Isolation, Structural Elucidation, Antioxidant and Hypoglycemic Activity of Polysaccharides of Brassica rapa L.

The aim of this study was to investigate the effects of microwave ultrasonic-assisted extraction (MUAE) on the content, structure, and biological functions of Brassica rapa L. polysaccharide (BRP). Response surface methodology (RSM) was used to optimize the parameters of MUAE, and it obtained a polysaccharide with yield of 21.802%. Then, a neutral polysaccharide named BRP-1-1 with a molecular weight of 31.378 kDa was isolated and purified from BRP using DEAE-650 M and Sephadex G-100. The structures of the BRP-1-1 were elucidated through a combination of FT-IR, GC-MS, NMR, and methylation analysis. The results showed that BRP-1 consisted of mannose (Man) and glucose (Glu) in a molar ratio of 7.62:1. The backbone of BRP-1-1 mainly consisted of →6)-α-D-Glup-(1→4-β-D-Glup-(1→2)-α-D-Manp-(1→2)-α-D-Glup-(1→, the branch was [T-α-D-Manp-(1]n→. BRP-1-1 intervention significantly inhibited α-glucosidase activity; an inhibition rate of 44.623% was achieved at a concentration of 0.5 mg/mL. The results of the in vitro biological activity showed that BRP-1-1 has good antioxidant and hypoglycemic activity, suggesting that BRP-1-1 could be developed as a functional medicine.


Introduction
Brassica rapa L. is part of the Cruciferae Brassica family, and is widely distributed in mainland China; local residents call it "Chamaguer" or "Man Jing" in the Xinjiang Uygur Autonomous Region, China. This plant is extensively used in medicine and food because it helps with digestion, diuresis, and cough suppression [1]. The active ingredients in Brassica rapa L. have been documented to principally include saponins, flavonoids, polysaccharides, alkaloids, amino acids, and proteins [2]. Polysaccharides act as important bioactive components, and have gradually become a research hotspot in medicine and nutrition in recent years because of their health-promoting effects [3,4]. Studies have shown that BRP has numerous biological functions, including immune regulation, anti-hypoxia, anti-tumor, anti-oxidation, and anti-fatigue activities [5][6][7][8][9].
Extraction methods can result in large differences in polysaccharide contents, structures and biological functions [10,11]. Ultrasound-assisted extraction (UAE) is an extraction technique that disrupts the plant cell wall structure through ultrasonic treatment, resulting in the faster release of polysaccharides; however, it has weak thermal effects and takes a long time to reach the desired temperature levels during the extraction process [12,13]. Microwave ultrasonic-assisted traction (MUAE) is an improvement based on the UAE method, with advantages of a short consumption time and low extraction temperature; it has been demonstrated to be an effective novel technique for polysaccharide extraction [14,15]. However, microwaves may alter the chemical structure and biological activity of polysaccharides [16]. There are currently no relevant studies on the extraction process and product properties of BRP using MUAE.
In this experiment, the response surface methodology (RSM) was used to optimize the extraction parameters (ratio of water to material, microwave-ultrasound time, extraction temperature and ultrasound power) of MUAE. The physicochemical parameters and major chemical structure of BRP-1-1 were determined through NMR and methylation after further separation and purification. Finally, the antioxidant and hypoglycemic activity of BRP-1-1 was tested in vitro. To our knowledge, this is the first time MUAE has been used to extract polysaccharides from Brassica rapa L., and the structure and biological applications of BRP fractions have been thoroughly investigated. These results may provide a reference for the integrated use of BRP polysaccharides in health products.

Single-Factor Experiments Analysis
Through experiments with controlled variables, we found that the BRP yield reached a maximum (16.377%) when ratio of water to material was 25:1 mL/g (Figure 1a), the dissolution of BRP rate had reached equilibrium, and the continuous addition of extractant could not produce more polysaccharides, but might lead to the loss of polysaccharides [17]; the BRP yield reached a considerable value (20.130%) at 75 • C, after which the yield gradually decreased (Figure 1b), and the high temperature and the obvious increase in water evaporation may have affected the dissolution of polysaccharides [18]; the yield of BRP reached the maximum (21.843%) at 9 min (Figure 1c), and then began to decline sharply, and the structures and properties of BRP polysaccharides changed (degradation to monosaccharides) under prolonged microwave sonication, causing this result [19]. We also found that the highest BRP yield (21.843%) was obtained at 300 W of ultrasound power (Figure 1d), after which it started to decrease. Higher ultrasound power reduced the cavitation phenomenon, which caused insufficient bubble collapse [20], resulting in a decrease in polysaccharide yield.
process and product properties of BRP using MUAE.
In this experiment, the response surface methodology (RSM) was used to optim the extraction parameters (ratio of water to material, microwave-ultrasound time, extr tion temperature and ultrasound power) of MUAE. The physicochemical parameters a major chemical structure of BRP-1-1 were determined through NMR and methylation ter further separation and purification. Finally, the antioxidant and hypoglycemic activ of BRP-1-1 was tested in vitro. To our knowledge, this is the first time MUAE has be used to extract polysaccharides from Brassica rapa L., and the structure and biological a plications of BRP fractions have been thoroughly investigated. These results may provi a reference for the integrated use of BRP polysaccharides in health products.

Single-Factor Experiments Analysis
Through experiments with controlled variables, we found that the BRP yield reach a maximum (16.377%) when ratio of water to material was 25:1 mL/g (Figure 1a), the d solution of BRP rate had reached equilibrium, and the continuous addition of extracta could not produce more polysaccharides, but might lead to the loss of polysaccharid [17]; the BRP yield reached a considerable value (20.130%) at 75 °C, after which the yie gradually decreased (Figure 1b), and the high temperature and the obvious increase water evaporation may have affected the dissolution of polysaccharides [18]; the yield BRP reached the maximum (21.843%) at 9 min (Figure 1c), and then began to decli sharply, and the structures and properties of BRP polysaccharides changed (degradati to monosaccharides) under prolonged microwave sonication, causing this result [19]. W also found that the highest BRP yield (21.843%) was obtained at 300 W of ultrasou power (Figure 1d), after which it started to decrease. Higher ultrasound power reduc the cavitation phenomenon, which caused insufficient bubble collapse [20], resulting in decrease in polysaccharide yield. Therefore, we set the ratio of water to material of 25 mL/g, extraction temperature 75 °C, microwave-ultrasound time of 9 min and ultrasound power of 300 W as the cen point of RSM experiments.

Optimization of Extraction Parameters by RSM
As shown in Table S1 (See Supplementary Materials), 29 runs were performed combining four single-factor parameters (A, B, C and D); the BRP content ranged fro 14.750% to 21.873%. The model data were analyzed by regression fitting, and the qua ratic multiple regression equations of A, B, C and D with the BRP content (Y) were o tained as follows: The analysis of variance (ANOVA) results of the secondary regression model shown in Table S1 (See Supplementary Materials). The model F = 103.48, p < 0.0001 in cated that the equation obtained from this model reached a highly significant level. T correlation coefficient R 2 = 0.9405 and the adjusted correlation coefficient R 2 Adj = 0.88 indicated that the experimental and predicted values of the equation had a high corre tion [17][18][19][20]. Meanwhile, the F = 8.25 (p = 0.82 > 0.05) was a misfitting term, which indica non-significance and that the experimental error caused by it was small. In addition, coefficient of variation (C.V) was an important indicator to evaluate the repeatability the model. C.V % = 4.150% (C.V < 5%), indicating good model repeatability. Finally, factor significance analysis of the model coefficients showed that p < 0.05 for B, C, AD, B A 2 , B 2 , C 2 , and D 2 , showing that these factors had a significant effect on the yield of BR In addition, because the magnitude of F value reflects the strength of the influence of ea factor on the BRP yield, the order of the influence of each factor on the yield is: B > C > > A. In summary, the proposed model has high accuracy and credibility and can be us for BRP extraction, analysis and prediction.

Analysis of Response Surface
The interactions between the factors and their effects on the BRP yields are shown Figure 2. It is known that the interactions between the factors and their influence on BRP yield can be determined from the shapes of the three-dimensional (3D) response s face plots and contour plots. The steeper the 3D response surface plots, the higher Therefore, we set the ratio of water to material of 25 mL/g, extraction temperature of 75 • C, microwave-ultrasound time of 9 min and ultrasound power of 300 W as the center point of RSM experiments.

Optimization of Extraction Parameters by RSM
As shown in Table S1 (See Supplementary Materials), 29 runs were performed by combining four single-factor parameters (A, B, C and D); the BRP content ranged from 14.750% to 21.873%. The model data were analyzed by regression fitting, and the quadratic multiple regression equations of A, B, C and D with the BRP content (Y) were obtained as follows: The analysis of variance (ANOVA) results of the secondary regression model are shown in Table S1 (See Supplementary Materials). The model F = 103.48, p < 0.0001 indicated that the equation obtained from this model reached a highly significant level. The correlation coefficient R 2 = 0.9405 and the adjusted correlation coefficient R 2 Adj = 0.8811 indicated that the experimental and predicted values of the equation had a high correlation [17][18][19][20]. Meanwhile, the F = 8.25 (p = 0.82 > 0.05) was a misfitting term, which indicated non-significance and that the experimental error caused by it was small. In addition, the coefficient of variation (C.V) was an important indicator to evaluate the repeatability of the model. C.V % = 4.150% (C.V < 5%), indicating good model repeatability. Finally, the factor significance analysis of the model coefficients showed that p < 0.05 for B, C, AD, BC, A 2 , B 2 , C 2 , and D 2 , showing that these factors had a significant effect on the yield of BRP. In addition, because the magnitude of F value reflects the strength of the influence of each factor on the BRP yield, the order of the influence of each factor on the yield is: B > C > D > A. In summary, the proposed model has high accuracy and credibility and can be used for BRP extraction, analysis and prediction.

Analysis of Response Surface
The interactions between the factors and their effects on the BRP yields are shown in Figure 2. It is known that the interactions between the factors and their influence on the BRP yield can be determined from the shapes of the three-dimensional (3D) response surface plots and contour plots. The steeper the 3D response surface plots, the higher the influence of the factors on the BRP yield. In addition, if the contour plot is elliptical, it indicates a significant interaction between the factors. As shown in Figure 2c,d, the 3D plots are quite steep and the contour plots are elliptical. This indicates that the interaction between AD and BC is significant and affects the polysaccharide yield to a higher extent. This is consistent with the findings presented in Table S2 (See Supplementary Materials).
Based on the regression equation, the optimal conditions for the RSM-optimized BRP extraction process were obtained as A:24.67 (g/mL), B:76.45 °C, C:9.7 min, and D:292 W. The optimal extraction conditions were adjusted according to the actual operation of the extraction process as follows: A:25 mL/g, B:76.5 °C, C:9.7 min, and D:292 W. To verify the accuracy of the predicted results, a validation experiment was conducted. As a result, the actual yield was 21.802 ± 0.682%. In contrast, MUAE has been successfully applied to the extraction of BRP (Table S3, See Supplementary Materials). For example, the extraction temperature was lower and the process was 26.60 times shorter when using MUAE than when using hot water extraction (HWE) for similar BRP yields [7]; moreover, the difference in BRP yields between MUAE and UAE extraction was 15.03-fold, mainly due to the different origins [8].
The results of the monosaccharide composition analysis showed that BRP-1-1 consisted of Man and Glu in a molar ratio of 7.62:1. The above results significantly differ from previous reports on BRPs from Urumqi, Xinjiang, China [7], probably because MUAE reduces the BRP molecular weight and thus alters the structures of BRPs. Based on the regression equation, the optimal conditions for the RSM-optimized BRP extraction process were obtained as A:24.67 (g/mL), B:76.45 • C, C:9.7 min, and D:292 W. The optimal extraction conditions were adjusted according to the actual operation of the extraction process as follows: A:25 mL/g, B:76.5 • C, C:9.7 min, and D:292 W. To verify the accuracy of the predicted results, a validation experiment was conducted. As a result, the actual yield was 21.802 ± 0.682%. In contrast, MUAE has been successfully applied to the extraction of BRP (Table S3, See Supplementary Materials). For example, the extraction temperature was lower and the process was 26.60 times shorter when using MUAE than when using hot water extraction (HWE) for similar BRP yields [7]; moreover, the difference in BRP yields between MUAE and UAE extraction was 15.03-fold, mainly due to the different origins [8].

FT-IR Spectroscopy Analysis
The high absorption peaks of about 3452 and 2925.76 cm −1 which can be seen in the FT-IR spectra are caused by the stretching vibrations of -OH and -CH ( Figure 3d). The absorption peak generated by water is 1628.76 cm −1 ; the absorption peak generated by the bending vibration of -CH2-is 1422.96 cm −1 . We speculate that the absorption peak of 1053.71 cm −1 implies the possible presence of pyranose in the sample [21]. In addition, we found two bands of 836.4 cm −1 and 873.1 cm −1 , which imply that the sugar linkages are αtype and β-type glycosidic bond structures, indicating that BRP-1-1 has a typical polysaccharide absorption peak.

Methylation Analysis
The data related to the total ion rheology of BRP-1-1 are shown in Figure S1 (See Supplementary Materials). The types of the obtained BRP-1-1 are shown in Table 1, including →T-Manp、→2-D-Manp、→2-D-Glup、→4-D-Glup, and →6-D-Glup. This is consistent with the monosaccharide composition analysis result of BRP-1-1. The results of the monosaccharide composition analysis showed that BRP-1-1 consisted of Man and Glu in a molar ratio of 7.62:1. The above results significantly differ from previous reports on BRPs from Urumqi, Xinjiang, China [7], probably because MUAE reduces the BRP molecular weight and thus alters the structures of BRPs.

FT-IR Spectroscopy Analysis
The high absorption peaks of about 3452 and 2925.76 cm −1 which can be seen in the FT-IR spectra are caused by the stretching vibrations of -OH and -CH (Figure 3d). The absorption peak generated by water is 1628.76 cm −1 ; the absorption peak generated by the bending vibration of -CH 2 -is 1422.96 cm −1 . We speculate that the absorption peak of 1053.71 cm −1 implies the possible presence of pyranose in the sample [21]. In addition, we found two bands of 836.4 cm −1 and 873.1 cm −1 , which imply that the sugar linkages are α-type and β-type glycosidic bond structures, indicating that BRP-1-1 has a typical polysaccharide absorption peak.

NMR Analysis
The NMR spectrum of BRP-1-1 is shown in Figure 4. In the 1 H NMR spectrum of BRP-1-1 (Figure 4a), three proton signals appear in the heterogeneous proton region. Among them, the signal at 5.24-5.46 ppm was attributed to the α-glycoside conformation; the β-glycoside conformation was characterized with the signal at 4.55-4.68 ppm [22]. Furthermore, in the 13 C NMR spectrum of BRP-1-1 (Figure 4b), five isomeric carbon signals at 94.93, 94.98, 98.96, 101.98, and 106.86 ppm were labeled as residues A to E, respectively.
We also explored the coupling between proton and carbon signals using HSQC spectroscopy ( Figure 4d). By combining methylation analysis, NMR data and the cited literature [23,24], the (H1/C1) cross peaks of residues A to E appeared at (5.43/100.79), (5.43/106.59), (5.42/94.95), (4.66/98.63), and (5.24/94.81), respectively. The above results fully demonstrate that all residues in BRP-1-1 were in α-configuration, except for residue D, which was in β-configuration. This was in strong agreement with the FT-IR results, and the 1 H and 13 C chemical shifts are presented in Table S4 (See Supplementary Materials).
In addition, we needed to assess the HMBC and NOESY spectra of the residues to clarify their sequences [25,26]. In the HMBC and NOESY spectrum (as shown in Figure 4c

Morphological Properties
Purified BRP-1-1 is a highly water-soluble mucopolysaccharide, and its scanning electron micrographs at magnifications of 20 KX are shown in Figure 5a. BRP-1-1 is composed of many pebble-like particles; this is highly different from other kinds of plant polysaccharides in morphological characteristics, which may be due to the different preparation, extraction and purification methods of the product.

XRD Analysis
The XRD pattern of BRP-1-1 is shown in Figure 5b. There is a weak and broad steamed bun peak around 21°, and a sharp and strong diffraction peak near 29°, indicating that there are not only microcrystals in the structure of BRP-1-1, but there is also a polycrystalline system in which crystalline and amorphous coexist [27].

Congo Red Test Analysis
The acid dye Congo red chemically reacts with polysaccharides with a triple helix structure at pH > 7 to produce a Congo red-polysaccharide complex with red-shifted

Morphological Properties
Purified BRP-1-1 is a highly water-soluble mucopolysaccharide, and its scanning electron micrographs at magnifications of 20 KX are shown in Figure 5a. BRP-1-1 is composed of many pebble-like particles; this is highly different from other kinds of plant polysaccharides in morphological characteristics, which may be due to the different preparation, extraction and purification methods of the product.

Morphological Properties
Purified BRP-1-1 is a highly water-soluble mucopolysaccharide, and its scanning electron micrographs at magnifications of 20 KX are shown in Figure 5a. BRP-1-1 is composed of many pebble-like particles; this is highly different from other kinds of plant polysaccharides in morphological characteristics, which may be due to the different preparation, extraction and purification methods of the product.

XRD Analysis
The XRD pattern of BRP-1-1 is shown in Figure 5b. There is a weak and broad steamed bun peak around 21°, and a sharp and strong diffraction peak near 29°, indicating that there are not only microcrystals in the structure of BRP-1-1, but there is also a polycrystalline system in which crystalline and amorphous coexist [27].

Congo Red Test Analysis
The acid dye Congo red chemically reacts with polysaccharides with a triple helix structure at pH > 7 to produce a Congo red-polysaccharide complex with red-shifted

XRD Analysis
The XRD pattern of BRP-1-1 is shown in Figure 5b. There is a weak and broad steamed bun peak around 21 • , and a sharp and strong diffraction peak near 29 • , indicating that there are not only microcrystals in the structure of BRP-1-1, but there is also a polycrystalline system in which crystalline and amorphous coexist [27].

Congo Red Test Analysis
The acid dye Congo red chemically reacts with polysaccharides with a triple helix structure at pH > 7 to produce a Congo red-polysaccharide complex with red-shifted wavelength. When the polysaccharide Congo red complex is in a certain pH range, the complex exhibits a transfer region; the triple helix structure of the polysaccharide is disrupted with increasing pH conditions, which leads to a decrease in the redshift effect of the complex [28]. Figure 5c shows the variation in λ max of the Congo red-polysaccharide complex detected with UV light under the effect of different NaOH concentration gradients. The maximum absorption wavelength of BRP-1-1 for the red shift occurred at a concentration of 0.05 mol/L of NaOH. When the NaOH concentration exceeded 0.4 mol/L, the maximum absorption wavelength of BRP-1-1 decreased, confirming the existence of a triple helix structure of BRP-1-1, which may have anti-cancer and anti-tumor functions to some extent [29,30]. In the presence of ethanol, DPPH produces stable nitrogen-containing radicals (purple color in solution) with a characteristic absorption peak at 517 nm [31,32]. Figure 6a shows that the DPPH + clearance activities of BRP, BRP-1-1, and control substances were correlated with concentration, and the scavenging rate varied with different concentrations. The scavenging rate of BRP-1-1 (the IC 50 value of BRP-1-1 was 2.772 mg/mL), BRP and Vc were 40.313%, 32.697%, and 90.010% at 2.0 mg/mL, respectively. Among them, the DPPH + clearance by BRP-1 was much greater than that reported for BRP-1-1 in HWE (22.370%) [7], which may be because BRP-1-1 possesses a lower molecular weight (1510 kDa) than the latter.

α-Glucosidase Inhibitory Activity
The human intestine contains α-glucosidase and α-amylase, which catalyze the hydrolysis of starch and oligosaccharide (1→4)-glycosidic bonds to produce monosaccharides. Among them, α-glucosidase is secreted by small intestinal epithelial cells, whereas α-amylase is mainly secreted by the salivary glands or pancreas. By inhibiting the activity of these digestive enzymes, the absorption of dietary carbohydrates by the cells of the small intestine can be slowed down, resulting in a lowering of blood glucose. Therefore, the development of inhibitors is considered to be a new strategy for the treatment of type 2 diabetes [34,35].

Hydroxyl Scavenging Rate
Hydroxyl radicals are extremely powerful reactive oxygen species that can be produced in the human body, and excess hydroxyl radicals can react with biological macromolecules and harm human health. We found that the hydroxyl scavenging rate of BRP, BRP-1-1 and the reference substances varied at low concentrations, and eventually converged to equilibrium (Figure 6b). In ethanol reaction system, samples with excessive concentrations can induce flocculent precipitation in the system, causing interference [33]. The scavenging rate of BRP-1-1, BRP, and Vc were 47.863%, 9.235%, and 99.590% at 0.6 mg/mL, respectively. Among them, the scavenging rate of ascorbic acid (99.590%) against hydroxyl radicals was only 2.08 times higher than that of BRP-1-1 (47.863%). This finding suggests that BPRs can act as electron or hydrogen donors to scavenge hydroxyl radicals.

ABTS Scavenging Rate
It can be seen that the ABTS scavenging rate of BRP, BRP-1-1, and the control substance (ascorbic acid) was strongly concentration-dependent (Figure 6c). The scavenging rate of BRP-1 (The IC 50 value of BRP-1-1 was 7.112 mg/mL), BRP and Vc were 25.533%, 21.216%, and 90.244% at 0.9 mg/mL, respectively. This finding suggests that BPRs can act as antioxidants to scavenge ABTS + . In conclusion, BRP-1-1 extracted by MUAE with a small molecular weight showed strong antioxidant activity and could be studied in follow-up experiments.
2.11. Hypoglycemic Activity 2.11.1. α-Glucosidase Inhibitory Activity The human intestine contains α-glucosidase and α-amylase, which catalyze the hydrolysis of starch and oligosaccharide (1→4)-glycosidic bonds to produce monosaccharides. Among them, α-glucosidase is secreted by small intestinal epithelial cells, whereas αamylase is mainly secreted by the salivary glands or pancreas. By inhibiting the activity of these digestive enzymes, the absorption of dietary carbohydrates by the cells of the small intestine can be slowed down, resulting in a lowering of blood glucose. Therefore, the development of inhibitors is considered to be a new strategy for the treatment of type 2 diabetes [34,35].
The inhibitory activity of BRP and BRP-1-1 on α-glucosidase was not concentrationdependent (Figure 7a). At low, medium and high doses, the inhibition of BRP was negative, suggesting that it promotes α-glucosidase to hydrolyze more monosaccharides; at medium doses (0.5 mg/mL), the α-glucosidase inhibition of BRP, BRP-1-1 and acarbose was −69.467%, 44.623%, and 79.100%, respectively. Among them, the considerable variability in the inhibitory activities of BRP and BRP-1-1 may be due to the low molecular weight of BRP-1-1, which has (1→4) and (1→6) glycosidic bonds in its main chain and branched chains, respectively. This is consistent with the results for Rosa roxburghii Tratt polysaccharide (RTFP-3); most polysaccharides with hypoglycemic activity have (1→3), (1→4), and (1→6) glycosidic bonds [36]. Furthermore, for the negative values of BRP inhibition, not all plant polysaccharide fractions exhibit hypoglycemic activity [37]; on the other hand, they may be veiled or influenced by polysaccharide components with the same source activity (BRP-1-1). This finding suggests that BRP-1-1 is a potential inhibitor of α-glucosidase.

Extraction process
The root powder of B. rapa L. was refluxed with 95% (v/v) ethanol 3 times to remove

α-Amylase Inhibitory Activity
The inhibitory activities of BRP and BRP-1-1 on α-amylase were not concentrationdependent (Figure 7b). At low, medium, and high doses, the inhibition rates of both BRP and BRP-1-1 were positive, which indicated that they could both inhibit α-amylase to hydrolyze more starch; at medium doses (0.3 mg/mL), the α-amylase inhibition rates of BRP, BRP-1-1, and acarbose were 28.610%, 6.1%, and 55.120%, respectively. Among them, BRP has higher inhibitory ability than BRP-1-1, which may partly be because BRP has more active components, such as flavonoids, saponins, and alkaloids [38]; it may also be because the glycosidic bond of BRP-1-1 is broken during the reaction. In conclusion, BRP-1-1 exerts a highly hypoglycemic activity.

Materials
Brassica rapa L. was planted in Atushi County (39 •

Extraction Process
The root powder of B. rapa L. was refluxed with 95% (v/v) ethanol 3 times to remove lipids and pigments; 1 g of the powder was taken and added to deionized water with a certain ratio of material. Additionally, polysaccharide extract was obtained after the liquid was extracted in a combined microwave ultrasonic synthesizer/extractor (XH-300A, Beijing Xianghu Technology Development Co., Beijing, China), and suction-filtered before centrifugation. All the extracts were combined and concentrated at 50 • C under reduced pressure with a rotary evaporator. The concentrated solution was precipitated with a 3-fold greater volume of ethanol for 12 h, then centrifuged (8000 rpm, 20 min, 4 • C) and freezedried to obtain crude polysaccharide. The crude polysaccharide was dissolved in distilled water and dialyzed for 48 h against deionized water, then treated with macroporous resin (AB-8) to remove colors and proteins, then freeze-dried. The BRP content (%) was determined with the phenol-sulfuric acid technique [22] and calculated as follows:

RSM Experimental Design
Based on the test studies presented in Section 3.2.1, the microwave power was fixed at 440 W, and the effects of ratio of water to material (A: 10-30 mL/g), extraction temperature (B: 70-90 • C) microwave-ultrasound time (C: 3-11 min), and ultrasound power (D: 200-400 W) on the content of BRP were investigated. The experimental optimization design was based on previous studies [16,38]. The complete design comprised 29 experimental points that were executed at random (Table S1, See Supplementary Materials).

Purification of Polysaccharides
After centrifugation, the supernatant of BRP (0.1 g) was sampled on a DEAE-650M cellulose column (4.0 × 20 cm) and gradually eluted with 0, 0.2, and 0.4 M NaCl solution at a flow rate of 2 mL/min. The fraction (18.0 mL) was collected by CombiFlashRF+ (Teledyne Technologies, Inc., Lincoln, NE, USA) and monitored with anthracone-sulfuric acid technique for drawing the elution curve. The fractions were collected according to tube number, then concentrated and lyophilized to obtain polysaccharides. Finally, the BRP-1 (30 mg) was sampled on a Sephadex G-100 column (1.6 × 80 cm) and gradually eluted with distilled water at a flow rate of 0.4 mL/min. The fraction (2.0 mL) was collected according to the conditions mentioned above.

Molecular Weight
The molecular weight (Mw) of BRP was determined based on a previous study [42]. Polysaccharide and standard dextran solutions (1 mg/mL) were filtered through a filter membrane (0.22 µm) and detected using HPLC (Shimadzu Corporation, Kyoto, Japan) apparatus fitted with a column (TSK-GEL-G3000SWXL) and a differential refractive index detector (RID-20A), and then eluted with distilled water at an elution rate of 0.6 mL/min. The standard linear regression equation was used to estimate the Mw of BRP (Mw: 1, 5, 25, 50, 100, 250, and 500 kDa).

Monosaccharide Composition
The monosaccharide composition of BRP was also studied based on a previous study [10]. Polysaccharide samples (10 mg) were hydrolyzed with 4 mL trifluoroacetic acid (2M) at 120 • C for 6 h, 3-4 mL of methanol was then added, and rotary evaporation was repeated several times to remove excess TFA. Subsequently, 1 mL chloroform was added to the residue and fully dissolved before filtration (0.22 µm). The pretreated samples were analyzed with gas chromatography-mass spectrometry (7890A-5975C, Agilent Technologies Ltd., Santa Clara, CA, USA).

Infrared Spectral Analysis
The polysaccharide sample was mixed with potassium bromide powder (1:200), crushed and pressed into pellets. FTIR measurements were performed using a Fourier transform infrared spectrophotometer (2600, Shimadzu Corporation, Kyoto, Japan) in the wavenumber range of 4000-400 cm −1 .

Methylation Analysis
The methylation analysis of BRP-1-1 was based on a previous study, with slight adjustments [43]. Briefly, the sample was dissolved in 3 mL DMSO overnight. Subsequently, 20 mg of sodium hydroxide and methyl iodide (0.3 mL) were put in the reaction unit, stirred intermittently, and the solution reacted for 3 h in an ice bath and under light. Finally, the reaction was terminated by adding 4 mmol/L Na 2 S 2 O 3 (1 mL) solution, and chloroform was added to the reaction solution for extraction and drying to obtain methylated polysaccharides. It could then be judged whether the methylation of BRP-1-1 was complete through the disappearance of OH stretching at 3200-3700 cm −1 in the FT-IR spectrum. Finally, the resulting product was hydrolyzed by TFA, reduced by NaBD 4 , acetylated with acid anhydride-pyridine, and analyzed for methylation by GC-MS (7890B-7000C, Agilent Technologies Ltd., Santa Clara, CA, USA).

Congo Red Test
Equal amounts of 1 mL of Congo red solution (80 µmol/L) and BRP-1-1 solution (1 mg/mL) were mixed. Some NaOH solution and water was added, making the volume constant to 4 mL; the final concentrations of NaOH were 0, 0.05, and 0.1-0.5 mol/L. The solution reacted at 25 • C for 10 min, followed by scanning at 480-520 nm with a UV spectrophotometer (2550, Shimadzu Corporation, Kyoto, Japan).

Scanning Electron Microscopy
The apparent characteristics of BPPS were observed using a SUPRA 55VP scanning electron microscope (SEM). A small amount of sample was fixed uniformly on a thin copper plate, and a layer of gold powder was vaporized on the surface of the sample using an ion sputterer. Subsequently, the polysaccharides were observed under vacuum conditions at 20.0 kV.
3.13. Antioxidant Activity Assays In Vitro 3.13.1. DPPH Scavenging Rate The scavenging rate of DPPH by BRP was based on a previous study [44]. A 0.5 mL mixture of the sample (0.1-2.0 mg/mL) with 1.5 mL of DPPH (1 × 10 −3 mol/L) was reacted for 30 min at 25 • C under dark condition. The absorbance of the mixture at 517 nm was measured using ascorbic acid (Vc) as a positive control. The percentage clearance (%) was calculated as follows: where A 0 and A 1 are the absorbance of the control group (DPPH + H 2 O) and the test group (DPPH+BRPs/Vc), respectively.

Hydroxyl Scavenging Rate
The scavenging rate of hydroxyl radicals by BRP was obtained based on a previous study [45]. The 0.5 mL sample (0.1-0.8 mg/mL), 0.5 mL FeSO 4 (9 × 10 −3 mol/L) solution, and 0.5 mL salicylic acid (9 × 10 −3 mol/L) solution were mixed thoroughly. Then, 0.5 mL H 2 O 2 (88 × 10 −3 mol/L) was added and shaken well before reacting at 37 • C for 30 min. The absorbance of the mixture at 510 nm was measured using Vc as a positive control, and the scavenging rate (%) was calculated as follows:

ABTS Scavenging Rate
The scavenging rate of ABTS free radicals of BRP was based on a previous study [46]. For this assay, 1 mL of ABTS (7.2 × 10 −3 mol/L) solution and potassium persulfate (2.45 × 10 −3 mol/L) were mixed in equal volumes and reacted at 25 • C for 24 h and under light to produce ABTS free radical (ABTS + ) solution. Next, ABTS + was diluted with PBS buffer to yield an absorbance of 0.70 ± 0.02 at 734 nm. Then, 1 mL solutions of the samples (0.05-1.0 mg/mL) were mixed well with 9 mL ABTS + , and the mixture reacted for 10 min at 25 • C. Finally, Vc was used as a positive control, and the scavenging rate (%) was calculated using Equation (1).

Hypoglycemic Activity Assays In Vitro
To investigate the in vitro hypoglycemic activity of BRPs, their α-glucosidase [16] and α-amylase inhibitory activities [37] were determined by the respective reported methods, with acarbose used as a positive control.

Statistical Analysis
All experiments were carried out three times in parallel, and the data were averaged and statistically analyzed using SPSS (26.0), with a p-value < 0.05 indicating statistical significance. RSM analysis was performed here using Design Expert (Version 8.0.6.1). Graphics are drawn using software such as OriginPro 2021b (64-bit) SR2.