Anti‑Inflammatory and Antioxidant Activity of the Traditional Herbal
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MOLECULAR MEDICINE REPORTS 13: 4365-4371, 2016 Anti‑inflammatory and antioxidant activity of the traditional herbal formula Gwakhyangjeonggi‑san via enhancement of heme oxygenase‑1 expression in RAW264.7 macrophages SOO-JIN JEONG1,2, OHN-SOON KIM1, SAE-ROM YOO3, CHANG-SEOB SEO3, YEJI KIM3,4 and HYEUN-KYOO SHIN3 1KM Convergence Research Division, Korea Institute of Oriental Medicine, Daejeon 34054; 2Korean Medicine Life Science, University of Science and Technology, Yuseong-gu, Daejeon 34113; 3K-herb Research Center, Korea Institute of Oriental Medicine, Daejeon, Chungcheong 34054; 4Mucosal Immunology Laboratory, Department of Convergence Medicine, University of Ulsan College of Medicine/Asan Medical Center, Seoul 05505, Republic of Korea Received April 8, 2015; Accepted December 21, 2015 DOI: 10.3892/mmr.2016.5084 Abstract. Gwakhyangjeonggi-san (GHJGS) is a mixture of Introduction herbal plants, including Agastache rugosa, Perilla frute‑ scens, Angelica dahurica, Areca catechu, Poria cocos, Gwakhyangjeonggi-san (GHJGS) is a traditional Korean Magnolia officinalis, Atractylodes macrocephala, herbal formula composed of the following 13 medicinal herbs, Citrus reticulata, Pinellia ternata, Platycodon grandiflorum, Agastache rugosa, Perilla frutescens, Angelica dahurica, Glycyrrhiza uralensis, Ziziphus jujuba and Zingiber offici‑ Areca catechu, Poria cocos, Magnolia officinalis, nale. GHJGS has been used for treating diarrhea-predominant Atractylodes macrocephala, Citrus reticulata, Pinellia ternata, irritable bowel syndrome in traditional Korean medicine. Platycodon grandiflorum, Glycyrrhiza uralensis, In the present study, the anti‑inflammatory and antioxidant Ziziphus jujuba and Zingiber officinale. It has been used for effects of GHJGS were investigated using the RAW 264.7 treating diarrhea-predominant irritable bowel syndrome (1). murine macrophage cell line. GHJGS significantly reduced In addition, GHJGS has been identified as an effective treat- production of the proinflammatory cytokines, tumor necrosis ment for allergies (2), respiratory (3) and cardiovascular (4) factor-α, interleukin-6 and prostaglandin E2 in lipopolysac- diseases, and bacterial infections (5). However, to the best charide (LPS)-stimulated macrophages. GHJGS markedly of our knowledge, there have been no reports to date on the suppressed LPS-induced phosphorylation of mitogen-acti- anti‑inflammatory effect of GHJGS. vated protein kinases, whereas it had no effect on nuclear Inflammation is a protective response against various factor-κB activation. Furthermore, GHJGS enhanced expres- harmful stimuli, such as pathogens, damaged cells and sion of heme oxygenase-1 and prevented the generation of irritants (6). This response is controlled by production of reactive oxygen species in RAW 264.7 cells. These results proinflammatory biomolecules (7,8). Overproduction of the indicate that GHJGS is a viable therapeutic agent against proinflammatory cytokines, tumor necrosis factor‑α (TNF-α) inflammation and oxidative stress-associated disorders. and interleukin‑6 (IL‑6), and the proinflammatory mediator, prostaglandin E2 (PGE2) may result in inflammatory disor- ders accompanied by fever, tissue destruction or pain (7,8). Therefore, targeting these proinflammatory cytokines or PGE2 is considered to be a potential therapeutic approach for treating inflammatory disorders. Mitogen‑activated protein kinase (MAPK) and/or nuclear factor-κB (NF-κB) signaling pathways are important in the regulation of inflammatory responses, including triggering the initiation of proinflamma- Correspondence to: Dr Hyeun-Kyoo Shin, K-herb Research Center, Korea Institute of Oriental Medicine, 1672 Yuseong-daero, tory cytokine production (9). Additionally, previous studies Yusung-gu, Daejeon, Chungcheong 34054, Republic of Korea have reported a link between anti-inflammatory and anti- E-mail: [email protected] oxidative regulation using various natural products through activation of heme oxygenase-1 (HO-1), an enzyme with Key words: Gwakhyangjeonggi-san, anti-inflammation, antioxidant, antioxidant effects (10-12). heme oxygenase-1, macrophages In the present study, the anti‑inflammatory and antioxidant activity of GHJGS was investigated using the murine macro- phage cell line, RAW 264.7. The inflammatory reaction was induced by lipopolysaccharide (LPS) stimulation and the 4366 JEONG et al: ANTI-INFLAMMATORY AND ANTIOXIDANT PROPERTIES OF GHJGS production of TNF-α, IL-6, and PGE2 was examined using (glycyrrhizin), 280 nm (liquiritin, hesperidin and 6‑gingerol), enzyme-linked immunosorbent assays (ELISAs). In addition, and 330 nm (rosmarinic acid). The sample injection volume the effects of GHJGS on activation of MAPK and NF-κB was 10 µl. signaling pathways, and the expression of HO-1 in RAW 264.7 cells were investigated. Cell culture. The murine macrophage cell line, RAW 264.7, was obtained from the American Type Culture Collection Materials and methods (Rockville, MD, USA). The cells were cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Plant materials. The 13 herbs that form GHJGS were Inc., Waltham, MA, USA) supplemented with 5.5% heat‑inac- purchased from Kwangmyungdang Medicinal Herbs (Ulsan, tivated fetal bovine serum (Gibco; Thermo Fisher Scientific, South Korea). The taxonomic classification of the 13 herbs was Inc.), penicillin (100 U/ml; HyClone Laboratories, Inc., verified by Professor Je‑Hyun Lee from Dongguk University Logan, UT, USA), and streptomycin (100 µg/ml; HyClone (Gyeongju, South Korea). Voucher specimens (2012-KE32-1 Laboratories, Inc.) in an incubator with 5% CO2 at 37˚C. to KE32-13) were deposited at the K-herb Research Center, Korea Institute of Oriental Medicine (Daejeon, South Korea). Cytotoxicity assay. Cell viability assay was performed to deter- mine the cytotoxicity of GHJGS using a Cell Counting Kit‑8 Chemicals and reagents. Liquiritin, glycyrrhizin and (CCK‑8; Dojindo Molecular Technologies, Inc., Kumamoto, 6-gingerol were purchased from Wako Pure Chemical Japan). Cells were plated onto a 96-well microplate at 3x103 Industries, Ltd. (Osaka, Japan). Hesperidin and rosmarinic cells/well and treated with 0, 15.625, 31.25, 62.5, 125, 250, 500 acid were purchased from Acros Organics (Morris, NJ, USA) or 1,000 µg/ml GHJGS for 24 h. Following incubation with and Sigma-Aldrich (St. Louis, MO, USA), respectively. The CCK‑8 reagent for 4 h, optical density (OD) at a wavelength purity of each component was determined to be ≥98% using of 450 nm was determined using a Benchmark plus micro- high-performance liquid chromatography (HPLC) analysis. plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The chemical structures of the five marker compounds are Cell viability was calculated using the following equation: presented in Fig. 1A. HPLC-grade reagents, methanol, acetoni- Cell viability (%) = mean ODGHGJS-treated cells / mean ODuntreated trile and distilled water were obtained from J.T. Baker; Avanto cells x100 Performance Materials (Phillipsburg, NJ, USA). Acetic acid was obtained from Merck KGaA (Darmstadt, Germany). ELISAs for TNF‑α, IL‑6, and PGE2. Cells were pretreated with 0, 250, 500 or 1,000 µg/ml GHJGS for 4 h and stimu- Preparation of GHJGS decoction. GHJGS was composed lated with LPS (1 µg/ml) for an additional 20 h. Production of 13 herbs (Table I; total weight, 5.0 kg, ~148.15 times the of TNF-α, IL-6 and PGE2 in the culture supernatants was composition of a single dose) and extracted in distilled water measured using commercial ELISA kits from R&D Systems, at 100˚C for 2 h under 98 kPa pressure using a COSMOS‑660 Inc. (Minneapolis, MN, USA), BD Biosciences (San Jose, electric extractor (KyungSeo Machine Co., Incheon, South CA, USA) and Cayman Chemical Company (Ann Arbor, MI, Korea). The extracted solution was filtered using a standard USA), respectively. Indomethacin (2.5 ng/ml; Sigma-Aldrich) sieve (no. 270; mesh size, 53 µm; Chung Gye Sang Gong Sa, was used as a positive control. Seoul, Korea) and freeze-dried. The yield of the extract was 12.89% (644.5 g). The lyophilized GHJGS extract (40 mg) was Western blotting. Whole cell extract (WCE) was prepared dissolved in 50% methanol (20 ml) and mixed for quantitative by suspending cells using the Mammalian Cell Lysis kit analysis. The solution was filtered through a 0.2‑µm SmartPor (Sigma-Aldrich) containing protease inhibitor cocktail GHP syringe filter (Woong Ki Science Co., Ltd., Seoul, South (Roche Diagnostics, Indianapolis, IN, USA). Nuclear extract Korea) prior to being injected into a HPLC column. (NE) was isolated using NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Inc., Rockford, Quantitative analysis of GHJGS. The quantitative deter- IL, USA) according to the manufacturer's protocol. The mination was performed using a Prominence LC-20A protein concentration was determined using the Bio-Rad series HPLC system (Shimadzu Corporation, Kyoto, Japan) Protein Assay kit II (Bio-Rad Laboratories, Inc.). Equal consisting of a solvent delivery unit (LC-20AT), online quantities of cell extract (30 µg) were resolved by 4‑20% degasser (DGU-20A3), column oven (CTO-20A), auto sample sodium dodecyl sulfate-polyacrylamide gel electrophoresis injector (SIL-20AC), and photodiode array (PDA) detector (Bio-Rad Laboratories, Inc.) at 100 v for 1 h and transferred (SPD-M20A). Data were collected and processed using LC to a polyvinylidene fluoride membrane (GE Healthcare Life solution software (version 1.24; Shimadzu Corporation). A Sciences,