Polysaccharides for Biomedical ApplicationsView this Special Issue
Structural Characterization, Antioxidant Activity, and Biomedical Application of Astragalus Polysaccharide Degradation Products
To study the antioxidant capacity of Astragalus polysaccharides (APS) with different molecular weights, we used hydrogen peroxide to degrade original Astragalus polysaccharide (APS0) with an initial molecular weight of 11.03 kDa and obtained three degraded polysaccharides with molecular weights of 8.38 (APS1), 4.72 (APS2), and 2.60 kDa (APS3). The structures of these polysaccharides were characterized by 1H NMR, 13C NMR, FT-IR, and GC/MS. The degradation process did not cause significant changes in the main chain structure of APS. The monosaccharide component of APS before and after degradation was slightly changed. The antioxidant ability in vitro (removing hydroxyl and ABTS radicals and reducing ability) and in cells (superoxide dismutase and malondialdehyde generation) of these polysaccharides is closely related to their molecular weight. If the molecular weight of APS is very high or low, it is not conducive to their activity. Only APS2 with moderate molecular weight showed the greatest antioxidant activity and ability to repair human kidney epithelial (HK-2) cells. Therefore, APS2 can be used as a potential antistone polysaccharide drug.
Radix Astragali is one of the most popular medicinal herbs in China, and it has been used for more than 2000 years. One of its main active ingredients is Astragalus polysaccharides (APS), which show antioxidation, antitumor, and antiaging properties and cardiovascular, liver, and kidney protective effects [1, 2].
The polysaccharide property is closely related to its molecular weight, acid group content, monosaccharide composition, glycosidic linkage type, and main chain structure [3–6]. Given the different varieties, qualities, and processing methods, a certain difference exists in its monosaccharide composition and structure . Wang et al.  separated APS with a molecular weight of 3.6 × 104 Da from A. membranaceus roots by hot water extraction method. The main chain of APS is made up of α–D–(1 → 4)–Glc and (1 → 6)–α–D–Glcp, and the branching point is located at O-6. Fu et al.  obtained APS with a molecular weight of approximately 3.01 × 105 Da from Mongolian Astragalus using low concentration of ethanol for precipitation and gel chromatography for purification. Spectral analysis results of 1H NMR and 13C NMR showed that the APS backbone has a 1,3-linked β-D-Gal residue and the branched portion has β-Glc, 1,6-linked α-Gal; 1,5-linked β-Xyl; 1,4-linked β-Gal; β-D-Gal, 1,2-linked α-Rha; and 1,2,4-linked α-Rha residues.
The molecular weight affects the antioxidant ability of the polysaccharide. In addition, optimal antioxidant ability will vary depending on the type of polysaccharide. Liu et al.  reported that Ganoderma lucidum polysaccharide (GLPL1) with low molecular weight (5.2 kDa) has a higher ability to scavenge free radicals, superoxide radicals, and hydrogen peroxide than the component (GLPL2) with high molecular weight (15.4 kDa). At a concentration of 10 mg/mL, the scavenging rates of GLPL1 to hydroxyl radical and superoxide anion are close to 75% and 90%, respectively, whereas the scavenging ability of GLPL2 is only 50% and 60%, respectively. Ma et al.  isolated four kinds of polysaccharides, namely, GLP1 (>10 kDa), GLP2 (8–10 kDa), GLP3 (2.5–8 kDa), and GLP4 (<2.5 kDa), from G. lucidum by ultrasonic method. At the concentration of 0.5 mg/mL, the reducing ability was in the following order: GLP2 > GLP1 > GLP3 > GLP4, showing that the GLP2 polysaccharide with moderate molecular weight had the best reducing ability. Sheng and Sun  obtained four polysaccharides, namely, CPA-1, CPA-2, CPA-3, and CPA-4, from Athyrium multidentatum (Doll.) Ching by H2O2 degradation method, and their molecular weights were 14.53, 12.37, 11.55, and 6.40 kDa, respectively. The DPPH scavenging rates of the four polysaccharides with a concentration of 30 μg/mL were 63.35%, 50.23%, 42.45%, and 30.01%, respectively, showing that the polysaccharide with high molecular weight had the greatest scavenging activity.
Free radical-induced oxidative stress is thought to be one of the major causes of kidney disease, including kidney stones . Polysaccharides can remove free radicals in vitro and act as antioxidants to protect living organisms from oxidative damage . Khan  certified that free radicals can cause renal epithelial cell damage and allow urine crystals to attach to the damaged epithelial cell layer through simulation of the kidney environment in vitro. This research also proved that renal cell injury induced by free radicals can produce an environment conducive to crystal growth, accelerating the formation of stones. Antioxidant activity means that some antioxidants can protect cells against the damage of reactive oxygen species , such as superoxide, singlet oxygen, and hydroxyl radicals. Thus, research and exploring potent natural compounds with antioxidant activities and low cytotoxicity from plants have become very important in the field of biomedicine.
Based on this fact, the effects of the molecular weight of APS on its antioxidant capacity and cell repair ability were studied. The results of this study will provide enlightenment for screening for the optimal active polysaccharides.
2. Experimental Method
2.1. Reagents and Instruments
Astragalus polysaccharides (95% purity) produced by Shaanxi Ciyuan Biology Co. Ltd.; 30% hydrogen peroxide; anhydrous ethanol; trichloroacetic acid (CCl3COOH); o-phenanthroline; K4[Fe(CN)6]; ferrous sulfate (FeSO4•7H2O); potassium bromide (KBr); D2O (99.9%, Sigma); ascorbic acid (Vc); and ferric chloride (FeCl3) were all purchased from Guangzhou Chemical Reagent Factory.
Human kidney proximal tubular epithelial (HK-2) cells were purchased from Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum were purchased from HyClone Biochemical Products Co. Ltd. (UT, USA). Cell culture plates were purchased from Wuxi NEST Biotechnology Co. Ltd. (Wuxi, China). Malondialdehyde (MDA) kit and superoxide dismutase (SOD) kit were purchased from Jiancheng Institute of Biotechnology (China). Hematoxylin-eosin (HE) kit was purchased from Shanghai Beyotime Biotech Co. Ltd. (Shanghai, China).
The apparatus used in this paper were the following: KQ2200DB Ultrasonic Cleaning Instrument (Kunshan Ultrasonic Instruments Co. Ltd.), Ubbelohde capillary viscometer (0.45 mm, Qihang Glass Instrument Factory, Shanghai, China), conductivity meter (DDS-11A, LEICI, Shanghai, China), ultraviolet-visible spectrophotometer (Cary 500, Varian, Palo Alto, CA, USA), Fourier-transform infrared spectrometer (Equinox 55, Bruker, Karlsruhe, Germany), enzyme mark instrument (Safire2, Tecan, Männedorf, Switzerland), nuclear magnetic resonance (Varian Bruker-600 MHz, Bruker, Germany), 7890A-5975C gas chromatography-mass spectrometer (Agilent, USA), and fluorescence microscope (22DI-E-D282, Leica, Solms, Germany).
2.2. Experimental Methods
We followed the methods of Bhadja et al. .
2.2.1. Degradation of Polysaccharides
About 1.2 g original Astragalus polysaccharide (APS0) was weighed and dissolved in 20 mL distilled water. H2O2 solution was added to the reaction system—90°C water bath degradation for 2 h. The degradation reaction was cooled at room temperature, at which point the solution pH was adjusted to 7.0 by adding 2 mol/L NaOH solution. The degraded solution was then concentrated to one-third of its original volume at 60°C. The product was precipitated by adding three volumes of anhydrous ethanol. After filtration, the degraded polysaccharide was obtained by drying. The above experimental procedure was repeated by changing the H2O2 concentration at 4%, 6%, and 14%; degraded Astragalus polysaccharides with different molecular weights were obtained.
2.2.2. Measurement of Molecular Weights of Polysaccharides
According to the literature , the molecular weight was determined by Ubbelohde viscosity method at 25 ± 0.2°C. After determining the drop time of each polysaccharide in the viscometer, the relative viscosity and the specific viscosity were calculated, where and , where and are the drop time of polysaccharide solution and deionized water. According to the one-point method, the intrinsic viscosity , where is the concentration of the sample to be tested. The molecular weights of each polysaccharide before and after degradation were calculated based on values. The relationship between the intrinsic viscosity of the polymer solution and the molecular weight of the polymer can be expressed by the empirical equation of Mark-Houwink: , where and are the two parameters of the empirical equation and are constants related to polymer morphology, solvent, and temperature.
2.2.3. Analysis of Carboxylic Group Content
The carboxyl group (−COOH) content of APS was measured by conductometric titration. The conductivity titration curve was plotted using the conductivity value as the Y-axis and the used NaOH volume as the X-axis. The conductivity titration curve can be divided into three parts, a conductivity reduction stage (A), an equilibrium stage (B), and a conductivity increase stage (C). Three tangent lines are constructed from the three-stage curve, and the intersection is a stoichiometric point. The intersection of lines A and B gives the volume of NaOH () that excessive HCl and −SO3H consumed; the intersection of lines B and C gives the volume of NaOH () that excessive HCl and −SO3H and the –COOH of the APS consumed together; so, (platform portion) is the NaOH volume that the –COOH of the APS consumed alone. The −COOH content can be obtained according to the following formula : wherein (mol/L) represents the molar concentration of NaOH, (g/L) represents the mass concentration of the polysaccharide, 45 g/mol is the molar mass of −COOH, and 40 mL is the solution volume of polysaccharide. The final value is the average of three parallel experiments.
2.2.4. Fourier-Transform Infrared Spectroscopy (FT-IR) Analysis
A dried polysaccharide sample (2.0 mg) was mixed with KBr (200 mg). After grinding and pressing into KBr pellet, scanning was performed between the ranges of 4000 cm−1 to 400 cm−1 with a resolution of 4 cm−1.
2.2.5. 1H NMR and 13C NMR Detection of APS
According to the literature , 20 mg of fully dried APS1 polysaccharides was weighed and added to a NMR tube containing 0.5 mL of deuterated water (D2O), which is completely dissolved and placed in the magnetic field of the nuclear magnetic resonance spectrometer for detection.
2.2.6. Monosaccharide Component Detection of APS by GC-MS
According to the literature , 10 mg of APS1 polysaccharide was added to a 121°C sealed container containing 2.5 mol·L−1 trifluoroacetic acid (TFA) (2 mL) for 90 minutes. The solution was concentrated to dryness under reduced pressure, and then the TFA was removed with MeOH to a neutral solution and concentrated to dryness under reduced pressure. The residue was dissolved in 2 mol/L NH4OH (1 mL) and 1 mol·L−1 fresh NaBD4 (1 mL). The reaction was carried out at room temperature for 2.5 h and stirring was done at room temperature. Then two drops of acetic acid were added to decompose excess NaBD4 until no bubbles were produced. The solution was concentrated to dryness under reduced pressure. The filtrate was added with MeOH to remove boric acid and dried in vacuo. 1 mL of acetic anhydride was added and acetylated at 100°C for 2.5 h. The acetylated product was extracted with dichloromethane. The organic layer was washed with distilled water, dried, and analyzed by GC-MS. The HP-5MS capillary column (15 m × 250 μm × 0.25 μm) was programmed, and the temperature was raised from 135°C to 180°C at 0.5°C/min, then to 190°C at 10°C/min, and up to 310°C at 40°C/min. Helium acts as carrier gas, with a column flow rate of 0.6 mL/min. The acetylated product was identified by debris ions in GC-MS and relative retention times in GC. The structure is identified by peaks and assessed by peak area. Standard monosaccharides (rhamnose, arabinose, fucose, xylose, mannose, glucose, and galactose) are used as reference.
2.2.7. Hydroxyl Radical (•OH) Scavenging Activity of APS with Different Molecular Weights
The •OH scavenging ability of polysaccharide in vitro was detected by H2O2/Fe system method . The four different molecular weight polysaccharide solutions (60 μg/mL, 1 mL) were incubated with phenanthroline (2.5 mmol/L, 1 mL), ferrous sulfate (2.5 mmol/L, 1 mL), and hydrogen peroxide (20 mmol/L, 1 mL) in phosphate buffer (20 mmol/L, 1 mL, pH 7.4) for 90 min at 37°C. The absorbance measured at 536 nm was designated . The absorbance when hydrogen peroxide (H2O2) was replaced with distilled water and polysaccharide solution was and , respectively. Ascorbic acid (Vc) was used as the positive control group. The ability to scavenge hydroxyl radicals was calculated using the following equation:
2.2.8. ABTS Radical Scavenging Activity of APS with Different Molecular Weights
ABTS was oxidized to produce a stable blue-green cationic radical ABTS+. The antioxidant reacts with ABTS+ to cause the reaction to fade and initiate a change in the absorbance of a particular wavelength segment to determine the size of the antioxidant to remove ABTS free radicals .
The ABTS radical scavenging activity of polysaccharides was performed according to  with slight modification. 7 mmol/L ABTS solution was mixed with 2.45 mmol/L potassium persulfate aqueous solution, and then the mixture was incubated in the dark at room temperature for 12–16 h. Then 3 mL ABTS+ solution was added to 1 mL polysaccharide solutions of various concentrations in a test tube. After reacting for 6 min at room temperature, the absorbance was measured at 734 nm. where is the control group without polysaccharide, is the experiment group, and is the blank group without reagents (the absorbance of polysaccharide solution was 0).
2.2.9. Reducing Power of APS with Different Molecular Weights
Using the Prussian blue method , the polysaccharide sample may reduce the positive Fe3+ ions into Fe2+ ions in potassium ferricyanide (K4[Fe(CN)6]) to form Prussian blue and develop at 700 nm. That is, the greater the absorbance at 700 nm is, the stronger is the reduction of polysaccharide .
2 mL APS with different molecular weights (60 μg/mL) was mixed with 2 mL phosphate buffer solution (PBS) (pH = 6.6) and 2 mL 1% K4[Fe(CN)6]. The mixture was incubated at 50°C for 20 min. 2 mL 10% trichloroacetic acid was added to the mixture which was then centrifuged for 10 min at 3000 r/min. The supernatant (2 mL) was mixed with 0.5 mL FeCl3 (0.1%, w/v) solution and 2 mL distilled water. The mixture was shaken well and left to stand for 10 min. Then the absorbance was measured at 700 nm. Phosphate buffer was used as negative control, and Vc was used as positive control for comparison.
2.2.10. Cell Culture
HK-2 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin, and100 μg/mL streptomycin antibiotic with a pH of 7.4 and cultured in a 5% CO2-humidified environment of 37°C. Cells were passaged by trypsin digestion. Upon reaching 80%–90% confluent, cells were gently blown after trypsinization to form a cell suspension for the following cell experiments.
2.2.11. Superoxide Dismutase (SOD) Activity Detection
SOD activity was assessed using a commercially available kit based on the autooxidation of hydroxylamine. The cell suspension was inoculated in 24-well plates with a cell concentration of 5 × 104 cell/mL and 500 μL per well and was incubated for 24 h in an incubator at 37°C. The experimental model was divided into three groups: (1) control group: only serum-free medium was added, (2) injury group: serum-free medium containing 2.6 mmol/L oxalate was added for 3.5 h, and (3) repair group: 60 μg/mL of APS solutions with different molecular weights was added to the injured cells and repaired for 10 h. At the indicated time points, the treated cells were homogenized in 100 mmol/L Tris-HCl buffer and centrifuged at 10,000 rpm for 20 min and then the SOD activity was determined using assay kits. The absorbance of the supernatant was then measured directly by a microplate reader at 550 nm with a reference wavelength of 600 nm.
2.2.12. Malondialdehyde (MDA) Detection
For lipid peroxidation assay, we used a commercial kit to quantify the generation of MDA according to the manufacturer’s protocol. Cell culture and polysaccharide treatment were performed using a method similar to that of SOD measurement. After the repair effect was completed, the cells were harvested by trypsinization and cellular extracts were prepared by sonication in ice-cold buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 1 mM DTT). After sonication, lysed cells were centrifuged at 10000 rpm for 20 min to remove debris. The supernatant was subjected to the measurement of MDA levels by detecting the absorbance at 532 nm.
2.2.13. Hematoxylin and Eosin (HE) Staining
Cell suspension with a cell concentration of 5 × 104 cells/mL was inoculated per well in 12-well plates, 1 mL of DMEM containing 10% fetal bovine serum was added per well, and the cells were incubated for 24 h. Cell culture and polysaccharide treatment were performed using a method similar to that of SOD measurement. After the repair was completed, the supernatant was removed by aspiration and washed 2 times with PBS. Then the cells were fixed with 4% polyoxymethylene at room temperature for 15 min. After fixation, the cells were stained with hematoxylin stain for 15 minutes. The cells were then washed with distilled water for 2 min to remove excess stains. Thereafter, the cells were stained with an eosin staining solution for 5 min. The cells were washed with pure water for 2 min to remove excess eosin. After treatment, the cells were observed under a microscope, the nuclei were stained purple, and the cytoplasm was stained pink.
2.2.14. Statistical Analysis
Experimental data were expressed as mean ± SD. The experimental results were analyzed statistically using SPSS 13.0 software. The differences of means between the experimental groups and the control group were analyzed by Tukey’s test. indicates significant difference; indicates extremely significant difference.
3.1. Degradation of APS
Original Astragalus polysaccharide (APS0) with a molecular weight of 11,033 Da was degraded by H2O2 with concentrations of 4%, 6%, and 14%. Three kinds of degraded APS, namely, APS1, APS2, and APS3, with the molecular weights of 8376, 4716, and 2600 Da, respectively, were obtained.
3.2. Change in the Contents of the –COOH in APS before and after Degradation
The content of the –COOH in APS was measured by conductivity titration. The results are shown in Table 1. The carboxyl content of three degraded APS was between 16.2% and 17.2%, which only slightly changed (16.8%) before degradation.
The carboxyl group content of APS2 (17.2%) increased slightly. The reason is that hydroxyl radicals produced by the H2O2 degradation system break the sugar chain, exposing the carboxyl groups. Thus, high levels of carboxyl were detected in polysaccharides with low molecular weight after degradation . Chang et al.  degraded the original polysaccharide with a molecular weight of 1.29 × 102 kDa by ethanol precipitation and obtained two degraded polysaccharides with molecular weights of 60.0 and 52 kDa. The content of uronic acid was increased from 70.8% before degradation to 79.5% and 86.2%.
The carboxyl group content (16.9%) of APS3 decreased slightly, probably because free radicals generated by high concentration of H2O2 (14%) led to oxidative decarboxylation of polysaccharides, allowing the carboxyl groups in the polysaccharide chain to be converted into other groups, such as –OH .
3.3. Structural Characterization of APS by FT-IR Spectra
Figure 1 shows the FT-IR spectra of four APS. The FT-IR spectra of polysaccharides before and after degradation were similar, and no new peaks appeared, which indicated that the degradation of H2O2 did not cause significant effect on the overall structure of polysaccharides.
The absorption peaks of all four APS are shown in Table 1. The strong absorption band at 3392.7 cm−1 corresponds to the absorption peak of the stretching vibration of –OH in the polysaccharide. The absorption band at 2932.9 cm−1 corresponds to the stretching vibration of C–H . The two distinct absorbance peaks, at about 1616.7 and 1417.9 cm−1, suggested the presence of uronic acids. The absorption peaks at 1113.7 and 1027.7 cm−1 indicate that the polysaccharide contains α-glucose residues .
As the polysaccharide of each sample has the same mass (2.0 mg), the intensity of the absorption peak can reflect the content of the characteristic functional groups (such as the –COOH) . Compared with the original polysaccharide before degradation, the absorption peaks of the –OH and –COOH at 3408 and 1620 cm−1 were enhanced and the peaks of APS2 were the strongest, which was probably due to the exposure of the –OH and –COOH of polysaccharides after degradation, thus increasing their content [32, 33]. The intensity change of the absorption peak of the –COOH is consistent with the change rule of the measured content of the –COOH (Table 1).
3.4. Structural Analysis of APS by 1H NMR and 13C NMR
The polysaccharides were characterized by 1H and 13C NMR spectroscopy and typical spectra are shown in Figure 2.
3.4.1. 1H NMR
Table 2 lists the attribution of each line in the 1H NMR spectra of APS1 and APS2. The 1H NMR spectrum of APS1 was similar to that of APS2 (Table 2). For APS2, the signal peaks at δ5.31 and δ4.87 ppm correspond to the chemical shifts of H-1 of (1 → 4)–α–D–Glcp and (1 → 6)–α–D–Glcp, respectively. The highly overlapping peak at δ3.42–3.88 ppm belongs to the H-2, H-3, H-4, H-5, and H-6 signals of (1 → 4)–α–D–Glcp and (1 → 6)–α–D–Glcp of polysaccharides . The proton signals at δ5.12 and 5.08 ppm belong to the H-1 signal of (1 → 6)–α–D–Gal and (1→)–α–D–Rha, respectively, of polysaccharide.
3.4.2. 13C NMR Spectrum
Table 3 lists the attribution of the lines in the 13C NMR spectra of APS1 and APS2. The 13C NMR spectrum of APS1 was similar to that of APS2. For APS2, the signal at δ103.7 ppm belongs to the C-1 signal peak of β–D–Glcp of polysaccharide. The signals at δ99.6, δ71.5, δ73.2, δ76.7, δ71.2, and δ60.4 ppm are attributed to C-1, C-2, C-3, C-4, C-5, and C-6 signals of (1 → 4)–α–D–Glcp, respectively. The signals at δ71.9, δ73.1, δ69.3, δ70.4, and δ65.6 ppm are attributed to C-2, C-3, C-4, C-5, and C6 signals of (1 → 6)–α–D–Glcp, respectively [34, 35]. The signals at δ100.2, δ72.7, δ72.3, δ81.2, and δ73.3 ppm are attributed to C-1, C-2, C-3, C-4, and C-5 signal peaks of (1 → 4)–GalA, respectively . The signals at δ92.1, δ72.2, δ70.1, δ83.7, and δ69.0 ppm correspond to the C-1, C-2, C-3, C-4, and C-5 signal peaks of (1→)–α–D–Rha, respectively. The signals at δ70.5, δ72.4, and δ63.7 ppm are attributed to the C-4, C-5, and C-6 signal peaks of (1 → 6)–α–D–Gal, respectively.
3.5. Monosaccharide Composition of APS
The GC spectra of APS1, APS2, APS3, and 7 standard monosaccharides after derivatization were obtained by GC-MS, as shown in Figure 3. The retention time of each monosaccharide peak in APS is consistent with the retention time of standard monosaccharides of glucose, arabinose, rhamnose, and galactose (Figure 3(a)). According to the peak area, the molar ratio of monosaccharides in APS1 can be calculated as 11.4 : 7.8 : 0.7 : 1 (Figure 3(b)). The molar ratio of monosaccharides in APS2 is 13.9 : 8.2 : 1.2 : 1 (Figure 3(c)). The molar ratio of monosaccharides in APS3 is 13.3 : 8.4 : 1.1 : 1 (Figure 3(d)).
3.6. Differences in Scavenging Capacity of •OH Radicals of Polysaccharides with Different Molecular Weights
Among the reactive oxygen free radicals in the body, hydroxyl radicals (•OH) are known as the strongest oxidants, which can induce damage of surrounding biological macromolecules, such as certain proteins, nucleic acids, and unsaturated fatty acids. Such injuries can cause aging, cancer, and other diseases .
The •OH scavenging capacity of the four kinds of APS is shown in Figure 4. The •OH scavenging rate increased with the increase in APS concentration from 0.15 mg/mL to 3 mg/mL. The IC50 of APS0, APS1, APS2, APS3, and the positive control group (Vc) were 3.1, 2.7, 2.4, 4.2, and 0.06 mg/mL, respectively, which indicated that all APS had the ability to remove •OH radicals and APS2 with moderate molecular weight had the strongest scavenging capacity. At a concentration of 2.5 mg/mL, the scavenging rate of APS2 (63.4%) was twice as high as that of APS3 (31.7%) with the lowest molecular weight and it was also obviously higher than that of APS0 (45.3%) with the highest molecular weight. However, the scavenging rates of the four polysaccharides were all less than that of Vc (97.8%).
3.7. Differences of ABTS Radical Scavenging Capacity of APS with Different Molecular Weights
Four kinds of APS with different molecular weights all had an obvious scavenging effect on ABTS free radicals (Figure 5) and showed a dose-dependent relationship in the concentration range of 0.15–3 mg/mL. At the concentration of 2.5 mg/mL, the scavenging rates of APS0, APS1, APS2, and APS3 were 88.8%, 93.5%, 95%, and 83.2%, respectively, which indicated that APS2 with moderate molecular weight had the highest antioxidant activity and was slightly lower than Vc (98%).
3.8. Differences of Reducing Ability of APS with Different Molecular Weights
The reducing ability of materials in vivo provides hydrogen atoms to destroy the free radical reaction chain, so as to achieve antioxidation. Therefore, the reducing ability is an important indicator of the potential antioxidant capacity of antioxidants.
As shown in Figure 6, the reducing ability of each polysaccharide showed a concentration-dependent manner. When the concentration was 2.5 mg/mL, the ability of the four polysaccharides to reduce Fe3+ was in the following order: APS2 (1.06) > APS1 (0.8) > APS0 (0.76) > APS3 (0.6). APS2 had the highest reducing ability.
3.9. Changes of SOD Activity in Damaged Cells and APS-Repaired Cells
The reduction of SOD activity in organism implies decreased ability to resist free radical-induced damage in organism . Figure 7 shows the changes of SOD activity in damaged HK-2 cells after polysaccharide repair for 10 h. It can be seen that the activity of intracellular SOD increases first and then decreases with the decrease of APS molecular weight. The SOD activity in the control group was 8.5 ± 0.1 U/mL, and it decreased to 4.34 ± 0.25 U/mL after 2.6 mmol/L oxalate damage. After damaged cells were repaired by APS0, APS1, APS2, and APS3 for 10 h, the SOD activity increased to 5.95 ± 0.38, 6.3 ± 0.36, 6.87 ± 0.37, and 5.39 ± 0.29 U/mL, respectively. APS2 with moderate molecular weight has the strongest ability to enhance SOD activity.
3.10. Changes of MDA Content in Damaged Cells and APS-Repaired Cells
The change of MDA content usually reveals the level of lipid peroxidation in vivo and indirectly reflects the degree of cell injury . After the cells were injured by 2.6 mmol/L oxalate, the release of MDA increased from 0.86 ± 0.16 nmol/mL (control group) to 7.2 ± 0.3 nmol/mL (Figure 8), indicating that HK-2 cells suffered oxidative damage. When the damaged cells were repaired by APS0, APS1, APS2, and APS3 for 10 h, the generation content of MDA decreased from 7.2 ± 0.3 nmol/mL to 4.7 ± 0.3, 4.0 ± 0.3, 3.08 ± 0.28, and 5.15 ± 0.23 nmol/mL, respectively. That is, APS inhibit the release of MDA to the outside of the cells. Especially, APS2 with moderate molecular weight has the strongest ability to inhibit MDA release.
3.11. Cell Morphology Repair of APS with Different Molecular Weights in HK-2 Cells
The morphological changes of renal proximal tubule epithelial cells (HK-2) after repair by APS with different molecular weights were observed by hematoxylin and eosin staining (Figure 9). Normal HK-2 grows tightly and is plump. After oxidative damage by 2.6 mmol/L oxalic acid, the inherent form of HK-2 cells was lost. The cell volume was reduced, and nuclei staining was enhanced. Many dense apoptotic bodies were formed. After repair in injured HK-2 cells by APS with different molecular weights, the cell number increased and the cell morphology recovered similar to normal cells in varying degrees. Especially for the APS2-repaired cells, the morphology is the closest to normal cells. By contrast, APS3 with a lower molecular weight and APS0 with a higher molecular weight were less effective than APS2.
4.1. Chemical Structure of APS
The structure of polysaccharides is the basis for their biological activity. Many factors affect the biological activity of polysaccharides, including molecular weight, acid group content, monosaccharide composition, ligand type, and main chain structure [3–6]. In the present study, the monosaccharide composition and type of sugar residues of APS were analyzed by 1H NMR, 13C NMR, GC-MS, and FT-IR spectroscopy.
From the results of comprehensive analysis (Figures 1–3), α- and β-glycosidic bonds exist in the four kinds of APS. The main structure is that the main chain is composed of (1 → 4) connected Glcp and the branch point is located at the C-6 position of (1 → 6) connected Glcp, both containing 1,4-linked glucuronic acid fragments (Figure 10). The main structure is basically consistent with the structure illustrated by Li and Zhang . Fu et al.  also found that APS mainly consist of glucose, rhamnose, xylose, and galactose, which is inconsistent with our results in that the monosaccharides of APS consisted of rhamnose, arabinose, fucose, sugar, mannose, glucose, and galactose. Even with the same species of polysaccharides, differences will exist in their monosaccharide components due to their origin, processing method, separation process, and purification conditions .
The main chain structures of the four polysaccharides were similar from FT-IR results (Figure 1), but the absorption peak intensities of characteristic functional group of the polysaccharides before and after degradation were different (Table 1). This finding is mainly because the hydroxyl radicals produced by H2O2 degradation can attack the polysaccharide glycosidic bonds and cleave them, resulting in the change of side chain structure but not the main chain structure in polysaccharides. For example, Tian et al.  analyzed the chitosan structure before and after H2O2 degradation by infrared spectroscopy and nuclear magnetic resonance. The depolymerization only leads to changes of side groups, and no distinct change takes place in the structures of the main chain.
4.2. Effects of Molecular Weight on the Activity of APS Polysaccharides
4.2.1. Reasons for the Low Activity in Low-Molecular Weight APS
For different types of plant polysaccharides, the range of molecular weights that exhibit the best bioactivity is different. When the molecular weight of APS is very low, an active polymeric structure might not form, resulting in the loss of polysaccharide activity . In low-molecular weight polysaccharide, the unique bond linking method of polysaccharide and their three-dimensional structure (conformation) based on intramolecular hydrogen bond of polysaccharide will be destructed. The carbonyl group in low-molecular weight polysaccharide changed to be in an open chain, whereas in high-molecular weight polysaccharide, it was attached to a ring molecule. The destruction of the ring structure will break the hydrogen bond structure of the polysaccharide molecule, leading to reduced bioactivity.
Polysaccharide has two mechanisms of clearing the hydroxyl radical: the first is to remove the already-generated hydroxyl radicals and the second is to inhibit the generation of hydroxyl radicals . The former mechanism is related to that hydroxyl radical can quickly capture the hydrogen atom of the C-H chain of polysaccharide and form H2O, whereas, the carbon atom of polysaccharide generates a single electron and turns into carbon free radical which then will be oxidized to be a peroxy radical that decomposes harmless products to organism . The latter mechanism is related to the transition of metal ions that catalyze the generation of hydroxyl radicals, so the chelating ability may affect the hydroxyl radicals .
Hydroxyl radical can quickly capture the hydrogen atom of the C-H chain of polysaccharide and form H2O . In addition, the acid group of polysaccharide (such as −COOH) can decrease the production of hydroxyl radicals through the inactivating Fenton reaction. The carboxyl group content has considerable effect on scavenging •OH radicals . APS contained many hydroxyl groups and carboxyl groups in each monomer unit. Polysaccharides with low molecular weights would have more reductive hydroxyl group or carboxyl group terminals (on per unit mass basis) to accept and eliminate the free radicals . The chain structure of APS3 with the lowest molecular weight suffered significant destruction, and its molecular structure was the most loose. Therefore, the ability of APS3 to clear free radicals was the lowest. Chen et al.  reported that when the hydroxyl groups from polysaccharide are replaced with sulfuric acid groups, the antioxidant activity of polysaccharides decreases, which also indicates that the reduction in the number of hydroxyl groups will reduce the antioxidant activity of polysaccharides. Lai et al.  extracted two molecular weights of mung bean polysaccharides by ethanol precipitation. At a concentration of 0.8 mg/mL, the DPPH free radical scavenging rate (70.2%) of mung bean polysaccharide with low molecular weight (45 kDa) was weaker than that of mung bean polysaccharide (91.6%) with high molecular weight (83 kDa).
4.2.2. Reasons for the Low Activity in High-Molecular Weight APS
APS0 has high molecular weight, molecular volume, and viscosity and relatively low solubility, which affect its biological activity. The biological activity of polysaccharides is closely related to the single-helix structure of the main chain and hydrophilic group (hydroxyl group) located on the surface of the spirochetes . Another reason is because the polysaccharide with high molecular weight has a more compact structure and stronger intramolecular hydrogen bonds, resulting in less exposure of effective active groups. Qi et al.  found that the polysaccharide chain has a reduction and a nonreducing end. The greater the molecular weight of polysaccharides is, the lesser the reduction and nonreducing end contents are and the weaker the antioxidant activity is.
Polysaccharide degradation can effectively improve its antioxidant capacity. After degradation of APS0 to the polysaccharide with a suitable molecular weight, the branching degree of the polysaccharide becomes small, providing sufficient spatial extent to form a regular helical structure, which favors the exposure of more hydroxyl groups to the surface of the helical structure and is good for exerting bioactivity.
For example, Lycium barbarum polysaccharides with low molecular weight (10.2 kDa) have anticancer activity, whereas those with high molecular weight (6.50 × 103 kDa) have no anticancer activity . Similarly, Ligusticum chuanxiong polysaccharide with low molecular weight has a strong reducing ability . At a concentration of 5 mg/mL, the IC50 values of reduction capacity of three kinds of Rhizoma chuanxiong polysaccharides with molecular weights of 2.83 × 104, 1.23 × 104, and 6.31 × 104 Da are 1.05, 1.1, and 0.84 mg/mL, respectively. However, the immunoregulatory effect of Opuntia polysaccharides with high molecular weight (133 kDa) on macrophages is better than that of Opuntia polysaccharides with low molecular weight (168 kDa) .
4.2.3. APS with Moderate Molecular Weight Has the Strongest Activity
As shown in Figures 4–6, the antioxidant capacity of APS was in the following order: APS2 > APS1 > APS0 > APS3, and APS2 had the strongest antioxidant capacity. APS with a very high (APS0) or low (APS3) molecular weight had weakened antioxidant capacity. The ability of each polysaccharide to repair damaged HK-2 cells also showed the same activity sequence.
A number of studies have also confirmed that polysaccharides with moderate molecular weight have more conducive activity [54, 55]. For example, Im et al.  studied the immunomodulatory and antitumor functions of aloe polysaccharides with different molecular weights. The results showed that the inhibitory rate of aloe polysaccharide G2E1DS2 (Mw = 5–400 kDa) with moderate molecular weight on the growth of S180 cells in mice was 91.53%. The inhibition rates of G2E1DS3 (>400 kDa) with the highest molecular weight and G2E1DS1 (<5 kDa) with the lowest molecular weight were only 14.87% and 15.89%, respectively. Fan et al.  isolated three polysaccharides (DDP1, DDP2, and DDP3) with molecular weights of 51.5, 26.1, and 6.95 kDa, respectively, from Dendrobium denneanum by hot water extraction. At the concentration of 2 mg/mL, the scavenging ability of the three polysaccharides to •OH, DPPH, and ABTS radicals was in the following order: DDP2 > DDP1 > DDP3. Moreover, the antioxidant capacity of DDP2 with moderate molecular weight (26.1 kDa) was the strongest.
Chen et al.  obtained three kinds of APS, namely, APS1-1, APS2-2, and APS3-3, by DEAE resin column chromatography, with molecular weights of 112, 98.3, and 20.4 kDa, respectively, and with the uronic acids content of 0.0%, 5.9%, 11.6%, respectively. Their corresponding total antioxidant capacities were 1152.8, 1566.3, and 1580.4 μmol/L, respectively, at the concentration of 2 mg/mL. These findings showed that polysaccharides with a high uronic acid content and a low molecular weight have great antioxidant ability. There are two variables (molecular weight and uronic acid content) in this study, and it is difficult to determine which factor is the most important in affecting antioxidant activity. In our study, we obtained four different molecular weight APS by H2O2 degradation method and the differences of their carboxyl content were small (ranging from 16.2% to 17.2%). Therefore, the influence of the carboxyl group content on the antioxidant ability can be avoided. At the same time, the molecular weight of APS used in our study ranged from 2.60 to 1.03 kDa, which is much less than what Chen et al.  reported. We also found that only APS with moderate molecular weight showed the greatest antioxidant capacity.
Three degraded APS with molecular weights of 8.38, 4.72, and 2.60 kDa were obtained by degradation of primitive APS (APS0, Mw 11.03 kDa) with different concentrations of H2O2. The results of 1H NMR, 13C NMR, FT-IR, and GC-MS showed no significant difference in the main chain structure of the four polysaccharides. All four polysaccharides had (1 → 4)–D–glucose as the main chain and (1 → 6)–D–glucose linkage as the branch chain. The monosaccharide component of APS before and after degradation was slightly changed. APS with four molecular weights had the ability to scavenge hydroxyl and ABTS radicals and good reducing ability. The antioxidant capacity of APS was in the following order: APS2 > APS1 > APS0 > APS3, that is, the antioxidant effect of APS2 with moderate molecular weight was the best. Moreover, the polysaccharides showed good ability to enhance SOD activity, inhibit MDA release, and recover cell morphology. APS, especially APS2, can be used as potential antioxidative drugs for renal protection.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare no competing financial interest.
This research work was granted by the National Natural Science Foundation of China (nos. 21701050 and 81670644) and the China Postdoctoral Science Foundation (no. 2017M612837).
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