42 2014 4 FENXI HUAXUE Chinese Journal of Analytical Chemistry 4 552 ~ 558 DOI 10. 3724 /SP. J. 1096. 2014. 30594 α- 1 2 * 1 1 * 1 1 130022 2 100039 LC-ESI-MS n α- 4- -α-d- PNPG α- 0. 027 0. 050 g /L α- LC-ESI-MS n 12 7 16 7 5 4 α- α- 1 1-3 I II II 90% α- II 4 5 α- 6-7 8 α- 9 α- 10 - LC-ESI-MS n α- 2 α- α- LC-MS n 11 12 16 α- 2 2. 1 Waters H-class Waters Finnigan LTQ Thermo Fisher Tecan CENios TECAN ALPHA 2-4 Christ 2013-06-24 2013-12-19 Nos. 81373952 81073040 21005075 No. 2012IM030600 * E-mail mslab20@ ciac. jl. cn liuzq@ ciac. ac. cn
4 α- 553 Centrifuge 5810R Eppendorf YM-1000 Millipore α- EC 3. 2. 1. 18 Sigma 4--α-D- 4-Nortiphenyl-α-Dgluocpynoaside PNPG EC 223. 189. 3 Sigma Acarbose Bayer Fluck Fisher Milli-Q 2. 2 1 g 10 ml - 70 30 V /V 10 min 4000 g 10 min 100 g /L 0. 2 mol /L ph = 6. 8 10 g /L α- 0. 2 mol /L ph = 6. 8 0. 04 U /ml α- 10 mmol /L ph = 6. 8 20 μmol /L 0. 45 μm PNPG 0. 2 mol /L ph = 6. 8 0. 5 mmol /L 2. 3 2. 3. 1 Agilent SB-C 18 150 mm 4. 6 mm 5 μm 30 0. 5 ml /min 10 μl 1 A B 0. 5% 0 ~ 10 min 20% ~ 40% A 10 ~ 30min 40% ~ 100% A 30 ~ 35min 100% A 2 A B 0. 5% 0 ~ 35 min 5% ~ 15% A 2. 3. 2 Finnigan LTQ m /z 100-1000 4. 0 kv N 2 50 arb 5 arb 250 2. 4 2. 4. 1 α- PNPG 96 α- 10 α- 0. 04 U /ml 40 μl 37 5 min PNPG 0. 5 mmol /L 20 μl 37 30 min Na 2 CO 3 0. 1 mol /L 100 μl 200 μl 405 nm 16 Inhibition ratio IR IR % = A blank - A 0 - A /A Blank 100% 1 A blank A 0 A 2. 4. 2 10 g /L 100 μl 100 μl 20 μmol /L α- 37 30 min 100 μl 10 kda YM-10 10 mmol /L ph = 6. 8 100 μl 10000 g 10 min 3-50 50 V /V 150 μl 13000 g 15 min 3 40 μl - 50 50 V /V LC-ESI-MS n 3 3. 1 α- α- IC 50 IC 50 0. 027 0. 050 g /L IC 50 0. 12 g α-
554 42 3. 2 - - LC-ESI-MS n 10 α- α- 12 α- ESI-MS n 1 1 α- 1a 1b 1 α- Table 1 LC-MS n data in negative ion mode of α-glucosidase inhibitors from polygoni cuspidati rhizoma et radix extract Retention UV λ Peak max time No. nm min Formula M - H - MS m /z 2 MS 3 Compounds 1 13. 9 303 C 20 H 22 O 8 389 389 227 100 227 185 100-4'-O- Resveratrol-4'-O-glucoside 2 14. 7 278 C 22 H 18 O 10 441 441 289 100 245 205 179 165 289 245 100 205 179 165-3-O- Catechin-3-O-galloyl 3 15. 6 312 C 20 H 22 O 8 389 389 227 100 227 185 100 Polydatin 4 16. 8 213 302 C 27 H 26 O 12 541 541 313 100 227 313 169 100 125 169-3 5 4'- - 4'-O-β-D- 2 -O- Trans-3 5 4'-trihydroxystilbene-4'-O-β-D- 2 -Ogalloyl gulcopyranoside 5 17. 1 213 302 C 27 H 26 O 12 541 541 313 100 227 169 313 169 100 125-3 5 4'- - 4'-O-β-D- 6 -O- Trans-3 5 4'-trihydroxystilbene-4'-O-β-D- 6 -Ogalloyl gulcopyranoside 6 18. 9 216 305 C 14 H 12 O 3 227 227 185 100 143 185 143 100 Resveratrol 7 21. 6 283 C 21 H 20 O 10 431 431 269 100 241 225 269 241 225 100-8-O- Emodin-8-O-glucoside 8 22. 2 236 264 C 19 H 22 O 9 407 407 245 100 245 230 100 9 24. 5 275 C 21 H 20 O 10 431 431 269 100 241 225 269 241 225 100-8-O- Torachryson-8-O-glucoside -1-O- Emodin-1-O-glucoside 10 26. 2 225 270 C 22 H 22 O 10 445 11 26. 8 222 270 C 24 H 24 O 11 487 445 283 100 268 240 487 283 100 268 240 12 31. 7 221 265 C 15 H 10 O 5 269 269 241 225 100 283 268 240 100 283 268 240 100-8-O- Physcion-8-O-glucoside -8-O- 6'- Physcion- 8-O- 6'-acetyl glucoside 225 210 100 182 Emodin 6 m /z 227 C 2 H 2 O m /z 185 C 2 H 2 O m /z 143 6 1 3 m /z 449 m /z 389 CH 3 COOH 1 3 M - H - m /z 389 m /z 389 C 6 H 10 O 5 m /z 227 1 3 m /z 227 1 3 13 1-4'-O- 3
4 α- 555 1 Fig. 1 Liquid chromatograms of polygoni cuspidati rhizoma et radix extract with active a and inactivation b α-glucosidase after performing ultrafiltration 1 Peak numbers are the same as in Table 1 2 m /z 441 m /z 289 m /z 289 m /z 289 CO 2 15 C 2 H 2 O 2 C 6 H 6 O 2 C 7 H 8 O 2 m /z 245 179 165 18 2 4 5 m /z 541 m /z 313 4 5 m /z 313 m /z 169 m /z 169 CO 2 m /z 125 m /z 313 O-β-D- 2 -O- 5-3 5 4'- -4'-O-β-D- 6 -O- 2 4 3 2-3 5 4'- -4'-O-β-D- 2 -O- Fig. 2 Negative ESI-MS 2 and ESI-MS 3 spectra for trans-3 5 4'-trihydroxystilbene-4'-O-β-D- 2 -O-galloyl gulcopyranosid 12 m /z 269 CO CO 2 m /z 241 225 m /z 225 CH 3 m /z 210 m /z 210 CO m /z 182 12 7 9 m /z 431 m /z 269 m /z 269 7 9 8 15 17 4-3 5 4'- -4'- 13 15 7-8-O- 9-1-O- 8 m /z 407 m /z 245 m /z 245 CH 3 m /z 230
556 42 3 Fig. 3-3 5 4'- -4'-O-β-D- 2 -O- Fragmentation pathways of trans-3 5 4'-trihydroxystilbene -4'-O-β-D- 2 -O-galloyl gulcopyranoside proposed according to its MS n data 13 15 8-8-O- 10 m /z 445 m /z 283 m /z 283 CH 3 m /z 268 m /z 268 3 CO m /z 240 212 184 m /z 283 13 15 10-8-O- 11 m /z 487 m /z 283 m /z 13 14 11-8-O- 6'- 3. 3 4 α- 4a 4b 4 7 α- 14-16 18 7 2 12 7 α- 13-18 4 Fig. 4 Liquid chromatograms of Rhei radix et rhizoma extract with active a and inactivation α-glucosidase b after performing ultrafiltration 2 Peak numbers are the same as in Table 2
4 α- 557 2 α- Table 2 LC /MS n data in negative ion mode of α-glucosidase from Rhei radix et rhizoma extract Retention UV λ Peak max time No. nm min Formula 1 11. 1 210 275 C 15 H 14 O 6 289 M - H - m /z MS 2 MS 3 Compounds 289 245 100 245 227 203 100 205 179 165 187 161 Catechin 2 13. 6 215 305 C 14 H 12 O 3 227 227 185 100 143 185 143 100 Resveratrol 3 18. 9 225 270 C 22 H 22 O 10 431 445 283 100 283 268 240 100 268 240-8-O- Physcion-8-O-glucoside 4 20. 5 216 275 C 23 H 26 H 11 477 477 313 100 169 313 169 100 125 Lindleyin 5 22. 2 220 260 C 15 H 10 O 5 269 269 240 100 240 211 100 Aloe-emodin 6 30. 0 223 265 C 16 H 12 O 5 283 283 268 240 100 268 240 100 240 212 100 184 Physcion 7 31. 6 221 265 C 15 H 10 O 5 269 269 241 225 100 225 210 100 182 Emodin 16 References 1 Holman R R Paul S K Bethel M A Matthews D R Neil H A W.New England J. Medicine 2008 359 15 1577-1589 2 Gaede P Lund-Andersen H Parving H H Pedersen O. New England J. Medicine 2008 358 6 580-591 3 GU Jin-Ning NIU Jiu PI Zi-Feng YUE Hao WU Sui-Sheng LIU Shu-Ying. Chinese J. Anal. Chem. 2013 41 3 371-376. 2013 41 3 371-376 4 Nathan D M Buse J B Davidson M B Ferrannini E Holman R R Sherwin R Zinman B. Diabetes Care 2009 32 1 192-203 5 Matsui T Tanaka T Tamura S Toshima A Tamaya K Miyata Y Tanaka K Matsumoto K. J. Agric. Food Chem. 2007 55 1 99-105 6 FENG Cheng-Gen CHEN Lin LIU Xia. Chinese J. New Drugs. 2005 14 6 669-672. 2005 14 6 669-672 7 de Melo E B Gomes A D Carvalho I. Tetrahedron 2006 62 44 10277-10302 8 GAO Xiao-Ping ZHANG Wei-Yu ZOU Wen-Jun SU Zhi JI Yuan-Qiao LIU Zhong-Rong. Natural Product Research and Development 2003 15 6 536-538. 2003 15 6 536-538 9 SHEN Zhong-Ming LI Ying JIANG Hong YIN Jian-Wei XU Zhao-Long. Chinese J. Biochemical Pharmaceutics 2000 21 2 69-70. 2000 21 2 69-70 10 Zhou H Li H L Zheng Z Song F R Xing J P Liu Z Q Liu S Y. J. Liquid Chromatogr. Relate Technol. 2012 35 1-4 1-14 11 XIE Yu-Ping TIAN Jing GAO Peng XU Guo-Wang FEI Xu WANG Yi. Chinese J. Anal. Chem. 2013 41 1 36-41. 2013 41 1 36-41 12 PENG Si-Yuan ZHANG Jie TIAN Mei-Ping WANG Zhan-Lin SHEN He-Qing.Chinese J. Anal. Chem. 2012 40 9 1588-1592. 2012 40 9 1588-1592 13 DONG Jin WANG Hong WAN Le-Ren DUAN Yu-Shu CHEN Shi-Zhong. Chinese Journal of Chromatography 2009 27 4 425-430. 2009 27 4 425-430
558 42 14 Liang Z T Sham T Yang G Y Yi L Chen H B Zhao Z Z. Anal. Bio. Chem. 2013 405 12 4199-4212 15 Wei J Ying F W Ge R L Shi H M Jia C Q Tu P F. Rapid Commun. Mass Spectrom. 2007 21 14 2351-2360 16 Li G B Chen X G Liu M C Hu Z D. Analyst 1998 123 7 1501-1505 17 Wei J Ge R L Wei Q J Bao T Y Shi H M Tu P F. J. Chromatogr. A 2006 1132 1-2 320-324 18 LIU Guo-Qiang DONG Jing WANG Hong WAN Le-Ren DUAN Yu-Shu CHEN Shi-Zhong. Chem. J. Chinese Universities 2009 30 8 1566-1570. 2009 30 8 1566-1570 Screening of α-glucosidase Inhibitors from Polygoni Cuspidati Rhizoma et Radix and Rhei Radix et Rhizoma Using Ultrafiltration LC-ESI-MS n YANG Dong 1 2 LIU Shu * 1 SONG Feng-Rui 1 LIU Zhi-Qiang * 1 1 Changchun Center of Mass Spectrometry Changchun Institute of Applied Chemistry Chinese Academy of Sciences Changchun 130022 China 2 University of Chinese Academy of Sciences Beijing 100039 China Abstract Centrifugal ultrafiltration vitro α-glucosidase inhibition assay and liquid chromatography coupled to electrospray ionization tandem mass spectrometry LC-ESI-MSn were combined to screen α-glucosidase inhibitors from two kinds of Chinese herb medicine Polygoni cuspidati rhizoma et radix and Rhei radix et rhizoma which are rich in anthraquinones. First a vitro α-glucosidase inhibition assay was carried on based on 4-nitrophenyl-α-d-glucopyranoside PNPG as substrate. As a result Polygoni cuspidati rhizoma et radix and Rhei radix et rhizoma extracts showed 50% α-glucosidase inhibition at the concentration of 0. 027 g / L and 0. 050 g /L respectively and the α-glucosidase inhibitory activities of them were stronger than those of control drug acarbose. Then the centrifugal ultrafiltration LC-ESI-MSn was used to screen and identify α-glucosidase inhibitors from Chinese herb medicine extracts. As a result 12 and 7 kinds of samll-molecule active compounds were successfully identified as α-glucosidase inhibitors from Polygoni cuspidati rhizoma et radix and Rhei radix et rhizoma respectively totally 16 kinds of active compounds. Seven kinds of the compounds belonged to anthraquinones five compounds belong to polyphenols and four compounds had a galloxyl group. The results showed that anthraquinones had good α-glucosidase inhibition activity which laid the foundation for the further application. Keywords α-glucosidase inhibitor Ultrafiltration mass spectrometry Polygonum cuspidatum Rheum Inhibitors Received 24 June 2013 accepted 19 December 2013 This work was supported by the Innovation Method Fund of China No. 2012IM030600 the National Natural Science Foundation of China Nos. 81373952 81073040 21005075