Structure characterization of novel heteropolysaccharides from Pteridium revolutum with antioxidant and antiglycated activities

Highlights • Novel water-soluble polysaccharides, PRP0, PRP1, and PRP2 were isolated from Pteridium revolutum.• Molecular weight and monosaccharide composition of three polysaccharides were analyzed.• The structures of PRP1 and PRP2 were systematically characterized by a combination of chemical and spectra methods.• PRP1 and PRP2 are highly branching polysaccharides with a backbone of α-galactose and α-mannose units.• All polysaccharides showed significant antioxidant and antiglycated activity.


Introduction
Bracken ferns have more than 12,000 species widely distributed in uplands and marginal areas throughout Asia, Australia, Europe, North and South America, with about 2600 species in China (Kardong, Upadhyaya, & Saikia, 2013;Xu et al., 2009).Certain ferns species (Pteridium) are used as food or plant medicine to treat ailments in several countries (Chen et al., 2015;Wang, & Wu, 2013).Many chemical compositions such as flavonoids, terpenoids, and steroids with various bioactivities including antioxidant, antibacterial, anti-Alzheimer's disease, antiosteoporosis, hypolipidemic and hypoglycemic activities have been isolated from Pteridium genus (Castillo et al., 2003;Song et al., 2017;Zhao et al., 2022).Pteridium revolutum (Blume) Nakai (Pteridiaceae family), widely distributed in south of Asia and north of Australia, has been used as food and ethnic medicine in traditional Chinese medicine.It has the functions of removing dampness, diuresis, antipyretic and insect repellent.
Plant polysaccharides have drawn considerable attention from the world because of their potential application value in various industries (Sun et al., 2008).Previous works have demonstrated that polysaccharides possess essential pharmacological activities including antioxidant, antiglycated, anti-inflammatory, hypolipidemic, antimicrobial and immunoregulatory (Chen et al., 2019(Chen et al., , 2023;;Long et al., 2022;Song et al., 2019;Wang et al., 2022;Zhu et al., 2019).Polysaccharides isolated from the Pteridaceae family were also reported have antioxidant and immunomodulatory activity (Xu et al. 2009;Song et al., 2017;Zhao et al., 2022).So far, the detailed structure of polysaccharides from P. revolutum still unclear.Therefore, the present work aimed to investigate the extraction, separation, structure characterization and the bioactivity of polysaccharides from this plant.The results exhibit that the novel polysaccharides have strong antioxidant and antiglycated activities in a concentration dependent manner.It could be used as a functional ingredient in food to reduce the formation of glycosylation products of protein.

Polysaccharides preparation from P. revolutum
The sample of P. revolutum was collected from Suichang County, Zhejiang province, China.A voucher specimen was deposited in Zhejiang Gongshang University, Hangzhou, China.The dried aerial part of P. revolutum was ground to pass a 60-mesh sieve and extracted by refluxing ethanol (95% v/v, 2 × 2 h) to remove liposoluble compounds.Then, the sample was extracted with hot distilled water (10 vol, 90 • C, 3 × 3 h).The extracts obtained were vacuum concentrated at 55 • C by a rotary evaporator (Buchi R-210, BUCHI Labortechnik AG, Switzerland) and precipitated with ethanol (75% v/v, 4 • C, 24 h).The sample were centrifuged (8,000 g, 5 min).The precipitate was re-dissolved in ultrapure water to remove the protein using the Sevage method (n-butanol: chloroform, 1:4 v/v).The aqueous phase was concentrated, dialyzed (3,500 molecular weight cut off (MWCO)) against distilled water for at least 2 d.Finally, the solution was vacuum concentrated (55 • C) and lyophilized to offer the crude polysaccharide (cPRP).

Partial acid hydrolysis
The purified polysaccharides (each 10 mg) were hydrolyzed by TFA (2.0 M, 100 • C, 2 h) (Song et al., 2017).The product was dialyzed (MWCO 3,500) in ultra-pure water for 2 d.The dialyzable fraction (outside of the bag, O) and the non-dialyzable fraction (inside of the bag, I) were lyophilized separately, offering the partially hydrolyzed samples PRP0-O, PRP1-O, PRP2-O, PRP0-I, PRP1-I, and PRP2-I, respectively.Monosaccharide compositions of these six samples were tested by GC as Section 2.2.

Periodate oxidation and Smith degradation
Twenty milligrams of PRP0, PRP1, or PRP2 sample were oxidized with NaIO 4 (15 mM, 25 mL, 120 h), respectively.Ethylene glycol (1 mL) was used to destroy the excess NaIO 4 .The consumption of periodate was detected by Ultraviolet spectroscopy (UV, 223 nm).The generated products of formic acid were titrated by NaOH (0.01 M).After reduced by NaBH 4 and neutralized with HOAc to pH 7.0, the solution was then hydrolyzed in TFA (2 M, 120 • C, 3 h) and analyzed the monosaccharide compositions by GC (Zhao et al., 2022).

Methylation analysis
The samples of PRP1 and PRP2 (each 20.0 mg) were methylated with the method reported (Song et al., 2017) and detected by Infrared spectroscopy (IR).The methylated samples were hydrolyzed, reduced and acetylated, successively.The obtained alditol acetates were detected by Gas chromatography-mass spectrometry (GC-MS).PRP0 did not methylate because of no more sample.GC-MS analysis: Agilent 5975C system (Agilent Technologies Inc., USA), DB-17 MS column.The starting temperature of the column is 50 • C, then raised to 230 • C (4 • C/min), finally reached to 280 • C (10 • C/min, maintain 15 min).Ion-source: 230 • C. N 2 : 1.0 mL/min.

BSA-Glucose glycation model
The anti-glycation ability of PRPs was assayed using Bovine serum albumin-Glucose (BSA-Glu) model according to Hafsa's method (Hafsa et al., 2016) with some modifications.The total 5.0 mL of reaction mixture consisted of phosphate buffer (pH 7.0, 0.1 M, 0.2% NaN 3 ), BSA (10 mg/mL), glucose (0.5 M), and samples with different concentrations (0.25 ~ 2.0 mg/mL).Negative control: no aminoguanidine (AG) and polysaccharide.Positive control: AG instead of polysaccharide.All the mixtures were reacted in dark (100 • C, 40 min).The mixture is cooled in ice water bath and stored at 4 ℃ for the following experiments.The fluorescence of the solution was measured using at an excitation/emission wavelength of λ 380 /λ 450 nm (LS55 spectrofluorometer, Perkin-Elmer, USA).The inhibition activity was calculated as following equation (Eq).
F 0 : Fluorescence intensity of negative control.F 1 : Fluorescence intensity of sample/AG solution.

Analysis of amadori products
The amadori products were determined by method reported before (Zhu et al., 2019).0.1 mL glycated BSA was added to 0.1 mL 0.15 mM NBT reagent (Dissolved in 100 mM sodium carbonate buffer, pH 10.35).The mixture was incubated for 30 mins at room temperature and the absorbance was tested at λ 530 nm on a Syhergy H1 Microplate Reader (BioTek, USA).

Analysis of dicarbonyl compounds
The content of dicarbonyl compounds were measured by Girard-T assay (Zhu et al., 2019).0.4 mL glycated BSA was added into the mixed solution containing Girard-T stock solution (0.2 mL, 500 mM) and sodium formate (3.4 mL, 500 mM, pH 2.9) and then reacted for 1 h at room temperature.The absorbance was determined at λ 294 nm (UV-1880 spectrophotometer, Shimadzu Corporation, Japan).Glyoxal was used a standard to make a calibration curve.

Analysis of pentosidine
The content of pentosidine was determined by a LS55 spectrofluorometer (Sun et al., 2010).The fluorescence of the glycated solution was determined at an excitation/emission wavelength of λ 335 /λ 385 nm.The effect on pentosidine was calculated as following Eq.
F 0 : Fluorescence intensity of negative control.F 1 : Fluorescence intensity of sample/AG solution.

Statistical analysis
Statistical analysis was analyzed by T-test to assess the significance (SPSS 20.0) of difference between groups and all data were given as mean ± SD.

Preparation, molecular weight and monosaccharide composition determination
cPRP was obtained from the P. revolutum by hot distilled water extraction, precipitated in 75% EtOH (v/v), deproteinated and freezedried.cPRP gave four peaks on the DSFF CC for purification (S-Fig.1).The three major fractions, eluted with ultra-pure water, 0.1 and 0.2 M NaCl solution, were further chromatographed by Sephadex G-200 (S-Fig.2) to provide three purified novel polysaccharides (named PRP0, PRP1, and PRP2).
The homogeneity and average molecular weight of three novel polysaccharides was identified by HPGPC (S-Fig.3).The average MW was 1.04 × 10 6 , 8.39 × 10 5 , and 7.37 × 10 5 Da, which calculated using the Standard curve of Dextran molecular weight (S-Fig.4), respectively.As showed in UV spectra (S-Fig.5), three polysaccharides were free of protein because of the weak absorption at 260-280 nm.

Structural characterization
The chemical structures of three purified polysaccharides were then systemically characterized by chemical and spectroscopy methods.
Additionally, the substitution of H atom of OH on the sugar ring results in the chemical shift of corresponding C atom downfield shifting (Roslund et al., 2011;Yao et al., 2021) 3.
By comprehensive analyses of the information of partial acid hydrolysis, periodate oxidation, methylation, and NMR spectra, the predicted structures of two novel polysaccharides PRP1 and PRP2 are shown in Fig. 1.

Scavenging activity on DPPH
The scavenging activity of four polysaccharides on DPPH free radicals were shown in Fig. 2A.The polysaccharides showed significant effects against DPPH free radicals.PRP0, PRP1, and PRP2 have better activity than cPRP.PRP2 has the best scavenging activity increased from 7.94 ± 1.87% to 86.92 ± 1.62% following the concentration increased from 0.05 to 3.2 mg/mL in a concentration-dependent manner.As a control, Vc showed higher activity than all polysaccharides, reaching 98.11 ± 1.09% at 3.2 mg/mL.
The scavenging activity on DPPH free radicals of PRP2 is better than reported polysaccharides PLP and PAP-3 from P. aquilinum (Xu et al., 2009;Zhao et al., 2022).The activity of all the isolated polysaccharides was lower than that of the oligosaccharides derived from P. aquilinum reported by Wang (Wang & Wu, 2013).The activity of PAP-3 is slightly better than that of PLP.The molecular weight may be an important factor antioxidant affecting activity.

Scavenging ability on • OH
The scavenging activity of four polysaccharides on • OH were shown in Fig. 2B.The ability was enhanced according the concentration increased from 0.05 to 3.2 mg/mL.The scavenging activities of four polysaccharides could be ordered as PRP1 > PRP2 > PRP0 > cPRP, and PRP1 displayed remarkable scavenging rate of 83.37 ± 0.98% at a concentration of 3.2 mg/mL, while Vc showed higher scavenging rate of 98.34 ± 0.21% at the same concentration.

Antiglycation capability of PRPs in the BSA-Glu model
The inhibitory activity of PRPs on glycation was assayed using BSA-Glu model.In the early stage of glycation, the carbonyl group of glucose react with compounds containing free amino groups to form unstable Schiff bases through carbonyl amine condensation, which undergo irreversible rearrangement to form more stable Amadori products.Amadori products can react with NBT reagent to produce a colored substances, which can be measured at λ 530 nm.The inhibition rate on Amadori products iomproved gradually with increasing concentration  of PRPs and the positive AG (Fig. 3A).Among the tested inhibitors, the inhibitory activity was in the order of PRP1 > PRP0 > AG > cPRP > PRP2.At the concentration of 2.0 mg/mL, the inhibition rate of cPRP, PRP0, PRP1, PRP2 was 40.26 ± 1.72%, 45.66 ± 1.18%, 53.26 ± 0.59%, and 35.22 ± 1.03%, respectively.In the intermediate stage, the dicarbonyl compounds were formed from Amadori products after oxidation and dehydration.In this research, PRPs showed inhibition activity on the formation of dicarbonyl compounds, and the inhibition rate gradually increased with the concentration increasing from 0.25 to 2.0 mg/mL.Among them, cPRP and PRP1 have better inhibitory activity than positive control AG, and cPRP exhibited the best inhibition ability with inhibition rate 60.28 ± 1.48% at 2.0 mg/mL (Fig. 3B).
In the last phase, the polymerization of carbonyl compounds with amino proteins to form advanced glycation end products (AGEs), and pentosidine is a representative compound of fluorescent AGEs.As shown  in Fig. 3C and 3D, all PRPs had strong inhibitory effect on the formation of pentosidine and fluorescent AGEs.The ability of the samples to inhibit pentosidine was in the order of AG > cPRP > PRP1 > PRP2 > PRP0, while to fluorescent AGEs formation was in the order of AG > PRP0 > cPRP > PRP1 > PRP2.
The contents of PCO and free thiol group were used to evaluate the protein oxidation induced by AGEs.As shown in Fig. 3E, the carbonyl content of glycated BSA was 14.43 nmol carbonyl/mg proteins.The level of PCO decreases significantly with the PRP sample is added to glucose-glycated BSA.At the concentration of 2.0 mg/mL, cPRP, PRP0, PRP1, PRP2 displayed 5.66 ± 0.66, 3.56 ± 0.51, 8.91 ± 0.82, 7.41 ± 0.48 nmol carbonyl/mg proteins, respectively, and there was no significant difference between the activity of PRP0 and aminoguanidine (P < 0.01).
The effects of PRPs on protein thiol group level in glucose-glycated BSA were shown in Fig. 3F.The protein thiol group content was significant decrease in glycated BSA comparing to BSA solution.The addition of PRPs can increase the thiol content in glycated BSA solution.
At the concentration of 2.0 mg/mL, all PRPs showed significant improvement compared to the glycated BSA, but still had a significant difference from the positive control AG (P < 0.01).
Many factors can induce the formation of AGEs and protein damage.The production of reactive oxygen species and free radical is one of the mechanisms (Hafsa et al., 2016;Shen, Xu, & Sheng, 2017).In our study, PRPs showed inhibitory activity against the formation of protein carbonyl groups and protective effect on protein thiol groups may be related to their antioxidant activities of scavenging free radicals and reactive oxygen species.

Conclusions
The preparation and structure characterization from P. revolutum polysaccharides were reported for the first time.The spectrometry and spectroscopy analyses indicated that PRP1 consists of (1 → 3,6)-linked D- Man and (1 → 3)-linked D-Glc, while PRP2 consists of (1 → 2,4)-linked D-Man and (1 → 3)-linked D-Gal on main chain.Interestingly, compare the antioxidant activities between PRP1 and PRP2, PRP1 has better hydroxyl radical scavenging ability, while PRP2 has better DPPH scavenging ability.These differences may be resulted in the physiochemical and structural properties of the two polysaccharides, such as the molecular weight, monosaccharide contents, glycosidic linkage, main-chain, branched-chain, and water solubility.The difference in the content of L-Arabinose, L-Rhamnose, and D-Galactose may be an important factor for the activity.Moreover, polysaccharide with smaller MW usually has better performance on antioxidant activity because of better solubility.Consequently, in future research, a comprehensive understanding of the structure-activity relationships of P. revolutum polysaccharides will be studied.
In the BSA-Glu model, PRPs displayed inhibitory activity on the formation of Amadori products, dicarbony compounds, pentosidine, fluorescent AGEs, PCO and have a protective effect on protein thiol group.These results suggest that PRPs can be used as an natural antioxidants and the inhibitors of AGEs.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Table 1
Results of monosaccharide composition (MC) and partial acid hydrolysis of PRPs (Molar ratio).

Table 2
GC-MS data for methylation analysis of PRP1 and PRP2.