Preparation method and application of ya-pa-1 yacca polysaccharide

Abstract

The application provides a preparation method and application of YAPa-1 yacon polysaccharide, and belongs to the field of food biotechnology and natural polysaccharide development. The main chain of the molecular structure of YAPa-1 yacon polysaccharide is composed of galactose and mannose in alternation, and the side chain is arabinose, glucose and rhamnose; the molar ratio of galactose, mannose, glucose, arabinose and rhamnose is 1.00:0.82:0.25:0.11:0.25. The molecular weight of YAPa-1 yacon polysaccharide is 10873 Da, has a triple helix structure, and the microscopic morphology presents a sponge-like porous network structure, and the alpha-glucosidase / alpha-amylase inhibition mechanism thereof can be explained by the'solid-state slow-release inhibition'. The application obtains high-activity single YAPa-1 yacon polysaccharide through cellulase-assisted hydrothermal extraction and two-step column chromatography purification process. YAPa-1 yacon polysaccharide can effectively inhibit the activities of alpha-glucosidase and alpha-amylase, the inhibition effect is close to that of the commonly used hypoglycemic drug acarbose, and the YAPa-1 yacon polysaccharide has no side effects common in synthetic drugs, and can be applied to the development of hypoglycemic products.

Description

Technical Field

[0001] This invention belongs to the field of food biotechnology and natural polysaccharide development, specifically relating to a method for preparing yacon polysaccharide YAPa-1 and its application. Background Technology

[0002] Diabetes is a prevalent metabolic disease worldwide. A 2023 report by the International Diabetes Federation (IDF) indicated over 540 million adults globally suffering from diabetes, a number projected to exceed 783 million by 2045. While clinically synthesized hypoglycemic drugs (such as acarbose) can inhibit α-glucosidase / α-amylase, they easily cause gastrointestinal side effects such as bloating and diarrhea, and long-term use increases the burden on the liver and kidneys, leading to poor patient compliance. Plant polysaccharides, due to their low toxicity and high biocompatibility, have become a core research direction for natural hypoglycemic agents.

[0003] Yacon tubers are rich in polysaccharides, and the hypoglycemic activity of their crude polysaccharides has been preliminarily confirmed. However, existing research has key technological gaps and is fundamentally different from the core innovation of this invention.

[0004] 1. Limitations of extraction and purification processes: Traditional hot water extraction of yacon polysaccharides yields low yields, and high temperatures easily damage the polysaccharide structure; the crude extract contains many protein and pigment impurities, and no process has been established for the targeted acquisition of highly active single polysaccharide components;

[0005] 2. Gaps in Molecular Structure Research: Although existing studies employ conventional structural analysis methods such as ion chromatography and FT-IR (which are consistent with the methods used in this invention), they can only obtain information on the monosaccharide composition or simple functional groups of crude polysaccharides. They have never isolated a single polysaccharide fragment with clear dual-enzyme inhibitory activity (such as YAPa-1), let alone resolved the complete molecular structure of this type of polysaccharide (including glycosidic bond linkage, repeating units, and higher-order structures). In other words, conventional methods have not yielded a novel molecular structure of YAPa-1.

[0006] 3. Lack of structure-activity relationship: Because the molecular structure of the active polysaccharide is not clear, the relationship between the polysaccharide structural characteristics and the dual-enzyme inhibitory activity cannot be elucidated. Only a rough inhibitory effect can be inferred, which cannot provide a theoretical basis for the targeted development of polysaccharides.

[0007] Based on the technical problems existing in the above-mentioned technologies, the present invention provides a process for extracting and purifying the highly active polysaccharide YAPa-1 from yacon, and uses systematic characterization technology to fully resolve the molecular structure of the polysaccharide, thereby expanding the application of yacon polysaccharide in the preparation of natural hypoglycemic products. Summary of the Invention

[0008] Technical Problem to be Solved: To address the aforementioned technical problems, the purpose of this invention is to provide a method for preparing yacon polysaccharide YAPa-1 and its application. Through an "enzyme-assisted hydrothermal extraction – two-step column chromatography purification" process, highly active single yacon polysaccharide YAPa-1 is directionally obtained, and its molecular structure is obtained using structural characterization techniques. The yacon polysaccharide YAPa-1 can significantly inhibit the activity of α-glucosidase and α-amylase, with an inhibitory effect close to that of the clinical drug acarbose, while avoiding the side effects of chemically synthesized drugs. This provides a core raw material for developing novel hypoglycemic products that combine high efficiency and safety, possessing significant social health value and promising industrial application prospects.

[0009] Technical solution: A yacon polysaccharide YAPa-1, the molecular structural formula of which is:

[0010]

[0011] The main chain of the above molecular structure is composed of alternating galactose and mannose, and the side chains are arabinose, glucose and rhamnose; the molar ratio of galactose, mannose, glucose, arabinose and rhamnose is 1.00:0.82:0.25:0.11:0.25.

[0012] The preparation method of the above-mentioned yacon polysaccharide YAPa-1 includes the following steps:

[0013] S1. Add water and cellulase to yacon powder, enzymatically hydrolyze in water bath, centrifuge and collect the supernatant, add anhydrous ethanol to the supernatant, let stand and centrifuge to collect the precipitate, redissolve the precipitate in water, add 15% trichloroacetic acid, let stand and centrifuge to collect the supernatant.

[0014] S2. Add pretreated NKA-2 macroporous resin to the supernatant, shake to adsorb pigments, filter and collect the filtrate, concentrate the filtrate and dialyze it, freeze-dry the retentate to obtain Yacon crude polysaccharide YAP.

[0015] S3. Yacon crude polysaccharide YAP was prepared by DEAE-52 cellulose column chromatography to obtain yacon polysaccharide YAPa;

[0016] S4. Yacon polysaccharide YAPa-1 was prepared by Sephadex G-200 gel column chromatography.

[0017] Preferably, in step S1, the mass-to-volume ratio of snow lotus fruit powder to water is 1:10~60 g / mL.

[0018] Preferably, the cellulase activity in step S1 is 100-1100 U / mL.

[0019] Preferably, the enzymatic hydrolysis temperature in step S1 is 40~90℃, and the enzymatic hydrolysis time is 1.5~4 h.

[0020] Preferably, in step S2, the oscillation speed is 100~500 r / min, the oscillation temperature is 25~45℃, and the oscillation time is 3~12 h.

[0021] Preferably, the molecular weight of the dialyzed molecules in step S2 is 1000~10000 Da.

[0022] Preferably, in step S3, the loading concentration of Yacon crude polysaccharide YAP in DEAE-52 cellulose column chromatography is 10~50 mg / mL, and the loading volume is 5~30 mL.

[0023] Preferably, in step S3, the eluent for DEAE-52 cellulose column chromatography is a 0~1.0 mol / L NaCl solution, and the elution rate is 0.2~1.5 mL / min.

[0024] Preferably, in step S3, the loading concentration of yacon polysaccharide YAPa in Sephadex G-200 gel column chromatography is 10~50 mg / mL, and the loading volume is 5~30 mL.

[0025] Preferably, the eluent for the Sephadex G-200 gel column chromatography in step S3 is deionized water, and the elution rate is 0.1~0.5 mL / min.

[0026] The application of the above-mentioned yacon polysaccharide YAPa-1 in inhibiting the activity of α-glucosidase and α-amylase.

[0027] The application of the above-mentioned yacon polysaccharide YAPa-1 in the preparation of hypoglycemic drugs.

[0028] Beneficial effects:

[0029] 1. High efficiency of the process: This invention provides an "enzyme-assisted hydrothermal-two-step column chromatography" process, which achieves a yield of 8.26% of yacon polysaccharide YAPa-1 and a purity of over 90%, solving the problems of low efficiency and high impurity content in traditional processes.

[0030] 2. Structural novelty: This invention obtained the molecular structure of YAPa-1 by structural analysis of the purified yacon polysaccharide YAPa-1, and clarified the specific residue linkage mode of YAPa-1 such as α-Araf-(1→6)-β-Galp and →3,6)-β-Manp-(1→3)-β-Galp, filling the technical gap in the detailed analysis of residue linkage in yacon polysaccharide.

[0031] 3. Clear structure-activity relationship: Based on the molecular structure of Yacon polysaccharide YAPa-1, it is elucidated that "high ratio of Gal / Man (galactose / mannose) (1.00:0.82), coexistence of α / β glycosidic bonds, and triple helix structure" are the key to dual enzyme inhibitory activity, providing a precise target for polysaccharide structure modification and efficient derivative development.

[0032] 4. Significant application value: The α-glucosidase and α-amylase inhibitory activities of yacon polysaccharide YAPa-1 are comparable to those of the clinical drug acarbose (IC50). 50 (No significant difference), and without the side effects of synthetic drugs, it can be directly used in the development of hypoglycemic products, possessing both social (meeting the needs of diabetic patients) and economic (industrialization of natural functional ingredients) value. Attached Figure Description

[0033] Figure 1 The effects of extraction process on the extraction efficiency of yacon polysaccharide in Example 1 are as follows: (a) Effect of extraction solvent on the yield of yacon polysaccharide; (b) Effect of extraction solvent on the inhibitory activities of α-glucosidase and α-amylase in yacon polysaccharide; (c) Effect of extraction process on the yield of yacon polysaccharide; (d) Effect of extraction process on the inhibitory activities of α-glucosidase and α-amylase in yacon polysaccharide; (e) Effect of biomimetic enzyme on the yield of yacon polysaccharide; (f) Effect of biomimetic enzyme on the inhibitory activities of α-glucosidase and α-amylase in yacon polysaccharide.

[0034] Figure 2 The effects of extraction process conditions on the extraction efficiency of yacon polysaccharide in Example 2 are as follows: (a) Effect of enzymatic hydrolysis time on the yield of yacon polysaccharide; (b) Effect of enzymatic hydrolysis temperature on the yield of yacon polysaccharide; (c) Effect of material-liquid ratio on the yield of yacon polysaccharide; (d) Effect of enzyme activity on the yield of yacon polysaccharide.

[0035] Figure 3 This is a response surface plot showing the effect of the interaction between enzyme activity and enzymatic hydrolysis temperature on the yield of yacon polysaccharides in Example 2.

[0036] Figure 4 This is a response surface plot showing the effect of the interaction between enzyme activity and enzymatic hydrolysis time on the yield of yacon polysaccharides in Example 2.

[0037] Figure 5 This is a response surface plot showing the effect of the interaction between enzyme activity and material-liquid ratio on the yield of yacon polysaccharides in Example 2.

[0038] Figure 6 This is a response surface plot showing the effect of the interaction between enzymatic hydrolysis temperature and enzymatic hydrolysis time on the yield of yacon polysaccharides in Example 2.

[0039] Figure 7The response surface plot shows the effect of the interaction between enzymatic hydrolysis temperature and material-liquid ratio on the yield of yacon polysaccharides in Example 2.

[0040] Figure 8 The response surface plot shows the effect of the interaction between enzymatic hydrolysis time and material-to-liquid ratio on the yield of yacon polysaccharides in Example 2.

[0041] Figure 9 Elution curves of crude yacon polysaccharide YAP and yacon polysaccharide YAPa on the chromatography column in Example 3; wherein: (a): elution curve of crude yacon polysaccharide YAP on DEAE-52 column; (b): elution curve of yacon polysaccharide YAPa on Sephadex G-200;

[0042] Figure 10 Chemical composition and purity analysis of Yacon polysaccharide YAPa-1 in Example 4; wherein: (a): standard curve of glucose content; (b): standard curve of protein content; (c): standard curve of uronic acid content;

[0043] Figure 11 The chromatogram of yacon polysaccharide YAPa-1 in Example 4 is shown below; where: (a): IC chromatogram of YAPa-1; (b): HPGPC chromatogram of YAPa-1;

[0044] Figure 12 Here are the FT-IR spectra of yacon polysaccharides in Example 4; where: (a): comparison of FT-IR spectra of crude yacon polysaccharide YAP, yacon polysaccharide YAPa and YAPa-1; (b): FT-IR spectrum of yacon polysaccharide YAPa-1;

[0045] Figure 13 The images show the UV spectrum and Congo red staining results of yacon polysaccharide YAPa-1 in Example 4; where: (a): UV spectrum of YAPa-1; (b): Congo red staining results of YAPa-1;

[0046] Figure 14 Gas chromatography-mass spectrum of methylated yacon polysaccharide YAPa-1;

[0047] Figure 15 Yacon polysaccharide YAPa-1 1 H-NMR spectrum;

[0048] Figure 16 Yacon polysaccharide YAPa-1 13 C-NMR spectrum;

[0049] Figure 17 HSQC-NMR spectrum of yacon polysaccharide YAPa-1;

[0050] Figure 18 COSY-NMR spectrum of yacon polysaccharide YAPa-1;

[0051] Figure 19 HMBC-NMR spectrum of yacon polysaccharide YAPa-1;

[0052] Figure 20 This is a schematic diagram of the molecular structure of yacon polysaccharide YAPa-1;

[0053] Figure 21 SEM images of Yacon polysaccharide YAPa-1 at different magnifications: (a): ×200, showing the overall morphological characteristics; (b): ×1000, showing the distribution of porous structure; (c): ×5000, showing the details of porous structure; (d): ×10000, revealing the microscale aggregation characteristics.

[0054] Figure 22 The inhibitory activities of α-glucosidase and α-amylase on yacon polysaccharides of Examples 3 and Comparative Examples 7-12 are as follows: (a): inhibition rate of α-glucosidase by YAP, YAPa, YAPb and YAPc; (b): inhibition rate of α-amylase by YAP, YAPa, YAPb and YAPc; (c): inhibition rate of α-glucosidase by YAPa, YAPa-1 and YAPa-2; (d): inhibition rate of α-amylase by YAPa, YAPa-1 and YAPa-2. Detailed Implementation

[0055] The present invention will be further described below with reference to embodiments. These embodiments are illustrative of the present invention, but the present invention is not limited to these embodiments:

[0056] The pectinase, cellulase, acidic protease, neutral protease, and alkaline protease involved in the following examples and comparative examples were all purchased from Shanghai Yuanye Biotechnology Co., Ltd., with enzyme activities of 50, 50, 50, 50, and 200 U / mg, respectively; α-glucosidase and α-amylase were both purchased from Beijing Solarbio Technology Co., Ltd., with enzyme activities of 100 U / mg.

[0057] Example 1

[0058] This embodiment illustrates the effect of extraction process on the extraction efficiency of yacon polysaccharides, including the following steps:

[0059] S1. Extraction solvent screening: Using the yield of yacon polysaccharides and the inhibitory activities of α-glucosidase and α-amylase as indicators, 1.0 g of yacon powder was weighed and extracted with deionized water, 0.05 mol / L HCl, and 0.05 mol / L NaOH at a material-to-liquid ratio of 1:25 (g / mL) in a 60℃ constant temperature water bath for 2 h. After extraction, the supernatant was collected by centrifugation at 6000×g for 15 min. The polysaccharide content in the supernatant was determined by the phenol-sulfuric acid method and the yield of yacon polysaccharides was calculated. The supernatant was freeze-dried and reconstituted with deionized water to 5 mg / mL. The inhibition rate of the crude polysaccharide solution on α-glucosidase and α-amylase was determined.

[0060] S2. Screening of extraction processes: Using deionized water as solvent, the effects of three extraction techniques were compared. Traditional hydrothermal extraction was performed by a constant temperature water bath at 60℃ for 2 h, ultrasonic-assisted hydrothermal extraction was performed by 400 W ultrasound + 60℃ water bath for 2 h, and microwave-assisted hydrothermal extraction was performed by 400 W microwave for 5 min + 60℃ water bath for 115 min (total duration 2 h). After extraction, the supernatant was collected by centrifugation at 6000×g for 15 min. The polysaccharide content in the supernatant was determined by the phenol-sulfuric acid method, and the yield of yacon polysaccharide was calculated. The supernatant was freeze-dried and reconstituted with deionized water to 5 mg / mL. The inhibition rate of the crude polysaccharide solution on α-glucosidase and α-amylase was determined.

[0061] S3. Screening of biomimetic enzymes: Using deionized water as solvent, traditional hydrothermal extraction was performed, with pectinase, cellulase, acidic protease, neutral protease, and alkaline protease added respectively (enzyme activity gradient 100~1100 U / mL). Enzymatic hydrolysis was carried out at 60℃ for 2 h. After extraction, the supernatant was collected by centrifugation at 6000×g for 15 min. The polysaccharide content in the supernatant was determined by phenol-sulfuric acid method, and the yield of yacon polysaccharide was calculated. The supernatant was freeze-dried and reconstituted with deionized water to 5 mg / mL. The inhibition rate of the crude polysaccharide solution on α-glucosidase and α-amylase was determined.

[0062] like Figure 1 a and Figure 1 As shown in b, the yield of yacon polysaccharides, the inhibition rate of α-glucosidase, and the inhibition rate of α-amylase in the deionized water group were significantly higher than those in the 0.05 mol / L HCl group and the 0.05 mol / L NaOH group (P < 0.05). Furthermore, deionized water can avoid the hydrolysis and destruction of polysaccharide glycosidic bonds by acids and bases. Therefore, deionized water was determined to be the extraction solvent.

[0063] like Figure 1 c and Figure 1As shown in Figure d, the yield of yacon polysaccharides was in the order of ultrasound-assisted extraction > microwave-assisted extraction > traditional hydrothermal extraction. However, the order of inhibitory activity against α-glucosidase and α-amylase was: traditional hydrothermal extraction > microwave-assisted extraction > ultrasound-assisted extraction. The yield and activity trends of yacon polysaccharides showed opposite directions, indicating that ultrasound and microwaves, on the one hand, disrupt cell walls, increasing polysaccharide dissolution, and on the other hand, break glycosidic bonds, causing partial hydrolysis of polysaccharides. Traditional hydrothermal extraction yielded the lowest yacon polysaccharide yield but the strongest activity. Compared to ultrasound extraction, which yielded the highest yacon polysaccharide, the yield was only 6.33% lower, but the activity increased by 122.58%. Therefore, traditional hydrothermal extraction was chosen as the final extraction technique.

[0064] like Figure 1 e and Figure 1 As shown in f, the highest yield of yacon polysaccharides in the cellulase group reached 7.12±0.25% (corresponding to an enzyme activity of 900 U / mL), and the inhibition rates of α-glucosidase and α-amylase were significantly higher than those in the pectinase group, neutral protease group, acidic protease group, and alkaline protease group (P<0.05). This indicates that cellulase can efficiently degrade the cell wall of yacon and release more highly active polysaccharides. Therefore, cellulase was selected as the biomimetic enzyme.

[0065] Example 2

[0066] This embodiment describes an optimized method for extracting polysaccharides from yacon, comprising the following steps:

[0067] S1. Single-factor experiment: Using deionized water as solvent, traditional hydrothermal extraction was performed with the addition of cellulase to extract yacon polysaccharides. The yield of yacon polysaccharides was used as the response value. The effects of cellulase activity (100, 300, 500, 700, 900, 1100 U / mL), enzymatic hydrolysis temperature (40, 50, 60, 70, 80, 90℃), enzymatic hydrolysis time (1.5, 2, 2.5, 3, 3.4, 4.0 h), and solid-liquid ratio (1:10, 1:20, 1:3, 1:40, 1:50, 1:60 g / mL) on extraction efficiency were investigated using the "fixed three factors, optimized one factor" method. After extraction, the supernatant was collected by centrifugation at 6000×g for 15 min. The polysaccharide content in the supernatant was determined by the phenol-sulfuric acid method, and the yield of yacon polysaccharides was calculated.

[0068] S2. Response Surface Experiment: Based on the results of the single-factor experiment in step S1, a response surface experiment with four factors and three levels was designed. The optimal extraction parameters were obtained by analyzing the yield of yacon polysaccharide, and a response surface regression equation model was established. The coded values ​​are shown in Table 1.

[0069] like Figure 2As shown, the yield of yacon polysaccharide was highest when the cellulase activity was 900 U / mL. The yield decreased after exceeding this activity, suggesting that excessive enzyme would hydrolyze the released polysaccharide. The highest yield was achieved at an enzymatic hydrolysis temperature of 70℃. Low temperatures resulted in insufficient enzyme activity, leading to low yields, while high temperatures caused enzyme inactivation and destroyed the polysaccharide structure. The highest yield was achieved at an enzymatic hydrolysis time of 3.0 h. Excessive time would lead to polysaccharide degradation. The highest yield was achieved at a material-to-liquid ratio of 1:40 (g / mL). Excessively high material-to-liquid ratios would dilute the polysaccharide concentration and reduce extraction efficiency. Based on this, the optimization range for each factor was determined.

[0070] Table 1 Response surface factors and levels

[0071]

[0072] Table 1 shows the response surface methodology (RSM) factors and levels. The RSM extraction process conditions for yacon polysaccharides were optimized using the RSM method. Based on the results of the single-factor experiments and the central composite design principle of Box-Benhnken, the RSM method was used to optimize the extraction conditions of yacon polysaccharides at four factors and three levels.

[0073] Table 2 Response Surface Experimental Design and Results

[0074]

[0075] As shown in Table 2, the data analysis using the RSREG (response surface regression) procedure in Design-Expert 12 showed significant regression analysis and variance, indicating a reliable experimental design that was statistically significant. The lack-of-fit term (P=0.7621>0.05) indicates that the lack-of-fit term in the regression model was not significant. The correlation coefficient R0 of the model... 2 =0.9782 and Radj=0.9547, indicating that the model has a good fit and the predicted values ​​have a good correlation with the actual values. It can explain 97.82% of the changes in the response values ​​and can be used to analyze and predict the optimal process and yield of yacon polysaccharide extraction.

[0076] Table 3 Response Surface Model Analysis

[0077]

[0078] As shown in Table 3, the formula for predicting the yield of yacon polysaccharides is as follows: Y = 7.580902 - 0.3547A + 0.0537B + 0.0772C + 0.0462D - 0.04042AB - 0.2760AC + 0.0484AD - 0.6844BC + 0.0839BD - 0.0671CD - 0.8621A 2 -1.7452B 2 -1.3927C 2 -0.6803D2 Regression tests on the coefficient model showed that the p-values ​​for factors B (enzymatic hydrolysis temperature), C (enzymatic hydrolysis time), and BC (enzymatic hydrolysis temperature - enzymatic hydrolysis time) were <0.01, indicating that the effects of enzymatic hydrolysis temperature, enzymatic hydrolysis time, and their interaction on the yield of yacon polysaccharides were highly significant. The F-values ​​showed that the order of influence of each factor on the polysaccharide yield was: enzymatic hydrolysis time > enzymatic hydrolysis temperature > enzyme activity > material-to-liquid ratio.

[0079] like Figures 3-8 As shown, response surface methodology analysis yielded the following optimal extraction parameters for yacon polysaccharides: enzyme activity of 932.58 U / mL, hydrolysis temperature of 49.16℃, hydrolysis time of 2.977 h, and a solid-liquid ratio of 1:40.65 g / mL. The theoretical predicted yield of yacon polysaccharides was 7.44%. Based on actual experimental conditions, the optimal process conditions were determined to be: enzyme activity of 930 U / mL, hydrolysis temperature of 72℃, hydrolysis time of 3.0 h, and a solid-liquid ratio of 1:40 g / mL. Under these conditions, three parallel experiments verified an actual yield of 8.26% for yacon polysaccharides, which was not significantly different from the theoretical value. This indicates that the model can be used to predict the extraction of yacon polysaccharides, and that cellulase-assisted water extraction is far superior to water bath extraction in terms of energy consumption and time cost, making it suitable for industrial application.

[0080] Example 3

[0081] This embodiment describes a method for preparing yacon polysaccharide YAPa, including the following steps:

[0082] S1. Weigh 100 g of yacon powder, add 4 L of deionized water at a material-to-liquid ratio of 1:40 (g / mL), then add 930 U / mL of cellulase, and enzymatically hydrolyze in a constant temperature water bath at 72℃ for 3.0 h, stirring once every 30 min (100 r / min).

[0083] S2. After enzymatic hydrolysis, centrifuge at 6000×g for 15 min to remove residue, collect about 3.8 L of supernatant, add 4 times the volume of anhydrous ethanol (15.2 L) to the supernatant, let stand at 4℃ for 24 h to allow the polysaccharide to fully precipitate, and centrifuge under the same conditions to collect the precipitate.

[0084] S3. Redissolve the precipitate with 1 L of deionized water, add an equal volume of 15% trichloroacetic acid, let stand at 4℃ for 24 h to denature the protein, centrifuge at 6000×g for 15 min to discard the protein precipitate, and collect the supernatant.

[0085] S4. Add 120 g of pretreated NKA-2 macroporous resin (soaked in distilled water for 24 h → defatted with 95% ethanol → washed with distilled water until neutral → dried at 60℃) to the supernatant. Shake at 250 r / min and 35℃ for 6 h to adsorb pigments. Collect the filtrate by suction filtration. Concentrate the filtrate to 50 mL using a rotary evaporator at 50℃ and -0.09 MPa. Place it in a 3500 Da dialysis bag and dialyze with distilled water for 48 h (changing the water every 12 h) to desalinate. Finally, freeze-dry the dialysis retentate to obtain Yacon crude polysaccharide YAP.

[0086] S5. DEAE-52 cellulose was soaked in 0.5 mol / L NaCl for 24 h, washed with distilled water until neutral, and then packed into a Φ2.6×30 cm chromatography column. The column was equilibrated with deionized water until the UV 280 nm absorbance was <0.02. The crude yacon polysaccharide YAP prepared in S4 was prepared into a 10 mg / mL solution with deionized water. 5 mL of the solution was loaded onto the column and eluted with 0 mol / L NaCl solution (deionized water) at a flow rate of 1 mL / min. 5 mL of the eluent was collected in each tube and freeze-dried to obtain yacon polysaccharide YAPa.

[0087] S6. The Sephadex G-200 was soaked in deionized water for 48 h until it was completely swollen. It was then packed into a Φ1.5×30 cm chromatography column and equilibrated with the same buffer until the baseline was stable. The yacon polysaccharide YAPa prepared in S5 was prepared into a 10 mg / mL solution with deionized water. 5 mL of the solution was loaded onto the column, and deionized water was used as the eluent. The column was eluted at a flow rate of 0.25 mL / min. 5 mL of the eluent was collected in each tube, and the elution curve was plotted to obtain two fractions with significantly different molecular weight distributions: YAPa-1 and YAPa-2.

[0088] Comparative Example 1

[0089] The difference between this comparative example and Example 3 is that the eluent in this comparative example is a 0.1 mol / L NaCl solution, while the other steps are the same as in Example 3, to obtain yacon polysaccharide YAPb.

[0090] Comparative Example 2

[0091] The difference between this comparative example and Example 3 is that the eluent in this comparative example is a 0.2 mol / L NaCl solution, while the other steps are the same as in Example 3.

[0092] Comparative Example 3

[0093] The difference between this comparative example and Example 3 is that the eluent in this comparative example is a 0.3 mol / L NaCl solution, while the other steps are the same as in Example 4.

[0094] Comparative Example 4

[0095] The difference between this comparative example and Example 3 is that the eluent in this comparative example is a 0.4 mol / L NaCl solution, while the other steps are the same as in Example 3.

[0096] Comparative Example 5

[0097] The difference between this comparative example and Example 3 is that the eluent in this comparative example is a 0.5 mol / L NaCl solution, while the other steps are the same as in Example 3.

[0098] Comparative Example 6

[0099] The difference between this comparative example and Example 3 is that the eluent in this comparative example is a 0.6 mol / L NaCl solution, while the other steps are the same as in Example 3.

[0100] Comparative Example 7

[0101] The difference between this comparative example and Example 3 is that the eluent in this comparative example is a 1.0 mol / L NaCl solution, while the other steps are the same as in Example 3, to obtain yacon polysaccharide YAPc.

[0102] DEAE-52 ion exchange chromatography provides excellent separation for polysaccharides because polysaccharides carry a certain electrical charge. The anion exchange matrix binds to the negatively charged polysaccharides, which are then retained on the column. By preparing eluents with different salt concentrations, the polysaccharides adsorbed on the column can be eluted sequentially, with weaker binding components being eluted and separated first. Figure 9 Figure a shows the elution curve of crude yacon polysaccharide YAP on a DEAE-52 column. After gradient elution with NaCl solution, three distinct elution peaks appeared: YAPa, YAPb, and YAPc. These three polysaccharide components were obtained by elution with deionized water, 0.1 mol / L NaCl solution, and 1 mol / L NaCl solution, respectively.

[0103] like Figure 9 Figure b shows the elution curves of yacon polysaccharide (YAPa) on Sephadex G-200. Both elution peaks exhibit a typical symmetrical Gaussian distribution, indicating low diffusion effect and no tailing during separation. This reflects the good separation efficiency of the chromatography column and the compatibility between the sample and the gel matrix. Notably, the elution peak of YAPa-1 has a long plateau period (elution volume 45–60 mL), indicating the high homogeneity of this component. This plateau period typically originates from the characteristics of highly homogeneous polysaccharides within the gel channels under equilibrium partitioning conditions. In summary, these results confirm that Sephadex G-200 can achieve high-resolution fractionation purification of YAPa, laying the foundation for subsequent functional studies and component structure characterization.

[0104] Example 4

[0105] This embodiment describes the structural characterization and identification of yacon polysaccharide YAPa-1, including the following steps:

[0106] S1. Chemical composition and purity analysis:

[0107] (1) The polysaccharide content in YAPa-1 was determined using the phenol-sulfuric acid method;

[0108] (2) The protein content in YAPa-1 was determined using the Coomassie brilliant blue method;

[0109] (3) The content of uronic acid in YAPa-1 was determined by the m-hydroxybiphenyl method;

[0110] S2. Monosaccharide composition analysis (ion chromatography): Weigh 5 mg YAPa-1, add 2 mL of 3 mol / L trifluoroacetic acid (TFA), and hydrolyze at 120℃ for 3 h; after hydrolysis, dry under nitrogen, reconstitute with 5 mL of deionized water, take 50 μL and dilute to 1 mL, centrifuge at 8000×g for 5 min; take the supernatant and analyze it using an ion chromatograph (ICS5000), with a mobile phase of 0.1 mol / L NaOH, a flow rate of 1 mL / min, and a column temperature of 30℃;

[0111] S3. Molecular weight analysis (HPGPC): A 1 mg / mL YAPa-1 solution was prepared, filtered through a 0.45 μm PES membrane, and analyzed by high performance gel permeation chromatography (HPGPC) using a TSKgel G4000PWXL column (7.8 × 300 mm). The mobile phase was 0.05 mol / L NaCl, the flow rate was 0.5 mL / min, and the column temperature was 35℃. A standard curve was plotted using dextran standards (1000~100000 Da), and the average molecular weight and polydispersity index of YAPa-1 were calculated.

[0112] S4. Functional group and glycosidic bond analysis (FT-IR): 2 mg YAPa-1 and 200 mg KBr (spectrally pure) were mixed and ground for 10 min, compressed into a pellet (10 MPa, 30 s), and then scanned from 4000 to 400 cm⁻¹ using a Fourier transform infrared spectrometer (Nicolet 5700). -1 ;

[0113] S5. Ultraviolet analysis: Prepare a 0.5 mg / mL YAPa-1 solution and perform a full-wavelength ultraviolet scan in the range of 200~600 nm;

[0114] S6. Helical structure analysis (Congo red experiment): Take 5 test tubes, add 1 mL of 0.5 mg / mL YAPa-1 solution and 1 mL of 0.1 mg / mL Congo red reagent to each tube, and then add 1 mL of 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaOH solutions respectively. Incubate at 37℃ for 30 min, measure the absorption spectrum in the wavelength range of 400~700 nm, and record the maximum absorption wavelength (λmax). Use gel polysaccharide with a typical triple helix structure as a positive control to analyze the helical structure of yacon polysaccharide YAPa-1.

[0115] S7. Glycosidic bond type analysis (methylation-GC-MS): 25 mg YAPa-1 was weighed and dissolved in 2 mL DMSO, and sonicated for 30 min until completely dissolved; 10 mg NaOH powder was added, and the mixture was sonicated for 10 min under nitrogen purging and then placed in an ice bath for 10 min; 2 mL iodomethane was slowly added dropwise, and the mixture was sonicated for 1 h to carry out the methylation reaction; after the reaction was completed, 1 mL of water was added to quench the reaction, and the residual iodomethane was removed by concentration under reduced pressure. The methylated product was extracted three times with chloroform (5 mL each time) to collect the methylated product; 100 μL of 2 mol / L LTFA was added to the methylated product, and the mixture was hydrolyzed at 121 °C for 90 min, aminated, and then reduced with NaBD4 for 2.5 h, followed by acetylation with acetic anhydride; the analysis was performed using a gas chromatography-mass spectrometry (Agilent 7890A) column with a DB-5MS column and a column temperature program of 80 °C → 280 °C (10 °C / min).

[0116] S8. Residue Linkage and Repeat Unit Analysis (NMR): 50 mg of YAPa-1 was dissolved in 0.55 mL of D2O (99.9%) and allowed to stand at room temperature for 12 h to fully dissolve; 1D was measured using a 500 MHz NMR spectrometer (Bruker Avance III) at 25 °C. 1 ¹H NMR: δ 0~10ppm; 13 C NMR: δ 0~130 ppm) and 2D (COSY, HSQC, HMBC) spectra;

[0117] S9. Microscopic Morphology Observation (SEM): The dried YAPa-1 powder was fixed to the sample stage with conductive tape, followed by gold plating (45 seconds, 10 mA). The sample was then placed on the scanning electron microscope stage, and the microstructure was observed using a Zeiss EVO 15 scanning electron microscope at an accelerating voltage of 10 kV.

[0118] like Figure 10 As shown in figure a, the polysaccharide content in YAPa-1 is 90.03±1.03% (standard curve y=7.9595x, Ra). 2 =0.9991); such as Figure 10 As shown in b, the protein content in YAPa-1 is 0.24 ± 0.01% (standard curve y = 8.6272x + 0.3516, R0). 2 =0.9997); for example Figure 10 As shown in c, the uronic acid content in YAPa-1 is 1.31 ± 0.17% (standard curve y = 0.0098x, R0). 2 =0.9993), all of the above results indicate that the purity of yacon polysaccharide YAPa-1 is very high.

[0119] like Figure 11 As shown in a, yacon polysaccharide YAPa-1 is composed of five monosaccharides: galactose (Gal), mannose (Man), glucose (Glc), arabinose (Ara), and rhamnose (Rha), with a molar ratio of 1.00:0.82:0.25:0.11:0.25.

[0120] like Figure 11 As shown in b, the average molecular weight of yacon polysaccharide YAPa-1 is 10873 Da, and the polydispersity index (Mw / Mn) is 1.0151, indicating that its molecular distribution is uniform.

[0121] like Figure 12 As shown in figure a, the Fourier transform infrared (FT-IR) spectra of crude yacon polysaccharide YAP, yacon polysaccharide YAPa, and YAPa-1 are all at 3372, 2941, 1612, 1421, and 1043 cm⁻¹. -1 The presence of similar characteristic absorption peaks indicates that the functional groups of yacon polysaccharide remain stable during the separation and purification process.

[0122] like Figure 12 As shown in b, the Fourier transform infrared (FT-IR) spectrum of yacon polysaccharide YAPa-1 shows a characteristic absorption peak corresponding to a specific functional group: 3372 cm⁻¹. -1 The strong absorption peak at 2941 cm⁻¹ is attributed to the OH stretching vibration of the hydroxyl group, a typical characteristic of intramolecular and intermolecular hydrogen bonding in polysaccharides. -1 2902 cm -1 and 2837 cm -1 The three splitting peaks at 1612 cm⁻¹ correspond to the CH stretching vibrations of the methylene (-CH₂-) and methylene (-CH-) groups on the sugar ring, indicating the presence of alkyl structures in the polysaccharide backbone. -1 The absorption peak at 1043 cm⁻¹ corresponds to the asymmetric stretching vibration of the carboxylate ion (-COO⁻), while the peak at 1043 cm⁻¹ corresponds to the asymmetric stretching vibration of the carboxylate ion (-COO⁻). -1 The strong peak at 826 cm⁻¹ is a characteristic peak of the COC stretching vibration of the glycosidic bond in the pyranose ring. It is worth noting that... -1 and 896cm-1 The absorption peak at the end indicates that its terminal carbon has both α and β configurations, confirming that YAPa-1 is a pyran-type polysaccharide containing both α and β glycosidic bonds.

[0123] like Figure 13 As shown in Figure a, within the wavelength range of 230–400 nm, the yacon polysaccharide YAPa-1 component exhibits the following characteristics: No characteristic absorption is observed near 260 nm, indicating extremely low residual nucleic acid content in YAPa-1. Since the conjugated double bonds of nucleic acid bases (purine / pyrimidine) exhibit strong absorption in the 250–280 nm range, while YAPa-1 shows no significant absorption in this range, it confirms that the purification process effectively removed nucleic acid contaminants. No significant absorption is observed at 280 nm, indicating a low protein content in YAPa-1. The benzene ring conjugated system of aromatic amino acids (tryptophan / tyrosine) in the protein produces characteristic absorption at 280 nm; the absence of absorbance in this band confirms the high purity of YAPa-1. The above results are consistent with the typical UV spectral characteristics of purified polysaccharides, providing key quality assurance for subsequent bioactivity studies: the removal of nucleic acids / proteins avoids their interference with α-glucosidase inhibition experiments (such as non-specific binding of nucleic acids or effects on protease activity); the low UV background absorption confirms the effectiveness of the purification strategy of using DEAE-52 in combination with Sephadex G-200, meeting the purity requirements for functional polysaccharide studies.

[0124] like Figure 13 As shown in b, the λmax of the complex formed by yacon polysaccharide YAPa-1 and Congo red first increased (0.1~0.2 mol / L) and then decreased (0.3~0.5 mol / L) with increasing NaOH concentration. The trend of change is consistent with that of gellan gum, which has a known triple helix structure, confirming that YAPa-1 has a typical triple helix higher order structure.

[0125] like Figure 14 As shown, the GC-MS chromatogram of methylation of yacon polysaccharide YAPa-1 revealed seven partially methylated sugar alcohol acetate peaks (PMAAs) in YAPa-1. By comparing with the CCRC (Center for Complex Carbohydrates) spectral database, seven glycosidic bonds in YAPa-1 were identified as Rhap-(1→, Araf-(1→, →4)-Glcp-(1→, →3)-Galp-(1→, →6)-Manp-(1→, →3,6)-Manp-(1→, →3,6)-Galp-(1→, →3,6)-Galp-(1→). Detailed data are shown in Table 4. The molar ratio of the seven glycosidic bonds was 0.18:0.21:0.18:1.00:0.75:0.23:0.25, with galactose and mannose residues accounting for more than 60% of the total sugar residues. This aligns with the monosaccharide composition analysis (…). Figure 11 a) The results are consistent.

[0126] Table 4. Methylation analysis of YAPa-1

[0127]

[0128] like Figures 15-19 As shown, 1 α / β terminal hydrogen signals were detected by ¹H NMR in the δ 4.60–5.40 ppm range. 13 Terminal carbon signals were detected by C NMR in the δ 99–110 ppm range. Figure 12 ); HSQC spectroscopy identified 7 terminal hydrogen-carbon cross peaks ( Figure 13 ), combined with the adjacent proton coupling information of the COSY spectrum ( Figure 14 Based on the results of methylation analysis, the chemical shifts of seven sugar residues can be determined, and the specific data are shown in Table 5; HMBC spectroscopy further clarifies the linkage relationships between residues through long-range CH correlation signals. Figure 16 Examples include α-Araf-(1→6)-β-Galp (corresponding to δ 5.42 / 70.17 ppm) and →3,6)-β-Manp-(1→3)-β-Galp. The final main chain repeating unit of YAPa-1 is →3,6)-β-Manp-(1→3)-β-Galp-(1→6)-β-Manp-(1→3)-β-Galp-(1→), with side chains containing α-Araf-(1→) and α-Rhap-(1→). Its molecular structure is as follows: Figure 20 As shown.

[0129] Table 5. YAPa-1 in D2O 1 H and 13 C NMR chemical shift assignment

[0130]

[0131] like Figure 21 As shown, YAPa-1 forms a sponge-like porous network structure composed of smooth, uniformly cross-linked nanofibers. This morphology is consistent with the typical aggregation mode of neutral polysaccharides—the porous network originates from the self-assembly of molecular chains, while the uniform fibrous structure is related to the regular stacking of β-conformation sugar rings. The three-dimensional nanofiber network and submicron-scale porous topology of YAPa-1 share commonalities with many reported substances possessing hypoglycemic activity, and its structural characteristics directly contribute to its inhibitory effects on α-glucosidase and α-amylase. Specifically:

[0132] Nanofiber networks slow down substrate diffusion, reducing the effective contact frequency between the substrate and the two enzymes. This nanofiber-mediated substrate diffusion delay enhances enzyme inhibitory activity.

[0133] Submicron porous topologies impose spatial constraints on enzyme conformation: inhibiting the flexible folding of α-amylase active sites (such as the catalytic residues of aspartic acid 206 / 300) while constraining the spatial active conformation of α-glucosidase.

[0134] The smooth fiber surface exposes abundant hydroxyl groups, which can competitively bind to the catalytic residues of two enzymes (such as aspartic acid 205 / aspartic acid 352 in α-glucosidase and the catalytic center residue in α-amylase), thereby achieving enzyme inhibition through active site competition mediated by the hydroxyl network.

[0135] In summary, this cross-species evidence suggests that the interwoven nanofiber framework enhances substrate diffusion resistance, submicron pores inhibit enzyme conformational changes through topological confinement, and exposed hydroxyl / carboxyl groups on the smooth surface directly compete for the enzyme's active site. These three mechanisms synergistically constitute a universal "solid-state sustained-release inhibition mechanism," providing structural-biological theoretical support for the hypoglycemic activity of YAPa-1.

[0136] Comparative Example 8

[0137] The difference between this comparative example and Example 3 is that this comparative example uses Yacon crude polysaccharide YAP, and the preparation method of Yacon crude polysaccharide YAP is described in Example 3.

[0138] Comparative Example 9

[0139] The difference between this comparative example and Example 3 is that this comparative example uses yacon polysaccharide YAPa, and the preparation method of yacon polysaccharide YAPa is described in Example 3.

[0140] Comparative Example 10

[0141] The difference between this comparative example and Example 3 is that this comparative example uses yacon polysaccharide YAPb, and the preparation method of yacon polysaccharide YAPb is described in Comparative Example 1.

[0142] Comparative Example 11

[0143] The difference between this comparative example and Example 3 is that this comparative example uses yacon polysaccharide YAPc, and the preparation method of yacon polysaccharide YAPc is described in Comparative Example 7.

[0144] Comparative Example 12

[0145] The difference between this comparative example and Example 3 is that this comparative example uses yacon polysaccharide YAPa-2, and the preparation method of yacon polysaccharide YAPa-2 is described in Example 3.

[0146] Comparative Example 13

[0147] The comparative example uses acarbose as a positive control.

[0148] like Figure 22As shown in a~b, compared with crude yacon polysaccharides YAP (Comparative Example 7), YAPb (Comparative Example 2), and YAPc (Comparative Example 10), yacon polysaccharide YAPa (Comparative Example 9) exhibited the highest inhibitory activity against α-glucosidase and α-amylase, with its α-glucosidase half-maximal inhibitory concentration (IC50) being the highest. 50 The concentration was 0.1034 mg / mL, and the α-amylase IC50 was... 50 It is 0.2125 mg / mL.

[0149] like Figure 22 Figure c shows the inhibitory effects of yacon polysaccharides YAPa (Comparative Example 9), YAPa-1 (Example 3), and YAPa-2 (Comparative Example 12) on α-glucosidase activity, with acarbose (Comparative Example 13) as a positive control. Within a concentration range of 0–0.5 mg / mL, all three yacon polysaccharide components exhibited concentration-dependent inhibition of α-glucosidase. YAPa-1 showed the strongest inhibitory activity: its inhibition rate rapidly increased in the low concentration range (0–0.1 mg / mL) and approached the inhibition rate of acarbose (Comparative Example 13) at 0.5 mg / mL. The IC50 of YAPa-1 against α-glucosidase is shown in Figure c. 50 The concentration was 0.0532 mg / mL, significantly lower than YAPa and YAPa-2 (P<0.05), and similar to acarbose (IC50). 50 There was no significant difference (P > 0.05) between the two groups (0.0325 mg / mL).

[0150] like Figure 22 As shown in d, the inhibitory activity order of yacon polysaccharides against α-amylase is YAPa-1 > YAPa > YAPa-2, which is consistent with their inhibitory activity order against α-glucosidase. The IC50 of YAPa-1 against α-amylase... 50 The concentration was 0.1158 mg / mL, a decrease of 46.48% compared to YAPa (0.2164 mg / mL).

[0151] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A yacon polysaccharide YAPa-1, characterized in that: Its molecular structural formula is: 。 2. The yacon polysaccharide YAPa-1 according to claim 1, characterized in that: The main chain of the molecular structure is composed of alternating galactose and mannose, and the side chains are arabinose, glucose and rhamnose; the molar ratio of galactose, mannose, glucose, arabinose and rhamnose is 1.00:0.82:0.25:0.11:0.

25.

3. The yacon polysaccharide YAPa-1 according to claim 1, characterized in that: Its molecular weight is 10873 Da and its polydispersity index is 1.0151.

4. A method for preparing yacon polysaccharide YAPa-1 according to any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Add water and cellulase to yacon powder, enzymatically hydrolyze in water bath, centrifuge and collect the supernatant, add anhydrous ethanol to the supernatant, let stand and centrifuge to collect the precipitate, redissolve the precipitate in water, add 15% trichloroacetic acid, let stand and centrifuge to collect the supernatant. S2. Add pretreated NKA-2 macroporous resin to the supernatant, shake to adsorb pigments, filter and collect the filtrate, concentrate the filtrate and dialyze it, freeze-dry the retentate to obtain Yacon crude polysaccharide YAP. S3. Yacon crude polysaccharide YAP was obtained by DEAE-52 cellulose column chromatography, with deionized water as the eluent. S4. Yacon polysaccharide YAPa was prepared by Sephadex G-200 gel column chromatography with deionized water as the eluent.

5. The preparation method according to claim 4, characterized in that: In step S1, the mass-to-volume ratio of snow lotus fruit powder to water is 1:10~60 g / mL; the unit enzyme activity of cellulase is 100~1100 U / mL; the enzymatic hydrolysis temperature is 40~90℃, and the enzymatic hydrolysis time is 1.5~4 h.

6. The preparation method according to claim 4, characterized in that: In step S2, the oscillation speed is 100~500 r / min, the oscillation temperature is 25~45℃, and the oscillation time is 3~12 h; the molecular weight of the dialysis is 1000~10000 Da.

7. The preparation method according to claim 4, characterized in that: In step S3, the loading concentration of Yacon crude polysaccharide YAP in DEAE-52 cellulose column chromatography is 10~50 mg / mL, the loading volume is 5~30 mL, and the elution rate is 0.2~1.5 mL / min.

8. The preparation method according to claim 4, characterized in that: In step S4, the loading concentration of Yacon polysaccharide YAPa in Sephadex G-200 gel column chromatography is 10~50 mg / mL, the loading volume is 5~30 mL, and the elution rate is 0.1~0.5 mL / min.

9. The use of the yacon polysaccharide YAPa-1 according to any one of claims 1 to 3 in the preparation of a drug that inhibits the activity of α-glucosidase and α-amylase.

10. The use of the yacon polysaccharide YAPa-1 according to any one of claims 1 to 3 in the preparation of hypoglycemic drugs.

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