ARTICLE IN PRESS Microbiological Research 163 (2008) 136—139 0944-5013/$ - s doi:10.1016/j. �Correspond fax: +55 43337 E-mail addr www.elsevier.de/micres The in vitro antiviral activity of an aliphatic nitro compound from Heteropteris aphrodisiaca Fernando L. Meloa, Fabricio J. Benatia, Walter Antonio Roman Juniorb, João Carlos Palazzo de Melloc, Carlos Nozawaa, Rosa Elisa Carvalho Linharesa,� aDepartamento de Microbiologia/CCB, Universidade Estadual de Londrina, Caixa Postal 6001, CEP 86051-990 Londrina-Paraná, Brazil bPPG em Ciências Farmacêuticas, Universidade Estadual Paulista Jùlio de Mesquita Filho, Araraquara-SP, Brazil cDepartamento de Farmácia e Farmacologia, Universidade Estadual de Maringá, Maringá-PR, Brazil Accepted 16 March 2006 KEYWORDS Heteropteris aphrodisiaca; Aliphatic nitro compound; Poliovirus; Antiviral activity; Bovine herpesvirus ee front matter & 2006 micres.2006.03.011 ing author. Tel.: +55 433 14465. ess: relin@uel.br (R.E.C Summary We investigated the antiviral activity of an aliphatic nitro compound (NC) isolated from Heteropteris aphrodisiaca O. Mach. (Malpighiaceae), a Brazilian medicinal plant. The NC was tested for its antiviral activity against poliovirus type 1 (PV-1) and bovine herpes virus type 1 (BHV-1) by plaque reduction assay in cell culture. The NC showed a moderate antiviral activity against PV-1 and BHV-1 in HEp-2 cells, and the 50% inhibitory concentration (IC50) were 22.01 mg/ml (selectivity index (SI) ¼ 2.83) and 21.10 mg/ml (SI ¼ 2.95), respectively. At the highest concentration of the drug (40 mg/ml) a reduction of approximately 80% in plaque assay was observed for both viruses. The treatment of cells or virus prior to infection did not inhibit the replication of virus strains. & 2006 Elsevier GmbH. All rights reserved. Introduction Viral diseases remain as an important worldwide problem, 50 yr after the beginning of the works with antiviral agents, due in part to the toxicity of Elsevier GmbH. All rights rese 3714617; . Linhares). many drugs developed (Ernst and Franey, 1998; De Clercq, 2001) and rapid emergence of drug resis- tant strains (Gilbert et al., 2002; Field, 2001; Zoulim, 2001). In order to find new alternatives and efficient antiviral compounds, many traditional medicinal plants have been screened (Vlietinck and Vanden Berghe, 1991; Jassim and Naji, 2003), including Brazilian medicinal plants (Simões et al., 1999; Schmitt et al., 2001; Esquenazi et al., 2002). rved. www.elsevier.de/micres dx.doi.org/10.1016/j.micres.2006.03.011 mailto:relin@uel.br ARTICLE IN PRESS R4O R3O R2O OR6 OR1 O R1=H R2=R3=R4=R6=3NPA O -CCH2CH2NO2 =3NPA || Figure 1. Chemical structure of 2,3,4,6-tetra-O-(3-ni- tropropanoyl)-O-a _ -D-glucopyranoside. In vitro antiviral activity of an aliphatic nitro compound 137 A great diversity of compounds isolated from plants show antiviral activity including flavonoids, tan- nins, proteins, polysaccharides, alkaloids and others (Jassim and Naji, 2003). Bovine herpesvirus type 1 (BHV-1), a DNA virus member of the family Herpesviridae, is responsible for infections in bovines, such as those involved with the upper respiratory (rhinotracheitis) and genital tracts (vulvovaginitis and balanopostitis) and is resistant to acyclovir (Hinkley et al., 1998; Babiuk et al., 1983). Poliovirus is an RNA virus, member of the family Picornaviridae and is an appropriate model for study of viral replication. The virus is the etiological agent of poliomyelitis, disease under control in most of the countries; however, it has been faced as a serious threat worldwide. Besides, the virus belongs to the genus Enterovirus, in which members are found causing a wide array of illnesses, such as, meningitis, myocarditis, ence- phalitis and respiratory diseases (Mueller et al., 2005). Heteropteris aphrodisiaca O. Mach. (Malpighia- ceae) is a plant, endemic to the Brazilian scrubland regions, traditionally used in folk medicine as an aphrodisiac, stimulant and in the treatment of nervous weaknesses (Pott and Pott, 1994). Mattei et al. (2001) demonstrated that its extract, BST0298, reduced the oxidative stress in young and old rat brains. Galvão et al. (2002) found out that the treatment with the same extract improved learning and memory in aged rats. In this work we reported the antiviral activity of a nitro compound (NC) from H. aphrodisiaca against poliovirus and bovine herpesvirus in cell culture. To our knowl- edge this biological activity was never studied for this NC. Materials and methods Plant material Roots of H. aphrodisiaca O. Mach. (Malpighia- ceae) were collected in October 2000, in Santo Antônio do Leverger, Mato Grosso State, Brazil, and identified by Prof. Miramy Macedo (Universidade Federal de Mato Grosso, UFMT). A voucher speci- men (UFMT-22181) was deposited at the UFMT Central Herbarium in Cuiabá, Mato Grosso, Brazil. Extraction and isolation The roots (500 g) were macerated with acetone for 1 week at room temperature of approximately 25 1C. The extract was filtered and the solvent evaporated under vacuum resulting in the crude extract (13 g). One portion of the extract (10 g) was chromatographed on silica gel by vacuum liquid chromatography using toluene and CHCl3/MeOH (97:3; 19:1; 9:1; 17:3; 1:1) to yield six fractions. Fractions (200ml) were collected and checked by thin-layer chromatography [silica gel 60 F254 plates, using solvent systems: n-BuOH:CHCl3:MeOH (55:40:5)]. Fraction 4 (1.0 g) was crystallized with methanol:water to yield pure compound 2,3,4,6- tetra-O-(3-nitropropanoyl)-O-a _ -D-glucopyranoside (200mg) (Roman Jùnior et al., 2005) (Fig. 1). Cells and virus HEp-2 cells (human larynx carcinoma, ATCC, CCL- 23) were cultured at 37 1C with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2mM glutamine, Penicillin (100 IU/ml), Streptomycin (100 mg/ml) and Ampho- tericin B (0.25 mg/ml). The clinical isolate strain of bovine herpes virus type 1 (BHV-1) was supplied by Prof. A. Alfieri, DMVP-UEL. The poliovirus type 1 (PV-1) (ATCC, VR- 58) was provided by Departamento de Virologia, IMPPG-UFRJ. Both virus strains were propagated in HEp-2 cells and stored at �80 1C. The virus titers were determined by plaque assay. Cytotoxicity test The NC cytotoxicity activity was evaluated by the alteration cell morphology and by trypan blue exclusion staining. Briefly, HEp-2 cells grown in 96-well culture plates were submitted to treatment with the compound at different concentrations and observed daily for cytotoxic alterations. Alterna- tively, cell cultures submitted to 48 h treatment with NC were dispersed with 0.2% trypsin and after staining with trypan blue the percentage of viable cells was calculated as follows: cell viability (%) ¼ (viable cell number/total cell number)� 100. ARTICLE IN PRESS F.L. Melo et al.138 Plaque reduction assay Confluent cell monolayers grown in 24-well plates were inoculated with 50–100pfu/ml of virus strains and after 1 h adsorption at 37 1C cells were washed with phosphate-buffered saline, pH 7.3, and over- laid with DMEM with 0.75% agarose containing two- fold dilutions of the NC, and for poliovirus 25mM MgCl2 were added (Wallis et al., 1966). After 48h, the monolayers were fixed with 10% formaldehyde and nutrient agarose layer removed. Cell mono- layers were stained with 0.5% crystal violet in 20% ethanol, and plaques counted. The antiviral activity was determined as the percent of plaque inhibition in comparison to controls. Assay of NC either on virus strains or on the cells before infection The stock of virus strains (106 pfu/ml) was diluted with equal volumes of DMEM containing 10, 20, 30 or 40 mg/ml of the NC, and incubated for 1 h at 37 1C. Virus strains without treatment were used for control. Virus titers were determined, as Table 1. The 50% cytotoxic concentration (CC50) and the 50% inhibitory concentration (IC50) of a nitro compound from Heteropteris aphrodisiaca against bovine herpesvirus type 1 (BHV-1) and poliovirus type 1 (PV-1) in HEp-2 cells by plaque reduction assay Virus CC50 (mg/ml)a IC50 (mg/ml)b SI (CC50/IC50) c PV-1 62.29 22.01 2.83 BHV-1 62.29 21.10 2.95 a50% Cytotoxic concentration. b50% Inhibitory concentration. cSelectivity index. ** ** ** ** 0 0 20 40 60 80 100 10 20 30 40 Concentration (ug/ml) In hi bi tio n (% ) (A) Figure 2. Dose–response curve of the inhibition of poliovir aliphatic nitro compound from Heteropteris aphrodisiaca, in (n ¼ 4). **P ¼ 0:01, no treatment vs. different concentration before. Confluent cell monolayers were incubated during 1 h at 37 1C with DMEM containing the same concentrations of the NC. Cells were washed with PBS and followed by infection as described before for plaque reduction assay. Statistical analysis All experiments were performed in quadrupli- cate, and data were analyzed by ANOVA, followed by Dunnett’s test. P ¼ 0:05 values were considered significant. The 50% cytotoxic concentration (CC50) and the 50% inhibitory concentration (IC50) were calculated by regression analysis of the dose–re- sponse curves. Results and discussion The antiviral activity of plant extracts against poliovirus and members of the Herpesvirida family has been demonstrated by several authors. Medic- inal plants from Brazilian Atlantic tropical forest showed antiviral activity against herpes simplex type 1 (HSV-1) and poliovirus type 2 (PV-2) (Andrighetti-Fröhner et al., 2005). The antiviral activity of an aqueous extract of Phyllanthus orbicularis against BHV-1 and HSV-2 was found to be dependent on the extract concentration in tissue culture (Barrio and Parra, 2000). NCs, such as hiptagin, were found in several plants of the genus Heteropteris, but no biological activity had been attributed to them (Stermitz et al., 1975). However, a synthetic NC, 5-nitropyrimi- dine derivative, was demonstrated to possess activity against HSV-1 suggesting its effect in the stage of virus attachment or fusion (Schmidtke et al., 2002). 0 10 20 30 40 Concentration (ug/ml) ** ** ** 0 20 40 60 80 100 In hi bi tio n (% ) (B) us type 1 (A) and bovine herpesvirus type 1 (B) by the HEp-2 cell cultures. Data are expressed as mean7S.D. s of the nitro compound. ARTICLE IN PRESS In vitro antiviral activity of an aliphatic nitro compound 139 We found no cytotoxicity of the NC at concentra- tions lower than 50 mg/ml. However, at 50 mg/ml cells demonstrated discrete cytopathic changes revealed by abnormal granules and vacuoles. Nevertheless, trypan blue staining resulted in CC50 of 62.29 mg/ml (Table 1). The effect for both virus strains was evaluated at the concentrations of the NC below 40mg/ml. Table 1 shows the IC50 and selectivity index (SI) defined by the ratio CC50/IC50. The NC showed a moderate antiviral activity with selectivity indexes of 2.83 and 2.95 for PV-1 and BHV-1, respectively. The dose–r- esponse curves are shown in Fig. 2. We demonstrated a direct correlation of compound concentrations and viral inhibition, and at the highest drug concentration (40mg/ml) a reduction of approximately 80% in the number of plaques was observed for both viruses. No effect was observed when both viruses and cells were pre-treated (data not shown), suggesting that the NC neither inactivated virus particles directly (both strains) nor protected the cell from infection, but interfered with the virus replication after the penetration step. Nevertheless, further experiments are required to elucidate the mechanism of action for this compound. Acknowledgments FLM was recipient of scholarship granted by CNPq, and this work is part of his M.Sc. manuscript. The authors wish to thank to CNPq, CAPES, Fundac-ão Araucária and PROPPG/UEL for financial support. References Andrighetti-Fröhner, C.R., Sinceroa, T.C.M., da Silva, A.C., Savia, L.A., Gaidoa, C.M., Bettega, J.M.R., Almeida, M.T.R., Mancini, M., Barbosa, R.A., Farias, M.R., Barardi, C.R.M., Simões, C.M.O., 2005. Antiviral evaluation of plants from Brazilian Atlantic tropical forest. Fitoterapia 76, 374–378. Babiuk, L.A., Acres, S.D., Misra, V., Stockdale, P.H.G., De Clercq, E., 1983. Susceptibility of bovid herpesvirus 1 to antiviral drugs: in vitro versus in vivo efficacy of (E)-5-(2-bromovinyl)-20-deoxyuridine. Antimicrob. Agent. Chemother. 23, 715–720. Barrio, G., Parra, F., 2000. Evaluation of the antiviral activity of an aqueous extract from Phyllanthus orbicularis. J. Ethnopharmacol. 72, 317–322. De Clercq, E., 2001. Antiviral drugs: current state of the art. J. Clin. Virol. 22, 73–89. Ernst, M.E., Franey, R.J., 1998. Acyclovir- and ganciclo- vir-induced neurotoxicity. Ann. Pharmacother. 32, 111–113. Esquenazi, D., Wigg, M.D., Miranda, M.M.F.S., Rodrigues, H.M., Tostes, J.B.F., Rozental, S., Silva, A.J.R., Alviano, C.S., 2002. Antimicrobial and antiviral activities of polyphenolics from Cocos nucifera Linn. (Palmae) husk fiber extract. Res. Microbiol. 153, 647–652. Field, H.J., 2001. Herpes simplex virus antiviral drug resistance – current trends and future prospects. J. Clin. Virol. 21, 261–269. Galvão, S.M.P., Marques, L.C., Oliveira, M.G.M., Carlini, E.A., 2002. Heteropterys aphrodisiaca (extract BST0298): a Brazilian plant that improves memory in aged rats. J. Ethnopharmacol. 79, 305–311. Gilbert, C., Bestman-Smith, J., Boivin, G., 2002. Resis- tance of herpesviruses to antiviral drugs: clinical impacts and molecular mechanisms. Drug Resist. Update 5, 88–114. Hinkley, S., Hill, A.B., Srikumaran, S., 1998. Bovine herpesvirus-1 infection affects the peptide transport activity in bovine cells. Virus Res. 53, 91–96. Jassim, S.A.A., Naji, M.A., 2003. Novel antiviral agents from plants. J. Appl. Microbiol. 95, 412–427. Mattei, R., Barros, M.P., Galvão, S.M.P., Bechara, E.J.H., Carlini, E.a., 2001. Heteropteris aphrodisiaca O. Macha- do: effects of extract BST 0298 on the oxidative stress of young and old rat brains. Phytother. Res. 15, 604–607. Mueller, S., Wimmer, E., Cello, J., 2005. Poliovirus and poliomyelitis: a tale of guts, brains, and an accidental event. Virus Res. 111, 175–193. Pott, A., Pott, V.J., 1994. Plantas do Pantanal. EMBRA- PA–SPI, Corumbá, Brasil, p. 101. Roman Jùnior, W.A., Vilegas, W., Nakamura, C.V., Dias Filho, B.P., Mello, J.C.P., 2005. A new antimicrobian, 2,3,4,6-tetra-O-(3-nitropropanoyl)-O-b-D-glucopyra- noside, from the roots of Heteropteris aphrodisiaca, Acta Farm. Bonaerense 24, 543–545. Schmidtke, M., Riabova, O., Dahse, H.M., Stelzner, A., Makarov, V., 2002. Synthesis, cytotoxicity and antiviral activity of N,N_-bis-5-nitropyrimidyl derivatives of dispirotripiperazine. Antiviral Res. 55, 117–127. Schmitt, A.C., Ravazzolo, A.P., Von Poser, G.L., 2001. Investigation of some Hipericum species native to southern of Brazil for antiviral activity. J. Ethnophar- macol. 77, 239–245. Simões, C.M.O., Falkenberg, M., Mentz, L.A., Schenkel, E.P., Amoros, M., 1999. Antiviral activity of South Brazilian medicinal plant extract. Phytomedicine 6, 205–214. Stermitz, F.R., Hnatyszyn, O., Bandoni, A.L., Rondina, R.V.D., Coussio, J.D., 1975. Screening of Argentine plants for aliphatic nitro compounds: hiptagin from Heteropteris angustifolia. Phytochemistry 14, 1341–1345. Vlietinck, A.J., Vanden Berghe, D.A., 1991. Can ethno- pharmacology contribute to the development of antiviral drugs? J. Ethnopharmacol. 32, 141–153. Wallis, C., Morales, F., Powell, J., Melnick, J.L., 1966. Plaque enhancement of enteroviruses by magnesium chloride, cysteine and pancreatin. J. Bacteriol. 91, 1932–1935. Zoulim, F., 2001. Detection of hepatitis B virus resistance to antivirals. J. Clin. Virol. 21, 243–253. The in vitro antiviral activity of an aliphatic nitro compound from Heteropteris aphrodisiaca Introduction Materials and methods Plant material Extraction and isolation Cells and virus Cytotoxicity test Plaque reduction assay Assay of NC either on virus strains or on the cells before infection Statistical analysis Results and discussion Acknowledgments References