Chemical Composition and Antifungal Activity of the Essential Oil of Hyptis ovalifolia Benth . ( Lamiaceae )

As folhas de Hyptis ovalifolia Benth. (Lamiaceae) foram submetidas à hidrodestilação e a fração volátil foi investigada por CG/EM. O componente principal, (R)-6-[(Z)-1-heptenila]-5,6-diidro-2Hpiranona (1), representando 60% do óleo essencial, foi isolado por cromatografia em coluna e identificado por métodos espectroscópicos. O composto mostrou atividade in vitro contra 4 espécies (60 isolados clínicos) dos dermatófitos: Microsporum canis, Microsporum gypseum, Tricophyton mentagrophytes e Tricophyton rubrum com concentração inibitória mínima variando de 125-7,8 μg mL.


Introduction
The genus Hyptis Jacq.][4] In the course of our program aimed to isolate new antifungal compounds from Brazilian Cerrado plants, the essential oil of Hyptis ovalifolia Benth., popularly known in Brazil as 'malva do cerrado', was found to show a strong activity against dermatophytes.In this paper we report the isolation, structural determination and antifungal activity of the major constituent of this essential oil against Microsporum canis, Microsporum gypseum, Tricophyton mentagrophytes, and Tricophyton rubrum using the agar dilution method 5 to determine the minimal inhibitory concentrations (MICs).

General experimental procedures
The GC/MS analyses were carried out using a QP5050 GC/MS (Shimadzu), equipped with J&W Scientific DB-5 fused silica capillary column (30 m x 0.25 mm x 0.25 µm).The oven temperature was programmed as follows: 60 °C for 2 min with an increase of 3 °C min -1 to 240 °C, followed by 10 °C min -1 to 280 °C for 2 min.Carrier gas: Helium (1 mL min -1 ).The injector was operated in split mode with ratio 1:50 at 220 °C and interface temperature at 240 °C.The retention index were obtained by co-injecting the oil sample with an n-alkanes series 6 and used to identify the compounds present, together with the comparison to the NIST (NIST 21 and NIST 107) mass spectral library.NMR spectra were recorded with a Varian Gemini 2000 spectrometer to operating at 300 MHz for 1 H and at 75.5 MHz for 13 C. CDCl 3 was used as the solvent, with Me 4 Si (TMS) as internal standard for the 1 H NMR spectrum and CDCl 3 as internal standard for the 13 C NMR spectrum.Methyl, methylene, methyne, and carbon non-bonded to hydrogen were discriminated using DEPT-135° and DEPT 90° spectra (Distortionless Enhancement by Polarization Transfer).2D NMR spectroscopy was performed with standard H, H correlation and H, C correlation pulse sequences available in the spectrometer.

Extraction and isolation of the essential oil compounds
Hyptis ovalifolia Benth.(Lamiaceae) specimens were colleted in Goiânia, state of Goiás, Brazil.Voucher specimens (20.283) were deposited in the Herbarium of the Universidade Federal de Goiás (UFG).

Assay for antifungal testing
In vitro antifungal activity of the compound 1 was evaluated against dermatophytes using a serial dilution agar technique.The compound 1 was solubilized in 1mL of dimethyl sufoxide (DMSO) and serially two-fold diluted in Mycobiotic agar medium (Difco, Detroit, Mich.) to obtain a concentration range of 3.9-1000 µg mL -1 .Dermatophyte suspensions in a sterile 0.85% saline solution containing Tween 80 (0.05%) were adjusted to give a final concentration of 1x10 6 CFU mL -1 .The holes in the solid agar media were inoculated with 10 mL of the dermatophyte suspensions and plates were incubated at 25 °C for 5 days.Negative control plates containing only diluted DMSO, as well as positive control plates impregnated with Itraconazole (0.06-250 µg mL -1 ) were included in each assay.The minimal inhibitory concentration (MIC) was defined as the lowest concentration of the compound 1 that inhibited any visible fungus growth.

Results and Discussion
In a preliminary screening, the essential oil obtained from the leaves of H. ovalifolia was found to be an efficient antifungal agent against the dermatophytes M. canis, M. gypseum, T. rubrum and T. mentagrophytes.It inhibited these fungi at a concentration of 31.2 µg mL -1 and most often at even lower concentrations (Table 1).The analysis of the essential oil by GC/MS has enabled the identification of the following compounds (relative concentrations and retention index given in parentheses): αcopaene (0.84%; 1386), β-bourbonene (1.58%; 1405), (Z)caryophyllene (0.74%; 1409), γ-elemene (4.38%; 1438), α-humulene (1.05%; 1474), γ-cadinene (6.60%; 1506), viridiflorol (6.08%; 1599), caryophyllene oxide (4.98%; 1606) and α-cadinol (0.74%; 1674), among other minor constituents.The identification of the compounds was based on the comparison of their mass spectra with those of the NIST and by comparison of their retention indices with those provided in the literature. 7However, the main peak representing 60% of the total oil composition could not be identified by the mass spectra and retention index, thus it was isolated for further investigations.The unknown compound was obtained by the fractionation of the essential oil on a silica gel column. 13C NMR and DEPT

Table 1 .
In vitro antifungal activity of compound 1 obtained from the essential oil of H. ovalifolia leaves minimal inhibitory concentration; b N (number of strains); PC (positive control -Itraconazole); EO (Essential Oil); numbers into ( ) means percentage of strains. a