Effect of media milling on anti-inflammation of Ganoderma tsugae

靈芝產品為台灣的市場上相當受歡迎的健康食品，文獻指出松杉靈芝(G. tsugae)可藉由減少肺和呼吸道中發炎細胞滲入及發炎介質分泌，改善支氣管肺泡之發炎反應(bronchoalveolar inflammation)。三萜類(triterpenoids)為靈芝主要生物活性成分之一，具有抗發炎與免疫調節等功效。介質研磨(media milling)為一種製造超細微粒的方法，微粒表面積增加可有效增加活性物質萃取率與其生物利用率。本研究利用介質研磨技術製備奈米/次微米靈芝液，並評估樣品粒徑下降對抗發炎反應的影響，將其結果與靈芝粗三萜萃取液對照，推測其生理活性之機制。實驗結果顯示，靈芝經3小時研磨後，其粒數平均粒徑(number average diameter)為0.10 μm，與細碎靈芝(number average diameter 3.33 μm)相比，具顯著下降。熱水浴組和研磨組靈芝上清液之粒數平均粒徑分別為110 nm和70 nm。粗三萜含量方面，研磨組和熱水浴處理組之粗三萜萃取率分別為3.44 mg/g和2.60 mg/g；介質研磨可以增加粗三萜萃取率與萃取物中粗三萜所占比例。將奈米/次微米靈芝上清液與預先經LPS (lipopolysaccharide)誘導之巨噬細胞RAW264.7共同培養後，顯著減少一氧化氮(NO)之釋放及調節細胞激素TNF-α之分泌。分別以不同濃度之靈芝萃取液處理LPS誘導之巨噬細胞RAW264.7，結果顯示於低濃度時，能夠降低TNF-α含量與NF-κB (p65)表現量，高濃度時可以抑制NO分泌與iNOS表現量。Ganoderma product is a popular healthy food in Taiwan. Literatures showed that G. tsugae supplementation alleviates bronchoalveolar inflammation by decreasing the infiltration of inflammatory cells and the secretion of inflammatory mediators into local lung and airway tissues. Triterpenoids is one of major bioactive compounds of G. tsugae. Literatures indicated it posses anti-inflammatory and immunoregulatory activities. Media milling is an economic method to produce ultrafine particles. The ultrafine particles might had enhanced bioactive compounds extraction efficacy and bioavailability. The objective of this article was to explore the feasibility of preparing nano/submicron Ganoderma suspension by using media milling, and to evaluate the effect of size reduction on anti-inflammatory activities. The number average diameter was dramatically reduced from 3.33 μm to 0.10 μm via media milling. Number average diameters of hot water extract and milled supernatant were 110 nm and 70 nm, respectively. Yields of triterpenoids extracted by milled and hot water samples 3.44 mg/g and 2.60 mg/g. Media milling can increase the efficiency of triterpenoids extraction and the triterpenoids content. Crude triterpenoids of nano/submicron Ganoderma suspension were fractionalized to treat RAW264.7 cells which were stimulated by lipopolysaccharide (LPS). The results can be used to evaluate the secretion of pro-inflammatory cytokines. NO and TNF-α concentration were decreased when LPS-induced RAW264.7 were treated with nano/submicron G. tsugae. Furthermore, treatment with different concentration G. tsugae extract on LPS-induced RAW264.7 showed the inflammatory inhibition through decrease of TNF-α concentration and NF-κB (p65) expression in low dosage, while decreased NO concentration and iNOS expression in high dosage