The medium used for cultivation of myxomycete plasmodia was adapted from the research of Henney and Henney [23]. We attempted to replace glucose in the original medium with other carbon sources (e.g., oyster mushroom powder [since the oyster mushroom is one of the favorite food sources of some myxomycete plasmodia in the nature], rice bran and galactose). However, preliminary results showed that Ph. polycephalum preferred glucose and Phy. oblonga grew better in water agar without glucose (Phy. oblonga has agar hydrolytic activity). As such, for slime track and EPS production, typical plasmodia of Ph. polycephalum and Phy. oblonga were transferred to nutrient and water agar, respectively, and incubated under dark condition at 25 °C for 7 days (Fig 1). The amounts of slime track material and EPSs obtained are presented in Table 1. The amounts of slime track material obtained from both species were higher than those of EPSs still in contact with the plasmodium.
Fig. 1Plasmodium and slime track
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Table 1 Amounts of slime track and EPSs isolated from cultures of Phy. oblonga and Ph. polycephalumFull size table
The carbohydrate, protein and sulfate contents of EPSs are listed in Table 2. The total carbohydrate content of the samples varied from 55 to 82% according to the phenol-sulfuric acid method. Sulfated groups and protein made up small proportions (Table 2). In general, the EPS and slime track of Phy. oblonga had greater amounts of carbohydrate compared to those of Ph. polycephalum. However, the samples from the latter species had higher percentages of sulfate content. When comparisons are made between the slime track and EPS samples of each species, the amounts of carbohydrates of the slime tracks were higher than that of the EPS, and this applied for both species.
Table 2 Total carbohydrate, protein and sulfate contents of the slime track and EPSsFull size table
The slime tracks and EPSs were depolymerized by using the TFA hydrolysis method. The monosaccharide compositions of the EPSs produced by Phy. oblonga and P. polycephalum were detected by TLC, and their quantities were measured by GC-FID analysis. The data obtained data are displayed in Fig. 2 and Table 3.
Fig. 2Chromatograms of GC analysis of the monosaccharide composition of slime tracks and EPSs. The chromatogram of Phy. oblonga EPS (a), Phy. oblonga slime trạck (b), Ph. polycephalum EPS (c), Ph. polycephalum slime track (d) and standard sugars (e) was developed using values of GC. Galactose (Gal), glucose (Glc), rhamnose (Rha) were used as standard sugars. Inositol (IS) was used as internal reference
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Table 3 Monomer compositions of crude EPSs obtained from cultures of Ph. polycephalum and Phy. oblongaFull size table
Table 3 showed that the slime track and EPS samples contained glucose, galactose and rhamnose and rhamnose was the major monosaccharide of the EPS from Phy. oblonga and the slime tracks of both species, for which it accounted for 66.37%, 62.58% and 71.46%, respectively. In contrast, EPS from Ph. polycephalum was composed mainly of glucose (50.87%).
The present study is the first to determine the monomer compositions of EPSs isolated from Phy. oblonga. However, with Ph. polycephalum, the results reported have varied from one study to another. Extracellular slime from broth cultures (containing glucose as the carbon source) of Ph. polycephalum was found to contain galactose, sulfate, and trace amounts of rhamnose [19]. However, Simon and Henny [20] found that slime production of Ph. rigidum, Ph. flavicomun and Ph. polycephalum contained a single sugar component of galactose when cultured on media containing glucose as the carbon source. Similar results for Ph. polycephalum were also reported by Farr et al. [24]. In general, monomer compositions and their ratios in microbial EPSs are influenced by the carbon source in the culture medium [25, 26]. However, with the myxomycetes, there would appear to be some other factors involved. Ph. polycephalum in our study was cultured on a glucose-based solid medium, but the monomer composition was completely different from what has been reported in other studies. It is possible that plasmodia produce different kinds of slime material as compared to microplasmodia.
Antimicrobial activities of the EPS and slime track samples as determined by the agar diffusion method are presented in Table 4.
Table 4 Antimicrobial activities of EPS and slime track samples from Phy. oblonga and Ph. polycephalumFull size table
The results indicate that there were significant differences in antimicrobial activities among the samples. The slime tracks of both two species did not exhibit any inhibitory activity against the strains of microbes tested. This could be explained by the theory that myxomycete plasmodia leave slime tracks behind when migrating simply to mark the area which has been exploited for food resources [12]. In contrast, isolated EPS from plasmodia showed promising activities towards S. aureus and C. albicans, whereas C. albicans was found to be the most susceptible to the EPSs from both species (zone of inhibition ≥20 mm) (Table 4). The antimicrobial activities of the EPSs which are still in contact with the plasmodia would be explained by the possibility that these compounds would protect the plasmodia from external factors, including other microorganisms.
The results obtained for antimicrobial activities in the present study agree with those reported in some previous studies relating to the antimicrobial property of microbial EPSs. Asgari and Henney [22] found that the cell growth and division of Bacillus subtilis (a gram positive bacterium) was inhibited by slime secreted by Ph. flavicomun. The degradation of the cell wall caused morphological changes such as swollen cells or cell lysis. Li et al. [27] found that EPS from Lactobacillus plantarum exhibited inhibitory activities against S. aureus and C. albicans. EPS from Enterobacter faecalis showed significantly high activity toward C. albicans [28].
The MIC values of the EPS samples from Ph. polycephalum and Phy. oblonga were studied against C. albicans and S. aureus. The data obtained are shown in Table 5.
Table 5 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) of EPS and slime track samples from Phy. oblonga and Ph. polycephalumFull size table
With respect to their ability against S. aureus, the MIC value of the Ph. polycephalum EPS was almost the same with that of Phy. oblonga. However, EPS from Ph. polycephalum showed much better antifungal activity, since the MIC value (1280 μg/mL) of the EPS from this species against C. albicans was just about a half that from Phy. oblonga (2560 μg/mL) and twice when compared with the standard antifungal drug (640 μg/mL) (Table 5). However, this EPS is not yet purified. Nevertheless, EPS from Ph. polycephalum appears to have the potential for treatment of C. albicans. However, it should be noted that the MBC or MFC values are higher than the MIC values. This suggests that the compound would easily inhibit microbial growth at low concentrations, but leading to actual microbial death would require higher doses.
In this part of our study, in vitro antioxidant activities of the EPS samples with the concentration range of 0–6.0 mg/mL from Phy. oblonga and Ph. polycephalum were determined by DPPH assay and compared with that of ascorbic acid. Figure 3 illustrates that there was not a major difference observed between radical scavenging ability of slime track and EPS extracts from Phy. oblonga and Ph. polycephalum at an initial concentration 1.0 mg/mL. However, at the higher sample concentrations, EPS isolated from a plasmodium showed higher radical scavenging ability than EPS isolated from the slime track material in both species. EPS from Phy. oblonga showed maximum DPPH scavenging activity (80.41%) at a concentration of 6 mg/ml, whereas that of ascorbic acid was 99.56%.
Fig. 3Antioxidant activities of EPSs of Phy. oblonga and Ph. polycephalum in vitro. POS and POP are slime track and EPS samples from Phy. oblonga, whereas PPS and PPP are slime track and EPS samples from Ph. polycephalum
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The EC50 is the concentration of antioxidant needed to obtain a 50% antioxidant effect, and is typically used as a parameter to express or compare the antioxidant capacity of different compounds. Lower EC50 values show a higher antioxidant activity [28]. EC50 values of the EPS samples and ascorbic acid are displayed in Table 6.
Table 6 EC50 values of the slime track and EPS samples from Phy. oblonga and Ph. polycephalumFull size table
According to the EC50 data, the slime track and EPS samples from Phy. oblonga showed higher scavenging abilities than those from Ph. polycephalum. These data also indicated that EPSs and slime tracks from Ph. polycephalum and Phy. oblonga have comparable antioxidant capacity with some common edible mushrooms [29,29,32]. However, the antioxidant activities these samples were far smaller than ascorbic acid.
In this experiment, crude EPS and slime track samples from Phy. oblonga and Ph. polycephalum were subjected to in vitro cytotoxicity SRB assay with fibroblast and cancer cell lines. Cells were treated with EPSs ranging from 0.25 to 1.5 mg/mL and incubated for 48 h, and then the cell inhibitory rate was measured by using a spectrophotometer. The data obtained data are shown in Fig. 4.
Fig. 4Growth inhibition of MCF-7 (a) and HepG2 (b) cancer cell lines by treating with crude EPS extracts from Phy. oblonga and Ph. polycephalum in comparison with camptothecin standard (CPT) with the concentration of 0.005 μg/mL via SRB assay. POS, POP, PPS and PPP were represented in the Phy. oblonga slime track, Phy. oblonga EPS, Ph. polycephalum slime track and Ph. polycephalum EPS
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The results indicate that EPSs possess different levels with respect to their toxicity effects against the cancer cell lines. At low concentrations (0.25–0.5 mg/mL), none of negatives effect on the proliferation of cancer cell lines were observed. However, EPSs were found to show anti-proliferation when the concentration increased from 0.75 to 1.5 mg/mL.
EPSs isolated from a plasmodium showed higher inhibition rates against the cancer cell lines than EPSs isolated from the slime tracks. Most notably, EPS from Phy. oblonga showed significantly higher inhibitory activities against MCF-7 and HepG2 when compared to that of Ph. polycephalum.
The half inhibitory concentrations (IC50) of the EPS sample from Phy. oblonga toward MCF-7 and HepG2 were found as 1.22 and 1.11 mg/mL, respectively. However, these activities are not comparable to the positive control (camptothecin).
Microbial EPS have been found to have anti-proliferation effects against HepG2 and MCF-7 cells. Wang et al. [9] reported that at the concentration of 600 μg/ml, purified EPS from Lactobacillus plantarum could suppress proliferation of HepG2 cells by 56.34% when treated for 72 h. In addition, Osama et al. [5] found that (IC50) of purified EPS from Bacillus marinus in the MCF-7 was 118.0 μg/mL after 48 h.