毛豆葉萃取物之抗氧化活性及抗發炎活性探討

Abstract

毛豆為蔬菜用大豆的一種，屬於豆科（Leguminosae）大豆屬（Glycine）植物，為台灣外銷重要的經濟作物，主要在春、秋兩季種植，在台灣毛豆葉一般都以廢棄物處理或堆肥。在先前研究中發現毛豆葉及大豆葉萃取物含有豐富的酚類化合物且有許多生理活性對人體有益。因此本研究目的為探討不同溶劑萃取毛豆葉，比較其總酚及總類黃酮含量，並評估不同萃取物之抗氧化活性與抗發炎的能力，且了解其抗發炎的機制。將新鮮毛豆葉乾燥後磨成粉，用極性由高至低的溶劑水、70%甲醇、70%乙醇及乙酸乙酯萃取，得萃取率方面依序以水(25%)、甲醇(19%)、乙醇(18%)及乙酸乙酯(5%)，而萃取物之總酚及類黃酮含量，均以70%甲醇及70%乙醇有較高的含量。在抗氧化評估方面，DPPH自由基清除率的結果顯示，甲醇及乙醇萃取物濃度為2.5 mg．mL-1時可達80%以上清除率，而對ABTS+自由基清除力及螯合亞鐵離子能力，以水及70%甲醇萃取物有較好的效果。 毛豆葉萃取物對是否有LPS誘導之RAW264.7巨噬細胞之細胞毒性評估顯示，水、甲醇及乙醇萃取物在1000 mg．L-1濃度下皆未顯示任何細胞毒性。於抑制一氧化氮生成方面，水萃物以600 mg．L-1、甲醇及乙醇萃取物以200 mg．L -1處理的一氧化氮生成量顯著低於正控制組，且具有濃度效應，隨著萃取物濃度上升，一氧化氮的抑制效果有顯著上升的趨勢，尤其在70%甲醇萃取物的抑制效果最為明顯且抑制率可達86%，與一氧化氮合成酶表現量有相同的趨勢，以甲醇及乙醇萃取物的iNOS表現量最低，推測毛豆葉可能藉由抑制iNOS來調控一氧化氮生成，另外，在COX-2基因表現量也有明顯降低。對於LPS誘導之RAW264.7細胞之TNF-α、IL-1β及IL-6生成量之結果表示，70%甲醇萃取物能顯著降低TNF-α、IL-6及IL-1β的產生，另外，也能提高細胞內GSH / GSSG ratio，由8增加至12.8。根據研究結果，毛豆葉具有抗氧化能力且對以LPS誘導之巨噬細胞生成之發炎相關介質具有抑制效果，因此毛豆葉具有發展成保健食品之潛力。

Edamame (Glycine max (L.) Merr.) is a kind of vegetable soybean, belonging to Leguminosae family Glycine genus, is an important export economic crop in Taiwan, which is mainly grown in spring and autumn. In Taiwan, edamame leaves are generally treated as waste or fertilizer. Previous studies have found that edamame leaf and soybean leaf extracts are rich in phenolic compounds and have many physiological activities that are beneficial to the human body. The purpose of this study was to explore the extraction of edamame leaves with different solvents, then comparing their total phenol and total flavonoid content. Its antioxidant activity and anti-inflammatory capacity of different extracts, and anti-inflammatory mechanisms were experimented. Fresh edamame leaves were dried and ground to powder, and then respectively extracted with polarity high-to-low solvent: water, 70% methanol, 70% ethanol, and ethyl acetate. The sequentially extractabilities were water (25%), methanol (19%), ethanol (18%) and ethyl acetate (5%). The total phenol and flavonoid content of the extracts in 70% methanol and 70% ethanol were higher. In terms of antioxidant evaluation, the results of DPPH radical scavenging rate showed that the methanol and ethanol extract concentration was 2.5 mg．mL-1, and the scavenging rate could reach more than 80%. Water extract and 70% methanol extract exhibited better effect in scavenging ABTS + radicals and chelating ferrous ion. The potential cytotoxicities of edamame leaf extracts were evaluated in RAW264.7 cells in the presence or absence of LPS. Water, methanol and ethanol extracts did not cause any cytotoxicity at concentrations up to 1000 mg．L -1. In terms of inhibiting the formation of nitric oxide, 600 mg．L -1 water extract and 200 mg．L -1 methanol and 200 mg．L -1 ethanol extract were significantly lower than the positive control group and also showed concentration-effect. As the concentration of the extract increased, there was a significant upward trend in inhibitory effect on nitric oxide biosynthesis, especially on 70% methanol extract in which the inhibition rate was up to 86%, and nitric oxide synthase (iNOS) expressed the same trend. As the result, it speculated that edamame leaves might regulate nitric oxide production by inhibiting iNOS. In addition, the expression level of COX-2 gene was also significantly reduced. The results of LPS-induced production of TNF-α, IL-1β, and IL-6 in RAW264.7 cells showed that 70% methanol extract could significantly reduce the production of TNF-α, IL-6, and IL-1β, which could also increase the intracellular GSH / GSSG ratio from 8 to 12.8. According to the research results, edamame leaves had anti-oxidant ability and had inhibitory effect on the inflammation-related mediators produced by LPS-induced macrophages. Therefore, edamame leaves have the potential to be the new kind of raw material of healthy foods.