Antineoplasic activity of Agaricus brasiliensis basidiocarps on different maturation phases.

The fungus Agaricus brasiliensis is a Basidiomycete studied because of its immunomodulation and/or antitumor substances. The objective of this study was to verify the Agaricus brasiliensis antineoplasic activity in vivo on different basidiocarp maturation phases on Sarcoma 180 cells implanted in mice. Sarcoma cells were implanted in mice and after seven days mice were divided in three groups. The first group was treated with saline solution, the second group was treated with closed basidiocarp extract solution and the third group was treated with opened basidiocarp extract solution. After 30 days of being daily orally treated with these three solutions all animals suffered euthanasia, and the splenic index, tumor mass and volume were determined. No significant differences of the tumor growth inhibition in function of the different basidiocarp maturation phases for the Agaricus brasiliensis strain were observed. The in vivo basidiocarp antineoplasic average activity was 89.22%.

INTRODUCTION

Agaricus brasiliensis Wasser et al. (A. blazei Murrill ss. Heinemann) (25), also denoted A. subrufescens Peck (10), is a Basidiomycete that has been reported because of its antitumoral substances (2,9, 16). Reports have demonstrated that the use of Agaricus blazei basidiocarp aqueous extract induced the production of specific cytotoxic cells such as T lymphocytes and natural killer cells as well as gamma-interferon what consequently inhibited tumor growth in rodents (22, 27). Menoli et al. (14) reported a decrease of the micronuclear frequency in the presence of A. blazei basidiocarp aqueous extract and Rodrigues et al. (21) reported a bioantimutagenic action of A. blazei suggesting an action on one or more DNA damage-repair systems. Liu et al. (12) studied the immunostimulatory activity of A. brasiliensis basidiocarp extract in human volunteers and observed a natural killer activity increase on the immunological system of patients.

A. brasiliensis antineoplasic activity on Sarcoma 180 tumor cells has been reported from vegetative mycelium (16) as well as reproductive mycelium, the basidiocarp (18). However it is usual to find out variations among strains of the same species as well as among different growth phases of the basidiocarp in the same strain. Each A. brasiliensis basidiocarp growth phase shows biochemical differences such as different protein and beta-D-glucan concentrations which are the substances considered to be responsible for the antineoplasic effect (4).

A. brasiliensis has absence of universal veil and presence of inner veil which characterize basidiocarp maturation. Before the rupture of the inner veil the basidiocarp is immature and spores are in development. At this phase the basidiocarp is called “closed cap”. After the rupture of the inner veil the basidiocarp is mature and spores are developed, ready to be spread and the senescence phase begins. At that phase the basidiocarp is called “opened cap” (4, 15).

Commercially the basidiocarps are cropped before the rupture of the veil (closed cap) mainly due to sensory characteristics and firmness what makes cropping easier and reduces fragmentation during the processing in Brazil (2). Opened caps are considered visually less attractive for consumers because of the dark spores released which gives a dirty appearance to mushrooms. Other commercial claim in Brazil is that closed caps have a better biological activity than opened caps so they get a higher price even when the final purpose is to make capsules. However Camelini et al. (4) and Firenzuoli et al. (7) suggest that opened caps are likely to have better biological activity because they have higher concentration and diversity of glucans and proteins.

Whereas active principles of some Basidiomycetes such as Lentinula edodes (8), Pleurotus ostreatus (1) and Agaricus blazei (4) are extracted from the vegetative mycelium or immature basidiocarps, others such as Ganoderma lucidum have their active principles extracted mainly from spores, where higher concentrations of these substances are found (13). Pinheiro et al. (20) evaluated chemoprevention in rat liver cells using different A. blazei strains and basidiocarps with closed or opened caps and reported that the fungus protective activity depends on strain and period of basidiocarp crop. However there are no conclusive reports on the in vivo antineoplasic activity of A. brasiliensis basidiocarps on different maturation phases on sarcoma 180 cells.

Due to the functional importance of this fungus and the absence of published researches comparing the in vivo biological activity of the fungus on different basidiocarp maturation phases on sarcoma 180 the objective of this study was to verify the Agaricus brasiliensis antineoplasic activity on different basidiocarp maturation phases on sarcoma 180 cells implanted in mice.

MATERIALS AND METHODS

The research was approved by the Animal Ethics Committee at the Paranaense University. Agaricus brasiliensis (Agaricus blazei 97/11) obtained from the Molecular Biology Laboratory at Paranaense University was used in the research. Mushrooms were cropped on different maturation phases: before the rupture of the inner veil (closed cap) and after its rupture (opened cap). Mushrooms were washed with a sponge under flowing tap water and dehydrated in an oven with air circulation at 65 °C. After that they were grounded, placed in a double polypropylene bag that was thermically sealed and kept in a freezer at -70 °C.

A mix of grounded mushrooms and ultrapure water in a proportion of 1:10 was kept in a glass flask with lid and immersed at 90 °C for 12 h. After this period the solution was filtered with Qualy brand filter paper 80 g/m2, thickness 205 μm, 14 μm pores. The filtered solution was stored in a refrigerator at 4 °C in individual aliquots to be used daily.

The strain TG180 of murine sarcoma cells were obtained from the peritoneum of mice previously inoculated and kept for 10 days according to Desmonts and Remington (5). Cell concentration was determined in a Newbauer chamber and it was adjusted to 106 with isotonic saline solution (NaCl 0.18%).

Female Swiss mice with 21 days of age and weight of 25 g ± 5 g from the Biotery at Paranaense University were subcutaneously inoculated on their backs with 106 sarcoma cells. Animals were kept in conventional plastic boxes in a room with controlled temperature at 24 °C ± 2 °C in a cycle of 12 h of light per 12 h of dark regime. After seven days the rodents were divided into three groups: “Control” group with eight animals that received NaCl 0.18%, “Closed” group with eight animals that received closed cap extracted solution and “Opened” group with eight animals that received opened cap extracted solution. Extracted solutions were taken from the refrigerator and kept at room temperature (around 26 °C) for 30 minutes before use. Each mouse received a daily oral dose of 0.1 mL.

After 30 days of the beginning of the treatments the animals were killed by anesthetic overdose in a chamber saturated of ether vapor. Spleen and tumoral mass were removed and measured. Tumor volume was measured by a millimeter ruler and calculated considering its minimum (a) and maximum (b) diameter (tumor volume = 4/3 π (a2 b)/2). The inhibition rate was calculated considering the volume of the tumor in the Control group as 100% (11).The splenic index was calculated considering the spleen mass per animal body mass. Comparison among groups was evaluated by variance analysis and significant differences were determinate by Tukey’s test with a significance level of p < 0.05.

RESULTS AND DISCUSSION

One mouse in the Closed group and two mice in the Opened group did not show any subcutaneous tumoral mass, thus they were not considered in the experiment. Results of the splenic index and tumoral mass (width, length and volume) are shown in Table 1.

Table 1

Evaluation of neoplasm growth in Swiss mice inoculated with sarcoma 180 cells and treated with oral saline solution 0.18% (Control) or with Closed or Opened Agaricus brasiliensis basidiocarp extract.

Tumor measurements*	Control group	Closed group	Opened group
Splenic index	0.0134a μ 0.0033	0.0064a μ 0.0004	0.0108a μ 0.0014
Mass (g)	3.0400a μ 1.1492	0.2180b μ 0.0557	0.9333b μ 0.4228
Width (mm)	1.4400a μ 0.2298	0.7200b μ 0.0567	0.7017b μ 0.0744
Length (mm)	2.3286a μ 0.3519	1. 1040b μ 0.0793	1.3667ab μ 0.2297
Volume (mm3)	13.9248a μ 5.0425	1.2804b μ 0.2953	1.7213b μ 0.7219

*Means followed by different letters indicate significant differences among the groups according to Tukey’s test (p < 0.05)

There was no significant difference in the splenic index among the experimental groups (Table 1). The tumor volume of Closed and Opened groups treated with A. brasiliensis extracts was significantly (p < 0.05) smaller than Control group. However there was no significant difference between Closed and Opened groups (Table 1.). The rate of tumor growth inhibition compared with Control group was 90.80% on Closed group and 87.64% on Opened group, with average of 89.22% between them. Based on these data closed caps have the same biological activity than opened caps. Thus the claim in the Brazilian market about higher prices for closed caps based on higher biological activity has no confirmation on this study. However other commercial advantages related to sensory and processing characteristics remains for that choice. Another relevant aspect is that because there is no difference of active principle concentration between closed and opened caps it is suggest that the main active principle is not concentrated in spores as occurs in Ganoderma lucidum (13). Thus the production of vegetative mycelium in liquid medium is likely to be more advantageous for active principle production in large scale than basidiocarps.

Camelini et al. (4) and Firenzuoli et al. (7) reported differences in the concentration of A. brasiliensis glucans and proteins at different basidiocarp growth phase (closed or opened cap), suggesting that mushrooms with opened cap would have higher quantities of antitumoral active principles and probably higher antineoplasic activity. However according to our results the differences in the basidiocarp maturation were not enough to affect fungal antineoplasic activity in vivo (Table 1). It suggests that beta-glucan even found in higher amounts in opened caps (4) may not be the only or the main responsible substance for the biological activity for this fungus. It is possible that other factors may play a concomitant role either helping or interfering on the activity of antitumor substances of A. brasiliensis.

Zang et al. (26) reported that the presence of protein or mannose produced higher immunomodulatory action against sarcoma 180 neoplasms. Mizuno et al. (17), Wang et al. (24) and Eo et al. (6) reported several biologically active compounds obtained from fungi such as polysaccharides, glycoproteins and glycopeptides acting synergistically in the immunomodulatory activity. On the other hand Walton et al. (23) reported that basidiocarps of the genus Agaricus have a substance called Agaritine which shows cancerigenous activity when extracted by cold infusion. However it was not found reports about this substance in the vegetative mycelium.

Camelini et al. (4) reported higher quantity and structural diversity of glucans in opened basidiocarps. Other authors (3, 19) reported that quantity and structural diversity of fungal glucans are related to their biological activity in the immune system. They suggest that opened caps would be more indicated for finding out glucans with higher antineoplasic activity. However according to Mizuno et al. (16) the antitumoral activity is related to a higher amount of water soluble glucans but it is not necessarily related to a specific basidiocarp maturation phase. Besides that our results did not confirm a better in vivo antineoplasic activity on opened basidiocarps (Table 1). Apparently there is a tendency of decreasing the antineoplasic activity in vivo of opened basidiocarps what is the opposite of Camelini et al. (4) and Firenzuoli et al. (7) suggestions. New studies should be carried out in order to verify the association between in vivo biological activity and beta glucan, protein and other substances such as triterpens concentration, in different basidiocarp maturation phases and in different A. brasiliensis strains in order to amplify the knowledge in this area.

In conclusion, our results suggest that there are no significant differences of tumor growth inhibition in function of different basidiocarp maturation phases for the Agaricus brasiliensis strain, being the in vivo basidiocarp antineoplasic activity average of 89.22%. These findings will contribute to the development of the biological activity research on Agaricus brasiliensis strains.