Antifungal, cytotoxic and chemical
analyses of essential oils of Lippia
origanoides H.B.K grown in Colombia

Liliana Betancur-Galvis¹, Bibiana Zapata¹, Armando Baena², Juan Bueno³,
Carlos Alberto Ruíz-Nova², Elena Stashenko⁴, Ana Cecilia Mesa-Arango¹

1. Grupo de Investigación Dermatológica (GRID), Facultad de Medicina, Universidad de Antioquia
2. Grupo Infección y Cáncer, Facultad de Medicina, Universidad de Antioquia.
3. Grupo de Micobacterias, Instituto Nacional de Salud, Bogotá, D.C., Colombia
4. Centro de Investigación en Biomoléculas, CIBIMOL. Universidad Industrial de Santander ]]> Correspondencia: Liliana Betancur-Galvis. Spc, MsC, PhD. Chemist. Group of Investigative Dermatology (GRID), School of Medicine, University of Antioquia, Medellín, Colombia.
Cra. 51D # 62 -29 Lab 283. Phone: (+574) 2196064. A.A. 1226. Medellín, Antioquia, E-mail: labeta@quimbaya.udea.edu.co.
Recibido: 14 de febrero 2011- Aceptado: 10 de agosto de 2011

ABSTRACT

Introduction: Aspergillus fumigatus is most commonly associated to invasive aspergillosis. Strong antifungal activity against A. fumigatus of L. origanoides essential oil gives a new added value to this natural product from Boyacá-Colombia. Aims: The increase in fungal infections, the development of resistance and toxicity of wide-spectrum antifungals have led to a constant search for therapeutic alternatives. The chemical composition, antifungal and cytotoxic activity of nine essential oils obtained from L. origanoides were evaluated and the relationship between the antifungal activities of the oil and of its major components were explored. Methods and Results: Antifungal activity was determined following the protocols AFST-EUCAST for Candida krusei and C. parapsilosis, and CLSI-M38A for Aspergillus fumigatus and A. flavus. The GC-MS analysis identified three chemotypes: thymol, carvacrol and p-cymene/trans-beta-caryophyllene. The essential oil of the thymol chemotype was the most active in antifungal assays with MIC values of 157.5, 198.4, 125 and 31 μg ml-1 against C. parapsilosis, C. krusei, A. flavus and A. fumigatus, respectively. The major components carvacrol and thymol were not active against A. fumigatus at concentrations below 157.5 μg ml-1. In general, the oils were not cytotoxic. Conclusions: The essential oil of the thymol chemotype of L. origanoides from the region of Boyacá- Colombia showed the highest antifungal activity against A. fumigatus among all the oils and major components tested. Salud UIS 2011; 43 (2): 141-148

Keywords: Lippia origanoides, essential oil, antifungal activity, cytotoxicity, monoterpenes

Actividad antifúngica, citotóxica y composición
química de aceites esenciales de Lippia
origanoides H.B.K recolectadas en Colombia

RESUMEN

Palabras Clave: Lippia origanoides, aceite esencial, actividad antifúngica, citotoxicidad, monoterpenos

INTRODUCTION

The AIDS epidemic, chemotherapy in cancer patients, neutropenia and immunosuppressant therapies in recipients with transplants have increased the number of cases of mycotic infections^1,2. Species of the genera Candida, Cryptococcus, Aspergillus, Histoplasma, Rhizopus, Mucor, Acremonium, and Fusarium are the most frequent causes of these infections¹. Although C. albicans is the most frequent species, the prevalence of other non-albicans species has been increasing recently³. Aspergillus infections have also grown in importance in the last years^4,1. A. fumigatus and A. flavus are the most widespread causes of invasive aspergillosis⁴.
The development of resistance of some species to the antifungal drugs available and the toxicity of widespectrum antifungals, such as amphotericin B, have led to a constant search for therapeutic alternatives². Many aromatic plants are frequently used in traditional medicine as antimicrobial agents, and the essential oils recovered from some of them have demonstrated antibacterial and antifungal effects^5,6. In addition, some oils have shown activity against fluconazole and itraconazole resistant Candida spp.⁵
Lippia origanoides H.B.K. (Verbenaceae) is a shrub up to 3 m tall that grows wild in Central America (Mexico, Guatemala, Cuba) and northern South America (Guiana, Venezuela, Brazil, Colombia)7,8. In Colombia, it is found at altitudes between 500 and 800 m in several Andean states and in the northern peninsula of Guajira. Its green oval leaves are employed as a condiment (due to their thymol and carvacrol content) and as a traditional medicine (flower and leaf infusions)^9,8. In Mexico, L. origanoides is called "oregano", and the Mexican Pharmacopoeia recognizes it as a substitute for common oregano – L. graveolens Kunth⁸. Essential oils with high thymol content have showed MIC values of 3.3 mg ml-1 against C. albicans¹⁰. Oliveira et al.⁸ studied the antifungal activity of the essential oil of L. origanoides which presented high carvacrol content and showed active against human pathogenic fungi of genera Candida.
The composition and biological activity of essential oils from L. origanoides H.B.K. grown in Colombia have not been studied. In the present work, the chemical composition, antifungal and cytotoxic activity of nine essential oils obtained from L. origanoides, collected from four Colombian regions were evaluated and the relationship between the antifungal activities of the oil and of its major components were explored.

MATERIALS AND METHODS

Plant material and essential oil extraction
Nine samples (1 kg each) of L. origanoides stems and leaves were collected from four regions in Colombia, as part of a survey conducted by CENIVAM, a Research Center devoted to the study of aromatic plants and essential oils in Colombia. The taxonomic identification of the botanical samples was performed by Dr. José Luís Fernández at "Herbario Nacional de Colombia (COL)", Institute of Natural Sciences, School of Sciences, "Universidad Nacional de Colombia" (Bogotá), where exsiccate of each plant remain as permanent samples. The essential oils were extracted from dried L. origanoides stems and leaves (300 g) by microwave-assisted hydrodistillation as described previously¹¹. Anhydrous sodium sulfate (Merck, Darmstadt, Germany) was added as a drying agent to the decanted essential oil. The voucher numbers, the region of plant collection, date of collection and the codes assigned to the essential oils obtained are presented in (Table 1). ]]> Analysis of essential oils -Compound identification was based on mass spectra (EI, 70 eV), obtained with a gas chromatograph (Agilent Technologies 6890 Plus, Palo Alto, CA, USA), equipped with a mass selective detector (Agilent Technologies 5973), a split/splitless injector (split ratio 1:50), and a data system (HP ChemStation 1.05) with WILEY 138K, NIST 2002 and QUADLIB 2004 mass spectra libraries, as described previously¹². Individual components were identified by comparing their retention indices¹¹ determined using a linear scale on the DB-5MS (60m x 0.25mm x 0.25μL, J and W Scientific, Folsom, CA, USA) column, and the mass spectra of each GC component to those of standard substances (Sigma- Aldrich, USA; Table 2). The hydrodistillation process ensured endotoxin-free material, since such a technique is unable to remove high-molecular mass molecules such as endotoxins (10 kDa) from plant material, and the molecular weight of essential oil components does not surpass 0.3 kDa. Stock solutions of oils were prepared in DMSO and frozen at -70°C until required.

Monoterpenes and drug
Thymol, carvacrol, p-cymene, trans-beta-caryophyllene, gamma-terpinene and beta-myrcene and other standard substances for the analysis of essential oils were from Sigma (Chemical Company St Louis, MO, USA). Stock solutions of both oils and monoterpenes were prepared in DMSO for cytotoxicity and antifungal assays. Amphotericine B and itraconazole were from Sigma.

Antifungal activity assay
The antifungal activity of the oils and monoterpenes was evaluated following the Clinical and Laboratory Standards Institute M38-A protocol¹³ for filamentous fungi, and the standard method proposed by the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (EUCAST)¹⁴ for fermentative yeasts, as described previously¹². Candida parapsilosis (ATCC 22019), C. krusei (ATCC 6258), Aspergillus flavus (ATCC 204304) and A. fumigatus (ATCC 204305) served to evaluate antifungal activity. For the EUCAST method, MIC were determined after 24 hours of incubation, and defined as the lowest concentration that resulted in a 90% reduction of growth. For the CLSI M38-A method, the MICs were determined after 48 hours of incubation, and defined as the lowest dilution of essential oils that resulted in total inhibition of visible growth. Susceptibility testing was performed in duplicate in three different assays. Essential oils and monoterpenes were considered active when they presented MIC values below 500 μg ml-1.

Cytotoxicity assay
Cercopithecus aethiops African green monkey kidney cells (Vero cell line ATCC CCL-81) were used. The cytotoxicity of the essential oils and their major components were examined in vitro with an MTT (dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) (Sigma, New Jersey, USA) assay as described in Mesa-Arango et al.¹². The concentration of compounds that induces 50% growth inhibition in 48h was expressed as IC₅₀. Vincristine was used as positive control (Sigma-Aldrich, Co, MO, USA).
Data analysis - The IC₅₀ values for each compound were obtained by lineal regression analysis of the dose– response curves generated from the absorbance data with the statistical package R (Development Core Team, Vienna, Austria, 2008). IC₅₀ values are expressed as the Mean ± S.E.M. of at least four dilutions performed in quadruplicates in two different assays. MIC values are expressed as geometric means (GM-MIC) of tests performed in duplicate in three different assays against each of the fungal species.
To test the association of oil activity with chemical composition of the major components, the Cox model with the determination of the hazard ratio was applied; a 95% confidence interval was adopted. Statistical analyses were conducted using the statistical package R (Development Core Team, Vienna, Austria, 2008).

RESULTS

As can be observed in (Table 3), only the 4D oil did not show activity for the fungi_evaluated. The 3C oil showed the highest antifungal activity against C. parapsilosis, C. krusei and A. flavus but not against A. fumigatus with MG-MIC values of 157, 198, 125, 63 μg ml-1, respectively. The 7G oil showed the highest antifungal activity against A. fumigatus, with MG-MIC value of 31 μg ml-1. In general, A. fumigatus was more sensitive to essential oils than A. flavus and Candida species. The monoterpenoids, trans-beta-caryophyllene, gammaterpinene, and beta-myrcene did not show activity at concentrations of 500 μg ml-1 for the fungi evaluated. Among the six compounds tested, carvacrol and thymol were the most active against the fungi evaluated.
To examine the correlation between oil activity and the major components in situ, percentage composition of the major components of the oils were screened with oil antifungal activity in a Cox model. (Table 4) shows the results of these analyses that were adjusted for each fungus. Thymol and carvacrol were the major components that were associated with oil antifungal activity for Candida species. For A. fumigatus and A. flavus, thymol/beta-myrcene and thymol/carvacrol/pcymene were the monoterpenoids associated with oil antifungal activity, respectively.

DISCUSSION

Lippia origanoides H.B.K. Fam. (Verbenaceae) is a slender, very aromatic shrub⁷. Oliveira et al.⁸ reported the composition of L. origanoides essential oil from Oriximiná - Brazil, in which carvacrol was the main constituent (38%), followed by thymol (18%) and p-cymene (10%). Rojas et al.¹⁵ compared the compositions of L. origanoides oils from leaves collected during the rainy and dry seasons in Venezuela; thymol (61.9 and 44.7%) and carvacrol (7.9 and 16.8%) were the main components in these oils, respectively. Oliveira et al.⁸ suggests the existence of two different chemotypes for this species – thymol and carvacrol.
In the present study, the chemical composition of nine essential oils obtained of L. origanoides collected from four Colombian regions were evaluated. The GC/MS analysis of the oils revealed the presence of three chemotypes: thymol, carvacrol, and p-cymene/ trans-beta-caryophyllene. The p-cymene/trans-betacaryophyllene chemotype was reported previously¹⁶, and it was observed that the composition of the oil was not caused by microclimate or geobotanical differences. The findings suggest that there are different chemotypes for this species, in a way similar to what happens with Lippia alba⁵. In spite of the existence of several L. origanoides chemotypes in Colombia, there had been no evaluation of the antifungal and cytotoxic activity of their essential oils.
With respect to antifungal activity there is no agreement on the acceptance level of activity for plant material when compared to standard drugs¹². Duarte et al.¹⁷ indicated a strong activity of L. alba essential oils against C. albicans with a MIC value of 60 μg ml-1, when nistatin was used as positive control (MIC of 50 μg ml-1). According to these criteria the 7G oil was very active against A. fumigatus with a MIC value of 31 μg ml-1. The 3C oil showed a strong antifungal activity against the four fungi evaluated. Both oils were thymol chemotypes. In contrast the oils 2B and 4D, p-cymene-/ trans-beta-caryophyllene chemotype, showed moderate and weak activity, respectively.
Studies of antifungal activity of essential oils with high thymol and carvacrol have been carried out on Candida species. Botelho et al.¹⁸ evaluated the essential oil activity from L. sidoides of the thymol (56.7%)/ carvacrol (16.7%) chemotype against C. albicans. The resulting value of the MIC was about a thousand times higher than the ketoconazole value. Oliveira et al.⁸ showed the activity of the thymol (18.5%)/carvacrol (38.6%) chemotype of L. origanoides essential oil against Candida species. In contrast, in the present study A. fumigatus was more sensitive to essential oils of L. origanoides than Candida species. The most active oil with content of thymol (54.5%) and carvacrol (1.7%) showed a MIC value against A. fumigatus about ten times higher than the Amphotericin B value. Interestingly, the 6F oil obtained by extraction for 15 min and with the highest thymol concentration was less active than 7G oil obtained of the same plant by extraction for 30 min. This finding shows the importance of the percentage composition of components of oils.
To determine the contribution of each major component to L. origanoides oil activity, the antifungal activity of the thymol, carvacrol, p-cymene, trans-beta-caryophyllene, gamma-terpinene and beta-myrcene were evaluated. The lack of activity of p-cymene and gamma-terpinene is in agreement with previous studies¹⁹. However, Tampieri et al.²⁰ reported that monoterpenes as gammaterpinene, p-cymene and carvacrol displayed good antifungal activity at 100 μg ml-1 against C. albicans. In our study, only thymol and carvacrol were active against the fungi evaluated, however, their MIC values were higher than those found for the 3C and 7G oils. ]]> It is difficult to attribute the activity of a complex mixture as it is an essential oil to some particular constituent. Therefore, to confirm the correlation of oil activity with the percentage_of their major components in situ, in a Cox model, percentage composition of the major components of the oil were screened to evaluate a possible relationship with oil antifungal activity. Thymol and carvacrol were the major components that were more closely associated with oil antifungal activity for Candida species. However, for species of Aspergillus fumigatus these analyses suggest that there is a concentration-dependent effect among the monoterpenes thymol, carvacrol, and p-cymene.
A. fumigatus is the most prevalent species in the genus and it is most commonly associated to invasive aspergillosis²¹. In our study, essential oils from Boyacá-Colombia L. origanoides have shown a marked antifungal activity against A. fumigatus and their activity could give a new added value to these natural products. Essential oil from Boyacá-Colombia L. origanoides is promising candidate to evaluate their gaseous contact activity against filamentous fungi and could be employed to sterilize hospital rooms of immunosuppressant patients.

ACKNOWLEDGMENTS

Financial support from COLCIENCIAS, Bogotá, Colombia (Grant RC 432-2004 and 245-2011) is gratefully acknowledged.

ETHICAL CONSIDERATIONS

Ethical compliance is not applicable to this study.

CONFLICT OF INTEREST

The authors have no conflicts of interest to declare. Liliana Amparo Betancur Galvis certify that: The manuscript represents valid work and that neither this manuscript nor one with substantially similar content has been published under my responsibility or is being considered for publication elsewhere. There are no financial interests in relation to this manuscript. All material and financial support for this work is clearly expressed in the manuscript.

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