Chinese Medicine, 2011, 2, 171-177
doi:10.4236/cm.2011.24027 Published Online December 2011 (http://www.SciRP.org/journal/cm)
171
Antimicrobial Activity of Terminalia catappa L. Leaf
Extracts against Some Clinically Important Pathogenic
Microbial Strains
Sumitra Chanda*, Kalpna Rakholiya, Rathish Nair
1Phytochemical, Pharmacolo gi cal an d Mi crobiological Laborat ory , De partme nt of Biosciences,
Sauras h t ra University, Rajkot, India
Received July 27, 2011; revised Se ptember 2, 2011; accepted September 14, 2011
Abstract
Objective: The present study was undertaken to evaluate in-vitro antimicrobial activity of methanol, acetone
and N, N-dimethylformamide extracts from leaf of Terminalia catappa L. (Combretaceae). Methods: In vi-
tro antimicrobial activity of all the extracts was done by agar disc diffusion assay. 91 clinically important
strains were used for the study, which were both clinical isolates as well as identified strains. Piperacillin and
gentamicin were used as standards for antibacterial assay, while nystatin and flucanazole were used as stan-
dards for antifungal assay. Antimicrobial activity was determined by measurement of inhibition zone around
each paper disc. For each extract three replicate trials were conducted against each organism. Results: The
antibacterial activity was more pronounced against bacteria than fungal strains. The Gram positive bacteria
were more susceptible than Gram negative bacteria. The methanol extract showed best antibacterial activity.
T. catappa leaf extracts showed better antibacterial activity than commercially used antibiotics. Conclusion:
Demonstration of antimicrobial activity of T. catappa provides the scientific basis for the use of this plant in
the traditional treatment of diseases and may help to discover new chemical classes of antibiotic substances
that could serve as selective agents for infectious disease chemotherapy and control. This investigation has
opened up the possibility of the use of this plant in drug development for human consumption possibly for
the treatment of various infections caused by microbes.
Keywords: Terminalia catappa, Antibacterial Activity, Antifungal Activity, Clinical Strains,
Organic Solvents
1. Introduction
Traditional medicine has been practiced for many centu-
ries in many parts of the world, including India especi-
ally in rural areas due to availability and low cost. Nature
has provided a source of medicinal agents for thousands
of years and an impressive number of modern drugs have
been isolated from natural sources, many based on their
use in traditional medicine [1]. There has been an incr-
easing incidence of multiple resistances in human path-
ogenic microorganisms, largely due to the indiscriminate
use of commercial antimicrobial drugs commonly empl-
oyed in the treatment of infectious diseases [2]. The de-
velopment of bacterial resistance to presently available
antibiotics has necessitated the search for new antibacte-
rial agents. Numerous studies have been conducted with
the extracts of various plants, screening antimicrobial ac-
tivity as well as for the discovery of new antimicrobial
compounds [3-6]. The efforts of scientists in establishing
plants with promising antimicrobial property is yielding
fruitful results as a number of plants with high antim-
icrobial property have been elucidated [7-13].
Terminalia catappa L. belongs to the family Combre-
taceae. T. catappa is used primarily as an ornamental, sh-
ade, and salt-tolerant street tree, but the leaves provide
food for the Tasar silkworm, and the seeds are edible like
almonds with similar oils. On the Malay peninsular and
through the Canary islands this tree is known as the tro-
pical almond. T. catappa has been claimed to have the-
rapeutic effects for liver related diseases [14]. In Java, it
is attributed with cholagogue action. In India, it is used
as cardiac stimulant. Its leaves are widely used as a folk

S. CHANDA ET AL.
172
medicine in Southeast Asia for the treatment of dermato-
sis and hepatitis [15]. More and more pharmacological
studies have reported that the extract of T. catappa lea-
ves and fruits have anticancer, antioxidant, anti-HIV re-
verse transcriptase, anti-inflammatory, antidiabetic effe-
cts and hepatoprotective activities [16-19] but the effecti-
ve components and related mechanisms remain unkno-
wn.
In the present work, antimicrobial activity of T. ca-
tappa leaf extracts were investigated against an array of
clinically isolated as well as standard microbial cultures.
2. Material and Methods
2.1. Plant Material
The leaves of T. catappa were collected in February,
2005 from Rajkot in the State of Gujarat Western India
and identified by comparison with specimens PSN 291
available at the Herbarium of the Department of Bio-
sciences, Saurashtra University, Rajkot, Gujarat, India.
2.2. Extraction
The leaves of T. catappa were air dried and then po-
wdered in a homogenizer and 10 g was used for different
solvent extraction N, N-dimethylformamide (DMF), ace-
tone and methanol, the sample was extracted in solvent
kept on a rotary shaker overnight, and then the filtrate
was collected and centrifuged at 5000 rpm. The solvent
was then evaporated to dryness under reduced pressure
and the extracted compound left was used for the antim-
icrobial assay. The percentage yield of N, N-dimethyl-
formamide (DMF), acetone and methanol extracts were
20.92, 4.96 and 14.48 respectively.
2.3. Microorganisms Studied
91 clinically important microbial strains which included
20 Gram positive, 55 Gram negative and 16 fungal str-
ains were studied for the antimicrobial activity. These
strains included both clinical isolates as well as identified
strains. The identified strains were obtained from Natio-
nal Chemical Laboratory (NCL), Pune, India and clinical
isolates were obtained from Spandan Diagnostic and Mi-
crocare Diagnostic Laboratory, Rajkot, Gujarat, India
(Tables 1-5). The bacteria were grown in the nutrient
broth and maintained on nutrient agar slants at 4˚C while
fungal strains were grown in Sabouraud dextrose broth
and maintained on MGYP slants for yeast and potato de-
xtrose agar slants for mould at 4˚C.
Table 1. Antibacterial activity of Terminalia catappa leaf extracts against some Gram positive bacteria.
Zone of inhibition (mm)a
Sr.
No. Strain (Location of collection) TME TAE TDE G Pc
1 Staph-1 (Sputum) 14.67 ± 0.33 9.66 ± 0.33 10 ± 0.58 - -
2 S. aureus (Pus) 14 ± 0 11± 0.58 9 ± 1.15 18.67 ± 0.33 17.33 ± 0.33
3 S. aureus (Urine) 13 ± 0.58 9 ± 0.58 8 ± 0.58 - -
4 S. aureus (Pus) 16 ± 0.58 8 ± 0.58 14 ± 0.58 - -
5 Staph-2 (Pus) - - - - -
6 S. aureus (Sputum) - - - - -
7 S. aureus (Tracheal) 15 ± 0.58 10 ± 0.58 9.67 ± 0.33 - -
8 S. aureus (Tracheal) 15 ± 0.58 12 ± 0.59 13 ± 0.58 - -
9 Staph-3 (Sputum) 14.33 ± 0.66 12.33 ± 0.88 10 ± 1.73 14.67 ± 0.33 -
10 S. aureus (Ear swab) 16.67 ± 1.53 14 ± 2.89 10 ± 1.73 - -
11 S. aureus (Sputum) 18.67 ± 0.33 14 ± 0.58 13 ± 0.58 20.67 ± 0.33 -
12 S. aureus (Pus) - - - - -
13 S. aureus (Pus) - - - 10.33 ± 0.33 -
14 S. aureus (ATCC25923) 14.5 ± 0.28 8.5 ± 0.86 10 ± 1.73 - -
15 S. epidemidies (ATCC12228) 11 ± 0.58 - - - -
16 S. subflava (NCIM2178) 19 ± 0.58 13.5 ± 1.44 11.5 ± 0.28 - 20.17 ± 0.44
17 B. cereus (ATCC11778) 11.5 ± 0.28 9.5 ± 0.28 11 ± 0.58 20.17 ± 0.16 18.83 ± 0.16
18 B. subtilis (ATCC6633) 9 ± 1.15 8.5 ± 0.86 - 18.33 ± 0.33 17.83 ± 0.93
19 B. mega (ATCC9885) - - - - -
20 M. flavus (ATCC10240) 14 ± 0.58 8.5 ± 0.86 15 ± 1.15 27.67 ± 0.33 12.67 ± 0.33
aValues are Mean ± SEM, n = 3, zone includes disc diameter 7 mm; G―Gentamicin (10 µg/disc); Pc―Piperacillin (100 µg/disc); TME―Methanol extract;
TAE―Acetone extract; TDE―N, N-dimethylformamide (DMF) extract; “-” means no activity; Staph―Staphylococcus species.

173 S. CHANDA ET AL.
Table 2. Antibacterial activity of Terminalia catappa leaf extracts against some Pseudomonas species.
Zone of inhibition (mm)a
Sr.
No. Strain (Location of collection) TME TAE TDE G Pc
1 Ps. aeruginosa (ATCC27853) - - - 17 ± 1.15 12.33 ± 0.66
2 Ps. aeruginosa (Sputum) - - - 16.67 ± 0.67 -
3 Ps. aeruginosa (Pus) - - - 19.67± 0.33 -
4 Ps. fluorescence (Tracheal) 8.67 ± 0.33 - 12.67± 1.44 - -
5 Ps. fluorescence (Pus) 13.67 ± 3.18 8 ± 0.58 - - -
6 Ps. fluorescence (Urine) - - - - -
7 Ps. testosterone (NCIM5098) - - - 22.33 ± 0.66 -
8 Ps. pseudoalcaligene s (ATCC17440) 15.5 ± 0.28 12.5 ± 0.86 14.5 ± 028 19.33 ± 0.6 -
9 Pseudo-1 (Sputum) 11 ± 2.31 13 ± 0.58 11.67 ± 0.33 14 ± 0.58 -
10 Pseudo-2 (Pus) 13.67 ± 3.18 8 ± 0.58 - - -
11 Pseudo-3 (Urine) 14.67 ± 1.45 16 ± 0.58 14.67 ± 0.33 - -
12 Pseudo-4 (Pus) 14 ± 0.58 10.6 ± 2.34 9.33 ± 1.23 - -
13 Pseudo-5 (Tracheal) - - - - -
14 Pseudo-6 (Wound swab) - - - - -
15 Pseudo-7 (Pus) 16 ± 0.56 10 ± 0.58 12 ± 1.15 - -
16 Pseudo-8 (Tracheal secretion) 14 ± 1.15 9 ± 1.15 9 ± 1.15 - -
17 Pseudo-9 (Pus) 11.67 ± 0.88 9.33 ± 1.20 - - -
18 Pseudo-10 (Sputum) 17 ± 0.58 12 ± 0.33 13.67 ± 0.88 - -
19 Pseudo-11 (Sputum) 18.33 ± 0.33 16.33 ± 1.45 13 ± 0.58 20 ± 0.58 -
aValues are Mean ± SEM, n = 3, zone includes disc diameter 7 mm; G―Gentamicin (10 µg/disc); Pc―Piperacillin (100 µg/disc); TME―Methanol extract;
TAE―Acetone extract; TDE―N, N-dimethylformamide (DMF) extract; “-” means no activity; Pseudo―Pseudomonas species.
Table 3. Antibacterial activity of Terminalia catappa leaf extracts against some E. coli isolates.
Zone of inhibition (mm)a
Sr.
No. Strain (Location of collection) TME TAE TDE G Pc
1 E. coli (Pus) 10 ± 1.53 8.66 ± 0.88 7.66 ± 0.33 - -
2 E. coli (Urine) 12.33 ± 2.73 9.66 ± 1.45 - - -
3 E. coli (Urine) 16 ± 0.58 12.33 ± 0.88 11.67 ± 0.33 - -
4 E. coli (Urine) 15 ± 0.88 10 ± 0.33 13 ± 0.58 - -
5 E. coli (Urine) 15 ± 0.88 11 ± 0.58 14 ± 0.33 - -
6 E. coli (Pus) 10 ± 0.58 14 ± 0.88 13 ± 1.15 - -
7 E. coli (Urine) 14.33 ± 1.20 12 ± 0.58 14 ± 1.15 - -
8 E. coli (Stool) 15.67 ± 0.33 10.67 ± 0.33 13 ± 0.58 21 ± 0.58 -
9 E. coli (Pus) 12 ± 0.58 11.33 ± 0.88 14.67 ± 0.33 - -
10 E. coli (Urine) 14.33 ± 0.33 10.67 ± 0.33 14 ± 0.58 18.67 ± 0.33 -
11 E. coli (Pus) 12.67 ± 0.66 11.67 ± 0.33 11.33 ± 0.66 - -
12 E. coli (Urine) 15.33 ± 0.88 12.67 ± 0.33 14 ± 0.58 20.33 ± 0.33 -
13 E. coli (Vaginal swab) 13.5 ± 0.28 - 12.67 ± 0.33 - -
14 E. coli (Urine) - - - - -
15 E. coli (Blood) 14.5 ± 0.28 - - - -
16 E. coli (ATCC25922) 14 ± 0.58 10 ± 1.73 - 17.83 ± 0.16 14.5 ± 0.50
aValues are Mean ± SEM, n = 3, zone includes disc diameter 7 mm; G―Gentamicin (10 µg/disc); Pc―Piperacillin (100 µg/disc); TME―Methanol extract;
TAE―Acetone extract; TDE―N, N-dimethylformamide (DMF) extract; “-” means no activity.

S. CHANDA ET AL.
174
Table 4. Antibacterial activity of Terminalia catappa leaf extracts against some Gram negative bacteria.
Zone of inhibition (mm)a
Sr.
No. Strain (Location of collection) TME TAE TDE G Pc
1 Ent-1 (Tracheal) 8.33 ± 0.88 - - - -
2 Ent-2 (Tracheal) 11 ± 1.15 - 8 ± 0.58 19.67 ± 0.88 -
3 E. aerogenes (ATCC 13048) - - - - -
4 Kleb-1 (Urine) 13.67 ± 0.88 11 ± 0.58 11 ± 0.58 22 ± 0.58 -
5 Kleb-1 (Sputum) 14 ± 0.58 10.33 ± 0.33 10 ± 0.58 - -
6 K. aerogenes (Pus) 8 ± 0.58 - 8.67 ± 0.88 - -
7 Kleb-2 (Urine) 14 ± 0.58 12.33 ± 0.33 14.67 ± 0.33 - -
8 K. aerogenes (Urine) 13.67 ± 0.33 10.67 ± 0.33 13.33 ± 0.33 - -
9 K. pneumoniae (NCIM2719) - - - - 24.67 ± 0.33
10 P. mirabilis (Wound swab) 18 ± 1.20 10.33 ± 0.33 12.67 ± 0.33 - 14 ± 0.58
11 Prot-1 (Pus) 14.67 ± 0.33 10 ± 0.58 13.33 ± 0.33 - -
12 P. mirabili s (NCIM2241) - - - 18.67 ± 0.33 -
13 P. vulgaris (NCTC8313) 14.5 ± 0.28 - - 18 ± 1.00 -
14 P. morganii (NCIM2040) - - - - -
15 P. rettgeri (Pus) 16.33 ± 0.88 10.67± 0.33 11.67 ± 0.33 - -
16 Citro-1 (Pus) 12 ± 0.58 9 ± 0.58 10 ± 1.16 - -
17 C. freundii (Pus) - - - 12.33 ± 0.33 -
18 C. freundii (ATCC10787) - - - - -
19 A. fecalis (ATCC8750) - - - 18.33 ± 0.66 -
20 S. typhimurium (ATCC23564) 12 ± 0.58 8.5 ± 0.86 10.5 ± 0.86 18.5 ± 0.28 -
aValues are Mean ± SEM, n = 3, zone includes disc diameter 7 mm; G―Gentamicin (10 µg/disc); Pc―Piperacillin (100 µg/disc); TME―Methanol extract;
TAE―Acetone extract; TDE―N, N-dimethylformamide (DMF) extract; “-” means no activity; Ent―Enterobacter species; Kleb―Klebsiella species;
Citro―Citrobacter species; Prot―Proteus species.
Table 5. Antifungal activity of Terminalia catappa leaf extracts.
Zone of inhibition (mm)a
Sr.
No.
Fungus
(Location of collection) TME TAE TDE Fu Ns
1 Candida spp. (Sputum) - - - - 14 ± 0.58
2 C. albicans (Urine) - 7.5 ± 0.29 10 ± 1.73 - 11.33 ± 0.33
3 C . albicans (Sputum) - - - - 18 ± 0.58
4 Candida spp. (Sputum) - - - - 14 ± 0.58
5 Candida spp. (Urine) - - - - 10 ± 0.58
6 C. albicans (ATCC2091) 8.5 ± 0.87 8.5 ± 0.87 - 17.67 ± 0.33 13 ± 0.58
7 C. albicans (ATCC18804) - - - - 14.33 ± 0.33
8 C. glabrata (NCIM3448) - - - 39.67 ± 0.88 22 ± 0.58
9 C. tropicalis (ATCC4563) - - - - 8.33 ± 0.33
10 C. apicola (NCIM3367) 19.33 ± 0.33 13 ± 1.15 14.33 ± 0.33 - 21.33 ± 0.88
11 C. neoformans (ATCC34664) - - - 21.33 ± 0.33 17 ± 0.58
12 C. luteolus (ATCC32044) 17.5 ± 2.60 8.5 ± 0.86 - 23.66 ± 0.88 17.66 ± 0.88
13 T. beigelii (NCIM3404) 12 ± 0.58 12 ± 0.58 7.5 ± 0.29 - -
14 A. flavus (NCIM538) - - - - -
15 A. candidus (NCIM883) - - - - -
16 A. niger (ATCC6275) - - - - -
aValues are Mean ± SEM, n = 3, zone includes disc diameter 7 mm; Ns―Nystatin (100 units/disc); Fu―Fluconazole (10 µg/disc); TME―Methanol extract;
TAE―Acetone extract; TDE―N, N-dimethylformamide (DMF) extract; “-” means no activity; Fu―Fluconazole; Ns―Nystatin.

S. CHANDA ET AL.
175
2.4. Antimicrobial Assay
The N, N-Dimethyl formamide extract (TDE), acetone
extract (TAE) and methanol extract (TME) were dissol-
ved in DMSO. The antimicrobial activity was evaluated
at a concentration of 250 g/disc. Antimicrobial activity
was performed by agar disc diffusion method [20,21].
The bacterial strains were grown in nutrient broth while
fungal strains were grown in MGYP (Malt glucose yeast
peptone) broth. Mueller Hinton agar No. 2 was the media
used to study the antibacterial susceptibility while Sab-
ouraud dextrose agar was used to study the antifungal
susceptibility test. The cultures were grown for 24 h, and
the turbidity of the culture was maintained according to
the 0.5 MacFarland standards. The inoculum’s size was 1
× 108 cells/ml. The media Mueller Hinton Agar No. 2
and MRS media and the test bacterial cultures were
poured into Petri dishes Hi-Media. The test strain 200 µl
was inoculated into the media inoculums size 108
cells/ml when the temperature reached 40˚C - 42˚C. The
test compound 20 µl was impregnated in to sterile discs 7
mm Hi-Media and was then allowed to dry. The disc was
then introduced into medium with the bacteria. For each
microbial strain negative controls were maintained where
pure solvent DMSO was used instead of the extract since
it does not possess any antimicrobial effect [22] and for
positive control the standard antimicrobics Gentamicin
10 µg/disc and piperacillin 100 µg/disc for bacteria, nys-
tatin 100 units/disc and flucanazole 10 µg/disc Himedia
Labs for fungus were used for comparative studies. The
plates were incubated overnight at 37˚C for bacterial
strains and 42˚C for fungal strains. The experiment was
performed under strict aseptic conditions. Microbial gro-
wth was determined by measuring the diameter of the
zone of inhibition. The experiment was performed in tri-
plicates and the mean values of the result are shown in
Tables 1-5.
3. Results and Discussion
Herbal medicine in developing countries is commonly
used for the traditional treatment of health problems [23].
In recent years multiple drug resistance in human patho-
genic microorganisms have developed due to the indis-
criminate use of commercial antimicrobial drugs com-
monly used in the treatment of infectious diseases [24].
In addition to this problem, antibiotics are sometimes
associated with adverse effects on host including hyper-
sensitivity, immunosuppression and allergic reactions
[25]. Therefore there is a need to develop alternative
antimicrobial drugs for the treatment of infections ob-
tained from various sources such as medicinal plants [26,
27].
In the present study T. catappa leaf extracts extracted
in DMF (TDE), acetone (TAE) and methanol (TME)
were investigated for their antimicrobial potentiality ag-
ainst 91 clinically important microbial strains. Drug re-
sistance is a new problem, but it is not a new phenome-
non. Soon after the introduction of penicillin, Staphylo-
cocci were found to be very resistant to many of the an-
tibiotics. Although recognized earlier that antibiotics
resistance was only in the hospitals, now resistance in the
community is also seen. Bacteria such as Staphylococcus
have emerged with resistance to six and more different
antibiotics [28].
All the three extracts of T. catappa TDE, TAE and
TME were active against 70% of the total Gram positive
bacteria studied while only 63% of Gram negative bacte-
ria were inhibited Tables 1-4, on the other hand, the three
extracts of T. catappa were active against only 25% of
fungal strains Table 5. The best antibacterial activity was
shown by the methanol extract. Similar results were also
shown by Babayi et al. [29] and Kaneria et al. [30]. The
Gram positive bacteria were more susceptible than Gram
negative bacteria. This is in agreement with previous
reports that plant extracts are more active against Gram
positive bacteria than Gram negative bacteria [31-33].
These differences may be attributed to the fact that the
cell wall in Gram positive bacteria is of a single layer,
whereas the Gram negative cell wall is multilayered stru-
cture [34].
The most striking feature of the present findings is that
many of the clinical isolates were resistant to the stan-
dard antimicrobics used while the plant extracts showed
moderate to good antibacterial activity. The need of the
hour is to find new antimicrobics because the microor-
ganisms are getting resistant to the existing antibiotics
[35,36]. The persistent increase in multi drug resistant
strains compels the search for more potent new antibiot-
ics. Thus there is a need for a continuous search for new
effective and affordable antimicrobial drugs. The results
of present study signify the potentiality of T. catappa
leaf as a source of therapeutic agents which may provide
leads in the ongoing search for antimicrobial botanicals.
4. Conclusions
Present study showed that the T. catappa leaf extracts
possessed significant in vitro antimicrobial property
against 91 clinical isolate as well as identified strains.
The methanol extract exhibited strongest inhibitory ef-
fect on bacteria as compared to standard antibiotics ag-
ainst the tested microorganisms. It is necessary to carry
out a bioassay guided fractionation of the extract in a bid
to isolate and identify the compounds responsible for the
antimicrobial activity. An elucidation of the mechanisms

S. CHANDA ET AL.
176
of action of these extract must be followed by toxicity
and in vivo tests to determine the therapeutic applicabil-
ity of such compounds in combination therapy. These are
subjects of on-going investigation in our research group.
5. Acknowledgements
Financial support to R. Nair from UGC (DSA project),
New Delhi and supply of clinical isolates by Micro Care
and Spandan Diagnostic Laboratories, Rajkot are grate-
fully acknowledged.
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