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I HC THI NGUYN

TRNG I HC S PHM
---------------------------------

BI VN BNH

NGHIN CU HO HC V
NHN DNG MT S NHM CHT
C TRONG CY NGN
(CRATOXYLUM PRUNIFOLIUM KURTZ)

LUN V N TH C S HO HC

THI NGUYN - 2008

I HC THI NGUYN
TRNG I HC S PHM
---------------------------------

BI VN BNH

NGHIN CU HO HC V
NHN DNG MT S NHM CHT
C TRONG CY NGN
(CRATOXYLUM PRUNIFOLIUM KURTZ)
Chuyn ngnh: Ho hu c
M s: 60.44.27

LUN V N TH C S HO HC

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

THI NGUYN - 2008

DANH MC CC CH VIT TT DNG TRONG LUN VN

SKLM

Sc k lp mng

UV

Ultraviolet spectrocopy

MS

Mass Spectroscopy

EI-MS

Electron Impact Mass Spectroscopy

LC-MS

Liqud chromatography - Mass Spectroscopy

FT-IR

Fourier Transform Infrared Spectroscopy

NMR

Nuclear Magnetic Resonance

H-NMR :

13

C-NMR :

H-Nuclear Magnetic Resonance

13

C- Nuclear Magnetic Resonance

DEPT

Distortionless Enhancement by Polarisation Transfer

COSY

Correlated Spectroscopy

HSQC

Heteronuclear Spectroscopy- Quantum Coherence

HMBC

Heteronuclear multiple - Bond Correlation

HIV

Human Immunodeficiency Virus

Unit

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M U
Vit Nam nm trong vng nhit i, gi ma, ma thun gi ho nn
h thc vt rt phong ph, a dng. l ngun ti nguyn sinh hc qu gi
thuc loi c th ti to c. Ngay t xa xa cha ng ta bit khai thc
ngun ti nguyn qu bu ny lm n, thc phm, thuc cha bnh, vt
liu xy dng v nhin liu phc v cuc sng.
Trong th gii thc vt y c nhng loi cung cp thc n cho chng ta,
c nhng loi cung cp vt liu, c loi cung cp hng thm, qu ngt, c
nhiu loi c dng lm thuc cha bnh, thuc tr su, cung cp nguyn
liu cho cc ngnh cng nghip thc phm, m phm v dc phm.
Vit nam c mt v tr thun li v thin nhin nh vy nn nn Y hc
dn tc c truyn pht trin t lu i, chim v tr c bit trong vic bo v
sc kho nhn dn trong sut hng ngn nm u tranh dng nc v gi
nc. Tip thu truyn thng qu bu ca cc th h cha ng, ngy nay ng
v Nh nc ta xy dng nhiu chnh sch, nhiu hnh thc ng vin
nhm k tha v pht huy tt ngun ti nguyn qu bu c th ti to c
phc v con ngi c hiu qu nht.
Nhiu cy c c trng dng lm thuc, nhiu loi dng lm
nguyn liu cung cp tinh du cho cng nghip dc phm v m phm nh
bc h, thanh hao hoa vng, hoa hi, c loi c dng lm thc phm
chc nng ung nh cc loi tr, cc loi sm v.v... Trong s c cy
ngn (Cratoxylum prunifolium) thuc loi cy mc hoang di v ph bin
khp cc vng trn lnh th nc ta. c bit cy ngn c nhiu cc
tnh min ni pha Bc, Ty Nguyn v Nam B.

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Cy ngn v ang c dng lm thuc v lm nc ung
trong phm vi dn gian mt cch kh ph bin cc nc chu , Vit
Nam c bit vng ng bo dn tc min ni pha Bc nc ta. Gn y
ngi ta pht hin dch chit ca l cy ngn c tc dng cha bnh gan,
c tc dng tt vi h thn kinh v tc dng ca n khng thua km g cc
thuc nhp ngoi.
Cy ngn c dng trong dn gian c t lu, nghin cu ho
thc vt cy ngn th mi ch c cc nh khoa hc ch n trong mt
s nm gn y, gp phn lm r thm thnh phn ho thc vt ca cy
ngn to thun li cho vic dng, s dng cy thuc ny lm dc liu v
nguyn liu cho cc mc ch khc, chng ti chn ti: Nghin cu ha
hc v nhn dng mt s nhm cht c trong cy ngn (Cratoxylum
prunifolium). i tng nghin cu l cy ngn mc hoang, thu hi vo
thng 11 nm 2007 ti x Thnh c thnh ph Thi Nguyn .

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Chng 1

TNG QUAN
1.1. M t thc vt
Theo ti liu v phn loi thc vt, cy ngn cn gi l cy thnh
ngnh, lnh ngnh tu thuc vo tng a phng tn khoa hc l
Cratoxylum prunifolium Kurtz thuc h Ban Hypericaceae [1, 2, 5, 8, 11]
Cy ngn phn b rng ri vng Nhit i, Cn Nhit i, n i,
ch yu vng n i bn cu Bc. Chi Cratoxylum Vit Nam c 5 loi:
Cratoxylum maingayi Kurz c tn l lnh ngnh nh, phn b Bc Trung B
v Lm ng; Cratoxylum prunifolium Kurz lnh ngnh l hp phn b
vng ng Nam B trn lnh th VIt Nam; Cratoxylum prunifolium Kurtz
cy ngn, cn gi l thnh ngnh p mc ph bin vng ng Bc
nc ta; Cratoxylum polyanthum Korth hay Cratoxylum ligustrinum Blume
lnh ngnh; Cratoxylum cochinchinense Blumer cn gi l lnh ngnh Nam
B. Hai loi cui ch yu mc Nam B song cng kh ph bin cc tnh
ng Bc B.
Cy ngn - Cratoxylum prunifolium Kurtz thng thy mc t nhin
hu ht cc tnh min ni v trung du pha Bc Vit Nam c nhiu Xun
Mai - Ba V, H Ty, Sapa, Ph Th, Thi Nguyn, Bc Cn, Lng Sn, Vnh
Ph. Cy ngn cng phn b ph bin cc nc Chu nh: Trung
Quc, Malaysia, Philippin, n , Indonexia, Myanma, Thi Lan.
c im sinh trng v pht trin ca cy ngn:
Cy ngn l loi cy nh, thn g, c gai gc (trong rng lu nm
cy c th cao v to) cnh non c lng t, mu nn gi l ngn [11]. L
hnh mc di 12 - 13 cm, rng 3,5 - 4 cm, mc i xng, cung ngn 3 - 5
mm. Mt gn chnh mu n 1/3 l non, gn l v l c mu n qu

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na. Hoa mc trn nhng cnh k l mu trng hoc hng c lng mu ta.
Qu nang di 15mm, rng 7 - 8mm. Ht hnh trng di 6mm, rng 3mm

Hnh 1.1. nh cy ngn (Cratoxylum Prunifolium Kurt, Hypericaceae)


1.2. Mt s cng dng ca chi Cratoxylum
Hu ht cc loi thc vt thuc chi Cratoxylum u c nhn dn
s dng lm cc bi thuc dn gian cha bnh, chm sc v bi
dng sc kho.
Cy ngn Cratoxylum prunifolium mi thy dng trong phm vi
nhn dn, lm thuc kch thch tiu ho, n ngon hng ngy hoc sau khi au
yu, sau khi sinh. Kinh nghim cho thy ngy ung chng 15 - 30 g l kh
di hnh thc sc hoc pha nh tr. C khi phi hp vi l vi nu nc
ung cho tiu cm em li hiu qu cao cho sc kho con ngi [11].
Nhn dn ta cc vng thu hi l cy ngn vo dp tt oan Ng phi
kh nu nc ung mi khi cm thy c th mt mi..

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Trong nhng nm gn y mt s tc gi nghin cu tc dng cha
bnh ca dch chit ton phn l v thn cy ngn. Nhm nghin cu ca
Hc vin Qun Y - H Ni cho bit cy ngn t c, dnh chit ton phn
ca l v thn cy ngn c tc dng chng xi ho tt, hot tnh t 69%
trong khi tanakan ch t 48% [9] v c tc dng hot huyt, lm lu thng
mu, gim ng nhng trng hp tng ng [12].
B mn Dc hc qun s - Hc Vin Qun Y trn c s nghin cu lm
sng dnh chit ca l ngn Cratoxylum prunifolium trn chut nht trng v
th c so snh vi thuc tanakan do hng Beaufour Ipsen ca Php sn xut
a ra kt lun:
Dch chit ton phn l ngn v dch chit etylaxetat l ngn u
c nh hng ln hot ng ca h thn kinh cc mc khc nhau:
- Dch chit etylaxetat v dch chit ton phn l ngn, Tanakan u
c tc dng hot ho h thn kinh thc vt, th hin s tng r hm lng
Catecholamin trong mu ng vt th nghim sau khi ung cc thuc ny.
- Dch chit etylaxetat l ngn c tc dng tng ng Tanakan
gy hot ho ng b cc t bo no th thc nghim, th hin lm gim
thnh phn sng chm delta, tng thnh phn sng alpha trn in no , cn
dch chit ton phn l ngn khng th hin r tc dng hot ho h thn
kinh trung ng.
- Dch chit etylaxetat l ngn c tc dng lm tt c hai qu trnh
hng phn v c ch c iu kin no b ng vt th nghim (thng qua
hot ng phn x c iu kin), tng ng vi Tanakan v tt hn tc
dng ny ca dch chit ton phn l ngn.
- Mc nh hng lm tng cng chc nng h thn kinh trung
ng ca cc cht theo th t gim dn: dch chit etylaxetat l ngn,
Tanakan, dch chit ton phn l ngn [4].

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i vi cao nc (cn tng) 30 - 80 mg/kg c hot tnh khng khun
vi cc vi khun staphylococcus auresu, Escherichia coli, Salmonella typhi.
Dch chit tng s t l cy Cratoxylum cochinchinense Thi Lan
c ng-i ta th nghim v cho thy chng c tc dng chng nhim khun,
chng xi ho, nhng hot ng ca t bo [22, 23, 24].
Singapo dch chit cy Cratoxylum cochinchinense c s dng
trong nhn dn c tc dng chng cc gc t do cha cc bnh lin quan n
s cng thng, mt mi [25].
Ti Nht Bn, t r cy ca mt s loi thuc chi Cratoxylum ngi ta
bo ch thnh thuc lm tng tr nh, chng lo ho, mt ng ngi gi.
Trung Quc, l ca cy ngn ngi ta ch bin thnh tr pha
nc ung hng ngy v s dng mt cch rng ri. T phn dch chit
etylaxetat ca l cy ngn tch c cc xanthone c tc dng khng u
trng ca mui gy st da vng hn c cht rotenon, khng nhng th cc
cht ny cn c nhiu trin vng lm thuc chng mi [28].
Phn dch chit etylaxetat ca Cratoxylum cochinchinense c tc dng
km hm peroxit ho lipit mng t bo gan, km hm s xut hin lipit c tnh
gy vim gan v c tc dng bo v gan tt [27].
Theo cc lng y, cy ngn c v ngt, tnh mt b phn dng l
non, v cy, v r. Ngi b au bng n khng tiu, ung nc nu ca l
lnh ngnh gip tiu ho tt thng dng 100g l non nu mt lt nc, thay
nc ung hng ngy. Khi b cm nng, st th dng l non 50g nu vi 1 lt
nc ung.
Trong dn gian Vit Nam, cy ngn mi thy dng trong phm vi nhn
dn lm thuc kch thch tiu ha, phc hi sc kho khi m au, sinh , bo v
thnh mch, chng lo ho, tng tr nh ngi cao tui. Ngi ta thu hi l
ngn vo dp Tt mng 5 thng 5 (Tt oan Ng) lm thc vt nu nc

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ung hoc c th sau em phi kh mi dng. Gn y c mt vi tc gi
Vit Nam nghin cu v cho thy l ngn c tc dng cha bnh, c tc
dng chng gc t do, bo v thnh mch, bo v t bo gan chng m lo,
chng xi ho v min dch invitro kh r [7].
Ti c s sn xut kinh doanh thuc thnh phm, thc phm chc nng
Y hc c truyn Thi Nguyn, Bc s Hong Sm s dng dch chit ca l
ngn lm thc phm chc nng, thay ch lm nc ung, cha cc bnh
nan y nh: nga, gh l, ronan thn kinh, mt ng, ming ng n khng
ngon, gim tr nh
1.3. Tnh hnh nghin cu ha hc thc vt chi Cratoxylum
1.3.1. Cc hp cht c khung triterpen
Theo ti liu [19] t dch chit n-hexan ca thc vt Cratoxylum
cochinchinense tch c ngoi cc triterpen c 5 vng ngng t nhm
lupeol v -mangostin cn phn lp c cc triterpen c hai vng ngng t.
Sau khi tch bng cc phng php sc k v da vo kt qu phn tch cc
ph ca cc cht c tch ra ngi ta ngh cng thc cu to ca 7 hp
cht triterpen hai vng l:

I.a: (13E,17E)-polypoda-8 (26),13,17,21-tetraen-3-ol; (R= -OH).


I.b: -polypodatetraene; (R= H).

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27

28

29
30

25
26

II.a: (13E,17E)-polypoda-7,13,17,21-tetraen-3-ol; (R= - OH).


II.b: - polypodatetraene; (R = H).
II.c: (13E,17E)-3-axetoxypolypoda-7,13,17,21-tetraene; (R= - OAc).
II.d: (13E,17E)-polypoda-7,13,17,21-tetraen-3-one; (R= O).

III.a: Polypoda-13,17,21-trien-8 -ol; (R=H).


III.b: Polypoda-13,17,21-trien-3-one; (R = - OH).
III.c : Polypoda-13,17,21-trien-3, 8 -diol; (R= O).
1.3.2. Cc cht axit hu c
T l mt loi rau thuc loi Cratoxylum formosum Thi Lan Pilchaon
Maisuthisakul [23] phn lp c hai axit hu c IV, V v cho bit cc
hp cht y u c hot tnh chng oxi ha. Sau khi tch, phn lp tc gi
ngh cu trc ca n c cng thc l:

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COOH
HO
O
HO

O
OH
OH
OH

IV: Acid chlorogenic

OH

HO
OH

O
O

O
O

HO

HO

HO

OH

V: Acid dicaffeoylquinic
1.3.3. Cc cht c khung xanthone
Cc xanthone l nhm cht ln nht, ph bin nht c hu ht cc
loi thuc chi Cratoxylum [6, 14, 17, 19, 21, 22, 23, 24, 25, 26, 27, 29, 30].
pht hin ra l cc xanthone v dn xut di dng C-glycoside ca
chng. T loi Cratoxylum cochinchinense [25] Soon Yew Tang cng vi cc
cng s phn lp c C-glicoside ca xanthone v xc nh c cng
thc cu to ca n c dng VI.

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HO

OH

O
HO

OH

HO
OH

OH

VI:

1,3,6,7-tetrahydroxyxanthone- C2--D-glycoside.

T loi Cratoxylum formosum Nawong Boonnak cng cc cng s [22]


cng phn lp c cc xanthone c cu trc VII di y:

OH

OH

MeO

O
HO

HO

R1

OCH3

VII.b: -mangostin; (R1 = OMe)

VII.a: Dulxis-xanthone

VII.c: -mangostin; (R 1 = OH)

OH
R1

OH

OH

VII.d:

Formoxanthone A; (R 1 =

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R1

OH

MeO

HO

VII.e:

3-isomangostin; (R 1 =

).

VII.e1: 3,4-dihidro- 5,9-dihidroxy-8-methoxy-7-(3-methoxy-3-methylbutyl)OMe

2,2- dimethyl-2H,6H-pyrano-[3,2-] xanthen-6-one; (R1 =


VII.e2:

).

3,4-dihidro-5,9-dihidroxy-7-(3- hidroxy-3-methybutyl)-8-methoxyOH

2,2- dimethyl-2H,6H-pyrano-[3,2-] xanthen-6-one; (R1 =


O

).

OH
R1

HO

R2

OH

VII.f:

Isocudraniaxanthone B;

(R1 = H; R2 = OMe).

VII.f1: Gerontoxanthone I;

(R1 =
O

R2

OH

O
OH

VII.g: 10-O-methylmaclur-xanthone;
VII.g1: Macluraxanthone;

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; R2 = OH).

O
R1

(R1 =
(R1 =

; R2 = OMe).
; R2 = OH).

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VII.g2: Xanthone V 1.

(R1 =

VII.g3: 6 - deoxyjacreubin;

(R1 = H; R2 = H).
O

; R2 = OH).

OH
R1

OH

VII.h: Formoxanthone B;

(R1 =

).

T loi Cratoxylum cochinchinese ca min Nam nc ta L Th Diu


Nguyn v Leslie J. Harrison [19] phn lp c ngoi cc triterpen I, II v
III. Hai nh nghin cu xc nh c cc xanthone c cu trc VIII.a,VIII.b.
O

OH

HO

OH

VIII.a: 1,3,7-trihydroxy-2,4-di (3-methyl-but-2-enyl) xanthone; (R = prenyl).


VIII.b: 2-geranyl-1,3,7- trihydroxy-4-3- methylbut-2-enyl) xanthone; (R=geranyl).
T loi Cratoxylum maingayi [14] ngi ta cng phn lp c mt s
hp cht xanthone (IX), cc cht ny c cu trc n gin nhng u c tc
dng nh cc cht c hot tnh chng oxi ho tt.

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O

OH

HO

O
R

IX.a: 1-7- dihidroxyxanthone; (R = H).


IX.b: 1,7- dihidroxy - 4- methoxyxanthone; (R = OMe).
OH
7
6

OMe

8'

9'

OH

2
5 10'
O

4'

IX.c: 2,8 - dihidroxy - 1- methoxyxanthone.


OMe
RO
7
6

8
5

O
8'

OMe
9'

OMe

2
3

10'

4'

OMe

IX.d: 7-hidroxy-1,2,3,8 -tetramethoxyxanthone; (R = H).


T loi Cratoxylum formosanum ngi ta cng pht hin c cc
xanthone c cu trc phn t nh sau [21, 22] .
R3

OMe
OMe

R1

O
R2

X.a: 1,7-dihidroxy-5,6-dimethoxy xanthone; (R1 = R2 =R3 = OMe).


X.a1: 1,2,3,8-tetramethoxy xanthone; ( R1 = R3 = OH; R2 = H).
X.a2: 1,2,3,4,8-pentamethoxy xanthone; (R1=R3=OMe;R2=H).

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R2

OH

HO

O
R1

X.b: 1,7-dihidroxy-4-methoxy xanthone; (R1 = OMe; R2 = H).


X.b1: 1,7-dihidroxymethoxy xanthone; (R1 = R2 = H).
X.b2: 1,7-dihidroxy-8-methoxy xanthone; ( R1 = H; R2 = OMe).
O

OH

OMe

MeO
HO

OH

MeO

O
OMe

X.c: Maclurathone

X.d: 1,5,6,7-tetramethoxy xanythone


OMe

OMe

HO

OH

X.e: 2,7-dihidroxy-1,8-dimethoxy xanthone.


OMe

OH

HO

O
OH

X.f: 1,4,7-trihidroxy-8-methoxy xanthone.

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O

OH

OH

HO

HO

OH

OH

OH

X.g: 1,4,7-trihydroxy xanthone.

X.h: 1,3,5,6-tetrahidroxy xanthone.

2'

O
1

H3CO

8'

B
4''

HO

1'

9'

4'

OCH3

'''

OH

1''

X.i: Fruniflorone;

3''

(R=

5''

2''

) [22]

1.3.4. Mt s i din ca khung anthraquinon


T loi Cratoxylum formosum ngoi vic xc nh cc xanthone
Nawong Boonnak, Chatchanok Karalai v cc cng s [22] cn phn lp
c cc anthraquinon c cc cu trc XI. Cc tc gi cn cho bit cc cht
ny c hot tnh sinh hc rt cao. Chng c tc dng tt trong vic iu tr
bnh tiu ng, vim nhim, h ng huyt [20].
OH

OH

OR
O

XI.a: 3-geranyloxy-6-methyl-1,8-dihidroxy-anthraquinon;
(R=
XI.a1: madagascin;

)
(R =

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XI.a2: physcion;

(R = Me)

XI.a3: emodin;

(R = H )

OH

OMe
O

XI.b: 11-hidroxyl-5-methoxy-2,2,9-trimethyl-2H-anthra-[1,2-] pyran-7, 12-dion.


OH

OH

HO

OMe

OH

OH

XI.c: Vismiaquinon

XI.d: 3,4 - dihidrojacareubin

1.3.5. Mt s i din ca khung flavonoit


T loi Cratoxylum cochinchinense, Cratoxylum formosanum nhiu tc
gi phn lp c 2 flavonoit XII.a v XII.b [18, 13, 15]. Chng c hot
tnh km hm peroxit ho lipit mng t bo gan, c tc dng tng kh nng
thi c ca gan, c tc dng km hm s xut hin lipit c tnh.
OH
HO

HO

OH

OH
OH

OH

OH

XII.a: ihirokampherol

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OH

XII.b: Taxifolin

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1.4. Nhng nghin cu ho thc vt loi Cratoxylum prunifolium.
T l ca loi Cratoxylum prunifolium, Xue-Li-Cao, Yoichiro Ito
dng phng php sc k kh lng tc cao phn lp c epigallocatechin
gallat (XIII.a) v epicatechin gallat (XIII.b) [27].
OH

OH

HO

OH
OH
O

OH

C
O
OH
OH

XIII.a:

Epigallocatechin-3-O- gallate

HO

OH

O
OH

OH
OH

C
O
OH
OH

XIII.b:

Epicatechin-3-O-gallate

Theo Nguyn Lim v cc cng s [10], bng cc phn ng nh tnh


cho bit Cratoxylum prunifolium c cha cc flavonoit, tanin pyrocatechic
phitosterol saponin triterpen v cc axit hu c, ng kh. Tc gi cn cho
bit cy ngn ca Vit Nam khng thy c ancaloit v anthraquinon
T v cy ngn Cratoxylum prunifolium. Phm nh Hng, Nguyn
Diu Lin Hoa v cc cng s ti i hc Quc gia thnh ph H Ch Minh
phn lp c macluraxanthone, 1,7-ihidroxanthone v hai xanthone mi

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[6] v cho bit chng u c tc dng dit mui gy st rt v c tc dng tt
trong vic dit mi.
T v v r cy Cratoxylum prunifolium trng tnh Nng Khai min
Nam Thi Lan, Nawong Boonnak v cc cng s chit bng iclometan,
phn lp cc cht bng sc k tch c 10 xanthone vi s khc nhau ch
l cc mch nhnh (R 1 v R2) [22] v tc gi gi tn chng l cc
prunifloron c nh s t 1 n 10. Cng thc ca chng c cu trc XIV.
R1

OH

RO

R2

HO

OR

Prunifloron XIV; R1 R2 v u l gc ankyl hoc dn xut ca n.


R: l H hoc CH3
Tn ca chng ln lt l: XIV.1 Pruniflorone A; XIV.2 Pruniflorone
B; XIV.3 Pruniflorone C; XIV.4 Pruniflorone D; XIV.5 Pruniflorone E;
XIV.6 Pruniflorone F; XIV.7 Pruniflorone G; XIV.8 Pruniflorone H; XIV.9
Pruniflorone I; XIV.10 Pruniflorone J.
Cng t r ca thc vt Cratoxylum prunifolium cc tc gi Thi Lan
li pht hin c mt anthraquinon m cu trc ca n c tc gi m t c
dng XV.
OH

OH

OH
O
O

XV: Anthraquinon

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Chng 2

PHN THC NGHIM


2.1. i tng v phng php nghin cu
2.1.1. Thu mu cy, xc nh tn khoa hc v phng php x l mu
Nguyn liu nghin cu gm l cy ngn, thu hi vo thng 11
nm 2007 ti x Thnh c - Thnh ph Thi Nguyn.
Cy ngn, cn gi l cy thnh ngnh p, lnh ngnh p, c tn
khoa hc l: Cratoxylum prunifolium Kurtz , h Hyperaceas hay h Ban.
Mu cy ti thu hi gm c cnh nh v l c em sy 800C
dit men, sau sy kh nhit 500C cho ti khi kh hon ton. Mu kh
c chia ring thnh cc phn l, v cnh. Chn mu l lm i tng
nghin cu. Nghin nh mu ngm, chit trong etanol 90o nhit phng
nhiu ln.
Sau khi ct loi dung mi, cn c c di dng xiro, chit ln lt
bng cc loi dung mi c phn cc tng dn: clorofom, etylaxetat,
n-butanol, metanol, cc dch chit c bay hi dung mi bng thit b ct
quay nhit 500C di p sut thp. Cc cn th c phn chia bng sc
k ct 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 .
2.1.2. 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 ngn chng ti phi hp s dng cc phng php sc k
v kt tinh li trong dung mi thch hp, cc phng php gm:
- Sc k lp mng (SKLM).
- Sc k ct silicagen thng dng Merck 63- 200nm.
- Kt tinh phn on v kt tinh li.

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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 tnh cht vt l c
trng: mu sc, mi v, h s Rf, im nng chy, ghi cc loi ph nh: ph t
ngoi (UV), ph hng ngoi (FT-IR), ph cng hng t ht nhn 1H- NMR,
ph 13C- NMR, tu theo tng loi cht. Cc s liu ph thc nghim ca cc
cht sch c dng nhn dng cu trc ho hc ca chng.
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 hay dng
trong phn tch l loi tinh khit phn tch (PA).
Sc k lp mng dng tm mng nhm DC - Alufolien Kiesegel 60
F254 Art.5554 trng sn, dy 0,2mm c s dng xc nh s b s
thnh phn c trong cc dich 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

90:10

H A

2. n-Hexan - etylaxetat

95:10

H B

3. Clorofom - metanol

90:10

H C

4. Clorofom - metanol

50:10

H D

5. Clorofom - metanol

20:10

H E

Cc tm SKLM sau khi sy kh c soi di n t ngoi (UVBIOBLOCK) bc sng = 254nm v 365nm. Thuc th hin mu l
vanilin 1% trong dung dch metanol-H2SO4 , sau sy nhit trn 1000C

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

chiu di di chuyn ca cht th


chiu di di chuyn ca dung mi

2.2.2. Thit b nghin cu


- Nhit nng chy o trn my Boetus (c) hoc trn my
Eletronthermal IA - 9200.
- Ph hng ngoi ghi trn my IMPACT - 410.
- Ph UV o trn my UV 1700 Phamaspec.
- Ph khi ghi trn my MS- Engine - 5989- HP ion ho bng va chm
eletron (LC- MS) 70ev v s dng ngn hng d liu DATABASE/
WILLEY 250L.
- Ph 1H- NMR v 13C- NMR ghi trn my Bruker 500MHz ni chun
TMS, dung mi CDCl3, MeOD.
2.3. Cc dch chit t cy ngn (Cratoxylum prunifolium Kurtz)
2.3.1. Cc dch chit
L cy ngn sy kh, nghin nh c ngm chit kit bng
etanol 900 nhit phng cho n khi thu c dch khng mu. Dch chit
c ct loi ht dung mi p sut gim cho n dng cao lng, xc nh
khi lng cn kh, sau thm nc vo cn v ln lt chit vi cc loi
dung mi clorofom, etylaxetat, n-butanol, cui cng ui ht nc v ho tan
bng metanol.
Cc dch chit trn c lm kh bng Na2SO4 lc v ct dung mi
bng thit b ct quay di p sut gim nhit 500C. Cn c sy kh
v cn xc nh trng lng.

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Nh vy t l cy ngn thu nhn c 04 phn on cn l: cn
trong clorofom (C), cn trong etylaxetat (E), cn trong butanol (B) v
cn trong metanol (M). S 2.1
MU KH
1. Etanol
2. Cn trong nc
CHCl3

EtOAc

Cn CHCl3
(C)

Cn EtOAc
(E)

BuOH

MeOH

Cn BuOH
(B)

Cn MeOH
(M)

E1

C1

C2

C3

S 2.1. Quy trnh ngm chit


Bng 2.1. Khi lng cht tng s c chit tng phn on l cy
ngn (Cratoxylum prunifolium Kurtz)
Khi lng mu l

Cn chit

(gam)

Clorofom

Etylaxetat

n-Butanol

Metanol

787,4

52,264

34,654

40,411

26,604

100%

6,63%

4,40%

5,13%

3,38%

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2.3.2. Kho st nh tnh cc dch chit
Dng cc thuc th c hiu pht hin cc nhm hp cht thin
nhin c trong hot tnh sinh l cao trong thc vt [3] chng ti thu c kt
qu th nh tnh vi cc nhm cht, kt qu c ch ra bng 2.2.
Bng 2.2. Pht hin cc nhm cht trong cy ngn
Stt Nhm cht

Thuc th c hiu

Hin tng

Kt qu

Saponin

To bt

Bt bn trong axit ++

Glicozttim

Kelle-Kiliani

Vng nu

Cumarin

Axit v kim

Kt ta bng

++

Poliphenol

(FeCl3+K3[Fe(CN)6]) 1% Xanh thm

Flavonoit

Xianidin

T hng n

++

Steroit

Liberman-Bourchard

Mu xanh vng

++

Ancaloit

Dragendorf

Mu vng da cam -

ng kh Felinh

Cho kt ta mu
gch

+++

++

Ghi ch: Du (+) l c phn ng dng tnh, (++) l c phn ng tnh


rt r, (-) l khng c phn ng.
2.3.3. Th hot tnh sinh hc
Th hot tnh vi sinh vt kim nh bng nh tnh theo phng php
khuych tn trn thch, s dng khoang giy lc tm cht th theo nng
tiu chun ti b mn Vi Sinh trng i hc Y Thi Nguyn.
Cc chng vi sinh vt th gm i din cc nhm:
Vi khun Gr (-) Escherichia coli.
Vi khun Gr (+) Staphylococcus auresu.
Vi khun: Salmonella typhi.

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Hnh 2.1. nh c gy c ch xung quanh ging thch

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Chng ti cn tin hnh th cc ng dng lm thc phm chc nng
ti c s sn xut kinh doanh thuc thnh phm, thc phm chc nng Y hc
c truyn Thi Nguyn. Kt qu th hot tnh cc dch chit th c trnh
by trong bng 2.3.
Bng 2.3. Kt qu th hot tnh sinh hc ca dch chit th
t l cy ngn (Cratoxylum prunifolium)
ng knh vng c ch xung quanh ging thch(mm)
Dch chit

Cn tng (H2O)
Cn EtOAc

Staphylococcus auresu Escherichia coli


ng
knh
(mm)
30
29

Hot tnh
+
+

ng
Hot
knh
tnh
(mm)
26
+
17

Salmonella typhi
ng
knh
(mm)
27
18

Hot
tnh
+
+

Ch : Du (+) l phn ng c hot tnh.


2.4. Chit sut, phn lp v tinh ch cc cht t cy ngn
T 787,4g l Cratoxylum prunifolium kh chit nhiu ln bng etanol
900 n khi dch chit trong sut khng mu, loi b bt dung mi ru bng
my ct quay nhit 500C n khi dch chit cn li dng xiro c, thm
nc, lc cn tng s ny vi clorofom n khi thu c tt c cc cht c th
tan c trong CHCl3 u c ly ra ht, c cn dung dch CHCl3 nhit
thp thu uc 52,264g cht (k hiu cc cht C).
Phn cn khng tan trong CHCl3 c lc nhiu ln vi EtOAc cho n
khi dch chit EtOAc hon ton khng mu v trong sut, c cn dch EtOAc
trong my ct quay thu c 34,654g cht tng s (k hiu l cc cht E).
Phn khng tan c trong EtOAc, c lc vi n-butanol sau
cng c cn trong iu kin p sut thp c 40,410g cht (k hiu l cc
cht B).

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Cc cht cn cn li khng tan trong cc dung mi trn cho tan trong
MeOH thu oc 32,346g cht (k hiu l cc cht M).
2.4.1. Dch chit clorofom
T 11g cn t dch clorofom ca l ngn k hiu (C) c phn
chia trn sc k ct silicagel vi cc h dung mi n-hexan - clorofom vi cc
t l tng dn clorofom t 0% n 100% kim tra cc phn on trn sc k
lp mng, cc phn on ging nhau em gp li v ct loi dung mi thu
c ba cht C-1, C-2, C-3.
2.4.1.1. Taraxeron (Friendoolean - 14- en-3-on) (C1)
Ra gii vi cc h dung mi n- hexan-clorofom (50:1), sau khi ct loi
dung mi, cn thu c kim tra trn SKLM trong h dung mi B v pht
hin bng vanilin 1% trong dung dch CH3OH- H2SO4, kt tinh li trong dung
mi trn thu c 21mg cht rn, tinh th hnh kim, mu trng c nhit
nng chy 278-2800C v cho cc c trng ca mt tritecpen.
Ph FT-IR (KBr); max (cm-1): 1720 (C=O); 1500 (C=C); 3050 (CH3);
2940 (CH).
Ph LC- MS, m/z (%) 424 [M+]; 408 [M-O]+ .
PH 1H- NMR (500MHz, CDCl3): (ppm) 0,762ppm (3H, s, CH3);
0,84ppm (3H, s, CH3); 0,885ppm (3H, s, CH3); 0,996ppm (3H, s, CH3);
1,011ppm (3H, s, CH3); 1,02ppm (3H, s, CH3); 1,07ppm (3H, s, CH3)
5,48ppm (1H, dd, 3,21 v 8,17Hz).
Ph

C - NMR (500MHz, CDCl3); (ppm): 38,35 (Cl); 33,08 (C2);

13

217,3 (C3); 47,56 (C4); 55,77 (C5); 19,91 (C6); 33,57 (C7); 38,87 (C8); 48,80
(C9); 33,57 (C10); 17,44 (C11); 36,67 (C12); 37,74 (C13); 157,59 (C14); 117,19
(C15); 34,12 (C16); 35,57 (C17); 48,70 (C18); 40,64 (C19); 28,79 (C20); 35,11
(C21); 37,69 (C22); 29,56 (C23); 21,57 (C24); 14,79 (C25); 25,56 (C26); 29,92
(C27); 26,12 (C28); 33,39 (C29); 21,47 (C30).

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2.4.1.2. Stigmast- 5,22- ien-24R-3-ol
Ra gii ct vi h dung mi n-hexan- clorofom (50:5), sau khi ct loi
dung mi cn thu c kim tra trn SKLM trong h dung mi A, pht hin
bng vanilin 1% trong dung dch CH3OH- H2SO4, kt tinh li trong h dung
mi trn thu c 29mg tinh th hnh kim, khng mu, khng mi, c nhit
nng chy 152-1550C, Rf 100 =61.
Ph FT-IR (KBr); max (cm-1): 3429,2 (OH); 2864,7 (C-H); 1651 (C=C).
Ph EI- MS; m/z (%): 412 [M+] (7); 300 (7); 255 (11); 231 (4); 213
(8); 173 (7); 145 (20); 133 (20); 83 (49,3).
Ph

H- NMR (500MHz, CDCl3); (ppm): 5,33 (1H, dd, j = 5Hz v

2H, H6 ); 515 (1H, dd, j22,23 = 15, Hz, j22,20= 5Hz, H-22); 5,01 (1H, dd, j23,22 =
15Hz, j23,24 = 5Hz, H-23).
Ph 13C- NMR (500MHz, CDCl3); (ppm): 36,4 (t, C1); 29,67 (t, C2);
71,8 (d, C3); 42,3 (t, C1); 139,2 (s, C5); 121,4 (d, C6); 37,3 (t, C7); 31,85 (d,
C8); 51,15 (d, C9); 36,12 (s, C10); 24,35 (t, C11); 42,18 (t, C12); 31,4 (s, C13);
56,83 (d, C14); 25,38 (t, C15); 31,6 (t, C16); 55,9 (d, C17); 12,01 (q, C18); 18,95
(q, C19); 40,47 (d, C20); 21,03 (q, C21); 138,4 (d, C22); 129,2 (d, C23); 50,01 (d,
C24); 33,9 (t, C25); 21,19 (q, C26); 19,79 (d, C27); 28,89 (q, C28); 12,22 (q, C29).
2.4.1.3. - Sitosterol
Tip tc ra gii trn ct vi h dung mi n-hexan- clorofom (40:10),
sau khi ct loi dung mi, cn thu c kim tra trn sc k lp mng trong h
dung mi B, pht hin n bng vanillin 1% trong dung dch CH3OH- H2SO4,
kt tinh li thu c 27mg cht rn, tinh th hnh kim, nng chy 140- 1410C,
Rf .100 = 71.
Ph FT-IR (KBr); max (cm-1): 3450 vn rng (H32, C3); 3010- 1650
(lin kt i).

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Ph EI- MS; m/z (%): 414 [M+] (20); 413 [M-H]+ (41); 398 (28); 397
(100); 395 (32); 383 (11); 361 (11); 257 (3); 255 (6,3); 151 (5,6); 139 (11).
Ph 1H-NMR (500MHz, CDCl3 ); (ppm): 0,68 (3H, s, CH3-18); 1,01
(3H, s, CH3-19); 0,81 (3H, d, j, 7Hz, CH3-26); 0,88 (3H, d, j, 7Hz, CH3-27);
0,83 (3H, t, 7,32Hz, CH3 -29); 0,92 (3H, d, j, 10Hz, CH3-21); 3,52 (1H, m, H3); 5,42 (1H, d, j, 5Hz, H-6).
Ph

13

C- NMR (500MHz, CDCl3); (ppm): 141,1 (s, C5); 121,8 (d,

C6); 71,8 (d, C3); 56,8 (d, C14); 56,1 (d, C17); 50,2 (d, C9); 45,7 (d, C24); 42,5
(s, C13); 42,4 (t, C4); 39,8 (t, C12); 37,3 (t, C1); 36,5 (s, C10); 36,2 (d, C20);
33,9 (d, C8); 31,9 (t, C7); 31,7 (t, C2); 29,2 (d, C25); 28,3 (t, C16); 26,2 (t, C23);
24,2 (t, C15); 21,1 (t, C11); 19,8 (q, C26); 19,4 (q, C19); 19,1 (q, C27); 18,8 (q,
C21); 11,9 (q, C29); 11,9 (q, C18); 23,2 (t, C28).
2.4.2. Dch chit trong etylaxetat (E)
T 15g cn chit etylaxetat ca l cy ngn (k hiu E) c tin
hnh tch cc cht trn sc k ct silicagel, ra gii ct sc k bng h dung
mi clorofom- metanol c t l theo tng dn ca dung mi phn cc,
metanol 0% -100%, kim tra cc phn on trn sc k lp mng, thuc th
pht hin (F eCl3 + K3[Fe(CN)6]) 1% sau gp cc phn on ging nhau,
ui ht dung mi v kt tinh li thu c cht (E1).
* Axit gallic (E1)
Ra gii trn ct bng h dung mi clorofom- metanol (90:10), sau khi
kim tra bng SKLM vi h dung mi C, ct loi dung mi, thu c cn th,
kt tinh li trong h dung mi trn thu c 70mg cht rn mu vng, tinh th
hnh kim, c tn/c khong 248- 2580C.
Ph FT-IR (KBr); max (cm-1): 1700 (C=O); 3100 (OH) rt t.
Ph 1H-NMR (500MHz, MeOD); (ppm): 7,092 (2H, C2 v C6).
PH 13C-NMR (500MHz, MeOD); (ppm): 121,960 (C1); 139,559 (C4);
110,349 (C2, C6); 146,345 (C3, C5); 170,364 (C7).

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Chng 3

THO LUN KT QU NGHIN CU


3.1. Nguyn tc chung
Trong qu trnh nghin cu ha thc vt cn phi tn trng nguyn tc
chung l khng c lm thay i cu trc ha hc ca cc cht sn c trong
thc vt v khng lm nh hng n thnh phn ho hc ca chng ti thi
im ly mu. Nh vy, ngay sau khi mu thu hi xong phi c dit men
trnh s chuyn ho do cc qu trnh sinh tng hp xy ra thc vt, sy
kh nhit thch hp, bo qun mu trong iu kin kh ro.
tch cc cht ra khi thc vt c th c tin hnh bng nhiu cch
khc nhau: p ly du bo, ct li cun bng hi nc tch cc tinh du,
ngm chit bng dung mi hu c v.v Tuy nhin c hai phng php ph
bin hn c tch cc cht ra khi thc vt:
Cch 1: Trc tin chit bng dung mi ru-nc tch hu ht cc
cht ra khi thc vt. Sau chit sng lc bng cc dung mi t khng phn
cc n cc dung mi c phn cc tng dn. Cc dch chit cha cc hp
cht c phn cc gn nhau s c ct ui dung mi, bo qun dng cho
cc qu trnh tch tip theo.
Cch 2: Chit v phn lp cc cht mu thc vt bng cc loi dung
mi t khng phn cc n c phn cc tng dn nh ln lt l: n-hexan,
clorofom, etylaxetat, n-butanol, metanol(etanol), etanol-nc.
Vic chit ly cht t l thc vt (Cratoxylum prunifolium) c thc
hin theo cch 1 (s 2.1).
Cc dch chit tng s v cc phn chit th em th nghim vi cc
tc ng sinh hc v c t t bo nhm gip cho vic nh hng tm kim
cc cht c hot tnh sinh l cao trong nhng dch chit.
Kt qu th hot tnh vi vi sinh vt kim nh nu trong bng (2.2)

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3.2. Phn tch nh tnh v pht hin cc nhm cht
Dng cc thuc th c hiu pht hin cc nhm hp cht thin
nhin c hot tnh sinh l cao trong thc vt [3] cho thy trong dch chit
bng clorofom ca l cy ngn c cha cc axit hu c, streroit,
triterpenoit. dch chit bng etylaxetat ca l cy ngn c cha cc hp
cht polyphenol, cc hp cht flavonoit, cng phn dch chit ny tch
c axit gallic v dn xut ca n. Trong dch chit n-butanol c cha cc
tanin, cc ng kh. Khng pht hin thy c ancaloit, cc xianua trong cc
dch chit ca l ngn. Kt qu ca cc phn ng nh tnh xc nh cc
nhm hp cht thin nhin c hot tnh sinh l cao c ch ra bng 2.2.
3.3. Phn lp v nhn dng cc hp cht c trong cc dch chit khc nhau
ca cy ngn
Cc dch chit t cy ngn (Cratoxylum prunifolium) u l nhng
hn hp phc tp cha cc hp cht khc nhau. phn lp tng cht ra khi
hn hp s dng cc phng php sc k ct, cht hp ph dng l
silicagel, cc h dung mi ra gii thch hp v thng phi lp li nhiu ln.
Vic tinh ch cc cht thng dng phung php kt tinh li trong
dung mi hoc h dung mi thch hp. Nh cch lm thu c cc n
cht c tinh khit cao, p ng cc yu cu kho st tnh cht ho l v
xc nh quang ph ca chng.
Khi phn lp cc thnh phn ho hc t l cy ngn c thc hin
nh trong s 2.1. Bng phng php phn lp trn, t dch chit bng
clorofom ca l cy ngn chng ti thu c 3 hp cht sch l: mt
tritecpen v hai steroit; cn dch chit etylaxetat phn lp c axit gallic v
hn hp cc xanthone vi flavonoit.
3.3.1. Taraxeron ( hay Frendoolean-14-en-3-on) (C-1)
Cht C-1 l cht rn tinh th hnh kim, mu trng c nhit nng
chy 278-2800C n c tch t phn cn chit clorofom. Bng sc k ct

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silicagen vi h dung mi n-hexan:clorofom thu c cht C-1, cht ny
cho cc phn ng nh tnh ca terpenoit.
Khi lng phn t ca cht C-1 c xc nh bng ph khi LCMS cho bit khi lng phn t ca [M+] m/z bng 424,2 tng ng vi khi
lng phn t bng 424 u v tng ng vi cng thc phn t C30H48O.
Trong ph LC-MS cn xut hin pic m/z 408,2 iu ny tng ng vi
mnh [M-O]+ vi m/z bng 408
Ph FT-IR: cho thy cc vn hp th ca nhm xeton (C=O) max =
1720cm-1, lin kt i (C=C) max = 1500cm-1, cc nhm CH3 max =
3050cm-1 v cc nhm CH max = 2940cm-1, khng pht hin thy cc vn
hp th ca nhm OH. iu ny c th khng nh c hp cht C-1
khng c chc ru m l c chc xeton.
Ph 1H-NMR v ph DEPT: cho bit hp cht C-1 c tn hiu ca 8
nhm CH3 u dng singlet (xem hnh 3.2 v 3.4) c cc chuyn dch
ho hc nh sau: chuyn dch = 0,762ppm (3H, s, CH3); = 0,841ppm
(3H, s, CH3); = 0,847ppm (3H, s, CH3); = 0,885ppm (3H, s, CH3); =
0,996ppm (3H, s, CH3);

= 1,011ppm (3H, s, CH3); = 1,021ppm (3H, s,

CH3); = 1,07ppm (3H, s, CH3). dch chuyn ho hc = 5,48ppm


(1H, d, J = 3,21 v 8,17Hz) l ca proton lin kt ti cacbon lin kt i
(nhm = CH ti C15).
Ph

13

C-NMR: nhn thy nguyn t cacbon trng thi lai ho sp2

thuc nhm =CH c chuyn dch ho hc = 117,19ppm. Nguyn t


cacbon bc 4 cng trng thi lai ho sp2 tham gia vo lin kt i vi
nguyn t ny c chuyn dch ho hc = 157,59ppm.
Ph 13C-NMR cn cho mt tn hiu trng rt yu tng ng vi
chuyn dch ho hc = 217,43ppm c trng cho cacbon trong nhm
cacbonyl ti v tr C-3.

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32
Nh vy, ph NMR hon ton ph hp vi ph FT-IR khng thy c
vn c trng ca nhm OH m li thy xut hin vn c trng ca C=O.
Cc nguyn t H i vi nhm C=O thy ch c 2H vi = 2,50ppm (2H,
dd, J = 7,1 v 11Hz), cn c 2 proton v tr ca nhm =CH (ti C16) c
chuyn dch ho hc = 2,27ppm (2H, dd, J = 3,3 v 4,08 v 6,39).
Trn c s phn tch cc tnh cht ho hc, nht l phn tch cc thng
tin c c t ph LC- MS, FT-IR, ph cng hng t ht nhn 1 H-NMR,
13

C-NMR, ph DEPT, ph HMBC, HSQC, ph H-H COSY v phn mm

ACD/HNMR ACD/CNMR m phng ph NMR cng cho php chng ti


quy kt hp cht C-1 l mt triterpen phi c cng thc cu to nh sau:

29

23

30

19 20
18

12

11
1

8 14

22

17

28
16

15

13

27

9
10

2
3

24

21

6
25

26

Taraxeron (hay Frendoolean-14-en-3-on)

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33
Bng 3.1. Ph 13C-NMR v 1H-NMR ca taraxeron (C-1) t l ngn
V tr
cacbon
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30

13

C-NMR
(-ppm)
38,35
33,08
217,3
47,56
55,77
19,91
33,57
38,87
48,8
33,57
17,44
36,67
37,74
157,59
117,19
34,12
35,57
48,7
40,64
28,79
35,11
37,69
29,92
14,79
29,86
21,34
25,56
26,12
33,36
21,47

1
ACD/13CH-NMR
NMR (-ppm)
(-ppm)
38,4
Vng (1,2 - 1,6)
34,1
2,5 (2H; dd; 7,1 v 11,0)
217,3
47,6
55,8
(1,99)
20,0
Vng (1,2 - 1,6)
35,2
Vng (1,2 - 1,6)
38,9
48,7
(1,78 - 1,87)
37,6
17,5
Vng (1,2 - 1,6)
35,8
Vng (1,2 - 1,6)
37,7
157,6
117,2
5,48 (1H; d; 3,21; 8,17)
36,7
2,27 (2H; dd; 3,3; 4;1; 6,4)
37,7
48,8
(1,78 - 1,87)
40,7
Vng (1,2 - 1,6)
28,8
33,6
Vng (1,2 - 1,6)
33,1
Vng (1,2 - 1,6)
29,9
14,8
29,9
21,5
0,76 - 1,07
(24H ca 8 nhm CH3)
25,6
26,2
33,4
21,4

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Hnh 3.1

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35

Hnh 3.2

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Hnh 3.3

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Hnh 3.4

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38
3.3.2. Stingmast -5,22-dien-24R-3-ol (C-2)
Cht C-2 l cht rn kt tinh, c nhit nng chy 1520-1550C,
tch c t dch chit clorofom, phn lp bng sc k ct silicagen, ra gii
bng h dung mi n-hexan-clorofom (90:10).
Ph EI-MS cho pic ion phn t [M+] 412 m/z. Cc ph FT-IR, 1HNMR v 13C-NMR u khng nh s c mt ca nhm hydroxyl, ph FT- IR
c vn 3410cm-1 rng, cn ph NMR cho H-3 = 3,47ppm v C-3 =
71,6ppm). Phn t c hai ni i khng lin hp (IR cho max 1622cm-1).
Ph 1H-NMR ca cc proton thuc nhm metyl (CH) cng khng
nh iu y, H-6 = 5,33ppm (1H, dd, j =15Hz v 2Hz); H-22 = 5,15ppm (1H,
dd, j =15Hz v 5Hz); H-23 = 5,01ppm (1H, dd, j =15Hz v 5Hz). Ph 13CNMR cho thy C-5 = 139,2ppm; C-6 = 121,4ppm; CC-22 = 138,4ppm v C-23
= 129,2ppm. So snh cc s liu ph 1H-NMR v 13C-NMR ca cht C-2
vi s liu ph NMR ca stingmasterol [16] hon ton tng t nhau v c
ch ra bng 3.2.
Da trn phn tch cc s liu v ph EI-MS, FT-IR v cc ph NMR ca
hp cht C-2 hon ton ph hp vi cu trc ca cht Stingmast -5,22-dien24R-3-ol.
29
28
21
19
12
18

11

14

17

22

23

27

24

25
26

16
15

HO

13

20

10

5
4

Stingmast -5,22-dien-24R-3-ol

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39
Bng 3.2. Ph 13C-NMR ca cc cht C-2 v stingmasterol [16]
Ph 13C-NMR ca Stigmasterol
(C-2)
V tr
13
C-NMR(-ppm)
cacbon
1
36
2
29
3
71
4
42
5
140
6
122
7
37
8
31
9
51
10
36
11
24
2
42
13
40
14
57
15
25
16
30
17
56
18
12
19
19
20
40
21
19
22
138
23
129
24
50
25
32
26
21
27
21
28
19
29
12

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Ph 13C-NMR ca Stigmasterol
[16]
V tr
13
C-NMR(-ppm)
cacbon
1
37,28
2
31,66
3
71,78
4
42,31
5
140,76
6
121,69
7
31,91
8
31,91
9
50,18
10
36,52
11
21,09
2
39,70
13
42,22
14
56,88
15
24,38
16
28,92
17
55,97
18
19,40
19
12,06
20
40,50
21
21,23
22
138,31
23
129,29
24
51,25
25
31,91
26
21,09
27
19,00
28
25,41
29
12,25

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40

Hnh 3.5

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Hnh 3.6

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42

Hnh 3.7

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43

Hnh 3.8

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44
3.3.3. -sitosterol (C-3)
Cht C-3 l cht rn tinh th hnh kim, khng mu, nng chy 1401410C, n c tch t dch chit clorofom, trong h dung mi n-hexanclorofom (40:10).
Ph EI- MS, m/z (%) cho [M+] = 414 (23), [M-1]+ = 413 (41).
T ph EI-MS cho pic [M+] = 414 ng vi cng thc phn t C29H50O.
Ph hng ngoi, ph 1H-NMR v ph 13C-NMR cho thy trong phn t
c nhm OH ti v tr C-3 (IR c vn 3450 cm-1 rng, H-3 = 3,57ppm v C-3
= 72,1ppm). Mt lin kt i ti C 6 (ph IR c max= 3010cm-1 v 1650cm-1
ng vi dao ng ho tr ca lin kt i; cn trn ph 1H-NMR cho H-6 =
5,42ppm (1H, d, J, 5,2Hz), ph

13

C-NMR cho C-5 = 141,1ppm v C-6 =

121,7ppm). So snh cc s liu ph 1H-NMR v 13C-NMR ca cht C-3 vi


s liu ph NMR ca -sitosterol [16] hon ton tng t nhau v c ch
ra bng 3.3.
Da trn phn tch cc s liu v ph EI-MS, FT-IR v cc ph NMR ca
hp cht C-3 hon ton ph hp vi cu trc ca cht -sitosterol.
29
28
21
19
12
18
1

14

17

22

23

27

24

25
26

16
15

HO

13

11

20

10

5
4

-sitosterol

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45
Bng 3.3. Ph 13C-NMR ca cc cht C-3 v -sitosterol [16]
Ph 13C-NMR ca -sitosterol
(C-3)
V tr
13
C-NMR(-ppm)
cacbon
1
37
2
31
3
71
4
42
5
140
6
121
7
32
8
32
9
50
10
36
11
21
12
40
13
42
14
57
15
24
16
28
17
56
18
12
19
19
20
36
21
19
22
34
23
26
24
46
25
29
26
19
27
19
28
23
29
12

S ha bi Trung tm Hc liu i hc Thi Nguyn

Ph 13C-NMR ca -sitosterol
[16]
V tr cacbon
1
2
3
4
5
6
7
8
9
10
11
2
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29

13

C-NMR(-ppm)
37,27
32,54
71,77
42,29
140,75
121,69
31,92
31,92
50,15
36,51
21,10
39,80
42,23
56,78
24,31
28,25
56,08
19,39
11,89
36,16
18,80
33,96
26,11
45,85
29,18
18,82
19,05
23,08
11,99

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46

Hnh 3.9

Hnh 3.9

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47

Hnh 3.10

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48

Hnh 3.11

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49

Hnh 3.12

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50
3.3.4. Axit gallic (E-1)
Cc cht thu c t dch chit etylaxetat (E) c phn chia trn sc
k ct vi cht hp ph silicagel, ra gii bng hn hp dung mi clorofommetanol vi t l metanol tng dn t 0% - 100% thu c nhiu phn on
khc nhau trong c phn on CHCl3:MeOH (9:1) cha mt cht sch
c k hiu l cht E-1.
Cht E-1 l cht rn, tinh th hnh kim c nhit nng chy
2480 C-2500 C.
Ph IR cho vn hp th mnh max= 1700cm-1 c trng cho nhm
cacboxyl c max = 1700cm-1, vi vn rt t c s sng max = 3100cm-1 c
trng cho nhm OH ca phenol.
Ph 1H-NMR cho tn hiu 7,092ppm ng vi hai proton hai v tr C2
v C6.
Ph 13C-NMR cho thy cc tn hiu: C-1 = 121,960ppm; C-4 =139,559
ppm; C-2 = C-6 =110,349ppm; C-3 = C-5 =146,345ppm; C-7 =170,364ppm.
Nhng s liu v ph NMR ca cht E-1 hon ton ph hp vi phn
mm ACD/NMR ca axit gallic (xem bng 3.4).
Bng 3.4. S liu ph NMR ca E-1 v s liu ph NMR trong
phn mm ACD/NMR ca axit gallic
V tr
cacbon
1
2
3
4
5
6
7

Ph ca cht E-1
13

C-NMR
(-ppm)
121,96
110,35
146,35
139,56
146,35
110,35
170,36

H-NMR
(-ppm)
7,092
7,092
-

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Phn mm ACD ca axit gallic


ACD/13CNMR (-ppm)
123,88
111,35
147,44
138,18
147,44
111,35
167,37

ACD/ 1H-NMR
(-ppm)
7,11
7,11
-

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51
Trn c s phn tch cc s liu v ph FT-IR, NMR v phn mm
ACD/NMR. Cu trc ca axit gallic l:

7COOH
1
6

HO

OH

4
OH

Axit gallic
T dch chit etylaxetat, ngoi axit gallic cn thu c mt s phn
on khng sch khc c cha cc cht cho phn ng mu vi xianidin; cho
phn ng to mu xanh en vi FeCl3. Cc mu ny c cha hn hp cc
flavonoit, cc polyphenol c h s R f rt gn nhau v vic tch chng bng
ct silicagen cha t c s phn chia tt.
Khi ra gii bng CH3OH 100% thu c phn on cho cc phn ng
mu ging dch chit tng s bng n-butanol. Khi thc hin phn ng thu
phn cc mu ny trong axit HCl 2N thu c glucoz v axit gallic. T kt
qu thu phn ny c th cho rng cc phn on chit bng etylaxetat v
bng n-butanol u cha cc tannin v l galotanin. Tuy nhin cc tannin
y c to ra bi lin kt C-glycoside hay O-glycoside cu trc v th t
lin kt ca chng nh th no, cn cha c lm r v cha tch chng
thnh cc on sch.

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52

Hnh 3.13

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53

Hnh 3.14

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54

Hnh 3.15. Ph 1H-NMR ca cht axit gallic

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Hnh 3.16. Ph 13C-NMR ca cht axit gallic

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56
3.4. Th hot tnh sinh hc
Cc cht tng s c chit, tch bng dung mi c phn cc khc
nhau sau khi lm kh kit, loi b hon ton dung mi c gi ti phng th
nghim vi sinh trng i hc Y khoa Thi Nguyn th tc dng sinh hc
ca chng. Kt qu c nu bng 2.3 v hnh 2.1 cho thy tt c cc cht
thu c t cc dch chit ca l cy ngn u c tc dng khng khun
i vi khun Staphylococcus, E.coli, Salmonella typhi.
Chng ti cng gi mu chit bng etylaxetat v chit bng etanol
sau khi lm sch dung mi n c s sn xut kinh doanh thuc thnh
phm, thc phm chc nng Y hc c truyn Thi Nguyn. Cc mu u c
tc dng tt i vi vic chm sc sc kho con ngi, cc cht c trong cc
dch chit trn u khng gy c i vi con ngi.

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57

KT LUN
1. Nghin cu sng lc ho thc vt ca l cy ngn (Cratoxylum
prunifolium) pht hin thy nhiu nhm hp cht thin nhin c hot tnh
sinh l cao; l ng kh, tritecpenoit, steroit, saponin, cumarin, axit hu
c, cc flavonoit, cc cht polyphenol, cc tannin, cc cht xanthone.
2. T l cy ngn ln u tin phn lp v da vo cc c trng
ho l, cc s liu ph EI-MS, NMR, FT-IR nhn dng c cu trc ca 4
hp cht hu c thuc cc nhm polyphenol, steroit, terpenoit.
3. Phn tch cc ph IR, UV, MS v NMR cc cht tinh khit c
tch ra ni trn nhn dng c chng l: taraxeron, stigmasterol , sitosterol v axit gallic.
4. T dch chit tng ca l ngn (Cratoxylum prunifolium) chng
hon ton khng gy c hi vi con ngi, c th ch bin thnh thc ung
hng ngy nh mt thc phm chc nng c tc dng khng cc khun
Staphylococcusauresu (T cu vng), E.coli (Thc khun ng rut) v
Salmonella typhi (Thng hn).

KIN NGH
Cy ngn l mt cy thuc qu c nhiu tc dng trong y hc v vy
ti ngh vi cc cp c thm quyn tip tc cho nghin cu mt cch su
rng hn v cy ngn nhm phc v tt trong i sng nhn dn.

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58

TI LIU THAM KHO


Ting vit
1. V Vn Chi (1999), T in cy thuc Vit Nam, Nxb Y hc, trang 969.
2. Trn nh i (1998), Khi qut v h thc vt Vit Nam, Hi tho
Vit - c v ho hc cc hp cht thin nhin, H N i, trang 17-27.
3. Nguyn Vn n (1997), Cc phng php nghin cu cy thuc, Nxb
Y-Dc, TP H Ch Minh .
4. ti cp b quc phng ca Hc vin Qun Y (2002) , Nghin cu tc
dng ca dch chit t cy ngn ln mt s chc nng ca h thn
kinh trn ng vt thc nghim, H Ni
5. Phm Hong H (1999), Cy c Vit Nam, Nxb Tr, H Ni.
6. PGS.TS Phm nh Hng (2007), Nghin cu xc nh cu trc cc hp
cht c hot tnh sinh hc cao, (M s ti 510402).
7. Hong Thanh Hng, Cm Th ch, H Vit Sn (2006), Kho st phn
dch chit etylaxetat ca l thnh ngnh, tp ch Ho hc, T.44(1), trang
71-75.
8. L Kh K (1977), Cy c thng thy Vit Nam, tp 2, Nxb Khoa hc
v k thut, trang 189.
9. Nguyn Lim, Triu Duy it, Vn Bnh (1996), Nghin cu tc dng
chng oxi ho (Antioxidant invitro) ca mt s cy thuc Vit Nam, Cng
trnh nghin cu khoa hc qun s s 3, trang 30-33.
10. Nguyn Lim v cng s (1996), Nghin cu tc dng chng oxi ho
(antioxydant), ca cy ngn phng v cha cc bnh v ri lon
huyt ng. ti cp B quc phng, H Ni.
11. Tt Li (2001), Nhng cy thuc Vit Nam, Nxb Y hc, trang 122-123.
12. Trn Cm Vinh (1997), Tc dng ca cy ngn trong qu trnh ng
mu, Cng trnh nghin cu y hc qun s, s 3, trang 7- 9.

S ha bi Trung tm Hc liu i hc Thi Nguyn

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59
Ting Anh
13. A.K.Ratty, N.P.Das (1988), Biochemical medicine and metabolic
biologo, 39, 69-79.
14. Anake Kijjoa, Maria Jose, T.G.Gonzalez, Madalena M.M.Ana M.Damas,
Ing-on Mondranondra, Llartur M.S.Silva and Werner Herz (1998),
Xanthones from Cratoxylum Maingayi. Phytochemistry Vol 49, No.7,
2159-2162.
15. G.M.Kitanov, I.Assenov I and Dam The Van (1988), Flavonols and
Xanthones from Cratoxylum prunifloum kurz, Pharnazie 43, 879 .
16. Goat J.L, Akihisa T.Analysis of steroit frested, pp.324. Chapman & Hall.
London, New York - Tokyo.
17. Guat-Lee Sia, Graham J.Bennett, Leslie J.Harrison and Keng-Yeow Sim
(1995), Minor xanthones from the Bark of Cratoxylum cochinchinense.
Phytochemistry, Vol 38, No.6, pp.1521-1528.
18. J. Graham (1993), et al, triterpenoids, tocotrienols and xanthones from the
Bark of cratoxylon cochinchinense, phytochemistry, Vol 32, No 5, 1245.
19. Lien Hoa Dieu Nguyen, Le Li J. Harrison (1998), ritecpenoid and
xanthone constituents fo Cratoxylum conchinchinese, phytochemisty
50, 471-476.
20. Macias, M.Ulloa, M.Ulloa, M. Gamboa, A. and Mata, R. Phytotoxic
comopounds from the new coprophilous fungus Gnanomyces polytlrix.
21. Munekazu Iinuma, Hideki, Ttero Ito, Toshiyuki Tanaka and Domingo
A.Madulid (1996), Two xanthones from roots of Cratoxylum
formosanum. Phytochemistry, Vol, 42, No 4, PP.1195-1198.
22. Nawong Boonnak, Chatchanok Karalai, Suchada Chantrapromma, Channita
Ponglimanont, Hoong-Kun Fun (2006), Bioac tive prenylated xanthones
and

anthraquinones

from Cratoxylum formosun ssp.pruniflorum.

Tetrahedron 62 ,8850-8859.

S ha bi Trung tm Hc liu i hc Thi Nguyn

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60
23. Pitchaon Maisuthisakul, Rungnaphar Pongsawatmanit, Michael H.Gordon
(2007), Charaterization of the phytochemicals and antioxidant properties
of extracts from Teaw (Cratoxylum fromosum Dyer). Food chemistry
100 1620-1629.
24. Sompong Boonsri, Chatchanok Karalai, Chanita Ponglimanont, Akkharawit
Kanjana-Opas, Kan Chantrapromma (2006), Anibac terial and cytotoxic
xanthones from the roots of Cratoxylum fomosum. Phytochemistry 67,723-727.
25. Soon Yew Tang, Matthew Whiteman, Zhao Feng Peng, Andrew Jeniner, Eu
Leong Yong and Barry Halliwell (2004), Charcterition of Antioxidant and
Antiglycation Properties and Isolation of Active Ing redients from
Traditional Chinese medicines. Free Radical Biology & Medicine,
Vol.36, No.12, pp.1575-1587, .
26. Soon Yew Tang, Matthew Whiteman, Andrew Jeniner, Zhao Feng Peng
and Barry Halliwell (2004), Mechanisin of cell death induced bi an
antioxidant extract of Cratoxylum cochinchinense, Free Radical
Biology and Medicine, Vol 36, No.12,pp, 1588-1611.
27. Xue- Li Cao, Yu Tian, Tian-You Zhang, Yoichiro Ito (2000), Supercritical
fluid extraction of catechins Cratoxylum prunifolium Dyer. Joumal of
Chromatography A, 898, 75-81.
28.

Xue- Li Cao, Yu Tian, Tian-You Zhang and Yoichiro Ito (2000),


Beijing

Institute

of

New

Technology

Application,

Xizhimen

Southstree No.16, Beijing 100035, China.


29. W. Mahabusaraka, W. Nuangnaowarat, W.C. Taylor (2006), xanthone
derivativens from Cratoxylum cochinchinense roots, Phytochemistry 67
470- 474.
30. K. Tawaraya, Y. Takaya, M. Turjaman, S.J. Tuah, S.H. Limin, Y. Tamai,
J.Y. Cha, T. Wagatsuma, M. Osaki (2003), Arbuscular mycorrhizal
colonization of tree species grown in peat swamp forests of central
kalimantan, indonesia, Forest Ecology and Management 182, 381- 386.

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PH LC
I. Cht taraxeron

Trang

Ph LC-MS ca taraxeron. 62
Ph FT-IR ca taraxeron................................................................................65
Ph 1H-NMR ca taraxeron66
Ph 13C-NMR ca taraxeron...71
Ph C13CPD & DEPT ca taraxeron.73
Ph C13CPD-COSYGP ca taraxeron...78
Ph C13CPD-HSQC ca taraxeron81
Ph C13CPD-HMBC ca taraxeron...82
II. Cht stigmasterol
Ph EI-MS ca stigmasterol........84
Ph FT-IR ca stigmasterol ...85
Ph 1H-NMR ca stigmasterol86
Ph 13C-NMR ca stigmasterol...87
III. Cht - Sitosterol
Ph EI-MS ca - Sitosterol88
Ph FT-IR ca - Sitosterol.89
Ph 1H-NMR ca - Sitosterol90
Ph 13C-NMR ca - Sitosterol...95
Ph C13CPD & DEPT ca - Sitosterol.97
IV. Cht axit gallic
Ph FT-IR caaxit gallic 98
Ph t ngoi UV ca axit gallic.................................................................. 99
Ph 1H-NMR ca axit gallic..100
Ph 13C-NMR ca axit gallic101

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63

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Ph FT-IR ca taraxeron

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Ph 1H-NMR ca taraxeron

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67

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69

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Ph 13C-NMR ca taraxeron
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Ph DEPT ca taraxeron

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Ph COSYGP ca taraxeron

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Hnh

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Ph HSQC ca taraxeron
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Hnh

Ph HMBC ca taraxeron
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83

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89

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Ph 1H NMR ca - Sitosterol
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91

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Ph 13C NMR ca - Sitosterol


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Ph DEPT ca - Sitosterol
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Ph IR ca axit gallic
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Ph UV ca axit gallic
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Ph 1H NMR ca axit gallic


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Ph 13C NMR ca axit gallic


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MC LC
M U.................................................................................................... 1
Chng 1. TNG QUAN........................................................................... 3
1.1. M t thc vt .................................................................................. 3
1.2. Mt s cng dng ca chi Cratoxylum............................................... 4
1.3. Tnh hnh nghin cu ha hc thc vt chi Cratoxylum ...................... 7
1.3.1. Cc hp cht c khung triterpen ................................................. 7
1.3.2. Cc cht axit hu c .................................................................. 8
1.3.3. Cc cht c khung xanthone ....................................................... 9
1.3.4. Mt s i din ca khung anthraquinon.................................. 15
1.3.5. Mt s i din ca khung flavonoit......................................... 16
1.4. Nhng nghin cu ho thc vt loi Cratoxylum prunifolium. .......... 17
Chng 2. PHN THC NGHIM ........................................................ 19
2.1. i tng v phng php nghin cu ............................................ 19
2.1.1. Thu mu cy, xc nh tn khoa hc v phng php x
l mu ...................................................................................... 19
2.1.2. 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................................................................................... 20
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 .................................................................. 21
2.3. Cc dch chit t cy ngn (Cratoxylum prunifolium Kurtz) ........ 21
2.3.1. Cc dch chit .......................................................................... 21
2.3.2. Kho st nh tnh cc dch chit .............................................. 23
2.3.3. Th hot tnh sinh hc.............................................................. 23

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2.4. Chit sut, phn lp v tinh ch cc cht t cy ngn................... 25
2.4.1. Dch chit clorofom.................................................................. 26
2.4.1.1. Taraxeron (Friendoolean - 14- en-3-on) (C1) ................... 26
2.4.1.2. Stigmast- 5,22- ien-24R-3-ol .......................................... 27
2.4.1.3. - Sitosterol....................................................................... 27
2.4.2. Dch chit trong etylaxetat (E) ............................................... 28
Chng 3. THO LUN KT QU NGHIN CU.............................. 29
3.1. Nguyn tc chung........................................................................... 29
3.2. Phn tch nh tnh v pht hin cc nhm cht ................................ 30
3.3. Phn lp v nhn dng cc hp cht c trong cc dch chit khc
nhau ca cy ngn ............................................................................ 30
3.3.1. Taraxeron ( hay Frendoolean-14-en-3-on) (C-1) .................... 30
3.3.2. Stingmast -5,22-dien-24R-3-ol (C-2) .................................... 38
3.3.3. -sitosterol (C-3) ................................................................... 44
3.3.4. Axit gallic (E-1) .................................................................... 50
3.4. Th hot tnh sinh hc .................................................................... 56
KT LUN .............................................................................................. 57
KIN NGH ............................................................................................. 57
TI LIU THAM KHO........................................................................ 58

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DAN H MC C C B N G
Bng 2.1. Khi lng cht tng s c chit tng phn on l cy
ngn (Cratoxylum prunifolium Kurtz) ..................................... 22
Bng 2.2. Pht hin cc nhm cht trong cy ngn ................................. 23
Bng 2.3. Kt qu th hot tnh sinh hc ca dch chit th t l cy
ngn (Cratoxylum prunifolium) ................................................... 25
Bng 3.1. Ph 13C-NMR v 1H-NMR ca taraxeron (C-1) t l ngn...... 33
Bng 3.2. Ph 13C-NMR ca cc cht C-2 v stingmasterol [16] ............... 39
Bng 3.3. Ph 13C-NMR ca cc cht C-3 v -sitosterol [16] .................. 45
Bng 3.4. S liu ph NMR ca E-1 v s liu ph NMR trong phn
mm ACD/NMR ca axit gallic .................................................. 50

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DAN H MC C C H N H
Hnh 1.1. nh cy ngn (Cratoxylum Prunifolium Kurt, Hypericaceae) ... 4
Hnh 2.1. nh c gy c ch xung quanh ging thch............................ 24
Hnh 3.1 ................................................................................................... 34
Hnh 3.2 ................................................................................................... 35
Hnh 3.3 ................................................................................................... 36
Hnh 3.4 ................................................................................................... 37
Hnh 3.5 ................................................................................................... 40
Hnh 3.6 ................................................................................................... 41
Hnh 3.7 ................................................................................................... 42
Hnh 3.8 ................................................................................................... 43
Hnh 3.9 ................................................................................................... 46
Hnh 3.10 ................................................................................................. 47
Hnh 3.11 ................................................................................................. 48
Hnh 3.12 ................................................................................................. 49
Hnh 3.13 ................................................................................................. 52
Hnh 3.14 ................................................................................................. 53
Hnh 3.15. Ph 1H-NMR ca cht axit gallic ............................................. 54
Hnh 3.16. Ph 13C-NMR ca cht axit gallic ............................................. 55

DAN H MC C C S
S 2.1. Quy trnh ngm chit ................................................................ 22

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