Nghiên cứu khả năng ức chế DNA Gyrase của một số dẫn xuất Chalcon tổng hợp


H 


KHịAăLUNăCăNHÂNăSINHăHC


 tài:
NGHIểNăCUăKHăNNGăCăCHă
DNA GYRASE CAăMTăSăDNăXUTă
CHALCONăTNGăHP

Khoa: CỌNGăNGHăSINHăHC
Chuyên ngành: VI SINH _SHPT
GVHD: PGS.ăTS.ăTrnăCátăông
SVTH: NguynăThănhăChi
MSSV: 1053010061
Khóa: 2010- 2014



 Chí Minh - Tháng 6 
LI CM N
    hoàn thành   
a c
 

, n thành .
ThS
- -

 và giú.
ThS, ThS 
            -
- .
Q        



viên giúp 
con 



 
 i
CHăVITăTT
ATCC
American Type Culture Collection
ATP
Adenosin Triphosphat
CFU
Colony Froming Unit

CLSI
Clinical Laboratory Standards Institute
DMSO
Dimethyl Sulfoxid
DNA
Desoxyribonucleic Acid
EDTA

Ethylendiamintetraacetic acid
KS
Kháng sinh
LB
Luria  Bertani
MHA
Muller  Hinton Agar
MHB
Mueller  Hinton Broth
MIC
Minimum Inhibitory Concentration

MRSA
Methicillin  Resistant Staphylococcus aureus
Staphylococcus aureus kháng methicillin
MSSA
Methicillin  Sensitive Staphylococcus aureus
Staphylococcus aureus y methicillin
OD
Optical density
VRSA
Vancomicin  Resistant Staphylococcus aureus
Staphylococcus aureus kháng vancomicin

SDS
Sodium Dodecyl Sulfat
SSB
Single Stranded Binding
TSA
Tryptic Soy Agar

TSB
Tryptic Soy Broth
 
 ii
DANHăMCăHỊNH
 3
Hình 1.2  3
 4
 8
Hình 1.5  9
Staphylococcus aureus 10
Staphylococcus aureus 10
E. coli  25
  26
 27
và E. coli 28
E. coli 28
 MRSA, MSSA và E. coli 28
E. coli 29
 trên gel agarose 30
 31
Hình 3.10  31
 32
se 33
 34
DANHăMCăBNG
 16
 25
 29
 

 iii
MC LC
CH VIT TT i
DANH MC HÌNH ii
DANH MC BNG ii
T V 1
CHNGă1. TNG QUAN 2
1.1. CHALCON 3
1.1.1.  3
1.1.2.  4
1.1.3.  4
1.1.4.  7
1.2. ENZYM DNA GYRASE (DNA TOPOISOMERASE II) 8
1.2.1.  8
1.2.2.  9
1.3. TNG QUAN V STAPHYLOCOCCUS AUREUS 10
1.3.1.  10
1.3.2.  11
1.3.3.  12
1.3.4.   12
CHNGă2. VT LIU 15
2.1. NG NGHIÊN CU 16
2.1.1.  16
2.1.2.  17
2.1.3.  17
2.1.4.  17
2.1.5.  18
2.1.6.  18
2.2. U 19
2.2.1.  19

2.2.2.  19
2.2.3.  20
2.2.4. Ly trích DNA plasmid 21
2.2.5.  22
2.2.6. Tt tính DNA gyrase 22
CHNGă3. KT QU VÀ BÀN LUN 24
3.1. NH TÍNH KH N 25
 
 iv
3.2. NH N C CH TI THIU CA CÁC CHT
TH VÀ KHÁNG SINH 26
3.3. KT QU TÁCH CHIT PLASMID 30
3.4. KT QU TINH SCH PLASMID 30
3.5. KT QU TH NGHIM KH C CH DNA GYRASE
CA CHALCON 32
3.5.1.  32
3.5.2.  33
CHNGă4. KT LU NGH 38
4.1. KT LUN 39
4.2. KIN NGH 39
TÀI LIU THAM KHO 40




 
 1
TăVNă
Flavonoid là    ên phâ   ã   , có  
 

 [10, 27, 29, 31].   

enzym DNA gyrase trong 
DNA. Ngoài ra chalcon có 
oxy hóa, trung hòa các  [7, 33].

tính  -  vòng 
    vòng benzen (vòng      vòng  
pyridin, thiophen, furan vòng 
              
 úng tô  "NghiênăcuăkhănngăcăchăDNA
gyraseăcaămtăsădnăxutăchalcon tngăhp
  DNA        ung

- 
- DNA 
 
 2
CHNG 1. TNG QUAN
 

 3
1.1. CHALCON
1.1.1. Tngăquanăvăchalcon
Flavonoid t chu  C
6
C
3
C
6

hay nói
vòng 
carbon (Hình 1.1) [13].
A
B


Hình 1.1 Cuătrúcăflavonoidă(diphenylăpropan)
Tùy th
3
C
6
C
3
C
6

chia thành 3 phân nhóm: eucoflavonoid, isoflavonoid, neoflav
phân nhóm eucoflavonoid [6].
Chalcon có 2 vòng 
vòng            
vòng  vòng B, vòng 
(Hình 1.2)


Hình 1.2 CuătrúcăchalconăvƠăquyătcăđánhăsăcaăchalcon
Trong các vòng 
vòng 
vòng (heterocyclic chalcon) [24].
              vòng 

flavanon, flavon, auron, benzothiazepin và pyrazolin (Hình 1.3) [19, 24].
 

 4


Hình 1.3 Dnăchtădăvòng qua trung gian chalcon
1.1.2. Phnăngăđnhătính
ch KOH vòng C 
vòng A và B
 [6].

2
SO
4
cid H
2
SO
4
khi n
 [6].

3

 [6].
 
dung 
H
+
hoc OH

-

 .
Tác d        
5
trong
CCl
4

vòng 
5

2
SO
4
[6].
1.1.3. Hotătínhăsinhăhcăcaăcácădnăxutăchalcon

 [39],  [39, 41], kháng viêm [16,
 

 5
39] [38] [2]
.
López và cng s (2001) 
         t       
kháng

 [26].
Nielsen và cng s (2004)       on.




có tính a    -    có   kém, còn
s -
 [33].
Azad và cng s (2007) 

       cao trên Escherichia coli, Pseudomonas
aeruginosa, Aspergilus niger, Candida albican[12]


          Escherichia coli, Pseudomonas
aeruginosa, Aspergilus niger, Candida albicans[19].
E. coli, S. aureus
              
   vòng      2-hydroxy-4,6-dimethoxy-3-(1-
methylpiperidin-4-            

 [25].
Prasad và cng s (2008) n       

 

 6

3

 


lai [37].
Hugo 

-
-
 [11].
Swamy và (
           -(3-
hydroxy benzofuran-2-yl)-3-aryl-2-propen-1-on và -(3-hydroxy benzofuran-2-yl)-3-
(4-methoxy-aryl)-2-propen-1-        Aspergillus
flavus và Candida albican  [42].
 và cng s 

khôn

trên Staphylococcus aureus [5].


cao trên S. aureus, Bacillus subtilis, Escherichia coli và Pseudomonas aeruginosa
[32].

vòng Staphylococcus aureus,
Escherichia coli, Streptococcus faecalis[7].

              
nhóm   gram          Streptococcus
mutans  [15].
 

 7


         chalcone '4-hydroxy-3-(3-p-
tolylacryloyl)-2H-chromen-2-one [21].

ol
 
[23].
1.1.4. Căchăkhángăkhunăcaăchalcon

 DNA DNA 
t     nucleotic        DNA
 a các nucleotic, DNA gyrase giúp
DNA vòng 
vòng DNA 
 DNA gyrase [18].  ng phân t iên cu rõ
ràng.
chalcon quinoline
E.coli, K.aerogenes, S. typhimurium, S. aureus,
S. pyogenes, và B. subtilis.  quan

 
 acid LEU735,
GLN788, ASP686, SER734, VAL737, VAL787, ARG739, VAL685, LEU836 và
VAL784. Cht A4, A6 có vòng quinoline liên kt cht ch vi Ile736, Val737 và
Val787 th hi   c; nhóm N i nhóm hydro ca
Asp686 và Leu735; mt khác, nhánh Cl ca vòng piridi Val737 và
Gln788, nhóm Br  v i Val784 n
nh ca hp cht [30].
 

 8
1.2. ENZYM DNA GYRASE (DNA TOPOISOMERASE II)
1.2.1. TngăquanăvăDNAăgyraseă
Trong quá trình sao chép, hDNA ->

Protein SSB (single stranded binding protein) giúp cho
DNA 
DNA 
vòng 
cvà DNA  [14, 22].

Hình 1.4 QuáătrìnhătháoăxonăDNA
DNA topoisomerase II có tên là DNA gyrase. DNA topoisomerase
II 
t DNA 
siêu n trái (-DNA Hình 1.5). 
              
nucleotid   [14,
22].
 

 9






Hình 1.5 HotăđngăcaăDNA topoisomeraseăIIăăchcăbaăsao
 eukaryote, DNA 

 [14, 22].
1.2.2. CácănghiênăcuăvăkhănngăcăchăDNA gyrase
Ohemeng 
DNA Escherichia coli [34].
Aiello và c
DNA- p acid

 [9].
           
fluoroquinolone     N-benzoyl-3-(4-bromophenyl)-1H-pyrazole-5-
carbohydrazide gram 
gram   Escherichia coli, S. aureus, Streptococcus faecali, Streptococcus
mutans, Pseudomonas aeruginosa, Aspergilus niger, Candida albican, Bacillus
subtili, M. tuberculosis…DNA gyrase [14, 17, 25, 43].
 

 10
1.3. TNGăQUANăVăSTAPHYLOCOCCUS AUREUS
1.3.1. Hìnhăthái,ăđcăđimăsinhăhóa
 Hình thái
Staphylococcus aureus 
gram - 
u nhau (Hình 1.6).


Hình 1.6 căđimăhìnhătháiăcaăStaphylococcus aureus

gram (Hình 1.7).

Hình 1.7 HìnhănhumăGramăcaătăcuăStaphylococcus aureus

Staphylococcus aureus 
 S. aureus có màu
- 1,5 mvòng sá-

2
TeO
3

lethinase) [28, 40].
 

 11


 [28].
 c đim sinh hóa
S. aureus 

S. aureus 
H
2
O
2
H
2
O + O
2
S.aureus             

S. aureus  [17].
DNA
S. aureus 

 [8].
 dòng S. aureus vòng
vòng 
S. aureus 
- 2 ngày

 [17].
S. aureus 

o
- 37
o
C
[8].
1.3.2. Căchăđăkhángăkhángăsinhă
              -
            
 [35].
 

 12
         -lactam (do
Staphylococcus aureus          
 [35].
Staphylococcus aureus 
-Multi Drug Resistance pumps) [35].

1.3.3. Tínhăđăkhángăkhángăsinh
S. aureus 

òng MRSA (Methicilin resistant Staphylococcus aureus)

Enterococcus
faecalis S. aureus. Ngoài ra, S. aureus 
 [43].



               
kháng vancomycin VRSA (Vancomycin Resistant Staphylococcus aureus) [43].
Theo k 
 (2006S. aureus 
    n, 52,9% kháng ciprofloxacin, 52% kháng
amoxillin và 12,5% kháng getamicin [3].
1.3.4. Khănngăgơyăbnh


 h khác nhau [1]:
- Nhimăkhunăda
 chân
lông, chân   :
 

 13


 

- Nhimăkhunăhuyt


             



- Viêmăphi
Vi



- NhimăđcăthcănăvƠăviêmărutăcp


 


- 


- Nhimăkhunăbnhăvinădoătăcu



- Hiăchngădaăphngărpă(Scaldedăskinăsyndrome)
 

 14



- Hiăchngăscănhimăđcă(toxicăshockăsyndrome)

ng


 

 15
CHNG 2. VT LIU VÀ
PHNG PHÁP NGHIÊN CU

 

 16
2.1. IăTNGăNGHIểNăCU
2.1.1. CácăchtăthătrênăDNA gyrase
 : các chalcon          - Khoa
- (2.1).
Kháng sinh ofloxacin (O8757- 1G) 
Bngă2.1 Cácădnăxutăchalconădăvòng dùngătrongăthănghim
Kí
hiu
Danh pháp
Côngăthc
Kíăhiu
Danh pháp
Côngăthc
H1
1-(2-

hydroxyphenyl)-
3-(pyridin-2-yl)-
2-propen-1-on


F16
-dihydroxychalcon
O
OH HO



H2
1-(2-
hydroxyphenyl)-
3-(pyridin-3-yl)-
2-propen-1-on


P1
1-(pyridin-2-yl)-3-
(2-hydroxyphenyl)-2-
propen-1-on


F2
1-(furan-2-yl)-3-
(3-
hydroxyphenyl)-
2-propen-1-on

N11
1-(furan-2-yl)-3-
(4-N,N
dimethyaminophenyl)-
2-propen-1-on



F12
5 -
hydroxychalcon



T2
1-(thiophen-2-yl)-3-
(3-hydroxyphenyl)-2-
propen-1-on





 

 17
2.1.2. Các chng vi khunădùngăkhoăsát


- Staphylococcus aureus 
- Staphylococcus aureus kháng methicillin ATCC 43300 (MRSA).
- Escherichia coli ATCC 25922.
2.1.3. Môiătrngăthănghim

- 
- 
- - Hinton Agar (MHA).
- - Bertani (LB).
2.1.4. Dungămôi,ăhóaăchtăvƠăthucăth
Hóaăchtăchit plasmid
- 
Glucose 50mM
EDTA 10mM
Tris- Cl, pH 8 25mM
- - SDS:
NaOH 1M 2ml
SDS 20% 0.5ml
dH
2
O 7.5 ml
- Ac (kali acetate) 5M, pH 4,8:
KOH 5M 60 ml
 11,5 ml
dH
2
O 28,5 ml
HóaăchtătinhăschăDNA
 

 18
-  ® SV Gel and PCR Clean- Up System (A9282)
công ty Promega.
HóaăchtăthănghimăhotătínhăGyrase
- Enzym gyrase (E. coli), DNA gyrase 1X pH 7,5
 - HCl 35 mM, KCl 24 mM, MgCl
2
4 mM, DTT 2 mM, spermidin
5mM, 30 µg/ml tRNA, BSA 0,1 mg/ml, glycerol 6,5%.
- Enzym gyrase (E. coliDNA gyrase 1X pH 7,5
bao - HCl 35 mM, KCl 24 mM, MgCl
2
4 mM, DTT 2 mM, ATP 1,75
mM, spermidin 5mM, 30 µg/ml tRNA, BSA 0,1 mg/ml, glycerol 6,5%.
2.1.5. Dngăcătrangăthităb
- Máy OD Gene Quant
- Máy ly tâm Sigma 6K15
- Máy vortex Labnet
-  
- 
- 
- ESCO AHC 4A1
- -20
o
C
- Cân phân tích Satorius
- 
- 
- 



2.1.6. Bătrí thíănghim

- vòng 
MRSA, MSSA :
 

 19
+  u
+ 
- TDNA plasmid.
- DNA 
2.2. PHNGăPHÁPăNGHIểNăCU
2.2.1. Chunăbăviăkhunăthănghim
 
o

  
o
C trong 2-

 
-  
1-2x10
8
(CFU/ml).
 
2.2.2. Phngăphápăkhuchătánătrênăđaăthch
       
- 

 - 30 phút.
 que
 

o
 - 5 phút cho

  . 

         
2%.
 Hút             
