World Journal of Microbiology & Biotechnology (2005) 21: 12311236

DOI 10.1007/s11274-005-1479-6

Springer 2005

Isolation, identication and study of antimicrobial property of a bioactive compound

in an Indian medicinal plant Acalypha indica (Indian-nettle)
R.D. Jebakumar Solomon*, Subramaniam Kallidass and Jayaraj Vimalan
Department Of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai 625 021,
Tamilnadu, India
*Author for correspondence: Tel.: +91-452-2459480, Fax: +91-452-2459105, E-mail: jsolomon@mrna.tn.nic.in
Received 26 March 2004; accepted 13 January 2005

Keywords: Acalypha indica, antimicrobial compound, Aspergillus niger, bioactive compound, Candida albicans,
Clotrimazole, Escherichia coli, HPLC, TLC

Summary
Fresh, dried and powdered samples of leaf, stem and root of Acalypha indica were subjected to fractional distillation
in a soxhlet apparatus using solvents such as hexane, chloroform, acetone and methanol. The plant extracts and a
synthetic antifungal compound, Clotrimazole (authentic standard) were subjected to TLC and HPLC analyses. The
Rf (relative front) value of Clotrimazole was 0.371. The plants leaf, root and stem extracts also gave distinct spots
respectively at Rf value of 0.371 0.0009. In HPLC, the TLC-separated active compound and Clotrimazole
resolved at 1.90 0.2 min (retention time). The amounts of active compound present in root, leaf and stem
extracts were 538, 415 and 171 lg/g respectively. From the results of our study, we infer that the active compound
isolated from Acalypha indica is more potent in controlling Candida albicans, Aspergillus niger and Escherichia coli.
The Active compound present in the plant had more than 100% activity when compared to standard Clotrimazole.

Introduction
Many plants are known to possess fungicidal substances
(Shekhawat & Prasad 1971; Khanna & Chandra 1972;
Ganesan & Krishnaraju 1995). During recent years
considerable attention has been directed towards
exploitation of plant products as botanical pesticides for
control of dierent microbial infestations (Varma et al.
2002). Some active principles of plants, viz. azadirachtin
from Azadirachta indica, pyrethrum from Chrysanthemum cinerariaefolium and carvone from Carum carvi
have been formulated and are in regular use in the
management of dierent agricultural pests (Varma &
Dubey 1999; Varma et al. 2002). Much work has been
carried out on the eects of medicinal and aromatic plant
extracts against various fungi (Lu et al. 1999), but their
eect upon dermatophytes and related fungi is unexplored. Dermatophytes are known to cause supercial
skin infections like scabies (Ganapathiraman et al. 1979).
Candida species have become common opportunistic
pathogens in compromised hosts. The predominant
clinical isolate, Candida albicans is the fth most common cause of nosocomial blood stream infection (Edwards 1991). Oropharyngeal candidiasis is the most
common opportunistic fungal infection in human
immunodeciency virus (HIV)-infected patients and
other immunocompromised hosts (Pelletier et al. 2000).
Apart from Candida spp. and Cryptococcus neoformans

which cause invasive opportunistic mycotic infection

(Hajjeh et al. 1995; Kao et al. 1999; Rees et al. 1998;
Pfaller et al. 2002), serious infections due to Aspergillus
spp. and other lamentous fungi are emerging as prominent causes of infectious morbidity and mortality worldwide (Groll et al. 1996; Denning et al. 1998; Groll &
Walsh 2001; Perfect & Schell 1996; Pfaller et al. 2002). So
there is an immediate need for an eective antimicrobial
agent to control above the pathogens. Clotrimazole is a
broad-spectrum antifungal compound reported to control many dermatophytic fungal infections (Prasad et al.
1993). Clotrimazole usage has grown substantially since
its discovery in 1968, far from its initial limited use as a
systemic antifungal agent. Clotrimazole is now utilized
extensively as a front-line topical antifungal agent for
prevention or treatment of mucosal candidiasis in
immunocompromised patients (Pelletier et al. 2000).
With the continued emergence of antibiotic resistance
and drug-resistant pathogenic microbial strains, we are
forced to search for further powerful antimicrobial
compounds. It is reported that various organic solvent
extracts of Acalypha indica leaves exhibited signicant
antibacterial activity against both gram-negative and
gram-positive bacteria (Rao & Krishna 1982). Acalypha
indica solvent extracts also have post-coital antifertility
activity in female albino rats (Hiremath et al. 1999).
Therefore in the present investigation Acalypha indica
belonging to the family Euphorbiaceae (Stone 1970;

1232

R.D. Jebakumar et al.

Swarbrick 1997) was selected for the isolation and

identication of its active compound from leaf, stem, and
root extracts. The active compound isolated from these
extracts is compared with Clotrimazole (commercial
antifungal compound) for its antifungal activity against
Aspergillus niger, Candida albicans and antibacterial
activity against Escherichia coli.

Materials and methods

Sample preparation
Fresh Acalypha indica plants collected from the
Tamilnadu Agricultural University Madurai were
washed thoroughly in tap water and with distilled water
and air dried in the shade at room temperature for ve
days. Dried leaves, stems and roots were taken separately and powdered for extraction. The antifungal
compound Clotrimazole commercially available as
Candid B was obtained from Glenmark Pharmaceuticals Ltd., Nasik, Bombay, India.
Active compound extraction
Seven grams of powdered leaf, stem and root of
Acalypha indica were taken separately and extracted
with 250 ml of acetone, chloroform, hexane and methanol separately in soxhlet apparatus for 810 h. The
plant extracts were collected and excess solvents were
evaporated through rotary vacuum ash evaporator and
the crude residues were collected.
Separation of bioactive compound
Thin layer chromatography was carried out using
0.25 mm thickness silica gel-G coated plates dried at
room temperature (28 1 C). Ten microlitres of acetone, chloroform, hexane and methanol extracts of leaf,
stem and root were applied to the TLC plate separately.
Similarly 10 ll of (mg/ml) Clotrimazole (authentic
standard) was also loaded. The solvent system used in
the TLC analysis was chloroform/methanol (10:1, v/v).
After complete elution, the spots were identied by
passing iodine vapour. The Rf values of the synthetic
Clotrimazole and the active compound in the crude
extracts of the leaf, stem and root extracts of acetone,
chloroform, hexane and methanol are given in Table 1.
Table 1. Rf values of bioactive compound in Acalypha indica from
dierent solvent extracts resolved by thin layer chromatography (TLC)
in chloroform/methanol (10: v/v).
Sample

Leaf
Stem
Root

Rf values in cm
Acetone

Chloroform

Hexane

Methanol

0.3710.009
0.3710.009
0.3650.015

0.3700.010
0.3720.008
0.3680.012

0.3710.009
0.3710.009
0.3690.011

0.3700.010
0.3720.008
0.3640.016

HPLC analysis of bioactive compound

The expected active compound spot resolved from the
crude extracts was scraped o aseptically and part of it
was used for antimicrobial assay. The remaining part
was dissolved in the respective solvents such as acetone,
chloroform, hexane and methanol and serially diluted
with the HPLC mobile phase. This was subjected to
HPLC analysis using a Shimadzu HPLC system with
Shim pack CLC ODS (4.6 mm 15 cm) column, guard
column, Liquid pump LC 6 AD, system controller
SCL 6B, detector (u.v.VIS) SPD-6 AV, data processor CR-5A and the detection at 254 nm with mobile
phase (methanol: double distilled water 95:05, v/v) ow
rate 1 ml/min. Identication and quantication of each
peak was made by comparing with the retention time of
the authentic standard peak and the related concentration.
Antifungal activity
The antifungal activity of the plants leaf, root and stem
extracts and the reference standard Clotrimazole was
examined by the Kirbey-Bauer disc diusion method.
Candida albicans and Aspergillus niger cultivated on
YPS broth, Candida albicans starting inoculam size
106 c.f.u./ml and Aspergillus niger starting inoculam size
104 c.f.u./ml were prepared by the spectrophotometric
method recommended by NCCLS (NCCLS 1992).
These cultures were spread-plated on Mueller-Hinton
agar separately. The spots of the expected active compound resolved in TLC, in respective solvents acetone,
chloroform, hexane and methanol were scraped o and
200 lg of this powder was dissolved in 1000 ll of sterile
double distilled water. This was treated as stock. From
this, 50 ll was loaded onto sterile Hi-media discs and
dried aseptically. Similarly 10 lg of the reference standard Clotrimazole (Candid B) dissolved in 1000 ll of
double distilled water was taken as stock and from that
20, 30, 40 and 50 ll was loaded on separate sterile
Himedia discs and dried aseptically. These discs were
placed onto the plates, inoculated with test organisms
respectively, each disc being placed 5 cm apart. Control
discs with sterile double distilled water were also placed
on the respective plates and were incubated at 37 C for
2436 h.
Antibacterial activity
Antibacterial activity of the plants leaf, root and stem
extracts and reference standard Clotrimazole was
examined by the Kirbey-Bauer disc diusion technique
against E. coli. Log phase culture of E. coli, cultured on
nutrient broth was spread-plated on Mueller-Hinton
agar. The spots of the expected active compound
resolved in TLC, in the respective solvents acetone,
chloroform, hexane and methanol were scraped o and
200 lg of this powder was dissolved in 1000 ll of sterile
double distilled water. This was treated as stock. From

Antimicrobial property of A. indica

1233

this, 50 ll was loaded onto sterile Hi-media discs and

dried aseptically. Similarly 10 lg of authentic standard
Clotrimazole (Candid B) was dissolved in 1000 ll of
double distilled water. This was taken as stock and from
it 20, 30, 40 and 50 ll was loaded onto separate sterile
Himedia discs and dried aseptically. These discs were
placed 5 cm apart onto the plates inoculated with
E. coli. The control disc with sterile double distilled
water was also placed on the respective plates and
incubated at 37 C for 24 h.

Results and discussion

Qualitative analysis of the bioactive compound
Thin layer chromatography results revealed that the Rf
value of Clotrimazole was 0.371. The other extracts of
Acalypha indica in respective solvents (acetone, chloroform, hexane and methanol) formed distinct spots at Rf
value of 0.371 0.0009 which is close to Clotrimazole
(Table 1).
The TLC-separated active compound and the
authentic standard (Clotrimazole) injected into the
HPLC system resolved at 1.90 0.002 min. The retention times of the active compound in leaf, stem and root
extracts in the solvents acetone, chloroform, hexane and
methanol were given in Table 2 and Figure 1. The HPLC
retention times of bioactive compound and authentic
standard (Clotrimazole) agreed well with the ndings of
Controller of Publications, Government Of India (1996).
The total amount of active compounds in leaf, stem and
root were calculated from the peak area obtained in the
HPLC analysis in comparison with the authentic standard were presented in Table 3.
Among the solvents acetone, chloroform, hexane and
methanol used for extracting active compound from
Acalypha indica, acetone and methanol were more ecient in extracting the active compound than the other
solvents tested.
Quantitative analysis of the bioactive compound
The amount of active compound in TLC-separated
solvent extracts of leaf, stem and root determined by
HPLC is presented in Table 3. The solvent methanol
had maximum eciency in extracting the bioactive
compound from leaf, methanol extract of leaf contained
Table 2. Retention time of bioactive compound of Acalypha indica
from dierent solvent extracts in HPLC in methanol/water (95:5 v/v)
as described in Section Methods.
Sample

Leaf
Stem
Root

Retention time in minutes

Acetone

Chloroform

Hexane

Methanol

2.0350.013
1.8900.002
1.9910.005

1.7890.001
1.9740.006
1.9000.015

1.9670.002
1.9900.004
1.8390.003

1.9200.008
1.8510.007
1.7190.003

Figure 1. Elution pattern of Acalypha indica solvent extracts in HPLC.

(A) A. indica leaves extracted with methanol, (B) A. indica leaves
extracted with acetone, (C) A. indica stem extracted with acetone and
(D) A. indica root extracted with acetone.

Table 3. Quantitative estimation of bioactive compound of Acalypha

indica from various solvent extracts by HPLC.
Sample

Leaf
Stem
Root

Quantity of bioactive compound (lg/g)

Acetone

Chloroform

Hexane

Methanol

103.840.12
171.000.23
538.000.04

83.090.05
9.050.03
13.110.01

49.090.04
0.800.01
0.480.02

415.440.16
51.490.04
120.980.25

415.44 0.16 lg/g of bioactive compound. Further,

acetone extract of stem and root contained a high
amount of bioactive compound. Stem extract in acetone
had 171.00 0.23 lg/g of bioactive compound and
root extract in acetone had 538.00 0.16 lg/g of bioactive compound.
From these results we infer that the solvents methanol
and acetone were more ecient in extracting the bioactive compound. In leaf, stem and root extracts of
Acalypha indica, leaf extract had the highest concentration of bioactive compound in methanol, chloroform
and hexane extractions. In acetone extraction, root
extract had the highest concentration of bioactive
compound 538.00 0.16 lg/g.
Antimicrobial activity
The antimicrobial activities of the test organisms against
reference standard Clotrimazole were given in Table 4.
The antimicrobial activity pattern of active compounds
of various solvent extracts toward Candida albicans,
Aspergillus niger and E. coli shown in diameter of the
clearing zones around the discs are presented in
Tables 57 respectively. Control discs with sterile

1234

R.D. Jebakumar et al.

Table 4. Antimicrobial activity pattern of standard Clotrimazole against

the test organisms Aspergillus niger, Candida albicans and E. coli.
Clotrimazole

200
300
400
500

Zone of inhibition in mm

ng
ng
ng
ng

Aspergillus niger

Candida albicans

E. coli

10
18
20
22

11
14
16
20

14
16
19
22

double distilled water did not show any zone of inhibition. The results revealed that the active compound in
the methanol extract had the highest antifungal and
antibacterial activity against Candida albicans, Aspergillus niger and Escherichia coli. Our ndings are in
agreement with ndings of Singh & Singh (1997), Rao
& Krishna (1982); Perumal Samy et al. (1999) and
Hiremath et al. (1999). This indicates that methanol
extracts of Acalypha indica contained more antimicrobial substance (bioactive compound).
It was estimated by HPLC that 1 g of TLC-scraped
powder of the active compound contained approximately 500 lg of bioactive compound. We have dissolved 200 lg of that powder in 1000 ll of sterile

distilled water and from which 50 ll was loaded in each

disc, so each disc had 5.0 ng of active compound.
Similarly 10 lg of reference standard was dissolved in
1000 ll of sterile distilled water from which each disc
was loaded 20, 30, 40 and 50 ll respectively so each disc
had 200, 300, 400 and 500 ng respectively. (The maximum antimicrobial activity is observed around the disc
with 500 ng Clotrimazole.)
The percentage of activity of active compound in
comparison with reference standard is also given in
Tables 57 respectively. The quantity of active compound taken for testing is 10)2 of the reference standard
(5 ng active compound is loaded per disc:500 ng of
Clotrimazole is loaded per disc).
With the widespread use of prophylactic or empirical
antifungal therapy, more drug-resistant Candida species
have gained prominence (Nguyen et al. 1996). Current
diagnostic and therapeutic approaches fall short in
addressing the problem of lamentous fungal infections
(Denning et al. 1998; Groll & Walsh 2001; Klont et al.
2001; Pfaller et al. 2002). Although Amphotericin B
remains the standard therapy for these infections, therapeutic outcomes are suboptimal (Caillot et al. 2001;
Groll & Walsh 2001; Tolleman et al. 2001; Wingard
et al. 1999; Pfaller et al. 2002). Clearly, there is a need

Table 5. Antimicrobial activity pattern of the bioactive compound separated from various solvent extracts of Acalypha indica against C. albicans.
Solvent

Acetone
Chloroform
Hexane
Methanol

Leaf

Stem

Root

Zone of inhibition in mm

% of activity

Zone of inhibition in mm

% of activity

Zone of inhibition in mm

% of activity

13
20
20
25

61
95
95
119

20
18
19
20

95
85
90
95

14
18
16
23

66
85
76
109

Table 6. Antimicrobial activity pattern of the bioactive compound separated from various solvent extracts of Acalypha indica against
Aspergillus niger.
Solvent

Acetone
Chloroform
Hexane
Methanol

Leaf

Stem

Root

Zone of inhibition in mm

% of activity

Zone of inhibition in mm

% of activity

Zone of inhibition in mm

% of activity

15
22
18
14

71
104
85
66

15
15
13
18

71
71
61
85

11
13
15
12

52
61
71
57

Table 7. Antimicrobial activity pattern of the bioactive compound separated from various solvent extracts of Acalypha indica against E. coli.
Solvent

Acetone
Chloroform
Hexane
Methanol

Leaf

Stem

Root

Zone of inhibition in mm

% of activity

Zone of inhibition in mm

% of activity

Zone of inhibition in mm

% of activity

15
16
25
23

71
76
119
109

18
22
16
20

85
104
76
95

14
22
20
25

66
104
95
119

Antimicrobial property of A. indica

for alternative antifungal agents to address these serious
infections (Walsh et al. 1996; Groll & Walsh 1997;
Dismukes 2000; Groll & Walsh 2001; Lin et al. 2001;
Pfaller et al. 2002). Clotrimazole is extensively used for
topical therapies and in localized and non-extensive
lesions (Pelletier et al. 2000). Due to the emergence of
resistance to Clotrimazole, the use of Clotrimazole is
being discontinued and systemic antifungal agents, such
as ucanozole, are utilized instead.
From these studies we understand that active compound isolated from Acalypha indica is more potent in
controlling Candida albicans, Aspergillus niger and
E. coli. This active compound is a best alternative for the
antibiotics to which resistance has been developed. It is
very clear that 1 unit of active compound isolated from
the plant has 7090% activity compared with 100 units
of authentic standard Clotrimazole. Further structural
studies on the bioactive compound extracted from
Acalypha indica are going on in our laboratory.

Acknowledgements
The authors thank the International Atomic Energy
Agency, Vienna, Austria for the support. We thank the
Bioinformatics Centre, School of Biotechnology,
Madurai Kamaraj University, Madurai for access to its
facilities.
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