M UVit Nam nm trong vng nhit i m, gi ma nn c h thc vt rt phong ph v a dng vi hn 12.000 loi, bao gm hn 300 h v 1.200 chi. Ngun thc vt phong ph ny cung cp cho con ngi nhiu sn phm thin nhin c gi tr. Cc sn phm thin nhin c hot tnh sinh hc c ng dng rt ln trong nhiu lnh vc khc nhau ca cuc sng, c bit dng lm thuc cha bnh. Cc loi thuc tho mc t gy tc dng ph v t c hi cho ngi s dng. Chng c dng nh tc nhn iu tr trc tip, lm cht d sinh ho lm sng t nguyn l dc hc hoc lm cht chun pht trin cc loi thuc mi. Chnh v vy vic nghin cu ho hc cng nh hot tnh sinh hc ca cc loi cy thuc c ngha quan trng cho vic s dng mt cch hp l v c hiu qun nht ngun ti nguyn thin nhin ny. Chi Croton l mt chi ln thuc h Thu du (Euphorbiaceae) v c quan tm nghin cu trn th gii. Chnh v hot tnh l th ca cc ent-kauran diterpenoid t cy kh sm Bc B Croton tonkinensis (Euphorbiaceae) m lun vn ny c cc nhim v sau:1. Tin hnh kho st qui trnh chit tch ent-kauran diterpenoid t l cy kh sm

Bc B.2. Thc hin mt s phn ng chuyn ha vi hp cht ent-kauran diterpenoid

phn lp c gm: Phn ng epoxi ha ni i v thy phn epoxit. Phn ng thy phn. Phn ng axetyl ha. Phn ng oxi ho.

3. Tin hnh th hot tnh sinh hc vi cc sn phm chuyn ha nhn c.

1

Chng 1

TNG QUAN1.1 Cy kh sm Bc B (Croton tonkinensis Gagnep.) 1.1.1 Thc vt hc [1, 2] Cy kh sm Bc B c tn khoa hc l Croton tonkinensis Gagnep. thuc h Thu du (Euphorbiaceae).

Hnh 1: Cy kh sm Bc B (Croton tonkinensis Gagnep., Euphorbiaceae) Kh sm Bc B l cy nh, cao 0,7-1 m. L mc cch hay gn nh mc i, c khi mc thnh vng gi gm 3-6 l. L hnh mi mc, mp nguyn di 5-10 cm, rng 1-3 cm. C hai mt l u c lng hnh khin ng nh (kiu lng l cy nht) nhng mt di nhiu hn mt trn. Khi phi kh mt l di c mu trng bc, mt trn c mu nu en. Cm hoa mc k l hay u cnh, lng tnh hay n tnh.

2

Hoa c c 5 l i,12 nh. Hoa ci c 5 l i, 3 vi nh. Qu gm 3 mnh v mu hung . 1.1.2 Ni sng v thu hi [1] Cy kh sm Bc B mc hoang v c trng khp ni, trong cc vn gia nh hoc vn thuc, ch yu cc tnh pha bc Vit Nam. Thng trng bng gieo ht hay trng bng cnh vo ma xun. Thu hi khi cy ang c hoa, em phi hoc sy kh, khi dng em sao vng. 1.1.3 Hot tnh v cng dng ca cy kh sm Bc b [1, 2] Cy kh sm Bc B c dng trong dn gian lm thuc cha mn nht, l nga, vim mi, l, au bng, vim lot d dy, t trng, tiu ho km... Ngy dng 15-20 l sao vng, dng thuc sc, dng ngoi ly nc sc c ra. Gn y, cy kh sm Bc B c ch nhiu do l cy dng trong n thuc cha bnh au d dy kt hp vi cc v thuc khc (l b cng anh Lactuca indica v l khi Ardisia sylvestris) hoc dng ring di dng nc sc. Nc sc kh sm Bc B trong thc nghim c tc dng khng mt s nm ngoi da. Kh sm Bc B c tc dng khng sinh i vi trc khun l ng thi c tc dng khng l amip, lm cho n bo co thnh kn. Tc dng i vi k sinh trng st rt: nc sc ca bi thuc gm Kh sm v v Bi c tc dng c ch k sinh trng st rt mnh trn ng vt th nghim c gy nhim st rt, nhng ti pht trong thi gian 10 ngy theo di trn m hnh thc nghim chut nht nhim Plasmodium berghei v g nhim Plasmodium gallinaceum. 1.1.4 Cc ent-kauran diterpenoid ca cy kh sm Bc b Nm 2000, Phan Tng Sn v cng s cng b quc t mt diterpen mi t l cy kh sm. Bng cc phng php ph xc nh cu trc ca diterpen ny l ent-7-hydroxy-15-oxo kaur-16-en-18-yl axetat (1) [5, 33]. Cht ny c hot tnh

3

khng k sinh trng st rt Plasmodium falciparum in vitro. Nhm tc gi trn tip tc nh gi hot tnh ca diterpen vi k sinh trng st rt Plasmodium falciparum (vi c chng nhy v chng khng cloroquin) tc dng gy c i vi mt s t bo ung th (Hep-G2, RD, FI, VR), tc dng c ch s pht trin ca cc vi sinh vt kim nh [7].CH3 H H CH3 OH O CH2

CH3COOCH2

1 Trong nghin cu tip theo ca Phan Minh Giang v cng s [19] bn entkaurane diterpen gm hai cht bit, ent-7,14-dihydroxykaur-16-en-15-one (2) v ent-18-acetoxy-7-hydroxykaur-16-en-5-one (3) v hai cht mi, ent-1-acetoxy-7, 14-dihydroxykaur-16-en-15-one (4) v ent-18-acetoxy-7,14-dihydroxykaur-16-en15-one (5) c phn lp t l cy kh sm Bc B. Cc ent-kauranoid ny c ch nhn t phin m NF-B v s sn xut NO c hot ho bng LPS trong i thc bo RAW264.7 ca chut IC50 = 0,07 - 0,42 g/ml.R1

2 R1 =H, R2 =H, R3 =OHR3 OH R2 O

3 R1 =H, R2 =OAc, R3 =H 4 R1 =OAc, R2 =H, R3 =OH 5 R1 =H, R2 =OAc, R3 =OH

Nm 2004, ent-7-hydroxy-15-oxokaur-16-en-18-ol (6) cng c phn lp t cy kh sm Bc B [30].

4

H H OH O

HO

6 Tip , Phan Minh Giang v cng s cng b cu trc ca 4 ent-kauran diterpen mi, ent-1,14-diacetoxy-7-hydroxykaur-16-en-15-one (7), ent-1,7diaxetoxy-14-hydroxykaur-16-en-15-one (8), ent-18-acetoxy-14-hydroxykaur-16en-15-one (9) v ent-(16S)-18-acetoxy-7-hydroxy kauran-16-en-15-one (10) [20].R1 R4 O R3 AcO H

H H

H

O OH

R2

R1

R2

R3 OH H

R4 OAc OH

10

7 OAc H 8 OAc H 9 H OAc

OAc OH

Nm 2005, c 6 ent-kauran diterpenoid mi c phn lp t l cy kh sm Bc B l cc cht ent-7 -acetoxy-11-hydroxykaur-16-en-15-one (11), ent-18acetoxy-11-hydroxykaur-16-en-15-one (12), ent-11-acetoxykaur-16-en-18-oic acid (13), ent-15,18-dihydroxykaur-16-ene (14), ent-11,18-diacetoxy-7 -hydroxykaur-

5

16-en-15-one (15), and ent-(16S)-1,14a-diacetoxy-7 -hydroxy-17-methoxykauran15-one (16) [21].R5 R1

R4 H O R3 R2 H

Ba

diterpenoid

R1 R2 R3 R4 R5 11 H H OAc H OH 12 H OAc H H OH 15 H OAc OH H OAc vi cu trc ent-kaur-16-ene, en-11-acetoxy-7,

14

dihydroxykaur-16-en-15-one (17), ent-18hydroxykaur-16-ene (18) v ent-kaur-16- en15-oxo-18-oic acid (19) c phn lp t phn chit metanol l kh cy kh sm Bc B bng phng php tch sc k ct ln lt trn silica gel, ODS gel, v HPLC pha o [18].AcO

H H OH

OH O HOOC H

H O HO H

H

17 1.2 Vi nt v terpen v diterpen [3, 25]

18

19

Khi nim terpen c dng ch mt lp hp cht thin nhin c cu trc c th phn chia ra thnh cc n v isopren. Cn c vo s n v isopren hp thnh ngi ta phn bit cc monoterpen C10, sesquiterpen C15, diterpen C20, sesterterpen C25, triterpen C30, tetraterpen C40 v polyterpen.

6

Bn cnh thut ng terpen ngi ta cn dng khi nim terpenoit bao hm rng ri cc sn phm thoi bin t nhin, th d cc ionon v cc dn xut t nhin cng nh tng hp ca cc terpen, th d cc terpen - ancol, -andehit, -xeton, axit, - este, - epoxit, v.v Tuy nhin trong khi s dng thng khng c s phn chia ranh gii tht r rt gia cc khi nim terpen v terpenoid nh va nu. Thut ng terpen nhiu khi c dng bao hm cc terpen hidrocacbon ln cc dn xut ca chng. Cc diterpenoid hp thnh mt nhm ln cc hp cht C-20 c dn ra t geranyl geraniol pyrophotphat. Chng ch yu xut hin trong cc loi nm hoc trong thc vt v bao gm cc axt nha v cc hocmon sinh trng thc vt nhm gibberellin. Cc diterpenoid c mt s nt ring c trng. S oxi ho thng hay xy ra hn cc nhm trong phn t v ta thy xut hin nhiu phn ng to ete hoc lacton. Cc tng tc gia cc nhm khng lin kt trc tip vi nhau thng lm cho khung cacbon b bin dng v dn ti nhiu chuyn v l th. a s cc diterpen l hp cht vng. i vi cc diterpen ch yu kiu ng vng hnh thnh cc dn xut ca perhidronaphtalen v perhidrophenantren. C hai kiu ng vng : Kiu 1: ng vng do s proton ho ni i ca n v isopropyliden xut pht. Kiu th nht thuc v cng dy lp th vi steroid. Kiu 2: Nhm pyrophotphat ri khi phn t cho mt cacbacation v khai mo cho phn ng ng vng. T cc tin thn khng vng, thng qua cc kiu ng vng khc nhau m dn n cc h thng khung v cng phong ph ca cc terpen cng nh diterpen. Chng hn ngy nay ta bit n trn 170 khung cacbon ca cc diterpen, trong s c 7

7

khung (hnh 1) chim khong 50% cc diterpen c bit, khung labdan chim s lng ln nht.

cem bran

labdan

clerodan

kauran

pim aran

abietan

gibberellan

Hnh 2: Mt s khung ca cc diterpen Cc diterpen c hot tnh sinh hc v cng phong ph: in hnh l cc hocmon sinh trng thc vt dy gibberellin. Ngi ta phn lp v tinh ch c axt gibberellic t nm Gibberella fujikuroi tc dng c trng cho s pht trin bnh thng ca cy. Cc axt nha thuc vo s cc sn phm thin nhin xut hin vi lng ln. Chng c tc dng c kt cc si g, c bit l cc axt nha c nhm phenolic c hot tnh khng sinh nh v bo v cho g khi b vi sinh vt lm mc nt (axt podocarpic v axt ferruginol). Nha copan hnh thnh khi mt s cy nhit i mc nt c tc dng bo v cy. Diterpen pleu-romutilin phn lp t cy Pleurotus mutilus c hot tnh khng khun.

8

Ngoi ra nhiu hp cht diterpenoid cn c tc dng km hm s pht trin ca khi u (v d quinon-methin), hoc l nhng hp cht c c tnh nguy him (v d taxin 1 cy thu tng). T nha ca Fabiana densa Remy var. ramulosa Wedd tch c hai diterpen mi: ent-beyer-15-en-18-O-succinat (20) v ent-beyer-15-en-18-O-oxalat (21). Hai hp cht ny c hot tnh khng vi trng i vi Escherichia coli (ATCC 8739), Klebsiella pneumoniae, Salmonella aviatum (ATCC 2228), Pseudomonas aerug-inosa (ATCC 14207), Staphylococcus aureus (ATCC 6538P), Micrococeus flavus (ATCC 10290), Bacillus subtilis (ATCC 6633), Saccharomyces cerevisiae [16].

R O H2C

CH3

Phn chit CH2Cl2 ca l Croton zambesicus (Euphorbiaceae) gy c t bo i vi dng t bo ung th biu m t cung. T phn chit ny tch c mt trachyloban diterpen, ent-trachyloban-3-ol (22) vi hot tnh c t bo dng t bo Hela, IC50=7,3 g/ml [14].

H HO H

22

9

1.3 Khi qut v kauran v ent-kauran Cc kauran tn ti hai dng: dng ent v phyllocladane. Phn ln kauran dng ent, t ph bin hn l phyllocladan c cu metylen v tr i din so vi nhm metyl C-10.20 2 3 1 10 5 11 9 12 13 14 8 7 15 16 17 2 3 1 11 20 10 5 9 12 13 14 8 7 15 16 17

H6

H6

4

4

19

18

H

19

18

H

ent-kauran 1.3.1 Sinh tng hp cc ent-kauran

phyllocladan

Ent-kauran c hnh thnh t geranylgeranyldiphosphat (GGPP) di tc dng xc tc ca 2 enzym [22]. 1. Enzym ent-kaurene synthase (A) (copalyl PP synthase) xc tc ng vng to thnh (-)-copalyl PP (ent-copalyl diphosphat).

OPP

enzym (A) H H

OPP

GGPP

(-)-copalyldiphosphat

2. Enzym ent-kaurene synthase (B) xc tc (-)-copalyl PP (ent-copalyl diphosphat) tip tc ng vng v phn b li.

10

OPP GGPP i H ( ) copalyl diphosphat (ent copalyl diphosphat ) H

ii H

+

H

+

+

H H H ent kaurene H H H

H

1.3.2 Cc nghin cu chuyn ha cc ent-kauran diterpenoid Nm 2007, Ronan Batista cng cc ng s tng hp thnh cng mt entkauran glycosid mi bng phn ng Koeings-Knorr t axit kaurenoic v 2,3,4,6-tetraO-acetyl-glucopyranosyl bromid, theo s thy phn nhm axetat [13].

Hnh 3: S tng hp alcol kauran 25 v 26

11

Hnh 4: S tng hp cc dn xut kauran 32a/b t alcol kauran 25

12

Hnh 5: S tng hp cc dn xut kauran 33-38 t alcol kauran 26 Nm 2008, Silvia Marquina cng vi cc cng s tin hnh hydroxyl ha cc diterpen axit ent-kaur-16-en-19-oic v axit ent-beyer-15-en-19-oic bng phng php vi sinh s dng vi nm Aspergillus niger [29].

13

H H H

HO

Aspergillus nigerH H H COOH H COOH H OH

Hp cht Eriocalyxin B (39) c phn lp t loi Isodon (Rabdosia), sau c cc nh ha hc Trung Quc tin hnh chuyn ha to thnh 19 dn xut mi ca Eriocalyxin B c tc dng gy c trn t bo ung th [42].O H

O H OH OH

O

39

14

Hnh 6: S chuyn ha Eriocalyxin B (a): NaBH4, MeOH, 1:1; (b) NaBH4, MeOH, 1:3; (c) H2, Pd/C

15

46: R=CH2Br 47: R=CH2=CH2

Hnh 7: S chuyn ha Eriocalyxin B (a): PCC, CH2Cl2; (b): tc nhn Jones, axeton; (c): Ac2O, BF3.Et2O, THF; (d): axyl clorid, NaH, pyridin, THF; (e): Ac2O, DMAP, pyridin

16

51: R=CH3 52: R=CH2Br 53: R=CH2CH2Cl 54: R=CH2CH2CH2Cl Hnh 8: S chuyn ha Eriocalyxin B (a): TsOH, THF; (b): axyl clorid, CH2Cl2 Nm 2005, Liang Xu tin hnh bn tng hp ent-karan (55) theo hng epoxi ha ni i ca ent-kauran diterpenoid, thng qua 7 bc bao gm cc phn ng: aldol ha, epoxi ha, oxi ha v qu trnh BaeyerVilliger thu c sn phm dioxan t hiu sut 19% [39].

17

Ent-kauran (55) c phn tch t phn chit etanol ca l cy Isodon eriocalyx.

55

18

Hnh 9: S chuyn ha ent-kauran 55 Nm 2002, Bruno cng vi cc cng s tin hnh cc chuyn ha bn tng hp t linearol (68) thu c cc dn xut ent-kauran [15].

19

+HO OAc OH AcO OH

69H2/Pd, C

70

OH

O

O3OH

O

+HO OAc OH HO OAc OH

O

O

HO

68

OAc

74MCPBA

Zn/AcOH

75

O

O

O

+HO OAc OH AcO OH OH

(CH3)2CO/pyridin

AcO

OAc

71

72

73

OAc

Hnh 10: S chuyn ha bn tng hp linearol to thnh cc ent-kauran 1.3.2 Hot tnh sinh hc ca cc ent-kauran diterpenoid t nhin v bn tng hp Cc ent-kauran t nhin c hot tnh sinh hc rt r rt. Mt s ent-kauran diterpenoid th hin tnh khng khun nh axit kaurenic, c kh nng c ch vi khun Gram (+) . V d nh diterpen kauran axit (ent-16-kauren-19-oic axit) dng kh trng, cha cm lnh v cm [36]. Mt s ent-kauran diterpenoid th hin hot tnh gy c t bo nh ent-kauran diterpenoid t cy I. eriocalyx var. laxiflora. Mi hp cht ny c th c tnh trn t bo u ngi K562, T24 l laxiflorin E, laxiflorin C, eriocalyxin B th trn dng K562 (IC50 = 0,077; 0,569 v 0,373 g/ml), cn hp cht laxiflorin E v eriocalyxin th trn dng T24 (IC50 = 0,709 v 0,087 g/ml) cn laxiflorin D v maocrystal u c

20

hot tnh chng li c hai dng t bo ung th trn vi gi tr IC50 nh hn 6,5 g/ml [31]. Hp cht 16,17-dihydroxy-ent-kaurane-19-oic acid (75) c tch t qu Annona squamosa c hot tnh chng s sao chp HIV trong t bo bch cu H9 vi gi tr ED50 = 0,8 g/ml [35].R216 17

R3 H H R1

H H

19

R

74 87

R COOH CH2OH

75 76 77 78 79 80 81 82 83 84 85 86

R1 COOH CH2OH COOH COOH COOH COOH CHO CHO CHO CHO OH OH

R2 OH OH H CH2OH CH2OH H H OH H OH H OH

R3 CH2OH CH2OH CH2OH H OH CH2OAc COOH CH2OH CH2OH CH2OAc COOH CH2OH

Hnh 11: Cc ent-kauran t qu Annona squamosa Nm 1999, cc nh khoa hc c nghin cu loi a tin Lepidolaena taylorii phn lp c bn hp cht 8,9-secokauran mi gy c t bo, 11 hp cht 8,9-seco-kauran (88) v 6 hp cht c cu trc ca kauren-15-on. Hp cht 8,9secokauran (88) l hp cht gy c t bo chnh cng vi dn xut dihydro v epoxy (89 v 90) t c hn nng thp hn [32].

21

12 20 1 4 18 19 10 5 11 14 9

13

H16 17

13

H16 17

H H14

O OH

15

O H

O OH

H O H

O8

O

H

OH

88

89

90

T phn chit etyl axetat ca Isodon sculponeata tch c 4 ent-kauran diterpenoid mi, cc sculponeatin F-1 (91-94) v 6 hp cht ent-kauran diterpenoid bit (95-100). Hp cht 93, 94, 96, 97, 100 c nh hng c ch trc tip ln dng t bo ung th K562, IC50=3,2 g/ml 8,2 g/ml. Hp cht 91, 96 c ch dng t bo ung th T24 vi gi tr IC50 = 18,3 g/ml v 15 g/ml. Khng c hp cht no trong s cc hp cht trn gy c i vi dng t bo A549 [26].R3 O7 15 14 17 16

O O O

OEt O O H O

R3

O2 3 1

20 10 5

8 9

H11

13 12

H O R1 R2 H O H

O6

R118

4

R2 OH AcO

H19

H OH

91 R1= H, R2= OH, R3= -OH 92 93 R1= H, R2= -OH, R3= =O 95 R1= OH, R2= H, R3= -Oac 94 R1= OH, R2= H, R3= =O 96 R1= H, R2= OH, R3= =O 99 R1= H, R2= H, R3= =O 97 R1= H, R2= OH, R3= -OH 100 R1= H, R2= H, R3= -OH 98 R1= OH, R2= H, R3= =O 101 R1= H, R2= -OH, R3= =O Hnh 12: Cc ent-kauran t Isodon sculponeata Cc nh khoa hc Trung Quc tch mt ent-kauran diterpen, pseudoirroratin A v mt diterpen bit, pseurata A. Hp cht pseudoirroratin A gy c t bo i vi dng t bo ung th Lu1, SW626, LNCaP, KB, v HOS vi gi tr IC50 = 0,26 (0,75), 0,20 (0,57), 0,90 (2,59), 0,90 (2,59) v 0,5 (1,44) g/ml (M) [41].

22

OH O HO H O H OH

pseudoirroratin A T ngn ca cy Isodon adenantha Trung Quc tch c 11 diterpenoid mi, cc adenanthin B-L (102-112), cng 5 hp cht khc, calcicolin B (113), adenanthin (114), weisiensin A (115), nervosanin (116) v forrestin C (117). Hp cht 103, 104, 115 c hot tnh c ch ln dng t bo K562 vi IC50 l 3,3; 3,6 v 3,3 g/ml [27].

23

AcO R1

HO R1

HO HO

H HO H OAc

R2

H R2 H OAc

OH

H AcO H OAc R

O

102 R1 = AcO R2 = -OH 103 R1 = AcO R2 = = O 104 R1 = AcO R2 =-AcO 105 R1 = OH R2 = = O

106 R1 = OH R2 = AcO 108 R1 = AcO R2 = OH

107 R = H 109 R = -OH

AcO AcO

AcO R1H

HO HO

OH

HO

H R1

OAc

R2

H HO H R2 O

R3AcO H

H O

110 R1= = O R2= -OH 114 R1= = O R2 = =O 115 R1= -OH R 2= =O 116 R1= = O R2 = -AcO 117 R1= -OH R2 = OH

111 R1 = AcO R2 = OH R3= -AcO 113 R1= OH R2 = AcO R3 = = O

112

Hnh 13: Cc ent-kauran t Isodon adenantha Cc nh khoa hc bang Carolina tin hnh chuyn ha bn tng hp t linearol thu c 26 dn xut ent-kauran. Cc dn xut ny c nghin cu cho hot tnh chng HIV. Kt qu l 5 hp cht 118, 119, 120, 121 v 122 c hot tnh chng li s sao chp HIV trong t bo bch cu H9 vi gi tr EC 50 = 0,1 - 3,11 g/ml v TI l 163 v 184. Hp cht 118, 121 trong tng lai c th pht trin nh l tc nhn chng ung th [15].

24

Hnh 14: Cc ent-kauran bn tng hp t linearol T phn chit etanol ca l Isodon xerophilus tch c 3 ent-kauran diterpenoid (cc xerophilusin L-N, 123-125) v ponicidin (128). Hp cht 128 c kh nng gy c t bo mnh nht ln hai dng t bo ung th K562 v T24 vi gi tr IC50 ln lt l 0,09 v 0,32 g/ml. Hp cht 125 gy nh hng c ch yu IC50 > 15,6 g/ml [28].

25

125

128

Hai diterpen axit spasmolytic, ent-kaur-16-en-19-oic acid (129) v ent-beyer-15en-19-oic acid (130) c tch ra t thn ca Viguiera hypargyrea c hot tnh chng li Staphylccoccus aureus, Enterococcus feacalis v Candida albicans. Hp cht 130 c ch nng 63,54% v khng ph thuc vo nng , gi tr ED 50 = 4,9 g/ml. Hp cht 129 th hin c ch 8,04%, liu dng l 10 g/ml [40].

Nghin cu ha thc vt thn ca Bruguiera gymnorrhiza tch c 3 entkauran diterpenoid mi, 13, 16, 17-trihydroxy-ent-9(11)-kauren-19-oic acid (131), 16 , 17-dihydroxy-ent-9(11)-kaurene-19-al (132), 17-chloro-13, 16 -dihydroxy-entkauran-19-al (133) cng 9 hp cht ent-kaurane diterpen bit. Hu ht cc hp cht c th c t bo vi dng t bo L-929, K562 v dng t bo ung th c t cung Hela. Cc hp cht 16H-17,19-ent-kauranediol (134), 13-hydroxy-16-ent-kauren-19al (135), 16-ent-kauren-19-ol (136) chng li hai dng t bo ung th K562 v L-929. Hp cht 16-ent-kauren-19-ol (136) chn lc nht cho hot tnh ny IC50 = 6,8 g/ml [23]. Cc loi Rabdosia Trung Quc c s dng trong y hc c truyn nh tc nhn chng ung th v chng vim nhim. Cc nh khoa hc tin hnh nghin cu thnh phn ha hc ca l v thn cng cc hot tnh sinh hc i vi cc loi Rabdosia khc nhau, c bit l Rabdosia rubescens (Hemsl.) Hara. Cc kauran

26

diterpen nh oridonin (137) v ponicidin (138) l nhng cht c t bo chnh trong cc loi Rabdosia [32].

137

138

Cc hp cht 139, 140, 144, 145 v 146 tch ra t ngn ca Isodon enanderianus gy c t bo mnh ln dng t bo K562 vi IC50 ln lt l 0,67; 0,16; 0,59; 0,13; 0,87 g/ml. Cc hp cht 141 v 142 khng gy c t bo i vi dng t bo K562 [38].r1 2 3 4 18 10 5 9

r

1 20

2

11

12 13

r14

316

R1r17

R2 OAc H H H H OAc

R3 OH OH OH H H H

R4 H H H

R5 =O =O -OH

h8

139 140 143 144 145 146

OH OAc OAc OAc OAc OH

o6 7

15

4

h19

oh

r

5

OH -OH OH H =O =O

Hnh 15: Cc ent-kauran t Isodon enanderianus Nm ent-kauran diterpenoid mi, cc ludongnin F-J (147-151) cng vi 10 hp cht bit, cc guidongnin A-C (152-154), angustifolin (155), 6-epiangustifolin

27

(156), sculponeatin J (157) v gardenin D c tch ra t l ca Isodon rubescens var. lushiensis. Tt c cc hp cht 147-159 c nh gi nh hng c ch ln t bo ung th K562. Hp cht 151-152, 155-158 gy nh hng c ch mnh vi gi tr IC50 ln lt l 0,18; 0,30; 0,23; 0,87; 0,83; 0,25 g/ml. Trong khi cc hp cht cn li 147-150, 153-154 khng c hot tnh (IC50 > 50 g/ml). iu ny xc nhn rng cu hnh cyclopentanon vi mt nhm exomethylen l trung tm hot ng gy hot tnh. Hp cht 151 gy c t bo i vi dng t bo ung th gan CA v dng t bo ung th c t cung Hela vi gi tr IC50 ln lt l 0,09 v 0,70 g/ml [24].3 18 19 4 2r 2 11 1 13

R116 17

R2 H H H H H OH H OH OH

R3 =O =O =O =O =O -OH =O =O =O

R4 -CH3 -CH3 -CH3 -CH3 =CH2 =CH2 -CH3 -CH3 =CH2

h5 6

10 20

9

h8 7

14 15

r4

14 7 14 8 14 9 15 0 15 1 15 2 15

-OCH3 -OCH3 -OCH3 -OCH3 -OCH3 =O =O =O -OCH3

o

r1

o

o

r3

h o o140

h o

3 15 4 15 5o

o

28

15 6 15 7 15 8

-OCH3 =O =O

OH OH H

=O =O =O

=CH2 =CH2 -CH3

Hnh 16: Cc ent-kauran tch ra t l Isodon rubescens var. lushiensis Nm 2006, ln u tin trn th nghim hot tnh khng vi khun Staphylococus aureus khng methicillin c Phan Minh Giang v cng s thc hin trn cc entkauran diterpenoid c phn lp t Croton tonkinensis [22]. Cc diterpenoid hot ng nht l ent-18-acetoxy-11-hydroxykaur-16-en-15-one (MIC 32 g/ml), ent-18acetoxy-7-hydroxykaur-16-en-15-one 32 (500 g/ml) v ent-18-acetoxy-7, 14dihydroxykaur-16-en-15-one 32 (125 g/ml).

29

Chng 2

KT QU V THO LUN2.1 i tng v phng php nghin cu 2.1.1 Nguyn liu thc vt Mu thc vt c thu hi vo thi im thch hp trong nm. Mu ti sau khi ly v c ra sch, hong kh ni thong mt v sy kh 50oC. Sau , xay mu kh thnh dng bt mn. Bt nguyn liu thc vt c ngm chit vi cc dung mi nh metanol, ri phn b chn lc trong cc dung mi thch hp (n-hexan, diclometan) thu c cc phn chit. 2.1.2 Cc phng php phn tch, phn lp cc hp cht phn tch v phn tch cc phn chit ca cy kh sm Bc B cng nh phn lp cc cht s dng cc phng php sc k nh sc k lp mng (TLC), sc k ct thng (CC), sc k ct nhanh (FC) v cc phng php kt tinh phn on. Sc k lp mng c thc hin trn bn silica gel trng sn DC-Alufolien 60 F254, dy 0,2 mm (Merck). Hin mu cc vt bng thuc th dung dch vanilin/H2SO4 1%, Dragendoff-Munier v soi n t ngoi bc sng 254 nm. Sc k ct (CC) c thc hin di trng lc ca dung mi di p sut kh quyn. Cht hp ph cho sc k ct l silica gel Merck cc c ht, c nhi theo phng php nhi t. 2.1.3 Cc phng php kho st cu trc cc hp cht Cu trc ca hp cht phn lp c c kho st nh s kt hp cc phng php ph: ph khi lng (EI-MS), ph hng ngoi (IR), ph cng hng t ht nhn mt chiu (1D NMR) l 1H NMR, 13C NMR, DEPT.

30

2.1.4 iu ch cc phn chit t l cy kh sm Bc B Bt kh l cy kh sm Bc B c ngm chit 2 ln vi metanol khan nhit phng. Dch chit metanol c ct loi dung mi xung cn 1/10 th tch ri pha long bng nc ct theo mt t l thch hp. Dch metanol-nc c chit ln lt vi dung mi khng hoc t phn cc nh n-hexan, diclometan. Sau khi c lm kh, ct loi dung mi cc dch chit cho cc phn chit tng ng n-hexan, diclometan (D). S 1: Qui trnh chit l cy kh sm Bc B Bt l cy kh sm Bc B 1. Chit vi metanol 2.Gp c dung mi

Phn chit MeOH

Phn b

1.Chit vi n-hexan 2. Lm kh dch chit bng Na2SO4 3. Ct loi dung mi Phn chit n-hexan (H) Dch nc 1.Chit vi diclometan 2. Lm kh dch chit bng Na2SO4 3. Ct loi dung mi

Phn chit diclometan (D)

Dch nc

31

2.2 Qui trnh phn lp cc ent-kauran diterpenoid t l cy kh sm Bc B (Croton tonkinensis Gagnep., Euphorbiaceae) phn lp cc ent-kauran diterpenoid, ta i t phn chit D (xem 3.3.2). 15 gam phn chit D c tm vi 10 gam silica gel (Merck, 0,063-0,100mm) thu c hn hp bt ti mu nu sm. Phn tch hn hp ny bng sc k ct (CC) vi h dung mi ra gii l n-hexan-axeton thu c 8 phn on t D1 n D8. Nhm phn on D2 sau c phn tch li bng sc k ct thng (CC) trn cht hp ph silica gel (Merck, 0,0630,200mm) vi h dung mi ra gii l n-hexan-axeton, ta thu c 3 nhm phn on D2.1, D2.2, D2.3. Nhm phn on D2.2 thy c tinh th hnh kim mu trng kt tinh, ly ra kt tinh li trong axeton thu c mt cht tinh khit CH1 (1). Nhm phn on D2.3 thu c mt hn hp hai tinh th ng kt tinh rt kh tch k hiu l hn hp 2 cht CH2 (7) v CH3 (8).12 11 1 2 3 4 5H

13 14 16 15O OH

17

OAc

OAc

10H

9

8 7

OAc

OH

6

HOH H

O H

HOAc

O

AcO

18

19

1

7

8

32

S 2: Qui trnh phn tch phn chit diclometan (D)

D1 1 25

26 39

D2.1 P 1- 9

Ra, n-hexan - axeton

1

33

7+8

Kt tinh li, axeton

D (15 g)CC, silica gel (0,063 0,100 mm), 80 p (150 ml/ p) D2 D3 40 80 CC, silica gel (0,063 0,100 mm), 35 p (15 ml/ p) D2.2 1015 D2.3 2635

2.3 Chuyn ho cc ent-kauran diterpenoid Cc cht ent-kauran diterpenoid c phn lp t cy kh sm Bc B (Croton tonkinensis Gagnep., Euphorbiaceae) c hot tnh kh phong ph. Cc cht ny c hot tnh khng k sinh trng st rt Plasmodium falciparum in vitro. ng thi chng cng c tc dng c ch r rt i vi mt s dng t bo ung th (Hep-G 2, RD, FI, VR) v c tc dng c ch i vi mt s vi sinh vt nh vi khun. Vi mc ch thc hin chuyn ho mt s nhm chc ca cc cht ny nhn c cc hp cht mi c hot tnh tt hn, chng ti tin hnh nghin cu mt s phn ng chuyn ho ca chng.

HAcO

O OAc

H

166

Ac2O, pyridin

KOH/MeOH HHO O OH

1

K2Cr2O7/ H2SO4AcO

HO

O

H

H

164

MCPBA

167

O

OH OH

HAcO

O OH O

NaOH/MeOH HHO O OH

O

H

H

159

162

Hnh 17: S cc phn ng chuyn ha cht 1

34

OH OH

HOH H

O

165

KOH/MeOHO OH

OAc OAc

OAc

OAc OH

O OAc

OAc

HH OH

+H

O

H

MCPBAH

O OAc

HOH

O

+H

HOAc

O

7

8 CrO3/H2SO4

160

161 BF3.Et2O/CHCl3

OAc OAc

OAc

O OH

HO H

O H

HOH

O

168

163

Hnh 18: S cc phn ng chuyn ha hn hp cht 7 v 8 2.3.1 Phn ng epoxi ha Cn 0,5 g cht u vo bnh cu phn ng c dung tch 50 ml. Ha tan ht cht u trong 16 ml CH2Cl2. Cho 0,4 g MCPBA vo bnh, ha tan hon ton trong 16 ml CH2Cl2. Lm lnh bnh phn ng n -10oC. Lm lnh dung dch peaxit n -5oC. Nh git dung dch peaxit vo bnh phn ng, khuy u trong thi gian 40. Khuy tip hn hp phn ng nhit phng, ri yn trong 3 ngy. Cho hn hp sau phn ng ln phu chit, lc hn hp vi dung dch NaHCO 3 bo ha (3 ln, mi ln 30 ml), chit gi li lp di CH2Cl2. Cui cng ra bng nc

35

4 ln n pH trung tnh. Lm khan dung dch nhn c bng Na 2SO4. Ct loi dung mi cho sn phm th c mu trng ng. Sn phm th c tinh ch bng sc k ct trn silica gel (Merck, c ht 0,063 -0,100 mm), h dung mi n-hexan-axeton 8:1 thu 10 ml/ phn on cho cc sn phm epoxit tng ng. T cht u l cht (1) thu c sn phm chnh l A3 (159). T cht u l hn hp 2 cht (7) v (8) thu c sn phm chnh l F (160 v 161). Phn ng ny c mc ch oxi ha ni i C-16 v C-17 thnh oxit. Phng trnh phn ng:O

O

HAcO

O OH

MCPBAAcO

HOH O

H

H

1

159O OAc O OH

OAc OAc

OAc

OAc OH

OAc

HH OH

+H

O

H

MCPBAH

O OAc

HOH

O

+H

HOAc

O

7

8

160

161

159-

Tinh th mu trng, nc 176 178oC. Rf = 0,35 (TLC, silica gel, h dung mi n-hexan-etyl axetat 1:1), vt cht c mu tm hng khi phun vi thuc th vanilin/H2SO4 1%. Vt c mu vng sm khi phun thuc th Dragendoff-Munier.

Ph 1H NMR ca 159 cho thy s c mt ca mt nhm cacbonyl [C 170,4 (s)] mt nhm metin c oxy ha [C 76,9 (d), C-7], mt nhm metylen mang oxi C-18 [C 72,1 (t); H 3,65 (d, J=11,5 Hz) v 3,89 (d, J=11,5 Hz)] v mt nhm axetat [ C

36

121,1 (q); H 2,07 (s)]. Mt vng epoxit hnh thnh v tr c ca ni i C-16/C-17 [C 51,4 (t); H 2,59 (d, J=6,0 Hz)], vng ny c lin kt vi mt vng lacton [C 170,4 (t)] bng mt cu axetat [C 91,1 (s)]. 160: Ph 1H-NMR cho thy s c mt ca hai nhm axetat v tr s 1 v 14 [H 1,98 (s); 1,99 (s)] v mt nhm OH v tr s 7 [H 4,17 (brs)] ng thi c mt vng epoxit c hnh thnh v tr c ca ni i [H, 3,26 (d, J=10,5 Hz)]. 161: Ph 1H-NMR cho thy s c mt ca hai nhm axetat v tr s 1 v 7 [ H 2,01 (s); 2,02 (s)] v mt nhm OH v tr s 14 [H 5,04 (s)] ng thi c mt vng epoxit c hnh thnh v tr c ca ni i [H, 3,18 (d, J=11,0 Hz)]. 2.3.2 Phn ng m vng epoxit A3 bng thy phn kim Cn 100 mg cht A3 cho vo bnh cu phn ng c dung tch 25 ml. un hi lu hn hp phn ng vi dung dch NaOH 2,5% trong MeOH (10 ml) trn ni nc nng trong 2h. Sau phn ng thm nc ct vo bnh phn ng. Chit hn hp phn ng 3 ln bng diclometan (mi ln 6 ml). Gp cc dch chit diclometan li, ra bng nc ct n mi trng trung tnh. Lm khan dung dch nhn c bng Na2SO4, lc v ct loi diclometan, thu c cn th. Cn th c tinh ch bng sc k ct trn silica gel (Merck, c ht 0,063 -0,100 mm), h dung mi n-hexan-axeton (3:1, v/v) thu 8 ml/ phn on cho tinh th L (162). Phn ng ny c mc ch thy phn epoxit thnh diol. Phng trnh phn ng:O OH OH

O

NaOH/MeOH

O H

HOH AcO O HO H

OH

O

H

159

162

37

162 Tinh th mu trng hnh kim. Rf = 0,65 (TLC, silica gel, h dung mi n-hexan-axeton (1:2, v/v)), vt cht c mu vng khi phun vi thuc th vanilin/ H2SO4. Ph 13C NMR v DEPT ch ra s c mt ca 22 nguyn t C, c mt nhm cacbonyl [C 180,3 (s)], c bn nguyn t C bc 4 [C 36,9; 38,2; 82,7; 87,9], mt nhm metylen mang oxi C-18 [C 70,0; H 3,76 (d, J=10,5 Hz) v 3,95 (d, J=10,5 Hz). Mt vng epoxit v tr C-16/C-17 [C 62,4; H 3,0 (d, J = 11,0 Hz)], vng ny c lin kt vi mt vng lacton [C 180,3 ] bng mt cu axetat [C 87,9 (s)] 2.3.3 Phn ng thy phn hn hp epoxit F bng phc BF3.Et2O Cho 20 mg epoxit A1 c ha tan trong CHCl3 khan vo bnh cu phn ng c khuy t trong iu kin c lm lnh bng . Thm 5 git BF 3.Et2O vo bnh phn ng. Sau 5 pht, hn hp phn ng c ra bng dung dch NaHCO 3, c lm khan trn Na2SO4, lc v ct loi dung mi di p sut gim thu c cn th. Cn th c phn tch bng sc k ct trn silica gel (Merck, c ht 0,063 -0,100 mm), h dung mi n-hexan-axeton (10:1, v/v) thu 5 ml/ phn on cho tinh th F2 (163).OAc O OAc OH O OAc OAc O

OH

HOAc H

O

+H

HOH

O H

HOH

O

161 163

160

163

Tinh th mu trng dng bt.

38

Rf = 0,32 (TLC, silica gel, h dung mi n-hexan-axeton (3:1, v/v)), vt cht c mu vng khi phun vi thuc th vanilin/ H2SO4. Ph13C NMR ch ra s c mt ca mt nhm cacbonyl [C 217,1], c nm nguyn t C bc 4 [C 21,5; 32,9; 42,8; 61,9; 62,4], ph 1H NMR ch ra s c mt ca mt nhm axetat [H 2,01 (s)], mt vng epoxit v tr C-16/C-17 [C 62,4; 48,3 H 2,91 (d, J = 11,0 Hz)]. 2.3.4 Phn ng thy phn Cn 200 mg cht u cho vo bnh cu phn ng c dung tch 25 ml. Thm 5 ml metanol cha 15% KOH vo bnh phn ng. Hn hp phn ng c un hi lu nhit khong 70 80oC trong 12 gi. Sau khi phn ng, thm 30 ml nc ct vo hn hp trn v ct loi metanol. Chit dung dch ny 3 ln bng diclometan (mi ln 5ml). Ra bng nc n pH = 7, gp cc dch chit diclometan li, lm khan v ct loi diclometan, thu c mt cn th. Cn th ny c phn tch bng sc k ct trn silica gel (Merck, c ht 0,040 0,063 mm), h dung mi l gradient n-hexan-axeton thu 5 ml/ phn on cho cc sn phm thy phn tng ng. T cht u l cht 1 thu c sn phm chnh l 164. T cht u l hn hp 2 cht 7 v 8 thu c sn phm chnh l K2 (165). Phn ng ny c mc ch thy phn cc nhm axetat thnh nhm hydroxyl. Phng trnh phn ng:

H O AcO H OH HO H OH H O

39

1OAc OH OAc OAc

164OH OH

HOAc H

O

+H

HOH

O

HOH H

O

7 165

8

165

Tinh th mu trng hnh kim. Rf = 0,53 (TLC, silica gel, h dung mi n-hexan-axeton (3:1, v/v)), vt cht c mu vng khi phun vi thuc th vanilin/ H2SO4. Ph13C NMR ch ra c nm nguyn t C bc 4 [C 72,7; 32,9; 43; 61,6; 146,0], ni i ngoi vng gia C-16 v C-17 [C 146,0; 117,7], ph 1H NMR cng ch ra khng c nhm axetat trong phn t. 2.3.5 Phn ng axetyl ha cht 1 Cn 50 mg cht 1 cho vo bnh cu phn ng c dung tch 25 ml. Thm 0,2 ml anhidrit axetic, sau cho 0,5 ml pyridin ( c lm khan k bng KOH rn v chng ct trc khi dng) vo bnh phn ng. Phn ng c tin hnh nhit phng trong 3 ngy. Sau phn ng, thm nc ct v dung dch HCl 7M vo bnh phn ng. Chit hn hp phn ng khong 7 ln bng diclometan (mi ln 6 ml). Gp cc dch chit diclometan li, ra bng nc n mi trng trung tnh. Lm khan v ct loi diclometan, thu c mt cn th. Cn th ny c phn tch bng sc k ct trn silica gel (Merck, c ht 0,0630,100 mm), h dung mi n-hexan-axeton (6:1, v/v) thu 5ml/ phn on cho tinh th 166.

40

2.3.6 Phn ng oxi ho cht 1 Cn 50 mg cht 1 cho vo bnh cu phn ng c dung tch 25 ml. Thm 1 ml clorofoc vo trong bnh ho tan cht. Sau cho vo bnh dung dch A (40mg K2Cr2O7 trong 2,5 ml nc ct) v nh git t t 0,1 ml axit sunfuric c vo. Hn hp phn ng c khuy v un hi lu nhit 50 60oC trong 3 ngy. Sau phn ng chit dung dch thu c 5 ln bng clorofoc (mi ln 7 ml). Ra bng nc ct n pH = 7, gp cc dch chit clorofoc li, lm khan v ct loi clorofoc, thu c mt cn th. Cn th ny c phn tch bng sc k ct trn silica gel (Merck, c ht 0,040 - 0,063 mm), h dung mi l gradient n-hexan-axeton thu 5 ml/ phn on cho tinh th 167.

2.3.7 Phn ng oxi ho hn hp 7 v 8 bng tc nhn Jones Cn 10 mg cht u vo bnh cu phn ng c dung tch 25 ml. Thm 10 ml axeton (c lm lnh n -20oC) vo bnh ho tan cht. Sau thm t t vo bnh dung dch 1 ml tc nhn Jones (tc nhn Jones c iu ch t 27g CrO3 v 23 ml H2SO4 c sau thm 20 ml nc ct) sao cho nhit phn ng khng vt qu

41

-5oC. Sau 5pht phn ng kt thc, thm isopropanol ph hy lng d tc nhn. Sau phn ng hn hp phn ng vo nc, chit dung dch thu c khong 3 ln bng diclometan (mi ln 5 ml). Gp cc dch chit diclometan li, ra bng dung dch Na2CO3 bo ha n pH = 7. Lm khan v ct loi diclometan, thu c mt cn th. Cn th ny c phn tch bng sc k ct trn silica gel (Merck, c ht 0,0630,100 mm), h dung mi n-hexan-axeton (6:1, v/v) thu 5ml/ phn on cho tinh th hnh kim mu trng O1 (168). Phn ng ny c mc ch oxy ho nhm ancol bc hai C-7 thnh xeton.OAc OH OAc OAc OAc OAc

HOAc H

+

O H

HOH

O H

HO

O

7 168 Tinh th mu trng hnh kim.

8

168

Rf = 0,41 (TLC, silica gel, h dung mi n-hexan - axeton (3:1, v/v)), vt cht c mu vng khi phun vi thuc th vanilin/ H2SO4. Ph 13C NMR ch ra ni i ngoi vng gia C-16 v C-17 [C 145,5; 118,7], c hai nhm cacbonyl v tr s 7 v 15 [ C 201,5; 205,1], ph 1H NMR cng ch ra s c mt ca 2 nhm axetat v tr s 1 v 14 [H 1,96; 1,99]. 2.4 Hot tnh khng khun v khng nm ca cc dn xut ent-kauran diterpenoid Chng vi khun Escherichia coli (E. Coli) ng hng u trong nhng cn nguyn gy bnh a chy, vim ng tit niu, vim ng mt, gy nhim khun huyt, ngoi ra cn l nguyn nhn ca nhng bnh khc nh vim phi, vim mng

42

no, gy nhim trng vt thng. Chng vi khun Staphylococcus aureus (cn gi l t cu) gy nhim c thc n, gy vim rut cp, gy ra nhng nhim trng cc bnh ngoi da. Pseudomonas aeruginosa cng l chng vi khun gy bnh c iu kin, khi c th ngi suy gim min dch vi khun ny gy vim m in hnh, vim ph tng (xng, ng tit niu...), nhim trng bnh vin kh ph bin. Mt s loi nm si khi nhim vo thc n c kh nng gy c t, trong c chng nm mc Aspergillus niger v Fusarium oxysporum. Cc chng nm men, ngoi tc dng ln men khi c s dng trong cng nghip thc phm nh chng Saccharomyces cerevisiae, cn c th gy ra nhim trng ni tng, nhim trng ng tit niu nh chng Candida albicans [10, 11]. Nh vy, vic kho st hot tnh khng khun v khng nm ca cc phn chit v cc hp cht c phn lp l rt cn thit v hu ch. Hot tnh khng sinh ca cc ent-kauran diterpenoid thin nhin ent-7hydroxy-15-oxo kaur-16-en-18-yl axetat (1), ent-1,14-diacetoxy-7-hydroxykaur16-en-15-one (7), ent-1,7-diaxetoxy-14-hydroxykaur-16-en-15-one (8), ent-18acetoxy-7,14-dihydroxykaur-16-en-15-one (CT2), v cc dn xut bn tng hp: sn phm epoxi ha cht 1 l A3 (159), sn phm epoxi ha hn hp cht (7+8) l F (160 v 161), sn phm m vng epoxit A3 (159) l L (162), sn phm thy phn hn hp epoxit F (160 v 161) l F2 (163), sn phm thy phn cht 1 l 164, sn phm thy phn hn hp cht (7+8) l K2 (165), sn phm axetyl ha cht 1 l 166, sn phm oxi ha cht 1 l 167, sn phm oxi ha hn hp cht (7+8) l 168 c th nghim trn phin vi lng 96 ging theo phng php ca Vander Berghe v Vlietlinck (1991) [35]. Cc chng vi sinh vt c th nghim bao gm hai chng vi khun Gram (-), Escherichia coli v Pseudomonas aeruginosa, hai chng vi khun Gram (+), Bacillus subtilis v Staphylococcus aureus, hai chng nm mc Aspergillus niger v Fusarium oxysporum, v hai chng nm men Candida albicans v Saccharomyces cerevisiae.

43

Kt qu hot tnh khng vi sinh vt kim nh ni trn xc nh cc tc nhn khng khun v khng nm mi thuc dy ent-kauran diterpenoid. Cc hot tnh tp trung vo cc chng vi khun E. coli v S. aureus vi cc gi tr MIC tng ng l 25 v 50 g/ml O1(168). Dn xut 167 (MIC 12,5 g/ml), 166, CT2, 7 (MIC 50 g/ml) khng S. aureus. Cc hot tnh khng nm A. niger c pht hin cho 166 (MIC 25 g/ml); 167 (50 g/ml), 1 (50 g/ml) v khng nm S. cerevisiae cho 1 (50 g/ml).

44

Chng 3

PHN THC NGHIM3.1 i tng v k thut thc nghim 3.1.1 Nguyn liu thc vt Nguyn liu thc vt l ton b phn l ca cy kh sm Bc B. Cy c thu hi H Ni vo thng 9 nm 2004. Mu cy c ra sch, hong trong bng rm, sau tut l, nht b cnh v em sy nhit 40 oC. Khi mu l cy kh hon ton, em xay thnh bt mn. Bt nguyn liu thc vt c ngm chit vi metanol, ri phn b chn lc trong cc dung mi thch hp n-hexan, diclometan v etyl axetat thu c cc phn chit. 3.1.2 Dung mi Cc dung mi (n-hexan, axeton, cloroform, diclometan, metanol v etyl axetat) u c lm khan v chng ct li. Sau pha h dung mi theo t l ph hp vi yu cu s dng trong sc k. Lc k cho cc dung mi trn u nhau trong h ri mi cho vo bnh khai trin y bng c np nhm kn. yn dung mi n khi bo ha mi s dng chy sc k lp mng. 3.1.3 Cc thuc th pht hin cc lp cht Dng nh sng n UV ( 254 nm) v cc thuc th vanilin/axit sunfuric 1%, thuc th Dragendoff Munier v thuc th FeCl3 - iu ch thuc th vanilin/axit sunfuric: Ho tan hon ton 1,86 g vanilin trong 100 ml axt sunfuric c thu c dung dch thuc th vanilin/axit sunfuric - iu ch thuc th Dragendoff Munier Pha ch dung dch gc t cc dung dch sau:

45

Dung dch A Bi(NO3)5 17g

Dung dch B KI 160g

Axit tactric 200g Nc 800ml

Nc 400ml

Trn ln hai dung dch A v B vi nhau thu c dung dch gc c mu da cam. Dung dch gc ny cn c bo qun trong chai mu nu, trnh nh sng v lnh. Khi cn s dng ta pha dung dch gc thnh dung dch thuc th nh sau: Dung dch gc Axit tactric Nc 50ml 200g 400ml

3.1.4 Cc phng php phn tch, phn tch v phn lp cc hp cht Sc k lp mng (TLC) Sc k lp mng c thc hin trn bn silica gel trng sn DC-Alufolien 60 F254, dy 0,2 mm (Merck). Cch a cht ln lp mng: ly mt lng nh cn chit ho tan bng 2 3 git dung mi thch hp, sau dng capilla chm cht ln lp mng. Dung mi: Cc dung mi (n-hexan, axeton, clorofoc, benzen, v metanol) u c lm khan v chng ct li. Sau pha cc h dung mi theo t l ph hp. Lc k cho cc dung mi trn u nhau trong h ri cho vo bnh trin khai y bng c np nhm kn. yn dung mi n khi bo ho mi s dng chy sc k lp mng. Sc k ct (FC v CC)

46

Trong sc k ct nhanh (FC) p dng khng kh nn vi p lc t 0,2 0,3 bar y dung mi chy qua ct sc k. Cht hp ph dng y l silica gel 60 Merck ( c ht 0,015 0,040 mm hoc 0,040 0,063 mm ). Ct tch sc k l mt ng thu tinh hnh tr, pha di c kho. Ct c ng knh tu theo yu cu s dng. Tc ra gii l 1 1,5 ml/pht. Sc k ct thng (CC) c dng vi cht hp ph silica gel 60 Merck (c ht 0,040 0,063 mm, 0,063 0,100 mm, hoc 0,063 0,200 mm). Qu trnh chy ct c thc hin di trng lc ca dung mi. Dung mi s dng trong sc k ct l n-hexan, etyl axetat (EtOAc), toluen, axeton, clorofoc, metanol... Cc dung mi u c lm khan, chng ct li v bo qun trong chai kn. Cht cn phn tch bng sc k ct c ho tan trong dung mi, thm mt lng nh cht hp ph. Trn u hn hp v lm bay hi ht dung mi, thu c mt hn hp dng bt ti. Khi a cht ln ct cht hp ph phi dn u v dng bng thu tinh hoc ct ph ln b mt cht. Nhi ct sc k theo phng php nhi kh hay nhi t. Trong qu trnh nhi ct phi loi b trit cc bt kh bng cch cho dung mi chy qua ct nhiu ln v g nh vo thn ct ct c nn u sau yn qua m cho ct n nh. 3.1.5 Cc phng php kho st cu trc cc hp cht *Ph hng ngoi (IR) c ghi trn thit b Impact 410 Nicolet FTIR. *Ph khi lng (EI-MS v HR-EI-MS) thit b Hewlett Packard 5989 B MS v Varian MAT 44S. *Ph cng hng t ht nhn (NMR)1

H NMR c ghi trn my Brucker Avance 500.

47

13

C NMR (vi chng trnh DEPT) c ghi trn my Brucker Advance 500.

TMS (tetrametyl silan) (1H NMR) hoc tn hiu ca dung mi (13C NMR) l cht chun ni. chuyn dch ho hc c biu th bng ppm. im nng chy c o trn b dng c Boetius (c). 3.2 iu ch cc phn chit t cy kh sm Bc B (Croton tonkinensis Gagnep., Euphorbiaceae) 1 kg bt l kh ca cy kh sm Bc B c ngm chit 2 ln vi metanol khan nhit phng, mi ln 3-4 lt metanol, cch nhau 3 ngy. Tin hnh lc trn phu Buchne, mi ln trng b l bng 100ml metanol. Dch lc sau 2 ln chit, c gom li ri tin hnh c quay di p sut thp cn li 650ml dch chit metanol. Ly 100 g b l sau khi lc ln hai ngm tip vi mt lng va metanol, sau 3 ngy ly dch lc kim tra trn sc k bn mng. Hin mu cc vt bng soi n t ngoi bc sng 254nm v phun vi cc thuc th vanillin/H 2SO4 1%, Dragendoff Munier, so snh vi cht chun thy khng cn vt cht chun nn coi qu trnh ngm chit hon thnh. Pha long dch metanol bng nc ct theo mt t l thch hp (metanol/ nc: 1/1) v chit ln lt vi cc dung mi c phn cc tng dn l n-hexan v diclometan. Dch n-hexan thu c l 1l v dch diclometan l 1l. Dch diclometan c lm khan bng Na2SO4 v loi kit dung mi di p sut gim thu c phn chit diclometan (D).

48

3.3 Phn tch ent-kauran diterpenoid t l cy kh sm Bc B 3.3.1 Kho st phn chit diclometan D bng phng php sc k lp mng (TLC) S dng bn mng trng sn DC-Alufolien 60 F254 (Merck). H dung mi nhexan-axeton 3:1 v/v. Pht hin vt cht trn lp mng bng nh sng t ngoi, phun thuc th vanilin/H2SO4 1% v thuc th Dragendorff-Munier. Bng 1: Kt qu kho st TLC phn chit diclometan (D) (TLC, silica gel, h dung mi n-hexan-axeton (2:1, v/v)STT 1 2 3 4 5 6 7 Rf 0,86 0,69 0,47 0,36 0,32 0,27 0,24 n UV ( 254 nm) pht quang hng xanh hng vng nu nu nht Thuc th vanilin/ axit sunfuric tm tm hng tm xanh tm sm tm nht xanh

Ghi ch: Vt (-) l khng c mu

3.3.2 Qui trnh phn tch phn chit diclometan D bng phng php sc k ct 15 gam phn chit D c tm vi 10 gam silica gel (Merck, c ht 0,0630,100mm) thu c hn hp bt ti mu nu sm. Phn tch hn hp ny bng sc k ct (CC) vi h dung mi ra gii l n-hexan-axeton thu c 8 phn on t D1 n D8. Nhm phn on D2 sau c phn tch li bng sc k ct thng (CC) trn cht hp ph silica gel (Merck, 0,0630,200 mm) vi h dung mi ra gii l n-hexan-

49

axeton, ta thu c 3 nhm phn on D2.1, D2.2, D2.3. Nhm phn on D2.2 c tinh th hnh kim mu trng kt tinh, ly phn tinh th ra kt tinh li trong axeton thu c mt cht tinh khit, k hiu l cht 1. T nhm phn on D2.3 thu c mt hn hp hai tinh th ng kt tinh khng phn tch c bng phng php sc k ct l 7 v 8.

Hng s vt l v cc d kin ph ca cht 1: Tinh th mu trng hnh kim, nc. 135 138oC; Rf = 0,46 (TLC, silica gel, h dung mi axeton-diclometan (10:1, v/v), vt cht c mu tm hng khi phun vi thuc th vanilin/ H2SO4.1

H-NMR (CDCl3, , ppm): (ppm) 0,74 (1H, tdq, J = 13Hz, 40 Hz, 0,9 Hz, H-

1), 0,84 (3H, s, 3H-19), 1,14(3H, d, 3H-20), 1,23 (1H, brd, J = 8,5 Hz, H-9), 1,28 (1H, dd, J = 1,26 Hz, 1,8 Hz, H -5), 1,35 (1H, m, H-3), 1,38 (1H, brd, J = 4,2 Hz, H-3), 1,45 (1H, q, J = 12,0 Hz, H-6), 1,47 (1H, m, H-11), 1,50 (1H, m, H-2), 1,63 (1H, s, OH), 1,65 (1H, m, H-2), 1,70 (1H, ddd, J = 12,0 Hz, 4,4 Hz, 1,6 Hz, H-6), 1,70 (1H, m, H-12), 1,79 (1H, ddd, J = 3,5 Hz, 3,5 Hz, 13 Hz, H-1), 1,96 (1H, tdd, J = 13,0 Hz, 6,2 Hz, 2,7 Hz, H-12), 2,07 (1H, brd, H-14), 2,10 (3H, s, OCOCH3), 3,10 (1H, m, H13), 3,66 (1H, d, J = 10,8 Hz, H-18A), 3,87 (1H, d, J = 10,8 Hz, H-18B), 4,05 (1H, dd, J = 4,4 Hz, 12,0 Hz, H-7), 5,29 (1H, t, J = 1,1 Hz, H-17A), 5,97 (1H, t, J = 1,1 Hz, H17B).13

C-NMR (CDCl3, , ppm): (ppm) 17,5 (t, C-2), 17,5 (q, C-19), 18,0 (t, C-11),

18,2 (q, C-20), 21,1 (q, OCOCH3), 27,7 (t, C-6), 27,9 (t, C-14), 32,8 (t, C-12), 35,4 (t, C-3), 36,4 (s, C-4), 37,6 (d, C-13), 38,9 (t, C-1), 39,6 (s, C-10), 46,3 (d, C-5), 51,8 (d, C-9), 58,3 (s, C-8), 70,8 (d, C-7), 72,3 (t, C-18), 115,0 (t, C-17), 149,2 (s, C-16), 171,2 (s, OCOCH3), 209,7 (s, C-15). 3.4 Cc phn ng chuyn ha cc ent-kauran

50

3.4.1 Phn ng epoxi ha Cn 0,5 g cht u vo bnh cu phn ng c dung tch 50 ml. Ha tan ht cht u trong 16 ml CH2Cl2. Cho 0,4 g MCPBA vo bnh, ha tan hon ton trong 16 ml CH2Cl2. Lm lnh bnh phn ng n -10oC. Lm lnh dung dch peaxit n -5oC. Nh git dung dch peaxit vo bnh phn ng, khuy u trong thi gian 40. Khuy tip hn hp phn ng nhit phng, ri yn trong 3 ngy. Cho hn hp sau phn ng ln phu chit, lc hn hp vi dung dch NaHCO 3 bo ha (3 ln, mi ln 30 ml), chit gi li lp di CH2Cl2. Cui cng ra bng nc 4 ln n pH trung tnh. Lm khan dung dch nhn c bng Na 2SO4. Ct loi dung mi cho sn phm th c mu trng ng. Sn phm th c tinh ch bng sc k ct trn silica gel (Merck, c ht 0,063-0,100 mm), h dung mi n-hexan-axeton 8:1 thu 10 ml/ phn on cho cc sn phm epoxit tng ng. T cht u l cht 1 thu c sn phm chnh l A3 (159) (0,41 g). Hiu sut phn ng l 73%. T cht u l hn hp 2 cht 7 v 8 thu c sn phm chnh l F (hn hp 160 v 161) (0,42 g). Hiu sut phn ng l 81,5%.

Hng s vt l v cc d kin ph ca cht A3:-

Tinh th mu trng, nc 176 178oC. Rf = 0,35 (TLC, silica gel, h dung mi n-hexan-etyl axetat 1:1 (v/v), vt cht c mu tm hng khi phun vi thuc th vanilin/H2SO4 1%. Vt c mu vng sm khi phun thuc th Dragendorff-Munier.

1

H-NMR (CDCl3): (ppm): 0,86 (3H, s, Me-19), 0,92 (H, dt, J = 13,2 Hz, 3,6

Hz, H-1a), 1,12 (3H, s, Me-20), 1,36(2H, m, 2H-3), 1,40 (1H, m, H-5), 1,48 (1H, q, J = 12,5 Hz, H-6a), 1,58 (1H, m, H-2a), 1,59 (1H, brs, H-9), 1,63 (1H, m, H-2b), 1,65 (1H, m, H-12a), 1,80 (1H, dd, J = 12,5 Hz, H-6b), 1,87 (1H, m, H-1b), 1,92 (2H, m, 2H-11), 1,96 (1H, m, H-12b), 2,0 (1H, brs, H-13), 2,07 (3H, s, OCOCH3), 2,42 (1H, brs, H-13),

51

2,52 (1H, d, J = 6,0 Hz, H-17a), 2,80 (1H, d, J = 6,0 Hz, H-17b), 3,65 (1H, d, J = 11,5 Hz, H-18a), 3,83 (1H, dt, J = 12,0 Hz, 4,0 Hz, H-7), 3,89 (1H, d, J = 11,5 Hz, H-18b).13

C-NMR (CDCl3): (ppm): 17,5 (t, C-2), 17,8 (t, C-11), 17,84 (q, C-19), 18,4

(q, C-20), 21,0 (q, -OCOCH3), 24,8 (t, C-14), 26,8 (t, C-6), 26,9 (t, C-12), 33,1 (d, C13), 35,2 (t, C-3), 36,3 (s, C-4), 39,7 (t, C-1), 40,0 (s, C-10), 46,2 (d, C-5), 51,38 (d, C-9), 51,4 (t, C-17), 55,9 (s, C-8), 72,1 (t, C-18), 76,9 (d, C-7), 91,1 (s, C-16), 170,4 (s, C-15), 171,1 (s, - OCOCH3).

Hng s vt l v cc d kin ph ca cht F: Bt v nh hnh mu trng. Rf = 0,59 (TLC, silica gel, h dung mi n-hexan-axeton 2:1 (v/v)), vt cht c

mu vng khi phun vi thuc th vanilin/ H2SO4. 160: 1H-NMR (CDCl3): (ppm): 0,91 (3H, s, CH3-19), 0,95 (3H, s, CH3-18), 1,29 (3H, s, CH3-20), 1,98 (3H, s, 14-OAc), 1,99 (3H, s, 1-OAc), 2,91 (1H, d, J = 10,5 Hz, H-17a), 3,26 (1H, d, J = 10,5 Hz, H-17b), 4,17 (1H, brs, H-7), 4,89 (1H, s, H-1), 6,16 (1H, s, H-14). 161: 1H-NMR (CDCl3): (ppm): 0,88 (3H, s, CH3-19), 0,97 (3H, s, CH3-18), 1,18 (3H, s, CH3-20), 2,01 (3H, s, 1-OAc), 2,02 (3H, s, 7-OAc), 2,94 (1H, d, J = 11,0 Hz, H-17a), 3,18 (1H, d, J = 11,0 Hz, H-17b), 4,89 (1H, s, H-1), 5,04 (1H, s, H-14), 5,46 (1H, dd, J = 12,0 Hz, 4,0 Hz, H-7).

3.4.2 Phn ng m vng epoxit A3 bng thy phn kim Cn 100 mg cht A3 cho vo bnh cu phn ng c dung tch 25 ml. un hi lu hn hp phn ng vi dung dch NaOH 2,5% trong MeOH (10 ml) trn ni nc nng trong 2h. Sau phn ng thm nc ct vo bnh phn ng. Chit hn hp phn ng 3 ln bng diclometan (mi ln 6 ml). Gp cc dch chit diclometan li, ra bng

52

nc ct n mi trng trung tnh. Lm khan dung dch nhn c bng Na2SO4, lc v ct loi diclometan, thu c cn th. Cn th c tinh ch bng sc k ct trn silica gel (Merck, c ht 0,063 -0,100 mm), h dung mi n-hexan-axeton (3:1, v/v) thu 8 ml/ phn on cho tinh th L (162) (60 mg). Hiu sut phn ng t 42,4%.

Hng s vt l v cc d kin ph ca cht L (162): Tinh th mu trng hnh kim. Rf = 0,65 (TLC, silica gel, h dung mi n-hexan-axeton (1:2, v/v)), vt cht c mu vng khi phun vi thuc th vanilin/ H2SO4.1

H-NMR (CDCl3 + CD3OD, , ppm): 0,73 (3H, brs, CH3-19), 1,0 (3H, brs, CH3-

20), 2,54 (1H, brs, H-13), 3,0 (1H, d, J = 11,0 Hz, H-17a), 3,38 (1H, d, J = 11,0 Hz, H17b), 3,76 (1H, d, J = 10,5 Hz, H-18a), 3,77 (1H, dd, J = 11,0 Hz, 5,4 Hz, H-7), 3,95 (1H, d, J = 10,5 Hz, H-18b).13

C-NMR (CDCl3 + CD3OD, , ppm): 17,4 (C-19), 17,5 (C-18), 17,6 (C-2), 17,9

(C-11), 26,3 (C-6), 27,3 (C-12), 28,4 (C-14), 34,9 (C-3), 36,9 (C-4), 38,2 (C-10), 39,6 (C-1), 34,8 (C-13), 44,8 (C-5), 54,9 (C-9), 62,4 (C-17), 70,0 (C-18), 73,9 (C-7), 82,7 (C-8), 87,9 (C-16), 180,3 (C-15).

3.4.3 Phn ng thy phn hn hp epoxit F bng phc BF3.Et2O Cho 20 mg epoxit F c ha tan trong CHCl3 khan vo bnh cu phn ng c khuy t trong iu kin c lm lnh bng . Thm 5 git BF 3.Et2O vo bnh phn ng. Sau 5 pht, hn hp phn ng c ra bng dung dch NaHCO 3, c lm kh trn Na2SO4, lc v ct loi dung mi di p sut gim thu c cn th.

53

Cn th c phn tch bng sc k ct trn silica gel (Merck, c ht 0,063 -0,100 mm), h dung mi n-hexan-axeton (10:1, v/v), thu 5 ml/ phn on cho tinh th F2 (163) (7 mg). Hiu sut phn ng l 27%.

Hng s vt l v cc d kin ph ca cht F2 (163):

Bt mu trng v nh hnh. Rf = 0,32 (TLC, silica gel, h dung mi n-hexan-axeton 3:1 (v/v), vt cht c mu vng khi phun vi thuc th vanilin/ H2SO4.1

H-NMR (CDCl3, , ppm): 0,91 (3H, s, CH3-19), 0,97 (3H, s, CH3-18), 1,17

(3H, s, CH3-20), 2,01 (3H, s, 1-OAc), 2,29 (1H, brs, H-13), 2,91 (1H, d, J=11,0 Hz, H17a), 3,18 (1H, d, J=11,0 Hz, H-17b), 4,34 (1H, dd, J=12,5 Hz, 4,0 Hz, H-7), 4,88 (1H, brs, H-14), 5,04 (1H, J=1,5 Hz, H-1).13

C-NMR (CDCl3, , ppm): 18,2 (C-11), 18,6 (C-20), 21,3 (C-19), 21,5 (1-

OAc), 22,7 (C-2), 27,6 (C-6), 28,0 (C-12), 32,9 (C-4), 33,3 (C-20), 34,9 (C-3), 42,8 (C10), 42,9 (C-13), 45,5 (C-9), 47,2 (C-5), 48,3 (C-17), 61,9 (C-8), 62,4 (C-16), 72,8 (C1), 74,2 (C-7), 75,4 (C-14), 170,1 (1-OAc), 217,1 (C-15). 3.4.4 Phn ng thy phn Cn 200 mg cht u cho vo bnh cu phn ng c dung tch 25 ml. Thm 5 ml metanol cha 15% KOH vo bnh phn ng. Hn hp phn ng c un hi lu nhit khong 70 80oC trong 12 gi. Sau khi phn ng, thm 30 ml nc ct vo hn hp trn v ct loi metanol. Chit dung dch ny 3 ln bng diclometan (mi ln 5 ml). Ra bng nc n pH = 7, gp cc dch chit diclometan li, lm khan v ct loi diclometan, thu c mt cn th. Cn th ny c phn tch bng sc k ct trn silica gel (Merck, c ht 0,040 0,063 mm), h dung mi l gradient n-hexan-axeton thu 5 ml/ phn on cho cc sn phm thy phn tng ng.

54

T cht u l cht 1 thu c sn phm chnh l 164 (90 mg). Hiu sut phn ng l 75%. T cht u l hn hp 2 cht 7 v 8 thu c sn phm chnh l K2 (165) (83 mg). Hiu sut phn ng l 74%.

Hng s vt l v cc d kin ph ca cht K2 (165):

Tinh th mu trng hnh kim. Rf = 0,53 (TLC, silica gel, h dung mi n-hexan-axeton (3:1, v/v)), vt cht c mu vng khi phun vi thuc th vanilin/ H2SO4.1

H-NMR (CDCl3, , ppm): 0,89 (3H, s, CH3-19), 0,96 (3H, S, CH3-18), 1,09

(3H, s, CH3-20), 3,08 (1H, s, H-13), 3,69 (1H, brs, OH), 4,37 (1H, dt, J = 12,5 Hz, 4,0Hz, H-7), 4,91 (1H, s, H-1), 5,29 (1H, s, H-14), 5,41 (1H, s, H-17a), 6,18 (1H, s, H17b),13

C-NMR (CDCl3, , ppm): 18,6 (C-20), 21,4 (C-19), 33,2 (C-18), 117,7 (C-17),

146,0 (C-16), 206,5 (C-15), 76,1 (C-14), 44,2 (C-13), 32,3 (C-12), 16,6 (C-11), 43,0 (C-10), 48,1 (C-9), 61,6 (C-8), 72,9 (C-7), 27,3 (C-6), 47,6 (C-5), 32,9 (C-4), 35,0 (C3), 22,7 (C-2), 72,7 (C-1).

3.4.5 Phn ng axetyl ha cht 1 Cn 50 mg cht 1 cho vo bnh cu phn ng c dung tch 25 ml. Thm 0,2 ml anhidrit axetic, sau cho 0,5 ml pyridin (lm khan k bng KOH rn v chng ct trc khi dng) vo bnh phn ng. Phn ng c tin hnh nhit phng trong 3 ngy.

55

Sau phn ng, thm nc ct v dung dch HCl 7M vo bnh phn ng. Chit hn hp phn ng khong 7 ln bng diclometan (mi ln 6 ml). Gp cc dch chit diclometan li, ra bng nc n mi trng trung tnh. Lm khan v ct loi diclometan, thu c mt cn th. Cn th ny c phn tch bng sc k ct trn silica gel (Merck, c ht 0,0630,100 mm), h dung mi n-hexan-axeton (6:1, v/v) thu 5ml/ phn on cho tinh th 166 (47mg). Hiu sut phn ng l 89,5%.

Hng s vt l v cc d kin ph ca cht 166: Tinh th hnh kim, mu trng. Rf = 0,63 (TLC, silica gel, h dung mi n-hexan-etyl axetat (2:1, v/v), vt cht

c mu tm hng khi phun vi thuc th vanilin/ H2SO4.1

H-NMR (CDCl3, , ppm): 0,82 (3H, s, Me-19), 1,15 (3H, s, Me-20), 1,91 (3H,

s, 7-OAc), 2,13 (3H, s, 18-OAc), 3,10 (1H, sbr, H-13), 3,60 (1H, d, J = 11,0 Hz, H18A), 3,85 (1H, d, J = 11.0 Hz, H-18B), 5,10 (1H, dd, J = 11,0 Hz, 4,5 Hz, H-3), 5,29 (1H, s, H-17A), 5,96 (1H, s, H-17B).13

C-NMR (CDCl3, , ppm): 17,6 (t, C-2), 17,7 (q, C-19), 17,9 (t, C-11), 18,2 (q,

C-20), 21,0 v 21,0 (q, 7-OAc, 18-OAc), 24,4 (t. C-6), 29,2 (t, C-14), 32,6 (t, C-12), 35,7 (t, C-3), 36,4 (s, C-4), 35,7 (d, C-13), 39,0 (t, C-1), 39,7 (s, C-10), 45,9 (d, C-5), 51,9 (d, C-9), 56,2 (s, C-8), 72,0 (d, C-7), 73,1 (t, C-18), 115,2 (t, C-17), 148,8 (s, C16), 169,5 (s, 7-OAc), 171,4 (s, 18-OAc), 207,4 (s, C-15). 3.4.6 Phn ng oxi ho cht 1 Cn 50 mg cht 1 cho vo bnh cu phn ng c dung tch 25 ml. Thm 1 ml clorofoc vo trong bnh ho tan cht. Sau cho vo bnh dung dch A (40mg K2Cr2O7 trong 2,5 ml nc ct) v nh git t t 0,1 ml axit sunfuric c vo. Hn hp phn ng c khuy v un hi lu nhit 50 60oC trong 3 ngy.

56

Sau phn ng chit dung dch thu c 5 ln bng clorofoc (mi ln 7 ml). Ra bng nc ct n pH = 7, gp cc dch chit clorofoc li, lm khan v ct loi clorofoc, thu c mt cn th. Cn th ny c phn tch bng sc k ct trn silica gel (Merck, c ht 0,040 - 0,063 mm), h dung mi l gradient n-hexan-axeton thu 5 ml/ phn on cho tinh th 167 (42 mg). Hiu sut phn ng t 82,5%.

Hng s vt l v cc d kin ph ca cht 167: Bt mu trng v nh hnh. Rf = 0,69 (TLC, silica gel, h dung mi n-hexan-etyl axetat (2:1, v/v), vt cht

c mu vng da cam khi phun thuc th vanilin/ H2SO4.1

H-NMR (CDCl3, , ppm): 0,85 (3H, s, Me-19), 0,94 (3H, s, Me-20), 2,11 (3H,

s, 18-OAc), 2,40 (1H, dd, J = 18,5 Hz, 11,5 Hz, H-6A), 2,71 (1H, dd, J = 18,5 Hz, 8,0 Hz, H-6B), 3,02 (1H, dd, J = 8,5 Hz, 4,5 Hz, H-13), 3,64 (1H, d, J = 11,0 Hz, H-18A), 3,82 (1H, d, J = 11,0 Hz, H-18B), 5,39 (1H, s, H-17A), 5,92 (1H, s, H-17B).13

C-NMR (CDCl3, , ppm): 14,8 (q, C-20), 17,2 (q, C-19). 17.5 (t, C-2), 17,7 (t,

C-11), 21,0 (q, 18-OAc), 27,7 (t, C-6), 30,2 (t, C-14), 35,1 (d, C-13), 35,8 (t, C-3), 36,8 (s, C-4), 38,2 (s, C-10), 38,4 (t, C-12), 39,2 (t, C-1), 44,0 (d, C-5), 54,9 (d, C-9), 63,8 (s, C-8), 72,2 (t, C-18), 116,9 (t, C-17), 151,2 (s, C-16), 171,5 (s, 18-OAc), 203,2 (s, C7), 210,7 (s, C-15).

3.4.7 Phn ng oxi ho hn hp 7 v 8 bng tc nhn Jones Cn 20 mg cht u vo bnh cu phn ng c dung tch 25 ml. Thm 10 ml axeton (c lm lnh n -20oC) vo bnh ho tan cht. Sau thm t t vo bnh dung dch 1 ml tc nhn Jones (tc nhn Jones c iu ch t 27g CrO3 v 23

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ml H2SO4 c sau thm 20 ml nc ct) sao cho nhit phn ng khng vt qu -5oC. Sau 5 pht phn ng kt thc, thm isopropanol ph hy lng d tc nhn. Sau phn ng hn hp phn ng vo nc, chit dung dch thu c khong 3 ln bng diclometan (mi ln 5 ml). Gp cc dch chit diclometan li, ra bng dung dch Na2CO3 bo ha n pH = 7. Lm khan v ct loi diclometan, thu c mt cn th. Cn th ny c phn tch bng sc k ct trn silica gel (Merck, c ht 0,063 0,100 mm), h dung mi n-hexan-axeton (6:1, v/v) thu 5ml/ phn on cho tinh th hnh kim mu trng O1 (168) (16 mg). Hiu sut phn ng t 78,5%.

Hng s vt l v cc d kin ph ca cht O1:

Tinh th mu trng hnh kim. Rf = 0,41 (TLC, silica gel, h dung mi n-hexan-axeton (3:1, v/v)), vt cht c mu vng khi phun vi thuc th vanilin/ H2SO4.1

H-NMR (CDCl3, , ppm): 0,90 (3H, s, CH3-19), 0,91 (3H, s, CH3-18), 1,41

(3H, s, CH3-20), 1,96 (3H, s, 14-OAc), 1,99 (3H, s, 1-OAc), 3,12 (1H, brs, H-13), 4,97 (1H, s, H-1), 5,43 (1H, s, H-17a), 5,76 (1H, s, H-14), 6,19 (1H, s, H-17b).13

C-NMR (CDCl3, , ppm): 16,1 (C-11), 17,2 (C-20), 20,8 (1-OAc), 20,9 (14-

OAc), 21,1 (C-19), 22,5 (C-2), 31,9 (C-12), 32,3 (C-18), 33,2 (C-4), 34,8 (C-3), 38,1 (C-6), 42,2 (C-10), 43,1 (C-13), 46,6 (C-5), 48,6 (C-9), 68,7 (C-8), 72,9 (C-1), 76,2 (C14), 118,7 (C-17), 145,5 (C-16), 170,0 (1-OAc), 171,0 (1H-OAc), 201,5 (C-7), 205,1 (C-15).

3.5 Kho st hot tnh khng khun v khng nm ca cc dn xut. Hot tnh khng vi sinh vt kim nh c tin hnh nh gi hot tnh khng sinh ca cc mu chit c thc hin trn cc phin vi lng 96 ging (96-well

58

microtiter plate) theo phng php hin i ca Vander Bergher v Vlietlinck (1991), v MCKane, L., & Kandel (1996). Cc chng vi sinh vt kim nh:-

Vi khun Gr (-): Escherichia coli, Pseudomonas aeruginosa. Vi khun Gr (+): Bacillus subtilis, Staphylococcus aureus. Nm si: Aspergillus niger, Fusarium oxysporum. Nm men: Candida albicans, Saccharomyces cerevisiae.

Chng dng tnh: + Ampicilin cho vi khun Gr (+) + Tetracylin cho vi khun Gr (-) + Nystatin hoc Amphotericin B cho nm si v nm men Thuc thng sinh c pha trong DMSO 100% vi nng thch hp: Ampicilin: 50 mM; Tetracylin: 10 mM; Nystatin: 0,04 mM. Chng m tnh: Vi sinh vt kim nh khng trn ln khng sinh v cht th Mi trng nui cy vi sinh vt: Mi trng duy tr v bo tn ging: Sabouraud Dextrose Broth (SDB)-Sigma cho nm men v nm mc. Vi khun trong mi trng Trypcase Soya Broth (TSB)Sigma. Mi trng th nghim: Eugon Broth (Difco, M) cho vi khun, Mycophil (Difco, M) cho nm. Tin hnh th nghim:

59

Cc chng kim nh c hot ha v pha long ti nng 0,5 n v Mc Fland ri tin hnh th nghim. c kt qu: Kt qu c sau khi cc phin th nghim trong t m 37 oC/24 gi cho vi khun v 30oC/48 gi i vi nm si v nm men. Kt qu dng tnh l nng m khng c vi sinh vt pht trin. Khi nui cy li nng ny trn mi trng thch a kim tra, c gi tr CFU< 5. Nng c ch ti thiu (MIC-Minimum Inhibitory concentration) ca cht c hot tnh: Cc mu c hot tnh c sng lc ban u c pha long theo cc thang nng thp dn t (5- 10) thang nng tnh gi tr nng ti thiu m vi sinh vt b c ch pht trin gn nh hon ton. Mu th c MIC 200g/ml; mu tinh c MIC 50g/ml l c hot tnh. Kt qu th hot tnh c nu phn 2.4.

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KT LUN

Thc hin ni dung nghin cu phn lp v chuyn ho ent-kauran diterpenoid t cy kh sm Bc B (Croton tonkinensis Gagnep., Euphorbiaceae) v kho st hot tnh sinh hc ca cc sn phm nhn c, lun vn thu c cc kt qu nh sau: 1. phn lp c cc ent-kauran diterpenoid 1 v hn hp 7 + 8 lm cc cht u cho chuyn ha. 2. Cc cht u ny c chuyn ha v tr ca cc ni i v cc nhm chc hidroxy v cacbonyl to thnh cc dn xut mi. thc hin thnh cng phn ng epoxi ha ni i ca cc ent-kauran diterpenoid v m vng epoxit to thnh nh thy phn hoc botriflorua eterat; thc hin cc phn ng thy phn, axetyl ha v oxi ha cc ent-kauran diterpenoid thnh cc dn xut dihydroxy, diaxetyl v dixeton tng ng. Cu trc ca cc sn phm chuyn ha c xc nh bng cc phng php ph. 3. th nghim hot tnh khng vi sinh vt vi cc sn phm nhn c. Sn phm oxi ha ent-kauran diterpenoid cho kt qu c ch tt i vi mt s vi khun v nm.

61

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