Chemical composition , antimicrobial and antifungal activities of essential oils of the leaves of Aegle marmelos ( L . ) Correa growing in Egypt

Nabaweya A. Ibrahim, Fatma S. El-Sakhawy, Magdy M. D. Mohammed, Mohamed A. Farid, Nayera A.M. AbdelWahed, Doaa A.H. Deabes Pharmacognosy Department, Pharmaceutical and Drug Industries Research Division, National Research Center, Dokki-12311, Cairo, Egypt. Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Giza, Egypt. Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug Industries Research Division, National Research Center, Dokki12311, Cairo, Egypt.


INTRODUCTION
Aegle marmelos (L.) Correa is a tree belongs to family Rutaceae commonly known as bael; it is native to Northern India but widely found throughout Indian Peninsula and in a Ceylon, Burma, Bangladesh, Thailand and Indo-China (Rahman and Parvin, 2014).It is traditionally used for treatment of various diseases such as dysentery, fever, diabetes, asthma, heart problems, ophthalmia, haemorrhoids and urinary problems (Bansal and Bansal 2011).Moreover,thealcoholic leaves extract used as anti-ulcer (Shenoy et al., 2012), anti-diabetic (Bhatti et al., 2012),antibacterial and antifungal activities (Venkatesan et al., 2009, Kothari et al., 2011).
In addition it has antiproliferative and antioxidant activities (Chockalingam et al., 2012).The leaf extracts significantly inhibits the dermatophytic fungilike Trichophyton mentagrophytes, T. rubrum, Microsporum canis, M.gypseum and Epidermophyton floccosum (Balakumar et al., 2011).Various phytochemical constituents have been isolated from the different extracts of the leaves i.e., alkaloids, cardiac glycosides, terpenoids, saponins, tannins, flavonoids and steroids (Sekar et al., 2011),in addition to coumarins, polysaccharides, seed oil and carotenoids from different parts of the tree (Dhankhar et al., 2011).The antifungal activity of essential oil isolated from leaves was evaluated using spore germination assay.The oil exhibited variable efficacy against different fungi and the inhibition of spore germination was 100% at 500 µg/mL (Rana et al., 1997).The insecticidal, larvicidal and nematocidal activities of the oil isolated from A. marmelos leaves of Nepal sources due to the presence of high level of limonene(64.1%)(Satyal et al., 2012).Furthermore, the oil isolated from A. marmelos leaves of Cuba sources contains δ-cadinene (12.1%) and β-caryophyllene (10%)as major compounds (Pinoet al., 2005).On the other hand, the oil isolated from A. marmelosleaves growing in India composed mainly of α-phellandrene (39.2%) and limonene (26.8%) (Raju et al., 1999).The aims of the present study were to isolate the essential oil of the leaves of Aegle marmelos (L.)Correa growing in Egypt in order to identify its constituents and to evaluate its antibacterial and antifungal activities.

Plant materials
Fresh leaves of Aegle marmelos (L.) Correa were collected from El-Zohrya Garden, Giza, Egypt during flowering stage in April 2008.The plant materials were identified by Mrs. Therese Labib, consultant of taxonomy at the ministry of agriculture and the former director of El-Orman Botanical Garden, Giza, Egypt.A voucher specimen (No.00017 1Ac 04-02-05-17) of the plant was kept at the Herbarium of El-Orman Botanical Garden.

Essential oil isolation
Fresh leaves of A. marmelos (L.)Correa(300 g)were subjected to hydrodistillation method using Clevenger apparatus (Egyptian Pharmacopoeia, 2005), which was repeated three times and then the mean percentage of each yield was calculated.The obtained oil was dried over anhydrous sodium sulphate and stored at -20ºC.The essential oil was diluted (1:10 hexane, v/v), 0.2 μL of the oil was analyzed by GC-MS using the following conditions.

Analysis of the essential oil
The oil was analyzed by trace GC ultra-Gas Chromatographs (THERMO Scientific Corp., USA), coupled with a THERMO mass spectrometer detector (ISQ Single Quadrupole Mass spectrometer).The capillary column of fused silica (30m x 0.25mm ID x0.25μm (film thickness) was used.Analyses were carried out using helium as carrier gas at a flow rate of 1.0 mL/min at a split ratio of 1:10 and the following temperature program:50ºC for 1 min; rising at 5.0ºC/min to 180ºC and held for 6 min; rising at 5ºC/min to 250ºC and held for 1 min the injector and detector were held at 250ºC and 240ºC, respectively.Mass spectra were obtained by electron ionization (EI) at 70 eV using a spectral range of 40-450m/z.The identification of the components was based on comparison of their mass spectral fragmentations pattern with those of the data reported in Wiley and NISTLibraries and those described by (Adams, 2001).

Microorganisms
The bacteria used in this study were Gram-positive bacterial strains i.e., Bacillus subtilis NRRL 543, Staphylococcus aureus NRRL B-313,Sarcina lutae NRRL B-1018,Streptococcus faecalis NRRL 537and Arthrobacter citreus NRRL B-1258,Gramnegative bacterial strains i.e., Escherichia coli NRRL B-210, Klebsiella pneumonia NRRL B-117and Pseudomonas aeruginosa NRRL B-23.Fungi i.e., Aspergillus niger NRRL 599, Fusarium oxysporum NRRL 28184 and Candida albicans NRRL Y-477.These micro-organisms obtained from Northern Utilization Research and Development Division, United State Department of Agriculture, Peoria, Illinois, USA.The bacterial strains were revived for bioassay by sub-culturing in fresh nutrient broth medium for 24 hours before test.While fungi were cultured on potato dextrose agar (PDA) (2.5% w/v agar) for 7 days at 28°C before the experiment was carried out.

Agar diffusion method
The antibacterial and antifungal activities of the essential oil were tested using agar diffusion method (Linday, 1962).0.1 mL suspension of 24 hours bacterial cultures in sterile distilled water was added to 40 mL sterile nutrient agar media.For fungal strains, 0.1 mL suspension of seven days cultures was added to 40 mL sterile PDA (potato dextrose agar) at 45°C.The mixture was transferred to sterile Petri dishes and allowed to solidify.Holes of 9 mm in diameter were made using a cork borer.Amounts of 0.1mL of the diluted essential oil with paraffin oil were poured inside the holes.A hole filled with paraffin oil only was also used as control.
The plates were left for 1 hour at room temperature as a period of pre-incubation diffusion.The diameters of the inhibition zone were measured and compared with that of the standard and the values were tabulated.Plate cultures were kept in an incubator at 28 o C for 48 hours for fungi and at 37 o C for 24 hours for bacteria.
Erythromycin, methicillin, oxacillin, bacitracin and nystatin were included during the experiment as references antibiotics.Following incubation, the zone of inhibition for each sample was recorded in mm (including the hole).Microorganisms with zone size ≥ 28 mm were classified as strongly sensitive, with a zone diameter of < 28 to 16 mm as moderately sensitive, with a zone diameter of < 16to 12 mm as weakly sensitive and isolates with zone diameter of < 12 mm as resistant (Elgayyar et al., 2001).Plates were done in triplicate and an average + SD was recorded (Table 2).

Estimation of Minimal Inhibitory Concentration
The minimal inhibitory concentration (MIC) of the oil was determined by broth dilution method previously established by (Carson et al., 1995) in test tubes containing 5mLnutrient broth.Serial two fold dilutions of stock solution of essential oil were prepared over the range of1.0-200µL/mL.A 50µL spore suspension of each test strain was inoculated in the test tubes and incubated for 24-72 hours at 37°C.The control tube containing the same medium was inoculated only with microorganism's suspension without essential oil.The minimum inhibitory concentrations at which no visible growth observed (100% inhibition) were defined as the MICs which were expressed in µL/mL (Table 3) all dilutions were performed in duplicate.

Spore germination assay
Spore germination assay was carried out according to (Rana et al., 1997).Three concentrations of the essential oil (4, 2, 1 % v/v) and controls (one sterile distilled water and other 0.1 % (v/v) methanol in sterile distilled water) were tested for spore germination of Aspergillus niger and Fusarium oxysporum.Aliquots of 0.1 mL from each sample were mixed with fungal spores obtained from 10 days cultures of the fungi and placed on separate glass slides in triplicate.Slides containing the spores were incubated in a moist chamber at 28 °C for 24 hours.Each slide was then fixed in lactophenol-cotton blue and observed under the microscope for spore germination (Table 4)a spore was considered germinated when the germ tube length was 1.5 times the spore diameter (Plascencia-Jatomea et al., 2003).
On the other hand, our obtained results were different from the leaves oil isolated from Cuba sources, which constituted mainly of sesquiterpenes i.e., δ-cadinene (12.1%) and βcaryophyllene (10%)as reported by (Pinoet al., 2005).Satyal et al.,(2012) reported that, the essential oil isolated from Nepalsources mainly contain slimonene (64.1%).The qualitative and quantitative variations between our results and the previous reports for the constituents of the leaves oil may be attributed to the difference in geographical location, climate conditions and time of harvest.Which comes in accordance with Verma et al., (2014) who reported that the growing location and time of harvest (season) had a close relation to the yield and quality of A. marmelos essential oil.
The lowest minimal inhibitory concentration (MIC) value (Table 3) was against the most susceptible species of A. niger and C. albicans marked 50µL/mL, this was followed by E. coli and B. subtilis with 100 µL/mL and 150 µL/mL respectively.These values suggested that the essential oil possessed bacteriostatic and bactericidal activities and demonstrated great potential as an antifungal compound with potent in-vitro fungicidal activity against C. albicans, an opportunistic pathogen responsible for both superficial and systemic mycoses.Furthermore, the essentiall oil of A. marmelos (L.)Correa leaves (Table 4) demonstrated a significant inhibition of A. niger at concentration (4, 2, 1 % v/v) and F. oxysporum spores at concentrations (4, 2 % v/v) with no response at concentration 1% (v/v).Previous studies reported the antifungal activity of essential oil isolated from the leaves of Aegle marmelos (L.) Correa, with inhibition of the growth of dermatophytes and Fusarium species at concentration of 500 µg/mL (Rana et al., 1997).Our obtained results for the antifungal activity of the oil come in accordance with Balakumar et al., (2011) who reported that the essential oil of A. marmelos (L.) Correa leaves have potent antifungal activity against the clinical isolates of dermatophytes.The mode of action of the essential oil as antimicrobial agents may be due to inhibition of respiration and disrupting the permeability barriers of the cell membrane structures (Cox etal.,2000).Furthermore, İşcan et al., (2012) reported that αphellandrene is the responsible for the antimicrobial activity, while the antibacterial and fungicidal activities Wedelia prostrata was mainly attributed to α-pinene.

CONCLUSION
The biological activity of the essential oil isolated from the leaves of Aegle marmelos (L.)Correa was due to the synergism between the total components of the oil and mainly attributed to the presence of α-phellandrene and α-pinene as major constituents.The broad spectrum antimicrobial and antifungal activities of the oils against variety of pathogenic fungi and bacteria, can recommend its incorporation in different pharmaceutical preparations.

Table 1 :
Chemical compositions of the essential oil of A. marmelos (L.) Correa leaves.

Table 2 :
Antimicrobial activities of the essential oil from A. marmelos(L.)Correa.

Table 3 :
MICs of the essential oil of A. marmelos (L.) Correa leaves.

Table . 4
:Effect of essential oil from A. marmelos (L.)Correa leaves on spore germination of A. niger and F. oxysporum.