Application of prunella vulgaris polysaccharide in preparation of medicine for treating oxidative liver injury caused by heavy metal exposure

Abstract

The invention belongs to the technical field of biological medicine, and relates to application of prunella vulgaris polysaccharide in preparation of a medicine for treating oxidative liver injury caused by heavy metal exposure, and the prunella vulgaris polysaccharide has the effects of protecting liver, resisting inflammation, resisting oxidation and regulating intestinal flora at the same time. The prunella vulgaris polysaccharide is extracted and purified from prunella vulgaris, and in-vivo and in-vitro experiments are carried out to verify that the prunella vulgaris polysaccharide has the effects of protecting liver, resisting inflammation, resisting oxidation, regulating intestinal flora and the like, so that treatment of oxidative liver injury caused by exposure of heavy metals is facilitated.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and relates to application of a polygonum sophorae polysaccharide in preparation of a medicine for treating oxidative liver injury caused by heavy metal exposure. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing an understanding of the general background of the application and does not necessarily constitute an admission or a recognition that the information forms part of the prior art that is already known in this field.

[0003] Oxidative liver injury caused by heavy metal exposure has become an important public health problem. As a typical hexavalent chromium compound, K2Cr2O7 directly attacks liver cells with excessive ROS, triggering lipid peroxidation, damaging cell membrane structure and function, and triggering a series of complex inflammatory cascade reactions. This vicious cycle of oxidative stress and inflammation is a key pathological mechanism leading to liver cell damage, necrosis, and even liver fibrosis and liver failure.

[0004] Although traditional antidotes such as chelating agents can alleviate heavy metal toxicity, their clinical application has obvious limitations. For heavy metal poisoning, clinicians mainly rely on chelating agents (such as EDTA, DMSA, DMPS, etc.) for detoxification treatment. This type of drug forms stable complexes by combining with free heavy metal ions, promoting their excretion from the body. However, chelation therapy has significant limitations: 1. Non-specific chelation may chelate essential trace elements (such as zinc, copper, calcium, etc.) in the body, leading to nutritional imbalance and secondary damage. 2. Unable to remove the generated ROS, chelating agents only target metal ions themselves and have no direct removal or inhibition effect on the excessive ROS generated by heavy metals and the oxidative damage and subsequent inflammatory response caused by them. 3. Potential nephrotoxicity, some chelating agents or their metal complexes may cause burden to the kidneys. In addition, more and more studies have shown that intestinal flora homeostasis plays a key role in heavy metal toxicity (especially liver injury). Heavy metal exposure can significantly destroy the intestinal flora structure (dysbiosis), resulting in a decrease in the abundance of beneficial bacteria (such as lactobacilli and bifidobacteria) and an increase in potentially pathogenic bacteria; this imbalance leads to impaired intestinal barrier function, increased harmful metabolites, and weakened host antioxidant defenses. However, existing chelating agents and other antidotes do not have intestinal flora regulation function, thereby affecting the treatment effect of oxidative liver injury caused by heavy metal exposure. SUMMARY

[0005] In order to solve the problems in the prior art, the present application aims to provide an application of Prunella vulgaris polysaccharide in the preparation of a medicine for treating oxidative liver injury caused by heavy metal exposure, the Prunella vulgaris polysaccharide is obtained by extraction and purification from Prunella vulgaris, and in-vivo and in-vitro experiments are conducted to verify that the Prunella vulgaris polysaccharide has the effects of liver protection, anti-inflammation, antioxidation and intestinal flora regulation, thereby facilitating the treatment of oxidative liver injury caused by heavy metal exposure.

[0006] In order to achieve the above-mentioned purpose, the technical scheme of the present application is as follows: The application of Prunella vulgaris polysaccharide in the preparation of a medicine for treating oxidative liver injury caused by heavy metal exposure, the Prunella vulgaris polysaccharide has the effects of liver protection, anti-inflammation, antioxidation and intestinal flora regulation.

[0007] Prunella vulgaris is a perennial plant, which is rich in bioactive substances such as polysaccharides, flavonoids, triterpenoids and phenolic acids. It is often used as a health food and a traditional Chinese medicine for the treatment of jaundice, hepatitis, gonorrhea, tuberculosis and diabetes. The present application found that there is a Prunella vulgaris polysaccharide in Prunella vulgaris, which has the effects of liver protection, anti-inflammation, antioxidation and intestinal flora regulation, and the specific performance is as follows: The liver protection is specifically manifested as follows: the hepatitis is induced by K2Cr2O7, and the improvement of the symptoms related to hepatitis includes inhibition of body weight loss, reduction of liver weight increase, regular arrangement of liver cords, improvement of K2Cr2O7-mediated reduction of liver fibroblasts and type I collagen fibers, and reduction of ALT and AST levels; The anti-inflammation is specifically manifested as follows: improvement of the symptoms related to hepatitis caused by heavy metal ions, and reduction of the expression levels of IL-6, TNF-α and IL-1β; The antioxidation is specifically manifested as follows: promotion of the expression of T-AOC, SOD and GSH-Px; The intestinal flora regulation is specifically manifested as follows: reduction of the abundance of Brevundimonas, Lactobacillus, Lactobacillus, Nitrifying Spheres and Abnormal Spheres, and increase of the abundance of Unclassified Lachnospiraceae and Unclassified Erysipelotrichaceae.

[0008] Meanwhile, the Prunella vulgaris polysaccharide does not bind to essential trace elements in the body, and can avoid nutritional imbalance and secondary damage.

[0009] Therefore, the Prunella vulgaris polysaccharide having the effects of liver protection, anti-inflammation, antioxidation and intestinal flora regulation is beneficial to the treatment of oxidative liver injury caused by heavy metal exposure.

[0010] In order to obtain the Prunella vulgaris polysaccharide, a preparation method thereof includes the following steps: S1, lipid-soluble substances are removed from pretreated Prunella vulgaris by ethanol reflux extraction to obtain a residue; S2, boiling the residue prepared in step S1 with water and then performing extraction treatment to obtain a water extract, removing insoluble components by centrifugation, and collecting the supernatant; S3, performing alcohol precipitation treatment on the supernatant obtained in step S2, and collecting the precipitate, i.e., crude polysaccharide; S4, obtaining the crude polysaccharide in step S3 after removing proteins.

[0011] The present application has the following beneficial effects: The present application extracts a Prunella vulgaris polysaccharide from Prunella vulgaris, which has the effects of protecting the liver, anti-inflammation, anti-oxidation, and regulating intestinal flora. In vitro experiments show that the Prunella vulgaris polysaccharide can reduce MDA, increase T-AOC, SOD, and GSH-Px, and has anti-oxidation function; in vitro experiments can improve total antioxidant capacity, ABTS free radical clearance rate, and DPPH free radical clearance rate. In vivo experiments on a K2Cr2O7-induced mouse hepatitis model show that the Prunella vulgaris polysaccharide can not only improve K2Cr2O7-induced liver damage, make the liver cords align neatly, and reduce the expression levels of serum liver function indicators ATL and AST, achieving the effect of protecting the liver; but also can reduce the expression levels of mouse serum IL-6, TNF-α, and IL-1β, achieving the effect of anti-inflammation; and can significantly regulate K2Cr2O7-induced intestinal flora disorder, increase the diversity of the intestinal flora of the mice, reduce the abundance of Brevundimonas, Lactobacillus mucus, Lactobacillus, Nitrifyingcoccus, and Parvimonas, and increase the abundance of unclassified Lachnospiraceae and unclassified Erysipelotrichaceae. The Prunella vulgaris polysaccharide is conducive to the treatment of oxidative liver damage caused by heavy metal exposure. At the same time, the Prunella vulgaris polysaccharide is easy to obtain, safe, has universality, and is easy to produce on a large scale. BRIEF DESCRIPTION OF DRAWINGS

[0012] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and the illustrative embodiments of the present application and their description are used to explain the present application, and do not constitute improper limitations on the present application.

[0013] Figure 1 FIG. 1 is a graph of the in vitro antioxidant activity of the Prunella vulgaris polysaccharide in the embodiments of the present application; A is total antioxidant capacity, B is ABTS free radical clearance rate, and C is DPPH free radical clearance rate.

[0014] Figure 2Figure for the effect of Prunella vulgaris L. polysaccharide on body weight, liver weight and pathological changes in K2Cr2O7-induced liver injury in the embodiments of the present application; A is the experimental mode diagram, B is the average body weight of mice, C is the liver weight index, D is the Sirius red staining, E is the collagen fiber area; wherein, the small letters a, b, c on different groups, the same letter on different groups indicates that there is no significant difference between groups (P>0.05), and different letters indicate that there is a significant difference between groups (P<0.05), and when the letters a, b, c are marked with **, it means that the difference is extremely significant (P<0.001); in B, ** means that there is an extremely significant difference between the test groups at the same time point (P<0.01), and ns means no significant difference (P>0.05).

[0015] Figure 3 Figure for the effect of Prunella vulgaris L. polysaccharide on K2Cr2O7-induced liver function indexes, antioxidant enzymes and inflammatory factors in mice in the embodiments of the present application; A is ALT, B is AST, C is T-AOC, D is GSH-px, E is SOD, F is IL-6, G is TNF-α, H is IL-1β, I is IL-10; a, b, c different groups are marked with the same letter, which means that there is no significant difference between groups (P>0.05), and different letters indicate that there is a significant difference between groups (P<0.05), and when the letters a, b, c are marked with *, it means that the difference is extremely significant (P<0.01), and abc is marked with **, which means that the difference is extremely significant (P<0.001).

[0016] Figure 4 Figure for the alpha diversity analysis of the effect of Prunella vulgaris L. polysaccharide on K2Cr2O7-induced intestinal flora changes in mice in the embodiments of the present application, A is ACE, B is Chao1, C is Simpson, D is Shannon, E is PD_whole_tree; a, b on different groups, the same letter on different groups indicates that there is no significant difference (P>0.05), and different letters indicate that there is a significant difference between groups (P<0.05), and when the letters a, b, c are marked with *, it means that the difference is extremely significant (P<0.01), and a, b, c are marked with **, which means that the difference is extremely significant (P<0.001); for example, the blank group in A is marked with ab, which means that it has no significant difference with the model group and the Prunella vulgaris group (P>0.05), but the Prunella vulgaris group and the model group are marked with a and b respectively, which means that the difference is significant (P<0.05).

[0017] Figure 5 Figure for the effect of Prunella vulgaris L. polysaccharide on K2Cr2O7-induced intestinal flora changes in mice in the embodiments of the present application; A is the sparse curve, B is the rank abundance curve.

[0018] Figure 6Figure of the effect of Prunella vulgaris L. polysaccharide on K2Cr2O7-induced intestinal marker flora of mice in the embodiment of the present application; A is ASV Wean diagram, B is PCA, C is NMDS, and D is PCoA.

[0019] Figure 7 Figure of the effect of Prunella vulgaris L. polysaccharide on K2Cr2O7-induced intestinal marker flora of mice in the embodiment of the present application; A is ASV Wean diagram, B is PCA, C is NMDS, and D is PCoA. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed description is merely exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0021] It should be noted that the terms used herein are merely for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, the singular form is intended to include the plural form unless the context clearly indicates otherwise, and it should also be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of the features, steps, operations, devices, components and / or combinations thereof.

[0022] In view of the defects of existing chelating agents and other antidotes in treating oxidative liver damage caused by heavy metal exposure, the present application proposes an application of Prunella vulgaris L. polysaccharide in preparing a medicine for treating oxidative liver damage caused by heavy metal exposure.

[0023] In a typical embodiment of the present application, an application of Prunella vulgaris L. polysaccharide in preparing a medicine for treating oxidative liver damage caused by heavy metal exposure is provided, wherein the Prunella vulgaris L. polysaccharide has the effects of protecting liver, anti-inflammation, anti-oxidation and regulating intestinal flora.

[0024] In some embodiments, the Prunella vulgaris polysaccharide is prepared according to the following steps: S1, the pretreated Prunella vulgaris is extracted with an ethanol aqueous solution to remove lipid-soluble substances, and a residue is obtained; S2, the residue obtained in step S1 is boiled with water and then extracted to obtain a water extract, and the insoluble components are removed by centrifugation, and the supernatant is collected; S3, the supernatant obtained in step S2 is subjected to alcohol precipitation treatment, and the precipitate is collected as crude polysaccharide; S4, the crude polysaccharide in step S3 is removed of protein to obtain.

[0025] Specifically, the pretreatment includes one or more of rinsing, crushing, and drying. The drying is drying at a temperature of 40-60°C.

[0026] The ethanol aqueous solution refers to a mixed solution of ethanol and water in any ratio, and specifically, the volume fraction of ethanol in the ethanol aqueous solution is 94-96%. Under this condition, the lipid-soluble substances can be removed better.

[0027] Specifically, the number of times of reflux extraction is 1-5, and the time of each reflux extraction is 1-5 hours. Under this condition, the lipid-soluble substances can be removed better.

[0028] Specifically, during the boiling extraction with water, the solid-liquid ratio is 1:1-10, kg / L, and preferably 1:4.5-5.5, kg / L.

[0029] Specifically, in the alcohol precipitation treatment, an ethanol solution with a volume fraction of not less than 80% is used, and preferably an ethanol solution with a volume fraction of 94-96% is used.

[0030] Specifically, in the alcohol precipitation treatment, the volume ratio of the supernatant to the ethanol solution is 1:2.7-3.3.

[0031] Specifically, the time of alcohol precipitation treatment is 10-40 hours. Preferably, it is 23-25 hours.

[0032] Specifically, the method for removing protein is the Sevage method. More specifically, the Sevage reagent used in the Sevage method is a mixed solution of chloroform (CHCl3) and n-butanol (BuOH), and preferably, the volume ratio of CHCl3 to BuOH is 3.6-4.4:1. More specifically, the number of times of removing protein by the Sevage method is 5-8.

[0033] Specifically, after removing protein, freeze-drying is performed.

[0034] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in conjunction with specific examples.

[0035] Example 1 A preparation method of a polygonum multiflorum polysaccharide, comprising the following steps: S1, the polygonum multiflorum is rinsed with water to remove impurities, dried in the air, then crushed, and then dried at 50℃, and the dried polygonum multiflorum is extracted with 95% ethanol by volume for 3 times, each time for 2 hours, to remove lipid-soluble substances, to obtain a residue.

[0036] S2, the residue obtained in step S1 is added with water according to a solid-liquid ratio of 1:5 (kg / L) and boiled to extract, to obtain a water extract; wherein the boiling extraction is performed for 3 times, each time for 2 hours; the water extract is centrifuged to remove insoluble components, and the supernatant is collected.

[0037] S3, the supernatant obtained in step S2 is added with 95% ethanol by volume (the volume ratio of the supernatant to 95% ethanol by volume is 1:3) to stand (i.e. alcohol precipitation treatment), and the standing time is 24 hours; after standing, the precipitate, i.e. crude polysaccharide, is collected.

[0038] S4, the protein in the crude polysaccharide in step S3 is removed by Sevage method using Sevage reagent (CHCl3: BuOH = 4:1, v / v), and the removal of protein is performed for 5 times; after freeze-drying, the polygonum multiflorum polysaccharide is obtained.

[0039] The sugar content of the prepared polygonum multiflorum polysaccharide is 88.06% determined by phenol-sulfuric acid method.

[0040] The polygonum multiflorum polysaccharide is suitable for examples 2-5.

[0041] Example 2 This example is used to determine the in vivo hepatoprotective effect of the polygonum multiflorum polysaccharide in example 1. 45 KM mice (n=15) of 4 weeks old (24.56±0.75 g) were allowed free access to water and light was on for 12 h. After three days of acclimatization, the animals were randomly divided into three groups (AC, AM, AXK). The AXK group of mice was given Sp of Pr, at a dose of 75 mg / kg, and the AC and AM groups were given the same amount of normal saline for 32 days. AM and AXK were simultaneously injected with K2Cr2O7 (25 mg / kg) (2 mL / kg, 1:1 v / v olive oil mixture) to induce hepatitis. That is, the AC group was the blank group, the AM group was the model group, and the AXK group was the Sp of Pr group. All mice were euthanized by carbon dioxide method after 24 hours of fasting and water deprivation. The serum, liver and rectal specimens were collected and stored at -80°C for further study, such as Figure 2

[0042] Mouse weight determination: The mice were weighed daily two weeks after gavage, 24 hours after the last administration.

[0043] Histological observation of liver: A part of the liver tissue was immersed in formalin fixative for fixation, and the tissue was stained with Sirius red, and the collagen fiber area was calculated.

[0044] Determination of serum liver function indicators: After the eyeball blood of each group of mice was taken, it was placed at room temperature for 30 min, and centrifuged at 3000 r / min for 10 min. The supernatant was taken for use. The contents of ALT and AST were detected according to the ELISA kit instructions.

[0045] The experimental results show that Sp of Pr can increase the weight induced by K2Cr2O7, as shown in Figure 2 B; compared with the AM group, it can significantly reduce the liver weight ( P <0.01), as shown in Figure 2 C; it can alleviate the liver damage caused by K2Cr2O7, as shown in Figure 2 D; compared with the AM group, AXK can significantly reduce the area of liver fibroblasts and type I collagen fibers ( P <0.01), as shown in Figure 2 E; the levels of ALT and AST are extremely significantly reduced ( P <0.01), as shown in Figure 3 A and B.

[0046] Example 3 This example is used to determine the anti-inflammatory activity of the Sp of Pr in Example 1: Animal treatment method, same as Example 2.

[0047] ​Effects of Prunella vulgaris polysaccharide on the levels of inflammatory factors in mice: Blood was collected from the eyeballs of mice in each group, incubated at room temperature for 30 min, and centrifuged at 3000 r / min for 10 min. The supernatant was collected for later use. IL-6, TNF-α, IL-1β, and IL-10 were detected according to the ELISA kit instructions.

[0048] The experimental results showed that, compared with the AC group, the serum levels of IL-6, TNF-α, and IL-1β in the AM group were significantly increased. P <0.05); IL-10 levels were significantly reduced, while this trend was significantly reversed in the AXK group ( P >0.05), such as Figure 3 The F, G, H and I symbols are shown in the figure.

[0049] Example 4 This embodiment is used to determine the in vitro and in vivo antioxidant activity of Prunella vulgaris polysaccharide from Example 1: To verify the in vitro antioxidant activity, the Prunella vulgaris polysaccharide was serially diluted according to the kit instructions, and the total antioxidant capacity, ABTS free radical scavenging rate, and DPPH free radical scavenging rate were determined according to the kit instructions.

[0050] In vitro antioxidant experiments showed that Prunella vulgaris polysaccharides exhibited a significant concentration-dependent antioxidant effect. In the T-AOC assay, when the polysaccharide concentration was >5 mg / mL, the total antioxidant capacity reached a stable plateau (4.02 ± 0.15 μmol / L). Figure 1 As shown in A in the figure. DPPH and ABTS free radical scavenging experiments showed that the scavenging rate reached 0.81 ± 0.03% at a critical concentration of 0.055 mg / mL, and thereafter remained stable with increasing concentration, as shown in Figure A. Figure 1 As shown in B and C in the figure. This result suggests that Prunella vulgaris polysaccharide can achieve a free radical scavenging saturation effect at a low concentration (0.055 mg / mL).

[0051] The animal handling method is the same as in Example 2.

[0052] Effects of Prunella vulgaris polysaccharide on antioxidant factor levels in mice: Blood was collected from the eyeballs of mice in each group, incubated at room temperature for 30 min, and then centrifuged at 3000 r / min for 10 min. The supernatant was collected for later use. T-AOC, SOD, and GSH-Px were detected according to the ELISA kit instructions.

[0053] Experimental results showed that the levels of T-AOC, GSH-Px, and SOD were significantly reduced in the AM group. P <0.05, MDA level significantly increased ( P <0.05%. After treatment with Prunella vulgaris polysaccharide, the levels of T-AOC, GSH-Px, and SOD significantly increased ( P<0.05), MDA level was significantly reduced ( P <0.05), as shown in Figure 3 C, D and E.

[0054] Example 5 This example is used to determine the microbial regulation effect of example 1 Prunella vulgaris polysaccharide: Animal grouping and administration, same as example 2.

[0055] K2Cr2O7 induced hepatitis, and the rectum of each group of mice was taken for the study of fucoidan microbial regulation. The extraction of rectal microbial DNA was carried out according to the kit instructions, the V3-V4 region was amplified, the library was constructed, and the Illumina NovaSeq platform was sequenced.

[0056] Regulation of Prunella vulgaris polysaccharide on the alpha diversity of mouse rectal microbiota: QIIME2 is used to analyze the alpha diversity of intestinal microorganisms.

[0057] Regulation of Prunella vulgaris polysaccharide on the flora structure of mouse rectal microbiota: non-metric multidimensional scaling, partial least squares discriminant analysis and principal coordinate analysis are used to analyze the flora structure.

[0058] The sequencing results show that a total of 4117 ASVs are detected in the three groups (1628 in the AC group, 907 in the AM group, and 1582 in the AXK group). The three groups share 117 ASVs, AC and AM share 156, and AC and AXK share 218, as shown in Figure 6 A of the above table. There are a total of 1048787 original sequences, after quality control, 49871-73263 sequences are retained in the AC group, 49700-73577 in the AXK group, and 47169-72867 in the AM group.

[0059] Alpha diversity analysis shows that the model group is lower than the blank group, the Prunella vulgaris polysaccharide group is between the blank and model groups and tends to the blank group. Among them, the ACE index and Chao1 index show that the model group and the Prunella vulgaris polysaccharide group are significantly different ( P <0.05), the PD-whole-tree index shows that the model group and the blank group are extremely significantly different ( P <0.01), and the Prunella vulgaris polysaccharide group has no difference with the blank group, as shown in Figure 4 A, B, C, D and E of the above table. Sparse curve and rank abundance curve show that the sequencing depth is sufficient, as shown in Figure 5 A and B of the above table. Beta diversity analysis shows that the relationship between the three groups of microorganisms is that the dispersion degree between the blank group and the model group is large, and the blank group is between the model group and the blank group, as shown in Figure 6 B, C and D of the above table. The dominant genera are Lactobacillus, Limosilactobacillus and Lactobacillus.

[0060] One differential phylum and 11 differential genera were found among the three groups after treatment with Prunella vulgaris polysaccharides P <0.05). At the phylum level, the abundance of Bacteroidetes in the AXK group was significantly higher than that in the AM and AC groups P <0.05). At the genus level, the abundance of Parvibacter, Lachnospiraceae UCG_006 group, Acidovorax, Pseudomonas, Bacteroidetes Vadina HA17 group, Aristipetes, Subdoligranulum, Fusobacterium, Streptococcus, and Rhodobacter in the AM group was significantly lower than that in the AC group P <0.05), and Prunella vulgaris polysaccharides could reverse the decrease of these bacteria P <0.05), as shown in A, B, C, D, E, F, G, H, I, J, K, and L in FIG. 6. Figure 7

[0061] The preferred embodiments of the present application have been described above with the preferred embodiments, but not for limiting the present application. Those skilled in the art can make various modifications and changes to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​

Claims

1. Use of a polygonatum multiflorum polysaccharide in the preparation of a drug for treating oxidative liver damage caused by heavy metal exposure, wherein the polygonatum multiflorum polysaccharide has the effects of protecting the liver, anti-inflammation, anti-oxidation and regulating intestinal flora.

2. Use according to claim 1, wherein the compound is ###0002### The polygonatum multiflorum polysaccharide is prepared according to the following steps: S1. Removing fat-soluble substances from pretreated polygonatum multiflorum by reflux extraction with an ethanol aqueous solution to obtain a residue; S2. Extracting the residue obtained in step S1 by boiling with water to obtain a water extract, removing insoluble components by centrifugation, and collecting the supernatant; S3. Alcohol precipitation treatment of the supernatant obtained in step S2 to collect the precipitate, which is a crude polysaccharide; S4. Obtaining the polygonatum multiflorum polysaccharide by removing protein from the crude polysaccharide in step S3.

3. Use according to claim 2, wherein the compound is ###0002### The pretreatment includes one or more of rinsing, crushing and drying.

4. The use according to claim 2, wherein the compound is ###0002### The volume fraction of ethanol in the ethanol aqueous solution is 94-96%.

5. The use according to claim 2, wherein the compound is ###0002### The number of times of reflux extraction is 1-5, and the reflux extraction time is 1-5 hours each time.

6. The use according to claim 2, wherein the compound is ###0002### In the process of boiling extraction with water, the solid-liquid ratio is 1:4.4-5.5, kg / L.

7. The use according to claim 2, wherein the compound is ###0002### In the alcohol precipitation treatment, the volume fraction of ethanol is 94-96%.

8. The use according to claim 2, wherein the compound is ###0002### The alcohol precipitation treatment time is 23-25 hours.

9. The use according to claim 2, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The method for removing protein is the Sevage method.

10. The use according to claim 9, wherein the compound is ###00003### or a pharmaceutically acceptable salt thereof. The Sevage reagent used in the Sevage method is a mixed solution of CHCl3 and BuOH, and the volume ratio of CHCl3 to BuOH is 3.6-4.4:1.

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