key: cord-283984-jch0ja1o
authors: Loizzo, Monica R.; Saab, Antoine M.; Tundis, Rosa; Statti, Giancarlo A.; Menichini, Francesco; Lampronti, Ilaria; Gambari, Roberto; Cinatl, Jindrich; Doerr, Hans Wilhelm
title: Phytochemical Analysis and in vitro Antiviral Activities of the Essential Oils of Seven Lebanon Species
date: 2008-03-20
journal: Chem Biodivers
DOI: 10.1002/cbdv.200890045
sha: 
doc_id: 283984
cord_uid: jch0ja1o

The chemical composition of the essential oils of Laurus nobilis, Juniperus oxycedrus ssp. oxycedrus, Thuja orientalis, Cupressus sempervirens ssp. pyramidalis, Pistacia palaestina, Salvia officinalis, and Satureja thymbra was determined by GC/MS analysis. Essential oils have been evaluated for their inhibitory activity against SARS‐CoV and HSV‐1 replication in vitro by visually scoring of the virus‐induced cytopathogenic effect post‐infection. L. nobilis oil exerted an interesting activity against SARS‐CoV with an IC (50) value of 120 μg/ml and a selectivity index (SI) of 4.16. This oil was characterized by the presence of β‐ocimene, 1,8‐cineole, α‐pinene, and β‐pinene as the main constituents. J. oxycedrus ssp. oxycedrus oil, in which α‐pinene and β‐myrcene were the major constituents, revealed antiviral activity against HSV‐1 with an IC (50) value of 200 μg/ml and a SI of 5.

Introduction. -In the past decades, besides a variety of synthetic antiviral drugs with different molecular targets, a large number of phytochemicals have been recognized to control infections caused by viruses. Recently, the anti-herpesvirus activity of several essential oils of different plant sources as well as of various constituents of essential oils was demonstrated [1] [2] .

The severe acute respiratory syndrome (SARS) is a febrile respiratory illness primarily transmitted by respiratory droplets or close personal contact. The causative organism has been identified as a novel coronavirus, i.e., SARS-CoV [3] . The overriding clinical feature of SARS is the rapidity with which many patients develop symptoms of acute respiratory distress syndrome (ARDS). Currently, there are no approved or universally recommended therapies for SARS. Treatment for the disease is mainly supportive.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen that causes localized skin infections of the mucosal epithelia of the oral cavity, the pharynx, the oesophagus, and the eyes. The virus may establish an acute primary infection, followed by the development of a latent, lifelong infection [4] . Presently, the only aspect of the A total of 48 compounds (82.39% of the total oil) were identified in J. oxycedrus ssp. oxycedrus berry oil. a-Pinene (27.4%) and b-myrcene (18.9%) were the major constituents. Other identified compounds were a-phellandrene (7.1%), limonene (6.7%), epibicyclosesquiphellandrene (2.3%), and d-cadinene (2.2%). Forty-one components, representing 80.91% of the total, were identified in S. thymbra oil, in which p-cymene (10.76%), a-pinene (10.15%), thymol (9.92%), sabinene (8.64%), gterpinene (7.56%), carvacrol (4.98%), trans-caryophyllene (3.67%), b-pinene (2.90%), and linalool (2.81%) were the main abundant compounds. C. sempervirens ssp. pyramidalis oil was characterized by 19 components, representing 90.45% of the total oil. The main components were a-pinene (53.56%), a-terpinene (18.90%), thymol (3.84%), and terpinolene (3.15%). Twenty-six compounds were identified in S. officinalis (94.39% of the total oil) in which 1,8-cineole (43.62%), a-thujone (12.99%), sabinene (6.97%), camphor (5.71%), a-pinene (4.72%), a-humulene (3.41%), a-terpineol (3.18%), and b-pinene (3.01%) were the major components. P. palaestina oil was characterized by 29 components, representing 79.82% of the total oil. The main components were sabinene (17.08%), limonene (8.56%), b-pinene (6.48%), g-terpinene (6.33%), p-cymene (6.01%), and aromadendrene (3.99%).

Antiviral Activities. In this study, we report the antiviral activity of seven essential oils obtained from berry, fruits, and leaves of different species collected in Lebanon. Results are summarized in Table 3 . Our results demonstrated how L. nobilis berries oil exhibited an IC 50 value of 120 mg/ml against SARS-CoV with a selectivity index (SI; TC 50 /IC 50 ) of 4.2. An interesting activity with an IC 50 value of 60 mg/ml was found when L. nobilis berry oil was incubated with HSV-1 virus. Armaka et al. reported the ability of isoborneol to completely inhibit HSV-1 replication, without affecting viral adsorption [11] . The content of this monoterpene in L. nobilis berry oil was found in low percentage, and probably, therefore, it is not able to exert antiviral activity against HSV-1.

A certain activity against SARS-CoV was found for T. orientalis and J. oxycedrus ssp. oxycedrus oils with IC 50 values of 130 and 270 mg/ml, and a SI of 3.8 and 3.7, respectively. Interestingly, J. oxycedrus ssp. oxycedrus oil exhibited the highest activity against HSV-1 with a IC 50 value of 200 mg/ml and a SI of 5. On the other hand, T. orientalis oil did not show any antiviral activity against HSV-1 when it was incubated under the same conditions. HSV-1 Growth was inhibited also when S. thymbra oil was used (IC 50 of 220 mg/ml and SI of 4.6). The C. sempervirens oil did not exhibit any activity against HSV-1 (IC 50 > 1000 mg/ml). This results may be related to the inactivity of the main component a-pinene as we have previously demonstrated [2] . A weak activity against SARS-CoV was found when C. sempervirens ssp. pyramidalis and S. officinalis essential oils were applied in virus culture (IC 50 700 and 870 mg/ml, resp.). P. palaestina essential oil was inactive against SARS-CoV (IC 50 > 1000 mg/ml) and less active against HSV-1 (IC 50 500 mg/ml). Interestingly, L. nobilis, T. orientalis, and J. oxycedrus ssp. oxycedrus oils exhibited higher potencies to inhibit SARS-CoV and a great margin of safety compared to the positive control glycyrrhizin (IC 50 641 mg/ml; SI 1.2).

Cytotoxic Activity. Cytotoxic effects of the essential oils were tested in confluent layers of Vero cells by MTT assay. Assessment of cytotoxicity is clearly an important aspect of the evaluation of a potential antiviral agent, because a useful oil should be selective for virus-specific processes with no or only few effects on cellular metabolism. In Vero cells the TC 50 value of tested samples was in a range of 120 to 1000 mg/ml.

Conclusions. -Severe acute respiratory syndrome (SARS) is an emerging disease that created international anxiety because of its relatively high infectious, rapid progression and relatively high death rate. The fact that no conventional medicine was used for the treatment of SARS was based on the evidence that natural products from plants exhibited antiviral activity to other coronaviruses although the mechanism of action of these herbal products is mainly through inhibition of viral replication [12] .

In this paper, we presented the first evidence for a strong antiviral activity of L. nobilis oil against SARS-CoV, and we also reported the interesting anti-herpetic activity of J. oxycedrus ssp. oxycedrus and S. thymbra oils providing a potential use of these oils for treatment of viral infectious diseases.

Biology Department, Faculty of Sciences II, Lebanese University, and deposited with the Herbarium of Faculty of Sciences II, Lebanese University. Isolation of Essential Oils. The fresh aerial parts (200 g of each of the above mentioned species) were submitted to hydrodistillation for 3 h using a Clevenger-type apparatus as described in [2], yields in percent are listed in Table 1. The white-yellow essential oils were dried (anh. Na 2 SO 4 ) to remove traces of moisture and stored at 4 -88 in bottles covered with aluminium foil to prevent the negative effect of light. GC/MS Analysis. To determine the essential oils composition, analyses were carried out using a GC system

Cytotoxicity of the essential oils towards Vero cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye reduction in active mitochondria using monkey kidney cell line Vero (ATCC, Manassas, VA) as described in [2]. The drug concentration that reduced the cell growth by 50% is expressed as TC 50

Germany) replication was tested as follows. Vero cells were seeded in 96-well plates and infected with HSV-1 or SARS-CoV at multiplicity of infection (MOI) of 0.01. The viruses were propagated in Vero cells as described in [2] [14]. In accordance with WHO recommendations, all work involving infectious SARS-CoV was performed under biosafety level (BSL)-3 conditions in a BSL-3 facility. Acyclovir (ACV) was used as a control for antiviral activity against HSV-1, while glycyrrhizin (GLZ) was used against SARS-CoV. Both positive control compounds were purchased from Sigma-Aldrich, D-Munich. Virus and the tested essential oils of different concentrations were added at the same time in MEM supplemented with 2% FBS. For each dilution step, 8 wells were used in parallel. Virus infection was assessed by visually scoring of the virus-induced cytopathogenic effect (CPE) 72 h (HSV-1) or 48 h (SARS-CoV) post-infection. The effective concentration inhibiting 50% of virus growth (IC 50 ) was determined as concentration of compound required to inhibit the CPE effect to 50% of the control value. The selective index (SI), also known as a therapeutic ratio or margin of safety

Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy