Antiviral Activity of Melissa Officinalis Essential Oils Linked to Their Terpenes Composition

Introduction

Herpes simplex virus type 1 and type 2 are one of the most common and most contagious pathogens. the virus is transmitted through Mouth, respiratory system, Direct person-to-person contact, saliva, sexual contact. Primery infection usually apears in the form of labialis on skin or Mucous membrane. These viruses can cause damage in liver, adrenal glands, eyes of infants and people with immunodeficiency disorders. It can also causes encephalitis, meningitis, and death with an attack on central nervous system and brain. In most cases, the virus tends to in latent form in sensory ganglions. An antiviral treatment for HSV infection is Acyclovir that still the most commonly used chemotherapy. In recent years, due to long-term treatments with acyclovir, there have been reports of the emergence of drug-resistant mutants. Drug resistant Mutants of virus without Thymidine kinase are isolated from immunocompromised patients that can be a major problem in future years. Due to these issues finding new drugs with low side effects is felt. Medicinal plant extracts and essential oils are increasingly of interest as antimicrobial and antiviral agents and have been widely used in traditional medicine. Medicinal plants produce a variety of chemical components with the potential to inhibit viral replication with low side effects. these plants represent a massive source of new bioactive component for discovering new drugs with low side effects.

Melissa officinalis (lemon balm) is a member of the Lamiaceae family and plants of this family are well known in antiviral phytotherapy. Melissa officinalis is a perennial herbaceous plant in the mint family Lamiaceae and native to south-central Europe, the Mediterranean Basin, Iran, and Central Asia. The main components of lemon balm essential oil are citral (citronellol, linalool) and geraniol and trepenes. antioxidant and antibacterial effects of Extracts and essential oils of Lamiaceae family are reported in many studies. An antiviral activity of lemon balm aqueous extracts and essential oils has been described previously but details about main effective component of extract and essential oil are not available.

In this study we compared antiviral activity of melissa officinalis essential oils and two component of that and the mode of antiviral action is analysed at different steps in the viral infection cycle.

Melissa officinalis essential oil was purchased from Primavera Life, Sulzberg, Germany that previously analysed with gas chromatography by Dr. Schnitzler and we used their analaysis for our study. Melissa officinalis oil was dissolved in ethanol and added to the cell culture medium at a maximum final concentration of 1% ethanol.

In this study we used A549 cell line to evaluate cytotoxicity and antiviral activity of our compunds. Monolayer of A549 cells (human lung carcinoma) were grown with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% fetal calf serum (FCS), 100 U/ml penicillin and 100 mg/ml streptomycin. Herpes simplex virus type 1 (HSV-1) were used for experiments and viruses were cultivated on A549 cells. Acyclovir was purchased from Sigma-Aldrich (Sigma-Aldrich, Germany) and dissolved in Dimethyl sulfoxide (DMSO) a maximum final concentration of 1% DMSO. at first cytotoxisity of compunds on A549 cell were evaluated. for this porpuse, A549 cells were seeded in 96-well tissue culture plate and medium containing serial dilutions of the essential oil and compounds and were added onto A549 cells in eight replicates for each concentration of the drugs. Wells containing a medium with 1% ethanol but no compounds were also included on each plate as controls. After 72 h of incubation, the cytotoxicity of the essential oil and other compounds were determined by MTT assay. The cytotoxic concentration of the drug which reduced the viable cell number by 50% (CC50) was determined from dose-response curves.

Inhibition of HSV replication was examined by plaque reduction assays in 12-well culture plates. 25×104 cells was seeded in each well and incubated until reaching at least 90% confluency, then virus was added in multiplicity of infection (MOI) 0. 0002 to each well in 4 different phases, cells were pretreated with essential oil before viral infection, viruses were incubated with essential oil before cell infection, essential oils and compounds was added after penetration of the virus into the cells or during penetration of virus into cells, cell pre-treatment, cells and viruses were incubated together during adsorption, cells were pretreated with Serial dilutions of essential oil before viral infection, viruses were incubated with essential oil before cell infection or after penetration of the virus into the host cells. For virus pre treatment, cell pretreatment and treatment during penetration. After adsorption, the remaining inoculum was removed and the infected cells were overlayed with medium containing 1% methylcellulose and for post infction phase overlayed with medium containing 1% methylcellulose and serial dilution of essential oil and compunds was added. for each plate there were 2 positive control that infected with virus and treated with acyclovir, 2 mock control wells that no virus and compound were added and 2 untreated control wells that was not treated with essential oils and compunds were used as untreated control to determain plaque reduction of serial dilutions of essential oil and compounds. positive control, untreated control and mock control always contained 1% ethanol to evacuate effect of ethanol on cells or virus. After 3 days of incubation, cells were fixed by 10 % formalin and stained with 1% crystal violet and then plaque were counted. concentration of the test compound which inhibited the plaque number by 50% (IC50) was determined from dose-response curves. The selectivity index (SI) was determined as the ratio of CC 50 /IC 50.

Result

Melissa Officinalis oil and some selected terpenic compounds and mixture of them were serially diluted in ethanol and added to the cell culture medium to examine the effect on the growth and viability of tissue culture cells, always resulting in an ethanol concentration below 1% which had no effect on cells and viruses. Cell monolayers were grown in medium containing different concentrations of these drugs. After 3 days of incubation, the cell viability of A549 cells was determined with the MTT assay. The maximum noncytotoxic concentrations of these drugs were determined 40 μg/mL for citral and 50 μg/mL for beta-Caryophyllene, 0. 004 % for Melissa oil and 0. 006 % for mixture of Citral and Beta-Caryophyllene. The potential antiviral effect of different essential oils and some selected components was determined against herpes simplex virus type 1 (HSV-1) in vitro. HSV-1 was incubated for 1 h at room temperature with various concentrations of Melissa oil, citral and beta-caryophyllene mixture, Beta-Caryophyllene and citral. In all assays untreated virus-infected cells were used as a control. Subsequently, aliquots of each dilution were added on the cells for 1 h, afterwards the cells were washed and overlaid with drug-free medium and incubated for 3 days at 37 ºC. The 50% inhibitory concentrations (IC50) for HSV-1 were determined in a wide range below the maximun no cytotoxic concentration.

The results are presented as virus reduction and represent the average of three independent experiments. In plaque reduction assays, all essential oils and terpenes exhibited a concentration-dependent antiviral effect, Beta-Caryophyllene was the most effective drug. Selectivity indices for oils and different terpenes were calculated as the TC50/IC50 ratio, the highest selectivity index of 30 was determined for Beta-Caryophyllene.

Herpesvirus replication is characterized by a complex sequence of different steps at which antiviral agents might interfere. In order to investigate the inhibitory effects on herpes simplex virus in detail, all drugs were added at different stages during viral infection For comparison, all untreated controls contained the same concentration of ethanol as the drug-treated viruses, in order to exclude any infl uence of ethanol. When the host cells were pretreated with drugs prior to infection, some of the tested drugs showed minor effects on viral infection. On the other hand, pretreatment of HSV-1 with the oils or terpenic compounds for 1 h prior to infection caused a significant reduction in plaque formation. Acyclovir showed the highest antiviral activity when added during the replication period with inhibition of the viral replication of 98. 6%. This drug inhibits specifically the viral DNA polymerase during the replication cycle when new viral DNA is synthesized. However, only minor effects on viral infection were detected when cells or viruses were pretreated with acyclovir (data not shown). In contrast, when the oils or compounds were added to the overlay medium after penetration of the viruses into the host cells, plaque formation was not significantly reduced.

Drugs were applied to HSV-1 infected cells after penetration of the viruses into cells for 3 days. Number of virus plaques was determined 3 days after infection and compared with untreated control. Results are expressed as percentage of plaque reduction. These experiments were repeated independently and data presented are the mean of three experiments. HSV was incubated for 1 h at room temperature with maximum noncytotoxic concentrations of drugs. The number of virus plaques was determined 3 days after infection and compared with untreated control. Results are expressed as percentage of plaque reduction. These experiments were repeated independently and data presented are the mean of three experiments.

Prior to viral infection, the cells were incubated with maximum noncytotoxic concentrations of drugs for 1 h at 37 °C. The number of virus plaques was determined 3 days after infection and compared with the untreated control. The results are expressed as percentage of plaque reduction. These experiments were repeated independently and data presented are the mean of three experiments.

Discussion

The pharmaceutical industry is increasingly targeting medicinal plants with the aim of identifying lead compounds, focusing particularly on suitable alternative antiviral agents. Several drugs are currently available for the management of HSV infections such as acyclovir. Topical treatment of herpes labialis infection is standard, for the most part carried out with acyclovir creams, but also with phytotherapeutic preparations containing sage oil and lemon balm oil. Both plant essential oils were shown to be signifi - cantly superior to placebo and equivalent to acyclovir. In the present study, the inhibitory effects Melissa Officinalis essential oil against herpes simplex virus infection were compared with the antiviral potential of their major terpenic compounds. essential oil and terpenes exhibited high levels of antiviral activity against HSV-1 in viral suspension tests. High antiviral activity was observed for essential oil and isolated terpenes when herpesvirus was incubated with these drugs prior to host cell infection.

These results suggest that the investigated drugs directly inactivate herpes virus and might interfere with virion envelope structures or mask viral structures which are necessary for adsorption or entry into host cells. Thus different mechanisms of antiviral activity of different essential oils seem to be present. The inhibition of HSV by the tested essential oils and terpenes appears to occur before adsorption but not after penetration of the virus into the cell. It remains to be determined whether the inhibitory effect of essential oils is due to binding of the essential oil to viral proteins involved in host cell adsorption and penetration. Acyclovir inhibits virus replication by interference with the DNA polymerase inside the cell, whereas essential oils and terpenes probably inactivate HSV before it enters the cell. Viral resistance to acyclovir represents a particular problem, the prevalence of resistance in acyclovir-treated immunocompromised individuals is approximately 4% to 7%. The most important predictive value for future application of these drugs is their selectivity index, Amoros et al. (1992) recommended a selectivity index of at least 4 as appropriate. According to this suggestion, essential oil and terpenes and mixture of terpenes might be suitable agents.

Conclusion

The result showed that essential oil of melissa officinalis and other drugs have higher antiviral activity when virus were incubated by drugs than drug were added during virus attachment or adding them to HSV-1 infected cell. it means Essential oil and their compounds have high virusidal activity against hsv-1. the result of antiviral activity of essential oil of thymus vulgaris and their related compounds showed that essential oil of thymus vulgaris have higher antiviral activity when they added to HSV-1 infected cell than when drug were added during vurus attachment or incubated by virus before infect cells. the results indicated that probably beta-Caryophyllene was the main factor of antiviral activity.