Black Garlic Extract on Dyslipidemia and Hepatitis in Chronic Carbon Tetrachloride Intoxicated Swiss Albino Mice
Single clove garlic is the product of atypical bulbing process of garlic under specific conditions. Therefore, the number of researches on single clove garlic bioactivity is limited. Recently, the hepatoprotective effect of single clove garlic has been demonstrated. In this study, we investigated amelioration of single clove black garlic aqueous extract, a processed product from single clove garlic, on dyslipidemia and hepatitis induced by chronic administration of CCl4. Mice were randomly divided into four groups: control, extract control, CCl4 intoxication, and coadministrated CCl4 and extract group. Mice were orally given a dose of 1 ml/kg body weight of CCl4 for 28 days twice a week to establish chronic liver injury model. To evaluate the hepatoprotective effect of single clove black garlic, mice were cotreated with CCl4 and single clove black garlic extract (200 mg/kg body weight) via gastric gauge for 30 days. Cotreatment with CCl4 and extract could improve the changes of body weight, liver weight, and relative liver weight as compared to CCl4 intoxicated mice. Single clove black garlic ameliorated dyslipidemia and the elevation of ALT and AST levels induced by chronic CCl4 intoxication. Histological studies revealed that single clove black garlic could prevent mononuclear cells infiltration and hepatocyte necrosis.
Liver is the vital organ in human body due to its important role in metabolism of endogenous and exogenous molecules, such as lipids, proteins and carbohydrates, and its detoxification functions. Liver is vulnerable to a variety of liver diseases including hepatic steatosis, hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma [1, 2]. It has been suggested that free radicals, reactive oxygen species, and lipid peroxidation serve a pivotal role in pathogenesis of liver diseases . Therefore, antioxidant activity is considered as the key mechanism underlying the protective effect of traditional medicines which prevent and ameliorate hepatic damage in chronic liver diseases. Therefore, a large number of researches investigating identification and isolation natural hepatoprotective compounds have been documented in recent years .
For screening potential hepatoprotective medicines, toxic chemicals and xenobiotics such as carbon tetrachloride, thioacetamide, paracetamol, and alcohol have been used to generate pathological models [5–8]. Among these substances, carbon tetrachloride is the most common used toxicant since it provides an ideal model for studying oxidative hepatic damage due to its distinctive hepatotoxicity and its rapid metabolisms. Furthermore, carbon tetrachloride has also been used in industry such as refrigerant, fire suppression agent, and cleaning solvent; thus the risk of CCl4 exposure has been considered. In liver, carbon tetrachloride is metabolized by hepatic cytochrome P450 leading to production of hepatotoxic metabolites such as trichloromethyl and peroxy radicals, which lead to lipid peroxidation, alteration of cell membrane permeability, and cell death . It has been well documented that CCl4 administration not only leads to fatty liver and hepatocyte necrosis, but also induces accumulation of triglycerides, decrease of reduced glutathione level, membrane damage, and loss of enzyme activity . Furthermore, Hsu and collaborators also suggested that carbon tetrachloride induced liver cirrhosis response was similar to human liver cirrhosis .
Garlic (Allium sativum L.) is a traditional herbal spice and well-known functional food in Vietnamese and Asian cuisines. It has been documented that garlic possesses many bioactive compounds, such as alliin, allicin, allyl-sulfides, ajoene, and 1,2-vinyldithiin, which account for many health benefits such as anticancer, antithrombotic, anti-inflammatory, antioxidant, antimicrobial, cardioprotective, and immune-modulatory activities . Obioha and collaborators have indicated that garlic exerts potential hepatoprotective effect through lowering lipid peroxidation and activating antioxidant defense system . Recently, garlic also is reported for its antihyperglycemic, antihyperlipidemic, and anti-inflammatory effects in type 2 diabetes mellitus associated with obesity patients . However, utilization of raw garlic is strictly limited due to its peculiar flavor and its involvement in hemolytic anemia and gastrointestinal mucosa damage [14, 15].
Black garlic, the processed product which is generated by fermentation process in high temperature and high humidity, has appeared in markets for decades. Black garlic has sweet taste and eliminates unpleasant odor of raw garlic. It possesses many bioactivities including inhibition of colon and gastric cancer cells growth, antioxidant, alteration of lipid profile in diabetes, antiobesity, anti-inflammatory, and antiallergic activities [16–18]. Furthermore, black garlic extract has proven its ameliorating effect on Aβ-induced neurotoxicity and cognitive impairment . Some reports also indicate that black garlic and its bioactive constituent, S-allyl-cysteine, employ hepatoprotective effects in alcohol, D-galactosamine, high fat diet, and acute carbon tetrachloride intoxicated models [20–22]. Of note, the quality and bioactive value of black garlic are diverse depended on processing method and garlic cultivars [18, 23, 24].
Due to cultivation practices and climate conditions, the bulbs of garlic sometime are not divided into cloves and generates single clove of garlic, known as single clove garlic, solo garlic, and pearl garlic. The number of researches of single clove garlic bioactivity is limited. Recently, Naji et al. (2017) suggested that single clove garlic exerted the higher hepatoprotective effect than normal garlic, which was known as “multiclove garlic” in CCl4 intoxicated rabbit model . Furthermore, the beneficial effect of multiclove black garlic on acute carbon tetrachloride intoxicated rat was proved . To our knowledge, the effects of single-clove black garlic (SBE) on dyslipidemia and liver injury in hepatic pathology model have not been studied yet. In this study, we investigated the hepatoprotective effect of single clove black garlic aqueous extract on dyslipidemia and hepatitis induced by chronic administration of carbon tetrachloride in Swis
Results and Discussions
3.1. Determination of S-Allyl-Cysteine Content in Single Clove Black Garlic Extract
There is a growing body of evidences linking black garlic with amelioration of human diseases, especially in liver disease. In previous study, Seo et al. (2009) suggested that black garlic extract could improve the lipid profile in type 2 diabetes mellitus . Moreover, Jung et al. (2014) reported that consumption of black garlic extract increased HDL-cholesterol as well as ratio of LDL-cholesterol/lipoprotein B and exerted cardioprotective effect in hypercholesterolemic patients . Kim et al. (2011) also proved that black garlic extract could protect liver from liver damage induced by chronic alcohol consumption . Hepatoprotective effect of black garlic in carbon tetrachloride, D-galactosamine, and high fat diet treated rodents was demonstrated in previous report . However, single clove black garlic aqueous extract composition and the effect of long-term administration have not been considered yet. S-Allyl-cysteine, an important sulfur-containing constituent of garlic, accumulates during manufacturing process of black garlic and exerts many biofunctions . Therefore, S-allyl-cysteine content is an excellent indicator to evaluate the quality and bioactive value of black garlic. In this study, we examined S-allyl-cysteine concentration for primary evaluation of bioactive value of single clove black garlic produced by our institutional procedure. It has been found that S-allyl-cysteine existed in retention time about 7.5 min with 162.06 [M+H] and 145.03 / [M-H2O+H]. The mass spectra diagram at retention time 7.5 min is presented in Figure 2. S-Allyl-cysteine concentration in extract ( μg/g dry weight) is comparable with concentration of S-allyl-cysteine concentration of black garlic juice ( μg/g dry weight) which was produced from previous report and exhibited antidiabetic activity . Furthermore, Kodai et al. (2015) have proven that S-allyl-cysteine, an important sulfur-containing constituent of black garlic, prevents liver fibrosis induced by carbon tetrachloride in rats . Taken together, the data imply that single clove black garlic extract may have the beneficial effect for prevention of liver injury.
3.2. Effect of Single Clove Black Garlic Extract on Body Weight and Relative Liver Weight
In this study, we observed that there was no significant difference between body weight, liver weight, and relative liver weight of normal mice ( g, , and , resp.) and SBE administrated mice (, , and , resp., ). These data implied that SBE administration did not cause the change of body weight, liver weight, and relative liver weight. On the contrary, administration of CCl4 caused a marked reduction of body weight ( g) along with a notable increase of liver weight ( g) as compared to normal mice (). Moreover, relative liver weight of CCl4 treated group was significantly increased versus control group (% versus % resp., ). These results were consistent with those reported in previous studies [26, 27, 31, 32]. Of note, SBE treatment resulted in the recovery of body weight ( g) and reversed the change of liver weight ( g) of CCl4 intoxicated mice (). A remarkable decrease of relative liver weights (%) was observed in CCl4 and SBE cotreated mice as compared to CCl4 treated mice (). Moreover, no significant difference of body weight, liver weight, and relative liver weight between control group and coadministered CCl4 and extract group was observed (Table 1). These results shed light on the protection effect of black garlic extract in the changes of body weight, liver weight, and relative liver weight induced by chronic CCl4 administration.
3.3. Effect of Single Clove Black Garlic Extract on Plasma Lipid Profile
A growing body of evidences from previous reports showed that CCl4 intoxication caused alteration on plasma lipid profile [31–33]. In our study, CCl4 treatment resulted in significant increase of TG (195.10 ± 20.34 mg/dl), TC (212.29 ± 12.47 mg/dl), and LDL-cholesterol (124.18 ± 6.17 mg/dl) levels accompanied with a remarkable decrease of HDL-cholesterol (49.09 ± 6.41 mg/dl) versus control group (123.55 ± 16.63, 134.04 ± 8.58, 41.67 ± 2.97, and 67.66 ± 4.46 mg/dl, resp., ). Of note, coadministration SBE and CCl4 to mice caused a significant decrease in plasma TG, TC, and LDL-cholesterol accompanied with a notable increase in plasma HDL-cholesterol level in comparison with CCl4 group. Furthermore, SBE could improve the LDL-cholesterol level in CCl4 treated mice but LDL-cholesterol level in coadministered CCl4 and SBE group was higher than control group ( mg/dl versus mg/dl, resp., ). We also observed a significant difference in plasma total cholesterol levels between coadministered CCl4 and SBE mice versus normal mice ( mg/dl versus mg/dl, resp., ). Collectively, these results suggested that black garlic extract administration could improve in lipid profile of CCl4 intoxicated mice and decreased plasma TG level in control mice (Table 2). Furthermore, Asdaq (2015) proved that aged garlic extract and its constituent, S-allyl-cysteine, exerted antioxidative and hypolipidemic effects on high fat diet treated rats . Therefore, the high concentration of S-allyl-cysteine in extract may elucidate the improvement of single clove black garlic on the alteration of blood lipid profile in SBE and CCl4 treated. To our knowledge, the effect of single clove black garlic extract on lipid profile in CCl4 intoxication has not been elucidated yet. Thereby, these results proved in the first time the amelioration of single clove black garlic on the change of lipid profile induced by chronic CCl4 administration
In conclusion, our results demonstrated that single clove black garlic aqueous extract exerted the beneficial effects on dyslipidemia and hepatitis induced by chronic carbon tetrachloride administration, a common toxin induced liver disease model, and provided more information about the bioactivity of single clove black garlic.
Research Article | Open Access
Volume 2018 |Article ID 9383950 |
Gia-Buu Tran,Sao-Mai Dam, and Nghia-Thu Tram Le
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