Journal of Ethnopharmacology 74 (2001) 113123

Antimicrobial and phytochemical studies on 45 Indian

medicinal plants against multi-drug resistant human
pathogens
Iqbal Ahmad *, Arina Z. Beg
Department of Agricultural Microbiology, RAK Institute of Agricultural Sciences, Aligarh Muslim Uni6ersity,
Aligarh 202002, India
Received 8 February 2000; received in revised form 10 August 2000; accepted 15 August 2000
Abstract
Ethanolic extracts of 45 Indian medicinal plants traditionally used in medicine were studied for their antimicrobial
activity against certain drug-resistant bacteria and a yeast Candida albicans of clinical origin. Of these, 40 plant
extracts showed varied levels of antimicrobial activity against one or more test bacteria. Anticandidal activity was
detected in 24 plant extracts. Overall, broad-spectrum antimicrobial activity was observed in 12 plants (L. inermis,
Eucalyptus sp., H. antidysentrica, H. indicus, C. equistifolia. T. belerica, T. chebula, E. ofcinalis, C. sinensis, S.
aromaticum and P. granatum). No correlation was observed between susceptibility of test strains with plant extracts
and antibiotic resistance behaviour of the microbial strains (Staphylococcus aureus, Salmonella paratyphi, Shigella
dysenteriae, Escherichia coli, Bacillus subtilis, Candida albicans). Qualitative phytochemical tests, thin layer chro-
matography and TLC-bioautography of certain active extracts demonstrated the presence of common phytocom-
pounds in the plant extracts including phenols, tannins and avonoids as major active constituents. 2001 Published
by Elsevier Science Ireland Ltd.
Keywords: Medicinal plants; Antimicrobial activity; Multidrug resistance; TLC-bioautography
www.elsevier.com/locate/jethpharm
1. Introduction
Infectious diseases are the worlds leading cause
of premature deaths, killing almost 50 000 people
every day. In recent years, drug resistance to
human pathogenic bacteria has been commonly
reported from all over the world (Piddock and
Wise, 1989; Singh et al., 1992; Mulligen et al.,
1993; Davis, 1994; Robin et al., 1998). However,
the situation is alarming in developing as well as
developed countries due to indiscriminate use of
antibiotics. The drug-resistant bacteria and fungal
pathogens have further complicated the treatment
of infectious diseases in immunocompromised,
AIDS and cancer patients (Rinaldi, 1991; Dia-
mond, 1993). In the present scenario of emergence
of multiple drug resistance to human pathogenic
organisms, this has necessitated a search for new
* Corresponding author.
0378-8741/01/$ - see front matter 2001 Published by Elsevier Science Ireland Ltd.
PII: S0378- 8741( 00) 00335- 4
I. Ahmad, A.Z. Beg / Journal of Ethnopharmacology 74 (2001) 113123 114
antimicrobial substances from other sources in-
cluding plants.
Traditionally used medicinal plants produce a
variety of compounds of known therapeutic
properties (Iyengar, 1985; Chopra et al., 1992;
Harborne and Baxter, 1995). The substances
that can either inhibit the growth of pathogens
or kill them and have no or least toxicity to
host cells are considered candidates for develop-
ing new antimicrobial drugs. In recent years, an-
timicrobial properties of medicinal plants are
being increasingly reported from different parts
of the world (Grosvenor et al., 1995; Ratnakar
and Murthy, 1995; Silva et al., 1996; David,
1997; Saxena, 1997; Nimri et al., 1999; Saxena
and Sharma, 1999). It is expected that plant ex-
tracts showing target sites other than those used
by antibiotics will be active against drug-resis-
tant microbial pathogens. However, very little
information is available on such activity of
medicinal plants (Hasegawa et al., 1995; Lee et
al., 1998). In the present study, we have selected
45 Indian medicinal plants to be screened
against multi-drug resistant bacteria including
Staphylococcus aureus, Salmonella paratyphi, Es-
cherichia coli, Shigella dysenteriae and Candida
albicans. The selection of medicinal plants is
based on their traditional uses (32 plants) in In-
dia and our reported antimicrobial activity of 13
plants (Chopra et al., 1992; Ahmad et al., 1998;
Mehmood et al., 1999). However most of these
plants were not previously screened against
multi-drug resistant, pathogenic organisms. Phy-
tochemical analysis of active plant extracts for
their major group of phytoconstituents and the
active group of certain extracts is also reported
here.
2. Materials and methods
2.1. Plant material
Thirty-ve authenticated plant samples were
collected locally and 10 plant samples were
kindly provided by the Himalaya Drug Co.
(New Delhi, India). All the plant materials were
further identied in the Department of Botany,
AMU, Aligarh, India by a senior plant tax-
onomist, Professor Wazahat Hussain. Voucher
specimens have been deposited in the Depart-
ment of Agricultural Microbiology, RAK Insti-
tute of Agricultural Sciences, AMU, Aligarh,
India. The details of medicinal plants along with
their acquisition code numbers are listed in
Table 1.
2.2. Preparation of plant extracts
Plant extracts were prepared by the method of
Alade and Irobi (1993) with minor modication.
Briey 100 g of each powdered plant material
were soaked in 100 ml of 70% alcohol for 72 h.
Each mixture was stirred every 24 h using a
sterile glass rod. At the end of extraction each
extract was passed through Whatman lter pa-
per No. 1 (Whatman, UK). The alcoholic
ltrates obtained was concentrated in vacuo at
30C and stored at 4C until further use.
2.3. Microorganisms used
The test organisms used included E. coli UP
2566, resistant to M, Cx, Do, Nv, (Central
Drug Research Institute, Lucknow, India),
Bacillus subtilis MTCC-121 (Microbial Institute
of Technology, Chandigarh, India) and drug-re-
sistant clinical isolates of S. aureus IOA-106
(Am, Cu, Cx, Cj, Nal, Co), S. paratyphi IOA-
107 (M, Nv, Cx, Do), S. dysenteriae IOA-108
(M, Tc, Do) and C. albicans IOA-109 (Fu, Ns) ,
kindly provided by the Department of Medical
Microbiology, JN Medical College, AMU, Ali-
garh, India. The clinical isolates were biochemi-
cally and serologically characterized by standard
methods.
2.4. Culture media and inoculum
Sabouraud Dextrose (SD) and Soyabean
Casien Digest (SCD) media (Hi-Media Pvt.
Ltd., Bombay, India) were used for C. albicans
and test bacteria, respectively. Microbial cul-
tures, freshly grown at 37C were appropriately
diluted in sterile normal saline solution to ob-
tain the cell suspension at 10
5
CFU/ml.
I. Ahmad, A.Z. Beg / Journal of Ethnopharmacology 74 (2001) 113123 115
T
a
b
l
e
1
E
t
h
n
o
b
o
t
a
n
i
c
a
l
a
n
d
p
h
y
t
o
c
h
e
m
i
c
a
l
d
a
t
a
o
f
4
5
I
n
d
i
a
n
m
e
d
i
c
i
n
a
l
p
l
a
n
t
s
K
n
o
w
n
p
h
y
t
o
c
o
n
s
t
i
t
u
e
n
t
s
o
f
p
a
r
t
B
o
t
a
n
i
c
a
l
n
a
m
e
;
f
a
m
i
l
y
,
v
o
u
c
h
e
r
C
o
m
m
o
n
n
a
m
e
T
r
a
d
i
t
i
o
n
a
l
u
s
e
s
(
C
h
o
p
r
a
e
t
a
l
.
,
P
a
r
t
u
s
e
d
S
p
e
c
i
m
e
n
n
o
.
s
p
e
c
i
m
e
n
u
s
e
d
1
9
9
2
)
R
h
i
z
o
m
e
B
a
c
h
/
V
a
j
G
l
u
c
o
s
i
d
e
a
c
o
r
i
n
,
a
l
k
a
l
o
i
d
,
E
m
e
t
i
c
,
s
t
o
m
a
c
h
-
a
c
h
e
,
n
e
r
v
e
A
c
o
r
u
s
c
a
l
a
m
u
s
L
.
;
A
r
a
c
e
a
e
,
1
.
1
.
5

3
.
5
%
e
s
s
e
n
t
i
a
l
o
i
l
,
m
e
t
h
y
l
H
D
C
O
-
1
6
6
/
2
9
7
d
y
s
p
e
p
s
i
a
,
c
o
l
i
c
,
t
o
n
i
c
i
n
i
s
o
e
u
g
e
n
o
l
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
;
b
r
o
n
c
h
i
t
i
s
,
d
y
s
e
n
t
e
r
y
i
n
c
h
i
l
d
r
e
n
H
a
r
b
o
r
n
e
a
n
d
B
a
x
t
e
r
,
1
9
9
5
)
L
e
a
v
e
s
A
l
l
i
u
m
c
e
p
a
L
.
;
L
i
l
i
a
c
e
a
e
,
P
i
y
a
z
2
.
M
e
t
h
y
l
a
l
l
y
l
,
d
i
a
l
l
y
l
d
i
m
e
t
h
y
l
,
E
x
p
e
c
t
o
r
a
n
t
p
r
o
p
a
n
e
t
h
i
o
l
,
p
h
l
o
r
o
g
l
u
c
i
n
o
l
,
I
O
A
-
0
1
/
9
9
p
r
o
p
a
n
e
t
h
i
o
l
(
H
a
r
b
o
r
n
e
a
n
d
B
a
x
t
e
r
,
1
9
9
5
)
C
a
r
m
i
n
a
t
i
v
e
,
c
o
u
g
h
s
i
n
f
e
v
e
r
s
,
L
a
s
a
n
A
l
l
i
u
m
s
a
t
i
6
u
m
L
.
;
L
i
l
i
a
c
e
a
e
,
3
.
A
l
l
i
c
i
n
0
.
0
6

0
.
1
%
,
d
i
a
l
l
y
l
B
u
l
b
c
a
t
e
c
h
o
l
s
,
p
r
o
t
o
c
a
t
e
c
h
u
i
c
a
c
i
d
I
O
A
-
0
2
/
9
9
j
u
i
c
e
u
s
e
d
i
n
s
k
i
n
d
i
s
e
a
s
e
s
,
e
a
r
a
c
h
e
s
,
a
t
o
n
i
c
d
y
s
e
p
s
i
a
,
c
o
l
i
c
A
l
l
i
s
t
i
n
I
&
I
I
,
a
j
o
e
n
e
,
a
l
l
y
l
p
r
o
p
y
l
s
u
l

d
e
(
H
a
r
b
o
r
n
e
a
n
d
B
a
x
t
e
r
,
1
9
9
5
;
R
a
t
n
a
k
a
r
a
n
d
M
u
r
t
h
y
,
1
9
9
5
;
N
a
g
e
n
a
w
a
e
t
a
l
.
,
1
9
9
6
)

A
.
s
a
t
i
6
u
m
L
.
;
I
O
A
-
0
3
/
9
9
L
a
s
a
n
L
e
a
v
e
s

4
.
T
o
n
i
c
,
a
s
t
r
i
g
e
n
t
,
a
n
t
i
p
e
r
i
o
d
i
c
5
.
A
z
a
d
i
r
a
c
h
t
i
n
,
m
a
r
g
o
s
i
c
a
c
i
d
B
a
r
k
N
e
e
m
A
z
a
d
i
r
a
c
h
t
a
i
n
d
i
c
a
A
.
J
u
s
s
.
;
s
n
a
k
e
p
e
r
i
o
d
i
c
b
i
t
e
0
.
0
4
%
n
i
m
b
i
n
,
0
.
0
0
1
%
n
i
m
b
i
n
i
n
M
e
l
i
a
c
e
a
e
,
I
O
A
-
0
4
/
9
9
0
.
4
%
n
i
m
b
i
d
i
n
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
B
e
t
a
6
u
l
g
a
r
i
s
L
.
;
C
h
e
n
p
o
d
i
a
c
e
a
e
,
C
h
o
k
u
n
d
a
r
C
o
o
l
i
n
g
,
d
i
a
p
h
o
r
e
t
i
c
B
e
t
a
m
i
c
a
c
i
d
,
P
r
e
b
e
t
a
n
i
n
,
6
R
o
o
t
V
u
l
g
a
x
a
n
t
h
i
n
I
&
I
I
(
H
a
r
b
o
r
n
e
I
O
A
-
0
5
/
9
9
a
n
d
B
a
x
t
e
r
,
1
9
9
5
)
C
a
l
o
t
r
o
p
i
n
,
C
a
l
o
t
r
o
p
a
g
e
n
i
n
,
C
a
l
o
t
r
o
p
i
s
p
r
o
c
e
r
a
L
.
;
M
a
d
a
r
L
e
a
v
e
s
F
e
v
e
r
7
.
A
s
c
l
e
p
e
d
i
a
c
e
a
e
,
I
O
A
-
3
5
/
9
9
l
a
t
e
x
-
u
s
c
h
a
r
i
n
,
C
a
l
o
t
o
x
i
n
,
C
a
l
a
c
t
i
n
L
e
a
v
e
s
C
a
f
f
e
i
n
e
,
t
h
e
a
i
n
e
,
T
h
e
o
b
r
o
m
i
n
e
,
8
.
C
a
m
e
l
i
a
s
i
n
e
n
s
i
s
L
.
;
T
h
e
a
c
e
a
e
,
C
h
a
i
A
s
t
r
i
g
e
n
t
,
d
i
u
r
e
t
i
c
s
t
i
m
u
l
a
n
t
A
s
s
a
m
T
e
a
C
h
o
w
k
L
t
d
.
C
h
l
o
r
g
e
n
i
c
a
c
i
d
,
M
y
r
i
c
e
t
i
n
,
E
p
i
-
g
a
l
l
o
t
a
n
n
i
n
s
,
3
g
a
l
a
c
t
o
s
i
d
e
s
o
f

a
v
o
n
e
s
a
n
d

a
v
o
n
o
l
s
(
H
a
r
b
o
r
n
e
a
n
d
B
a
x
t
e
r
,
1
9
9
5
)
C
a
s
u
a
r
i
n
a
e
q
u
i
s
t
i
f
o
l
i
a
L
.
;
9
.
A
s
t
r
i
g
e
n
t
,
u
s
e
f
u
l
i
n
d
i
a
r
r
h
o
e
a
a
n
d
J
a
n
g
l
i
-
s
a
r
u
B
a
r
k
C
a
s
u
a
r
i
n
,
6

1
8
%
T
a
n
n
i
n
s
d
y
s
e
n
t
e
r
y
C
a
s
u
a
r
i
n
a
c
e
a
e
,
I
O
A
-
0
7
/
9
9
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)

D
e
c
o
c
t
i
o
n
u
s
e
d
i
n
c
o
l
i
c
L
e
a
v
e
s
C
.
e
q
u
i
s
t
i
f
o
l
i
a
L
.
;
I
O
A
-
0
8
/
9
9
J
a
n
g
l
i
-
s
a
r
u
1
0
.
R
i
n
d
M
u
s
a
m
b
i
C
i
t
r
u
s
s
i
n
e
n
s
i
s
L
.
;
R
u
t
a
c
e
a
e
,
C
a
r
m
i
n
a
t
i
v
e
,
t
o
n
i
c
u
s
e
d
f
o
r
a
c
n
e
1
1
.
T
a
n
g
e
t
i
n
,
S
i
n
e
n
s
e
t
i
n
,
L
i
m
o
n
e
n
e
d
e
c
y
c
l
i
c
a
l
d
e
h
y
d
e
,
L
i
n
a
l
o
o
l
I
O
A
-
0
9
/
9
9
d
i
t
e
r
p
i
n
e
o
l
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
;
H
a
r
b
o
r
n
e
a
n
d
B
a
x
t
e
r
,
1
9
9
5
)
L
e
a
v
e
s

1
2
.
C
o
r
d
i
a
d
i
c
h
o
t
a
m
a
L
.
;
B
o
r
a
g
i
n
a
c
e
a
e
,
L
a
s
o
r
a
U
s
e
d
f
o
r
h
e
a
d
a
c
h
e
s
a
n
d
u
l
c
e
r
s
;
I
O
A
-
1
0
/
9
9
d
e
c
o
c
t
i
o
n
u
s
e
d
f
o
r
s
o
r
e
t
h
r
o
a
t
I. Ahmad, A.Z. Beg / Journal of Ethnopharmacology 74 (2001) 113123 116
T
a
b
l
e
1
(
C
o
n
t
i
n
u
e
d
)
C
o
m
m
o
n
n
a
m
e
T
r
a
d
i
t
i
o
n
a
l
u
s
e
s
(
C
h
o
p
r
a
e
t
a
l
.
,
B
o
t
a
n
i
c
a
l
n
a
m
e
;
f
a
m
i
l
y
,
v
o
u
c
h
e
r
S
p
e
c
i
m
e
n
n
o
.
P
a
r
t
u
s
e
d
K
n
o
w
n
p
h
y
t
o
c
o
n
s
t
i
t
u
e
n
t
s
o
f
p
a
r
t
u
s
e
d
1
9
9
2
)
s
p
e
c
i
m
e
n
V
i
t
a
m
i
n
C
,
p
h
o
s
p
h
a
t
i
d
e
s
,
s
e
e
d
s
F
r
u
i
t
s
A
m
l
a
E
m
b
l
i
c
a
o
f

c
i
n
a
l
i
s
G
a
e
r
t
h
.
;
A
c
r
i
d
,
c
o
o
l
i
n
g
,
r
e
f
r
i
g
e
r
a
n
t
d
i
u
r
e
t
i
c
,
1
3
.
c
o
n
t
a
i
n
t
a
n
n
i
n
s
,
C
h
e
b
u
l
i
n
i
c
A
c
i
d
s
u
s
e
d
i
n
d
i
a
r
r
h
o
e
a
,
d
y
s
e
n
t
e
r
y
,
E
u
p
h
o
r
b
i
a
c
e
a
e
,
I
O
A
-
1
1
/
9
9
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
;
H
a
r
b
o
r
n
e
a
n
d
a
n
a
e
m
i
a
,
j
a
u
n
d
i
c
e
a
n
d
c
o
u
g
h
B
a
x
t
e
r
,
1
9
9
5
)
0
.
9

1
.
2
%
o
i
l
:
C
i
n
e
o
l
e
,
P
i
n
e
n
e
L
e
a
v
e
s
A
n
t
i
s
e
p
t
i
c
,
i
n
f
e
c
t
i
o
n
s
o
f
u
p
p
e
r
E
u
c
a
l
y
p
t
u
s
E
u
c
a
l
y
p
t
u
s
s
p
.
;
M
y
r
t
a
c
e
a
e
,
1
4
I
O
A
-
1
2
/
9
9
r
e
s
p
i
r
a
t
o
r
y
t
r
a
c
t
,
s
k
i
n
d
i
s
e
a
s
e
s
,
S
e
s
q
u
i
t
e
r
p
e
n
e
a
l
c
o
h
o
l
s
,
A
s
t
r
g
i
n
b
u
r
n
s
,
r
h
e
u
m
a
t
i
s
m
e
u
d
e
s
m
a
l
,
a
-
p
h
e
l
l
a
n
d
r
e
n
e
(
H
a
r
b
o
r
n
e
a
n
d
B
a
x
t
e
r
,
1
9
9
5
)
L
e
a
v
e
s
1
5
.
A
c
i
d
a
n
t
F
i
c
u
s
c
a
r
i
c
a
L
.
;
M
o
r
a
c
e
a
e
,
F
i
c
u
s
i
n
a
n
d
b
e
r
g
a
p
t
e
n
e
0
.
0
6
%
A
n
j
i
r
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
I
O
A
-
1
3
/
9
9
L
e
a
v
e
s
B
e
r
g
a
p
t
o
l
a
n
d
b
e
r
g
a
p
t
e
n
(
S
w
a
m
i
P
u
r
g
a
t
i
v
e
1
6
.
F
i
c
u
s
r
e
l
i
g
i
o
s
a
L
.
;
M
o
r
a
c
e
a
e
,
P
i
p
a
l
I
O
A
-
1
4
/
9
9
a
n
d
B
i
s
h
t
,
1
9
9
6
)
H
e
m
i
d
e
s
m
u
s
i
n
d
i
c
u
s
R
.
B
r
.
;
0
.
1
8
%
2
-
O
H
,
4
M
e
t
h
y
l
A
n
a
t
a
m
u
l
1
7
.
R
o
o
t
s
D
e
m
u
l
c
a
n
t
,
t
o
n
i
c
,
d
i
a
p
h
o
r
e
t
i
c
,
s
k
i
n
d
i
s
e
a
s
e
s
,
s
y
p
h
i
l
i
s
,
a
n
d
b
l
o
o
d
p
u
r
i

e
r
A
s
c
l
e
p
i
a
d
a
c
e
a
e
,
H
D
C
O
-
2
0
4
/
7
6
b
e
n
z
a
l
d
e
h
y
d
e
,
s
t
e
r
o
l
s
,
g
l
u
c
o
s
i
d
e
s
,
r
e
s
i
n
i
c
a
c
i
d
,
t
a
n
n
i
n
s
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
B
a
r
k
H
o
l
a
r
r
h
e
n
a
a
n
t
i
d
y
s
e
n
t
e
r
i
c
a
R
.
;
D
r
o
p
s
y
,
d
i
a
r
r
h
o
e
a
A
l
k
a
l
o
i
d
s

C
o
n
e
s
s
i
n
e
,
K
u
r
c
h
i
n
e
,
1
8
.
K
u
r
a
c
h
i
K
u
r
c
h
i
c
i
n
e
,
H
o
l
a
r
h
i
m
i
n
e
(
C
h
o
p
r
a
A
p
o
c
y
a
n
a
c
e
a
e
,
H
D
C
O
-
1
2
0
/
2
3
5
e
t
a
l
.
,
1
9
9
2
)
L
e
a
v
e
s
E
s
s
e
n
t
i
a
l
o
i
l
c
a
r
m
e
n
e
,
i
s
o
c
a
m
e
r
e
n
e
,
1
9
.
D
e
c
o
c
t
i
o
n
u
s
e
d
i
n
m
a
l
a
r
i
a
,
a
t
o
x
y
,
L
a
n
t
a
n
a
c
a
m
a
r
a
L
.
;
V
e
r
b
e
n
a
c
e
a
e
,
G
h
a
n
e
r
i
r
h
e
u
m
a
t
i
s
m
I
O
A
-
1
9
/
9
9
m
i
c
r
a
m
e
n
e
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
H
e
n
a
/
M
e
h
d
i
H
e
a
d
a
c
h
e
,
b
u
r
n
i
n
g
o
f
s
k
i
n
,
2
0
.
L
a
w
s
o
n
i
a
i
n
e
r
m
i
s
L
.
;
L
y
t
h
r
a
c
e
a
e
,
L
e
a
v
e
s
G
l
u
c
o
s
i
d
e
,
h
e
n
n
o
t
a
n
n
i
c
a
c
i
d
,
d
e
c
o
c
t
i
o
n
u
s
e
d
f
o
r
s
o
r
e
t
h
r
o
a
t
L
a
w
s
o
n
e
(
H
a
r
b
o
r
n
e
a
n
d
B
a
x
t
e
r
,
I
O
A
-
1
5
/
9
9
1
9
9
5
)
p
-
C
r
e
s
o
l
,
p
h
e
n
o
l
,
m
o
r
i
n
(
H
a
r
b
o
r
n
e
2
1
.
D
e
c
o
c
t
i
o
n
u
s
e
d
f
o
r
s
o
r
e
t
h
r
o
a
t
S
h
a
h
t
u
t
M
o
r
u
s
a
l
b
a
L
.
;
M
o
r
a
c
e
a
e
,
I
O
A
-
1
6
/
9
9
L
e
a
v
e
s
a
n
d
B
a
x
t
e
r
,
1
9
9
5
)
S
t
e
m

E
p
i
l
e
p
s
y
,
d
i
a
r
r
h
o
e
a
,
d
y
s
e
n
t
e
r
y
2
2
.
K
a
l
a
M
u
s
a
p
a
r
a
d
i
s
i
a
c
a
L
.
;
M
u
s
a
c
e
a
e
,
I
O
A
-
1
7
/
9
9
N
e
l
u
m
b
o
n
u
c
i
f
e
r
a
G
a
e
r
t
h
.
;
A
l
k
.
N
e
l
u
m
b
i
n
e
,
a
m
m
o
n
a
i
n
e
A
s
t
r
i
g
e
n
t
,
d
i
a
r
r
h
o
e
a
c
h
o
l
e
r
a
,
f
e
v
e
r
F
l
o
w
e
r
s
2
3
.
K
a
m
a
l
p
r
o
n
u
c
i
f
e
r
i
n
e
(
H
a
r
b
o
r
n
e
a
n
d
N
y
m
p
h
a
c
e
a
e
,
H
D
C
O
-
0
1
/
9
9
B
a
x
t
e
r
,
1
9
9
5
)
N
e
r
i
u
m
i
n
d
i
c
u
m
M
i
l
l
.
;
A
p
o
c
y
a
n
a
c
e
a
e
,
G
l
u
c
o
s
i
d
e
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
S
w
e
l
l
i
n
g
a
n
d
s
k
i
n
d
i
s
e
a
s
e
s
K
a
n
e
r
2
4
.
L
e
a
v
e
s
I
O
A
-
1
8
/
9
9
G
l
u
c
o
s
i
d
e
,
m
e
l
a
n
t
h
i
n
,
s
a
p
o
n
i
n
E
r
u
p
t
i
o
n
s
o
f
s
k
i
n
S
e
e
d
s
K
a
l
o
n
g
i
2
5
.
N
i
g
e
l
l
a
s
a
t
i
6
a
L
.
;
R
a
n
u
n
c
u
l
a
c
e
a
e
,
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
H
D
C
O
-
1
1
1
/
1
9
4
N
y
c
t
a
n
t
h
e
s
a
r
b
o
r
t
r
i
s
t
i
s
L
.
;
O
l
e
a
c
e
a
e
,
A
l
k
a
l
o
i
d
,
r
e
s
i
n
s
,
g
l
u
c
o
s
i
d
e
H
a
r
s
i
n
g
a
r
F
e
v
e
r
,
r
h
e
u
m
a
t
i
s
m
,
o
b
s
t
r
i
n
a
t
e
s
c
i
a
t
a
2
6
.
L
e
a
v
e
s
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
I
O
A
-
2
0
/
9
9
W
h
o
l
e
p
l
a
n
t
7
1
.
3
%
E
u
g
e
n
o
l
,
3
.
7
%
c
a
r
v
a
c
r
o
l
2
7
.
O
c
i
m
u
m
s
a
n
c
t
u
m
L
.
;
L
a
b
i
a
t
a
e
,
G
a
s
t
r
i
c
d
i
s
o
r
d
e
r
s
b
r
o
n
c
h
i
t
i
s
,
e
a
r
T
u
l
s
i
a
c
h
e
a
n
t
i
s
e
p
t
i
c
,
d
i
a
p
h
o
r
e
t
i
c
,
h
e
p
a
t
i
c
H
D
C
O
-
2
3
/
2
8
p
l
a
n
t
2
0
.
4
%
M
e
t
h
y
l
e
u
g
e
n
o
l
1
.
7
%
a
f
f
e
c
t
i
o
n
s
C
a
r
y
o
p
h
y
l
l
e
n
e
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
I. Ahmad, A.Z. Beg / Journal of Ethnopharmacology 74 (2001) 113123 117
T
a
b
l
e
1
(
C
o
n
t
i
n
u
e
d
)
P
a
r
t
u
s
e
d
K
n
o
w
n
p
h
y
t
o
c
o
n
s
t
i
t
u
e
n
t
s
o
f
p
a
r
t
T
r
a
d
i
t
i
o
n
a
l
u
s
e
s
(
C
h
o
p
r
a
e
t
a
l
.
,
B
o
t
a
n
i
c
a
l
n
a
m
e
;
f
a
m
i
l
y
,
v
o
u
c
h
e
r
S
p
e
c
i
m
e
n
n
o
.
C
o
m
m
o
n
n
a
m
e
1
9
9
2
)
u
s
e
d
s
p
e
c
i
m
e
n
P
l
u
m
b
a
g
o
z
e
y
l
a
n
i
c
a
L
.
;
P
l
u
m
b
a
g
i
n
(
B
r
u
n
e
t
o
n
,
1
9
9
5
)
U
s
e
d
i
n
s
k
i
n
d
i
s
e
a
s
e
s
,
d
y
s
p
e
p
s
y
,
C
h
i
t
a
2
8
.
R
o
o
t
p
i
l
e
s
,
l
e
p
r
o
s
y
a
n
d
s
k
i
n
d
i
s
e
a
s
e
s
P
l
u
m
b
a
g
i
n
a
c
e
a
e
,
H
D
C
O
-
4
3
/
6
4
A
p
p
l
i
c
a
t
i
o
n
i
n
e
r
y
s
i
p
e
l
a
s
,
i
n
f
u
s
i
o
n
P
l
a
n
t
C
h
o
t
a
-
l
u
n
y
a
2
9
.

P
o
r
t
u
l
a
c
a
q
u
a
d
r
i
f
o
l
i
a
L
.
;
P
o
r
t
u
l
a
c
a
c
e
a
e
,
I
O
A
-
2
1
/
9
9
u
s
e
d
a
s
d
i
u
r
e
t
i
c
a
n
d
i
n
d
y
s
u
r
i
a
L
e
a
v
e
s
E
u
g
e
n
o
l
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
A
s
t
r
i
n
g
e
n
t
,
f
o
r
b
o
w
e
l
s
,
u
s
e
d
f
o
r
3
0
.
A
m
r
u
d
P
s
i
d
i
u
m
g
u
a
j
a
6
a
L
.
;
M
y
r
t
a
c
e
a
e
,
I
O
A
-
2
2
/
9
9
d
i
a
r
r
h
o
e
a
,
u
l
c
e
r
s
,
p
i
l
e
s
,
c
h
o
l
e
r
a
,
v
o
m
i
t
i
n
g
W
a
t
e
r
s
o
l
u
b
l
e
y
e
l
l
o
w
p
i
g
m
e
n
t
D
i
a
r
r
h
o
e
a
a
n
d
d
y
s
e
n
t
e
r
y
R
i
n
d
A
n
a
r
-
k
e
-
p
e
r
3
1
.
P
u
n
i
c
a
g
r
a
n
a
t
u
m
L
.
;
P
u
n
i
c
a
c
e
a
e
,
I
O
A
-
2
3
/
9
9
(
C
h
u
l
a
s
u
r
i
,
1
9
9
7
)
M
e
t
h
y
l
m
e
r
c
a
p
t
a
n
(
H
a
r
b
o
r
n
e
a
n
d
R
a
p
h
a
n
u
s
s
a
t
i
6
u
s
L
.
;
B
r
a
s
s
i
c
a
c
e
a
e
,
M
o
u
l
i
U
r
i
n
a
r
y
c
o
m
p
l
a
i
n
t
s
,
p
i
l
e
s
,
R
o
o
t
3
2
.
g
a
s
t
r
o
d
y
n
a
m
i
c
p
a
i
n
s
B
a
x
t
e
r
,
1
9
9
5
)
I
O
A
-
2
4
/
9
9

S
a
p
i
n
d
u
s
s
p
.
;
S
a
p
i
n
d
a
c
e
a
e
,
E
x
p
e
c
t
o
r
a
n
t
,
e
m
e
t
i
c
,
e
p
i
l
e
p
s
y
,
R
i
t
h
a
F
r
u
i
t
3
3
.
I
O
A
-
2
5
/
9
9
a
s
t
h
m
a
p
u
r
g
a
t
i
v
e
A
l
k
.
S
a
u
s
s
u
r
i
n
e
,
b
i
c
y
c
l
i
c
l
a
c
t
o
n
e
,
T
o
n
i
c
,
s
p
a
s
m
o
d
i
c
i
n
c
o
u
g
h
,
c
h
o
l
e
r
a
,
R
o
o
t
K
u
t
h
3
4
.
S
a
u
s
s
u
r
e
a
l
a
p
p
a
C
.
B
.
C
l
a
r
k
e
;
C
o
m
p
o
s
i
t
a
e
,
H
D
C
O
-
1
0
2
/
1
7
5
k
u
s
h
t
i
n
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
;
c
h
r
o
n
i
c
s
k
i
n
d
i
s
e
a
s
e
s
H
a
r
b
o
r
n
e
a
n
d
B
a
x
t
e
r
,
1
9
9
5
)
L
a
u
n
g
S
t
i
m
u
l
a
n
t
,
c
a
r
m
i
n
a
t
i
v
e
u
s
e
d
i
n
S
y
z
g
i
u
m
a
r
o
m
a
t
i
c
u
m
L
.
;
M
y
r
t
a
c
e
a
e
,
3
5
.
B
u
d
E
u
g
e
n
o
l
,
e
u
g
e
n
i
i
n
,
C
a
s
u
a
r
j
i
-
c
i
t
i
n
d
y
s
p
e
p
s
y
I
O
A
-
2
6
/
9
9
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
O
i
l
T
o
o
t
h
a
c
h
e
a
n
d
c
o
n
s
t
i
p
a
t
i
o
n
L
a
u
n
g
3
6
.
S
.
a
r
o
m
a
t
i
c
u
m
L
.
;
M
y
r
t
a
c
e
a
e
,
D
a
b
u
r
E
u
g
e
n
o
l
,
v
a
n
i
l
l
i
n
(
H
a
r
b
o
r
n
e
a
n
d
B
a
x
t
e
r
,
1
9
9
5
)
I
n
d
i
a
L
t
d
.
B
a
r
k
J
a
m
b
o
s
i
n
e
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
3
7
.
A
s
t
r
i
g
e
n
t
,
u
s
e
d
f
o
r
s
o
r
e
t
h
r
o
a
t
,
S
y
z
g
i
u
m
c
u
m
i
n
i
L
.
;
M
y
r
t
a
c
e
a
e
,
J
a
m
u
n
I
O
A
-
2
7
/
9
9
d
i
a
r
r
h
o
e
a

D
y
s
e
n
t
e
r
y
L
e
a
v
e
s
3
8
.
S
.
c
u
m
i
n
i
L
.
;
I
O
A
-
2
8
/
9
9
B
a
r
k
A
r
j
u
n
i
n
e
,
L
a
c
t
o
n
e
,
A
r
j
u
n
e
t
i
n
3
9
.
A
s
t
r
i
g
e
n
t
,
b
i
l
i
o
u
s
a
f
f
e
c
t
i
o
n
s
,
h
e
a
r
t
T
e
r
m
i
n
a
l
i
a
a
r
j
u
n
a
W
.
&
A
.
;
A
r
j
u
n
C
o
m
b
r
e
t
a
c
e
a
e
,
I
O
A
-
2
9
/
9
9
t
a
n
n
i
n
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
d
i
s
e
a
s
e
s
B
a
h
e
r
a
A
n
t
i
p
y
r
e
t
i
c
,
l
e
p
r
o
s
y
,
d
i
a
r
r
h
o
e
a
,
F
r
u
i
t
T
e
r
m
i
n
a
l
i
a
b
e
l
e
r
i
c
a
R
o
x
b
.
;
4
0
.
1
7
%
t
a
n
n
i
n
s
,
t
r
i
t
e
r
p
e
n
o
i
d
(
C
h
o
p
r
a
e
t
a
l
.
,
1
9
9
2
)
C
o
m
b
r
e
t
a
c
e
a
e
,
H
D
C
O
-
2
4
/
2
9
d
r
o
p
s
y
T
e
r
m
i
n
a
l
i
a
c
h
e
b
u
l
a
R
e
t
z
.
;
C
h
e
b
u
l
i
n
i
c
a
c
i
d
,
t
a
n
n
i
c
a
c
i
d
H
a
r
i
r
4
1
.
F
r
u
i
t
L
a
x
a
t
i
v
e
,
u
l
c
e
r
s
,
u
s
e
d
i
n
c
a
r
i
o
u
s
2
0

4
0
%
,
A
n
t
h
r
o
q
u
i
n
o
n
e
c
h
e
b
u
l
a
g
i
c
C
o
m
b
r
e
t
a
c
e
a
e
,
H
D
C
O
-
7
0
/
1
1
6
t
e
e
t
h
,
p
i
l
e
s
a
c
i
d
,
C
o
r
i
l
a
g
i
n
(
H
a
r
b
o
r
n
e
a
n
d
B
a
x
t
e
r
,
1
9
9
5
)
V
i
t
i
s
6
i
n
i
f
e
r
a
L
.
;
V
i
t
a
c
e
a
e
,
4
2
.
D
i
a
r
r
h
o
e
a
A
n
g
u
r
L
e
a
v
e
s
p
-
O
H
b
e
n
z
o
i
c
a
c
i
d
(
H
a
r
b
o
r
n
e
a
n
d
I
O
A
-
3
0
/
9
9
B
a
x
t
e
r
,
1
9
9
5
)
L
e
a
v
e
s
W
a
t
t
a
k
a
6
o
l
u
b
i
l
i
s
L
.
;
A
s
c
l
e
p
i
a
d
a
c
e
,
F
e
v
e
r
s
,
b
o
i
l
s
,
a
b
s
c
e
s
s
e
s
4
3
.
G
l
u
c
o
s
i
d
e
,
d
r
e
g
e
i
n
(
C
h
o
p
r
a
e
t
a
l
.
,
M
a
d
u
m
a
l
a
t
i
1
9
9
2
)
I
O
A
-
3
1
/
9
9
P
l
a
s
t
e
r
f
o
r
s
t
r
a
n
g
u
r
y
,
e
y
e
s
o
r
e
s

L
e
a
v
e
s
4
4
.
B
e
r
Z
i
z
y
p
h
u
s
j
u
j
u
b
a
L
.
;
R
h
a
m
n
a
c
e
a
e
,
I
O
A
-
3
2
/
9
9
B
e
r
Z
.
j
u
j
u
b
a
L
.
;
I
O
A
-
3
3
/
9
9
D
i
a
r
r
h
o
e
a

4
5
.
B
a
r
k
I. Ahmad, A.Z. Beg / Journal of Ethnopharmacology 74 (2001) 113123 118
2.5. Antibiotic resistance of test strains
Antibiotic sensitivity of test strains was deter-
mined by the standard Disc diffusion method of
Baur et al. (1966) against a number of antibi-
otics including two antifungal drugs. The po-
tency of antibiotics per disc are as follows.
Amoxycillin (Am), Cefuroxime (Cu), Cefaclor,
Chloramphenicol, Doxycycline (Do), Nalidixic
acid (Nal), Novobiocin (Nv), Tetracycline, (Tc)
(30 mg/disc each); Cloxacillin (Cx), Methicillin
(M), Fluconazole, (Fu) (10 mg/disc each); Nys-
tatin (Ns), (100 mg/disc) and Nitofurantoin (300
mg/disc). E. coli B (a sensitive strain) was used
to check the potency of the discs. All antibiotic
discs were purchased from the Hi-Media Pvt.
Ltd. (Bombay, India).
2.6. Antimicrobial assay
The agar well diffusion method (Perez et al.,
1990) as adopted earlier (Ahmad et al., 1998)
was used; 0.1 ml of diluted inoculum (10
5
CFU/
ml) of test organism was spread on SDA/SCD
agar plates. Wells of 8 mm diameter were
punched into the agar medium and lled with
100 ml (150 mg/ml) of plant extract, solvent
blanks and antibiotic (chloramphenicol, 100 mg/
ml conc.) to which the test bacteria were sensi-
tive. The plates were incubated for 18 h at
37C. Antimicrobial activity was evaluated by
measuring the zone of inhibition against the test
organism.
2.7. Phytochemical analysis
Phytochemical analysis for major phytocon-
stituents of the plant extracts was undertaken
using standard qualitative methods as described
by various authors (Vogel, 1958; Kapoor et al.,
1969; Rizk and Bashir, 1980; Fadeyi et al.,
1989; Odebiyi and Sofowora, 1990). The plant
extracts were screened for the presence of bio-
logically active compounds like glycosides, phe-
nolics, alkaloids, tannins, avonoids, saponins
and steroids.
2.8. Thin layer chromatography (TLC) of plant
extracts
TLC of the 11 plant extracts with strong an-
timicrobial activity was carried out. Different
solvent systems were used for different classes of
compounds based on the polarity of the organic
solvents. TLC microslides were prepared as de-
scribed by Harborne (1984) with silica gel G.
About 20 ml of plant extract was applied to the
TLC chromatogram. Tannic acid, resorcinol,
and anthrone were used as control. Solvent sys-
tems used were (1) petroleum ether and benzene,
1:1, (2) benzene and chloroform 1:1, (3) benzene
and ethyl acetate 2:1, (4) acetone and alcohol,
1:1, and (5) methanol and water 1:1. Individual
R
f
for each spot was measured. TLC spots were
visualized under UV light and adequate TLC
reagents were used to detect the phytocon-
stituent.
2.9. TLC-bioautography
For direct bioautographic assay, agar overlay
assay as described by Slusarenko et al. (1998)
was used with minor modication. Drug-resis-
tant S. aureus was used as test strain. About 10
ml of plant extract was spotted on preparative
Merck 38 cm chromatographic silica gel-60
plates. Only one solvent system (acetone and
ethanol, 1:1) was used. One milliliter of (10
5
CFU/ml) broth culture was used for every 10 ml
of nutrient agar. The developed chromatogram
was placed in sterilized Petri plates. Culture was
added to 42C nutrient agar, mixed and poured
over the chromatograms as a thin layer. Plates
were incubated at 37C for 24 h. The zone of
inhibition of bacterial growth could be seen
around the active chromatogram spot.
3. Results and discussion
Emergence of multi-drug resistance in human
and animal pathogenic bacteria as well as unde-
sirable side effects of certain antibiotics has trig-
gered immense interest in the search for new
antimicrobial drugs of plant origin. In the
I. Ahmad, A.Z. Beg / Journal of Ethnopharmacology 74 (2001) 113123 119
present study alcoholic extracts of 45 traditionally
used Indian medicinal plants have been tested
against drug-resistant bacteria and a pathogenic
yeast, C. albicans. Ethnobotanical and phytochem-
ical data, plant parts used along with their acquisi-
tion code number are given in Table 1. The
antimicrobial activity of the extracts and their
potency was quantitatively assessed by the pres-
ence or absence of inhibition zone and zone diame-
ter, respectively as given in Table 2. Only the
alcoholic extract was tested, as alcohol was found
to be a better solvent for extraction of antimicro-
bially active substances compared to water and
hexane (Ahmad et al., 1998). The results of screen-
ing are encouraging as out of the 45 plants, 40
extracts showed antibacterial activity against one
or more test bacteria while 24 extracts showed
anticandidal activity. Twelve plants namely L.
inermis, Eucalyptus sp., H. antidysenterica, H. indi -
cus, C. equistifolia, T. belerica, E. ofcinalis, C.
sinensis, S. aromaticum and P. granatum demon-
strated broad spectrum antibacterial activity. Sim-
ilar reports on antibacterial and anticandidal
activities of certain Indian medicinal plants such as
Nigella sati6a, Plumbago zeylanica, P. granatum,
Citrus sisensis, Allium sati6um, Azadirachta indica,
Lantana camara, Psidium guaja6a, Camelia sisensis,
T. belerica, Zizyphus jujuba, Acorus calamus and
Ocimum sanctum were also reported by other
workers (Anesini and Perez, 1993; Belachew Desta,
1993; Youraj et al., 1995; David, 1997; Saxena,
1997; Ahmad et al., 1998; Nimri et al., 1999).
However antimicrobial activity of some Indian
plants namely Beta 6ulgaris, Sapindus sp., Casuar-
ina equistifolia, Nelumbo nucifera, Portulaca
quadrifolia, Vitis 6inifera, Cordia dichotoma and
Nyctanthes arbortristis is reported here for the very
rst time. Overall percent inhibition by active plant
extracts against test bacteria is shown in Fig. 1.
Sensitivity of test strains was, in decreasing order:
S. aureus\S. dysenteriae\E. coli \S. paraty-
phi \C. albicans\B. subtilis. In the case of test
bacteria, the basis for their differences in suscepti-
bility might be due to the differences in the cell wall
composition of Gram +ve and Gram ve bacte-
ria. (Grosvenor et al., 1995). B. subtilis was least
sensitive compared to other test bacteria, which
may be due to their ability to form highly resistant
resting stages called endospores. Drug-resistant
strains of bacteria and C. albicans were found to
be sensitive to the tested plant extracts. This has
clearly indicated that antibiotic resistance does not
interfere with the antimicrobial action of plant
extracts and these extracts might have different
modes of action on test organisms. Thirteen plants
in this study were also screened previously against
other test strains (Ahmad et al., 1998; Mehmood
et al., 1999) and showed similar results to this study
with varying degrees of potency. The difference in
potency may be due to the stage of collection of the
plant sample, different sensitivity of the test strains
and method of extraction (Nimri et al., 1999).
Phytochemical analysis of 40 active extracts
demonstrated the presence of common phytocon-
stituents like phenols (79.5%), epi/gallotannins or
condensed salt tannins (77%), glycosides (49%),
saponins (38%), avonoids (28%) and alkaloids
(25%). The presence of these compounds was also
detected by thin layer chromatography (TLC).
TLC analysis also differentiated between
monomeric and dimeric forms of avonoids. Our
phytochemical analyses are in agreement with the
reports of other workers (Iyengar, 1985; Chopra et
al., 1992; Bruneton, 1995; Harborne and Baxter,
1995; Cai and Wu, 1996). To locate the major
active constituents responsible for antimicrobial
activity against the most sensitive test strains (S.
aureus), TLC-bioautography was performed
against six highly active plant extracts (Amla,
Anar, Behera, Eucalyptus, Harir and Hena). In
Fig. 1. Antibacterial spectrum of plant extracts.
I. Ahmad, A.Z. Beg / Journal of Ethnopharmacology 74 (2001) 113123 120
T
a
b
l
e
2
A
n
t
i
m
i
c
r
o
b
i
a
l
a
n
d
p
h
y
t
o
c
h
e
m
i
c
a
l
p
r
o
p
e
r
t
i
e
s
o
f
a
l
c
o
h
o
l
i
c
e
x
t
r
a
c
t
o
f
m
e
d
i
c
i
n
a
l
p
l
a
n
t
s
a
A
n
t
i
m
i
c
r
o
b
i
a
l
a
c
t
i
v
i
t
y
P
a
r
t
u
s
e
d
P
l
a
n
t
n
a
m
e
;
f
a
m
i
l
y
P
h
y
t
o
c
o
m
p
o
u
n
d
s
C
o
m
m
o
n
n
a
m
e
P
T
S
S
A
B
S
E
C
S
P
S
D
C
A
A
F
G

+
1
+

1
+
+

3
+
2
+

R
h
i
z
o
m
e
B
a
c
h
/
V
a
j
1
.
A
c
o
r
u
s
c
a
l
a
m
u
s
L
.
;
A
r
a
c
e
a
e
+
+

3
+

2
.
A
l
l
i
u
m
c
e
p
a
L
.
;
L
i
l
i
a
c
e
a
e

P
i
y
a
z
L
e
a
v
e
s

+
+

2
+

2
+

2
+

3
.
A
l
l
i
u
m
s
a
t
i
6
u
m
L
.
;
L
i
l
i
a
c
e
a
e
L
a
s
a
n
L
e
a
v
e
s

L
a
s
a
n

2
+

2
+

2
+
2
+
B
u
l
b
+
+
4
.
A
.
s
a
t
i
6
u
m
L
.
;
L
i
l
i
a
c
e
a
e

N
e
e
m
+
+
+
2
+

2
+

B
a
r
k

5
.
A
z
a
d
i
r
a
c
h
t
a
i
n
d
i
c
a
A
.
J
u
s
s
.
;
M
e
l
i
a
c
e
a
e

2
+

2
+

6
.
B
e
t
a
6
u
l
g
a
r
i
s
L
.
;
C
h
e
n
o
p
o
d
i
a
c
e
a
e

R
o
o
t
C
h
o
k
u
n
d
a
r
N
T
N
T
N
T

7
.
C
a
l
o
t
r
o
p
i
s
p
r
o
c
e
r
a
L
.
;
A
s
c
l
e
p
i
a
d
a
c
e
a
e

M
a
d
a
r
L
e
a
v
e
s
N
T
N
T
N
T
+
+
+
3
+
2
+
3
+
2
+
+
2
+
C
h
a
i

8
.
C
a
m
e
l
i
a
s
i
n
e
n
s
i
s
L
.
;
T
h
e
a
c
e
a
e

L
e
a
v
e
s
+
J
a
n
g
l
i
s
a
r
u
+
+

3
+
2
+
3
+
3
+
2
+
2
+
L
e
a
v
e
s
+

9
.
C
a
s
u
a
r
i
n
a
e
q
u
i
s
t
i
f
o
l
i
a
L
.
;
C
a
s
u
a
r
i
n
a
c
e
a
e
+
J
a
n
g
l
i
s
a
r
u
+
+
+
2
+
2
+
2
+
3
+
3
+
3
+
B
a
r
k
+

1
0
.
C
.
e
q
u
i
s
t
i
f
o
l
i
a
L
.
;
C
a
s
u
a
r
i
n
a
c
e
a
e
+
+

2
+

2
+
3
+

2
+
1
1
.
C
i
t
r
u
s
s
i
n
e
n
s
i
s
L
.
;
R
u
t
a
c
e
a
e
3
+

R
i
n
d
M
u
s
a
m
b
i
+
+

3
+

1
2
.
C
o
r
d
i
a
d
i
c
h
o
t
a
m
a
L
.
;
B
o
r
a
g
i
n
a
c
e
a
e
2
+
L
a
s
o
r
a

L
e
a
v
e
s

+
+
+
+
4
+
2
+

4
+
+
2
+

2
+
+
1
3
.
E
m
b
l
i
c
a
o
f

c
i
n
a
l
i
s
G
a
e
r
t
h
.
;
E
u
p
h
o
r
b
i
a
c
e
a
e
A
m
l
a
F
r
u
i
t
s

E
u
c
a
l
y
p
t
u
s
+
+

3
+
2
+
2
+
3
+
3
+
2
+
L
e
a
v
e
s
+

1
4
.
E
u
c
a
l
y
p
t
u
s
s
p
.
;
M
y
r
t
a
c
e
a
e
+
A
n
j
i
r

2
+

L
e
a
v
e
s

1
5
.
F
i
c
u
s
c
a
r
i
c
a
L
.
;
M
o
r
a
c
e
a
e
+
+

2
+

2
+
2
+
+
2
+
1
6
.
F
i
c
u
s
r
e
l
i
g
i
o
s
a
L
.
;
M
o
r
a
c
e
a
e
2
+

L
e
a
v
e
s
P
i
p
a
l
+
+

2
+
2
+
2
+
3
+
4
+
1
7
.
H
e
m
i
d
e
s
m
u
s
i
n
d
i
c
u
s
R
.
B
r
.
;
A
s
c
l
e
p
i
a
d
a
c
e
a
e
5
+
A
n
a
n
t
a
m
u
l
R
o
o
t
+

+
+

2
+
2
+
2
+
3
+

2
+
+
2
+
+
1
8
.
H
o
l
a
r
r
h
e
n
a
a
n
t
i
d
y
s
e
n
t
e
r
i
c
a
R
.
;
A
p
o
c
y
a
n
a
c
e
a
e
K
u
r
a
c
h
i
B
a
r
k

G
h
a
n
e
r
i
+
+
+
2
+
2
+

3
+
2
+

L
e
a
v
e
s

1
9
.
L
a
n
t
a
n
a
c
a
m
a
r
a
L
.
;
V
e
r
b
e
n
a
c
e
a
e
+
M
e
h
d
i
+
+
+
4
+
2
+
2
+
4
+
2
+
4
+
L
e
a
v
e
s
+

2
0
.
L
a
w
s
o
n
i
a
i
n
e
r
m
i
s
L
.
;
L
y
r
t
h
a
c
e
a
e

2
+

2
+
+
3
+
2
1
.
M
o
r
u
s
a
l
b
u
s
L
.
;
M
o
r
a
c
e
a
e

L
e
a
v
e
s
S
h
a
h
t
u
t
N
T
N
T
N
T

2
2
.
M
u
s
a
p
a
r
a
d
i
s
i
a
c
a
L
.
;
M
u
s
a
c
e
a
e

K
a
l
a

S
t
e
m
N
T
N
T
N
T
+
+

3
+
2
+
1
+

2
+

2
+
+
2
3
.
N
e
l
u
m
b
o
n
u
c
i
f
e
r
a
G
a
e
r
t
h
.
;
N
y
m
p
h
a
c
e
a
e
K
a
m
a
l
F
l
o
w
e
r
s
N
T
K
a
n
e
r
N
T
N
T
N
T

L
e
a
v
e
s
N
T
N
T
2
4
.
N
e
r
i
u
m
i
n
d
i
c
u
m
M
i
l
l
.
;
A
p
o
c
y
a
n
a
c
e
a
e

K
a
l
o
n
g
i
+
+
+
3
+

2
+
2
+

S
e
e
d
s

2
5
.
N
i
g
e
l
l
a
s
a
t
i
6
a
L
.
;
R
a
n
u
n
c
u
l
a
c
e
a
e
+
+

2
+

2
+

2
6
.
N
y
c
t
a
n
t
h
e
s
a
r
b
o
r
t
r
i
s
t
i
s
L
.
;
O
l
e
a
c
e
a
e

L
e
a
v
e
s
H
a
r
s
i
n
g
a
r
+
+

2
+

1
+

2
7
.
O
c
i
m
u
m
s
a
n
c
t
u
m
L
.
;
L
a
b
i
a
t
a
e
4
+
T
u
l
s
i
3
+
W
h
o
l
e
p
l
a
n
t

2
+

2
+
3
+

2
+
C
h
i
t
a
3
+
2
8
.
P
l
u
m
b
a
g
o
z
e
y
l
a
n
i
c
a
L
.
;
P
l
u
m
b
a
g
i
n
a
c
e
a
e
+
+
R
o
o
t

C
h
o
t
a
l
u
n
i
y
a

2
+

S
t
e
m
/
l
e
a
v
e
s

2
9
.
P
o
r
t
u
l
a
c
a
q
u
a
d
r
i
f
o
l
i
a
L
.
;
P
o
r
t
u
l
a
c
a
c
e
a
e
+
A
m
r
u
d
+
+
+
2
+

L
e
a
v
e
s

3
0
.
P
s
i
d
i
u
m
g
u
a
j
a
6
a
L
.
;
M
y
r
t
a
c
e
a
e
+
+

4
+
2
+
2
+
4
+
+
4
+
3
1
.
P
u
n
i
c
a
g
r
a
n
a
t
u
m
L
.
;
P
u
n
i
c
a
c
e
a
e
3
+
+
+
R
i
n
d
A
n
a
r
N
T
N
T
N
T

3
2
.
R
a
p
h
a
n
u
s
s
a
t
i
6
u
s
L
.
;
B
r
a
s
s
i
c
a
c
e
a
e

M
o
u
l
i

R
o
o
t
s
N
T
N
T
N
T

+
2
+
2
+

2
+
+
3
3
.
S
a
p
i
n
d
u
s
s
p
.
;
S
a
p
i
n
d
a
c
e
a
e
R
i
t
h
a
F
r
u
i
t
s
+
K
u
t
h
+
+

2
+

3
+
3
+
4
+
3
+
R
o
o
t
s
+
+
3
4
.
S
a
u
s
s
u
r
e
a
l
a
p
p
a
C
.
B
.
C
l
a
r
k
e
;
C
o
m
p
o
s
i
t
a
e
+
L
a
u
n
g
+
+

3
+
2
+
3
+
3
+
4
+
5
+
B
u
d

+
3
5
.
S
y
z
g
i
u
m
a
r
o
m
a
t
i
c
u
m
L
.
;
M
y
r
t
a
c
e
a
e
+

2
+
3
+
3
+
5
+

3
+
3
6
.
S
.
a
r
o
m
a
t
i
c
u
m
L
.
;
M
y
r
t
a
c
e
a
e
5
+
+

O
i
l
L
a
u
n
g
I. Ahmad, A.Z. Beg / Journal of Ethnopharmacology 74 (2001) 113123 121
T
a
b
l
e
2
(
C
o
n
t
i
n
u
e
d
)
A
n
t
i
m
i
c
r
o
b
i
a
l
a
c
t
i
v
i
t
y
P
l
a
n
t
n
a
m
e
;
f
a
m
i
l
y
C
o
m
m
o
n
n
a
m
e
P
a
r
t
u
s
e
d
P
h
y
t
o
c
o
m
p
o
u
n
d
s
P
T
S
S
A
B
S
E
C
S
P
G
S
D
C
A
F
A
+
+

1
+

1
+
1
+
3
+
3
7
.
S
y
z
g
i
u
m
c
u
m
i
n
i
L
.
;
M
y
r
t
a
c
e
a
e

J
a
m
u
n
B
a
r
k

+
+
+
2
+

1
+
1
+

4
+

3
8
.
S
.
c
u
m
i
n
i
L
.
;
M
y
r
t
a
c
e
a
e
J
a
m
u
n
L
e
a
v
e
s

+
+

3
+

2
+
3
+
3
9
.
T
e
r
m
i
n
a
l
i
a
a
r
j
u
n
a
W
.
&
A
.
;
C
o
m
b
r
e
t
a
c
e
a
e
2
+
A
r
j
u
n

B
a
r
k

+
+
+
3
+
2
+
2
+
3
+
+
2
+
4
0
.
T
e
r
m
i
n
a
l
i
a
b
e
l
e
r
i
c
a
R
o
x
b
.
;
C
o
m
b
r
e
t
a
c
e
a
e
2
+
+

F
r
u
i
t
s
B
a
h
e
r
a

+
+
+
+
3
+
2
+
2
+
3
+
2
+
2
+
H
a
r
i
r
4
1
.
T
e
r
m
i
n
a
l
i
a
c
h
e
b
u
l
a
R
e
t
z
.
;
C
o
m
b
r
e
t
a
c
e
a
e
F
r
u
i
t
s

+
+

3
+

2
+

+
2
+

4
2
.
V
i
t
i
s
6
i
n
i
f
e
r
a
L
.
;
V
i
t
a
c
e
a
e
A
n
g
u
r
L
e
a
v
e
s
N
T
M
a
d
u
m
a
l
a
t
i
N
T
N
T
N
T

L
e
a
v
e
s
N
T
N
T
4
3
.
W
a
t
t
a
k
a
6
o
l
u
b
i
l
i
s
L
.
;
A
s
l
e
p
i
a
d
a
c
e
a
e
+
+

2
+

2
+
2
+
3
+
2
+
4
4
.
Z
i
z
y
p
h
u
s
j
u
j
u
b
a
L
.
;
R
h
a
m
n
a
c
e
a
e
B
e
r
L
e
a
v
e
s

+
+

2
+
2
+

3
+
+

2
+

4
5
.
Z
.
j
u
j
u
b
a
L
.
;
R
h
a
m
n
a
c
e
a
e
B
e
r
B
a
r
k
T
o
t
a
l
n
u
m
b
e
r
o
f
a
c
t
i
v
e
p
l
a
n
t
s
3
2
3
1
1
4
3
6
1
7
2
9
2
7
3
3
2
4
1
0
1
2
1
9
a
P
h
y
t
o
c
o
m
p
o
u
n
d
k
e
y
:
A
,
a
l
k
a
l
o
i
d
;
F
,

a
v
o
n
o
i
d
;
G
,
g
l
y
c
o
s
i
d
e
;
P
,
p
h
e
n
o
l
;
T
,
t
a
n
n
i
n
;
S
,
s
a
p
o
n
i
n
;
N
T
,
n
o
t
t
e
s
t
e
d
.
A
c
t
i
v
i
t
y
k
e
y
:

,
n
o
i
n
h
i
b
i
t
i
o
n
;
1
+
,
B
1
0
m
m
z
o
n
e
o
f
i
n
h
i
b
i
t
i
o
n
;
2
+
,
1
0

2
0
m
m
;
3
+
,
2
1

3
0
m
m
;
4
+
,
3
1

4
0
m
m
;
5
+
,
4
1

6
5
m
m
.
O
r
g
a
n
i
s
m
k
e
y
:
S
A
,
S
.
a
u
r
e
u
s
;
B
S
,
B
.
s
u
b
t
i
l
i
s
;
E
C
,
E
.
c
o
l
i
;
S
P
,
S
.
p
a
r
a
t
y
p
h
i
;
S
D
,
S
.
d
y
s
e
n
t
e
r
i
a
e
;
C
A
,
C
.
a
l
b
i
c
a
n
s
.
I. Ahmad, A.Z. Beg / Journal of Ethnopharmacology 74 (2001) 113123 122
the majority of the plants tested by TLC and
TLC bioautography, phenols and tannins were
observed as the most common active con-
stituents. These ndings correlate with the ob-
servations of Tanaka et al. (1991) and Silva et
al. (1996). Monomeric avonoids were detected
as the active constituent in E. ofcinalis. It is
expected that more active compounds might
have been detected by TLC bioautography if
different solvent systems, microbial strains and
more plant extracts were used. Thus our antimi-
crobial screening results also justify the tradi-
tional uses of these plants in various ailments
including infectious diseases. Further, the active
phytocompounds of these plants against multi
drug-resistant bacteria and C. albicans has to be
characterized and the efcacy of non-toxic ex-
tracts/preparations has to be evaluated in vivo.
Study of the synergistic interaction of active
phytocompounds with antibiotics is required to
exploit these potential plant extracts in the com-
bination therapy of infectious diseases caused by
multi drug-resistant organisms.
Acknowledgements
The authors wish to thank the Director of the
RAK Institute of Agricultural Sciences, Aligarh
Muslim University Aligarh, India for providing
facilities for this work. We also thank Dr S.
Farooq (Director, The Himalaya Drug Co.,
New Delhi) for providing some of the plant
samples used in this study and Shaba Ansari
and Fazlur-Rahman (A. M. U., Aligarh) for
their technical help.
References
Ahmad, I., Mehmood, Z., Mohammad, F., 1998. Screening of
some Indian medicinal plants for their antimicrobial prop-
erties. Journal of Ethnopharmacology 62, 183193.
Alade, P.I., Irobi, O.N., 1993. Antimicrobial activities of crude
leaf extracts of Acalypha wilkensian. Journal of Ethnophar-
macology 39, 171174.
Anesini, C., Perez, C., 1993. Screening of plant use in Argen-
tine folk medicine for antimicrobial activity. Journal of
Ethnopharmacology 39, 119128.
Baur, A.W., Kirby, W.M.M., Sherris, J.C., Turch, M., 1966.
Antibiotic susceptibility testing by a standardized single
disc method. American Journal of Clinical Pathology 45,
494496.
Belachew Desta, K.H., 1993. Antimicrobial activity of
Plumbago zeylanica. Journal of Ethnopharmacology 39,
129139.
Bruneton, J., 1995. Pharmacognosy, Phytochemistry of Medic-
inal Plants. Lavoisler, France, pp. 265380.
Cai, L., Wu, C.D., 1996. Compounds of Syzygium aromaticum
possessing growth inhibitory activity against oral patho-
gens. Journal of Natural Products 59 (9), 987990.
Chopra, R.N., Nayer, S.L., Chopra, I.C., 1992. Glossary of
Indian Medicinal Plants, 3rd edn. Council of Scientic and
Industrial Research, New Delhi, pp. 7246.
Chulasuri, M., 1997. A water soluble component with antimi-
crobial activity from pomegranate rind: An ingredient in
antiseptic liquid soap. Thai Journal of Phytopharmacy 4
(1), 25.
David, M., 1997. Antimicrobial activity of garlic. Antimicro-
bial Agents and Chemotherapy 41, 2286.
Davis, J., 1994. Inactivation of antibiotic and the dissemina-
tion of resistance genes. Science 264, 375382.
Diamond, R.D., 1993. The growing problem of mycoses in
patients infested with human immunodeciency virus. Re-
view of Infectious Diseases 13, 480486.
Fadeyi, M.Q., Adeoye, A.E., Olowokudejo, J.D., 1989. Epi-
dermal and phytochemical studies in the genus Boerha6ia
(Nyctanginaceae) in Nigeria. International Journal of
Crude Drug Research 29, 178184.
Grosvenor, P.W., Supriono, A., Gray, D.O., 1995. Medicinal
plants from Riau Province, Sumatra, Indonesia. Part 2,
Antibacterial and antifungal activity. Journal of
Ethnopharmacology 45, 97111.
Harborne, S.B., 1984. A Guide to Modern Techniques of
Plant Analysis. Chapman and Hall, London, pp. 480.
Harborne, S.B., Baxter, H., 1995. Phytochemical Dictionary.
A Handbook of Bioactive Compounds from Plants. Taylor
and Francis, London.
Hasegawa, H., Matsumya, S., Yamasak, K., 1995. Reversal of
efux mediated tetracycline resistance in Staphylococcus
aureus clinical isolates by Ginseng prosaponenins. Phy-
totherapy Research 9 (4), 260263.
Iyengar, M.A., 1985. Study of Crude Drugs, 2nd edn. College
of Pharmaceutical Sciences, Manipal, pp. 1378.
Kapoor, L.D., Singh, A., Kapoor, S.L., Srivastava, S.N.,
1969. Survey of Indian medicinal plants for saponins,
alkaloids and avonoids. Lloydia 32, 297302.
Lee, C.K., Kin, H., Moon, K.H., Shun, K.H., 1998. Screening
and isolation of antibiotic resistance inhibitors from herb
materials resistance inhibition of volatile components
of Korean aromatic herbs. Archives of Pharmaceutical
Research 21 (1), 6266.
Mehmood, Z., Ahmad, I., Mohammad, F., Ahmad, S., 1999.
Indian medicinal plants: A potential source of anticandidal
drugs. Pharmaceutical Biology 37, 237242.
I. Ahmad, A.Z. Beg / Journal of Ethnopharmacology 74 (2001) 113123 123
Mulligen, M.E., Murry-Leisure, K.A., Ribner, B.S., Standi-
ford, H.C., John, J.F., Karvick, J.A., Kauffman, C.A., Yu,
V.L., 1993. Methicillin resistant Staphylococcus aureus.
American Journal of Medicine 94, 313328.
Nagenawa, R., Twata, N., Ishikawa, K., Suzuki, A., 1996.
Inhibition of microbial growth by ajoene, a sulfur com-
pound derived from garlic. Applied and Environmental
Microbiology 62 (11), 42384242.
Nimri, L.F., Meqdam, M.M., Alkofahi, A., 1999. Antibacte-
rial activity of Jordanian medicinal plants. Pharmaceutical
Biology 37 (3), 196201.
Odebiyi, A., Sofowora, A.E., 1990. Phytochemical screening of
Nigerian medicinal plants. Part III. Lloydia 41, 234246.
Perez, C., Pauli, M., Bazerque, P., 1990. An antibiotic assay
by the well agar method. Acta Biologiae et Medicine
Experimentalis 15, 113115.
Piddock, K.J.V., Wise, R., 1989. Mechanisms of resistance to
quinolones and clinical perspective. Journal of Antimicro-
bial Chemotherapy 23, 475483.
Ratnakar, P., Murthy, P.S., 1995. Purication and mecha-
nisms of action of antitubercular principle from garlic
(Allium sati6um) active against isoniazid susceptible and
resistant Mycobacterium tuberculae H37RV. Indian Jour-
nal of Clinical Biochemistry 10, 1418.
Rinaldi, M.G., 1991. Problems in the diagnosis of invasive
fungal diseases. Review of Infectious Diseases 13, 493495.
Rizk, A.M., Bashir, 1980. A chemical survey of sixty plants.
Fitoterapia 53, 3544.
Robin, E.H., Anril, W., Alexander, M., Loeto, M., Keith, K.,
1998. Nasopharyngeal carriage and antimicrobial resis-
tance in isolates of Streptococcus pneumoniae and
Heamophilus inuenzae Type b in children under 5 years of
age in Botswana. International Journal of Infectious Dis-
eases 3 (1), 1825.
Saxena, K., 1997. Antimicrobial Screening of Selected Medici-
nal Plants from India. Journal of Ethnopharmacology. 58
(2), 7583.
Saxena, V.K., Sharma, R.N., 1999. Antimicrobial activity of
essential oil of Lantana aculeata. Fitoterapia 70 (1), 5960.
Silva, O., Duarte, A., Cabrita, J., Gomes, E., 1996. Antimicro-
bial activity of Guinea Bissau traditional remedies.
Journal of Ethnopharmacology 50, 5559.
Singh, M., Chaudhry, M.A., Yadava, J.N.S., Sanyal, S.C.,
1992. The spectrum of antibiotic resistance in human and
veterinary isolates of Escherichia coli collected from 1984
1986 in Northern India. Journal of Antimicrobial
Chemotherapy 29, 159168.
Slusarenko, A.J., Longland, A.C., Whitehead, I.M., 1998.
Convenient, sensitive and rapid assay for antibacterial
activity of phytoalexins. Botanica Helvetica 99 (2), 203
207.
Swami, K.D., Bisht, N.P.S., 1996. Constituents of Ficus reli -
giosa and F. infectoria and their biological activity. Journal
of the Indian Chemical Society 73 (11), 631.
Tanaka, T., Moriita, A., Nanaka, G., 1991. Tannins and
related compounds C III. Isolation and characterisation of
new monomeric, dimeric and trimeric ellagitannins, cala-
manisanin and calamanins A, B, and C from Terminalia
caamansani. Chemical Pharmacy Bulletin 38, 60.
Vogel, A.I., 1958. A Textbook of Practical Organic Chemistry.
Longman, London, pp. 9092.
Youraj, R.S., Padmaja-Kaimal, A.V., Ranjan, M.B., 1995.
Prophylactic therapy Salmonella septicemia in mice with
traditionally prescribed drug formulation. Journal of
Ethnopharmacology 45, 141147.
.