Artículo original

 

Chemical composition and modifying activity of essential oil of Lantana camara L. (wild sage) against Candida strains

Composición química y actividad modificadora del aceite esencial de Lantana camara L. (camará) contra cepas de Candida

 

Felicidade Caroline Rodrigues1
José Weverton Almeida Bezerra1*
Nadghia Figueiredo Leite2
Antonia Eliene Duarte2
Adrielle Rodrigues Costa3
Kleber Ribeiro Fidelis3
Juliana de Lima Silva3
Aline Augusti Boligon4
Maria Ivaneide Rocha3
Luiz Marivando Barros5
Henrique Douglas Melo Coutinho5
Jean Paul Kamde5
Maria Flaviana Bezerra Morais-Brag5

 

1 Posgraduate Program Plant Biology, Recife, PE, Brazil.
2 Microbiology and Molecular Biology Laboratory, Regional University of Cariri (URCA), Crato, CE, Brazil.
3 Microscopy Laboratory, Regional University of Cariri (URCA), Crato, CE, Brazil.
4 Departamento de Ciências Biológicas, Universidade Regional do Cariri (URCA), Crato, CE, Brazil.
5 Microbiology and Molecular Biology Laboratory, Regional University of Cariri (URCA), Crato, CE, Brazil.

*Autor para la correspondencia. Correo electrónico:weverton.almeida@urca.com

 

 

ABSTRACT

Introduction: Fungal infections, particularly those caused by the genus Candida, are an important public health problem. Lantana camara L. stands out among the medicinal species with abundant biological and pharmacological activity.
Objective: Analyze the phytochemical profile of essential oil of L. camara and evaluate its antioxidant capacity and modifying antifungal potential against various Candida strains.
Methods: Identification of the chemical components present in the essential oil of L. camara was performed by gas chromatography / mass spectrometry (GC/MS). Antioxidant activity was determined by elimination of free DPPH radicals. Antifungal activity of the essential oil of L. camara leaves was evaluated by microdilution against Candida albicans, C. krusei and C. tropicalis.
Results: It was found that the essential oil of L. camara leaves is rich in terpenes, mainly sesquiterpenes, (E)-caryophyllene (25.80 %), germacrene D (17.36 %), γ-elemene (7.62 %) and sabinene (6.61 %). The essential oil displayed very low activity in DPPH radical elimination, while it displayed antimicrobial activity against the study strains (MIC ≥ 1 024 μg/ml), but it significantly modified the effect of fluconazole against Candida kruzei and C. tropicalis.
Conclusions: The essential oil of L. camara was found to be a good modifier of fluconazole, but it does not have any relevant antioxidant activity.

Key words: modifying activity, antioxidant activity, wild sage, Lantana camara.

RESUMEN

Introducción: Las infecciones fúngicas, particularmente las causadas por el género Candida, son un importante problema de salud pública. Entre las especies medicinales con abundante actividad biológica y farmacológica se destaca Lantana camara L.
Objetivo: Analizar el perfil fitoquímico del aceite esencial de L. camara, así como evaluar su capacidad antioxidante y el potencial modificador antimicótico contra diferentes cepas de Candida.
Métodos: Se identificaron los componentes químicos presentes en el aceite esencial de L. camara mediante GC/MS. Se comprobó la acción antioxidante mediante la actividad de eliminación de radicales libres de DPPH. Se evaluó la propiedad antifúngica del aceite esencial de hoja de L. camara mediante el método de microdilución contra Candidia albicans, C. krusei y C. tropicalis.
Resultados: Se demostró que el aceite esencial de la hoja de L. camara es rico en terpenos, principalmente sesquiterpenos, (E)-cariofileno (25,80 %), germacreno D (17,36 %), γ-elemeno (7,62 %) y sabineno (6,61 %). El aceite esencial mostró muy baja actividad para eliminar radicales DPPH y demostró actividad antimicrobiana contra las cepas probadas (CIM ≥ 1024 μg/mL), pero modificó significativamente el efecto del fluconazol contra Candida kruzei y C. tropicalis.
Conclusiones: Se evidenció que el aceite esencial de L. camara es un buen modificador de fluconazol, pero no presenta ninguna actividad antioxidante relevante.

Palabras clave: actividad modificadora; actividad antioxidante; camará, Lantana camara.

 

 

Recibido: 02/05/2018
Aprobado: 19/07/2018

 

 

INTRODUCTION

Fungal infections have been growing in recent years, mainly due to the microbial resistance, acquired from abusive use of the first choice drugs. The frequency of such infections and the continued growth of multiresistant organisms has motivated a continuous search for new drugs that exhibit biocidal property with reduced or without side effects.1-3 Therefore, natural products from plant origin appear to be an alternative source for the development of potential drugs with these characteristics.4,5 They have been shown to possess a variety of secondary metabolites that act against fungi.6,7 In addition, numerous studies have demonstrated that the combination of plant derived compounds with conventional antimicrobial drugs resulted in improved effect in comparison to that obtained with antimicrobial drugs alone.3,8-11 Although the quantity of antifungal medicines has increased in recent years, this number still represents a small amount compared to standard antibiotics.12

Yeasts of the genus Candida is known to cause various infections, and it is considered the major challenge in medical practice. In humans, species of this genus can reach the mucosal and cutaneous surfaces and can cause oral, vaginal, onychomycosis, and intertrigo candidiasis. In some cases, they be invasive.13

One of the most frequently used plant components is essential oils (EOs) derived from the secondary metabolism of plants, since they have several biological activities, such as bactericidal, fungicidal, antiseptic and others.14 However, in spite of the fact that they are generally regarded as safe without any side effects, studies are necessary to determine their chemotypes, since they may be toxic to the organism.15

Free radicals are constantantly produced in living organisms as by-products of the mitochondrial respiration, and they have been reported to be implicated in the pathophysiology of numerous diseases including infectious diseases, cardiovascular and neurodegenerative diseases. Free radicals are molecules that have lost atoms in their metabolic processes and require a new atom to pair and become stable. In order to stabilize, they cause damage to lipids, proteins and DNA, leading to the alteration of cell structure and function.16,14 However, the organism is equipped with enzymatic antioxidants (catalase, superoxide dismutase and glutathione peroxidase) that act by inhibiting or retarding the action of these free radicals18. In addition to endogeneous antioxidants, other antioxidants can come from exogeneous source like ascorbic acid, which is widely found in vegetables and fruits, particularly in the fruits of the Rutaceae family.19.As in this taxon, some botanical families, such as Verbenaceae, possess secondary compounds of the EOs, that exhibit a variety of pharmacological activities, among which antioxidant activity.20,21

Lantana is a genus composed of about 150 species,22 and it is known mainly for the fact that they are almost immune to herbivory and contain a great diversity of natural substances.23 Lantana camara L. belonging to the Verbenaceae family, is a shrub native to America and Africa and has been cultivated as an ornamental plant in many countries . L. camara, popularly known as "camara", "cambará" or "camará de espinho", is used in the treatment of ulcers, rheumatism, asthma, hypertension, cancer, fever, toothache, bronchitis and as an antiseptic.21,22,24.It has been reported that L. camara exhibit leishmanicidal and antibacterial potential25.in addition to its anti-inflammatory and antitumor action.26,27

Based on the abovementionned information, this study aims to analyze the phytochemical profile of the essential oil of L. camara, a species with several biological and pharmacological activities, as well as to evaluate its antioxidant capacity and antifungal modulator potential against different Candida strains.

 

 

METHODS

Plant Material

The leaves of L. camara were collected in the municipality of Crato, Ceará, Brazil, with the following coordinates 7 ° 22'S; 39 ° 28'W, 492 m above sea level, at 9:00 in June 2012. The plant material was identified and a specimen was deposited in the Herbarium Caririense Dárdano de Andrade Lima - HCDAL of the Regional University of Cariri - URCA, under the number 7,518. The study was carried out in accordance with the existing ethical regulations and principles for biodiversity rights, and the application number of the System of Authorization and Information on Biodiversity - SISBIO for the collection of the botanical material was 64034-6.

Obtention of L. camara leaf essential oil

The EO of the leaves of L. camara was obtained according to the methodology proposed by Guenther,28 in a Clevenger type apparatus with some modifications. The leaves were dried in the shade and then crushed to increase the contact area, placed in a volumetric flask where 300 mL of distilled water was added. Immediately after, the flask was placed in Clevenger type apparatus and heated on a heating blanket until the material boiled within 2 hours.

Analysis of the chemical composition of L. camara essential oil

The chemical composition of the EO of L. camara dried leaves was performed by Gas Chromatography coupled to Mass Spectrometry (GC/MS) using a Shimidzu, QP2010 Series equipment. The capillary column used was Rtx-5MS type measuring 30 mm in length by 0.25 mm in diameter and 0.25 mm in thickness of the film. Helium gas was used as carrier at a rate of 1.5 mL/min. The temperature of the injector was 250 °C and the detector was 290 °C. The temperature of the column initially varied from 60 to 180 °C, increasing by 5 ° C/min, then ranged from 180 to 280 ° C by increasing by 10 °C/min. The EO was diluted 1: 200 in chloroform and the injected volume was 1 μL. The mass spectrophotometer was set to an ionization energy of 70eV. The identification of the individual components was based on their mass spectrum fragmentation according to their NIST Mass 08 spectral library, retention indices and comparison with published data.29,30

Strains of Candida and standard antifungal drugs

The antifungal activity of L. camara leaf EO was investigated against three strains of Candida: C. albicans ATCC 40006, C. krusei ATCC 40095, and C. tropicalis ATCC 40042.

For comparative purpose, the standard antifungal drugs, Nystatin and Fluconazole (Sigma Co., St. Louis, USA) were used at concentration of 2048 μg/mL. The drugs were dissolved in sterile water.

Determination of minimum inhibitory concentration (MIC)

The microdilution method was used to determine the MIC value of L. camara leaf EO. To reach the 1024 μg/mL concentration to be used in the tests, 0.105 g of the EO was diluted in 1 mL of dimethylsulfoxide (DMSO), which was then diluted in distilled water. The inoculum was diluted in 10% of Brain Heart Infusion (BHI) to a concentration of 105 CFU/mL. 100 μL of the BHI and the inoculum were distributed into each well of a 96-well plate and then the serial microdilution was performed with the 100 μL solution of the extract, varying from concentrations of 2 to 1.024 µg/mL. The plates were taken to the incubator for 24 hours at 37 °C.31 The MIC of fungi was determined by turbidity. The MIC was defined as the lowest concentration where no growth can be observed.32

Evaluation of the modulatory potential of the essential oil

In order to analyze whether the EO would have the potential to modify the action of the antibiotics against the strains tested, the methodology proposed by Coutinho et al.33 was used, where the oil solutions were tested at subinhibitory concentration (MIC/8) of 128 μg/ml. 100 μL of a solution containing BHI, inoculum and EO were distributed in each well in the alphabetical direction of the plate. Then, 100 μL of the drug was mixed into the first well, serially microdiluted, in a ratio of 1:1 to the penultimate cavity. Antifungal concentrations ranged from 0.5 to 512 μg/mL.

In vitro antioxidant activity

The radical scavenging ability of the EO of L. camara (EOLC) was performed using the free radical DPPH (1,1-diphenyl-2-picrylhydrazyl), where 50 μL of the EO at different concentrations (1-80 μg/mL), were mixed with 100 μL of 0.3 mM DPPH in ethanol. The plate was then held in the dark at room temperature for 30 min and read, which was measured by monitoring the absorption drop at 517 nm using a microplate reader (SpectraMax, Sunnyvale, CA, USA). Ascorbic acid (1-80 μg/mL was used as the standard compound

Statistical analysis

Data were expressed as mean ± standard error of the mean (SEM). The analyzes were done with GraphPad Prism 6 software, using One-way ANOVA, followed by the Bonferroni test.

 

 

RESULTS

Chemical composition of L. camara leaf EO

Table shows the chemical composition of L. camara leaf EO analyzed by GC/MS. A total of 29 components were found, totalizing 94.44 % of the chemical composition identified. The major components were (E)-caryophyllene (25.80 %), germacrene D (17.36 %), γ-elemene (7.62 %) and sabinene (6.61 %); while the compounds found in small amounts were α-tujene (0.22 %), Β-cymene (0.24 %), β-terpineol (0.32 %) and borneol (0.34 %).

 

 

Modulation

EOLC, tested against strains of Candida alone was not clinically relevant since it showed a MIC value ≥1,024 μg/mL (data not shown). However, when associated with fluconazole at the sub-inhibitory concentration, it significantly potentiated the effect of the drug, againstC. krusei ATCC 40095 (Figure 1) and C. tropicalis ATCC 40042 (Figure 2).

 

In contrast, its association with the antifungal drug nystatin, did not show any significant change in the MIC value against all the Candida strains tested (Figure 3).

In vitro antioxidant activity of L. camara leaf essential oil

The potential antioxidant activity of L. camara leaf essential oil evaluated by its capacity to scavenge DPPH radical is shown in Figure 4. Our results show that L. camara leaf EO exhibited very low antioxidant activity at all the concentrations tested, in comparison to ascorbic acid used as standard antioxidant (IC50 = 42.43 μg/mL).

 

 

DISCUSSION

There is a constant interest in finding in the plant kingdom, organisms that have therapeutic efficacy in the treatment of various diseases. L. camara from a medicinal point of view is one of the most important plants in the world34 and, therefore, has been one of the most studied species in this sense. The present study determined the potential antifungal modulator of the EO of L. camara, as well as its antioxidant properties and its phytochemical content.

The chemical components of L. camara leaf EO obtained in our study was similar with that obtained by Barros et al.,21 but in different amount. Barros et al.21 found (E)-caryophyllene (23.75 %), bicyclogermacrene (15.80 %), germacrene D (11.73 %), terpinolene (6.01 %), and sabinene (5.92 %) as the major components in L. camara leaf EO, thus confirming that the plant is rich in mono and sesquiterpenes. However, our results are different to that obtained by Costa et al., 35 in which the major components of L. camara leaf EO were bicyclogermacrene (19.42 %), isocharyophyllene (16.70 %), valencene (12.94 %) and germacrene D (12.34 %).

Many factors can justify the variability of compounds found in L. camara leaf EO, including the geographic origin, genetic variability36,37 collection time, age and development of the plant, temperature, water availability, ultraviolet radiation, nutrients, altitude, and atmospheric pollution. In addition, induction by mechanical stimuli or attack of pathogens and variation during the light/dark cycle of the plant can also alter the chemical composition of the species38 and may thus interfere with its antimicrobial activit 39. As in the study of Barros et al.,21 the leaves were collected in the same region and at the same time, which may explain at least in part the reason why there were no significant difference in the composition of our oil compared to that obtained by Barros et al.21

Previous reports have shown that plants of the Verbenaceae family have similar chemical composition and that these are potential sources of the compounds (E)-caryophyllene and germacrene D.37 In addition, Sousa et al.24 reported that germacrene D and bicyclogermacrene compounds are common constituents of parts of the genus Lantana.

In the current study, it was possible to observe that the EO in combination with fluconazole significantly potentiated the antifungal effect of the drug against C. krusei and C. tropicalis. The combination between the natural product and the antifungal standard alters the permeability of the cellular membrane of the fungi, favoring in this way the intracellular access of the drug that in turns inhibits the synthesis of ergosterol, an important component of the fungal membrane, thus causing strains to die at low concentrations.1

The association between fluconazole and the natural product may also be a way of reducing the side effects of the antibiotics used, since the minimum inhibitory concentration of the antifungal drugs would be lower, which would reduce the doses of its use.1,40 In addition, studies have demonstrated the potentiating effect of EOs with some antifungal agents at the sub-inhibitory concentrations (MIC/8 = 128 μg/mL).41

Our results demonstrated that there was no synergism or antagonism when combining the EO with nystatin against the Candida strains tested. Only the combination of fluconazole with the EO against C. albicans did not result in any significant change. This can be explained by several factors, one of which is the genomic plasticity characteristic of C. albicans strains, as seen for example in aneuploidies, which, for many, is associated with antifungal resistance gain.42

Another fact related to resistance to fluconazole is the expression of genes that encode mechanisms that confers resistance to fungi, such as the development of efflux pumps, a mechanism capable of expelling substances that are considered harmful to cells; alterations of the target enzyme of the azol components preventing the attachment of these azoles to the enzyme site; genetic changes such as chromosome duplication or even the action of several of these mechanisms together.43 In addition, fluconazole is a fungistatic drug and is always the drug of first choice, which favors the development of antifungal resistance.42

Razo et al.44 reported that in natural products, the IC50 results are in the range of 1-500 μg/mL, suggesting that L. camara EO has very low antioxidant activity, since its concentrations tested did not inhibit 50% of DPPH radical.

EOs are complex mixture of various components, in which one or more compounds can be responsible for their biological activities. 45-47 In the study by Benites et al.,48 the EO of L. camara showed high antioxidant activity and the authors attributed this activity to carvone (75.9 %) and limonene (16.9 %), which were the major components found in the oil. In our study carvone was not present and limonene represented only 3.43 % of L. camara leaf EO. Thus, it is possible that the lower antioxidant activity of L. camara leaf EO (IC50/DPPH > 500μg/mL) obtained in this study is due at least in part, to the absence of the carvone.

Our results suggest that the essential oil of L. camara is a good modulator of fluconazole, but it does not present any relevant antioxidant activity, in addition, EO is rich in mono- and sesquiterpenes.

 

 

Conflicto de intereses

Los autores expresan que no existen conflicto de intereses.

 

 

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