Abstract
Although the use of macrofungi in popular medicine is very common, especially in East Asia, the knowledge about their pharmacological properties is poorly investigated. The aim of this work was to evaluate the pharmacological potential of six species of macroscopic fungi: Phellinus rimosus (Berk.) Pilát (Hymenochaetaceae), Pycnoporus sanguineus (L.) Murrill (Poyporaceae), Hymenochaete rheicolor (Mont.) Lév. (Hymenochaetaceae), Hexagonia papyracea Berk. (Polyporaceae), Datronia caperata (Berk.) Ryvarden (Polyporaceae), and Lepiota sp. (Agaricaceae), collected in the state of Bahia, in north-eastern Brazil. Extracts of these species were obtained and tested to determine their cytotoxicity in normal mouse spleen cells. Immunomodulatory, antineoplasic and antiparasitic activities were investigated. Four macrofungi extracts (Phellinus rimosus, Hymenochaete rheicolor, Lepiota sp., and Datronia caperata) inhibited the lymphoproliferative response stimulated by concanavalin A in 65–96% of inhibition in mitogen-induced lymphoproliferation assay, three (Hymenochaete rheicolor, Pycnoporus sanguineus, and Lepiota sp.) inhibited 60–70% of production of nitric oxide by J774 activated by IFN-γ and LPS, and two (Phellinus rimosus and Pycnoporus sanguineus) had antimalarial activity against chloroquine-resistant Plasmodium falciparum (over 60%). We did not find inhibition greater than 60% to the growth of both Leishmania amazonensis and Trypanosoma cruzi. To our knowledge, this is the first description of immunomodulatory activity of Hymenochaete rheicolor (HR), Datronia caperata (DC) and Pycnoporus sanguineus (PS). These results indicate that macrofungi species from the Brazilian north-east have pharmacological activity and are thus a potential source of natural products with medicinal interest.
Introduction
The use of mushrooms in traditional oriental therapies has a well established history. Modern clinical practice in Japan, China, Korea and other East Asian countries continues to rely on mushroom-derived preparations (CitationZaidman et al., 2005). Several pharmacologically active compounds have been identified and isolated from Basidiomycota with a wide spectrum of biological activities, including immunomodulatory, antineoplasic, antibacterial, antiviral, and anti-inflammatory activities (CitationBobek et al., 1998; CitationGunde-Cimerman, 1998; CitationKino et al., 1989; CitationJang et al., 2004; CitationLee et al., 2005; CitationShon et al., 2003; CitationWasser, 2002; CitationZhuang & Mizuno, 1999; CitationZjawiony, 2004).
The state of Bahia is situated in north-eastern Brazil and has an area of 567,295.3 km2. The climate is very complex due to its diverse physiographic and rainfall patterns. Almost all of the Brazilian tropical biomes are represented in this state: Atlantic forest, mangroves, cerrado (woodland savannah) and caatinga (dry scrub forest), although more than 50% of its area is situated in the semi-arid region. These biomes display a rich fungal diversity with a significant lack of information on its pharmacological potential. The aim of this work was to assess the pharmacological potential of six species of macrofungi using in vitro assays. The investigation included native polypore and agaric mushrooms collected in Bahia, Brazil.
Materials and methods
Macrofungi
Six macrofungi species (Basidiomycota) were studied and their basidiomata were collected from private properties, with permission of each owner, in different localities in Bahia (). Specimens were identified following a descriptive study of external and internal morphology and using a key for neotropical polypores (CitationRyvarden, 2004). Specimen identifications were authenticated by A. Góes Neto, Departamento de Ciências Biológicas, Universidade Estadual de Feira de Santana, Bahia, Brazil. Voucher specimens were deposited at the Herbarium of Universidade Estadual de Feira de Santana (HUEFS).
Table 1. Macrofungi species collected in the State of Bahia, Brazil.
Preparation of extracts
Basidiomata were washed with tap water and sliced into small pieces, dried, and powdered. Powdered material of all species was extracted in a hydroalcoholic solution of 80% methanol. Macrofungi extracts were filtered and dried on a rotatory evaporator under reduced pressure. Masses of powdered macrofungi utilized, and the extract yields obtained, are shown in .
Cell lines and parasite cultures
Human chronic myeloid leukemia cell K562 and human Burkitt’s lymphoma cell Daudi were maintained in RPMI 1640 (Life Technologies, GIBCO-BRL, Gaithersburg, MD). Murine macrophage cell line J774 was maintained in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT). Both media were supplemented with 10% fetal bovine serum (FBS, Cultilab, Campinas, Brazil) and 40 μg/mL gentamycin (Novafarma, Anápolis, Brazil). Cell cultures were maintained at 37ºC and 5% CO2.
Plasmodium falciparum (strain W2, chloroquine-resistant, mefloquine-sensitive) was maintained in human A+ erythrocytes in RPMI 1640 (GIBCO-BRL) supplemented with 10% human plasma at 37ºC in a 5% CO2-air mixture.
Trypanosoma cruzi (epimastigote form of Y strain) and Leishmania amazonensis (promastigote form of MHOM/BR88/BA-125 Leila strain) were maintained at 25ºC in liver infusion tryptose medium (Difco, Detroit, MI) supplemented with 10% FBS, 1% hemin (Sigma, St. Louis, MO), 1% R9 medium (Hyclone) and 5% sterile human urine.
Animals
BALB/c mice (6-8 weeks old) were used as spleen donors for cytotoxicity and lymphoproliferation assays. Animals received water and food ad libitum. This work was approved by the Ethics Committee for Animal Use of the Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz.
Assessment of cytotoxicity
BALB/c spleen cells obtained from normal mice were used to determine non-toxic concentrations. Cells were plated at 5 × 106 cells/well to 96-well plates with or without macrofungi extracts and 1 μCi/well [3H]-thymidine (Amersham, Little Chalfont, UK). Saponin (Sigma) was used as a positive control. Plates were incubated at 37ºC and 5% CO2 for 24 h. Cells were harvested using a Filtermate 196 cell harvester and the radioactivity in dried filters was counted using a β-counter (Packard, Meriden, CT). Mean values of the triplicates expressed in counts per minute (CPM) were calculated and cytotoxicity was given as the percentage of control values. Concentrations used were 1, 10, and 100 μg/mL and extract concentrations with values smaller or equal to 30% were considered non-toxic (CitationLeite et al., 2006).
Mitogen-induced lymphoproliferation assay
BALB/c spleen cells obtained from normal mice were used in this assay. Cells were added at 5 × 106 cells/well to 96-well plates with or without macrofungi extracts, in non-toxic concentrations, and in the presence of concanavalin A (2 μg/mL, Sigma). Plates were incubated for 48 h at 37ºC and 5% CO2. One μCi of 3H-thymidine was added to each well, and plates were incubated for additional 18 h. Uptake of 3H-thymidine was determined as described above. Mean values of the triplicates expressed in CPM were calculated, and lymphoproliferation inhibition was given as the percentage of control values (CitationSoares et al., 2006).
Nitric oxide production assay
Nitric oxide (NO) production was estimated by the levels of nitrite determined by the Griess method (CitationPadgett & Pruett, 1992). J774 macrophages were added at 1 × 105 cells/well to 96-well plates with or without macrofungi extracts, in non-toxic concentrations, in the presence of 5 ng/mL gamma-interferon (IFN-γ, BD Biosciences PharMingen, San Diego, CA) and 1 μg/mL lipopolysaccharide (LPS, Escherichia coli serotype 0111:B4, Sigma). Plates were incubated at 37ºC and 5% CO2 and, after 24 h, 50 μL aliquots of supernatants were collected and mixed with an equal volume of Griess reagent in 96-well plates. Absorbance was measured at 570 nm using microplate reader Spectramax 190 (Molecular Devices, Sunnyvale, CA). Quantitative analysis was performed by optical density (OD) comparison with standard solutions freshly prepared NaNO2 in culture medium and mean values of the triplicates were calculated and inhibition of the nitric oxide production was given as percentage of control values (CitationMorazzoni et al., 2005).
Antineoplasic assay
Cancer cells were added to 96-well plates (Daudi at 5 × 104 cells/well and K562 at 1 × 105 cells/well) with or without macrofungi extracts. Positive control used in the half-maximal inhibitory response (IC50) determination was doxorubicin. Plates were initially incubated at 37ºC and 5% CO2 for 24 h. Then, [3H]-thymidine was added and plates were incubated for additional 18 h. Uptake of 3H-thymidine was determined as described above. Mean values of triplicates expressed in CPM were calculated and the growth inhibition of the neoplasic cells was given as percentage of control values.
Antimalarial assay
Antimalarial effect of macrofungi extracts was measured by the [3H]-hypoxanthine incorporation assay. Trophozoite stages at 1% to 2% parasitemia and 2.5% hematocrit were incubated with or without macrofungi extracts, in non-toxic concentration, in culture medium without hypoxanthine; a mefloquine control (as a reference antimalarial drug) was used in IC50 determination. Parasites were harvested using a Packard Filtermate 196 cell harvester and the radioactivity in dried filters was counted using a β-counter. Mean values of the triplicates expressed in CPM were calculated and growth inhibition was given as the percentage of control values (CitationAndrade-Neto et al., 2004; CitationZalis et al., 1998).
Anti-Leishmania amazonensis and anti-Trypanosoma cruzi assays
Epimastigotes of T. cruzi and promastigotes of L. amazonensis were plated in 96-well plates at 1 × 107 and 5 × 106 parasites/well, respectively, with or without macrofungi extracts, in non-toxic concentrations to mouse spleen cells. After incubation at 25ºC for 24 h, the number of viable parasites was evaluated by counting in a Neubauer chamber using a light microscope. Mean values of the triplicates were calculated and growth inhibition was given as the percentage of control values.
Data analyses
All bioassays were performed in triplicate in three independent experiments and inhibition percentage was calculated as follows:
% inhibition = 100 − (extract value × 100)/control value
where extract value corresponds to CPM, OD or number of parasites in the presence of extracts, and control value corresponds to CPM, OD or number of parasites in the absence of extracts. Extracts with inhibition percentage greater than 60% were used for the IC50 calculation by curve-fitting.
All data were expressed as mean ± SD of three independent experiments. ANOVA followed by Tukey´s multiple comparison test were used to assess statistical significance and P < 0.01 was considered significant. All analyses were conducted in Prism 4 for Windows (Graphpad Software Inc., San Diego, CA).
Results
Cytotoxicity
Macrofungi extracts were tested at 1, 10, and 100 μg/mL in mouse spleen cell cultures (). Concentrations causing an inhibition greater than 30% were arbitrarily considered cytotoxic. Thus, macrofungi extracts were subsequently used in the bioassays in the following concentrations: extracts PR, HR, HP and DC were tested at 100 μg/mL, whereas PS and LP were tested at 10 μg/mL.
Table 2. Cytotoxicity of macrofungi extracts in normal spleen cells.
Immunomodulatory activity
Inhibition of proliferation of splenocytes and nitric oxide production by macrophages were used as indicators of immunosuppressive activity of the macrofungi extracts (). In lymphoproliferation assays, the macrofungi extracts PR, HR, LP and DC had inhibitory activities greater than 65%, while PS and HP inhibited below 50%. The former extracts were then tested to determine the IC50 values (). LP had the lowest IC50 value (2.6 μg/mL). The inhibition of nitric oxide production was above 60% only when PS, HR and LP were added to the culture, with IC50 of 1.7, 34.4, and 2.2 μg/mL, respectively ().
Table 3. Pharmacological activity of the macrofungi extracts in different assays.
Table 4. IC50 of macrofungi extracts with inhibition percentage greater than 60% in different assays.
Antineoplasic activity
Antineoplasic potential of the macrofungi extracts was evaluated using human leukemic cells lines K562 and Daudi (). HR and HP extracts inhibited more than 60% of the proliferation of both K562 and Daudi cells, while PS extract inhibited only K562 cell growth. The IC50 values of extracts PS, HR and HP against K562 cells were 4.1, 30.9, and 39.1 μg/mL, respectively. HR inhibitory activity in Daudi cells was 40.8 μg/mL ().
Antiparasitic activity
Effects of the macrofungi extracts in cultures of erythrocytic forms of P. falciparum, promastigotes of L. amazonensis and epimastigotes of T. cruzi were evaluated (). The extracts tested did not inhibit more than 11% of the growth of L. amazonensis; however, extracts PR, PS and HP inhibited 50-58% the growth of T. cruzi (). Extracts PR and PS had antimalarial activity of 88% and 60%, respectively, while the others inhibited less than 42%. PR and PS extracts had IC50 of 4.4 and 0.3 μg/mL, respectively ().
Discussion
The results obtained in this work indicate that non-cultivated macrofungi from Bahia have pharmacological activities, as demonstrated using different bioassays in vitro. The evaluation of cytotoxicity in normal spleen cells prior to testing the extracts in the experimental models was important to determine non-toxic concentrations, warranting that the pharmacological activity was not due to toxic effects. Only Pycnoporus sanguineus (PS) and Lepiota sp. (LP) presented cytotoxicity greater than 30% at 100 μg/mL.
Mytogen-induced lymphoproliferation and nitric oxide production by macrophages stimulated with LPS and IFN-γ are assays commonly used to evaluate the immunomodulatory potential of natural products (CitationMeselhy, 2003; CitationMorazzoni et al., 2005; CitationNicholl et al., 2001; CitationSoares et al., 2006). Phellinus rimosus (PR) extract had the most potent inhibitory activity on lymphoproliferation, although it had little effect on nitric oxide production. This suggests that the extract acts on lymphocytes but not on macrophages. It is also possible that its free radical scavenging properties interfere with the assay (CitationAjith & Janardhanan, 2001), considering the indirect measurement of nitric oxide by nitrite by the Griess method. To our knowledge, this is the first description of immunomodulatory activity of Hymenochaete rheicolor (HR), Datronia caperata (DC) and Pycnoporus sanguineus (PS).
Cell lines K562 (chronic myelogenic leukemia) and Daudi (Burkitt’s lymphoma) are derived from neoplasies of different origins and are commonly used in the screening of new antineoplasic agents. This difference may explain the antineoplasic activity of P. sanguineus in K562 cells but not in Daudi cells, with an IC50 value of 4.1 μg/mL, which was close to the doxorubicin IC50 value (2.8 μg/mL; P > 0.05). The hydroalcoholic extract of P. rimosus did not have activity against the tumoral cell lines assayed, although, in a previous work of CitationAjith and Janardhanan (2003), methanol and ethyl acetate extracts of P. rimosus exhibited cytotoxic and antitumor activities against Dalton’s lymphoma ascites (DLA) and Ehrlich’s ascites carcinoma (EAC) cell lines. To our knowledge, this is the first description of antineoplasic activity of the Hexagonia papyraceae (HP) and Hymenochaete rheicolor (HR).
Leishmanicidal, antibacterial, antiviral and cytotoxic activities of substances isolated from P. sanguineus have been described before (CitationCorrea et al., 2006; CitationSmânia et al., 1995; CitationSmânia Jr et al., 2003). Despite the activity against L. (Viannia) panamensis described by CitationCorrea et al. (2006), P. sanguineus (PS) did not show any inhibition to L. amazonensis, possibly due to differences between species of the parasite utilized in the assays and/or the collection site, since in the aforementioned study the fungus was obtained from Colombian mycota. In addition, we observed a moderate activity against T. cruzi and P. falciparum. Hexagonia papyracea (HP) extract had a significant anti-T. cruzi activity and Phellinus rimosus (PR) had an inhibition of more than 85% against P. falciparum, demonstrating for the first time the antiparasitic activity of this macrofungus.
In conclusion, our results suggest that extracts of the studied macrofungi collected in the Brazilian north-eastern region have marked biological effects, including immunomodulatory, antiparasitic, and antineoplasic activities. Therefore, these macrofungi may be sources of natural products with medicinal interest, requiring chemical investigations in these extracts to identify the active principles.
Acknowledgements
This work was supported by MCT/CNPq - Ministério da Ciência e Tecnologial/Conselho Nacional de Desenvolvimento Científico e Tecnológico; IMSEAR - Instituto do Milênio do Semi-árido; FAPESB - Fundação de Amparo à Pesquisa do Estado da Bahia; PPBIO SEMI-áRIDO - Programa de Pesquisa em Biodiversidade do Semi-árido; RENORBIO - Rede Nordeste de Biotecnologia; and FIOCRUZ - Fundação Oswaldo Cruz.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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