Study of bioactive compounds in crude extracts of Tartary buckwheat

韃靼蕎麥(Fagopyrum tataricum (L.) Gaertn)於台灣屬小量栽種的類穀物(pseudo-cereal)，全植株均含有豐富的類黃酮成分，如蘆丁(rutin)、檞皮素(quercetin)、山柰酚(kaempferol)等。本試驗中擬以國產韃靼蕎麥為試驗材料，分別於幼苗期(seedling)、莖生長期(stem elongation)、乳熟期(milky)、開花期(flowering)及成熟期(matured)採收，並依植株部位分為葉、莖、花、種實(成熟或未熟)作樣本。清潔後，進行熱風乾燥(60℃)和凍乾處理並磨碎成粉末，以80%乙醇萃取後測定其總類黃酮與總酚含量。結果顯示，韃靼蕎麥開花期之花以熱風乾燥後有最高的總類黃酮含量(157.27±3.01 mg rutin eq./g powder, d.b.)及總酚含量(121.60±3.20 mg gallic acid eq./g powder, d.b.)，其次為成熟期的葉子；而各個時期中均以莖部位的總酚和總類黃酮含量為最低。花雖然含有豐富的機能性成分，但其僅占新鮮植株之重量百分比0.3%，且樣品收集困難，故後續機能成分分析及應用之樣品以成熟期的葉子及日常用作糧食之成熟期種實為主。韃靼蕎麥熱風乾燥的成熟期葉子與種實之80%乙醇粗萃取液，以HPLC-PDA分析後得知，葉子和種實乙醇粗萃取液之主要成分為蘆丁，其次為山奈酚-3-O-芸香糖苷(kaempferol-3-O- rutioside)，再者為檞皮素、異檞皮素(isoquercetin)和山奈酚。韃靼蕎麥葉子和種實之乙醇粗萃取液於1000 μg/mL亞鐵離子螯合能力分別為40%和89%，而清除DPPH自由基的IC50分別為63.51±2.15和85.73±4.60 μg/mL。韃靼蕎麥葉子和種實粗萃液之醣類消化酵素α-葡萄糖苷酶(α-glucosidase)活性抑制試驗結果得知，其IC50分別為283.38±10.62和180.63±13.52 μg/mL，與正控制組阿卡波糖(acarbose，為目前治療第二型糖尿病藥物)之IC50 (670.78±66.29 μg/mL)相較，有較佳的效果。本試驗研究結果顯示，韃靼蕎麥植株與種實具有良好抗氧化力以及抑制α-葡萄糖苷酶的活性，甚至比市售藥物阿卡波糖效果更佳，應可作多方面的生產利用。Tartary buckwheat (Fagopyrum tataricum Gaertn) is a pseudo-cereal which are planted with a small area in Taiwan. It has been reported that the whole plant of Tartary buckwheat contains abundant flavonoids, such as rutin, quercetin, kaempferol, etc. In this study the contents of total flavonoids and total phenolics at different growth stages of Tartary buckwheat plant were determined. The growth stages of Tartary buckwheat plant for investigation includes seedling, stem elongation, milky, flowering and matured stages. Each stage, the plant was separated into four parts, including leaf, stem, flower and grain (immature and matured). After sample collecting, hot-air drying (60℃) and lyophilized were applied to dry the sample. The dried sample was then milled into fine powders. The flower part contained the highest amount of total flavonoids and total phenolics than other parts, which were 157.27±3.01 mg rutin eq./g powder, d.b. and 121.60±3.20 mg gallic acid eq./g powder, d.b., respectively. Following the flower, high amount of total flavonoids and total phenolics could be found in leaf. The stem contained the lowest amount of total flavonoids and total phenolics compared to other parts at all growth stages. Although the flower had the highest amount of bioactive compounds, it is impractical to use the flower due little amount of flower (0.3 w/w% of whole plant) and difficulty of sample collection. Therefore, the matured seed and its plant parts were chosen as the samples for this study. Tartary buckwheat leaf and grain at matured stage were dried by hot-air (60℃) and then extracted with 80% ethanol. The major components of 80% ethanol crude extraction included rutin, followed by kaempferol-3-O-rutinoside, quercetin, isoquercetin and kaempferol analyzed by HPLC-PDA. The chelated ferrous ion ability of ethanol crude extraction from leaf and grain were 40% and 89% at 1000 μg/mL, respectively. The IC50 of scavenging DPPH radicals were 63.51±2.15 and 85.73±4.60 μg/mL for the ethanol crude extraction from leaf and grain, respectively. The inhibition of α-glucosidase activity expressed as IC50 were 283.38±10.62 and 180.63±13.52 μg/mL for the ethanol crude extraction from leaf and grain, respectively. However, the IC50 of the inhibition of α-glucosidase activity of acarbose, a medical for type II diabetes, was 670.78±66.29 μg/mL. The ethanol crude extraction from matured Tartary buckwheat shows a better inhibition effect than acarbose. The ethanol crude extraction of Tartary buckwheat leaf and grain at matured stage had good antioxidative ability and superior α-glucosidase activity inhibition with great potential for the prevention of type II diabetes.口試委員會審定書……………………………………………………………………Ⅰ 誌謝……………………………………………………………………………………Ⅱ 中文摘要………………………………………………………………………………Ⅲ 英文摘要………………………………………………………………………………Ⅳ 目錄……………………………………………………………………………………Ⅵ 圖目錄…………………………………………………………………………………Ⅷ 表目錄…………………………………………………………………………………Ⅸ 第一章、前言……………………………………………………………………………1 第二章、文獻回顧………………………………………………………………………3 一、韃靼蕎麥之簡介……………………………………………………………3 二、韃靼蕎麥之化學組成………………………………………………………5 三、韃靼蕎麥之安全評估……………………………………………………14 四、韃靼蕎麥之生理活性……………………………………………………15 五、韃靼蕎麥之藥理活性……………………………………………………17 第三章、材料與方法…………………………………………………………………24 一、試驗架構…………………………………………………………………24 二、試驗材料…………………………………………………………………25 三、試驗方法…………………………………………………………………27 第四章、結果與討論…………………………………………………………………32 一、不同生長時期韃靼蕎麥植株之變化……………………………………32 1. 不同生長時期各部位之水分含量……………………………………32 2. 乾燥方式對植株粉末色澤之影響……………………………………34 3. 植株不同生長時期熱風乾燥和凍乾各部位之機能性成分…………35 二、韃靼蕎麥之一般成分分析………………………………………………38 三、韃靼蕎麥粗萃取液之化學成分分析……………………………………39 1. 韃靼蕎麥葉和種實之萃取率…………………………………………39 2. 韃靼蕎麥葉和種實乙醇粗萃取液之主要機能性成分………………40 四、韃靼蕎麥粗萃取液之抗氧化能力分析…………………………………44 1. 清除DPPH自由基能力………………………………………………44 2. 亞鐵離子螯合能力……………………………………………………46 五、韃靼蕎麥粗萃取液抑制醣解酵素活性之分析…………………………47 第五章、結論…………………………………………………………………………52 第六章、參考文獻……………………………………………………………………5