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Ethnobotanical
Leaflets 12: 1090-95. 2008. In vitro Phytochemical Screening and Antibacterial
Activity of Aqueous and Methanolic Leaf Extracts of
Tridax procumbens
against Bovine
Mastitis Isolated Staphylococcus aureus *R. Dhanabalan, A. Doss, M. Jagadeeswari,
C.M. Reena Josephine, R. Vaidheki
and K. Kalamani Department of Microbiology, Centre for Post
Graduate Studies in Microbiology RVS *Corresponding author:
bharathi.dhanabalan@gmail.com Issued Abstract Tridax procumbens Linn
is a tropically distributed medicinal plant. Antimicrobial activity of
aqueous and methanol extracts of this plant was investigated by agar disc and
well-diffusion method against bovine mastitis causing Staphylococcus aureus strains. The plant extracts showed inhibitory
activity against the tested organisms. Phytochemical
screening of the plant revealed the presence of tannins, flavonoids,
saponins and alkaloids. The study scientifically
validates the use of this plant in traditional and ethnoveterinary
medicine. Key words: Tridax procumbens,
Staphylococcus aureus, Ethnoveterinary
medicine, Anti-mastitis and Flavonoids. Introduction Tridax procumbens Linn (compositae)
is a common plant found in tropical areas of all countries, growing primarily
during raining season. It is a common weed in Tamilnadu
present along with economically important crops. It habitats waste places,
road sides and hedges throughout In In view of the dearth of all above information’s, the present study was undertaken to investigate the effects of aqueous and methanolic extracts of leaves of T. procumbens. This study is to elucidate the mechanism of in vitro antibacterial action of plant material against mastitis isolated contagious Staphylococcus aureus. To our knowledge, no reports or studies exist relating to in vitro application of T. procumbens extracts in bovine mastitis works. This is the first report on T. procumbens antibacterial action against bovine mastitis isolated contagious pathogens. MATERIALS
Plant
collection Fresh plant leaves were
collected randomly from the villages of Plant
extraction For aqueous extraction, 10 g of air-dried
powder was mixed with 100 ml distilled water and stand at room temperature
for 48 h. It was then filtered through 8 layers of muslin cloth and
centrifuged at 5000 g for 10 min. The supernatant was collected and
stored at 4oC. For solvent extraction, 10 g of air dried powder
was mixed with 100 ml of organic solvent (methanol) in a conical flask,
plugged with cotton and then kept on a rotary shaker at 190 - 220 rpm for 24
h. After 24 h, it was filtered through 8 layers of muslin cloth and
centrifuged at 5000 g for 10 min. The supernatant was collected and
the solvent was evaporated using rotary vaccum pump
and stored at 4oC in air-tight bottles. Bacterial
strains Bacterial strains used in this study were the multidrug resistant contagious bovine mastitis S.aureus isolated from the mastitis infected
dairy cows of four different breeds (Jersey, Holstein-Friesian, Zebu and
Cross breeds). All the strains were confirmed by cultural, biochemical
characteristics (Klastrup, O, 1975) and screened for their antibacterial
resistance (Bauer et al., 1966).
Among 21, only four multidrug resistant S.aureus
resistant to Methicillin and other Penicillin
derivatives but susceptible to Cloxacillin were
selected. Each strain was denoted by J.Sau, HF.Sau, Z.Sau and CB.Sau as they were isolated from Antibacterial
activity The antibacterial assay of aqueous and methanolic extracts was performed by two methods. The
agar disc diffusion method (Bauer et
al., 1966; Parekh and Chanda,
2006) and agar well diffusion method (Perez et al., 1990; Nair and Chanda, 2005).
The media (Mueller Hinton Agar No.2), along with the inoculum
(108 cfu/ml)
was poured into the Petri plate (Hi-Media). For the agar disc diffusion
method, the disc (0.7 cm) (Hi-Media) was saturated with 100 μl of the test compound, allowed to dry and then
placed on the upper layer of the seeded agar plate. For the agar well
diffusion method, a well was prepared in the plates with a cup-borer (0.85
cm) and 100 μl of the test compound was pipetted directly into the well. The plates were
incubated overnight at 37°C. Antibacterial activity was determined by
measuring the diameter of the zone of inhibition (mm) surrounding bacterial
growth. For each bacterial strain, controls were included that comprised pure
solvents instead of the extract (Parekh and Chanda, 2007b). The experiments were repeated three times
and the mean values are presented with ± Standard Deviation (SD). Phytochemical screening Chemical tests were carried out on the aqueous
extract and on the powdered specimens using standard procedures to identify
the phytoconstituents as described by Sofowara (1993), Trease and
Evans (1989) and Harborne (1989). Results and
Discussion T. procumbens has shown significant antibacterial action
against bovine mastitis isolated S.aureus.
Our present investigation proved that the methanol extracts of this plant
showed maximum activity (8.2 ± 0.836) against CB Sau followed by
J Sau
& Z.Sau (8.0 ± 0.707) and HF Sau isolates (7.8 ± 1.30) Table 1. The aqueous extract
also showed antimastitis activity but lesser when
compared to methanolic extract. The phytochemical
screening revealed the presence of Alkaloids, Tannin, Saponin
and Falvonoids (Table 2). Most of the secondary metabolites
were identified in the polar (methanol and water) extracts. The concentration
of polar metabolites is higher than non-polar metabolites in leaves of these
species. Alkaloids are one of the characteristic secondary metabolites in
leaves of this genus. Flavonoids are known to be
synthesized by plants in response to microbial infection. Hence it should not
be surprising that they have been found to be in vitro effective antibacterial substances against a wide array
of infectious agents (Jamine et al., 2007). Tannins (commonly referred to as tannic acid) are
also known antimicrobial agents. They are water-soluble polyphenols
and precipitated proteins present in many plant foods. Tannins have been
reported to prevent the development of microorganisms by precipitating
microbial protein. The growth of many fungi, yeasts, bacteria, and viruses
were inhibited by this compound. They have been reported to have various
physiological effects like anti-irritant, antisecretolytic,
antiphlogistic, antimicrobial and antiparasitic effects. Phytotherapeutically,
tannin-containing plants are used to treat nonspecific diarrhoea,
inflammations of mouth, throat and slightly injured skins (R. Naveen Prasad et al.,2008).New commercial
synthetic antimastitis drugs can bring biohazards
such as consumer health problem, Bulk Tank Milk storage problem, emergence of
multidrug resistant strains. Cow as a grazing
animal T. procumbens
with all phytoconstituents can be directly fed
to the animal which has no side effect as it is commonly grazed along with
grasses. This process will be a natural remedy to cure mastitis in dairy
cows. Further studies may be necessary to elucidate the phytochemistry
of the active principles in the leaf extract of the plant Tridax
procumbens. References 1.
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Yadawa RN Saurabh K (1998). A new flavones glycoside:
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Nat. Prod. Res. 1(2):147-52. Table
1.
Antibacterial activity of methanolic and aqueous
extracts of Tridax procumbens
against bovine mastitis Staphylococcus aureus
J.Sau- Staphylococcus aureus
isolated from HF.Sau- Staphylococcus aureus
isolated from Holstein-Friesian cow Z.Sau- Staphylococcus aureus
isolated from Zebu cow CB.Sau- Staphylococcus aureus
isolated from Cross breed cow Table 2.
Phytochemical
screening of Tridax procumbens.
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