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1 32 8 OOD & MACHINERY 食品与机械 Vol.32No.8 Aug.2016 DOI: /j.issn α- Synthesisandcharacterizationofbrewer sgrainspeptidewith α-glucosidaseinhibitoryactivity FANG Yu-wen WU Jin-hong HE Jia-jun CHEN Shan-shan WANG Zheng-wu MA Hong-cheng GAN Li SHI Hai-ming ( ) (Food Scienceand EngineeringSchoolof Agricultureand Biology ShanghaiJiaotong UniversityShanghai200240China) : Fmoc fluorescence quenching assays. Therefore a fast method of SS-5( :Ser-Pro-Asp-Arg-Ser) obtainingbrewer sgrainswithα-glucosidaseinhibitory (HPLC) 95% activityinquantitywasproposedinthispaper. SS-55α- Keywords:solid-phasesynthesisalpha-glucosidaseinhibitorsyn- SS-5 theticinteraction SS-5α- 1 [1] α- 2 SS-5 : α- SS-5 [2] Abstract:Brewer sgrains(sequence:ser-pro-asp-arg- Ser)wassynthesized bysolid-phasesynthesis with Fmocprotect group.thenthecrudesyntheticpeptidewaspurifiedanddesaltedby HPLCandasaresultthepuritywasover95%.Theinhibitoryac- tivityofsynthetictoα-glucosidase wasdetectedand theresultsshowedthatitwasinfluencedbysaltionicconcentration and the lower concentration presented higher inhibility. Furthermoretheinteractionbetweensyntheticandα- glucosidasewasconfirmedbycirculardichroismspectrum(cd)and [4] α- : 863 ( :2013AA102207) β ( : ) ( :2016YFD ) α- [5] : : [1] α- α- [3] : α- : (1961 ) α- zhengwuwang@sjtu.edu.cn (1978 ) wujinhong@sjtu.edu.cn (78% ) (14% ) 1

2 α SS-5 [6] [7] C ( )N ( ) [8] 9- YuZhi-peng [9] (Fmoc) Fmoc 10min [10]16-28 [15] α- C α- SS-5 : ( [11] 26.43% ) (Fmoc) α- (TFA) α % Fmoc NN - (DCCDicyclohexylcarbodimide) [12] [13] [14] Fmoc (HF) [10]43-44 α SS-5 SS-5 :Ser-Pro-Asp-Arg-Ser C18 Fmoc SS-5 α- (30mm 250mm) 50mg/mL α- 30mL/min 220nm :A 100% B100% :0~20 min14% A~ 39% A20.0~20.1 min39% ~80% A20.1~60.0 min 1 2% 20 min 1.1 (30%~80%) HPLC Kromasil α- (EC ):19.3 U/mg C18-5(4.6mm 250mm) 10μL 1.0mL/min Sigma-Aldrich -α-d - (pnpg): Sigma- Aldrich : 100% A25.1~30.0min100% A :883BasicICplus Metrohm :Vario MICRO cube Elementar :R-205 Buchi :FreeZone Labconco SS-5 α- :UV-2802S [17] α- pnpg :HH-S 405nm :BCD-189JB-S :MS3 α- (0.175U/mL) SS-5 ph :Delta320 METTLER-TOLEDO ( ) 30min 250 mmol/l Na2CO3 :ME104E METTLER-TOLEDO 405nm α- ( ) 2 :QM/TM/IM PTI R = :JASCOJ-815 SS-5 [16] 80% A 2% 220nm :A 0.01% B 0.01% : 0.01~25.00 min1% ~26% A25.0~25.1 min26% ~ SS-5 JY/T JY/T nm 37 ph 6.8 (50 mmol/l) 30 min pnpg (10 mmol/l) (1) : A -A A 100% (1)

32 8 : α- : R % A A 1.2.5 α- SS-5 200~250nm 1.4 U/mL α- 5 mg/ml SS-5 37 50nm/min 1s 1nm 3 2.5mmol/L PBS 1.2.6 SS-5α- 50mmol/L ph6.8 PBS 1.4mL0.5 U/mL α- 0.7 ml 1.7860.8930.4460.179 0.

3 32 8 : α- : R % A A α- SS-5 200~250nm 1.4 U/mL α- 5 mg/ml SS nm/min 1s 1nm 3 2.5mmol/L PBS SS-5α- 50mmol/L ph6.8 PBS 1.4mL0.5 U/mL α- 0.7 ml mmol/L V 5nm 240nm/min280nm 300~400nm SS-5 SS SS-5 1 Ser-Pro-Asp-Arg-Ser Figure1 Propertiesofsynthetic pi SS-5 80% SS min ( 2) %95% 3 SS SS-5 SS-5 Figure2 2.3 SS SS % N N N : = SS-5 C28H44N6O !a" #$%!b" & ( SS-5 Reversed-phasehigh-performanceliquidchroma- tographyofsynthetic = 80.84% 1 SS-5 Table1 Thesequenceandpropertyofsynthetic 5 SPDRS ph= % Ser-Pro-Asp-Arg-Ser 2 SS-5 Table2 RP-HPLCanalysisresultofsynthetic / / / min % (mv min) / mv

4 Figure3 3 SS-5 Massspectrumofsynthetic (CD) α- α- SS-5 CD 5 5 α- CD nm [17-18] α- SS-5 α nm CD ( ) α- α- SS-5α- 5 SS-5α- Table5 Theinhibitionactivityofsyntheticon α-glucosidase SS-5 / (mg ml -1 ) /% ± ± ± SS-5 Figure4 Ionchromatography(IC)ofsynthetic ± ± SS-5 Table3 ICanalysisofsynthetic / / / /min ( μ S cm -1 min) ( μ S cm -1 )(mg L -1 ) SS-5 Table4 Contentsofsaltandelementinsynthetic /% 2.4 SS-5α- SS-5α SS-5α- 5 SS-5 α- α- 334nm SS-5α- SS mg/ml SS-5 α- 8.43% 2.5 (CD) 4 5 a.α- + SS-5 b.α- SS-5α- Figure5 CDspectraofα-glucosidasewithorwithoutsyn- thetic 2.6 SS-5α- [19] 3 280nm 300~400nm [10]49 3 Fmoc SS-5 SS-5

5 32 8 : α- [12]. [J]. a~e mg/mL SS (4): SS-5α- [13]. I(cTnI) [J] (29): Figure6 InteractionbetweenSS-5 withvariousconcentra- scencespectra [J] (3): % [16]. [J]. SS-5α (6): mg/mL SS-5α- 8.43% [17]OHTA TSASAKISOOHORITetal.α-Glucosidaseinhib- itoryactivityofa70% methanolextractfromezoishige(pelve- SS-512.3% tiababingtonidetoni)anditsefectontheelevationofblood glucoselevelinrats[j].biosci.biotechnol.biochem (7): α- SS-5 [18]DUZhi-yunLIU Rong-rongSHAO Wei-yanetal.α-Gluco- α- sidaseinhibitionofnaturalcurcuminoidsandcurcuminanalogs [J].Eur.J.Med.Chem (2): ( ) [19]. tions and α-glucosidaseinvestigated by fluore- [1]GUNAWAN-PUTERIMKAWABATAJ.Novelalpha-glucosi- daseinhibitorsfrom Macarangatanariusleaves[J].FoodChemis- try (2): [2].2 LDL-C [D]. : 2013:1-2. [3]. α- [J] (9): [4].α [J] (9): [5]BHANDARI M RJONG-ANURAKKUN NGAO Hong KAWABATA J. Alpha- glucosidase and alpha-amylase inhibitory activities of Nepalese medicinal herb Pakhanbhed (Bergeniaciliata Haw.)[J].Food Chemistry (1): [6]WANG HuiDU Yang-jiSONG Hua-can.Alpha-Glucosidase andalpha-amylaseinhibitoryactivitiesofguavaleaves[j].food Chemistry (1):6-13. [7]KIMJSHYUN T KKIM MJ.Theinhibitoryefectsofetha- nolextractsfromsorghumfoxtailmiletandprosomiletonal- pha-glucosidase and alpha - amylase activities [J]. Food Chemistry (4): [8]CHENJingCHENG Yong-qiangYAMAKIKetal.Anti-al pha-glucosidase activity of Chinese traditionaly fermented soybean (douchi)[j].foodchemistry (4):1091- [9]YUZhi-pengYIN Yong-guangZHAO Wen-zhuetal.Novel peptidesderivedfromeggwhiteproteininhibitingalpha-glucosidase[j].foodchemistry (4): [10]. α- [D]. : [11]. [J] (19): [14]. PreS1 PreS1 [J] (5): [15].Fmoc(9- ) [J] (5): %~20% ( ) ( : 5

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