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INTRODUCTION
Garlic (Allium sativum) has been used as a medicinal ingredient in folk remedies since the old times. It contains various bio-functional properties affecting health [1,2,3,4,5], but was not enjoyed by many due to its peculiar smell. Various food processing methods have been employed to get rid of the smell of garlic. Black garlic is made by ripening raw garlic at high temperature and humidity, which removes the peculiar pungent odor [6]. Black garlic gets its name from the black hue of the cloves, but retains the same shape as raw garlic. During the ripening process, the glyco-component and amino acids of the garlic undergo a non-enzymatic browning reaction, producing melanoidins and water-soluble components such as S-allylcysteine (SAC) and S-allyl melcaptocystein (SAMC), and nearly removing all volatile substances [7]. In black garlic extracts, the contents of water soluble components, such as SAC and SAMC, are known to increase significantly during the aging process, unlike raw garlic, which may play a key role in antilipidemic action as well as conferring potent antioxidant activity [8,9].
Not all garlic preparations can be assumed equivalent in composition. Studies on garlic powder and garlic oil failed to show any lipid lowering or hypoglycemia effects [11,12]. The inconsistent results of animal studies may be due at least in part to differences in the types of garlic studied (i.e. raw garlic, cooked garlic or black garlic).
Excessive intake of fat alters lipid metabolism, which can act as a risk factor for various diseases including fatty liver disease, hypertension, hyperlipidemia, and arteriosclerosis [10]. Elevation of blood lipids levels due to excessive fat intake stimulates SREBP-1c, the key transcription factor involved in fat metabolism, which leads to the activation of lipogenetic enzymes for triglyceride and cholesterol synthesis, as well as inhibition of lipolytic enzymes [13,14,15]. SREBP-1c also mediates the action of insulin on hepatic gene expression, while its transcriptional activity is also regulated by insulin [16].
Several studies have shown that the lipid-lowering mechanisms of fresh garlic, not black garlic, involve many gene factors such as AMP-activated protein kinase (AMPK) and enzymes such as ACAT and FAS [17,18]. Limited study has been carried out regarding the possible mechanism of the action of black garlic on lipid metabolism [19]. Thus, the objectives of this study were to determine the effects of black garlic on blood lipids and cholesterol profiles, and the expression of related hepatic genes in rats fed a high fat diet.
MATERIALS AND METHODS
Animals and Diets
Thirty-two male Sqrague-Dawley rats aged 4 weeks were randomly divided into four groups (n = 8) by weight, and fed the following diets: normal-fat (NF, 7% fat); high-fat (HF, 20% fat); high-fat with 0.5% black garlic extract (HFBG0.5); or high-fat with 1.5% black garlic extract (HFBG1.5). The compositions of the experimental diet were based on the AIN-93G diet (Table 1).
Black garlic extract was generously provided by the Korea Euisung Agricultural Association (Gyeongsangbuk-do, Republic of Korea). The black garlic extract used in this study was 60 brix concentrate. In the experimental group, the HFBG0.5 group was fed 0.5% black garlic extract (5 g extract/kg diet) and the HFBG1.5 group was fed 1.5% black garlic extract (15 g extract/kg diet). All experiments were approved by the guidelines of the Animal Testing Ethics Committee of Dankook Univeristy (approval code: 12-004).
Sample Preparation
After 5 weeks of feeding, the experimental animals were fasted for 12 hr and then anesthetized with ethylether. Blood samples were drawn from the hearts and centrifuged for 30 minutes at 3,000 rpm, after which the plasma was stored at -70℃ until use. After scarification of the rats, the liver, kidney, thymus, spleen, internal fat, kidney fat, and epididymal fat were removed and rinsed with 0.9% NaCl solution, then dried with filter paper. After measuring the weights of the organs and adipose tissue, all samples were stored at -70℃ until further analysis. Stool samples were individually collected twice daily and completely dried by heating at 105℃.
Analysis of plasma biochemical parameters
Plasma glucose concentration was measured with an assay kit using the glucose oxidase method (Asan Pharmaceutical Co., Seoul, Korea). Plasma insulin concentrations were measured via a rat insulin ELIZA kit (Shibayagi Co., Shibukawa, Japan). Plasma AST (aspartate aminotransferase) and ALT (alanine aminotransferase) were measured with commercial kits (Asan Pharmaceutical Co., Seoul, Korea), and the absorbance at 505 nm was determined with a spectrophotometer.
Lipid measurements in plasma, liver and feces
Plasma total lipids were determined as described by Frings and Dunn [20], with slight modifications. Plasma total cholesterol, TG, and HDL were measured enzymatically using a commercial kit (Asan Pharmaceutical Co., Seoul, Korea). Total lipids in the liver and feces were extracted with chloroform: methanol (2 : 1, v/v) using a modified method described by Folch et al. [21]. After extraction, the total lipid, TG, and cholesterol were measured using the same methods as for plasma lipid determination.
Total RNA isolation
Total liver RNA was isolated using TRI-reagent (Sigma aldrich, MO, USA) according to the manufacturer's protocol. Liver samples (0.2 g) were homogenized in 1 mL TRI Reagent and then kept at room temperature for 5 min. The homogenate was then mixed with 200 µL chloroform (Sigma aldrich, MO, USA), shaken vigorously, left for 10 min, and then centrifuged at 12,000 g for 10 min. After transferring the supernatant, 500 µL of isopropanol was added and kept at room temperature for 5 min, after which the sample was centrifuged at 12,000 g for 10 min at 4℃.
After removing the supernatant, the RNA pellet was washed with 75% ethanol followed by centrifugation at 7,500 g for 5 min at 4℃. After removing the ethanol wash, samples were dried thoroughly and then the RNA was dissolved in 50 µL RNase-free dH2O/0.1 mmol/L EDTA through a pipette tip. The purity of each RNA preparation was evaluated by measuring the ratio of the absorbance at 260 nm to that at 280 nm. The final preparations of total RNA were essentially free of DNA and proteins, and had 260/280 ratios of 1.7-2.0.
Reverse Transcription (RT)
cDNA was synthesized using 3 µg of total RNA with Super-Script II reverse transcriptase (Invitrogen, CA, USA). In 1.5 mL tubes, 3 µg total RNA from the samples and 1 µL of oligo DT (0.5 µg/µL, Invitrogen, CA, USA) were added and the final volume was brought to 12 µL with DEPC water, followed by incubation at 70℃ for 10 min [15]. A reaction buffer (4 µL 5 X first standard buffer, 2 µL 0.1 mol/L DTT, 1 µL 10 mmol/L NTP) was added to the mixture and incubated at 42℃ for 5 min. After incubation, 0.5 µL superscript II reverse transcriptase (Invitrogen, CA, USA) was added and incubated at 42℃ for 100 min, and then 70℃ for 15 min. Finally, 80 µL Rnase DEPC water was added to the mixture, which was stored at -20℃ until subsequent analysis.
Realtime PCR
Real-time PCR was performed using the modified method recently described in detail [15]. Aliquots of 2 µL of the prepared cDNA samples were mixed with 10 µL SYBR green Master mix (Applied biosystems, CA, USA), 1 µL of each sense/antisense primer (Table 2), and 6 µL DEPC water. The initial activation of the primer at 95℃ for 10 min was followed by 40 cycles of 95℃ for 15 min and 60℃ for 1 min. Expression of mRNA was analyzed with an Applied Biosystems StepOne Plus RT-PCR system (Applied biosystems, CA, USA). Fold difference of gene expression was calculated using the 2-ΔΔCT method with the endogenous control gene.
Statistical analysis
Statistical analysis was performed using the Statistical Analysis System software (SAS Institute, Cary, NC, USA). Data were expressed as means with standard error, and statistically significant differences among groups were evaluated using one way-ANOVA (analysis of variance). Statistically significant differences among the means of groups were tested at α = 0.05 using Duncan's multiple range tests.
RESULTS
Body weight, food intake, and organ weight in rats fed black garlic extract
Final body weight and weight gain in the HFBG1.5 group were significantly lower than the HF and HFBG0.5 groups. Significant differences in the food efficiency ratios (FER) were not observed among the groups (Table 3).
No significant differences in liver, kidney, spleen, and thymus weights were found between the HF group and the HFBG0.5 or HFBG1.5 groups (Table 4). Among adipose tissues, kidney fat and epididymal fat were significantly decreased in the HFBG1.5 group compared to both the HF and HFBG0.5 groups. Total fat of the adipose tissues weighed 9.47 g in the HFBG1.5 group, which was lower than the 11.04 g measured for the HF group, though the difference was not significant.
Plasma biochemical parameters in rats fed black garlic extract
The blood biochemical values are shown in Table 5. Both the HFBG0.5 and HFBG1.5 groups showed significantly lower blood glucose levels than the NF or HF groups. In addition, the insulin level in the HFBG1.5 group was significantly decreased compared to the HF and HFBG0.5 groups. The AST levels in the HF group were significantly elevated due to administration of the HF diet, but the levels were significantly decreased in the HFBG1.5 group in comparison. Significant decrease in the ALT levels was observed in both the HFBG0.5 and HFBG1.5 groups, compared to the HF group.
Lipid profiles of the plasma, liver, and feces in rats fed black garlic extract
The levels of total lipids, cholesterol, and HDL-cholesterol in the plasma of the HFBG0.5 group were significantly lower than in the HF group, while HDL-cholesterol was significantly higher. Plasma total lipids, TG, and HDL-cholesterol in the HFBG1.5 group were also significantly improved. Total hepatic lipids in the HFBG1.5 group were significantly decreased compared to the HF group. Significant increases of total fecal lipids and TG were also observed in the HFBG1.5 group compared to the HF group (Table 6).
The levels of mRNA expression of transcription factors in rats fed black garlic extract
The mRNA expressions of ACC, FAS, and SREBP-1C were significantly lower in both the HFBG1.5 and HFBG0.5 group, compared to the HF group, with higher reduction in the HFBG1.5 group than in the HFBG0.5 group (Fig. 1). The mRNA expression of G6PDH was significantly lower in the HFBG1.5 group, not in the HFBG0.5 group, compared to the HF group. The expression of CPT1 mRNA in the HF group was significantly decreased compared to the NF group (Fig. 2); however, the expressions were significantly higher than HF in both the HFBG1.5 and HFBG0.5 groups. The hepatic mRNA expressions of HMG-CoA reductase and ACAT were significantly lower than HF in both the HFBG1.5 and HFBG0.5 groups, with much greater reduction observed in the HFBG1.5 group (Fig. 3).
DISCUSSION
Based on previous animal studies regarding the effects of garlic on lipid metabolism, the hypolipidemic actions of fresh garlic were encouraging when less than 2% of garlic were administrated to rats [4,5,6,22,23]. In our prelimary study, similar effectiveness was observed for 0.5% and 1.0% black garlic extract. Therefore, 0.5% (5 g/kg diet) and 1.5% (15 g/kg diet) black garlic extract were selected as the applicable high and low dosage for supplementation in rats fed a high fat diet in this study.
Final body weights in the HFBG1.5 group were significantly lower than that in both the HF and HFBG0.5 groups. It could be suspected that the unique flavor and smell of the black garlic had a higher effect on the appetites of the rats in the HFBG1.5 group, resulting in a decrease in food intake and weight reduction. However, there were no significant differences in the total dietary intakes or food efficiency ratio between the HFBG1.5 group and the HF or HFBG0.5 groups. Therefore, the observed weight reduction may be mediated by other variables in the black garlic extract, rather than food intake.
Like the previous reports [9,24,25,26], this study showed the hypoglycemic effects of black garlic extract in rats fed a high fat diet. The effect was more potent with the addition of 1.5% black garlic extract than 0.5%, demonstrated by decreases in blood glucose levels and increases in plasma insulin level herein.
The changes of serum AST and ALT levels, which are serum markers of liver damage, are generally monitored in high fat diets [9]. Several studies reported that the alterations in AST and ALT due to liver toxicity were significantly prevented by prior administration of 0.2-1.0% fresh garlic extract for a short period of time [4,22,27]. In the present study, the AST levels in the HF group were significantly elevated compared to the NF group, while both 0.5% and 1.5% black garlic extract showed favorable hepatoprotective effects. Therefore, the present study suggests that the doses of 0.5-1.5% are safe for black garlic extract usage in animal study.
The importance of plasma TG and cholesterol levels in the pathogenesis of atherosclerosis has generally been accepted. It was clearly demonstrated herein that black garlic, dose-dependently, has hypolipidemic effects. These results were consistent with many previous studies [18,20,23,24,25,26]. However, the amounts of garlic supplemented in previous animal studies were quite different from those used herein. Some studies reported that 3-5% fresh garlic or black garlic extract significantly decreased blood cholesterol and triglyceride (TG) levels in diabetic or hypercholestemic mice [23,24,25,26]. The amounts of 3-5% garlic in the rat's diet are quite high, and are not applicable for the normal diet of humans [28]. Therefore, the results of this study are meaningful in that even low dosage, such as 0.5 or 1.5% black garlic extract, is beneficial to improve hyperlipidemia caused by high fat diet.
Allicin in fresh garlic was initially identified as the active compound responsible for the anti-atherosclerotic effect. Unfortunately, this study did not measure the bio-active compound content of the black garlic extract used; however, studies reported that the main constituents of black garlic are water soluble compounds, such as SAC and SAMC, which are produced during the ripening process of black garlic [6,7,8,9]. These water soluble compounds, which are rare in raw garlic, are thought to be responsible for black garlic's anti-hyperlipidemic effects [9,22,29].
In evaluating the mechanism of how black garlic lowers the lipid profiles in plasma and liver in rats, we measured the expressions of hepatic SREBP-1c mRNA, a key transcription factor of lipid metabolism, and decreased expression of SREBP-1c was observed in the rats treated with black garlic extract. As a consequence of the fall in hepatic SREBP-1c, the levels of ACC, FAS, and G-6PDH mRNA, target genes of SREBP-1c, were strongly reduced to the levels observed in the normal fat mice, suggesting that inhibition of the expression of these genes causes decrease of fat synthesis in the liver. This was associated with a decrease in the plasma TG concetration. Similar results were reported, in that the methanol extract of black garlic in rats fed a high-cholesterol diet inhibited lipid accumulation via the up-regulation of AMPK and the down-regulation of SREBP-1c, ACC, and FAS [16,17,18,30,31].
Increased high fat diet results in the production of plenty of acetyl-CoA and malonyl CoA in the liver. The increased level of malonyl CoA, as a fuel for TG synthesis, will inhibit the expression of CPT-1, a key enzyme for lipolysis, thus decreasing fatty acid oxidation [32]. As shown in this study, the supplementation of high fat diet with black garlic extract led to increased expression of CPT-1 mRNA, causing more TG to break down (degrade) in order to supply free fatty acid as an energy source.
The extent of hepatic SREBP-1c levels is also believed to activate the enzymes, HMG-CoA reductase and ACAT, which regulate cholesterol synthesis and catalyzes esterification of cholesterol, respectively [15,29]. In rats fed black garlic extract, the plasma cholesterol concentration was markedly decrased which was related to the decreased expressions of HMG-CoA reductase and ACAT.
In this study, the supplementation of black garlic extract significantly increased the fecal excretion of lipids, both TG and cholesterol, which suggests that the mechanism of black garlic extract is not only related to the mechanisms of fat synthesis, but also could be related to another mechanisms leading to increased fecal excretion or the absorption rate of dietary fat, resulting in a significant reduction in the plasma concentration of lipid profiles in rats fed black garlic extract. Therefore, further studies regarding the mechanisms related to fecal excretion of lipids are needed to clarify this matter.
Garlic and its preparations have been widely recognized as agents for the prevention and treatment of cardiovascular diseases. However, it is commonly known that excessive consumption of garlic can cause problems such as odor of the breath and skin, occasional allergic reactions, and toxicity. The main constituents of black garlic extract are water soluble compounds, thus it lacks the toxicity and has no peculiar garlic smell. Therefore, it can be taken by humans for a long time without presenting toxic side effects or contraindications with medications to prevent or reduce hyperlipidermia.
In conclusion, administration of 0.5% or 1.5% black garlic extract was effective, dose-dependently, in improving blood lipid profiles, especially TG, and blood glucose levels in rats fed a high fat diet, as evidenced by decreased expressions of SREBP-1c and its related enzymes, along with increased expression of CPT-1. Thus, appropriate intake of black garlic will be beneficial in the prevention of hyperlipidemia and hyperglycemia caused by a high fat diet.
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