利用大孔树脂从葛根中分离纯化总黄酮_英文_刘火安

1、Journa l o f Chinese Pha mr aceutica l Science s 2006, 15(2):121-126121Optmi ization of Preparative Separation and Purification of TotalFlavonoids from Radix Puerariae by M acroporous Resin M ethodLIU H uo-an1, 2 , WANG Bo-chu1 , DA I Chuan-yun1* , SHAO Zhi-yong3 , HE Cong-lin1 ,and JIA Yun2(1. Coll

2、ege of B ioeng ineering,Chongqing Un iversity, Chongqing 400030, China;2. College of Chem istry and B iology,Chongqing Poly techn ic College, Chongqing 400050, Ch ina;3.Interdepartm ent of Genetics, Iowa S ta te Un iversity,Iowa 50011 , USA)Abstract:A imTo screen the optim um m acroporous resinand c

3、onditions fo r theisola tion andpurification of flavonoids from Radix Puerariae. M ethodsThe staticand dynam ic adso rption /desorp-tion m ethods w ere used, andthesepara tion and purification process was evalua ted by m easuringtheconcentration of to tal flavonoidinthe frac tions w ith UV spectroph

4、o tom e ter. R esults The SP70 m acro-porous re sin w as the m ost effective com pared w ith othe r m acroporousresins. The optim um cond itionsw ere sc reened, which we re 0.5 g m L - 1 corresponding tocrude d rugfor concentra tion o f extrac t,pH5 - 6, and appended 60 tim es the vo lum e of the re

5、sin bed (BV) w ith the adso rp tion speed 2BV h- 1 ,and the vo lum e o f aq. 70% (V /V) ethano l as e luan tw as 5 BV w ith desorption speed 2 BVh- 1. Byth ism ethod,the final contents of tota l flavonoids exceeded 80%.ConclusionThe SP70 m acropo rousresin is the m ost e ffective onefo r large-sca l

6、e iso lationand purification o f flavono ids from Rad ix Puerar-ia, w hich m ee ts industrial needs.K ey w ords:Radix Puerariae;to tal flavono ids;m acropo rous resin;separa tion;purificationCLC number:R284. 2D ocum ent code:AArticle ID:1003-1057(2006)2-121-06IntroductionPueraria lobata (W illd. ) O

7、hw i is a perennial le-gum inous plan t na tive to eastern A sia. The ex tract ofRad ix Puerariae is rich inisoflavones andhas beenw ide ly used as antipyre tic,antidiarrhoetic,diaphore t-ic, and antiem etic agents intrad itiona l Chinese m edi-cine 1,andhas recently been used asa possib lerem edy a

8、gainst alcoho lism 2 .Pue rarin (daidze in 8-C-glucoside),daidzin(da idzein 7-O-gluco side), anddaidze in arethe m a jo r iso flavono ids in Radix P uerari-ae 3 .In recent years,there has been an increasing in-tere stinthe study of purified active com ponen ts. H ow-ever,this approach has been restr

9、icted p rim arily be-Received date:2005-09-25.Foundation item:S cience and Technology Comm ittee of Chongqing in China(CSTC. 2004BB5122).* Correspond ing au thor:Tel86-23-89126756, E-m ail cydai 126. comcause the traditional separation m ethods used so farw ere lim ited tosim ple techniques suchasex

10、 tractionand precipitationleading tolowrecoveries.Thustherecovery of the m a in com ponent of Radix Puerarie, pu-e ra rin, w as reported to beve rylow ,abou t 1%,al-though w ith a purity as highas 97%4, 5 .Traditional-ly,the ch rom a tograph ic procedures used we re based onthe adsorben ts w ith low

11、 purity g rade,in comb inationw ith o ther un it operations such as filtration and ex trac-tion 6 . This is w hy a m ore e fficient and simp le purifi-cation m ethod for puerarin is required.M acropo rous resin is a po rous h igh mo lecular m a-terial, wh ich has porosity and b iggish specific surfa

12、cearea. For separation and purifica tionofnaturalcom-pounds,it adso rbs o rganic substanceselective lyfromso lu tionthrough physica laction.Thepurificationm ethod of m ac roporous resinis a newp rom isingtech-no logy in pharm aceutica l industry,and has beensuc-122Jou rna l of Ch inese Pharm aceutic

13、a l Sc iences 2006,15(2):121-126cessfu lly appliedin the ex traction,separation and puri-A =0.095 7+70.92C (r =0.999 9). Sam ple w asfication o f flavono ids, saponins and a lkaloids 7, 8 . Thequantified by using the sam e m ethods.resu lts o f som einvestiga tions 9, 10show ed that m acro-Preparati

14、on of crude drug solutionporous resin AB-8 can be used to separate and purify the total flavonoid from the extracts of Rad ix Pueraria.The powdered Radix P uerariae (500 g) w as ex-trac ted 9 tim es w ith (W V/) 30% aqueous ethanol un-The presen t repo rt show s SP70 to iso late and purify thede r r

15、eflux for 1 h 3 to a ffo rd ex tract so lutions and w astota l flavono ids from the extrac ts of Rad ix Pueraria w assuperior to AB-8 and has som e o ther m erits such assim ple techniques and low cos.tM aterials and M ethodsM aterials and instrum entsRadix Puerariae w as obta ined from W ukuaishi M

16、 edicinalM ate ria lsM arke t o f Chengdu, Ch ina (iden-then filtered.Thefiltrate each tim e w as co llec ted, con-centrated to 0.5 gmL- 1 crude drug, and then centrifugedat 12 000rm in- 1 fo r 15 m in. The superna tant w asquantifiedfor to tal flavono ids, whose contentw as 47.0m g m L- 1.Results a

17、nd D iscussionsScreening of macroporous resintified by Pro .f JiaM R, Chinese M edicine Un ive rsity of Chengdu, China). Pue ra rin w as purchased from N a-Static adsorption /desorptionThirty m illilitreso f crude drug so lu tion w as pu t into10 g of p re treatedtiona l Institute fo r Con tro l of

18、Pharm aceutica l and B io-m acroporous resin and shaked 5 m in one tim e for 2 h,log ica l Produc ts, Beijing, China (N o:752-200108). A ll reagents w ere of ana ly tical g rade and obta ined fromand the resin w as then filtered. The filtra te w as collect-ed and the resin w as desorbed w ith 30 mL

19、of 70% a-Be ijing Chem icals Industry,B eijing, Ch ina. M acro-porous resin w as purchasedfrom M itsubish i C o.Ltd.of Japan for SP825, SP850, SP207, SP700,andSP70, and from Chengguang A cademy of Chem ical In-queous e thanol. The conten t of to tal flavonoids of Ra-dix Puerariae in filtrates afte r

20、 both adsorp tion and dep-sorption w e re de term ined.Resu lts showed. (F igure 1)tha t there are 8 k inds ofresin, AB-8, X-5, H P20,dustry, Chengdu, C hina, for D4020, AB-8, X-5, D141, D140, and D100, and from N ankaiUniversity, Tianjin, China fo r HP20, HP2MG, HP2MGL, NKA-9, and NKA -12.The ultra

21、vio le t absorption m easu rem ents w ere car-ried out on a UV-1601 spectropho tom e ter (Sh im adzu, Japan).D eterm ination of tota l flavonoids in Rad ix Pu-erariaeRe ference standards of the puera rin (7.43 m g) w as accurate ly we ighed and dissolved in 250 mL vo lu-m etric flasks w ith m e than

22、o l as stock so lution (29.72 g m L - 1 ). These stock solutionsw ere used to prepareseries o f standard solutions w ith m e thano l tog ive finalconcentrations of 2.972,5.944, 7.430,8.916,11.888, and 14.860 g mL - 1 ,respectively,of wh ichOD250 was m easured w ith UVspectrophotom eter.Therew as a l

23、inear rela tionship betweenthe concen trations andabsorbance, w hich are between 0.002 972 and 0.014 860 m g mL - 1. The fo llow ing equations gove rned the re-lationships be tw een the absorbance and concen trations:SP850, SP207, SP70,D141, and D140, w hosead-sorption rates are m orethan 90% and de

24、sorptionratsare m ore than 60%.Figure 1 Adsorption rate and desorption rate of m acro-porous resin for total flavonoids. a:D4020;b:AB-8;c:X-5;d:NKA-9;e:NKA-12;f:HP 20;g:SP825;h:SP850;i: H P2MG; j:SP207; k: SP700;1:H P2MG L;m:SP70;n: D141;o:D 140;p:D100D ynam ic adsorptionTen m illilite rs of the abo

25、ve 8 kinds of p re treated resinsw ere separatedly put into g lass co lum n (2 cm 20 cm ) and then the extrac t so lution o fR ad ix Puerari-L IU Huo-an, et a l:O ptim ization of P repa ra tive Sepa ra tion and Purification of T o ta l123F lavonoids from Rad ix Puerariae by M ac ropo rous R esin M e

26、thodae w as added w ith2 BVh- 1 adsorbeddynam ica lly.The e luting so lu tion w asco llec tedacco rdingtothenum ber o f tim es the volum e of theresinbedandtheconten t of to tal flavonoidsw as de te rm inedas the resu ltsshow n in Figure 2.From Figure 2,it isseenthattheadso rbed process of SP70 w as

27、 the best and D141 is the second fo r tota l flavonoids.co lum n (2 cm 20 cm ) w ith 20 m L of P70resin.A fter 2 BV h- 1adsrop tion, it w as successive lyelutedw ith 5 BV w ater and 75% aqueous e thanol by 2 BV h- 1. The e lu ting so lu tion w as co llected and the conten t o f to tal flavono ids w

28、as de term ined as the results shownin Figure 4.It is shownthat desorption ra te reaches thehighest whenthe drugso lution w as dilu ted 15tim es,bu t the so lu tion w ithou t dilution reached a lsoto m orethan 90%. Therefo re, it is im p lied that d ilution is un-necessary, and orig ina l m edicine

29、(co rresponding to 0.5 g mL - 1 ) can be added into resin.F igure 2L eakage curve of dynam ic absorptionFigure 4 E ffect of diluting multiple on desorption rate and content of tota l flavonoidspH valueTen m illiliters of crude drug solutionw as ad justed fo r pH va lue to 1 - 2, 3 - 4, 5 - 6,7 -8,9

30、- 10 w ith HC l or NaOH ,respective ly,and w asF igure 3Results of dynam ic desorptionthen put in to glass co lum n (2 cm 20 cm ) w ith 20mL of P70 resin.A fter 2 BVh- 1 adsorption,it w asDynam ic desorptionsucce ssive ly e luted w ith 5 BV w ater and 75% aqueoush - 1.The e luting so lu tion w as co

31、llect-Ten m illiliters of pretrea ted SP70and D141resine thano l by 2 BVseparated ly put in to g lass co lum n (2cm 20cm )ed and the con tent of to tal flavonoidsw as determ ined asand then 25 m L of the ex tract so lu tion o fRad ix Puerar-theresu ltsshow nin Figu re 5.From Figure5itis- 1show n tha

32、tdeso rptionrateisthe highest whenpHiae w ere added w ith 2 BV h adsorbed dynam ica lly,- 1is 5 - 6.w hich w as e lu ted w ith 5 BV w ater by 2 BV h,andAbsorption speedw ith 75% aqueous e thanol by 2 BVh - 1 deso rbed dy-nam ica lly. The e luting solution w as co llected acco rdingTen m illilite rs

33、of crude drug so lution w as adjustedfor pH 5 - 6 w ith HC l,and w as then put into g lass co l-to number o f tim e s the vo lum e o f the resin bed and theum n (2 cm20 cm )w ith 20 m L o f P70 resin.conten t o f to tal flavono id w as determ ined as the resu ltsA fter1,2, 3, 4and 5 BVh- 1adsorption

34、 separated ly,itshow ns in F igure 3. From Figu re 3,it is seenthat de-sorp tion proce ss o f SP70 is be tte r thanthat of D141.w as successively e lu ted w ith 5 BV w ater and w ith 75%aqueous ethano l by2 BVh- 1.The elutingsolutionSelection of optmi um separating processw as co llected andtheconte

35、nt ofto ta l flavono ids w asConcentration of extract solutionTen m illili-ters o f crude drug so lu tion w as diluted 0,5, 10,15,de term ined as the resu lts show n in Figure 6. From Fig-ure 6 it is show n that absorption effect is the bestwhenand 20tim es and w as then pu t intosepara tedly g lass

36、124Jou rna l of Ch inese Pharm aceutica l Sc iences 2006,15(2):121-126abso rption speed is 2 BV h- 1.Amount of append ing solutionTen m illilite rsof crude drug so lution w as adjusted for pH 5 -6, andw as then put in to glass column (2cm 20 cm ) w ith20 mL of P70 resin. A fte r 2 BV h- 1 adso rp ti

37、on,itw asConcentration of ethanolTen m illilite rs of crude drug so lution w as adjusted for pH 5 - 6, and w as then put in to glass co lum n (2cm 20 cm ) w ith 20 m L o f P70 resin. A fter 2 BVh- 1 adso rption,it w as e luted successively w ith 5 BVeluted w ith 5 BV w ater by 2 BV h- 1.The e lu tin

38、g so-w ater and then w ith 10, 30, 50, 70,and 90% aque-lu tion w as co llected and the conten t of to tal flavonoids w as de term ined as the resu lts shown in Figu re 7. F romous ethano l by 2 BVh - 1 ,re spective ly.The elutingso lu tion w as collectedandthe con tento f to tal fla-Figure 7 it can

39、be seen thatw hen the am ount of appen-ding sam ple w as 40 BV, it began to leak out, and it reached adsorption saturation on 65 tim es the vo lum e of the resin bed. So, 60 tim es the vo lum e of the resin bed w as suitab le for the am ount of appending sam ple.vono idsw as de term ined as the resu

40、lts show n in Figure 8. F rom Figure 8 it is show n that w ash-off effect w as the bestw hen the concen tra tion o f aqueous ethano lw as 70%.F igure 5 E ffect of pH value on desorption ra te and content of total flavonoidsFigure 8 E ffect of ethanol on desorption rate and content of total favonoids

41、F igure 6 E ffect of absorption speed on desorption rate and content of the total flavono idsF igure 7Expermi enta l results of amont of appending sampleEluting speedTen m illilite rs o f crude drug so lu-tion w as adjusted for pH 5- 6, and w as then put intog lass co lum n (2 cm20cm )w ith 20 mL of

42、 P70 re s-in pretreated. A fte r 2 BVh- 1 adsorption itw as elutedsucce ssive ly w ith 5BV w a te r by 2 BVh- 1 , and thenw ith 70% aqueousethanol by 1, 2, 3,4 and 5 BVh- 1. The e lu ting so lu tion w as co llected and the conten t o f to tal flavono ids w as de term ined as the results shown in Fig

43、ure 9. F rom Figu re 9 it is show n that the desorp-tion rate w as the highest (abou t 90%) when the e lu-ting speed w as 1 BV h- 1 , w hich was no t higher than tha t o f 2 BV h- 1. Fo r the sake o f h igh efficiency, 2 BV h- 1 w as used.D eterm ination of eluting term ina lSaturated resin w as elu

44、ted w ith 70% aqueous e th-ano l by 2 BVh- 1.The e lu ting so lu tion w as collectedand the con tent oftota l flavonoids w as dete rm ined asthe resu lts show n in Figu re 10. From F igure 10,it canbe seen thatthe to tal flavonoids w ere eluteden tirelywhen the dosage of eluting so lvent w as 5 BV.m

45、 adzu, Japan) is m ost effective. W hen the ex tract so-lu tion o f Rad ix Puerariae is in the concentra tion of 0. 5L IU Huo-an, et a l:O ptim ization of P repa ra tive Sepa ra tion and Purification of T o ta l125F lavonoids from Rad ix Puerariae by M ac ropo rous R esin M ethodis 60 BV w ith the a

46、dsorp tion speed 2 BVh- 1 , and thevo lum e of aqueous. 70% (V /V ) e thano l as eluant is 5BV w ith deso rp tion speed 2 BV h- 1.The conten t o ftheto tal flavonoids can reach m o re than80% accord-ingto the above optim a l technical conditions.Acknow ledgem entsThe authors gratefully acknow ledget

47、hefinancial sup-port of the Science and Technology Comm ittee ofChongqingF igure 9 E xperim ental results of the eluting speedin China (G rantNumber:CSTC. 2004BB5122).Figure 10Content of flavonoids under different eluting volumesPurity of prepared tota l flavonoids from Radix PuerariaeReferences 1F

48、ang Q. Som e current study and re search app roaches re la-tingto the use of plan ts in the trad itiona l Ch inese m edicine J .J E thnopharmacol,1980, 2 (1):57-63. 2K eung WM , V a llee BL.Therapeu tic lessons from tradi-tiona l orienta l medic ineto contem po ra ry occidenta l pha r-maco logy J .E

49、XS, 1994,71:371-381. 3K eung WM ,V a llee BL.D aidzin andda idze in suppressfree-choice ethanol in take by Syrian go lden ham sters J .Proc N atl Acad SciUSA,1993,90 (21):10008-10012. 4R oeck le in JC, Leung PS.Aprofile of econom ic plants M . N ew Brunsw ick,USA:T ransaction Book, 1987. 5Lin RC, G uth rie S,X ieCY,et a .lIso flavonoid com-Ten m illiliters o f crude drug so lu tion w as ad justedpounds extrac