Vietnam J. Agri. Sd. 2016. Vd. 14. No. 9:1368-1376 Tap chi KH Nfing nghifp Vift Nam 2016, tfp 14, s6 9:136S-BSI www.vnuaei !•
BANH 6 U HIEU SUAT CHin VA TAC DUNG CUA CAO CHIET TllT GO Td MfiC {CaesalplaiasappaifL^ TRONG DUNG MOI ETHANOL VOll VI KHUAN CsehericUam
Nguyen Tan Dat, Nguyin Bfi Tilp*
Khoa Thil y, Hpe vien Nong nghifp Viet Nam Email: [email protected]
Ngay gui bai: 30.05.2016 Ngay chfip nhan: 16.10.2016 T6M TAT
Chift xuft thi> nghifm gd To mfc che nhfi bang phuong phfip ngfim l<ift vd^l dung mfii ethanol 96% vd cAitt hfii cdn fir nhift dfi 80 - 90°C cho hifu suft chift trung binh 6,80%. Dung djch chift pha lofing cua cao T6 mOc5|4 vfi 10 pg dfu cfi tfic dgng uc chf vi Idiuin Escherichia coli (E. coli) nhu'ng l<fim hon sg vfiii cfic khfing sint Dgxycydine. Qprgflgxadn vfi Colistin. Hogt tinh khfing khuin cua djch chift cao Tfi mfc vfi cfic khfing sl*
Doxycydine, Ciprofloxacin vfi Colistin vd-i chung £ coli chuin cao hon so vfi'i chung E. coli phfin Ifp tif gfi bfnh. Tt dgng khfing khufn phg thufc vfio hfim lugmg cao chift. Chf phim cao Tfi mfc 10% cfi tac dyng phfing vfi trj lifti do E. co//trfin gfi Lining Phugng gfiy nhiim thgc nghifm. Kft qufi nfiy Ifi ca sfi' khoa hgc cho ofic nghiSn cihi Si theg v f tfic dyng phdng vfi tri bfnh cua cfic chit chift tU Tfi mfc.
Tirkhda: E. col, gfi Luorng Phugrng, hifu suit chift khfing khuin, Tfi mfc.
Assessment of Extraction Efficacy and Antibacterial Activity of Ethanol Extract of Caesalpinia sappan L. against Escherichia coli
ABSTRACT
^IMaceration extraction of Caesalpinia sappan L. (CS) in ethanol solvent (96%) follovi/ed by steam distillation alS - 90 C resulted in an average extract efficacy of 6.80%. Ten percent extract sollution showed inhibitory effects on£
coli. The effects were concentration-dependent and higher on standard E coli strain in comparison to those»
chicken-derived isolates. The strain dependent inhibition was similarly observed for doxycydine, dprofloxadn l i colistin. The 10% sollution of CS extrad exerted preventive and treatment effects on experimental infection wilh£
coll in Yellow-feather chidfens. The results can be considered as sdentific basis for further studies on preventionii treatment potential of CS extracts.
Keyvrards: Antibacterial effect Caesalpinia sappan L., E coli. extract efficacy, Yellow-feather chickens.
1. DAT VAN DE ^ ' ' ^ ^ ' ^''ortman vfi Mukhopadhyay, 2016). Tron.
_, khi dd, stt dung cac khang sinh mdi, khang fflj' Da 80 khang sinh dugc sU dung dfu nhfim phf rfing lai cd chi phi cao. TU nhOng nh*
didu tri nhifm triing do vi khuin. Tuy nhien, dilm tren cho thfy cfin tim kifm cfic chf pbir vigc su dijng khfing sinh khdng diing each dfi phd'i hdp hofic thay t h f khfing sinh trong die dfin ddn tmh trang khfing khfing sinh vfi lfim tri. Khang sinh ngudn gfib thUc vfit lfi mfttKft p a m , tham ehi mfi't hieu qua difu tri eua chiing. nhflng nhdm chf p h i m dap iing ydu eiu lu Ngoai ra. su dung khang sinh cdn gay ra cac tac (Wisatre, 2005; Hemalatha et al, 2016). >
dgng khfing mong muon nhu qua min, suy giam dung thao dUdc lfi mfit p h i n trong nfn y to mifin dich, ton du trong san p h i m ddng vfit gay Viet Nam, vfla mang tinh tu phat vfi d^i chur.
lfl 11)
!6S-
l^/oerdenbag er a i . 2012). Vdi xu hudng chti _^gng ngudn nguyen Ueu dUdc Ueu Irong nUdc, da Wlii nhifu nghien cflu vf kha nfing khang khufn Ja nhflng cay thufib Viet Nam (Woerdenbag et i . 2012; Vu e t a i , 2015).
Vi thufib Td mgc la gd phdi khd ctia cay Gf ang hay cfiy Td mfic (Caesalpinia sappan L.;
inh 1), c6 nhifu d cac tinh phia Bfic (Dd Tft di, 2015). Sau khi thu hoach. phin gfi gific j|, -ang dugc got bd. Itfy phin gS dd hen trong. cUa
lfinh khiic va che ra thanh manh nhd, phdi ofic sfi'y khd (Duge d i & Viet Nam IV, 2009) Tfi
>6c vi nggt, binh. khdng dgc, tac dung van 3 j^inh tam. can vfi ti, tae dung hoat huyft. thfing j.mh boat lac, khU ii, chi thong, tfin phong hfia 2 uyet, dugc dung lfim thufib sfin da vfi cim mau l o n g cfic trudng hdp xufi't huyft tit cung, mat -:iau sau sinh, choang vang, hoa mfit. Theo 'bdng Ddng y thtic nghifm, Vien Vi trung Vift
^ am (trich theo D5 Tfi't Ldi, 2015), nudc sfie Td IOC cd tfic dung khang vdi vi triing taphylocoecus 209P (vdng vfi khuin 1,2cm) almonella typhi (0,4 cm), Shiga flexneri (0,7 n), Shigella sonnei (0,2 cm). Shigella
•vsenteria shiga (1 cm) vfi BaciVus subtUis (1 n). Tac dung khfing sinh cua Tfi mgc lai khfing ifl't di dudi tac dung cua dieh vi vfi dich tuy dn nay, chUa cfi nhifu kft qua nghien cflu vf .'c dung ciia Tfi mfic trong thu y. Nghien cflu ay nhfim muc dich danh gia hifu qua cua ihuong phap ngam kift de chift xufi't cao Tfi
«fc vfi d ng dung dich cao Tfi mfic phdng vfi tri Inh do E. coli tren gfi Ludng Phudng.
Nguyin Tfin £)at, Nguyin Bfi Tifp
2 VAT LEEU VA PHlTONG PHAP 2.1. Vat lieu
Zl.l. Vat Ueu cho chiet xuat To moc - Dugc Ueu to rode: 200 kg (tieu chuin Dugc Dien Viet Xam IV) vdi cac dac diem: thanh gg To mdc hinh tru hay nika tru trdn cd dudng kinh tiJf 3 - 12 cm, dfii khoang 10 cm, mau dd vfing den do nau, thUctag cd khe nflt dgc; mat cfit ngang hoi bdng. vdng tuoi thfiy ro ret vdi mfiu da cam. cfi the thiy mfiu nau tm. co cac Id mach go n h ^ df tach thanh tflng manh theo thd gfi.
tuy CO 16 ro, cfi the CO nhflng chd co mau nhat hay dfim hdn; chfi't gfi cflng. nang. khdng mui. vi hdi se (Hinh 2).
- Ethanol 96%
- Thift hi: Bdn ngam chiet; thiet hi chiet siia't. thiet bi CO cao vfi thu hdi dung mfii bfing noi cft 3 ldp chft Ueu Inox SusSlO.
2.1.2. Vat Ueu thUc hien khang sinh do - Cao chiet Tfi mdc thanh phim.
- E. coh ginng gdb (E. cob .\TCC 25922) do cdng t> TNHH TM - SX \ u Pha cung cfi'p.
- Vi k h u i n E. coli phan Ifip tfl gfi mfic benh do £. tx,li dugc Trudng dai hgc Ndng U m thfinh phg Ho Chi Minh cimg cfp.
- Mfii trudng thue hien khang smh dd: .\IHA (Mueller Hmton Agar) do TITAN BIOTECH LTD san rufit.
- Dia gifiy t i m khang sinh (Cdng ty TNHH Nam Khoa cung cfi'p) gfm: AmoxidlUn (\x) Ciprofloxacin (Ci), CoUstin (Co), DoxycyUne (Dx)
Hinh 1. La va q u a To moc
1369
l i y u u n y iiiwi euBan^VJ,,
Bfinh gifi hifiu suft chift vfi tfic dung ciJa cao chift tU gS tfi m f c (Caes^pinia sappan L.) trong dung mfii ^anolvdi vi khuan EschericNa coH
- Dia giay t i m dung dich cao Tfi mgc d cac ndng do khfic nhau (sau khi cfi san phfim chiet) cfi dudng kinh bfing dudng kinh cua cfic dia khfing sinh.
2.1.3. Ga thi nghiem
Gfi Ludng PhUdng 1 - 3 7 ngfiy tudi do trai chfin nufii An Do cung cap. Gfi dUde nufii bfing thflc fin tu trgn. khong chfla khang sinh, dUdc chiing ngiia benh Newcastle va viem phe quan truyen nhiim (IB) vfio 7 vfi 14 ngay tuoi: phdng Gumboro vfio 10 vfi 21 ngfiy tufii.
2.2. Phi^clng p h a p nghien cufu
2.2.1. Chiet ngam kiet va bao che cao To moc Mdi me dUde Ufu Tfi mgc (20 kg) dugc ngfim trong 80 kg ethanol 96% (tUdng dUdng 101.4 lit) trong 72 gid d nhift do 70°C, sau dfi tach dich phfin chiet (budc nfiy dUdc Ifip lai 6 lin). Gdp tfi't ca dich chift cua 6 lin dem chung cat thu hdi dung mdi d nhiet do 80 - SOfC thu ducjc cao chift Td mgc. Trong thi nghifm nfiy, 200 kg Td mgc dUdc ngfim ehie't vfi chung cfi't sau 10 me. Cfi dfic dich chiet cua 10 me se thu duge cao Tfi mfic khfi tofin phin. Hofin tfi't san phim cao Tfi mgc khfi tofin phin thfinh dang dung dich 10% cao chift theo cfing thflc phfii hdp (Bang 1) vdi cac budc:
(i) Cho 500 g propylene glycol vfio 400g nude cat;
(ii) Gia nhift hdn hdp dung mdi dfn 80°C; (iii) Hda tan lUdng cao ehie't vfio h5n htjp dung mfii cho den khi tan hft; (iv) Lam mat dung dich ve nhift dfi phdng; (v) Bd sung lugng propylene glycol den 1.000 ml vfi (vi) Dfing chai HDPE efi dung tieh 500 ml.
Bang 1. Cong thiifc bao che cao To moc 10%
Ttiinh ph4n Luvng
Cao chiit T6 m$c toin phin Propylen glycol Nin>ccit Propylen glycol
105 gam 500 gam 400 gam
VCra du. ain 1.000 ml
2.2.2. Danh gia tac dung ciia cao chiet xuat To moc vdi E. coli
D ng phUdng phap khufch tfin tren dia
MHA), theo hudng dfin cua CLSI (CUnfcal and Laboratory Standards Institute, 2008). Dia giav cd dfldng kinh 6 mm dfldc h i p tift trflng (121*0 trong 15 ph t) vfi sfi'y khd. Sau dd, dia giS'y iiuc tfim dung dich cao chiet d hai hfim lUdng (i) .i I dung dich cao chift tfldng dfldng vdi 5 g dugc Ueu va (ii) 10 1 dung dich cao chift tUdng dfldng vdi 10 g dUdc Ufu (sik dung micropipet d l hut dich chiet da pha loang d ndng dd thich hdp nhd tren dia gify). Vdng vd khuin do dung dich cao chiet dUdc so sanh vdi vdng vd khuiin ciia cac khang sinh Amoxicillin (10 g).
Ciprofloxacin (5 g), CoUstin (10 g) va Doxycydine (30 g).
2JZ.3. Danh gia hieu qua phong va trj b^nh cia che pham cao To mgc 10% trSn git
Ga thi nghiem nudi dfh 28 ngfiy tuoi si phan Id thi nghiem. Ga Id thi nghifm cho udiig dich ehie't tfi mgc d ngfiy thfl 28, sau dd dfn ng4y 30 cho ca 3 Id ufi'ng dung dich vi khuin E. cdi gay dung huyft phan lap tfl gfi bfnh vdi Ulu 3 ml/con. Moi 1 ml dung dich vi khuin E. coh chfla khoang 1 - 3x10' tf hfio vi khufi'n (chpn 3 khuin lac cd kich thudc ddng deu tren dia NA cho vfio dng nghifm vd triing cd chfla 3 ml nd*:
sinh 1 vd tr ng, trdn bfing vortex. Sau dd do dJ due vdi d'ng Mc Farland 0,5 trdn ndn td giJfy trfing cd vach den vfi dieu chinh vdi nUdc muoi sinh 1^). Svt dung syringe Sml vd trflng bdm 3ml dung dich chfla vi k h u i n E. coli vfio v ng sau h i u tCtng con gfi. Theo doi ty le bfnh, ty le chft ciia gfi sau 7 ngfiy gay bfnh; md kham va phin Ifip vi khufi'n E. coli nhiing gfi chet.
Bo tri thi nghiem: Bfin lfi gfi 28 ngfiy tufi (10 con/18) bao gdm: Ld TNl ufng chf phim To mdc 10% vdi Ufu 1 ml/lOkg/ngfiy, bfit diu tii ngay 28 cho dfn ngfiy 30 (hda tan trong nUdc ufi'ng, vdi Ifldng nfldc uong vfla dii ufnp trong hai tieng doi vdi gfi thi nghifm). Sau 2 gid. cho uong vi khuin E. coh vdi lieu 3 ml/con. Lo TN2 uong lieu 3 ml vi khuin E. coli/con; cho udng chf phim cao Td mdc 10% vdi Ueu 1 ml/lOkg 6 gid sau khi gay nhilm. Ld chflng dfldng cho uong vi khuin E. coli vdi Ufu 3 ml/eon (uong 1 lin duy nhfi't). Ld chflng am khfing cho uong "
khuin E. coli hay dich cao Tfi mfc 10"!.. Thi
Nguyin T i n B a t Nguyen Bfi Tifp
•4,4;
iliiiil^
*8ii!i
Tudlga (ng^y)
J B a ^ 2 . Bo t r i p h o n g v a t r i b e n h bfing e h e p h ^ To m o c 10% t r e n ga
Can thi^p
30 30-37
T6 mac 10% (1 ml/IOkg)
Ufing vi khuSn £ CO* (1 - 3x10" t« bao/ml) Old phfim To m6c 10% (i ml/IOkg)
L6TN1 (n = 10)
L6TN2
(n =10) €>^' Chung duong (n = 10) Co
Co Khdng
£)fii chi/ng am (n = 10) Ktidng
Co Co
Khong Co Khong
Khong Khdng Khong
voij^
nioi
iM{i
12.2.4. P h a n tich so lieu
Sf Ufu dugc phfin tieh hfing phin mem GraphPad Prism 5.0 San Diego CA. Sai khac cd y nghia dfldc xac dinh d mflc tin cfiy > 95%.
3. K^T QUA VA THAO LUAN
nm^'^' ^^* """^ *=*''^* * " » t cao dUdc Ueu To mgc (cao toan p h a n )
^.3.1.1. Hieu suSi chiet xuat difOc liSu iBii ^ D l x c l n g san phfi'm ciia 10 lin chift xufi't i,ll|tu du<?c lifu Td mdc (20 kg dfldc Ufu/lin) dUdc ifelitrinh bfiy qua bang 3.
n Bj Khdi lugng cao chift thu dudc thfi'p nhfi't lfi nlkl,23 kg (lin chift thfl 8), cao nhfi't d lin chift 2 ii!(l,47 kg) vdi hifu sufi't chift tfldng flng lfi iii 6,40% vfi 7,35%. Khdng ed bifn ddng ldn cua a.whieu sufi't chift. Nhfl vfiy, qui trinh chift xuft riifcd tfnh dn dinh.
!"»• Tfng khdi ludng cao ehie't thu dflgc qua 10
«.lfin chiet xufi't lfi 13,5 kg. Vfiy, hieu sufi't chift
trung binh d^t dfldc 6,8%. Ke't qua nfiy thfi'p hdn chi sfi'tfii thie'u (khdng dudi 8,5% tinh theo dfldc Ufu khd kiet theo qui dinh cua Dudc dien Viet Nam IV, 2009). Hifu sufi't chift thfp hdn so vdi tieu chuin cfi t h i do dfldc Ueu Tfi mdc khdng dat tinh khd kift Day lfi dilm cin chu y trong cac lin chift xufi't tifp theo nhfim nang cao hieu sufi't chift vfi giam chi phi san xufi't.
Dfic dilm cam quan cua san phfi'm (cao khd tofin phin): Mfiu nau do anh vfing, de lau ngofii khdng khi khi cdn ndng se chuyin sang mfiu nau dfim. Cao ndng cd tinh deo, de ngudi kft rfin cfi t h i tan bfit (Hinh 3). Dung djch cao 10% vdi nude cfi't, lgc nfing d 80°C cfi chi so cfin khfing hfia tan <0,l°o.
3.1.2. San xuat che pham cao To mpc 10%
San phim thfl nghifm dudc bao chf dang dung dich chfla 10% cao chiet. San phfi'm dfl^c dfing gdi bao quan trong chai nhUa chfi't lieu HDPE vdi dung tich 500 ml. Chf phfi'm dat dfldc tinh on dinh theo cfing thflc bfio chf. Chf phfi'm B i n g 3. Ket qua chift dfltfc lifu To moc tdi cao toan p h i n
i-^nchi4t Khil lugng dl^,c ligu (kg) Khfii i i « n n . , . .h";;-:^::^;:;^
20,0 20.0 20.0 20.0 20.0 20,0 20,0 20,0
3 20,0 '0 20.0 Tfing khfii luvng cao tofin phin thu difgc (kg) Higu suit ctiiil (%)
Higu suit chigt (%) 1,34
1,47 1,31 1,31 1,26 1,42 1,30 1,28 1 38 1,43
13.5 J 0.7 6.75 ± 0.36
6,70 7,35 6,55 6,55 6,30 7,10 6,50 6,40 6,90 7,15
1371
^
€)finh gifi hifiu suat chift vfi tfic dung cua cao chift tCr gfi tfi mfic {Caesalpinm sappan L.) nong oung moi etnanoi « i VI khuan Escherichia cdi
nfiy dfldc sfl dung nghien ciiu kha nfing phdng va tri benh do \-i khuin E. coh tren gfi thi nghiem (Hlnh 4 vfi 5).
3.2. Ket qua k h a n g sinh do cua cao chiet Td mgc doi vdii vi k h u a n £, coli
3.2.1. Tinh khang khuan cua cao chiet va 4 loai khang sinh vdi E. coU chuan (ATCC 25922)
Ke't qua danh gia vdng vd khuan ciia chd' phim va mot so khang sinh vdi E. coh ATCC 25922 (Bang 4) cho thfi'y dUdng kinh vdng vd khuin nhd nhfi't vdi cao chift ndng dd 5 g/dia dat 10 mm; vdi cao chiet ndng dd 10 g/dia lfi 12 mm. Dudng kinh vdng vfi khuin trung binh cua Tfi mgc ndng do 5 g/dIa lfi 10,02 mm; vdi Td mgc ndng dd 10 g/<Ea lfi 12,02 mm. Dudng kinh vdng cd khuin cua Td mgc tha'p hdn 3 khang sinh: Amoxicillin, Ciprofloxacin vfi Doxycydine.
Mgt dieu thu vi lfi dfldng kinh vdng vd khuin eua chat chift Td mgc 10 g/(Ba cfi gifi tri tUdng dudng vdi gifi tri nfiy cua khfing sinh Colistin.
So sfinh vdi chi sd' khfing khuin tieu chuan (CLSI, 2008) thi kha nfing khang khufin cua ca bfi'n lo^i khfing sinh deu cfi tfic dung dift khuin tren vi khuan E. coii chufin (ATCC 25922).
3.2.2. Kha ning kbang khuan cUa cao ehie't va 4 lo^i khiing sinh v&i vi khuan E. coli phan lap tifga Li/dng PbiiOng bi benh
Cac chung vi k h u i n chuin thfldng du^t dflng d l danh gia tac diing khang khuin ctii khang sinh, dac biet lfi khang sinh thfl n g h i ^ Tuy nhien, khong phai khang sinh cd tac dung tfi't tren chung vi khufin chufin deu cfi tac dtjuv dfii vdi vi k h u i n gay benh tren ddng vfit. Chlnh vi vfiy, tac dung cua cao chift Td mdc vdi \i k h u i n E. coli gay benh tren gfi dfldc xfic dinh cung vdi mdt so'khang sinh khac (Bang 5).
Ke't qua khang sinh do bang 5 cho thfy vi k h u i n E. coli phan Ifip tfl ga bfnh da df khaag vdi AmoxicilUn va Colistin (dfldng kinh vdng vo khuin thfi'p nhfi't cua Amoxicillin lin lUdt Ifi 13 mm va 10 mm. Vi khufin nhay cam cao vSi Ciprofloxacin (vdi dudng kinh vdng vd khuin thfi'p nhfi't lfi 23 mm) va vdi Doxycydine (dit^
kinh vdng vo khuin thfi'p nhfi't lfi 11 mm). Kha nfing de khang thuoc nay cd the do tinh tr?ng si dung khang sinh trong dieu tri khfing diing li|u trinh, thdi gian s i dung khang sinh qua ngjn, cung nhu vifc bfi' sung khang sinh vdi lieu thip thfldng xuyen trong thflc fin nhfim mgc didi phdng bfnh da tao ra cac chung vi khuin cfi siic
de khang cao. M Cung trong thi nghifm nfiy, dfldng kfnh
vdng vd khuin d hai hfim Ifldng cao chift To rape chi dat 7,04 mm vdi hfim Ifldng 5 g vfi d?t miic 8,02 vdi hfim Ifldng 10 g. Nhfl vfiy, cd thi cho rfing tac dung khang sinh cua cao chift to in9C thfi'p hdn cila cac loai khang sinh kilm tra.
Hinh 3. Cao chiet To moc t r o n g hgp thiiy t i n h
Hinh 4. Cao chiet To mgc t h a n h p h a m t r o n g chai
nhtfa HDPE 500 ml
Hinh 5. Dich To mgc W' d u n g cho thi nghifm
-'X
Umg in\ ilAil vdi If III
Hm
Nguyin Tfn Ogt, Nguyfn Ba Tifp
B a n g 4. Dlfldlng kinh vong vo khuan ciia cao chift To moc va mot so khang sinh vdi E. coU ATCC 25922 (dan vi: mm)
U n thi>
1 2 3 4 5 6 7 8 9 10 Tnjng binh
TdmScSpg 10.0 10.0 10.0 10,0 10,0 10,0 10,0 10,0 10,2 10,0 10,02 ± 0,06
T6 mgc 10 pg 12.0 12,0 12.0 12,0 12,0 12,2 12,0 12,0 12,0 12,0 12,02 ± 0,06
Amoxidfiin 20,0 21,0 20.0 20.0 20,4 20.0 20,0 20,2 20,0 20.0 20,16 ± 0,32
Ciprofloxacin 40,0 40,0 40,0 40.5 40,0 40.2 40,0 40.0 40.2 40.2 40.11 ±0.17
Colistin 12.0 12,0 12,0 12,2 12.0 12.0 12.0 12,0 12.0 12,0 12,02 ± 0,06
Doxycydine 25,0 25,0 25,0 25,0 25,0 25,0 25,0 25,0 25,0 25,0 25,0 ± 0,00
Theo hilu bift eua chung tfii, chfi't chift xuft tfl cay Td mdc chfla dfldc sil dung rfing rai ' ^ ^ ^ d i f u tri nhilm khuin ma chi nghidn cflu trong phdng thi nghifm nen ve ly thuyft dudng I kinh trung binh vdng vd khuin cua chfi't chiet tfl Td mge trdn vi k h u i n E. cob chuin ATCC 25922 vfi vi khuin E cob dugc phan Ifip tfl ga bfnh se khdng khfic nhau vi vi khuin chua khang vdi cac chft chfla trong Td mdc. Tuy nhien, ke't qua (Bang 6) cho thfiy dfldng kinh trung binh vdng vd khufi'n cua chfi't chift tren vi khuin E cob chuin ATCC 25922 vfi vi khuin E cob dudc
phfin lap tfl gfi bfnh cd sfl khac biet. Nhu vfiy, Sfl khac nhau vf cfic dfic tinh sinh hgc khac gifla Chung E. cofi chuin vfi chiing gay benh thilc dia cd the d i n dfn sU khae nhau vf tinh mfin cam cua vi khuin vdi cfic khfing sinh, cac chfi't chifi't vfi cfic hfia chfi't. Han chf ctia nghien cflu nay lfi chfla xac dinh dfldc ndng do fle che tfii thifi'u ciia chfit chift Td mfic dfn vi khuin E. cob.
Su khfic nhau gifla dfldng ki'nh vdng vd khuin cua cao chift Td moc va cae loai khang sinh kiem tra dfii vdi chung E. cob gay benh vfi chung E. cob chuin duge tfim tfit d bang 6.
Bang 5. DuTctag kinh vong vo k h u i n ciia cao chiet To moc
Lin thi>
1 2 3 4 S 6 7 8 9 10 Tmng binh
TfimOc 5|Jg 7.20 7.00 7.20 7.00 7.00 7.00 7.00 7.00 7.00 7.00 7.04 ± 0.08
TdmOc 10 Mg 8.00 8,00 8.00 8.00 8,00 8,00 8,20 8,00 8.00 8.00 8.02 ± 0,06
Amoxicillin 13,00 13.00 13,50 13,00 13,00 13,20 13,M 13.00 13,00 13,00 13,07 ±0.16
Ciprofloxacin 23,00 23,40 23,00 23,20 23,00 23,00 23.00 23.00 23,00 23,00 23.06 ±0.13
Colistin 10.00 10,00 10,20 10.00 10.00 10.00 10.00 10.00 10.00 10.00 10.02 ± 0,06
11)
Doxycydine 11,00 11,00 11,00 11,00 11,00 11,00 11,00 11,20 11,00 11.00 11,02 ±0,06
1373
Ofinh dfi hifu suft tiiift vfi tac dgng cua cao chift tfl g f tfi m f c (Caesaifiniia aaijfjan i../ a\mg aung moi emanai vM vi IdiuTn Escherichia coH
Bang 6. DUdng k i n h t r u n g binh vong vo k h u a n c u a cao c h i ^ t To moc vtk k h a n g sinh vdi vi k h u a n E. coli (ddn vi: mm)
(^fit chifitMifing sinh E CO* chufin (ATCC 25922) E. coli phfin Ifip tir gfi b$nh Td m4c 5 pg
Td mOc 10 pg Amoxicillin Ciprofloxacin Colistin Doxycyline
10.02 ± 0.06 12.02 ±0.06 20,16 ±0.32 40,11 ±0,17 12.02 ± 0.06 25,00 ± 0,00
7,04 ± 0,08 8,02 ± 0,06 13,07 ± 0,16 23,06 ±0.13 10.02 ± 0.06 11.02 ±0.06
Theo Srinivasan et aL (2012), ham lUdng 5 ng cao chiet trong dung mfii ethanol. Tfi mgc eho dfldng kinh vdng vd khuin 6.0 = 0,3 mm den 15,0
± 1,4 mm. Kft qua danh gia cua chiing tdi ciing dat mflc khang khuin trong thi nghifm cua Senthilkumar et aL (2011) vdi vdng vd khuin lfi 6 mm d hfim Ifldng cao chift 50 ppm vfi 8 mm d hfim Iflgng cao chift 100 ppm (dung mdi chift methanol). Cd the cho thfy ring khdng cd sfl khfic nhau vf ho^t tfnh khang E. cob cua chft chiet Td mdc bftng cfic dung mfii ethanol vfi methanol.
Nghien cflu ciia Srinivasan et aL (2012) eung cho rftng chfi't ehie't Tfi mdc cd ho^t tfnh khfing sinh manh hdn tren cac vi khuin gram dUdng nhu Streptixoecus feacabs vfi Staphylococcus aureus so vdi tac dgng tren vi khuin gram fim nhu E. cob, SalmoneUa typhi
Trong thf nghifm nfiy, dfldng kfnh vdng vd khufin cua ca 4 loai khfing sinh kiem tra tren chung vi khuin gfiy bfnh deu thfi'p hdn so vdi chung vi khuin chuin. Oidnh Ifch cao nhfi't doi vdi Doxycydine (tfl 25 mm vdi chiing E. cob chuin so vdi 11,02 mm vdi chiing E. cob gay bfnh); tifp theo lfi Ciprofloxacin, AmoxicilUn vfi Colistin. Nhu vfy, de danh gia kha nfing khang
k h u i n cua mgt chfi't hay nhdm chfi't cd nguin gfib thflc vft nhfl cao chiet Td mdc, dfldng kinh vd khuin cung chi dfldc xem nhfl mot ti&i chuin xac dinh.
3.3. Tac diing c u a c h e p h a m cao To moc 10% t r e n ga gay n h i l m E. coli thflc nghifm D l danh gia tfnh an tofin cua chf phjm trUdc thf nghiem, cho 3 con gfi 28 ngfiy tuS ud'ng chd' phim cao Td mdc 10% vdi li& 1 ml/lOkg t h i trgng vfi theo doi gfi sau 7 ngfiy. TU nghifm chinh thflc dfldc thiie hifn khi ca 3 (i nfiy dfu khde manh. Kft qua 3 lin Ifp l?i thi nghifm dfldc trinh bfiy d bang 7.
Sau 24 gid gay bfnh, gfi cd bilu hifn u rii, xfl ldng, bd fin. Gfi d i u tidn chet sau 72 gii Bfnh tfch chu yfu lfi xoang bao tim tfch djch vfing, mfing bao tim dfiy due hofc vidm ming bao tim. Sau khi kft thuc thf nghifm, ty 1$ gi bfnh cua cfic 16 TNI, TN2, ddi chflng dfldng ciii ca 3 d(?t thf nghifm lin Iflijt lfi 23,33%; 23,33%
vfi 73,33%. Sai cd ^ ngUa gifla ti If mftc bfnh cua Id TNl vfi Id TN2 so vdi hai 16 dfi chflng dfldng vfi dfii chflng fim dfldc xfic djnh (P < 0,01).
Bang 7. Ket qua theo doi gfi thi n g h i f m sau 7 ngay gay n h i l m
OgtTN SfigfiHA
/dgi (con) SAgibSnh Tyl$(%) ^figfi benh
Bfii Chung dif ong od chi>ng 4m I T:>l«(%) Sfi gfi
b$nh T!>l«(%) Sfi gfi b$nh Tyi«(%) 1
2 3 Tfing
10 10 10 30
4 2 1 7
40 20 10 23,33
4 1 2 7
40 10 20 23,33
8 7 7 22
80 70 70 73,33
0 0 0 0
0 1
0
0 f
00,00
itTo ttic Nguyin Tfn Oat Nguyfn Bfi Tifp Bang 8. Ty le ga chet sau 7 ngay gay benh
Dtfl TN
_LaTN1(n=10)x3 Gfi chit
L a T N 2 ( n = 1 0 ) x 3
1 2 3 Tfing
4 (40%) 1(10%
1(10%
6 (20%)
6 d chfit do E. CO*
3 (30%) 1 (10%) 1 (10%) 5 (16,66%)
Ga chfit Ga chfit do
£ c o «
Chung dutmg (n = 10) x 3 Ga chfil
tairnig am (n = 10) X 3 Ga chfit do
E.COS Ga chfit
3(30%) 1 (10%) 2 (20%) 6 (20%)
Gfi chfit do £ CO*
3 (30%) 1(10%) 10.00
2(20%) 6 (20%) Ghi chii: Cbtt aff trong mii 6 chl sfgi cbgt (con): ty le chgt
6(60%) 4(40%) 4 (40%) 14 (46.66%)
6(60%) 4(40%) 4 (40%) 14 (46,66%) duac ghi trong 0
0(0%) 0(0%) 0(0%) 0(0%) 0 (0%) 0 (0%) 0(0%) 0(0%)
itTo«i5t ^^ * ^ " ^ ^ "^t --ang cho ufng ehe phfi'm c im'i- ';" ^^ " ^ c 10% trudc vfi sau gfiy nhilm E. cob
deu cfi tae dung vdi vi khuin nfiy. Tac dvmg phfing bfnh cua chf phim vdi Ueu" 1 phlfflB,:"!'^l°''e'"8^y C6 1) tUdng dUdng tac dung tri E.MMt,,''!"'' f ^^- ^ ^ " '' °S^y gSy nhilm, md' kham vfi I ; ' ^ " • ^ S ^ - "'"' ">• e^ -"^e bfnh CO trieu chflng
7m r '^"^' ""^"^ '''^ *^" ^'"^ ^^ i^^^t <!"« PMn
'^"^phfin lap dfldng tinh vdi E cob (Bang 8). Sau 7
"8 ngfiy thf nghifm, ty le gfi chft ca 3 ddt cua 16 TNl ga CO ftivfi TN2 lfi 20%. T^ If gfi chft ca 3 d^t cua Id chflng ftdSaidfldng lfi 46,66%, cao hdn ti If chet ciia cac Id gfi ng ta liiidfl^fc udng chf phim Td mfc 10% (P < 0,05).
km Kit qua md- khfim gfi chft vfi phan Ifip vi
« # k h u f t n E cob Bfnh tfch ehu yfi'u lfi xoang bao tim jMKttfch djch vfing, vifim bao tim. Gfi chft do E cobd timid TNl cua 3 d^lt thf nghifm chifm ty 16 thfp nhfi't gia.fili«(16,66%) vfi ty If cao nhfi't d Id chflng dfldng ,rtlil(46.66?4) (P < 0,05). Qua dd cd t h i nhfn xfit rfing
;sj(iifchfi phfi'm cao Td mfc 10% cfi tac dymg lfim giam Uia nfing xfim nhfp vfi gfiy bfnh cua E. coh.
{B Ho^t tfnh sinh hgc cua chfi't chift Td mfc da
—^lUiJc mgt sfiT tfic gia chflng minh nhu chd'ng oxi
^ 6 a (Badami et al., 2003); khfing mft so' tfic , » hhfin kfch thfch phfit trie'n khfii u (Benabadji et
^^-^1-. 2004): khfing khufi'n (Xu et al., 2004;
• Saravanakuma et al., 2013); bao ve vfi tftng 1 --Udng chfle nftng gan (Sathya Srilakshmi et al.,
> 2010) vfi tfic dfng dfin bd' t h i (Sarumathy et aL, I iOl 1). Tuy nhidn, nghidn cflu nfiy lin diu tidi^
xac dinh kha nfing phdng va tri bfnh do E. cob tren ga thi nghifm.
Hifn nay ehan nuoi "xanh", han chf khang sinh trong phdng benh vfi tri bfnh cho vfit nudi dang ngfiy dUdc khuye'n khich vi nhflng mfit trai eua su dimg khang sinh. Van di nfiy dfldc dfic bift quan tam d cac nfldc dang phat triln, ndi khang sinh da vfi dang bi lam diing vi lgi nhufin trflde mfit cua mgt so' nha san xufi't. Sfl dung cac chfi't cd ngudn gob thflc vfit lfi mdt trong'nhflng bifn phap hflu hifu d l ban chf nhflng tac ddng khong tot cua tinh trang nfiy. Sfl dung cac chfi't cfi ngufin gfib thuc vfit trong dd cfi cay Tfi moc nhfim phdng vfi tri benh lfi mdt trong cac bien phfip dfi. Nhflng kft qua nfiy mdi sfing loc, dinh tinh, chua xae dinh dugc ndng df fle chf tdi thieu vfi kha nfing kft hgp vdi khfing sinh. Kft qua nghien cflu nfiy cd t h i coi la cd sd cho cfic nghien eflu tifp theo nhfim tao ra cac chf phim tfl To mgc sfl diing phdng vfi difu tri bfnh do E coh nfii Chung vfi mgt so' bfnh do vi khuin khac trong chfin nufii. Thi nghiem tiep theo danh gia hifu qua kmh tf cua phUdng phfip dung cao chiet To mfic trong phdng vfi difu tri bdnh do E cob tren gfi cung cin dUde thuc hifn.
4. KET LUAN
Qui trinh chift xufi't dugc lieu Td mdc vdi dung mfii ethanol 96% sau dfi tifn hfinh cft thu hfii dung mfii d nhiet do 80 - 90°C. Hieu sufi't chiet xufi't dat trung hinh 6,75%.
Cao chift Td mdc cfi tac dung tren vi khuin E. cob. Hoat tinh khfing k h u i n phu thudc vfio ham lugng eao chift vfi chung vi khuin.' Hoat
1375
• M
Dfinh afi hifu suft chift vfi tfic dgng oia cao chi^ tu gd tfi mfc (Caesalpinia sappan L.) trong dung mfii ethanol vd, vi l(huln Escherichia cot
tfnh khfing k h u i n cua cao Td mfc tr&i chung E.
cob c h u i n cao hdn tren chung E. cob phfin Ifip tfl ga bfnh. Su sai khac nay cung tfldng tfl ddi vdi cac khfing sinh Doxycychne. Ciprofloxacin vfi OiUstin.
Chf p h i m cao Td mgc 10% eo tfic dung phdng vfi tri bfnh do E. cob gay nhiem tren ga vdi lieu gfiy n h i l m 3 ml/eon.
Ke't qua nfiy la cd sd cho cae nghien cflu phan tfch danh gifi kha nfing khang k h u i n cua cao chiet Tfi mfic khi dflng ket hgp vdi mgt so dugc Ufu khfic vfi danh gifi tfic dung phdng tri bfnh do E. cob ciia cao chift Td mge.
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