Screening and Studying Anti-inflammatory Effects of Various Herbal Extracts in Mouse Macrophages
許貴媄、蔡明勳
E-mail: [email protected]
ABSTRACT
The inflammatory response is a part of the human immune system, and is also a necessary process of cure. However, when a persistent chronic inflammation happen, it will not only cause organ/tissue damage but also promote the proliferation of abnormal cells, then leading to many common diseases by scientific studies. Therefore, if we can prevent or treat inflammation, we can prevent or cure these diseases. However, some anti-inflammatory drugs have significant toxicity or side-effects. Accordingly, natural or herbal extracts with anti-inflammatory effects have become a good choice for anti-inflammatory pharmaceutical research and development. In this study, we evaluate twenty-one ethanol extracts of Chinese herbals or plants as research subjects. We use lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophage as inflammatory cell model. We screened all these extracts to select which with anti-inflammatory activity according to their inhibition rates of NO productions. Preliminary results showed that four extracts, including Schisandra chillensis, Artemisia indica, Colocasia esculenta leaves and Alpinia officinarum, at concentration of 250 μg/mL exhibited significant anti-inflammatory activities. Therefore, we analyzed the anti-inflammatory effects of these four extracts at various concentrations (6.25~25 μg/mL) in LPS-induced macrophages. Results show that both extracts of Schisandra chillensis and Colocasia esculenta leaves not only promote the production of an anti-inflammatory interleukin-10 (IL-10), but also suppress LPS-induced inflammatory factors including Tumor Necrosis Factor-α (TNF-α), NO, Cyclooxygenase-2 (COX-2) and Prostaglandin E2 (PGE2). Extract of Artemisia indica suppresses LPS-indused NO, and reduces PGE2 production by inhibiting LPS-induced COX-2 level, but it doesn’t promote IL-10 and suppress LPS-indused TNF-α. Extract of Alpinia officinarum promotes IL-10 and suppresses the LPS-induced NO and PGE2, but it doesn’t suppress LPS-induced TNF-α and COX-2 level.
In brief, we identify that ethanol extracts of Schisandra chillensis, Artemisia indica, Colocasia esculenta leaves and Alpinia officinarum have anti-inflammatory activities in LPS-induced RAW264.7 mouse macrophage model. The anti-inflammatory mechanisms of extracts from Schisandra chillensis and Colocasia esculenta leaves are clearer than the other two extracts. These four extracts may have potential to develop anti-inflammatory foods or drugs. Further researches are needed to decipher their
anti-inflammatory mechanisms and effective concentrations.
Keywords : RAW264.7 mouse macrophages、anti-inflammatory activity、herbal and plant extracts Table of Contents
封面內頁 簽名頁 中文摘要 iii 英文摘要 v 誌謝 vii 目錄 viii 圖目錄 xi 表目錄 xiv 1.緒論 1 2.文獻回顧 3 2.1發炎與巨噬細胞 3 2.2脂多醣(LPS) 4 2.3 NO與NO合成?(NO synthases, NOSs) 7 2.4腫瘤壞死因子-α (TNF-α) 9 2.5前列腺素E2 (PGE2)與環氧合?
(COX) 10 2.6介白素-10 (IL-10) 11 2.7植物萃取液介紹 12 2.7.1五味子 12 2.7.2艾草 14 2.7.3芋頭葉 15 2.7.4高良薑 16 3.材料與 方法 18 3.1實驗材料 18 3.1.1植物萃取液 18 3.1.2細胞株 18 3.1.3藥品 19 3.1.4 Enzyme-Linked Immunosorbent Assay (ELISA) Kit 21 3.1.5抗體 21 3.1.6其它耗材及設備 22 3.2細胞培養與保存 22 3.2.1以粉末配製DMEM培養基(以1 L為例) 22 3.2.2 RAW264.7巨噬細胞株之培養 23 3.2.3 RAW264.7巨噬細胞株之保存 23 3.3細胞存活率分
析3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay 24 3.4發炎反應相關檢測分析 24 3.4.1樣品製備 24 3.4.2亞硝酸鹽測定(Nitrite assay) 25 3.4.3 IL-10與TNF-α測定 26 3.4.4 PGE2測定 27 3.5偵測COX-2蛋白表現量 28 3.5.1細胞 全蛋白萃取液製備 28 3.5.2蛋白質濃度測定 29 3.5.3以SDS polyacrylamide gel electrophoresis (SDS-PAGE)進行蛋白質電泳分 析 30 3.5.4轉印 32 3.5.5蛋白質免疫偵測（西方雜合法）33 3.6統計分析 33 4.結果與討論 34 4.1 73種乙醇萃取物對LPS刺激 巨噬細胞分泌NO之影響 34 4.2不同濃度的五味子萃取物對巨噬細胞發炎反應之影響 36 4.3不同濃度的艾草萃取物對巨噬細 胞發炎反應之影響 44 4.4不同濃度的芋頭葉萃取物對巨噬細胞發炎反應之影響 52 4.5不同濃度的高良薑萃取物對巨噬細胞 發炎反應之影響 60 5.結論 68 參考文獻 73 附錄 79 圖目錄 圖2.1 LPS在格蘭氏陰性菌的位置和結構圖 5 圖2.2巨噬細胞經 由LPS活化調控發炎相關傳遞路徑與基因之途徑 5 圖2.3巨噬細胞分泌的各種細胞激素對其他免疫相關細胞的交互作用影響 6 圖2.4五味子果實照片 13 圖2.5艾草植株照片 14 圖2.6芋頭植株及其葉子照片 15 圖2.7高良薑根莖切片的照片 17 圖3.1三明 治轉漬夾示意圖 32 圖4.1不同濃度的五味子萃取物對RAW264.7細胞存活率之影響 38 圖4.2不同濃度的五味子萃取物對LPS 誘導RAW264.7細胞分泌NO之影響 39 圖4.3不同濃度的五味子萃取物對LPS誘導RAW264.7細胞分泌IL-10之影響 40 圖4.4不 同濃度的五味子萃取物對LPS誘導RAW264.7細胞分泌TNF-α之影響 41 圖4.5不同濃度的五味子萃取物對LPS誘
導RAW264.7細胞分泌PGE2之影響 42 圖4.6不同濃度的五味子萃取物對LPS誘導RAW264.7細胞表現COX-2蛋白質之影響 43 圖4.7不同濃度的艾草萃取物對RAW264.7細胞存活率之影響 46 圖4.8不同濃度的艾草萃取物對LPS誘導RAW264.7細胞分 泌NO之影響 47 圖4.9不同濃度的艾草萃取物對LPS誘導RAW264.7細胞分泌IL-10之影響 48 圖4.10不同濃度的艾草萃取物 對LPS誘導RAW264.7細胞分泌TNF-α之影響 49 圖4.11不同濃度的艾草萃取物對LPS誘導RAW264.7細胞分泌PGE2之影響 50 圖4.12不同濃度的艾草萃取物對LPS誘導RAW264.7細胞表現COX-2蛋白質之影響 51 圖4.13不同濃度的芋頭葉萃取物 對RAW264.7細胞存活率之影響 54 圖4.14不同濃度的芋頭葉萃取物對LPS誘導RAW264.7細胞分泌NO之影響 55 圖4.15不同 濃度的芋頭葉萃取物對LPS誘導RAW264.7細胞分泌IL-10之影響 56 圖4.16不同濃度的芋頭葉萃取物對LPS誘導RAW264.7細 胞分泌TNF-α之影響 57 圖4.17不同濃度的芋頭葉萃取物對LPS誘導RAW264.7細胞分泌PGE2之影響 58 圖4.18不同濃度的 芋頭葉萃取物對LPS誘導RAW264.7細胞表現COX-2蛋白質之影響 59 圖4.19不同濃度的高良薑萃取物對RAW264.7細胞存 活率之影響 62 圖4.20不同濃度的高良薑萃取物對LPS誘導RAW264.7細胞分泌NO之影響 63 圖4.21不同濃度的高良薑萃取 物對LPS誘導RAW264.7細胞分泌IL-10之影響 64 圖4.22不同濃度的高良薑萃取物對LPS誘導RAW264.7細胞分泌TNF-α之 影響 65 圖4.23不同濃度的高良薑萃取物對LPS誘導RAW264.7細胞分泌PGE2之影響 66 圖4.24不同濃度的高良薑萃取物 對LPS誘導RAW264.7細胞表現COX-2蛋白質之影響 67 圖5.1五味子與芋頭葉萃取物之抗發炎可能機制示意圖 70 圖5.2艾草 萃取物之抗發炎可能機制示意圖 71 圖5.3高良薑萃取物之抗發炎可能機制示意圖 72 ? 表目錄 表3.1 SDS-PAGE配製比例 31 表4.1初始NO抑制率達90%以上的萃取物之NO抑制率統計 35
REFERENCES
1.弓德?、何衍彪、谷會、武?霞、王松標、詹儒林。2010。20種植物提取物對芒果炭疽病菌的抑制作用研究。安徽農業科學 :5149-5151
。 2.王蘭、張麗娟、張芳?、樊錦慧。2010。?柏、苦參、陳皮、高良薑等中藥材的體外防霉活性研究。中國衛生檢驗雜誌 20（8
）:1942-1943。 3.江睿玲、謝明村、周泰廷、郭槐秋、張永勳。1997。五味子中schizandrin及gomisin A定量及抗肝損傷之研究。中醫藥 雜誌 8（3）:155-164。 4.吳秀慧。2004。Leflunomide類似物之合與生物活性:第25-26頁。國防醫學院碩士論文。台北。 5.巫玉琳。2008
。香菜萃出物對脂多醣體誘發小鼠RAW264.7巨噬細胞發炎反應之探討:第4-10頁。中山醫學大學碩士論文。台中。 6.林旻儒、杜宇晨、
謝旻儒、郭宗甫。2011。五味子的藥物療效介紹。中華傳統獸醫學會會刊 15（2）:51-60。 7.林興中。1992。類固醇在基層醫療之使用
。臺灣醫界 35（10）:68-76。 8.洪毓珮。2007。中醫治病最愛用的植物--艾草 。中國醫訊 53:62-64。 9.范少怡、詹國靖、朱燕華。2010
。五味子萃取物對倉鼠血脂質及肝臟脂質之影響。臺灣農業化學與食品科學 48（4）:189-196。 10.徐雅芬。2006。全球植物藥產業概況 及市場分析。農業生技產業季刊 5:1-5。 11.張倩芝、蔡明招、盧志毅。2006。高良薑與大高薑姜精油中活性物質的比較。中草藥 37（8
）:1151-1152。 12.陳姵吟。2006。Gamma-次亞麻油酸與亞麻油酸對脂多醣體誘發小鼠RAW264.7巨噬細胞發炎反應之效應及相關機轉:
第4-8頁。中山醫學大學碩士論文。台中。 13.陳素靜。2007。非類固醇類抗發炎止痛藥 在創傷治療扮演的角色 。中國醫訊 49:58-59。
14.陳艷芬、江濤、唐春萍、馮毅凡、楊超燕。2009。高良薑總?酮抗炎?痛作用的實驗研究。廣東藥學院學報 25（2）:188-191。 15.曾堯 人、顏兆熊。2010。老人慢性疼痛之藥物治療。家庭醫學與基層醫療 25（2）:44-52。 16.黃智生。2008。發炎反應－常見文明病的始作 俑者。科學發展 422:6-10。 17.蔡佩珊。2003。Ccy1a-c1影響巨噬細胞株RAW264.7生成一氧化氮的研究:第8-10頁。中山醫學大學碩士論 文。台中。 18.鄭文玲。2003。共軛亞麻油酸在脂多醣體誘RAW264.7巨噬細胞發炎反應相關事件之影響:第19-22頁。中山醫學大學碩士 論文。台中。 19.薛守英。2006。硫磺菌固體發酵及其生物活性之研究:第10-11頁。南台科技大學碩士論文。台南。 20.謝世川。2007。
魚針草(anisomeles indica)抗發炎活性成分之分離純化探討:第12-19頁。朝陽科技大學碩士論文。台中。 21.謝廷芳、黃晉興、謝麗娟、胡 敏夫、柯文雄。2005。植物萃取液對植物病原真菌之抑菌效果。植物病理學會刊 14（1）。 22.謝強敏、卞如濂、楊秋火、唐法娣、王 硯。1999a。艾葉油的呼吸系統藥理研究I:支氣管擴張、鎮咳和祛痰作用。中國現代應用藥學 16（4）:16-19。 23.謝強敏、唐法娣、王硯
、楊秋火、卞如濂。1999b。艾葉油的呼吸系統藥理研究-II:抗過敏作用。中國現代應用藥學 16（5）:3-7。 24.醫學全在線（2012a）。芋 葉。檢索自: http://www.med126.com/pharm/2009/20090113051817_91394.shtml 25.醫學全在線（2012b）。高良薑。檢索自:
http://www.med126.com/mingzu/2009/20090512114749_152929.shtml 26.魏國會、杜梅素、宋寧、岳春景。2010。艾葉油的平喘作用研 究——小鼠卵蛋白復制法。時珍國醫國藥 21（1）:86-87。 27.Dong, Z., and I.J. Fidler. 2002. Macrophages. In Encyclopedia of Cancer (Second Edition). R.B. Editor-in-Chief: Joseph, editor. Academic Press, New York. 77-88. 28.Duan, W., J. Zhou, S. Zhang, K. Zhao, L. Zhao, K.
Ogata, T. Sakaue, A. Mori, and T. Wei. 2011. ESeroS-GS modulates lipopolysaccharide-induced macrophage activation by impairing the assembly of TLR-4 complexes in lipid rafts. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1813:772-783. 29.Gordon, S. 1998.
Macrophage Activation. In Encyclopedia of Immunology (Second Edition). J.D. Editor-in-Chief: Peter, editor. Elsevier, Oxford. 1642-1649.
30.Hammes, L.S., R.R. Tekmal, P. Naud, M.I. Edelweiss, N. Kirma, P.T. Valente, K.J. Syrjanen, and J.S. Cunha-Filho. 2007. Macrophages, inflammation and risk of cervical intraepithelial neoplasia (CIN) progression—Clinicopathological correlation. Gynecologic Oncology.
105:157-165. 31.Handa, S.S., A. Sharma, and K.K. Chakraborti. 1988. Natural products and plants as liver protecting drugs. Fitoterapia.
21:307-345. 32.Hikino, H., Y. Kiso, H. Taguchi, and Y. Ikeya. 1984. Antihepatotoxic actions of lignoids from Schizandra chinensis Fruits. Planta Medica. 50:213-218. 33.Huang, Y.H., P.S. Tsai, and C.J. Huang. 2008. Bupivacaine inhibits COX-2 expression, PGE2, and cytokine production in endotoxin-activated macrophages. Acta anaesthcsiologica Scandinavica. 52:530-535. 34.Kang, S.-M., K.-N. Kim, S.-H. Lee, G. Ahn, S.-H.
Cha, A.-D. Kim, X.-D. Yang, M.-C. Kang, and Y.-J. Jeon. 2011. Anti-inflammatory activity of polysaccharide purified from AMG-assistant extract of Ecklonia cava in LPS-stimulated RAW 264.7 macrophages. Carbohydrate Polymers. 85:80-85. 35.Kiso, Y., M. Tohkin, H. Hikino, Y. Ikeya,
and H. Taguchi. 1985. Mechanism of antihepatotoxic activity of wuweizisu C and gomisin A1. Planta Medica. 51:331-334. 36.Kou, X., S. Qi, W.
Dai, L. Luo, and Z. Yin. 2011. Arctigenin inhibits lipopolysaccharide-induced iNOS expression in RAW264.7 cells through suppressing JAK-STAT signal pathway. International Immunopharmacology. 11:1095-1102. 37.Laskin, D.L., C.R. Gardner, and J.D. Laskin. 2010. 5.06 - Phagocytes. In Comprehensive Toxicology (Second Edition). A.M. Editor-in-Chief: Charlene, editor. Elsevier, Oxford. 133-153. 38.Oh, B.K., J.
Mun, H.W. Seo, S.Y. Ryu, Y.S. Kim, B.H. Lee, and K.-S. Oh. 2011. Euonymus alatus extract attenuates LPS-induced NF-κB activation via IKK β inhibition in RAW 264.7 cells. Journal of Ethnopharmacology. 134:288-293. 39.Park, C.M., J.Y. Park, K.H. Noh, J.H. Shin, and Y.S. Song.
2011. Taraxacum officinale Weber extracts inhibit LPS-induced oxidative stress and nitric oxide production via the NF-κB modulation in RAW 264.7 cells. Journal of Ethnopharmacology. 133:834-842. 40.Park-Min, K.H., T.T. Antoniv, and L.B. Ivashkiv. 2005. Regulation of macrophage phenotype by long-term exposure to IL-10. Immunobiology. 210:77-86. 41.Ramaiah, S.K., and R.E. Rose. 2010. 9.08 - Endotoxin-Induced Hepatotoxicity. In Comprehensive Toxicology (Second Edition). A.M. Editor-in-Chief: Charlene, editor. Elsevier, Oxford. 613-625. 42.Sautebin, L. 2000. Prostaglandins and nitric oxide as molecular targets for anti-inflammatory therapy. Fitoterapia. 71, Supplement 1:S48-S57. 43.Squadrito, M.L., and M. De Palma. 2011. Macrophage regulation of tumor angiogenesis: Implications for cancer therapy. Molecular Aspects of Medicine.
32:123-145. 44.Stachowska, E., M. Ba?kiewicz-Masiuk, V. Dziedziejko, G. Adler, J. Bober, B. Machali?ski, and D. Chlubek. 2007. LIPIDS. In Conjugated Linoleic Acids Can Change Phagocytosis of Human Monocytes/Macrophages by Reduction in Cox-2 Expression. Vol. 42. 707-716.
45.Xiao, C., J. Li, X. Dong, X. He, X. Niu, C. Liu, G. Zhong, R. Bauer, D. Yang, and A. Lu. 2011. Anti-oxidative and TNF-[alpha] suppressive activities of puerarin derivative (4AC) in RAW264.7 cells and collagen-induced arthritic rats. European Journal of Pharmacology. 666:242-250.
46.Yu, T., Y.J. Lee, H.-J. Jang, A.R. Kim, S. Hong, T.W. Kim, M.-Y. Kim, J. Lee, Y.G. Lee, and J.Y. Cho. 2011. Anti-inflammatory activity of Sorbus commixta water extract and its molecular inhibitory mechanism. Journal of Ethnopharmacology. 134:493-500.
