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Sn xut cc hp cht th cp tnui cy t bo thc vtNguyn Hong Lc

Vin Ti nguyn, Mi trng v Cng ngh sinh hc, i hc Hu

1. Cc hp cht th cp thc vtThc vt l ngun cung cp cc hp cht dng lm dc liu hoc ph gia thc phm

c gi tr. Nhng sn phm ny c bit nh l cc cht trao i th cp, thng c hnhthnh vi mt lng rt nh trong cy v chc nng trao i cht cha c bit y .Chng dng nh l sn phm ca cc phn ng ha hc ca thc vt vi mi trng hoc ls bo v ha hc chng li vi sinh vt v ng vt.Nhng nghin cu v cc hp cht thcp c ngun gc thc vt pht trin t cui nhng nm 50 ca th k 20 (Rao v cs 2002).Cc cht trao i th cp c th xp trong ba nhm chnh l alkaloid, tinh du v glycoside.

Cc alkaloidc dng tinh th l cc hp cht cha nitrogen, c hot tnh sinh l trntt c ng vt v c s dng trong cng nghip dc. H alkaloid bao gm: codein,nicotine, caffeine v morphine. Mt s loi thc v

- - (hSolanaceae). Ngi ta thng gp trong mt cy tp hp cc alkaloid c cu trc ha hc gnging nhau. i khi ton c

- -

(hTaxaceae).

dng ln mch mu (hydrastin, ephedrin...), mt s khc tc dng

dng lm thuc cha bnh (Misawa, 1994).

Cc tinh ducha hn hp terpenoid, c s dng nh cht mi, cht thm v dungmi. Ging nh nhng

, v d monoterpenecha 2 n v isoprene, sesquiterpene cha 3 n v isoprene, diterpene cha 4 n visoprene (Lee 2001).

Cc glycosidebao gm cc hp cht phenol v flavonoid, saponin v cc cyanogenicglycoside, mt s trong chng c s dng lm thuc nhum, cht mi thc phm v dcphm (Lee 2001).

2. Nui cy t bo thc vtK thut nui cy t bo thc vt tiu biu cho tim nng ci thin cc hp cht c gi

tr trong y dc, gia v, hng liu v mu nhum m khng th sn xut chng t cc t bovi sinh vt hoc tng hp bng con ng ha hc. Nhng nm gn y, s pht trin ca cchp cht th cp quan trng trong thng mi l kt qu c mong inht trong lnh vcnghin cu ny. u thv mtnguyn l ca k thut nui cy t bo thc vt l c th cung

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cp lin tc ngun nguyn liu tch chit mt t l ln lng hot cht t t bo thc vtnui cy (Mulbagal and Tsay, 2004).

Mt trong nhng nhn t quan trng nh hng n vic sn xut cc hp cht th cp

t t bo thc vt l s phn ha hnh thi. Nhiu hp cht th cp c sn xut trong sutqu trnh phn ha t bo, v th chng c tm thy trong cc m c kh nng phn ha caonh r, l v hoa. Do s phn ha hnh thi v s trng thnh khng xut hin trong nui cyt bo nn cc cht th cp c khuynh hng ngng to thnh trong qu trnh nui cy. Tuynhin, cc t bo khng phn ha trong nui cy huyn ph thng to thnh mt khi vitrm t bo, cc t bo gia khi c s tip xc vi mi trng khc vi cc t bo bnngoi nn s phn ha s xut hin ti mt mc no trong khi to thnh cc cht thcp (Lee 2001).

Nui cy t bohuyn ph thng khi u bng cch t cc khi callus d v vntrong mi trng lng chuyn ng (lc hoc khuy). Trong qu trnh nui cy, cc t bo s

dn dn tch ra khi mu do nhng chuyn ng xoy ca mi trng. Sau mt thi gian ngn trong dch huyn ph s ccc t bo n, cc cm t bo vi kch thc khc nhau, cc munui cy cn tha cha pht trinv cc t bo cht. Tuy nhin, cng c nhng dch huynph hon ho, cha t l cao cc t bo n vt l nh cc cm t bo. Mc tch ri cat bo trong nui cy ph thuc vo c tnh ca cc khi t bo xp v c th iu chnh bngcch thay i thnh phn mi trng (Misawa, 1994).

Bn cnh nui cy t bo huyn ph, nui cy callus (trn mi trng rn) c u iml thao tc th nghim n gin, d vn chuyn mu nhng nhc im l th tch nui cy bnn kh pht trin quy m cng nghip, mu nui cy ch tip xc c mt mt vi ngundinh dng, nhng sn phm do mu nui cy to ra trong qu trnh trao i cht s tch txung quanh dn n lm chm s sinh trng ca t bo. V th, nui cy t bo huyn ph

thch hp hn cho vic sn xut sinh khi t bo thc vt v c th duy tr v thao tc tng tvi cch thng ln men vi sinh vt ngp chm trong mi trng lng.

3. S tch ly cc hp cht th cp trong t bo thc vtS tin b vt bc ca cng ngh sinh hc trong nui cy m v t bo thc vt gip

nhn ging cc cy trng c gi tr v tch chitcc ha cht qu himmang li nhiu nghav mt thng mi. Phng php ny s m rng v tng kh nng thu hi cc ha cht gi trc ngun gcthc vt, mt s thay tht quy m nng nghip truyn thng ln quy m cngnghip trong sn xut cc hp cht th cp (Dicosmo v Misawa 1995). K thut nui cy t bo c khi xng t cui nhng nm 60 ca th k 20 nh l mt cng c hu ch

nghin cu v sn xut hp cht th cp thc vt. K thut ny c pht trin vi mc tiuci thin hiu sut cc sn phm chot tnh sinh hc. u im ca chng l c th cung cpsn phm mt cch lin tc v ng tin cyda trn nhng l do sau: (1) tng hp cc hpcht th cp c gi trdin ra di s iu khin cc yu t mi trng nui cy, c lp vikh hu v iu kin t trng; (2) ph nh nh hng sinh hc n cc sn phm l hp chtth cp trong t nhin (vi sinh vt v cn trng); (3) c th chn lc cc ging cy trng chonhiu loi hp cht th cp khc nhau; (4) vi vic t ng ha iu khin s sinh trng ca

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t bo v iu ha qu trnh chuyn ha, chi ph c th gim v lng sn phm tng ln. Bncnh , nhng kt qu nghin cu gn y cho thy nui cy t bo huyn ph ca thc vtcng c s dng sn xut ccsn phm protein ti t hp (Fisher v cs 1999).

Trong nui cy t bo, vic chn la cn thn cc t bo c kh nng pht trin v iukin nui cyti us gip tng kh nngtch ly mt vi sn phm mc cao hn. thuc hiu sut cao cho khai thc thng mi, ngi ta s dng nhiu phng php khcnhau trong nlc tp trung vo vic kch thch hot ng sinh tng hp ca cc t bo nuicy (Rao 2000, Dixon 1999). T bo nui cy tch ly mt lng ln hp cht th cp ch khi nhngiu kin c bitnh (1) chn la thnh phn mi trng v iu kin nui cythch hp, (2) chn la cc dng t bo nng sut cao, (3)b sung tin cht nui cy v (4) cccht kch khng bo v thc vt (Mulbagal and Tsay 2004).

Ti u ha iu kin nui cy. Cc thng s ha hc v vt l nh thnh phn v pHmi trng, cht iu ha sinh trng, nhit nui cy, s thng kh, s lc hoc khuy, vnh sng nh hng n hm lng cc hp cht th cp c nghin cu nhiu

(Goleniowski v Trippi 1999, Lee v Shuler 2000, Wang v cs 1999). Mt vi sn phm tchly trong t bo mc cao hn so vi trong cy trng t nhin khi c nui cy iukin ti u. Cc thng svt l v yu t dinh dng trong mt m c th gn nh l yu tc bn cho vic ti u ha hiu sut nui cy.

Chn lc cc dng t bo cho nng sut cao . Cc t bo thc vt trong nui cy lmt tp hp cc c im sinh l c lp. Chn lc t bo da vo kh nng tng hp mt vihp cht c gi tr cao trong nui cy c Berlin v Sasse cng b nm 1985, v sau phng thc ny c ng dng rng ri. Chng hn, mt dng t bo ca cy bt tin(Euphorbia milli) sau 24 ln chn lc tch ly gp khong 7 ln lng anthocyanincsn xut t nui cy t bo b m (Yamamoto v cs 1982) . Yamada v Sato (1981) chnlc c mt dng t boca Coptis japonicac kh nng sinh trng gp 6 ln trc y sau

3 tun nui cy v lng berberin t ti 1,2 g/L.Cung cp tin cht (precursor feeding). B sung cc tin cht ca qu trnh sinh tng

hp ni bo vo mi trng nui cy cng c th tng lng sn phm mong mundo mt shp cht trung gian nhanh chng bt u sinh tng hp cc hp cht th cp v v th lm tnglng sn phm cui cng. Phng php ny hu chkhi dng cc tin cht c gi thnh r.Tng cngkch thch hoc b sung tin cht hoc cc hp cht tng t mang li hiu qutrong nhiu trng hp (Silvestrini v cs 2002; Moreno v cs 1993). Chng hn, b sungphenylalanine khi nui cy t bo huyn ph cy Salvia officinalis kch thch to rarosmarinic acid, cung cp ferulic acid trong nui cyt bo cyVanilla planifolia tng tchly valnillin, hocb sung leucine dn n vic tng cc monoterpen d bay hi trong nuicy Perilla frutiscens (Mulbagal and Tsay 2004).

S kch khng bo v thc vt (elicitation). Thc vt sn xut cc hp cht th cptrong t nhin nh mt b my bo v chng li cc yu t gy bnh. Cht kch khng bo vthc vt (elicitor) bo hiu vic hnh thnh cc hp cht th cp. S dng cc elicitor ca bmy bo v cy, tc s kch khng bo v thc vt, lphng thc thu c cc sn phmhp cht th cp c hot tnh sinh hcmt cch hiu qu nht. S dng cc elicitor sinh hcv phi sinh hc(c phn loi da trn ngun gcca chng) kch thch hnh thnh cc

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hp cht th cp trong qu trnh nui cy t bo, c th gip rt ngnthi gian v t hiu sutcao (DiCosmo v Tallevi, 1985).

4. ng dng nui cy t bo thc vt trong sn xut cc hot cht sinh hcNhng nm gn y, thuc truyn thng tr thnh mt ti quan trng mang tnh

ton cu. Mc d cc nc pht trin ngi ta thng s dng tn dc trong iu tr nhngcc loi thuc c ngun gc tho mc vn c dng ph bin do yu t lch s v v n ha.Theo cc nh gi v mt khoa hc, nhiu loi tho mc c th ng dng trong y hc. Vn t ra l vng sinh trng ca cy thuc ang bin mt nhanh chng do s khng n nh caiu kin mi trng v cc yu t khc. Nh vy, tht kh c mt ngun nguyn liu ln tch chit cc hp cht th cp dng trong dc phm. iu ny cnh bo cho ngnh cngnghip cng nh cc nh khoa hc cn tnh n tim nng ca k thut nui cy t bo thcvt nh mt s thay th khc cung cp nguyn liu cho ngun dc phm ny. Bng 2 giithiu sn phm ca mt s cy dc liu quan trng c sn xut t nui cy callus v tbo huyn ph (Mulbagal and Tsay 2004).

Ngay t nm 1971, Wani v cc cng s tm ra mt diterpene amide mi c khnng chng ung th gi ltaxol chit t cy thng Pacific (Taxus brevifolia). n nm1983, taxol c Cc qun l Dc phm v Thc phm Hoa K (FDA) ng a vo thnghim giai on Iiu tr cho ung th bung trng. Sau , FDA cho php s dngtaxol trong iu trcc trng hp ung th bung trng v ung th v . Ngoi ra, taxol cng ctc dng i vi cc bnh nhn c khi u c tnh, ung th phi v cc dng u bu khc(Wickremesinhe v Arteca 1993 v 1994), v n c xem nh l cht u tin ca mt nhmmi trong ha tr liu ung th (Cragg v cs 1993). Tuy nhin, s dng taxol trong iu tr bhn ch do ch tch chitc mt lng rt tt v ca cy thng t nhin. Lp v mngny cha khong 0,001% taxol tnh theo khi lng kh. cy 100 nm tui trung bnh chthu c 3 kg v(khong 300 mg taxol), lng ny ng vi mt liu trong ton t iu trung th. S dngun taxol khan him nh vy l do cc cy t nhin sinh trng rt chm(Cragg v cs 1993). Do , cn c nhng ngun khc thay th mi p ngcnhu cus dng ngy cng tngtrong y hc.

Nui cy t bo cc loi Taxusc xem nh l mt phng php u th cung cpn nh ngun taxol v dn xut taxane ca n (Slichenmyer v Von Hoff 1991). Hin nay,vic sn xut taxol bng nui cy t bo cc loi Taxus tr thnh mt trong nhng ngdng rng ri ca nui cy t bo thc vtv angto ra cc gi tr thng mi to ln. Fett-Neto v cs (1994) nghin cu nh hng ca cc cht dinh dng v mt s yu t khcln s tch ly taxol trong nui cy t boT. cuspidata. Srinivasan v cs (1995) nghin cu

qu trnh sn xut taxol bng nui cy t bo ca T. baccata. Lee v cs (1995) nghin cusn xut taxol bng nui cy t bo huyn ph ca cy T. mairei, mt loi c tm thy tii Loan cao 2000m so vi mc ncbin. Cc dng t bo thu ct callus c ngungc thn v l, v mt trong nhng dng ny sau khi c b sung cc tin cht vo mitrng nui cy, th sau 6 tunc mt ltdch huyn ph t bos c khong 200 mg taxol.

Tsay v cs (1994) nghin cu sn xut imperatorin t nui cy t bo huyn ph cacyAngelica dahurica var. Formosana. y l mt loi cybn a lu nm i Loan, c

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s dng cha chng au u v bnh vy nn. Imperatorin c xem l thnh phn hotng chnh trong iu tr cc bnh v da.Nu sn xut cyAngelica dahurica var. formosanabng phng php nhn ging truyn thngs mt mt thi gian di mi c th p ng c

nhu cu. V vy,phng php nui cy t bo huyn ph sn xut imperatorin c chnla s dng.Nghin cu cho thy trong chu k sinh trng ca t bo huyn ph, sn phmimperatorin t gi tr cc i trong khong gia 10 v 14 ngy. Benzylamino purine nng t 0,5-1,0 mg/L kch thch tng hp imperatorin, mt tl thch hp ammonium nitratev nitrate (2:1) cng nh tng nng phosphate t 1-2 mM slm tng lng impertatorin.Glucose l ngun carbon tt hn saccharose v fructose v hiu qu sn xut imperatorin. Vaitr ca elicitor cng c kho st, b sung thm vanadyl sulphate trong mi trng stng tch ly imperatorin, quyt nh nng v thi gian sinh trng ca t bo. B sungvanadyl sulphate nng 30 mg/L vo mi trng cho hiu qu tt nht sau 10 ngynui cy. Hoc b sung 20 g/L cht hp ph amberlite XAD-7 vo mi trng, qu trnh tnghp imperatorin cng tng mnh ngy nui cy th 10. Hm lng imperatorin sn xut bi

phng thc ny t 460 g khi lng ti cao hn i chng 140 ln.Berberine l mt isoquinoline alkaloid c trong h r ca cy Coptis japonicav vca cy Phellondendron amurense. Berberine chloride c s dng cha bnh ri lontiu ha. thu c nguyn liu th t r cy Coptis phi mt 5-6 nm. Yamada v Sato(1981) C. japonica. Sau

, cng ty ha du Mitsui (Nht Bn) ci thin c nng sut bng cch thm 10 -8 Mgibberellic acid vo mi trng, hiu sut rt nhiuti 1,66 g/L (Misawa1994).

R ca cy Panax ginseng, mt loi tho dc lu nm cn gi l nhn sm c sdng rng ri nh mt v thuc b, mt dc phm qu gi, c tc dng cha bnh ri lontiu ha, bnh i ng,

sapogenin khc nhau. Trong , ginsenoside-Rb c hot tnh an thn, cn Rg c hot tnh kchthch. T 1973, Furuya v cs nui cy m callus P. ginseng phn lp saponins vsapogenins. Nm 1994, Choi bt u nghin cu nui cy P. ginseng trn quy m cngnghip.n nay, y l mt trong cc i tng c cc nh khoa hc trn th gii tp trungnghin cu nhiu nht.

Merkli v cs (1997) nui cy r t ca cy Trigonella foenum-graecum bng cchgy nhim chng A4caAgrobacterium rhizogenes. Cc r t ny sn xut diosgenin, mtspirostanol quan trng cho s bn tng hp (semi-synthesis) ca cc hormone steroid. Hmlng diosgenin thu c cao nht l 0,040 % khi lng kh gn gp 2 ln so vi cc rkhng bin np chng A4 8 thng tui (0,024 %). Cc tc gi ny nghin cu nh hngca cholesterol, pH mi trng v chitosan n kh nng sn xut diosgenin. Kt qu cho thy

b sung 40 mg/L chitosan vo mi trng nui cy s tng hm lng diosgenin ln gp 3 lnso vi i chng.

Yeh v cs (1994) nghin cu sn xut diosgenin bng nui cy t bo huyn ph cacyDioscorea doryophora. Phng php ny c s dng nh mt cch thay th qu trnhtng hp steroid. Nui cy t bo huyn ph c thit lp bng cch a callus vo mitrng c 0,2 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D). Nng saccharose ti thch chotng hp diosgenin l 3%. Lng diosgenin thu c trong trng hp ny t ti 3,2% khi

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lng kh. Snxut diosgenin t cy D. doryophorabng nui cy t bo huyn ph hin nay c ng dng trn quy m cng nghip.

Gentiana davidii var. formosanal tho mc bn a sng lu nm i Loan. T xa

n c s dng nh mt loi thuc th s trong y hc c truyn Trung Quc nhm ngnchn bo ph v lo ha, bo v phi khi cc cht c (Zheng v cs 1997). Secoiridoidglycoside l hp cht chnh vi c tnh y hc trong r ca chi Gentiana (Skrzypczak v cs1993). Chueh v cs (2000) nghin cuti u ha iu kin nui cy t bo huyn ph ca G.davidii var.formosa sn xut gentipicroside v swertiamarin, hai dc cht quan trng. Tbo huyn ph sinh trng ti u khi nui cy callus trong mi trng b sung 1,2 mg/Lkinetin v 3% saccharose, pH 4,2-5,2, tc lc 80-100 vng/pht. S tch ly cao nht 2 hpcht swertiamarin v gentipicroside trong t bo c xc nh ln lt sau 12 v 24 ngynui cy.

Miyasaka v cs (1989) nghin cu sn xut cryptotanshinone t nui cy callus cySalvia miltiorrhiza. Salvial mt chi quan trng ca h Lamiaceae v mt vi loi ca Salvia

mc hoang di trn khp th gii dng lm thuc dn gian. Hiu qu ca BA i vi vic hnhthnh cryptotanshinone trong nui cy callus S.miltiorrhiza c kho st. Callus s cpc to ra t nui cy mnh l trong ti trn mi trng b sung 1,0 mg/L 2,4-D. Callussau pht trin nhanh hn trn mi trng c 1,0 mg/L 2,4-D v 0,5 mg/L BA. Kt qu phntch HPLC cho thy callus cha mt lng nh cryptotanshinone (0,26 mg/g khi lng kh).Loi b 2,4-D khi mi trng nui cy cho kt qu l lng cryptotanshinone trong callustng ln. Nng cao nht ca cryptotanshione thu c trong callus nui cy trn mi trngc 0,2 mg/L BA trong 6 ngy l 4,59 mg/g khi lng kh (Wu v cs 2003).

chi thuc h Solanaceaenh:Duboisia, Scopolia, Atropa, Hyoscyamus vDatura.

nhngnm 1960 (Misawa, 1994). Tuy nhin, hm lng ca scopolamine v hyoscyamine trong tbo nui cy thng rt thp. Sau ny, Tabata v cs (1971) b sung tropic acid vo dchhuyn ph nui cy Scopolia japonicaln 15 ln.

Shikonin, mt loi sc t c kh nng dit khun, c trong r cy Lithospermumerythrorhizon . Tuy nhin, ngi ta toc dng t bo r cy Lithospermum c kh nng tch ly n 15% shikonin v honchnh cng ngh nui cy t bo sn xut shikonin. Cng ngh ny cho php trong mt chu knui cy thu hoch ti 5 kg hot cht v gip gim nhiu gi thnh ca shikonin.

Podophyllotoxin l mt aryltetralin lignan chng khi u c tm thy cc cyPodophyllum peltatum v Podophyllum hexandrum.N cng c dng tng hp cc dnxut etoposide v teniposide, s dng rng ri trong iu tr chng khi u (Issell v cs 1984).Tuy nhin, trong t nhin nhng cy ny sinh trng rt chm v v th hn ch vic cungcp podophyllotoxin, bt buc chng ta phi hng ti mt phng thc thay th khc. Nuicy tbo sn xut podophyllotoxin c Kadkade v cs thc hin ln u tin vo nm1981 v 1982. Woerdenberg v cs (1990) dng mt phc hp precursor l coniferyl alcohol

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v b-cyclodextrin bsung trong mi trng nui cy t bo huyn ph ca P. hexandrum. Bsung phc hp 3 mM coniferyl alcohol tng hiu sut podophyllotoxin ln 0.013% theokhi lng kh, trong khi cc nui cy khng c precursor ch sn xut c 0.0035%

podophyllotoxin. Smollny v cs (1992) thng bo callus v t bo huyn ph ca Liliumalbum sn xut c0.3% podophyllotoxin.

Kt lunng dng nui cy t bo thc vt sn xut cc hp cht th cp to ra mt bc

tin xa trong khoa hc thc vt. Vic pht trin v s dng cc cng c di truyn cng nh shiu bit ngy cng su sc hn vbn cht ca t bo v cc phng thc iu ha qu trnhchuyn ha trao i cht th cp l c s cho vic sn xut chng quy m thng mi.

Do nhu cu s dng cc sn phm t nhin trong y-dc ngy cng tng nhng snlng ca chng cy trng t nhin li rt thpthc y s pht trin khng ngng cacng ngh nui cy t bo quy m ln. Tuy nhin, cc con ng sinh tng hp cc hpcht th cp mong mun trong thc vt cng nh trong nui cy t bo quy m ln l rtphc tp. V vy, cc thng tin mc t bo v phn tca cc qu trnh chuyn ha l rtcn thit cho s pht trin ca sn xut cng nghip.Nhiunghin cu c thc hin cc iu kin khc nhau gii thch cc hin tng xut hin trong qu trnh sn xut cccht trao i th cp t cc t bo thc vt nui cy in vitro. Cc kt qu ny cng cho thycc h thng nui cy t bo thc vt c tim nng rt ln cho vic khai thc thng mi cccht trao i th cp.

Bng 2. Cc hp cht th cp c sn xut t nui cy m v t bo thc vt (Mulbagal andTsay 2004)

Loi thc vt Hp cht th cp Dng nuicy

Tham kho

Agave amaniensis Saponins Callus Andrijany v cs 1999

Ailanthus altissima Alkaloids Huyn ph Anderson v cs 1987

Ailanthus altissima Canthinone alkaloids Huyn ph Anderson v cs 1986

Allium sativum Alliin Callus Malpathak v David 1986

Aloe saponaria Tetrahydroanthracene

glucosides

Huyn ph Yagi v cs 1983

Ambrosia tenuifolia Altamisine Callus Goleniowski v Trippi

1999

Anchusa officinalis Rosmarinic alkaloids Huyn ph De-Eknamkul v Ellis1985

Brucea javanica Canthinone alkaloids Huyn ph Liu v cs 1990

Bupleurum falcatum Saikosaponins Callus Wang v Huang 1982

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Camellia sinensis Theamine, dn xut ca-glutamyl

Huyn ph Orihara v Furuya 1990

Canavalia ensiformis L-Canavanine Callus Ramirez v cs 1992

Capsicum annuum Capsaicin Huyn ph Johnson v cs 1990

Cassia acutifolia Anthraquinones Huyn ph Nazif v cs 2000

Catharanthus roseus Indole alkaloids Huyn ph Moreno v cs 1993

Catharanthus roseus Catharanthine Huyn ph Zhao v cs 2001

Choisya ternata Furoquinoline alkaloids Huyn ph Sejourne v cs 1981

Chrysanthemum

cinerariaefolium

Pyrethrins Callus Rajasekaran v cs 1991

Chrysanthemum

cinerariaefolium

Chrysanthemic acid v

pyrethrins

Huyn ph Kuel v cs 1985

Cinchona L. Alkaloids Huyn ph Koblitz v cs 1983

Cinchona robusta Robustaquinones Huyn ph Schripsema v cs 1999

Cinchona sp. Anthraquinones Huyn ph Wijnsma v cs 1985

Cinchona succirubra Anthraquinones Huyn ph Khouri v cs 1987

Citrus sp. Naringin, Limonin Callus Barthe v cs 1987

Coffea arabica Caffeine Callus Waller v cs 1983

Cornus kousa Polyphenols Huyn ph Ishimaru v cs 1993

Corydalis ophiocarpa Isoquinoline alkaloids Callus Iwasa v Takao 1982

Croton sublyratus Plaunotol Callus Morimoto v Murai 1989

Cruciata glabra Anthraquinones Huyn ph Dornenburg v Knorr1996

Cryptolepis buchanani Cryptosin Callus Venkateswara v cs 1987

Digitalis purpurea Cardenolides Huyn ph Hagimori v cs 1982

Dioscorea deltoidea Diosgenin Huyn ph Heble v Staba 1980

Dioscorea doryophora Diosgenin Huyn ph Huang v cs 1993

Duboisia leichhardtii Tropane alkaloids Callus Yamada v Endo 1984

Ephedra spp. L-Ephedrine, D-pseudoephedrin Huyn ph ODowd v cs 1993

Eriobotrya japonica Triterpenes Callus Taniguchi v cs 2002

Eucalyptus tereticornis Sterol v hp chtphenolic

Callus Venkateswara v cs 1986

Eucommia ulmoides Chlorogenic acid Huyn ph Wang v cs 2003

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Fumaria capreolata Isoquinoline alkaloids Huyn ph Tanahashi v Zenk 1985

Gentiana sp. Secoiridoid glycosides Callus Skrzypczak v cs 1993

Ginkgo biloba Ginkogolide A Huyn ph Carrier v cs 1991

Glehnia littoralis Furanocoumarin Huyn ph Kitamura v cs 1998

Glycyrrhiza echinata Flavonoids Callus Ayabe v cs 1986

Glycyrrhiza glabra var.

glandulifera

Triterpenes Callus Ayabe v cs 1990

Hyoscyamus niger Tropane alkaloids Callus Yamada v Hashimoto

1982

Isoplexis isabellina Anthraquinones Huyn ph Arrebola v cs 1999

Linum flavum 5-Methoxypodophyllo-

toxin

Huyn ph Uden v cs 1990

Lithospermum erythrorhizon Dn xut ca shikonin Huyn ph Fujita v cs 1981

Lithospermum erythrorhizon Dn xut ca shikonin Huyn ph Fujita v cs 1990

Lycium chinense Cerebroside Huyn ph Jang v cs 1998

Morinda citrifolia Anthraquinones Huyn ph Zenk v cs 1975

Morinda citrifolia Anthraquinones Huyn ph Bassetti v cs 1995

Mucuna pruriens L-DOPA Huyn ph Wichers v cs 1993

Mucuna pruriens L-DOPA Callus Brain K. R. 1976

Nandina domestica Alkaloids Callus Ikuta v Itokawa 1988

Nicotiana rustica Alkaloids Callus Tabata v Hiraoka 1976

Nicotiana tabacum Nicotine Huyn ph Mantell v cs 1983

Nothapodytes foetida Camptothecin Callus Thergane v cs 2003

Ophiorrhiza pumila Camptothecin dngalkaloids

Callus Kitajima v cs 1998

Panax ginseng Saponins v sapogenins Callus Furuya v cs 1973

Panax notoginseng Ginsenosides Huyn ph Zhong v Zhu 1995

Papaver bracteatum Thebaine Callus Day v cs 1986

Papaver somniferum Morphine, Codeine Huyn ph Siah v Doran 1991Peganum harmala -Carboline alkaloids Huyn ph Sasse v cs 1982

Phytolacca americana Betacyanin Huyn ph Sakuta v cs 1987

Picrasma quassioides Quassin Huyn ph Scragg v Allan, 1986

Podophyllum hexandrum Podophyllotoxin Huyn ph Uden v cs 1989,Chattopadhyay v cs

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2002

Polygala amarella Saponins Callus Desbene v cs 1999

Portulaca grandiflora Betacyanin Callus Schroder v Bohm 1984

Polygonum hydropiper Flavonoids Huyn ph Nakao v cs 1999

Ptelea trifoliata Dihydrofuro [2,3-b]

quinolinium

alkaloids

Callus Petit-Paly v cs 1987

Rauwolfia sellowii Alkaloids Huyn ph Rech v cs 1998

Rauwolfia serpentina Reserpine Huyn ph Yamamoto v Yamada,1986

R. serpentina Rhazya

stricta

3-Oxo-rhazinilam Callus Gerasimenko v cs 2001

Ruta sp. Acridone, furoquinoline

alkaloids v coumarins

Callus Baumert v cs 1992

Salvia fruticosa Rosmarinic acid Callus vhuyn ph

Karam v cs 2003

Salvia miltiorrhiza Lithospermic acid B v

rosmarinic acid

Callus Morimoto v cs 1994

Salvia miltiorrhiza Cryptotanshinone Callus Morimoto v cs 1994

Sapium sebiferum Tannin Callus v

huyn phNeera v Ishimaru, 1992

Scopolia parviflora Alkaloids Callus Tabata v cs 1972Scutellaria columnae Phenolics Callus Stojakowska v Kisiel

1999

Solanum chrysotrichum Spirostanol saponin Huyn ph Villarreal v cs 1997

Solanum laciniatum Solasodine Huyn ph Chandler v Dodds 1983

Solanum paludosum Solamargine Huyn ph Badaoui v cs 1996

Stizolobium hassjoo L-DOPA Huyn ph Huang v cs 2002

Tabernaemontana

divaricata

Alkaloids Huyn ph Sierra v cs 1992

Taxus spp. Taxol Huyn ph Wu v cs 2001Taxus baccata Taxol, baccatin III Huyn ph Cusido v cs 1999

Taxus cuspidata Taxoids Huyn ph Ketchum v cs 2003

Tecoma sambucifolium Phenylpropanoid

glycosides

Callus Pletsch v cs 1993

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Thalictrum minus Berberin Huyn ph Kobayashi v cs 1987

Thalictrum minus Berberin Huyn ph Nakagawa v cs 1986

Torreya nucifera var.

radicans

Diterpenoids Huyn ph Orihara v cs 2002

Trigonella foenumgraecum Saponins Huyn ph Brain v Williams 1983

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