Plant Extracts Antiviral Activity against Herpes simplex

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PLANT EXTRACTS ANTIVIRAL ACTIVITY AGAINST HERPES SIMPLEX VIRUS TYPE 1 AND AFRICAN SWINE FEVER VIRUS

ABSTRACT

Twenty-eight extracts prepared from plants used in African traditional medicine and from Rhamnus glandulosa Ait. of the Portuguese flora, were screened in order to assay their antiviral activity against Herpes simplex virus type 1 (HSV-1) and African swine fever virus (ASFV). Twelve of these extracts revealed virucidal activity against HSV-1 whereas only six have the same activity against ASFV. Further studies showed that thirteen of the tested extracts inhibited HSV-1 infection, some of which had a significant activity against this virus such as Senna podocarpa (Guill. & Pert.) Lock, Cassia sieberiana DC., Guiera senegalensis J.F. Gmel., Piliostigma thonningii (Schum.) Milne-Redhead, Rhamnus glandulosa Ait. and Uvaria chamae P. Beauv. Four of the twenty-one tested extracts inhibited ASFV infection.

INTRODUCTION

Twenty plants, belonging to fifteen plant families, used in folk medicine by Guinea-Bissau traditional healers to treat several infectious diseases, were screened for in vitro antiviral activity against a clinical strain of Herpes simplex virus type I (HSV-1) and a strain of African swine fever virus (ASFV). After determination of the maximum tolerated dose (MTD) against Vero cells, ethanol extracts were evaluated for overall virucidal activity and for the ability to inhibit specific steps of viral infection (viral replication and viral adsorption). This is the first report on the antiviral activity against HSV-1 and ASFV from these plants. These experiments are of importance because African swine fever is an endemic disease in Portugal and Spain and no vaccine nor effective therapy is available (Costa, 1990).

MATERIALS AND METHODS

Plant Materials

Plants were collected in Guinea-Bissau and identified by Dr. A. Diniz; voucher herbarium specimens have been preserved in LISC Herbarium of the "Centro de Botânica Tropical", Lisbon, Portugal; Rhamnus glandulosa leaves were collected by Mr. N¢brega from a plant specimen of "Jardim Botânico do Funchal", Madeira Island, Portugal.

Extracts Preparation

Different parts of the dried plants (Table 1) were extracted with ethanol at room temperature and concentrated under reduced pressure (temperature < 40øC). The dried extracts were dissolved in dimethylsulfoxide (DMSO) at a concentration of 100 mg/ml.

Cells Used

Vero cells were obtained from the American Type Culture Collection and were grown as monolayers in Dulbecco's Modified Eagle Medium (DME) (Gibco, Scotland) supplemented with 10% of New-born Calf Serum (NCS) (Gibco, Scotland) and gentamicin (50 æg/ml).

Viruses

African swine fever virus (ASFV), strain Lisbon 60, was originally obtained in 1960 from an infected pig in Lisbon (Ribeiro and Azevedo, 1961), adapted to grow in monkey cells and was cloned by four successive plaque purification in Vero cells, as described by Enjuanes et al. (1976). Herpes simplex virus type 1 (HSV-1, clinical strain) stocks were prepared in Vero cells in DME supplemented with 2% NCS. Only the extracellular viruses were used, after low speed centrifugation of cell debris. When necessary, vires suspensions were concentrated by centrifugation at 11000 G for 6 h at 4øC. Plaque titrations were done as described by Enjuanes et al. (1976).

Antiviral Assays

Cytotoxicity assays -- As a first step, the cytotoxicity of the extracts was evaluated in order to determine their maximum tolerated concentration (MTC) to Vero cells. Vero Cell monolayers in 96-well plates (Nunc, England) were incubated with decreasing concentrations of the extracts (dissolved in DMSO) for 48 h at 37øC. After 48 h, a dye uptake assay (Finter, 1969) was performed to determine the percentage of living cells in relation to the control (cells incubated with DMSO).

Virucidal activity of plant extracts -- The direct effect of plant extracts on HSV-1 and ASFV was determined by incubating both viruses for 1 h at 37øC with the extracts at the MTC. The extracts were removed by centrifugation (53,000 G or 100,000 G) and virucidal activity was determined by plaque titration. Controls were performed by incubating the viruses with Phosphate Buffered Saline-A (PBS-A) solution (Dulbecco and Vogt, 1954).

Effect of plant extracts on viral replication -- Vero cell monolayers were incubated for 30 min at 37øC with the extracts at MTC. The extracts were removed and the cells washed with PBS-A or DME2 (DME supplemented with 2% NCS) at the same temperature. Cell monolayers were infected with HSV-1 or ASFV at a multiplicity of infection (m.o.i.) of 1 in the presence of the extracts. After adsorption of the viruses for 2 h at 37øC, the remaining viruses were removed and cells were again washed with PBS-A. The plates were incubated at 37øC until an extensive cytopathic effect was observed in control (without extract) wells. The media from all infected wells were recovered and titrated for virus.

Effect of plant extracts on viral adsorption -- Vero cell monolayers were pre-cooled at 4øC for 15 min after which they were infected with HSV-1 in presence of the extracts. After viral adsorption for 2 h at 4øC, the remaining virus was removed and cells washed with PBS-A. They were then incubated for a further 24 h at 37øC with DME2 and later the progeny virus was plaque titrated.

All the experiments were effectuated in duplicate.

RESULTS

Different pans of twenty medicinal plants, belonging to fifteen plant families (Table 1) were tested for their antiviral activity. We have considered the antiviral activity to be significant if the difference between control viral yield (without the extract) and the experimental viral yield (with the extract) would be higher than a 0.7 log(10) which corresponds to 80% of inhibition of viral yield.

The results of 28 plant extracts tested against HSV-1 and ASFV are given in Tables 2 and 3, respectively. As demonstrated in Table 2, thirteen of the plant extracts inhibited HSV-1 (yield in the host cells > 80%) and twelve of the tested extracts had virucidal activity (> 80%) against HSV-1. Only Cassia sieberiana and Guiera senegalensis (aerial parts) specifically inhibited HSV-1 adsorption (Table 2) (the others showed virucidal activity).

Four extracts (Adansonia digitata fruit; Cochlospermum angolense root; Lippia chevalieri root and Piliostigma thonningi stem bark) inhibited ASFV yield in the host cells (> 80%) (Table 3) and only six extracts (Cassia sieberiana aerial part; Gardenia ternifolia root; Pavetta oblongifolia stem and root; Rhamnus glandulosa leaf and Sarcocephalus latifolius root) have virucidal activity (> 80%) against this virus.

Extracts from five plants (Cassia sieberiana, Guiera senegalensis, Pavetta oblongifolia, Rhamnus glandulosa and Sarcocephalus latifolius) had virucidal effect on both viruses, and four plant extracts (Cochlospermum angolense, Lippia chevalieri, Pavetta oblongifolia and Piliostigma thonningii) inhibited the replication of both viruses.

DISCUSSION

The need for new disinfectants and antiseptics makes importance of studying the plants with virucidal activity. A great number of the tested extracts have shown virucidal activity and one of tested plants (Guiera senegalensis), with significant virucidal effect against HSV-1 (100% inhibition), is now under investigation to determinate the active component(s).

The rhizome extract of Cochlospermum tinctorium and the root extract of Cryptolepis sanguinolenta are used in Guinea-Bissau to treat and prevent liver disease (liver damage by hepatitis and jaundice) but neither of the two plant extracts had any virucidal activity nor affected viral adsorption of HSV-1. However, they inhibited the viral replication which suggests that these plant extracts might be inhibiting an event post-adsorption during the infective cycle of HSV-1.

Cochlospermum tinctorium has been the subject of several recent studies about phytochemical compounds of its rhizome and biological activities [anti-hepatotoxic (Diallo et al., 1987); inhibitor of skin-tumour viral promotion (Diallo et al., 1989)], but it has not been studied for the antiviral activities against HSV-1 or ASFV. Concerning Cryptolepis sanguinolenta, in a recent publication Cimanga et al. (1991) did not find any antiviral properties for cryptolepine, the main alkaloid of this species.

Concerning the inhibitory effect on ASFV replication (four extracts with 80% viral replication inhibition effect) as well as the virucidal activity on this virus (only one extract with 100% virucidal inhibition effect), our results suggest that so far these effects are less specific than those observed with HSV-1 (six extracts with 100% viral replication inhibition effect and six with 100% virucidal inhibition effect). Work is in progress in order to screen these plant extracts on other viruses.

ACKNOWLEDGEMENTS

The authors express their thanks to JNICT-Portugal for the financial support through the PMCT/C/SAU/749/90 project; Dr. Irene Neves for supply of Herpes simplex virus type 1 clinical strain and Dr. J.D. Vigário for the supply of African swine fever virus strain Lisbon 60.

Keywords: Antiviral activity, African swine fever virus (ASFV), Herpes simplex virus type 1 (HSV-1), medical plants.

Accepted: October 19, 1996

REFERENCES

Cimanga K., Pieters L., Clayes M., Vanden Berghe D. and Vlietnick A.J. (1991) Biological activities of cryptolepine, an alkaloid from Cryptolepis sanguinolenta. Planta Med. 57: suppl. 2, A98-A99.

Costa J.V. (1990) African Swine Fever Virus. In: Molecular Biology of Iridoviruses (Eds. G. Darari). Kluwer Academic Publishers, Boston.

Diallo B., Vanhaelen M., Kiso Y. and Hikino H. (1987) Antihepatotoxic actions of Cochlospermum tinctorium rhizomes. J. Ethnopharmacol. 20: 239-243.

Diallo B., Vanhaelen-Fastre R., Konoshima I., Kosuka M. and Okuda H. (1989) Studies on inhibitors of skin-tumor promotion. Inhibitory effects of triterpenes from Cochlospermum tinctorium on Epstein-Barr virus activation. J. Nat. Prod. 52: 879-881.

Dulbecco R. and Vogt M. (1954) Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Experi. Med. 99: 167-182.

Enjuanes L., Carrascosa A.L., and Viuela E. (1976) Isolation and properties of the DNA of African swine fever virus. J. Gen. Virol. 32: 479-492.

Finter N.B. (1969) Dye uptake methods for assessing cytopathogenicity and their application to interferon assay. J. Gen. Virol. 5: 419-427.

Ribeiro J.M. and Azevedo J.A.R. (1961) La peste porcine africaine au Portugal. Bull. Off Int. Épizooties. 55: 88-106.

Swets & Zeitlinger.

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By O. Silva; S. Barbosa; A. Diniz; M.L. Valdeira and E. Gomes

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