Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Effect of Pine (Pinus densiflora) Needle Extracts on Synthesis of Collagen in Osteoblastic MC3T3-E1 Cells

적송잎 추출물이 조골세포의 collagen 합성에 미치는 영향

  • Received : 2010.03.25
  • Accepted : 2010.04.08
  • Published : 2010.04.30
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Abstract

Osteoporosis is a disease involving a decrease in bone mineral density and an increased risk of fractures. The MC3T3-E1 pre-osteoblastic cell line is a well-accepted model of osteogenesis in vitro. Pine needles have long been used as a traditional health-promoting medicinal food in Korea. In this study, MTT assay, the alkaline phosphatase (ALP) activity and collagen synthesis of osteoblast cells were investigated to determine the effects of pine needle extracts on cell proliferation and differentiation. Pine needle extracts were prepared using hexane, ethanol and water. The effects of the pine needle extracts were examined by comparing the results with those of commercial agents, such as proanthocyanidin. The MC3T3-E1 cells exposed to proanthocyanidin showed increased proliferation in a concentration-dependent manner. The cells exposed to the hexane extract showed a similar increase in proliferation to that observed with proanthocyanidin. The hexane extract showed the highest ALP activity. Moreover, a supplement of pine needle extracts induced collagen synthesis in MC3T3-E1 cells. The pine needle extract produced the highest level of collagen synthesis at concentrations of $10{\sim}50\;{\mu}g/ml$. These results indicate that pine needle extracts have an anabolic effect on bone by promoting osteoblastic differentiation, and may be used in the treatment of common metabolic bone diseases.

솔잎은 항산화력이 우수한 것으로 알려져 있으며, mouse calvaria 유래의 MC3T3-E1 osteoblastic cell은 골세포의 세포 활성과 관련된 연구에서 유용하게 이용되어왔다. 따라서 본 연구에서는 MC3T3-E1 세포를 이용하여 적송잎 용매별 추출물이 세포 증식에 미치는 영향과 ALP 활성 및 조골세포의 골 형성을 위한 필수 인자인 콜라겐 합성의 영향에 대해 검토하였다. 적송잎 추출물의 농도(1, 10 50, $100\;{\mu}g/ml$)에 따른 조골세포 성장에 미치는 영향을 MTT assay로 분석한 결과, proanthocyanidin의 경우 $50\;{\mu}g/ml$ 이상 첨가한 군에서 대조군보다 급격한 증식률을 나타내었다. 에탄올 추출물을 첨가하였을 때 유의적인 증식률이 나타나지 않은 반면, 적송잎 헥산 추출물 처리군에 있어서는 $10\;{\mu}g/ml$ 농도 이상에서 유의적으로 세포증식이 촉진되었다. 적송잎 추출물이 ALP 활성에 미치는 영향을 조사한 결과, 적송잎 추출물이 조골세포의 ALP의 활성을 증가시켜 조골세포의 분화에 영향을 줄 가능성이 제시 되었다. 또한 적송잎의 열수 및 에탄올 추출물보다 헥산 추출물에서 조골세포의 ALP 활성이 증가하여 적송잎의 ALP 활성에 영향을 주는 성분은 수용성 성분보다 지용성 성분인 것으로 추측되었다. 적송잎 추출물이 조골세포의 콜라겐 합성에 미치는 실험결과에서 헥산 추출물뿐만 아니라 열수 추출물에서도 높은 콜라겐 합성능력을 나타내었다. 따라서 단일성분에 의한 것보다 복합적 성분들의 상승작용에 의해 조골세포의 콜라겐 합성이 촉진된 것으로 추측할 수 있다. 적송잎 추출물이 조골세포의 증식, ALP 활성 및 콜라겐 합성을 촉진하여 골 생성에 영향을 줄 수 있는 것이 확인되었으며, 그에 대한 구체적인 기작 연구를 위하여 향후 분자생물학적인 차원에서의 in vitro 및 in vivo 실험을 통한 구체적인 연구가 수행되어야 할 것으로 사료된다.

Keywords

References

  1. Aldercreutz, H. and W. Mazur. 1996. Phyto-estrogens in relation to cancer and other humam health risks. Proc. Nutr. Soc., 55, 399-417 https://doi.org/10.1079/PNS19960038
  2. Canalis, E. 1983. The hormonal and local regulation of bone formation. Endocr Rev. 4, 62-77. https://doi.org/10.1210/edrv-4-1-62
  3. Canalis, E. 1985. Effect of growth factors on bone cell replication and differentiation. Clin Orthop. Related Res. 193, 246-263.
  4. Centrella, M., T. L. Mcartht, and E. Canalis. 1987. Transforming growth factor B is a bifunctional regulator of replication and collagen synthesis in osteoblast-enriched cell cultures from fatal rat bone. J. Biol. Chem. 262, 2869-2874.
  5. Choi, E. J., C. Lee, T. J. Rhim, B. C. Cha, and H. J. Park. 1997. Antimicrobial activities of pine needle (pinus densiflora) extract. Korean J. Appl. Microbiol. Biotechnol. 25, 293-297.
  6. Choi, E. M., K. S. Suh, Y. S. Kim, R. W. Choue, and S. J. Koo. 2001. Soybean ethanol extract increase the function of osteoblastic MC3T3-E1 cells. Phytochemistry 56, 733-739. https://doi.org/10.1016/S0031-9422(00)00484-2
  7. Choi, J. H., D. I. Kim, S. H. Park, D. W. Kim, J. H. Lee, and H. S. Kim. 2001. Investigation of anti-aging effect and experiment. IV. Effects of butanol fraction on oxygen radicals and their scavenger enzymes in brain of SD rats. Korean J. Gerontol. 11, 7-13.
  8. Chung, Y. J., M. W. Bae, M. I. Chung, J. S. Lee, and M. S. Chung. 2002. Cytotoxin effect of the distilled pine-needle extracts on several cancer cell lines in vitro. Korean J. Food Sci. Nutr. 31, 2-13.
  9. Dragsted, L. O., M. Strube, and J. C. Larsen. 1993. Cancer-protective factor in fruits and vegetables. Biocheminal and biological background. Pharmacol. Toxicol. 72, 116-135.
  10. Hsu, T., S. Sheu, E. Liaw, T. Wang, and C. Lin. 2005. Anti-oxidant ctivity and effect of pinus morrisonicola Hay. on the survival of leukemia cell line U937. Phytomedicine 12, 663-669. https://doi.org/10.1016/j.phymed.2004.03.013
  11. Jang, Y. J. 2008. Effects of Acanthopanax senticosus vat. Subinermis (ASVS) leaf extract on promoting cell proliferation, differentiation, mineralization and osteogenesis-related gene expression in ROS 17/2.8 cells. Department of Food and Nutrition. Graduate School, Younsei University. M. S. thesis, Korea.
  12. Jilka, R. L. 1998. Cytokines, bone remodeling and estrogen deficiency: a 1998 update. Bone 23, 75-81. https://doi.org/10.1016/S8756-3282(98)00077-5
  13. Kalu, D. 1991. The ovariectomized rat model on postmenopausal bone loss. Bone and Mineral 15, 175-192. https://doi.org/10.1016/0169-6009(91)90124-I
  14. Kang, S. R. 2009. Effect of ecklonia cava extracts on lipids and bone turnover markers in menopausal women. Department of Food Nutrition. Graduate School, Silla University. M. S. thesis, Korea.
  15. Kang, S. R., Y. K. Kim, S. G. Kim, S. H. Lee, and M. H. Kim. 2009. The Effect of Pine (pinus densiflora) Needle Extracts on Blood Flow and Serum Lipid Improvement. J. Life Science 19, 508-513. https://doi.org/10.5352/JLS.2009.19.4.508
  16. Kimble, R. B., J. L. Vannice, and D. C. Bloedow. 1994. Interleukin-I receptor antagonist decrease bone loss and resorption in ovariectomized rats. J. Clin. Invest. 93, 1959-1967. https://doi.org/10.1172/JCI117187
  17. Kim, E. J., K. P. Choi, S. S. Ham, and H. Y. Kang. 1998. Inhibitory effect of pine needle extracts on the chemical induces mutagenicity. Korean J. Food Sci. Technol. 30, 450-455.
  18. Kim, E. J., S. W. Jung, K. P. Chio, and S. S. Ham. 1998. Cytotoxic effect of the pine needle extracts. Korean J. Food Sci. Technol. 30, 213-217.
  19. Lee, H. J. 2004. Studies on biological activities of the pine-needle distilled water extract. School of Biotechnology and Bioengineering Graduate School, Kangwon National University. M. S. thesis, Korea.
  20. Lee, J. W. 2004. Effect of Solidago Virga-aurea var. giagantea Mig. Root extract on the activity of osteoblastic cells and bone metabolism. Department of Food Science and Technology, Graduate School of Keimyung University, Daegu, Korea. M. S. thesis, Korea.
  21. Lee, O. H. K. Y. Kim, M. K. Jang, K. H. You, S. G. Kim, M. H. Kim, and S. H. Lee. 2008. Evaluation of proanthocyanidin contents in total polyphenolic compounds of pine (pinus densiflora) needle extracts and their antioxidative activities. J. Life Science 18, 213-219. https://doi.org/10.5352/JLS.2008.18.2.213
  22. Lee, Y. H., Y. S. Choi, and S. Y. Lee. 1996. The cholesterol-lowering effects of the extract from Pinus strobus in chickens. Korean J. Soc. Food Nutr. 25, 188-192.
  23. Manolagas, S. C. 2000. Basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Birth and death of bone cells. Endocr. Rev. 21, 115-137. https://doi.org/10.1210/er.21.2.115
  24. Newmark, H. L. 1996. Plant phemolics as potential cancer prevention agents. Adv. Exp. Med. Biol. 401, 25-34. https://doi.org/10.1007/978-1-4613-0399-2_3
  25. Noriyoshi, K., I. Seichi, K. Mamoru, H. Yoshiyuki, I. Katsumi, and K. Masuoshi. 1986. Effect of 1,25-dihydroxyvitamin D3on osteoblastic MC3T3-E1 cell. Endocrinology 118, 940-947. https://doi.org/10.1210/endo-118-3-940
  26. Owen, T. A., M. Aronow, and V. Shahoub. 1990. Progressive development of the rat osteoblast phenotype in vitro: relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J. Cell Physiol. 143, 420-430. https://doi.org/10.1002/jcp.1041430304
  27. Pack, C. S. 1998. Antibacterial activity of ethanol extract of pine needle against pathogenic bacteria. Korean J. Postharvest Sci. Technol. 5, 380-385.
  28. Park, G. Y., H. S. Lee, I. D. Hwang, and H. S. Jung. 2006. The functional effects of fermented pine needle extract. Korean J. Biotechnol. Bioeng. 21, 376-383.
  29. Park, H. M. 1995. Role of systemic hormones and local factors in regulation of bone remodeling. Korean J. Medical Association 38, 8-19.
  30. Pfeilschifter, J. and O. Wolf. 1990. Chemotactic response of osteoblastic cells to transforming growth factor B. J. Bone Miner. Res. 5, 815-823. https://doi.org/10.1002/jbmr.5650050804
  31. Shin, H. K. 1997. The development of functional food and research trends. Food Sci. and Industry 30, 2-13.
  32. Shin, J. M., C. K. Park, E. J. Shin, T. H. Jo, and I. K. Hwang. 2008. Effects of scutellaria radix extracts on osteoblast differentiation and osteocalst formation. Korean J. Food Sci. Technol. 40, 674-679.
  33. Solt, D. B. 1991. The pathogenesis, oral manifestations, and implications for dentistry of metabolic bone disease. Curr. Opin. Dent. 1, 783-791.
  34. Stein, G. S., J. B. Lian, A. J. van Wifnen, and M. Montechino. 1996. Transcriptional control of osteoblast growth and differentiation. Physiol. Rew. 76, 593-629.
  35. Wlodarski, K. H. 1990. Properties and origin of osteoblast. Clin. Orthop. Relat. Res. 252, 276-293.
  36. Woessner, J. F. 1961. The determination of hydroxyproline in tissue and protein sample containing small proportion of this imino acid. Arch. Biochem. Biophys. 93, 440-447. https://doi.org/10.1016/0003-9861(61)90291-0
  37. Yoon, H. G., J. T. Woo, J. W. Kim, Y. S. Kim, K. Y. Kim, Y. K. Choi, and K. S. Seo. 1993. Osteoblastic cell line MC3T3-E1 cell to the triiodothyronine. Korean J. Medicine 44, 49-58.