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:
bharathi.dhanabalan@gmail.com Issued
Abstract 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. Introduction 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
plants’. 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 Spathodea campanulata and Tridax procumbens. This study is to elucidate the antibacterial action of plant material against bovine mastitis causing pathogens. Plant Description 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 MATERIALS
Plant
collection Fresh
plant leaves were collected from the villages of Plant
extraction 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. 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). Bacterial
strains Bacterial strains used in this study were
isolated from clinical cases of bovine mastitis such as coagulase
positive Staphylococcus aureus, coagulase
negative Staphylococcus aureus ( 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 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 The
results on antibacterial activity of S.
campanulata and T. procumbens were shown in table 1. Methanol extracts of
S. campanulata
showed significant activity against Streptococcus
agalactiae (7.6±0.547)
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
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campanulata and Tridax procumbens against bovine mastitis
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NA-No Activity; (±) Mean of three
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screening of Spathodea campanulata and Tridax procumbens.
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