Antiviral properties of a crude extract from a green alga Caulerpa taxifolia (Vahl) C. Agardh

PHYTOTHERAPY RESEARCH Phytother. Res. 13, 245–247 (1999)

SHORT COMMUNICATION

Antiviral Properties of a Crude Extract from a Green Alga Caulerpa taxifolia (Vahl) C. Agardh E. Nicoletti,1 F. Della Pieta’,2* V. Calderone,3 P. Bandecchi,4 M. Pistello,1 I. Morelli5 and F. Cinelli2 1

Dipartimento di Biomedicina, Centro Retrovirus sezione di Virologia, Universita` degli Studi di Pisa, via S. Zeno 35, 56127 Pisa, Italy Dipartimento di Scienze dell’Uomo e dell’Ambiente, Universita` degli Studi di Pisa, via A. Volta 6, 56126 Pisa, Italy Istituto Policattedra di Discipline Biologiche, Universita` degli Studi di Pisa, via Bonanno 6, 56126 Pisa, Italy 4 Dipartimento di Patologia Animale, Universita` degli Studi di Pisa, via delle Piagge 2, 56124 Pisa, Italy 5 Dipartimento di Chimica Bioorganica, Universita` degli Studi di Pisa, via Bonanno 33, 56126 Pisa, Italy 2 3

A crude extract of Caulerpa taxifolia was tested for its antiviral activity. The chloroform–methanol residue showed an interesting inhibitor effect in vitro toward the feline immunodeficiency virus (FIV), a valid model for studying the acquired immunodeficiency syndrome. This extract reduced the virus-induced syncytia in the cultured cells, the viral reverse transcriptase activity and the viral capsid protein P24 expression. Copyright # 1999 John Wiley & Sons, Ltd. Keywords: Caulerpa taxifolia; green algae; crude extract; antiviral activity; FIV.

INTRODUCTION C. taxifolia (Vahl) C. Agardh (Caulerpales), a tropical green alga recently introduced into the Mediterranean, is spreading along the French, Italian and Spanish coasts of the Western Mediterranean. This alga is very different from tropical C. taxifolia because of its vigorous development, gigantism and resistance to winter temperatures (Boudouresque et al., 1992; Meinesz et al., 1993). C. taxifolia contains secondary metabolites, terpenoids, that demonstrated toxicity against microorganisms (Paul and Fenical, 1986; Guerriero et al., 1993), mammalian cells, cell proliferation in sea urchin eggs (Leme´e et al., 1993), and the single neuron of the leech Hirudo medicinalis (Scuri et al., 1996). The treatment of acquired immunodeficiency syndrome (AIDS) is a current problem, so it is very important to identify some drugs that treat or prevent this disease. The feline immunodeficiency virus (FIV) is a valid model for studying human immunodeficiency virus type 1 (HIV-1) (Bendinelli et al., 1995; Gardner and Luciw, 1989). This virus induces syncytia in the feline kidney Crandell cells cultured in vitro. We report the results of a study on the possible antiviral activity of an extract from the green alga C. taxifolia.

MATERIALS AND METHODS Extract. C. taxifolia was collected in July 1996 near Imperia Italy and identified in the Dipartimento di Scienze dell’Uomo e dell’Ambiente, Pisa, Italy. An algal * Correspondence to: F. Della Pieta’, Dipartimento di Scienze dell’ Uomo e dell’ Ambiente, Universita´ degli Studi di Pisa, via A Volta 6, 56126 Pisa, Italy.

CCC 0951–418X/99/030245–03 $17.50 Copyright # 1999 John Wiley & Sons, Ltd.

sample is deposited in this Department. The fresh material was washed with deionized water, freeze-dried, powdered and extracted exhaustively with a Soxhlet apparatus with solvents of increasing polarity: hexane, CHCl3, CHCl3 –MeOH (9:1). The CHCl3 –MeOH (9:1) extract was dissolved in DMSO (dimethyl sulphoxide, 1%). Cells. Crandell feline kidney cells (CrFK), furnished by the Centro Substrati Cellulari, Istituto Zooprofilattico, Brescia, Italy, were propagated in MESN medium (Eagle’s minimum essential medium, essential and nonessential amino acids, biotin, glutamine, sodium pyruvate, vitamins, antibiotics, NaHCO3 2.225 g/L, NUserum IV-5%), as described by Tozzini and Bandecchi. (1985). Viruses. Feline immunodeficiency viruses of the prototype Californian Petaluma strain were used. This virus induces formation of syncytia in the CrFK cells. The 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 (10ÿ4 cells, suspended in 1.5 mL of medium) were cultivated in the wells. Then 0.4 mL of medium containing the FIV were added, to obtain a viral concentration of 100 SFU /mL. The extract was dissolved in a volume of 0.1 mL and immediately administered (time 0). Different concentrations of extract (from 3  10ÿ5 mg/mL, with 3-fold increasing steps) were tested. In a consequent set of experiments, the different concentrations of plant extract were added 6 h (time 6 h), 12 h (time 12 h) or 24 h (time 24 h) after the viral infection. Furthermore, in a third set of experiments, a fixed concentration of extract (10ÿ3 mg/mL) was administered Accepted 13 August 1998

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to the CrFK cells, immediately after infection by different viral concentrations (100, 300 and 500 SFU). In all procedures, parallel control groups received the vehicle DMSO (1%). All the tests were performed in triplicate, and repeated at least four times. Evaluation of the virus-induced syncytia. After an incubation time of 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 of at least seven nuclei were counted. The vitality of the extract-treated cells was also considered. Viral reverse transcriptase (RT) activity. The assay was based on the viral RT-induced 3H-thymidine inclusion in the cells, as described (Ongradi et al., 1990), both in the absence and in the presence of the plant extract. 3H-thymidine was detected as cpm, by a bcounter. Viral capsid 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 the plant extract. The assay was performed by an in-house capture ELISA immunoenzymatic method, as described by Lombardi et al. (1994). Data analysis. 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 the control condition (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 Prism 2,0), which calculated 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 = ÿlogIC50. Both the RT activity and the P24 expression values, in the presence of extracts 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.

Figure 1. Virus-induced syncytia (as % of the control, left yaxis), in the presence of increasing concentrations of the extract, expressed as log (mg/mL), administered at time 0 (~), at time 6 h (^), at time 12 h (!) and at time 24 h (*). The value of viable cells (&), expressed as % of the control (right y-axis) following the administration of increasing concentrations of the extract, is also shown. The vertical bars indicate the SEM.

When added at time 0, the extract showed a pIC50 value of 3.62  0.058, while this value was significantly lower for the extract added at time 6 h, at time 12 h and at time 24 h (respectively 3.23  0.016, 3.16  0.026 and 3.08  0.052). However, in all the cases, the Caulerpa taxifolia extract concentration of 10ÿ3 mg/mL almost fully abolished the viral activity (Fig. 1). The antiviral effect elicited by the extract (10ÿ3 mg/ mL) was significantly reduced, but not abolished by increased viral concentration:the values of syncytia induced by 100, 300 and 500 SFU were respectively 1.52%  0.59%, 12.51%  0.87% and 22.40%  2.46% of the control (Fig. 2). RT activity The enzymatic activity was significantly reduced by the extract. The recorded values were 61.87%  3.47%, 36.07%  2.31% and 22.37%  2.68% of the control, respectively for the extract concentrations 3  10ÿ4, 10ÿ3 and 3  10ÿ3 mg/mL (Fig. 3).

RESULTS Cytotoxicity As shown in Fig. 1, extract concentrations lower than 3  10ÿ3 mg/mL did not have any significant toxicity. Syncytia inhibition The extract induced a marked and significant reduction of the virus (100 SFU)-induced formation of syncytia. Copyright # 1999 John Wiley & Sons, Ltd.

Figure 2. 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. 13, 245–247 (1999)

ANTIVIRAL PROPERTIES OF CAULERPA TAXIFOLIA

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Figure 3. Reduction of the RT activity (as % of the control), induced by increasing concentrations of the extract, expressed as log (mg/mL). The vertical bars indicate the SEM.

Figure 4. Reduction of the P24 expression (as % of the control), induced by increasing concentrations of the extract, expressed as log (mg/mL). The vertical bars indicate the SEM.

P24 expression

present work reports the first results of an inhibitory activity of C. taxifolia towards the cytopathogenicity induced by the feline immunodeficiency virus. What is noteworthy about antiviral activity is that it is already at work, even before the administration of the algal extract has taken place. After the extract administration, the RT activity and capsid protein P24, two important viral parameters, underwent a remarkable decrease. We conclude that the C. taxifolia chloroform–methanol (9:1) extract contains one or more compounds which are not completely cytotoxic and show a strong antiviral activity. Studies are in progress in order to understand the exact mechanism of action of the extract. Furthermore, chemical investigations are being carried out to isolate and identify the active principles.

The administration of the extract caused a marked and significant decrease of the capsid protein P24. The evaluated values were 8.47%  2.05%, 5.20%  0.47%, 1.52%  0.21% of the control, respectively for the extract concentration of 3  10ÿ4, 10ÿ3 and 3  10ÿ3 mg/mL (Fig. 4).

DISCUSSION Many studies were carried out on secondary metabolites from Caulerpa taxifolia and their biological activity. The

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Phytother. Res. 13, 245–247 (1999)