Physicochemical Characterization and Biological Properties of Polysaccharides from Alpiniae oxyphyllae Fructus

Polysaccharides (AOPs) were extracted from Alpiniae oxyphyllae fructus using three distinct methods: hot water (AOP-HW), hydrochloric acid (AOP-AC), and NaOH/NaBH4 (AOP-AL). This study systematically investigated and compared the physicochemical properties, structural characteristics, antioxidant activities, and α-amylase inhibitory activities of the extracted polysaccharides. Among the three AOPs, AOP-AC exhibited the highest yield (13.76%) and neutral sugar content (80.57%), but had the lowest molecular weight (121.28 kDa). Conversely, AOP-HW had the lowest yield (4.54%) but the highest molecular weight (385.42 kDa). AOP-AL was predominantly composed of arabinose (28.42 mol%), galacturonic acid (17.61 mol%), and galactose (17.09 mol%), while glucose was the major sugar in both AOP-HW (52.31 mol%) and AOP-AC (94.77 mol%). Functionally, AOP-AL demonstrated superior scavenging activities against DPPH, hydroxyl, and ABTS radicals, whereas AOP-AC exhibited the strongest inhibitory effect on α-amylase. These findings indicate that the extraction solvent significantly influences the physicochemical and biological properties of AOPs, thus guiding the selection of appropriate extraction methods for specific applications. The results of this study have broad implications for industries seeking natural polysaccharides with antioxidant and enzymatic inhibitory properties.


Introduction
Alpiniae oxyphyllae Miq., belonging to the species of Zingiberaceae, is an herbal medicine distributed between the Yangtze River and the Nan Mountains in China.The dried mature fruit of Alpiniae oxyphyllae (A.oxyphyllae) Miq. was considered to have the potential to warm the kidney, reduce the urine, and store essential substances in traditional Chinese medicine theory [1][2][3].In China, A. oxyphyllae fructus has been used on a large scale as a Chinese medicine material.A. oxyphyllae fructus mainly contains polysaccharide, essential oil, terpene, flavonoid, and heptane derivatives [4].Modern pharmacological studies have proved that A. oxyphyllae fructus polysaccharide (AOP) shows a good efficacy in urinary incontinence, anti-neuro inflammatory activity, antiviral activity, and immunoregulation [1,[5][6][7][8].Yang et al. characterized the structure of A. oxyphyllae fructus polysaccharide and found that it could significantly increase the phagocytic activity of RAW264.7 macrophages [9].Generally, the bioactivity of polysaccharides depends on the chemical composition and structural features, which varies with a range of factors, especially the extraction solvent employed [10][11][12].
Extraction conditions, including temperature, pH, and the type and concentration of the extracting solvents, can directly influence the structure of the obtained polysaccharide.Solvents have significant effects on the biological activities and physicochemical features of polysaccharides [13,14].Water, acid, and alkali are common extracting agents, selected based on the nature of the polysaccharide.Hot water extraction is widely utilized for polysaccharide extraction because it is simple, low-cost, safe, and environmentfriendly [15,16].Acidic and alkali solutions are also usually used to extract polysaccharides.It has been reported that acidic solvents are effective in cleaving glycoside linkages within polysaccharides, leading to increased yields of small-molecule polysaccharides [17,18].Moreover, several relevant studies have found that alkali-extracted polysaccharides possess higher bioactivities, such as anti-inflammatory and antioxidant properties [19,20].Zhang, Zhu, Zhang, Yang, Ni, Zhang, Liu, and Zhang [21] extracted polysaccharides from green tea leaf using different extraction solvents and found that alkali-extracted polysaccharide showed a higher viscosity and better gelling properties than hot-water-and acid-extracted polysaccharides.Therefore, it is necessary to compare the physicochemical and biological properties of polysaccharides with different solvents.Thus far, the research about polysaccharides from A. oxyphyllae fructus has all involved water extraction [1,5,6].The effect of different extraction solvents on the physicochemical and biological properties of polysaccharides of A. oxyphyllae fructus is still unclear.
In summary, the objective of this study was to evaluate the effects of different solvents (hot water, hydrochloric acid, and NaOH/NaBH 4 ) on the physicochemical (chemical composition, molecular weight (Mw), morphology, and crystal structure) and biological (antioxidant and α-amylase inhibitory ability) properties of polysaccharides extracted from A. oxyphyllae fructus.This work might provide valuable information for the utilization of AOPs in the food and dietary supplements industries.

Extraction Yields
In general, the yield of polysaccharides from different sources depends on the extraction parameters and raw materials.As shown in Table 1, the yield of AOPs varies with the extraction solvent.AOP-AC had the highest yield (13.76%), followed by AOP-AL (8.88%) and AOP-HW (4.54%).The higher yields of AOP-AC and AOP-AL may result from the disruption of the cell wall structure under acidic and alkali conditions, which accelerated the release of soluble polysaccharides into the extract [22].During the extraction process, hot water only dissolves long-chain soluble polysaccharides, resulting in the lowest extraction yield of AOP-HW.Zhang, Zhu, Zhang, Yang, Ni, Zhang, Liu, and Zhang [21] also found that the yield of green tea leaf polysaccharides extracted by alkali and acid solution was higher than that by hydrothermal extraction.In the acidic extract, the glycosidic bond between biopolymers was disrupted by H+, and the soluble molecules quickly dissolved towards the extracellular space under the electrostatic stress, resulting in a significant increase in the production of polysaccharides [23].The samples AOP-HW, AOP-AC, and AOP-AL represent polysaccharides extracted from A. oxyphyllae fructus using hot water, hydrochloric acid, and NaOH/NaBH 4 , respectively.Different letters in the same column represent significant differences (p < 0.05).Mw, molecular weight.

Chemical Composition
The uronic acid, neutral sugar, and protein contents of the three AOPs are shown in Table 1.The neutral sugar contents of AOP-AC and AOP-HW were 80.57% and 69.62%, respectively, significantly higher than that of AOP-AL (28.28%).This result may be interpreted as the fact that the glycosidic bonds in the AOPs are more sensitive, and the acidic environment promotes its hydrolysis [22].The uronic acid contents of AOP-AL, AOP-HW, and AOP-AC were 63.13%, 19.51%, and 13.15%, which indicated that the three AOPs were all anionic polysaccharides.This result also indicated that AOP-AL was an acidic polysaccharide.This finding may be due to the fact that alkaline conditions were conducive to the release of uronic acid, thus increasing the content of uronic acid in the AOP-AL.All AOPs were contaminated with protein, but the protein content was low (1.12-3.37%).It was possible that the obtained polysaccharides were deproteinized by the Sevag reagent and the remaining protein was closely connected to the framework of polysaccharides.
The monosaccharide composition results of the three AOPs were presented in Table 2 and Figure 1.There were certain differences in the relative abundance of each polysaccharide.Glucose was the predominant sugar found in AOP-HW (52.31 mol%) and AOP-AC (94.77 mol%), which was in line with Han's study (96.8 mol%) [1].Glucose was found to account for an exclusively high proportion of the hot-water-extracted polysaccharides from the same Zingiberaceae family, such as Kaempferia galanga L (34.10 mol%) and Zingiber officinale (96.00 mol%) [24,25].However, the main monosaccharides of AOP-AL were determined as arabinose (28.42 mol%), galactose (17.64 mol%), and galacturonic acid (17.09mol%), indicating that AOP-AL was likely to be hemicellulose [26].And the monosaccharides of the AOP-HW sample mainly included arabinose, glucose, and xylose, which was speculated to be araboxylan [27].As the glucose content of AOP-AC exceeds 90%, it was presumed to be glucan.This finding was aligned with a previous study which showed that the polysaccharides extracted from Alpiniae oxyphyllae fructus were composed of glucose alone [9].Their exact structure has yet to be further resolved by means such as nuclear magnetic resonance.The above results indicated that extraction solvents affected the chemical composition of the AOPs.Samples AOP-HW, AOP-AC, and AOP-AL represent polysaccharides extracted from A. oxyphyllae fructus using hot water, hydrochloric acid, and NaOH/NaBH 4 , respectively.Different letters in the same column represent significant differences (p < 0.05).Man, mannose; Rha, rhamnose; GlcA, glucuronic acid; GalA, galacturonic acid; Glc, glucose; Gal, galactose; Xyl, xylose; Ara, arabinose; N.D., not detected.

Mw of AOPs
The Mw of AOPs detected by high-performance size exclusion chromatography is displayed in Table 1 and Figure 2A.The molecular weight profiles of AOP-HW, AOP-AC, and AOP-AL all contained a single RI peak, and their Mw values were 385.42, 121.28, and 232.40 kDa, respectively.The lower average Mw of AOP-AL and AOP-AC indicated that polysaccharides were degraded during the extraction process due to harsh extraction conditions.A similar trend was found in the previous study; the Mw decreased with the increase in H + /OH -concentration, and polysaccharides gradually degraded from large molecules to small-size polymers [28].Both AOP-HW and AOP-AL exhibited symmetrical peaks, while AOP-AC had a broad molecular mass distribution.The relatively wide Mw distribution of AOP-AC might be due to the cleavage of glycosidic bonds and intramolecular hydrogen bonds of AOP-AC to form a variety of glucose polymers during the extraction process, inferring this polysaccharide may have a highly branched and heterogeneous structure [19].

FT-IR Spectroscopy Analysis
The FT-IR spectra of AOP-HW, AOP-AC, and AOP-AL are depicted in Figure 2B.The absorption peak between 3100 and 3700 cm −1 corresponds to the O−H stretching vibration caused by inter-and intramolecular hydrogen bonds [29].The band at 2800-3000 cm −1 is associated with the stretching vibration of C−H in unconstrained carbohydrates [5].The strong absorption peak at 1615-1653 cm −1 is accounted for by the C=O asymmetric stretching vibration of the carbonyl group, while the weak band at 1420 cm −1 reflects the C−O stretching vibration of the carbonyl group [30].The peak at 1090 cm −1 indicates the existence of a pyranose ring in the main chain [25].The characteristic absorption at 1020 cm −1 might represent an α-1,6 linkage [30,31].The three AOPs by FT-IR analysis demonstrated the typical absorption peaks of polysaccharides.A small shoulder at ca. 1730 cm -1 is visible for AOP-HW which is accounted for by the C=O stretching vibration of the methyl esterified carboxylic group.However, the C=O stretching vibration of the methyl esterified carboxylic group is not shown in the FT-IR profile of AOP-AC and AOP-AL, which indicates that de-esterification possibly occurs during acidic or alkali extraction.

FT-IR Spectroscopy Analysis
The FT-IR spectra of AOP-HW, AOP-AC, and AOP-AL are depicted in Figure 2B.The absorption peak between 3100 and 3700 cm −1 corresponds to the O−H stretching vibration caused by inter-and intramolecular hydrogen bonds [29].The band at 2800-3000 cm −1 is associated with the stretching vibration of C−H in unconstrained carbohydrates [5].The strong absorption peak at 1615-1653 cm −1 is accounted for by the C=O asymmetric stretching vibration of the carbonyl group, while the weak band at 1420 cm −1 reflects the C−O stretching vibration of the carbonyl group [30].The peak at 1090 cm −1 indicates the existence of a pyranose ring in the main chain [25].The characteristic absorption at 1020 cm −1 might represent an α-1,6 linkage [30,31].The three AOPs by FT-IR analysis demonstrated the typical absorption peaks of polysaccharides.A small shoulder at ca. 1730 cm -1 is visible for AOP-HW which is accounted for by the C=O stretching vibration of the methyl esterified carboxylic group.However, the C=O stretching vibration of the methyl esterified carboxylic group is not shown in the FT-IR profile of AOP-AC and AOP-AL, which indicates that de-esterification possibly occurs during acidic or alkali extraction.

Morphological Properties
The SEM images provide the surface morphological properties of the AOPs.The morphologies of the three AOPs presented significant differences in shapes and sizes (Figure 3).The microstructure of AOP-HW appeared an irregular and slightly rough layer with uneven dimensions (Figure 3A), which was similar to that reported by Shi, Zhong, Zhang, and Yan [5].AOP-AL possessed many thick and smooth slice patches, and some filamentous connections were found on the surface (Figure 3C).However, AOP-AC exhibited a block appearance with uneven dimensions, which was composed of many corrosive hill-like and irregular pores (Figure 3B).Based on the principle of hydrochloric acid extraction, it could be inferred that the polysaccharide molecules were effectively decomposed into small segments by H + [32], which was in line with the abovementioned Mw distribution and yield of AOP-AC.

XRD Analysis
XRD was applied to examine the crystalline structure of the three AOPs.Generally, sharp and narrow peaks reflect crystalline structures, while broad peaks indicate amorphous structures.As shown in Figure 4, AOP-HW and AOP-AL displayed no particularly obvious diffraction peaks between 5° and 50°, indicating that these two polysaccharides were amorphous polymers.Most studies have reported that water-extracted polysaccha-

XRD Analysis
XRD was applied to examine the crystalline structure of the three AOPs.Generally, sharp and narrow peaks reflect crystalline structures, while broad peaks indicate amorphous structures.As shown in Figure 4, AOP-HW and AOP-AL displayed no particularly obvious diffraction peaks between 5 • and 50 • , indicating that these two polysaccharides were amorphous polymers.Most studies have reported that water-extracted polysaccharides from plants are amorphous structures without obvious crystallization peaks [33,34].Meanwhile, AOP-AL was likely to be derived from hemicellulose, because it contained a high content of arabinose, galactose, and galacturonic acid.Soluble hemicellulose is a group of complex plant polysaccharides arranged in disorder [35], thus AOP-AL was an amorphous structure.However, AOP-AC was a crystalline structure with major diffraction peaks at 14.45 • , 26.25 • , and 32.38 • .These diffraction peaks might be related to the complex and distinctive molecular organization of cellulose, which corresponds to the elevated abundance of glucose in the monosaccharide composition of AOP-AC.These observations suggest that the extraction process was closely related to the structure of the polysaccharides.
Polymers 2024, 16, x FOR PEER REVIEW 7 of 16 diffraction peaks at 14.45°, 26.25°, and 32.38°.These diffraction peaks might be related to the complex and distinctive molecular organization of cellulose, which corresponds to the elevated abundance of glucose in the monosaccharide composition of AOP-AC.These observations suggest that the extraction process was closely related to the structure of the polysaccharides.

DPPH Radical Scavenging Activity
The DPPH radical, a kind of stable free radical source, has been widely used in the evaluation of natural active compounds [36,37].Figure 5A shows the scavenging activity of the three AOPs against DPPH radicals.The DPPH radical scavenging activities of AOP-HW, AOP-AC, and AOP-AL increased with concentration, achieving peak values of 44.33%, 52.90%, and 82.51% at a concentration of 2.0 mg/mL, respectively.AOP-AL displayed a remarkably higher scavenging activity against DPPH radicals (IC50 of 0.56 mg/mL) than AOP-HW (IC50 of 2.16 mg/mL) and AOP-AC (IC50 of 1.74 mg/mL) (Table 3).These findings suggested that AOP-AL is a potential scavenger of DPPH radicals.

In vitro Antioxidant Activity 2.4.1. DPPH Radical Scavenging Activity
The DPPH radical, a kind of stable free radical source, has been widely used in the evaluation of natural active compounds [36,37].Figure 5A shows the scavenging activity of the three AOPs against DPPH radicals.The DPPH radical scavenging activities of AOP-HW, AOP-AC, and AOP-AL increased with concentration, achieving peak values of 44.33%, 52.90%, and 82.51% at a concentration of 2.0 mg/mL, respectively.AOP-AL displayed a remarkably higher scavenging activity against DPPH radicals (IC 50 of 0.56 mg/mL) than AOP-HW (IC 50 of 2.16 mg/mL) and AOP-AC (IC 50 of 1.74 mg/mL) (Table 3).These findings suggested that AOP-AL is a potential scavenger of DPPH radicals.The samples AOP-HW, AOP-AC, and AOP-AL represent polysaccharides extracted from A. oxyphyllae fructus using hot water, hydrochloric acid, and NaOH/NaBH4, respectively.Different letters in the same column represent significant differences (p < 0.05).

Hydroxyl Radical Scavenging Activity
The hydroxyl radical is a physiological free radical with the greatest lethality on biological components [38].As shown in Figure 5B, three AOPs exhibited a moderate scavenging ability of the hydroxyl radical.At the concentration of 2.0 mg/mL, the hydroxyl radical scavenging rate of AOP-AL was 1.84 and 2.33 times that of AOP-HW and AOP-AC, respectively.AOP-AL was a considerably effective hydroxyl radical scavenger with an IC50 value of 1.07 mg/mL, which was stronger than the alkali-extracted Laminaria japonica polysaccharide (IC50 of 1.75 mg/mL) [18] and Zingiber officinale Roscoe stem and leaf polysaccharide (IC50 of 2.426 mg/mL) [39].These results revealed that AOP-AL exhibited an excellent scavenging activity on hydroxyl radicals, which was consistent with the results of the DPPH radical scavenging activity.The samples AOP-HW, AOP-AC, and AOP-AL represent polysaccharides extracted from A. oxyphyllae fructus using hot water, hydrochloric acid, and NaOH/NaBH 4 , respectively.Different letters in the same column represent significant differences (p < 0.05).

Hydroxyl Radical Scavenging Activity
The hydroxyl radical is a physiological free radical with the greatest lethality on biological components [38].As shown in Figure 5B, three AOPs exhibited a moderate scavenging ability of the hydroxyl radical.At the concentration of 2.0 mg/mL, the hydroxyl radical scavenging rate of AOP-AL was 1.84 and 2.33 times that of AOP-HW and AOP-AC, respectively.AOP-AL was a considerably effective hydroxyl radical scavenger with an IC 50 value of 1.07 mg/mL, which was stronger than the alkali-extracted Laminaria japonica polysaccharide (IC 50 of 1.75 mg/mL) [18] and Zingiber officinale Roscoe stem and leaf polysaccharide (IC 50 of 2.426 mg/mL) [39].These results revealed that AOP-AL exhibited an excellent scavenging activity on hydroxyl radicals, which was consistent with the results of the DPPH radical scavenging activity.
Generally speaking, the types of antioxidants can be summarized as hydrogen atom transfer and single electron transfer [42].The antioxidant potential of polysaccharides is closely related to their structure characteristics, including chemical composition, type of glycosidic linkage, and monosaccharide composition and conformation [33].It was suggested that polysaccharides containing a high uronic acid content were effective antioxidants, because their electrophilic groups could promote the release of hydrogen from the O−H bond [43].In this study, AOP-AL exhibited the best effective antioxidant activity, which was probably due to it having the highest content of uronic acid.Moreover, AOP-HW demonstrated a superior scavenging activity compared to AOP-AC in both the ABTS and hydroxyl radical models.A possible explanation is that AOP-HW had a higher galactose content than AOP-AC.Previous studies have reported that the antioxidant activities of polysaccharides may be explained by their abundant galactose content [44,45].

Inhibitory Effects on α-Amylase Activity
The rise of postprandial blood glucose is related to the hydrolysis of starch by α-amylase.Therefore, the α-amylase inhibition assay can be considered as a rapid and simple strategy to evaluate the in vitro hypoglycemic activity [46].
As depicted in Figure 6, the inhibitory effects of the three AOPs on α-amylase activity intensified with higher concentrations.At the concentration of 10 mg/mL, the inhibitory abilities of AOP-AC, AOP-AL, and AOP-HW on α-amylase were 63.41%, 52.71%, and 41.73%, respectively, which were higher than those of polysaccharides extracted from Trifolium pretense L. and comfrey root (20-35%) [37,47].AOP-AC possessed the strongest inhibitory effect on α-amylase activity among the three AOPs.Amamou, Lazreg, Hafsa, Majdoub, Rihouey, Le Cerf, and Achour [41] reported that anti-hyperglycemic activity might be related to the high amounts of glucose in the polysaccharide structure.Meanwhile, the high content of uronic acid could also inhibit the enzyme, because the amino acid residues of α-amylase easily interact with free carboxyl groups [13], which verified that AOP-AL had a stronger α-amylase inhibitory activity than AOP-HW despite the low glucose content.

Polysaccharide Extraction and Isolation
Alpiniae oxyphyllae fructus were separately extracted using an acid solution, hot water, and alkaline solution.The specific extraction conditions were chosen based on a combination of referenced literature [48] and our preliminary experimental findings.In brief, 100.0 g of A. oxyphyllae fructus powder was degreased with n-hexane (1:5 w/v) for 6 h, and then extracted with 3 L of distilled water at 90 °C for 2 h, 3 L of hydrochloric acid (pH 1.5) at 90 °C for 2 h, and 3 L of 0.1 mol/L NaOH/20 mmol/L NaBH4 solution at 25 °C for 2 h, respectively.After centrifugation (5000 rpm, 5 min), the supernatant was neutralized and concentrated with a rotary evaporator.The crude polysaccharide was isolated by adding four volumes of ethanol, allowing the mixture to stand for 12 h, and then centrifuging it at 5000 rpm for 20 min.The polysaccharide solution was then deproteinized with Sevag reagent and dialyzed (8-14 kDa) for 3 days.The three dehydrated polysaccharides were obtained by freeze-drying (FD5 series, SIM International Group, LA, USA), namely, AOP-HW, AOP-AC, and AOP-AL.
The extraction yield of crude A. oxyphyllae fructus polysaccharide was calculated using Formula (1):

Polysaccharide Extraction and Isolation
Alpiniae oxyphyllae fructus were separately extracted using an acid solution, hot water, and alkaline solution.The specific extraction conditions were chosen based on a combination of referenced literature [48] and our preliminary experimental findings.In brief, 100.0 g of A. oxyphyllae fructus powder was degreased with n-hexane (1:5 w/v) for 6 h, and then extracted with 3 L of distilled water at 90 • C for 2 h, 3 L of hydrochloric acid (pH 1.5) at 90 • C for 2 h, and 3 L of 0.1 mol/L NaOH/20 mmol/L NaBH 4 solution at 25 • C for 2 h, respectively.After centrifugation (5000 rpm, 5 min), the supernatant was neutralized and concentrated with a rotary evaporator.The crude polysaccharide was isolated by adding four volumes of ethanol, allowing the mixture to stand for 12 h, and then centrifuging it at 5000 rpm for 20 min.The polysaccharide solution was then deproteinized with Sevag reagent and dialyzed (8-14 kDa) for 3 days.The three dehydrated polysaccharides were obtained by freeze-drying (FD5 series, SIM International Group, LA, USA), namely, AOP-HW, AOP-AC, and AOP-AL.
The extraction yield of crude A. oxyphyllae fructus polysaccharide was calculated using Formula (1): where C represents concentration of polysaccharides (mg/mL), V represents volume of extraction solution (mL), d denotes dilution ratio, and m represents weight of A. oxyphyllae fructus powders (g).

Determination of Chemical Composition
The contents of neutral sugar, uronic acid, and protein were determined according to the phenol-sulfuric method [49], carbazole-sulfuric acid method (galacturonic acid as the standard) [49], and spectrophotometric method (bovine serum albumin as the standard) [50], respectively.

Determination of Mw
The Mw of AOPs was analyzed by high-performance size exclusion chromatography (Agilent 1260, Agilent Technologies, Santa Clara, CA, USA) equipped with an RI detector and a PL aquagel-OH mixed-M column (7.5 × 300 mm, 8 µm) [51].The polysaccharide was dissolved in ultrapure water containing 0.02% NaN 3 and filtered through a 0.45 µm syringe filter.The chromatographic analysis was conducted under the following conditions: column temperature set at 30 • C, flow rate maintained at 0.5 mL/min, injection volume of 20 µL.In addition, calibration curves were established using dextran standards with known molecular weights (Mw: 4.32, 12.6, 60.6, 110, and 496 kDa).

FT-IR Spectroscopy
The functional groups of AOPs were analyzed by FT-IR spectroscopy [52].The sample was fully ground with dried KBr powder and then tabled into a transparent sheet.A Nicolet 5700 spectrometer (Thermo Nicolet Co., Waltham, MA, USA) was used to obtain spectra in the wavelength range of 4000-400 cm −1 .

Scanning Electron Microscopy Analysis (SEM)
The dried polysaccharide powder was pasted on a conductive pile and sputtered with a platinum layer [53].The SEM image was observed using a scanning electron microscope (JEOL Ltd., Akishima, Tokyo, Japan).The detailed measurement parameters were set to the acceleration voltage of 5 kV and the magnification of 5000-fold.

X-ray Diffraction Analysis (XRD)
The crystal structure of AOPs were analyzed using an X-ray diffractometer (D8 Advance, Bruker, Germany) [54].The measurement was carried out between 5 • and 50 • (2θ).The counting time and step size were set to 1 s/step and 0.02 • , respectively.
3.9.Antioxidant Qctivities 3.9.1.DPPH Radical Scavenging Activity The antioxidant activities of AOPs were assessed using the methodology outlined in a previous literature [33].Specifically, a mixture containing 1 mL of 0.2 mmol/L DPPH solution (dissolved in 50% ethanol) and 1 mL of polysaccharide solution (at concentrations ranging from 0 to 2 mg/mL) was observed at 25 • C in darkness for 30 min.Subsequently, the absorbance was measured at 517 nm using a TU-1810 spectrophotometer, with distilled water serving as the blank control.The results were computed based on Formula (2): where A 0 represents the absorbance of 1 mL distilled water plus 1 mL DPPH solution, and A 1 shows the absorbance of 1 mL polysaccharide solution to be measured plus 1 mL DPPH solution.

Hydroxyl Radical Scavenging Activity
The hydroxyl radical scavenging activities were analyzed according to the method reported by Wei and Zhang [38].In summary, an aliquot of 1 mL containing 2 mmol/L FeSO 4 and another aliquot of 1 mL with 6 mmol/L ethanol-salicylic acid were introduced to 1 mL of a polysaccharide solution.Subsequently, this amalgamation was combined with 1 mL of 6 mmol/L H 2 O 2 while being continuously agitated.Following this, the resulting mixture was subjected to incubation for a duration of 30 minutes at a temperature of 37 • C. The optical density was then measured at a wavelength of 510 nm using a TU-1810 spectrophotometer.The hydroxyl radical scavenging activity was calculated according to Formula (3): Scavenging activity (%) = [1 − (A 2 −A 1 )/A 0 ] × 100% where A 0 represents the absorbance of the reaction system containing 1 mL FeSO 4 , 1 mL ethanol-salicylic acid solution, 1 mL H 2 O, and 1 mL H 2 O 2 ; A 1 shows the absorbance of the reaction system containing 1 mL FeSO 4 , 1 mL ethanol-salicylic acid solution, 1 mL polysaccharide solution, and 1 mL H 2 O; and A 2 denotes the absorbance of the reaction system containing 1 mL FeSO 4 , 1 mL ethanol-salicylic acid solution, 1 mL polysaccharide solution, and 1 mL H 2 O 2 .

ABTS Radical Scavenging Activity
The ABTS radical scavenging activity was measured in accordance with the procedures outlined in the literature [31] with certain adjustments.Initially, the ABTS stock solution was formulated following the documented protocol and subsequently diluted using phosphate buffer (pH 7.0) to achieve an absorbance of 0.700 ± 0.030 at 734 nm.Subsequently, a mixture of 30 µL of polysaccharide solution and 970 µL of ABTS diluent was prepared in a 48-well microplate.Following a 6 min incubation period in darkness, the absorbance was measured at 734 nm using a microplate reader.The ABTS radical scavenging activity was estimated by Formula (4): Scavenging activity (%) = [1 − (A 0 −A 1 )/A 0 ] × 100% (4) where A 0 denotes the absorbance value of the blank control, while A 1 indicates the absorbance value of the sample.

Statistical Analysis
All experiments and analyses were replicated thrice, and the results were displayed as the mean ± standard deviation.The origin 2021 (OriginLab, Northampton, MA, USA) and SPSS 26.0 (SPSS Inc., Chicago, IL, USA) software were used to conduct draw the graph and one-way ANOVA, respectively.The p < 0.05 represents a significance difference between samples.

Figure 2 .
Figure 2. Molecular weight curves (A) and FT-IR spectra (B) of three polysaccharides extracted from A. oxyphyllae fructus with different extraction methods.The samples AOP-HW, AOP-AC, and AOP-AL represent polysaccharides extracted from A. oxyphyllae fructus using hot water, hydrochloric acid, and NaOH/NaBH4, respectively.

Figure 2 .
Figure 2. Molecular weight curves (A) and FT-IR spectra (B) of three polysaccharides extracted from A. oxyphyllae fructus with different extraction methods.The samples AOP-HW, AOP-AC, and AOP-AL represent polysaccharides extracted from A. oxyphyllae fructus using hot water, hydrochloric acid, and NaOH/NaBH 4 , respectively.

Figure 4 .
Figure 4. X-ray diffraction patterns of three polysaccharides extracted from A. oxyphyllae fructus with different extraction methods.The samples AOP-HW, AOP-AC, and AOP-AL represent polysaccharides extracted from A. oxyphyllae fructus using hot water, hydrochloric acid, and NaOH/NaBH4, respectively.

Figure 4 .
Figure 4. X-ray diffraction patterns of three polysaccharides extracted from A. oxyphyllae fructus with different extraction methods.The samples AOP-HW, AOP-AC, and AOP-AL represent polysaccharides extracted from A. oxyphyllae fructus using hot water, hydrochloric acid, and NaOH/NaBH 4 , respectively.

Figure 5 .
Figure 5.The antioxidant activities of three polysaccharides extracted from A. oxyphyllae fructus with different extraction methods.(A) DPPH radical scavenging activity, (B) hydroxyl radical scavenging activity, (C) ABTS radical scavenging activity.The samples AOP-HW, AOP-AC, and AOP-AL represent polysaccharides extracted from A. oxyphyllae fructus using hot water, hydrochloric acid, and NaOH/NaBH 4 , respectively.

Figure 6 .
Figure 6.Inhibitory effects of three polysaccharides extracted from A. oxyphyllae fructus with different extraction methods on the α-amylase activity.The samples AOP-HW, AOP-AC, and AOP-AL represent polysaccharides extracted from A. oxyphyllae fructus using hot water, hydrochloric acid, and NaOH/NaBH4, respectively.

Figure 6 .
Figure 6.Inhibitory effects of three polysaccharides extracted from A. oxyphyllae fructus with different extraction methods on the α-amylase activity.The samples AOP-HW, AOP-AC, and AOP-AL represent polysaccharides extracted from A. oxyphyllae fructus using hot water, hydrochloric acid, and NaOH/NaBH 4 , respectively.

Table 2 .
Monosaccharide ratio of three polysaccharides extracted from A. oxyphyllae fructus with different extraction methods (mol%).

Table 2 .
Monosaccharide ratio of three polysaccharides extracted from A. oxyphyllae fructus with different extraction methods (mol%).

Table 3 .
Scavenging activity of three polysaccharides extracted from A. oxyphyllae fructus with different extraction methods for DPPH, hydroxyl, and ABTS radicals.

Table 3 .
Scavenging activity of three polysaccharides extracted from A. oxyphyllae fructus with different extraction methods for DPPH, hydroxyl, and ABTS radicals.