| 摘要: | 本研究第一部分嘗試以烷基乙酯（alkyl acetates）分配萃取、聚醯胺 （polyamide）及木屑（柳安木和養菇木）吸附試驗，希望能以簡單且較安全的程序製備含低咖啡因之酯化型兒茶素（esterified catechins）精製物。在分配萃取試驗中，首先將茶粉以少量的水在低溫下潤濕，再以有機溶劑（乙基乙酯（乙酸乙酯；ethyl acetate, EA）和丁基乙酯（butyl acetate, BA））進行數次分配萃取，然後以高效能液相層析儀（high performance liquid chromatography, HPLC）檢視有機溶劑層之主要成分。其次再觀吸附試驗中，先將茶粉以熱水萃取，分別以自製聚醯胺粉末及酸鹼處理過的木屑進行吸附，進而用水將未吸附的成分（主要為咖啡因 （caffeine）、非兒茶素物質及兒茶素氧化物）洗掉，然後再將吸附的酯化型兒茶素以不同比例的乙醇濃度沖提出。由HPLC分析的結果顯示，乙基乙酯在精製酯化型兒茶素的能力較丁基乙酯好，但丁基乙酯移除咖啡因的能力卻較乙基乙酯佳，而且水的潤濕程序是必要的。聚醯胺粉末的吸附能力較木屑好。水-丁基乙酯分配萃取方法一般可移除生茶粉中80%的咖啡因，而酯化型兒茶素也能回收大約70%。另一方面，聚醯胺粉末及木屑皆能移除90%以上咖啡因，而回收80%以上酯化型兒茶素；就整體而言，聚醯胺粉末的吸附能力較木屑好。此外，聚醯胺粉末及木屑都能重複使用五次以上，而不會明顯喪失其吸附效率。
本研究的第二部分是將天然物（茶葉及諾麗果葉的萃取液）經HPLC分離後，以本實驗室所改良的兩種簡單的體外抗氧化能力檢驗程序，以96槽為平板配合酵素連結免疫吸附分析法（ELISA）檢測儀快速評估與篩選天然物萃取液中的（DPPH清除能力與Trolox當量抗氧化能力；TEAC）抗氧化活性成分。最後結果顯示這DPPH與ABTS‧+ （在30~50% 甲醇溶液中）皆適合於作為抗氧化分析用的檢測試劑。TEAC法由於靈敏度比較高反應快，不僅可應用於批次分析，更有可能設計成管柱後（post column）直接進行抗氧化活性檢測程序。
本研究進而以上述的TEAC程序，比較九種不同發酵程度茶品抗氧化活性成分的差異。由HPLC分析得知，未發酵及半發酵茶中酯化型兒茶素含量(以每g乾茶粉計) 較多 (38.7-59.9 mg)，紅茶 (28.1 mg) 及普洱茶 (1.3 mg) 則較低。咖啡因含量以椪風茶為最 (76.4 mg)，高山茶的46.2 mg為最低，但含量與發酵程度並沒有直接關係。比較九種不同發酵程度的茶葉之總抗氧化能力 (TEAC值)，一般在286-360 mg 的Trolox當量之間，紅茶 (259 mg) 及普洱茶 (239 mg) 則較低。茶葉熱水萃取液經HPLC分離所得各區劃之抗氧化能力顯示，未發酵及半發酵茶中除椪風茶外酯化型兒茶素類佔抗氧化能力50 % 以上，但紅茶及普洱茶的抗氧化能力不但較差，且幾乎都是來自以沒食子酸 (gallic acid) 為主的高親水性物質。
另一方面，利用同樣的程序進行海巴戟天（諾麗； Morinda citrifolia）葉95 % 乙醇萃取液抗氧化活性成分的篩選，結果顯示該萃取液中至少含有五種以上之抗氧化活性成分，其中主要成分經純化並與純品比對後初步鑑定為芸香素（rutin），每克乾諾麗葉的含量約為16 mg。
The first part of this research is a trial to get a refined tea preparation of high esterified catechins with low caffeine content by simple and safer processes. The procedures used including organic solvent (ethyl acetate, EA and butyl acetate, BA) partition, and polyamide or wood-dusts (Liu-An or material for mushroom growing) adsorptions. Firstly, tea powder was wetted with small amount of water (at low temperature), and then partitioned with the organic solvent (EA or BA) for several times, and then the composition in organic solvent layer was examined by HPLC. Secondly, tea infusion after hot water treatment was adsorbed with either treated wood-dusts or home-made polyamide powder. After washing out unbound components (mainly caffeine and free or oxidized catechins), esterified catechins were then desorbed with the aqueous alcohol solution of different concentration of methanol.
The results showed that EA was better than BA in isolating esterified catechins, but BA revealed higher ability to remove caffeine. On the other hand, the home-made polyamide powder was more efficient as the adsorbent as comparing to that of the acid/base treated-wood-dusts. In general, 80% or more of caffeine in raw tea can be removed by water-BA partition method, and the recovery of esterified catechins is about 70%. Polyamide powder and treated Liu-An wood-dust can eliminate more than 90% of caffeine, and the yield of esterified catechins is higher than 80%. Furthermore, both polyamine powder and treated Liu-An wood-dust can be used repeatedly for five or more times without apparently losing their binding ability.
The second part of this study is to optimizing two convenient in vitro antioxidant assay procedures (DPPH scavenging and TEAC activity). The 96-well micro-plate with ELISA (Enzyme-linked immunosorbent assay) reader was used for rapidly evaluating the anti-oxidative activity of the components in extract of natural product after HPLC. The results shown that both DPPH‧ and ABTS‧+ free radicals dissolved in 30~50 % of MeOH could be used as reagent in anti-oxidant survey. The sensitivity and reactivity of TEAC assay was higher than DPPH scavenging assay. TEAC assay can be applied not only for batch analysis, but may also be designed as a direct post-column assay procedure.
Anti-oxidative activity of nine different fermented tea products were compared with TEAC assay. HPLC data showed that un-fermented and semi-fermented tea products contained higher amount of esterified catechins (38.7-59.9 mg/g) than black tea (28.1 mg) and Pu-erh tea (1.3 mg). The caffeine content is higher in Pon-hong tea (76.4 mg) and high mountain tea (46.2 mg) is the lowest. Tea infusions from nine different fermented teas were detected for their TEAC value. In general, 286-360 mg Trolox equivalent for most infusions were obtained, except black tea (259 mg) and Pu-erh tea (239 mg) More than 50% of antioxidative ability came from esterified catechins in hot-water extract of unfermented and semi-fermented tea except Pon-fong tea, but the antioxidative ability of black and Pu-erh tea is mostly obtained from highly hydrophilic components, including gallic acid.
By using the same procedure, antioxidants in 95% ethanol extract from Noni’s leaf was surveyed, results shown that the extract contain more than five antioxidants. The main component was further identified as rutin. By referring the authentic compound, Noni contains as high as about 16 mg of rutin per gram of dried leaf. |
