Extraction method of Tibetan polygonatum polysaccharide and preparation process of capsule

Abstract

The invention discloses a Tibetan rhizoma polygonati polysaccharide extraction method and a Tibetan rhizoma polygonati polysaccharide capsule preparation process, relates to the technical field of biological extraction, and has the technical key points that the Tibetan rhizoma polygonati polysaccharide extraction method which is simple in extraction process, high in purity and clear in component is adopted, and the Tibetan rhizoma polygonati polysaccharide capsule preparation process is explored through the Tibetan rhizoma polygonati polysaccharide extraction method. And a solid theoretical basis is provided for medicine research for improving immunity, resisting anoxia and resisting fatigue in the future.

Description

Technical Field

[0001] This invention relates to the field of bio-extraction technology, specifically to an extraction method for Polygonatum sibiricum polysaccharides and a capsule preparation process. Background Technology

[0002] There are many species of Polygonatum, with my country having the richest variety, reaching 31 species, including 7 species from Tibet. However, the Tibetan Polygonatum commonly found in the market are mostly *Polygonatum verticillatum* (L.) All. and *Polygonatum cirrhifolium* (Wall.) Roylez., both belonging to the Liliaceae family, and are commonly used as both food and medicine. *Polygonatum cirrhifolium* (Wall.) Royle is a perennial herb belonging to the genus *Polygonatum* in the Liliaceae family, and its rhizome is used medicinally. This plant is highly adaptable, resistant to cold, and prefers a moist, shady environment; it does not grow well in dry areas. It thrives in deep, fertile, loose, well-drained, and water-retentive sandy soils, and its biological characteristics are well-suited to this plant.

[0003] According to ancient Tibetan medical texts such as the *Four Medical Classics* and *Tara Materia Medica*, as well as modern monographs, Tibetan Polygonatum has nourishing, longevity-promoting, anti-jaundice, stomach-warming, heat-clearing, and body-soothing effects. It is used to treat complications of "Peigen" and "Chiba," "jaundice," consumptive cough and asthma, fetal heat, indigestion, and sores and abscesses. Milk-processed Polygonatum is generally used in kidney-tonifying medicines, while cleaned Polygonatum is used in prepared medicines such as Coral Seventy-Flavor Pills and Pomegranate Sun Wheel Pills. Polygonatum is a medicinal and edible herb containing polysaccharides, flavonoids, unsaturated fatty acids, and trace elements, among other nutrients and bioactive substances. Polysaccharides are the most abundant bioactive substance, and sugars are the most abundant component of Polygonatum. Identification has shown that its main components are oligosaccharides and polysaccharides. Polygonatum polysaccharides are composed of glucose, mannose, and galacturonic acid in varying proportions, with a 2→6 linear linkage as the main chain and a molecular weight greater than 200,000. Polygonatum oligosaccharides are polymerized from 2 to 10 different numbers of fructose and glucose molecules, and are classified into three types (A, B, and C) based on their structural differences. Oligosaccharide A has a molecular weight of 1630 and a fructose to glucose molar ratio of 8:1; oligosaccharide B has a molecular weight of 862 and a fructose to glucose molar ratio of 4:1; and oligosaccharide C has a molecular weight of 474 and a fructose to glucose molar ratio of 2:1. Currently, research and development of Polygonatum both domestically and internationally mainly focus on Polygonatum multiflorum and Polygonatum yunnanense, with relatively little research on Polygonatum tsuifolia.

[0004] Therefore, the present invention aims to provide an extraction method for Tibetan Polygonatum polysaccharides and a capsule preparation process to solve the above problems. Summary of the Invention

[0005] The purpose of this invention is to solve the above-mentioned problems and provide an extraction method for Tibetan Polygonatum polysaccharides and a capsule preparation process. This invention provides a simple, high-purity, and clearly defined extraction method for Tibetan Polygonatum polysaccharides and explores the preparation process of Tibetan Polygonatum polysaccharide capsules using this method, providing a theoretical basis for subsequent research on drugs for improving immunity, resisting hypoxia, and combating fatigue.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides a method for extracting polysaccharides from Tibetan Polygonatum, characterized in that the extraction method comprises the following steps:

[0008] S1. Use 95% ethanol to extract the Tibetan Polygonatum residue by hot reflux, and then dry the Tibetan Polygonatum residue until there is no alcohol smell.

[0009] S2. Add the dried Tibetan Solomon's Seal residue powder to deionized water at a material-to-liquid ratio of 1:10 w / v, and extract at a constant temperature of 60℃ for 2 hours. Then filter the extract with gauze and concentrate the extract to 1 / 3 of the original volume under reduced pressure. Stir continuously and add 95% ethanol to make the final solution have a final concentration of 75%.

[0010] S3. Place the final solution in a refrigerator at 4℃ for 12 hours to settle. Centrifuge the precipitate at 3000 r / min for 10 min, dissolve and volatilize the alcohol, and freeze-dry to obtain crude tsampa extract polysaccharide.

[0011] The crude polysaccharide of *Tibetan Polygonatum* was subjected to deproteinization, dialysis, and purification to obtain *Tibetan Polygonatum* polysaccharide. The specific steps are as follows:

[0012] S301, Tibetan Polysaccharide (with protein removed)

[0013] Proteins in Tibetan Polygonatum polysaccharides were removed using a combined papain-TCA method. A 0.1 g / mL polysaccharide solution was prepared, and 0.2% papain was added. The solution was incubated at 60°C for 30 min, followed by enzyme inactivation at 100°C for 3 min. Finally, TCA (1 / 3 volume of polysaccharide) was added to remove free proteins. The solution was centrifuged at 3000 rpm for 10 min to remove the precipitate, and the supernatant was concentrated under reduced pressure. The concentrate was then freeze-dried to obtain the Polygonatum polysaccharide.

[0014] S302, Dialysis of Tibetan Ficus pumila crude polysaccharide

[0015] The protein-free sample was dialyzed with running water (MWCO = 4000 Da) for 48 h, then dialyzed with double-distilled water for 24 h, and finally dried using a freeze dryer to obtain Tibetan Polygonatum polysaccharide.

[0016] S303, DEAE-52 column purification

[0017] DEAE-52 cellulose ion exchange resin was fully swollen by adding deionized water. After removing impurities by decantation, it was activated by alternating soaking in 0.5M NaOH and HCl solutions. Finally, it was washed with deionized water until neutral, dried, and set aside. The activated column material was degassed by ultrasonication and then packed into the column. The column material was then eluted with deionized water to allow it to deposit and compact. After the polysaccharide was completely dissolved, it was slowly and evenly added dropwise to the chromatography column. After loading, it was eluted with NaCl aqueous solutions of concentrations of 0.05, 0.1, and 0.2M, respectively, to prepare Polysaccharide Polysaccharide (PSP).

[0018] This invention also provides a preparation process for Tibetan Polygonatum polysaccharide capsules. The preparation process is as follows: Tibetan Polygonatum polysaccharide and lactose monohydrate are mixed evenly in a 1:1 ratio, and then the mixture of Tibetan Polygonatum polysaccharide and lactose is mixed evenly with microcrystalline cellulose in a 1:1.5 ratio. Finally, the mixture of Tibetan Polygonatum polysaccharide, lactose and microcrystalline cellulose is weighed, and 2% talc powder is added and mixed evenly to obtain a mixed contents. The angle of repose of the capsule contents is less than 37 degrees. The mixed contents are then filled into No. 0 capsules to obtain the finished Tibetan Polygonatum polysaccharide capsules.

[0019] Compared with existing technologies, the beneficial effects of this solution are:

[0020] This invention provides a simple, high-purity, and clearly defined method for extracting Tibetan Polygonatum polysaccharides, and uses this method to explore the preparation process of Tibetan Polygonatum polysaccharide capsules, providing a solid theoretical foundation for future drug research on improving immunity, resisting hypoxia, and combating fatigue. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the extraction process of Polygonatum tigrinum polysaccharides in an embodiment of the present invention;

[0022] Figure 2 This is the elution curve of Tibetan Polygonatum polysaccharide purified by ion exchange resin chromatography in an embodiment of the present invention;

[0023] Figure 3 This is the 1H NMR spectrum of Polygonatum sibiricum polysaccharide in the embodiments of the present invention;

[0024] Figure 4 This is a schematic diagram illustrating the change in the maximum absorption wavelength of Polygonatum tsao-jīng polysaccharide in Congo red solution in an embodiment of the present invention;

[0025] Figure 5 This is a schematic diagram illustrating the effect of Polygonatum sibiricum polysaccharide on the immune organ (spleen) index of immunosuppressed mice in an embodiment of the present invention;

[0026] Figure 6 This is a schematic diagram illustrating the effect of Polygonatum tigrinum polysaccharide on the immune organ (thymus) index of immunosuppressed mice in an embodiment of the present invention;

[0027] Figure 7 This is a schematic diagram illustrating the histopathological effects of Polygonatum tigrinum polysaccharide on the immune organ (spleen) of immunosuppressed mice in an embodiment of the present invention.

[0028] Figure 8 This invention relates to the histopathological effects of Polygonatum tigrinum polysaccharide on the immune organ (thymus) of immunosuppressed mice in the embodiments of the present invention.

[0029] Figure 9 This invention relates to the effect of Polygonatum sibiricum polysaccharide on the phagocytic index of macrophages in immunosuppressed mice.

[0030] Figure 10 This invention relates to the effect of Polygonatum sibiricum polysaccharide on serum hemolysin in immunosuppressed mice.

[0031] Figure 11 This invention relates to the effect of Polygonatum sibiricum polysaccharide on IgA in immunosuppressed mice.

[0032] Figure 12 This invention relates to the effect of Polygonatum sibiricum polysaccharide on IgM in immunosuppressed mice.

[0033] Figure 13 This invention relates to the effect of Polygonatum sibiricum polysaccharide on IgG in immunosuppressed mice.

[0034] Figure 14 This invention relates to the effect of Polygonatum sibiricum polysaccharide on the DTH response (weight difference) in immunosuppressed mice in the embodiments of the present invention.

[0035] Figure 15 This invention relates to the effect of Polygonatum sibiricum polysaccharide on serum IL-2 in immunosuppressed mice.

[0036] Figure 16 This invention relates to the effect of Polygonatum sibiricum polysaccharide on serum IFN-γ in immunosuppressed mice.

[0037] Figure 17 This invention relates to the effect of Polygonatum sibiricum polysaccharide on serum NO in immunosuppressed mice.

[0038] Figure 18 This invention relates to the effect of Polygonatum sibiricum polysaccharide on key proteins in the MAPK pathway.

[0039] Figure 19 This invention relates to the effects of Polygonatum sibiricum polysaccharide on key proteins in the upstream and downstream signaling pathways of MAPK.

[0040] Figure 20 This is an example of the effect of Polygonatum sibiricum polysaccharide on mouse body weight in this invention embodiment;

[0041] Figure 21This invention relates to the effect of Polygonatum sibiricum polysaccharide on the swimming time of mice under load in the embodiments of the present invention;

[0042] Figure 22 This invention relates to the effect of Polygonatum sibiricum polysaccharide on biochemical indicators of weight-bearing swimming mice.

[0043] Figure 23 This is an example of the effect of Polygonatum sibiricum on the body weight of Kunming mice in this invention.

[0044] Figure 24 This invention relates to the effect of Polygonatum sibiricum polysaccharide on the normobaric hypoxia tolerance time of mice.

[0045] Figure 25 This invention relates to the effects of Polygonatum sibiricum on the biochemical indicators of normobaric hypoxia-tolerant mice. Detailed Implementation

[0046] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0047] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0048] Example:

[0049] 1. Extraction and purification of crude polysaccharides from Tibetan Polygonatum and preparation of Tibetan Polygonatum polysaccharide capsules

[0050] 1.1 Instruments, Materials, and Reagents

[0051] 1.1.1 Instruments

[0052]

[0053] 1.1.2 Materials and Reagents

[0054] Tibetan Polygonatum was harvested in Shannan City, Tibet. DEAE-52 cellulose exchange resin was purchased from Beijing Solarbio Science & Technology Co., Ltd. Anthrone, sulfuric acid, ethanol, sodium chloride, sodium hydroxide, trichloroacetic acid, etc. were all of analytical grade and purchased from Chengdu Kelong Reagent Co., Ltd.

[0055] 1.2 Extraction and purification of crude polysaccharides from Tibetan rhizome

[0056] 1.2.1 Extraction of Polysaccharides from Tibetan Polygonatum

[0057] The residue of *Tibetan Polygonatum sibiricum* extracted by reflux with 95% ethanol was dried until no alcohol odor remained. The dried *Tibetan Polygonatum sibiricum* residue powder was added to deionized water at a material-to-liquid ratio of 1:10 (w / v). Extraction was carried out at 60℃ with stirring for 2 hours. The extracts were filtered through gauze and combined, then concentrated under reduced pressure to 1 / 3 of the original volume. 95% ethanol was added continuously with stirring until the final concentration reached 75%. The mixture was allowed to settle at 4℃ for 12 hours. The precipitate was centrifuged at 3000 rpm for 10 minutes to dissolve and volatilize the alcohol, then freeze-dried to obtain crude *Tibetan Polygonatum sibiricum* polysaccharide.

[0058] 1.2.2 Purification of Polysaccharides from Tibetan Polygonatum

[0059] 1.2.2.1 Deproteinized Tibetan Polysaccharide

[0060] Proteins in Tibetan Polygonatum polysaccharides were removed using a combined papain-TCA method. A 0.1 g / mL polysaccharide solution was prepared, and 0.2% papain was added. The solution was incubated at 60°C for 30 min, followed by enzyme inactivation at 100°C for 3 min. Finally, TCA (1 / 3 volume of polysaccharide) was added to remove free proteins. The solution was centrifuged at 3000 rpm for 10 min to remove the precipitate, and the supernatant was concentrated under reduced pressure. The concentrate was then freeze-dried to obtain the Polygonatum polysaccharide.

[0061] 1.2.2.2 Dialysis of Polysaccharides from Tibetan Polygonatum

[0062] The protein-free sample was dialyzed with running water (MWCO = 4000 Da) for 48 h, then dialyzed with double-distilled water for 24 h, and finally dried using a freeze dryer to obtain Tibetan Polygonatum polysaccharide.

[0063] 1.2.2.3 DEAE-52 column purification

[0064] DEAE-52 cellulose ion exchange resin was fully swollen by adding deionized water. After removing impurities by decantation, it was activated by alternating soaking in 0.5M NaOH and HCl solutions. Finally, it was washed with deionized water until neutral, dried, and set aside. The activated column material was degassed by ultrasonication and then packed into the column. The column material was then eluted with deionized water to allow it to deposit and compact. After the polysaccharide was completely dissolved, it was slowly and evenly added dropwise to the chromatography column. After loading, it was eluted with NaCl aqueous solutions of concentrations of 0.05, 0.1, and 0.2M, respectively, to prepare Polysaccharide Polysaccharide (PSP).

[0065] 1.3 Determination of crude polysaccharide content in Tibetan rhizome

[0066] The content of the prepared Tibetan Polygonatum polysaccharide was determined by the anthrone-sulfuric acid method, and a standard curve was prepared using glucose solution.

[0067] 1.3.1 Preparation of reagents

[0068] Reference solution: Accurately weigh 33 mg of glucose dried to constant weight into a 100 ml volumetric flask, add water and dilute to the mark.

[0069] Tibetan Polygonatum Polysaccharide Solution: Accurately weigh 10 mg of Tibetan Polygonatum polysaccharide into a 100 ml volumetric flask, add water to dissolve and dilute to the mark.

[0070] 0.2% anthrone solution (w / v): Accurately weigh 0.2g of anthrone into a beaker, slowly add 100ml of concentrated sulfuric acid solution to dissolve it. The solution will be a yellow, transparent color. Prepare fresh before use.

[0071] 1.3.2 Configuration of Standard Curve

[0072] Accurately measure 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, and 0.6 ml of the reference solution into separate 10 ml stoppered graduated test tubes. Add water to each tube to a final volume of 2.0 ml, shake well, and slowly add 0.2% anthrone-sulfuric acid solution to the mark in an ice-water bath. Mix well, cool, and then incubate in a water bath for 10 minutes. Remove from the water bath and immediately cool in an ice-water bath for 10 minutes. Use the corresponding reagents as blanks. Measure the absorbance at 582 nm using UV-Vis spectrophotometry. Plot a standard curve with absorbance on the ordinate and concentration on the abscissa, and calculate the regression equation.

[0073] 1.3.3 Determination of Polysaccharide Content in Tibetan Polygonatum

[0074] Accurately measure 0.3 ml of Tibetan Polygonatum polysaccharide solution and place it in a 10 ml stoppered graduated test tube. Process the sample according to the determination steps under the standard curve section, measure the absorbance value of the sample solution, and calculate the content of Tibetan Polygonatum polysaccharide in the sample according to the regression equation.

[0075] 1.4 Structural Study of Polysaccharides from Tibetan Polygonatum

[0076] 1.4.1 NMR Analysis

[0077] Weigh 15 mg of Tibetan Polygonatum polysaccharide, dissolve it completely in 0.6 mL of D2O by shaking, and place it in a standard NMR tube. Perform NMR measurements using a Bruker 400 NMR spectrometer to obtain relevant spectra such as 1H and 13C.

[0078] 1.4.2 Congo Red Experiment

[0079] 5 mg of Tibetan Polygonatum polysaccharide was fully dissolved in 4 mL of Congo red reagent aqueous solution (40 μmol / L), and then different volumes of 1 mol / L NaOH solution were added. The final NaOH concentration was controlled to increase from 0 to 0.5 mol / L. The maximum absorbance of the solution was measured by scanning in the range of 400 to 800 nm under a UV-Vis spectrometer.

[0080] 1.5 Preparation of Tibetan Polygonatum Polysaccharide Capsules

[0081] Tibetan Polygonatum polysaccharide and lactose monohydrate were mixed evenly in a 1:1 ratio. Then, the mixture of Tibetan Polygonatum polysaccharide and lactose was mixed evenly with microcrystalline cellulose (model 301) in a 1:1.5 ratio. Finally, the mixture of Tibetan Polygonatum polysaccharide, lactose, and microcrystalline cellulose was weighed, and 2% talc powder was added and mixed evenly. The angle of repose of the capsule contents was measured to be less than 37 degrees. The contents were then filled into size 0 capsules to obtain the finished Tibetan Polygonatum polysaccharide capsules.

[0082] 2. Study on the in vivo immune-regulating effects of Polysaccharides from Tibetan Polygonatum

[0083] 2.1 Instruments, Materials, Reagents and Laboratory Animals

[0084] 2.1.1 Instruments

[0085]

[0086] 2.1.2 Materials and Reagents

[0087] Tibetan Polygonatum polysaccharide (processed from Tibetan Polygonatum harvested in Shannan City, Tibet); levamisole hydrochloride (LM) was purchased from Shanghai Maclean Biotechnology Co., Ltd.; Indian ink and 5% chicken erythrocyte suspension were purchased from Nanjing Senbega Biotechnology Co., Ltd.; guinea pig serum was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; immunoglobulin (Ig) and cytokine kits were purchased from Wuhan Genemei Co., Ltd.; 2,4-dinitrofluorobenzene (DNFB) was purchased from Shanghai Maclean Biotechnology Co., Ltd.; RIPA strong lysis buffer, protein phosphatase inhibitor, BCA protein concentration assay kit (enhanced version), SDS-PAGE gel rapid preparation kit, and transfer filter paper (7.5×10cm) were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.; other reagents were all of analytical grade and purchased from Chengdu Kelong Reagent Co., Ltd.

[0088] Tibetan Polygonatum polysaccharide samples were prepared into solutions with concentrations of 50, 100, and 200 mg / kg using physiological saline.

[0089] Weigh 200mg of CTX and prepare a fresh CTX solution of 4mg / mL with 50mL of physiological saline. Prepare and use immediately.

[0090] Weigh 50 mg of DNFB and dissolve it in 5 mL of pre-prepared acetone-sesame oil solution (acetone:sesame oil = 1:1) to prepare a 10 mg / mL DNFB solution. This solution should be sealed and stored, and should be prepared and used immediately.

[0091] 2.1.3 Laboratory Animals

[0092] Experimental animals: Balb / c mice at 6 - 8 weeks of age with standard body weight (60 mice, 30 males and 30 females), purchased from Wensville Animal Co., Ltd. (License number: SCXK(Xiang)2019 - 0004). They were housed in the Animal Experiment Center of West China School of Pharmacy, Sichuan University under SPF standards, at 24 ± 1°C, with 12 h of light and 12 h of darkness, humidity (55% - 60%), provided with basal diet, and free access to water. They were allowed to adaptively feed freely under these conditions for 7 days before the experiment began.

[0093] 2.2 Experimental methods

[0094] 2.2.1 Grouping of experimental animals

[0095] Sixty SPF - level Balb / c mice were randomly and evenly divided into 6 groups, including a normal group, a model group, a positive control group, and low, medium, and high dose groups of polygonatum cirrhifolium polysaccharide (PSP). Mice in the normal group and the model group were given normal saline (0.1 ml / 10 g) by gavage every day, the positive group was given levamisole hydrochloride (LM, 100 mg / kg / d) by gavage, and the administration groups were given polygonatum polysaccharide solutions at different doses by gavage. Mice in each group were gavaged for 28 days. Except for the blank control group, mice in other groups were intraperitoneally injected with cyclophosphamide (CTX) at 60 mg / kg on the 23rd, 24th, and 25th days for immunosuppressive modeling.

[0096] Table 1 Grouping and administration of animals

[0097]

[0098]

[0099] 2.3 Effects of polygonatum cirrhifolium polysaccharide on immune organs of immunosuppressed mice

[0100] 2.3.1 Analysis of immune organ indices

[0101] Twenty - four hours after the last administration, the mice were weighed, blood was collected, and after the mice were sacrificed, their thymuses and spleens were removed, blotted dry with sterile paper and weighed, and the organ indices were calculated according to the following formula:

[0102] Thymus (spleen) index = thymus (spleen) weight (mg) / body weight (g)

[0103] 2.3.2 Histopathological examination of thymus and spleen

[0104] Mice in each group were sacrificed, and their spleens and thymuses were taken. At room temperature, they were fixed in 4% paraformaldehyde (the tissue blocks were completely immersed in paraformaldehyde). The fixed tissues were dehydrated with gradient ethanol solutions, embedded in paraffin, sectioned, stained with hematoxylin - eosin staining method (H&E), and the tissue morphology was observed under an optical microscope.

[0105] 2.4 Effects of Tibetan Polygonatum Polysaccharide on Non-specific Immune Function in Immunosuppressed Mice

[0106] The carbon clearance assay can be used to determine the clearance activity of macrophages, and the clearance rate of carbon particles is positively correlated with the phagocytic capacity of cells. Twenty-four hours after the last administration, Indian ink diluted 10 times with physiological saline was injected into the tail vein of mice at a dose of 0.1 mL / 10 g bw. Blood samples (20 μL each) were collected at 2 min (t1) and 10 min (t2), diluted with 2 mL of 0.1% Na₂CO₃ solution, and the OD value at 600 nm was measured, using 0.1% Na₂CO₃ solution as a blank for zeroing. Mice were sacrificed, and the liver and spleen were harvested, cleaned of surface blood, and weighed. The clearance rate (K) and phagocytic index (α) were calculated using the following formulas:

[0107] K = (lgODt1 – lgODt2) / (t2 – t1)

[0108] Phagocytic index α = body weight / (liver weight + spleen weight) × K1 / 3

[0109] 2.5 Effects of Tibetan Polygonatum Polysaccharide on Humoral Immune Function in Immunosuppressed Mice

[0110] 2.5.1 Serum hemolysin generation experiment

[0111] Serum hemolysin formation experiments were conducted according to the references. On day 26 after drug administration, 0.2 mL of 5% chicken erythrocyte suspension was injected intraperitoneally. One hour after the last drug administration, blood was collected, centrifuged at 3500 r / min, and the supernatant serum was collected. 20 μL of serum was diluted 100 times with physiological saline, and 1 mL of 5% chicken erythrocyte suspension and 10% guinea pig complement were added and mixed. The mixture was incubated in a constant temperature water bath at 37℃ for 30 min, then placed in an ice-water bath, centrifuged, and the OD value of the supernatant was measured at a wavelength of 540 nm. A blank control without serum was also included.

[0112] 2.5.2 Determination of serum immunoglobulins

[0113] Overnight after the last administration, blood was collected from mice, centrifuged at 3500 rpm, and the supernatant serum was collected. The serum IgA, IgG, and IgM levels were measured using an enzyme-linked immunosorbent assay (ELISA) kit.

[0114] 2.6 Effects of Tibetan Polygonatum Polysaccharide on Cellular Immune Function in Immunosuppressed Mice

[0115] 2.6.1 DNFB-induced delayed-type hypersensitivity reaction experiment

[0116] Delayed-type hypersensitivity experiments were conducted according to the references. On days 24 and 25 after drug administration, the abdominal skin of mice was completely shaved, and 50 μL of DNFB (10 mg / mL) was evenly applied to a 2 cm × 2 cm area to induce sensitization. On day 27 after drug administration, 5 μL of DNFB was applied to both sides of the left ear of each mouse. The mice were sacrificed 24 hours after the attack, and the left and right ears of the mice were collected using a punch. The obtained ear pieces were weighed and their thickness was measured. The difference in ear piece weight indicated the degree of delayed-type hypersensitivity (DTH).

[0117] 2.6.2 Serum cytokine and NO assay

[0118] Overnight after the last administration, blood was collected from mice, centrifuged at 3500 rpm, and the supernatant serum was collected. The levels of IL-2, IFN-γ, and NO in the serum were determined according to the kit's operating procedure.

[0119] 2.7 Effects of Polygonatum sibiricum polysaccharide on the expression of MAPK signaling pathway proteins

[0120] The MAPK signaling pathway comprises three major protein kinase groups (ERK, JNK, and p38) and is a downstream pathway of TLR4. Studies have shown that MAPKs can activate the NF-κB pathway, a key mediator of immune activation and inflammatory responses.

[0121] 2.8 Effects of Polygonatum sibiricum polysaccharide on the expression of upstream and downstream proteins in the MAPK signaling pathway

[0122] Polysaccharides can often regulate the production of interferons and cytokines by inducing MyD88 to further activate TRAF6. NF-κB is a transcription factor that plays a key role in the production of pro-inflammatory cytokines and other mediators.

[0123] 3. Study on the anti-fatigue effects of Polygonatum sibiricum polysaccharides

[0124] 3.1 Instruments, Materials, Reagents and Laboratory Animals

[0125] 3.1.1 Instruments

[0126]

[0127] 3.1.2 Materials and Reagents

[0128] Tibetan Polygonatum polysaccharide (prepared from Tibetan Polygonatum harvested in Shannan City, Tibet); basic rodent feed was purchased from Wayneville Animal Co., Ltd.; blood urea nitrogen (BUN) test kit (BC1535) and glycogen content test kit were purchased from Beijing Solarbio Technology Co., Ltd.; Red Bull energy drink was purchased from Red Bull Vitamin Beverage Co., Ltd.; and physiological saline was purchased from Sichuan Kelun Pharmaceutical Co., Ltd.

[0129] 3.1.3 Laboratory Animals

[0130] Thirty SPF - level male Kunming mice were purchased from Wensville Animal Co., Ltd. (license number: SCXK(Xiang)2019 - 0004). They were housed in an animal experiment center with SPF standards. Under the conditions of 25 ± 1°C, with 12 hours of light and 12 hours of darkness each, humidity (55% - 60%), provided with basic feed, and free access to water. And they were adaptively fed freely under these conditions for 7 days before the start of the experiment.

[0131] 3.2 Grouping of experimental animals

[0132] Thirty Kunming mice were randomly and evenly divided into 5 groups, including a normal group, low - dose, medium - dose, high - dose polygonatum cirrhifolium polysaccharide groups, and a positive control group. Mice in the normal group and the model group were given normal saline (0.1 ml / 10 g) by gavage every day, the positive group was given Red Bull energy drink (0.1 ml / 10 g) by gavage, and the administration groups were given different doses of polygonatum cirrhifolium polysaccharide solution by gavage. Each group of mice was gavaged for 30 days.

[0133] Table 2 Grouping of experimental animals for anti - fatigue experiment

[0134]

[0135] 3.3 Weight - loaded swimming experiment of mice

[0136] 30 minutes after the last administration, a wire with a weight of 5% of the body weight was loaded at the root of the tail of each mouse. The mouse was placed in a swimming tank with a water depth of 40 cm and a water temperature of 30 ± 2°C for swimming, that is, the weight - loaded swimming experiment was carried out. The stopwatch was used to time the time from the mouse entering the water until its head was completely submerged in the water and could not surface within 10 s, which was used as the weight - loaded swimming time of the mouse.

[0137] 3.4 Treatment of various tissues of mice and analysis of biochemical indexes

[0138] After the weight - loaded swimming, the mice were bled by eye - ball extraction, and the serum was separated and stored in a - 80°C refrigerator for later use. After blood collection, the mice were sacrificed by cervical dislocation, and the muscles and livers of the hind limbs of the mice were taken. After removing the connective tissue, they were rinsed twice with normal saline at 4°C and then divided and stored in a - 80°C refrigerator for later use. According to the kit instructions, the contents of serum urea nitrogen, hepatic glycogen, and muscle glycogen in the serum were measured. The data were recorded and analyzed.

[0139] 4 Study on the anti - hypoxia effect of polygonatum cirrhifolium polysaccharide

[0140] 4.1 Instruments, materials, reagents and experimental animals

[0141] 4.1.1 Instruments

[0142]

[0143] 4.1.2 Materials and reagents

[0144] Polygonatum cirrhifolium polysaccharide (prepared from Polygonatum cirrhifolium picked in Shannan City, Tibet); the basic mouse feed was purchased from Winesville Animal Co., Ltd., the superoxide dismutase (SOD) detection reagent, malondialdehyde (MDA) content detection reagent, and catalase (CAT) content test kit were all purchased from Shanghai Beyotime Biotechnology Co., Ltd.; the normal saline was purchased from Sichuan Kelun Pharmaceutical Co., Ltd.

[0145] 4.1.3 Experimental animals

[0146] 30 SPF-grade male Kunming mice were purchased from Winesville Animal Co., Ltd. (license number: SCXK(Xiang)2019-0004). They were housed in an animal experiment center under SPF standards, at 25±1°C, with 12 h of light and 12 h of darkness, and a humidity of (55%-60%). They were provided with basic feed and free access to water. And they were adaptively fed freely under these conditions for 7 d before the start of the experiment.

[0147] 4.2 Grouping of experimental animals

[0148] The 30 SPF-grade male Kunming mice were randomly and evenly divided into 4 groups, including a normal group, low, medium, and high-dose groups of Polygonatum cirrhifolium polysaccharide. The mice in the normal group were given normal saline (0.1 ml / 10 g) by gavage every day, and the mice in the dosing groups were given different doses of Polygonatum cirrhifolium polysaccharide solution by gavage. The mice in each group were gavaged for 30 d.

[0149] Table 3 Grouping of experimental animals for hypoxia tolerance experiment

[0150]

[0151] 4.3 Normobaric closed hypoxia experiment

[0152] 30 min after the last administration, the mice in each group were placed separately in a 250 mL wide-mouth bottle containing 10 g of soda lime and filter paper (1 mouse per bottle), the bottle mouth was sealed with a bottle stopper and vaseline, and the survival time of the mice was recorded. Survival time criteria: Starting from sealing the bottle mouth, the last breath stop (when each mouse suddenly went limp after struggling and twitching, and the chest no longer rose and fell) was used as the death indication, and the weight of each mouse and the survival time under normobaric closed hypoxia were observed and recorded.

[0153] 4.4 Processing of mouse tissues and organs and analysis of biochemical indexes

[0154] Mice that underwent normobaric hypoxia tolerance experiments were glansively charged with blood collection. Serum was separated and stored at -80°C for later use. Mice were euthanized by cervical dislocation after blood collection, and their heart, spleen, kidneys, liver, and brain tissue were harvested. After removing connective tissue, the tissue was rinsed twice with 4°C physiological saline and then aliquoted and stored at -80°C for later use. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), and catalase (CAT) were measured according to the kit instructions. Data were recorded and analyzed.

[0155] Results analysis:

[0156] 1. Extraction and purification of crude polysaccharides from Tibetan Polygonatum and preparation of Tibetan Polygonatum polysaccharide capsules

[0157] 1.1 Content of Polysaccharides from Tibetan Polygonatum

[0158] The content of polysaccharides in Tibetan Polygonatum (calculated as glucose) was measured, and the crude polysaccharide content of Tibetan Polygonatum was calculated to be 52.6% based on the standard curve.

[0159] 1.2 Isolation and purification of Polysaccharides from Tibetan Polygonatum

[0160] Tibetan Polygonatum polysaccharides were purified using DEAE-52 cellulose ion exchange resin, yielding four distinct fractions. The neutral polysaccharide obtained by elution with deionized water had the highest content, measuring 64.6%.

[0161] 1.3 Nuclear Magnetic Resonance Analysis of Polysaccharides from Tibetan Polygonatum

[0162] In the 1H NMR spectrum, the chemical shifts of hydrogen atoms are all in the region of 3.3 to 5.5 ppm, which is a typical characteristic signal of polysaccharides. The resonance signal at chemical shift 1.20 ppm is the methyl proton signal of rhamnose residues. The absence of signal at chemical shift 5.7 ppm indicates that there is no uronic acid in this part of the polysaccharide.

[0163] 1.4 Determination of the triple helix structure of Polygonatum sibiricum polysaccharide

[0164] When the NaOH concentration was in the range of 0–0.5 mol / L, the maximum absorption wavelength of the complex formed by Tibetan Polygonatum polysaccharide and Congo red was significantly higher than that of the control group. However, when the NaOH concentration reached 0.6 mol / L, the maximum absorption wavelength decreased somewhat; and when the NaOH concentration reached 0.7 mol / L, the maximum absorption wavelength decreased rapidly, suggesting that Polygonatum polysaccharide possesses a triple-helix structure. Studies have shown that polysaccharides with a triple-helix structure have better activity than those without this structure. This indicates that Tibetan Polygonatum polysaccharide may be a polysaccharide with strong biological activity.

[0165] 1.5 Preparation of Tibetan Polygonatum Polysaccharide Capsules

[0166] After mixing Tibetan Polygonatum polysaccharide and lactose monohydrate evenly in a certain proportion, the mixture of Tibetan Polygonatum polysaccharide and lactose is then mixed evenly with microcrystalline cellulose (model 301), and 2% talc powder is added and mixed evenly. The angle of repose of the capsule contents is measured to be less than 37 degrees. The contents are then filled into size 0 capsules to obtain the finished Tibetan Polygonatum polysaccharide capsules.

[0167] 1.6 Summary

[0168] This invention achieves the extraction and purification of Polygonatum sibiricum polysaccharides. Crude Polygonatum sibiricum polysaccharides were prepared using the classic water extraction and alcohol precipitation method, purified by deproteinization, and the content of the prepared Polygonatum sibiricum polysaccharides was determined using the anthrone-sulfuric acid method, yielding a purity of 52.6%. The purified Polygonatum sibiricum polysaccharides were further separated and purified using ion exchange chromatography, obtaining the highest yield fraction, with a purity of 64.6%. Furthermore, some structural features of the Polygonatum sibiricum polysaccharides were preliminarily detected through nuclear magnetic resonance analysis and the Congo red assay.

[0169] 2. Study on the in vivo immune-regulating effects of Polysaccharides from Tibetan Polygonatum

[0170] 2.1 Effects of Tibetan Polygonatum Polysaccharide on Immune Organs in Immunosuppressed Mice

[0171] like Figure 5 As shown in Figure 6, compared with the normal group, the spleen and thymus indices in the model group were significantly reduced (p<0.005), indicating that the CTX-induced immunosuppression model was successfully established. Compared with the model group, the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide significantly increased the spleen and thymus indices in immunosuppressed mice (p<0.0001), and the low and medium dose groups of Tibetan Polygonatum polysaccharide showed slightly better repair of the thymus than the positive levamisole group, but the difference was not statistically significant. These results indicate that Tibetan Polygonatum polysaccharide can improve CTX-induced spleen and thymus atrophy and enhance the body's immune function.

[0172] To further investigate the effects of Tibetan Polygonatum polysaccharides on the spleen and thymus, H&E staining was used for histopathological observation. Figure 6 As shown in Figure 7, the red and white pulp of the spleen in the model group were mixed and the borders were disordered, indicating that CTX could significantly suppress the immune status of the spleen and form tissue lesions. In the Tibetan Polygonatum polysaccharide treatment group, the pathological damage caused by CTX was effectively improved with increasing dosage, and this improvement was dose-dependent. In the high-dose Tibetan Polygonatum polysaccharide group, the boundary morphology of the red and white pulp returned to normal. In the model group, the boundary between the thymic medulla and cortex was blurred, and the number of lymphocytes was significantly less than that in the normal group, indicating that CTX could significantly suppress the immune function of the spleen and form tissue lesions. In the Tibetan Polygonatum polysaccharide treatment group, the boundary between the cortex and medulla gradually returned to normal, and the number of lymphocytes in the cortex returned to normal, while no obvious abnormalities were observed in the medulla. This indicates that Tibetan Polygonatum polysaccharide has a good protective effect on the immune organs of mice with CTX-induced immunosuppression.

[0173] 2.2 Effects of Tibetan Polygonatum Polysaccharide on Non-specific Immune Function in Immunosuppressed Mice

[0174] like Figure 9 As shown, compared with the normal control group, the phagocytic index (α) of the model group mice was significantly lower than that of normal mice (p<0.0001), indicating that cyclophosphamide can significantly reduce the phagocytic activity of mouse macrophages. Compared with the model group, the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide significantly increased the phagocytic index of macrophages in cyclophosphamide-induced immunosuppressed mice (p<0.05). Compared with the positive control group, there was no statistically significant difference among the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide. The results indicate that Tibetan Polygonatum polysaccharide can improve the phagocytic capacity of macrophages in immunosuppressed mice and has a good effect on improving the non-specific immune function of CTX-induced immunosuppressed mice.

[0175] 2.3 Effects of Polygonatum sibiricum polysaccharide on specific (humoral) immune function in immunosuppressed mice

[0176] Serum hemolysin is an indicator of humoral immunity levels and can enhance specific responses to thymus-dependent cell antigens. Increasing research indicates that natural polysaccharides can promote humoral immune function by stimulating the secretion of IgG, IgA, and IgM from serum hemolysin. For example... Figures 10-13 As shown, compared with the normal group, the production of serum hemolysin and immunoglobulins in the model group mice was significantly reduced (p<0.01), indicating that CTX can significantly inhibit humoral immune function. Compared with the model group, the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide significantly promoted the production of serum hemolysin, IgA, IgM, and IgG in a dose-dependent manner, and the high dose group was superior to the positive control group. There was no statistically significant difference between the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide compared with the positive control group. The results indicate that Tibetan Polygonatum polysaccharide has a good repair ability on the formation of serum hemolysin and immunoglobulins in CTX-induced immunosuppressed mice and can enhance humoral immune function.

[0177] 2.4 Effects of Tibetan Polygonatum Polysaccharide on Specific (Cellular) Immune Function in Immunosuppressed Mice

[0178] DNFB-induced DTH response is a cellular immune response that activates T lymphocyte differentiation to produce Th1 and Th17 cells, reflecting the body's cellular immune level. For example... Figure 14As shown, compared with the normal group, the ear weight difference in the model group was significantly reduced (p<0.05), indicating that the DNFB-induced DTH model had been successfully established. Compared with the model group, the medium and high dose groups of Tibetan Polygonatum polysaccharide significantly improved the ear weight difference in immunosuppressed mice (P<0.005), and were superior to the positive control group. Tibetan Polygonatum polysaccharide can effectively improve the cellular immune function of immunosuppressed mice. Compared with the positive control group, there was no statistically significant difference between the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide.

[0179] IFN-γ and IL-2 are secreted by Th1 cells and play important roles in inflammation mediation. IFN-γ has antitumor, antiviral, and immunomodulatory effects and mediates various cellular immune responses. IFN-γ can activate NO production in macrophages. NO is a major effector molecule that can stimulate the immune system, participate in apoptosis, and destroy target cells by inhibiting DNA synthesis, electron transfer, and the tricarboxylic acid cycle in mitochondria. IL-2 is an important cytokine in the growth, proliferation, and differentiation of T cells. Results are as follows... Figures 15-17 As shown, compared with the normal group, the serum secretion of IFN-γ, IL-2, and NO in the model group mice was significantly reduced (p<0.01), indicating that CTX can significantly inhibit the body's humoral immune function. Compared with the model group, the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide significantly promoted the increase of serum IFN-γ, IL-2, and NO secretion (p<0.005), and the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide showed a dose-dependent relationship.

[0180] 2.5 Effects of Polygonatum sibiricum polysaccharide on the expression of MAPK signaling pathway proteins

[0181] The MAPK signaling pathway comprises three major proteokinase groups (ERK, JNK, and p38) and is a downstream pathway of TLR4. Studies have shown that MAPKs can activate the NF-κB pathway, a key mediator of immune activation and inflammatory responses. ImageJ software was used for quantitative analysis of western blot bands. Results are shown below. Figure 18 As shown, compared with the normal group, the phosphorylation levels of p38, ERK, and JNK in the model group mice were significantly reduced (p<0.01). Compared with the model group, mice in the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide showed dose-dependent induction of phosphorylation of p38, ERK, and JNK (p<0.05). Compared with the positive control group, there was no statistically significant difference in ERK and JNK phosphorylation in the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide. The phosphorylation of p38 protein in mice in the low and medium dose groups of Tibetan Polygonatum polysaccharide was lower than that in the positive group with statistically significant differences (p<0.01), while there was no statistically significant difference between the high dose group of Tibetan Polygonatum polysaccharide and the positive group.

[0182] 2.6 Effects of Polygonatum sibiricum polysaccharide on the expression of upstream and downstream proteins in the MAPK signaling pathway

[0183] Western blot was used to detect proteins in the upstream TRL4 marker protein (Myd88) and downstream NF-κB (p65) pathway of the MAPK signaling pathway. NF-κB is a transcription factor that plays a crucial role in the production of pro-inflammatory cytokines and other mediators. Figure 19 As shown, compared with the normal group, the expression levels of Myd88 and p-p65 / p65 proteins in the model group mice were significantly reduced (p<0.01). Compared with the model group, the mice in the medium and high dose groups of Tibetan Polygonatum polysaccharide showed a dose-dependent increase in the expression levels of Myd88 and p-p65 / p65 proteins (p<0.05).

[0184] 2.7 Summary

[0185] The immunomodulatory effects of Polygonatum tsao-ko polysaccharides on a CTX-induced immunosuppressed mouse model were investigated from the perspectives of immune organs, non-specific immunity, humoral immunity, and cellular immunity. Immune organ index and histopathological observation experiments revealed that Polygonatum tsao-ko polysaccharides significantly increased spleen and thymus indices, improved CTX-induced spleen and thymus atrophy, significantly alleviated histological lesions of the spleen and thymus in immunosuppressed mice, and thus protected the function of immune organs in immunosuppressed mice. Carbon clearance experiments showed that Polygonatum tsao-ko significantly enhanced the phagocytic index of macrophages in immunosuppressed mice, thereby enhancing non-specific immunity. Serum hemolysin production and serum immunoglobulin assays showed that Polygonatum tsao-ko significantly promoted serum hemolysin production and increased serum immunoglobulin levels, thereby enhancing humoral immunity in immunosuppressed mice. Delayed-type hypersensitivity reaction experiments and serum cytokine and NO assays showed that Polygonatum tsao-ko significantly reversed CTX-induced ear weight reduction, increased serum cytokine and NO levels in immunosuppressed mice, and thus enhanced cellular immunity in immunosuppressed mice. To further elucidate the mechanism of action of Tibetan Polygonatum polysaccharide, verify the key proteins in the classic TLR4-MAPK-NF-κB pathway, and reveal its molecular mechanism, it promotes the phosphorylation of MAPKs by inducing MyD88 upregulation, thereby activating the phosphorylation of proteins in the NF-κB channel, and thus promoting the secretion of cytokines, thereby exerting an immune-enhancing effect.

[0186] 3. Study on the anti-fatigue effects of Polygonatum sibiricum polysaccharides

[0187] 3.1 Effects of Polygonatum sibiricum polysaccharide on mouse body weight

[0188] SPF-grade mice were allowed to eat freely for 7 days, then randomly assigned to groups for the experiment. At the start of the experiment, the daily condition of each group of mice was observed, including food and water intake, activity level, agility, changes in weight, and coat color, and compared with a normal control group, with a primary focus on weight monitoring. Figure 20As shown, during the anti-fatigue efficacy study, no abnormalities such as biting or death were observed in any of the groups of animals, and their coat color remained normal. Body weight increased with the extension of feeding days. Within 30 days of the experiment, there was no significant difference in body weight between the Polygonatum polysaccharide-treated group and the positive control group compared to the normal control group, indicating that low, medium, and high doses of Polygonatum polysaccharide had no adverse effect on the weight gain of mice.

[0189] 3.2 Effects of Polygonatum sibiricum polysaccharide on swimming time under load in mice

[0190] Improved exercise endurance is the most powerful and intuitive indicator of fatigue resistance, and among all fatigue resistance models, weighted swimming is the most effective and common model. For example... Figure 21 As shown, compared with the normal group, the high-dose group of Tibetan Polygonatum polysaccharide significantly prolonged the time to exhaustion during swimming under load in weight-bearing mice (p<0.005), and this effect was dose-dependent. This indicates that Tibetan Polygonatum polysaccharide can improve the body's exercise capacity and alleviate physical fatigue caused by weight-bearing swimming in mice.

[0191] 3.3 Effects of Tibetan Polygonatum Polysaccharide on Serum and Tissue Biochemical Indicators in Mice

[0192] When the body exercises for an extended period, the energy released from the metabolism of carbohydrates and fats is insufficient to sustain the activity. The body then increases protein metabolism to meet its energy needs, leading to an increase in blood urea nitrogen (BUN). Changes in BUN levels indicate the state of nitrogenous substance metabolism in the body and are a sensitive indicator of the body's ability to withstand physical exertion under specific conditions. The body's important energy reserves are liver glycogen and muscle glycogen. During exercise, the body initially consumes a large amount of muscle glycogen. When muscle glycogen is depleted, the body accelerates the consumption of liver glycogen to maintain stable blood glucose levels. Figure 22 As shown, urinary nitrogen ( Figure 22 (A) Compared with the blank control group, serum urinary nitrogen levels were significantly reduced in the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide (p < 0.005). This indicates that Tibetan Polygonatum polysaccharide solution delays fatigue by reducing serum BUN production. There was no significant difference in the medium dose group of Tibetan Polygonatum polysaccharide compared with the positive control group. (Muscle glycogen...) Figure 22 (B) Compared with the blank control group, the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide were significantly higher than the blank group (p < 0.01), and the low, medium, and high dose groups of Tibetan Polygonatum polysaccharide showed a dose-dependent trend. (Liver glycogen) Figure 22 (C) Compared with the blank control group, the high-dose group of Tibetan Polygonatum polysaccharide was significantly higher than that of the blank group (p<0.0001), but there was no significant difference between the medium and low-dose groups and the blank control group. The low, medium and high-dose groups of Tibetan Polygonatum polysaccharide showed a dose-dependent trend.

[0193] 3.4 Summary

[0194] Fatigue is a complex physiological and pathological phenomenon involving many physiological and biochemical reactions in the body. From a biological perspective, it manifests as energy depletion and metabolite accumulation. The duration of weight-bearing swimming in mice is an important indicator for evaluating anti-fatigue effects. Results of weight-bearing swimming experiments showed that, in the same water tank with the same water temperature and surface height, mice administered saline by gavage swam significantly shorter than those administered Polygonatum sibiricum polysaccharide by gavage. This indicates that Polygonatum sibiricum polysaccharide can prolong the swimming time of mice and enhance their exercise endurance. Furthermore, the increase in swimming time becomes more pronounced with increasing polysaccharide dosage. Therefore, the anti-fatigue effect of Polygonatum sibiricum polysaccharide is dose-dependent, with the high-dose group being superior to the positive control group. One factor contributing to fatigue is insufficient energy supply. The energy storage and metabolic capacity of liver glycogen and muscle glycogen are the main manifestations of the body's endurance and are also important factors in maintaining stable blood glucose levels. Numerous studies have demonstrated that energy resources are depleted during physical exhaustion; therefore, the content of liver glycogen and muscle glycogen is a major indicator for measuring the degree of fatigue. The experimental results comparing serum urinary nitrogen, liver glycogen, and muscle glycogen content in each group showed that Tibetan Polygonatum polysaccharide can increase the synthesis of liver glycogen and muscle glycogen, which can replenish energy in a timely manner, thereby reducing protein catabolism and reducing the production of urea nitrogen.

[0195] 4. Study on the hypoxia tolerance effect of Polysaccharide from Tibetan Polygonatum

[0196] 4.1 Effects of Polygonatum sibiricum polysaccharide on mouse body weight

[0197] SP-grade mice were given free access to food for 7 days, then randomly divided into groups for the experiment. At the start of the experiment, the daily condition of each group of mice was observed, including food and water intake, activity level, agility, changes in weight, and coat color, and compared with a normal control group, with a primary focus on weight monitoring. Figure 23 As shown, during the hypoxia tolerance study, no abnormalities such as bites or deaths were observed in any of the groups, and their coat color remained normal. Body weight increased with the duration of feeding. Within the 30-day experimental period, there was no significant difference in body weight between the Tibetan Polygonatum polysaccharide treatment group and the positive control group compared to the normal control group, indicating that low, medium, and high doses of Tibetan Polygonatum polysaccharide had no adverse effect on the weight gain of mice.

[0198] 4.2 Effects of Polygonatum sibiricum polysaccharides on normobaric hypoxia tolerance time in mice

[0199] like Figure 23 As shown, compared with the normal group, the level of Tibetan Polygonatum polysaccharide was low, while the level in the medium-dose group showed an increasing trend, with no significant difference. The high-dose group of Tibetan Polygonatum polysaccharide significantly increased the hypoxia tolerance time of mice (p<0.01). This indicates that Tibetan Polygonatum polysaccharide can improve the body's hypoxia tolerance time and prolong the survival time of mice.

[0200] 4.3 Effects of Tibetan Polygonatum Polysaccharide on Serum and Tissue Biochemical Indicators in Mice

[0201] Under hypoxic conditions, a large amount of reactive oxygen species and free radicals are generated in the body, causing oxidative damage. Catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) are indicators of oxidation and antioxidation

[41] , so SOD and MDA were measured in normobaric hypoxic mice. Figure 25 As shown in A and B, compared with the blank model group, mice in the medium and high dose groups of Tibetan Polygonatum polysaccharide showed significantly increased dose-dependent SOD and CAT enzyme activities (p < 0.05). Figure 25 C, MDA showed no significant differences among the groups.

[0202] 4.4 Summary

[0203] Hypoxia is an adverse stimulus to the body, affecting its oxidative energy supply. Under hypoxic conditions, free radicals cannot be cleared in time, leading to the accumulation of oxygen free radicals in the body, which in turn causes a strong lipid peroxidation reaction, resulting in irreversible damage to the body. The results of normobaric hypoxia tolerance experiments showed that high doses of Tibetan Polygonatum polysaccharide significantly prolonged the normobaric hypoxia tolerance time in mice. Catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) are common indicators of oxidation and antioxidation. Among them, CAT and SOD are antioxidant enzymes in the body, which can defend against oxidative damage generated in the body. MDA is the final decomposition product of lipid peroxides, which can reflect the rate and intensity of lipid peroxidation in the body, and can also indirectly reflect the degree of tissue peroxidation damage. The levels of SOD, CAT, and MDA in mice in the normobaric hypoxia tolerance experiment were measured. The results showed that Tibetan Polygonatum polysaccharide increased SOD and CAT in mice in a dose-dependent manner, but there was no difference in MDA among the groups. It is speculated that Tibetan Polygonatum polysaccharides may play a role in hypoxia resistance by promoting the production of antioxidant enzymes in the body, but do not prevent the decomposition of lipid peroxides.

[0204] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for extracting polysaccharides from Tibetan Polygonatum, characterized in that: The extraction method includes the following steps: S1. Use 95% ethanol to extract the Tibetan Polygonatum residue by hot reflux, and then dry the Tibetan Polygonatum residue until there is no alcohol smell. S2. Add the dried Tibetan Solomon's Seal residue powder to deionized water at a material-to-liquid ratio of 1:10 w / v, and extract at a constant temperature of 60℃ for 2 hours. Then filter the extract with gauze and concentrate the extract to 1 / 3 of the original volume under reduced pressure. Stir continuously and add 95% ethanol to make the final solution have a final concentration of 75%. S3. The final solution was placed in a refrigerator at 4°C for 12 hours to settle. The precipitate was centrifuged at 3000 r / min for 10 minutes, dissolved and evaporated, and then freeze-dried to obtain crude polysaccharide of Tibetan flavonoids.

2. The method for extracting Tibetan Polygonatum polysaccharides as described in claim 1, characterized in that: After undergoing deproteinization, dialysis, and purification, the crude polysaccharide of Tibetan Polygonatum was obtained as Tibetan Polygonatum polysaccharide.

3. The preparation process of Tibetan Polygonatum polysaccharide capsules as described in claim 1, characterized in that: The preparation process is as follows: Tibetan Polygonatum polysaccharide and lactose monohydrate are mixed evenly in a 1:1 ratio, and then the mixture of Tibetan Polygonatum polysaccharide and lactose is mixed evenly with microcrystalline cellulose in a 1:1.5 ratio. Finally, the mixture of Tibetan Polygonatum polysaccharide, lactose and microcrystalline cellulose is weighed, and 2% talc powder is added and mixed evenly to obtain the mixed contents. The angle of repose of the capsule contents is less than 37 degrees. The mixed contents are filled into No. 0 capsules to obtain the finished Tibetan Polygonatum polysaccharide capsules.

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