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
*Corresponding author: email@example.com
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.
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 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.
Fresh plant leaves were
collected randomly from the villages of
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 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
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).
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.
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Table 1. Antibacterial activity of methanolic and aqueous extracts of Tridax procumbens against bovine mastitis Staphylococcus aureus
J.Sau- Staphylococcus aureus
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.