• Nghien
CIPU- Ky thuat
Opc tinh ban truong diin
Thulc thti- Vinatan khdng gay ddc tinh ban trudng diln tren chugt cing ulng lilu 0,36 g/kg/
ngay (lilu cd tac dung tuang duang vdi lilu dy kiln tren ngudi) va lilu eao gip 3 lin (1,08 g/kg/
ngay) lien tue trong 4 tuin.
- Tit ca cae ehl s l theo ddi v l tinh trang ehung, can nang, ehire nang tao mau, ehire nang gan, mire dp htiy hoai t l bao gan, chirc nang than, md benh hgc gan, than d i u n i m trong gidi han binh thudng, khdng cd sy khac biet se vdi Id chtrng.
Tai lieu tham khao
1. Gerhard Vogel H. (2008), Dmg discovery and evaluation Phamiacological assays. Springer
{Ngay nhan bai: 24/10/2016 - Ngay phan
2. Van den Heuvel M. J., Clark D. G., Fielder R. J., Koundakjian R R, Oliver G. JA., Relling D., Tomlinson N.
J. and Walker A. R (1990), "The international validation of a fixed-dose procedure as an alternative to the classical LDjj test. Fd", Chem. Toxicol., 28, 469-482
3. OECD (2001), Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experimental Animals Used in Safety Evaluation, acute oral toxicity Environmental Health and Safety Monograph Series on Testing and Assesment No 19.
4. World Health Organization (2000), Wori<ing group on the safety and efficacy of herbal medicine. Report of Regional Office for the Western Racific of the World Health Organization.
bien: 29/11/2016 - Ngay duyet dang: 02/12/2016)
Khao sat tac dong cua NL197 tren mo hinh gay cam img co giat bang acid N-methyl-D-aspartic
tren chuot nhat V
Nguyen T h | Kim Thoa'', Vo Phiing Nguyen^
Vu Thi Thuy Hang^, Tru-ong Thi Thu Trang^
'Khoa Duo'c - Dgi hoc Cong nghe TP. Ho Chi Minh 'Khoa Duac - Dgi hoc Y Duac TP. Ho Chi Minh 'Truang Cgo ddng Y te Tien Giang 'E-mail: nguyenthoapharm@gmail. com
Summary
The 4(3H)-Quinazolinone derivative NL197 was experimented in mice with N-methyl-D-aspartic acid- induced seizures. On the central nervous system, NL197 showed the following positive effects: Tranquilizant, anti-Parkinson, analgesic, anticonvulsant In addition, NL197 exhibited antioxidant effects and reduced the mortal'ity rate to 40%.
Keywords: NL197, NMDA, antioxidant, seizure.
Dat vin
d l
Cf Vigt Nam, mgt din chit ctia quinazolin la NL197, din xuit 3-(2-cloropyridin-3-yl)-2-methyl- 4(3H)-quinazolinon, da duge nghign eiru cho thiy nhu-ng tac ddng tren he thin kinh trung uang ''-^l Sau khi sang Igc cac tae ddng trgn he thin kinh trung ugng, NL197 cho thiy triln vpng ching co giat hu-u hieu. Vi vay, nghien ciru nay tiln hanh khao sat tae ddng ctia NL197 trdn md hinh gay cam irng co giat bing N- methyi-D-aspartic acid tren chudt nhit.
Nguyen lieu va phu-o-ng phap nghien ciru Nguyen lieu
Hoat chat thir nghiem
NL197 dugc phan tan diu trong ta duge (PEG 400 va acid citric), lilu 12,5 mg/kg, 25 mg/kg, 50 mg/kg, dung dudng ulng.
N-l\/lethyl-aspart:ic acid (NMDA) (Sigma- Aldrieh), 75 mg/kg tiem tTnh mach.
Diazepam (Roeh), phan tan diu trong NaCI 0,9%, lilu 5 mg/kg, dung dudng ulng.
TijiP rHT n i r % H o r - ] ipmti rsn 488 NAM ^6) 49
• Nghien
CIPU- Ky thuat
Dgng vat thir nghiem
Chugt n h i t t r i n g chung Swiss albino, khoe manh, gidi tinh dye, 5-6 t u i n t u l i , can nang tif 18-22 g do Vien Pasteur TP H i Chi IVIinh cung c i p . Chugt d u g c nudi on djnh trong hop n h y a 2 ngay trudc khi thu- nghiem vdi chu trinh 12 g i d sang t i l (7-19 gid). Chudt d u g e can ngay t r u d c khi lam thtr nghiem va l i l u d u g e tinh d y a trgn can nang. Thdi gian lam thtr nghiem tif khoang 9-15 gid m l i ngay
Phu-o-ng phap nghien cii-u
Khao sat tac dgng chong co giat cua NL197 tren mo hinh gay co giat bang NMDA 75 mg/kg Chudt duge chia ngiu nhien thanh 7 Id mdi Id 13 con.
Ld tring duge eho ulng dung djeh NaCI 0,9%, Id bgnh dugc eho ulng dung dieh NaCI 0,9%, Id 3 ulng ta duge. Id 5 - 7 dugc eho ulng NL197 d 3 lilu khao sat 30 phut va Id chtrng duang dugc eho ulng DZP 60 phiit trudc khi gay cam irng co giat bing NIVIDA.
Quan sat trong 30 phtit va ghi nhan eae thdng s l khao sat.
Chudt ttf vong trong qui trinh thu nghiem va edn sing sau thtr nghiem dugc gay me, gilt liy nao nguygn ven 3 miu d l nhudm md (bao quan trong formol 10%) va taeh nhanh liy hii hai ma 10 miu d diiu kien 4 °C, lam lanh ngay bing da COjVa bao quan d -80 "C din khi sti- dung de djnh lugng MDA va tl le GSH/GSSG.
Danh gia hoat tinh chong oxy hoa Phuong phap dinh luung malonyl dialdehyd (MDA)
Ra ddng md nao d nhiet do phdng trong 15 phuL thgm 300 pL dung djeh dem muli phosphat (PBS) sau dd nghiin tao djch ding t h i bing may tan sigu am Sonic Vibraeel cdng suit 15 Watt, tiln hanh d nhigt do 0-4 °C bing each dat eppendorf trong nudc da muli. Ly tam djch ding thi vdi t i c do 10000 vdng/phiit trong 3 phtit Liy 200 pL djch sau ly tam, them 500 pL nudc khtr kheang va 100 pL SDS 10 % rli u d 37 °C trong 30 phut. Them 500 pL HCI 0,1 N, lie kT 5 phiit. Ly tam liy 1 mL dich, thgm vao 250 pL TBA 50 mg/kg. Dun each thuy d 95 °C trong 20 phtit rli d l ngugi. Do quang d budc sdng 532 nm.
Phuang phap dinh luung GSH, GSSG va xac dinh ty le GSH/GSSG
Dich ding thi: md nao bao quan d -80 °C sau khi ra ddng d nhiet do phdng trong 15 phtit duge nghiin tao djch ding t h i trong 300 pL acid 5-suifesalieylic 5% bing may tan sieu am Sonic
Vibraeell cdng suit 15 V\fett, tiln hanh d nhigt do 0-4 °C bing each dat eppendorf trong nude da muoi. Ly tam (lOOOOg/phiit trong 3 phut) liy djch ddng t h i .
Djeh djnh lugng GSSG: 100 nL djch dong thi + 2 p.L 2-vinylpiridin + 6 ^iL triethanolamin, khuiy 1 phtit, de ygn 60 phtit.
Bang 1: Thanh phin phan img dinh luung GSH toan phin hay GSSG Dung djch phan Ong
Dung dich dem (NADPH 0,248 mg/mL) DTNB
DW (niroc cat)
Miu thii' Mau tring 140 nL 20 nL 35 nL
140 nL 20 nL 35 ^L Hilt dii 3 thanh ph§n tren vao dia 96 gieng de yen 10 phiit a nhiet do phong
Djch dong the hoac dich Smh lu-gng GSSG Dung dich acid 5 sulfosalicylic 5%
GSSG reductase Tong the tich
5jiL
-
2 111 202 nL
-
5nL 2nL 202 ML
Thay djch ddng the bang day ndng dg dung dich L-glutathion khu va djeh djnh lugng GSSG bing day nong do dung djeh L-glutathion oxi hoa d l xay d y n g d u d n g ehuan.
Do quang d 415 nm trong 5 phtiL L i y k i t qua tmng binh: AOD/1 phiiL
Danh gia: D y a tren d u d n g c h u i n xay dyng d u g c , tinh toan ham lugng GSH toan p h i n , GSSG dang oxy hda. Ojnh lugng prgtein toan p h i n (mg/
mL) trong m i u sau dd tinh toan n i n g do GSH/
lugng protein (pmol/mg), GSSG/lugng protein (pmol/mg), ty le GSH/GSSG.
Tinh ty Ig GSH/GSSG = (tGSH - 2 x GSSG)/
GSSG
(GSH: glutathione toan phan
Binh luung protein toan phin (phuung phap Bradford) Bang 2: Thanh phin hdn hgp phan ung dmh luung
protein toan phin
Thanh phan dng trang dngthir Coomassie Ix 980 pL 980 pL Niroc 20 pL OpL Djch sau ly tam (da pha loang 6 lan) hoac 0 pL 20 pL day nong do protein chuan
Tong cgng ImL ImL
U d nhiet do phdng trong 2 phtiL Oo quang d b u d c sdng 595 nm.
Nhudm md nao
Ngay sau khi mo lay nao, toan bg nao dugc ngam trong formol 10%. M i u d u g c nhudm eresyl violet Vj tri yeu e i u cat n i m trong khoang -1,28 d i n -3,8 tinh tir thdp nao.
50 TAP CHi Dl/OC " —
• Nghien
CLPU- Ky thuat
Quan sat dudi kinh hiln vi quang hgc vdi do phdng dai X100.
Phan tich thong ke ket qua
DO- lieu du-gc trinh bay d dang trung binh (IVIEAN) ± sai s l ehuin etia s l trung binh (S.E.M).
S l lieu dygc thing kg bing phin mim Minitab 16.0. Cac day s l lieu duge so sanh bing phep kilm Kruskal Wallis, nlu cd sy khac nhau se tilp tuc dugc so sanh tirng cap bing phep kiem Mann Whitney. D l thj dygc ve bing phin mim Sigma- AldriehPlot12.0.
Kit qua va ban luan
Khao sat tac dong cua NL197 tren mo hinh gay co giat bing NIVIDA lilu 75 mg/kg
Chudt dugc tigm tTnh mach dung djeh NaCI 0,9% khdng quan sat thiy eae bilu hign bit thudng. Chudt dugc tiem NMDA d lilu khao sat, 100% ehugtdlucd nhirng bieu hign khdi phat can CO giat va ttf vong. Nhu vay, md hinh nay cd t h i dugc ap dung d l khao sat sa bd tac dgng ching CO giat va bao ve ehudt khdi tu- vong ctia cae ehit.
Bang 3: Kit qua bao ve chudt tu vong cua NL197 va DZP tren chudt gay cam ung co giat bing NMDA IV
liiu 75 mg/kg
Kit qua thtf nghiem eho thiy: Tac ddng ehing eo giat etia NL197 d cac lilu khao sat cd tac dgng bao ve chugt gay eo giat bing NMDA khdi tti- vong.
Tuy nhien, eaSlilu NL197 sir dung diu khdng bao ve chudt hoan toan khdi ttr vong ma ehl lam giam tl le ttf vong xuing edn 40%.
Oanh gia hoat tinh chdng oxy hoa
Kit qua 6inh luong hoat tinh MDA va GSH/
GSSG
Bang 4: Kit qua (Tmh luang MDA trong thir nghiem khao sat tac ddng cda NL197 tren md hinh gay co giat
cip bing NMDA 75 mg/kg
Nong So MDA (pmol/mg protein)
STT Lo
1 Lo NaCI 0,9% + NaCI IV, 0,9% (n = 8 3,731 ±0,221 2 L6 NaCI 0.9% + NIVIDA (n = 9) 3,669"* + 0,933 3 Lo PEG, acid citric PO + NMDA (n = 9) 8 , 9 5 0 " ± 0,795 4 Lo DZP 5 mg/kg + NMDA (n = 10) 6,463'« 1:0,653
Lo NL197 12,5 mg/kg + NMDA mg/kg (n = 10)
4,014""*®
± 0,323 6 L6NL19725mg/kg + NMDA(n = 10)
±0,192 7 L6NL197 50mgfl<g + NMDA(n = 4,308"™® ±0,524
STT L6 1 LoNaCI0,9%+NMDA(n = 9) 2 16 PEG, acid citric PO + NMDA (n = 9) 3 Lo DZP 5 mg/kg + NMDA (n = 10) 4 L6NL19712,5mg/kg+ NMDA(n = 10) 5 Lo NL197 25 mg/kg + NMDA (n = 10) 6 L6NL197 50mg/kg + NMDA(n = 10)
Ti le tir vong (%) 100 100 80 70 60 40
*"p<0,001:so vdi 16 1; 'p<0,05,'"p<0,001: so voi 16 2
^p<0,05,^^p<0,001: so vailo 3; @p<0,05, »p<0,01: so vai 16 4
O cac Id diiu tri sti- dung DZP lilu 5 mg/kg, NL197 lilu 12,5; 25; 50 mg/kg lam giam ning do MDA cd y nghTa so vdi Id benh cho ulng nudc cit chtrng td tac dgng ehdng oxy hda cua DZP va NL197 lilu 12,5; 25; 50 mg/kg.
Kit qua dmh luong GSH/GSSG
Bang 5: Kit qua dinh luung GSH/GSSG trong thu nghiem khao sat tac ddng ciia NL197 tren md hinh gay benh cip bang NMDA IV 75 mg/kg
sn
Lo GSH/Proteintoan phan (pmol/mg)
GSSG/Protein
toan phin (pmol/mg) 77 so GSH/GSSG 1 Lo NaCI PO 0,9% + NaCI IV 0,9% (n = 10) 10,965 ± 0412 0,4358 ±0,0541 28,97 ± 3,640 2 LS NaCI PO 0,9% -i- NMDA IV 75 mg/kg (n = 4,696*** + 0,185 0,8072** ±0,094 6,447" ± 0,749 3 Lo ta droc PEG, add citric PO * NMDA IV 75 mg/kg (n = 9) 4,954*** ± 0,329 0,8225** ±0,C ± 0,464 4 Lo DZP PO 5 mg/kg + NMDA IV 75 mg/kg (n = 10) 5,167*** ± 0,204 6**+0,032 8,100*** ± 0,728 5 Lo NL197 P012,5 mg/kg + NMDA IV 75 mg/kg (n = 5,335'" + 0,159 0,5904+0,034 3,308'' • ± 0,616 6 Lo NL197 PO 25 mg/kg ± NMDA IV 75 mg/kg (n = 10) 8,053"
7 Lo NL197 PO 50 mg/kg + NMDA IV 75 mg/kg (n = 10) 7,042"
' ± 0 , 4 1 8 0,5501'*± 0,051 1 5 , 9 r ™ e e « * ± 1,830
±0,291 0,5657* ±0,043 1 3 1 2 " " ' * * * * * ± 1,170
*p<O.05**p<0,OJ,"p<0,(»1:sora'ei,'p<0,05>«,(«,*>«,001:soiae2*p<0,C)5,«p<0,OT^^
ep<0,05Wp<0,01,SMp<Qa«.sora'B4M05>«,01,"p<0,Om:sovolB5;'p<0,Cl5''p<0,OT^^
Qua thti- nghiem da eho thiy dugc tae ddng ehing oxy hda NL197 lilu 12,5; 25 va 50 mg/kg.
Tuy nhign, eae ehl s l v i n edn eao han so vdi mirc binh thudng.
DZP t h i hien tae dgng bao ve tofdc tac ddng
oxy hda eua NMDA; NL197 d 3 lilu thu- nghiem lam giam ning do MDA va lam tang tl le GSH/
GSSG so vdi Id benh ulng nude eit chtrng td tac ddng ching oxy hda eua DZP va NL197.
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TAPf-mnirrjfrH9|^-i7/7nififS!n 488 N A M =;6) 51