In vitro cytotoxic, antioxidant and antiviral effects of Pterocaulon alopecuroides and Bidens segetum extracts

Efeitos citotóxico, antioxidante e antiviral in vitro de extratos de Pterocaulon alopecuroides e Bidens segetum

ARTIGO

In vitro cytotoxic, antioxidant and antiviral effects of Pterocaulon alopecuroides and Bidens segetum extracts

Efeitos citotóxico, antioxidante e antiviral in vitro de extratos de Pterocaulon alopecuroides e Bidens segetum

Cristiane Silva Silveira^I; Fernanda Otaviano Martins^II; Clarice dos Santos Costa^I; Maria Teresa Villela Romanos^II; Maria Auxiliadora Coelho Kaplan^I; Fábio de Sousa Menezes^{III, *}

^INúcleo de Pesquisa em Produtos Naturais, Centro de Ciências da Saúde, Universidade Federal do 21941-590 Rio de Janeiro-RJ, Brazil

^IILaboratório Experimental de Drogas Antivirais e Citotóxicas, Departamento de Virologia, Instituto de Microbiologia Prof. Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro-RJ, Brazil

^IIISchool of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, University of Dublin, Dublin 2, Ireland

ABSTRACT

Pterocaulon alopecuroides (Lamark) De Candolle and Bidens segetum Mart. ex Colla are two species belonging to the Asteraceae family. Extracts from those two species were evaluated to their cytotoxic, antioxidant and antiviral activities. All the extracts assayed have shown a very high cytotoxity against RBL-2H3 cell line. The antioxidant assay pointed out a really high activity of the ethyl acetate extracts for B. segetum and P. alopecuroides. This can be partially explained due to the high content of coumarins, at least for P. alopecuroides. None of the total ethanol extracts from B. segetum showed significant activity against the two strains of Herpes simplex virus (Types 1 and 2 resistant to acyclovir). P. alopecuroides ethanol extract was also inactive against the Herpes simplex virus type 1 resistant to acyclovir. However, this extract presented inhibitory activity against the Herpes simplex virus type 2 resistant to acyclovir. From the ethanol crude extract of P. alopecuroides, it was possible to isolate 7-(2',3'-dihidroxy-3'-methylbutyloxy)-6-methoxycoumarin, which was tested in the same conditions, showing a viral inhibitory rate almost twice bigger than the P. alopecuroides sample for HSV-2-ACVr. The coumarin was also active against HSV-1-ACVr. Those results provide further evidence of the importance of Pterocaulon alopecuroides and Bidens segetum as medicinal plants.

Keywords: Pterocaulon alopecuroides, Bidens segetum, antiviral activity, cytotoxic activity, antioxidant activity, 7-(2',3'-dihidroxy-3'-methylbutyloxy)-6-methoxycoumarin.

RESUMO

Pterocaulon alopecuroides (Lamark) De Candolle e Bidens segetum Mart. ex Colla são duas espécies pertencentes à família Asteraceae. Os extratos dessas duas espécies foram avaliados quanto às suas atividades citotóxica, antioxidante e antiviral. Todos os extratos analisados apresentaram citotoxidade muito alta contra linhagens de células RBL-2H3. O ensaio de atividade antioxidante demonstrou uma alta atividade das frações em acetato de etila de B. segetum e P. alopecuroides. Isso pode ser parcialmente explicado pelo alto conteúdo de cumarinas, ao menos para P. alopecuroides. Nenhum dos extratos etanólicos totais de B. segetum mostraram atividade significativa contra o vírus Herpes simplex (Tipos 1 e 2 resistentes ao aciclovir). O extrato etanólico de P. alopecuroides também foi inativo contra o vírus Herpes simplex tipo 1 resistente ao aciclovir. Entretanto, este extrato apresentou atividade inibitória contra o vírus Herpes simplex tipo 2 resistente ao aciclovir. Do extrato etanólico bruto de P. alopecuroides foi possível isolar a 7-(2',3'-dihidroxi-3'-metilbutiloxi)-6-metoxicumarina, a qual foi testada nas mesmas condições, demonstrando um índice de inibição viral quase duas vezes maior do que o da amostra de P. alopecuroides para HSV-2-ACVr. A cumarina tabém foi ativa contra HSV-1-ACVr. Esses resultados evidenciam a importância de Pterocaulon alopecuroides e Bidens segetum como plantas medicinais.

Unitermos: Pterocaulon alopecuroides, Bidens segetum, atividade antiviral, atividade citotóxica, atividade antioxidante, 7-(2',3'-dihidroxi-3'-metilbutiloxi)-6-metoxicumarina.

INTRODUCTION

Pterocaulon alopecuroides (Lamark) De Candolle and Bidens segetum Mart. ex Colla are two species belonging to the Asteraceae family, one of the biggest families of Angiospermae (Da Costa et al., 2005). Species from Bidens genus occurs in tropical regions (Ballard, 1986; Agra et al., 2007 and 2008) and they are rich in quercetin (Hoffmann, 1989; Lastra Valdes, 2001) and other polyphenolic compounds (Rice-Evans, 1996). It has been reported antibacterial (Rabe and Van Staden, 1997), antimalarial (Oliveira et al., 2004), antioxidant and immunomodulatory effects for Bidens species (Abajo et al., 2004). P. alopecuroides is widely distributed in North and South America and Australia. Twelve species belonging to Pterocaulon genus have previously been mentioned as medicinal (Maes et al., 2006). Studies concerning pharmacological activities report antiviral activity against poliovirus and rhinovirus (Semple et al., 1998, 1999), antibacterial activity (Macleod and Rasmussen, 1999), DNA interaction and anti-tumor activities (Mongelli et al., 2000), anti-proliferative and differentiating properties on human leukemia cells (Riveiro et al., 2004) and antifungal activity (Stein et al., 2005). The main class of active coumponds found in Pterocaulon genus are terpenes, flavonoids and coumarins (Bohlmann et al., 1981; Debenedetti et al., 1997, 1999; Macleod and Rasmussen, 1999; Riveiro et al., 2004; Semple et al., 1999; Vera et al., 2001; Vilegas et al., 1995).

This work was undertaken to evaluate the in vitro antioxidant, cytotoxic and antiviral activities of Pterocaulon alopecuroides and Bidens segetum extracts, altogether with a coumarin isolated from P. alopecuroides ethanol extract.

MATERIAL AND METHODS

Plant material

Pterocaulon alopecuroides and Bidens segetum aerial parts were collected on april 2005, in Nova Friburgo, State of Rio de Janeiro, Brazil. The GPS coordinates were 22º23' 17.2" S and 42º17' 23.0" W for P. alopecuroides and 22º23' 19.7" S and 42º17' 18.6" W for B. segetum. The plant material was identified by Dr. Rosana C. Lopes (Department of Botany, CCS, Federal Universidade Federal do Rio de Janeiro). Voucher specimens were deposited in the Herbarium of the Botany Department – RFA.

Extract preparations

Bidens segetum was separated in flowers and leaves/stems, while for Pterocaulon alopecuroides, aerial parts were employed. Dried plant materials were powdered and submitted to exhaustive extraction with ethanol at room temperature. The obtained extracts were concentrated under reduced pressure. The residue was suspended in water and submitted to a sequential liquidliquid extraction with the following different solvents: hexane, dichloromethane, ethyl acetate and n-butanol, providing new extracts as in Matheus et al., 2005.

Isolation and identification of compounds

The ethanol extract from Pterocaulon alopecuroides (2.55g) was chromatographed by High Speed Counter-Current Chromatography (HSCCC) using C₆H₆-EtOAc-MeOH-H₂O (4:5:3:1.3) as solvent system. The upper phase was used as mobile phase. The flow rate was 2 mℓ/min and the rotation speed was 850 rpm. The fraction volume was 4 mℓ and a total of 109 fractions were obtained. The fractions were joined, followed by TLC using silica gel, and the fractions 78-87 were re-chromatographed in Sephadex LH-20® column eluted with MeOH. A total of 73 fractions were obtained and 90.5 mg of the 7-(2',3'-dihidroxy-3'-methylbutyloxy)-6-methoxycoumarin was isolated from fractions 13-17. The coumarin was identified based on its spectral data (¹H NMR 600MHz, ¹³C NMR 150MHz).

Cytotoxicity assay

The assay was performed with a cytotoxicity detection kit designed as a precise, fast and simple colorimetric alternative to quantify cytotoxicity based on the measurement of lactate dehydrogenase (LDH) activity released from damaged cells (RBL-2H3).

A sterile 96-wells tissue culture plate was filled according to Table 1, in triplicate wells for each control and sample. Plate was incubated at 37 °C for 24 hours, in atmosphere of 5% CO₂ and 90% humidity. Cells were removed from microplate by centrifuging them at 250 x g for 10 minutes and 100µℓ/well supernatant was carefully transferred into corresponding wells of an optically clear 96-wells flat bottom microplate. To determine the LDH activity in these supernatants, 100 µL of reaction mixture was added to each well and incubate for up to 30 minutes at 25 °C, protected from light. The absorbance of the samples was measured at 490nm using a microplate analyser Fusion Universal (Packard BioScience Co.).

To determine the percentage cytotoxicity (C%), the average absorbance values (ABS) of the triplicates were calculated. The resulting values are substituted in the following equation:

C% = {[(ABS)_SAMPLE -ABS_{BACKGROUND CONTROL} ) – ABS_{LOW CONTROL}] / ABS_{HIGH CONTROL} - ABS_{LOW CONTROL})} x 100

Antioxidant assay

This assay was performed by 2,2-diphenyl-1-picrilhydrazil (DPPH) stable free radical photocolorimetric method, according previously published papers (Menezes et al., 2005; Silva et al., 2005; Falcão et al., 2006; Vicentino and Menezes, 2007; Nunes et al., 2008). The ethanol extracts of Bidens segetum flowers, Bidens segetum leaves/stems, Pterocaulon alopecuroides aerial parts and their partition extracts in dichloromethane, ethyl acetate and n-butanol were evaluated. Results were compared to Ginkgo biloba standard, EGb 761®, which is known by its free radical scavenging property.

Antiviral assay

Extracts and pure compound were lyophilized and solved in water to a final concentration of 400 µg/ mℓ. Each solution was sterilized by filtration using a 0.22 µm Milipore membrane, aliquoted and stored at - 20 ºC.

In order to evaluate the antiviral activity, the extracts were assayed at the maximum non-toxic concentrations (MNTC), which is unable to produce any morphological alterations on the cells tested. Vero cells (African green monkey's kidney) were grown in Eagle's minimum essential medium (MEM) supplemented with 10% of fetal bovine serum (FBS) and maintained at 37 °C in atmosphere of 5% CO₂. Antiviral activity was determined by reduction of the virus titres. Logarithmical dilutions of virus suspension were added to the treated and untreated cell cultures and incubated at 37 °C in atmosphere of 5% CO₂. After that, the virus titres determination was performed using Reed and Muench (1938) statistical method and expressed as TCID₅₀ values. The results were expressed as percentage of inhibition (PI) and viral inhibition index (VII) according with described for Gonçalves et al. (2001).

RESULTS AND DISCUSSION

All the three extracts evaluated at the cytotoxicity assay have shown a very high cytotoxicity power against RBL-2H3 cells (Table 1). The extract of flowers of B. segetum and the extract of P. alopecuroides were almost as active as Triton X (high control) and about 30% higher than the terfenadine standard (positive control). These results offer evidence that B. segetum and P. alopecuroides extracts may have an important role in cancer treatment studies and the partition extracts of these two species must be evaluated as well.

Cancer is one of a series of diseases that can be originated by oxidative damage (Castro and Freeman, 2001). Compounds with scavenging properties have been studied in several chronic diseases caused by free radicals (Matheus et al., 2006). Very few systematic studies have been reported on structure-antioxidant activity correlations in coumarins, but their activity is probably due to their structural analogy with flavonoids and benzophenones (Farombi and Nwaokeafor 2005). In fact, as substitutions can occur at any of the six available sites of their basic molecular moiety (1,2-benzopyrone), coumarins possess a great structural diversity (Beillerot et al., 2008).

The antioxidant assay performed here pointed out a really high activity of the ethyl acetate extracts of B. segetum and P. alopecuroides (Table 2). This can occur due to the high content of coumarins of these extracts. Only the dichloromethane extract of P. alopecuroides, the dichloromethane extract of B. segetum leaves/stems, the ethanol extract of B. segetum flowers and its partition in n-butanol presented a higher CE₅₀ than the Ginkgo biloba standard (Table 2).

Coumarins are a group of natural compounds which are extremely variable in structure (Beillerot et al., 2008), leading to compounds displaying multiple biological properties as in vitro antiproliferative and in vivo antitumor activities (Kostova, 2005); antioxidant activity (Beillerot et al., 2008; Farombi and Nwaokeafor, 2005; Fylaktakidou et al., 2004; Girennavar et al., 2007; Kontogiorgis et al., 2004; Torres et al., 2006; Wua et al., 2007; von Kruedener et al., 1996); inhibitory activity against cytochrome P450 enzyme (Girennavar et al., 2007); neuroprotective, anticancer and antimutagenic activities (Borges et al., 2005; Stanczyk et al., 2005); anti-inflammatory activity (Borges et al., 2005; Khan and Sharma, 1993; Kontogiorgis et al., 2004; Stanczyk et al., 2005); vasorelaxant (Hoult and Paya, 1996) and anticoagulant activities (Khan and Sharma, 1993). Some studies reveal that the multiple pharmacological activities of coumarins may be related to their antioxidant properties (Borges et al., 2005; Stanczyk et al., 2005), as oxidative damage to biomolecules causes accelerated aging and many chronic diseases, including neurodegenerative diseases, cancer, cardiovascular diseases and inflammation (Castro and Freeman, 2001).

According to Schnitzler et al. (2008), an aqueous extract of Pelargonium sidoides, in which coumarins were identified as major constituents, showed efficacy against herpes virus. Some other plant extracts and pure compounds isolated from natural sources have been tested and can be considered an important tool on herpes infection treatment (Khan et al., 2005).

None of the total ethanol extracts from B. segetum showed significant activity and the total ethanol extract from P. alopecuroides did not show any inhibitory activity against the Herpes simplex virus type 1 resistant to acyclovir (HSV-1-ACVr) either. However, this extract showed inhibitory activity against the herpes simplex virus type 2 resistant to acyclovir (HSV-2-ACVr) with a percentage of inhibition higher than 70% (Table 3). This may be due to the fact that this sample is a crude extract, with low content of active compounds. Based on that, the isolated coumarin from this extract was tested in the same conditions, showing a viral inhibitory rate almost two times bigger than the P. alopecuroides sample for HSV-2-ACVr. The coumarin was also active against HSV-1-ACVr (Table 3).

Herpes simplex virus (HSV) is widely spread around the world and represents an important cause of orolabial and genital ulceration. It is also responsible for neonatal morbidity, a co-factor for HIV transmission, particularly in developing countries (Patel and Rompalo, 2005), and it is also involved in several ocular diseases (Liesegang, 2001; Pepose et al., 2006). The two forms of Herpes simplex virus (HSV-1 e HSV-2) are morphologically similar and produce indistinguishable characteristics during the initial infection. However, HSV-1 and HSV-2 have an anatomical tropism and site-dependent incidence of reactivation, thus HSV-1 is more likely to reactivate producing orofacial infection while genital infection is frequently produced by HSV-2 (Pepose et al., 2006).

The results obtained in this test provide further evidence of the importance of P. alopecuroides on Herpes simplex treatment. According to Semple et al. (1998,1999), Pterocaulon sphacelatum has antiviral activity suggesting that other species from Pterocaulon genus may also have antiviral activity. Coumarins have been related to HIV-1 protease inhibitory activity (Kirkiacharian et al., 2002). Our results suggest that 7-(2',3'-dihidroxy-3'-methylbutyloxy)-6-methoxycoumarin can be responsible to the Herpes simplex virus inhibitory activity. Other assays will take place as a continuation of this research to try to find out the mechanism of action of 7-(2',3'-dihidroxy-3'- methylbutyloxy)-6-methoxycoumarin against Herpes simplex virus.

ACKNOWLEDGMENTS

This study had the financial support of CNPq, CAPES and FAPERJ.

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Received 26 December 2008; Accepted 18 March 2009

Publication Dates

• Publication in this collection
  01 Sept 2009
• Date of issue
  June 2009

History

• Accepted
  18 Mar 2009
• Received
  26 Dec 2008