Phân tích xác định thành phần và nhóm hoạt chất của một số dược liệu bằng phương pháp sắc ký lớp mỏng kết hợp đo mật độ quang hấp thụ TLC, DENSITOMETRY SCANNING phục vụ công tác chuẩn hoá dược liệu các sản phẩm khoa học của đ

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BOYTE
SAN PHAM KHOA HOC CUA DE TAI
TEN DE TAI
PHAN TICH XAC DINH THANH PHAN VA NHOM HOAT CHAT
CUA MOT SO
DirOC
LIEU BANG PHl/ONG PHAP SAC KY
LOfP
MONG
• • •
KET
HOP
DO MAT DO QUANG HAP THU
(TLC,
DENSITOMETRY/

SCANNING)
PHUC VU CONG TAC
TIEU
CHUAN HOA
DU'OC
LIEU
Ha
Noi
2009
MUC LUC
SAN
PHAM KHOA
HOC
CUA
DE
TAI
TEN DE MUC
1.
Cac mau
dugc
lieu nghien
2.
Quy trinh phan tich va
nghien cuu, ky thuat TLC
BACH TRUAT
CHITir
COT TOAI BO
DIE?
HA CHAU DANG
DIA COT Bi

KIM NGAN HOA
MADE
NGU VI
Tir
RAU
MEO
THIEN MON DONG
3.
Cac bai bao
cuu
bp du lieu chuan
va TLC
ve
scanning
phan tich cua
cdc dugc
lieu
Trang
1
12
13
18
23
27
33
38
45
50
55
62

67
SAN PHAM KHOA HOC CUA DE TAI
1.
CAC MAU
DU'OC
LIEU NGHIEN
CLTU
Diroc
lieu bach truat
• • • •
Rhizoma Atractyloidis macrocephalae
Bach
I
rait
rtiod >
«l
lit
•
%«M r.«*«t«'
«»*W*«''^***^
•" *"
^ ,9 tip
wi».^
"a^ff
Mi»
J
i
:
BT
2«1

Bach
fru5t
>1»tt w
5 . BT
:f»5
Dirffc
lieu chi
tu*
Fructus Gardeniae
t
w
'<
« « «
>J
•
A
*«"*
*^ ^
Dirac
lieu cot toai bo
Rhizoma Drynariae
'/Jm
"*-1
w *•
••• •
tVii (oai |4
H^K
^^^H
1
.

1 ,0 " '}
11 —,
Cof
loii bd
^t-^r*^
»*t
m
%
It,
««»»•*
Diep ha chau dang
Herba Phyllanthi amari
Du-o-c lieu
dia cot bi
• •
•
Cortex Lycii
i
Dja
cut
b)
Z^dK
^_^''**^^^^^^^^^^^^^^^^^H
:mkM
mm.
Dja
c6t
bii
Mia
li

3
: Rf B 203
.&£>•
Bja cot bi
it/rlKX Ly<-*!i
VlJs »d
S :
»C» 2«5
Du'o'c
lieu kim ngan hoa
Flos Lonicerae
Kira
ngiSn b«a
^mM->'
i
Kim
iigan hou
Du-o-c lieu ma dl
Folium Plantaginis
Du-o-c lieu ngu vi tu-
Fructus Schisandrae
"U
rn ^.
\
kk»Mfimr
^Bt-
Du-g-c
lieu rau meo
Herba Orthosiphonis spiralis
^^^^1 ^

E^HI
^m
^'A
Kiiu men
»* *.
IW»JM**«*».
^«^.•.
Mi'.w «
t
WM :il
••^^^^B9
k^-U^
,c
^B^V
^mm^m
\f
C*
•fllK
w 1
At]
10
Du-ac
lieu thien mon dong
Radix Asparagi
ThiSn mdn
dOng
Thien mon ddng
Thiin
moB
tfSng

IWiMMSiTMUi^
11
SAN PHAM KHOA HOC CUA DE TAI
2.
QUI TRINH PHAN TICH VA BO
DlT
LIEU CHUAN VE PHAN
TICH CUA 10 DOI
TU'ONG DtTOC Ll£U
NGHIEN
ClTU
KY
THU
AT
TLC-TLC SCANNING
12
BACH TRUAT {Rhizoma Atractylodis macrocephalae)
1.
Nguon goc: Than re phai kho
ciia
cay bach truat Atractylodes macrocephala
Koidz., hg
Cue
{Asteraceae).
2.
Thu hai,
xu-
ly mau: Thu hai vao thang 10-12, dao lay than rl, bo rl con, co
the thai thanh lat mong, say nhe (nhiet do <
60°C)

den kho.
3.
Mo ta: Than re to (quen ggi la cu) co hinh dang thay doi, hinh chuy co nhieu
mau phinh ra, phia tren thot nho, hoac
timg khiic
map,
nae,
dai
5-10cm,
duong kinh
2-5cm. Mat ngoai mau nau nhat hoac xam, co nhieu mau, co van hinh hoa
cue,
co
nhieu nep nhan dgc. Chat cung, kho be gay, mat
cSt
khong bang, co mau vang den
nau nhat, mui thorn nhe.
4.
Dac diem vi hoc:
• •
* Dac diem bgt dugc lieu
Bgt mau nau vang, mui tham, vi hai man. Soi duai kinh hien vi thay: Mo mem gom
cac te bao thanh mong, co
thi
mang cac te bao mo cung. Te bao mo cung thanh
day, thiy ro cac
6ng
trao
d6i.
Tinh

thi calci
oxalat hinh kim, kich thuoc khoang
0,02-0,025 mm rieng le hay tap trung thanh dam. Manh mach
diim,
mach vach. Rai
rac
CO
sgi thanh day.
5. Dinh tinh:
Binh tinh terpenoid
Chudn bi mdu:
-
Mlu
thu:
0,5g
dugc lieu da xay nho, chilt vai 5 ml methanol, sieu am 30
phut, gan, lgc, dich lgc thu dugc dung dl chim
sic
ky. Lugng
ch4m
10-12 |il.
-
MIU
ddi chilu: Atractylenolide III 0,2mg/ml methanol, lugng
ch§m
5
^1.
L&p
mong: TLC Silica gel 60
F254

Merck, hoat hoa a
105^C
trong 1 gia truac khi
su dung.
He dung moi sdc ky:
- HDM
1:
n.Hexan: ethylacetat (2:1)
-^
Hinh
4.1
(A,B)
- HDM 2: Ether diu: ethylacetat
(50:1) ->
Hinh
4.1(C)
Phdt hien:
- TT
1:
DMAB - acid
sulphuric,Vis
/ UV366nm
-^
Hinh
4.1(A,
B)
TT 2: Vanillin - acid sulphuric , Vis
->
Hinh
4.l.C.

Kitqud:mr\hAA.{A,B,C).
13
At1
2345
At1
2345
Hinh
4.1:
S^c
ky dd dinh
tinh
thanh phan nhom hoat chat trong
diro-c
lieu bach truat
{Rhizoma Atractylodis
macrocephalae).
Ky hieu: Atractylenolide III (At); Saccharose (Sa); Cac
mlu
dugc lieu
bach truat nghien cuu (vet 1- 5).
SKD dinh tinh terpenoid (Hinh AVIS, Hinh B/UV366, Hinh C/ VIS);
SKD dinh
tinh
polysaccharid (Hinh D/ VIS).
14
Dinh
tinh
polysaccharid
Chudn bi mdu:
- Mau thu: 0,5g dugc lieu da xay nho, chilt vai 10 ml

h6n
hgp methanol, nuac
(ty le 4: 1), sieu am 30 phut, gan, lgc, dich lgc thu dugc dung dl chim sic ky.
Lugng cham 5
fil.
Mau doi chieu: Saccharose 0,2mg/ml methanol, lugng chim
5|al.
L&p
mong: TLC silica gel 60 F254 Merck.
He dung moi sdc ky: n.Propanol :
ethylacetat:
acid acetic : nuac
(4:3:1:2)
Phdt hien: TT a-Naphthol
Kitqud:\\MiAA.D.
6. Djnh lu'o'ng
Phuomg phdp
Chudn bi mdu
- Mau thu: Can 1.0 g dugc lieu da xay nho, chiet vai 20ml methanol x 2 lan,
sieu am 30 phut moi lan. Gan, gop dich chiet vao coc co mo, lam bay hai dung moi
tren
each
thuy den can. Hoa can thu dugc trong 1 ml methanol va 9ml nuac, cho
vao binh lang gan, lac vai 20mlx 3 lan chloroform. Gan, gop phan chloroform vao
r
r
r
f
r
y

coc
CO
mo, hoc hai thanh can. Hoa can thu dugc vua du 10ml methanol, lac deu.
Lgc qua giay lgc, bo 1ml dich lgc dau, lay l-2ml djch lgc tiep theo de cham sac ky.
- Mau doi chieu: Dung dich atractylenolide III 0,2mg/ml trong methanol.
L&p
mong: HPTLC Silica gel 60 F 254 Merck, kich thuac 20 x 10 cm, hoat hoa a
105°C
trong
1
gia truac khi su dung.
Chdm
mdu: Chim thang chuin:
2fil
,
4}il
x 2,
6|il
,
8|j.l
va
lOjil;
Mau
thii:
4-
12^1
(chim nhic lai
mlu
thu 2 - 3 lin). Chim
each

mep kinh 15mm (chieu X), 8 mm
(chilu Y), chieu rgng vet (8mm), khoang
each
giua cac vet (10mm).
He
dung
moi sdc ky: n.Hexan : ethylacetat (2:1), dl bmh sic ky bao hoa dung
moi 20 phut.
Phdt hien: Do TLC scanning, hap thu ,
k
220 nm.
Cdch tiin hdnh: Kinh sau khi da trien khai vai he dung moi neu tren, cao 85mm, liy
ra, dl bay hoi
hit
dung moi trong h6t, thai gian 30 phiit. Tiln hanh do TLC
scanning tren may CAMAG.TLC. SCANNER3, buac song
X
220 nm, t6c do quet
20mm/giay,
chi
do do remission. Chuang trinh dugc thuc hien duai su
diiu
khiln
ciia
phin
mim
wintcats. Dgc
kit
qua do tren may. Tinh ham lugng %
atractylenolide III co trong dugc lieu dugc tinh theo cong thiic sau:

m.V.100.100
X%=
u.lOOO.P(lOO-B)
Trong do :
15
X%
:
Ham lugng % atractylenolide III trong
dugc
lieu kho tuyet ddi
m
:
Lugng mau
thii
do dugc
(jig) (kit
qua trung binh
ciia
2-3 lin nhic lai)
\):
Lugng miu
thii
chim tren may
(|il)
V: The tich dich chilt miu
thii
(ml)
r
P
:

Khoi lugng dugc lieu dem can (g)
B
:
Do am cua dugc lieu
Ket qua dinh
liegmg:
Ket qua dinh lugng atractylenolide III trong dugc lieu bach truat dugc ghi a Bang
4.1
va Hinh
4.2.
r
Bang 4.1. Ket qua dinh lugng atractylenolide III trong dugc lieu bach truat.
TT
1.
2.
3.
4.
5.
Ten DU ky hieu
BT201
BT202
BT203
BT204
BT205
Ham
lugng
(%)
atractylenolide HI /DL
0,0670
0,0918

0,0626
0,0500
0,0778
Ghi chu
Mau thu hai
nt
nt
nt
nt
Ham lugng atractylenoide III trong dugc lieu bach truat kho tuyet doi nam trong
khoang tu 0,0500 -
0,0918%.
16
•^^i
Tn «C
n*
Atractylenolide III
B
•Ja
tn
»« tZ IE
*W~
Duac lieu bach truat
—
a.>——
f ae —
a^mw.
M
JM
.

4134
»•
•
X
•
-
B
WTH
ut
•
1«
• oe
tM
IM
3.K
Do thj xac djnh khoang tuyen ti'nh
Hmh 4.2.
sac
ky do TLC scanning va do thj xac dinh KTT
Dinh
lirgng
atractylenolide III trong
dirg-c
lieu bach truat
(Rhizoma Atractylodis macrocephalae)
17
CHI TU {Fructus Gardeniae)
l.Nguon goc: Qua chin phai hay say kho
ciia
cay danh danh Gardenia jasminoides

Ellis.,
hg ca phe {Rubiaceae).
2.
Thu hai,
xw
ly mau: Thu hoach vao thang
9-11,
hai lay qua chin chuyin mau
vang do, ngat bo cuong qua va loai tap, do den khi hat hoi phong len, lay ra phoi
hoac say kho.
3.
Mo ta: Qua hinh thoi
ho^c
hinh tning dai, dai 2-4,5cm, duang kinh l-2cm, mau
vang cam den do nau, co khi xam nau
din
dr xam, hai bong, c6 5-8 duang gd chay
dgc qua,
giiia
hai gd la ranh ro ret. Dinh
qu^
ldm cd 5-8 la dai tdn tai, thudng bj gay
cut. Gdc qua hep, cdn cd vet cudng qua. Vd qua mdng, gidn, hoi bong. Vd qua
giua
mau vang
due,
day han. Vd qua trong mau vang nga, bong, rat mdng, cd 2-3 vach
ngan gia. Hat nhd, mau vang cam, nau dd hoac nau den nhat, mat vd hat cd rat
nhieu hat min.
Miii

nhe. Vi hai chua va
d^ng.
4.
Dac diem vi hoc:
Dac diem bdt dugc lieu: Bdt mau vang nau hay nau dd, soi dudi kinh
hien vi
thiy: Sgi rieng le hay tap hgp thanh bd. Manh vd qua cau tao
tir
nhung te bao thanh
day, khoang tl bao rdng. Hoac hep. Be mat te bao (loai khoang rdng) thudng cd
nhung
Id
nhd. Md mem vd qua, manh ndi
nhii
chua cac chat du tru.
5. Dinh tmh:
Dinh
tinh
iridoid
Chudn bi mdu:
-
MIU
thii:lg
dugc lieu da dugc tan nhd, chilt vdi 10ml methanol, sieu am 20
phiit, gan lgc liy phin dich chilt, thu dugc dung dich
thii
dl dinh tinh
- Miu ddi chilu: Geniposide 0,25 mg/ml methanol, gardenoside 0,25 mg/ml
methanol.
L&p mong: TLC silica gel 60 F254 Merck, hoat hda d

105°C
trong 1 gid trudc khi
sii
dung.
Luang chdm mdu:
12^1
dung dich mau
thii;
6|al
mdi dung dich mau ddi chieu
geniposide va gardenoside.
He dung moi sdc ky:
n.Butanol:
acid acetic : nudc (7:2:1)
Phdt hien:
- Quan sat / UV 254
^
Hinh
4.3.A.
- Anisaldehyde- acid sulphuric (TT)
-^
Hinh 4.3.B.
Kit
qud:
mnh
4.3.
{A,
B).
18
-0,5

Ge Ga 1 2 3 Ge Ga 1 2 3 Ch 1 2 3 1 2 3
Hinh
4.3.Sac ky do dinh
tinh
thanh phan nhom hoat chat dugc lieu chi tir
{Fructus Gardeniae).
Ky hieu: Geniposide (Ge); Gardenoside (Ga); Acid chlorogenic (Ch); Cac mau
dugc lieu chi
tii
nghien curu (1,2,3).
SKD dinh tinh iridoid (Hinh. A/ UV254 , Hinh. B/ VIS); SKD djnh tinh
din
xuit acid caffeic (Hinh. C/ UV366); SKD dinh tinh sic td crocin (Hinh.D/VIS).
Dinh tinh dan xuat acid caffeic
Chudn bi mdu:
- Mau
thii
:lg dugc lieu da dugc tan nhd, chilt vdi
10ml
methanol, sieu am 20
phiit, gan lgc liy phin dich chilt, thu dugc dung dich
thii
dl chim sic ky.
- Miu ddi chilu: Acid chlorogenic acid 0,05 mg/ml methanol.
L&p
mong: TLC silica gel 60 F254 Merck, hoat hda d
105^C
trong 1 gid trudc khi
sii
dung.

Lucmg chdm mdu:
8|il
dung dich mau
thii;
12(xl
dung dich mau ddi chieu acid
chlorogenic.
He dung moi sdc ky:
Ethylacetat:
acid acetic : acid formic : nudc (10:1:1:2)
Phdt hien: Natural products- PEG 4000 ( TT)
Kitqud:Umh4.3.C.
Dinh tinh sdc td (crocin)
Chudn bi mdu:
- Miu
thii :lg
dugc lieu da dugc tan nhd, chilt vdi
10ml
methanol, sieu am 20
phiit, gan lgc liy phin dich chilt, thu dugc dung dich
thii
dl dinh tinh
L&p
mong: TLC silica gel 60 F254 Merck, hoat hda d
105°C
trong 1 gid trudc khi
sii
dung.
Lucmg chdm mdu:
6|il

dung dich miu thu.
He dung moi sdc ky: Ethylacetat: isopropanol: nudc
(6,5
: 2,5 : 1)
Phdt Men: Quan sat/ anh sang thudng
19
Ketqud:mnh4.3.D.
6.Dmh lu'o'ng:
Phuong phdp
Chudn bi mdu:
- Mau
thii:
Can 1,0 g dugc lieu da xay nhd, chilt vdi 25 ml methanol (x 3
lin),
sieu am 30 phiit mdi lin. Gan va gdp phin djch chilt va them methanol vua
dii
100ml. Pha loang
tiip
vdi methanol dl thu dugc dung djch cd ty le lg dugc lieu
trong 500ml dung mdi. Lgc qua giiy lgc, bd 2ml dich lgc diu, liy 2 - 3 ml dich lgc
tiep theo de cham sic ky.
- Mau ddi chilu: Dung dich geniposide 0,20 mg/ml trong methanol.
L&p mong: HPTLC Silica gel 60 F254 Merck, kich thudc kinh 20 x 10 cm, hoat
hda d
105°C
trong
1
gid trudc khi
sii
dung.

Chdm mdu: Chim thang chuin:
2^1
,
4\x\
x 2, 6}il ,
S\x\
va
12|il;
Miu
thii:
4-
10|il
(cham nhac lai miu
thii
2 -3 lin). Chim
each
mep kinh 15 mm (chilu X), 8 mm
>
^ f
r
(chieu Y), chieu rdng vet (8mm), khoang
each
giua cac vet
(10mm).
He dung mdi sac ky: Ethylacetat: isopropanol: nudc ( 6,5 : 2,5 : 1) de binh sac ky
bao hoa dung mdi 30 phiit.
Phdt hien: Do TLC scanning, hap
thu.
A,
235nm

Cdch tien hanh: Kinh sau khi da trien khai vdi he dung mdi neu tren, cao 85mm, lay
ra, de bay hoi het dung mdi trong hdt, thdi gian 30 phiit. Tien hanh do TLC
scanning tren may CAMAG.TLC. SCANNER3, budc sdng
A,
235 nm, tdc do quet
20mm/giay, che do do remission. Chuang trinh dugc thuc hien dudi su dieu khien
ciia
phan mem wintcats. Dgc ket qua do tren may. Tinh ham lugng % geniposide cd
trong dugc lieu dugc tinh theo cdng thiic sau :
m.V.100.100
X%=
u.lOOO.P(lOO-B)
Trong dd :
X% : Ham lugng % geniposide trong dugc lieu khd tuyet ddi
m : Lugng miu thu do dugc
(fig) (kit
qua trung binh
ciia
2-3 lin nhic lai)
u
: Lugng miu
thii
chim tren may
(|LI1)
V:
Thi
tich dich chilt miu
thii
(ml)
P : Khdi lugng dugc lieu dem can (g)

B : Do im
ciia
dugc lieu
Kit qua
Mnh
lucmg:
Kit
qua dinh lugng geniposide trong dugc lieu chi
tii
dugc ghi d Bang 4.2, va Hinh
4.4.
20
Bang 4.2. Ket qua dinh lugng geniposide trong dugc lieu chi
tii
TT
1.
2.
3.
Ky hieu mau
CT201
CT202
CT203
Ham
luptg
%
geniposide
6,0600
6,4100
5,2800
Ghi chil

Mau thu hai
nt
nt
Ham lugng geniposide trong dugc lieu chi
tii
khd tuyet ddi nam trong khoang
5,2800-6,4100%
21
Geniposide
B
.—-r/;
;«.r.^.^/
ii
:'K
«kj
("Vj""
Duoc lieu chi tu
D6
thi xac dinh khoang tuyen tinh
Hinh 4.4.
S^c
ky do TLC scanning va do thi xac dinh khoang tuyin tmh.
Dinh
lirong
geniposide trong
dirge
lieu chi tn
{Fructus
Gardeniae).
22