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Research Article Protective Effects of Ligularia Fischeri and Aronia Melanocarpa Extracts on Alcoholic Liver Disease (In Vitro and in Vivo Study)

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Research Article Protective Effects of Ligularia Fischeri and Aronia Melanocarpa Extracts on Alcoholic Liver Disease (In Vitro and in Vivo Study) Hindawi BioMed Research International Volume 2020, Article ID 9720387, 11 pages https://doi.org/10.1155/2020/9720387 Research Article Protective Effects of Ligularia fischeri and Aronia melanocarpa Extracts on Alcoholic Liver Disease (In Vitro and In Vivo Study) Chang-Won Pyun ,1 Tae-Su Seo,1 Dae-Jung Kim,2 Tae-Woo Kim,2 and Jung-Shik Bae1 1R&D Laboratory, Wellfine Co., Ltd, 2-6, 32, Soyanggang-ro, Chuncheon-si, Gangwon-do, Republic of Korea 2Well-being Bioproducts R&D Center, Kangwon National University, Gangwon 25209, 11, IT Valley-gil, Gonggeun-myeon, Hoengseong-gun, Gangwon-do, Republic of Korea Correspondence should be addressed to Chang-Won Pyun; fl[email protected] Received 26 December 2019; Accepted 20 March 2020; Published 15 April 2020 Academic Editor: Cristiano Capurso Copyright © 2020 Chang-Won Pyun et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Hepatic protective effects of Ligularia fischeri (LF) and Aronia melanocarpa (AM) against alcohol were investigated in vitro and in vivo test. LF, AM, and those composed mixing material (LF+AM) were treated in HepG2 cell. Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities were significantly increased in each singleness extract and mixed composite. The protective effect on alcoholic liver damage was investigated by animal models. Serum alcohol level and acetaldehyde level were significantly decreased by LF+AM treatment in acute experimental model. In the chronic mouse model study, we had found that the increased plasma liver damage index (alkaline phosphatase) by alcohol treatment was declined by oral administration of LF+AM extraction composite. As well as, it was identified that the protection effect was induced by increasing catalase activity and suppressing COX-2, TNF-α, MCP-1, and IL-6 mRNA expressions. CYP2E1 mRNA expression was also increased. These results suggest that oral ingestion of LF and AM mixed composite is able to protect liver against alcohol- induced injury by increasing alcohol metabolism activity and antioxidant system along with decreasing inflammatory responses. 1. Introduction Alcohol absorbed into the body moves through the stom- ach and small intestines and is subsequently metabolized in Alcohol consumption has become popular worldwide, par- the liver. Approximately 90% of alcohol absorbed in the liver ticularly for relieving stress and for socializing [1, 2]. How- is primarily converted into acetaldehyde by alcohol dehydro- ever, hangover after drinking may cause symptoms such as genase (ADH), which is then secondarily oxidized into acetic headache, vomiting, nausea, and muscle ache, while also acid by aldehyde dehydrogenase (ALDH). These metabolites causing mental and social detriment [3]. Moreover, excessive of alcohol are ultimately degraded into water and CO2 for drinking may cause ischemic heart disease, pancreatic can- excretion through various routes, such as urine or sweat cer, esophageal cancer, oral cancer, and diabetes [4]; more- [6]. As indicated, metabolism of alcohol is regulated by the over, it can be the cause of various liver diseases, including activities of ADH and ALDH, but when excessive amounts liver cirrhosis, hepatitis, and fatty liver [5]. Most habitual of alcohol are absorbed into the body, the metabolites accu- drinkers have alcoholic fatty liver, while 10-35% have alco- mulate inside the body due to the limited metabolic rate of holic hepatitis, and 10-20% have alcoholic liver cirrhosis. ADH and ALDH. Acetaldehyde produced during alcohol Consequently, the seriousness of alcoholic liver diseases has metabolism is the major cause of toxic action that is more become a major issue. In addition, the disease treatment is severe than the alcohol itself [7]. Acetaldehyde is a highly very costly, and thus, there is a growing trend in consumer reactive compound, which interferes with mitochondrial demand for materials and foods, which can improve liver respiration [8] and produces protein adducts, which are functions to prevent such diseases. involved in hepatotoxicity [9, 10]. Here, the adducts produced 2 BioMed Research International bind directly with tubulin present in hepatocyte to impair Advantec filter paper No. 2 (CA, USA), after which, the fil- material absorption and protein egress, which can induce trate was vacuum concentrated. The concentrated sample immune responses that lead to liver damage [11]. In addition, was dissolved in distilled water and freeze-dried for 72 h to significant amounts of reactive oxygen species produced prepare the LF and AM extracts, which were used for the during alcohol metabolism trigger lipid peroxidation, while further experiments. The yield of each freeze-dried extract the oxides cause damage to blood cells and cell membrane powders for dried raw materials was 10.1% and 39.8%, [12, 13] and produce reactive aldehydes to facilitate inflam- respectively, in a lab scale process. As well as, to investigate mation and liver fibrosis [14]. the synergistic effect of LF+AM composite on alcoholic liver Ligularia fischeri (LF) is a perennial plant belonging to disease, each extract was evenly mixed at a ratio from 1 : 9 to the Asteraceae family, which can grow up to 1 m tall, usually 9 : 1 (w/w) for use in the experiment and had compared with in wetlands deep in mountains. Its leaf is heart-shaped, and each singleness extract material. one stem usually has three leaves, growing up to 85 cm. Young leaves can be eaten raw as wraps or after slightly 2.2. HPLC-RP Analysis. LF was dispersed in 50% EtOH steaming and seasoning them in the Republic of Korea. LF (10 mg/mL) by vortex mixer. Dispersed solutions were soni- β consists of vitamins A, B1,B2, and C, as well as -carotene cated for 10 min and centrifuged. The supernatant was used and niacin. Among these, its vitamin A and β-carotene for HPLC analysis. AM was dissolved in 70% EtOH contents are known to be higher than in other vegetables (5 mg/mL) and directly used for HPLC analysis. All samples [15]. It has generally been used as a therapeutic herb for were filtered using 0.45 μm nylon syringe filter before inject- bruises, low back pain, sputum, and coughs, while it has also ing it into the HPLC system. been reported to have antioxidative [16], anticancer, anti- Chromatographic separations of caffeoylquinic acids inflammatory [17, 18], and nitric oxide inhibition [19] effects. (CQAs) were achieved using a Phenomenex Luna C18 col- LF contains several iso forms of chlorogenic acid component umn (250 mm × 4:6mmI.D, 100 Å). A reverse phase HPLC such as 4-caffeoylquinic acid (4-CQA), 3,4-dicaffeoylquinic was carried out by gradient conditions, and the flow rate of acid (3,4-dCQA), and 3,5-dicaffeoylquinic acid (3,5-dCQA) mobile phase was 1.0 mL/min. Mobile phases were composed etc. Those phenolic compounds are reported as an antioxida- in 0.1% triflouroacetic acid (TFA) in deionized water (A) and tive, antibacterial, and anti-inflammatory material [20]. methanol (B). The gradient program started in 15% B and Aronia melanocarpa (AM) is the name that collectively with the following composition: 0–3 min, 15% B; 3–7 min, refers to a shrub belonging to the Aronia family of Rosaceae 19% B; 7–15 min, 19% B; 15–28 min, 32% B; 28–33 min, family in the order Rosales and its berries. It is an ornamental 90% B; 33–38, 15% B; and 38–43, 15% B. Column oven was plant, which is also grown for its edible berries. Aronia can set at 30°C, and the detection of CQAs was performed at thrive in harsh environments, including subzero cold and 330 nm on a UV detector. strong ultraviolet ray environments. Reported reference C3G was determined by separating with a Phenomenex materials include polymeric proanthocyanidins, chlorogenic Luna C18 column (150 mm × 4:6mm, 100 Å), and the acid, neochlorogenic acid, cyanidin-3-O-galactoside, and mobile phases were composed of 0.4% of TFA in deionized cyanidin-3-O-arabinoside [21], while it has been reported water and 0.4% of TFA in 50% MeOH. The gradient program to have various physiological activities, including cardiopro- begun in 0% B: 0–2 min, 70%; 20–22 min, 100%, 22–30 min, tective, antioxidant, anticancer, antimutagenic, and hepato- 0% B; 30–35 min, 0% B. Temperature of separation was set protective activities [22]. Anthocyanins, which are major at 30°C, and the detection wavelength was 520 nm. physiologically active substances, are the most abundant in AM among berries and widely used as functional materials 2.3. HepG2 Cell Culture. Human hepatoma (HepG2) cells in various foods and dietary supplements. were obtained from the Korean Cell Line Bank (Seoul, However, it has not been reported that the effect of both Korea). HepG2 cells were cultured in Dulbecco’s Modified natural products (LF and AM) on alcohol induced liver Eagle Medium (DMEM) containing 10% heat-inactivated injury. Therefore, to investigate the effects of extracts pre- fetal bovine serum (FBS) and 1% penicillin-streptomycin pared from LF and AM on the alcoholic liver disease, the under 5% CO2 condition with temperature maintained present study conducted experiments under in vitro and at 37°C. in vivo systems. 2.4. Cell Viability Assay. Cell viability was measured using 2. Materials and Methods the MTT (3-[4,5-dime-thylthiazol-2-yl]-2,5-diphenyl-tetra- zolium bromide) reduction method as previously described 2.1. Preparation of Experimental Materials. Dried LF powder by [23]. After dispensing 100 μL each of HepG2 cells was purchased from an agricultural company Chorok Sarang (1×106 cells/mL) into a 96-well plate and incubating it (Jeongseon, Korea), and AM powder was purchased from for 24 h, samples with concentrations of 0.05, 0.125, 0.25, local farm (Goseong-gun, Gangwon-do, Korea).
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