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Inhibition of MMP-2 and -9 by Crude Extracts and Their Solvent-partitioned Fractions from the Halophyte Atriplex gmelinii

가는갯능쟁이(Atriplex gmelinii) 추출물과 용매분획물의 MMP-2와 MMP-9 활성 저해효과

  • Park, Min Jeong (Division of Marine Bioscience, Korea Maritime & Ocean University) ;
  • Kim, Junse (Ocean Science & Technology School, Korea Maritime & Ocean University) ;
  • Kong, Chang-Suk (Department of Food and Nutrition, College of Medical and Life Sciences Silla University) ;
  • Seo, Youngwan (Division of Marine Bioscience, Korea Maritime & Ocean University)
  • 박민정 (한국해양대학교 해양생명과학부) ;
  • 김준세 (한국해양대학교 해양과학기술전문대학원) ;
  • 공창숙 (신라대학교 식품영양학과) ;
  • 서영완 (한국해양대학교 해양생명과학부)
  • Received : 2019.04.10
  • Accepted : 2019.06.17
  • Published : 2019.06.30

Abstract

In this study, the inhibitory effect of Atriplex gmelinii C. A. Mey. against the activity of MMP-2 and MMP-9 secreted from phorbol-12-myristate-13-acetate (PMA)-stimulated HT-1080 cells was evaluated by gelatin zymography and enzyme-linked immunosorbent assay (ELISA), reverse transcription polymerase-chain reaction (RT-PCR), and Western blot assay. Specimens of the halophyte A. gmelinii were extracted twice for 24 hr with methylene chloride ($CH_2Cl_2$), and then twice with methanol (MeOH), in turn. Each extract significantly inhibited the enzymatic activities in gelatin zymography and MMP ELISA kit, and expression of MMP-2 and 9 in mRNA and protein levels. Two crude extracts were combined and then the combined crude extracts were fractionated into n-hexane, 85% aqueous methanol (85% aq.MeOH), n-butanol (n-BuOH), and water ($H_2O$) fractions, according to solvent polarity. Among solvent-partitioned fractions, the 85% aq.MeOH fraction showed the strongest inhibitory effect against MMP-2 and -9 in gelatin zymography and MMP ELISA kit. In RT-PCR, all solvent-partitioned fractions significantly suppressed mRNA expression of MMP-2 and -9. On the other hand, in Western blot assay, all solvent-partitioned fractions except $H_2O$ significantly reduced expression levels of protein. HT 1080 cell migration was most significantly inhibited by the n-BuOH fraction followed by the 85% aq.MeOH and $H_2O$ fractions. These results suggest that A. gmelinii could be used as a potential source to inhibit tumor cell metastasis.

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Fig. 1. Preparation of crude extracts and their solvent-patitioned fractions from A. gmelinii

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Fig. 2. Effect of A. gmelinii crude extracts (A) and their solvent fractions (B) on cell viability observed by MTT assay.

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Fig. 3. Inhibitory effects of A. gmelinii crude extracts and their solvent-partitioned fractions on secretion levels of MMP-2 (A and C) and MMP-9 (B and D) were observed by ELISA kit.

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Fig. 4. Inhibitory effects of the combined crude extracts (C.E) of A. gmelinii on enzymatic activity (A), mRNA expression (B), and protein (C) expression of MMP-2 and MMP-9 were observed by gelatin zymography, RTPCR, and Western blot assay, respectively.

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Fig. 5. Inhibitory effects of solvent-partitioned fractions from A. gmelinii crude extracts were observed in enzymatic activity (A), mRNA expression (B), and protein expression (C) of MMP-2 and MMP-9 by gelatin zymography, RT-PCR and Western blot assay, respectively, and in HT-1080 cell migration (D)

Table 1. Sequences of primers used for RT-PCR

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Acknowledgement

Supported by : 한국연구재단

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