Equisetum ramosissimum粗萃物之生理活性開發

Developments of bio-functional activities from Equisetum ramosissimum extracts

邱予品(Yu-Pin Chiu)

指導教授 ： 張耀南

共同指導教授 ： 王惠民(Hui-Min Wang)

國立虎尾科技大學/文理學院/生物科技研究所/碩士(2015年)

摘要

實驗之設計是以節節草(Equisetum ramosissimum)之甲醇粗萃物依序以正己烷(n-hexane；Hex)、乙酸乙酯(ethyl acetate；EA)、二氯甲烷(dichloromethane；DM)、甲醇(methanol；MeOH)及水(Water)進行萃取，並且針對五種粗萃取物進行篩選，以評估其還原能力、清除DPPH 自由基能力和亞鐵離子的金屬螯合之抗氧化能力評估。根據結果顯示，萃取物EA 為五種粗萃取物中具有最佳的抗氧化功效。接著，抑制黑色素瘤的生長是透過人類的的黑色素瘤細胞之體外模式，包括A375、A375.S2 及A2058 細胞的生長抑制能力，結果顯示EA 萃取物以50 μg/mL 以上的高濃度作用於A375 及A375.S2 細胞呈現顯著的抗增殖能力。而以各萃取物作用於人類正常的角質生成細胞及纖維母細胞時，細胞存活率測試皆證實其濃度都在可接受的安全範圍中，對於正常細胞皆不會造成強大的毒殺性。透過細胞型態之觀察，當萃取物濃度增加時，其A375、A375.S2 及A2058形態會有皺縮、死亡的情形; 藉由西方墨點法檢測EA 及DM 萃取物作用下，其細胞凋亡相關蛋白的表現量，證實於皮膚癌細胞之凋亡相關蛋白Caspase-3 及Caspase-9 蛋白表現量增加，說明EA 及DM 萃取物可抑制細胞的生長。而B16-F10 黑色素細胞則較不受萃取物之影響，於DM 萃取物作用時，其細胞存活率並無顯著的降低，透過細胞型態觀察及黑色素含量測定評估，其黑色素含量也未減少，反而增加。藉由西方墨點法檢測黑色素生成的相關蛋白發現，DM 萃取物具有刺激黑色素之生成，其相關蛋白MITF、tyrosinase、TRP-1 及TRP-2 皆有增加，因而增加黑色素的含量。根據以上結果顯示， E. ramosissimum 的EA 萃取物具有較明顯的活性功效，在抗氧化試驗也具有較佳的清除自由基能力，抑制黑色素瘤細胞的生長，也能達到減緩黑色素生成，達到美白的效果，有利於開發天然的抗癌製劑及驅黑淨白的美白產品。

關鍵字

Equisetum ramosissimum ； 抗氧化 ； 黑色素瘤細胞 ； 抗增殖 ； 黑色素抑制

並列摘要

This designed experiments of Equisetum ramosissimum extracts were separated with ethyl acetate (EA), dichloromethane (DM), n-hexane (Hex), methanol (MeOH), and Water, respectively. Five crude extracts were screened to evaluate the antioxidant effects, containing reducing power, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals scavenging ability and Fe2+-metal chelating power evaluations. According to our result, EA extract showed the best anti-oxidative properties of five crude extracts. All of the extracts were examined the inhibitory effectiveness against human melanoma cell lines, including A375, A375.S2, A2058, and B16-F10 cells, and EA extract also presented the better anti-proliferation abilities for A375, A375.S2 and A2058 cells above 50 μg/mL. The cell viability assay in two human keratinocytes (HaCaT) and fibroblasts at dosages ranging from 0 to 100 μg/mL for 24 hours was demonstrated an acceptable outcome in normal cell safeties. The pigment melaningenesis suppression was evaluated through the melanin content assay with mouse melanoma cell, B16-F10, in vitro platform to discover skin lightening extracts. The suppressive data indicated that EA extract inhibited melanin productions, meanwhile, DM extract possessed the stimulation ability of pigment enhancements. Western blot analytics demonstrated the increase of pro-apoptotic proteins such as caspase-3 and caspase-9 after treatment with E. ramosissimum extracts. In addition, western blotting was showed that DM extract increase the melaningenesis on B16-F10 cell, the protein of the MITF, tyrosinase, TRP-1 and TRP-2 were significantly increased while the high concentration.
The totally results were showed that EA extract from E. ramosissimum has obvious effect to improve anti-oxidation, inhibit the growth of melanoma cells and reduce the melanin production.

並列關鍵字

Equisetum ramosissimum ； Antioxidant ； Melanoma ； Anti-proliferation ； Melanin inhibition activity

參考文獻

26. Paulsamy S., Moorthy, D., Nandakumar K., Saradha M. (2013). Evaluation of in vitro antioxidant potential of methanolic extracts of the ferns, Actiniopteris radiata (Sw) Link. and Equisetum ramosissimum Desf. International Journal of Research and Development in Pharmacy and Life Sciences, 2(3), 5.

7. Channing, A., Zamuner, A., Edwards, D., Guido, D. (2011). Equisetum thermale sp. nov. (Equisetales) from the Jurassic San Agustin hot spring deposit, Patagonia: anatomy, paleoecology, and inferred paleoecophysiology. American Journal of Botany, 98(4), 680-697.

8. Cho, Soohyun., Kang, Geunho., Seong, PN., Park, Beomyoung., Kang SM. (2015). Effect of slaughter age on the antioxidant enzyme activity, color, and oxidative stability of Korean Hanwoo (Bos taurus coreanae) cow beef. Meat Science, 108(0), 44-49.

9. Chen, CY., Wu, PY., Huang, TS., Lin., CW., Li, YC., Chou, RH., Chang, HW., Wang, HM. (2009). The sour taste-modifying protein (Miraculin), tyrosinase inhibitors and antioxidants from Synsepalum dulcificum. Current Nutrition & Food Science, 5(3), 172-179.

10. Costin, GE., Hearing, VJ. (2007). Human skin pigmentation: melanocytes modulate skin color in response to stress. The FASEB Journal, 21(4), 976-994.