|
Ethnobotanical
Leaflets 13: 320-25. 2009.
Antimicrobial Activity of Sphaeranthus
indicus L.
V.
Duraipandiyan, P. Kannan and S. Ignacimuthu*
Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, India
600 034
*Corresponding author, e-mail: entolc@hotmail.com
Issued 15 February 2009
Abstract
Aerial
parts and flowers of Sphaeranthus
indicus were extracted with n-hexane, benzene, chloroform, ehtylacetate
and acetone. The extracts were screened for their antimicrobial activity
using in vitro disc diffusion
method at concentrations of 5, 2.5 and 1.25 mg/disc. The Minimum Inhibitory
Concentration (MIC) was tested using broth micro dilution method at
concentrations ranging from 5 to 0.039 mg/ml. Significant antibacterial and
antifungal activity was observed in hexane extract of flower and aerial
parts. The flower extract showed MIC as 0.15 mg/ml against Staphylococcus
aureus and the highest MIC (5 mg/ml) was noted for S. epidermidis.
The n-hexane extracts of flower and aerial parts showed MIC as 0.15 and 1.25
mg/ml respectively against Candida albicans. In conclusion, the S.
indicus flower n-hexane extract seems to be a promising antimicrobial
agent.
Key words: antibacterial activity, antifungal activity, minimum inhibitory
concentration, Sphaeranthus indicus.��
1. Introduction
Different
societies across the world have shown great interest in curing illnesses
using plants/plant based drugs. Spheranthus
indicus L. (Asteraceae) a medicinal plant is wide spread in India and
Malaysia. S. indicus has long been
used in the treatment of skin infection, bronchitis, jaundice and nervous
depression (Nadkarni 1976). The roots and seeds are considered anthelmintic.
The herb is also reported to be useful as a tonic to treat indigestion,
asthma, leucoderma and dysentery (Chopra et al. 1956). A
novel isoflavone glycoside from leaves (Yadava and Kumar 1999) and a new
sesquiterpene glycoside and sphaeranthanolide were isolated from the flowers
of S. indicus and it was found to
be an immune stimulant (Shekhani et al. 1990). Medicinal information from tribal healers indicated that S. indicus is used to treat skin
disease, cough and fever. The bark, ground and mixed with whey, is said to be
useful in treating piles. Flowers are credited with alterative, depurative
and tonic properties; leaf juice is boiled with milk and sugar-candy and
prescribed for cough. An aqueous extract of the whole plant was slightly
toxic to American cockroaches (Chopra et al. 1958). The
present study was undertaken to assess the antimicrobial property of the
solvent extracts of flowers and aerial parts of S. indicus.
MATERIALS AND
METHODS
Plant material
S. indicus was collected from paddy
fields from Kancheepuram district of Tamil Nadu, India. The plant was
identified and confirmed by a taxonomist and the voucher specimen (ERIB-D-73)
was deposited in the herbarium at Entomology Research Institute, Loyola
College, Chennai.
Preparation of plant extracts
The
flowers and the aerial parts were separated, shade dried and coarsely
powdered with electric blender.200 g powder of flowers and aerial parts were
soaked separately in 600 ml of n-hexane, in an aspirator bottle for 72 h. The
extracts were collected and concentrated at 40�C under reduced pressure using
rotary evaporator. The extract was stored at 4�C
until further use. The remaining plant residue was subsequently extracted
with benzene, chloroform and ethylacetate similar manner.�
Test concentrations�������������������������������������
The
crude extracts were dissolved in Dimethyl sulphoxide (DMSO) and extracts were
loaded on the 6 mm dia. sterile disc (Himedia, Bombay)
with the concentrations of 1.25, 2.5, and 5 mg/disc.
Antimicrobial assay
Test cultures
�������������������� Bacteria:
Bacillus subtilis MTCC 441, Staphylococcus aureus ATCC
25923, Staphylococcus epidermidis
MTCC 3615, Enterococcus faecalis ATCC
29212, Escherichia coli ATCC
25922, Pseudomonas aeruginosa 27853
and Klebsiella pneumoniae ATCC
15380 and fungi; Candida albicans
MTCC 227, Aspergillus niger MTCC
1344 and Botrytis cinerea.The National
Committee for Clinical Laboratory Standards (NCCLS 1999) recommended
antimicrobial susceptibility test strains acquired from Christian Medical
College, Vellore were used in this study. The bacterial cultures were
maintained in Nutrient Agar (NA) and fungal cultures were maintained in
Sabouraud Dextrose Agar (SDA) slants at 4�C.
The
bacterial cultures were inoculated in Mueller Hinton (MH) broth and incubated
at 37�C for 18 h at 150 rpm. The bacterial inoculum was standardized to 0.8
OD at 660 nm and it was used for disc diffusion method. The final inoculum
size of 1x105 CFU/ml for bacteria and 1x104 CFU/ml for Candida were used for broth micro dilution technique. Antifungal screening was carried
out by broth micro dilution method; the final inoculum size was 1 x 104 spores/ml.
Disc diffusion method
Preliminary
antibacterial screening was carried out using disc diffusion method (Bauer et
al. 1966). Discs with different concentrations of plant extracts were
placed on the preinoculated Mueller Hinton Agar (MHA)� plates with respective cultures and were
incubated at 37oC for 24 h. Streptomycin (10 �g/disc) and DMSO
were used as positive and negative control, respectively. The inhibition zone
around the disc (diameter) was measured and recorded.
Minimum Inhibitory Concentrations (MIC)
Broth
micro dilution method (NCCLS 1999) was used to determine the MIC. This was
carried out in 96 well microtitre plates containing 200 �l Mueller Hinton
Broth with different concentrations of plant extracts. The final concentration
of DMSO was maintained at 0.1% in the test broth. Triplicates were maintained
along with the negative control. Plates were incubated at 37�C for 12 h for
bacteria and at 27�C for fungi.� MIC
was determined as the complete inhibition of growth at lowest concentration.
Results
Flower
extracts with hexane gave pale yellow colour and other extracts were pale
brown to brown in colour. Extracts from aerial parts were dark green to dark
brown in colour. The yield of the flower extract fell in the range of 0.5-1%
(w/w) and that of aerial parts were in the range of 1-2% (w/w) of the dried
material.
Hexane
extract of S. indicus (flowers and
aerial parts) showed antimicrobial activity against most of the bacteria and
fungi tested. Flower extract showed higher activity than the aerial parts
against Gram positive bacteria such as B.subtilis,
S. aureus, S. epidermidis and E. faecalis which were comparable with
antibiotic Streptomycin (10 �g/disc) (Table
1). The fungi Aspergillus niger,
Botrytis cinerea and C. albicans
were inhibited by the extracts of both the flower and aerial parts. Benzene,
chloroform, ethyl acetate and acetone extracts of flower and aerial parts
showed some activity at higher concentration (5 mg/disc) against gram
positive bacteria.
The
hexane extract of flowers showed MIC at 0.31 mg/ml for B.subtilis,
0.15 mg/ml for S. aureus and 5
mg/ml for S. epidermidis. On the
other hand, aerial parts showed higher MIC at 2.5 mg/ml for B.subtilis 5mg/ml
for Staphylococcus spp. and 5 mg/ml
for E. faecalis compared to flower.
Most of the gram negative bacteria showed higher MIC (>5 mg/ml) for both
the extracts of flower and aerial parts (Table 3).
The
fungus C. albicans showed MIC at
0.15 mg/ml for flower extract and 1.25 mg/ml for hexane extracts of aerial
parts. The hexane extract of aerial parts of S. indicus showed promising antifungal activity against B. cinerea. 100% growth inhibition was
observed at 0.625 mg/ml concentration and determined as MIC (Table 2).
Discussion
Hexane
extracts of flowers and aerial parts of S.
indicus exhibited antibacterial and antifungal activity. The essential
oil of S. indicus has been reported
for its antifungal activity against plant pathogenic fungi (Rao et al.,
1971). A sesquiterpene lactone, 7-hydroxyfrullanolide isolated from S. indicus had antimicrobial activity
(Atta-ur-Rahman et al., 1989; Perumalsamy et al., 1999). The inhibition zone
of antibiotic streptomycin (10 �g/disc) was comparable with both the flower
extract (1.25 mg/disc) and aerial parts extract (2.5 mg/disc) against B.subtilis and S. aureus. Similar antibacterial activity was observed in other
plants of the same family)(Roose et al., 1998). Higher inhibition zone was
observed in Bacillus subtilis
at 5 mg/disc for hexane flower extract. The inhibition zone was directly
proportional to the concentration used. Hexane flower extract showed MIC at
0.31 mg/ml for Bacillus sp. whereas
the aerial part showed higher MIC at 2.5 mg/ml. The extracts of flower and
aerial parts showed inhibition against gram positive organisms but not
against gram negative organisms. Similar results were observed in Chrysanthemum coronarium flower
extract (Urzua and Mendosa, 2003). Sesquiterpene lactones from Vernonia colorata, possessed high
antibacterial activity primarily against Gram positive and low activity
against Gram negative species (Rabe et al., 2002) similar to our findings
here.
The
flower extract showed MIC at 0.15 mg/ml and aerial parts showed MIC at 1.25
mg/ml against C. albicans; this is the
first report on anti fungal activity against Candida as per the
available literature. The hexane extract of flower showed complete inhibition
against A. niger
and B. cinerae; the MIC was determined as 1.25
and 0.625 mg/ml respectively. The hexane extract of aerial parts showed MIC
as 2.5 and 0.625 mg/ml against A. niger
and B. cinerea respectively. The antifungal activity against Trichophyton spp., Epidermophyton floccosum and Microsporum cooki was reported in
thiophene compound isolated from Tagetes
patula (Asteraceae) (Romagnoli et al., 1998). Antifungal
activity was also reported for the ethanol extract of underground parts of Leuzea carthamoides (Asteraceae)
against C. albicans, A. fumigatus (Chobot et al., 2003), and Centaurea
hermanni (Asteraceae) (Sur-Altiner et al., 1997). which is
similar to our results.
Conclusion
����������� The S. indicus hexane extracts of flower and aerial parts showed good
antibacterial activity against gram positive organisms. Flower extracts were
more active than the aerial parts. It also possessed strong antifungal
activity against Candida and other
tested fungi. The findings of the present research may lead to the
development of natural antimicrobial agents
References
- Nadkarni KM. Indian
Materia Medica, Popular Prakshan, Bombay,
1976, Vol 1. pp. 1126.
- Chopra RN et al.,
(1956) Glossary of Indian Medicinal Plants, Publication and Information
Directorate, New Delhi, pp.
232.
- Yadava
RN and� Kumar S (1999) A novel
isoflavone glycoside from the leaves of Sphaeranthus indicus, Fitoterapia.70:127-9.
- Shekhani
MS et al An immunostimulant sesquiterpene glycoside isolated from Sphaeranthus indicus, Phytochem
1990; 29:2573-6.
- Chopra RN, Chopra
IC, Honda KL, Kapur LD. Indigenous drugs of India,
2nd edn., UN Dhur and Sons (P) Ltd, Calcutta,
1958.
- National Committee
for Clinical Laboratory Standards (NCCLS), Document M31- A performance
standards for antimicrobial disk and dilution susceptibility tests for
bacteria isolated from animals, Approved Standard, NCCLS, Villanova,
1999.
- Bauer AW, Kirby MDK,
Sherries JC, Turck M. Antibiotic susceptibility testing by standard
single disc diffusion method. Am J Clin Pathol 1966;45:493-6.
8.
Rao BG, Narasimha V, Joseph PL. Activity of some essential oil towards
phytopathogenic fungi. Riechstoffe Aromen Koerper-pflegemit 1971;21:405-10.
- Atta-ur-Rahman,
Shekhani MS, Perveen S, Habib-ur-Rehman, Yasmin A, Zia-ul Haq A, Sheikh
D. 7-hydroxyfrullanolide, an antimicrobial sesquiterpene lactone from Sphaeranthus indicus Linn.� J Chem Res (S)1989;68.
- Perumalsamy R, Ignacimuthu
S, Patric Raja D. Preliminary screening of ethanomedicinal plants from India.
J Ethanopharmacol 1999;66:235-40.
- Roos� G, Prawat H, Walter CU, Klaiber I,
Vogler B, Guse JH, Kraus W. New sesquiterpene lactone with antibacterial
activity from Vernonia fastigiata. Planta Medica 1998;64:673.
- Urzua
A, Mendoza L. Antibacterial activity of fresh flower heads of Chrysantemum
coronatium. Fitoterapia 2003;74:606-8.
- Rabe T,
Mullholland, Van Staden J. Isolation and identification of antibacterial
compounds from Vernonia colorata leaves. J Ethnopharmacol. 2002;80:91-4.
- Romagnoli C, Mares D, Sacchetti G, Bruni A. The
photodynamic effect of 5-(4-hydroxy-l-butinyl)-2,2'-bithienyl on
dermatophytes. Mycol Res 1998;102:1519-24.
- Chobot
V, Buchta V, Jahodarova H, Pour M, Opletal L, Jahodar L, Harant P.
Antifungal activity of a thiophene polyine from Leuzea carthamoides.
Fitoterapia 2003;74:288-90.
- Sur-Altiner D,
Gurkan E, Sarioglu I, Tuzlaci E, Ang O. The antibacterial and antifungal
effects of Centaurea hermannii.
Fitoterapia 1997;68:374.
Table 1 Antimicrobial
activity of flower extracts of Sphaeranthus
indicus by disc diffusion method
|
Tested
organisms
|
Zone of inhibition in diameter (mm)
|
|
Streptomycin
|
Hexane (mg/disc)
|
Benzene (mg/disc)
|
Chloroform (mg/disc)
|
Ethyl acetate (mg/disc)
|
Acetone (mg/disc)
|
|
10 �g/disc
|
1.25
|
2.5
|
5.0
|
1.25
|
2.5
|
5.0
|
1.25
|
2.5
|
5.0
|
1.25
|
2.5
|
5.0
|
1.25
|
2.5
|
5.0
|
|
Bacteria
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Bacillus
subtilis MTCC441
|
13
|
12
|
18
|
22
|
-
|
9
|
12
|
-
|
9
|
10
|
-
|
9
|
12
|
-
|
10
|
12
|
|
Staphylococcus
aureus ATCC 25923
|
12
|
11
|
15
|
17
|
-
|
11
|
12
|
-
|
9
|
10
|
-
|
11
|
13
|
-
|
-
|
10
|
|
Staphylococcus
epidermidis MTCC 3615
|
-
|
12
|
16
|
19
|
-
|
9
|
14
|
-
|
-
|
10
|
-
|
12
|
13
|
-
|
-
|
10
|
|
Enterococcus
faecalis
ATCC 29212
|
-
|
8
|
10
|
12
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
10
|
-
|
-
|
9
|
|
Escherichia
coli
ATCC
25922
|
13
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
Klebsiella
pneumonia
ATCC 15380
|
11
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
Pseudomonas
aeruginosa
ATCC 27853
|
13
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
Fungi
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Candida
albicans
MTCC 227
|
-
|
9
|
10
|
12
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-, no activity
Table 2.� Antimicrobial activity of the extracts of
aerial parts of Sphaeranthus indicus by disc diffusion method.
|
Tested organisms
|
Zone of inhibition in diameter (mm)
|
|
Streptomycin
|
Hexane (mg/disc)
|
Benzene (mg/disc)
|
Chloroform (mg/disc)
|
Ethyl acetate (mg/disc)
|
Acetone (mg/disc)
|
|
10 �g/disc
|
1.25
|
2.5
|
5.0
|
1.25
|
2.5
|
5.0
|
1.25
|
2.5
|
5.0
|
1.25
|
2.5
|
5.0
|
1.25
|
2.5
|
5.0
|
|
Bacteria
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Bacillus subtilis MTCC 441
|
13
|
10
|
13
|
18
|
-
|
-
|
11
|
-
|
-
|
12
|
-
|
9
|
14
|
-
|
-
|
13
|
|
Staphylococcus
aureus
ATCC 25923
|
12
|
11
|
13
|
16
|
-
|
-
|
10
|
-
|
-
|
10
|
-
|
8
|
11
|
-
|
-
|
12
|
|
Staphylococcus
epidermidis
MTCC 3615
|
-
|
9
|
12
|
14
|
-
|
9
|
12
|
-
|
9
|
11
|
-
|
-
|
13
|
-
|
-
|
9
|
|
Enterococcus
faecalis
ATCC 29212
|
-
|
8
|
10
|
13
|
-
|
-
|
-
|
-
|
-
|
9
|
-
|
8
|
11
|
-
|
-
|
-
|
|
Escherichia
coli
ATCC 25922
|
13
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
Klebsiella
pneumonia
ATCC 15380
|
11
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
Pseudomonas
aeruginosa
ATCC 27853
|
13
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
Fungi
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Candida
albicans
MTCC 227
|
-
|
8
|
10
|
11
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
|
-, no activity
Table 3.� Minimum Inhibitory Concentration (MIC) of
hexane extracts of Sphaeranthus indicus
by broth micro dilution method.
|
Tested
organisms
|
Minimum
Inhibitory Concentration (mg/ml)
|
|
Flower
|
Aerial part
|
|
Bacteria
|
|
|
|
Bacillus subtilis �MTCC 441
|
0.31
|
2.5
|
|
Staphylococcus aureus ATCC
25923
|
0.15
|
5.0
|
|
Staphylococcus epidermidis MTCC 3615
|
5.0
|
5.0
|
|
Enterococcus faecalis ATCC
29212
|
1.25
|
5.0
|
|
Escherichia coli ATCC 25922
|
>5.0
|
>5.0
|
|
Klebsiella pneumonia ATCC
15380
|
>5.0
|
>5.0
|
|
Pseudomonas aeruginosa ATCC
27853
|
>5.0
|
>5.0
|
|
Fungi
|
|
|
|
Candida albicans MTCC 227
|
0.15
|
1.25
|
|
Aspergillus niger
MTCC 1344
|
1.25
|
2.5
|
|
Botrytis cinerea
|
0.625
|
0.625
|
|
ATCC - American
Type Culture Collection Centre; MTCC - Microbial Type Culture Collection, India
|
|