Mechanism of herpes simplex virus type 2 suppression by propolis extracts.ABSTRACT
Genital herpes caused by herpes simplex virus type 2 (HSV-2) is a chronic, persistent infection spreading efficiently and silently as sexually transmitted disease through the population. Antiviral agents currently applied for the treatment of herpesvirus infections include acyclovir and derivatives. Aqueous and ethanolic extracts of propolis were phytochemically analysed, different polyphenols, flavonoids and phenylcarboxylic acids were identified as major constituents. The aqueous propolis extract revealed a relatively high amount of phenylcarboxylic acids and low concentrations flavonoids when compared to the ethanolic special extract GH 2002. The cytotoxic and antiherpetic effect of propolis extracts against HSV-2 was analysed in cell culture, and revealed a moderate cytotoxicity on RC-37 cells. The 50% inhibitory concentration ([IC.sub.50]) of aqueous and ethanolic GH 2002 propolis extracts for HSV-2 plaque formation was determined at 0.0005% and 0.0004%, respectively. Both propolis extracts exhibited high levels of antiviral activity against HSV-2 in viral suspension tests, infectivity was significantly reduced by >99% and a direct concentration-and time-dependent antiherpetic activity could be demonstrated for both extracts. In order to determine the mode of virus suppression by propolis, the extracts were added at different times during the viral infection cycle. Addition of these drugs to uninfected cells prior to infection or to herpesvirus-infected cells during intracellular replication had no effect on virus multiplication. However both propolis extracts exhibited high antiherpetic activity when viruses were pretreated with these drugs prior to infection. Selectivity indices were determined at 80 and 42.5 for the aqueous and ethanolic extract, respectively, thus propolis extracts might be suitable for topical therapy in recurrent herpetic infection.
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Herpes simplex virus type 2
Propolis is a resinous hive product collected by honeybees from various plant sources. It has a long history of being used in folk medicine (Ghisalberti 1979) and also has been reported to possess a wide range of biological activities. The chemical components of propolis are qualitatively and quantitatively variable, depending on the geographic origin and regional plant ecology. Since propolis possesses a broad spectrum of biologica activities, it is applied in popular folk medicine. It has also beer used as a health drink in Asian, European and American countries (Banskota et al. 2001). Pharmacological properties of different propolis preparations have been reported as antihepatotoxic (Gonzales et al. 1995), anticarcinogenic, (El-khawaga et al. 2003) antioxidative (Russo et al. 2002), neuroprotective (Nakajima et at 2007), anti-inflammatory (Borrelli et al. 2002), antimicrobial (Takaisi-Kikuni and Schilcher, 1994; Kujumgiev et al. 1999; Scheller et al. 1999; Koo et al. 2000; Marcucci 1995; Abd EI-Hadi and Hegazi. 2002; Kartal et al. 2003; Al-Waili 2005; Boyanova et al. 2005; Popova et al. 2005) and antiviral. A pharmacological activity against several viral infections has been demonstrated, e.g. influenza (Serkedjieva et al. 1992), HIV (Ito et al. 2001), adenovirus (Amoros et al. 1992a), and herpes simplex virus (Debiaggi et al. 1990; Amoros et al. 1994; Huleihel and Isanu 2002; Schnitzler et al. 2009). Therapeutic benefits have been reported for propolis extracts against respiratory tract infections in children (Cohen et al. 2004) and genital herpes (Vynograd et al. 2000). Propolis reveals a broad spectrum of biological activities, is used in food and folk medicine, thus there is a renewed interest in its antimicrobial and antiviral potential.
Herpes simplex virus (HSV) is differentiated into two antigenic types of type 1 (HSV-1) and type 2 (HSV-2) and infects mucocutaneous surfaces. In the acute stage of ganglionic infection, some sensory neurons undergo lytic virus infection and are destroyed, as are cells at the site of entry. After inoculation and limited replication at genital sites, HSV-2 ascends along neuronal axons to establish latent infection for life-time in the lumbosacral sensory ganglia. The latent virus is reactivated spontaneously or is induced to reactivate by a variety of stimuli. During the reactivation process, the virus is transported through the nerve cells axons to the original peripheral infection site, where HSV replication occurs. Infectivity is highest in pimary infections and virus excretion can persist for many weeks beyond clinical healing. While both types of HSV produce first episode genital infection, most cases of symptomatic primary disease are due to HSV-2 (Sucato et al. 1998). HSV-2 prevalence is usually higher in women than men in populations with higher risk sexual behavior (Howard et al. 2003; Smith and Robinson 2002). Genital herpes is one of the most prevalent sexually transmitted disease worldwide and is the most common cause of genital ulcers. What makes HSV so difficult to control is that most sexual and perinatal transmissions occur during unrecognized or asymptomatic shedding (Koelle and Wald 2000). The impact of genital herpes as a public health threat is augmented because epidemiological studies clearly demonstrate a strong link to the HIV epidemic. Genital herpes is a chronic, persistent infection that, on any given day, causes subclinical reactivation in about 1 to 2% of infected persons (Roizman and Sears 1995; Wald et al. 1995). HSV-2 can spread efficiently and silently as sexually transmitted disease through the population, A dramatic increase in the prevalence of HSV-2 infection was observed in younger age cohorts (Fleming et al. 1997). Genital herpes continues to be a public health problem in both developed and developing countries.
In the current study we have investigated the virus suppression by an aqueous and a special ethanolic propolis extract named GH 2002 against herpes simplex virus type 2. Both extracts were prepared from propolis of Middle Europe, well characterized in respect to its botanical and geographical origin as well as chemical composition.
Materials and methods
Aqueous and ethanolic propolis extracts
Propolis, the bee glue of Apis mellifera, was collected at Moravia, Czech Republic, has a defined composition, quality and provenance and contains flavonoids and phenylcarboxylic acids. Aqueous propolis extract was prepared by extracting propolis with 15% (v/v) ethanol, resulting in a viscous gold brown to brown extract with an aromatic odour with a drug to dried extract ratio of 15:1; the dried extract corresponds to about 7% of the primary raw propolis material. For experiments, an aliquot of the viscous aqueous extract was dissolved in 15% (v/v) etharnoi to obtain a 10% (v/v) stock solution (1:9 diluted native extract). This aqueous extract was further diluted in water for cell culture experiments, always resulting in an ethanol concentration below 1% final concentration which has no effect on cells and viruses as reported previously (Schnitzler et al. 2009). The ethanolic extract GH 2002 was prepared with a special procedure to remove the wax and resin components. Subsequently, propolis was extracted with 90% ethanol resulting in a viscous brown extract with a bitter flavour and a drug to dried extract ratio of 2:1; the dried extract corresponds to about 50% of the primary raw propolis material. For experiments, an aliquot of GH 2002 was dissolved in 90% (v/v) ethanol for preparation of a 10% (v/v) stock solution. For cell culture experiments, ethanolic propolis extract was further diluted resulting in a final ethanol concentration below 1% which is not toxic for cells and viruses.
Acyclovir was purchased from GlaxoSmithKline (Bad Oldesloe, Germany) and dissolved in sterile water.
Phytochemical analysis of aqueous and ethanolic propolis extracts by HPLC
Analytical HPLC of the aqueous and ethanolic extracts of propolis was run on HPLC (Shimadzu LC 10) equipped with a diode array detector Shimadzu SPD-M 10 Avp. Separation was performed on a Purospher Star RP-18 column (250 x 4.6 mm), particle size of 5 u.m, using a mobile phase of acidified water and acetonitril. The elution was carried out with a non-linear gradient and a flow rate of l.Oml/min, spectrophotometric detection was conducted at 220 nm. The components were identified by comparison of their retention times and UV-spectra with standards, acquired commercially. The content of phenols in the extracts was determined according to Pharm. Eur. 2.8.14.
Ceil culture and viruses
RC-37 cells (African green monkey kidney cells) were grown in monolayer culture with Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum (FCS), 100 u/ml penicillin and 100[mu]g/ml streptomycin. The monolayers were serially passaged whenever they became confluent, cells were plated into 96-weIl and 6-well culture plates for cytotoxicity and antiviral assays, respectively, and propagated at 37[degrees]C in an atmosphere of 5% [CO.sub.2] (Schnitzler et al. 2007). Herpes simplex virus type 2 (HSV-2) was used for experiments. Viruses were routinely grown on RC-37 cells as described previously (Koch et al. 2008).
Cytotoxicity and antiviral assay
For cytotoxicity assays, 6 x [10.sup.3] cells were seeded into 96-well plates per well and incubated for 24 h at 37[degrees]C. The medium was removed and fresh DMEM (Dulbecco's modified minimal essential medium) containing the appropriate dilution of propolis extracts were added onto subconfluent cells in eight replicates for each concentration of the drug. Wells containing medium with 1% ethanol but no drug were also included on each plate as controls. After 3 days of incubation, the growth medium was removed and viability of the drug treated cells RC-37 was determined in a standard neutral red assay (Soderberg et al. 1996). The mean OD of the cell-control wells was arbitrarily assigned to 100%. The cytotoxic concentration of the drug which reduced viable cell number by 50% ([TC.sub.50]) and the maximum noncytotoxic concentration of each drug were determined from dose-response curves. Inhibition of HSV replication was evaluated with plaque reduction assays. Usually 2 x [10.sup.3] plaque forming units (pfu) were incubated with different concentrations of propolis extracts or selected compounds for 1 h at room temperature, afterwards treated viruses were allowed to adsorb to RC-37 cells for 1 h at 37 [degrees]C. The residual inoculum was then discarded and infected cells were overlaid with medium containing 0.5% methylcellulose. Each assay was performed in six replicates. After incubation for 3 days at 37 [degrees]C, monolayers were fixed with 10% formalin, stained with 1% crystal violet and subsequently macroscopically clearly visible plaques were counted visually. By reference to the number of plaques observed in virus control monolayers without addition of drugs, the concentration of test compound which inhibited plaque numbers by 50% ([IC.sub.50]) was determined from dose-response curves (Koch et al. 2008).
Mechanism of HSV-2 suppression
In order to determine the mechanism of virus suppression for propolis extracts, cells were pretreated with propolis extracts before viral infection, viruses were incubated with these drugs before infection or infected cells were incubated together with propolis extracts after penetration of the virus into the host cells (Fig. 1). Aqueous and ethanolic propolis extracts were always used at the maximum non-cytotoxic concentration. Cell monolayers containing 5 x [10.sup.5] cells were pretreated with the drugs prior to inoculation with virus by adding these drugs to the culture medium and subsequent incubation for 1 h at 37[degrees]C After incubation, drugs were aspirated and cells were washed immediately before the HSV inocolum without drug was added. For pre-treatment of herpes simplex virus, HSV-2 was incubated in medium containing the maximum non-cytotoxic concentration of the drugs for 1 h at room temperature prior to infection of RC-37 cells. After lh of adsorption at 37[degrees]C, the inocolum was removed and cells were overlaid with medium containing 0.5% methylcellulose without drug. The effect of propolis extracts against HSV was also tested during the replication period by adding extracts after viral penetration to the overlay medium, as typical performed in antiviral susceptibility studies. Each assay was run in triplicate, the number of plaques of drug-treated cells or viruses were compared to untreated controls to calculate the extent of plaque reduction. All untreated controls were incubated without drugs, acyclovir was used as positive antiviral control.
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All experiments were performed in triplicate and statistical analysis was performed by SPSS software (SPSS for Windows, 11.0, 2001, SPSS Chicago, Illinois). The means and standard errors were recorded.
Phytochemical analysis of aqueous and ethanolic propolis extracts by HPLC
Propolis, the bee glue of Apis mellifera, was collected at Moravia, Czech Republic, has a defined homogeneous composition, quality and provenance and contains polyphenols, flavonoids and phenylcarboxylic acids. The chemical composition of the aqueous and ethanolic extracts has been characterized every year and demonstrated a highly conserved distribution and homogenous pattern for polyphenoles, flavonoids and phenylcarboxylic acids for many years (Table 1). HPLC analysis of the aqueous and ethanolic propolis extracts identified polyphenoles, flavonoids and phenylcarboxylic acids as main components (Figs. 2 and 3). With regard to flavonoids, both propolis extracts differed quantitatively and qualitatively. The ethanolic extract displayed a relatively high flavonoid content including quercetindihydrate, chrysin, pinocembrin and galangin. In contrast, the aqueous propolis extract demonstrated only a very low content of flavonoids lacking galangin and quercetindihydrate but revealed a high amount of phenylcarboxylic acids (Table 1). it could be demonstrated that both propolis extracts differed qualitatively and quantitatively concerning flavonoids, and quantitatively in respect to phenylcarboxylic acids. Structural formulas of major compounds in both extracts are shown in Fig. 4.
Table 1 Phytochemical HPI.C analysis of aqueous and ethanolic propolis extracts. compound aqueous ethanolic propolis propolis extract extract polyphenoles 13.1% 9.47% flavonoides quercetindihydrate n.d. 0.13% chrysin 0.22% 2.32% pinocembrin 0.16% 2.81% galangin n.d. 2.05% phenylcarboxylic acids benzoic acid 3.94% 1.50% cinnamic acid 0.34% 0.63% caffeic acid 1.52% 0.46% p-cumaric acid 6.00% 2.03% n.d.: not detected.
Aqueous and ethanolic propolis extracts were serially diluted and added to cell culture medium to examine the effect on the growth and viability of tissue culture cells. Propolis extracts were further diluted in medium for cell culture experiments, always resulting in an ethanoi 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, cell viability of RC-37 cells was determined with the neutral red assay. The maximum noncytotoxic concentrations of the tested drugs were determined at 0.03% for aqueous propolis extract, 0.001% for the ethanolic propolis extract, TC50 values are 0.04% and 0.017%, respectively. Experiments to assess the cytotoxicity of propolis extracts for cultured eucaryotic cells indicate a moderate toxic behaviour of these extracts in cell cultures.
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The potential antiviral effect of different propolis extracts and some components was determined against herpes simplex virus type 2 (HSV-2) in vitro. HSV-2 was pretreated for 1 hour with various concentrations of aqueous and ethanolic propolis extract. In all assays untreated virus-infected cells without addition of drugs were used as negative control. HSV-2 was incubated with serially diluted drugs for 1 hour at room temperature. Subsequently, aliquots of each dilution were added on the cells and incubated for 1 h at 37 [degrees]C. The 50% inhibitory concentrations ([IC.sub.50]) for HSV-2 of aqueous propolis extract and ethanolic propolis extract were determined at 0.0005% and 0.0004%, respectively (Fig. 5). The results are presented as a percentage of the control and represent the mean of three independent experiments. In plaque reduction assays, both propolis extracts exhibited a concentration-dependent antiviral effect. Using maximal noncytotoxic concentrations of aqueous and ethanolic extracts in viral suspension assays, plaque formation for HSV-2 was reduced by >99%, respectively (Fig. 5). Selectivity indices for aqueous and ethanolic propolis extracts were calculated as the [TC.sub.50]/[IC.sub.50] ratio and have been determined at SO and 42.5, respectively. In order to examine the time-dependence of the antiviral effect, HSV-2 was incubated with maximum noncytotoxic concentrations of the propolis extracts for different periods of time. After 1, 5, 10,15, 20, 30, 60 and 120 minutes, an aliquot was applied on confluent monolayers of RC-37 cells. A clearly time-dependent activity could be demonstrated for both propolis extracts, inactivation of herpes simplex virus and plaque reduction was faster achieved when the aqueous extract was used (Fig. 6). After 60 min of incubation with propolis extracts, the infectivity of HSV-2 was nearly completely abolished, the ethanolic propolis extract exhibited a less pronounced antiviral activity. A clearly time-dependent anti-HSV-2 activity could be demonstrated for both propolis extracts. In both cases, after 1 h of incubation plaque formation was reduced by >99%.
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Fig. 5. Determination of the 50% inhibitory concentration (IC.sub.50) of propolis extracts against HSV-2. Viruses were incubated for 1 hour at room temperature with increasing concentrations of the extracts and immediately tested in a plaque reduction assay. Experiments were repeated independently and performed in triplicate assays, data are presented as mean[+ or -] SD of three independent experiments.
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Fig. 6. Time-dependent activity aqueous and ethanolic propolis extracts against herpes simplex virus type 2. Virus was incubated with maximum noncytotoxic concentrations of these propolis extracts for indicated time periods. Experiments were repeated independently, data are presented as mean [+ or -]SD of three independent experiments.
Mechanism of HSV-2 suppression
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, propolis extracts and their major compounds were added at different stages during viral infection. Tested drugs did not show any effect on HSV when host cells were pretreated prior to infection (Table 2). Incubation of HSV-2 with the aqueous propolis extract and ethanolic propolis extract caused a significant suppression of HSV multiplication. At maximum noncytotoxic concentrations of the tested drugs, infectivity was reduced by >99%. In contrast, when the extracts were added to the overlay medium after penetration of the viruses into the host cells, plaque formation was not significantly reduced. For comparison, all untreated controls contained the same concentration of ethanol as the drug-treated viruses, in order to exclude any influence of ethanol. Acyclovir showed the highest antiviral activity when added during the replication period with inhibition of the viral replication of 98.8%. This drug inhibits specifically the viral DNA polymerase during the replication cycle when new viral DNA is synthesized.
Table 2 Suppression of herpes simplex virus type 2 (HSV-2) by acyclovir, aqueous and ethanolic propolis extract at different times during the viral infection cycle. acyclovir aqueous extract suppression (%)[+ or -]SD pretreatment cells 3.9[+ or -]0.5 16.8[+ or -]2.1 pretreatment virus 10.7[+.or.-]1.8 99.4[+ or -]2.7 replication 98.8[+ or -]3.1 22.9[+ or -]1.6 ethanolic extract pretreatment cells 29.1[+ or -]3.4 pretreatment virus 99.6[+ or -]2.5 replication 6.3[+ or -]0.7
Propolis reveals a broad spectrum of biological activities, is used in food and folk medicine, thus there is a renewed interest in its antimicrobial and antiviral potential (Banskota et al. 2001). It is well understood that bees collect propolis to seal their hive and to prevent the decomposition of creatures which have been killed by bees after an invasion of the hive. Thus, propolis is considered to possess antimicrobial activity. Bankova et al. (1995) examined the antibacterial activity of different fractions of propolis towards Staphylococcus aureus and observed that the antibacterial activity is mainly due to polar phenolic compounds. Genital herpes is a critical global health problem and is associated with the HIV epidemic. Only little information on the effects of propolis against viral infections is available, most reports on the inhibitory activity of essential oils against HSV are anecdotal reports. Several drugs Maximum noncytotoxic concentrations of propolis extracts were used for all experiments. Data represent percentages of virus suppression compared to untreated controls and are the mean of three independent experiments. are currently available for the management of HSV infections such as acyclovir. Acyclovir and related synthetic nucleosides interfere with viral DNA replication through activation by viral thymidine kinase (De Clercq 2004). Genital herpes is a chronic, persistent infection that might reactivate quite frequently. A dramatic increase in the prevalence of HSV-2 infection was observed in younger age cohorts (Fleming et al. 1997). Genital herpes continues to be a public health problem in both developed and developing countries. A therapeutic application of propolis against viral and bacterial infections has been described previously, propolis is effective against respiratory tract infections in children (Cohen et al. 2004) and genital infections (Vynograd et al. 2000). Patients with recurrent genital herpes infections participated in a multi-center study comparing the efficacy of ointment of Canadian propolis containing natural flavonoids with ointments of acyclovir and placebo. The healing process of HSV-2 infected patients was faster in the propolis-treated group.
Our results suggest that both propolis extracts interfere with virion envelope structures or are masking viral compounds which are necessary for adsorption or entry into host cells. Apparently, free herpesvirus is very sensitive to propolis extracts and the inhibition of HSV-2 appears to occur before entering the cell but not after penetration of the virus into the cell. It remains to be determined whether the inhibitory effect is due to binding of some constituents of the extracts to viral proteins involved in host cell adsorption and penetration or is due to damage of the virions, possibly their envelopes, thereby impairing their ability to infect host cells. Recently it has been reported that propolis demonstrated a similar antiviral effect on HSV-1 (Schnitzler et al. 2009). The positive antiviral control acyclovir showed the highest antiviral activity when added during the intracellular replication period. This drug inhibits specifically the viral DNA polymerase during the intracellular replication cycle when new viral DNA is synthesized. In contrast to propolis extracts, no effect on viral replication was detected when acyclovir cells or viruses were pretreated with acyclovir. Amoros et al. (1992b) investigated the in vitro antiviral activity of resin balsam against HSV and could detect a virucidal effect when herpesvirus was pretreated with propolis, but pre-treatment of cells with propolis did not inhibit viral replication. These data are in accordance with our results. On the other hand, when propolis was only added during intracellular replication, the plaque size was greatly reduced, this finding differs from our experimental experience. In contrast, Huleihel and Isanu (2002) reported a strong interaction between propolis extract and the surface of Vera cells but no direct interaction with herpesvirus particles. Administration of propolis before or at the time of infection yielded the most significant inhibitory effect. Thus the antiviral activity is probably due to prevention of virus adsorption to host cells and apparently different propolis preparations reveal different modes of antiviral action. Debiaggi et al. (1990) investigated propolis-derived flavonoids and reported concentration-dependent reduction of HSV replication for quercetin, when high drug concentrations were used. A synergy could be demonstrated when binary flavone-flavonol combinations of propolis compounds were tested against HSV, thus explaining why propolis is more active than its individual compounds (Scheller et al. 1999). Consequently the extracts containing many different components are more effective against herpes simplex virus than single isolated components and exhibited significant higher antiherpetic effects as well as higher selectivity indices than single isolated constituents (Schnitzler et al. 2009). Propolis extracts might be suitable for topical application against herpes infection. Propolis from different geographic origins and with different phytochemical compositions had been compared in their antiviral activity against avian influenza virus (Kujumgiev et al. 1999). Flavonoids and esters of phenolic acids in one propolis sample were responsible for the antiviral effect, whereas tropical propolis samples did not contain these compounds but were still active against this virus. Obviously, in different samples, different substance combinations are essential for the biological activity of propolis. Synthetic substances as esters of substituted cinnamic acids were synthesized and inhibited significantly influenza virus H3N2. Pretreatment of cells with this synthetic compound was not effective, but adsorption of the virus was inhibited (Serked-jieva et at. 1992). Probably, the antiviral impact of propolis is not only due to single compounds, but to a mixture of different constituents and therefore the propolis complex in the galenic preparation as extract is more effective.
A topical application of propolis for the treatment of oral herpetic infections with herpes simplex virus appears promising, especially for those patients suffering from frequent recurrences.
We would like to thank Dr. G. Darai for helpful discussions and continuing support.
Silke Nolkemper(a), (b), Jurgen Reichling (b,) Karl Heinz Sensch(c,) Paul Schnitzler(a) *
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(a) Department of Virology, Hygiene Institute, University of Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
(b) Department of Biology, Institute of Pharmacy and Molecular Biotechnology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
(c) Gehrlicher GmbH, Department Research and Development; Robert Koch .Sir. 5. 82547 Eurasbung, Germany
* Corresponding author. Tel: +496221 56 50 16; fax: +49 6221 56 50 03.
E-mail address: Paul_Schnitzler@med.uni-heiclelberg.de (P. Schnitzler).
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