Antibacterial and hypoglycemic activity in vitro of polysaccharide obtained from Periconia byssoides

Fungi are source of polysaccharides that can show biological activity. The objective of this research was obtained polysaccharides from Periconia byssoides and evaluate antibacterial and hypoglycemic activity in vitro. The number-average molecular weight and degree of polymerization were determined. The results show that the polysaccharide of P. byssoides has potential as hypoglycemic. Therefore, it would be interesting to conduct in vivo research with this polysaccharide, to know about its hypoglycemic

The fungus Periconia byssoides was first isolated from the marine species Aplysia kurodai (Yamada et al., 2007).
Subsequently, research with active metabolites of this fungal species identified cell adhering inhibitors (Yamada et al., 2005), that demonstrated significant inhibition on human cancer cells (Yamada et al., 2007).
Therefore, the objective of this research was obtained polysaccharides from Periconia byssoides and evaluate its antibacterial and hypoglycemic activity in vitro.

Micro-organism
The fungus Periconia byssoides (CCMB 07.07) was obtained from the Collection of Cultures of Micro-organisms of Bahia (UEFS) and was maintained in BDA agar medium at 28 °C in an incubator (IGO 150 Cell Life -Jouan).

Cultivation conditions
The fungus Periconia byssoides was cultivated in BDA medium for a period of 20 days at 28°C for biomass production. After, fungal mycelium discs (~ 1 cm) were transferred to Erlenmeyer flasks containing 100 mL of liquid culture medium (malt 2%, peptone 1% and glucose 2%). The vials were placed in agitation at 120 rpm in a rotary agitator. After 15 days, the medium was filtered and centrifuged (Centrifuge 5804R -Eppendorf, São Paulo, Brazil) at 8000 x g for 15 min at 4 °C, and the precipitate, which contained biomass of P. byssoides, used to extract polysaccharides (Dong et al. 2009).

Polysaccharide extraction
Forty mL of a 0.1 M sodium hydroxide (NaOH) solution was added to 2.0 g of dry biomass (20mL/g) and heated in a water bath at 100°C for 10 minutes. After the incubation time, the material was centrifuged at 9000 g for 15 min and the soluble polysaccharide in the alkaline solution was dried at 55 ºC until constant weight.

Purification of polysaccharide
A polysaccharide solution (15 mg/mL) was applied to a column of Sephacryl S-200 (Amersham Bioscience, USA) (48 x 1.2 cm) previously balanced with sodium phosphate buffer (0.05 M, pH 7.0), which was used to elude the product.
Fractions of 2.5 mL were collected and analyzed for polysaccharide totals by the phenol-sulfuric acid method (Dubois et.al, 1956).

The number-average molecular weight (MWn) and degree of polymerization (DPn)
The number-average molecular weight (MWn) and degree of polymerization (DPn) were determined by the measurement of the sugar reduction value by means of the 3.5-dinitrosaliclic (DNS) method (Miller, 1959)
The concentration capable of inhibiting 50% of enzymatic activity (IC50) was calculated from the inhibition value obtained from the serial dilution of the polysaccharide, expressed in a dose-response curve (1 -10 mg/mL). After bacteria growth in nutrient broth for 24 h to 37 °C, they were diluted in nutrient broth to 10 5 CFU/mL and incubated with polysaccharides (1, 3, 6, 10 mg/mL) for 24 h to 37 ºC in microplates. After, resazurine (0.01%) was added to the suspension and the MIC was determined as the lowest concentration where there was no bacterial growth after 1 h, indicated by the blue color.

Statistical analysis
The results on amylase activity were compared with acarbose by ANOVA and Dunnett's post-test with significance level of 5% using for Program Graphpad Prism 5.0.

Results and Discussion
The fungus Periconia byssoides CCMB 07.07 showed biomass production of 2.154 ± 0.020 g/100 mL of medium. Fang and Zhong (2002) observed biomass production value of the fungus Ganoderma lucidum of 1.67 g/100 mL of medium.
By analyzing the biomass production of the fungus Pleurotus sajor-cashew also through submerged fermentation, Confortin (2006) obtained a lower value than the present research (0.257 g/100 mL of medium). Lee et al. (2013) obtained values higher than the above-mentioned studies, observing biomass production equal to 2.31 g/100 mL of medium when analyzing the fermentation of the fungus Paecilomyces japonica.
The analysis of the content of total sugars and reducers sugars of the polysaccharides of P. byssoides were used to obtain the number-average molecular weight (MWn) and degree of polymerization (DPn), with values equal to 5.7 x 10 4 Da and 352, respectively. Hashemifesharaki et al. (2020) when studying the molar mass of a polysaccharide observed a slightly lower value (4.87 x 10 4 Da) than that of the polysaccharide analyzed in this study. Nuerxiati et al. (2019) obtained two polysaccharides in his research, which shows a molar mass of 25.57 and 11.64 kDa. The polysaccharide purification was conducted in a column (48 × 1.2 cm) of Sephacryl S-200 and it was possible to observe the presence of two symmetrical peaks ( Figure 1). Research, Society and Development, v. 10, n. 12, e493101218433, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i12.18433 Source: Authors.
The antibacterial and hypoglycemic activities of P. byssoides were analyzed with peak 1 of P. byssoides (fractions 5-15). The results shows that this polysaccharide exhibit good inhibition of the enzyme α-amylase (72.3%) concentration of 10 mg/mL and IC50 equal to 3.316 mg/mL. In addition, a significant difference was observed between all doses tested and acarbose, a positive control, as can be seen in Figure 2. A study published in 1980 showed that valienamine, a pseudo sugar isolated from fungal cultures, is a potent inhibitor of α-glucosidase, besides being one of the components of acarbose, a potent hypoglycemic agent (LAUBE et al., 1980). In addition, more current studies have also linked polysaccharides to hypoglycemic activity. Indeed, Yuan et al. (2020) showed that polysaccharides significantly reduced fasting blood glucose levels in mice with type 2 diabetes mellitus. Additionally, Ru et al. (2020) observed that a selenized polysaccharide reduced blood glucose levels and increased insulin levels, being considered a promising hypoglycemic compound. Wu et al. (2016) isolated polysaccharides from green tea (Camellia sinensis) and detected that the acid polysaccharide obtained showed an inhibiting activity of around 65% in relation to α-amylase. Wang et al. (2018) isolated three fractions of acid polysaccharides of the species Inonotus obliquus and observed that these fractions strongly inhibited the enzymes αamylose and α-glycosidase.
Researches with fungal metabolites are important due the possibility can lead to new therapies for the treatment of diabetes. A non-peptide fungal metabolite (L-783,281), for example, was isolated from the endophytic fungus Pseudomassaria sp., collected in a forest in the Democratic Republic of Congo (Strobel * and Daisy, 2003). Oral administration of this polysaccharide in two animal models of diabetes significantly reduced serum glucose levels. In addition, these compost acts as mimetic insulin and, unlike insulin, is not destroyed in the digestive tract, and can be administered orally (ZHANG et al., 2007).
On the other hand, the polysaccharide of P. byssoides did not present antibacterial potential against the evaluated strains (S. aureus, K. pneumoniae, P. aeruginosa, E. coli and P. mirabilis), because at the maximum tested concentration (10 mg/mL) the bacteria showed growth. Sharma et al. (2016) showed that the aqueous extract of the fungus Lentinula edodes showed small antibacterial activity against the bacteria Acinetobacter sp., E. coli, Klebsiella pneumoniae, Salmonella typhii and Vibrio cholerae.
Here, the antiproliferative activity of polysaccharide obtained from the fungus P. byssoides was also evaluated, however no such activity was observed in the tested concentration.

Conclusion
The results showed that the polysaccharide of P. byssoides has potential as hypoglycemic. Therefore, it would be valuable to perform in vivo studies with this polysaccharide aiming to confirm its hypoglycemic activity.