利用高效液相層析法分析蔬菜中類黃酮含量之研究

Abstract

類黃酮是植物性的天然抗氧化劑，主要存在於蔬果中，具有抗氧化、降低心血管疾病及抗癌等作用，隨著健康意識的抬頭，類黃酮在蔬果中含量成為一熱門研究主題。為了更了解蔬果中類黃酮的含量分布，本試驗以高效液相層析分析(HPLC)法分析類黃酮之研究並進而分析國內常見蔬菜中類黃酮之含量。 以C18管柱進行HPLC分析五種類黃酮，為apigenin、luteolin 、quercetin、kaempferol、myricetin，在移動相的選擇方面，不同比例30%、40%、50%之氰甲烷為移動相，偵測結果無法判讀。但利用0.025 M磷酸鉀溶液：55%甲醇(v/v)為移動相，能將標準品以分離且有良好成分峰解析度。 類黃酮以糖苷形式存在於細胞中，需進行水解得到個別的糖苷配基，以便定量。利用不同濃度之鹽酸溶液及水解時間進行水解，結果顯示，使用2N、1.5N之HCL進行水解，會破壞植物樣品類黃酮之含量；最適合之水解條件為1N HCL，則可以維持其含量。而水解時間2小時，效果達最佳化，水解效率為85.1%。 本試驗以55%甲醇(v/v)為移動相，1N HCL、加熱2小時進行水解，為類黃酮HPLC分析之條件，測定國內常見之蔬菜種類中含量最豐富的黃酮類的luteolin以及apigenin及黃酮醇類中的myricetin、quercetin、kaempferol，本試驗之結果可提供國人營養食用之參考。結果顯示，在黃酮類中的quercetin以洋蔥的含量最為豐富(1147.42 μg/g)，依次是翡翠椒(921.35 μg/g)、秋葵(542.78 μg/g)、大蒜(357.77 μg/g)；myricetin以洋蔥(559.65 μg/g)為最高、甘藷葉(156.85 μg/g)；kaempferol以青花菜(166.99 μg/g)為最高、牛角椒(131.81 μg/g)、翡翠椒(126.79 μg/g)。在黃酮醇類中的luteolin以大蒜(627.13 μg/g)為最高、朝天椒(212.27 μg/g)；apigenin以洋蔥(339.31 μg/g)為最高、萵苣(163.81 μg/g)。在總類黃酮含量上，洋蔥總類黃酮含量最豐富。每克乾重含量2046.38μg；其他如翡翠椒、大蒜、秋葵、萵苣的總類黃酮含量都超過400 μg/g；芫荽、番茄的總類黃酮含量則低於30 μg/g。

Flavonoids are a class of plant secondary metabolites found in many fruits and vegetables, and known for their anti-oxidation capacity. Flavonoids are important in the human diet since there is evidence that they act as antioxidant, and reduce the risk of cardiovascular diseases and cancers. With the consciousness of health, flavonoid content in fruits and vegetables becomes a popular research topic. To have better understanding of the distribution of flavonoids in horticultural crops, HPLC technique was studied and applied to analyze the flavonoids in common vegetables in Taiwan.C18 column is used to analyze the five flavonoids which are apigenin, luteolin, quercetin, kaempferol, and myrcetin. In the meantime, different concentrations (30%, 40%, and 50%) of HCN solution were used as mobile phase, but the results were undetectable. However, by the use of 45% 0.025 M KH2PO4:55% MeOH (v/v) solution as mobile phase, the standards could be well separated with good resolution Flavonoids are present in plant tissues as sugar conjugates which could not easily be quantified individually. Hence, hydrolysis of the flavonoid glycosides are required. Results showed that the use of 2 N, 1.5N HCl hydrolysis, would decrease the flavonoid contents of plant samples, 1N HCl can maintain the content. The optimum hydrolysis condition was 2 hours in 1N HCl, and the best hydrolysis rate was 85.1%. In this study, after 2 hours hydrolysis in 1N HCl, using 55% methanol (v/v) as mobile phase, was the conditions for analysis of flavonoids. To provide the dietary information, the content of luteolin, apigenin, myricetin, quercetin, and kaempferol in the daily vegetables in Taiwan are analyzed. The results showed that the highest content of quercetin occurred in onions (1147.42 μg/g), followed by green pepper (921.35 μg/g), okra (542.78 μg/g), garlic (357.77 μg/g); and the highest of myricetin also occurred in onion (559.65 μg/g), followed by sweet potato tips (156.85 μg/g); and the highest content of kaempferol happened in broccoli (166.99 μg/g), followed by green chili pepper (131.81 μg/g), green pepper (126.79 μg/g). As for the content of the luteolin and apigenin, the highest content occurred in garlic (627.13 μg/g) and onion (339.31 μg/ g), followed by cone pepper (212.27 μg/g) and lettuce (163.81 μg/g), respectively. The sum of flavonoid was the highest in onion (2046.38 μg/g). Green pepper, garlic, okra, lettuce contained more than 400 μg/g of flavonoids while coriander and tomato contained less than 30 μg/g.