Polysaccharide from stem and leaf of polygonatum sibiricum and preparation method and application thereof with improved efficacy of depression and sleep

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a polygonatum sibiricum Redoute stem and leaf polysaccharide with the effects of improving depression and sleep, and a preparation method and application thereof. The weight average molecular weight of the polygonatum sibiricum Redoute stem and leaf polysaccharide is 4000-5000 Da, and the polygonatum sibiricum Redoute stem and leaf polysaccharide comprises the following monosaccharide components: arabinose, galactose, glucose, xylose, mannose and fructose, wherein the percentage of fructose is the highest. The polygonatum sibiricum Redoute stem and leaf polysaccharide with high purity obtained by the present application is used for preparing health care products, drugs for improving sleep, or antidepressant drugs. Since natural plant raw materials are used, the dependence of a treatment object on the drugs can be reduced, and the harm to the body is relatively small.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a polygonatum stem and leaf polysaccharide with the effects of improving depression and sleep, and a preparation method and application thereof. BACKGROUND

[0002] Depression is a complex central nervous system disease, which is usually manifested as low mood, loss of interest, fatigue, difficulty concentrating, and sleep disorders and other symptoms, and seriously affects the quality of life of patients. Among them, sleep disorders are particularly common in depression, including difficulty falling asleep, difficulty maintaining sleep, and may also appear early awakening, sleep structure disorder and circadian rhythm disorder. There is a bidirectional correlation between sleep problems and depression, on the one hand, depression can lead to decreased sleep quality, on the other hand, long-term poor sleep can also exacerbate depression symptoms, forming a vicious cycle. In recent years, the incidence of depression and insomnia has been increasing year by year, and traditional drug treatment is effective, but often accompanied by side effects and addiction risk. Therefore, it has become a research hotspot to find safe, low-toxic and natural alternative treatment methods with significant effects on improving mood and sleep.

[0003] Polysaccharides are polymers composed of various monosaccharides, and are widely sourced and can participate in and regulate various cell life activities and physiological processes. In the field of polysaccharide research, plant polysaccharides have attracted much attention. Plants contain a variety of active substances, and polysaccharides have gradually become a research hotspot for treating central nervous system diseases due to their low toxicity, safety and diverse biological activities.

[0004] Polygonatum is a precious medicinal material in traditional Chinese medicine in China, and as an important member of food and medicine, its unique pharmacological effects and significant health effects are highly respected in the population. In recent years, under the guidance of the "big food concept", it has become an inevitable trend to develop food resources in all directions and through multiple channels to expand food sources. However, for a long time, the medicinal part of polygonatum has been mainly concentrated in the dried rhizome, which is used for tonifying qi and blood, nourishing yin and moistening dryness, etc., while the research and development of its stem and leaf part are relatively less. Polygonatum stem and leaf also contains a variety of bioactive components, such as polysaccharides, flavonoids and saponins, but its potential medicinal value and functional characteristics have not been fully explored and utilized. SUMMARY

[0005] The present application extracts, separates and purifies high-purity polysaccharides from polygonatum stem and leaf, and then studies the effects of polysaccharides on depression and sleep improvement, filling the research gap of natural by-products and polygonatum stem and leaf polysaccharides.

[0006] The present application provides a polygonatum stem and leaf polysaccharide, the weight average molecular weight of the polygonatum stem and leaf polysaccharide is 4000-5000 Da, and the polygonatum stem and leaf polysaccharide comprises the following monosaccharide components: arabinose, galactose, glucose, xylose, mannose and fructose, wherein the percentage of fructose is the highest.

[0007] Further, the glycosidic bond of the polysaccharide in stems and leaves of Polygonatum sibiricum includes: →1)-Fru f -(2→,→1,6)Fru f -(2→,→6)Fru f -(2→,Fru f -(2→,Ara f -(1→,Glc p -(1→,→4)-Glc p -(1→,→6-Glc p -(1→.

[0008] Further, the polysaccharide in stems and leaves of Polygonatum sibiricum includes the following mass percentage of monosaccharide components: arabinose 4.2%, galactose 1.1%, glucose 2.3%, xylose 1.7%, mannose 1.8%, and fructose 88.9%.

[0009] The present application also provides a preparation method of the polysaccharide in stems and leaves of Polygonatum sibiricum, which comprises the following steps:

[0010] (1) water extraction of stems and leaves of Polygonatum sibiricum to obtain a concentrated solution;

[0011] (2) alcohol extraction of the concentrated solution to obtain a crude extract;

[0012] (3) protein removal of the crude extract to obtain a crude polysaccharide in stems and leaves of Polygonatum sibiricum;

[0013] (4) preparation of a crude polysaccharide solution from the crude polysaccharide in stems and leaves of Polygonatum sibiricum and water, flowing the crude polysaccharide solution into an anion exchange column, sequentially performing gradient elution of 0 mol / L and 0.1 mol / L sodium chloride, collecting polysaccharide components eluted by 0.1 mol / L sodium chloride, dialysis, and freeze-drying to obtain a purified polysaccharide component in stems and leaves of Polygonatum sibiricum.

[0014] Further, in step (1), the water extraction specifically comprises: drying and crushing the stems and leaves of Polygonatum sibiricum, water-bath extraction after adding water, cooling to room temperature, filtering the supernatant, concentrating the obtained filtrate to obtain a concentrated solution;

[0015] Preferably, in step (1), the water-bath extraction is performed for 3-5 h at a temperature of 60-90°C, and water is added according to a solid-liquid ratio of 1:10-1:40 g / mL.

[0016] More preferably, in step (1), the drying is performed at a temperature of 50-60°C.

[0017] Further, in step (2), the alcohol extraction specifically comprises: adding anhydrous ethanol to the concentrated solution, standing and precipitating, centrifuging, collecting the precipitate, then adding distilled water to the obtained precipitate to remove insoluble impurities to obtain a crude extract.

[0018] Preferably, in step (2), the volume ratio of anhydrous ethanol to the concentrated solution is 3-5:1.

[0019] More preferably, in step (2), the time for standing and precipitating is 10-15 h.

[0020] Further, in step (3), the removing of protein specifically comprises: adding Sevage reagent to the above-mentioned crude extract, standing after centrifugation, discarding the lower organic layer and the middle protein layer, repeating the operation until the protein layer is removed, then subjecting the obtained supernatant to low-temperature and reduced-pressure distillation, dialysis, and freeze-drying of the obtained dialysate to obtain the polygonatum stem and leaf crude polysaccharide.

[0021] Further, in step (3), the volume ratio of Sevage reagent to the crude extract is 3-5:1; wherein the Sevage reagent is obtained by mixing chloroform and n-butanol at a volume ratio of 4:1.

[0022] In step (3), the temperature for low-temperature and reduced-pressure distillation is 50-60 DEG C.

[0023] In step (3), the dialysis is performed in a dialysis bag with a molecular weight of 1000 Da.

[0024] Further, in step (4), the concentration of the crude polysaccharide solution is 10 mg / mL.

[0025] In step (4), the flow rate of the crude polysaccharide solution flowing into the anion exchange column is 1 mL / min, and the time is 10 min.

[0026] The present application also proposes the use of the polygonatum stem and leaf polysaccharide prepared by any of the above-mentioned methods in the preparation of health-care products or drugs for improving sleep, or in the preparation of antidepressant drugs.

[0027] The present application has the following advantages:

[0028] The polygonatum stem and leaf polysaccharide prepared by the present application has a clear structure, and the monosaccharide components include arabinose, galactose, glucose, xylose, mannose and fructose, wherein the fructose glycosidic bond is the main connecting mode. The sugar content of the polysaccharide is as high as 97.6%, and the gel permeation chromatography shows that only a single peak appears, indicating that the polysaccharide is a high-purity and uniform polysaccharide component.

[0029] The preparation method of the polygonatum sibiricum leaves polysaccharide provided by the present application uses the stems and leaves of polygonatum sibiricum as raw materials, and a polygonatum sibiricum leaves polysaccharide with high purity is prepared through a specific method. Then the polygonatum sibiricum leaves polysaccharide is used for preparing a medicine with an anti-depression effect, or a health care product or medicine for improving sleep. Since the plant raw materials are used, the dependence of a treatment object on the medicine is reduced, the harm to the body is relatively small, and the full development and utilization of plant resources of traditional Chinese medicines are realized. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative labor.

[0031] Fig. 1 is a molecular weight determination spectrum of the polygonatum sibiricum leaves polysaccharide in test example 2.

[0032] Fig. 2 is a total ion current chromatogram of the polysaccharide glycosidic bond type detection of the polygonatum sibiricum leaves polysaccharide in test example 2.

[0033] Fig. 3a, Fig. 3b and Fig. 3c are secondary proton chromatograms of the polysaccharide glycosidic bond type detection of the polygonatum sibiricum leaves polysaccharide in test example 2.

[0034] Fig. 4 is a tail suspension test of each group of mice after the modeling is completed (a) and after the administration is completed (b) in test example 3.

[0035] Fig. 5 is a forced swimming test of each group of mice after the modeling is completed (a) and after the administration is completed (b) in test example 3.

[0036] Fig. 6 is an observation diagram of the hippocampus HE staining section of each group of mice at 400 times in test example 3.

[0037] Fig. 7 is a content change diagram of the anti-depression related indexes in the hippocampus of each group of mice in test example 3.

[0038] Fig. 8 is a content change diagram of the anti-depression related indexes in the colon of each group of mice in test example 3.

[0039] Fig. 9 is a PCoA, NMDS diagram in the beta diversity analysis of the intestinal flora of each group of mice in test example 3.

[0040] Fig. 10 is a relative abundance column chart of the top 10 phylum (a) and a relative abundance column chart of the top 20 genus (b) of each group of samples in test example 3.

[0041] Figure 11 is a diagram showing the content changes of sleep improvement related indicators in the serum (a, b) and colon (c-f) of mice in each group in Test Example 4.

[0042] Figures 4-8 In Figure 11, compared with the NC group, p <0.001; compared with the CRS group, # p <0.05, ## p <0.01, ### p <0.001. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. The embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0044] In one aspect, the present application provides a polygonatum stem and leaf polysaccharide, the weight average molecular weight of the polygonatum stem and leaf polysaccharide is 4000-5000 Da, and the polygonatum stem and leaf polysaccharide comprises the following monosaccharide components: arabinose, galactose, glucose, xylose, mannose and fructose, wherein the percentage of fructose is the highest.

[0045] The polygonatum stem and leaf polysaccharide provided in the embodiments of the present application has a clear structure, fructose glycosidic bond is the main connection mode, and it also contains arabinose and glucose and other glycosidic bonds. The sugar content of the polysaccharide is as high as 97.6%, the monosaccharide composition analysis shows that the total amount of each monosaccharide is 881.64 μg / mg, and the gel permeation chromatography detection result only has a single peak, indicating that the polysaccharide is a high-purity and uniform polysaccharide component.

[0046] In an embodiment of the present application, the glycosidic bond of the polygonatum stem and leaf polysaccharide comprises: →1)-Fru f -(2→, →1, 6) Fru f -(2→, →6) Fru f -(2→, Fru f -(2→, Ara f -(1→, Glc p -(1→, →4)-Glc p -(1→, →6-Glc p -(1→.

[0047] In the embodiments of the present application, the fructose type glycosidic bond of the polygonatum stem and leaf polysaccharide mainly comprises →1)-Fru f -(2→, →1, 6) Fru f-(2→, 6)Fru f -(2→, 6)Fru f -(2→, a small amount of other glycosidic bonds include Ara f -(1→, Glc p -(1→, 4)-Glc p -(1→, 6-Glc p -(1→.

[0048] In an embodiment of the present application, the polysaccharide in stems and leaves of Polygonatum sibiricum includes the following weight percentage of monosaccharide components: 4.2% of arabinose, 1.1% of galactose, 2.3% of glucose, 1.7% of xylose, 1.8% of mannose, and 88.9% of fructose.

[0049] In a preferred embodiment of the present application, the polysaccharide in stems and leaves of Polygonatum sibiricum has a weight average molecular weight of 4287 Da.

[0050] In another aspect, an embodiment of the present application provides a preparation method of the polysaccharide in stems and leaves of Polygonatum sibiricum, including the following steps:

[0051] (1) Water extraction is performed on the stems and leaves of Polygonatum sibiricum to obtain a concentrated solution;

[0052] (2) Alcohol extraction is performed on the concentrated solution to obtain a crude extract;

[0053] (3) Protein is removed from the crude extract to obtain a crude polysaccharide in stems and leaves of Polygonatum sibiricum;

[0054] (4) A crude polysaccharide solution is prepared from the crude polysaccharide in stems and leaves of Polygonatum sibiricum and water, the crude polysaccharide solution is flowed into an anion exchange column, gradient elution is performed in sequence using 0 mol / L and 0.1 mol / L sodium chloride, polysaccharide components eluted using 0.1 mol / L sodium chloride are collected, dialysis is performed, and freeze-drying is performed to obtain a purified polysaccharide component in stems and leaves of Polygonatum sibiricum.

[0055] In an embodiment of the present application, in step (1), the water extraction specifically includes: after the stems and leaves of Polygonatum sibiricum are dried and crushed, water extraction is performed in a water bath, and then the solution is cooled to room temperature, the supernatant is filtered, the obtained filtrate is concentrated to obtain the concentrated solution.

[0056] In an embodiment of the present application, in step (1), the water extraction is performed for 3-5 h at a temperature of 60-90°C, and water is added according to a solid-liquid ratio of 1:10-1:40 g / mL.

[0057] In an embodiment of the present application, in step (1), the stems and leaves of Polygonatum sibiricum are fresh stems and leaves of Polygonatum sibiricum, which are picked from the stems and leaves on the fresh Polygonatum sibiricum rhizomes in Liaoning Province, China. Polygonatum sibiricum Red.) on the fresh Polygonatum sibiricum rhizomes in Liaoning Province, China.

[0058] In one embodiment of the present application, in step (1), the drying temperature is 50-60°C. Preferably, the drying temperature is 60°C.

[0059] Further, the drying is specifically drying to constant weight in a constant temperature drying oven.

[0060] In one embodiment of the present application, in step (1), the concentration is rotary evaporation concentration.

[0061] In one embodiment of the present application, in step (1), the room temperature is 10-25°C.

[0062] In one embodiment of the present application, in step (1), step (1) further comprises that the residue obtained after filtering the supernatant is extracted in water bath again, and the filtrates obtained after 1-2 times of extraction are combined and then concentrated.

[0063] In one embodiment of the present application, in step (2), the alcohol extraction specifically comprises: adding anhydrous ethanol to the concentrated solution, standing and precipitating, centrifuging, collecting the precipitate, then adding distilled water to the obtained precipitate to remove insoluble impurities, and obtaining a crude extract.

[0064] In one embodiment of the present application, in step (2), the standing and precipitating time is 10-15 h. Preferably, in step (2), the standing and precipitating time is 12 h.

[0065] In one embodiment of the present application, in step (2), the volume ratio of anhydrous ethanol to concentrated solution is 3-5:1. Preferably, in step (2), the volume ratio of anhydrous ethanol to concentrated solution is 4:1.

[0066] In one embodiment of the present application, in step (3), the protein removal specifically comprises: adding Sevage reagent to the crude extract, standing after centrifugation, discarding the lower organic layer and the middle protein layer, repeating the operation until the protein layer is completely removed, then low-temperature and reduced-pressure distilling the obtained supernatant, dialyzing, freeze-drying the obtained dialysate, and obtaining the polygonatum stem and leaf crude polysaccharide.

[0067] In one embodiment of the present application, in step (3), the volume ratio of Sevage reagent to crude extract is 3-5:1. Preferably, in step (3), the volume ratio of Sevage reagent to crude extract is 4:1. The Sevage reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:1.

[0068] In one embodiment of the present application, in step (3), the centrifugation is specifically centrifugation after vortexing for 10 min with a vortex shaker.

[0069] In one embodiment of the present application, in step (3), the low-temperature and reduced-pressure distillation temperature is 50-60°C. The low-temperature and reduced-pressure distillation is mainly to remove chloroform and n-butanol.

[0070] In one embodiment of the present application, the dialysis in step (3) is performed in a 1000 Da dialysis bag.

[0071] In one embodiment of the present application, in step (3), the dialysis is performed using tertiary water until the conductivity of the obtained dialysate is close to that of the tertiary water.

[0072] In step (4) of the present application, different concentrations of sodium chloride are used for elution, wherein when the concentration of sodium chloride is 0.1 mol / L, the salt gradient can effectively compete and release the electrostatic adsorption of the stem and leaf polysaccharide of Polygonatum sibiricum to the DEAE anion exchange layer, and does not excessively increase the ionic strength to cause a large amount of impurities to be co-eluted, so that the stem and leaf polysaccharide component with the highest sugar content can be obtained under this condition.

[0073] In one embodiment of the present application, in step (4), the concentration of the crude polysaccharide solution is 10 mg / mL.

[0074] In one embodiment of the present application, in step (4), the crude polysaccharide solution is prepared using primary water.

[0075] In one embodiment of the present application, in step (4), the flow rate of the crude polysaccharide solution flowing into the anion exchange column is 1 mL / min, and the time is 10 min.

[0076] In one embodiment of the present application, step (4) further comprises passing the crude polysaccharide solution through a 0.45 μm water-based microporous filter membrane to remove impurities, and then flowing the crude polysaccharide solution into the anion exchange column.

[0077] In step (4) of the present application, the model of the DEAE-52 anion exchange column is DEAE-52 anion exchange column.

[0078] In one embodiment of the present application, in step (4), the dialysis is specifically dialysis using tertiary water in a 1000 Da dialysis bag.

[0079] In another aspect, the present application also provides the use of the stem and leaf polysaccharide of Polygonatum sibiricum prepared by any one of the above-mentioned preparation methods in the preparation of health care products, drugs for improving sleep, or in the preparation of antidepressant drugs. In the present application, the use in the preparation of antidepressant drugs is mainly the use in the preparation of drugs for relieving and / or improving depression.

[0080] The present application will be described in detail below with reference to the accompanying drawings.

[0081] Example 1 A preparation method of a stem and leaf polysaccharide of Polygonatum sibiricum, the specific steps are as follows:

[0082] (1) Water extraction: Fresh Polygonatum sibiricum leaves and stems were washed twice with distilled water to remove impurities, dried in a constant temperature drying oven at 60°C until constant weight, and then ground with a pulverizer. The material was extracted with water at a solid-liquid ratio of 1:30 (g / mL) for 3 h at 80°C in a water bath. After cooling to room temperature, the supernatant was filtered, and the residue was repeatedly extracted with water for 1 h. The filtrates from the two extractions were combined, concentrated by rotary evaporation, and a concentrated solution was obtained.

[0083] (2) Alcohol precipitation: Four times the volume of anhydrous ethanol was added to the concentrated solution, and the mixture was allowed to stand for 12 h. The precipitate was collected by centrifugation, redissolved in distilled water, and the insoluble impurities were removed to obtain a crude Polygonatum sibiricum leaf and stem polysaccharide extract.

[0084] (3) Protein removal: Four times the volume of Sevage reagent (V(chloroform):V(n-butanol)=4:1) was added to the crude extract obtained in step (2), and the mixture was shaken for 10 min using a vortex shaker and then centrifuged. The lower organic layer and the middle protein layer were discarded, and the operation was repeated more than 10 times until the protein was completely removed. Finally, the supernatant was combined, and the chloroform and n-butanol were removed by low-temperature vacuum distillation at 55°C. The solution was dialyzed against triple-distilled water in a 1000 Da dialysis bag until the conductivity of the dialysate was close to that of the triple-distilled water. The solution was then freeze-dried (-70°C, 72 h) to obtain a crude Polygonatum sibiricum leaf and stem polysaccharide sample.

[0085] (4) Purification by anion exchange column: A 10 mg / mL solution of the crude Polygonatum sibiricum leaf and stem polysaccharide was prepared using the above-mentioned crude polysaccharide and water, and the solution was filtered through a 0.45 μm water-based microporous filter to remove impurities. The sample obtained in step (3) was then loaded onto a DEAE-52 anion exchange column at a flow rate of 1 mL / min for 10 min. The column was eluted with a gradient of 0 mol / L and 0.1 mol / L sodium chloride, and the polysaccharide component eluted with 0.1 mol / L sodium chloride was collected. The solution was dialyzed against triple-distilled water in a 1000 Da dialysis bag, and then freeze-dried (-70°C, 72 h) to obtain a purified Polygonatum sibiricum leaf and stem polysaccharide component (referred to as PSLP1).

[0086] Comparative Example 1

[0087] The same as in Example 1, except that in step (4), the component eluted with 0 mol / L sodium chloride was collected.

[0088] Comparative Example 2

[0089] The same as in Example 1, except that in step (4), the column was eluted with a gradient of 0 mol / L, 0.1 mol / L, and 0.3 mol / L sodium chloride, and the component eluted with 0.3 mol / L sodium chloride was collected.

[0090] Comparative Example 3

[0091] The same as example 1, except that in step (4), 0 mol / L, 0.1 mol / L, 0.3 mol / L, 0.5 mol / L sodium chloride gradient elution was used in turn, and the component eluted by 0.5 mol / L sodium chloride was collected.

[0092] Test Example 1

[0093] The test example aims to explore the highest sugar content component of polygonatum stem and leaf polysaccharide. The best process is determined by studying the total sugar content of different components eluted by different concentrations of sodium chloride.

[0094] The total sugar content of the polygonatum stem and leaf polysaccharide prepared in each example was determined. The phenol sulfuric acid method was used for determination, and the ultraviolet wavelength for total sugar content determination was 490 nm. The calculation was carried out according to the standard curve y=0.0079x+0.0059.

[0095] The experimental results are shown in Table 1. The total sugar content of the polygonatum stem and leaf polysaccharide component in example 1 is the highest, so after elution by 0 mol / L sodium chloride, the component with the highest sugar content can be obtained by collecting the component with 0.1 mol / L sodium chloride concentration.

[0096] Table 1 Total sugar content of each example eluted by different concentrations of sodium chloride

[0097]

[0098] Test Example 2 Study on the structural characteristics of polygonatum stem and leaf polysaccharide

[0099] 1. Determination of physicochemical properties of polygonatum stem and leaf polysaccharide

[0100] 1.1 Determination of monosaccharide composition by ion exchange chromatography

[0101] Standard solution preparation: Take 16 kinds of monosaccharide standard (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, amino galactose hydrochloride, amino glucose hydrochloride, N-acetyl-D glucosamine, guluronic acid, mannuronic acid) to prepare standard mother solution. Take each monosaccharide standard solution to prepare concentration standard as mixed standard. According to the absolute quantitative method, the mass of different monosaccharides is determined, and the molar ratio is calculated according to the molar mass of monosaccharide. The peak time and peak area of each monosaccharide are shown in Table 2.

[0102] Table 2 Sequence of monosaccharide standard for monosaccharide composition in polysaccharide

[0103]

[0104] Sample preparation: 5 mg of the sample obtained in Example 1 was precisely weighed into an ampoule, 2 mL of 3M trifluoroacetic acid was added, and hydrolysis was carried out at 80°C for 2 h. The acid hydrolysis solution was accurately pipetted into a tube and blown dry with nitrogen, 5 mL of water was added and vortexed to mix, 50 μL was pipetted into 950 μL of deionized water, and centrifuged at 12000 rpm for 5 min. The supernatant was taken for ion chromatography (IC) analysis.

[0105] Chromatographic method: Chromatographic column: Dionex Carbopac TM PA20 (3*150 mm); mobile phase: A: H2O; B: 15 mM NaOH; C: 15 mM NaOH & 100 mM NaAc; flow rate: 0.3 mL / min; injection volume: 25 μL; column temperature: 30 °C; elution gradient: 0 min phase A / phase B / phase C (98.8:1.2:0, V / V), 18 min phase A / phase B / phase C (98.8:1.2:0, V / V), 20 min phase A / phase B / phase C (50:50:0, V / V), 30 min phase A / phase B / phase C (50:50:0, V / V), 30.1 min phase A / phase B / phase C (0:0:100, V / V), 46 min phase A / phase B / phase C (0:0:100, V / V), 46.1 min phase A / phase B / phase C (0:100:0, V / V), 50 min phase A / phase B / phase C (0:100:0, V / V), 50.1 min phase A / phase B / phase C (98.8:1.2:0, V / V), 80 min phase A / phase B / phase C (98.8:1.2:0, V / V). Detector: electrochemical detector.

[0106] The results obtained by testing the sample of Example 1 and the standard were compared, and the monosaccharide composition of the rhizoma polygonati stem and leaf polysaccharide obtained is shown in Table 3, in which the molar ratio of fructose is up to 88.9%, and the content is 791.69 μg / mg. The total content of each monosaccharide is 881.64 μg / mg, proving that the polysaccharide component used has a high sugar content.

[0107] Table 3 Monosaccharide composition of rhizoma polygonati stem and leaf polysaccharide

[0108]

[0109] 1.2 Molecular weight detection of rhizoma polygonati stem and leaf polysaccharide by gel permeation chromatography

[0110] Preparation of standard solution: 2 mg of each of 9 standards (dextran, see Table 4) was precisely weighed and dissolved in 1 mL of mobile phase solution to prepare a 2 mg / mL solution. The sample was transferred to a 1.8 mL sample vial.

[0111] Preparation of sample solution: 5 mg of the sample of Example 1 was precisely weighed and dissolved in 1 mL of mobile phase solution to prepare a 5 mg / mL solution. After vortex dissolution, centrifugation was performed at 12000 rpm for 10 min. The supernatant was aspirated and filtered with a 0.22 μm water-based microporous filter, and then the sample was transferred to a 1.8 mL sample vial.

[0112] Mobile phase: 0.05M NaCl solution; column: BRT105-103-101 series gel column (8 x 300 mm); flow rate: 0.7 ml / min; column temperature: 40 °C; sample size: 25 μl; detector: differential detector RID-20A; analysis time: 70 min.

[0113] The standards were placed in the sample tray and analyzed by the above chromatographic method to obtain the retention time and plot lgMw-RT (Mw weight average molecular weight), as shown in Table 3; the lgMw-RT correction curve equation was calculated as y = -0.208x + 11.86, R²= 0.992, wherein x is the response time (min) and y is lgMw.

[0114] Table 4 Specific data of each standard in molecular weight

[0115] Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Standard 8 Standard 9 Mw 1153 6941 9835 20600 47880 112200 240100 396400 883100 lgMw 3.06 3.84 3.99 4.31 4.68 5.05 5.38 5.60 5.95 Response time (min) 41.49 38.94 38.17 36.54 34.62 32.67 31.01 29.77 28.49

[0116] The chromatogram measured in Example 1 is shown in Figure 1. The molecular weight (Mw) can be obtained by substituting the retention time into the correction curve equation. The weight average molecular weight of the polysaccharide from the stems and leaves of Polygonatum sibiricum was calculated to be 4287 Da.

[0117] 1.3 Analysis and detection of the type of glycosidic bond of the polysaccharide from the stems and leaves of Polygonatum sibiricum by GC-MS method

[0118] A polysaccharide sample (2-3 mg) was weighed and placed in a glass reaction bottle, 1 mL of anhydrous DMSO was added, and a methylating reagent A solution was quickly added, sealed, dissolved under ultrasonic action, and then a methylating reagent B solution was added. The reaction was carried out in a magnetic stirring water bath at 30 °C for 60 min. Finally, 2 mL of ultrapure water was added to the above mixture to terminate the methylation reaction.

[0119] After methylation, 1 ml of 2M trifluoroacetic acid (TFA) was added to hydrolyze for 90 min, and then rotary evaporator was used to evaporate the water. 2 mL of distilled water and 60 mg of sodium borohydride were added to reduce for 8 hours, and then glacial acetic acid was added for neutralization. After rotary evaporation, the residue was dried in an oven at 101 degrees, and then 1 ml of acetic anhydride was added for acetylation at 100 degrees for 1 hour. Then 3 mL of toluene was added, and the mixture was concentrated and evaporated under reduced pressure. This process was repeated 4-5 times to remove excess acetic anhydride.

[0120] After acetylation, the product was dissolved in 3 mL of CH2Cl2 and transferred to a separatory funnel. A small amount of distilled water was added, and the mixture was shaken thoroughly. The upper aqueous layer was removed, and this process was repeated 4 times. The CH2Cl2 layer was dried with an appropriate amount of anhydrous sodium sulfate, concentrated to 1 mL, and placed in a liquid phase vial. The acetylated product sample was analyzed using a Thermo scientific, 1300-7000 gas chromatograph-mass spectrometer;

[0121] The GC-MS conditions were as follows: HP-INNOWAX column, 30 m*0.32 mm*0.25 um; programmed temperature conditions: initial temperature 140°C, increased to 230°C at 1°C / min; injection port temperature 250°C, detector temperature 250°C, carrier gas helium, flow rate 1 mL / min.

[0122] The ion current chromatogram and secondary proton chromatogram of GC-MS are shown in Figures 2 and Figure 3 a、 Figure 3 b、 Figure 3 c. According to the detection results of monosaccharide composition, the polysaccharides in the stems and leaves of Polygonatum sibiricum contain a large amount of fructose. During the derivation process, the reduction of 2-keto group of fructose leads to isomerization to form mannitol and glucitol. Therefore, the detected Man f / Glc f glycoside residue fragments were integrated, and the final determination results of the glycosidic bond type of the polysaccharides in the stems and leaves of Polygonatum sibiricum are shown in Table 5, and the total ion current chromatogram and secondary proton chromatogram are shown in Figure 2 According to the results in Table 5, it can be seen that the polysaccharides extracted by this method mainly contain fructose type glycosidic bonds, which are →1)-Fru f -(2→, →1, 6) Fru f -(2→, →6) Fru f -(2→ and Fru f -(2→, and Ara f -(1→, Glc p -(1→, →4)-Glc p -(1→ and →6-Glc p -(1→, which is significantly different from the polysaccharide structure of traditional Polygonatum sibiricum.

[0123] Table 5 Analysis of the type of glycoside linkage of the polysaccharides in the stems and leaves of Polygonatum sibiricum

[0124]

[0125] Test Example 3 Study on the antidepressant effect of polysaccharides in the stems and leaves of Polygonatum sibiricum

[0126] 1. Design of animal experiments

[0127] This experiment was reviewed by the Experimental Animal Ethics Committee of the Experimental Animal Center of South China Agricultural University [No. 2024B017], and the experimental plan complies with the principles of animal ethics and meets the ethical requirements.

[0128] Animal modeling and grouping: 40 male SPF C57BL / 6J mice, 5 weeks old, weighing 18-22 g, purchased from Hunan Slike Jingda Experimental Animal Co., Ltd. (Certificate No. SCXK (Xiang) 2021-0002). After one week of adaptive feeding, the mice were randomly divided into a healthy group (NC, n = 8), a model group (CRS, n = 8), a positive group (CRS+P, n = 8), a low-dose polysaccharide group (CRS+LD, n = 8), and a high-dose polysaccharide group (CRS+HD, n = 8).

[0129] The first 4 weeks were the modeling period. The NC group of mice could consume water and food at any time, and the other 4 groups of mice were restrained in custom centrifuge tubes every day for 7 hours a day for 4 consecutive weeks. During the restraint period, the mice were deprived of food and water. After the modeling was successful, the mice were given intragastric intervention. The intragastric time was set at 9-11 am every day, and the intragastric intervention was performed for 4 consecutive weeks: the NC and CRS groups were given 10 mL / kg of normal saline by gavage; the CRS+P group was given 5.2 mg / kg. BW of fluoxetine hydrochloride (Suzhou Zhonghua Pharmaceutical Industry Co., Ltd.); the CRS+LD group was given 50 mg / kg. BW of PSPL1; and the CRS+HD group was given 250 mg / kg. BW of PSPL1. The preparation method was to dissolve the prepared Polygonatum sibiricum stem and leaf polysaccharide lyophilized powder in sterile primary water according to the corresponding concentration, and then filter sterilize.

[0130] Two days before the experimental period, behavioral experiments were performed on the mice,

[0131] Tail suspension test (TST): The middle section of the tail of the experimental mouse was fixed downward with tape on a fixed rod with equally spaced iron hooks. The mice were separated by a blackboard to avoid interference with each other. The activity of the mice was recorded for 5 minutes, and the cumulative immobility time of the mice was recorded 4 minutes later.

[0132] Forced swim test (FST): Each mouse was placed individually in a 2L beaker (14 cm in diameter, 20 cm in height) filled with 1.5L water (24 ± 1℃) so that the mouse’s hind limbs could not touch the bottom to support the body. The swimming behavior of the mouse was observed within 5 min, and the cumulative immobility time of the mouse in the last 4 min was recorded. The mouse floating on the water surface without struggling or only with slight movement of the hind limbs to support the head floating on the water surface was considered to be in an immobile state. After the test, the mice were wiped with a dry towel and given comfort. The water was changed after each test.

[0133] After the end of the experimental period, all mice were fasted and deprived of water for 12 h. The blood of the mice was collected by orbital bleeding, and then the mice were anesthetized and euthanized. The mice were quickly dissected on ice, and the colon of the mice was taken out and wrapped in tin foil in a pathology bag. The brain tissue of each mouse was taken out, and half of it was placed in a 15 mL centrifuge tube containing 4% paraformaldehyde, and the other half was placed in a sterile EP tube. The cecal contents of each mouse were collected in different sterile EP tubes.

[0134] HE staining of hippocampus sections: The mouse half-brain sample was fixed in a 4% paraformaldehyde solution for 24 h. After dehydration, wax immersion, embedding, and sectioning, the sections were first placed in xylene for 20 min, 3 times. Then they were deparaffinized in 100%, 100%, 95%, 85%, and 75% ethanol, respectively, and then immersed in distilled water for 5 min. Then they were stained with hematoxylin reagent, washed with distilled water after staining, and returned to PBS blue; then they were stained with eosin reagent and washed with distilled water; finally, they were dehydrated in gradient alcohol (95-100%) for 5 min each (or directly baked). After taking out, they were placed in xylene, then sealed with neutral resin, scanned with a panoramic scanner, and observed the hippocampus region.

[0135] Biochemical index determination of hippocampus: About 20 mg of hippocampus tissue was separated from the brain tissue, and the blood was washed off with 1X PBS. It was cut into small pieces and placed in a tissue grinder (homogenizer tube) with 0.3 mL of 1X PBS to make a homogenate, which was then placed at -20℃ overnight. After repeated freeze-thaw treatment for 2 times to destroy the cell membrane, the tissue homogenate was centrifuged at 2-8℃ at 5000g for 5 minutes to obtain the supernatant. An appropriate amount of supernatant was immediately tested, and the contents of 5-HT (5-hydroxytryptamine), KYN (kynurenine), QUIN (quinolinic acid), and TRP (tryptophan) in the mouse hippocampus were determined according to the ELISA kit instructions.

[0136] Colonic tissue biochemical index determination: Take about 30 mg of colon tissue, wash off blood stains with 1X PBS. Cut into small pieces and put into a tissue grinder (homogenate tube), add 0.3 mL of 1X PBS, make homogenate, and then place at -20°C overnight. After treating the cell membrane by repeated freezing and thawing 2 times, centrifuge the tissue homogenate at 2-8°C 5000g for 5 minutes to obtain the supernatant. Take an appropriate amount of supernatant and immediately perform the experiment. According to the ELISA kit instructions, the contents of KYN (kynurenine) and QUIN (quinolinic acid) in the mouse colon are determined.

[0137] Determination of expression of colon / hippocampus-related proteins:

[0138] Protein extraction: Cut 0.025 g of mouse hippocampus / colon tissue, wash the tissue with ice-precooled PBS, add 270 μL of RIPA lysis buffer to the biological sample homogenizer, and repeatedly grind the tissue until no tissue pieces are visible; centrifuge at 4°C, 12000 rpm for 15 min after lysis on ice (pre-cool the centrifuge in advance); transfer the supernatant after centrifugation into a 1.5 mL centrifuge tube.

[0139] Gel preparation: Prepare 10% separating gel, shake immediately after adding TEMED to perform gel filling, and use isopropanol for gel sealing after completion. Slightly tilt the gel maker, and when the boundary line no longer changes, it indicates that the gel has solidified, wait for another 3 min to ensure complete solidification, then pour off the isopropanol on top of the gel and absorb it with filter paper. Next, prepare 4.8% concentrated gel, shake immediately after adding TEMED to perform gel filling. Insert the comb into the glass plate, fill the remaining space with concentrated gel, and wait for the gel to solidify.

[0140] Sample preparation: Take 200 μL of protein supernatant, add 50 μL of 5*loading buffer, mix well, boil in boiling water for 5 min, and place in an ice box for quick cooling.

[0141] Electrophoresis: According to the protein quantification results, add 2 μL of marker to the first well, and add 10 μL of denatured protein to the rest of the wells. Start electrophoresis with a constant voltage of 75 V and an electrophoresis time of 130 min. When bromophenol blue electrophoresis reaches the bottom of the gel, terminate electrophoresis.

[0142] Membrane transfer: Cut the gel according to the molecular weight, and the specific molecular weight is shown in the antibody information. Prepare 6 filter papers of the same size as the gel and 1 NC membrane, and place the NC membrane in the transfer buffer together with the filter papers until it is completely soaked. Place them in the order of 3 filter papers, NC membrane, gel, and another 3 filter papers, ensuring that there are no air bubbles in the middle. Cover the instrument, connect the power supply, and perform membrane transfer at a constant current of 300 mA. The membrane transfer time for each index is shown in the antibody information. After membrane transfer is complete, remove the membrane and wash it once in 1X PBST.

[0143] Blocking: Prepare 5% skim milk powder with 1*PBST, after immersing the membrane, place it at room temperature for 90 min.

[0144] Primary antibody incubation: Dilute the primary antibody according to a certain proportion (IDO1 1:1000, KMO 1:1000, TPH2 1:1000, GAPDH 1:5000) with 1*PBST, incubate the membrane with the primary antibody, 4°C overnight, the next day at room temperature for 30 min. After incubation, wash 3 times with 1*PBST.

[0145] Secondary antibody incubation: Dilute the HRP-labeled secondary antibody (1:5000) with 1*PBST, incubate the diluted secondary antibody with the membrane at room temperature for 90 min. After incubation, wash 3 times with 1*PBST.

[0146] Color development and exposure: ECL (Enhanced Chemiluminescence) color development and exposure: use ECL chemiluminescence solution to incubate the membrane for 1 min, absorb the liquid with filter paper, wrap the membrane with plastic film, and image with gel imaging system.

[0147] Mouse intestinal flora determination: Use second-generation full-length amplicon sequencing to determine the gene sequence of the intestinal flora of the mouse cecum contents.

[0148] Genomic DNA extraction and PCR amplification: Extract the genomic DNA of the sample using the CTAB or SDS method, then detect the purity and concentration of the DNA using agarose gel electrophoresis, take an appropriate amount of sample in a centrifuge tube, and dilute the sample to 1 ng / μL with sterile water. Use the diluted genomic DNA as a template, use specific primers with Barcode according to the selection of the sequencing region, Phusion® High-Fidelity PCR Master Mix with GC Buffer from New England Biolabs, and high-efficiency high-fidelity enzyme for PCR to ensure amplification efficiency and accuracy.

[0149] PCR product mixing and purification: The PCR product is detected by electrophoresis using a 2% concentration agarose gel; according to the concentration of the PCR product, equal amounts are mixed, and after thorough mixing, the PCR product is detected by electrophoresis using a 2% agarose gel, and the desired band is recovered using the gel recovery kit provided by Qiagen.

[0150] Library construction and sequencing: DNA ligase was used to connect sequencing adapters to both ends of the amplified DNA fragments, and AMpure PB magnetic beads were used to purify and select the DNA fragments to construct the SMRT Bell library. After the purified fragments were dissolved in buffer, BluePipin fragment screening was used to select fragments of a specific size, and AMpure PB magnetic beads were used to purify the DNA fragments. The constructed library was quantified by Qubit concentration, and the insert size was detected by Agilent 2100, followed by sequencing on the PacBio platform.

[0151] 2. Experimental results

[0152] (1) Depression behavioral test results

[0153] The experimental results of the forced swimming test and the tail suspension test of mice are shown in FIG. 4 and FIG. 5,

[0154] In the forced swimming test and the tail suspension test, the despair time of the mice in the remaining groups was significantly increased compared with the NC group after being restrained for 4 weeks P <0.001), and there was no significant difference between groups P > 0.001), indicating that restraint can significantly increase the negative time of mice when facing forced stress environment, and reduce the ability of mice to cope with difficulties, at this time the CRS mouse model is completed.

[0155] After being restrained and intragastrically administered for 4 weeks, i.e., the eighth week, the despair time of the mice in each drug intervention group in the forced swimming test and the tail suspension test was decreased to varying degrees compared with the CRS group, and was significantly lower than the CRS group P <0.001), and in the forced swimming test, the CRS+HD group had the best effect.

[0156] (2) Determination results of hippocampal HE staining sections

[0157] HE staining of the hippocampus can directly show neurons and structural integrity, can infer whether the hippocampal function is normal, and can indirectly reflect the antidepressant effect of polygonatum stem and leaf polysaccharide. The hippocampal tissue sections of the mice in each group are shown in FIG. 6. The hippocampal nerve cells of the mice in the NC group were arranged closely and orderly, and the cell morphology was normal, while the hippocampal nerve cells of the mice in the CRS group were arranged in disorder, the gap was large, and the nerve cells were severely damaged. The hippocampal nerve cell state of the mice in each intervention group of the CRS group was improved, and the arrangement was relatively compact, indicating that polygonatum stem and leaf polysaccharide can improve the nerve cell damage caused by CRS induction to a certain extent, and the effect is similar to that of fluoxetine hydrochloride.

[0158] (3) Determination results of antidepressant-related indicators in the hippocampus and colon

[0159] Changes in 5-HT (5-hydroxytryptamine), KYN (kynurenine), QUIN (quinolinic acid), TRP (tryptophan), IDO1 (indoleamine 2,3-dioxygenase 1), TPH2 (tryptophan hydroxylase 2), and KMO (kynurenine 3-monooxygenase) in the hippocampus, as well as KYN, QUIN, IDO1, and KMO in the colon, are closely associated with antidepressant mechanisms. In the hippocampus, these indicators directly influence the levels of 5-HT and the synthesis of related neurotransmitters in the central nervous system by regulating the balance of TRP conversion to 5-HT or KYN pathways, thereby regulating mood and neuroplasticity. Furthermore, increases in neurotoxic metabolites such as QUIN and upregulation of IDO1 and KMO in the hippocampus promote neuroinflammatory responses, further exacerbating depressive-like behaviors. In the colon, activation of the KYN pathway and increased expression of IDO1 and KMO indirectly affect central 5-HT levels and systemic inflammatory status mainly through the gut-brain axis mechanism, leading to reduced 5-HT synthesis in the central nervous system and increased neuroinflammation, thereby promoting the occurrence and development of depressive symptoms. As shown in Figures 7 and 8, the various indicators in the hippocampus and colon of mice in the CRS group were significantly different from those in the NC group ( P <0.001). After intervention, all indicators in the dose groups showed improvement to varying degrees, indicating that oral administration of Polygonatum sibiricum stem and leaf polysaccharide can improve depression in mice induced by chronic stress restraint, and the effect is comparable to that of the common drug fluoxetine hydrochloride.

[0160] (4) Intestinal flora analysis

[0161] In the analysis of intestinal flora, beta diversity is used to evaluate the differences or similarities in microbial communities between different samples. As shown in Figure 9, PCoA and NMDS analysis based on Bray-Curtis distance showed that the microbial communities of the CRS group and the NC group were significantly separated, indicating that chronic restraint stress changed the structure and composition of the intestinal flora. After oral administration of fluoxetine hydrochloride and Polygonatum sibiricum stem and leaf polysaccharides, the sample points of the dose group gradually moved from the CRS group to the NC group, especially the sample points of the CRS+HD group showed a certain degree of aggregation with the NC group, indicating that the flora structure was significantly improved.

[0162] To further investigate how Polygonatum sibiricum stem and leaf polysaccharides improve depression in mice induced by chronic restraint stress, the relative abundance of the top 10 phylum and top 20 genus in the intestinal flora of each group of mice was calculated to obtain a relative abundance bar graph (Figure 10). It can be found that the abundance of Verrucomicrobiota was significantly reduced at the phylum level in the CRS group, but after intervention with Polygonatum sibiricum stem and leaf polysaccharides, the abundance of Verrucomicrobiota was reversed and restored to a level close to that of the NC group. At the genus level, the abundance of Verrucomicrobiota in the CRS group was significantly reduced at the phylum level. Akkermansia 、 Lactobacillus 、Alistipes 、 Ileibacterium The relative abundance of the above-mentioned bacteria in the CRS+HD group increased and decreased to a certain extent compared with the NC group, but after the intervention of the polysaccharides in the stems and leaves of Polygonatum, the abundance of the above-mentioned bacteria in the CRS+HD group can be restored to the level close to that of the NC group. These results show that the polysaccharides in the stems and leaves of Polygonatum can reverse the intestinal flora disorder caused by the depression model and restore the intestinal flora of the depressed mice to a level close to that of normal mice.

[0163] Test Example 4 Study on the sleep improvement effect of polysaccharides in the stems and leaves of Polygonatum

[0164] 1. Animal experiment design

[0165] The chronic restraint stress (CRS) model can produce behaviors and emotional disorders similar to depression in animals through continuous stress treatment, accompanied by obvious abnormalities in sleep structure and quality, thereby providing the possibility of simultaneously studying anti-depression and sleep improvement intervention methods in the same model.

[0166] Therefore, the determination of the corresponding indicators was performed on the tissues of the mice in Test Example 3 except for the CRS+P group.

[0167] Serum biochemical index determination: After the collected blood was naturally settled for 1 h, it was placed at 4℃ for 3 h to separate the serum, and then the serum sample was obtained by centrifugation at 4℃ at 4000 rpm for 15 min. The content of 5-HT (5-hydroxytryptamine) and TRP (tryptophan) in the mouse serum was determined according to the ELISA kit instructions. The mouse colon tissue was treated in the same way as in Test Example 3, and the content of 5-HT (5-hydroxytryptamine) and TRP (tryptophan) in the mouse colon was determined according to the method of the ELISA kit instructions. The same protein expression determination method was used to determine the TPH1 protein expression in the mouse colon, and in the step of primary antibody incubation, the dilution ratio of TPH1 was 1:1000.

[0168] 2. Experimental results

[0169] The changes of 5-HT, TRP in serum and colon, and TPH1 (tryptophan hydroxylase 1) in colon are closely related to the improvement of sleep. In serum, 5-HT and TRP have an important influence on sleep quality by affecting the synthesis of melatonin and regulating circadian rhythm. In colon, TPH1 plays a key role in peripheral 5-HT synthesis, regulates the level of 5-HT in the intestine, and thus has an impact on the central 5-HT system through the gut-brain axis, indirectly promoting sleep regulation and improving sleep quality. As shown in FIG. 11, the indicators in the serum and colon of mice after CRS induction were significantly lower than those in the NC group, but after the intervention of polygonatum stem and leaf polysaccharide, the relevant indicators of each dose group were improved to a certain extent, especially in the CRS+HD group. The results show that oral administration of polygonatum stem and leaf polysaccharide can effectively improve the abnormal sleep quality of mice caused by CRS.

[0170] The above only is the preferred embodiment of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A preparation method of Polygonatum sibiricum leaves polysaccharide, characterized in that, The preparation method comprises the following steps: (1) water extraction of the stem and leaf of Polygonatum sibiricum, to obtain a concentrated solution; step (1) specifically comprises: drying and crushing the stem and leaf of Polygonatum sibiricum, then water bath extraction, cooling to room temperature, filtering the supernatant, concentrating the filtrate, to obtain the concentrated solution; (2) alcohol extraction of the concentrated solution, to obtain a crude extract; step (2) specifically comprises: adding anhydrous ethanol to the concentrated solution, standing and precipitating, centrifuging, collecting the precipitate, then adding distilled water to the precipitate, removing insoluble impurities, to obtain the crude extract; (3) removing protein from the crude extract, to obtain crude polysaccharides from the stem and leaf of Polygonatum sibiricum; step (3) specifically comprises: adding Sevage reagent to the crude extract, centrifuging and standing, discarding the lower organic layer and the middle protein layer, repeating the operation until the protein layer is completely removed, then low-temperature and reduced-pressure distillation of the supernatant, dialysis, and freeze-drying of the dialysate, to obtain the crude polysaccharides from the stem and leaf of Polygonatum sibiricum; (4) preparing a crude polysaccharide solution from the crude polysaccharides from the stem and leaf of Polygonatum sibiricum and water, flowing the crude polysaccharide solution into a DEAE-52 anion exchange column, sequentially performing gradient elution with 0 mol / L and 0.1 mol / L sodium chloride, collecting the polysaccharide components eluted with 0.1 mol / L sodium chloride, dialyzing, and freeze-drying, to obtain purified polysaccharide components from the stem and leaf of Polygonatum sibiricum.

2. The preparation method according to claim 1, wherein in step (1), the water bath extraction is performed for 3-5 h at a temperature of 60-90 ℃, and the water bath extraction is performed by adding water in a solid-liquid ratio of 1:10-1:40 g / mL.

3. The preparation method according to claim 2, wherein in step (1), the drying is performed at a temperature of 50-60 ℃.

4. The preparation method according to claim 1, wherein in step (2), the volume ratio of anhydrous ethanol to the concentrated solution is 3-5:

1.

5. The preparation method according to claim 4, wherein in step (2), the standing and precipitating is performed for 10-15 h.

6. The preparation method according to claim 1, wherein in step (3), the volume ratio of Sevage reagent to the crude extract is 3-5:1, and the Sevage reagent is a mixture of chloroform and n-butanol in a volume ratio of 4:1; in step (3), the low-temperature and reduced-pressure distillation is performed at a temperature of 50-60 ℃; and in step (3), the dialysis is performed in a dialysis bag with a molecular weight cutoff of 1000 Da.

7. The preparation method according to claim 1, wherein in step (4), the concentration of the crude polysaccharide solution is 10 mg / mL. ​ ​ ​ ​ ​ ​ ​ ​

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