Investigação da citotoxicidade e atividade antiviral dos extratos de Origanum vulgare e Rosmarinus officinalis.

Abstract

Origanum vulgare and Rosmarinus officialis are plants of the family Lamiaceae, appreciated by their aromatic properties. Because of the potential of the compounds present in their extracts and essential oils, they have several biological activities, such as antifungal, antibacterial and antiviral. The present study aimed at determining the chemical composition, the non-toxic concentration of the essential oils, aqueous and ethanolic extracts of Origanum vulgare and Rosmarinus officialis in different cell lineages and evaluating the antiviral activity against viruses of veterinary importance. The chemical identifications of the essential oils of Origanum vulgare and Rosmarinus officialis were carried out with gas chromatography with flame ionization and the compounds of the extracts were identified by high-performance liquid chromatography. In the cytotoxicity assay cell lineages MDBK, MDCK, CRFK and RK13 were used, and cellular viability was confirmed by MTT method. The presence of antiviral activities of the extracts was analyzed by titration of bovine viral diarrhea virus – BVDV, equine arteritis virus – EAV, equine influenza virus – EIV, feline calicivirus – FCV, canine distemper virus – CDV, canine adenovirus – CAV, and canine cororavirus – CcoV were carried out, in the presence or absence of each extract. The plates were kept in incubator with 5% of CO2 at 37 ºC for 72 hours until reading, based on the absence or presence of cytopathic effect. Titer was determined as infective dose for cell culture at 50% (DICC50/100μL). The found compounds in both extracts were carnosic acid, rosmarinic acid, luteolin, apigenin, kaempferol and quercetin. In the essential oil of oregano can be observe the α-terpinene, γ-terpinene, linalool, 4 - terpineol and thymol. In rosemary oil were found to α-pinene, camphene, 1,8-cineole and camphor. According to the results obtained in the determination of non-toxic concentration of the extracts and their chemical composition, the safe and efficient concentration of the extracts for antiviral tests was defined. The aqueous extract from 1600 μg/mL did not show cytotoxicity for all cellular lineages evaluated (MDBK, RK13, MDCK, and CRFK), and the ethanolic extract of Origanum vulgare was not toxic at 600 μg/mL. The maximum non-toxic concentration of essential oil of Rosmarinus officinalis against cells RK13, MDBK, MDCK andCRFK was 0,023μg/mL. Regarding the essential oil of Origanum vulgare front cells RK 13 and MDCK was 0,013μg/mL, using the cells MDBK. The result was 0,052μg/mL and in CRFK was 0,023μg/mL. With respect to aqueous extracts of Origanum vulgare and Rosmarinus officinalis, showed no concentration of toxic 50 μg/mL to cells MDBK, CRFK e MDCK. RK 13 cells already in the aqueous extract of Rosmarinus officinalis 12,5 μg/mL and aqueous extracts of Origanum vulgare was 3.125 μg/mL. Regarding ethanol extracts of Origanum vulgare and Rosmarinus officinalis, showed no toxic concentration of 3.125 μg/mL front CRFK cells, same result presented in RK 13 cells with ethanol extract of Rosmarinus officinalis, since MDBK and MDCK cells was 6, 25 μg/mL. The ethanol extract of Origanum vulgare was 12,5 μg/mL for MDBK and RK 13 and 25 μg/mL for MDCK cells. The essential oils of both plants showed results more toxic than extracts, and showed no antiviral action. The results of the essential oils of both plants were more toxic than the extracts and did not show antiviral action. On its turn, the ethanolic extract of Origanum vulgare, when added to EAV in RK13 cells, resulted in reduction of viral titer from 105.42TCID50/100 μL to 100.79 TCID50/100 μL. This indicates a significant antiviral action (p <0.05). The aqueous extract of O. vulgare when added together to EAV in RK13 cells, resulted in a reduction in viral titer of 105,42TCID50/100 μL for 102,09 TCID50/100 μL.Adding the aqueous extract of O. vulgare against CDV resulted in reduction of titers from 102.00TCID50/100 μL to 100.00 TCID50/100 μL, while in the presence of the ethanolic extract the reduction was from 102.00TCID50/100 μL to 101.50 TCID50/100 μL. No significant differences in titers regarding the other analyzed viruses were found. The protocol seeking virucidal activity was able to demonstrate strong action virucidal of the ethanol extract of Origanum vulgare about EAV, since after 1 hour incubation mean titers of EAV were reduced from 106 to 104 when incubated at 37 º C and 103 with incubation at 24 ° C. Incubation of EEO with EAV at 4 ° C resulted in no difference in viral titer. The results of this study evidenced the antiviral potentiality of aqueous and ethanolic extracts of Origanum vulgare against the EAV and CDV, confirming the virucidal action of the ethanol extract of O. vulgare to EAV, presenting a promising antiviral agent.