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 [
For screening potential hepatoprotective medicines, toxic chemicals and xenobiotics such as carbon tetrachloride, thioacetamide, paracetamol, and alcohol have been used to generate pathological models [
Garlic (
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 [
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 [
All reagents were provided by Sigma-Aldrich Chemical Company (St. Louis, MO, USA) unless otherwise noted. Carbon tetrachloride was obtained from Merck (1.02223.1000, Merck, Germany) and extra virgin olive oil was purchased from Sigma (W530191, Sigma, USA).
Single clove garlic (
The whole of single-clove garlic (diameter
Fermentation process of single black garlic. In brief, whole bulbs of single clove garlics were fermented at 75°C in relative humidity 90% for 20 days until they obtained the particular black color. The representative samples were collected after 5 days (a), 10 days (b), 15 days (c), and 20 days (d).
For screening the presence of S-allyl cysteine, a well-known bioactive compound of black garlic in aqueous extract of single clove black garlic, we performed LC/MS analysis with the given protocol. In brief, aliquots (20
Eight-week-old male Swiss albino mice were obtained from Pasteur Institute of Ho Chi Minh City, weighting approximately 30–32 g. The animals were randomly divided into polycarbonate cages with 5 mice for each cage. They were housed under standard husbandry conditions with 12 h light-dark cycle (8:00–20:00) for at least 1 week to acclimate with laboratory environment. They were supplied ad libitum with standard chow and distilled water. The experimental procedure was strictly compliance with Declaration of Helsinki (1964). Twenty healthy mice were randomly divided into 4 groups with 5 mice per group and treated as the protocol given below: Group 1 (control group): mice orally received equal volume of saline for 30 days. They also received olive oil at a dose 1 ml/kg body weight via gastric gavages twice per week for 28 days. Group 2 (extract control group): mice were orally received SBE (200 mg/kg body weight) for 30 days. They also received olive oil at a dose 1 ml/kg body weight via gastric gavages twice per week for 28 days. Group 3 (CCl4 intoxicated group): hepatic injury model was induced by CCl4 according to previous study with some modifications [ Group 4 (CCl4 and extract treated group): mice were treated with SBE (200 mg/kg body weight) daily via gastric gavages for 30 days. In addition, they were orally given CCl4 at the dose of 1 ml/kg body weight (in 50% in olive oil) twice per week via gastric gavages for 28 days. In CCl4 treated day, they were treated with SBE one hour before administration of mixture of CCl4 : olive oil.
At the end of experiment (30 days), all experimental animals were fasted overnight to reduce the difference of feeding. The body weights were directly measured using an electronic balance. Subsequently, they were anesthetized using diethyl ether. Blood was collected via cardiac puncture into heparinized tube for biochemical analysis. After that, livers were collected, washed with ice-cold saline, and weighted using an electronic balance. The relative liver weights were calculated by formula (relative liver weight (%) = liver weight/body weight × 100). Subsequently, livers were immediately fixed in 10% formalin for histological studies.
Blood was collected into heparinized tubes, and then the plasma was separated by centrifuge. Lipid profile including triglycerides (TG), total cholesterol (TC), high density lipoprotein cholesterol (HDL-cholesterol), low density lipoprotein cholesterol (LDL-cholesterol), and plasma levels of hepatic enzymes such as alanine aminotransferase (ALT) and aspartate transaminase (AST) were determined using commercial diagnostic kits (Diagnosticum Zrt, Hungary) according to manufacture instructions.
Livers were preserved in 10% formalin and processed for histological studies with Hematoxylin and Eosin staining. The specimens were dehydrated in different grades of alcohol, cleared in xylol, embedded in paraffin wax, sectioned at 4–6 um thick, and stained with Hematoxylin and Eosin. The liver sections then were examined under microscope for estimation of extent of hepatic damage.
Statistical analysis was performed using Statgraphics Centurion XVI software (Statpoint Technologies Inc., Warrenton, Virginia, USA). The data were presented as mean ± standard deviation. Differences between means of different groups were analyzed using ANOVA variance analysis followed with multiple range tests, and the criterion of statistical significance was set as
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 [
The mass spectrometry diagram of S-allyl-cysteine in single clove black garlic extract (7.5 min). The presence of bioactive compound such as S-allyl-cysteine was confirmed by LC/MS. S-Allyl-cysteine exhibited 2 peaks with
In this study, we observed that there was no significant difference between body weight, liver weight, and relative liver weight of normal mice (
Effect of SBE on the body weight, liver weight, and relative liver weight.
Control | SBE control | CCL4 treated | CCl4 + SBE treated | |
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Body weight (g) | | | | |
Liver weight (g) | | | | |
Relative liver weight (%) | | | | |
In previous study, Shin et al. (2014) also reported that administration with black garlic (200 mg/kg body weight) resulted in a significant decrease in liver weight as compared to acute CCl4 intoxicated rats [
A growing body of evidences from previous reports showed that CCl4 intoxication caused alteration on plasma lipid profile [
Effect of SBE on lipid profile in experimental mice.
Control ( | SBE control ( | CCL4 treated ( | CCl4 + SBE treated ( | |
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Triglyceride (mg/dl) | | | | |
Total cholesterol (mg/dl) | | | | |
HDL-cholesterol (mg/dl) | | | | |
LDL-cholesterol (mg/dl) | | | | |
Furthermore, there is no difference in TC, LDL-cholesterol, and HDL-cholesterol between control group and extract control group. However, chronic administration of black garlic extract reduced plasma TG level as compared to control group (
Hepatocellular toxicity is characterized by elevation of alanine transaminase (ALT) and alanine transaminase (AST), both enzymes involved in the transfer of amino groups of aspartate and alanine to ketoglutaric acid [
Effect of SBE on plasma ALT and AST levels in experimental mice.
Control ( | SBE control ( | CCL4 treated ( | CCl4 + SBE treated ( | |
---|---|---|---|---|
ALT | | | | |
AST (U/l) | | | | |
The histological analysis was conducted via HE staining and gross examination for further confirmation of hepatoprotective effects of single clove black garlic extract. In gross examination, livers of normal mice showed a normal appearance with redness and soft texture, and glistering and smooth surface. There is no remarkable difference in gross morphology between livers from normal and SBE control groups. CCl4 intoxication resulted in an enlargement of livers and changes in color (pale-brown), hardness (hard texture), and coarse surface of livers versus the livers from control group. Livers of coadministered CCl4 and SBE mice improved the redness and hardness but showed slightly coarse surface comparing with CCl4 intoxicated mice. The representative pictures of gross examination of livers from normal, SBE control, CCl4 intoxicated, and coadministered CCl4 and SBE mice are presented in Figure
Effect of black garlic extract on gross morphology of liver from experimental mice. In gross examination, livers of normal mice showed a normal appearance with redness and soft texture, and glistering and smooth surface (a). SBE control groups showed no remarkable difference in gross morphology with livers from normal mice (b). Livers of CCl4 intoxicated mice revealed an enlargement of livers and changed in color (pale-brown), hardness (hard texture), and coarse surface of livers as compared with livers from control group (c). Livers of coadministered CCl4 and SBE mice improved the redness and hardness (semihard texture) but appeared slightly coarse surface comparing with CCl4 intoxicated mice (d). Our data support the strong evidence of protective effect of black garlic extract on the change of liver morphology in CCl4 treated mice.
In microscope, liver sections from normal and SBE control mice stained with HE revealed normal and steady architecture of hepatocytes and portal space. CCl4 intoxication led to severe hepatic inflammation with mononuclear cell infiltration surrounding hepatic veins accompanied by partial destruction of adjacent hepatocytes. On the contrary, treatment of SBE improved inflammation response with mild mononuclear cell infiltration surrounding portal space and normal architecture of hepatocytes and portal space (Figure
Effect of black garlic extract on liver histology from experimental mice. Liver sections from normal stained with HE revealed normal and steady architecture of hepatic parenchymal cells and portal space (a). There is no significant difference between liver sections from normal and SBE control mice stained with HE (b). CCl4 intoxication led to severe hepatic inflammation with mononuclear cell infiltration surrounding hepatic veins accompanied by adjacent hepatocyte necrosis (c). Of note, treatment of SBE ameliorated inflammation response with mild mononuclear cell infiltration surrounding portal space with normal architecture of hepatocytes (d). The data demonstrated that black garlic extract attenuated hepatotoxicity and liver inflammation induced by CCl4 administration. The scale bar is presented as 100
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.
Single clove black garlic extract
Triglyceride
Total cholesterol
High density lipoprotein cholesterol
Low density lipoprotein cholesterol
Aspartate transaminase
Alanine transaminase
Hematoxylin-Eosin.
The data sets supporting the results of this article are included within the article and its supplementary files.
The authors declare that they have no financial and competing interests.
Gia-Buu Tran conceived and designed the study and drafted the manuscript. Gia-Buu Tran and Sao-Mai Dam performed the experiments. Nghia-Thu Tram Le handled the research data and conducted the statistical analysis of the data. Gia-Buu Tran interpreted the result, revised the manuscript, and resolved the queries of reviewers. All authors read and approved the final manuscript.
This research project received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
The authors would like to express gratitude to Dr. Thi-Huyen Tran, Dr. Hong-Thien Van, and Dr. Xuan-Uyen Thuy Phan for sharing their pearls of wisdom with them during the course of this research. The authors would like to thank their colleagues from their institution for their assistance during this project.
The raw data that demonstrated the effect of single clove black garlic extract on body weight, liver weight, and relative liver weight were available in Supplementary Data S1. The amelioration effects of single clove black garlic extract on lipid profile and hepatic enzyme were proved via data set in Supplementary Data S2 and S3. Solid material content, S-allyl-cysteine concentration, and pH of single clove black garlic extract were presented in Supplementary Data S4. S1: changes of the body weight, liver weight, and relative liver weight in experimental mice. S2: changes of lipid profile in experimental mice. S3: plasma ALT and AST levels in experimental mice. S4: concentration of S-allyl-cysteine in single clove black garlic extract.