blunt EM. Role of liver biopsy in nonalcoholic fatty liver disease. 2014.). In the present study, accumulation of fat and inflammatory cells were observed, confirming the production of fatty liver by applying fatty diet. In the hepatic tissue sections of the treated groups with 6-gingerol, the improvement in the liver tissue status was clearly visible. It has been shown that 6-gingerol could inhibit the adipogenesis (differentiation of adipocytes) and cytoplasmic accumulation of lipid droplets in the 3T3-L1 cell line (Tzeng, Liu, 2013Tzeng T-F, Liu I-M. 6-Gingerol prevents adipogenesis and the accumulation of cytoplasmic lipid droplets in 3T3-L1 cells. Phytomedicine. 2013;20(6):481-487.).

Many previous studies have used natural compounds to protect liver injury (Al-Rasheed, et al., 2018Al-Rasheed NM, El-Masry TA, Tousson E, Hassan HM, Al-Ghadeer A. Hepatic protective effect of grape seed proanthocyanidin extract against Gleevec-induced apoptosis, liver Injury and Ki67 alterations in rats. Brazilian Journal of Pharmaceutical Sciences. 2018;54(2). ). In the present study, silymarin was selected as the positive control as it has shown efficacy in the treatment of NASH (Solhi, et al, 2014Solhi H, Ghahremani R, Kazemifar AM, Yazdi ZH. Silymarin in treatment of non-alcoholic steatohepatitis: A randomized clinical trial. Caspian journal of internal medicine. 2014;5(1):9.). Herein, treatment with silymarin significantly improved the lipid profiles and the levels of insulin, glucose, and HOMA. Silybin, forming 50-60% of the silymarin, has shown to significantly reduce the levels of triglyceride, total cholesterol, LDL, and VLDL and increase the HDL level (Gobalakrishnan, Asirvatham, Janarthanam, 2016Gobalakrishnan S, Asirvatham SS, Janarthanam V. Effect of Silybin on Lipid Profile in Hypercholesterolaemic Rats. Journal of clinical and diagnostic research: JCDR. 2016;10(4):FF01.; Yao, Zhi, Minhu, 2011Yao J, Zhi M, Minhu C. Effect of silybin on high-fat-induced fatty liver in rats. Brazilian Journal of Medical and Biological Research. 2011;44(7):652-9). It has also been demonstrated that hydroalcoholic extract of silymarin improved lipid disorders created in diabetic rats (Sajedianfard, Behroozi, Nazifi, 2014Sajedianfard J, Behroozi Z, Nazifi S. The effects of a hydroalcoholic extract of silymarin on serum lipids profiles in streptozotocin induced diabetic rats. Comparative Clinical Pathology. 2014;23(3):779-784.), reduced blood glucose levels in type 2 diabetes (Sajedianfard, Behroozi, Nazifi, 2014; Voroneanu, et al., 2016Voroneanu L, Nistor I, Dumea R, Apetrii M, Covic A. Silymarin in type 2 diabetes mellitus: a systematic review and meta-analysis of randomized controlled trials. Journal of diabetes research. 2016;2016.), and decreased both plasma lipid and insulin levels in obese rats (Guo, et al., 2016Guo Y, Wang S, Wang Y, Zhu T. Silymarin improved diet-induced liver damage and insulin resistance by decreasing inflammation in mice. Pharmaceutical biology . 2016;54(12):2995-3000.). In accordance with previous studies, we demonstrated that silymarin could well improve the levels of leptin, adiponectin, antioxidant enzymes, and hepatic steatosis (Abdel-Moneim, et al., 2015Abdel-Moneim AM, Al-Kahtani MA, El-Kersh MA, Al-Omair MA. Free radical-scavenging, anti-inflammatory/anti-fibrotic and hepatoprotective actions of taurine and silymarin against CCl4 induced rat liver damage. PloS One. 2015;10(12):e0144509.; Guo, et al., 2016; Cacciapuoti, et al., 2013Cacciapuoti F, Scognamiglio A, Palumbo R, Forte R, Cacciapuoti F. Silymarin in non alcoholic fatty liver disease. World journal of hepatology. 2013;5(3):109.).

Along with sylimarin, polyphenolic 6-gingerol, in a dose-dependent mode, showed capability of improving non-alcoholic fatty liver by increasing the antioxidant enzymes (SOD and catalase) and adiponectin levels and also decreasing the insulin resistance, serum leptin level, and lipid profiles.

ACKNOWLEDGMENT:

This study has been performed in the Laboratory Complex of the Science and Research Branch of Azad University. Authors declare no conflict of study.

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