In vitro Antimicrobial Activities of Different Fractions of
Swertia chirata Ethanolic Extract
The aim of the present study was to evaluate the in
vitro antimicrobial activity of pet-ether, dichloromethane and methanol
fractions of Swertia chirata (Family: Gentianaceae) ethanolic extract.
Disc diffusion technique and food poison method were used for antibacterial
and antifungal activity, respectively. Dichloromethane fraction from both
leaf and stem showed significant antimicrobial activities against some
Gram-positive and Gram-negative bacteria and mild to moderate activity
against some fungi. A large zone of inhibition was observed (19 mm) against
Staphylococcus aureus. Test materials at a concentration of 400
μg disc-1 were used to evaluate the antimicrobial activity
while Kanamycin at a concentration of 30 μg disc-1 was
used as positive control in this study. Among different fractions, dichloromethane
fraction showed significant antimicrobial activity against Gram-positive,
Gramnegative and fungi. The most significant antimicrobial activity was
seen against Staphylococcus aureus which reflects it potentiality
to be used in skin infections.
The use of higher plants and preparations made from them to treat infections
is an old-age practice in a large part of the world population, especially in
developing countries, where there is dependence on traditional medicine for
a variety of diseases (Ahmad et al., 1998). In
recent time, interest with herbal medicine for antimicrobial activities has
been increased significantly. The economic crisis, high cost of industrialized
medicines, inefficient public access to medical and pharmaceutical care, in
addition to the side effects caused by synthetic drugs are some of the factors
contributing to the central role of medicinal plants in health care (Johann
et al., 2007). Hence, the present study was undertaken to evaluate
the antimicrobial activity of Swertia chirata (Family: Gentianaceae).
The plant is a native of temperate Himalayas, found at an altitude of 1200-3000
m (4000 to 10,000 ft), from Kashmir to Bhutan and in the Khasi hills at 1200-1500
m (4000 to 5000 ft) (Kirtikar and Basu, 1984; Clarke,
1885). It can be grown in sub-temperate regions between 1500 and 2100 m
altitudes (Bentley and Trimen, 1880). The plant has about
2-3 feet long stem, the middle portion is round, while the upper is four-angled.
The stems are orange brown (Anonymous, 1982) or purplish
in color (Bentley and Trimen, 1880) and contain large
continuous yellowish pith. The root is simple, tapering and stout, short, almost
7 cm long and usually half an inch thick (Clarke, 1885;
Bentley and Trimen, 1880). Flowers are small, stalked,
green-yellow, tinged with purple colour, rotate and tetramerous (Kirtikar
and Basu, 1984; Bentley and Trimen, 1880). The plant
is gathered during the late stages of flowering, commonly tied up in flattish
bundles about 3 ft long and 1.5 to 2 lbs in weight (Bentley
and Trimen, 1880) and is sold in the market as dried brownish stems with
root and leaves intact. The plant extract have been reported to possess antipyretic
(Bhargava et al., 2009), anti-viral (Verma
et al., 2008), anthelmintic (Iqbal et al.,
2006), anticarcinogenic (Saha et al., 2004),
hepatoprotective (Mukherjee et al., 1997) hypoglycemic
(Bajpai et al., 1991; Saxena
et al., 1993) activities. Early studies documented the presence of
flavonoids, xanthones, terpenoids, iridoid and secoiridoid glycosides in the
S. chirata plant (Pant et al., 2002).
In the present study, the antimicrobial properties of pet-ether, dichloromethane
and methanol fractions of leaf and stem of Swertia chirata Ethanolic
extract were investigated by disc diffusion technique and food poison method.
MATERIALS AND METHODS
Collection and preparation of plant materials: Fresh plant sample of Swertia chirata was collected from Chawk bazar, Dhaka in November 2007 and taxonomically identified by the National Herbarium of Bangladesh, Mirpur, Dhaka having the identification accession number -34333. Leaf, stem and root were separated from the plant and dried in shade. The dried leaf and stem were then ground in coarse powder using high capacity grinding machine in the Phytochemical Research Laboratory, Faculty of Pharmacy, University of Dhaka and preserved in an air tight containers.
Extraction and fractionation: Leaf and stem powder of Swertia chirata were extracted with ethanol by cold extraction procedure. The extracts were filtered and concentrated at 40°C with a Heidolph rotary evaporator until solid/semisolid mass were produced. The crude extract of leaf and stem were then dissolved in 10% water in methanol (100 mL) and partitioned between pet-ether, dichloromethane and methanol fractions.
Test organism: Both Gram-positive (Bacillus cereus, Bacillus
megaterium, Bacillus subtilis, Staphylococcus aureus, Sarcina
lutea) and Gram-negative bacteria (Escherichia coli, Pseudomonas
aeruginosa, Salmonella paratyphi, Salmonella typhi, Shigella
boydii, Shigella dysenteriae, Vibrio mimicus, Vibrio parahemolyticus),
as well as fungal strains (Candida albicans, Aspergillus niger, Sacharomyces
cerevacae) used for the experiment were collected as pure cultures from
the Institute of Nutrition and Food Science (INFS), University of Dhaka, Bangladesh.
Experimental procedure: The antimicrobial study of the test sample was
carried out by Disc diffusion technique (Monica, 2000)
for bacteria and Poison food technique (Dhingra and Sinclair,
1995) for fungi. Standard Kanamycin disc (30 μg disc-1)
and disc containing the test materials (400 μg disc-1) impregnated
with the respective solvents were used as positive and negative controls, respectively.
The antimicrobial potency of the test samples was measured by determined the
diameter of the zone of inhibition in millimeter.
The study showed that among different fractions, dichloromethane fraction of both leaf and stem have significant antimicrobial activities at a dose of 400 μg disc-1 against some Gram-positive and Gram-negative bacteria and moderate activity against some fungi, whereas other fractions showed mild to moderate antimicrobial activities (7-11 mm). The results are summarized in the Table 1.
Results of antibacterial screening: Among Gram-positive bacteria, the
most notable effect was seen against Staphylococcus aureus (19 mm with
dichloromethane fraction of both leaf and stem) in comparison to others. Dichloromethane
fraction of leaf and stem also showed marked antimicrobial activities against
Bacillus cereus (16 mm for dichloromethane fraction of leaf and 17 mm
for dichloromethane fraction of stem) and Bacillus subtilis (14 mm for
dichloromethane fraction of leaf and 13 mm for dichloromethane fraction of stem).
activity of different fractions of Swertia chirata
|PL: Pet-ether fraction of leaf, DCML: Dichloromethane fraction
of leaf, ML: Methanol fraction of leaf, PS: Pet-ether fraction of stem,
DCMS: Dichloromethane fraction of Stem, MS: Methanol fraction of stem, MS:
Methanol fraction of stem, Dose -400 μg disc-1 (Sample)
and 30 μg disc-1 (Kanamycin). -: Zone of inhibitions less
than 6 mm are not included in the table
Against Gram-negative bacteria, dichloromethane fraction of leaf and stem showed
highest activity against Escherichia coli in comparison to others. The
zone of inhibition against Escherichia coli was found to be 15 mm for
Dichloromethane fraction of leaf and 17 mm for stem fraction. Dichloromethane
fraction of stem and leaf also showed moderate antimicrobial activities (10-13
mm) against Gram-negative bacteria except Escherichia coli.
Result of antifungal screening: Against different fungi, dichloromethane fraction also showed moderate antimicrobial activity (12 mm for dichloromethane fraction of leaf and 12-13 mm for dichloromethane fraction of stem).
Herbal plants are an important source of new chemical substances with potential
therapeutic uses. Approximately 119 pure chemical substances extracted from
higher plants are used in medicine throughout the world (Farnsworth
et al., 1985). The increased interest on plant medicines in todays
world is from the belief that green medicine is safe and dependable, compared
with costly synthetic drugs that have adverse effects (Nair
and Chanda, 2006).
In the present investigation we have studied the antimicrobial activity of
Swertia chirata. As the result, dichloromethane fraction of Swertia
chirata was most active against Staphylococcus aureus in comparison
to other microorganisms tested. Among Gram-negative bacteria E. coli
also showed significant susceptibility to dichloromethane fraction. On contrary,
microorganisms were less susceptible to pet-ether and methanol fraction in relative
to dichloromethane fraction. This may be due to good solubility of active chemical
substance in dichloromethane fraction. The highest activity of dichloromethane
fraction against Staphylococcus aureus reflects its potentiality to treat
skin infections. Traditionally the plant is used for curing various skin diseases
in Indian subcontinent where the plant is indigenous (Joshi
and Dhawan, 2005). From the present investigation, the results obtained
confirmed the therapeutic potency of Swertia chirata used in traditional
medicine. The present study also set an important basis for further phytochemical
and pharmacological investigation on Swertia chirata.
The present study suggests that the plant extract certainly possess some chemical constituents with antimicrobial properties and this finding is very important in discovering new drugs for the therapy of infectious diseases. However, further studies can be subjected to isolate and characterize the active constituents responsible for the antimicrobial property of Swertia chirata.
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