Antimicrobial Activity of <i>Terminalia catappa</i> L. Leaf Extracts against Some Clinically Important Pathogenic Microbial Strains

doi:10.4236/cm.2011.24027

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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

E-mail: *[email protected]

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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