Nghien cim. Phdt trien yd Cns dung Cong nghi Vdt lieu (KC.02/06-10 >- Ha Noi 27/11/2009
NGHIEN CUT HOAT TINH KHANG KHUAN CUA VAT LIEU BAC-CHITOSAN N A N O C O M P O Z I T
Bo Trircmg Thien, Dirong Anh Vu, Tran Thj Y Nhi, Nguyen Tien An. Pham Thi Bich
H a n h . Nguyen T h i B o n g . Nguyen T h | T h a n h , P h a m T h i M a i V i e n Hoa hoc. Vien Khoa hoc va Cong nghe Viet N a m
18-Hoang Quoc Viet. Ha N o i , Viet Nam Tom tit Bdi bdo trinh bay kit qua nghiin ciru hoat tinh khdng khudn ciia vdt lieu nano bac- B-
chitosan nanocompozit theo phuong phdp pha lodng da nong dp. Hoat tinh cua vdt lieu duoc danh gid thdng qua cdc gid tri: ndng dp ire che tdi thieu (MIC): ndng dp ire chi 50% (ICso) va ndng dp diet khudn tdi thiiu (MBC). Cdc gid tri nay duoc tinh toan dua tren sd lieu do dp due ti bao bang may quang phd TECAN. Kit qua nghiin cim cho thay, tdc apng khdng sinh hiep luc cua chitosan vd nano bac lam cho vdt lieu bac- fi-chitosan nanocompozit cd tdc dung khdng sinh rat manh vdi cdc chirng vi sinh vdt.
I . M O D A U
Hien nay, vat lieu nano tren co so polysacarit, dac biet la chitosan duoc sir dung rong rai
trong cac linh vuc sinh. y va duoc hoc. Chitosan ((l-4)2-amino 2-deoxy (3-D-glucan) la san pham deaxetyl hoa chitin. mot polysacarit san co trong vo cac loai giap xac. Chitosan la mot polycation co nhieu tinh chat quy nhu tinh tucmg hop sinh hoc. khong doc. t u phan hiiy sinh h o c . v v .
Do co tinh khang khuan manh v o i kha nang diet v i khuan pho rong (gram am va gram duong) ciing nhu cac loai nam (nam moc va nam men) nen bac da duoc sir dung tir chien ttanh the gidi lan thu I I uong dieu tri vet thuemg. vet bong va mot so loai benh nhiem khuan khac duoi dang bac k i m loai, bac ion (bac nitrat). bac sulfadiazin...Ion bac co tac dung khang khuan b nong do rat thap (khoang 0.001 ppm). Do do. mac du bac la k i m loai nang. nhung v i sir dung d nong do rat thap nen no khong doc v d i te bao con ngudi va duoc coi la vat lieu an toan.
The ky 2 1 , cirng v d i su phat trien ciia cong nghe nano. nano bac duoc sir dung nhu mot dong khang sinh the he mdi (Silver nanoparticles as a new generation of antimicrobials) cho cac ung dung Uong cac linh vuc nhu sinh. y va duoc hoc. 6 kich thudc nano. bac the hien nhirng dac tinh ly, hoa, sinh hoc ky la, khac han so v o i bac d dang khdi. Chinh vi vay. nghien cuu tong hop va ung dung vat lieu bac - (3-chitosan nanocomposit tiep tuc duoc quan tam nghien ciru.
H. T H U C N G H I E M 1. Tong hop vat lieu bac-chitosan nanocompozit
Hoa tan hoan toan chitosan 0.6g (DA =0%, MH=30.000 Da) trong 30 ml dung dich axetic
axit 1%. Chuan bi dung dich A g N O ; v d i cac nong do 1.5. 10. 20 m M va dung dich N a B H t 0.1M. Nho tir tir 2 m l dung dich muoi bac co nong do xac dinh vao 4 m l dung dich chitosan, khuay tten may khuay tir trong 30 phiit d nhiet do phdng. Them 1 m l dung dich N a B H t vao hdn hop. tiep tuc khuay trong 2 gid. Cac m l u duoc ky hieu lan luot la M l . M 2 . M 3 . M 4 tucmg ung voi nong do muoi AgNO? sir dung la 1. 5. 10 va 2 0 m M .
Phan img khir ion bac bang tac nhan N a B H t v d i su co mat ciia chitosan lam chat on djnh va phan tan xay ra theo so do sau:
((3-Chitosan)Ag+ + [H] - » (p-Chitosan)Ag + H "
2. T h i r hoat t i n h k h a n g sinh
2.1. Cac chiing vi sinh vat kiem dinh.
Cac chiing vi sinh vat dai dien gay benh d ngudi gom:
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V i khuan: Gram (-) gom: Pseudomonas aeruginosa (Pa) A T C C 15442 va E. Coli (Ec) A T C C 25922. Gram (+) g6m Staphylococcus aureus (Sa) A T C C 13709; Bacillus subtilis (Bs) A T C C 6633; Lactobasillus fermentum N4v a Enterococus faecium B65Q.
2.2. Phuong phap thu hoat tinh khang v i sinh vat k i i m dinh.
Hoat tinh khang v i sinh vat kiem dinh duoc thuc hien dua tren phuong phap pha loang da nong do. Day la phuong phap thir hoat tinh khang v i sinh vat kiem dinh va nam nham danh gia muc do khang khuan manh yeu ciia cac mau thir thong qua cac gia tri the hien hoat tinh la M I C (nong do ire che toi thieu), I C5 0 (nong do ire che 50%), M B C (nong do diet k h u i n toi thieu).
2.3. M o i truong nuoi cay
M H B (Mueller-Hinton Broth), M H A (Mueller-Hinton Agar). TSB (Tryptic Soy Broth), T S A (Tryptic Soy A g a r ) cho v i k h u i n , S A B , SA cho nam.
2.4. Cach tien hanh
* Pha loang mau thir:
- M a u ban dau duoc pha loang Uong nuac cat vo trung thanh mot day 10 nong do thich hop theo yeu cau va muc dich thir.
- Nong do mau ban dau la nong do goc, nong do sau pha loang 1:2 cir the cho den nong do cuoi ciing ciia day 10 nong do.
* T h u hoat tinh:
- Chuan b i dung dich v i khuan hoac nam v o i ndng do 5.105CFU/ml k h i tien hanh thu.
Lay lOpl dung djch mau thir theo cac nong do da duoc pha loang, them 200pl dung dich v i khuan, i i 6 37°C. Sau 24h, doc gia tri M I C bang mat thudng. Gia t r i M I C dupe xac dinh tai gidng co nong dp chat thir thap nhat gay uc che hoan toan su phat trien ciia v i sinh vat. Gia tri IC50 dupe tinh toan dua tten so lieu do dp due te bao bang may quang pho T E C A N .
III. KET QUA VA THAO LUAN
1. Hoat tinh cua bac-chitosan nanocompozit voi khuan gram am
Hoat tinh ciia bac-chitosan nanocompozit v o i khuan gram am dupe trinh bay 6 bang 1.
Chung Pseudomonas aeruginosa ( A T C C 15442): (gram (-), true khuan mu xanh, gay nhiem trung huyet, cac nhiem trung 6 da va niem mac, gay viem duong tiet nieu, viem mang nao, mang trong t i m , viem rudt) cho thay: mau M 4 co hoat tinh manh nhat, v o i cac gia t r i IC50, IC90, M B C lan lupt la 6,02; 9,67 va 13,84 pg/ml (tinh toan theo nong dp tong- la nong dp bac- chitosan nanocompozit). Hoat tinh khang sinh ciia cac mau dupe sap xep theo thu tu:
M 4 > M 3 > M 2 > M 1 . Tuy nhien, gia tri M B C tinh toan theo nong dp bac trong cac mau M l , M 2 , M 3 , M 4 (tuong ung v o i kich thuoc nano bac la 3, 5, 10, 15 run) lan lupt la: 1,27, 1,50, 2,02, 2,94 pg/ml, co nghia kich thuoc hat cang nho hoat tinh cang tot.
Tuong tu voi chung E.coli, mlu M4 co hoat tinh manh nhat, voi cac gia tti IC50, IC90,
M B C lan lupt la: 26,9; 87,87; 102,68 pg/ml. Hoat tinh ciia bac-chitosan nanocompozit dupe xep theo thir t u M 4 > M 3 > M 2 > M 1 . Gia tti M B C tinh toan theo nong dp bac trong cac m l u M l , M 2 , M 3 , M 4 lan lupt la: 2,17; 3,02; 4,04; 6,74 (kich thuoc hat cang nho, hoat tinh cang tdt).
Chitosan sir dung dl tdng hpp bac-chitosan nanocompozit trong cong trinh nghien cuu nay
co kha nang uc c h i su hoat dong ciia toan bp v i sinh vat 6 nong dp > 200 pg/ml. Theo Pal va cong su (2007) thi kha nang ire c h i toan bp su phat trien ciia v i khuan gram am ciia nano bac co dang hinh cau la 12,5 pg/ml Tir k i t qua 6 bang 1 cho thay, kha nang khang k h u i n ciia bac- chitosan nanocompozit cao hon han so v o i k h i sir dung chi sir dung nano bac hay chi sir dung chitosan. D i l u nay dupe giai thich la do lop mang ngoai ciia nhom v i khuan gram am (bao gom ca E.coli) co mot lop lipopolysacarit chira nhom photphat va pyrophotphat lam cho mang
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te bao co dien tfch am, Chitosan la mot cation polysacarit se lien ket v o i mang te bao v i khuan i theo co che hut tinh dien) lam mang te bao kem on dinh va lam may doi tfnh tham ciia mang te bao. Chinh qua trinh nay lam cho su lien ket giua nano bac va protein chua nhdm thiol thuan l o i hon. nano bac se xam nhap vao te bao. pha huy va lam uc che su hoat dong ciia v i sinh vat.
2. Hoat t i n h ciia bac-chitosan nanocompozit v o i k h u a n g r a m d u o n g . Bang 1. Hoat tinh khdng khudn cua b-ac-chitosan nanocompozit vdi chiing gram dm
Pseudomonas aeruginosa
IC50 IC90 MBC
Kich Ndng Nong Nong
thudc Nong Ndng dp Nong Nong dp" Ndng Ndng dd Ten hat dd tdng do bac chitosan dp tdng dp bac chitosan dp tong dp bac chitosan mau (nm) (ug-'ml) i us ml) (us. ml ) i ug/ml) i ug/ml) (pg/ml) '. us m l1 1 pg/ml) i ug/ml) M l 2 19.51 0.52 19.29 25.39 0.6" 24,72 48.34 1.2" 47,07 M2 5 10.50 0.5- 9.96 20.91 1.07 19,84 29,34 1.50 2".S4 M3 10 9.39 1.31 s.os 14.15 1,97 12.18 14.51 2.02 12,49 M4 15 6.02 1.2S 4.~4 9.6" 2.06 ".61 13,84 2.94 10.90
Escherichia coli
IC50 IC90 MBC
Kich None Nong Nong
thudc Nong Ndng do" Nong Ndng do Nong Ndng do Ten hat do tong dd bac chitosan dp tong do bac chitosan dp tdng do bac chitosan miu r.m (pg/ml) ~s ml (pg/ml) (pg/ml) i pg/ml) i pg/ml) i us mT) 0rg/ml) 1 u.s ml •
M l 26.90 0.71 26.19 80,87 2.13 78.74 S2.6S 2,17 S0.51 M2 s 17.00 0.S" 16.13 46.69 2.39 44.30 59.00 3.02 55.98 M3 10 13.97 • _yf 12.02 26.11 3.64 22.4" 29.01 4.04 24.9"
M4 1 ^ 15,42 3.2S 12.14 31.29 6.65 2^.64 31.69 6.74 24,95 Bdng 2. Host tinh khdng khudn c ua bac-chi tosan nanocompozit vdi chung gram duong
Staphylococcus aureus
IC50 IC90 MBC
Kich : Ndng Ndng Nong
thudc Nong Ndng dp Ndng Ndng dp" Ndng Ndng do"
Ten hat do tdng do bac chitosan dp tong do bac chitosan dp tdng dp bac chitosan miu r.m us mh i^ug ml'i 1 pg/ml) (Ug'mli (pg/ml) (ua/mli t us. m l ' (pg/ml) i pg/ml) M l 3 23.86 0.63 23.23 96.05 2.53 93.52 116.05 3.05 113.00 M2 21.10 1,08 20.02 58."3 3.01 55.72 58.S9 3.02 55.87 M3 10 12.SI 1.79 11.02 29.31 4.09 25.22 31.69 27.2"
M4 1 ^ 9.6" 2. r ".61 23.62 5.02 IS. 60 29.62 6.30 23.32 Baci'.'.us subti'is
IC50 IC90 MBC
Kich Ndng Ndng Nong
thudc Ndng Nong do" Nong Ndng do" Nong Ndng do"
Ten hat dp tdng do bac chitosan dp tdng do bac chitosan dp tdng do bac chitosan miu (nm) i us.'mli (U£/mD
L Ai (pg/ml) (pg/ml) ijig'mli Qig/ml i 0rg/ml) (pg/ml) (pg/ml)
M l 2.02 74.99 97.55 2.56 Q-l O Q
7^*77 102.6S 2.70 99 98 M2 5 ~ 1,5S 3.67 6".91 9S.59 5.05 9;.^4 5.-0 99.95 M3 i n 66.SS 9.32 5".56 106.2 14.81 91.39 ' ' - " - 16 18 99.8"
M4 15 54 85 11.55 43.19 116 24.65 91.30 126 ."5 26.9^ 99,80
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Ket qua nghien cuu d bang 2 cho thay vat lieu bac-chitosan nanocompozit co tac dung
khang manh Staphylococcus aureus ( A T C C 13709): cau khuan gram (+), la v i khuan nguy hiem gay ra nhiem trung da nghiem Uong nhu loet, mun mu va nhot tren cac vet thuong va vet bong. Ngoai ra, day la chung gay viem hong, cac co quan noi tang va nhiem trung mau va la chiing v i khuan ngay cang khang khang sinh. Ket qua thu hoat tinh t u bang 2 cho thay, quy luat ve hoat tfnh khang sinh doi v o i Staphylococcus aureus va Bacillus subtilis cung tuong t u nhu v o i khuan gram am. Tuy nhien gia tri M B C a tat ca cac mau deu cao hon so v o i gia tri M B C doi v d i cac chiing gram am. Dieu nay dupe giai thich la do v i khuan Gram duong cd thanh te bao d i y , dang l u d i c i u tao bdi peptidoglycan chidm 80-90% thanh p h i n vach td bao.
Peptidoglycan la loai polime xdp, khdng tan, kha cirng va ben vung bao quanh te bao nhu mot mang ludi, do dd kha nang tan cong va xam nhap vao ben trong te bao ciia bac-chitosan nanocompozit khd khan hon so v d i v i k h u i n gram am.
IV. KET LUAN
- Vat lieu bac-chitosan nanocompozit cd hoat tinh khang sinh rit tdt ddi vdi 6 chiing vi khuin k i e m dinh va nam men.
- Hoat tinh khang sinh ty le thuan v d i ty le ion bac/chitosan - K i c h thudc hat nano cang be, hoat tinh khang sinh cang tdt.
L o i cam o n
Tap thd tac gia chan thanh cam on Ban Chii nhiem Chuong trinh "Nghien cuu, phat tridn va
ling dung cong nghe vat lieu", ma sd KC-02/06-10 va Van phdng Chuong trinh Khoa hoc Cong nghe trong diem cap Nha nude da rat quan tam va ling hp dd dd tai "Nghien cuu che tao vdt lieu nanochitosan, ung dung trong duoc pham va trong nong nghiep", ma sd K C - 0 2 - 09/06-10 dupe thuc hidn.
Tai lieu tham khao
1. Bao cao chuyen de "Nghien cuu tinh chdt khdng vi sinh vdt kiem dinh, khdng nam", ciia de tai "Nghien cuu che tao vat lieu nanochitosan, ung dung trong dupe pham va trong ndng nghiep", ma sd KC-02-09/06-10, thudc Chuong trinh khoa hoc va cong nghe trong diem cap Nha nude "Nghien cuu, phat trien va ung dung cong nghe vat lieu", ma sd K C - 02/06-10.
2. Hadacek, F., Greger, H . (2000), "Test o f antifungal natural products methodolagies, comparability of result and assay choise", Phytochem. Anal., 90, 137-147.
3. Pual Cos, Louis Maes, Jean-Bosco Sindambiwe, Arnold J. Vlietinck, D i r k Vanden Berghe; " Bioassay for antibacterial and antifungal activities"; Laboratory f o r Microbiology, Parasitology and Hygien, Faculty o f Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Belgium, 1-13 (2005).
4. Tran T h i Y N h i , D o Truong Thien, Nguyen Van Tuyen, Nguyen T h i Dong, Nguyen T h i Thanh, Pham T h i M a i , Nguyen Tien A n and Duong A N h V u , "synthesis and characterization o f nano silver-p%chitosan", Tap chi Khoa hoc va cong nghe, 45 (3 A ) , 214- 219, (2007).
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