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Chemical composition and antimicrobial activities of the essential oil from theseeds of Enterolobium contortisiliquum (leguminosae)Abdelaaty A. Shahat a; Gamal El-Barouty b; Rasmeia A. Hassan a; Fiza M. Hammouda a; Fawzia H. Abdel-Rahman c; Mahmoud A. Saleh c

a Phytochemistry Department, National Research Center, Cairo, Egypt b Faculty of Agriculture, University ofCairo, Cairo, Egypt c Department of Chemistry, Texas Southern University, Houston, Texas, USA

Online Publication Date: 01 August 2008

To cite this Article Shahat, Abdelaaty A., El-Barouty, Gamal, Hassan, Rasmeia A., Hammouda, Fiza M., Abdel-Rahman, Fawzia H.and Saleh, Mahmoud A.(2008)'Chemical composition and antimicrobial activities of the essential oil from the seeds of Enterolobiumcontortisiliquum (leguminosae)',Journal of Environmental Science and Health, Part B,43:6,519 — 525

To link to this Article: DOI: 10.1080/03601230802174714

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Journal of Environmental Science and Health Part B (2008) 43, 519–525Copyright C© Taylor & Francis Group, LLCISSN: 0360-1234 (Print); 1532-4109 (Online)DOI: 10.1080/03601230802174714

Chemical composition and antimicrobial activities of theessential oil from the seeds of Enterolobium contortisiliquum(leguminosae)

ABDELAATY A. SHAHAT1, GAMAL EL-BAROUTY2, RASMEIA A. HASSAN1, FIZA M. HAMMOUDA1,FAWZIA H. ABDEL-RAHMAN3 and MAHMOUD A. SALEH3

1Phytochemistry Department, National Research Center, Cairo, Egypt2Faculty of Agriculture, University of Cairo, Cairo, Egypt3Department of Chemistry, Texas Southern University, Houston, Texas, USA

Seeds of Enterolobium contortisiliquum were subjected to steam distillation to obtain a light yellow essential oil in a yield of 3 ml/kgof seeds. The major components of the oil were identified using gas chromatography/mass spectrometry (GC-MS) and were furfural,limonene, linalool, estragole, carvone, and apiole with carvone representing more than 50% of the total composition. Antimicrobialactivities of the essential oil were determined against four species of gram positive bacteria (Bacillus subtilis, Bacillus cereus, Staphy-lococcus aureus, Micrococcus luteus) and two gram negative bacteria (Klebsiella pneumoniae, Serratia Marcescencs). The essential oilinhibited the growth of all tested bacteria but was most effective against the gram positive bacteria. Chemicals that are responsiblefor the antibacterial effect of the essential oil were determined using the bio-autography thin layer chromatography (TLC) technique.The active compounds responsible for the activity were found to be carvone and estragole.

Keywords: Antibacterial activity; volatile compounds; terpenes; bioautography; GCMS.

Introduction

Essential oils and their chemical conistuents are widelyused in the manufacturing of medicinal products, cosmeticsfragrances and as food flavoring additives. Many essentialoils are known to exert antimicrobial activity; however, themechanism of action is not fully understood.[1] Pathogenicbacteria cause an estimated 6.5–33 million cases of humanillness and up to 9,000 deaths in the United States eachyear. The costs of human illness are estimated to be $9.3–$12.9 billion annually.[2] Losses from plant diseases causedby bacteria also can have a significant economic impact.Many pathogenic micro-organisms and plant pathogens arecontinuously developing resistance to commercial chemicalantibiotics, which may seriously hinder the managementof diseases and agricultural products. Therefore, there isa continuous need for alternative agents for the manage-ment of pathogenic micro-organisms. Green plants repre-sent a reservoir of effective chemotherapeutic agents and

Address correspondence to Mahmoud A. Saleh, Department ofChemistry, Texas Southern University, Houston, Texas 77004,USA; E-mail: saleh ma@tsu.eduReceived February 20, 2006.

provide valuable sources of naturally occurring bioactivecompounds either as drugs, antibiotics or pesticides.[3]

Enterolobium contortisiliquum (Vell.) Morong (Legumi-nosae) is a tree belonging to the Mimosoideae subfamilyand widely populated in Egypt. It is more than 20 m highwith black ear-shaped fruits and brownish colored seeds(Figure 1). Seeds have been thoroughly studied by chemicalanalysis for their proteins, carbohydrates and fats; however,nothing can be found in the literature about the essentialoil of the seeds.[4−7]

The objective of this study is to identify naturally occur-ring antimicrobial compounds against common pathogenicand food spoilage bacteria to utilize them as a novelleads for safer and environmentally friendly antimicrobialagents.

Materials and methods

Plant material and preparation of the essential oil

Seeds of Enterolobium contortisiliquum were collected dur-ing the month of April 2007 from trees grown at HelwanPark, 25 km south of Cairo, Egypt. The plant was identi-fied by Professor Ibrahim El-Garf, Department of Botany,

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520 Shahat et al.

Fig. 1. Seeds and fruit of Enterolobium contortisiliquum.

University of Cairo. The seeds were crushed and subjectedto hydrodistillation in a Clevenger-type apparatus.[8] Theessential oil was dried over anhydrous sodium sulfate re-sulting in pale yellow oil with a yield of 3 ml oil/kg of driedseeds.

Gas chromatography/mass spectrometry (GC/MS)analysis

GC/MS analysis of the essential oil was carried out usingan HP5890 Series II Gas Chromatograph, HP 5972 MassSelective Detector and Agilent 6890 Series Autosampler(Agilent Technologies, USA). A Supelco MDN-5S 30 m by0.25 mm capillary column with a 0.5 µm film thickness wasused with helium as the carrier gas at a flow rate of 1.0 ml/min. The GC oven temperature was programmed at an ini-tial temperature of 40◦C for 5 minutes, then heated up to140◦C at 5◦C /min and held at 140◦C for 5 min, then heatedto 280◦C at 9◦C/min and held for 5 additional minutes. In-jector and detector temperatures were set at 250◦C. Massspectrometry was run in the electron impact mode (EI) at70 eV. The identification of the chemical constituents of theoil was determined by their GC retention times, interpreta-tion of their mass spectra and confirmed by mass spectrallibrary search using the National Institute of Standards andTechnology (NIST) database.

Bioassay for antibacterial activity

Preparation of bacterial culturesSix different species of bacteria were purchased from theFisher-Co (Texas, USA). Four species of gram positive bac-

teria (Bacillus subtilis, Bacillus cereus, Staphylococcus au-reus, and Micrococcus luteus) and two gram negative bac-teria(Klebsiella pneumoniae andSerratia marcescencs) wereused for the antimicrobial assay. Bacteria were sub culturedon nutrient agar at 37◦C before being grown in nutrientbroth overnight. All overnight cultures were standardizedby matching to the McFarland 0.5 turbidity standard usingsterile saline to produce approximately 1.5 × 108 colonyforming units (cfu) per mL.

Disc diffusion bioassayThe antibacterial activity of the essential oil was carried outby the disc diffusion assay as described by Gulluce et al.[9]

Muller Hinton agar (MHA) plates were swabbed with therespective broth cultures of the organisms (diluted to 0.5McFarland Standard with saline). Sterile 6 millimeters di-ameter filter paper discs were impregnated with the appro-priate equivalent amount of the essential oil dissolved insterile dimethylsulfoxide (DMSO) at a concentration of 5mg/ml. Negative controls were prepared using the samesolvents employed to dissolve the essential oil. Chloram-phenicol (Sigma Co) was used as positive reference standardto determine the sensitivity of one strain in each bacterialspecies tested. Plates were incubated overnight at 37◦C. Theantimicrobial activity was evaluated by measuring the inhi-bition zones expressed as millimeters of inhibition againstthe tested organism. The assays were performed in trip-licate. The minimal inhibitory concentration (MIC) testswere determined according to Andrew,[10] the least concen-tration showing a clear zone of inhibition was consideredas the MIC.

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Antimicrobial activities of leguminosae seed oil 521

Thin layer chromatography (TLC) and bioautography

A series of three TLC plates were used in each experi-ment, three spots of 4 µL each of the neat essential oilwere applied on each plate using 10 × 20 cm GF 254plate (Merck, Darmstadt, Germany). The spots were sep-arated by at least 3 cm. The plates were then developedwith toluene- ethyl acetate (95: 5, v/v) and dried for com-plete removal of solvents. The dried TLC plates were thencut with a diamond knife into three strips each one rep-resenting one applied spot. One of the strips was sprayedwith vanillin/sulfuric acid mixture for spot visualizationand the second was used for the TLC bioautography, whilethe third strip was used for elution of the active constituent.TLC bioautography was carried out using the previouslyidentified four bacterial strains (B. subtilis, S.aureus, M.luteus, K. pneumoniae), which showed the highest effec-tiveness in the Disk Diffusion assay. Suspensions of thebacteria in Difco-nutrient broth containing 0.5% agar and0.1% iodonitrotetrazolum chloride media were individuallydistributed over the TLC plates (second strip); the plateswere then incubated at 37◦C for 48 h. Inhibition zones wereshown as clear areas against a pink-colored background.The third strip of the TLC plates was used for eluting theregion of the plate that showed the inhibition zones foridentifying the chemical composition of the active com-pound(s). TLC zones that showed inhibition activities werescraped from the plates, and eluted with dichloromethaneand filtrated followed by application of a nitrogen streamto concentrate to a final volume of 100 µL and analyzed byGCMS.

Results and discussion

GC/MS analysis

The light yellow essential oils of Enterolobium contor-tisiliquum were analyzed by GC-MS as shown in the ex-perimental section. Chemicals were identified by their GCretention times (Kovats retention indices), by interpretationof their mass spectra, and by matching of the mass spec-tra against those listed in commercial libraries (NIST05).Percentage composition was calculated using the peak nor-malization method. The total ion chromatogram (TIC) andthe chemical structure of the major compounds (>1% of thetotal) of the Enterolobium contortisiliquum essential oil areshown in Figure 2.

Antibacterial activity

The antibacterial activity of the Enterolobium contor-tisiliquum essential oil against the microorganisms consid-ered in the present study were qualitatively and quanti-tatively assessed by evaluating the presence of inhibitionzones and zone diameter measurements as shown in Fig-ure 3 and Table 1. The minimum inhibitory concentra-tions determined against six strains are also shown in Ta-ble 1. Among Gram-positive bacteria, S. aureus was themost sensitive organism to the oil, Gram-negative bacteriashowed relatively higher resistance. Figure 4 shows bioau-tography TLC, the TLC plates, which were sprayed withthe visualizing vanillin sulfuric acid reagents and the massspectra of the two antimicrobial zones (Rf values of 0.1and 0.26), which were eluted from the TLC plates and

Fig. 2. Total ions chromatogram of the essential oil of Enterolobium contortisiliquum, chemical structure, retention times and percentageof the major components of the oil are shown.

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522 Shahat et al.

Fig. 3. Diffusion disk antibacterial test of the essential oil againstthe six tested strains of bacteria.

examined by GCMS. The two active antibacterial com-pounds were identified as estragole and carvone, respec-tively. The identification of the active compounds was elu-cidated based on the interpretation of their mass spectraldata, retention times and the confidence level of the li-brary search that was used for matching their mass spectraldata.

Mass spectral interpretation

Based on the mass spectrum of the first active chemicalwith a TLC Rf value of 0.26 as shown in Figure 4, themolecular ion has a mass of 150. Applying the mass spec-tral “Rule of 13”, [11] a molecular formula of C10H14Owas predicted. Consideration of the carbon-hydrogen ra-tio suggests 4 degrees of unsaturation, implying the pos-sibility of the presence of a benzene ring in the structure.However, based on the study of the molecular ion’s rel-atively low abundance, the presence of a combination ofdouble bonds and/or a cyclic structure is present. Exami-

Table 1. Inhibition zone and minimum inhibition concentration of Enterolobium contortisiliquum essential oil against the testedbacteria.

Bacteria strains

Applied doses µg/disk B.cereus B.subtilis K.pneumoniae S.aureus M.luteus S.Marcescencs

20 4 4 3 3 4 360 6 6 5 5 6 590 8 8 6 7 8 7120 10 10 9 9 10 9150 13 13 12 12 13 11MIC for the oil µg/mL 100 100 150 150 100 150Chloramphenicol 10 µg/disk 10 10 10 10 10 10MIC for chloramphenicol µg/mL 20 20 20 20 20 20

nation of the fragmentation patterns shows various frag-ments of even mass suggesting rearrangement reactionsor multiple bond breaking. Odd mass ions are generallyproduced by single bond cleavage. Therefore, ions of m/z135 and 122 imply the loss of a methyl group and car-bon monoxide respectively. Ions of m/z 108, 94 and 54 (alleven masses) are consistent with fragmentations charac-teristic of McLafferty rearrangements[12]. The most abun-dant ion m/z 82 (base peak), is produced by the lossof an isoprene molecule by a retro Diels-Alder fragmen-tation. The chemical structures and fragmentation path-ways of all ion fragments are shown in Figure 5. All ofthe presented mass spectral interpretation is in agreementwith the suggested structure of carvone (CAS RegistryNumber 99-49-0). Confirmation was also obtained by amass spectral library search showing spectral matching of98%.

With regards to the second active chemical with a TLC Rfvalue of 0.1 (Figure 4), its mass spectrum showed a molec-ular ion of m/z 148 which was also the base peak in thespectrum. Applying the “Rule of 13” described above alsosuggested a possible molecular formula of C10H12O whichis consistent with 5 degrees of unsaturation which predictsthe presence of a benzene ring and one double bond in thestructure. The highly abundant molecular ion is consistentwith the presence of a benzene ring. The mass spectrum alsoshows that all fragment ions are of an odd mass, therefore,its production is consistent with single bond fragmentationand an absence of any rearrangement reactions. The pres-ence of a methoxy group is shown by the loss of a massof 31 to form an ion of m/z 117 which can easily lose anethylene molecule to form the benzylic ion of m/z 91. Thepresence of a benzene ring is also confirmed by the m/z ion77. The chemical structures and fragmentation pathwaysof all ion fragments are shown in Figure 6. All of the pre-sented mass spectral interpretations are in agreement withthe suggested structure of estragole (CAS Registry Num-ber 140-67-0). This was also confirmed by a mass spec-tral library search showing spectral matching of 90% orbetter.

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Antimicrobial activities of leguminosae seed oil 523

Fig. 4. Thin layer chromatogram (left), bioautography (right) and mass spectra of the antibacterial compounds of the Enterolobiumcontortisiliquum essential oil.

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524 Shahat et al.

Fig. 5. Mass spectrum interpretation of carvone.

Fig. 6. Mass spectrum interpretation of estragole.

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Antimicrobial activities of leguminosae seed oil 525

Conclusion

This is the first report of the presence of carvone and es-tragole as major constituents in the essential oil of any ofthe Leguminacea seeds. We[13] have also isolated carvonefrom the essential oil of Artemisia herba alba and proven tobe highly effective in controlling fungi. The antimicrobialactivity of carvone[14−17] and estragole[18,19] is well-knownand previously reported by several investigators. Carvoneis also reported to play a role in the interaction between in-sects and plants.[20] Schlyter et al.[21] reported a feeding de-terrent effect of carvone on insects and slugs. Estragole wasalso found to have insecticidal activity against coleopteranstored-product insects.[22]

Acknowledgments

This work was supported by National Aeronauticsand Space Administration/University Research Center(NASA/URC) grant NCC 9.165. and by the National In-stitutes of Health Research Centers in Minority Institution(RCMI) under grant R003045–17A

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