C. fragrans and S. chinensis plants were obtained from nurseries and grown in a controlled greenhouse at the Ben Gurion University, Beer-Sheva, Israel.

ACV [9-(2-hydroxyethoxymethyl) guanosine, Sigma] was used as a positive control drug.

Ethanol and aquatic extracts were prepared from leaves of C. fragrans and S. chinensis. Plant tissues were crushed, incubated at room temperature for 48 hours in appropriate solvent, centrifuged at 2000 rpm for 10 min and the supernatant was evaporated by lyophilizer. The pellet was dissolved in minimal amount of 95% ethanol (0.5 ml) or water and diluted with water to final concentration of 10mg/ml. The extracts were sterilized by filtration and diluted with medium containing 2% Newborn calf serum (NBCS) to the appropriate concentrations.

Extracts were separated into different fractions using reverse phase column with rising methanol gradient: 0%, 20%, 40%, 60%, 80% and 100% (RP-C18 sepack).

African green monkey kidney (Vero) cells were purchased from the American Type Culture Collection (ATCC), Rockville, MD, USA. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum (FCS), 1% glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin and incubated at 37° C in a humidified air containing 5% CO₂. HSV-1, HSV-2 and VZV were propagated in Vero cells and their concentration was estimated by a standard plaque assay [9Huleihel M, Ishanu V, Tal J, Arad (Malis) S. Antiviral effect of red microalgal polysaccharides on Herpes simplex and Varicella zoster viruses J Appl Phycol 2001; 13: 127-34.].

Vero cells were treated with various concentrations of leaf extracts and the toxicity of the extracts was tested by three different methods:

1. Direct count The cells were counted by Neubauer hemacytometer indicating their replication rate.
2. Morphological changes were observed daily by optical inverted microscope.
3. MTT assay was performed as previously described [10Shi Y, Kornovski BS, Savani R, Turley EA. A rapid, multiwell colorimetric assay for chemotaxis J Immunol Methods 1993; 164: 149-54.].

The antiviral activity of the tested plant extracts and their fractions was evaluated by plaque assay and cytopathic effect (CPE) development [9Huleihel M, Ishanu V, Tal J, Arad (Malis) S. Antiviral effect of red microalgal polysaccharides on Herpes simplex and Varicella zoster viruses J Appl Phycol 2001; 13: 127-34.].

In order to elucidate the antiviral mechanism of action of the tested extracts, the cells were treated with the appropriate doses of the extract for different periods of time at/ and or after infection with virus as follow:

1. Treatment only at the time of infection- The appropriate dose of the extract was added to the cells at the time of infection. At the end of 2h infection the medium containing the extract was removed and replaced with fresh medium without extract up to the end of the experiment.
2. Treatment at the time and after infection- The appropriate dose of the extract was added to the cells at the time of infection. At the end of 2h infection the medium containing the extract was removed and replaced with fresh medium containing the extract up to the end of the experiment.
3. Treatment only at the end of infection- The cells were infected with the virus without treatment with the extract. At the end of 2h infection the medium was removed and replaced with fresh medium containing the appropriate dose of the extract up to the end of the experiment.

For examining possible interactions between the examined extracts and the cells, Vero cells were incubated in a medium containing the appropriate dose of the extract at 37°C for 2h, washed 3 times with saline and infected with the virus without further treatment with the leaf extract. For elucidating possible direct effect of the tested extracts on the infectivity of the virus particles, the extracts were incubated with the virus at 4ºC for different periods of time (15, 30 and 45 minutes). Then these mixtures were diluted with fresh medium and cell monolayers were infected with the diluted mixture.

The selectivity index (SI) was determined as CC₅₀/EC₅₀, where CC₅₀ is the concentration found to cause 50% toxicity and EC₅₀ is concentration found to provide 50% prevention of plaque forming units (PFU).

All data were analyzed using Statistica for Windows software (StatSoft, Inc., Tulsa, Oklahoma), and P<0.05 was chosen as the minimal acceptable level of significance.

Vero cells were treated with the extracts at different concentrations (1-1500 µg/ml) for 4 days. Our results (Table 1) show that both ethanol and aquatic extracts of C. fragrans didn't show any toxicity at concentrations ≤ 500 µg/ml, however, the aquatic extracts were less toxic than ethanol extracts. Both ethanol and aquatic of S. chinensis leaf extracts had a high cytotoxicity.

Table 1

Toxicity of Leaf Extracts at Different Concentrations

Table 2

Effect of Time of Leaf Extract Addition on Herpes Viruses Infection

Table 3

Antiviral Activity of Crude Extracts and Fractions of C. fragrans Against Herpetic Viruses

Table 4

Antiviral Activity of Crude Extracts and Fractions of S. chinensis and ACV Against Herpetic Viruses

Vero cell monolayers were treated with increasing extract concentrations at the time of infection with 0.1 m.o.i. of HSV-1, HSV-2 or VZV. The treatment was terminated immediately post-infection (p.i) and the antiviral activity was evaluated by plaque assay.

Fig. (1) demonstrates that the best results were obtained with ethanol extract of C. fragrans which showed the highest SI against HSV-1, HSV-2 and HSV-2 mutant (resistant to ACV), while it was ineffective against VZV. The ethanol extract of S. chinensis was not effective against all tested viruses due to its high toxicity. Therefore, it was not included in the further experiments with the extracts. Although SI of all used extracts were lower than that of ACV (Table 3), these extracts nevertheless inhibited ACV-resistant HSV-2 mutant (Fig. 1).

Fig. (1) Selectivity index (SI) of plant extracts against HSV-1, HSV-2, VZV and HSV-2 mutant (resistant to ACV). SI was determined as CC50 /EC50. Values are means ± SD (n=5).

Fig. (2) Effect of pre-incubation of leaf extracts with HSV-1, HSV-2 and VZV on virus infectivity. (A) 1000 µg/ml of C. fragrans ethanol leaf extract, (B) 1000 µg/ml of C. fragrans aquatic leaf extracts and (C) 100 µg/ml of S. chinensis aquatic leaf extracts were incubated with the infecting virus (5m.o.i.) at 4°C for different periods of time (15, 30 and 45 minutes). Every mixture was diluted 104 times with fresh medium and the cells were infected with the diluted mixture. PFU were evaluated by the standard plaque assay and presented as means ± SD (n=5).

Fig. (3) Effect of leaf extracts removal on the development of herpes viruses cytophatic effect (CPE). Vero cells were infected with 0.05 m.o.i. of HSV-1and treated with 500 µg/ml of C. fragrans ethanol leaf extract at the end of infection. The treatment was terminated at 1, 3 or 10 days p. i. CPE development was evaluated every 24 h by inverted microscopy and expressed as the percentage of damaged cells in the culture. Data are means ± SD of three independent experiments.

Vero cells were infected with 0.1 m.o.i. of the examined herpes viruses and treated with 100 µg/ml of C. fragrans leaf extracts or 50 µg/ml of S. chinesis aquatic extract for various periods of time before, at, or after infection. When the cells were treated with the extracts only at the time of infection or both at the time and post-infection, the inhibition of all examined viruses was the highest (Table 2). It seems that pre-incubation of the cells with the extracts (without further treatment) had no significant effect on the development of infection induced by all examined viruses (p>0.1). However, treatment of the infected cells only p. i. caused partial inhibition of the viral infection (Table 2).

Plant extracts are thought to exert their inhibitory action at a very early stage in the viral infection cycle i.e. virus adsorption onto and/or penetration into the host cell.

In order to examine possible interactions between viral particles and leaf extracts, the tested viruses were pre-incubated with the extracts, then these mixtures were diluted with a fresh medium to antivirally inactive concentrations of the extract. The diluted mixtures were used for infecting Vero cell monolayers. Our results showed considerable inhibition of plaque formation by the highly diluted extracts mixtures (Fig. 2). Most significant inhibition of the tested viruses was obtained after 30-45 minutes of incubation with the different extracts (Fig. 2).

Vero cells were infected with 0.5 m.o.i. of HSV-1 and treated with 500 µg/ml ethanol extract of C. fragrans immediately post infection. At different periods of time p.i. (1, 3, 10 days) the treatment with the extract was terminated. It can be seen that continuous treatment almost prevented the CPE development of all examined viruses (about 95% prevention) during the period of experiment (Fig. 3). However, when the leaf extract was removed even at 10 days p.i., a moderate increase in CPE development was observed (Fig. 3). Similar results were obtained with other plant extracts and other viruses (data not shown). The continuous presence of the leaf extracts in the cell culture medium is apparently essential for continuous protection of Vero cells against viral CPE development.

Cytotoxicity and antiviral activity of several fractions from the plant extracts were examined. The obtained results (Table 3) showed that for C. fragrans, fraction 60%-MeOH of the ethanol extract strongly inhibited HSV-1 and HSV-2 but only slightly VZV, whereas fraction 20%-MeOH of the aquatic extract significantly inhibited all tested viruses. All fractions of both ethanol and aquatic extracts of S. chinensis showed relatively high SI but the 60%-MeOH fraction of the ethanol extract had the highest SI (Table 4).