2006 The Japan Mendel Society Cytologia 71(2): 101 106, 2006 Absence of Clastogenic Effects of the Extract from Medicinal Plant Rosmarinus officinalis L. on Wistar Rat Bone Marrow Cells Thamara Figueiredo Gaiani, José Carlos Tavares Carvalho, José Maurício Schneedorf Ferreira da Silva and Edson Luis Maistro* Faculdade de Farmácia, Universidade José do Rosário Vellano (UNIFENAS), Alfenas Campus, Alfenas, Minas Gerais, Brazil, 37130-000 Received November 30, 2005; accepted December 22, 2005 Summary The use of medicinal plants by the general population is an old and still widespread practice, which makes studies of their mutagenicity essential. Rosmarinus officinalis, long used in folk medicine, is used as an antispasmodic in renal colic and dysmenorrhoea, in relieving respiratory disorders, to stimulate growth of hair and has choleretic, hepatoprotective and antitumerogenic activity. The aim of this study was to evaluate the clastogenic potential of the Rosmarinus officinalis hidro-alcoholic extract in vivo on bone marrow cells of Wistar rats by evaluating the induction of chromosome aberrations and micronuclei induction on polychromatic erythrocytes. The extract was administered by gavage at doses of 6.43, 100 and 200 mg/kg body weight. Experimental and control animals were submitted to euthanasia 24 h after the treatment. R. officinalis extract did not induce statistically significant increases in the average numbers of micronucleus or chromosome aberrations in the test systems employed. Rosmarinus officinalis, Labiatae, Clastogenicity, Micronucleus test, Chromosome aber- Key words rations. The use of herbs as medicines has played an important role in nearly every culture on earth, including Asia, Africa, Europe and the Americas. Among the many reasons cited by the general public for use of herbal medicines is the belief that botanicals will provide some measure of benefit over and above traditional allopathic medical approaches (Wargovich et al. 2001). However, medicinal plants, and indeed plants in general, may synthesize toxic substances, which in nature act as a defense against infections, insects and herbivores, but which often affect the organisms that feed on them. Thus, an evaluation of their cytotoxic and mutagenic potential is necessary to ensure a relatively safe use of medicinal plants (Teixeira et al. 2003, Maistro et al. 2004, Espósito et al. 2005). Rosmarinus officinalis Linn., popularly known as rosemary or alecrim, is a common household plant grown in many parts of the world. It is used for flavouring food, a beverage drink, as well as in cosmetics; in folk medicine it is used as an antispasmodic in renal colic and dysmenorrhoea, in relieving respiratory disorders and to stimulate growth of hair. Extract of rosemary relaxes smooth muscles of trachea and intestine, and has choleretic, hepatoprotective, analgesic, antimicrobial and antitumerogenic activity (Newall 1996, Mangena and Muyima 1999, Al-Sereiti et al. 1999, Fahim et al. 1999, among others). Since R. officinalis has been long time used in folk medicinal without any mutagenic assessment the present study was carried out to investigate the clastogenic and cytotoxic potential of the R. officinalis hydroalcoholic extract in Wistar rat bone marrow cells in vivo by employing chromosomal aberrations and micronucleus test. * Corresponding author, e-mail: edson.maistro@unifenas.br
102 Thamara Figueiredo Gaiani et al. Cytologia 71(2) Materials and methods Plant material Plant material was obtained from Santos Flora Ltda business (Ervas Medicinais e Aromáticas) located in São Paulo town, state of São Paulo, Brazil (Catalog number ALER 04/02), the samples from Turkey, wich botanical identification as Rosmarinus officinalis Linn. A voucher specimen has been deposited in the Laboratório de Fitofármacos of the UNIFENAS, Alfenas, Minas Gerais, Brazil. After been dried out at room temperature, the leaves and stems were ground (approximately 5 kg) and macerated in 85% with hydroalcoholic solutions at room temperature for 15 d. The filtered hydroalcoholic extract was evaporated to drynesss, yielding about 50 g of the extract. Animals and assay procedures Experiments were carried out using six-week-old Wistar rats (Rattus norvegicus) weighing 90 110 g acquired from UNIFENAS animal house and kept in polyethylene boxes (n 6) in a climate-controlled environment (25 4 C, 55 5% humidity) with a 12 h light/dark cycle (07:00 h to 19:00 h) and fed Labina-Purina (Agribrands Purina do Brasil Ltda, Paulínia, São Paulo, Brazil) and water ad libitum. The rats were divided into 3 experimental and 2 control groups each containing 3 females (F 1 to F 3 ) and 3 males (M 1 to M 3 ). The Rosmarinus officinalis extract was administered in a single dose of 0.5 ml by gavage at concentrations of 6.43, 100 and 200 mg/kg body weight, chosen on the basis of the therapeutic dose of 6.43 mg/kg. The negative control group received distilled water by the same route as the experimental rats and the positive control group 30 mg of cyclophosphamide/kg body weight. Animals were injected intraperitoneally with 0.5 ml of 0.16% colchicine 90 min before euthanasia by carbon dioxide asphyxiation, which occurred 24 h after experimental treatment. Both femur bones were then excised and their bone marrow flushed into test tubes using a syringe. For the micronucleus (MN) assay, the bone marrow cells were prepared as recommended by Schimid (1976). The slides were coded, fixed with methanol and stained by Giemsa solution. Two thousand polychromatic erythrocytes (PCE) from each animal were scored for MN presence. Bone marrow preparations for the analysis of chromosome aberrations in metaphase cells were obtained by the technique of Ford and Hamerton (1956). One-hundred metaphases per animal (600 metaphases per group) were analyzed in order to determine the number of chromosomal aberrations in a blind test. Chromosomal aberrations were classified according to Savage (1975) as gaps, breaks, deletions, fragments, rings and dicentric chromosomes. Gaps were recorded but not included in the statistical analysis. The mitotic index was obtained by counting the number of mitotic cells in 1000 cells per animal. The UNIFENAS Animal Bioethical Committee approved the present study on 10th August 2004. The data were submitted to one-way analysis of variance (ANOVA) and the Tukey-Kramer multiple comparison test using the GraphPad Instat software version 3.01 (GraphPad Software, Inc., San Diego, USA). Results were considered statistically significant at p 0.05. Results and discussion Tables 1 and 2 summarize the results of the analysis of micronucleus and chromosome aberrations respectively, in bone marrow cells of Wistar rats following treatment with different concentrations of the R. officinalis extract and controls. Acute administration of R. officinalis extract result in a increase in the average number of polychromatic erythrocytes with micronuclei (MNPCE) but the data were no statistically significant (p 0.05) (Table 1). Comparisons between different dose groups showed no significant differences between MNPCE mean numbers (Tukey-Kramer test, p 0.05). The mitotic index values obtained from the analysis of 1000 cells/animal for a sample of 30
2006 The Clastogenic Potential of Rosmarinus officinalis Hydroalcoholic Extract 103 a) b) b f c) d) g r d Fig. 1. Some typical chromosome aberrations on bone marrow cells of Wistar rats after Cyclophosphamide treatment. a) Micronucleus on polychromatic erythrocyte (arrow); b) b chromatidic break, f fragment; c) g isochromatidic gap; d) d deletion, r ring. Table 1. Mean of polychromatic erythrocytes with micronuclei (MNPCE) observed in bone marrow cells of female (F) and male (M) Wistar rats treated with a Rosmarinus officinalis extract, and respective controls Treatments Dose mg/kg Number of MNPCE per animal MNPCE (mean SEM) F 1 F 2 F 3 M 1 M 2 M 3 Negative control (water) 0 1 2 0 1 1 0 0.83 0.31 R. officinalis extract 6.43 5 5 1 1 1 0 2.16 0.91 R. officinalis extract 100 9 0 5 8 0 8 5.00 1.67 R. officinalis extract 200 0 4 2 3 3 4 2.66 0.61 Positive control 30 12 15 10 10 11 16 12.33 1.05* (cyclophosphamide) Two thousand cells were analyzed per animal, for a total of 12000 cells per group. SEM standard error of the mean. * Significantly different from negative control (P 0.001). animals (n 6/group) ranged from 1.1 to 7.1% (means) and statistical analysis by the Tukey-Kramer test showed no significant differences (p 0.05) between the different treatments with R. officinalis extract, or between these treatments and their controls. These data indicate no cytotoxic effect of the R. officinalis extract at the doses tested (Table 2). The data obtained from 600 metaphases analyzed per treatment (100 metaphase cells/animal),
104 Thamara Figueiredo Gaiani et al. Cytologia 71(2) Table 2. Mitotic Index (MI) and distribution of the different types of chromosomal aberrations (CA) observed in female (F) and male (M) Wistar rat bone marrow cells treated with a Rosmarinus officinalis extract, and respective controls Chromosomal aberrations Treatments Sex MI (%) Breaks Gaps OA C IC C IC Total (CA) without gaps Negative control F 1 3.6 1 0 0 0 0 1 (water) F 2 3.3 0 0 0 0 1 r 1 F 3 1.7 0 0 1 0 0 0 M 4 2.9 0 0 0 1 0 0 M 5 1.1 0 0 0 0 0 0 M 6 2.8 0 0 0 0 1 f 1 mean SEM 2.56 0.39 0.50 0.22 R. officinalis extract F 1 1.6 0 0 1 0 0 0 (6.43 mg/kg) F 2 2.0 0 0 2 0 0 0 F 3 2.8 1 0 1 0 1 del 2 M 4 3.4 0 0 1 0 0 0 M 5 2.8 0 0 2 0 0 0 M 6 2.3 1 0 2 0 0 1 mean SEM 2.48 0.26 0.50 0.34 R. officinalis extract F 1 1.6 0 0 0 0 0 0 (100 mg/kg) F 2 3.2 0 0 1 1 0 0 F 3 1.5 0 0 1 0 0 0 M 4 4.5 0 0 0 0 0 0 M 5 6.8 2 0 1 0 0 2 M 6 3.4 1 0 0 0 1 f/1 r 3 mean SEM 3.50 0.81 0.83 0.54 R. officinalis extract F 1 6.2 0 0 3 1 1 r 1 (200 mg/kg) F 2 2.8 0 0 1 0 0 0 F 3 5.4 0 0 1 3 0 0 M 4 2.6 0 0 2 1 1 f/1 r 2 M 5 4.0 3 0 0 0 2 del 5 M 6 7.1 0 0 1 0 0 0 mean SEM 4.68 0.75 1.33 0.80 Positive control F 1 2.8 1 0 1 0 2 del/2 f 5 (cyclophosphamide) F 2 2.8 1 0 0 0 0 1 (30 mg/kg) F 3 6.2 4 0 1 3 4 del 8 M 4 4.7 2 0 0 2 1 del/1 r 4 M 5 2.1 2 0 0 2 1 del/1 r 4 M 6 1.4 0 0 1 1 2 del/2 r 4 mean SEM 3.33 0.73 4.33* 0.92 One hundred cells were analyzed per animal, for a total of 600 cells per treatment. C, Chromatid-type; IC, isochromatid-type; OA, other aberrations: del deletion; r ring; f fragments; SEM standard error of the mean. * Significantly different from negative control (P 0.001). also showed that there were no statistically significant differences between the mean number of chromosome aberrations of treated groups and of the negative control group. The most frequent types of aberrations of the treated groups were chromatid gaps, chromatid breaks, followed by of deletions, rings and fragments (Table 2). R. officinalis extract contains flavonoids, phenols, volatile oil, terpenoids (Collin and Charles 1987, Newall 1996), 2 abietane-type diterpenoid o-quinones called rosmaquinone A and rosmaquinone B (Mahmoud et al. 2005), among other less important compounds. Study of the embryotoxic effects of this mixed compounds obtained from aqueous extract of R. officinalis using Wistar rat pregnancy showed that the extract may present an anti-implantation effect without interfering
2006 The Clastogenic Potential of Rosmarinus officinalis Hydroalcoholic Extract 105 with the normal development of the concept after implantation (Lemonica et al. 1996). On the other hand, several other studies with rosemary extracts have shown potential positive health effects. Singletary and Nelshoppen (1991) observed that dietary supplementation with rosemary extract with its individual antioxidative constituents resulted in a significant decrease in mammary tumor incidence. Antioxidant vitamin mix consisting of ascorbic acid, alpha-tocopherol, lecithin and rosemary extract with carnosic acid and carnosol shown to exhibit strong antimutagenic effects in Ames tester strain TA102 (Minnunni et al. 1992). Since oxygen radicals are known to be involved in the multiprocess of carcinogenicity, the authors concluded that the antioxidants of this mix might exhibit anticarcinogenic properties. Carnosol and ursolic acid, constituents of the rosemary extract showed inhibition on tumor initiation and promotion in mouse skin (Huang et al. 1994). In the same way, Fahim et al. (1999) observed hepatoprotective and antimutagenic activities of the rosemary ethanolic extract and essential oil, respectively, and according to the authors, these effects are attributed to the presence of a relatively high percentage of phenolic compounds with high antioxidant activity. The clastogenic effect of Rosmarinus officinalis leaves and stems extract on the bone marrow of Wistar rats was studied for the first time in the present work. The results that the mixture of the compounds found in these extract did not induce a significant increase in the mean number of cells with micronuclei or chromosome aberrations and showed no cytotoxic effects when given at the doses of 6.43, 100 and 200 mg/kg body weight. These results regarding the cytotoxicity and clastogenicity of this plant extract provide valuable information about the safety of using them as therapeutic and chemopreventive agent. Acknowledgements This investigation was supported by UNIFENAS and FAPEMIG (Rede Mineira de Ensaios Toxicológicos e Farmacológicos de Produtos Terapêuticos, EDT 1879/02). References Al-Sereiti, M. R., Abu-Amer, K. M. and Sem, P. 1999. Pharmacology of rosemary (Rosmarinus officinalis Linn.) and its therapeutic potentials. Indian J. Exp. Biol. 37: 124 130. Collin, M. A. and Charles, H. P. 1987. Antimicrobial activity of carnosol and ursolic acid: two anti-oxidant constituents of Rosmarinus officinalis L. Food Microbiol. 4: 311 315. Espósito, A. V., Pereira, D. M. V., Rocha, L. M., Carvalho, J. C. T. and Maistro, E. L. 2005. Evaluation of the genotoxic potential of the Hypericum brasiliense (Guttiferae) extract in mammalian cell system in vivo. Genet. Mol. Biol. 28: 152 155. Fahim, F. A., Esmat, A. Y., Fadel, H. M. and Hassan, K. F. 1999. Allied studies on the effect of Rosmarinus officinalis L. on experimental hepatotoxicity and mutagenesis. Int. J. Food Sci. Nutr. 50: 413 427. Ford, C. E. and Hamerton, J. L. 1956. A colchicine, hypotonic citrate, squash sequence for mammalian chromosomes. Stain Technol. 31: 247 251. Huang, M. T., Ho, C. T., Wang, Z. Y., Ferraro, T., Lou, Y. R., Stauber, K., Ma, W., Georgiadis, C., Laskin, J. D. and Conney, A. H. 1994. Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid. Cancer Res. 54: 701 708. Lemonica, I. P., Damasceno, D. C. and Di-Stasi, L. C. 1996. Study of the embriotoxic effects of an extract of rosemary (Rosmarinus officinalis L.). Braz. J. Med. Biol. Res. 29: 223 227. Mahmoud, A. A., Al-Shihry, S. S. and Son, B. W. 2005. Diterpenoid quinones from Rosemary (Rosmarinus officinalis L.). Phytochem. 66: 1685 1690. Maistro, E. L., Carvalho, J. C. T. and Mantovani, M. S. 2004. Evaluation of the genotoxic potential of the Casearia sylvestris extract on HTC and V79 cells by the comet assay. Toxicology in Vitro 18: 337 342. Mangena, T. and Muyima, N. Y. 1999. Comparative evaluation of the antimicrobial activities of essential oils of Artemisia afra, Pteronia incana and Rosmarinus officinalis on selected bacteria and yest strains. Lett. Appl. Microbiol. 28: 291 296. Minnunni, M., Wolleb, U., Mueller, O., Pfeifer, A. and Aeschbacher, H. U. 1992. Natural antioxidants as inhibitors of oxy-
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