African Journal of Biotechnology Vol. 10(27), pp. 5398-5401, 15 June, 2011 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB10.2576 ISSN 1684–5315 © 2011 Academic Journals Full Length Research Paper Study of Salmonella typhimurium mutagenicity assay of (E)-piplartine by the Ames test Andreia de A. Morandim-Giannetti1,2*, Fernando Cotinguiba1, Luis O. Regasini1, Mariana C. Frigieri3, Eliana A. Varanda3, Aline Coqueiro1, Massuo J. Kato4, Vanderlan S. Bolzani1 and Maysa Furlan1 1Departamento de Química Orgânica, Instituto de Química, Universidade Estadual de São Paulo, Rua Francisco Degni, s/n, 14800-900 Araraquara-SP, Brazil. 2Departamento de Engenharia Química, Centro Universitário da FEI, Av. Humberto de Alencar Castelo Branco, 3972, 09850-901 São Bernardo do Campo-SP, Brazil. 3Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Rodovia Araraquara/Jaú Km 1, 14801-902 Araraquara-SP, Brazil. 4Departamento de Química Fundamental, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, 05508-000, São Paulo-SP, Brazil. Accepted 25 March, 2011 Phytochemical studies carried out with Piperaceae species have shown great diversity of secondary metabolites among which are several displayed considerable biological activities. The species Piper tuberculatum has been intensively investigated and a series of amides have been described. For instance, (E)-piplartine showed significant cytotoxic activity against tumor cell lines, especially human leukemia cell lines; antifungal activity against Cladosporium species; trypanocidal activity and others. Considering the popular use of P. tuberculatum and the lack of pharmacological studies regarding this plant species, the mutagenic and antimutagenic effect of (E)-piplartine was evaluated by the Ames test, using the strains TA97a, TA98, TA100 and TA102 of Salmonella typhimurium. No mutagenic activity was observed for this compound. Key words: Piperaceae, Piper tuberculatum, (Z)-piplartine, mutagenic activated, Ames test. INTRODUCTION Studies with plants from the Piperaceae have shown a great diversity of secondary metabolites such as pyrones, lignans, neolignans, terpenes, propenylphenols, chal- cones, flavones, benzopyranes, chromenes, lactones and amides, with biological activities (Morandim et al., 2010; Morandim-Giannetti et al., 2010; Regasini et al., 2009; Bezerra et al., 2008; Batista-Junior et al., 2008; Lopes et al., 2008; Parmar et al., 1998; Felipe et al., 2007; Parka et al., 2007; Navickiene et al., 2006). The Piper species are widely distributed in the tropical *Corresponding author. E-mail: andreia.morandim@gmail.com. Tel: +55-011-43532900. Fax: +55-01141095994. Abbreviations: TLC, Thin layer chromatography; 5-FU, 5- fluorouracil. and subtropical regions of the world and includes more than 1000 species, is often used as food flavouring agents, psychotropic, antimicrobial, antioxidant and in traditional medicines for treating many diseases including gynaecological maladies, vaginitis, intestinal disorders, psychotropic, antimicrobial, antioxidant, cytotoxic effects, asthma, bronchitis, fever, hemorrhoidal afflictions, gastro- intestinal diseases and rheumatism (Parmar et al., 1998; Felipe et al., 2007; Parka et al., 2007; Navickiene et al., 2006). Some preparations obtained from these plants have shown anti-inflammatory, insecticidal, anti-hypertensive, antidiabetics, immunomodulatory and antimutagenic effects, potential benzo(a)pyrene-induced DNA-damage, and neurotoxic properties. They also generate oxidative stress by changing thiol cellular status, and may be used as pest control agents (Parmar et al., 1998; Cotinguiba et al., 2009; Lago et al., 2004; Duarte et al., 2004; Silva et al., 2002; Navickiene et al., 2000; Varanda, 2006). The amide alkaloids, known as alkamides, are typical metabolites of this family, and are classified as isobutyl, pyrrolydine, pyridonil and piperidines with various biolo- gical effects. Indeed, the pharmacology of the these alkamides revealed interesting compounds with hormone- modulating, anticancer, anti-hypertensive, antioxidant, anti-lipidemic, enzyme-inhibiting, anxiolytic and antide- pressant, as well as anti-inflammatory effects. For example, piperine and piperdardine, isolated of Piper tuberculatum Jacq. which showed an important hypotensive activity in rats (Cotinguiba et al., 2009). (E)-Piplartine is an amide alkaloid component of Piper species. This secondary metabolite has shown significant cytotoxic activity against tumor cell lines, especially human leukemia cell lines, such as HL-60, K562, Jurkat and Molt-4, as well antifungal, antileishmanial, antitry- panosomal activities, anti-platelet aggregation, anxiolytic, antidepressant properties, genotoxic potential and the induction apoptosis in cultured mammalian cells, employing a permanent cell line derived from Chinese hamster lung fibroblasts (V79 cell line), as well as its mutagenic and recombinogenic potential in the simple eukaryote, Saccharomyces cerevisiae (Bezerra et al., 2008; Cotinguiba et al., 2009; Bernstein et al., 1982). The objective of this study was to investigate the effect of piplartine and piperine in combination with the chemotherapeutic agent 5-fluorouracil (5-FU) using both in vitro and in vivo experimental models. Hematological, biochemical, histopathological and morphological analy- ses of treated animals were performed to assess the toxicological aspects related to different treatments (Bezerra et al., 2008; Cotinguiba et al., 2009; Varanda, 2006). In spite of the proven pharmacological properties for several medicinal plants, several analyses should be made including the genotoxic activity, since their constituents can cause harmful changes in the DNA. The risks associated with genotoxic activity are significant when such alternative treatments are applied without criteria, without due attention to correct botanical identification, and not considering the correct part of the plant to be used or even the method of preparation and administration (Bezerra et al., 2008). Therefore, the present study has been conducted in order to evaluate the mutagenic effect (genotoxic activity) of (E)-piplartine by the Ames test, using TA97a, TA98, TA100 and TA102 strains of Salmonella typhimurium microsome assay. MATERIALS AND METHODS Plant material Specimen of P. tuberculatum Jacq. (Piperaceae) was obtained from greenhouse cultivation at the Institute of Chemistry, UNESP, Araraquara-SP, Brazil. Plant material was identified by Dr. Morandim-Giannetti et al. 5399 Guillermo E. D. Paredes (Universidad Pedro Ruiz Gallo, Lambayeque, Peru). The voucher specimen (Kato-163) was deposited at the Herbarium of the Institute of Bioscience, USP, São Paulo-SP, Brazil. Extraction and isolation of (E)-piplartitne The shade-dried and powdered leaves (147.0 g) of P. tuberculatum were extracted with CH2Cl2:MeOH (2:1) (3 x 800 ml), for two weeks at room temperature. The crude extract (3.6 g) were fractionated by repeated column chromatographic on silica gel followed by preparative thin layer chromatography (TLC), resulting in the isolation of (E)-piplartine (45.0 mg), as previously described (Batista-Junior et al., 2008; Lopes et al., 2008; Cotinguiba et al., 2009). Salmonella mutagenicity assay Mutagenicity was tested by the Salmonella/microsome assay, based on the preincubation method using the S. typhimurium test strains TA97a, TA98, TA100 and TA102, kindly provided by Dr. B.N. Ames (University of California, Berkeley, CA), with and without metabolization. The test strains obtained from frozen cultures were grown overnight for 12 to 14 h in Oxoid Nutrient Broth No. 2 (London, UK). (E)-Piplartine (100, 150, 200, 250 and 300 µg per plate for strains TA100, TA98, TA97a and TA102) dissolved in dimethyl sulfoxide were added to 100 ml of bacterial culture and 0.5 ml phosphate buffer (pH 7.4) or 0.5 ml of S9 mixture and incubated for 20 to 30 min at 37°C. After the incubation, 2 ml of top agar was added, mixed and then poured onto a plate having a minimum amount of agar. The plates were incubated at 37°C for 48 h, and His+ revertant colonies were enumerated with an automatic counter (ProtoCOL, Synbiosis, Cambridge, UK). All experiments were analyzed in triplicate. The concentrations were selected on the basis of a preliminary toxicity test. In all subsequent assays, the upper limit of the dose range tested was either the highest nontoxic dose or the lowest toxic dose determined in this preliminary assay. Toxicity was apparent either as a reduction in the number of His� revert ants or as an alteration in the autotrophic background (background lawn). Statistical analysis was performed with the Salanal computer program. The mutagenic index was also calculated for each dose, as the average number of revertants per plate divided by the average number of revertants per plate in the negative (solvent) control. A sample was considered positive when the mutagenic index was ≥2. The standard mutagens used as positive controls in each experiment were 2-aminoanthracene (1.25 µg per plate) and sodium azide (1.25 µg per plate) for TA100, 4 nitro-ophenylenediamine (10 µg per plate) and 2-aminoanthracene (1.25 µg per plate) for TA98 and TA97a, and mitomycin C (0.25 µg per plate) and 2- aminofluorene (5 µg per plate) for TA102. Dimethyl sulfoxide served as the negative (solvent) control (Bernstein et al., 1982; Lima et al., 2008). The mutagenicity ratio (MR) was calculated for each concentration. The final data obtained from the test were analyzed using the statistical program Salanal (Salmonella Assay Analysis) version 1.0 of the Research Triangle Institute, RTP, NC, USA. This program allows assessment of the dose-response effect by calculating the analysis of variance (ANOVA) between the mea- surements of the number of revertants tested at different concentrations and the negative control, followed by linear regression. This program also provides the slope of the linear part of the concentration-response curve, which is the number of revertants induced per unit of measurement of the sample. 5400 Afr. J. Biotechnol. Table 1. Mutagenic activity of (E)-piplartine from P. tuberculatum strains in the presence (+S9) and absent (-S9) of metabolic activation. Treatment (mg per plate) Number of revertants per plate in (E)-piplartine of P. tuberculatum TA98 TA97a TA100 TA102 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 0.00a 22±7 29±7 185±18 200±33 139±1 127±8 371±12 291±18 100.00 22±1(1.0) 37±3(1.3) 163±1(0.9) 244±8(1.2) 124±1(0.9) 141±11(1.1) 279±22(0.8) 289±29(1.0) 150.00 19±4(0.9) 22±1 (0.7) 150±6(0.8) 256±0(1.3) 121±5(0.9) 109±4(0.9) 302±32(0.8) 249±25(0.9) 200.00 21±2(0.9) 27±4(0.9) 138±31(0.7) 252±33(1.3) 115±6 (0.8) 152±1(1.2)* 337±49(0.9) 250±16(0.9) 250.00 21±2(0.9) 27±1(0.9) 124±25(0.7) 244±13(1.2) 137±2(1.0) 134±18(1.1) 323±55(0.9) 272±36(0.9) 300.00 19±6(0.9) 32±2(1.1) 132±3(0.7) 278±4(1.4)* 107±9(0.8) 143±1(1.1) 290±0(0.8) 246±53(0.8) C+b 2782±20 1014±309 1983±72 1206±14 1697±11 1180±48 2328±73 336±10 Data are mean ± SD values (mutagenic index). The negative control (0.0) was dimethyl sulfoxide (100.0 µL). In the presence of metabolic activation, the addition (+S9) was 2-aminoanthracene (1.25 µg per plate) for strains TA98, TA100 and TA97a and 2-aminofluorene (5.0 µg per plate) for strain TA102. In the absence of metabolic activation, the addition (-S9) was 4-nitro-o- phenylenediamine (10.0 µg per plate) for TA98 and TA97a, sodium azide (1.25 µg per plate) for TA100, and mitomycin C (0.25 µg per plate) for TA102. Statistically different values by ANOVA are indicated: *P < 0.05, **P < 0.01. RESULTS The Ames assay is commonly used to detect mutagenic and antimutagens activities and is a widely accepted method for identifying various chemicals and drugs that can cause gene muta- tions. It has a high predictive value for in vivo carcinogenicity and the most common test strains are TA97a, TA98, TA100 and TA102. Hence, the mutagenicity of (E)-piplartine was investigated by Ames mutagenicity assay in the most sensitive Salmonella test strains currently available (TA100, TA98, TA97a and TA102). This test was performed in both the absence and the presence of a rat liver metabolizing system (S9 mix), providing a very sensitive study of potentially mutagenic pathways for the metabolism of these materials. Increasing doses of (E)-piplartine were tested and compared with the results of solvent control. Analysis of mutagenic activities in (Z)-piplartine from P. tuberculatum showed no positive muta- genicity and the mutagenic index did not exceed 2 when compared with the standards and the solvent (Table 1). DISCUSSION There is a relationship between mutagenesis and carcinogenesis, in that, both show abrupt changes in a single cell, permanent and inherited by daughter cells. Because of this relationship, the mutagenicity evaluation for new drugs and also for medicinal plants should be highly recommended for detecting potential genotoxic compounds. The amide (E)-piplartine from P. tuberculatum presented a decrease in the amount of revertants in strains TA100, TA98, TA97a and TA102. The highest mutagenicity ratios for TA98 at a concen- tration of 100 µg/plate were 1.3 in the presence, and 1.0 in the absence of metabolic activation, respectively. For TA97, the highest detected ratio was 1.4 at 300 µg/plate and 0.9 at 100 µg/plate, either in the presence or absence of metabolic activation. Strains TA100 and TA102 showed maximum ratios in the presence of metabolic activation of 1.2 at a concentration of 200 mg/plate and 1.0 at a concentration of 100 mg/plate, respectively. In the absence of metabolic activation, maximum ratios at 250 mg/plate were 1.0 and 0.9, respectively. An increase in the number of revertant colonies would yield positive results. In the present case, no increase was observed and mutagenic index values were below the limit (≥2). These results indicated efficiency for protecting bacterial genetic material againt different types of damage caused by several mutagenic agents. The present data in conjunction with the absence of mutagenicity of (E)-piplartine indicate promising use of this species against tumor, fungi and Trypanossoma cruzi and showed that (E)-piplartine is a potential natural product for further development of new drugs. ACKNOWLEDGEMENTS The authors are grateful to FAPESP and CNPq for financial support. The pharmacological study was supported by Brazilian grants obtained from CNPq and FAPESP. REFERENCES Batista-Junior JMB, Lopes AA, Ambrósio DL, Regasini LO, Kato MJ, Bolzani VS, Cicarelli RM, Furlan M (2008). Natural chromenes and chromene derivatives as potential anti-trypanosomal agents. Biol. Pharm. Bull. 31: 538–540. Bernstein L, Kaldor J, McCann J, Pike MC (1982). An empirical approach to the statistical analysis of mutagenesis data from the Salmonella test. Mutat Res. 97: 267-281. Bezerra DP, Moura DJ, Rosa, RM, Vasconcellos MC, Silva ACR, Moraes MO, Silveira ER, Lima MAS, Henriques JPH, Costa-Lotufo LV, Saffib J (2008). Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells. Mutat. Res. 652: 164-174. Cotinguiba F, Regasini LO, Bolzani VS, Debonsi HM, Passerini GD, Cicarelli RMB, Kato MJ, Furlan M (2009). Piperamides and their derivatives as potential anti-trypanosomal agents. Med. Chem. Res.18: 703-711. Duarte CM, Verlia H, Araújo-Júnior JX, Medeiros IA, Barreiro EJ, Fraga CAM (2004). New optimized piperamide analogues with potent in vivo hypotensive properties. Eur. J. Med. Res.23: 363–369 Felipe FCB, Sousa Filho JT, Souza LEO, Silveira JA, Uchoa DEA., Silveira ER, Pessoa ODL, Viana GSB (2007). Piplartine, an amide alkaloid from Piper tuberculatum, presents anxiolytic and antidepressant effects in mice. Phytomedicine, 14: 605-612 Lago JHG, Ramos CS, Casanova DCC, Morandim AA, Bergamo DCB, Cavalheiro AJ, Bolzani VS, Furlan M, Guimarães EF, Young MCM, Kato MJ (2004). Benzoic acid derivatives from Piper species and their fungitoxic activity against Cladosporium cladosporioides and C. sphaerospermum. J. Nat. Prod. 67: 1783-1788. Lima ZP, Santos RC, Torres TU, Sannomiya M, Rodrigues CM, Santos LC, Pellizzon CH, Rocha LRM, Vilegas W, Brito ARMS, Cardoso CRP, Varanda EA, Moraes HP, Bauab TM, Carli C, Carlos IZ , Hiruma-Lima CA (2008). Byrsonima fagifolia: An integrative study to validate the gastroprotective, healing, antidiarrheal, antimicrobial and mutagenic action. J. Ethnopharmacol. 120: 149-160 Morandim-Giannetti et al. 5401 Lopes AA, López SN, Regasini LO, Batista-Júnior JM, Ambrósio DL, Kato MJ, Bolzani VS, Cicarelli RMB, Furlan M (2008). In vitro activity of isolated compounds from Piper crassinervium against Trypanosomacruzi. Nat. Prod. Res. 22: 1040-1046. Morandim AA, Pin AR, Pietro, NAS, Alécio AC, Kato MJ, Young CM, Oliveira JE, Furlan M (2010). Composition and screening of antifungal activity against Cladosporiumsphaerospermum and Cladosporiumcladosporioides of essential oils of leaves and fruits of Piper species.Afr. J. Biotechnol.: 6135-6139 Morandim-Giannetti AA, Pin AR, Pietro, NAS, Oliveira HC, Mendes- Giannini MJS, Alécio AC, Kato MJ, Oliveira JE, Furlan M (2010). Composition and antifungal activity against Candida albicans, Candida parapsilosis, Candida krusei and Cryptococcus neoformans of essential oils from leaves of Piper and Peperomia species. J. Med. Plants Research.: 1810-1814. Navickiene HMD, Alécio AC, Kato MJ, Bolzani VS, Young MCM, Cavalheiro AJ, Furlan M (2000). Antifungal amides from Piper hispidum and Piper tuberculatum. Phytochemistry, 55: 621-626. Navickiene HMD, Morandim AA, Alécio AC, Regasini LO, Bergamo DC, Telascrea M, Cavalheiro AJ, Lopes MN, Bolzani VS, Marques MO, Young MCM, Kato MJ (2006). Composition and antifungal activity of essential oil from Piper aduncum, Piper arboreum and Piper tuberculatum. Quim. Nova, 29: 467-470. Parka BS, Sonb DJ, Parkc YH, Kimd TW, Lee SE (2007). Antiplatelet effects of acidamines isolated from the fruits of Piper longum L. Phytomedicine, 14: 853-855. Parmar VS, Jain SC, Bisht KS, Jain R, Gupta S, Talwar S, Rajwanshi VK, Kumar R, Azim A, Malhotra S, Kumar N, Jain R, Sharma NK, Jha A, Tyagi OD, Lawrie SJ, Errington W, Howarth OW, Olsen CE, Singh SK, Wengel J (1998). Polyphenols and alkaloids from Piper species. Phytochemistry, 49: 1069-1078. Regasini LO, Cotinguiba F, Morandim AA, Kato MJ, Scorzoni L, Mendes-Giannini MJ, Bolzani VS, Furlan M (2009). Antimicrobial activity of Piper arboreum and Piper tuberculatum (Piperaceae) against opportunistic yeasts. Afr. J. Biotechnol. pp. 2866-2870 Silva RV, Navickiene HMD, Kato MJ, Bolzani VS, Méda CI, Young MCM, Furlan M (2002). Antifungal amides from Piper arboreum and Piper tuberculatum. Phytochemistry, 59: 521-527. Varanda EA (2006). Atividade mutagênica de plantas medicinais. Revista de Ciências Farmacêuticas Básica e Aplicada. 27: 1-7.