In Vitro Antifungal Effect of Essential Oils from N e p e t a m e y e r i Benth.

In Vitro Antifungal Effect of Essential Oils from Nepeta meyeri Benth.

Kordali, 6 $8VDQPD] $&DNLU $&DYXVR÷OX DQG6(UFLVOL

* Atatürk University, Faculty of Agriculture, Department of Plant Protection, 25240 Erzurum, Turkey ** Kilis $UDOÕN8QLYHUVLW\)DFXOW\RI6FLHQFHDQG$UWV'HSDUWPHQWRI Chemistry, 79000 Kilis, Turkey

(DVWHUQ$QDWRO൴D$JU൴FXOWXUDO5HVHDUFK,QVW൴WXWH(U]XUXP7XUNH\

**** Atatürk University, Faculty of Agriculture, Department of Horticulture, 25240 Erzurum, Turkey (Received: June 9, 2013 and Accepted: July 21, 2013)

A BST R A C T

(VVHQWLDORLOLVRODWHGIURP 1HSHWDPH\HUL %HQWKREWDLQHGE\ K\GURGLVWLOODWLRQ ZDVDQDO\]HGLQ*6DQG067RWDOO\  FRPSRQHQWV ZHUH LGHQWLILHG LQ WKH HVVHQWLDO RLO UHSUHVHQWLQJ  RI WKH LVRODWHG RLO 0DLQ HVVHQWLDO RLO FRPSRQHQWV ZHUH IRXQG WR EH D Į ĮDȕ-1HSHWDODFWRQH  D Į ĮDĮ±1HSHWDODFWRQH  WUDQV-3XOHJRO&LQHROH$QWLIXQJDODFWLYLWLHVRIWKHRLOZHUHWHVWHGRQWKHJURZWKRISK\WRSDWKRJHQLF IXQJL$QWLIXQJDO DVVD\V VKRZHG WKDW 1 PH\HUL RLO FRPSOHWHO\ LQKLELWHG P\FHOLDO JURZWK RI WKH  SK\WRSDWKRJHQLF IXQJL WKHLU DQWLIXQJDO HIIHFWV ZHUH KLJKHU WKDQ WKH FRPPHUFLDO IXQJLFLGH ³EHQRP\O´ (VVHQWLDO RLO RI 1 0H\HUL DW GLIIHUHQW FRQFHQWUDWLRQV   DQG  PJPO VLJQLILFDQWO\ SUHYHQWHG WKH GHYHORSPHQW RI DOO SODQW SDWKRJHQLF IXQJL$QWLIXQJDODFWLYLWLHVRIWKHRLOLQGLFDWHGWKDWWKHUHZDVDQLQKLELWLRQLQP\FHOLDOJURZWKRIDOOWHVWHGSDWKRJHQLF IXQJLUDQJHGIURPWRDWRLOWUHDWPHQWFRQFHQWUDWLRQVUDQJHGIURPWRPJPO 1PH\HULHVVHQWLDO RLOEORFNHGWKHSODQWSDWKRJHQLFIXQJLE\ZLWKJURZWK7KHILQGLQJVRIWKHSUHVHQWVWXG\VXJJHVWWKDW HVVHQWLDORLOVKDYHDSRWHQWLDOXVHDVIXQJLFLGHV

K ey words:

Nepeta meyeri

essential oil,Antifungal activity,

Fusarium, , Sclerotinia,

Turkey. IN T R O DU C T I O N

Substantial yield losses in food production have increased recently due to insect pests and plant diseases caused by fungi, bacteria and viruses (Fletcheret

et al

., 2006 and Ozturk and Ercisli, 2006). Fungi and bacteria have unfavorable effects on quality, safety and preservation of food. Synthetic chemicals are widely used for the control of plant diseases. However, they cause toxic residues in treated products (Isman, 2000). Synthetic pesticides cause also an environmental pollution owing to their slow biodegradation (Misra and Pavlostathis, 1997). In addition, the risk of developing resistance by microorganisms and high cost-benefit ratio are other disadvantages of synthetic pesticide usage (Brent and Hollomon, 1998).

The genus

Nepeta

(Lamiaceae) comprises 280 species that are distributed over large part of Central and Southern Europe, West, Central and Southern Asia. About half of the existing species have been recorded in Iran. The genus

Nepeta

are represented in Turkey by 33 species, 17 of these being endemic (Rechinger, 1982; Baser

et al

., 2000; Dirmenci, 2005 and Javidnia

et al

., 2011). The endemic and non-endemic Turkish species are commonly found in East Anatolia and the Taurus Mountains (Dirmenci, 2005).

Many of the

Nepeta

species have been reported to be biologically active and are widely used in folk medicine because of their antispasmodic, expectorant, diuretic, antiseptic, antitussive,

antiasthmatic, and febrifuge activities (Baser

et al

., 2000; Tepe

et al

., 2007; Sajjadi, 2005 and Micelia

et

al

., 2005).

This study was carried out to determine antifungal properties of the essential oil isolated from

N. meyeri

.

M A T E RI A LS A ND M E T H O DS Plant materials and isolation of essential oils

Nepeta meyeri

was collected at the flowering stage from Erzurum region, Turkey in 2007. Collected plant material was dried in shadow and ground in a grinder. The dried plant samples (500 g) were subjected to hydrodistillation (plant material in boiling water) using a Clevenger-type apparatus for 4 h. Hydrodistillation of

N. meyeri

0.11% (w/w) yielded the essential oil. The yield was based on dry materials of plant samples. The essential oils were stored in a freezer at 4 °C until further tests.

G C analysis

Analysis of the essential oil was performed using a Thermofinnigan Trace GC/A1300 (E.I.), equipped with a SGE/BPX5 MS capillary column  P î  PP LG  ȝP +HOLXP ZDV WKH carrier gas, at a flow rate of 1 ml/min. Injector temperature was set at 220 °C. The program used was 50±150 °C, at a rate of 3 °C/min, held isothermal for 10 min and finally raised to 250 °C at 10 °C/min. Diluted samples (1/100, v/v, in PHWK\OHQHFKORULGHRIȝOZHUHLQMHFWHGPDQXDOO\ and in the splitless mode.

G C±MS analysis

Analysis of essential oil was performed using the abovementioned technique. GC±MS detection was done by using an electron ionization system with ionization energy of 70 eV. Carrier gas was helium at a flow rate of 1 ml/min. Injector and MS transfer line temperatures were set at 220 and 290 °C, respectively. Oven temperature was programmed from 50 to 150 °C at 3 °C/min, then held isothermal for 10 min and finally raised to 250 °C at 10 °C/min. Diluted samples (1/100, v/v, in methylene chloride) of 1 ȝO ZHUH LQMHFWHG PDQXDOO\ LQ WKH VSOLWOHVV mode. Identification of individual compounds was based on comparison of their relative retention times with those of authentic samples on SGE-BPX5 capillary column, and by matching of their mass spectra of peaks with those obtained from authentic samples and/or the Wiley 7N and TRLIB libraries spectra and published data, and by comparison with the retention index of the components with published data (Adams, 2007).

Antifungal activity assays

16 agricultural pathogenic fungi were obtained from the culture collection at Atatürk University (Faculty of Agriculture, Department of Plant Protection, Erzurum), Turkey. Culture of each fungus was maintained on potato dextrose agar (PDA) and stored at 4 °C.

Antifungal activity was studied by using a contact assay (

in vitro

), which produces hyphal growth inhibition (Kordali

et al

., 2007). Briefly, potato dextrose agar (PDA) plates were prepared using 9-cm diameter glass Petri dishes. The essential oil was dissolved in dimethyl sulfoxide (DMSO) (Merck) at different concentrations (1%, v/v) (0.25, 0.5 and 1.0 mg/ml concentration) and required amounts of the solutions (20.0 mg/Petri dish) were added to each of the PDA plates containing 20 ml of agar at 50 °C. A disc (5 mm diameter) of the fungal species was cut from 1-week-old cultures on PDA plates and then the mycelial surface of the disc was placed upside down on the centre of a dish with fungal species in contact with growth medium on the dish. Then, the plates were incubated in the dark at 22±2 °C. Extension diameter (mm) of hyphae from centers to the sides of the dishes was measured at 24-h intervals for 6 days. Mean of growth measurements were calculated from four replicates of each of the fungal species. PDA plates containing DMSO±water solution (1%, v/v), without essential oil solutions were used as negative control. In addition, PDA plates treated with benomyl (20.0 mg/Petri dish or 1000 mg/l concentration) were used as positive control.

Percentage of growth inhibition by treatment was calculated using the following equation:

Inhibition (%) = C-T X 100 C

where:

C

is the mean of four replicates of hyphal extension (mm) of controls

T

is the mean of four replicates of hyphal extension (mm) of plates treated with essential oil and the compound solutions.

Statistical analysis

Variance analyses were carried out using SPSS 10.0 software package. Differences between means were tested by Duncan and LSD tests and values with

p

<0.05 were considered significantly different.

R ESU L TS A ND DISC USSI O N Chemical composition of the oils

The aerial parts of

N. meyeri

yielded 0.11% (w/w) of oil, where eleven components, representing 99.99 % of the total oil were detected and their percentages are shown in table (1).

GC: co-injection with standards; MS; tentatively identified based on computer matching of the mass spectra of peaks with Wiley 7N and TRLIB libraries and published data; RI: identification based on comparison of retention index with those of published data (Adams, 2007); tr: traces (less than 0.1%).

a: Retention index relative to

n

-alkanes on SG E-BPX5 capillary column

Quantitative data of the oil was obtained from FID area (Table 1). The major separated constituents were D Į ĮDȕ-Nepetalactone (80.32%), D Į ĮD Į ±Nepetalactone (10.32%), Table (1): Chemical composition of essential oil of

aerial parts of

N. Meyeri

RIa _Components Nepeta

meyeri oils Identification methods  -&LQHROH  *&065,  į-7HUSLQHRO  *&065,  7HUSLQHQ--RO  *&065,  Į±7HUSLQHRO  *&065,  WUDQV-3XOHJRO  *&065,  _{1HSHWDODFWRQH} DĮĮDĮ-  *&065,  ȕ-%RXUERQHQH  *&065,  _{1HSHWDODFWRQH} DĮĮDȕ-  *&065,  *HUPDFUHQH'  *&065, *URXSHGFRPSRQHQWV 2[\JHQDWHGPRQRWHUSHQHV  6HVTXLWHUSHQHK\GURFDUERQV  2WKHUV 7U 7RWDO 

trans

-Pulegol (3.13%), 1,8-cineole (2.95%), ȕ-Bourbonene (2.04%).

Nepetalactone isomers, which were the major constituens in the treated oil, were presented in the essential oil of several

Nepeta

spp. (Baser

et al

., 2000 and Sefidkon and Shaabani, 2004). It was found that nepetalactones and its isomers were responsible for the feline attractant properties of

Nepeta

species (Baser

et al

  DĮĮDĮ-Nepetalactone, which was the main component of this oil, was detected as the major one in four

Nepeta

species growing in Turkey (Baser

et al

., 2000 and Mutlu

et al

., 2010). According to the previous authors, the main components of these

Nepeta

species were found to be as follows:

Nepeta

glomerulosa

 Į-pinene (9.4%), geranyl acetate (9.3%), limonene (8.2%) and caryophyllene oxide (8.0%);

Nepeta fissa

 ȕ -caryophyllene (17.4%) and caryophyllene oxide (12.3%);

Nepeta

pogonosperma

 DĮ-Į-Dȕ ±nepetalactone (57.6%) and 1,8-cineole (26.4%) (Sefidkon and Akbarinia, 2003);

N. racemosa

 DĮ-Į-Dȕ -nepetalactone  DĮ-Į-DĮ-nepetalactone (25.6%) DQG DĮ-Dȕ -nepetalactone (24.4%) (Dabiri and Sefidkon, 2003a);

Nepeta crassifolia

, DĮ-Į-DĮ-nepetalactone (92.6%) (Dabiri and Sefidkon, 2003b);

Nepeta persica

,1,4-hexadiene- WHWUDPHWK\O DQG Dȕ -Į-7aĮ- nepetalactone (Javidnia

et al

., 2011);

N. ispahanica

,8 1,8-cineole (66.0%) (Rustaiyan and Nadji, 1999);

Nepeta binaludensis,

1,8-cineole (42.0%) and nepetalactone (25.0%) (Matloubi Moghadam and Hosseini, 1996);

Nepeta denudata

, 1,8-cineole

(48.0%) and myrtenol (5.0%). Antifungal activity of the oil

Antifungal bioassay was performed against the following fungal species:

Alternaria solani,

Fusarium verticilloides, F. semitectum, F.

culmorum, F. proliferatum, F. graminearum, F.

chlamydosporium, F. sambucinum, F. scirpi, F.

equiseti, Nigrospora oryzae, Phytophthora capsici,

Phoma

sp.,

Sclerotinia sclerotiorum, Sclerotonia

sp. and

S. rolfsii.

Results of antifungal activities of

N.

meyeri

at different concentrations are presented in table (2). The results of the antifungal tests revealed that the oil was inhibitory against all of the tested fungi, and the mycelial growth of all tested fungi was completely inhibited at 0.5 and 1.0 mg/ml dose of the oil. Inhibitory effects against the pathogenic fungi were also higher than the commercial fungicide benomyl (Table 2).

These results may be attributed to the high concentration of D Į ĮDȕ-Nepetalactone (80.32%), D Į ĮD Į ±Nepetalactone (10.32%),

trans

-Pulegol (3.13%), 1, 8-cineole (2.95%), ȕ-Bourbonene (2.04%), while the essential oil of

N.

meyeri

showed best antifungal effect on the teseted

Fusarium

and

Sclerotonia

species (Table 2). Development of pathogens due to treatment with

N.

meyeri

essential oil, 0.25 mg/ml dose, was inhibited by 18.5 to 65.8%, where 0.5 mg/ml concentration inhibition increased to 82.7-100%. Depending on the dose of the volatile oil, it was observed that it decreased the rate of inhibition of fungal pathogens (Table 2).

Table (2): Antifungal activities for different concentrations of N. meyeri essential oil on the growth

of plant pathogenic fungi

Fungal species 0.25 mg/ml Treateda _Inh. (mm) _(%) 0.5 mg/ml Treateda _Inh. (mm) _(%) 1.0 mg/ml Treateda _Inh. (mm) _(%) Benomyl(1mg/ml) Treateda _Inh. (mm) (%) Control Treateda (mm)

Alternaria solani 1.1 ± 0.4b 50** _{0.0± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{1.8 ± 1.3a 18.1 2.2± 1.0a} Fusarium verticilloides 1.6 ± 0.5b 54.2*** _{0.5± 0.4c 85.7}*** _{0.6 ± 0.5c 82.8}*** _{0.0 ± 0.0c 100}*** _{3.5 ± 1.3a} Fusarium semitectum 1.4 ± 0.5b 65.8*** _{0.0± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{0.4 ± 0.3c 90.2}*** _{4.1 ± 1.7a} Fusarium culmorum 1.7 ± 0.6b 41.3*** _{0.5± 0.5c 82.7}*** _{0.2± 0.3cd 93.1}*** _{0.0 ± 0.0d 100}*** _{2.9 ± 1.1a} Fusarium proliferatum 1.8 ± 0.8b 53.8*** _{0.0± 0.0c 100}*** _{0.1 ± 0.3c 97.4}*** _{0.0 ± 0.0c 100}*** _{3.9 ± 1.6a} Fusarium graminearum 1.6 ± 0.5b 56.7*** _{0.2± 0.4c 94.5}*** _{0.0 ± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{3.7 ± 1.5a} Fusarium chlamydosporium 1.5 ± 0.4b 53.1*** _{0.0± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{3.2 ± 1.3a} Fusarium sambucinum 1.9 ± 0.8b 58.6*** _{0.4± 0.4c 91.3}*** _{0.4± 0.4c 91.3}*** _{0.0 ± 0.0c 100}*** _{4.6 ± 2.4a} Fusarium scirpi 1.8 ± 0.7b 53.8*** _{0.0± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{3.9 ± 1.4a} Fusarium equiseti 1.2 ± 0.4b 61.2*** _{0.0± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{3.1± 1.6a} Nigrospora oryzae 2.6 ± 2.0a 18.5 0.0 ± 0.0b 100**** _{0.0 ± 0.0b 100}*** _{0.0 ± 0.0b 100}*** _{3.2 ± 2.7a} Phytophthora capsici 1.6 ± 0.6b 44.8*** _{0.3± 0.4c 89.6}*** _{0.2 ± 0.3c 93.1}*** _{0.0 ± 0.0c 100}*** _{2.9 ± 1.2a}

Phoma sp. 1.6 ± 0.7b 36*** _{0.0± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{2.5 ± 1.1a}

Sclerotinia sclerotiorum 3.1 ± 2.9b 34 0.0± 0.0c 100*** _{0.0 ± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{4.7 ± 3.4a}

Sclerotonia sp. 2.9 ± 1.9b 47.2*** _{0.0± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{5.4 ± 2.6a 1.18 5.5 ± 2.7a}

Sclerotium rolfsii 2.0 ± 1.9b 60*** _{0.0± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{0.0 ± 0.0c 100}*** _{5.0 ± 2.7a} a:Means in the same column by the same letter are not significantly different to the test of Duncan (Į = 0.05).

The major components in the tested essential oil are probably responsible for the resulted antimicrobial activity. As shown in table (1), the essential oil of

N. meyeri

contained mainly D Į ĮDȕ-Nepetalactone, DĮĮDĮ ±Nepetalactone,

trans

-Pulegol and 1,8-Cineole. Previous reports showed that Nepetalactone and 1,8-cineole possessed weak and/or no antifungal activity against phytopathogenic fungal species (Kordali

et al

., 2007 and Abd El-Moaty, 2010). As well, it had significant antifungal activity over a wide spectrum. Therefore, its antifungal activity cannot be attributed to these major compounds. On the other hand, it should be considered that minor components in essential oil, as well as synergistic and/or antagonistic interactions between the volatile components could also affect the antifungal properties of the essential oil. The essential oil of

N. meyeri

was also characterized to be rich in oxygenated monoterpenes (97.70%) of total oils. This is in agreement with the previous studies of Sonbolia

et al

. (2004), Kordali

et

al

.(2007) and Abd El-Moaty (2010) who stated that oxygenated monoterpenes such as D Į ĮDȕ-Nepetalactone (80.32%), D Į ĮD Į-Nepetalactone (10.32%),

trans

-Pulegol (3.13%), 1, 8-cineole (2.95%), ȕ-Bourbonene (2.04%), and essential oil containing relatively high amount of oxygenated monoterpenes possess antifungal activity. The pharmacological properties and various biological activities are usually ascribed to nepetalactone compounds primarily found in the essential oils of the

Nepeta

species (Nestorovic

et

al

., 2010).

The essential oil of

N. meyeri

was weak fungitoxic against

Nigrospora oryzae

(18.5%),

Sclerotinia sclerotiorum

(34%)

, Phoma

sp., (36%) and

Fusarium culmorum

(41.3%) among the tested phytopathogenic fungi at the dose of 0.25mg/ml (Table 2). Some literatures regarding the biological activity of their essential oils including antimicrobial and antifungal potential have been published (Saxena and Mathela, 1996; De Pooter

et al

., 2006 and Ljaljevic Grbic

et al

., 2008). Considering the literature regarding the biological activity of nepetalactones and the fact that these metabolites are the main constituents of

N. meyeri

essential oil

,

it was presumed that it may be responsible for antifungal activities. However, the possible synergistic or antagonistic effects of these compounds with other compounds present in trace amounts have to be considered.

R E F E R E N C ES

Abd El-Moaty, H. I. 2010. Essential oil and iridoide glycosides of

Nepeta septemcrenata

Erenb. J. Nat. Prod. 3:103-111.

Adams, R. P. 2007. Identification of essential oil components by Gas Chromatography/ Mass Spectrometry, Allured Publishing Corp, Carol Stream, Illinois, USA.

%DVHU.+&1.LULPHU0.UNoR÷OXDQG% Demirci 2000. Essential oils of

Nepeta

species growing in Turkey. Chem. Nat. Comp. 4:356-359.

Brent, K. J. and D. W. Hollomon 1998. Fungicide Resistance: The Assessment of Risk. Monograph No. 2. FRAC, Global Crop Protection Federation, Brussels, pp. 1±48.

Dabiri, M. and F. Sefidkon 2003a. Chemical composition of the essential oil of

Nepeta

racemosa

Lam. from Iran. J. Flavour Fragrance 18: 157-158.

Dabiri, M. and F. Sefidkon 2003b. Chemical composition of

Nepeta crassifolia

Boiss. & Buhse. oil from- Iran. Flavour Fragr. J. 18, 225-227.

De Pooter, H. L., B. Nicolai, J. De Laet, L. F. De Buyck, N. M. Schamp and P. Goetghebeur 2006. The essential oils of five

Nepeta

species. A preliminary evaluation of their use in chemotaxonomy by cluster analysis. Flavour Fragr J, 3: 155-159.

Dirmenci, T. 2005. A new subspecies of

Nepeta

(Lamiaceae) from Turkey. Bot. J. Linn. Soc. 147: 229-233.

Fletcher, J., C. Bender, B. Budawle, W. T. Cobb, S. E. Gold, C. A. Ishimaru, D. Luster, U. Melcher, R. Murch, H. Scherm, R. C. Seen, J. L. Sherwood, B. W. Sobral, and S. A. Tolin 2006. Plant pathogen forensics: capabilities, needs, and recommendations. Microbial Mol. Biol. Rev. 70: 450-471.

Isman, M. B. 2000. Plant essential oils for pest and disease management. Crop Protect. 19: 603-608. Javidnia, K., A. R. Mehdipour, B. Hemmateenejad,

S. R. Rezazadeh, M. Soltani, A. R. M Khosravi, A. R. and R., Miri 2011. Nepetalactones as chemotaxonomic markers in the essential oils of

Nepeta

species. Chem. Nat. Comp. 47: 843-847. Kordali, S., R. Kotan and A. Cakir 2007. Screening

of in vitro antifungal activities of 21 oxygenated monoterpenes as plant disease control agents. Allelopathy J. 19: 373-392.

LjaOMHYLü *UELü 0 0 6WXSDU - 9XNRMHYLü 0 6RNRYLü ' 0LãLü ' *UXELãLü DQG 0 5LVWLü 2008. Antifungal activity of

Nepeta rtanjensis

essential oil. J. Serb. Chem. Soc. 73: 961-965. Matloubi Moghadam, F. and M. Hosseini 1996.

Composition of the essential oil from

Nepeta

crassifolia

Boiss. & Buhse. Flavour Fragr

.

J

.

11:113-115.

Micelia, N., M. F. Taviano, D. Giuffrida, A. Trovato, O. Tzakouc and E. M. Galatia 2005. Anti-inflammatory activity of extract and

fractions from

Nepeta sibthorpii

Bentham

. J.

Ethnopharmacol.

97: 261-266.

Misra, G., and S. G. Pavlostathis 1997. Biodegradation kinetics of monoterpenes in liquid and soil-slurry systems. Appl. Microbiol. Biotechnol. 47: 572-577.

Mozaffarian, V. 1996. A Dictionary ofIranian Plant Names, Farhang-e Moaser, Tehran, 1996, pp. 360±361.

Mutlu, S., O. Atici, and M. Esim 2010. Bioherbicidal effects of essential oils of

Nepeta

meyeri

Benth. on weed spp. Allelopathy J. 26(2): 291-299

Nestorovic, J., D. Misic., B. Siler, M. Sokovic, J. Glamoclija, A. Ciric, V. Maksimovic and D. Grubisic 2010. Nepetalactone content in shoot cultures of three endemic

Nepeta

species and the evaluation of their antimicrobial activity. Fitoterapia. 81(6): 621-626.

Ozturk, S. and S. Ercisli 2006. The chemical composition of essential oil and in vitro antibacterial activities of essential oil and methanol extract of

Ziziphora persica

Bunge. J. Ethnopharmacol. 106 (2): 372-376.

Rechinger, K. H. 1982.

Nepeta

, In: Flora Iranica, No.150, Akademische Druck and Verlagsanstalt, Graz, Austria, pp. 108-216.

Rustaiyan, A., K. Nadji 1999. Composition of the essential oil of

Nepeta ispahanica

Boiss. and

Nepeta binaludensis

Jamzad from Iran. Flavour Fragr. J.14: 35±37.

Sajjadi, S. E. 2005. Analysis of the essential oil of

Nepeta sintenisii

Bornm. from Iran. Daru 13: 61-64.

Saxena, J., and C. S. Mathela 1996. Antifungal activity of new compounds from

Nepeta

leucophylla

and

Nepeta clarkei.

Appl. Environ. Microbiol. 702-704.

Sefidkon, F. and A. Akbarinia 2003. Essential oil composition of

Nepeta pogonosperma

Jamzad et Assadi from Iran. J. Essent. Oil Res. 15(3): 327-328

Sefidkon, F., and A. Shaabani 2004. Essential oil composition of

Nepeta meyeri

Benth. from Iran. Flavour Frag. Journal. 19(3): 236-238.

Sonbolia, A., P. Salehib and M. Yousefzadic 2004. Antimicrobial activity and chemical composition of the essential oil of

Nepeta crispa

Willd. From Iran. Verlag der Zeitschrift für Naturforschung Tübingen ± Mainz, 59c: 653-656.

Tepe, B., D. Daferera, A. S. Tepe, M. Polissiou and A. Sokmen 2007. Antioxidant activity of the essential oil and various extracts of Nepeta flavida Hub. Mor. Food Chem. 103: 1358-1364.