' Nghien ciru - Ky thuat
r
Nghien cmi tac dung cua dich chiet CO nho noi tren mo hinh gay ton thuong gan
bang paracetamol
Cd nhg ndi (Edipta prostrata L., Asteraceae) Id du'gc li$u s§n cd d ViOt Nam, td Idu da dugc sd dyng rOng rdi trong diiu trj cdc chdng huyit nhiOt, ho ra mdu, ndn ra mdu, ehdy mdu dirdi da, bdng huyit dong kinh, d m mdu trong sdt xuit huyit... vdi tdc dyng lu'ang huyit, chf huyit dd dugc nghidn cdu nhiiu trong nu-dc vd thi gidi'^-''.
Trong ddn gian, cd nhg ndi cdn dung du-di dgng cao todn tfnh d i diiu trj can thOn dm hu-, bdo vO vd phgc hdi chdc nang gan^. Tuy nhidn, nhdng nghidn cdu v i tdc dyng cda ed nhg ndi trdn gan cd rit it tdc gid nghidn cdu.
Trong bdi vlit ndy, chCing tdi nghiin cdu tdc dgng chdng oxy hda cua cao Idng ed nhg ndi (chiit nu-dc vd chiit cdn) trdn md hlnh gdy tdn thu-ang gan chuOt nhit tring bing paracetamol liiu cao.
D6\ t u ^ n g v d p h y a n g p h d p n g h i d n cipu
Nguyin v$t liOu
Cd nhg ndi khd mua v i du-gc kiim nghiOm ttieo Dirge diin ViOt Nam IV bdi Tmng tdm Kiim nghiOm Thudc vd My phlm Hd NOi.
Cao Idng cd nhg ndi chiit xuit bing dung mdi nirdc (cao nu-dc) ty 10 1:2 (1 ml cao chda 2g dirvc liOu) bing phu-ang phdp sac. Cao Idng cd nhg ndi chiit xuit bing dung mdi cdn 80°
(cao cdn) ty 10 1:2 (1 ml cao chda 2g duvc liOu) bing phirang phdp ngim kiOt. Quy trinh chiit xuit vd diiu chi cao Idng dirge thyc hiOn tgi bO mdn Duvc - Tnrdng Cao ding Y t i Hd NOi.
DOng v$t thf nghiOm
ChuOt nhit ti^ng chung Swiss, cd 2 gidng, khde mgnh, tivng lu'gng 20,0 ± 2.0 gam do ViOn
TAP CHi DlfOC HOT - m/?ftl ? «rt Jii M i u e-.^
L§ Th} Hai Yen\ Pham Thi Vin Anh^
' Trudng cao ddng Y te Hd Npi
^Tru&ng dai hpc YHd Ngi VO sinh Djch te Trung u-ang cung cip. Dgng vdt du-gc nudi trong dieu kiOn dly du ttidc an va Twsitc udng tgi phdng thf nghidm td 3 - 5 ngdy tiTjdc khi nghidn cdu vd trong sudt thdi gian nghidn cdu tgi Bd mdn Duvc ly - Tnrdng Dgi hgcYHdNgi.
Phuvng tifn nghidn cdu
Kit ^nh lu-gng edc enzym AST, ALT cua hang DIAU\B GmbH (Ao).
Cdc thudc dCing trong nghidn cdu: sylimarin (bidt dirge Legalon) dgng vidn ndn, hdm lirgng 70 mg cOa hang Madaus (Hdn Qudc) vd paracetamd vidn sCii 500mg (biOt duvc Efferalgan - Phdp). Hoa chit vd kinh hien vi quang hgc d i Idm gidi phlu vi thi gan tgi Tmng tdm phdt hign vd chin dodn sdm ung ttiu'. Kit qud do PGS.TS. Le Dinh Roanh ddnh gid vd nhdn S\r\\\.
PhuY^ng phdp nghiin cdu Xdc dinh thinh phin hda thyc v$^^:
* Dirdng khd: Liy 2ml cao Idng dirge liOu cho vdo dng nghlOm, thdm 5ml dung djch Fehling, dun sdi edch iMy niu cd tua mdu vdng hogc dd ttii xdc ^|nh ed du'dng khd.
+ Terpenoid: liy 2ml cao Idng dwgc ligu dio vdo dng nghiOm, hda vdi 2ml dorofomi, them ti>
td H2SO4 ddc vdo thdnh dng. Mdu tfm hdng chuyin din sang do ndu xuit hiOn d Idp phdn each chi ra cd terpenoids.
+ Alealoid: liy 2ml cao Idng dwgc liOu ti^On vdi vdi gigt acid acetic (TT), thdm 2-3 gigt thudc thd Dragendoff vdo dng nghiOm. Mdu dd vdng
» j l t hiOn chl ra ed alealoid.
+ Coumarin: liy 2ml cao Idng dirge liOu vdo dng nghiOm, thdm 1ml Na2C03 10%, dun edch ttiuy din sdi rdi Idm nguOi bdng nirdc dd. Nhd vdi gk?t ttiudc thd Diazo sd xuit hiOn mdu dd chdng td cd coumarin.
U
Nghien CLFU - Ky thuit
+ Acid hdu ca: liy 2ml cao Idng, ttiem 2 gigt ninhydrin 0,2% rdi dun ndng. Mdu xanh xuat hiOn chdng td cd add hdu co.
Tic dyng cua djch chiet co nhg n6i tren mo hinh gay ^n tfitrong gan bang paracetamol
Gdy dgc gan d p bdng udng paracetamol liiu cao'^"-^'.
ChuOt nhdt ti^ng duvc chia ngdu nhien thdnh 5 Id, moi Id 10 con.
Ld 1 (chdng sinh hgc): udng nu'dc cit 0,2ml/10gttiittgng
Ld 2 (gdy md hinh vidm gan): udng nu'dc elt + udng PfiR
Ld 3 (chdng duong): uwig silymarin 70 mg/kg + udng PAR
L6 4 (ttiudc ttid): Cao niritc 4g/kg + udng PAR L6 5 (thudc ttid): Cao cdn 4g/kg + udng PAR ChuOt duvc udng nu'dc hogc tiiude Iidn tyc trong 8 ngay. Ngdy ttid 8, sau udng thudc 3 gid, chudt duvc nhjn ddi 16 - 18 gid tnrde dd, gdy tdn tiiu-ang gan diuOt d cac Id td Id 2 din Id 5
Bdng 2: Anh hudng cQa cao long co nhg nSi len
bdng udng paraeetamol (PAR) liiu 400mg/kg vdi ttie tfch 0,2ml/10g. 48 gid sau khi udng paracetamol, liy mdu dOng mgeh cdnh cQa chugt de djnh luvng enzym AST vd ALT. ddng thdi liy gan de xdc djnh ttgng lirvng vd quan sdt md bOnh hge (dgi ttii, vi ttii) vd djnh lirgng MDA djch ddng t h i gan.
Ket qua nghien ciioi
Bdng 1: Kit qua phin tich thinh phin h6a thijc v$t trong cao long cd nhg noi
Hvp chit Alealoid Coumarin DiPdng khd
Terpenoid Acid hO-u CO
Cao cdn + + + + +
Cao nude +
+ +
Cd hai logi cao Idng diu ed chda alealoid, du-dng khd vd terpenoid. Tuy nhidn luvng terpenoid vd alealoid d cao cdn nhieu han so vdi cao nirdc. Ngodi ra trong cao cdn cdn chda acid hdu ca va coumarin.
trgng lu^g gan chu^t nhit L6 thi nghiem
LO 1 (ctiimg sinh hoc) LO 2 (MS hlnh) L6 3 Silymarin 70mg/I(g
Lfl 4 Cao niroc 4g/1(g LO 5 Cao c6n Aglitg
Tf^ng lu^g gan tuong d6t (H.J (X± SE) 48 ± 5
63 ± 8 59 ± 7 61 ± 7 59 ± 7
psov6>l2
<0.05
>0,05
>0,05
>0,05 Trgng luvng tirang ddi gan chuOt 6 Id 2
tdng rd rOt so vdi Id chdng sinh hgc (p<0,05).
- Cy cdc Id udng thudc (cd thudc thd vd thudc chdng dirang) bvng lirgng gan deu tdng cao so
vdi Id chdng sinh hge (p<0,01). Tuy nhidn cao nirdc vd cao cdn deu ed xu hirdng Idm hgn chi tdng tivng lirgng gan chudt so vdi Id 2 nhirng chira cd sy khde bidt (p>0,05)
Bdng 3: Anh hwdng cao Idng cd nhg ndi len ho^t dd AST tmng huyit thanh chu0t nhit L6 thi nghidm
Lfl 1 (chu-ng sinh hoc) Lfl 2 (M6 hlnh) Lfl 3 Silymarin lieu 70mg/kg
Lfl 4 Cao hLrcrc 4gykg Lfl 5 Cao cdn 4g/kg
Hoat Of AST (Ul/I) pC±SE) 118.74 ±37.16 391.55 ±84,92 179.82 ±71.77 302.97 ± 69.45 225,96 ± 63.89
PSOV&I2
<0.001
<0.001
-
>0.05
<0.06
Hogt dO AST d Id 2 (md hinh) gdy dgc bing paracetamol tdng cao ro rOt so vdi Id chdng sinh hgc (p < 0,001).
- Hogt dO AST d Id chuOt udng cao cdn liiu 4 g/kg vd silymarin lieu 70mg/kg trong 8 ngdy tnrde khi gdy dgc gan da gidm rd rOt so vdi Id 2
(p <0,001- 0,05) nhirng vin tdng han Id 1 chdng sinh hgc. Cao n u ^ liiu 4g/kg chf cd xu h u ^ g lam hgn chi tdn ttiirong gan thdng qua Idm hgn che tdng hogt dO AST nhung chira cd sy khde biOt(p>0.05).
TJiP CHi DirOC H p c - 03/2012 (S6 431 N A M 52)
' Nghien ciru - Ky thuat
BAng 4: Anh hwdmg cOa cac Ld thi nghidm LO 1 (chi>ng sinh hoc)
LO 2 (Mfl hlnh) LA 3 Silymarin 70mgykg
LO 4 Cao nude 4g/kg Lfl 5 Cao cdn 4g/kg
i I6ng cd nhQ ndi l§n ho^t &QALT tmng huyet thanh Hoirt dfALT (UW) (JTi SE)
101.80 ±27.81 522.51 ±131.54 397,56 ±113,59 219,91 ±49,98 172,02 ± 37,81
ChudtnhSt p so v6i 2
<0,001
<0,05
<0,01
<0,01 - Hogt dd ALT d id 2 md hinh gdy dgc bang
paracetamol tdng cao rd rOt so vdi Id chdng sinh hgc ( p < 0,001).
- Hogt dd ALT d cdc Id chugt udng cao nirCfc vd cao cdn l i i u 4g/kg vd silymarin l i i u 70mg/kg trong 8 ngdy t r u ^ ^ khi gdy dOc gan da gidm rd rOt so vdi Id
2 (p<0,01) nhung van tang han Id chdng sinh hge.
- Hogt dO AST d ede Id udng cao nirdc vd cao cdn l i i u 4g/kg deu t h i p han so vdi Id udng silymazJn. Cao cdn cd tdc dyng hgn che s y tdng hogt dO ALT tdt han cao nu-dc vd tdt han silymazin d l i i u 70mg/kg (p<0,05).
Bdng 5: Anh hifdng cQa cao Idng cd nhg noi lin him Itrgng MDA gan chu^t nhit LAthlnghl$m
LO 1 (chi>ng sinh hpc) LO 2 (MO hlnh) Lfl 3 Silymarin 70mg/kg
Lfl 4 cao nin>c 4g/kg Lfl 5 cao c6n 4g/kg
Him linimg MDA (nmoVgam gan) (X±SE) 0, 963 ± 0, 239
1,248 ±0,240 1,007 ±0,210 1,419 ±0,131 1,266 ±0,292
psovifl2
<0,05
<0,05
>0,05
>0.05 Hdm lirgng MDA d Id 2 md hinh gdy dgc tiong 8 ngdy tnj-dc khi gdy ddc da gidm so vdi Id 2 b i n g paracetamol tdng cao rd rOt so vdi Id (p<0.05). Hdm luvng IVIDA d cac Id chugt udng 1 chdng sinh hgc. cao cdn vd cao nirdc khdng Idm gidm d u v c hdm
Hdm li/gng MDA d cdc Id chudt udng silymarin luvng MDA djch ddng t h i gan (p>0,05).
B d n g 6: Anh hu^g cda cao Idng c6 nhg nil lin s ( / Uiay doi md bgnh hgc gan
L6 nghiin cihi Dflthi VI thi
Chdng sinh hgc
Gan mdu dd, m$t nhin, m$t dd mim, khfing phi^ n i , khdng sung huyit
T i t cd cdc m i u b$nh phim c6 c i u tnjc gan binh thu'dng, t l bdo gan khdng hogi td, kh3ng thodr hda, khdng cd x3m nh|lp vi6m vA khdng tdng liSn kit xcr.
Gan mdu dd thim, phCi n i , sung huyit, b i mdt khdng nhin, c6 did bj hogi t d vS bgc mdu. cd nhiiu chim xuit huyit
Cdc m l u b$nh phlm c6 nhiiu d vigm. 2/3 m i u b^nh phlm cd ttiodi hda, mi>c dd v i ^ tr nhu md gan.
Silymarin 70mg/kg
Gan mdu dd, sung huyit nh?, c6 mdt vdi diim tdn thu-cmg, mdt vdi diim bgc mdu.
2/3 m i u b^nh phim c6 tho^i hda te bdo gan mdc dd nh^ vd vda, tdng chi sd nhdn chia. 1/3 m i u b$nh phlm gan trong gidi hgn binh thirdng.
Gan mdu dd, sung huyet tdn Cao nirdc thirang mdc dd nhg vd vda, co 4g/k9 mdt sd diim t>gc mdu. B i m|it
nh§n.
2/3 cdc m l u b^nh phlm cd thodi hod vda vd nang t i t)ao gan. Trong dd 1/3 m i u b§nh phlm cd xdm nhdp vidm. 1/3 m i u bdnh phlm trong gidi hgn binh thu^mg.
Cao cdn 4g/kg
Gan mdu dd, sung huyet, b i mdt nhin, tdn thuong mdc dd nh^ vd vda.
2/3 cac mau b^nh phlm trong gidi hgn binh thirdng. 1/3 m§u b$nh phlm cd x3m nhdp vidm vd thodi hda nhg.
Bdn tu$n
D^ gdy tdn thirang gan trdn thyc nghiOm, ngu'di la dCing n h i i u c h i t hda hgc khde nhau nhu' paracetamd, carbon tettaehk}rid, D-galadosamin,
ethand erythromycin estolat, aflatoxin B, ttikacetamW... ^^•^. Mdi mdt md hinh gdy tdn thirang gan d i u cd ca c h i rieng dgc hiOu ^•^. Tuy nhien trong nghidn cdu ndy n h I m ddnh gid tdc
TAP CHi DlTQC HQC - 03/2012 (S6 431 NAM 521
' Nghien CIFU - Ky thuat
dyng chdng oxy hda nen dhung toi chgn tdc nhdn gdy tdn ttmong gan do sinh ra gdc ty do peroxy hda mdng t i bdo g ^ . Paracetanxil gdy tdn tiiuvng gan vdi ca die sinh ra gdc ty do nhu' CCI4, d lieu cao paracetamd con Idm can kigt hg thdng chong oxy hda cua ca thi OiO thing cac chit thiol) ^•^.
HOu qua din den tdn thuiTng cac ^ tiao gan. lam tang AST, ALT, MDA vd bien ddi eau tn:Je gan.
Kit qud nghien cdu cho thay paracetamol lam tang hogt dg AST, ALT huydt thanh vd ham lu-gng MDA rd rOt so vdi Id chdng.
Cao Idng 1:2 chiet cdn vd chiet nirdc cOa ed nhg ndi vdi lieu 4g/kg udng lien tyc 8 ngdy dd Idm hgn che sy tang AST, ALT vd hgn chi tdn ttiu-ang ciu tnlic vi t h i gan do paracetamol gay ra. Cao cdn ed xu hirdng Idm hgn che tdn thirang gan han cao nirdc nhung khdng cd sy khde biOt (p>0,05).
Cao cdn vd cao nu'dc khdng cd tdc dyng lam hgn chi tdng hdm lirgng MDA. Sy chuyin hda vd chia cit cac Iidn kit trong endoperoxyd cua cdc gdc ty do din din sy hlnh thdnh hogc Id malonyl diakJehyd (MDA) hodc pentan (Id nhdng sdn phlm eudi ciing cua qud trinh peroxy hda lipid). Trdn md hlnh gdy tdn thuwng gan bdng paraeetamol ed sy tham gia cOa hO thdng chdng oxy hda thid cua ca thi. Khi glutathion thliu thi d i i t tmng gian chuyen hda NAPQI cua paracetamol mdi tdn tgi de gdy ra qud trinh oxy hda, niu luvng glutattinn du d i Iidn hgp vdi NAPQI thi se khdng cd qua trinh tdn thu-ang.
Tdc dyng bdo vO gan cua cao cdn cd nhg ndi tdt han cao nusS-c diiu ndy ed t h i ly gidi bdi luvng terpenokl dirge biet din cd tdc dyng bdo vO gan d cao cdn nhiiu han cao niflW^.
Cdu hdi dirge dgt ra: tgi sao cd nhg ndi ed tdc dyng bdo vO gan nhung tgi sao Igi khdng ed tac dgng chdng oxy hda, khdng lam gidm MDA?
Sy hlnh thdnh MDA cdn chju nhieu anh huwig cua nhieu yiu td khac nhau. Mgt sd cac akiehyd khde Midng phdi MDA cOng cd thi tgo phdn dng vdi add thnbarbituric vd duvc hip thy d tnrde sdng 532nm. Do vOy danh gid ham lirgng MDA d n phdi kit hgp vdi ffjnh luvng cac enzym chdng oxy hda vd cdc chl sd khde nhu- TAS mdi cd tfii ddnh gid mdc dd ton tgi cua cdc gdc ty do trong ca thi vd mi>c dO peroxy hda lipid mdng ^ bdo
^•^. Nin d i khIng (f|nh 1^ c6 nhg ndi cd anh hu-dng qud trinh peroxkj hda mang tk bdo gan Chung tdi d n tiip tyc nghidn cdu sau han.
Ket luan
- Trdn md hinh gdy tdn thuwng gan bdng paracetamol, eao long (cdn vd nu-dc) c6 nhg ndi lieu 4g/kg cd tdc dgng bdo vO gan thdng qua Idm hgn che tdng trgng lirgng gan tutmg ddi vd hogt dO ALT, AST; hgn chi duvc mgt phan tdn thirang tren giai phlu vi thd gan.
- Cao Idng (cdn vd nirdc) co nhg ndi khdng cd tdc dgng chdng oxy hod do ktidng cd tdc dyng Idm giam ndng dd cua MDA djch ddng thi gan.
S u m m a r y
The aqueous and ethanol extracts from the leaves and whole plant of Edipta prostrata L (Asteraceae) were evaluated for antioxidant acdvities by liver-protecUve effects on mouse liver poisoned by paracetanK^. At daily doses of 4g/kg per oral route in a course of 8 consecutive days, the extracts exerted significant elects by decreasing t>oth serum AST and ALT concentration and paracetamol-indut^
histopathologycal injuries of the liver. However, these extracts showed no prowled andoxklant effects - without decreasing MDA concentration from the mouse liver homogenates.
Tai lif u tham khdo 1. Phgm Thj Vdn Anh, Nguyin Trgng Thdng, Phgm Thign Nggc vd c^ig sir; Nghidn cdu tdc dyng chdng oxy hda cua cao qua nhdu trdn hai md hlnh gdy tdn thircmg gan chupt nhit trdng bing carbon tetrachlorid (CCU) vd paracetamol; Tap chl Dtigc hgc (2007), sd 4, tr. 22-25.
2. Bd Y T i vd BO Gido Dgc & Ddo Tgo (1998).
Bai giing Duf?c /#eu,Tnrdng Dgi iigc Dirge Hd Ndi, BO mdn Duvc liOu.
3. Harbome J.B.; Phytochemica) Method: A Guide to Modem Technkjues of Plant Analysis, Chapman and Hall; London; UK.(1984)
4. Guwrksel senner. Hale Z. Tklu and al;
Protective effects of resveratral against acetaminophen induced toxKity in mice; Hepatology Research vd 35. 62-68.(2006}
5. Sunwka I., Matsi^a T. and Yamada K.
Acetaminophen - induced hepatotoxka^ Still an important insue; Ycnago Ada medicar. (2004); 47.17-28.
6. Sunita Dalai, Sudhir K Kataria. KV Sastry and SVS Rana; Phytochemk:^ Screening of Methanol'ic Extrad and Antibacterial Adivity of Active Principles of Hepatoprotective Hert, Ecflpta altia;
Ethnobotanical Leaflets 14: (2010) 248-258.
7. Vo Van CN; Td diin cdy thudc ViOt Nam; Nhi xuit bin Yhgc, (1997) trang 279-280.
16 T ^ CHI DlTQC HQC - 03/2012 (S6 431 N A M 52)