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


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.

HOGBB1_2019_v41n2_79_f0001.png 이미지

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

HOGBB1_2019_v41n2_79_f0002.png 이미지

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

HOGBB1_2019_v41n2_79_f0003.png 이미지

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.

HOGBB1_2019_v41n2_79_f0004.png 이미지

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.

HOGBB1_2019_v41n2_79_f0005.png 이미지

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

HOGBB1_2019_v41n2_79_t0001.png 이미지


Supported by : 한국연구재단


  1. National Institute of Biological Resources (2011) Biodiversity of the Korean Peninsula. Accessed 25 Mar 2019
  2. Kim S-K (2018) If you know the sea, you will see the future (Marine biological resources that have become economically useful due to the 4th industrial revolution). World Science, Seoul, 332 p
  3. Kim EK (2013) Halophytes of Korea. Nature and Ecology, Seoul, 368 p
  4. Bae JJ, Yoon HS, Choo YS, Song SD (2003) The responses of antioxidative enzymes and salt tolerance of Atriplex gmelinii. Korean J Ecol 26:273-280
  5. Bae MJ, Karadeniz F, Oh JH, Yu GH, Jang MS, Nam KH, Seo Y, Kong C-S (2017) MMP-inhibitory effects of flavonoid glycosides from edible medicinal halophyte Limonium tetragonum. Evid-Based Compl Alt 2017:6750274. doi:10.1155/2017/6750274
  6. Egeblad M, Werb Z (2002) New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2:161-174
  7. Giannelli G, Falk-Marzillier J, Schiraldi O, Stetler-Stevenson WG, Quaranta V (1997) Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science 277:225-228
  8. Ha KT, Kim JK, Kang SK, Kim DW, Lee YC, Kim HM, Kim CH (2004) Inhibitory effect of Sihoga-Yonggol-Moryo-Tang on matrix metalloproteinase-2 and-9 activities and invasiveness potential of hepatocellular carcinoma. Pharmacol Res 50:279-285
  9. Hadler-Olsen E, Fadnes B, Sylte I, Uhlin-Hansen L, Winberg JO (2011) Regulation of matrix metalloproteinase activity in health and disease. FEBS J 278:28-45
  10. Hu J, Van den Steen PE, Sang Q, Opdenakker G (2007) Matrix metalloproteinase inhibitors as therapy for inflammatory and vascular diseases. Nat Rev Drug Discov 6:480-498
  11. Jeong H, Kim H, Ju E, Kong C-S, Seo Y (2016) Antioxidant effect of the Halophyte Atriplex gmelinii. KSBB J 31:200-207
  12. Jeong H, Kim H, Ju E, Lee S-G, Kong C-S, Seo Y (2017) Antiinflammatory activity of solvent-partitioned fractions from Atriplex gmelinii C. A. Mey. in LPS-stimulated RAW264.7 macrophages. J Life Sci 27:187-193
  13. Kim H, Kong C-S, Seo Y (2017) Inhibitory activity of Ligustrum japonicum fructus on MMP-2 and MMP-9. KSBB J 32:328-334
  14. Kim J, Kong C-S, Seo Y (2018) Inhibitory effects of Carex pumila extracts on MMP-2 and MMP-9 activities in HT-1080 cells. Ocean Polar Res 40:249-257
  15. Legrand C, Gilles C, Zahm JM, Polette M, Buisson AC, Kaplan H, Birembaut P, Tournier JM (1999) Airway epithelial cell migration dynamics: MMP-9 role in cell-extracellular matrix remodeling. J Cell Biol 146:517-529
  16. Miller AB, Hoogstraten B, Staquet M, Winkler A (1981) Reporting results of cancer treatment. Cancer 47(1):207-214<207::AID-CNCR2820470134>3.0.CO;2-6
  17. Mook ORF, Frederiks WM, Van Noorden CJF (2004) The role of gelatinases in colorectal cancer progression and metastasis. BBA-Rev Cancer 1705:69-89
  18. Oh JH, Lee JI, Karadeniz F, Seo Y, Kong C-S (2018) 3,5-Dicaffeoyl-epi-quinic acid isolated from edible halophyte Atriplex gmelinii inhibits adipogenesis via AMPK/MAPK pathway in 3T3-L1 adipocyte. Evid-Based Compl Alt 2018:8572571. doi:10.1155/2018/8572571
  19. Sang QXA, Jin Y, Newcomer RG, Monroe SC, Fang X, Hurst DR, Lee SW, Cao Q, Schwartz MA (2006) Matrix metalloproteinase inhibitors as prospective agents for the prevention and treatment of cardiovascular and neoplastic diseases. Curr Top Med Chem 6:289-316
  20. Seo UK, Lee YJ, Kim JK, Cha BY, Kim DW, Nam KS, Kim CH (2005) Large-scale and effective screening of Korean medicinal plants for inhibitory activity on matrix metalloproteinase-9. J Ethnopharmacol 97:101-106
  21. Son J, Kim H, Kong C-S, Seo Y (2018) Anti-invasive effect of the solvent-partitioned fractions from viticis fructus in PMA-induced HT-1080 cells. J. Life Sci 28:293-299
  22. Son J, Kim J, Kim H, Seo Y (2019) Anti-invasion effects of Calystegia soldanella solvent extracts and partitioned fractions on PMA-stimulated fibrosarcoma cells. J Life Sci 29:287-294
  23. Sternlicht MD, Werb Z (2001) How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Bi 17:463-516
  24. Zhang C, Kim SK (2009) Matrix metalloproteinase inhibitors (MMPIs) from marine natural products: the current situation and future prospects. Mar Drugs 7:71-84
  25. Zheng L, Zhang Y-M, Zhan Y-Z, Liu C-X (2014) Momordica cochinchinensis seed extracts suppress migration and invasion of human breast cancer ZR-75-30 cells via down-regulating MMP-2 and MMP-9. Asian Pac J Cancer P 15:1105-1110
  26. Kang J-H, Han I-H, Sung M-K, Yoo H, Kim Y-G, Kim J-S, Kawada T, Yu R (2008) Soybean saponin inhibits tumor cell metastasis by modulating expressions of MMP-2, MMP-9 and TIMP-2. Cancer Lett 261:84-92
  27. Yang J-L, Lin J-H, Weng S-W, Chen J-C, Yang J-S, Amagaya S, Funayana S, Wood WG, Kuo C-L, Chung J-G (2013) Crude extract of Euphorbia formosana inhibits the migration and invasion of DU145 human prostate cancer cells: The role of matrix metalloproteinase-2/9 inhibition via the MAPK signaling pathway. Mol Med Rep 7:1403-1408