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Anti-Inflammatory Activity and Cytotoxicity of the Starfish Extracts

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Anti-Inflammatory Activity and Cytotoxicity of the Starfish Extracts l ch cina em di is e tr Wikarta et al., Med chem (Los Angeles) 2016, 6:5 M y Medicinal chemistry DOI: 10.4172/2161-0444.1000366 ISSN: 2161-0444 t Research Article Open Access Anti-Inflammatory Activity and Cytotoxicity of the Starfish Extracts on Cancer Cells in Culture Jumeri Mangun Wikarta1,2 and Sang Moo Kim2* 1Department of Agroindustrial Technology, Faculty of Agricultural Technology, Gadjah Mada University, Bulaksumur, Yogyakarta, Indonesia 2Department of Marine Food Science and Technology, Gangneung, Wonju National University, Gangneung 210-702, Republic of Korea Abstract Background: The starfishes were shown to containe the bioactive compounds which exhibit various biological activities including cytotoxicity, antiinflammatory, hemolytic, antibacterial, antiviral, and antifungal effects. Objective: To determine the anti-inflammatory activity and cytotoxicity of the extracts of four different starfishes. Materials and methods: The homogenized fresh body component of starfishes were extracted and concentrated. The EtOH extract of A. pectinifera with high extraction yield and total phenol was partitioned using different extraction solvents. The CHCl3 fraction was further fractionated using thin-layer chromatography, silica-gel chromatography, gel filtration, and recycling preparative HPLC, in that order. Results: Fraction SAP4.2.5 exhibited strong anti-inflammatory activity without cytotoxic effect up to a concentration of 25 µg/mL, and high cytotoxicity, with IC50 values of 19.4, 34.8, and 32.7 µg/mL against AGS, DLD-1, and HeLa cells, respectively. These biological activities could be due to the presence of lathosterol, 13-tetradecen-1-ol acetate, and stigmast-7-en-3-ol (3ß,5α,24S)-like compounds. Conclusion: Starfish has a great potential as a resource for natural health products because of its strong anti- inflammatory and anticancer activities. Keywords: Anti-inflammation; Cytotoxicity; Extract; Starfish; Ste- widely distributed in most of the oceans of the world. In recent years, roid starfish have attracted much attention due to their large scale outbreaks in coastal areas, which led to severe economic damage to the fishery and Introduction aquacultural grounds of benthic shellfishes, including mussels, oysters and scallops. Moreover, the industrial applications of the secondary Many new drugs derived from secondary metabolites have metabolites of starfish have received attention worldwide. Steroidal been applied in the treatment and/or prevention of various diseases. glycosides, which are composed of a polyhydroxylated steroidal However, these drugs have various and severe adverse effects such as aglycone and a carbohydrate portion containing only one or two nausea, vomiting, edema, and diarrhea. Therefore, naturally occurring monosaccharide units, are a growing subgroup of the active glycoside agents with high effectiveness and no side-effects are desirable chemical compounds that have been isolated from starfishes [5]. Starfishes have therapeutics. also been reported to have antibacterial [6], antiviral [7], cytotoxic [8] Inflammation is a complex pathophysiological process that is and apoptotic [9], neuritogenic [10], antifungal [11], antityrosinase mediated by a variety of signaling molecules produced by leukocytes, [12], anti-inflammatory [13], and anticancer [14,15] activities. macrophages, mast cells, and platelets, among other cells. Macrophages The objective of this study was to determine and compare the play an important role in inflammatory disease through releasing factors, such as the free radical nitric oxide (NO), prostaglandin anti-inflammatory activity and cytotoxicity against cancer cells of mediators, and cytokines, which are involved in the immune response four starfishes, Asterina pectinifera, Asterina amurensis (the purple [1]. Excessive production of the inflammatory mediators is associated and yellow types) and Distolasterias nipon. Because A. pectinifera with many diseases, such as rheumatoid arthritis, chronic hepatitis, starfish is abundant and ubiquitous and has high levels of polyphenol and pulmonary fibrosis [2]. Hence, inhibiting the production of and saponin, the anti-inflammatory and anticancer compounds of its inflammatory mediators in response to inflammatory stimuli might be species were purified, isolated, and identified. a useful therapeutic strategy in inflammatory diseases. Materials and Methods Nitric oxide (NO) is a diatomic free radical produced from L-arginine by constitutive (cNOS) and inducible nitric oxide synthase Animal material (iNOS) in numerous mammalian cells and tissues. Nitric oxide (NO), Specimens of Asterina pectinifera, purple-type Asterina amurensis, - - superoxide (O2 ) and their reaction product peroxynitrite (ONOO ) may be generated in excess during the host response against viral and antibacterial infections and may contribute to pathogenesis by *Corresponding author: Sang Moo Kim, Department of Marine Food Science and promoting oxidative stress, tissue injury and even cancer [3]. Technology, Gangneung, Wonju National University, Gangneung 25457, Republic of Korea, Marine organisms are excellent sources of structurally diverse Tel: +82336402343; Fax: +82336402850; E-mail: [email protected] molecules that are potentially valuable as drugs. Many natural marine Received May 03, 2016; Accepted May 13, 2016; Published May 18, 2016 products have been designated as sources of new anti-inflammatory Citation: Wikarta JM, Kim SM (2016) Anti-Inflammatory Activity and Cytotoxicity of factors and cancer chemopreventive agents [4]. In particular, the the Starfish Extracts on Cancer Cells in Culture. Med chem (Los Angeles) 6: 331- starfishes was considered as one of the most promising sources of such 338. doi:10.4172/2161-0444.1000366 agents due to the variety of species and applications of its products. Copyright: © 2016 Wikarta JM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits Starfish (sea star) is an invertebrate belonging to the class unrestricted use, distribution, and reproduction in any medium, provided the Asteroidea, phylum Echinodermata, of which over 1500 species are original author and source are credited. Med chem (Los Angeles) Volume 6(5): 331-338 (2016) - 331 ISSN: 2161-0444 Med chem (Los Angeles), an open access journal Citation: Wikarta JM, Kim SM (2016) Anti-Inflammatory Activity and Cytotoxicity of the Starfish Extracts on Cancer Cells in Culture. Med chem (Los Angeles) 6: 331-338. doi:10.4172/2161-0444.1000366 yellow-type Asterina amurensis, and Distolasterias nipon were collected with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at of the easterncoast off the Korean peninsula. The animals were brought 37°C in a humidified atmosphere of 5% CO2. The concentration of to the laboratory in a fresh condition and were rapidly dissected and RAW 264.7 cells was adjusted to 1 × 106 cells/mL and that of the AGS rinsed using tap water to eliminate contaminants, and were then stored cells, DLD-1 cells, and HeLa cells was adjusted to 1 × 105 cells/mL using at -40°C until use. Immediately before use, the starfishes were thawed at the same medium. 4°C overnight and then were sliced into small pieces using a knife. The starfish samples were placed in a stainless-steel container, frozen using Preparation and purification of starfish extract liquid nitrogen, and then ground using a blender. The flow chart for the purification of starfish bioactive compounds Reagents is shown in Figure 1. The homogenized fresh body component of the starfishes was extracted using 95% EtOH and 2H O in cold room for 6 Dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS) from h, which was repeated three times. The extracts were combined and Escherichia coli 0127:B8, NΨ-nitro-L-arginine methyl ester hydrochloride filtered. The filtrate was concentrated in vacuo at 40°C to the point of (L-NAME), Griess reagent (0.1% napthylethylenediamine and 1% dryness, yielding the crude extract. Because A. pectinifera is abundant and resulted in high extraction yield as well as high amount of total sulfanilamide in 5% H3PO4), and 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazoliumbromide (MTT) were purchased from the Sigma phenol (Table 1), this species was chosen for further analysis. The Chemical Co. (St. Louis, MO, USA). RPMI 1640 medium and fetal EtOH-extracted material of A. pectinifera was suspended in H2O. This bovine serum (FBS) were purchased from Lonza (Walkersville, MD, solution was partitioned using C6H14 to yield a C6H14 fraction. Next, USA). All of the other commercially available chemicals and reagents the aqueous layer was successively partitioned using CHCl3, EtOAc, that were used were of analytical grade. and BuOH, in that order. After removing each solvent by vacuum evaporation, five fractions were obtained and were hereafter referred to Cell lines and culture as the C6H14, CHCl3, EtOAc, BuOH, and H2O fractions. The macrophage cell line RAW 264.7 (Korean Cell Line Bank, Because the CHCl3 fraction exhibited the strongest anti- Seoul, Korea) was grown in plastic culture flasks in RPMI-1640 inflammatory and anticancer activities (Table 2), this fraction was medium containing L-glutamine and supplemented with 10% FBS, 100 coarsely fractionated using silica-gel (Merck KgaA, Darmstadt, U/mL penicillin, and 100 µg/mL streptomycin under 5% CO2 at 37°C. Germany) column (3.0 × 50.0 cm) chromatography, using step- After 3 days, the cells were removed from the culture flask by scraping gradient
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