V.Umamaheswara Rao et al.

/ Journal of Pharmacy Research 2012,5(5),2906-2909

Research Article
ISSN: 0974-6943

Available online through

www.jpronline.info

Antibacterial activity of leaf and stem extracts of Avicennia marina L.

V. Umamaheswara Rao1 *, N. Sharief Md 2 and A. Srinivasulu 2
Department of Botany & Microbiology, Acharya Nagarjuna University, Nagarjunanagar 522510, Guntur (Dt.), Andhra Pradesh, India.
2
Department of Biochemistry, V.S.Lakshmi Womens Degree & P.G. College, Kakinada- 533005, East Godavari (Dt.), Andhra Pradesh, India.
1

Received on:11-01-2012; Revised on: 17-02-2012; Accepted on:19-04-2012

ABSTRACT
Leaf and stem extracts of Avicennia marina L. were prepared in hexane, benzene, ethyl acetate, acetone, methanol and ethanol, and their antibacterial
activity was evaluated against Escherichia coli MTCC 64, Enterobacter aerogenes MTCC 111, Klebsiella pneumoniae MTCC 39, Pseudomonas
aeruginosa MTCC 424, Bacillus subtilis MTCC 121, Lactobacillus delbrueckii MTCC 911, Staphylococcus aureus MTCC 87 and Streptococcus pyogens
MTCC 1928 by agar well diffusion method. The leaf extract in ethanol and stem extracts in acetone and ethanol demonstrated best antibacterial activity.
However, extracts prepared in other solvents also showed antibacterial activity against the test organisms. The extracts that possessed antibacterial
activity were further subjected to the determination of the Minimum Inhibitory Concentration (MIC) using different concentrations viz., 1.25, 2.5 and
5.0mg /100l. The MIC value of different solvent extracts was found to be different but in the range of 1.25mg/100l to 5mg/100l. Further, the positive
antibacterial extracts were checked for their bactericidal or bacteriostatic nature. The present study reveals the potentiality of leaf and stem extracts of
Avicennia marina L as antibacterial agent.
Key words: Leaf extracts, Stem extracts, Avicennia marina L., Antibacterial activity.
INTRODUCTION
The discovery of antibiotics in the early twentieth century provided an
increasingly important tool to combat bacterial diseases. As antibiotics are
increasingly used and misused, the bacterial strains become resistant to antibiotics rapidly. The World health organization advocated the countries to
interact with traditional medicine with a view of identifying and exploiting
the aspects that provide safe and effective remedies for ailments of both
microbial and non microbial origins. Many pathogenic organisms are developing plasmid-mediated resistance to the prevailing drugs. Hence, there is a
need for novel natural compounds that can be obtained from the plants or
microorganisms. Plants in particular have been a source of inspiration for
novel drug compounds since days immemorable. Plants serve as a reservoir
of effective chemotheraputants and provide valuable sources of natural products in the control of several bacterial diseases. Many studies indicate that
plants contain bio-active compounds such as peptides, glycosides, alkaloids,
saponins, terpenoids, flavonoids etc with antimicrobial activity against bacterial, fungal and viral infections 1,2,3 . However, the antibacterial activity of
mangrove plants has still not been studied as extensively as most other plant
species. Avicennia marina L. plant varies from shrubby stunted individuals
to tall trees with broad trunk. In the present study, leaves and stem of A.
marina L were selected for extraction in different organic solvents to evaluate their antibacterial activity against the selected MTCC bacterial cultures.

with 0.01% mercuric chloride solution. The leaf and stem were chopped
separately into small pieces and shade dried at room temperature for seven
days.
Extraction
The extraction of leaf and stem was carried out with different solvents in
their increasing order of polarity viz., hexane, benzene, ethyl acetate, acetone, methanol and ethanol by soaking the plant material in the respective
solvents overnight at room temperature one after the other 4 . The contents of
each flask were subjected to reflux below the boiling point of the respective
solvents viz., Hexane (68o C), Benzene (80o C), Ethyl acetate (77o C), Acetone
(55o C), Methanol (65o C) and Ethanol (78o C) for 6-8h in order to extract the
active compounds into the solvent. Each extract was vacuum filtered and the
filtrates were concentrated by vacuum distillation. The concentrated extracts
were incubated at 37o C for 3-4 days to facilitate complete evaporation of the
volatile solvent leaving behind the dried plant extract. The dried plant extract
of100mg each was dissolved in 1000l of 1:10 diluted DMSO (in sterile
distilled water) so as to obtain the final concentration of 10mg /100l 5 .

Determination of antibacterial activity

The antibacterial activity of extracts against the bacterial strains viz., Escherichia coli MTCC 64, Enterobacter aerogenes MTCC 111, Klebsiella
MATERIALS AND METHODS
pneumoniae MTCC 39, Pseudomonas aeruginosa MTCC 424, Bacillus
subtilis MTCC 121, Lactobacillus delbrueckii MTCC 911, Staphylococcus
Plant material collection
aureus MTCC 87 and Streptococcus pyogens MTCC 1928 was tested by
The leaf and stem parts of healthy Avicennia marina L. plants were agar well diffusion method 6 , and zones of inhibition were measured. Each
collected from East Godavari mangroves at Corangi Reserved Forest, ( Geo- experiment was performed in triplicate and the average value of inhibition
graphically located between 16o 39 N longitude 17o N longitude and 82o zones and standard deviation were calculated. The zone of inhibition was
14 E latitude -82o 23E lattitude ) Kakinada, Andhra Pradesh, India. The compared with that of standard Gentaimicin concentration of 1mg/100l 7 .
plant materials were collected in new polythene bags and surface sterilized
Determination of MIC, Bactericidal and / or Bacteriostatic activity
Minimum Inhibitory Concentration (MIC) as well as bactericidal or bacte*Corresponding author.
riostatic activity were determined by broth dilution assay method. For the
Dr. V. Umamaheswara Rao
determination of MIC, the reconstituted extract in DMSO was serially diAsst. Professor
luted in Mueller Hinton broth medium to get the concentrations of 1.25,
Dept. of Botany & Microbiology
2.5and 5.0mg/100l 5 .
Nagarjunanagar 522510
Guntur District
For the determination of bactericidal and / or bacteriostatic activity, 0.1ml
Andhra Pradesh, India

Journal of Pharmacy Research Vol.5 Issue 5.May 2012

2906-2909

V.Umamaheswara Rao et al. / Journal of Pharmacy Research 2012,5(5),2906-2909

Antibacterial activity by the leaft extracts of Avicennia marina

Zone of inhibition in mm

Hexane

Benzene

Ethyl acetate

Acetone

Methanol

Ethanol

Gentamicin

20
18
16
14
12
10
8
6
4
2
0
Esch

S
E
S
erich nteroba Klebsiella Pseudom Bacillus Lactobac taphylo treptoco
c
cter
ia co
aero pneumo onas aer subtilis illus delb occus au ccus pyo
li
ruec
gene
ugino
gene
niae
reus
kii
s
s
sa
Test culture

Fig-1. Antibacterial activity of Leaf extracts in Hexane, Benzene, Ethyl acetate,Acetone,Methanol and Ethanol of
Avicennia marina on selected bacteria in comparison with Gentamicin.
Antibacterial activity by the Stem extracts of Avicennia marina

Zone of inhibition in mm

Hexane

Benzene

Ethyl acetate

Acetone

Methanol

Ethanol

Gentamicin

20
18
16
14
12
10
8
6
4
2
0
Esch

E
P
S
K
S
L
erich nteroba lebsiella seudom Bacillus actobac taphyloc treptoco
cter
i
o
ccu
l
ia co
o
l
s
p
n
u
cc
ubt
neu
as
s de
aero
li
lbrue us aure s pyoge
g e n e m o n i a e aerugin i l i s
us
nes
c
osa
kii
s
Test culture

Fig-2. Antibacterial activity of Stem extracts in Hexane, Benzene, Ethyl acetate, Acetone, Methanol and Ethanol of
Avicennia marina on selected bacteria in comparison with Gentamicin.
of culture medium from each broth tube showing no apparent growth was
picked upon and sub-cultured on fresh Mueller Hinton agar medium. After
incubation at 37o C for 24 hrs, plates showing no visible growth of bacteria
were considered for Bactericidal effect and plates with visible growth of
bacteria as Bacteriostatic 8 .
RESULTS AND DISCUSSION
The data on antibacterial activity of the Leaf and stem extracts of A. marina
L in, hexane, benzene, ethyl acetate, acetone, methanol, ethanol and gentamicin against eight bacterial species is given in Figs 1 and 2. Hexzane, benzene
and acetone extracts of A. marina L. leaf were effective only against gram
negative test cultures used, where as, ethyl acetate and methanol extracts are
active only on gram positive cultures used. While the ethanol extracts were
active on all the test cultures used except E. coli and E. aerogenes.
Leaf extract of A. marina L. in ethyl acetate and methanol were active only on
gram positive cultures used. The effect of these extracts is higher than gentamicin against gram positive test cultures except leaf extract in ethyl acetate
against B. subtilis and was found to be equivalent to gentamicin.. Hexane
and Acetone extracts of A. marina L leaves were active only against E. coli

and E. aerogenes, with same zone size. The efficacy of these extracts were
less than that of the gentamicin. Whereas, the benzene extracts were effective only against the gram negative test cultures used. The zone size in the
case of K. pneumonia and P. aeruginosa to ethanol extracts of leaf were
same and is less than that of the gentamicin. However, the effectiveness
against B. subtilis, L. delbrueckii, S. aureus and S. pyogenes is higher than
gentamicin.
The stem extracts of A. marina in hexane, ethyl acetate, acetone, methanol
and ethanol were effective on all the gram positive test organisms used. The
efficacy of the extracts on these organisms was higher than gentamicin.
Where as, benzene extract does not inhibit any one of the gram positive test
cultures used, but showed effect on the growth of gram negative test cultures
used. E. coli and E. aerogenes were senstive only to the stem extract in
benzene. Where as, K. pneumonia and P. aeruginosa were sensitive to the
stem extracts in benzene, acetone and ethanol. The size of the inhibition
zones in the case of K. pneumonia and P. aeruginosa were same to the
benzene and acetone extracts and are higher than that of gentamicin, however, same zone size is exhibited by the ethanol extract also but it is lower
than that of gentamicin.

Journal of Pharmacy Research Vol.5 Issue 5.May 2012

2906-2909

V.Umamaheswara Rao et al. / Journal of Pharmacy Research 2012,5(5),2906-2909

Table-1. MIC (mg/100l) of the Leaf extracts of Avicennia marina against the bacterial test organisms
Organism

Hexane

Benzene

Ethyl acetate

Acetone

Methanol

Ethanol

Escherichia coli MTCC64

Enterobacter aerogenes MTCC111
Klebsiella pneumoniae MTCC39
Pseudomonas aeruginosa MTCC424
Bacillus subtilis MTCC121
Lactobacillus delbrueckii MTCC 911
Staphylococcus aureus MTCC 87
Streptococcus pyogenes MTCC 1928

5.0
5.0
-

5.0
5.0
5.0
5.0
-

2.5
2.5
2.5
2.5

5.0
5.0
-

1.25
1.25
1.25
1.25

5.0
5.0
1.25
1.25
1.25
1.25

Table-2. MIC (mg/100l) of the Stem extracts of Avicennia marina against the bacterial test organisms
Organism

Hexane

Benzene

Ethyl acetate

Acetone

Methanol

Ethanol

Escherichia coli MTCC64

Enterobacter aerogenes MTCC111
Klebsiella pneumoniae MTCC39
Pseudomonas aeruginosaMTCC424
Bacillus subtilis MTCC121
Lactobacillus delbrueckii MTCC 911
Staphylococcus aureus MTCC 87
Streptococcus pyogenes MTCC 1928

2.5
2.5
2.5
2.5

5.0
2.5
2.5
2.5
-

2.5
2.5
2.5
2.5

1.25
1.25
2.5
2.5
2.5
2.5

1.25
1.25
1.25
1.25

2.5
2.5
1.25
1.25
1.25
1.25

Table-3. Bactericidal /Bacteriostatic activity of Leaf extracts of Avicennia marina against the bacterial test organisms
Organism

Hexane

Benzene

Ethyl acetate

Acetone

Methanol

Ethanol

Escherichia coli MTCC64

Enterobacter aerogenes MTCC111
Klebsiella pneumoniae MTCC39
Pseudomonas aeruginosa MTCC424
Bacillus subtilis MTCC121
Lactobacillus delbrueckii MTCC 911
Staphylococcus aureus MTCC 87
Streptococcus pyogenes MTCC 1928

BS
BS
-

BS
BS
BS
BS
-

BS
BS
BS
BS

BS
BS
-

BC
BC
BC
BC

BC
BC
BC
BC
BC
BC

BS Bacteriostatic

BC - Bactericidal

Table-4 Bactericidal /Bacteriostatic activity of Leaf extracts of Avicennia marina against the bacterial test organisms
Organism

Hexane

Benzene

Ethyl acetate

Acetone

Methanol

Ethanol

Escherichia coli MTCC64

Enterobacter aerogenes MTCC111
Klebsiella pneumoniae MTCC39
Pseudomonas aeruginosa MTCC424
Bacillus subtilis MTCC121
Lactobacillus delbrueckii MTCC 911
Staphylococcus aureus MTCC 87
Streptococcus pyogenes MTCC 1928

BS
BS
BS
BS

BS
BS
BS
BS
-

BS
BS
BS
BS

BS
BS
BS
BS
BS
BS

BC
BC
BC
BC

BC
BC
BC
BC
BC
BC

BS Bacteriostatic

BC - Bactericidal

The positive extracts for antibacterial activity, were further tested to determine the Minimum Inhibitory Concentration (MIC) at different concentrations viz., 1.25, 2.5 and 5.0 mg /100l and the data is given in tables 1 and
2. The value of MIC was found to be in the range of 1.25 to 5.0 mg /100l
for leaf and stem extracts of A. marina L. against all the bacteria tested. The
MIC of leaf extracts of A.marina L. in hexane, benzene acetone and ethanol
was found to be 5mg/100l for all the gram negative bacteria tested. Whereas,
the ethyl acetate extract exhibited 2.5mg/100l MIC value for the gram
positive test cultures. Leaf extracts of methanol and ethanol showed a MIC
value of 1.25mg/100l on gram positive test cultures.
The hexane, benzene and acetone extracts of stem showed the MIC value of
2.5mg/100l against all the gram positive bacteria tested, where as, methanol
and ethanol extracts exhibited a MIC value of 1.25mg/100l against the same
gram positive bacteria. Regarding gram negative bacteria, the benzene extract
showed a MIC value of 5.0mg/100l against E.coli and 2.5mg/100l on the
rest of the gram negative bacteria tested. The acetone and ethanol extracts of
stem exhibited the MIC values of 1.25mg/100l and 2.5mg/100l, respectively on K. pneumonia as well as P. aeruginosa.

Bactericidal and / or bacteriostatic nature

The bactericidal or bacteriostatic nature of the stem and root extracts that are
positive for antibacterial activity are given in tables 3 and 4. The leaf and
stem extracts in hexane, benzene, ethyl acetate and acetone were bacteriostatic in nature. Where as, methanol and ethanol extracts are bactericidal in
nature.
Leaf extracts of A. marina L. in ethanol and stem extract in ethanol and
acetone were active against all the bacteria tested except, E. coli and E.
aerogenes. Stem extract in hexane is active against only gram positive test
cultures used, whereas, stem extract in benzene active only against gram
negative test cultures used. Leaf and stem extracts in ethyl acetate were
active against gram positive test cultures used, but failed to inhibit gram
negative test cultures. This observation of our study was found to be almost
in concurrence with the earlier studies on A. marina by Mahasneh (2002)9 ,
who reported the inhibitory effect of ethanol extracts of aerial parts of A.
marina against E. coli, P. aeruginosa, B. cereus, S. aureus, Candida albicans
and Aspergillus flavus.
.
Abeysinghe and Wanigatunge (2006)10 , reported the antibacterial activity of

Journal of Pharmacy Research Vol.5 Issue 5.May 2012

2906-2909

V.Umamaheswara Rao et al. / Journal of Pharmacy Research 2012,5(5),2906-2909

A. marina leaf extracts in petroleum ether, chloroform, ethyl acetate and
ethanol against E. coli, Pseudomonas species, Proteus species, Shigella
species and Staphylococcus species. Satdive et al, (2003)11 screened for the
antimicrobial activity of Gymnema sylsestre leaf extract in ethanol and
reported that the leaf extract demonstrated antibacterial activity against Bacillus pumilis, B.subtilis, Staphylococcus aureus and Pseudomonas aeruginosa
but not inhibited the growth of Escherichia coli. Our present study results
are in agreement with the above findings.
The presence of biologically active substances such as alkaloids, steroids,
triterpenoids and flavonoids in the leaf extracts of A. marina may be responsible for the antibacterial activity against the test cultures used as revealed by
Abeysinghe et al. (2002)12 through phytochemical studies of leaf extracts of
A. marina.
Among the isolates tested, B. subtilis is more sensitive to stem extract of A.
marina in acetone and leaf extract in methanol. It has been reported that
acetone and methanol are the better solvents for the solubility of many
secondary metabolites. The differential effectiveness of leaf and stem extracts in ethyl acetate on Gram positive and Gram negative bacteria used can
be reasoned to morphological differences between Gram negative and positive bacteria with regard to the cell wall composition. The Gram negative
bacteria contain an outer polysaccharide membrane carrying the structural
lipopolysaccharide components. This makes the cell wall impermeable to
lipophilic solutes, whereas the Gram positive bacteria will be more susceptible due to the outer peptidoglycan layer which is not an effective permeability barrier13 .
Different plant extracts used in our study exhibited variation in effectiveness
towards antibacterial property against the tested bacteria. This antibacterial
activity may be due to the active compounds that are present in the plant
extracts. However, some plant extracts were unable to exhibit antibacterial
activity against tested bacterial strains. These bacterial strains may have
some kind of resistance mechanisms, for example, enzymatic inactivation,
target site modification and decreased intracellular drug accumulation.
The leaf and stem extracts that showed antibacterial activity were compared
with broad spectrum antibiotic gentamicin at a concentration of 1 mg/100l.
From this comparison, it was observed that majority of the extracts in crude
form itself were more effective than gentamicin. With the case of the positive
extracts exhibiting equal or less effectiveness in comparison to gentamicin,
there is every possibility for having more antibacterial activity than gentamicin when the bioactive compounds of these extracts were purified and tested.
Several reports are documented in literature on determination of MIC values
of several plant extracts. Different workers reported different ranges of MIC
values with respect to the solvents, plant parts and plants. Nkere and Iroegbu
(2005)5 studied the MIC values of the ethanol extracts of root and stem bark
of Picarlima nitida and reported the MIC values ranging from 6.25 to 50mg/
ml. Okoli and Iroegbu (2005)6 , from their studies, reported the MIC range
value of 3.125 to 12.50 mg/ml for the ethanol root extracts of Synclisa
scabrida.
In our study, the MIC value for all the positive extracts against the tested
bacteria were between 1.25mg/100l to 5mg/100l. Gram negative test
cultures showed higher MIC values than Gram positive text cultures to the
leaf and stem extracts in ethanol. However, stem extracts in acetone exhibited lower MIC value to Gram negative test cultures used when compared to
gram positive test cultures. This difference may be explained by susceptibility testing condition, physico chemical characters of the bioactive principle
present in the extract and even strain to strain difference. In comparison to
some of the earlier reports14,15 on MIC values of pure compounds, our MIC
may be higher. But this can be substantiated by the argument that this value
is for the crude extract. However, the purified form of bioactive compound of
the crude extract responsible for antibacterial activity may exhibit the inhibitory effect at a lower concentration.

So, the A. marina L. can also be strongly recommended for consideration as

a valuable source for identification, isolation and characterization of potential bioactive compounds with antibacterial property. Finally, there is a need
to explore this area further to understand the potentiality of the mangrove
plants towards the development of new era medicines.
ACKNOWLEDGEMENT
One of the authors, N.Sharief, Md, would like to express his deep thanks to
the Secretary and Correspondent of V.S. Lakshmi Womens Degree and P.G.
College, Kakinada, for the facilities provided to pursue the work in their
research centre of the college. Thanks are also due to the Director, P.G
courses of V.S. Lakshmi Colllege, Kakinada.
REFERENCES
1. Janathan, I., Yassin, M., Chin, C., Chen, L., and Sim, N. Antifungal
activity of the essential oils of nine Zingiberaceae species,
Pharmaceut Biol., 2003, 41: 392-97.
2. Khan, M.R., Kihara, M., and Omoloso, A.D. Broad spectrum
antibacterial activity of the leaves, stem and root barks of
Myristica subabulata, Natural product sciences., 2001, 7(1): 912.
3. Perez, R.M. Antiviral activity of compounds isolated from plants,
Pharmaceut Biol., 2003, 41: 105-57.
4. Choudhury, S., Sree, A., Mukherjee, SC., Patnaik, P. and Bapuji,
M. In Vitro Antibacterial Activity of Extracts of Selected Marine
Algae and Mangroves against Fish Pathogens, Asian Fisheries
Science., 2005;18 :285-294.
5. Nkere,CK.and Iroegbu, CU. Antibacterial screening of the root,
seed and stem bark extracts of Picralima nitida, African journal
of biotechnology., 2005, 4(6): 522-26.
6. Okoli, S. and Iroegbu, CU., In vitro antibacterial activity of
Synclisa scabrida whole root extracts, African journal of biotechnology., 2005, 4(9): 946-52.
7. Ahmad-reza, S., Iranshahi, M., Roohollah, M., Hossein, J.,
Gholamerza Abbas, S. Bioassay- Guided isolation and identification of an antibacterial compound from Ferula persica var persica
roots, DARU., 2005,13: 17-9.
8. Damintoti, K., Mamoudou, HD., Jacques, S.and Alfred ST. Antioxidant and antibacterial
activities of polyphenols from
ethnomedicinal plants of Burkina faso, African Journal of Biotechnology, 2005, 4 :823 8.
9. Mahasneh, AM. Screening of some indigenous Qatar medicinal
plants for antimicrobial activity, Phytotherapy research, 2002,
16(8):751-53.
10. Abeysinghe, PD. and Wanigatunge, RP. Evaluation of antibacterial activity of different
mangrove plant extracts, Ruhuna
Journal of Science., 2006, 1: 109-117.
11. Satdive, RK., Abhilash, P.and Fulzekle, DP. Antimicrobial activity of Gymnema sylvestre leaf extract, Fitoterapia, 2003,74(78):699-701.
12. Abeysinghe, PD., Withanawasam, M., Pathirana, RN. and
Abeysinghe, S. Preliminary in vitro screening of antibacterial
compounds of some mangrove plant extracts of clinical isolated
from different sources. Proceedings of the First Science Symposium, University of Ruhuna, 2002, 22-25.
13. Mann, CM., Cox, SD. and Markham, JL..The outer membrane of
Pseudomonas aeruginosa NCTC6749 contributes to its tolerance to the essential oils of Melaleuca alternifolia, Letters in
applied microbiology 2000, 30: 294-97.
14. Maria DC, Torrado, T., Maria, H., Sarragiotto,, Benicio, Alves.,
de Abreu, Filho, Celso VN, Benedito P, Dias F. In Vitro Antibacterial activity of a 7-O- -D-glucopyranosyl- nutanocoumarin
from Chaptalia nutans (Asteraceae). Mem inst Oswaldo Cruz,
Riode Janerio 2003, 98: 283-6.
15. Celso, VN., Tania, UB., Erika, B., Abraho, FNM., Diogenes,
AGC. and Benedito, PDF. Antibacterial activity of Ocimum
gratissimum L. Essential oil. Mem Inst Oswaldo Cruz, Rio de
Janeiro 1999, 94: 675-8.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 5.May 2012

2906-2909