Antiviral Properties of Supercritical CO₂ Extracts from Oregano and Sage

Abstract

The antiviral properties of supercritical CO₂ extracts obtained from oregano and sage were evaluated against the herpes simplex virus type 1 at different stages during virus infection. All of the extracts tested presented a moderate extracellular direct virucidal activity, although a pre-treatment of Vero cells, with 10 μg/mL of sage extracts before virus addition, inhibited 70% of the virus infection. Moreover, supercritical extracts of sage and oregano were able to significantly inhibit the in vitro virus replication, showing IC₅₀ values of 1.88 and 5.33 μg/mL, respectively. Carvacrol and thymol could be pointed out as the compounds responsible for the antiviral activity found in oregano supercritical extracts; meanwhile, borneol, camphor, and 1,8-cineole could be proposed as antiviral compounds in supercritical sage extracts. Results demonstrated that supercritical extraction was an appropriate technique to obtain antiviral extracts from oregano and sage.

Keywords:

• Antiviral activity
• Supercritical fluid extraction
• Oregano
• Sage
• Herpes simplex virus type 1

INTRODUCTION

Herpes simplex virus type 1 (HSV-1) is a highly prevalent pathogen among children and adults, causing primary infections that are presented clinically as herpes labialis or as primary gingivostomatitis. The virus is also able to establish a latent infection in the nervous system that can be reactivated quite frequently.[1] The major therapeutic agents for HSV infections are nucleoside analogues, such as acyclovir and vidarabine. However, the increased and prolonged use of these compounds, especially in immunocompromised patients, has led to viral resistance against most of these drugs.[2,3] The drug-resistant HSVs retained their pathogenicity and could be associated with progressive and relapsing disease. Thus, it is necessary to explore and discover novel potential antiherpetic approaches.

For a long time, medicinal plants have been used for the treatment of many infectious diseases, in most cases without a scientific background supporting their employment. On the contrary, at present, there is increasing emphasis on determining the scientific evidence and rationale use of preparations from medicinal plants. Thus, in the last several years, a large number of antiherpes screening experiments on medicinal plant extracts and plant-derived secondary metabolites (e.g., polyphenolics, glycosides, terpenes, polysaccharides, polyketides, pheophorbides, etc.) have been reported.[4] Further, the antiherpes activity of several essential oils of different plant sources, as well as of various constituents of essential oils, was demonstrated.[5 ⁻ 7] Many species of the Lamiaceae family are known for their antiviral activity. Among them, aqueous and ethanolic extracts of Salvia officinalis and Salvia coccinia revealed an important antiviral activity against HSV-1 and HSV-2.[8,9] Also, extracts of Melissa officinalis and its essential oil presented antiviral activity against herpes viruses.[10,11] Essential oils of Mentha piperita and Thymus vulgaris exhibited high levels of virucidal activity against herpes viruses.[7,12] This virucidal effect was also presented by aqueous extracts of several species of this family (Rosmarinus officinalis, Mentha piperita, Prunella vulgaris).[13]

In recent years, supercritical fluid extraction (SFE) has received increased attention as an important alternative to the traditional solvent extraction methods, since this technique provides a high speed and efficiency of extraction, eliminates concentration steps, and avoids the use of organic solvents that are potentially harmful in terms of environmental impact. SFE is an extraction/fractionation method that exploits the unique properties of gases above their critical points to extract soluble components from a raw material. Carbon dioxide is an ideal solvent for the extraction of some classes of natural substances from food because it is non-toxic, non-explosive, readily available, and easy to remove from extracted products. In that way, the quality of supercritical fluid extracts is higher than those obtained by extraction solvents or by water or steam distillation, since these methods can induce thermal degradation or present the problem of toxic residual solvent in the products.[14]

The goal of the present work was to study the in vitro antiviral activity of supercritical extracts obtained from oregano (Origanum vulgare) and sage (Salvia officinalis) against HSV-1. Simultaneously, the antiviral activity of the extracts at different steps during the viral infection cycle was also determined, and attempts were made to effectively correlate the antiviral activity with chemical profile of the extracts.

MATERIALS AND METHODS

Samples and Chemicals

The oregano (Origanum vulgare) and sage (Salvia officinalis) samples consisted of dried leaves obtained from a herbalist's shop (Murcia, Spain). Cryogenic grinding of the samples was performed under carbon dioxide. The size of the particle (between 999 and 500 μm) was determined by passing the ground plant material through sieves of appropriate size. The whole sample was stored at –20°C until use.

1,8-Cineole, camphor, borneol, sabinene hydrate, carvacrol, and thymol standards were purchased from Sigma (Madrid, Spain). CO₂ (N38 quality) was supplied from Air Liquid (Madrid, Spain).

Extraction Method

Extractions were carried out using a pilot-plant supercritical fluid extractor (model SF2000; Thar Technology, Pittsburgh, PA, USA) comprising a 2-L cylinder extraction cell and two different separators (S1 and S2), each of 0.5 L capacity, with independent control of temperature and pressure. For each experiment, the extraction vessel was packed with 0.6 kg of the plant. Extraction assays were performed at 30 MPa and 313 K, with a CO₂ flow rate of 50 g/min. Temperature was set to 313 K in both S1 and S2 separators. In the first separator (S1), the pressure was maintained at 10 MPa, while in the second separator (S2), the pressure was ambient pressure. The cascade decompression system produced two different extracts with different composition, which were collected in separator 1 (S1) and separator 2 (S2). According to previous kinetic studies, the overall extraction time was set to be 8 h.[15]

Antiviral Assays

Cells and viruses

Vero cells (African green monkey kidney cell line) were obtained from the Americam Type Culture Collections, Rockville, MD (ATCC number: CCL-81). They were used as the host for HSV-1. The cells were grown using Eagle's Minimum Essential Medium (MEM) supplemented with 5% fetal bovine serum (FBS), 1% penicillin-streptomycin, 1% hepes buffer 1M, 1% non essential amino acids, and 1% L-glutamine. Maintenance medium for Vero cells was as described above but with 2% FBS.

Herpes virus simplex type 1, sensitive to acyclovir, (HSV-1) (KOS) was obtained from the Americam Type Culture Collections, Rockville, MD (ATCC number: VR-1493D), prepared in aliquots and stored at –80°C until use. Virus titer was determined by plaque reduction assay in Vero cells and expressed as plaque forming units (pfu) per mL.

Cytotoxicity assays

The cytotoxic effect of the different extracts and standards on Vero cells was tested using 3-(4,5-dimethylthiazol-2-ol)-2,5-diphenyl tetrazolium bromide (MTT) assay, according to a published method.[16] MTT (Sigma, Spain) is a yellow water soluble tetrazolium dye that is reduced by live cells, but not dead to a purple formazan product that is insoluble in aqueous solutions. Monolayers of Vero cells in 24-multiwell plates were incubated with MEM containing different concentrations of the extracts for 48 h at 37°C. Cells were then washed with phosphate buffered saline (PBS) and 0.5 mg/ml of MTT were added to each well and incubated for 4 h at 37°C. Supernatants were discarded and formazan crystals dissolved in an extraction solution (10% sodium dodecyl sulphate in a mixture of dimethyl formamide and water 1:1 v/v, adjusted to pH 4.7 with acetic acid) overnight at 37°C. Formazan quantification was performed by measuring the optical density at 570 nm using a multiscanner autoreader (Sunrise, Tecan) with the extraction solution as a blank. The data were plotted as dose-response curves, from which the concentration required to reduce 50% of the number of viable Vero cells (CC₅₀) after 48 h of incubation with the different extracts were obtained.

Evaluation of virucidal activity

Virus samples containing 10⁵ pfu/ml were mixed and incubated at 37°C for 1 h with MEM containing different extracts or standards concentrations or MEM alone (control). Samples were then diluted and used to infect confluent Vero cells for 1 h at 37°C. After incubation, the virus inocula was removed, the cells washed with PBS, and then overlaid with maintenance medium (with 0.5% agarose) for 48 h at 37°C. The infected cells were fixed acetone:methanol (50:50) at 4°C, stained with a 1% solution of crystal violet, and the number of the plaques counted. The percentage of inhibition of plaque formation was calculated as follows: [(mean number of plaques in control) – (mean number of plaques in test)]/(mean number of plaques in control) × 100.

Influence of various treatment periods on the anti-HSV-1 activity of the extracts

Vero cells and viruses were incubated with the extracts at different stages during the viral infection cycle in order to determine the mode of antiviral action. (1) Cells pretreatment: monolayers of Vero cells in 24-multiwell plates were pretreated with MEM containing different concentrations of the extract or standard for 3 h at 37°C. Cells were then washed with PBS and infected with 120 pfu of HSV-1. After incubation for 1 h at 37°C, the virus inocula was removed, the cells washed with PBS, and then overlaid with maintenance medium (with 0.5% agarose) for 48 h at 37°C. The infected cells were fixed, stained, and the number of the plaques counted. Control consisted on untreated cells infected with HSV-1. (2) Adsorption period: cells were infected with 120 pfu of HSV-1 in the presence of different concentrations of the extracts for 1 h at 37°C. Then, the virus inocula and the extract were removed, the cells washed with PBS, and then overlaid with maintenance medium (with 0.5% agarose) for 48 h at 37°C. The infected cells were fixed, stained, and the number of the plaques counted. Control consisted on cells infected without extract. (3) Intracellular replication: cells were infected with 120 pfu of HSV-1. After incubation for 1 h at 37°C, the virus inocula was removed, the cells washed with PBS, and then overlaid with maintenance medium (with 0.5% agarose) containing different concentrations of the extracts. After incubation for 48 h at 37°C, the infected cells were fixed, stained, and the number of the plaques counted. The concentration of a substance required to reduce plaque number in Vero cells by 50% (IC₅₀) as compared to control, was calculated from the dose-response curves generated from the data.

GC-MS Analysis

Characterization of the supercritical sage and oregano extracts was carried out by a GC-2010 spectrophotometer (Shimadzu, Japan), equipped with a split/splitless injector, electronic pressure control, AOC-20i auto injector, GCMS-QP2010 Plus mass spectrometer detector, and a GCMS Solution software. The column used was a ZB-5 (Zebron) capillary column (30 m × 0.32 mm I.D. and 0.25 μm phase thickness). Helium, 99.996% was used as a carrier gas at a flow of 1 mL/min. Oven temperature programming was 60°C isothermal for 4 min, increased to 64°C at 1°C/min, then increased to 106°C at 2.5°C/min. Oven temperature was then increased from 106 to 130°C at 1°C/min, and then to 200°C at 5°C/min, and then to a final temperature of 250°C/min at 8°C/min, which was kept constant for 10 min. Sample injections (1 μL) were performed in split mode (1:20). The inlet pressure of the carrier gas was 57.5 KPa. Injector temperature was 250°C and MS ion source and interface temperatures were 230 and 280°C, respectively. The mass spectrometer was used in TIC mode, and samples were scanned from 40 to 500 amu. Compounds thymol, carvacrol, borneol, and linalool were identified by comparison with standard mass spectra obtained in the same conditions and compared with the mass spectra from the Wiley 229 library. The rest of the compounds were identified by comparison with the mass spectra from the Wiley 229 library and by their linear retention index.

Statistical Analysis

One-way analysis of variance was performed using a StatGraphics Centurion XVI for Windows software (Statistical Graphics Corporation, MD, USA). The mean comparison test used was Fisher's least significant differences procedure. All the presented values are the mean of four determinations ± standard deviation.

RESULTS AND DISCUSSION

Extraction of oregano and sage leaves by using supercritical carbon dioxide was carried out on a pilot scale plant. Usually, the first step was to determine both the working pressure and temperature, since the optimization of the experimental conditions represent a critical step in the development of a SFE process. In this work, the extraction pressure and temperature were set to 30 MPa and 313 K, respectively, using pure CO₂. These experimental conditions were selected based on previous studies done in our laboratory with rosemary, oregano, and laurel leaves.[17 ⁻ 20] As already mentioned, the extraction system employed in the present study allows a cascade depressurization providing two different extracts: S1 and S2. The main difference between S1 and S2 extracts was the fractionation pressure, which brought about a gradual precipitation of the extracted compounds on the basis of their solubility in the extracting agent at the conditions set in each separator. These two extracts are expected to present different characteristics in terms of both composition and functional activity. Therefore, in this work, the antiviral activity of S1 and S2 extracts of sage and oregano were investigated.

Cytotoxicity

Oregano and sage supercritical extracts (S1 and S2) were initially evaluated for cytotoxicity on preformed monolayers of Vero cells by MTT method. The CC₅₀ data obtained (Table 1) indicated similar cytotoxicity for all the extracts tested.

Table 1  Antiviral activities of supercritical extracts obtained from aromatic plants and pure standards against herpes simplex virus type 1

Sample	CC50 (μg/mL)	IC50 (μg/mL)	SI
Oregano S1	128.20 ± 5.36^b	5.33 ± 0.07^b	24.05
Oregano S2	121.20 ± 6.32^b	5.50 ± 0.05^a	22.03
Salvia S1	119.42 ± 4.02^b	1.88 ± 0.09^e	63.52
Salvia S2	122.34 ± 4.69^b	2.09 ± 0.07^d	58.53
1,8-cineole	46.02 ± 2.35^f	1.53 ± 0.06^f,g	30.08
Camphor	54.43 ± 3.21^e	1.41 ± 0.11^g	38.60
Borneol	65.92 ± 4.73^d	1.65 ± 0.09^f	39.95
Sabinene hydrate	254.91 ± 7.23^a	5.19 ± 0.07^c	49.12
Carvacrol	83.54 ± 3.78^c	1.80 ± 0.09^e	46.41
Thymol	76.45 ± 4.87^c	1.50 ± 0.07^g	50.97
CC50 (cytotoxic concentration 50%): concentration required to reduce 50% the number of viable Vero cells after 48 h of incubation with the compounds; IC50 (inhibitory concentration 50%): concentration required to reduce plaque number in Vero cells by 50%; SI (selectivity index): ratio CC50/IC50.
Each value is the mean of four determinations ± standard deviation. SI (selectivity index): ratio CC50/IC50.
Different superscript letters within a column indicated significant differences (p < 0.05) among data.

Virucidal Activity of Oregano and Sage Supercritical Extracts

In order to elucidate the possibility that oregano and sage supercritical extracts may act directly on the virus particle, a suspension of the virus was treated at 37°C for 1 h with different concentrations of these extracts. As shown in Fig. 1, pre-incubation of HSV-1 with extracts resulted in a dose-dependent reduction of remaining infectivity of the virus when compared with the untreated control. Data also indicated that sage supercritical extracts presented a higher virucidal activity than oregano extracts, with S1 being a little more effective than S2 in both cases. However, these samples exerted a moderate virucidal activity since 3 mg/mL of sage S1 extract were necessary to reduce 60% virus infectivity.

Figure 1 Virucidal effects of supercritical extracts against HSV-1. (a) Oregano extracts and its main components (carvacrol, thymol, and sabinene hydrate); (b) sage extracts and its main components (camphor, borneol, and 1,8-cineole). Each bar is the mean of four determinations ± standard deviation.

Influence of Various Treatment Periods on the Anti-HSV-1 Activity of Oregano and Sage Extracts

Vero cells were pre-treated for 3 h at 37°C with different concentrations of oregano and sage S1 and S2 extracts. Afterward, extracts were removed and cells were washed and infected with the HSV-1 virus. Results indicated that cells’ pre-treatment with oregano and sage extracts produced an important reduction of virus infectivity (Fig. 2), although sage extracts were more effective than oregano extracts. Thus, 10 μg/mL of sage S1 extract inhibits virus infection by approximately 60%, whereas 20 μg/mL of oregano S1 extract reduced the virus infectivity by only 50%. Also, in both cases, extracts obtained in separator 1 were more effective than those collected in separator 2. These data suggested that all extracts interfered with the HSV-1 infection process at the initial infection steps, perhaps by blocking virus attachment or adsorption to Vero cells.

Figure 2 Effect of cell pre-treatment with supercritical extracts and pure standards on HSV-1 infectivity. (a) Oregano extracts and its main components (carvacrol, thymol, and sabinene hydrate); (b) sage extracts and its main components (camphor, borneol, and 1,8-cineole). Each bar is the mean of four determinations ± standard deviation.

In order to investigate the influence of extracts on virus adsorption, cells were infected with HSV-1 in the presence of different concentrations of the extracts for 1 h at 37°C. Then, the virus inocula and the extract were removed, the cells washed with PBS, and maintained for 48 h at 37°C. The addition of 10 μg/mL of sage S1 and 20 μg/mL of oregano S1 extracts reduced virus infectivity by 70 and 60%, respectively (Fig. 3). Also, in that case, oregano extracts were less effective than sage ones. Comparing data obtained during the adsorption stage with those found in the pre-treatment step, when oregano and sage extracts were applied during the adsorption stage, at the same concentration, the reduction of the virus infectivity was increased by 10% indicating that extracts were more effective when applied during the adsorption period.

Figure 3 Effect of supercritical extracts and pure standards on HSV-1 adsorption period. (a) Oregano extracts and its main components (carvacrol, thymol, and sabinene hydrate); (b) sage extracts and its main components (camphor, borneol, and 1,8-cineole). Each bar is the mean of four determinations ± standard deviation.

The antiviral activity on the intracellular replication of the virus was evaluated by adding different concentrations of the extracts to previously HSV-1 infected Vero cells and incubated for 48 h at 37°C. All of the samples showed a dose-dependent inhibition of virus replication. In this assay, sage extracts were more efficient against HSV-1 replication than oregano ones, showing the lowest IC₅₀ values (1.88 μg/mL) (Table 1). However, oregano extracts also showed an important inhibition of virus intracellular replication (IC₅₀ = 5.33 μg/mL). Comparing these data with those obtained during adsorption, sage and oregano extracts seemed to be much more active when added after virus infection. Thus, sage and oregano supercritical extracts were effective as inhibitors of the intracellular virus replication as well as agents able to disrupt virus attachment to the cell. These results were different than those obtained by other authors when employing aqueous and ethanolic extracts or essentials oils from several plants and herbs. Schnitzler et al.[9] studied the antiviral activity of aqueous and ethanolic extracts of Salvia officinalis and reported that HSV-1 was considerably inactivated after treatment with the extracts prior to cell infection; meanwhile when the host cells were pretreated with the extracts prior to virus infection the activity of the extracts was lower, and when the extracts were added to infected cells no or only a moderate reduction of plaques was detected. In the same way, Nolkemper et al.[13] indicated that aqueous extracts from species of Lamiaceae family, including Salvia officinalis, exerted their antiviral effect on the free HSV and had no effect on the intracellular virus replication. Also, Koch et al.[7] reported that essential oils obtained from different plats and herbs significantly reduced plaque formation when HSV-2 was preincubated with essential oils. The different behavior of supercritical extracts from oregano and sage obtained in this work might be related with the composition of the extracts, since the extraction technique employed in this work was completely different to those used in the literature.

In order to better define the anti-HSV-1 compounds presented in supercritical extracts, they have been analyzed by GC-MS to identify potential antiviral components.

GC-MS Characterization of Supercritical Extracts from Oregano

In an attempt to identify the compounds responsible for the antiviral activity found in supercritical S1 and S2 extracts from Origanum vulgare, a characterization by GC-MS of these samples was performed. Results obtained are shown in Table 2, where a tentative identification has been performed based on the comparison of mass spectra and retention index (RI). As can be observed, 16 compounds were identified. Some of them were detected in large amounts like trans-sabinene hydrate, thymol, and carvacrol. The sum of these three compounds represented 77.3% of the S1 extract; meanwhile, extract obtained on separator 2 contained a smaller quantity (72.03%) of these compounds. In that sense, several authors[21 ⁻ 23] have reported the antimicrobial and antifungal activity of thymol and carvacrol, meanwhile only a few works has been published regarding their antiviral activity.[24] Thus, in order to correlate the antiviral activity found in the supercritical extracts with their chemical composition, the cytotoxicity and antiviral activity of pure standards of these three main components of the extracts (sabinene hydrate, thymol, and carvacrol) were also examined at the same conditions. The cytotoxicity assays revealed that sabinene hydrate was less cytotoxic than the supercritical extracts, meanwhile thymol and carvacrol standards presented higher cytotoxicity values (Table 1). Pre-incubation of HSV-1 with pure standards indicated that these standards presented a higher virucidal activity than oregano S1 and S2 extracts, being that carvacrol and thymol were more effective than sabinene hydrate (Fig. 1). When Vero cells were pre-treated with 20 μg/mL of carvacrol and thymol, the HSV-1 infection was inhibited by approximately 90% (Fig. 2), being that sabinene hydrate was less effective (60% virus inhibition). In contrast, when supercritical samples were applied at this concentration, only 50–40% inhibition was achieved. Moreover, if 20 μg/mL of carvacrol and thymol were applied during the virus adsorption period, the infectivity was reduced to 100% (Fig. 3), as compared with a reduction of 50–60% when supercritical extracts were used. The antiviral activity of standards on the intercellular replication of the virus was also evaluated, showing that carvacrol and thymol IC₅₀ values were smaller than sabinene hydrate and oregano extracts (Table 1). Consequently, carvacrol and thymol could be pointed out as the compounds responsible for the antiviral activity found in oregano supercritical extracts, although sabinene hydrate also contributes to this activity. Besides, the higher antiviral activity found in S1 extract could be explained since this extract presented a higher percentage of thymol than in S2, being that the percentages of carvacrol and sabinene hydrate were quite similar in the two extracts.

Table 2  GC-MS identification, peak area contribution (normalized area percent), and retention indices (RI) of compounds found in oregano supercritical extracts

			S1	S2
Retention time (min)	RI	Compound	normalized area (%)	Normalized area (%)
10.20	971	Sabinene	n.d.	1.04
12.52	1015	Alpha-terpinene	n.d.	0.74
12.94	1023	ρ-Cymene	5.70	1.78
13.19	1027	Limonene	n.d.	0.57
14.94	1057	γ-Terpinene	2.48	3.74
15.39	1065	Cis-sabinene hydrate	2.76	3.67
17.17	1096	Trans-sabinene hydrate	45.21	45.05
17.35	1100	Linalool	2.29	1.62
21.74	1175	4-Terpineol	2.60	5.14
22.52	1189	α-Terpineol	2.37	2.84
25.09	1231	NI	n.d.	0.73
25.61	1240	Thymyl methyl ether	1.33	2.09
26.17	1250	Trans-sabinene hydrate acetate	1.55	0.87
26.40	1254	Linalyl acetate	1.62	1.51
28.65	1291	Thymol	24.10	19.81
29.23	1300	Carvacrol	7.99	7.17
37.80	1412	Trans-caryophyllene	n.d.	1.63
NI: non-identified compound; n.d.: not detected.

GC-MS Characterization of Supercritical Extracts from Sage

A characterization by GC-MS of supercritical sage extracts was also performed. As can be observed in Table 3, 16 compounds were identified. The most abundant were camphor, 1,8-cineole, and borneol, representing 62.4% of S1 extract and 48.1% of S2 extract. As previously reported for oregano, the antimicrobial activity of these compounds has been extensively described,[25,26] meanwhile few studies reported their antiviral activity.[4] With regard to potential compounds responsible for the antiviral activity detected in both sage samples, the determination of antiviral activity of pure standards of these three compounds, in the same conditions of previously was carried out. Data obtained (Table 1) indicated that camphor, 1,8-cineole, and borneol presented cytotoxicity values higher than those of the supercritical extracts. Virucidal assays showed that these standards presented a higher virus inhibition when compared to those presented by supercritical extracts (Fig. 1). Pre-treatment assays indicated that camphor, borneol, and 1-8 cineole presented a similar virus inhibition, increasing 10–15% in the values obtained using the supercritical samples (Fig. 2). Moreover, during the virus adsorption period, 10 μg/mL of these substances reduced the infectivity by 85 to 80% (Fig. 3), meanwhile when sage extracts were applied the reduction was only 60–70%. Regarding the intracellular replication of the virus, camphor, borneol, and 1,8-cineole showed similar values of the IC₅₀, slightly better than those obtained with the extracts. After analyzing all of the data, borneol, camphor, and 1,8-cineole could be proposed as the compounds responsible of the antiviral activity found in supercritical sage S1 and S2 extracts. Data also explained the higher antiviral activity found in extract obtained in S1, since this fraction contained a higher quantity of borneol, camphor, and 1,8-cineole than extract S2.

Table 3  GC-MS identification, peak area contribution (normalized area percent), and retention indices (RI) of compounds found in supercritical extracts (S1 and S2) from sage (Salvia officinalis)

			S1	S2
Retention time (min)	RI	Compound	normalized area (%)	Normalized area (%)
13.33	1029	1,8 Cineole	13.34	4.64
17.38	1099	Linalool	1.16	1.10
19.64	1138	Cis-sabinol	2.05	n.d.
19.78	1140	Camphor	41.78	33.9
21.07	1163	Borneol	7.28	9.56
22.55	1188	Alpha-terpineol	1.16	1.81
26.44	1253	Linalyl acetate	4.86	5.81
28.19	1282	Endobornyl acetate	3.19	3.46
28.70	1291	Sabinyl acetate	5.00	5.94
32.60	1345	Alpha-terpinenyl	3.68	4.71
37.84	1412	E-cariophyllene	2.33	2.97
40.65	1484	Alpha-humulene	1.75	2.41
43.05	1535	Geranyl propionate	1.25	1.91
51.19	1575	Spathulenol	1.01	2.05
51.48	1579	Cariophyllene oxide	1.03	1.61
52.04	1586	Viridiflorol	2.00	4.02
56.61	1685	NI	2.11	5.06
NI: non-identified compound; n.d.: not detected.

CONCLUSIONS

Supercritical extracts from edible herbs like oregano and sage presented important antiviral activities against herpes simplex type 1, overall extracts obtained in separator 1. These extracts mainly inhibit HSV-1 intracellular replication, although they were also able to disrupt the virus attachment step. Carvacrol and thymol could be pointed out as the compounds responsible for the antiviral activity found in oregano supercritical extracts, although sabinene hydrate also contributes to this activity. Meanwhile, borneol, camphor, and 1,8-cineole could be proposed as antiviral compounds in supercritical sage extracts. These results demonstrated that supercritical extraction was an appropriate technique to obtain antiviral extracts from oregano and sage.

ACKNOWLEDGMENTS

This work has been supported by the program CONSOLIDER-INGENIO 2010 (CDS2007-00063) and the regional program ALIBIRD-CM S0505/AGR-0153 from the Comunidad de Madrid, Spain.