PHNG PHP NGHIN CU DC LIU

= PHNG PHP NGHIN CU HO HC CY THUC

 Tin trnh nghin cu thuc t dc liu

Thu thp mu

Xc nh tn khoa hc

Chit xut

- + Sng lc hot tnh

 Tin trnh nghin cu thuc t dc liu
Xc nh cc thnh
phn ho hc

+ Phn lp

K thut sc k

1H NMR, 13C NMR spectra + Xc nh cu trc

COSY, HSQC/HMBC ho hc
spectra

Nuclear magnetic Resonance

+ nh lng cc
thnh phn
Tin trnh nghin cu thuc t dc liu

Xc nh thnh phn ho hc

nh gi hot tnh in vitro,

in vivo, c tnh

Th nghim lm sng

Cng ngh bo ch, sn

xut cng nghip,
marketing..
Stt Ni dung S tit hc
L thuyt CBGD
Phng php, quy trnh chit xut hp cht t
1 12 C Hoi
nhin
2 i cng v sc k 2 C Linh
3 Sc k lp mng 2 C Linh
4 Sc k lc gel v sc k trao i ion 4 C linh
4 Sc k ct 9 C Hin
6 Sc k kh, sc kh lng hiu nng cao 4 C Ph
Cc phng php xc nh cu trc ca hp
7 12 Thy c
cht t nhin
Tng cng 45
 Ti liu tham kho

Gio trnh lu hnh ni b

Natural Products Isolation, edited by Richard J.P. Cannell,
1998.
Phng php nghin cu ho hc cy thuc, Nguyn Vn n,
Nguyn Vit Tu, 1985.
Phng php c lp cc hp cht t nhin, Nguyn Kim Phi
Phng, 2007.
 Phng php nghin cu
ho hc cy thuc

XC NH
CHIT XC NH
XUT PHN LP CU TRC
HM
LNG

7
 NGHIN CU CHIT XUT
HP CHT LACTON T CY
XUYN TM LIN THEO HNG
P DNG SN XUT
 XTL (Andrographis paniculata): cy thuc c truyn cha cm st,
vim h hp, vim rut, l, tiu chy, thi tr cht c
Hot cht trong XTL: Cc diterpen lacton c tc dng :
khng khun, khng virus,
chng ung th, kch thch min dch
bo v gan,
h ng huyt,
h huyt p
Ch phm t XTL VN:
Vin nn Xuyn tm lin t bt l:
iu tr tiu chy, vim d dy, vim gan, vim hng, chng
cm cm.
Vin nn Panilin t bt l:
Phi hp vi khng sinh iu tr lao phi c kq tt:
Isoniazid-Pyrazinamid-Panilin (IPP)
Isoniazid-Streptomycin-Panilin (ISP)
 TNH HNH BNH LAO KHNG THUC

Trn th gii: 2,2 t ngi nhim lao, 9 triu ca mi/nm, 2

triu ngi cht/nm
Vit Nam: th 13/22 nc nhiu bn lao nht: 260.000 ca
mi/nm
Trn th gii c khong 100 triu ngi nhim vi khun lao
khng thuc.
T l khng thuc Vit Nam khong 32,5% v t l khng a
thuc vo khong 2,3%.
Nhu cu tm kim cc thuc mi c hiu qu trong iu tr
lao khng thuc
CC DITERPEN LACTON TRONG XTL
 CC DITERPEN LACTON TRONG XTL

Diterpen lacton TP (UV-VIS): 6,13% /phn trn mt t

Andrographolid (HPLC): 1,24-3,35% /l
Dehydroandrographolid (HPLC): 0,02-0,21% /l
1,18% /phn trn mt t
Neoandrographolid (HPLC): 0,65% /phn trn mt t
 MC TIU

Chn c cc iu kin chit xut ph hp hon thin

phng php chit xut theo tiu ch:
C th p dng quy m sn xut
Ph hp iu kin trang thit b hin c
n nh cht lng sn phm
H gi thnh
 PHNG PHP NGHIN CU
nh tnh hp cht lacton: bng TLC
nh lng hp cht lacton ton phn trong dc liu v
trong sn phm chit xut bng HPLC.
Phn lp cc hp cht: CC silica gel
Xc nh cu trc cc cht phn lp c:
Tnc, UV, IR, MS, 1H-NMR, 13C-NMR
Xc nh tinh khit: HPLC
Kho st cc iu kin chit xut: thay i tng iu kin
trong khi gi cc iu kin cn li khng i.
 S CHIT XUT V PHN LP
Dc liu
chit cn/ 1,5h x 3

B dc liu Dch chit

C p sut gim

Cn TH Cao lng
Loi tp

Cao loi tp
Ct silicagel, n-hexan EtOAc (1:1)

XT1 (0,1g) XT2 (0,8g) XT3 (0,3g)

KT QU XC NH CU TRC CC CHT

O
15

14 16
13
O
HO

12
11
18
17
1 9
2 10 8

3 5 7
4 6
HO
H
20
19
OH

Andrographolid (XT2)
 KT QU XC NH CU TRC CC CHT

15 O O
O 15
15
16 16
14 16 14 13 14 13
13 O O
O
HO
12 12
12
11 11
11 18 18
18
17 17
17 9
9 1 1 9
1
2 10 8 2 10 8
2 10 8
3 5 7 3 5 7
3 5 7 4 6 4 6
4 6 HO
HO HO
H H H
20 20
19 20
19 19
OH OR
OH
OH

HO
R= O OH
Andrographolid (XT2) 14-deoxy 11,12-didehydro
OH
andrographolid (XT1)
neoandrographolid (XT3)
KQ NH LNG 3 HC LACTON TRONG DL

Andrographolid: 3,20%
Dehydroandrographolid: 0,46%
Neoandrographolid: 0,43%
Kt qu kho st cc dung mi chit xut
4,5

4 3,48

3,5
2,96
3

2,5

2 1,62

1,5

0,5

0
MeOH EtOAc EtOH 95%

Hiu sut chit (%)

Kt qu kho st chit xut bng cn/nc
cc nng khc nhau

3,5
3 2,96
2,72 2,61
2,5 2,4
2
1,5
1
0,5
0
EtOH 95% EtOH 80% EtOH 65% EtOH 50%

Hiu sut chit xut (%)

 Kt qu kho st t l dung mi/dc liu
3,5
2,94 2,79
3 0,13 0,1
2,5 0,63 0,56
Ln chit 3
2
Ln chit 2
1,5
Ln chit 1
1 2,18 2,13
0,5
0
T l dm/dl 10/1, 9/1, 9/1 T l dm/dl 7/1, 6/1, 6/1

Hiu sut chit (%)

Kt qu kho st kch thc dc liu

3,5
2,87 2,85
3

2,5

1,5

0,5

0
Khng xay Xay th

Hiu sut chit (%)

Kt qu kho st v nhit chit xut
3,5

2,72
3
2,37
2,5

1,5

0,5

0
Nhit phng Nhit si ca
dung mi
Hiu sut chit (%)
 IU KIN CHIT XUT C CHN

Dung mi chit: Cn 95%

T l dung mi/dc liu: 13L/kg
ln chit 1: 7L/kg
ln chit 2: 6L/kg
Nhit chit: 800C (hi lu dung mi)
Thi gian chit: 90 pht x 2 ln chit
 KQ CHIT XUT 1KG DL/M
Hiu sut thu c sn phm trung bnh : 2,78%
Hm lng hc lacton trong sn phm: 96,80%
Andrographolid: 95,92% (chit c 83,33% c trong dl)
Dehydroandrographolid: 0,88%
Neoandrographolid: 0%
 KQ KHO ST NGUYN LIU CHIT XUT

Stt Ngun gc dc liu Hiu sut chit (%)

1 TP HCM CHTP 4/2010 1,02

2 TP HCM CHTP 8/2010 1,67
3 TP HCM CHKA 4/2010 1,08
4 TP HCM CHKA 8/2010 2,33
5 Tn Kiu, ng Thp 1/2010 1,23
6 Tn Kiu, ng Thp 3/2010 2,52
7 Tn Kiu, ng Thp 2/2010 2,03
8 Thp Mi, ng Thp 9/2009 1,16
9 Thanh Tr, H Ni 8/2009 1,05
10 Bc Giang 8/2010 2,87
 KT LUN

kho st cc iu kin chit xut hon thin phng

php chit xut hp cht lacton t xuyn tm lin theo hng
c th p dng sn xut.
Hiu sut chit xut trung bnh l 1,02 - 2,87%.
Hm lng hp cht lacton trong sn phm (HPLC): >95%.
 Chit xut ton phn

CHIT
Chit xut phn on
XUT
Chit pha rn

Sc k lp mng

Sc k ct
PHN
LP
Sc k lp mng iu ch

Sc k lng iu ch (HPLC iu ch)

29
XC NH
Ph cng hng t ht nhn
CU TRC
Mt chiu: 1H-NMR, 13C-NMR, DEPT

Hai chiu: HMBC, HSQC, COSY, NOESY

Ph khi lng MS, HRMS, UV, IR, CD, X-ray

Phng php ho hc (to dn xut)

30
 Tin trnh
T tn khoa hc, kim tra ti liu, xc nh thnh phn ha hc, nh
hng lm theo cch no???
Ly nguyn liu lm cho nghin cu, ty loi dc liu, b phn dng,
hm lng, mc ch..
Lm chit tch phn lp, xc nh cu trc
nh lng? Phn lp nh cht chun
Th hot tnh in vitro, in vivo, lm sng
Xy dng quy trnh cng ngh hay ko?
Kim tra SKLM nh hng
Thnh phn
Hm lng
Kh nng tch
Cho phn lp:
SK ct
SKLM iu ch
HPLC iu ch
SKLM kim sot ton b qu trnh, nu c th HPLC
 QUY TRNH CHIT XUT - PHN LP

Bt dc n-hexan
liu H

B dc liu Ngm nhit

phng/MeOH
Chloroform
C
Dch chit/MeOH

T0, p sut C quay

gim Ethyl acetat
E
Cn
Phn tn/H2O
n-butanol
Dch H2O B
Lc phn on

Dich H2O
N 36
 HOT TNH GY C T BO CA DCH CHIT CC PHN ON
Phn on H Phn on C Phn on E
Nng (% c ch) (% c ch) (% c ch)
(g/ml) MDA-BA- MDA-BA-
MKN7 MDA-BA-321 MKN7 MKN7
321 321
100 104,47 100,56 99,12 97,89 98,59 98,05
20 93,68 91,42 97,72 96,92 79,65 80,37
4 37,72 39,09 94,83 74,84 31,32 32,82
0,8 24,12 22,34 36,49 38,13 18,33 21,77
IC50 3,27 3,42 0,971 1,305 8,44 8,08
Phn on B Phn on N Ellipticine
Nng (% c ch) (% c ch) (% c ch)
(g/ml) MDA-BA- MDA-BA-
MKN7 MDA-BA-321 MKN7 MKN7
321 321
100 95,70 97,35 74,91 76,55 90,77 99,78
20 48,16 51,21 29,47 32,08 59,72 65,72
4 27,19 30,02 21,84 23,49 19,76 33,33
0,8 21,14 20,46 14,47 17,82 -0,54 5,76
IC50 22,72 18,77 60,92 52,48 1,249 0,935
MDA-BA-321 : t bo ung th v di cn
MKN7 : t Phn
bo ungon
th dchloroform
dy c la chn phn lp 37
QUY TRNH 1 Bt dc liu
1. Ngm chit vi MeOH
2. Lc

B dc liu Dch MeOH

Thu hi dung mi di
p sut gim, nhit

Cn
1. Acid ha bng acid tartaric 3%
2. Loi tp dch acid thu c bng EtOAc

Dch acid Dch EtOAc

1. Base ha bng Na2CO3 n pH=10
2. Chit dch base thu c bng CHCl3

Dch CHCl3
Thu hi dung mi di
p sut gim,nhit

Alkaloid dng base

Cc loi SK ct
 C s chn cht nhi ct SK ph hp
Kh nng tch
iu kin kinh t
Lng cao cn chit, tch
Kh nng ha tan ca dch chit

Cc loi cht hay dng
Silicagel pha thng
Silicagel pha o
Silicagel gn NH2
Shephadex G, LH20
MIC gel HP 20
Dianion HP 20
.
 C
n- hexan Chit pha rn
100% HA 20:1 HA 10:1 HA 5:1 HA 1:1 aceton
100%
C1 C2 C3 C4 C5 C6

Chit pha rn
n- hexan ethyl acetat
100% HE 20:1 HE 10:1 HE 5:1 HE 1:1
100%
C2A C2B C2C C2D C2E C2F

SKC pha o methanol : nc (3:1)

C2B1 C2B2 C2B3 C2B4 C2B5 C2B6 C2B7

SKC pha thng n-hexan : ethyl acetat : aceton (20:1:0,5)

C2B6A C2B6B C2B6C C2B6D C2B6E

SKC Sephadex LH 20 methanol 80%
Hp cht 1
(UGC12)
45
 C
Chit pha rn
n- hexan
100% HA 20:1 HA 10:1 HA 5:1 HA 1:1 aceton 100%
C1 C2 C3 C4 C5 C6

SKC pha thng chloroform : methanol (40:1)

C5A C5B C5C C5D

SKC pha thng n-hexan: aceton (5:1)

C5A1 C5A2 C5A3 C5A4 C5A5 C5A6 C5A7

SKC pha o aceton : nc : acid formic (3:1:0,1) Bay hi dung mi
Lc ra bng
C5A2A C5A2B C5A2C C5A2D methanol
SKC Shephadex LH 20 methanol 80% SKC Shephadex Hp cht 4
LH 20 (UGC8)
C5A2B1 C5A2B2 C5A2B3 methanol 100%
Lc ra bng methanol Hp cht 3
Hp cht 2 (UGC5)
(UGLE1)
46
NHN DANG CU TRC CC HP CHT
PHN LP

47
D kin ph ca hp cht 2 (UGLE1)
Ph ESI-MS (UGLE1)

Pic ion gi phn t ti

m/z 313,4
CTPT C14H14O6

Ph 13C, Ph DEPT

48
Ph 1H-NMR (UGLE1)

5 proton thm
thuc nhm
benzoyloxy
2H cis olefinic 4H gn vi C lin
kt O

49
 Ph DEPT (UGLE1)

9 nhm CH

1 nhm CH2

7C thuc nhm benzoyloxy 2 nguyn t C olefinic

4 nguyn t C (IV)

1C ca nhm cacbonyl cng hng 4 nguyn t cacbon sp3 gn vi oxy

50
 Ph COSY (UGLE1) Ph HMBC (UGLE1)

H4 H3 H2 H4 H5 H2

C3
C2
C1
H3

H5

H4 C4

C6

Vng 6 cnh vi ni i ti v tr 4, 5
Nhm oxo v tr 6

HMBC Nhm C-7 ni vi C-1 v nhm benzoyloxy

51
 Cu hnh tng i ca UGLE1
H7b
H6 H3
H2 H7a H2

H2
H3

H5

H4

Ph NOESY (UGLE1) Hng s tng tc J

Hp cht tham kho 3-O-debenzoylzeylenon c []D = +12,5 cn UGLE1 c []D = -

12,0 nn UGLE1 l mt i quang ca 3-O-debenzoylzeylenon.

52
 Cu trc UGLE1 COSY 1H-NMR
NOESY
HMBC

DEPT
HSQC

13C-NMR
ESI-MS

()-3-O-debenzoylzeylenon

* Yoshifusa Takeuchi, Qing Wen Shi, Takeyoshi Sugiyama, Takayuki Oritani (2001),Polyoxygenated
Cyclohexenes from the Chinese tree, Uvaria purpurea, Bioscience Biotechnology and Biochemistry,
66(3), pp. 537-542. 53
Cu truc ha hc
Hp cht Tn khoa hc Cng thc cu to

UGC12 Uvarilactam

UGC5 Zeylenon

UGC8 Pipoxid

54
 HOT TNH GY C T BO CA CC HP CHT PHN LP TRN DNG T
BO LU-1*

Nng % c ch
(g/ml) Hp cht 1 Hp cht 2 Hp cht 3 Hp cht 4 Ellipticine
100 97,94 111,17 108,41 106,48 86,66
20 53,52 107,54 37,46 48,44 76,23
4 41,23 82,73 22,99 35,70 30,31
0,8 28,65 31,74 13,12 25,92 13,12
IC50 16,66 1,30 37,63 26,19 0,67
* LU-1: t bo ung th phi

Hp cht 2 (()-3-O-debenzoylzeylenon) c tc dng c ch

s pht trin t bo ung th dng LU-1

55
 HOT TNH GY C T BO CA HP CHT ()-3-O-debenzoylzeylenon

Nng % c ch
(g/ml) KB MDA-BA-231 LNCaP HepG2
UGLE1 Ellipticine UGLE1 Ellipticine UGLE1 Ellipticine UGLE1 Ellipticine
IC50 3.71 0.98 17.81 0.79 12.63 0.79 3.65 0.86
Nng % c ch
(g/ml) MKN7 SW480 HL-60 3T3 (t bo thng)
UGLE1 Ellipticine UGLE1 Ellipticine UGLE1 Ellipticine UGLE1 Ellipticine
IC50 2.40 0.94 3.13 0.78 1.73 0,72 7,19 0,31

KB: ung th biu m MKN7: ung th d dy

MDA-BA-321: ung th v SW480: ung th rut kt
LNCaP: ung th tuyn tin lit HL-60: ung th bch cu
HepG2:
ung th ganv tnh hng
an ton ch tt vi
3T3: ccthng
t bo dng t bo
ung th
khc nhau ca ()-3-O-debenzoylzeylenon

56
Ph HR-ESI-MS(+) (cht sch 1)

591,2412

C28H40Na1O12

17
 Ph 1H-NMR (cht sch 1)

12 H ca 4 nhm OCH3
H 3,77 (x6); 3,76 (x6)

H ca cc nhm CH2, CH
X H 1,94-2,77
X 3 H ca nhm
1H CH3 ca hp
anome phn -L-
4 H thm
H 4,69 rhamnosyl H
H 6,30 (x2);
(s) 1,28
6,32 (x2)

18
Ph 13C-NMR (cht sch 1)

28 C
X

19
 Ph DEPT (cht sch 1)
4 CH ca hp phn -L-rhamnosyl
CH - C 70,2-73,9 2 CH bo ho C
4 CH thm C
anome 40,9; 44,2
107,1 (x2); 107,2 (x2)
C 102,3

4 nhm OCH3 C 1 nhm CH3 ca hp

56,6 (x2); 56,7(x2) phn -L-rhamnosyl C
CH 11 17,9
CH3 5
CH2 4
2 nhm OCH2 C 62,9; 69,5 2 nhm CH2 C 36,5; 36,8
C bc cao 8

20
 Cc tng tc ca cht sch 1

Tng tc HMBC ( ), COSY ( ), NOESY ( ) chnh ca 1

Ph HMBC Ph COSY Ph NOESY

21
 Cu trc ca cht sch 1

-D-xylopyranosyl -L-rhamnosyl
(8S,8S)-4,4-dihydroxy-3,5,3,5-tetramethoxy-8,8-
butyrolignan-9-O--L-rhamnoside

Ssiorisidse Fauvelidse

22padus,
Kiyoshi Yoshinari, Yukata Sashida and Hiroko Shimomura (1989), Two new lignan xylosides from the barks of Prunus ssiori and Prunus
Chem. Pharm. Bull, 37 (12), 3301-3303.
NHN DNG CC HP CHT PHN LP C

Ph 1H-NMR (PPE1)

2H ca C gn vi 2
nguyn t O H
6,09 v

2H ca nhm methin
H 7,19; 7,76
6 H thm
H 6,75; 6,96; 7,39; 7,52;
7,54; 8,15
 Ph 13C-NMR (PPE1)

18 nguyn t C
 Ph DEPT (PPE1)

8 nhm methine

1 nhm methylen gn vi 2
nguyn t O C 102,0

9C khng gn vi nguyn t H
C 117,1-178,1
 Ph NMR 2 chiu (PPE1)

HSQC HMBC COSY

66
 Hp cht PPE1
* R.N.Khanna and T.R.Seshadri (1963), Pongaglabrone, a new component of the seeds of Pogamia glabra: Its
constitution and synthesis, Tetrahedron Vol.19, pp: 219225.
67
 Hp cht PPE3
Ovalichromene B c
phn lp ln u tin t chi
Pongamia
* Rajinder Kumar Gupta and M.Krishnamurti (1976), Chromenoflavanones from Milletia ovalifolia,
Phytochemistry, Vol.15, p.2011
68