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African Journal of Biomedical Research
Ibadan Biomedical Communications Group
ISSN: 1119-5096
Vol. 9, Num. 2, 2006, pp. 89-93
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African Journal of Biomedical Research, Vol. 9, Vol. 2, May, 2006, pp. 89-93
Full length Research Article
In-vitro Antimicrobial
Activities of Extractsof Launaea procumbens Roxb.
(Labiateae), Vitis vinifera L. (Vitaceae) and Cyperus rotundus L.
(Cyperaceae)
Jigna Parekh, and Sumitra Chanda*
Phytochemical,
Pharmacological and Microbiological Laboratory, Department of Biosciences,
Saurashtra University, Rajkot 360 005, India.
*Address for Correspondence (e-mail): sumitrachanda@yahoo.com
Received: November, 2005
Accepted (Revised): April,
2006
Published May
2006
Code Number: md06016
ABSTRACT
The aqueous and ethanolic
extracts of Launaea procumbens Roxb. (Labiateae), Vitis vinifera L. (Vitaceae)
and Cyperus rotundus L. (Cyperaceae) were evaluated for antimicrobial activity
against clinically important bacteria viz. Alcaligenes faecalis ATCC8750,
Bacillus cereus ATCC11778, Bacillus subtilis ATCC6633, Enterobacter aerogenes
ATCC13048, Escherichia coli ATCC25922, Klebsiella pneumoniae NCIM2719, Proteus
mirabilis NCIM224, Proteus vulgaris NCTC8313, Pseudomonas aeruginosa ATCC27853,
Pseudomonas pseudoalcaligenes ATCC17440, Salmonella typhimurium ATCC23564,
Staphylococcus aureus ATCC25923, Staphylococcus epidermidis ATCC12228, Staphylococcus
subfava NCIM2704 and Candida tropicalis ATCC4563. The in vitro antimicrobial
activity was performed by agar disc diffusion and agar well diffusion method.
The ethanolic extracts of all the plants were active against all the
investigated bacterial strains while all the aqueous extracts were inactive
except for Vitis vinifera L. S. typhimurium was the most resistant bacterial
strain against all the extracts
Keywords: antimicrobial activity, aqueous extract,
ethanolic extract, Launaea procumbens Roxb., Vitis vinifera L.
and Cyperus rotundus L.
INTRODUCTION
Plants
have a great potential for producing new drugs of great benefit to mankind.
There are many approaches to the search for new biologically active principles
in higher plants (Farnsworth and Loub, 1983). Many efforts have been done to
discover new antimicrobial compounds from various kinds of sources such as
soil, microorganisms, animals and plants. One of such resources is folk
medicine and systematic screening of them may result in the discovery of novel
effective compounds (Janovska et al., 2003). Further, scientific investigation
and information of the therapeutic potential of the plant material is limited.
Despite the existence of potent antibiotic and antifungal agents, resistant or
multi-resistant strains are continuously appearing, imposing the need for a
permanent search and development of new drugs (Silver, 1993). There is an
urgent need to systematically evaluate the plants used in traditional
medicine. Such research could lead to new drug discovery or advance the use of
indigenous herbal medicines for orthodox treatment. Now a day a renewed
interest in traditional medicine is observed and there has been an increasing
demand for more and more drugs from plant sources. This revival of interest in
plant-derived drugs is mainly due to the current widespread belief that green
medicine is safe and more dependable than the costly synthetic drugs many of
which have adverse side effects.
The need of the hour is to screen a number of medicinal plants for promising
biological activity. Considering the aforesaid, the three traditionally used
medicinal plants belonging to three different families were screened for their
antimicrobial properties. Launaea procumbens Roxb. (Labiateae) is used
as coolent, diuretic, demulcent, allergic infections. Vitis vinifera L.
(Vitaceae) is used in conditions like burning sensations, haemorrhages,
anaemia, leprosy, skin diseases, syphilis, asthama, jaundice, bronchitis
(Anjaria et al., 2002; Sriram et al., 2004). Cyperus rotundus L.
(Cyperaceae) is used in stomach disorders, diarrhoea, dysentery. The
roots and tubers are analgesic, antibacterial, antispasmodic, antitussive,
aromatic, astringent, carminative, diaphoretic, diuretic, emmenagogue,
litholytic, sedative, skin, stimulant, stomachic, tonic and vermifuge (Singh
and Kachroo, 1976; Lassak and McCarthy, 1978; Yeung, 1985; Bown, 1995). An
essential oil in the tubers has antibiotic activity and has been shown to
arrest the growth of Micrococcus pyrogenes (Chopra et al., 1986). The
purpose of this study was to screen for the aqueous and ethanolic extracts of
these medicinal plants that could be useful for the development of new tools as
antimicrobial agents for the control of infectious diseases.
MATERIAL AND METHODS
Plant
material: Fresh plant or plant parts
were collected randomly from the semi-arid region of Rajkot Gujarat, India. Whole
plant of Launaea procumbens Roxb., leaves of Vitis vinifera L.
and whole plant of Cyperus rotundus L. were taken for investigation of
antimicrobial property. The taxonomic identities of these plants were confirmed
by Dr. P. S. Nagar, Department of Biosciences, Saurashtra University, Rajkot
and the voucher specimen numbers of the plants were preserved. Fresh plant
material were washed under running tap water, air dried and then homogenized to
fine powder and stored in airtight bottles.
Preparation
of extracts: For aqueous extraction,
10 g of air-dried powder was added to distilled water and boiled on slow heat
for 2 hours. It was then filtered through 8 layers of muslin cloth and
centrifuged at 5000g for 10 min. The supernatant was collected. This procedure
was repeated twice. After 6 hours, the supernatant collected at an interval of
every 2 hours, was pooled together and concentrated to make the final volume
one-fourth of the original volume (Parekh et al., 2005). It was then autoclaved
at 121 °C temperature and at 15 lbs pressure and stored at 4 oC.
For solvent extraction, 10 g of air-dried powder was taken in 100 ml of organic
solvent (methanol or ethanol) in a conical flask, plugged with cotton wool and
then kept on a rotary shaker at 190-220 rpm for 24 h. After 24 hours the
supernatant was collected and the solvent was evaporated to make the final
volume one-fourth of the original volume (Parekh et al., 2005)and
stored at 4oC in airtight bottles.
Microorganisms: In vitro antimicrobial activity was examined for aqueous and
ethanol extracts from three medicinal plants used by traditional healers.
Microorganisms were obtained from National Chemical Laboratory (NCL), Pune,
India. Microorganisms were maintained at 4°C on nutrient agar slants (for bacteria)
and MGYP slants (for yeast).
Antimicrobial assay: The antimicrobial assay was performed by
two methods viz. agar disc diffusion method (Bauer et al., 1966)for
aqueous extract and agar well diffusion method (Perez et al., 1990)for
solvent extract. The molten Mueller Hinton Agar was inoculated with the 100 µl
of the inoculum (1 x 108 Cfu) and poured into the Petri plate
(Hi-media). For agar disc diffusion method, the disc (0.7cm) (Hi-Media) was
saturated with 100 µl of the test compound, allowed to dry and was introduced
on the upper layer of the seeded agar plate. For agar well diffusion method, a
well was prepared in the plates with the help of a cork-borer (0.85cm). 100 µl
of the test compound was introduced into the well. The plates were incubated
overnight at 37 °C. Microbial growth was determined by
measuring the diameter of zone of inhibition. For each bacterial strain
controls were maintained where pure solvents were used instead of the extract.
The result was obtained by measuring the zone diameter (Table-1). The
experiment was done three times and the mean values are presented.
RESULTS
The data reported in Table 1 presents the antimicrobial activity of the aqueous
and ethanolic extracts of Launaea procumbens Roxb., Vitis vinifera L. and Cyperus rotundus L. The results
indicate that the extracts from the medicinal plants studied showed
inhibition of growth of some of the tested micro organisms with to various
degrees.
Table 1. In
vitro Antimicrobial activity of aqueous and ethanolic extracts
of Launaea procumbens (Roxb.) (Labiateae), Vitis vinifera L.
(Vitaceae) and Cyperus rotundus L. (Cyperaceae).
Microorganisms |
L.
procumbens Roxb. |
Vitis
vinifera L. |
Cyperus
rotundus L. |
Antimicrobics |
Ae |
Ee |
Ae |
Ee |
Ae |
Ee |
C |
Ap |
|
Zone of inhibition (mm)* |
A.
fecalis ATCC8750 |
- |
10 |
10 |
10 |
- |
12 |
17 |
- |
B. cereus ATCC11778 |
- |
25 |
13 |
21 |
- |
18 |
17 |
- |
B. subtilis ATCC6633 |
- |
10 |
10 |
12 |
- |
12 |
16 |
- |
E. aerogenes ATCC13048 |
- |
11 |
12 |
14 |
- |
11 |
20 |
- |
E. coli ATCC25922 |
- |
10 |
- |
15 |
- |
- |
22 |
- |
K. pneumoniae NCIM2719 |
- |
14 |
14 |
16 |
- |
15 |
32 |
- |
P. mirabilis NCIM224 |
- |
17 |
10 |
16 |
- |
15 |
18 |
- |
P. vulgaris NCTC8313 |
- |
- |
- |
11 |
- |
11 |
21 |
- |
P. aeruginosa ATCC27853 |
- |
- |
- |
11 |
- |
- |
10 |
- |
P.pseudoalcaligenes ATCC17440 |
- |
- |
17 |
13 |
- |
11 |
28 |
- |
S. typhimurium ATCC23564 |
- |
- |
- |
- |
- |
- |
25 |
- |
S. aureus ATCC25923 |
- |
30 |
10 |
15 |
- |
13 |
20 |
- |
S. epidermidis ATCC12228 |
- |
21 |
11 |
12 |
- |
14 |
19 |
- |
S. subfava NCIM2704 |
- |
19 |
10 |
14 |
- |
13 |
18 |
- |
C. tropicalis ATCC4563 |
- |
18 |
- |
- |
- |
- |
- |
7 |
Ae: aqueous extract; Ee: ethanolic extract; Disc
diameter -7mm and cork-borer diameter 8.5 mm, Positive control: C: Chloramphenicol
(30 mcg/disc);
Ap: Amphotericin B (100 units/disc)
*Values are mean of three replicates;
--: no
inhibition zone
The ethanolic extract was found to be the most effective antimicrobial agent
as
compared to the aqueous extract. The ethanolic extract of Launaea
procumbens Roxb. was active against more than 70 percent of microorganisms
investigated while its aqueous extract was totally inactive against all the
microorganisms investigated. The ethanolic as well as the aqueous extract of Vitis
vinifera L. was active against more than 85 and 65 per cent of the studied
bacterial strains respectively. Both, ethanolic and aqueous extracts of
Cyperus rotundus L. showed similar activity as that of Launaea procumbens Roxb. Amongst all the three plant species, Launaea procumbens Roxb.
exhibited remarkable activity against some microorganisms.
B.
cereus was the most susceptible
gram-positive bacteria followed by S. aureus and S. epidermidis while B. subtilis was the least susceptible gram-positive bacteria. S.
typhimurium was the most resistant gram-negative bacterial strain followed
by P. aeruginosa, P. vulgaris and E.coli. against all the
extracts. K. pneumoniae was the most susceptible gram-negative
bacteria. None of the extracts except ethanolic extract of Launaea
procumbens Roxb. exhibited anticandidal activity against C. tropicalis.
The inhibitory activities of all the extracts reported in Table 1 are
comparable with standard antimicrobics chloramphenicol (30 mcg/disc) and
amphotericin B (100 units/disc).
DISCUSSION
Successful prediction of botanical compounds from plant material is largely
dependent on the type of solvent used in the extraction procedure. The
traditional healers or practitioners make use of water primarily as a solvent,
but our studies showed that methanol extracts of these plants were certainly
much better and powerful. This may be due to the better solubility of the
active components in organic solvent (de Boer et al.,
2005). These observations can be rationalized in terms of the polarity
of the compounds being extracted by each solvent and, in addition to their
intrinsic bioactivity, by their ability to dissolve or diffuse in the different
media used in the assay. The growth media also seem to play an important role
in the determination of the antibacterial activity. Lin et al. (1999)reported
that Muller-Hinton agar appears to be the best medium to explicate the antibacterial
activity and the same was used in the present study.
Amongst the gram-positive and gram-negative bacteria, gram-positive bacterial
strains were more susceptible to the extracts as compared to gram-negative
bacteria. This is in agreement with previous reports that plant extracts are
more active against gram-positive bacteria than gram-negative bacteria
(Vlietinck et al, 1995; Rabe and Van Staden, 1997).
The results of
present study supports the traditional usage of the studied plants and suggests
that some of the plant extracts possess compounds with antimicrobial properties
that can be used as antimicrobial agents in new drugs for the therapy of
infectious diseases caused by pathogens. The most active extracts can be
subjected to isolation of the therapeutic antimicrobials and carry out further
pharmacological evaluation.
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