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A Superoxide Dismutase Purified from the Rhizome of Curcuma aeruginosa Roxb. as Inhibitor of Nitric Oxide Production in the Macrophage-like RAW 264.7 Cell Line

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Abstract

Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme or antioxidant enzyme that catalyzes the disproportionation of the harmful superoxide anionic radical to hydrogen peroxide and molecular oxygen. Due to its antioxidative effects, SOD has long been applied in medicinal treatment, cosmetic, and other chemical industries. Fifteen Zingiberaceae plants were tested for SOD activity in their rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Curcuma aeruginosa were found to contain a significant level of SOD activity. The SOD enzyme was enriched 16.7-fold by sequential ammonium sulfate precipitation, diethylaminoethyl cellulose ion exchange, and Superdex 75 gel filtration column chromatography. An overall SOD yield of 2.51 % with a specific activity of 812.20 U/mg was obtained. The enriched SOD had an apparent MW of 31.5 kDa, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a pH and temperature optima of 4.0 and 50 °C. With nitroblue tetrazolium and riboflavin as substrates, the K m values were 57.31 ± 0.012 and 1.51 ± 0.014 M, respectively, with corresponding V max values of 333.7 ± 0.034 and 254.1 ± 0.022 μmol min−1 mg protein−1. This SOD likely belongs to the Fe- or Mn-SOD category due to the fact that it was insensitive to potassium cyanide or hydrogen peroxide inhibition, but was potentially weakly stimulated by hydrogen peroxide, and stimulated by Mn2+and Fe2+ ions. Moreover, this purified SOD also exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC50 = 14.36 ± 0.15 μg protein/ml).

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Acknowledgments

The authors thank the 90th Anniversary of Chulalongkorn University fund, the National Research University Project of CHE, the Ratchadaphiseksomphot Endowment Fund (AG001B, AM1019A, and AS613A), and the Thai Government Stimulus Package 2 (TKK2555), for financial support of this research, as well as the Institute of Biotechnology and Genetic Engineering for support and facilities. We also thank Dr. Robert Butcher (Publication Counseling Unit, Chulalongkorn University) for his constructive comments in preparing this manuscript.

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Authors and Affiliations

  1. Program in Biotechnology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok, 10330, Thailand

    Wanwisa Moon-ai & Ploypat Niyomploy

  2. Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok, 10330, Thailand

    Ruethairat Boonsombat & Aphichart Karnchanatat

  3. Department of Chemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok, 10330, Thailand

    Polkit Sangvanich

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  1. Wanwisa Moon-ai
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Correspondence to Aphichart Karnchanatat.

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Moon-ai, W., Niyomploy, P., Boonsombat, R. et al. A Superoxide Dismutase Purified from the Rhizome of Curcuma aeruginosa Roxb. as Inhibitor of Nitric Oxide Production in the Macrophage-like RAW 264.7 Cell Line. Appl Biochem Biotechnol 166, 2138–2155 (2012). https://doi.org/10.1007/s12010-012-9640-9

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