介質研磨對於枸杞蛋白質免疫調節能力之影響

Abstract

摘要 枸杞為東方傳統藥食同源中草藥，可以增添食品風味外，且具有許多生理功能，枸杞多醣-蛋白質複合物 (Lycium barbarum polysaccharide-protein complex, LBP) 是主要活性物質之一，具有免疫調節、降血糖、改善糖尿病、抗氧化、抗腫瘤和延緩老化等多種功能。以介質研磨 (Media milling)製備奈米/次微米枸杞懸浮液，有助於枸杞活性成分釋出。本研究之目的主要為評估介質研磨對於枸杞蛋白質免疫調節能力之影響。經過介質研磨 90 分鐘之枸杞，粒數平均粒徑為0.111 μm，小於 1 μm 之粒子數佔總粒子數的 99.9%，小於 0.1 μm 之粒子數佔總粒子數的 57.8%，電子顯微鏡影像亦可證實枸杞懸浮液具有奈米/次微米尺度之粒子，枸杞蛋白含量提高了 21.05%。利用陰離子交換層析，分離出枸杞蛋白質區分物一 (goji protein fraction 1, GPF1) 與枸杞蛋白質區分物二 (goji protein fraction 2, GPF2)，細碎組 GPF1 與 GPF2 比例為 17.59 : 82.41，介質研磨組別 GPF1 與 GPF2 比例為 5.12 : 94.88。以枸杞粗蛋白處理 RAW264.7 小鼠巨噬細胞株，結果顯示，研磨組刺激細胞增生之能力優於細碎組別。在 1000 μg/mL 濃度下，刺激一氧化氮產生能力研磨組比細碎組高了46.9%，刺激細胞產生 TNF-α 之能力研磨組比細碎組增加 13.86%，刺激細胞激素 IL-1β 之能力研磨組比細碎組提高 12.24%。以陰離子交換層析分離之枸杞蛋白質區分物處理 RAW264.7 小鼠巨噬細胞株，結果顯示，只有 GPF2 具有刺激細胞增生之能力。GPF1 與 GPF2 均具可刺激一氧化氮及 TNF-α 濃度上升，而 GPF2 在 500 μg/mL 濃度下，研磨組刺激 RAW264.7 產生一氧化氮比細碎組高了 15.91%，研磨組刺激 RAW264.7 產生 TNF-α 比細碎組高了 23.2%。只有 GPF2 可刺激 IL-1β 和 IL-12 濃度提升，在濃度 500 μg/mL 時，研磨組刺激 RAW264.7 產生 IL-12 比細碎組高了 16.25%。

Abstract Goji is a traditional medicine and food in Chinese and had multiple biological activities, Lycium barbarum polysaccharide-protein complex (LBP) are one of the main active compounds of goji. LBP have several positive health effects such as immunomodulating, hypoglycemic effect, anti-diabetes, antioxidant activity, antitumor activity and anti-aging. The objective of this study was to evaluate the effect of media milled on immunoregulatory response of goji protein and it’s different fractions. At first, media milled for 90 min, the number average diameter of goji suspension particle was reduced from 3.44 μm to 0.111 μm, the percentage of particle smaller than 1μm was 99.9% and the percentage of particle smaller than 0.1 μm was 57.8%. The quantity of goji prtein could be increased 21.05% by media milled. Blended and milled goji protein could be separated into protein fraction 1 (GPF1) and protein fraction 2 (GPF2) by DEAE. The protein content of GPF1 and GPF2 in blended goji protein was 17.59 : 82.41, and 5.12 : 94.88 in milled goji protein. In addition, goji was studied for its immunoregulatory response by using murine macrophages RAW264.7 cell line. In the first part, goji protein were treated with RAW264.7 and the results showed that milled goji protein could stimulate the proliferation of RAW264.7 better than blended goji protein. At 1000 μg/mL, milled goji protein had increased the concentration of nitric oxide, TNF-α and IL-1β compared with blended goji protein, by 46.9%, 13.86% and 12.24%. In the second part, two protein fractions which were separated by DEAE were treated with RAW264.7. Results revealed that only GPF2 could induce the proliferation of RAW264.7. However, both of the protein fractions had increased the concentration of nitric oxide and TNF-α, when treated GPF2 at 500 μg/mL, the nitric oxide and TNF-α concentration of milled sample was 15.91% and 23.2% higher than that of blended one, respectively. Only GPF2 can induce the secretion of IL-1β and IL-12, when treated GPF2 at 500 μg/mL, the IL-12 concentration of milled sample was 16.25% higher than that of blended one.