Iranian Journal of Pharmaceutical Research (2006) 1: 65-68 Received: January 2005 Accepted: February 2006

Copyright 2006 by School of Pharmacy Shaheed Beheshti University of Medical Sciences and Health Services

Original Article

Antimicrobial Activity and Composition of the Essential Oil of Cymbopogon Olivieri (Boiss.) Bor from Iran
Ali Sonboli*a, Mohammad Hossein Mirjalilib and Morteza Yousefzadic
of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran. bDepartment of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran. cDepartment of Ecology & Systematic, Research Institute of Applied Sciences, Tehran, Iran. Abstract
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Keywords: Cymbopogon Olivieri; Poaceae; Essential oil; Antimicrobial activity; Iran.

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The in vitro antimicrobial activity of the essential oil isolated from the aerial parts of Cymbopogon Olivieri (Boiss.) Bor, an aromatic grass of Iran was tested against three Gramnegative and four Gram-positive bacteria and also three fungi. The results of the bioassays showed that the oil has a remarkable antimicrobial activity. Bacillus subtilis and Candida albicans were more sensitive to the oil than other microorganisms with inhibition zones of 20 mm and MIC values of 3.75 mg/ml and 2.5 mg/ml, respectively. The Gram-negative bacteria, Pseudomonas aeruginosa was resistant and Klebsiella pneumoniae showed less sensitivity to the oil with MIC value of >15 mg/ml. GC-MS analysis of the oil conrmed the determination of 40 compounds representing 95.0% of the oil. The main identied constituent was piperitone (48.9%).

Introduction

The genus Cymbopogon Spreng. is an important member of aromatic grasses, belonging to the Poaceae family which are widely distributed and cultivated in tropical and subtropical regions in the world especially in southeast of Asia (1). The genus Cymbopogon comprises two perennial species in flora of Iran, C. Olivieri (Boiss.) Bor and C. parkeri Stapf., which are distributed in tropical regions of Iran including southern parts of Fars, Kerman, Hormozgan, Khuzestan, Bushehr and Baluchestan provinces (2, 3). C. Olivieri is known as Kah Makki, Potar and Nagerd in different areas which this plant is gathered
* Corresponding author: E-mail: a-sonboli@sbu.ac.ir

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(4). In traditional medicine, leaves and roots are widely used as antiseptic and for the treatment of stomachache (5). Evaluation of repellent activities of Cymbopogon essential oils against mosquito vectors of malaria, filariasis and dengue fever in India has been reported (6). The antimicrobial action of palmarosa oil (7) and antibacterial activity of the oil of C. densiflorus (8) has been the subject of earlier studies. As far as our literature survey could ascertain, the essential oil composition of the aerial parts of C. Olivieri has already been analyzed and piperitone (53.3%), -terpinene (13.6%) and elemol (7.7%) were found to be the major constituents (9). In another study, the composition and antimalarial efficacy of the essential oil of C. Olivieri have been reported and it has exhibited interesting activity against larvaes of Anophel stephensi (10). To the best

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Sonboli A, Mirjalili MH and Yousefzadi M / IJPR 2006, 1: 65-68

of our knowledge, the in vitro antimicrobial activity and composition of the essential oil of C. Olivieri reported here, has not been the subject of previous investigation. Experimental Plant Material Aerial parts of Cymbopogon Olivieri were collected at full flowering stage in April 2003, from Hormozgan: Bandar-Abbas - Hajiabad road, Siahoo village, at an altitude of 1900 m, Iran. A voucher specimen (MP-489) was deposited at the Medicinal Plants and Drugs Research Institute Herbarium, Shahid Beheshti University, Tehran, Iran. Essential Oil Isolation and Analysis Procedure The essential oil obtained by hydrodistillation from the air-dried aerial parts of plant using a Clevenger-type apparatus for 3 h. The distilled oil was dried over anhydrous sodium sulfate and stored in sealed vial at 4 oC until bioassay and GC-MS analyses. GC-MS analysis was carried out on a Thermoquest-Finnigan Trace GC-MS equipped with a fused silica capillary DB-1column (60 m 0.25 mm i.d., lm thickness 0.25 m). The oven temperature was raised from 60 C to 250 C at a rate of 5 C/min, then held at 250 C for 10 min.; transfer line temperature, 250 C. Helium was used as the carrier gas at a ow rate of 1.1 ml/min; split ratio, 1/50. The quadruple mass spectrometer was scanned over the 45-465 amu with an ionizing voltage of 70 eV and an ionization current of 150 A. Identication of individual compounds was made by comparison of their mass spectra with those of the internal reference mass spectra library Wiley 7.0 or with authentic compounds and conrmed by comparison of their retention indices with authentic compounds or with those of reported in the literature (11). For quantication purposes relative area percentages obtained by FID were used.

Table 1. Essential oil composition of C. olivieri from Iran. No. 1 2 3 4 5 6 7 8 9 tricyclene -pinene camphene methyl-4-methylene-bicyclo [3.2.1] oct-2-en myrcene -terpinene p-cymene limonene (Z)-ocimene Compound % 0.6 1.2 2.2 1.7 0.7 13.8 0.4 6.3 0.1 0.1 0.5 1.0 t 0.7 0.5 0.4 0.5 1.5 0.3 0.3 RI 924 933 948 964 980 999 1013 1023 1035 1073 1083 1110 1116 1126 1154 1161 1165 1175 1182 1192

10 fenchone 11 linalool 12 (Z)-p-menth-2-en-1-ol 13 2-carene

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15 borneol 16 (E)-carveol 17 4-terpineol 18 -trpineol 19 (Z)-piperitol 20 (E)-piperitol 21 piperitone 24 thymol 25 isoborneol 26 geranyl actate 27 -elemene 28 -yelangen 29 -caryophyllene 30 germacrene D 31 valencene 33 -cadinene 34 -cadinene 35 elemol 36 spathulenol 38 -eudesmol 39 guaiol 40 -eudesmol

14 (E)-p-menth-2-en-1-ol

48.9 1237 0.1 0.1 0.1 t 0.4 1.4 0.1 0.1 0.3 0.2 0.2 0.4 0.2 3.8 0.3 2.2 0.7 0.8 1.9 1240 1254 1263 1270 1355 1389 1418 1423 1480 1486 1491 1509 1515 1537 1570 1610 1624 1626 1642

22 thymol methyl ether 23 p-mentha-1,3-dien-7-al

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32 bicyclogermacrene

37 -cadinol isomer=1

Bioassay Procedure The in vitro preliminary antimicrobial activity of the essential oil (dissolved in 0.1%

Total identified 95.0 a RI, retention indices relative to C - C n-alkanes on a DB-1 6 24 column; MS, mass spectrum; Co-I, co-injection with authentic compound. b t, trace (<0.1%).

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Antimicrobial Activity and Composition of the Essential Oil of Cymbopogon Olivieri (Boiss.) Bor from Iran

Table 2. Antimicrobial activity of Cymbopogon olivieri essential oil. Microorganism Bacillus subtilis Enterococcus faecalis Staphylococcus aureus Staphylococcus epidermidis Pseudomonas aeruginosa Escherichia coli Klebsiella pneumoniae Candida albicans Aspergillus niger Essential oila IZb 20 11 15 13 12 8 20 14 MICc 3.75 15 7.5 7.5 nt 15 >15 2.5 5 Inhibition zone (mm)b Ampicillin (10 g/disc) 14 11 13 19 9.7 12 nt nt nt Nystatine (30 g/disc) nt nt nt nt nt nt nt 18 16 17

tween 80, 1:1) was evaluated by disc diffusion method using Mueller-Hinton Agar for bacteria and Sabourod Dextrose Agar for fungi (12) with determination of inhibition zones. Three Gram-negative and four Gram-positive bacteria and also three fungi used were as follows: Bacillus subtilis ATCC 9372, Enterococcus faecalis ATCC 15753, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27852, Klebsiella pneumoniae ATCC 3583, Candida albicans ATCC 5027, Microsporium gypsium ATCC 5070 and Aspergillus niger ATCC 16404. All bacteria strains as well as two yeasts, Candida albicans and Microsporium gypsium were sub-cultured from their original lyophilized stocks (ATCC) on nutrient agar and then the obtained single colonies were used for future bioassays. For Aspergillus niger a concentration of 1 106 /ml spore was used. Ampicillin for bacteria and nystatine for fungi were used as positive controls. Tween 80 (0.1%) was used as negative control. The incubation conditions used were 24 h at 37 C for bacteria and 48 h at 24 C for fungi. Minimum inhibitory concentration (MIC) was defined as the lowest concentration of the oil that resulted in a complete inhibition of visible growth in the broth which was measured by microdilution broth susceptibility assay recommended by NCCLS (13).

The yield of the essential oil of C. Olivieri was 0.6% (w/w) and 0.9% (v/w) based on the dry weight of plant. The oil was yellow in colour. GC-MS analysis of the oil confirmed the determination of 40 compounds representing 95.0% of the oil (Table 1). The main identified constituents were piperitone (48.9%), terpinene (13.8%), limonene (6.3%) and elemol (3.8%), which constituted 72.9% of total oil. The results obtained from essential oil analysis showed some quantitative and qualitative differences with the previous investigation (9), which could be attributed to geographical origin and chemotype of them. Antimicrobial activity of the oil measured by disc diffusion and minimal inhibitory concentration (MIC) methods showed that the oil of C. Olivieri was active against most of the tested microorganisms except for Pseudomonas aeruginosa that was resistant to the oil. Table 2 shows in vitro antimicrobial property, growth inhibitory zones and MIC values, of the oil of C. olivieri and the inhibition zones formed by standard reference antibiotic discs. The oil exhibited moderate to high activity towards the microorganisms among which B. subtilis and C. albicans with inhibition zones of 20mm and MIC values of 3.75mg/ml and 2.5mg/ml, respectively, being more sensitive than the others. Klebsiella pneumoniae exhibited less

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15 10 Microsporium gypsium a Essential oil tested at a concentration of 15 l/disc for bacteria and 30 l/disc for fungi. bDiameter of inhibition zones including diameter of sterile disc (6 mm). c Minimal inhibitory concentration, values are given as mg/ml (-), Inactive; (7-14), moderately active; (>14), highly active; nt, not tested.

Results and Discussion

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Sonboli A, Mirjalili MH and Yousefzadi M / IJPR 2006, 1: 65-68

sensitivity to the oil with inhibition zone of 8mm and MIC value of >15mg/ml. The results revealed that the oil exhibited the same type of activity compared to positive standard controls (Table 2). The essential oil composition and the observed antimicrobial property showed that the essential oil of plant has a good potential for use in aromatherapy and pharmacy and supports its uses in traditional medicine. References
(1) Nair EVG. Promotional aspects of lemongrass. In: Atal CK and Kapur BM (eds) Cultivation and Utilization of Aromatic Plants. CSIR, Jammu-Tawi (1981) 314-317 (2) Bor NL. Cymbopogon. In: Rechinger, K.H. (ed.) Flora Iranica, No. 70, Akademische Druck- und Verlagsanstalt, Graz (1982) 543-544 (3) Mozaffarian V. A Dictionary of Iranian Plant Names. Farhang Moaser, Tehran (1996) 172 (4) Mobayen S. Flora of Iran, Vol. 1, Tehran University, Tehran (1974) 264-266 (5) Zargari A. Medicinal Plants, Vol. 4, Tehran University, Tehran (1991) 738 (6) Tyagi BK, Shahi AK and Kaul BL. Evaluation of repellent activities of Cymbopogon essential oils against mosquito vectors of malaria, filariasis and dengue fever in India. Phytomedicine (1998) 5: 324329

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(7) Prashar A, Hili P, Veness RG and Evans CS. Antimicrobial action of palmarosa oil (Cymbopogon martinii) on Saccharomyces cerevisiae. Phytochemistry (2003) 63: 569-575 (8) Takaiasi-Kikuni NB, Tshilanda D and Babady B. Antibacterial activity of the essential oil of Cymbopogon densiflorus. Fitoterapia (2000) 71: 69-71 (9) Norouzi-Arasi H, Yavari I, Ghaffarzadeh F and Mortazavi MS. Volatile constituents of Cymbopogon Olivieri (Boiss.) Bor from Iran, Flavour Fragr. J. (2002) 17: 272-274 (10) Hadjiakhoondi A, Vatandoost H, Jamshidi A and Ebrahim Bagherj A. Chemical constituents and efficacy of Cymbopogon Olivieri (Boiss.) Bor essential oil against malaria vector, Anopheles stepensi. Daru (2003) 11: 125-128 (11) Shibamoto T. Retention indices in essential oil analysis. In: Sandra P and Bicchi C. (eds.) Capillary Gas Chromatography in Essential Oil Analysis. Huethig Verlag, New York (1987) 259-274 (12) Baron EJ and Finegold SM. Methods for testing antimicrobial effectiveness. In: Stephanie M. (ed.) Diagnostic Microbiology. Mosby, Baltimore (1990) 171-194 (13) National Committee for Clinical Laboratory Standards (NCCLS) Performance Standards for Antimicrobial Susceptibility Testing, 9th International Supplement. The Committee, Wayne (1999) M100-S9
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