• Nghien CLFU - Ky thuat
Dieu che va thiet lap chat chuan crinamidin
Nguyin Tlii Ngoc Tram^ Du-ong Thi Thuy Nga', Nguyin Thi Tu-ong Vy' Pham Quynh Khoa', Ha Dieu Ly^ Ha H6iS Nguyin Minh Dire'*
'Khoa Dugc, Dgi hgc Y Dugc TP. Ho Chi Minh 'Cong ty TNHH Thien Dugc, tinh Binh Ducmg
'Vien Kiem nghiem Thuoc TP. Ho Chi Minh 'E-mail: [email protected]
Summary
Alkaloid crinamidin known as the major principle for medicinal virtue of Cnnum latifolium L (Amaryliidace) responsible for curative effects was extracted, isolated from the plant leaves and bulbs and purified to establish a staridard substarice for quality control of the commercial preparations from the plant materials for treatment of ulcers, tumors and cancers, especially of prostate and ovary, etc..
The reference standards of the obtained crinamidin were validated by physico-chemical tests and Inter- laboratory trials at three independent GLP/ISO laboratories based on guidelines of ISO 13528:2005 and ISO Guide 35.
Keywords: Crinum latifolium, crinamidin, reference standard, ISO Guide 35, ISO 13528, inter- laboratory validation.
Dat van de
Trinh nu- hoang cung {Crinum latifolium L. - Amaryllldaceae) la mdt cay thulc dan gian dung d l chua cac ehd'ng u budu. Theo kinh nghiem dan gian, djeh sac la cay trinh nu' hoang cung (TNHC) dung dudng ulng d l dllu trj cae chirng u vu, u nang buIng tru-ng, u gan, phoi ... Thanh phin hda hpc chu y l u ciia la TNHC gdm cae alkaloid nhu ambellin, lycorin, pratorin, crinamidin, powellin, erinan-3a-ol, 9-octadeeenamid, dlhydro- oxa-demethoxyhaemanthamln, augustamln, oxoassoanln, buphanidrin, undulatin... P"'. Hien nay nhilu san phim tu- TNHC dang luu hanh rdng rai tren thj trudng dudi dang thule hay thuc phim chu-e nang. Tuy nhien, chit lugng ciia eae san phim chira TNHC tren thj trudng v i n cdn nhilu bit cap vl thilu cac bien phap kilm tra chit lugng trong qua trinh trong trpt, san xuit, luu tru-, phan phii va luu hanh.
Muc tieu cua d l tai la dllu ehl va thilt lap chit ehuin crinamidin - mdt trong cac alkaloid chu y l u cua TNHC - nhim phuc vu chp cac muc tieu nghien cu-u va kilm nghiem d l dam bap va nang cao chit lugng ciia dugc lieu TNHC va cae c h i phim.
Ddi tu'O'ng va phu'O'ng phap nghien
CLPU
Nguyen lieu
Nguyen lieu dung de chilt xuit phan lap la bdt la khd cay trinh nu' Crila, ed ten khoa hpe da dugc xac djnh la Crinium latifolium L. var. crilae Tram & Khanh, var. n.Pi. Bpt la cd kich thudc 1-3 mm dupe eung c i p bdi Trai Dugc lieu Long Thanh, tinh Ddng Nai, do Cdng ty TNHH Thien Dugc quan ly.
Trang t h i l t bj va phu'ang phap nghien cii'u Phwang phap chung
Dilm chay (khdng hieu chinh) do tren may do dilm chay Gallenkamp (Anh). Pho hing ngoai do bang may Shimadzu FTIR-8201PC (Nh§t) bang ky thuat vien nen KBr. Pho cdng huo'ng tir hat nhan dugc thuc hien bang may Brucker 500 MHz NMR (My), dung mdi do 1^ CDCI3 vdl chuan ndi la TMS, dp ddi hda hpc (5) tinh thep ppm v^
hing sd ghep J tinh theo dan vj Herzt (Hz). Pho khii dugc do tren may Brucker pQ-TOF MS (My) d- 70eV. S i c ky Idng hieu nang eao (HPLC) dugc thuc hien tren may Shimadzu LC-20A (Nhat), detector PDA, cdt Phenemenex C8 (250 x 4,6 mm, kich thudc hat 5 pm). Sac ky cpt thuc hiSn tren silica gel (Merck, Du'e), kieh thud-e 230-400
52 TAP CHI DU'OC HOC - 1/2016 rSO 477 N A M «/;^
mesh, s i e ky ldp mdng thue hien tren ban mdng trang s i n Kieselgel 60 F^^^ (Merck, Diec), phat hien bing each sol UV d budc sdng 254 va 365 nm, sau dd phun thulc thii- Dragendorff.
Tham djnh va thiit lap crinamidin dieu che - Djnh tinh va xae djnh c i u true: S i c ky Idp mdng, HPLC phan tich, dilm chay pho IR, pho MS va pho NMR.
- Xay dung va danh gia quy trinh djnh lupng crinamidin bing HPLC: Danh gia eae ehl tieu bao gdm tinh tuang thich he thing, tinh dae hieu, tinh tuyin tinh, dp lap lal va dp dung.
- Ddng gdl va danh gia ddng nhit Ip theo ISO Guide 35'^'. Chit chuan dllu ehl dugc ddng gdl tai Vien Kiem nghiem Thulc TP. Ho Chi Minh trong diiu kien ddng gdl tieu chuin.
- Danh gia chat chuin thdng qua lien phdng thi nghiem: Cac phdng thi nghiem duge lua ehpn phai dat GLP hoae ISO 17025, trong dd cd it nhit mdt phdng thi nghiem cd dii thim quyln va y nghTa phap ly ddi vdi Viet Nam, mdi phdng se dugc gui sau Ip ngiu nhien kem theo chit chuin, quy trinh phan tich va cac tai lieu khac cd lien quan. Ba phdng thi nghiem tham gia danh gia chit chuan gim:
+ Phdng kilm tra chit lupng Cdng ty Co phin Hda - Dupc phim Mekophar
+ Khoa thilt lap chit chuan va chit ddi chilu, Vien Kilm nghiem Thule TP Hd Chi Minh.
+ Khoa kiem nghiem cac dang bao ehl, Vien
Kiem nghiem Thuoc TP. He Chi Minh.
- Xae djnh gia trj an djnh va dp khdng dam bao do theo ISO 13528'^'.
Kdt qua nghien CLPU
Chilt xuit phan lap crinamidin tir la TNIHC Bpt la TNHC (100 kg) dugc ehilt ngim kiet vdl ethanol 96%. Djch chilt dugc ed giam ap thu h l l dung mdi cho 21 kg cao ethanol. Cao ethanol dupe hda tan trong nud-c va acid hda bing acid acetic d i n pH = 4. Lac phan b l vdl dlcloromethan, cd thu hdi dung mdi, thu dugc cao chilt pH = 4 (2,5 Kg). Djch chilt acid cdn lai dupe kilm hda bing amoniac pH = 9, sau dd lie phan b l vdl dlcloromethan, cd thu hdi dung mdi thu dugc cao ehilt pH = 9 (210 g).
Cao pH = 9 (130 g) dugc sac ky cdt nhanh (VLC) tren silica gel vd-i he dung mdi lin lugt la dielerometan, dlcloromethan methanol (98:2) va dicloromethan:methanol (80:20). Tiln hanh sac ky Idp mdng d l theo ddi qua trinh phan lap va gom cae phan doan cd thanh phin gilng nhau, thu dugc 9 phan doan tu- A-H. Tlln hanh sic ky cdt silica gel lan lugt 3 phan doan D, E va F vdi he dung mdi dlclorometan - methanol vdl dp phan cue tang din. Thu liy phan doan chira crinamidin, cd thu h l l dung mdi va kit tinh nhilu lin trong methanol. Kit qua thu dugc 1,5 g crinamidin dudi dang tinh t h i hinh kim mau tring.
Hinh '\:Sicky dd ban mdng cac phari doan sac ky cdt nhanh.
He dung mdi: Ethyl acetat - methanol - nudc - ammoniac (8:1:1:0,5).
C: Crinamidin chuin; T: Cao pH 9; A-H: Cac phan doan tuA-H
TAP CHi DlTOC HOC - 1/2016 (SO 477 NAM 56) 53
• Nghien CLFU - Ky thuat
Djnh tinh va xac djnh cau true Sac ky l&p mdng
1 2 3 1 2 3
U V 2 5 4 n m Thuoc thitDragendoff Hinh 2: SSc ky dd sac ky Idp mdng cririamidiri
vdi 3 he dung mdi khac nhau He dung mdi:
1. Ether d i u hda - ethyl acetat (2:8).
2. Cloroform - methanol (9:1).
3. Ethylacetat - methanol - nudc - ammoniac (8:1:1:0,5).
Phat hlen:Soi UC 254 nm va phun thuoc thii' Dragendorff.
Kit qua sac ky Idp mdng vdi 3 he dung mdi khae nhau cd dp phan cue tang din cho thiy crinamdin dllu ehl chi eho mdt v i t duy nhit.
Diim chay
Kit qua xac djnh dilm chay ciia crinamidin dllu ehl: 215 - 217°C, phu hpp vdl tai lieu ^ l
PhoIR
Php IR cua crinamidin diiu e h l cd cac bang hip thu cue dai (cm') a 3200 -3400 (-0H);
1499, 1278 (epoxy), 1035, 918 (-0CH20-), 802 (epoxy), phu hgp vdl eae du' lieu pho IR eua hgp chat crinamidin theo tai lieu'^'.
Pho UV-Vis
Abs
1.5-
1.0-
0.5-
0.0-
'— ' ' ^ j I I I 1 I I n m 200 250 300 350
Hinh 4: Phd UV-Vis cririamidin diiu chi
Pho UV cho thiy crinamidin dllu e h l ed budc sdng hip thu cue dai 6- 220 va 285 nm.
Pho HR-MS
Hinh 3: Phd IR ciia cririamidiri diiu chi.
Hinh 5: Phd HR-MS cririamidin diiu chi Phd HR-MS ciia crinamidin dllu c h i cho manh ed s l khii 318,1369 m/z (100% rel. int) tuang irng vdl manh ion phan tu- gia [M+Hf phu hgp vdl edng thu-c C,^H^gN05 cua crinamidin.
Pho NMR
DO- lieu pho NMR ciia crinamidin dllu ehl trong bang 1 phii hgp vdl du- lieu NMR ciia crinamidin theo tai lieu'^l
54 T A P C H I D l / O C H O C - 1/2016 r.SO 477 M A M -
Bang 1 : So sanh du lieu phd '^C-NMR (125 MHz, 5 = ppm, CDCI J va 'H-NMR (500 MHz, 5 = ppm, CDCI J ciia cririamidiri diiu chi vdi cririamidiri (CDCQ theo tai lleLf^>
C 1 2 3 4 4a 6 6a
7 8 9 10 10a 10b 11 12 13 14
^.
53,9 56,6 65,7 29,9 61,2 58,8 117,8 141,3 133,6 148,4 96,6 139,0 41,8 39,3 527 100,8
59,3
Crinamidin dieu che
^
3.75(1H,d,J = 4,0Hz) 3,28 (1H,m) 4,51 (1H;m) 1,59 (m;2H) 3,23(ni;1H) 3,73 (d;J= 17,5 Hz;1H)
4,15 (d;J= 17,0; IH)
- - -
6,64 (1H,s)
- -
2,0 (IH; m)
2,38(1 H; ddd: J =12,5:11; 5,5 Hz) 2,79 (1H;m)
3,17 (1H,m)
5,86 - 5,87 (2 H; 2d; J = 1,5 Hz; -OCH,0) 3,59 (3 H. s, 7-OMe)
^
53,8 56,4 65,5 29,7 61,0 58,6 117,5 141,1 133,4 148,1 96,4 138,7
41,6 39,2 52,5 100,7
59,1
Crinamidin'"
s„
3,75(1H,d,J = 4,0Hz) 3.26 (IH; ddd; J = 4,0; 2,5; 1,5 Hz) 4,48 (IH, ddd; J = 3,0; 2,5; 2,0 Hz) 1,56 (IH, ddd; J= 13,5; 12,5; 3,0 Hz; HP) 1,61 (1 H, dddd; J = 13,5; 5,5; 2,0; 1,55 Hz, Ha)
3,17(1 H;dd; J = 12,5; 5,5 Hz) 3,71(1H,dJ=17,5Hz;H|3) 419(1 H,d;J= 17,5 Hz; Ha)
- - - -
6,61 (1H;s)
- -
2,0 (1 H; ddd; J = 12,5; 9,0; 5,0 Hz; H-llendo) 2,37 (1 H; ddd; J = 12,5; 10,5; 5,5 Hz; H-llexo) 2,77 (1 H; ddd; J = 12,5; 9,0; 5,5 Hz; H-12endo) 3,17 (1 H; ddd:J= 12,5; 10,5; 5,0 Hz; H-12exo) 5,85 - 5,86 (2 H; 2d; J = 1,5 Hz; -OCH,0-)
3,59 (3 H, s, 7-OMe) TCf cae ket qua tren, cd the ket luan hgp chat
thu dugc ehinh la crinamidin vdl cong thirc c l u
tao trong hinh 6. lis
Hinh 6: Cdng thuc cririamidiri Tham djnh quy trinh djnh lu'gng
Diiu kien s i c ky HPLC: Cdt: C8 (250 x 4,6 mm, kich thuac hat 5 pm); pha dpng: MeOH - HjPO^ 0,1% (35:65); tie dp ddng: 1 ml/ phut;
detector: PDA; nhiet dp: 30 °C; t h i tieh m i u bam:
20 pi; dung mdi pha miu: pha dpng.
Quy trinh djnh lugng HPLC da thilt lap tren dugc danh gia cac chT tieu gdm tinh tuang thieh he thing, tinh tuyin tinh, dp dae hieu, dp lap lai va dp dung. T i t ca cae chi tieu tren d i u dap img yeu c i u phan tich.
Trong dieu kien HPLC da thilt lap, crinamidin diiu ehl ehl cho chu y l u mot dinh cd thai gian luu Rt ~ 6,8 phut.
Hinh 7: Sic ky dd HPLC dirih lugng cririamidiri Dong goi va danh gia dong nhat lo Ddng gdl
Khii lugng ban d i u : 1,523 g.
Khdi lugng ddng lp: 10 mg/ Ip.
s l Ip ddng duge (N): 100 lp.
Khil lugng edn lal: 0,523 g, ddng thanh 1 lp d l tham djnh va sir dung sau.
Danh gia dong nhat Ig
S l lp_cin liy d l danh gia ddng nhit Ip:
g = ^ . v ^ l = 11 lp
Lay ngiu nhien cac Ip theo phin mim Excel, s l thu- tu cac Ip se liy d l djnh lupng la 84, 72, 70, 25, 1, 93, 19, 96, 57, 89, 73; mil Ip kiem tra 2 lan.
T.AP CHI DlTOfC HOC - 1/2016 (SO 477 NAM 5fi^ 55
• Nghien CLFU - Ky thuat
M i u thu- 8 M i u thu- 9 M i u thif 10 M i u thu-11 Mau thu-12 M i u th LP 13 M i u thtr 14 M i u thir 15 Mau thir 16 M i u thir 17 M i u thLP 18 Trung binh Dp lech s
x * m 6 i S'lTlCfi
99,85 99,85 99,85 99,85 99,85 99,85 99,86 99,87 99,88 99,88 99,88 99,85 0,02 99,85
0,01 99,85 99,85 99,85 99,85 99,85 99,85 99,86 99,87 99,87 99,87 99,87 99,85 0,01 99,85
0,02 99,85 99,85 99,85 99,85 99,85 99,85 99,86 99,87 99,87 99,87 99,87 99,85 0,01 99,85 0,02
0,07 0,07 0,07 0,07 0,07 0,07 0,69 1,32 1,32 1,32 1,32
99,85 99,85 99,85 99,85 99,85 99,85 99,86 99,87 99,88 99,88 99,88 99,85 Bang 3: Ket qua dmh lugng danh gia ddng nhat id
crinamidin diiu chi STT
1 2 3 4 5 6 7 8 9 10 11
Loso 84 72 70 25 1 93 19 96 57 89 73
Lan 1 (%) 99,79 99,83 99,85 99,84 99,83 99,84 99,88 99,85 99,83 99,88 99,88
Lan 2%
99,85 99,85 99,85 99,85 99,86 99,84 99,87 99,75 99,74 99,73 99,45
Phan tich phuang sai mdt y l u t l theo ANOVA.
Bang 4: Kit qua phan tich danh gia ddng nhit Id cririamidin theo ANOVA
Tong do
Bien thien lech bmh „ • Phwang sai F Bac
• twdo ^ phwang
Giua cac nhom Trong cung
0,003436 0,00335
10 0,000343636 1,128 2,854
nhom
TSng cong 0,006786 21
11 0,000304545
Nhan xet F = 1,128 < F^^^ = 2,854, khi dd ta cd:
- ^ = 0,07 < p^^ = 0,0044
Kit qua phan tieh chu-ng td cac lp crinamidin diiu ehl da ddng gdl cd ham lugng dong n h i t
Xac djnh gia trj I n djnh, do khong dam bao do va gia trj cong b l tren philu kilm nghiem ciia crinamidin
Bang 4: Klf qua tinh gia tri in dinh ham lugng (%,) crinamidin (n = 18).
So lan thay doi
6 = 1,5x8*
x * - 5 x* + 8 Mau thLP 1 Mau thip 2 Mau thLP 3 M i u thLP 4 M i u thu- 5 Mau thu- 6 Mau thCp 7
X * fO)
99,79 99,83 99,83 99,83 99,84 99,84 99,84
X*
IV
0,02 99,83 99,87 99,83 99,83 99,83 99,83 99,84 99,84 99,84
X*
0,02 99,82 99,87 99,83 99,83 99,83 99,83 99,84 99,84 99,84
z-score
-1,18 -1,18 -1,18 -1,18 -0,56 -0,56 -0,56
xchqn(%)
99,79 99,83 99,83 99,83 99,84 99,84 99,84
Nhan xet: Sau 2 lan thay doi, gia tri s* khdng thay doi (0,02), khi dd gia trj x* mdi dugc chpn la 99,85%.
Tu- kit qua tren, tinh z - score. T i t ca gia trj ciia 18 miu thii'diu ed \z\ < 2. Tinh trung binh 18 miu, thu duge kit qua nhu sau:
Ham lugng trung binh: 99,85% (n = 18).
Dp lech s: 0,01.
Op khdng dam bao do p: 0,0006.
Nhu vay gia trj i n djnh, hay kit qua edng b l trong philu kilm nghiem eua ehit ehuan crinamidin dllu ehl la 99,85% tinh theo nguyen trang.
K§t luan
Tu' la trinh nu' Crila {Crinium latifolium L. var.
crilae Tram & Khanh, var. n.), ehung tdi da chilt xuit va phan lap crinamidin vdl lugng hang gam d l sif dung lam chit chuin d i l ehiiu. Quy trinh chilt xuit kha dgn gian va on djnh, cd the tilp tue ap dung d l dllu e h l crinamidin ehuan d l eung eip eho thj trudng phue vu cong tae kilm nghiem. Chit chuan crinamidin dllu ehl dugc thim djnh d i y dii theo cae tieu chi chit chuin va danh gia lien phdng thi nghiem tai 3 phdng thi nghiep ddc lap dat heu chuan GLP va hudng d i n ciia ISO 13528 (2005) va ISO Guide 35 (2006). K i t qua cho thiy crinamidin dieu ehl dat tieu ehuin chuin g l c (primary) vo'i ham lugng dugc xae djnh la 99,85% tinh theo nguyen trang (n 18, s = 0,01, p = 0,0006). Chit chuin crinamidin da dllu ehl va thilt lap se gdp phin vap viec kilm nghiem chit lugng trinh nu' hoang cung va cac ehl pham.
56 TAP CHI D l / O C H O C - 1/2016 (Hn 477 M A »
Tai lieu tham khao
1. ISO 13528 (2005), Statistical methods for use in proficiency testing by interiaboratory comparisons, ISO.
2. ISO Guide 35 (2006), Reference materials General and statistical principles for ceriification, ISO.
3. Nguyen Thi Ngoc Tram, Maya Mitova, Vassya Bankova, Nedyalka Handjieva and Simeon S. Popov (2002), "GC-MS of Crinum latifolium L. alkaloids", Z Naturforsch., 57c, 239-242.
4. Nguyen Thi Ngoc Tram, Tz. V. Tltorenkova, V. St.
Bankova, N. V. Handjieva, S. S. Popov (2002), 'Crinum L. (Amaryllidaceae)", Fitoterapia, 73, 183-208.
5. Nguyin Thj Ngoc Tram, Trin Cong Khanh, "Trinh nO' Crila- Crinum latifolium L. van crilae Tram & Khanh, var. n.: Mgt thir mdi cua loal trlnh nu hoang cung - Crinum latifolium L. (Ho Nang - Amaryllldaceae) a Viet Nam", Tap chi Sinh hoc, 2012, 34(2), 190-193
6. V. Francesc, R. A. Giovanna, C. C. Carles, B.
Jaume, E. C. William, M. Shaheed (1996), "Alkaloids from Bmnsvigia orientalis", Phytochemistry, 43(6), 1379-1384.
{Ngay nhan bar 21/12/2015 - Ngay duyet dang: 06/01/2016)
K h a o s a t t a c d u n g . . . (TUP theo trang 51)
Bang 5: So sanh tdrig sd vi khuiri (CFU) trudc va sau khi rim tay
TrwoKrira Sau rira Giam Log CFU/ml 1,475x10"^ 07 X W
(0,1-5x10°) ( 0 - 4 x i y ) 4,32 CFUiarf 25x 10' 0175
Sir dung ndng dp tinh d u g c ciia vl sinh vat (CFU/ml) c' thd-i d i l m t r u d c khi rii-a tay d l tinh toan s u thay del ciia ndng dp vl sinh vat sau khi rifa tay b i n g gia trj log^g. Ghi lal s u thay doi d u a l dang Log reduction.
Log reductlon= Log^^^R^ - Log^^R, LogJ1,475* IC)- LogJ0,7* 10') = 4,32 Vay t / le diet k h u i n la: (1-16,95.10'^) x 100%
= 99,99%
Ban luan
Hien nay, d l ve sinh chan tay, t h u d n g dung cac san pham xa phdng diet k h u i n hoac cac dung djch rira tay sat k h u i n n h u ethanol, dung djch clohexldin... Tuy nhien, sii- dung xa phdng diet khuan phai dung nude, khdng phai luc nao cung s i n cd dae biet trong trudng h g p k h i n clp. Ngoai ra, n l u sir dung dung djch diet k h u i n clohexldin khdng phai la tuyet d i l an toan va cung phai dimg nudc. V a i gel nano bac, quy trinh rira tay dan gian va tien lgi han. Cimg v d l cdng tac tuyen t r u y i n sau rpng trong edng dong trong nhu-ng nam g i n day va viee thuc hien nhu-ng du- an v l xay dung thdi quen rira tay cua to ehire Y t l t h i gidi tai Viet Nam, thdi quen hO'u ich nay dang ngay cang d u g c thuc hien mdt each cd hieu qua. Chlnh vl vay, thj trudng cac e h l pham n u d c rii-a tay cung dang d u g c da dang hda v l hinh thu-c, dang dung, hoat ehit... Gel rira tay nano
bae khdng dung n u d c la ehe pham cd nhieu tien ich: vu'a khdng phai rira lai b i n g nude, lai cd t h i mang theo de dung b i t cir liic nao. Ngoai viec phuc vu cho cdng tae phdng c h i n g djch benh, nhu'ng san p h i m nay cdn ed the d u g c dung rdng rai d khap nai: tai gia dinh, t r u d n g hpc, khach san, nai ccng s d . . .
K§t luan
Qua khao sat cho t h i y cae vl sinh vat thudng gap tren da ban tay trud-c khi rii-a la: Staphylococcus epidermidis, Steptococcus nhdm A tan mau beta, Enterococcus sp. (lien c l u d u d n g ruptj, Klebsiella spp., E coil, tu c i u vang va n i m Aspegillus sp.. Sau khi rira tay vd-i gel sat k h u i n nano bae, cae m i u cd n h i l m vi sinh vat thi chii y l u la Staphylococcus epidermidis va E. coll Sau khi danh gia s l lugng vi sinh vat tren ban tay nhan vien y t l (n = 108) trudc va sau khi rira vdi gel rira tay sat k h u i n nano bac, hieu qua diet k h u i n cua gel rira tay sat khuan nanp bac la 99,99%.
Tai lieu tham khao
1. Baldlnl N. C , Gutman E. L., Keefe E. et al.
(2001), "Standard test method for evaluation of health care personnel handwash formulation by utilizing fingernail regions". In: American society for testing and materials, Baltimore, MD: American Society for Testing and Materials, 599-602.
2. Belongia E. A., Chyou P., Greenlee R. T et al (2003), "Diarrhea incidence and farm-related riskfactors for Esc/ieric/i/aco/; 0157:H7 and Campylobacter jejuni antibodies among rural children", J. Infect. Dis., 187, 1460-1468.
3. Centers for Disease Control and Prevention (1999), Outbreak of Escherichia coli 0157:H7 and Campylobacter among attendees of the Washington county fair. New York, Morb Mortal Wkly Rep., 48(36), 803-804.
{Ngay nhan bai: 15/12/2015 - Ngay duyet dang: 06/01/2016)
TAP CHI D U O C H O C - 1/2016 (SO 477 NAM 5fiN 57
