Antimicrobial activity of ethyl acetate extract of Citrullus lanatus seeds

Purpose: To determine the antimicrobial activity and chemical constituents of ethyl acetate extract of Citrullus lanatus seeds. Methods: Antimicrobial activity of the ethyl acetate extract of the seeds of C. lanatus was evaluated against Staphylococus aureus ATCC 25923, Escherichia coli ATCC 25922, Bacillus subtilis NCTC8236, Pseudomonas aeruginosa ATCC 10145 and Candida albicans ATCC 24433 using standard microbiological method. Chemical constituents of the extract were determined with Gas chromatography and mass spectrometry (GC MS) systems. Results: An oily, yellowish orange coloured extract with a yield of 18.7 % was obtained. The extract inhibited the growth of all test microorganisms at minimum inhibitory concentration range of 0.313 -2.5 mg/mL and minimum bactericidal concentration of 0.313-5 mg/mL. GC MS profile of the extract showed the presence of oleic acid (31.22 %), saturated fatty acid (23.85 %) and gamma tocopherol (8.79 %). Other organic compounds accounted for 36.16 % of the extract Conclusion: Ethyl acetate extract of the seeds of Citrullus lanatus has antimicrobial effect against some Gram positive and Gram negative bacteria as well as C. albicans. Twenty-one compounds including fatty acids, hexamethyl-2-ethylacridine, gamma tocopherol and methylphthalate have been identified in the seeds.


INTRODUCTION
Plant derived substances have become of great interest owing to their versatile application [1].It has been estimated that 14.28 % of higher plant species are used medicinally and that 74 % of pharmacologically active plant derived components were discovered after following up on ethnomedicinal use of the plants [2].
Citrullus lanatus is a plant species that belongs to the family, Cucurbitaceae.It is a vine-like (scrambler and trailer) flowering plant which originated from West Africa and often cultivated for its fruits.This species has two cultivars namely, watermelons (C.lanatus (Thunb.)var lanatus) and citron melons (C.lanatus var citroides (LH Bailey) Mansf), In Chinese medicine, the rind of C. lanatus fruit is used for the treatment of diabetes, hypertension and acne [3].Water melon red pulp has been extensively studied for its antioxidant and cancer preventing properties [4].The seeds are crushed and made into an emulsion with water for treatment of catarrhal infections, bowel disorders and as worm expeller [5].Although different solvent extracts of the leaves, roots, flower, fruit pulp and stems of this plant have been evaluated for useful phytochemicals around the world, the few reports that exist on the seeds as a potential new source of antimicrobial agent in Nigeria were preliminary and unstandardized methods for evaluation of antimicrobial susceptibility pattern of crude extracts of other variates of C. lanatus seeds were used [6][7][8][9].
This study is aimed at determining the antimicrobial property and chemical components of the ethyl acetate extract of C.lanatus var.lanatus seeds using the standard agar well diffusion and agar dilution methods as well as gas chromatography and mass spectrometry methods.

EXPERIMENTAL
C. lanatus fruits was purchased from a local market (Tamboga market) in Benin City and identified by Prof JO Bamidele of the Department of Plant Biology and Biotechnology, University of Benin, Benin City.A voucher specimen was deposited in the local Departmental herbarium.The seeds were air dried for five days and 1 kg was pulverized and macerated in ethyl acetate for 72 hr.Resultant extract was filtered with Whatmann filter paper no-1 and the filtrate was concentrated, weighed and stored at 4 o C in an airtight container.

Antimicrobial susceptibility tests
Staphylococus aureus, Escherichia coli, Bacillus stubtilis and Pseudomonas aeruginosa were subcultured from stock into sterile nutrient agar plates while or Candida albicans was subcultured from stock into sterile sabouroud dextrose agar plates.The plates with bacteria were then incubated for 48 hr at 37 o C while those with fungus were incubated for 48 hr at 28 o C. Identical colonies from overnight plates were suspended in sterile broth for 12 h and adjusted to 0.5 McFarland standard to give an inoculum size of approximately 10 8 cfu/mL.Adjusted inocula were diluted 1:100 to give inoculum size of 10 6 cfu/mL as previously reported [10].
Antimicrobial susceptibility test was carried out using modified agar well diffusion method [11].Standardized inoculum of each microorganism (200µL) was mixed thoroughly with 30 mL of sterile Mueller Hintons agar (at 45 -50 o C) for bacteria or sabouroud dextroxse agar for fungus and poured into sterile plates to set.A cork borer (10 mm) was used to bore six wells in each agar plate and the agar in the well was removed, and each of the sealed wells was filled with 200 µL (containing 100mg) ethyl acetate extract.A similar procedure was carried out with ciprofloxacin (0.5 mg/mL) and ketoconazole (1 mg/mL) standard drugs.Exactly 200 µL of each standard drug was emptied into each well to give 100 µg and 200 µg per well for ciprofloxacin and ketoconazole respectively.Negative controls (without extract and standard drugs) and positive control (viability test for microorganisms used) were carried out for each set of experiment.All seeded plates remained on the bench for 30 min, before incubation in an upright position for 18-24 hr at 37 0 C for bacterial and 25-28 o C for 48 h in the case of C. albicans.Inhibition zone diameters (IZD) were measured in millimetres (mm).All experiments were carried out in triplicates.

Determination of minimum inhibitory concentration (MIC)
Agar dilution method [12] was used for determination of MIC of the extract using ciprofloxacin and ketoconazole on susceptible test microorganisms (S. aureus ATCC 25923, E. coli ATCC 25922, B. subtilis NCTC8236, P. aeruginosa ATCC 10145 and C. albicans ATCC 24433.Mueller Hintons agar was prepared according to manufacturer's instructions and placed in water bath at 50 o C. Stock solution of extract was incorporated into the molten agar at different volumes to obtain a concentration range of 3.125 -400 mg/ mL.Serial dilution of the stock solution of ciprofloxacin (0.5 mg/mL) was also carried out to obtain a concentration range of 0.01562 -5 µg/mL.Similarly a concentration range of 0.2 -200 µg/mL was prepared from a stock solution of ketoconazole (1 mg/mL).Agar extract and agar ciprofloxacin/ketoconazole mixtures of different concentrations were poured into separate sterile plates and allowed to set.Each of the test microorganisms was radially streaked onto the prepared plates.Negative and positive controls were set up for each set of experiments.All plates were incubated at 30 o C for 18-24 h except for C. albicans which was incubated at 25 -28 o C for 48 h.Experiments were carried out in triplicates and the MICs were defined as the lowest concentrations of extract and standard drugs that inhibited growth of test microorganisms.

Determination of minimum bactericidal (MBC) and minimum fungicidal concentration (MFC)
Plates that showed no visible microbial growth following MIC determination were swabbed with moistened sterile sticks and inoculated onto sterile Mueller Hinton agar plates containing 5 % Tween 80 [13].All plates were incubated for 18 -24 h at 37 o C except C. albicans which was incubated for 48 h at 25 -28 o C. Agar plates with least concentration without visible growth were considered as MBC/MFC values.

GC-MS analysis
Determination of the chemical constituents was carried out on Agilent technologies 6890N network GC system and Agilent technology 5973 network mass selective detector coupled with 7683B series injector.Model number of the column and capillary specifications used were; Agilent 122-5533, stationary phase (5% phenylmethylsiloxane) internal diameter (0.25 mm x 30 m), and film thickness (1um).Helium at a flow rate of 1.2 mL/min was used as carrier gas.An initial inlet temperature of 230 o Cwas programmed at 50 o C for 5minutes and then increased to 300 o C at a rate of 10 o C/min ending with 25 min for a total run time of 45 min.An electron ionization mode of 70 eV was used.Total ion count was used for compound identification and quantification.The peaks were identified using Phero base software which compared the relative peaks with standard data base of known compounds saved in National Institute of Standards and Technologies (NIST02) reference spectra library.

Data analysis
Inhibition zone diameters for antimicrobial assay were expressed as mean ± SEM using Minitab 13.One way ANOVA was used to compare activity of extract with standard drugs.Data analysis and peak area measurementof raw GC-MS data was carried out using Agilent Chemstation and Phero base software.

RESULTS
In this study all the test microorganisms were susceptible to inhibitory effect of the ethyl acetate extract of C. lanatus seeds (Table 1).The pattern of MIC values (Table 2) for E. coli, B. subtilis and C. albicans were observed to be similar to those of MBC/MFC (Table 3).GC MS chromatogram showed several peaks with their corresponding retention time (Figure 1) and on comparison of this mass spectral with NIST02 reference spectral library, 21 phytocompounds were identified (Table 4).The predominant compounds identified with known antimicrobial properties were fatty acids (Palmitic, Lauroleic and oleic), hexamethyl-2-ethylacridine, dibutyl phthalate, and vitamin E.

CONCLUSION
The findings of this study demonstrate the antimicrobial action of the ethyl acetate extract of C. lanatus seeds against some bacteria and fungi.Twenty one chemical compounds including fatty acids, hexamethyl-2-ethyl acridine, gamma tocopherol and methyl phthalate have been identified in the seeds.This study offers a scientific proof for the traditional use of C. lanatus seed extract in the treatment of infections, having been shown to be active against some bacteria and fungi associated with respiratory, gastrointestinal and urinary tract infections.

Acknowledgement
The corresponding author is grateful for the access to Professor Abiodun's TETFUND and STEP-B laboratory facilities where all the physicochemical studies on the extract were carried out. Trop

Figure 1 :
Figure 1: GC MS chromatogram of ethyl acetate fraction of C. lanatus extract

Table 1 :
Inhibition zone diameters of extracts against test microorganisms

Table 2 :
Minimum inhibitory concentrations of extract and standard drugs against the test microorganisms

Table 3 :
Minimum bactericidal/fungicidal concentration (mbc/mfc) of extract and standard drugs against the test microorganisms

Table 4 :
Chemical composition of ethyl acetate extract of Citrullus lanatus seeds

Peak no. Retention time (min) Area (%) Name of compound
Antibacterial and antifungal activities of fatty acid methyl esters of Blind-your eye mangrove from Indian.Brazilian J. Microbiol 2007;38:739-742.16.Al-Bari MM, Sayeed Ma, Rahman MS and Mossadik MA.