N MN HC................................................................................................2 LI M U........................................................................................................2 TNG QUAN........................................................................................................3 KT LUN..........................................................................................................40 TI LIU THAM KHO........................................................................................

40

 N MN HC ti : Tng quan v thnh phn ha hc v hot tnh sinh hc ca cy Bch Trinh Bin (Hymenocallis litoralis) thuc h Thy Tin ( Amaryllidaceae) LI M U t nc Vit Nam vo vng kh hu nhit i gi ma, cho nn thin nhin ban tng cho chng ta mt thm thc vt phong ph trong c hn 3.800 loi cy c th dng lm thuc cha bnh phc v sc khe nhn dn. Trong 200 loi bo ch thnh thuc ang c cc thy thuc ng y dng cha bnh. Khong 400 loi trong nhn dn dng m chng ta gi l thuc Nam. Cn hn 3000 loi ch trong cc cng ng dn tc thiu s bit v s dng mt t cha bnh, cn li cha c iu kin nghin cu y . Thc vt h Thy Tin vi s a dng v chng loi l ngun nguyn liu ln cc hp cht t nhin. Dch chit ca chng thng c hot tnh sinh hc cao. V vy t lu, thc vt h Thy Tin c dng lm thuc cha bnh [1]. Vit Nam, mt s loi trong h thc vt Thy Tin c nghin cu v phn lp cc hot tnh sinh hc. Trong thi gian gn y, hu ht mi ngi u bit n cy Trinh n hong cung vi tc dng iu tr cc bnh v ung bu, u x rt c hiu qu. Cy Bc trinh bin cng l mt loi thuc h Thy Tin di thc vo nc ta, thng c trng lm cnh v gn y cng c nghin cu cc hp cht c hot tnh chng ung th, gy c t bo, ngn chn s sao chp ca virut HIV.Vi cc c tnh qu bu trn, em lm ti

TNG QUAN

1.

Gii thiu v thc vt h Thy tin (Amaryllidaceae) H Thy Tin (danh php khoa hc: Amaryllidaceae) l mt h trong

thc vt c hoa. H ny ch yu l cc loi cy thn tho sng lu nm, mc ra t thn hnh, thng thng c hoa sc s. H ny cha khong 60 70 chi, vi khong trn 800 loi. Mt vi chi hay c trng trong vn, chng hn nh hoa loa kn , hu thuc chi Amryllis, cy trinh n hong cung thuc chi Crinum [2]. Tr mt s t ging mc min n i, cn li cc i din chnh ca h ny ch yu phn b cc min tho nguyn ca vng nhit i v cn nhit i ca c hai bn cu [3] L cy xp lm 2 hng, mc xung quanh thn cy. Hoa sc s thng mc t mt tn ca gc l thun di. Cung hoa c bc bi cc l bc c th c mu sc s. Hoa c 6 cnh bng hoc tng t nhau v thng c 6 nh, bu nh c 3 ngn. Qu nang, thnh thong c qu mng. Mt s loi c bit c ht c cnh mng v mt s khc c cc ht nhiu mu quanh gc, khng th bo qun c v phi trng ngay khi va chn [4].

2. 2.1.

Gii thiu v thc vt chi Bch trinh (Hymenocallis) Gii thiu chung

2.1.1. V tr ca chi trong phn loi thc vt Chi Bch trinh thuc : Gii : plantae - thc vt

Phn gii : tracheobionta Ngnh : spermatophyta

- thc vt bc cao - ht kn

Division : magnoliophita mc lan - cy c hoa Lp : liliopsida Phn lp : liliiae Order : liliales H : Amaryllidaceae - thy tin - mt l mm

2.1.2. c im hnh thi chung ca cc cy thuc chi Hoa ln, mi thm, hoa hnh ming ni la ph hp loi su bm th phn. C hnh trn hoc thun, c bao ph trong 1 ci v, r dy mu trng. C ny c th ln n 15cm trong nhng loi ln ca chi nhng nh hn loi Ismene v Leptochiton. Ismene v Leptochiton rng l sm. Hymenocallis vng nhit i l thng xanh trong khi vng kh cn thng sm rng l. Cc l khc nhau v s lng v chiu di v thng sp xp theo hng tuy nhin H. eucharidifolia c s sp xp theo hnh hoa th. L thng rng khong 5-7mm, khng cung, thng hng v c mu lc lam. Ring H.graminifolia l rng 15cm, c cung, cy H.speciosa l hnh mi mc v mu xanh l ti sng. Trong Ismene gc l hnh g , cao ln n 50cm. Hoa c th c 1 hoc nhiu bng mc trn thn cy ni ln t bn trong l. Thn cy b kp gia hai vin l vng chc, chng c sinh ra vo u ma sinh trng hoc sm hn.

2.2.

Phn b

Hymenocallis c khong 70 loi phn phi trn ton ng Nam Hoa K, Mexico, Trung M, Ty n v min bc Nam M. Mexico l khu vc c s lng ln nht cc loi v khu vc c s lng loi ln th 2 l M. C 3 loi l loi c hu ca Nam M l H.littoralis, H.pedalis v H.tubiflora. Cc loi c chia thnh 6 lin minh da trn nhng c tnh hnh thi hc ca Traub (1962).

2.3.

Mt s loi thuc chi ny

Hymenocallis acutifolia c ngun gc t Mexico. N c tn l hp v hoa n t cui ma h qua ma thu (hnh 2.3.1).

Hnh 2.3.1 Hymenocallis acutifolia Hymenocallis azteciana ngun gc t ty Mexico (hnh 2.3.2).

Hnh 2.3.2: Hymenocallis azteciana Hymenocallis caribaea variegata c tn l xanh xm hp dn, rng v vin mu trng kem (hnh 2.3.3).

Hnh 2.3.3: Hymenocallis caribaea variegata Hymenocallis eucharidifolia l c ngun gc t Mexico. Mt bng hoa xinh p trn mt cy p. N r cng mt lc nh H. sonorensis. (hnh 2.3.4)

Hnh 2.3.4: Hymenocallis eucharidifolia

Hymenocallis durangoensis l mt loi t Mexico. (hnh 2.3.5).

Hnh 2.3.5: Hymenocallis durangoensis Hymenocallis harrisiana l mt loi rng l t Mexico. Cc l hnh di c mu luc lam, v n xut pht t mt khu vc kh cn. Hoa c mt vng ho quang nh c mi thm (vng min).Mi hoa ch n trong thi gian mt m (hnh 2.3.6).

Hnh 2.3.6: Hymenocallis harrisiana Hymenocallis liriosme pht trin trong trung tm Lady Bird Johnson Wildflower. N c ngun gc Texas cng nh mt s cc tiu bang khc pha Nam. nh ca cy n hoa c chp vo thng 4 nm 2004 bi Lee Poulsen. Thm mt hnh nh c ca cy ny v ht ging (hnh 2.3.7b) Texas. Hai trong s nhng ht ging ny mm (hnh 2.3.7a).

Hnh 2.3.7a: Hymenocallis liriosme

Hnh 2.3.7 b: c ca loi Hymenocallis liriosme Hymenocallis littoralis l mt loi c mu xanh mnh m, ngun gc t Mexico v Guatemala. Chng pht trin mnh m trong nc vng kh hu m p. N cn phi c bo v trong ma ng lnh. Nhng bng hoa trng ng yu c cnh hoa di v hp np ng sau mt cc trung tm. Hoa mc trn mi 70-90 cm ca thn (hnh 2.3.8)

Hnh 2.3.8 : Hymenocallis littoralis

Hymenocallis maximillianii (syn. hymenocallis maximiliani) c ngun gc t Mexico. N c l nh gn v hp, dng ng v hoa duyn dng cn i (hnh 2.3.9) .

Hnh 2.3.9 : Hymenocallis maximillianii Hymenocallis occidentalis c ngun gc t pha ng M v n hoa vo cui ma h. Ty thuc vo cy v mi trng pht trin m l c th vng mt, ging nh mt Lycoris, hoc khi chp bc nh hin ti vo thi im n hoa (hnh 2.3.10).

Hnh 2.3.10 : Hymenocallis occidentalis Hymenocallis riparia c ngun gc t Mexico. l mt cy nh vi tn l hp (hnh 2.3.11).

Hnh 2.3.11: Hymenocallis occidentalis Hymenocallis Sister of Tropical Giant c tn l rng v xm mu xanh thay v mu xanh m (hnh 2.3.12).

10

Hnh 2.3.12: Hymenocallis Sister Hymenocallis sonorensis c tm thy ven sui v khp thung lng Sonora, Mexico. Bc nh u tin chp thng chn nm 2003, nhng cc cy ny tng trng thm ch cn ln hn v c vi cnh hoa vo thng Chn nm 2004 (hnh 2.3.13).

Hnh 2.3.13: Hymenocallis sonorensis Hymenocallis traubii l mt loi n hoa vo ma xun c ngun gc t Florida. N l mt cy nh vi nhng bng hoa ln (hnh 2.3.14).

11

Hnh 2.3.14: Hymenocallis traubii

-

Hymenocallis 'Tropical Giant' c l rng, bng, cng, mu xanh m v

hoa c o cn i (hnh 2.3.15).

Hnh 2.3.15: Hymenocallis 'Tropical Giant' 2.4. Loi Vit Nam

Vit Nam, theo tc gi Ng Tin Bn v Nguyn Th ,chi ny c 2 loi. l H .littoralis.Salisb (Bch Trinh bin) v H. speciosa.Salisb (Bch Trinh p).[2]

12

Bch trinh bin c tn khoa hc l Hymenocallis littoralis Salisb,thuc: Gii : Plantae (Thc vt) H : Amaryllidaceae(Thy Tin) Chi : Hymenocallis (Bch Trinh) Loi : H.littoralis Salisb

Ngnh : Angiosperm (Ht kn) Lp B : Monocotyledon ( 1 l mm) : Asparagales

Bch trinh bin (H. littoralis) (hnh 2.4) l cy c nhiu nm, thn hnh, hnh cu, ng knh khong 5-10cm, c o bao mng bn ngoi. L xp 2 hng gc, khng cung. Phin l hnh di hoc hnh kim,kch thc khong 60cm x 4cm. L dy,mu xanh m, lng bong, gn gia mt trn lm thnh hnh mng, mt di li ln thnh g rng, c nhiu gn dc song song khng r.Chp l nhn, gc m ly nhau. Cm hoa tn,gm 7-15 hoa,trn mt cung hoa chung,di 60-80cm mu xanh,thng ng,dp v c.L bc tng bao dng mo,kch thc 5-8cm x 34cm, mu xanh mng, 2 chic tn ti.Hoa u, lng tnh, mu trng, thm. Gc mi hoa c mt l bc nh mu trng xanh, mng, cung ngn. Bao hoa 6 mnh, dng trng, phn di dnh vo nhau thnh ng, di 8-12cm. Phn trn 6 thy hnh di, gn bng nhau, xp 2 vng.Cc thy vng ngoi dng m nhn. Nh 6, gc ch nh dnh co nhau thnh dng phu. Pha di mu xanh cao 2,5-4cm, mng, mp cc rng nhn xen k,nh hng ng bao hoa. Phn trn dng si mu xanh di 4-5cm, ri nhau. Bao phn nh gc, di, hng trong v m bng khe dc. Bu h di c 1cm,gm 3 , mi mt non nh gn y , vi nhy di t 10-11cm dng si mnh pha di mu trng pha trn mu xanh. Qu mng ng knh 1,2-1,5cm nt bn,c 1 ht.Ht to v rt dy, xp,ni nh nc bao ly phi nh.

13

Hnh 2.4: Hymenocallis litorallis Vi nt c bn v c im sinh thi,phn b v gi tr s dng Bch trinh bin ra hoa vo ma h, ra qu vo ma thu, ma ng, l loi mc t nhin Chu M, sng nhng ni t m nh ven sui, ven song, ven rng. Chng c a vo Vit Nam trng trong vn hoa, chu hoa, nhn ging bng thn hnh con. Bch trinh bin phn b khp cc chu lc trn th gii trong nhiu nht l Bc M v Trung M.Ngoi ra chng cn c mt Chu Phi,Chu i Dng mt s nc Chu nh Trung Quc, n ,Malaixia, Indonexia v Vit Nam. Hin nay,Bch trinh bin c trng ch yu lm cy cnh.Tuy nhin trong mt s nghin cu gn y pht hin trong Bach trinh bin c mt s ankaloid qu him v c th s dng lm thuc.Bi vy vic s dng Bch trinh bin nh l dc liu ang c nghin cu trong thi gian gn y. 3. Gii thiu v cy Bch trinh bin (Hymenocallis litoralis) 3.1.M t cy

14

Cy thn c sng lu nm do c c ln. L mc st t, nhiu, dy c, dng thun di, mu xanh bng, nhn. Nhng bng hoa mu trng p c nhng cnh hoa di vi nhng np un hp ng sau i trung tm. Cc bng hoa pht trin trn cc pht hoa di t 70 n 90 cm [5]. ng hoa di 14 n 17cm hoc hn. Qu nang.[6] 3.2. Phn b cy

Cy Bch trinh bin c ngun gc cc vng ven bin min nam Mexico v Trung M, v hai qun th hoang d c bo co c tm thy trn b bin pha Ty ca Florida [6]. Chng pht trin rt tt nhng vng nhit i v cn nhit i vi khi hu m p. 3.3. Thnh phn ha hc ca cy

Nghin cu thc vt hc ca c v hoa ca cy Bch trinh bin Ai Cp c thc hin cho kt qu chit tch vi 4 nhm alkaloid: lycorine (1), hippeastrine (2), 11-hydroxyvittaine (3), v (+)-8-O-demethylaritidine (4); v 2 cht flavonoids: quercetin 3-O-glucoside (5), v rutin (6). Cc thnh phn d bay hi ca cy hoa c phn tch ln u tin bng GC/MS cho thy c 26 hp cht bit (Bng 1). T , hot tnh khng vi sinh vt trong dch chit ca hoa cy Bch trinh bin c pht hin. [7]
Me N 11c
12 1 3a 5 5a

H HO O H O 1 N O O

11a 9

11b

OH

O O 2

15

O O

10a

OH OH H N
MeO HO
9 10a

1 10b

11 4a

OH
12

6a

H
8 6a

N5

4
OH

OH HO HO HO
7 9 10 4

OH

HO
1''

O
3' 4'

OH O
2 3 1'

OH
OH
O

O O
1'''

1''

OH

OH

HO HO

OH

HO OH 6

OH

Bng 1: Cc thnh phn d bay hi chit tch t hoa cy Bch trinh bin St t 1 Citronellal Cht CT PT C10 H18 2 Hydroxycitronellel O C10 H20 3 4 5 6 Farnesene Cyclohexanone 6-Methylhept-5-en-2one Geranyl acetone O2 C15 H24 C6H O C8H
10 14

Mr [D a] 15 4 17 2 20 4 98 12 6 19 4
16

O C13

H22

Methyl 4methoxycinnamate

O C11 H12 O3 C15 H24 C18 H30 O C17 H34 O2 C18 H36 O2 C16 H32 O2 C19 H34 O2 C20 H36 O2 C18 H32 O2 C22 H46 C15 H26 O C21

19 2 20 4 26 2 27 0 28 4 25 6 29 4 30 8 28 0 31 0 22 2 32
17

8 9

Germacrene A Farnesyl acetone C

10

Methyl palmitate

11

Ethyl palmitate

12

Palmitic acid

13

Methyl linoleate

14

Ethyl linoleate

15

Linoleic acid

16 17

Docosane Farnesol 2

18

3-Methylbutyl

hexadecanoate 19 Geranyl linalool

H42 O2 C20 H34 O C23 H48 C24 H50 C25 H52 C26 H54 C28 H58 C29 H60 C31 H62 O

6 29 0 32 4 33 8 35 2 36 6 39 4 40 8 45 0

20 21 22 23 24 25 26

Tricosane Tetracosane Pentacosane Hexacosane Octacosane Nonacosane Palmitone

Cc cht trn c chia thnh 3 lp cht ln, trong monoterpene chim 9.12%, sesquiterpene chim 2.08%, diterpene alcohol chim 0.8%, hp cht benzene chim 0.19%, acid hoc cht bo no bo ha chim 13.08%, acid hoc cht bo no khng bo ha chim 21.35%, hydrocacbon mch thng chim 29.7% v cc cht khc chim 2.18%. Cc thnh phn chnh nh tricosane chim 12.16%, pentacosane chim 10.64%, methyl linoleate chim 8.45%, palmitic acid chim 8.3%, citronellal chim 7.12% v linoleic acid chim 7.1%. Bng 2: Cc lp cht chnh v cc thnh phn chnh trong dch chit hoa cy Bch trinh bin Lp cht Hm Cht chnh Hm
18

lng (%) 1. Monotetpene hydrocarbon Alcohol Aldehyde Ketone Tng 2. Sesquiterpene Hydrocarbon Alcohol Aldehyde Ketone Tng 3. Diterpene alcohol 4. Hp cht benzene 5. Chui hydrocarbon 6. Acid/este no bo ha 7. Acid/este no khng bo ha 8. Cc cht khc Tng hydrocarbon Tng hp cht cha oxi Tch chit c Phn khng tch c 8.48 0.64 9.12 0.59 0.22 1.27 2.08 0.8 0.19 29.7 13.09 21.35 2.18 30.29 48.22 78.51 21.49 Citronellal Geranyl acetone Germacrene A Farnesol 2 Farnesyl acetone C Geranyl linalool Methyl 4methoxycinnamte n-tricosane Palmitic acid Methyl linoleate Palmitone

lng (%) 7.12 0.64 0.32 0.22 1.27 0.8 0.19 12.1 6 8.3 8.45 1.53

mt s cng trnh nghin cu khc cng pht hin ra mt vi thnh phn khc ca cy Bch trinh bin c trng Bangkok, Thi Lan nh littoraline (7), tazettine (8), pretazettine (9), macronine (10), homolycorine (11), lycoramine (12), haemanthamine (13), vittatine (14), 5,6-hihydrobicolorine (15), secoisolariciresinol (16). [8]

19

3 2 1 4a

OMe H Me N O 8 OH

O 11 O
10

12 12a

12b 6a 8a 8

OH Me N H
6

O O

OMe Me
O

OMe

O O 9 O OH

H N O 10 O H

Me

MeO MeO

Me N H H O

H O

11

MeO MeO

Me N H H

H O

MeO MeO

Me N H H

H O

H O MeO N 14

OH H

12

OH

13

OMe

Me

20

OMe O H O 15 N OH
O H O 16 N

OH

O O 17 N Me

MeO MeO

CH2 OH CH2 OH

OMe 18
4.

OH

Cc nghin cu v hot tnh ca h Thy tin (Amaryllidaceae)

v cy Bch trinh bin (Hymenocallis litoralis) 4.1. Hot tnh ca h Thy tin

4.1.1. Hot tnh chung Cc cy thuc h Thy Tin c c cha rt nhiu alkaloid. Nhng alkaloid ny c c nhng cht c li v c hi cho con ngi. Cc cht chit sut t h thc vt ny Nam Phi c cha nhng cht alkaloid c, khi ung vo c th gy o gic, thm tr dng qu liu 1 lng nh cng c th gy ra t vong. Nhng cy thuc h Thy Tin c s dng lm thuc t hng nghn nm, nhng ngi Hy Lp c i bit gi tr cha bnh ca chng. Cc cht chit sut t c ca cy h ny c nghin cu hot tnh khong gn 200 nm. Lycorine l alkaloid u tin c c lp v c nghin cu vi hot tnh chng ung th.

21

H HO O H O 1 N
O

OH OH

OH NH

OH OH

OH NH

OH

OH HO OH

OH HO OH

O
OH NH

OH NH

OH

Trong hai thp k qua th cc hp cht lycoricidine (2), narciclasine (3), pancratistatin (5), v 7-deoxypancratistatin (4) c phn lp kim chng thy hot ng chng ung th v tng hp bi mt nhm nghin cu. Cu trc ha hc, hot tnh sinh hc, cng nh phng thc tng hp cc ankaloid ca h Amaryllidaceae tng c nghin cu nhiu. Cho ti nay th cc alkaloid ny ch c sn trong t nhin vi s lng nh, tng lai n s ng vai tr l ngun ch yu cha ung th. C lp vi s lng ln cc alkaloid ny l khng thc t. Ngoi ra cng c trng hp cho vic tng hp hoc bn tng hp cc alkaloid ny, dn xut ca chng v cc tin cht tim nng. Mt s alkaloid
22

c cng th hin kh nng chng glycosidic (khng virut) do c s ging nhau gia hp cht oxi ha ca chng vi cc loi ng t nhin. Sau y l qu trnh tng hp narciclasine, ent-7-deoxypancratistatin bng ba phng php tip cn khc nhau. Tt c u tng hp bt u t cc biooxidation ca mt hp cht thm. Nghin cu v cc alkaloid ca h Thy tin bt u t nm 1877 vi s phn lp c Lycorine 7 t Narcissius pseudonarcissus, k t nhm hp cht t nhin ny cng hp dn bi hot tnh chng ung th v khng virut. Cho n nay c khong 150 loi cy ca h ny thuc vo 36 chi c kho st v alkaloid. Cu trc ca cc alkaloid h Thy tin tuy khc nhau nhng u bt ngun t 3 nhn c bn l: N-(3,4- dioxybenzyl)- 4oxy-phenetylamin Pyrrolo [d,e] phenanthridin 5, 10b- etanophenanthridin Bng cch bin i cc nhn ny nh oxi ha, dehidro ha, dehidrat ha, ng vng, metyl ha O- v N-, cng hp v tch loi m cc kiu alkaloid khc nhau c hnh thnh.

4.1.2. Mt s ankaloid c phn lp Mt s alkaloid c tm thy trong chi Bch trinh nh: Tazettine, Lycorine, Pseudolycorine, Adenosine, Amisine, Pancratistatine, Narciclasine. Hp cht Pancratistatin phn lp c t Hymenocallis littoralis c kh nng gy cht t bo ca khi u c tnh ngi [14]. Kt qu nghin cu ch ra rng Pancratistatin c hiu qu gy cht t bo ca vi loi ung th. Ngi ta nh gi hiu qu ca Pancratistatin thuc ngoi vi ca t bo mu n nhn t
23

15 bnh nhn mi b bnh bch cu n nhng bnh nhn c s quan thip ca phng php chun on lm sng. Kt qu Pancratistatin gy cht t bo ung th bch cu [15]. Trong sut qu trnh nghin cu hn ch s sao chp nghch o ca virut HIV, gy c t bo v chng st rt ca cc hp cht t thc vt, alkaloid chit xut t loi Hymenocallis littoralis c kh nng gy c t bo. Littoraline 8 c kh nng km hm s sao chp nghch o ca HIV, Lycorine 7 v Haemanthamine 9 c kh nng gy c t bo [16].
OH HO H O H O N

OMe

OH NMe O O H

7 Lycorine
OMe

8 Littoraline

O
H NMe O O O OH

OH O O N

CH3

10 Tazettine.

9 Haemanthamine

23 dn xut Lycorine v cc hp cht t nhin c cu trc lin quan n Lycorine c kim tra v kh nng ca chng trong vic hn ch sinh tng hp vitamin C ( acid ascorbic) trong ca khoai ty [17]. Lycorine 7 c ni l km hm qu trnh tng hp protein v DNA t bo ca chut v trong th nghim pht trin cy ghp khi u xng c trng chut. Trong th nghim Lycorine c kh nng lm gim t bo ung th.
24

Lycorine- 1- O- glucoside c kh nng l mt tc nhn kch thch min dch. Lycorine c tc dng km hm s pht trin v phn chia t bo thc vt bc cao, to v men. Lycorine-1-O-glucoside cn c kh nng kch thch r cy pht trin. Lycorine c kh nng chm dt s tng hp cc virut bi lit, tin thn ca virut bi lit v men polipepted v c kh nng chng virut. Lycorine cn km hm s tng hp protein trong t bo nhn in hnh bng cch cn tr to thnh lin kt peptid. Cc alkaloid cng c hot tnh tng t l Dihydrolycorine, Pseudolycorine, Narciclasin, Haemanthamine v Pretazettine. Cc Alkaloid ny lm ngng s pht trin ca t bo Hela, ngn chn s tng hp ca t bo c trng, lm chm s tng hp DNA [18]. Hippadine 11 gy ra s km hm thun nghch i vi kh nng sinh sn ca chut c [18].
OH

OMe
N

O O

N Me

11 Hippadine.

12 Galanthamine

Tnh cht gim au ca cc alkaloid h Thy tin c cho l c s tng t gia khung ca n vi khung ca morphine v codeine. Cc alkaloid thuc nhm pyrrolo[de]phenanthridine nh galanthine, nhm lycorenine v pretazettine c tnh u vit hn cc alkaloid nhm dibenzofuran nh galanthamine 12 v nhm 5, 10b-ethanophenanthridine v t c hn [18]. Nhng nghin cu sm ch ra rng mt s alkaloid c kh nng lm gim huyt p tm thi . Sau cc nh nghin cu khng tit l tm quan trng trng ca s nh hng ti huyt p ca cc Amaryllidaceae alkaloid. Tuy nhin , (+)- narwedine, galanthamine, epi- galanthamine c tc dng ln
25

hiu qu lm gim huyt p chut. iu th v l ngi ta cn ch ra kh nng khng emzym cholinesteraza ca galanthamine, n c kh nng khuch i h thn kinh tt hn epi- galanthamine l mt cht khc c tc dng dc l rt ng ch [18]. Quercetin- 3- O- glucoside 6 l mt flavonoid c phn lp t h Thy tin c kh nng gy c t bo, gy c thc vt, chng vi trng v tc dng chng oxy ha [19]. Cc hp cht Narciclasine 13, Lycoricidine 14, Pancratistatine l cc Isocarbostyril Alkaloids ca loi Narcissus c tim nng gy c t bo ung th v c hot tnh chng ung th in vivo vi chut M-5076 v bnh bch cu P388 [20].
OH OH H O O O NH OH

OH OH H O O O NH OH

13 Narciclasine

14 Lycoricidine

4.2.

Hot tnh ca cy Bch trinh bin

4.2.1. Hot tnh chung Chi Bch trinh c nghin cu ln u tin vo nm 1920 v cho kt qu l phn lp c hp cht lycorine. Mc d c mt lot cc tc ng sinh hc km theo cc ankaloid ca chi ny nhng n vn cha c khoa hc quan tm cho n nm 1993 khi Pancratium littorale Jacq phn lp c ankaloid pancratistatin c hot tnh chng ung th.
26

T lu,trong dn gian, dch chit t l cy Trinh n hong cung (Crinum latifolium ) mt loi thuc h Amaryllidaceae vi Bch trinh bin- c s dng cha ung th d dy,ung th t cung v ung th tuyn tin lit. Theo nhng kho st gn y, dch chit ny giu cc ankaloid c kh nng gy c t bo v c ch kh nng pht trin ca cc t bo ung th. Khi nghin cu v cc ankaloid chit xut t loi H.littoralis ,Long-ze Lin cng cc cng s ti Trng i hc Illinois pht hin ra rng dch chit t thn hnh ca loi ny c hot tnh sinh hc cao c bit l kh nng gy c t bo,tr st rt v c ch qu trnh sao chp ca virus HIV.[3] Cng trong thi gian trn, G.Pettit cng cc cng s trng i hc Arizona vi s gip ca NCI tin hnh nghin cu c lp v ch ra rng: dch chit phn thn hnh t cy H.littoralis c kh nng c ch s pht trin ca t bo ung th mnh. Bi vy mt s hp cht c chit xut t H.littoralis nh Pancratistatin, Lycoridine, Narcislasine v cc dn xut bn tng hp ca chng ang c nghin cu s dng lm thuc cha ung th. [4].Nghin cu su v hot tnh cha ung th, nm 2008 cc nh khoa hc B tin hnh th in Vitro cc hot tnh ny.Kt qu thu c kh lc quan khi cc hp cht t nhin nh Pancratistatin, Lycoridine, Narcislasine c hot tnh mnh i vi cc dng t bo th trong khi cc hp cht bn tng hp li c hot tnh rt cao,hn hn cc loi thuc cha Ung th ang dng nh Cisplatin,Doxorubicin.[5] Comp ounds Cancer Cell Lines Leu kem ia P38 8 Pan cre as BX PC B re as t M C C N S S F 2 L u n g N Col on KM 20L 2 Pr ost ate Du 14
27

F7 Narcic lasine 0.04 2 0.0 11 0. 0 1 0

6 8 0. 0 1 0

S C 0 . 0 2 7 0.0 11

0.0 11

Lycori cidine

0.06 5

0.2 4

0. 1 6

0. 4 1

0 . 1 8

0.2 9

0.1 7

Pancra tistatin

0.05 2

0.0 61

0. 0 7 1

0. 0 4 3

0 . 0 9 8

0.0 77

0.0 49

Transdihydr o Narcic lasine Transdihydr o Lycori cidine

0.00 78

0.0 39

0. 0 1 7

0. 0 6 5

0 . 0 3 0

0.0 49

0.0 21

0.09 9

0.1 6

0. 1 2

0. 2

. 0 . 1 5

0.1 7

0.1 4

Bng 1: Bng gi tr IC50(M) cc ankaloid chit xut t H.littoralis

28

Sau nhng tn hiu kh quan ca kt qu th in vitro mt s dng t bo ung th,mt s hp cht c em i th in vivo v cc c tnh bn trng din v trng din.Pancratistatin v Narciclasine l 2 hp cht cho kt qu tt nht.Tuynhin cc hp cht ny li km tan trong nc bi vy s gy kh khn trong cc trng hp dng tim tnh mch.c tnh ny c th tng ln trong cc dung mi hu c nh Dimethylformamide, dimethylsulfoxide hay cc ru mch thng t cacbon nh MeOH,EtOH nhng khi s lm gim hot tnh ca thuc v xut hin thm cc tnh cht khng mong i.Do xu hng ngy nay ang l bn tng hp cc dn xut d tan hn ca chng v c hot tnh mnh hn lm thuc iu tr ung th.Trong thi gian gn y Pancratistatin v Narciclasine ang c s dng nh l cc hp cht tin thuc v thu ht s quan tm ,nghin cu ca th gii. Ngoi cc hot tnh tuyt vi k trn,nm 2008,Khi nghin cu v thnh phn ha hc ca dch chit t hoa H.littoralis cc nh khoa hc Ai Cp m ng u l Shawky Eman pht hin ra hot tnh khng khun i vi mt s dng vi khun gram (+) nh Staphylococcus aureus v gram(-) nh Pseudomonas aeruginosa.Hot tnh ny cn cao hn so vi mt s thuc khng sinh,khng khun ang dng nh Cephalosporin, Ciprofloxacine. 4.2.2. Mt s ankaloid c phn lp Trong qu trnh phn tch cc hp cht c trong c ca cy Bch trinh bin, ngi ta phn lp c Lycorine (1) , hippeastrine (2) , 11 hydroxyvittatine (3) , v (+)-8-O-demethylmaritidine (4) . Ngoi ra, hai cht flavonoid ni ting c phn lp t nhng bng hoa ca cy ny l : quercetin 3'-O-glucoside (5) v rutin (6).

29

H HO O H O 1 N
12

Me N 11c O O
9 11a 11b

3a 5 5a

OH

O O 2

O O

10a

OH OH H N
MeO HO
9 10a

1 10b

11 4a

OH
12

6a

5
8 6a

H 4

N5

OH

OH
HO O

OH

HO HO HO
7 9 10

1''

O
3' 4'

OH O
4 2 3 1'

OH
OH
O

O O
1'''

1''

OH

OH

HO HO

OH

HO OH 6

OH

Dch chit ether ca H. littoralis cho thy hot tnh khng khun i vi Staphylococcus aureus Gram dng v Gram m, Pseudomonas aeruginosa, nhng khng c hot ng chng li Escherichia coli v Candida albicans ( Bng 3 ). Tng ng vi nng c ch ti thiu (MIC) v nng dit khun ti thiu (MBC) gi tr l 15,6 v 62,5 mg / ml (S. aureus), v 250 v 500 mg / ml (P. aeruginosa), tng ng, DMF v ampicillin lm kim sot tiu cc v tch cc, tng ng.

30

5.

Phng php chit v phn lp cc cht trong cy Bch trinh

bin (Hymenocallis litoralis) chit v phn lp cc cht trong cy Bch trinh bin, ta c th p dng nhiu cch bit nh chit nng bng my chit lin tc Shoxlet hoc chit hi lu; chit nng hoc chit nhit thng; chit lin tc hoc chit phn onsau kt hp cc phng php lc, kt tinh thu sn phm tinh; da vo cc phng php hin i nh sc k lp mng, phn tch cu trc bng ph xc nh thnh phn cht tch c. Vi cc alkaloid c th chit t dc liu kh tn bt. Nguyn liu chit cn lm sch, sau khi tn bt ta ngm vi ete du hoc ete trong vi gi n 1 ngy, sau chit lin tc bng Shoxlet hoc hi lu vi ete du 1 2 gi. C th dng dung mi hu c nh clorofoc, ete, etyl clorua.. hoc chit bng dung dch nc axit hoc cn nh axetic, sunfuric, nitric, ty theo loi alkaloid. Sau tch ring alkaloid bng phn b gia 2 pha dung mi c phn cc khc nhau v khng tan ln nhau, mi phn on tch ring tip tc tinh ch bng kt tinh phn on. phn tch alkaloid ta c th dng cc phng php nh: Dng thuc th cho phn ng mu hoc kt ta vi alkaloid nh thuc th Dragendorft, thuc th Mayer; Dng phn tch ph hng ngoi, ph cng hng t ht nhn Vi cc flavonoid, c th tch s b hn hp cha nhiu flavonoid bng cch dng dung mi c phn cc khc nhau v khng ha ln tch phn on hoc tch phn on bng cc dung dch c kim khc nhau. Ngoi ra cn c cc phng php hin i khc nh dng sc k ct vi cc cht hp ph thng dng nh polyamide, silicagel, xenlulozo, sau dng cc dung mi ra gii nh ete du, benzene, CHCL3. tinh ch flavonoid thu c ta lc qua ct cht hp ph, kt tinh trong cn ( EtOH, MeOH) hoc cn axeton, hn ch dng than hot v than hot hp ph rt mnh flavonoid. Phn tch flavonoid da vo cc phn ng mu ca flavonoid vi ammoniac, hay phn ng Xianidin ca
31

Wilstatter, phn ng Marini Bettolo., p dng sc k lp mng, phn tch cu trc bng ph 5.1. Phn lp t c ca cy Bch trinh bin

Phng php ca Long-ze Lin v cc cng s [8]: Bm nh 8.5kg c hnh ti ca cy, em chit vi 95% EtOH lm 2 ln, ln u dng 15l EtOH, ln 2 dng 10l EtO. Gp tt c dch lc lm mt, c c cn 125ml dch. Bng ha dch thu c 56g cht rn th. Tn nh cht rn vi 1% HCl cho n khi dung dch thu c c phn ng yu vi thuc th Dragendorff. Dung dch axit thu c sau khi chit vi CHCl 3 ( chit 5 ln, mi ln 2l), c bazo ha bng dung dch NH3 n pH=9 v li chit tip vi dung dch CHCl3 (5 ln, mi ln 1l). Gp tt c dch chit li v ra 2 ln vi nc, sau lm khan vi Na2SO4, ha hi dung mi thu c hn hp bazo th. Hn hp ny a ln ct v chy sc k bng hn hp dung mi CHCl3:MeOH theo phn cc tng dn cui cng ra gii hon ton bng dd acid HCl 1%.Qu trnh sc k ct c kim tra cc phn on bng TLC vi pha ng l h dung mi CHCl3:MeOH = 9:1 (or 49:1) hoc h Cyclohexan: EtOAc: Et2NH2 = 6:4:1.Cc phn on c Rf thch hp c th c gom li vi nhau tinh ch mt s cht t 1-14 nh bng trn. Phng php ca Amina H. Abou Donia v cc cng s [7] 6. Phng php ha l hin i phn tch cc cht tinh khit phn

lp c t h Thy tin (Amaryllidaceae) 6.1. Cc phng php ph

6.1.1. Cc loi ph thng dng thng dng y l phng php thng thng rt hay c s dng xc nh cu to cc hp cht ha hc. xc nh cu trc ca mt hp cht ta phi s dng kt hp cc phng php ph: Ph cng hng t ht nhn (H1NMR, C13, DEPT), Ph

32

khi ( MS), Ph hng ngoi (IR). Nhng ch yu ch dng ph cng hng t ht nhn v ph khi. a. Phng php ph khi (MS / GC-MS)

Nguyn tc l da vo s phn mnh ca cc ion phn t di s bn ph ca cc chm ion bn ngoi. Ph EI-MS: Da vo s phn mnh ion di s bn ph ca chm ion vi nng lng khc nhau.Thng l 70ev. Ph ESI-MS: gi l ph khi phun m in t.y l phng php ch yu c s dng Vit Nam.Ph ny c thc hin vi nng lng bn ph thp hn nhiu so vi EI-MS nn ph thu c ch yu l pic ion phn t, cc pc c trng cho s ph v cc lin kt c nng lng thp,d b ph v (Peak c bn) Ph GC-MS: l phng php tch hp gia sc k kh v khi ph. Phng php ny c s dng xc nh thnh phn hn hp, kh nng phn tch cc thnh phn trong hn hp v cng thc cu to mt s cht trong hn hp . b.
1

Phng php ph cng hng t ht nhn (NMR)

H-NMR: Ph proton: Trong ph ny, dch chuyn ha hc ca cc proton

xc nh trong thang o 0-14ppm.Cht chun s dng thng l TMS.Da vo dch chuyn ha hc c trng v cc tng tc spin m ta c th xc nh c cng thc ha hc ca cc cht.
13

C-NMR: Ph cacbon: Ph ny cho ta tn hiu ca cacbon trong nhng

nhm th khc nhau. dich chuyn ha hc cc C xc nh trong thang o 0230ppm. DEPT: thng s dng DEPT-90 hoc DEPT-135.Ph ny cho ta cc tn hiu phn bit cc C cc bc khc nhau da vo mnh ca tn hiu, dch chuyn ha hc ca C v pha ca pc. V d nh ph DEPT-90 ch xut hin cc tn hiu ca nhm CH trong khi trong ph DEPT-135 cc tn hiu CH,
33

CH3 nm pha trn trc dch chuyn ha hc,cc tn hiu CH 2 nm pha di v cc tn hiu C bc 4 bin mt. 2D-NMR: Ph 2 chiu cng hng t ht nhn, thng s dng khi cc cht cn xc nh c cu trc phc tp hoc cn kim nh chnh xc cng thc cu to d on.Cc dng ph hay s dng nh COSY, HMQC, HMBC, NOESY. c. Ph hng ngoi (IR)

y l phng php xc nh nhm chc da vo tn s hp th ca cc vch do cc dao ng ha tr v dao ng bin dng gy ra.y l phng php n gin xc nh li cc nhm chc trong phn t ch khng c nhiu ngha trong vic xc nh cng thc cu to ca cc hp cht.

6.1.2. Mt s hot cht c xc nh cu trc Alcaloit c lp t cy Zephyranthes concolor chlidanthine (1), v galanthamine (2) c cu trc c xc nh nh sau:

O
OR3
9 8 7 10

2 1 3 10b

R1 R2
4

B
10a 6a 11

A
4a 12

D
6 5

NR4

1: R1 = OMe, R2 = H,R3 = H, R4 = Me ax 2: R1 = OH, R2 = H, R3 = Me, R4 = Me eq

Chlidanthine (1). Tinh th khng mu hnh kim (kt tinh t acetone). []D20 = 129 (EtOH, c. 3.0 mg/mL).

34

IR (CCl4 , film) max cm-1 2923, 2853, 1604, 1460, 1254, 1088. EI-MS m/z (%): 287(100) M+( C17H21NO ), 272(9) M+ Me, 256(65) M+OMe, 244(10), 230(6), 225(14), 212(29), 202(45) (C17H21NO2), 115(42), 77(39). i vi ph 1H v 13 C-NMR d liu ph xem bng : C17H21NO2 Bng 1: cc d liu quang ph ca chlidanthine (1)
V tr 1H (J, Hz) 1 2 2 3 4 4a 6 4.5(td,J=3.3,1,2) 1.99(ddd,J=-15.9,5.8,3.3) 2.59(ddd,J=15.9,3.3,1.5) 3.79(m,J=5.8,1.5) 5.98(ddd,J=10.4,4.5,1.2) 6.17(dt,J=10.4,1.2) 3.62(d,J= -15.0) NOESY H(2),H(2), H(11) H(1),H(3) H(1) H(2) H(4a) H(4),H(6),H(12) H(7),H(12),NMe 70.0 s C(4),C(1),OMe 13 C 87.2.s 28.1 t HMBC C(11)

124.3 s C(2),C(10b) 129.4 s C(1),C(3),C(10b) 60.6 t C(10a),C(6a),C(7), C(12),NMe

4.09(d,J= -15.0)

H(4a),H(12)

C(10a),C(6a),C(7), C(12),NMe

6a 7 8 9 10 10a 10b 11 11 12 12 2.10(dt,J= -13.3,3.0 1.52(ddd,J=13.3,3.8,1.6) 3.03(d,J= -14.3) 3.28(d,J= -14.3,1.4) H(1) H(12) H(6),H(11), NMe H(4a),H(6) 6.47(d,J= 8.0) 6.47(d,J= 8.0) H(6),H(8) H(7)

128.7s 121.9d 115.3d 145.4s 140.1s 132.1s 48.6s 34.6t C(10b) C(10),C(10a),C(6) C(9),C(10),C(6a)

54.0t

35

OMe 3.37s NMe 2.36s

56.8q H(6),H(11), H(12 ) 41.9q

C(3) C(6),C(12)

Ph 1H c th hin nh sau:
4.01

O
5.35

2.08;1.83

3.30

H 2.79
1.70;1.45 2.00;1.75 2.41;2.31

HO
1.69;1.59 6.52 6.54 3.64;3.61

2.26

Ph C13 c th hin nh sau :

O
HO
47.3 140.3 145.8 131.5

37.7 76.5 82.6

57.1

H
29.5

26.0 33.4 112.1 119.0 125.2 63.0

59.6

N
44.4

36

Galanthamine (2). Ph 1H- v 13C-NMR d liu ph c bo co bi Carrol et al. [27]. Galanthamine N-oxide (3). Ph 1H- v 13 C-NMR d liu ph c bo co bi Kobayashi et al. [30].

Ankaloid Lycorine (3) c phn lp t cy Bch trinh bin c xc nh cu trc nh sau:

OMe
3 4 2 12 11 10 12a 8a 9 8 1 12b 6a 6

OH NMe H

37

D liu ph 1 H:
3.30

0.86 2.79 1.70;1.45

1.91;1.66

OH

2.26

O
6.07

2.00;1.75 0.86 7.06

2.50;2.25

O
6.95 4.68;4.58

3.31

38

D liu ph C13
57.1

29.8 6.8

78.1

37.8

OH

40.6

O
101.2

109.7 147.6

15.0 6.8

60.2

103.4 78.9

141.1 129.4

56.4

145.9 107.4 71.1

6.2.

Cc phng php khc

39

KT LUN TI LIU THAM KHO

40