Polygonatum sibiricum oligosaccharide, preparation method thereof and application of medicine for treating depression

Abstract

The invention belongs to the technical field of plant polysaccharides, and particularly discloses rhizoma polygonati oligosaccharide, a preparation method thereof and application of a medicine for treating depression. The polygonatum sibiricum oligosaccharide has a structure as shown in a formula (I), is a novel acidic homogeneous oligosaccharide separated from a natural traditional Chinese medicinal material polygonatum sibiricum for the first time, can effectively improve depression-like behaviors and sleep disorders of CUMS mice, can reverse the change of depression-related biochemical indexes of the CUMS mice, and has a good application prospect. The anti-depression effect of the polygonatum sibiricum oligosaccharide is equivalent to that of the existing classic drug fluoxetine, but the side effect is obviously lower, and the polygonatum sibiricum oligosaccharide is expected to be developed into a new anti-depression drug. # imgabs0 # is represented by formula (I).

Description

Technical Field

[0001] The invention belongs to the technical field of plant polysaccharides, and specifically discloses polygonatum oligosaccharide, a preparation method thereof, and medicinal use thereof for treating depression. Background Art

[0002] Polygonatum sibiricum is a perennial herbaceous plant of the genus Polygonatum in the Liliaceae family. Its dried rhizome is a traditional Chinese medicinal herb with a long history of medicinal use. According to Traditional Chinese Medicine, Polygonatum sibiricum is mild in nature, sweet in flavor, and enters the spleen, lung, and kidney meridians, making it a representative herb for "tonifying" deficiency. Modern research indicates that Polygonatum sibiricum contains saponins, oligosaccharides, and polysaccharides, with oligosaccharides and polysaccharides considered to be its primary pharmacological agents.

[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.Mechanistic studies have shown that all three can downregulate the expression of key proteins in the TLR4 / MyD88 / NF-κB pathway, and PET can specifically activate the Nrf2 antioxidant pathway (Zhang Ming, Li Zhenhua, Wang Xue, Chen Li. Comparative study on the immunomodulatory mechanism of multimodal extracts of Polygonatum sibiricum. Chinese Journal of Traditional Chinese Medicine. 2023;48(9):2356-2363.). It is worth noting that POW showed an abnormal increase in liver coefficient at a high dose (400 mg / kg), suggesting that attention should be paid to the safety and control of the extraction process. Chen Xiao et al. used a graded alcohol precipitation method to separate three oligosaccharide components (POS-I, POS-II, and POS-III) from Polygonatum sibiricum. HPAEC-PAD analysis found that their monosaccharide compositions were significantly different: POS-I had glucose (82.3%) as the main chain, POS-II contained characteristic galacturonic acid (17.8%), and POS-III showed a high mannose ratio (34.5%). In vitro experiments showed that POS-II had a significantly better DPPH free radical scavenging rate (IC50=1.28 mg / mL) than Vc. p <0.05), while POS-III can reduce LPS-induced IL-1β secretion in THP-1 cells by 61.4% by inhibiting NLRP3 inflammasome activation (Chen Xiao, Wu Jun, Zhou Min, et al. Analysis of the characteristics and activity differences of Polygonatum sibiricum oligosaccharides based on graded alcohol precipitation. Food Science. 2022;43(17):87-94.). The above studies confirmed that the biological functions of Polygonatum sibiricum oligosaccharides are closely related to their structural characteristics, including the connection order of specific sugar units, branching degree, and modification groups, and the extraction method directly affects the structure-activity characteristics of the oligosaccharide components.

[0004] However, Polygonatum oligosaccharide is rich in isomers and has a very complex structure. Currently, there are few Polygonatum oligosaccharides whose structures and functions have been elucidated. Discovering more Polygonatum oligosaccharides with new structures and exploring their new biological functions are of great significance for comprehensively explaining the structure-activity relationship of Polygonatum oligosaccharide, clarifying the material basis of its biological functions, and deeply exploring the medicinal value of Polygonatum oligosaccharide. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a Polygonatum sibiricum oligosaccharide, a preparation method, and a pharmaceutical use for treating depression. This invention isolates Polygonatum sibiricum oligosaccharide PCO-A1 from dried Polygonatum sibiricum. A multi-dimensional characterization system, including ultraviolet spectroscopy, infrared spectroscopy, HPGPC, Congo red assay, and ion chromatography, ensures product purity and structural homogeneity and successfully elucidates its primary structure. Studies on its significant antidepressant efficacy and mechanism lay a solid foundation for the development of new antidepressant drugs made of Polygonatum sibiricum oligosaccharide.

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

[0007] Formula (I).

[0008] In some embodiments, the weight average molecular weight of polygonatum oligosaccharide is 1000-1200 kDa.

[0009] In a second aspect, the present invention provides a preparation method of the aforementioned polygonatum oligosaccharide, comprising: extracting polygonatum with hot water, concentrating the extracted solution, and then sequentially performing defatting, deproteinizing, re-concentrating and alcohol precipitation; re-dissolving the precipitate obtained by alcohol precipitation in water, separating it by cellulose column chromatography, gradient eluting it with water and 0.1-0.5 mol / L NaCl solution in sequence, collecting the eluate obtained by water elution, concentrating and freeze-drying to obtain a primary purified product; re-dissolving the primary purified product in water, adding ethanol to complete the precipitation, performing solid-liquid separation and collecting the liquid, concentrating and freeze-drying to obtain a secondary purified product; re-dissolving the secondary purified product in water, performing gel column chromatography purification, eluting it with water, collecting the eluate, concentrating and freeze-drying to obtain polygonatum oligosaccharide.

[0010] In some embodiments, dried Polygonatum sibiricum is used for hot water extraction.

[0011] In some embodiments, the extraction conditions include: the temperature of the hot water is 90-100° C., the ratio of Polygonatum sibiricum to hot water is 1 g:3-5 mL, the extraction time is 45-60 min, and the number of extractions is 1-3 times.

[0012] In some embodiments, each concentration is performed by rotary evaporation until the volume is reduced by 3 / 5 to 4 / 5.

[0013] In some embodiments, petroleum ether is used for extraction and degreasing, and the aqueous phase is collected. Sevage reagent (chloroform to n-butanol, volume ratio: 4-6:1) is then mixed with the extract at a volume ratio of 1:3-5. After thorough mixing, the mixture is allowed to stand for 0.5-1.5 hours before separation to remove proteins. Combining petroleum ether degreasing with the Sevage method significantly improves deproteinization efficiency.

[0014] In some embodiments, 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.

[0015] In some embodiments, the conditions for cellulose column chromatography separation include: DEAE-52 as the cellulose column filler, a sample loading concentration of 20-25 mg / mL, and an elution flow rate of 1-3 mL / min. A gradient elution procedure can achieve efficient separation of oligosaccharide components.

[0016] 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.

[0017] In a third aspect, the present invention provides a pharmaceutical composition comprising the aforementioned Polygonatum sibiricum oligosaccharide and a pharmaceutically acceptable excipient or carrier.

[0018] 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.

[0019] 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.

[0020] The beneficial effects of the present invention are: 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.

[0021] 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

[0022] Figure 1 This is a flow chart of the preparation process of Polygonatum oligosaccharide PCO-A1 in Example 1 of the present invention; Figure 2A This is the HPGPC diagram of PCO-A in Example 1 of the present invention; Figure 2B This is the HPGPC diagram of PCO-A0 in Example 1 of the present invention; Figure 3 This is the HPGPC diagram of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; Figure 4A This is the HPGPC diagram of the sample prepared in Comparative Example 1 of the present invention; Figure 4B This is the HPGPC diagram of the sample prepared in Comparative Example 2 of the present invention; Figure 5AThis is the UV spectrum of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; Figure 5B This is the infrared spectrum of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; Figure 6 This is the Congo red image of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; Figure 7 This is a scanning electron micrograph of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; Figure 8A This is a particle size diagram of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention. The components on the horizontal axis refer to PCO-A1 measured three times in parallel; Figure 8B This is a potential diagram of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention. The components on the horizontal axis in the figure refer to five different concentrations of 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; E is 0.00005 mg / ml; Figure 9 This is a pH diagram of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention. The components on the abscissa in the figure refer to PCO-A1 measured in parallel three times; Figure 10 Ion chromatography analysis results of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; 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 =15.334min;Gal, t R =17.859 min;Glc t R =20.392 min;GlcNAc t R =22.109 min;Xyl t R =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; Figure 11A This is the hydrogen spectrum of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; Figure 11B This is the carbon spectrum of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; Figure 11C This is the COSY graph of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; Figure 11D This is the HSQC chart of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; Figure 11E This is the HMBC chart of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; Figure 12A is the inferred structural formula of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; Figure 12B This is the inferred structure diagram of Polygonatum oligosaccharide PCO-A1 prepared in Example 1 of the present invention; 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); 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; Figure 13C is the percentage of sugar water preference test before treatment in each group of CUMS mice; 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; Figure 14A Comparison of REM latency in each group of CUMS mice. * P <0.05 vs.Control, # P <0.05 vs. CUMS; Figure 14B Comparison of awake times of CUMS mice in each group. * P <0.05 vs.Control, # P <0.05 vs. CUMS; Figure 14C Comparison of sleep duration of each group of CUMS mice. * P <0.05 vs.Control, # P <0.05vs.CUMS; Figure 14D Comparison of NREM duration in each group of CUMS mice. * P <0.05 vs.Control, # P <0.05vs.CUMS; Figure 14E Comparison of sleep duration percentages of CUMS mice in each group. * P <0.05 vs.Control, # P <0.05vs.CUMS; Figure 14F Comparison of the percentage of NREM duration in each group of CUMS mice. * P <0.05 vs.Control, # P <0.05vs.CUMS; Figure 15A Serum CORT levels of CUMS mice in each group were detected by ELISA. * P <0.05 vs.Control, # P <0.05vs.CUMS; 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; 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; 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

[0023] 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.

[0024] Example 1 Polygonatum oligosaccharide PCO-A1, preparation process as follows Figure 1 As shown, the following steps are included: (1) Extraction of crude oligosaccharides from Polygonatum sibiricum: According to the material-liquid ratio of 1g:4mL, accurately weigh 100g of Polygonatum sibiricum and add 400mL of distilled water. Put it into a pot and boil for 1 hour. Collect the decoction. Repeat this operation twice. Boil for the last time for 45 minutes. Collect the extract. Combine the three extracts and concentrate them by rotary evaporation to 50mL for later use. Add petroleum ether to the concentrated extract for degreasing. Collect the aqueous phase. Repeat this operation 3 times and collect the degreased extract. Use sevage reagent (chloroform and n-butanol in a volume ratio of 5:1) and mix with the extract in a volume ratio of 1:4. After complete mixing, let it stand for 1 hour for separation and protein removal. Repeat this step 3 times. The treated extract was rotary evaporated at 70°C, and the concentrated extract was mixed with anhydrous ethanol in 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, the extract was centrifuged at 4750 rpm for 15 minutes, and the precipitate was filtered. The precipitate was completely dissolved in pure water and placed in a freeze dryer for freeze-drying to obtain the freeze-dried powder of Polygonatum sibiricum oligosaccharide PCO.

[0025] (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%.

[0026] (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.

[0027] (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-200 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.

[0028] Comparative Example 1 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.

[0029] Comparative Example 2 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.

[0030] Experimental Example 1 Analysis of the physicochemical properties of Polygonatum oligosaccharide PCO-A1 (1) UV absorption of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1 was measured by UV scanning. 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.

[0031] (2) Analyzing the characteristic groups of Polygonatum oligosaccharide obtained in Example 1 using infrared spectrometer 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 .

[0032] 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.

[0033] (3) The homogeneity of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1 was determined by HPGPC method 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.

[0034] like Figure 3 As shown, after testing, the polygonatum oligosaccharide PCO-A1 obtained in Example 1 is a uniform oligosaccharide. (4) UV scanning was used to determine the triple helical structure of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1 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.

[0035] 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 6 As 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.

[0036] (5) Scanning electron microscopy was used to measure the surface ultrastructure of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1 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.

[0037] 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.

[0038] (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.

[0039] (7) Use Starter3100 pH meter to measure the pH of PCO-A1, as shown in Figure 9 shown.

[0040] (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.

[0041] Experimental Example 2 Chemical Structure Identification of Polygonatum Oligosaccharide PCO-A1 (1) Ion chromatography analysis of the monosaccharide composition of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1 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.

[0042] (2) Determination of the chemical structure characteristics of the Polygonatum oligosaccharide PCO-A1 obtained in Example 1 by NMR 45 mg of oligosaccharide was completely dissolved in 99.9% D2O, placed in a nuclear magnetic resonance tube, and subjected to NMR determination.

[0043] Detection conditions: VNMRS600 superconducting nuclear magnetic resonance spectrometer; One NMR probe (5mm); 1 H spectrum operating frequency 599.81MHz; 13 C spectrum working frequency 150.84MHz; measured at 25℃ 1 H spectrum ( Figure 11A ), measured at 25℃ 13 C( Figure 11B )、COSY( Figure 11C )、HSQC( Figure 11D )、HMBC spectrum( Figure 11E ).

[0044] The results showed that the polygonatum oligosaccharide PCO-A1 obtained in Example 1 is composed of β-D-Fru-6, 1-β-D-Fru-2, 1-β-D-Fru-2 and 1-Fru-D-β main chains, and 1-α-D-Glc and 1-α-D-Glc-6 side chains. The primary structure of the polygonatum oligosaccharide PCO-A1 is as follows: Figures 12A and 12B shown.

[0045] The specific NMR spectrum structure analysis is shown in Table 1: Table 1 NMR analysis of Polygonatum oligosaccharide PCO-A1

[0046] Experimental Example 3 Experimental study on the antidepressant and anti-sleep disorder effects of Polygonatum oligosaccharide PCO-A1 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.

[0047] 2. Experimental Methods 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.

[0048] 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.

[0049] 2.3 Sugar Water Preference Experiment: Sugar water preference experiments were conducted before and after modeling. Before the sugar water preference experiment, sugar water preference training was conducted. On the first day, two water bottles of identical specifications, both containing 1% sugar water, were placed. On the second day, two bottles of the same specifications were placed, one containing pure water and the other containing 1% sugar water. After one hour, the two bottles were swapped. On the third day, water was withheld for 24 hours, after which the test officially began. The specific operation was as follows: a bottle of 1wt% sugar water and a bottle of pure water of the same specifications were placed. The lights were turned off and the subjects remained silent during the period. After one hour, the two bottles were swapped. After two hours, the sugar water consumption rate (%) was measured.

[0050] 2.4 EEG / EMG Acquisition and Analysis: After isoflurane anesthesia, the mice were fixed to a stereotaxic apparatus. The skin on the top of the head was cut along the midline and wiped with 3% hydrogen peroxide using a cotton ball to remove the periosteum. Four skull pins were placed on the outside of the mouse skull at a depth of 1-1.2 mm. C-shaped electrodes were wrapped around the pins to record EEG activity. Two electrodes were placed in the neck muscles to record EMG activity. One week after postoperative recovery, the mice began to adapt to the sleep recording system. The Medusa system was used to record and store EEG / EMG signals for further analysis. After data export, Sirenia Sleep Pro software was used to analyze the sleep / wakefulness of the mice. Based on the EEG / EMG spectral characteristics of the recording time (9:00-21:00), the mice were classified into wake, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep. The wakeful state is characterized by asynchronous, low-amplitude EEG rhythms and high levels of EMG activity with phasic bursts. NREM sleep is characterized by synchronized, high-amplitude, low-frequency (0.5-4 Hz, delta) EEG activity and low EMG activity. REM sleep is characterized by continuous theta wave (4-8 Hz) oscillations and very low EMG activity. The time from the start of sleep monitoring to the first transition to REM sleep is the REM sleep latency. The sum of NREM and REM sleep is the total sleep time.

[0051] 3. Detection indicators After the behavioral testing, mice in each group rested for 24 hours and were anesthetized with 5% isoflurane gas. Blood was collected by rapid decapitation. The blood was placed in a centrifuge tube at room temperature for 2 hours and then centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was slowly aliquoted (50 μL / aliquot) into clean centrifuge tubes. Furthermore, serum levels of CORT (serum cortisol), IL-1β (interleukin-1β), and IL-10 (interleukin-10) biochemical markers were measured in each group using ELISA. Specific assay procedures for each marker were performed according to the ELISA kit instructions.

[0052] 4. Results: 4.1 Polygonatum oligosaccharide improves depressive-like behavior in CUMS mice The results of the tail suspension test and forced swim test were as follows: Figure 13A 、 13BAs shown in the results, compared with the control mice, the immobility time of CUMS mice was significantly increased. For 14 consecutive days, at 9 am every day, CUMS mice were given high, medium and low doses of Polygonatum sibiricum oligosaccharide by gavage at 120 mg / kg, 60 mg / kg and 30 mg / kg respectively, and fluoxetine at a dose of 10 mg / kg. The immobility time of CUMS mice was reduced to varying degrees, and there was a significant difference between the high dose, medium dose and fluoxetine. The results of the sugar water preference experiment are shown in the figure below. Figure 13C 、 13D As shown, there was no difference in the percentage of sugar water preference among the animals in each group before treatment. After treatment, the sugar water preference of CUMS mice was significantly reduced, and there were significant differences between the high and medium doses and fluoxetine. These results indicate that Polygonatum oligosaccharide can improve the depressive-like behavior of CUMS mice, and its antidepressant effect is similar to that of fluoxetine.

[0053] 4.2 Polygonatum oligosaccharide improves sleep disorders in CUMS mice The results of EEG / EMG acquisition and analysis showed that compared with Control mice, the REM latency of CUMS mice ( Figure 14A ), duration of each sleep ( Figure 14C ), duration of each NREM Figure 14D ), sleep duration percentage ( Figure 14E ) and percentage of NREM time ( Figure 14F ) were significantly reduced, and the number of awake times ( Figure 14B ) were significantly increased. After giving different doses of Polygonatum oligosaccharide and fluoxetine, the REM latency, sleep duration, NREM duration, sleep duration percentage and NREM duration percentage of CUMS mice were increased, and the number of awake times of CUMS mice was reduced. Only the low dose and fluoxetine had no difference in the NREM duration, and the rest had significant differences.

[0054] 4.3 Polygonatum oligosaccharide improves biochemical indicators in CUMS mice like Figures 15A-15D As shown in the results of ELISA experiments, compared with control mice, the serum levels of CORT, IL-1β, and TNF-α in CUMS mice were significantly increased, and the 5-HT content was significantly reduced. After administration of different doses of Polygonatum oligosaccharide, the above biochemical indicators were reversed to varying degrees. Among them, high, medium, and low doses had a significant effect on reducing serum CORT and TNF-α in CUMS mice, and high and medium doses had a significant effect on increasing serum 5-HT and reducing IL-1β in CUMS mice. These results suggest that Polygonatum oligosaccharide can reverse the changes in depression-related biochemical indicators in CUMS mice.

[0055] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

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, concentrating the extracted solution, and then defatting, deproteinizing, concentrating again and precipitating with alcohol 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, 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 extraction conditions include: the temperature of the hot water is 90-100° C., the dosage ratio of polygonatum to hot water is 1 g:3-5 mL, the extraction time is 45-60 min, and the number of extractions is 1-3 times.

5. 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.

6. 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.

7. The preparation method according to claim 3, characterized in that The gel column chromatography purification conditions include: the gel column filler is Sephadex G-25, the sample concentration is 8-12 mg / mL, and the elution flow rate is 0.1-0.3 mL / min.

8. 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.

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

10. The use according to claim 9, 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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