Han the (Co Vitexin)

S ha bi Trung tm Hc liu i hc Thi Nguyn http://www.lrc-tnu.edu.vn

1

I HC THI NGUYN TRNG I HC S PHM

-----------------------------

NGHIM TH HNG

NGHIN CU THNH PHN HO HC C TRONG

CY HN THE (DESMODIUM HETEROPHYLLUM)

H CNH BM (PAPILIONACEAC)

LLUUNN VVNN TTHHCC SS HHOO HHCC

Ngi hng dn khoa hc: PGS.TS. PHM VN THNH

Thi Nguyn - 2009


2

I HC THI NGUYN TRNG I HC S PHM

-----------------------------

NGHIM TH HNG

NGHIN CU THNH PHN HO HC C TRONG

CY HN THE (DESMODIUM HETEROPHYLLUM)

H CNH BM (PAPILIONACEAC)

Chuyn ngnh: HO HU C M s: 60.44.27

LLUUNN VVNN TTHHCC SS HHOO HHCC

Ngi hng dn khoa hc: PGS.TS. PHM VN THNH

Thi Nguyn - 2009


3

MC LC

M U .......................................................................................................... 1

CHNG I: TNG QUAN ................................................................................. 3

1.1. M t thc vt ...................................................................................... 3

1.2. Mt s cng dng ca chi Desmodium .................................................. 4

1.3.Tnh hnh nghin cu ho hc thc vt ca chi Desmodium .................. 5

1.3.1. Mt s i din cc ancaloit. .......................................................... 5

1.3.1.1. Cc ancaloit c b khung indol ................................................ 5

1.3.1.2. Cc ancaloit khng cha d vng hay cn gi l cc baz amin8

1.3.1.3. Cc ancaloit c b khung pirol .............................................. 10

1.3.2. Mt s i din hp cht flavonoit ............................................... 11

1.3.3. Mt s i din nhm dn xut polyphenol .................................. 16

CHNG II: THC NGHIM ......................................................................... 18

2.1. Nguyn tc chung............................................................................... 18

2.2. i tng v phng php nghin cu .............................................. 18

2.2.1. Thu mu cy, xc nh tn khoa hc v phng php x l mu ......... 18

2.2.2. Phng php phn lp cc hp cht t cc dch chit ................. 19

2.1.3. Phng php kho st v xc nh cu trc ho hc cc hp cht ...... 19

2.2. Dng c, ho cht v thit b nghin cu ............................................ 20

2.2.1. Dng c v ho cht .................................................................... 20

2.2.2. Thit b nghin cu ...................................................................... 20

2.3. Cc dch chit t cy hn the (Desmodium heterophyllum - Papilionaceac) .......... 21

2.3.1. Cc dch chit .............................................................................. 21

2.3.2. Kho st nh tnh cc dch chit ................................................. 22

2.3.2.1. Xc nh ng kh. ............................................................. 22

2.3.2.2. Xc nh nh tnh ankaloit. ................................................... 22

2.3.2.3. Xc nh nh tnh steroit....................................................... 23


4

2.3.2.4. Xc nh nh tnh flavonoit .................................................. 23

2.3.2.5. Xc nh nh tnh poliphenol. ............................................... 23

2.3.2.6. Xc nh nh tnh cumarin.................................................... 23

2.3.2.7. Xc nh nh tnh glycozit tim. ............................................. 24

2.3.2.8. Xc nh nh tnh saponin. ................................................... 24

2.4. Phn lp, tinh ch cc cht t cy hn the (Desmodium heterophyllum

Papilionaceac) ............................................................................................ 24

2.4.1. Dch chit n-hexan ....................................................................... 24

2.4.1.1. Cht PH1 (Stigmasterol) ........................................................ 25

2.4.1.2. Cht PH2: Glycozit ca stigmasterol ..................................... 25

2.4.2. Dch chit Clorofom (PC1) .......................................................... 25

2.4.3. Dch chit Etylaxetat (PE1) ......................................................... 26

CHNG III: THO LUN KT QU NGHIN CU ....................................... 28

3.1. Xc nh nh tnh cc nhm cht thin nhin trong cy hn the

(Desmodium heterophyllum Papilionaceac) ................................................ 28

3.2. Phn lp v nhn dng cc hp cht c trong cc dch chit khc nhau

ca cy hn the.......................................................................................... 29

3.2.1. Cht PH1: Stigmast-5,22-dien-24R-3 -ol .................................... 29

3.2.2. Cht PH2: 3-O--D- glucopyranozyl stigmasterol ....................... 32

3.2.3. Cht PC1: -sitosterol-3--D-glucopyranozit .............................. 34

3.2.3. Cht PE1 :8-C--D-glucopyranozyl apigenin (Vitexin) ................ 36

KT LUN..................................................................................................... 40

KIN NGH ................................................................................................... 40

TI LIU THAM KHO .................................................................................. 41


5

DANH MC CC BNG BIU, HNH, S

Bng 2.1: Khi lng cht tng s c chit tng phn on ca cy hn

the (Desmodium heterophyllum Papilionaceac) ............................................. 22

Bng 3.1: Pht hin cc nhm cht trong cy hn the .................................. 28

Bng 3.2: S liu ph 13C-NMR (DMSO-C13CPD) ca PE1 ........................ 37

Hnh 1.1: nh cy Hn the Desmodium heterophyllum, Papilionaceac .............. 3

Hnh 3.1:Ph 1H-NMR ca Stigmast-5,22-dien-24R-3 -ol ......................... 31

Hnh 3.2: Ph 1H-NMR ca: 3-O--D- glucopyranozyl stigmasterol .......... 33

Hnh 3.3: Ph 1H-NMR ca: -sitosterol-3--D-glucopyranozit. ................ 35

Hnh 3.4: Ph 1H-NMR ca:8-C--D-glucopyranozyl apigenin ................... 38

Hnh 3.5: Ph 13C-NMR ca:8-C--D-glucopyranozyl apigenin .................. 39

S 2.1: Quy trnh ngm chit mu ........................................................... 22


1

M U

Vit Nam ta l mt nc nhit i, nng, m v ma nhiu, c ngun

dc liu rt phong ph, a dng v mt nn y hc dn tc pht trin lu i.

T xa xa, ng cha ta bit cch s dng nhiu loi tho dc trong vic

dng thng, tr bnh v bi b c th. Nh vy, nhng cy thuc dn gian

ng vai tr ht sc quan trng trong i sng hng ngy ca con ngi.

Ngy nay, khi thuc bit dc ca nn y hc hin i c s dng

rng ri, nhiu loi cy c trong t nhin vn c s dng trong dn gian

cha bnh rt c hiu qu. Rt nhiu loi bnh tt c cha khi nh tho

dc, rt nhiu thc vt c dng ch bin thnh thc phm chc nng

qu gi.

Trong thi gian qua, nhng hp cht t nhin c phn lp t cy c

c ng dng rng ri trong rt nhiu ngnh cng nghip, nng nghip,

chng c dng sn xut thuc cha bnh, thuc bo v thc vt, lm

nguyn liu cho ngnh cng nghip thc phm v m phm v.v... Mc d

cng ngh tng hp ho dc ngy nay pht trin mnh m, to ra cc bit

dc khc nhau s dng trong cng tc phng, cha bnh, song nhu cu s

dng cy c xung quanh lm thuc cng ngy cng tng ln, v khoa hc

hin i chng minh c trong chng c cha nhng bit dc rt kh

tng hp, v hu nh rt t khi c tc dng ph.

C nhiu b mn khoa hc nghin cu v cy thuc ra i. Vic

nghin cu cy thuc gip cho chng ta hiu r v thnh phn v cu trc

ha hc, hot tnh sinh hc, tc dng dc l ca cy thuc. Trn c s cc

nghin cu c th to ra cht mi c hot tnh sinh hc cao nh mong

mun lm thuc cha bnh.


2

Cy hn the l loi cy c mc hoang khp ni. Trong Y hc dn tc

cy ny c nhn dn dng lm thuc cha nhiu loi bnh nh: lm

thuc trong cha st nng, ho c m, tiu sng, tiu vim, cm mu. Dng

ngoi gi nt p vt thng. R lm thng hi, b, li tiu, l li sa. Cha

cc chng lu ra mu, i but, b tiu tin do c th b nhit qu, dng gii

nhit cha st v ho kh kh, au d dy, vim lot hnh t trng... Cy hn

the cn c dng phi hp vi cc v thuc khc. Dng ton thn cy sc

nc ung cha b tiu tin, cha si thn rt c hiu qu.

Thc vt hn the c nhiu ng dng quan trng nhng gn y mi

c mt vi nh khoa hc cc nc quan tm chn lm i tng nghin

cu, cn nc ta hu nh khng c cng trnh nghin cu no v thnh

phn ha hc v dc l hc ca cy hn the.

Vi mc ch nghin cu v tm hiu thnh phn ha hc cc hp cht

c hot tnh sinh hc ca cy hn the, gp phn lm tng thm s hiu bit v

thnh phn ho hc ca ngun thc vt lm thuc phong ph v qu gi ca

Vit Nam. Chng ti chn cy hn the (Desmodium heterophyllum,

Papilionaceac) lm i tng nghin cu cho cng trnh ny. Tn ti

l:Nghin cu thnh phn ha hc c trong cy hn the (Desmodium

heterophyllum) h cnh bm (Papilionaceac)


3

CHNG I

TNG QUAN

1.1. M t thc vt

Cy hn the (cn gi l Sn lc u) c tn khoa hc l: Desmodium

heterophyllum, thuc h Cnh bm- Papilionaceac [4]

Phn b a l: L loi cy mc hoang di cc bi c, ven b rung

khp ni nc ta. Trn th gii cn thy mc hoang nhiu nc nhit i

vng ng Nam . ng c c nhiu cy hn the c coi l ng c tt.

Hnh 1.1: nh cy Hn the Desmodium heterophyllum, Papilionaceac

M t cy: Hn the l loi cy mc b st t, phn cnh t gc, cnh

tri ra mt t. L mc so le, thng c 3 l cht, cc l di ch c 1 l cht,

l cht hnh tri xoan ngc, mt trn xanh sm, nhn, mt di mu nht, l

km hnh tri xoan nhn, c nhiu vn. Cm hoa rt ngn, mc nch l, t

hoa, khng cung, hoa nh mu tm hng. Qu thun khng cung, c 4-5

t, mi t cha 1 ht.


4

Thu hi v ch bin: Nhn dn ta dng ton cy dng ti hay phi

kh, sy kh. Ma thu hi gn nh quanh nm [4].

1.2. Mt s cng dng ca chi Desmodium

Hu ht cc loi thc vt thuc chi Desmodium u c nhn dn s dng

lm cc bi thuc dn gian cha bnh, chm sc v bi dng sc kho.

Cy hn the (Desmodium heterophyllum): Theo ng y (Y hc c truyn

Tu Tnh), l hn the c v hi chua cht, tnh mt. Trong nhn dn, cy hn

the c dng lm thuc trong cha st nng, ho c m, tiu sng, tiu

vim, cm mu. Dng ngoi gi nt p vt thng. R lm thng hi, b, li

tiu, l li sa. Cy hn the c dng cha cc chng lu ra mu, i but,

b tiu tin do c th b nhit qu, dng gii nhit cha st v ho kh kh, au

d dy, vim lot hnh t trng... Cy hn the cn c dng phi hp vi cc

v thuc khc (cy huyt d, bng m , c tranh, cy k u nga) c

cc lng y ch thnh rt nhiu v thuc hay, c bit trong cha bnh tit

niu, si thn. Dng ton thn cy sc nc ung cha b tiu tin, mn nga

tiu c, cc bnh v thn.

Vit Nam, cng chi vi cy hn the l cy mt tru (Desmodium

styracifolium). Cy mt tru c cc lng y khp ni dng lm thuc

cha bnh, dn gian dng cy v l mt tru cha cc chng bnh nh gii

c, tiu vim, tiu sn, gh nga Do c nhiu cng dng nn cy mt tru

c nhiu nh khoa hc nghin cu v tm ra nhiu cht ho hc c

trong loi ny. Y hc c truyn ng dng v sn xut i tr cc v thuc

c tn Kim tin tho dng cha nhiu loi bnh v c kt qu tt.

Trn th gii, t nhiu loi ca chi Desmodium, cc nh khoa hc

nghin cu v th hot tnh sinh hc ca nhiu cht c phn lp trong chi ny c

mt s tc dng dc l ng ch . V d:

T loi Desmodium pulchellum tch c mt s cht c tc dng khng

c, c kh nng gy o gic ging nh ma tu [10]


5

Ti Nht Bn, t r cy ca mt s loi thuc chi Desmodium ngi ta

bo ch thnh thuc cha tm thn phn lit, cht lm tng hng phn thn

kinh, thuc cha cc bnh ni tng nh gan, mt, thn..[20]

1.3.Tnh hnh nghin cu ho hc thc vt ca chi Desmodium

1.3.1. Mt s i din cc ancaloit.

Ancaloit l 1 nhm ln cc hp cht hu c trong phn t cha nit,

c tnh kim, c tch ra t thc vt, vi sinh vt v ng vt . Nhiu

ancaloit c tc dng sinh l i vi c th sng ca ngi v ng vt, nn

mt s ancaloit c bo ch sn xut thuc cha bnh, cht gy m,

thuc kch thch.

Cc ancaloit c nhiu trong thc vt chi Desmodium, c trong l, thn,

hoa, qu, r. Hm lng ancaloit cng thay i theo ma, thi tit, kh hu...

Mt s ancaloit c tm ra trong thc vt chi Desmodium nh sau:

1.3.1.1. Cc ancaloit c b khung indol

Theo ti liu [8], t loi Desmodium pulchellum, cc tc gi U.S.Pat,

1993 v Lam.Y.K.T, 1994 phn lp c 1 ankaloit c b khung indol, cu

trc c xc nh l: 3-(Dimethylaminomethyl)indole, c cng thc phn t

l C11H14N2, c im chy c xc nh l 138-1390C. C tc dng nh mt

tc nhn khng c.

Cc tc gi Moriyasu.M,1997 v Roseghini.M,1976 v cc cng s [9]

li tch c cng t loi Desmodium pulchellum hp cht N-Oxide, cng

thc phn t C12H16N2O im chy o c l 123-1280C, dng tinh th ngm

nc. N-Oxide cn c phn lp trong loi Desmodium triflodium v

Desmodium other. Cu trc ca 2 cht ny c xc nh l:


6

1. 3-(Dimethylaminomethyl)indole 2. N-Oxide

Nm 1958, t loi Desmodium pulchellum, Wilkinson.S.J.C.S v cc cng

s [10] phn lp c hp cht 5-Methoxy-N,N-dimethyltryptamine, cng

thc phn t C13H18N2O, dng tinh th kt tinh trong (C2H5)2O/ n-C6H14, cng

l mt ankaloit khung indol. c xc nh c tc dng nh mt tc nhn gy

c, to ra chng chng mt, hoa mt, chong vng.

3. 5-Methoxy-N,N-dimethyltryptamine.

Theo tc gi Moriyasu.M, nm1997, t loi Desmodium arundo donax,

Mimosa hostilis [11] phn lp c cht N,N-Dimethyltryptamine, cng

thc phn t C12H16N2, c im chy rt thp 48- 49

0C, c xc nh l cht

c kh nng gy o gic, d b lm dng nh lm dng ma tu, c c tnh

cao. Bng cc phng php quang ph tc gi xc nh c cu trc ho

hc ca n l:

4. N,N-Dimethyltryptamine


7

T cc loi Desmodium pulchellum v Desmodium caudatum, tc gi

Roseghini.M v cc cng s phn lp c cht Bufotenine N-oxide, cng

thc phn t l C12H16N2O2 , dng tinh th c nhit nng chy cao 214-

2150C. Bufotenine N-oxide cn c tm thy trong nc ca con cc, trong

nm amanita v mt s loi thc vt khc [12]. Cu trc c xc nh l:

5. Bufotenine N-oxide

Theo Cahill.W.M v cc cng s [13], t ht cy Desmodium

tiliaefolium phn lp c cht N-metyltryptophan, cng thc phn t

C12H14N2O2 c kh nng tan trong H2O, C2H5OH nhng khng tan trong ete

(C2H5)2O. Tc gi xc nh c cu trc l:

6. N-metyltryptophan

Mt cht c xc nh l c kh nng c ch tng trng thc vt,

tn l N,N-Dimethyltryptophan, cng thc phn t C13H16N2O2, c tc gi

Mandava.N phn lp t loi Desmodium triflorum [14]. Dng tinh th tan

trong C2H5OH, c im chy cao: 243-2450C, cu trc ho hc l:

7. N,N-Dimethyltryptophan


8

1.3.1.2. Cc ancaloit khng cha d vng hay cn gi l cc baz amin

Theo tc gi Yamaguchi v cc cng s, phn lp t cy Desmodium

gangeticum c cht (N,N-Dimethylamino)acetophenone, cng thc phn t

C10H13NO, c nhit si 130-1320C [15], c cng thc cu to c xc

nh l:

8. (N,N-Dimethylamino)acetophenone

T loi Desmodium styracifolium, Yang J.et al,Yaoxue Xuebao phn

lp c mt amit m cu trc ca n c xc nh l 3-Acetamidodihydro-

4,5-dimethyl-2(3H)-furanone. Cn c gi l Desmodilactone, cng thc

phn t C8H13NO3, l cht c nhit nng chy tng i thp, 84-850C [16]

9. 3-Acetamidodihydro-4,5-dimethyl-2(3H)-furanone

T loi Desmodium triflorum, Konno.C cng vi cc cng s phn

lp c cht coryneine, cng thc phn t C11H18NO2+, l cht c xc

nh c kh nng l mt tc nhn kch thch giao cm [17]

10. Coryneine


9

Theo Ranieri.R cng vi cc cng tc vin [18] phn lp c t

loi Desmodium tiliaefolium, c cht 3,4-Dimethoxy phenethylamine. Tinh

th kt tinh trong C6H6 hoc ete petrol, cng thc phn t C10H15NO2. C

nhit nng chy 1240C, l cht c hot tnh sinh hc cao. Cng thc cu

to c nhn dng l mt amin bc nht.

11. 3,4-Dimethoxy phenethylamine

Theo cc tc gi Ghosal.S. Bruhn, J.G v Ranieri.R.N, t ngun

Desmodium tiliaefolium phn lp c cht Normacromerine, cu trc

c xc nh l N-Methyl-3,4-dimethoxy-hydroxy phenethylamine, cng

thc phn t C11H17NO3 [19]. Normacromerine cn c phn lp t cc loi

Coryphantha macromeris, Coryphantha calipensis v Dolichothele

longimamma.

12. Normacromerine. N-Methyl-3,4-dimethoxy-hydroxy phenethylamine

c phn lp t cc ngun Desmodium several, trichocereus

candicans, trichocereus spachianus, tc gi Gven.K.C [20] tch c hp

cht N-Me, cn c tn l candicine, cng thc phn t C11H18NO+, c xc


10

nh l cht c kh nng c ch hch thn kinh, lm co mch, nh hng n

hot ng thn kinh, c tnh ging nh nicotin, c cu trc c xc nh l:

13. N-Me

Theo tc gi Rondest.J v cc cng s, hp cht c tn 4-Hydroxy

phenethylamine, cng thc phn t C8H11NO l cht c xc nh l c kh

nng gip co mch mu, l cht ph bin sinh vt, c tm thy nhiu

loi cy nh: Desmodium v Magnolia, Pisum sativum, m ng vt ang

phn hu, cn c tm thy trong nc tiu ca bnh nhn bnh parkison v

trong nhiu loi cy khc [21]. Cu trc c xc nh l:

14. 4-Hydroxy phenethylamine

1.3.1.3. Cc ancaloit c b khung pirol

i din cho ancaloit khung pirol l cht Desmodimine, cng thc phn

t C12H15NO4, c tc gi Yang.J.S v cc cng s phn lp t loi

Desmodium styracifolium [22]. Bng cc phng php quang ph nhn

dng c cu trc ho hc ca n l:


11

15. Desmodimine

1.3.2. Mt s i din hp cht flavonoit

T thn v r loi Desmodium canum, tc gi Botta.B [23], phn

lp c cc hp cht Desmodianone D v Desmodianone E, u c cng

cng thc phn t: C26H28O6, c 2 u tn ti dng cht rn v nh hnh,

chng c ph UV rt ging nhau nhng c nhit nng chy khc nhau.

Ngi ta phn lp v nhn dng c chng u l cc iso flavanon c

vng A ging nhau nhng vng B c cc nhm th v tr 4 , khc nhau.

Botta. B t tn cho cho chng l Desmodianone D v E vi cng thc

cu to tng ng l:

,

16. Desmodianone D

17.Desmodianone E


12

Theo tc gi Ahluwalia.V, t loi preferred genus name Desmodium,

phn lp trong dch chit etylaxetat c cht 2',4',5-Trihydroxy-7-methoxy-

6-methylisoflavanone, cng thc phn t C17H16O6, tn ti dng tinh th hnh

kim vng nht [24]

18. 2',4',5-Trihydroxy-7-methoxy-6-methylisoflavanone

Ngoi hp cht trn, theo ti liu [25] cng t loi preferred genus name

Desmodium, cc tc gi Bohlmann.F v Kalidhar.S.B tch c mt iso

flavanonol khc c cng thc phn t C20H20O6, dng tinh th hnh phin khi

kt tinh trong C6H6, cu trc c nhn dng l:

19. 3,4,,5.7-Tetrahydroxy-8-preylflavanone

Nm 2003, t loi Desmodium canum, tc gi Botta.B, phn lp

c hp cht 6-Methyltetrapterol A, c cng thc phn t C26H24O6 , l cht

dng bt v nh hnh [26], bng cc phng php ph hin i tc gi xc

nh c cu trc nh sau:


13

20. 6-Methyltetrapterol A

Cng nm 2003, tc gi Tasanuo.M.K v cc cng tc vin tch c

t loi Desmodium uncinatum [27] hp cht c xc nh l 6-(2,4-

Dihydroxyphenyl)-2,3,6,7-tetrahydro-4-hydroxy-2-(1-methylethenyl)-5H-furo[3,2-

g][1]benzopyran-5-one), (tn khc: Uncinanone B), cng thc phn t C20H18O6 v

cht 2'-Me ether (Uncinanone C) cng thc phn t C21H20O6. Cu to ca

Uncinanone Bv C l:

21. Uncinanone B

22. Uncinanone C

Theo cc tc gi Mizuno.M v Lin.Y-L, t thnh phn ca l loi

Desmodium oxyphyllum, phn lp c 4 cht dng bt, c th l

Desmoxyphyllin B, c cng thc phn t C16H10O6;


14

7-O-D-Glucopyrano side, c cng thc phn t C22H20O11 ; 5- hidroxy, c

cng thc phn t C16H10O7, dng bt mu vng v 5-hidrroxy,7-0-D-

glucopyranoside, cng thc phn t C22H20O12 cng dng bt mu vng.

[28]. Cu trc phn t c xc nh ln lt l :

23. Desmoxyphyllin B 24. 5-hidroxy

25. 7-O-D-Glucopyrano side

T loi Desmodium canum, tc gi Monache.G.D v cc cng s

[29] phn lp c 2 hp cht l Desmodianone A, c cng thc phn t

C26H28O6 v Desmodianone C, c cng thc phn t C26H30O6, c 2 cht u

c tc dng nh mt tc nhn khng khun. Nh cc phng php ph hin

i, tc gi a ra cng thc cu to ca 2 cht l:


15

26. Desmodianone A

27. Desmodianone C

Theo tc gi Ueno.A t loi Desmodium caudatum phn lp c cht

Desmodol, cht dng bt mu vng c cng thc phn t C21H18O6 , c im

chy tng i cao 281-2820C v t loi Desmodium sequax,tc gi Khan.H

phn lp c cht: Lanceolatin B. Cng thc phn t : C17H10O3, tinh th

kt tinh trong hn hp C6H6/C6H12, c im chy thp hn: 138-1400C.[30]

28. Desmodol 29. Lanceolatin B


16

T nm1973, trn tp ch Soedin, tc gi Chernobrovaya.N.V thng bo

phn lp t loi Desmodium canadense, tch c 2 cht cng thc c

ch ra l 2''-O--D-Xylopyranosyl (tn khc:Homoadonivernite), cng thc

phn t: C26H28O15 v 2''-O--D-Xylopyranosyl( tn khc: Desmodin), cng

thc phn t: C26H28O14, c 2 cht c im chy tng ng 198-2030C [7].

Cu trc c xc nh l:

30. Homoadonivernite 31. Desmodin

1.3.3. Mt s i din nhm dn xut polyphenol

Theo cc tc gi Purushothaman.K.K, Brin.A.J v Kitagawa.I, t loi

Desmodium gangeticum, phn lp c 4 cht c th l: Gangetin, cng thc

phn t C26H28O5, tn ti dng tinh th kt tinh trong hn hp dietylete/hexan,

cht c tn Gangetinin, cng thc C26H26O5 , Desmodin c cng thc phn t

C22H22O6 v 3-Me ether (Desmocarpin), cng thc C16H14O5, dng tinh th

khng tan trong H2O v hexan, tan trong CH3OH, CH3COOC2H5 [31]


17

32. Gangetin 33. Gangetinin

34. Desmodin 35. Desmocarpin


18

CHNG II

THC NGHIM

2.1. Nguyn tc chung

phn lp c cc hp cht trong bt k mt thc vt no m khng

lm nh hng ti thnh phn ho hc c trong n th trc khi ngm chit

bng dung mi hu c, mu thc vt phi c a i kh men ngay sau khi

thu mu v sy kh iu kin thch hp.

V nguyn tc vic ngm chit mu thc vt c th tin hnh theo 2

cch ph bin sau:

1. Chit v phn lp cc hp cht t mu thc vt bng cc loi dung

mi c phn cc tng dn: ete du ha hoc n-hexan, clorofom, etylaxetat,

metanol hoc etanol vv...

2. Chit tng bng cc ancol (metanol, etanol) hay h dung mi ancol :

nc. Sau sng lc cc hp cht bng cc loi dung mi c phn cc

tng dn nh phng php 1 thu c cc dch chit c cha cc hp cht

c phn cc tng i ging nhau.

Vic chit mu thc vt l cy hn the (Desmodium heterophyllum -

Papilionaceac) c thc hin theo phng n 2 (S 2.1).

2.2. i tng v phng php nghin cu

2.2.1. Thu mu cy, xc nh tn khoa hc v phng php x l mu

Nguyn liu nghin cu gm ton b phn trn mt t ( thn, l)

ca cy hn the, thu hi vo thng 11, 12 nm 2008 ti cc bi c trong khun

vin Trng i hc S phm Thi Nguyn, Thnh ph Thi Nguyn.

Cy hn the (cn gi l Sn lc u) c cc nh khoa hc khoa Sinh

trng HSP Thi Nguyn xc nh c tn khoa hc l: Desmodium

heterophyllum, thuc h Cnh bm- Papilionaceac.


19

Mu cy ti sau khi thu hi gm c thn, l c ra sch, sau sy

kh nhit 40-500C cho ti khi kh hon ton. Mu kh c ct nh v

ngm chit trong etanol 90o nhit phng nhiu ln, lin tc trong 15

ngy, 1 ngy chit dch 1 ln.

Sau khi ct loi dung mi bng my ct quay, p sut thp, nhit

500C cn c c n c di dng xir. Tip theo, cn c lc, chit ln

lt bng cc loi dung mi c phn cc tng dn: n-hexan, clorofom

etylaxetat v n-butanol. Cc dch chit c ui kit dung mi bng thit b

ct quay nhit 500C di p sut thp. Cc cn th c phn chia bng

sc k ct nhi silicagel vi cc h dung mi ra gii c phn cc tng dn

phn lp cc cht c phn cc gn ging nhau, kt tinh phn on v

kt tinh li trong h dung mi thch hp thu c cc cht sch .

2.2.2. Phng php phn lp cc hp cht t cc dch chit

pht hin, phn lp c nhng hp cht sch t cc dch th khc

nhau ca cy hn the chng ti phi hp s dng cc phng php sc k

v kt tinh li trong dung mi thch hp. C th cc phng php gm:

- Sc k lp mng (SKLM).

- Sc k ct silicagel, dng silicagel Merck 63- 200nm.

- Kt tinh phn on v kt tinh li.

2.1.3. Phng php kho st v xc nh cu trc ho hc cc hp cht

Cc cht kt tinh phn lp ra c xc nh nhng hng s vt l c

trng: mu sc, mi v, h s Rf, im nng chy, ghi cc loi ph nh: ph

hng ngoi (FT-IR), ph cng hng t ht nhn 1H- NMR, ph 13C- NMR

v cc ph phn gii cao. Cc s liu ph thc nghim ca cc cht sch c

dng nhn dng cu trc ho hc ca chng.


20

2.2. Dng c, ho cht v thit b nghin cu

2.2.1. Dng c v ho cht

Cc loi dung mi dng ngm chit mu l cc loi tinh khit (pure),

cn cc loi dung mi dng sc k ct, sc k lp mng, dng kt tinh

li l ho cht loi tinh khit phn tch (PA).

Sc k lp mng dng bn mng nhm DC Alufolien Kiesegel 60

F254 trng sn, dy 0,2mm c s dng xc nh s b s thnh phn

c trong cc dch chit, cc phn on chy ct v kim tra s b sch ca

sn phm thu c.

Cc h dung mi khai trin SKLM:

1. n-hexan etylaxetat 85: 15 H A

2. n-hexan etylaxetat 20:80 H B

3. clorofom metanol 90:10 H C

4. clorofom metanol 30:10 H D

5.n-butanol- axit axetic -nc 4:1:5 H E

6. dung dch HCl 2N

Cc bn SKLM sau khi sy kh c soi di n t ngoi (UV-

BIOBLOCK ) bc sng = 254nm v 365nm. Thuc th hin mu l

vanilin 1% trong dung dch metanol-H2SO4 5%, sau sy trn 1000C .

Cc gi tr Rf trong h dung mi trin khai c biu thc:

2.2.2. Thit b nghin cu

- Nhit nng chy o trn my Boetus (c), hoc trn my

Eletronthermal IA9200.

chiu di di chuyn ca cht th chiu di di chuyn ca dung mi

Rf =


21

- Ph hng ngoi ghi trn my IMPACT - 410 dng vin nn KBr.

- Ph 1H-NMR v 13C-NMR ghi trn my Bruker 500MHz, ni chun

TMS, dung mi CDCl3, DMSO, aceton-D6.

2.3. Cc dch chit t cy hn the (Desmodium heterophyllum Papilionaceac)

2.3.1. Cc dch chit

Ton b phn trn mt t ca cy hn the phi kh ni thong t

nng to, nghin nh c ngm chit kit bng etanol nhit phng cho

n khi thu c dch khng mu. Dch chit c ct loi ht dung mi p

sut gim n dng cao kh, xc nh khi lng cn kh, sau thm nc

vo cn v ln lt chit vi cc loi dung mi: n-hexan, clorofom, etylaxetat

v n-butanol.

Cc dch chit ni trn c lm khan bng Na2SO4, lc v ct kit

dung mi bng ct quay di p sut gim nhit 500 C. Cn c sy

kh v cn xc nh trng lng. Nh vy t ton b phn trn mt t

(thn, l) ca cy hn the thu nhn c 4 phn on (4 loi cn) l n-

hexan, clorofom, etylaxetat v n-butanol vi cc k hiu tng ng l: cn

trong n-hexan (PH), cn trong clorofom (PC), cn trong etylaxetat (PE) v cn

trong n-butanol (PB).


22

S 2.1: Quy trnh ngm chit mu

Bng 2.1: Khi lng cht tng s c chit tng phn on ca cy

hn the (Desmodium heterophyllum Papilionaceac)

Khi lng

nguyn liu kh

(g)

Khi lng

cn etanol

tng s (g)

Khi lng cn chit (g)

n-Hexan Clorofom Etylaxetat n-Butanol

1150 220,4 40,07 24,45 20,28 49,28

2.3.2. Kho st nh tnh cc dch chit

2.3.2.1. Xc nh ng kh.

- Ly 4-5 ml dch chit trong etanol ( cn tng) vi 4-5 ml thuc th Felinh,

un si 2-3 pht, nu c kt ta da cam ca Cu2O chng t c mt ng.

2.3.2.2. Xc nh nh tnh ankaloit.

- Xc nh bng thuc th Dragendooc. Thuc th Dragendooc c

chun b t hai dung dch sau:

Mu kh

ct nh

Cn n-hexan

PH

Cn CHCl3

PC

Cn EtOAc

PE Cn n-BuOH

PB

PH1 PH2 PC1 PE1

1. Etanol

2. Cn trong nc


23

Dung dch 1: Ly 0,85 gam NaHSO3 ho tan trong 40ml nc ct v

10ml axit axetic bng

Dung dch 2: Ly 8 gam KI ho tan trong 20ml nc ct.

Ly 2ml dung dch 1 trn vi 5ml dung dch 2 v thm 20ml axit axetic

sau thm nc n 100ml l c thuc th Dragendooc.

Nu c mt ancaloit th thy xut hin mu da cam sng khi cho dch

chit tc dng vi thuc th Dragendooc.

2.3.2.3. Xc nh nh tnh steroit

- Phn ng mu Libecman-Bcsa. Thc hin theo 2 cch:

Cch 1: Thc hin theo Ston: Cho vi ml dch chit tng vo ng nghim,

thm vi git CH3COOH c v vi ml hn hp (CH3CO)2O : H2SO4 (50:1). Mu

thay i t hng sang xanh l cy.

Cch 2: Thc hin theo Brietcoc: cho vi mg dch chit trong CHCl3, thm

vo 2 ml thuc th (CH3CO)2O : H2SO4 (20:1). Mu s chuyn t - hng- xanh

l cy- chm tu thuc vo cc loai steroit.

2.3.2.4. Xc nh nh tnh flavonoit

- Phn ng Xianidin: ly 1 ml dch chit etylaxetat cho v ng nghim, thm

vi git HCl c + 1 mnh Mg, un nh, dung dch chuyn sang mu hng.

- Phn ng vi NH3: chm cn dch chit etylaxetat vo mnh giy lc

ri h trn l ng NH3 c, mu vng ch chm m hn chng t c

flavonoit.

2.3.2.5. Xc nh nh tnh poliphenol.

- Phn ng vi dung dch FeCl3 1%: Ly khong 1 ml dch chit

etylaxetat phn ng vi FeCl3 1% , nu phn ng to ra mu xanh lc tc l

dch chit c cht c gc phenol.

2.3.2.6. Xc nh nh tnh cumarin.

- Cho vo 2 ng nghim 2 ml dch chit trong etanol, ng 1 thm 0,5 ml


24

NaOH 10%. Sau un c 2 ng nghim trn bp cch thu n si ri lm lnh.

Thm vo mi ng 4 ml nc ct. Quan st thy cht lng ng 1 trong sut hn so

vi ng 2 v khi axit ho bng vi git HCl c nu c kt ta bng th kt lun c

cumarin.

2.3.2.7. Xc nh nh tnh glycozit tim.

- Phn ng Kelle-Kiliani: Thuc th Kelle-Kiliani gm 2 dung dch:

Dung dch 1: 100 ml CH3COOH long + 1ml FeCl3 5%

Dung dch 2: 100 ml H2SO4 c + 1ml FeCl3 5%

Ly vi mg cn dch chit cho vo ng nghim, thm 1 ml dung dch 1,

lc u cho tan, thm t t 1-2 ml dung dch 2 theo thmh ng nghim, quan

st s xut hin mu gia 2 lp cht lng. Khng xut hin mu l phn ng

m tnh vi glycozit tim.

2.3.2.8. Xc nh nh tnh saponin.

- S to bt: Cho vo 2 ng nghim, ng 1 cho 5 ml dung dch HCl

0,1N. ng 2 cho 5 ml dung dch NaOH 0,1N. Cho vo mi ng 3-5 git

dch chit tng, lc u, yn, quan st. Nu bn ca ci bt ng 1

bn hn ng 2 chng t mu nghin cu c saponin tritecpen, ngc li

c saponin steroit.

- Phn ng mu Liberman: Dung dch chit + 2 ml (CH3CO)2O, lc u

ri cho t t H2SO4 c t y ln, xut hin mu tm nu c saponin.

2.4. Phn lp, tinh ch cc cht t cy hn the (Desmodium heterophyllum

Papilionaceac)

2.4.1. Dch chit n-hexan

T 25g cn dch n-hexan ca ton b phn trn mt t (thn, l) ca

cy hn the, k hiu (PH) c tin hnh phn lp cc cht trn sc k ct

silicagel. H dung mi ra gii l n-hexan : etylaxetat vi t l theo tng

dn ca phn cc dung mi t 0 n 100% etylaxetat ( C th: h dung


25

mi n-hexan:etylaxetat ln lt cc t l 95-5, 90-10, 85-15n 0-100) Cc

phn on c thu tng l nh, c kim tra bng sc k lp mng (bn

mng), cc phn on ging nhau em gp li v ct loi dung mi thu

c cht sch.

2.4.1.1. Cht PH1 (Stigmasterol)

Ra gii ct bng h dung mi n-hexan - etylaxetat (95:5). Sau khi ct

loi dung mi, cn thu c kim tra bng sc k lp mng trong h dung mi

A,(n-hexan: etylaxetat 85:15), pht hin bng vanilin 1% trong dung dch

metanol-H2SO4 5%, thu c cht rn dng tinh th hnh kim, khng mu,

khi lng 25 mg. Nhit nng chy o c l 156-1580C, Rf = 0,8 trong

h dung mi A.

Tin hnh o ph cht PH1 v so snh cc thng tin ph o c

chng ti thy cc thng s tng ng ph ca stigmasterol.

2.4.1.2. Cht PH2: Glycozit ca stigmasterol

Tip tc ra gii ct bng h dung mi n-hexan - etylaxetat (30:70).

Sau khi ct loi dung mi, cn thu c kim tra bng sc k lp mng trong

h dung mi B (n-hexan: etylaxetat 20:80), pht hin n bng vanilin 1%

trong dung dch metanol-H2SO4 5%, thu c 15 mg tinh th dng v nh

hnh mu trng. o nhit nng chy c 288-2900C, Rf = 0,65 trong h

dung mi B. So snh cc thng tin trn ph 1H-NMR vi ph gc, phn tch

cc dung dch thu phn v phn gennin vi cc cht mu phng th nghim

chng ti kt lun cht PH2 l Stigmasterol.3--D- glucopyranozyl.

2.4.2. Dch chit Clorofom (PC1)

- T 17,5 g cn dch Clorofom ca ton b phn trn mt t (thn, l)

ca cy hn the, k hiu (PC1) c tin hnh phn lp cc cht trn sc k ct

silicagel. H dung mi ra gii l etylaxetat : metanol vi t l theo tng

dn ca phn cc dung mi t 0 n 100% etylaxetat (C th l h dung


26

mi etylaxetat : metanol ln lt cc t l 95-5, 90-10, 85-15n 0-100).

Cc phn on c thu tng l nh, c kim tra bng sc k lp mng

(bn mng), thuc th pht hin l vanilin 1% trong dung dch metanol-

H2SO4 5%. Kt qu: h dung mi ra gii etylaxetat: metanol (70-30) Sau

khi ct loi dung mi, cn thu c kim tra bng sc k lp mng trong h

dung mi D (cloroform metanol -30:10) v h E (4:1:5), thu c 10 mg

tinh th dng bt mu vng nu. Rf = 0,65 trong h dung mi D, Rf = 0,45

trong h dung mi E. Nhit nng chy o c l 275-2780C.

Tin hnh o ph 1H-NMR, kt qu cho thy cht o cha c tinh

khit, chng ti thu trong axit cht thu c, phn tch dch thu phn cho

thy c ng glucoz, phn gennin cho ph ph hp vi ph 1H-NMR tng

t ph ca -sitosterol, t kt qu chng ti d on cht (PC1) l glycozit

ca -sitosterol.

2.4.3. Dch chit Etylaxetat (PE1)

- Lm tng t nh cc phn 2.4.1 v 2.4.2, t 20,4 g cn dch chit

EtOAc em tch trn ct silicagel, ra gii ct bng h dung mi etylaxetat:

metanol tng dn theo phn cc (0-100% metanol), kim tra cc phn on

trn sc k lp mng ( 5-10ml/phn on).Thuc th pht hin bng vanilin

1% trong dung dch metanol-H2SO4 5%, thu c khong 35 mg cht bt mu

vng. Lng cht bt ny c tinh ch li trn ct silicagel, ra gii ct bng

h dung mi etylaxetat: metanol t 97-3, 95-5, 90-10, 85-15. Kim tra cn thu

c bng h dung mi C (cloroform : metanol 90:10) v kim tra bng hi

NH3 c, thu c 12 mg cht bt mu vng. Nhit nng chy o c l

271-2740C, Rf = 0,55 trong h dung mi C.

- Tin hnh o ph cht bt vng ny thu c cc thng tin ph nh sau:

Ph 1H-NMR (500MHz, DMSO-1H). (ppm): 13,15 (1H, S, OH); 8,01

(2H, d, J= 8, H-2 v H-6

); 6,90 (2H, d, J =10, H-3

, H-5

); 6,26 (1H, S, H-6);


27

6,76 (1H, S, H-3); 4,96 (1H, d, J= 11, H-1); 4,6 ( 1H, t, H-2

); 3,82 (1H, m,

H-6); 3,70 (1H, t, H-3

); 3,52 (1H, m, H-4

); 3,45 (1H, m, H-5

)

Ph 13C-NMR (DMSO-C13CPD): 163,78 (C-2); 103,8 (C-3); 181,88

(C-4); 160,31(C-5); 98,04 (C-6); 160,97 (C-7); 104,48 (C-8); 154,82 (C-9),

102,34 (C-10); 121,51 (C-1); 128,69 (C-2

); 115,71 (C-3

); 160,98 (C-4

);

115,71 (C-5); 160,98 (C-6

); 73,38 (C-1

); 70,84 (C-2

); 78,59 (C-3

); 70,53

(C-4); 81,62 (C-5

); 61,14 (C-6

).


28

CHNG III

THO LUN KT QU NGHIN CU

3.1. Xc nh nh tnh cc nhm cht thin nhin trong cy hn the

(Desmodium heterophyllum Papilionaceac)

Trc khi tin hnh phn lp v xc nh cu trc ca cc cht hp

cht hu c thin nhin c trong thc vt, vic u tin l phi th nh tnh

nhn dng cc nhm cht. xc nh nh tnh cc nhm hp cht hu c

c hot tnh sinh hc c trong thc vt, thuc th thng dng l phn tch

bng cc phn ng c trng i vi mi nhm cht, c th c m t

phn 2.3.2. Kt qu th nh tnh cc nhm cht trong cy hn the

(Desmodium heterophyllum) nh sau:

Bng 3.1: Pht hin cc nhm cht trong cy hn the

STT Nhm

cht

Thuc th

c hiu Hin tng

Kt

qu

1 ng kh Felinh Cho kt ta mu gch ++

2 Ankaloit Dragendooc Mu vng da cam +

3 Steroit Libecman-Bcsa T hng n xanh l cy ++

4 Flavonoit

Xianidin

T vng da cam, n hng,

n ++

NH3 c Mu vng m hn ++

5 Poliphenol FeCl3 1% Mu xanh lc ++

6 Cumarin Axit v kim Kt ta bng ++

7 Glycozit tim Kelle-Kiliani Khng c hin tng g -

8 Saponin Liberman Mu tm ++

To bt Bt bn trong kim ++

Ghi ch: Du (+): Phn ng dng tnh. Du: (++): Phn ng dng tnh

rt r. Du (-): Khng c.


29

T kt qu ny c th nhn thy trong cy (Desmodium heterophyllum)

t nht c cha ti 7 nhm hp cht thin nhin quan trng l: ng kh,

ankaloit, steroit, flavonoit, poli phenol, cumarin v saponin. Khng pht hin

thy glycozit tim c trong cc mu th. T kt qu phn tch nh tnh cng

s b cho thy cy hn the l thc vt cha nhiu cht c hot tnh sinh hc

cao. Trong c nhiu hp cht c th c tc dng cha bnh, ng nh kinh

nghim ca cc lng y dn gian.

3.2. Phn lp v nhn dng cc hp cht c trong cc dch chit khc

nhau ca cy hn the

Cc dch chit phn on thu c t cy hn the (Desmodium

heterophyllum) theo s 2.1 u l nhng hn hp phc tp cha cc hp cht

khc nhau, c phn cc gn nhau. phn lp tng cht ra khi hn hp,

chng ti s dng phng php sc k ct vi cht hp ph silicagel, cc

h dung mi ra gii c phn cc tng dn, ph hp v thng phi lp li

nhiu ln. Vic tinh ch cc cht thng dng l kt tinh li trong dung mi

hoc h dung mi thch hp v th sc k lp mng vi nhiu h th khc

nhau. Nh mi thu c cc hp cht c tinh khit cao p ng cc

nhu cu kho st tnh cht ha l v quang ph ca chng. chnh l nhng

yu t quan trng trong qu trnh nhn dng v xc nh cu trc ha hc ca

cc cht phn lp c t cc i tng nghin cu ni trn.

Vic phn lp cc thnh phn ha hc t cy hn the c thc hin

nh s 2.1. Kt qu thu c cc hp cht sch gm cc nhm cht:

steroit, glycozit ca -sitosterol v glycozit ca flavonoit.

3.2.1. Cht PH1: Stigmast-5,22-dien-24R-3 -ol

Thu c t cn n-hexan ca thc vt hn the cht rn dng tinh th

hnh kim, khng mu, khi lng 25 mg. Nhit nng chy o c l

156-1580C, Rf = 0,8 trong h dung mi A.


30

Ph FT-IR cho php khng nh s c mt ca nhm OH axm = 3406

cm-1

, cn ph 1H-NMR cho 3H = 3,58 ppm. C 2 ni i khng lin hp vi

axm = 1621 cm-1

, cn trong ph 1H-NMR c 3 H cc nhm metin (C-H)

lin kt vi cacbon cha no cc ni i trn vi H-6 = 5,37 v H-22 = 5,15

ppm, J = 15HZ v 7 HZ v H-23 = 5,02, J = 15 v 7 HZ)

So snh cc d liu ph FT-IR, ph 1H-NMR, nhit nng chy, cc

hng s vt l khc ca cht PH1 vi stigmasterol ca phng th nghim,

chng ti nhn thy chng hon ton tng t nhau. V vy, chng ti c th

quy kt cht PH1 l stigmast-5,22-dien-24R-3 -ol, cn gi l stigmasterol.

Stigmast-5,22-dien-24R-3 -ol (stigmasterol)


31

Hnh 3.1:Ph 1H-NMR ca Stigmast-5,22-dien-24R-3 -ol


32

3.2.2. Cht PH2: 3-O--D- glucopyranozyl stigmasterol

Cht PH2 l cht rn v nh hnh, tan c trong etylaxetat, trong

metanol, d tan trong hn hp dung mi n-hexan-etylaxetat (2:8), c nhit

nng chy 288-2900C.

Thu phn cht PH2 trong dung dch HCl 2N thu c stigmasterol, ph

1H-NMR ca sn phm sau thu phn khng cn nhng tn hiu ca phn

ng v hon ton tng t nh ph ca stigmasterol. Phn dung dch c

phn tch bng sc k, so snh vi cc ng mu chun ca ca phng th

nghim cho thy tng ng vi ng glucoz.

Phn tch ph 1H-NMR ca cht PH2 cng cho thy cc phn r rt tng

ng vi ph 1H-NMR ca stigmasterol v phn ng tng ng vi cc

chuyn dch ho hc ca cc H trong phn ng ( 3H t 3,24 n 4,5 ppm).

T cc d kin thc nghim trn chng ti cho rng cht PH2 l 3-O--D-

glucopyranozyl stigmasterol

3-O--D- glucopyranozyl stigmasterol


33

Hnh 3.2: Ph 1H-NMR ca: 3-O--D- glucopyranozyl stigmasterol


34

3.2.3. Cht PC1: -sitosterol-3--D-glucopyranozit

Hp cht PC1 c phn lp trong cn dch clorofom bng sc k ct

nhi silicagel ra gii bng h dung mi etylaxetat : metanol (70-30), thu

c 10 mg tinh th dng bt mu xm. Rf = 0,65 trong h dung mi D, Rf =

0,45 trong h dung mi E. Nhit nng chy o c l 276-2800C.

Phn tch ph 1H-NMR ca cht PC1 thy rt ging vi ph ca cht PH2

tc l ging vi ph ca stigmasterolglucozit ngoi tr khng pht hin thy

H =5,15 v H =5,03 ca cc proton lin kt C-H ti cc v tr C22 v C23,

iu ny gi cho bit c th cht PC1 l glycozit ca -sitosterol.

Khi thu phn cht PC1 bng dung dch HCl 2N ta thu c cht kt ta

m c Rf ln ph 1H-NMR ca n u trng lp vi ph v Rf ca -sitosterol.

Phn dung dch thu phn c nhn dng ng glucoz bng phng php

sc k giy c so snh vi glucoz ca phng th nghim thy cho cc s liu

trng nhau. T kt qu trn c th quy kt cht PC1 l -sitosterol-3--D-

glucopyranozit.

-sitosterol-3--D-glucopyranozit.


35

Hnh 3.3: Ph 1H-NMR ca: -sitosterol-3--D-glucopyranozit.


36

3.2.3. Cht PE1 :8-C--D-glucopyranozyl apigenin (Vitexin)

Hp cht PE1 thu c di dng cht rn v nh hnh mu vng, khi

lng 12 mg, thu c cn dch etylaxetat khi ra gii h dung mi

etylaxetat : metanol (9:1). Nhit nng chy o c l 271-2740C.

Ph UV v ph NMR ca PE1 c dng ph ca hp cht flavonoit

glycozit. C tn hiu rng singlet cng hng vng trng rt thp ( =

13,15 ppm) gi v s c mt ca nhm OH v tr C-5 (vng A) v tham

gia lin kt hidro vi oxi trong nhm C=O v tr C-4 (vng C). Hai tn hiu

mnh singlet khc = 6,76 c trng cho proton ni i ti C-3 ca cc

flavon. vng B ch c 1 nhm OH v tr C-4 cng c minh chng trong

ph 1H-NMR vi tn hiu doublet = 8,0; J = 8,1 Hz (2H c trng cho cc

H-2 v H-6

). Mt tn hiu doublet na = 6,9 vi J = 11 c trng cho

proton H-3 v H-5. Proton c tn hiu = 6,26 l ca proton ca H-6.

Cc proton ca phn ng cng c pht hin trong vng tn hiu = 3,4-

4,96. ng ch l proton cacbon anome (C-1) c gi tr cao hn c = 4,96 (J =

11Hz) so vi cc gi tr tng ng trong trng hp 0-glycozit. iu ny cho php

ngh n lin kt glycozit hp cht PE1 l lin kt C-glycozit.

Ph 13C-NMR xut hin tn hiu tng ng ca 15 C thuc khung flavon v

6 cacbon ca mch ng. Nhm cacbonyl c nhn dng bi tn hiu = 181,88.

Vng B c 2 tn hiu cao gp i cc tn hiu khc = 128,69 v = 115,71.

chnh l cc tn hiu ca C-2, C-6 v ca C-3, C-5. V tr C-4 c nhn ra vi tn

hiu = 160,98. Cn tn hiu ca C-1 = 121,51.

Cc gi tr C ca phn t ng l 61,19 (C-6); 70,53 (C-4

); 70,84

(C-2); 73,38 (C-1

); 78,5 (C-3

) v 81,62 (C-5

) l hon ton ph hp vi cc

tn hiu ca ng glucopyranozit c lin kt C-C [ti liu 33]. Khi so snh

cc gi tr ph v tnh cht vt l khc ca cht PE1 vi cht 8-C--D-

glucopyranozyl apigenin (Vitexin) cng cho kt qu rt ph hp vi [5]

Trn ph HSQC v HMBC ca cht PE1 cng cho php gn dch

chuyn ho hc ca cc v tr trong cng thc cu to ca cht PE1.


37

Tng tc ca H-1 vi C-2, C-3, C-5, C-6, C-7, C-8 khng nhng

khng nh thm cc gi tr dch chuyn ho hc ca cc v tr trn m cn

khng nh s to lin kt C-glycozit v tr C-8. Tng tc HMBC ca H-6

( = 6,26) vi C-8 v C-10; tng tc ca H-3 vi C-10 v C-1 cng cho

bit hai nhm OH phi v tr C-5 v C-7 v ni i C-2 vi C-3

T cc lp lun trn v t kt qu ca cc ph NMR c th quy kt cho

cc v tr nh bng 3.2

Bng 3.2: S liu ph 13C-NMR (DMSO-C13CPD) ca PE1

V tr C

(ppm)

n ppm

(J.Hz) V tr

C

(ppm)

n ppm

( J.Hz)

2 163,78 1

121,51

3 103,80 6,76- S 2

128,69 8,0 ; d ; J = 8

4 181,88 3

115,71 6,4 ; d ; J = 9

5 160,31 4

160,98

6 98,09 6,26- S 5

115,71 6,4 ; d ; J = 9

7 160,97 6

128,69 8,0 ; d ; J = 8

8 104,48 1

73,38 4,46 ; d ; J = 11

9 154,82 2

70,84 4,68 ; d ; J =9

10 102,34 3

78,59 3,75 ; br

4

70,53 3,5 ; br

-OH 13,155 5 81,62 3,4 ; br

6

61,19 3, 8 ; br

8-C--D-glucopyranozyl apigenin


38

Hnh 3.4: Ph 1H-NMR ca:8-C--D-glucopyranozyl apigenin


39

Hnh 3.5: Ph 13C-NMR ca:8-C--D-glucopyranozyl apigenin


40

KT LUN

1. Ln u tin, cy hn the (Desmodium heterophyllum Papilionaceac)

mc hoang ti Thi Nguyn c nghin cu sng lc ha thc vt, thit

lp c quy trnh ngm chit mu hp l thu c 4 dch chit chn lc.

2. T ton b phn trn mt t (thn v l) cy hn the (Desmodium

heterophyllum Papilionaceac), bng phng php phn tch nh tnh khng

nh trong cy hn the t nht c cha 7 nhm hp cht thin nhin quan trng

l: ng kh, ankaloit, steroit, flavonoit, poliphenol, cumarin v saponin.

3. Ln u tin, phn lp v xc nh c cu trc ha hc ca 4 hp

cht c trong cy hn the l Stigmast-5,22-dien-24R-3 -ol; 3-O--D-

glucopyranozyl stigmasterol; -sitosterol-3--D-glucopyranozit v 8-C--D-

glucopyranozyl apigenin .

KIN NGH

Cy hn the v ang c s dng nh mt dc liu lm thuc cha

bnh trong nhiu bi thuc y hc c truyn. Nhng nhng nghin cu ho hc

v loi thc vt ny cn mc rt t. Trong khun kh ca ti, chng ti

mi ch thc hin mt s nghin cu ban u v mt ho hc tm hiu cc

hp cht c trong cy thc vt hn the. Chng ti ngh tip tc nghin cu

su hn na v bn cht ho hc cc hp cht c trong cy hn the, nghin

cu hot tnh sinh hc ca n lm r cc c s khoa hc cho vic s dng

cy hn the lm thuc cha bnh.


41

TI LIU THAM KHO

A. Ti liu ting Vit

1. V Vn Chi (1997), T in cy thuc Vit Nam, NXB Y hc -TPHCM, 1997.

2. Nguyn Vn n (1997), Cc phng php nghin cu cy thuc, NXB Y-

Dc, Tp. H Ch Minh.

3. Nguyn Hu nh- Trn Th (1999), ng dng mt s phng php

ph nghin cu cu trc phn t. NXB Gio dc.

4. Tt Li ( 1999), Nhng cy thuc v v thuc Vit Nam, NXB Y hc

H Ni, 1999.

5. Nguyn Th Hng Vn, Phan Vn Kin, Chu Vn Minh... Cc hp cht

flavonoit phn lp t l cy b kt, Tp ch Dc hc s 379 (11/2007).

B. Ti liu ting Anh

6. Agrawat.P.K. Cacbon-13 NMR of flavonoids. Elevier Science Publishers

B.V 1989 p 324-326.

7. Chernobrovaya, N.V., Khim. Prir. Soedin., 1973, 9, 801; Chem. Nat.

Compd. (Engl. Transl.), 1973, 9, 767 (2''-xylosyl)

8. U.S. Pat., 1993, 5 233 050; CA, 119, 179344 (isol, deriv)

Lam, Y.K.T. et al., J. Antibiot., 1994, 47, 724 (isol, deriv)

9. Moriyasu, M. et al., J. Nat. Prod., 1997, 60, 299-301 (Trimethyltryptamine)

Roseghini, M. et al., Z. Naturforsch., C, 1976, 31, 118-120

(Trimethyltryptamine)

10. Wilkinson, S., J.C.S., 1958, 2079-2081 (5-Methoxy-N-methyltryptamine, isol)

Patchter, I.J. et al., J.O.C., 1959, 24, 1285-1289 (5-Methoxy-N,N-

dimethyltryptamine)

Ghosal, S. et al., J.O.C., 1966, 31, 2284-2288 (5-Methoxy-N-

methyltryptamine, 5-Methoxy-N,N-dimethyltryptamine, 5-Methoxy-N,N-

dimethyltryptamine oxide)


42

11. U.S. Pat., 1993, 5 233 050; CA, 119, 179344 (isol, deriv)

Lam, Y.K.T. et al., J. Antibiot., 1994, 47, 724 (isol, deriv)

12. Roseghini, M. et al., Z. Naturforsch., C, 1976, 31, 118-120 (Bufoviridine,

Bufotenidine O-sulfate)

Erspamer, V. et al., Biochem. Pharmacol., 1959, 2, 270 (Bufoviridine)

13. Cahill, W.M. et al., J. Biol. Chem., 1938, 126, 29-36 (isol, config)

14. Mandava, N. et al., Phytochemistry, 1974, 13, 2853 (Dimethyltryptophan)

15. Yamaguchi, K. et al., J. Agric. Food Chem., 1979, 27, 847 (isol)

Ghosal, S. et al., Planta Med., 1972, 22, 434 (isol, deriv)

Shure, K.B. et al., J. Agric. Food Chem., 1994, 42, 350 (isol)

16. Yang, J. et al., Yaoxue Xuebao, 1993, 28, 197-201; CA, 119, 156209m

(Desmodilactone)

17. Agurell, S. et al., J. Nat. Prod., 1971, 34, 183 (isol)

Konno, C. et al., Planta Med., 1979, 35, 150 (isol)

18. Ranieri, R. et al., J. Nat. Prod., 1976, 39, 172-174 (isol, N-Me)

Bruhn, J.G. et al., J. Nat. Prod., 1976, 39, 175-177 (isol)

Lindgren, J.E. et al., J. Nat. Prod., 1976, 39, 464-466 (isol, struct, derivs)

Pardanani, J.H. et al., J. Nat. Prod., 1977, 40, 585-590 (isol)

19. Ghosal, S. et al., Phytochemistry, 1973, 12, 193 (isol, deriv)

Bruhn, J.G. et al., J. Pharm. Sci., 1974, 63, 574 (isol, derivs)

Ranieri, R.L. et al., J. Nat. Prod., 1976, 39, 172 (deriv)

20. Gven, K.C. et al., Phytochemistry, 1970, 9, 1893 (isol, Phyllophora)

Rao, K.V., Planta Med., 1975, 27, 31 (isol, deriv)

21. Rondest, J. et al., Bull. Soc. Chim. Fr., 1968, 2411-2414 (N-4-

hydroxycinnamoyl)

Stuart, K.L. et al., Phytochemistry, 1971, 10, 460 (isol, derivs)

22. Yang, J.S. et al., Yaoxue Xuebao, 1993, 28, 197; CA, 119, 156209m


43

23. Botta, B. et al., Phytochemistry, 2003, 64, 599-602 (isol, cd, uv, pmr, cmr, ms)

24. Ahluwalia, V.K. et al., Indian J. Chem., 1966, 4, 250 (isol)

25. Bohlmann, F. et al., Phytochemistry, 1980, 19, 1815 (isol)

Kalidhar, S.B. et al., J. Indian Chem. Soc., 1984, 61, 561 (isol)

Souza, M.P. et al., Phytochemistry, 1989, 28, 2467 (isol)

26. Botta, B. et al., Phytochemistry, 2003, 64, 599-602 (isol, cd, uv, pmr, cmr, ms)

27. Tsanuo, M.K. et al., Phytochemistry, 2003, 64, 265-273 (isol, pmr, cmr, ms)

28. Mizuno, M. et al., Phytochemistry, 1992, 31, 361 (isol, cmr, pmr, struct)

Lin, Y.-L. et al., Heterocycles, 1993, 36, 1501 (Oblongin, Oblonginol)

29. Monache, G.D. et al., Phytochemistry, 1996, 41, 537 (isol, uv, ir, cd, pmr,

cmr, ms)

30. Ueno, A. et al., Chem. Pharm. Bull., 1978, 26, 2411 (isol)

Khan, H. et al., Tetrahedron, 1974, 30, 2811 (isol, pmr)

31. Purushothaman, K.K., Phytochemistry, 1975, 14, 1129 (isol, uv, pmr, ms, struct)


44

H

ong


45


46

Documents

Han the (Co Vitexin)