Chemical Composition and Antimicrobial Activity of Essential Oil of ...

Jeobp 12 (5) 2009 pp 557 - 561

557

ISSN 0972-060X

Chemical Composition and Antimicrobial Activity of Essential Oil of Echium italicum L. Katayoun Morteza-Semnani 1*, Majid Saeedi 2 and Mohammad Akbarzadeh 3 1

Department of Medicinal Chemistry, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran 2 Department of Pharmaceutics, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran 3 Research Center of Agriculture and Natural Resources of Mazandaran, Sari, Iran Received 31 January 2009; accepted in revised form 24 June 2009

Abstract: The essential oil of Echium italicum L. (Boraginaceae) collected from the suburb of Geloogah, north of Iran, in June 2008, was isolated by hydrodistillation and analyzed by means of GC and GC-MS. Twenty-two components were identified in this oil. The major constituents of the essential oil were hexadecanol (27.1 %) and pulegone (8.8 %). The antimicrobial activity of E. italicum oil was studied using the disk diffusion method and determination of minimal inhibitory concentration (MIC) values against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Aspergilus niger and Candida albicans. The E. italicum oil exhibited concentration-dependent antimicrobial activity on all microorganisms tested. Key Words: Echium italicum, Boraginaceae, essential oil composition, hexadecanol, pulegone. Introduction: The Boraginaceae is very large group comprising about 100 genera and 2000 species in the tropical and temperate regions of the world 1. The genus Echium (Boraginaceae) comprises 4 species, which are found wild in many regions of Iran 2. Echium italicum L. is an aromatic plant, which grows wild in Azerbaijan, Fars, Golestan, Khorasan, Kurdestan and Mazandaran provinces of Iran 3. Some species of Echium have been used as cough suppressant, expectorant and diuretic in traditional medicine of Iran 4-6. A literature survey has shown that there is no report on the volatile constituents and antimicrobial activity of E. italicum oil. The medicinal properties attributed to the genus Echium, prompted us to investigate the chemical constituents of the oil of E. italicum and its antimicrobial activity for the first time. *Corresponding author (Katayoun Morteza-Semnani) E- mail: < [email protected] >

Katayoun Morteza-Semnani et al. / Jeobp 12 (5) 2009 pp 557 - 561

558

Experimental Plant material: The aerial parts of E. italicum L. were collected in June 2008 from the suburb of Geloogah, Mazandaran Province, North of Iran and identified by Mohammad Akbarzadeh (Department of Botany, Research Center of Agriculture and Natural Resources of Mazandaran). Voucher specimen (herbarium No. 435) was deposited in the Herbarium of the Department of Botany, Research Center of Natural Resources of Mazandaran. Oil isolation: The oil of air-dried floweing aerial parts of E. italicum (100 g) was isolated by hydrodistillation in Clevenger-type apparatus for 3 h, with the addition of pentane (5 mL) for trapping the liberated oil. The oil was dried over anhydrous sodium sulfate and kept at 4°C in a sealed brown vial until the GC and GC-MS analyses. Analysis: Gas chromatographic analysis was carried out on a Perkin-Elmer 8500 gas chromatograph with FID detector and a DB-5 capillary column (30 m x 0.25 mm; film thickness 0.25 μm). The operating conditions were as follows: carrier gas helium with a flow rate of 2 mL/min, split ratio was 1:30, the oven temperature was programmed 4 min. Isothermal at 60°C and then 60 - 220°C at 4°C/min., injector and detector temperatures were set at 240°C. Gas Chromatography-Mass Spectrometry (GC-MS) was carried out on Hewlett Packard 6890 series, using a DB-5 capillary column (30 m x 0.25 mm, film thickness 0.25 μm) which was programmed as follows: 60°C for 5min. and then up to 220°C at 4°C/min. The carrier gas was helium at a flow rate of 2 mL/min; split ratio, 1: 40; ionization energy, 70 eV; scan time, 1 s; acquisition mass range, m/z 40-400. The components of the oil were identified by their retention time, retention indices relative to C9-C28 n-alkanes, computer matching with the WILEY275. L library and as well as by comparison of their mass spectra with those of authentic samples or with data already available in the literature 7-9. Antimicrobial assay: Bacillus subtilis PTCC 1023, Staphylococcus aureus PTCC 1112, Escherichia coli PTCC 1330, Salmonella typhi PTCC 1639, Pseudomonas aeruginosa PTCC 1074, Aspergilus niger PTCC 5011 and Candida albicans PTCC 5027 were used for testing the antimicrobial activity. Diffusion method using filter paper disk (6 mm) was used for the screening of oil antibacterial and antifungal activities 10. Bacterial and fungal strains were tested on Muller-Hinton agar and Sabouraud dextrose agar, respectively. Sterilized paper disks were loaded with different amounts of E. italicum oil (250, 500, 1000, 2000, 4000 and 8000 μg /disk) and applied on the surface of agar plates. All plates were incubated at 37°C for 24 h for bacteria; at 25°C for 24 h for C. albicans; and at 25°C for 3 days for A. niger. Inhibition zone diameters were measured after conventional incubation period. Gentamycin (50 μg/disk), Amikacin (3 μg/disk) and Amphotericin B (100 μg/disk) (obtained from Sigma) were used as positive reference standards. The estimation of the minimal inhibitory concentration (MIC) was carried out by the broth dilution method. Dilutions of essential oil from 6.4 to 0.1 mg/mL were used. MIC values were taken as the lowest essential oil concentration that prevents visible microbe


559

growth after the incubation period (described above) 11. Gentamycin, Amikacin and Amphotericin B (128, 64, 32, 16, 8, 4 and 2 μg/mL) were used as positive reference standards and control with no essential oil was used. Each experiment was made three times. Results and discussion Chemical analyses of essential oil: The hydrodistillation of the dried flowering aerial parts of E. italicum gave light yellowish oil with yield of 0.2 % (w/w). As shown in Table 1, twenty-two compounds were identified in the oil of E. italicum, which presented about 90.1 % of the total composition of the oil; the major components of this oil were hexadecaol (27.1 %) and pulegone (8.8 %). The oil of E. italicum comprised 4 monoterpenoids (17.1 %), one sesquiterpene (3.8 %) and 17 non-terpenoids (69.2 %). The essential oil of the flowering aerial parts of E. italicum was rich in non-terpenoids. In 2003, Ghassemi et al. reported δ-cadinene (24.2 %), n-hexadecane (8.7 %), npentadecane (5.6 %), viridiflorol (4.9 %), α-muurolene (4.5 %), ledene (3.8 %), α-calacorene (3.0 %) and γ-cadinene (2.9 %) as main compounds of the oil of E. amoenum collected from Ghazvin Province of Iran 5. In 2005, thymol (19.5 %) and carvacrol (7.5 %) were identified as the major compounds from the essential oil of Echium amoenum Fisch. & C.A. Mey. collected from the suburb of Sari, Mazandaran Province, north of Iran 6. There is no report on the volatile constituents of E. italicum; therefore, we were unable to investigate variations of oil components due to differences in climate and geographic areas. Antimicrobial activity: The E. italicum oil exhibited concentration-dependent antimicrobial activity on all microorganisms tested. Tables 2 and 3 show the antimicrobial activity (inhibition zone and MIC) of E. italicum oil against microorganisms tested.

1. 2. 3. 4. 5.

6. 7. 8.

References Özcan, T. (2008). Analysis of the total oil and fatty acid composition of seeds of some Boraginaceae taxa from Turkey. Plant Syst. Evol., 274: 143-153. Mozaffarian, V. (1996). A Dictionary of Iranian Plant Names. Farhang Moaser, Tehran, Iran, p. 198. Rechinger, K.H. (1967). Flora Iranica. Akademische Druck-U.Verlagsanstalt, GrazAustria, No. 48, p. 214. Zargari, A. (1989). Medicinal Plants. Tehran University Publications, Tehran, Iran, 3, pp. 538-541. Ghassemi, N., Sajjadi, S.E., Ghannadi, A., Shams-Ardakani, M. and Mehrabani, M. (2003). Volatile constituents of a medicinal plant of Iran, Echium amoenum Fisch. and C.A. Mey. Daru, 11: 32-33. Morteza-Semnani, K. and Saeedi, M. (2005). Essential oils composition of Echium amoenum Fisch. & C.A. Mey., J.f Essent. Oil Bearing Plants. 8: 61-64. Adams, R.P. (2001). Identification of Essential Oil Components by Gas Chromatography/Quadrupole Mass Spectroscopy. Allured Publishing Corporation, IL. Davies, N.W. (1990). Gas chromatographic retention indices of monoterpenes and


9.

10.

11.

560

sesquiterpenes on methyl silicone and carbowax 20M phases. J. Chromatogr., 503: 1-24. Engel, R., Gutmann, M., Hartisch, C., Kolodziej, H. and Nahrstedt, A. (1998). Study on the composition of the volatile fraction of Hamamelis virginiana. Planta Med., 64: 251-258. Dehghan, Gh., Solaimanian, R., Shahverdi, A.R., Amin, Gh., Abdollahi, M. and Shafiee, A. (2007). Chemical composition and antimicrobial activity of essential oil of Ferula szovitsiana D. C. Flavour Fragr. J., 22: 224-227. Hernandez, T., Canales, M., Teran, B., Avila, O., Duran, A., Garcia, A.M., Hernandez, H., Angeles-Lopez, O., Fernandez-Araiza, M. and Avila, G. (2007). Antimicrobial activity of the essential oil and extracts of Cordia curassavica (Boraginaceae). J. Ethnopharmacol., 111: 137-141. Table 1. The chemical constituents (%) of the oil of Echium italicum No.

Component

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

3-Octanol acetate Menthone cis-Isopulegone trans-Isopulegone 1-Dodecane Pulegone 4E-Decen-1-ol 2E-Decen-1-ol Spathulenol n-Pentadecanol n-Hexadecanol Cyclohexadecanol n-Eicosane n-Heneicosane n-Docosane n-Tricosane n-Tetracosane n-Pentacosane n-Hexacosane n-Heptacosane n-Octacosane n-Nonacosane Total

a

KI = Kovats index on DB-5 column.

KIa

GC Area %

1124 1154 1169 1170 1191 1238 1263 1272 1579 1775 1877 1937 2000 2100 2200 2300 2400 2500 2600 2700 2800 2900

1.8 0.9 3.8 3.6 1.3 8.8 0.8 1.3 3.8 4.1 27.1 3.6 0.9 1.6 1.4 2.9 3.8 3.2 4.4 3.2 4.1 3.7 90.1

250 500 1000 2000 4000 8000 50 3 100

Conc. (μ μg/disc)

7.8 ± 0.5 8.9 ± 0.75 11.3 ± 2.1 13.1 ± 1.6 14.7 ± 1.8 17.2 ± 1.8 29.8 ± 1.9 21.8 ± 1.55 -

7.2 ± 0.4 8.1 ± 0.75 9.1 ± 1.4 9.9 ± 1.6 10.8 ± 1.8 37.3 ± 2.5 24.9 ± 3.1 -

7.6 ± 0.5 8.5 ± 0.7 9.6 ± 1.2 10.2 ± 1.5 11.1 ± 1.3 31.6 ± 3.2 23.8 ± 2.5 -

7.25 ± 0.51 8.6 ± 0.75 29.0 ± 2.5 16.8 ± 3.1 -

nd: Not Determined

Bacillus subtilis (G+) Staphylococcus aureus (G +) Escherichia coli (G -) Salmonella typhi (G -) Pseudomonas aeruginosa (G -) Candida albicans Aspergilus niger

Strains 0.4 0.8 0.8 6.4 0.8 3.2 3.2

Essential oil

32 × 10-3 8 × 10-3 16 × 10-3 32 × 10-3 32 × 10-3 nd nd

4 × 10-3 4 × 10-3 2 × 10-3 8 × 10-3 8 × 10-3 nd nd

MIC (mg/mL) Gentamycin Amikacin

7.2 ± 0.3 8.3± 0.7 9.0 ± 1.2 22.7 ± 2.1

nd nd nd nd nd 64 × 10-3 32 × 10-3

Amphotericin B

7.4 ± 0.4 8.2 ± 0.6 9.1 ± 1.1 22.3 ± 2

Table 3. Minimal inhibitory concentration (MIC) of essential oil of Echium italicum

7.25 ± 0.51 8.6 ± 0.75 10.9 ± 1.3 12.3 ± 1.6 14.1 ± 1.6 31.02 ± 1.2 15.8 ± 1.12 -

Diameter of zone of inhibition (mm) (Mean ± S.D.) Bacteria Fungi Bacillus Staphylococcus Escherichia Salmonella Pseudomonas Candida Aspergilus subtilis (G+) aureus (G +) coli (G -) typhi (G -) aeruginosa(G -) albicans niger

Values are inhibition zone (mm) and an average of triplicates; -, no inhibition.

Gentamycin Amikacin Amphotericin B

E. italicum oil

Sample

Table 2. Antimicrobial activity of the essential oil of Echium italicum

Katayoun Morteza-Semnani et al. / Jeobp 12 (5) 2009 pp 557 - 561 561