triển khai phương pháp đánh giá tác dụng ức chế xanthin oxidase in vitro trên đĩa...

B Y T

TRNG I HC DC H NI

NGUYN TH HU

TRIN KHAI PHNG PHP NH

GI TC DNG C CH XANTHIN

OXIDASE IN VITRO TRN A 96

GING V P DNG VI MT S

CY THUC C TIM NNG KHAI

THC CA NG BO PAKO VN

KIU

KHA LUN TT NGHIP DC S

Ngi hng dn:

T.S. Nguyn Thy Dung

Ni thc hin:

B mn Dc lc

H NI - 2013

LI CM N

hon thnh kha lun ny, ti nhn c s gip qu bu ca cc

tp th, cc thy c gio, bn b v nhng ngi thn ca ti.

Trc ht, ti xin by t lng bit n su sc ti TS. Nguyn Thy Dng,

ngi thy hng dn tn tnh v quan tm gip ti trong sut qu trnh thc

hin kha lun.

Ti xin trn trng cm n ng y, Ban Gim hiu v cc b mn trng

i hc Dc H Ni to iu kin thun li cho ti trong thi gian hc tp v

nghin cu.

Ti xin gi li cm n chn thnh ti:

Cc thy c v anh ch k thut vin b mn Dc lc, trng i hc Dc

H Ni gip v to iu kin cho ti hon thnh kha lun.

PGS.TS. Nguyn Th Hoi, Khoa Dc, trng i hc Y Dc Hu.

TS. Phng Thin Thng, Khoa Phn tch tiu chun, Vin Dc liu.

Li cui cng, ti xin cm n ti gia nh, bn b, ngi thn - nhng ngi

ng h, chia s, ng vin ti nhng lc kh khn v gip ti hon thnh kha

lun ny.

H Ni, thng 5, nm 2013

Sinh vin

Nguyn Th Hu

MC LC

LI CM N..............................................................................................................i

DANH MC CC K HIU, CH VIT TT.......................................................iv

DANH MC CC BNG.........................................................................................v

DANH MC CC HNH..........................................................................................vi

T VN ............................................................................................................1

CHNG 1. TNG QUAN......................................................................................3

1.1. Tng quan v bnh gt......................................................................................3

1.1.1. nh ngha........................................................................................................3

1.1.2. Mi lin quan gia bnh gt v s tng acid uric mu, vai tr ca enzym

xanthin oxidase...........................................................................................................3

1.1.3. Thuc c ch xanthin oxidase.........................................................................5

1.2. Tng quan v xanthin oxidase.........................................................................6

1.2.1. Ngun gc, phn b.........................................................................................6

1.2.2. Cu trc, c ch hot ng..............................................................................7

1.2.3. ng hc enzym.............................................................................................9

1.2.4. Cc yu t nh hng n hot enzym.......................................................9

1.3. Phng php nh gi tc dng c ch xanthine oxidase in vitro.............11

1.3.1. Phng php o quang....................................................................................12

1.3.2. Phng php o p..........................................................................................13

1.3.3. Phng php o s dng HPLC vi detector hunh quang..........................14

1.4. Cc dc liu c tim nng c ch xanthin oxidase......................................14

1.5. Thng tin v mt s dc liu ca ng bo Pako- Vn Kiu.....................15

CHNG 2. I TNG, PHNG TIN V PHNG PHP NGHIN

CU..........................................................................................................................17

2.1. i tng nghin cu.......................................................................................17

2.1.1. Dc liu nghin cu......................................................................................17

2.1.2. Chun b cc mu nghin cu........................................................................18

2.2. Phng tin nghin cu...................................................................................18

2.2.1. Thuc v ha cht...........................................................................................18

2.2.2. Thit b v dng c.........................................................................................19

2.3. Ni dung nghin cu........................................................................................19

2.4. Phng php nghin cu.................................................................................20

2.4.1. Trin khai phng php nh gi tc dng c ch xanthin oxidase in vitro

trn a Costar 96 ging............................................................................................20

2.4.2. p dng nh gi tc dng c ch xanthin oxidase in vitro trn a Costar

96 ging ca cc cy thuc ca ng bo Pako Vn Kiu...................................24

2.4.3. Phng php thng k....................................................................................25

CHNG 3: KT QU V BN LUN..............................................................26

3.1. Kt qu..............................................................................................................26


trn a Costar 96 ging...........................................................................................26


96 ging ca cc cy thuc ca ng bo Pako Vn Kiu....................................31

3.2. Bn lun............................................................................................................37

3.2.1. V trin khai phng php nh gi tc dng c ch xanthin oxidase in vitro

trn a Costar 96 ging............................................................................................37

3.2.2. V p dng nh gi tc dng c ch xanthin oxidase in vitro ca cc cy

thuc c tim nng khai thc ca ng bo Pako Vn Kiu..................................39

KT LUN V XUT.....................................................................................42

TI LIU THAM KHO........................................................................................vii

DANH MC CC K HIU, CH VIT TT

ABTS : 2,2-azino-di(3-ethylbenzthiazoline-6-sulphonate)

AHP : 2-amino-4-hydroxypteridin

DMSO : dimethyl sulfoxid

HPLC : sc k lng hiu nng cao (high-performance liquid chromatography)

IC50 : nng c ch 50% (the half maximal inhibitory concentration)

IXP : isoxanthopterin

NSAIDs : thuc chng vim khng steroid (non steroidial anti inflammatory drugs)

DANH MC CC BNG

Trang

Bng 1.1. Thng tin v mt s cy thuc ca ng bo Pako

Vn Kiu 16

Bng 2.1. Danh sch cc cy thuc nghin cu 17

Bng 3.1. Kt qu nh gi tc dng c ch xanthin oxidase

ca allopurinol cc nng khc nhau 28


ca Quercetin cc nng khc nhau 30


ca 11 mu dc liu nng 100g/mL 32






ca cy mn a theo tng nng khc nhau 35

DANH MC CC HNH

Trang

Hnh 1.1. S chuyn ha base purin 4

Hnh 1.2. Cu trc xanthin oxidase 7

Hnh 1.3. C ch hot ng ca xanthin oxidase 8

Hnh 1.4. th hm s ngc 1/v i vi [S] trong s c mt

v vng mt cht c ch 11

Hnh 2.1. Quy trnh th nghim kho st ng hc enzym 21

Hnh 2.2. Quy trnh th nghim xy dng th phng trnh

Linewweaver Burk 22

Hnh 2.3. Quy trnh th nghim nh gi tc dng c ch xanthin

oxidase ca mt mu th 23

Hnh 2.4. Quy trnh th nghim xc nh c ch c ch xanthin

oxidase 24

Hnh 3.1. th biu din s ph thuc theo thi gian ca

lng acid uric to thnh (biu din thng qua OD)

vi cc dung dch enzym nng khc nhau 26

Hnh 3.2. th Lineweaver Burk ca xanthin oxidase 27

Hnh 3.3. th biu din tc dng c ch xanthin oxidase ca

allopurinol theo nng 29

Hinh 3.4. th biu din tc dng c ch xanthin oxidase ca

quercetin theo nng 30

Hnh 3.5. th biu din tc dng c ch xanthin oxidase ca

cao ton phn Mn a theo nng 36

Hnh 3.6 th biu din c ch c ch xanthin oxidase ca cao

phn mn a 37

1

T VN

Gt l mt bnh chuyn ha, c lin quan trc tip n ch n ung giu

acid nucleic nh tht (c bit l ni tng), cc loi u, mt s loi hi sn v nm

men. Bnh xy ra mi dn tc, mi iu kin a d v kh hu, c chiu hng

pht trin thnh th v cc tng lp c mc sng cao [1]. Trn th gii hin nay, t

l mc gt ngi trng thnh l 1%, y cng l bnh vim khp ph bin nht

nam gii [14]. Vit Nam gn y, i sng ngi dn ngy cng cao li c

quan tm chn on nn t l pht hin mc gt ngy cng ln [12].

Trc tnh hnh bnh gt ngy cng ph bin, cc nghin cu pht trin

thuc iu tr gt ngy cng c quan tm. Bn cnh vic pht trin cc thuc tng

hp ha hc, thc vt v dc liu cng l mt ngun nguyn liu to ln tm ra

cc hng i cho qu trnh iu tr bnh gt. Nhiu nghin cu sng lc v tm kim

dc liu c tim nng iu tr gt c tin hnh vi c th nghim in vivo ln in

vitro. Trong , th nghim in vitro th hin li th vi u im nhanh v tn

km hn. c bit, nh gi tc dng c ch xanthin oxidase in vitro l hng

nghin cu c nhiu tc gi la chn nhm tm kim cc dc liu c tim nng

iu tr gt [30]. C kh nhiu phng php nh gi tc dng c ch xanthin

oxidase, trong , phng php o quang da trn nh lng acid uric l phng

php ph bin, c th thc hin d dng cc iu kin trong nc. Bn cnh ,

Vit Nam cng l mt t nc c nhiu ti nguyn cy c, cng vi nn y hc c

truyn pht trin vi ngun tri thc s dng thuc dn gian ca 54 dn tc khc

nhau. Mi dn tc u c nhng kinh nghim s dng dc liu khc nhau, l

ngun d liu to ln nghin cu v pht trin cc thuc iu tr bnh ni chung

v bnh gt ni ring.

Trc nhng yu cu v iu kin nh vy, ti nghin cu Trin khai

phng php nh gi tc dng c ch xanthin oxidase in vitro trn a 96

ging v p dng vi mt s cy thuc c tim nng khai thc ca ng bo

Pako Vn Kiu c thc hin vi hai mc tiu sau:

2

1. Trin khai c phng php nh gi tc dng c ch xanthin oxidase in

vitro trn a Costar 96 ging.

2. nh gi kh nng c ch v xc nh c IC50 ca cy thuc c tc

dng c ch xanthin oxidase in vitro mnh nht trong cc cy thuc c tim nng

khai thc ca ng bo Pako Vn Kiu.

3

CHNG 1. TNG QUAN

1.1. Tng quan v bnh gt

1.1.1. nh ngha

Gt l mt bnh ri lon chuyn ha lin quan n tnh trng tng acid mu

[1],[7],[20]. Bnh biu hin bi cc cn vim khp cp ti din lp i lp li lin

quan n tinh th urat kt tinh, bnh thn do gt, si thn, s lng ng ca natri

urat (tophi) sn, gn, mng hot dch v nhng m khc ca c th [20].

1.1.2. Mi lin quan gia bnh gt v tng acid uric mu, vai tr ca xanthin

oxidase

Mi lin quan gia bnh gt v tng acid uric mu

Acid uric l sn phm thoi ging cui cng ca nucleoprotein c cha nhn

purin [1],[3],[20]. Khi nng acid uric vt qu gii hn ti a ha tan urat trong

dung dch c cng nng natri nh huyt tng tc l tnh trng tng acid mu,

lin quan trc tip n bnh gt. Trong mi trng hp, tng acid uric mu dn n

tch ly tinh th urat ti cc m, to nn cc ht microtophi. S lng ng urat ti

cc m khc nhau gy ra cc biu hin khc nhau. Ht tophi ti sn khp b v s

khi pht cn gt cp; s lng ng vi tinh th cnh khp, trong mng hot

dch,trong m sn v m xng s dn n bnh xng khp mn tnh do gt; s c

mt vi tinh th urat ti m mm, bao gn to ht tophi; vim thn k (bnh thn do

gt) l do tinh th urat lng ng ti t chc k ca thn. Acid uric niu tng v s

toan ha nc tiu dn n si tit niu trong bnh gt [7],[20].

Tng acid uric mu c xc nh l yu t nguy c ca gt [20]. Nng

acid uric mu cng cao th nguy c mc gt cng ln [37]. Bt k yu t no lm

tng qu trnh tng hp hoc gim s thi tr acid uric u c th lm tng nng

acid uric mu [20].

Vai tr ca xanthin oxidase trong sinh chuyn ha acid uric

Xanthin oxidase l mt enzym c vai tr quan trng trong vic hnh thnh

acid uric. Enzym ny c vai tr xc tc cho phn ng chuyn hypoxanthin v

4

xanthin thnh sn phm l acid uric ( hnh 1.1). Bt k tc ng no nh hng n

hot ng ca xanthin oxidase cng nh hng n s to thnh acid uric [3].

Hnh 1.1.

S chuyn ha base purin

1.1.3. Thuc c ch xanthin oxidase trong iu tr gt

Hin nay, chng tng acid uric mu v hu qu ca n l bnh gt ang ngy

cng ph bin. V vy, c kh nhiu nghin cu c tin hnh nhm tm ra cc loi

thuc mi c ng dng trong iu tr gt. Mt s thuc iu tr gt c bn ang

c s dng hin nay l thuc chng vim v thuc h acid uric mu. Thuc chng

vim nh colchicin hay NSAIDs thng c dng ch yu iu tr cc t cp

ca gt vi tc dng gim au, chng vim [4],[11]. Gt l mt bnh mn tnh m

5

nguyn nhn l chng tng acid uric mu. S dng thuc nhm mc ch gim nng

acid mu trong c th s gp phn ci thin c gt v ngn nga ti pht. C 2

con ng thc hin c iu ny, mt l tng o thi, hai l gim tng hp

acid uric. Thuc tng o thi acid uric cng c s dng trong lm sng nhng

chng c tc dng h acid uric mu yu v d gy ra si tit niu [2]. Trong khi ,

c ch tng hp acid uric t xanthin v hypoxanthin nh s xc tc ca xanthin

oxidase kh r rng. c ch xanthin oxidase s c ch qu trnh tng hp acid

uric v lm gim nng acid uric mu, ci thin tnh trng bnh gt [3]. Hin nay

c mt s thuc c ch xanthin oxidase c s dng trong lm sng kh ph

bin trong iu tr gt.

Allopurinol

- C ch tc dng:

Allopurinol lm gim nng acid uric mu do c ch cnh tranh xanthin

oxidase. Ngoi ra, thuc lm tng bi xut cc tin cht ca acid uric qua nc tiu

v lm n nh cc ht tophi [2].

- S dng trong iu tr gt:

Allopurinol l thuc c s dng ph bin nht h nng acid uric

mu. Thuc c dng trong iu tr gt mn v cc trng hp tng acid mu [2].

Thuc c gi tr c bit nhng bnh nhn c tnh trng tng acid uric mu tng

hp, bnh nhn c tophi, bnh nhn khng p ng vi cc thuc tng o thi acid

uric v bnh nhn c si acid uric [4],[11].

- Tc dng khng mong mun:

- Gy kch ng ng tiu ha, c vi gan v d ng vi da.

- C th gp cn gt cp giai on u iu tr [2].

Febuxostat

Febuxostat l mt thuc c tc dng c ch chn lc xanthin oxidase c

Lin minh chu u s dng gn y iu tr tnh trng tng acid uric mn c

xut hin s lng ng tinh th urat. Thuc c ch hot ng ca enzym thng qua

vic kha lin kt ca enzym vi c cht [22]. So vi allopurinol, febuxostat c ch

6

xanthin oxidase v lm gim nng acid uric mu hiu qu hn trong c th

nghim tin lm sng v lm sng [15].

Thuc c th c mt s tc dng khng mong mun nh gy ri lon chc

nng gan, au u, bun nn, chng mt. Ngoi ra, thuc c th gy cc nguy c v

bnh tim mch [22].

1.2. Tng quan v xanthin oxidase

Xanthin oxidase l mt enzym c cu trc phc tp, c bit n cch y

hn 100 nm v c xem l enzym cha kha trong chuyn ha purin. N xc tc

cho phn ng chuyn hypoxanthin v xanthin thnh urat. S tng xanthin oxidase

lin quan n nhiu c ch bnh sinh nn hiu bit v enzym, ng hc v kim

sot enzym l thc s cn thit. Tuy nhin, do cu trc phc tp v s phn b c

bit trong cc m, cc chc nng ca enzym ny vn cha c hiu bit y

[24].

1.2.1. Ngun gc, phn b

Xanthin oxidase c tm thy trong cc loi ng vt c v, gan b v sa

b nhng cng c trong chim, cn trng v vi khun [8]. Trong cc loi ng vt c

v, enzym c phn b rng ri trong cc m nhng nng cao nht l gan v

rut. Xanthin oxidase cng c tm thy b mt cc t bo ni m ca b v ln

nh, v c b mt ca t bo ni m gan chut. Enzym ny chit xut t sa b l

c trng nht, tuy nhin cng c th c chit t gan chut, g, g ty v t tuyn

v ca chut [24].

1.2.2. Cu trc, c ch hot ng

Cu trc

Xanthin oxidase l mt protein c khi lng khong 300 kDa, gm 2 tiu

n v. Mi tiu n v cha bn th oxi ha kh : mt phn ph molybden, mt

FAD, 2 Fe2S2 [8],[24]. Phn ph molybden bao gm mt dn xut pterin hu c tr

vi 2 nguyn t lu hunh ca molybdopterin, 2 nguyn t oxi v vi 1 nguyn t

lu hunh khc [24]. (Hnh 1.2)

7

A B

C

Hnh 1.2.

Cu trc xanthin oxidase

A. Molybdopterin

B. Molybden cofactor

C. Cu trc khng gian ca xanthin oxidase

C ch hot ng

Hot ng xc tc cho phn ng to thnh acid uric t xanthin xy ra trung

tm molybden ca xanthin oxidase. trng thi oxi ha, trung tm molybden ca

enzym c th c xy dng nh m hnh LMoVIOS(OH) vi L l i in cho

ng yu t pyranopterin chung cho cc enzym molybden v vonfram n nhn.

C ch hot ng ca enzym nh sau: proton t nhm Mo-OH tn cng

trung tm i nhn ti v tr C-8 ca c cht, ng thi vn chuyn hydro gii phng

ra t v tr C-8 ti nhm Mo=S to ra LMoIVO(SH)(OR) (Giai on 1). Sn phm

trung gian ny b ph v bi s vn chuyn electron cc trung tm oxi ha kh khc

trong enzym, gii phng H+, to ra LMoVOS(OR) (Giai on 2). Sau , hydro t

8

dung mi thay th nhm R, quay v trng thi ban u LMoVIOS(OH) ca enzym

(Giai on 3) [18] (Hnh 1.3).

Hnh1.3.

C ch hot ng ca xanthin oxidase

1.2.3. ng hc enzym

Thng s ng hc enzym (Km v Vmax) c xc nh bng hai phng

php o quang (UV) v phng php sc k lng hiu nng cao (HPLC). Gi tr Km

thu c nm trong khong 5,32 13,8M [26].

1.2.4. Cc yu t nh hng n hot ng enzym

Nng c cht

Xanthin oxidase xc tc cho phn ng chuyn hypoxanthin thnh xanthin v

xanthin thnh acid uric. Khi nng ca xanthin trong c th tng th tc ca

phn ng chuyn ha s tng, acid uric to ra s cng nhiu. Tuy nhin, n mt

mc no , enzym s bo ha c cht v tc phn ng khng tng ln na [3].

V vy, cn la chn nng xanthin thch hp trong phn ng tc phn ng

ti a m li khng lng ph c cht.

Nng enzym

Khi nng enzym tng ln, tc phn ng cng tng. Tuy nhin, khi

nng enzym tng n mt mc no , sn phm to ra qu nhiu s tc ng

9

vo trung tm d lp th ca enzym, phn ng bo ha v tc phn ng s khng

tng ln na [3]. Nng enzym cng l mt yu t quan trng khi xc nh ng

hc enzym, thi gian dng phn ng thch hp.

Nhit

Nhit c tc dng lm tng tc phn ng enzym ln cc i nh c

ch tng nng lng cung cp cho cc phn t c cht. Mi khi tng nhit ln

10C th tc phn ng s tng ln 2 ln. Tuy nhin, n mt nhit qu cao,

enzym s b bin tnh do b ph v cc lin kt trong cu trc. Mi enzym u c

mt nhit thch hp nht cho hot ng xc tc, nhit phn ng enzym

t ti tc cao nht [3]. Nhit cng l mt yu t trong qu trnh bo qun

enzym. Xanthin oxidase bo qun nhit 4C s khng b gim hot tnh trong

vng su thng. nhit -20C, enzym n nh trong t nht mt nm. C tc gi

cho rng, nhit hot ng ti u ca enzym l 65C [33]. Tuy nhin, theo mt

nghin cu so snh gia xanthin oxidase t sa b vi xanthin oxidase t sa d,

nhit ti u cho xanthin oxidase t sa b l 10C v t sa d l 20C [21].

pH

Enzym rt nhy cm vi pH ca mi trng, v vy pH c tc dng rt ln

i vi tc phn ng enzym. Mi enzym c mt gi tr pH ph hp nht cho

hot ng ca mnh. Mt s thay i nh so vi pH ti u cng dn n s gim

hot enzym do n lm thay i s ion ha ca cc nhm chc trong trung tm

hot ng ca enzym [3]. Vi xanthin oxidase, pH thch hp l 4,7 [8]. Tuy nhin,

c tc gi cho rng pH thch hp cho xanthin oxidase l 7,5 - 8 [33]. Cng c

nghin cu khc li cho thy, pH thch hp cho xanthin oxidase t sa b l 7,5 v

t sa d l 7,2 - 7,4 [21].

Ion kim loi

Cc ion kim loi nng c th c ch xanthin oxidase (V d: Hg2+, Ag+...).

Tc dng ca ion kim loi rt phc tp v n cn nh hng ti c trung tm hot

ng v c ch xc tc ca enzym [3].

10

Cc cht c ch

Xanthin oxidase b c ch bi ure, rt nhiu purin, pyrimidin v cc hp cht

d vng khc nh purin-6-aldehyd, 2-amino-4-hydroxypteridin-6-aldehyd. Xanthin

oxidase cn b c ch bi mt s thuc v mt s dch chit t cc loi thc vt.

Xanthin oxidase b bt hot khng thun nghch bi xyarit [8].

Hai c ch c ch enzym l c ch cnh tranh v c ch khng cnh tranh.

Cht c ch cnh tranh l nhng cht c cu trc tng t c cht, kt hp vi

enzym trung tm hot ng, ngn chn enzym kt hp vi c cht. c ch khng

cnh tranh l s c ch th hin qua lin kt cht c ch vi enzym t do hay phc

hp enzym c cht, mc c ch khng ph thuc nng c cht [3].

C ch c ch c xc nh thng qua vic xy dng th phng trnh

Lineweaver Burk, l th hm s ngc 1/v i vi [S] vi s c mt v vng

mt cht c ch [3]. (Hnh 1.4)

A B

Hnh 1.4.

th hm s ngc 1/v i vi [S] vi s c mt v vng mt cht c ch

A: c ch khng cnh tranh

B: c ch cnh tranh

11

1.3. Phng php nh gi tc dng c ch xanthin oxidase in vitro

nh gi tc dng c ch xanthin oxidase, cn so snh hot ca enzym

trong mi trng c v khng c cht th . C kh nhiu phng php xc nh

hot enzym [19]:

- Da vo s gim ca cc tc nhn oxi ha thch hp nh oxy, xanh

methylen, cytocrom.

- Da vo gim ca c cht hay s to thnh ca sn phm nh acid uric.

Tng ng vi cc nguyn tc l cc php o p, o nhit lng, o

hunh quang, o quang, o bc x hay o tch in [32].

1.3.1. Phng php o quang

Phng php o quang da trn nh lng acid uric

c p dng ph bin nht l phng php ca Noro [31], nguyn tc da

trn phn ng :

Xanthin + H2O + O2 Acid uric + H2O2

Hot xanthin oxidase c xc nh thng qua vic nh lng acid uric

to thnh c o bc sng 290nm 25C. Mt n v enzym c nh ngha

l tng lng enzym sn xut ra 1 mol acid uric trong mi pht nhit 25C.

Phng php ny c nhiu nh nghin cu p dng v c sa i cho ph

hp iu kin, ch yu s dng my quang ph UV v mt s t dng h thng

ELISA [16],[30],[31],[35]. Dung dch m thng c s dng l m phosphat

c pH t 7,5 - 8, v m phosphat khng nh hng n hot ng ca xanthin

oxidase [19], bc sng o cng dao ng t 290-295nm.

Quy trnh o thng c p dng vi hn hp th bao gm dung dch m

v dung dch enzym trong m c trong 15 pht 25C. Sau , thm dung

dch c cht (xanthin). Tip tc trong 30 pht nhit 25C. Dng phn ng

bng acid HCl 1N. Mu trng chun b tng t nhng enzym cho vo sau khi

cho HCl. Tin hnh o quang xc nh hp th ca tng mu [31].

Phng php ny c u im l thun tin, nhanh v nhy i vi enzym

tinh khit ( thng dng trong in vitro), tuy nhin khng th p dng phng php

12

ny o hot enzym trong t chc v c s c mt ca uricase [25]. C th

khc phc hn ch ny bng cch cho vo hn hp th kali oxonat 0,1 mM, acid

uric to thnh s khng b nh hng bi uricase [25].

Phng php o quang da trn cht ABTS (2,2-azino-di(3-ethylbenzthiazoline-

6-sulphonate)

y l mt phng php mi s dng cht mu ABTS nh mt cht hp th

mt quang thng qua vic s dng 2 enzym uricase v peroxidase. Phng php

ny c nhy kh cao, c th xc nh c hot ln ti 20 U/mL v c th p

dng cho cc bnh l huyt thanh khc [29].

Nguyn tc ca phng php ny da trn chui phn ng:

Hypoxanthin + 2 H2O + 2 O2 uric acid + 2 H2O2

Acid uric + O2 allantoin + H2O2 + CO2

H2O2 + ABTSred ABTSox + 2 H2O

Hot ca xanthin oxidase c xc nh thng qua vic o hp th ca

ABTSox to thnh sau 10 pht bc sng 410nm [29].

1.3.2. Phng php o p

Trong phn ng, xanthin oxidase ng vai tr nh mt cht cho electron nn

c th xc nh hot enzym thng qua vic nh gi s kh ca cc cht nhn

electron ph hp nh oxi, xanh methylen v cytochrom C.

Phng php o t l tiu th oxi cng c mt s tc gi p dng. Tuy

nhin, ng dng ca phng php ny c mt s hn ch nu trong gan c s tiu

th oxi vi t l cao m khng c c cht [19].

Quy trnh o c p dng vi hn hp gm mt lng gan c lm

ng nht v p lnh, m phosphat pH 8,6, dung dch natri xanthat, dung dch

KOH. Oxi tiu th c o p 37C [19].

13

nh gi tc dng c ch xanthin oxidase in vitro, thng dng enzym

tinh khit phn ng vi c cht cng s c mt ca cht th trong dung dch m

[30].

1.3.3. Phng php o s dng HPLC vi detector hunh quang

Nguyn tc da trn phn ng oxi ha 2-amino-4-hydroxypteridin (AHP)

thnh isoxanthopterin (IXP) di s xc tc ca xanthin oxidase. Lng IXP to ra

c o bi h thng HPLC s t l vi hot xanthin oxidase [32].

Trn thc nghim, c th tin hnh phng php ny nh sau: hn hp phn

ng gm m phosphat, dung dch AHP, dung dch 2,6-diclorophenonlindophenol

natri, enzym v nc th tch; hn hp c trong 10 pht 37C; dng phn

ng bng HClO4 v lm lnh trong 10 pht. Ph hunh quang kch thch v pht x

ca IXP c o 343 nm v 410 nm [32].

Phng php ny c u im n gin v nhy (nhy hn phng php

hunh quang thng thng khong 100 ln). Mt khc, hn hp phn ng c

phn tch trc tip bi HPLC khng cn phi tch IXP nh trong phng php

phng x [32].

1.4. Cc dc liu c tim nng c ch xanthin oxidase

Cc thuc tng hp ha hc ang c s dng kh ph bin trong iu tr

gt. Tuy nhin, cc thuc tng hp ha hc c nhiu tc dng ph, trong khi , gt

l mt bnh mn tnh cn iu tr lu di. Do , vic nghin cu tm nhm tm ra

thuc iu tr gt t dc liu thin nhin ngy cng c u tin v c t tc dng

khng mong mun. Bn cnh , hng nghin cu da trn c ch c ch xanthin

oxidase ang c quan tm gn y. V vy, trn th gii cng nh trong nc,

ngy cng c nhiu nghin cu c tin hnh tm hiu tc dng c ch xanthin

oxidase ca cc dc liu, t tm ra phng hng cho vic pht trin thuc iu

tr gt t dc liu. C kh nhiu dc liu c nghin cu v th hin tim

nng c ch xanthin oxidase.

cc nc trn th gii, c kh nhiu nghin cu sng lc cc dc liu

c tc dng c ch xanthin oxidase. Trong mt nghin cu sng lc cc cy thuc

14

n , c 14 dch chit th hin tc dng c ch xanthin oxidase nng 100

g/mL trong 18 dch chit; trong c 10 dch chit c ch hn 50% vi IC50 b

hn 100 g/mL; nhng cy thuc c tim nng nht tip tc c nh gi tc dng

h acid uric mu [34]. Mt nghin cu khc nghin cu trn 95 dch chit cc cy t

New Caledonia v Vanuatu, 82 % trong s th hin tc dng c ch xanthin

oxidase; mt s dch chit th hin tc dng tt vi IC50 thp [17]. Mt nghin cu

trn 122 dch chit methanol ca dc liu Trung Quc, c 69 dch chit th hin

tc dng c ch xanthin oxidase nng 100 g/mL vi 29 cy c ch hn 50%,

58 dch chit th hin tc dng nng 50 g/mL vi 15 dch chit c ch hn

50%. Cc cy tim nng nht bao gm Qu (IC50 = 18 /mL), Cc hoa (IC50 = 22

g/mL), C Gpxi (IC50 = 26 g/mL) [27].

Trong nc cng c mt s nghin cu sng lc c thc hin. Mt

nghin cu ca Nguyn Th Thanh Mai v cc ng s tin hnh nh gi tc dng

ca 288 dch chit c ly t 96 dc liu chn lc, thu c mt s kt qu

kh quan. Trong 288 dch chit, c 188 dch chit (65,3%) th hin tc dng c ch

xanthin oxidase nng 100 g/mL, trong c 46 (24,5%) c ch vi t l hn

50%. nng 50 g/mL, c 168 (58,3%) dch chit th hin tc dng vi 21

(12,5%) c ch hn 50%. Ti nng 25 g/mL, c 146 (50,7%) dch chit th

hin tc dng v 8 (5,5%) c ch vi t l hn 50%. Trong khi , nng 10

g/mL, c 126 (43,8%) dch chit th hin tc dng c ch vi 2 (1,6%) dch chit

c ch hn 50%. Tng cng c 49 dch chit c IC50 di 100 g/mL [30].

1.5. Thng tin v mt s dc liu ca ng bo Pako- Vn Kiu.

Vit Nam l quc gia c 54 dn tc sinh sng vi nhiu ngn ng khc nhau.

Mi dn tc li c mt nhm cc cy thuc chm sc sc khe v cha bnh c

th ca dn tc mnh. Tc ngi Pako v Vn Kiu l nhng c dn c mt sm

nht vng Trng Sn tri di t Qung Nam n Qung Bnh, nhiu nht l

a bn min ni Qung Tr vi hn 70% ngi Pako - Vn Kiu sinh sng. Qua

thi gian h tch ly c nhng kinh nghim, tri thc v s dng cc loi thc

vt lm thuc cha bnh. Trong thi gian qua, c mt s cng trnh nghin

15

cu bo tn ti nguyn cy thuc, tuy nhin, nhng kt qu ny mi ch dng li

iu tra kinh nghim s dng cy thuc ca cng ng, ch cha c nhng nh gi

kim chng khoa hc v tc dng ca cc loi ny [9],[10]. Trong ti ny, 10 cy

thuc ca ng bo Pako - Vn Kiu c la chn t cc cy thuc c s dng

nhiu nht trong dn gian vng ng bo sinh sng nhng cha c cc nghin cu

c th kim chng thnh phn ha hc v hot tnh. Cc cy thuc ny ch yu c

tc dng chng vim, gim au, li tiu dng trong cc bnh xng khp hay cc

bnh v ng tit niu. Mt s thng tin thu thp c t nhng ngi dn bn x

v 10 cy thuc c tng hp trong bng 1.1.

Bng 1.1.Thng tin v mt s cy thuc ca ng bo Pako Vn Kiu

STT Tn khoa hc Tn Vit

Nam Kinh nghim dn gian

1 Leea rubra Blume ex

Spreng Gi hc

au nhc xng khp, chng vim

2 Archidendron clyearia

(Jack.)I. Niels

Mn a

Chng vim tt, vim cu thn, vim gan, vim phn

ph

3 Erythropalum

scandens Blume

Dy

hng Li tiu, chng vim

4 Ficus fulva Reinw. ex

Blume Nga lng

Cha au nhc xng khp, li tiu, cha ph

5 Nepenthes mirabilis

(Lour.) Druce Np m

Li tiu, cha si ng tit niu, cao huyt p

6 Helminthostachys

zeylanica (L.) Hook

Sm bng

bong Cc bnh xng khp, bnh

lin quan n tiu tin

7 Flemingia stricta

Roxb.ex Ait.f

Tp m thng

Cha si thn

8 Stachytarpheta

jamaicensis (L.) Vahl

ui chut

Cha i but, i ra si v vim ng tit niu

9 Phyllanthus

reticulatus Poir Phn en

Cha tiu tin kh, i tiu ra mu

10 Caesalpinia

pulcherrima (L.) Sw

Kim

Phng Li tiu, cha si thn

16

CHNG 2. I TNG, PHNG TIN V PHNG

PHP NGHIN CU

2.1. i tng nghin cu

2.1.1. Dc liu nghin cu

Dc liu nghin cu bao gm 10 cy thuc ca ng bo dn tc Pako -

Vn Kiu Qung Tr v ang dng phng v cha bnh lin quan n tc

dng chng vim, chng oxy ho v chng ung th.

Cc mu cy nghin cu c PGS. TS. Ninh Khc Bn, ThS. Nguyn Th

Cng Vin Sinh thi v Ti nguyn Sinh vt Vin Khoa hc v Cng ngh

Vit Nam xc nh tn khoa hc.

Cc mu c thu hi bi PGS.TS Nguyn Th Hoi Khoa Dc i hc

Y Dc Hu.

Danh sch dc liu nghin cu c th hin bng 2.1.

2.1.2. Chun b cc mu nghin cu

Chit xut dc liu: mu cy thuc c phi sy kh, xay nh thnh bt

mn, sau chit vi methanol tuyt i bng php ngm lnh (48 gi/ln x 3 ln).

Gp dch chit methanol v ct thu hi dung mi di p sut gim thu c cao.

Cao ton phn c sy chn khng n khi lng khng i.

Chun b mu th hot tnh: ha tan cao methanol trong dimethyl sulfoxid

(DMSO), pha long bng m thnh cc nng khc nhau s dng cho nghin

cu.

17

Bng 2.1. Danh sch cc cy thuc nghin cu

STT

K

hiu mu

Tn khoa hc H Tn ph

thng

B phn dng

1 AV08 Leea rubra Blume ex

Spreng Leeaceae Gi hc

Cnh

mang l,

r

2 BM 87

Archidendron

clyearia (Jack.) I.

Niels

Mimosaceae Mn a

Cnh

mang l

3 BM

130

Erythropalum

scandens Blume Erythropalaceae

Dy

hng Phn trn mt t

4 BM

135

Ficus fulva Reinw.

ex Blume Moraceae

Nga

lng

Cnh

mang l

5 BM

157

Nepenthes mirabilis

(Lour.) Druce Nepenthaceae Np m

Phn trn mt t

6 DK 40 Helminthostachys

zeylanica (L.) Hook Ophioglossaceae

Sm bng

bong

Phn trn mt t

7 DK 56 Flemingia stricta

Roxb.ex Ait.f Fabaceae

Tp m thng

Cnh

mang l

8 QT01

Stachytarpheta

jamaicensis (L.)

Vahl

Verbenaceae ui chut

Phn trn mt t

9 QT02 Phyllanthus

reticulatus Poir Euphorbiaceae Phn en

Phn trn mt t

10 QT03

Caesalpinia

pulcherrima (L.)

Sw

Fabaceae Kim

Phng Cnh

mang l

18

2.2. Phng tin nghin cu

2.2.1. Thuc v ha cht

- Allopurinol (Sigma Aldrich).

- Xanthin, 99% (Sigma Aldrich).

- Xanthin oxidase (9 n v/mg protein, 8mg protein/ml; Sigma Aldrich).

- Dimethyl sulfoxid (Meck).

- Ha cht pha m Na2HPO4, KH2PO4 (Vel, B).

- Mt s dung mi, ha cht, thuc th khc dng trong chit xut v ha cht

t tiu chun dc dng khc.

2.2.2.Thit b v dng c

- My o pH (EUTECH).

- Cn phn tch AY 220 (SHIMADZU).

- H thng my ELISA gm my dc khay vi tinh th (Biotek, M) v my

lc khay (Awareness, M).

- My ct nc 2 ln (Halminton, M).

- My khuy t ( Heidolph, c).

- My ly tm (HERMILE Z300).

- Micro pipet mt u knh 2-20l, 10-100l, 100-1000l (Eppendorf, c).

- Micro pipet a knh 30-100l (Eppendorf, c).

- a Costar 96 ging 3596 (Corning, M).

- Cc dng c khc s dng trong thc nghim: u cn, micro tube cc loi,

pipet, bnh thy tinh, cc c m, a thy tinh....

2.3. Ni dung nghin cu

t c nhng mc tiu nghin cu t ra, ti nghin cu c

thit k vi cc bc tin hnh nh sau:

Thc hin mc tiu 1: Trin khai c phng php nh gi kh nng c ch

xanthin oxidase in vitro trn a 96 ging:

- Kho st ng hc enzym: nhm mc ch

+ La chn hot enzym thch hp cho kho st.

19

+ La chn thi gian dng phn ng thch hp.

- Xy dng th Lineweaver Burk.

- Thm nh phng php thng qua nh gi tc dng ca allopurinol v quercetin.

Thc hin mc tiu 2: Xc nh c IC50 ca cy thuc c tc dng c ch

xanthin oxidase in vitro ln nht trong cc cy thuc c tim nng khai thc ca

ng bo Pako Vn Kiu.

- Chit xut cao ton phn ca cc cy thuc.

- nh gi tc dng c ch xanthin oxidase ca cc cao ton phn 3 nng 10

g/mL, 50 g/mL, 100 g/mL.

- Xc nh IC50 ca cy thuc tim nng nht.

- Xc nh s b c ch c ch enzym ca cy thuc tim nng nht.

2.4. Phng php nghin cu

Phng php nh gi tc dng c ch xanthin oxidase trn a 96 ging

c trin khai theo phng php m t bi Nguyn Th Thanh Mai [30] v c

thay i cho ph hp vi iu kin th nghim.

Nguyn tc ca phng php da trn s to thnh acid uric t xanthin nh

s xc tc ca xanthin oxidase. Lng acid uric to ra t l vi hot enzym, c

nh lng bi vic o quang 290nm. Mu c cht th c so snh vi mu

khng c cht th nh gi tc dng c ch enzym ca cht th.


trn a Costar 96 ging

Chun b cc ha cht cn thit

- m phosphat 70mM, pH=7,5.

- Enzym: xanthin oxidase vi cc dung dch c hot khc nhau trong m

phosphat.

- C cht : dung dch xanthin 260M trong m phosphat.

- Ha cht dng phn ng: dung dch HCl 1N.

20

Kho st ng hc enzym

-B tr th nghim:

Lc, trong 15 pht 25C.

Thm 60l dd xanthin

o mt quang nhng pht th 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, 31.

Hnh 2.1.

Quy trnh th nghim kho st ng hc enzym

Th nghim c lp li 3 ln.

- nh gi kt qu:

V th biu din mi lin quan gia thi gian v s to thnh acid uric

(da trn mt quang) ca tng hot enzym, t la chn hot enzym

thch hp m ti s hnh thnh acid uric tuyn tnh theo thi gian nht, ng thi

la chn thi gian dng phn ng thch hp.

Ging chng:

115l m

6 ging th :

85l m,

30l enzym (vi 6

hot thay i t

0.01U/mL

0.1U/mL)

21

Xy dng th Lineweaver Burk ca xanthin oxidase

- B tr th nghim:

Lc, trong 15 pht 25C

Thm 60 l xanthin (vi cc nng thay i t 50 M-100 M)

trong 5 pht

Dng phn ng bng 25 l HCl 1N

o quang 290nm.

Hnh 2.2.

Quy trnh th nghim xy dng th phng trnh Lineweaver Burk

Song song vi mi mu th c mt mu trng c tin hnh tng t

nhng enzym c cho vo sau khi cho HCl 1N.

- nh gi kt qu:

Xy dng th tuyn tnh gia 1/V (biu th bi 1/OD) v 1/[S]. Trong

:

Cc ging

th: 85l

m,

30 l dung

dch enzym

0,04 U/mL

22

[S]: nng c cht.

V : tc phn ng.

OD: OD th - OD trng.

Thm nh phng php thng qua nh gi tc dng ca allopurinol v

quercetin

- B tr th nghim:

trong 15 pht

Thm 60 l dd xanthin 260 M, 30 pht


o quang 290 nm.

Hnh 2.3.

Quy trnh th nghim nh gi tc dng c ch xanthin oxidase ca mu th.

Ging th c p dng vi mu th l allopurinol cc nng 0.2 g/mL

0.7 g/mL v quercetin cc nng t 15 g/mL 40 g/mL.

Ging chng

85 l m,

30 l enzym

Ging th

65 l m,

20 l cht th,

30 l enzym

23



Th nghim c lp li 3 ln.

- nh gi kt qu:

Tnh I (% c ch) theo cng thc :

Trong :

ODchng = ODchng - ODtrng chng.

ODth = ODth - ODtrng th

2.4.2. p dng nh gi tc dng c chxanthin oxidase in vitro trn a Costar

96 ging ca cc cy thuc ca ng bo Pako Vn Kiu

nh gi tc dng c ch xanthin oxidase ca 11 mu dc liu ba nng

khc nhau

B tr th nghim nh m t hnh 2.3 vi cht th l dch chit cc cy

thuc 3 nng 10 g/mL, 50g/mL, 100 g/mL.

- nh gi kt qu:

Tnh I (% c ch) theo cng thc (1).

Xc nh IC50 ca cy thuc tim nng nht.

T kt qu sng lc trong cc dc liu trn, tm ra dc liu c tim nng

c ch xanthin oxidase cao nht.

B tr th nghim nh m t hnh 2.3 vi cht th l cc nng khc

nhau ca dc liu tim nng.

T kt qu, dng phn tch probit tm ra IC50 ca cy thuc.

24

Xc nh s b c ch c ch xanthin oxidase ca cy thuc tim nng nht

- B tr th nghim:

Lc, trong 15 pht 25C

Thm 60 l xanthin (vi cc nng thay i t 50 M-100 M)

trong 5 pht


o quang 290nm

Hnh 2.4.

Quy trnh th nghim xc nh c ch c ch xanthin oxidase



- nh gi kt qu:

Xy dng th phng trnh Lineweaver Burk cho nng cht th, da

vo th xc nh c ch c ch enzym ca dc liu.

Cc ging th

65 l m,

20 l cht th

nng 200

g.mL,

30 l dung dch

enzym.

Cc ging chng

85 l m,

30 l dung dich

enzym.

25

2.4.3. Phng php thng k

S liu c lu tr v x l bng phn mm Microsoft Office Excel 2007

v phn mm SPSS 13.0.

S liu c biu din di dng gi tr trung bnh lch chun (Xtb

SD). S dng One-way ANOVA vi test hu kim Dunnett (post hoc) so snh

gi tr trung bnh gia cc mu. S khc bit c ngha thng k nu p< 0,05.

S dng phn tch probit tm gi tr c ch 50% hot ng enzym IC50.

Gi tr IC50 c biu din vi khong tin cy 95%.

26

CHNG 3. KT QU V BN LUN

3.1. Kt qu


trn a Costar 96 ging

Kho st ng hc enzym

la chn c nng enzym thch hp m ti lng acid uric to

thnh tuyn tnh theo thi gian nht, th nghim c tin hnh vi 6 dung dch

enzym c hot khc nhau. S ph thuc ca lng acid uric to thnh theo thi

gian c biu din di dng th. Kt qu th hin hnh 3.1.

Hnh 3.1.

th biu din s ph thuc theo thi gian ca lng acid uric to thnh (biu

din thng qua OD) vi cc dung dch enzym c hot khc nhau.

27

Nhn xt:

- hot 0,01 v 0,02 U/mL, enzym hot ng kh yu, lng acid uric

tng chm theo thi gian. Vi cc hot 0,06, 0,08 v 0,1 U/mL, enzym hot ng

mnh, lng acid uric tng nhanh v t n tnh trng bo ha khi cha ht thi

gian phn ng. hot 0,04 U/mL, enzym hot ng tt, lng acid uric to

thnh tuyn tnh theo thi gian. V vy, hot ny c la chn cho cc th

nghim tip theo ca ti.

- Sau thi gian 30 pht, phn ng t bo ha i vi hu ht cc hot

enzym. Do , trong cc th nghim tip theo, phn ng s c dng ti thi im

30 pht bng HCl 1N.

Xy dng th Lineweaver Burk ca xanthin oxidase

Tin hnh th nghim vi cc dung dch c cht c nng khc nhau. Kt

qu c biu din th hnh 3.2.

Hnh 3.2.

th Lineweaver Burk ca xanthin oxidase trn a Costar 96 ging

28

Thm nh phng php thng qua nh gi tc dng ca allopurinol v

quercitin.

- Allopurinol l mt cht th c chng minh c tc dng c ch enzym

xanthin oxidase in vitro v in vivo. thm nh phng php nh gi tc dng c

ch hot ng xanthin oxidase in vitro ca dc liu trn a 96 ging, chng ti

tin hnh nh gi tc dng ca cht ny. Tin hnh th nghim vi cht th l cc

dung dch allopurinol nng khc nhau. Kt qu c biu din bng 3.1 v

hnh 3.3.

Bng 3.1. Kt qu nh gi tc dng c ch xanthin oxidase ca allopurinol cc

nng khc nhau.

Nng allopurinol

(g/ml)

OD % hot

xanthin

oxidase

% c ch hot xanthin

oxidase

0 0,539 0,066 100

0,2 0,479 0,018 89,1 10,9

0,3 0,452 0,025 84,1 15,9

0,4 0,353 0,018 65,5 34,5

0,5 0,255 0,065 47,1 52,9

0,6 0,079 0,048 14,7 85,3

0,7

0,037 0,014 6,9 93,1

29

Hnh 3.3.

th biu din tc dng c ch xanthin oxidase ca allopurinol theo nng

Nhn xt:

Allopurinol cc nng khc nhau th hin s c ch r rt hot ng ca

xanthin oxidase, nng cng ln cao, kh nng c ch cng ln. Ti nng 0,7

g/mL, kh nng c ch t gn n 100%.

Theo kt qu phn tch probit, nng allopurinol c ch 50% hot ng

xanthin oxidase l 0,456 (0,416-0,497) g/mL.

- Quercetin l mt flavonoid c chit xut t dc liu v cng c

chng minh c tc dng c ch hot ng ca xanthin oxidase in vitro v in vivo.

Chng ti cng s dng cht ny thm nh phng php nh gi tc dng c

ch xanthin oxidase in vitro trn a Costar 96 ging. Tin hnh th nghim vi cht

th l cc dung dch quercetin cc nng khc nhau. Kt qu c biu din

bng 3.2 v hnh 3.4.

30

Bng 3.2. Kt qu nh gi tc dng c ch xanthin oxidase ca quercetin cc

nng khc nhau.

Nng quercetin (g/mL)

OD % hot xanthin

oxidase

% c ch hot xanthin oxidase

0 0,610 0,016 100

15 0,496 0,023 81,3 18,7

20 0,380 0,006 62,3 37,7

25 0,300 0,022 49,2 50,8

30 0,261 0,030 42,8 57,2

35 0,193 0,021 31,7 68,3

40 0,141 0,008 23,1 76,9

Hnh 3.4. th biu din tc dng c ch xanthin oxidase ca quercetin theo

nng

31

Nhn xt:

Quercetin th hin tc dng c ch xanthin oxidase r rt theo tng nng ,

nng cng cao, kh nng c ch xanthin oxidase cng ln.

Theo kt qu phn tch probit, IC50 ca quercetin l 26,779 (24,953 - 28,543)

g/mL.

Nh vy, s dng phng php nh gi tc dng c ch xanthin oxidase in

vitro trn a Costar 96 ging cho kt qu ph hp vi allopurinol v quercetin.


96 ging ca cc cy thuc ca ng bo Pako - Vn Kiu

3.1.2.1. nh gi tc dng c ch xanthin oxidase ca 10 cy thuc ba nng

khc nhau

Phng php sau khi c thm nh c p dng vi mu th l cao ton

phn ca 10 cy thuc ca ng bo Pako Vn Kiu. Tin hnh th nghim vi 3

nng khc nhau 10 g/mL, 50 g/mL, 100 g/mL cho mi mu cao ton phn

xc nh tc dng c ch xanthin oxidase in vitro.

- Ti nng 100 g/mL

Nng 100 g/mL thng l nng gii hn trn c nhiu nh nghin

cu s dng nghin cu tc dng c ch xanthin oxidase in vitro ca cc dch

chit dc liu. Kt qu c th hin trong bng 3.3.

32

Bng 3.3. Kt qu nh gi tc dng c ch xanthin oxidase ca 11mu cy thuc

nng 100 g/mL.

Cy thuc OD % hot

xanthin oxidase

% c ch hot xanthin

oxidase

Chng 0,529 0,056 100

Mn a 0,332 0,012 * 62,8 37,2

Np m 0,518 0,011 98,1 1,9

Nga lng 0,424 0,023* 80,3 19,7

Gi hc (cnh mang l)

0,481 0,023 91 9

Gi hc (r) 0.485 0,035 91,9 8,1

Phn en 0,469 0,058 88,9 11,1

ui chut 0,454 0,030 86 14

Tp m thng 0,499 0,039 94,5 5,5

Sm bng bong 0,511 0,013 96,6 3,4

Kim phng 0,509 0,012 96,4 3,6

Dy hng 0,456 0,037 86,4 13,6

Ghi ch: *: p

33

- Ti nng 50g/mL.

Kt qu c th hin bng 3.4.

Bng 3.4. Kt qu nh gi tc dng c ch xanthin oxidase ca 11mu cy thuc

nng 50 g/mL.

Dc liu OD % hot

xanthin oxidase


Chng 0,529 0,056 100

Mn a 0,434 0,016* 82,2 17,8

Np m 0,519 0,016 100 0

Nga lng 0,446 0,029* 84,5 15,5

Gi hc (cnh mang l)

0,505 0,048 95,7 4,3

Gi hc (r) 0,530 0,023 100 0

Phn en 0,480 0,030 91 9

ui chut 0,497 0,045 94,1 5,9

Tp m thng 0,478 0,022 90,6 9,4

Sm bng bong 0,503 0,012 95,3 4,7

Kim phng 0,465 0,044 88,1 11,9

Dy hng 0,479 0,009 90,7 9,3

Ghi ch: *: p< 0,05 khi so vi mu chng (mi th nghim c lp li 3 ln)

Nhn xt:

nng 50 g/mL, mn a v nga lng vn tip tc th hin tc dng c

ch xanthin oxidase vi t % c ch tng ng l 17,8% v 15,5% (p

34

-Ti nng 10g/mL.

Kt qu c th hin bng 3.5.

Bng 3.5. Kt qu nh gi tc dng c ch xanthin oxidase ca 11 cy thuc

nng 10g/mL

Dc liu OD % hot

xanthin oxidase


Chng 0,510 0,009 100

Mn a 0,502 0,007 98,4 1,6

Np m 0,512 0,019 100,4 -

Nga lng 0,536 0,031 105,3 -

Gi hc 0,522 0,022 102,3 -

Phn en 0,516 0,024 101,7 -

ui chut 0,531 0,039 104,1 -

Tp m thng 0,527 0,037 103,3 -

Sm bng bong 0,510 0,021 100

Kim phng 0,467 0,013 91,8 8,2

Dy hng 0,512 0,013 99,4 0,6

Gi hc (r) 0,507 0,020 99,6 0,4

Ghi ch: mi th nghim c lp li 3 ln

Nhn xt:

nng 10 g/mL, khng c cy thuc no th hin tc dng c ch

enzym r rt, k c 2 cy mn a v nga lng.

35

Xc nh IC50 ca cy thuc tim nng

Sau khi kho st 3 nng , nhn thy mn a th hin tc dng c ch

xanthin oxidase n nh v r rt nht. V vy, mn a c kho st mt ln na

xc nh IC50. Th nghim c b tr vi cht th l dch chit mn a cc

nng 10 g/mL, 20 g/mL, 50 g/mL, 100 g/mL, 200 g/mL, 300 g/mL. Kt

qu c th hin trong bng 3.6.

Bng 3.6. Kt qu nh gi tc dng c ch xanthin oxidase ca cy mn a theo

tng nng khc nhau

Nng (g/mL)

OD % hot

xanthin oxidase


0 0,360 0,013 100

10 0,359 0,014 99,7 0,3

20 0,343 0,005 95 5

50 0,293 0,012 81,1 18,9

100 0,239 0,042 66,4 33,6

200 0,134 0,047 37,2 62,8

300 0,033 0,035 9,2 90,8

36

Hnh 3.5.

th biu din tc dng c ch xanthin oxidase ca mn a theo nng

Nhn xt :

Tuy khng bng allopurinol v quercetin nhng tc dng c ch xanthin

oxidase ca cao ton phn mn a cng th hin r v tng theo s tng ca nng

.

Theo phn tch probit, IC50 ca cao ton phn mn a l 161,7 (129,6-

203,3) g/mL.

Xc nh s b c ch c ch ca dc liu tim nng

xc nh c ch c ch enzym ca cao ton phn cy mn a, th nghim

c b tr vi cc dung dch c cht c nng thay i. Kt qu c biu din

dng th phng trnh Lineweaver Burk cho mu khng c v c cht c ch

(hnh 3.6).

37

Hnh 3.6.

th phng trnh Lineweaver-Burk vi mu c v khng c c ch (mn a)

Nhn xt :

Da vo th cho thy c ch c ch xanthin oxidase ca cao ton phn

mn a l kiu hn hp.

3.2. Bn lun

3.2.1. V trin khai phng php nh gi tc dng c ch xanthin oxidase in

vitro trn a Costar 96 ging

Xu hng nghin cu v tc dng c ch xanthin oxidase ca dc liu ang

ngy cng pht trin, trn th gii cng c kh nhiu phng php nh gi hot

xanthin oxidase cng nh phng php nh gi tc dng c ch xanthin oxidase.

Mt s tc gi s dng phng php o p [19], c tc gi s dng phng php

hunh quang [32], c tc gi li dng phng php da trn cht mu ATBS [29]

nhng a phn u s dng phng php o quang da trn vic nh lng acid

38

uric theo m t ca Noro [31]. Tuy nhin, phng php ny thng c cc nh

nghin cu p dng o quang trn cuvet [16],[34],[35], ch c mt s t tc gi trong

nc hay nc ngoi p dng trn h thng ELISA [30]. V vy, chng ti tin

hnh ti vi ni dung trin khai phng php nh gi tc dng c ch xanthin

oxidase in vitro trn a 96 ging.

Xanthin oxidase thng c bo qun 4C m bo hot tnh. Tuy

nhin trong qu trnh pha v tin hnh th nghim, enzym c th hot ha v sau

gim hot ng. V th, cn phi gi nhit thp cho dung dch enzym trong c

qu trnh lm th nghim bng cch ngm cc ng enzym trong nc . Enzym

v c c cht u ch c pha ngay trc khi dng. Cc nghin cu trc y

thng s dng dung dch enzym c hot 0,01 U/mL 0,1 U/mL [30],[31],[35].

Trong ti ca chng ti, sau khi kho st ng hc enzym, dung dch enzym c

hot 0,04 U/mL c la chn s dng cho cc th nghim tip theo bi v ti

hot ny enzym hot ng xc tc tt, lng acid uric to ra t l tuyn tnh nht

theo thi gian trong 30 pht u ca phn ng. Phn ng n pht th 30 t bo

ha nn chng ti cng la chn thi im sau 30 pht l thi im dng phn ng.

Trong cc nghin cu tin hnh trc, cng la chn thi im sau 30 pht l thi

im dng phn ng [30],[31].

Dc liu c s dng trong th nghim nh gi tc dng c ch xanthin

oxidase thng c chit bng methanol v ha tan trong DMSO lm dch th

hoc chit bng nc v ha tan trong m hay hn hp ethanol v tween 80

[16],[30],[31],[34],[35]. Cc cy thuc c s dng trong th nghim ca chng

ti cng c chit bng methanol, c ha tan trong DMSO. Khi pha long mu

thnh cc nng khc nhau, mu c pha long bng m trnh nh hng

qu nhiu ca DMSO.

Phng php nh gi tc dng c ch xanthin oxidase in vitro trn a 96

ging c thm nh thng qua nh gi tc dng ca allopurinol v quercetin.

Allopurinol l mt thuc c s dng rng ri trong gt, c tc dng h acid uric

mu thng qua c ch c ch xanthin oxidase, n thng c s dng lm cht

39

chng trong cc th nghim nh gi tc dng c ch xanthin oxidase. Bn cnh ,

quercetin l mt flavonoid c chit ra t dc liu c tc dng chng oxi ha v

c ch hot ng ca enzym xanthin oxidase [13]. Trong phng php ny,

allopurinol v quercitin u th hin tc dng c ch xanthin oxidase r rt vi IC50

ln lt l 0,456 (0,416-0,497) g/mL v 26,779 (24,953-28,543) g/mL. Tuy

nhin gi tr IC50 ny kh cao so vi cc th nghim khc [17],[28]. iu ny c th

do phng php s dng a Costar 96 ging 3596 (Corning, M). Ging ny

thng c s dng o quang bc sng ln hn 340 nm trong khi bc

sng ca th nghim ny l 290 nm. iu ny c th gy sai s cho php o. Tuy

phng php cn nhiu hn ch nhng n c nhng u im nh nhanh, lp li

kh n nh, c th lm c nhiu mu cng mt lc, thch hp vi kiu sng lc

tc dng. Bn cnh , kinh ph cho vic s dng phng php ny cng kh hp

l. Tuy gi tr IC50 ca allopurinol v quercetin cao nhng vn c th dng i

chiu trc tip vi cc gi tr c ch IC50 ca cc cy thuc.

3.2.2. V p dng nh gi tc dng c ch xanthin oxidase in vitro ca cc cy

thuc c tim nng khai thc ca ng bo Pako Vn Kiu

3.2.2.1. V tc dng c ch xanthin oxidasein vitro ca 10 cy thuc a vo

nghin cu

Phng php sau khi c thm nh, c tin hnh p dng vi 10 cy

thuc (11 mu) ca ng bo Pako- Vn Kiu. y l cc cy thuc thng c s

dng lm thuc trong cng ng ngi Pako- Vn Kiu, c kho st v thu thp

thng tin bi nhm nghin cu ca PGS.TS Nguyn Th Hoi. Chng c s dng

nhiu trong dn gian vi cc tc dng ch yu l chng vim, gim au, li tiu.

Gi hc, sm bng bong hay nga lng thng c dng trong cc bnh au nhc

xng khp, cc cy cn li dng trong cc bnh ng tit niu nh vim,

si...Cc cy thuc ny c nghin cu sng lc tc dng chng oxy ha v

chng ung th [5]. Thng cc cy c tc dng chng oxy ha s c nghi ng c tc

dng c ch xanthin oxidase. V vy, chng ti cng tin hnh nh gi tc dng c

ch xanthin oxidase ca 10 cy thuc ny. Trong 10 cy thuc (11 mu), ch c mn

40

a (Archidendron clypearia (Jack.) I. Niels) v nga lng (Ficus fulva Reinw. Ex

Blume) th hin tc dng c ch xanthin oxidase. Mn a th hin tc dng r nht

c 2 nng 100 g/mL v 50 g/mL. Trong nghin cu sng lc tc dng chng

oxi ha, cy mn a cng l cy c tim nng chng oxi ha cao nht vi ED50 l

2,18 [5].

3.2.2.2. V kh nng c ch xanthin oxidase ca mn a

Tc dng c ch enzym ca mt mu th c th hin thng qua IC50 ca

n. Trong th nghim nh gi tc dng c ch xanthin oxidase, mn a th hin

tim nng c ch xanthin oxidase mnh nht, c a vo xc nh IC50. Gi tr

IC50 theo phn tch probit ca cao ton phn mn a l 161,7 (129,6-203,3) g/mL,

allopurinol dng lm i chiu c IC50 l 0,45 (0,416-0,497) g/mL. Mn a l

mt cy thuc c tc dng chng vim tt, thng c s dng trong dn gian

cha cc bnh v vim nh vim cu thn, vim gan, vim phn ph. Ngoi ra, mn

a cn c dng tr bnh thy u, tr sng, ho, p tr tri r [6]. Tuy vy, cy

thuc ny vn ch ang mc dng theo kinh nghim ch cha c nghin cu

su. Trong ti ny, mn a th hin tc dng c ch xanthin oxidase tt, y

l tin xut cho nhng nghin cu su hn v tc dng ca mn a.

Cc cht hay dc liu khi th hin tc dng c ch enzym s c nghin

cu su hn v c ch c ch enzym ca n. C ch c ch c th l c ch cnh

tranh hoc c ch khng cnh tranh, c xc nh thng qua th phng trnh

Lineweaver Burk [3]. th ny cng c p dng trong ti ca chng ti

xc nh c ch c ch xanthin oxidase ca cao ton phn mn a. Tuy nhin, cao

methanol mn a khng th hin r rt c ch cnh tranh hay khng cnh tranh.

Chng ti tm xp c ch c ch ca mn a l theo kiu hn hp. C ch ny

cng l dng c ch hay gp khi nghin cu v tc dng c ch xanthin oxidase

[31],[35]. Chng ti ngh n gi thuyt, cao ton phn dc liu thng cha

nhiu hot cht c th c tc dng c ch xanthin oxidase, mi hot cht c ch

theo cc c ch khc nhau dn n c ch ca cao ton phn thng l c ch hn

hp.

41

KT LUN V XUT

KT LUN

Sau mt thi gian tin hnh thc nghim kha lun thu c mt s kt

qu nh sau:

1. - trin khai c phng php nh gi tc dng c ch xanthin oxidase in

vitro trn a Costar 96 ging vi hot xanthin oxidase ti u l 0,04 U/mL, thi

im dng phn ng l sau 30 pht. Bng phng php ny, xc nh c IC50 ca

allopurinol l 0,456 (0,416 0,497) g/mL, IC50 ca quercetin l 26,779 (24,953

28,543) g/mL.

2. - Trong 10 cy thuc ca ng bo Pako - Vn Kiu, cao methanol ca cy mn

a v nga lng th hin tc dng c ch xanthin oxidase nng 100 g/mL vi

% c ch ln lt l 37,2% v 19,7%, nng 50 g/mL vi % c ch ln lt l

17,8% v 15,5% .

- Xc nh c IC50 ca cao methanol mn a l 161,7 (129,6-203,3) g/mL.

- Bc u xc nh c cao methanol mn a c ch xanthin oxidase in vitro

theo c ch hn hp.

XUT

T nhng kt qu thu c, ti xin xut :

- Tip tc trin khai phng php nh gi tc dng c ch xanthin oxidase in

vitro trn a 96 ging c kh nng o bc sng thp hn, bao gm c 290 nm.

- Tip tc xc nh kh nng h acid uric mu thc nghim ca cy mn a c

th hng ti kh nng s dng trong iu tr gt v h acid uric mu ca cy thuc

ny.

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