南瓜多糖降糖有效部位的提取分离及降糖作用的研究_英文_熊学敏

Rev Respir Dis,1990,141:456-465.9Eric Schultze A.Emeis JJ.and Ro th RA.Celluar fi-bro ncctin and yo n Willebra nd fac tor co ncentra tio n in pla sma o f rats tr ea ted with mo no cro taline py rr olc.B ioche Pha rmaco l,1996,51:187-191.10Ono S,et al.PA F antag onist inhibit monocr o ta line-in-duccd lung injur y a nd puhno nary hy pe rtensio n.J ApplPhysiol.1991,71(6):2483-2492.11Chao uat A.Weitzenblum E.H ig enbo ttam T.The ro le o f thro mbosis in sev ere puh no na ry hyper tensio n.Eur Respir J1996:9:356-363.收稿日期:2000-08-16This program w as app roved by J iangxi Provnicial Science and Technology Committee in2000.Ref.No.2000A-10*1998年度江西省中医药科研计划项目,编号:200A10熊学敏,男,43岁,江西省卫生系统跨世纪学科带头人.副主任药师,中药室副主任.Study of Extraction and Isolation of Effective Pumpkin Polysaccharide Component and its Reducing Glycemia Function*Xiong XueMin,Cao Jue(J iangx i Prov incial Institute of T raditional Chinese Mdeicine and Materia Medica,N anehang P. R.China330077)ABSTRACT　OBJECTIVE:T o study decr ea sing g lysemia effec t of the effectiv e pumpkin po lysaccha ride co mpo nent thro ugh ex trac tio n a nd isola tio n.METHODS:Cucurbia t mo schata Duch pumpkin wa s wa ter-ex tracted,alco hol-pro cessed,precipita ted and iso la ted by io n ex cha ng e collumn,Sephero se and Sephadex A-200,the n poly saceharide co mponent wa s o btained,coo led and dried into w hite polysaccharide pow der.U sing rats that to ok Xiao ke pill(a kind of Chinese medicine to reliev e thir st)as positiv e co ntro l g ro up,th e w hite polysaccharide pow de r wa s giv en to a llox an induced diabetic r ats(3.8g/kg)fo r21running da ys.Fasting blood w as ta ken fo r the determina tio n of g lycemia chang es.RESULTS:This pumpkin polysaccha ride had r e-mar ka bly bet ter effect of reducing g lycemia on a llox an induced dia betic rats tha n that on Xiao ke pill g roup(P<O.01).CON-CLUSION:The effectiv e pumpkin polysaccha ride com po ne nt has sig nificant g lycemia reductio n effec t o n allonxa n induced dia-betic ra ts.KEY WORDS　Pumpkin Polysaccharide,Effec tive Co mpo nent,Allox a n,Glycemia Decline,Diabetic Ra ts M odel南瓜多糖降糖有效部位的提取分离及降糖作用的研究*熊学敏　曹　珏1(南昌330077江西省中医药研究所;1南昌330006江西医学院)摘要　目的:对南瓜多糖有效部位进行提取、分离并进行降糖作用的研究。

方法:将葫芦科植物Cucurbia t mo scha ta Duch南瓜由水提、醇沉后经大孔径离子柱和Sephe ro se柱、Sepha dex A-200柱分离,获得多糖组份,冷冻干燥后得白色粉末多糖,该多糖经给予四氧嘧啶糖尿病造模大鼠(3.8g/kg)连续21天后测定其空腹血糖的变化情况,以中药消渴丸作为阳性对照组,结果该有效部位有明显降低四氧嘧啶糖尿病大鼠血糖的作用,且效果优于消渴丸对照组(P<0.01)。

结论:南瓜多糖有效部位有明显的降低糖尿病大鼠血糖效果。

关键词　南瓜多糖;有效部位;降血糖;糖尿病造模;四氧嘧啶 Pum pkin,the fruit o f Cucurbiat mo schata Duch,co n-taining a va riety o f t race elements,vitamines,pro teins, starch a nd fibrin,is r ega rded as a health fo od.In tra ditio nal Chinese medicine,pumpkin is believ ed to hav e th e function o f inv ig o rating the splee n and benefitting th e lung[1].Its liq-uid ex tract processed with alco hol has o bvio us g tycemia de-cline effect o n r ats[2].Folks used pumpkin,Her ba po rtul-ca l,Stig ma ma ydis to tr ea t diabetes and had g ood result[3]. How ev e r,th ere has been no r epo rt o n o bserv a tions of effec-tiv e pumpkin polysaccha ride com po ne nt Her e,a preliminer y study o f effec tiv e pumpkin polysaccha ride component is in-tr oduced.1　Instruments and agents1.1　Instrument:4-ho le wa ter steamer,dig ital elec tric stir-re r;1.2　Agents:All ag ents used in this ex perim ent w ere ana-·266·Chin J M AP,2001Augus t,Vol.18No.4 中国现代应用药学杂志2001年8月第18卷第4期ly zed purity.1.3　Ex perimental drugs:Xiao ke pill(No.O ne Gua nzhou Pha rmaceutical Facto ry,No:9702013),allo x an(Sig ma Company,No:36H0804),N S(J iang xi Zh uhu Phar ma ceuti-ca l Facto ry,N o:970511)2　Experimental Methods2.1　Ex traction o f semifinish ed pumpkin polysaeeharide: Fresh pumpkin belo ng ing to the f ruit o f Cucurbia t moschata Duch w as taken,tested,peeled a nd sliced. 5.0kg w as w eighed o ut o f it,w ashed with0.02%N a HS03fo r30min, then clea ned under tap w ater,made into paste with a bea t-er,pur e wa ter added at th e ratio of6,5,5times,under75～80℃,stir red and ex tr acted fo r3times,th e r espectiv ely limited time:6h,4h,4h,filter ed and reg ulated to pH3.0～3.2by HCI6mol/L,immediately centrifug ed(r=2500r/m, t=30min.),the filtra te super na tent was ta ke n and neut ral-ized with5mo l/L HCI,p H(6.5～7.0),and0.5%(w/w) activ ated carbo n a dded,heated,decolourized,filter ed a nd dailyzed with(w/w).Par tially the dia ly zed liquid wa s de-compressed and concentra ted(t=40～50℃,p=-0.1 M Pa),r eprecipita ted with95%etha no l,cent rifug ed,pr e-cipitated,then the precipita te w as dehydra ted with acetone and ethanol,free ze-dried and white semifinished pumpkin polysaccha ride pow de r w as o btained.(A)2.2　Iso la tion a nd purificato n o f pumpkin poly saccharide'sg lycemia decline component:Disso lv e the abov e-mentio ned semifinished pumpkin po lysaccha ride in N aCI(0.1mo l/L)to r each sa turation,place it a into a f reezer(5～8℃)fo r12h, centrifug e,r emov e pr ecipitation,add40%t richlor acetie acid to r each8%co nce nt ration,place it into the free zer(5～8℃)ag ain fo r12h,centrifug e,mix e prcipita tio n with0. 1mo l/L Na CI,dissolved,test by Sephar ose CL-B4,washe o ut with0.1mo l/L N aCI,isolate,make fo llow ing surv ey with sulphurie acid-enzo l,collec te this mo th er liquo r co n-taining po ly sa echaride co mpo nents(collec tion ra te:45%); which wa s nut ralized with0.1mol/L N aO H to pH6.5～7. 0,pr oper ly co ncentra te this liquid,dialy ze with distilled wa-ter,test with Sephadex A-200,iso la te,purify,wa sh out with0.1mo l/L N a CI(wa shing speed:6m/9min),co llect liq-uid-like po ly sa eeha ride.Deco mpresse,co ncentra te,f reeze-dried,and o btain white pumpkin polysaeeharide's g ly cemia decline compo nent(purified po lysaeeharide).(B)2.3　Purifica tio n test of po ly saceha ride's effectiv e pumpking ly eemia deteline co mpo nent2.3.1　PC a nalysis:Test the purification deg ree of purified polysaecahride w ith PC.Cut PC paper into2cm×20cm.Ac-cording to T LC principles in95Editio n,sample sizes r anked r espectiv ely in5μg,10μg,20μg,and dev eloped o n the uppe r line,ag ents(chlo rofo rm∶methy l alcoho l∶5%acelate8∶2∶0.5)and(n-butyl alcoho l∶ace tone∶wa ter4∶5∶1)dis-tance:16～18cm,dried a nd spra yed the plate with sulphurie acid-benzo l liquid,no mo re tha n3purplish red spots sho w ed r espectiv ely.2.3.2　Acetate fiber paper ele tro pho resis test:Cut W ater-man G F/C aceta te fiber paper into2cm×20cm,sample was spo tted on a no ther acetate fiber paper(1cm×10cm),adh ere the sample paper nex t to the ba sic line,ma ke sample seeped dow n to eleetr ohporisic paper,remov e sample pa per,v= 400v,t=20min,buffe r:0.025mo l/L bo ric a cid liquid(P H9.3),r emov e piece o f paper,natura lly dry,sho we co lo ur by sprayingα-N aph tho l-sulfuric acid test so lution,bake fo r 15min(100℃),and o bse rv e.L ess than3pur plish r ed spots should appea r.3　Making diabetic rats'model groups3.1　M aking dia betic r ats'model g ro ups:70Wistar rats (each w eig hed258g～275g),male,w ere pr ovided by Ex-perim enta l Animal Depa rtment o f J ia ng xi M edical Co lleg e. 10o ut of them we re randomly taken a s a contr ol g r oup,and 70mmo l/L allo xa n wa s injected into th e r est60ra ts'tail v ein.72ho urs la ter,cut the tail and g o t fasting bloo d,de-ter mine glycemia.Tho se w ith g lycemia ov er18mmol/L w ere selected for ex perimentation.50qualified diabetic rats w ere dev ided into5g ro ups at r ando m a s mo del g roups: polysaceharide(A),no n-polysaccha ride,polysaccha ride(B) and Xia oke(10ra ts/g ro up,g lycemia's av erag e v alue be-tw een g r oups wa s<0.65mmo l/L).Ea ch g ro up wa s giv en N S,pumpkin polysaccha ride ex tra ct,pumpkin polysaccha-ride(B),pumpkin no n-polysaccha ride pow de r——a co mpo-nent disso lv ed in alcoho l(6.88g/kg)a nd Xia oke suspension (0.75g/kg).All drugs w ere giv en by gast ric irriga tio n,ex-cept po ly saccharide po wder by mixing with fodder and giv en to fa sting ra ts,2times a day,4ml each time,fo r21running da ys.3.2　Testing index:On the22nd day,aft er a fast o f12 hour s,tested fasting glycemia a nd o bse rv ed the g eneral co n-ditio ns ev ery day,such as a nimals'hair,ing estio n,drink-ing,qua ntity of urine,body w eight,brea th,mov ements of limbs etc.3.3　Sta tistic me tho ds:Deter mined sig nificant differ ences accor ding to v ariance ana ly sis a nd Q-test.4　Results4.1　Determina tio n o f fasting g lycemia:After21day s of tr eatment,the lev el o f g lycemia in the mo del g ro up r e-mained high,that of the3g r oups trea ted w ith drug s de-clined obv iously(P<0.01)and show ed sig nificant differ-ences;but,no r emarkable differences in comparison be-·267·中国现代应用药学杂志2001年8月第18卷第4期 Chin J M AP,2001Augus t,V ol.18No.4tw een pumpkin po ly saccharide g ro up and pumpkin no n-polysaccha ride g ro up;yet bette r fing ding s(p<0.01)when compar ed with Xia oke g ro up.It indicated tha t pumpkin polysaccha ride had glycemia decline effect o n allox an induced diabetic rats w ith better r esult tha n o n tho se taking Xia oke pill.See table1.Table1.Pumpkin po ly saccharide's g lycemia decline effects o n diabetic r at mo del g roupsGroup Dosage(g/kg)Aanimal(No)M ean value of glycemia(mmol/L)(X±S)M odel(NS)0.071018.3±2.72 Xiaoke0.571017.01±3.14 Potysaccharide(B) 3.81010.25±3.10Non-polys aecharicd 6.881011.06±2.07 Polysaceharide(A) 6.881010.34±4.21 Control(N S)50ml108.45±2.994.2　O bserva tio ns o f g eneral co nditions:Allo xa n w as inject-ed into60ra ts.O n the1st da y,all rats w er e quie t.O n the 2nd day(24ho urs la ter),they beg an to eat,drink,and the quantity o f urine incr ea sed.With co ntinuo us injectio n fo r3 w eeks,improv ements appea red in the g r oups of pumpkin non-po ly saccharide and pumpkin polysaccha ride:hair brig ht and smo o th,drinking and urinatio n reduced.But the mo del g ro up w as under wo rse co ndition,hair ro ug h and untidy, the body w eak and no tably mor e urina tion.5　Discussion5.1　Allox an is a specificβcell tox ic agent.It can selectiv e-ly dest roy theβcell of many kinds o f a nimals[5]and cause ex-perime ntal diabetes.Allo xa n induced dia be tic rats ar e cho sen as co mmo n animal models fo r studying the effectiv eness o f diabetic dr ug s.T his ex periment prov es tha t pumpkin polysaccha ride can decline fa sting g ly cemia of allox an in-duced diabetic ra ts,imply that pum pkin po ly saccharide can efficie ntly co ntro l diabetic sympto ms w ith g oo d sho rt te rm thera peutic effect.5.2　Cur rent study show s[6]tha t pumpkin is a low-suga r and lo w-calo r y fo od,containing lo ts of tr ace elements,o f which Co.is the most typical a nd indispensable fo r islet cells to stimula te nor mal insulin secr etio n o f diabetic r ats so as to o btain g lycemia decline functio n.5.3　Fur ther study is required for the mechanism of pump-kin polysaccha ride's glycemia decline[7].Theo retica lly,pro-tein and a mino-a cids absor ptio n ma y stimulate insulin secr e-tio n.It is sugg ested tha t pumpkin po ly saccharide migh t ex-ist in the fo r m o f g lycopro tein and contain abundent v arieties o f amino-acids,stimula te insulin secr etio n and hav e g lycemi-a decline functio n.T his will fa cilitate fur ther appr oach into the mechanism of g lycemia decline.5.4　Acco rding to th e r esults of animal ex periments,it prov ed tha t pum pkin po ly sacchride's effec tive compo nent has better effect o f g ly cemia decline than that o f pum pkin polysaceharide a nd Xia oke g ro ups.This indicates tha t the g ly cemia decrea se intensity o f pumpkin po ly saccahride's ef-fectiv e co mponent is high er than tha t in pumpkin polysac-cha ride g ro up,ie.,not all components in pum pkin are effec-tiv e in g lycemia decline.5.5　T his tea m will set up o ther items to study th e pum pkin polysaccahride's effect,th e relatio nship be tween its struc-ture a nd effect,the mechanism o f pumpkin po ly saccah ride B in stimulating diabetic r ats to reach no rmal insulin secre tion and th e mor pho lo gic 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edicine.1997,17(9):569.7Chen ZhiM in.The Effect of Pumpkin Pow der's Glycemia and Hypertension Decline on Human.J iangxi J ournal of Traditional Chinese M edicine.1994,25(2):114收稿日期:2000-09-18·268·Chin J M AP,2001Augus t,Vol.18No.4 中国现代应用药学杂志2001年8月第18卷第4期。