Garlic (Allium sativum
L.) has been used as a spice and traditional medicine for centuries. Many studies have shown that garlic has numerous beneficial effects for human health including antioxidant, anti-inflammation, anti-cancer, lipid regulation, reduction of blood pressure, and improvement of blood glucose control [1
]. Additionally, garlic and its derivatives exhibit hepatoprotective effects against alcoholic hepatic injury, drug-induced hepatic injury, fibrogenic liver disorders, etc. [7
]. For instance, to improve hypoglycemic and hypolipidemic activities, taking 350 mg/kg body weight of garlic extract can significantly decrease the levels of blood glucose, triglycerides, and cholesterol in diabetic rabbits [8
]; for the hepatoprotective effect, male albino rats treated with heated garlic juice at 100 mg/kg/day for four weeks could suppress hepatic oxidative stress via the Nrf2/Keap1 pathway [9
However, the consumption of unprocessed raw garlic is limited due to its characteristic odor and spicy taste, causing the gastric mucosal cells injury with excessive intake [10
]. Fermented black garlic, a kind of heat processed garlic, has been reported for its biological activities such as anti-cancer activity, anti-obesity activity, hepatoprotective activity, and anti-inflammatory activity [1
]. For anti-cancer activity, 100 mg/mL age black garlic (ABG) extract could induce apoptosis in human gastric cancer cells (SGC-7901) [11
]; for anti-obesity activity, the mice given 400 mg/kg BG significantly decreased in body weight, abdominal fat weight, abdominal adipocyte diameters, and abdominal fat pad thickness [12
]; and 200 mg/kg ABG demonstrated hepatoprotective activity by decreasing ALT and AST levels in the liver of Sprague–Dawley rats [13
]. Nevertheless, knowledge of the ingredients or compounds possessing those bioactivities remains unclear.
In our previous study, five solvents (n
-hexane, dichloromethane, ethyl acetate, n
-butanol, and water) were used to separate compounds with different polarity in black garlic [1
]. Results showed that black garlic promoted gastrointestinal motility mainly in the n
-butanol and water fractions. In this study, a similar system was adopted to evaluate whether black garlic extracts may have a hepatoprotective effect.
The development of fibrosis, particularly cirrhosis, is associated with morbidity and mortality [14
]. An animal model of hepatic injury induced by CCl4
has been commonly used to study the hepatoprotective effects of natural medicine, and refers to an ordinary method for the investigation of hepatoprotective activity of different kinds of medicine [15
The increase in serum AST and ALT levels was attributed to impaired liver structure. Generally, cell deaths were associated with plasma permeability as the levels of AST and ALT became outstanding due to hepatic structure damage [16
]. As a result, hepatic injury can be determined by analyzing serum AST and ALT levels. In our study, the levels of AST, ALT, and ALP in serum increased after CCl4
was introduced. This outcome can be attributed to hepatic damage, resulting in an increased rate of synthesis or release of functional enzymes from biomembranes [19
]. Usually, ALT and AST are used to assess liver function; ALT is very accurate in monitoring hepatocellular status, and AST is a sensitive indicator of mitochondrial problems, especially in centrilobular areas of the liver [20
]. Lipid peroxidation has been categorized as one of the most important causes of CCl4
-induced hepatic injury [21
Inflammation was triggered by CCl4
-induced hepatotoxicity, followed by the release of proinflammatory cytokine such as IL-lβ and TNF-α. IL-1β and TNF-α are key cytokines in inflammation and the content of IL-1β and TNF-α increases during the development of hepatic injury [22
]. IL-1β is a proinflammatory cytokine that plays an important role in regulating liver NO synthesis [23
]. TNF-α is produced by Kupffer cells, activates T cells and macrophages, and stimulates secretion of other inflammatory cytokines and NO. Our results indicated that the production of TNF-α and IL-1β was significantly reduced after feeding the BA and WS layers, demonstrating that BA and WS have a hepatoprotective effect on CCl4
-induced damage. MDA was formed due to free radicals attacking the plasma membrane and refers to the end product of lipid peroxidation. In addition, MDA has been widely used as an indicator of lipid peroxidation damage [24
In the latest study, an increase in the hepatic MDA level suggested the enhancement of lipid peroxidation, consequently leading to hepatic damage as well as the inactivation of the antioxidant defense system. To estimate the effect of BA and WS pretreatment on CCl4-induced liver lipid peroxidation, MDA was monitored. In the study, MDA levels were significantly reduced by treatment with BA at 0.2 g/kg and 0.5 g/kg, silymarin (200 mg/kg), and WS (0.2 and 0.5 g/kg).
Reactive oxygen species (ROS) formed in the CCl4
-induced hepatic damage model could significantly decrease through the expressions of antioxidant enzymes such as SOD, GSH-Px, and GSH-Rd. SOD, GSH-Px, and GSH-Rd are antioxidative enzymes that are known to be easily inactivated by lipid peroxides or ROS when CCl4
is administrated [16
]. Therefore, an increase in antioxidant activity and the inhibition of free radical generation is positively correlated with hepatic protection. GSH is a main non-enzymatic anti-oxidant that decreases hydrogen peroxide, hydroperoxide, and xenobiotic toxicity through the oxidation of GSH transforming to GSSG [26
]. GSH content was significantly decreased, which is an important indicator of CCl4
toxicity due to CCl4
administration being prone to reduce the levels of GSH. In this study, the data showed that the level of GSH was increased significantly by treatment with BA, WS, and silymarin.
In the enzymatic defense system, SOD dissimulates superoxide to H2
in order to prevent oxygen toxicity. Both GSH-Px and GSH-Rd are GSH-related enzymes and play a role in detoxification, while antioxidant activity in cellular defense undergoes conjugation with glutathione or the reduction of free radicals [24
]. GSH-Px works with GSH to metabolize H2
, which allows a harmful toxin to become a nontoxic product, while GSH-Rd catalyzes the reduction of GSSG to GSH. Our results revealed that the activities of SOD, GSH-Px, and GSH-Rd were significantly decreased during the development of CCl4
-induced acute hepatic injury [29
], the outcome of which agreed with those of previous studies. In this study, the activities of SOD, GSH-Px, and GSH-Rd improved after treatment with BA and WS. These results suggest that BA and WS reduce ROS production by increasing hepatic antioxidant activities and against hepatotoxicity in order to provide hepatic protection [25
]. The suppression of MDA production is likely to promote the activities of SOD, GSH-Px, and GSH-Rd. Furthermore, an increase in SOD activity not only increases the superoxide anion scavenging capacity, but also prevents peroxynitrite production.
The present study suggests that the black garlic extract with n-butanol fraction can exhibit hepatoprotective activity to attenuate CCl4-induced acute hepatic injury in mice, and this result was proven by the decreased AST and ALT levels and liver histopathological analysis. There was a lack of significant difference in EA treatment. The hepatoprotective mechanisms of black garlic extraction were likely to be related to the decreased MDA level and increased GSH level by increasing the activities of anti-oxidative enzymes such as SOD, GSH-Px, GSH-Rd, and GST. In addition, BA increased the physiological antioxidant activities to render a reduction of hepatic injury caused by free radical (ROS and peroxide) attacks during the metabolism of CCl4. In our research results, both BA and WS could significantly decrease CCl4-induced acute hepatic injury, which caused the reduction in the content of ALT, AST, and MDA, and also significantly decreased liver inflammatory response. Moreover, in our research, black garlic exhibited a hepatoprotective effect by increasing antioxidant enzymatic activity (SOD, GSH-Px, and GSH-Rd) and increasing the content of CAT as well as decreasing CCl4-induced acute hepatic injury.
Furthermore, the flavor, nutritional, and functional properties of the garlic change during the thermal processing of black garlic by the Maillard reaction [30
] as black garlic produced in a high temperature, humid environment matures and reduces the spicy and irritation qualities from raw garlic under control conditions [31
]. γ-glutamyl-S-allyl-L-cysteines (GSACs) and S-allyl-L-cysteine sulfoxide (alliin) are the major sulfur-containing compounds in intact garlic [32
]. After cutting or squeezing, alliinase can be released from the garlic, which hydrolyzes alliin to allicin. However, allicin is an unstable compound; it decomposes to yield organic lipid-sulfur-containing compounds, e.g., diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), and allyl methyl sulfide (AMS), ajoene. Moreover, water-soluble sulfur compounds (S-allylcysteine, SAC) are also formed [5
]. Previous studies have shown that the SAC content in black garlic is six-fold higher than that in fresh garlic. Studies have demonstrated that SAC improved liver disease via regulation of the peroxisomal proliferator activator receptor α (PPAR-α), sterol regulatory element binding protein 1c (SREBP-1c), serum adiponectin levels, liver MDA, reactive oxygen species (ROS) content, serum ALT, serum AST, GSH content, GSH-Px and CAT activities, and the inhibition of lipolysis. Additionally, SAC can significantly inhibit N-nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis [4
]. We further analyzed the SAC content of EA, BA, and WS where, from the data, it was found that SAC only appeared in BA (497.58 ± 43.78 μg/g, Table 1
Therefore, we surmise that SAC is a major active compound in BA [1
]. However, there was no SAC in WS, but our result showed that WS had a hepatoprotective effect. In previous research, we found that WS has abundant polysaccharides as well as great antioxidant activities, e.g., DPPH radical scavenging ability. Thus, we surmised that the main ingredients of hepatoprotective effect in black garlic might be SAC and polysaccharides that inhibit CCl4
-induced hepatic injury by inhibiting lipid peroxidation and inflammation.