Antimbacterial activity of mangosteen hull extract against Listeria monocytogenes

Pussadee Tangwatcharin +, Nutwara Meannui and Arunrat Onyen

Faculty of Technology and Community Development, Thaksin University, Phatthalung Campus, Phatthalung, 93110, Thailand

Abstract. The objective of this study was to investigate the in vitro activities of crude methylene chloride (CH2Cl2) extract and α–mangostin from hulls of mangosteen (Garcinia mangostana L.) against Listeria monocytogenes TU1, isolated from pig carcasses, by determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill method and transmission electron microscopy. The result was shown that the crude CH2Cl2 extract at > 0.53 mg/disc exhibited ability of inhibition of L. monocytogenes TU1. Furthermore, MIC and MBC of crude CH2Cl2 extract were 17.00 and 136.00 mg/ml, respectively. In time-kill studies, MBC of the crude CH2Cl2 extract produced a bactericidal effect. This resulted in a loss and change of the cytoplasm and membrane cells of the bacterium.

Keywords: crude mangosteen extract, α–mangostin, Listeria monocytogenes

1. Introduction Listeria monocytogenes has caused considerable concern for the food industry, health regulatory officials,

and consumers. Center for Disease Control and Prevention estimates that there are 2500 cases of listeriosis annually, which lead to approximately 500 deaths [1]. L. monocytogenes is a concern to the meat industry because the organism can grow at refrigerated temperatures, persist for long periods of time in food processing environments, and is found in relatively high incidence (6.7%) in sliced vacuum packaged ready-to-eat (RTE) meat and poultry products [2]. Hence, new prototype antimicrobial agents are needed to address this situation. The antimicrobial compound from plants may inhibit microbial growth by different mechanism than those presently used antimimicrobial agents and may have significant clinical value in treatment of resistant microbe [3].

Garcinia mangostana L. of the family Clusiaceae (Guttiferae) is a tree found in Thailand and other South East Asian countries. Treatments of diarrhea, dysentery, skin infections and as an anti-inflammatory agent are some of the medicinal application of this plant. Xanthones, terpenoids and sugars have been reported from the fruit hulls and leaves of G. mangostana, and some of them have shown a variety of biological activities [4, 5, 6].

This encouraged us to evaluate G. mangostana as a source of antimicrobial agent against L. monocytogenes TU1, base on their methylene chloride (CH2Cl2) use.

2. Materials and Methods

2.1. Bacterial strain Listeria monocytogenes TU1 was previously isolated from pig carcasses in Southern Thailand abattoirs

by the standard procedure [7] and its identity was confirmed by the Department of Medical Sciences,

+ Corresponding author. Tel.: + 1100-66-74-693996; fax: + 1100-66-74-693996.

E-mail address: putang3009@hotmail.com.

2012 4th International Conference on Bioinformatics and Biomedical Technology IPCBEE vol.29 (2012) © (2012) IACSIT Press, Singapore

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Ministry of Public Health of Thailand. These organisms were maintained on Mueller Hinton agar (MHA) (Merck, Germany). The overnight cultures were prepared by inoculating approximately 2 ml Mueller Hinton broth (MHB) (Merck, Germany) with 2-3 colonies taken from MHA. Broths were incubated overnight at 35oC. Inocula were prepared by diluting overnight culture in saline to 108 CFU/ml (McFarland standard of 0.5). These suspensions were further diluted with saline as required. The initial concentration of approximately 5x105 CFU/ml was adopted for susceptibility test, synergy and kill-time methods.

2.2. Antimicrobial agents Hulls of mangosteen were collected from various areas in Chumporn province in 2009. Crude extract

and purified α-mangostin were prepared at Department of Community Medicine and Family Medicine, Faculty of Medicine, Thammasat University, Pathum Thani province. Briefly, dried and ground hulls were macerated in hexane for 24 h. The resulting hull was subsequently macerated in CH2Cl2 for 24 h. The CH2Cl2 extract was then recrystallized, and ground into powder.

To obtain α-mangostin, the crude extract was chromatographed on a silica gel column and eluted with increasing percentages of CH2Cl2 in hexane (0-30%). A hexane-CH2Cl2 (4:1) eluate was selected based on the thin layer chromatography profile. The selected fraction was further identified as α–mangostin by using high performance liquid chromatographic (HPLC).

2.3. Susceptibility test methods Susceptibility tests were performed by the disc diffusion method of Bauer et al. [8] with MHA. The

crude extract was dissolved in sterile solution of 20% (v/v) dimethyl sulfoxide (DMSO, Merck, Germany) in water and subsequent two-fold serial dilutions were performed in the culture medium. DMSO was used as a negative control. Final concentrations of the test samples on disc ranged from 0-136 for crude extract. Zones of inhibition were measured after 18 h of incubation at 35oC.

The minimal inhibitory concentration (MIC) was determined by a broth microdilution method [9] for each isolate. Serial two-fold dilution of the test substances were mixed with MHB in microtiter plates. Final concentrations of the test samples in broth ranged similarly in disc diffusion method. MIC was recorded as the lowest concentration that limited the turbidity of the broth to < 0.05 at absorbance of 600 nm by UVM 340 Microplate Reader (Biochrom Ltd., Cambridge, UK).

The minimal bactericidal concentration (MBC) was determined by comparing the number of remaining viable bacteria with the initial number of bacteria. All wells from the MIC experiments that showed no visible turbidity were spread onto MHA plates for viable cell counting. MBC was then recorded as the lowest concentration that killed at least 99.99% of the initial number of bacteria. All MIC and MBC experiments were repeated three times.

2.4. Kill-time methods The effect of the crude extract (MBC and 2MBC) on the cell viability of L. monocytogenes TU1 was

evaluated by the viable cell count procedure. For this, 8 ml of MHB was inoculated with 1 ml of the bacterial inoculum and 1 ml of extract solutions and incubated at 35oC [10]. At different time intervals (0, 5, 10, 15, 25 and 30 min), cells capable of growth on solid selective media were enumerated using spread plate count on Listeria selective agar (Oxoid, UK) to which Listeria selective supplement (Oxoid, UK) and MHA to determine the culturable and total culturable population, respectively. The cell numbers (CFU) were determined following incubation at 35oC for 48 h. The culturable and total culturable populations were determined by plating on Listeria selective agar and MHA, respectively. The densities of stressed and non-stressed cell sub-populations were determined using the assumptions and calculations described in Table 1.

Table 1. Scheme of determination of sub-population density [11, 12]

Sub-population Calculation criteria Stressed cells = Total culturable cellsa – culturable cellsb Non-stressed cells = Culturable cells

a Determined by plating on MHA 192

b Determined by plating on Listeria selective agar

2.5. Transmission Electron Microscopy (TEM) TEM was performed using a modification of the methods described by Chaveerach et al. [13], Zakaria et

al. [14] and Tangwatcharin et al. [12]. L. monocytogenes TU1 samples for TEM were centrifuged at 16,000 × g for 5 min and the supernatant was diskarded. The pellets were washed 3 times with Sorensen’s phosphate buffer (SPB). The pellets were subsequently fixed in 2.5% glutaraldehyde in SPB for 1-2 h and kept at 4oC, followed by 1% osmium in SPB for 1-2 h. The sample was washed 3 times with SPB between fixatives. The pellets were dehydrated by passage through a graded ethanol series [3 × 5 min each at 50, 70, 80, 90 and 95 and 2 × 10 min at 100% (v/v)] and then stored overnight. Ethanol was replaced with propylene oxide, which was gradually replaced with Spurr’s resin (Polysciences, Warrington, PA). Following polymerization, specimen blocks were thin sectioned (70 - 90 nm). Sections were stained with 5% uranyl acetate and 10% lead citrate for examination with a JEM-2010 transmission electron microscope (JEOL Ltd., USA) operated at 160 kV.

2.6. Statistical analysis Data were presented as means and standard deviations. All statistical computations were performed to

determine significant differences (P < 0.05) by ANOVA followed by Duncan’s new multiple range test [15].

3. Results and Discussion

3.1. Susceptibility test

The results of antibacterial activity of crude CH2Cl2 extract tested by disc diffusion method against L. monocytogenes TU1 is given in Fig. 1. The crude extract exhibited a favorable activity against the bacteria tested. The blind control (20% (v/v) DMSO) did not inhibit strain. It was inhibited at > 0.53 mg/disc of crude extract. This may be due to the yield of alpha-mangostin content about 316.40+32.36 μg/mg of dry matter of extract (31.64%) from dried inner mangosteen hull extraction with CH2Cl2. Mahabusarakam et al. [16] reported that the order of the efficacy determined by the MIC was found to be α–mangostin > γ-mangostin > 1-isomangostin > 3-isomangostin > gartanin against S. aureus. Furthermore, MIC and MBC of antibacterial against strain were 8.50 and 17.00 mg/ml, which MBC was two-fold higher than the corresponding MIC. Similarly, eariler study found MIC and MBC of crude ethanolic extract against L. monocytogenes in a range of 0.05-50.00 and 0.10-100 mg/ml, respectively [17, 18]. Canillac and Mourey [19] reported that if the MBC/MIC ratio was found to be less than or equal to the bacteria was considered to be susceptible; on the other hand, if this ratio was greater than 4, it was considered to be tolerant.

Fig. 1: Antibacterial activity (zone of inhibition) of crude CH2Cl2 extract against L. monocytogenes TU1. The results are presented as means of three independent experiments and standard deviations (bar). a-d Different letters indicate that

values are significantly different (P < 0.05).

3.2. Kill-time methods

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To determine the rates at which bacteria were killed, L. monocytogenes was exposed to crude CH2Cl2 extract in MHB is given in Fig. 2. Addition of MBC crude extracts (17.00 mg/ml) to broth caused a sharp drop in the total and non-stressed bacterial counts after 10 min and under one log cycle were maintained in the remainder evaluated times. In case of non-stressed cells can be induced to mostly change to stressed cells (P < 0.05). The crude extract completely killed the bacteria within 20 min at MBC concentration. This result might shows that antimicrobial effect depends on time. Similarly, the bacteriostatic and bactericidal effects of mangosteen extract against S. mutans at 2MBC (2.5 μg/ml) required at least 90 and 120 min, respectively, for the exposure time [20].

Fig. 2: Survivors curves for total culturable, non-stressed and stressed cells of L. monocytogenes TU1 in MHB at 35oC

as a function of antibacterial concentration at 17.00 mg/ml.

3.3. Transmission Electron Microscopy (TEM)

Cells treated with 17.00 mg/ml of crude CH2Cl2 extract underwent considerable morphologic alterations in comparison with the control when studied by TEM (Fig. 3). Untreated cells (control) appear as rod and smooth (Fig. 3A). For treated cells, there was some loss and change of the membrane and cytoplasm in cells of the bacterium following exposure to crude CH2Cl2 extract (Fig. 3B).

Fig. 3: Transmission electron micrographs of L. monocytogenes TU1 in MHB containing antimicrobials: (A) control, (B) 17.00 mg/ml of crude CH2Cl2 extract at 35oC for 20 min. Membrane cells were disturbed and leaked (solid arrow) and

subsided (hatched arrow). Bars = 0.2 μm.

4. Acknowledgements

This study was supported by grants from the General Research Fund of Thaksin University in 2011. The authors would like to thank the Thai Government for support through the General Research Fund of Thaksin University. The authors also thanks Assoc. Prof. Dr. Arunporn Itrat for crude CH2Cl2 extract.

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B) A)

0.2µm 0.2µm

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