Danh gia doc tinh cap va tac dung khang Ithuan cua bai thuoc
'Thong xoang HV" tren thuc nghiem
EVALUATING THE EFFECT OF ACUTE TOXICITY ANDANTIBACTERIAN OF THONG XOANG HVRFMEDY OF EXPERIMENTAl ANIMALS
Phgm Dinh Qu^n C30 hoc 9 - Hoc vien Y Duoc hoc co truven Viet Nam
TOM TAT
Bdi thudc Thdng xoang HVgdm 5 vi (NgU tinh thdo, Kinh gidi tui, Td tU, La bac tH vd Ktm ngdn hoa) di diiu tri viem miii xoang man tinh (VMXMT). Nghiin ciiu dUdc th^Cc hiin nham ddnh gid dgc tinh cdp vd tdc dung khdng khudn cua bdt thudc.
Kit qud: Bdc tinh cdp: chtia xdc dinh dUdcLBSO cua bdi thudc mac dii chuot da udngdin liiu tdi da 37S g/
kg/24h. Khdng khudn: xdc dinh dU(?c ndng dd iic chi tdi thiiu cda Thdng xoang HV vdi Staphylococcus aureus la 1/16 (0,273 g/ml), vdi Streptococcus pneumonia vdPseudomonas aeruginosa Id 1/8 (0,546 g/ml).
Tii khda: Thong xoang HV, dde tinh cdp, khdng khudn.
ABSTRACT
Thang xoang HV was madeftom Houttuynia cordata Thumb, Schinozepeta tenuifolia Briq, Penlla ocymoides L, Raphanus sativus L and Lonicera japonica Thumb for treating chronic sinusitis. The study was on the acute toxicity on mice and antibacterian of this remedy.
Result: The acute toxicity not identify the LDSO on mice although they were taken the highest dose b 37S gj kgbody weight. Jheminimum inhibitory concentration of ThongxoangHV with Staphylococcus aureus is 1/16 (0,273 g/ml), both Streptococcus pneumonia and Pseudomonas aeruginosa are 1/8 (0,546 g/ml).
Key words: Thong xoang HV, acute toxicity, antimicrobial.
Ngay nhan bai 20/12/2018 Ngay phan bien 24/12/2018 Ngay chdp nhdn dong 24/12/2018
26 I TAP CHIY DUOC C 6 TRUYEN VIET NAM ^
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DAT VANDE
Viem mui xoang man tinh la mdt benh man tinh phd biln khdng ehi d Vilt Nam ma d Dfle, My ti ll mac binh cung khdng he thap (10 - 20%
trong cpng ddng dan cfl) [2]. Benh khdng trflc tiep de dpa din tinh mang nhflng inh hfldng din chit Ifldng sdng cua ngu'di binh.
Trong eae nguyen nhan giy VMXIVIT thi nhilm khuan li nguyin nhin quan trpng va khd giai quyet, can load trfl nguyen nhin nay dl giii quyet tan gdc VXMMT Vile dilu tri chu dich hfldng den vi khuan gay benh dac thu rieng cho tflng nhiem khuan la cin thilt [4]. Dfla trin cic nghien cflu trong va ngoai nflde, chflng tdi nhan thay mpt sd vi khuan cd ti le gap cao trong VMXMT va trong qui trinh dieu tri tren lam sang, chung tdi nhan thay bai thudc Thdng xoang HV cd tic dung cii thien cac trieu chflng khd chiu cfla benh nhan VMXMT. Bai thudc gdm 5 vi, chu ylu la thudc nam, dl sfl dung nhifng chifa ed nghiin eflu khoa hpe chflng minh.
Do do, chflng tdi bfldc diu tiln hanh nghien cflu thflc nghiim vdi muc tieu:
1. Dinh gii dde tinh cip cua bai thudc.
2. Danh gia tac dung khang khuan cua bii thudc tren mdt sd vi khuan hay gap trong VMXMT.
CHAT UEU, OOi VSm VA PHUONG PHAP NGHIEN Cifu
Dia di^m va thdi gian n ^ l n cflu Bd mdn Dfltfe ly - Trfldng Dai hpc Y Ha Ndi va Bd mdn Vi sinh Benh vien Nhiet ddi Trung flcfng.
Thdi gian tfl thing 10 din thing 12 nam 2018.
Chat lieu nghien cflu
Bai thudc Thdng xoang HV gdm 5 vi, cung cap bdi Khoa Difpc Benh viln Y hpc cd truyen Tmng flcfng, dat tieu chuan Dfldc dien Viet Nam IV:
Ngfl tinh thao (Houttuynia cordata Thumb) 35g Kinh gidi tue (Schinozepeta tenuifolia Briq) 25g
Td tfl (Perilla ocymoides L) lOg La bac tfl (Raphanus sativus L) ISg
¥jmngin.hoa.(Lonicerajaponica Thumb) 15g Mdi thang Thdng xoang HV dflcfc sic va cd dae edn 20 ml vdi nghiin cflu dpc tinh cap va 22,88 ml vdi nghiin cflu tic dung khang khuan.
Ddi tiifdng nghien cflu
Chuot nhat trang chung Swiss trpng lifpng 18- 22g do Vien Ve sinh dieh tl Trung flcfng cung cap vi 3 vi khuan chuin qudc te: Staphylococcus aureus, Streptococcus pneumonia vd Pseudomonas aeru^nosa.
Phfltfng phap nghien cflu Boc tinh cdp
Thfl dpc tinh cap va xac dinh LD50 cua thudc tren ehudt nhat trang theo phiftfng phap Litchfied- WUcoxon va hifdng din cfla WHO:
Tnfdc khi tiln hinh thi nghiem, cho chuot nhin an qua dim.
Chia ehudt thanh cac Id khac nhau, mdi Id 10 con. Cho ehudt udng cao Idng Thong xoang HVvdi lieu tang dan (30 ml/kg den 7SmI/kg) trong cung mdt the tich dl xac dinh hlu thip nhat giy chet 100% ehudt va lieu cao nhat khdng gay chet ehudt (gay chet 0% chuot).
Theo ddi tinh trang chung cua ehudt, qua trinh diln bien ed dau hieu nhiem dpc (nhfl ndn, co giat, kich ddng...) vi sd lifpng ehudt ehlt sau 72 gid sau khi udng thude. Tat ca ehudt chet diftfc md de danh gia tinh trang tdn thUtfng dai the. Tfl do xay difng md hinh tuyen tinh de xac dinh LDSO cua thudc thfl. Sau dd tilp tuc theo ddi tinh trang cua ehudt den ngay thfl 7 sau khi udng thudc [l],[6].
Khdngkhud'n Nguyin tac
Pha loang che pham thudc thfl d 5 ndng dp khac nhau: 1/2,1/4,1/8, l/16va 1/32 trong mdi tnfdng thach. Ciy cic ehung vi khuan thfl nghiim vao cic dilm khac nhau trin dia thach. De 37°C/ 24
TAP CHfY DUOC C 6 TRUYfN VI^T NAM 27
gid. Dpc ket qui ndng dp fle chi tdi thieu cua thudc (gia tri MIC: Mimmal inhibitory concentration) ddi vdi cic chung vi khuan thfl nghiem.
Ky thudt tiin hdnh:
Nudi ciy, phan lip, dinh danh vi khuan tfl miu thfl:
- Dung que cay lay 1 lfltfng dung dieh cin thfl eay lln mdi tnfdng thach miu, uri select. U d 37''C/18-24h.
- Dinh danh vi khuan neu moc. Chi thflc hiln eac bifdc tilp theo neu khdng mpc trong sd nhiing chflng thfl nghiim.
Xie dinh boat tinh khang khuan, khang nam:
-Dung pipet bflt 1 ml dung dieh thfl nghiem vao dng tube vd trung.
- Pha loangbac 2 cic dung dich thfl nghiem vao cac dng tube (l, 2, 4, 8, 16...) bang nifdc mudi sinh ly vd trung, dinh dau tflng dng tube vdi mdi dp pha loang.
-1 dng chflng la dng chi cd nfldc mudi sinh ly.
- Chuan bi huyen dieh:
Chung vi khuan can thfl dfltfe nudi eay thuan, qua dim.
Dung que cay liy 3-5 khuan lae hda tan vao dng tube dflng nifdc mudi smh ly vd trung 0,9 %, dilu chinh de dat huyen dich cd nong dp 0,5 Mac Farland (tiftfngdfltfng 108 CPU/lml).
Trong vdng 15 phut sau, tiep tuc pha loang huyen dich trin bang canh thang Muller-Hmton vd trflng(MHB) (ddi vdi vi khuan dempc)hoac canh thang Muller-Hinton cd 2,5 -5 % mau cflu (ddi vdi S. pneumoniae va S. agalactiae), de dat ndng dp 5x 105 CFU/lml (pha loang 1:100 dl dat ndng dp 106 CFU/ Iml, sau do tiep tue pha loang 1:2 de dat ndng d p 5 x l 0 5 CFU/lml).
Huyen dich difpc pha loing tiep 1:100 sau dd 1:2 bang canh thang RPMI 1640 de d:it ndng dp 5x103-2,5x104 tibio/lml.
- Dung pipet hut 1ml huyen dieh sau khi pha
loang d bfldc cudi cung cho vao dng chflng, tilp den la tiing dng tube chfla dung dich thfl nghiem da pha loang tfl ndng dp thap din ndng dp cao.
iSThif vay, mdi dng se chfla 2 ml, ndng dp tie nhan can thfl va ndng dp vi khuan deu la 1:2.
- U cac dng tube d 35X/18-24 h.
- Sau thdi gian u am tren, dung que cay lay mgt liftfng dich & mdi tube cay vao tflng dia thach mau, danh diu cae ifia thach tfldng flng vdi tflng ndng dp pha loang. \J cac dia thach d 37°C/18-24 h, cd hoEic khdng cd CO^ tuy thudc vao ehung vi khuan can thfl.
- Kiem tra cic dia thach miu xem sfl mpc cua vi khuan: dat khi d dia chflng, vi khuan mpc tdt vi d tit ci cac dia vi khuin mpc thuin nhat, khi dd mdi tim ndng dp flc che tdi thieu: la ndng dp ma tiftfng flng tai dd vi khuan khdng mpc hoac mpc < 10 % so vdi dia chflng (bi flc che >90 %).
Trong nghien eflu vdi mdi chung vi khuan deu pha loang 5 nong dd: 1/2,1/4, 1/8,1/16 va 1/32 so vdi dung dich gdc. Mdi ndng dp it nhat n=5 cho mdi chflng vi khuan.
Nhin dinh kit qui: tim dif tfc ndng dp thip nhit cd tic dung flc ehe sfl phat trien cua vi khuan khi it nhat 4 dia vi khuan khdng mpc hay mpc it htfn 10%
so vdi dia chuiig [3],[5].
Phfldng phap xfl Iy so lieu
Cac sd Ueu thu thip dfltfe xfl ly bing phan mem Microsoft Exel 2013 vi cic test phu hcfp. Kit qui dfltfe trinh biy dfldi dang; X ± SD. Sfl khic bilt co y nghia thdng ke khi gia tri p < 0,05.
Quy ifdc vdi md hinh khang khuan, khang nim:
Vi khuan khdng mpc hoac moc it hdnlO% so vdi dia chflng:
Vi khuan moc tren 10% so vdi cRa chflng:
I -
Vi khuan mpc khdng du tilu chuan so sinh:
dl trdng
28 I TAP CHI Y DUDC C 6 T R U Y E N V I E T NAM ^
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KET QUA NGHllN CDU Doc tinh cap
Bdng 1 Kit qud nghien cdu dgc tinh cdp
LochuQt
L61 L62 L63 L64
"
10 10 10 10
Li^u (ml/kg)
30 45 60 75
Li^u (gdU<?c lieu/kg)
150 225 300 375
Tyl4cha(%)
0 0 0 0
Dau hieu bat thiidng khac Khong Khong Khong Khong Qua Bing 1 cho thiy; Khi ehudt udng cao long Thdng xoang HV& Ueu tii 30 ml/kg tflcfng difdng 150 gam dfldc lieu/kg den Ueu tdi da 75ml/kg tiftfng dUtfng 375g dUdc Ueu/kg khdng cd chuot nio chet hay cd bilu hiln bit thfldng.
Khang khu^n
Bdng 2. Xdc dinh tf li pha loang cda cao long Thdng xoang HV lin stXphdt triin ciia chiing vi khudn S. aureus
Ndng dp pha loang
1/2 1/4 1/8 1/16 1/32
S. aureus Lan 1
+
Lan 2
+
Lan 3
+
Lan4
+
*
LanS
+
Vdi chung vi khuan S. A wreus, eao Idng Thdng xoang HV flc chi sfl phat trien cfla vi khuan lan lUtft d eac ty le pha loang Vi, "4,1 /8 va 1 /16. Do do, xac dmh dfltfe ndng dp flc ehe tdi thieu MIC cua cao long Thdng xoang HV vdi chung vi khuan nay la 1/16 hay 0,273 g/ml.
Bdng 3. Xdc dinh ty le pha loang cda cao long Thdng xoang HV len sii phdt triin cua chung vi khudn S. pneumoniae
Ndng dd pha loang
1/2 1/4
S. pneumoniae
L ^ l Lan 2 Lan 3 Lan4 LfinS
TAP CHIY DUOC CO TBUYEN VIET NAM lil«l»MJ;li:ti!JilEI 1 29
1/8 1/16 1/32
+ +
+
*
+
*
+
+
*
Vdi chung vi khuin S. pneumoniae, eao long Thdng xoang HV flc che sfl phat trien cua vi khuan d 3 ndng dp pha loang 1/2, 1/4, 1/8. Vi vay, xac dinh diftfc ndng dp flc che tdi thieu MIC cua cao long Thdng xoang HV vdi chung vi khuan nay li 1/8 hay 0,546 g/ml.
Bdng4XdcdinhtyliphalodngciiacaoldngThdngxoangHVlinsiiphdttrtinciiachungvikhudnP.aeru^nosa
Ndng do pha loang
1/2 1/4 1/8 1/16 1/32
P. aeruginosa L ^ l
+ +
Lan 2
+ +
Lan3
+ +
L a n 4
+ +
LanS
+
*
Vdi chung vi khuan P.aeruginosa, cao Idng Thdng xoang HV flc che sfl phat trien cua vi khuan d 3 ty le pha loang 1/2, 1/4, 1/8. Vi vay, xie dinh difdc ndng dd flc cbl tdi thilu MIC cua cao Idng Thdng xoang HV vdi chung vi khuan nay 1 /8 hay"
0,548 g/ml.
BAN LUAN Dgc tinh cap
Khi cho ehudt nhat tring udng eao long Thdng xoang HV tii Ueu 150 g/kg den Ulu 375 g/kg. Ld chuot da udng din lilu 0,25 ml/1 Og, 3 lin trong 24 gid dung dich dam dac nhat, theo ddi ehudt trong cac Id, ehung tdi thiy cac Ueu cao Idng Thong xoang HVkhdng lam ehudt cd bilu hiln gi khic thfldng, ehudt khdng xuat hien trieu chflng bat thfldng nao trong 72 gid va trong sudt 7 ngay theo ddi sau udng
thude thfl. Lieu 375g/kg la hlu tdi da chung tdi ed thi dung difdc bang difdng udng.
So sanh vdi Ueu thfldng dung tren ngfldi la lOOg dfldc Ueu/ngifdi/ngay hay 2g dUtfc Ulu/kg/24h (tinh trpng lifdng trung binh ngfldi ldn trfldng thanh la 50kg) thi ehudt nhat trang da udng din Ulu gap 15,625 lan so vdi Ueu thudng dung tren ngfldi (da tinh he sd 12 trin chuot nhat trang) nhifng khdng xuat hien dpc tinh cap.
Khang khuan
PhUtfng phap xie dinh ndng dp flc cbl tdi thieu de danh gia tae dung khang khuan cua thudc la mdt phfltfng phap de sfl dung, the hien tinh khoa hpc va mfle tui esiy cao, phu hdp vdi mue dieh va ddi tifdng nghiin eflu li 3 chung vi khuan hay gap trong VMXMT. Chung tdi xie dmh dfltfe ndng dd flc che tdi thieu cua Thdng xoang HV vdi Tu cau
30 I TAP CHfY DUQC CO TRUYEN VIET NAM WR
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vang [Staphylococcus aureus) d ti ll pha loang 1/16 hay 0,273 g diftfe Ueu/ml, Phe cau (Streptococcus pneumonia) va Trflc khuin mu xanh (Pseudomonas aeruffnosa) la l/8hayO,546gdfltfcheu/ml.
KETLUAN Doc tinh cap
Cao long Thdng xoang HV khdng cd bieu hien dde tinh cap tren ehudt nhat tring khi udng din
heu 375 g dfltfe Ueu/1^, khdng xie dinh difdc LDjg cua Jhong xoang HV.
Tac dung khang khuan
Cao Idng Thong xoang HV the hien tac dung khing khuan tren 3 chung vi khuan Staphylococcus aureus. Streptococcus pneumonia, Pseudomonas aeruginosa thdng qua xac dinh dfldc ndng dp fle chi tdi thieu vdi till pha loing lan lifdt la 1/16 (0,273g/
ml), 1/8,1/8 (0,546 g/ml).
TAI LIEU T H A M KHAO
1. BQ Y tl (2005). "Bli thdo htidng ddn thii doc tinh ciia thudc", Cac phifdng phap thfl dde tinh cap - OECD, Phu luc 2.
2. Allen M. Seiden et al (2002). "Otolarynology: the essentials", Thieme, 77-85.
3. J. M. Andrews (2001). "Betermination of minimum inhibitory concentrations", Jdumal of Antunicrobial Chemotherapy 48, (suppll), 5-16.
4. Brucke A. Cxuiha,MD,MACP (2016). "Hudngddn dieu tn khdngsinh theo kinh nghiem", Nhixuitbin Yhpc, Ha Npi.
5. Jump up Davison, HC; Low, JC;, Woolhouse, ME (2000). "What is antibiotic resistance and how can we measure it?", Trends ui Microbiology, 8, (12), 554-559.
6. World Health Organization (2013). "Working group on the safety and efficacy of herbal medicine", Report of regional office for the Western Pacific ofthe World Health Organization.
TAP CHIY DUOC CO TRUYEN VIET NAM L-ltl»Mil:li JniJilbl I 3 1
