Proceedings of International Conference on Clean Green and Sustainable
Technology in Biotechnology and Chemical Engineering www.stmjournals.com
*
Department of Biotechnology, Acharya Institute of Technology, Bengaluru
The antibacterial activity of aqueous and methanol extracts of Punica granatum L was investigated against bovine mastitis causing bacterial pathogens- Streptococcus agalactiae, Staphylococcus aureus, Streptococcus uberis, Escherichia coli, Streptococcus dysagalactiae and coagulase negative Staphylococcus aureus by agar disc diffusion method [3] isolated locally from clinical cases. The results obtained in the present study scientifically proved and suggest the use of Punica granatum for treating mastitis causing organism. The phytochemical screening of the plant revealed the presence of Alkaloids,
Tannins, Coumarins, Flavonoids, Steroids, Phenolic compounds, Saponins, Terpenoids,
Catechin, Anthraquinone and Quinone. The proximate analysis was carried out.
Antimicrobial activity of methanolic and aqueous extracts of flavonoids was done and its presence in methanolic and aqueous extracts of P.granatum was confirmed by TLC (Thin
Layer Chromatography).
Keywords : Punica granatum, bovine mastitis pathogen, Disc Diffusion method, Solvents,
Antibacterial activity, Phyto-chemical screening, Proximate analysis
* Author for Correspondence E-mail: suneethatb@acharya.ac.in
Bovine Mastitis, the inflammation of mammary gland associated with bacterial infection, continues to be the costliest disease to the dairy industry all over the world [1]. Clinical and sub clinical cases of mastitis are usually treated with antibiotics administered via intra-mammary and parental route. The continuous use of antibiotics for a long period may lead to multi-drug resistance in causative organisms and also leads to the danger of increasing amounts of antibiotic residues in milk- a potential hazard [2]. Antimicrobials of plant origin have enormous therapeutic potential and have been proved effective in the treatment of infectious diseases without any side-effects which are often associated with synthetic antibiotics. Punica granatum Linn commonly known as Pomegranate belongs to the family Punicaceae. The plant has been used as an antispasmodic and anti-helminthic. In the context the aim of this work was to study the effect of aqueous and methanolic extracts of
P.granatum
in the prevention and control of BM and further on the effects of its isolated phytocompounds. There were no reports available relating to the in-vitro applications of P.granatum
L. extracts in Bovine mastitis studies.
All the solvents and reagents used in the study were analog grade sourced from Himedia.
Collection and Extraction of Plant Material
The plant was collected and, along with its leaves, rinsed with sterilized water. Leaves were removed and separated. They were air dried for 3 weeks and then crushed with mortar and pestle and kept in air tight glass container at 4 0 C until further use [3, 4].
Proximate Analysis
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Chemical Profiling Suneetha et al.
Proximate analysis was carried out to determine the moisture content, total ash value, acid insoluble ash value, and alcohol and water extractive values.
Preparation of Crude Extracts
Aqueous extract was prepared by using 500g of crushed leaves and 500 ml of distilled water in soxhlet apparatus and the apparatus was allowed to run for 10 hours. Similarly the methanol extract was prepared [5].
Bacterial strains used in this study were isolated from clinical cases of BM namely Streptococcus agalactiae, Staphylococcus aureus, Streptococcus uberis , Escherichia coli, Streoptococcus dysagalactiae and Coagulase negative Staphylococcus aureus. All the strains were maintained in nutrient agar slants at 4ºC for further use.
Antibacterial Activity
The antibacterial assay of aqueous and methanolic extracts was performed by agar disc diffusion method [6]. The molten Mueller Hinton agar was inoculated with 100μl of the inoculums (1*106
CFU/ml) and poured into the petriplate (Himedia). For agar disc diffusion method, the disc (0.7cm),
(Himedia) was saturated with 100μl of the test compound, allowed to dry and was introduced on the upper layer of the seeded agar plate. The plates were incubated overnight at 37ºC. Microbial growth was determined by measuring the diameter of the zone of inhibition of each bacterial strain.
Phytochemical Analysis
Phytochemical analysis for major phyto constituents of the plant extracts was undertaken using standard qualitative methods as described by various authors [6, 7]. The plants extracts were screened for the presence of biologically active compounds like glycosides, alkaloids, phenolics, tannins, flavonoids, saponins and steroids.
Estimation and Extraction of Phytocompounds
Alkaloids, Flavanoids, Saponins, Terpenoids and Tannins were extracted from the crude extract using standard methods. The content of each one was found out using the formula Content (%) = (Weight of phytocompounds)/ (weight of crude extract) * 100
Identification of Bioactive Compounds
Flavanoids of aqueous and methanolic extracts were applied onto a glass plate coated with silica gel for TLC (Thin Layer Chromatography) analysis. The solvent system used was chloroform: methanol
(15:1) and the colouring reagent used was ethanolic aluminium chloride and this was subsequently viewed under UV light.
The proximate analysis showed 6.5% moisture content,22 water soluble extractive value, 26% alcohol soluble extractive value,4.9% total ash value and 0.63% Acid insoluble ash value (Table-1).
Table 1: Proximate Analysis Punica granatum L. (Leaf)
Parameters Values (%)
Moisture content
Water soluble extractive value
Alcohol soluble extractive value
Total ash value
Acid insoluble ash value
6.5
22
26
4.9
0.63
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Proceedings of International Conference on Clean Green and Sustainable
Technology in Biotechnology and Chemical Engineering www.stmjournals.com
Results obtained in the present study revealed that the tested plant, Punica granatum extract possess potential antibacterial activity against Streptococcus agalactiae , Staphylococcus aureus,
Streptococcus uberis, Escherichia coli , coagulase negative Staphylococcus aureus and Streptococcus dysagalactieae (Table 2).
Antimicrobial activity of Punica granatum – Zone of Inhibition
(M- Methanol, W- Water)
Organisms
Zone of inhibition (mm)
M W
Streptococcus agalactiae
Staphylococcus aureus
Streptococcus uberis
Escherichia coli
29.2
36
25
27
29
36
25
32
Coagulase negative Staphylococcus aureus 32 30
Streptococcus dysagalactiae 28 28
The most pronounced activity with inhibition zones of more than 36 mm was shown by methanol as well as water extracts against Staphylococcus aureus. The minimum activity was observed in
S treptococcus uberis from aqueous and methanol extracts. The phytochemical screening revealed the presence of alkaloids, tannins, carbohydrates, flavanoids, phytosterols, phenols, sterols, terpenes, and volatile oils (Table 3). Most of the secondary metabolites were identified in the polar extracts.
Alkaloids are one of the characteristic secondary metabolite in leaves of this genus found in aqueous extract. Tannins are water soluble polyphenols known as tannic acid acts as antimicrobial agents.
Presence of tannins is to prevent the development of microorganism by precipitating microbial proteins in many microorganisms. Phyto-therapeutically, tannin containing plants are used to treat non-specific diarrhoea, inflammations of mouth, throat and injured skins. 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 agent [8].
Table-3: Phyto-chemical Analysis of Punica granatum L.
Phytoconstituents
Alkaloids
Tannins
Coumarins
Flavonoids
M
+
+
W
+
+
Steroids
Phenolic compounds
Saponins
Terpenoids
Catechin
Anthraquinone
-
-
+
+
-
+
-
+
+
+
-
-
-
+
-
-
Quinone - +
(+ indicates the Presence of Phytocompound; − Indicates the absence of phytocompound).
Table 4: Antibacterial Activity of each Phytocompound against causative organisms
(in terms of mm diameter of zone of inhibition)
Organism
E.coli
S.uberis
S.aureus
CONS
Phytocompounds
Alkaloids Flavonoids Saponins Tannins Terpenoids
M W M W M W M W M W
0 0 14 15 0 0 0 0 0 0
0
0
0
0
0
0
11
12
16
16
12
13
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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Chemical Profiling Suneetha et al.
Flavonoids isolated from methanolic and aqueous extracts of Punica granatum showed antibacterial activity against the causative organisms of Bovine mastitis (Table 4). The highest inhibition zone was observed by methanolic extracts of Punica granatum against Coagulase negative Staphylococcus aureus (CONS) and aqueous extracts of P. granatum against S. uberis (16 mm) and the least was observed by methanolic extracts of Punica granatum against S. uberis (11 mm). The presence of flavonoids was tested for in the methanolic and aqueous extracts of P.granatum using the Thin Layer
Chromatography technique. Figure 1 shows the run of methanolic and water extracts on TLC plate respectively.
Figure 1: TLC Plate.
All plant pots synthesize some chemicals by themselves to perform their physiological activity. In the present study, Punica granatum exhibited different kinds of secondary metabolites. The medicinal value of these secondary metabolites is due to the presence of chemical substances that produce a definite physiological action on the human body. The most important of these substances include carbohydrates, phenols, phytosterols, sterols, terpenes and volatile oil for cell growth, replacement, and body building describes today’s traditional medicine as undoubtedly the oldest form of medicine and probably had evolved simultaneously with the evolution of human beings. With the traditional knowledge in the background potential plants can be prospected to reach bioactive compounds which can be further formulated. Further studies are suggested to explore the specific phyto-active compounds in the leaf extract of plant Punica granatum.
Aqueous and methanolic extracts of flavonoids have remarkable antimicrobial potential against
Bovine mastitis causing organism.
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