臺灣博碩士論文加值系統

黑豆(Glycine max)富含植化素，植化素可作為黑豆之化學特徵，用以鑑別不同品種之黑豆。本研究建立黑豆植化素的分析平台，探討黑豆中植化素組成資訊。使用超高效能液相層析串聯質譜儀(UHPLC-DAD-ESI-MS)分別進行花青素及酚酸的定性分析，共鑑定出黑豆中6種花青素及10種酚酸。使用高效能液相層析串聯光二極體陣列檢測器(HPLC-DAD)定量分析異黃酮、花青素及酚酸，結果顯示，黑豆中異黃酮以丙二醯型及醣苷型為主，而乙醯型及苷元型含量稀少，其中又以genistein苷元結構之總含量最高；花青素定量分析發現cyanidin-3-O-hexoside含量最多，佔了超過60%的花青素總含量；酚酸有游離態與結合態兩種型態，游離態酚酸中有高比例的p-coumaric acid，結合態酚酸主要為protocatechuic acid，其次為syringic acid及vanillic acid，總酚酸則是以p-coumaric acid含量最高。以UHPLC-ESI-MS分析黑豆指紋圖譜，搭配主成分分析可將不同品種之黑豆分群，並區分出不同產地。本研究建立之分析平台能夠測定黑豆中的植化素，利用其化學資訊可以鑑別不同品種之黑豆。

Black soybean (Glycine max) is rich in phytochemicals. Phytochemicals could be used to diffentiate black soybean according to species. The characteristic chemical composition could be used to evaluate its quality. In this research, a method for the analysis of phytochemicals in black soybean was developed. Qualititative analysis of anthocyanin and phenolic acid was done by UHPLC-DAD-ESI-MS. A total of six anthocyanins and ten phenolic acids were identified in black soybean. Quantitative analysis of isoflavone, anthocyanin and pheolic acid was done by HPLC-DAD. Results showed that malonylglucoside and glucoside isoflavone were the major form in black soybean with less amount of acetylglucoside and aglycone isoflavone. Among the three aglycone structures, genistein had the highest amount. The analysis of anthocyanin indicated that cyanidin-3-O-hexoside was the main compound in black soybean, containing over 60% of total anthocyanin. Phenolic acid could be divided into free form and bound form. In this study, p-coumaric acid was the main compound among the free form phenolic acids and protocatechuic acid was the main compound among the bound form phenolic acids. The content of p-coumaric acid was the highest among all phenolic acids. Fingerprinting of black soybean was done by UHPLC-ESI-MS. Data from the fingerprinting was extracted and analysed by principal component analysis to group the black soybeans according to species. Differentiation of the origin of black soybean was also achieved. In conclusion, this study developed a method for the analysis of phytochemicals in black soybean and the chemical information could differentiate black soybean according to species.

總目錄
摘要 I
Abstract II
總目錄 III
圖目錄 V
表目錄 VII
壹、前言 1
貳、文獻回顧 2
一、黑豆 2
二、黑豆之植化素 2
(一) 類黃酮(flavonoids) 2
(二) 酚酸(phenolic acids) 11
三、分析方法 14
(一) 高效能液相層析(High performance liquid chromatography, HPLC) 14
(二) 檢測器 14
四、指紋圖譜 17
參、研究目的與實驗架構 19
肆、材料與方法 20
一、實驗材料 20
二、實驗藥品 20
(一) 標準品 20
(二) 化學試劑及藥品 21
三、儀器設備 22
(一) 實驗耗材 22
(二) 一般儀器設備 22
(三) 高效能液相層析與質譜儀 22
四、實驗方法 23
(一) 總酚含量測定 23
(二) 總黃酮含量測定 23
(三) 總單體花青素含量測定法 24
(四) 異黃酮分析 24
(五) 花青素分析 25
(六) 酚酸分析 28
(七) 指紋圖譜分析 30
(八) 統計分析 32
伍、結果與討論 33
一、異黃酮HPLC-DAD分析 33
二、花青素分析 42
(一) UHPLC-DAD-ESI-MS定性分析 42
(二) HPLC-DAD定量分析 54
三、酚酸分析 60
(一) UHPLC-DAD-ESI-MS定性分析 60
(二) HPLC-DAD定量分析 74
四、指紋圖譜分析 84
陸、結論 92
柒、參考文獻 93

衛生福利部食品藥物管理署。膠囊錠狀食品中大豆異黃酮之檢驗方法。2014年8月21日。
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。
Abu-Reidah, I.M.; Ali-Shtayeh, M.S.; Jamous, R.M.; Arráez-Román, D.;Segura-Carretero, A. HPLC–DAD–ESI-MS/MS screening of bioactive components from Rhus coriaria L. (Sumac) fruits. Food Chem. 2015, 166, 179-191.
Acosta-Estrada, B.A.; Gutiérrez-Uribe, J.A.; Serna-Saldívar, S.O. Bound phenolics in foods, a review. Food Chem. 2014, 152, 46-55.
Alberts, P.; Stander, M.A.; Villiers, A. Advanced ultra high pressure liquid chromatography–tandem mass spectrometric methods for the screening of red wine anthocyanins and derived pigments. J. Chromatogr. A. 2012, 1235, 92-102.
Alu’datt, M.H.; Rababah, T.; Ereifej, K.; Alli, I. Distribution, antioxidant and characterisation of phenolic compounds in soybeans, flaxseed and olives. Food Chem, 2013, 139, 93-99.
Bataglion, G.A.; Silva, F.M.A.; Eberlin, M.N.; Koolen, H.H.F. Determination of the phenolic composition from Brazilian tropical fruits by UHPLC–MS/MS. Food Chem. 2015, 180, 280-287.
Biesaga, M.; Pyrzynska, K. Liquid chromatography/tandem mass spectrometry studies of the phenolic compounds in honey. J. Chromatogr. A. 2009, 1216, 6620-6626.
Box, J.D. Investigation of the Folin-Ciocalteau phenol reagent for the determination of polyphenolic substances in natural waters. Water Res. 1983, 511-525.
César, I.C.; Braga, F.C.; Vianna-Soares, C.D.; Nunan, E.A.; Moreira-Campos, G.A.P.L.M. Quantitation of genistein and genistin in soy dry extracts by UV-visible spectrophotometric method. Quim. Nova. 2008, 31, 1933-1936.
Chen, G.G.; Lai, P.B.S. 2012. Novel apoptotic regulators in carcinogenesis. Berlin: Springer Science & Business Media.
Cho, K.M.; Ha, T.J.; Lee, Y.B.; Seo, W.D.; Kim, J.Y.; Ryu, H.W.; Jeong, S.H.; Kang, Y.M.; Lee, J.H. Soluble phenolics and antioxidant properties of soybean (Glycine max L.) cultivars with varying seed coat colours. J. Funct. Food. 2013, 5, 1065-1076.
Choung, M.G.; Baek, I.Y.; Kang, S.T.; Han, W.Y.; Shin, D.C.; Moon, H.P.; Kang, K.H. Isolation and determination of anthocyanins in seed coats of black soybean (Glycine max (L.) Merr.). J. Agric. Food Chem. 2001, 49, 5848-5851.
Correa, C.R.; Li, L.; Aldini, G.; Carini, M.; Chen, C.Y.O.; Chun, H.; Cho, S.; Park, K.; Russell, R.M.; Blumberg, J.B.; Yeum, K. Composition and stability of phytochemicals in five varieties of black soybeans (Glycine max). Food Chem. 2010, 123, 1176-1184.
Eldridge, A.C. Determination of isoflavones in soybean flours, protein concentrates, and isolates. J. Agric. Food Chem. 1982, 30, 353-355.
Fang, C.; Wan, X.; Tan, H.; Jiang, C. Identification of isoflavonoids in several kudzu samples by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. J. Chromatogr. Sci. 2006, 44, 57-63.
Ghasemzadeh, A.; Ghasemzadeh, N. Flavonoids and phenolic acids: role and biochemical activity in plants and human. J. Med. Plants Res. 2011, 5, 6697-6703.
Ha, T.J.; Lee, J.H.; Shin, S.O.; Shin, S.H.; Han, S.I.; Kim, H.T.; Ko, J.M.; Lee, M.H.; Park, K.Y. Changes in anthocyanin and isoflavone concentrations in black seed-coated soybean at different planting locations. J. Crop Sci. Biotech. 2009, 12, 79-86.
Hamed, A.I.; Al-Ayed, A.S.; Moldoch, J.; Piacente, S.; Oleszek, W.; Stochmal, A. Profiles analysis of proanthocyanidins in the argun nut (Medemia argun—an ancient Egyptian palm) by LC–ESI–MS/MS. J. Mass Spectrom. 2014, 49, 306-315.
He, X.; Li, J.; Zhao, W.; Liu, R.; Zhang, L.; Kong, X. Chemical fingerprint analysis for quality control and identification of Ziyang green tea by HPLC. Food Chem. 2015, 171, 405-411.
Hong, J.L.; Qin, X.Y.; Shu, P.; Wang, Q.; Zhou, Z.F.; Wang, G.K.; Lin, B.B.; Wang, Q.; Qin, M.J. Comparative study of isoflavones in wild and cultivated soybeans as well as bean products by high-performance liquid chromatography coupled with mass spectrometry and chemometric techniques. Eur. Food Res. Technol. 2011, 233, 869-880.
Ito, C.; Oki, T.; Yoshida, T.; Nanba, F.; Yamada, K.; Toda, T. Characterisation of proanthocyanidins from black soybeans: Isolation and characterisation of proanthocyanidin oligomers from black soybean seed coats. Food Chem. 2013, 141, 2507-2512.
Jiang, Y.; David, B.; Tu, P.; Barbin, Y. Recent analytical approaches in quality control of traditional Chinese medicines—A review. Anal. Chim. Acta. 2010, 657, 9-18.
Kim, E.H.; Kim, S.L.; Kim, S.H.; Chung, I.M. Comparison of isoflavones and anthocyanins in soybean [Glycine max (L.) Merrill] seeds of different planting dates. J. Agric. Food Chem. 2012, 10196-10202.
Kim, E.H.; Lee, O.K.; Kim, J.K.; Kim, S.L.; Lee, J.; Kim, S.H.; Chung, I.M. Isoflavones and anthocyanins analysis in soybean (Glycine max (L.) Merill) from three different planting locations in Korea. Field Crops Res. 2014, 156, 76-83.
Kim, E.H.; Ro, H.M.; Kim, S.L.; Kim, H.S.; Chung, I.M. Analysis of isoflavone, phenolic, soyasapogenol, and tocopherol compounds in soybean [Glycine max (L.) Merrill] germplasms of different seed weights and origins. J. Agric. Food Chem. 2012, 60, 6045-6055.
Kim, M.Y.; Jang, G.Y.; Lee, Y.; Li, M.; Ji, Y.M.; Yoon, N.; Lee, S.H.; Kim, K.M.; Lee, J.; Jeong, H.S. Free and bound form bioactive compound profiles in germinated black soybean (Glycine max L.). Food Sci. Biotechnol. 2016, 25, 1551-1559.
Koh, K.; Youn, J.E.; Kim, H.S. Identification of anthocyanins in black soybean (Glycine max (L.) Merr.) varieties. J. Food Sci. Technol. 2014, 51, 377-381.
Lee, J.; Durst, R.W.; Wrolstad, R.E. Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study. J. AOAC Int. 2005, 88, 1269-1278
Lee, J.; Rennaker, C.; Wrolstad, R.E. Correlation of two anthocyanin quantification methods: HPLC and spectrophotometric methods. Food Chem, 2008, 110, 782-786.
Lee, J.H.; Cho, K.M. Changes occurring in compositional components of black soybeans maintained at room temperature for different storage periods. Food Chem. 2012, 131, 161-169.
Lee, J.H.; Kang, N.S.; Shin, S.O.; Shin, S.H.; Lim, S.G.; Suh, D.Y.; Baek, I.Y.; Park, K.Y.; Ha, T.J. Characterisation of anthocyanins in the black soybean (Glycine max L.) by HPLC-DAD-ESI/MS analysis. Food Chem. 2009, 112, 226-231.
Lee, S.J.; Ahn, J.K.; Kim, S.H.; Kim, J.T.; Han, S.J.; Jung, M.Y.; Chung, I.M. Variation in Isoflavone of Soybean Cultivars with Location and Storage Duration. J. Agric. Food Chem. 2003, 51, 3382-3389.
Lee, S.J.; Kim, J.J.; Moon, H.I.; Ahn, J.K.; Chun, S.C.; Jung, W.S.; Lee, O.K.; Chung, I.M. Analysis of isoflavones and phenolic compounds in korean soybean [Glycine max (L.) Merrill] seeds of different seed weights. J. Agric. Food Chem. 2008, 56, 2751-2758.
Lee, S.J.; Seguin, P.; Kim, J.J.; Moon, H.I.; Ro, H.M.; Kim, E.H.; Seo, S.H.; Kang, E.Y.; Ahn, J.K.; Chung, I.M. Isoflavones in Korean soybeans differing in seed coat and cotyledon color. J. Food Comp. Anal. 2010, 23, 160-165.
Liang, X.M.; Jin, Y.; Wang, Y.P.; Jin, G.W.; Fu, Q.; Xiao, Y.S. Qualitative and quantitative analysis in quality control of traditional Chinese medicines. J. Chromatogr. A. 2009, 1216, 2033-2044.
Lin, F.; Giusti, M.M. Effects of solvent polarity and acidity on the extraction efficiency of isoflavones from soybeans (Glycine max). J. Agric. Food Chem. 2005, 53, 3795-3800.
Lin, P.Y.; Lai, H.M. Bioactive compounds in legumes and their germinated products. J. Agric. Food Chem. 2006, 54, 3807-3814.
López, B.G.C.; Schmidt, E.M.; Eberlin, M.N. Sawaya, A.C.H.F. Phytochemical markers of different types of red propolis. Food Chem. 2014, 146, 174-180.
Manchón, N.; D’Arrigo, M.; García-Lafuente, A.; Guillamón, E.; Villares, A.; Ramos, A.; Martínez, J.A.; Rostagno, M.A. Fast analysis of isoflavones by high-performance liquid chromatography using a column packed with fused-core particles. Talanta. 2010, 82, 1986-1994.
Medic, J.; Atkinson, C.; Hurburgh Jr., C.R. Current knowledge in soybean composition. J. Am. Oil Chem. Soc. 2014, 91, 363-384.
Navas, M.J.; Jiménez-Moreno, A.M.; Bueno, J.M.; Sáez-Plaza, P.; Asuero, A.G. Analysis and antioxidant capacity of anthocyanin pigments. part III: an introduction to sample preparation and extraction. Anal. Chem. 2012a, 42, 284-312.
Navas, M.J.; Jiménez-Moreno, A.M.; Bueno, J.M.; Sáez-Plaza, P.; Asuero, A.G. Analysis and antioxidant capacity of anthocyanin pigments. part IV: extraction of anthocyanins. Anal. Chem. 2012b, 42, 313-342.
Nikkhah, E.; Khayamy, M.; Heidari, R.; Jamee, R. Effect of sugar treatment on stability of anthocyanin pigments in berries. J. Biol. Sci. 2007, 7, 1412-1417.
Pellati, F.; Orlandini, G.; Pinetti, D.; Benvenuti, S. HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts. J. Pharm. Biomed. Anal. 2011, 55, 934-948.
Peng, H.; Li, W.; Li, H.; Deng, Z.; Zhang. B. Extractable and non-extractable bound phenolic compositions and their antioxidant properties in seed coat and cotyledon of black soybean (Glycinemax (L.) merr). J. Funct. Food. 2017, 32, 296-312.
Quifer-Rada, P.; Vallverdú-Queralt, A.; Martínez-Huélamo, M.; Chiva-Blanch, G.; Jáuregui, O.; Estruch, R.; Lamuela-Raventós, R. A comprehensive characterisation of beer polyphenols by high resolution mass spectrometry (LC–ESI-LTQ-Orbitrap-MS). Food Chem. 2015, 169, 336-343.
Robbins, R.J. Phenolic acids in foods: an overview of analytical methodology. J. Agric. Food Chem. 2003, 51, 2866-2887.
Rostagno, M.A.; Villares, A.; Guillamón, E.; García-Lafuente, A.; Martínez, J.A. Sample preparation for the analysis of isoflavones from soybeans and soy foods. J. Chromatogr. A. 2009, 1216, 2-29.
Rubert, J.; Lacina, O.; Zachariasova, M.; Hajslova, J. Saffron authentication based on liquid chromatography high resolution tandem mass spectrometry and multivariate data analysis. Food Chem. 2016, 204, 201-209.
Sanz, M.; Simón, B.F.D.; Cadahía, E.; Esteruelas, E.; Muñoz, A.M.; Hernández, T.; Estrella, I.; Pinto E. LC-DAD/ESI-MS/MS study of phenolic compounds in ash (Fraxinus excelsior L. and F. americana L.) heartwood. Effect of toasting intensity at cooperage. J. Mass Spectrom. 2012, 47, 905-918.
Savarese, M.; Marco, E.D.; Sacchi, R. Characterization of phenolic extracts from olives (Olea europaea cv. Pisciottana) by electrospray ionization mass spectrometry. Food Chem. 2007, 105, 761-770.
Schütz, K.; Persike, M.; Carle, R.; Schieber, A. Characterization and quantification of anthocyanins in selected artichoke (Cynara scolymus L.) cultivars by HPLC–DAD–ESI–MSn. Anal. Bioanl. Chem. 2006, 384, 1511-1517.
Shen, Y.; Jin, L.; Xiao, P.; Lu, Y.; Bao, J. Total phenolics, flavonoids, antioxidant capacity in rice grain and their relations to grain color, size and weight. J. Cereal Sci. 2009, 49, 106-111.
Stalikas, C.D. Extraction, separation, and detection methods for phenolic acids and flavonoids. J. Sep. Sci. 2007, 30, 3268-3295.
Tistaert, C.; Dejaegher, B.; Heyden, Y.V. Chromatographic separation techniques and data handling methods for herbal fingerprints: A review. Anal. Chim. Acta. 2011, 690, 148-161.
Tsai, H.S.; Huang, L.J.; Lai, Y.H.; Chang, J.C.; Lee, R.S.; Chiou R.Y.Y. Solvent effects on extraction and HPLC analysis of soybean isoflavones and variations of isoflavone compositions as affected by crop season. J. Agric. Food Chem. 2007, 55, 7712-7715.
Valls, J.; Millán, S.; Martí, M.P.; Borràs, E.; Arola, L. Advanced separation methods of food anthocyanins, isoflavones and flavanols. J. Chromatogr. A. 2009, 1216, 7143-7172.
Wally-Vallim, A.P.; Vanier, N.L.; Zavareze, E.R.; Zambiazi, R.C.; Castro, L.A.S.; Schirmer, M.A.; Elias, M.C. Isoflavone aglycone content and the thermal, functional, and structural properties of soy protein isolates prepared from hydrothermally treated soybeans. J. Food Sci. 2014, 79, E1351-E1358.
Wu, X.; Gu, L.; Prior, R.L.; Mckay, S. Characterization of anthocyanins and proanthocyanidins in some cultivars of Ribes, Aronia, and Sambucus and their antioxidant capacity. J. Agric. Food Chem. 2004, 52, 7846-7856.
Xia, J.; Wishart, D.S. Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis. Curr. Protoc. Bioinform. 2016, 55, 14.10.1-14.10.91.
Xian, F.; Hendrickson, C.L.; Marshall, A.G. High resolution mass spectrometry. Anal. Chem. 2012, 84, 708-719.
Xu, B.; Chang, S.K.C. Antioxidant capacity of seed coat, dehulled bean, and whole black soybeans in relation to their distributions of total phenolics, phenolic acids, anthocyanins, and isoflavones. J. Agric. Food Chem. 2008, 56, 8365-8373.
Zhang, R.F.; Zhang, F.X.; Zhang, M.W.; Wei, Z.C.; Yang, C.Y.; Zhang, Y.; Tang, X.J.; Deng, Y.Y.; Chi, J.W. Phenolic composition and antioxidant activity in seed coats of 60 Chinese black soybean (Glycine max L. Merr.) varieties. J. Agric. Food Chem. 2011, 59, 5935-5944.
Zhang, Z.; Kou, X.; Fugal, K.; Mclaughlin, J. Comparison of HPLC methods for determination of anthocyanins and anthocyanidins in bilberry extracts. J. Agric. Food Chem. 2004, 52, 688-691.
Žilić, S.; Akıllıoğlu, H.G.; Serpen, A.; Perić, V.; Gökmen, V. Comparisons of phenolic compounds, isoflavones, antioxidant capacity and oxidative enzymes in yellow and black soybeans seed coat and dehulled bean. Eur. Food Res. Technol. 2013, 237, 409-418.