ii

Mc lc

DANH MC CC CH VIT TT.................................................3 T VN .......................................................................................4 Chng 1 TNG QUAN..................................................................51.1. C IM THC VT..............................................................................5

1.1.1.V tr phn loi: [20][21]...............................................................5 1.1.2.Hnh thi thc vt..........................................................................6 1.1.3.Phn b, sinh thi..........................................................................6 1.1.4.B phn dng, thu hi v ch bin................................................61.2. THNH PHN HA HC..........................................................................7 1.3. TC DNG DC L..............................................................................12

Chng 2 I TNG V PHNG PHP NGHIN CU 172.1. I TNG NGHIN CU....................................................................17

2.1.1.Nguyn liu.................................................................................17 2.1.2.Dung mi, ha cht.....................................................................17 2.1.3.Thit b, dng c.........................................................................172.2. PHNG PHP NGHIN CU..............................................................17

Chng 3 KT QU NGHIN CU:..........................................20 3.1. Phng php 1: THY PHN ISOFLAVONE T DCH CHIT U NNH TON PHN[31]..........................................203.2 Phng php 2: THY PHN ISOFLAVONE SAU KHI PHN LP TNG ISOFLAVONE GLYCOSID RING[34]...........................................34

Chng 4 BN LUN....................................................................37 Chng 5 KT LUN V NGH...........................................38 TI LIU THAM KHO.................................................................38

3

DANH MC CC CH VIT TTEtOH: Ethanol HDL-C: lipoprotein t trng cao ton phn HPLC: sc k lng hiu nng cao LDL-C: lipoprotein t trng thp ton phn MeOH: Methanol SFE: chit bng cht lng siu ti hn

4

T VN u nnh v nhng ch phm t u nnh nh u ph, sa u nnh t lu c ngi chu s dng nh mt thc phm giu cht dinh dng. Trong nhng thp k gn y, nhiu nghin cu dch t c thc hin v cho thy vic s dng u nnh lm gim nguy c ca nhiu bnh ung th[1] nh ung th v[2][3], ung th tuyn tin lit[4], ung th rut kt[5], long xng[6][8], triu chng hu mn kinh[6][7][8] v bnh ng mch vnh[7][9] Chnh v vy, u nnh tr thnh ti thu ht nhiu nh nghin cu trn th gii, nht l trong bi cnh hin nay, nhu cu s dng estrogen thc vt thay cho tr liu hormone truyn thng i vi ph n mn kinh hoc sau mn kinh ngy cng gia tng[10][11]. T , hng lot cc nghin cu v thnh phn hot cht v phng php phn lp chng ra khi u nnh c tin hnh. Kt qu cho thy u nnh l mt trong nhng ngun cung cp isoflavone di do nht[12]. Isoflavone trong u nnh tn ti ch yu dng glycoside: 6 - O malonylglucosides, 6 - O - acetyl glucoside, -glucoside v him khi dnh aglycone[13] [14][15][16][17]. Bn cnh , nhng nghin cu lm sng cng cho thy isoflavone dng aglycone c s hp thu nhanh hn v nhiu hn so vi dng glycoside cng liu dng[18]. iu gi rng mt ch phm giu isoflavone dng aglycone s tt hn mt cht phm giu isoflavone dng glycoside. Do , trong nghin cu ny, chng ti s kho st iu kin ti u chit xut isoflavone dng aglycone t u nnh.

5

Chng 1 TNG QUAN1.1. C IM THC VTTn gi: Tn thng gi: u tng, u nnh Tn khoa hc: Glycine max (L) Merr. H: u (Fabaceae)[19]

1.1.1. V tr phn loi: [20][21]Theo h thng phn loi Takhtajan, cy u nnh c v tr phn loi nh sau: Ngnh Ngc Lan (Mangnoliophyta)

Lp Ngc Lan (Magnoliopsida)

B u (Fabales)

H u(Fabaceae)

Hnh 1: cy u nnh

Chi Glycine

Glycine max S 1: v tr phn loi ca u nnh

6

1.1.2. Hnh thi thc vt Cy tho hng nm, c thn mnh, cao 50-150 cm, c lng, cc cnh hng ln trn. L mc so le, c 3 l cht hnh tri xoan, gn mi nhn, gai hi khng u. Hoa trng hay tm, xp thnh chm nch. Qu thng, hnh lim, c nhiu lng mm, mu vng lc hoc xanh nht, tht li gia cc ht. C 2-5 ht, hnh cu hoc hnh thn, c mu sc thay i ty theo ging[22]. Ra hoa vo thng 6-7, cho qu vo thng 7-9.[22]

1.1.3. Phn b, sinh thi Cy c xut x t Trung Quc[22]. Trng ph bin khp Vit Nam, nht l cc tnh Trung du, min ni pha Bc: Lai Chu (Ta Cha, Mng Lan, Qunh Mai), Lo Cai(Mng Khng, Bc H), Cao Bng, Lng Sn, Bc Giang(Lc Ngn) v cc tnh min ng Nam B. Ngoi ra, cn c trng nhiu nc nhit i v cn nhit i trn th gii, c bit Trung Quc v Hoa K[20].

1.1.4. B phn dng, thu hi v ch binB phn dng : ht

Hnh 2: ht u nnh ti

7

1.2. THNH PHN HA HCTrong ht u nnh c cc thnh phn bit: Protid 40%, lipid 12-25%, glucid 1015%; cc mui khong nh Ca, Fe, Mg, P, K, Na, S; cc vitamin A, B1, B2, D, E, F; cc men, sp, nha, cellulose. Theo GS. Tt Li[20], ton cy cha 12% nc, 16% glucid, 14-15% protein, 6% mui khong v cc cht khc khng c nit.

- Protein.Nhiu nghin cu ch ra rng, ht u nnh c gi tr dinh dng rt cao. Thnh phn protein trong ht u nnh cha 8 loi amino axit thit yu : tryptophan, threonine, isoleuxin, valin, lysin, methionin, phenylalanin v leucin. Hm lng ca cc cht amino axit ny tng ng vi hm lng amino acid ca trng g, c bit l tryptophan, gn gp ri ca trng[23]. Protein trong ht u nnh cha khong trn 38% tu loi. Hin nay, nhiu ging u nnh c hm lng protein c bit cao (ti 40-50%). C nhng ch phm ca u nnh mang ti 90-95% protein, y l ngun protein thc vt c gi tr cao cung cp cho con ngi[24]. Protein ca u nnh d tiu ho, khng c cholesterol v t cht bo bo ho nh thng c tht ng vt. Hm lng protein ca u nnh cng cao hn c tht c v gn gp i cc loi u khc[23]. -

Vitamin v cht khong.

Khng ch giu protein hn bt c loi thc phm no, k c tht ng vt. u nnh cn rt giu vitamin v cht khong. Theo GS. Tt Li[20], ht u nnh cha trung bnh 8% nc, 4-5% cht v c, trong Kali 2%, Natri 0,38%, Canxi 0,23%, photpho 0,65%, Magi 0,24%, lu hunh 0,45%. u nnh cha cc vitamin tan trong nc nh: cc vitamin B1, B2, B3, B6; vitamin PP v cha cc vitamin tan trong du nh: vitamin A, vitamin D, vitamin E (trong u nnh chu v chu M), vitamin K, vitamin F khng c vitamin C. Cc vitamin A v D xut hin khi ht mi chn, sau

8 b men oxy ho ph hu. Trong nhm vitamin B, u nnh cha lng vitamin B 1 gp 3 lng vitamin B1 trong sa bt v trong bt nhng loi u khc. Lng vitamin B2 cha t hn trong sa bt khong 1/3 nhng li gp 6 ln so vi mt s loi u khc. -

Cht bo v cht cholesterol.

u nnh l thc phm nhiu protein nhng li t calorit, t cht bo bo ho v hon ton khng c cholesterol[23]. Cht bo chim khong 15-20%, c khi t ti 23%. T l phn trm ca cc glycerid axit bo: linolein 49,3%, olein 32%, linolenin 2%, panmitin 6,5%, stearin 4,2%, aracgidin 0,7%, lignoxerin 0,1% v 0,5% axit panmitoleic[20]. So snh vi cc loi u khc th u nnh c hm lng acid bo thit yu cao hn. Tng lng cht bo chim khong 18%, cn thnh phn cacbonhydrat chim khong 31%[22].

- Du u nnhDu u nnh thuc loi du kh nh du ht lanh, cha khong 14% cht bo bo ho, 59% cht bo khng bo ho nhiu ni i, 23% cht bo khng bo ho mt ni i. Trong s lng cht bo khng bo ho nhiu ni i li cha 8% linoleic acid (omega 3- fatty acid)[22][25], him c trong cc thc phm ngun gc thc vt[20] [23]. Ngoi ra, trong du u nnh cn c cc phospholipid, ch yu l lecitin (1-5%). Ngoi ra, du bo cn c cc cht steron nh: stigmasteron, sitosteron v mt s sapogenol khc[25] Isoflavone Isoflavone l thnh phn hot cht chnh ca u nnh. Qua phn tch, cho thy isoflavone trong u nnh l mt hp cht phenolic gm c: aglycone (daidzein, genistein v glycitein); glucosid (genistin, daidzin, glycitin); glycosid kt hp vi nhm malonyl ( 6 - O malonyldaidzin, 6 O

9 malonylgenistin v 6 O malonylglycitin); - glycosid kt hp vi nhm acetyl ( 6 O axetyldaidzin, 6 O - axetylgenistin v 6 O axetylglycitin) [27]. Trong ht u nnh cc aglycone chim mt lng nh, l cc dn xut malonyl v dn xut acetyl ca glycosid. Ngoi ra, cn cha cc aglycone : sissotrin, ononin; cc dn xut acetyl ca glycosid : 6 acetylsissotrin, 6 acetylononin; cc dn xut malonyl ca glycosid : 6 - malonylsissotrin, 6 malonylononin. Nm 2006 cc nh khoa hc Hn Quc thc hin mt nghin cu so snh thnh phn isoflavone trong phi, l mm, ht v v ht u nnh. Kt qu nhn c, tng t l trung bnh ca isoflavone l 2887g/g trong phi, 575g/g trong ht, 325g/g trong l mm, 33g/g trong v ht. Cc nh khoa hc cng phn tch c 12 ng phn isoflavone. Cu trc c bn ca isoflavone gm 2 vng bezen: A v B ni vi mt d vng pyron.

10 Bng 1: Cu trc ha hc ca isoflavones

11 Tnh cht l ha ca cc isoflavone:

L tnh ca Isoflavone:

1.Trng thi: -Dng glycosid thng kh kt tinh. -Dng genin thng kt tinh, im chy cao. 2.Mu sc : Isoflavone: khng mu n mu vng nht 3. ha tan: tan ca genin: -Tan/dung mi phn cc n phn cc trung bnh. -Km tan trong dung mi km phn cc. -Tan / kim long, km tan/ dung dch acid. tan ca glycosid: -Tan trong MeOH, EtOH -Mch ng di/nhiu: c th tan trong nc nng. 4.Ph UV: Bng II: 245-275 (nm) 5.Ph IR: -Thng dng nh danh (so vi ph chun). -Ch cc bng trong cc vng: 3300-3400( OH linh ng ) 3000( Vng thm ) 1450-1650( Vng pyron) 1050-1150( Carbinol; C-O )

Bng I: 310-330 (nm)

Ha tnh nhm Isoflavone:

1.Kim phn Isoflavone:

12 Trong mi trng kim, cc isoflavone c H-5 s b mt mt carbon C-2, to deoxybenzoin v formiat. 2.Ha tnh ca nhm OH: -Tnh acid yu. -To glycosid vi ng. -To mui vi kim long: => phenolat km bn (tng mu) -To phc vi ion kim loi(Al+++, Fe++, Mg++, Pb++, Zn++, Zr++) => chuyn dch max. 3.Ha tnh ca vng -pyron: -Tnh kim: kim yu, vi acid to mui km bn. -Tnh oxy ha kh: Tnh oxy ha: vng -pyron=> vng -pyrillium (tng mu, quan trng/ nh

tnh) Tnh kh: to OH phenol

4.Ha tnh ca cc vng thm: Phn ng th azoic: to mu, dng nh tnh.

1.3. TC DNG DC LTc dng ca cc hot cht chnh trong u nnh:[25][26][28] Ch yu c tc dng trn tr triu chng mn kinh, trn xng, trn tim mch v ung th. 1.3.1. Protease inhibitor: Protease inhibitor c tc dng chng li s pht trin mm ung th kt trng, ung th phi, ung th ming[24][29] Ngn nga s tc ng ca mt s gen di truyn gy nn chng ung th. N cng bo v cc t bo c th khng cho h hi, gy nn bi s tc ng ca mi trng xung quanh nh tia nng phng x v cc cht c th tn cng ADN[24][29]. 1.3.2. Phytate Cc nh khoa hc chng minh phytate khng nhng c tc dng ngn nga mm ung th m cn c kh nng ngn nga bnh tim mch.

13 Phytates c tc dng ging nh cht antioxydant, vitamin C, Beta carotin. 1.3.3. Phytosterol: Phytosterol ch c trong cc thc phm rau, u. Phytesterol c tc dng ngn nga cc bnh v tim mch qua vic cnh tranh hp thu vi cholesterol rut. Do cholesterol khng vo mu c m phi bi tit ra ngoi, lng cholesterol trong mu gim, mc gim tu tng c th. Phytosterol cng c kh nng lm gim s pht trin cc bu ung th kt trng v chng li ung th da. 1.3.4. Saponin Saponin l c c tnh ging nh antioxidant, trc tip ngn cn s pht trin ung th kt trng ng thi lm gim lng cholesterol trong mu. 1.3.5. Phenolic acid: Phenolic acid c hot tnh chng oxy ho v phng nga cc nhim sc th AND khi b tn cng bi nhng t bo ung th. 1.3.6. Lecithin: Lecithin ng mt vai tr quyt nh trong vic kch thch s bin dng khp cc t bo c th. Lecithin c kh nng lm gia tng tr nh bng cch nui dng tt cc t bo no v h thn kinh, lm vng chc cc tuyn v ti to cc m t bo c th. Ngoi ra, lecithin c tc dng ci thin h thng tun hon, b xng v tng cng sc khng. Khi h thn kinh thiu nng lng, cht lecithin u nnh s phc hi nng lng mt. 1.3.7. Browman Birk Inhibitor (BBI) : BBI l mt cht mi nht tm thy trong u nnh, c kh nng ngn cn tin trnh pht trin mm ung th vi hai dng tinh ch PBBI v c c BBIC. Theo bo co kt qu tng trnh ti hi ngh khoa hc th gii v vai tr ca u nnh trong vic phng v tr bnh (1996) th PBBI v BBIC kim sot c s pht trin tin trnh ung th ming, v, rut gi, gan, phi, thc qun c cc t bo trong ng th nghim ln cc con chut bch v chut ng. Hin nay BBI c dng trn con ngi vi trung tm nghin cu v kt qu s b rt kh quan. BBIC c thm nh l loi thuc mi bi c quan F.D.A (the U.S.Food and Drug Administration).

14 1.3.8. Acid bo Omega 3 : Axit bo Omega 3 l loi cht bo khng bo ho c kh nng lm gim lng LDL-C, gia tng lng HDL-C trong mu, gip ngn chn s pht trin ca cc bnh tim mch. 1.3.9. Isoflavone: Cc hot cht c tc dng phytoestrogen trong ht u nnh gm ch yu l daidzin, genistin. Tc dng ca isoflavone u nnh trong phng v iu tr bnh: Nghin cu thm d trn lm sng cho thy nhng tc dng c li ca u nnh trn cc triu chng vn mch tui mn kinh: isoflavone trong u nnh lm gim cng bc ho, gim s ln m hi m, mang li li ch hin nhin nng cao cht lng sng ca ph n sau mn kinh. Isoflavone u nnh khng gy thay i c ngha v FSH hoc v dy ca mng trong t cung. Khng c ngi no phn nn v ri lon v, khng thy tng cc tc dng ph khng mong mun nh estrogen. V vy, c th khuyn co dng isoflavone u nnh cho ph n no c chng ch nh dng liu php hormon thay th hoc khng mun dng liu php hormone thay th v l do c nhn. Isoflavone u nnh cn cho thy c ci thin trn qu trnh dinh dng da. Tuy nhin, khng nn dng liu qu cao trnh gy ra nhng tc dng estrogen khng mong mun. 1.3.9.1. Tc dng trn chuyn ho ca xng: Hiu lc ca isoflavone u nnh trn qu trnh dinh dng ca xng ph n sau mn kinh cho thy tng c ngha v mt khong xng ti cc t sng L2 L4 v lm gim nguy c long xng nh c ch c hot tnh ca hu ct bo, nn c tc dng hip ng chng tiu xng nh hot tnh estrogen ca thuc ny. 1.3.9.2. Tc dng trn tim mch: Ch dinh dng giu u nnh s lm gim nguy c bnh ng mch vnh. Isoflavone u nnh c nhng tc dng khc nhau chng ri lon lipid mu ngi mn kinh dn n gim cc nguy c tim mch. u nnh v chit xut ca u nnh cng c nhng tc dng khc c li cho tim mch, c s kt bi nhm cc

15 chuyn gia Hi Mn kinh Bc M nh: lm gim huyt p tm trng, gim cholestreron ton phn, gim LDL C, gim triglycerid, tng HDL C, chng kt tp tiu cu, ngn chn s tin trin ca cc mng x va, ci thin tnh n hi ng mch, chng oxy ho, loi b cc gc t do, i khng vi tc hi ca s lipoperoxy ho ca lecithin v ca LDL C sn sinh ra cc sn phm cui cng c hi, v cc sn phm ny c i lc rt cao vi thnh ng mch v gy ra nhng mng va x. Da vo cc kt qu trn, c quan Thc phm v Dc phm (FDA) Hoa K chp nhn t nm 1999, dng u nnh cng ch dinh dng ngho acid bo no, ngho cholesterol lm gim nguy c bnh ng mch vnh. 1.3.9.3. Tc dng trn si thn NXB y hc , trang 930 -932,2004.: Vi ngi mc bnh thn hay chc nng thn suy yu, gim bi tit m l mt phng thc tr liu gim nng nhc cho thn. Khi thay vic cung cp m ng vt bng m thc vt nh t u nnh th s lng protein trong nc tiu gim. iu chng t vic gim gnh nng cho thn. u nnh cng lm gim nguy c bnh si thn bng cch khng canxi tht thot qua nc tiu. 1.3.9.4. Tc dng trn cc khi u ph thuc hormon: Cc nghin cu v thc nghim, lm sng v v dch t hc chng minh hiu lc ca phytoestrogen lm gim nguy c mt s loi u ph thuc hormon ( nh u mng trong t cung, v v bung trng ). Tuy kt qu cha hon ton chc chn, c th do isoflavone u nnh ch yu kch thch -estrogen receptor, m -estrogen receptor li him gp cc c quan nhy cm nht vi estrogen.1.4. Cng dng:

Thng dng lm thc n bi b c th, nht l i vi tr em, ngi b i tho ng, ngi lm vic qu sc, thiu khong v lm vic tr c, ngi mi m dy, ngi b thp khp, thng phong. Ngi ta thng dng ch thc phm nh: u ph, chao, bt u nnh, hoc ch sa u nnh, bt u nnh trn vi bt ng cc, ca cao dng lm bt dinh dng cho tr s sinh.

16 m u tng tr bnh phong hn ngoi cm, st khng ra m hi, nhc u, au mt, thng tiu, bng y kh chu, mt mi, ng khng c, nn ma. Ngy dng 16-20g dng thuc bt hoc thuc sc[22].

17

Chng 2 I TNG V PHNG PHP NGHIN CU2.1. I TNG NGHIN CU 2.1.1. Nguyn liuBt u nnh: sn phm ca cng ty Sigma Aldrich Isoflavone: genistein, daidzein v glycitein tinh khit 99% t LC Labs. Ethanol (96%): ca hng Riedel de Hae acid hydrocloric 37% v acid acetic: ca hng Fisher Scientific Ethanol tinh khit (Merck) v methanol (Mallinck-rodt) dng cho HPLC. Nc: cung cp bi h thng Milli-Q (Millipore) Carbon dioxide (CO2, 99,9%): t Beiji ng Analytical Instrument Factory. Methanol tinh khit: Merk

2.1.2. Dung mi, ha cht-

-

-

2.1.3. Thit b, dng c-Thit b: h thng HPLC Agilent 1100 (gm c h thng tim mu, my ht chn khng, h thng bm mu t ng v u d UV-DAD), ct pha o C18 (Phenomenex, 5m, 250 x 4,6 mm i.d) - HPLC phn tch: h thng sc k (DIONEX, Germany) gm: bm HPLC P680, h thng bm mu t ng ASI-100, bung n nh nhit TCC-100, u d dy diode quang PDA-100 v b phn ghi nhn tn hiu Chromeleon. - HPLC iu ch: bm Waters 515, u d 2487, phn mm N2000 (Zhejiang University, Hangzhou, China). - H thng chit bng cht lng siu ti hn: Suprex HA.111-05-20 ( cng ty TNHH Hua An SFE., thnh ph Nan Tong, Jiang Su Province, China). - Ct polymethacrylate 4 x 70 ,Toso Hass CG71.

2.2. PHNG PHP NGHIN CUTrong ht u nnh, isoflavone tn ti ch yu dng glycozid,v mc ch ca nghin cu l phn lp isoflavone dng aglycone nn cn thit phi qua giai on thy phn.

18 C nhiu phng php thy phn isoflavone dng glycozid, trong hai tc nhn thy phn thng dng l acid[31] hay enzyme (-glucosidase)[32]. y, v vn gii hn v thi gian, nn ch kho st tc nhn thy phn, v tc nhn c chn l acid v kh nng thy phn han ton ca n so vi -glucosidase ( ch thy phn c glucosyl isoflavone v khng c tc dng vi malonyl-isoflavone, hn na hot tnh enzyme s b c ch bi mt s ion kim loi nh l Hg2+, Cu3+)[33]. C nhiu phng php thu c isoflavone dng aglycone tinh khit, c th thy phn dch chit ton phn sau phn lp tng isoflavone c th[31] hay l phn lp cc isoflvone dng glycoside, sau mi tin hnh thy phn tng phn on ring bit[34]. C hai phng php trn ln lt c kho st di y. 2.1.4. Phng php 1: THY PHN ISOFLAVONE T U DCH CHIT TON PHN[31]: Quy trnh chit xut gm 4 bc chnh: Chit xut

Thy phn Tinh ch

Phn lp

S 2: quy trnh tng qut -Bc 1: chit xut isoflavone t bt u nnh bng dung mi hu c: + Bt u nnh c chit vi EtOH 40-99,99% vi cc t l EtOH-bt t 1:1 n 10:1 (tt/kl)

19 +Hn hp thu c un nng cc nhit khc nhau (40-900C) trong 2-24 gi, c khuy trn. +Sau , hn hp c lc qua chn khng vi mng lc giy s 1 (Whatman), loi cc cu t khng tan. -Bc 2: giai on thy phn, nhm mc ch chuyn cc cu t isoflavone dng glycosid thnh dng aglycon.+

Dch chit sau khi lc c thy phn vi acid HCl 37% hay acid acetic tinh khit,

v pH ca hn hp cui cng l khong 1-5. +Hn hp c un nng nhit 40-900C trong 1-12 gi, c khuy trn. -Bc 3: giai on tinh ch: + Sn phm thy phn c trn vi nc ct vi t l 1:15 n 1:1 (ml/ml), khuy trn nhit phng. +:Ta tinh th c tch bng cch lc qua my lc chn khng, mng lc 0,2m (Millipore). Cht rn thu c trn mng lc c lm kh v ha tan vo EtOH tinh khit. -Bc 4: phn lp bng h thng HPLC ch ha. 2.2.2. Phng php 2: THY PHN ISOFLAVONE SAU KHI PHN LP TNG ISOFLAVONE GLYCOSID RING[34]. Mt cch khc phn lp isoflavone dng aglycone, thay v thy phn dng glycoside ngay sau khi chit bng cn,ta c th dng sc k ct tch ring tng phn on isoflavone trong dch chit cn lc u, sau khi thu c tng isoflavone dng glycoside tinh khit mi tin hnh thy phn thu c dng aglycone tng ng. Cch thc hin: Bt u nnh c chit bng cn ( MeOH hay EtOH) Dch chit bt u nnh ton phn c pha long vi nc ct cho n khi t cn cui cng l 20%. Hn hp thu c c lc loi nhng phn t khng tan.

20 Bc tip theo, tin hnh phn lp isoflavone: v hn hp thu c khng ch cha isoflavone m cn ln cc tp tan trong cn 20% nh l cc phenol, protein, cc ng n hay ng phc Nhm loi c nhng tp khng mong mun ny, ng thi gi li cc isoflavone, hn hp c np ln ct pha o (polymethacrylate hay C-18). Da vo c ch hp ph: cc isoflavone c i lc mnh vi pha tnh s c hp ph li trn ct trong khi nhng cht i lc thp hn s b ragii khi ct. Pha ng l nc-alcol vi cc t l khc nhau. Hai alcol c a chung l methanol v ethanol. Ch rng ta phi tng dn t l dung mi mnh ( y l alcol, v y l ct pha o nn dung mi km phn cc l dung mi mnh) so vi nc ch khng c lm ngc li v nu mt lng ln alcol c s dng ngay t u, nhng isoflavone c i lc mnh hn vi pha tnh, thay v c hp ph, cng s b ragii khi ct cng vi nhng isoflavone c i lc thp hn v khi vic phn lp s khng hiu qu. Sau khi thu c cc phn on khc nhau, c thu hi dung mi tng phn on, ta thu c cc sn phm th. Bc tip theo v cng l bc cui cng trong quy trnh l kt tinh v tinh ch cc isoflavone. u tin, sn phm th thu c t tng phn an bc trn c ha tan trong mt lng dung mi thch hp (khong 1 mg/ml). Dung mi c a chung l methanol, mc d vy ethanol cng vn c s dng. hn hp c ty mu bng than hot v c lc loi than hot. dung dch thu c c c thu hi dung mi v sau kt tinh li trong methanol nhit thp (trong t lnh). Tinh th c lc qua phu thy tinh v lm kh.

Chng 3 KT QU NGHIN CU: 3.1. Phng php 1: THY PHN ISOFLAVONE T DCH CHIT U NNH TON PHN[31]3.1.1. Chit xut:

21 Nhm mc ch tm ra iu kin ti u chit xut v phn lp isoflavone t u nnh ta tin hnh thm d cc iu kin chit xut khc nhau gm loi dung mi, thi gian v nhit trong qu trnh chit xut. Do b phn dng y l ht, c cha nhiu cht bo d tr, loi bt tp km phn cc, trc khi chit nn loi cht bo khng phn cc trong bt u nnh bng cch chit bt vi cht lng siu ti hn (CO2, 99,9%) trong thit b Suprex HA.111-0520[35]. Lai dung mi: u tin, cn kho st tm loi dung mi chit xut ph hp nhm mc ch thu c nhiu isoflavone ng thi hn ch gi thnh sn phm. Dung mi c chn chit xut isoflavone t u nnh l cn, v tnh thn thin vi mi trng, kinh t, cng l dung mi thng c s dung chit xut nhm hot cht flavonoid. Ta cn kho st nng cn ti u cho hiu qu kinh t cao nht. Cc mu bt u nnh ln lt c chit bng EtOH vi cc cn khc nhau t 4099,99% v vi cc t l EtOH-bt t 1:1 n 10:1 (tt/kl). Sau , hm lng isoflavone c nh gi bng h thng HPLC. Kt qu cho thy: Trong s nhng dung mi s dng, dch chit t EtOH tinh khit c hm lng isoflavone ton phn cao hn nhiu so vi dch chit EtOH-nc (70%) (ch yu l genistein v daidzein dng malonyl v glycosyl). Trong EtOH-nc 96% cho dch chit c hm lng isoflavone tng t nh EtOH tinh khit, nn chn EtOH-nc 96% lm dung mi chit xut mu ln do gi thnh r hn. T l dung mi: Trong qu trnh chit xut, quan st thy hm lng isoflavone ton phn ph thuc vo t l th tch EtOH v khi lng bt u nnh. Kho st t l dung mi thch hp bng cch chit cc mu bt u nnh nhit 62 0C trong 8 gi vi cc t l dung mi- bt khc nhau t 3:1 n 8:1( Hnh 3:A). Kt qu cho thy: t l dung mi-bt thp 3:1 cho hm lng isoflavone cao hn v t tn dung mi hn t l 5:1 v 8:1 nn c chn la cho quy trnh chit mu ln.

22 Tip tc kho st thi gian chit xut ti u. Cc mu ln lt c chit cc khong thi gian khc nhau: 2, 4, 6, 8 v 11 gi, nhit 650C, t l dung mi- bt u nnh l 3:1 Thi gian chit xut: Kho st vi t l dung mi- bt u nnh l 3:1, nhit 650C vi thi gian chit khc nhau ln lt l 2, 4, 6, 8 v 11 gi. Kt qu cho thy lung isoflanone thu c tng theo thi gian chit. Lc u lng isoflavone tng nhiu khi thi gian chit tng, nhng sau thi gian chit 8 gi th lng isoflavone gn nh t cci, khi tip tc ko di thi gian chit th lng isoflavone tng khng ng k (hnh 3: B). Cn nhc gia lng isoflavone thu c v s tiu tn thi gian, nhn lc chn thi gian chit l 8 gi lm iu kin ti u. Nhit : Nhit cng nh hng nhiu n hiu sut chit. Kho st bng cch chit cc mu bt u nnh cc nhit khc nhau (65-100 0C), t l dung mi-bt u nnh l 3:1 trong 8 gi. Kt qu cho thy: Hm lng isoflavone tng ln khi tng nhit chit xut (hnh 3: C). Lng isoflavone thu c ti a khi nhit chit xut l 80 0C, chit nhit cao hn khng cho lng isoflavone nhiu hn, thm ch cn thy c s gim st v tng lng isoflavone thu c, do chn nhit 800C lm nhit ti u. Tm li: iu kin chit xut ti u c xc nh l dung cn 960, chit nhit 800C trong 8 gi vi t l dung mi-bt u nnh l 3:1.

23

Hnh 3: kt qu kho st quy trnh chit xut A: hm lng isoflavone (aglycone tng ng) t u nnh (g isolavone/g bt u nnh) bng cch chit vi cc t l dung mi khc nhau, t0=620C, trong 8 gi. B: hm lng isoflavone vi cc khong thi gian chit khc nhau, t l dung mi-bt u nnh l 3:1, nhit 650C. C: chit cc nhit khc nhau, t l dung mi-bt u nnh l 3:1 trong 8 gi.

24 Hm lng Glycitein c b qua v hm lng trong isoflavone ton phn qu thp (thng di 5%). 3.1.2. Kho st iu kin thy phn: Thnh phn isoflavone trong dch chit u nnh gm hn hp aglycon, epigenin, v dng glycosid ( gm cc -glycosid, 6-O-acetyl--glycosid, 6-O-Malonyl-glycosid). thu c dng aglycon, phi qua phng php thy phn. Vic thy phn phi m bo sn phm thu c khng phi l artifact, phi loi c hon ton tc nhn thy phn ra khi sn phm cui cng, ng thi hiu sut thu c phi l cao nht. Do lin quan n nhiu vn phc tp nh tc nhn thy phn, thi gian v nhit trong qu trnh thy phn nn y ta kho st nhng iu kin thy phn khc nhau nhm mc ch tim ra iu kin thy phn ti u. Cc mu ln lt c thy phn di nhng iu kin khc nhau v nh hng ca cc thng s thy phn ln lng aglycon ton phn c phn tch bng h thng HPLC. Hm lng aglycone ( ch yu l genistein v daidzein) c lin quan vi nng acid, thi gian, v nhit thy phn (hnh 4), trong , thng s quyt nh l nng acid v thi gian thy phn. Nng acid: u tin cc mu c thy phn vi cc nng acid khc nhau, nhit 800C trong 2 gi. Tc nhn thy phn c chn l acid HCl (37%). Kt qu cho thy s thy phn khng hiu qu khi s dng nng acid thp, hm lng aglycon thu c tng theo nng acid s dng. Tuy nhin, nng acid cao gy kh khn trong quy trnh loi acid d trong sn phm kt tinh, iu nay nh hng n tinh khit ca sn phm thu c. T kt qu kho st (hnh 4: A), cn nhc gia hiu sut thy phn v d lng acid trong sn phm thu c, chn nng acid HCl khong 0,13 mol/L. Nhit thy phn: Cc mu c thy phn vi nng acid l 1 mol/lit trong 2 gi cc nhit khc nhau 600C, 700C v 800C.

25 Kt qu cho thy s thy phn khng hiu qu nhit thp (hnh 4: B), hiu sut thy phn tng theo nhit s dng ( nghin cu ny ch kho st ti nhit 800C) . Do , y ta chn nhit 800C lm iu kin thy phn ti u. Thi gian thy phn: S dng nng acid HCl 0,13 mol/L v nhit 800C kho st thi gian chit ti u , kt qu cho thy 6 gi l thi gian chuyn hu ht dng glycosid sang dng aglycone (hnh 4:C). Vy: iu kin thy phn c chn l nng acid 0,13 mol/lt, nhit 80 0C trong 6 gi.

26

Hnh 4: hiu sut thy phn ( phn trm glycosid chuyn sang aglycone) A: vi nng acid khc nhau nhit 800C trong 2 gi. B: nhit khc nhau vi nng acid l 1 mol/L trong 2 gi. C: thi gian thy phn khc nhau vi nng acid l 0,13 mol/L 800C. Hiu sut ca glycitein c b qua bi v hm lng qu nh trong isoflavone ton phn (thng nhp hn 5%).

27 3.1.3. Tinh ch: Mt trong nhng phng php thu c tinh th l s dng dung mi m cht mun kt tnh km tan trong . Isoflavone l mt cht kh km phn cc, nn kt tinh trong dung mi phn cc, y chn nc v nc ct l dung mi rt phn cc, kinh t ng thi v hi cho mi trng v con ngi. V t l nc ct s dng c nh hng trc tip n lng kt tinh thu c, nn cn kho st lng nc thch hp. Kho st vi cc t l nc ct-dch thy phn khc nhau t 2:1 n 10:1(hnh 5). Trong th nghim ny cho thy lng kt tinh thu c nhiu hn khi s dng lng nc nhiu hn. Mt d vy, lng nc qu ln dn n s hao ht nhiu giai on lc, gy kh khn cho quy trnh sn xut. T l cui cng c chn l 4:1 bi v vi t l nc cao hn khng cho lng kt tinh nhiu hn ng k (hnh 5). y, t l 4:1 c kh nng thu hi n 83% genistein v 65% daidzein. Kt lun: kt tinh li isoflavone dng aglycone bng nc ct, t l nc ct-hn hp thy phn l 4:1.

Hnh 5: hiu sut thu hi isoflavone vi t l nc ct-hn hp thy phn khc nhau. Hiu sut ca glycitein c b qua do hm lng trong isoflavone ton phn qu thp (thng thp hn 5%).

28

Bt u nnh

SFE (CO2, 99,9%) Bt u nnh sau khi loai cht bo EtOH-bt u nnh=3:1 T=800C, 8 gi

Dch chit EtOH 96%

Acid HCl 0,13 mol/l t= 800C, 6 gi

Dch thy phn

Nc ct-dch thy phn: 4:1

Kt tinh isoflavone ton phn (dng aglycone)

S 3: s chit xut v phn lp isoflavone dnh aglycone t u nnh.

29

nh gi hm lng tng isoflavone trong isoflavone ton phn sau khi ti u ha: Chun b mu: Isoflavone chun c pha trong ethanol tinh khit vi hai dy nng khc nhau: Genistein v daidzein: 62,5-500 g/ml Glycitein: 12,5-100 g/ml

Phn tch HPLC: -Thit b: h thng HPLC Agilent 1100 (gm c h thng tim mu, my ht chn khng, h thng bm mu t ng v u d UV-DAD), ct pha o C18 (Phenomenex, 5m, 250 x 4,6 mm i.d) -Th tch bm mu l 10 l. - Pha ng: nuc ct vi 0,1% acid acetic (A) v methanol (B). Dung mi ragii thay i t l tuyn tnh t 30 n 50% B bt u t 0 pht n 45 pht. -Tc dng l 1,0 ml/pht. -Nhit ca ct c gi 400C. -u d UV bc sng 255 nm. Vic nhn dng cc isoflavone da vo vic so snh vi thi gian lu v ph UV ca cht chun. Vic nh lng s dng ng cong chun. Dung dch chun c bm v o 3 ln lin tip. ng cong chun c xy dng bng nhng gi tr trung bnh din tch peak ca dung dch chun nhng nng xc nh. Cc thng s ca ng cong nh l dc (b), khong gii hn (a), h s tng quan (R2) c s dng nh gi nng isoflavone ca mi mu v th hin nhy phn tch (AS), gii hn pht hin (LOD), v gii hn nh lng (LOQ). nh lng hm lng isoflavone trong mu chit. Sc k ca cc isoflavone trong dch chit bng phng php ti u ha c so snh c th hin hnh 5B. Nhng peak tng ng vi 3 isoflavone chnh (genistein, daidzein, glycitein) c ragii khi ct theo th t v thi gian lu so vi mu chun (33,8 pht vi daidzein; 37,0

30 pht cho glycitein; 43,7 pht cho genistein). Sau c khng nh li bng cch so snh ph hp thu vi mu chun (s liu khng c th hin). Da vo ng cong chun ta tnh c hm lng mi aglycon trong sn phm ca quy trnh sau khi c ti u ha (hnh 5B): 0,942 mg/ml vi genistein, 0,641 mg/ml vi daidzein v 0,071 mg/ml vi glycitein. Vi mu 60 ml sn phm, t 160 g bt u nnh c sn xut theo quy trnh ti u ha. Trong mu ny c 56,5 mg genistein, 38,5 mg daidzein, 4,3 mg glycitein v tng hm lng isoflavone c tnh ton l 0,62 mg/g bt. Da vo sc k HPLC (hnh 5B), thnh phn ca sn phm chng ti kh n gin. Genistein v daidzein l thnh phn chnh, vi genistein chim khang 57% tng lng isoflavone v daidzein chim khong 39%. bc sng 255 nm, lng tp trong sn phm l rt t.

Hnh 6: Phn tch HPLC genistein, daidzein v glycitein trc v sau khi ti u ha.

31

Kt qu nh lng cho thy vic s dng iu kin chit xut v tinh ch ti u trn, lng genistein thu c nhiu hn 2 ln v lng daidzein thu c nhiu hn 2,5 ln so vi ban u (hnh 7). Ni cch khc, lng isoflavone thu c gp i sau khi c ti u ha.

Hnh 7: lng isoflavone t u nnh (g isoflavone/g bt u nnh) trc v sau khi ti u ha (S liu c th hin bng gi tr trung bnh lch chun ca sn phm isoflavone t 5 mu th khc nhau). 3.1.4. Phn lp cc isoflavoneoid bng HPLC iu ch[35]: u tin, s dng h thng HPLC iu ch phn lp tng isoflavone (genistein, daidzein, glycitein) ra khi hn hp isoflavone ton phn. Sn phm trc v sau khi phn lp c phn tch bng h thng HPLC phn tch.

32 Cc thit b: H thng HPLC iu ch: Cc thng s sc k:-

Ct YWG C18 (20,0 x 250 mm, ng knh trong 10 m (Hanbon Science and Technology CO., Ltd. Jiang Suprovi nce, Trung Quc) Th tch bm mu: 750 l Pha ng: MeCN-nc-AcOH (35:65:2 v/v/v) Tc dng: 4,5 ml/pht Nhit : 250C o UV bc sng: 260 nm

-

-

H thng HPLC phn tch: Thng s sc k:-

Lichrospher C18 (6,0 x 150 mm, ng knh trong: 5 m) (Hanban Science, Jiang Suprovince, Trung Quc). Pha ng: MeOH nc AcOH (45:55: 2 v/v/v). Tc dng: 1,0 ml/pht Nhit : 250C o UV bc sng: 260 nm

-

-

Hn hp isoflavone ton phn c phn tch bng h thng HPLC trc khi tin hnh phn lp (hnh 8:E), so snh vi sc k ca cht chun, xc nh c th t, thi gian lu ca tng cht, th t ra khi ct l daidzein, glycitein v genistein. T , dng pha ng xc nh trn ragii tng cht ra khi ct HPLC ch ha v hng cc thi im khc nhau, thu c tng cht ring bit. Sn phm phn lp c phn tch li vi h thng HPLC phn tch (hnh 8:F, G, H), kt qu cho thy mi phn on ch c mt pic ring v tinh khit t 99%.

33

Hnh 8: sc k HPLC: E: isoflavone ton phan; F: genistein; G: glycitein v H: daidzein.

34

3.2 Phng php 2: THY PHN ISOFLAVONE SAU KHI PHN LP TNG ISOFLAVONE GLYCOSID RING[34]3.2.1. Chit xut: Khong 909 g bt u nnh sau khi c loi cht bo c chit vi 2 lt methanol nhit 550C trong 30 pht, khuy bng con c khuy t. Sau , cc phn t rn c loi ra khi dch chit bng cch lc. B u nnh thu c chit li 4 ln na vi 2 lt methanol/ln, iu kin chit xut nh ln u. Sau khi lc loi b, 5 dch chit c gm li thnh dch chit ton phn th. Dch chit th c phn tch bng h thng HPLC, kt qu cho thy trong dch chit th c khong 485 mg daidzin v 636 mg genistin. Hai pic ra trc hn c xc nh l 6-O-malonyl daidzin v 6-O-malonyl genistin. 3.2.2. Phn lp bng sc k ct pha o: Khong 9 lt dch chit methanol c ha vi nc ct n khi t hn hp 20% methanol trong nc. Sau mu c np ln ct polymethacrylate 4 x 70 v ra gii vi pha ng c phn trm methanol trong nc tng dn l 50%, 60% v 70%. Th tch hng l 100 ml/ ln v c nh s th t 0,1; 0,2; 0,3; 0,4 cc phn on c em phn tch thnh phn nhm pht hin isoflavone bng h thng HPLC. t l pha ng u tin, ta quan st thy s hin din ca cc isoflavone ln lt cc phn on thu c: 6-O-malonyl daidzin: phn on 0-1,3 6-O-malonyl genistin: phn on 1-1,6 Daidzin: phn on 3,5-6,1 Genistin c ragii khi ct t l methanol-nc l 60%, phn on 7,010,0. Cc phn on ln lt c c thu hi dung mi cho n khi cn kh, ta c: 396 mg daidzein v 515 mg genistin. 3.2.3. Kt tinh:

35 Genistein th ( tinh khit l 63%) v daidzin th ( tinh khit 62%) ln lt c ha tan trong methanol, sau thm than hot loi mu dung dich.Than hot c loi ra khi dung dch bng cch lc. Dung dch sau c c thu hi dung mi cho n cn kh, ha tan tr li trong lng MeOH va v kt tinh trong t lnh. Thu kt tinh bng cch lc qua phu thy tinh sp. Kt qu: tinh th daidzin thu hi c c tinh khit l 97% v t l phc hi l 80%. Genistin vi tinh khit l 97% v t l phc hi l 72%. 3.2.4. Thy phn tinh th daidzin v genistin: Deidzin v genistin c un hi lu vi mt lng HCl 4N trong 5 gi 105050C. daidzein v genistein (sn phm thy phn ca daidzin v genistin) km tan trong nc, nn kt tinh li. Lc thu daidzein v genistein. T l phc hi ca daidzein vf genistein sau thy phn ln lt l 92% v 75%. Sn phm thu c sau c ha tan li v kt tinh trong MeOH. Kt qu: Thu c daidzein v genistein c tinh khit l 99%, t l phc hi ln lt l 77% v 63%.

36

S 4: s phn lp isoflavone t u nnh (phng php 2)

37

Chng 4 BN LUNTrong nghin cu ny, isoflavone c chit xut v tinh ch t bt u nnh. V isoflavone tn ti ch yu trong u nnh dng glycoside[36], nhng glycone c tc dng sinh hc mnh hn v c hp thu d dng hn so vi dng glycoside tng ng[18]. Do , thy phn l mt bc cn thit nng cao tc dng sinh hc cng nh kh nng hp thu ca cc ch phm t u nnh. Dung mi chit xut c chn l EtOH v MeOH, do kh nng ha tan v s bn vng ca nhm isoflavone trong alcol. Tc nhn thy phn trong c hai phng php u c la chn l acid HCl v hiu qu thy phn ca chng cng nh kh nng c loi b d dng trong nhng bc tip theo trong quy trnh phn lp. Tuy nhin, m bo acid HCl c loi b hon ton khi sn phm cui cng, th nng acid s dng khng c qu cao (trong khi nu nng acid thp qu thi s thy phn khng hiu qu), v nng acid ti u sau khi c kho st c khuyn co nn l 0,13 mol/l[31]. Trong phng php 2[34] , nng acid c s dng cao hn rt nhiu: 4N. thu c thu c sn phm tinh khit, tc gi phi chit li vi diethyl ether v sn phm thu c c tinh khit rt cao (99%)[34]. Mt phng php thy phn isoflavone dng glycoside thnh dng aglycone cng c cc nh khoa hc nghin cu rt nhiu l thy phn bng enzyme -glucosidase (mt enzyme c trong u nnh)[32]. u im ca phng php ny l an ton, khng cn lo s d lng acid tn li trong sn phm cui cng, tit kim c nng lng ( do nhit thp- khong 500C). Nhng mt nhc im ca n l ch thy phn c acetyl-glycosid m khng tc ng trn malonylglycosid[33], do khng th thy phn hon ton, hn na phng php ny ph thuc vo hm lng -glucosidase trong u nnh, iu ny khng c li cho sn xut cng nghip do tc nhn thy phn khng n nh, kh t ng nht gia cc l. Qu trnh tinh ch sn phm: phng php 1: thc hin n gin bng cch kt tinh isoflavone ton phn trong nc ct. Nguyn tc l da vo tnh tan ca isoflavone

38 trong cc dung mi khc nhau. tan ca isoflavone dng aglycone trong nc km hn nhiu so vi tan trong EtOH.

Chng 5 KT LUN V NGHNhn chung, phng php 1 thc hin n gin hn phng php 2. Trc ht, phng php 1, ta thy phn isoflavone dng glycoside ngay t u, iu ny c li v ta ch cn thy phn 1 ln duy nht cho tt c cc isoflavone, do ta c th tit kim c thi gian v cng sc. Th hai, phng php th nht ta s dng nhng k thut n gin nhng hiu qu nh l kt tinh trong nc, s dng phng php lc loi tp thay v dng nhng phng tin tn km hn nh: chit lc nhiu ln, sc k, ly tm v th thch hp vi quy m sn xut cng nghip hn.

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