MC LC

MC LC..................................................................................................................... i PH LC...................................................................................................................... i GII THIU................................................................................................................1 CHNG 1: ..........................................................................................................2 TNG QUAN...............................................................................................................2 KT LUN................................................................................................................. 54 TI LIU THAM KHO.........................................................................................55

PH LC
Ph lc 1: Tra cu cc bng Bng 1: nh hng ca nng ng sucrose n s nui cy huyn ph t bo ..................................................................................................................................... 53 Ph lc 2: Tra cu cc hnh Hnh 1 : 1. Cnh ang n hoa; 2.Qu; 3.Ht.............................................................4 Hnh 2: Ht ca cy C.roseus......................................................................................4 Hnh 3: Ba ging C.roseus...........................................................................................5

Hnh 4: Cng thc ha hc ca leurosidine, vincristine, vinblastine, leurosine, catharine, vinamidine................................................................................................12 ..................................................................................................................................... 12 Hnh 5: Cng thc ha hc ca ajmalicin................................................................12 Hnh 6: Cng thc ha hc ca vinflunine...............................................................13 Hnh 7: Cng thc ha hc ca vinblastine................................................13 Hnh 8: Cng thc ha hc ca vincristine..............................................................14 Hnh 9: Con ng chuyn ha thnh vinblastine v vincristine..........................16 Hnh 10: Con ng sinh tng hp Vinblastine...............................................17 Hnh 11: Qu trnh sinh tng hp vincristine t vinblastine ..............................18 Hnh 12: C ch c ch s hnh thnh cu trc vi ng ca vinca alkaloid............20 Hnh 13: Cu trc ha hc ca vinblastine sulfate..................................................21 Hnh 14: Sn phm v cu trc ha hc ca vincristine sulfate.............................24 Hnh 15: S qu trnh chit tch bng SFE........................................................28 Hnh 16: Bnh Soxhlet......28 Hnh 17: S mt b chng ct li cun hi nc...............................................30 Hnh 18: Sn phm m so v cy mc t ht nhn to.........................................49 Hnh 19: Sn phm r t...........................................................................................50 Ph lc 3: Tra cu cc th th 1: ng cong tng trng ca dch huyn ph t bo trong mi trng c b sung cc nng ng khc nhau.....................................................................54

ii

GII THIU
Da cn Catharanthus roseus. G. Don thuc h Trc o Apocynaceae l mt trong nhng loi cy cnh ph bin ng thi cng l mt loi dc tho dn gian. Ngi ta khm ph ra c kh nng cha bnh ca loi cy ny l nh n cha nhiu loi hp cht alkaloid. T da cn ngi ta c th chit c cht cha ung th nh vinblastine, vincristine v cha cao huyt p nh ajmalicin, serpentin. Hin nay, nhu cu v C.roseus trn th gi rt cao, nhng thc t, trong iu kin t nhin s nhn ging C.roseus bng con ng sinh sn hu tnh rt chm v cn ph thuc nhiu vo mi trng. Mt khc, lng alkaloid thu c t t nhin l rt thp nn khng p ng c nhu cu th trng. V vy em chn ti Cy da cn Catharanthus roseus v nhm hp cht Vinca alkaloid vi mc ch l tm hiu v dc tnh, cch thu nhn v ng dng ca n hin nay. Nh c gi tr dc tnh nn C.roseus tr thnh i tng ca nhiu nghin cu sinh hc. Nhng nghin cu ny ch yu lin quan ti iu kin nui cy in vitro ca nhng c quan, m v t bo khc nhau, qu trnh tch nhng indole alkaloid v xc nh cu trc ha hc ca n cng nh qu trnh sinh tng hp v ha tng hp ca n.

CHNG 1:

TNG QUAN
1.1 Catharanthus Ging Catharanthus (thuc h trc o Apocynaceae) gm c 8 loi, hu ht l cy thn tho lu nm. Trong s ch c loi Catharanthus pusillus c ngun gc t n , cn tt c cc loi cn li c ngun gc t Madagascar. S lng nhim sc th cho tt c cc loi Catharanthus u l 2n=16. Tm loi thuc ging ny l: Catharanthus coriaceus Markgr. Madagascar. Catharanthus lanceus (Bojer ex A.DC.) Pichon. Madagascar. Catharanthus longifolius (Pichon) Pichon. Madagascar. Catharanthus ovalis Markgr. Madagascar. Catharanthus pusillus (Murray) G. Don. India subcontinent. Catharanthus roseus (L) G. Don. Madagascar. Catharanthus scitulus (Pichon) Pichon. Madagascar. Catharanthus trichophyllus (Baker) pichon. Madagascar Nhng ging Catharanthus c ngun gc t Madagascar thng c trng lm cnh. c bit n nhiu nht l loi Madagascar Periwinkle hay cn gi l Vinca nh vo kh nng chu hn v chu nng ca n. Ngoi vic c bit n nh mt loi cy cnh, t lu dch chit alkaloid t Catharanthus roseus c s dng trong y hc dn gian nh mt loi thuc chng i tho ng, li tiu, cha tiu chy, xut huyt, gip vt thng mau lnh, chng vim mt, lm se da.v ngy nay ngi ta cn tm ra c mt cng dng ht sc quan trng ca loi Catharanthus roseus l kh nng tr bnh ung th rt hiu qu.[6]

1.2 Catharanthus roseus (L).G.Don [1] 1.2.1 Tn gi Tn khoa hc: Catharanthus roseus (L) G. Don; Vinca rosea L; Lochnera rosea Reich. Tn khc: bng da, hoa hi ng, trng xun hoa, phjc pt ng (Ty), dng gic, Madagascar periwinkle H: Trc o (Apocynaceae). 1.2.2 c im thc vt Cy da cn l cy thn tho sng lu nm, cao 40-60 cm, phn nhiu cnh, cy c b r pht trin, thn g pha gc, mm phn trn. Mc thnh bi dy. L mc i, thun di, u l hi nhn, cung l hp nhn, di 4-6cm, rng 2-3 cm, hai mt nhn, mt trn sm bng, mt di nht. Hoa trng hoc hng, c mi thm. Hoa mc ring l k l gn ngn. Qu gm 2 i, di 2-4cm, rng 2-3cm, mc thng ng, hi ng sang hai bn, trn v c vch dc, u qu hi t, trong qu cha 12-20 ht nh mu nu nht, hnh trng, trn mt ht c cc ht ni thnh ng chy dc. Ma hoa qu gn nh quanh nm.

Hnh 1 : 1. Cnh ang n hoa; 2.Qu; 3.Ht

Hnh 2: Ht ca cy C.roseus 1.2.3 Phn b Chi Catharanthus G.Don c ngun gc Madagascar. Loi da cn c di nhp sang nhiu nc nhit i Nam cng nh ng Nam trong c Vit Nam v o Hi Nam Trung Quc. Vo gia th k 18, da cn c trng Paris, sau c mt nhiu vn thc vt khc Chu u. Vit Nam, da cn l cy hoang di, c vng phn b t nhin tng i c trng t tnh Qung Ninh n Kin Giang dc theo vng ven bin, tp trung cc tnh

min Trung nh Thanh Ha, Ngh An, Tha Thin-Hu, Qung Nam, Nng, Bnh nh v Ph Yn. Ngoi ra cn c Cn o v Ph Quc. nhng vng phn b t nhin ven bin, da cn c khi mc gn nh thun loi trn cc bi ct di rng phi lao, trng c cy bi thp, c kh nng chu ng iu kin t ai kh cn ca vng ct ven bin. Da cn cn c trng khp ni trong nc lm cnh v lm thuc. 1.2.4 Phn loi [6] Catharanthus roseus G.Don. l ngun giu alkaloid thuc chng loi alkaloid terpenoid indole c c lp t 3 ging cy khc nhau: roseus vi hoa mu tm hoc hng. albus vi hoa mu trng. ocellatus vi hoa mu trng nhy . Trong hoa mu tm c hm lng vincristine v vinblastine cao nht.

Hnh 3: Ba ging C.roseus 5

1.2.5 Trng trt v thu hoch Da cn l loi cy a sng, a m v c kh nng chu c hn. Trong iu kin trng trt, khi c cung cp y iu kin cn thit, cy sinh trng pht trin mnh, khi lng cht xanh thu c c th cao gp i cy mc t thin nhin. Cy mc t ht trong t nhin vo khong 40%, nu c x l c th tng ln 90% [9]. Cy trng t ht ra hoa sau 4-5 thng. Trong thi k sinh trng mnh, nu b ct, cy ti sinh chi khe. Ngun da cn mc t nhin Vit Nam tng i di do. Trc nm 1975, min Bc tng xut khu sang ng u 1-3 tn/nm. Nhng nm gn y, lng xut khu sang Php (khong trn 10 tn/nm) thng xuyn hn, nhng ch yu l t cy trng ti Ph Yn. [1] Vo nm 1970, Vin Dc Liu tin hnh nghin cu k thut trng da cn trn quy m sn xut. Cy c nhn ging bng ht, mi hecta cn gieo 500-700gr ht trong vn m. Thi v gieo ht vo thng 9-10 hoc thng 1-2. Cn ngm ht 34 gi, vt ra ro ri gieo ln lung vn m c chun b k. Sau ph rm ri ti nc. Sau khong mt tun, ht ny mm, cn tho b rm r. Khi cy c 3-4 i l tht (khong 40-45 ngy sau khi gieo) nh i trng. C th gieo thng nhng cch ny tn cng chm sc. [1] Da cn a t pha ct, t ph sa, hi chu hn nhng km chu ng. t cn lm k, ln lung cao 20cm, mt lung rng 50-60cm, dung 10-15 tn phn chung hoai mc v 120-150 kg super ln bn lt. Trng vi khong cch 30x30cm, sau khi trng cn ti ngay cy mau bn r. Ti thc cho mi hecta 100-200kg ure, ti 2 ln, cch nhau 1 thng. Mc d cy chu c hn nhng phi gi m thng xuyn. Ch thot nc nhanh sau khi ma ln. Khi mi trng, cy thng b su bm ph hoi. Cy trong vn c th b Phytophthora lm cho cht hng lot. Cn ta bt cho t thong v phun phng bng Boxdeaux. [1] Sau khi trng 3-4 thng, cy cho thu hoch, cnh mang l di 10-15cm c ct v phi sy kh. t thot nc v chm bn tt c th thu hoch nhiu la. Trung bnh 1 hecta thu c 1-1,2 tn l kh mi la. Ta c th thu r chit ajmalicin.

1.2.6 Mt s cng dng ch yu Theo kinh nghim dn gian Vit Nam v Trung Quc, c ni dng thn v l phi kh sc ung thng tiu tin, cha bnh i tiu v t, lm thuc iu kinh, ty giun, cha st, sn da, cha bnh ngoi da. Nam Chu Phi, n , chu c, qun o Antilles ngi dn dng tr bnh i tho ng. C ni dng cha tiu ha km v l. Chnh nh thc nghim trn chut m cc nh khoa hc Canada pht hin tc dng lm gim bch cu ca mt s cht tch c t da cn v dn n s pht hin ra cht vincaleucoblastin v 3 alkaloid khc cng c tc dng chng u l leurosin, leurocristin v leurosidin. Ngoi ra ngi ta cn pht hin ra tc dng ty giun kh mnh, tc dng li tiu ca catharanthin, vindolinin v vindolidin. Nhng th nghim dng trn ngi bnh c bt u vo nhng nm 1960 M, Php v mt s nc khc, tuy nhin c nhiu kin khc nhau. Mc d vy, v hin nay cha c loi thuc no khc tt hn, nn nh cu v da cn vn c tng ln. Cng v mc ch cha cc khi u nn khi mua da cn, ngi ta c bit ch ti hm lng alkaloid ton phn, v trong s alkaloid ton phn y c hm lng vincaleucoblastin l bao nhiu.[1][6]

CHNG 2:

CC HP CHT ANKALOID TRONG CY C.roseus

2.1 Alkaloid trong loi Catharanthus roseus C.roseus thu c rt nhiu s quan tm t cng nghip dc phm, c khong 70 loi alkaloid hu ch c pht hin (G.H.Svobda v cng s (1991). Trong chim nhiu nht l hai loi alkaloid vinblastine (vincaleukoblastine) v vincristine (leurocristine).[1] 2.1.1 Alkaloid 2.1.1.1 Khi nim Alkaloid l nhng cht hu c c cha d vng nit, v c tnh base. Thng gp trong nhiu loi thc vt v i khi cn tm thy trong mt vi loi ng vt. c bit, alkaloid c hot tnh sinh l rt cao i vi c th con ngi v ng vt, nht l i vi h thn kinh. Vi ch mt lng nh alkaloid l cht c gy cht ngi nhng li c khi n l thn dc tr bnh c hiu. Hm lng alkaloid c th t ti 10% trong cc loi rau qu thng dng nh khoai ty, ch, c ph.[7] 2.1.1.2 Phn loi Cc alkaloid thng thng c phn loi theo c trng phn t chung ca chng, da theo kiu trao i cht c s dng to ra phn t. Khi khng bit nhiu v tng hp sinh hc ca alkaloid, th chng c gp nhm theo tn ca cc hp cht bit. V d: do cc cu trc phn t xut hin trong sn phm cui cng nn cc alkaloid thuc phin i khi cn c gi l cc phenanthren. Hay gi tn da theo nhm ng/thc vt m t ngi ta chit xut ra cc alkaloid v d nh cc alkaloid chit t cy da cn vinca th c gi chung l cc vinca alkaloid.

Khi ngi ta bit nhiu hn v mt alkaloid c th no , th vic gp nhm thng ly theo tn gi ca amin quan trng v mt sinh hc v ni bt nht trong tin trnh tng hp. Cc nhm ankaloid hin nay gm c: Nhm pyridin: piperin, coniin, trigonellin, arecaidin, guvacin, pilocarpin, cytisin, nicotin, spartein, pelletierin. Nhm pyrrolidin: hygrin, cuscohygrin, nicotin Nhm tropan: atropin, cocain, ecgonin, scopolamin . Nhm quinolin: quinin, quinidin, dihydroquinin, dihydroquinidin, strychnin, brucin, veratrin, cevadin Nhm isoquinolin: Cc alkaloid gc thuc phin nh : morphin, codein, thebain, papaverin, narcotin, sanguinarin, narcein, hydrastin, berberin Nhm phenethylamin: mescalin, ephedrin, dopamin, amphetamin Nhm indol: Cc tryptamin: DMT, N-metyltryptamin, psilocybin, serotonin. Cc ergolin: Cc ancaloit t ng cc/c nh ergin, ergotamin, acid lysergic. Cc beta-cacbolin: harmin, harmalin, yohimbin, reserpin, emetin. Cc alkaloid t chi Ba gc (Rauwolfia): reserpin. Nhm purin: Cc xanthin ( caffein, theobromin, theophyllin). Nhm terpenoit: Cc alkaloid aconit: aconitin. Cc steroit: solanin, samandari (cc hp cht amoni bc bn): muscarin, cholin, neurin. Cc vinca alkaloid: Vinblastin, vincristin.. 2.1.1.3 Tnh cht ca cc alkaloid a. Tnh cht vt l Phn t lng: khong 100-900. 9

Cc alkaloid khng cha cc nguyn t xy trong cu trc thng thng l cht lng iu kin nhit phng (v d nicotin, spartein, coniin). Cc alkaloid vi cc nguyn t xy trong cu trc ni chung l cc cht rn kt tinh iu kin nhit phng (v d: berberin). Hu ht alkaloid base gn nh khng tan trong nc nhng tan trong cc dung mi hu c nh CHCl3, eter, cc alcol dy carbon ngn. Mt s alkaloid do c thm nhm phn cc nh OH, nn tan c mt phn trong nc hoc trong kim (Morphin, Cephalin). Ngc li vi base, cc mui alkaloid ni chung tan c trong nc v alcol, hu nh khng tan trong dung mi hu c. C mt s ngoi l nh Ephedrin, Colchixin, Ecgovonin cc base ca chng tan c trong nc, ng thi cng kh tan trong dung mi hu c, cn cc mui ca chng th ngc li.

Alkaloid c N bc 4 v N- oxid khc tan trong nc v trong kim, rt t tan trong dung mi hu c. Cc mui ca chng c tan khc nhau ty thuc vo gc acid to ra chng.

b. Tnh cht ha hc Alkaloid l cc base yu, a s lm xanh giy qu tm. Vi acid thng to mui tan trong nc v kt tinh. Tnh kim ph thuc vo kh nng sn c ca cc cp in t n c trn nguyn t nit & v kiu khc (d) vng cng cc phn thay th. Tnh base gim dn theo th t mui amoni bc 4, amin bc 1, amin bc 2, amin bc 3. Mui ca alkaloid rt bn, nhng chng b phn hy bi tia sng mt tri hoc tia t ngoi. Phn ng alkaloid c v ng . To ta vi cc dung dch acid phosphotungstic, phosphomolipdic, picric Ngoi tnh base, cc alkaloid c phn ng tng t nhau nh i vi mt thuc th, gi tn chung l cc thuc th alkaloid. 2.1.2 Alkaloid trong catharanthus [1]

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Alkaloid ton phn c l da cn vi hm lng 0.37-1.15%, thn 0.40%, r chnh 0.7-2.4%, r ph 0.9-3.7%, hoa 0.14-0.84%, v qu 1.14%, ht 0.18%. C khong 150 loi alkaloid c chit t Catharanthus roseus. Trong s c bit ch l nhm 20 alkaloid dimeric, l nhng nhm c hot tnh chng ung th, bao gm vincristine v vinblastine.

Vinblastine c l vi hm lng 0.013-0.063%, b phn trn mt t 0.0015%, r 0.23%. Nu cy b bnh asteryllow-virus th s khng c vinblastine. Vincristine c hm lng thp hn 0.0003-0.0015%.

L da cn thu thp nhiu a phng khc nhau cha 0.7-1.2% alkaloid ton phn, cao nht Ph Yn (1.21-1.62%). Vinblastine c vi hm lng 1.6-2 phn vn l. Thi gian thu hi nguyn liu tt nht trn cy c hm lng hot cht cao l vo cui thng 8 n gia thng 9 dng lch. Theo L Th Tuyt Anh v cng s, hm lng alkaloid ton phn cy da cn thu thp hoang di Ph Yn t cao nht l 0.892-0.982% l v 1.38-1.445% r vo ma kh.

Ngoi hai alkaloid chnh l vinblastine v vincristine c chit xut, nhiu tc gi bn tng hp c vinblastine t cathanthin v vindolin c da cn v vincristine (c vi hm lng thp) t vinblastine (c vi hm lng cao hn).

Mai Ngc Tm v cng s, 1997 chng minh r da cn vng Nha Trang cha ajmalicin 0,18%, serpentin 0,27%, tetrahydroalstonin, tabersonin, lochnericin, catharanthin v akuamin.

Nhiu tc gi khc (Phm Thanh K v cng s, 1995; Trn Vn Thanh v cng s, 1996 v 1999) chit xut ajmalicin t r da cn. Trn Vn Thanh v cng s, 1996 v 1999 chit xut alkaloid ton phn t r, lm giu ajmalicin bng phng php hydro ha serpentin, sau mi chit xut ajmalicin, to hiu sut chit xut cao hn gp 2 ln.

V nhng alkaloid ny ch l thnh phn nh ca cy (vincristine thu c t cy thuc th ch t hiu sut 0,0002%), v vy nu mun sn xut th ta phi cn s lng rt ln nguyn liu th trch ly.

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Hnh 4: Cng thc ha hc ca leurosidine, vincristine, vinblastine, leurosine, catharine, vinamidine

Hnh 5: Cng thc ha hc ca ajmalicin

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Hnh 6: Cng thc ha hc ca vinflunine 2.2 Cc vinca alkaloid chnh 2.2.1 Vinbastine 2.2.1.1 Cng thc

Hnh 7: Cng thc ha hc ca vinblastine Cng thc phn t: C48H58N4O9 Tn ha hc: dimethyl (2,3,4,5,12,19)- 15-[(5S,9S)- 5-ethyl- 5-hydroxy9-(methoxycarbonyl)- 1,4,5,6,7,8,9,10-octahydro- 2H- 3,7 methanoazacycloundecino[5,4-b]indol- 9-yl]- 3-hydroxy- 16-methoxy- 1-methyl- 6,7didehydroaspidospermidine- 3,4-dicarboxylate Tn gi khc: vincaleukoblastine Tn thng mi: Cytoblastin

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2.2.1.2 c im L tinh th hnh kim (kt tinh t methanol), im chy 211-2160. Khng tan trong nc, ether du ha, tan trong alcol, aceton, ethyl acetat, chloroform. [1] Hp thu: Vinblastine hp thu nhanh chng theo ng tim tnh mch. Phn b: thuc phn b nhanh vo cc m ca c th. Thuc lin kt nhiu vi protein huyt tng. Vinblastine t qua hng ro mu no v khng t nng iu tr trong dch no ty. Chuyn ha: Vinblatine c chuyn ha nhiu, ch yu gan thnh desacetyl vinblastine l cht c hot tnh mnh hn vincristine, tnh trn c s khi lng. Thi tr: thuc thi tr qua mt vo phn v nc tiu, mt s o thi di dng thuc khng bin i. 2.2.2 Vincristine 2.2.2.1 Cng thc

Hnh 8: Cng thc ha hc ca vincristine Cng thc phn t: C46H56N4O10 Tn ha hc: methyl (1R,9R,10S,11R,12R,19R)- 11-(acetyloxy)- 12-ethyl- 4[(13S,15S,17S)- 17-ethyl- 17-hydroxy- 13-(methoxycarbonyl)- 1,11 diazatetracyclo [13.3.1.04,12.05,10]nonadeca- 4(12),5,7,9-tetraen- 13-yl]- 8-formyl- 10-hydroxy- 5-

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methoxy- 8,16-diazapentacyclo [10.6.1.01,9.02,7.016,19] nonadeca- 2,4,6,13-tetraene- 10carboxylate Tn gi khc: leurocristine Tn thng mi: Oncovin, Vincasar, Vincrex v cng c th gi l sunlfat vincristine.

2.2.2.2 c im L tinh th hnh phin, im chy: 218-2200 Phn b: sau khi tim, vincristine nhanh chng phn b vo cc m c th v gn vi cc yu t mu hnh thnh, c bit l hng cu v tiu cu. Vincristine khng xm nhp h thn kinh trung ng vi mc ng k. Chuyn ha v bi tit: vincristine c chuyn ha nhiu gan. Con ng thi tr chnh l qua ng mt i ra phn. Mt phn ba liu dng c th c phc hi trong phn trong vng 24 gi u v hai phn ba trong vng 72 gi. Ch c 12% liu c bi tit qua thn. Khong mt na liu c phc hi trong phn v nc tiu di dng cht chuyn ha 2.2.3 Sinh tng hp [6] Vinblastine v vincristine c to thnh t s ghp ni ca hai monomer alkaloid: catharanthine (indole) v vindoline (dihydroindole), c hai u xut hin t do trong cy. Vincristine cng c cu trc tng t nh vinblastine nhng thay nhm fromyl bng mt nhm methyl trn phn t nitrogen indole ca vindoline.

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Hnh 9: Con ng chuyn ha thnh vinblastine v vincristine Nhng alkaloid ny c hnh thnh bi s kt hp ca hai na: mt na l indole v mt na l dihydroindole. V th, chng c bit n vi tn gi l dimer alkaloid hoc bisindole alkaloid. S khc nhau ca Catharanthus alkaloid ph thuc vo loi alkaloid terpenoid indole. Chng gm hai na bt ngun t hai qu trnh chuyn ha ring bit- qu trnh mevalonate cho na khng cha tryptophan; v na tryptophan nhn c t qu trnh tryptophan. Cu trc phc tp ca nhng alkaloid ny lun c mt hai nguyn t nit. Mt l indole nit (na bt ngun t tryptophan). V nguyn t nit th hai c to thnh t s tch ri ca hai carbon ti v tr ca vng indole. Na khng c tryptophan bt ngun t acid mevalonic v n l mt C10-geraniol (monoterpenoid). Geraniol c to thnh , thng qua mt chui chuyn ha s chuyn thnh dng loganin v sau l secologanin (mt monoterpenoid glucoside). Cha kha trung gian trong thuyt pht sinh sinh hc ca nhng alkaloid monoterpene indole l 3 (S)-strictosidine, to thnh t s ngng hot tnh enzyme ca trytamine v secologanin. Enzyme chu trch nhim cho phn ng quan trng ny

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l strictosidine synthase. Strictosidine sau s hnh thnh cu trc cathenamine (alkaloid loi coryanthe). Enzyme lin quan y l cathenamine synthase. Cathenamine sao phi tri qua mt chui cc phn ng dn n s hnh thnh catharanthin (alkaloid loi iboga) v vindoline (alkaloid loi aspidosperma). Catharanthine v vindoline l cc alkaloid monomeric indole, xut hin t do trong cy. 3,4-Anhydrovinblastine l cha khoa trung gian t s ghp ni ca catharanthine v vindoline v cc enzyme lin quan l nhng peroxidase. Sau n c chuyn thnh vinblastine.

Hnh 10: Con ng sinh tng hp Vinblastine Vinblastine c to thnh t s ghp ni ca hai monomer alkaloid: catharanthine (indole) v vindoline (dihydroindole)

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Mc d nhu cu s dng vincristine nhiu hn so vi vinblastine nhng cy li sn xut t l vinblastine nhiu hn. May mn thay, gi y ta c th bin i vinblastine thnh vincristine bng phng php ha hc hay thng qua phng php vi sinh hc microbiological N-demethylation s dng Streptomyces albogriseolus.

Hnh 11: Qu trnh sinh tng hp vincristine t vinblastine 2.2.4 Tc dng dc l [1], [5], Vinblastine hay cn gi l vincaleucoblastine, l mt cht c ch cu trc vi ng. Vinblastine c ng a vo iu tr bi t chc Food and Drug Administration (FDA) nm 1961 v tr thnh mt thnh phn chnh ca phng php ha tr liu

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iu tr cc t bo mm ca t bo ung th, khi u c tnh v mt s loi lymphoma cp cao, bao gm u lymphoma Hodgkin, u lymphoma khng Hodgkin, u si dng nm, t bo ung th phi nh, ung th v, ung th tinh hon tin trin, bu tht Kaposi, bnh m bo huyt, ung th nhau, chriocarcinoma (mt loi ung th t cung), Theo ghi ch trong lch s s dng vinblastine th vinblastine c th s dng mt mnh hoc kt hp vi cc tc nhn khc cha bu tht Kaposi v ung th bng quang, ung th v v mt vi loi u c tnh no. Hot tnh iu tr ung th ca vinblastine cng hiu qu nh hot tnh ca vincristine, song n li l mt c t thn kinh. Desacetyl vinblastine (vindesine) c xem nh l dn xut ca vinblastine c bn tng hp bi Potier v cng s. Vinorelbine (5-norhydro Vinblastine), c hot tnh chng ung th rng hn v gim phn ng ph l gy c thn kinh. N l cu trc c sa i trn nhn catharanthine, kt qu l tng ng k lng lipophilicity (cht c kh nng ha tan trong cht bo v dung mi khng phn cc) hn so vi alkaloid Vinca. N c hiu qu trong s kt hp vi ha tr liu nh anthracycline, fluorouracil v taxol. N c chp nhn M trong vic iu tr t bo ung th phi nh, c th s dng ring l hoc kt hp vi cisplatin, v n cng c dng cho bnh nhn b ung th v. Vincristine hay cn gi l leurocristine, thng c dng iu tr khi u c tnh tr em, n c kh nng chng li s kt hp rt nhy cm ca bnh khi u c tnh tr em i vi vincristine v cho tc dng tt hn khi dng ng liu lng dnh cho tr em. Mt khc, c ngi ln v tr em, vincristine l mt thnh phn cn thit trong liu php ha hc cha vim bch cu cp tnh, s ln cn lm v lymphoid ca bch cu myeloid mn tnh (chronic myeloid leukemia), v hai bnh lymphoma Hodgkin v lymphoma khng Hodgkin. N cng ng vai tr trong mt vi liu php iu tr khi u Wilms, bu tht Ewing, u nguyn bo thn kinh v rhabdomyosarcoma, cng tt nh trong iu tr a u ty (multiple myeloma) v t bo ung th phi nh (small-cell lung cancer) ngi ln.

19

C ch tc dng: vinblastin v vincristin, l nhng cht c ch mnh s phn bo. Cc alkaloid ny lin kt c hiu vi tubulin, l protein vi ng thoi phn bo v ngn cn s kt hp ca nhng cu trc hnh ng c trong nguyn sinh cht ca nhiu t bo di ng, ngn cn s tng ln v s lng trong k gia gin phn ca t bo.

Hnh 12: C ch c ch s hnh thnh cu trc vi ng ca vinca alkaloid Vinblastin c tc dng chng ung th cn do tc ng i vi chuyn ho ca glutamate v aspartate. V vincristin tc dng chng ung th cn do ngn cn s tng hp RNA v cc protein. nng cao, thuc dit c t bo, cn nng thp lm ngng phn chia t bo. Vinca alkaloid c tc dng lm tng nng cc phosphat acid v kim trong tinh hon v tuyn tin lit ca chut cng trng. iu ny chng t cao da cn lm bin i chc nng chuyn ho v hot tnh ca phosphatase cc c quan ny ca chut. Vinca alkaloid cn c tc dng c ch mnh hot tnh ca protease ca c hai chng T5 v T12 ca nm da Trichophyton rubrum. Tc dng c ch hon ton c ngha nhng nng cao. nhng nng thp, hoc khng c ch, hoc tc dng 20

c ch trn protease. C ch khng nm u l c ch s h hp ca si nm ca c 2 chng T.rubrum. Nhng iu ny c th nghim trn chut: Khi cho chut cng trng ci th tinh ung cao da cn, thy liu cao khng gy tai bin cho chut m nhng c du hiu ngn cn s pht trin ca thai. Khi cho chut cng ung liu trung bnh vo ngy 6 13 ca thi k thai nghn th 50% chut bnh thng, 15% chut c c t cung khng bnh thng, 35% cn li khng c du hiu th thai. Trong nghin cu, nh hng ca vinblastine chit t da cn Vit Nam ln b nhim sc th ca t bo ty xng chut nht thy c tc dng gy sai lc nhim sc th v s lng, ch yu gy nn nhng t bo a bi th (t bi), gy sai lc v cu trc nhim sc th, i n tiu nhim sc th v gy c ch s gin phn ca t bo 2.2.5 ng dng trong y hc 2.2.5.1Vinblastin [1] Tn bit dc: DBL Vinblastin Injection Dng bo ch: dung dch tim. Thnh phn: Vinblastine sulfate

Hnh 13: Cu trc ha hc ca vinblastine sulfate

21

L thnh phn ca mt phi hp 3 thuc, l thuc la chn th nht iu tr ung th biu m tinh hon. N l 1 thnh phn ca nhng phi hp thuc l la chn th hai iu tr bnh Hodgkin, ung th rau, ung th biu m t bo c vy u v c, ung th biu m t bo thn. N l mt trong nhng thuc la chn th ba iu tr u nguyn bo thn kinh, ung th v, ung th c v ung th dng nm da. N cng c dng cha bnh sacoma limpho, sacoma bch huyt bo, bnh da sacoma, chy mu Kaposi, bnh sacoma t bo di. Mt s cng thc s kt hp ca Vinblastine vi cc thnh phn khc: ABVD (adriamycin, bleomycin, vinblastine, dacarbazine)

ChIVPP/EVA (chlorambucil, oncovin, procarbazine, prenisone , etoposide, vinblastine, adriamycin) Stanford-V (adriamycin, mustargen , bleomycin, vinblastine , oncovin , etoposide, prednisone) Khng c s khng cho gia vinblastin v cc loi chng ung th khc. Tc dng ph: Vinblastine c th lm gim vic sn xut t bo bch cu ca ty xng, lm cho bnh nhn d b nhim trng. iu ny c th c hiu lc sau 7 ngy dng thuc, v kh nng b nhim trng cao nht l sau 10-14 ngy sau khi ha tr. Cc tc dng nh bun nn, nn, nhc u v cm xy ra sau khong 4 - 6 gi v ko di trong 2 - 10 gi. Cc hin tng tiu chy, to bn, tc rut, lit, chn n, vim ming cng c th xy ra v thng bo trc nhng tc dng c v thn kinh nh nhc u nng, kh chu, trm cm, d cm v mt nhng phn x gn su. c tnh thn kinh xy ra trong 22

5 - 20% trng hp, ty thuc vo liu lng. Tn thng h thn kinh trung ng i khi c tnh lu di, khi dng liu qu cao xy ra m v t vong. Chng rng tc c th xy ra cho khong 30 - 60 % ngi dng, nhng tc s pht trin tr li khi phng php iu tr c hon thnh. S c ch nh ty xng vi s gim bch cu xy ra bnh nhn vi t l cao, v cn ngng dng thuc. D bm tm, chy mu bt thng (mi, ming, m o hoc trc trng). Nhng tiu cu t b nh hng, t xy ra thiu mu, cn m s lng huyt cu hng tun. Thn trng lc dng: Thuc c tc dng c hi ti ch , cn trnh s trn thuc ra ngoi v c th gy nhim tnh mch ni tim. Nu vinblastine v tnh d r vo m xung quanh, da hoc c bp c th b hng nng. C th xy ra s tit bt thng cht ni tit t khng tit niu. Thuc c tc dng gy qui thai trn ng vt, do khng c dng 3 thng th nht vo thi k thai nghn. Liu dng: Ngi ln tim tnh mch, ngy u tim 0,1 mg/kg, 7 ngy sau v mi tun sau , liu thuc c tng mi ln 0,05 mg/kg, cho ti khi s lng bch cu gim xung ti 3.000 bch cu/mm3, ung th thuyn gim, hoc ti khi t mt liu ti a 0,5mg/kg (bnh thng liu cui cng l 0,15 - 0,2mg/kg). Sau , liu lng duy tr ti mc gim bt tr s gia tng so vi liu cui cng v c tim nhng khong cck t 1 - 2 tun l. Mt s chuyn gia dng mt liu duy tr 10mg, 1-2 ln trong 1 thng. Vinblastin c th gy c cho thai, nn ch dng thi k mang thai nu tnh trng bnh e da tnh mng hoc bnh nng m cc thuc an ton hn khng c hiu lc. i vi tr em, tim tnh mch 0,1 - 2 mg/kg, mt ln trong mt tun.

23

iu kin lu tr: t lnh t 36 46 0F (gia 2,2 7,80C). Thuc c th c bo qun trong 30 ngy k t ngy sn xut. 2.2.5.2 Vincristine [1] Tn bit dc: Vincran Dng bo ch: dung dch tim Thnh phn: Vincristine sulfate

Hnh 14: Sn phm v cu trc ha hc ca vincristine sulfate Vincristine c s chp thun ca Food and Drug Administration (FDA) ca M vo thng 7/1963 nh Oncovin. L mt trong nhng thuc chng ung th c dng rng ri nht, c bit c ch i vi cc bnh ung th mu, thng c dng lm thuyn gim bnh bch cu lympho cp. N c dng trong liu php phi hp thuc, l la chn hng u iu tr bnh Hodgkin, u bch huyt khng Hodgkin, ung th biu m phi, u Wilm (mt loi u thn ph bin tr em), bch cu ty, sarcoma Ewing v sarcoma c vn. Phi hp thuc cha vincristine l la chn hng th hai cho ung th biu m v, ung th c t cung, u nguyn bo thn kinh v bnh bch cu lympho mn tnh. Mt s chuyn gia a dng vincristine ch lm thuyn gim v khng dng trong iu tr duy tr v vic s dng ko di s gy c hi thn kinh. S khng vincristine c th pht trin trong qu trnh iu tr. Vincristine gy gim bch cu nn phi m s lng bch cu trc mi liu.

24

 Tc dng ph: Thng bt u vi bun nn, nn, to bn, co cng c bng, st cn nhng s phc hi nhanh. Thuc cng c th gy nhng phn ng chm phc hi nh rng tc v bnh thn kinh ngoi bin. Nhng tai bin nng v thn kinh c th xy ra nh mt nhng phn x gn su, vim au thn kinh, t cc chi, nhc u, mt iu ha. Nhng khuyt tt th gic, lit nh hoc bi lit v teo mt s c dui c th xy ra chm. Lit nhng dy thn kinh s 2, 3, 6 v 7 cng c th xy ra. Cc tai bin thn kinh c th ko di trong nhiu thng. Tng huyt p nng, tnh trng kch ng hoc trm cm cng c th xy ra nht thi. Thuc gy c ti ch, cn trnh s trn thuc ra ngoi, tt nht nn cho dng thuc bng cch tim truyn tnh mch. Vincristine c nhanh chng thi tr khi mu, 70% thuc c thi trong mt. Trong bnh vng da tc mt, c tnh thuc ln hn v cn gim liu. Khong 12% thuc c thi tr trong nc tiu. Vincristine khng vo trong no, do khng dng cho bnh bch cu h thn kinh trung ng. Thn trng lc dng: Vincristine gy c hi cho thai. i vi ph n cn kh nng sinh , cn dng cc bin php trnh thai. Ph n ang iu tr vi Vincristine cng nh Vinblastine khng c cho con b. Vinscristine khc vi hu ht cc thuc chng ung th khc ch s c ch ty xng khng xy ra thng xuyn, do n c dng trong cc phi hp thuc. Tuy vy c gim bch cu v phi m s lng bch cu trc mi liu. Vic s dng thng b hn ch bi tc dng c v thn kinh. Liu dng: Tim tnh mch mi tun mt ln trong bnh bch cu lymph cp tnh ca tr di 12 tui, bt u 0,03 - 0,075 mg/kg cho tr em 10 tui v 0,05 - 0,15 mg/kg cho tr em 1 tui, 1 liu trong tun u,tip theo bi nhng tr s gia tng hng tun 0,025 mg/kg cho ti 1 liu ti a 0,15mg/kg. Sau dng 1 thuc khc duy tr, nhng bnh nhn t 12 20 tui, tim tnh mch 1,5 4,5mg/m2 din tch b mt c th mt ln trong 1 tun cng vi prednisone (ung 40mg/m2, 1 ln trong mi ngy), cho ti khi t s thuyn gim bnh. Dng nhng thuc khc duy tr. Ngi ln: 0,025 0,075 mg/kg, mi tun 1 ln, ty theo nhng tc dng ph v hiu qu iu tr m iu chnh liu khi cn thit. iu kin lu tr: gi 2-80C, bo qun lnh, khng lm ng, trnh nh sng.

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CHNG 3:

PHNG PHP CHIT TCH

3.1 Phng php chit tch dng cht lu siu ti hn 3.1.1 Khi nim chit sut siu ti hn: L qu trnh phn tch mt hay mt s cht t hn hp (dc liu, hn hp nguyn liu) bng cch s dng cht lng CO2 siu ti hn nh mt dung mi. Khi trng thi ny CO2 c c tnh v tan tng t nh mt cht lng ng thi c kh nng khuch tn v nht gn vi cht kh, nh vy chng c kh nng khuch tn v ha tan nhanh cc hot cht trong nguyn liu. Gore (1891) l ngi u tin pht hin ra kh nng ha tan tt ca Naphtalen v Camphor trong CO2 lng. Sau Andrews (1875) nghin cu v c tnh ca CO2 trng thi siu ti hn. Tuy nhin, ti u nhng nm 1970 cng ngh chit xut cc hp cht t nhin bng dung mi CO2 siu ti hn (SC-CO2) mi thc s pht trin v i vo ng dng trong cng nghip thc phm, m phm, dc phm nh: loi cafein trong c ph v ch xanh, chit xut du vng en, chit polyphenol t ch xanh, loi b cholesterol trong thc phm, loi alcol trong ung, chit xut phm mu, chit xut cc hot cht chng oxy ha, chit xut tinh du, hng liu t thc vt s dng trong m phm, thc phm 3.1.2 u im ca SC-CO2 trong chit xut hot cht t nhin: CO2 c im ti hn thp (nhit gn nh nhit phng, p sut thp) v vy cc hot cht t b oxy ho hay phn hu bi nhit v oxy ho tan, ngoi ra vn thit k h thng chit xut m bo p lc siu ti hn cng d dng hn nn kh nng ng dng cho quy m sn xut cng nghip cng thun li. Sn phm sau khi chit khng cn tn d dung mi v CO2 d dng chuyn sang trng thi kh v bay hi ton b sau khi gim p sut, nhit xung di im ti hn. V vy, chit xut theo phng php ny rt ph hp i vi cc sn phm dng lm thc phm, thuc, m phm. 26

Kh nng chit xut chn lc do:

tan ca SC-CO2 thay i khi p sut v nhit t siu ti hn thay i

vy n s ho tan chn lc cc cht khc nhau nhit , p sut tng ng. Thng thng cc tinh du d bay hi c th c chit p sut di 100bar, trong khi cc cht bo c chit p sut cao hn.

Dung mi SC-CO2 s phn cc hn khi c ho trn vi cc dung mi ph

tr phn cc nh: methanol, ethanol v vy kh nng ho tan cc hp cht s a dng hn. Tuy nhin cc dung mi ph tr c th lm thay i im ti hn ca CO2 do trong thc nghim cn phi kho st t l dung mi ph tr thch hp t nh hng n im ti hn. Thi gian chit xut ngn: Do cht lng siu gii hn c h s khuch tn cao hn cht lng, trong khi nht thp, sc cng b mt nh nn kh nng khuch tn ca dung mi vo trong t bo nhanh hn v vy thi gian chit c rt ngn hn cht lng thng thng. Khng thay i hoc mt hng thm, mu sc t nhin ban u ca hot cht. Khng to ra mi ,v l do SC-CO2 l cht tr, khng mi v v bay hi hon ton khi thay i trng thi siu ti hn. CO2 khng n mn thit b, khng gy chy n trong qu trnh vn hnh, an ton, thn thin vi mi trng, gi thnh r, d kim, ngoi ra c th ti s dng trong thi gian di. Trong trng hp ca vinca alkaloid, vindoline c tch chit nh SFE. Theo nh mt nghin cu nhm ti u ha trong qu trnh thc hin, mt lng ng k vindoline (58% lng cht kh) c tch chit t l ca Catharanthus roseus s dng SFE di iu kin nhit l 350C v p sut 300 bar. [6]

27

Hnh 15: S qu trnh chit tch bng SFE

3.2 Phng php chit tch dng bnh chit Soxhlet [6] (Soxhlet extraction) Qu trnh chit cc ankaloid c thc hin trn my Soxhlet gm c: bnh cu, tr chit, ng sinh hn v bp in. Nguyn liu c nghin nh, cho vo ti giy lc hoc dng ng hnh tr cho mu vo. Cho ti nguyn liu vo tr chit, lp tr chit vo bnh cu, cho dung mi vo bnh chit n ngp ti nguyn liu, lp ng lm lnh, ngm nguyn liu trong dung mi mt vi gi. t my Soxhlet vo ni cch thy. Ta s thu c alkaloid.

Hnh 16: Bnh Soxhlet

28

3.3 Phng php chit tch solid liquid [6] Phng php solid-liquid l qu trnh loi b mt hoc nhiu cht tan t mt cht rn bng cch s dng dung mi lng. Phng php chit tch ny c s dng trong cng nghip ha cht khi m cc phng php c hc v nhit khng th p dng. Cht chit t cy C.roseus l mt hn hp phc tp ca cc alkaloid vi mt phm vi phn cc rng. Quy trnh ca phng php chit tch solid-liquid truyn thng cho Catharanthus alkaloid bt u t vic ngm nguyn liu trong mi trng acid, ch yu da trn c tnh tng hp ca chng. Cc alkaloid s hnh thnh dng mui khi ngm trong mi trng acid ci thin tan v tng s n nh ti gi tr pH thp. Thm vo , proton trong dung dch acid s h tr trong vic b gy cc lin kt nn s gii phng analyte d dng hn. 3.4 Phng php tch chit bng nc nng [6] (hot water extraction-HWE) HWE l mt trong nhng phng php dng tinh sch cc hp cht hu c t nhin. Phng php ny ang ngy cng c nghin cu v p dng. l dng nc nng ang dng lng, iu kin nhit v p sut c th khc nhau t 1000C-3740C v 1 bar -219 bar. Tuy nhin qu trnh thng c thc hin trong khong nhit 100-1800C trn nhng p lc bo ha tng ng 2-30bar cc pha lng c duy tr. Khi hi nc i qua mu, n mang cc hp cht ha tan c trong n i theo, sau chng c ngng t thu c cht mong mun.

29

Hnh 17: S mt b chng ct li cun hi nc 3.5 Sc k lng cao p hiu nng cao [4] Cng vi s ra i ca cc vt liu hiu qu cao, cng nh s tin b v trang thit b cho php thc hin y nng lc ca cc vt liu ny, k thut HPLC pht trin v cng mnh m v vng chc hn so vi cc phong php sc k khc. 3.5.1 nh ngha: HPLC l k thut tch hn hp trn ct oc nhi bng cc ht c kch thoc 10 m. Trong , ngoi ta dng mt bm c p sut cao 300atm y pha ng qua ct vi tc dng khong vi ml/pht v cho php phn gii nhanh mt long mu nh khong 20g. u im: Trong sc k kh, cc hn hp oc kho st trong pha hi, v vy cn phi to mt cht hi n nh t hn hp cn phn tch, hoc chuyn cc cht trong hn hp thnh cc dn cht bn vi nhit. Ch c khong 20% cc hp cht ho hc thch hp cho sc k kh m khng cn phi bin i mu thnh mt dng khc, s cn li u khng bn vi nhit v khng bay hi. Hn na, cc cht c cc nhm chc rt phn

30

cc hay c th ion ho thong c khuynh hong ko ui, khng thch hp khi p dung sc k kh. V th HPLC l mt k thut tt hn i vi cc i phn t, cc loi cht v c v ion ho, cc hp cht thin nhin khng bn, cc hn hp thuc v cc cht ho sinh. Trong sc k kh ch c mt pha (pha tnh lng hay rn) l sn sng tong tc vi cc phn t ca mu. V pha ng l mt cht kh, tt c hi ca mu u tan trong cht kh ny. Trong HPLC, c pha tnh ln pha ng c th tng tc mt cch chn lc vi mu. Cc tng tc nh to phc hay lin kt hydrogen khng c trong pha ng ca sc k kh c th xy ra trong pha ng ca HPLC. Nhiu loi tng tc chn lc khc nhau ny cng c th c gia tng bi s bin i ho hc thch hp ca b mt silica, v th HPLC l mt k thut linh hot hn sc k kh v thng c th thc hin c trn cc phn t tch kh hn. Nhc im: Xt c v u t trang thit b ln ph tn vn hnh, HPLC l mt k thut t tin, hn c sc k kh. 3.5.2 Cu to v hot ng ca my sc k lng cao p HPLC: Thit b c bn my HPLC bao gm: Bnh cha pha ng Bm to p lc cao v h thng to ra vic ra gii ng mi hay ra gii tim tin ( pump + gradient system) B phn tim mu (injection unit) a mu vo ct. Ct oc nhi vi pha tnh c hiu qu cao. Ct c th c t trong mt b phn iu nhit. B phn pht hin v h thng x l d liu My ghi (recorder) hoc my vi tnh ghi hoc lu gi sc k

31

Pha tnh trong HPLC l cc phn t c kch thoc rt nh, oc gi l cht nhi vi tiu phn, chng thng ng dng, c hnh cu hoc hnh dng bt nh, ong knh 10, 5, hay 3 m. C ch phn tch c th oc thc hin bng cc nhm ho hc lin kt vo b mt ca cc tiu phn silica to ra cc pha lin kt. Hin nay, pha lin kt thng dng nht oc s dng trong HPLC l ODS, trong nhm ankyl 18 carbon c gn vo b mt ca cc tiu phn silica. Khi c nhi vo ct, kch thc nh ca cc vi tiu phn dn n s khng ng k vi s chy ca dung mi, v th pha ng phi c bm qua ct bng mt p lc cao. Ct v ton b h thng ct phi chu c p sut s dng v cng phi khng v mt ho hc vi cc dung mi pha di ng, chng thng phi c ch to bng thp khng g. Pha ng c bm qua ct vi vn tc vi ml/pht. Nu thnh phn pha ng khng i, phong php c gi l ra gii ng mi. Ngc li, nu thnh phn ca pha ng c th c lm thay i theo mt cch nh trc trong lc phn tch, phng php c gi l ra gii tim tin. Ra gii tim tin thch hp cho cc mu th nghim cha cc cht c phn cc khc nhau nhiu, cn n s thay i phn cc ca hn hp dung mi trong qu trnh tch. Sau khi qua ct, cc cht tan tch ra c ghi nhn bng mt b phn pht hin lp t trong h thng. Thng tin a ra ca b phn pht hin l mt tn hiu in, s bin i ca tn hiu ny c biu hin trn mt my o th nng, mt dng c tch phn vi tnh, hoc mt mn hnh ca my tnh. Phn ln cc b phn pht hin ph bin trong HPLC l b phn pht hin chn lc, chng khng th p ng vi tt c cht tan c trong hn hp. Do vy, s c mt s cht tan khng c pht hin trong HPLC, chng cn phi c chuyn thnh mt dng khc c th pht hin c sau khi ra khi ct, phng php ny gi l phng php to dn cht sau ct ( post-column derivatisation).

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CHNG 4:

PHNG PHP NUI CY IN VITRO T BO C.roseus

4.1 Nui cy m v ngha ca nui cy m [2] 4.1.1 Gii thiu Nhn ging in vitro hay nui cy m u l thut ng m t cc phng thc nui cy cc b phn thc vt trong ng nghim c cha mi trng xc nh iu kin v trng. Mi trng c cc cht dinh dng thch hp nh mui khong, vitamin, cc hormone tng trng v ng. K thut nui cy m t bo thc vt da trn c s l lun khoa hc v tnh ton nng v kh nng phn ha, phn phn ha ca t bo thc vt. K thut nui cy m cho php ti sinh chi hoc c quan (s pht sinh c quan) t cc m nh: l, thn, hoa hoc r 4.1.2 Tnh u vit ca nui cy m t bo thc vt

33

So vi cc phng php nhn ging t nhin khc th phng php nui cy m l mt ng lc thc y cng ngh sinh hc v nng nghip pht trin v phng php ny c nhng u im sau: - Khng ph thuc vo thi tit, thi v, qu trnh c thc hin trn din tch khng ln, bt k a th no, c th kim tra khng ch cc hng nghin cu theo mun. - To c mt s lng ln cy con trong mt thi gian ngn vi tnh ng nht cao v mt di truyn, ph hp vi nhu cu th trng. - L phng php phc trng ging cy b nhim bnh, thoi ha to ging cy khe mnh. - Gip bo qun ging cy d dng bng cch to ra cc th phi hay bo qun lnh. - To ra cc dng t bin, cc dng siu sn xut, tng tn s t bin trong di truyn t bin thc vt bc cao. Nui cy m dung hp protoplast c th kt hp nhiu tnh trng mong mun trong chn ging cy trng. - Gim chi ph vn chuyn t ni ny n ni khc gip vic trao i ging gia ni ny n ni khc c d dng. - To ra cc sn phm t nhin nh cc cy bnh thng nh dc phm, thuc nhum, hng liu

4.1.3 Mt s k thut nui cy m t bo thc vt. 4.1.3.1 Nui cy c quan C th s dng k thut ny nui cy cc b phn ca cy nh l, rTuy nhin hng ng dng ny khng c p dng rng ri. K thut nui cy c quan thng c p dng trong nc nh: ct l, cnh t l ng nghim ti sinh cy, cy chuyn sang cc l ng nghim tip theo. 4.1.3.2 Nui cy m phn sinh (meristem) M phn sinh thng l cc m nh chi v cnh c kch thc 0.1mm 1cm. Cc m phn sinh dng nui cy thng tch t cc mm non, cc chi mi hnh thnh hoc cc cnh non.

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i vi nui cy m phn sinh s cn bng gia cc cht iu ha sinh trng rt quan trng. Mun kch thch to chi cn b sung cytokinine hoc t hp cytokinine vi auxin. Mun to r th b sung cc auxin nh NAA, IAA,... Nui cy m phn sinh c s dng loi virus to cy sch virus v nhn ging in vitro. Nui cy m phn sinh cn c s dng nghin cu qu trnh hnh thnh c quan, to cy a bi qua x l colchicin. K thut ny c dng rt nhiu trong cng tc phc trng cy trng b nhim virus nn cn c gi l phng php to cy sch bnh. Phng php ny c t nm 1937, tuy nhin n nm 1949 Linesset v Cornuet mi bt u tin hnh nui cy nh sinh trng cy thuc l. Sau , Morel v Martin nui cy thnh cng nh sinh trng cy thc dc (1952) v Cymbidium (1960). 4.1.3.3 Nui cy m so (callus) M so l mt khi t bo khng c t chc, hnh thnh t cc m hoc c quan phn ha di cc iu kin c bit ( vt thng, x l vi cc cht iu ha sinh trng thc vt) nui cy m so, ngi ta c th s dng hu ht cc c quan ang cn sng ca thc vt nh: thn, l, cungTuy nhin, ngi ta thng s dng cc mu cy cn non qu trnh cm ng ca mu xy ra d dng hn.

Nui cy m so c p dng trong nhiu trng hp: Nhn ging in vitro i vi loi thc vt m phng php nhn ging bng nh sinh trng c hiu qu khng cao. Lm nguyn liu cho nui cy t bo n, thu nhn cc hp cht th cp. Nghin cu cc qu trnh hnh thnh c quan.

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4.1.3.4 Nui cy huyn ph t bo Huyn phu t bao la mt mi trng long c lc lin tuc, trong o co s hin din cua nhng t bao soma c lp hoc nhng cum nho cac t bao co kha nng sinh phi co th tai sinh thanh mt thc vt nguyn ven ( Henshaw v cng s, 1965; Torres 1989). S tao huyn phu t bao co kha nng sinh phi trong mi trng long c lc lin tuc va s tai sinh thc vt t huyn phu t bao nay la hai giai oan quan trong cn thit cho phep thc hin s nhn ging in vitro mc cng nghip. Ngoai ra cac giai oan nay cung cn thit cho vic s dung cac tac nhn vt ly, hoa hoc nhm tuyn chon cac c tinh thich hp phuc vu cho nhu cu sng cu sng cua con ngi ma c bit la cac ky thut chuyn gen nh bn DNA, dung hp t baocung nh moi thao tac mc t bao. 4.1.3.5 To phi soma to ht nhn to Trong mi trng nui cy thch hp th m cy hay t bo c th pht trin thnh phi v tnh. Phi c bc bng lp v bc cha ni nh nhn to thnh ht nhn to. C hai cch to phi v tnh l: pht sinh phi trc tip v pht sinh phi gin tip. S ghi nhn u tin v nui cy phi l cng trnh ca Charles Bonnet th k XVIII. ng tch phi Phascolus v Fagopyrum trong trong t v nhn c cy nhng l cy ln. T u th k XX cc cng trnh nui cy phi dn c hon thin hn. T cc cng trnh nghin cu trc , Knudson (1922) nui cy thnh cng phi cy lan trong mi trng cha ng v khm ph ra mt iu l nu thiu ng th phi khng th pht trin thnh protocorm. Raghavan (1976, 1980) cng b rng phi pht trin qua hai giai on d dng v t dng. giai on d dng (tin phi) cn c cc cht iu ho sinh trng pht trin. Trong giai on t dng s pht trin ca phi khng cn cht iu ho sinh trng.

36

i vi nui cy phi, nh bit ng ng vai tr rt quan trng. Trong nhiu trng hp th ng sucrose cho kt qu tt hn cc ng khc. Ngoi ra mt s cht t nhin nh nc da, nc chit malt, casein thu phn, l nhng cht rt cn trong nui cy phi. Cc cht kch thch sinh trng nh GA3, auxin, cytokinine thng c dng nhiu trong nui cy phi Cc yu t ngoi cnh nh nhit , nh sng cng nh hng n s pht trin ca phi nui cy in vitro. Thng phi nui cy cn nhit v nh sng thp hn phi pht trin t nhin. 4.1.3.6 Nui cy ht phn v bao phn n bi Nguyn liu cho phng php ny l bao phn hoc ht phn tch ri, tnh trng vt l ca cy v kiu gen nh hng ng k n s thnh cng ca vic nui cy bao phn. Ngoi ra, trng thi ca ht phn cng rt quan trng. 4.1.3.7 Nui cy t bo trn T bo trn (protoplast) l t bo b loi b vch cng, ch cn li mng t bo bao bc xung quanh. Ngi ta c th tin hnh lai, dung hp gia cc dng t bo trn vi nhau, sau li ti to mng v vch t bo. Vic dung hp t bo trn gip to cc c th lai m phng php lai hu tnh khng th tin hnh c. 4.1.4 Cht iu ha sinh trng [2] Thut ng cht iu ho sinh trng thc vt (Plant growth regulator, PGR) c dng rt nhiu bi cc cng ty nng dc ch cc cht iu ho sinh trng tng hp. nh ngha ca Van Overbreek v cng tc vin (1954) vn cn c dng n ngy nay. Cht iu ho sinh trng thc vt l nhng hp cht hu c khc vi nhng cht dinh dng, vi mt hm lng nh kch thch, c ch, hoc b sung bt k mt qu trnh sinh l no trong thc vt. Trong sinh l thc vt khi nim v cht kch thch sinh trng bao gm cc hp cht c tc dng iu tit cc hot ng sinh trng v pht trin ca thc vt. Nhm 37

hp cht ny bao gm cc hormone t nhin (auxin, gibberellin, cytokinin, acid abscicic, ethylene, cc hp cht phenol iu tit sinh trng, hormone ra hoa). 4.1.4.1 Auxin Bn cht ha hc ca auxin t nhin trong t bo thc vt l acid indol acetic (IAA) v n l dng auxin u tin, ch yu v quan trng nht trong tt c cc loi thc vt. Trong thc vt n khng ch tn ti dng t do m cn dng lin kt khng c hot tnh sinh hc nh IAA-glucose, IAA-myoinositol, IAA-glucan, IAAaspartateCc dn sut khc ca indol cng th hin hot tnh ca auxin l indol tryptamine, indol acetaldehyde, indol pyruvate, indol ethanol. Auxin c tng hp tt c cc thc vt bc cao, to, nm, vi khun v ch yu nh chi ngn ri di chuyn xung cc b phn non ca c th thc vt: l, r v cc m d trAuxin gm c auxin t nhin v auxin tng hp (IBA, NAA, 2,4-D) Auxin c nhiu vai tr khc nhau trong i sng thc vt, lin quan ti hng lot cc qu trnh sinh l: tc sinh trng, trng thi ng, s ra hoa, s kt tri, sinh trng qu, s chn ca qu, s rng qu v rng l, to c, s lo hang nhin, do hormone thc vt tc ng ln sinh trng thng qua mi tng quan nng gia cc loi hocmone khc nhau, nn cc qu trnh trn y khng ch nh hng ca auxin m cn ca cc hormone khc. Ty thuc vo nng tc dng m cc m thc vt c cc kiu phn ng khc nhau i vi auxin. Phn ng ch yu v nhanh chng nht i vi x l auxin l lm tng ko di ca t bo thng qua tc dng trc tip ln s gin n ca vch t bo. Cc cht iu ha sinh trng thuc nhm auxin gm mt vi hp cht c s dng t rt lu trong cng nghip. Ch sau mt thi gian ngn sau khi IAA c tm thy trong t nhin, n c tng hp v tr thnh mt hp cht c gi tr. Nhng IAA khng c li dng trong nng nghip bi n d dng b phn hy thnh cc hp cht mt hot tnh di nh hng ca nh sng v vi sinh vt. Mt trong nhng tc dng ca auxin l kch thch s hnh thnh r ca nhng lt ct thn. Mt s hp cht tng hp nhn to c vai tr tng t nh IAA, trong c IBA. IBA l hp cht c hot tnh auxin yu nhng n c kh nng n nh v v hiu h enzyme lm mt hot tnh ca auxin. 4.1.4.2 Cytokinin Phn ln cytokinin l dn xut ca purine. Loi cytokinin u tin pht hin c v cng l dng ph bin nht l zeatin tch t ht ng. Ngoi ra cn c hng lot cytokinin khc nh kinetin, dihydrozeatin, benzyladenin, chlorephenylurea, trong kinetin khng c mt trong t nhin, m ngi ta thu nhn bng cch x l nhit ADN. 38

Chng minh v kh nng ngn cn s vng l ca benzyladenin (BA) l mt pht hin thu ht nhiu nh sinh l hc t nhng nm 1950. Nhng nm 1960, cc nh nghin cu thy rng BA c th kch thch nhiu qu trnh, BA c s dng trong nui cy m ko di chi v pht sinh phi vi cc nng khc nhau ty theo i tng thc vt nui cy v mc ch nui cy. Cytokinin c mt trong mi thc vt, vi hm lng cao nht trong phi v trong qu ang pht trin. Hot tnh ca chng c tng cng khi chng tng tc vi myo-inositol, nhng c th b mt khi kt hp trong thnh phn ca cc glycoside. Cng nh auxin, cytokinin tham gia iu ha cc phn ng trong cy, ng thi lm tng cc qu trnh trao i acid nucleic v protein. Cytokinin iu chnh sinh trng bng nhiu cch: iu chnh tc tng hp ADN khi phn chia t bo. Lm chm s lo ha ca l. Gp phn ph v trng thi ng ca ht, kch thch ht ny mm, kch thch ra hoa v sinh trng ca qu. - Gy nn s hnh thnh chi mm trong nhiu m, bao gm m so trong nui cy m. - Trong cy, cytokinin vn ng t r n chi thng qua xylem kh d dng, nhng chng hu nh khng linh ng trong l, ngoi tr mt phn c dn truyn nh hng thng qua cung l. 4.1.4.3 Gibberellin L mt nhm cc cht iu ha sinh trng thc vt gm hn 80 hp cht khc nhau. Cc hp cht ny c im ging nhau cu trc ha hc l sn gibbane. Gibberellin c lin quan n nhiu qu trnh sinh l trong cy. Tuy nhin nhng chi, loi vi nhng yu t khc nhau s quyt nh gibberellin c hiu hiu qu nht. Gibberellin nh hng n nhiu qu trnh sinh trng v pht trin ca thc vt nh s pht trin thn, s ny mm ca ht, min trng, tr hoa, phn ha gii tnh, trinh qu sinh, u tri v lo ha. nh hng trn s pht trin ca thc vt sng: Cc gibberellin bit u c kh nng kch thch s vn di ca thn hay s phn chia t bo. S kch thch vn di ca GA th hin rt r trn nhng cy non hoc b phn non, cy trng thnh hay c quan gi th nh hng s km i. Nhn chung, GA kch thch s sinh trng ca nhiu loi cy c bit l nhng cy ln. nh hng ln tnh trng ln: C nhiu bin d thiu sinh tng hp GA c pht hin. y l tnh trng n gene, kch thc ca cy bin d c th ch bng 39 -

mt phn nm cy bnh thng v s ln ch yu l do lng b ngn li. Cc dng t bin ln nh t bin bp ln (Zea Mays L.) d1 v d5 v la ln (Oryza Sativa L.) Tan-ginbozu v Waito-C. GA ni sinh kim sot hot ng ca bp v la l GA1. Vic x l GA ngoi sinh lm cho cc cy ny cao tr li bnh thng. nh hng ln s ny mm ca ht v min trng: Hin nay GA c bit l nhng cht c kh nng kch thch ny mm v ph v min trng trn nhiu loi cy trng. GA c th kch thch hot ng ca cc enzyme thy phn hydrolase trong ht ng cc. GA ngoi sinh tc ng ln lp aleurone ca ht ng cc v kch thch s sn sinh enzyme -amylase tc ng ln s phn hy tinh bt thnh ng n. Tc ng ny c tc dng kch thch ny mm v ph v min trng. nh hng ln s tr hoa: Gibberellin c kh nng thc y qu trnh tr hoa trong nhiu loi thc vt. i vi nhng cy cn yu cu ngy di hay tri qua iu kin lnh trc tr hoa th khi x l GA trong nhng iu kin khng cm ng chng s tng hoa v tr hoa. nh hng ny c lin quan n s kch thch qu trnh phn chia t bo v vn di t bo. nh hng ln s phn ha gii tnh, trinh qu sinh, u tri v lo ha: GA c th lm thay i gii tnh ca hoa tng t nh auxin, cytokinin v ethylene. Tuy nhin, GA c hiu qu ngc vi auxin v ethylene. GA lm tng s hoa c trn da leo. GA cng gy nn hin tng trinh qu sinh v to nn tri khng ht. GA cng gip cho tri to v tr hon s lo ha. 4.1.4.4 Acid abscisic L mt cht iu ha sinh trng thc vt t nhin c to ra trc tip t acid mevalonic hoc do s phn gii caroteniod. Acid abscisic c sinh tng hp trong cc lp th (Neill v Horgan, 1984; Milborrow, 1974). Vai tr quan trng ca ABA nh l mt cht cm ng vi stress c bit trong nhiu nm qua. X l ABA ngoi sinh gy ng kh khu trong iu kin sng v duy tr cho n khi ABA b chuyn ha. Trong iu kin stress do thiu nc ABA c th gia tng ln 20 ln.

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Hm lng ABA gia tng khi cy b stress do mn, lnh v nng. Nhng s bin i ny l nguyn nhn ca s thiu nc. Vic x l ABA ngoi sinh c th lm cho mt s loi cy chng li iu kin lnh v mn. Trong iu kin ngy ngn, hm lng ABA gia tng trong l v mm chi dn n s min trng. Tuy nhin cng c trng hp trong iu kin ngy ngn gy ra s min trng trong vi loi li khng c s gia tng ABA ni sinh. Vic x l ABA ngoi sinh ln mm chi v ln ht kch thch min trng ca chng. T khi mi c pht hin, ABA c xem l cht gy nn s rng l, tri v hoa. Tht ra iu khng hon ton ng v ABA khng trc tip nh hng ln s rng. ABA c th tc ng gin tip ln qu trnh lo ha trc trng thnh v lm gia tng s sn sinh ethylene v ethylene nh thc mt s gene lin quan n s rng. Ngy nay ngi ta bit rng ABA c nhiu nh hng v mt sinh l, sinh ha v phn t trong ht cng nh c mt ph bin trong lc ht pht trin. Tuy nhin vai tr trc tip ca ABA i vi cc qu trnh ny vn cn cha r. 4.1.4.5 Ethylene Nhng ngi Ai Cp khng bit rng ethylene chnh l tc nhn gy chn tri khi h to vt thng trn tri. Ngy nay, ethylene c bit l hormone iu ho s chn tri. Nm 1901, Neljubow chng minh ethylene c ch s vn di, kch thch s pht trin theo chiu ngang. Ngy nay, ngi ta bit c ethylene c kh nng kch thch hoc c ch s vn di ca thn, r hoc nhng c quan khc. Ethylene cng c biu hin kch thch s vn di ca thn hoc r vi tc chm. p ng b ba kch thch bi ethylene c th hot ng nh mt c ch tn ti trong cy con. p ng b ba da trn tnh sinh trng ngang, s phng ln v c ch s pht trin chiu cao thn. V d: khi mt cy u H Lan gp phi b mt cng ca t, th n s p ng vi s sinh trng theo kiu b ba, cho php m thng hoc i chung quanh chng ngi, vn ti b mt t v tip tc pht trin. Ethylene trong vng mc cu ca cy con gip n duy tr hnh mc cu cht ch, vt qua p lc vt l khi va chm vi t m khng b tn thng. Khi cy con 41

nhn c nh sng , s sn sinh ethylene s gim. Kt qu l mc m. iu ny hot ng nh mt c ch an ton, ngn cn s m trc ca mc v gim thng tn n cy con trc khi nh ln khi mt t. Qu trnh rng rt quan trng trong nng nghip bi v n nh hng n nng sut v hiu qu sn xut. Ethylene c vai tr t nhin trong vic iu ho tc rng. Nm 1985, Reid a ra bng chng: ethylene sn sinh gia tng trc khi rng trong nhiu c quan ca cy ang rng; X l ethylene kch thch s rng thc vt; Nhng cht c ch s sinh tng hp ethylene hoc c ch hot ng ca ethylene s c ch s rng. Nhiu nm qua ngi ta quan st khi t g thc y s tr hoa trong cy khm v xoi, thnh phn chnh ca khi l ethylene. Ethylene trong phn ln trng hp c ch qu trnh tr hoa; Ngc li, i vi cy khm, xoi v vi, ethylene kch thch tr hoa. Cy c th c x l trc tip vi ethylene hoc gin tip thng qua vic s dng nhng cht phng thch ethylene Lo ho c c trng bi s phn r dip lc t, protein, hoc RNA. C l v hoa khi c x l vi ethylene ngoi sinh u gia tng qu trnh lo ho v gia tng s sn sinh ethylene ni sinh. Cn ch rng c nhng trng hp ngoi l, ethylene khng n l lin quan n qu trnh lo ho: nhiu cy khng gia tng sn sinh ethylen trc khi lo ho. Ethylene cng cng c lin quan n quang hp, h hp, vn chuyn, min trng, s ny mm ca ht, nh chi, u th chi ngn, sinh trng t bo, nui cy m, thnh lp phi, s nghing, s khi sinh r, nhng c quan d tr, s thnh lp g, s c ch mm hoa, s pht trin gii tnh, a hng ng, s r nha v s thnh lp nha m ca cy. 4.1.5 Mi trng nui cy[2] i vi k thut nui cy m, s nui cy in vitro i hi nhng iu kin v mi trng nui cy v mi trng xung quanh. Nhng iu kin ny c th thay i trong qu trnh nui cy v iu ny cho php ngi ta ch ng k thut mt cch t nh nht. Mc d thnh phn mi trng nui cy m thc vt thay i ty theo loi b 42

phn nui cy, s pht trin phn ha ca m cy v ty theo ta mun duy tr m trng thi m so, to rm thay i t nhiu nhng tt c cc mi trng nui cy bao gi cng bao gm 5 thnh phn chnh sau: ng lm ngun carbon. Cc mui khong a lng. Cc mui khong vi lng. Cc vitamin. Cc cht kch thch sinh trng.

Ngoi ra, cc tc gi cn thm mt s cht hu c c thnh phn ha hc xc nh ( amino acid, EDTA) hoc khng xc nh (nc da, nc chit nm men, nc chit chui, nc chit khoai ty, c chua) 4.1.5.1 ng (ngun carbohydrate) Trong nui cy nhn to, ng c s dng nh ngun carbon m, t bo thc vt tng hp nn cc cht hu c gip t bo phn chia, tng sinh khi ca m, nh hng n sinh trng pht trin ca chi. Hai dng ng thng c s dng nht hin nay l saccharose v glucose, nhng saccharose c s dng ph bin hn. Ty theo mc ch nui cy m nng ng c s thay i. Nhu cu cacbohydrate trong mi trng nui cy l 2-3% i vi ng saccharose hay thp hn i vi ng glucose [10] 4.1.5.2 Cc mui khong a lng Nhu cu mui khong ca m t bo thc vt tch ri khng khc nhiu so vi cy trng t nhin. Cc nguyn t a lng cn thit l N, P, K, Fe, Ca, Mg. 4.1.5.3 Cc mui khong vi lng Nhu cu mui khong vi lng ca m thc vt trong nui cy cn rt t c nghin cu, mc d chng l thnh phn khng th thiu c cho s pht trin ca m v t bo thc vt. Nhng an ton, cc tc gi cung cp hu ht cc yu t vi lng cho m nui cy. 4.1.5.4 Cc vitamin v cc cht kch thch sinh trng kch thch cho s sinh trng pht trin ca m thc vt nui cy, ngi ta cn b sung thm mt s loi vitamin: myo-inositol, vitamin PP, vitamin B1, B6v cc cht kch thch sinh trng thuc nhm auxin, cytokinin, gibberelin...

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4.1.5.5 Cc cht b sung a. Agar (thch) Agar l thnh phn quyt nh trng thi vy l ca mi trng. Hm lng agar dng cho mi trng nui cy dao ng t 0,6-1,0% theo khi lng. Agar l nguyn liu ph bin nht dng cho vic pha cc mi trng bn rn hay mi trng rn. Nu b sung agar vi nng cao th mi trng tr nn cng, s khuch tn ca cc cht dinh dng cng nh hp th ca m gp kh khn (Bhojwani v Razdan; 1983). a s nui cy phi c thc hin trn mi trng c agar nhng ph thuc vo cy m s dng cho ph hp. [11] b. Than hot tnh Than hot tnh c s dng trong mi trng nui cy nh mt cht ph gia v than hot tnh c kh nng hp ph cc cht tit ra t m cy, v bao phnv lm tng hiu sut sinh phi (Fridborg et al, 1978; Ammirato, 1983). Than hot tnh khng phi l cht iu ha sinh trng nhng c th lm thay i thnh phn ca mi trng, hp thu nhiu hp cht khc nhau, m cc cht ny c th hnh thnh trong qu trnh hp mi trng, hp thu cc cht tit ra t mu cy cng nh cc cht iu ha sinh trng khi b sung vo mi trng, ngn chn s pht trin m so khng mong i. Ngoi ra than hot tnh cn gp phn y mnh s pht sinh phi v s hnh thnh r. [12] Trong mt s nghin cu v nh hng ca than hot tnh khi b sung vo mi trng nui cy, thy s hin din ca than hot tnh trong mi trng nui cy rt quan trng trong vic lm tng chiu cao cy, chiu di r, trng lng ti v trng lng kh, v lm gim s hnh thnh chi nch t m cy mt cch r rt [10]. Ngoi ra, mu cy c nui cy trong mi trng c than hot tnh khe mnh, c mu xanh m, cn mi trng khng c than hot tnh mu cy yu hn v l hi vng. [13] c. Cc cht hu c khc Bn cnh cc cht iu ha sinh trng, ngi ta cn s dng nhiu dung dch hu c phc tp c thnh phn khng xc nh nh nc da, dch chit chui xanh, khoai ty, c chua v dch chit nm men, casein thy phn nhn ging nhm tng cng s sinh trng v pht trin ca m cy. Hiu qu thc y qu trnh tng trng ny c gii thch bng nhng vitamin, amino acid v nhng cht iu ha khc cha trong chng (Perik, 1987). Nc da

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Nm 1941, Van Ovebeck xc nhn tc dng kch thch ca cy da trong nui cy phi h C. Nm 1948, Steward cng thu c kt qu nh vy m c rt. Nc da v trng c Trung tm Sm Vit Nam dng lm mt thnh phn trong mi trng nui cy m to sinh khi m Sm K5 thnh cng t nm 1983. Ngoi ra s dng trong nui cy thnh cng trong cc loi cy khc nh ng sm di thc, phong lan Nhiu cng trnh nghin cu xc nh trong nc da ngoi khi lng nc chim 90% cn c cc cht ng, amino acid, lipid, vitamin, cc nguyn t vi v a lng, acid hu c, cc cht kch thch sinh trng. Theo Tuleke, amino acid nc da xanh chim hn 40% amino acid tng s l glutamine, nc da gi cng c mt s lng ln acid glutamic, glutamine, serin, leucin, acid amino butyric. Ngoi ra trong nc da cn c cc acid hu c c cha acid hu c c cha acid malic, sikimic, quinic v cc ngun hydrate carbon khc nh saccharose, glucose, fructose. Theo Vandebelt (1954), trong nc da c nhiu vitamin nh acid nicotinic, acid pantotenic, biotin, riboflavin, acid folic, thiamin v pyridoxine. Theo Paris, Duhamat (1953) trong nc da c cha IAA. Cn Radiey (1958) pht hin c gibberelin trong dch thc vt ny. Dch chit chui Chui l cy n qu lu i gn lin vi cuc sng gia nh hng triu dn vng nhit i ni chung v c ph bin nc ta. Hm lng cht dinh dng trong qu chui rt cao, c bit ng chim 10-22% v nhiu loi vitamin khc nh vitamin B1, B2, PP, C v c bit vitamin A chim t 250-320 n v. Theo kt qu nghin cu ca Atwater, thnh phn dinh dng trong qu chui rt cao: protein 1.3%, m 0.6%, bt ng 22%, tro 0.8%. Khoai ty Thnh phn ca khoai ty bao gm: magnesium 10%, thyamin 15%, riboflavin 3%, vitamin C 50-70%, Dietary Fibre 10-15%, phosphorus 5%, vitamin B6 13-19%, acid forlic 7%, Fe 6-14%, protein 6.5%, iodine 14%, acid pantothenic 4-7%, calcium 6%, niacin 10%, km 5% [14] 4.2 Phng php nui cy in vitro t bo C.roseus [8] Trc y nhng nguyn liu th dng sn xut alkaloid thng c thu nhn hu ht ti nhng vng m C.roseus xut hin t nhin hoc ti nhng vng n c trng trt. iu kin thi tit v c tnh ca t mt s nc chu u th bt li cho vic gieo trng C.roseus. V th n ch c th c trng nh l mt loi cy 45

hng nm trong nh knh v nhng nh mi vng lm bng nha. Mt khc, trong khi nhu cu v alkaloid rt ln v gi ca chng rt cao, nhng hm lng alkaloid trong nguyn liu th thu nhn t cy trong t nhin l rt thp. Do , vic thu nhn indole alkaloid t m v c quan thc vt, v t nhng cy c nhn ging in vitro l nhng hng i mi cn c pht huy. M so, huyn ph t bo v r t c nui trong bioreactor c s dng trong nhiu th nghim. Nhng m c nui cy t nhng c quan khc nhau tng hp rt nhiu hp cht th cp v c tnh di truyn n nh.Nhiu trin vng ha hn bao gm vic sn xut nhng indole alkaloid nh : ajmalicine trong m so, catharanthine trong l c nui cy trong bnh lc v bioreactor c sc kh, v vinblastine t chi v r t. 4.2.1 Nui cy dch huyn ph t bo Nhiu nghin cu c th nghim trong vic thu nhn huyn ph t bo nui cy cho nng sut thu indole alkaloid cao, c bit l vinblastine v vincristine . Phng php sn xut catharanthine v ajmalicine trong dch huyn ph c nui cy trong bioreactor thnh cng. Moreno v cng s (1996) nghin cu nh hng ca cc cht cm ng ln nhng con ng chuyn ha khc nhau lin quan n qu trnh chuyn ha hp cht th cp trong nui cy huyn ph t bo C.roseus. Dch trch t bo nm Phytium aphanidermatum c tit trng c dng trong nghin cu ny. Nhng t bo C.roseus ngay lp tc bin i qu trnh chuyn ha ca chng phn ng li tc ng ca nm. Zhao v cng s (2001) th nghim nhiu cht cm ng ca nm nhn c t 12 loi nm v nh hng ca chng ln vic ci thin sn xut indole alkaloid huyn ph t bo C.roseus. Nhng kt qu ny cho thy rng nhng cht chit t nm khc nhau kch thch tch ly nhng indole alkaloid khc nhau, c th gp 2 n 5 ln nui cy thng. Zhao v cng s (2001) tng sn phm catharanthine trong t bo nui cy C.roseus bng cch kt hp cht cm ng nui trong bnh lc v bioreactor. Tc ng tng hp ln s tch ly alkaloid trong t bo nui cy c theo di khi chng c x l vi mt vi s kt hp gia cht cm ng ca nm v ha cht. S kt hp gia tetramethyl ammonium bromide v nm si Aspergillum niger cho hiu sut thu ajmalicine cao nht v ci thin s tch ly catharanthine. Trong khi s kt hp gia cht cm ng malate v sodium alginate cho kt qu hiu sut catharanthine cao nht v cng tch ly ajmalicine cao trong t bo nui cy. Cc tc gi sau pht trin quy trnh ny nng cao vic sn xut catharanthine t bo C.roseus nui trong bnh lc v bioreactor. Sau 10 ngy nui cy thu c catharanthine vi hiu sut theo th t l 25 mg/l, 32 mg/l v 22 mg/l trong bnh lc 500 ml, 1,000 ml v bioreactor dng sc kh 20 lt. Tc gi cng quan st thy rng s kt hp gia vic 46

x l malate v alginate kch thch s phn ng li, v d nh l s oxy ha lipid xy ra hu ht qu trnh nui cy C.roseus v iu ny c th gin tip sn xut indole alkaloid thng qua con ng jasmonate. 4.2.2 Nui cy m so Nhng m so c th l ngun cung cp alkaloid chnh, ph bin nht, cung cp vt liu khi u cho s thit lp qu trnh nui cy huyn ph hoc cho vic cm ng nhng c quan nh chi hoc r. Nhng kho st u tin c lin quan n s hnh thnh ca r t m so C.roseus c cng b bi Dhruva v cng s (1977). Vic ti sinh nhng cy C.roseus khe mnh c th thc hin t nhng m so thng qua vic lm ly nhim nhng cy ny vi mycoplasma-like organisms (MLOs). Mollers v Sarkar (1989) lm nhim nhng ch ct t thn cy C.roseus vi 3 loi MLOs khc nhau v nui chng trn mi trng dinh dng MS rn. M so c hnh thnh trn nhng m b nhim, ri c phn vo cc cy, sau chuyn sang mi trng MS c b sung 1-naphthyl acetic acid (NAA) 1.0 mg/l, 6-benzyl aminopurine (BAP) 0.25 mg/l v gentamycin 10.0 ml/l. Nhng cy ny sau d dng thch nghi vi iu kin trong nh knh ni chng s c nui trong 1 nm. Vic nhum mu l vi fluorochoromes DAPI (4,6-diamidino-2-phenylindole) v berberine sulfate chng minh s vng mt ca MLOs trong cy ti sinh. Ramavat v cng s (1978) m t qu trnh hnh thnh chi non C.roseus t m so. S ti sinh cy t nhng t bo m so n bi hay lng bi C.roseus cng ang c kt hp vi vic s dng nhng cht iu ha sinh trng thc vt nh kinetin v -indolylacetic acid (IAA) c thc hin bi Abou-Mandour v cng s (1979). Phng php cho tn s cy C.roseus ti sinh cao bng cch l to phi soma c m t bi Kin v cng s (1994). Tc gi thu c nhng m so t bao phn ca cy C.roseus s dng mi trng MS rn b sung NAA 1.0 mg/l v kinetin 0.1 mg/l (MSNK). Nhng phi soma c hnh thnh sau khi chuyn nhng m so ny vo mi trng MSNK lng. Nhng cy c trng nh vy s c cng s nhim sc th nh l cy trng t ht (2n=16). Pietrosiuk v cng s (1999) thnh lp hai dng m so C.roseus trng v xanh (hnh 17d, e) trn mi trng Gamborg rn b sung vi 0.1 mg/l kinetin v 1.0 mg/l IAA. M so c ly t nhng on tr di l mm ca cy C.roseus ging. Dng m so trng c chn t nhng m xanh pht trin trong pha tnh. Tri ngc vi s rn chc ca dng m so mu xanh, dng mu trng li mn v mm. Phng php HPLC phn tch cc cht chit t m so ca c hai dng m so 47

xanh v m so trng ch cho thy cc indole alkaloid tn ti dng vt nhng khng c dc tnh. 4.2.3 Nui ht nhn to Krueger v cng s (1982) to ra c cy c l nui cy t ht C.roseus ny mm v trng trn mi trng Murashige v Skoogs Revised Tobacco (MS RT) c b sung benzyladenine (BA), vi c im l nhn ging nhanh chng v sn xut c cc alkaloid nh vindoline, ajmalicine, sitsiriline, tetrahydroalstonine v serpentine. Qu trnh nui cy cho thy s pht trin v sn xut vindole nhanh chng v rt nhiu hn hp alkaloid khc, bao gm c nhng alkaloid khng c tm thy cy nguyn vn. Miura v cng s (1988) c lp c vinblastine t nhng chi non C.roseus nui cy c ly t nhng cy con ny mm t ht. Lng vinblastine thu c l 15g/g cht kh v cao hn trong m so (Miura et al. 1987). Kt qu ny cho thy rng l s sn xut vinblastine th lin quan n s hnh thnh chi. 4.2.4 Nui cy m phn sinh S nhn ging cy t nhng m phn sinh hin nay l mt phng php hu ch sn xut nhanh sinh khi phc v cho y hc. N cho php kh nng ti to mt kiu gen n nh v khng thay i. Alen v cng s (1995) xng vic nhn ging in vitro C.roseus bng cch nui dng m phn sinh ngn v chi nch t nhng cy c nui trong nh knh. Cc tc gi s dng mi trng MS c bn c b sung BA v 2,4-D nng thp. Nhng chi nch ny s hnh thnh chi ngn sau 4-5 tun nui cy. Mt phn nhng cnh nhn ging c chuyn vo nui cy tip trong mi trng t do. Sau chng c ct v cho to r in vitro trong mi trng MS c b sung vi NAA v pht trin trong nh knh vi m tng i 100%. Trong c 2 trng hp u thnh cng. Furmanowa v cng s (1991, 1994) thnh cng trong vic p dng phng php ny cho s nhn ging cy C.roseus. Tc gi ti sinh nhng cy con t chi ngn v chi nch. Nhng chi ngn c ct t nhng cy con 7 ngy tui v nui trong mi trng rn Nitsch v Nitsch (NN) (Nitsch v Nitsch 1969) c b sung thm kinetin, bezyladenine, indole-3-butyric acid (IBA) v IAA kt hp vi nhau. Sau 2 thng nui cy nhng cy con c r c ct v chuyn vo mi trng mi. Nhng cy ny phi gm c chi ngn hoc chi nch. Khong 200 cy con ang mc r (hnh 17a, b) c thu t mt cy con. Sau chng s pht trin trong nh mi vng bng nha v trong nh knh (hnh 1c). Sau 5 thng sinh trng chng c thu nhn, sy v cn. Vic phn tch thnh phn ha hc ca l c thc hin. Vindoline 48

v catharanthine chim u th trong giai on cn non ca cy (trc khi ra hoa). Hm lng alkaloid tng trong l ca nhng cy c nui cy in vitro ri mi chuyn sang mi trng t th cao hn nhng cy trng ch trong t.

Hnh 18: Sn phm m so v cy mc t ht nhn to

(a) Cy C.roseus mc r pht trin t m phn sinh ngn trn mi trng NN b sung vi 0.1 mg/l kinetin v 0.5 mg/l IBA sau 1 tun nui cy. (b) Cy C.roseus ang ra hoa pht trin t m phn sinh ngn trn mi trng NN b sung vi 0.1 mg/l kinetin v 0.5 mg/l IBA sau 6 ngy chuyn qua. (c) Cy C.roseus ang ra hoa trong nh knh c nhn ging in vitro t m phn sinh ngn. (d) Dng m so mu trng nui cy trn mi trng B5 b sung vi 0.5 mg/l kinetin v 1.0 mg/l IAA (sau 96 ngy chuyn qua). (e) Dng m so mu xanh nui cy trn mi trng B5 c b sung 0.1 mg/l kinetin v 1.0 mg/l IAA (sau 96 ngy chuyn qua)

4.2.5 Nui cy r t Trong nhng nm gn y, phng php nui cy r, ch yu l r t ca C.roseus tng ln ng k hn c mong i, nh l mt ngun tim nng ca nhng hp cht c gi tr cha bnh ny. S pht trin ca k thut vi nhn ging cy v phng php m rng vic nui cy r in vitro l rt quan trng v c th tr thnh gii php quan trng gii quyt vic thu nhn indole alkaloid t nguyn liu th. Nhng yu t di truyn ca r t, thu c bng s nhim vi Agrobacterium 49

rhizogenes, sn xut hp cht th cp nhiu hn l nhng cy t nhin. Hm lng alkaloid trong r t c th tng ng hoc cao hn khi i chiu vi hm lng o c trong nghin cu r t nhin. Nhng alkaloid chim u th trong r t l ajmalicine, serpentine, vidoline v catharanthine, c tm thy vi hm lng cao hn trong r t nhin.

Hnh 19: Sn phm r t (a) R t ca C.roseus xut hin sau 7-10 ngy nhng ch c cm ng vi Agrobacterium rhizogenes chng ATCC 15843. (b) R t ca C.roseus nui cy trong mi trng lng B5 m khng c cht iu ha tng trng (11 ngy chuyn qua) (c) Cy C.roseus c chuyn gen ti sinh t r t trong mi trng lng B5 khng c cht iu ha sinh trng. (d) Ht nhn to thu c t on r t C.roseus cha trong gel calcium alginate (e) R t pht trin t ht nhn to C.roseus nui cy trong mi trng rn NN c b sung IBA 0.5 mg/l v kinetin 0.1 mg/l

50

(f) R t pht trin t ht nhn to C.roseus nui cy trn mi trng rn NN b sung vi IBA 0.5 mg/l v BAP 0.1 mgl/l

4.3 Cc yu t nh hng n qu trnh nui cy t bo C.roseus 4.3.1 nh hng ca cht iu ha sinh trng Hirata v cng s (1994) chng minh rng phytohormone (hormone thc vt) rt quan trng cho s tng trng v phn chia hnh thi ca m, dn n s hnh thnh v pht trin ca chi C.roseus, v cng lm a dng ha cc alkaloid v lm tng hm lng ca chng Tt c chi non nui cy ca C.roseus c cm ng hon ton vi tn s cao t nhng ht ny mm trn mi trng Murashige v Skoog (MS) (Murashige v Skoog 1962) c b sung thm BA 1.1 mg/l (Hirata v cng s 1987). Kt qu nui cy mc d ch gm c m so mc trc tip trn mi trng rn v c mt s chi non vi mt vi l nh. Theo thng s thu c t phng php phn tch sc k lng hiu sut cao (HPLC) ca dch trch nui cy cho thy rng vindoline v catharanthine hin din ch yu trong cnh non, c bit l trong l, trong khi ajmalicine (0.072 mg/g cht kh) tch ly chnh trong cc m so. Hm lng catharanthine (0.370 mg/g cht kh) trong l cy nui cy cao hn vi ln trong l ca nhng cy i chng (0.035 mg/g cht kh), trong khi hm lng vindoline th gn nh bng nhau trong c hai trng hp (ln lt l 1.8 mg/g v 1.2 mg/g cht kh). Khi chuyn qu trnh nui cy vo mi trng khng c BA kch thch s pht trin ca chi non v dn n s gia tng sn xut vindoline v catharanthine ng thi vi vic c ch sn xut ajmalicine. Satdive v cng s (2003) nghin cu nh hng nhng nng IAA v BA khc nhau ln s sn xut ajmalicine trong bnh lc (shake flasks) s dng chi C.roseus nui cy. Nhng chi c nui trong mi trng MS c b sung IAA nng cao v BA nng thp, tch ly mt lng ln ajmalicine. Vi IAA nng thp v BA nng cao th chi s gii phng nhiu ajmalicine vo mi trng. Qu trnh sn xut ajmalicine t chi nui cy th khng tng quan vi tc tng trng. Vinblastine v vincristine th khng hin din trong chi nui cy. S sinh tng hp alkaloid xy ra trong lc lp. Theo , s hnh thnh nhng dimeric alkaloid c th ph thuc vo mc khc nhau ca lc lp trong t bo nui cy in vitro. Loyola-Vargas v cng s (1986) th nghim nhng cht iu ha sinh trng khc nhau v nh hng ca chng ln vic cm ng m so xanh trong mt dng t bo C.roseus 3 nm tui. Cc tc gi xc nh c c vinblastine v vincristine trong c hai dng m so trng v xanh. Hm lng ca nhng alkaloid 51

ny trong dng mu trng cao hn xp x 2 ln trong dng mu xanh. Trong mi trng c b sung 3 mg/l benzylaminopurine (BAP) m khng c mt auxin sn xut hm lng cht dip lc cng vi vic gia tng hm lng dimeric alkaloid. iu kin nui cy m so rt quan trng cho vic sn xut alkaloid. Morris (1986) thit lp qu trnh nui cy m so t l ca nhng cy C.roseus s dng 3 loi mi trng: mi trng Gamborg (B5), mi trng MS v Zenk vi nhng thay i khc nhau (B5 vi 2,4-D 1.0 mg/l v kinetin 0.1 mg/l; B5 vi IAA 1.0 mg/l v kinetin 0.1 mg/l; MS vi NAA1.0 mg/l v kinetin 0.1 mg/l, mi trng Zenk b sung IAA 0.175 mg/l v BAP 1,125 mg/l, v ch mt mnh NAA, 2,4-D v zeatin 1.0 mg/l). Cc tc gi cng th tin hnh nui cy trong 2 iu kin sng v ti. Cc m so hnh thnh c c trng v mu sc, hnh thi v hm lng alkaloid. Bn loi alkaloid: catharanthine, vindoline, ajmalicine v serpentine c mt trong m so nui cy trn mi trng c b sung IAA v BAP. Serpentine l alkaloid chnh tch ly trong iu kin sng v ajmalicine tch ly trong iu kin ti. Miura v cng s (1987) c lp vinblastine t m so C.roseus hnh thnh t nhng on l non. Mi trng MS c b sung NAA 1.0 mg/l v kinetin 0.1 mg/l c s dng. Cc m so c nui cy trong mi trng ti. S hin din ca vinblastine trong m so ca nhng r khc nhau c xc nh bng phng php sc l lng cao p (HPLC) v php o ph khi lng. Lng vinblastine (1g/g cht kh), thp hn so vi cy m. Marfori v Alejar (1993) cho thy kh nng sn xut alkaloid ca nhng m so c ngun gc t r, thn, l v hoa ca hai ging C.roseus, trng v hng tm. Cc tc gi thu c tm dng m so bng cch s dng mi trng LS b sung BA 3.0 mg/l, 2,4-D 0.5 mg/l v nc da. Sau khi cc m c hnh thnh th c chuyn vo mi trng LS khng c cht iu ha sinh trng v c nui trng trong 1 nm. Hm lng alkaloid cao nht l trong cy mu hng tm v trong m so c ngun gc t r. Theo bo co ca Bi Vn L, Nguyn Ngc Hng ng trn tp ch pht trin khoa hc v cng ngh th m so xanh c cm ng t l cy da cn in vitro c nui trong mi trng MS (Murashige v Skoog) lng c b sung cc cht iu ha tng trng thc vt khc nhau gm c auxin v cytokinin. nng 1 mgl-1 naphthaleneacetic acid (NAA) v 0,5 mgl-1 kinetin (Kin) thu nhn c sinh khi v alkaloid ton phn cao nht trong khi cng mi trng ny nhng thay th NAA bng 2,4-dichlorophenoxyacetic acid (2,4-D) cng nng thu c sinh khi v alkaloid ton phn thp. 4.3.2 nh hng ca cng chiu sng.

52

Yu t mi trng cng nh hng n chng, v d nh l cng nh sng hay bc x t ngoi UV. Hirata v cng s (1991, 1992) pht hin ra rng m so xanh c cm ng t l cy da cn in vitro c nui trong mi trng MS (Murashige v Skoog) lng c b sung cc cht iu ha tng trng thc vt khc nhau gm c auxin v cytokinin. nng 1 mgl-1 -naphthaleneacetic acid (NAA) v 0,5 mgl-1 kinetin (Kin) thu nhn c sinh khi v alkaloid ton phn cao nht trong khi cng mi trng ny nhng thay th NAA bng 2,4-dichlorophenoxyacetic acid (2,4-D) cng nng thu c sinh khi v alkaloid ton phn thp. Nhng kt qu ny c th cho thy rng nh sng NUV r rng c th kch thch s tng hp leurosine v cng tng hp vinblastine t vindoline v catharanthine nhng vi mc t hn. 4.3.3 nh hng ca nhit Ten Hoopen v cng s (2002) cho thy rng nhit cng l mt yu t quan trng nh hng n s tng trng v sn xut ajmalicine t huyn ph C.roseus. Nhit ti u cho c hai qu trnh thu sinh khi v sn xut hp cht th cp l 27.50 C. Yuan v Hu (1994) nghin cu nh hng ca nhng s kt hp khc nhau ca auxin, cytokinin v cng chiu sng ln s hnh thnh ca chi cy C.roseus trong nui cy in vitro. Cc tc gi chng minh rng cht iu ha sinh trng BA v NAA c thm vo mi trng MS theo th t l 7.0 mg/l v 1.0 mg/l c tc dng kch thch s hnh thnh chi non, trong khi 2,4-dichlorophenoxyacetic acid (2,4-D) c ch s phn ha v hnh thnh ca chng. Cng chiu sng 550700lux cho thy l c nh hng tt khi kt hp ng thi vi mi trng MS b sung BA 2 mg/l v NAA 0-1.0 mg/l. 4.3.4 nh hng ca nng ng Theo bo co ca Bi Vn L, Nguyn Ngc Hng ng trn tp ch pht trin khoa hc v cng ngh th m so c c a vo mi trng MS c cht iu ha tng trng thch hp v b sung cc nng ng khc nhau. Th nghim c b tr nh bng di. Bng 1: nh hng ca nng ng sucrose n s nui cy huyn ph t bo

53

 th 1: ng cong tng trng ca dch huyn ph t bo trong mi trng c b sung cc nng ng khc nhau Theo ta thy rng nng ng l 60g/l cho lng sn phm l cao nht

KT LUN
ng dng nui cy t bo C.roseus sn xut cc hp cht th cp c ch to ra mt bc tin xa trong khoa hc thc vt. Do nhu cu s dng cc sn phm t nhin trong y dc ngy cng tng nhng sn lng ca chng cy trng t nhin li rt thp thc y s pht trin khng ngng ca cng ngh nui cy t bo quy m ln. Tuy nhin, cc con ng sinh tng hp cc hp cht th cp mong mun trong thc vt cng nh trong nui cy t bo quy m ln l rt phc tp. V vy, cc thng tin mc t bo v phn t ca cc qu trnh chuyn ha l rt cn thit cho s pht trin ca sn xut cng nghip.

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TI LIU THAM KHO

Ti liu tham kho ting Vit

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