Antiviral activity of fractions from leaves of Piper regnelli var. pallescens

Antiviral activity of fractions from leaves of Piper regnelli var. pallescens

Jéssica W. Bertol^II; Patricia R. Santos^I; Juliana Rodrigues^I; Diógenes A. G. Cortez^I; Benedito P. D. Filho^I,II; Celso V. Nakamura^I,II; Tania Ueda-Nakamura^I,II,*

^IPrograma de Pós-graduação em Ciências Farmacêuticas, Universidade Estadual de Maringá, Brazil

^IIDepartamento de Farmácia, Universidade Estadual de Maringá, Brazil

ABSTRACT

This study investigated the antiviral potential of fractions and eupomatenoid-5 obtained from Piper regnelli (Miq.) C. DC., Piperaceae, leaves against bovine herpesvirus-1 (BHV-1) and poliovirus. VERO cell monolayers in 96-well cell culture plates were infected with BHV-1 or poliovirus and incubated in the presence and absence of samples for 48 h. The cells were then fixed and stained with sulforhodamine B, and the virus-induced cytopathic effect was measured in a 96-well plate reader at 530 nm. Cytotoxicity was assessed by incubating the cell monolayers with samples for 72 h. The hexane, chloroform, chloroform/ethyl acetate (19:1), and chloroform/ethyl acetate (9:1) fractions showed activity against BHV-1. The chloroform, chloroform/ethyl acetate (19:1), chloroform/ethyl acetate (9:1), chloroform/ethyl acetate (1:1), and ethyl acetate fractions were active against poliovirus. The chloroform/ethyl acetate (9:1) fraction presented the best selectivity index for both viruses. The present study reports the antiviral activity of the extract and fractions of P. regnelli leaves.

Keywords: antiviral, bovine herpesvirus, Piper regnelli, poliovirus

Introduction

The Piperaceae family comprises a great number of species with medicinal uses and economic and commercial importance. It is used for essential oil production and spices and in the pharmaceutical and insecticide industries.

Piper regnelli var. pallescens (C. DC.) Yunck., popularly known in Brazil as "pariparoba," is an herbaceous plant found in tropical and subtropical regions of the world (Cronquist, 1981). Its leaves and roots are used in folk medicine in the form of crude extracts, infusions, and poultices for the treatment of wounds, swelling, and skin irritations (Yuncker, 1972). The extract and eupomatenoid-5, the neolignan isolated from the leaves of P. regnelli, have antimicrobial activity, including antibacterial (Pessini et al., 2003), antifungal (Koroishi et al., 2008), antileishmanial (Vendrametto et al., 2010), and trypanocidal (Luize et al., 2005) effects.

Bovine herpesvirus type 1 (BHV-1) is a member of the Herpesvirales order, Herpesviridae family, Alphaherpesvirinae subfamily, and Varicellovirus genus (Davison et al., 2009). BHV-1 is an important cattle pathogen that causes economic losses worldwide. BHV-1 is mainly implicated in respiratory infection (e.g., infectious bovine rhinotracheitis), genital disease (e.g., vulvovaginitis/pustular balanoposthitis), and abortion (Kahrs, 2001). This virus may be transmitted via respiratory and venereal routes and by artificial insemination with virus-contaminated semen, whereby samples of a single ejaculate may be inseminated into many females (Afshar & Eaglesome, 1990).

Poliovirus is a member of the Enterovirus genus and Picornaviridae family (Melnick, 1996). It is the etiological agent of poliomyelitis (Landsteiner & Popper, 1909), a disease that has been brought under control in most countries. Picornaviruses encompass numerous important human pathogens, including the enteroviruses polio, Coxsackie A and B, and echo and rhinoviruses, but no single antipicornavirus agent has been approved for clinical use (De Clerq, 2012). Although well studied, poliovirus remains one of the most appropriate models for the study of viral replication (Mueller et al., 2005).

Our previous studies found that the hydroalcoholic extract of P. regnelli leaves exhibited antiviral activity against BHV-1 but not against poliovirus. In the present study, we investigated the antiviral activity of fractions and eupomatenoid-5 (1) obtained from the leaves of Piper regnelli var. pallescens against BHV-1 and poliovirus.

Materials and Methods

Cell culture and viruses

African green monkey kidney (VERO) cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Gibco) and 50 μg/mL gentamicin in a humidified 5% (v/v) CO₂ incubator at 37 ^oC. BHV-1 and vaccinal poliovirus were provided by Dr. Rosa Elisa Linhares, Microbiology Department, State University of Londrina. The viruses were titrated by inoculating the cells with 10-fold dilutions using the endpoint dilution technique (De Clercq, 1982).

Plant material

Piper regnelli var. pallescens (C. DC.) Yunck., Piperaceae, leaves were collected from the Profa. Irenice Silva Garden of Medicinal Plants, State University of Maringá. The plant material was identified by Marília Borgo, Botanical Department, Federal University of the Paraná, and a voucher specimen (no. HUM 8392) was deposited in the Herbarium of the State University of Maringá, Paraná, Brazil. The hydroalcoholic extract (crude extract), fractions, and pure compound eupomatenoid-5 (1) were obtained as described by Vendrametto et al. (2010). Briefly, the extract was obtained by extraction with ethanol/water (9:1, v/v) and evaporation under vacuum at 40 ^oC. This extract (275.0 g) was lyophilized, and the residue was partitioned with ethyl acetate. The organic layer was evaporated to yield the ethyl acetate extract (25.0 g). This extract was then subjected to vacuum column chromatography silica gel and eluted with hexane (2.8 g), chloroform (11.1 g), chloroform/ethyl acetate (19:1, 1.1 g), chloroform/ethyl acetate (9:1, 0.4 g), chloroform/ethyl acetate (1:1, 0.9 g), ethyl acetate (0.3 g), acetone (0.5 g), methanol (1.7 g), and methanol/water 9:1 (0.2 g), providing the corresponding fractions. The chloroform fraction was separated by column chromatography on silica gel 60 to yield eupomatenoid-5. The samples were stored at -20 ^oC until use. All of the compounds were dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium prior to use. The final concentration of DMSO did not exceed the minimum noncytotoxic concentration of 1% (v/v).

Antiviral assays

Confluent VERO cells in 96-well cell culture plates were washed with phosphate-buffered saline (PBS), infected with 25 μL/TCID₈₀/well of BHV-1 or poliovirus, and incubated at 37 ^oC in humidified 5% (v/v) CO₂ for 1 h. After incubation, 75 μL/well of culture medium was added, followed by 100 μL/well of culture medium that contained different concentrations of the fractions or eupomatenoid-5 (1).

The cells were incubated for 48 h. Afterward, the medium was removed, and the cells were washed with 100 μL/well of PBS. The monolayer was then fixed with 50 μL/well of 10% trichloroacetic acid for 1 h at 4 ^oC and subsequently washed four times with deionized water. The microplates were left to dry at room temperature for at least 1 h and then stained with 50 μL/well of 0.4% sulforhodamine B (SRB) in 1% acetic acid for 30 min. The microplates were then washed four times with 1% acetic acid. Bound SRB was solubilized with 150 μL/well of 10 mM buffered Tris-base solution, and the plates were left on a plate shaker for at least 15 min. Absorbance was read in a 96-well plate reader at 530 nm (Bio-Tek, Power XS). The virus-induced cytopathic effect (CPE) is expressed as a percentage of the optical density compared with the parallel virus control and cell control. The concentration that reduced 50% of the CPE compared with the virus control was estimated from the plots of the data and defined as the EC₅₀ value (Papazisis et al., 1997).

Cytotoxicity assay

The cytotoxicity assay was performed as previously described, with some modifications (Skehan et al., 1990). Confluent VERO cell monolayers in 96-well cell culture plates had the medium replaced with 100 μL of serum-free medium. Subsequently, 100 μL/well of culture medium that contained different concentrations of fractions or eupomatenoid-5 was added. The plates were incubated at 37 ^oC in humidified 5% (v/v) CO₂. After 72 h, the plates were fixed and stained, and the optical density was determined as described above. The toxic concentration for 50% of the cells (CC50) is expressed as a percentage of the optical density of the control.

Results and Discussion

The fractions, and eupomatenoid-5 (1) obtained from the crude extract of Piper regnelli var. pallescens (C. DC.) Yunck., Piperaceae, leaves were evaluated against BHV-1 and poliovirus. Cytotoxicity was also tested. The results are summarized in Table 1.

Some Piper species have exhibited antiviral activity, such as the effects of the methanolic and aqueous extracts of P. cubeba fruits against hepatitis C virus (Hussein et al., 2000) and effects of the crude extract of P. aduncun leaves and flowers against poliovirus (Lohézic-Le Dévéhat et al., 2002). The methanolic extract of P. lanceafolium against Herpes simplex virus type 1 (HSV-1) and poliovirus (Lopez et al., 2001) and extracts of different parts of P. methysticum against HSV-1 (Locher et al., 1995) showed no antiviral activity.

As mentioned above, the hydroalcoholic extract (i.e., crude extract) from the leaves of Piper regnelli had antiviral activity against BHV-1, with an EC50 6.0 μg/mL, but no antiviral activity against poliovirus, with an EC50 >100 μg/mL. The results of the antiviral activity of the fractions and eupomatenoid-5 obtained from this extract are shown in Table 1.

The anti-BHV-1 activity of the hydroalcoholic extract can be attributed to the components present in the hexane, chloroform, chloroform/ethyl acetate (19:1), and chloroform/ethyl acetate (9:1) fractions, with EC50 values that ranged from 26.2 to 3.6 μg/mL. The chloroform, chloroform/ethyl acetate (19:1), chloroform/ethyl acetate (9:1), chloroform/ethyl acetate (1:1), and ethyl acetate fractions were shown to be effective against poliovirus, although the crude extract had no effect. This issue should be assessed carefully in future investigations. The best activity against BHV-1 and poliovirus was obtained with the chloroform/ethyl acetate (9:1) fraction, with selectivity indices of 6 and 5 respectively. These results appear to show that this fraction contains the largest amount of the active substance and consequently is the fraction that exhibited the best antiviral activity against both tested viruses.

The antimicrobial activity of eupomatenoid-5 isolated from Piper regnelli (Pessini et al., 2003) was not observed against either BHV-1 or poliovirus, indicating that this compound has no antiviral activity.

Many active compounds have been isolated from this plant species, including amides, alkaloids, lignans, and neolignans, which have central nervous system depressant, antipyretic, analgesic, antiinflammatory, insecticidal, larvicidal, molluscicidal, anti-platelet-activating factor (PAF), and antimicrobial effects (Parmar et al., 1997; Santos et al., 2001; Sengupta & Ray, 1987). A phytochemical study of Piper regnelli isolated phenylpropanoids and dihydrobenzofuran neolignans. Previous studies indicated the presence of (+)-conocarpan as a major compound, in addition to eupomatenoid-6, eupomatenoid-5, and eupomatenoid-3 (Koroishi et al., 2008; Luize et al., 2006; Pessini et al., 2003).

Benzofuran neolignans are a subclass of neolignans with various biological activities, including anti-PAF, antifungal, antibacterial, insecticidal, trypanocidal, and leishmanial effects (Sartorelli et al., 2001). Many 2-arylbenzofuran derivatives, whose skeleton is present in the lignans isolated from P. regnelli, exhibit antiviral activity (Charlton, 1998; Craigo et al., 2000; Leung et al., 2000). Therefore, these compounds may be responsible by the antiviral activity of P. regnelli. The isolated eupomatenoid-5 (1) was also analyzed against BHV-1 and poliovirus. The chloroform fraction was chosen because of the greater amount of material provided by chromatography.

New vaccines have been developed against BHV-1 to ensure effective protection for calves. Rispoval 4 has presented good results under experimental conditions (Salt et al., 2007). Additionally, alternative therapeutics for the treatment of infection in calves have been interesting, especially in the presence of low serum neutralizing antibody titers.

The possibility of new outbreaks in the future and lack of specific and effective drugs encourage the resumption of the search for antipoliovirus compounds (Collett et al., 2008). The possibility that these compounds are also effective against other viruses of the Enterovirus genus cannot be ignored. The members of the Enterovirus genus cause a wide array of illnesses, such as meningitis, myocarditis, encephalitis, and respiratory diseases (Rotbart et al., 1998).

The antiviral potential of Piper regnelli was demonstrated in the present study. Many fractions obtained from P. regnelli leaves exhibited antiviral activity against BHV-1 and poliovirus, demonstrating the potential antiviral activity of Piper regnelli var. pallescens.

Although eupomatenoid-5 has been shown to have trypanocidal (Pelizzaro-Rocha et al., 2011), antibacterial (Pessini et al., 2003), antifungal (Koroishi et al., 2008), and antileishmanial (Vendrametto et al., 2010) effects, it was not active against both viruses.

Future purifications and studies are needed to determine the substance or substances that are responsible for the antiviral activity and mechanism of action of these compounds.

Acknowledgment

This work was supported by Fundação Araucária, CNPq, CAPES and FINEP.

Received 17 Apr 2012

Accepted 13 Jul 2012

• Afshar A, Eaglesome MD 1990. Virus associated with bovine semen. Vet Bull 60: 93-109.
• Charlton JLJ 1998. Antiviral Activity of Lignans. J Nat Prod 61: 1447-1451.
• Collett MS, Neyts J, Modlin JF 2008. A case for developing drugs against polio. Antiviral Res 79: 179-187.
• Craigo J, Callahan M, Huang RCC, Delucia AL 2000. Inhibition of human papillomavirus type 16 gene expression by nordihydroguaiaretic acid plant lignan derivatives. Antivir Res 47: 19-28.
• Cronquist A 1981. An Integrated System of Classification New York: Columbia University Press, p. 670.
• Davison AJ, Eberle R, Ehlers B, Hayward GS, McGeoch DJ, Minson AC, Pellett PE, Roizman B, Studdert MJ, Thiry E 2009. The order Herpesvirales. Arch Virol 154: 171-177.
• De Clercq E 1982. Comparative efficacy of antiherpes drugs in different cell lines. Antimicrob Agents Ch 21: 661-663.
• De Clercq E 2012. Highlights in antiviral drug research: antivirals at the horizon. Med Res Rev doi: 10.1002/med.21256.
• Hussein G, Miyashiro H, Nakamura N, Hattori M, Kakiuchi N, Shimotohno K 2000. Inhibitory effect of Sudanese medicinal plant extract on hepatitis C virus (HCV) protease. Phytother Res 14: 510-516.
• Kahrs RF 2001. Infectious bovine rhinotracheitis and infectious pustular vulvovaginitis. In Viral Diseases of Cattle Ames, IA: Iowa State University Press, p. 159-170.
• Koroishi AM, Foss SR, Cortez DAG, Ueda-Nakamura T, Nakamura CV, Filho BPD 2008. In vitro antifungal activity of extracts and neolignans from Piper regnellii against dermatophytes. J Ethnopharmacol 117: 270-277.
• Landsteiner K, Popper E 1909. Übertragung der poliomyelitis acuta auf affen. Z Immunitatsforsch 2: 377-390.
• Leung C, Charlton JL, Cow C 2000. Antiviral activity of arylnaphthalene and aryldihydronaphthalene lignans. Can J Chem 78: 553-561.
• Locher CP, Burch MT, Mower HF, Berestecky J, Davis H, Van Poel B, Lasure A, Vanden Bergue DA, Vlietinck AJ 1995. Antimicrobial activity and anti-complement activity of extract obtained from selected Hawaiian medicinal plants. J Ethnopharmacol 49: 23-32.
• Lohézic-Le Dévéhat F, Bakhtiar A, Bézivin C, Amoros M, Boustie J 2002. Antiviral and cytotoxic activities of some Indonesian plants. Fitoterapia 73: 400-405.
• Lopez A, Hudson JB, Towers GHN 2001. Antiviral and antimicrobial activities of Colombian medicinal plants. J Ethnopharmacol 77: 189-196.
• Luize PS, Tiuman TS, Morello LG, Maza PK, Ueda-Nakamura T, Filho BPD, Cortez DAG, Mello JCP, Nakamura CV 2005. Effects of medicinal plant extracts on growth of Leishmania (L.) amazonensis and Trypanosoma cruzi. Braz J Pharm Sci 41: 85-94.
• Luize PS, Ueda-Nakamura T, Filho BPD, Cortez DAG, Nakamura CV 2006. Activity of neolignans isolated from Piper regnelli (MIQ.) C. DC. var. pallescens (C. DC.) Yunck against Trypanosoma cruzi. Biol Pharm Bull 29: 2126-2130.
• Melnick JL 1996. Enteroviruses: polioviruses, coxsackieviruses, echoviruses, and newer enteroviruses. In Fields BN (Ed.) Virology New York: Raven Press, p. 655-712.
• Mueller S, Wimmer E, Cello J 2005. Poliovirus and poliomyelitis: A tale of guts, brain and an accidental event. Virus Res 111: 157-193.
• Papazisis KT, Geromichalos GD, Dimitriadis KA, Kortsaris AH 1997. Optimization of the sulforhodamine B colorimetric assay. J Immunol Methods 208: 151-158.
• Parmar VS, Jain CS, Bisht KS, Taneja P, Jha A 1997. Phytochemistry of the genus Piper. Phytochemistry 46: 597-673.
• Pelizzaro-Rocha KJ, Veiga-Santos P, Lazarin-Bidoia D, Ueda-Nakamura T, Filho BPD, Ximenes VF, Silva SO, Nakamura CV 2011. Trypanocidal action of eupomatenoid-5 is related to mitochondrion dysfunction and oxidative damage in Trypanosoma cruzi. Microbes Infect 13: 1018-1024.
• Pessini GL, Dias BPF, Nakamura CV, Cortez DAG 2003. Antibacterial activity of extracts and neolignans from Piper regnelli (Miq.) C. DC var. pallescens (C. DC.) Yunck. Mem I Oswaldo Cruz 98: 1115-1120.
• Rotbart HA, OConnel JF, McKinlay MA 1998. Treatment of human enteroviruses infections. Antivir Res 38: 1-14.
• Salt JS, Thevasagayam SJ, Wiseman A, Peters AR 2007. Efficacy of a quadrivalent vaccine against respiratory diseases caused by BHV-1, PI3V, BVDV and BRSV in experimentally infected calves. Vet J 174: 616-626.
• Santos PRD, Moreira DL, Guimarães EF, Kaplan MAC 2001. Essential oil analysis of 10 Piperaceae species from the Brazilian Atlantic forest. Phytochemistry 58: 547-551.
• Sartorelli P, Benevides PJC, Ellensohn RM, Rocha MVAF, Moreno PRH, Kato MJ 2001. Enantioselective conversion of p-hydroxypropenyl benzene to (+)-conocarpan in Piper regnelli. Plant Sci Lett 161: 1083-1088.
• Sengupta S, Ray AB 1987. The chemistry of Piper species: a review. Fitoterapia 63: 147-166.
• Skehan P, Storeng R, Scudeiro D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR 1990. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer I 82: 1107-1112.
• Vendrametto MC, Santos AO, Nakamura CV, Filho BPD, Cortez DAG, Ueda-Nakamura T 2010. Evaluation of antileishmanial activity of eupomatenoid-5, s compound isolated from leaves of Piper regnelli var. pallescens. Parasitol Int 59: 154-158.
• Yuncker TG 1972. The Piperaceae of Brazil I: Piper Group I, II, III and IV. Hoehnea 2: 19-366.

Publication Dates

• Publication in this collection
  18 Sept 2012
• Date of issue
  Dec 2012

History

• Received
  17 Apr 2012
• Accepted
  13 July 2012