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老 CNAL94-01 2: Bowman-Birk 量麗行理行 94 年 07 01 94 年 12 31 (Bowman-Birk protease inhibitor, BBI) 異益理料 BBI 行量年 BBI 量 BBI 量年不不 BBI 量不 (p<0.05) 年 BBI 量 BBI 量參不不更留 SDS-PAGE 易 (World Trade Organization, WTO) 行 (WTO2002) 了力行量精行度 (Glycine max L.)( 連 2005) 更年來 (1998; 連 1999) 來冷 88 % ( 連 2004) 異力益理 (protease 1

inhibitors) 異 (isoflavone) BBI BBI 量劣 (Lang et al., 2003) 良廉 () 力行 BBI 量兩異不年不異量不年 BBI 量料良 13 7() 料 4 ()BBISDS-PAGE 量 (Tris-EDTA-Triton-X100-PMSF, ph7.0) (McCulloch MD41010) 來量參 Bradford (1976) BSA (Bovine serum albumin; Pierce 23209) 利量 SDS-PAGE Laemmli (1970) 15%SDS-PAGE 行 BBI (T9777, Sigma-Aldrich Corporation) 3 µg 量 Gel-Code (Pierce 24590) Coomassie brilliant blue G-250 BBI Towbin transfer buffer (Towbin, 1979, 1984) BBI Anti-Moust IgG AP coujugate Promega (S372B) 利 AP (Western Blue R, Promega S3841) (UMAX Astra 1200S) 理 (SigmaGel TM Gel Analysis Software; Sigma Z36, 561-0, Jandel) 行 BBI 量 BBI 量 (µg/mg ) 數 ( Sigmagel ) x 3µg (BBI 量 ) BBI 量數 ( Sigmagel ) x 0.015 mg ( 量 ) () 2

SPSS 10.0 (Jandel) One Way ANOVA (analysis of variance) LSD (least significant difference) 異論 BBI 70-80 硫量 8 kda (Lajolo and Genovese, 2002) SDS-PAGE 來量 BBI 量 BBI BBI 行量 15% 20%SDS-PAGE 不數量 BBI 行 Sigma Chemicals (T-9777) BBI 量 >6.5 kda 量 >16 kda( 1A) 20%SDS-PAGE 量量 8.1 kda 量 14.4 kda ( 1B) 度 BBI 易 ( 1A) ( 1B)(Ford et al., 1982; Birk, 2003) 理 SDS (Millar et al., 1969) 降 BBI 度量理 ( 1B)BBI BBI 15%SDS-PAGE 行 1C D 5 BBI 量 8 kda (Sessa and Wolf, 2001) 量不量不 ( 1D) BBI 量 9~10 kda ( 1D ) 量不 () BBI 量 2002 年 BBI 量 BBI 量不 () 六 BBI 量 ( 茶 ) BBI 量 BBI 量 168.29~201.79 (µg/mg ) 2002 年 2003 年 BBI 量行 2003 年 BBI 量 ( 2) 3

了 2002 年 BBI 量 2003 年 () 不年 BBI 量不年 BBI 量異 ( 3) BBI 量 2001 年 (mg) 143.8 (µg) BBI 2003 年 246.92 µg/mg 兩異 1.7 () BBI 量不 BBI 量異 ( 4) BBI 量 (p<0.001) 2003 年行 ( 2) 論 BBI 量 () BBI 量不料量不行 BBI 不 (Data not shown.) 100 20 量異行益料 Bowman-Birk 行 SDS-PAGE 量 BBI 量都量 () BBI 量年 BBI 量年 BBI 量不 BBI 量 BBI 量見良理 BBI 量異 BBI 量度不念益不 4

說益立良良林理利行參 1. 連 2004 來 2111-14 http://www.tndais.gov.tw/dryland/ 來.pdf 2. 連隆 2005 省 92 年 NSC92-3114-P-055-001-Y 良 3. 連 19993 良 3514-24 4. 麗連 2005Bowman-Birk 量 3945-60 5. 1998 力異論 134pp. 6. 1997 論 110pp. 7. Bradford, M. M. (1976). A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72:248-254. 8. Birk, Y. (2003). Plant protease inhibitors. Springer-Verlag, Berlin, Germany. 9. Eldridge, A.C. and W.F. Kwolek. 1983. Soybean isoflavones : effect of environment and variety on composition. Journal Agricultural Food Chemistry, 31:394-396. 10. Ford, D. E., Gutay, P. A., Ladin, B., Beachy, R. N., and Larkins, B. A. (1982). In vitro synthesis of the Bowman-Birk and related soybean proteinase inhibitor. Plant Molecular Biology, 1:227-243. 11. Head KA (1998) Isoflavones and other soy constituents in human health and disease. Alternative Medicine Review, 3: 433-450. 12. Huang, LJ (2003) Studies on the Genetic and Breeding Behaviors of Quality Characters of the Soybean Varieties Released in Taiwan, p.73. MS Thesis, National Chiayi University, Chiayi, Taiwan, ROC. 13. Lang, J. T., ONeill, K. M., and Hallman, W. K. (2003). Expertise, trust, and communication about food biotechnology. AgBioForum, 6:185-190. 14. Laemmli, U. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227:680-685. 15. Lajolo, F. M., and Genovese, M. I. (2002). Nutritional significance of lectins and enzyme inhibitors from legumes. Journal Agricultural Food Chemistry, 50: 6592-6598. 16. Lee, S.J., K.A. Joung, H.K. Seung, T.K. Jung, J.H. Sang, Y.J. Mun and M.C. Ill. 2003. Variation in isoflavone of soybean cultivars with location and storage duration. Journal Agricultural Food Chemistry, 51:3382-3389. 5

17. Millar, D. B. S., Willick, G. E., Steiner, R. F., and Frattali, V. (1969). Soybean inhibitors. IV. The reversible self-association of a soybean proteinase inhibitor. Journal Biological Chemistry, 244:281-284. 18. Nakamura Y, A Kaihara, K Yoshii, Y Tsumura, S Ishimitsu, Y Tonogai (2001) Content and composition of isoflavonoids in mature or immature beans and bean sprouts consumed in Japan. Journal of Health Science, 47: 394-406. 19. Sessa, D. J. and Wolf, W. J. (2001). Bowman-Birk inhibitors in soybean seed coats. Industrial Crops and Products, 14:77-83. 20. Towbin, H., Staehelin, T., and Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings National Acadamic Science USA, 79:4350-4354. 21. Towbin, H., and Gordon, J. (1984). Immunoblotting and dot immunoblotting: current status and outlook. Journal of Immunology Methods, 72:313-340. 22. Wan, X.S. T.C. Hamilton, J.H. Ware, J.J. Donahue, A.R. Kennedy. 1998. Growth inhibition and cytotoxicity induced by Bowman-Birk inhibitor concentration in cisplatin-resistant human ovarian cancer cells. Nutrition and Cancer, 31:8-17. 23. Wang, H. and P.A. Murphy. 1994a. Isoflavone content in commercial soybean foods. Journal Agricultural Food Chemistry, 42:1666-1673. 24. Wang, H. and P.A. Murphy. 1994b. Isoflavone composition of American and Japanese soybeans in Iowa: effect of variety, crop year, and location. Journal Agricultural Food Chemistry, 42:1674-1677. 6

SDS-PAGE Analysis of Soybean (Glycine max L.) Bowman-Birk Protease Inhibitor (BBI) Lee-Ju Cheng and Jin-Jia Fan Department of Applied Life Science and Health Chia-Nan University of Pharmacy and Science, Tainan, Taiwan (ROC) Abstract Soybean functional ingredients, such as: protease inhibitors, lectin, phytic acid, saponin and isoflavones all have been proved useful in human health and some physiological functions. In these studies, we first analyzed and compared several domestic and commercial soybeans with their Bowman-Birk protease inhibitor (BBI) content using SDS-PAGE protein gel electrophoresis and western blot. Investigation of varieties, cultivation year, and plant area effect to the BBI content for soybeans were performed. Results indicated that the amount of BBI in soybean seed is determined by varieties and planting year. The manifestation difference (p<0.05) of BBI content in vegetable soybean Kaohsiung No. 5 that collected from Tainan and Kaohsiung was mainly due to their plant area. In fact cultivation year and plant area are obviously correlated and interacted to each others. In general, the BBI content in the black soybeans is higher than non-black soybeans, however, the amount of BBI in vegetable soybean Tainan selected No. 1 is comparable to the black soybeans. Key words: Bowman-Birk protease inhibitor (BBI), SDS-PAGE, western blotting, soybean 7

1 BBI 量 Table 1. Domestic and commercial soybeans used for BBI analysis Variety Number Crop Use Crop year Source Remarks Tainan selected #1 (TS85-21V) TNS1-2002 Green soybean 2002 TDARES* Brown seed Tainan selected #1 TN1-2003 Green soybean 2003 TDARES Brown seed Tainan #2 TN2-2002 Soybean 2002 AVRDC** Tainan #3 TN3-1998 Black soybean 1998 TDARES Tainan #3 TN3-1999 Black soybean 1999 TDARES Tainan #3 TN3-2000 Black soybean 2000 TDARES Tainan #3 TN3-2001 Black soybean 2001 TDARES Tainan #3 TN3-2002 Black soybean 2002 TDARES Tainan #3 TN3-2003 Black soybean 2003 TDARES Tainan #4 TN4-2002 Soybean 2002 TDARES Yellow seed coat Tainan #5 TN5-1998 Black soybean 1998 TDARES Tainan #5 TN5-2002 Black soybean 2002 TDARES Tainan #5 TN5-2003 Black soybean 2003 TDARES Tainan #6 TN6-2002 Soybean 2002 TDARES Black seed coat Tainan #6 TN6-2003 Soybean 2003 TDARES Black seed coat Kaohsiung #5 KS5-2002-TN Green soybean 2002 TDARES Kaohsiung #5 KS5-2002-KS Green soybean 2002 KSARES*** Kaohsiung #5 KS5-2003 Green soybean 2003 KSARES Kaohsiung #6 KS6-2002 Green soybean 2002 KSARES Kaohsiung #6 KS6-2003 Green soybean 2003 KSARES Kaohsiung #7 KS7-2002 Green soybean 2002 KSARES Black seed coat Kaohsiung #7 KS7-2003 Green soybean 2003 KSARES Black seed coat Kaohsiung selected #10 KS10-2002 Soybean 2002 AVRDC Five Leaf WYB Black soybean 2002 TDARES Taiwan native Heng Chun HCB Black soybean 2002 TDARES Taiwan native X Sun Foods RS Soybean Purchased in 2002 USA GMO X-Mei Foods YS Soybean Purchased in 2002 Australia Non GMO X Sun Foods RBS Black soybean Purchased in 2002 China Green seed coat X Hong ZBS Black soybean Purchased in 2002 South East Asian Green seed coat X Eagle MBS Black soybean Purchased in 2002 China Green seed coat XX Cruz Organic Foods SOBS Black soybean Purchased in 2003 USA Organic soybean Kaohsiung #8 KS8 Azuki bean 2002 KSARES Phaseolus vulgaris PV - 2003 Local market *TDARES: 良 ; **AVRDC: ; ***KSARES: 良 8

22002 2003 年 BBI 量 Table 2. Analysis of BBI content among domestic soybeans collected in 2002 and 2003 BBI 量 (µg/mg ) 2002 年 2003 年 1 TNS1 217.65 ± 7.12 251.32 ± 5.52 2 TN2 188.66 ± 8.88 ND 3 TN3 168.29 ± 7.99 246.92 ± 1.84 4 TN4 201.79 ± 6.17 202.94 ± 5.45 5 TN5 173.41 ± 6.58 244.06 ± 7.41 6 TN6 153.78 ± 3.60 b) ND 7 KS5-TN 183.58 ± 2.10 ND 8 KS5-KS 169.99 ± 3.32 170.08 ± 2.46 9 KS6 284.16 ± 8.05 175.87 ± 8.00 10 KS7 248.24 ± 6.41 178.22 ± 4.20 11 KS10 148.61 ± 7.03 ND 12 WYB ND 151.09 ± 5.90 13 HCB ND 246.53 ± 5.98 F values c) 115.64*** 156.01*** a) Data are means ± S.D. for 3 trials b) n=2 c) Levels of significance of ANOVA; ***, significant at 0.1 % 3 不年 BBI 量 Table 3. The content of BBI on black soybean variety of Tainan 3 in different crop years 年 BBI 量 ( 藍量 ) (µg/mg ) a) 1999 206.77 ± 9.94 2000 204.41 ± 8.46 TN3 2001 143.80 ± 5.19 2002 168.29 ± 7.99 2003 246.92 ± 1.84 F values b) 88.62*** a) Data are means ± S.D. for 3 trials b) Levels of significance of ANOVA; ***, significant at 0.1 % BBI (µg) BBI (µg) kda M 5 10 15 kda M 1 3 5 5 5 A 20.4 B C D 16.9 14.4 8.1 6.2 BBI 2.5 BBI 14.4 BBI BB 6.2 BBI BB 1 BBI SDS-PAGE 藍 9

Fig 2. SDS-PAGE protein gel electrophoresis and western analysis of soybean BBI A. 15% SDS-PAGE B. 20% SDS-PAGE C. 15%SDS-PAGE D. M = protein markera B BBI 量 C D BBI 量 3 µg 5 量 15 µg (µg/mg protein) 350 300 年年 250 度 200 150 100 50 0 TN1 TN3 TN5 TN6 KS5 KS6 KS7 22002 年 2003 年 BBI 量 Fig 2. Comparison of the BBI content among domestic soybeans in two crop years 10