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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 72  |  Page : 738-744  

Isolation, structural characteristics, and in vitro and in vivo antioxidant activity of the acid polysaccharide isolated from Pholiota nameko


1 Department of Landscape Plants, Sichuan Agricultural University, Chengdu 610041, China
2 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China

Date of Submission24-Mar-2020
Date of Decision06-May-2020
Date of Acceptance03-Dec-2020
Date of Web Publication16-Feb-2021

Correspondence Address:
Aoxue Luo
Department of Landscape Plants, Sichuan Agricultural University, Chengdu 611130
China
Gang Wang
Department of Landscape Plants, Sichuan Agricultural University, Chengdu 611130
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pm.pm_109_20

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   Abstract 


Background: Previous studies have revealed that edible fungi are rich in polysaccharides, which typically have strong biological activity. Pholiota nameko is one of the main cultivated edible fungi and an important economic crop. Objectives: In order to screen a polysaccharide with antioxidant activity from Pholiota nameko. Methods and Materials: A water-soluble acidic P. nameko polysaccharide (PNP) was found from extraction with alkaline water. After purification by chromatography, the structural features of PNP were analyzed by chromatographic, spectral, & nuclear magnetic resonance (NMR) spectroscopic analyses and the content of sulfate & uronic acid in PNP was determined concurrently. Results: The content of sulfate and uronic acid was 1.756 and 7.173 w/w, respectively. The molecular weight of PNP was 1.05 × 106 Da by gel permeation chromatography. The Fourier transform infrared (FTIR) and NMR spectra displayed that the following sugar chains might be present in PNP: β-1,4-D-Manp, →4)-β-D-Manp-(1→, β-1,3-D-Glcp, β-D-Xylp-(1→. Evaluation of in vitro antioxidant activity showed that PNP had a strong scavenging ability to scavenge ABTS+, hydroxyl, and superoxide anion radicals. The results of our in vivo experiments exposed that PNP might significantly lessen the formation of malondialdehyde. High levels of PNP can meaningfully increase the activity of catalase and superoxide dismutase activity in mice serum. In addition, PNP can significantly increase the activity of GSH-Px, effectively protect the structure and function of the cell membrane. Conclusion: PNP has antioxidant effect and can be explored as a potential antioxidant.

Keywords: Acid polysaccharide, antioxidant, free radical, Pholiota nameko, structural characteristics


How to cite this article:
Fan Y, Chun Z, Wang G, Pu S, Pan Y, Ma J, Miao R, Luo A. Isolation, structural characteristics, and in vitro and in vivo antioxidant activity of the acid polysaccharide isolated from Pholiota nameko. Phcog Mag 2020;16:738-44

How to cite this URL:
Fan Y, Chun Z, Wang G, Pu S, Pan Y, Ma J, Miao R, Luo A. Isolation, structural characteristics, and in vitro and in vivo antioxidant activity of the acid polysaccharide isolated from Pholiota nameko. Phcog Mag [serial online] 2020 [cited 2021 Feb 28];16:738-44. Available from: http://www.phcog.com/text.asp?2020/16/72/738/309300



SUMMARY

  • Pholiota nameko is a popular pleasant edible fungus and is extensively cultivated in China. It is opulent in polysaccharides, which display biological activity. In order to extraction and characterization of an acid polysaccharide with antioxidant activity from Pholiota nameko, a water-soluble acidic P. nameko polysaccharide (PNP) was found from extraction with alkaline water. The results exhibited that the content of sulfate and uronic acid was 1.756 and 7.173 w/w, respectively. The molecular weight was 1.05 × 106 Da. The following sugar chains might be present in PNP: β-1,4-D-Manp, →4)-β-D-Manp-(1→, β-1,3-D-Glcp, β-D-Xylp-(1→. Antioxidant activity showed that PNP had a strong scavenging ability to scavenge ABTS+, hydroxyl, and superoxide anion radicals, and might significantly lessen the formation of malondialdehyde, increase the activity of catalase, superoxide dismutase activity and GSH-Px.




Abbreviations Used: PNP: Polysaccharide of Pholiota nameko; IR: Infrared spectra; NMR: Nuclear Magnetic Resonance; OH•: Hydroxyl radicals; ABTS+: 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); CAT: Catalase; SOD: Superoxide dismutase; GSH-Px: Glutathione peroxidase; LPS: Lipopolysaccharide; DPPH: 1,1-Diphenyl-2-picrylhydrazyl; HPGPC: High-performance Gel Permeation Chromatography; GC-MS: Gas chromatography–mass spectrometry.


   Introduction Top


Polysaccharides can produce more than 10 monosaccharide units after hydrolysis. It is extensively scattered across almost all organisms such as animals, plants, micro-organisms, and algae. It is one of the four basic substances establishing a life and is closely related to the upkeep of life function. It is used as an energy source and is a basic material of various organisms. However, the authors have established that polysaccharides from different sources have complex biological structures and that glycoconjugates are strictly related to many diseases, such as cancer and bacterial and viral infections.[1] Therefore, polysaccharides have attracted greater attention among various groups of researchers. Current studies have revealed that many polysaccharides are allied with various properties such as immunomodulatory effect,[2],[3] antitumor effect,[4],[5] antioxidant activity,[6] and anti-inflammatory activity[7] as a treatment for lupus nephritis,[8] and hypoglycemic activity,[9],[10] and some active polysaccharides also play an vital role in preventing oxidative damage.[11],[12]

Previous studies have exposed that when the level of free radicals increases, it can cause biofilm damage, protein denaturation, enzyme inactivation, and DNA replication errors, thereby accelerating cell aging and death.[13] Polysaccharides from edible mushrooms can scavenge free radicals, surge antioxidant enzyme activity, inhibit lipid peroxidation, protect biofilm from external stress, and deferral aging. Tian[14] found that water-soluble polysaccharides from Houttuynia cordata Thunb established good antioxidant activity.

Pholiota nameko is a popular pleasant edible fungus and is extensively cultivated in China. It is opulent in polysaccharides, which display hypolipidemic,[15] anti-inflammatory,[16] and immunological effects.[17] It also comprises proteins that show antitumor activity.[18] However, we intended to extract, isolate, and conduct a structural analysis of the acidic polysaccharide from P. nameko (PNP) and assess its antioxidant capacity under in vitro and in vivo conditions.


   Materials and Methods Top


Materials and chemicals

Dextrans were purchased from Pharmacia Co. (Uppsala, Sweden). AB-8 and ADS-7 were procured from the Chemical Plant of Nankai University (Tianjin, China). 1,1-Diphenyl-2-picrylhydrazine (DPPH) radical; lipopolysaccharide (LPS); 2,2-azobis (2-methylpropionamidine)-dihydrochloride (AAPH); trolox; 3- [4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT); and Vitamin C were procured from Sigma (St. Louis, USA). 2,2'-azinobis(3-ethylbenzthiazoline) 6-sulphonic acid (ABTS) was procured from Merck, (Darmstadt, Germany). Ethanol, petroleum ether, pyridine, hydroxylamine hydrochloride and all other chemicals and reagents were of analytical grade.

Extraction and purification of Pholiota nameko polysaccharide

PNP was extracted according to the method of Xiao et al.[19] First, 50 g of the powder of P. nameko was added into a round-bottomed flask with 500 mL petroleum ether (b.p. 60°–90°), and the mixture was refluxed in a water bath for 2 h, filtered, and the filtrate was dried until a continuous mass was found. To the dried residue, 0.2 M sodium hydroxide (500 mL) was added to extract the polysaccharide and mixed at room temperature for 6 h. Then, the filtrate was collected and neutralized by adding 0.2 M hydrochloric acid (500 mL). The protein was extracted by Sevage reagent (n-butanol: trichloromethane = 1:4) for four times.[20] The solvent is concentrated to 100 mL, and anhydrous ethanol is added slowly until the volume of ethanol in the system reaches 70%. Finally, the precipitate was collected by centrifugation.

The crude PNP was more purified based on the method described by Luo et al.[21] with minor alterations. First, the polysaccharide was dissolved in deionized water, filtered with 0.45 μm nucleolar pore, and purified on AB-8 macroporous resin column (26 mm × 300 mm) to eliminate the pigment. Next, the eluant was purified with ADS-7 ion exchange resin (26 mm × 300 mm) to confiscate the residual protein. Then, 80 mg of crude PNP was dissolved in 8 mL of ddH2O and loaded onto DEAE-cellulose column and then serially eluted with ddH2O, 0.05 M NaCl, 0.1 M NaCl, and 0.2 M NaCl solution and the eluate was collected by the fully automatic partial collector. A total of 30 tubes (6 mL each, elution speed 1 mL/min) were collected for each elution gradient. The polysaccharide content was observed by the sulfuric acid phenol method. The purified PNP samples were collected based on the elution peak (PNPw, PNP, PNPx, and PNPn were obtained by gradient elution). Finally, the purified sample was dialyzed with deionized water for 72 h, frozen, and dried.

Structural characteristics of Pholiota nameko polysaccharide

The content of protein in PNP was evaluated according to the method of Bradford.[22] The total flavone content in PNP was determined by AlCl3 method described by Zuo et al.[23] The total phenolic content was analyzed by the Folin phenol method based on the explanation.[24] The content of sulfate radical in the PNP was assessed by the phenol sulfuric acid method with K2SO4 as the standard.[25] The uronic acid content was analyzed by carbazole sulfuric acid method using D-glucuronic acid as the standard.[26]

The molecular weight (MW) of PNP was determined by using the high-performance gel permeation chromatography (HP-GPC) according to the Yamamoto method.[27] Samples and series of standard dextran solutions (2500; 4600; 7100; 10,000; 21,400; 41,100; 84,400; 133,800; 200,000; 555,500; 1,110,000; and 2,990,000 Da) were analyzed by HP-GPC using refractive detector. The analytical columns were UltrahydrogelTM 2000 and UltrahydrogelTM 250 (ID: 300 × 7.8 mm) in series at room temperature. We used 0.2 mol/L phosphate buffer solution as the mobile phase with a flow rate of 0.7 mL/min.

The structural features of PNP were measured by Fourier-transform infrared spectroscopy (FTIR).[28] Briefly, 1.0 mg PNP was ground consistently with KBr powder at a ratio of 1:20 (W: W) and then scanned by using FTIR spectrophotometer (Perkin-Elmer Corp., USA) in the scanning range of 4000–500 cm−1. The monosaccharide composition in the PNP was analyzed by GC-MS, 1H-NMR, and 13C-NMR spectral analysis according to the method defined by Luo.[29]

In vitro antioxidant activity

ABTS free-radical scavenging assay

In this experiment, the free-radical scavenging activity of PNP against ABTS+ was determined according to the method of Re[30] with slight alterations. ABTS powder (0.0192 g) was weighed in a centrifuge tube and 5 mL of phosphate-buffered saline (PBS) (0.01 mol/L) was added to dissolve it. After this, potassium persulfate (0.0033 g) was precisely weighed and added to it and entirely dissolved. The tube was closed and placed in dark for 16 h at room temperature for ABTS+ to be formed. 100 mL of the reaction mixture was withdrawn and diluted by adding 0.01 mol/L PBS to the solution so that the absorbance value at 734 nm was 0.70 + 0.02. The diluent reacts at room temperature for 30 minutes. Standard solution of Vitamin C (0.1 mL) and PNP were added into the test tube; blank control was prepared with the same amount of deionized water. Next, 1.0 mL of ABTS solution (A734 = 0.70 + 0.02) was added to each test tube and permissible to react at room temperature for 20 min. The absorbance of the solution at 734 nm was assessed by ultraviolet spectrophotometer. The scavenging effects were considered based on the following formula:

ABTS radical scavenging activity (%) = [Ac − (A − Ab)]/Ac × 100.

Where Ac is the absorbance of ABTS solution without the added sample at 734 nm, Ab is the absorbance of sample without the addition of ABTS, and A is the absorbance of sample and ABTS.

DPPH free-radical scavenging activity

DPPH free-radical scavenging capability of various fractions was assessed using the method of Fan et al.[31] with some alterations. Briefly, 0.3 mL of standard solution (Vitamin C) and PNP solution with various concentrations (50, 100, 200, 400, 800, 1600, and 3200 μg/mL) were taken in the test tube; blank control was prepared by adding the same amount of deionized water. Then, 0.9 mL of DPPH methanol solution (0.1 mmol/L) was added to each test tube and the reaction was conducted at room temperature for 30 min. After the reaction, the absorbance of the reaction mixture was read at 517 nm by UV-Vis spectrophotometer. The DPPH free-radical scavenging ability was considered as follows:

DPPH radical scavenging activity (%) = [Ac − (A − Ab)]/Ac × 100.

Where Ac is the absorbance of DPPH without sample at 517 nm, Ab is the absorbance of the sample without DPPH, and A is the absorbance of sample and DPPH.

Hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity of PNP samples was measured according to the method of Luo et al.[32] Briefly, 0.3 mL PNP and Vitamin C at with various concentrations (50, 100, 200, 400, 800, 1600, and 3200 μg/mL) were incubated with 0.5 mL of EDTA–Fe (2 mM), 1.0 mL of H2O2 (3% v/v), and 0.36 mg/mL crocus in 4.5 mL sodium phosphate buffer (150 mM, pH 7.4) for 35 min at 37°C and the hydroxyl radicals were distinguished by monitoring the absorbance at 520 nm. The hydroxyl radical scavenging activity of samples was considered as follows:

Hydroxyl radical scavenging activity (%) = [(Ac − A)/Ac] × 100.

Where Ac is the absorbance of the control solution and A is the absorbance of the sample solution.

Superoxide anion free-radical scavenging activity

Determination of superoxide anion radical scavenging activity of PNP was conducted according to the method described by Wang et al.[33] with minor alterations. Briefly, 4.5 mL of 50 mmol/L Tris-HCl (pH 8.2) and 1.0 mL of PNP or Vitamin C at various concentrations (50, 100, 200, 400, 800, 1600, and 3200 μg/mL) were mixed in tubes. The reaction mixture was incubated for 20 min at 25°C. At the same time, 0.4 mL of pyrogallol (25 mmol/L) was added. After 4 min, the reaction was completed by adding 0.1 mL of 8.0 mol/L HCl solution. The absorbance of the sample was assessed by the UV-Vis spectrophotometer at 325 nm. The scavenging activity of PNP against superoxide anion was examined by the following equation:

Superoxide anion scavenging activity (%) = (Ac − A)/Ac × 100.

Where Ac is the absorbance without sample and A is absorbance with a sample at 325 nm.

In vivo antioxidant activity

Specific-pathogen-free Kunming mice (male: female = 50:50, provided by Sichuan Academy of Medical Sciences) weighing 18–22 g were employed in this study. The mice were randomly separated into four groups: control group (normal saline group), high-concentration PNP group (200 mg/mL), medium-concentration PNP group (100 mg/mL), and low-concentration PNP group (50 mg/mL). After 28 days, the mice were forfeited and serum from the eye socket was used to observe the concentrations of GSH-PX, superoxide dismutase (SOD), and malondialdehyde (MDA).

Statistics analysis

The test results were articulated as mean ± standard deviation (SD). Statistical analysis was conducted using SPSS software version 23.0 and significant difference, very significant difference, and extremely significant difference were uttered by P < 0.05, P < 0.01, and P < 0.001, respectively.


   Results Top


Preparation and structural characteristics of Pholiota nameko polysaccharide

The polysaccharides PNPw, PNP, PNPx, and PNPn were gotten by gradient elution of ultrapure water, 0.05 M NaCl, 0.1 M NaCl, and 0.2 M NaCl on DEAE-cellulose column (26 mm × 300 mm) [Figure 1]. PNP was found as a gray powder after purification from the macroporous adsorption resin and ion-exchange chromatography; the crop was 5.8%. According to the results, PNP did not encompass phenols and flavonoids. However, it satisfied of sulfuric radical (1.756 w/w) and uronic acid (7.173 w/w) in PNP.
Figure 1: Isolated of crude polysaccharide by DEAE-cellulose column (26 mm × 300 mm)

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The MW of PNP is carefully related to its biological activity. If the MW is too big, it may damagingly disturb the solubility of PNP and its biological activity. Therefore, we verified the MW of PNP by comparing it with the standard curve of dextran attained by high-performance gel chromatography. Based on this, the MW of PNP was found to be 1.05 × 106 Da.

Infrared spectrum of PNP [Figure 2] demonstrations that the absorption peak of −OH group of the sugar molecule was recorded at 3401 cm−1, the absorption peak of C-H was recorded at 2924 cm−1, and the absorption peak of C = O was recorded at 1646 cm−1, which displays that PNP is a polysaccharide in nature. The characteristic absorption at 891 cm−1 indicates the β-configuration of the sugar units.[34] In addition, there were two absorption peaks between 1120 and 1010 cm−1, which shows a furanose form of sugar in PNP. From the infrared spectrum of PNP, it can be contingent that it is mainly unruffled of β-form of furanose.
Figure 2: Fourier-transform infrared spectra of the polysaccharide Pholiota nameko polysaccharide

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We found four peaks for the standard monosaccharides in the GC-MS spectrum of PNP, among which the peak area of glucose derivative was the uppermost, representing that glucose is the most important monosaccharide in PNP. The peak area of mannose is the second largest, signifying that the content of mannose is great. However, the peak area of xylose was smaller, demonstrating that the content of PNP is fewer. According to the calculation of peak area, the molar ratio of four monosaccharades in PNP is as trails: mannose: glucose: galactose: xylose = 28.05 mol%:41.23 mol%:16.92 mol%:3.86 mol%.

This demonstrates the monosaccharide type of PNP, the configuration of glycosidic bond, and the position of sugar connection from carbon spectrum and hydrogen spectrum [Figure 3]a and [Figure 3]b. In 1H-NMR, the proton signal of heterocyclic carbon is mostly concentrated in the region less than δ5.0 ppm, designating that the sugar molecules in PNP were mainly of β configuration. However, there were also some absorption peaks in the region greater than δ5.0 ppm, specifying that a small amount of sugar molecules in PNP was of α-configuration.[35],[36] The signal of δ102.55 ppm in 1H-NMR and δ4.53 in 13C-NMR presented that the glucose residue in PNP was mostly of β-configuration. The absorption peaks at δ3.88 ppm and δ3.75 ppm in 1H-NMR are typical signals of mannose. The absorption peaks at δ4.532 ppm and δ3.28 ppm in 1H-NMR are typical signals of glucose. The absorption peaks at δ3.546 ppm and δ3.88 ppm in 1H-NMR are typical signals of galactose. The absorption peak at δ4.7 ppm in 1H-NMR is β-D-Xylp-1 → heterocephalic proton.[37] There is no absorption peak in the 13C-NMR spectrum from δ61 ppm to δ68 ppm, demonstrating that there is no 1 → 6 bond in PNP. 13C-NMR spectra show absorption peaks in the range of δ70.7 ppm to δ73 ppm, indicating that there is a C4 bond in PNP. In 13C-NMR spectra, there are absorption peaks in the range of δ76.8 ppm to δ80 ppm, showing that PNP has branching at C3 and C4 positions and there may be 1 → 3 and 1 → 4 bonds.[38] Based on the articles, we can say that the above-mentioned chains are the main sugar chains in PNP [Table 1].
Figure 3: Nuclear magnetic resonance test of Pholiota nameko polysaccharide. (a) For 13C-NMR test of Pholiota nameko polysaccharide and (b) for 1H-nuclear magnetic resonance test of Pholiota nameko polysaccharide

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Table 1: The chemical shifts (1H nuclear magnetic resonance and 13C nuclear magnetic resonance) of the polysaccharide Pholiota nameko polysaccharide

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In vitro antioxidant activities

The free-radical scavenging activity of PNP was verified on superoxide anion, hydroxyl, DPPH, and ABTS radicals [Figure 4]. The scavenging capacities of PNP were found to be related to its concentration, viewing a certain concentration dependence.
Figure 4: Antioxidant activities of Pholiota nameko polysaccharide in vitro. (a) The scavenging effects of Pholiota nameko polysaccharide on ABTS radicals, (b) The scavenging effects of Pholiota nameko polysaccharide on DPPH radicals, (c) The scavenging effects of Pholiota nameko polysaccharide on OH radicals, (d) The scavenging effects of Pholiota nameko polysaccharide on superoxide anion radicals

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In the ABTS free-radical experiment [Figure 4]a, the positive control (Vitamin C) presented a strong dose-dependent scavenging activity. The maximum scavenging was recorded at 3200 μg/mL (89.9%). PNP also established a good scavenging effect on ABTS free radicals. The effect was not clear at lower concentration but augmented with increasing concentration. The maximum outcome was recorded at 3200 μg/mL (76.5%), which was close to that of the positive control. Thus, we can say that PNP displays a significant effect on ABTS radical scavenging (P < 0.05).

[Figure 4]b displays the scavenging effect of PNP on DPPH free radicals. The positive control (Vitamin C) and PNP exhibited dose-dependent scavenging ability. With the increase in the concentration, the scavenging activity of each sample presented an upward trend. The maximum activity was recorded at 100 μg/mL for Vitamin C (90.5%). On the contrary, the scavenging ability of PNP on DPPH radical was not noteworthy (P > 0.05). Even at a high concentration (3200 μg/mL), its scavenging capability was only 30.1%. Thus, we can say that PNP has no significant scavenging effect on DPPH free radicals.

[Figure 4]c confirms the scavenging activity of Vitamin C against hydroxyl radicals. We established low concentrations (0–800 μg/mL) and high concentrations (800–3200 μg/mL) and the maximum activity was recorded at 3200 μg/mL (94.5%). PNP established slowly increasing activity in the range of 0–1600 μg/mL and reached the maximum value (98.6%) at 3200 μg/mL (P < 0.05). There was no important difference between PNP and Vitamin C. According to our results, PNP proved significant scavenging ability against hydroxyl radicals.

[Figure 4]d expresses the results of scavenging superoxide anions [Figure 4]d. According to our results, the scavenging effect of PNP and Vitamin C is comparable. With the increase in the concentration, the scavenging activity gradually upsurges. There was no statistically significant difference between the scavenging effect of PNP and Vitamin C at 400 μg/mL concentration (P > 0.05), indicating that PNP had a very sturdy superoxide anion scavenging activity.

Antioxidant activities in vivo

GSH-Px is a vital antioxidant enzyme, which scavenges hydrogen peroxide in vivo. It can also precisely catalyze the reduction of reduced glutathione to hydrogen peroxide and defend the structure and function of the cell membrane. As shown in [Table 2], compared to the control, 100 mg/mL PNP suggestively augmented the content of GSH-PX in the serum of mice (P < 0.05). However, at low concentrations (50 mg/mL), there was no significant change between PNP and the control. With the increase in the concentration, the effect of PNP on GSH-PX increased progressively. The maximum activity was recorded at 200 mg/mL. PNP might suggestively recover the activity of GSH-PX (P < 0.01).
Table 2: Antioxidant activities of the Pholiota nameko polysaccharide in vivo

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SOD mostly scavenges superoxide anion radicals under in vivo conditions. The reduction in the activity of SOD might lead to lipid peroxidation and deposition of lipofuscin in cells, resultant in cellular dysfunction. [Table 2] demonstrates that PNP might significantly increase the activity of SOD in mice (P < 0.05). Compared with the control group, PNP expressively increased the activity of SOD at low concentration (50 mg/mL) (P < 0.05). With the increase in PNP levels, the effect of PNP on the activity of SOD is superior.

Hydrogen peroxide can induce lipid peroxidation of cell membranes, abolish the structure of cell membranes, induce apoptosis, and contribute in a variety of pathological mechanisms. Catalase (CAT) can exactly eliminate hydrogen peroxide from the body and avoid oxidative damage. According to our results, a high concentration of PNP might significantly increase the activity of CAT (P < 0.05), but a medium-to-low concentration of PNP had no significant consequence on CAT activity.

MDA is an imperative metabolite in the process of lipid peroxidation. It is produced by the oxidation of unsaturated fatty acids on the cell membrane under the direct action of radiation and hydroxyl radical. Therefore, the quantity of MDA formed can specify the degree of lipid peroxidation and it is one of the vital indicators to judge the damage of free radicals. The results showed that compared with the control group, low concentration of PNP may intentionally remove MDA in mouse serum, and the higher the concentration of PNP, the more obvious the effect of removing MDA.


   Conclusion Top


In this study, PNP was prepared by alkali water extraction and ethanol precipitation. The content of the sulfuric radical and uronic acid in the polysaccharide PNP was confirmed, after purifying it on a macroporous resin. According to the findings, the level of sulfuric radical was 1.756 w/w% and the level of uronic acid was 7.173 w/w%. The MW of PNP was 1.05 × 106 Da using the HPGPC method. The results of GC-MS display that PNP comprises four monosaccharides and glucose is the most plentiful monosaccharide in PNP. The molar ratio of four monosaccharides in PNP was as follows: mannose: glucose: galactose: xylose = 28.05 mol%:41.23 mol%:16.92 mol%:3.86 mol%. The outcomes of Fourier-transform infrared spectroscopy indicate that PNP chiefly checked furanose in the β-configuration form and that there may be the following sugar chains in PNP: s β-D-Xylp-(1 → heterocephalic proton,β-1,4-D-Manp, →4)-β-D-Manp-(1→,β-1,3-D-Glcp, β-D-Xylp-(1→.

PNP displayed a resilient scavenging activity against ABTS free radicals, hydroxyl free radicals, and superoxide anion free radicals under in vitro circumstances. The antioxidant effect of PNP was comparable to that of Vitamin C. However, the DPPH scavenging activity of PNP was fragile.

In vivo findings established that PNP significantly abridged the content of MDA in mice compared with the control group, signifying that PNP scavenges lipid peroxidation products under in vivo conditions. High concentrations of PNP can significantly increase the activity of CAT and SOD in mice, which augments the role of endogenous free-radical scavenging system and lessens the production of lipid peroxides. At the same time, PNP can suggestively increase the activity of GSH-PX and effectively protect the structure and function of cell membrane, which displays that PNP has antioxidant effect in vivo.

Previous studies have revealed that many plant polysaccharides have immunopotentiating activity and P. nameko polysaccharides can significantly progress the immune function of tumor mice.[39] Concurrently, there are reports on polysaccharides with regard to their antioxidant activity and protection of cell membrane integrity. Some polysaccharides can struggle with the damage of immune cells induced by reactive oxygen species and play a part in the immunoregulation by refining the antioxidant activity of enzymes in immune cells and preserving the integrity of their cell membrane. In this study, PNP confirmed antioxidant activity under in vitro and in vivo conditions and played an important part in increasing the immunomodulatory ability. Thus, it can be employed as a potential antioxidant molecule.

Financial support and sponsorship

This research was funded by the National Natural Science Foundation of China (grant number: 31700302), International Cooperation and Exchange Research Program of Sichuan Province (grant number: 2017HH0095), and Science and Technology Project of Sichuan Province (grant number: 2017SZ0022).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

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