versión On-line ISSN 1981-528X
Rev. bras. farmacogn. v.18 n.1 João Pessoa ene./mar. 2008
Atividade imunomoduladora de extratos de Cordia superba Cham. and Cordia rufescens A. DC. (Boraginaceae), espécies de plantas nativas do semi-árido brasileiro
José Fernando Oliveira CostaI; Juceni P. L. DavidII; Jorge M. DavidIII; Ana M. GiuliettiIV; Luciano P. QueirozIV; Ricardo R. SantosI; Milena Botelho P. SoaresI, *
ILaboratório de Engenharia Tecidual e Imunofarmacologia, Centro de Pesquisas Gonçalo Moniz, FIOCRUZ/BA, Rua Waldemar Falcão, 121, 40296-750 Salvador-BA, Brazil
IIDepartamento do Medicamento, Faculdade de Farmácia, Universidade Federal da Bahia, 40170-280 Salvador-BA, Brazil
IIIDepartamento de Química Orgânica, Instituto de Química, Universidade Federal da Bahia, 40170-280 Salvador-BA, Brazi
IVDepartamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, 44031-060 Feira de Santana-BA, Brazil
The family Boraginaceae is widely distributed in Brazil and in the Northeastern region some species are popularly used to treat symptoms of rheumatism, painful menstruation and dyspepsia. In this work we studied Cordia superba Cham. and C. rufescens A. DC., native from Brazilian Semi-arid region, in order to investigate their immunomodulatory activity. Six extracts were prepared from aerial parts of C. superba and C. rufescens. The cytotoxicity was evaluated using splenocytes from BALB/c mice. The immunomodulatory activity was determined by in vitro assays using activated mouse macrophages and lymphocytes. Peritoneal macrophages obtained from BALB/c mice were stimulated with IFN-g and LPS in the presence/absence of the samples. The NO production was measured indirectly through Griess method. Three samples inhibited the production of nitric oxide in values near 50% at a concentration of 100 µg/mL. To evaluate the effects of the extracts on lymphocytes, splenocytes from BALB/c mice were incubated with the samples and concanavalin A. Proliferation inhibition was determined by analysis of 3H-thymidine uptake. Samples from the two species had a strong inhibitory activity on lymphocyte proliferation and IL-2 production. Two chloroform extracts prepared from aerial parts of C. rufescens had the lowest IC50 values (7.6 and 11.0 µg/mL).
Keywords: Cordia, Boraginaceae, immunomodulatory activity.
A família Boraginaceae é amplamente distribuída no Brasil e na região nordeste algumas espécies são usadas popularmente no tratamento de reumatismo, dores menstruais e dispepsias. Neste trabalho foram estudadas as espécies Cordia superba Cham. and C. rufescens A. DC., nativas da região semi-árida brasileira, objetivando investigar a atividade imunomoduladora. Seis extratos foram preparados a partir de partes aéreas das espécies. A citotoxicidade foi avaliada usando culturas de esplenócitos de camundongos BALB/c. A atividade imunomoduladora foi determinada por ensaios in vitro usando macrófagos e linfócitos murinos ativados. Macrófagos peritoneais obtidos de camundongos BALB/c foram estimulados com IFN-g and LPS na presença/ausência das amostras. A produção de NO foi medida indiretamente através do método de Griess. Três amostras inibiram a produção de NO em valores próximos a 50% (100 µg/mL). Os efeitos das amostras sobre os linfócitos foram avaliados cultivando esplenócitos de camundongos BALB/c em presença destas amostras e de concanavalina A. A proliferação foi determinada pela análise da incorporação de 3H-tritiada. Amostras de duas espécies apresentaram uma forte atividade inibidora sobre a proliferação de linfócitos e sobre a produção de IL-2. Dois extratos clorofórmicos (partes aéreas de C. rufescens) tiveram os menores valores de IC50 (7,6 and 11,0 µg/mL).
Unitermos: Cordia, Boraginaceae, atividade imunomoduladora.
The family Boraginaceae comprises about 2740 species and 148 genera distributed in temperate and tropical zones of Europe, Asia, Africa, Australia, and Americas (Langstrom and Chase, 2002). Cordia, with more than 200 species, is one of the largest genera (Gottschling et al., 2005). It was recorded 46 species of Boraginaceae in the Brazilian semi-arid, 26 belonging to Cordia (Stapf, 2006).
Species of Cordia are largely employed in folk medicine worldwide. In Northeastern Brazil these plants are used to treat symptoms of rheumatism, painful menstruation and dyspepsia (Silva et al., 2004; Agra et al., 2007; Biavatti et al., 2007). Cordia rufescens is a shrub popularly known in the same region as "ramela-de-velho" and along with other species from genus Cordia is used in popular medicine as abortive, anti-inflammatory and to treat dysmenorrhea (Silva et al., 2004).
Immunopathologies such as autoimmune and allergic processes are diseases with high incidence and to which the currently available drugs, although efficient in most cases, cause undesirable side effects, resulting in complications to patients, especially after long term use. The search for new bioactive molecules in plants is of great interest, considering the diversity of chemical entities produced by them (Gottlieb et al., 1996) and the need of new medicines for more effective treatment of pathologies with lower toxic effects. In this report we evaluated the immunomodulatory activity of C. rufescens and C. superba extracts on inhibition of lymphoproliferation and nitric oxide production.
MATERIAL AND METHODS
Preparation of extracts
Plant specimens were collected in semi-arid areas of the Bahia State, Brazil (Table 1) in authorized areas by IBAMA (Brazilian Institute for the Environment and Natural Resources), dried at 40 ºC and received botanic identity. Vouchers were prepared and stored at the Universidade Estadual de Feira de Santana Herbarium (HUEFS). Material from one individual of C. superba and from two individuals of C. rufescens were studied. Dried stem and leaves of C. superba (200 and 90 g, respectively) and C. rufescens (183 and 235 g of stems; 100 and 57 g of leaves) were individually extracted with MeOH. Thus, 1.32 g (0.66%) of MeOH extract was obtained from 200 g of stem and 10.5 g (11.67%) from 90 g of leaves of C. superba. Also, 891 mg (0.49%) and 8.5 g (8.5%) of MeOH extract were obtained from 183 g and 100 g of stems and 6.1 g (2.6%) and 6.0 g (10.53%) from 235 g and 57 g of leaves of C. rufescens specimens. Separately, the crude extracts obtained were partitioned with CHCl3/MeOH:H2O (6:4). These procedures allowed the production of 538.2 mg (40.8%) and 7.4 g (70.5 %) of CHCl3 extract from C. superba (stem and leaves) and 83.2 mg (9.3%) and 1.8 g (21.1 %) of one sample and 108.7 mg (1.8 %) and 1.2 g (20 %) from the other sample of C. rufescens. The hydroalcoholic fraction, after the evaporation of MeOH under vacuum, gave rise to the aqueous phases and was further partitioned with EtOAc:H2O. In the latter procedure 121.4 mg (9.2%) and 838.5 mg (8%) of C. superba were obtained (stem and leaves, respectively) and 215.3 mg (24.2%) and 2.2 g (25.9%) of C. rufescens and 82 mg (1.34%) and 138.0 mg (2.3%)from the other sample of the same specie. The final extracts were dissolved using dimethyl sulfoxide (Sigma, St. Louis, MO) adjusting a final concentration for 10 mg/mL. The samples were sterilized with 60,000 rad of gamma radiation (CIS International IBL437C, France) for use in biological assays.
To determine the cytotoxicity of the samples, splenocytes from BALB/c mice (6 x 105 cells/well) were cultured in 96 well plate in Dulbeccos Modified Eagles Medium (DMEM, Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal calf serum (Cultilab, Campinas, SP, Brazil) and 50 µg/mL of gentamycin (Novafarma, Anápolis, GO, Brazil). Each extract was evaluated in five concentrations starting at 100 µg/mL, in triplicates. Cells were incubated in the presence of 1 µCi/well [methyl-3H] thymidine (Amersham, Little Chalfont, England) during 24 h at 37 ºC and 5% CO2. After this period, cultures were harvested using a cell harvester (Filtermate 196, Packard, Groningen, Netherlands) to determine the 3H-thymidine incorporation using a beta radiation counter (b-matrix 9600, Packard, Groningen, Netherlands). The viability of the cells was determined by the thymidine incorporation and the cytotoxicity was calculated in relation to the 3H-thymidine incorporation of untreated cultures.
Nitric oxide assay
Peritoneal exudate cells were obtained by washing the peritoneal cavity of mice with cold Hanks balanced salt solution (HBSS; Life Technologies, GIBCO-BRL, Gaithersburg, MD) five days after injection of 3% thioglycollate in saline (1.5 mL per mouse). Peritoneal cells were washed twice with HBSS and re-suspended in RPMI medium (GIBCO-BRL) supplemented with 10% fetal calf serum (Cultilab, Campinas, SP, Brazil), L-glutamine (2 mM), RPMI 1640 vitamins solution (1%) (Sigma), sodium pyruvate (1 mM), HEPES (10 mM), 2-mercaptoethanol (50 µM), and gentamycin (50 µg/mL) (Sigma, St. Louis, MO). Cells were plated (2 x 105 cells/well) in 96 well plates. After one hour of incubation at 37 ºC, non-adherent cells were removed by washing with complete RPMI. Cultures were then stimulated with 1 µg/mL lipopolysaccaride (LPS from Escherichia coli serotype 0111:B4, Sigma) in combination with 5 ng/mL interferon-g (IFN-g; PharMingem, S. Diego, CA) and treated with various concentrations of extracts, as described in figure legends, in a final volume of 200 µL. Cell-free supernatants were collected at 24 h of culture for determination of nitrite concentration using the Griess method, as described previously (Ding et al., 1988).
BALB/c splenocytes suspension were prepared in complete RPMI medium and cultured in 96 well plates at 6 x 105 cells/well in 200 µL, in triplicates, in the presence of concanavalin A (Con A; 2 µg/mL), alone or various concentrations of plant extracts, as described in figure legends. After 48 h, plates were pulsed with 1 µCi of [methyl-3H] thymidine for 12 h, and proliferation was assessed by measurement of 3H-thymidine uptake. The percentage of inhibition of lymphocyte proliferation by the extracts was determined in relation to untreated controls.
Myxed lymphocyte reaction (MRL)
BALB/c (H-2d) mice were weekly immunized with C57BL/6 (H-2b) splenocytes intraperitoneally (107 cells/mouse). After 3 weeks of immunization, mice were sacrificed for spleen cell preparation in DMEM medium supplemented as described above. Spleen cells were cultured in 96 well plates at 5 x 105 cells/well in the absence or in the presence of irradiated C57BL/6 splenocytes at 106cells/well (dose of 3000 rad) and extracts, in triplicates. After 72 h of culture, plates were pulsed with 3H-thymidine for 12 h for proliferation assessment, as described above.
EC50 and LC50 calculations and statistical analyses
The lethal concentration for 50% of cells (LC50), the effective concentrations for 50% (EC50) of lymphoproliferation and MLR were calculated based in a nonlinear regression (curve fit). The statistical analyses were made by one-way ANOVA with Dunnetts post test using Graph Pad Prism version 4.0 (Graph Pad Software, San Diego, CA, USA). Differences were considered significant when p values were < 0.05.
Samples and cytotoxicity values
Two species of the genus Cordia were analyzed, C. superba and C. rufescens. The extracts were obtained using chloroform and ethyl acetate as solvents. Six different extracts were analyzed in different concentrations. Stem and aerial parts of the plants were used and, from C. rufescens, two collects were done in different localities, generating two extracts of the same part and with the same solvent (Table 1). Samples were assayed in five different concentrations, starting at 100 µg/mL. As shown in Table 2, the lethal concentration for 50% (LC50) could be determined only for two samples, CR1 and CR3 obtained from C. rufescens (34.2 and 32.8 µg/mL, respectively) The other four samples did not present cytotoxicity to allow the calculation the LC50. The cytotoxicity of the samples is shown in Figure 1a.
Immunomodulatory activity assays
First we tested the activity of the extracts on cultures of activated macrophages. The samples tested did not have a high inhibitory activity on the nitric oxide production. The maximum inhibition was achieved with CS1, CR1 and CR3, which had, respectively, 54.8, 52.9 and 44.9% when assayed at 100 µg/mL (Figure 1b; p < 0.05).
The extracts had strong inhibitory activity in lymphocyte cultures, CR1 and CR3 have the lowest EC50 values for proliferation inhibition of lymphocytes stimulated with concanavalin A (Table 2). These samples had high inhibitory activity even when assayed at concentrations below 100 µg/mL. CR2 and CR4, samples extracted from the same specie (C. rufescens) but using different parts of the specimen, had higher values of EC50. CS1 and CS2, extracts from C. superba, presented values of inhibition up to 70% (Figure 1c). All the samples inhibited IL-2 production higher than 50% when assayed at 100 µg/mL, being CR2 and CR4 the most potent samples (80.7 and 84.8%, respectively; p < 0.05).
Similar results were obtained in cell cultures of mixed lymphocyte reaction (MLR). CR1 and CR3 had also a low EC50 values (6.0 and 7.9 µg/mL) in this assay. In addition, CR4 had the third lowest EC50 (8.8 µg/mL) in MLR assay (Table 2). All samples inhibited the proliferation in MLR in percentuals above 90%, when assayed at 100 µg/mL (Figure 1d).
A great variety of uses in ethnopharmacology for species of Cordia sp. has been reported (Ficarra et al., 1995; Reddy et al., 2002; Agra et al., 2007). C. globosa and C. verbenacea have been used in the folk medicine for treatment of rheumatism, painful menstruation and gastric ulcer (Reddy et al., 2002; Al-Awadi, 2001). Some pharmacologically active metabolites with analgesic, antiinflammatory, antiarthritic and larvicidal activities were already purified from Cordia sp., emphasizing an immunomodulatory activity of this genus (Sertié et al., 1991; Silva et al., 2003; Arrebola et al., 2004; Santiago et al., 2005). The species Cordia superba Cham. and Cordia rufescens A. DC (Boraginaceae), endemic in the Brazilian semi-arid, were investigated for the first time regarding their biological activities.
In general the samples had low cytotoxicity. Only two samples had their LC50 calculated (Table 2). The others could not be determined because the results obtained did not permit the calculation of the lethal doses, indicating that LC50 values for these samples are superior to 100 µg/mL. It is interesting to note that LC50 values of CR1 and CR3 extracts are similar (Table 2). The samples were extracted from the same specie, prepared with aerial parts of different specimens, collected in different localities (Sento Sé and Morro do Chapéu) and the same solvent was used (Table 1).
Three samples (CS1, CR1 and CR3) inhibited NO production next to 50% when assayed at 100 µg/mL. However, CR1 and CR3 had high cytotoxicity at 100 µg/mL, and thus only CS1 activity on NO production can be considered.
In addition to the effect on the NO production, the immunomodulatory potential for the samples was also measured in lymphoproliferation assay using concanavalin A as stimulus for the splenocytes or allogenic stimulation in mixed lymphocyte reaction. CR1 and CR3 had the highest values of lymphocyte proliferation inhibition (up to 100%), when assayed at 100 and 33 µg/mL in both assays (Figures 1c and 1d). Consequently, these samples had the lowest EC50 values for these parameters. These data are relevant if observed that LC50 for them were very superior to these values (Table 2), suggesting an inhibition of proliferation and not toxicity-induced cell death. These data reinforce again the hypothesis of production of bioactive metabolites soluble in chloroform by Cordia rufescens.
CS1, CS2 and CR2, in spite of having the most elevated EC50 values, had not a detectable LC50 or they are superior to 100 µg/mL (Table 2). Two samples had a high percentual of lymphocyte proliferation inhibition with low EC50, CR1 and CR3 (Table 2). IL-2, an important cytokine produced upon lymphocyte activation, was also inhibited. Corroborating the immunosuppressive potential of these two samples, both of them had higher values of LC50 when compared with EC50 values obtained. Thus, these results indicate a real effect on inhibition of proliferation and not due cell toxicity.
The results confirm the basis of the utilization of some Cordia species by traditional medicine in pathologies mediated by the immune system. Studies should be continued with the development of more specific assays and in vivo evaluation models for immune-mediated pathologies, followed by fractionation of extracts in order to identify the active compounds.
This work was supported by Millenium Institute of Semi-Arid (MCT), National Council on Scientific and Technological Development (CNPq), FAPESB and FIOCRUZ.
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Received 17 September 2007; Accepted 2 February 2008