Application of gynostemma pentaphylla polysaccharide in preparation of medicine for treating ulcerative colitis

Abstract

The invention discloses application of fiveleaf gynostemma herb polysaccharide in preparation of medicine for treating ulcerative colitis, the main structure of the fiveleaf gynostemma herb polysaccharide is shown in the specification, research proves that the prepared fiveleaf gynostemma herb polysaccharide has an excellent treatment effect on ulcerative colitis for the first time, activation of NLRP3 inflammasome is inhibited by activating an NRF2 anti-oxidation pathway, and the curative effect of the fiveleaf gynostemma herb polysaccharide on ulcerative colitis is improved. The bacterial flora abundance of bacteroides and thick-walled mycophylum is increased, and the bacterial flora abundance of proteobacteria is reduced, so that the ROS level is reduced, the inflammatory response is reduced, and the intestinal mucosal barrier repair of DSS mice is promoted.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of Gynostemma pentaphyllum polysaccharide in the preparation of drugs for treating ulcerative colitis. Background Technology

[0002] Ulcerative colitis (UC), a subtype of inflammatory bowel disease, is characterized by chronic inflammation of the colonic mucosa, barrier disruption, and recurrent episodes of mucus-containing bloody stools, and carries a risk of malignant transformation. While existing treatments (such as corticosteroids, anti-inflammatory drugs, and immunosuppressants) can alleviate symptoms, they suffer from numerous side effects and limited efficacy. Patients often experience mucosal immune homeostasis dysregulation and persistent inflammatory damage. Therefore, there is an urgent need to find long-term, relatively safe medications for treating UC.

[0003] Natural polysaccharides have attracted much attention due to their immunomodulatory, antioxidant, and intestinal protective effects. Astragalus polysaccharides enhance the activity of antioxidant enzymes, licorice polysaccharides restore intestinal flora diversity, and Polygonatum polysaccharides alleviate ulcerative colitis (UC) by regulating inflammatory immune responses. Gynostemma pentaphyllum polysaccharide (GPP) is a heteropolysaccharide extracted from the cucurbitaceous plant Gynostemma pentaphyllum. It possesses antioxidant, anti-inflammatory, and immunomodulatory activities. Furthermore, Li Shizhen's *Compendium of Materia Medica* from the Ming Dynasty records Gynostemma pentaphyllum as a plant resource with both medicinal and edible properties; however, there are no reports on the therapeutic effects of GPP on UC. Summary of the Invention

[0004] The purpose of this invention is to provide the use of Gynostemma pentaphyllum polysaccharide (GPP) in the preparation of a medicament for treating ulcerative colitis.

[0005] This invention is achieved through the following technical solutions:

[0006] The application of Gynostemma pentaphyllum polysaccharide in the preparation of drugs for treating ulcerative colitis; the main structure of the Gynostemma pentaphyllum polysaccharide is as follows.

[0007] The extraction method of Gynostemma pentaphyllum polysaccharide is as follows: Take dried Gynostemma pentaphyllum raw material, add distilled water, with a solid-liquid ratio of 1-2 g: 10 mL, soak for 20-30 min, reflux at 100℃ for 2-4 h, filter to remove residue, keep the supernatant, and after deproteinization, decolorization, alcohol precipitation, vacuum filtration, and freeze-drying, crude Gynostemma pentaphyllum polysaccharide is obtained; after centrifugation of crude polysaccharide, the supernatant is loaded onto a DEAE-52 cellulose column (2.6×30 cm), and NaCl solution gradient elution is performed (flow rate: 4 mL / min). The elution peak is monitored by phenol-sulfuric acid method (490 nm), and the corresponding main peak fraction is collected (retention time: 15–25 min). The fraction is concentrated to 1 / 5 of the original volume by rotary evaporation, freeze-dried, and purified by ion exchange. After reconstitution, the sample is purified by Sephadex G-100 column (1.6×100 cm), and the fractions are automatically collected.

[0008] Preferably, the specific steps are as follows: Take 250 g of dried Gynostemma pentaphyllum raw material, add distilled water at a solid-liquid ratio of 1 g: 10 mL, soak for 30 min, reflux at 100℃ for 3 h, filter to remove residue, keep the supernatant, concentrate, then use Sevag reagent to centrifuge and separate the protein, add 4 times the volume of anhydrous ethanol, mix and stir, precipitate overnight, pour onto Brinell's funnel lined with filter paper, cover with plastic wrap, filter by suction, and freeze-dry to obtain crude Gynostemma pentaphyllum polysaccharide; dissolve the crude polysaccharide to prepare a stock solution, centrifuge, load the supernatant onto a DEAE-52 cellulose column (2.6 × 30 cm), perform gradient elution with NaCl solution (flow rate: 4 mL / min), monitor the elution peak using the phenol-sulfuric acid method (490 nm), collect the corresponding main peak fraction (retention time: 15–25 min), concentrate by rotary evaporation to 1 / 5 of the original volume, freeze-dry; after ion exchange purification, reconstitute the sample and then pass it through Sephadex. Purification was performed using a G-100 column (1.6 × 100 cm), with automated fraction collection (12 mL / tube). The total sugar content was analyzed, the total sugar content was measured, and the elution curve was plotted. The main peak fractions were combined, concentrated, and freeze-dried to obtain purified Gynostemma pentaphyllum polysaccharide (GPP).

[0009] Preferably, the drug may also include a pharmaceutically acceptable carrier or excipient, and be formulated into different dosage forms, such as injections, oral solutions, powder injections, aqueous injections, decoctions, or sustained-release preparations.

[0010] The beneficial effects of this invention are as follows:

[0011] 1) This invention is the first to confirm that the prepared Gynostemma pentaphyllum polysaccharide has excellent therapeutic effects on ulcerative colitis. By activating the NRF2 antioxidant pathway, inhibiting the activation of NLRP3 inflammasomes, increasing the abundance of Bacteroidetes and Firmicutes and reducing the abundance of Proteobacteria, it can reduce ROS levels, reduce inflammatory response, and promote the repair of intestinal mucosal barrier in DSS mice. Experimental studies have shown that it has the following effects: (1) It improves the intestinal barrier function of mice with ulcerative colitis, inhibits the inflammatory response, enhances the activity of antioxidant enzymes and reduces ROS accumulation, and improves the symptoms of colitis induced by DSS in mice; (2) It regulates the composition of intestinal microbiota by increasing the abundance of Firmicutes and reducing the level of Proteobacteria, and acts as a natural prebiotic; (3) Further in-depth exploration of the possible mechanism of Gynostemma pentaphyllum polysaccharide in treating ulcerative colitis at the gene level may be related to the targeted inhibition of NLRP3 inflammasome activation and activation of the NRF2 / HO-1 antioxidant pathway, thereby reducing the production of ROS.

[0012] 2) Gynostemma pentaphyllum polysaccharide is a traditional Chinese medicine ingredient that is both food and medicine. It has no toxic effects, is widely distributed and abundant in my country, and has fewer side effects and a price advantage compared to traditional Western medicine. Attached Figure Description

[0013] Figure 1 GPP improves DSS-induced colitis symptoms in mice. The left graph represents the disease activity index; the middle graph represents the spleen index; the right graph represents colon length; CON represents the normal group, MOD represents the DSS model group, Mesalazine represents the DSS model + positive control group, GPPL represents the DSS model + low-dose GPP group, GPPM represents the DSS model + medium-dose GPP group, and GPPH represents the DSS model + high-dose GPP group.

[0014] Figure 2 GPP enhances intestinal wall integrity. The left image shows the relative expression level of the ZO1 gene mRNA; the middle image shows the relative expression level of the Occludin gene mRNA; and the right image shows the relative expression level of the claudin gene mRNA.

[0015] Figure 3 GPP reduces the expression level of NLRP3 inflammasome-related factors.

[0016] Figure 4 GPP activates the NRF2 pathway to alleviate oxidative stress in mice with DSS-induced acute colitis.

[0017] Figure 5 NRF2 knockdown reduces GPP-induced NRF2. - / -Protective effect in mice. The left figure shows changes in body weight; the others, from left to right, represent spleen index, colon index, and colon length.

[0018] Figure 6 GPP can also improve the richness of fecal microbiota and protect UC mice; the left figure shows the abundance of microbiota in each group at the phylum level; the right figure shows the structure of microbiota in each group at the family level.

[0019] Note: Compared with the control group, #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.0001; compared with the model group, *P < 0.05, **P < 0.01, **P < 0.01, ***P < 0.001, ****P < 0.0001. Detailed Implementation

[0020] The following is a further description of the invention, but not a limitation thereof.

[0021] 250 g of Gynostemma pentaphyllum raw material was sourced from Kangmei Pharmaceutical Co., Ltd.

[0022] Sixty male C57BL / 6 mice (8 weeks old, 20–25 g) were purchased from Shenzhen Zhuhai Beston Biotechnology Co., Ltd. NRF2 gene knockout mice parental offspring were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd. The Nfe2l2 gene was knocked out of C57BL / 6 mice using CRISPR / Cas9 technology. Genotyping was verified by PCR (primer sequences: F: 5'-GAAGCATTATCATTTGGTGGGAAC-3', R: 5'-TGCACCAGAGTTCAAAGGTAGC-3'). Homozygous mice were obtained through offspring genotyping and selection for experiments. All mice were housed in an SPF-grade barrier facility (temperature: 23–25°C, humidity: 50 ± 5%, free access to food and water).

[0023] Example 1: GPP can improve DSS-induced colitis symptoms in mice.

[0024] Experimental methods

[0025] (1) Preparation of Gynostemma pentaphyllum polysaccharide: Take 250 g of dried Gynostemma pentaphyllum raw material, add distilled water at a ratio of 1 g: 10 mL (w / v), soak for 30 min, reflux extract at 100℃ for 3 h, filter to remove residue, keep the supernatant, and then concentrate. Then use Sevag reagent to centrifuge and separate the protein, add 4 times the volume of anhydrous ethanol, mix and stir, and precipitate overnight. Pour onto Brinell's funnel lined with filter paper, cover with plastic wrap, filter the liquid with a water pump, and freeze dry to obtain 13.15 g of crude Gynostemma pentaphyllum polysaccharide (extraction rate: 5.26 g / 100 g). Crude polysaccharide was dissolved to prepare a stock solution, centrifuged at 10,000 × g for 10 min, and the supernatant was loaded onto a DEAE-52 cellulose column (2.6 × 30 cm). The supernatant was subjected to gradient elution with NaCl solution (flow rate: 4 mL / min), and the elution peak was monitored by the phenol-sulfuric acid method (490 nm). The corresponding main peak fraction was collected (retention time: 15–25 min), concentrated to 1 / 5 of the original volume by rotary evaporation, and lyophilized. After reconstitution of the ion-exchange purified sample, it was purified by Sephadex G-100 column (1.6 × 100 cm), and the fractions were automatically collected (12 mL / tube). The total sugar content was analyzed, the total sugar content was measured, and the elution curve was plotted. The main peak fractions were combined, concentrated, and lyophilized to obtain purified GPP. Monosaccharide composition and molecular weight analysis of GPP: 10 mg of GPP sample was taken, 1 mL of 2 M trifluoroacetic acid was added, and hydrolysis was carried out at 121 °C for 2 h. The hydrolysis product was dried under nitrogen, washed three times with methanol, and the residue was dissolved in sterile water and transferred to an HPLC vial for analysis. Separation was performed on a Sugar-D column (4.6 × 250 mm, 5 μm) with acetonitrile-water (75:25, v / v) as the mobile phase, at a flow rate of 1.0 mL / min and a column temperature of 30 °C. Further comparison with standard ion chromatography revealed that the obtained GPP is a polysaccharide, with galactose (Gal), arabinogalactose (Ara), rhamnose (Rha), mannose (Man), xylose (Xyl), glucose (Glc), fucose (Fuc), galacturonic acid (Gal-UA), and glucuronic acid (Glc-UA) as its main components, accounting for 42.96%, 25.41%, 9.79%, 4.94%, 3.38%, 2.10%, 1.50%, 6.38%, and 3.55%, respectively. The molecular weight of GPP was determined using high performance size exclusion chromatography coupled with multi-angle laser light scattering (HPSEC-MALLS). The main molecules were concentrated in the range of ~47.6 kDa (Mp), but the average molecular weight was significantly increased (Mw ~160.3 kDa) due to the influence of a small amount of extremely high molecular weight components (Mz ~993.5 kDa).

[0026] GC-MS was used to analyze the polysaccharide bonding structure, and FT-IR spectra were recorded to detect GPP-specific functional groups. Further NMR measurements of 1H and 13C spectra, as well as two-dimensional COSY, HSQC, HMBC, and NOESY spectra, were used to predict the anomeric configuration, glycosidic bond linkage mode, and linkage sequence of the GPP polysaccharide. The main structure of Gynostemma pentaphyllum polysaccharide was finally obtained as follows: .

[0027] The prepared Gynostemma pentaphyllum polysaccharide was prepared according to the experimental animal equivalent drug dose, and was mixed with pure water in the proportions of 1 times the adult equivalent drug dose (400 mg / kg), 0.5 times the adult equivalent drug dose (200 mg / kg), and 0.25 times the adult equivalent drug dose (100 mg / kg) to prepare high-dose, medium-dose, and low-dose solutions. After preparation, the solutions were stored in a refrigerator at 4 degrees Celsius and each storage period should not exceed 2 days. Before use, the solutions were warmed to room temperature.

[0028] (2) Grouping and intervention of experimental animals

[0029] Sixty male C57BL / 6J mice were randomly divided into six groups using a random number table: normal control group (CON), DSS model group (MOD), DSS model + positive control group (Mesalazine), DSS model + low-dose GPP group (GPPL), DSS model + medium-dose GPP group (GPPM), and DSS model + high-dose GPP group (GPPH), with 10 mice in each group. Except for the normal control group (CON), which received an equal volume of physiological saline by gavage, the other groups were allowed free access to a sterile aqueous solution containing 3% sodium dextran sulfate (DSS) to establish the UC mouse model. The DSS model group received physiological saline by gavage, the DSS model + positive control group received mesalazine enteric-coated tablets 100 mg / kg daily by gavage, the DSS model + low-dose GPP group received GPP 100 mg / kg by gavage, the DSS model + medium-dose GPP group received GPP 200 mg / kg by gavage, and the DSS model + high-dose GPP group received GPP 400 mg / kg by gavage.

[0030] (3) Observation and material collection

[0031] The control group was given sterile water freely for 7 days, while the other groups were given (sodium dextran sulfate) DSS freely for 7 days, administered via gavage daily at regular intervals during this period, with the administration occurring simultaneously with model establishment. After 7 days, mice were fasted for 12 hours, then anesthetized and euthanized, and tissues were collected. Body weight was recorded before dissection, and colon length, colonic index (colon weight / body weight), and spleen index (spleen weight / body weight) were measured. Results are shown below. Figure 1 Tissue samples (serum, colon contents, mucosa) should be stored at −80°C or fixed in 4% paraformaldehyde.

[0032] (4) Disease Activity Index (DAI)

[0033] During the experiment, the mice's general condition, weight, fecal consistency, and occult blood were recorded daily. The DAI (Digital Injection Assessment) score was the average of the scores for weight loss, fecal consistency, and rectal bleeding. See the results below. Figure 1 .

[0034] (5) Immunofluorescence staining

[0035] Cells were cultured to a certain density. The old culture medium was discarded, and cells were washed three times with PBS for 5 minutes each time. Cells were fixed with 4% paraformaldehyde, permeated with 0.1% Triton X-100 for 10 minutes, and blocked with 3% BSA for 1 hour. For paraffin-embedded tissue sections, the sections were dewaxed and antigen retrieval was performed by incubation with proteinase K at 37°C for 30 minutes. The sections were then washed three times with PBS for 5 minutes each time, followed by blocking with 3% BSA for 30 minutes. Diluted primary antibodies (anti-NRF2, anti-HO-1, anti-ZO-1, anti-Occludin, anti-NLRP3, anti-IL-1β) were added to the cells and tissue sections and incubated overnight at 4°C. The next day, the sections were washed three times with PBS for 5 minutes each time. Secondary antibodies were then added, and the sections were incubated in the dark at room temperature for 1 hour. Cell nuclei were stained with DAPI. The sections were then mounted with an anti-fluorescence quencher. Images were captured under a fluorescence microscope, and fluorescence intensity was analyzed using ImageJ software.

[0036] (6) RT-qPCR

[0037] Total RNA was extracted from cells and tissues using TRIzol, cDNA was synthesized using the PrimeScript RT kit, and PCR amplification was performed using SYBR Green (10 μL TB Green Premix Ex Taq™, 0.4 μM primers, 2 μL cDNA (50 ng / μL)).

[0038] (7) Western Blotting

[0039] Total protein was extracted from drug-treated colonic mucosa and cultured cells using RIPA lysis buffer containing PMSF. After quantification using the BCA method, the protein was denatured at 95°C for 15 minutes with 5× loading buffer, separated by SDS-PAGE, and transferred to a PVDF membrane. After sealing, diluted primary antibodies (anti-NRF2, anti-HO-1, anti-ZO-1, anti-Occludin, anti-NLRP3, anti-IL-1β, anti-ASC, anti-Caspase-1, and anti-β-actin) were added and incubated overnight at 4°C. The membrane was washed four times with TBST for 5 minutes each time, and incubated at room temperature with HRP-conjugated secondary antibody for 1 hour. Protein bands were detected by chemiluminescence.

[0040] 2. Experimental Results

[0041] (1) GPP can improve DSS-induced colitis symptoms in mice.

[0042] During the experiment, compared with the normal control group, the DSS-induced colitis group had higher DAI scores, which decreased after GPP treatment. Spleen index results showed that high-dose GPP treatment significantly improved DSS-induced splenomegaly. Simultaneously, GPP also demonstrated an important role in protecting the colon from DSS-induced shortening. These data suggest that GPP plays a crucial role in alleviating the symptoms of DSS-induced colitis.

[0043] (2) GPP can enhance intestinal wall integrity, reduce the expression level of NLRP3 inflammasome-related factors, and activate the NRF2 pathway to alleviate oxidative stress in mice with DSS-induced acute colitis.

[0044] RT-qPCR analysis showed that DSS-induced colitis significantly downregulated the mRNA expression levels of ZO-1, Occludin, and claudin-1 in colonic tissue. Figure 2 Meanwhile, the GPP treatment group significantly downregulated the expression of NLRP3 inflammasome core components (NLRP3, Caspase-1, ASC) and IL-1β at the protein level. Figure 3 The results of the immunofluorescence double staining experiment showed that the fluorescence intensity of Nrf2 / HO-1 in the DSS group was the weakest. Figure 4 ).

[0045] Example 2: NRF2 knockdown reduced GPP-induced NRF2 in DSS. - / - Protective effect in mice

[0046] 1. Experimental Methods

[0047] The control group received the same treatment as above. Knockout mice were divided into two groups: NRF2 - / - Model group (NRF2) - / -+DSS): Free access to sterile water containing 3% DSS; daily gavage with sterile water; NRF2 - / - GPPH treatment group (NRF2) - / - +GPPH): Administer a sterile aqueous solution containing 3% DSS via oral administration, followed by daily gavage administration of 400 mg / kg of GPP. Other treatments are the same as above.

[0048] 2. Experimental Results

[0049] (5) NRF2 knockdown reduced the effect of GPP on DSS-induced NRF2. - / - Protective effect in mice

[0050] We further used high-dose GPP to treat DSS-treated Nrf2 gene knockout mice. GPP in Nrf2 - / - There was no significant therapeutic effect in mice. Compared with the control group, there was no significant difference in DAI and body weight loss rate between the DSS-treated group and the DSS+GPPH group. Regarding spleen index, colon index, and colon length, the protective effect disappeared in the GPPH group, and more severe tissue edema was observed. These results indicate that Nrf2 plays a crucial role in the GPP-mediated remission of DSS-induced colitis.

[0051] Example 3: GPP can also improve fecal microbiota richness and protect UC mice.

[0052] Experimental methods

[0053] 16S rRNA sequencing analysis

[0054] Total genomic DNA was extracted from colonic microbiome samples using the TGuide S96 Magnetic Soil / Fecal DNA Extraction Kit (Tiangen Biotech (Beijing) Co., Ltd.) according to the manufacturer's instructions. The hypervariable regions V3-V4 of the bacterial 16S rRNA gene were amplified using primer pairs 338F: 5'-ACTCCTACGGGAGGCAGCA-3' and 806R: 5'-GGACTACHVGGGTWTCTAAT-3'. PCR products were examined on agarose gels and purified using the Omega DNA Purification Kit (Omega Inc., Norcross, GA, USA). The purified PCR products were collected and sequenced at both ends (2 × 250 bp) on an Illumina Novaseq 6000 platform.

[0055] Experimental results: GPP can also improve fecal microbiota richness and protect UC mice.

[0056] Phylum-level analysis showed that the abundance of pathogenic bacteria such as Proteobacteria and Deferribacteres increased in the DSS-induced group, while the abundance of Bacteroidetes and Firmicutes decreased. Family-level analysis further showed that the abundance of pathogenic bacteria such as Enterobacteriaceae increased in the DSS-induced group, while the abundance of Lachnospiraceae and Bacteroidetes decreased. After GPP administration, the abundance of pathogenic bacteria decreased with increasing dose, beneficial bacteria gradually recovered, and the gut microbiota structure approached that of the control group. GPP significantly improved the gut microbiota imbalance in UC mice by reshaping the gut microbiota structure, increasing beneficial bacteria, and inhibiting pathogenic bacteria, thus exerting a prebiotic-like effect.

[0057] In summary, this study is the first to demonstrate that GPP reduces ROS levels, decreases inflammatory responses, and promotes intestinal mucosal barrier repair in DSS mice by activating the NRF2 antioxidant pathway, inhibiting NLRP3 inflammasome activation, increasing the abundance of Bacteroidetes and Firmicutes, and decreasing the abundance of Proteobacteria. These findings not only expand the medicinal value of Gynostemma pentaphyllum resources but also provide a new direction for developing IBD treatment strategies based on natural polysaccharides.

Claims

1. The application of Gynostemma pentaphyllum polysaccharide in the preparation of drugs for treating ulcerative colitis, wherein the main structure of the Gynostemma pentaphyllum polysaccharide is as follows: ； The extraction method of Gynostemma pentaphyllum polysaccharide is as follows: Take dried Gynostemma pentaphyllum raw material, add distilled water, with a solid-liquid ratio of 1-2 g: 10 mL, soak for 20-30 min, reflux at 100℃ for 2-4 h, filter to remove residue, keep the supernatant, and after deproteinization, decolorization, alcohol precipitation, vacuum filtration, and freeze drying, crude Gynostemma pentaphyllum polysaccharide is obtained; after centrifugation of crude polysaccharide, the supernatant is loaded onto a DEAE-52 cellulose column, and NaCl solution gradient elution is performed. The elution peak is monitored by phenol-sulfuric acid method, and the main peak fraction with the corresponding retention time of 15-25 min is collected. The fraction is concentrated to 1 / 5 of the original volume by rotary evaporation, freeze-dried, and after ion exchange purification, the sample is reconstituted and purified by Sephadex G-100 column, and the fractions are automatically collected.

2. The method according to claim 1, characterized in that, DEAE-52 cellulose columns are 2.6 × 30 cm in size, and Sephadex G-100 columns are 1.6 × 100 cm in size.

3. The method according to claim 1, characterized in that, The specific steps for extracting Gynostemma pentaphyllum polysaccharides are as follows: Take 250 g of dried Gynostemma pentaphyllum raw material, add distilled water at a solid-liquid ratio of 1 g: 10 mL, soak for 30 min, reflux at 100℃ for 3 h, filter to remove residue, keep the supernatant, concentrate, then use Sevag reagent to centrifuge and separate the protein, add 4 times the volume of anhydrous ethanol, mix and stir, precipitate overnight, pour onto Brinell's funnel lined with filter paper, cover with plastic wrap, filter by suction, and freeze-dry to obtain crude Gynostemma pentaphyllum polysaccharides; dissolve the crude polysaccharides to prepare a stock solution, centrifuge, load the supernatant onto a DEAE-52 cellulose column, perform gradient elution with NaCl solution, monitor the elution peaks using the phenol-sulfuric acid method, collect the main peak fraction with a retention time of 15–25 minutes, concentrate to 1 / 5 of the original volume by rotary evaporation, and freeze-dry; After reconstitution of the ion-exchange purified sample, it was purified by Sephadex G-100 column, automatically fractionated and collected, and the main peak fractions were combined, concentrated and freeze-dried to obtain purified Gynostemma pentaphyllum polysaccharide.

4. The method according to claim 1, characterized in that, The drug may also include pharmaceutically acceptable carriers or excipients, and be formulated into different dosage forms.

5. The method according to claim 4, characterized in that, Dosage forms include injections, oral solutions, powder injections, water injections, decoctions, or sustained-release preparations.

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