PHYTOTHERAPY RESEARCH Phytother. Res. 17, 560 – 562 (2003) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1163 John Wiley & Sons, Ltd.
SHORT COMMUNICATION Research Article
Antiviral Potency of Mistletoe (Viscum album ssp. album) Extracts against Human Parainfluenza Virus Type 2 in Vero Cells Ali Karagöz, Evren Önay, Nazlı Arda* and Avni Kuru
ANTIRIRAL POTENCY OF MISTLETOE EXTRACTS
Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, 34459 Vezneciler-Istanbul, Turkey
Various extracts from the leaves of mistletoe (Viscum album L. ssp. album) were investigated for their antiviral activity on human parainfluenza virus type 2 (HPIV-2) growth in Vero cells. Plant extracts were prepared using distilled water, 50% ethanol, petroleum ether, chloroform and acetone. The 50% effective dose (ED50) of aqueous extract for HPIV-2 replication was 0.53 ± 0.12 µg /mL, and the antiviral index (AI), which was based on the ratio of the 50% inhibitory concentration (CD50) for host cell viability to the ED50 for parainfluenza virus replication, was 10.05. The aqueous extract was found to be the most selective inhibitor. Furthermore, the aqueous extract at a concentration of 1 µg /mL was found to inhibit HPIV-2 replication and the virus production was suppressed to more than 99% without any toxic effect on host cells. The chloroform extract was also found to be moderately active. In an effort to further analyse the mechanism of antiviral activity, the effectiveness of the aqueous extract on different steps of virus replication was examined. The antiviral activity could neither be attributed to the direct inactivation of the HPIV-2 nor to the inhibition of adsorption to Vero cells. The active aqueous extract has shown a dose-dependent antiviral activity on virus replication. Copyright © 2003 John Wiley & Sons, Ltd. Keywords: antiviral activity; mistletoe; Viscum album L. ssp. album; human parainfluenza virus.
INTRODUCTION
MATERIALS AND METHODS
Mistletoe (Viscum album L. ssp. album) (Loranthaceae) is a semi-parasitic plant, which grows on dicotyledonous trees. It has been used as a traditional medicinal plant in the treatment of many diseases such as hypertension, epilepsy, asthma, vertigo, lumbago, etc. for centuries (Duke, 1987). Many herbal preparations containing Viscum extracts are used in complementary cancer therapy (e.g. Iscador®, Helixor®, Isorel®) (Zarkovic et al., 1997), especially in Europe. Viscum extracts have also been reported to be effective as immunomodulators for adjuvant AIDS therapy (http://www.immunet.org/immunet/ atn.nsf/page/ZQX09203.html). A number of biological activities of the plant have been reported (Stein et al., 1998), but only few studies have examined antiviral activity. Mistletoe extracts have been used in HIV-positive and healthy individuals and indirect results were discussed (Stoss and Gorter, 1999). Since our laboratory carries out routine analysis of the antiviral activity of selected medicinal plants in Turkey (Turan et al., 1996; Arda et al., 1997; Karagöz et al., 1999), the antiviral potency of this plant on HPIV-2 was investigated, as well as the possible mechanism of activity in this study.
Preparation of plant extracts. Viscum album L. ssp. album growing on lime trees was collected from Belgrad Forest, Bentler Region (Istanbul) in February 2001. A voucher specimen (ISTF No. 34486) was deposited in the Herbarium of the Section of Botany, Department of Biology, Faculty of Science, Istanbul University. Professor Tuna Ekim identified the plant material. Two separate samples of the air-dried and powdered leaves of the plant material (20 g of each) were extracted with distilled water (150 mL) and 50% ethanol (150 mL) at room temperature using a Waring blender. Alternatively, a sample of the plant material (20 g) was macerated with petroleum ether, chloroform and acetone (100 mL of each) successively in a shaker (190 rpm, 40 °C) overnight. Plant residues were removed by centrifugation (15 000 rpm, 20 min, 4 °C) and the supernatant was evaporated to dryness under reduced pressure and/or lyophilized. In this way, five different crude extracts were obtained: aqueous extract (AE), 50% ethanol extract (EE), petroleum ether extract (PEE), chloroform extract (CE) and acetone extract (ACE). AE was dissolved in the medium (Eagle’s mimimum essential medium, EMEM), EE in dimethyl sulphoxide (DMSO) and the others in corresponding solvents (PEE in petroleum ether; CE in chloroform; ACE in acetone). They were then prepared at various concentrations in EMEM. The solvent used in the preparation of the extract was added to the control medium.
* Correspondence: Dr N. Arda, Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, 34459 VeznecilerIstanbul, Turkey. E-mail:
[email protected] Contract/grant sponsor: The Research Fund of Istanbul University; Contract/grant number: 1339/280799, 1603/30042001.
Copyright © 2003 John Wiley & Sons, Ltd.
Received 14 September 2001 Accepted 13 November 2001
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Cell and virus. The test system consisting of Vero cells and human parainfluenza virus type 2 (HPIV-2) (Toshiba strain) used in this study was evaluated as described earlier (Karagöz et al., 1999). Cytotoxicity tests. The cytotoxic effects on Vero cells were examined as reported earlier (Karagöz et al., 1999). The concentration of extract that reduced the viability of the Vero cells by 50% was estimated as the 50% cytotoxic dose, CD50. Plaque assay. Confluent monolayer cultures of Vero cells in a 60 mm plastic dish were washed twice with Dulbecco’s phosphate buffer saline (PBS) (Sigma, pH 7.2) and exposed to HPIV-2 at a MOI of 0.0001. After adsorption for 90 min at 37 °C, the cells were washed twice with PBS and then overlaid with EMEM containing 0.9% agarose and plant extracts prepared at different concentrations. The cultures were incubated at 37 °C for 3–4 days. At the end of this period the cell monolayers were fixed in formol-saline for 15 min and stained with amido black (Nagata et al., 1990). The concentration of extract that was required to inhibit the virus plaque number by 50% was estimated as the 50% effective dose (ED50). The antiviral index (AI) was calculated by dividing (CD50) by (ED50). Investigation of possible mechanism of antiviral activity. The effectiveness of the aqueous extract (AE) on different steps of virus replication was analysed, in order to examine possible mechanism of the antiviral activity, using the following procedures.
RESULTS AND DISCUSSION The effect of each extract was examined both on the growth rate and the cell viability of Vero cells. Neither the growth rate nor the cell viability were affected by EE, PEE or ACE at a concentration of 25 µg/mL or by the AE at a concentration of 1 µg/mL, or CE at a concentration of 10 µg/mL (data not shown). The CD50 values of AE and CE obtained by counting viable cells were found to be 5.33 ± 2.05 and 39.6 ± 4.5 µg/mL, respectively. The AE and CE dose-dependently inhibited the plaque formation on HPIV-2 in Vero cells (Fig. 1), whereas the other extracts (EE, PEE and ACE) were found to be inactive. The dose required to reduce the plaque number by 50%, ED50, was calculated and the mean ED50, CD50 and AI (CD50 /ED50) values were found. The ED50 of AE and CE for HPIV-2 were 0.53 ± 0.12 µg /mL and 24.3 ± 5.31 µg/mL, respectively (Table 1). When the cytotoxicity was evaluated by the reduction of cell viability (50% cytotoxic dose), AE and CE were toxic to Vero cells at a concentration of 5.33 ± 2.05 µg/mL and 39.6 ± 4.5 µg/mL, respectively. The antiviral index (AI), which was based on the ratio of CD5 0 /ED50, of AE was much higher than those of CE. The antiviral indices for Viscum extracts, AE and CE were 10.05 and 1.63, respectively. Thus, the most potent extract against
Direct pre-infection incubation (DPI) assay. Equal volumes of AE, prepared in EMEM containing 2% fetal calf serum (EMEM2) at a 10 µg /mL concentration, and the virus stock were mixed and incubated for 6 h, at 37 °C, 25 °C and 4 °C, to test whether AE had a direct virucidal effect on HPIV-2. At the end of the incubation, virus titres were determined by plaque assay. The experiment was performed in triplicate. Effect on the adsorption of virus to cells. In the experiments carried out to determine whether AE has any direct action on the adsorption of HPIV-2 to Vero cells, the following experiment was employed (Dargan and Subak-Sharpe, 1985). Vero cells were grown to confluence in 60 mm dishes. Viscum extract (AE) was added to the dishes (final concentration, 5 µg/mL) and incubated at 37 °C for 4 h. In order to determine the effect of AE on the adsorption of virus to the cells, the cells were washed with EMEM2 and inoculated with HPIV-2 at a MOI of 0.001 at 37 °C for 90 min. The inocula were recovered, dishes were washed three times with EMEM2, and each wash was harvested. The unadsorbed viruses in these samples were determined by plaque assay. The experiment was performed in triplicate. Virus yield reduction assay. Vero cell cultures were inoculated at a MOI of 0.01. After adsorption, the infected cells were overlaid with EMEM2 either extract free or containing 1 µg/mL AE. At the indicated times after adsorption the culture fluids were harvested and disrupted by three cycles of freezing and thawing. Virus yields were determined by plaque assay. Copyright © 2003 John Wiley & Sons, Ltd.
Figure 1. Effect of Viscum extracts on HPIV-2 plaque formation [ aqueous extract (AE); chloroform extract (CE)]. Inactive extracts (EE, PEE and ACE) are not shown.
Table 1. Comparative potency of Viscum extracts against HPIV-2 in Vero cells Viscum extract Aqueous (AE) EtOH (EE) Petroleum ether (PEE) Chloroform (CE) Acetone (ACE)
ED50 (µg/mL)a 0.53 ± 0.12 ND ND 24.3 ± 5.31 ND
CD50 (µg/mL)b 5.33 ± 2.05 >50 >50 39.6 ± 4.5 >50
AIc 10.05 – – 1.63 –
a Fifty-percent effective dose, or concentration required to inhibit virus plaque formation by 50%. b The concentration of extract, which reduced the viability of the Vero cells by 50%. c Antiviral index: CD50 / ED50. ND: Not determined Values of ED50 and CD50 are averages and standard deviations for 3 independent experiments.
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HPIV-2 replication was the aqueous extract while the chloroform extract also had moderate activity. Aqueous extract (AE) at a concentration of 10 µg /mL was tested for its virucidal effect, using the DPI assay but no activity was found (data not shown). In the experiments performed to determine whether AE has any effect on adsorption, the number of viruses that were not bound to monolayers was compared with those recovered from untreated cells. There were no significant differences between the residual infectivity (p.f.u/mL) in each sample. The fact that virus adsorption to the cells pretreated with the extract was almost the same as that of the untreated cells suggests that the extract did not act at this early stage in virus replication cycle. These results suggest that AE interferes with a subsequent stage of the virus replication cycle within the cell. The differential sensitivity of HPIV-2 to the aqueous extract was further studied by virus yield reduction assay in Vero cells. Production of the infectious virus decreased over 72 h in the culture with added AE (Fig. 2). This result, possibly reflects a greatly reduced rate of infectious viral synthesis compared with the control. In the presence of 1 µg/mL AE, the total amount of HPIV-2 produced was only 0.3% of the total amount of the control, which corresponds to an inhibition of 99.7%. Since the antiviral activity was not related to
Figure 2. HPIV-2 replication in the presence of aqueous extract. HPIV-2 growth curve was obtained without extract () or in the presence of 1 µg/mL aqueous extract (AE) ().
the virucidal effect or virus adsorption, progeny virus released from infected cells into the surrounding, extract containing medium, was not inactivated or reduced. Therefore, it seems likely that the antiviral activity of AE is dependent on the inhibition of HPIV2 replication in Vero cells. In summary, the aqueous extract prepared from Viscum album L. ssp. album growing on lime trees appears to have a potent anti-parainfluenza virus activity.
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Copyright © 2003 John Wiley & Sons, Ltd.
Stein GM, Schietzel M, Büssing A. 1998. Mistletoe in immunology and the clinic (short review). Anticancer Res 18: 3247 – 3250. Stoss M, Gorter RW. 1999. No evidence of IFN-gamma increase in the serum of HIV-positive and healthy subjects after subcutaneous injection of non-fermented Viscum album L. extract. Nat Immun 16: 157–164. Turan K, Nagata K, Kuru A. 1996. Antiviral effect of Sanicula europaea L. leaves extract on influenza virus-infected cells. Biochem Biophys Res Commun 225: 22 –26. Zarkovic N, Zarkovic K, Grainca S, Kisel D, Jurin M. 1997. The Viscum album preparation Isorel inhibits the growth of melanoma B16F10 by influencing the tumor-host relationship. Anticancer Drugs 8: 17 –22.
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