International Journal of Drug Development and Research

Key words

Manilkara zapota, Antimicrobial, Stem bark and Leaves.

Introduction

Natural products either as pure compounds or as standardized plant extracts provide unlimited opportunities for new drug [1]. Many infectious diseases are known to be treated with herbal remedies throughout the history of mankind. Even today, plant materials continue to play a major role in primary health care as therapeutic remedies in many developing countries [2]. To promote the proper use and to determine their potential as sources for new drugs, it is essential to study the medicinal plants. Therefore, researchers are increasingly turning their attention to folk medicine to develop better drugs against microbial infections [3].

Manilkara zapota (L.) P. Royen, rarely found in Bangladesh as sofeda, is a small to medium evergreen tree of slow growth. This plant has antioxidative property [4] and its fruit is preventive against biliousness and attacks of fever whereas seeds are diuretic [5]. The major constituents isolated from fruits of Manilkara zapota are polyphenols (methyl chlorogenate, dihydromyricetin, quercitrin, myricitrin, (+)-catechin, (-)-epicatechin, (+)-gallocatechin, and gallic acid) [6]. From perusal of literature it was found that the leaf and stem bark of Manilkara zapota had not been subjected to screening for antibacterial and antifungal properties so far. The present study was conducted to evaluate the antimicrobial activity and cytotoxic effect of ethyl acetate extract of leaves and stem bark of Manilkara zapota against microorganisms and brine shrimp nauplii (Artemia salina), respectively.

Material and Methods

Plant materials

Leaves and stem bark of Manilkara zapota (Family: Sapotaceae) were collected in the month of October, 2009 from Rajshahi district of Bangladesh. The plant material was taxonomically identified by Professor A.T.M Naderuzzaman, Department of Botany, University of Rajshahi and a voucher specimen was deposited under the accession number DACB-23801 at the Bangladesh National Herbarium.

Extraction

The collected leaves and stem bark were cleaned and shade-dried. The dried leaves and stem bark were then pulverized into a coarse powder by a grinding machine (FFC-15, China). The powdered leaves (150 gm) and stem bark (290 gm) was extracted with ethyl acetate at room temperature. These two extracts were then filtered through filter papers and filtrates were evaporated under reduced pressure at 40°C using a rotary evaporator to have 4.1 and 3.5 g ethyl acetate extracts of stem bark and leaves, respectively.

TLC Screening

Both extracts were run on pre-coated silica gel plate using the mixture of n-hexane, petroleum ether and ethyl acetate in different proportion as the mobile phase and vanillin-H2SO4 reagent was used as spray reagent. Rf values were also calculated for every spots [7].

Tests for Antimicrobial Activity

Four Gram positive (Bacillus subtilis BTCC19, Bacillus megaterium BTCC18, Bacillus cereus ATCC258 and Sarcina lutea ATCC27803), five Gram negative (Escherichia coli ATCC25922, Shigella sonnei ATTC8992, Shigella shiga ATCC27853, Shigella dysenteriae ATCC561 and Salmonella typhi ATCC14228) pathogenic bacterial strains and five fungal strains (Aspergillus flavus ACCT10558, Aspergillus fumigatus ATTC10231, Candida albicans ATTC25889, Vasianfactum sp ATTC235561, and Fusarium sp ACCT56390) were collected from the Institute of Biological Science (IBSC), University of Rajshahi, Bangladesh. The ethyl acetate extracts of leaves and stem bark of Manilkara zapota were tested separately for antibacterial activity by disc diffusion assay method [8]. Kanamycin disc (30 ìg/disc) and Fluconazole disc (100 ìg/disc) were used as positive antibacterial and antifungal control, respectively. Blank disc impregnated with the respective solvent was used as negative control. The antibacterial and antifungal activities of each extract were tested against each microorganism at concentrations of 300 ?g/disc, 600 ?g/disc and 900?g/disc. Antibacterial assay plates were incubated at 37±1°C for 24h, whereas antifungal assay plates were incubated at 37±1°C for 48h. Each experiment was carried out in triplicates and diameter of the zone of inhibition surrounding each disc was recorded. The minimum inhibitory concentration for the extracts having antimicrobial activity, were also determined by serial dilution technique [9].

Brine Shrimp Lethality Bioassay

The experiment was carried out using the method described by Meyer et al. [10]. In brief, Artemia salina Leach (brine shrimp eggs) was allowed to hatch and mature as nauplii (Larvae) in seawater for 48 h at 25°C. Serially diluted test solutions (80 ìL in DMSO from a st°Ck solution of 5 mg/mL DMSO) were added to the seawater (5 mL), containing 10 nauplii. After incubation for 24 h at 25°C, the number of survivors was counted. The LC50 (50% lethal concentration, ìg/ml) was determined from triplicate experiments using Probit analysis as described by Finney [11]. Ampicillin trihydrate was used as positive control.

Results and Discussion

Phytochemical screening of the leaves and stem bark of Manilkara zapota (L.) revealed that the plant contain terpenoids, flavonoids and glycosides (Table 1).

In antibacterial study, the efficacy of ethyl acetate extract of stem bark and leaves of Manilkara zapota (L.) to inhibit the growth of four gram (+) positive bacteria and five gram (–) negative bacteria is shown in table 2. Ethyl acetate extract of stem bark showed moderate activity against all the pathogenic bacteria used in this study and produced inhibition zone ranging from 08 to 15 mm. Ethyl acetate extract of leaves showed mild activity against only Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, Escherichia coli and Salmonella typhi with inhibition zones in the range of 06-09 mm.

Minimum inhibitory concentration (MIC) values were also evaluated against four Gram positive and four Gram negative bacteria. The lowest MIC values were 256 ìg/ml and 512 ìg/ml for ethyl acetate extract of stem bark and leaves (Table 4).

In antifungal activity test, ethyl acetate extract of stem bark produced zone of inhibition between 08 to 13 mm against Aspergillus flavus, Fusarium sp and Vasianfactum sp (Table 3). Ethyl acetate extract of leaves had no antifungal activity.

The pattern of above results indicated that ethyl acetate extract of stem bark and leaves contained antibacterial constituents at very low concentrations and so both of them showed antibacterial activity at high doses (300, 600 & 900 ?g/disc doses). The similar type of results has been reported by Kumaraswamy et al. [12] when they evaluated antimicrobial activity of Woodferdia fruticosa

In literature, it has been found that the presence of terpenoids, flavonoids and glycosides like chemicals in crude extract plays an important role for producing antimicrobial activity [13-15]. The presence of terpenes flavonoids and glycosides as predicted by TLC profile may be the cause of antimicrobial activity of ethyl acetate extract of stem bark and leaves of Manilkara zapota (L.).

Ethyl acetate extracts of both stem bark and leaves showed cytotoxicity against brine shrimp nauplii (Artemia salina) (Table 5). Among the samples, ethyl acetate extract of leaves (LC50: 16.17 ìg/ml) showed the highest toxicity, whereas ethyl acetate extract of stem bark (LC50: 50.26 ìg/ml) exhibited mild activity in comparison with ampicillin trihydrate (LC50: 12.38 ìg/ml). The mortality was not shown in the negative control experiment. Several studies have shown that brine shrimp bioassay has been an excellent method to screen the cytotoxic property of medicinal plants and for the isolation of a great variety of biologically active compounds [16]

The results of this study therefore, offer a scientific basis for isolation and purification of bioactive principles from stem bark and leaves of Manilkara zapota. Our future studies to isolate these active phytochemicals and determine their activities against microorganisms, are in progress.

Conflict of Interest

NIL

Source of Support

NONE

Tables at a glance

Table 1 Table 2 Table 3 Table 4 Table 5

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