Purification, characterization, and antioxidant ability of polysaccharides from Phascolosoma esculentas

Abstract The polysaccharide was extracted from Phascolosoma esculenta (PEP). Two purified polysaccharides (PEP‐1 and PEP‐2) were obtained by the column chromatography separation method. The molecular weights of PEP‐1 and PEP‐2 were 33.6 and 5.7 × 103 kDa, respectively. PEP‐1 and PEP‐2 had the same monosaccharides composition, but their molar ratios varied. The in vitro antioxidant activity of the PEP, PEP‐1, and PEP‐2 were investigated by scavenging free radicals like 3‐ethylbenzoth‐iazoline‐6‐sulfonic acid (ABTS), •OH, and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH). Additionally, the in vivo antioxidant activity of PEP‐1 was examined using the Caenorhabditis elegans (C. elegans) organism. Results showed that PEP‐1 was much more effective than PEP and PEP‐2 at scavenging DPPH, •OH, and ABTS radicals. Additionally, PEP‐1 strengthened C. elegans' ability to endure oxidative stress. PEP‐1 possessed the in vivo antioxidant capacity, including the reactive oxygen species (ROS) content reducing, and protective effect on antioxidant enzyme activities in C. elegans. In summary, PEP, PEP‐1, and PEP‐2 might have the potential to develop as functional foods and clinical medications.

Phascolosoma esculenta (P.esculenta), a member of the Phascolosoma genus, is an important food resource in China (Wu et al., 2020).P. esculenta is also known as "jelly seaworm," which contains mostly collagen.Thus, it can keep its beauty and health.
The most essential bioactive component of P. esculenta is polysaccharides.Recently, several researchers extracted polysaccharides from P. esculenta and discovered that the polysaccharides of P. esculenta exhibited antioxidant properties (Liang, 2008;Wu et al., 2020).However, there were limited researches on the antioxidant capacity and structural study of the purified polysaccharides from P. esculenta.Much more attention is required for the development and extension of P. esculenta polysaccharide applications.
In this study, two purified polysaccharides were obtained from P. esculenta.The characterization of the polysaccharides was tested.
Additionally, the polysaccharides' antioxidant properties were evaluated.This research would contribute to the development of polysaccharides from P. esculenta as a functional food and natural drug source that can prevent and treat chronic diseases.

| Extraction and purification
Fresh P. esculenta was washed, and combined with sodium acetate buffer (0.1 M, 1:30 w/v), and then homogenized.After that, the homogenate was mixed with 5% papain.The mixture was stirred and incubated in a water bath (DK-600 Shanghai Feiyue Instrument Co., Ltd., Shanghai, China) at 55°C for 6 h.After the incubation, the mixture was kept at 95°C for 10 min.Afterward, the mixture was centrifuged at 4°C, 7606 g for 25 min.The supernatant was combined with sevag reagent (n-butanol: chloroform = 1:4, v/v) to remove the protein.The extract was mixed with 95% ethanol in a volume ratio of 1:3 (v/v) and then kept in a refrigerator (4°C, 12 h).The polysaccharides of P. esculenta (PEP) were obtained after freeze-drying (FDU-2110, Eyela Co., Ltd., Tokyo Japan).
PEP (5 mg/mL) was combined with distilled water and purified using Q Beads 6FF column (2.6 × 60 cm).Distilled water and a gradient NaCl solution (0.1-0.5 mol/mL) were used to elute the column.
The flow rate was 3 mL/min.Each tube has a capacity of 10 mL.
The polysaccharide content was analyzed as reported by Yang et al. (2020).The eluate was then dialyzed (3500 Da) and freezedried to produce P. esculenta purified polysaccharides (PEP-1 and PEP-2).Using the following formula, the extraction yield of polysaccharides was determined.
A i : the weight of the dried polysaccharides; A 0 : the weight of dried P. esculenta.

| Molecular weight and monosaccharide composition analysis
The molecular weights of the PEP-1 and PEP-2 were determined using high-performance gel permeation chromatography (HPGPC, 1260 Infinity MDS, Agilent, USA) with a deferential refraction detector and a multi-angle laser light scatterer (Guo et al., 2020).The content of monosaccharide was analyzed by high-performance liquid chromatography (1200, Agilent, USA) as reported by Liu et al. (2020).

| Fourier transform infrared (FT-IR) spectroscopy analysis
Potassium bromide (100 mg) was combined with polysaccharides (1 mg) before being formed into a disk.The FT-IR spectral analysis was performed with a Fourier transform infrared spectrophotometer (Nicolet IS50, Thermo Fisher, USA) in the 4000 to 400/cm range.

Extraction yield( % ) =
A i is the absorbance of DPPH solution with the sample solution, A 0 is the absorbance of DPPH solution without the sample solution, and A j is the absorbance of the sample solution.
A i is the sample absorbance, A 0 and A j are the distilled water absorbance instead of sample or hydrogen peroxide, respectively.

| ABTS radical scavenging activity
The ABTS radical scavenging activity was assayed as described by Wei et al. (2022).Simply, the same volume of potassium persulfate solution (5 mL, 2.45 mM) and ABTS solution (5 mL, 7 mM) were combined to start the reaction for 12 h at 25°C.At 734 nm, the absorbance of the mixture was adjusted to 0.7 ± 0.05, using distilled water.
Additionally, the 96-well plate was placed in an incubator at 25°C for 6 min.At 734 nm, the absorbance was measured.Using the following formula, the ABTS scavenging activity was determined.
A i is the absorbance of ABTS solution with the sample solution, A 0 is the absorbance of ABTS solution without sample solution, and A j is the absorbance of the sample solution.

| Caenorhabditis elegans and culture conditions
Caenorhabditis elegans was fed E. coli OP50as the food source.
Nematode growth medium (NGM) plates were used to cultivate the C. elegans.The incubation temperature for C. elegans was 20°C.
C. elegans were treated with phosphate buffer saline, and then homogenized with an ice-cold tissue grinder.The bicinchoninic acid (BCA), GSH-Px, MDA, and SOD were determined separately based on the kit instructions (Nanjing Jiancheng Biotechnology Co., Ltd., Nanjing, China).

| Statistical analysis
GraphPad Prism 10 was used to process the images.Differences were considered significant at p < .05.All data were expressed as mean ± standard deviation of three replicates.

| Yield
PEP was obtained from P. esculenta, resulting in a yield of 7.91%.As shown in Figure 1, the PEP was separated by Q Beads 6FF column and the purified polysaccharides of P. esculenta (PEP-1 and PEP-2) were obtained.PEP-1 eluted by distilled water with a yield of 3.75%.
PEP-2 eluted by 0.1 mol/L NaCl with a yield of 2.12%.

| FT-IR spectrum analysis
The chemical bonds of PEP-1 and PEP-2 were investigated by FT-IR (Figure 3).The broad band at 3415/cm was attributed to O-H stretching vibration, and the band at 2933/cm was attributed to C-H stretching vibration.The absorption peaks at 1655/cm were assigned to the stretching vibrations C=O, indicating the presence of uronic acid, which was associated with the monosaccharide components (Fu et al., 2020).The peak near 1540/cm is the absorption peak of C-OH bending vibration.A characteristic absorption peak of 930 and 1078/cm may suggest the presence of β-glycosidic linkage.
Simultaneously, these results indicated that the structure of PEP-1 and PEP-2 displayed typical skeletons of polysaccharides.

| SEM analysis
SEM is a straightforward approach to investigating polysaccharides morphology.As shown in Figure 4, there were no significant

| In vitro antioxidant activity assays
The in vitro antioxidant activities of PEP, PEP-1, and PEP-2 were investigated in this research.The scavenging activity against radicals of ABTS, •OH, and DPPH were evaluated.Figure 5a

| Effect of polysaccharides on the survival of C. elegans
Caenorhabditis elegans.survival rate was shown in Figure 6.In comparison to the control group, the survival rate of C. elegans treated with PEP-1 increased by 13.01%, 25.67%, and 38.33% at dosages of 0.25, 0.5, and 1 mg/mL, respectively.C. elegans survival rate exhibited a dose-response relationship with the polysaccharides concentration.Polysaccharides of P. esculenta exhibited protective effects against oxidative damage to C. elegans induced by H 2 O 2 .

| DISCUSS ION
The enzyme has been applied to deproteinate the polysaccharide substances.Polysaccharides from marine resources such as abalone and sea cucumber are frequently extracted using papain (Guo et al., 2020;Zhang et al., 2011).Therefore, papain was used to obtain polysaccharides from P. esculenta.Presently, ABTS, •OH, and DPPH assays are frequently used to evaluate an antioxidant's potential (Wang et al., 2018;Zhang et al., 2016).In this research, two purified polysaccharides (PEP-1 and PEP-2) from P. esculenta were harvested.The in vitro antioxidant activity results demonstrated that PEP, PEP-1, and PEP-2 had scavenging activities against ABTS, •OH, and DPPH radicals.The free radical scavenging ability of polysaccharides was intimately related to their chemical structures, including the position and type of glycosidic bonds, monosaccharide conformation, and molecular weight (Ferreira et al., 2015;Ji et al., 2017;Wang et al., 2019).In this research, PEP-1 and PEP-2 both contained 12 monosaccharides.
In comparison to PEP, the PEP-1 displayed better antioxidant activities.Low molecular weight of polysaccharides would exhibit stronger free radicals scavenging activity (Wang et al., 2019).PEP-1, with a low molecular weight, demonstrated higher antioxidant activity than PEP-2.Similar phenomena were found by Shao et al. (2023).PEP-1, with a high galactose content, exhibited superior antioxidant activity compared to PEP-2, aligning with Sun's findings that polysaccharides rich in galactose possess enhanced free radical scavenging ability (Sun et al., 2020).Furthermore, some studies suggested that the βglycosidic bond configuration contributed to the antioxidant ability of polysaccharides (Song et al., 2018;Wang et al., 2018).
ROS homeostasis is critical for recovery from oxidant stress.
Excessive ROS can cause oxidative damage to organisms, reflecting Figure5c, the polysaccharides from P. esulenta exhibited scavenging activities against ABTS radicals.At the same concentration, the PEP-1 had the highest ABTS radicals scavenging rate compared to PEP and PEP-2, and the PEP-2 had the lowest ABTS radicals scavenging rate.PEP-1 had better in vitro antioxidant activity than PEP and PEP-2.Therefore, PEP-1 was used for the in vivo antioxidant activity assays.

Figure 7
Figure 7 illustrated that the C. elegans in control group exhibited strong fluorescence intensity.It might suggest that the C. elegans were in a situation of oxidative stress.Compared to control group, the PEP-1 could decrease the fluorescence intensity of C. elegans.The fluorescence intensity of C. elegans decreased with higher contraction of PEP-1.C. elegans treated with 1 mg/mL PEP-1 had the lowest fluorescence intensity.There are significant variations among different contractions of PEP-1 on ROS level in C. elegans (p < .05, Figure 8).The increase in

F
Immunofluorescence staining of ROS in Caenorhabditis elegans.(a) 0 mg/mL of PEP-1, (b) 0.25 mg/mL of PEP-1, (c) 0.5 mg/mL of PEP-1, and (d) 1 mg/mL of PEP-1.F I G U R E 8 ROS level of Caenorhabditis elegans.Values marked by different letters are significantly different (p < .05).

F
Oil red O staining of Caenorhabditis elegans.(a) 0 mg/mL of PEP-1, (b) 0.25 mg/mL of PEP-1, (c) 0.5 mg/mL of PEP-1, and (d) 1 mg/mL of PEP-1.F I G U R E 1 0 Relative fat level of Caenorhabditis elegans.Values marked by different letters are significantly different (p < .05).F I G U R E 11 The antioxidant enzymes activities and MDA content.Values marked by different letters are significantly different (p < .05).