Polygonatum kingianum polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses polygonatum kingianum polysaccharide and a preparation method thereof, the polygonatum kingianum polysaccharide is composed of fructose and glucose in a molar ratio of 18.23: 1, is a Graminan type fructan, and has a structure shown as a formula I; the preparation method comprises the following steps: (1) carrying out alcohol extraction and impurity removal, water extraction and alcohol precipitation, redissolution and freeze drying on polygonatum kingianum powder to obtain polygonatum kingianum water extraction crude polysaccharide; (2) performing combined deproteinization by a papain-Sevage method, and dialyzing to obtain polygonatum kingianum refined polysaccharide; (3) carrying out primary separation by adopting DEAE-cellulose DE-52 anion exchange chromatographic column chromatography to obtain polygonatum kingianum polysaccharide washing component powder; and (4) carrying out purification by using a Sephadex G-100 gel chromatographic column, so as to obtain the purified polygonatum kingianum polysaccharide. Polygonatum kingianum polysaccharide can be used for preparing medicines, foods or functional foods with the effects of resisting fatigue and regulating intestinal flora.

Description

Technical Field

[0001] This invention relates to the field of plant polysaccharide technology, and in particular to a polysaccharide of Polygonatum odoratum and its preparation method and application. Background Technology

[0002] Polysaccharides are widely found in the cell walls of plants, animals, and microorganisms. Natural polysaccharides have various biological effects, such as lowering blood lipids, lowering blood sugar, antioxidation, antitumor, immunomodulation, and anti-inflammation. They can directly affect the body's metabolism of substances and energy, and have important medicinal value. Polygonatum contains abundant polysaccharide components with complex chemical structures. The content, structure, and activity of polysaccharides vary considerably among different sources and varieties of Polygonatum.

[0003] In recent years, Polygonatum polysaccharides, as the main active ingredient of Polygonatum, have attracted much attention due to their significant pharmacological activities. Studies have shown that polysaccharides from plants of the Polygonatum genus possess a variety of biological activities, including anti-inflammatory, immunomodulatory, anti-osteoporosis, and anti-diabetic effects, which may be closely related to their unique structural characteristics.

[0004] Fatigue is a complex physiological and pathological phenomenon involving various physiological and biochemical reactions within the body. From a biological perspective, it manifests as energy depletion, metabolite accumulation, and free radical metabolic disorders. While some drugs currently used clinically to treat fatigue have certain effects in combating fatigue, long-term use can lead to side effects. For example, long-term use of caffeine-containing drugs can not only keep the nerves in a state of constant excitement, leading to nervous system disorders, but also cause addiction. Therefore, finding a safe, effective, and low-side-effect anti-fatigue agent is urgently needed. Research on the anti-fatigue effects of Polygonatum polysaccharides is still very limited, and further exploration of its application prospects in this field is warranted. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, this invention provides a polysaccharide of Polygonatum odoratum, its preparation method, and its application.

[0006] The technical solution of the present invention is as follows: The first objective of this invention is to provide a polysaccharide of Polygonatum odoratum, named DYHJSC, which is composed of fructose and glucose in a molar ratio of 18.23:1. It is a Graminan-type fructan, and its structure is shown in Formula I.

[0007] Formula I In the formula, n=3; Fru f Represents furanylfructose, Glc p It represents glucose pyranose.

[0008] In one embodiment of the present invention, the weight-average molecular weight of the Polygonatum odoratum polysaccharide is 4855 Da.

[0009] The second objective of this invention is to provide a method for preparing the above-mentioned Polygonatum odoratum polysaccharide, comprising the following steps: (1) The powder of Polygonatum odoratum was subjected to alcohol extraction to remove impurities, water extraction and alcohol precipitation, redissolved and freeze-dried to obtain crude polysaccharide extracted from Polygonatum odoratum by water extraction; (2) The polysaccharide of Polygonatum odoratum obtained by deproteinization and dialysis using papain-Sevage method; (3) The refined polysaccharide of Polygonatum odoratum was dissolved in deionized water and initially separated by DEAE-cellulose DE-52 anion exchange chromatography column. The polysaccharide was eluted with sodium chloride solution of different concentrations, the elution peaks were combined and concentrated, dialyzed with pure water, and freeze-dried to obtain the water-washed component powder of Polygonatum odoratum polysaccharide. (4) Dissolve the water-washed fraction powder of Polygonatum yunnanensis polysaccharide in deionized water and purify it using Sephadex G-100 gel chromatography column with distilled water as the eluent. Collect 2 mL of each tube and detect the polysaccharide content using the anthrone-sulfuric acid colorimetric method. One fraction is obtained after purification. Combine the peak fractions, concentrate, dialyze, and freeze dry to obtain purified Polygonatum yunnanensis polysaccharide (DYHJSC).

[0010] In one embodiment of the present invention, in step (1), the method of removing impurities by alcohol extraction is as follows: remove impurities three times with 10 times the amount of 70% ethanol, each time for 1 hour.

[0011] In one embodiment of the present invention, in step (1), the water extraction and alcohol precipitation method is as follows: the water extract is concentrated at 50-60°C, and 95% ethanol is added until the alcohol concentration in the solution is 80% to obtain polysaccharide precipitation, that is, water-extracted crude polysaccharide is obtained.

[0012] In one embodiment of the present invention, in step (3), the DEAE-cellulose chromatography column is prepared as follows: DEAE-cellulose column packing is used, and after soaking in pure water overnight, the supernatant is removed to prepare for column loading; before loading the column, 1 / 3 of pure water is added to the column in advance, and then cellulose turbid liquid is slowly added along the wall to allow it to precipitate naturally, thus completing the column packing; the size of the chromatography column is 2.6 cm × 67.5 cm.

[0013] In one embodiment of the present invention, in step (3), after the polysaccharide is dissolved in deionized water, filtered through a 0.22 μm microporous membrane, it is loaded onto a DEAE-52 cellulose anion exchange chromatography column. The elution program consists of distilled water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L NaCl solutions, with 8 mL collected per tube. The polysaccharide content is determined using the anthrone-sulfuric acid method. The elution gradient is changed when no more polysaccharides are eluted. After dialyzing with a dialysis bag with a molecular weight cutoff of 500 Da for 72 h, the polysaccharide is freeze-dried at -80℃ to obtain the water-washed fraction powder of Polygonatum odoratum polysaccharide.

[0014] In one embodiment of the present invention, in step (4), after preparing 50 mg / mL of Polygonatum odoratum polysaccharide by washing the fraction with deionized water, the fraction is filtered through a 0.22 μm microporous membrane and loaded onto the sample; after dialyzing with a dialysis bag with a molecular weight cutoff of 500 Da for 72 h, the fraction is freeze-dried at -80℃ to obtain Polygonatum odoratum polysaccharide DYHJSC.

[0015] The third objective of this invention is to provide an application of the above-mentioned Polygonatum odoratum polysaccharide for the preparation of pharmaceuticals, foods, or functional foods with anti-fatigue effects.

[0016] The beneficial technical effects of this invention are as follows: Large-leaved Polygonatum yunnanense, a variant of the original Polygonatum yunnanense, has a significant advantage in yield. Research on its activity is crucial for expanding the production and application of Polygonatum yunnanense. This invention, using a mouse weight-bearing swimming model, found that large-leaved Polygonatum yunnanense polysaccharides significantly increased the exhaustion swimming time in mice, with higher doses resulting in longer durations. Subsequent studies on the in vivo anti-fatigue effects of large-leaved Polygonatum yunnanense polysaccharides revealed that low-dose groups also demonstrated their anti-fatigue ability by slowing the increase in lactic acid levels and reducing the accumulation of urinary nitrogen metabolites during exercise. Attached Figure Description

[0017] Figure 1 Molecular weight analysis of the polysaccharide DYHJSC of Polygonatum odoratum in this invention.

[0018] Figure 2 FT-IR analysis of DYHJSC, a polysaccharide from Polygonatum yunnanensis of the present invention.

[0019] Figure 3 The total ion current gas chromatogram of DYHJSC polysaccharide from *Polygonatum yunnanensis* after methylation is shown in this invention.

[0020] Figure 4 This invention relates to the polysaccharide DYHJSC of Polygonatum odoratum. 1 H-NMR spectra; fragments A, B, C, and D are shown in Table 1.

[0021] Figure 5 This invention relates to the polysaccharide DYHJSC of Polygonatum odoratum. 13 C-NMR.

[0022] Figure 6 This is the DEPT135 spectrum of the polysaccharide DYHJSC from *Polygonatum yunnanensis* of this invention.

[0023] Figure 7 This is the HSQC correlation spectrum of the polysaccharide DYHJSC of Polygonatum odoratum of the present invention.

[0024] Figure 8 This invention relates to the polysaccharide DYHJSC of Polygonatum odoratum. 1 H- 1 HCOSY related spectra.

[0025] Figure 9 This is the HMBC diagram of the polysaccharide DYHJSC of Polygonatum odoratum of the present invention.

[0026] Figure 10 The NOESY diagram shows the polysaccharide DYHJSC of Polygonatum odoratum of this invention.

[0027] Figure 11 This is a scanning electron microscope image of the polysaccharide DYHJSC from Polygonatum odoratum of the present invention.

[0028] Figure 12 The structural formula of the polysaccharide DYHJSC of Polygonatum odoratum of the present invention is shown below.

[0029] Figure 13 This invention relates to the effect of the polysaccharide DYHJSC from *Polygonatum yunnanensis* on exercise fatigue in mice. Figure A shows the time to exhaustion during swimming, Figure B shows changes in liver glycogen levels, Figure C shows changes in blood urea nitrogen levels, and Figure D shows changes in lactate levels. Data are presented as mean ± SD (n = 12). Intergroup comparisons were performed using... t test, P <0.05 indicates a significant difference. P A difference of <0.01 indicates a highly significant difference, which is statistically significant.

[0030] Figure 14 This invention relates to the effect of DYHJSC polysaccharide from *Polygonatum yunnanensis* on the overall structure of the intestinal flora in mice. A represents diversity analysis: Chao, Simpson, Shannon, and Sobs index plots; B represents nonmetric multidimensional scaling (NMDS) plots; and C represents Venn plots. Data are presented as mean ± SD (n = 6), and t-tests were used for comparisons between groups. P <0.05 indicates a significant difference. P A difference of <0.01 indicates a highly significant difference, which is statistically significant.

[0031] Figure 15 This invention relates to the effects of the polysaccharide DYHJSC from *Polygonatum yunnanensis* on the intestinal flora of mice at the phylum and genus levels. Figure A shows the relative abundance changes of flora at the phylum level; Figure B shows the compositional differences between Firmicutes and Bacteroidetes; Figure C shows the differences at the genus level; and Figure D shows the LEfSe analysis results. Data are presented as mean ± SD (n = 6). Intergroup comparisons were performed using t-tests and ANOVA tests. P <0.05 indicates a significant difference. P A difference of <0.01 indicates a highly significant difference, which is statistically significant. Detailed Implementation

[0032] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0033] Materials and reagents used in this invention: The Polygonatum in the experimental material was collected from Chongqing and identified by Researcher Si Jinping of Zhejiang Agriculture and Forestry University as Polygonatum macrocephalae. DEAE 52 anion exchange chromatography column and Sephadex G 100% dextran gel (Beijing Solarbio Science & Technology Co., Ltd.). Whole blood lactate kit: Nanjing Jiancheng Bioengineering Institute 20230218; Blood urea nitrogen kit: Nanjing Jiancheng Bioengineering Institute 20221216; Liver / muscle glycogen kit: Nanjing Jiancheng Bioengineering Institute 20221214; TransStart Fastpfu DNA Polymerase: Beijing TransGen Biotechnology Co., Ltd. AP221-02; AxyPrep DNA gel recovery kit: AXYGEN. Unless otherwise specified, all other reagents are analytical grade.

[0034] Example 1 A method for preparing polysaccharide of Polygonatum yunnanense includes the following steps: 1) After washing and removing the rootlets of fresh Polygonatum yunnanense rhizomes, the rhizomes are cut into thin slices, dried and pulverized, and passed through a 60-mesh sieve to obtain Polygonatum yunnanense rhizome powder with uniform particle size.

[0035] 2) Take an appropriate amount of Polygonatum powder, remove impurities three times with 10 times the amount of 70% ethanol, 1 hour each time, and extract the filter residue by reflux with 10 times the amount of water three times, 1 hour each time. Combine the filtrates, concentrate them at 55℃, add 95% ethanol to make the alcohol concentration in the solution 80%, filter and wash with water three times to obtain water-extracted crude polysaccharide, freeze-dry to obtain water-extracted crude polysaccharide powder.

[0036] 3) Dissolve the crude polysaccharide powder in pure water and then react it with Sevage reagent (V 氯仿 ∶V 正丁醇 Mix at a volume ratio of 4:1 (=4∶1), mix thoroughly, let stand and separate into layers, then discard the white gel-like substance between the two phases; Repeat the above steps multiple times until no protein layer remains in the solution. After removing the organic reagents by rotary evaporation under reduced pressure, the solution is placed in a 500 Da dialysis bag and dialyzed with running water for 5 days, then dialyzed with pure water for 1 day. Finally, the aqueous solution is concentrated by rotary evaporation and then freeze-dried to obtain refined polysaccharide from Polygonatum odoratum.

[0037] 4) Weigh 200 g of refined polysaccharide from *Polygonatum odoratum*, dissolve it in 5 mL of deionized water to prepare a solution with a concentration of 40 mg / mL, and perform preliminary separation using DEAE-cellulose DE-52 column chromatography (2.6 cm × 67.5 cm). Elute sequentially with distilled water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L NaCl solutions, collecting 8 mL of each solution. Determine the polysaccharide content using the anthrone-sulfuric acid method. Replace with the next elution gradient when no more polysaccharide is eluted. Plot the elution curve, combine the elution peaks for concentration, dialyze against a dialysis bag with a molecular weight cutoff of 500 Da for 72 h, and then freeze-dry at -80℃ to obtain the washed polysaccharide powder from *Polygonatum odoratum*.

[0038] 5) Prepare 1 mL of a 50 mg / mL *Polygonatum yunnanense* polysaccharide water-washed fraction, filter it through a 0.22 μm microporous membrane, and load it onto a Sephadex G-100 gel chromatography column for purification. Use distilled water as the eluent, and collect 2 mL from each tube. Determine the polysaccharide content using the anthrone-sulfuric acid colorimetric method; plot the elution curve. Only one fraction was obtained. Combine the peak fractions, concentrate the fraction, dialyze it through a dialysis bag with a molecular weight cutoff of 500 Da for 72 h, and then freeze-dry it at -80℃ to obtain *Polygonatum yunnanense* polysaccharide DYHJSC.

[0039] Test case 1. Molecular weight test Preparation of standard solution: Accurately weigh 5 mg of each standard (dextran), dissolve it in 1 mL of mobile phase solution to prepare a 5 mg / mL solution, and transfer the sample to a 1.8 mL vial.

[0040] Sample solution preparation: Accurately weigh 5 mg of the sample prepared in Example 1, dissolve it in 1 mL of mobile phase solution to prepare a 5 mg / mL solution, sonicate for 10 min, and centrifuge at 12000 rpm for 10 min. Aspirate the supernatant, filter it through a 0.22 μm aqueous microporous membrane, and then transfer the sample to a 1.8 mL vial.

[0041] Mobile phase: 0.05 mol / L NaCl solution; Column: BRT105-103-101 tandem gel column (8×300 mm); Flow rate: 0.8 mL / min; Column temperature: 40 ºC; Injection volume: 25 μL; Detector: RID-20A differential detector; Analysis time: 60 min.

[0042] According to the standard curve equation of dextran (lgMw-RT correction curve equation is: Y = -0.222 X + 11.45 R 2 =0.994) Calculate the Mw of the sample. For example Figure 1 As shown, DYHJSC only showed one elution peak at a retention time of 34.97 min, and the peak shape was relatively symmetrical and uniform, with a Mw of 4855 Da.

[0043] 2. FT-IR analysis FT-IR: Accurately weigh 2 mg of sample and 200 mg of potassium bromide, compress them into tablets, and place them in a Fourier transform infrared spectrometer for scanning and recording.

[0044] FT-IR analysis of DYHJSC polysaccharides is shown in Figure 2, with absorption bands in the range of 3600-3200 cm⁻¹. -1 This is the absorption peak of the stretching vibration of -OH, and the absorption peak in this region is a characteristic peak of carbohydrates. Additionally, at 2935 cm⁻¹... -1 and 1419 cm -1 The absorption peaks at 1646 cm⁻¹ are related to the stretching vibrations of CH and CO, respectively; -1 There is an absorption peak at 1128 cm⁻¹, which may be attributed to water of crystallization. -1 and 1027cm -1 These two absorption peaks may be the result of the stretching vibration of the COC glycosidic bond in the pyran ring; at 1270 cm⁻¹ -1 Location, 1213 cm -1 and 869 cm -1 The absorption peak may be attributed to the OH-angle vibration; 930 cm⁻¹ -1 and 812 cm -1 The significant absorption peak near the [specific location] indicates the presence of a furanose ring with a β-glycosidic bond. Based on the above results, DYHJSC possesses the characteristic functional groups of a typical polysaccharide compound and exhibits both pyranose and furanose conformations.

[0045] 3. Methylation analysis Methylation: After methylation, hydrolysis, and acetylation, the samples were measured by GC-MS and compared with a standard mass spectrum library.

[0046] The total ion-phase gas chromatogram of methylated sugar alcohol acetyl esters of DYHJSC polysaccharide is shown below. Figure 3 As shown, the mass spectrum was compared with the PMAA standard mass spectrum to determine its linkage mode. The methylation results of DYHJSC are shown in Table 1 below. It can be seen that DYHJSC actually contains four types of glycosidic bonds, with glucose linked by the terminal sugar Glcp-(1→) and fructose linked by three linkage modes: Fruf-(2→, →1)-Fruf-(2→ and →2,6)Fruf-(1→). Therefore, it can be inferred that this polysaccharide is fructan.

[0047] Table 1

[0048] 4. Nuclear magnetic resonance analysis Weigh 50 mg of the polysaccharide sample, dissolve it in 0.5 mL of heavy water, and freeze-dry. Then, redissolve the freeze-dried powder in 0.5 mL of heavy water and continue freeze-drying, repeating this process several times. Dissolve the sample in 0.5 mL of heavy water and measure its content at 25°C using a 600 MHz nuclear magnetic resonance spectrometer. 1 H NMR spectrum, 13 C NMR spectrum, DEPT135 one-dimensional spectrum and two-dimensional spectrum.

[0049] DYHJSC 1 In the H-NMR spectrum δ A weak signal peak was observed at 5.30 ppm, and based on the methylation analysis results, it was inferred to be a terminal proton of α-D-Glcp-1→. Figure 4 As shown. This result is consistent with the results of FT-IR analysis. And... δ A strong signal was present in the range of 3.5–4.2 ppm, which is characteristic of β-D-fructose; in addition, 1 Some characteristic signals from ¹H NMR can also be used to determine whether polysaccharides contain certain sugar residues or groups. For example, the methyl proton signal of an acetyl group generally appears in the low-field region of 1.8-2.2 ppm, and the polysaccharide DYHJSC... 1 ¹H NMR indicates that it does not contain acetyl groups or methyl ester groups.

[0050] exist 13 In the C NMR spectrum, for the glycosidic bond on the pyranose residue, the chemical shift of the terminal carbon in the β configuration is... δ In the range of 100~106 ppm, the α-configuration end carbon is in δ The concentration of DYHJSC polysaccharides is in the range of 93~100 ppm. 13 C-NMR, such as Figure 5 As shown, there are two signal peak regions in the terminal carbon signal region, namely... δ105.31, 105.00, 104.55 ppm and δ 93.30 ppm, so in δ The signal peak at 93.30 corresponds to the main terminal carbon signal in DYHJSC. Based on the methylation analysis results of DYHJSC, it can be seen that... δ The signal peak at 93.30 should belong to glucose residues. δ The signal peaks at 105.31, 105.00, and 104.55 ppm showed a significant downward shift, indicating that DYHJSC contains fructose residues with β-glycosidic bonds.

[0051] DEPT (Distortionless Enhancement by Polarization Transfer) spectroscopy is a detection technique in carbon NMR spectroscopy primarily used to distinguish between primary, secondary, tertiary, and quaternary carbons. For example... Figure 6 The inverted methylene peaks at 61.76, 63.06, and 64.52 are presumably the C1 and C6 of fructose and the C6 of glucose, respectively.

[0052] Figure 7 The HSQC correlation spectrum of DYHJSC is shown. Figure 8 For DYHJSC 1 H- 1 HCOSY related spectra; in 1 H- 1 In the H COSY two-dimensional spectrum, located at δ 5.30 / 3.44 δ The four chemical shifts at 3.44 / 3.67 represent the correlations of H1-H2 and H2-H3 on glucose residues, respectively, corresponding to carbon spectra of 93.32, 73.57, and 73.96 ppm according to HSQC. Furthermore, located at... δ 4.09 / 3.97 δ 3.97 / 3.72, δ The three chemical shifts at 3.72 / 3.64 ppm represent the correlations of H3-H4, H4-H5, and H5-H6 on fructose residues, respectively. The corresponding carbon signals are... δ 79.47, 76.58, 83.33 ppm.

[0053] according to Figure 7 , Figure 8 to its main residues 1 H NMR and 13 The chemical shifts of the C10 NMR spectra were assigned, as shown in Table 2 below.

[0054] Table 2

[0055] In the HMBC spectrum, a correlation peak exists between C2 of the glycosidic bond β-D-Fruf-2→ and H6 of →1,6-β-D-Fruf-2→, indicating the presence of β-D-Fruf-1→1,6-β-D-Fruf-2→. In the HMBC spectrum, a strong cross-peak exists between the matrix carbon at the →1-β-D-Fruf-2→ glycosidic bond and its own H1 resonance region: C2(→1-β-D-Fruf-2→)–H1(→1-β-D-Fruf-2→), indicating the presence of α-D-Glcp-1→2-β-D-Fruf-1→2-β-D-Fruf-1→. In the HMBC spectrum, a correlation peak exists between C2 of the glycosidic bond →1-β-D-Fruf-2→ and H1 of →1,6-β-D-Fruf-2→, indicating the presence of →2-β-D-Fruf-1→1,6-β-D-Fruf-2→, as shown in the figure. Figure 9 As shown.

[0056] NOESY spectroscopy reveals the spatial proximity relationships between all protons within a molecule. If there is correlation between two nuclei, cross-peaks will appear in the spectrum. In the NOESY spectrum, the terminal hydrogen of α-D-Glcp-1→ and its H1 of →1-β-D-Fruf-2→ show correlation peaks, indicating the presence of α-D-Glcp-1→1-β-D-Fruf-2→. Figure 10 As shown.

[0057] In summary, the main chain of the polysaccharide DYHJSC of *Polygonatum yunnanense* in this invention is →1-β-D-Fruf-2→1-β-D-Fruf-2→, while the side chains are through →1,6-β-D-Fruf-2→. O -6 is linked on the main chain, as shown in Equation I above.

[0058] 5. Scanning electron microscope Approximately 5 mg of dried sample was adhered to a conductive carbon film containing double-sided adhesive and placed in the sample chamber of an ion sputtering instrument for approximately 40 seconds of gold sputtering. After removal, the sample was placed in the observation chamber of a scanning electron microscope with an accelerating voltage of 5 kV for observation. The results are as follows. Figure 11 As shown, under 150x and 300x magnification, the DYHJSC sample exhibits an irregular shape, appearing as spheres or thin sheets. The spherical surfaces are smooth or have small protrusions. At 600x magnification, small protrusions are visible on the spheres, with smooth surfaces, indicating cracks on the surface of the spheres. Figure 11 ) Continuing to magnify to 1200x, it can be seen that the surface of the sphere is smooth, the cracks are clear, and small spheres are embedded in the surface, but the surface is smooth.

[0059] Application example: In vivo study of the effect of DYHJSC on improving post-exercise fatigue in mice: Animals and experimental design: 154 male Balb / c mice, weighing 18 g - 20 g, were Specific Pathogen Free (SPF) - level animals, sourced from the Peking University Health Science Center (Laboratory Animal Science Department), with the experimental animal license number: SCXK (Beijing) 2021 - 0013. The SPF - level animals were housed in a barrier environment, 5 or 6 per cage, with free access to conventional solid feed and pure water. The animal facility license: SYXK (Beijing) 2018 - 0022.

[0060] 1. Anti - fatigue effect 1.1 Animal rearing and grouping First batch: After 2 days of adaptive feeding in the SPF animal house, 60 Balb / c mice were randomly divided into 5 groups of 12 mice each. They were the blank control group, the low - dose (DYHJSC_L) group of Polygonatum kingianum polysaccharide (0.2 g / kg), and the high - dose (DYHJSC_H) group of Polygonatum kingianum polysaccharide (0.3 g / kg). Under the same rearing conditions, each drug - administered group was intragastrically administered different concentrations of the drug (ig. 0.2 mL / 10 g), and the normal group was intragastrically administered an equal amount of pure water once a day for 42 consecutive days. Mouse swimming training was carried out in the last week of drug administration, and the swimming exhaustion time was measured.

[0061] Second batch: The grouping and feeding conditions were the same as those in the first batch. Samples were taken from the mice after weight - bearing swimming or non - weight - bearing swimming according to the content indexes of blood lactic acid, serum urea nitrogen, and liver glycogen.

[0062] 1.2 General observation The conditions of mice in each group were observed daily, and the body weights of the mice were weighed weekly.

[0063] [[ID=2Q]]It can be found that the average body weight of mice in each group showed a gradually increasing trend with the extension of feeding time, and there was no significant difference in weight gain between the control group and other experimental groups. The results are shown in Tables 3 and 4; Table 3 shows the effect of Polygonatum kingianum polysaccharide on the body weight of the first batch of mice ( , g), and Table 4 shows the effect of Polygonatum kingianum polysaccharide on the body weight of the second batch of mice ( , g). It shows that feeding with Polygonatum kingianum polysaccharide has no effect on the body weight of mice, does not cause mouse obesity, and does not affect the results of subsequent weight - bearing swimming tests.

[0064] Table 3

[0065] Note: Compared with the blank control group, P <0.05.

[0066] Table 4

[0067] Note: Compared with the blank control group, P <0.05.

[0068] 1.3 Swimming Exhaustion Test Thirty minutes after the last gavage, a weight of 7% of the mouse's body weight was applied to the tail near the base of the tail, as determined in the preliminary experiment. The mouse was placed in a water tank with a depth of 30 cm and a water temperature of 25.0 ± 1.0 ℃. The time from the start of swimming until the mouse's head sank underwater for 10 seconds and could not float to the surface was recorded as the weighted swimming time.

[0069] Results of the weight-bearing swimming test on mice using Polygonatum yunnanense polysaccharide, such as... Figure 13 As shown in Figure A, the average swimming time in the CK group was 361 s, while the average swimming times in the DYHJSC_L and DYHJSC_H groups were 445 s and 590 s, respectively. Compared with the CK group, the DYHJSC_L and DYHJSC_H groups significantly prolonged the exhaustive swimming time of the mice, which was statistically significant. These results indicate that the water-extracted polysaccharide from *Polygonatum odoratum* can improve the exercise endurance of mice, and within a certain range, the higher the concentration of *Polygonatum odoratum* polysaccharide fed to the mice, the better their exercise endurance, demonstrating an anti-exercise fatigue effect.

[0070] 1.4 Blood lactate detection Blood samples of 0.05 mL were collected 30 min after the last gavage, 6 min after swimming in 30°C water with a 3% body weight of tin wire, and 20 min after rest. After adding protein precipitant, mixing, and centrifuging, the supernatant was used to determine the blood lactate content. The rate of increase and elimination of blood lactate were calculated based on the measured values.

[0071] The accumulation of metabolic byproducts in the body is also a significant factor contributing to fatigue, primarily including lactic acid and nitrogenous compounds. Excessive accumulation of blood lactic acid can lower the pH of muscles and blood, inhibiting the production of glycolysis-related enzymes and ATP synthesis, thus leading to muscle fatigue. Furthermore, the H+ produced by the dissociation of blood lactic acid... + It can inhibit Ca 2+ Its binding to skeletal muscle troponin leads to impaired muscle contractility. The result is as follows: Figure 13 As shown in Figure C, compared with the CK group, the rate of increase in blood lactate in the DYHJSC_L and DYHJSC_H groups was significantly reduced, at 0.12 mmol / L / min and 0.26 mmol / L / min, respectively, but only the DYHJSC_L group showed a statistically significant difference. Figure 13 D).

[0072] 1.5 Serum urea nitrogen and liver glycogen assay Thirty minutes after the last gavage, the mice were placed in a swimming tank at 30°C and swam without weight for 90 minutes. Blood was collected immediately after swimming, and the mice were then sacrificed and their livers were harvested. Serum urea nitrogen and liver glycogen levels were measured according to the kit instructions.

[0073] The liver is the direct organ for energy storage and utilization. The energy required for exercise initially comes from glycogenolysis, and later from glycogen and non-esterified fatty acids released from the liver circulation. Therefore, increased liver glycogen reserves contribute to endurance and exercise capacity; experimental results are as follows... Figure 13 As shown in Figure B, the liver glycogen content in all treatment groups was lower than that in the blank control group after 90 minutes of non-weight-bearing swimming, with the DYHJSC_L group showing a significant decrease. This indicates that low-dose water-extracted polysaccharides from Polygonatum odoratum not only did not increase the body's liver and muscle glycogen content to provide more energy and relieve fatigue, but also promoted the breakdown and utilization of glycogen.

[0074] Blood urea nitrogen (BUN) is also a sensitive indicator of fatigue. During high-intensity exercise, energy from carbohydrate and fat metabolism becomes insufficient. To compensate, proteins and amino acids exhibit high catabolism, leading to a rapid increase in BUN. Experimental results are as follows... Figure 13 As shown in Figure D, the urea nitrogen level in CK mice after exercise was 1.03 mmol / L. In contrast, the urea nitrogen levels in mice in the DYHJSC_L and DYHJSC_H groups were lower, at 0.74 mmol / L and 0.89 mmol / L, respectively, with the difference in the DYHJSC_L group being statistically significant. Figure 13 C).

[0075] The above results indicate that the low-dose group of polysaccharide extracted from Polygonatum odoratum can achieve anti-fatigue effects in mice by slowing down the rate of increase in blood lactate and reducing the metabolism and accumulation of urea nitrogen.

[0076] 2. 16S rRNA analysis of gut microbiota: Experimental Procedure: Feces from the first batch of experimental mice were collected after 42 days of feeding, and 16S rDNA sequencing was performed. To obtain the species classification information corresponding to each ASV, the classify-sklearn (Naive Bayes) algorithm was used to perform taxonomic analysis on the representative ASV sequences, obtaining the classification information of ASVs at the kingdom, phylum, class, order, family, genus, and species levels. The abundance of microbial species is directly proportional to the number of ASVs in the sample.

[0077] result: Figure 14 The α diversity (Chao, Simpson, Shannon, Sobs index) analysis results shown in Figure A indicate that DYHJSC_H improved the species richness and diversity of the mouse bacterial community. Figure 14The NMDS analysis shown in Figure B indicates that the gut microbiota of the DYHJSC_H group differs significantly from that of the CK group compared to the DYHJSC_L group. Figure 14 As shown in Venn analysis (C), 518 common ASVs were found in the three groups of mice, while 506, 763, and 1384 unique ASVs were detected in the CK group, DYHJSC_L group, and DYHJSC_H group, respectively. Diversity analysis results indicate that DYHJSC_L and DYHJSC_H improved the microbial diversity and richness of the intestinal bacteria in mice.

[0078] Secondly, DYHJSC improved the imbalance of the mouse gut microbiota structure at different levels. Phylum-level studies showed that Firmicutes, Bacteroidetes, Proteobacteria, Patescibacteria, Actinobacteria, and Desulfobacterota were the major phyla in the mouse gut microbiota. Figure 15 A). In DYHJSC, Firmicutes decreased and Bacteroidetes increased, especially in the DYHJSC_H group, where there were significant differences. Figure 15 B). The decreased ratio of the relative abundance of Firmicutes to Bacteroidetes indicates that DYHJSC has the potential to become a functional anti-obesity component. Genus-level analysis showed an increase in DYHJSC. Alistipes , Bacteroides The abundance of the same genera decreased. Novosphingobium genus abundance ( Figure 15 C). This indicates that a certain dose of DYHJSC can promote the growth of specific beneficial bacteria, thereby helping to regulate the gut microbiota structure; however, excessive doses may have an inhibitory effect. LEfSe analysis results showed that there were 7, 10, and 10 significantly different species in the CK, DYHJSC_L, and DYHJSC_H groups, respectively. Figure 15 D).

[0079] In summary, the above data collectively demonstrate that DYHJSC can significantly improve the gut bacterial composition of mice at both the phylum and genus levels.

[0080] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A polysaccharide from Polygonatum yunnanense, characterized in that, Named DYHJSC, it is composed of fructose and glucose in a molar ratio of 18.23:1, and is a Graminan-type fructan, with the structure shown in Formula I: Formula I In the formula, n=3; Fru f Represents furanylfructose, Glc p It represents glucose pyranose.

2. The Polysaccharide of Polygonatum yunnanense according to claim 1, characterized in that, The weight-average molecular weight is 4855 Da.

3. A method for preparing the polysaccharide of Polygonatum odoratum according to claim 1, characterized in that, Includes the following steps: (1) The powder of Polygonatum odoratum was subjected to alcohol extraction to remove impurities, water extraction and alcohol precipitation, redissolved and freeze-dried to obtain crude polysaccharide extracted from Polygonatum odoratum by water extraction; (2) The polysaccharide of Polygonatum odoratum obtained by deproteinization and dialysis using papain-Sevage method; (3) The refined polysaccharide of Polygonatum odoratum was dissolved in deionized water and initially separated by DEAE-cellulose DE-52 anion exchange chromatography column. The polysaccharide was eluted with sodium chloride solution of different concentrations, the elution peaks were combined and concentrated, dialyzed with pure water, and freeze-dried to obtain the water-washed component powder of Polygonatum odoratum polysaccharide. (4) Dissolve the water-washed fraction powder of Polygonatum odoratum polysaccharide in deionized water and purify it using Sephadex G-100 gel chromatography column with distilled water as the eluent. Collect 2 mL of each tube and detect the polysaccharide content using the anthrone-sulfuric acid colorimetric method. One fraction is obtained after purification. Combine the peak fractions, concentrate, dialyze, and freeze dry to obtain purified Polygonatum odoratum polysaccharide.

4. The preparation method according to claim 3, characterized in that, In step (1), the method for removing impurities by alcohol extraction is as follows: remove impurities three times with 10 times the amount of 70% ethanol, each time for 1 hour.

5. The preparation method according to claim 3, characterized in that, In step (1), the water extraction and alcohol precipitation method is as follows: the water extract is concentrated at 50-60℃, and 95% ethanol is added until the alcohol concentration in the solution is 80% to obtain polysaccharide precipitation, that is, water-extracted crude polysaccharide.

6. The preparation method according to claim 3, characterized in that, In step (3), the DEAE-cellulose chromatography column is prepared as follows: DEAE-cellulose column packing is used, and after soaking in pure water overnight, the supernatant is removed to prepare for column loading; before loading the column, 1 / 3 of pure water is added to the column in advance, and then cellulose turbid liquid is slowly added along the wall to allow it to precipitate naturally, thus completing the column packing; the size of the chromatography column is 2.6 cm × 67.5 cm.

7. The preparation method according to claim 3, characterized in that, In step (3), after the polysaccharide was dissolved in deionized water, filtered through a 0.22 μm microporous membrane, it was loaded onto a DEAE-cellulose DE-52 anion exchange chromatography column. The elution program consisted of distilled water, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L NaCl solutions, with 8 mL collected per tube. The polysaccharide content was determined using the anthrone-sulfuric acid method. The next elution gradient was replaced when no more polysaccharides were eluted. After dialyzing with a dialysis bag with a molecular weight cutoff of 500 Da for 72 h, the polysaccharide was freeze-dried at -80℃ to obtain the water-washed fraction powder of Polygonatum odoratum polysaccharide.

8. The preparation method according to claim 3, characterized in that, In step (4), after washing the fraction with 50 mg / mL of Polygonatum odoratum polysaccharide using deionized water, the fraction was filtered through a 0.22 μm microporous membrane and loaded onto the sample. After dialyzing with a dialysis bag with a molecular weight cutoff of 500 Da for 72 h, the sample was freeze-dried at -80℃ to obtain Polygonatum odoratum polysaccharide DYHJSC.

9. An application of the Polygonatum odoratum polysaccharide according to claim 1, characterized in that, Used in the preparation of medicines, foods or functional foods with anti-fatigue effects.

10. An application of the Polygonatum odoratum polysaccharide according to claim 1, characterized in that, Used to prepare pharmaceuticals, foods, or functional foods that regulate gut microbiota.

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