Dihydromyricetin inclusion compound-bioadhesive microsphere and ionizing radiation protection application thereof

Abstract

The invention discloses a gastrointestinal tract bioadhesive drug delivery system, and particularly relates to a dihydromyricetin clathrate-bioadhesive microsphere, a raw material dihydromyricetin clathrate, an optimized process of the dihydromyricetin clathrate, and application of the dihydromyricetin clathrate in ionizing radiation injury protection of nuclear radiation, nuclear medicine, tumor radiotherapy and the like. Compared with a traditional oral administration mode, the dihydromyricetin-bioadhesive administration prolongs the residence time of the medicine in gastrointestinal tracts and improves the membrane permeability of the medicine, the bioavailability of the medicine is remarkably improved, and the dihydromyricetin-bioadhesive administration can be used for preparing the protective medicine for preventing / treating ionizing radiation injury and has a wide application prospect. The invention particularly relates to application in preparation of drugs for preventing / treating radioactive intestinal injury and the like.

Description

Invention Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a gastrointestinal bioadhesive drug delivery system, particularly dihydromyricetin inclusion complex-bioadhesive microspheres, as well as the raw material dihydromyricetin inclusion complex, its preparation process, and its use in the preparation of drugs for the prevention / treatment of ionizing radiation protection. Background Technology

[0002] Ionizing radiation, as opposed to non-ionizing radiation such as ultraviolet radiation, includes nuclear radiation, radiation related to nuclear medicine and tumor radiotherapy.

[0003] Dihydromyricetin (DMY), also known as ampelopsin (AMP), is a dihydroflavonol compound extracted from vine tea. It has various pharmacological activities, such as anti-inflammatory, antioxidant, antitumor, antithrombotic, hypoglycemic, hypolipidemic, and hepatoprotective effects.

[0004]

[0005] The structure of dihydromyricetin

[0006] However, the poor water solubility, poor bioavailability, and easy oxidative degradation of dihydromyricetin have limited its further application.

[0007] Currently, there are numerous literature reports on enhancing the solubility of dihydromyricetin in the gastrointestinal tract and improving its oral bioavailability through new formulation technologies and dosage forms. These mainly include liposomes, self-microemulsions, micelles, inclusion complexes, phospholipid complexes, hydrogels, cocrystals, and intragastric floating formulations. Liposomes have a biomembrane-like structure, which gives them good cell affinity and tissue compatibility.

[0008] However, the oil-water partition coefficient (LogP = 1.17) of dihydromyricetin revealed that DMY is a relatively difficult drug to encapsulate. If dihydromyricetin is prepared into liposomes, the encapsulation efficiency and drug loading will be low. Self-microemulsions can spontaneously form transparent or semi-transparent oil-in-water dispersions under gastrointestinal peristalsis or gentle stirring at suitable temperatures. These dispersions are characterized by small particle size and rapid dispersion, and can increase drug permeability through the gastric and intestinal walls by reducing surface tension, thereby increasing absorption and bioavailability. However, they also suffer from problems such as easy interaction between the active ingredient and the capsule material, relatively high cost, and the tendency for crystallization or stratification during storage. Some literature has used three cyclodextrin derivatives as excipients to prepare three amorphous DMY inclusion complexes via freeze-drying, improving solubility and bioavailability; however, these complexes still have drawbacks such as rapid in vivo metabolism and short duration of action.

[0009] Radiation-induced intestinal injury (RIII) is an adverse intestinal event caused by radiotherapy in patients with abdominopelvic malignancies. The injury can affect the small intestine, colon, and rectum, and mainly manifests as abdominal pain, diarrhea, rectal bleeding, tenesmus, and obstruction. Severe cases significantly impact quality of life. RIII not only affects patients' quality of life but also limits radiotherapy dosage and clinical efficacy. According to statistics from the National Cancer Center, approximately 850,000 new cases of abdominopelvic malignancies (such as cervical cancer, prostate cancer, rectal cancer, and bladder cancer) are diagnosed annually in my country. During peri-abdominal radiotherapy (RT), healthy intestinal tissue is easily damaged by radiation, leading to radiation-induced intestinal injury. While precise stereotactic radiotherapy techniques can reduce the degree of radiation damage to tissues within the irradiated field, the presence of radiation bystander effects inevitably causes adverse reactions to surrounding normal tissues and organs. Furthermore, due to the rapid proliferation of intestinal epithelial cells, they are highly sensitive to ionizing radiation (IR), resulting in a greater than 75% risk of RIII after abdominopelvic radiotherapy. Currently, Japan is discharging all its nuclear wastewater into the sea, a process that will continue for 30 years, further increasing the incidence of radiation-induced intestinal injury. The exact causes of RIII are not yet fully understood, but may be related to radiotherapy techniques, radiation dose, radiation range, and individual differences. Its pathogenesis is highly complex, primarily involving intestinal mucosal stem cell death, vascular endothelial damage, intestinal flora imbalance, and abnormal expression of inflammatory factors.

[0010] Currently, the main clinical methods for preventing and treating RIII include drug therapy, hyperbaric oxygen therapy, and surgery. The high cost of hyperbaric oxygen therapy and surgery limits their widespread application. Furthermore, the timing and method of surgery should be carefully considered to minimize surgical mortality and complications, and improve prognosis and long-term quality of life. Drug therapy mainly includes two categories: Western medicine and traditional Chinese medicine. Western medicine for RIII mainly includes anti-inflammatory drugs, antibiotics, probiotics, antioxidants, and antidiarrheal drugs. While these drugs can provide some relief, long-term use can lead to serious adverse reactions such as dizziness, nausea, abdominal pain, and diarrhea. Traditional Chinese medicine, due to its unique advantages such as fewer side effects and good long-term efficacy, has attracted increasing attention and interest in the treatment of complex diseases. In recent years, the synergistic effects of multiple components and targets, as well as the low toxicity of traditional Chinese medicine, have made it a hot topic in the research and development of new anti-RIII drugs. Currently, commonly used traditional Chinese medicine compound formulas for treating RIII include Sijunzi Decoction, Baitouweng Decoction, and Tongxie Yaofang; single Chinese medicines include vine tea, sea buckthorn, immature bitter orange, wolfberry, propolis, trifoliate orange, centella asiatica, nutmeg, and pomegranate peel; and single Chinese medicine components include dihydromyricetin, baicalin, puerarin, silymarin, and (-)-epicatechin; their mechanisms of action mainly include inhibiting oxidative stress, regulating the expression of inflammatory factors, improving intestinal cell damage, regulating mitochondrial apoptosis pathways, and improving intestinal flora.

[0011] Gastrointestinal bioadhesive drug delivery system (GBDDS) refers to a drug delivery system that uses bioadhesive materials as carriers to bioadhere to the mucus layer of the gastrointestinal mucosa, prolonging the drug's retention time and allowing it to enter the circulatory system through the contacted mucosal epithelium to exert its therapeutic effect.

[0012] β-CD: Betacyclodextrin,

[0013] HP-β-CD: Hydroxypropyl betacyclodextrin;

[0014] SBE-β-CD: Sulfobutyl betacyclodextrin. Summary of the Invention

[0015] The applicant's research shows that dihydromyricetin has a certain protective effect against ionizing radiation (nuclear radiation, nuclear medicine, tumor radiotherapy, etc.) and can effectively alleviate gastrointestinal damage caused by ionizing irradiation. Experiments show that dihydromyricetin can significantly scavenge radiation-induced reactive oxygen species (ROS), reduce oxidative stress damage to cells, and exert a radiation protection effect.

[0016] This application aims to prepare inclusion complex-bioadhesive microspheres from dihydromyricetin, thereby increasing retention and permeability to improve bioavailability and enhance its gastrointestinal radiation protection effect.

[0017] This invention provides a dihydromyricetin inclusion complex-bioadhesive microsphere, which is made of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan. The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is (20-100):(2-8):(0.2-2); preferably, the mass ratio is (40-80):(4:6):(0.5-1.5).

[0018] Furthermore, a more preferable mass ratio of the three is 60:5:1, for example:

[0019] 1.5g of dihydromyricetin inclusion complex

[0020] Sodium alginate 0.125g

[0021] 0.025g of konjac glucomannan.

[0022] This invention relates to the preparation of dihydromyricetin inclusion complexes. The preparation process of dihydromyricetin inclusion complexes was investigated through multi-factor analysis. The freeze-drying method used in this invention for preparing dihydromyricetin inclusion complexes was optimized.

[0023] The dihydromyricetin inclusion complex is composed of dihydromyricetin and cyclodextrin, wherein the cyclodextrin can be selected from β-CD, HP-β-CD, or SBE-β-CD.

[0024] Phase solubility and molecular docking experiments were conducted using β-CD, HP-β-CD, and SBE-β-CD. It was found that β-CD, HP-β-CD, and SBE-β-CD can all form effective inclusion complexes. Meanwhile, HP-β-CD showed the best inclusion complexe effect for dihydromyricetin, followed by sulfobutyl betacyclodextrin. HP-β-CD is more economical and has been included in the Chinese Pharmacopoeia.

[0025] This invention prepares DMY-HP-β-CD inclusion complexes via freeze-drying. Solubility experiments were conducted to explore the solubilizing effects of β-CD, HP-β-CD, and SBE-β-CD on DMY, revealing that HP-β-CD exhibited the best solubilizing effect. Molecular docking techniques were used to investigate the binding energies of DMY with β-CD, HP-β-CD, and SBE-β-CD. The lowest binding energy was found with HP-β-CD, at -5.24 kcal / mol. Therefore, HP-β-CD was selected as the inclusion material. The optimal preparation process for the dihydromyricetin inclusion complex was optimized using a star-point design-response surface methodology, resulting in a molar ratio of 1:1, a temperature of 60℃, and a time of 4.5 h. Microscopic observation revealed that the prepared inclusion complex was plate-like with an average particle size of 163.04 nm and a PDI of 0.271. Infrared spectroscopy and X-ray diffraction characterized the inclusion complex, confirming the successful preparation of the dihydromyricetin inclusion complex. Water solubility experiments demonstrated that preparing dihydromyricetin into an inclusion complex significantly increased its water solubility. Influencing factor experiments and pH stability experiments further demonstrated that preparing dihydromyricetin into an inclusion complex could further increase its stability.

[0026] The dihydromyricetin inclusion complex of the present invention, wherein the raw materials are in the following proportions by molar amount: dihydromyricetin With ring paste Refined The molar ratio is 1:(0.5~2); preferably 1:1.

[0027] The dihydromyricetin inclusion complex was prepared using conventional methods under the following conditions: inclusion time 3–6 h; reaction temperature 40–80 °C. Further, the preparation conditions for the dihydromyricetin inclusion complex were: inclusion time 4–5 h; reaction temperature 50–70 °C. Even further, the preparation conditions for the dihydromyricetin inclusion complex were: inclusion time 4.5 h; reaction temperature 60 °C.

[0028] This invention also provides the preparation of dihydromyricetin inclusion complex-bioadhesive microspheres. Through multi-factor investigation, this invention optimizes the preparation process of dihydromyricetin inclusion complex-bioadhesive microspheres.

[0029] This application employs a two-step method to prepare dihydromyricetin inclusion complex-bioadhesive microspheres. In the first step, dihydromyricetin inclusion complexes are prepared using HP-β-CD as the inclusion material to improve solubility and stability. The complexes are then characterized to ensure successful preparation of the DMY-HP-β-CD inclusion complex. Furthermore, the particle size of dihydromyricetin is significantly reduced after inclusion, increasing permeability. In the second step, the dihydromyricetin inclusion complex is extruded to form sodium alginate bioadhesive microspheres. Because adhesive drug delivery systems can prolong drug retention time, increase local drug concentration, and promote drug absorption, thereby improving drug bioavailability, the DMY-HP-β-CD inclusion complex can adhere to the gastrointestinal tract and exert its effect, increasing retention and permeability, thus improving bioavailability and enhancing radiation protection against gastrointestinal radiation.

[0030] The preparation method of the dihydromyricetin inclusion complex-bioadhesive microspheres of this invention is as follows: Sodium alginate and konjac glucomannan are added to water and stirred. The dihydromyricetin inclusion complex is then added and stirred. The solution is then added dropwise to a calcium chloride solution to obtain the microspheres. Example:

[0031] Take sodium alginate and konjac glucomannan, add water, stir, add dihydromyricetin inclusion complex, stir, and add to calcium chloride solution to obtain the final product.

[0032] The dihydromyricetin inclusion complex-bioadhesive microspheres of this invention can also be prepared by the following method:

[0033] Take 0.125g sodium alginate and 0.025g konjac glucomannan, add 10mL water, stir, add 1.5g dihydromyricetin inclusion complex, stir, add dropwise to 3% calcium chloride solution, crosslink for 2min-30min, freeze dry to obtain the product.

[0034] The dihydromyricetin inclusion complex-bioadhesive microspheres of the present invention can also be prepared by the following method:

[0035] Weigh 0.125 g of sodium alginate and 0.025 g of konjac glucomannan into a beaker, add 10 mL of ultrapure water, and stir in a magnetic stirrer. Sonicate until bubbles are eliminated, then add 1.5 g of dihydromyricetin inclusion complex. Stir and sonicate until bubbles are eliminated, then add dropwise to a 3% calcium chloride solution using a syringe. Crosslink for 5-10 min, remove, and freeze-dry to constant weight. Weigh, grind, dissolve, and dilute to an appropriate concentration. Measure the DMY content and further calculate the encapsulation efficiency and drug loading. Drug loading and encapsulation efficiency are the evaluation indicators in this study. The preparation method for blank microspheres is the same as above, except that the dihydromyricetin inclusion complex is not added.

[0036] The konjac glucomannan / sodium alginate microspheres of this invention have good biocompatibility, and no surfactants or organic solvents are used in the microsphere preparation process, so they will not cause secondary damage to radiation-induced intestinal injury.

[0037] Swelling capacity is an important parameter for evaluating the adhesive properties of bioadhesive microspheres. The swelling capacity of a microsphere is equivalent to its ability to absorb water from mucosal tissue, thereby enabling adhesion between the microsphere and the mucosa. Therefore, the swelling capacity of a microsphere can reflect its adhesive properties to a certain extent.

[0038] XRD analysis showed that dihydromyricetin existed in an amorphous state within the microspheres. The in vitro drug release from the dihydromyricetin inclusion complex-bioadhesive microspheres conformed to the Ritger-Peppas model, with the drug release mechanism being Fick diffusion, meaning the drug first dissolves into a solution before diffusing from the formulation into the medium. Stability experiments demonstrated that the microspheres remained stable during storage.

[0039] This invention optimizes the formulation and preparation process of the dihydromyricetin inclusion complex, and also optimizes the formulation and preparation process of the dihydromyricetin inclusion complex-bioadhesive microspheres. The resulting dihydromyricetin inclusion complex-bioadhesive microspheres exhibit high bioavailability and outstanding characteristics.

[0040] The dihydromyricetin inclusion complex-bioadhesive microspheres described in this invention can be used in fields such as nuclear radiation injury, nuclear medicine, and tumor radiotherapy, specifically in the preparation of protective drugs for the prevention / treatment of ionizing radiation injury, particularly in the preparation of drugs for the prevention / treatment of radiation-induced intestinal injury.

[0041] Instruction manual illustrations

[0042] 1. Figure 1 Appearance and properties of dihydromyricetin inclusion complex, etc.: (a) HP-β-CD powder, (b) DMY powder, (c) lyophilized DMY-HP-β-CD inclusion complex;

[0043] 2. Figure 2 Solubility diagram of inclusion phase;

[0044] 3. Figure 3 3D surface plots and contour plots showing the impact of each factor on the overall score;

[0045] 4. Figure 4 The particle size distribution of the DMY-HP-β-CD inclusion complex is shown.

[0046] 5. Figure 5 Microscopic images of HP-β-CD (a), DMY (b), inclusion complex (c), and physical mixture (d);

[0047] 6. Figure 6 Fourier transform infrared spectra: (a) HP-β-CD, (b) DMY, (c) inclusion complex, (d) physical mixture;

[0048] 7. Figure 7 X-ray polycrystalline diffraction patterns: (a) HP-β-CD, (b) DMY, (c) inclusion complex, (d) physical mixture;

[0049] 8. Figure 8 This is a comparison of the stability of inclusion compounds with the stability of physical mixtures;

[0050] 9. Figure 9 The optimal conformation for molecular docking of the DMY / HP-β-CD inclusion complex;

[0051] 10. Figure 10 The optimal conformation for molecular docking of the DMY / SBE-β-CD inclusion complex;

[0052] 11. Figure 11 Appearance and properties of inclusion complex-bioadhesive microspheres: (a) blank microspheres, (b) DMY-HP-β-CD inclusion complex-bioadhesive microspheres;

[0053] 12. Figure 12 The encapsulation efficiency and drug loading curves of microspheres under different polysaccharide concentrations are shown.

[0054] 13. Figure 13 The encapsulation efficiency and drug loading curves of microspheres under different blending ratios are shown.

[0055] 14. Figure 14 The encapsulation efficiency and drug loading curves of microspheres under different dosage conditions are shown.

[0056] 15. Figure 15 The encapsulation efficiency and drug loading curves of microspheres under different calcium chloride concentrations are shown.

[0057] 16. Figure 16 X-ray diffraction patterns of DMY-HP-β-CD inclusion complex microspheres (a), blank microspheres (b), and DMY (c);

[0058] 17. Figure 17 The in vitro release curve of dihydromyricetin inclusion complex-bioadhesive microspheres;

[0059] 18. Figure 18 The mean plasma drug concentration-time curve after oral administration in rats;

[0060] 19. Figure 19 Comparison of small intestinal villi in mice from different drug administration groups;

[0061] 20. Figure 20 Comparison of the number of mouse crypt survivors;

[0062] twenty one, Figure 21 The levels of the inflammatory cytokine TNF-α in each group of mice;

[0063] twenty two, Figure 22 The levels of the inflammatory cytokine IL-6 in each group of mice. Detailed Implementation

[0064] Reagents and instruments:

[0065] Reagent: Commercially available.

[0066] Table 1. Main instruments, their models, and manufacturers.

[0067]

[0068] Example 1: Preparation of DMY-HP-β-CD inclusion complex (1:1)

[0069] 15.41 g of HP-β-CD was accurately weighed and dissolved in ultrapure water, then diluted to a 100 mL volumetric flask to prepare the HP-β-CD stock solution. 3.20 g of DMY was accurately weighed and dissolved thoroughly in anhydrous ethanol, then diluted to a 100 mL volumetric flask to prepare the DMY stock solution. The DMY stock solution and HP-β-CD stock solution were mixed at a molar ratio of 1:1, stirred magnetically at 60 °C for 4.5 h, and the anhydrous ethanol was removed by rotary evaporation. The solution was then redissolved in ultrapure water, filtered through a 0.45 μm microporous membrane to remove free DMY, pre-frozen at -80 °C for 2 h, and freeze-dried for 8 h to obtain the dihydromyricetin inclusion complex. Results are shown below. Figure 1 .

[0070] like Figure 1 As shown, DMY monomer is a pale yellow powder, and HP-β-CD is a white amorphous powder. After DMY is encapsulated by HP-β-CD, its color changes from pale yellow to yellowish-white.

[0071] Example 2: Preparation of DMY-HP-β-CD inclusion complex (1:2)

[0072] 15.40 g of HP-β-CD was accurately weighed and dissolved in ultrapure water, then diluted to a 100 mL volumetric flask to prepare the HP-β-CD stock solution. 1.60 g of DMY was accurately weighed and dissolved thoroughly in anhydrous ethanol, then diluted to a 100 mL volumetric flask to prepare the DMY stock solution. The DMY stock solution and HP-β-CD stock solution were mixed at a molar ratio of 1:2, stirred magnetically at 60 °C for 4.5 h, and the anhydrous ethanol was removed by rotary evaporation. The solution was then reconstituted with ultrapure water, filtered through a 0.45 μm microporous membrane to remove free DMY, pre-frozen at -80 °C for 2 h, and freeze-dried for 8 h to obtain the dihydromyricetin inclusion complex.

[0073] Example 3: Preparation of DMY-HP-β-CD inclusion complex (1:0.5)

[0074] 15.40 g of HP-β-CD was accurately weighed and dissolved in ultrapure water, then diluted to a 100 mL volumetric flask to prepare the HP-β-CD stock solution. 6.40 g of DMY was accurately weighed and dissolved thoroughly in anhydrous ethanol, then diluted to a 100 mL volumetric flask to prepare the DMY stock solution. The DMY stock solution and HP-β-CD stock solution were mixed at a molar ratio of 1:0.5, stirred magnetically at 60 °C for 4.5 h, and the anhydrous ethanol was removed by rotary evaporation. The solution was then reconstituted with ultrapure water, filtered through a 0.45 μm microporous membrane to remove free DMY, pre-frozen at -80 °C for 2 h, and freeze-dried for 8 h to obtain the dihydromyricetin inclusion complex.

[0075] Example 4: Phase Solubility Screening – Inclusion Molar Ratio Determination

[0076] Weigh appropriate amounts of β-CD, HP-β-CD, and SBE-β-CD, dissolve them in 50 mL of deionized water to prepare a 14 mM solution, and then serially dilute to prepare cyclodextrin solutions of different concentrations (2, 4, 6, 8, 10, 12, 14 mM). Measure 5 mL of each solution into a 10 mL centrifuge tube, add excess DMY, shake well, and sonicate for 30 min. Then, maintain constant temperature and shake at 25℃, 37℃, and 50℃ for 72 h to allow the solution to reach equilibrium. Centrifuge at 3000 r / min, filter the supernatant through a 0.45 μm microporous membrane, and dilute appropriately. Determine the concentration of dihydromyricetin, calculate the content of dihydromyricetin, and perform three parallel experiments. Calculate the solubility of dihydromyricetin in a series of concentrations of beta-cyclodextrin and its two derivatives in aqueous solutions at different temperatures. Plot phase solubility curves with the molar concentration of cyclodextrin as the x-axis and the molar concentration of DMY as the y-axis. Results are shown below. Figure 2 .

[0077] from Figure 2 As can be seen, the phase solubility diagrams of the reaction system of DMY with beta-cyclodextrin and its two derivatives in aqueous solution at 25℃, 37℃, and 50℃ are as follows: Figure 2 As shown in the figure. The results indicate that DMY increases linearly in aqueous solutions of beta-cyclodextrin and its two derivatives, exhibiting an AL-type pattern, indicating that within the concentration range of 0-14 mM of cyclodextrin and its derivatives, DMY forms inclusion complexes with β-CD, HP-β-CD, and SBE-β-CD, respectively, with a molar ratio of 1:1.

[0078] Example 5: Phase Solubility Experiment – ​​Inclusion Thermodynamics Study

[0079] A phase solubility diagram was obtained by plotting the concentrations of beta-cyclodextrin and its two derivatives in aqueous solutions on the x-axis and the solubility of DMY in these solutions on the y-axis. The inclusion stability constant (K) can be calculated using formula (1-1). S This is an index for evaluating the affinity between DMY and beta-cyclodextrin and their two derivatives, where S0 is the intrinsic solubility of DMY, slope is the gradient, and other thermodynamic parameters can be calculated from temperature and inclusion stability constant. R is the universal gas constant (8.314 J / mol). -1 k -1 ), where T is the Kelvin temperature.

[0080]

[0081] ΔG=-RTlnK S (1-2)

[0082] ΔG=ΔH-TΔS (1-3)

[0083] From equations (1-2) and (1-3), we can obtain:

[0084]

[0085] With lnK S Perform linear regression on 1 / T, and calculate the thermodynamic parameters ΔH and ΔS based on the slope and intercept of the line.

[0086] K in the reaction system of DMY with beta-cyclodextrin and two of its derivatives at three different temperatures S The values ​​of Gibbs free energy (ΔG), enthalpy change (ΔH), and entropy change (ΔS) are shown in Table 2. Among them, K... S The size of the inclusion complex is correlated with the stability of the inclusion complex, K S The larger the Ks value, the higher the stability of the inclusion complex. Based on Ks values ​​at different temperatures, the stability order of the inclusion complexes formed by dihydromyricetin with beta-cyclodextrin and its two derivatives is: DMY-HP-β-CD inclusion complex > DMY-SBE-β-CD inclusion complex > DMY-β-CD inclusion complex. Ks is highest at 25℃ and lowest at 50℃, indicating that the formation of the three inclusion complexes is an exothermic reaction, and increasing the temperature is detrimental to the reaction.

[0087] Table 2 shows K obtained from the phase solubility diagram. S ΔG, ΔH, ΔS

[0088]

[0089] Example 6: Optimization of the preparation of DMY-HP-β-CD inclusion complex using response surface methodology

[0090] Based on the results of the single-factor investigation, the highest encapsulation efficiency and drug loading under optimal conditions were investigated using the Box-Behnken response surface methodology, taking into account three factors: the molar ratio between DMY and HP-β-CD, the reaction system temperature, and the system reaction time. The results are shown in Table 3.

[0091] Table 3 Factor-Level Coding Table

[0092] Coding Molarratio Temperature / ℃ Time / h -1 0.5 37 1 0 1.25 53.5 3 1 2 70 5

[0093] Using encapsulation efficiency and drug loading as response values, the experimental results are shown in Table 4. Model fitting and analysis of variance: Design-Expert 11.0 software was used to perform quadratic polynomial regression fitting on the data in the table. The comprehensive score (OD) of the quadratic polynomial fitting equation is: 89.21 + 8.78 × molar ratio + 3.01 × temperature + 0.8428 × stirring time - 0.0369 × temperature × molar ratio + 2.54 × stirring time × molar ratio + 0.6777 × temperature × stirring time - 15.84 × molar ratio 2 -4.49 × temperature 2 +1.33 × mixing time 2 According to the binomial fitting equation, the order of importance of factors affecting the inclusion rate is: molar ratio > stirring time > temperature. Regression parameters of the effect surface were obtained using analysis of variance, and the results are shown in Table 4. Figure 3 .

[0094] Table 4. Box-Benhnken design and experimental results for inclusion molar ratio, inclusion temperature, and inclusion time.

[0095]

[0096] Example 7: Optimal Formulation and Preparation Process of Inclusion Compound

[0097] Based on the above experiments and related experimental data, the preparation process of the inclusion compound of the present invention was further optimized as follows:

[0098] DMY∶HP-β-CD molar ratio = 1:1;

[0099] Encapsulation time: 4.5 hours;

[0100] The reaction temperature is 60℃.

[0101] Example 8: Process Validation of Inclusion Compound

[0102] Three batches of DMY-HP-β-CD inclusion complex samples were prepared for verification, and the results are shown in Table 5.

[0103] Table 5 shows that the process for preparing the DMY-HP-β-CD inclusion complex exhibited good reproducibility. The average drug loading was 18.47%, with an RSD of 0.92%, and the average encapsulation efficiency was 87.65%, with an RSD of 0.46%.

[0104] Table 5. Results of the verification experiment (n=3 batches)

[0105]

[0106] Particle size analysis of inclusion complex in Example 9

[0107] The DMY-HP-β-CD inclusion complex (Example 1) was diluted with ultrapure water and poured into a particle size analyzer. The particle size distribution and polydispersity index (PDI) were determined using a Brookhaven nanolaser particle size analyzer with dynamic light scattering. Results are shown below. Figure 4 .

[0108] The average particle size of DMY-HP-β-CD is 163.04 nm, and the PDI is 0.271, indicating that the system has good polydispersity. From... Figure 4 It can be seen that the particle size distribution is concentrated and there are no abnormal particle sizes, which also proves that the prepared inclusion complex has good polydispersity and stability.

[0109] Example 10 Characterization by optical microscopy

[0110] Take a small amount of DMY, HP-β-CD, DMY-HP-β-CD inclusion complex (Example 1), and DMY / HP-β-CD physical mixture onto a glass slide, spread them evenly with a spatula to make them as thin as possible to avoid affecting observation. Observe under a 200x microscope. The results are as follows. Figure 5 As shown.

[0111] from Figure 5 As can be seen from the observation, the microscopic image of DMY raw material is clear, showing irregular needle-like crystals; HP-β-CD shows irregular granules or spheres; the physical mixture DMY / HP-β-CD clearly shows the microscopic image characteristics of DMY raw material crystals with HP-β-CD; the DMY-HP-β-CD inclusion complex clearly shows plate-like crystals with moderate particle size, without obvious needle-like crystals or spheres, which indicates that the inclusion complex has been formed.

[0112] Example 11 Fourier Transform Infrared Spectroscopy (FTIR)

[0113] The dried KBr solid and the sample were thoroughly ground and then compressed into a pellet. The wavenumber range for scanning was 4000–400 cm⁻¹. -1The test results for HP-β-CD, DMY, DMY-HP-β-CD inclusion complex (Example 1), and DMY / HP-β-CD physical mixture were recorded separately. The results are shown in [the table below]. Figure 6 .

[0114] from Figure 6 It can be seen that in the range of 500-1500cm -1 Between them, some small absorption peaks of DMY were almost masked by HP-β-CD, which suggests that some weak natural interactions occurred between them during the formation of the DMY-HP-β-CD inclusion complex.

[0115] Example 12 X-ray diffraction analysis (XRD)

[0116] X-ray diffraction analysis was performed on HP-β-CD, DMY, DMY-HP-β-CD inclusion complex (Example 1), and a physical mixture of DMY / HP-β-CD, respectively. A Cu target was used, the tube voltage was 40 kV, the diffraction angle was 5°–90°, and the scan rate was 10° / min. The XRD patterns were obtained, and the results are shown below. Figure 7 .

[0117] from Figure 7 It can be seen that the diffraction pattern of DMY shows multiple specific crystalline diffraction peaks, while the diffraction pattern of HP-β-CD shows no obvious crystalline peaks, indicating that it exists in an amorphous form. For the physical mixture of DMY and HP-β-CD, the diffraction pattern exhibits both the amorphous state of HP-β-CD and multiple diffraction peaks of DMY, but the peak intensities are significantly weakened or disappear. This indicates that no new crystals are formed between the two physical mixtures; they are simply a physical mixture. Compared to DMY and HP-β-CD alone, the diffraction pattern of the DMY-HP-β-CD inclusion complex is similar to that of HP-β-CD, with the characteristic peaks of DMY almost completely disappearing. This is because DMY is incorporated into the cavity of HP-β-CD, and the crystalline characteristics of DMY disappear.

[0118] Example 13 Determination of water solubility of inclusion complex

[0119] Weigh an excess of DMY-HP-β-CD inclusion complex (Example 1) into a test tube, add ultrapure water to prepare a supersaturated solution, shake at room temperature for 24 h, let stand for 24 h, centrifuge and filter, dilute with ultrapure water to a suitable concentration, and calculate the saturated solubility.

[0120] The solubility of the DMY-HP-β-CD inclusion complex was obtained using the saturated solution method, and it was found to be as high as [insert value here].

[0121] The concentration of 112.88 mg / mL is much higher than that of DMY, indicating that the solubility of DMY is significantly improved after inclusion.

[0122] Example 14 Stability Influencing Factors Experiment

[0123] The DMY-HP-β-CD inclusion complex (Example 1) and a physical mixture were placed under high temperature (60°C), high humidity (92.5% relative humidity), and strong light (5000 Lx) conditions for 0 and 10 days, and the changes in DMY content were observed. The measurements were repeated three times. The results are shown in Table 6, where all data are the average of the three measurements.

[0124] Table 6. Comparison of stability of inclusion compounds and physical mixtures (n=3)

[0125]

[0126] Example 15 pH stability experiment

[0127] Equal amounts of the DMY inclusion complex and physical mixture (DMY concentration 100 μg / mL) were placed at room temperature, pH 6.8, and pH 8.0 for 9 h, and the changes in dihydromyricetin content were observed. The measurements were repeated three times. Results are shown below. Figure 8 .

[0128] Figure 8 The results showed that the amount of dihydromyricetin in the inclusion complex increased by 57.52% compared with the amount in the physical mixture.

[0129] Example 16 Molecular docking comparison

[0130] The molecular-level interaction mechanisms between dihydromyricetin and β-CD, HP-β-CD, and SBE-β-CD were investigated using molecular docking. The study employed Autodock 4.2 with a genetic algorithm. The 2D structures of DMY and β-CD were downloaded from the PubChem database, and energy optimization was performed using the MM2 force field with ChemDraw 21.0.0 and Chem3D 21.0.0 software. Finally, Autodock Tools were used.

[0131] 1.5.6 Add polar hydrogen and charge. Finally, plot the optimal conformation using PyMoL 1.7.6, as shown in the figure. Figure 9 and Figure 10 As shown.

[0132] from Figure 9 and Figure 10Molecular docking results show that DMY achieves effective encapsulation in β-CD, HP-β-CD, and SBE-β-CD, and the encapsulation structures exhibit good stability and spontaneous binding. When DMY binds to β-CD, DMY is completely encapsulated by β-CD, with DMY located at the center of β-CD, forming 6 intermolecular hydrogen bonds with β-CD. The lowest binding energy of inclusion is -5.14 kcal / mol. When DMY binds to HP-β-CD, DMY is partially encapsulated by HP-β-CD, located in the middle of HP-β-CD. However, due to steric hindrance, one end of the branch chain is encapsulated in the cavity, and some groups still exist outside the cavity. DMY forms 5 intermolecular hydrogen bonds with HP-β-CD, and the lowest binding energy of inclusion is -5.24 kcal / mol. When DMY binds to SBE-β-CD, DMY is partially encapsulated by SBE-β-CD, located in the middle of SBE-β-CD. However, due to steric hindrance, one end of the branch chain is encapsulated in the cavity, and some groups still exist outside the cavity. DMY forms 5 intermolecular hydrogen bonds with SBE-β-CD, and the lowest binding energy of inclusion is -4.64 kcal / mol.

[0133] Example 17 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (60:5:1)

[0134] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 60:5:1.

[0135] Weigh 0.125g sodium alginate and 0.025g konjac glucomannan into a beaker, add 10mL ultrapure water, stir in a magnetic stirrer, and sonicate until the bubbles are eliminated. Then add 1.5g of dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, and then add dropwise to a 3% calcium chloride solution using a syringe. Crosslink for 10 minutes, remove, and freeze-dry to constant weight to obtain the product. Appearance is as shown. Figure 11 As shown.

[0136] from Figure 11 As can be seen, the blank microspheres are white, while the DMY-HP-β-CD inclusion complex-bioadhesive microspheres are pale yellow. The particle size of the DMY-HP-β-CD inclusion complex-bioadhesive microspheres is significantly increased compared to the blank microspheres.

[0137] Example 18 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (20:2:0.2)

[0138] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 20:2:0.2.

[0139] Weigh 0.15g sodium alginate and 0.015g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 1.5g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 10min, remove, freeze dry to constant weight, and the product is obtained.

[0140] Example 19: Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (20:2:2)

[0141] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 20:2:2.

[0142] Weigh 0.15g sodium alginate and 0.15g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 1.5g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 5min, remove, freeze dry to constant weight, and the product is obtained.

[0143] Example 20: Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (20:8:2)

[0144] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 20:8:2.

[0145] Weigh 0.6g sodium alginate and 0.15g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 1.5g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 15min, remove, freeze dry to constant weight, and the product is obtained.

[0146] Example 21 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (20:8:0.2)

[0147] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 20:8:0.2.

[0148] Weigh 0.6g sodium alginate and 0.015g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 1.5g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 10min, remove, freeze dry to constant weight, and the product is obtained.

[0149] Example 22 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (100:2:0.2)

[0150] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 100:2:0.2.

[0151] Weigh 0.12g sodium alginate and 0.012g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 6.0g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 30min, remove, freeze dry to constant weight, and the product is obtained.

[0152] Example 23 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (100:2:2)

[0153] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 100:2:2.

[0154] Weigh 0.12g sodium alginate and 0.12g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 6.0g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 10min, remove, freeze dry to constant weight, and the product is obtained.

[0155] Example 24 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (100:8:0.2)

[0156] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 100:8:0.2.

[0157] Weigh 0.48g sodium alginate and 0.012g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 6.0g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 5min, remove, freeze dry to constant weight, and the product is obtained.

[0158] Example 25 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (100:8:2)

[0159] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 100:8:2.

[0160] Weigh 0.48g sodium alginate and 0.12g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 6.0g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 10min, remove, freeze dry to constant weight, and the product is obtained.

[0161] Example 26 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (40:4:0.5)

[0162] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 40:4:0.5.

[0163] Weigh 0.15g sodium alginate and 0.019g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 1.5g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 15min, remove, freeze dry to constant weight, and the product is obtained.

[0164] Example 27 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (40:6:1.5)

[0165] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 40:6:1.5.

[0166] Weigh 0.225g sodium alginate and 0.056g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 1.5g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 60min, remove, freeze dry to constant weight, and the product is obtained.

[0167] Example 28 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (40:4:1.5)

[0168] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 40:4:1.5.

[0169] Weigh 0.15g sodium alginate and 0.056g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 1.5g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 10min, remove, freeze dry to constant weight, and the product is obtained.

[0170] Example 29 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (80:6:0.5)

[0171] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 80:6:0.5.

[0172] Weigh 0.113g sodium alginate and 0.01g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 1.5g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 10min, remove, freeze dry to constant weight, and the product is obtained.

[0173] Example 30: Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (80:6:1.5)

[0174] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 80:6:1.5.

[0175] Weigh 0.113g sodium alginate and 0.028g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 1.5g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 15min, remove, freeze dry to constant weight, and the product is obtained.

[0176] Example 31 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (80:4:1.5)

[0177] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 80:4:1.5.

[0178] Weigh 0.075g sodium alginate and 0.028g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 1.5g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 10min, remove, freeze dry to constant weight, and the product is obtained.

[0179] Example 32 Preparation of dihydromyricetin inclusion complex-bioadhesive microspheres (80:4:0.5)

[0180] The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 80:4:0.5.

[0181] Weigh 0.075g sodium alginate and 0.01g konjac glucomannan into a beaker, add 10mL ultrapure water, place in a magnetic stirrer and stir, sonicate until the bubbles are eliminated, then add 1.5g dihydromyricetin inclusion complex from Example 1, stir, sonicate until the bubbles are eliminated, then add dropwise to 3% calcium chloride solution with a syringe, crosslink for 5min, remove, freeze dry to constant weight, and the product is obtained.

[0182] Example 33: Investigation of Adhesive Microsphere Process – Effect of Polysaccharide Concentration on Drug Loading and Encapsulation Efficiency

[0183] With a fixed blending ratio (konjac glucomannan, KGM: sodium alginate, SA) of 1:6, a dosage (amount of dihydromyricetin inclusion complex) of 0.5 g, and a CaCl2 solution concentration of 3%, bioadhesive gel microspheres were prepared according to the method of Example 15 under polysaccharide concentrations of 0.75%, 1.00%, 1.50%, 2.00%, and 2.50%. The results are as follows... Figure 12 As shown.

[0184] from Figure 12 It can be seen that with the increase of polysaccharide concentration, the drug loading and encapsulation of microspheres first increase and then decrease. This indicates that the encapsulation efficiency and drug loading are highest when the polysaccharide concentration is 1%. Based on the analysis of this experimental result, we selected three conditions—polysaccharide concentrations of 0.75%, 1.00%, and 1.50%—for orthogonal analysis.

[0185] Example 34: Investigation of Adhesive Microsphere Process – Effect of Blending Ratio on Drug Loading and Encapsulation Efficiency

[0186] With a fixed polysaccharide concentration of 1.5%, a dosage (amount of dihydromyricetin inclusion complex) of 0.5 g, and a CaCl2 solution concentration of 3%, gel microspheres were prepared according to the method in Example 15 under polysaccharide blending ratios of 1:3, 1:4, 1:5, and 1:6. The results are shown in [Figure 15]. Figure 13 .

[0187] from Figure 13 It can be seen that as the proportion of sodium alginate in the polysaccharide increases, the drug loading and encapsulation of the microspheres first increase and then decrease. This indicates that the encapsulation efficiency and drug loading are highest at a blending ratio of 1:4. Based on the analysis of this experimental result, three blending ratios of 1:3, 1:4, and 1:5 were selected for orthogonal analysis.

[0188] Example 35: Investigation of Adhesive Microsphere Process – Effect of Dosage on Drug Loading and Encapsulation Efficiency

[0189] With a fixed polysaccharide concentration of 1.5%, a fixed blending ratio (KGM:SA) of 1:4, and a CaCl2 solution concentration of 3%, gel microspheres were prepared according to the method of Example 15 at dosages of 0.5 g, 1.0 g, and 1.5 g. The results are as follows... Figure 14As shown.

[0190] from Figure 14 It can be seen that as the dosage increases, the drug loading of the microspheres first increases and then decreases. The microsphere encapsulation continuously increases. When the dosage reaches 1.5g, sodium alginate tends to become very viscous. When the dosage is 1.8g, it is difficult to mix evenly. Therefore, based on the analysis of the experimental results, orthogonal analysis was conducted using dosages of 0.5g, 1.0g, and 1.5g.

[0191] Example 36: Investigation of Adhesive Microsphere Process – Effect of Calcium Chloride Solution Concentration on Drug Loading and Encapsulation Efficiency

[0192] With a fixed polysaccharide concentration of 1.5%, a blending ratio (KGM:SA) of 1:4, and a dosage (amount of dihydromyricetin inclusion complex) of 0.5 g, gel microspheres were prepared according to the method in Example 15 under CaCl2 solution concentrations of 1.0%, 3.0%, 5.0%, and 7.0%. The results are shown in [Figure 15]. Figure 15 .

[0193] from Figure 15 It can be seen that with the increase of calcium chloride concentration, the drug loading and encapsulation of microspheres first increase and then decrease. Based on the analysis of the experimental results, we selected three conditions with calcium chloride concentrations of 3.0%, 5.0%, and 7.0% for orthogonal analysis.

[0194] Example 37 Investigation of the Adhesive Microsphere Process – Orthogonal Experimental Design

[0195] Table 7 Factor-Level Table

[0196]

[0197] The results of the orthogonal experiment are shown in Table 8. The order of influence of the four factors on microsphere formation is: dosage (C) > calcium chloride solution concentration (D) > blending ratio (B) > polysaccharide concentration (A). The optimal process formulation was obtained as C3D1B3A3.

[0198] Table 8 Results of the orthogonal experiment

[0199]

[0200] Example 38: Optimal Preparation Process of Dihydromyricetin Inclusion Complex-Bioadhesive Microspheres

[0201] Based on the above experiments and related experimental data, the preferred preparation process of the inclusion complex-bioadhesive microspheres of the present invention is as follows:

[0202] 1.5g of dihydromyricetin inclusion complex;

[0203] Sodium alginate 0.125g;

[0204] Konjac glucomannan 0.025g;

[0205] Calcium chloride concentration 3%;

[0206] Crosslinking time: 5-10 min.

[0207] Example 39 Study on the formulation and process of adhesive microspheres—Verification of the optimal process

[0208] Process validation was performed by preparing three batches of dihydromyricetin inclusion complex-bioadhesive microspheres according to the formulation process of Example 21, measuring drug loading and encapsulation efficiency, and the results are shown in Table 9.

[0209] Table 9 shows that under the following process conditions—an inclusion complex dosage of 1.5 g, a polysaccharide concentration of 1.5%, a konjac glucomannan to sodium alginate ratio of 1:5, and a calcium chloride concentration of 3%—the average drug loading was 16.13%, with an RSD of 1.53%, and the average encapsulation efficiency was 76.50%, with an RSD of 1.04%. The results indicate that the three batches of prepared bioadhesive microspheres exhibited stable processing and good reproducibility.

[0210] Table 9. Results of the verification experiment

[0211]

[0212] Example 40: X-ray diffraction (XRD) of adhered microspheres

[0213] Samples of dihydromyricetin, blank microspheres, and drug-containing microspheres were prepared by grinding. X-ray diffraction analysis was performed. A Cu target was used, with a tube voltage of 40 kV, a diffraction angle of 5°–90°, and a scan rate of 10° / min. The XRD patterns of the samples were recorded, and the results are shown below. Figure 16 .

[0214] from Figure 16 It can be seen that the diffraction pattern of DMY has multiple specific crystal diffraction peaks, while the diffraction patterns of DMY-HP-β-CD inclusion complex-microspheres and blank microspheres do not show obvious crystal peaks.

[0215] Example 41 Determination of the swelling properties of adhesive microspheres

[0216] Weigh 100 mg of microspheres and place them in a PBS buffer solution at pH 4.5, then incubate at 37°C with shaking. Periodically remove the microspheres, wipe off surface moisture, and quickly weigh them to calculate the swelling ratio.

[0217] The dihydromyricetin inclusion complex-bioadhesive microspheres reached their maximum swelling rate of (54.08±2.64)% within 3 minutes.

[0218] Example 42 Evaluation of the in vitro adhesion performance of the adhesive microspheres

[0219] Male SD rats were fasted for 12 hours but allowed free access to water. They were then injected intraperitoneally with 10% chloral hydrate. The intestines were dissected, cut open, and thoroughly rinsed with physiological saline before being fixed onto a glass slide. Thirty microspheres were evenly sprinkled onto the intestinal mucosa and placed in a sealed container maintained at 92.5% relative humidity (saturated potassium nitrate solution) to allow for full hydration and swelling. After 20 minutes, the slide was removed. The glass slide was fixed on a 45° support, and the burette was adjusted to flush the intestinal tissue with pH 4.5 PBS buffer at a rate of 6 mL / min for 10 minutes. The microspheres in the flushing solution were collected and counted, and the retention rate of the composite microspheres in the rat intestinal mucosa was calculated.

[0220] Dihydromyricetin inclusion complex-bioadhesive microspheres exhibit strong adhesion, with an average retention rate of (95.45±1.78)%.

[0221] Example 43: Study on in vitro drug release characteristics

[0222] The dissolution profile of dihydromyricetin inclusion complex-bioadhesive microspheres in phosphate buffer (pH 4.5) was determined using the basket method. Microspheres were added to 900 mL of dissolution medium at 75 rpm and 37 °C. Samples (5 mL) were taken at 2, 5, 10, 20, 40, 60, 90, 120, 150, 210, 270, and 330 min, and the same volume of dissolution medium was replenished promptly. HPLC analysis was performed to determine the cumulative drug release rate, and time-cumulative release curves were plotted. The results are shown in Table 10. Figure 17 .

[0223] from Figure 17 It can be seen that the microspheres release DMY relatively quickly because the preparation of the dihydromyricetin into an inclusion complex increases its solubility. The cumulative release rate of the microspheres was fitted using zero-order release, first-order release, Higuchi, and Ritger-Peppas models. The fitting results are shown in Table 10. The results indicate that the in vitro release mechanism of DMY from the microspheres is more consistent with the Ritger-Peppas release model, showing a higher degree of fit. 2 It is 0.9241.

[0224] Table 10 Microsphere Fitting Results

[0225] Model Fitting results <![CDATA[R 2 ]]> Zero-order dynamics Q(t) = 0.108x + 66.93 0.5740 First-order dynamics Q(t) = 92.92(1-e-0.17x) 0.8369 Higuchi model Q(t) = 2.59 x 1 / 2 + 56.40 0.7602 Ritger-Peppas model Q(t) = 47.31(x^0.133) 0.9241

[0226] Example 44: Stability Experiment of Bioadhesive Microspheres

[0227] This study conducted a long-term experiment to explore the dihydromyricetin inclusion complex-bioadhesive microspheres. Three batches of microspheres were placed under conditions of (25±2)℃, (60±5)%RH, and protection from light for stability testing. The appearance and color were observed and the content was determined at 0, 15, and 30 days. The results are shown in Table 11.

[0228] Table 11 Stability Experiment

[0229] Sampling time (days) Appearance Liquidity content(%) 0 pale yellow granules good 99.54 15 pale yellow granules good 97.82 30 Yellow granules good 96.79

[0230] In summary, the konjac glucomannan / sodium alginate microspheres of the present invention have good biocompatibility, and no surfactants or organic solvents are used in the microsphere preparation process, thus avoiding secondary damage to radiation-induced intestinal injury.

[0231] Swelling capacity is an important parameter for evaluating the adhesive properties of bioadhesive microspheres. The swelling capacity of a microsphere is equivalent to its ability to absorb water from mucosal tissue, thereby enabling adhesion between the microsphere and the mucosa. Therefore, the swelling capacity of a microsphere can reflect its adhesive properties to a certain extent.

[0232] XRD analysis showed that dihydromyricetin existed in an amorphous state within the microspheres. The in vitro drug release from the dihydromyricetin inclusion complex-bioadhesive microspheres conformed to the Ritger-Peppas model, with the drug release mechanism being Fick diffusion, meaning the drug first dissolves into a solution before diffusing from the formulation into the medium. Stability experiments demonstrated that the microspheres were stable during storage.

[0233] Example 45: Preliminary in vivo pharmacokinetic evaluation of dihydromyricetin inclusion complex-bioadhesive microspheres

[0234] The pharmacokinetic characteristics of the prepared dihydromyricetin inclusion complex-bioadhesive microspheres were studied. The total icariin content in the plasma of rats was determined by UPLC-MS / MS after gavage administration of DMY raw material (100 mg / kg), three dose groups of microspheres (low (50 mg / kg), medium (100 mg / kg), and high (200 mg / kg), and after intravenous administration of DMY raw material (50 mg / kg).

[0235] Preparation of gavage solution. Preparation of DMY raw material gavage solution: Accurately weigh 250mg of DMY raw material, slowly add 25mL of 0.5% CMC-Na solution, grind and suspend, and prepare a CMC-Na suspension with a DMY concentration of 10mg / mL.

[0236] Low-dose DMY microsphere group: Accurately weigh 0.94g of DMY inclusion complex microspheres, slowly add 30mL of 0.5% CMC-Na solution, suspend, and prepare a CMC-Na suspension with a DMY concentration of 5mg / mL.

[0237] Medium-dose group of DMY microspheres: Accurately weigh 1.88g of DMY inclusion complex microspheres, slowly add 30mL of 0.5% CMC-Na solution, suspend, and prepare a CMC-Na suspension with a DMY concentration of 10mg / mL.

[0238] High-dose DMY microsphere group: Accurately weigh 3.76g of DMY inclusion complex microspheres, slowly add 30mL of 0.5% CMC-Na solution, suspend, and prepare a CMC-Na suspension with a DMY concentration of 20mg / mL.

[0239] Preparation of DMY intravenous injection solution: Accurately weigh 125 mg of DMY raw material, add 2.5 mL of ethanol and 2.5 mL of dimethyl sulfoxide (DMSO) to dissolve it, then add 20 mL of physiological saline to prepare an intravenous injection solution with a DMY concentration of 5 mg / mL.

[0240] Plasma sample collection. At 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 8, and 10 minutes after drug administration, 0.25 mL of blood was collected from the orbital venous plexus and placed in centrifuge tubes containing the anticoagulant sodium heparin and the stabilizer vitamin C. The tubes were shaken well and centrifuged at 12,000 rpm for 5 minutes at 4°C. The plasma was separated and stored at -20°C for later use.

[0241] Blood drug concentration curve in rats.

[0242] like Figure 18 As shown, the mean plasma drug concentration-time curves of rats in the low, medium, and high dose groups of DMY microspheres after gavage administration and the gavage and intravenous administration of DMY raw material are presented. Compared with the DMY raw material group, at the same dosage, DMY microspheres significantly increased the peak plasma drug concentration and the area under the curve. This figure intuitively illustrates that DMY microspheres play a positive role in improving the oral absorption of DMY.

[0243] Calculation and results of pharmacokinetic parameters.

[0244] Blood drug concentration data of DMY microspheres administered by gavage at low, medium, and high doses, DMY raw material administered by gavage, and DMY raw material administered intravenously were processed using Phoenix 8.1.0 software to obtain AUC. (0-10h) C max T max The pharmacokinetic parameters are shown in Table 12. After oral administration of DMY raw material, the AUC... (0-10) The mean residence time (MRT) was 23.5 ± 3.26 h mg / L.(0-10) The duration was 4.08 ± 1.03 h, C max The concentration was 6.88 ± 0.540 mg / L. After oral administration of the same dose of DMY microspheres, the AUC was... (0-10) The concentration was 37.80 ± 5.99 h* mg / L, MRT (0-10h) It was 5.47 ± 0.98 h; C max The concentration was 10.30±1.00 mg / L. Compared with the oral raw material group, the pharmacokinetic parameters increased by 1.61, 1.34, and 1.50 times, respectively, and the absolute bioavailability increased from 26.1% to 41.9%. These results indicate that DMY bioadhesive microspheres can effectively improve the absolute bioavailability of DMY, prolong its retention time in vivo, and slow down the metabolism of DMY in vivo.

[0245] Table 12. Major pharmacokinetic parameters of different rat samples after oral administration (n=5)

[0246]

[0247] This invention used liquid chromatography-mass spectrometry (LC-MS) to determine the plasma concentration of DMY in rats. Preliminary pharmacokinetic experiments in rats were conducted to study the pharmacokinetic characteristics of DMY administered by gavage, DMY inclusion complex-bioadhesive microspheres administered by gavage, and DMY active pharmaceutical ingredient administered by intravenous injection. The maximum plasma concentration C0 in the medium-dose group administered by gavage with DMY inclusion complex-bioadhesive microspheres was determined. max Area under the curve (AUC) during drug administration (0-t) and mean residence time (MRT) (0-t) The data were all higher than those of the DMY raw material group administered by gavage, increasing by 1.50 times, 1.61 times, and 1.34 times, respectively.

[0248] The final results showed that dihydromyricetin inclusion complex-bioadhesive microspheres increased the local concentration of DMY, thereby increasing the concentration absorbed into the bloodstream and improving bioavailability. In the DMY inclusion complex-bioadhesive microsphere group administered by gavage, the pharmacokinetic parameters differed significantly among the low, medium, and high dose groups, suggesting a correlation between the therapeutic effect and the administered dose within the concentration range designed for this experiment. Calculations showed that the absolute bioavailability of the low-dose group and the relative bioavailability of the medium-dose group were 41.9% and 161%, respectively. The high bioavailability of the dihydromyricetin inclusion complex-bioadhesive microspheres is a significant advantage.

[0249] Example 46: Study on the protective effect of dihydromyricetin inclusion complex-bioadhesive microspheres on the intestine.

[0250] Modeling: 10 animals per group were given a single local abdominal irradiation of 12 Gy of 137Cs γ-rays at a dose rate of 1 Gy / min.

[0251] Grouping: The subjects were divided into 5 groups: blank control group, irradiation group, irradiation + free dihydromyricetin group, irradiation + dihydromyricetin preparation group (dihydromyricetin inclusion complex-bioadhesive microsphere group), and irradiation + positive control drug amifostine group.

[0252] Drug administration: Mice in each group were administered the drugs for 5 days before and 3 days after irradiation. On the day of irradiation, the drugs (including dihydromyricetin and amifostine) were administered 30 minutes beforehand. The volume of drugs administered by gavage and intraperitoneal injection was 50 mg / kg. Mice in each group were sacrificed on day 3.5 after irradiation, and samples (whether mouse small intestine was collected) were used for subsequent experimental testing.

[0253] See results Figure 19 and Figure 20 It can be seen that, compared with the irradiation group, the free dihydromyricetin group can effectively increase the number of mice. Small intestinal The dihydromyricetin preparation group (dihydromyricetin inclusion complex-bioadhesive microsphere group) showed more significant effects on villous length and crypt survival, and was superior to the positive control group.

[0254] Example 47: Study on the inhibitory effect of dihydromyricetin inclusion complex-bioadhesive microspheres on inflammatory factors.

[0255] Based on Example 31, serum samples from mice in each group were collected 3.5 days after irradiation, and the expression levels of inflammatory factors TNF-α and IL-6 were detected by ELISA.

[0256] See results Figure 21 and Figure 22 It can be seen that, compared with the irradiation group, the levels of inflammatory factors TNF-α and IL-6 in mice in the free dihydromyricetin group were significantly reduced, while the dihydromyricetin preparation group (dihydromyricetin inclusion complex-bioadhesive microsphere group) showed more significant inhibition of inflammatory factors.

[0257] In summary, compared with traditional oral administration, the dihydromyricetin-bioadhesion drug delivery method of this invention prolongs the residence time of the drug in the gastrointestinal tract and improves the membrane permeability of the drug, significantly improving the bioavailability of the drug. It can be used to prepare drugs for the prevention / treatment of ionizing radiation damage, especially for the preparation of drugs for the prevention / treatment of radiation-induced intestinal injury.

Claims

1. A dihydromyricetin inclusion complex-bioadhesive microsphere, the raw materials of which include: Dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan.

2. The dihydromyricetin inclusion complex-bioadhesive microspheres according to claim 1, wherein: The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is (20-100):(2-8):(0.2-2).

3. The dihydromyricetin inclusion complex-bioadhesive microspheres according to claim 1, wherein: The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is (40-80):(4:6):(0.5-). 1.5)。 4. The dihydromyricetin inclusion complex-bioadhesive microspheres according to claim 1, wherein: The mass ratio of dihydromyricetin inclusion complex, sodium alginate, and konjac glucomannan is 60:5:

1.

5. The dihydromyricetin inclusion complex-bioadhesive microspheres according to any one of claims 1-4, wherein the dihydromyricetin inclusion complex is composed of dihydromyricetin and cyclodextrin.

6. The dihydromyricetin inclusion complex-bioadhesive microspheres according to claim 5, wherein the cyclopaste is selected from any one or more of β-CD, HP-β-CD, and SBE-β-CD; preferably HP-β-CD.

7. The dihydromyricetin inclusion complex-bioadhesive microspheres according to claim 5, characterized in that... The molar ratio of dihydromyricetin to cyclodextrin is 1:(0.5-2).

8. The dihydromyricetin inclusion complex-bioadhesive microspheres according to claim 5, characterized in that... The molar ratio of dihydromyricetin to cyclodextrin is 1:

1.

9. The dihydromyricetin inclusion complex according to claim 5, characterized in that: Inclusion time: 3–6 h; reaction temperature: 40–80 °C.

10. The dihydromyricetin inclusion complex according to claim 9, characterized in that: Inclusion time: 4–5 h; reaction temperature: 50–70 °C.

11. The dihydromyricetin inclusion complex according to claim 10, characterized in that: Inclusion time: 4.5 h; reaction temperature: 60 °C.

12. A method for preparing the dihydromyricetin inclusion complex-bioadhesive microspheres according to any one of claims 1-4: Take sodium alginate and konjac glucomannan, add water, stir, add dihydromyricetin inclusion complex, stir, and add to calcium chloride solution to obtain the final product.

13. The method of claim 12, characterized in that: Take 0.125g sodium alginate and 0.025g konjac glucomannan, add 10mL water, stir, add 1.5g dihydromyricetin inclusion complex, stir, add dropwise to 3% calcium chloride solution, crosslink for 2min-30min, freeze dry to obtain the product.

14. The use of the dihydromyricetin inclusion complex-bioadhesive microspheres according to any one of claims 1-4 in the preparation of drugs for the prevention / treatment of ionizing radiation protection.

15. The use of the dihydromyricetin inclusion complex-bioadhesive microspheres of claim 13 in the preparation of drugs for the prevention / treatment of radiation-induced intestinal injury.

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