Antileishmanial activity of Eugenol-rich essential oil from Ocimum gratissimum
Introduction
Protozoan parasites of the genus Leishmania cause visceral, cutaneous, and mucosal diseases in humans, which are collectively referred to as leishmaniasis. These diseases affect more than 12 million people worldwide and are responsible for high rates of mortality in tropical and subtropical countries. A major emerging problem is coinfection of Leishmania with human immunodeficiency virus, especially because there is no effective treatment for these patients. The leishmanial diseases are transmitted to the vertebrate host by the bite of a sandfly. The promastigote in the stationary growth phase is the infective form of the parasite, it penetrates a mononuclear phagocyte, differentiates into an amastigote and proliferates intracellularly [1].
The number and efficacy of drugs available for the treatment of human and animal trypanosomiasis, leishmaniasis, amoebiasis and malaria are limited. Considering the side effects and the resistance that pathogenic protozoan parasites develop against these drugs, more attention should be given to the extracts and biologically active compounds isolated from plant species commonly used in herbal medicine [2], [3], [4].
Ocimum gratissimum L. (Lamiaceae) is used to treat a variety of diseases, e.g., upper respiratory tract infections, diarrhea, headache, fever, ophthalmic and skin diseases, and pneumonia [5], [6]. The oil of O. gratissimum is also active against several species of bacteria (Escherichia coli, Shigella, Salmonella, and Proteus), yeasts (Candida albicans, C. tropicalis, C. parapsilosis, and C. krusei), and dermatophyte fungi (Trichophyton rubrum and T. mentagrophytes) [7], [8], [9], [10], [11], [12].
Eugenol is the major component of essential oil of O. gratissimum. Eugenol is an important chemical constituent of the essential oils of many aromatic plants, such as Eugenia caryophyllus, Dicipelium cariophyllatum, Pimenta dioica, Croton zehntneri var. eugenoliferum, and Croton zehntneri [13]. Eugenol is used as an aromatic agent for food and fragrance in the cosmetic industry; it is also commonly used in dentistry for sedation of toothache, pulpitis, and dental hyperalgesia. Several biological actions of eugenol have previously been reported. For instance, this compound induces central nervous system effects in mammals such as hypothermia, decrease in spontaneous motor activity, anticonvulsant, and general anaesthesia. Eugenol and its analogue methyl–eugenol are potent depressants of peripheral nervous activity and of excitation–contraction coupling in skeletal muscle [14]. Both molecules induce myorelaxant, antispasmodic and vasorelaxant effects on both the rat and rabbit thoracic aorta, as well as on the rat mesenteric vascular bed [15], [16].
Nitric oxide (NO) is the principal effector molecule mediating intracellular killing of Leishmania, although the mechanisms by which phagocytes kill microorganisms are still not completely understood. Undoubtedly, phagocyte-derived reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important molecules in this process. Some molecular targets are inhibited by NO, such as cisaconitase [17], glyceraldehyde-3-phosphate dehydrogenase [18], and cysteine proteinases [19]. Also, NO may be involved in initiating programmed cell death [20].
The present study reports the effects of the essential oil from O. gratissimum on growth, viability, and ultrastructural alterations of amastigote and promastigote forms of L. amazonensis, as well as on the interaction of these flagellates with mouse peritoneal macrophages, concomitant with nitric oxide production by the infected macrophages. These results strongly suggest that the essential oil from O. gratissimum and its major component eugenol may be used in studies for the development of new drug therapies for leishmaniasis.
Section snippets
Plant material
O. gratissimum was collected in Maringá, Paraná, Brazil, in March 2003 and identified. A Voucher No. Sp.P1.1197.175 is deposited in the Herbarium of the Instituto Agronômico de Campinas, São Paulo, Brazil. Fresh leaves from the plant were cut into pieces and subjected to steam distillation. The distillate was then extracted with petroleum ether, which was removed carefully, and the essential oil was obtained. The oil was then divided into aliquots and stored at − 20 °C until use.
Isolation and identification of major constituent
The essential
Inhibition of parasite growth
The minimum inhibitory concentration (MIC) of O. gratissimum essential oil for both promastigotes and amastigotes was 150 μg/ml.
Antileishmanial activity
The effects of the essential oil of O. gratissimum on the viability of L. amazonensis were tested. The time courses of the viability of L. amazonensis promastigotes, amastigotes and mouse peritoneal macrophages in the absence or in the presence of O. gratissimum essential oil are shown in Fig. 1. After 60 min treatment all the parasites were destroyed by essential oil
Discussion
Medicinal plants are distributed worldwide, but they are most abundant in tropical countries [28], [29]. The approximately 80,000 species of higher plants described, in Brazil offer enormous prospects for discovering new compounds with therapeutic properties. Several species and varieties of plants of the genus Ocimum yield oils of a diverse nature, commonly known as basilic oils. Eugenol, isolated from Ocimum gratissimum, shows antibacterial [9] and antihelminthic activity [30]. We demonstrate
Acknowledgments
This study was supported by grants of the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), PRONEX/Fundação Araucária, and Programa de Pós-graduação em Ciências Farmacêuticas da Universidade Estadual de Maringá.
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