FENXI HUAXUE Chinese Journal of Analytical Chemistry. LC-MS n Thermo Christ

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1 FENXI HUAXUE Chinese Journal of Analytical Chemistry ~ 558 DOI /SP. J α- 1 2 * 1 1 * LC-ESI-MS n α- 4- -α-d- PNPG α g /L α- LC-ESI-MS n α- α I II II 90% α- II 4 5 α α- 9 α LC-ESI-MS n α- 2 α- α- LC-MS n α Waters H-class Waters Finnigan LTQ Thermo Fisher Tecan CENios TECAN ALPHA 2-4 Christ Nos No. 2012IM * mslab20@ ciac. jl. cn liuzq@ ciac. ac. cn

2 4 α- 553 Centrifuge 5810R Eppendorf YM-1000 Millipore α- EC Sigma 4--α-D- 4-Nortiphenyl-α-Dgluocpynoaside PNPG EC Sigma Acarbose Bayer Fluck Fisher Milli-Q g 10 ml V /V 10 min 4000 g 10 min 100 g /L 0. 2 mol /L ph = g /L α mol /L ph = U /ml α- 10 mmol /L ph = μmol /L μm PNPG 0. 2 mol /L ph = mmol /L Agilent SB-C mm 4. 6 mm 5 μm 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 Finnigan LTQ m /z kv N 2 50 arb 5 arb α- PNPG 96 α- 10 α U /ml 40 μl 37 5 min PNPG 0. 5 mmol /L 20 μl min Na 2 CO 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 g /L 100 μl 100 μl 20 μmol /L α min 100 μl 10 kda YM mmol /L ph = μl g 10 min V /V 150 μl g 15 min 3 40 μl V /V LC-ESI-MS n α- α- IC 50 IC g /L IC g α-

3 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 C 20 H 22 O '-O- Resveratrol-4'-O-glucoside C 22 H 18 O O- Catechin-3-O-galloyl C 20 H 22 O Polydatin C 27 H 26 O '- - 4'-O-β-D- 2 -O- Trans-3 5 4'-trihydroxystilbene-4'-O-β-D- 2 -Ogalloyl gulcopyranoside C 27 H 26 O '- - 4'-O-β-D- 6 -O- Trans-3 5 4'-trihydroxystilbene-4'-O-β-D- 6 -Ogalloyl gulcopyranoside C 14 H 12 O Resveratrol C 21 H 20 O O- Emodin-8-O-glucoside C 19 H 22 O C 21 H 20 O O- Torachryson-8-O-glucoside -1-O- Emodin-1-O-glucoside C 22 H 22 O C 24 H 24 O C 15 H 10 O O- Physcion-8-O-glucoside -8-O- 6'- Physcion- 8-O- 6'-acetyl glucoside Emodin 6 m /z 227 C 2 H 2 O m /z 185 C 2 H 2 O m /z 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 m /z '-O- 3

4 4 α 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 m /z 541 m /z m /z 313 m /z 169 m /z 169 CO 2 m /z 125 m /z 313 O-β-D- 2 -O '- -4'-O-β-D- 6 -O '- -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 m /z 225 CH 3 m /z 210 m /z 210 CO m /z m /z 431 m /z 269 m /z '- -4' O- 9-1-O- 8 m /z 407 m /z 245 m /z 245 CH 3 m /z 230

5 Fig '- -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 O- 10 m /z 445 m /z 283 m /z 283 CH 3 m /z 268 m /z CO m /z m /z O- 11 m /z 487 m /z 283 m /z O- 6' α- 4a 4b 4 7 α α 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

6 4 α 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 C 15 H 14 O M - H - m /z MS 2 MS 3 Compounds Catechin C 14 H 12 O Resveratrol C 22 H 22 O O- Physcion-8-O-glucoside C 23 H 26 H Lindleyin C 15 H 10 O Aloe-emodin C 16 H 12 O Physcion C 15 H 10 O Emodin 16 References 1 Holman R R Paul S K Bethel M A Matthews D R Neil H A W.New England J. Medicine Gaede P Lund-Andersen H Parving H H Pedersen O. New England J. Medicine GU Jin-Ning NIU Jiu PI Zi-Feng YUE Hao WU Sui-Sheng LIU Shu-Ying. Chinese J. Anal. Chem Nathan D M Buse J B Davidson M B Ferrannini E Holman R R Sherwin R Zinman B. Diabetes Care Matsui T Tanaka T Tamura S Toshima A Tamaya K Miyata Y Tanaka K Matsumoto K. J. Agric. Food Chem FENG Cheng-Gen CHEN Lin LIU Xia. Chinese J. New Drugs de Melo E B Gomes A D Carvalho I. Tetrahedron GAO Xiao-Ping ZHANG Wei-Yu ZOU Wen-Jun SU Zhi JI Yuan-Qiao LIU Zhong-Rong. Natural Product Research and Development SHEN Zhong-Ming LI Ying JIANG Hong YIN Jian-Wei XU Zhao-Long. Chinese J. Biochemical Pharmaceutics Zhou H Li H L Zheng Z Song F R Xing J P Liu Z Q Liu S Y. J. Liquid Chromatogr. Relate Technol XIE Yu-Ping TIAN Jing GAO Peng XU Guo-Wang FEI Xu WANG Yi. Chinese J. Anal. Chem PENG Si-Yuan ZHANG Jie TIAN Mei-Ping WANG Zhan-Lin SHEN He-Qing.Chinese J. Anal. Chem DONG Jin WANG Hong WAN Le-Ren DUAN Yu-Shu CHEN Shi-Zhong. Chinese Journal of Chromatography

7 Liang Z T Sham T Yang G Y Yi L Chen H B Zhao Z Z. Anal. Bio. Chem Wei J Ying F W Ge R L Shi H M Jia C Q Tu P F. Rapid Commun. Mass Spectrom Li G B Chen X G Liu M C Hu Z D. Analyst Wei J Ge R L Wei Q J Bao T Y Shi H M Tu P F. J. Chromatogr. A LIU Guo-Qiang DONG Jing WANG Hong WAN Le-Ren DUAN Yu-Shu CHEN Shi-Zhong. Chem. J. Chinese Universities 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 China 2 University of Chinese Academy of Sciences Beijing 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 g / L and 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. 2012IM the National Natural Science Foundation of China Nos

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