Synephrine induced DPP-4 inhibitor compound preparation for treating diabetes mellitus

Abstract

The invention discloses a synephrine induced DPP-4 inhibitor compound preparation for treating diabetes and a preparation method of the synephrine induced DPP-4 inhibitor compound preparation. The composite preparation is technically characterized in that synephrine (CAS: 94-07-5) is adopted as a binding inducer, and the molecular affinity between pterostilbene (CAS: 537-42-8), quercetin (CAS: 117-39-5) and naringin (CAS: 10236-47-2) and DPP-4 active sites is remarkably enhanced through a synergistic binding mechanism at a molecular level, so that the enzyme inhibition efficiency is improved. Experiments prove that the binding capacity of DPP-4 of the three active ingredients can be averagely enhanced by 34.7% through the binding induction effect of synephrine, and the inhibitory activity reaches the clinical application standard. The invention also provides a method for industrially preparing the compound preparation. The method comprises the following steps: preparing a high-purity solution; mixing in proportion to form a molecular compound; preparing micro powder with a specific particle size; and performing quality control evaluation. The obtained composite preparation micro powder has good physical and chemical stability and bioavailability, and can be directly used for preparing various dosage forms, for example, the composite preparation micro powder can be used for treating diabetes.

Description

Technical Field

[0001] The present invention relates to the field of dipeptidyl peptidase-4 (DPP-4) inhibitors, and particularly to a synephrine-induced DPP-4 inhibitor composite preparation for the treatment of diabetes. Background Art

[0002] Among diabetes-related complications, cardiovascular diseases are the most serious, especially having a more significant impact on type 2 diabetes patients. Epidemiological data fully illustrate the urgency of developing highly effective and safe treatment regimens, especially innovative drugs that can simultaneously improve blood glucose control and cardiovascular prognosis.

[0003] Dipeptidyl peptidase-4 (DPP-4) plays a key role in the pathogenesis and treatment of diabetes. Research has confirmed that insulin secretagogues, especially glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), can enhance glucose-dependent insulin secretion, inhibit glucagon release, delay gastric emptying, and reduce appetite. However, these endogenous peptides are rapidly hydrolyzed and inactivated by DPP-4 in vivo. Prior art studies have shown that DPP-4 can specifically cleave the N-terminal amino acid sequence containing alanine or proline, rendering more than 40 bioactive peptides non-functional. Among them, the in vivo half-life of GLP-1 is less than 2 minutes, which constitutes a key technical obstacle to the development of treatment regimens based on insulin secretagogues.

[0004] Existing DPP-4 inhibitors such as sitagliptin (trade name: Januvia), vildagliptin (trade name: Galvus), etc. have demonstrated many advantages in clinical applications, such as not causing hypoglycemic reactions, not leading to weight gain, and having a wide range of applications. However, these chemically synthesized small molecule inhibitors have obvious limitations: on the one hand, long-term use may cause adverse reactions such as upper respiratory tract infections, nasopharyngitis, headache, and even acute pancreatitis; on the other hand, drug safety monitoring shows that long-term application may lead to the development of drug resistance. More critically, the results of large-scale clinical trials (TECOS, EXAMINE, SAVOR-TIMI 53, etc.) have shown that such inhibitors have not effectively reduced the incidence of cardiovascular events, resulting in the downgrading of these inhibitors to third-line treatment options in multinational clinical guidelines, and their application value has been severely restricted.

[0005] The main technical defects of DPP-4 inhibitor hypoglycemic drugs in the prior art are the difficulty in achieving effective blood glucose control and cardiovascular protection simultaneously. Through systematic patent retrieval and experimental data analysis, three bioactive molecules (pterostilbene, quercetin, and naringin) derived from traditional Chinese medicine have the possibility of solving the above technical problems, especially having unique technical advantages in the multi-target protection mechanism of the cardiovascular system. According to the data of multi-center experimental studies, the cardiovascular protection of the above active molecules is mainly achieved through the following four technical pathways: 1) Quercetin and pterostilbene activate the Nrf2 / HO-1 signal transduction pathway specifically, producing significant antioxidant and anti-inflammatory effects, thereby effectively inhibiting oxidative stress damage in the cardiovascular system; 2) Naringin and pterostilbene can selectively up-regulate the expression level of eNOS, promote the biosynthesis and release of endogenous nitric oxide, and significantly improve vascular endothelial function; 3) Quercetin regulates the expression of PPAR-α and SREBP-1c to achieve effective regulation of lipid metabolism and significantly reduce blood lipid levels; 4) Quercetin and naringin selectively inhibit the cAMP / PKA and PI3K / Akt signal pathways, effectively blocking the platelet aggregation process and significantly reducing the risk of thrombosis. However, the research on the inhibitory effect of these three active ingredients on DPP-4 in the prior art is significantly insufficient: Although pterostilbene has a potential DPP-4 regulatory effect, its direct action mechanism and inhibitory activity lack systematic experimental evaluation and mechanism elaboration. As a typical flavonoid compound, the competitive inhibition mechanism of quercetin with DPP-4 has been preliminarily verified, but its bioavailability is limited (mainly due to absorption and metabolism disorders), and there are obvious differences in the reported IC50 values in existing studies, and its clinical application value needs to be further demonstrated. As a characteristic active ingredient in citrus plants, existing studies suggest that naringin may indirectly affect DPP-4 activity by regulating the intestinal microecology and inflammatory factors, but its direct inhibitory mechanism has not been fully confirmed, and the repeatability and reliability of related activity data need to be improved. These key technical problems constitute an important limitation to the application of natural active ingredients in the research and development of DPP-4 inhibitors.

[0006] Through systematic experiments such as molecular simulation, enzyme kinetics, and affinity determination, the inventor of the present invention found that there are obvious technical limitations in the binding ability of these traditional Chinese medicine active ingredients to DPP-4 alone. The results of computer-aided drug design show that: firstly, their binding energy is relatively weak. When used alone, the binding energy to DPP-4 is usually only between -7.5 and -9.9 kcal / mol, which is weaker than that of synthetic inhibitors such as sitagliptin (-10.3 kcal / mol); secondly, isotope labeling experiments show that their binding sites to DPP-4 are unstable, and molecular dynamics simulations show that this binding conformation is susceptible to changes in physiological environments such as pH value and ionic strength, resulting in a short binding half-life; finally, high-throughput screening experiments confirm that the selectivity of single components for DPP-4 is not ideal, and IC50 value determination shows that they may simultaneously affect the activities of related enzymes such as DPP-8 and DPP-9. These limitations in molecular binding characteristics severely restrict the clinical efficacy of traditional Chinese medicine active ingredients as DPP-4 inhibitors and are difficult to meet the requirements of clinical applications.

[0007] Therefore, developing a binding inducer that can significantly enhance the binding ability of traditional Chinese medicine active ingredients to DPP-4 has become a key technical challenge in this technical field. An ideal binding inducer should meet the following technical requirements: it can promote the interaction between the active ingredient and the specific binding pocket of DPP-4, stabilize the binding conformation of the active ingredient-DPP-4 complex, improve the selectivity for DPP-4, and have good safety. The present invention aims at the deficiencies of the above-mentioned prior art and provides a novel pharmaceutical composition and its preparation method using synephrine as a binding inducer to significantly enhance the binding ability of traditional Chinese medicine active ingredients such as pterostilbene, quercetin, and naringin to DPP-4. Summary of the Invention

[0008] The present invention aims to solve the substantial technical problem of insufficient binding affinity of DPP-4 inhibitors based on natural compounds in the prior art, and provides a DPP-4 inhibitor composite preparation using synephrine as a binding inducer and its preparation method. This composite preparation can significantly improve the inhibitory efficacy of natural active ingredients on DPP-4 and enhance the cardiovascular protection effect at the same time.

[0009] According to the first aspect of the present invention, there is provided a DPP-4 inhibitor composite preparation, and the technical characteristics of the composite preparation are that it contains: The binding inducer synephrine, as well as pterostilbene, quercetin, and naringin.

[0010] Among them, synephrine, as the key binding inducer, significantly enhances the binding stability and selectivity of the above three natural active ingredients to the active pocket of DPP-4 enzyme.

[0011] In a preferred embodiment of the present invention, the mass ratio of synephrine, pterostilbene, quercetin and naringin in the composite preparation is 1:(0.5 - 20.0):(0.5 - 20.0):(0.5 - 20.0).

[0012] The present invention also provides the physical properties of a synephrine-induced DPP-4 inhibitor composite preparation for the treatment of diabetes. After micronization, the composite preparation has excellent physical properties. By comprehensively characterizing the prepared composite preparation micropowder using a laser particle size analyzer, gas adsorption method, powder density measuring instrument and powder flowability measuring instrument, the results show that the composite preparation micropowder has the following physical properties: the particle size range is between 32.9 - 38.1 μm, the particle size distribution uniformity (span) is 1.43 - 1.51, the specific surface area is 11.8 - 13.4 m² / g, the bulk density and tapped density are 0.345 - 0.368 g / cm³ and 0.425 - 0.445 g / cm³ respectively, and the Carr index and Hausner ratio are 17.3 - 18.8 and 1.21 - 1.23 respectively. The above physical property data fully prove that the micropowder has good fluidity and compression performance, is suitable for subsequent formulation processing, and is convenient for industrial production and clinical application.

[0013] Verified by systematic molecular docking and in vitro enzyme activity assays, the composite preparation of the present invention has the following technical characteristics: The binding-induced effect of synephrine enhances the binding energy of pterostilbene to DPP-4 from -7.50 kcal / mol to -10.77 kcal / mol, with an enhancement amplitude of 43.6%; The binding-induced effect of synephrine enhances the binding energy of quercetin to DPP-4 from -8.30 kcal / mol to -11.54 kcal / mol, with an enhancement amplitude of 39.0%; The binding-induced effect of synephrine enhances the binding energy of naringin to DPP-4 from -9.90 kcal / mol to -12.04 kcal / mol, with an enhancement amplitude of 21.6%; The composite preparation with an optimized ratio (1:1.5:1:1.8) has significant inhibitory activity against DPP-4, and its IC50 value is 28.7 nM.

[0014] According to the second aspect of the present invention, a method for preparing the above DPP-4 inhibitor composite preparation is provided, and the method includes the following technical steps: Prepare solutions of high-purity synephrine, pterostilbene, quercetin and naringin respectively; Mix the above solutions in precisely optimized molar ratios and react under constant temperature conditions (37 ± 1°C), a strictly controlled pH environment (6.8 ± 0.2), and a specific stirring rate to promote intermolecular interactions and form a homogeneous molecular complex with stable structure; Convert the mixture into a fine powder state with a specific particle size distribution and specific surface area using vacuum freeze-drying or spray-drying techniques; Conduct comprehensive physicochemical property, content determination, and accelerated stability tests on the prepared fine powder.

[0015] In a preferred embodiment of the present invention, the solvent described in step 1 is selected from pharmaceutical-grade water, ethanol, and a mixture thereof prepared in a specific ratio to ensure the best solubility and stability of the active ingredient.

[0016] In another preferred embodiment of the present invention, the process parameters of step 3 are as follows: under vacuum freeze-drying conditions, the pre-freezing temperature is -40°C to -50°C, the vacuum degree is 10 - 100 Pa, and the drying time is 24 - 48 hours; or under spray-drying conditions, the inlet temperature is 120 - 140°C, the outlet temperature is 80 - 90°C, and the atomization pressure is 0.2 - 0.4 MPa.

[0017] The present invention has the following remarkable technical effects: Through the synephrine-binding induction mechanism, the binding affinity between the natural active ingredient and DPP-4 is significantly improved, making the binding energy reach or exceed the level of the synthetic inhibitor sitagliptin (-10.3 kcal / mol), fundamentally solving the key technical problem of insufficient activity of natural inhibitors; Compared with synthetic inhibitors such as sitagliptin (IC50 is approximately 19 nM), the composite preparation of the present invention improves the DPP-4 inhibition efficiency of the natural active ingredient to the level required for clinical application (IC50 is approximately 28.7 nM) through the binding induction of synephrine, while maintaining better safety and selectivity; The micronization preparation technology based on the principle of molecular interaction ensures the physical and chemical stability, molecular uniformity, and bioavailability of the preparation, and is suitable for the development of various clinical dosage forms. The composite preparation can be made into dosage forms such as tablets, capsules, granules, or oral liquids.

[0018] This technological breakthrough provides a feasible path for the development of new diabetes treatment drugs based on natural ingredients, and at the same time has a cardiovascular protection effect, providing a new option for the comprehensive management of diabetes and its complications. Description of the Drawings

[0019] Figure 1:Schematic diagram of the synergistic molecular binding of synephrine with pterostilbene, quercetin, and naringin. A, Schematic molecular structures of synephrine, pterostilbene, quercetin, and naringin; B, Molecular docking model of the synergistic binding of synephrine and pterostilbene to the DPP-4 active site; C, Molecular docking model of the synergistic binding of synephrine and quercetin to the DPP-4 active site; D, Molecular docking model of the synergistic binding of synephrine and naringin to the DPP-4 active site. Detailed implementation method

[0020] Example 1: Verification of the enhancing effect of synephrine as a binding inducer on the binding energy of traditional Chinese medicine active ingredients to DPP-4 In this example, through computer-aided molecular docking technology and in vitro enzyme activity assay methods, the enhancing effect of synephrine as a binding inducer on the binding affinity of pterostilbene, quercetin, and naringin to DPP-4 enzyme was systematically verified.

[0021] 1.1 Materials and reagents Synephrine (purity ≥ 99.0%), pterostilbene (purity ≥ 98.5%), quercetin (purity ≥ 98.0%), and naringin (purity ≥ 97.0%) used in the experiment were all purchased from Sigma-Aldrich; recombinant human DPP-4, DPP-8 / 9 enzymes (specific activity ≥ 25 U / mg) were purchased from R&D Systems; the fluorescent substrate Gly-Pro-AMC (purity ≥ 98.0%) was purchased from Bachem. All reagents were of analytical grade or higher purity.

[0022] 1.2 Molecular docking method Molecular docking studies were carried out using AutoDock Vina 1.2.0 software. The crystal structure of human DPP-4 (PDB ID: 5t4e) was used as the receptor model, and synephrine, pterostilbene, quercetin, and naringin were used as ligands respectively. First, the binding energies of each active ingredient to DPP-4 were evaluated separately, and then the changes in the binding energies of the ternary complexes formed after adding synephrine were evaluated under optimized conformational conditions.

[0023] 1.3 Binding energy measurement results (see attachment Figure 1 ) The binding energy of pterostilbene alone to DPP-4 was -7.50 ± 0.11 kcal / mol, and it was significantly enhanced to -10.77 ± 0.09 kcal / mol in the presence of synephrine; The binding energy of quercetin alone to DPP-4 was -8.30 ± 0.13 kcal / mol, and it was significantly enhanced to -11.54 ± 0.08 kcal / mol in the presence of synephrine; The binding energy of naringin alone with DPP-4 was -9.90 ± 0.15 kcal / mol, and in the presence of synephrine, the binding energy was significantly enhanced to -12.04 ± 0.10 kcal / mol.

[0024] 1.4 Determination of DPP-4 enzyme inhibitory activity An enzyme activity assay method was established using the fluorescent substrate Gly-Pro-AMC (λex = 380 nm, λem = 460 nm) and carried out in a 96-well black microplate according to the literature method. The reaction system (100 μL) contained: 50 mM HEPES buffer (pH 7.4), 0.1 mg / mL BSA, 100 mM NaCl, 50 μM Gly-Pro-AMC, 2.5 ng recombinant human DPP-4, and different concentrations of the test compound. After incubation at 37°C for 60 minutes, the inhibition rate of each component alone and in combination on DPP-4 enzyme activity was measured using a SpectraMax M5 multi-functional microplate reader, and the IC50 value was calculated: Synephrine: The IC50 value > 1000 μM, and no obvious inhibitory activity was shown within the test concentration range; Pterostilbene: The IC50 value was 100.3 ± 4.7 μM; Quercetin: The IC50 value was 203.5 ± 9.2 μM; Naringin: The IC50 value was 452.8 ± 17.6 μM; After synephrine was compounded with pterostilbene, quercetin, and naringin at the optimal mass ratio (1:1.5:1:1.8): The IC50 value was 28.7 ± 2.1 nM; Positive control sitagliptin: The IC50 value was 25.7 ± 1.3 nM.

[0025] Example 2: Preliminary screening study on the DPP-4 inhibitory activity of synephrine compound preparation The purpose of this example was to study the effect of the ratio of synephrine to the other three natural active ingredients on DPP-4 inhibitory activity by systematically optimizing the ratio, so as to provide a scientific basis for the formulation design and development of compound preparations.

[0026] 2.1 Experimental method On the premise of ensuring the constant total mass of the active ingredients, six compound preparation formulas with different ratios were designed, numbered F1 - F6. The formula design was based on the mass ratio range of synephrine, pterostilbene, quercetin, and naringin of 1:(0.5 - 20.0):(0.5 - 20.0):(0.5 - 20.0), and the specific ratios are shown in the following table: 2.2 Experimental results According to the standard experimental procedures described in Example 1, the DPP-4 inhibitory activity of each formulation was determined, and its corresponding IC50 value was calculated. Meanwhile, surface plasmon resonance technology (SPR, Biacore T200) was used to accurately determine the kinetic parameters (ka, kd, and KD) of each formulation with DPP-4 to comprehensively evaluate the correlation between its binding affinity and inhibitory activity, thereby verifying the scientificity and rationality of the formulation design. The following are the experimental results of each formulation: The results showed that formulation F2 had relatively high DPP-4 inhibitory activity (IC50 = 35.0 nM) and exhibited excellent binding affinity and kinetic parameters, verifying its scientificity and rationality in the compound preparation.

[0027] Example 3: Confirmation of the optimized formulation F0 of the DPP-4 inhibitor compound preparation Based on Example 2, this example aims to conduct an optimization study on the formulation of the compound preparation through the orthogonal experimental design method to obtain the optimal component ratio with the best DPP-4 inhibitory activity.

[0028] 3.1 Formulation optimization design The L9(34) orthogonal experimental design was adopted, with synephrine (A), pterostilbene (B), quercetin (C), and naringin (D) as the key factors to be investigated. Each factor was set at 3 levels, namely 0.5 times, 1.5 times, and 3 times. Taking the inhibitory activity (IC50 value) of the compound preparation against DPP-4 as the evaluation index, the influence degree of each factor on the activity was analyzed by analysis of variance (ANOVA) and range analysis to determine the optimal formulation combination.

[0029] Test temperature: Controlled at 25 ± 2 °C to ensure data reproducibility; Test design: Each formulation was independently prepared 3 times, and the activity of each prepared sample was measured 3 times; Orthogonal design table: The mass ratios of each factor (synephrine, pterostilbene, quercetin, and naringin) were arranged according to L9(34) to ensure the scientificity and representativeness of the experiment.

[0030] 3.2 Determination and verification of the best formulation Analysis of variance and significance test of the above orthogonal test results showed that the order of the influence of each component on DPP-4 inhibitory activity was B (pterostilbene) > D (naringin) > C (quercetin) > A (synephrine), and the F value of factor B reached a significant level (p < 0.05). After further optimization, the optimal mass ratio was determined to be synephrine:pterostilbene:quercetin:naringin = 1:1.5:1:1.8, denoted as the optimized formula F0. The composite preparation prepared with this optimized formula was assayed for DPP-4 enzyme activity, and its IC50 value was 28.7 ± 2.1 nM. Compared with when each component was used alone, the inhibitory activity was increased by at least 1000 times. Statistical analysis showed a significant synergistic effect (p < 0.001).

[0031] Example 4: Verification of the Synergistic Effect of Different Combinations of Active Ingredients To systematically verify the contributions and synergistic effects of each active ingredient, this example studied the DPP-4 inhibitory activities of different combinations of synephrine and other components.

[0032] 4.1 Experimental Method Seven different combination schemes were designed, namely: synephrine alone (A), synephrine + pterostilbene (AB), synephrine + quercetin (AC), synephrine + naringin (AD), synephrine + pterostilbene + quercetin (ABC), synephrine + pterostilbene + naringin (ABD), synephrine + quercetin + naringin (ACD), and all four components (ABCD). Each component was formulated according to the optimal ratio F0 (1:1.5:1:1.8), and the total mass concentration was kept consistent. The IC50 values of each combination against DPP-4 were determined using the method described in Example 1, and the synergy index (CI) was calculated: CI < 0.9 indicates a synergistic effect, CI = 0.9 - 1.1 indicates an additive effect, and CI > 1.1 indicates an antagonistic effect.

[0033] 4.2 Results and Analysis * The formula for calculating the synergy index (CI) is CI = (D1 / Dx1) + (D2 / Dx2) +... + (Dn / Dxn), where D1, D2...Dn are the concentrations of each component in the combination, and Dx1, Dx2...Dxn are the concentrations required for each component to produce the same effect when used alone. ** Relative activity: The relative ratio with the activity of the combination of all four components (ABCD) as 100%.

[0034] 4.3 Conclusion Experimental data show that in the two-component combination tests, the synergistic effects of synephrine with other components are all significant (CI values are all less than 0.9), among which the synergistic effect of synephrine and pterostilbene is the most prominent (CI = 0.82 ± 0.05); in the three-component combination tests, the synephrine-pterostilbene-quercetin (ABC) combination shows the optimal synergistic effect (CI = 0.64 ± 0.03) and significant relative activity (62.0% ± 3.7%); the four-component complete combination (ABCD) achieves the optimal effect (IC50 = 28.7 ± 2.1 nM) in the DPP-4 inhibitory activity assay and shows the most significant synergistic effect (CI = 0.45 ± 0.02). The above systematic research results fully confirm that the component ratio of the four-component compound preparation described in the present invention has clear theoretical basis and reliable experimental data support. This optimized formula not only verifies the scientificity of the synergistic effects among the components, but also provides the key technical basis for the optimization of the process parameters and industrial development of the compound preparation of the present invention.

[0035] Example 5: Research on the preparation process of micronized compound preparation 5.1 Preparation process Step 1: Weigh accurately 2000 mg (±0.1 mg) of synephrine, pterostilbene, quercetin and naringin. Step 2: Dissolve the above four active ingredients in suitable solvents respectively: synephrine is dissolved in ethanol solution filtered through a 0.22 μm filter membrane (prepare 100 ml, concentration is 20 mg / mL), pterostilbene and quercetin are dissolved in aqueous solution filtered through a 0.22 μm filter membrane (each prepare 100 ml, concentrations are each 20 mg / mL), and naringin is dissolved in ethanol-water (6:4, v / v) solution filtered through a 0.22 μm filter membrane (prepare 100 ml, concentration is 20 mg / mL). Step 3: Under nitrogen protection, mix the four solutions according to the solute ratios F0 (1:1.5:1:1.8), F1 (1:0.5:0.5:0.5), F4 (1:10:10:10), F6 (1:20:20:20), stir with a magnetic stirrer (300 rpm) for 45 minutes, and ultrasonically treat for 10 minutes (power 300 W, intermittent mode) until completely homogeneous. Step 4: Use a BÜCHI B-290 type spray drying equipment to prepare micronized powder under the following optimized parameter conditions: inlet temperature 130 ± 2 °C, outlet temperature 80 ± 2 °C, nozzle diameter 0.7 mm, atomization air pressure 0.22 MPa, feeding rate 4 mL / min, air extraction rate 32 m³ / h. Step 5: Transfer the obtained powder to a vacuum dryer, vacuum dry at 40 ± 1 °C for 12 hours (vacuum degree ≤ 50 Pa), cool to room temperature and then seal and store to obtain a standardized micronized preparation, with a total yield of 89.6 ± 1.5%.

[0036] 5.2 Physical Property Characterization The prepared composite preparation micropowders were comprehensively characterized using a laser particle size analyzer (Malvern Mastersizer 3000), gas adsorption method (Micromeritics ASAP 2460), powder density analyzer (ERWEKA SVM 121), and powder flowability analyzer (ERWEKA GTB). The results showed that: All formulations indicated that the micropowders had good flowability and compression properties, suitable for subsequent formulation processing.

[0037] Example 6: Stability Study of the Composite Preparation According to the requirements of the ICH Q1A(R2) guideline, the prepared composite preparation micropowders (F0(1:1.5:1:1.8), F1(1:0.5:0.5:0.5), F4(1:10:10:10), and F6(1:20:20:20)) were dispensed into 10 mL brown glass bottles and placed under different stability test conditions: long-term stability (25 ± 2°C / 60 ± 5%RH), intermediate stability (30 ± 2°C / 65 ± 5%RH), and accelerated stability (40 ± 2°C / 75 ± 5%RH). At time points of 0, 1, 3, and 6 months, the contents of each active ingredient were detected using a validated HPLC-MS / MS method (Agilent 1290-6460), and the DPP-4 inhibitory activity was also determined. The results showed that after storage at 25°C / 60%RH for 6 months, the contents of synephrine, pterostilbene, quercetin, and naringin in the four formulations were 98.3%, 96.8%, 95.7%, and 97.2% of the original, respectively, meeting the quality standard (≥90.0%); the retention rate of DPP-4 inhibitory activity was 94.8% with no significant decrease (p > 0.05). Even after storage at accelerated conditions (40°C / 75%RH) for 3 months, the contents of each component remained above 92.0%, indicating that the composite preparation had good chemical and functional stability.

[0038] Example 7: Preparation and Quality Control of the Synephrine Composite Preparation Capsule Dosage Form Based on the composite preparation micropowders F0(1:1.5:1:1.8), F1(1:0.5:0.5:0.5), F4(1:10:10:10), and F6(1:20:20:20) prepared in Example 5, this example developed a capsule dosage form suitable for oral administration and established a complete quality control system.

[0039] 7.1 Capsule Formulation 7.2 Preparation Method Step 1: Pass the compound preparation micropowder, microcrystalline cellulose, hypromellose, and crospovidone through a 40-mesh sieve. After mixing evenly, granulate them by wet granulation with a 60-mesh sieve (the binder is a 5% aqueous HPMC solution). Step 2: Dry the obtained granules in an oven at 50°C until the water content does not exceed 3.0%. Step 3: Add silicon dioxide and magnesium stearate, and mix them in a three-dimensional mixer for 10 minutes. Step 4: Fill them into No. 1 hard capsules with an automatic capsule filling machine, and control the single-dose weight at 400 ± 20 mg.

[0040] 7.3 Quality Indicators and Analytical Methods Establish the following quality control standards according to the Chinese Pharmacopoeia 2020 Edition and ICH guidelines: Assay: F0(1:1.5:1:1.8): synephrine 37.9 mg / capsule, pterostilbene 56.8 mg / capsule, quercetin 37.9 mg / capsule, naringin 68.2 mg / capsule; F1(1:0.5:0.5:0.5): synephrine 80.0 mg / capsule, pterostilbene 40.0 mg / capsule, quercetin 40.0 mg / capsule, naringin 40.0 mg / capsule; F4(1:10:10:10): synephrine 6.5 mg / capsule, pterostilbene 64.5 mg / capsule, quercetin 64.5 mg / capsule, naringin 64.5 mg / capsule; F6(1:20:20:20): synephrine 3.3 mg / capsule, pterostilbene 65.6 mg / capsule, quercetin 65.6 mg / capsule, naringin 65.6 mg / capsule; Determined by HPLC-DAD method, chromatographic conditions: Agilent ZORBAX SB-C18 column (4.6×250mm, 5μm), gradient elution, flow rate 1.0 mL / min, column temperature 30°C, detection wavelengths: synephrine 275 nm, pterostilbene 320 nm, quercetin 370 nm, naringin 280 nm; Disintegration time: not exceeding 15 minutes; Dissolution: the dissolution of each component is not less than 80% within 45 minutes; Microbial limit: shall comply with the requirements of the General Principles of the Fourth Part of the Chinese Pharmacopoeia 2020 Edition; Heavy metals and residual solvents: comply with the requirements of ICH Q3 guidelines.

[0041] 7.4 Quality Evaluation Results The test results of three batches of samples (batch numbers: 20240501, 20240502, 20240503) showed that: For the F0 (1:1.5:1:1.8) formulation: the synephrine content was 36.8 - 38.5 mg per capsule, the pterostilbene content was 55.0 - 58.2 mg per capsule, the quercetin content was 36.5 - 38.9 mg per capsule, and the naringin content was 66.5 - 69.8 mg per capsule; For the F1 (1:0.5:0.5:0.5) formulation: the synephrine content was 78.5 - 81.2 mg per capsule, the pterostilbene content was 39.0 - 41.2 mg per capsule, the quercetin content was 38.5 - 41.0 mg per capsule, and the naringin content was 38.8 - 41.5 mg per capsule; For the F4 (1:10:10:10) formulation: the synephrine content was 6.3 - 6.7 mg per capsule, the pterostilbene content was 62.8 - 65.8 mg per capsule, the quercetin content was 62.5 - 66.0 mg per capsule, and the naringin content was 63.0 - 66.2 mg per capsule; For the F6 (1:20:20:20) formulation: the synephrine content was 3.2 - 3.5 mg per capsule, the pterostilbene content was 63.8 - 67.2 mg per capsule, the quercetin content was 63.5 - 67.0 mg per capsule, and the naringin content was 64.0 - 67.5 mg per capsule; The disintegration time of the capsules of the four formulations was 9.4 - 11.2 minutes; The dissolution rate at 45 minutes was 84.3 - 89.6%; The microbial limit and heavy metal tests all met the requirements.

[0042] 7.5 Conclusion Based on the above experimental data, the following technical effects were obtained in Example 7 of the present invention: (1) The preparation process of the present invention has high stability and controllability. Through the test verification of three batches of samples, the content fluctuation ranges of synephrine, pterostilbene, quercetin, and naringin in each batch were strictly controlled within ±2.5%, fully confirming the reproducibility of the process; (2) The prepared capsule dosage form met the requirements of the pharmacopoeia standards and ICH guidelines in terms of key quality indicators such as disintegration time, dissolution rate, microbial limit, and heavy metal content; (3) Through the systematic study of the preparations with four different ratios (F0, F1, F4, F6), it was shown that the compound preparations exhibited good physical and chemical stability under different ratio conditions, and all the indicators of the prepared capsules met the quality standard requirements, verifying the scientificity and rationality of the formula design of the present invention; (4) The preparation process and quality control method adopted in the present invention are simple to operate, the process parameters are controllable, meeting the industrialization requirements, and having the technical basis for large-scale production. The above research results fully confirmed the innovation and practicality of the present invention in terms of preparation process, product quality, and large-scale production.

[0043] Example 8: In vitro inhibitory activity study of synephrine compound preparations with different formula ratios This example aims to systematically evaluate the differences in in vitro inhibitory activities and the characteristics of the mode of action between synephrine compound preparations with different formulation ratios and existing marketed DPP-4 inhibitors, in order to confirm the technical innovation points and substantial improvements of the present invention.

[0044] 8.1 Experimental design and methodology In this study, a variety of clinically common marketed DPP-4 inhibitory drugs (specific enzyme inhibitors) with a purity ≥ 95% were selected as control drugs, and were strictly compared in parallel with four synephrine compound preparations with different formulation ratios prepared in Example 3: F0 (synephrine:pterostilbene:quercetin:naringin = 1:1.5:1:1.8, batch number CF20240105), F1 (synephrine:pterostilbene:quercetin:naringin = 1:0.5:0.5:0.5, batch number CF20240106), F4 (synephrine:pterostilbene:quercetin:naringin = 1:10:10:10, batch number CF20240107), F6 (synephrine:pterostilbene:quercetin:naringin = 1:20:20:20, batch number CF20240108). Using the recombinant human DPP-4 enzyme inhibitory activity assay method described in Example 1, at pH 7.4 and 37 °C, the half-maximal inhibitory concentration (IC50) of each sample against DPP-4 was measured at 10 concentration gradients (0.1 nM - 100 μM), with 3 replicates for each concentration, and the experiment was repeated 3 times.

[0045] 8.2 Results and analysis Selectivity index value: IC50 of DPP-4 / IC50 of DPP-8 or 9 subtype.

[0046] The selectivity indices (132 - 183) of the four formulated synephrine compound preparations were all significantly higher than those of the commercially available DPP-4 inhibitors (42 - 68), indicating that this compound preparation has higher target selectivity for DPP-4, reducing the inhibition of homologous enzymes such as DPP-8 / 9, thereby reducing the risk of potential adverse reactions. As the ratios of pterostilbene, quercetin, and naringin increased, the selectivity index showed an upward trend, and the F6 formulation (1:20:20:20) had the highest selectivity index (183 ± 15).

[0047] 8.3 Study on inhibition persistence The enzyme-inhibitor complex dilution method was used to evaluate the inhibition persistence. After incubating DPP-4 with each inhibitor at 10×IC50 concentration for 30 minutes, it was rapidly diluted 100-fold to a concentration far lower than IC50, and the recovery of enzyme activity over time was measured. The results showed that: The inhibitory effects of sitagliptin and linagliptin synthetic inhibitors rapidly weakened within 30 - 60 minutes after dilution, and the activity recovery rate was 75 - 85%; The inhibitory effect of the F0(1:1.5:1:1.8) formulation remained after 240 minutes of dilution, and the activity recovery rate was 32%; The inhibitory effect of the F1(1:0.5:0.5:0.5) formulation remained after 240 minutes of dilution, and the activity recovery rate was 38%; The inhibitory effect of the F4(1:10:10:10) formulation remained after 240 minutes of dilution, and the activity recovery rate was 28%; The inhibitory effect of the F6(1:20:20:20) formulation remained after 240 minutes of dilution, and the activity recovery rate was 25%; All four formulations indicated that the synephrine compound preparation had significant long-acting inhibitory characteristics, and the F6 formulation had the best persistence.

[0048] 8.4 Conclusion Verified by the systematic research of this example, through the quantitative determination and comparative analysis of the in vitro inhibitory activity of the synephrine compound preparation of four formulations, the results showed that: Although the apparent IC50 value (28.7 - 84.5 nM) of the compound preparation was higher than that of the synthetic DPP-4 inhibitor (1 - 23 nM) in the prior art, its DPP-4 / DPP-8, 9 selectivity index (132 - 183) was significantly better than that of the prior art (58 - 63), and it showed significant inhibitory persistence under 100-fold dilution conditions (activity recovery rate of 25 - 38% at 240 min). Further research found that with the increase of the mass ratio of pterostilbene, quercetin and naringin to synephrine, the selectivity index and inhibitory persistence of the compound preparation showed a significant positive correlation. The above technical features fully confirmed the significant technical effects of the present invention in reducing the risk of long-term drug use adverse reactions and inhibiting the occurrence of drug resistance, and provided an innovative technical solution for the development of new DPP-4 inhibitors.

[0049] Example 9: Pharmacodynamic evaluation study on the ratio of F0 to F6 in db / db mouse model This example aimed to evaluate the in vivo pharmacodynamic characteristics of the synephrine compound preparation in the db / db diabetic mouse model and comprehensively verify its regulatory effects on key indicators such as blood glucose metabolism, GLP-1 level and insulin secretion.

[0050] 9.1 Experimental animals and grouping Forty-eight 8-week-old male db / db mice (C57BLKS / J-Leprdb / Leprdb), weighing 25 - 30 g, were purchased from a certain biotechnology company. Animal ethics: AECBNUZ2024010. After 1 week of adaptive feeding, they were randomly divided into 6 groups (n = 8) according to fasting blood glucose and body weight: Model control group: Given an equal volume of solvent Positive control group: Sitagliptin (10 mg / kg / d); F0 formulation group (1:1.5:1:1.8) (75 mg / kg / d); F0 formulation group (1:1.5:1:1.8) (150 mg / kg / d); F6 formulation group (1:20:20:20) (75 mg / kg / d); F6 formulation group (1:20:20:20) (150 mg / kg / d); Meanwhile, 8 C57BL / 6J mice of the same age were set as the normal control group. All animals were raised in an SPF-level environment, with free access to food and water, a light cycle of 12h / 12h, a temperature of 22±2°C, and a relative humidity of 55±5%.

[0051] 9.2 Administration protocol and index detection Continuous administration for 4 weeks, with intragastric administration once a day. The following indexes were monitored during this period: Basic physiological indexes: body weight change, food intake, water intake (recorded every 3 days); Blood glucose regulation: fasting blood glucose (measured once a week), 2-hour postprandial blood glucose (measured once a week), glycated hemoglobin HbA1c (measured at the beginning and end of the experiment); Oral glucose tolerance test (OGTT): Conducted in the 2nd and 4th weeks of the experiment, intragastrically administered with 2g / kg glucose solution, and blood samples were collected at 0, 15, 30, 60, 90, and 120 minutes to measure blood glucose, and the area under the blood glucose curve (AUC) was calculated; Insulin sensitivity: Insulin tolerance test (ITT) was conducted in the 4th week of the experiment. Insulin (0.75U / kg) was injected intraperitoneally, and blood samples were collected at 0, 15, 30, and 60 minutes to measure blood glucose, and the proportion of blood glucose decrease was calculated; GLP-1 level: At the end of the experiment, portal vein blood was collected 30 minutes after oral glucose load, and the active GLP-1 concentration was measured by ELISA method; Insulin secretion: At the end of the experiment, blood samples were collected 15 minutes and 30 minutes after oral glucose load to measure serum insulin levels; DPP-4 activity: At the end of the experiment, serum was collected to measure DPP-4 enzyme activity.

[0052] 9.3 Experimental results 9.3.1 Changes in basic physiological parameters *P<0.01 vs normal control group; #P<0.05 vs model control group.

[0053] The results showed that, compared with the model control group, all dose groups of the synephrine compound preparation could significantly relieve the symptoms of polyphagia, polydipsia and weight gain in db / db mice, and showed an obvious dose-dependence. The F0 formulation group had a significantly better effect than the F6 formulation group, indicating that a lower proportion of pterostilbene, quercetin and naringin might have a better balance and synergistic effect.

[0054] 9.3.2 Blood glucose regulation Fasting and postprandial blood glucose (at the end of the 4th week) *P<0.01 vs normal control group; #P<0.05 vs model control group.

[0055] Results of oral glucose tolerance test (OGTT) (at the 4th week) *P<0.01 vs normal control group; #P<0.05 vs model control group.

[0056] 9.3.3 GLP-1 and insulin-related indicators *P<0.01 vs normal control group; #P<0.05 vs model control group.

[0057] 9.5 Conclusion In this example, the in vivo pharmacodynamic characteristics of the synephrine compound preparation were systematically evaluated through a db / db diabetic mouse model, and the experimental results showed that: After 4 weeks of drug administration, the glycemic metabolism indexes of the experimental group mice were significantly improved, manifested as a decrease in fasting blood glucose (21.46±2.15 mmol / L vs 12.59±1.35 mmol / L, P<0.05), a decrease in postprandial blood glucose (28.73±2.54 mmol / L vs 16.93±1.77 mmol / L, P<0.05), and a decrease in glycated hemoglobin level (10.83±0.97% vs 7.42±0.68%, P<0.05); Mechanism studies showed that the compound preparation improved pancreatic islet β-cell function by significantly inhibiting the activity of DPP-4 enzyme (inhibition rate 79.3±7.6%), thereby increasing the activity of portal vein GLP-1 (22.85±2.31 pmol / L) and postprandial insulin level (1.39±0.16 ng / mL); The dose-effect relationship study confirmed that the F0 formulation (synephrine:pterostilbene:quercetin:naringin = 1:1.5:1:1.8) had a better hypoglycemic effect than the F6 formulation (1:20:20:20), and the pharmacodynamic indexes of its high-dose group (150 mg / kg / d) were comparable to those of the positive control sitagliptin (10 mg / kg / d); Statistical analysis results showed that the high-dose group of F0 formulation (150 mg / kg / d) was comparable to the clinical first-line DPP-4 inhibitor sitagliptin (10 mg / kg / d) in terms of blood glucose control, GLP-1 level elevation, and insulin secretion promotion (P>0.05). It is worth noting that although the component ratios of each group in the F6 formulation were relatively high, it still exhibited definite hypoglycemic activity and DPP-4 inhibitory effects.

[0058] Example 10: Pharmacodynamic evaluation study on different formulation ratios in db / db mouse models This example aimed to evaluate the in vivo pharmacodynamic characteristics of synephrine compound preparations with different ratios in db / db diabetic mouse models and comprehensively verify their regulatory effects on blood glucose metabolism.

[0059] 10.1 Experimental animals and grouping Sixty-four 8-week-old male db / db mice (C57BLKS / J-Leprdb / Leprdb), weighing 25-30 g, were purchased from a certain biotechnology company. Animal ethics: AECBNUZ2024011. After 1 week of adaptive feeding, they were randomly divided into 8 groups (n = 8) according to fasting blood glucose and body weight: Model control group: Given an equal volume of solvent Positive control group: Sitagliptin (10 mg / kg / d); F1 formulation group (1:0.5:0.5:0.5) (150 mg / kg / d); F0 formulation group (1:1.5:1:1.8) (150 mg / kg / d); F2 formulation group (1:1:1:1) (150 mg / kg / d); F3 formulation group (1:5:5:5) (150 mg / kg / d); F4 formulation group (1:10:10:10) (150 mg / kg / d); F5 formulation group (1:15:15:15) (150 mg / kg / d); F6 formulation group (1:20:20:20) (150 mg / kg / d).

[0060] Meanwhile, 8 C57BL / 6J mice of the same age were set as the normal control group. All animals were raised in an SPF-level environment, with free access to food and water, a light cycle of 12h / 12h, a temperature of 22±2°C, and a relative humidity of 55±5%.

[0061] 10.2 Administration regimen and index detection Continuous administration for 4 weeks, with gavage once a day. The following indexes were detected: Fasting blood glucose (measured once a week); 2-hour postprandial blood glucose (measured once a week); Glycated hemoglobin HbA1c (measured at the end of the experiment).

[0062] 10.3 Experimental results 10.3.1 Blood glucose control effect *P < 0.01 vs normal control group; #P < 0.05 vs model control group.

[0063] 10.4 Conclusions Data analysis of the research according to this example shows that: After statistical analysis, each formulation group of the compound preparation showed blood glucose regulation activity with statistical significance (P < 0.05); Among them, the F0 formulation (synephrine:pterostilbene:quercetin:naringin = 1:1.5:1:1.8) showed the best hypoglycemic efficacy, and its therapeutic effect was comparable to that of the clinical control drug sitagliptin (10 mg / kg / d); Through systematic evaluation, it was found that with the decrease in the proportion of the synephrine component (F2 - F6 formulation series), the hypoglycemic activity of the compound preparation showed a significant dose-dependent decreasing trend; Experimental data further confirmed that when the excipient ratio was lower than the optimal ratio (such as the F1 formulation), its therapeutic effect was significantly lower than that of the F0 formulation, indicating that the synergistic effect of synephrine and other active ingredients has a key impact on the overall pharmacodynamic effect of the compound preparation.

[0064] Example 11: Safety evaluation This example mainly evaluated the safety indicators of four formulations (F0, F1, F4, F6) of the synephrine compound preparation in db / db diabetic mice at different dose levels, including liver and kidney functions, hematological parameters, and other biochemical indicators.

[0065] 11.1 Experimental design The grouping scheme described in Example 9 was followed, including a normal control group, a model control group, a sitagliptin group (10 mg / kg / d), an F0 formulation group (1:1.5:1:1.8), an F1 formulation group (1:0.5:0.5:0.5), an F4 formulation group (1:10:10:10), and an F6 formulation group (1:20:20:20). Each formulation group was divided into a low-dose group (50 mg / kg / d), a medium-dose group (100 mg / kg / d), and a high-dose group (150 mg / kg / d). At the end of the 4-week drug administration, blood samples of the mice were collected to measure liver and kidney functions and blood biochemical indicators.

[0066] 11.2 Biochemical safety indicators *P < 0.05 vs normal control group; #P < 0.05 vs model control group.

[0067] 11.3 Electrolytes and hematological parameters 11.4 Cardiac safety evaluation During the administration period, the changes in electrocardiogram and blood pressure of mice were monitored, and no obvious abnormalities were observed. No obvious prolongation of QT interval or other electrocardiogram abnormalities were found in all formulation groups and at each dose level. *P < 0.05 vs normal control group; #P < 0.05 vs model control group.

[0068] 11.5 Dose-dependent safety analysis To comprehensively evaluate the safety of synephrine compound preparations, the safety indexes of four formulations at different doses (50, 100, 150 mg / kg / d) were systematically analyzed in this experiment. The results showed that no obvious dose-dependent toxic reactions occurred at each dose level of all formulations. There were no statistical differences in safety parameters between the high-dose group (150 mg / kg / d) and the low-dose group (50 mg / kg / d) (P > 0.05). Formulation F0 showed obvious protective effects on liver and kidney functions and cardiovascular system at each dose level, and the effects were the best in the high-dose group.

[0069] 11.6 Conclusion Liver and kidney functions: None of the synephrine compound preparation formulations caused significant liver and kidney function damage. Among them, formulation F0 (1:1.5:1:1.8) had obvious improvement effects on the liver and kidney indexes of diabetic mice at each dose level, and showed a dose-dependent effect. Formulation F4 (1:10:10:10) was the second, and formulations F1 (1:0.5:0.5:0.5) and F6 (1:20:20:20) had weaker effects; Hematological parameters: No obvious hematological abnormalities were observed during the administration period of the four formulations at each dose level, and there were no changes in the indexes related to bone marrow suppression. Among them, the parameters of the formulation F0 group were close to those of the normal control group at each dose, and the effects were the best in the high-dose group; Cardiovascular safety: No obvious electrocardiogram abnormalities occurred in all compound preparation formulation groups at each dose level. Among them, formulations F0 and F4 had obvious improvement effects on the blood pressure increase caused by diabetes, and the effects were close to those of sitagliptin, and the effects were enhanced with the increase of dose; Dose-dependence: None of the four formulations showed obvious dose-dependent toxic reactions, and the safety window was good. All safety indexes were within the acceptable range at the highest dose (150 mg / kg / d). In particular, formulation F0 still maintained excellent safety and improvement effects at high doses.

[0070] The results of Example 11 showed that the four synephrine compound formulations all exhibited good safety profiles within the experimental dose range (50 - 150 mg / kg / d), and no obvious organ toxicity or adverse reactions were observed. Through systematic evaluation, the safety parameters of the F0 formulation (synephrine:pterostilbene:quercetin:naringin = 1:1.5:1:1.8) in Example were better than those of other formulations at each dose level, and it had significant hypoglycemic activity (P < 0.05). At the same time, it had a statistically significant improvement effect on liver and kidney function abnormalities and elevated blood pressure related to diabetes (P < 0.05). It was speculated that this mechanism of action might be related to its cardiovascular protection effect. The comprehensive evaluation of the F4 formulation (1:10:10:10) was second, while the F1 formulation (1:0.5:0.5:0.5) and F6 formulation (1:20:20:20) had acceptable safety, but their therapeutic effects were relatively weak. The dose escalation test data showed that within the dose range studied in this example, the four formulations all exhibited good safety windows, and no obvious dose-dependent toxic reactions were observed. The above experimental data fully confirmed that the component ratios of the synephrine compound formulation of the present invention had a significant impact on its efficacy and safety. Among them, the F0 formulation had outstanding safety and efficacy advantages as a new type of diabetes treatment agent, and good safety parameters could still be maintained at higher treatment dose levels.

[0071] Example 12 Study on the Influence of Complex Formation Conditions of F0 and F6 Groups To study the influence of pH and temperature on the formation of synephrine compound formulations of F0 group (1:1.5:1:1.8) and F6 group (1:20:20:20), the following detailed experiments were carried out: 12.1 Influence of pH Conditions Under the condition of 37°C, the formation of complexes in the pH range of 5.0 - 8.0 was investigated by dynamic light scattering method: F0 group, pH 5.0 - 6.0: The complex formation rate was moderate. After 24 h, the complexation rate reached 75 ± 5%, and about 20 ± 3% of free synephrine was detected in the solution. F6 group, pH 5.0 - 6.0: The complex formation rate was slow. After 24 h, the complexation rate was only 55 ± 5%, and about 40 ± 3% of free synephrine was detected in the solution; F0 group, pH 6.8 ± 0.2: The complex formation was the most complete, and the complexation rate could reach 95 ± 3% within 4 h, with a uniform particle size distribution (PDI < 0.2). F6 group, pH 6.8 ± 0.2: The complex formation was good, and the complexation rate reached 85 ± 3% within 4 h, with a relatively uniform particle size distribution (PDI < 0.3); Group F0 at pH 7.5 - 8.0: Slight precipitation occurred. The 72 - h stability test showed that the complex content decreased by 10 ± 2%, with a small amount of precipitation. Group F6 at pH 7.5 - 8.0: Obvious precipitation occurred. The 72 - h stability test showed that the complex content decreased by 20 ± 2%, with a large amount of precipitation formed.

[0072] 12.2 Influence of temperature conditions Under the condition of pH 6.8, the influence of different temperatures on the formation of the complex was studied by high - performance liquid chromatography: At 25 °C: Group F0: The complex formation rate was medium, and the complexation rate was 80 ± 4% after 48 h. Group F6: The complex formation rate was slow, and the complexation rate was 65 ± 4% after 48 h; At 37 ± 1 °C: Group F0: The complex formation rate was moderate, reaching a complexation rate of 95 ± 2% in 4 h, and having good stability for 30 days (degradation < 2%). Group F6: The complex formation rate was relatively slow, reaching a complexation rate of 82 ± 2% in 4 h, and having general stability for 30 days (degradation < 5%); At 45 °C: Group F0: The complexation rate reached 97 ± 2% within 2 h, and the degradation rate of pterostilbene was 5 ± 1% after 12 h. Group F6: The complexation rate reached 93 ± 2% within 2 h, and the degradation rate of pterostilbene reached 12 ± 1% after 12 h; The above research results show that for both Group F0 and Group F6, pH 6.8 ± 0.2 and temperature 37 ± 1 °C are the optimal preparation conditions. Under these conditions, Group F0 shows more excellent formation kinetics (complexation rate > 95% within 4 h) and thermodynamic stability (degradation rate < 2% in 30 days), while the indicators of Group F6 are relatively poor.

[0073] Example 13 Particle size and particle size distribution study of Group F0 and Group F6 The particle size analysis of the composite preparations of Group F0 and Group F6 under different preparation process conditions was carried out using a laser particle size analyzer (Malvern Mastersizer 3000). Samples with different particle size ranges were obtained by adjusting the atomization pressure (0.2 - 0.4 MPa) and the temperature difference between the inlet and outlet (ΔT = 30 - 60 °C) of spray drying. The dissolution rate was measured using a USP II dissolution tester (100 rpm, 37 ± 0.5 °C), and the bioavailability was evaluated in rats (n = 6). The main research results are as follows.

[0074] 13.1 Results of Group F0 (1:1.5:1:1.8): Sample group of 20 - 30 μm: Preparation conditions: Atomization pressure 0.4 MPa, ΔT = 60 °C; Fast dissolution rate (dissolution rate of 98% in 2 h), but uneven particle size distribution (span = 1.9); Bioavailability: 59.5 ± 14.2%, with significant individual differences (CV = 24%).

[0075] Sample group of 30 - 40 μm: Preparation conditions: atomization pressure 0.3 MPa, ΔT = 45 °C; Dissolution rate is moderate (dissolution rate at 4 h is 88.3 ± 2.8%); Particle size distribution is uniform (span = 1.38 ± 0.07); Bioavailability: 75.6 ± 7.2%, with small individual differences (CV = 9.5%).

[0076] Sample group of 40 - 50 μm: Preparation conditions: atomization pressure 0.2 MPa, ΔT = 30 °C; Dissolution rate is slow (dissolution rate at 4 h is 68%); Bioavailability: 48.3 ± 12.5%, with incomplete absorption.

[0077] Results of group F6 of 13.2 (1:20:20:20): Sample group of 20 - 30 μm: Preparation conditions: atomization pressure 0.4 MPa, ΔT = 60 °C; Dissolution rate is fast (dissolution rate at 2 h is 92%), but particle size distribution is uneven (span = 1.7); Bioavailability: 55.1 ± 16.2%, with significant individual differences (CV = 26%).

[0078] Sample group of 30 - 40 μm: Preparation conditions: atomization pressure 0.3 MPa, ΔT = 45 °C; Dissolution rate is moderate (dissolution rate at 4 h is 82.9 ± 3.6%); Particle size distribution is uniform (span = 1.46 ± 0.11); Bioavailability: 70.0 ± 9.6%, with small individual differences (CV = 14%).

[0079] Sample group of 40 - 50 μm: Preparation conditions: atomization pressure 0.2 MPa, ΔT = 30 °C; Dissolution rate is slow (dissolution rate at 4 h is 62%); Bioavailability: 42.1 ± 14.9%, with incomplete absorption.

[0080] 13.3 Conclusion: The research shows that for the F0 group and the F6 group, the composite preparations with an average particle size of 30 - 40 μm and a uniform particle size distribution (span < 1.51) have the best dissolution characteristics and bioavailability, and the indicators of the F0 group are slightly better than those of the F6 group.

[0081] As described above, it is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present application should be covered within the protection scope of the present application.

Claims

1. A synephrine-inducible DPP-4 inhibitor compound preparation for the treatment of diabetes, characterized in that: The compound formulation comprises the binding inducer synephrine and the active ingredients pterostilbene, quercetin and naringin.

2. The composite formulation according to claim 1, characterized in that: The mass ratio of synephrine, pterostilbene, quercetin and naringin is 1:(0.5-20.0):(0.5-20.0):(0.5-20.0).

3. The composite formulation according to claim 1, characterized in that: The composite preparation is in the form of micro powder, and the micro powder of the composite preparation has an average particle size of 30-40 μm.

4. A method for preparing the composite formulation according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) preparing high-purity solutions of synephrine, pterostilbene, quercetin and naringin respectively; (2) mixing the above solutions in a predetermined mass ratio to form a uniformly dispersed molecular complex under specific temperature and pH conditions; (3) converting the mixture into a micronized powder by vacuum freeze drying or spray drying technology; (4) Conduct physical and chemical property determination and stability test on the prepared micropowder.

5. The method according to claim 4, characterized in that: The solvent in step (1) is selected from pharmaceutical grade water, ethanol or a mixture thereof, and the purity of the ethanol is not less than 98%.

6. The method according to claim 4, characterized in that: The specific temperature (37±1°C) and pH (6.8±0.2) conditions in step (2) are as follows.

7. The method according to claim 4, characterized in that: The process parameters of step (3) are: under vacuum freeze-drying conditions, the pre-freezing temperature is -40°C to -50°C, the vacuum degree is 10-100Pa, and the drying time is 24-48 hours; or under spray drying conditions, the inlet temperature is 120-140°C, the outlet temperature is 80-90°C, and the atomization pressure is 0.2-0.4MPa.

8. Use of the composite preparation according to any one of claims 1 to 3 or the preparation method according to any one of claims 4 to 7 in the preparation of a pharmaceutical preparation for preventing or treating diabetes.

Images