Antimicrobial Activity of <i>Terminalia catappa</i> Extracts Against Some Pathogenic Microbial Strains

doi:10.4236/pp.2011.24038

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Pharmacology & Pharmacy, 2011, 2, 299-305

doi:10.4236/pp.2011.24038 Published Online October 2011 (http://www.SciRP.org/journal/pp)

299

Antimicrobial Activity of Terminalia catappa

Extracts against Some Pathogenic Microbial

Strains

Abul Manzur1*, Arifuddin Raju1, Shahedur Rahman2

1Department of Microbiology, North South University, Dhaka, Bangladesh; 2Department of Biotechnology and Genetic Engineering,

Islamic University, Kushtia, Bangladesh.

Email: *[email protected]

Received September 4th, 2011; revised September 27th, 2011; accepted October 20th, 2011.

ABSTRACT

The methanol, acetone and N,N-dimethylformamide extracts of Terminalia catappa L. leaf were evaluated for antibac-

terial and antifungal activity. Piperacillin and gentamicin were used as standards for antibacterial assay, while nystatin

and flucanazole were used as standards for antifungal assay. 91 clinically important strains were used for the study

which were both clinical isolates as well as identified strains. The antimicrobial activity of all the extracts was deter-

mined by agar disc diffusion method. The antibacterial activity was more pronounced against bacteria than fungal

strains. The Gram positive bacteria were more suscep tible than Gram negative bacteria. The methanol extract showed

best antibacterial activity. T. catappa leaf extracts showed better antibacterial activity than commercially used antibi-

otics.

Keywords: Terminalia catappa, Antibacterial Activity, Antifungal Activity, Clinica l Strains, Organic Solvents

1. Introduction

Traditional medicine has been practiced for many centu-

ries in many parts of the world, including Bangladesh

especially 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 increasing incidence of multiple resistances in

human pathogenic microorganisms, largely due to the

indiscriminate use of commercial antimicrobial drugs

commonly employed in the treatment of infectious dis-

eases [2]. The development of bacterial resistance to

presently available antibiotics has necessitated the search

for new antibacterial agents. Numerous studies have been

conducted with the extracts of various plants, screening

antimicrobial activity as well as for the discovery of new

antimicrobial compounds [3-6]. The efforts of scientists

in establishing plants with promising antimicrobial prop-

erty is yielding fruitful results as a number of plants with

high antimicrobial property have been elucidated [7-13].

Terminalia catappa L. belongs to the family Combre-

taceae. T. catappa is used primarily as an ornamental,

shade, 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

tropical almond. T. catappa has been claimed to have

therapeutic effects for liver related diseases [14]. In Java,

it is attributed with cholagogue action. In India, it is u sed

as cardiac stimulant. Its leaves are widely used as a folk

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 leaves

and fruits have anticancer, antioxidant, anti-HIV reverse

transcriptase, anti-inflammatory, antidiabetic effects and

hepatoprotectiv e activities [16-19] but the effective com-

ponents and related mechanisms remain unknown.

In the present work, antimicrobial activity of T. catappa

leaf extracts were investigated against an array of clini-

cally isolated as well as standard microbial cultures.

2. Material and Methods

2.1. Plant Material

The leaves of T. catappa were collected from the Boro

Parulia, Gopalgonj and Arappur, Madaripur of Bangla-

Antimicrobial Activity of Terminal i a c at a p p a Extracts against Some Pathogenic M i c r ob i a l Strai ns

300

desh, in January 2009 and identified by Dr. Masudur

Rahman, Bangladesh National Herbarium, Dhaka.

2.2. Extraction

The leaves of T. catappa were air dried and then pow-

dered 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

strains were studied for the antimicrobial activity. These

strains included both clinical isolates as well as identified

strains. The identified strains were provided by Depart-

ment of Microbiology, North So uth University and clini-

cal isolates were obtained from Lab Aid Diagnostic

Laboratory, Dhaka, Bangladesh (Tables 1-5). The bacte-

ria were grown in the nutrient broth and maintained on

nutrient agar slants at 4˚C while fungal strains were

grown in Sabouraud broth and maintained on MGYP

slants yeast and potato dextrose agar slants mould at 4˚C.

2.4. Antimicrobial Assay

The Dimethylformamide extract TDE, acetone extract

TAE and Methanol extract TME were dissolved 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 fun-

gal 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 Sa-

broaud agar was us ed to study the antifungal susceptibil-

ity test. The cultures were grown for 24 h, and the turb id-

ity of the culture was maintained according to the 0.5

MacFarland standards. The inoculum’s size was 1 × 108

cells/ml. The med ia Mueller Hinton Agar No. 2 and MR S

media and the test bacterial cultures were poured into

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. 6 7 ± 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 1 8.83 ± 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 7mm, 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.

Antimicrobial Activity of Terminal i a c at a p p a Extracts against Some Pathogenic Mi cr o bi a l Strai ns301

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. 6 6

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. testosteroni (NCIM 5 098 ) - - - 22.33 ± 0.66 -

8 Ps. pseudoalcaligenes (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 1 4 .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 sec retion) 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 ± 1.4 5 - - -

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 1 8.67 ± 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 2 0.33 ± 0.33 -

13 E. coli ( V aginal 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 7mm, 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.

Antimicrobial Activity of Terminal i a c at a p p a Extracts against Some Pathogenic M i c r ob i a l Strai ns

302

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. mirabilis (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 7mm, 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.

Petri dishes Hi-Media. The test strain 200 µl was inocu-

lated into the med ia ino cu lums 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 intro-

duced into medium with the bacteria. For each microbial

strain negative controls were maintained where pure sol-

vent DMSO was used instead of the extract since it does

not possess any antimicrobial effect [22] and for positive

control the stand ard antimicr ob ics Gentamicin 10 µg/d isc

and piperacillin 100 µg/disc for bacteria, nystatin 100

units/disc and flucanazole 10 µg/disc Himedia Labs for

fungus were used for comparative studies. The plates

were incub ated ov ern ight at 37˚C for bacterial strains and

42˚C for fungal strains. The experiment was performed

under strict aseptic conditions. Microbial growth was de-

termined by measuring the diameter of the zone of inhibi-

tion. The experiment was performed in triplicates 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, immune suppression and allergic reactions

[25]. Therefore there is a need to develop alternative an-

timicrobial drugs for the treatment of infections obtained

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 th eir antimicro b ial poten tiali ty again st 91

clinically important microbial strains. Drug resistance is

a new problem, but it is not a new phenomenon. Soon

after the introduction of penicillin, Staphylococci were

found to be very resistant to many of the antibiotics. Al-

though recognized earlier that antibiotics resistance was

only in the hosp itals, now resistance in the community is

also seen. Bacteria such as Staphylococcus have emerged

with resistance to six and more different antib iotics [28].

Antimicrobial Activity of Terminal i a c at a p p a Extracts against Some Pathogenic M i c r ob i a l Strai ns 303

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

4 C. albicans (Sputum) - - - - 18 ± 0.58

5 Candida spp.(Sputum) - - - - 14 ± 0.58

6 Candida spp. (Urine) - - - - 10 ± 0.58

7 C. albicans (ATCC2091) 8.5 ± 0.87 8.5 ± 0.87 - 17.67 ± 0.33 13 ± 0.58

8 C. albicans (ATCC18804) - - - - 14.33 ± 0.33

9 C. glabrata (NCIM3448) - - - 39.67 ± 0.88 22 ± 0.58

10 C. tropicalis (ATCC4563) - - - - 8.33 ± 0.33

11 C. apicola (NCIM3367) 19.33 ± 0.33 13 ± 1.15 14.33 ± 0.33 - 21.33 ± 0.88

12 C. neoformans (ATCC34664) - - - 21.33 ± 0.33 17 ± 0.58

13 C. luteolus (ATCC32044) 17.5 ± 2.60 8.5 ± 0.86 - 23.66 ± 0.88 17.66 ± 0.88

14 T. beigelii (NCIM3404) 12 ± 0.5 8 12 ± 0.58 7.5 ± 0.29 - -

15 A. flavus (NCIM538) - - - - -

16 A. candidus (NCIM883) - - - - -

17 A. niger (ATCC6275) - - - - -

aValues are Mean ± SEM, n = 3, zon e includes dis c diameter 7mm, Ns: N ystatin (100 un its/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.

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 Table 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]. 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 [30-32]. These differences may

be attributed to the fact that the cell wall in Gram posi-

tive bacteria is of a single layer, whereas the Gram nega-

tive cell wall is multilayered structure [33]. The most

striking feature of the presen t finding s is that many of th e

clinical isolates were resistant to the standard antimicro-

bics used while the plant extracts showed moderate to

good antibacterial activity. The need of the hour is to find

new antimicrobics because the microorganisms are get-

ting resistant to the existing antibiotics [34,35]. The per-

sistent increase in multi drug resistant strain s compels the

search for more potent new antibiotics. Thus there is a

need for a continuous search for new effective and af-

fordable antimicrobial drug s. The results of present study

signify the potentiality of T. catappa leaf as a source of

therapeutic agents which may provide leads in the ongo-

ing search for an ti microbial botanicals .

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