探討臺灣水鹿與紐西蘭鹿茸中免疫調節之保健成分

Abstract

鹿茸來自雄鹿未完全鈣化的鹿角，在亞洲做為傳統中國中藥材已超過兩千年，有許多研究指出鹿茸具有抗發炎、促進傷口癒合、抗關節炎、抗腫瘤、改善骨質疏鬆和抗氧化等機能性功效。由臺灣特有亞種臺灣水鹿取得新鮮的鹿茸，先前本實驗室的研究已證實臺灣水鹿鹿茸具有免疫調節的功效，如抗發炎、抑制呼吸道的過敏反應與抗金黃色葡萄球菌感染的抗菌能力；鹿茸可由許多不同的鹿種取得，目前最主要提供鹿茸為紅鹿、梅花鹿、麋鹿與馴鹿等。國外研究多著重於紅鹿、梅花鹿和麋鹿鹿茸成分功能的比較，但不同鹿種所提供的鹿茸對於機能性的關係目前並未明瞭，因此本研究以體外實驗探討臺灣水鹿鹿茸與紐西蘭紅鹿鹿茸進行比較其免疫調節功效的差異，並利用二維膠體電泳與液相層析串聯式質譜儀 (LC MS/MS) 分析比對兩種鹿茸是否含有可能影響機能性的成分。 首先，將臺灣水鹿與紐西蘭紅鹿的鹿茸冷水萃取物以不同濃度與小鼠巨噬細胞 RAW 264.7細胞共培養，利用脂多醣 (lipopolysaccharide, LPS) 誘導小鼠巨噬細胞產生發炎反應，發現到給予萃取物的小鼠巨噬細胞顯著降低促發炎細胞激素：腫瘤壞死因子 (tumor necrosis factor alpha, TNF-alpha) 與細胞介白素 (interleukin, IL)-6的分泌量，且給予高濃度的臺灣水鹿鹿茸冷水萃取物顯著較紐西蘭紅鹿降低TNF-alpha的生成 (p < 0.05)，臺灣水鹿與紐西蘭紅鹿萃取物皆可有效調節巨噬細胞，顯著較控制組刺激細胞分泌IL-10 (p < 0.05)。進一步以二維電泳與LC MS/MS分析鹿茸萃取液中蛋白質組成，發現部分蛋白質種類與含量不同，但大部分的蛋白質無文獻指出具有調節免疫的能力。將臺灣水鹿鹿茸冷凍乾燥切片以-20℃、4℃與25℃存放6個月，將其冷水萃取物與RAW 264.7細胞共培養後發現，鹿茸經各溫度儲藏後仍具有免疫調節之能力，有隨著時間的增加而調節免疫的能力有逐漸下降的趨勢，其中以-20℃有最佳的抗發炎能力。再以二維電泳分析經過儲存後蛋白質冷水萃取物中蛋白質組成，發現蛋白質僅有濃度上有些微差異，然而，與新鮮鹿茸冷水萃取物蛋白質電泳膠片比較，發現在分子量小於20 kDa的蛋白質有減少的現象。 綜合上述實驗結果，臺灣水鹿鹿茸可能有比紐西蘭紅鹿有較佳的免疫調節能力，經過儲存可得知適當的儲藏條件可減緩免疫調節能力的下降與機能性成分的降解，且推測免疫調節相關蛋白質可能為小分子蛋白質。

Velvet antler (VA), a traditional Chinese Medicine in Asia for more than 2000 years, was collected from deer no calcified antler. The numerous benefits of VA have been found such as anti-inflammation, accelerating wound healing, anti-arthritis, and anti-oxidant. In Taiwan, antlers from Formosan sambar deer (Rusa unicolor swinhoei), an indigenous subspecies, demonstrated immunomodulatory effects including anti-allergy and anti-inflammation. Various deer species could provide velvet antler, however, the differences in health benefits among different velvet antler remain unknown. Thus, in the present study, we would like to investigate the immunomodulatory effect of cold water extracted VA from sambar deer and red deer in vitro. The possible components were also evaluated by two dimensional electrophoresis and Liquid Chromatography-Mass spectrometer. First, we co-cultured VA extraction with mice macrophage RAW 264.7 and found that both VA from sambar deer and red deer could significantly reduce the levels of proinflammatory cytokines, tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 after stimulated by lipopolysaccharide (LPS) . The high dosage of sambar deer showed a significantly lower TNF-alpha production than red deer (p < 0.05). For regulatory T cell, IL-10, the significantly higher levels were observed in both VA extractions from sambar deer and red deer when compared with negative control (p < 0.05) . Further analysis both cold water extracted VA by two dimensional electrophoresis and LC-MSMS indicated that several proteins and chemicals were different in volumes and components. Additional analysis is required to identify those compounds. Storage under different temperatures in 6 months still had the inhibitory ability to cytokine production. We found that the interaction between temperature and storage period existed. By the increasing of storage period, the functionality of VA had a downward trend. According to the protein profiles of stored VA water extraction, some protein spots under 20 kDa were invisible. The storage showed a significant influence in the stability of VA components. In summary, Formosan sambar deer VA might have better effect on immunomodulation than red deer. Possible beneficial components might be small molecule protein under 20 kDa. The proper storage for VA is necessary to maintain its health benefits.