Varaprasad Bobbarala et al.

/ Journal of Pharmacy Research 2009, 2(6),1019-1021

Research Article
Available online through
ISSN: 0974-6943
www.phresearchjournal.info
Antimicrobial Potentialities of Mangrove Plant Avicennia marina
Varaprasad Bobbarala*, Varahala Rao Vadlapudi ** and K. Chandrasekhar Naidu**
*For U Biosciences, A/4A, Park lane Residency, East point colony, Visakhapatnam, A. P-530017, India.
** Department of Botany, Andhra University, Visakhapatnam (A.P.)
Received on: 22-02-2009; Accepted on:26-03-2009
ABSTRACT
In this present study Antimicrobial activity of aerial parts of Avicennia marina were evaluated against the pathogens belong to aquatic,
human and plant origin, Soxhlet extraction method used to get the corresponding extracts of hexane, chloroform and methanol. The antimicrobial
activities of the organic solvent extracts on the various test microorganisms, including bacteria and fungi investigated using agar well
diffusion technique. The length of inhibition zone was measured in millimeters from the edge of the well to the edge of the inhibition zone.
Methanol and chloroform extracts exhibited promising antimicrobial activity than hexane extracts. Among all microorganisms studiedErwinia
caratovara and Streptococcus mutans showed the considerable growth inhibition with chloroform and methanolic extracts.

Keywords: Phytopathogens, Antimicrobial activity, Folkloric medicine, Acanthaceae

INTRODUCTION
Natural products can be selected for biological screening
based on ethno medical use of plants, because many infectious
diseases are known to have been treated with herbal remedies
throughout the history of mankind. Antimicrobial properties of
medicinal plants are being increasingly reported from different parts
of the world [1, 2, 3]. It has been reported that the higher plants have
shown to be a potential source for the new antimicrobial agents [4].
Even today, plant materials continue to play a major role in primary
health care as therapeutic remedies in many developing countries [5,
6]. Studies on antibacterial and antifungal activity of the plants and
their prospects for use in different systems require scientific
experimentation. In recent years, drug resistance to pathogenic
microorganisms has been commonly and widely reported in literature
[7, 8] therefore antimicrobials may have a significant clinical value in
treatment of resistant microbial strains [9]. Because of the side effects
and the resistance that pathogenic microorganisms build against
antibiotics, many scientists have recently paid attention to extracts
and biologically active compounds isolated from plant species used
in herbal medicines [10].
Mangrove and mangrove associates contain biologically
active antiviral, antibacterial and antifungal compounds [11]. They
provide a rich source of steroids, triterpenes, saponins, flavonoids,
alkaloids and tannins. Therefore, it is worth to screen mangrove plants
for the presence of new antibacterial compounds to combat the patho-
genic bacteria and fungi. A. marina (Forssk.) is commonly known as
gray mangrove tree classified in the plant family Avicenniaceae, and
is commonly used for ulcers. So the main objectives of this study was
to screen antimicrobial activity of the organic solvent extracts of A.
marina mangrove plant species against pathogenic and antibiotic
*Corresponding author.
Tel.: +91-9949129539
E-mail: varaprasadphd@rediffmail.com
resistant bacterial strains and also to isolate and characterization of
chemical components which are responsible for the claimed activity.
MATERIALS AND METHODS
Plant material and extraction:
A. marina is commonly known as gray mangrove and ver-
nacular name is Tella mada belongs to the family Aviceniaceae, grow
as a shrub or tree to a height of three to ten metres, or up to 14 meters
in tropical regions, growing in the saline intertidal zones of sheltered
coast lines. It has been reported to tolerate extreme weather condi-
tions, high winds. The material was taxonomically identified and the
Voucher specimen is stored. The aerial plant parts were collected from
Coringa Mangrove Wetland, Andhra Pradesh, India. The plant mate-
rial were dried under shade with occasional shifting and then pow-
dered with a mechanical grinder and stored in an airtight container.
The powder obtained was subjected to successive soxhlet extraction
with organic solvents with increasing order of polarity i.e. Hexane,
Chloroform and Methanol respectively.
Test microorganisms:
The antibacterial activity of the extracts was assessed against
microbial strains of clinical, plant and aquatic origin i.e. Aeromonas
hydraophylla (MTCC 646), Alternaria alternate (MTCC 1362),
Ustilago maydis (MTCC 1474), Asperigellus niger (MTCC 2723),
Acremonium strictum (MTCC 3072), Pencillium expansum (MTCC
2006), Fusarium oxysporum (MTCC 1755), Xanthomonas compestries
(MTCC 2286), Erwina caratovara (MTCC 3609), Lactobacillus aci-
dophilus (MTCC 447), Pseudomonas marginalis (MTCC), Pseudomo-
nas syringae (MTCC 1604), Pseudomonas aeruginosa (MTCC 1688),
Streptococcus mutans (MTCC 890), Steptococcus salivarious (MTCC
1938) and Staphylococcus aureus (MTCC 96) including both fungi
and bacteria were procured from Microbial Type Culture Collection
(MTCC), Chandigarh. Active cultures were generated by inoculating a
loopful of culture in separate 100mL nutrient/potato dextrose broths
and incubating on a shaker at 37oC overnight. The cells were har-

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 Varaprasad Bobbarala et al. / Journal of Pharmacy Research 2009, 2(6),1019-1021

vested by centrifuging at 4000 rpm for 5 min, washed with normal pathogens tested S. aureus was the most resistant bacteria and E.
saline, spun at 4000 rpm for 5 min again and diluted in normal saline to caratovara was the most sensitive strain. No inhibition of the growth
obtain 5 x 105cfu/mL. of pathogens tested was observed in extracts in hexane extract of A.
Determination of antimicrobial activity: marina. The degree of antimicrobial activity of extracts decreased
The crude extracts of the different plant parts of different with time for all tested microbial strains. According to phytochemical
species were subjected to antimicrobial assay using the agar well screening, mature leaves of A. marina contained alkaloids, steroids
diffusion method of Murray et al., [12] modified by Olurinola [13]. 20 and flavonoids.
ml of nutrient agar was dispensed into sterile universal bottles these
Table 1: MIC assays of chloroform and methanol extracts Avicennia
were then inoculated with 0.2 ml of cultures mixed gently and poured
into sterile petri dishes. After setting a number 3-cup borer (6mm) marina
diameter was properly sterilized by flaming and used to make three to Plant Name Chloroform Methanol
five uniform cups/wells in each petri dish. A drop of molten nutrient 100 50 25 100 50 25
agar was used to seal the base of each cup. The cups/wells were filled Aeromonas hydraophylla 14 13 12 19 15 13
Alternaria alternate 11 10 - 15 10 10
with 50µl of the extract concentration of 500mg/ml, 250mg/ml and 100mg/ Asperigellus niger 10 10 - 15 16 10
ml and allow diffusing for 45 minutes. The solvents used for reconsti- Acremonium strictum 12 - - 14 - -
tuting the extracts were similarly analyzed. The plates were incubated Erwinia caratovara 15 13 - 25 18 -
at 37°c for 24 hours for bacteria. The above procedure is allowed for Fusarium oxysporum 13 10 - 17 15 16
Lactobacillus acidophilus 12 12 8 21 20 10
fungal assays but except the media potato dextrose agar instead of Pencillium expansum 10 - - 14 - -
nutrient agar and incubates at 25°c for 48 hours. The zones of inhibi- Pseudomonas marginalis 12 13 10 17 17 18
tion were measured with antibiotic zone scale in mm and the experi- Pseudomonas syringae 14 13 - 18 17 -
ment was carried out in duplicates. The extracts and the Pseudomona aeruginosa 11 12 10 23 21 21
phytochemicals that showed antimicrobial activity were later tested Steptococcus mutans 13 - - 25 21 19
Steptococcus salivarious 12 - - 20 - -
to determine the Minimal Inhibitory Concentration (MIC) for each Stephylococcus aureus 10 - - 13 - -
bacterial and fungal sample Ustilago maydis 12 10 - 14 18 16
Minimum inhibitory concentration (MIC) assays: Xanthomonas compestries 15 - - 20 14 9
Based on the preliminary screening chloroform and
Volume per well: 50µl, Borer size used: 6mm, Drug concentration in mg/
methanolic extracts were found to have potent antimicrobial activity
and Minimum Inhibitory Concentrations (MIC) of the extracts was ml, Zone of inhibition in mm
determined according to Elizabeth, [14]. A final concentration of 0.5%
(v/v) Tween-20 (Sigma) was used to enhance crude extract solubility. DISCUSSION
A series of two fold dilution of each extract, ranging from 0.2 to 100 The results of the present study clearly showed that man-
mg/ml, was prepared. After sterilization, the medium was inoculated grove plant A. marina extracts showed antimicrobial activity against
with 3ìl aliquots of culture containing approximately 105 CFU/ml of tested pathogenic strains including antibiotic resistant strains. The
each organism of 24 hours slant culture in aseptic condition and trans- effectiveness of the active compounds present in plant extracts cause
ferred into sterile 6 inch diameter petri dishes and allowed to set at the production of growth inhibition zones that appear as clear areas
room temperature for about 10 minutes and then kept in a refrigerator surrounding the wells. Antibacterial activity may be due to active
for 30 minutes.
components which are present in plant extracts. However, some plant
After the media solidified a number 3-cup borer (6mm) diam-
extracts were unable to exhibit antibacterial activity against tested
eter was properly sterilized by flaming and used to make three to five
uniform cups/wells in each petri dish. A drop of molten nutrient agar bacterial strains. These bacterial strains may have some kind of resis-
was used to seal the base of each cup. Different plant crude extracts tance mechanisms e.g. enzymatic inactivation, target sites modifica-
ranging from 0.2 to 100 mg/ml were added to the cups/wells of each tion and decrease intracellular drug accumulation [15] or the concen-
petri dish and the control plates without plant extract. Inhibition of tration of the compound used may not be sufficient. No inhibition was
organism growth in the plates containing test crude extracts was judged observed with controls, which proves that solvents could not act as
by comparison with growth in blank control plates. The MICs were antibacterial agents. In almost all tests, crude methanolic extracts
determined as the lowest concentration of extracts inhibiting visible showed better inhibition against all tested bacterial strains, indicating
growth of each organism on the agar plate. Similarly the MICs of that active ingredients in plant materials could be extracted into metha-
methanolic extracts were determined against all other microorganisms. nol. However, highest antibacterial activity was observed against
The results were given in Table-1.
Erwinia caratovara . The results obtained from preliminary phy-
tochemical screening are comparable with the results reported earlier
RESULTS
In the present study, chloroform and methanol extract exhib- by Abeysinghe et al., [16] according to the studies the presence of
ited different degree of growth inhibition against tested bacterial and secondary metabolites such as alkaloids, flavonoids and steroids may
fungal strains (Table 1). According to Table 1, methanolic extracts of exert antibacterial activity against tested bacterial strains. Further re-
A. marina exhibited considerable antimicrobial activity against tested search is necessary for successful separation, purification and char-
microbial strains. Methanolic extracts of A. marina showed more inhi- acterization of biologically active compounds using chromatographic
bition than chloroform extracts of A. marina against S. mutans, E. methods and spectroscopic techniques. Further studies are being
caratovara, P. aeruginosa and L. acidophilus (Table 1). Whereas carried out in order to separate the individual components that are
chloroform extracts of A. marina exhibited more pronounced inhibi- present in plant extracts of A. marina using column chromatography.
tion against X. compestris and E. caratovara (Table 1). Among the all
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Source of support: Nil, Conflict of interest: None Declared

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