• Nghifin CIFU - Ky thu$t
emulsificatlon stining speed, coacen/atton pH value, concentration of formaldehyde solution, ratio of gelatin/ arabk: gum, weight of PVA, and the stability of mkmencapsules on the mbmcapsulatbn yield, encapsul^kin efficiency, particle sia and morphology of microcapsules were Inve^igated.
The suitable condHtons were detennined and rational formulatkin vmre established.
Tdi il^u tham khdo
1. Alavi T. H. et al. (2010), "Optimization of morphology and geometry of encapsulatedHypophthalmichthya molltrix oil", Iranian Journal of
Fisheries Sciences, 9(2), p. 199-206.
2, BO Y td. (2009), Phu Igc 10.10: "Djnh luyng vitamin A, phuang phdp 5: Phuong phdp sdc ky ldng sau khi chidt tdch vitamin A". Duvc <wn Vi$t Nam IV, Nhd xudt bdn Y hpe, Hd NQI, tr. PL ie0-PLig3.
3, James S et al. (2007), "Microencapsulation Technology, Encyclopedia of Phannaceuticat Technolog/", Informa Healthcare, New York. 3(4), p.
2315-2332.
4, Junyaprasert, V. B. et al. (2001), •Effect of Process Variables on the Microencapsulation of Vitamin A Palmltate by Gelattn-Acada Coacervafion", Dmg Devefopment and Industnal Pharmacy, 27(6), p, 561-566,
Tac dong khang cholinesterase ciia ac-ti-s6, tra xanh va hutmg nhu tia lien quan den kha nang chong suy giam tri nhd tren chuot nhat trang
Dat van de
Higu qua chdng suy gidm tri nhd (SGTN) cua cac eao chidt ac-ti-sd {Cynara scolymus.
Asteraeeae), trd xanh {Camellia sinensis, Theaceae) vd huang nhu tia {Ocimum sanctum, Lamiaeeae) da d^rcrc ehung minh thdng qua md hinh gay SGTN do trimethyltin (TMT). TMT Idm giam eholin acetyltransferase (ChAT), enzym cd vai trd sinh tdng h y p acetyleholin (Ach), tren nao chudt nhgt trdng 3 ngdy sau khi tidm TMT '^'.
Nhieu nghien euu cung dd ghi nhdn s y tdn t h u o n g neuron cholinergic tren nao chudt do Tf^T gay ra '^l Nghidn c u u ndy d u y c thyc hidn nhdm danh gid hdm l u y n g acetyleholin, mdt chat trung gian hda hpe ed vai trd rdt quan trpng doi vdi qua trinh hinh thdnh hogt dpng nhdn thuc vd ghi nhd, Ddng thai, hogt tinh men acetylcholinesterase (AchE), men thuy phdn acetyleholin tgi synap, eholin aeetyltransferase (ChAT) cung da d u y c djnh l u y n g , gdp mpt phan vao qua trinh tim hieu ea che tac dung cua cae
Trkn Phi Hodng Yen, Dinmg Phirdc An, Nguyen Ngpc Khfii, TrSn LS Tuylt Chfiu Khoa Du(rc - Dgi hpe YDu^c TP. Ho Chi Minh h y p ehdt da d u y e ehung minh ed vai trd bdo v$
ndo, chdng SGTN tren m d hinh ehudt thyc nghidm.
D6i t u v n g , nguydn v$t li$u vd phLPcng phdp nghien ci>u
E)6l tu>yng
Chu0t nhdt trdng (dye), gidng Swiss alt»no, tn?ng luyng 20-25g (khodng 5-6 tudn tudi), do Vi^n Vaedn vd Sinh phdm Y td Nha Trang cung cdp.
Nguydn v|it li$u
Trimethyltin (TMT) clorid - Sigma Aldrieh (S6 Id 146498), dung djch n u d c mudi Saline (Sal), galanthamin (Gal).
Kit thu n g h i ^ acetylcholine/ acetyl choiinesterase A m p l e x ® R e d ( A 1 2 2 1 7 )
Cao chiet ac-ti-sd (Art), cao chidt trd xanh, (CS), va cao ehiet h u a n g nhu tia (OS) tieu ehudn ca s d do nhdm nghien c d u t h y c hi^n.
Phuonng p h a p n g h i e n c u u
Chupt d u y e phan chia thdnh cde 16 nhu sau:
TAP CHl DUOC HOr-nq/ini? (86 437. 52)
Nghien ciyu - Ky thugt
Bing La
1 : Phan Id chudt thir nghiim
Thudc tShl chd
1 Sal 2
3 4 5 6 7 8 9
Sal + TtwlT 2.4 mg/kg. i.p Gal (lOmg/kg, p.c x 5 ngdy) + TMT 2.4 mg/kg, I.p Art190 (190 mg/kg, p.o x 9 ngdy)* TMT 2.4 mg/kg, I.p Art380 (380 mg/kg, p.o x 9 ng4y)+ TMT 2.4 mg/kg, I.p CS80 (SOmg/kg. p.o x 9 ngdy)* TMT 2.4 mg/kg, I.p CS160 (160 mg/kg. p.o x 9 ngSy)+ TMT 2,4 mg/kg, i p
OS50 (50 mg/kg, p.o X 9 ngdy)+ TMT 2.4 mg/kg. i p OS100 (100 mg/kg. p.o x 9 ngSy)+ TMT 2.4 mg/kg. i.p
TMT tISm Hdu duy nhfit Dl6u trj tmdc 3 ngSy. sau 2 ngdy TMT Ol6u trj trudc 7 ngdy, sau 2 ngdy TMT Didu trj trudc 7 ngdy, sau 2 ngdy TMT Oidu trj tnrdc 7 ngdy, sau 2 ngdy TMT Didu trj trudc 7 ngdy, sau 2 ngdy TMT Didu trj tnfdc 7 ngdy, sau 2 ngdy TMT Oidu trj tn/dc 7 ngdy, sau 2 ngdy TMT Hai ngdy sau khi tidm TMT, tdt ed chupt
d u y c md, tdch Idy vung hdi ma vd djnh luyng Ach vd AChE
Djnh lu>yng Ach vd AchE
Nguydn tde: Aeh se bi AchE chuydn thanh eholin, sau dd nd bi eholin oxydase oxi hda thdnh betain va H202. Dudi s y xue tde cua horseradish peroxydase, H202 phdn ung vdi 10 - aeetyl-3, 7-dihydroxyphenoxazin (Amplex Red Ach/AChE Idt) tao ehat cd mdu hong. Do dd hdp thu d 563nm.
Dmh Iwgng Aeh
Chudn bj ede dung djch Aeh chuan: Hda tan dung djch me Ach lOOmM trong dung djch dem phdn ung 1X de d u y e eae dung djeh ed nong dd: 0; 0 , 1 ; 0.2; 0,5; 1; 2; 5, 10 nmol/IOOpl.
Chuan bj mdu; 30 pi dich ddng the md va 70 pi dung djch dem phan dng I X de d u y c 100 pi m l u .
Hdn hyp phan ung: 400 pM Amplex Red ehua 2 lU/ml HRP; 0,2 lU/ml eholin oxydase va 1 lU/mlAchE.
Bang 2: Thinh phan hon hop phin Cmg djnh luong Ach
Thinh phan Thi tich ddng cho hdn hg^ phan Cmg 100 miu djnh Itrqmg Ach Amplex Red reagent 200^1
Dung dich m^HRP 100 pi 100 |J|
Dung dich me
acetylcholinesterase 100 ^Jl Dung dich ddm
phdn ung 1X
Tien hanh phan ung: eho 100 pi dung djch idm vigc vao moi gieng ed ehda san 100 pi mdu.
u eho phdn dng xdy ra hodn todn d nhidt dp phdng trong 60 phut vd trdnh dnh sdng.
Do m l u d b u d c sdng 563 nm.
Ghi nhdn kdt qud: (nmol) Aeh / (mg) pnatein.
Dinh gii ho^t tfnh AchE
Chudn bj eae dung djeh chudn AchE: hda tan dung djch mg AchE 100 lU/ml trong dung djeh d0m phdn ung I X dd thu d u y e edc dung djeh AchE d ede ndng dp: 0; 1; 2; 3; 4; 5; 10; 20 (mlU/IOOpI).
Chuan bj m l u : ldy 5 pi (djeh ddng the md da d u y e pha loang 10 Idn trong dung dieh PBS) them 95 pi dung dich dgm phdn ung I X de d u y e 100 pi mdu.
Hon h y p phdn ung 400 pM Amplex Red reagent chua 2 lU/ml HRP, 0,2 lU/ml eholin oxydase va 100 pM Aeh.
Bang 3: Thanh phin hdn hep phin img dinh lu^ng AchE
Thinh phan h6n hijrp phin img Amplex Red reagent
Dung djch me HRP Cholin oxydase Dung djch m§ AchE
Dung djch dem phdn ung IX
Thi tich ddng cho 100 miu dinh /upng AchE
200^1 100 Ml 100^1 100 ^JI 9,5 ml Tien hanh phan ung: Lay 100 pi hon hap phdn ung eho them vdo mdi gieng ed ehua 100 pi mau djnh l u y n g . 0 eho phan ung xay ra trong 60 phut d nhiet dp phdng.
Do mau d b u d c sdng 563 nm.
Ghi nhdn ket qua: (mlU) AchE hoat dpng/(mg) protein.
TAP CHI Dira
Nghidn CIFU - Ky thujit
Dinh gii ho^t tfnh ChAT^^
Hdn hyp phdn Cpng gdm 40 pL djch ddng thd vd 10 pL: 0,5 M dung djch ddm natri phosphat (pH 7,2). acetyl-CoA (6,2x10'^ M), eholin clorid (1 M), neostigmin sulfat (7,6x10"^ M). NaCI (3 M) and EDTA (1,1 x 10"^ M), thdm nude edt vi>a dii 200 pL. 0 d 37°C trong 5 phdt. Enzym d u y c thdm vdo vd tidp tge u 37°C trong 20 phiit, Oun sdi d bdp cdch thOy 2 phOt dd dCmg phdn dng vd thdm 0,4 mL n u d c (dfl logi oxy). Ly tdm dd logi protein dd kdt tua. Thdm 6 pL 4,4'-dithlopyridln (10"^ M) vdo 200 pL dieh ddng thd md. Do dd hdp thu d 324 nm sau 15 phut. Mdt dan vj hogt tinh enzym d u y c djnh nghTa bdng 1 pmol eoenzym sinh ra trong 1 phut/ mitigam protein.
Phuvmg phip xv ly thdng ki Cde kdt qud eua nghidn c u u d u y e bidu didn bdng MEAN±S,E vd d u y e x u ly bdng phuang phdp phdn tieh phuang sai 1 ydu td vdi Fischer's PLSD test, S y khde bidt d u y e xem Id cd y nghTa thdng kd khi p<0,05.
Ket qua vd bdn l u | n
Tdc d^ng cua cdc cao chidt ddn sy thay ddi ham luyng acetyleholin trong viing hdi ma
S \ T Sit'
Hinh 1 : Si/ thay ddi him li/Qmg Aeh (nmol/mg pmtein) giua nhdm chimg vi cic nhdm tiim TMT cd ho$e khong cd dliu tii vdi Art190, An380.
CS80. CS380. OS50. vi OS100. ^p<0.001 so vdr nhdm Sal; ''p<0,05. '^p<0,01. '"*'p<0,001 so vdn nhom TMT, ''p<0.05, '^p<0.01, °^p<0,001 so vdri nhom Gal. (SO- dung phuxmg phip phSn tich phuxmg sai 1 yeu td vdi Fischer's PLSD test).
Ham l u y n g acetyleholin trong vung hdi ma, vung nao lien quan den vigc nhan thuc va nhd cua chupt nhat, giam sau khi tiem trimethyltin t u 6 gid, 12 gid, 1 ngay, giam thap nhdt sau 2 ngay va cd s u hoi phue sau 3 ngay tiem TMT
12
(khdng trinh bdy kdt quA). Do dd, t h d i didm sau 2 ngdy d y y c dp dgng tfd khdo sdt dnh hudng cua cdc cAc cao chidt ddn s y thay ddi hdm l u y n g acetyleholin d vCing hdl md nfio ehudt nhdt trdng do TMT gdy ra ( n h u bdng 1).
Tic d ^ n g cOa c4c cao chiAt dAn s y thay d 6 l h o f t t i n h e n z y m A C h E t r o n g v i i n g hdi mS
0
bU
X r
bbb n bbb,il
^ «* f ] fll
z Sillnt M (M ArtlN ArtltS CSV CW 06N O W
Hinh 2: Si/ thay ddi ho^t ttnh AchE (mU/mg protein) gii/a nhdm chifng vi cie nhdm tiim TMT cd hoic khdng cd diiu trj vdi ArtlQO, ArtSeO, CS80. CS380, OS50. vi OS100.
"'p<0,001 so vdi nhdm Sal: ''p<0.05. '^p<0.001 so vdi nhdm TMT, ''p<0,05 so vdi nhdm M190 (SCr dung phwong phip phin Hch phuemg sa 1 ydu td vdi Fischer's PLSD test).
Tdc d$ng cua cdc cao chidt ddn sy thay ddi hogt tinh enzym ChAT trong viing hdi ma
2.400
X
i J c. J L H
I J L X
o SM^ Sd <M ArtIM ArtlH CS30 C3N O850 0S1H TMT2,4msniB
Hinh 3: Si/ thay ddi ho^t tinh ChAT (mU/mg protein) glu^ nhdm chimg vi cic nhdm tiim
TMT cd hoic khdng cd diiu trj vdi Art190.
Art38Q, CS80, CS380, OS50, vi OS100-
"^°p<0,001 so vdi nhdm Sal (SO dijng phuong phip phin tich phwcmg sai 1 ydu td v&
Fischer's PLSD test).
Hdm l u y n g ACh gidm, ddng thdi hogt tinh eua AChE tang d thdi diem 2 ngdy sau khi tidm TMT lieu duy nhdt 2,4 mg/kg. Kdt qud ndy phO
TAP CHf DirOC HOC-09/2012 (S6 437 NAM 52)
' Nghi§n ci>u - Ky thugt
hyp vdt nh&ng nghidn cuu dd duyc cdng bd vd tdc ddng cOa TMT Idn hd cholinegic ndo chudt.
Cao ehidt ae-ti-sd thd hidn khd ndng uc chd sy gia tdng hogt tinh cOa cholinesterase d cd 2 lidu thu nghidm (190 mg/kg x14 ngdy vd 380 mg/kg x14 ngdy), vd cd sy gia tdng tuydn tinh giO>a lidu vd hidu qud khdng cholinesterase. Hdm luyng Ach gia tdng 6 nhdm didu tri vdi cao chidt ac-ti- sO, tuy nhidn, hogt tfnh cga ChAT khdng thay ddi dd chi>ng td sy gia tdng Aeh Id do hi$u qud i>c ehd hogt tinh cholinestergse eua eao chidt ae-ti-sd. Cao chidt trd xanh cQng thd hidn kdt qud tuyng ty cao ehidt ac-ti-sd, tuy nhidn, chua cd sy khdc bidt vd khd ndng khdng cholinesterase giua 2 lidu eao b^ xanh su dgng trong nghidn euu ndy (30 vd 60 mg/kg x 14 ngdy). Vdi cao ehidt huang nhu tia, hidu qud khdng cholinesterase rdt thdp, chT phdt hidn d lidu cao (100 mg/kg x 14 ngdy).
Hogt tinh cCia ChAT gidm cd ^ nghTa d nhdm tidm TMT, tuy nhidn, chua phdt hidn khd ndng hdi phgc vide tdn thuang Idn sy hinh thdnh ACh do TMT gdy ra d ede nhdm cd didu trj vdi galanthamin vd cde cao chidt ae-ti-sd, trd xanh vd huang nhu tfa. Didu ndy chung td galantamin vd 3 cao ehidt thu nghidm ddu khdng cd khd ndng Idm gia tdng sinh tdng hyp ACh, md chl dnh hudng ddn hogt tfnh cua AChE, tu dd Idm gidm bdt sy phdn huy ACh trong vCing hdi ma nao chudt nhdt trdng.
Ket lu$n
Kdt qud vd hogt tinh khdng cholinesterase da gdp phdn Idm sdng td vai trd bdo vd nao, chdng igi sy suy gidm trf nhd do TMT gdy ra trong dieu kidn thyc nghidm. Ddc tfnh cua TMT ddi vdi cde neuron cholinergic Id mdt md hinh rdt cd gid trj eho cdc thu nghidm tim kiem cdc hyp chdt khdng cholinesterase, hudng ddn chdng suy gidm tri nhd, didu tri cho cdc tidnh lidn quan den
suy gidm tri nhd nhu bdnh alzheimer, Parkinson, hungtinton...
Nghidn cuu ndy dd eht>ng minh cdc eao ehidt ac-ti-sd, trd xanh vd huang nhu tfa cd khd ndng dc ehd hogt tinh cua cholinesterase trong didu kidn cd sy gia tdng hogt tinh enzym ndy trdn ndo chudt nhdt, Id ca sd cho edc nghidn cuu sdu han nhdm tim kidm cdc hyp chdt cd ngudn gde ty nhidn su dgng trong didu trj cdc bdnh SGTN.
Summary
The extracts of Cynara scolymus. Camellia sinensis and Ocimum sanctum showed to exert anticholinesterase effects in experimental mice with tnmethyltin-induced increase in the brain enzymic cholinesterase activity. Our findings may lay bases for further researches to screen natural compounds of therapeutic efficacy for memory impairment
Key words: Cynara scolymus, Camellia
sinensis, Ocimum sanctum, trimethyttin-induced memory impairment, anticholinesterase effect.
Jh\ li^u tham khao 1. Kim M. J., Choi S. J., Urn S. T., Kim H. K. et al (2007), "Ferulic acid supplementation prevents trimethyltin-induced cognitive deficits in mice", Bioscl BiotechnolBbch., Apr-71(4), p. 1063-1068.
2. Roy A., Agrawal A. K., Husaln R., Dubey M. P., Seth P. K. (1999), "Chdlnergic and serotonergic alterations in the rat hippocampus following trimethyltin exposure and fetal neural transplantation", Neurosci Lett., Jan-15, 259(3), p. 173-176.
3. Chao L. P., Wolfgram F (1972),
"Spectrophotometnc assay for choline aeetyltransferase", AnaiBtochem, 46, p. 114-118.
4. Aas P., Pagenhart A , Eriksen S., Kolderup J., Fonnum F, (1996), "The effect of trimethyltin on acetylcholine release in the guinea-pig trachea".
Environ Toxicol Pharmacol, Jul-15, 1(4), P. 257-268
P h a n l a p . . . CT/ep f/ieo trang 70;
cdu true hda hpe cua ede ehdt phdn Igp duyc xdc djnh dya vdo ede phuang phdp phd edng hudng td hgt nhdn (*H, "C-NMR vd cdc phd
DEPT 90, DEPT13S) vd phd khdi {ESl-MS).Summary
From the biomasses of Taxus wallichiana
Zucc, three derivatives of taxuyunnanine C were isolated by column chromatography and their structure were elucidated by ^H-NMR, "C- NMR. DEPT and ESl-MS with reference to the literature data.
(Xem tiep trang 67)
TAP CHi mrntf- unt- _ nonni •> ^giS Ati N ^ M S2)