Ethnobotanical Leaflets 13: 131-39. 2009.
Phytochemical Screening and Antibacterial Activity of Aqueous and Methanolic Leaf Extracts of Two Medicinal Plants against Bovine Mastitis Bacterial Pathogens
M. P. Ayyappa Das1, R. Dhanabalan2*, A. Doss2 and M. Palaniswamy3
1Department of Biotechnology, Centre for Post
Graduate Studies in Biotechnology, RVS
2Department of Microbiology, Centre for Post
Graduate Studies in Microbiology, RVS
3Department of Microbiology,
*Corresponding author: email@example.com
Spathodea campanulata P. Beauv is extensively used in Indian traditional and folklore medicines to cure various human ailments. Tridax procumbens Linn is a tropically distributed medicinal plant. Antimicrobial activity of aqueous and methanol extracts of two plants were investigated by agar disc and well-diffusion method against bovine mastitis bacterial pathogens. 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 plant in traditional and ethnoveterinary medicine.
Key words: Spathodea campanulata; Tridax procumbens; Antibacterial activity; Bovine mastitis; Ethnoveterinary medicine.
Ever since the dawn of
civilization man has used plants for his food, shelter and fodder for his
animals. Plants were also identified for use to cure him from innumerable ailments
which struck his physical being. They designated these plants as ‘medicinal
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 Spathodea campanulata and Tridax procumbens. This study is to elucidate the antibacterial action of plant material against bovine mastitis causing pathogens.
Spathodea campanulata is a species belonging to the Bignoniaceae
family, native from equatorial
The stem bark preparations are employed against enemas, fungus skin diseases, herpes, stomach aches and diarrhea (Mendes et al., 1986; Jardim et al., 2003). Hypoglycemic, anti-HIV and antimalarial activities were also observed in stem bark extracts (Makinde et al., 1988; Niyonzima et al., 1999). The leaves are used against kidney diseases, urethra inflammations and as an antidote against animal poisons. In vitro antimalarial activity against Plasmodium falciparum and antibacterial activity of bovine mastitis causing S.aureus were evaluated using leaf extracts of S.campanulata (Dhanabalan et al., 2008). The leaves have been found to contain spathodol, caffeic acid, other phenolic acids and flavonoids (Ngouela et al., 1991; Subramanian et al., 1973; El-Hela, 2001a; El-Hela, 2001b). In vitro antibacterial activity of leaf extracts of this plant against standard strains was evaluated (Parek and Chanda, 2007). A qualitative fungitoxic activity of S. campanulata roots against Cladosporium herbarum CCT 0279 has been evaluated and reported (Pianaro et al., 2007).
Tridax procumbens Linn
(compositae) is a common grass found in tropical
areas of all countries, growing primarily during rainy season. It is a common
weed in Tamilnadu present along with economically
important crops. It habitats waste places, road sides and hedges throughout
plant leaves were collected from the villages of
For aqueous extraction, 10 g of air-dried powder was mixed with 100 ml distilled water and kept at room temperature for 48 h. It was then filtered through 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 220 rpm for 24 h. After 24 h, it was filtered through muslin cloth and centrifuged at 5000 g for 10 min. The supernatant was collected and the solvent was evaporated using rotary vacuum pump and stored at 4oC in air-tight bottles.
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).
Bacterial strains used in this study were
isolated from clinical cases of bovine mastitis such as coagulase
positive Staphylococcus aureus, coagulase
negative Staphylococcus aureus (
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 l 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 l 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, 2007). The experiments were repeated three times and the mean values are presented.
Results and Discussion
results on antibacterial activity of S.
campanulata and T. procumbens were shown in table 1. Methanol extracts of
showed significant activity against Streptococcus
followed by Streptococcus uberis (7.2±0.447), Escherichia coli (7.2±0.836), coagulase positive Staphylococcus aureus (7.0±1.0). Whereas only a
moderate activity was observed against coagulase
negative S. aureus
On the other hand the methanol extracts of T. procumbens showed significant activity against coagulase positive S. aureus (8.0±0.707). But only least antibacterial activity was observed on other selected bacterial strains. The aqueous extracts of T. procumbens showed no pronounced antibacterial activity against Streptococcus uberis and K. pneumonia.
The phytochemical screening revealed the presence of alkaloids, tannin, saponin, steroids, terpenoid 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 effective as antibacterial substances against a wide array of infectious agents (Jamine et al., 2007). Tannins (commonly referred to as tannic acid) are also known as 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 (Prasad et al., 2008). 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 (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 can be directly fed with S. campanulata and T. procumbens with all phytoconstituents 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 S. campanulata and T. procumbens.
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Table 1. Antibacterial activity of methanolic and aqueous extracts of Spathodea campanulata and Tridax procumbens against bovine mastitis pathogens.
NA-No Activity; (±) Mean of three replicates
Table 2. Phytochemical screening of Spathodea campanulata and Tridax procumbens.