Mc lc

Mc lc
Bn cc t vit tt ................................................................................................................................... 1
KHO ST S C CH ACETYLCHOLINESTERASE TRN SC K LP MNG ............. 2
1. TNG QUAN ................................................................................................................................. 2
1.1 BNH ALZHEIMER ................................................................................................... 2
1.2 S LC V ACETYLCHOLINESTERASE V CHT C CH
ACETYLCHOLINESTERASE ............................................................................................... 2
1.2.1 Acetylcholinesterase .............................................................................................. 2
1.2.2 Cht c ch acetylcholinesterase ........................................................................... 3
1.3 GII THIU SC K LP MNG
[7]
........................................................................... 6
2. KHO ST S C CH ENZYME ACETYLCHOLINESTERASE TRN TLC ............... 7
2.1 TH NGHIM TRN TLC VI THUC TH MUI FAST BLUE B
[8]
................... 7
2.1.1 C s l thuyt ...................................................................................................... 7
2.1.2 Nguyn liu............................................................................................................ 8
2.1.3 Dng c, ha cht v cch pha ha cht ................................................................ 8
2.2.3.1 Dng c v ha cht ........................................................................................... 8
2.2.3.2 Cch pha cc dung dch ...................................................................................... 8
2.1.4 Quy trnh thc hin ............................................................................................... 9
2.1.5 Kt qu .................................................................................................................. 9
2.2 TH NGHIM TLC VI THUC TH ELLMAN .................................................. 11
2.2.1 Kho st s c ch cho kt qu dng tnh tht
[9]
............................................... 11
2.2.3.3 C s l thuyt .................................................................................................. 11
2.2.3.4 Nguyn liu ...................................................................................................... 12
2.2.3.5 Ha cht, dng c v cch pha ha cht ........................................................... 12
2.2.3.6 Phng php .................................................................................................... 13
2.2.3.7 Kt qu ............................................................................................................. 14
2.2.2 Kho st s c ch cho kt qu dng tnh gi
[10]
................................................ 16
2.2.3 Kho st s c ch enzyme bng sc k bn mng hiu nng cao
[11,12,13]
............... 19
3. TNG KT ................................................................................................................................... 22
TI LIU THAM KHO ................................................................................................................... 23






1


Bn cc t vit tt

ACh
AChE
AChI
ATC
ATCI
BSA
DTNB
HPTLC
TLC
DTNB
acetylcholine
acetylcholinesterase
cht c ch acetylcholinesterase
acetylthiocholine
acetylthiocholine iodide
bovine serum albumin
5,5- dithiobis-(2-nitrobenzoic acid)
sc k lp mng hiu nng cao
sc k lp mng
5-thio-2-nitrobenzoate


2

KHO ST S C CH ACETYLCHOLINESTERASE TRN
SC K LP MNG
1. TNG QUAN
1.1 BNH ALZHEIMER
Bnh Alzheimer l bnh thoi ha thn
kinh ph bin c c chng bi s gim
nng cht dn truyn thn kinh
acetylcholine (ACh) trong no b. Mt s iu
tr lm sn ca bnh Alzheimer l kch thch
th th tit ra ACh hoc ko di s tn ti ca
ACh trong cc khe thn kinh sinap bng cch
s dng cc cht c kh nng tch tr v lm
tng nng ACh. c tnh c khong 50-60
thuc c a vo th nghim lm sn trong
vic iu tr bnh Alzheimer v vic s dng
cht c ch enzyme acetylcholinesterase (c
vai tr thy phn ACh) ha hn y tim nng
trong vic iu tr bnh Alzheimer
[1]
.

1.2 S LC V ACETYLCHOLINESTERASE V CHT C CH
ACETYLCHOLINESTERASE
1.2.1 Acetylcholinesterase
Enzyme acetylcholinesterase (AChE) l mt enzyme rt quan trong i vi ng
vt k c loi ngi. AChE c nhim v chuyn ha cht dn truyn thn kinh (ACh)
sau khi ACh truyn thng tin. Nu cht dn truyn thn kinh khng c chuyn
ha sau khi n hon thnh nhim v th cc c c lin quan s khng th ngh v iu
c th gy ra cc cn co tht , t lit v mt s vn khc.
AChE c tm thy trong khe synap, gia cc t bo thn kinh thng qua cc
lung thng tin. Khi ACh i qua, AChE s chuyn ha ACh thnh choline v acid
acetic.
Hnh 1. Bnh Alzheimer


3

AChE chuyn ha ACh ch trong

s, rt nhanh gi cho khe synap lun r

rng thng tin hn hp khng xy ra
[2]
.
1.2.2 Cht c ch acetylcholinesterase
Cht c ch acetylcholinesterase (AChI) l nhng hp cht c kh nng c ch
phn ng thy phn acetylcholine, c chia lm hai nhm l cht c ch thun
nghch v cht c ch khng thun nghch (cht c ch thun nghch l cht c ch to
lin kt vi cc enzyme bng lin kt khng ni nh lin kt hydrogen, lin kt Van
der waals, ni lin kt ion; cht c ch bt thun nghch l cht c ch to lin kt vi
enzyme bng lin kt cng ha tr
[1]
.
Hu nh cc cht c ch c tm c u cha nitrogen, c 3 mc c ch
[3]
:
c ch mnh : phn trm c ch enzyme ln hn 60 %.
c ch trung bnh: phn trm c ch enzyme t 15 - 50 %.
c ch yu: phn trm c ch enzyme nh hn 15 %.
Mt s hp cht c ch enzyme c s dng trong vic iu tr bnh
Alzheimer:


Hnh 2. Cng thc ca mt s hp cht c ch
[1]



4


Tacrine (1) l thuc u tin c cc qun l dc v thc phm cho php p
dng vo vic iu tr bnh Alzheimer (1993), l mt cht c ch khng cnh
tranh thun nghch.
Donepezil (2) l thuc th hai c cc qun l dc v thc phm cho php p
dng cho vic iu tr bnh Alzheimer (1996), l cht c ch thun nghch.
Rivastigmine (3) l cht c ch khng cnh tranh thuc nhm carbamates, lin
kt vi b mt mang nhm ester ca enzyme, l thuc c cc qun l dc v
thc phm cho php s dng vo nm 2000.
Galathamine (4) l mt phenanthrene alkaloid, mt cht c ch cnh tranh
thun nghch, c cc qun l dc v thc phm cho php s dng vo nm
2001.
Huperzine A (5) v huperzine B (6) l hai alkaloid c c lp t loi tho
mc Huperzia serrate. C hai hp cht ny u l cht c ch thun nghch y
tim nng v c tnh chn lc cao, c cho php s dng Trung Quc.
Physostigmine (7) (eserine) l cht c ch thun nghch c c cu l mt amine
bc 3.
Eptastigmine (8) l mt dn xut ca physostigmine, trong nhm
carbamoylheptyl c thay bng nhm carbamoylmethyl ti v tr th 5 ca
vng indole
[1]
.
Dae Keun Kim v cng s nm 2004 c lp c 4 hp cht isoquinoline
alkaloid t Corydails speciosa c hot tnh c ch AChE thu c gi tr IC
50
(c ch
enzyme 50%): corynoxidine (89 M) (9), protopine (16.1 M) (10), palmatine (5.8
M) (11), berberine (3.3 M) (12)
[4]
.
Ngoi cc alkaloid l galanthamine, huperzine A v huperzine B cn c 3
alkaloid khc: sanguinine (0.1 0.01 M) (13), 11-hydroxygalanthamine (1.61
0.21 M) (14), epinorgalanthamine (9.60 0.65 M) (15); 3 lycorine: oxoassoanine
(47.21 1.13 M) (16), assoanine (3.87 0.24 M) (17), pseudolycorine (152.32
32.06 M) (18) cng c kh nng c ch AChE
[5]
.


5











6

M. Iqbal Choudhary v cng s nm 2006 nghin cu mt s hp cht
triterpennoid alkaloid c lp t cy Buxus hyrcana c hot tnh c ch AChE:
hyrcanone (145.0 11.5) (19), buxabenzacinine (468.0 5.0 M) (20), N
b-
dimethylcycloxobuxoviricine (310.0 22.1 M) (21), hyrcamine (83.0 1.3 M) (22),
buxidine (210.6 8.3 M) (23), buxandrine (175.4 11.0 M) (24), buxippine-K
(25)
[6]
.







1.3 GII THIU SC K LP MNG
[7]

Sc k lp mng (TLC) da ch yu vo hin tng hp thu trong pha ng l
mt dung mi hay hn hp cc dung mi, di chuyn ngang qua pha tnh l mt cht
hp thu tr, th d nh: silica gel hoc nhm oxid. Pha tnh ny c trng thnh mt
lp mng, u, ph ln mt nn phng nh tm king, tm nhm hoc tm plastic. Do
cht hp thu c trng thnh mt lp mng nn phng php ny c gi l sc k
lp mng.
u v nhc im ca sc k lp mng:
u im: ch cn mt lng rt t mu phn tch; c th phn tch ng
thi mu v cht chun i chng trong cng iu kin phn tch, tt c cc hp


7

cht trong mu phn tch c th c nh v tr trn sc k lp mng, dng c ln,
kch thc mu ln c th kim sot c.
Nhc im: cht em i phn tch phi c tnh km bay hi, hiu qu tch
cht nh lng v kh nng tch lng ln khng cao.
2. KHO ST S C CH ENZYME ACETYLCHOLINESTERASE
TRN TLC
kho st s c ch AChE bng phng php sc k lp mng (TLC), c rt
nhiu cch thc hin trong hai cch tiu biu nht l s dng thuc th mui fast
blue B v thuc th Ellman (phng php Ellman, ngoi TLC c th dng phng
php 96 ging vi thuc th Ellman).
2.1 TH NGHIM TRN TLC VI THUC TH MUI FAST BLUE B
[8]

2.1.1 C s l thuyt


Hnh 3. Phn ng ca acetylcholinesterase vi 1-naphthyl acetate v nhum mu tm trn th nghim
sc k lp mng.

-naphthyl acetate b thy phn vi xc tc enzyme AChE thnh sn phm l -naphthol. Sau ,
sn phm ny tip tc phn ng vi thuc th mui fast blue B cho hp cht azo c mu tm.


8

Ti nhng v tr enzyme b c ch th trn bn mng s quan st thy xut hin cc im mu
trng trn nn tm do khng xy ra phn ng gia -naphthol v thuc th mui fast blue B sau khi
bn mng c chm mu, gii ly, phun enzyme v phun thuc th.

2.1.2 Nguyn liu
C ca h Amaryllidaceae: Galanthus nivalis (Champagne, Switzerland),
Leucojum vernum (Vuillens, Switzerland), Narcissus pseudonarcissus (Oron-la-Ville,
Switzerland), c ca Ismene festalis, Nerine bowdenii, Crinum powellii v Sprekelia
formosissima (Charmois Garden Centre, Savigny, Switzerland) v phn ngn ca
Chelidonium majus (Papaveraceae, Dixa AG (St. Gallen, Switzerland)).
10 g nguyn liu kh v nhuyn c trch bng phng php ngm dm vi 250
ml EtOH 80% trong 12 gi. Dch chit c c quay p sut thp thu c cao
EtOH. Cao th EtOH c pha thnh dung dch c nng 1 mg/ml thc hin sc
k lp mng.
2.1.3 Dng c, ha cht v cch pha ha cht
2.2.3.1 Dng c v ha cht
Bn mng silica gel 60 F254 (Merck) v bn Al2O3 60 F254 trung tnh loi E
(Merck).
Enzyme AChE (Sigma), galanthamine (Sigma), tris-HCl, bovine serum albumin
(BSA) v -naphthyl acetate (Merck), mui fast blue B, physostigmine (eserine),
chlorogenic acid, quinine, caffeine (Fluka), v mt s dung mi thng dung nh
ethanol, acetone, isopropanol.
2.2.3.2 Cch pha cc dung dch
Dung dch enzyme AChE: ha tan dung dch AChE 1000 U vo 150 ml dung
dch m tris-HCl 0.05 M (pH 7.8). Sau thm 150 mg BSA vo dung dch trn
n nh enzyme AChE cho n khi tin hnh th nghim (c gi 4 ).
Dung dch -naphthyl acetate: ha tan 250 mg -naphthyl acetate trong 100 ml
ethanol.


9

Dung dch mui fast blue B: ha tan 400 mg mui fast blue B trong 160 ml nc
ct.
Dung dich thuc th: trn 10 ml -naphthyl acetate vo 40 ml mui fast blue B.
Khng pha trc hai dung dch mui fast blue B v -naphthyl acetate khi cha s
dng trnh hin tng thy phn.
2.1.4 Quy trnh thc hin
Ra sch bn mng vi acetone
v sy kh bn mng.
Chm 15 l cht chng dng
galanthamine v mu cn kho st ln
bn mng sau gii ly bn mng
trong h dung mi CHCl
3
-MeOH-H
2
O
(65:35:5)
Phun dung dch enzyme AChE
ln bn mng cha mu gii ly v
sy kh li. Sau , tin hnh bn
mng 37 trong 20 pht.
Phun dung dch thuc th ln
bn mng sau khi tin hnh
enzyme v i trong 1-2 pht
phn mng enzyme ri quan st bn
mng.

2.1.5 Kt qu
A. Marson v cng s (2001) dng TLC kho st s c ch AChE vi 2 cht i
chng dng v thu c kt qu sau.
i vi physostigmine, nng ti thiu c th quan st c s c ch AChE
(gii hn pht hin) l 0.001 g, cn galanthamine th mc 0.01 g. (hnh 4).
Hnh 4. S quy trnh thc hin th nghim c ch
AChE vi thuc th mui fast blue B bng sc k lp
mng


10

Mt s alkaloid khc nh boldine, caffeine v quinine c ch AChE nng ti
thiu ln lt l 0.1 g, 1 g v 10 g. Mt flavonoid l kaempferol cng cho hot c
ch 10 g. Tt c cc hp cht ny u cho hot tnh km.
Tt c cc dch chit t cao EtOH ca cc cy kho st u th hin hot tnh c
ch AChE ngoi tr cao ca Sprekelia formosissima.



Hnh 6. Bn mng sau khi vi enzyme v phun hn hp dung dch thuc th c thc hin trn: 1
galathamine , v mt s cao EtOH 80% (15 g) nh 2 Galanthus nivalis, 3 Leucojum vernum, 4 Narcissus
pseudonarcissus, 5 Chelidonium majus, 6 Ismene festalis, 7 Nerine bowdenii, 8 Crinum powellii, 9 Sprekelia
formosissima.





11


Hnh 5. Bn mng sau khi vi enzym v phun hn hp dung dch thuc th c thc hin trn
hai cht i chng: (a) galanthamine (0.0001-1 g) v (b) physostigmine (0.00001-0.1 g).
Dung mi gii ly: CHCl
3
-MeOH-H
2
O (65:35:5).


2.2 TH NGHIM TLC VI THUC TH ELLMAN
2.2.1 Kho st s c ch cho kt qu dng tnh tht
[9]

2.2.3.3 C s l thuyt


Hnh 7. S xc nh hot tnh ca enzyme bng phng php Ellman


12

Acetylthiocholine (ATC) b thy phn vi xc tc enzyme AChE thnh sn phm
l thiocholine. Sau , sn phm ny tip tc phn ng vi thuc th 5,5-dithiobis-(2-
nitrobenzoic acid) (DTNB) cho sn phm 5-thio-2-nitrobenzoate c mu vng.
Ti nhng v tr enzyme b c ch th trn bn mng s quan st thy xut hin cc
im mu trng trn nn vng do khng xy ra phn ng gia thiocholine v thuc th
Ellman sau khi bn mng c chm mu, gii ly, phun enzyme v phun thuc th.
2.2.3.4 Nguyn liu
1, Hymanocallis festails Zwanenburg; 2, Chlidanthus fragrans Herb; 3,
Narcissus Avalanche (Tazetta); 4, Nerine bowdenii; 5, Narcissus Grand Soleil
dOr(Tazetta); 6, Zephyranthes candida (Lindl) Herb; 7, Crinum powellii Baker; 8,
Polianthes tuberosaL; 9, Amaryllis belladonna L; 10, Eucharis amazonica Linden
exPlanch; 11, Hippeastrelia; 12, Habranthus robustus Herb. Ex Sweet; 13,
Rhodophiala bifida (Herb) Traub; 14, Hymencallis Sulphur Queen; 15, Sprekelia
formosissima (L.) Herb.
2.2.3.5 Ha cht, dng c v cch pha ha cht
2.2.3.5.1 Ha cht v dng c
Bn silica gel 60 F254 (Merck).
Enzyme AChE, tris-HCl, DTNB (Sigma), ATCI (Sigma), BSA, galanthamine
hydrobromide, physostigmine (eserine), carbachol (Sigma); pancracine, lycorine,
tazettine (Dr. Serap Whitmer).
2.2.3.5.2 Cch pha cc dung dch
Dung dch m A: tris-HCl 50 mM pH 8.
Dung dch m B: m A cha 0.1 % BSA.
Dung dch enzyme: t mu enzyme AChE c ng c chn khng, pha
long vi m A thu c dung dch enzyme gc c nng 1000 U/ml. Tip tc
pha long dung dch enzyme vi m B thu c dung dch enzyme AChE c nng
3 U/ml dng cho sc k lp mng.
Dung dch cht nn: m A cha 1 mM acetylthiocholine iodide (ATCI).


13

Hnh 5. S quy trnh thc hin th nghim c ch
AChE vi thuc th Ellman bng sc k lp mng
Thuc th Ellman: m A cha 1 mM DTNB v 1 mM ATCI.
2.2.3.6 Phng php
2.2.3.6.1 Mu chit
Phi kh v nghin nhuyn nguyn liu ca 15 loi cy Amaryllidaceae thnh
dng bt.
Bt cy ca tng loi (1 g) c trch bng phng php ngm dm vi 10 ml
methanol v toluene ring bit nhit phng trong 24 gi cho mi ln trch v qu
trnh c lp li trong mt tun. Cc dch chit c c quay di p sut thp thu
c cao methanol v cao toluene.
2.2.3.6.2 Quy trnh thc hin
Pha mu cao methanol v toluene
trong methanol c mu c nng
10 mg/ml.
2.5 l ca tng loi dung dch mu
ni trn v dung dch i chng
galanthalmine c dng chm ln
bn mng ri gii ly bn mng trong
h dung mi CHCl
3
-MeOH (8:2).
Phun dung dch thuc th Ellman
ln bn mng sy kh cho n khi
bn mng bo ha dung dich thuc th.
Sy kh bn mng trong 3-5 pht
ri phun dung dch enzyme AChE 3
U/ml ln bn mng.
Sau khong 5 pht th bn mng s chuyn sang mu vng v c nhng im
trng th hin kh nng c ch enzyme AChE. Ghi nhn kt qu trong 15 pht t khi
bn mng chuyn mu v sau 20-30 pht th kt qu s bin mt.
Tng t nh cc bc trn, chm 1.5 l galanthamine, physostigmine, pancracine,
lycorine, tazettine, carbachol vi cc nng khc nhau (5 mM 5 M) ln bn mng


14

xc nh nng ti thiu c th quan st c s c ch enzyme AChE l
nhng im trng trn nn mu vng.

2.2.3.7 Kt qu
S dng lng dung dch phun phi ph hp. Nu phun qu nhiu th cc im
trng trn nn vng s b lan rng v ko di ra khi ta s kh quan st c kt qu.
V bn mng s c lm kh sau khi phun cht nn ri mi phun dung dch enzyme
AChE.
In Kyung Rhee v cng s (2001) th nghim trn cao methanol v toluene ca 15
loi cy thuc h Amaryllidaceae v mt s cht i chng dng thu c cc kt
qu sau:




Hnh 7. Kt qu c ch AChE trn TLC ca 15 cao methanol nguyn liu thuc h
Amaryllidaceae (1, Hymanocallis festails Zwanenburg; 2, Chlidanthus fragrans Herb;
3, Narcissus Avalanche (Tazetta); 4, Nerine bowdenii; 5, Narcissus Grand Soleil
dOr(Tazetta); 6, Zephyranthes candida (Lindl) Herb; 7, Crinum powellii Baker; 8,
Polianthes tuberosaL; 9, Amaryllis belladonna L; 10, Eucharis amazonica Linden
exPlanch; 11, Hippeastrelia; 12, Habranthus robustus Herb. Ex Sweet; 13, Rhodophiala
bifida (Herb) Traub; 14, Hymencallis Sulphur Queen; 15, Sprekelia formosissima (L.)
Herb) v cht i chng galanthamine.



15



Hnh 8 : Kt qu c ch AChE trn TLC ca 15 cao toluene nguyn liu thuc h
Amaryllidaceae (1, Hymanocallis festails Zwanenburg; 2, Chlidanthus fragrans Herb; 3, Narcissus
Avalanche (Tazetta); 4, Nerine bowdenii; 5, Narcissus Grand Soleil dOr(Tazetta); 6, Zephyranthes
candida (Lindl) Herb; 7, Crinum powellii Baker; 8, Polianthes tuberosaL; 9, Amaryllis belladonna
L; 10, Eucharis amazonica Linden exPlanch; 11, Hippeastrelia; 12, Habranthus robustus Herb. Ex
Sweet; 13, Rhodophiala bifida (Herb) Traub; 14, Hymencallis Sulphur Queen; 15, Sprekelia
formosissima (L.) Herb) v cht i chng galanthamine.

Bng 1. Nng ti thiu c th quan st c kh nng c ch enzyme ca mt s cht
i chng trong phng php Ellman
Akaloid Gii hn pht hin (g)
Galanthamine 0.01
Physostigmine 0.01
Pancracine 0.1
Carbachol 0.1
Lycorine 0.2
Tazettine >0.5

Khi s dng nhiu loi bn mng khc nhau: bn silica gel, bn nhm oxid, bn
pha o, bn tinh bt, bn polyamide rt ra c cc kt qu[8]:
i. Bn mng nhm oxid cho nn vng r hn nhng im trng li ko di
hn so vi bn silica gel.


16

ii. Bn silica gel cn lng enzyme nhiu hn c th quan st c s
c ch.
iii. Sau khi gii ly vi h dung mi ph hp th trn bn nhm oxid s quan
st thy hin tng ko di ui. B dy ca bn silica gel cng nh hng n kt
qu. Th d nh vi b dy 1 mm th nhn c nn vng r hn nhng li cn
mt lng enzyme v mu ln.
2.2.2 Kho st s c ch cho kt qu dng tnh gi
[10]

Ngoi nhng cht cho kh nng c ch thc s th bn cnh c nhng cht tuy
cho kt qu dng tnh nhng thc t khng c hot tnh c ch AChE. Da vo
phng php Ellman c th pht hin nhng cht cho kt qu dng tnh gi bng th
nghim vi mt s aldehyde (heptanal, decanal, cinnamaldehyde, 4-
dimethylaminobenzaldehyde, 3-metoxy-4-hyroxybenzaldehyde) v mt s amine
(diethylamine, triethylamine, ethanolamine, triethanolamine, hexylamine, trypamine
hydrochloride v tyramine hydrocloride). Thc hin song song hai quy trnh:

Quy trnh 1 Quy trnh 2


Quy trnh 1: ging nh quy trnh khi kho st cht c ch dng tnh tht.
Chm cc mu aldehyde v amine c pha trong methanol vi cc nng
khc nhau trn trn hai loi bn mng silica gel v nhm oxid. Sau phun hn hp


17

thuc th DTNB-ATCI (1mM DTNB v 1mM ATCI pha trong m 50 mM tris-
HCl (pH = 8 )) cho n khi bn mng bo ha, kh bn khong 3-5 pht ri phun
dung dch enzyme AChE 3 U/ml. Sau 5 pht, bn mng s xut hin vt mu trng
trn nn mu vng.
Quy trnh 2: ging nh quy trnh 1 nhng th t phun thuc th khc nhau. Sau
khi bn mng cha mu gii, phun dung dch c trn dung dch enzyme
AChE 3 U/mL v ATCI 1 mM ( dung dch thiocholine) ri bn mng trong 15
pht cui cng l phun dung dch DTNB 1 mM ln bn mng s thu c cc dim
trng trn nn vng ca bn mng
Nu l cht c ch tht (nh galanthamine v physostigmine) th khi phun thuc
th DTNB ln bng mng theo quy trnh 2 s khng quan st thy im trng trn nn
vng cn cht c ch gi s cho thy im trng trn nn vng.
Kt qu TLC theo quy trnh 1 cho thy c s c ch AChE v c ch phn ng
gia ATC vi DTNB. Kt qu TLC theo quy trnh 2 ch cho thy c s c ch phn
ng gia ATC vi DTNB. So snh kt qu TLC theo hai quy trnh c th phn bit
nhm hp cht no c hot tnh c ch enzyme AChE hay c ch phn ng gia ATC
vi DTNB.
In K. Rhee v cng s (2001) nh gi s c ch dng tnh gi bng TLC vi
mt s aldehyde, amine, cc dch chit t cao ethanol cua cy Nerrine bowdenii



Hnh 9. Cc phn on chit cao toluene ca N. bowdenii





18

Bng 2. Kt qu xc nh dng tnh gi trong th nghim c ch enzyme AChE


p dng phng php ny c th xc nh c cc hp cht hoc cao th no
mi thc s c hot tnh c ch AChE.
Mt th d l trch lng-lng cao toluene ca Nerine bowdenii vi cc dung mi
hexane, chloroform, methanol 50% (methanol pha trong nc) thu c cc dch
tng ng (hnh 9).
Cc dch chit hexane, chloroform, methanol 50% c tin hnh theo hai quy
trnh nh gi hot tnh c ch AChE.
Hp cht
Nng
[mM (%)]
Bn silica gel Bn nhm oxid
c ch do
phn ng
ha hc
c ch do
enzyme
c ch do
phn ng
ha hc
c ch do
enzyme
Heptanal 720 (10%) + + + +
Decanal 530 (10%) + + + +
Cinanamaldehyde 790 (10%) + + + +
Anisaldehyde 820 (10%) + + + +
Benzaldehyde 100 + + - -
4-dimethylaminobenzaldehyde 100 - + - +
3-etoxy-4-hydroxybenzaldehyde 100 - + - +
Diethylamine 960 (10%) - + - +
Triethylamine 717 (10%) - + - +
Ethanolamine 1670 (10%) - - - -
Triethanolamine 750 (10%) - + - +
Hexylamine 756 (10%) + + + +
Tryptamine 50 + + + +
Tyramine 50 - + - +
Galanthamine 1 - + - +
Physostigmine 1 - + - +


19



Hnh 10. Th nghim xc nh kh nng c ch AChE v hin tng dng tnh gi ca cc dch chit t Nerine
bowdenii. Bn mng c chm cc mu dch chit toluene (1), hexane (2), chloroform (3), methanol 50% v
c gii ly trong h dung mi CHCl
3
- methanol (10:1).
Hnh A: vt mu trng trn nn vng th hin s c ch khi phun DTNB 1 mM/ ATCI 1 mM trong tris-
hydrochloride 50 mM ri phun 3 units/mL AChE.
Hnh B: vt mu trng trn nn vng th hin s c ch gi khi phun 3 units/mL AChE/ ATCI 1 mM
(thiocholine) ri phun DTNB 1 mM.
Hnh C : hin hnh vi thuc th anisaldehyde

T kt qu thu c (hnh 10) cho thy mc d cc dch chit c cha nhiu hp
cht c ch, tuy nhin ch c mt s hp cht c hot tnh c ch. Trong 4 dch chit
ch c dch chit chloroform c cha nhiu hp cht c ch nht. T c th s dung
cao chit chloroform c lp cc hp cht c ch c trong Nerine bowdenii.

2.2.3 Kho st s c ch enzyme bng sc k bn mng hiu nng cao
[11,12,13]

Ngoi k thut TLC, k thut sc k lp mng hiu nng cao (HPTLC) cng c th
p dng kho st s c ch AChE. K thut HPTLC c dng nh tnh, th
tinh khit , c th bn nh lng v nh lng.
V nguyn tc c bn HPTLC khng khc nhiu so vi TLC. im khc ch yu
l qu trnh chm sc k c iu khin v thc hin bng my, cht hp thu pha tnh
c kch thc nh hn, s vt, lng dch chit s chm trn bn mng c xc lp
trn my vi tnh. Qu trnh chm dch chit thc hin vi s h tr ca my nn kh
hoc bnh kh nn. Sau khi trin khai, sc k c chp nh qua bung chp
(reprostar 3) v kt qu c nh gi bng phn mm Videoscan.
A B C


20

HPTLC cho php chm nhanh nhiu ln nhng dung dch pha long cht cn phn
tch. Bn mng c trng sn cht hp thu c cht pht quang thch hp.
Khi lm sc k lp mng bn nh lng, chnh xc ca kt qu phn tch ph
thuc rt nhiu vo chnh xc ca lng cht th a ln bn mng, tc l th tch
dung dch chm ln bn mng. Do , vi nhng trng hp phn tch bn nh lng
phi dng cc mao qun nh mc chnh xc. Khi khng cn nh lng dng
micropipet hoc ng mo qun thng.
Theo nghin cu ca Christel Weins v cng s nm 1996 s dng k thut
HPTLC nh gi s c ch AChE thu c kt qu bng 3.

Bng 3. Gii hn xc nh ca cc hp cht c ch cholinesterase trong HPTLC
C cht Gii hn xc nh (ng)
Parathion-ethyl sau khi oxi ha 0.045
Paraoxon-ethyl 0.013
Paraoxon-methyl 0.400
Mevinphos 0.200
Dichlorvos 0.200
Carbaryl 0.200
Aldicarb 0.400
Butoxycarboxim 0.100
Butocarboxim 0.800
Oxamyl 0.800


21

Pentachlorophenol 20.000


Hnh 10. S k thut HPTLC



22

3. TNG KT
Cc hp cht cho hot tnh c ch enzyme AChE thng c cha nitrogen.
Mc d TLC l phng php nh tnh vi nhy khng cao nhng phng php
cng c th gip nhn bit c cc hot tnh c ch enzyme vo giai on u nghin
cu. T c th chn cao th no c cha nhiu hp cht cho hot tnh c ch d c
lp.
Bn cnh pht hin cc hp cht cho hot tnh c ch tht th c th pht hin ra
cc hp cht c hot tnh c ch gi bng phng php Ellman.
Vi thuc th mui fast blue B th c th xc nh hp cht cho hot tnh c ch
AChE nhng cng khng th kt lun c cc hp cht khng cho kt qu dng
tnh gi v cha tm thy c ti liu no vn . y c th l hng nghin cu
mi trong vic kho st s c ch AChE.
K thut HPTLC cng c th nh gi s c ch ca enzym AChE, nhanh hn k
thut TLC thng thng tit kim thi gian hn, c th dng bn nh lng v
nh lng.




23

TI LIU THAM KHO
[1] arka tpnkov
*,a
and Karel Komers
b
, (2008). Cholinesterase and
cholinesterase Inhibitors. Current Enzymee Inhibition. 4, 160-171.
[3] E. A. Adewusi
1
, N. Moodley
2
and V.Steekamp
1*
, (2010). Medicinal plants with
cholinesterase inhibitory activity: A Review. African Journal of Biotechnology.
9(49),8257-8276.
[4] Dae Keun Kim, Ki Taek Lee, Nam-In Baek
1
, Sung-Hoon Kim
2
, Hee Wook
Park, Jong Pil Lim, Tae Yong Shin, Dong Ok Eom, Jae Heon Yang, and Jae Soon Eun,
(2004). Acetylcholinesterase inhibitors from the aerial parts of Corydalis speciosa.
Archibes of Pharmacal Research. 27(11), 1127-1131.
[5] Susana Lo1pez, Jaume Bastida, Francesc Viladomat, Carles Codina*, (2002).
Acetylcholinesterase inhibitory activity of some Amaryllidaceae alkaloids and
Narcissus extracts. Life Sciences. 71, 2521-2529.
[6] M. Iqbal Choudhary
a)b)*
, Salma Shahnaz
a)
, ShehnazParveen
a)
, Asaad Khalid
a)
,
M. Ahmed Mesaik
b)
, S. Abdul Majeed Ayatollahi
c)
,and Atta-ur-Rahman
a)b)
, (2006).
New cholinesterase-inhibiting triterpenoid alkaloids from Buxus hyrcana. Chemistry
and Biodiversity. 3, 1039-1051.
[7] Nguyn Kim Phi Phng, Phng Php C Lp Hp Cht Hu C. i Hc
Quc Gia Thnh Ph H Ch Minh, (2007).
[8] A. Marston, J. Kissling and K. Hostettmann, (2002). A Rapid TLC
Bioautographic Method for the detection of Acetylcholinesterase and
Butyrylcholinesterase inhibitors in plants. Phytochem. Anal. 13,51-54.
[9] In Kyung Rhee
*
, Michiel van de Meent, Kornkanok Ingkanianan, Robert
Verpoorte, (2001). Screening for acetylcholinesterase inhibitors from Amaryllidaceae
using silica gel thin-layer chromatography in combination with bioactivity staining.
Journal of Chromatography A. 915, 217-223.
[10] In K. Rhee, Richard M. van Rijin and Robert Verpoorte, (2003). Qualitative
determination of false-possive effects in the Acetylcholinesterase assay using thin layer
chromatography. Phytochem. Anal. 14, 127-131.
[11] Christel Weins*, Hellmut Jork1, (1996). Toxicological evaluation of harmful
substances by in situ enzymeatic and biological detection in high-performance thin-
layer chromatography. Journal of Chromatography A. 750, 403-407.


24

[12] ManMohan Srivastava, (2011). High-Performance Thin-Layer
Chromatography (HPTLC). Springer-Verlag Berlin Heidelberg.
PHN TI LIU TRN INTERNET
[2] http://www.rcsb.org/pdb/101/motm.do?momID=54(28/04/1012).
[3] http://www.elmhurst.edu/~chm/vchembook/573inhibit.html (19/05/2012).
[13] http://duoclieu.net/Dlieuhoc/Tools/Phuongphap/HPTLC.htm (23/06/2012.