Antiviral activity of Sanicula europaea L. extracts on multiplication of human parainfluenza virus type 2

PHYTOTHERAPY RESEARCH Phytother. Res. 13, 436–438 (1999)

SHORT COMMUNICATION

Antiviral Activity of Sanicula europaea L. Extracts on Multiplication of Human Parainfluenza Virus Type 2 Ali Karago¨z,1 Nazl Arda,1 Nezhun Go¨ren,2 Kyosuke Nagata3 and Avni Kuru1* 1

Molecular Biology Section, Department of Biology, Faculty of Science, University of Istanbul, Vezneciler, 34459-Istanbul, Turkey Department of Biology, Yildiz Technical University, C¸ukur Saray, Bes¸iktas¸, 80750-Istanbul, Turkey 3 Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan 2

The antiviral activity of Sanicula europaea L. extracts against human parainfluenza virus type 2 (HPIV2) was examined. The extract prepared from the leaves of the plant and a fraction separated from the crude extract with gel filtration chromatography were found to inhibit HPIV-2 replication without any toxic effect on Vero cells. The acidic fraction obtained from the crude extract of S. europaea leaves was found to be the most active fraction with plaque inhibition assay at non-cytotoxic concentrations. Unfortunately, antiviral activity was not detected in the molecules purified from the crude ethanol extract of Sanicula leaves. Copyright # 1999 John Wiley & Sons, Ltd. Keywords: Sanicula europaea L.; Apiaceae (Umbelliferaea); antiviral activity; parainfluenza.

INTRODUCTION

EXPERIMENTAL

S. europaea has been used as a traditional medicinal plant in the treatment of dermatological, gastrointestinal and respiratory system diseases since the 12th century (Winkelmann, 1951). Several biological activities of the plant have been reported (Trzenschik et al., 1967; Hiller and Linzer, 1967; Lamaison et al., 1990). Of interest is that Sanicula extracts have been shown to have antiviral activities. The first evidence was obtained as an antiphage activity (Karago¨z et al., 1995; Turan and Kuru, 1996; Karago¨z et al., 1997). Furthermore, it was shown that the Sanicula extract has an antiinfluenza virus activity (Turan et al., 1996). Also the 50% ethanol extract exhibited the anti-HIV activity and a new triterpene saponin glycoside, saniculoside N, and phenolic acids including rosmarinic acid and caffeic acid were isolated from this extract as major components; only rosmarinic acid was identified as the principal active substance (Arda et al., 1997). The present study describes the effects of Sanicula extracts prepared by several solvents, and the fractions and some pure substances isolated from these extracts on the multiplication of human parainfluenza type 2 (HPIV2) in Vero cells.

Preparation of plant extract. Air-dried leaves of Sanicula europaea L. [Umbelliferaea (Apiaceae)] were used as plant material. The plants were collected from the Botanical Garden of the University of Istanbul and from Arhavi (Northeast Turkey) in early autumn 1994 and identified by Professor A. Aydin (Herbarium no. 864). The air-dried leaves were extracted with bidistilled water at room temperature. Half of the supernatant was frozen and lyophilized, and a dry powder (the crude aqueous extract) was obtained. A part of the crude aqueous extract was separated by gel filtration chromatography with Sephadex G-100 (Pharmacia) and the elution of the column with distillled water yielded two fractions (fraction I and II) as reported earlier (Karago¨z et al., 1997). The residue of the crude supernatant was precipitated by adding an equal volume of 10% trichloroacetic acid (TCA). The precipitate was dialysed against bidistilled water at 4 °C overnight, lyophilized and used as an acidic fraction. The 50% ethanol extract and pure substances (saniculoside N, rosmarinic acid and caffeic acid) from this extract have been obtained previously (Arda et al., 1997).

* Correspondence to: Dr. A. Kuru, Molecular Biology Section, Department of Biology, Faculty of Science, University of Istanbul, Veznealer, 34459Istanbul, Turkey. Contract/grant sponsor: Research Fund of the University of Istanbul; Contract/grant number: T-1/170395. Contract/grant sponsor: Biodesign Research Programme, Riken.

CCC 0951–418X/99/050436–03 $17.50 Copyright # 1999 John Wiley & Sons, Ltd.

Cell and virus. Vero cells were grown and maintained in Eagle’s minimum essential medium (EMEM) with Earle’s saline, supplemented with an antibiotic–antimycotic mixture [penicillin (100 U/mL), streptomycin (100 mg/mL), amphotericin B (0.25 mg/mL)] (Sigma), and 10% (v/v) fetal calf serum (JRH Biosciences). Cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. Human parainfluenza virus type 2 Accepted 21 October 1998

ANTIVIRAL ACTIVITY OF SANICULA EUROPAEA

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the logarithmic phase of the cell growth, the number of viable cells were determined by the trypan blue-exclusion test. The concentration of extract that reduced the viability of the Vero cells by 50% was estimated as the 50% cytotoxic dose (CD50). A plaque inhibition assay was performed according to De Rodriguez et al. (1990) and the 50% effective dose (ED50) was determined from a dose response curve. Antiviral indices (AIs) were also calculated by dividing CD50 by ED50.

RESULTS AND DISCUSSION Figure 1. The effect of Sanicula extracts on HPIV-2 plaque formation. Plaque formation assay was carried out in the presence of increasing concentrations of Sanicula extracts [aqueous (^), ethanol (&), acidic (~), fraction I (&), fraction II (*)].

Figure 2. HPIV-2 replication in the presence of acidic extract. HPIV-2 growth curve was obtained without extract (^) or in the presence of 100 mg/mL acidic extract (&).

(Toshiba strain) was obtained from Mie University, School of Medicine, Japan. Approximately 106 plaque forming units (pfu) of virus in a minimal medium were allowed for the adsortion to Vero cells with gentle rocking of the tissue culture flask every 10 min for 90 min. The virus solution was then removed and freshly prepared media, EMEM containing 2% fetal calf serum (EMEM2), was added. The infected cells were allowed to grow until the cytopathic effect was completely obtained (usually in 48–72 h). The supernatant was removed, quickly frozen and stored at ÿ80 °C until use. The infectivities were determined by plaque assay on Vero cell monolayers and expressed as plaque forming unit per milliliter (pfu/mL). Cytotoxicity tests. Prior to the investigation of the effect of Sanicula extracts on the HPIV-2 multiplication, cytotoxic effects on Vero cells were examined by monitoring cell viability and cell growth rate. For the cell viability test, the Vero cells were collected by trypsinization, and resuspended in Dulbecco’s phosphate buffer saline (Sigma), pH 7.2. The number of viable cells were counted according to the trypan blue-exclusion method (Nagata et al., 1990). For the growth rate test, cells were seeded in a 24-well plate and incubated at 37 °C in the presence or absence of each extract. During Copyright # 1999 John Wiley & Sons, Ltd.

The effects of the extracts and the fractions were examined both on the growth rate and the cell viability of Vero cells. Neither the growth rate nor the cell viability were affected by the aqueous extract, fraction I and the acidic extract at a concentration of 100 mg/mL, or by the ethanol extract and fraction II at a concentration of 50 mg/mL. The results of the plaque inhibition test and the virus yield reduction assay are shown in Fig. 1 and Fig. 2, respectively. According to Fig. 1, the addition of Sanicula extract dose-dependently inhibited the plaque formation of HPIV-2 in Vero cells. Plaque formation was inhibited to different extents by the aqueous extract, fraction I, ethanol extract and acidic extract. In contrast, pure substances (saniculoside N, rosmarinic acid and caffeic acid) isolated from the ethanol extract previously (Arda et al., 1997) were found to be inactive (data not shown) as was fraction II. The virus yield reduction assay (Fig. 2) confirmed the results of the plaque assay. The mean ED50, CD50 and AI (CD50/ED50) values are shown in Table 1. Of the extracts tested, the ethanol extract was consistently the most toxic with a CD50 of 200 mg/mL and the antiviral indices for Sanicula extracts ranged from 10 to 37.5. The most potent extract affecting HPIV-2 replication was the acidic extract, followed by fraction I, the aqueous extract and the ethanol extract. The antiviral effect of Sanicula extracts could neither be attributed to direct inactivation of the human parainfluenza virus type 2 (HPIV-2) nor to inhibition of their adsorption to the host cells as was shown in influenza A/ PR/8/34 (Turan et al., 1996). Since the inhibition of RNA polymerase activity by the extracts in vitro is correlated with the inhibition of the multiplication of influenza A/ PR/8/34 virus (Turan et al., 1996), it seems likely that Sanicula extracts inhibit the RNA polymerase activity of

Table 1. Antiviral indices of Sanicula europaea L. extracts against HPIV-2 in Vero cells Sanicula extract

Aqueous Fraction I Fraction II Acidic 50% EtOH

ED50 (mg/mL)a

20 15

ND

12 20

CD50 (mg/mL)b

500 >500 250 450 200

AIc

25 >33.5 ± 37.5 10

A summary of antiviral experiments composed of mean values derived from at least 3 experiments are presented. a The concentration of extract required to inhibit the virus plaque number by 50%. b The concentration of extract which reduced the viability of the Vero cells by 50%. c AI:CD50/ED50. ND: Not determined. Phytother. Res. 13, 436–438 (1999)

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HPIV-2. The HIV-RT bioassay of the 50% EtOH extract (Arda et al., 1997) also supports this idea. All the results so far obtained suggest that Sanicula extracts contain anti-HPIV-2 substances. However, the mechanism of action and the nature of the active molecule(s), except pure substances, remain to be elucidated. Further analyses of Sanicula extract, including additional purification and chemical characterization, along with further antiviral testing should permit identification of this interesting compound(s) possessing

specific antiviral activity. Also tests on animals should be performed.

Acknowledgements This work was supported by the Research Fund of the University of Istanbul, project number T-1/170395 (A. Karago¨z, A. Kuru) and in part by a grant for Biodesign Research Programme from Riken (K. Nagata).

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Phytother. Res. 13, 436–438 (1999)