In this study it was evaluated whether the phenological phase that the plant is in at the time of collection influences the antibiofilm capacity of Candida spp. It was noticed that all extracts of the leaves and bark showed inhibitory effect on both biofilm formation and pre-formed biofilms. Furthermore, it was noticed that with regard to biofilm formation, the extracts from the second collection showed greater inhibitory effect than those from the first collection. In the case of pre-formed biofilms, the leaf extracts from the first collection were better than those from the second, and the bark extract from the second collection was more effective than the first. The presence of gallic acid was also verified in the barks and leaves of C. leptophloeos, and the barks contained a significantly higher amount than the leaves, although with no difference between the collections. In the leaves, the difference in the amount of gallic acid is noticeable, since in the fruiting season (second harvest) the leaves contained more gallic acid than when in the vegetative season (first harvest).
Determination Of The Minimum Inhibitory Concentration (MIC) And Minimum Fungicidal Concentration (MFC)
The MIC of the extracts ranged from 1 mg/ml to 2 mg/ml (Table 1). The lowest MIC values obtained were 1.75 mg/ml for extracts EHF1 and EEC1 against C. albicans URM 4387, 1 mg/ml for extract EEF2, against C. tropicalis URM 4262 and 1.5 mg/ml for all extracts against C. kruzei URM 6352.
All extracts (from both harvests) showed fungicidal effect on the strains studied, with concentration equal to or greater than 2 mg/ml (Table 2).
Biofilm Forming Capacity
All strains were previously tested for their biofilm forming ability, and classified as strong (OD > 0.3), moderate (OD > 0.2 < 0.3) or weak (OD > 0.1 < 0.2). In this regard, C. albicans URM 4387 (OD = 0.35) and C. krusei URM 6352 (OD = 0.31) were considered as strong biofilm formers, while C. tropicalis URM 4262 (OD = 0.29) was considered as moderate biofilm formers.
Effect Of Extracts On Biofilm Formation
The effect of ethanolic and hexanolic extracts of C. leptophloeos leaves and bark on Candida spp. biofilm formation was observed through quantification of biofilm biomass, which was determined by colorimetric technique with crystal violet and subsequent reading of the respective Optical Densities (ODs).
The OD values indicate the amount of biomass of fungal biofilms formed in each well of the microdilution plate. Thus, it is considered that the lower the OD reading, the lower the biomass present, consequently, the better the action of the extract in inhibiting biofilm formation.
The effect of the extract EEL1 on the C. krusei strain was significant (Fig. 2a). Although the OD value of the EEL1 extract was higher than that of the control, there is an indirect relationship between the concentrations of the extract and the observed biomass. However, it is not possible to see this pattern when analyzing the effect of EEL1 extract on C. albicans and C. tropicalis strains (Fig. 2a). For these yeasts, the opposite happens: in the higher concentrations of the extracts the OD values are higher when compared to the lower concentrations of the extracts. Thus, one of the possible explanations for the results obtained by this technique is that the effect of the extract was not inhibitory, but favored the growth of the biofilm.
The extract HEL1 was also efficient when tested against C. krusei URM 6352 (Fig. 2b). This time, the OD value of all concentrations tested were lower than the control, showing the inhibitory effect of this extract. Thus, we highlight that the extract HEL1 was the only one that, when compared to the control, inhibited the development of biofilms by C. kruzei URM 6352.
When tested against C. albicans URM 4387 and C. tropicalis URM 4262, the extract HEL1 showed a higher reading than the control, contrary to what was expected, that is, also causing a possible favoring of biofilm growth by these yeasts.
The EEB1 extract showed higher OD value than all controls of the tested yeasts (Fig. 2c), besides there was no inverse relationship between the concentration of the extract and the amount of biomass of the biofilms.
The ethanolic extract of leaves from the second collection (EEL2) was able to inhibit the biofilm development of C. krusei and C. tropicalis strains (Fig. 3a), as the OD values were lower than those of the controls.
The hexanic extract of leaves from the second collection (HEL2) (Fig. 3b) was efficient against the formation of biofilms of C. albicans, in this case it is noticed that besides the OD values being lower than the control, the readings are indirectly related to the concentration, that is, the higher the concentration of the extract, the lower the amount of biofilm biomass for this yeast. For the other two Candida strains, this relationship is not observed.
The efficiency of the ethanolic extract of bark from harvest 2 (EEB2) (Fig. 3c) is perceived on the formation of biofilms of C. albicans URM 4387, which in the same way as HEL2, showed lower OD values than the control, and with the ratio OD value/concentration of the extract met.
Effect Of Extracts On Preformed Biofilms
For this analysis, the ODs of the preformed biofilms without action of the extracts were read, i.e., the positive controls and are used as a comparative standard to analyze the effects of the extracts on preformed biofilms of the studied yeasts.
It was observed that the extract EEL1 (Fig. 4a) presented effect against the preformed biofilms of C. tropicalis URM 4262, because, except for the concentration of 2 mg/ml, the OD readings were lower in the presence of the extracts, when compared to the positive control. For this same extract, the OD values of C. albicans and C. kruzei biofilms were all higher than the positive control, and here there may have been a positive influence of this extract on biofilm development.
The OD values after action of the HEL1 extract (Fig. 4b) were lower than all the controls of the tested strains. However, the expected correlation was not presented, that is, the more concentrated the extract, the lower the biofilm biomass.
Extract EEB1 (Fig. 4c) also showed lower OD values than controls for C. albicans and C. tropicalis strains, again without the expected pattern. This pattern, however, is observed when the extract is in contact with the biofilm of C. krusei, whose reading increases as the concentration decreases, even though the OD is higher than that of the control.
The EEL2 extract (Fig. 5a) showed a significant effect against the pre-formed biofilms of C. albicans URM 4387 and C. tropicalis URM 4262, however we see that the highest concentration had a higher OD reading than the other concentrations, again suggesting that the extracts favored the development of the biofilms.
The extract HEL2 (Fig. 5b) showed antibiofilm effect against the C. krusei strain URM 6352. Here we notice the opposite effect of extract EEL2 (Fig. 5a), because against this yeast the extract showed OD value lower than the control, especially at the highest concentration (2 mg/ml) showing its ability to decrease the biofilm biomass. In EEB2 extract (Fig. 5c), the OD values were lower than all controls for the three yeasts studied, indicating the inhibitory effect of this extract.
Morphological Analysis Of The Yeasts
It was observed that in the presence of the extracts (EEL1, HEL1, EEB1, EEL2, HEL2 and EEB2), the yeasts did not multiply, thus showing a smaller number of cells, visually, when compared to the positive control (cells without the extract). This control showed a large amount of dividing cells (budding), while the cells of the yeasts that were subjected to the action of the extracts showed a much smaller amount of buds and pseudo hyphae (Fig. 6).
Cellular Viability - Methylene Blue
This methodology allows the observation of the quantity of non-viable cells, through methylene blue staining and cell count. Thus, it is considered that the greater the number of non-viable cells, the greater the antifungal and antibiofilm capacity of the extract.
On Biofilm Formation
All extracts, from both harvests, were able to decrease the formation of biofilms of Candida spp. strains (Tables 3, 4 and 5). In extract EEL1, its strong staining at the highest concentration (2mg/ml) prevented the visualization of cells, so it was not possible to count the cells affected by this extract at this concentration. However, it can be seen that the 1mg/ml concentration was able to decrease more than 50% of the cells of all the yeasts studied, having the greatest effect on C. tropicalis URM 4262, decreasing more than 86% of the cells (Table 5).
The extract HEL1, at the concentration of 2mg/ml decreased 84.6% of the cells of C. albicans URM 4387 (Table 3), 94.6% of the cells of C. krusei URM 6352 (Table 4) and 87.6% of C. tropicalis URM 4262 (Table 5). When the effects of ethanolic (EEB1) and hexanic extract of leaves (HEL1) on yeasts were compared, the HEL1 extract had a greater effect at all concentrations (Tables 3, 4 and 5). The EEB1 extract was also more efficient against C. krusei URM 6352 yeasts (Table 4), making 96.3% of the cells inviable. In addition, the bark extract from the first collection showed greater effect than the leaf extract (EEL1) on the tested yeasts (Tables 3, 4 and 5).
Extract EEL2 was most efficient against C. tropicalis URM 4262, decreasing more than 89% of the cells at the 1mg/ml concentration (Table 5). Extract HEL2 was most efficient at the 2mg/ml concentration against all Candida strains (Tables 3, 4 and 5). When comparing the effects of the two extracts on yeast, extract HEL2 showed better results in all concentrations.
Similarly, the EEB2 extract also showed greater efficiency at the highest concentration (2mg/ml) against C. krusei URM 6352 (Table 5) and C. tropicalis URM 4262 (Table 5), but against C. albicans URM 4387 the concentration of 0.5mg/ml was more efficient, as there was no statistically significant difference compared to the higher concentrations. When compared to the leaf extract, the EEB2 extract was more efficient in inhibiting biofilm formation.
When comparing the differences in the effects of the extracts from different collections, it is noticed that even at the lowest concentration, i.e. 0.125mg/ml, the extract EEB2 had a greater effect than EEB1. Among the leaf extracts, the inhibitory capacity of the ethanolic extract of the second collection (EEL2) was higher than that of the first (EEL1). However, in the hexanic extracts, the opposite occurs; the extract EHF1 has a greater effect on yeasts than HEL2.
This implies that the phenological phase of the plant is not the only factor that influences the potentiality of the extracts. Although all had some effect on Candida spp. it is necessary to stick to the part of the plant and the solvent used.
On Preformed Biofilms
When tested against the pre-formed biofilm of C. albicans URM 4387 (Table 6), extract EEL1 was able to decrease the biofilm cells by 52% at the 1mg/ml concentration. Again, it was not possible to observe the amount of viable cells at the 2 mg/ml concentration due to the staining of the extract. However, at the 1 mg/ml concentration the efficiency of the extract is noticeable, as it was able to decrease more than 70% the viable cells of C. krusei URM 6352 (Table 7) and C. tropicalis URM 4263 (Table 8) biofilms.
Extract HEL1 (Table 6) was more efficient against C. albicans URM 4387 biofilm decreasing viable biofilm cells by more than 70% at the concentration of 1 mg/ml. When the comparison between the leaf extracts, EEL1 and HEL1 was made, it was noticed that on pre-formed biofilms the ethanolic extract had a greater inhibitory effect. The EEB1 extract was able to reduce more than 80% of the C. albicans biofilm (Table 6) and C. tropicalis (Table 8), and when compared with the effect of the leaf extract, EEB1 showed better effect in all concentrations.
The EEL2 extract was more efficient against C. albicans (Table 6) and C. krusei (Table 7) biofilms with more than 70% reduction of these biofilms at the concentration of 1 mg/ml. The biofilm of C. albicans URM 4387 suffered the greatest reduction (67% of biofilm) in contact with the extract HEL2 (Table 7) when compared to the other yeasts. As in the first collection, HEL2 was more efficient than HEL2 when tested against preformed biofilms. In contact with the EEB2 extract, C. krusei (Table 7) had a greater reduction, since the extract turned 90% of the biofilm cells non-viable at the concentration of 2 mg/ml. This extract was also more effective than the leaf extract (EEL2).
There was a difference between the effects of the extracts in different collections. Among the bark extracts, EEB2 was found to have greater antibiofilm capacity than EEB1, even at the lowest concentrations. Among the leaf extracts, the extracts from the first harvest (EEL1 and HEL1) had the greatest effect on the pre-formed biofilms.
Thus, as with biofilm formation, it is evident that one cannot consider phenological phase as the only determining factor influencing biological activity. The plant part and the solvent should also be taken into consideration to obtain a better action of the extracts.
Determination Of The Presence Of Gallic Acid By High Performance Liquid Chromatography (HPLC):
The HPLC analysis was performed in order to determine if there was a difference in the amount of gallic acid in the extracts obtained depending on the collection season. The presence of gallic acid was verified in the ethanolic extracts of leaves and bark, in both collections (Fig. 7). However, the leaf extract showed a lower concentration of gallic acid in the first harvest (0.0003 mg/ml) when compared to the second one (0.0023 mg/ml) (Fig. 7b). The concentration of gallic acid in the cuttings varied from 0.0160 mg/ml (harvest 1) and 0.0218 mg/ml (harvest 2), however this difference was not significant, indicating that this compound does not vary temporally in this part of the plant.