Professional Documents
Culture Documents
3
Somasekhar Penumajji
1
For U Biosciences, A/4A, Park lane Residency, East point colony, Visakhapatnam-17, A. P, India
2
Department of Botany, Andhra University, Visakhapatnam-3.
3
Vivimed labs Limited, 2nd, 4th Floor, Veeranag towers, Habsiguda, Hyderabad, A.P.
Abstract: We have examined antimicrobial activities of forty-nine medicinal plants extracts that have been
popularly used as folk medicines. Hexane, chloroform and methanolic extracts were investigated in vitro for
antibacterial activity against Pantoea agglomerans using agar well diffusion technique. The plant extracts inhibited
the growth of all the test organisms. P. pterophorus appears to be more effective as an antibacterial agent than all
the medicinal plant extracts tested. This study has to some extent validated the medicinal potential of medicinal
plants. Hence this plant can be further subjected to isolation of the therapeutic antimicrobials and further
pharmacological evaluation.
Keywords: Medicinal plants, Folk medicines, Antimicrobials, P. pterophorus, Pantoea agglomerans
of healthcare known to mankind. This situation, ml of nutrient agar was dispensed into sterile
the undesirable side effect of certain antibiotics, universal bottles these were then inoculated with
and the emergence of previously uncommon 0.2 ml of cultures mixed gently and poured into
infections, has forced scientists to look for new sterile petri dishes. After setting a number 3-cup
antimicrobial substances from various sources, borer (6mm) diameter was properly sterilized by
such as medicinal plants [6, 7]. A large number flaming and used to make three to five uniform
of plants in different location around the world cups/wells in each petri dish. A drop of molten
have been extracted and semi-purified to nutrient agar was used to seal the base of each
investigate individually their antimicrobial cup. The cups/wells were filled with 50µ of the
activity [8]. extract concentration of 500mg/ml, 250mg/ml
and 100mg/ml and allow diffusing for 45
Materials and Methods minutes. The solvents used for reconstituting the
extracts were similarly analyzed. The plates were
The medicinal plants belong to forty nine
incubated at 37°c for 24 hours for bacteria. The
different plant species were collected selectively
above procedure is allowed for fungal assays but
from Andhra University surroundings and
except the media potato dextrose agar instead of
nearest villages, Viskhapatnam, Andhra Pradesh,
nutrient agar and incubates at 25°c for 48 hours.
India. The authenticated samples of plant parts
The zones of inhibition were measured with
were shade dried, cut into small pieces and
antibiotic zone scale in mm and the experiment
powdered in a mixer grinder the residues (crude
was carried out in duplicates. The extracts and
extracts) obtained were finally dried under
the phytochemicals that showed antimicrobial
vacuum.
activity were later tested to determine the
The extraction method employed here is a Minimal Inhibitory Concentration (MIC) for each
known amount of coarsely powdered plant bacterial and fungal sample.
materials of different plant species were
successively extracted with organic solvents like Minimum Inhibitory Concentration (MIC) Assays
hexane, chloroform and methanol basing on order
Based on the preliminary screening chloroform
of their polarity using soxhlet apparatus. The
and methanolic extracts were found to have
different extracts obtained were subsequently
potent antimicrobial activity and Minimum
concentrated under reduced pressure to get their
Inhibitory Concentrations (MIC) of the extracts
corresponding residues. The extracts were
was determined according to Elizabeth, A final
screened for antimicrobial activity using the
concentration of 0.5% (v/v) Tween-20 (Sigma)
method described under the section
was used to enhance crude extract solubility. A
Pantoes agglomerans was procured from series of two fold dilution of each extract, ranging
Microbial Type Culture Collection MTCC, from 0.2 to 100 mg/ml, was prepared. After
IMTECH, Chandigarh. Active culture was sterilization, the medium was inoculated with 3µl
generated by inoculating a loop full of culture in aliquots of culture containing approximately 105
separate 100mL nutrient broth and incubating on CFU/ml of each organism of 24 hours slant
a shaker at 37 o C overnight. The cells were culture in aseptic condition and transferred into
harvested by centrifuging at 4000 rpm for 5 min, sterile 6 inch diameter petri dishes and allowed
washed with normal saline, spun at 4000 rpm for to set at room temperature for about 10 minutes
5 min again and diluted in normal saline to obtain and then kept in a refrigerator for 30 minutes.
5 x 105cfu/mL.
After the media solidified a number 3-cup
borer (6mm) diameter was properly sterilized by
Determination of Antimicrobial Activity flaming and used to make three to five uniform
The crude extracts of the different plant parts of cups/wells in each petri dish. A drop of molten
different species were subjected to antimicrobial nutrient agar was used to seal the base of each
assay using the agar well diffusion method of cup. Different plant crude extracts ranging from
Murray et al., [9] modified by Olurinola [10] 20 0.2 to 100 mg/ml were added to the cups/wells
Biological Control of Phytopathogenic Bacteria Pantoea Agglomerans using Medicinal Plant Extracts 115
of each petri dish and the control plates without Results and Discussion
plant extract. Inhibition of organism growth in (Graph 1a&b) In the present study different
the plates containing test crude extracts was concentrations of methanol extracts of inhibited
judged by comparison with growth in blank activity but their effectiveness varied. The zones
control plates. The MICs were determined as the of inhibition ranged from 7 to 24 mm.
lowest concentration of extracts inhibiting visible
growth of each organism on the agar plate. Graph 2 Minimum inhibitory concentration
Similarly the MICs of methanolic extracts were (MIC) (Volume per well: 50ìl, Borer size used:
determined against all other microorganisms. The 6mm, ‘x’ axis = Medicinal plants, ‘y’ axis =0-300
results were given in = Extract concentration in mg/ml)
0-25: Zone of Inhibition in mm; A: 100mg/ml; B: 300mg/ml; C: 500mg/ml; Includes Diameter of Well (6 mm)
0-25: Zone of Inhibition in mm; A: 100mg/ml; B: 300mg/ml; C: 500mg/ml; Includes Diameter of Well (6 mm)
116 International Journal of Pharmagenesis, 1(1) 2010
[6] Marchese A., Shito G. C. Resistance Patterns of Lower [8] Draughon F. A. Use of Botanicals as Bio Preservatives
Respiratory Tract Pathogens in Europe. Int J. in Foods. Food Technol. 2004; 58: 20-28.
Antimicrobial Agents 2001; 16: 25-29. [9] Murray P. R., Baron E. J., Pfaller M. A., Tenover F. C.,
[7] Poole K. Overcoming Antimicrobial Resistance by Yolken H. R. Manual of Clinical Microbiology, 6 th
Targeting Resistance Mechanisms. J. Pharmacy and Edition. ASM Press, Washington. DC 1995. 15-18.
Pharmacol, 2001; 53: 283-284. [10] Olurinola P. F. A Laboratory Manual of Pharmaceutical
Microbiology. Idu, Abuja, Nigeria 1996; 69-105.