In vitro antiviral effects of an aqueous extract ofArtemisia verlotorum Lamotte (Asteraceae)

PHYTOTHERAPY RESEARCH, VOL. 12, 595–597 (1998)

SHORT COMMUNICATION

In vitro Antiviral Effects of an Aqueous Extract of Artemisia verlotorum Lamotte (Asteraceae) Vincenzo Calderone,1* Elisa Nicoletti,2 Patrizia Bandecchi,3 Mauro Pistello,2 Paola Mazzetti,2 Enrica Martinotti1 and Ivano Morelli4 1 Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Facolta` di Farmacia, Universita` degli Studi di Pisa, via Bonanno 6, 56126 Pisa, Italy 2 Centro Retrovirus e Sezione Virologia, Dipartimento di Biomedicina, Universita` degli Studi di Pisa, Pisa, Italy 3 Dipartimento di Patologia Animale, Profilassi e Igiene degli Alimenti, Universita` degli Studi di Pisa, Pisa, Italy 4 Dipartimento di Chimica Bioorganica, Universita` degli Studi di Pisa, Pisa, Italy

Plants represent a possible source of new interesting antiviral drugs. An aqueous extract of Artemisia verlotorum exhibited, in vitro, strong activity against the feline immunodeficiency virus (FIV), which can be considered a reliable model of the human immunodeficiency virus type 1, the aetiological agent of AIDS. The extract determined a decrease of the virus-induced syncytia in the cultured cells, an inhibition of the viral reverse transcriptase activity and a reduction of the viral capsid protein P24 expression. # 1998 John Wiley & Sons, Ltd. Phytother. Res. 12, 595–597 (1998) Keywords: Artemisia verlotorum Lamotte; FIV; antiviral activity.

INTRODUCTION Several natural products from plants are under evaluation as possible new antiviral drugs. Human immunodeficiency virus type 1 (HIV-1), the aetiological agent of AIDS, probably represents the most important target of such research, and many plant species, such as Hyssopus officinalis (Gollapudi et al., 1995; Kreis et al., 1990), Formitella supina, Phellinus rhabarbarinus, Trichaptum perrottettii, Trametes cubensis (Walder et al., 1995), Swertia franchetiana, Maprounea africana and Cetraria islandica (Pengsuparp et al., 1995), showed interesting antiviral activity against HIV-1. The aim of this work was to evaluate, in in vitro cultured Crandell feline kidney cell line (CrFK) cells, the antiviral effects of an aqueous extract of Artemisia verlotorum Lamotte (Asteraceae), against the feline immunodeficiency virus (FIV), a retrovirus (subfamily lentivirus) which resembles HIV1 in many similarities (Bendinelli et al., 1995) and represents a reliable animal model of AIDS infection (Gardner and Luciw, 1989).

MATERIALS AND METHODS Extract. Artemisia verlotorum was collected wild, in the country around Pisa, Italy, and was identified by Professor Paolo Tomei (Dipartimento di Scienze Botaniche, Universita` degli Studi di Pisa). The plant was collected in October 1996. A plant sample is deposited in the Herbarium Horti Pisani. An infusion was obtained by making a suspension of fresh leaves in boiling distilled * Correspondence to: V. Calderone, Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, Facolta` di Farmacia, Universita` degli Studi di Pisa, via Bonanno 6, 56126 Pisa, Italy.

CCC 0951–418X/98/080595–03 $17.50 # 1998 John Wiley & Sons, Ltd.

water (1 g/10 mL) for 10 min, and filtering the extract. The infusion was lyophilized in a freeze dryer. The lyophilized extract was dissolved in bi-distilled water, at a concentration required by the experimental protocol, on the day of the experiments. Cells. CrFK cells were obtained by the Centro Substrati Cellulari, Istituto Zooprofilattico, Brescia, Italy. The cells were routinely propagated in MESN medium (composition: Eagle’s minimum essential medium, essential and non-essential amino acids, biotin, glutamine, sodium pyruvate, vitamins, antibiotics, NaHCO3 2.25 g/L, NUserum IV 5%), as described (Tozzini and Bandecchi, 1985). Viruses. Feline immunodeficiency viruses of the prototype Californian Petaluma strain were used. This virus induces the formation of syncytia in CrFK cells. Titration of the viral stocks was performed as described by Tozzini et al. (1992). The viral titres were expressed as syncytium forming units (SFU)/mL. Experimental protocol. The CrFK cells (104 cells, suspended in 1.5 mL of medium) were added to the wells. Then, 0.4 mL of medium containing the FIV were added, to obtain a viral concentration of 100 SFU/mL. The extract, dissolved in a volume of 0.1 mL, was immediately administered (time 0). Different concentrations of extract (10ÿ6–1 mg/mL, with 3-fold increasing steps) were tested. In a second set of experiments, the different concentrations of plant extract were added 6 h after the infection (time 6 h). Furthermore, in a third set of experiments, a fixed concentration of extract (10ÿ3 mg/mL) was administered to the CrFK cells, immediately after the infection by different viral concentrations (100, 300 and 500 SFU). Received 15 December 1997 Revised 20 April 1998 Accepted 4 June 1998

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In all the above procedures, parallel control groups did not receive any concentration of extract. All the tests were performed in triplicate, and repeated at least four times. Virus-induced syncytia. After an appropriate incubation time (5 days), the medium was discarded and the cells were stained by crystal violet (0.5%) in methanol (30%). The virus-induced syncytia were observed (100 magnification) (Tozzini et al., 1992). Only the syncytia composed by at least seven nuclei were counted. The vitality of the extract-treated cells was also considered, as viable cells as a % of the control. The number of viable cells was determined by the conventional trypan blue dye exclusion method (Walder et al., 1995). Viral reverse transcriptase (RT) activity. The assay was based on the viral RT-induced 3H-thymidine inclusion in the cells (Ongradi et al., 1990), both in the absence and in the presence of three different concentrations of plant extract (3  10ÿ4, 10ÿ3 and 3  10ÿ3 mg/mL). The 3H-thymidine included was detected as cpm (count/min), by a b-counter. Viral capsid protein P24 expression. The expression of the viral capsid protein P24 was detected as anti-P24 antibodies produced by the infected cells, both in the absence and in the presence of three different concentrations of plant extracts (3  10ÿ4, 10ÿ3 and 3  10ÿ3 mg/mL). The assay was performed by an in-house capture ELISA immunoenzymatic method (Lombardi et al., 1994). Data analysis. The extract concentrations were expressed as mg of lyophilized extract/mL of solution. The values of virus-induced syncytia in the CrFK cells, in the presence of the plant extract, were expressed as a % of those induced in control conditions (syncytia %). The correlation between the calculated values of syncytia % and the respective concentrations of extract, was analysed by a non-linear regression (computer program Graph-Pad Prism1 2.0), which obtained the value of IC50, representing the extract concentration determining a half-reduction of the virus-induced syncytia in the cells. The IC50 values were expressed as pIC50 = ÿlog IC50. Both the RT activity and the P24 expression values, in

Figure 1. Viable cells (as % of the control), following the administration of increasing concentrations of the extract, expressed as log (mg/mL). The vertical bars indicate the SEM. # 1998 John Wiley & Sons, Ltd.

Figure 2. Virus-induced syncytia (as % of the control), in the presence of increasing concentrations of the extract, expressed as log (mg/mL), administered at time 0 (&) and at time 6 h (~). The vertical bars indicate the SEM.

the presence of the extract, were expressed as a % of the respective values recorded in control conditions. Statistical analysis. All data were expressed as mean  SEM. The significance of differences was evaluated by means of ANOVA and Student’s t-test for unpaired data. A value of p lower than 0.05 was considered as significant.

RESULTS Cytotoxicity As shown in Fig. 1 extract concentrations lower than 10ÿ1 mg/mL did not show any significant toxicity in the CrFK cells. Syncytia inhibition The extract induced a remarkable and significant reduction of the virus (100 SFU)-induced formation of syncytia. When added at time 0, the extract showed a

Figure 3. Virus-induced syncytia (as % of the control), in the presence of a ®xed concentration of the extract (10ÿ3 mg/mL) and of increasing viral concentrations (100, 300 and 500 SFU/ mL). The vertical bars indicate the SEM. Phytother. Res. 12, 595–597 (1998)

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induced by 100, 300 and 500 SFU were respectively 4.41%  2.26%, 13.70%  1.00% and 35.23%  1.36% of the control (Fig. 3). Viral RT activity The enzymatic activity was significantly reduced by the extract. The recorded values were 50.23%  12.13%, 33.27%  5.81% and 27.03%  6.50% of the control, respectively for the extract concentrations of 3  10ÿ4, 10ÿ3 and 3  10ÿ3 mg/mL (Fig. 4A). Viral capsid protein P24 expression The administration of the extract caused a marked and significant decrease of the expression of the capsid protein. The evaluated values were 6.79%  1.20%, 4.88%  1.34% and 1.14%  0.26% of the control, respectively for the extract concentrations of 3  10ÿ4, 10ÿ3 and 3  10ÿ3 mg/mL (Fig. 4B).

DISCUSSION

Figure 4. Reduction of the RT activity (A) and of the P24 production (B) (both expressed as % of the control), induced by increasing concentrations of the extract, expressed as log (mg/mL). The vertical bars indicate the SEM.

pIC50 value of 4.00  0.080, while this value was significantly lower (3.60  0.030) for the extract added at time 6 h. However, in both cases, the Artemisia verlotorum extract concentration of 10ÿ3 mg/mL almost fully abolished the viral activity (Fig. 2). The antiviral effect elicited by the extract (10ÿ3 mg/ mL) was significantly reduced, but not abolished, by increased viral concentrations: the values of syncytia

To our knowledge, this study represents the first demonstration of an inhibitory activity of Artemisia verlotorum against the cytopathogenicity induced by the feline immunodeficiency virus. Such a concentrationdependent activity avoids the virus-induced syncytia formation and is yet present, albeit 3-fold reduced in potency, when the administration of the extract is delayed with respect to the infection. Following the plant extract administration, the two important viral parameters of RT activity and capsid protein P24 expression underwent a remarkable decrease. It is concluded that Artemisia verlotorum could contain one or more water soluble and almost non-cytotoxic compounds, showing a strong antiviral activity. Further studies are in progress with the aim of understanding the exact mechanism of action of the plant and of isolating and identifying the active principle(s).

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Phytother. Res. 12, 595–597 (1998)