256 2O12 99%)was from Acros Organics lnc., USA:standard mOnOsaccha rides in- jng,pOlySacCha rjdes and proteins are tents of the purified Opuntia milpa alta components were measured by modi— lfed pheno1.sulfuric acid method[1a,glu. cose was set as the standard sample. Analysis on the mOnosaccharide compositions and alditol acetate derivatives Amino acid derivative of biological macromolecules with differ— ent quantity of electric charges.and cluding mannose (Man),galactose (Ga1),rhamnose sugar(Rham),arabi— nose(Ara),xylose(Xy1),glucose(GIc), glucuronic acid(GIcUA),galacturonic acid(GalUA),fibrinogen were alI pur- chased fr0m Sigma;thrombin of ln— strumentation Laboratory Inc.,U.S.: ion exchange chromatography can separate and purify different biological macromolecules with diferent charges based on ion exchangers,which have diferent binding forces to a variety of ions or ionic compounds. high-performance liquid chromatogra. phy was used to measure the monosa- de compositions f 13].chromato. Accurate 500ma ofOpun妇milpa cchariacetonitrile(chromatographic grade); ta CP were taken to dissolve in graphic conditions:ZORBAX Eclipse TC.TG assay kit of Beijing Zhong- al 0 000 r/min centrifuged for 2O sheng Biological Engineering High— ddH2O,1tech Company;HDL-C assay kit of Wenzhou Eastern Biotechnology min to remove fnsolubles.and then the solution was gone through DEAE— XDB-C18 column(250 mmx4.6 mm,5 um):mobile phase was 0.1 mol/L phosphate(pH 6.7)buffer/acetOnitnIe Company;APTT,PT,TT assay kit of Beijing Bio—lab Materials Institute. Instruments and equipments 721 spectrophotometer was purchased frOm Shanghai Precision Scientific In- strumenl Co..Ltd.:TDL240B high- speed centrifuge of Xiangyi Centrifuge lnstrument Co..Ltd.:Waters 600 HPLC Of Waters Corporation.USA; SPECTRAmax PLUS384 Microplate Reader of Molecular Device Company, U.S.:Coatron M1 single pass coagu- Iometer of Germany TECO Company; GC-MS analyzer(Finnigan Trace MS) frOm Finnigan Corporation.U.S. Methods Preparation of OpunUa milpa alta pOIVsaccharides Fresh Opunf『a milpa alta was washed by water after removing the stick and peeling,cut into 2 mm filaments.immersed in 80% ethanoJ and extracted for three times. for removing insoluble substances. Residue was dried to evaporate the ethanol,ground and screened through a 200-mesh sieve.obtaining Opun“a milpa alta powder.A certain amount of Opun妇milpa alta powder was used for the second extration under diferent ext.raction conditions.The aqueous extracts were combined with water ex- tract.then subjected to reduced pres— sure condense,removed protein by Sevag method.Next four times of the 95%ethanol was added to the solu・ tion.resulting in a Iarge number of white precipitate,centrifuged to collect precipitate.The precipitate was eluted by absolute ethanol,acetone,and an- hydrous ethyl for twice,respectively, the crude polysaccharide (CP)was obtained by vacuum drying(Fig.1). Grading purification and purlty 、dentification of Opuntia mIIpa alta PoIVsaccharides Generally speak- Agricultural Science&Technology Sepharose CL-6B column(926 mmx30 (83:1 7,V/ :column temperature of 3O cm),washed by 0.05 mol/L phosphate ℃:detection wavelength of 250 nm: buf-fer(pH 5.9),then added 0—1。8 mol/L flow rate was 1 ml/min:injection vo1. NaCI for gradient elution.collected by ume of20 u1. step collector(6 mI/min,1 0 ml/tube): AIditol acetate derivatives temper— detecting the p0lysaccha ride content ament analysis【14].GC:OV1701 cap— tube by tube,the single peak compo— iIlary column ( 0_25 mm×30 m,州m nents were collected separately,dialy・ thickness of 0_25 mm1.temperature sis desalted,freeze-dried. programming:initiaI temperature of 1 5 High Performance GeI Filtration ℃increased to 250℃with 3℃/m_n_ Chromatography(HPGPC)was used remain 1 OmIn.MS:EI source.70 ev. for the purity identification[11].Impure mass spectra scanning of 30——650 components were gone through the amu/sec,interface temperature of 250 Sepharose CL-6B column(中1 6 mm× oC.Ion source temperature of 200℃。 1 0O cm1 again with 0。2 mol/L NaCI for multiplier of voltage 350V,helium. elution,collected by step collector(40 In vitro anticOaguIant activity de- ml/h,1 0 ml/tube);detecting polyphenol tection of polVsaCCharides Antico— content,single peak components were agulation components and fibrinolytic collected separately,dialysis desalted, system in the blood can controli/ n vivo freeze—dried.Then the purity and coagulation balance by inhibiting the weight-average molecular weight was activity of clotting component 一s or hy. measured by HPGPC. drolyzing fibrin,maintaining blood vis Measurement of the pOIySacCha ride cosity,and preventing the organism contents The pOlySaCCha ride con- frOm excessive blood clotting.such as lee Fl【ter I'CS idLIc Remove the peeL,cut into fi[amsD.ts(2ⅡⅡI1),wei t Exttaction Fitter Opuntia milpa a』ta / Rm ̄we the Lipid,dry and grind(60 meshes) 妲00 “ 。e e卜\ F l1L r rl e uce pressco ̄xtense el e 圳l H ’I] (Add 4 t iⅡBs the VO[LUm of 95%ethano[.iaSt 48 h throw away the ftoatirg substantes) Eiut ion for twice by absolute ethanol,acetOl-le, absotute ethyl ether.respect ive[y c 。℃,l vacu m 一r i Crude poiysacchar[de of Opuntia m lpa i』ia Fig.1 Preparation scheme of crude polysaccharides from Opuntia milpa alta Vo1.1 3,No.2,201 2 2012 257 stroke.myocardiaI jnfarction.Activa- tion of clotting components is the trig- ghted.fully dis- time(APTT)determination:take 50 uI WSP2a-pH was weiof blood,37 oC pre heat for 3 minthen 。solved in DMSO,added NaOH dry powder and methyIjodide respectively ger mechanism leading to blood co- agulation.According to diferent trigger mechanisms,the coagulation process is divided jnto the intrinsic coagulation add 50 ul Of PT reagent which has been preheated in 37℃.37 oC pre- to conduct methylation reaction,the re— heat for 5 min,then add 50 uI Of 37℃ action liquid was extracted by dichlo- umn, pre-heated 0.025 mol/L CaCl2 solution, romethane,through Na2SO4 colrecording the measured results. Determination of the inhibition ratio collecting the filtrate,dried by N2;O.5 mI of acetic anhydride was added and derived into Partially Methylated AIditol pathway(intrinsic pathway),extrinsic coagulation pathway(extrinsic path- way(common pathway).ATPP,PT and TT are three indicators to deter- mine the coagulation pathway in medi- caI science:the determination of ATPP nogen-thrombin conversion way)and common coagulation path- of fibriFibrinogen。thrombin and polysac- charide samples were accurately measured and dissolved in pH 7.2 Acetate(PMAA)at 1 O0℃. Resu lts and Analysis buffer (0.O5 mol/L Tris-HCI,0.1 2 The yield rate of crude polysaccha- is the screening test of overalI blood coagulation status of all the blood co- agulation components in the jntrinsic coagulation system;the determination of PT is the screening test of overall blood coagulation status of alI the blood coagulation components jn ex- trinsic coagulation system:while de— termination of TT is the screen siz- ing test of the common coagulation pathway. The preparation of platelet poor plasma(PPP)and platelet rich plasma 《PRP1:rabbits which did not take as— pirin and any drugs of platelet inhibition in two weeks were selected to take venous blood;the blood was mixed with 3.8%(m/v)sodium citrate by vol- ume ratio of 1:9 to make it anticoagu- lated.centrifuged in 3 000 r/min for 1 5 arin and 1 000 r/min for 10 min。resp- ectively,the supernatants were taken and packed to the plastic centrifuge tube,preserved in refrigerator,obtain- ing PPP and PRP.37℃preheated for 3 min when used。the corresponding jndexes were measured quickly ̄’同. The Opuntia milpa alta polysac- charide samples were taken and dis- solved in saline to measure the antico- agulation activity/n vitro.The samples were fully mixed with the PPP of 1:4 volume ratios. Saline with same amount of the potysaccharide sample was set as the contro1. Thrombin time( determination: take50 ofblood,37℃pre—heatf0r3 min,and then add 50 Ul of TT reagent which has been preheated in 37℃. recording the measured results. Prothrombin time(PT)determina- tion:take 50 uI of blood,37 oC pre-heat for 3 min。and then add 50 ul of PT reagent which has been preheated in 37℃.recording the measured results. Activated partial thromboplastin mmol/L NaCl1 to make the mass frac— ridesfrom Opuntiamilpaalta tion of fibrinogen 1 g/L,pOlysaccharide The obtained Opuntia milpa alta 1 g/L,thrombjn 1.25口/L.140 uI of fib— powder was condensed vacuum dried, rinogen,40 UI of pOIysaccha ride,5 I Of obtaining Opuntia milpa alta CP.The thrombin were added to the holes of yield rate was 0.655%(wet basis). 96-well micro-plate,respectively,mixed, Purified poIysaCcha ride compo・ 37 oC preheated for 2 min,measuring nents and analysis on the monosa- absOrbance at 405 nm.A blank contro1 ccharide composition was set up for each welI using buffer Three water_s0lubIe polysaccha- instead of thrombin.Value was taken rides(WSP)were obtained affer the after the system absorbance became Opun妇milpa alta CP was purified by stable(1 0 min),the inhibition ratio of DEAE Sepharose CL-6B. namely the sample to fibrinogen conversion was determined by the fol ̄owing WSP1,WSP2,WSP3(Fig.2A).Treat- ed with dialysis,freeze-dried,weighed, formula[1 ̄: the calculated amount of the three ac- y/%=( r J/( r Ao)xl 00 countedfor11.59%.45.21%and20.48% Here,A。is the system ab- of totaI CP,respectively,total recovery sorbance without inhibitor;A is system was 77.3%.indicating that there were absorbance after adding polysaccha- at Ieast three kinds of p0lysaccharides rides;Ao is the system absorbance in Opunf『amilpaa, 伺. with buffer(pH=7.2). WSP1 was obtained by direct Mapping was done by using the elution using 0.05 mol/L phosphate time as the abscissa,the calculated In- buffer(pH 5.9),therefore,the compo- hibition ratio as vertical COOrdinate. nent of this group was neutraI polysac- WSP2a partial acid hydrolysis, charide【’6l:DEAE—Sepharose CL・6B methylation and GC-MS analysis system exchanged resins at stable, According to the method of Yuan-lin weak anion agar sugar ion at pH 2-1 2, SUN[14],20 mg of WSP2a was taken and with the size exclusion effect.WSP2 put in the stopper.dissolved in 4 mI of and WSP3 were obtained by the gra- 0.2 mol/L TFA solution.hydrolyzed at dient elution of 0-1.8 mol/L NaCI,sug- 10O℃for 1 h.Hydrolyzates were gesting the polysaccharide sizes and dialysed with water(molecular weight charges vary between WSP2,WSP3. cutoff 3 50o).obtaining Permeate liq- MOlecuIar sieve p rinCiple waS廿1e uid with low motecutar weight and re- basjs fOr HPGPC meagurement tentate fraction with high molecular weight—average m0IecuIar wejght Of weight.After blowing with N2 to re— p0Iysaccharide 0r sugar cOmpOund. move the excess TFA,the high The weight-aVerage m0lecular weight molecular weight retention fraction (Mw)of poIysaccharide or sugar com— was re-dissolved in water,freeze- pound and the eIuti0n voIume on dried。obtaining WSP2a partial acid the gel cOlumn and distnbutlOn cOeffI- hydrolysis products(WSP2a・pH):then cient K exisled the relatiOn§hip 0f = 5 mg of WSP2a-pH was dissolved in + InMlw= 一8 ( , is the D2o,added CMC to fully dissolve,ad— v0IUme 0f mObile phaSe in particIeS justing pH to 4.75,added NaBD4 solu- and Out Of particIes,respectiveIy, ,8 tion to conduct carboxyI reduction, Is cOnstant1.The sIandard cun,e relat- dned by N2;3 mg of carboxyI reduced ed with relative m0IecuIar weight, V0I.1 3,NO.2,201 2 AgncuIturaI Science&TechnOIogy 258 2.5 2012 2.O 吕。 1.5。u皇10sq《 墨 窨 墨 1.0 5 0 一晷 § 基 The number of tubes The number of tubes A:Elution curve of CP on DEAE Sepharose CL一6B;B:Elution curve ofiNSP2 on Sepharose CL・6B Fig.2 Elution profiles of CP and WSP2 on DEAE-Sepharose CL一6B column 童 口 Retention t【me I/min Fi售g.。3 HPGPC Chr §皇J0苎 omatograms of WSP1 WSP3 and WSP2a which was obtained after treated with seriesofdextranstandardsandEmpow- er chromatography workstation,elu- tion volume and retention time was used to detect the homogeneity and molecularweightofpolysaccharidc ̄17-18]. HPGPC results (Fig.3)showed that the weight—average molecular weight of WSP1,WSP3 was 2.32xl 00, 7.92xl 00,respectively,which was sim- ilar to Majdoub’s report[19】_The purity Of WSP2 was not high.and two sub- components WSP2a,WSP2b(Fig.2B) were obtained after purified by Sepha. rose CL.6B.The weight.average mo1. ecular weight of WSP2a.WSP2b was 1.24xl 06,5 979,respectively,after dial- ysis,freeze-dried,weighed,the calcu- Iated amount of the two was account. ed for 56.82%and 1 4.35%of the WSP2 sugar weight,the recovery was 71.2%.WSP2b(1ittle amount,HPGPC figure not shown1 may be the protein components with weak binding of polysaccharide.protein complexes[19-2o]. The WSP2a content in CP was 45.21%x56.82%=25.68%. After derivatization by the PMP, according to HPLC analysis(Table 1), the m0nOsaccharide compositions of WSP1,WSP3,WSP2a were mainly arabinose, galactose and xylose; Agricultural Science&Technology g = IIⅡH Time rain Fig.4 Inhibition of WSP1.WSP2 and WSP3 to the conversion of fibrinogen into thrombin in purified system WSP1 was mainly composed of gal- marizes that the blood coagulation is actose with close arabinose and xylose accomplished by two pathways of in- content,which was consistent with trinsic pathway and extrinsic Pathway. what Maidoub et a1.had reported : three stages of the f0rmation of pro— diferent fr0m WSP1. WSP3 and thrombin activator,activation of pro- WSP2a were acidic pOIysaccha rides, thrombin into thrombin,conversion of of which the galactose and arabinose fibrinogen into fibrin[m-m].The conver. content ratio was 1.15 tO 1.07.and the sion of fibrinogen into fibrin is the Iast results were similar to the study of step in both intrinsic and extrinsic Matsuhiro et aP 1.1ngredient diference pathways.The fibrinogen cross.1inked of Opuntia m pa alta CP may be reIal— and then turned into thrombus. ed to diferences in extraction technol- As shown in Fig.4,with the 1 m ml ogy,pOIysaccha ride purlty,cactus re。 mass fraction of the pOlVsaccha ride glen,age,and harvest season.And component.when the thrOmbin acted the uronic acid content was highest in on fibrinogen conversion system for 1 0 WSP2a(4.4%). min.the inhibition of WSP2a.WSP3 Effects of pOIysaccharIde compo- and WSP1 to the conversion of fibrino. nents on antiCoagUIant activity gen iirte fibrin became stable;the jnhi— The measurement results of bition ratio was 73. .48.5%and 2.2%. ATPP.PT and TT were as shown in respectively.1t indicates that WSP2a Table 2.it indicated that the APTT and has strong anticOaqulant activity. TT diference was statistically signifi- As shown in Table 1 and Fig.4. cant after adding WSP2,WSP3 com- the mOn0SaCCharide compositions and ponents,WSP2 prolonged the AP-r-i- proportion of WSP3 and WSP2a were and TT,while had no significant effect diferent,resulting in quite diferent On PT in vitro.indicating that anticoag— uronic acid content:the one with high- ulant activiyt of WSP2 was worked er uronic acid content had high jnhibi— by affecting the intrinsic Coagulation tion activity on the conversion of fib— system. rinogen into fibrin:The anticoagulant Effects of poIVsaccharide on fib- aetiviyt of non.・sulfate plant polysac-, rinogen—-fibrin conversion charides was related with the uronic The”Cascade hypothesis”sum— acid content that after the reduction of Vo1.1 3,No.2,201 2 j‘i『0=蛊201 2—显 日Ⅱ圈 丑互口互薹 I田五口.E!丹■ 259 .—means not detected. Table2 EffectofCP,WSP1,WSP2andWSP3onAP1_r,PTandTT『n vitro susceptible to volatilization and able to be conducted GC analysis.The peak sequence and major ion fragments of the corresponding mass spectra were analyzed bV GC-MS,making it accu- rately in determining the composition, configuration, relative content of monosaccharide and the connection type of each mOnOsaccha ride residue. Compared with the saline group,…indicates P<0-O1;~indicates P<0・05; indicates P< After partial acid hydrolyzed.the 0.1O.Data in the table are x ̄n=5. acid—unstable Iow molecular weight Table 3 Monosaccharide composition of WSP2a-・pH and carboxyl reduced・-WSP2a-・pH fragments in the periphery of WSP2a mole.% were filtered through the dialysis bag, trapping the main chain components of WSP2a,which was WSP2a-pH.After carboxyI reduction,methylation。PM从 derivatization,the reduced WSP2a_DH was compared with the Spectral Data- bases of Partially Methylated AIditol Acetate(PMAA)Derived from a Gly— cosidically Linked Sugar Residue【矧. Table 4 Methylation analysis of WSP2a-pH and carboxyl reduced WSP2a-pH The comparison results were as shown in Table 4. From the comprehensive analysis of Table 3 and 4。it can be concluded the follows: I.The— )Gal(1 of WSP2a-pH was trace detected。the molar ration increased after carboxyI reduction;Gal A decreased by 27%,which was equaI to the increase amount of GaI(27.3%), indicating that the residue was galac— turonic acid.probably located in the main chain; --i++represents large amounts;++represents a small amount;+represents trace lI.The other two galactose-con— uronic acid,the corresponding biologi- powefful means to analyze the Struc- taining residues were 6-O-methyl-galac・ caI actMty became very IOw[盈刮.There- ture of carbohydrate[ ̄J.It first methy- tose terminal group and 1,6-galactose; ….T-XyI f1—}terminal xylose was fore.the next step is to further study lates alI the free hydroxyls of various detected,indicating that the proportion the mechanism of the anticoagulant mOnOsaccha ride residues in polysac- was Iess; activity of WSP3 and WSP2a,such as charides,and then hydrolyzes the gly- IV.There was no GaI(not Iisted) the impact on platelet aggregation,the cosidic bonds of the pOIVSaCcha ride. in the dialysis bag,three galactose effect on intrinsic and extrinsic coagu- The Iocation of the hydroxyl of the hy- connection types were detected in the lation pathways.and the relationship drolysed compounds js the connection WSP2a-DH methylated carboxyl re. between the function and structure of point of the original glycosy1.Based on duction product,inferring that galac- pOIysaccha rjde,with the aim to provide the diferent proportion of methylated turonic acid was in the main chain,not usefuI references for the study of non- m0n0saCcha rides.the proportion of the branched・chain; sulfate plant polysaccharides antico- this connection bond in the repeated V.Rhamnose and galactose only agulant activity and cactus function pOIVsaccha ride structures could be in. had one connection type that rham- products. ferred(Table 3).Methylated monosac- nose。-4 was connected with the galac- StructuraI analysis of WSP2a-pH charides can be further acetylated into tose一6.Combined with the molar ratio Methylation analysis is the most methylated alditoI acetates which were Of mOnOsaccha ride.the general struc. Vo1.1 3,No.2,201 2 Agricultural Science&Technology 2012 ture of the main chain was speculated as shown in Fig.5. 1,4-GalA,1,2-Rha and 1,2,4-Rha. Processing(农产品加工学刊),2009, 1 75(6):72-79. 【1 1】QIN CG(钦传光),HUANG KX(黄开勋), -÷4)GatA(1 n÷2)Rha(1_÷4)GatA(1_÷2)n1a【1-÷4)Ga LA(1-÷2)II}1a(1--)4)GatA【1 2)P,ha(1 4XU HB(徐辉碧).Component analysis and molecular weight determination of misgurnus anguillicaudatus polysac- charides by gel filtration chromatogra— 4 t .r。a60 4个 。 u 个 个 phy(凝胶过滤色谱法测定泥鳅多糖的 组成及分子量)【J】.Chinese Journal of Ga Fig.5 Schematics structure of WSP2a-OH Gil Analytical Chemisty(r分析化学),2002, 3O(41:41 1一甜_8. 【12】DONG Q(薰群),ZHENG LY(郑丽伊), FANG JN(方积年).Modifiedphenol sulfuricacid method for determination of the content of oligo and polysac— Conclusions WSP1. wSP2a and WSP3 p0Iysaccha ride components were ob- References 【1】WANG YG(q:有国).Erythrcyote viscosi・ t、,Ieading to brain infarction( ̄粘导致脑 tained frOm the CP bv DEAE Sepha- 梗阻)fJ1.Chinese JournaI of Cardio- rose CL-6B anion-exchange chro- vascular Review(中国心血管病研究杂 志),2O01(2):1 9—20. matography and Sepharose CL-6B gel f21 THOMAS DP.Does Iow molecular wei- filtration chromatography purification, ght heparin cause less bleeding【J]. through the gradient elution of phos- Thromb Haemost.1 997.78:1 422— phate buffer solution and NaCI solu— 1425. tion,dialysis desalination and freeze- [3】ZHOU YG(周永国),YANG YD(杨越冬), drying;the weight-average molecular WANG SY(王树元).Research progress weight of the three was 2.32×1 00. 0f natural active polysaccharides for ap- plication in the biomedicaI field f天然活 1.24 xl 00 and 7.92×1 00.respectively. 性多糖在生物医药领域中的研究进展1 and the contents of the three account— 【J】.Chinese Polymer Bulletin(高分子通 ed for 1 1.59%.25.68%and 20.48%in 报),2006(9):1 6-23. CP,respectively;amino acid derivative f41 JI H(季慧).Compositions analysis and of high-performance liquid chro- products investigation of opuntia Milpa matography was used to measure the alta(“米邦塔”仙人掌成分分析及产品开 mOnOsaccha—de compositions.the re- 发)[D】.Wuxi:Jiangnan University( ̄锡: 江南大学).2006. suits indicated that WSP1 was mainly f51 TREJO GONZLLEZ A,GABRIEL OR. composed of galactose;components TlZ G.PUEBLA PEREZ AM.A purified WSP2a and WSP3 were composed of extract frOm prickly pear cactus fOpuntia arabinose, galactose and xylose, fuliginosa1 controls experimentally in- rhamnose,mannose,glucose and duced diabetes in rats fJ1.Ethnophar- uronic acid。 macol,1 996,55(1):27—33. The three indicators in the coagu- 『61 GALATI EM,MONFORTE MT,TRIP— ODO MM.Antiulcer activity of Opun妇 Iation pathway showed that WSP2 pro- ficus indica(L.)Mil1.(Cactaceae):ultra- Ionged the APTT and TT,while has no structuraI study[J].Journal of Ethnopha— significant effect on the(PT)/n vitro,in- rmacology,2001,76(1):1—9. dicating that anticoagulant activity of 『71 FERNANDEZ ML,VEGA S。AYALA MT, WSP2 works by affecting the intrinsic er a1.Pectin fsolated fr0m prickly pear coagulation system;turbidity test re- (Opunf『a sp.)modifies low density lipoprotein metabolism in chOIesterOI・fed suits of the conversion of fibrinogen in- to fibrin showed that components guinea pigs[J].Journal of Nutrition,1 990, 120:1283—1290. WSP2a and WSP3 can inhibit the 『81 AHAMD A,DAVIES J,RANDALL S,ef conversion of fibrinogen into fibrin. a AntiviraI properties of extract of OP— when the mass fraction was 1 ma/ml, untia streptacantha『J1.AntiviraI Resea— the inhibition rates were 73.3%and rch,1 996.30:75-85. 48.5%.initiaI displaying that the Op— 【9】FANG T(方涛),ZHU cc(褚翠翠),LU N untia milpa alta pOIysaccha rides (陆宁).Development of research on ed— ible cactus’functionaI compounds(食用 WSP2a and WSP3 had anticoagulant 仙人掌功能性成分研究进展)[J].Food activity. and Machinery(食品与机械),2007,23 Comparison of WSP2a.which (1):148-154. had been subiected to partial acid hy- n0】JIANG CM(蒋长明),TANG MZ(唐盂 drolysis.methylated alditoI acetate 忠),LIU sx(刘树兴).Study on optimum derivatization,with PMAA database, extraction process for the polysaccha- rides frOm opuntia with orthogonal de- the results indicated that the main sign(仙人掌多糖提取工艺的研究)【J】. chain of WSP2a was constituted of Academic PeridoicaI of Farm Products Agricultural Science&Technology Vo1.1 3,No.2,201 2 cha—rides(改良的苯酚一硫酸法测定多 糖和寡糖含量的研究)[J].Chinese PharmaceuticaJ JournaJ f中国药学杂 志),1 996(9):1 55-1 58. f131 YANG XB,ZHAO Y,WANG QW,efa Analysis of the mOnOsaccha ride com- ponents in Angelica p0IVsaccha rides by high performance Iiquid chromatog— raphy[J].Analytical Science,2005,21: 1177-118O. 【14】SUN YL(孙元琳),SHEN RL(申瑞玲), TAIXIG J(汤坚).Methylation analysis of pOIysaccharide ASP3 fr0m Angelica sinensis(0liv.)Diels(当归多糖ASP3 的甲基化分析)『J1.Chemical Journal of Chinese Universities(高等学校化学学 报),2008,29(7):1 367—1 370. 『151 YAN WG。WANG Z,XU SY.A new method for determination of antithrom. botic activity of egg white protein hy- drolysate by microplate reader『J1. Chinese Chemical Letter,2007(1 8): 449-451. 【1 6】MATSUHIRO B,LILLO LE,SAENZ C, et aL ChemicaI characterization of the mucilage frOm fruits of Opunf『a ifcus in— dica[J].Carbohydrate Polymers,2006. 63:263-267. 【17】NIE SP(聂少平),XlE MY(鲥明勇), SHEN MY(申明月).Determination of tea pOIVSaCCha ride by high perfor. mance liquid chromatography f应用高 效液相色谱法测定茶iJ- 多糖)[J].Food Science(食品科学),2006,27(4):177— 181. 【1 8】DAWKINS JV.High performance gel permeation chromatography of poly— mers[J].Pure and Applied Chemistry, 1 982,54(2):282—292. [1 9】MAJD0UB H,ROUDESLI S,PICTON L.ef a1.Prickly pear nopals pectin’s frOm Opuntia ficus indica physico— chemicaI study in dilute and semi-di— Iute solutions fJ].Carbohydrate Poly— mers,2001。46:69-79. 【20】MAJDOUB H,ROUDESLI S,DER. ATANl A,ef a POIvsaccharides frOm prickly pear peel and nopals of Opuntia ficus indica:extraction,characterization and polyelectrolyte behaviour[J】.Poly- mer International,2001,50:552-560. (Continued on page 264) 中华补血草 ntaxin基因的克隆 张莹,陈世华 ,韩会玲,窦伟红,尹海波,赵吉强,郭善利 (烟台大学生命科学学院,山东烟台264005) 摘要【目的]对中华补血草Synta ̄in基因进行克隆。【方法】以中华补血草叶片为材料,提取总RNA并进行反转录反应。依据已知Syntaxin基 因5’端EST序列信息设计巢式引物,以反转录得到的cDNA为模板,利用2轮PCR扩增cDNA 3’末端(3’RACE),获得Syntaxin基因3’端序列。 【结果]获得1 090 bp的DNA片段。分析表明,该片段编码LsSyntaxin全长基因,其中开放阅读框长816 bp,编码271个氨基酸。该基因编码的 Syntaxin蛋白相对分子量为30 254-3 Da,理论等电点为5.55。[结论】为研究Syntaxin基因在补血草中的功能及其在补血草泌盐过程的作用奠定 了基础。 关键词中华补血草;3’Race;Syntaxin基因;克隆 基金项目 国家自然科学基 ̄(30870199);山东省自然科学基金(Y2007D34);山东科技攻关项目(2010GNC10937 o 作者简介 张莹(1985一),女,山东济宁人,在读硕士,研究方向:植物抗逆分子生物学。 共同通讯作者,副教授,博士,从事植物发育分子生物学 研究(E—mail:csh—yantai@163.com);郭善利,教授,博士,从事植物发育分子生物学与基因工程研究,E-ail:gsl@ytu.edu.cn。 收稿日期 201l-11-10 修回日期2011-12—16 (Continued from page 260) 『21】MATSUHIRO B,LILLO LE,SAENZ C, ef a^Chemical characterization of the mucilage from fruits of Opuntia ficus and its function as a biochemical am- with anticoagulant activity mediated by plifier[J].Nature,1 964。202:498-499. I241 YOON SJ,PERElRA MS,HWANG JK, 甜 The medicinaI plant Porana volu- bilis contains pOIVsaccharides with an- ticoagulant activity mediated by hep- antithrombin[J].Thrombosis Research。 2003,112《31:151—158. 【26]ZHANG WJ(张惟杰).Glycoconjugate biochemist ̄technology research f糖 indica fJ].Carbohydrate Polymers, 2006,63(2):263-267. 『221 DAVID EW,RARNOFF OD.WaterfalI sequence for intrinsic blood clotting[J]. 【25】YOON SJ,YU MA,PYUN YR。ef a Science,1964,145(18):1310-1312. 『23】MACFARLANE RG.An enzyme cas- cade in the blood clotting mechanism arin cofactor II『J].Thrombosis Re- search,2002,1 06(1):51-58. The nontoxic mushroom Auricularia auricula contains a pOIysaccha de 复合物生化研究技术)【M】.The 2 ed (第2版).Hangzhou Zhejiang Univer- sity Press(杭州:浙江大学出版卒t), ’998. Responsible editor:Na LI Responslble proofreader:Xiaoyan WU 仙人掌多糖结构分析及抗凝血活性研究 蔡为荣’,谢亮亮 ,陶鹏飞2崔武卫 (1.安徽工程大学生化与化学工程学院,安徽芜湖241000;2.南陵县质量技术监督局,安徽南陵242400; 3-力口拿大农业与农业一食品部圭尔夫食品研究中心,加拿大安大略湖州N1G5C9) 摘要[目的]分析仙人掌多糖组分和结构,并研究其抗凝血活性。[方法]以米邦塔仙人掌为试验材料,通过热水浸提、醇沉制得粗多糖cP,经交 换层析和凝胶层析纯化,研究其单糖组成和重均分子质量分布与主链结构,并对多糖组分抗凝血活性进行了初步研究。【结果】对仙人掌粗多糖分 离纯化后共得到3个多糖组分WSPI、WSP2a和WSP3,重均分子量分别为2.32x10 、I.24x106和7.92xl06。对氨基苯甲酸(PMP1衍生化高效液相色 谱法检测表明,WSPI主要成分为半乳糖;WSP2a由鼠李糖、阿拉伯糖、木糖、甘露糖、葡萄糖、半乳糖、葡萄糖醛酸和半乳糖醛酸组成,含量分别为 6.3%、32.3%、12.9%、1.5%、4.8%、37.1%、0.64%和4.4%;WSP3主要成分为阿拉伯糖、半乳糖和木糖以及少量鼠李糖、甘鳐糖、糖醛酸。WSP2能延长 活化部分凝血酶原时间(A 和凝血酶时间(1Tr),而对凝血酶原时间( 的影响不显著,说明其是通过影响内源性凝血系统而发挥抗凝血 作用。纤维蛋白原转化纤维蛋白的抑制试验初步表明WSP2a和WSP3具有抗凝血活性。甲基化分析显示WSP2a含有1,4一连接的半乳糖酸,1,2一 连接的鼠李糖和l,2,4一连接的鼠李糖构成的主链。[结论]该研究为仙人掌多糖资源优化和深度开发提供了试验依据。 关键词米邦塔仙人掌;抗凝血;多糖结构 基金项目 国家自然科学基金资助项目(31171753),安徽省国际科技合作计划项目(10080703035 o 作者简介 蔡为 ̄(1963一),男,安徽芜湖人,教授,博士,主要从事天然产物功能因子的研究。E—mail;weirongcai0781@sina.con。十通讯作者,教授 主要从事多糖结构与功能。 收稿日期 201 1-l1—15 修回日期201 1-12—04 Cambridge Scientific Abstracts(CSA) Cambridge Scientific Abstracts(CSA)is a retireval system published by Cambridge Information Group.With a history of more than 30 years,the company is an important publisher of publishing abstracts and indexes with agent centers in Britain,France,Australia,Japan,Netherlands,New Zealand,and Hong Kong China.The products include traditional printed abstracting and indexing journals,a variety of electronic database which are retrieved through remote on—line and CD・ROM.Recently,the CSA launched network based(IDS)services,which can retireve hundreds of databa¥es provided by CSA and CSA publishing pa ̄nem. Agricultural Science&Technology Vo1.1 3。No.2,201 2