Nghien CLFU - Ky thuat
4. Silhankova L, Smid F, Cerna M, Davidek J &
Velisek J. Mutagenicity of glycerol chlorohydrines and of their esters with higher fatty acids present in protein hydrolysate, Mutat. Res.,(1982) 103, 77-81.
5 Velisek, J,D,, Davidek, J., Kubelka, V,, Janicek, G , Svobodova, Z. & Simicova, Z,, New chlorine containing organic compounds in protein hydrolysates, J. Agric. Food C^em.,(1980) 28, 1142- 1144.
6 Lynch BS, Bryant DB, Hook GJ, Nestmann ER
& Munro IC, Carcinogenicity of monochloro-1,2- propanediol (alpha-chlorohydrin, 3-MCPD), Int. J.
Toxicol. (1998) , 17,47-76.
7. Sunahara G, Perrin I & Marchesini M.
Carcinogenicity study on 3-monochloropropane-1,2- diol (3-MCPD) administered in drinking water to Fischer 344 rats. Unpublished report No, RE- SR93003 submitted to WHO by Nestec Ltd, Research
& Development, (1993) Switzerland,
8. Zeiger E, Anderson B, Haworlh S, Lawlor T &
Mortelmans K. Salmonella mutagenicity tests; IV.
Results from the testing of 300 chemicals. Environ.
Moi. Mutag . (1988) 11 (SuppI 12), 1-158,
9 Cho W-S, Han BS, Lee H, Kim C, Nam KT, Park KD, Choi M, Kim SJ, Kim SH, Jeong J, Jang DD.
Subchronic toxicity study of 3-monochloropropane- 1,2-diol administered by drinking water to B6C3F1 mice. Food & Chem. Toxicol. (2008) 46: 1666-1673
10. Marchesini M & Stalder R, Toxicity of 3- chloro-1,2-propanedio1 in a 4 weeks gavage study on rats. Part I. Unpublished report No. LA 70/1082 from the Societe d'Assistance Technique Pour Produits Nestle SA, Switzerland (1983)
11, Marchesini M, Stalder R & Perrin I. Subchronic toxicity of 3-chloro-1,2-propanediol, 90 days administration in drinking water of Fischer F344 rats.
Unpublished report No. 1264 from Nestec Ltd Research Centre, Nestle, Switzerland (1989)
Nh&ng nghien cii'u biro'c dau ve du'O'c tinh va thanh phan cua noc ran doc Hon So'n
{Cryptelytrops hosonesis Grismer)
Hoang Ngoc Anh', Pham Nguyen Dong Yen', Liru Cong Thinh^ Vo Phung Nguyen^ Nikitin Ilya^ Utkin Yuri^
' Vien Khoa hgc Veil lieu img dung, Vien KHdcCNVN.
-Dgi ligc Y Dugc TP. HCM 'Shenivaliin-Ohchinuiliov Institute of Dioorganic Chemistry,
Russian Academy oJ Sciences
Dat van de
Npe rdn chira nhilu thanh p h i n cd hoat tinh sinh hoc, nhirng chdt nay la vu khi d l lam te liet hodc tieu diet con mdi. V l ca ban rdn dde ehia Idm hai loai tuy theo tae ddng eiia npe dde len he t h i n kinh hay len mach mau. Tat ca cae loai noe rIn luc (Viperidae) d i u ed tac ddng len mach mdu vd gdy rdi loan qua trinh ddng e l m mau '", edn noe rdn ho (Elapidae) thi gdy dde v l t h i n kinh '^' Ngay nay ngudi ta dd nghien ciru cdc thdnh phan ciia ngc rdn d l Idm edng eu nghien ciPu thude ngdn chan benh ddt quy vd d i l u tri ede benh tim mach '^' Mdt sd edc toxin ciia noe rdn lue duge sir dung d l phd eac mach
mau nudi t l bao ung thu vd d i n d i n viee ire e h i su phat trien eiia t l bao ung thu Vi vdy hien nay, viee nghien ciru cae toxin mdi trong npe rdn d l irng dung trong y duac dang phat triln,
Rdn luc Hdn San [Cryptelytrops honsonensis Grismer) Id loai rdn mdi dugc xdc dmh ten tai dao Hdn San tinh Kien Giang '"' Ngudi ddn tren dao hay bi rdn c i n gay sung vii, dau nhirc, nang thi phai thao khdp ngdn tay n l u khdng se bj hoai tip ddn d i n tir vong. Ddy la loai rdn ddc mdi phat hien nen viec nghien ciPU ddc tinh vd thanh phin ciia nd ehua duge t i l n hdnh, Trong bai bao nay chimg tdi t i l n hanh nghien cu'u dde tinh cap len ddng vdt va cdn triing, tdc ddng
TAP C l i i DUQC H Q C - 8 / 2 0 1 1 (SO 424 NAM 51) 37
Nghien CLFU - Ky thuat >4
chdng eo giat gdy ra bdi strychnin sulfat va thanh phin eiia npe rdn luc Hdn San,
N g u y e n l i e u v a p h u ' O ' n g p h a p n g h i e n CLFU
Noc rdn
Rdn luc Cryptelytrops honsonensis Grismer duac bit tai dao Hdn San tinh Kien Giang. Lan ddu tien rdn duac thir nghiem nudi vd ldy ngc, thirc dn ciia rdn ehii y i u la mdi, Ngc dugc tilt ra bdng cdch xoa bdp rIn bdng tay, sau dd dem ddng khd vd giir d nhiet dd -20°C cho d i n khi diing.
Dong vat thiF nghiem
Chudt nhlt trdng due khde manh gidng Swiss albino, duge eung e l p bdi Vien Pasteur thanh phd Hd Chi Minh. Chudt mua v l duge nudi dn dinh it nhlt trong 2 ngdy trong phdng thi nghiem Vien Khoa hpc Vat lieu irng dung trudc khi tiln hanh nghien ciru, Chudt cd the trgng 20
± 02 g/eon duge dung lam thi nghiem,
D l Achetus sp, dugc mua d trai d l Cii Chi, TpHCM. D l mua ve dugc chgn nhirng con khoe manh va d l dn djnh 1 ngdy trong phdng thi nghiem. D l ed t h i trpng 0,3 - 0,9 g duge diing lam thi nghiem,
Nghien cii'u doc tinh d p vo'i d o n g vat du-dng tiem tinh mach tren chudt '^'
Nghien ciru sa khai: Dua tren nhirng khao sat ban d i u da dugc thi nghiem trudc day, khdi dau thi nghiem tip lilu gdy tir vong chugt hodn todn la 5mg/kg, Xdc dinh lilu LDQ la lilu tdi da khdng gay c h i t chudt ndo vd lieu LDioo Id lilu thdp nhlt gay c h i t 100% chudt.
Nghien ciru xae dinh: Chudt trdng dat tieu chuin nghien ciru duge ehia ngdu nhien lam 4 Id, moi Id it nhlt 5 con, cho sip dung thude d cac lilu trong khoang LDQ vd LD100, chia theo cdp sd. 6"
nhu'ng lieu g i n LD50 tdng sd lugng chudt len de su do ludng duge chinh xdc han, Quan sdt chudt trong 24 gid sau khi diing thuoc, ghi nhdn cdc phan ung xay ra tren chudt vd sd lugng chudt sdng, chit d mdi Id. Theo ddi chudt cdn sdng sau 14 ngdy k l tip khi dung thude. LD50 dugc tinh theo phuang phdp Karber-Behrens.
Phwang phap Karber-Behrens
LD50 dugc tinh theo phuang phap Karber- Behrens vdi cdng thipc sau:
Trong dd:
D,: lilu toi thieu Idm chet tdt ca ddng vdt a: chi so trung binh ciia sd ddng vdt chet d hai lilu k l tiep. , ';
b: hieu sd giira hai l i l u k l t i l p .
n: sd ddng vdt diing d mdi lieu hodc sd trung binh ciia nhirng trj sd tren
Nghien ciru doc t i n h d p v 6 i con trung du'd'ng tiem vao ben b u n g d§ '^^
Nghien ciru sa khai: Dua tren nhii'ng khao sdt ban dau dd dugc thi nghiem trudc ddy, khdi d i u thi nghiem tip lieu gdy tip vong d l hodn toan Id 50 |.ig/g, sau dd giam lilu xudng. Xdc dinh lilu LDo id lieu tdi da khdng gay c h i t d l ndo vd lieu LD100 Id lieu t h I p nhat gdy chet 100% d l trong Id thi nghiem.
Nghien ciru xae dinh: D l can ndng tip 0,3- 0,9 g duge chia ngau nhien lam 8 Id, mdi 16 7 con, cho sir dung thude d cac l i l u trong khoang LDo va LD100, cdng theo 1 Id chipng tiem nudc elt. Thude dugc tiem vdo trong mdng bung d l , lugng tiem chi dugc khoang 1 6 |il. Thulc dugc pha Idm nhilu ndng do: 1; 1,5; 2; 2,5; 3; 4;
5 |.ig/(il, sir dung cho cdc lieu khde nhau, de dam bao lugng tiem trong khoang cho phep.
Quan sdt d l trong 24 gid sau khi tiem thuoc.
Ghi nhdn cdc phan ipng xay ra tren d l vd s6 lugng d l sdng, c h i t a mdi Id. LD50 dugc tinh theo phuang phap Karber-Behrens vdi cdng thiPc tinh nhu d phdn tren.
ThLP nghiem c h d n g co giat gay bdi strychnin sulfat cua noc ran luc Hdn San
Dung djch ngc rdn l i l u 0,135 mg/kg (bdng 1/20 lilu LD50) d u g c tiem tinh mach de khao sat tac dung chdng co giat. Dung djch strychnin sulfat lieu 2 mg/kg d u g c tiem dudi da chudt de gay hien tugng co cirng, co giat. Chudt dugc chia ngau nhien thanh 3 nhdm,tiem thuoc vdi the tich 0,1 ml/10g the trong. Trong dd:
Nhdm chipng (n=8) d u g c tiem dudi da bdng dung djch sinh ly va sau 30 phiit duoc tiem dudi da dung djch strychnin sulfat
Nhdm ddi chipng (n=8) d u g c uong dung dich diazepam lieu 5 mg/kg vd sau 60 phiit duge tiem dudi da dung djch strychnin sulfat;
Nhdm thir (n=8) d u g c tiem tTnh mach bdng dung djch ngc l i l u 0,135 mg/kg vd sau 15 phiit dugc tiem dudi da dung djch strychnin sulfat.
Ghi nhdn thdi gian chudt bdt dau co giat vS thdi diem chudt tCr vong, sd can co giat va thdi
Nghien
CLFU- Ky thuat
gian co gidt So sdnh ede trj sd tren giipa ede nhdm bdng thdng ke phan tich. Su tang thdi gian khdi phdt can co giat vd giam ti le chudt tir vong cua nhdm thir so vdi nhdm chipng cho thIy tdc ddng chdng co giat ciia c h i t thip.
Phan tich thdng ke
Dir lieu duge trinh bdy dudi dang sd MEAN (sd trung binh) ± SEM (sai sd chuan eiia sd trung binh). Su khde biet giira cae nhdm duge phdn tich bdng phuang phap Mann-Whitney vdi phin m I m Minitab 14, p<0,05 dugc xem la khac nhau ed y nghTa thdng ke.
Phu'O'ng phap tach cac peptid trong noc rin luc Hdn Sen
Cae phan doan peptid dugc tdch ra tip ngc rIn bdng phuang phdp lge qua gel tren cdt Superdex 75 HR (1 x 30 em), Cdt dugc can bdng trudc trong dem ammoni acetat 0,1 M (pH 6,2), Lugng npe chay moi l i n 0,165 mg, tdc dp rira cdt 0,5 ml/phiit, T i l p dd nhu-ng phdn doan ndy duge ddng khd vd tilp tue tdeh bdng phuang phdp sdc ky Idng eao dp tren cdt phan tich Jupiter C18 (4 x 250 mm), tdc dd rira cdt Iml/phiit, Cdt sdc ky dugc ripa bdng gradient tuyIn tinh cua edc dung djch sau: A Id 0,1% TFA trong nude vd B Id 0,1%
TFA trong acetonitril, Thdi gian rira cdt tip 2% B d i n 52% B trong 50 phiit. Su ed mat eiia protein trong eac phan doan dugc xde djnh bdng mat dd quang tai bude sdng 226 nm, Khdi lugng phan tir eiia edc peptid tdch ra duge xde dinh tren may khdi pho MALDI MS (Bruker Daltonik, Dire).
K^t qua va ban luan
LD50 noc rdn luc Hdn Sen du'd'ng tiem tinh mach len chudt
Cae bieu hien eua chudt dugc quan sdt trong vdng 48 gid sau khi tiem tTnh mach dudi npe rdn lue Hdn San: sau vdi phiit eac chudt d i u xci Idng, thd manh, mdt sd chudt thi chay nude milng, quay m i y vdng chudt tir vong trong vdng 30-40 phiit,
Xae dinh LDg va LD^oo noc ran luc Hon San len chupt
K i t qua xdc dinh LDo vd LDioo len chudt duac trinh bdy d bang 1
Bang 1: LDg va LD100 ciia noc rin luc Hdn San len chudt
Dwdng tiem thuoc Tinh mach
Lieu (mg/kg) LDo LD,
Xae dinh LDso noc ran luc Hon San len chugt
Bang 2: Ty le tir vong cua chudt sau 24 gid tiem tinh mach noc rin luc Hdn San
L i l u (mg/kg) So chupt/16 So chupt tir vong/ld
S6 chuot s6ng/l6 Ty le c h i t (%) Bang 3: Tinh kit Karber-Behrens
A 1,5 B 0,9 Ab 1,35
1,6 5 0 5 0 qua
2,5 7 3 4 43 theo
3,5 0,5 1,75
3,0 7 4 3 57
3,9 7 7 0 100 phwang phap
4,5 0,9 4,05 Tip dd cd: l a b = 7,15; n,b = 24/4 = 6 vd tip cdng thirc cua Karber-Behrens ed:
LD,„ = L£)„ lab
Tip dd suy ra LD50 = 3,9- ( 7 , 1 5 :6) = 2,708 mg/kg
D l tinh sai sd chuan ciia LD50 chiing tdi dd dung dd thi su phu thude giua ty le chudt c h i t vd lilu thude tiem. Tip dd tinh duge phan phoi chuan s = 0,75 vd budc nhay trung binh d,b = 0,77. Vdi hdng sd Karber-Behrens k = 0,564 chiing tdi dd tinh ra sai sd chuan eiia LD50 Id 0,285. Nhu vdy chiing tdi dd xde dinh dde tinh cap eiia npe rdn lue Hdn San len chudt Id LD50 = 2,708 + 0,285 mg/kg.
LD50 noc rdn luc Hdn San len cdn trung Cae bilu hien eiia d l duge quan sat trong vdng 24 gid sau khi tiem ngc rdn luc Hdn Son vdo ben bung d l , Sau vdi gid mdt sd d l kem boat ddng va c h i t trong 24 gid.
Xae dinh LDg va LD100 ngc rin luc Hon San len de
K i t qua xdc djnh LDo vd LDmo len d l dugc trinh bay trong bang 4,
Bang 4: LDQ va LDwo ciia noc rin luc Hdn San len di
Noc rdn Igc Hdn San 1,6 3,9
Dwdng tiem thuoc
L i l u (pg/g) Noc ran luc Hdn San
Tiem phan ben ciia bung
LDo LDioo 5 30
TAP CHi DUQC HQC - 8/2011 (SO 424 NAM 51) 39
Xae dinh LD^Q ngc ran luc Hon San len de
Bang 5: Ty le tir vong ciia di sau 24 gid tiem noc rin luc Hdn Son Lieu (/jg/g)
Ndng do noc (|ig/|il) So de/ld So de tir vong/16 Ty le tir vong (%)
5 1 7 0 0
7,5 1,5 7 1 14
10 2 7 2 29
15 2,5 7 3 43
17,5 3 7 4 57
20 4 7 5 71
25 5 7 6 86
30 5 7 7 100
Nwac cat
7 0 0 Bang 6: Tinh kit qua theo phwang phap
Karber-Behrens A
B Ab
0,5 1,5 2,5 2,5 1,25 3,75
2,5 5 12,5
3,5 2,5 8,75
4,5 2,5 11,25
5,5 5 27,5
6,5 5 32,5 Tip dd cd: Zab = 97,5
ciia Karber-Behrens ed:
Y.ab
ntb = 7 vd tip cdng thiPC
LD,„ = LD„ Tip dd suy ra LD50 = 30 • (97,5 :7)= 16,07|jg/g
Oe tinh sai sd chuan cua LD50 chung tdi dd
Bang 7: Tac dung chdng co giat gay bdi strychnin sulfat ciia noc rin luc Hon San
dung do thj s u phu thude giipa ty le de chet vd lilu tiem ngc. Tip dd tinh d u g c phan phdi chuan s = 8,47, budc nhay trung binh dtb = 3,75. Vdi hdng sd Karber-Behrens k = 0,564 chung tdi da tinh ra sai sd chuan cua LD50 la 1,56. Nhu vay chimg tdi da xdc djnh ddc tinh cdp cua npe rdn luc Hdn San len de LD50 = 16,07 ± 1,56 |ag/g.
K§t qua khao sat tac dong chdng co giat cua noc rdn luc Hon S a n
Sau khi tiem strychnin sulfat vdo chudt vd ghi tdc dung cua thuoc len chudt trong bang sau.
Ld Chirng Diazepam 5 mg/kg, PC Noc ran luc lieu 0,135mg/kg
Thai diem bat dau co giat (giay)
365 ± 67 672,88 ± 71 **
296,1 ± 104 ##
Thai diem chet (giay) 451,6 ± 100
> 1800 381,3 ± 100
Thai gian co giat (giay)
66,75 ± 4 1 106,88 ± 5 4 64 ± 44 ## **
Ty le chet (%) 100%
0% **
100%##
(*'), p<0,01, khac CO y nghia thong ke so vdi 16 chdng (#tt), p<0.01, khac CO y nghTa thong ke so vdi 16 diazepam Qua khao sdt cho thdy sir dung ngc rdn d
lilu 0,135 mg/kg khdng lam thay doi thdi gian khdi phdt can eo giat vd thdi d i l m tip vong cung nhu ty le tip vong cd y nghTa so vdi Id chirng.
Nhu vdy sip dung noc ran luc Hdn San d lilu nhu tren khdng ed tae ddng chdng eo giat gdy ra bdi strychnin sulfat.
Ket qua tach va xae djnh khdi lu'O'ng phan tup ciia cac peptid trong noc rdn Hdn San
Bdng phuang phdp sdc ky Igc gel tren cdt Superdex 75 HR chiing tdi dd tach ngc rdn luc Hdn San C. honsonensis ra Idm 5 phdn doan cd khdi lugng phdn tir khde nhau (xem hinh 1).
Tiep theo de tdch eac peptid trong phan doan 5 chiing tdi dd sir dung phugng phdp sdc ky Idng hieu nang cao. K i t qua phdn tich, tip phan doan 5 chiing tdi dd tdch va xae dinh khdi lugng phan tir eiia 3 peptid. Cac peptid nay cd khdi lugng phdn tip Id 1213 Da, 1520 Da vd 1741 Da,Trong nhirng nghien ciPu t i l p theo ehiing tdi se t i l n hanh khao sdt boat tinh sinh hoe eua cac peptid da tach ra tip ngc rdn luc Hdn San.
San Hinh 1: Sac ki noc ran luc Hdn C.honsonesis tren cot Superdex 75 HR
Ket luan
Ngc rdn luc Hdn San cd ddc tinh cap LD50 dudng tiem tTnh mach tren chudt nhdt trdng Swiss albino, the trgng 18-24 g, 3-4 tuan tuoi theo phuang phdp Karber-Behrens Id 2,708 + 0,285 mg/kg. Ngc rdn ndy cd ddc tinh cap LD50 dudng tiem ben bung d l Achetus sp., the trpng 0,3-0,9g, theo phuang phap Karber-Behrens 1^
16,07 ± 1,56 |.ig/g. Ngc rdn luc Hdn San d lilu
40
TAP CHi DUQC HQC 8/2011 (SO 424 NAM 51)
Nghien CLFU - Ky thuat
0,135 mg/kg khdng cd tdc dung chdng eo giat gdy ra bdi strychin sulfat.
Tip ngc rdn lue Hdn San dd tdeh vd xde dinh dugc khoi lugng phdn tir eua ba peptid id 1213 Da, 1520 Da vd 1741 Da, Tinh c h i t ly sinh eua cae peptid nay se duge khao sdt trong nhirng nghien ciPU t i l p theo.
Summary
Venoms collected from the snakes Cryptelytrops honsonensis, inhabiting Hon Son Island, Raeh Gia bay and Kien Giang province of the Southern Vietnam were investigated for toxicity and peptide components. The LD^ of C.
honsonensis venoms were 2.708 ± 0.285 mg/kg B.W mouse by intravenous injection and 16.07 ± 1.56 f.ig/g B.W. cricket by intraperitoneal injection at dose of 0,135 mg/kg. The venoms did not prevent convultions induced by strychnin sulfate in mice. Three venom peptides of molecular masses 1213 Da, 1520 Da and 1741 Da were isolated by combination of gel-filtration on Superdex 75 HR and reversed-phase HPLC on a C18 column. The physiological activities of these peptides are to be characterizated in a further study.
Keywords: Cryptelytops honsonensis, peptide, LDso, anti-convultion activity, snake, venom, toxin, gel-filtration, HPLC, molecular mass.
Tai lieu tham khao
1 Panfoli 1., Calzia D., Ravera S., Morelli A.
Inhibition of hemorrhagic snake venom components: old and new approaches. Toxins, (2010), 2, pp. 417-427.
2. Utkin Yu.N., Osipov A.V. Snake venom polypeptides affecting the central nervous system.
Central Nervous System Agents in Medicinal Chemistry, (2007), 7, pp. 97-107.
3. Andew R.K., Kamiguti AS., Berlanga 0,, Leduc M,, David R.G., Steven T., Watson P. .The use of snake venom toxins as tools to study platelet receptors for collagen and von Willebrant factor.
Haemostasis, (2001), 31, pp. 417-427.
4. Grismer L.L., Ngo Van Tri, Grismer J.L. A new species of insular pitviper of the genus Cryptelytrops (Squamata : Viperidae) from southern Vietnam.
Zootaxa. (2008), 1715, pp. 57-68,
5. Do Trung Oam,'Phuang phap xae dinh doc tinh clp ciia thuoc. NXB Y hoc Ha Npi, (1996).
6. Starkov V.G., Osipov A,V,, Utkin Yu,N, Toxicity of venoms from vipers of Pelias group to crickets Gryllus assimilis and its relation to snake entomophagy. Toxicol. (2007), 49, pp. 995-1001.
Xay dyng phircrng phap dmh lu'O'ng carboplatin trong thuoc tiem dong kho carboplatin b3ng sSc ky long hieu nang cao
Dat van de
Hien nay edn benh ung thu ngdy cdng gia tdng manh me md gid thdnh eiia nhirng loai thude trj ung thu lai rat cao, ddc biet Id cac thuoc nhdp khau, chinh vi vay d l tai nghien ciPU san xult thude tiem ddng khd carboplatin diing dilu trj ung thu Id mdt nhiem vu cdp thiet cua Cdng ty Dugc - TTBYT Binh Ojnh nhdm giam phin nao gdnh ndng chi phi dilu trj eho ca benh nhdn vd xd hdi. O l ddnh gia dugc chat lugng cua san pham trong sudt qua trinh nghien ciPU cdng ty dd xdy dung phuang phdp djnh
Phan Thj My Hoang, Banh Thi Nggc Quynh, Ly Thi My Dung, Mai Tong Ba
Cong ty Dugc - TTBYT Binh Dinh lugng Carboplatin trong e h i pham ddng khd bdng cdc trang thilt bi hien ed ciia dan vj dua tren su tham khao cdc dugc diln nudc ngodi.0 6 i tu'O'ng va phu'O'ng phap
Odi tu'O'ng nghien cii'u
Thude tiem ddng khd carboplatin 50mg vd 150mg do Cdng ty Dugc - TTBYT Binh Oinh nghien ciru san xult,
Phu'O'ng phap nghien CIPU
Xay dung phuang phap dinh lugng bdng phugng phdp sdc ky Idng hieu nang cao (HPLC)'^"'^'
TAP CHi DUQC HQC - 8/2011 (SO 424 NAM 51)