Journal of Ethnopharmacology 99 (2005) 403407

Short communication

In vitro anti-rotavirus activity of some medicinal plants used in Brazil against diarrhea
J.L.S. Gonc alves a , R.C. Lopes b , D.B. Oliveira c , S.S. Costa c , M.M.F.S. Miranda a, 1 , M.T.V. Romanos a , N.S.O. Santos a , M.D. Wigg a,
a

Departamento de Virologia, Instituto de Microbiologia Professor Paulo de G oes, Universidade Federal do Rio de Janeiro Centro de Ci encias da Sa ude (CCS), Bloco I, Caixa Postal 68040, CEP 21941-590, Rio de Janeiro, Brazil b Instituto de Biologia, Departamento de Bot anica, Universidade Federal do Rio de Janeiro, CEP 21941-590, Rio de Janeiro, Brazil c N ucleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, CEP 21941-590, Rio de Janeiro, Brazil Received 28 December 2004; received in revised form 28 December 2004; accepted 14 January 2005 Available online 3 May 2005

Abstract Acute diarrhea, especially in children, is a very common disease with worldwide distribution and with a signicant public health impact. Rotaviruses have been recognized as the major agents of diarrhea in infants and young children in developed as well as developing countries. In Brazil, diarrhea is one of the principal causes of death, mainly in the infant population. To ght diarrhea, traditional Brazilian medicine uses a great variety of plants. In this work, 12 medicinal plant species were screened for simian (SA-11) and human (HCR3) rotaviruses inhibition in vitro. At non-cytotoxic concentrations, the extracts from Artocarpus integrifolia L. (Moraceae) bark (480 g/ml) and Spondias lutea L. (Anacardiaceae) leaves (160 g/ml) had antiviral activity against both viruses. They showed inhibition of 99.2% and 97%, respectively, for human rotavirus, and 96.4% and 96.2% for simian rotavirus. The extracts from Myristica fragrans Houtt (Myristicaceae) seeds (160 g/ml) and Spongias lutea bark (40 g/ml) inhibited human rotavirus (90% and 82.2% inhibition, respectively), whereas the extracts from Anacardium occidentale L. (Anacardiaceae) leaves (4 g/ml) and Psidium guajava L. (Myrtaceae) leaves (8 g/ml) showed activity only against simian rotavirus (82.2% and 93.8% inhibition, respectively). Our results indicate that the extracts of Artocarpus integrifolia, Myristica fragrans and Spongias lutea can be useful in the treatment of human diarrhea if the etiologic agent is a rotavirus. 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Antiviral activity; Rotavirus; Cytotoxicity; MA-104 cells; Diarrhea; Plant extracts

1. Introduction In Brazil, acute diarrhea is a signicant public health problem, especially in newborn humans, due to the high mortality rate. Rotaviruses (Reoviridae) are the major etiologic agents of diarrhea in infants and young children under 2 years of age throughout the world (Pereira et al., 1983; Bern and Glass, 1994). Rotaviruses are human and animal pathogens, nonenveloped viruses with a triple-layered double capsid and
1

Corresponding author. Tel.: +55 21 2562 6749; fax: +55 21 2560 8344. E-mail address: marcia.wigg@micro.ufrj.br (M.D. Wigg). In memoriam.

11 segments of double-stranded RNA. The fecaloral route transmits them, and the disease is a consequence of the virus replication in the small intestine (Davidson et al., 1975; Ball et al., 1996). The clinical manifestation is acute gastroenteritis with fever, vomiting, abdominal pain, diarrhea and dehydration. Although progress has been made during the past few years in developing various live vaccine candidates, up to now, there is no effective one (MMWR, 1999). The only treatment of rotavirus gastroenteritis is the replacement of uids and electrolytes lost by vomiting and diarrhea (Kapikian et al., 2001). However, for patients who are suffering from prolonged diarrhea, virus-specic treatment will be necessary.

0378-8741/$ see front matter 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.01.032

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Some in vitro studies have shown the activity of synthetic compounds, such as 1--d-ribofuranosyl-1,2,4-triazole-3carboxamide (ribavirin) and 3-deazaguanine (Smee et al., 1982), and isoprinosine (Linhares et al., 1989), against simian rotavirus. As natural products, theaavins, which are polyphenolic compounds extracted from black tea, have been found to be inhibitory to human and bovine rotavirus (Clark et al., 1998). Nevertheless, these compounds are not available for human use, and the viral gastroenteritis is still largely uncontrolled. Thus, alternative methods to control rotavirus infection are extremely desirable. Brazilian medicinal plants have many folk uses, and some of them have shown to be efcient to control diarrhea (Carib e and Campos, 1991; Di Stasi et al., 1994). In this study, we evaluated 12 medicinal plants popularly used in Brazil to treat diarrhea towards simian (SA-11) and human (HCR3) rotavirus propagation inhibition in vitro.

2.3. Preparation of extracts Leaves, owers, seeds and bark were air-dried under shade and subsequently powdered. Afterwards, 8 g of each material was mixed with 100 ml of boiling distilled water, and cooled to room temperature. The mixtures were strained, the marcs pressed and the extracts claried by ltration using Whatman no. 1 lter paper with the aid of a suction pump. Before the tests, the ltrates were diluted in Eagle-MEM without serum and sterilized by ltration using 0.22 m millipore membrane. For standardization, the ltrates were lyophilized (Labconco, USA), and the percent (w/w) yields in terms of dry starting material are presented in Table 1. 2.4. Phytochemical tests Freshly prepared extracts were tested for the presence of avonoids, tannins, saponins, nitrogenous compounds, alkaloids, and terpenes by thin-layer chromatography (TLC). The analysis was performed on Merck silica gel 60 F254 aluminum plates, which were developed with butanol/acetic acid/water (8:1:1) solution. The TLC plates were sprayed either with cerium sulphate, ninhydrin or Dragendorff reagent, and then visualized under UV light at 254 nm and 365 nm. 2.5. Determination of the maximum non-toxic concentration The cytotoxicity of each extract was determined by a method similar to that described by Walker et al. (1971), with small modications (Miranda et al., 1997). This method is based on cellular morphologic alterations. Several concentrations of each extract were placed in contact with conuent MA-104 cell monolayers and incubated in a 5% CO2 atmosphere at 37 C for 4 days. At the end of this time, the cells were examined using an inverted optical microscope (Leitz) whereby we compared treated and untreated cultures (control). The higher concentration of the extract showing no cellular morphologic changes was considered as the maximum non-toxic concentration (MNTC). 2.6. Antiviral assay Antiviral activity was assessed by the reduction of virus titers using TCID50 determinations. The MA-104 cell monolayers cultivated in 96-well microplates were treated with the extracts at the maximum non-toxic concentration in presence of 3 g/ml trypsin. Afterwards, logarithmic dilutions of activated rotaviruses were added to treated and untreated cell cultures and incubated in a 5% CO2 atmosphere at 37 C. Virus titers (TCID50 /ml) were calculated after 4 days of incubation. Experiments were carried out in triplicate. The antiviral activity was expressed in terms of percentage of inhibition (PI) (Nishimura et al., 1977) using antilogarithm values of TCID50 , as follows: PI = [1 (T antilogarithm/C antilogarithm)] 100. An extract was considered active when it

2. Materials and methods 2.1. Cell culture and viruses African Rhesus monkey kidney cells (MA-104) were cultivated in Eagles minimum essential medium (Eagle-MEM) supplemented with 10% fetal bovine serum (FBS), 50 g/ml garamicin and 2.5 g/ml amphotericin B (Schmidt, 1979). Cell cultures were prepared in 96-well plastic microtiter plates and incubated in a 5% CO2 atmosphere at 37 C. Simian and human rotaviruses were activated with 10 g/ml trypsin for 30 min at 37 C and propagated in MA-104 cells monolayers in presence of 3 g/ml trypsin. Virus titrations were performed by the end-point titration assay. The virus titers were estimated from cytopathogenicity and expressed as 50% tissue culture infectious doses (TCID50 /ml) by the Reed and Muench (1938) statistical method. 2.2. Plant material The medicinal plants tested are listed in Table 1. They were collected from the Rio de Janeiro State area, Brazil, in 2000. Voucher specimens were identied by Dr. Rosana C. Lopes and deposited at the Herbarium of the Institute of Biology, Federal University of Rio de Janeiro, Brazil. Spondias lutea L. (Anacardiaceae), Anacardium occidentale L. (Anacardiaceae) and Cassia ocidentalis L. (Caesalpiniaceae) are native of Brazil. The other ones were introduced in Brazil a long time ago and are very well acclimated species. DaonTM and quebracho were used as standard compounds. The former was a gift from Servier do Brasil Ltda. (Brazil), and it is constituted of two avonoids (90% purity): diosmin and hesperidin (9:1). Quebracho is constituted of condensed tannins extracted from Schinopsis sp. (Anacardiaceae) bark using acetone/water (7:3) solution, and were analysed by high performance liquid chromatography (HPLC) system (Merck, USA).

J.L.S. Gon calves et al. / Journal of Ethnopharmacology 99 (2005) 403407 Table 1 Cytotoxicity and anti-rotavirus activity of medicinal plants used in Brazil against diarrhea Plant family, scientic name and voucher specimen number Annonaceae Annona squamosa L. (RFA 30816) Anacardiaceae Anacardium occidentale L. (RFA 30820) Spondias lutea L. (RFA 30818) Lauraceae Cinnamomum zeylanicum Breyn (RFA 30704) Caesalpiniaceae Cassia occidentalis L. (RFA 30705) Lythraceae Punica granatum L. (RFA 30703) Moraceae Artocarpus integrifolia L. (RFA 30819) Myristicaceae Myristica fragrans Houtta Myrtaceae Syzygium jambos (L.) Alston (RFA 30706) Psidium guajava L. (RFA 30817) Oxalidaceae Averrhoa carambola L. (RFA 30834) Rosaceae Rosa canina L. (RFA 30707) DaonTMb Quebrachob Brazilian name Plant part Yield % (w/w) MNTC (g/ml) PI HCR3 fruta-de-conde cajueiro cajazeiro Leaves Leaves Leaves Bark Leaves Bark Leaves 25.0 16.2 22.8 21.7 19.6 5.7 26.7 22.2 14.6 17.8 18.4 17.2 20.3 16.4 22.6 9.3 35.8 20.0 4.0 160.0 40.0 40.0 4.0 320.0 2.0 480.0 160.0 20.0 20.0 8.0 4.0 20.0 4.0 2.0 200.0 6.2 43.8 25.9 97.0 82.2 32.4 33.9 68.4

405

SA-11 0 84.5 96.2 53.2 16.8 55.3 0

canela-do-Ceil ao

fedegoso

rom azeiro jaqueira

Leaves Bark

0 99.2 90.0 20.6 62.0 47.5 53.2 66.8 53.2 46.3 90.0 0

0 96.4

noz-moscada jambeiro goiabeira

Seeds Leaves Bark Leaves Bark Leaves Bark Flowers

0 0 16.8 93.8 0 43.8 0 56.3 53.2 80.0

caramboleira

roseira

MNTC: maximum non-toxic concentration; PI: percentage of inhibition; HCR3: human rotavirus; SA-11: simian rotavirus. a From Jordely Com ercio de G eneros Aliment cios Ltda., Brazil. b Standard compounds.

showed percentage of inhibition higher than 80%, at the MNTC.

3. Results Twelve plant species belonging to 10 families used by the Brazilian population to treat diarrhea were included in this study. Seventeen aqueous plant extracts were evaluated for cytotoxicity and antiviral activity against SA-11 (simian) and HCR3 (human) rotaviruses propagated in MA-104 cell culture. The MNTC of the extracts varied in the range of 480 g/ml and 2 g/ml. The less cytotoxic extract was obtained from Artocarpus integrifolia (Moraceae) bark (480 g/ml). Extracts of Punica granatum (Lythraceae) leaves and Rosa canina (Rosaceae) owers were the most cytotoxic, with MNTC of 2 g/ml.

The antiviral activity of each plant extract at the MNTC can be observed in Table 1. Plant extracts showing inhibition higher than 80% were considered to have anti-rotavirus activity. According to this criterion, the extracts of Artocarpus integrifolia bark (99.2%), Myristica fragrans (Myristicaceae) seeds (90%), Spongias lutea leaves (97%) and bark (82.2%) were able to inhibit the propagation of human rotavirus in vitro. Extracts of Anacardium occidentale leaves (84.5%), Artocarpus integrifolia bark (96.4%), Psidium guajava (Myrtaceae) leaves (93.8%), and Spongias lutea leaves (96.2%) had activity against simian rotavirus.

4. Discussion For decades, researchers have been seeking new drugs through the knowledge gained by popular medicine. Natural products have been selected from biological screenings

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mainly based on the ethnobotanic use, although other approaches such as random studies or chemotaxonomic data have been used (Vlietinck and Vanden Berghe, 1991). Phytotherapy is widespread in some regions of Brazil, being sometimes the only alternative treatment for those who do not have access to trade mark drugs. Nevertheless, many plants have not been studied for the claimed biological activity. For this reason, we have participated in a program aiming to screen the antiviral activity of plants cultivated in Brazil. In this work, we studied 12 plant species used in the country in the treatment of diarrhea. This work was focused on their potential activity against rotavirus cell propagation. The cytotoxic level of the extracts was evaluated before carrying out the antiviral tests. In general, plant extracts show different degrees of toxicity depending on the extraction method. Higher toxicity has been observed in ethanol than in aqueous extracts (Abad et al., 1999; Bedoya et al., 2001). For this reason and because people take either plant decoction or infusion, we chose water to prepare the extracts. All extracts showed some level of toxicity for MA-104 cells. Nevertheless, this result was not expected, as there is no report in the literature on toxic effects associated with the use of those plants in the treatment of abdominal pain and diarrhea. At maximum non-toxic concentrations (CMNT), extracts of Artocarpus integrifolia bark and Spongias lutea leaves showed activity against both rotaviruses, whereas the extracts of M. fragans seeds and Spongias lutea bark inhibited human rotavirus. Extracts of Anacardium occidentale and Psidium guajava leaves had effect only on simian rotavirus. These data suggest that there might be different bioactive compounds in the extracts. Preliminary phytochemical analysis revealed the following compounds in the active extracts: Anacardium occidentale leaves (tannins, avonoids, terpenes and saponines); Spondias lutea leaves and Myristica fragrans seeds (avonoids); Spongias lutea bark and Psidium guajava leaves (tannins and avonoids); Artocarpus integrifolia bark (avonoids, terpenes and nitrogen compounds). Alkaloids were not detected in any active extracts (data not shown). Bae et al. (2000) demonstrated that some avonoids detected in plant extracts were able to inhibit the rotavirus propagation, protecting the cell cultures against virus invasion. Considering that avonoids were found in all active extracts, we could possibly claim that these compounds could be inhibitors of rotavirus propagation in MA-104 cells. However, our extracts also contain tannins, terpenes, saponins and nitrogenous compounds. Thus, ongoing experiments are being performed to identify the anti-rotavirus bioactive compound(s). Psidium guajava leaves are used to stop diarrhea in Brazil (Di Stasi et al., 1994) and Mexico (Jaiarj et al., 1999), with the avonoid quercetin and its glycosides thought to be the active substances (Lozoya et al., 1994). Some compounds, such as avonoids, from herbal preparations reach the small intestine without processing and are further metabolized in

the large intestine by the human intestinal micro-ora (Bae et al., 2000). As rotaviruses cause severe diarrhea resulting in damage to cells in the small intestine, these bioactive substances should be delivered intact to the target site, and they could protect against rotavirus infection to some degree. Interestingly, some plants used in the treatment of diarrhea were either weakly active or ineffective in our in vitro model. This data can lead to some hypotheses: (1) the plant has this effect acting against an etiologic agent other than rotavirus; (2) the diarrhea is caused either by physiopathological or by biochemical disturbs. Finally, extracts of Artocarpus integrifolia bark, Myristica fragrans seeds, and Spongias lutea leaves and bark inhibited human rotavirus cell propagation. Although a number of people use plants to treat diarrhea without knowing the causes underlying this disease, we believe that these plants can be useful in the treatment of human diarrhea if the etiologic agent is a rotavirus.

Acknowledgements This research was supported by Conselho Nacional de Deo senvolvimento Cient co e Tecnol ogico (CNPq), Fundac a ` Pesquisa do Estado do Rio de Janeiro (FAPERJ), de Amparo a and Conselho de Ensino Para Graduados (CEPG/UFRJ).

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