32 8 OOD & MACHINERY 2016 8 食品与机械 Vol.32No.8 Aug.2016 DOI:10.13652/j.issn.1003-5788.2016.08.001 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 ( 200240) (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 ( :3147162321276154) ( :2016YFD0400206) α- [5] : :2016-05-09 [1] α- α- [3] : α- : (1961 ) α- E-mail:zhengwuwang@sjtu.edu.cn (1978 ) E-mail:wujinhong@sjtu.edu.cn (78% ) (14% ) 1
2016 8 1.2 α- 1.2.1 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- 11.52% Fmoc NN - (DCCDicyclohexylcarbodimide) [12] [13] [14] Fmoc (HF) [10]43-44 α-1.2.2 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%) 1.1.1 HPLC Kromasil α- (EC3.2.1.20):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 1.1.2 :883BasicICplus Metrohm :Vario MICRO cube Elementar :R-205 Buchi :FreeZone Labconco 1.2.4 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% ~ 1.2.3 SS-5 JY/T020 1996 JY/T017 1996 405nm 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 0.089mmol/L 37 400 V 5nm 240nm/min280nm 300~400nm 2 2.1 SS-5 SS-5 1 1 SS-5 1 Ser-Pro-Asp-Arg-Ser Figure1 Propertiesofsynthetic 560.56 pi7.4 1.3 SS-5 80% SS-5 2.2 10.313min ( 2) 1 98.4%95% 3 SS-5 561.58 560.58 SS-5 SS-5 Figure2 2.3 SS-5 4 3 SS-5 12.3% N N N :14 6 560.70=0.1498 SS-5 C28H44N6O6 560.70 2!a" #$%!b" & ( SS-5 Reversed-phasehigh-performanceliquidchroma- tographyofsynthetic 0.1211 0.1498= 80.84% 1 SS-5 Table1 Thesequenceandpropertyofsynthetic 5 SPDRS 560.56 7.4 ph=7 0.0 1.3 80% Ser-Pro-Asp-Arg-Ser 2 SS-5 Table2 RP-HPLCanalysisresultofsynthetic / / / min % (mv min) / mv 1 9.550 0.2647 13658 1991 2 9.993 0.5679 29306 4766 3 10.087 0.7867 40594 10813 4 10.313 98.3807 5076760 442225 100.0000 5160318 459795 3
2016 8 Figure3 3 SS-5 Massspectrumofsynthetic (CD) α- α- SS-5 CD 5 5 α- CD 209222nm [17-18] α- SS-5 α- 209222nm CD ( ) α- α- SS-5α- 5 SS-5α- Table5 Theinhibitionactivityofsyntheticon α-glucosidase SS-5 / (mg ml -1 ) /% 5.0 3.921±0.049 0 2.5 2.887±0.008 0 1.0 2.692±0.012 3.27 4 SS-5 Figure4 Ionchromatography(IC)ofsynthetic 0.5 2.554±0.063 8.21 2.783±0.021 3 SS-5 Table3 ICanalysisofsynthetic / / / /min ( μ S cm -1 min) ( μ S cm -1 )(mg L -1 ) 1 3.827 0.2327 1.007 12.092 2 7.425 0.1028 0.267 2.373 4 SS-5 Table4 Contentsofsaltandelementinsynthetic 10.309 2.023 12.11 53.48 7.709 /% 2.4 SS-5α- SS-5α- 6 5 6 SS-5α- 5 SS-5 α- α- 334nm SS-5α- SS-5 0.5 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
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