高血壓模式下大鼠血漿中異黃酮代謝物之分析

The analysis of plasma isoflavone metabolites in hypertensive rat model

陳兆聰(Sio-Chong Chan)

指導教授 ： 陳宏彰

共同指導教授 ： 邱家琪

國立臺灣大學/生物資源暨農學院/食品科技研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902969

摘要

黃豆[Glycine max (L.) Merr.]富含植化素，為大豆異黃酮之主要來源；大豆異黃酮具有多種生物活性，且以苷元型式的異黃酮具有良好的生物利用率。本研究分析以自行分離的Lactobacillus rhamnosus S1.所發酵之豆漿，測定發酵後豆漿中異黃酮之含量；並將此發酵豆漿餵食高血壓大鼠後，評估大鼠血漿中異黃酮代謝物之含量。結果顯示，利用UPLC-ESI-HRMS分析發酵豆漿，在發酵豆漿中丙二醯型，乙醯型及醣苷型式之異黃酮含量顯著低於未發酵豆漿，苷元型式之異黃酮經發酵後則顯著上升(p<0.05)，上升幅度約1.5-3.8倍不等。血漿中的異黃酮代謝物經UPLC-ESI-HRMS分析，利用精確分子量、二次質譜碎片，並利用標準品或文獻比對後，可鑑定21種異黃酮代謝物。利用此分析平台進行不同處理下高血壓大鼠血漿中異黃酮代謝物之半定量分析，結果顯示血漿中的異黃酮代謝物主要以phase II 代謝物為主，且以葡萄糖醛酸硫酸結合物為主，亦檢測到4-ethylphenyl sulfate。6種代謝物Daidzein-glucuronide (r=0.45, p=0.0059)，Daidzein-7-glucuronide-4’-sulfate (r=0.47, p=0.0040)，Daidzein Disulfate (r=0.49, p=0.0025)，Daidzein sulfate (r=0.57, p=0.0003)，Genistein sulfoglucuronide (r=0.56, p=0.0004)及Genistein glucuronide (r=0.50, p=0.0019)的含量與收縮壓呈中度正相關，顯示異黃酮代謝物的含量與血壓的調整有正相關。本研究藉由高解析質譜建立了動物體內血漿中21種異黃酮代謝物的分析平台，其能運用於豆漿及動物體中代謝轉換之分析，並進一步可探討代謝物含量與疾病治療之關聯性。

關鍵字

異黃酮 ； 發酵豆漿 ； 高血壓 ； 異黃酮代謝物 ； 高解析質譜

並列摘要

Soybean [Glycine max (L.) Merr.] is rich in phytochemicals, which can be the source of isoflavones. Soy bean isoflavones have a variety of biological activities and the aglycone form have a better bioavailability. In this study, the content of isoflavones in soymilk fermented by Lactobacillus rhamnosus S1 was determined. Moreover, the content of isoflavone metabolites in the plasma of hypertensive rat was determined. The UPLC-ESI-HRMS was used to analyze the fermented soymilk. Results showed that the content of malonylglucoside, acetylglucoside and glucoside form of isoflavone in the fermented soymilk was significantly lower (p<0.05) than those in unfermented-soymilk. The aglycone form isoflavones significantly increased after fermentation (p<0.05), and its increase ranged from 1.5 to 3.8 times. The isoflavone metabolites in plasma were analyzed by UPLC-ESI-HRMS according to the mass accuracy and fragement of MS2. A total of 21 isoflavone metabolites were identified by standard or compare to literature reported. Semi-quantitation was used to quantify the isoflavone metabolites in rat plasma. Results showed that the phase II metabolites were the major metabolites in plasma. The main metabolites were glucuronic acid or sulfate conjugates, and 4-ethylphenyl sulfate was also detected. A positive Pearson correlation between the six metabolites including Daidzein-glucuronide (r=0.45, p=0.0059), Daidzein-7-glucuronide-4’-sulfate (r=0.47, p=0.0040), Daidzein Disulfate (r=0.49, p=0.0025), Daidzein sulfate (r=0.57, p=0.0003), Genistein sulfoglucuronide (r=0.56, p=0.0004) and Genistein glucuronide (r=0.50, p=0.0019) was positively correlated with systolic blood pressure and may be positively correlated with the regulation of blood pressure. This study established a high-resolution mass spectrometry platform which can be applied to the analysis of metabolic conversion in soymilk and animal plasma. Furthermore, to explore the relationship between metabolite contents and treatment.

並列關鍵字

isoflavones ； fermented soy milk ； hypertension ； isoflavone metabolites ； high resolution mass spectrometry

參考文獻

1. FAOSTAT: http://www.fao.org/faostat/en/#data/QC/visualize

2. 臺灣質譜學會 (2015)。質譜分析技術原理與應用。新北市：全華。

3. 衛生福利部食品藥物管理署。食品化學檢驗方法之確效規範【第二次修正】。2013 年 9 月 9 日。

4. Aguiar, C. L.; Haddad, R.; Eberlin, M. N.; Carrão-Panizzi, M. C.; Tsai, S. M.; Park, Y. K., Thermal behavior of malonylglucoside isoflavones in soybean flour analyzed by RPHPLC/DAD and eletrospray ionization mass spectrometry. LWT - Food Sci Technol 2012, 48, 114-119.

5. Braune, A.; Blaut, M., Deglycosylation of puerarin and other aromatic C-glucosides by a newly isolated human intestinal bacterium. Environ Microbiol 2011, 13, 482-494.