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NH GI C TNH THUN CHNG V HOT TNH KHNG

KHUN CA CY C MC (ECLIPTA PROSTRATE) V
CY DIP H CHU THN XANH (PHYLLANTHUS NIRURI)
NG BNG SNG CU LONG
Hunh Kim Diu1 v L Th Loan Em

ABSTRACT
Total 30 samples of Phyllanthus niruri and 30 samples of Eclipta prostrate cultivated in
different provinces of Mekong delta were collected for protein electrophoresis employing
the SDS-PAGE method and tested for the antibacterial susceptibilities expressed as
minimum inhibitory concentrations (MIC) of eight selected bacteria strains
Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Pseudomonas
aeruginosa, Salmonella spp. and Aeromonas hydrophila, Edwardsiella ictaluri and
Edwardsiella tarda. There were 8 lines of Phyllanthus niruri and 11 lines of Eclipta
prostrate were discovered. Their protein bands were 0.11% and 0.1% polymorphic while
the polymorphic individuals were 0.4% and 0.07%, the phenotypic diversity value (Ho) =
5.31 and 2.61, the genetic diversity value (HEP) = 0.71 and 0.6 and sum of the effective
number alleles SENA = 2.42 and 1.52 respectively. The antibacterial activity of
Phyllanthus niruri was divided into 7 groups, all of them had the strong antibacterial
activities against Edwardsiella tarda with MIC = 64-512 g/ml and Aeromonas
hydrophila MIC=512-1024 g/ml. Eclipta prostrate was divided into 3 groups and had
strong antibacterial susceptibilities against Edwardsiella tarda (MIC=256-512 g/ml),
Edwardsiella
ictaluri
(MIC=512
g/ml)
and
Aeromonas
hydrophila
(MIC=256-512 g/ml).
Keywords: Phyllanthus niruri, Eclipta prostrate, protein electrophoresis, minimum
inhibitory concentration
Title: Evaluation the genetic diversity and anti-bacterial activity of Phyllanthus niruri
and Eclipta prostrate in the Mekong Delta

TM TT
30 mu Ch Thn Xanh (CTX) v 30 mu C Mc thu thp nhiu tnh thuc ng
bng sng Cu Long, c in di protein bng phng php SDS-PAGE v th hot
tnh khng khun (xc nh nng c ch ti thiu MIC) trn 8 chng vi khun
Staphylococcus aureus, Streptococcus faecalis, Escherichia coli, Pseudomonas
aeruginosa, Salmonella spp. v Aeromonas hydrophila, Edwardsiella ictaluri v
Edwardsiella tarda. Kt qu cho thy cc mu CTX v C Mc c t l c th a hnh
ln lt 0,11% v 0,1%, t l bng protein a hnh 0,4% v 0,07%, v s allele hiu qu
SENA = 2,42 v 1,52, ch s ch a dng v kiu gen HEP = 0,71 v 0,6 v r nht l a
dng v kiu hnh Ho = 5,31 v 2,61. Cy CTX v C Mc khng thun chng: CTX
c 8 dng v C Mc c 11 dng. Hot tnh khng khun ca cc dng CTX v C Mc
trn cc chng vi khun th nghim khng ging nhau. CTX chia 7 nhm nhng tt c
cc dng u tc ng rt mnh trn Edwardsiella tarda (MIC = 64-512 g/ml), k n
Aeromonas hydrophila (MIC=512-1024 g/ml); cc dng C Mc u c kh nng tc
ng trn cc vi khun th nghim v c th chia lm 3 nhm v u tc ng mnh trn

Khoa NN & SHD, Trng i hc Cn Th

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Edwardsiella tarda (MIC=256-512 g/ml), k n Edwardsiella ictaluri (MIC=512

g/ml) v Aeromonas hydrophila (MIC=256-512 g/ml).
T kha: ch Thn Xanh, C Mc, in di protein, nng c ch ti thiu

1 T VN
Cy C Mc cn gi l cy Nh Ni, thng c dng cm mu bn trong v
bn ngoi, cha ho ra mu, lao phi l ra mu; cng c dng cha ho, bng,
chng vim nhim trong cc trng hp cm st, cm, ban si, nhim khun
ng h hp, tr mn nht, vim c l nga, au mt, sng rng, au d dy; iu
tr nm da, eczema, vt lot, vim da; (V Vn Chi et al., 1999). Cao lng l C
Mc c dng iu tr bnh nhn b vim m o do tp khun, do nm v do
Trichomonas, v c cho c c tnh rt thp, gii hn an ton rng, cm mu
tt, trong vi trng hp c bit, tc dng ny ca C Mc th hin r rt hn c
tc dng ca vitamin K (r rt trong cc trng hp suy gan).
Bn cnh y, cy Ch thn xanh (CTX) cng l cy mc hoang nh cy C
Mc v cng c dn gian s dng rt nhiu bo v gan, lm gim mc x
gan, st khun (p cc vt thng sng ty v lot, mn nht, l nga ngoi da,
tr l, bnh lu), cc bnh ca h niu sinh dc, v i tho ng. Thuc c tc
dng li tiu tr si mt v si thn, chng oxy ha v c tnh thp, c an ton
cao. CTX cng c tc dng gy h ng mu, h p v li tiu ngi (
Huy Bch et al., 2004). Tc dng ni bt nht ca cy CTX l cha suy gan;
chng vim gan vng da hay x gan c trng (Trn Xun Thuyt, 2003).
Hai cy thuc ny c s dng nhiu trong dn gian, tuy nhin, cha c
nghin cu no cho bit s thun chng ca hai cy ny. gp phn tm hiu v
nhng cy thuc ny, nghin cu v s thun chng ca cy C Mc v Ch
thn xanh c thc hin. Mc ch tng bc chn lc ra nhng dng c hot
tnh cao.
2 VT LIU V PHNG PHP TH NGHIM
2.1 Vt liu
- CTX v C Mc: s dng ton cy (tr r).
- Cy hoang di c thu hi mt s huyn thuc tnh An Giang, Hu Giang,
Tin Giang v Cn Th.
- Cc chng vi khun c s dng:
+Chng vi khun ngun gc t vin Pasteur Tp H Ch Minh:
Staphylococcus aureus (Staph.), Streptococcus faecalis (Strep.), Escherichia coli
(E.coli), Pseudomonas aeruginosa (Pseu.), Salmonella spp.(Sal.), Edwardsiella
tarda (Ed. tarda ) v Aeromonas hydrophila (A. hydrophila).
+Chng vi khun ngun gc t khoa Thy Sn (i hc Cn Th):
Edwardsiella ictaluri(Ed. ictaluri).
2.2 Phng php th nghim
2.2.1 in di protein
- 30 mu CTX v 30 mu C Mc dng in di protein c thu t 30 h dn
khc nhau (cch nhau ti thiu 1km).
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- in di protein CTX v C Mc c tin hnh theo phng php SDS-PAGE

(Laemmli, 1970).

S a dng v di truyn c nh gi da trn nhng thng s Ho (a dng v

kiu hnh), HEP (a dng v kiu gen) v SENA (tng ca s alen hiu qu: sum of
the effective number of alleles) (Hub and Ohnishi, 2002; Thanh et al., 2003):
Ho= -filnfi,
HEP= 1-fi2, SENA= (1/ fi2- 1)
Trong :
- fi l tn s xut hin dy bng protein i. Qui nh tn s ca nhng dy bng
protein c thy bng mt thng, nu c hin din cho im l 1, nu khng
hin din cho im l 0.
- n l s dy bng protein hin din.
- Nu Ho = 0 chng t tnh thun chng cao, nu gi tr Ho ln chng t c s a
dng v di truyn, tc cy khng thun chng.
- HEP bin thin t 0 n 1, nu tr s HEP nh chng t tnh thun chng cao, nu
tr s HEP ln chng t c s a dng v di truyn.
- SENA c tnh ton da vo xc nh s allele hiu qu.
- S a dng v hnh thi ca c th hay ca cc dy bng protein c ghi nhn
khi s bin i nhng dy bng protein ca n < 90%.
2.2.2 Th hot tnh khng khun
Cc cy c s khc bit v dy bng protein c trng li trong cng iu kin
chm sc, dinh dng. Sau khong 4 thng, cy c thu hoch th hot tnh
khng khun.
- CTX v C Mc c sy kh, chit bng phng php ngm dm vi
methanol v loi b dung mi bng my c quay n cn, c cao th, dng th
hot tnh khng khun MIC (minimum inhibitory concentration) (Nguyn Vn n
v Nguyn Vit Tu, 1985).
- Dng phng php phng php trong thch xc nh MIC (Trng Cng
Quyn et al., 1986; T Minh Kong et al., 2001).
3 KT QU V THO LUN
3.1 S a dng v di truyn
Trong 30 mu CTX v 30 mu C Mc bng phng php in di protein SDSPAGE pht hin c CTX c 18 dy bng protein v C Mc c 10 dy bng
protein c s khc bit (Hnh 1 v Hnh 2).
Nhng thng s biu th s a dng v di truyn ca CTX v C Mc c trnh
by qua Bng 1. Qua kt qu Bng 1 cho thy t l c th a hnh ca CTX v C
Mc ln lt l 0,11% v 0,1%, t l bng protein a hnh l 0,4% v 0,07%, v s
allele hiu qu SENA = 2,42 v 1,52, r nht l ch s ch a dng v kiu gen
HEP = 0,71 v 0,6 v a dng v kiu hnh Ho = 5,31 v 2,61. Nh vy, cy CTX
v C Mc khng thun chng m gm nhiu dng (line), nhng cng loi
(species). Theo Rao et al. (1992), kt qu cu trc nhng dy bng protein gia
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cc dng trong cng loi c khc bit nhng vn tiu biu cho mi loi v gia cc
loi, khi in di bng SDS-PAGE s cho cc dy bng protein khc nhau v s
lng ln trng khi.
Da vo kt qu in di protein cho thy CTX c 8 dng v C Mc c chia
lm 11 dng khc nhau.
Cy CTX v C Mc l cy mc hoang khp ni v c s a dng v di truyn.
Cy CTX mc hoang di rt nhiu ni, c hoa n tnh, hoa c v hoa ci trn
cng 1 cy, nh gi hoc theo dng nc m c s th phn cho to nn s a
dng gia cc dng.
C Mc c hoa n hoc lng tnh (hoa ci ngoi v hoa lng tnh gia)
mc hoang di nhiu ni trong t nhin nn d dng xy ra hin tng th phn
cho, v th C Mc c nhiu dng.
Bng 1: Nhng thng s a dng v di truyn ca cy CTX v cy C Mc

Thng s
C th a hnh (%)
Bng protein a hnh (%)
a dng v kiu hnh Ho
a dng v kiu gen HEP
SENA
1

CTX
0,11
0,4
5,31
0,71
2,42
2

C Mc
0,1
0,07
2,61
0,6
1,52
9 Marker
116 KDa

91,1 KDa

66,2 KDa
45 KDa

40 KDa
35 KDa

35 KDa

27 KDa

25 KDa

21,7 KDa
18,4 KDa
14,4 KDa
Hnh 1: Ph in di protein C Mc

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Marker

116 KDa

106,1KDa

66,2 KDa
55,6 KDa
45 KDa

45 KDa

35 KDa
28,5 KDa
25 KDa
20,6 KDa
18,4 KDa
Hnh 2: Ph in di protein ca CTX

3.2 Th hot tnh khng khun

Cc cy c s khc bit cc dy bng protein, c trng trong cng iu kin
chm sc sau 4 thng, l cc nhm cy ny c th hot tnh khng khun, kt
qu c trnh by qua bng 2 v bng 3.
Bng 2: Nng c ch ti thiu (MIC) i vi vi khun ca cc dng CTX (MIC, g/ml)

Vi khun

Dng
CTX

Staph.

Strep.

E. coli

Pseu.

Sal.

1
2
3
4
5
6
7
8

1024
2048
1024
1024
1024
2048
4096
2048

4096
4096
4096
4096
4096
4096
4096
4096

2048
4096
4096
4096
4096
4096
4096
4096

1024
1024
2048
1024
1024
1024
4096
2048

1024
2048
4096
2048
2048
1024
4096
2048

A.
hydrophila
512
512
512
1024
1024
512
1024
512

E.
ictaluri
2048
1024
1024
2048
2048
2048
2048
2048

E.
tarda
256
128
64
256
256
256
512
256

Qua bng 2, cho thy hot tnh khng khun ca cc dng CTX trn cc chng vi
khun th nghim khng ging nhau (ch c dng 4 v 5 ging nhau), nhng tt c
cc dng u tc ng rt mnh trn Edwardsiella tarda (MIC = 64-512 g/ml), k
n Aeromonas hydrophila (MIC=512-1024 g/ml) v cng tc ng yu trn
Streptococcus faecalis v E. Coli (MIC= 2048- 4096 g/ml).

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Bng 3: Nng c ch ti thiu (MIC) i vi vi khun ca cc dng C Mc (MIC,

g/ml)

Dng
C
Mc
1
2
3
4
5
6
7
8
9
10
11

Staph. Strep.
2048
1024
1024
1024
1024
2048
1024
1024
1024
1024
1024

2048
2048
2048
2048
2048
2048
2048
2048
2048
2048
2048

E. coli

Pseu.

Vi khun
Sal.

4096
2048
2048
2048
2048
4096
2048
2048
2048
2048
2048

2048
2048
2048
2048
2048
2048
2048
2048
2048
2048
2048

4096
2048
2048
2048
2048
4096
2048
2048
2048
2048
2048

A.
hydrophila
2048
1024
1024
1024
1024
2048
1024
1024
1024
1024
1024

E.
ictaluri
512
512
512
512
512
512
512
512
512
512
512

E. tarda
256
512
512
256
256
256
256
256
256
256
256

Kt qu bng 3 cho thy cc dng C Mc u c kh nng tc ng trn cc vi

khun th nghim v c th chia lm 3 nhm (dng 1 ging dng 6; dng 2 ging
dng 3; dng 4, 5, 7, 8, 9, 10, 11 ging nhau). Cc dng C Mc u tc ng rt
mnh trn Edwardsiella tarda (MIC=256-512 g/ml), k n Edwardsiella ictaluri
(MIC=512 g/ml), Staphylococcus aureus v Aeromonas hydrophila (MIC=10242048 g/ml).
S a dng v di truyn ca cy CTX v C Mc cng nh hng n hot tnh
khng khun ca chng vi s khc bit ch s MIC. Kt qu in di gip chn lc
dng c hot tnh khng khun mnh trn vi khun th nghim.
Theo kinh nghim dn gian s dng CTX tr lin cu, t cu khun vng,
trc khun bch hu, ( Huy Bch et al., 2004); dng C Mc phng tr nhim
khun, lm chng lnh vt m trong phu thut, tc dng tt trong nhim khun
ng h hp, tr mn nht, vim da, dng ngoi lm thuc st trng vt thng v
vt lot gia sc. Nh vy kt qu MIC gii thch c s dng hiu qu cc
cy CTX v C Mc trong tr bnh ca dn gian. Bn cnh y cn pht hin cc
cy thuc ny c kh nng tc ng rt tt trn vi khun gy bnh trn c l
Edwardsiella tarda gy p xe gan thn, gy bnh trn tm cng xanh (Quinn,
1994), Edwardsiella tarda cn ly nhim t c sang ngi gy tiu chy, vim h
thng niu, vim mng no, vim ni tm mc, vim d dy rut, p xe vi trng,
p xe vng chu; gy nhim khun d dy rut, vim rut gi, p xe gan v bnh
kit l ngi (Janda et al., 1991). Edwardsiella ictaluri gy bnh nhim trng
mu, bnh gan thn m c tra v Aeromonas hydrophila gy bnh m trn
c. Cc mm bnh ny khng rt nhiu khng sinh mnh v gy thit hi ng
k cho cc nh nui trng thy sn (Tu Thanh Dung et al., 2008). Mt khc, vic
s dng khng sinh iu tr gy chi ph cao v s tn d khng sinh trong sn
phm thy sn nh hng n sc khe con ngi, cn l ro cn cc doanh
nghip Vit Nam trong xut khu. Do , pht hin kh nng khng cc vi khun
gy bnh trn c ca cy CTX v C Mc s gp phn khng nh trong lnh vc
nui trng thy sn.
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Thng qua kt qu in di protein cng gip chn lc dng CTX v C Mc

c hot tnh khng khun cao. Cc dng c hot tnh cao ny hy vng s l tim
nng thay th khng sinh trong tng lai.
4 KT LUN
Ch thn xanh v C Mc u khng thun chng, chng c nhiu dng (Ch
thn xanh 8 dng v C Mc c 11 dng) v cc dng ny c s khc bit v
hot tnh khng khun, cc dng Ch thn xanh v C Mc u c kh nng tc
ng trn vi khun th nghim, c bit tc ng rt mnh trn vi khun gy bnh
ng vt thy sinh (Ch thn xanh mnh nht trn Edwardsiella tarda, C Mc
tc ng mnh nht trn Edwardsiella ictaluri).
TI LIU THAM KHO
Huy Bch, ng Quang Chung, Bi Xun Chng, Nguyn Thng Dong, Trung
m, Phm Vn Hin, V Ngc L, Phm Duy Mai, Phm Kim Mn, on Th Thu,
Nguyn Tp v Trn Ton (2004), Cy Thuc V ng Vt Lm Thuc Vit Nam, Tp
II, NXB Khoa Hc V K Thut.
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Powers, Robert P. Kokka, and K. Tamura (1991), Pathogenic Properties of Edwardsiella
Species, Journal of Clinical Microbiology 9th ser.
Laemmli U.K. (1970), Cleavage of structural proteins during the assembly of the head of
bacteriophage T4, Nature 227: 680 - 685.
Nguyn Vn n v Nguyn Vit Tu (1985), Phng php nghin cu ha hc cy thuc,
NXB Y hc Tp H Ch Minh.
Quinn P. (2004), Clinical veterinary microbiology, Elseviers Health Sciences Rights,
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Rao R., Vaglio M.D., Paino D'Urzo M. and Monti L. (1992), Identification of Vigna spp.
through specific seed storage polypeptides, Euphytica 62:39-43.
Thanh V.C., Nguyen T.N., Hirata Y. and Thuong N.V. (2003), Antenna protein diversity of
prawns (Macrobrachium) in the MeKong Delta, Biosphere Conservation 5:11-17.
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H Ni.
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Antimicrobial susceptibility pattern of Edwardsiella ictaluri isolates from natural
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