Bupleurum and white peony root polysaccharide composition, preparation method and application

Abstract

This invention discloses a polysaccharide composition of Bupleurum chinense and Paeonia lactiflora, its preparation method, and its applications, belonging to the field of macromolecular polysaccharides in traditional Chinese medicine. The composition comprises Bupleurum chinense polysaccharide CHTD-1 and Paeonia lactiflora polysaccharide BSTD-2 in a mass ratio of (1:3) to (3:1). The crude polysaccharides of Bupleurum chinense and Paeonia lactiflora are extracted using a water extraction and alcohol precipitation method, which yields high efficiency and does not easily alter the polysaccharide structure. Proteins in the crude polysaccharides are removed using the Sewage method. Refined polysaccharide fragments, CHTD-1, CHTD-2, BSTD-1, and BSTD-2, are obtained by passing the polysaccharides through a DEAE-52 cellulose column and a Sephadex G-100 gel column. The polysaccharides are combined, with the combination CB-2 (composed of CHTD-1 and BSTD-2) showing the best effect. Pharmacological experiments revealed that the combined use of CB-2 polysaccharides significantly enhances antidepressant effects and improves polysaccharide utilization, providing another feasible approach for the treatment of depression.

Description

Technical Field

[0001] This invention relates to a polysaccharide composition of Bupleurum chinense and Paeonia lactiflora, as well as its preparation method and application, belonging to the field of macromolecular polysaccharides in traditional Chinese medicine. Background Technology

[0002] Bupleurum polysaccharides, as the effective components of Bupleurum, have been confirmed by current research to possess various effects, including anti-inflammatory, immunomodulatory, anti-ulcer, anti-tumor, and anti-radiation properties. The modern pharmacological effects of Paeonia lactiflora mainly include: anti-inflammatory, analgesic, antipyretic, antispasmodic, antidepressant, antidiabetic, antitumor, antibacterial, antiviral, and antioxidant properties. It can also improve learning and memory abilities, regulate the body's immunity, affect the digestive system, affect the cardiovascular system, and has a certain protective effect on liver tissue.

[0003] Depression is a serious mental disorder. Currently, the pathogenesis of depression is not fully understood, and Western medicine still plays a dominant role in its treatment. However, Western medicines often have drawbacks such as a narrow antidepressant spectrum, significant side effects, high relapse rates, and long treatment cycles. Furthermore, the pathogenesis of depression is complex, and drug treatment targeting a single aspect is unlikely to achieve satisfactory results. Traditional Chinese medicine (TCM) has a long history of treating emotional disorders, and the clinical manifestations of "depression syndrome" are very similar to modern depression. The main method of TCM treatment for depression is to soothe the liver and relieve depression. Bupleurum and white peony are a commonly used classic herbal pair for soothing the liver and relieving depression, but their antidepressant active substances have not yet been elucidated, especially the pharmacological effects of their polysaccharide combination. This is the innovative point of this invention and also provides a basis for analyzing TCM herbal combination theory. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a polysaccharide composition of Bupleurum chinense and Paeonia lactiflora, which has a synergistic antidepressant effect.

[0005] Meanwhile, this invention provides a method for preparing a polysaccharide composition of Bupleurum chinense and Paeonia lactiflora, using traditional Chinese medicines Bupleurum chinense and Paeonia lactiflora as raw materials, which has the characteristics of significant effect, low toxicity and side effects, clear pharmacological effect and low cost.

[0006] Meanwhile, this invention provides the application of a Bupleurum and Paeonia lactiflora polysaccharide composition in the preparation of antidepressant drugs.

[0007] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A polysaccharide composition of Bupleurum and Paeonia lactiflora, comprising Bupleurum polysaccharide CHTD-1 and Paeonia lactiflora polysaccharide BSTD-2 in a mass ratio of (1:3) to (3:1).

[0008] The mass ratio of Bupleurum polysaccharide CHTD-1 to Paeonia lactiflora polysaccharide BSTD-2 is 2:1.

[0009] The monosaccharide composition of Bupleurum polysaccharide CHTD-1 is: galacturonic acid: xylose: galactose: glucuronic acid = 1:1.26:5.58:10.06.

[0010] The monosaccharide composition of Paeonia lactiflora polysaccharide BSTD-2 is: galacturonic acid: xylose: glucose: galactose = 1:1.81:6.38:9.96.

[0011] A method for preparing a polysaccharide composition of Bupleurum chinense and Paeonia lactiflora includes the following steps: S1, Extraction: Weigh 1.5 kg of Bupleurum chinense or Paeonia lactiflora powder separately and place them in a 20 L distillation flask. Add 15 L of distilled water at a material-to-liquid ratio of 1:10. After the liquid boils, heat under reflux for 2 hours and filter the liquid. Repeat the same process twice with the same material-to-liquid ratio and heating time for the residue. Discard the residue and combine the liquids from the three processes. Concentrate the liquid using a rotary evaporator until it becomes viscous and adheres to the walls, exhibiting a semi-fluid state. Place the concentrated liquid in a 5 L container, add 4 L of 95% ethanol, stir, and allow it to precipitate for 48 hours. Filter to obtain the precipitate, air-dry the precipitate for 24 hours, and then dry it in an oven to obtain Bupleurum chinense crude polysaccharide and Paeonia lactiflora crude polysaccharide, respectively. S2, protein removal: Sewage reagent was prepared according to the ratio of chloroform: n-butanol = 4:1; the crude polysaccharide powders of Bupleurum chinense and Paeonia lactiflora were respectively made into aqueous solutions with a concentration of 10 mg / ml, transferred to a 500 ml separatory funnel, added an equal amount of Sewage reagent, shaken, and allowed to stand overnight. The upper aqueous solution was taken, extracted three times, the extracts were combined, concentrated, and freeze-dried to obtain deproteinized polysaccharides of Bupleurum chinense and Paeonia lactiflora. S3, enrichment and purification Weigh 1g of deproteinized polysaccharide from Bupleurum chinense, dissolve it in distilled water, filter, and pour the filtrate into a DEAE-52 cellulose anion and cation exchange column. Elute with distilled water and 0.3 M NaCl solution, respectively. Identify whether the eluent contains polysaccharide using the sulfuric acid-phenol colorimetric method. Collect the polysaccharide solution, concentrate it to 50ml using a rotary evaporator, pour the concentrate into a square box with a liquid thickness of 1cm, and freeze it in the freezer for 24h. Place the frozen polysaccharide sample in a lyophilizer, freeze-dry it, and collect the sample. Obtain the water-washed fraction CH-1 and the salt-washed fraction CH-0.3 of the polysaccharide component. Similarly, enrich 1g of deproteinized polysaccharide from Paeonia lactiflora, and obtain the water-washed fraction BS-1 and the salt-washed fraction BS-0.3 of the polysaccharide component. S4, Sephadex G-100 gel enrichment: Weigh 300 mg of Bupleurum polysaccharide CH-1, dissolve in distilled water, sonicate for 5 min, filter, load, and perform Sephadex G-100 gel column chromatography. Elute with distilled water and 0.4 M NaCl solution, collecting 100 tubes of each solution using an automatic collector. Perform a phenol-sulfuric acid colorimetric reaction on even-numbered tubes and measure the absorbance at 490 nm. Plot the polysaccharide elution curve to obtain the water-eluted fraction CHTD-1 and the salt-eluted fraction CHTD-2. After drying, obtain Bupleurum polysaccharide CHTD-1. After dialyzing and drying, obtain Bupleurum polysaccharide CHTD-2. Similarly, 300 mg of Paeonia lactiflora polysaccharide BS-1 was enriched to obtain water-eluted fraction BSTD-1 and salt-eluted fraction BSTD-2, respectively; after drying, Paeonia lactiflora polysaccharide BSTD-1 was obtained; after dialysis and drying, Paeonia lactiflora polysaccharide BSTD-2 was obtained. S4, mix Bupleurum polysaccharide CHTD-1 and Paeonia lactiflora polysaccharide BSTD-2 to obtain a composition.

[0012] In S1, the rotary evaporator concentrates at a pressure of 60 Pa, uses tap water for condensation, has a water bath temperature of 55 °C, and a rotation speed of 30 r / min.

[0013] In S3, the rotary evaporator is concentrated at a pressure of 60 Pa, using tap water for condensation, with a water bath temperature of 55℃ and a rotation speed of 3000 r / min; the freeze dryer is preset to a pressure of 29 Pa, a temperature of -40℃, and a freeze-drying time of 48 h.

[0014] In S3, the specifications of the DEAE-52 cellulose anion and cation column are: 60cm × 3.5cm, and the filler is 500g.

[0015] In S4, the specifications of the Sephadex G-100 gel column chromatography are: 60cm × 3.5cm, and the packing material is 500g.

[0016] Application of a polysaccharide composition of Bupleurum chinense and Paeonia lactiflora in the preparation of antidepressant drugs.

[0017] The research group received funding from the National Natural Science Foundation of China (Study on the pharmacodynamic substances and mechanisms of Bupleurum-Paeonia lactiflora in the fight against depression based on "serum pharmacochemistry-pharmacokinetics-metabolomics", No:81803711). The group evaluated the antidepressant effects of Bupleurum-Paeonia lactiflora, Bupleurum, and Paeonia lactiflora extracts and found that Bupleurum and Paeonia lactiflora polysaccharides have significant antidepressant activity.

[0018] This invention studies the synergistic antidepressant effect of a specific ratio of Bupleurum and Paeonia lactiflora polysaccharides.

[0019] This invention provides a polysaccharide composition of Bupleurum chinense and Paeonia lactiflora, along with its preparation method and applications. The technical solution adopted in this invention utilizes the antidepressant effect of a polysaccharide combination. Crude polysaccharides of Bupleurum chinense and Paeonia lactiflora are obtained by water extraction and alcohol precipitation, a method with high yield and minimal alteration to the polysaccharide structure. Proteins and pigments are removed from the crude polysaccharides using the Sewage method. Refined polysaccharide fragments, CHTD-1, CHTD-2, BSTD-1, and BSTD-2, are obtained by passing the polysaccharides through a DEAE-52 cellulose column and a Sephadex G-100 gel column. Polysaccharide components CHTD-1, CHTD-2, BSTD-1, and BSTD-2 were combined to form CHTD-1 combined with BSTD-1 (CB-1), CHTD-1 combined with BSTD-2 (CB-2), CHTD-2 combined with BSTD-1 (CB-3), and CHTD-2 combined with BSTD-2 (CB-4) for antidepressant experiments. The polysaccharide combination CB-2 showed the best effect, and its structure was preliminarily characterized. CB-2 is composed of CHTD-1 and BSTD-2. HPLC-ELSD analysis revealed a single peak with symmetrical peak shape, indicating that the purified polysaccharide component is homogeneous and of high purity.

[0020] The beneficial effects of this invention are: the polysaccharide is extracted using water extraction and alcohol precipitation, resulting in a high polysaccharide content. The polysaccharide component is obtained using DEAE-52 cellulose and Sephadex G-100 gel. Pharmacological experiments have shown that the combined antidepressant effect of the polysaccharide is significant, improving the utilization rate of the homogeneous polysaccharide and providing another feasible method for the treatment of depression. Attached Figure Description

[0021] Figure 1 The peak is CH-1 in liquid chromatography. Figure 2 The peak is BS-1 in liquid chromatography. Figure 3 The values ​​represent the rat's body weight and food intake. Figure 4 The upright time of the rat; Figure 5 This represents the amount of sucrose consumed by the rats. Figure 6 The immobile swimming time of the rat; Figure 7 To investigate the body weight and food intake of CB-2 rats; Figure 8 To investigate the upright time of CB-2 rats; Figure 9 To investigate the sucrose consumption rate of CB-2 rats; Figure 10 To investigate the immobility time of CB-2 rats during swimming; Figure 11 High performance liquid chromatograms of CHTD-1 and BSTD-2; Figure 12 HPLC chromatogram of PMP derivatization of mixed monosaccharide standard; Figure 13 The HPLC chromatogram of PMP derivatization of CHTD-1 sample hydrolysate; Figure 14 The HPLC chromatogram of PMP derivatization of BSTD-2 sample hydrolysate; Figure 15 These are the UPLC chromatograms for each group in positive ion mode; Figure 16 The UPLC chromatograms for each group are shown in negative ion mode. Figure 17 PCA score charts for each group. Detailed Implementation

[0022] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Example

[0023] 1. Polysaccharide extraction and content determination 1.1 Extraction The extraction of polysaccharides from Bupleurum chinense (white peony root) was performed using a heated reflux extraction followed by high-concentration ethanol precipitation. 1.5 kg of crude Bupleurum chinense (white peony root) powder was weighed and placed in a 20 L distillation flask. 15 L of distilled water was added at a solid-liquid ratio of 1:10. After the liquid boiled, it was heated under reflux for 2 hours, and the liquid was filtered. The residue was processed twice more using the same solid-liquid ratio and heating time. The residue was discarded, and the three liquids were combined to obtain approximately 45 L of liquid. This liquid was concentrated using a rotary evaporator (pressure 60 Pa, tap water condensation, water bath temperature 55℃, rotation speed 30 r / min) until it became viscous and adhered to the walls, reaching a semi-fluid state of approximately 1 L. The concentrated liquid was placed in a 5 L container, and 4 L of 95% ethanol was added. After stirring, precipitation was carried out for 48 hours. The precipitate was obtained by suction filtration, air-dried for 24 hours, and then dried in an oven to obtain 277.6 g of crude Bupleurum chinense polysaccharide (243.1 g of crude white peony root polysaccharide).

[0024] 1.2 Content Determination 1.2.1 Determination of polysaccharide content Sugar content is generally determined using the phenol-sulfuric acid colorimetric method, which is simple to operate, highly controllable, and highly sensitive. Weigh 22 mg of Glc standard, place it in a volumetric flask, dissolve and dilute to volume, and shake well. Add the reagents according to the amounts in Table 1 to seven stoppered test tubes, shake well, and incubate in an 80°C water bath for 20 min.

[0025] Table 1. Data for plotting the glucose standard curve

[0026] The test tube reaction was cooled to room temperature, and the absorbance at 490 nm was measured and recorded. A calibration curve was plotted with the concentration of the Glc standard solution on the x-axis and the absorbance at 490 nm on the y-axis. The standard curve equation was obtained as y = 13.039x + 0.0448, R0 2 =0.9953.

[0027] Determination of polysaccharide content: 12.1 mg of crude polysaccharide from Bupleurum chinense was weighed, dissolved in distilled water, and prepared into a solution of 59 ug / ml. The solution was measured three times in parallel according to the method of test tube No. 3. The polysaccharide content of Bupleurum chinense was 46.61% and that of Paeonia lactiflora was 41.35% by calculating the average value through the standard curve of sugar content.

[0028] 1.2.2 Determination of uronic acid content The uronic acid content was determined using the common carbazole-sulfuric acid colorimetric method. A 0.1% carbazole solution (50 mg carbazole, 50 ml 95% ethanol) was prepared and shaken well. 10.1 mg of standard GlcA was weighed, dissolved in distilled water, and shaken well. The reagents were added to seven stoppered test tubes according to the amounts shown in Table 2. 6 ml of concentrated sulfuric acid was added, and the tubes were incubated in a water bath at 85°C for 25 min until the standard reaction was complete. The corresponding amount of carbazole reagent was then added to each test tube, and the mixture was allowed to develop color at room temperature for 2 h.

[0029] Table 2. Data for plotting the glucuronic acid standard curve

[0030] After complete color development, pour 2 / 3 of the solution into a glass cuvette, measure the absorbance at 530 nm, record the data, and plot a standard curve. The equation of the standard curve is y = 3.1174x + 0.0215, R0. 2 =0.99.

[0031] Determination of polysaccharide uronic acid content: Accurately weigh 20.1 mg of sample, prepare a 0.43 mg / ml solution with distilled water, and measure three times according to the standard method and calculate the average value. The content of saiko uronic acid in the sample was 24.36% and the content of paeony uronic acid was 17.28%.

[0032] 1.2.3 Protein content determination Coomassie Brilliant Blue is a commonly used dye for measuring protein content. This dye is red in its free state with a maximum absorption wavelength of 465 nm, and turns cyan after binding to proteins. The bound protein complex has a maximum absorption wavelength of 595 nm, and the reaction is rapid and stable. The absorbance value is directly proportional to the concentration within a certain range.

[0033] Prepare the required Coomassie Brilliant Blue reagent according to the ratio of Coomassie Brilliant Blue dye: 95% ethanol: concentrated phosphoric acid: distilled water = 2:1:2:25.

[0034] Preparation of bovine serum albumin (BSA) standard solution: Weigh 10.1 mg of protein standard and place it in a 100 ml volumetric flask. Dissolve and dilute with distilled water to prepare a protein standard solution with a concentration of 0.101 mg / ml.

[0035] According to Table 3, add protein standard solution, distilled water and G250 reagent to the test tube in sequence, incubate at 30°C for 5 minutes, and then cool to room temperature.

[0036] Table 3. Data for plotting protein standard curves

[0037] The absorbance of the protein-dye complex was measured at the wavelength of maximum absorption. A standard curve equation was fitted with protein content on the x-axis and absorbance on the y-axis. The resulting standard curve equation is y = 8.8544x + 0.0243, R0. 2 = 0.9929.

[0038] Accurately weigh 49.9 mg of sample and place it in a 50 ml volumetric flask. Dissolve the sample in distilled water and dilute to a volume of 1 mg / ml. Perform the test according to the procedure for plotting a standard curve. Perform the test three times in parallel and calculate the average value through the standard curve. The results show that the bupleurum protein content of the sample is 6.05% and the white peony protein content is 8.69%.

[0039] After heating and reflux followed by alcohol precipitation, 277.6 g of crude polysaccharide from Bupleurum chinense (yield 18.51%) and 243.1 g of crude polysaccharide from Paeonia lactiflora (yield 16.2%) were obtained. The sugar, uronic acid, and protein contents of the polysaccharides were determined using the sulfuric acid-phenol method, the sulfuric acid-carbazole method, and Coomassie Brilliant Blue G-250 staining method. The results showed that Bupleurum chinense polysaccharide content was 46.61%, uronic acid content was 24.36%, and protein content was 6.05%; while Paeonia lactiflora polysaccharide content was 41.35%, uronic acid content was 17.28%, and protein content was 8.69%.

[0040] 2. Polysaccharide isolation and purification 2.1 Protein Removal The Sewage method was used to deproteinize the crude polysaccharides of Bupleurum chinense and Paeonia lactiflora. Sewage reagent was prepared according to a chloroform:n-butanol ratio of 4:1. The crude polysaccharide powders of Bupleurum chinense and Paeonia lactiflora were dissolved in water to a concentration of 10 mg / ml. The solution was transferred to a 500 ml separatory funnel, and an equal volume of Sewage reagent was added. The mixture was shaken, allowed to stand overnight, and the supernatant was collected. The mixture was extracted three times, and the extracts were combined, concentrated, and freeze-dried to obtain the deproteinized polysaccharides of Bupleurum chinense and Paeonia lactiflora.

[0041] 2.2 Polysaccharide enrichment and purification 2.2.1 Enrichment of cellulose by DEAE-52 column chromatography Weigh 1g of Bupleurum chinense deproteinized polysaccharide, dissolve it in distilled water, filter, and pour the filtrate into a DEAE-52 cellulose anion and cation exchange column (medium-pressure column specifications: 60cm×3.5cm, packing material 500g). Elute with distilled water and 0.3M NaCl solution respectively. Identify whether the eluent contains polysaccharide by the sulfuric acid-phenol colorimetric method. Collect the polysaccharide solution and concentrate it to about 50ml using a rotary evaporator (pressure 60pa, tap water condensation, water bath temperature 55℃, rotation speed 3000r / min). Pour the concentrate into a square box with a liquid thickness of 1cm and freeze it in the freezer for 24h. Place the frozen polysaccharide sample in a lyophilizer with preset parameters of pressure 29pa, temperature -40℃, and freeze-dry for 48h. Collect the sample. Obtain polysaccharide components CH-1 (washed with water) and CH-0.3 (washed with 0.3mol / L NaCl). 0.26 g of CH-1 was obtained, with a calculated conversion rate of 26%; 0.31 g of CH-0.3 was obtained, with a calculated conversion rate of 31%. The purity of CH-1 was tested, such as... Figure 1 As shown, CH-1 is not a single chromatographic peak. The next step is to separate CH-1 by column chromatography using a gel column.

[0042] 1g of deproteinized polysaccharide from Paeonia lactiflora was similarly enriched to obtain polysaccharide fractions BS-1 (washed with water) and BS-0.3 (washed with 0.3mol / L NaCl). 0.18g of BS-1 was obtained, with a calculated conversion rate of 18%; 0.14g of BS-0.3 was obtained, with a calculated conversion rate of 14%. The purity of BS-1 was determined, as shown below... Figure 2 As shown, BS-1 is not a single chromatographic peak. The next step is to separate BS-1 by column chromatography using a gel column.

[0043] 2.2.2 Sephadex G-100 gel enrichment Weigh 300 mg of Bupleurum polysaccharide CH-1, dissolve in distilled water, sonicate for 5 min, filter, load, and perform Sephadex G-100 gel column chromatography (medium-pressure column specifications: 60 cm × 3.5 cm, packing material 500 g). Elute with distilled water and 0.4 M NaCl solution, collecting 100 tubes of each solution using an automatic collector. Perform a phenol-sulfuric acid colorimetric reaction on even-numbered tubes and measure the absorbance at 490 nm. Plot the polysaccharide elution curve to obtain CHTD-1 (eluted with water) and CHTD-2 (eluted with 0.4 M NaCl). The water-eluted fractions are CHTD-1 (51 mg, conversion rate 17%) and CHTD-2 (73 mg, conversion rate 24%).

[0044] Similarly, 300 mg of Paeonia lactiflora polysaccharide BS-1 was enriched to obtain BSTD-1 (eluted with water) and BSTD-2 (eluted with 0.4 M NaCl). BSTD-1 (77 mg, conversion rate 26%) and BSTD-2 (64 mg, conversion rate 21%) were obtained.

[0045] Using DEAE-52 cellulose and Sephadex G-100 gel as carriers, gradient elution was performed with water and different concentrations of NaCl. After dialysis and freeze-drying, four polysaccharide components CHTD-1, CHTD-2, BSTD-1, and BSTD-2 were obtained.

[0046] 3. Pharmacodynamic screening of polysaccharide combinations for antidepressant effects The polysaccharide components CHTD-1, CHTD-2, BSTD-1, and BSTD-2 were combined to form CHTD-1 combined with BSTD-1 (CB-1), CHTD-1 combined with BSTD-2 (CB-2), CHTD-2 combined with BSTD-1 (CB-3), and CHTD-2 combined with BSTD-2 (CB-4), with the dosage ratio referring to the ratio of Bupleurum chinense to Paeonia lactiflora in the research group (1:1), and an antidepressant experiment was conducted.

[0047] 3.1 Behavioral Testing Experiment 3.1.1 Extract Preparation The preparation methods for polysaccharide components CHTD-1, CHTD-2, BSTD-1, and BSTD-2 are described in "2. Polysaccharide Isolation and Purification".

[0048] 3.1.2 Grouping After a 7-day acclimatization period before the experiment, each rat was tested using the open-field test (OFT). Rats with similar scores were randomly divided into 11 groups of 10 rats each (5 males and 5 females). These groups were: Model group, Control group, Fluoxetine positive control group (PD), CB-1, CB-2, CB-3, CB-4, CHTD-1, CHTD-2, BSTD-1, and BSTD-2.

[0049] 3.1.3 Model Replication A rat depression model was established by referring to the modeling methods in the literature and improving upon the original methods based on practical experience. The control group was housed together with their parents, with normal food and water, and no stimulation was given. Except for the control group, the other groups were equipped with a rat depression model by chronic unpredictable stressors (CUMS) combined with solitary rearing. Nine stimulation methods were used: 24-hour food deprivation, 24-hour water deprivation, ice water swimming, reversed day-night cycle, tail clamping, damp bedding, empty bottle fear, noise, and electric shock to the soles of the feet. One to two combinations of the modeling methods were randomly selected each day without repetition. The same stimulation was used no more than 8 times in total. A modeling cycle was 7 days, and the modeling was completed after 8 weeks (56 days).

[0050] The specific methods of stress stimulation are as follows: (1) Deprivation of food for rats for 24 hours: food deprivation began at 8:30 am and ended at 8:30 am the next day.

[0051] (2) Deprivation of water bottles for rats for 24 hours: Deprivation of water bottles began at 8:30 am and ended at 8:30 am the next day.

[0052] (3) Ice water swimming: At 9:00 a.m., the rats were placed in a bucket of ice water at about 4±1℃. The specifications were: height 50cm, diameter 21cm, water depth 20cm. Each rat swam for 5 minutes. The rats' toes should not touch the bottom of the bucket. At the end, the rats were dried with a towel to avoid frostbite. After the rats had moved steadily, they were put back into their original cages.

[0053] (4) Reversing day and night: Cover the rat cage with a black cloth at 8:30 am, ensuring ventilation. 12 hours later, at 8:30 pm, remove the black cloth and turn on the fluorescent lights in the room. This creates an environment that is dark during the day and bright at night.

[0054] (5) Tail clamping: At 8:30 a.m., fix the rat's body, expose the tail, and use surgical forceps to gently clamp the rat 1 cm away from the tail root. The clamping time is calculated from the time the rat makes its first cry, and the clamping time is 1 min.

[0055] (6) Moist bedding: At 8:30 a.m., the rat bedding was soaked in water until 8:30 a.m. the next day to create a moist environment for the rats.

[0056] (7) Empty bottle fear method: The rat was bound and placed in a 550mL mineral water bottle, and its free movement was restricted for 2 hours.

[0057] (8) Noise stimulation: Use an ultrasonic cleaner to create noise stimulation for 2 hours.

[0058] (9) Foot shock: At 8:30 a.m., the rats were placed in a foot shock box with a voltage of 32V. The shock was given once every 10 seconds for 2 seconds each time, for a total of 10 times.

[0059] 3.1.4 Administration The drug was administered while the model was being created. During the modeling process, except for the control group and the model group, each group was administered the drug by gavage for 56 consecutive days, with a positive drug (PD) dose of 1.8 mg / kg.

[0060] The dosages for each group are calculated based on the extraction rate as follows: The dosages were as follows: CB-1 0.142 g / kg, CB-2 0.117 g / kg, CB-3 0.192 g / kg, CB-4 0.167 g / kg, CHTD-1 0.074 g / kg, CHTD-2 0.124 g / kg, BSTD-1 0.068 g / kg, and BSTD-2 0.043 g / kg.

[0061] To avoid differences in therapeutic effects due to different dosages, the dosage for all groups in this experiment was uniformly set at 0.192 g / kg.

[0062] 3.1.5 General Indicator Observation Observe the rats' mental state, respiration, coat color, feces, endocrine secretions, and spontaneous movement ability every day; record the rats' weight and food intake once a week at the same time.

[0063] 3.1.6 Behavioral Testing Methods Behavioral tests were performed on rats in each group after they were given the drug for 56 consecutive days.

[0064] (1) Open field test: The open field device was made of opaque material and the test was conducted in a room at 8:30 am. Absolute silence was maintained during the test. The rat was slowly placed in the center of the open field device. After acclimatization for 1 minute, the number of times the rat stood upright in 5 minutes was recorded (two front paws leaving the ground by 1 cm or climbing the wall was counted as one time). Each rat was tested in the open field only once. After the test, the open field device was thoroughly wiped with 10% alcohol before the next rat was tested to avoid residual feces and odor affecting the accuracy of the experiment. The open field test was used to observe the rat's spontaneous activity behavior and exploration ability.

[0065] (2) Sugar water consumption experiment: After the administration of the drug, the rats were trained to prefer sugar water. In a quiet and independent room, each rat was housed alone and provided with two bottles of 1% sucrose water to adapt to the sucrose solution. On the first day, two bottles of 1% sugar water were provided to train the rats to adapt to drinking sugary water; on the second day, one bottle of 1% sucrose water and one bottle of pure water were provided; on the third day, one bottle of 1% sucrose water and one bottle of pure water were provided; on the fourth day, a sugar water preference test was conducted by giving the rats one bottle of pre-prepared 1% sucrose water and one bottle of pure water, which were removed after 24 hours, and the consumption of sugar water and pure water was measured respectively.

[0066] (3) Forced swimming test: The forced swimming test was conducted on day 56 of the experiment. Rats were placed in a self-made rat test tank with a diameter of 25 cm and a height of 60 cm. The water temperature was (25±1) ℃ and the water depth was about 20 cm. During the process, it was ensured that the rats' hind feet could not touch the bottom of the tank. The day before the forced swimming test (i.e., day 55), each group of rats first swam for 15 minutes (pre-swimming can eliminate the acute stress caused by stress on rats), and then returned to their original cages. One day later, the forced swimming test was started. After 1 minute of adaptation, the total time that the rats stopped swimming during the 5-minute forced swimming test was recorded (the total time that the rats stopped swimming was the sum of the time when the rats' heads floated on the surface of the water and remained still or when their limbs made slight paddling movements).

[0067] 3.1.7 Behavioral test results (1) Growth status of rats Throughout the experiment, the control group rats exhibited the best mental state, good nutritional status, gradual weight gain, smooth and shiny fur, and heightened responsiveness. They drank and ate normally, urinated normally (pale yellow), and had formed, odorless feces. One week after modeling, the model group showed signs of lethargy, listlessness, limb weakness, disheveled and dull fur, loss of appetite, and significantly slower weight gain compared to other groups.

[0068] After the experiment ended on day 56, the mental state of rats in all treatment groups, especially the fluoxetine and polysaccharide groups, was significantly improved compared to the model group. Their water and food intake were relatively normal, and their individual development was close to that of the control group. Compared to the control group, the body weight and food intake of rats in the model group were significantly reduced (P<0.01), indicating successful replication of the CUMS model. The rats exhibited decreased appetite, stunted growth, and symptoms of depression. Compared to the model group, the body weight and food intake of rats in all treatment groups were significantly increased (P<0.01, P<0.05). Figure 3 As shown, the polysaccharide combinations CB-1, CB-2, CB-3, and CB-4 are superior to the single polysaccharide combinations CHTD-1, CHTD-2, BSTD-1, and BSTD-2.

[0069] (2) Open field experiment like Figure 4 As shown, compared with the control group, the number of upright movements in the model group rats on day 56 was significantly reduced (P < 0.01), indicating decreased motor and exploratory abilities and exhibiting core symptoms of depression. After drug administration, compared with the model group rats, the positive control group (P < 0.01), polysaccharide combination group (P < 0.01, P < 0.05), and each single polysaccharide group (P < 0.05) all significantly increased the number of upright movements in depressed rats. Furthermore, the polysaccharide combination was significantly superior to the single polysaccharide groups.

[0070] (3) Experiment on sugar water consumption like Figure 5As shown, the sucrose preference results on day 56 indicated that, compared with the control group, the sucrose consumption in the model group rats was significantly reduced (P < 0.01). After drug administration, compared with the model group, the sucrose consumption in the positive control group (P < 0.01), the polysaccharide combination group (P < 0.01, P < 0.05), and each single polysaccharide group (P < 0.05) were all significantly increased. The results indicate that the polysaccharide combination can significantly reverse the anhedonia phenomenon in depressed rats. Furthermore, the polysaccharide combination is significantly superior to the single polysaccharide groups.

[0071] (4) Forced swimming experiment like Figure 6 As shown, the results of forced swimming cessation time showed that, compared with the blank group, the forced swimming cessation time of rats in the model group was significantly prolonged (P < 0.01). After drug administration, compared with the model group, the positive drug group (P < 0.05), high-dose group (P < 0.01), polysaccharide combination group (P < 0.01), and each single polysaccharide group (P < 0.05) all significantly shortened the cessation time of CUMS rats; and the polysaccharide combination was significantly better than the single polysaccharide group.

[0072] Summary of behavioral experiments: This study used the CUMS method to establish a depression model. After 56 days of CUMS stimulation, all rats developed a depressive state. Compared to the control group, the model group rats showed significantly reduced food intake, number of times they stood upright, sucrose consumption, and time spent motionless during forced swimming, along with slower weight gain, indicating successful establishment of the depression model.

[0073] In the observation of general indicators and food intake test of rats, compared with the blank group, the rats in the model group had poor mental state, messy and dull fur, significantly decreased food intake, and decreased activity. Compared with the model group, the individual development of each drug administration group was close to that of the blank group. Among them, the rats in the CHTD-1 combined with BSTD-2 (CB-2) polysaccharide group had the best mental state and significantly increased food intake.

[0074] Decreased appetite leading to weight loss is one of the main causes of depression. In the weight loss experiment, all drug-treated groups were able to regulate the weight of rats, with the polysaccharide group showing a more significant increase in the weight of depressed rats. The regulatory effect was stronger than that of the positive control drug, and there was no significant difference between the groups.

[0075] Decreased motor and exploratory abilities are typical symptoms of depression. In the open field test, the number of upright movements in the model group rats decreased, while all drug-treated groups significantly increased the number of upright movements. The polysaccharide group showed a better recovery effect than the positive control group, with no significant difference between groups. This indicates that the polysaccharide combination can alleviate depressive behavior in rats and increase their motor and exploratory behaviors.

[0076] Anhedonia, caused by loss of interest in sweet substances, is one of the core symptoms of depression. In a sucrose consumption experiment, compared with the control group, the model group rats consumed less sucrose and less fresh water, indicating that the rats exhibited depressive symptoms due to anhedonia; compared with the model group, all treatment groups showed a significant reversal of this trend.

[0077] Experiencing hopelessness in daily life is also a significant factor in the development of depression. In the forced swimming experiment, the immobility time of rats in the model group was significantly prolonged, while the polysaccharide group significantly reduced the immobility time, with therapeutic effects comparable to the positive control group, indicating that polysaccharides can effectively improve depressive hopelessness behaviors in depressed rats. Among these, the polysaccharide combination with CB-2 showed the best effect, and CB-2 will be selected for further validation experiments in the next step.

[0078] 4. Pharmacodynamic Study of the Polysaccharide Combination CB-2 for Antidepressant Effect In the early stages of this study, pharmacodynamic experiments were conducted on the CHTD-1 and BSTD-2 dosage ratios of 1:1, 1:2, 2:1, 1:3, and 3:1, as well as on single doses of both. Ultimately, the optimal dosage of CHTD-1:BSTD-2 = 2:1 was selected.

[0079] 4.1 Behavioral Testing Experiment 4.1.1 Extract Preparation For the preparation method of polysaccharide components, see "2 Polysaccharide Isolation and Purification".

[0080] 4.1.2 Grouping The experiment was divided into: blank group, model group, positive drug group, high (CB-2H), medium (CB-2M), and low (CB-2L) polysaccharide combination groups, CHTD-1 group, and BSTD-2 group.

[0081] 4.1.3 Model Replication Same as "3.1.3 Model Replication".

[0082] 4.1.4 Administration Except for the control group and the model group, all groups were administered the drug by gavage for 56 consecutive days, with a positive control (PD) dose of 1.8 mg / kg. The dosages for the high (CB-2H), medium (CB-2M), and low (CB-2L) polysaccharide groups, converted according to their extraction rates, are as follows: The dosages were 0.96 g / kg, 0.64 g / kg, and 0.32 g / kg, respectively; the CHTD-1 dosage was 0.31 g / kg; and the BSTD-2 dosage was 0.29 g / kg.

[0083] To avoid differences in therapeutic effects due to different dosages, the unified dosage of CHTD-1 and BSTD-2 is 0.96 g / kg.

[0084] 4.2 Behavioral test results 4.2.1 Body weight and food intake like Figure 7 As shown, compared with the model group, the body weight and food intake of each treatment group increased significantly, specifically the positive control group (P<0.01), high-dose group (P<0.01), medium-dose group (P<0.01, P<0.05), and low-dose group (P<0.05). The results indicate that all treatment groups can increase the body weight and food intake of rats, with the high-dose group showing a better regulatory effect than the medium and low-dose groups. The polysaccharide combination is superior to the CHTD-1 and BSTD-2 dosage groups.

[0085] 4.2.2 Open Field Experiment like Figure 8 As shown, compared with the control group, the number of upright movements in the model group rats on day 56 was significantly reduced (P < 0.01), indicating decreased motor and exploratory abilities, exhibiting core symptoms of depression. After drug administration, compared with the model group rats, the positive control group (P < 0.01), and the high, medium, and low dose groups (P < 0.01) all significantly increased the number of upright movements in depressed rats. The results indicate that the polysaccharide combination can improve open field activity in depressed rats, with a recovery rate comparable to the positive control group, and no significant difference between groups. Furthermore, the polysaccharide combination is significantly superior to the CHTD-1 and BSTD-2 dose groups.

[0086] 4.2.3 Experiment on sugar water consumption like Figure 9 As shown, on day 56, the sucrose preference results indicated that, compared with the control group, the sucrose consumption in the model group rats was significantly reduced (P < 0.01). After drug administration, compared with the model group, the sucrose consumption in the positive control group (P < 0.01), and the high, medium, and low dose drug pairs (P < 0.01) were all significantly increased. The results indicate that polysaccharides can significantly reverse the anhedonia phenomenon in depressed rats. Furthermore, the polysaccharide combination was significantly superior to the CHTD-1 and BSTD-2 dose groups.

[0087] 4.2.4 Forced Swimming Experiment like Figure 10 As shown, the results of forced swimming cessation time on day 56 showed that, compared with the control group, the forced swimming cessation time of rats in the model group was significantly prolonged (P < 0.01). After drug administration, compared with the model group, the positive control group (P < 0.05), high (P < 0.01), medium-dose drug pair group (P < 0.05), and low-dose drug pair group (P < 0.05) all significantly shortened the cessation time of CUMS rats. The cessation time of rats in the high-dose drug pair group was comparable to that of the positive control group. The results indicate that polysaccharides can effectively reverse the despair phenomenon in rats. Furthermore, the polysaccharide combination was significantly superior to the CHTD-1 and BSTD-2 dose groups.

[0088] In summary, the high, medium, and low doses of the polysaccharide combination CB-2 significantly improved depressive behavior, demonstrating a significant antidepressant effect. Furthermore, the CB-2 polysaccharide combination was significantly superior to the CHTD-1 and BSTD-2 dose groups.

[0089] 5. Preliminary structural characterization of polysaccharide CB-2 Preliminary structural characterization of the CHTD-1 and BSTD-2 polysaccharide components was performed to provide a basis for polysaccharide quality control.

[0090] 5.1 Uniformity Detection Chromatographic conditions: mobile phase, water; column: TSKgel G4000PWXL 7.8mm ID*30cm, 10μm.

[0091] Prepare a 1 mg / ml aqueous solution of CHTD-1 and BSTD-2 and inject it into the HPLC system. Figure 11 As shown, the chromatographic peak is a single peak, indicating that the component is a homogeneous polysaccharide.

[0092] 5.2 Monosaccharide Composition Analysis (1) Preparation of reference solution Accurately weigh eight monosaccharide reference standards: mannose, rhamnose, galacturonic acid, glucuronic acid, glucose, galactose, arabinose, and xylose, and dissolve them in double-distilled water to prepare a solution of 1 mmol·L⁻¹. -1 A mixed monosaccharide reference solution. Separately, accurately weigh the monosaccharide reference standards and dissolve them separately in double-distilled water to prepare 1 mmol·L⁻¹ solutions. -1 The standard monosaccharide reference solution.

[0093] (2) Preparation of polysaccharide acid hydrolysis sample solution Weigh 10 mg of purified polysaccharide and place it in a 10 mL glass bottle. Add 2 mol·L⁻¹ -1 2 mL of trifluoroacetic acid (TFA) solution was added to a tube sealed with N2 and hydrolyzed at 120 °C for 160 min to obtain a hydrolyzed sample solution. Methanol was added and the solution was evaporated to dryness under reduced pressure using a rotary evaporator. The addition of methanol was repeated several times until the TFA was completely evaporated and removed. The solution was then diluted with deionized water to 2 mL and set aside for later use.

[0094] (3) PMP derivatization of standard monosaccharide, mixed monosaccharide and polysaccharide hydrolysate samples Accurately transfer 1.4 mL each of the standard monosaccharide reference, mixed monosaccharide reference, and polysaccharide hydrolysis sample solution into separate 10 mL centrifuge tubes. Add 700 µL of PMP methanol solution and 700 µL of 0.3 mol·L⁻¹ methanol solution sequentially. -1NaOH solution was heated in a 70°C water bath for 30 min, then removed and allowed to stand at room temperature for 10 min. 700 µL of 0.3 mol·L⁻¹ solution was then added. -1 Neutralize with hydrochloric acid, mix well, and extract twice with an equal volume of isoamyl acetate. Discard the supernatant, extract once with an equal volume of chloroform, and filter the methanol-aqueous phase through a 0.45µm microporous membrane for later use.

[0095] (4) Chromatographic conditions The chromatographic column was DIKMA Diamonsil C10. 18 Column (250 mm × 4.6 mm, 5 μm); Mobile phase A: 0.1 mol·L⁻¹ -1 Phosphate buffer (NaH₂PO₄-Na₂HPO₄, pH 6.7), mobile phase B: acetonitrile; column temperature: 30℃; flow rate: 1.0 mL / min -1 Detection wavelength: 245nm; Injection volume: 10μL. Elution conditions are shown in Table 4 below.

[0096] Table 4 HPLC Chromatographic Conditions

[0097] PMP pre-column derivatization high-performance liquid chromatography results The molar ratio of monosaccharides in polysaccharides was calculated using the internal standard plus correction factor method. The results are shown in Table 5 below. The HPLC chromatogram of the PMP derivatized mixed monosaccharide standard is shown below. Figure 12 The HPLC chromatogram of PMP derivatization of CHTD-1 sample hydrolysate is shown below. Figure 13 The HPLC chromatogram of PMP derivatization of BSTD-2 sample hydrolysate is shown below. Figure 14 .

[0098] Table 5. Molar ratio of monosaccharide composition in homogeneous polysaccharides

[0099] Summary: High-performance liquid chromatography (HPLC) confirmed the polysaccharide fraction to be homogeneous. PMP pre-column derivatization HPLC analysis results: CHTD-1 Glucuronic acid:galacturonic acid:xylose:glucose = 1:1.26:5.58:10.06; BSTD-2 galacturonic acid:xylose:glucose:galactose = 1:1.81:6.38:9.96 6. Metabolomics PCA Analysis Since PCA analysis in metabolomics can reflect the efficacy of the drug-treated group, this method is used for pharmacodynamic verification to make the antidepressant effect of the polysaccharide combination CB-2 more precise.

[0100] (1) UPLC-TOF / MS metabolometry analysis of samples The sample was analyzed by UPLC / Q-TOF metabolomics. The total ion current was normal, indicating that the analysis system is stable and reproducible, and can meet the requirements of metabolomics research. Figure 15 The retention times, ionic intensities, and other information of the chromatographic peaks in positive ion mode are displayed. Figure 16 The graphs display the chromatographic peak retention times, ionic intensities, and other information in negative ion mode. Observing the graphs reveals that the analytical system is stable and the sample retention times are reproducible.

[0101] (2) Principal Component Analysis (PCA) Results like Figure 17 As shown in the PLS-DA plot, the control group, positive drug group, model group, and polysaccharide group were significantly separated, indicating significant differences in their metabolic profiles. The results demonstrate metabolic dysfunction and changes in metabolite concentrations in depressed rats, confirming the success of the model. The distribution areas of the drug-treated group and the model group showed a clear separation trend, indicating quantitative changes in endogenous metabolites after drug intervention. This suggests that the drug acts on different metabolic pathways in rats, leading to changes in metabolite concentrations. After establishing the stress model, the rats' metabolism was disrupted, and the groups showed a separation trend, with the drug-treated group approaching the normal group, indicating that the polysaccharide combination has an antidepressant effect. Green: control; blue: model; yellow: high dose; black: medium dose; red: low dose.

[0102] It should be understood that, in order to simplify this disclosure and aid in understanding one or more of the various aspects of the invention, features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof in the above description of exemplary embodiments of the invention. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than expressly recited in each claim. Rather, as reflected in the claims, inventive aspects lie in fewer than all the features of the foregoingly disclosed embodiments. Therefore, the claims, following the detailed description, are hereby expressly incorporated into that detailed description, wherein each claim itself is a separate embodiment of the invention.

[0103] Although the invention has been described with reference to a limited number of embodiments, those skilled in the art will understand from the foregoing description that other embodiments are conceivable within the scope of the invention described herein. Furthermore, it should be noted that the language used in this specification has been chosen primarily for readability and edibility purposes, and not for the purpose of interpreting or limiting the subject matter of the invention. Therefore, many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the appended claims. The disclosure of the invention is illustrative and not restrictive, and the scope of the invention is defined by the appended claims.

[0104] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a Bupleurum and Radix Paeoniae Alba polysaccharide composition in the preparation of an antidepressant drug, characterized in that, This includes Bupleurum polysaccharide CHTD-1 and Paeonia lactiflora polysaccharide BSTD-2 in a mass ratio of 1:3 to 3:1; The monosaccharide composition of Bupleurum polysaccharide CHTD-1 is: galacturonic acid: xylose: galactose: glucuronic acid = 1:1.26:5.58:10.06; The monosaccharide composition of Paeonia lactiflora polysaccharide BSTD-2 is: galacturonic acid: xylose: glucose: galactose = 1:1.81:6.38:9.96; The method for preparing the composition, Includes the following steps: S1, Extraction: Weigh 1.5 kg of Bupleurum chinense or Paeonia lactiflora powder separately and place them in a 20 L distillation flask. Add 15 L of distilled water at a material-to-liquid ratio of 1:

10. After the liquid boils, heat under reflux for 2 hours and filter the liquid. Repeat the same process twice with the same material-to-liquid ratio and heating time for the residue. Discard the residue and combine the liquids from the three processes. Concentrate the liquid using a rotary evaporator until it becomes viscous and adheres to the walls, exhibiting a semi-fluid state. Place the concentrated liquid in a 5 L container, add 4 L of 95% ethanol, stir, and allow it to precipitate for 48 hours. Filter to obtain the precipitate, air-dry the precipitate for 24 hours, and then dry it in an oven to obtain Bupleurum chinense crude polysaccharide and Paeonia lactiflora crude polysaccharide, respectively. S2, protein removal: Sevage reagent was prepared according to the ratio of chloroform: n-butanol = 4:1; the crude polysaccharide powders of Bupleurum chinense and Paeonia lactiflora were respectively made into aqueous solutions with a concentration of 10 mg / ml, transferred to a 500 ml separatory funnel, added an equal amount of Sevage reagent, shaken, and allowed to stand overnight. The upper aqueous solution was taken, extracted three times, the extracts were combined, concentrated, and freeze-dried to obtain deproteinized polysaccharides of Bupleurum chinense and Paeonia lactiflora. S3, enrichment and purification Weigh 1g of deproteinized polysaccharide from Bupleurum chinense, dissolve it in distilled water, filter, and pour the filtrate into a DEAE-52 cellulose anion and cation exchange column. Elute with distilled water and 0.3 M NaCl solution, respectively. Identify whether the eluent contains polysaccharide using the sulfuric acid-phenol colorimetric method. Collect the polysaccharide solution, concentrate it to 50ml using a rotary evaporator, pour the concentrate into a square box with a liquid thickness of 1cm, and freeze it in the freezer for 24h. Place the frozen polysaccharide sample in a lyophilizer, freeze-dry it, and collect the sample. Obtain the water-washed fraction CH-1 and the salt-washed fraction CH-0.3 of the polysaccharide component. Similarly, enrich 1g of deproteinized polysaccharide from Paeonia lactiflora, and obtain the water-washed fraction BS-1 and the salt-washed fraction BS-0.3 of the polysaccharide component. S4, Sephadex G-100 gel enrichment: Weigh 300 mg of Bupleurum polysaccharide CH-1, dissolve in distilled water, sonicate for 5 min, filter, load, and perform Sephadex G-100 gel column chromatography. Elute with distilled water and 0.4 M NaCl solution, collecting 100 tubes of each solution using an automatic collector. Perform a phenol-sulfuric acid colorimetric reaction on even-numbered tubes and measure the absorbance at 490 nm. Plot the polysaccharide elution curve to obtain the water-eluted fraction CHTD-1 and the salt-eluted fraction CHTD-2. After drying, obtain Bupleurum polysaccharide CHTD-1. After dialyzing and drying, obtain Bupleurum polysaccharide CHTD-2. Similarly, 300 mg of Paeonia lactiflora polysaccharide BS-1 was enriched to obtain water-eluted fraction BSTD-1 and salt-eluted fraction BSTD-2, respectively; after drying, Paeonia lactiflora polysaccharide BSTD-1 was obtained; after dialysis and drying, Paeonia lactiflora polysaccharide BSTD-2 was obtained. S5, take Bupleurum polysaccharide CHTD-1 and Paeonia lactiflora polysaccharide BSTD-2 and mix them to obtain a composition.

2. Use according to claim 1, characterized in that, The mass ratio of Bupleurum polysaccharide CHTD-1 to Paeonia lactiflora polysaccharide BSTD-2 is 2:

1.

3. Use according to claim 1, characterized in that, In S1, the rotary evaporator concentrates at a pressure of 60 Pa, uses tap water for condensation, has a water bath temperature of 55 °C, and a rotation speed of 30 r / min.

4. Use according to claim 1, characterized in that, In S3, the rotary evaporator is concentrated at a pressure of 60 Pa, using tap water for condensation, with a water bath temperature of 55℃ and a rotation speed of 3000 r / min; the freeze dryer is preset to a pressure of 29 Pa, a temperature of -40℃, and a freeze-drying time of 48 h.

5. The use according to claim 1, characterized in that, In S3, the specifications of the DEAE-52 cellulose anion and cation column are: 60cm × 3.5cm, and the filler is 500g.

6. The use according to claim 1, characterized in that, In S4, the specifications of the Sephadex G-100 gel column chromatography are: 60cm × 3.5cm, and the packing material is 500g.

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