Intern
ati
o
n
a
l
Jo
urn
a
l
o
f
P
u
b
lic Hea
l
th Science (IJ
P
HS)
V
o
l.4
,
No
.2
,
Jun
e
2
015
, pp
. 81
~87
I
S
SN
: 225
2-8
8
0
6
81
Jo
urn
a
l
h
o
me
pa
ge
: h
ttp
://iaesjo
u
r
na
l.com/
o
n
lin
e/ind
e
x.ph
p
/
IJPHS
TLC-Bi
o
autography Prof
ile of Et
hyl Acet
at
e E
x
t
r
act of
5 B
a
cteria Is
ol
at
ed f
r
om
Ficus carica
L Rhizosphere
Nanik Sulistya
n
i, Iin Na
rw
a
n
ti
Faculty
of Phar
macy
, Univ
ersitas Ahmad Dahlan, Yog
y
ak
arta, I
ndonesia
Article Info
A
B
STRAC
T
Article histo
r
y:
Received Dec 30, 2014
Rev
i
sed
Feb
20
, 20
15
Accepted Apr 26, 2015
Research h
a
s been conducted on
the 5 is
olates o
f
bacter
ia isolated from the
rhizosphere of
Ficus
car
i
ca
L
as
a producer
of antibio
tics
.
T
h
e previous
s
t
ud
y
s
howed the
y
hav
e
NRP
S
gene profil
es
that differ from
each other. Th
is
stud
y
aim
s
to de
term
ine th
e
TL
C Rf
spots having inhibition
activity
agains
t
the growth of
Staphylo
coccus aureus
ATCC 25923 and
Escherichia coli
ATCC 25922. The stud
y
was conducted using
th
e 5 bacter
ia isolates, namely
T19,
T24,
T25
,
T37
and
T41
.
Al
l
is
ol
at
es
were
ferm
ent
e
d at
room
tem
p
eratur
e for
14 da
y
s
. Furth
e
r the e
ach bro
t
h cultur
e
was filte
red and
extracted using
eth
y
l acetate. C
o
mponent
s in the extr
act wer
e
separated
b
y
thin la
yer chro
m
a
tograph
y
(
T
LC) with the m
obile phase of chloroform
-
methanol (7: 3),
followed b
y
b
i
o
a
utograph
y
test against th
e
S. au
reus
and
E.
coli
to
de
term
in
e th
e
chrom
a
togr
am
spots conta
i
n
i
ng an
tibio
tics.
T
L
C resul
t
s
showed all isolates had differ
e
nt chro
matogram profiles. Th
e bio
a
utograp
h
y
res
u
lts
s
howed
that
onl
y
is
olat
e T25
c
a
n pro
duce
antib
ioti
cs
aga
i
ns
t
S.
aureus
. The antibiotic spot was at Rf 0.
9 in the use of ch
loroform-methanol
(7: 3) solv
ent
s
y
stem
.
Keyword:
An
tib
i
o
tics
Bio
a
u
t
og
r
a
ph
y
E. c
o
li
S. a
u
re
us
Copyright ©
201
5 Institut
e
o
f
Ad
vanced
Engin
eer
ing and S
c
i
e
nce.
All rights re
se
rve
d
.
Co
rresp
ond
i
ng
Autho
r
:
Nan
i
k
Su
listyan
i,
Faculty of Pha
r
m
acy,
Uni
v
ersiats
Ahmad Da
hlan,
Jal
a
n
P
r
o
f
.
D
r
. Soe
pom
o,
S
.
H
.
,
Ja
nt
ura
n
,
W
a
r
u
n
g
bot
o, Um
bul
ha
rj
o 5
5
6
1
2
, Yo
gy
aka
r
t
a
, In
do
nesi
a.
Em
a
il: n
a
n
i
k
s
ulistyan
i@g
m
ai
l.co
m
1.
INTRODUCTION
Th
e em
erg
e
n
c
e of
p
a
tho
g
e
n
i
c micro
b
i
al
resistan
ce to
v
a
rio
u
s
an
tib
io
tics (m
u
ltiresistan
t
) is a
seriou
s
p
r
ob
lem
in
th
e
treat
m
e
n
t
o
f
infectio
u
s
d
i
seases. Mu
ltires
istan
t
of m
i
cro
b
i
al p
a
thog
en
s i
n
creases th
e m
o
rbid
ity
an
d m
o
rtality
d
u
e
to
i
n
fectiou
s
d
i
seases. Th
is m
u
ltiresis
t
a
n
t
en
cou
r
ag
e
research
ers in
th
e wo
rl
d
t
o
fi
n
d
n
e
w
an
d m
o
re sensitiv
e an
tib
i
o
tics ag
ainst resistan
t m
i
cro
b
i
al p
a
th
og
ens [1
]-[3
]
.
On
e
p
o
t
en
tial so
urce of
n
e
w an
tib
i
o
tic m
o
lecu
les is
Actin
o
m
ycetes. Histo
r
ically, Acti
n
o
m
ycetes
pr
o
duce t
h
e l
a
rgest
num
ber
of cl
asses
of a
n
t
i
b
i
o
t
i
c
s suc
h
as t
e
t
r
acy
cl
i
n
e, am
i
nogl
y
c
o
s
i
d
es, ce
phal
o
s
p
o
r
i
n
s
and m
acrolide
s
[4]. Actinomycetes
i
s
also a so
urce
o
f
ne
w ant
i
b
i
o
t
i
c
s and com
p
o
u
nds
wi
t
h
vari
ous
p
h
a
rm
aco
lo
g
i
cal activ
ities g
u
id
e [4
],[5
]. Ogu
n
m
won
y
i et al. [6
] stated
t
h
at ap
prox
im
atel
y 7
0
%
of an
tibio
tics
were
fo
u
nd t
o
ori
g
i
n
at
e f
r
om
Act
i
nom
y
cet
es, so
Act
i
nom
ycet
es becom
e
a sou
r
ce
of
pot
e
n
t
i
a
l
for e
x
pl
o
r
at
i
on
p
r
od
u
c
e drug
s, p
a
rticu
l
arly n
e
w an
tib
io
tics
[7
].
M
a
ny
scree
n
i
n
gs
have
bee
n
carri
ed
o
u
t
an
d res
u
l
t
e
d i
n
t
h
o
u
sa
n
d
s o
f
b
i
oact
i
v
e m
o
l
e
cul
e
s f
r
om
m
i
crobes. T
h
e
r
efo
r
e a ne
w ap
pr
oac
h
i
s
need
ed t
o
re
d
u
ce t
h
e l
i
k
el
i
hoo
d o
f
redi
sc
ove
ry
of
kn
ow
n c
o
m
poun
d
s
[8]
.
Sy
st
em
at
ic ap
pr
oac
h
or
a
new
t
ech
no
l
ogy
i
n
t
h
e
se
arch
f
o
r
ne
w
dr
u
g
ca
ndi
dat
e
com
p
o
u
n
d
s
[
9
]
-
[
1
1]
shoul
d
be
applied, so that t
h
e
processes
are carri
ed
o
u
t
no
vai
n
. T
h
i
s
ap
pr
oac
h
ca
n
be d
o
n
e
wi
t
h
t
h
e
ap
p
lication
o
f
an
alysis to
i
d
en
tify and
elimin
ate s
econd
ary
m
e
tab
o
lites are alread
y
k
n
o
w
n
i
n
t
h
e research
process
as ea
rly as possi
ble.
This a
n
alysis is also
called
d
e
rep
licatio
n.
Evaluation Warning : The document was created with Spire.PDF for Python.
I
S
SN
:
2
252
-88
06
I
J
PH
S Vo
l. 4
,
N
o
. 2
,
Jun
e
201
5
:
8
1
–
87
82
Dere
pl
i
cat
i
on i
s
a pr
ocess
f
o
r
screeni
ng
by
c
o
m
p
ari
ng c
o
m
p
o
u
nds
o
r
sec
o
nda
ry
m
e
t
a
bol
it
es t
h
at
are
alread
y
k
nown to
th
e alleg
e
d
n
e
w co
m
p
ou
nd
s, so th
ere is
n
o
rep
e
titio
n
of
research
for
t
h
e resu
lts o
r
the
sam
e
com
pou
nd
[
1
2
]
. Dere
pl
i
cat
i
on can
be a
ppl
i
e
d t
o
e
x
t
r
act
natu
ral m
a
terial
s th
at con
t
ain certain
com
p
ounds or
metab
o
lites [12
]
, m
i
cro
b
i
al
(eg Actino
m
y
cetes) as
well as
g
e
n
e
s th
at
en
co
d
e
m
e
ta
b
o
lites
[13
]
.
Vari
o
u
s
app
r
oaches
ha
ve bee
n
car
ri
e
d
o
u
t
,
am
ong
ot
he
rs de
repl
i
c
at
i
on i
n
m
o
l
ecul
a
r, s
p
ect
r
o
sc
opy
,
or c
o
m
b
i
n
at
i
o
n
s
th
ereo
f.
Mo
lecu
lar
o
r
g
e
no
m
i
c ap
p
r
o
a
ch
es sh
ow rem
a
rk
ab
le p
r
og
ress i
n
recen
t years [1
4
]-[1
9
]
.
One
app
r
oach i
s
m
o
st
l
y
do
ne
by
anal
y
s
i
s
of t
h
e
pol
y
k
et
i
d
e sy
nt
hase
(P
KS
)
and
n
o
n
-ri
bo
so
m
a
l
pept
i
d
e sy
nt
et
ase
(NR
P
S)
ge
nes
[1
3]
,[
2
0
]
-
[
21]
.
PK
S a
n
d
NR
PS
gene
s a
r
e t
h
e
gen
e
s e
n
co
di
n
g
pol
y
k
e
t
i
d
es an
d
n
o
n
-ri
b
o
som
a
l
p
e
p
tid
es.
Po
ly
k
e
tid
es and
no
n-ri
b
o
so
m
a
l p
e
p
tid
es are th
e co
llectio
n o
f
m
u
ltifu
n
c
tio
n
a
l pro
t
ein
s
th
at
syn
t
h
e
size seco
nd
ary m
e
tab
o
lites [22
]
. The d
i
fferen
ce i
n
th
e
profile o
f
t
h
ese
g
e
nes sho
w
ed
a
gro
u
p
o
f
secon
d
a
ry
m
e
t
a
b
o
lites p
r
o
d
u
c
ed
d
i
fferen
ces.
Approac
h
with spectroscopic
analys
i
s
pl
ay
s an im
port
a
nt
rol
e
i
n
t
h
e p
r
ocess o
f
t
h
e d
i
scove
ry
of
an
tib
io
tics. Th
is an
alysis can
b
e
u
s
ed
early in
th
e pro
cess to detect the presence of
certain
co
m
p
o
und
s
in
th
e
ext
r
act
. Seve
ra
l
spect
r
o
sc
opi
c
m
e
t
hod
s have
bee
n
use
d
t
o
dere
pl
i
cat
i
on s
u
ch
as HPLC
[2
3]
, NM
R
[
1
2]
an
d
LC-M
S [
2
4]
-[
2
5
]
.
Infra
red ra
diation
(IR
) ca
n
potentially be used to
de
re
plication. This
is because
two m
o
lecules
of
d
i
fferen
t
co
m
p
o
und
s ch
em
ica
l
stru
ctu
r
e
will p
r
o
d
u
ce a d
i
fferen
t IR sp
ectru
m
. Th
is is
d
u
e
t
o
th
e d
i
fferent
bo
n
d
t
y
pes o
r
di
ffe
re
nt
vi
b
r
at
i
on f
r
eq
ue
nci
e
s. Al
t
h
ou
g
h
t
h
e sam
e
ki
nds
of
bo
n
d
s, b
u
t
di
ffe
re
nt
com
pou
n
d
s,
the differe
n
t
freque
ncy
because of th
e c
o
mm
on bond bei
n
g in a
differe
n
t
environm
ent [26]. In
gene
ral, t
h
e
m
o
lecu
les h
a
ve
m
a
n
y
b
o
n
d
s
an
d
each
b
ond can
pro
d
u
ce so
m
e
k
i
n
d
of
IR-
activ
e v
i
b
r
at
io
n
s
. I
R
sp
ectra ar
e
com
p
lex and t
h
ere
are m
a
ny abs
o
rptio
n ba
nds
of overla
pping and
distin
ct
ive for each com
pound.
The
r
efore
the spect
rum
of
IR s
p
ectra
is use
d
to i
d
e
n
tify com
p
ou
nd
s
b
y
co
m
p
ar
i
n
g th
e
I
R
sp
ectra with
referen
ce
com
pounds (finge
r printi
ng) [27].
Until recently, IR
spectroscopy has not
b
een
use
d
for m
e
ta
bolit
e
dere
plication
of Actinom
ycetes. The
r
efore the use
of IR
sp
ectro
scop
y meth
od
is an
app
licatio
n
of the n
e
w
m
e
t
hod t
o
do
dere
pl
i
cat
i
on i
n
ant
i
b
i
o
t
i
c
expl
o
r
at
i
o
n
.
It
i
s
expect
e
d
t
h
at
t
h
i
s
can be a st
anda
r
d
m
e
t
hod
i
n
t
h
e
next
pe
ri
o
d
(
f
i
nge
r pri
n
t
i
n
g
)
[
29]
.
Pre
v
i
o
us st
u
d
i
e
s [2
8]
-
[
2
9
]
h
a
ve s
u
ccessf
ul
l
y
obt
ai
ne
d 1
3
i
s
ol
at
es
of
Act
i
nom
y
cet
es whi
c
h ar
e
expect
e
d
t
o
pr
od
uce a
n
t
i
b
i
o
t
i
c
s. B
a
sed
on
R
F
LP anal
y
s
i
s
of the
NRPS
gene
, they can be classified i
n
to
5
g
r
ou
p
s
. Each
gr
oup
can
b
e
tak
e
n
1
iso
l
ate fo
r
f
u
r
t
h
e
r
study, so
th
er
e ar
e
5
iso
l
ates to
b
e
f
u
r
t
h
e
r
in
vestig
ated.
Hence
t
h
i
s
res
earch
i
s
i
m
por
t
a
nt
t
o
be c
o
nt
i
nue
d s
o
t
h
at t
h
e a
n
tibiotic c
a
n
be c
h
ara
c
terized a
n
d e
x
pe
cted to
obt
ai
n
ne
w a
n
t
i
bi
ot
i
c
s.
2.
R
E
SEARC
H M
ETHOD
Materials: Starch Nitrate
Aga
r
/Brot
h
,
Nu
trien
t
Ag
ar, Mu
eller Hin
t
on
Agar,
St
a
phyl
o
co
ccus aur
eu
s
A
T
CC259
23
,
Escheric
hia c
o
li
ATCC25922, ethyl acetate,
TLC plate.
Procedure:
1.
Ferm
en
tatio
n
1 l
o
op
(l
o
o
p
)
act
i
nom
y
cet
es col
o
ni
es was
i
noc
ul
at
ed i
n
5 m
L
of
m
e
di
um
St
arch-
N
i
t
r
at
e B
r
ot
h a
nd
i
n
cu
bat
e
d o
n
a rot
a
ry
sha
k
e
r
at
200
-
2
5
0
r
p
m
for 5 day
s
at
room
t
e
m
p
erat
ure
.
The c
u
l
t
u
re
was t
h
e
n
i
noc
ul
at
ed i
n
t
o
1
00 m
l
Li
qui
d
St
arch
-
N
i
t
r
at
e i
n
50
0 m
L
Erl
e
nm
ey
er and i
n
cu
bat
e
d
at
r
o
om
t
e
m
p
erat
ure
sha
k
er
14
day
s
. The
n
t
h
e c
u
l
t
u
re
was t
r
a
n
sfe
rre
d t
o
a
coni
cal
t
u
be a
nd ce
nt
ri
f
u
ged
at
30
00
rpm
1
5
min
u
t
es. Th
e su
p
e
rn
atan
t
was tak
e
n as
a source
o
f
second
ary
m
e
tab
o
lites.
2.
Ex
traction
o
f
seco
nd
ary m
e
ta
b
o
lites
The supernata
n
t containi
ng
seconda
ry
m
e
t
a
bolites was e
x
tracted
with
ethyl acetate
(1: 1) (v/v) by
sh
ak
ing
fo
r
10
m
i
n
u
t
es. Th
e
to
p
layer
was t
a
k
e
n
with
a
Pasteu
r
p
i
p
e
tte inserted
in th
e t
u
b
e
. Ex
traction
was ca
rrie
d
out 2 tim
e
s. Ethyl acetate phase was
co
llec
t
ed and
passe
d through a c
o
lum
n
of s
odi
um
su
lfate th
at h
a
s b
een
filled
.
Su
b
s
equ
e
n
tly, t
h
e filtrate ev
apo
r
ated
to
a v
o
l
u
m
e o
f
1
m
L
an
d
st
o
r
ed
in
the
r
e
fr
ig
er
ato
r
fo
r th
e
n
e
x
t
test.
3.
Thin Layer C
h
rom
a
togra
phy
(TLC)
TLC of et
hyl acetate extracts was perf
orm
e
d with the stationary
phase
of
silica gel F254
(E. Merc
k) and
m
obi
l
e
phase of c
h
l
o
r
o
fo
rm
-m
et
hanol
(
7
:
3
)
. C
h
rom
a
t
ogr
am
det
ect
i
on i
s
do
ne by
ul
t
r
a vi
ol
et
ray
s
2
54
an
d 366
n
m
4.
Pre
p
aration of test
bacteria
1 ose bacterial colonies of
S
.
aur
eus
an
d
E. coli
we
re
gr
o
w
n i
n
1 m
L
of
B
H
I,
a
n
d
i
n
c
u
ba
t
e
d f
o
r
24
ho
ur
s
at 37
o
C
.
S
ubse
que
nt
l
y
, i
t
was t
a
ken 1
0
0
uL
and
p
u
t
i
n
t
o
1
m
L
of B
H
I m
e
di
um
, i
n
cubat
e
d f
o
r
4 h
o
u
r
s a
t
37
o
C. Th
en
it
was
d
ilu
ted wit
h
0
.
9
%
NaCl t
o
turb
id
ity equal to
th
e Mc Farland
stan
d
a
rd
(10
8
CFU/ m
L
).
The test
bacterial suspe
n
si
on
was
s
p
re
ad
o
n
M
u
el
l
e
r Hi
nt
o
n
m
e
di
um
.
Evaluation Warning : The document was created with Spire.PDF for Python.
I
J
PH
S I
S
SN
:
225
2-8
8
0
6
TLC-Bi
o
au
togra
p
h
y
Pro
file of Eth
y
l Aceta
t
e
Extra
c
t o
f
5 Bacteria
Iso
l
a
t
ed
fro
m
.... (Nan
ik Su
listya
n
i
)
83
5.
Bio
a
u
t
og
r
a
ph
y
The
bi
oa
ut
o
g
r
aphy
agai
nst
t
e
st
bact
eri
a
o
f
TLC
pl
at
e c
o
n
t
ai
ni
ng
pat
c
hes
ch
rom
a
t
ogra
m
was carri
e
d
out
on
M
u
el
l
e
r
Hi
nt
o
n
a
g
ar m
e
di
um
. The exa
m
i
n
at
i
on wa
s
per
f
o
r
m
e
d by
t
ouc
hi
n
g
t
h
e
T
L
C
pl
at
e f
o
r
3
0
min
u
t
es o
n
M
u
eller Hin
t
o
n
ag
ar m
e
d
i
u
m
were
p
r
ev
iou
s
l
y
cu
ltiv
ated
th
e b
acteria test. Su
b
s
equ
e
n
tly, it
was i
n
c
ubat
e
d
at
37°C
f
o
r 1
8
-
2
4
h
o
u
rs
. The
exi
s
t
e
nce o
f
s
t
eri
l
e
zone (
n
o
gr
owt
h
o
f
bac
t
eri
a
l
col
oni
es
)
aft
e
r i
n
c
u
bat
i
o
n
reveal
s t
h
at
t
h
e c
h
r
o
m
a
t
ogr
am
spot
s c
o
nt
ai
n ant
i
b
i
o
t
i
c
co
m
poun
ds
3.
R
E
SU
LTS AN
D ANA
LY
SIS
Thi
s
st
u
d
y
use
d
5 sel
ect
ed i
s
ol
at
es havi
ng
d
i
ffere
nt
NR
P
S
(N
on R
i
bos
om
al
Pept
i
d
e Sy
nt
het
a
se)
gen
e
pr
ofi
l
e
s c
o
ded
i
s
ol
at
e T1
9,
T
2
4
,
T
2
5
,
T
3
7 a
n
d
T4
3.
T
h
e fe
rm
ent
a
t
i
on pr
o
cess i
s
car
ri
ed
out
o
n
eac
h i
s
ol
at
es
at
room
t
e
m
p
erat
ure
fo
r 1
4
d
a
y
s
. A t
o
t
a
l
of
10 m
L
st
art
e
r was cul
t
u
re
d f
o
r
5 day
s
at
ro
om
t
e
m
p
erat
ure wi
t
h
ag
itatio
n
in
100
m
L
o
f
SNB (Starch
N
i
t
r
at
e
B
r
ot
h
)
m
e
di
um
. Aft
e
r 5
day
s
, t
h
e starter
was tran
sferred
in
to
1
L
SNB
m
e
di
um
and
t
h
e i
n
cu
ba
t
i
on
was c
ont
i
nue
d
fo
r
5
day
s
. A
t
o
t
a
l
o
f
3
0
0
m
l
of cul
t
u
r
e
, was
t
h
e
n
i
n
c
ubat
e
d
i
n
3
L
SNB
m
e
di
um
for
1
4
da
y
s
.
The culture was then filtere
d and
extracte
d
with ethyl acetate. Th
e use of ethyl acetate has been
wid
e
ly app
lied in
v
a
ri
o
u
s research
o
f
an
tib
i
o
tics iso
l
a
tio
n
.
Th
is is b
ecau
s
e an
tib
io
tics are g
e
n
e
rally semip
o
l
ar
and easily isolated with et
hyl acetat
e
[21],[30].
T
h
e product of extr
action
was t
h
en e
v
aporated with
rot
a
e
v
ap
orat
or
and
obt
ai
ne
d e
x
t
r
act
s. T
h
e ex
t
r
act
was t
h
en
per
f
o
r
m
e
d TLC
-bi
o
a
u
t
o
g
r
ap
hy
. TLC
i
s
do
ne wi
t
h
t
h
e st
at
i
ona
ry
pha
se o
f
si
l
i
ca gel
F
2
54 a
n
d
t
h
e m
obi
l
e
pha
se o
f
c
h
l
o
r
o
fo
r
m
m
e
t
h
anol
(
7
:
3).
TLC
resul
t
s are
prese
n
t
e
d
i
n
Fi
gu
re 1.
Fi
gu
re
1.
T
h
e
TLC
ch
rom
a
t
ogram
s un
de
r
U
V
25
4
nm
(l
eft
)
a
n
d
U
V
3
6
6
nm
(ri
ght
)
The Rf val
u
es
as res
u
lts of T
L
C to
1.25 m
g
of et
hyl acetate extracts
of liqui
d
c
u
ltures of each
isolate
can
be calc
u
lated
for eac
h s
pot and are
summarized i
n
Ta
ble
1.
Evaluation Warning : The document was created with Spire.PDF for Python.
I
S
SN
:
2
252
-88
06
I
J
PH
S Vo
l. 4
,
N
o
. 2
,
Jun
e
201
5
:
8
1
–
87
84
Tabl
e 1.
R
f
Va
l
u
es of TLC
c
h
rom
a
t
ogram
s
isolat
e
Rf
(uv254)
Rf
(uv366)
T
24 0
0
0.
15
0.
28
0.
56
0.
43
0.
84
0.
84
0.
91
0.
91
T
43 0
0
0.
19
0.
19
0.
54
0.
80
0.
91
0.
91
T
37 0
0
0.
21
0.
21
0.
70
0.
70
0.
83
0.
82
0.
89
0.
89
0.
93
T
19 0
0
0.
25
0.
63
0.
76
0.
82
0.
79
0.
93
0.
91
T
25 0
0
0.
91
0.
91
B
a
sed o
n
R
f
v
a
l
u
es o
f
TLC
s
pot
s
(Ta
b
l
e
1
)
,
i
t
can be
ob
se
rve
d
t
h
e
di
ve
rs
i
t
y
of i
s
ol
at
es t
e
st
ed. T
h
er
e
are 2 sp
ot
s i
n
T2
5, 3 sp
ot
s i
n
T43,
5 sp
ot
s i
n
T1
9 an
d T2
4
,
whi
l
e
T3
7 ha
d 6 sp
ot
s. Al
t
h
o
u
gh T
19 a
n
d T2
4
have t
h
e sam
e
num
ber
of s
p
o
t
s, but
t
h
e R
f
val
u
e
respect
i
v
e di
ffe
re
nt
. It
i
ndi
cat
ed t
h
at
t
h
e sp
ot
s a
ppea
r
ed
i
n
al
l
i
s
ol
at
es var
y
bot
h t
h
e
nu
m
b
er of s
pot
s
and t
h
ei
r R
f
va
l
u
es. T
h
i
s
i
s
c
onsi
s
t
e
nt
wi
t
h
t
h
e res
u
l
t
s
o
f
p
r
evi
o
u
s
st
udi
es
base
d
on
NR
PS
ge
ne
p
r
o
f
i
l
e
t
h
at
s
h
ows
t
h
e
di
f
f
er
e
n
ce
bet
w
ee
n al
l
i
s
ol
at
es t
e
st
ed
[2
8]
. T
h
e
res
u
l
t
o
f
the analysis
of the scatter
pl
ot bet
w
een the num
b
er
of
patch
e
s an
d Rf v
a
lu
es as list
e
d
in Figur
e
2 (
U
V
det
ect
i
on at
2
5
4
nm
) and Fi
g
u
re
3 (
UV
det
ect
i
on at
36
6
nm
), i
ndi
cat
ed
t
h
at
t
h
e T25 i
s
t
h
e m
o
st
di
fferent
iso
l
ate. T25 only sh
ow
ed
2
spo
t
s,
bu
t th
e
o
t
her
s
h
a
s m
o
r
e
than
2
spo
t
s.
Fi
gu
re
2.
Scat
t
e
r
pl
ot
of t
h
e
n
u
m
b
er o
f
s
p
ot
s an
d R
f
val
u
es
at
det
ect
i
o
n
u
n
d
er
U
V
l
i
g
ht
of
2
5
4
nm
Evaluation Warning : The document was created with Spire.PDF for Python.
I
J
PH
S I
S
SN
:
225
2-8
8
0
6
TLC-Bi
o
au
togra
p
h
y
Pro
file of Eth
y
l Aceta
t
e
Extra
c
t o
f
5 Bacteria
Iso
l
a
t
ed
fro
m
.... (Nan
ik Su
listya
n
i
)
85
Fi
gu
re
3.
Scat
t
e
r
pl
ot
of t
h
e
n
u
m
b
er o
f
s
p
ot
s an
d R
f
val
u
es
at
det
ect
i
o
n
u
n
d
er
U
V
l
i
g
ht
of
3
6
6
nm
All of the TLC
profiles showe
d
that
each ext
r
act contain
only less
than
5 s
pots and se
parated clearly.
It also
o
ccurred
in
m
a
n
y
o
f
micro
b
i
al
m
e
ta
b
o
lite [31
]
, [32]. Th
erefo
r
e, it
mak
e
s easy to
p
u
rify th
e co
mp
on
en
t
o
f
in
terest. Gen
e
rally, TLC is u
s
ed
to
do
early sep
a
ra
t
i
o
n.
Ho
we
ver, i
t
al
so can
be
use
d
t
o
get
pu
ri
fi
e
d
com
pou
nd
s i
f
t
h
e s
p
ot
ha
ve
b
een
det
e
rm
i
n
ed as t
h
e
p
u
re
co
m
poun
ds.
The test results bioautography ethy
l acetate
extracts of liquid culture
s
of each isolate presented in
Fi
gu
re 4.
Fi
gu
re
4 B
i
oa
u
t
og
rap
h
y
t
e
st
r
e
sul
t
s
agai
nst
S. au
reu
s
(left) and
E. coli
(
r
ig
ht).
The
ar
ro
w
shows
the clea
r z
one
an
d ind
i
cates th
e inh
i
b
ition
of
S. a
u
reus
gr
o
w
t
h
by
t
h
e
co
m
poun
d i
n
t
h
e
act
i
v
e TLC
-s
p
o
t
The
bi
oa
ut
o
g
ra
phy
on T
L
C
sh
owe
d
one cl
ea
r
zone
o
n
l
y
fr
o
m
t
h
e et
hy
l
acet
a
t
e
ext
r
act
of
T2
5 agai
nst
S. a
u
re
us
. T
h
e
clear zone a
p
peared at Rf 0.91 in the sol
v
en
t
sy
st
em
of chl
o
r
o
f
o
rm
-
m
et
hanol
(
7
:
3)
. The
r
efo
r
e
,
it rev
eals th
at t
h
e spo
t
con
t
ains an
tib
io
tic th
at can
in
h
i
b
it the
S. a
u
re
us
growth. T
h
e inhibition zone appears
because the antibiotic can di
ffuse int
o
the medium a
nd inhi
bit the growth of
S. aure
u
s
. S
o
m
e
of
S. aure
u
s
were
dea
d
and s
h
owe
d
the
clear area
on the m
e
diu
m
th
a
t
w
a
s
p
r
ev
io
u
s
ly sp
r
e
ad
ed
b
y
th
e
e
x
amin
a
tion
b
acteria [33
]
. Actu
ally, th
e
oth
e
r iso
l
ates al
so
h
a
d
spo
t
at aroun
d
0
.
91
,
bu
t th
ere
was
no
inh
i
b
ition
zon
e
ov
er
t
h
ere.
It
i
n
di
ca
t
e
d t
h
at
t
h
e c
o
m
poun
d i
n
t
h
e
spot
a
r
e
di
ffe
re
nt
al
t
h
o
u
gh t
h
e
R
f
val
u
e i
s
sa
m
e
. B
a
sed o
n
t
h
e R
f
v
a
lu
e
o
f
th
e activ
e spo
t
and th
e so
lv
en
t syste
m
u
s
ed
in th
is stud
y, it can
b
e
co
n
c
l
u
d
e
d
t
h
at th
e
activ
e
com
pound is a
non
pola
r
. T
h
e
non
pola
r
com
pound will be
eluted fa
ster then the
polar in the chl
o
roform
-
meth
an
o
l
(7
: 3)
so
lv
en
t
system
.
B
a
sed
on
t
h
e
bi
oa
ut
o
g
ra
p
h
y
res
u
l
t
s
o
f
T2
5, t
h
e a
n
t
i
b
i
o
t
i
c i
n
T
2
5
has
na
rr
ow
s
p
ect
r
u
m
act
i
v
i
t
y
because the spot only inhi
bits the growt
h
of
S. a
u
re
us
an
d
di
dn
’t
i
nhi
bi
t
t
h
e gr
owt
h
o
f
E. coli
.
S. a
u
re
us
is a
Gram
p
o
s
itiv
e b
acteria, wh
ile
E. c
o
li
is
Gram
n
e
g
a
tiv
e.Thu
s
, p
e
rh
ap
s th
e an
tib
i
o
tic on
ly su
itab
l
e to kill th
e
Gram
p
o
s
itiv
e b
acteria, bu
t n
o
t
to
Gram
n
e
g
a
tiv
e. In
app
licatio
n
s
,
u
s
ag
e of n
a
rro
w
sp
ectru
m
an
tib
io
tic is
b
e
tter th
en
t
h
e
b
r
o
a
d
,
b
ecau
s
e on
ly certain
bacteria will b
e
k
illed
.
Th
e li
mita
tio
n
of t
h
is research
is on
l
y
u
s
e
2
ki
n
d
s of bact
e
r
i
a
(
S. aure
u
s
and
E. c
o
li
.).
Actu
ally th
ere are still
m
a
n
y
k
i
nd
s
o
f
o
t
h
e
r Gram
p
o
s
itive and
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I
S
SN
:
2
252
-88
06
I
J
PH
S Vo
l. 4
,
N
o
. 2
,
Jun
e
201
5
:
8
1
–
87
86
n
e
g
a
tiv
e
b
acteria. So
it is v
e
ry
i
m
p
o
r
tan
t
to
perfo
r
m
it in
th
e n
e
x
t
ex
p
e
rim
e
n
t
. Nev
e
rth
e
less, th
is resu
lt is v
e
ry
i
n
t
e
rest
i
ng a
n
d
im
port
a
nt
t
o
b
e
cont
i
n
ue
d i
n
t
h
e su
bse
que
nt
pu
ri
fi
cat
i
on
pr
ocess t
o
get
t
h
e pu
re ant
i
b
i
o
t
i
c
of
in
terest fro
m
T2
5.
4.
CO
NCL
USI
O
N
TLC resu
lts sho
w
ed
th
at all bacteria iso
l
ates h
a
d
c
h
r
o
m
a
t
ogram
pro
f
i
l
e
s t
h
at
di
f
f
er eac
h
ot
her
.
The
bi
oa
ut
o
g
ra
p
h
y
res
u
l
t
s
sh
ow
e
d
t
h
at
o
n
l
y
T25
i
s
ol
at
e can
pr
o
duce
ant
i
bi
ot
i
c
s a
g
ai
nst
S. a
u
re
us
. T
h
e
s
pot
appea
r
e
d
at
R
f
0.
9 i
n
t
h
e
sol
v
e
n
t
sy
st
em
of ch
l
o
r
o
f
o
rm
-
m
et
hanol
(
7
:
3
)
.
REFERE
NC
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