Exploring the Nutritional Profiling and Therapeutic Effect of Fermented Garlic on Alcohol-Induced Liver Injury in Animal Model

Abstract

Black garlic, a fermented product of fresh garlic, has shown promising potential as a culinary ingredient and a medicinal remedy. This study examined the microbiological makeup, nutritional profile, and health advantages of black garlic to better understand its health-endorsing properties. Thermus, Corynebacterium, Streptococcus, and Brevundimonas were among the prominent taxa found when the microbial diversity in black garlic samples was investigated using Illumina MiSeq sequencing. This provided insight into the complex interactions between microorganisms during the fermentation process and clarified the distinctive qualities of black garlic. This study expanded its scope to include black garlic’s therapeutic potential, specifically in relation to liver function and hangovers caused by alcohol, in addition to its microbial complexity. Significant liver damage was revealed in alcohol-treated rats by serum biochemical indicators and histological stains; this damage was lessened by the administration of black garlic, particularly at higher dosages. Furthermore, black garlic showed hepatoprotective effects attributed to its high phenolic and flavonoid contents. These results offer a novel understanding of the medicinal qualities of black garlic as they lay out possibilities for the creation of functional drugs to treat alcohol-induced liver damage. Conclusively, black garlic’s diverse microbial composition also advances our knowledge of its nutritional makeup and health advantages. In summary, this research highlights the potential of black garlic as a flexible medical tool, having implications for both gastronomic and therapeutic uses.

1. Introduction

Empirical evidence suggests that excessive alcohol consumption can lead to adverse physical and psychological effects, such as an increased risk of cancer, liver disease, and experiencing hangovers [1]. Hangovers are unpleasant experiences characterized by headache, diarrhea, exhaustion, anxiety, and short-term memory loss [2]. In this context, several studies have been conducted to find a cure for hangovers. Most conventional remedies in this regard only promote ethanol oxidation, leaving behind recovery from ethanol-induced liver damage. Thus, it is necessary to develop a new functional chemical that is effective in reducing hangovers and repairing liver damage [3].

The metabolism of ethanol is carried out by two liver enzymes, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). ADH breaks down alcohol into acetaldehyde, a carcinogen, while ALDH quickly converts acetaldehyde to acetate. Acetate is further broken down into carbon dioxide and water [4]. However, the microsomal ethanol-oxidizing system (MEOS) and catalase can metabolize 10–20% of the ingested ethanol, producing reactive oxygen species (ROS) that damage the liver. Ethanol oxidation by the MEOS can be aided by the activation of ADH, which protects the liver from oxidative stress caused by free radicals [5].

It has been noticed that ethanol-induced liver injury can lead to liver diseases such as fatty liver, hepatitis, and hepatocirrhosis, an irreversible condition with a 50% lower survival rate in five years [1]. The demand for alternative treatments is increasing due to the limitations of steroid and pilin treatments, which have numerous adverse effects. Natural bioactive compounds derived from items such as pine needles, bamboo, bean sprouts, and raisin trees have been studied for their potential to ease hangovers or correct liver damage [6].

Ethanol-induced liver damage is caused by the generation of free radicals resulting from three metabolic pathways: alcohol dehydrogenase, the microsomal ethanol oxidation system, and catalase [7]. In addition, microsomal ethanol oxidation in the mitochondria is catalyzed by cytochrome P450 2E1 (CYP2E1) isoenzymes, which generate reactive oxygen species (ROS) and a large amount of H₂O₂ [8]. Peroxisomal activity also contributes to ethanol oxidation in the liver, particularly in individuals who consume high amounts of ethanol, leading to the accumulation of fatty acids in the liver due to increased fatty acid peroxisomal oxidation. The oxidative stress caused by ethanol metabolism contributes to the development of hepatotoxic effects [9].

The process of bioconversion, which employs microbes or enzymes present in food, has been found to enhance human health by producing low molecular weight compounds that can be readily absorbed and used in various metabolic functions [10]. Black garlic, a form of garlic that is made by aging the whole garlic at high humidity and temperatures, has been identified as having higher antioxidant properties than raw garlic and has been proven to aid in the prevention of cancer, diabetes, and other non-communicable diseases [11]. Additionally, it has been observed to provide a protective effect against chronic liver impairment induced by alcohol in rats [12].

Numerous bioactive chemicals found in black garlic are thought to have preventive properties against liver damage. One of black garlic’s main active ingredients, S-allyl cysteine (SAC), has been demonstrated to have strong anti-inflammatory and antioxidant capabilities [13]. Studies have shown that SAC can defend the liver against a variety of injuries, including those brought on by chemicals, alcohol, and non-alcoholic fatty liver disease [14]. SAC accomplishes these benefits by reducing liver inflammation and oxidative stress while also encouraging liver cell regeneration. Additionally, black garlic includes a variety of flavonoids, such as S-allyl mercaptocysteine (SAMC), diallyl sulfide (DAS), and diallyl disulfide (DADS), which cooperate with organosulfur compounds including kaempferol, myricetin, apigenin, and luteolin [15].

In this research paper, we aim to provide new insights into the therapeutic properties of black garlic and its potential use as a functional substance for liver protection and hangover relief. This study will contribute to the development of new natural remedies for liver injury induced by alcohol consumption, which could have significant implications for public health.

2. Materials and Methods

2.1. Materials and Reagents

Fresh garlic was obtained from the local market, while the reagents, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,20-azinobis-3-ethylbenzothiazoline-6-sulfonic acid, were procured from Sigma-Aldrich Co (North Brunswick, NJ, USA). Additionally, all other chemicals and solvents used in this study were of analytical grade and were acquired from Fisher industrial Co., Ltd. (Tustin, CA, USA).

2.2. Preparation of Black Garlic

Garlic cloves were purchased from the local market and cleaned to remove any dirt and extra skin. The method mentioned in Wang’s paper was followed to produce black garlic with some modifications [16]. The garlic was placed in a temperature-controlled chamber at 70 °C and a 90% relative humidity for 45 days and then stored at −20 °C for further use.

To produce black garlic powder, the following procedure was followed. Initially, 100 g of black garlic was prepared by chopping and grinding it into a fine form, followed by drying it in a hot-air oven at 60 °C for a few hours and then grinding it into a fine powder. The resulting powder was stored at −80 °C until further experiments were required to proceed. In a conical flask, the garlic powder was dissolved in 100 mL of methanol, and the resulting mixture was filtered with filter paper to obtain a 100% concentration extract. The filtrate was then stored at 4 °C until use, while the residue was weighed, which amounted to 10 g. The concentration of the black garlic extract was equivalent to 90 g of garlic in 100 mL of distilled water, which is 0.9 g per 1 mL.

2.3. Proximate Analysis of Black Garlic

Using the recommended techniques from the Association of Official Analytical Chemists (AOAC), the nutritional content of fermented garlic was assessed. These techniques included measuring the amount of moisture, ash, protein, and fat [17]. Equations (1) and (2) can be used to estimate the energy and carbohydrate content.

Total carbohydrate (g∕100 g) = 100 − (water + ash + protein + fat)

(1)

Energy (kcal∕g) = 9 (fat) + 4 (protein) + 4 (carbohydrate)

(2)

2.4. Determination of Antioxidant Content

2.4.1. DPPH Analysis

The DPPH assay was used to analyze the antioxidant activity of the black garlic extract. A mixture of a 0.2 mM DPPH solution and different concentrations of the extract (ranging from 10 to 50 µg/mL) was prepared by adding 1 mL of the black garlic extract. The mixture was then incubated in a dark room at room temperature and shaken. After incubation, the absorbance was measured at 517 nm using a spectrophotometer (Agilent Technologies, Camspec Model M550) (Santa Clara, CA, USA). The fraction of inhibition activity was calculated using the following equation:

$$\begin{array}{cc}\hfill DPPHscavenging& Activity\left(RSA\right)\hfill \\ \hfill =& (Acontrol-Asample)/\left(Acontrol\right)\times 100\hfill \end{array}$$

(3)

The control signifies the absorbance of the DPPH radical in the absence of any sample. The sample refers to the absorbance of the DPPH radical at various concentrations of black garlic. A graph was plotted using the scavenging activity against the concentration of the black garlic extract, and the concentration that caused 50% inhibition was determined. The experiment was conducted in triplicate, and the mean values were considered for the final data interpretation [18].

2.4.2. Total Phenolic Content

The total phenolic content of black garlic was determined by following the standard method mentioned in [19]. In this protocol, the Folin–Ciocalteau reagent was used according to the method using gallic acid as a standard phenolic compound. A 0.1 mL extract solution containing 1.0 g of the extract was added to a volumetric flask filled with 46 mL of distilled water. Then, 1.0 mL of the Folin–Ciocalteau reagent was added and mixed thoroughly. After 3 min, 3 mL of sodium carbonate was added to the solution, and it was kept in a shaker for 3 h. The absorbance of the blue color was measured at 760 nm. The concentration was determined by estimating the µg of gallic acid equivalent using an equation obtained from standard gallic acid.

2.4.3. Antioxidant Activity

To conduct the antioxidant activity measurement, a range of concentrations of black garlic (10 to 50 µg/mL) were prepared. For each concentration, ethanol and a sample were combined with a mixture consisting of 2.88 mL of a 2.5% linoleic acid solution and 9 mL of a 40 mM phosphate buffer in a vial. The vial was then incubated at 40 °C for 96 h. At 12 h intervals during the incubation period, 0.1 mL of each vial was diluted with 9.7 mL of 75% ethanol, 0.1 mL of ammonium thiocyanate, and 0.1 mL of FeCl₂. The absorbance of the resulting samples was measured at 500 nm, and the percentage inhibition (i.e., the capacity to inhibit peroxide formation in linoleic acid) was calculated using a specified equation [20].

2.4.4. Fe³⁺-Reducing/Antioxidant Power

The procedure described in [21] was followed. The ferric-reducing antioxidant power reagent was prepared by mixing 300 mM acetate buffer, 10 mL of TPTZ in 40 mM HCl, and 20 mM FeCl₃·6H₂O in a ratio of 10:1:1 at 37 °C. Trolox was used as the standard and distilled water as the blank control. The Trolox concentration was selected under the condition of absorbance values ranging from 10 to 50 µg/mL to draw a standard curve. A freshly prepared FRAP reagent (900 µL) was mixed with either 90 µL of water or a 10 to 50 µg/mL concentration of a sample with a standard blank solution. The maximum absorption at 595 nm was read every 15 s using a spectrophotometer, while the temperature was maintained at 37 °C. The readings were noted, and after 30 s, the readings were included in this study.

2.5. Metagenomic Analysis

A DNA extraction kit was used to obtain DNA. Black garlic samples were first homogenized in a NaCl solution. The extracted DNA was kept at −20 °C after being eluted in a buffer to improve the samples’ bacterial communities. Buffering peptone water was combined with 1 g of each sample. The samples were incubated for 24 h at 35 °C at 200 rcf of shaking. Following that, the rich culture samples were centrifuged at 5000 rcf for 5 min. The pellets were again suspended in an enzyme-containing buffer solution. The material was incubated at 37 °C for the entire night, and the Liu method was used to extract the DNA [22]. Following that, primers were used to conduct metagenomic analysis in accordance with protocols, and built-in software (Gene Mark S2) was used to examine the results.

2.6. Animal Experiment

All animals were purchased from the Institute of Biochemistry and Biotechnology at the University of Veterinary and Animal Sciences, Lahore, and housed at 23–24 °C with a 12/12 h light/dark cycle. Albino rats weighing between 250 and 300 g and aged 8 weeks were used in this study. All the animals were housed in appropriate cages in a controlled environment and properly labeled. Standard feed pellets and water were provided to all the rats for 2 weeks. The rats were divided into 4 groups, each group consisting of 8 rats, and their weight and feed were monitored every week throughout the trial. Ethanol was used to induce liver damage in the diseased groups by administering ethanol, which was calculated based on their weight (15 mL/kg of body weight). The control group was given a saline solution instead of ethanol as a non-induced control. The other groups were given ethanol and black garlic powder throughout the trial. The black garlic dosage was estimated using the human equivalent dosage (HED). The dose was administered to rats using the gavage technique. The trial duration period was 4 weeks. After the end of the trial, the rats fasted for 12 h, they were anesthetized during the postabsorptive period, and their blood was centrifuged to separate the plasma from the blood for further testing. The livers were washed with saline and fixed with 10% neutral-buffered formalin for histopathological analysis.

2.7. Serum Analysis

The lipid profiles, including total cholesterol, low-density lipoprotein (LDL), very-low-density protein (VLDL), high-density lipoprotein (HDL), and triglycerides, in the sera were evaluated using commercial kits from Sigma Aldrich. The absorbance of the samples was measured at 505/670 nm using a UV spectrophotometer, and the results were reported in mg/dL. Liver function parameters such as aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), albumin (ALB), bilirubin, and the AG ratio were determined using kits from Sigma Aldrich, following the instructions provided by the manufacturer. The evaluation of antioxidant enzyme activities, including superoxide dismutase (SOD; EC 1.15.1.1) and glutathione peroxidase (GPx; EC 1.11.1.9), was conducted.

2.8. Histopathological Slides

The tissue specimens were fixed in 10% formalin overnight, followed by dehydration in ethanol with increasing concentrations from 70% to 100%, and clearance in xylene. Next, the tissues were embedded in paraffin wax blocks. The samples were sectioned to a thickness of 4 μm by a microtome (mrc-HIS-2268) and were mounted on glass slides. To remove the paraffin, the slides were immersed in xylene for 3 min, followed by a sequence of ethanol concentrations: 100% for 1 min, 96% for 1 min, 80% for 1 min, and 60% for 1 min. The slides were then stained with hematoxylin and eosin and mounted using the DPX mounting medium under coverslips. Finally, the histological changes in the tissue sections were examined and captured using the BA410E light microscope.

2.9. Statistical Analysis

The data analysis was conducted using GraphPad Prism 8.0 and SPSS version 25. One-way analysis of variance (ANOVA) was performed, followed by a post hoc Dunnett’s test for multiple comparisons. A statistically significant outcome was defined as a p-value of less than 0.05.

3. Results

3.1. Proximate Analysis

The proximate analysis of black garlic presented herein provides a comprehensive breakdown of its constituent components, offering valuable insights into its nutritional profile. The moisture content, representing the percentage of water in the samples, was determined to be 25.53%. This finding underscores the significance of water as a major component in black garlic. The ash content, indicative of the inorganic mineral residue remaining after complete combustion, was measured at 1.80%, reflecting the mineral composition of the garlic. Crude protein, a key nutritional component, constituted approximately 7.11% of black garlic. While this denotes a moderate protein concentration, it contributes to the overall nutritional value of the product. The presence of dietary fiber was evident, with a crude fiber content of 2.42%, highlighting the potential health benefits associated with the consumption of black garlic. In contrast, the fat content in black garlic was notably low, with crude fat accounting for only 0.02%. This characteristic aligns with the broader understanding of garlic products being low in fat. Carbohydrates, comprising 64% of the total, formed a substantial portion of black garlic, serving as a significant energy source.

These results offer crucial information that paves the way for further exploration into the health advantages and culinary applications of black garlic. The comprehensive nature of the proximate analysis allows for a nuanced understanding of the nutritional composition of this unique garlic variant.

3.2. DPPH Analysis

The findings show that the samples’ antioxidant activity increased in a dose-dependent manner, with greater activity occurring at higher concentrations. The samples showed an %RSA value of 77.19% at the highest concentration tested (50 g/mL), suggesting high antioxidant activity. High antioxidant activity was also observed at the standard value (ascorbic acid), with %RSA values ranging from 43.1% to 66.5%. These results indicate that the samples may be used as a natural antioxidant in a variety of applications. The samples had high antioxidant activity, with increasing efficacy at higher doses, according to the results of the DPPH study (Figure 1).

3.3. Total Phenolic Content

As the concentration of black garlic increased, the TPC values also increased. The highest TPC value of 1.202 mg of GAE/g of the dry plant extract was observed at the highest concentration of 50 µg/mL. However, the gallic acid standard exhibited significantly higher TPC values than black garlic at all the concentrations tested, with values ranging from 0.612 mg to 2.05 mg of GAE/g of dry plant extract. Similarly, the GAE/mL values for black garlic also increased with the increasing concentration, indicating the presence of higher concentrations of phenolic compounds in the samples. The highest GAE/mL value for black garlic was recorded as 27.31 µg/mL at a concentration of 50 µg/mL (Figure 2).

3.4. Antioxidant Activity

According to the findings, black garlic and BHT both had %RSA values that rose with the concentration. The %RSA values for black garlic and BHT were 62.1% and 72.1%, respectively, at the maximum concentration of 50 g/mL. At all tested concentrations, black garlic’s %RSA values were lower than those of BHT. The %RSA values for black garlic, however, were more than half that of BHT at the maximum dose of 50 g/mL, indicating substantial antioxidant action. Overall, the findings imply that black garlic has the potential to be employed as a natural antioxidant and demonstrates modest antioxidant activity (Figure 3).

3.5. Fe³⁺-Reducing/Antioxidant Power

With an increasing concentration, the percentage inhibition levels for both black garlic and Trolox dropped. Black garlic’s percent inhibition value was 5.7% at the maximum concentration of 50 g/mL, compared with Trolox’s of 13.0%. The black garlic sample results rose with the concentration, peaking at 0.336 at 50 g/mL. At all tested concentrations, it was discovered that the standard value for ferrous chloride was greater than the values for both black garlic and Trolox. Overall, the findings show that black garlic’s antioxidant activity is less effective than Trolox’s at reducing Fe³⁺ (Figure 4)

3.6. Metagenomic Analysis

A diversified microbial population was revealed by the metagenomic study of black garlic, offering insights into the bacterial colonies that flourish in this fermented foodstuff. Staphylococcus epidermidis was the most common species among those found, appearing in several samples, with colony counts ranging from 19 to 80. Though in lesser quantities, Staphylococcus warneri and Staphylococcus capitis were also observed. With species including Bacillus sonorensis, Bacillus methylotrophicus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus megaterium, Bacillus mojavensis, Bacillus pumilus, and Bacillus altitudinis, the Bacillus genus was one of the most widely distributed taxa. Different samples showed different patterns for these bacterial communities, indicating that the microbiota of black garlic is influenced by a range of environmental factors and fermentation conditions. This metagenomic snapshot lays the groundwork for future investigations into the microbial dynamics that give black garlic its distinct qualities (Figure 5).

3.7. Hepatoprotective Trial of Black Garlic

Effect of Black Garlic on Weight of Animals

The objective of this study was to investigate the effects of fermented garlic powder on the weight of rats with alcohol-induced liver injury over a 6-week period. As shown in

Table 1. Effect of fermented garlic on body weight of rats with alcohol-induced liver injury.

Group	1st Week	2nd Week	3rd Week	4th Week	5th Week	6th Week
G-I	250.23 ± 2.1 b	258.00 ± 0.53 b	267.00 ± 1.8 b	273.00 ± 1.2 b	288.00 ± 0.9 b	291.63 ± 0.74 b
G-II *	250.52 ± 0.75 a	255.88 ± 0.83 a	262.88 ± 1.3 a	270.88 ± 1.5 a	274.88 ± 1.2 a	285.00 ± 1.4 a
G-III	255.12 ± 1.3 b	260.00 ± 0.75 b	266.00 ± 1.1 b	272.00 ± 0.9 b	278.00 ± 1.1 b	281.75 ± 0.75 b
G-IV *	250.25 ± 2.1 a	256.63 ± 0.91 a	264.63 ± 1.1 a	269.63 ± 1.2 a	276.63 ± 0.9 a	278.75 ± 1.2 a

The values are means ± SD (n = 8). Values marked with different lower-case letters in superscript format indicate a significant difference at p-value ˂ 0.01 by Duncan’s multiple-range test; values marked with the same lower-case letters in superscript format indicate no significant difference. G-I (control), group II (high-fat diet), group III (high-fat diet + fermented garlic powder (level 1)), and group IV (high-fat diet + fermented garlic powder (level 2), * significant value.

, the results revealed a significant difference (F = 321.10; p ˂ 0.001) in weight between the groups. The rats in group II had the lowest weight throughout this study compared with the control group. The rats in groups III and IV showed a moderate increase in weight over time, with no significant difference between them. Overall, there was a significant increase in the weight of rats given fermented garlic. By the end of week 6, group IV showed a significant improvement in weight compared with group III.

3.8. Serum Lipid Profiles

Table 2. Effect of fermented garlic on serum lipid profiles of rats with alcohol-induced liver injury.

Group	Cholesterol (mg/dL)	Triglycerides (mg/dL)	LDL Cholesterol (mg/dL)	HDL Cholesterol (mg/dL)	Non-HDL Cholesterol (mg/dL)
G-I	63.50 ± 0.41 a	92.00 ± 0.45 a	70.13 ± 0.44 c	23.88 ± 0.27 c	37.88 ± 0.51 a
G-II	165.25 ± 0.42 c	130.75 ± 0.45 d	91.25 ± 0.43 d	14.50 ± 0.27 a	63.75 ± 0.52 d
G-III	80.00 ± 0.41 b	114.38 ± 0.45 c	63.00 ± 0.44 b	19.63 ± 0.27 b	54.75 ± 0.51 c
G-IV	72.88 ± 0.41 b	109.25 ± 0.45 b	59.63 ± 0.44 a	22.00 ± 0.28 b	51.88 ± 0.51 b

The values are means ± SD (n = 8). Values marked with different lower-case letters in superscript format indicate a significant difference at p-value ˂ 0.01 by Duncan’s multiple-range test; values marked with the same lower-case letters in superscript format indicate no significant difference. G-I (control), group II (high-fat diet), group III (high-fat diet + fermented garlic powder (level 1)), and group IV (high-fat diet + fermented garlic powder (level 2)).

shows the lipid profiles of the four different groups of rats. It shows significant differences in cholesterol (F = 13,426.76; p ˂ 0.001), triglycerides (F = 1217.35; p ˂ 0.001), HDL cholesterol (F = 203.56; p ˂ 0.01), LDL cholesterol (F = 1019.95; p ˂ 0.001), and non-HDL cholesterol (F = 454.10; p ˂ 0.001). The first group, which served as a control, had the lowest levels of cholesterol, triglycerides, LDL cholesterol, and non-HDL cholesterol, while the HDL cholesterol levels were the highest. The second group, which was the diseased group, had significantly higher levels of all the lipid parameters compared with the control group, with the highest levels of triglycerides and non-HDL cholesterol. The third and fourth groups, which received black garlic, showed improvement in their lipid profiles compared with the diseased group. Both groups had lower levels of cholesterol, triglycerides, LDL cholesterol, and non-HDL cholesterol compared with the diseased group, and the levels were closer to those of the control group. However, G-III showed slightly better results compared with G-IV, with lower levels of HDL cholesterol, which is considered good cholesterol, but also with slightly higher levels of non-HDL cholesterol. Overall, the results suggest that the treatment with black garlic (level 2) may have the potential to improve lipid profiles in rats with lipid abnormalities.

3.9. Liver Biomarker Assessment

The results for several liver function test parameters (bilirubin, ALT, AST, alkaline phosphate, protein, albumin, and the AG ratio) in various groups are shown in the following

Table 3. Effect of fermented garlic on liver function parameters of rats with alcohol-induced liver injury.

Group	Bilirubin (mg/dL)	ALT (U/L)	AST (U/L)	Alkaline Phosphate (U/L)	Protein (g/dL)	Albumin (g/dL)	AG Ratio
G-I	0.06 ± 0.09 a	35.50 ± 0.23 a	97.75 ± 0.25 a	198.88 ± 0.21 a	6.95 ± 0.03 d	5.03 ± 0.04 a	1.15 ± 0.01 c
G-II	0.46 ± 0.9 b	81.38 ± 0.22 d	175.63 ± 0.25 d	300.75 ± 0.21 d	4.10 ± 0.03 a	4.85 ± 0.04 d	2.62 ± 0.01 d
G-III	0.08 ± 0.09 a	39.75 ± 0.23 c	120.75 ± 0.25 c	269.50 ± 0.21 c	5.65 ± 0.03 c	4.25 ± 0.04 c	1.03 ± 0.01 b
G-IV	0.09 ± 0.09 a	37.38 ± 0.23 b	114.63 ± 0.25 d	245.63 ± 0.21 d	5.41 ± 0.03 b	4.06 ± 0.04 b	0.96 ± 0.01 a

The values are means ± SD (n = 8). Values marked with different lower-case letters in superscript format indicate a significant difference at p-value ˂ 0.01 by Duncan’s multiple-range test; values marked with the same lower-case letters in superscript format indicate no significant difference. G-I (control), group II (high-fat diet), group III (high-fat diet + fermented garlic powder (level 1)), and group IV (high-fat diet + fermented garlic powder (level 2)).

. There were significant differences noticed in bilirubin (F = 435.61; p ˂ 0.001), ALT (F = 11,705.26; p ˂ 0.001), AST (F = 17,272.12; p ˂ 0.001), alkaline phosphate (F = 40,315.10; p ˂ 0.001), protein (F = 988.984; p ˂ 0.001), albumin (F = 2963.26; p ˂ 0.001), AG ratio (F = 3096.33; p ˂ 0.001) in the given table. Except for protein and albumin, group I exhibited the lowest levels across all measures, pointing to normal liver function. Protein and albumin levels were greater in group II, indicating liver damage brought on by a disease induced by alcohol toxicity. Apart from alkaline phosphate levels, which were greater and could indicate cholestasis or bile flow restriction, group III revealed results that were identical to those of group 1 for other measures. With readings identical to those of group III but with, to some extent, lower alkaline phosphate and improved ALT and AST levels, group IV may have had improved liver function parameters compared with group II.

3.10. Liver Antioxidant Enzymes

According to the findings presented in Figure 6, all groups’ levels of SOD (F = 60.765; p ˂ 0.001) and GPX (F = 97.350; p ˂ 0.001) were greater than those in the control group. The greatest levels of SOD (2.67) and GPX (16.25) were found in group III, which was followed by group IV, with a SOD level of 2.53 and a GPX level of 18.375. With a SOD level of 2.30 and a GPX level of 18.5, group I had the greatest concentrations of both enzymes compared with the control group. With a SOD level of 1.81 and a GPX level of 11.75, group II had the lowest levels of both enzymes of any group. The considerable drop in these enzymes’ levels in group II could be a result of oxidative stress brought on by the intervention in this group. Overall, these outcomes show that the intervention was effective in maintaining oxidative stress.

3.11. Histopathological Examination

Microscopic analysis of the hepatic parenchyma in both the control and black garlic-treated groups revealed a conventional lobular architecture characterized by the distribution of hepatocytes within hepatic cords emanating from the central vein and segregated by clearly defined blood sinusoids. The sinusoidal endothelium comprised endothelial lining cells, which incorporated phagocytic Kupffer cells, thereby underscoring their crucial role in hepatic function. The individual hepatocytes were polyhedral in shape, with a central round nucleus, and some of them displayed binucleation (as shown in Figure 7a). Meanwhile, the administration of cyclophosphamide caused multiple histopathological changes, including leukocytic infiltration (as illustrated in Figure 7b), venous congestion, cytoplasmic vacuolation, and interlobular hemorrhage. Some hepatocytes exhibited pyknosis, while others displayed necrosis. However, the examination of rats treated with aged black garlic revealed a remarkable improvement in hepatic tissues compared with the other groups. The hepatocytes were rearranged in radiating strands, the nuclei were normal, and the cytoplasm did not show any vacuoles or fat droplets (as depicted in Figure 7c,d).

4. Discussion

The antioxidative activity of black garlic extracts was analyzed by their capability to lacerate a violet-colored compound, DPPH, which is a free radical that estimates the hydrogen-donating capacity of natural products. The DPPH-scavenging activity of black garlic extract was found to be three times higher than that of the standard, which can be attributed to the presence of organosulfur compounds and their oxidoreduction properties. Although garlic also exhibited DPPH-scavenging activity, its ability to reduce DPPH levels was much lower than that of the aged garlic extract, which is consistent with previous reports [23]. The contents of total polyphenols in black garlic (ranging from 55 to 273 GAE/g) were not only found to be significantly higher compared with those in raw garlic [24] but also displayed a significant increase compared with a previous study [25]. These augmented phenolic acids could possibly be linked to the increase in the total acid contents in black garlic. According to [26], the heating action of phenolic compounds leads to a rise in the free content of phenolic acids while simultaneously diminishing the ester and its related bound fractions, which leads to a hike in free phenol forms.

In the ferric-reducing antioxidant power potential (FRAP) assay, which is a colorimetric assay used to estimate the capacity of sample electrons, the black garlic extract exhibited a similar electron-donating capacity to raw garlic [27]. This suggests that the metal chelation efficacy of raw and aged garlic extracts is contingent on phytochemicals and phenolic compounds, as opposed to organosulfur compounds. Nonetheless, this result specifies that black garlic extract has slightly higher FRAP activity, which suggests that its phenolic content is also high [16,28]. In contrast, a phylogenetic study of black garlic processing samples based on OTU abundance showed a different picture [29]. There were notable distinctions between the two methods as the microbiome of these samples was primarily made up of individuals from four genera: Thermus, Corynebacterium, Streptococcus, and Brevundimonas. At the same time, we used PICRUSt to forecast, using KEGG, the metabolic profiles, and functional capacities of microbial communities in various sample groups. PICRUSt demonstrated value by utilizing evolutionary modeling with data from 16S rRNA marker gene sequencing and a reference genome database. According to the research conducted by [29], there was an average correlation of approximately 0.8 between the gene content discovered using metagenomic sequencing and the inferred gene families. The method showed consistent prediction accuracy in a variety of datasets, including soil and human data.

Lipid peroxidation and its byproducts in liver diseases are a major cause of morbidity. Therefore, we investigated the ability of black garlic to reduce lipid peroxidation by using the ferric thiocyanate (FTC) method. Black garlic extract was found to have the highest activity (60%) at a concentration of 50 µg/mL in the FTC method compared with previous studies [30]. It has been demonstrated that black garlic has a greater antioxidant potential compared with raw garlic [31]. This might be because the fermentation method used to create black garlic results in several chemical reactions that give rise to novel substances with strong antioxidant properties, like melanoidins and Maillard reaction products. These substances, which are absent in fresh garlic, may help explain why dark garlic exhibits higher antioxidant activity than white garlic. Additionally, the fermentation procedure may improve the antioxidants in black garlic’s bioavailability, making it easier for the body to ingest and use them.

The liver metabolizes alcohol in a detoxification process, primarily via alcohol dehydrogenases (ADH) that require NAD+ as a cofactor. When the alcohol concentration exceeds the liver’s metabolic capability, the reduced form of NAD+ (NADH) accumulates, causing hepatic NADH accumulation. This accumulation results in the synthesis of more fatty acids and triglycerides while impeding the β-oxidation of fatty acids, increasing oxidative stress and the toxicity to hepatic cells [32]. This study indicated that continual feeding with high concentrations of ethanol led to increased levels of ALT, ALP, and AST, as well as elevated levels of triglycerides and cholesterol, which are indications of alcohol intoxication in the liver [33]. The histological images also revealed accretions of fatty droplets in the hepatocytes, indicating ethanol-induced liver damage. Treatment with black garlic powder reduced the accumulation of fats in the rats’ livers and reduced the levels of AST, ALP ALT, bilirubin, and protein, along with SOD and GPX. Moreover, the groups treated with black garlic showed the most substantial improvement in steatosis and the greatest reduction in toxicity. Based on these findings, the researchers hypothesized that black garlic could potentially offer liver protection against liver damage [34].

Our study has yielded a significant finding that merits attention. Our results show that rats treated with black garlic demonstrated an enhanced lipid profile in comparison with the rats that were not treated. Notably, it was discovered that although black garlic treatment did not cause any changes in total cholesterol levels, it led to a substantial reduction in serum triglyceride and LDL cholesterol levels while increasing HDL levels in the serum. Our findings align with prior studies that have shown that aged garlic intake mitigates obesity-induced alterations in lipid profiles in both experimental animals and humans [34,35]. However, it is worth noting that a recent clinical study revealed that aged garlic treatment did not lead to an improvement in the lipid profile of type 2 diabetic patients [36]. This suggests that the effectiveness of black garlic in enhancing lipid profiles may depend on different pathological contexts.

The outcomes of this study provide compelling evidence that black garlic exhibits remarkable hepatoprotective activity in rats afflicted with liver damage induced by alcohol. The ability of black garlic-treated rats to withstand the deleterious effects of oxidative stress instigated by alcohol can be attributed to its antioxidant capacity, which could be partly due to its high total phenolic and flavonoid contents [37]. Notably, the hepatoprotective effects of the black garlic powder were found to be concentration-dependent, with a concentration of 300 mg/kg body weight being the most effective in restoring liver damage to near-normal levels. However, even different extract concentrations of black garlic exhibited moderate liver protection activities [37]. It is noteworthy that all the concentrations used in this study were lower than the highest concentration employed in an acute oral toxicology study (5000 mg/kg body weight) [38]. Given the favorable safety profile of black garlic, it holds great promise as a natural hepatoprotective agent against liver damage.

5. Conclusions

Our research suggests that black garlic has significant antioxidant activity that can help prevent disorders resulting from reactive oxygen species. Black garlic has different microbial communities. Black garlic processing showed that Thermus, Corynebacterium, Streptococcus, and Brevundimonas predominate. Bacillus and Pseudomonas are more common in garlic. Furthermore, this study confirms that black garlic has hepatoprotective properties against liver damage induced by alcohol. This was observed in the significant reduction in liver enzymes, decreased oxidative damage, and suppressed inflammatory responses. This study also demonstrates that the administration of black garlic recovers hepatocytes by maintaining histopathological changes, which confirms its health-promoting effects. Our findings support the notion that black garlic is an excellent antioxidant and has the potential to manage liver pathogenesis. Thus, we recommend the consumption of black garlic as a potential therapeutic remedy. Additionally, we suggest isolating and characterizing the main constituents present in black garlic that contribute to its hepatoprotective potential.