TRNG I HC Y DC HU

KHOA DC- B MN TVD-DL-DCT

BO CO TIU LUN

SNG LC CC LOI THO DC HN QUC IU TR

ALZHEIMERS THNG QUA C CH ENZIM CHOLINESTERASE

Nhm 4- D3A

Thnh vin:

Hunh Th Tho Uyn

L Th Phng Uyn

Bi Th Vn

inh Th Thy Vn

Nguyn Th Vn

Nguyn Th Thy Vn

Nguyn Quc Vit

V nh Vinh

H Quc Vng
 MC LC
T VN
CHNG 1 : TNG QUAN
1.1 Vi nt v bnh Alzheimer v enzym cholinesterase ................

1.1.1 Khi nim v bnh Alzheimer v c ch sinh bnh ................

1.1.2Mi lin quan gia bnh Alzheimers v enzim

chonineseterase................................................................................
.

1.1.3 Enzym Acetylcholinesterase

( AChE ) .......................................

1.2 Mt s thuc v dc liu c ch cholinesterase ......................

1.2.1 Cc thuc c ch AChE ang c lu hnh ........................

1.2.2 Cc nghin cu v pht trin dc liu c tc
dng c ch AChE ............................................................

CHNG 2: I TNG V PHNG PHP NGHIN

CU

2.1 Nguyn liu nghin cu : ............................................................

2.2 Phng tin nghin cu ...........................................................................

2.3 Qu trnh chit xut ....................................................................

2.4 Quy trnh nghin cu hot tnh sinh hc cho tc dng chng AChE
.......................................................................................................
 2.4.1 Cch tin hnh .............................................................................

2.4.2 Tnh ton kt qu ......................................................................

CHNG 3: KT QU V BN LUN
3.1 Kt qu
.............................................................................................
3.1.1 Kt qu thu thp d liu v cc dc liu a vo sng
lc ....
3.1.2 Kt qu sng lc tc dng c ch AChE invitro ca cc dc
liu ................................................................................................
3.1.3 Xc nh IC50 ca cc dc liu tim nng c ch AChE ......
3.2 Bn lun ..........................................................................................
3.2.1 Thng tin ca cy thuc a vo nghin cu ...........................
3.2.2 V kt qu sng lc c ch AChE invitro .................................
3.2.3 V kh nng c ch AChE ca cy thuc tim nng ...............

CHNG 4 KT LUN V KIN NGH

 T V N

T l u c c s n ph m t nhi n ng vi tr qun tr ng trng i u tri b nh.

Cu ng vi s i mi trng phng ph p i u tri b ng c c s n ph m t
nhi n, c y thu c v nhng ch ph m t thc v t c qun t m nhi u
hn bi nhng u i m s vi h dc,ng y c ng c nhi u nghi n cu
thu c mi t dc li u trng i u tri b nh.

Nghi n cu ph t tri n thu c mi l m t qu trnh gin nn t n nhi u thi

gin v chi ph bi qu trnh n y tr i qu nhi u gii n. M c du c nhi u
hng ti p c n nghi n cu kh c nhu nhng qu trnh nghi n cu thng
c b t u b ng vi c nh gi h t tnh cu m t lng ln c c m u dc
li u h c hp ch t. Gii n n y mng tnh ch t inh hng v c g i l
gii n nghi n cu s ng l c. gii n n y phng ph p th in vitr
c u ti n s du ng bi c u i m l ti n h nh ng thi nhi u m u,
lng m u c n ch th nghi m t, ch k t qu nhnh v chi ph th p. M c
du v y, c th x y dng c m t phng ph p th in vitr th ch
ph n t c b nh ( prtin, zim, thu th ) ph i c x c inh.

ctychlinstrs( Ch) l nzim c m t kh sinp cu h th n kinh

trung ng v c vi tr duy tr s n inh n ng cu ch t d n truy n
th n kinh ctylchlin. Vi vi c Whithus l n u ti n r gi thuy t
v vi tr cu h chlinrgic i vi b nh lzhimr v n m 1982, Ch
cu ng c x c inh l m t trng nhng ch ph n t i vi b nh n y. Tr n
c s m t v i phng ph p th in vitr c x y dng. Trng
phng ph p qung s du ng thu c th llmn c s du ng kh ph
bi n trng nghi n cu s ng l c t c du ng c ch Ch in vitr cu dc li u
v hp ch t hi n ny.

G n y, k t qu nghi n cu s ng l c dc li u ch th y dich chi t Mthnl

cu 7 c y th dc H n Qu c( Acorus calamus, Acorus gramineus,
Bupleurum facaltum, Dioscorea batatas, Epimedium koreanum, Poria cocus
v Zizyphi jujube) c s du ng c s du ng trng y h c c truy n H n
Qu c c i thi n tr nh v nh n thc tu i gi c ki m tr v tnh
c ch chlinstrs b ng phng ph p qung cu llmn. N ng c
ch ng k nzym 200 mcg/ml c x t trng dich chi t t .
clmus v . krnum. C c s s c uy tn tng kh s t nhng c y
n y h c nhng l i c y th m c kh c r th lu n d tr n
nhng i u tr trc trng h t ng h h c v sinh h c.

CHNG 1 : TNG QUAN

1.1 Vi nt v bnh Alzheimer v enzym cholinesterase

1.1.1 Khi nim v bnh Alzheimer v c ch sinh bnh

A . Khi nim v cc vn lin quan :

- Bnh Alzheimer (AD) l mt bnh thoi ha no nguyn pht,
cn nguyn cha r rng, biu hin lm sng bng trng thi
mt tr tin trin, khng phc hi, thng khi pht la tui
trn 65.
- T l bnh Alzheimer ngi trn 65 tui l khong 5% v
nhm ngi trn 85 tui l 20%.
- V lm sng, trng thi mt tr tin trin thng khi u
bng ri lon tr nh, cc ri lon ngn ng, ri lon hot ng,
ri lon tr tu.
- Cc bin i c trng trong no l s gim st ng k cc
nron, teo lan ta v no, gin rng no tht.
- Phn loi bnh :
+ Alzheimer tp 1 khi pht la tui sau 65, triu chng
ch yu l ri lon tr nh, bnh tin trin chm.
+ Alzheimer tp 2 khi pht la tui trc 65, bnh tin
trin nhanh.
B . Cc c ch sinh bnh Alzheimer :
- Cc nh khoa hc a ra mt s gi thuyt c gng gii thch
nguyn nhn ca bnh, trong c gi thuyt cholinergic thit
lp c phng php tr liu bnh trc tip. Gi thuyt ny
xut rng AD l do gim tng hp ca cht truyn thn kinh
acetylcholin. Tuy nhin gi thuyt cholinergic khng c
duy tr h tr rng ri, v thuc dng iu tr thiu ht
acetylcholine thc s khng c hiu qu i vi bnh nhn.
- Nm 1991, ngi ta mc nhin cng nhn gi thuyt amyloid
beta (A)l nguyn nhn c bn ca bnh. C s ca nh
ny xut pht t v tr ca gen sn xut protein tin cht
amyloid beta (APP) trn nhim sc th 21 . Gi thuyt v amyloid ch
ra s tch t ca peptid amyloid beta l s kin trng tm kch hot vic thoi ha n
ron. S tch t ca cc vi si (fibril) amyloid, c coi l dng c c t lm ngn
cn cn bng ion canxi trong t bo, kch hot s cht t bo theo chng
trnh (apoptosis). ng thi, A cng tch t nhiu hn trong ti th t bo ca no
bnh nhn Alzheimer, v n cng c ch chc nng ca mt s enzym v vic s
dng glucose trong n ron
- Mt s qu trnh vim v cc cytokine c th ng vai tr trong bnh hc ca
AD. Vim l du hiu chung ca vic hy hoi m bt c bnh no, v c th l
qu trnh th cp sau khi m b hy hoi hoc l 1 du hiu ca p ng min
dch vi bnh
- AD cn c coi l bnh do protein tau (tauopathy) do s tch t bt thng
ca protein tau. Mi t bo n-ron u c mt b khung xng t bo, mt cu trc
bn trong gp phn to nn cu trc t bo gi l vi ng (microtubule). Cc vi
ng ny hot ng nh cc loi ng ng dn cho cc cht dinh dng v phn t
t thn t bo n u kia ca trc axon v quay ngc tr li. Mt protein gi
l tau gip n nh cu trc vi ng khi c photphorylate ha, v v th c gi
l protein lin h vi vi ng. bnh Alzheimer, tau b thay i v mt ha hc, tr
nn photphorylate ha qu nhiu; n bt cp vi cc si khc, to thnh cc m ri
si thn kinh v lm tan r h thng vn chuyn ca n-ron
- S thay i trong vic phn b cc neurotrophin (cht iu ha s sng, pht
trin v chc nng ca n-ron) v trong vic biu hin cc th th ca chng v
d nh cht neurotrophin thu c t no (brain derived neurotrophic factor -
BDNF) cng c m t trong bnh Alzheimer
l mt s c ch cng nh nguyn nhn dn n bnh AD , trong bi bo co
ny chng ti tp trung nghin cu v c ch u tin l do gim tng hp cc
cht dn truyn thn kinh Acetylcholin.
1.1.2Mi lin quan gia bnh Alzheimers v enzim chonineseterase
Mt trong nhng cht dn truyn thn kinh c vai tr quan trng trong AD
chnh l acetylcholine: ngi bnh AD, acetylcholine gim n 90%
(Whitehouse v cs. 1982). Acetylcholine tham gia vo qu trnh tr nh v nh
hng n hot ng ca nron ca hi c nga v v no .Trong h thng
thn kinh trung ng ngi ta nhn thy Acetylcholin ng mt vai tr quan trng
trong cc hot ng cao cp ca v no nh cc qu trnh nhn thc , qu trnh tr
nh , ch ,... v vy Ach c lin quan cht ch vi bnh Alzheimer .
 AChE ch yu c mt trong h thn kinh trung ng, xc tc thy phn
cht dn truyn ACh. Qa trnh ny cn thit chuyn t bo thn kinh
cholinergic t trng thi hot ng sang trng thi ngh. bnh nhn
Alzheimers cho thy s suy gim trm trng nng cht dn truyn
thn kinh Ach. Tnh trng ny gy suy gim nhn thc i vi ngi
bnh. Gi thuyt v vai tr ca h cholinergic trong AD c a ra
bi Whitehouse nm 1982. Theo gi thuyt ny, nhng cht c ch hot
ng AChE lm tng nng v thi gian hot ng ca ACh khe
synap thn kinh t ci thin triu chng bnh.
Khi nao b cua chung ta phai hoat ng qua mc hoc khng
c nghi ngi ung mc, thi se dn n tinh trang cac cht
trung gian hoa hoc c bit la Acetylcholin can kit hoc thiu
ht khin nao b bi mt moi biu hin nh giam tri nh, kem tp
trung, s ly cac tinh hung kho khn va chm chap. Vi vy
ngi ta khuyn nhng ngi hoat ng tri oc nhiu nn s
dng cac thao dc co tac dng lam c th tng tit
Acetylcholin. Theo vin y dc c truyn Hn Quc s dng thnh cng 7 loi
tho dc c sng lc ci thin chc nng nhn thc cng nh trong iu tr
bnh Alzheimer .

1.1.3 Enzym Acetylcholinesterase ( AChE ) :

Cu to :
- Acetylcholinesterase (AChE ) l mt enzym xc tc cho phn ng chuyn ha ,
thy phn cht dn truyn thn kinh acetylcholine , t lm ngng li s
hot ng ca chng ti khe synap cholinergic c thn kinh trung ng v
ngoi bin . AChE l mt enzym gn vo mng sau sinap bng lin kt cng ha
tr vi nhm glicolipid . Enzym ny c s quay vng hoatj ng rt nhanh v n
t n s hon thin v ng hc xc tc cao . AChE l mt trong nhng
enzym thy phn nhanh nht. Hot tnh ca n mnh gp khong 10 ln so vi
serin protease hoc butyrylcholinesterase cng iu kin nhit v pH.

- Cu trc khng gian ba chiu ca enzym AChE c xc nh ln u tin vo

nm 1991 . Trung tm hot ng xc tc phn ng thy phn ca enzym cso
cu to gm500 aa nm su trong cu trc v b n di y ca mt rnh hp
v su . Khi nghin cu cu trc ca enzym ny ngi ta thy rng cu trc ca
trung tm hot ng tng t nh cu trc ca enzym xc tc thy phn -
hydroxylase .

c im ca enzym :

- Hai c tnh ca enzym AChE l kh nng xc tc cho phn ng thy

phn acetylcholine xy ra nhanh , v kh nng khu tr tc dng ca
n ti synap thn kinh cholinergic . Cc cht dn truyn thn kinh
c a n cc khe synap trong cc bc nh . khi n mng trc
synap th cc ACh c gii phng vo khe synap , ti n lin kt
vi cc receptor , tip enzym AChE s thy phn Ach thnh
choline v acid acetic . Choline c bm tr li vo mng trc
synap nh cht mang v c ti s dng cho qu trnh tng hp
tip theo .

- Enzym acetylcholinesterase b bt hot bi gc phosphate hu c ,

carbamate v cc cht c thn kinh . Trong trng hp ng c
thuc tr su hay ha cht bo v thc vt nhm phosphate hu c ,
carbamate th nng AChE gim , c bit trong trng hp ng
phosphate hu c nng AChE gim mnh .

- Theo gi thuyt cholinergic , cc cht i khng cholinergic gy ra

suy gim tr nh v kh nng nhn thc ca con ngi cn cc cht
i khng muscarinic th c tc dng ngc li . Do vy vic c ch
acetylcholinesterase s duy tr nng v thi gian hot ng ca
acetylcholine ti khe synap , t duy tr kh nng ghi nh . S suy
gim nng acetylcholine thng gp cc bnh nhn alzheimer ,
theonghin c ca salviana v cng s th trong no ca bnh nhn
alzheimer c s thiu ht 90% cht dn truyn thn kinh ny . Trong
khi nguyn nhn gy bnh cn cha c cc nh khoa hc lm r
th cc cht c ch AChE l la chn hng u cho cc bnh nhn
Alzheimer , thng qua vic duy tr nng Ach trong no cc cht
ny c tc dng lm gim triu chng v ngn chn s tin trin ca
bnh .

C ch thy phn Ach :

Cholinesterase l enzym thu phn lm mt tc dng ca acetylcholin. Mt

phn t acetylcholin s gn vo hai v tr hot ng ca enzym; v tr anion
(anionic site) s gn vi cation N + ca acetylcholin, cn v tr gn este
(esteratic site) gm mt nhm base v mt nhm acid proton ( --H) to nn
mt lin kt hai ha tr vi nguyn t C ca nhm carboxyl ca este:

1.2 Mt s thuc v dc liu c ch cholinesterase

1.2.1 Cc thuc c ch AChE ang c lu hnh

Thuc c ch AChE l nhm thuc chnh trong iu tr bnh Alzheimer . Hin
nay trn th trng c cc loi thuc c lu hnh nh su :

Cht c ch Cholinesterase gip ngn nga s gim hm lng

acetylcholine , mt cht dn truyn thn kinh quan trng cho qu trnh ghi
nh v hc hi. Bng cch gi cho hm lng acetylcholine mc cao,
nhng loi thuc ny s h tr cho qu trnh truyn tn hiu gia cc t bo
thn kinh.
Cc loi cht c ch cholinesterase thng s dng l :

+ Tacrine (Cognex, Park-Davis) : Tacrine l thuc p ch

cholinesterase u tin c cng nhn cha bnh Alzheimer (1993)

Phn ng ph ng k nht ca Tacrine l c tnh i vi gan

Tacrine thng c tng tc thuc vi theophylline v cimetidine v
n c chuyn ha bng cyp P450 . Khi dng tacrine phi ung ngy 4
ln t nht 1h trc khi n v thc n lm gim hot tnh ca thuc t 30-
40% .

+ Donepezil (Aricept) : l mt cht c ch cholinesterase th hai c

s dng trong trng hp mt tr nh trung bnh hoc nng ngi c
bnh Alzheimer

Thi gian bn thi ca donezepil di t 70 ti 80 gi nn thuc ny c

li im ch cn dng mt liu mi ngy, ngoi ra n c t phn ng ph.
Khc vi tacrine, n khng c c tnh vi gan
Donepezil hydrochloride l cht c ch acetylcholinesterase c trng v
thun nghch ca cholinesterase, chim u th trong no.
Donepezil hydrochloride l cht c ch enzyme c hiu nghim gp 1.000 ln
hn buturylcholinesterase, enzyme hin din ch yu bn ngoi h thn kinh
trung ng . Tc ng c ch acetylcholinesterase ca Donepezil
hydrochloride (AChE) t bo hng cu tng ng vi cc tc ng ca
n v no .Cn lu rng thuc ny khng phi l thuc iu tr tit cn.
Tt c bnh nhn s tip tc biu hin du hiu tin trin c bn ca bnh
hc thn kinh.
Donepezil c th c tng tc vi nhng thuc x dng c cng mt
li chuyn ha nh phenytoin, carbamazepine, dexamethasone, rifampin
v phenobarbital, ketoconazole v quinidine c chng minh l ngn
chn s chuyn ha ca donepezil trong phng th nghim. Donepezil
khng c tng tc thuc vi theophylline, cimetidine, warfarin v
digoxin. N c th lm chm nhp tim, c bit i vi nhng bnh nhn
c nhng xo trn v s truyn dn trong tim. i khi bnh nhn c th
ngt xu khi dng thuc ny.
+ Rivastigmine (Exelon) :
Thuc Exelon c mt trn th trng t thng 4 nm 2000
iu tr bnh nhn sa st tr tu nh hoc trung bnh kiu Alzheimer,
cng cn gi l c kh nng bnh Alzheimer, hoc bnh Alzheimer .
 Rivastigmine, mt cht c ch chn lc acetylcholinesterase no kiu
carbamate c cho l lm d dng cho dn truyn thn kinh ca
acetylcholine bng cch lm chm thoi bin acetylcholine do cc t bo thn
kinh c chc phn nguyn vn tit ra. Rivastigmine lm tng c chn lc
acetylcholine trong v no v hi hi m , c tc dng ci thin thiu ht
nhn thc qua trung gian acetylcholine trong bnh Alzheimer. Ngoi chc
nng c ch cholinesterase thuc c th lm chm s to thnh cc mnh
protein tin cht amyloidogenic -amyloid (APP) v do lm chm s pht
trin ca bnh AD .
Rivastigmine tng tc vi enzym ch ca n bng cch to thnh
mt phc hp gn ng ha tr lm enzym b bt hot tm thi .
Khc vi tacrine v donepezil, rivastigmine khng b chuyn ha
bi diu t cytochrome P450 , v vy nhng tc dng tng tc trn lm
sng l khng ng k v n cng khng c i vi gan .
Thuc exelon ni chung dung np tt, nhng vn c vi tc dng
ph nh bun nn, nn, chm nhp tim.

+ Galantamine (Razadyne) : L thuc mi trong iu tr bnh

Alzheimer , lm chm tin trnh suy thoi thn kinh .

Galantamine l mt alkaloid bc 3 , l mt thuc c ch cnh tranh , chn lc

v c th khng AChE . Ngoi ra galantamine cnlm gia tng tc ng ni
ti ca Ach trn receptor nicotinic c th l do gn vo v tr allosteric ca
receptor kt qu l c th ci thin c tnh trng bnh Alzheimer .
Galantamine tc dng bng cch c ch sn sinh AChE mt khc n lm tng
p ng cu no vi Ach , c ch tc dng th hai l lm tng s gii phng
 Ach tc l lm cho t bo no tit ra nhiu acetylcholin hn . Thuc khng
cha khi bnh AD nhng lm gim cc triu chng ca bnh

Memantine (Namenda) : gip iu ha hot ng ca glutamate, mt loi cht

chuyn dn truyn thn kinh khc lin quan n qu trnh ghi nh v hc hi.
Loi thuc ny c dng iu tr bnh Alzheimer's trong giai on va v
nng.
Namenda (memantine) l thuc mi nht gp mt trn th trng M
t thng 10 nm 2003. y l thuc duy nht thuc loi chng th th N-
methyl-D-aspartate (NMDA) dng iu tr Alzheimer .

C ch cho rng s kch thch qu th th NMDA bi hot ng

bt thng ca glutamate c th gi mt vai tr quan trng trong bnh
AD . Memantine (Namenda) gip lm gim hot tnh ca th th NMDA cn
thit cho hc hi v tri thc . Nhng cn ch rng thuc khng c tc dng
lm gim s thoi ha t bo thn kinh ca bnh Alzheimer .

Ngoi ra, cn s dng cc thuc nh nivalin, gliatylin cng cho kt qu

kh quan. Cc thuc trn ch c tc dng lm chm s tin trin ca
bnh Alzheimer ch khng iu tr khi bnh.

1.2.2 Cc nghin cu v pht trin dc liu c tc

dng c ch AChE

Nghin cu sng lc dc liu c tc dng c ch enzyme acetylcholinesterase

bang phng php o quang v sc k lp mng s dng cht hin mu sinh hc

- Alzheimer l mt trong nhng nguyn nhn hng u gy t vong ngi cao

tui trn th gii, l mt dng thng gp ca bnh sa st tr tu. S tch t
mng amyloid trong no l mt nguyn nhn gy bnh alzheimer, theo gi thuyt
cholinergic, vic pht sinh bnh alzheimer c lin quan n s thiu ht cht dn
truyn thn kinh acetylcholine trong no ti gn 90% cc thuc iu tr
alzheimer hin nay ch yu l cc cht c ch enzyme acetylcholinesterase
 (AChE) t duy tr nng acetylcholine, ng thi gim thiu s hnh thnh
mng amyloid trong no. Cc thuc ny hu ht u c ngun gc dc liu, v
vy chng ti tin hnh sng lc cc dc liu c s dng trong y hc c
truyn c tc dng c ch AChE nhm tm ra thuc mi iu tr alzheimer.
c th sng lc nhanh cc loi c kh nng c ch enzym AChE lm thuc iu
tr alzheimer, ta xy dng phng php c ch AChE da trn phng php o
quang ca Ellman. Sau tin hnh th tc dng c ch trn bn sc k lp
mng vi thuc th sinh hc nhm pht hin ra cc vt cht trong dch chit ton
phn hoc cc phn on c tinh ch c kh nng c ch AChE phn lp
v xc nh cu trc cc cht c tc dng sinh hc cho nghin cu tip theo.
Da trn nguyn tc nh gi hot tnh enzyme acetylcholinesterase ca Ellman,
ta xy dng phng php sng lc dc liu c tc dng c ch enzym
acetylcholinesterase bng phng php o quang v sc k lp mng vi thuc
th hin mu sinh hc (enzym acetylcholinesterase (AChE), c cht
acetylthiocholine iodide (ATCI) v TT Ellman Acid 5,5 -dithiobis-(2-nitrobenzoic)).
- Chit xut Methanolic ca 7 loi tho dc gm (Acorus calamus ,
Acorus gramineus , Bupleurm facaltum , Dioscorea batatas ,
Epimedim koreanum , Poria cocos and Ziziphi jujuba ) s dng trong
y hc c truyn Hn Quc ci thin tr nh v kh nng nhn thc
ca ngi ln tui c kim tra v tnh c ch cholinesterase bng
phng php o quang ca Ellman . S quan st cho cc chit xut t
A. Calamus v E. Koreanum cho kt qu v s c ch ng k enzyme
nng 200 g / ml . Kt lun ny da vo s hiu bit v cc loi
tho mc ny v cc loi tho mc khc c th nghim di s xem
xt ca nhng nghin cu trc y v ha hc v hot ng sinh hc
ca chng.
- Thuc c ch acetylcholinesterase (AchE) c s dng trong iu
tr bnh Alzheimer (AD). Hin nay phng thc hu hiu nht iu
tr bnh Alzheimer l tc ng ln h cholinergic tc l s dng cc
cht chng cholinesterase . AD l mt chng bnh thoi ha khp ph
bin nh hng n dn s cao tui v triu chng chnh l mt tr nh
. Mt s loi thuc tng hp, v d nh tacrine, donepezil v sn phm
thin nhin- c gc l rivastigmine v galantamine c dng iu
tr ri lon nhn thc v mt mt lin quan n AD. Cc loi thuc ny
b hn ch s dng bi cc tc dng ph bt li ca chng nh cc ri
 lon ng tiu ha v sinh kh dng thp . Vic tm kim cc cht c
ch AchE tt hn vn ang l mi quan tm ln.
- Hng ngn nm nay, cc loi thc vt c s dng ci thin
kh nng nhn thc cc nc ng Nam . Mt s nghin cu
dc l chng minh rng c rt nhiu kh nng pht hin ra cc
loi khng cholineterase mi t nhng loi thc vt ny. Panax
ginseng ( y l mt loi nhn sm ch mc Hn Quc) c tc dng
nng cao nhn thc v huperzine A t Huperzia serrata c khng
nh l c hiu qu trong iu tr AD (Ma v Yu, 1990, huang, 1999).
Mt s polyme alkylpyridinium t Reniera sarai dehydroevodiamine
t Evodia rutaecarpa, v protopine t Corydalis ternata c khng
nh l cht c ch AchE.
- Trong nghin cu ny, chng ti iu tra cc hot ng c ch AchE
ca mt s loi thuc tho dc-Acorus calamus (AC), Acorus
gramineus (AG), bupleurum falcatum (BF), Dioscorea batatas (DB),
Epimedium koreanum (EK), poria cocos (= Pachyma hoelen, PC) v
zizyphus jujuba (ZJ). Tt c chng u c s dng ph bin Hn
Quc v BF, DB, EK, PC v ZJ c chnh thc lit k trong t in
dc hc ca Hn Quc (Korea FDA, 2002a). Mt cch truyn thng,
by loi thuc tho dc c s dng ci thin chc nng tr
nh cc nc phng ng v khng c mt bo co v hot ng
chng cholinesterase ca by loi tho dc ny.

CHNG 2: I TNG V PHNG PHP NGHIN CU

2.1 Nguyn liu nghin cu :

Mu nghin cu l 7 loi tho dc ca Hn Quc : Cc loi thc vt gm thn

r cy Acorus calamus ( cy thy xng b ACR) , thn r ca cy Acorus
gramineus (cy thch xng b AGR) , r ca cy Bupleurum falcatum (BFR) ,
thn r cy Dioscorea batatas ( cy c mi DBR), cy Epimedium koreanum
(EKH) , hch nm ca nm Poria cocos (PCS) v qu ca cy Zizyphi jujuba
(ZJF) c sy kh di nh nng mt tri v mua t mt ca hng thuc

tho dc Hn Quc ti Seoul.

BFR, DBR, EKH, PCS v ZJF ph hp vi cc c im ca t in dc hc

Hn Quc (Cc Qun l Thc phm v Dc phm Hn Quc, 2002a, b). Tt
c cc loi thuc tho dc ny c ghi nhn bi gio s Whan Kyun Whang
ti trng i hc dc, i hc Chung-Ang, ni cc mu chng t c lu
gi (nm 2001-11-17) . Mi chit xut c xc nhn bng cch i chiu TLC
v HPLC vi cc d liu c cung cp trong t in dc hc Hn Quc hoc
B lut Dc phm Hn Quc.

2.2 Phng tin nghin cu

CC LOI HA CHT V THUC TH

Ha cht Acetylcholinesterase (AchE) t hng cu ngi, acetylthiocholine,

thuc th axit 5,5'-dithio-bis- (2-nitrobenzoic) (DTNB) v physostigmine c
mua t Sigma (Poole, Anh quc)

2.3 Qu trnh chit xut

- EKH v PCS c ct nh v c chit xut ba ln vi methanol nhit

phng trong 5 ngy, mi ln v ta thu c dch cht chit xut . Ct r ca AC,
AG, DB v qu ca ZJ c chit xut bng methanol bng cch s dng my
ngng t nhit 65oC trong 5h ba ln v thu c cht chit xut .

- . Cc chit xut khc nhau c c ng bng my c quay chn khng dn

n khi lng kh. Cc chit xut methanolic c th nghim cho hot tnh
chng cholinesterase ca chng c m t v liu iu tr ca tt c cc chit
xut l 200 /ml .
- ACR v EKH hot ng tng i mnh c phn chia gia nc v CH2Cl2
(DCM) v cc phn cng c kim tra cho xt nghim sinh hc .

Qu trnh chi t xu t c t m t t trng s nh su :

Dc liu tho
dc
Chi t sxhlt b ng mthnl kh ng 10-20C (24h)

Dch chit 1 B dc liu

Chi t sxhlt b ng mthnl kh ng 10-20C (24h)

Dch chit 2 B dc liu

Chi t sxhlt b ng mthnl kh ng 10-20C

Dch chit 3 B dc liu

Qu trnh chi t xu t KH v PCS

Chu y : i vi c c dc li u r cu C, G, DB v qu cu ZJ th i u ki n

chi t l trng m y ngng tu nhi t l 65 C trng v ng 5h

Dch chit 1,2,3

C c bng my c quay chn

khng

Sy lnh ( phng php ng kh )

- Dch chit kh c

2.4 Quy trnh nghin cu hot tnh sinh hc cho tc dng chng AChE

2.4.1 Cch tin hnh :

- c ch AchE c nh gi bng mt phng php ci tin ca phng php

o mu ca Ellman.

- AChE 40 l (0,36 U ml-1 m pH 8) v dung dch chit xut thc vt 20 l

c thm vo 2,0 ml m pH 8 v trn (4oC) trong 30 pht.

- Cc ng tip theo cng c x l theo cch ny vi s c mt ca dung dch

physostigmine 0,1 mM 20 l cho php nh gi v kim sot s thy phn
ACh khng phi do hot ng ca AChE .

- Phn ng c bt u bng cch thm 0.5 mM DTNB 20 l v 0.6 mM Ach

20 l, v dung dch c 37oC trong 20 pht.

- Dung dch Physostignine (0.1 mM) 20 l c thm vo chm dt phn

ng v dung dch c t trong mt quang ph k 25oC.

- Ngay sau ta o mt quang ti bc sng 412 nm . Tnh t l phn trm

% c ch v tnh ton . Physostigmine c s dng nh mt cht mu i
chiu dng tnh .

Phng php o quang xc nh hot ca enzym

acetylcholinesterasea (phng php ca Ellman 1961 ):
- Phng php ny s dng c cht l acetylthiocholin iodid (ATCI )
v thuc th hin mu l 5,5-dithiobis-nitrobenzoic acid (DTNB)
- Nguyn tc : c cht ATCI b thy phn di xc tc ca
cholinesterase to thnh thiocholin . Thiocholine phn ng vi
thuc th DTNB gii phng ra hp cht 5-thio-2-nitrobenzoic acid
c mu vng . Hp cht ny c xc nh bng cch o hp
th ca dung dch bc sng 412 nm .
- Phng trnh phn ng :

+ + - - +
CH3-CO-S-CH2CH2N (CH3)3 + H2O Ch (CH3)3N CH2CH2-S + CH3COO + 2H
 ctylthichlin thichlin

+ -
(CH3)3N CH2CH2-S +

thichlin thu c th DTNB

+
(CH3)3N CH2CH2-S- S NO2 + O2N S-

COOH COOH

5-thi-2-nitrbnznzic cid

M u v ng

2.4.2 Tnh ton kt qu v x l s liu :

- Phn trm c ch AChE c tnh theo cng thc :
I% =(1 - Ath /Ai chng ) * 100%
- Gi tr IC50 : l gi tr nng ti mu th c ch 50%
s to thnh thiocholin trong hn hp phn ng so vi
mu chng .
CHNG 3: KT QU V BN LUN
3.1 Kt qu
3.1.1 Kt qu thu thp d liu v cc dc liu a vo sng lc
D liu hin ti cho thy cc chit xut ca AGR, DRB v ZJF khng c tc dng c ch
trn AChE. Tuy nhin, nhng iu ny c s dng cho bnh lin quan n tr nh
theo truyn thng. Tc ng nng cao nhn thc ca nhng loi loi ny c th tc ng
n cc hot ng khc lin quan n bnh Alzheimer (AD) nh cht chng oxy ho, cc
tc dng chng vim hoc oestrogen.
 - Mt s quan st ng h gi thuyt rng oxy ha ng mt vai tr quan
trng trong qu trnh sinh bnh ca AD . Idebone, hot ng nh mt
cht nhn in t v cht thi gc t do, cho thy hiu qu trong sa
st tr tu v thoi ho mch mu.
- Cc thuc khng vim khng cha steroid cho thy hiu qu nhn
thc tt hn bnh nhn AD. Cc gia v mu vng, curcumin ( ngh),
c c hot tnh chng oxy ha , chng vim v ngn nga s suy gim
tr nh do A gy ra . lm gim mt sau synap v gim lng A
trong t bo no .
- C mt s bng chng cho thy rng liu php thay th estrogen
(ERT) c th c tc dng ngn chn chng li s pht trin v tin
trin ca AD v ERT ph n c lin quan n vic gim s pht trin
ca AD v phng thc iu tr thay th estrogen vi cn bnh ny
c bo co l gip tng cng kh nng nhn thc.
- Cht Aluminum cng c cp nhm nhn mnh n bnh sinh
ca AD. Gim s hp th hoc s ng nng nhm trong m no
cng cung cp cc c hi iu tr c th lm chm tc tin trin
lm sng ca bnh bng cch s dng deferoxamine, cht nhm
chelating agent.
AGR cha mt loi du d bay hi ch yu gm -asarone v -asarone , n

c cho l c tc ng dc l trn h thn kinh trung ng (CNS) bao

gm tc dng an thn v chng co git. Cc thuc sc, chit nc hoc du
d bay hi c th gim au do AGR gy ra, lm gim hot tnh t pht, thi
gian ng do pentobarbital gy ra, v co git do pentylenetetrazol gy ra i
vi chut. Tinh du t cy c ch s kch thch glutamate gy ra v kch hot
bo v thn kinh chng li c tnh gy ra bi NMDA.
Dioscorea batatas (DBR) cha saponin steroid, dioscin v cc hp cht
phenolic nh batatasin I, II, III gy bun ng. Mt s dn xut
tetrainsoquinolin t Dioscorea batatas c cha steroid saponin, dioscin v
cc hp cht phenolic ging nh batatasin I, II, III c tc dng gy bun
ng. Mt s dn xut tetrainsoquinoline c bo co. Dioscin l mt
steroid saponin quan trng ca thc vt c s dng trong tng hp ca
hormone steroid . Kt qu t mt s th nghim trong ng nghim chng
minh rng trong thn r Dioscorea batatas cha tinh th c hot tnh chng
 oxy ho. Dioscorins, cc protein lu tr phn ln ca DB cng c bo
co c c dehydroascorbate reductase v monodehydroascorbate hot ng.
ZJ l mt loi thc vt c cha mt lng ln cc hp cht ha hc v ch
yu c s dng nh mt loi thuc b v thuc an thn. Mt s alkaloid
c chit xut t ZJ v cc phn phn on alkaloid c dng sn
xut thuc an thn. Dammarane-loi triterpene oligoglycosides, jujubosides
A1 v C v acetyljujuboside B1 cng c chit xut v chng minh tc
dng trong vic c ch histamine t t bo dch tit phc mc chut gy ra
phn ng khng nguyn-khng th.
Tc dng c ch cn au bi BFR v PCS c th hin l 24,8% v
27,8%, tng ng, ti 200 g/ml (bng 1). Bupleurum gc l mt loi thuc
hng u trong y hc phng ng v rt nhiu cc triterpene saponin ,
sapogenins c chit xut v cc hp cht ph khc .
- Tc dng ca saponin trong BFR theo ng ung chut c
chng minh l c tc dng an thn, gim au v h st.
- Cht chit xut t Bupleurum c th c ch s gia tng thuc tnh
thm mao mch gy ra bi histamine hoc 5-hydroxytryptamine v
saikosaponin D c th kch thch yu t phng thch corticotropin
(CRF) v adrenocorticotropin t s tit ra vng di i .
Saikosaoponins cng cho thy mt tc ng ng k trn h thng t
bo to ra cc cht chuyn ha cyclooxygenase v lipoxygenase . T
nhng kt qu ny , BFR c s dng trong y hc c truyn iu
tr vim. N lm gim ng k nng cc cytokine vim trong huyt
thanh t cc phn ng vim.
- Cc axit carboxylic triterpen t chit xut methanol ca PCS, bao gm
axit pachymic v axit polyporenic, c tm thy c hot ng c
ch chng li s suy gim cc t bo hng cu do AAPH gy ra.
 Cc chit xut methanolic ca ACR v EKH v nng phn chia vi

H2O v CH2Cl2 cho thy hiu qu c ch mnh trn AChE . ACR-MeOH

c tm thy nh hot tnh mnh nht chng li AChE mc d khng c
AGR-MeOH nhng c hai loi ny u c s dng iu tr bnh
ng kinh, thn kinh, mt ng v cc bnh khc.
- Cc tinh du t AC v AG c bo co l c tc dng bo v thn
kinh . Trong AC c 3% v AG c 0.5-0.9% l tinh du v vy chng c
cc hot ng chng li cholinergic . -Asarone, -asarone v cc hp
cht khc cng c phn lp t cc loi tinh du ny .
- Thnh phn chnh ca EK l glycosides prenylflavone nh icarin,
epimedoside A, v korepimedoside A . Chc nng trn h min dch ,
tc dng h huyt p v hiu qu kch thch tnh dc cng c bo
co l tc dng dc l ca Epimedium . Epimedium koreanum c tc
dng chng co git do tc ng c ch h thn kinh trung ng v
c s dng trong iu tr chng suy nhc thn kinh v t lit tr
em .
- Magnoflorine, mt alkaloid loi aporphine, c c lp t r
Epimedium. Mt s phytosterol tm thy trong cc b phn di mt
t ca EK . S c mt ca cc hp cht ny c th gii thch c phn
no hot ng chng AChE ca EK

3.1.2Kt qu sng lc tc dng c ch AChE invitro ca cc dc

liu

- Cc hot ng c ch trn AChE i vi cc loi thc vt c kim tra c

nh gi v t l phn trm c ch c th hin trong Bng 1 nh sau :
Bng 1. sng lc dch chit methanolic t cc dc liu tho dc ca Hn Quc
cho thuc c ch AChE 200 g/ml (n = 3)

Tn thc vt B phn dng % c ch

 Acorus calamus ( Araceae ) Thn r 53.7 5.5

Acorus gramineus ( Araceae ) Thn r 7.2 6.4

Bupleurum falcatum R 24.8

( Umbellifeae ) 15.5

Dioscorea batatas (Dioscoreaceae Thn r 7.6 13.2

)

Epimedium koreanum Tho dc ??? 47.5 4.1

(berberidaceae )

Poria cocos (polyporaceae ) Hch nm 27.8 9.2

Zizyphus jujuba ( Rhamnaceae ) Qu 2.4

2.6
3.1.3 Xc nh IC50 ca cc dc liu tim nng c ch AChE

S c ch AChE ph thuc vo lng chit xut methanol ca ACR v

EKH, chit nc ca ACR v EKH, v chit xut DCM ca EKH. Khng
c s c ch ph thuc vo nng c th thu c bng chit DCM ca
ACR.
EKH-DCM c xc nh l hot ng cao nht vi IC50 c tnh l 82
g/ml nhng phn nc ca EKH li t hot ng hn (bng 2)
Gi tr IC50 ca ACR v EKH c th hin trong bng 2 nh sau :
Bng 2: Gi tr IC50 cho s c ch acetylcholinesterase bi ACR v EKH

Tho dc Gi tri IC50 ( g/ml )

ACR-MeOH 188
ACR-DCM ----
ACR-water 174
EKH-MeOH 258
EKH-DCM 82
EKH-water 507
Physostigmine 1.25 M ( 0.07 g/ml )

3.2 Bn lun
3.2.1 Thng tin cy thuc a vo nghin cu