Polygonatum sibiricum stem and leaf polysaccharide with effects of improving depression and sleep as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to rhizoma polygonati stem and leaf polysaccharide with the depression and sleep improving effect and a preparation method and application of the rhizoma polygonati stem and leaf polysaccharide. The weight-average molecular weight of the polygonatum sibiricum stem and leaf polysaccharide is 4000-5000 Da, the polygonatum sibiricum stem and leaf polysaccharide comprises the following monosaccharide components: arabinose, galactose, glucose, xylose, mannose and fructose, and the fructose accounts for the highest percentage. The obtained rhizoma polygonati stem and leaf polysaccharide with higher purity is used for preparing health care products and medicines for improving sleep or preparing antidepressant medicines, and plant raw materials with natural sources are adopted, so that the dependence of a treated object on the medicines can be reduced, and the harm to a body is relatively less.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to polygonatum sibiricum stem and leaf polysaccharide with the efficacy of improving depression and sleep, and a preparation method and application thereof. Background Art Depression is a complex disease of the central nervous system, usually manifested by a variety of symptoms such as low mood, loss of interest, fatigue, difficulty concentrating, and sleep disorders, which seriously affect the quality of life of patients. Among them, sleep disorders are particularly common in depression, including not only difficulty falling asleep and maintaining sleep, but also early awakening, sleep structure disorders, and circadian rhythm disorders. There is a bidirectional correlation between sleep problems and depression. On the one hand, the depressive state can lead to decreased sleep quality. On the other hand, long-term poor sleep can also aggravate depressive symptoms, forming a vicious circle. In recent years, the incidence of depression and insomnia has increased year by year. Although traditional drug treatments are effective, they are often accompanied by side effects and addiction risks. Therefore, finding safe, low-toxic natural alternative treatments that can significantly improve mood and sleep has become a research hotspot.

[0002] Polysaccharides are polymers formed by connecting multiple monosaccharides. They have a wide range of sources and can participate in and regulate a variety of cellular 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 the treatment of central nervous system diseases due to their low toxicity, safety and diverse biological activities.

[0003] Polygonatum sibiricum is a precious medicinal material in traditional Chinese medicine. As an important member of the medicine-food homology, its unique pharmacological effects and significant health-care effects are highly respected by the public. In recent years, under the guidance of the "big food concept", it has become an inevitable trend to develop food resources in an all-round and multi-channel manner and expand food source channels. However, for a long time, the medicinal part of Polygonatum sibiricum has been mainly concentrated on the dried rhizomes, which are used for the effects of replenishing qi and blood, nourishing yin and moistening dryness, while the research and development of its stems and leaves is relatively small. The stems and leaves of Polygonatum sibiricum are also rich in a variety of bioactive ingredients, such as polysaccharides, flavonoids and saponin compounds, but their potential medicinal value and functional characteristics have not yet been fully explored and utilized. Summary of the invention The invention extracts, separates and purifies high-purity polysaccharides from the stems and leaves of polygonatum, and then studies the anti-depression and sleep-improving effects of the polysaccharides, thus filling the research gaps in the utilization of natural by-products and the polysaccharides from the stems and leaves of polygonatum.

[0004] The invention provides a polygonatum stem and leaf polysaccharide, wherein the weight average molecular weight of the polygonatum stem and leaf polysaccharide is 4000-5000Da, and the polygonatum stem and leaf polysaccharide comprises the following monosaccharide components: arabinose, galactose, glucose, xylose, mannose and fructose, among which fructose accounts for the highest percentage. Furthermore, the glycosidic bonds of the Polygonatum cyrtonema stem and leaf polysaccharide include: →1) -Fruf -(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→.

[0005] Furthermore, the Polygonatum sibiricum stem and leaf polysaccharide includes the following monosaccharide components in the following mass percentages: arabinose 4.2%, galactose 1.1%, glucose 2.3%, xylose 1.7%, mannose 1.8%, and fructose 88.9%.

[0006] The present invention also provides a method for preparing any of the above-mentioned Polygonatum cyrtonema stem and leaf polysaccharides, comprising the following steps: (1) extracting the stems and leaves of Polygonatum cyrtonema with water to obtain a concentrated solution; (2) extracting the concentrated solution with alcohol to obtain a crude extract; (3) removing protein from the crude extract to obtain crude polysaccharide from stems and leaves of polygonatum; (4) A crude polysaccharide solution was prepared using the crude polysaccharide from the stem and leaves of Polygonatum cyrtonema and water. The crude polysaccharide solution was flowed into an anion exchange column and gradient eluted with 0 mol / L and 0.1 mol / L sodium chloride, respectively. The polysaccharide components eluted with 0.1 mol / L sodium chloride were collected, dialyzed, and freeze-dried to obtain a purified polysaccharide component from the stem and leaves of Polygonatum cyrtonema.

[0007] Furthermore, in step (1), the water extraction specifically comprises: drying and crushing the stems and leaves of Polygonatum cyrtonema, adding water for water bath extraction, cooling to room temperature, filtering the supernatant, and concentrating the obtained filtrate to obtain a concentrated solution; Preferably, in step (1), the water bath extraction time is 3 to 5 h; the water bath extraction temperature is 60 to 90°C; during the water bath extraction, the solid-liquid ratio is 1:10 to 1:40 g / mL; More preferably, in step (1), the drying temperature is 50-60°C. Furthermore, in step (2), the alcohol extraction specifically comprises: adding anhydrous ethanol to the above-mentioned concentrated solution, allowing the solution to settle, centrifuging, collecting the precipitate, and then adding distilled water to the obtained precipitate to remove insoluble impurities to obtain a crude extract; Preferably, in step (2), the volume ratio of anhydrous ethanol to the concentrated solution is 3-5:1; More preferably, in step (2), the static sedimentation time is 10 to 15 hours. Furthermore, in step (3), the protein removal specifically comprises: adding Sevage reagent to the above crude extract, centrifuging and then standing, discarding the lower organic layer and the middle protein layer, repeating the operation until the protein layer is completely removed, distilling the obtained supernatant under low temperature and reduced pressure, dialyzing, and freeze-drying the obtained dialyzate to obtain crude polysaccharides from polygonatum stems and leaves.

[0008] Furthermore, in step (3), the volume ratio of Sevage reagent to crude extract is 3-5:1; wherein the Sevage reagent is obtained by mixing chloroform and n-butanol in a volume ratio of 4:1; In step (3), the temperature of low temperature and reduced pressure distillation is 50-60°C; In step (3), the dialysis is performed in a 1000 Da dialysis bag. Further, in step (4), the concentration of the crude polysaccharide solution is 10 mg / mL; In step (4), the crude polysaccharide solution flows into the anion exchange column at a flow rate of 1 mL / min for 10 min.

[0009] The present invention also proposes the use of any of the above-mentioned Polygonatum sibiricum stem and leaf polysaccharides or the Polygonatum sibiricum stem and leaf polysaccharides prepared by any of the above-mentioned preparation methods in the preparation of health products and medicines for improving sleep, or in the preparation of antidepressant drugs. The present invention has the following advantages: The polysaccharide from the stem and leaves of Polygonatum cyrtonema proposed by the present invention has a clear structure, and the monosaccharide components include arabinose, galactose, glucose, xylose, mannose, and fructose, wherein the fructose glycosidic bond is the main connection mode. The sugar content of the polysaccharide is as high as 97.6%, and gel permeation chromatography shows that only a single peak appears, indicating that the polysaccharide is a high-purity and uniform polysaccharide component.

[0010] The method for preparing the stem and leaf polysaccharide of polygonatum sibiricum proposed by the present invention uses the stem and leaf of polygonatum sibiricum as raw materials, and prepares the stem and leaf polysaccharide of polygonatum sibiricum with high purity by a specific method. Then, the polysaccharide is used to prepare a drug with antidepressant effect, or a health product or drug for improving sleep. Since the plant raw materials from natural sources are used, the dependence of the treated object on the drug can be reduced, and the harm to the body is relatively less, thereby realizing the full development and utilization of the plant resources of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0012] FIG. 1 is a molecular weight determination spectrum of Polygonatum cyrtonema stem and leaf polysaccharide in Experimental Example 2.

[0013] Figure 2 is the total ion current diagram of the detection of glycosidic bond types of Polygonatum cyrtonema stem and leaf polysaccharides in Experimental Example 2.

[0014] Figures 3a, 3b and 3c are secondary proton chromatograms of the detection of glycosidic bond types of Polygonatum cyrtonema stem and leaf polysaccharides in Experimental Example 2.

[0015] FIG. 4 shows the tail suspension experiment of mice in each group after model establishment (a) and after drug administration (b) in Experimental Example 3.

[0016] FIG. 5 shows the forced swimming test of mice in each group after modeling (a) and after drug administration (b) in Experimental Example 3.

[0017] FIG. 6 is a picture of HE-stained hippocampal sections of mice in each group in Experimental Example 3 observed at 400× magnification.

[0018] FIG. 7 is a graph showing changes in the content of antidepressant-related indicators in the hippocampus of each group of mice in Experimental Example 3.

[0019] FIG8 is a graph showing changes in the content of antidepressant-related indicators in the colon of each group of mice in Experimental Example 3.

[0020] Figure 9 is the PCoA and NMDS graphs of the β diversity analysis of the intestinal flora of each group of mice in Experimental Example 3.

[0021] FIG10 is a bar graph of the relative abundance of the top 10 at the phylum level (a) and the top 20 at the genus level (b) of each group of samples in Experimental Example 3.

[0022] FIG11 is a graph showing changes in the levels of sleep-related indicators in the serum (a, b) and colon (cf) of each group of mice in Experimental Example 4.

[0023] Figure 4-Figure 8 Compared with the NC group, *** p <0.001; compared with the CRS group, # p <0.05, ## p <0.01, ### p <0.001. DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. In the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0025] On the one hand, an embodiment of the present invention provides a Polygonatum sibiricum stem and leaf polysaccharide, wherein the weight average molecular weight of the Polygonatum sibiricum stem and leaf polysaccharide is 4000-5000 Da, and the Polygonatum sibiricum stem and leaf polysaccharide includes the following monosaccharide components: arabinose, galactose, glucose, xylose, mannose, and fructose, among which fructose accounts for the highest percentage. The polysaccharide from the stem and leaves of polygonatum proposed in the embodiment of the present invention has a clear structure, with fructose glycosidic bond as the main connection mode, and also contains other glycosidic bonds such as arabinose and glucose. The sugar content of the polysaccharide is as high as 97.6%, and the monosaccharide composition analysis shows that the total amount of each monosaccharide is 881.64 μg / mg, and the gel permeation chromatography test result shows that only a single peak appears, indicating that the polysaccharide is a high-purity and uniform polysaccharide component.

[0026] In one embodiment of the present invention, the glycosidic bonds of the Polygonatum cyrtonema stem and leaf polysaccharide include: →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→.

[0027] In the embodiment of the present invention, the fructose type glycosidic bonds of the Polygonatum cyrtonema stem and leaf polysaccharide mainly include →1)-Fru f -(2→、→1,6)Fru f -(2→、→6)Fru f -(2→、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→.

[0028] In one embodiment of the present invention, the polysaccharide from the stems and leaves of Polygonatum sibiricum comprises the following monosaccharide components in the following weight percentages: 4.2% arabinose, 1.1% galactose, 2.3% glucose, 1.7% xylose, 1.8% mannose, and 88.9% fructose.

[0029] In a preferred embodiment of the present invention, the weight average molecular weight of Polygonatum cyrtonema stem and leaf polysaccharide is 4287 Da.

[0030] On the other hand, the present invention provides a method for preparing a polysaccharide from stems and leaves of polygonatum, comprising the following steps: (1) extracting the stems and leaves of Polygonatum cyrtonema with water to obtain a concentrated solution; (2) extracting the concentrated solution with alcohol to obtain a crude extract; (3) removing protein from the crude extract to obtain crude polysaccharide from stems and leaves of polygonatum; (4) A crude polysaccharide solution was prepared using the crude polysaccharide from the stem and leaves of Polygonatum cyrtonema and water. The crude polysaccharide solution was flowed into an anion exchange column and gradient eluted with 0 mol / L and 0.1 mol / L sodium chloride, respectively. The polysaccharide components eluted with 0.1 mol / L sodium chloride were collected, dialyzed, and freeze-dried to obtain a purified polysaccharide component from the stem and leaves of Polygonatum cyrtonema. In one embodiment of the present invention, in step (1), the water extraction specifically comprises: drying and crushing the stems and leaves of Polygonatum cyrtonema, adding water for water bath extraction, cooling to room temperature, filtering the supernatant, and concentrating the obtained filtrate to obtain a concentrated solution.

[0031] In one embodiment of the present invention, in step (1), the time of water bath extraction is 3 to 5 h; the temperature of water bath extraction is 60 to 90° C.; during water bath extraction, the solid-liquid ratio is added at 1:10 to 1:40 g / mL.

[0032] In one embodiment of the present invention, in step (1), the stems and leaves of Polygonatum cyrtonema are fresh stems and leaves of Polygonatum cyrtonema. Polygonatum sibiricum Red.) on the stems and leaves.

[0033] In one embodiment of the present invention, in step (1), the drying temperature is 50-60° C. Preferably, the drying temperature is 60° C.

[0034] Furthermore, the drying is specifically drying in a constant temperature drying oven to a constant weight.

[0035] In one embodiment of the present invention, in step (1), the concentration is concentrated by rotary evaporation.

[0036] In one embodiment of the present invention, in step (1), the room temperature is 10-25°C.

[0037] In one embodiment of the present invention, in step (1), step (1) further includes filtering the supernatant and extracting the residue again in a water bath, and combining the filtrates obtained from the two extractions and concentrating them. In one embodiment of the present invention, in step (2), the alcohol extraction specifically comprises: adding anhydrous ethanol to the above-mentioned concentrated solution, allowing the solution to stand for precipitation, centrifuging, collecting the precipitate, and then adding distilled water to the obtained precipitate to remove insoluble impurities to obtain a crude extract.

[0038] In one embodiment of the present invention, in step (2), the static precipitation time is 10 to 15 hours. Preferably, in step (2), the static precipitation time is 12 hours.

[0039] In one embodiment of the present invention, in step (2), the volume ratio of anhydrous ethanol to the concentrated solution is 3 to 5: 1. Preferably, in step (2), the volume ratio of anhydrous ethanol to the concentrated solution is 4: 1.

[0040] In one embodiment of the present invention, in step (3), the protein removal specifically comprises: adding Sevage reagent to the crude extract, centrifuging and then standing, discarding the lower organic layer and the middle protein layer, repeating the operation until the protein layer is completely removed, distilling the obtained supernatant at low temperature and reduced pressure, dialyzing, and freeze-drying the obtained dialyzate to obtain crude polysaccharides from polygonatum stems and leaves.

[0041] In one embodiment of the present invention, in step (3), the volume ratio of Sevage reagent to crude extract is 3 to 5: 1. Preferably, in step (3), the volume ratio of Sevage reagent to crude extract is 4: 1. The Sevage reagent is obtained by mixing chloroform and n-butanol in a volume ratio of 4: 1.

[0042] In one embodiment of the present invention, in step (3), the centrifugation is specifically performed by shaking with a vortex shaker for 10 minutes and then centrifuging.

[0043] In one embodiment of the present invention, in step (3), the temperature of low-temperature vacuum distillation is 50-60° C., and the low-temperature vacuum distillation is mainly for removing chloroform and n-butanol. In one embodiment of the present invention, in step (3), the dialysis is performed in a 1000 Da dialysis bag.

[0044] In one embodiment of the present invention, in step (3), during dialysis, tertiary water is used for dialysis until the conductivity of the obtained dialysate is close to the conductivity of tertiary water.

[0045] In step (4) of the embodiment of the present invention, different concentrations of sodium chloride are used for elution. When the concentration of sodium chloride is 0.1 mol / L, the salt gradient can effectively compete with and release the electrostatic adsorption of the Polygonatum sibiricum stem and leaf polysaccharide and the DEAE anion exchange layer without excessively increasing the ionic strength to cause a large amount of co-elution of impurities. Therefore, under this condition, the Polygonatum sibiricum stem and leaf polysaccharide component with the highest sugar content can be obtained.

[0046] In one embodiment of the present invention, in step (4), the concentration of the crude polysaccharide solution is 10 mg / mL.

[0047] In one embodiment of the present invention, in step (4), primary water is used to prepare the crude polysaccharide solution.

[0048] In one embodiment of the present invention, in step (4), the crude polysaccharide solution flows into the anion exchange column at a flow rate of 1 mL / min for 10 min.

[0049] In one embodiment of the present invention, step (4) further comprises passing the crude polysaccharide solution through a 0.45 μm water-based microporous filter membrane to remove impurities, and then passing the crude polysaccharide solution into an anion exchange column.

[0050] In one embodiment of the present invention, in step (4), the model of the DEAE-52 anion exchange column is DEAE-52 anion exchange column.

[0051] In one embodiment of the present invention, in step (4), the dialysis is specifically performed using tertiary water in a 1000 Da dialysis bag.

[0052] On the other hand, the embodiments of the present invention also propose the use of any of the above-mentioned Polygonatum sibiricum stem and leaf polysaccharides or the Polygonatum sibiricum stem and leaf polysaccharides prepared by any of the above-mentioned preparation methods in the preparation of health products and medicines for improving sleep, or in the preparation of antidepressants. In the embodiments of the present invention, the use in the preparation of antidepressants is mainly the use in the preparation of drugs for relieving depression and / or improving depression.

[0053] Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

[0054] Example 1 A method for preparing polygonatum stem and leaf polysaccharide, the specific steps are as follows: (1) Water extraction: Take fresh stems and leaves of Polygonatum cyrtonema, wash them twice with distilled water to remove impurities, dry them in a constant temperature drying oven at 60 degrees to constant weight, crush them with a grinder, extract them in a water bath at 80 degrees for 3 hours with a solid-liquid ratio of 1:30 (g / mL), cool them to room temperature, filter the supernatant, repeat the water bath extraction of the residue once, combine the filtrates after the two extractions, and concentrate them by rotary evaporation to obtain a concentrate; (2) Alcohol precipitation: add 4 times the volume of anhydrous ethanol to the concentrate, let it stand for 12 h, collect the precipitate after centrifugation, add distilled water again, remove insoluble impurities after re-dissolving, and obtain the crude extract of Polygonatum cyrtonema stem and leaf polysaccharide; (3) Protein removal: Add 4 times the volume of Sevage reagent (V (chloroform): V (n-butanol) = 4:1) to the crude extract obtained in step (2), vortex for 10 min, centrifuge, let stand, discard the lower organic layer and the middle protein layer, and repeat the operation for more than 10 times until the protein is completely removed. Finally, combine the obtained supernatants, remove chloroform and n-butanol by low-temperature vacuum distillation at 55°C, dialyze with tertiary water in a 1000 Da dialysis bag until the conductivity of the dialysate is close to that of tertiary water, take out the solution and freeze-dry (-70°C, 72 h) to obtain a crude polysaccharide sample of Polygonatum cyrtonema stems and leaves.

[0055] (4) Purification by anion exchange column method: The crude polysaccharide from the stem and leaves of Polygonatum cyrtonema was prepared with water to obtain a 10 mg / mL crude polysaccharide solution. After impurities were removed by filtering through a 0.45 μm water-based microporous filter membrane, the sample was loaded at a flow rate of 1 mL / min for 10 min. The sample obtained in step (3) was flowed into a DEAE-52 anion exchange column and eluted with 0 mol / L and 0.1 mol / L sodium chloride gradients, respectively. The polysaccharide fraction eluted with 0.1 mol / L sodium chloride was collected and dialyzed with grade tertiary water in a 1000 Da dialysis bag. After freeze-drying (-70°C, 72 h), the purified polysaccharide fraction from the stem and leaves of Polygonatum cyrtonema (abbreviated as PSLP1) was obtained. Comparative Example 1 The same as Example 1, except that in step (4), the components eluted with 0 mol / L sodium chloride are collected.

[0056] Comparative Example 2 The same as Example 1, except that in step (4), 0 mol / L, 0.1 mol / L, and 0.3 mol / L sodium chloride gradient elution is used in sequence, and the fraction eluted with 0.3 mol / L sodium chloride is collected.

[0057] Comparative Example 3 The same as Example 1, except that in step (4), 0 mol / L, 0.1 mol / L, 0.3 mol / L, and 0.5 mol / L sodium chloride gradient elution is used in sequence, and the fraction eluted with 0.5 mol / L sodium chloride is collected. Test Example 1 The purpose of this experiment is to explore the component with the highest sugar content in the stem and leaf polysaccharides of Polygonatum cyrtonema, and to determine the optimal process by studying the total sugar content of different components eluted at different sodium chloride concentrations.

[0058] The total sugar content of the Polygonatum cyrtonema stem and leaf polysaccharide prepared in each example was determined by phenol-sulfuric acid method, the total sugar content was determined at an ultraviolet wavelength of 490 nm, and was calculated according to the standard curve y=0.0079x+0.0059.

[0059] The experimental results are shown in Table 1. The total sugar content of the Polygonatum cyrtonema stem and leaf polysaccharide component in Example 1 is the highest. Therefore, after elution with 0 mol / L sodium chloride, collecting the component with a sodium chloride concentration of 0.1 mol / L can obtain the Polygonatum cyrtonema stem and leaf polysaccharide component with the highest sugar content.

[0060] Table 1 Total sugar content of each example under different sodium chloride concentration elution

[0061] Test Example 2 Study on the structural characteristics of the polysaccharide from the stems and leaves of Polygonatum cyrtonema 1. Determination of the physicochemical properties of Polysaccharides from the stems and leaves of Polygonatum cyrtonema 1.1 Determination of monosaccharide composition using ion exchange chromatography Standard solution preparation: 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, mannuronic acid) were prepared into standard mother solution. Each monosaccharide standard solution was precisely prepared with concentration standard as mixed standard. According to the absolute quantitative method, the mass of different monosaccharides was determined, and the molar ratio was calculated according to the molar mass of the monosaccharide. The peak time and peak area of ​​each monosaccharide are shown in Table 2.

[0062] Table 2 Monosaccharide standard sequences of monosaccharide composition in polysaccharides

[0063] Sample preparation: 5 mg of the sample obtained in Example 1 was accurately weighed and placed in an ampoule, 2 mL of 3M trifluoroacetic acid was added, and hydrolysis was performed at 80°C for 2 h. Accurately aspirate the acid hydrolysis solution and transfer it to a tube and blow it dry with nitrogen, add 5 mL of water and vortex to mix, aspirate 50 μL and add 950 μL of deionized water, and centrifuge at 12000 rpm for 5 min. Take the supernatant for ion chromatography (IC) analysis.

[0064] Chromatographic method: Column: Dionex Carbopac TMPA20 (3*150 mm); mobile phase: A:H2O; B:15mMNaOH; C: 15mM NaOH&100mM NaAc; flow rate: 0.3mL / min; injection volume: 25µL; column temperature: 30 ºC; elution gradient: 0min A phase / B phase / C phase (98.8:1.2:0, V / V), 18min A phase / B phase / C phase (98.8:1.2:0, V / V), 20min A phase / B phase / C phase (50:50: 0, V / V), 30min A phase / B phase / C phase (50:50: 0, V / V), 30.1min A phase / B phase / C phase (0:0:100, V / V), 46min A phase / B phase / C phase (0:0:100, V / V), 46.1min Phase A / B / C (0:100: 0, V / V), 50 min Phase A / B / C (0:100: 0, V / V), 50.1 min Phase A / B / C (98.8:1.2:0, V / V), 80 min Phase A / B / C (98.8:1.2:0, V / V). Detector: electrochemical detector.

[0065] The results of the sample in Example 1 and the standard product test were compared, and the calculated monosaccharide composition of the Polygonatum cyrtonema stem and leaf polysaccharide was shown in Table 3, where the molar ratio of fructose was 88.9% at most, and the content was 791.69 μg / mg. The total content of each monosaccharide was 881.64 μg / mg, proving that the polysaccharide component used had a high sugar content.

[0066] Table 3 Monosaccharide composition of Polysaccharide in the stems and leaves of Polygonatum cyrtonema

[0067] 1.2 Molecular weight determination of Polygonatum cyrtonema stem and leaf polysaccharides by gel permeation chromatography Preparation of standard solution: Accurately weigh 2 mg of each of 9 standards (dextran, see Table 4), dissolve in 1 mL of mobile phase solution to prepare a 2 mg / mL solution, and transfer the sample to a 1.8 mL injection vial.

[0068] Sample solution preparation: Accurately weigh 5 mg of the sample from Example 1 and dissolve it in 1 mL of mobile phase solution to prepare a 5 mg / mL solution. After vortexing to dissolve, centrifuge at 12,000 rpm for 10 min. Pipette the supernatant, filter it with a 0.22 μm water microporous filter membrane, and then transfer the sample to a 1.8 mL injection vial.

[0069] Mobile phase: 0.05M NaCl solution; chromatographic column: BRT105-103-101 series gel column (8× 300 mm); flow rate: 0.7ml / min; column temperature: 40 ºC; injection volume: 25 μl; detector: differential detector RID-20A; analysis time: 70min.

[0070] The standard was placed in the sample tray, and the above chromatographic method was selected for analysis to obtain the retention time and plot lgMw-RT (Mw weight average molecular weight), as shown in Table 3; the lgMw-RT calibration curve equation was calculated to be: y = -0.208x + 11.86, R² = 0.992, where x is the response time (min) and y is lgMw.

[0071] Table 4 Specific data of each standard in molecular weight Standard 1 Standard 2 Standard 3 Standard 4 Standard 5 Standard 6 Standard 7 Standard 8 Standard 9 M 1153 6941 9835 20600 47880 112200 240100 396400 883100 lm w 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 The chromatogram measured in Example 1 is shown in Figure 1. Substituting the retention time into the calibration curve equation can obtain the molecular weight (Mw). The weight average molecular weight of the Polygonatum cyrtonema stem and leaf polysaccharide is calculated to be 4287 Da.

[0072] 1.3 GC-MS method was used to analyze the glycosidic bond type of Polygonatum cyrtonema stem and leaf polysaccharides Weigh the polysaccharide sample (2-3 mg) and place it in a glass reaction bottle. Add 1 mL of anhydrous DMSO, quickly add methylation reagent A solution, seal, dissolve under ultrasound, and then add methylation reagent B solution. React in a magnetic stirring water bath at 30°C for 60 minutes. Finally, add 2 mL of ultrapure water to the above mixture to terminate the methylation reaction.

[0073] Take the methylated polysaccharide, add 1 ml of 2M trifluoroacetic acid (TFA) to hydrolyze for 90 min, and evaporate to dryness on a rotary evaporator. Add 2 mL of double distilled water to the residue, reduce it with 60 mg of sodium borohydride for 8 hours, add glacial acetic acid to neutralize, rotary evaporate, dry it in an oven at 101 degrees, then add 1 ml of acetic anhydride to acetylate at 100 °C for 1 hour, and cool it. Then add 3 mL of toluene, concentrate and evaporate to dryness under reduced pressure, and repeat 4-5 times to remove excess acetic anhydride.

[0074] The acetylated product was dissolved in 3 mL of CH2Cl2 and transferred to a separatory funnel. After adding a small amount of distilled water and shaking thoroughly, the upper aqueous solution was removed. This 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 samples were analyzed using a Thermo scientific, 1300-7000 gas chromatograph-mass spectrometer; GC-MS conditions: HP-INNOWAX column 30m*0.32mm*0.25um; programmed temperature conditions: starting temperature 140°C, heating to 230°C at 1°C / min; injection port temperature 250°C, detector temperature 250°C, carrier gas helium, flow rate 1mL / min.

[0075] The ion current diagram and secondary proton chromatogram of GC-MS are shown in Figures 2 and Figure 3 a. Figure 3 b. Figure 3 As shown in c, according to the monosaccharide composition test results, the polysaccharide of Polygonatum cyrtonema contains a large amount of fructose. During the derivatization process, the reduction of fructose 2-keto group leads to isomerization to form mannitol and glucitol. f / Glc f The glycoside residue fragments were integrated, and the final determination results of the glycosidic bond types of Polygonatum cyrtonema stem and leaf polysaccharides are shown in Table 5. The total ion current and secondary proton chromatogram are shown in Figure 2 As shown in Table 5, the polysaccharides extracted by this method are mainly composed of fructose-type glycosidic bonds, namely →1)-Fru f -(2→、→1,6)Fru f -(2→、→6)Fru f -(2→ and Fru f -(2→, and there is 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.

[0076] Table 5 Analysis of glycoside types of Polygonatum cyrtonema stem and leaf polysaccharides

[0077] Test Example 3 Study on the antidepressant effect of Polysaccharide from Polygonatum cyrtonema stems and leaves 1. Animal Experiment Design This experiment was reviewed by the Experimental Ethics Committee of the Experimental Animal Center of South China Agricultural University [No.: 2024B017], and the experimental protocols followed the principles of animal ethics and met the requirements of ethical standards.

[0078] Animal modeling and grouping: 40 male SPF-grade C57BL / 6J mice, 5 weeks old, weighing 18-22 g, were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. (certificate number: SCXK (Xiang) 2021-0002). After one week of adaptive feeding of mice, they were randomly divided into a healthy group (NC, n=8), a model group (CRS, n=8), a positive group (CRS+P, n=8), and low- and high-dose groups of Polygonatum cyrtonema stem and leaf polysaccharides (CRS+LD, CRS+HD, n=8).

[0079] The first 4 weeks were the modeling period. The mice in the NC group could take in water and food at any time. The mice in the other 4 groups were restrained in a customized centrifuge tube every day for 4 consecutive weeks, 7 hours a day, and fasted and deprived of water during the restraint period. After the modeling was successful, the mice were given gavage intervention at 9-11 am every morning for 4 consecutive weeks: 10 mL / kg normal saline was given to the NC group and the CRS group; 5.2 mg / kg. BW fluoxetine hydrochloride (Suzhou Sinochem Pharmaceutical Industry Co., Ltd.) was given to the CRS+P group; 50 mg / kg.BW PSPL1 was given to the CRS+LD group; 250 mg / kg.BW PSPL1 was given to the CRS+HD group; the preparation method was to dissolve the prepared Polygonatum cyrtonema stem and leaf polysaccharide freeze-dried powder in sterilized first-grade water according to the corresponding concentration, and sterilize it by membrane.

[0080] Two days before the experimental period, behavioral experiments were conducted on mice. Tail suspension test (TST): The middle part of the tail of the experimental mouse was fixed downwards on a fixed rod with equidistant iron hooks using tape. The mice were separated by a blackboard to avoid mutual interference. The activities of the mice were recorded within 5 minutes, and the cumulative immobility time of the mice in the last 4 minutes was recorded.

[0081] Forced swim test (FST): Each mouse was placed in a 2L beaker (14 cm in diameter, 20 cm in height) containing 1.5L of water (24 ± 1℃), so that the mouse's hind limbs could not touch the bottom of the cup to support the body. The mouse's swimming situation was observed within 5 minutes, and the cumulative immobility time of the mouse in the last 4 minutes was recorded. The mouse was considered immobile if it floated on the water without any struggle or only made some slight swings with its hind limbs to support its head floating on the water. After the experiment, each group of test mice was wiped with a dry towel and comforted, and the water was changed after each test.

[0082] After the experimental period, all mice were fasted and deprived of water for 12 h. The blood of the mice was collected by orbital bleeding. The mice were then anesthetized and euthanized. The mice were quickly dissected on ice, and the colons of the mice were taken out and wrapped in tin foil and stored in pathology bags. The brain tissue of each mouse was taken out, half of which 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.

[0083] HE staining of hippocampal slices: Mouse hemibrain samples were fixed in 4% paraformaldehyde solution for 24 h. After dehydration, wax immersion, embedding and slicing, the slices were placed in xylene for 20 min, 3 times. Then they were placed in 100%, 100%, 95%, 85% and 75% ethanol for dewaxing, and then rinsed in distilled water for 5 min. Then they were stained with hematoxylin reagent, rinsed with distilled water after staining, and PBS turned blue; then they were stained with eosin reagent and rinsed with distilled water; finally, they were dehydrated in gradient alcohol (95-100%), 5 min per level (or the slices were directly dried). After taking them out, they were placed in xylene, then sealed with neutral gum, and scanned with a panoramic scanner to observe the hippocampal area.

[0084] Determination of biochemical indicators of hippocampus: About 20 mg of hippocampus tissue was separated from brain tissue and washed with 1X PBS to remove blood stains. Cut into small pieces and put into a tissue grinder (homogenizer tube), add 0.3mL 1×PBS to make a homogenate, and then place it at -20℃ overnight. After repeated freeze-thaw treatment twice to destroy the cell membrane, the tissue homogenate was centrifuged at 2-8℃ 5000g for 5 minutes to take the supernatant. Take an appropriate amount of supernatant and conduct the experiment immediately. According to the instructions of the ELISA kit, the content of 5-HT (5-hydroxytryptamine), KYN (kynurenine), QUIN (quinolinic acid), and TRP (tryptophan) in the mouse hippocampus was determined.

[0085] Determination of biochemical indicators of colon tissue: Take about 30 mg of colon tissue and wash away blood stains with 1X PBS. Cut into small pieces and put into a tissue grinder (homogenizer tube), add 0.3 mL of 1×PBS to make a homogenate, and then place it at -20℃ overnight. After repeated freeze-thaw treatments to destroy the cell membrane, centrifuge the tissue homogenate at 2-8℃ and 5000g for 5 minutes to take the supernatant. Take an appropriate amount of supernatant and conduct the experiment immediately. According to the instructions of the ELISA kit, the content of KYN (kynurenine) and QUIN (quinolinic acid) in the mouse colon was determined.

[0086] Determination of colon / hippocampus-related protein expression: Protein extraction: Cut 0.025 g mouse hippocampus / colon tissue, wash the tissue with ice-cold PBS, add 270 μL RRIPA lysis buffer and grind the tissue repeatedly in a biological sample homogenizer until no tissue chunks are visible; after lysis on ice for 10 min, centrifuge at 4 °C and 12,000 rpm for 15 min (pre-cool the centrifuge in advance); transfer the supernatant after centrifugation into a 1.5 mL centrifuge tube.

[0087] Glue preparation: Prepare 10% separation gel, add TEMED and shake well immediately for glue filling, and use isopropyl alcohol to seal the glue after completion. Slightly tilt the glue dispenser. When the dividing line no longer changes, it means that the glue has solidified. Wait for another 3 minutes to ensure full solidification, then pour off the isopropyl alcohol on the top of the glue and dry it with filter paper. Next, prepare 4.8% concentrated gel, add TEMED and shake well immediately for glue filling. Insert the comb into the glass plate, fill the remaining space with concentrated gel, and wait for the gel to solidify.

[0088] 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 cool in an ice box at medium speed for later use.

[0089] Electrophoresis: According to the protein quantification results, 2 μL marker was added to the first well, and 10 μL of denatured protein was added to each of the remaining wells. Electrophoresis was started, and the constant voltage was set to 75 V and the electrophoresis time was set to 130 min. When the bromophenol blue electrophoresis reached the bottom of the gel, the electrophoresis was terminated.

[0090] Transfer: Cut the gel according to the molecular weight. For specific molecular weight, see the antibody information. Prepare 6 filter papers of the same size as the gel and 1 NC membrane. Place the NC membrane and filter paper in the transfer buffer until they are completely soaked. Place 3 filter papers, NC membrane, gel, and another 3 filter papers in order to ensure that there are no bubbles in the middle. Cover the instrument, turn on the power, and transfer the membrane at a constant current of 300 mA. For the transfer time of each indicator, see the antibody information. After the transfer is completed, take out the membrane and wash it once in 1×PBST.

[0091] Blocking: Prepare 5% skim milk powder with 1*PBST, immerse the membrane in it, and leave it at room temperature for 90 min.

[0092] Primary antibody incubation: dilute the primary antibody in a certain ratio (IDO1 1:1000, KMO 1:1000, TPH2 1:1000, GAPDH 1:5000) with 1*PBST, incubate the membrane with the primary antibody at 4°C overnight, and place at room temperature for 30 min the next day. After incubation, wash 3 times with 1*PBST.

[0093] Secondary antibody incubation: dilute HRP-labeled secondary antibody (1:5000) with 1*PBST, and incubate the membrane with the diluted secondary antibody at room temperature for 90 min. After incubation, wash 3 times with 1*PBST.

[0094] Color exposure: ECL (Enhanced Chemiluminescence) color exposure: Use ECL chemiluminescence solution to incubate the membrane for 1 min, absorb the liquid with filter paper, wrap the hybridization membrane with plastic film, and image with a gel imaging system.

[0095] Mouse intestinal flora determination: Next-generation full-length amplicon sequencing was used to determine the gene sequence of the intestinal flora of mouse cecal contents.

[0096] Extraction and PCR amplification of genomic DNA: The genomic DNA of the sample was extracted using the CTAB or SDS method, and then the purity and concentration of the DNA were detected by agarose gel electrophoresis. An appropriate amount of sample was placed in a centrifuge tube and diluted to 1 ng / μL with sterile water. The diluted genomic DNA was used as a template, and according to the selection of the sequencing region, specific primers with barcodes, Phusion® High-Fidelity PCRMaster Mix with GC Buffer from New England Biolabs, and high-efficiency and high-fidelity enzymes were used for PCR to ensure amplification efficiency and accuracy.

[0097] Mixing and purification of PCR products: PCR products were detected by electrophoresis using 2% agarose gel. Equal amounts of samples were mixed according to the concentration of the PCR products. After sufficient mixing, PCR products were detected by 2% agarose gel electrophoresis. The target bands were recovered using the gel recovery kit provided by Qiagen.

[0098] 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 fragments were used to screen 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 using Agilent 2100, and then sequenced using the PacBio platform.

[0099] 2. Experimental results (1) Results of depression behavior test The experimental results of the mouse forced swimming test and tail suspension test are shown in Figures 4 and 5. In the forced swimming test and tail suspension test, after 4 weeks of restraint, the despair time of mice in the other groups was significantly increased compared with the NC group ( P <0.001), there was no significant difference between the groups ( P >0.001), indicating that restraint can significantly increase the passive time of mice when facing a forced stress environment and reduce the ability of mice to cope with adversity. At this time, the CRS mouse model was established.

[0100] After 4 weeks of combined restraint and gavage intervention, i.e., the eighth week, compared with the CRS group, the despair time of mice in the forced swimming test and tail suspension test in each drug intervention group decreased to varying degrees, and was significantly lower than that in the CRS group ( P <0.001). In the forced swimming test, the CRS+HD group had the best effect.

[0101] (2) Results of HE-stained hippocampal sections HE staining of the hippocampus can directly show the integrity of neurons and structures, can infer whether the hippocampal function is normal, and can indirectly reflect the antidepressant effect of Polygonatum sibiricum stem and leaf polysaccharide. The hippocampal tissue sections of each group of mice are shown in Figure 6. The hippocampal neurons of the NC group mice are arranged tightly and orderly with normal cell morphology, while the hippocampal neurons of the CRS group mice are arranged in a disordered manner with larger gaps and severe neuronal damage. Compared with the CRS group, the state of the hippocampal neurons of the mice in each intervention group has improved and is arranged more compactly, indicating that Polygonatum sibiricum stem and leaf polysaccharide can improve the neuronal cell damage induced by CRS to a certain extent, and the effect is similar to that of fluoxetine hydrochloride.

[0102] (3) Results of determination of antidepressant-related indicators in the hippocampus and colon 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 related to antidepressant mechanisms. In the hippocampus, these indicators directly affect the level of 5-HT and the synthesis of related neurotransmitters in the central nervous system by regulating the conversion balance of TRP to 5-HT or KYN pathways, thereby regulating emotions and neuroplasticity. In addition, the increase of neurotoxic metabolites such as QUIN and the upregulation of IDO1 and KMO in the hippocampus promote neuroinflammatory responses and further aggravate depressive-like behaviors. In the colon, the activation of the KYN pathway and the increased expression of IDO1 and KMO indirectly affect the central 5-HT level and systemic inflammatory state 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 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 the intervention, all indicators of the dose groups were improved to varying degrees, indicating that oral administration of Polygonatum sibiricum stem and leaf polysaccharide can improve the depression of mice caused by chronic stress restraint, and the effect is comparable to that of the common drug fluoxetine hydrochloride.

[0103] (4) Intestinal flora analysis In the analysis of intestinal flora, beta diversity is used to evaluate the differences or similarities of 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.

[0104] In order to further study how Polygonatum sibiricum stem and leaf polysaccharides can improve depression in mice caused by chronic restraint stress, the relative abundance of the top 10 phylum level and the top 20 genus level of 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 in the CRS group at the phylum level, 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. Akkermansia , Lactobacillus , Alistipes , Ileibacterium Compared with the NC group, the relative abundance of the above bacteria in the CRS+HD group increased and decreased to a certain extent, but after intervention with Polygonatum sibiricum stem and leaf polysaccharide, we can see that the abundance of the above bacteria in the CRS+HD group can be restored to a level close to that of the NC group. These results show that Polygonatum sibiricum stem and leaf polysaccharide can reverse the intestinal flora disorder of mice caused by the depression model and restore the unbalanced intestinal flora of depressed mice to a level close to that of normal mice.

[0105] Test Example 4 Study on the efficacy of Polygonatum cyrtonema stem and leaf polysaccharide in improving sleep 1. Animal Experiment Design The chronic restraint stress (CRS) model causes animals to develop depressive-like behaviors and emotional disorders through continuous stress treatment, accompanied by obvious abnormalities in sleep structure and quality, thus providing the possibility of simultaneously studying anti-depressant and sleep-improving interventions in the same model.

[0106] Therefore, the corresponding indicators were measured on the mouse tissues except the CRS+P group in Experimental Example 3.

[0107] Determination of serum biochemical indexes: After the collected blood was naturally settled for 1 hour, it was placed at 4 ° C for 3 hours to precipitate the serum, and then centrifuged at 4 ° C 4000 rpm for 15 minutes to obtain serum samples. According to the instructions of the ELISA kit, the content of 5-HT (5-hydroxytryptamine) and TRP (tryptophan) in mouse serum was determined. The mouse colon tissue was treated in the same way as in Experimental Example 3, and the content of 5-HT (5-hydroxytryptamine) and TRP (tryptophan) in the mouse colon was determined according to the method in the instructions of the ELISA kit. The same protein expression determination method was used to determine the expression of TPH1 protein in the mouse colon, in which the dilution ratio of TPH1 was 1:1000 in the primary antibody incubation step.

[0108] 2. Experimental results Changes in indicators such as 5-HT, TRP in serum and colon, and TPH1 (tryptophan hydroxylase 1) unique to the colon are closely related to improving sleep. In serum, 5-HT and TRP regulate circadian rhythms by affecting the synthesis of melatonin, which in turn has an important impact on sleep quality. In the colon, TPH1 plays a key role in peripheral 5-HT synthesis, regulating the level of 5-HT in the intestine, thereby affecting the central 5-HT system through the gut-brain axis, indirectly promoting sleep regulation and improving sleep quality. As shown in Figure 11, the indicators in the serum and colon of mice after CRS induction were significantly lower than those in the NC group, but after intervention with Polygonatum sibiricum stem and leaf polysaccharides, the relevant indicators of each dose group improved to a certain extent, especially the CRS+HD group. The results show that oral administration of Polygonatum sibiricum stem and leaf polysaccharides can effectively improve the abnormal sleep quality of mice caused by CRS.

[0109] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A polysaccharide from stems and leaves of polygonatum, characterized in that The weight average molecular weight of the polygonatum stem and leaf polysaccharide is 4000-5000 Da, and the polygonatum stem and leaf polysaccharide includes the following monosaccharide components: arabinose, galactose, glucose, xylose, mannose, and fructose, among which fructose accounts for the highest percentage.

2. The polysaccharide from stems and leaves of polygonatum according to claim 1, characterized in that The glycosidic bonds of Polygonatum cyrtonema stem and leaf polysaccharides include: →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→.

3. The polygonatum sibiricum stem and leaf polysaccharide according to claim 1, characterized in that The polysaccharide of Polygonatum sibiricum stem and leaf includes the following monosaccharide components in weight percentage: arabinose 4.2%, galactose 1.1%, glucose 2.3%, xylose 1.7%, mannose 1.8%, and fructose 88.9%.

4. The method for preparing the polysaccharide from the stems and leaves of polygonatum according to any one of claims 1 to 3, comprising the following steps: (1) extracting the stems and leaves of Polygonatum cyrtonema with water to obtain a concentrated solution; (2) extracting the concentrated solution with alcohol to obtain a crude extract; (3) removing protein from the crude extract to obtain crude polysaccharide from stems and leaves of polygonatum; (4) A crude polysaccharide solution was prepared using the crude polysaccharide from the stem and leaves of Polygonatum cyrtonema and water. The crude polysaccharide solution was flowed into an anion exchange column and gradient eluted with 0 mol / L and 0.1 mol / L sodium chloride, respectively. The polysaccharide components eluted with 0.1 mol / L sodium chloride were collected, dialyzed, and freeze-dried to obtain a purified polysaccharide component from the stem and leaves of Polygonatum cyrtonema.

5. The preparation method according to claim 4, characterized in that: In step (1), the water extraction specifically comprises: drying and crushing the stems and leaves of Polygonatum cyrtonema, adding water for water bath extraction, cooling to room temperature, filtering the supernatant, and concentrating the obtained filtrate to obtain a concentrated solution; Preferably, in step (1), the water bath extraction time is 3 to 5 h; the water bath extraction temperature is 60 to 90°C; during the water bath extraction, the solid-liquid ratio is 1:10 to 1:40 g / mL; More preferably, in step (1), the drying temperature is 50-60°C.

6. The preparation method according to claim 4, characterized in that: In step (2), the alcohol extraction specifically comprises: adding anhydrous ethanol to the above-mentioned concentrated solution, allowing the solution to stand for precipitation, centrifuging, collecting the precipitate, and then adding distilled water to the obtained precipitate to remove insoluble impurities to obtain a crude extract; Preferably, in step (2), the volume ratio of anhydrous ethanol to the concentrated solution is 3-5:1; More preferably, in step (2), the static sedimentation time is 10 to 15 hours.

7. The preparation method according to claim 4, characterized in that: In step (3), the protein removal specifically includes: adding Sevage reagent to the above crude extract, centrifuging and then standing, discarding the lower organic layer and the middle protein layer, repeating the operation until the protein layer is completely removed, distilling the obtained supernatant at low temperature and reduced pressure, dialyzing, and freeze-drying the obtained dialyzate to obtain crude polysaccharides from polygonatum stems and leaves.

8. The preparation method according to claim 7, characterized in that: In step (3), the volume ratio of Sevage reagent to crude extract is 3-5:1; wherein the Sevage reagent is obtained by mixing chloroform and n-butanol in a volume ratio of 4:1; In step (3), the temperature of low temperature and reduced pressure distillation is 50-60°C; In step (3), the dialysis is performed in a 1000 Da dialysis bag.

9. The preparation method according to claim 1, characterized in that: In step (4), the concentration of the crude polysaccharide solution is 10 mg / mL; In step (4), the flow rate of the crude polysaccharide solution into the anion exchange column is 1 mL / min.

10. Use of the polygonatum stem and leaf polysaccharide according to any one of claims 1 to 3 or the polygonatum stem and leaf polysaccharide prepared by the preparation method according to any one of claims 4 to 9 in the preparation of health products and medicines for improving sleep, or in the preparation of antidepressant drugs.

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