Huangqin oligosaccharide, preparation method thereof and use of the oligosaccharide in preparation of a medicine for treating depression

Abstract

The application belongs to the technical field of plant polysaccharides, and specifically discloses a polygonatum sibiricum oligosaccharide, a preparation method thereof and application of the polygonatum sibiricum oligosaccharide in preparation of a medicine for treating depression. The polygonatum sibiricum oligosaccharide has a structure as shown in formula (I), is a novel structural acid-uniform oligosaccharide isolated from natural traditional Chinese medicinal materials polygonatum sibiricum for the first time, can effectively improve the depression-like behavior and sleep disorder of CUMS mice, and can reverse the depression-related biochemical index changes of the CUMS mice. The anti-depression effect of the polygonatum sibiricum oligosaccharide is equivalent to that of the existing classic medicine fluoxetine, but the side effect is obviously lower, and the polygonatum sibiricum oligosaccharide is expected to be developed into a new anti-depression drug.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant polysaccharides, and specifically discloses a polygonatum sibiricum oligosaccharide, a preparation method thereof and an application of the polygonatum sibiricum oligosaccharide in preparation of a medicine for treating depression. BACKGROUND

[0002] Polygonatum sibiricum is a perennial herb of the genus Polygonatum in the Liliaceae family, and its dried rhizome is a traditional Chinese medicinal material and has a long history of medicinal use. According to traditional Chinese medicine, polygonatum sibiricum is neutral in nature, sweet in taste, and belongs to the spleen, lung and kidney channels, and is one of the representatives of 'tonifying deficiency' medicinal materials. Modern studies have shown that polygonatum sibiricum contains saponins, oligosaccharides, polysaccharides and other substances, and it is believed that polygonatum sibiricum oligosaccharides and polysaccharides are the main medicinal substances.

[0003] Currently, research on Polygonatum sibiricum mainly focuses on the chemical and pharmacological activities of its polysaccharides. Oligosaccharides are natural compounds composed of fewer than ten monosaccharides linked by glycosidic bonds. They participate in numerous biological processes in living cells and possess diverse biological activities, such as hypoglycemic and lipid-lowering, anti-tumor, anti-inflammatory, antioxidant, antiviral, and immunomodulatory properties. They have become a focus of modern medicine and functional food chemistry. However, there are few reports on the oligosaccharides in Polygonatum sibiricum, particularly regarding their composition, structure, and pharmacological activities. Liu et al. isolated four novel cyclic oligosaccharides (POS-A to POS-D) from the rhizome of Polygonatum sibiricum. Using 2D NMR and X-ray crystallography, they revealed that POS-C possesses a unique [→4)-α-D-Glcp-(1→6)-β-D-Manp-(1→]6 cyclic structure. Notably, POS-D is acetylated at the C-3 position, a rare modification that enhances its inhibitory activity against α-glucosidase by 3.2-fold (IC50 = 58 μM) (Liu Y, Tanaka K, Zhang J, et al. Cyclic oligosaccharides from Polygonatum sibiricum: Structural elucidation and bioactivity evaluation. Carbohydrate Polymers. 2020;231:115735.). Zhang et al. reported five novel ferulic acid-oligosaccharide conjugates (FOS-1 to FOS-5), of which FOS-3 esterifies ferulic acid to a tetrasaccharide core. Anti-inflammatory experiments showed that FOS-3 inhibited COX-2 expression in a dose-dependent manner (79.2% inhibition at 1 μM) and selectively regulated the abundance of intestinal Bifidobacterium (+38.7%), suggesting its dual anti-inflammatory and prebiotic effects (Zhang HH, Wang Z, Li MX, et al. Novel feruloylated oligosaccharides from Polygonatum sibiricum with dual anti-inflammatory and prebiotic effects. Food Chemistry). 2019;300:125165.). Zhang Ming et al. systematically compared the regulatory effects of different polarity extracts of Polygonatum sibiricum on the cyclophosphamide-induced immunosuppression model and found that the water extract (POW), 50% ethanol extract (PET), and supercritical CO2 extract (SFE) could significantly increase serum IgG levels (p < 0.01). Among them, PET at a dose of 200 mg / kg restored the spleen index to 92.3% of the normal group.Mechanism studies have shown that all three can down-regulate the expression of key proteins in the TLR4 / MyD88 / NF-κB pathway, and PET can specifically activate the Nrf2 antioxidant pathway (Zhang M, Li ZH, Wang X, Chen L. Comparative study on the immunomodulatory mechanisms of polygonatum multiflorum polysaccharide extracts. Chinese Journal of Chinese Medicine. 2023; 48(9): 2356-2363.). It is worth noting that POW showed abnormal increase in liver coefficient at high dose (400 mg / kg), suggesting that the safety of the extraction process needs to be concerned and controlled. Chen X et al. isolated three oligosaccharide components (POS-I, POS-II, POS-III) from polygonatum multiflorum by fractional alcohol precipitation method. HPAEC-PAD analysis found that there were significant differences in the monosaccharide composition of the three oligosaccharide components: POS-I was mainly composed of glucose (82.3%), POS-II contained characteristic galacturonic acid (17.8%), and POS-III showed a high mannose ratio (34.5%). p <0.05), and POS-III could reduce the secretion of LPS-induced THP-1 cell IL-1β by 61.4% by inhibiting the activation of NLRP3 inflammasome (Chen X, Wu J, Zhou M, et al. Analysis of the characteristics and activities of polygonatum multiflorum oligosaccharide components based on fractional alcohol precipitation method. Food Science. 2022; 43(17): 87-94.). The above studies confirmed that the biological function of polygonatum multiflorum oligosaccharide is closely related to its structural characteristics such as specific sugar unit connection order, branching degree and modification group, and the extraction method directly affects the structure-activity characteristics of oligosaccharide components.

[0004] However, polygonatum multiflorum oligosaccharide isomers are rich and the structure is very complex. At present, there are few polygonatum multiflorum oligosaccharides with known structure and function. It is of great significance to discover more new structures of polygonatum multiflorum oligosaccharides and explore their new biological functions for fully elucidating the structure-activity relationship of polygonatum multiflorum oligosaccharides, clarifying the material basis of their biological functions, and deeply mining the medicinal value of polygonatum multiflorum oligosaccharides. SUMMARY

[0005] In view of the shortcomings of the prior art, the present application provides a polygonatum multiflorum oligosaccharide and a preparation method thereof and a use in preparing a medicine for treating depression. The polygonatum multiflorum oligosaccharide PCO-A1 is isolated from dried polygonatum multiflorum, and a multi-dimensional characterization system including ultraviolet spectrum, infrared spectrum, HPGPC, Congo red experiment and ion chromatography is used to ensure the purity and structural uniformity of the product and successfully analyze its primary structure. The significant anti-depression efficacy and mechanism research of the polygonatum multiflorum oligosaccharide lay a solid foundation for the development of new anti-depression drugs.

[0006] In a first aspect, the present application provides a polygonatum multiflorum oligosaccharide having a structure as shown in formula (I):

[0007]

[0008] Formula (I).

[0009] In some embodiments, the weight average molecular weight of the oligosaccharide is 1000-1200 kDa.

[0010] In a second aspect, the present application provides a preparation method of the oligosaccharide, comprising: extracting the polygonatum sibiricum with hot water, concentrating the extracted solution, and then sequentially performing defatting, deproteinization, re-concentration, and alcohol precipitation; resuspending the precipitate obtained by alcohol precipitation in water, and then performing cellulose column chromatography separation, sequentially eluting with water and 0.1-0.5 mol / L NaCl solution, collecting the eluate obtained by water elution, and then concentrating and freeze-drying to obtain a first purified product; resuspending the first purified product in water, and then adding ethanol to make the precipitate complete, performing solid-liquid separation, and collecting the liquid, and then concentrating and freeze-drying to obtain a second purified product; resuspending the second purified product in water, and then performing gel column chromatography purification, eluting with water, collecting the eluate, and then concentrating and freeze-drying to obtain the oligosaccharide.

[0011] In some embodiments, the polygonatum sibiricum is dried before hot water extraction.

[0012] In some embodiments, the extraction conditions include that the temperature of the hot water is 90-100°C, the ratio of the polygonatum sibiricum to the hot water is 1 g:3-5 mL, the extraction time is 45-60 min, and the extraction times is 1-3 times.

[0013] In some embodiments, the concentration is performed by rotary evaporation each time, and the volume is reduced by 3 / 5-4 / 5 each time.

[0014] In some embodiments, petroleum ether is used for defatting, and the water phase is collected; the sevage reagent (chloroform and n-butanol in a volume ratio of 4-6:1) is mixed with the extraction liquid in a volume ratio of 1:3-5, and after complete mixing, the liquid is allowed to stand for 0.5-1.5 hours for separation, and the protein is removed. The combination of petroleum ether defatting and the sevage method significantly improves the deproteinization efficiency.

[0015] In some embodiments, the alcohol precipitation conditions include that anhydrous ethanol is added to a concentration of 70%-90% v / v, and the precipitate is allowed to stand at 3-5°C for 20-30 hours.

[0016] In some embodiments, the cellulose column chromatography separation conditions include that the cellulose column filler is DEAE-52, the loading concentration is 20-25 mg / mL, and the elution flow rate is 1-3 mL / min. The gradient elution program can realize efficient separation of oligosaccharide components.

[0017] In some embodiments, the conditions for gel column chromatography purification include: the gel column filler is Sephadex G-25, the loading concentration is 8-12 mg / mL, and the elution flow rate is 0.1-0.3 mL / min.

[0018] In a third aspect, the present invention provides a pharmaceutical composition comprising the aforementioned Polygonatum sibiricum oligosaccharide and a pharmaceutically acceptable excipient or carrier.

[0019] In a fourth aspect, the present invention provides the use of the aforementioned Polygonatum oligosaccharide or pharmaceutical composition in the preparation of a drug for treating depression.

[0020] In some embodiments, the aforementioned drug is a drug that improves depressive-like behavior, improves sleep disorders, or improves changes in depression-related biochemical indicators.

[0021] The beneficial effects of the present invention are:

[0022] The Polygonatum sibiricum oligosaccharide provided by the present invention is a novel structurally acidic uniform oligosaccharide isolated from the natural medicinal material Polygonatum sibiricum. It can effectively improve the depressive-like behavior and sleep disorders of CUMS mice, and can reverse the changes in depression-related biochemical indicators of CUMS mice. The antidepressant efficacy of the Polygonatum sibiricum oligosaccharide is comparable to that of the existing classic drug fluoxetine but with significantly lower side effects, and is expected to be developed into a new antidepressant drug.

[0023] The preparation method provided by the present invention extracts crude polygonatum oligosaccharides through water extraction and alcohol precipitation, and realizes efficient separation and purification of oligosaccharide components through three-stage purification of cellulose column chromatography, ethanol recrystallization and gel column chromatography. In particular, an ethanol recrystallization step is specially added between the cellulose column chromatography separation and the gel column chromatography purification, thereby obtaining uniform polygonatum oligosaccharides. The preparation method has high yield, is simple and easy to operate, operates under mild conditions, and is suitable for large-scale production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a flow chart of the preparation process of Polygonatum oligosaccharide PCO-A1 in Example 1 of the present invention;

[0025] Figure 2A This is the HPGPC diagram of PCO-A in Example 1 of the present invention;

[0026] Figure 2B This is the HPGPC diagram of PCO-A0 in Example 1 of the present invention;

[0027] Figure 3 This is the HPGPC diagram of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention;

[0028] Figure 4A This is the HPGPC diagram of the sample prepared in Comparative Example 1 of the present invention;

[0029] Figure 4B HPGPC chart of the sample prepared for Inventive Example 2;

[0030] Figure 5A UV spectrum chart of the polygonati oligosaccharide PCO-A1 prepared for Inventive Example 1;

[0031] Figure 5B IR spectrum chart of the polygonati oligosaccharide PCO-A1 prepared for Inventive Example 1;

[0032] Figure 6 Congo red chart of the polygonati oligosaccharide PCO-A1 prepared for Inventive Example 1;

[0033] Figure 7 SEM chart of the polygonati oligosaccharide PCO-A1 prepared for Inventive Example 1;

[0034] Figure 8A Particle size chart of the polygonati oligosaccharide PCO-A1 prepared for Inventive Example 1, wherein the horizontal coordinate component refers to three parallel determinations of PCO-A1;

[0035] Figure 8B Potential chart of the polygonati oligosaccharide PCO-A1 prepared for Inventive Example 1, wherein the horizontal coordinate component refers to five different concentrations of the PCO-A1 solution: A is 0.5 mg / ml; B is 0.05 mg / ml; C is 0.005 mg / ml; D is 0.0005 mg / ml; and E is 0.00005 mg / ml;

[0036] Figure 9 pH chart of the polygonati oligosaccharide PCO-A1 prepared for Inventive Example 1, wherein the horizontal coordinate component refers to three parallel determinations of PCO-A1;

[0037] Figure 10 Ion chromatography analysis results of the polygonati oligosaccharide PCO-A1 prepared for Inventive Example 1; black: Fuc, t R = 6.034 min; GalN t R = 11.975 min; Rha, t R = 12.609 min; Ara t R = 13.784 min; GlcN t R = 15.334 min; Gal, t R = 17.859 min; Glc t R = 20.392 min; GlcNAc t R = 22.109 min; Xyl tR =24.067min;Man t R =25.234 min;Fru t R =28.125 min;Rib t R =29.109 min;GalA t R =43.717 min;GulA t R =44.417 min;GlcA t R =46.409 min;ManA t R =48.934 min; red: Glc t R =20.392min; Fru t R =28.125 min;

[0038] Figure 11A This is the hydrogen spectrum of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention;

[0039] Figure 11B This is the carbon spectrum of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention;

[0040] Figure 11C This is the COSY graph of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention;

[0041] Figure 11D This is the HSQC chart of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention;

[0042] Figure 11E This is the HMBC chart of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention;

[0043] Figure 12A is the inferred structural formula of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention;

[0044] Figure 12B This is the inferred structure diagram of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention;

[0045] Figure 13A The results of the immobility time test of the CUMS mice in each group are shown in Figure 2. * P <0.05 vs.Control (i.e. * indicates that compared with the Control group, P <0.05), # P <0.05 vs. CUMS (# indicates that compared with the CUMS group, P <0.05);

[0046] Figure 13B Results of the immobility time test of the forced swimming test for CUMS mice in each group. * P <0.05 vs.Control, # P <0.05 vs. CUMS;

[0047] Figure 13C is the percentage of sugar water preference test before treatment in each group of CUMS mice;

[0048] Figure 13D is the percentage of sugar water preference after treatment in each group of CUMS mice, * P <0.05 vs.Control, # P <0.05 vs. CUMS;

[0049] Figure 14A Comparison of REM latency in each group of CUMS mice. * P <0.05 vs.Control, # P <0.05 vs. CUMS;

[0050] Figure 14B Comparison of awake times of CUMS mice in each group. * P <0.05 vs.Control, # P <0.05 vs. CUMS;

[0051] Figure 14C Comparison of sleep duration of each group of CUMS mice. * P <0.05 vs.Control, # P <0.05vs.CUMS;

[0052] Figure 14D Comparison of NREM duration in each group of CUMS mice. * P <0.05 vs.Control, # P <0.05vs.CUMS;

[0053] Figure 14E Comparison of sleep duration percentages of CUMS mice in each group. * P <0.05 vs.Control, #P <0.05vs.CUMS;

[0054] Figure 14F Comparison of the percentage of NREM duration in each group of CUMS mice. * P <0.05 vs.Control, # P <0.05vs.CUMS;

[0055] Figure 15A Serum CORT levels of CUMS mice in each group were detected by ELISA. * P <0.05 vs.Control, # P <0.05vs.CUMS;

[0056] Figure 15B The serum 5-HT levels of CUMS mice in each group were detected by ELISA. * P <0.05 vs.Control, # P <0.05vs.CUMS;

[0057] Figure 15C The serum IL-1β levels of CUMS mice in each group were detected by ELISA. * P <0.05 vs.Control, # P <0.05vs.CUMS;

[0058] Figure 15D Serum TNF-α levels in each group of CUMS mice were detected by ELISA. * P <0.05 vs.Control, # P <0.05vs.CUMS. DETAILED DESCRIPTION

[0059] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0060] Example 1 Polygonatum oligosaccharide PCO-A1, preparation process as follows Figure 1As shown, comprising the following steps:

[0061] (1) Extraction of Polygonatum sibiricum crude oligosaccharides: 100 g of Polygonatum sibiricum was precisely weighed according to the ratio of 1 g:4 mL, and was added to 400 mL of distilled water in a pot and boiled for 1 hour. The decoction was collected, and the operation was repeated twice. The last time was boiled for 45 min, and the extraction liquid was collected. The three extraction liquids were combined and concentrated to 50 mL by rotary evaporation for standby. The concentrated extraction liquid was added to petroleum ether for extraction to remove fat, and the water phase was collected. After repeating the operation 3 times, the extraction liquid was collected. The sevage reagent (chloroform and n-butanol with a volume ratio of 5:1) was mixed with the extraction liquid at a volume ratio of 1:4, and after complete mixing, it was placed for 1 hour for liquid separation to remove protein. This step was repeated 3 times. The treated extraction liquid was rotary evaporated at 70°C, and then the concentrated extraction liquid and anhydrous ethanol were mixed at a volume ratio of 1:4 to make the ethanol concentration of the system 80% v / v. After alcohol precipitation at 4°C for 24 hours, centrifugation was performed at a speed of 4750 rpm for 15 min, and the precipitate was filtered. The precipitate was completely dissolved in pure water and then placed in a freeze dryer to obtain Polygonatum sibiricum crude oligosaccharide freeze-dried powder PCO.

[0062] (2) Cellulose column chromatography separation: Weigh 230 g of cellulose DEAE-52 and soak it evenly in pure water. Let it stand for 2 days to ensure that the cellulose fully absorbs water and expands, which is convenient for subsequent column loading. Load the soaked cellulose into the chromatography column, ensuring that the column is packed tightly and evenly to avoid the generation of bubbles. Accurately weigh 405 mg of the lyophilized powder of Polygonatum sibiricum oligosaccharide PCO obtained in step (1), dissolve it in 18 ml of pure water, and stir it thoroughly until it is completely dissolved to obtain a Polygonatum sibiricum oligosaccharide solution. Prepare 700 ml of 0.1 M, 0.2 M, 0.3 M, and 0.5 M sodium chloride solutions as eluents. Slowly add the prepared Polygonatum sibiricum oligosaccharide solution to the top of the packed cellulose column (3.5×30 cm), ensuring that the solution evenly penetrates the column. Connect a constant flow pump, set the flow rate to 1.8 mL / min, and start the elution process. Use water, 0.1 M, 0.2 M, and 0.3 M sodium chloride solutions in sequence for elution. Fractions eluted from each sodium chloride solution were collected and labeled with the corresponding salt concentration. The resulting polysaccharide extract was concentrated and lyophilized to yield four fractions: PCO-A (eluted with water), PCO-B (eluted with 0.1 mol / L NaCl solution), PCO-C (eluted with 0.2 mol / L NaCl solution), and PCO-D (eluted with 0.3 mol / L NaCl solution). A 6% phenol solution was prepared by adding 3 g of phenol to 47 ml of pure water. The elution solution, phenol solution, and concentrated sulfuric acid were then added sequentially in a phenol-sulfuric acid ratio of 1:1:5. Color development was performed using the phenol-sulfuric acid method. After color development, the separation efficiency of the different concentrations was evaluated using ultraviolet light. Experimental results showed that the pure water group had the highest polysaccharide content and was a pure white flocculent substance, while the polysaccharides from the other groups were yellow. A total of 132.4 mg of lyophilized sample was collected, with a calculated yield of 30.21%.

[0063] (3) Ethanol recrystallization: Accurately weigh 60 mg of PCO-A and place it in a clean glass beaker. Use a pipette to add 2 mL of pure water, place the beaker on a magnetic stirrer, and stir until completely dissolved. While stirring continuously, slowly add 10 mL of ethanol to the solution. Observe the precipitation (precipitation) in the solution and continue stirring until the precipitation is complete. Transfer the solution containing the precipitate to a centrifuge tube. Use a centrifuge to centrifuge at a speed of 5000 r / min for 10 minutes. After centrifugation, carefully pour out the supernatant, concentrate, and freeze-dry to obtain PCO-A0. The HPGPC diagrams of PCO-A and PCO-A0 are shown below. Figure 2A 、 2B As shown, by comparison, it can be seen that ethanol recrystallization can effectively remove polygonatum polysaccharides and greatly enrich the proportion of polygonatum oligosaccharides.

[0064] (4) Gel column chromatography purification: Weigh 50 mg of PCO-A0 and dissolve it in 5 mL of pure water to ensure that it is fully dissolved. Weigh 20 g of dextran gel G-25 (particle size 20-80 μm) and add enough pure water to soak it overnight to remove suspended impurities and bubbles. The soaked dextran is filtered to remove bubbles. Slowly pour the treated dextran gel G-25 into a chromatography column (16 × 1000 mm) fixed on an iron stand, taking care to avoid generating bubbles. Use pure water as the eluent and equilibrate the chromatography column at a flow rate of 0.2 mL / min until the effluent is stable. Slowly load the prepared PCO-A0 solution into the equilibrated chromatography column. Use pure water as the eluent and elute at a flow rate of 0.2 mL / min. Collect the effluent, collecting 2 mL per tube until enough eluent is collected. Draw an elution curve using the phenol-sulfuric acid method to determine the elution peak of the polysaccharide. The eluates corresponding to the elution peaks were combined, concentrated, and freeze-dried to obtain uniform Polygonatum oligosaccharide PCO-A1. Figure 3 shown.

[0065] Comparative Example 1

[0066] The difference between this comparative example and Example 1 is that step (3) is removed. The experimental results are as follows: Figure 4A As shown, the sample obtained is mainly a mixture of polygonatum polysaccharide and polygonatum oligosaccharide. After recrystallization with ethanol, the solubility of polygonatum polysaccharide in the solution with a higher proportion of ethanol is greatly reduced, resulting in precipitation. As a result, the mother liquor contains polygonatum oligosaccharide components, which removes the obstacles for further purification of uniform oligosaccharides.

[0067] Comparative Example 2

[0068] The only difference between this comparative example and Example 1 is that step (4) uses dextran gel G-200 (particle size 40-120 μm). The experimental results show that: by the phenol-sulfuric acid method, color begins to appear in the 40th tube and disappears until the 84th tube. 46 tubes of samples were collected. The tailing is a bit serious, such as Figure 4B This indicates that after replacing the column with G-200 dextran gel, the column efficiency is reduced and uniform Polygonatum oligosaccharide cannot be obtained.

[0069] Experimental Example 1 Analysis of the physicochemical properties of Polygonatum oligosaccharide PCO-A1

[0070] (1) UV absorption of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1 was measured by UV scanning.

[0071] like Figure 5A As shown, the obtained Polygonatum sibiricum oligosaccharide has no absorption peaks of pigments, proteins and nucleic acids in the UV scanning spectrum. The instrument used is a SHIMADZU UV-2550 UV-visible spectrophotometer with a scanning range of 200~800 nm.

[0072] (2) Analyzing the characteristic groups of Polygonatum oligosaccharide obtained in Example 1 using infrared spectrometer

[0073] Weigh about 2.0 mg of dried oligosaccharide sample, mix with 200 mg of KBr powder, press into pellets, and then scan and analyze on L1600400Spectrum TWO FT-IR spectrometer with a scanning range of 4000 cm -1 ~400cm -1 .

[0074] As shown in Figure 5 B, the 3400 cm -1 The strong absorption peak around 1200~1000 cm indicates that the sample contains OH functional groups. -1 The absorption in the region is COC, CC and C-OH stretching vibration. Among them, 1000~1150 cm -1 The three signals in between are assigned to the stretching vibration of the pyranose ring.

[0075] (3) The homogeneity of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1 was determined by HPGPC method

[0076] Test conditions: Waters 2695 system; pure water as eluent, column temperature 35°C; detection wavelength: 245 nm; injection volume 10 μL; flow rate 1.0 mL / min.

[0077] like Figure 3 As shown, after testing, the polygonatum oligosaccharide PCO-A1 obtained in Example 1 is a uniform oligosaccharide.

[0078] (4) UV scanning was used to determine the triple helical structure of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1

[0079] Prepare a 1 mg / mL solution of Polygonatum oligosaccharide and mix it with a 100 μM Congo red solution at a 1:1 (v:v) ratio. Then, add NaOH solutions at concentrations of 0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, and 0.5 mol / L, respectively, and scan the reaction solution under UV light at a wavelength of 400–600 nm.

[0080] The triple helical structure of PCO-A1 was characterized by the Congo red experiment. Under alkaline conditions, if the polysaccharide has a triple helical structure, it can form a complex with the Congo red reagent, and the maximum absorption wavelength of the complex will be red-shifted compared to the Congo red solution. As the alkaline concentration increases, the maximum absorption wavelength of the polysaccharide-Congo red complex decreases as the triple helical structure is destroyed. Figure 6As shown, the maximum absorption wavelength of the complex is 504 nm, which shows a slight red shift compared with 501 nm of Congo red solution, indicating that PCO-A1 does not have a triple helical structure.

[0081] (5) Scanning electron microscopy was used to measure the surface ultrastructure of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1

[0082] 2-3 mg of freeze-dried PCO-A1 powder was adhered to the conductive adhesive on the sample stage, placed in a vacuum coater and sprayed with gold. The sample was then observed and imaged using a JSM.7001F thermal field emission scanning electron microscope.

[0083] like Figure 7 As shown in the results, the scanning electron microscope of Polygonatum oligosaccharide at 2 kV and 5 kV mostly showed spherical shape with smooth surface, and PCO-A1 mainly showed microspheres with a particle size of 20-30 μm in the solution, suggesting that PCO-A1 may be taken up by endocytosis and then participate in related metabolic processes in the body.

[0084] (6) The particle size distribution of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1 was measured using a nanoparticle analyzer. Figure 8A As shown, the potential analysis is as follows Figure 8B shown.

[0085] (7) Use Starter3100 pH meter to measure the pH of PCO-A1, as shown in Figure 9 shown.

[0086] (8) The molecular weight of PCO-A1 was determined by ion chromatography, and the molecular weight of the oligosaccharide was found to be 1023.38 Da.

[0087] Experimental Example 2 Chemical Structure Identification of Polygonatum Oligosaccharide PCO-A1

[0088] (1) Ion chromatography analysis of the monosaccharide composition of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1

[0089] In view of the difficulty in studying monosaccharide composition, that is, the traditional PMP method can only detect aldoses (such as glucose, mannose, etc.) but cannot detect ketoses (such as fructose, etc.), the present invention uses ion chromatography analysis to determine the monosaccharide composition and content in Polygonatum oligosaccharide PCO-A1, which greatly improves the accuracy of structural analysis. Figure 10 As shown, after testing, the polygonatum oligosaccharide PCO-A1 obtained in Example 1 is composed of fructose and glucose in a molar ratio of 2:1.

[0090] (2) Determination of the chemical structure characteristics of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1 by NMR

[0091] The oligosaccharide 45 mg was completely dissolved in 99.9% D2O, and was loaded into a nuclear magnetic tube for NMR determination.

[0092] Detection condition: VNMRS600 superconducting nuclear magnetic resonance spectrometer; One NMR probe (5mm) probe; 1 H spectrum working frequency 599.81 MHz; 13 C spectrum working frequency 150.84 MHz; 25℃ respectively 1 H spectrum ( Figure 11A ), 25℃ respectively 13 C ( Figure 11B ), COSY ( Figure 11C ), HSQC ( Figure 11D ), and HMBC spectrum ( Figure 11E ).

[0093] It was detected that the oligosaccharide PCO-A1 obtained in Example 1 was composed of β-D-Fru-6, 1-β-D-Fru-2, 1-β-D-Fru-2 and 1-Fru-D-β main chain, and 1-α-D-Glc, 1-α-D-Glc-6 branch chain, and the primary structure of the presumed oligosaccharide PCO-A1 is shown in Figures 12A and 12B .

[0094] Specific nuclear magnetic spectrum structure analysis is shown in Table 1.

[0095] Table 1 Nuclear magnetic analysis of oligosaccharide PCO-A1

[0096]

[0097] Experimental Example 3 Anti-depression and anti-sleep disorder experimental study of oligosaccharide PCO-A1

[0098] 1. Experimental Animals and Treatments: Male C57BL / 6 mice, 2-3 months old, weighing 20-25 g, were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. [License No.: SCXK (Zhejiang) 2021-0009]. Mice were housed in an environment with a 12 h / 12 ​​h light / dark cycle, a temperature of 22 ± 2°C, and a relative humidity of 45-65%. Food and water were available ad libitum. The animals were divided into the following groups: normal control group (Control), model group (CUMS), model group + high-dose HJGT-H group (CUMS + HJGT-H), model group + medium-dose HJGT-M group (CUMS + HJGT-M), model group + low-dose HJGT-L group (CUMS + HJGT-L), and model group + fluoxetine (CUMS + Flu). Modeling Methods: Chronic Unpredictable Mild Stress (CUMS) was used to induce depressive-like behavior in mice. This model uses multi-dimensional random stressors, including fasting, social isolation, and temperature fluctuations, to continuously stimulate over several weeks, simulating the pathological characteristics of long-term stressful environments in humans. Dosing Methods: For 14 consecutive days, at 9:00 AM daily, different groups of animals were given the corresponding drugs by gavage. The high, medium, and low doses of Polygonatum oligosaccharide administered were 120 mg / kg, 60 mg / kg, and 30 mg / kg, respectively, and the fluoxetine dose was 10 mg / kg. The normal control and model groups received an equal volume of distilled water.

[0099] 2. Experimental Methods

[0100] 2.1 Tail Suspension Test: Mice are suspended by their tails. After a period of trying to escape but failing, they eventually give up and enter a state of immobility similar to depression. The duration of immobility is recorded to reflect the degree of depression. The mice are fixed 2 cm from their tails, hanging upside down, with their heads approximately 50 cm above a table, and shielded from view by boards on either side. The mice struggle in this state, but gradually cease struggling out of despair, exhibiting intermittent immobility. The duration of immobility within the first 4 minutes after 6 minutes is compared between the normal control group and the model group.

[0101] 2.2 Forced Swim Test: Experimental animals are placed in water and struggle to escape. After a period of attempts, they fail to escape and exhibit a "state of immobility," where they give up swimming. This is known as a "state of behavioral despair." The duration of immobility during this period can also reflect depression. Individual mice are placed in water (25°C) and allowed to swim for 6 minutes. After removal, they are air-dried in a 32°C room. The duration of the mouse's 6-minute swim is recorded, and the duration of immobility for the next 4 minutes is observed.

[0102] 2.3 Sugar water preference test: Sugar water preference test was detected before modeling and after modeling was completed. Before the sugar water preference test was performed, sugar water preference training was performed, that is, on the first day, water bottles of the same specifications were placed, all of which were 1% sugar water. On the second day, water bottles of the same specifications were placed, one of which was pure water and one of which was 1% sugar water, and after one hour, the positions of the two were exchanged. On the third day, water was prohibited for 24 hours, and then the test was officially started. The specific operation is as follows: the same specifications of one 1wt% sugar water bottle and one pure water bottle were placed, the light was turned off during the period, and the quiet was maintained, 1 hour later the positions of the two were exchanged, and 2 hours later the sugar water consumption rate (%) was measured.

[0103] 2.4 Electroencephalogram / electromyogram collection and analysis: After isoflurane anesthesia of the mouse, the head of the mouse was fixed on the stereotaxic instrument, the scalp on the top of the head was cut along the midline, and the bone membrane was wiped with a cotton ball dipped in 3% hydrogen peroxide to remove the bone membrane. Four skull screws were installed outside the skull of the mouse with a depth of 1-1.2 mm. C-type electrodes were wound on the skull screws for recording EEG activity. Two electrodes were placed in the neck muscles to record the EMG activity of the mouse. One week after the operation, the mouse was adapted to the sleep recording system. The Medusa system was used to record and store the electroencephalogram / electromyogram signals for further analysis. After the data was exported, the Sirenia Sleep Pro software was used to analyze the sleep / wake state of the mouse. According to the frequency spectrum characteristics of the electroencephalogram / electromyogram during the recording time (9:00-21:00), the state of the mouse was divided into wake period (Wake), non-rapid eye movement (NREM) sleep period and rapid eye movement (REM) sleep period. The wake state was characterized by asynchronous, low-amplitude electroencephalogram rhythm and high-level electromyogram activity with phase bursts. NREM sleep was characterized by synchronous, high-amplitude and low-frequency (0.5-4 Hz, δ) electroencephalogram and low electromyogram activity. REM sleep was characterized by continuous θ wave (4-8 Hz) oscillation and extremely low electromyogram activity. The time required from the start of sleep monitoring to the first conversion to REM sleep state was the REM sleep latency. The sum of NREM and REM sleep was the total sleep time.

[0104] 3. Detection index

[0105] After the animal behavior test, the mice in each group were rested for 24 hours, and then anesthetized with 5% isoflurane gas. After anesthesia, the blood was quickly collected by decapitation. The blood was placed in a centrifuge tube at room temperature for 2 hours, centrifuged at 3000 rpm / min for 15 minutes at 4°C, and the supernatant was slowly taken and aliquoted (50 μL / portion) into clean centrifuge tubes. Further, the levels of CORT (serum cortisol), IL-1β (interleukin-1β) and IL-10 (interleukin-10) biochemical indicators in the serum of mice in each group were detected by ELISA method. The specific detection steps of each index were performed according to the operation instruction manual of ELISA kit.

[0106] 4. Results:

[0107] 4.1 Huangjing oligosaccharide improves the depressive-like behavior of CUMS mice

[0108] The results of the tail suspension test and forced swimming test are shown in Figure 13A , 13B Compared with the Control mice, the immobility time of the CUMS mice was significantly increased. After the CUMS mice were given Huangjing oligosaccharide at a high dose of 120 mg / kg, a medium dose of 60 mg / kg, or a low dose of 30 mg / kg, or fluoxetine at a dose of 10 mg / kg by gavage at 9:00 am for 14 consecutive days, the immobility time of the CUMS mice was reduced to varying degrees, and there were significant differences between the high and medium doses and fluoxetine. Figure 13C 13D As shown in Figure 13C , 13D , there was no difference in the sugar water preference percentage of the animals in each group before treatment. After treatment, the sugar water preference percentage of the CUMS mice was significantly reduced, and there were significant differences between the high and medium doses and fluoxetine. The above results show that Huangjing oligosaccharide can improve the depressive-like behavior of CUMS mice and has an antidepressant effect similar to that of fluoxetine.

[0109] 4.2 Huangjing oligosaccharide improves sleep disorders in CUMS mice

[0110] The results of the electroencephalogram / electromyogram collection and analysis show that compared with the Control mice, the REM latency ( Figure 14A ), the duration of each sleep ( Figure 14C ), the duration of each NREM ( Figure 14D ), the percentage of sleep duration ( Figure 14E ), and the percentage of NREM duration ( Figure 14F ) of the CUMS mice were significantly reduced, and the number of awakenings ( Figure 14B ) was significantly increased. After being given different doses of Huangjing oligosaccharide and fluoxetine, the REM latency, the duration of each sleep, the duration of each NREM, the percentage of sleep duration, and the percentage of NREM duration of the CUMS mice were increased, and the number of awakenings was reduced. Only the low dose and fluoxetine had no difference in the duration of each NREM, and the rest had significant differences.

[0111] 4.3 Huangjing oligosaccharide improves the detection of biochemical indicators in CUMS mice

[0112] As shown in Figures 15A-15DAs shown, the ELISA experiment results show that compared with the Control mice, the serum CORT, IL-1β and TNF-α levels of the CUMS mice are significantly increased, the 5-HT content is obviously reduced, after administration of different doses of polygonatum oligosaccharides, the above biochemical indicators are reversed to different degrees, wherein the high, medium and low doses have a significant effect on reducing the serum CORT and TNF-α of the CUMS mice, the high and medium doses have a significant effect on increasing the serum 5-HT and reducing IL-1β of the CUMS mice. The above results suggest that polygonatum oligosaccharides can reverse the changes of depression-related biochemical indicators of the CUMS mice.

[0113] The above describes the preferred embodiments of the present application in detail, but the present application is not limited to the specific details in the above embodiments, within the technical concept range of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection range of the present application.

Claims

1. A Polygonatum oligosaccharide, characterized in that The polygonatum oligosaccharide has a structure as shown in formula (I): Formula (I).

2. The Polygonatum oligosaccharide according to claim 1, characterized in that The weight average molecular weight of the polygonatum oligosaccharide is 1000-1200 kDa.

3. A method for preparing polygonatum oligosaccharide according to claim 1 or 2, characterized in that: The preparation method comprises: Extracting Polygonatum sibiricum with hot water at a temperature of 90-100°C, with a ratio of 1 g of Polygonatum sibiricum to hot water of 3-5 mL, for 45-60 min, and 1-3 extractions; concentrating the extracted solution, and then defatting, deproteinizing, re-concentrating, and ethanol precipitation are performed in sequence; The precipitate obtained by alcohol precipitation was redissolved in water and then subjected to cellulose column chromatography, which was then eluted with water and 0.1-0.5 mol / L NaCl solution in a gradient manner. The eluate obtained by water elution was collected, concentrated, and freeze-dried to obtain a primary purified product. The primary purified product was redissolved in water and then ethanol was added to completely precipitate, and the solid-liquid separation was performed and the liquid was collected. After concentration and freeze-drying, the secondary purified product was obtained; The secondary purified product is redissolved in water and then purified by gel column chromatography, with Sephadex G-25 as the filler. The product is eluted with water, and the eluate is collected, concentrated, and freeze-dried to obtain the polygonatum oligosaccharide.

4. The preparation method according to claim 3, characterized in that The alcohol precipitation conditions include: adding anhydrous ethanol to an ethanol concentration of 70% to 90% v / v, and standing at 3 to 5° C. for 20 to 30 hours to allow precipitation.

5. The preparation method according to claim 3, characterized in that The conditions for the cellulose column chromatography separation include: the cellulose column filler is DEAE-52, the sample concentration is 20-25 mg / mL, and the elution flow rate is 1-3 mL / min.

6. The preparation method according to claim 3, characterized in that The gel column chromatography purification conditions include: a sample concentration of 8-12 mg / mL and an elution flow rate of 0.1-0.3 mL / min.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polygonatum oligosaccharide according to claim 1 or 2 and a pharmaceutically acceptable excipient or carrier.

8. Use of the polygonatum oligosaccharide according to claim 1 or 2 or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating depression.

9. The use according to claim 8, characterized in that The drug is a drug that improves depressive-like behavior, improves sleep disorders, or improves changes in depression-related biochemical indicators.

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