In vitro cytotoxic capacity against tumor cell lines and antioxidant activity of acidic polysaccharides isolated from the Andean Patagonian fungus Phylloporia boldo

Abstract Fungal polysaccharides possess a broad biological activity, including cytotoxic and antioxidant activities. This work aimed to evaluate the cytotoxic and antioxidant activity of the acidic polysaccharides of Phylloporia boldo strain (named PBAP40). Cytotoxic activity of polysaccharide was evaluated determining the viability of three tumor cell lines by MTT assay. The effect of acidic polysaccharide on the cell cycle of HL-60 cell line was evaluated by flow cytometry, and the antioxidant activity was determined by DPPH and ABTS assays. PBAP40 showed cytotoxic effects in tumor cell lines. Results suggest that P. boldo acidic polysaccharides arrested tumor cells in the cell cycle Sub G1 phase. The acidic polysaccharides of PBAP40 strain were not cytotoxic for the non-tumor cell line. PBAP40 also showed excellent antioxidant activity. The FT-IR analysis of the acidic polysaccharides indicated the presence of glucans bearing α- and β- type glycosidic bonds. Graphical Abstract


ABSTRACT
Fungal polysaccharides possess a broad biological activity, including cytotoxic and antioxidant activities.This work aimed to evaluate the cytotoxic and antioxidant activity of the acidic polysaccharides of Phylloporia boldo strain (named PBAP40).Cytotoxic activity of polysaccharide was evaluated determining the viability of three tumor cell lines by MTT assay.The effect of acidic polysaccharide on the cell cycle of HL-60 cell line was evaluated by flow cytometry, and the antioxidant activity was determined by DPPH and ABTS assays.PBAP40 showed cytotoxic effects in tumor cell lines.Results suggest that P. boldo acidic polysaccharides arrested tumor cells in the cell cycle Sub G1 phase.The acidic polysaccharides of PBAP40 strain were not cytotoxic for the non-tumor cell line.PBAP40 also showed excellent antioxidant activity.The FT-IR analysis of the acidic polysaccharides indicated the presence of glucans bearing aand btype glycosidic bonds.

Introduction
Acidic fungal polysaccharides constitute a relevant group of bioactive molecules (Wang et al. 2011).These biopolymers show strong biomedical effects (Li et al. 2020).For example, the acid polysaccharide fraction extracted from Cordyceps sinensis displayed remarkable modulating effects on murine macrophage cell line RAW264.7,as stimulation of phagocytosis, NO production and secretion of cytokines (Chen et al. 2010;Wang et al. 2011).
Polypore fungi of the Hymenochaetaceae Donk family comprises a group known to include several forest pathogens (Rajchenberg et al. 2019) as well as species with biomedical applications (Yang et al. 2016;Wu et al. 2019).The genus Phylloporia Murril (1904) includes a large variety of polypore fungi with a worldwide distribution (Qin et al. 2018;Wu et al. 2019).Phylloporia boldo Rajchenb & Pildain (Hymenochaetaceae) has been recently described in the south of Chile (Rajchenberg et al. 2019).This fungus was found associated with the stem of the endemic Chilean species Peumus boldus Molina (1782) (common name 'boldo') (Monimiaceae, Laurales).Studies about bioactive compounds in P. boldo are very limited (Riquelme et al. 2020).
In this study, we isolated acidic polysaccharides from a strain of P. boldo from the Bio B ıo region, Chile.Also, we examined the bioactivity of these polysaccharides by means of cytotoxicity against human tumor cell lines and the effect on their cell cycle.We also evaluated antioxidant capacity using DPPH and ABTS methods.
Interestingly, the cytotoxicity of the PBAP40 on the non-tumor primary HGF-1 cell line is rather mild (Figure 1D).In the presence of 0.04 mg mL À1 of PBAP40, the survival rate of HGF-1 cells was 86 ± 2.64%.Moreover, as the concentrations of PAPB40 increased, the viability of the non-tumor primary HGF-1 cells showed survival percentages above 100%, compared with the cells not treated with the acidic polysaccharides.
Our results show that PBAP40 significantly reduces the viability of the human tumor cell lines in a dose-dependent manner, while induces significant proliferation of the non-tumor cell line.This observation suggests a selective effect of PBAP40.Several studies have evaluated the bioactivity of crude fungal polysaccharides, reporting significant cytotoxic effect at a low concentration of polysaccharides (Liu et al. 2016, Mei et al. 2015).However, these samples are mixtures of acidic and neutral polysaccharide, hence their enhanced activity (Figueroa et al. 2020).The cytotoxic activity showed bv PBAP40 against tumor cells may be due to the presence of a and b-glucans, which have been identified using FT-IR analysis (Figure S1) (Nie et al. 2012).b-glucans are one of the main bioactive components present in fungal polysaccharides and strong anticancer activities have been attributed to them (Kozarski et al. 2011;Del Corno et al. 2020).

Effect of PBAP40 on the cell cycle of HL-60 tumor cell line
The effect of low concentration acidic polysaccharides, IC 50 values (127.85 mg mL À1 ) and 1 = 4 IC 50 values (31.96 mg mL À1 ), on HL-60 cell line showed no significant differences when compared with cells not treated with PBAP40 (negative control (n.c.)), data not shown.The highest concentrations of PBAP40 assayed (4X IC 50 ¼ 511.4 mg mL À1 ) caused a moderate increase in Sub G1 phase events percentage reaching 24.90 ± 3.57%, over the negative control (13.49± 0.87%) (Figure S2 A and B).Moreover, a smaller reduction of G0/G1 phase events (PBAP40 ¼ 40.10 ± 3.1%; n.c.¼ 54.45 ± 1.01%) and S/G2/M phase events (PBAP40 ¼ 29.00 ± 0.56%; n.c.¼ 32.06 ± 1.88%) was observed.Our results suggest that PBAP40 arrest the cell cycle of the tumor cell tested at the Sub G1 phase, inducing the death of HL-60 cells.Other studies have shown a similar effect; as an example, the percentage of HeLa cells in G0/G1 phase significantly increased from 27.11% to 48.17% when treated with polysaccharide from Phellinus baumii (Liu et al. 2016).

Antioxidant capacity of PBAP40
PBAP40, evaluated at a concentration of 30 mg mL À1 , showed antioxidant activity percentages of 24.53 ± 1.36% on DPPH and 10.20 ± 1.04% on ABTS (Table S1), which are greater than the negative control result.Also, the antioxidant activity of PBAP40 was substantially lower compared to the positive control.When the antioxidant activity of PBAP40 was contrasted with a Trolox calibration curve, it was determined that the acidic polysaccharide did not exceed the activity of 1 mg mL À1 Trolox (Table S1).
Studies performed with the fungi Phellinus linteus and P baumii have shown that their total polysaccharides it reaches an 86.9% antioxidant activity at a 10 mg mL À1 concentration and 80.29% at a 1.2 mg mL À1 concentration, respectively, against the free radical DPPH (Kozarski et al. 2011;Jin et al. 2016).Crude polysaccharides can be conjugated with other components, such as proteins, pigments, or polyphenols.These residues may be responsible for the radical reducing effect, causing crude polysaccharides to show a more significant antioxidant activity than after fractionation of the samples (Wang et al. 2016;Figueroa et al. 2020).

Experimental
The detailed description is provided in the supplementary material.

Conclusions
Phylloporia boldo acidic polysaccharides PBAP40 was highly effective and selective to reduce the viability of human colorectal carcinoma HCT-116, mammary adenocarcinoma MCF-7 and promyelocytic leukaemia HL-60 cell lines.Interestingly, at the concentrations tested, PBAP40 did not show cytotoxicity against non-tumor primary HGF-1 cell line.Analysis by flow cytometry revealed the effect of PBAP40 on the cell cycle -Sub G1 phase arrest.PBAP40 showed a strong antioxidant capacity by means of DPPH and ABTS assays.Further studies are required to determine the mechanisms by which these acidic polysaccharides exert their cytotoxic and antioxidant activities.