Uniform polysaccharide from Xiaoyao powder as well as preparation method and application thereof

Abstract

The invention discloses a homogeneous polysaccharide from Xiaoyao powder as well as a preparation method and application of the homogeneous polysaccharide. The homogeneous polysaccharide is composed of arabinose, galactose, mannose, glucose and fructose, and the weight-average molecular weight Mw of the homogeneous polysaccharide is 293.223 kDa. The polysaccharide is separated and purified from Xiaoyao powder through water extraction, alcohol precipitation, ion exchange column chromatography, gel column chromatography and other preparation methods. Experimental results show that the homogeneous polysaccharide can improve cortisol-induced zebra fish depression model behavioral performance and improve exercise activity, has anti-depression activity, and can be used for preparing drugs for treating or preventing depression.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a uniform polysaccharide from Xiaoyao San and a preparation method and application thereof. BACKGROUND

[0002] Depression is a common complex mental disorder, which is widely considered as a serious mental health problem. The clinical features of depression are long-term low mood, loss of appetite and social dysfunction, and in severe cases, it can lead to self-injury and suicide behavior of patients. It brings huge economic burden and social problems to the society. The clinical treatment mainly chooses selective serotonin reuptake inhibitors (sertraline, paroxetine, fluoxetine, citalopram, etc.), which has long onset time, strong drug dependence, liver and kidney damage, cardiovascular damage and other adverse reactions. Therefore, further exploring and developing safer and more effective drugs for the prevention and treatment of depression is an urgent problem to be solved.

[0003] Multiple mechanisms are involved in the pathological process of the occurrence and development of depression, including neurotransmitter hypothesis, inflammation hypothesis, oxidative stress hypothesis, HPA axis dysfunction hypothesis, and gut microbiota imbalance hypothesis. Research shows that intestinal health is closely related to the occurrence and development of depression. Basic research has also confirmed that the expression levels of central pro-inflammatory factors IL-1β, IL-6 and TNF-α are increased and the inflammatory pathway is activated in various animal models of depression. Studies have shown that Xiaoyao San can inhibit the expression of pro-inflammatory factors such as IL-6 and TNF-α in the colon and block their migration to the periphery and the central nervous system by improving the composition and structure of the intestinal flora, restoring the metabolic disorder of the flora, and strengthening the intestinal barrier. Traditional Chinese medicine polysaccharides are one of the effective components of traditional Chinese medicines, which play an important role in maintaining the health of the body and treating diseases. In recent years, research has shown that polysaccharides have anti-inflammatory, immunoregulatory, antitumor, hypoglycemic, and neuroprotective effects.

[0004] With the change of the research direction of new drugs in the world from the development of single chemical drugs and their preparations to the development of biological technology and natural plant drugs, the research direction of drugs tends to be diversified. At present, there are many reports on the research of small molecule compounds of compound Xiaoyao San, but there is no report on the isolation and preparation of anti-depression active uniform polysaccharides from Xiaoyao San. From the perspective of improving the efficacy of drugs and reducing adverse reactions of drugs, the polysaccharides from Xiaoyao San described in the present application effectively solve the above problems. SUMMARY

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present application is to provide a uniform polysaccharide from Xiaoyao San.

[0006] Another purpose of the present application is to provide a preparation method of the polysaccharide from Xiaoyao San.

[0007] Another object of the present application is to provide the use of the uniform polysaccharide in the preparation of a medicament for preventing or treating depression.

[0008] The present application is realized by the following technical means: A uniform polysaccharide from Xiaoyan San, characterized in that the uniform polysaccharide is composed of arabinose, galactose, mannose, glucose and fructose, and the uniform polysaccharide comprises a repeating unit structure as follows:

[0009] wherein m=5 and n=5.

[0010] The uniform polysaccharide has a weight average molecular weight Mw of 293.223 kDa, and the molar percentage of arabinose, galactose, glucose, mannose and fructose is 1.61:1.79:56.77:1.62:38.21.

[0011] The preparation method of the uniform polysaccharide from Xiaoyan San comprises the following preparation steps: S1. Take the following raw materials: Radix Bupleuri, Angelica sinensis, Atractylodes macrocephala Koidz, Radix Paeoniae Alba, Poria cocos, Radix Glycyrrhizae Praeparata Cum Melle, Mentha haplocalyx, and Zingiber officinale Roscoe; S2. Water extraction and concentration: add an appropriate amount of water, heat extraction, and concentrate the extract under reduced pressure to a suitable volume; S3. Alcohol precipitation and freeze-drying: take the concentrated solution obtained in step S2, add an alcohol solution, precipitate, dry the alcohol precipitate to obtain a crude polysaccharide dry powder of Xiaoyan San, remove impurities, and obtain a dry powder of Xiaoyan San polysaccharide; S4. Ion exchange column chromatography: dissolve the dry powder of Xiaoyan San polysaccharide obtained in step S3 in pure water, centrifuge to obtain the supernatant, perform ion exchange column chromatography gradient elution, collect the polysaccharide part, concentrate, dialysis purification, dry, and obtain the polysaccharide part; S5. Gel column chromatography: add an appropriate amount of pure water to the polysaccharide part obtained in step S4, centrifuge to obtain the supernatant for gel column chromatography, elute with pure water, collect the polysaccharide part eluent, and concentrate and dry to obtain the uniform polysaccharide from Xiaoyan San.

[0012] Further, the weight parts of the raw materials in step S1 are as follows: Radix Bupleuri 5-15 parts, Angelica sinensis 5-15 parts, Atractylodes macrocephala Koidz 5-15 parts, Radix Paeoniae Alba 5-15 parts, Poria cocos 5-15 parts, Radix Glycyrrhizae Praeparata Cum Melle 4-12 parts, Mentha haplocalyx 1-3 parts, and Zingiber officinale Roscoe 5-15 parts. Preferably, Radix Bupleuri 10 parts, Angelica sinensis 10 parts, Atractylodes macrocephala Koidz 10 parts, Radix Paeoniae Alba 10 parts, Poria cocos 10 parts, Radix Glycyrrhizae Praeparata Cum Melle 8 parts, Mentha haplocalyx 2 parts, and Zingiber officinale Roscoe 10 parts.

[0013] Further, in step S2, an appropriate amount of water is added, and the extraction is heated multiple times, and the extract is combined and concentrated under reduced pressure.

[0014] Preferably, the extraction is performed three times, the first time by adding 4-8 times the weight of the medicinal material in water, extracting for 1-3 hours, the second time by adding 5-10 times the weight of the medicinal material in water, extracting for 1-3 hours, and the third time by adding 5-8 times the weight of the medicinal material in water, extracting for 1-2 hours, and then combining the three extracts and concentrating under reduced pressure to a suitable volume. More preferably, the extraction is performed three times, the first time by adding 5 times the weight of the medicinal material in water, extracting for 2 hours, the second time by adding 8 times the weight of the medicinal material in water, extracting for 2 hours, and the third time by adding 7 times the weight of the medicinal material in water, extracting for 1 hour, and then combining the three extracts and concentrating under reduced pressure to a suitable volume.

[0015] Further, in step S3, the alcohol solution is an ethanol solution, and preferably, 95% ethanol is added to a concentration of 80% by volume. Further, in step S3, the impurity removal is performed by adding pure water to the dried Xiaoyao San crude polysaccharide powder to dissolve it, adding papain, and performing overnight enzymatic hydrolysis, then adding a macroporous resin to the water phase, mixing well, adsorbing overnight, and then removing small molecule components by dialysis, and drying to obtain the dried Xiaoyao San polysaccharide powder; preferably, the macroporous resin is macroporous resin AB-8, and the dialysis is performed using a 3000 kDa dialysis bag.

[0016] Further, in step S4, the gradient elution is performed using pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions in sequence, at a flow rate of 4 ml / min, and using the sulfite phenol method to track the elution curve, and the dialysis bag dialysis time is 48-72 h.

[0017] Preferably, in step S4, the ion exchange column chromatography dialysis bag has a molecular weight cut-off of 3000 Da.

[0018] Further, in step S5, the gel column chromatography is performed at a flow rate of 1 ml / min, using pure water to elute 1.5 times the column volume, and using the sulfite phenol method to track the elution curve; and the gel chromatography filler is Sephacryl S-400HR.

[0019] The present application also provides the use of the uniform polysaccharide from Xiaoyao San in the preparation of a medicament for treating or preventing depression.

[0020] Compared with the prior art, the present application has the following beneficial effects: 1. The product structure is novel, the uniformity is high, and the quality is controllable. The present application first separates and confirms a novel uniform polysaccharide from Xiaoyao San, and determines its molecular weight, monosaccharide composition, connection mode, main side chain structure, and repeating unit structure by high performance liquid chromatography, infrared spectroscopy analysis, and nuclear magnetic resonance analysis. The clear structural information provides a core basis for standardized production and quality control, and lays a foundation for the study of its targeted pharmacological activity and structure-activity relationship.

[0021] 2. The antidepressant activity is significant, the zebra fish depression model is established, the normal control group, the model group (cortisol induction), the positive control group (fluoxetine), the uniform polysaccharide low, medium and high dose groups from the free and easy powder, the free and easy powder group, the free and easy powder crude polysaccharide group and the small molecule group are set, and the experimental results show that the efficacy of the uniform polysaccharide is better than that of the free and easy powder group. The uniform polysaccharide from the free and easy powder can effectively antagonize the nerve inhibition effect of cortisol, improve the exploration behavior and motor activity of zebra fish under the stress state, so that the antidepressant effect is played. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Gel purification elution curve of the uniform polysaccharide from the free and easy powder Figure 2 Infrared spectrum of the uniform polysaccharide from the free and easy powder Figure 3 Electron microscope graph of the uniform polysaccharide from the free and easy powder Figure 4 Methylation total ion flow graph of the uniform polysaccharide from the free and easy powder Figure 5 Nuclear magnetic resonance hydrogen spectrum graph of the uniform polysaccharide from the free and easy powder Figure 6 Nuclear magnetic resonance DEPT135 spectrum graph of the uniform polysaccharide from the free and easy powder Figure 7 Nuclear magnetic resonance carbon spectrum graph of the uniform polysaccharide from the free and easy powder Figure 8 Nuclear magnetic resonance NOESY spectrum graph of the uniform polysaccharide from the free and easy powder Figure 9 Nuclear magnetic resonance HSQC spectrum graph of the uniform polysaccharide from the free and easy powder Figure 10 Nuclear magnetic resonance HMBC spectrum graph of the uniform polysaccharide from the free and easy powder Figure 11 Bright field movement distance of the uniform polysaccharide from the free and easy powder in the zebra fish depression model Figure 12 Schematic diagram of the bright field movement of the uniform polysaccharide from the free and easy powder in the zebra fish depression model DETAILED DESCRIPTION

[0023] The present application will be further described by specific embodiments, and the present application is not limited to the following embodiments. The changes, combinations or replacements made to the present application within the scope of the present application or without departing from the content, spirit and scope of the present application are obvious to those skilled in the art, and are included in the scope of the present application.

[0024] Preparation method and structure analysis of the uniform polysaccharide from the free and easy powder Example 1: Preparation of uniform polysaccharide from Xiaoyao San (XYSP1-2) Bupleurum 10 parts, Angelica 10 parts, Atractylodes 10 parts, White Peony Root 10 parts, Poria 10 parts, Fried Licorice 8 parts, Mint 2 parts, and Fresh Ginger 10 parts were selected. Five times the weight of the medicinal materials was added to water for the first time, heated for extraction for 2 hours; eight times the weight of water was added for the second time, heated for extraction for 2 hours, and seven times the weight of water was added for the third time, heated for extraction for 1 hour. The three extraction solutions were combined and concentrated under reduced pressure to a suitable volume. The concentrated solution was added to 95% ethanol to a volume concentration of 80%. The precipitate was freeze-dried to obtain a crude polysaccharide powder of Xiaoyao San. The crude polysaccharide powder of Xiaoyao San was added to pure water to dissolve thoroughly, and papain was added for overnight enzymatic hydrolysis. Macroporous resin AB-8 was added to the water phase, mixed thoroughly, and adsorbed overnight. The small molecule components were removed by dialysis using a 3000 kDa dialysis bag, dried, and a dried powder of Xiaoyao San polysaccharide was obtained. The dried powder of Xiaoyao San polysaccharide was dissolved in pure water, centrifuged for 10 min, and the supernatant was collected. Ion exchange column chromatography was performed with a gradient elution at a flow rate of 4 ml / min. Pure water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions were used for gradient elution in sequence. The elution curve was tracked using the sulfuric acid phenol method, and the polysaccharide eluate was collected. The polysaccharide eluate was concentrated, dialyzed for 48-72 h to remove salt, and freeze-dried to obtain a polysaccharide fraction. The polysaccharide fraction was dissolved in pure water, centrifuged for 10 min, and the supernatant was collected. Gel chromatography was performed with a gradient elution. The gel chromatography filler was Sephacryl S-400HR, and the flow rate was 1 ml / min. Pure water was used for elution at 1.5 times the column volume. The polysaccharide eluate was collected, and the elution curve was tracked using the sulfuric acid phenol method. The gel purification elution curve was plotted (as shown in FIG. 1), and the polysaccharide was concentrated and freeze-dried to obtain a uniform polysaccharide from Xiaoyao San (XYSP1-2). Figure 1

[0025] Example 2: Preparation of uniform polysaccharide from Xiaoyao San ​Select Bupleurum 5 parts, Angelica 5 parts, Atractylodes 5 parts, White Peony Root 5 parts, Poria 5 parts, Fried Licorice 4 parts, Mint 1 part, Fresh Ginger 5 parts, the first time to add 4 times the amount of water of the weight of medicinal materials, heating extraction 1 hour; the second time to add 5 times the amount of water, heating extraction 1 hour, the third time to add 5 times the amount of water, heating extraction 1 hour, combine three times of extraction liquid to reduce pressure concentration to the appropriate volume, the concentrated liquid obtained is added to 95%, the volume concentration of ethanol is 80%, precipitate, freeze-dried alcohol deposition after the material to obtain the coarse polysaccharide powder of Xiaoyao San. Add pure water to the coarse polysaccharide powder of Xiaoyao San and dissolve thoroughly, add papain and enzymolysis overnight. Add macroporous resin AB-8 to the water phase, mix thoroughly, adsorb overnight, and then dialyze to remove small molecular components using a 3000 kDa dialysis bag. Dry to obtain the polysaccharide powder of Xiaoyao San. Dissolve the polysaccharide powder of Xiaoyao San in pure water, centrifuge for 10 min, take the supernatant, and perform ion exchange column chromatography gradient elution at a flow rate of 4 ml / min. Use pure water, 0.1M, 0.2M and 0.3M Nacl solution, etc. gradient elution in sequence, and use the sulfuric acid phenol method to track the elution curve. Collect the polysaccharide part eluent, concentrate, dialyze for 48-72h to remove salt using a 3000 Da dialysis bag, and freeze-dry to obtain the polysaccharide part. Take the obtained polysaccharide part, add an appropriate amount of pure water, centrifuge for 10 min, take the supernatant, and perform gel chromatography gradient elution. The gel chromatography filler is Sephacryl S-400HR, and the flow rate is 1 ml / min. Elute with pure water for 1.5 times the column volume, collect the polysaccharide part eluent, use the sulfuric acid phenol method to track the elution curve, draw the gel purification elution curve, and concentrate and freeze-dry to obtain the uniform polysaccharide derived from Xiaoyao San.

[0026] Example 3 Preparation of the uniform polysaccharide derived from Xiaoyao San Select Bupleurum 15, Angelica 15, Atractylodes 15, White Peony Root 15, Poria 15, Fried Licorice 12, Mint 3, Fresh Ginger 15, the first time to add 8 times the amount of water by weight of medicinal materials, extraction 3 hours; the second time to add 10 times the amount of water, extraction 3 hours, the third time to add 8 times the amount of water, extraction 2 hours; combined three times of extraction liquid reduced pressure concentration to the appropriate volume, the concentrated liquid obtained by adding 95%, the volume concentration of ethanol is 80%, precipitate, freeze-dried alcohol deposition after the material to obtain the powder of the coarse polysaccharide of Xiaoyao Powder. To the powder of the coarse polysaccharide of Xiaoyao Powder, add pure water to dissolve, add papain, overnight enzymolysis. To the water phase, add macroporous resin AB-8, mix well, adsorb overnight, then dialyze with a 3000 kDa dialysis bag to remove small molecular components, dry to obtain the dried powder of Xiaoyao Powder polysaccharide. Dissolve the dried powder of Xiaoyao Powder polysaccharide in pure water, centrifuge for 10 min, take the supernatant, and perform ion exchange column chromatography gradient elution at a flow rate of 4 ml / min, using pure water, 0.1M, 0.2M and 0.3M Nacl solution, etc. gradient elution, using the phenol-sulfuric acid method to track the elution curve, collect the polysaccharide part of the eluate, concentrate, dialyze for 48-72h with a 3000Da dialysis bag to remove salt, and freeze-dry to obtain the polysaccharide part. Take the obtained polysaccharide part, add an appropriate amount of pure water, centrifuge for 10 min, take the supernatant, and perform gel chromatography gradient elution, using Sephacryl S-400HR as the gel chromatography filler and a flow rate of 1 ml / min; elute with pure water for 1.5 times the column volume, collect the polysaccharide part of the eluate, use the phenol-sulfuric acid method to track the elution curve, draw the gel purification elution curve, and concentrate and freeze-dry to obtain the uniform polysaccharide derived from Xiaoyao Powder.

[0027] Example 4 Preparation of the uniform polysaccharide derived from Xiaoyao Powder Ten parts of Bupleurum chinense, Angelica sinensis, Atractylodes macrocephala, Paeonia lactiflora, Poria cocos, Glycyrrhiza uralensis (processed), Mentha haplocalyx, and Zingiber officinale were selected. For the first extraction, 8 times the weight of the herbs in water was added, and the mixture was heated for 3 hours. For the second extraction, 10 times the weight of water was added, and the mixture was heated for 3 hours. For the third extraction, 8 times the weight of water was added, and the mixture was heated for 1.5 hours. The three extracts were combined and concentrated under reduced pressure to a suitable volume. The concentrated solution was then mixed with 95% ethanol to achieve a volume concentration of 80%, allowing precipitation. The ethanol precipitate was freeze-dried to obtain Xiaoyao San crude polysaccharide powder. The Xiaoyao San crude polysaccharide powder was dissolved in pure water, and papain was added for enzymatic hydrolysis overnight. Macroporous resin AB-8 was added to the aqueous phase, mixed thoroughly, and allowed to adsorb overnight. The mixture was then dialyzed using a 3000 kDa dialysis bag to remove small molecule components. The powder was dried to obtain Xiaoyao San polysaccharide powder. Dissolve the dried polysaccharide powder of Xiaoyao San in pure water, centrifuge for 10 min, and collect the supernatant. Perform gradient elution using an ion exchange column at a flow rate of 4 ml / min, sequentially using pure water, 0.1 M, 0.2 M, and 0.3 M NaCl solutions. Use the sulfuric acid-phenol method to track the elution curve. Collect the polysaccharide eluent, concentrate it, and dialyze it through a 3000 Da dialysis bag for 48-72 h to remove salts and purify it. Freeze-dry the polysaccharide fraction. Take the obtained polysaccharide fraction, add an appropriate amount of pure water, centrifuge for 10 min, collect the supernatant, and perform gradient elution using gel chromatography. Use Sephacryl S-400HR gel chromatography packing material at a flow rate of 1 ml / min. Elute with 1.5 column volumes of pure water, collect the polysaccharide eluent, and use the sulfuric acid-phenol method to track the elution curve. Plot the gel purification elution curve (e.g., ...). Figure 1 As shown), the homogeneous polysaccharide (XYSP1-2) derived from Xiaoyao San was obtained by concentration and freeze-drying.

[0028] Comparative Example 1: Preparation of Xiaoyao San Extract Select 10 parts of Bupleurum chinense, 10 parts of Angelica sinensis, 10 parts of Atractylodes macrocephala, 10 parts of Paeonia lactiflora, 10 parts of Poria cocos, 8 parts of Glycyrrhiza uralensis (processed), 2 parts of Mentha haplocalyx, and 10 parts of Zingiber officinale. First, add 5 times the weight of the herbs in water and extract for 2 hours; second, add 8 times the weight of the herbs in water and extract for 2 hours; third, add 7 times the weight of the herbs in water and extract for 1 hour. Combine the three extracts and concentrate under reduced pressure to a suitable volume to obtain Xiaoyao San extract.

[0029] Comparative Example 2: Preparation of crude polysaccharide extract of Xiaoyao San Ten parts of Bupleurum chinense, Angelica sinensis, Atractylodes macrocephala, Paeonia lactiflora, Poria cocos, Glycyrrhiza uralensis (processed), Mentha haplocalyx, and Zingiber officinale were selected. For the first extraction, five times the weight of the herbs in water were added, and the mixture was extracted for 2 hours. For the second extraction, eight times the weight of the herbs in water were added, and the mixture was extracted for 2 hours. For the third extraction, seven times the weight of the herbs in water were added, and the mixture was extracted for 1 hour. The three extracts were combined and concentrated under reduced pressure to a suitable volume. The concentrated solution was then precipitated with 95% ethanol to a volume concentration of 80%. The precipitate was freeze-dried to obtain the crude polysaccharide powder of Xiaoyao San.

[0030] Preparation of small molecule extract of Comparative Example 3 Select 10 parts of Radix Bupleuri, 10 parts of Angelica sinensis, 10 parts of Atractylodes macrocephala, 10 parts of Paeonia lactiflora, 8 parts of Radix Glycyrrhizae, 2 parts of Mentha haplocalyx, 10 parts of Zingiber officinale, add 5 times the amount of water of the weight of the medicinal materials for the first time, extract for 2 hours; add 8 times the amount of water for the second time, extract for 2 hours, add 7 times the amount of water for the third time, extract for 1 hour; combine the three times of extract and concentrate under reduced pressure to a suitable volume. Add 95% ethanol to the obtained concentrated solution for alcohol precipitation, so that the volume concentration of ethanol is 80%, precipitate, collect the supernatant, concentrate and dry to obtain the small molecule extract.

[0031] (I) Structural analysis of uniform polysaccharide (XYSP1-2) from Xiaoyao San (1) Monosaccharide composition analysis Determined by high performance liquid chromatography, the monosaccharide composition of uniform polysaccharide from Xiaoyao San is arabinose, galactose, glucose, mannose and fructose, as shown in Table 1 below.

[0032] Table 1 Monosaccharide composition

[0033] (2) Infrared spectrum detection Infrared spectrum results Figure 3 show that the absorption band at 3600-3200 cm -1 is the stretching vibration absorption peak of -OH, and the absorption peak in this region is the characteristic peak of sugar. Specifically, 3305.54 cm -1 is the stretching vibration absorption peak of -OH, which is the characteristic peak of sugar, as shown in Figure 2 .

[0034] (3) Electron microscope observation The uniform polysaccharide (XYSP1-2) sample from Xiaoyao San was passed through a 100-mesh sieve, a small amount was taken on a conductive carbon tape, treated with gold spraying, and then scanned by an electron microscope, with a magnification of 500-10000 times, as shown in Figure 3 .

[0035] (4) Determination of glycosidic bond composition by methylation analysis The GC-MS analysis results after methylation and hydrolysis are shown in Figure 4 and Table 2 below: Table 2 Glycosidic bond composition

[0036] (5) Analysis of polysaccharide configuration by nuclear magnetic resonance A for →4)-a-D-Glcp-(1→, B for →1)-β-D-Fruf-(2→, C for a-D-Glcp-(1→, D for →4,6)-a-D-Glcp-(1→, and E for β-D-Fruf-(2→, as shown in Table 3 below. The uniform polysaccharide from Xiaoyao San of the present patent application is mainly formed by →4)-a-D-Glcp-(1→, →1)-β-D-Fruf-(2→, and →4,6)-a-D-Glcp-(1→, etc. connected to each other to form a main chain, and the branches are mainly composed of a-D-Glcp-(1→ connected at the O-4 position of the sugar residue →4,6)-a-D-Glcp-(1→, etc. The nuclear magnetic resonance analysis spectrum is shown in Figures 5-10

[0037] Table 3 Sugar residues 1 H and 13 C chemical shifts

[0038] From the above results and comprehensive analysis of the spectrum, it can be concluded that the uniform polysaccharide from Xiaoyao San contains a repeating unit structure formula

[0039] wherein m = 5, n = 5, the weight average molecular weight Mw is 293.223 kDa, the number average molecular weight Mn is 158.721 kDa, and the highest peak molecular weight Mp is 114.828 kDa.

[0040] (II) Anti-depression activity of the uniform polysaccharide from Xiaoyao San (XYSP1-2) (1) Concentration screening of the uniform polysaccharide from Xiaoyao San (XYSP1-2) The experimental method is as follows: wild type AB strain zebrafish, 30 tails / 3 mL system. Set up normal control group, model control group (cortisol induction), experimental group (Xiaoyao San-derived uniform polysaccharide prepared in Example 1, Xiaoyao San extract prepared in Comparative Example 1, Xiaoyao San crude polysaccharide extract prepared in Comparative Example 2, and small molecule extract prepared in Comparative Example 3). Except for the normal control group, each experimental group was given cortisol to establish a zebrafish depression model. After 48 h of treatment at 28°C, the zebrafish in each experimental group was counted to determine the maximum tolerance (MTC) of the sample, which was 125 μg / mL.

[0041] (2) Anti-depression activity evaluation of the uniform polysaccharide from Xiaoyao San (XYSP1-2) The experimental method is as follows: ​Experimental grouping: a total of 9 groups, setting normal control group, model group (cortisol induction), positive control group (fluoxetine), low-dose group of uniform polysaccharide (XYSP1-2) prepared from Xiaoyao San (31.25 μg / mL) prepared in Example 1, medium-dose group of uniform polysaccharide (XYSP1-2) prepared from Xiaoyao San (62.5 μg / mL) prepared in Example 1, high-dose group of uniform polysaccharide (XYSP1-2) prepared from Xiaoyao San (125 μg / mL) prepared in Example 1, Xiaoyao San extract group (125 μg / mL) prepared in Comparative Example 1, Xiaoyao San crude polysaccharide group (125 μg / mL) prepared in Comparative Example 2, and small molecule group (125 μg / mL) prepared in Comparative Example 3.

[0042] Experimental method: wild-type AB strain zebrafish were used, 30 fish per 3 mL system. Except for the normal control group, each experimental group was given cortisol to establish a zebrafish depression model. After 48 h of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group, and the open field movement distance of the zebrafish was measured using a behavior analyzer. The statistical analysis results of this index were used to evaluate the antidepressant efficacy of the samples.

[0043] The experimental results are shown in Figure 11 and Table 4. Behavioral analysis showed that compared with the normal control group, cortisol exposure significantly reduced the movement distance of zebrafish in the open field environment ( P <0.00001). However, after treatment with uniform polysaccharides derived from Xiaoyao San, the movement inhibition phenomenon was significantly improved. Specifically, compared with the model group, the open field movement distance of zebrafish in the uniform polysaccharide treatment group was significantly restored, which was comparable to the effect of the positive drug control group, and there was a clear dose-effect relationship. The open field movement distance of the medium-dose group and the high-dose group of uniform polysaccharides was better than that of the model group ( P <0.01, P <0.001). And it was observed that at the same concentration, the efficacy of uniform polysaccharides was significantly better than that of Xiaoyao San group ( P <0.01), and also better than that of Xiaoyao San crude polysaccharide group ( P <0.05). This result fully proves that the uniform polysaccharide can effectively antagonize the nerve inhibition effect of cortisol and improve the exploration behavior and movement activity of zebrafish under stress, thereby exerting its antidepressant effect. The open field movement schematic diagram of each group in the zebrafish depression model is shown in Figure 12 .

[0044] Table 4 Open field movement distance of each group in the zebrafish depression model

[0045] The follow-up research experiment results show that the uniform polysaccharide from the Xiaoyao Powder has more significant antidepressant activity compared to other structural polysaccharides reported in the literature.

[0046] Finally, it should be noted that the above-described embodiments are merely an explanation of the present application and are not a limitation thereof, and for those skilled in the art, after reading the present application, modifications can be made to the present embodiments without creative contribution, such as equivalent changes to the structure, shape, principle of the present application, or equivalent replacement of some or all of the technical features, etc., all of which fall within the scope of the present application.

Claims

1. A homogeneous polysaccharide derived from Xiaoyao San, characterized in that, The homogeneous polysaccharide is composed of arabinose, galactose, mannose, glucose, and fructose, and the repeating unit structure of the homogeneous polysaccharide is as follows: ； Where m=5, n=5.

2. The homogeneous polysaccharide derived from Xiaoyao San according to claim 1, characterized in that, The homogeneous polysaccharide has a weight-average molecular weight (Mw) of 293.223 kDa, and the molar percentages of arabinose, galactose, glucose, mannose, and fructose are 1.61:1.79:56.77:1.62:38.

21.

3. A method for preparing a homogeneous polysaccharide derived from Xiaoyao San according to any one of claims 1-2, characterized in that, The preparation steps include the following: S1. Take the following ingredients: Bupleurum, Angelica sinensis, Atractylodes macrocephala, Paeonia lactiflora, Poria cocos, Glycyrrhiza uralensis (processed), Mentha haplocalyx, and Zingiber officinale; S2. Water extraction and concentration: Add an appropriate amount of water, heat and extract, and concentrate the extract under reduced pressure to a suitable volume; S3. Alcohol precipitation and freeze drying: Take the concentrated liquid obtained in step S2, add alcohol solution, precipitate, dry the alcohol precipitate to obtain Xiaoyao San crude polysaccharide dried powder, remove impurities to obtain Xiaoyao San polysaccharide dried powder. S4. Ion exchange column chromatography: Dissolve the dried Xiaoyao San polysaccharide powder obtained in step S3 in pure water, centrifuge to collect the supernatant, perform gradient elution by ion exchange column chromatography, collect the polysaccharide fraction, concentrate, dialyze to purify, dry, and obtain the polysaccharide fraction. S5. Gel column chromatography: Add an appropriate amount of pure water to the polysaccharide fraction obtained in step S4, centrifuge and take the supernatant for gel column chromatography, elute with pure water, collect the eluent of the polysaccharide fraction, concentrate and dry to obtain the homogeneous polysaccharide derived from Xiaoyao San.

4. The preparation method according to claim 3, characterized in that, In step S1, the raw materials are in the following weight parts: Bupleurum chinense 5-15 parts, Angelica sinensis 5-15 parts, Atractylodes macrocephala 5-15 parts, Paeonia lactiflora 5-15 parts, Poria cocos 5-15 parts, Glycyrrhiza uralensis (processed) 4-12 parts, Mentha haplocalyx 1-3 parts, and Zingiber officinale 5-15 parts; preferably, Bupleurum chinense 10 parts, Angelica sinensis 10 parts, Atractylodes macrocephala 10 parts, Paeonia lactiflora 10 parts, Poria cocos 10 parts, Glycyrrhiza uralensis (processed) 8 parts, Mentha haplocalyx 2 parts, and Zingiber officinale 10 parts.

5. The preparation method according to any one of claims 3-4, characterized in that, In step S2, add an appropriate amount of water and extract by heating multiple times. Combine the extracts and concentrate under reduced pressure. Preferably, extract three times: first, add 4-8 times the weight of the medicinal material in water and extract for 1-3 hours; second, add 5-10 times the weight of the medicinal material in water and extract for 1-3 hours; third, add 5-8 times the weight of the medicinal material in water and extract for 1-2 hours. Combine the three extracts and concentrate under reduced pressure to a suitable volume. More preferably, first, add 5 times the weight of the medicinal material in water and extract for 2 hours; second, add 8 times the weight of the medicinal material in water and extract for 2 hours; third, add 7 times the weight of the medicinal material in water and extract for 1 hour. Combine the three extracts and concentrate under reduced pressure to a suitable volume.

6. The preparation method according to any one of claims 3-5, characterized in that, In step S3, the alcohol solution is an ethanol solution, preferably, 95% ethanol is added until the ethanol volume concentration is 80%; The impurity removal process involves: adding pure water to the Xiaoyao San crude polysaccharide dry powder and dissolving it completely; adding papain and hydrolyzing overnight; adding macroporous resin to the aqueous phase and mixing thoroughly; adsorbing overnight; dialysis to remove small molecule components; and drying to obtain Xiaoyao San polysaccharide dry powder. Preferably, the macroporous resin is macroporous resin AB-8, and the dialysis is performed using a 3000kDa dialysis bag.

7. The preparation method according to any one of claims 3-6, characterized in that, In step S4, the gradient elution is performed sequentially with pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions at a flow rate of 4 ml / min. The elution curve is tracked using the sulfuric acid-phenol method. The dialysis time of the dialysis bag is 48-72 h.

8. The preparation method according to any one of claims 3-7, characterized in that, In step S4, the molecular weight cutoff of the ion exchange column chromatography dialysis bag is 3000 Da.

9. The preparation method according to any one of claims 3-8, characterized in that, In step S5, the gel column chromatography flow rate is 1 ml / min, pure water is used for elution of 1.5 column volumes, and the elution curve is tracked using the sulfuric acid-phenol method; the gel chromatography packing material is Sephacryl S-400HR.

10. The use of the homogeneous polysaccharide derived from Xiaoyao San according to any one of claims 1-9 in the preparation of a medicament for treating or preventing depression.

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