Application of radices stellariae dichotomae polysaccharide in preparation of medicine for treating chronic non-atrophic gastritis, radices stellariae dichotomae polysaccharide gastric floating tablet and preparation method thereof

Abstract

The invention discloses application of radices stellariae dichotomae polysaccharide in preparation of medicine for treating chronic non-atrophic gastritis, radices stellariae dichotomae polysaccharide gastric floating tablets and a preparation method thereof, and relates to the technical field of natural medicine preparations. System experiments prove that the radices stellariae dichotomae polysaccharide can play a role in treating chronic non-atrophic gastritis (CNAG) through triple mechanisms of regulating inflammatory response, improving oxidative stress and intervening a key signal channel for the first time; meanwhile, aiming at the problem of low gastrointestinal tract bioavailability of radices stellariae dichotomae polysaccharide, the gastric floating tablet preparation is designed and optimized, and the optimal prescription is determined by combining a wet granulation tabletting method with a Box-Behnken response surface method, so that long-acting retention and stable release of the medicine in the stomach are realized. The preparation has the advantages of high safety, strong targeting property, lasting curative effect and the like, and a novel natural medicine selection and a matched preparation technology are provided for clinical treatment of CNAG.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of natural medicine preparation, and more particularly to application of silver radix bupleuri polysaccharide in preparation of medicine for treating chronic non-atrophic gastritis, and silver radix bupleuri polysaccharide gastric floating tablet and a preparation method thereof. BACKGROUND

[0002] Chronic non-atrophic gastritis (CNAG) is the type of chronic gastritis with the highest clinical incidence, accounting for 59.3% of chronic gastritis cases, and is more common in people aged 16-59 years. Its causes are related to bile reflux, Helicobacter pylori (Hp) infection, alcohol abuse, long-term use of non-steroidal anti-inflammatory drugs, etc. The clinical manifestations of CNAG are irregular upper abdominal pain, early satiety, acid reflux and belching, etc. Although it can be alleviated after treatment, it is prone to recurrence and long-term development, which may progress to gastric mucosal atrophy, intestinal metaplasia and even gastric cancer, and requires long-term management.

[0003] At present, the clinical treatment of CNAG mainly uses chemical drugs, such as acid suppressants (omeprazole, ranitidine), gastric mucosa protectants (bismuth potassium citrate) and Hp eradication quadruple therapy. However, long-term use of chemical drugs can easily lead to adverse reactions such as insufficient gastric acid secretion and intestinal flora imbalance, and the patient's compliance is low. Therefore, finding natural, low-toxicity and high-efficiency treatment drugs has become a research hotspot.

[0004] As a natural macromolecular substance, plant polysaccharides have pharmacological activities such as anti-inflammatory, antioxidant and immunoregulatory effects, and have small toxic and side effects. Existing studies have confirmed that astragalus polysaccharide and taraxacum polysaccharide can treat chronic gastritis by regulating inflammatory factors and improving oxidative stress, but the CNAG treatment activity and mechanism of silver radix bupleuri polysaccharide (SRP) have not been clarified. Silver radix bupleuri is a native medicinal material in Ningxia, and it has been found that its polysaccharide has anti-acute alcoholic gastric mucosal injury activity. Based on this, the present application further explores the treatment effect and mechanism of SRP on CNAG.

[0005] At the same time, polysaccharide components are easily degraded in the gastrointestinal tract and have low bioavailability. Gastric retention system (GRDDS) can prolong the residence time of drugs in the stomach and improve the local drug concentration, and is suitable for the treatment of gastric diseases. As the main dosage form of GRDDS, gastric floating tablets achieve floating by the hydration and swelling of hydrophilic matrix materials and the generation of gas by foaming agents, but the prescription design (such as the type and amount of excipients) has a significant impact on the floating performance and release rate, and needs to be systematically optimized to ensure the stability and effectiveness of the preparation. SUMMARY

[0006] Therefore, the present application provides a silver radix bupleuri polysaccharide in the preparation of a chronic non-atrophic gastritis medicine and a silver radix bupleuri polysaccharide gastric floating tablet and a preparation method thereof.

[0007] In order to achieve the above purpose, the present application adopts the following technical solutions: Use of silver radix bupleuri polysaccharide in preparation of a drug for treating chronic non-atrophic gastritis.

[0008] The mechanism of action is as follows: Silver radix bupleuri polysaccharide regulates inflammatory response by reducing the content of IL-1beta and IL-6 in the serum of CNAG model animals (P<0.05 or P<0.01), increasing the content of PGE2 in the serum (P<0.05 or P<0.01), and reducing gastric mucosal hyperemia, edema and inflammatory cell infiltration.

[0009] Silver radix bupleuri polysaccharide improves oxidative stress by reducing the level of malondialdehyde (MDA) in the gastric tissue of CNAG model animals (P<0.05 or P<0.01), increasing the activity of superoxide dismutase (SOD) in the gastric tissue (P<0.05 or P<0.01), and reducing oxidative damage to the gastric mucosa.

[0010] Silver radix bupleuri polysaccharide regulates the expression of differentially expressed genes / proteins (such as Tff1, Gkn1, Iqgap2, etc.) in the gastric tissue by regulating the PI3K / Akt signaling pathway, the MAPK signaling pathway and the phospholipase D signaling pathway, thereby exerting a therapeutic effect.

[0011] Another object of the present application is to provide a silver radix bupleuri polysaccharide gastric floating tablet, each tablet being composed of a main drug and excipients, the main drug being 100 mg of silver radix bupleuri polysaccharide, and the excipients including: 90-150 mg of a hydrophilic matrix material, 30-70 mg of a foaming agent, 20-60 mg of a floating aid, and 20-60 mg of a pore-forming agent, with the tablet weight being 300-400 mg.

[0012] Further, the excipients further include a lubricant accounting for 1% of the total mass of the tablet.

[0013] Further, the hydrophilic matrix material is HPMC K4M; the foaming agent is NaHCO3; the floating aid is octadecanol; the pore-forming agent is PVPk30; and the lubricant is magnesium stearate.

[0014] Further, the quality indicators of the gastric floating tablet meet the following requirements: ① Appearance: light yellow tablets with uniform color and smooth and complete surface, free of spots and cracks; ② Weight difference: within ±5%; ③ Brittleness: <1%, free of cracking or crushing; ④ Polysaccharide content: RSD 27.84%±1.26%; ⑤ Floating performance: floating time <20 s and floating duration >8 h; ⑥In vitro release: In 0.1 mol / L hydrochloric acid medium, 37±0.5℃, using a small cup method, rotation speed 50r / min, 6h cumulative release >80%, release curve conforms to Higuchi equation.

[0015] The present application also aims to provide a preparation method of the above-mentioned silver radix bupleuri polysaccharide stomach floating tablet, comprising the following steps: (1) Extraction and purification of silver radix bupleuri polysaccharide: (11) Crude extraction: Take silver radix bupleuri medicinal materials, crush and sieve, add distilled water, heat reflux extraction, centrifuge after extraction, and combine supernatant; (12) Alcohol precipitation: Concentrate the supernatant to 1 / 5 of the original volume by rotary evaporation, add 3 times the volume of anhydrous ethanol, stand, centrifuge, and collect the precipitate; (13) Protein removal: Redissolve the precipitate to a concentration of 20mg / mL with distilled water, add 1 / 4 volume of Sevage reagent, shake, stand, discard the lower layer, and repeat the operation 6 times to collect the upper layer solution; (14) Color removal: Chromatograph the protein-removed solution through a pretreated macroporous adsorption resin AB-8 chromatographic column, with a sample flow rate of 2BV / h and a sample volume of 1BV, elute with 20% ethanol, and collect the eluate; (15) Purification: Concentrate the eluate, chromatograph through a DEAE-52 cellulose column, elute with a gradient of 0~0.5mol / L NaCl solution, monitor the elution peak by phenol-sulfuric acid method, collect the main fraction, dialyze, concentrate, and vacuum freeze-dry to obtain silver radix bupleuri polysaccharide; (2) Pre-treatment of excipients: Screen HPMC K4M, NaHCO3, octadecanol, and PVPk30 through 100 mesh sieves, and screen magnesium stearate through an 80 mesh sieve for standby; (3) Prescription screening and optimization: (31) Single-factor experiment: Fix the dosages of other excipients, respectively investigate the effects of HPMC type, NaHCO3 dosage, octadecanol dosage, and PVPk30 dosage on the floating performance and in vitro release of the tablet, and determine the types and dosage ranges of the excipients; (32) Box-Behnken response surface method optimization: Take HPMC K4M dosage A, NaHCO3 dosage B, and octadecanol dosage C as independent variables, take 2h release Y2, 6h release Y5, 10h release Y10, and floating performance score F as response values, adopt a three-factor three-level design, fit a regression equation through DesignExpert13 software: Y=10.06+1.97A-1.72B+1.33C-0.135AB+1.23AC-0.5825BC+2.22A²+1.59B²+2.42C², R²=0.9831, and determine the optimal excipient dosage; (4) Granulation and tableting: (41) Mixing: Weigh out the polysaccharide and excipients of Stellaria dichotoma according to the optimal prescription, and mix them in a three-dimensional mixer. The mixing uniformity RSD is less than 5% to avoid uneven distribution of the main drug and excipients. (42) Preparation of soft material: Add 75% ethanol as a wetting agent, and control the amount of wetting agent to 15%~20% of the total weight of the material to ensure that the soft material "can be clumped when squeezed and dispersed when lightly pressed", and avoid the particles being too hard or too soft. (43) Granulation: The soft material is granulated through a 40-mesh sieve, the wet granules are dried in a constant temperature oven, and then granulated through a 30-mesh sieve. (44) Total mixing: Add 1% magnesium stearate to the dry granules and mix for 5 min; (45) Tableting: Use a basket press, set the pressure to 4~5kg, and the tableting speed to 30 tablets / min to prepare gastric floating tablets of bupleurum polysaccharide; too low pressure will easily lead to loose tablets (loose when floating), and too high pressure will easily lead to hard tablets (floating time is extended by more than 2min).

[0016] (5) Quality inspection: Check the appearance, weight difference, friability, content, floating performance and in vitro release according to the quality indicators, and package after passing the inspection.

[0017] Quality control system Based on the 2020 edition of the Chinese Pharmacopoeia, Volume IV, and the guidelines for sustained-release formulations, a comprehensive quality control process was established: Intermediate control: Moisture content of wet granules is 3%~5%, and particle size of dry granules is >95% (passing rate through 30 mesh sieve).

[0018] Further, in step (11), the sieving is through a 40-mesh sieve, the material-to-liquid ratio of distilled water is g:mL=1:20, the hot reflux extraction is 90℃ hot reflux extraction twice, each time for 2 hours, and the centrifugation is 8000rpm for 10 minutes. In step (12), after adding anhydrous ethanol, the final concentration is 80%. The standing time is 12 h at 4 °C, and the centrifugation time is 10 min at 8000 rpm.

[0019] Furthermore, the Sevage reagent mentioned in step (13) includes chloroform: n-butanol in a volume ratio of 4:1, with a shaking time of 30 min and a standing time of 10 min; In step (14), the macroporous adsorption resin AB-8 chromatography column has a size of 4cm×60cm, and the eluent volume used during elution is 2BV. In step (15), the dialysis time is 24 hours, the molecular weight cutoff is 8000~14000 Da; the purity of the silver bupleurum polysaccharide is ≥93%, the protein content is ≤0.6%, and the weight average molecular weight is 90842 Da (determined by HPGPC method).

[0020] Furthermore, the pretreatment of macroporous adsorption resin AB-8 in step (14) is as follows: wash with distilled water until there are no impurities, soak in anhydrous ethanol for 24 hours, wash with distilled water until there is no alcohol odor, and adjust the pH to 7.0 to ensure adsorption efficiency.

[0021] Furthermore, the DEAE-52 cellulose column mentioned in step (15) is first pretreated. The pretreatment method is as follows: weigh 100g of DEAE-52 packing material, wash it with deionized water until there are no floating impurities, and soak it for 24h until it is fully swollen; then soak it in 0.5mol / L NaOH solution for 2h, wash it with deionized water until neutral, soak it in 0.5mol / L HCl solution for 2h, wash it with deionized water until neutral, repeat the NaOH treatment step once, and pack the column after vacuum degassing.

[0022] Furthermore, in step (32), the dosage of HPMC K4M is A: 90~150mg, the dosage of NaHCO3 is B: 30~70mg, and the dosage of octadecyl alcohol is C: 20~60mg.

[0023] Furthermore, the mixing time in step (41) is 15 min; In step (43), the drying is carried out at 40°C, and the moisture content is controlled at 3%~5%. Too high a moisture content will cause the tablets to stick together, and too low a moisture content will cause the tablets to be brittle and unqualified.

[0024] As can be seen from the above technical solution, compared with the prior art, the present invention has the following technical effects: The gastric floating tablets prepared by this invention enhance the therapeutic effect by prolonging the drug's retention time in the stomach (>8h), increasing the local drug concentration in the gastric mucosa, reducing drug degradation in the intestine; The therapeutic effects of the gastric floating tablets prepared in this invention on CNAG model animals are as follows: it improves symptoms of thinning and weakened elasticity of the gastric wall, reduces scattered bleeding points in the gastric mucosa; the arrangement of gastric mucosal epithelial cells tends to be more regular, and the edema of the mucosal layer and inflammatory infiltration of the lamina propria are reduced; serum IL-1β and IL-6 levels are decreased (P<0.05 or P<0.01), and PGE2 levels are increased (P<0.05 or P<0.01); gastric tissue MDA levels are decreased (P<0.05 or P<0.01), and SOD levels are increased (P<0.05 or P<0.01).

[0025] This invention clarifies for the first time the CNAG therapeutic activity of Stellaria dichotoma polysaccharide and its triple mechanism of action of "anti-inflammatory-antioxidant-regulation of signaling pathways", enriching the theoretical basis for the treatment of gastric diseases by plant polysaccharides; This invention develops new pharmaceutical applications for Stellaria dichotoma polysaccharide, providing a natural and low-toxicity novel drug for the treatment of CNAG, thus compensating for the shortcomings of chemical drugs; The gastric floating tablet formulation designed in this invention is scientifically optimized using the Box-Behnken response surface methodology to ensure stable floating performance and release rate, thus solving the problem of low bioavailability of polysaccharide drugs. This invention establishes a complete process of "extraction-purification-granulation-tableting-quality control", with clearly defined key parameters, suitable for industrial production, and provides technical support for the industrialization of *Stellaria dichotoma* polysaccharide preparations. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0027] Figure 1 The elution curve of DeAE-52 cellulose column chromatography for Stellaria dichotoma polysaccharide shows that the elution peak of 0 mol / L NaCl is the main peak (yield 83.25%). Figure 2 The standard curve for glucose (Y = 0.006315X + 0.1093, R0) is shown. 2 =0.9986), used for polysaccharide content determination; Figure 3 The standard curve for bovine serum albumin (Y = 3.9943X + 0.0246, R0) is shown. 2 =0.995), used for protein content determination; Figure 4 The effect of *Clerodendrum chinense* polysaccharide on the body weight of CNAG rats (A: male, B: female) showed that the body weight of the treatment group tended to be normal. Figure 5 The effect of Stellaria dichotoma polysaccharide on the morphology of gastric tissue in CNAG rats (A: blank group, B: model group, C: positive control group, D: low-dose group, E: medium-dose group, F: high-dose group) showed that the gastric mucosal damage was reduced in the treatment group; Figure 6 The effect of Stellaria dichotoma polysaccharide on the pathological characteristics of gastric tissue in CNAG rats (HE staining, 200 μm) showed that the epithelial cells in the treatment group were arranged in a regular manner. Figure 7 The image shows the appearance of the Stellaria dichotoma polysaccharide gastric floating tablets, which are pale yellow and have a smooth surface. Figure 8 The effect of *Bupleurum chinense* polysaccharide gastric floating tablets on gastric tissue of CNAG rats (A / B: blank group, C / D: model group, E / F: gastric floating tablet group) showed that the gastric floating tablet group had a significant effect on gastric mucosal repair. Figure 9The Box-Behnken response surface methodology is used to plot the effects of pairwise interactions of HPMC K4M dosage (A), NaHCO3 dosage (B), and stearyl alcohol dosage (C) on the overall effect of Yin Chai Hu polysaccharide gastric floating tablets. (a) Response surface methodology. Figure 1 (b) Contour plot: A (HPMC K4M) × B (NaHCO3) interaction; (c) Response surface Figure 2 (d) Contour plot: A (HPMCK4M) × C (octadecyl alcohol) interaction; (e) Response surface methodology Figure 3 (f) Contour plot: B (NaHCO3) × C (octadecyl alcohol) interaction; Figure 10 shows the KEGG enrichment analysis of differentially expressed genes in Control_VS_Model; Figure 11 shows the KEGG enrichment analysis of differentially expressed genes in Model_VS_SRP; Figure 12 shows the KEGG enrichment analysis of differentially expressed proteins in Control_VS_Model; Figure 13 shows the KEGG enrichment analysis of differentially expressed proteins in Model_VS_SRP. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] This invention, through systematic experiments, confirms the CNAG therapeutic activity of Stellaria dichotoma polysaccharide at the animal model and molecular mechanism levels, as detailed below: Animal model validation A CNAG rat model was established using a combination of chemical stimulation and a starvation-satiety disorder method. Rats had free access to 0.05% ammonia water daily and were subjected to a starvation-satiety disorder method of "2 days of adequate feeding + 1 day of fasting". Rats were administered 20 mmol / L sodium deoxycholate solution by gavage on a non-fasting stomach and 40% ethanol solution (2 mL / kg) by gavage on an empty stomach for 12 weeks. After successful model establishment, rats were treated with low (10 mg / kg), medium (20 mg / kg), and high (40 mg / kg) doses of Stellaria dichotoma polysaccharide for 14 days, with bismuth potassium citrate (80 mg / kg) as a positive control.

[0030] Improvement in general condition: The model group rats showed lethargy, disheveled fur, and weight loss. After administration of Stellaria dichotoma polysaccharide, the rats' appetite increased, their mental state improved, and their weight returned to normal. Figure 4 ); Gastric tissue morphology repair: In the model group, the gastric wall of rats was thinner, less elastic, and had fewer mucosal folds with scattered bleeding points. In the treatment group with Stellaria dichotoma polysaccharide, the gastric wall thickness and elasticity were improved, and the mucosal damage was reduced. Figure 5 ); Pathological improvement: In the model group, gastric mucosal epithelial cells showed disordered arrangement, shedding and necrosis, submucosal congestion and edema, and inflammatory cell infiltration in the lamina propria; in the treatment group, epithelial cells were regularly arranged, and edema and inflammatory infiltration were reduced. Figure 6 ).

[0031] Regulation of inflammatory factors Serum IL-1β, IL-6, and PGE2 levels in rats were detected using enzyme-linked immunosorbent assay (ELISA). Compared with the control group, the serum IL-1β and IL-6 levels in the model group were significantly increased (P<0.01), and the PGE2 level was significantly decreased (P<0.01). Compared with the model group, the levels of IL-1β and IL-6 in each dose group of Stellaria dichotoma polysaccharide were significantly decreased (P<0.05 or P<0.01), while the levels of PGE2 in the medium and high dose groups were significantly increased (P<0.01), and the results were dose-dependent.

[0032] Improvement of oxidative stress Detection of MDA levels and SOD activity in rat gastric tissue: The MDA content in the gastric tissue of the model group was significantly increased (P<0.01), and the SOD activity was significantly decreased (P<0.01). The MDA level in the *Stellaria dichotoma* polysaccharide treatment group was significantly reduced (P<0.05 or P<0.01), and the SOD activity was significantly increased (P<0.05 or P<0.01), suggesting that it can protect the gastric mucosa by enhancing antioxidant capacity.

[0033] Molecular mechanism analysis Transcriptomics and DIA quantitative proteomics techniques were used to analyze differentially expressed genes (DEG) and proteins (DEP) in the gastric tissue of CNAG rats: Transcriptomics results: 337 DEGs were screened from the control group and model group (208 upregulated / 129 downregulated), and 372 DEGs were screened from the model group and high-dose SRP group (110 upregulated / 262 downregulated). GO enrichment analysis showed that DEGs are mainly involved in biological processes such as "epithelial structure maintenance" and "interleukin-6 production". KEGG enrichment analysis showed that DEGs were significantly enriched in the PI3K / Akt signaling pathway and the MAPK signaling pathway. Figure 10 , 11 ).

[0034] Figure 10The study primarily showcases the pathway enrichment characteristics of differentially expressed genes (DEGs) between the control group and the CNAG model group. Primary pathways encompass cellular processes and environmental information processing. DEGs are mainly enriched in pathways related to drug metabolism and morphine dependence. Key pathways associated with gastritis include the PI3K-Akt signaling pathway, the MAPK signaling pathway, and the TNF signaling pathway, confirming the abnormal activation of inflammation-related pathways in the model group.

[0035] Figure 11 In the study, pathway enrichment of DEGs was observed in both the model group and the SRP treatment group, with the primary pathway types consistent with those in Figure 10. DEGs were enriched in gastrointestinal metabolic pathways such as gastric acid secretion and bile secretion. Core pathways related to gastritis included the PI3K-Akt signaling pathway, the Ras signaling pathway, and the Rapl signaling pathway, suggesting that SRP may improve gastritis by regulating these pathways.

[0036] Proteomics results: 204 DEPs were screened from the control and model groups (174 upregulated / 30 downregulated), and 500 DEPs were screened from the model and high-dose SRP groups (378 upregulated / 122 downregulated). GO enrichment analysis showed that DEPs are mainly involved in processes such as "inositol phosphate-mediated signal transduction" and "defensive respiratory burst." KEGG enrichment analysis showed that DEPs were also enriched in the PI3K / Akt and MAPK signaling pathways. Figure 12 , 13 ).

[0037] Figure 12 The study presented the pathway distribution of differentially expressed proteins (DEPs) between the control group and the model group. Primary pathways were mainly cellular processes and environmental information processing. DEPs were enriched in circadian rhythms and the TGF-β signaling pathway. The core inflammation-related pathways included the AMPK signaling pathway, FoxO signaling pathway, PI3K-Akt signaling pathway, and MAPK signaling pathway, which corresponds to the transcriptomic enrichment results and reflects abnormalities in inflammatory pathways at the protein level.

[0038] Figure 13 The study shows the pathway enrichment of DEPs in the model group and the SRP treatment group. Primary pathways include metabolism and cellular processes. DEPs are enriched in pathways such as protein secretion and butyrate metabolism. Core pathways related to gastritis include the PI3K-Akt signaling pathway, the Ras signaling pathway, and the MAPK signaling pathway, indicating that SRP can exert its therapeutic effect by regulating these protein pathways.

[0039] Combined analysis results: DEG / DEP, which showed a consistent expression trend between the model group and the SRP group, was mainly enriched in the phospholipase D signaling pathway, involving key molecules such as Tff1, Gkn1, and Iqgap2, suggesting that Stellaria dichotoma polysaccharide may exert its therapeutic effect by inhibiting the PI3K / Akt and MAPK pathways and regulating the phospholipase D signaling pathway.

[0040] The formulation design of the *Stellaria dichotoma* polysaccharide gastric floating tablets of this invention To address the issue of low gastrointestinal bioavailability of *Stellaria dichotoma* polysaccharides, this invention designs a gastric floating tablet formulation. The optimal formulation was determined through single-factor experiments combined with Box-Behnken response surface methodology, as detailed below: Selection of auxiliary materials Hydrophilic framework materials: HPMC K4M, K15M and K100M were investigated. The results showed that the tablets prepared by HPMC K4M had the shortest floating time (14.10s), the highest release rate (>80%) at 6h, and the floating duration was >12h. Therefore, HPMC K4M was selected. Foaming agent: NaHCO3, Na2CO3 and CaCO3 were examined. NaHCO3 reacted with gastric acid the fastest, with a floating time of 13.73s, which was significantly shorter than Na2CO3 (21.47s) and CaCO3 (66.97s). Therefore, NaHCO3 was selected. Bleeding aids: Stearyl alcohol and stearic acid were tested. The tablets in the stearyl alcohol group floated for more than 12 hours, while those in the stearic acid group floated for only 11 hours. Furthermore, the in vitro release of the stearyl alcohol group was more stable. Therefore, stearyl alcohol was selected. Pore-forming agents: PVPk30 and CMS-Na were investigated. CMS-Na interfered with the determination of polysaccharide content by the phenol-sulfuric acid method, while PVPk30 did not interfere and could improve the release rate. Therefore, PVPk30 was selected.

[0041] The optimal formulation was determined using HPMC K4M dosage (A), NaHCO3 dosage (B), and stearyl alcohol dosage (C) as independent variables. The Box-Behnken response surface methodology was employed for optimization, resulting in the following formulation per tablet: | Component | Dosage (mg) | Action | |------|------------|------|| *Stellaria dichotoma* polysaccharide | 100 | Main drug, exerting anti-inflammatory and antioxidant effects || HPMC K4M | 108 | Hydrophilic framework material, hydrating to form a gel barrier, prolonging release || NaHCO3 | 68 | Foaming agent, reacting with gastric acid to produce CO2, providing buoyancy || Stearyl alcohol | 36 | Floating aid, enhancing hydrophobicity, maintaining buoyancy || PVPk 30 | 38 | Pore-forming agent, increasing tablet porosity, improving drug release || Magnesium stearate | 3.5 (1%) | Lubricant, improving particle flowability, preventing sticking || Total | 350 | - The order of influence of excipients was determined by analysis of variance (Table 1). The order of influence of excipients on tablet performance was: HPMC K4M (A) > NaHCO3 (B) > stearyl alcohol (C). Among them, the amount of HPMC K4M had the most significant effect on release rate (F=90.73, P<0.0001), and NaHCO3 had the most significant effect on flocculation performance (F=69.44, P<0.0001).

[0042] Table 1. Results of Analysis of Variance

[0043] The specific implementation method is as follows: Example 1: Extraction and purification of *Stellaria dichotoma* polysaccharides Medicinal material processing: Take 350.07g of Bupleurum chinense, pulverize it and pass it through a 40-mesh sieve, then dry it in a forced-air drying oven at 60℃ until constant weight; Crude extraction: Add 7001.4 mL of distilled water at a material-to-liquid ratio of 1:20, and extract twice at 90℃ for 2 hours each time. After extraction, centrifuge at 8000 rpm for 10 minutes and combine the supernatants. Alcohol precipitation: The supernatant was concentrated to 1400 mL by rotary evaporation, 4200 mL of anhydrous ethanol (final concentration 80%) was added, and the mixture was allowed to stand at 4℃ for 12 h. After centrifugation at 8000 rpm for 10 min, the precipitate was collected. Protein removal: Redissolve the precipitate with distilled water to 20 mg / mL, add 1 / 4 volume of Sevage reagent (chloroform: n-butanol = 4:1), shake for 30 min, let stand for 10 min and discard the lower layer. Repeat 6 times and collect the upper layer solution. Pigment removal: Pass the supernatant solution through a pretreated macroporous adsorption resin AB-8 chromatography column (4cm×60cm) at a flow rate of 2BV / h and a loading volume of 1BV (approximately 700mL). Elute with 1400mL of 20% ethanol and collect the eluent. Purification: The eluent was concentrated to 200 mL and subjected to DEAE-52 cellulose column chromatography (100 g packing material). Gradient elution was performed with 0, 0.1, 0.3, and 0.5 mol / L NaCl solutions, collecting one tube every 10 min. The absorbance at 490 nm was measured using the phenol-sulfuric acid method, and the elution curve was plotted. The 0 mol / L NaCl elution peak (major peak) was combined. The mixture was dialyzed for 24 h (molecular weight cutoff 8000~14000 Da), concentrated, and then freeze-dried under vacuum to obtain 4.51 g of *Stellaria media* polysaccharide, with a yield of 1.29%, purity of 93.33%, protein content of 0.57%, and weight-average molecular weight of 90842 Da.

[0044] Example 2: Preparation of Stellaria dichotoma polysaccharide gastric floating tablets Excipients weighing: Weigh out 100g of Stellaria dichotoma polysaccharide, 108g of HPMC K4M, 68g of NaHCO3, 36g of stearyl alcohol, 38g of PVPk30, and 3.5g of magnesium stearate according to the optimal prescription (calculated as 1% of the total weight). Mixing: Place the polysaccharide of Stellaria dichotoma, HPMC K4M, NaHCO3, stearyl alcohol, and PVPk30 in a three-dimensional mixer and mix at 20 rpm for 15 min. Take a sample to determine the mixing uniformity (RSD = 3.2% < 5%). To prepare the soft material: Gradually add 75% ethanol (about 80 mL) while stirring, until the soft material "can be formed into a ball when squeezed by hand, but can be easily dispersed when lightly pressed"; Granulation: The soft material is granulated through a 40-mesh sieve. The wet granules are dried in a 40℃ constant temperature oven for 4 hours. The moisture content is measured to be 4.1%. The granules are then sized by passing them through a 30-mesh sieve. Total mixing: Add magnesium stearate to the dry granules and mix at 15 rpm for 5 min using a three-dimensional mixer; Tableting: Using a basket-type tablet press, the pressure was set to 4.5 kg and the speed to 30 tablets / min, 1000 tablets of Yinchaihu polysaccharide gastric floating tablets were obtained (each tablet weighs 350 mg). Quality inspection: 20 tablets were randomly selected for inspection. The appearance was light yellow with a smooth surface. The weight difference was ±3.8%, the brittleness was 0.52%, the polysaccharide content was 27.84%, the initial floating time was 13.05s, the floating time was >8h, and the cumulative release amount in 6h was 80.59%, all of which met the requirements.

[0045] Example 3: Pharmacodynamic Experiment of Stellaria dichotoma Polysaccharide Gastric Floating Tablets Animal grouping: Forty SPF-grade male SD rats, weighing 180-220g, were randomly divided into blank group, model group, Stellaria dichotoma polysaccharide powder group (40mg / kg), and Stellaria dichotoma polysaccharide gastric floating tablet group (40mg / kg SRP), with 10 rats in each group; Model establishment: Except for the control group, the other groups were modeled for 12 weeks according to the "free drinking of 0.05% ammonia water + irregular hunger and satiety + deoxycholate sodium / ethanol gavage" method. Drug administration: The blank group and the model group were administered normal saline by gavage, while the other groups were administered the corresponding drugs by gavage, once a day for 14 days; Testing indicators: Pathological examination: After treatment, rats were sacrificed and the gastric antrum tissue was stained with hematoxylin and eosin (HE). The gastric mucosal injury score of the model group (8.2±1.3) was significantly higher than that of the blank group (1.1±0.4), and the score of the gastric floating tablet group (3.5±0.8) was significantly lower than that of the powder group (5.1±1.0) (P<0.05). Inflammatory factors: Serum IL-1β (125.3±15.2 pg / mL) and IL-6 (210.5±22.3 pg / mL) in the gastric floating tablet group were significantly lower than those in the model group (280.6±30.1 pg / mL, 450.8±42.5 pg / mL) (P<0.01), while PGE2 (180.2±18.5 pg / mL) was significantly higher than that in the model group (85.3±10.2 pg / mL) (P<0.01). Oxidative stress: The gastric tissue MDA in the gastric floating tablet group (3.2±0.5 nmol / mgprot) was significantly lower than that in the model group (6.8±0.9 nmol / mgprot) (P<0.01), while the SOD (120.5±12.3 U / mgprot) was significantly higher than that in the model group (65.8±8.5 U / mgprot) (P<0.01).

[0046] Based on the above experiments, the following conclusions can be drawn: Gastric tissue repair: Gastric wall thickness and elasticity are significantly improved, mucosal bleeding points are reduced, epithelial cells are arranged in a regular manner, and inflammatory infiltration is reduced. Figure 8 ); Regulation of inflammatory factors: Serum IL-1β and IL-6 levels were significantly decreased (P<0.01), while PGE2 levels were significantly increased (P<0.01). Improvement of oxidative stress: The level of MDA in gastric tissue was significantly reduced (P<0.01), and the activity of SOD was significantly increased (P<0.01). The efficacy was comparable to that of the high-dose group of Stellaria dichotoma polysaccharide and superior to that of ordinary Stellaria dichotoma polysaccharide powder (because the floating tablets prolong the retention time in the stomach, resulting in a higher local drug concentration).

[0047] Formulation advantages Highly targeted: Float time > 8h, prolonging drug retention time in the stomach, increasing local drug concentration in the gastric mucosa, and reducing systemic distribution; Stable release: The cumulative release over 6 hours is >80%, and the release curve conforms to the Higuchi equation (sustaining release characteristics), avoiding local irritation caused by sudden drug release; The process is feasible: the wet granulation and tableting method is adopted, the equipment is conventional and suitable for industrial production, and the excipients are all listed in the pharmacopoeia, which ensures high safety. High patient compliance: Tablets are convenient to take, have no obvious adverse reactions, and can reduce the frequency of administration (once a day), thus improving patient compliance.

[0048] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0049] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of Stellaria dichotoma polysaccharide in the preparation of drugs for treating chronic non-atrophic gastritis.

2. A gastric floating tablet containing *Stellaria media* polysaccharide, characterized in that... Each tablet consists of a main drug and excipients. The main drug is 100mg of Stellaria dichotoma polysaccharide, and the excipients include: 90-150mg of hydrophilic skeleton material, 30-70mg of foaming agent, 20-60mg of bleaching agent, 20-60mg of pore-forming agent, and a tablet weight of 300-400mg.

3. The *Stellaria media* polysaccharide gastric floating tablet according to claim 2, characterized in that, The excipients also include a lubricant comprising 1% of the total weight of the tablets.

4. The *Stellaria media* polysaccharide gastric floating tablet according to claim 3, characterized in that, The hydrophilic skeleton material is HPMC K4M; the foaming agent is NaHCO3; the bleaching agent is octadecyl alcohol; the pore-forming agent is PVPk30; and the lubricant is magnesium stearate.

5. The *Stellaria media* polysaccharide gastric floating tablet according to claim 4, characterized in that, The quality indicators of the gastric floating tablets meet the following requirements: ① Appearance: Pale yellow tablets with uniform color, smooth and intact surface, free of spots and cracks; ② Weight difference: within ±5%; ③ Friability: <1%, with no breakage, cracking, or pulverization; ④ Polysaccharide content: RSD 27.84% ± 1.26%; ⑤Floating performance: Floating time < 20s, floating duration > 8h; ⑥ In vitro release rate: In 0.1 mol / L hydrochloric acid medium at 37±0.5℃, the release rate was measured using the small cup method at a rotation speed of 50 r / min. The cumulative release rate over 6 hours was >80%, and the release curve conformed to the Higuchi equation.

6. The method for preparing a gastric floating tablet of *Stellaria media* polysaccharide as described in claim 5, characterized in that, Includes the following steps: (1) Extraction and purification of Stellaria dichotoma polysaccharides: (11) Crude extraction: Take the medicinal material of Stellaria dichotoma, crush it and sieve it, add distilled water, extract by hot reflux, centrifuge after extraction, and combine the supernatants; (12) Alcohol precipitation: The supernatant was concentrated to 1 / 5 of its original volume by rotary evaporation, 3 times the volume of anhydrous ethanol was added, the mixture was allowed to stand, centrifuged, and the precipitate was collected. (13) Protein removal: The precipitate was reconstituted with distilled water to a concentration of 20 mg / mL, 1 / 4 volume of Sevage reagent was added, shaken, and the lower layer was discarded after standing. The operation was repeated 6 times and the upper layer solution was collected. (14) Pigment removal: The protein-free solution is passed through a pretreated macroporous adsorption resin AB-8 chromatography column at a flow rate of 2 BV / h and a loading volume of 1 BV. It is eluted with 20% ethanol and the eluent is collected. (15) Purification: After concentrating the eluent, the eluent was subjected to DEAE-52 cellulose column chromatography with gradient elution of 0~0.5mol / L NaCl solution. The elution peak was monitored by phenol-sulfuric acid method. The main fraction was collected, dialyzed, concentrated and then freeze-dried under vacuum to obtain Stellaria dichotoma polysaccharide. (2) Pretreatment of excipients: HPMC K4M, NaHCO3, octadecanol and PVPk30 are passed through a 100-mesh sieve and magnesium stearate is passed through an 80-mesh sieve for later use. (3) Prescription screening and optimization: (31) Single-factor experiment: With other excipients fixed, the effects of HPMC type, NaHCO3 dosage, octadecanol dosage and PVPk30 dosage on tablet floating performance and in vitro release were investigated to determine the type and dosage range of excipients. (32) Box-Behnken response surface methodology optimization: HPMC K4M dosage A, NaHCO3 dosage B, and octadecanol dosage C were used as independent variables, and the release rates at 2h (Y2), 6h (Y5), and 10h (Y6) were used as the values ​​of release rate. 10 The floating performance score F was used as the response value. A three-factor, three-level design was adopted, and the regression equation was fitted using DesignExpert 13 software. Y = 10.06 + 1.97A - 1.72B + 1.33C - 0.135AB + 1.23AC - 0.5825BC + 2.22A² + 1.59B² + 2.42C², R² = 0.9831, determine the optimal amount of auxiliary materials; (4) Granulation and tableting: (41) Mixing: Weigh out the polysaccharide and excipients of Stellaria dichotoma according to the optimal prescription, and mix them in a three-dimensional mixer. The mixing uniformity RSD is less than 5%. (42) To make soft material: add 75% ethanol as a wetting agent and gradually add it until it is in the state of "being able to be clumped by hand and easily dispersed by light pressure"; (43) Granulation: The soft material is granulated through a 40-mesh sieve, the wet granules are dried in a constant temperature oven, and then granulated through a 30-mesh sieve. (44) Total mixing: Add 1% magnesium stearate to the dry granules and mix for 5 min; (45) Tableting: Using a basket press, the pressure was set to 4~5 kg and the tableting speed was 30 tablets / min to prepare gastric floating tablets of Stellaria dichotoma polysaccharide; (5) Quality inspection: Check the appearance, weight difference, friability, content, floating performance and in vitro release according to the quality indicators, and package after passing the inspection.

7. The method for preparing a gastric floating tablet of *Stellaria media* polysaccharide according to claim 6, characterized in that, In step (11), the sieving is sieving through a 40-mesh sieve, the material-to-liquid ratio of distilled water is g:mL=1:20, the hot reflux extraction is reflux extraction at 90℃ twice, each time for 2 hours, and the centrifugation is centrifugation at 8000rpm for 10 minutes. In step (12), after adding anhydrous ethanol, the final volume concentration is 80%. The standing time is 12 h at 4 °C, and the centrifugation time is 10 min at 8000 rpm.

8. The method for preparing a gastric floating tablet of *Stellaria media* polysaccharide according to claim 6, characterized in that, The Sevage reagent mentioned in step (13) includes V 氯仿 V 正丁醇 =4:1, oscillation time 30min, settling time 10min; In step (14), the macroporous adsorption resin AB-8 chromatography column has a size of 4cm×60cm, and the eluent volume used during elution is 2BV. In step (15), the dialysis time is 24 hours, the molecular weight cutoff is 8000~14000 Da; the purity of the silver bupleurum polysaccharide is ≥93%, the protein content is ≤0.6%, and the weight average molecular weight is 90842 Da.

9. The method for preparing a gastric floating tablet of *Stellaria media* polysaccharide according to claim 6, characterized in that, The DEAE-52 cellulose column described in step (15) is first pretreated. The pretreatment method is as follows: weigh 100g of DEAE-52 packing material, wash it with deionized water until there are no floating impurities, and soak it for 24h until it is fully swollen; then soak it in 0.5mol / L NaOH solution for 2h, wash it with deionized water until neutral, soak it in 0.5mol / L HCl solution for 2h, wash it with deionized water until neutral, repeat the NaOH treatment step once, and pack the column after vacuum degassing.

10. The method for preparing a gastric floating tablet of *Stellaria media* polysaccharide according to claim 6, characterized in that, The mixing time in step (41) is 15 min; In step (43), the moisture content during drying is controlled at 3%~5%.

Images