TP CH PHT TRIN KH&CN, TP 10, S 04 - 2007

NHN GING IN VITRO CY BT RUI DROSERA BURMANNI VAHL THU NHN MT HP CHT QUINONE C HOT TNH SINH HC
Quch Dim Phng, Bi Vn L Trng i hc Khoa hc T nhin, HQG-HCM
(Bi nhn ngy 18 thng 08 nm 2006, hon chnh sa cha ngy 15 thng 03 nm 2007)

TM TT: nhn ging in vitro cy Drosera burmanni Vahl, ht c kh trng v nui cy trn mi trng MS c bn c hm lng khang a lng gim 1/2 (MS 1/2), b sung ng (30 g/l), than hot tnh (1 g/l), Casein hydrosylate (100 mg/l) v pH 5,8. t thn t cy con D. burmanni Vahl c nui cy trong mi trng MS 1/2 rn v lng c b sung ng (30 g/l), than hot tnh (1 g/l), Casein hydrosylate (100 mg/l). Hp cht quinone c ly trch t dch chit ethanol ca bt cy. Cc sinh trc nghim c dng xc nh hot tnh ca cht ny. Xc nh cu trc ca hp cht c lp bng cc phng php: ph khi MS, ph hng ngoi IR, ph cng hng t ht nhn 1 chiu 1H-NMR, 13C-NMR. 1.M U

Drosera (Droseraceae) l nhm thc vt bt mi ln nht, bao gm 170 loi trn ton th gii. Vit Nam, hin nay ch c 3 loi Drosera c tm thy: Drosera peltata J.E.Sm.var. lunata Clarke ex Hook. F, Drosera indica L., Drosera burmanni Vahl. Drosera l nhng loi thc vt mang nhiu c im khc l, p mt v c tim nng kinh t to ln trn th trng hoa cnh. Mt khc, Drosera c tm quan trng to ln gi tr dc tnh ca chng. Drosera cha cc hp cht quinone c nhiu tc dng tr liu khc nhau nh khng khun, khng nm, chng ung th, chng lao, vim phi, ho g, Nhng trong t nhin, qun th thc vt thuc h Droseraceae ngy cng tr nn khan him (chng c lit vo danh sch cc thc vt b e da ca Chu u), dn n tr ngi khi s dng cy trong thin nhin lm ngun cung cp hp cht c hot tnh sinh hc. Trong bi ny, chng ti nhn ging in vitro cy Drosera burmanni Vahl nhm ly trch v nghin cu hp cht quinone t dch chit ca cy [2,3].
2. VT LIU V PHNG PHP 2.1. Vt liu Ht cy Drosera burmanni Vahl thu c t khu du lch sinh thi Bc Bnh, Bnh Thun. Mi trng nui cy: mi trng mui khong c bn ca Murashige v Skoog (1962) c hm lng khong a lng gim 1/2 b sung ng (30 g/l), than hot tnh (1 g/l), Casein hydrosylate (100 mg/l) v pH 5,8 (mi trng MS 1/2). iu kin nui cy: chiu sng 16 gi/ngy vi cng 3000 lux, nhit 22-25OC. 2.2. Phng php 2.2.1.Kh trng ht Tri cha ht c em kh trng trong dung dch Javel vi cc nng thay i, b sung 0.01% (v/v) Tween-20, trong thi gian 5, 10, 15, 20 pht, sau ra li bng nc ct nhiu ln. Ht tch khi tri c gieo trn mi trng MS 1/2. Ht ny mm sau 3 tun.

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Science & Technology Development, Vol 10, No.04 - 2007 2.2.2.Tng sinh D. burmanni Vahl Cy con sau khi ny mm, c ct t v nui cy trong mi trng MS 1/2 rn (b sung 7 g/l agar) v lng (khng b sung agar). Cy trong mi trng lng c lc 120 vng/ pht trong cng iu kin nui cy. 2.2.3.Ly trch v c lp hp cht Ton cy ti em sy kh 500C, xay nhuyn c bt cy kh. Ngm dm bt cy kh trong ethanol nhit phng, sau t nht 24 gi em lc thu dch lc v c quay chn khng thu c cao ethanol. T cao ethanol tin hnh c lp hp cht bng phng php sc k ct silica gel v tinh ch bng sc k bn mng. nhn bit s hin din ca hp cht quinone, cc phn on thu c t phng php sc k ct c chm ln bn mng sc k, mi vt 10 l, song song vi cht i chiu 2-methyl-5hydroxy-1,4-naphthoquinone. Gii ly bn sc k vi h dung mi eter du ha: benzen (3:7), v tr cc vt c quan st di n UV 254 nm hay phun bn vi thuc th Borntraeger 5% KOH trong methanol ( thy vt ha ). 2.2.4.Kho st hot tnh sinh hc ca hp cht c lp Kho st tc dng khng khun: dng ethanol ha tan v tm 50 g hp cht c lp c trn mi a giy v trng (ng knh 6 mm). Thc hin cng vi 2 mu i chng l nc ct v trng v ethanol vi cng th tch dng tm hp cht trn mi a giy. Cc a giy c t trn cc a petri mi trng LB tri cc chng vi khun: Bacillus subtilis, Bacillus pumilus, Sarcina lutea (gram dng) ; Escherichia coli, Salmonella typhi, Shigella spp. (gram m). ng knh vng khng khun c quan st sau 24 gi 370C. 2.2.5.Xc nh cu trc ca hp cht c lp Cu trc ha hc ca hp cht c lp c xc nh bng cc phng php: ph khi MS, ph hng ngoi IR, ph cng hng t ht nhn 1 chiu 1H-NMR, 13C-NMR. 3. KT QU V THO LUN 3.1. Kh trng ht Drosera burmanni Vahl Ht cy Drosera burmanni Vahl kh trng thnh cng vi dung dch: Javel 10% (v/v) + Tween-20 (0,01%) trong thi gian 10 pht, to c 94,09% ht v trng (bng 1). Ht ny mm sau 3 tun gieo trn mi trng MS 1/2. Cy mm c dng lm nguyn liu nui cy in vitro. (hnh 1)

Hnh 1. Ht ny mm sau 3 tun nui cy

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TP CH PHT TRIN KH&CN, TP 10, S 04 - 2007

Hnh 2. S pht trin ca Drosera burmanni Vahl trong mi trng MS 1/2 lng v MS 1/2 rn. A: MS 1/2 lng; B: MS 1/2 rn

Bng 1. T l ht ny mm (% SE) sau 4 tun kh trng Nng Javel (% v/v) 5 10 15 20 5 6,99 5,68 40,32 8,19 58,81 11,89 25,64 6,21 Thi gian (pht) 10 15 10,42 4,79 25,07 6,93 37,37 9,42 94,09 6,45 27,60 9,15 12,46 4,47 13,91 6,20 1,05 2,35 20 11,96 4,91 4,84 5,58 0,00 0,00 0,00 0,00

3.2. Tng sinh D. burmanni Vahl Drosera burmanni Vahl c kh nng sng trong mi trng lng. Kt qu cho thy khi nui cy trong mi trng lng (khng c agar), 100% mu cy u cn sng, l tng trng xanh tt v to nhiu chi. T l chi/ mu cy mi trng lng cao hn trong mi trng rn (bng 2), nh vy mi trng lng ci thin kh nng nhn chi ca Drosera burmanni Vahl. Bng 2. So snh kh nng sng ca cy trong mi trng MS 1/2 lng v rn. Mi trng MS 1/2 lng Ch tiu so snh MS 1/2 rn

S chi/mu cy

3,18 0,68

2,06 0,36 - L xanh tt - Thi gian cy tng trng chm hn - Cy ra r bnh thng, thi gian lu hn

Tnh trng mu cy

- L xanh tt - Thi gian cy tng trng nhanh - Cy ra r khe v nhanh hn

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Science & Technology Development, Vol 10, No.04 - 2007 3.3. Ly trch v c lp hp cht quinone Cao th ethanol dng thc hin sc k ct vi cc n dung mi eter du ha, benzen, chloroform, ethyl acetate v c quay thu hi dung mi c nhiu phn on. Mi phn on c gii ly trn sc k bn mng cng vi cht i chiu (2-methyl-5-hydroxy-1,4naphthoquinone) v hin hnh vi thuc th 5% KOH trong methanol. Trn bn sc k gii ly vi h dung mi eter du ha: benzen (3:7), ch c phn on th 2 trong phn on cao benzen c 1 vt mu vng cam, trn, m nt, vi Rf= 0,33. Sau khi cho hin hnh vi thuc th Borntraeger 5% KOH trong methanol, vt ny chuyn thnh mu (hnh 3). Phn on ny c chn tinh ch hp cht quinone. Hp cht c tinh ch nhiu ln bng sc k iu ch bng dung mi cloroform cho n khi sn phm thu c ch cn mt vt trn r khi kim tra bng sc k bn mng. Hp cht c lp c kt tinh li trong chloroform c dng tinh th hnh kim mu cam (hnh 4).

Hnh 3. Sc k bn mng: quan st bng mt thng (A), quan st di n UV (B), phun xt bn mng bng thuc th Borntraeger (C); 1, 2, 3, 4, 5, 6: cc phn on trong phn on cao benzen; P: cht i chiu

Hnh 4. Sc k iu ch: A: Bn sc k iu ch; B: kim tra tinh sch ca hp cht; C: tinh th ca hp cht

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TP CH PHT TRIN KH&CN, TP 10, S 04 - 2007 3.4. Kho st hot tnh sinh hc ca hp cht quinone c lp Hp cht c lp th hin tnh khng vi 6 chng vi khun th nghim gm 3 chng gram m, 3 chng gram dng (ng knh vng khng khun t 12- 35 mm). Trong , hp cht c kh nng khng khun mnh nht i vi chng Escherichia coli (hnh 5).

Hnh 5.Vng khng khun do tc dng ca hp cht c lp c trn 6 chng vi khun th nghim A: Escherichia coli; B: Salmonella typhi ; C: Shigella spp. D: Bacillus pumilus ; E: Bacillus subtilis; F: Sarcina lutea 1: i chng nc v trng; 2: i chng ethanol; 3: Hp cht kho st

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Science & Technology Development, Vol 10, No.04 - 2007

Hnh 6a. Kt qu ph MS

Hnh 6b. Kt qu ph IR

Hnh 6c. Kt qu ph DMSO- 1H

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TP CH PHT TRIN KH&CN, TP 10, S 04 - 2007

Hnh 6d. Kt qu ph DMSO- C13CPD

3.5. Xc nh cu trc ca hp cht quinone c lp c Kt qu kt hp ph khi MS, ph hng ngoi IR, ph cng hng t ht nhn 1 chiu 1HNMR, 13C-NMR, cho bit hp cht c lp c thuc nhm anthraquinone c khi lng phn t M= 530 vi cng thc phn t l C36H50O3:

4. KT LUN

- Nng kh trng ht Drosera burmanni Vahl thch hp l 10% (v/v) Javel, b sung 0.01% (v/v) Tween-20, trong thi gian 10 pht. - Cy D. burmanni Vahl pht trin tt c trong mi trng MS 1/2 rn v lng. Nui cy lng gp phn ci thin kh nng nhn ging cy. - Hp cht c lp c xc nh l hp cht thuc nhm anthraquinone, cha tng c cng b c trong cc loi Drosera t trc n nay. - Hp cht c nh gi l c hot tnh sinh hc vi tc dng khng khun cao.

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Science & Technology Development, Vol 10, No.04 - 2007

ISOLATING A BIOACTIVE QUINONE FROM IN VITRO CULTURED DROSERA BURMANNI VAHL

Quach Diem Phuong, Bui Van Le University of Natural Sciences, VNU-HCM ABSTRACT: To establish in vitro cultivation of Drosera burmanni Vahl, steriled seeds were germinated on MS basal medium but decreasing of 1/2 macro salts content (MS 1/2) supplemented with saccharose (30 g/l), activated carbon (1 g/l), Casein hydrosylate (100 mg/l) at pH 5.8. Stem node of D. burmanni Vahl were cultured on MS 1/2 medium (solid and liquid) with saccharose (30 g/l), activated carbon (1 g/l), Casein hydrosylate (100 mg/l) at pH 5.8. A quinone compound was isolated from ethanol extract of dry plants. Bioassays were used to detect the activities of this compound. A quinone compound was identified by mass spectrum, IR spectrum and 1H-, 13C-NMR spectrum. TI LIU THAM KHO

[1]. Quch Ng Dim Phng. Kho st hp cht Naphthoquinone c hot tnh sinh hc c tch chit t cy bt rui Drosera indica L. nui cy in vitro. Lun vn thc s sinh hc (2006). [2]. Jayaram K., Prasad M.N.V.Rapidly in vitro multiplied Drosera as reliable source for plumbagin bioprospection. Current science, vol 89, 447- 448 (2005) [3]. Marczak L., Kawiak A., Lojkowska E., Stobiecki M., Secondary Metabolites in in vitro Cultured Plants of the Genus Drosera. Phytochem. Anal. 16, 143-149 (2005). [4]. Nguyn Kim Phi Phng. Cc phng php nhn danh, trch ly v c lp cc hp cht hu c. Gio trnh cao hc chuyn ngnh Ha hu c, H Khoa hc T nhin, TP. HCM (2001).

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