Peimisine for inhibiting gastric cancer as well as application and composition of peimisine

Abstract

The invention discloses peimisine for inhibiting gastric cancer as well as application and a composition thereof, and relates to the technical field of application of peimisine compositions. The peimisine has the effect of inhibiting gastric cancer by inhibiting a PI3K / Akt signal channel. The preparation method comprises the following steps: mixing pregelatinized starch, a composite auxiliary material, lactose and a peimisine microcapsule, adding polyethylene glycol, sodium carboxymethyl starch and low-substituted hydroxy propyl cellulose, mixing, and tabletting to obtain the peimisine microcapsule tablet. The application proves that peimisine can be used for inhibiting gastric cancer, and peimisine micro-capsule tablets prepared from the composition of peimisine can form pH responsive release, reduce gastric mucosa stimulation, improve bioavailability and facilitate absorption of peimisine.

Description

Technical Field

[0001] This invention relates to the field of fritillaria cirrhosa composition application technology, specifically a fritillaria cirrhosa for inhibiting gastric cancer and its application and composition. Background Technology

[0002] Gastric cancer is one of the most common malignant tumors in terms of incidence and mortality worldwide. Despite continuous advancements in medical technology and improvements in treatment methods such as surgery, chemotherapy, and targeted therapy, there are still serious shortcomings in terms of drug efficacy, drug resistance, and toxic side effects. Therefore, the search for and development of novel, highly effective, and low-toxicity anti-gastric cancer drugs from natural products remains one of the important directions in current anti-tumor research.

[0003] Natural products of the genus Fritillaria are rich in various isosteroidal alkaloids. The paper "CN201110297762.9 Novel Uses of Total Alkaloids and Compounds of Fritillaria in the Preparation of Anticancer Drugs" discloses the anticancer uses of certain total alkaloids or some compounds of Fritillaria. However, the potential application of specific monomeric components, such as Peimisine, in the treatment of gastric cancer remains unknown, mainly in the following aspects: Firstly, existing technologies only preliminarily suggest that Peimisine has certain cytotoxicity or antibacterial toxicity, but whether Peimisine can effectively inhibit the activity of specific malignant tumors, especially gastric cancer, and how strong the activity is, is currently unknown, lacking quantitative or targeted research data to support this. Secondly, existing observations of the in vitro activity of certain fritillaria alkaloids, especially fritillarin, are limited to the initial stage of cell survival rate. In particular, systematic pharmacodynamic evaluations of the malignant biological behaviors of gastric cancer cells (such as proliferation, migration, invasion, and colony formation) cannot reveal whether they can effectively intervene in the key phenotypes of gastric cancer development.

[0004] Third, and most importantly, the existing technology lacks research on the mechanism of action of Peimisine. The potential anti-tumor effect is not specifically studied in terms of which intracellular signaling pathways it is transmitted through, or whether it acts on key molecular targets or pathways that are recognized as being closely related to the progression of gastric cancer.

[0005] Therefore, research on the uses, mechanisms of action, and targeted applications of Fritillaria cirrhosa and specific combinations for inhibiting gastric cancer is lacking. In conclusion, addressing these issues is of great significance for the development of Fritillaria cirrhosa for inhibiting gastric cancer, as well as its applications and combinations. Summary of the Invention

[0006] The purpose of this invention is to provide a fritillaria cirrhosa for inhibiting gastric cancer, its application, and a composition thereof, in order to solve the problems mentioned in the background art.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A fritillary compound for inhibiting gastric cancer, wherein the fritillary compound exerts its inhibitory effect on gastric cancer by inhibiting the PI3K / Akt (phosphatidylinositol 3-kinase / protein kinase B) signaling pathway.

[0008] Ideally, the half-maximal inhibitory concentration (IC50) of the fritillaria cirrhosae against MKN-45 gastric cancer cells is 30-50 μM.

[0009] An application of fritillaria cirrhosa for inhibiting gastric cancer, wherein the fritillaria cirrhosa is used in the preparation of drugs for treating and / or preventing gastric cancer.

[0010] More preferably, the dosage form of the drug includes one of an oral preparation, an injectable preparation, a topical preparation, or a novel delivery system; the oral preparation is a tablet or a capsule.

[0011] A composition comprising fritillaria microcapsules prepared from the fritillaria.

[0012] A more optimized method for preparing the Fritillaria cirrhosa microcapsules is as follows: Step 1: Add cyclodextrin MOF (metal-organic framework) and fritillaria to an ethanol-water solution, stir, centrifuge to precipitate, and dry to obtain drug-loaded powder; add the drug-loaded powder and vinyl silane coupling agent to an ethanol-water solution, react at 60~70℃ for 3~5h, cool, and dry to obtain vinyl drug-loaded powder. Step 2: Under ultraviolet light irradiation, vinyl drug-loaded powder, hyaluronic acid-thiol group, and photoinitiator are added to a dimethylformamide-phosphate buffer mixed solvent. The reaction is carried out under ultraviolet light irradiation at a wavelength of 360~370nm and a power of 5~7W for 10~30min. After cooling, purification and drying, hyaluronic acid drug-loaded powder is obtained. Step 3: Under a nitrogen atmosphere, add hyaluronic acid drug-loaded powder to a 4-6 wt% L-glutamine aqueous solution, adjust the pH to 5.5, add EDC and NHS, react at room temperature for 6-8 hours, purify and dry to obtain Fritillaria cirrhosa microcapsules.

[0013] In a more optimized manner, the mass ratio of cyclodextrin MOF to fritillary in the raw materials of the drug-loaded powder is 1:(0.9~1.1); and the mass ratio of drug-loaded powder to vinyl silane coupling agent in the raw materials of the vinyl drug-loaded powder is (3~5):0.05. In the raw materials of the hyaluronic acid drug-loaded powder, the mass ratio of vinyl drug-loaded powder, hyaluronic acid-mercapto, and photoinitiator is 1:(0.6~0.8):(0.05~0.1); The raw materials for the Fritillaria cirrhosa microcapsules contain hyaluronic acid drug-loaded powder, 4-6 wt% L-glutamine aqueous solution, EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), and NHS (N-hydroxysuccinimide) in a mass ratio of 1:(10-15):(0.08-0.12):(0.1-0.15).

[0014] Ideally, the relative molecular mass of the hyaluronic acid-thiol group is 50 kDa to 100 kDa.

[0015] In a more optimized manner, the raw materials of the composition include the following components: by mass parts, 9-11 parts of Fritillaria cirrhosa microcapsules, 1-2 parts of pregelatinized starch, 2.5-4 parts of compound excipients, 0.5-1 part of lactose, 0.1-0.2 parts of polyethylene glycol, 0.5-1 parts of sodium carboxymethyl starch, and 0.8-1.3 parts of low-substituted hydroxypropyl cellulose; The composite excipients include modified microcrystalline cellulose and sodium carboxymethyl cellulose in a mass ratio of (2.2~3):(0.8~1).

[0016] In a more optimized manner, the modified microcrystalline cellulose is prepared by adding microcrystalline cellulose and carboxyl-polyethylene glycol-silane in a mass ratio of 1:(0.3~0.5) sequentially to an aqueous ethanol solution, reacting at 60~70℃ for 3~5h, cooling and drying to obtain modified microcrystalline cellulose. The relative molecular mass of the polyethylene glycol segment in the carboxyl-polyethylene glycol-silane is 1500~2500.

[0017] In a further embodiment, the cyclodextrin MOF is prepared according to the paper "Preparation and Properties Study of Astragaloside A Supported by γ-Cyclodextrin-Metal-Organic Framework", specifically by adding γ-cyclodextrin and potassium hydroxide in a molar ratio of 1:6 to deionized water, filtering, and then adding 8 mg·mL⁻¹ in a water bath. -1 A polyethylene glycol (molecular weight 20000)-methanol solution was centrifuged, washed, and precipitated to obtain metal powder. The metal powder was then added to 200 mL of dichloromethane for 72 h, and washed with anhydrous ethanol every 24 h. After centrifugation and precipitation, the precipitate was dried to obtain cyclodextrin MOF.

[0018] Compared with the prior art, the beneficial effects achieved by the present invention are: this application conducts targeted research on the application of Fritillaria cirrhosa in gastric cancer and the composition thereof; (1) This application is the first to use a standard experimental method (CCK-8 method (cell counting kit-8)) to confirm that fritillaria has an inhibitory effect on the proliferation of human gastric cancer MKN-45 cells, and to determine its half-maximal inhibitory concentration (IC50 value). This data fills the research gap in the prior art and provides a quantitative benchmark for evaluating its activity intensity and guiding subsequent optimization studies (such as structure-activity relationship and dosage form design).

[0019] (2) The experiments in this application show that fritillaria syringa can significantly inhibit the long-term clonogenic ability of gastric cancer cells at incomplete killing concentrations. This proves that fritillaria syringa can not only inhibit cell viability in the short term, but also interfere with the self-renewal and long-term proliferation potential of tumor cells, demonstrating a new use of fritillaria syringa in inhibiting gastric cancer.

[0020] (3) Fritillaria cirrhosa can inhibit the activation of the PI3K / Akt signaling pathway in gastric cancer cells. The inhibition of this pathway can manifest phenotypic effects such as anti-proliferation. Associating the activity of Fritillaria cirrhosa with a well-defined and abnormally active classical pathological pathway in gastric cancer provides an important mechanistic reference and directional guidance for its subsequent development as a candidate drug for gastric cancer (such as biomarker exploration and combination drug strategies).

[0021] (4) The Fritillaria cirrhosa composition consists of specific components such as Fritillaria cirrhosa microcapsules, pregelatinized starch, and composite excipients, which can improve the dissolution stability and drug loading effect of Fritillaria cirrhosa, reduce gastric mucosal irritation, and improve bioavailability. In Fritillaria cirrhosa microcapsules, a γ-cyclodextrin-potassium ion metal-organic framework is formed via a solvothermal method, which is activated to provide a larger specific surface area and nanopores, thereby increasing drug loading and dispersing the drug in an amorphous state, thus improving the solubility of Fritillaria cirrhosa. A pH-responsive coating layer is formed by sequentially vinylsilanizing and clicking chemistry with hyaluronic acid-thiol groups on the surface of the drug-loaded particles. This coating layer shrinks and densifies in gastric acid (pH 1.0–3.0) to protect the drug; it ionizes and swells in the intestinal environment (pH 5.5–7.0) to control release. Simultaneously, hyaluronic acid, as a ligand for the CD44 receptor, synergistically inhibits gastric cancer cells with Fritillaria cirrhosa. Finally, L-glutamine modification partially neutralizes gastric acid, forming a buffer microenvironment locally, which helps reduce direct drug irritation to the gastric mucosa and improves medication compliance.

[0022] It is important to note that the molecular weight of the hyaluronic acid-thiol group needs to be controlled between 50 and 100 kDa. If the molecular weight is too low, the film-forming properties are poor, which can easily lead to premature leakage of the drug in the stomach; if the molecular weight is too high, the steric hindrance is large, the coating is uneven, and the protective and release effects are affected.

[0023] However, even with fritillaria cirrhosa metal microcapsules and other excipients alone, uniform dispersion is still not possible. This results in a lack of binding force between particles during tableting, which easily leads to loose or cracked tablets, affecting their dispersion and release performance.

[0024] Therefore, this invention introduces a composite excipient composed of modified microcrystalline cellulose and sodium carboxymethyl cellulose in a specific mass ratio. The modified microcrystalline cellulose, through flexible polyethylene glycol-carboxyl chains grafted onto its surface, exhibits affinity with the surface of the fritillaria microcapsules, providing a certain degree of occlusion and ensuring uniform dispersion; simultaneously, it provides a certain binding force and compressibility, which is beneficial for improving tablet stability. Sodium carboxymethyl cellulose provides disintegration capability. The two, in a specific mass ratio, form a composite excipient that synergistically improves tablet encapsulation efficiency, creates a specific slow release, and enhances bioavailability. Attached Figure Description

[0025] Figure 1 Curves showing changes in the viability and IC50 values ​​of human gastric cancer MKN-45 cells after treatment with different concentrations of fritillaria cirrhosae; Figure 2 The graph shows the time-dependent inhibitory effect of fritillaria cirrhosa on the viability of MKN-45 cells. Figure 3 This is a diagram showing the invasion status of MKN-45 cells in the control group. Figure 4 Image showing the invasion status of MKN-45 cells in the fritillaria cirrhosa treatment group; Figure 5 A bar chart to quantitatively count the number of invasive cells; Figure 6 Figure showing the effect of fritillaria cirrhosa on the phosphorylation level of Akt protein in MKN-45 cells as detected by Western blot. Figure 7 Statistical graph showing the quantitative analysis of p-Akt protein expression in MKN-45 cells after treatment with fritillaria cirrhosa. Detailed Implementation

[0026] 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.

[0027] In the following embodiments, the parts are by weight; it should be noted that there are no special restrictions on the manufacturers of the raw materials involved in this invention, and they include, exemplarily, pregelatinized starch (CAS No. 9005-25-8); lactose (CAS No. 63-42-3); polyethylene glycol (molecular weight 5000); sodium carboxymethyl starch (CAS No. 9063-38-1); low-substituted hydroxypropyl cellulose (CAS No. S24695-250g, CAS No. 9004-64-2); fritillaria cirrhosae (CAS No. 19773-24-1); and vinyl silane coupling agents. The product name is JPHA-3-50k (KH-570), containing hyaluronic acid-mercapto-based hyaluronic acid with a molecular weight of 50 kDa; photoinitiator (2,2-dimethoxy-2-phenylacetophenone); γ-cyclodextrin (CAS No. 17465-86-0); microcrystalline cellulose (CAS No. 9004-34-6); sodium carboxymethyl cellulose (CAS No. 112412); carboxylated polyethylene glycol-silane (CAS No. PS2-CML-2K) with a molecular weight of 2000; and hyaluronic acid-mercapto-based hyaluronic acid (CAS No. JPHA-3-10k) with a molecular weight of 10 kDa.

[0028] In the examples described below, the human gastric cancer MKN-45 cell line is commercially available, and the Peimisine standard can be isolated and purified from natural products or obtained through chemical synthesis, the structure of which has been confirmed by nuclear magnetic resonance spectroscopy and mass spectrometry. Conventional cell culture reagents, diagnostic kits, and antibodies can all be purchased from biotechnology companies.

[0029] Example 1: Quantitative determination of the anti-proliferative activity of Fritillaria cirrhosa against gastric cancer cells: Step 1: Cell Culture and Inoculation MKN-45 gastric cancer cells in the logarithmic growth phase were routinely cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37°C and 5% CO2. After digestion and cell counting, cells were seeded at a density of 5 × 10³ cells per well into 96-well cell culture plates with 100 μL of medium per well, and incubated for 24 hours to allow for full cell adhesion.

[0030] Step 2: Drug processing and concentration setting Peimisine was dissolved in dimethyl sulfoxide (DMSO) to prepare a high-concentration stock solution, which was then serially diluted with complete culture medium to form a series of drug solutions of different concentrations. A control group containing only an equal volume of DMSO was also prepared. The old culture medium in the 96-well plates was discarded, and each concentration of drug-containing medium or control medium was added, with five replicates for each concentration. The plates were then returned to the incubator and incubated for another 48 hours.

[0031] Step 3: Cell viability assay After incubation, add 10 μL of CCK-8 detection solution to each well. Incubate the plate in an incubator for another 2–4 hours. Then, measure the optical density (OD) of each well at 450 nm using a microplate reader. To evaluate the time-dependent effect of Peimisine on the inhibition of MKN-45 cell proliferation, cell viability was assessed at 24, 48, and 72 hours at fixed concentrations. Figure 2 As shown, time (h) is plotted on the x-axis and OD value (450nm) on the y-axis. The OD value of the experimental group was significantly lower than that of the control group, a statistically significant difference (p<0.05). Therefore, under the detection conditions, Peimisine can effectively inhibit the activity of gastric cancer cells and has a short-term inhibitory effect on proliferation. This result is consistent with... Figure 1 The results were consistent with the IC50 results, further supporting the inhibitory effect of Peimisine on the growth of gastric cancer cells.

[0032] Step 4: Data processing and IC50 calculation, such as Figure 1 As shown; where drug concentration (Log[peimisine concentration (μM)]) is the x-axis and cell viability (%) is the y-axis; Calculate the average OD value for each drug concentration treatment group. Cell viability (%) = (OD value of drug group - OD value of blank well) / (OD value of solvent control group - OD value of blank well) × 100%. Using nonlinear regression curve fitting in software such as GraphPad Prism, the drug concentration required to inhibit 50% cell viability, i.e., the half-maximal inhibitory concentration (IC50), can be calculated.

[0033] Conclusion: Cell viability decreased in a dose-dependent manner with increasing peimisine concentration. This method clearly established that the IC50 of peimisine against MKN-45 cells was 40.68 μM, a quantitative finding that confirms for the first time that peimisine has a clear growth-inhibiting activity against this gastric cancer cell line. Example 2: Effect of Fritillaria cirrhosa on the clonogenic ability of gastric cancer cells Step 1: Cell seeding and drug administration MKN-45 cells were digested to prepare a single-cell suspension and seeded at a low density in 6-well plates. After cell attachment, the medium was replaced with either a medium containing a concentration of Peimisine below IC50 or a control medium.

[0034] Step 2: Cloning Culture and Staining Cells were cultured in an incubator for 10–14 days in the presence of the drug, with the medium replaced with fresh drug-containing or control medium every 3 days. Culture was terminated when visible cell clones (usually >50 cells) appeared in the control wells. The medium was discarded, and the cells were gently washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde for 15 minutes, and then stained with 0.1% crystal violet for 30 minutes.

[0035] Step 3: Results Analysis and Efficacy Wash away excess dye slowly with running water and let it air dry. Photograph and count the clones that have formed.

[0036] Conclusion: Compared with the solvent control group, the number of clones formed in the Peimisine-treated group was significantly reduced, indicating that the compound can continuously inhibit the long-term proliferation and clonal survival of gastric cancer cells at subtoxic concentrations, further confirming its anti-proliferative efficacy.

[0037] Example 3: Transwell (cell migration / invasion chamber) cell invasion assay, test results are as follows Figures 3-5 As shown; Figure 3 This is a diagram showing the invasion status of MKN-45 cells in the control group. Figure 4 Image showing the invasion status of MKN-45 cells in the fritillaria cirrhosa treatment group; Figure 5 A bar chart was created to quantitatively count the number of invasive cells, with the control group and Peimisine on the x-axis and the number of invasive cells on the y-axis. in conclusion: Figures 3-5 The effect of Peimisine on the invasive ability of gastric cancer cells was evaluated using a Transwell invasion assay (using matrix gel to simulate the basement membrane). As shown in the figure, a large number of cells in the control group penetrated the matrix gel and invaded the lower chamber membrane, while the number of cells penetrating the membrane was significantly reduced in the Peimisine-treated group. Quantitative analysis showed that Peimisine significantly inhibited the invasive ability of MKN-45 cells, suggesting its potential role in preventing gastric cancer metastasis.

[0038] Example 4: Study on the inhibitory mechanism of fritillaria on the PI3K / Akt signaling pathway: Step 1: Cell treatment and protein extraction MKN-45 cells were seeded in culture dishes. When the cell density reached 70-80%, the medium was replaced with either Peimisine-containing medium or a control medium, and treated for a specific time. After treatment, the cells were washed with pre-cooled PBS, and RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors was added. The cells were lysed on ice, and the supernatant was collected by centrifugation, which is the total protein sample. Protein concentration was determined using the BCA method.

[0039] Step 2: Western Blot Analysis Equal volumes of protein samples were taken and separated by SDS-polyacrylamide gel electrophoresis. The proteins were then transferred to a PVDF (polyvinylidene fluoride) membrane. After blocking with 5% skim milk at room temperature for 1 hour, the membrane was incubated overnight at 4°C with a specific primary antibody. Key primary antibodies used in this invention include: anti-phosphorylated Akt antibody, anti-total Akt antibody (t-Akt), and anti-PI3K p85 subunit antibody. The following day, the membrane was washed with TBST and incubated at room temperature for 1 hour with a corresponding horseradish peroxidase-labeled secondary antibody.

[0040] Step 3: Chemiluminescence imaging and data analysis The membrane was developed using an ECL chemiluminescent substrate, and the signal was acquired using a chemiluminescence imaging system. The relative changes in p-Akt protein levels between the treatment group and the control group were compared by analyzing the band grayscale values.

[0041] Step 4: Results Analysis and Working Principle Experimental results show that, Figures 6-7 As shown; Figure 6 This figure shows the effect of Peimisine on Akt protein phosphorylation levels in MKN-45 cells as detected by Western blot. The left lane represents the control group, and the right lane represents Peimisine. From top to bottom, the following proteins were measured: total Akt protein (t-Akt), phosphorylated Akt protein (p-Akt), and internal reference protein (β-actin).

[0042] Figure 7 A statistical graph showing the quantitative analysis of p-Akt protein expression in MKN-45 cells after treatment with Peimisine; where the control group (Control) and Peimisine (Control Peimisine) are plotted on the x-axis, and the activation level of protein kinase B (Akt) (p-AKT / t-AKT) is plotted on the y-axis. Conclusion: Compared with the control group, the protein expression level of p-Akt was significantly reduced in MKN-45 cells treated with Peimisine, while the total Akt protein level remained unchanged. This indicates that Peimisine specifically inhibits the phosphorylation activation process of Akt protein. Since Akt activation is a core marker for the initiation of the PI3K / Akt signaling pathway, the decrease in p-Akt levels indicates that Peimisine can inhibit the activity of this pathway. The PI3K / Akt pathway is a key pathway regulating cell proliferation, survival, metabolism, and invasion, and it is often abnormally activated in gastric cancer. Therefore, Peimisine inhibits the activation of the PI3K / Akt signaling pathway, thereby weakening downstream pro-proliferation, pro-survival, and pro-invasive effects, ultimately resulting in the inhibition of gastric cancer cell proliferation and invasion. The elucidation of this mechanism provides a molecular-level theoretical basis for the applications of this invention.

[0043] Example 5: A composition: The composition is prepared into Fritillaria cirrhosa microcapsule tablets, comprising the following steps: The cyclodextrin MOF was prepared according to the "Preparation and Properties Study of Astragaloside A Supported by γ-Cyclodextrin-Metal-Organic Framework". Specifically, γ-cyclodextrin and potassium hydroxide in a molar ratio of 1:6 were added to deionized water, filtered through a 0.45 μm filter, added to methanol, clarified in a water bath at 50 °C, and the mixture was kept in the water bath for another 20 min. Then, 8 mg / mL of the solution was added. -1 A polyethylene glycol (molecular weight 20000)-methanol solution was incubated overnight in an ice-water bath, centrifuged, washed, and precipitated to obtain metal powder. The metal powder was then added to 200 mL of dichloromethane for 72 h, with the dichloromethane being replaced every 24 h. The solution was washed with anhydrous ethanol, centrifuged to precipitate, and then vacuum dried at 50 °C to obtain cyclodextrin MOF. The preparation method of Fritillaria cirrhosa microcapsules is as follows: Step 1: Add cyclodextrin MOF and Fritillaria cirrhosa to a 60wt% ethanol aqueous solution at a mass ratio of 1:1, stir, centrifuge to precipitate, and dry to obtain drug-loaded powder; add drug-loaded powder and vinyl silane coupling agent to a 60wt% ethanol aqueous solution at a mass ratio of 4:0.05, react at 65℃ for 4h, cool, and dry to obtain vinyl drug-loaded powder; Step 2: Under ultraviolet light irradiation, vinyl drug-loaded powder, hyaluronic acid-mercapto, and photoinitiator were added to a dimethylformamide-phosphate buffer mixed solvent (the volume ratio of dimethylformamide to phosphate buffer was 1:3). (The mass ratio of vinyl drug-loaded powder, hyaluronic acid-mercapto (50kDa), and photoinitiator was 1:0.7:0.07). The mixture was irradiated with ultraviolet light at a wavelength of 365nm and a power of 6W for 20min. After cooling, purification, and drying, hyaluronic acid drug-loaded powder was obtained. Step 3: Under a nitrogen atmosphere, hyaluronic acid drug-loaded powder was added to a 5wt% L-glutamine aqueous solution, the pH was adjusted to 5.5, and EDC and NHS were added (the mass ratio of hyaluronic acid drug-loaded powder, 5wt% L-glutamine aqueous solution, EDC, and NHS was 1:12.5:0.1:0.12). The mixture was reacted at room temperature for 7 hours, purified, and dried to obtain Fritillaria cirrhosa microcapsules. The modified microcrystalline cellulose is prepared by reacting microcrystalline cellulose with carboxyl-polyethylene glycol-silane (molecular weight 2000) in a 60wt% ethanol aqueous solution at 65℃ for 4h (the mass ratio of microcrystalline cellulose to carboxyl-polyethylene glycol-silane is 1:0.4), and then cooling and drying to obtain modified microcrystalline cellulose. Mix 1.5 parts pregelatinized starch, 3.2 parts compound excipients (composed of modified microcrystalline cellulose and sodium carboxymethyl cellulose in a mass ratio of 2.7:0.9), 0.8 parts lactose, and 10 parts Fritillaria cirrhosa microcapsules. Add 0.15 parts polyethylene glycol (molecular weight 5000), 0.8 parts sodium carboxymethyl starch, and 1.1 parts low-substituted hydroxypropyl cellulose, and compress the mixture into tablets to obtain Fritillaria cirrhosa microcapsule tablets.

[0044] Example 6: A composition: The composition is prepared into Fritillaria cirrhosa microcapsule tablets, comprising the following steps: The cyclodextrin MOF was prepared according to the "Preparation and Properties Study of Astragaloside A Supported by γ-Cyclodextrin-Metal-Organic Framework". Specifically, γ-cyclodextrin and potassium hydroxide in a molar ratio of 1:6 were added to deionized water, filtered through a 0.45 μm filter, added to methanol, clarified in a water bath at 50 °C, and the mixture was kept in the water bath for another 20 min. Then, 8 mg / mL of the solution was added. -1 A polyethylene glycol (molecular weight 20000)-methanol solution was incubated overnight in an ice-water bath, centrifuged, washed, and precipitated to obtain metal powder. The metal powder was then added to 200 mL of dichloromethane for 72 h, with the dichloromethane being replaced every 24 h. The solution was washed with anhydrous ethanol, centrifuged to precipitate, and then vacuum dried at 50 °C to obtain cyclodextrin MOF. The preparation method of Fritillaria cirrhosa microcapsules is as follows: Step 1: Add cyclodextrin MOF and Fritillaria cirrhosa to a 60wt% ethanol aqueous solution at a mass ratio of 1:1, stir, centrifuge to precipitate, and dry to obtain drug-loaded powder; add drug-loaded powder and vinyl silane coupling agent to a 60wt% ethanol aqueous solution at a mass ratio of 4:0.05, react at 65℃ for 4h, cool, and dry to obtain vinyl drug-loaded powder; Step 2: Under ultraviolet light irradiation, vinyl drug-loaded powder, hyaluronic acid-mercapto, and photoinitiator were added to a dimethylformamide-phosphate buffer mixed solvent (the volume ratio of dimethylformamide to phosphate buffer was 1:3). (The mass ratio of vinyl drug-loaded powder, hyaluronic acid-mercapto (50kDa), and photoinitiator was 1:0.7:0.07). The mixture was irradiated with ultraviolet light at a wavelength of 365nm and a power of 6W for 20min. After cooling, purification, and drying, hyaluronic acid drug-loaded powder was obtained. Step 3: Under a nitrogen atmosphere, hyaluronic acid drug-loaded powder was added to a 5wt% L-glutamine aqueous solution, the pH was adjusted to 5.5, and EDC and NHS were added (the mass ratio of hyaluronic acid drug-loaded powder, 5wt% L-glutamine aqueous solution, EDC, and NHS was 1:12.5:0.1:0.12). The mixture was reacted at room temperature for 7 hours, purified, and dried to obtain Fritillaria cirrhosa microcapsules. The modified microcrystalline cellulose is prepared by reacting microcrystalline cellulose with carboxyl-polyethylene glycol-silane (molecular weight 2000) in an aqueous ethanol solution at 65°C for 4 hours (the mass ratio of microcrystalline cellulose to carboxyl-polyethylene glycol-silane is 1:0.4), and then cooling and drying to obtain modified microcrystalline cellulose. Mix 1.5 parts pregelatinized starch, 3.2 parts compound excipients (composed of modified microcrystalline cellulose and sodium carboxymethyl cellulose in a mass ratio of 2.7:0.9), 0.8 parts lactose, and 10 parts Fritillaria cirrhosa microcapsules. Add 0.15 parts polyethylene glycol (molecular weight 5000), 0.8 parts sodium carboxymethyl starch, and 1.1 parts low-substituted hydroxypropyl cellulose, and compress the mixture into tablets to obtain Fritillaria cirrhosa microcapsule tablets.

[0045] Example 7: A composition: The composition is prepared into Fritillaria cirrhosa microcapsule tablets, comprising the following steps: The cyclodextrin MOF was prepared according to the "Preparation and Properties Study of Astragaloside A Supported by γ-Cyclodextrin-Metal-Organic Framework". Specifically, γ-cyclodextrin and potassium hydroxide in a molar ratio of 1:6 were added to deionized water, filtered through a 0.45 μm filter, added to methanol, clarified in a water bath at 50 °C, and the mixture was kept in the water bath for another 20 min. Then, 8 mg / mL of the solution was added. -1 A polyethylene glycol (molecular weight 20000)-methanol solution was incubated overnight in an ice-water bath, centrifuged, washed, and precipitated to obtain metal powder. The metal powder was then added to 200 mL of dichloromethane for 72 h, with the dichloromethane being replaced every 24 h. The solution was washed with anhydrous ethanol, centrifuged to precipitate, and then vacuum dried at 50 °C to obtain cyclodextrin MOF. The preparation method of Fritillaria cirrhosa microcapsules is as follows: Step 1: Add cyclodextrin MOF and Fritillaria cirrhosa to a 60wt% ethanol aqueous solution at a mass ratio of 1:1, stir, centrifuge to precipitate, and dry to obtain drug-loaded powder; add drug-loaded powder and vinyl silane coupling agent to a 60wt% ethanol aqueous solution at a mass ratio of 4:0.05, react at 65℃ for 4h, cool, and dry to obtain vinyl drug-loaded powder; Step 2: Under ultraviolet light irradiation, vinyl drug-loaded powder, hyaluronic acid-mercapto, and photoinitiator were added to a dimethylformamide-phosphate buffer mixed solvent (the volume ratio of dimethylformamide to phosphate buffer was 1:3). (The mass ratio of vinyl drug-loaded powder, hyaluronic acid-mercapto (50kDa), and photoinitiator was 1:0.7:0.07). The mixture was irradiated with ultraviolet light at a wavelength of 365nm and a power of 6W for 20min. After cooling, purification, and drying, hyaluronic acid drug-loaded powder was obtained. Step 3: Under a nitrogen atmosphere, hyaluronic acid drug-loaded powder was added to a 5wt% L-glutamine aqueous solution, the pH was adjusted to 5.5, and EDC and NHS were added (the mass ratio of hyaluronic acid drug-loaded powder, 5wt% L-glutamine aqueous solution, EDC, and NHS was 1:12.5:0.1:0.12). The mixture was reacted at room temperature for 7 hours, purified, and dried to obtain Fritillaria cirrhosa microcapsules. The modified microcrystalline cellulose is prepared by reacting microcrystalline cellulose with carboxyl-polyethylene glycol-silane (molecular weight 2000) in a 60wt% ethanol aqueous solution at 65℃ for 4h (the mass ratio of microcrystalline cellulose to carboxyl-polyethylene glycol-silane is 1:0.4), and then cooling and drying to obtain modified microcrystalline cellulose. Two parts of pregelatinized starch, four parts of compound excipients (composed of modified microcrystalline cellulose and sodium carboxymethyl cellulose in a mass ratio of 3:1), one part of lactose, and nine parts of Fritillaria cirrhosa microcapsules were mixed. Then, 0.1 parts of polyethylene glycol (molecular weight 5000), 0.5 parts of sodium carboxymethyl starch, and 0.8 parts of low-substituted hydroxypropyl cellulose were added and mixed. The mixture was then compressed into tablets to obtain Fritillaria cirrhosa microcapsule tablets.

[0046] Comparative Example 1: Based on Example 5, the molecular weight of hyaluronic acid-thiol group is too small (10 kDa); otherwise, it is the same as Example 5.

[0047] Comparative Example 2: Based on Example 5, the hyaluronic acid-thiol group was adjusted to be directly added to a 2 mg / mL sodium alginate aqueous solution; the rest was the same as in Example 5.

[0048] Comparative Example 3: Based on Example 5, tablets were prepared and then coated with hyaluronic acid; the rest was the same as in Example 5.

[0049] Comparative Example 4: Based on Example 5, the composite excipient was changed to sodium carboxymethyl cellulose; the rest was the same as in Example 5.

[0050] Comparative Example 5: Based on Example 5, the mass ratio of modified microcrystalline cellulose and sodium carboxymethyl cellulose in the composite excipient was adjusted (the mass ratio of modified microcrystalline cellulose and sodium carboxymethyl cellulose was 1:1); the rest was the same as in Example 5.

[0051] Comparative Example 6: Based on Example 5, the modified microcrystalline cellulose in the composite excipient was changed to microcrystalline cellulose; the rest was the same as in Example 5.

[0052] Performance Test 1: 10 mg of each of the Fritillaria cirrhosa microcapsules prepared in Examples 5-7 and Comparative Examples 1-3 was added to a 50 mL volumetric flask, accurately weighed, and its mass recorded. The weighed sample was transferred to a 50 mL volumetric flask, and an appropriate amount of methanol was added. The mixture was sonicated for 30 minutes to ensure complete destruction of the microcapsule structure and full dissolution of Fritillaria cirrhosa. After cooling to room temperature, the volume was adjusted to the mark with methanol and shaken well. After filtration, the solution was used as the sample solution. The drug concentration was calculated using the following formula: Drug loading (%) = (Total mass of Fritillaria cirrhosa in 50 mL solution / Total mass of Fritillaria cirrhosa microcapsule sample) × 100%. Three batches were tested for each example, and the average value was taken as the actual drug loading. The test results are shown in Table 1. The absorbance of the supernatant of the above sample solution was measured at 420 nm using a spectrophotometer, and the content of unencapsulated free fritillary was calculated according to the standard curve. The encapsulation rate was calculated using the formula: Encapsulation rate = (Total fritillary content - Free fritillary content) / Total fritillary content × 100%. Three batches were tested for each example, and the average value was taken as the actual drug loading. The test results are shown in Table 1. Table 1

[0053] Conclusions: The data in Table 1 show that the Fritillaria cirrhosa microcapsules prepared in this application have high drug loading and encapsulation efficiency, meeting practical requirements. Comparative Example 1 shows that the molecular weight of hyaluronic acid-thiol groups is too small (10 kDa), resulting in poor film-forming properties and premature drug leakage from the stomach, significantly reducing the encapsulation efficiency. Comparative Example 2 shows that adjusting the hyaluronic acid-thiol groups to be directly added to a 2 mg / mL sodium alginate aqueous solution, compared to hyaluronic acid coating, sodium alginate, through weak negative bonding on the surface of the drug-loaded powder, is far inferior to hyaluronic acid. Furthermore, the different structure and density of the sodium alginate gel lead to a significant decrease in overall performance. Comparative Example 3 shows that preparing tablets before hyaluronic acid coating, in the case of Fritillaria cirrhosa microcapsules, does not actually result in microcapsules, but merely drug-loaded powder, leading to a significant decrease in overall performance.

[0054] Performance Test 2: The Fritillaria cirrhosa microcapsules prepared in Examples 5-7 and Comparative Examples 1-6 were placed in water for 2 hours and in intestinal fluid for 45 minutes, respectively. The in vitro dissolution rate of the Fritillaria cirrhosa microcapsules was tested, as shown in Table 2. Table 2

[0055] Conclusion: Data from Table 2 shows that the Fritillaria cirrhosa microcapsules prepared in this application dissolve well in fish water, exhibit specific pH responsiveness, and have a moderate release rate in intestinal fluid, meeting the requirements for drug use. Data from Comparative Example 1 shows that the molecular weight of hyaluronic acid-sulfhydryl groups is too small (10 kDa), resulting in rapid release. Data from Comparative Example 2 shows that adjusting the hyaluronic acid-sulfhydryl groups to be directly added to a 2 mg / mL sodium alginate aqueous solution resulted in poor solubility in water, forming a gel barrier and slow release; in intestinal fluid, the hyaluronic acid-sulfhydryl groups weakly bind to the drug, leading to still slow and incomplete release. Data from Comparative Example 3 shows that the tablets prepared... After the agent was coated with hyaluronic acid, the release rate was too fast. Data from Comparative Example 4 shows that adjusting the composite excipient to sodium carboxymethyl cellulose resulted in a lack of modified microcrystalline cellulose, leading to an accelerated dissolution rate in water. Data from Comparative Example 5 shows that adjusting the mass ratio of modified microcrystalline cellulose to sodium carboxymethyl cellulose in the composite excipient (1:1) resulted in insufficient modified microcrystalline cellulose, decreased synergy with Fritillaria cirrhosa microcapsules, and reduced overall performance. Data from Comparative Example 6 shows that changing the modified microcrystalline cellulose in the composite excipient to pure microcrystalline cellulose resulted in decreased pH response performance and reduced overall performance.

[0056] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A fritillaria cirrhosae compound for inhibiting gastric cancer, characterized in that: The fritillaria syrup is used to inhibit gastric cancer; the fritillaria syrup exerts its anti-gastric cancer effect by inhibiting the PI3K / Akt signaling pathway.

2. The Fritillaria cirrhosa extract for inhibiting gastric cancer according to claim 1, characterized in that: The half-maximal inhibitory concentration (IC50) of the fritillary bulb extract against MKN-45 gastric cancer cells was 30-50 μM.

3. An application of Fritillaria cirrhosa for inhibiting gastric cancer, characterized in that: The fritillaria cirrhosa is used to prepare drugs for the treatment and / or prevention of gastric cancer.

4. The application of Fritillaria cirrhosa for inhibiting gastric cancer according to claim 3, characterized in that: The dosage form of the drug includes one of oral preparations, injectable preparations, topical preparations, and novel delivery systems; the oral preparation is a tablet or capsule.

5. A composition, characterized in that: The raw materials of the composition include fritillaria microcapsules prepared according to claim 1.

6. The composition according to claim 5, characterized in that: The preparation method of the Fritillaria cirrhosa microcapsules is as follows: Step 1: Add cyclodextrin MOF and fritillaria to an ethanol-water solution, stir, centrifuge to precipitate, and dry to obtain drug-loaded powder; add the drug-loaded powder and vinyl silane coupling agent to an ethanol-water solution, react at 60~70℃ for 3~5h, cool, and dry to obtain vinyl drug-loaded powder. Step 2: Under ultraviolet light irradiation, vinyl drug-loaded powder, hyaluronic acid-thiol group, and photoinitiator are added to a dimethylformamide-phosphate buffer mixed solvent. The reaction is carried out under ultraviolet light irradiation at a wavelength of 360~370nm and a power of 5~7W for 10~30min. After cooling, purification and drying, hyaluronic acid drug-loaded powder is obtained. Step 3: Under a nitrogen atmosphere, add hyaluronic acid drug-loaded powder to a 4-6 wt% L-glutamine aqueous solution, adjust the pH to 5.5, add EDC and NHS, react at room temperature for 6-8 hours, purify and dry to obtain Fritillaria cirrhosa microcapsules.

7. The composition according to claim 6, characterized in that: In the raw materials of the drug-loaded powder, the mass ratio of cyclodextrin MOF to fritillaria thiocyanate is 1:(0.9~1.1); in the raw materials of the vinyl drug-loaded powder, the mass ratio of drug-loaded powder to vinyl silane coupling agent is (3~5):0.

05. In the raw materials of the hyaluronic acid drug-loaded powder, the mass ratio of vinyl drug-loaded powder, hyaluronic acid-mercapto, and photoinitiator is 1:(0.6~0.8):(0.05~0.1); The raw materials for the Fritillaria cirrhosa microcapsules contain hyaluronic acid drug-loaded powder, 4-6 wt% L-glutamine aqueous solution, EDC, and NHS in a mass ratio of 1:(10-15):(0.08-0.12):(0.1-0.15).

8. The composition according to claim 6, characterized in that: The relative molecular mass of the hyaluronic acid-thiol group is 50kDa~100kDa.

9. The composition according to claim 5, characterized in that: The raw materials of the composition include the following components: by mass parts, 9-11 parts of Fritillaria cirrhosa microcapsules, 1-2 parts of pregelatinized starch, 2.5-4 parts of compound excipients, 0.5-1 part of lactose, 0.1-0.2 parts of polyethylene glycol, 0.5-1 parts of sodium carboxymethyl starch, and 0.8-1.3 parts of low-substituted hydroxypropyl cellulose; The composite excipients include modified microcrystalline cellulose and sodium carboxymethyl cellulose in a mass ratio of (2.2~3):(0.8~1).

10. The composition according to claim 9, characterized in that: The modified microcrystalline cellulose is prepared by adding microcrystalline cellulose and carboxyl-polyethylene glycol-silane in a mass ratio of 1:(0.3~0.5) sequentially to an aqueous ethanol solution, reacting at 60~70℃ for 3~5h, cooling and drying to obtain modified microcrystalline cellulose. The relative molecular mass of the polyethylene glycol segment in the carboxyl-polyethylene glycol-silane is 1500~2500.

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