Cytotoxic activity of the crude polysaccharides/exopolysaccharides of Coprinus comatus and Coprinellus truncorum

Abstract Fungi are an important source of polysaccharides (PSH) and phenolic compounds (PC). Numerous studies have highlighted the beneficial effects of fungal consumption, but the impact of submerged cultivated mycelia (M) and filtrate (F) has not been fully investigated. We aimed to investigate the cytotoxic activity of isolated crude PSH and exopolysaccharides (ePSH) of submerged cultivated M and F of edible Coprinus comatus and Coprinellus truncorum species. Both PSH and ePSH exhibited significant cytotoxic activity towards HepG2 cancer cells of human origin (three-way ANOVA). The C. truncorum PSH/ePSH was more efficient inducing maximal reduction in cell viability (≈50% at 450 µg/mL) after 24 h while C. comatus PSH/ePSH needed 72 h to reach similar effect (≈60% at 450 µg/mL). Partial least square regression (PLSR) analysis indicated that specific phenolic composition of the PSH/ePSH could be responsible for the difference in their activity. Graphical Abstract


Introduction
Fungi are an important source of bioactive compounds, including polysaccharides (PSH) and phenolic compounds (Thu et al. 2020). Besides fruiting body, submerged cultivated mycelia (M) and extracellular mediumfiltrate (F), also contains PSH and exopolysaccharides (ePSH) (Mahapatra and Banerjee 2013). The Coprinus comatus (O.F. M€ ull.) Pers. 1797, and the Coprinellus truncorum (Scop.) Redhead, Vilgalys and Moncalvo 2001, are widespread, edible species common in Europe, Asia and North America. Their nutritional and medicinal values date from Chinese traditional medicine, and in recent decade, increment for investigation of antitumor/cytotoxic activity of PSH and ePSH have been observed (Elisashvili 2012). In order to meet the increasing demands of PSH and ePSH production, the submerged cultivation is gaining more attention. The present work is concerned with the isolation of PSH and ePSH from submerged cultivated M and F of the species C. comatus and C. truncorum, their evaluation and characterisation of the present phenolic compounds in the crude extracts, as well as consideration of their cytotoxic effects on human hepatocellular (HepG2) cell line.

Fourier transform infrared spectra of PSH extracts
The FTIR spectrum of PSH extract isolated from C. comatus M shows sharp, strong bands relating to polysaccharides. The band at about 890 cm À1 is relevant for b-glycosidic bond. The intense absorption at 1062 cm À1 and the weaker at about 1160 cm À1 refer to the ring-stretching vibrations and glycosidic bonds, respectively (Ka cur akov a et al. 2000).
The FTIR spectrum of ePSH isolated from submerged C. comatus F indicates the absorption at about 1150 cm À1 related to the stretching vibrations of the glycosidic bonds, while the sharp intense band at 1057 cm À1 refers to the C-C and C-O vibrations of the carbohydrate ring. The area of 1310-1410 cm À1 is indicative to OH groups of the phenolic compounds (Kozarski et al. 2012).
The FTIR spectrum of PSH C. truncorum M indicates slow-intensity absorption at 890 cm À1 , which is indicative of b-glycosidic bonds, while the band at 932 cm À1 refers to pyranose ring vibrations. The band at 1069 cm À1 is relevant for the C-O bending vibrations, while the frequency at about 1150 cm À1 refers to the C-O-C vibrations of the glycosidic bond .
The FTIR spectrum of ePSH of C. truncorum F submerged culture indicates the band at 763 cm À1 , which is characteristic of the bending vibrations of the C-H bond and is related to a-glycosidic bonds. The intense band at 1057 cm À1 corresponds to the C-C and C-O vibrations of the stretching carbohydrate ring, the absorption at about 1153 cm À1 refers to the C-O-C stretching of the glycosidic bond (Mathlouthi and Koenig 1987).

HPLC-MS/MS profile of PSH/ePSH extracts
A total of 20 compounds were identified and quantified in the 4 examined extracts (Table S1 see supplementary material). The most prevalent compound was amentoflavone detected in all investigated samples. It has been reported that flavonoid amentoflavone induces apoptosis in human breast MCF-7 cancer cells, as well as in human cervical (SiHa and CaSki) cancer cells (Pei et al. 2012;Liu et al. 2017). Besides amentoflavone, cinnamic acid was the second most abundant compound found in C. comatus PSH and ePSH samples. The cinnamic acid is also known as an anti-proliferative agent (Heleno et al. 2014). Comparing phenolic contents of methanol and ethanol extracts of C. comatus and C. truncorum species with the crude PSH and ePSH extracts of same species amentoflavone and cinnamic acid were less represented in methanol and ethanol extracts (Karaman et al. , 2020.

Cytotoxicity of PSH and ePSH
PSH and ePSH of submerged cultivated C. comatus and C. truncorum differently affected the cell viability of investigated cell line. The ANOVA analysis revealed that viability of the HepG2 cells significantly decrease (p < 0.0001) both with the increase of the dose and time of exposure to the extracts (Figure 1). Detailed pairwise comparisons of individual samples (Figure 1) showed that PSH and ePSH of C. truncorum had the fastest effects, reached maximal cytotoxic activity on the HepG2 cells already after 24 h of exposure, at all applied doses. PSH and ePSH of C. comatus showed delayed effects and induced maximal reduction of the HepG2 cells viability after 72 h of exposure. As a result, 450 mg/mL of all extracts induced large and statistically significant decrease in the HepG2 cells viability of around 60% after 72 h of treatment.
Differential effects of C. comatus and C. truncorum extracts can be partially explained by the large differences in their phenolic content. Partial least squares regression (PLSR) model showed existence of statistically significant positive correlation between phenolic compounds characterised either by total absence, or significantly lower concentrations in PSH/ePSH of C. truncorum compared to those of C. comatus ( Figure S2 and Table S1).

Conclusion
The isolated PSH and ePSH from submerged cultivated edible C. comatus and C. truncorum species showed significantly decrease the viability of the HepG2 cancer cells both with the increase of the dose and time of exposure (ANOVA analysis p < 0.0001). As a result, 450 mg/mL of all extracts induce large and statistically significant decrease in the HepG2 cells viability, around 60% after 72 h of treatment. According to the results of PLSR analysis, the differences in PSH/ePSH cytotoxic activity may be attributed to their variances in the present phenolic compounds.