Gp phn nghin cu cc hp cht c hot tnh sinh hc trong thn r cy ngi tin Bousigon (Hedychium Bousigonianum Pierre ex Gagn)

Th Hin
Trng i hc Khoa hc T nhin Lun vn ThS. ngnh: Ha hu c; M s: 60 44 27 Ngi hng dn: PGS.TS. Vn Ngc Hng Nm bo v: 2012
Abstract. Nghin cu tinh du ca thn r cy ngi tin bousigon. Nghin cu qui trnh chit chn lc cc hp cht c hot tnh sinh hc trong thn r cy ngi tin bousigon. Kho st hot tnh khng vi sinh vt kim nh ca cc sn phm chit c. Nghin cu phn lp cc - lacton , khng no trong cc cn chit t thn r cy ngi tin bousigon. Kho st invitro hot tnh gy c t bo ca 3 loi ng th ca cc sn phm phn lp c. Xc nh cu trc phn t ca cc cht phn lp c. Keywords. Ha hu c; Hp cht; Cy ngi tin; Hot tnh sinh hc

Content Nc ta nm trong vng kh hu nhit i gi ma nn h thc vt v cng a dng v phong ph c bit l cy c tinh du v cy lm thuc. Theo thng k bc u, nc ta c hn 600 loi cy c tinh du, nhng phn ln cha c nghin cu y , h thng v trit . H thc vt phong ph v a dng ca mi vng, mi quc gia c xem l mt trong nhng ngun ti nguyn qu gi, c quan h trc tip ti i sng con ngi. Ha hc cc hp cht thin nhin vi vai tr nghin cu thnh phn ha hc v tm hiu hot tnh sinh hc ca cy thuc mang ngha khoa hc v thc tin. Vic nghin cu cc hp cht c hot tnh sinh hc trong cy thuc dn tc l vn khng nhng hp dn v khoa hc m cn ng gp cho vic s dng cy thuc c hiu qu hn, chnh xc hn. Gp phn phong ph cho h thc vt Vit Nam, li rt gn gi vi i sng nhn dn phi k n h gng (Zingiberaceae). Nhu cu s dng cc sn phm t cc cy h gng lm thuc v hng liu trn th gii v trong nc ngy cng tng. Cc nghin cu v cc cy h gng n nay l rt nhiu nhng n lun l iu hp dn vi cc nh nghin cu. Trong h gng c mt chi m Vit Nam cha c nghin cu rng ri l chi ngi Hedychium. Trong khi th gii vi rt nhiu cc nghin cu bo co rng y l mt chi rt hp dn bi hng thm ca hoa v nhng hp cht c hot tnh sinh hc th v thuc dy cc - lacton diterpen , khng no. y l nhng hp cht c hot tnh chng ung th, chng vim rt mnh ang c cc nh khoa hc th gii quan tm mt cch c bit. Vi

nh hng nh vy chng ti chn ti: Gp phn nghin cu cc hp cht c hot tnh sinh hc trong thn r cy ngi tin bousigon ( Hedychium bousigonianum Pierre ex Gagn) Hedychium (chi ngi) l mt chi thuc h gng (Zingiberaceae) gm nhng cy lu nm ph bin vi chiu cao khi pht trin khong 120 180 cm. Chi ny thng c gi vi ci tn l chi ca nhng cy hoa loa kn gng v chi ca cc loi cy thn tho, thn r mp v phn nhnh. Chi ny c ngun gc t nhng vng t nhit i Chu v dy Himalaya. Cc loi ca chi ny thng c hoa rt p, rc r v hp dn bi mi hng. Ti Nam , chi Hedychium c hn 80 loi. Cc nghin cu gn y bo co c 41 loi n , trong c 17 loi c hu ca n . Ba loi mi ca chi Hedychium t Thi Lan mi c nhn dng v phn lp nm 1995 l H. samuiense, H. tomentosum v H. biflorum. Theo cun Cy c Vit Nam ca Phm Hong H th Vit Nam chi Hedychium c 12 loi, phn b hu ht cc tnh t Bc ch Nam. V thnh phn tinh du mi ch c mt cng trnh ca Nguyn Th Thy v cng s nghin cu thn r cy Ngi tin. Cy Ngi tin (Hedychium coronarium Koenig) mc t nhin ti huyn Sapa, tnh Lo Cai, c em v trng ti tri Thc nghim ca vin Sinh thi v Ti nguyn Sinh Vt (C Nhu, T Lim, H Ni) t nm 2000 v thu hoch cui nm 2002 nghin cu. Thnh phn ho hc ca tinh du c bit gm cc thnh phn chnh l 1,8 - Cineol - 43,32%, - Pinen - 25,45%, Pinen - 10,32%, 3- Cyclohexen- 1-methanol - 5,29%. [5] Mi y, vo thng 11 nm 2011, trn tp ch Bioorganic & Medicinal Chemistry Letters, Phan vn Kim v cc cng s cng b nghin cu mi nht v cc hp cht c phn lp t thn r ngi tin Sapa Vit Nam. Trong bo co ny, h phn lp c ba labdane loi diterpenes mi, t tn l coronarins G,H,I v 7 hp cht c bit n l: coronarin D, coronarin D methyl ether, hedyforrestin C, (E)-nerolidol, -sitosterol, daucosterol, v stigmasterol. Cc hp cht t cy c tc dng c ch sn xut cc cytokine ca t bo ui gai c ngun gc t ty xng l cc tc nhn gy vim hoc tin gy vim c gii phng ra khi b kch thch bi LPS (lypopolysacchride). Trong s cc hp cht ny, cc hp cht coronarins D, G, H l cht c ch ng k ti LPS kch thch TNF-, IL-6 v IL-12 p40 sn xut vi IC50 khc nhau, t 0,19 0,11 ; 10,38 2,34 M.[16]

Ba hp cht mi c phn lp (1-3 : coronarins G,H,I) Cy ngi tin bousigon, tn khoa hc l Hedychium bousigonianum Pierre ex Gagn, thuc h Gng ( Zingiberaceae). Hedychium bousigonianum Pierre ex Gagn c ngun gc l t vng Himalayas ca Nepal v n sau pht tn ti khu vc Nam chu Phi v Nam M. Loi cn phn b Nam Trung Quc, Malaixia, c v Vit Nam. Cy mc nhng vng c kh hu mt lnh. Thn c, cao 1-1,2 m. Cn hnh to 6 7 mm. L c phin thon hp, nhn, di 30 50 cm, rng 7 cm, khng lng. Gi tha, di 20 cm; l hoa c lng, di 2,5 cm; hoa to, vng; tiu nhy lp hp, di 4cm; mi xoan, ch n ; non so c lng. Thng mc trong rng Lt.

Hnh 1.1: Thn r cy ngi tin Bousigon Cc loi trong chi Hedychium rt hp dn bi hng thm ca hoa v nhng hp cht c hot tnh sinh hc th v thuc dy cc - lacton diterpen , khng no. c bit l cc lacton , khng no thuc dy labdan. Cc hp cht ny theo Kumakara AB v cng s l nhng hp cht khng ch c tc dng c ch s to thnh tc nhn gy ng th v nhiu bnh nguy him khc nh l NF-kB hot ng m cn c tc dng c ch s hot ng ca tc nhn ny gy bnh tt. Chnh c tnh lng dng ny m cc -lacton , khng no thuc dy labdan ditecpen ang l i tng c cc nh khoa hc trn th gii c bit quan tm nghin cu. Chng ti cng quan tm cc hp cht ny v tm kim n trong cc cy thuc chi Ngi (Hedychium). Trong cng trnh trc chng ti nghin cu cc labdan ditecpen c hot tnh gy c t t bo ng th trong cy ngi tin (Hedychium coronarium Koen) [6]. Trong cng trnh ny chng ti chn cy ngi tin Bousigon v tn ti: Gp phn nghin cu cc hp cht c hot tnh sinh hc trong thn r cy ngi tin bousigon ( Hedychium bousigonianum Pierre ex Gagn) . Mc tiu ca ti a. Nghin cu thnh phn ha hc ca thn r cy ngi tin bousigon. b. Nghin cu, phn lp, xc nh cu trc phn t v hot tnh chng ung th ca cc - lacton , khng no dy labdan diterpene trong thn r ngi tin bousigon. . Nhim v ca ti 1. Nghin cu tinh du ca thn r cy ngi tin bousigon. 2. Nghin cu qui trnh chit chn lc cc hp cht c hot tnh sinh hc trong thn r cy ngi tin bousigon. 3. Kho st hot tnh khng vi sinh vt kim nh ca cc sn phm chit c. 4. Nghin cu phn lp cc - lacton , khng no trong cc cn chit t thn r cy ngi tin bousigon. 5. Kho st invitro hot tnh gy c t bo ca 3 loi ng th ca cc sn phm phn lp c. 6. Xc nh cu trc phn t ca cc cht phn lp c. Mu thc vt v phng php x l: Nguyn liu thn r cy ngi tin bousigon (Hedychium bousigonianum Pierre ex Gagn) c Tin s Nguyn Quc Bnh Bo Tng thin nhin Vit Nam cung cp v gim nh. Mu thn r ngi tin bousigon c ly ti tnh Vnh Phc vo thng 4 nm 2011. Sau khi thu mua v, mu c ra sch t ct, loi b phn thi ra, h hng v thi

ngang thn r thnh lt dy 1- 3 mm, ri nghin thnh bt c ht 1 2 mm v bo qun trong t lnh tin hnh nghin cu. - T 0,2 kg nguyn liu kh chun b cho nghin cu phn tinh du. - T 3,6 kg nguyn liu ti chun b cho nghin cu cc hot cht sinh hc. iu ch tinh du thn r ngi tin bousigon. a/ Cho 0,2 kg nguyn liu ( chun b phn 2.3.1) cng 2,5 lt dung dch NaCl 10% cho vo bnh ct A ca thit b chng ct c bit (hnh 2.1) c by tinh du D bng 60ml dung mi toluen.

A: Bnh ct B: ng ni C: Sinh hn D: By tinh du

Hnh 2.1: Thit b ct cun hi nc hi lu. un hi lu 03 gi, ly toluen trong by ra v ct ly dch du nc cho n khi m tnh vi KmnO4 0,2%. b/Bo ha dch du-nc ct c bng NaCl ri chit 03 ln bng toluen mi ln 100ml. c/ Gp lng toluen ly trong by chit vi lng dch chit toluen t dch du nc,, lm kh bng Na2SO4, ct loi dung mi, thu hi tinh du, gi trong l kn v bo qun trong t lnh trc khi xc nh thnh phn ho hc v cc nghin cu khc. Hm lng tinh du c tnh bng t l phn trm khi lng tinh du trn khi lng mu kh (gam). Kt qu c 0,5g tinh du. Tnh hiu sut l 0,25%. Tinh du c mu vng chanh nht, mi thm d chu, hp dn. Chit cc hp cht c hot tnh sinh hc trong thn r ngi tin bousigon : a/ T 3,6 kg nguyn liu ti chun b phn 2.3.1, ngm vo 10 lt etanol 96% trong ba ngy, lc Busne, ngm tip 2 ln na, mi ln 1,5 lt EtOH 96% lc Busne. Gp dch chit etanol, thu c 10 lt dch lc. b/ C dch lc bng cch un cch thy n cn 1/6 th tch . Thm vo 4 th tch nc bo ha mui v chit 3 ln bng n hexan. Ln 1 : 150 ml Ln 2: 150 ml Ln 3: 150 ml. Gp dch chit n hexan, lm kh bng Na2SO4, ct loi dung mi bng cch thy thu c cn H, 3gam. Hiu sut 0.088% tnh theo nguyn liu ti. c/ Sau khi chit bng n hexan, dch c chit tip bng diclometan, cng tin hnh 3 ln, mi ln 100 ml, thu c dch diclometan. Lm kh bng Na2SO4 khan, ct cch thy loi dung mi thu c cn C khi lng 4 gam, hiu sut 0.11 % tnh theo nguyn liu ti. d/ Sau khi chit bng diclometan, dch c chit tip bng etylaxetat, cng tin hnh 3 ln, mi ln 100 ml, thu c dch etyaxetat. Lm kh bng Na2SO4 khan, ct cch thy

E: Kha

loi dung mi thu c cn E khi lng 1 gam, hiu sut 0.027 % tnh theo nguyn liu ti. Th hot tnh khng vi sinh vt kim nh (Antimicrobial activity) Theo phng php hin i ca Vanden Bergher v Vlietlink (1994) tin hnh trn phin vi lng 96 ging, khng sinh kim nh bao gm: Ampixilin, Tetracylin, Amphoterilin B v Nystatin.Cc chng vi sinh vt kim nh bao gm cc nhm: - Vi khun Gr (-): Escherichia coli (ATCC 25922);Pseudomonas aeruginosa (ATCC 25923). - Vi khun Gr (+) : Bacillus subtillis (ATCC 27212); Staphylococcus aureus (ATCC 12222) - Nm mc : Aspergillus niger (439); Fusarium oxysporum (M42). - Nm si : Candida albicans (ATCC 7754); Saccharomyces cerevisiae (SH20) Phn tch v nhn bit cc thnh phn ca tinh du thn r ngi tin bousigon: i vi cht lng d bay hi v nhit si thp nh tinh du th phng php hin i nht phn tch v nhn bit cc thnh phn l phng php sc k kh kt hp vi khi ph. Theo phng php ny, chy theo chng trnh nhit : Nhit u 900C, gia nhit 50C /pht n 2800C trn my GC-6890 MS-5973, chng ti xc nh trong tinh du thn r ngi tin bousigon kh c 45 thnh phn (xem ph lc s 1), chng ti nhn dng c 21 thnh phn l nhng thnh phn c trng lp v ph khi trn 90% so vi ph khi cc cht trong th vin my (xem bng 3.1). Bng 3.1: Cc thnh phn chnh ca Tinh du thn r cy ngi tin bousigon vng Vnh Phc Thi gian Hm trng STT Tn cht lu lng (%) lp(%) 1 4.29 Iso-amylacetate 0.27 90 2 8.11 99 1,8-cineole 6.49 3 9.38 Linalool oxid 0.67 91 4 9.87 Trans-Linalool oxid 0.60 91 5 10.32 97 Linalool 22.31 6 12.43 Borneol L 1.15 90 7 12.80 14,66-terpinen-4-ol 1.1 93 8 13.27 91 -terpineol 3.0 9 13.39 15,56-myrtenal 0.84 97 10 13.45 Myrtenal bicyclo 1.84 96 11 16.83 19,94 perilla ancol 0.41 95 12 17.09 20,14 carvacrol 0.69 93 13 24.40 Piperitenone 0.28 93 14 26.38 12-oxabicyclo[9,1,0]dodeca 1.06 91 15 27.07 Widdrene cyclopvopa[d]naphth 0.50 90 16 27.96 97 -tumerone 4.90 17 28.79 36,48 -germacrone 1.01 99 18 29.90 91 37,94-zerubone 25.15 19 30.08 Drim-8-en-11-all-1-naphthalen 0.57 90 20 30.15 38,47 oxobisabolene 0.34 97 21 35.60 1,2-benzenedicarboxylic axit 0.65 94 Cng 73.83 Kt qu trn cho thy s thnh phn xc nh c chim 73.83%; cn 26.17% trong tinh du cha xc nh c thnh phn. y l mt iu th v i vi tinh du ngi tin bousigon, cn c nghin cu tip tc v n chim trn 1/4 lng tinh du. Khi so snh thnh phn chnh ca tinh du thn r cy ngi tin bousigon trng ti Vnh Phc vi thnh phn chnh ca tinh du ngi tin trng ti Sapa, chng ti thy c s

ging v khc nhau v thnh phn v hm lng cc thnh phn. iu ny c th hin trn bng 3.2. Bng 3.2: So snh mt s thnh phn ca tinh du thn r ngi tin Sapa v tinh du thn r ngi tin bousigon Vnh Phc Thnh phn Loi (%) Tinh du Tinh du ngi tin SaPa Tinh du ngi tin bousigon Vnh phc Linalool 37,94Nerollidol Terpineol tumerone zerubone 12,14 3,0 4,9 11,86 25,15

1,8-cineole 2,23 6,49

23,44 22,31

S khc bit ny c th do phng php iu ch, cng c th do th nhng v kh hu, ma ly mu v loi. Chit chn lc cc cht c hot tnh sinh hc trong thn r ngi tin bousigon. Sau khi chit chn lc cc cht c hot tnh sinh hc trong thn r cy ngi tin bousigon bng cc dng mi n-hexan, diclometan v etylaxetat cho thy trong thn r cy ngi tin bousigon hm lng cc cht khng phn cc hay km phn cc rt thp, 0,088% tnh theo nguyn liu ti; trong khi hm lng cc cht c phn cc trung bnh cao: 0,11% v 0.027% tnh theo nguyn liu mu ti. y cng l iu mong c ca chng ti. Kho st hot tnh khng vi sinh vt kim nh ca cn chit ca thn r ngi tin bousigon Chng ti tin hnh kho st hot tnh khng khun ca mu cc cn chit H, C, E t thn r ngi tin i vi 8 loi vi sinh vt kim nh (VSVK) thuc 4 nhm khc nhau: Gram (+), Gram (-), nm mc, nm men theo phng php th hin i Vanden Bergher v Vliethin Gah. Kt qu th c ch ra trn bng 3.3. Bng 3.3 : Hot tnh khng VSVK ca cc cn chit ca thn r ngi tin bousigon VSVK Mu Cn C Cn H Cn E Vi khun Gr(-) P. E. Arugin coli osa 200 (-) 200 (-) 200 (-) Nng c ch ti thiu (MIC: g/ml) Vi khun Gr(+) Nm mc B. subtillis 100 100 100 S. aureus (-) (-) (-) Asp. niger (-) (-) (-) F. Oxysporum (-) (-) (-) Nm men S. C. cerevis Albicans iae (-) (-) (-) (-) (-) (-)

Du (-) : khng c hot tnh khng vi sinh vt kim nh. Kt qu trn cho cc cn chit t thn r cy ngi tin Bousigon u c hot tnh chng 2 loi vi khun gy bnh ng rut l E.coli gy bnh tiu chy v B.subtullis gy bnh vim trng. Kho st cc cn chit H v C bng SKLM. chng ti tin hnh kho st cn chit H bng SKLM vi h dung mi n hexan/EtOAc, 9/1,v/v; cn C vi h dung mi n hexan/diclometan, 1/9,v/v v hai thuc hin vanilin/H2SO4 1% v FeCl3 1M (pH = 4). Kt qu c ch ra trong bng 3.4 v 3.5.

TT 1 2 3 4 5 6 7 8

Rf 0,0 0,33 0,37 0,46 0,50 0,80 0,83 0,93

Bng 3.4: Kt qu kho st cn H bng SKLM Vanilin/H2SO4 FeCl3 1M Ngui Nng Ngui Nng Vng Nu en Khng mu Nu Xanh sm en Khng mu Xanh m Xanh sm en Khng mu Nu Nu en Khng mu Nu Hng Xanh en Khng mu Xanh m Hng Tm hng Khng mu Khng mu Nu en Khng mu Khng mu en en Khng mu m Bng 3.5. Kt qu kho st cn C bng SKLM Vanilin/H2SO4 FeCl3 Ngui Nng Ngui Xanh en en Khng mu Xanh en en Khng mu Tm Tm hng Khng mu Nu Xanh m Khng mu Khng mu Tm m Khng mu Khng mu Xm Khng mu Hng Hng tm Khng mu

TT 1 2 3 4 5 6 7

Rf 0,0 0,23 0,42 0,46 0,57 0,61 0,75

Nng Khng mu Khng mu Khng mu Xanh m Khng mu Khng mu Khng mu

Kt qu trn cho thy cn chit H phc tp, c 8 thnh phn nhng ch c 2 thnh phn l lacton dng este c Rf = 0,5 v 0,33 xanh en vi FeCl3 1M (pH =4) khi nng. Cn cn C th t phc tp hn, ch c 7 thnh phn v c ti 1 thnh phn hin mu xanh en vi FeCl3 khi un nng, khng mu khi ngui, cc thnh phn ny c th l lacton hoc este. Phn lp cc cht tinh khit trong cc cn chit H v cn C. i vi hp cht c nhit si cao, phng php phn lp tt nht l phng php sc k ct. Hiu qu ca phng php ny ph thuc vo nhiu yu t: t l cc cht cn phn tch trn cht hp ph, hot tnh ca cc cht hp ph, kch thc ct, phng php ra ct v dung mi ra ct Chng ti phn lp cc cht lacton trong cn H chit t thn r cy ngi tin bousigon, kch thc ct 2,5 x 70 cm v dung mi ra ct l n- hexan/EtOAc 9/1, v/v, tc ra 2 ml/pht. Kim tra v thu gom sn phm bng SKLM. Kt qu chng ti phn lp c 4 cht sch k hiu HB1, HB2, HB3 v HB5. Tng t nh trn nhng vi dung mi ra ct l n hexan/diclometan, 1/9,v/v t cn chit C chng ti phn lp c mt cht HB7, c Rf = 0,76 dung mi CH2Cl2/CH3OH, 7/3, v/v. Nh vy chng ti phn lp c 5 cht tinh khit t cc cn chit t thn r cy ngi tin bousigon. Xc nh cu trc phn t ca hp cht HB3 Hp cht HB3, dng du khng mu, Rf =0,45 (n- hexan/EtOAc 9/1, v/v), hin mu xanh en vi FeCl3 1M (pH=4). Ph IR ca HB3 cho thy phn t c mt vng -lactone c v tr , - cha no c C=O 1762 cm-1 v C=C 1681 cm-1; nhm exo- methylene C=C 1641 cm-1 , C-H 954 cm-1 v nhm ete C O-C = 1118cm-1 Ph 1HNMR) thy tn hiu ca cc nhm methyl (H = 0,68 ppm; 0,78ppm; v 0,85ppm) v H ca exo-methylene (H = 4,32ppm; 4,37ppm v 4,77ppm).

Quan st nhng ph 13C NMR cho thy 8 Cacbon olefin vng 107 147 ppm l 4 cp i cacbon olefin lin k c cng cng . Tng s cacbon ca hai ng phn ny l 42. Trong c 8 nhn CH, 15 nhm CH2, 8 nhm CH3). Tt c cc d kin trn cho php ngh n y l cp ng phn epime ca metyl coronarin D ete. iu ny c khng nh thm nh trn ph khi ca HB3 c pic ion phn t ti m/z = 331,7 (M+) , cng vi 1 phn mnh c im ion m/z = 300,7 (M+ CH3OH). Tt c tha mn vi cng thc phn t C21H32O3. S liu ph ca HB3 c ghi trn bng 3.8 v cng thc cu to ca HB3 c ch ra trn hnh.

12 11 1 2 10 4 19 5 H 18 6 20 9 13

O 16 O O 21

14 17 15 8 7

Hnh 3.1. Cng thc cu to ca HB3 metyl coronarin D ete Bng 3.6. S liu ph 13C ca hp cht HB3 S liu ph 13C ca hp cht HB3 Nguyn t C HB3/a HB3/b 39,19 39,35 1 19,231 19,231 2 41,919 41,919 3 32,844 32,798 4 55,300 55,253 5 24,020 23,999 6 37,704 37,704 7 147,731 147,974 8 56,056 56,056 9 39,348 39,197 10 25,327 25,327 11 142,562 142,700 12 124,148 124,079 13 32,844 32,798 14 100,934 100,934 15 169,538 169,538 16 107,249 107,560 17 33,308 33,458 18 21,630 22,540 19 14,005 14,231 20 14,260 14,849 21

Xc nh cu to phn t ca hp cht HB5 Hp cht HB5 l tinh th hnh kim, mu trng v Rf= 0,79 ( n- hexan/EtOAc, 3/7,v/v), hin mu xanh en vi FeCl3 1M (pH = 4) khi un nng. Ph IR ca HB5 cho thy c vng - lacton , khng no (C=O 1751 cm-1, v lin kt i 3 nhm th c C=C 1643 cm1 , C-H =829 cm-1); c lin kt i 2 nhm th dng E ( exo metylen) vi C=C 1603 cm-1, C-1 H 900 cm . Trn ph 13 C NMR v DEPT cho thy r phn t HB5 c 20 nguyn t C, trong c 3 nhm metin olefin vng trng yu 120 145 ppm, 2 nhm metin ca Cacbon bc 3 no vng 55 65 ppm, 6 nhm metylen v c bit nhm exo metylen c C = 108,40 ppm. Khi ph ca HB5 cho pic M+ = 300,6 . T cc s liu trn suy ra cng thc phn t ca HB5 l C20H28O2 v bi lin kt l 7. V phn t hp cht HB5 c 20 nguyn t cacbon, c vng - lacton , khng no, nhm exo metylen v 3 nhm metyl vng trng mnh nn c th suy ra y l diterpen v da vo qui lut u ui v c im cc ph chng ti xy dng khung labdan. Nhng d kin trn cho php khng nh HB5 l Villosin c cng thc nh hnh 3.1. Cc s liu ph 1H v 13C-NMR c ch ra trong bng 3.6. S liu ph HB5 tng ng vi s liu ph ca villosin cng b trong ti liu 26.

Hnh 3.2 . Cng thc cu to ca HB5 - Villosin Bng 3.7. S liu ph 1H v 13C-NMR ca hp cht HB5 V C (ppm) bi, J(Hz) C (ppm) ca H (ppm) tr C ca HB5 ca villosin [26] ca HB5 HB5 1 40,83 40,9 1,00; 1,40 m 2 19,11 19,1 1,49; 1,46 m 3 42,30 42,3 1,51; 1,21 m 4 33,57 33,6 5 54,75 54,8 1,09 dd(2,68; 2,68) 6 23,38 23,4 1,71; 1,38 m 7 36,75 36,8 2,45; 2,08 m 8 149,40 149,4 9 62,22 62,2 2,37 m 10 39,29 39,3 11 136,87 136,9 6,89 dd(10,13; 10,13) H (ppm) ca villosin [26] 1,00; 1,45 1,40; 1,51 1,18; 1,40 1,09 1,71; 1,39 2,08; 2,44 2,37 6,90

12 13 14 15 16 17 18 19 20

120,66 129,55 142,34 69,56 172,31 108,40 33,38 21,93 15,05

120,7 129,6 142,3 69,6 172,3 108,4 33,6 22,0 15,1

6,10 7,14 4,8 4,5; 4,76 0,89 0,84 0,87

d (15,84) br_s d(1,6) d(1,7); d(1,7) s s s

6,11 7,15 4,80; 4,51; 4,76 0,89 0,84 0,87

Xc nh cu trc phn t ca hp cht HB7 Hp cht HB7 l cht rn, mu trng, im nng chy 164oC v Rf= 0,76 (CH2Cl2/CH3OH, 7/3,v/v), hin mu xanh en vi Vanilin/H2SO4 Ph IR ca HB7 OH= 3542.06 cm-1, 3391.40 cm-1 C=O =1710.80cm-1; lin kt i 3 nhm th C=C =1640.30 cm-1 ; C-O-C ca vng piran khng no 1073.52 cm-1. Trn ph 13 C NMR v DEPT cho thy r phn t HB7 c 17 nguyn t C, trong c 10 nhm CH, 3 nhm CH2, 1 nhm CH3 v c 3C khng lin kt trc tip vi H. Khi ph ca HB7 cho pic M+ = 387.4 T cc s liu trn suy ra cng thc phn t ca HB7 l C17H24O10. Qua phn tch ph 13 C NMR ca HB7 thy r 4 nguyn t C ca nhn pyrano-Dglucose trong vng dch chuyn t 70-77ppm, cacbon anome 98.6, H anome c H=4.53ppm v nhm etylencacbinol H=3.06ppm, C=66.99ppm. iu ny cho thy HB7 l 1 glucosit. Ly cng thc phn t ca HB7 ( C17H24O10) tr i phn glycon C6H11O5 ta c phn cn li l C11H13O5. Trong c cc nhm chc: OH, C=O, C=C ba nhm th, C-O-C ca vng pyran khng no, 5 nhm CH, 2 nhm CH2, 1 nhm CH3 v 3 C khng lin kt vi H. Tng s vng v lin kt i trong aglycose l 5. Ph 1 H NMR ca HB7 cho thy c 2 nhm proton olefin l H=7.46ppm H=5.68ppm v c 2 nhm metoxyl H=3.64ppm C=51.02ppm. V c nhm >C=O nn s vng v s lin kt i cn li l 4, c 1 vng piran. Vy c th l vng khng no. T d kin trn chng ti xy dng c cng thc ca aglycon nh hnh
COOCH3

O HO H O*

Hnh 3.3: Cu to ca agycon Ghp vi phn D-glucosit ta c cng thc ca HB7 nh hnh

11 O 4 3 1 O

12 CH3

6 7 8 HO 10

5 9

6' OH O 5' OH 4' 2' OH 3' OH

O 1'

Hnh 3.4: Cu to ca HB7 Cu to ca HB7 th hin r trn ph: IR, FAB-MS, 1H-NMR, 13C-NMR, DEPT, HSQC, HMBC. y chng ta thy r trc ht l lin kt glucosit ca C1, vi C1 qua H th hin trn ph HMBC c tng tc H1, vi C1 v H1 vi C1 v hiu ng thun t ca in t v in t cha chia s ca oxi m cacbon ca nhm >C=O trng thp nht, cn nhm CH v tr 3 trng cao hn H-3=7.46ppm v C-3=151.58ppm. Di nh hng ca in t ca lin kt i m nhm metin v tr 7 cng trng yu: H-7=5.68ppm v C-7=125.55ppm. Cng l lun tng t C-4=110.95ppm v C-8=144.07ppm. Cc s liu 1H-NMR, 13C-NMR ca HB7 ch ra trong bng Bng 3.8. S liu ph 1H v 13C-NMR ca cht HB7 13 1 V tr C-NMR H-NMR C C:ppm H bi 1 95.76 5.12 dd 3 151.59 7.46 br.s 4 110.95 5 34.58 3.10 brdd 6 37.88 3.70 m 7 125.51 5.68 br,s 8 144.07 9 45.88 2.64 t 10 59.35 4.13 br d 3.97 br d 11 166.82 12 51.03 3.64 s 1 98.63 4.53 d 2 73.31 4.71 t 3 76.63 5.03 t 4 69.99 4.46 t 5 77.24 4.7 m 6 60.99 3.06 dd

Nhm CH CH CH CH2 CH CH CH CH CH3 CH CH CH CH CH CH2

J.Hz 6.8; 7.2

6.3; 7.3

7.3 14.5 14.6

8.0 5.4 5.9 5.7 4.9; 5.3

Theo cng thc ny ngoi 5C bt i trong phn glucose cn c 3C bt i trong phn aglycon: C-1, C-5, C-9. Nh vy s ng phn quang hc l rt ln. xt cu dng ca HB7 chng ta xt ph 1H-COSYGP (ph lc 25a, 25b, 25c, 25d). Ph ny cho thy r tng tc ca H-5 vi H-9 (hnh vung ABCD) c J=6.8 Hz v ca H-9 vi H-1 (hnh vung AEFG) c J=7.3 Hz. S tng tc Vicial spin-spin ny c hng s tng tc J ln nn chng

l tng tc axial-axial. iu ny c ngha l cc proton v tr axial c hnh 3.4a v 3.4b. Nh th HB7 c th dng thuyn (hnh 3.4a) v dng gh (hnh 3.4b). Chng ti cha c s liu khng nh dng no nhng c th suy on HB7 dng 3.4a v dng ny lin i dng E, dng ny 2 nhm th ln nm v tr trans vi nhau nn c s nh hng hiu ng khng gian l t hn, do bn hn v d tn ti hn. Vy HB7 chnh l geniposide.
OH 6' H OH 6 5 9 H H3C O O 4 H 3 H 1 7 10 8 H 1' O 2O O 2' OH 3' OH 5' 4' OH

Hnh 3.4a
OH H 7 6 5 H H 4 H3C 11 O O 8 10 H 9 H 3 1 O 2 O 1' O 2' OH 3' OH HO 6' 5' 4' OH

Hnh 3.4b Kho st hot tnh chng ung th ca hp cht HB5(villosin) phn lp c t thn r ngi tin bousigon. Villosin c nhiu cng trnh nghin cu hot tnh chng ung th. Cc loi ung th nh: ung th gan Hep-G2, ung th phi A 549, ung th thn kinh SK N SH, ung th v MCF 7, ung th c t cung HeLa [18, 33] c th nghim. Nhng cc dng ung th phi Lu, ung th c vn tim RD cha c th nghim. Chng ti kho st hot tnh gy c t bo ung th ca villosin phn lp c t thn r cy ngi tin bousigon Vnh Phc vi 3 dng t bo ung th: ung th gan (Hep-G2), ung th phi (Lu) v ung th c vn tim (RD) theo phng php ca Skehan v cng s (1993), Likhiwitayawuid v cng s (1993); cc phng php ny hin nay ang c p dng ti vin ung th quc gia ca M (NCI). Kt qu c ch ra trong bng 3.9 v bng 3.10. Bng 3.9. Hm lng t bo ung th sng st sau khi th Cc t bo cn sng st CS (%) K hiu mu Hep-G2 Lu RD DMSO 100,00,0 100,00,0 100,00,0 Chng (+) 2,20,0 1,80,04 0,300,03 HB5 0,00,0 8,40,10 0,000,00

TT 1 2 3

Kt lun HB5 dng tnh tnh c 3 dng

TT 1 2

Bng 3.10. Cng gy c t bo ung th Nng gy c IC50 (g/ml) K hiu mu Hep-G2 Lu RD Chng (+) 0,257 0,312 0,188 HB5 0,788 0,969 0,675 0,325 0,675 0,476

Kt lun HB5 dng tnh c 3 dng

T kt qu bng trn cho thy, mu HB5 (villosin) m chng ti phn lp c c tc dng gy c t bo ung th mnh i vi c 3 dng ung th ngi l ung th gan Hep-G2 ( IC50 0,78 microgam/ml), ung th phi Lu(IC50 0,96 microgam/ml) v ung th c vn tim RD ( IC500,67 microgam/ml). KT LUN Ti liu th gii v nghin cu ngi tin bousigon hu nh cha c, chng ti l ngi u tin nghin cu ngi tin bousigon (Hedychium bousigonianum Pierr ex Gagn) trng ti Vnh Phc v c kt lun sau: 1. xy dng quy trnh ct tinh du thn r ngi tin bousigon cho hiu sut tt 0,25% tnh theo nguyn lu kh. 2. phn tch thnh phn tinh du thn r ngi tin bousigon v nhn bit c 21 thnh phn trong s 45 thnh phn ca tinh du, c 2 thnh phn c hm lng ln nht l: Linalool: 22.31% v 37,94-zerubone: 25.15%. 3. Xy dng quy trnh chit phn lp cc cht trong thn r ngi tin bousigon theo phn cc tng dn ca dung mi n-hexan, diclometan v etylaxetac v kho st hot tnh khng vi sinh vt kim nh ca cc cn chit. Kt qu cho thy cc cn chit u c tc dng chng vi khun ng rut l E.coli v B.subtillis. 4. kho s cc cn chit bng sc k lp mng v phn lp c 5 cht tinh khit t cn chit n-hexan v diclometan bng sc k ct. 5. xc nh c cu trc phn t ca 3 trong 5 cht phn lp c bng cc phng php ph hin i l villosin, epimer ca ete metyl coronarin D v geniposide. Kho st hot tnh gy c t bo ca villosin i vi 3 dng t bo gy ng th gan Hep-G2, ung th phi Lu v ung th c vn tim RD v nhn thy Villosin c hot tnh gy c t bo mnh i vi c 3 dng ung th trn.

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