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標題: | 鑑定臺灣水鹿鹿茸冷水萃取物中免疫調節有效成分之研究 Identification of immunomodulatory components from cold water extraction of velvet antler of Formosan sambar deer |
作者: | Chun-yu Chien 簡群育 |
指導教授: | 陳明汝(Ming-Ju Chen) |
關鍵字: | 臺灣水鹿,鹿茸,冷水萃取,免疫調節, Formosan sambar deer,velvet antler,cold water extraction,immunomodulatory, |
出版年 : | 2014 |
學位: | 碩士 |
摘要: | 鹿茸為中國傳統名貴藥材及健康食品,具有增強體魄與提升免疫力之功效。本實驗室先前的研究中,於細胞試驗及小鼠試驗均證實臺灣水鹿鹿茸萃取粉之免疫調節能力,具有刺激免疫反應(促進細胞增生、細胞吞噬活性)及抑制免疫反應(抗發炎反應)的效果,因此本研究目的為鑑定臺灣水鹿鹿茸冷水萃取粉中具有免疫調節功效的有效成分。在鹿茸水萃物基本組成分中,粗蛋白質高達83.3 %,灰分10.4%。為了更了解萃取物的有效成分,藉由濃縮離心管將鹿茸冷水萃取粉依分子量分為(1) 大於10 kDa; (2) 介於5-10 kDa 之間(3) 小於 5 kDa三個部分。經由體外試驗可得知,分子量介於5-10 kDa和小於5 kDa的處理組,可有效抑制巨噬細胞株RAW 264.7產生前發炎反應細胞激素,腫瘤壞死因子-α及介白素-6,並具有促進細胞增生的能力。此外,當經100 °C加熱15分鐘後,鹿茸水萃物仍然具有抗發炎反應的能力。
接著進一步藉由十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE)以及液相色譜-質譜聯用,對分子量小於10 kDa的部分進行蛋白質身分鑑定,並於 MASCOT 資料庫比對蛋白質的身分,當中比對結果相似程度最高者為牛(Bos Taurus)的profilin-1,其他比對到可能性較高的蛋白質還包括紅鹿(Cervus elaphus)的fatty acid-binding protein-4,和單峰駱駝(Camelus dromedarius)的泛素(ubiquitin)。此外,利用高效能液相層析法成功將臺灣水鹿鹿茸萃取物分離,並收集純化出不同的片段。經體外試驗證實,許多片段仍然具有抑制巨噬細胞株RAW 264.7產生前發炎反應細胞激素的能力,並經質譜鑑定,結果顯示這些片段大多為鹿茸血中的蛋白質或其胜肽片段。 綜上所述,有效成分可能為分子量小於10 kDa,並具有耐熱性的水溶性蛋白質或胜肽片段,以及鹿茸血中的蛋白質成分。未來我們還會與其他鹿種來源的鹿茸比較,以找出臺灣水鹿鹿茸萃取物中所特有的有效成分。 Velvet antler (VA) is one of the most valuable Chinese traditional medicines and commercial functional foods with enhancing vital energy and immune system. In our previous studies, we had demonstrated the anti-inflammatory and immunomodulatory effects of cold water extraction of VA from Formosan samber deer (Rusa unicolor swinhoii). Thus, the purpose of this study is to identify the active components in the cold water extraction. The proximate composition of VA cold water extraction powder was 83.3% of crude protein and 10.4 % of ash. To further identify the active components in the extraction, the cold water extraction powder of VA was separated into three populations by ultra-filtration concentrator based on molecular weight: (1) >10 kDa; (2) 5-10 kDa and (3) <5 kDa, and evaluated their anti-inflammatory effects in-vitro using RAW 264.7 macrophage-like cells stimulated with lipoteichoic acid (LTA). The results of pro-inflammatory cytokines showed that the production of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by LTA stimulation were significantly suppressed after co-cultured with 5-10 kDa and < 5 kDa samples of VA extracts. Besides, heat treatment at 100°C for 15 minutes did not affect the anti-inflammatory effect of VA extract samples. We further analyzed the protein profile by sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and liquid chromatography (LC)-Mass spectrophotometer (MS)/MS of the cold water extractions. Three possible proteins were identified as profilin-1 from bovine (Bos taurus), fatty acid-binding protein-4 from red deer (Cervus elaphus), and ubiquitin from dromedary (Camelus dromedarius). Additionally, we also used high-performance liquid chromatography (HPLC) to separate and purify the fractions from VA cold water extraction. We found that some fractions separated from VA cold water extraction powder still had ability to suppress the pro-inflammatory cytokines production. After further identify the fractions, we found most of them were the proteins or peptides from antler blood. According to the findings, the active components in VA water extraction from Formosa sambar deer might be heat resistance and water soluble proteins or peptides under 10 kDa, and the proteins or peptides from antler blood. We will further compare the VA from Formosan samber deer with other species, to find the unique functional components. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18722 |
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顯示於系所單位: | 動物科學技術學系 |
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