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Hepatoprotective effect of licorice, the root of Glycyrrhiza uralensis Fischer, in alcohol-induced fatty liver disease
- Jae-Chul Jung†1,
- Yun-Hee Lee†2,
- Sou Hyun Kim3,
- Keuk-Jun Kim4,
- Kyung-Mi Kim1,
- Seikwan Oh5 and
- Young-Suk Jung3Email author
© Jung et al. 2016
Received: 20 September 2015
Accepted: 12 January 2016
Published: 22 January 2016
Our previous study suggested that licorice has anti-inflammatory activity in lipopolysaccharide-stimulated microglial cells and anti-oxidative activity in tert-butyl hydroperoxide–induced oxidative liver damage. In this study, we evaluated the effect of licorice on chronic alcohol-induced fatty liver injury mediated by inflammation and oxidative stress.
Raw licorice was extracted, and quantitative and qualitative analysis of its components was performed by using LC–MS/MS. Mice were fed a liquid alcohol diet with or without licorice for 4 weeks.
We have standardized 70 % fermented ethanol extracted licorice and confirmed by LC-MS/MS as glycyrrhizic acid (GA), 15.77 ± 0.34 μg/mg; liquiritin (LQ), 14.55 ± 0.42 μg/mg; and liquiritigenin (LG), 1.34 ± 0.02 μg/mg, respectively. Alcohol consumption increased serum alanine aminotransferase and aspartate aminotransferase activities and the levels of triglycerides and tumor necrosis factor (TNF)-α. Lipid accumulation in the liver was also markedly induced, whereas the glutathione level was reduced. All these alcohol-induced changes were effectively inhibited by licorice treatment. In particular, the hepatic glutathione level was restored and alcohol-induced TNF-α production was significantly inhibited by licorice.
Taken together, our data suggests that protective effect of licorice against alcohol-induced liver injury may be attributed to its anti-inflammatory activity and enhancement of antioxidant defense.
Accumulating lines of evidence show that licorice has anti-inflammatory, anticancer, antioxidant, and anti-microbial effects [1, 4, 7–9]. In particular, recent studies on hepatoprotective effects of licorice suggest that it can reduce liver injury by enhancing antioxidant and anti-inflammatory capacity [7, 10]. Administration of licorice extract prevented CCl4-induced hepatotoxicity by increasing antioxidant enzyme activity and decreasing TNF-α production . Jung et al.  investigated the hepatoprotective effects of 18β-glycyrrhetinic acid, one of the active compounds in licorice, in a CCl4-induced liver injury model. Treatment with 18β-glycyrrhetinic acid inhibited the increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and hepatic lipid peroxidation in a dose-dependent manner. In addition, 18β-glycyrrhetinic acid significantly protected against glutathione (GSH) depletion. Although these studies show a promising effect of licorice in preventing liver injury, their limitation was that the chemically induced acute hepatotoxicity model used was not very relevant to clinical situations.
Alcohol abuse causes a range of acute and chronic health problems worldwide, which lead to morbidity and mortality. Depending on overall alcohol consumption and drinking patterns, chronic exposure to alcohol is harmful to the central nervous system and many organs, including the liver. Among alcohol-induced liver diseases, fatty liver is the most common histopathologic condition in drinkers. Although alcohol-induced fatty liver is widely considered to be benign and to have a very low risk of progression, clinical studies have provided evidence that it is an important pathogenic factor in the development of liver disease [13–15]. Specifically, the authors suggested that both oxidative stress and inflammation as second hits are critical factors in the pathological progression from simple fat accumulation to liver disease. Recently, we reported that licorice extract had an anti-inflammatory effect in lipopolysaccharide-stimulated microglial cells and acted as an antioxidant in a tert-butyl hydroperoxide–induced oxidative liver injury model . Therefore, it was of interest to examine the effects of licorice on chronic alcohol-induced fatty liver, which is more relevant to clinical situations. In this study, we examined the preventive effect of licorice in alcoholic fatty liver by administering its extract to mice exposed to alcohol for 4 weeks.
Analysis of licorice extract
Analytical condition of LC-MS/MS
Condition for LC-MS/MS analysis of Licorice extracts
Luna C18 RP column (2.0 × 150 mm, 5 μm)
Turbo spray (Negative)
- 4.0 kV
Animals and treatments
Male C57BL/6 mice were purchased from Orient Bio (Sungnam, Korea). The use of animals was in compliance with the guidelines established and approved by the Institutional Animal Care and Use Committee in Pusan National University (PNU-2014-0568). Animals were acclimated to temperature (22 ± 2 °C) and humidity (55 ± 5 %) controlled rooms with a 12-h light/dark cycle for 1 week prior to use. The diets were purchased from Dyets Inc. (Bethlehem, PA, USA). Mice were fed a Lieber–DeCarli liquid diet with or without ethanol for 4 week. For the control liquid diet, 35 % of energy was derived from fat, 18 % from protein, and 47 % from carbohydrates; the liquid ethanol diet contained 35 % of energy from fat, 18 % from protein, 11 % from carbohydrates, and 36 % from ethanol.
Serum biochemistry and histopathologic evaluation
Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total serum triglyceride (TG) were measured using Automated Chemistry Analyzer (Prestige 24I; Tokyo Boeki Medical System, Tokyo, Japan). For histopathologic evaluation, a cross section of the left lateral lobe of the liver was sliced at 10 μm, immersed in propylene glycol for 5 min, and stained with Oil red O for 7 min. After rinsing with 85 % propylene glycol and distilled water, the sections were counterstained with hematoxylin for 2 min before microscopic examination.
Measurement of serum TNF-α
The levels of serum TNF-α were determined by enzyme-linked immunosorbent assay using a commercially available kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instruction.
Determination of hepatic triglyceride contents
Total lipids of the liver were extracted from homogenate prepared from 100 mg of mouse liver using the mixture of chloroform/methanol (2:1, v/v). Triglycerides in total lipid were determined enzymatically using a commercially available enzymatic kit (Sigma Chemical Co.) according to the manufacturer’s protocol.
Measurement of hepatic glutathione (GSH)
Liver was homogenized in a four-fold volume of ice-cold 1 M perchloric acid. After the denatured protein was removed by centrifugation at 10,000 g for 10 min, the supernatant was assayed for the total GSH concentration using a HPLC separation/fluorometric detection method .
Real time RT-PCR
Total RNA was purified from liver tissue using the RNeasy kit (Qiagen, Valencia, CA, USA). cDNA synthesis was accomplished with iScript™ cDNA Synthesis system (Bio-Rad, Hercules, CA, USA). Real time RT-PCR was performed using Thunderbird SYBR qPCR mix (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer’s protocol. Relative values of gene expression were normalized to 18S ribosomal RNA. Primer sequences and full name of the genes are provided in Additional file 1: Table S1.
All results expressed as mean ± s.d. were analyzed by one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple range test (parametric). The acceptable level of significance was established at P < 0.05.
Linearities, regression equation, correlation coefficients, limit of detection (LOD), and limit of quantitation (LOQ) for glycryrrhizic acid (GA), liquiritin (LQ) and liquiritigenin (LG)
Linear range (ng/ml)
Glycyrrhizic acid (GA)
Y = 0.0004x – 0.0055
Y = 0.0059x + 0.0451
Y = 0.0289x + 0.1574
We prepared the calibration curves for GA, LQ, and LG according to the concentration-dependent ESI method in LC-MS/MS analysis. We found that the correlation coefficient (r2) values were 0.9999, 0.9997, and 0.9998, respectively, which showed good linearity of the calibration curves (Table 2). The limit of detection (LOD) was 4.29 ng/mL, 1.27 ng/mL, and 0.54 ng/mL, whereas the limit of quantitation (LOQ) was 13.99 ng/mL, 3.86 ng/mL, and 1.64 ng/mL, respectively.
Analytical LC-MS/MS data of licorice extracts
Glycyrrhizic Acid (GA)
Although the mechanisms of the induction of fatty liver by alcohol appear to be complicated, accumulating lines of evidence suggest contribution of both oxidative stress and inflammation. On the basis of our recent findings that licorice protects cells against inflammation and oxidative stress , we hypothesized that licorice would alleviate alcohol-induced fatty liver injury.
In mice fed a standard Lieber–DeCarli alcohol diet for 4 weeks, hepatic triglyceride levels increased and GSH content decreased with concomitant increases in serum ALT and AST activities, triglycerides, and TNF-α. Supplementation of the alcohol diet with licorice for the same time period significantly reversed the changes in liver injury markers and effectively abrogated fat accumulation. Thus, we suggest that the hepatoprotective effect of licorice is associated with an augmentation of antioxidant defense and anti-inflammatory response.
GSH, a thiol-containing tripeptide, plays a major antioxidant and detoxification role in the liver. Alcohol increases the levels of intracellular reactive oxygen species and depletes mitochondrial GSH, and therefore induces oxidative stress . Although the contribution of oxidative injury to the development of alcoholic fatty liver remains to be elucidated, enhancement of antioxidant capacity using some compounds ameliorates alcoholic fatty liver [19–22]. In line with these results, overexpression of superoxide dismutase prevents the accumulation of lipid droplets in hepatocytes, whereas double knockout of glutathione peroxidase-1 and catalase aggravates alcohol-induced liver injury [23–25]. Our results thus indicate that an improvement in the antioxidant capacity in alcohol-fed mice via recovery of the hepatic GSH pool could make licorice valuable in the treatment of alcoholic liver disease.
Direct inflammatory and cytotoxic effects of TNF-α in alcoholic liver disease are well characterized. Chronic drinking of alcohol increases the level of bacterial endotoxin, which stimulates resident liver macrophages to produce free radicals and cytokines . NADPH oxidase plays critical roles in the generation of oxidants in resident liver macrophages after alcohol intake. Activation of NF-kB by oxidant generation leads to an increase in the TNF-α level, which causes tissue injury . Moreover, TNF-α is suggested to induce lipolysis in adipose tissue followed eventually by fatty liver. Earlier studies showed that TNF-α causes free fatty acid release from adipocytes, stimulates lipogenesis in the liver, and inhibits β-oxidation of free fatty acids [28–30]. Moreover, in a more recent report, TNF-α was suggested to increase intrahepatic fat deposition by affecting hepatic lipogenic metabolism that involves SREBP-1c . Indeed, TNFR1 knockout almost completely blocks the development of alcohol-induced fatty liver  . In agreement with these reports, the present study demonstrated that licorice significantly inhibited up-regulation of Srebf1 by chronic alcohol drinking. Importantly, increase of gene expression involved in lipid uptake such as Cd36, Lpl, and Fatp4 is also effectively reduced by licorice treatment. Considering the importance of TNF-α in the development of alcoholic fatty liver, suppression of TNF-α secretion by licorice may contribute to its overall preventive effect in alcoholic liver injury.
We found that licorice is effective in preventing alcoholic fatty liver in mice. An important issue in the management of alcoholic liver disease is the progression of simple fat accumulation to alcoholic hepatitis. Licorice treatment restored hepatic GSH content and inhibited TNF-α secretion, and also inhibited lipid accumulation in the liver of chronic alcohol-fed mice. Therefore, licorice is a promising candidate to prevent the progression of alcoholic liver injury, which probably acts by enhancing anti-oxidative and anti-inflammatory capacity.
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT&Future Planning (NRF-2014R1A1A1005435). This research was also financially supported by the Ministry of Trade, Industry and Energy (MOTIE) and the Korea Institute for Advancement of Technology (KIAT) through Promoting Regional Specialized Industry (R0002317).
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