GVHD: GS.TS. NGUYN KIM PHI PHNG
Mc lcMc lcBn cc t vit tt1Kho st s c ch Acetylcholinesterase trn
sc k lp mng21.TNG QUAN21.1BNH ALZHEIMER21.2S LC V
ACETYLCHOLINESTERASE V CHT C CH
ACETYLCHOLINESTERASE21.2.1Acetylcholinesterase21.2.2Cht c ch
acetylcholinesterase31.3GII THIU SC K LP MNG[7]62.KHO ST S C CH
ENZYME ACETYLCHOLINESTERASE TRN tlc72.1TH NGHIM TRN TLC VI THUC TH
MUI FAST BLUE B[8]72.1.1C s l thuyt72.1.2Nguyn liu82.1.3Dng c, ha
cht v cch pha ha cht82.2.3.1Dng c v ha cht82.2.3.2Cch pha cc dung
dch82.1.4Quy trnh thc hin92.1.5Kt qu92.2TH NGHIM TLC VI THUC TH
ELLMAN112.2.1Kho st s c ch cho kt qu dng tnh tht[9]112.2.3.3C s l
thuyt112.2.3.4Nguyn liu122.2.3.5Ha cht, dng c v cch pha ha
cht122.2.3.6Phng php132.2.3.7Kt qu142.2.2Kho st s c ch cho kt qu
dng tnh gi[10]162.2.3Kho st s c ch enzyme bng sc k bn mng hiu nng
cao[11,12,13]193.TNG KT22TI LIU THAM KHO23
Bn cc t vit ttACh AChE AChI ATC ATCI BSA DTNB HPTLC TLCDTNB
acetylcholine acetylcholinesterasecht c ch acetylcholinesterase
acetylthiocholineacetylthiocholine iodidebovine serum albumin5,5-
dithiobis-(2-nitrobenzoic acid)sc k lp mng hiu nng caosc k lp
mng5-thio-2-nitrobenzoate
Kho st s c ch Acetylcholinesterase trn sc k lp mng1. TNG QUAN1.1
BNH ALZHEIMER
Hnh 1. Bnh AlzheimerBnh 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
ACETYLCHOLINESTERASE1.2.1 AcetylcholinesteraseEnzyme
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. AChE chuyn ha
ACh ch trongs, rt nhanh gi cho khe synap lun r rng thng tin hn hp
khng xy ra [2].1.2.2 Cht c ch acetylcholinesteraseCht 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]
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 IC50 (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].
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),
Nb-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 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.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 liuC 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 cht2.2.3.1 Dng c v ha
chtBn 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 dchDung 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.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 CHCl3-MeOH-H2O (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. Hnh 4. S quy trnh thc hin th nghim
c ch AChE vi thuc th mui fast blue B bng sc k lp mngPhun 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 quA. 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). 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.
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: CHCl3-MeOH-H2O
(65:35:5).
2.2 TH NGHIM TLC VI THUC TH ELLMAN2.2.1 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
EllmanAcetylthiocholine (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 liu1, 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 cht2.2.3.5.1 Ha cht v dng
cBn 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 dchDung 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). Thuc th Ellman: m
A cha 1 mM DTNB v 1 mM ATCI.2.2.3.6 Phng php2.2.3.6.1 Mu chitPhi 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 hinPha 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 CHCl3-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.
Hnh 5. S quy trnh thc hin th nghim c ch AChE vi thuc th Ellman
bng sc k lp mngSy 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 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 quS 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.
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 EllmanAkaloidGii hn pht hin (g)
Galanthamine 0.01
Physostigmine0.01
Pancracine0.1
Carbachol0.1
Lycorine0.2
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. 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 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 mngNu 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
Bng 2. Kt qu xc nh dng tnh gi trong th nghim c ch enzyme AChEHp
chtNng [mM (%)]Bn silica gelBn nhm oxid
c ch do phn ng ha hcc ch do enzymec ch do phn ng ha hcc ch do
enzyme
Heptanal720 (10%)++++
Decanal530 (10%)++++
Cinanamaldehyde790 (10%)++++
Anisaldehyde820 (10%)++++
Benzaldehyde100++--
4-dimethylaminobenzaldehyde100-+-+
3-etoxy-4-hydroxybenzaldehyde100-+-+
Diethylamine960 (10%)-+-+
Triethylamine717 (10%)-+-+
Ethanolamine1670 (10%)----
Triethanolamine750 (10%)-+-+
Hexylamine756 (10%)++++
Tryptamine50++++
Tyramine50-+-+
Galanthamine1-+-+
Physostigmine1-+-+
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.
ABCHnh 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 CHCl3 - 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.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
HPTLCC chtGii hn xc nh (ng)
Parathion-ethyl sau khi oxi ha0.045
Paraoxon-ethyl0.013
Paraoxon-methyl0.400
Mevinphos0.200
Dichlorvos0.200
Carbaryl0.200
Aldicarb0.400
Butoxycarboxim0.100
Butocarboxim0.800
Oxamyl0.800
Pentachlorophenol20.000
Hnh 10. S k thut HPTLC
3. TNG KTCc 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.
TI LIU THAM KHO[1]arka tpnkov*,aand Karel Komersb, (2008).
Cholinesterase and cholinesterase Inhibitors. Current Enzymee
Inhibition. 4, 160-171.[3]E. A. Adewusi1, N. Moodley2 and
V.Steekamp1*, (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 Baek1 ,
Sung-Hoon Kim2 , 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 Choudharya)b)*, Salma
Shahnaza), ShehnazParveena), Asaad Khalida), M. Ahmed Mesaikb) , S.
Abdul Majeed Ayatollahic) ,and Atta-ur-Rahmana)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.[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
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