Black Garlic Ameliorates Obesity Induced by a High-fat Diet in Rats

Black garlic (also called aged garlic) is a type of fermented garlic product from fresh garlic that is often used as a food ingredient and a functional food in Asian countries. The aim of this study was to investigate whether black garlic ameliorates obesity induced by a high-fat diet in rats. Male Wistar rats were fed a normal diet or a highfat diet (HFD) (30% lard, w/w) combined with 0, 0.2, 0.6, or 1.2% black garlic (BG) (w/w) for a period of six weeks. The results demonstrated that body weight, tissue weights of liver, peritoneal fat, and epididymal fat, serum triglycerides, and hepatic lipid profiles (total lipids, triglycerides, and cholesterol) in the HFD+BG groups were significantly decreased compared with those in the HFD group. BG also reduced hepatic oxidative stress (reduced GSSG and enhanced TEAC, GSH, GRd, and GPx) in HFD-induced obese rats. These results suggest that black garlic may be useful for the treatment of obesity.


Introduction
Obesity is a serious health problem and is closely associated with lifestyle-related diseases such as hypertension, arteriosclerosis, hyperlipidemia, dyslipidemia, type 2 diabetes mellitus, cancer, respiratory complications, and osteoarthritis [1].Excessive amounts of body fat have health complications associated with major risk factors for several serious chronic diseases, such as metabolic syndrome.Obesity is also a public health concern and is associated with an increased risk of morbidity and mortality [2].Obesity-related dyslipidemia plays a crucial role in the development of atherosclerosis and cardiovascular diseases [3].Therefore, the prevention and treatment of obesity plays an important role in healthy life and longevity [4].In antiobesity drugs, some medical treatments for obesity have serious side effect, such as fenfluramine, sibutramine, and rimonabant [5].Many natural compounds have been used to treat obesity, including gallic acid, epigallocatechin 3-gallate, rutin, o-coumaric acid, and garlic [6,7,8,9].
The pharmacological actions of garlic (Allium sativum L.) include antiobesity, antibacterial, antiviral, antihypertensive, blood glucose lowering, antithrombotic, antimutagenic, and antiplatelet actions [9,10,11,12,13,14].Sheen et al. [15] indicated that the garlic oil and its organosulfur compounds can be beneficial for the suppression of high-fat diet (HFD)-induced body weight gain in rats.Moreover, bioconversion technology is a technology in which some organic compounds are modified using the specific reactions to change them into specific compounds for special use.Black garlic (also called aged garlic) is a type of fermented garlic product from raw garlic that is used for food ingredients and functional foods in Asian countries, such as Taiwan, Japan, and Korea.Kang et al. [16] indicated that black garlic powder can be used in the treatment of the atherosclerotic process and the improvement of hyperlipidemia.Seo et al. [17] indicated that aged garlic extract has beneficial effects on reducing weight and visceral fat gain and cholesterol lowering in rats fed with HFD.Ried et al. [18] indicated that aged garlic extract reduced the blood pressure in patients with uncontrolled hypertension.Jung et al. [19] indicated that the intake of fermented aged black garlic can be beneficial for the prevention of HFD-induced diabetic complications.However, the literature indicates that the antiobesity effects of black garlic in high-fat diet-fed rats remains unclear.
The aim of this study was to investigate the anti-obesity effect of black garlic using HFD-induced obese rats.In addition, growth parameters, serum biochemical parameters, organ and adipose tissue weights, histology, and the antioxidant defense system were measured in rats fed a normal diet (ND) and a HFD with or without black garlic powder.

Materials
The powders of black garlic were provided by Professor Chin-Yin Tseng (Chung Chou University of Science and Technology, Changhua County, Taiwan).All other chemicals used were of the highest pure grade available.

Animals, Diets, and Experimental Design
Ten-week-old male Wistar rats were purchased from the BioLASCO Taiwan Corp., Ltd (Ilan, Taiwan).Animals were housed individually in stainless steel cages in an airconditioned room at 23±2°C, 55-60% relative humidity, and a 12 h light/dark cycle and were given a laboratory rodent chow diet for 1 week.The rats were divided into normal and obese groups and then fed normal diets (NDs) and high fat-diets (HFDs), respectively.The HFD group (30% lard, w/w) (n=8/group) was then divided into four groups according to whether the animals received supplemental black garlic (BG) for six weeks: the HFD group, HFD+BG-LD (low dose, LD), HFD+BG-MD (medium dose, MD), and HFD+BG-HD (high dose, HD) at levels of 0%, 0.2%, 0.6 %, and 1.2% (w/w), respectively.These animals should normally be able to consume 5% of their body weight daily.The diets were stored in a 4°C cold chamber.Body weights, food intakes, and food efficiency were measured every day for six weeks.After overnight fasting, blood was withdrawn from the abdominal aorta under carbon dioxide anesthesia, and serum was harvested.The visceral tissues were immediately excised, rinsed, weighed, and frozen in liquid nitrogen.All experimental procedures involving animals were conducted in accordance with the guidelines of the National Institutes of Health (NIH).This experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of Chung Shan Medical University (IACUC Approval No: 894) in Taichung, Taiwan.

Measurement of Serum Parameters
Blood was placed into a sterile Vacutainer plastic tube (BD Vacutainer, Plymouth, UK).Serum was separated by centrifugation (4000×g, 10 min) and transferred to Eppendorf tubes.The serum concentrations of triglycerides, total cholesterol, high-density lipoprotein (HDL) cholesterol, aspartate aminotransferase (AST), alanine aminotransferase (ALT), uric acid, creatinine, Na + , K + , and Cl -were measured with commercial kits (Bayer Corporation, Tarrytown, NY, USA).The concentration of ketone bodies was measured with an abundant ketone body kit (Randox Laboratories Ltd., UK).

Hematoxylin and Eosin (H&E) Staining
Liver and fat tissue samples were collected following euthanasia, fixed in 10% formalin buffered solution, and cut into 5-µm sections.Hematoxylin and eosin (H&E) staining was performed using standard techniques.

Hepatic Lipid Analysis
Hepatic lipid was extracted according to the methods used by [20], and concentrations of triglycerides and cholesterol were measured using a TG assay kit (Teco Diagnostics, USA) and a cholesterol commercial kit (Randox Laboratories Ltd., UK), respectively.

Trolox Equivalent Antioxidant Capacity (TEAC) Assay
Determination of TEAC was conduction using the method of Arnao et al. [21].ABTS •+ is generated by the interaction of ABTS (100 µmol/L), H 2 O 2 (50 µmol/L), and peroxidase (4.4 U/mL).To measure antioxidant activity, 0.25 mL of serum was mixed well with an equal volume of ABTS, H 2 O 2 , peroxidase, and 1.5 mL of deionized water.The absorbance was measured at 734 nm after interacting with the sample solution for 10 min.The decrease in absorption at 734 nm after the addition of the reactant was used to calculate the TEAC value.A dose-response curve was plotted for trolox, and antioxidant ability was expressed as the TEAC.The higher the TEAC value of a sample, the stronger the antioxidant activity.

Determination of GSH and GSSG in the Liver
The levels of GSH and GSSG were determined by a GSH assay kit (Cayman Chemical Company, Ann Arbor, MI) according to the procedure of the manufacturer.Absorbance was measured spectrophotometrically in a VersaMax tunable microplate reader (Molecular Devices, Sunnyvale, CA) at 405 nm, and the concentrations of GSH and GSSG were calculated as nanomoles per milligram of protein.

Determination of Antioxidant Enzymes in the Liver
All antioxidant enzyme activities were determined after the hepatic tissue was homogenized with phosphate-buffered saline at a pH of 7.0.The GSH peroxidase (GPx) activity was determined according to the method of Lawerence and Burk [22].Liver homogenate solution (100 µL) was mixed with 800 µL of 100 mmol/L potassium phosphate buffer (pH 7.4) containing 1 mmol/L EDTA, 1 mmol/L NaN 3 , 0.2 mmol/L NADPH, 1 U/mL GSH reductase, and 1 mmol/L GSH.After 5 min, 2.5 mmol/L H 2 O 2 (100 µL) was added to start the reaction.The absorbance change at 340 nm was recorded over the course of 3 min.Enzyme activity was calculated by E 340 =6220/M per cm, and the result is expressed as units of nmol NADPH/mg protein/min.
The GSH reductase (GRd) activity was determined according to the method of Bellomo et al. [23].The liver homogenate solution (100 µL) was mixed with 900 µL of 100 mmol/L potassium phosphate buffer (pH 7.0) containing 1 mmol/L MgCl 2 .6H 2 O, 50 mmol/L GSSG, and 0.1 mmol/L NADPH and was incubated for 3 min at room temperature.The absorbance change at 340 nm was recorded over the course of 3 min.The enzyme activity was calculated by E 340 =6220/M per cm, and the result is expressed as units of nmol NADPH/ mg protein/min.Superoxide dismutase (SOD) activity was determined by a SOD assay kit-WST (Dojindo Molecular Technologies Inc., Maryland, USA) as specified by the manufacturer.The absorbance was measured spectrophotometrically in a VersaMax tunable microplate reader (Molecular Devices, Sunnyvale, CA) at 450 nm.The value (%) is expressed as SOD activity of the ND group at 100%.

Statistical Analysis
The data were analyzed using analysis of variance (ANOVA).Differences were considered significant at the 0.05 probability level, and differences between treatments were evaluated using the Least Significant Difference (LSD) test.All statistical analyses were performed using SAS (SAS Institute Inc., 2002).

Effect of Black Garlic on Body Weight, Food Intake, and Energy Intake of Rats with Obesity Induced by a HFD
In the present study, the HFD groups (high fat-diet containing 30% lard, w/w) were divided into four groups and supplemented with black garlic [0, 0.2, 0.6, and 1.2% (w/w)] for six weeks.After six weeks of feeding, body weights in the HFD groups supplemented with black garlic (0.2-1.2% black garlic in diet) were significantly decreased compared with those in the HFD group (Table 1).Moreover, after six weeks, food intake was significantly higher in the HFD group compared with the ND group.Food intake and energy intake in the HFD groups supplemented with black garlic were not significantly different (p>0.05).

Effect of Black Garlic on the Weights of
Organs and Adipose Tissue of Rats with Obesity Induced by a HFD The organ and adipose tissue weights of the five groups are depicted in Table 2.There were no significant differences in the weights of the heart, spleen, lung, and kidney among the five groups.The organ weights of liver (0.2, 0.6, and 1.2% black garlic in diet) and adipose tissue [peritoneal fat (0.2, 0.6, and 1.2% black garlic in diet) and epididymal fat (0.6 and 1.2% black garlic in diet)] in the HFD groups supplemented with black garlic was significantly decreased compared with those of the HFD group.The changes of liver and adipose tissue morphology are depicted in Figure 1 and Figure 2. As demonstrated in Figure 1, hematoxylin and eosin staining revealed hepatic macrovesicular fat accumulation in the HFD group.The HFD groups supplemented with black garlic (0.2-1.2% black garlic in diet) exhibited microvesicular fat accumulation.As shown in Figure 2, the sizes of the peritoneal fat in the HFD groups supplemented with black garlic (0.2-1.2% black garlic in diet) were smaller than those in the HFD group.

Effect of Black Garlic on the Serum Biochemical Indicators of Rats with Obesity Induced by a HFD
As shown in Table 3, serum levels of triglycerides (0.2-1.2% black garlic in diet), AST (0.2-1.2% black garlic in diet), and ketone bodies (0.6 and 1.2% black garlic in diet) in the HFD groups supplemented with black garlic were significantly decreased compared with those in the HFD group.Serum HDL-cholesterol levels in the HFD groups supplemented with black garlic at 0.2-1.2% were significantly increased compared with those in the HFD group.Moreover, there were no significant differences in the serum levels of total cholesterol, ALT, uric acid, creatinine, Na + , K + , or Cl − among the five groups.

Effect of Black Garlic on Hepatic Lipid
Profiles and Antioxidant Enzymes of Rats with Obesity Induced by a HFD garlic were significantly increased compared with those in the HFD group.The level of GSSG in HFD groups supplemented with black garlic at 0.2-1.2% was significantly decreased compared with those in the HFD group.There were no significant differences in the levels of SOD among the HFD groups supplemented with black garlic.

Discussion
The protective effects of black garlic have been investigated in the context of the immune system, oxidative stress, and inflammation.Dietary fat is an important environmental factor contributing to obesityrelated diseases, such as hyperlipidemia, hypertension, arteriosclerosis, type 2 diabetes mellitus, cancer, respiratory complications, and osteoarthritis [1].However, we examined the effect of black garlic on growth parameters, serum biochemical parameters, organ and adipose tissue weights, histology, and the antioxidant defense system in HFD-induced obese rats.Many studies have reported that obesity is induced in mice and rats by feeding the animals a high-energy diet containing 30% lard [24,25].Black garlic was then given as a supplement at levels of 0, 0.2, 0.6, or 1.2% in diets for a period of six weeks.Our data indicated diets containing black garlic provided over six weeks suppressed the increases in body weight and tissue weights of liver, peritoneal fat, and epididymal fat induced by a HFD (Table 1 and Table 2).The reports indicated that aged garlic extract can reduce body weight gain, visceral fat, and liver weight gain in high-fat diet-induced obese rats [17].Obesity is associated with a high incidence of steatosis, such as non-alcoholic fatty liver disease (NAFLD), and has been recognized as one of the most common causes of chronic liver disorders [26].Over-accumulation of hepatic lipids and the oxidation of fatty acids are important sources of reactive oxygen species in fatty liver [27].Morphologically, the livers of HFD rats exhibited abundant and large lipid droplets and an obvious increase in liver derangement compared with those of ND rats.The HFD groups supplemented with black garlic exhibited microvesicular fat accumulation in liver and small adipocyte sizes in peritoneal fat (Figure 1 and Figure 2).
Previous research demonstrated that the oral administration of phytochemicals (such as gallic acid, epigallocatechin gallate, rutin, o-coumaric acid, and curcumin) prevents HFD-induced dyslipidemia in animal models and clinical trials [6,8,28,29].We found that a HFD supplemented with black garlic resulted in significantly decreased serum levels of triglyceride, AST, and ketone bodies (Table 3).In addition, serum level of HDL-cholesterol in a HFD group supplemented with black garlic was significantly increased compared to those in the HFD group.Lavie and Milani [30] indicated that obesity has adverse effects on health and adversely affects the levels of plasma lipids, including high levels of triglyceride and low levels of HDL-cholesterol.In vivo studies indicated that obesity is associated with decrease antioxidant activities and increased oxidative stress in plasma and organ tissue [31,32].Oxidative stress plays an important role in the risk factors of hyperlipidemia and atherosclerosis [33].Antioxidant enzymes can scavenge free radicals, balance of oxidative stress, and prevent reactive oxygen species formation [34].Our data indicated that the HFD group supplemented with black garlic exhibited significantly decreased levels of hepatic lipid profiles (total lipid, triglyceride, and cholesterol) and GSSG and increased levels of antioxidant enzymes (TEAC, GSH, GRd, and GPx) (Table 4).

Conclusion
Rats fed diets containing black garlic for six weeks suppressed the increases in body weight, the tissue weights of liver, peritoneal fat, and epididymal fat, the levels of serum lipid triglycerides, and the hepatic lipid profiles induced by a HFD.We also found that the HFD supplemented with black garlic exhibited significantly decreased level of GSSG and increased levels of antioxidant enzymes (TEAC, GSH, GRd, and GPx).These results demonstrate that the intake of black garlic can be beneficial for the suppression of HFD-induced obesity in rats.

Figure 1 .Figure 2 .
Figure 1.Effect of black garlic (BG) on hepatosteatosis in rats with obesity induced by a high-fat diet.Livers were stained with hematoxylin and eosin (H&E).Original magnification: 200 ×

Table 2 . Effect of black garlic on the weights of organs and adipose tissue of rats with obesity induced by a high-fat diet
The reported values are the mean ± SEM (n=8).Mean values with different letters were significantly different (p < 0.05). *

Table 3 . Effect of black garlic on the serum biochemical parameters of rats with obesity induced by a high-fat diet
*The reported values are the mean ± SEM (n=8).Mean values with different letters were significantly different (p < 0.05).HDL, high-density lipoprotein; AST, aspartate aminotransferase; ALT, alanine aminotransferase.

Table 4 . Effect of black garlic on the hepatic lipid profiles and antioxidant enzymes of rats with obesity induced by a high-fat diet
The reported values are the mean ± SEM (n=8).Mean values with different letters were significantly different (p < 0.05).TEAC, Trolox equivalent antioxidant capacity; GSH, glutathione; GSSG, glutathione disulfide; GRd, glutathione reductase; GPx, glutathione peroxidase; SOD, superoxide dismutase. *

Table 4
presents the effect of black garlic on the hepatic lipid profiles and antioxidant enzymes of rats with obesity induced by a HFD.Hepatic lipid profiles of total lipids (0.2-1.2% black garlic in diet), triglycerides (0.6 and 1.2% black garlic in diet), and cholesterol (0.2-1.2% black garlic in diet) in the HFD groups supplemented with black garlic were significantly decreased compared with those in the HFD group.The levels of TEAC (0.2-1.2% black garlic in diet), GSH (0.6 and 1.2% black garlic in diet), GRd (1.2% black garlic in diet), and GPx (0.2-1.2% black garlic in diet) in the HFD groups supplemented with black