Application of hydroxypropyl-beta-cyclodextrin as synergistic antioxidant capacity component in preparation of phloretin preparation, phloretin preparation and preparation method of phloretin preparation

Abstract

The invention provides application of hydroxypropyl-beta-cyclodextrin serving as a synergistic antioxidant capacity component in preparation of a phloretin preparation, the phloretin preparation and a preparation method of the phloretin preparation. According to the phloretin preparation, a water saturation method is adopted, phloretin is wrapped in a cavity of hydroxypropyl-beta-cyclodextrin and interacts with the hydroxypropyl-beta-cyclodextrin through a hydrogen bond, so that an aromatic ring of the phloretin is located at one narrow end of the cavity of the hydroxypropyl-beta-cyclodextrin to form a clathrate compound, and the clathrate compound is used for preparing the phloretin preparation. According to the clathrate compound, the water solubility of the phloretin is greatly improved, so that the free radical scavenging capacity of the phloretin is improved, meanwhile, due to the action of the clathrate compound, the reducing capacity of the phloretin is greatly improved, and the oxidation resistance of the phloretin is improved.

Description

Technical Field

[0001] The present invention relates to the field of biochemical technology, and in particular to an application of hydroxypropyl-β-cyclodextrin as a component for enhancing antioxidant capacity in the preparation of a phloretin preparation, a phloretin preparation and a preparation method thereof. Background Art

[0002] Phloretin (PT) belongs to the dihydrochalcone class of small molecule compounds. Its chemical name is 3-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)-propanone, and its molecular formula is C 15 H 14 O5, with a molecular mass of 274.17 and a melting point between 270-272°C, is a light pink powder at room temperature. It is almost insoluble in water and easily soluble in organic reagents such as methanol, ethanol, and acetone.

[0003] PT has multiple biological activities, including hypoglycemic, anti-inflammatory, antioxidant, and whitening properties. Because PT can increase the activity of antioxidant enzymes in the body, such as superoxide dismutase, catalase, and glutathione peroxidase, it can effectively scavenge harmful free radicals and maintain intracellular redox balance. Studies have shown that PT significantly reduces intracellular reactive oxygen species and malondialdehyde levels, increases superoxide dismutase and glutathione peroxidase-1 activity, and restores mitochondrial membrane potential. Other studies have shown that PT can significantly increase catalase activity, thereby achieving antioxidant effects and reducing oxidative stress. Therefore, PT can be used in the prevention and adjuvant treatment of antioxidant-related diseases.

[0004] However, since PT is a poorly soluble small molecule with low solubility in water or physiological fluids, its solubility and dissolution rate are insufficient to meet the requirements for effective absorption in the body, resulting in low bioavailability. Specifically, the solubility of a drug is an important factor affecting its dissolution rate, and the speed of the dissolution rate is directly related to the absorption efficiency of the drug in the body. When the solubility of a drug is low, its dissolution rate will also slow down accordingly, which will prolong the absorption time of the drug in the gastrointestinal tract and limit absorption. In addition, due to low solubility, the distribution and metabolism of the drug in the body may also be affected, further affecting its efficacy and safety. Therefore, improving the solubility and dissolution rate of poorly soluble small molecule drugs is the key to improving their bioavailability.

[0005] To address this issue, researchers have conducted extensive research. For example, a Chinese patent with publication number CN118121544A discloses a phloretin-liposome composition based on a molecular inclusion complex, which contains embedded carrier particles and molecular inclusion complexes loaded on the embedded carrier particles; wherein the embedded carrier particles are liposomes; the molecular inclusion complex contains phloretin and cyclodextrin and / or cyclodextrin derivatives. The phloretin-liposome composition based on the molecular inclusion complex can achieve efficient and stabilized delivery of phloretin, which can not only effectively solve the pain points of phloretin in cosmetics, health foods, health products, medical beauty products and biopharmaceutical applications, but also improve the stability, compatibility and bioavailability of phloretin.

[0006] Although current research can improve the bioavailability of phloretin to a certain extent, thereby exerting its antioxidant properties, its oxidative properties still cannot meet the needs of high-efficiency antioxidant drugs. Summary of the Invention

[0007] The present invention aims to address the problem of insufficient antioxidant capacity of existing phloretin and provide a phloretin preparation, which improves the antioxidant performance of phloretin by encapsulating phloretin in the cavity of hydroxypropyl-β-cyclodextrin and forming an inclusion complex with hydroxypropyl-β-cyclodextrin.

[0008] According to a first aspect of the present invention, there is provided a use of hydroxypropyl-β-cyclodextrin as a component for enhancing antioxidant capacity in the preparation of a phloretin preparation.

[0009] As an optional embodiment, phloretin is encapsulated in the cavity of hydroxypropyl-β-cyclodextrin and cooperates with hydroxypropyl-β-cyclodextrin to form an inclusion complex, and the antioxidant property of phloretin is improved by the formed inclusion complex.

[0010] According to a second aspect of the present invention, a phloretin preparation is provided, which comprises phloretin (PT) and hydroxypropyl-β-cyclodextrin (HP-β-CD), wherein the phloretin is located in the cavity of hydroxypropyl-β-cyclodextrin, and the phloretin and hydroxypropyl-β-cyclodextrin cooperate to form an inclusion complex, and the antioxidant properties of the phloretin are improved by the formed inclusion complex.

[0011] As an optional embodiment, phloretin and hydroxypropyl-β-cyclodextrin form the inclusion complex through hydrogen bonding interaction.

[0012] As an optional embodiment, in the inclusion compound, the aromatic ring of phloretin is located at one end of the narrow side of the hydroxypropyl-β-cyclodextrin cavity.

[0013] As an optional embodiment, in the inclusion complex, the molar ratio of phloretin to hydroxypropyl-β-cyclodextrin is 1:1.

[0014] According to a third aspect of the present invention, there is provided a method for preparing the aforementioned phloretin preparation, comprising the following steps:

[0015] Dissolving hydroxypropyl-β-cyclodextrin in ultrapure water to form a hydroxypropyl-β-cyclodextrin solution; dissolving phloretin in anhydrous ethanol to form a phloretin solution;

[0016] The phloretin solution is added to the hydroxypropyl-β-cyclodextrin solution, and the mixture is stirred to react, so that the phloretin enters the cavity of the hydroxypropyl-β-cyclodextrin, and the phloretin and the hydroxypropyl-β-cyclodextrin cooperate to form an inclusion complex;

[0017] After the reaction, the mixture was cooled to room temperature and refrigerated in a 4°C refrigerator for 24 h, and then the solvent was removed by rotary evaporation and vacuum dried. The free phloretin was then washed to remove the free phloretin, dried, and sieved to obtain phloretin-hydroxypropyl-β-cyclodextrin inclusion complex (PT-CDs) powder.

[0018] As an optional embodiment, the mass ratio of phloretin to hydroxypropyl-β-cyclodextrin is 1:(4-12).

[0019] As an optional embodiment, the phloretin solution is added to the hydroxypropyl-β-cyclodextrin solution, and the stirring reaction conditions include: stirring at a temperature of 35-55° C. and a rotation speed of 250-550 rpm for 90-150 min.

[0020] As an optional embodiment, the conditions for removing the solvent by rotary evaporation include: rotary evaporation at a temperature of 60 to 80° C. and a rotation speed of 80 to 120 rpm.

[0021] Compared with the prior art, the present invention has the following significant beneficial effects:

[0022] The present invention adopts a water saturation method to wrap phloretin in the cavity of hydroxypropyl-β-cyclodextrin, and interacts with hydroxypropyl-β-cyclodextrin through hydrogen bonds, so that the aromatic ring of phloretin is located at the narrow end of the hydroxypropyl-β-cyclodextrin cavity to form an inclusion compound, so that the water solubility of phloretin is greatly improved, thereby improving the free radical scavenging ability of phloretin. At the same time, due to the action of the inclusion compound, the reducing ability of phloretin is greatly improved. In this way, the antioxidant performance of phloretin is improved through the inclusion of hydroxypropyl-β-cyclodextrin. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is a schematic structural diagram of the inclusion compound PT-HP-β-CD formed by the coordination of phloretin and hydroxypropyl-β-cyclodextrin according to the present invention.

[0024] Figure 2It is the influence of each factor on the comprehensive score of the inclusion compound in the single factor experiment in the example of the present invention; wherein A is the preparation ratio, B is the preparation time, C is the preparation temperature, D is the preparation speed; E is the rotary evaporation speed, and F is the rotary evaporation temperature.

[0025] Figure 3 These are the characterization spectra of PT and PT-HP-β-CD in the examples of the present invention; wherein A is FT-IR, B is PXRD, C is TGA, and D is DSC.

[0026] Figure 4 1 is the SEM image of PT and PT-HP-β-CD in the examples of the present invention; wherein A is PT, B is HP-β-CD, C is PM, and D is PT-HP-β-CD.

[0027] Figure 5 is a structural exploration test diagram of PT-HP-β-CD in the example of the present invention; wherein A is 1 H NMR spectrum, B is the 2D NOESY spectrum, C is the Job's plot, and D is the inclusion structure diagram.

[0028] Figure 6 This is a powder dissolution test diagram of PT and PT-HP-β-CD in the example of the present invention; wherein A is ultrapure water, B is PBS solution, C is pH = 6.5 buffer solution, and D is pH = 1.2 buffer solution.

[0029] Figure 7 This is a pH conversion dissolution test chart of the PT-HP-β-CD tablet in the example of the present invention.

[0030] Figure 8 1 is a statistical graph of Caco-2 cell survival rate in an example of the present invention; wherein A is PT, B is PT-HP-β-CD, C is indomethacin, D is verapamil, E is reserpine, and F is EDTA.

[0031] Figure 9 This is a statistical graph of the resistance of Caco-2 monolayer cells after 21 days in an example of the present invention.

[0032] Figure 10 is the P of PT and PT-HP-β-CD at different temperatures in the present invention. app Value; where A is PT and B is PT-HP-β-CD.

[0033] Figure 11 In the present invention, PT and PT-HP-β-CD were added with different efflux protein inhibitors. app Value; where A is PT and B is PT-HP-β-CD.

[0034] Figure 12is the Ka value of PT and PT-HP-β-CD unidirectional intestinal perfusion in the example of the present invention; wherein A is the ileum and B is the colon.

[0035] Figure 13 are the Ka values ​​of PT and PT-HP-β-CD in the examples of the present invention when different efflux protein inhibitors are added; wherein A is PT and B is PT-HP-β-CD.

[0036] Figure 14 This is a test chart of PT and PT-HP-β-CD scavenging free radicals in the examples of the present invention; wherein A is the ABTS method and B is the DPPH method.

[0037] Figure 15 This is a test chart of the reduction performance of PT and PT-HP-β-CD in the examples of the present invention. DETAILED DESCRIPTION

[0038] In order to better understand the technical content of the present invention, specific embodiments are given and described below with reference to the accompanying drawings.

[0039] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which a number of illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to be comprehensive. It should be understood that the various concepts and embodiments described above, as well as those described in more detail below, can be implemented in any of a number of ways.

[0040] [Phloretin preparations]

[0041] In an exemplary embodiment of the present invention, a phloretin preparation is provided. The preparation comprises phloretin (PT) and hydroxypropyl-β-cyclodextrin (HP-β-CD).

[0042] Combine Figure 1 As shown, phloretin is located in the cavity of hydroxypropyl-β-cyclodextrin, and the aromatic ring of phloretin is located at one end of the narrow side of the hydroxypropyl-β-cyclodextrin cavity. Through hydrogen bond interaction, phloretin and hydroxypropyl-β-cyclodextrin cooperate to form an inclusion complex, and the antioxidant properties of phloretin are improved by the formed inclusion complex.

[0043] In an optional example, in the formed inclusion compound structure, the molar ratio of phloretin to hydroxypropyl-β-cyclodextrin is 1:1.

[0044] In an optional example, the encapsulation efficiency of phloretin by hydroxypropyl-β-cyclodextrin can reach over 90%.

[0045] [Preparation method]

[0046] In another exemplary embodiment of the present invention, there is provided a method for preparing the aforementioned phloretin preparation, comprising the following steps:

[0047] Dissolving hydroxypropyl-β-cyclodextrin in ultrapure water to form a hydroxypropyl-β-cyclodextrin solution; dissolving phloretin in anhydrous ethanol to form a phloretin solution;

[0048] Slowly adding the phloretin solution to the hydroxypropyl-β-cyclodextrin solution, stirring and reacting at a certain temperature, so that the phloretin enters the cavity of the hydroxypropyl-β-cyclodextrin and forms an inclusion complex with the hydroxypropyl-β-cyclodextrin;

[0049] After the reaction, the mixture was cooled to room temperature and refrigerated in a refrigerator at 4°C for 24 hours, and then the solvent was removed by rotary evaporation and vacuum drying. The mixture was then washed several times with anhydrous ethanol, acetone and petroleum ether to remove free phloretin. The mixture was dried in a vacuum drying oven again to obtain a loose coarse powder, which was then ground and sieved to obtain phloretin-hydroxypropyl-β-cyclodextrin inclusion complex (PT-CDs) powder.

[0050] In an optional example, the mass ratio of phloretin to hydroxypropyl-β-cyclodextrin is 1:(4-12), particularly preferably 1:(8-12), wherein the most preferred mass ratio is 1:(10-11).

[0051] In an optional example, the phloretin solution is added to the hydroxypropyl-β-cyclodextrin solution, and the stirring reaction conditions include: stirring at a temperature of 35 to 55° C. and a rotation speed of 250 to 550 rpm for 90 to 150 minutes.

[0052] In one preferred example, the stirring reaction conditions are: stirring at a temperature of 45 to 50° C. and a rotation speed of 400 to 410 rpm for 120 to 130 minutes.

[0053] In an optional example, the conditions for removing the solvent by rotary evaporation include: rotary evaporation at a temperature of 60 to 80° C. and a rotation speed of 80 to 120 rpm.

[0054] In one preferred example, the conditions for removing the solvent by rotary evaporation are: rotary evaporation at a temperature of 70° C. and a rotation speed of 100 rpm.

[0055] It can be understood that in the preparation process of phloretin preparations, the optimization of preparation conditions can be achieved by using commonly used technical means in this field. For example, the extreme value is first determined by a single-factor experiment, and then the Plackeet-Burman experiment Design-Expert13 software is used for analysis to screen out four more significant influencing factors: preparation temperature, preparation time, preparation speed, and preparation time. Then, a Box-Behnken design of a four-factor three-level experiment is performed, and the optimal preparation conditions for the inclusion of PT and HP-β-CD are obtained using Design-Expert 13 software analysis. Finally, through the verification experiment, the response surface optimization model is successful, and the optimal preparation conditions are obtained; non-significant influencing factors can be determined by single-factor experiments; among them, the evaluation index is a comprehensive score based on the encapsulation rate and yield.

[0056] In another exemplary example of the present invention, there is also provided an application of hydroxypropyl-β-cyclodextrin as a synergistic antioxidant capacity ingredient in the preparation of phloretin preparations. Phloretin is encapsulated in the cavity of hydroxypropyl-β-cyclodextrin and combined with hydroxypropyl-β-cyclodextrin to form an inclusion complex, thereby improving the antioxidant properties of phloretin through the formed inclusion complex.

[0057] For better understanding, the present invention is further described below with reference to several specific examples, but the preparation process is not limited thereto, and the content of the present invention is not limited thereto.

[0058] Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0059] The following examples were prepared as follows:

[0060] Dissolving hydroxypropyl-β-cyclodextrin in ultrapure water to form a hydroxypropyl-β-cyclodextrin solution; dissolving phloretin in anhydrous ethanol to form a phloretin solution;

[0061] The phloretin solution was slowly added to the hydroxypropyl-β-cyclodextrin solution, stirred and reacted at a certain temperature. After the reaction was completed, the mixture was cooled and allowed to stand at room temperature and refrigerated in a refrigerator at 4°C for 24 hours. The solvent was then removed by rotary evaporation and vacuum drying. The mixture was then washed several times with anhydrous ethanol, acetone and petroleum ether to remove free phloretin. The mixture was then dried in a vacuum drying oven to obtain a loose coarse powder, which was then ground and sieved to obtain phloretin-hydroxypropyl-β-cyclodextrin inclusion complex (PT-CDs) powder.

[0062] [Example 1]

[0063] {Single-factor experiment}

[0064] (1) Preparation ratio

[0065] Accurately weigh samples with a PT:HP-β-CD mass ratio of 1:4, 1:6, 1:8, 1:10, and 1:12, and maintain the preparation temperature at 45°C, the preparation speed at 400 rpm, the preparation time at 120 min, the rotary evaporation temperature at 70°C, and the rotary evaporation speed at 100 rpm.

[0066] (2) Preparation time

[0067] Accurately weigh a sample with a PT:HP-β-CD mass ratio of 1:4, maintain the preparation temperature at 45°C, the preparation speed at 400 rpm, the rotary evaporation temperature at 70°C, and the rotary evaporation speed at 100 rpm, and prepare the inclusion complex at different preparation times of 30, 60, 90, 120, and 150 min.

[0068] (3) Preparation temperature

[0069] Accurately weigh a sample with a PT:HP-β-CD mass ratio of 1:4. Keep the preparation speed at 400 rpm, the preparation time at 120 min, the rotary evaporation temperature at 70°C, and the rotary evaporation speed at 100 rpm unchanged, and prepare the inclusion complex at different preparation temperatures of 35, 45, 55, 65, and 75°C.

[0070] (4) Preparation speed

[0071] Accurately weigh a sample with a PT:HP-β-CD mass ratio of 1:4, maintain the preparation temperature at 45°C, the preparation time at 120 min, the rotary evaporation temperature at 70°C, and the rotary evaporation speed at 100 rpm, and prepare the inclusion complex at preparation speeds of 250, 400, 550, 700, and 850 rpm.

[0072] (5) Rotary evaporation temperature

[0073] Accurately weigh a sample with a PT:HP-β-CD mass ratio of 1:4, maintain the preparation temperature at 45°C, the preparation time at 120 min, the preparation speed at 400 rpm, and the rotary evaporation speed at 100 rpm, and prepare the inclusion complex at rotary evaporation temperatures of 50, 60, 70, 80, and 90°C.

[0074] (6) Rotary evaporation speed

[0075] Accurately weigh a sample with a PT:HP-β-CD mass ratio of 1:4, maintain the preparation temperature at 45°C, the preparation time at 120 min, the preparation speed at 400 rpm, and the rotary evaporation temperature at 70°C, and prepare the inclusion complex at rotary evaporation speeds of 80, 90, 100, 110, and 120 rpm.

[0076] {Test Results}

[0077] Accurately weigh 10 mg of PT-CDs, add 10 mL of methanol and sonicate for 30 min. Remove and cool to room temperature. Determine the PT content in the inclusion complex using HPLC, and calculate the encapsulation efficiency and yield:

[0078] Encapsulation efficiency = measured PT content / input PT content × 100%

[0079] Yield = actual mass of PT-CDs obtained / total amount of PT and HP-β-CD input × 100%

[0080] The comprehensive score of yield and inclusion rate is used as the index for screening the optimal process conditions of inclusion compounds. Since the loss will have a certain impact during the preparation process, the two indicators are combined into a comprehensive score, and the weight coefficient is encapsulation rate: yield 0.8:0.2. The comprehensive score is calculated as follows:

[0081] Comprehensive score = encapsulation efficiency × 0.8 + yield × 0.2.

[0082] like Figure 2 The results of A preparation ratio showed that as the mass ratio of PT to HP-β-CD increased, the comprehensive score of the inclusion complex reached the highest when the mass ratio was 1:10; the results of preparation time showed that ( Figure 2 B): Encapsulation time ( Figure 2 C) reaches the highest comprehensive score when the inclusion compound is 120min; the preparation temperature results show that ( Figure 2 D): When the encapsulation temperature is 45℃, the comprehensive score of the inclusion compound reaches the maximum; the preparation speed results show that ( Figure 2 D): The highest comprehensive score is obtained at 400 rpm; the rotary evaporation speed results show that ( Figure 2 E): The highest comprehensive score is obtained at 100 rpm; the rotary evaporation temperature results show ( Figure 2 F): The inclusion complex has the highest comprehensive score at 70°C.

[0083] [Example 2]

[0084] (1) Plackeet-Burman experiment to screen single factors

[0085] Based on the single-factor experiment, six factors were preliminarily identified: preparation temperature, preparation speed, preparation ratio, preparation time, rotary evaporation speed, and rotary evaporation temperature. Their value ranges are shown in Table 1. The experiment used an experimental design of n=3 and set three virtual variables to estimate the error.

[0086] Table 1

[0087]

[0088] The important factors affecting the comprehensive score of inclusion complexes are shown in Table 2. The selected factors were tested according to the experimental design method, and the results are shown in Table 2.

[0089] Table 2

[0090]

[0091] The significance analysis of influencing factors was performed using the software Design-Expert 13.0. As shown in Table 3, the factors that have a greater impact on the comprehensive score of the inclusion compound are: preparation ratio, preparation speed, preparation time, and preparation temperature (P < 0.05), a total of four factors. This model is significant, R 2 =0.9696, indicating a good correlation, and the corrected determination coefficient R Adj 2 =0.9332, indicating that 93.32% of the variability in the experimental data can be explained by this regression model.

[0092] Table 3

[0093]

[0094] (2) Box-Behnken response surface experiment

[0095] Based on the Plackett-Burman experiment, four important factors were identified. Taking the comprehensive score of inclusion complexes as the indicator, a response surface analysis experiment with four factors and three levels, totaling 27 experimental points, was designed. The values ​​of each factor level, experimental design, and results are shown in Table 4.

[0096] Table 4

[0097]

[0098] DesignExpert13.0 software was used to perform multivariate linear regression linear fitting, and a quadratic regression equation with the comprehensive evaluation of inclusion complexes as the index value (Y) was obtained:

[0099] Overall score = 93.90 + 0.08 × A + 1.15 × B + 1.75 × C + 1.85 × D - 1.30 × AB + 3.75 × AC + 0.21 × AD - 0.39 × BC - 0.56 × BD - 0.25 × CD - 7.51 × A 2 -7.64×B 2 -8.11×C 2 -7.71×D 2

[0100] The results of the study are shown in Table 5: the created entity model has a significant impact (P < 0.05); the lack of fit item P > 0.05, indicating that the error value is not statistically significant, R 2=0.9808, the fit is good, and the adjusted determination coefficient R2adj=0.9584, which can explain 95.84% of the response value shift.

[0101] Preparation temperature, preparation time and preparation ratio are the significant factors affecting PT inclusion complexes, among which preparation ratio has the greatest impact, followed by preparation time, and preparation temperature and preparation speed have the least impact.

[0102] Table 5

[0103]

[0104] A study of the effects of the four factor interactions on the overall score showed that the more tortuous the response surface and the more dramatic the color change, the stronger the interaction between the two factors. The largest interactions were found between the preparation speed and the preparation time, preparation temperature, and preparation ratio; the interaction between the preparation ratio and preparation time was significant; and the interaction between preparation time and preparation temperature was minimal. The predicted optimal preparation conditions are shown in Table 6.

[0105] Table 6

[0106]

[0107] (3) Response surface validation experiment

[0108] The preparation conditions in Table 6 were appropriately adjusted based on actual conditions. When the preparation speed was 405 rpm, the preparation temperature was 46°C, the preparation time was 123 min, and the preparation ratio was 1:10.232, the model-predicted inclusion complex achieved the highest overall score, with a predicted value of 94.14. The validation experimental results, shown in Table 7, had an average of 94.44, close to the model-predicted value of 94.14, indicating successful model establishment.

[0109] Table 7

[0110]

[0111] [Example 3]

[0112] Hydroxypropyl-β-cyclodextrin was dissolved in ultrapure water to form a hydroxypropyl-β-cyclodextrin solution; phloretin was dissolved in anhydrous ethanol to form a phloretin solution; the mass ratio of phloretin to hydroxypropyl-β-cyclodextrin was 1:10.232.

[0113] The phloretin solution was slowly added to the hydroxypropyl-β-cyclodextrin solution, and the mixture was stirred at 405 rpm for 123 min at a temperature of 46°C.

[0114] After the reaction, the mixture was cooled to room temperature and refrigerated in a refrigerator at 4°C for 24 h. The solvent was removed by rotary evaporation at 70°C at a speed of 100 rpm, and the mixture was placed in a vacuum drying oven at 25°C for 24 h to fully remove the solvent. The mixture was then washed with anhydrous ethanol, acetone and petroleum ether to remove free phloretin, and dried in a vacuum drying oven at 25°C for 24 h to obtain a loose coarse powder, which was then ground through an 80-mesh sieve to obtain PT-CDs powder (PT-HP-β-CD).

[0115] [Example 4]

[0116] {Characterization of PT-CDs}

[0117] (1)FT-IR analysis

[0118] Figure 3 A is the infrared spectra of PT, HP-β-CD, physical mixture and PT-HP-β-CD.

[0119] The FT-IR of HP-β-CD mainly showed the -1 OH stretching vibration at 2928.7cm -1 -CH2- stretching vibration; the main characteristic peak of PT in FT-IR spectrum is 3261cm -1 -OH stretching vibration peak at 1636 cm -1 The -C=O stretching vibration peak at 100 nm was observed; the FT-IR spectrum of the physical mixture is simply a superposition of the absorption spectra of the individual components; the absorption spectra of PT and HP-β-CD are very similar, as the absorption of HP-β-CD and the guest (PT) molecule overlap in most spectral regions; and the -OH group of PT completely disappears in the PT-HP-β-CD spectrum. These results indicate that PT has entered the cavity of the HP-β-CD molecule.

[0120] (2) PXRD analysis

[0121] Figure 3 B is the PXRD patterns of HP-β-CD, PT, physical mixture (PM) and PT-HP-β-CD.

[0122] The spectrum of HP-β-CD shows that it is amorphous; the spectrum of PT shows obvious sharp peaks, indicating that it is crystalline; the spectrum of the physical mixture shows that there are both amorphous and crystalline substances, indicating that this is just a simple physical overlap of the spectra of the two samples; in contrast, the spectrum of PT-HP-β-CD has no sharp peaks, and the spectrum is similar to that of amorphous, without any diffraction peaks corresponding to PT, indicating that PT and HP-β-CD form an inclusion complex.

[0123] The powder X-ray diffraction pattern further confirmed the inclusion of PT by HP-β-CD.

[0124] (3) TGA analysis

[0125] like Figure 3 As shown in Figure C, PT begins to decompose at 262°C, the melting point of PT. After inclusion by HP-β-CD, the entire inclusion complex begins to decompose at 325°C. The inclusion by HP-β-CD improves the thermal stability of PT to a certain extent, further indicating that an inclusion complex is formed between PT and HP-β-CD. At 100°C, the mass of the inclusion complex decreases slightly, indicating that the prepared inclusion complex contains a certain amount of water.

[0126] Thermogravimetric analysis showed that the thermal stability of PT was enhanced to a certain extent after being encapsulated by HP-β-CD.

[0127] (4)DSC analysis

[0128] like Figure 3 As shown in Figure D, pure PT has an endothermic peak at 261°C, which is the characteristic peak of the melting point of PT; PM has a peak at around 256°C corresponding to the characteristic peak of PT, indicating that PM is a physical superposition of PT and HP-β-CD; it can be seen from the curve of the inclusion complex that the characteristic peak of PT does not appear, which may be due to the coverage of PT molecules after entering HP-β-CD; based on this, it can be considered that the disappearance of the characteristic peak of the guest molecule in the DSC spectrum can be used as evidence of the successful formation of the inclusion complex.

[0129] (5) SEM analysis

[0130] like Figure 4 These are typical SEM images of PT, HP-β-CD, physical mixture, and PT-CDs.

[0131] Figure 4 A is the microscopic morphology of PT, which is a regular needle-shaped crystal; Figure 4 B shows the morphological appearance of HP-β-CD, which is spherical particles with cavity structures of different sizes; Figure 4 C is the shape and appearance of the physical mixture, which includes both spherical particles of HP-β-CD (red arrows) and regular needle-shaped crystals of PT. Figure 4 In D, PT-HP-β-CD appears as an organized solid mass, which is significantly different from the physical mixture. These results indicate that HP-β-CD and PT form a new complex.

[0132] (6) 1 H NMR and 2D NOESY analysis

[0133] The results of the study were compared between PT and PT-HP-β-CD and between HP-β-CD and PT-HP-β-CD. 1 H NMR spectrum ( Figure 5 A) The chemical shifts of PT and HP-β-CD proton H in the free compound and PT-HP-β-CD are shown in Tables 8 and 9. The induced shifts were calculated by the following formula: Δ = inclusion complex - free compound.

[0134] It can be seen that the chemical shifts of H-3 and H-5 protons located in the HP-β-CD cavity are relatively large (0.0017ppm and 0.0056ppm, respectively), while H-2 and H-4 located outside the cavity have no obvious changes (Δ<0.0010ppm). The chemical position changes of H-11, 12 and H-20 protons in PT are more obvious (-0.0098ppm and -0.0057ppm, respectively). Since H-3 is close to the wide side of the cavity and H-5 is close to the narrow side of the cavity in HP-β-CD, according to 1 H NMR results predicted that HP-β-CD should include PT on the narrow side of the cavity.

[0135] In order to further verify the spatial compactness and relative position of HP-β-CD and PT during inclusion complex formation, 2D NOESY experiments were performed. Figure 5 B shows the NOESY spectrum of PT-HP-β-CD, where clear correlations can be observed between the H-11, 12, and H-20 protons of PT and the H-5 protons of HP-β-CD. These results further indicate that the aromatic ring of PT is located near the narrow side of the HP-β-CD cavity, and its inclusion structure is as follows: Figure 5 As shown in D, the host-guest molecular ratio of PT to HP-β-CD is 1:1.

[0136] Table 8

[0137]

[0138] Table 9

[0139]

[0140] (7) Stoichiometry: Job's plot

[0141] The stoichiometry of the complex was determined by the Job's plot method, which plotted the relationship between the absorbance of the physical property and the mole fraction.

[0142] Weigh pure PT and HP-β-CD to keep the total molar concentration of PT and HP-β-CD at 4×10 -4The molar ratio of M and PT (R = [PT] / [PT] [HP-β-CD]) was varied between 0.0 and 1.0. After stirring at 37°C for 4 h, the UV absorbance at 286 nm was recorded, and the absorbance difference (ΔA × mole fraction) in the presence and absence of HP-β-CD was plotted against the mole fraction R.

[0143] like Figure 5 As shown in Figure C, a maximum value is observed at a mole fraction of 0.5, which indicates that the stoichiometric ratio of PT-HP-β-CD is 1:1, which is consistent with the phase solubility and H NMR spectroscopy studies.

[0144] As can be seen from the above, the interaction between PT and HP-β-CD in the preparation of the present invention forms an inclusion complex, PT enters the cavity from the narrow side of the HP-β-CD cavity, and the molar ratio of PT to HP-β-CD in the inclusion complex is 1:1, which proves that the present invention successfully prepared PT-HP-β-CD.

[0145] [Example 5]

[0146] {Solubility Study}

[0147] (1) Powder dissolution

[0148] The dissolution curve of the drug powder was determined using a dissolution apparatus under non-sink conditions. 100 mg of PT and an equal amount of PT-HP-β-CD were accurately weighed and placed in 900 mL of PBS solution (pH = 7.4), pH = 6.5 buffer solution, pH = 1.2 solution, and ultrapure water for dissolution. The conditions were: slurry speed: 75 rpm, temperature: 37 ° C, and 0.3 mL of samples were taken at 15, 30, 60, 120, and 240 min, respectively, and the same amount of isothermal dissolution medium was added. The sample was centrifuged at 13,000 rpm for 3 min, the supernatant was diluted with methanol, and the drug concentration of the sample was determined by HPLC.

[0149] The results are as follows Figure 6 As shown, the results show that PT-HP-β-CD powder is Figure 6 A), PBS solution with pH = 7.4 ( Figure 6 B), pH = 6.5 ( Figure 6 C) and pH = 1.2 ( Figure 6 D) buffer solution, dissolving rapidly. After approximately 15 minutes, the cumulative release rate of PT reached nearly 100%, significantly higher than that of the API. Furthermore, the cumulative release of PT from the physical mixture increased over time in different environments. This is likely due to the continuous binding of PT to HP-β-CD during the dissolution process, which is equivalent to a slow preparation process, allowing it to dissolve slowly in the solvent.

[0150] It can also be seen from the figure that in an acidic environment, the cumulative dissolution of PT reaches a plateau when it reaches 25% and no longer dissolves, while in an alkaline environment, the cumulative dissolution of PT can reach 60% within 4 hours.

[0151] The overall results showed that due to the inclusion of PT by HP-β-CD, PT-HP-β-CD could achieve 100% cumulative release in a very short time regardless of whether it was in an alkaline or acidic environment, making PT-HP-β-CD have both high solubility and high dissolution rate.

[0152] (2) pH conversion two-step dissolution

[0153] Preparation of tablets: 100 mg each of PT and PT-HP-β-CD (containing an equal amount of PT) were mixed with corn starch, magnesium stearate, sodium carboxymethyl cellulose and other excipients through an 80-mesh sieve, and the mixture was directly compressed into tablets using a tablet press.

[0154] The dissolution curve of the tablets was determined using a dissolution apparatus under non-sink conditions. The powder amount was 100 mg, and it was first dissolved in 300 mL (pH = 1.4) of dissolution medium for 1 hour. Then, the pH of the dissolution medium was adjusted to 6.5 with 450 mL of Na2HPO4 solution and the dissolution experiment was continued for 4 hours. Dissolution conditions: slurry speed: 75 rpm, temperature: 37 ° C, sampling time was 15, 30, 60, 75, 90, 120, 180 and 240 min, 0.3 mL was sampled each time, the sample was centrifuged at 13000 rpm for 3 minutes, the supernatant was diluted with methanol, and the drug concentration of the sample was determined by HPLC.

[0155] like Figure 7 The results showed that the solubility of PT-HP-β-CD was 85% in an environment of pH = 1.4 after 60 minutes, while the API reached saturation at only 20% within 60 minutes; in an environment of pH = 6.5, the cumulative dissolution of the API increased from 20% to 50%, while PT-HP-β-CD basically maintained at 85%.

[0156] From the whole dissolution process, it can be seen that PT dissolves poorly in acidic environment and dissolves well in alkaline environment, which is highly dependent on pH value. The area under the PT dissolution curve (AUC 0-5h) is 13537 (μg mL -1 min -1 ), while PT-HP-β-CD was 25616 (μg·mL -1 min -1 ), and its AUC was twice that of PT. PT-HP-β-CD was superior to PT in terms of both dissolution environment and cumulative dissolution rate.

[0157] The above experiments demonstrate that PT-HP-β-CD powder dissolves rapidly in ultrapure water, PBS solution at pH 7.4, and buffer solutions at pH 6.5 and pH 1.2. Not only is its solubility high, but its dissolution rate is also rapid. Furthermore, PT-HP-β-CD exhibits similar solubility to pure PT in the gastrointestinal tract. This demonstrates that inclusion of HP-β-CD significantly enhances the solubility of PT.

[0158] [Example 6]

[0159] {Study on transmembrane transport of PT and PT-HP-β-CD in Caco-2 cells}

[0160] (1) CCK-8 cytotoxicity test

[0161] ① Take Caco-2 cells in the logarithmic growth phase.

[0162] ② Add 10 μL of drugs of different concentrations to each well. The blank control is to add the same amount of cell culture medium without adding drugs or cells; the positive control is to add drugs of different concentrations.

[0163] ③ Add 10 μL of CCK-8 reagent to each well, incubate for 30 min, select 450 nm wavelength to measure the light absorbance of each well on a microplate reader, record the results, and calculate the cell survival rate of different drugs at different concentrations according to formula (1).

[0164]

[0165] Wherein, Survival: cell survival rate; Sample A: OD value of the experimental group; Sample B: OD value of the negative control group; Sample C is the OD value of the blank group.

[0166] The cytotoxicity was measured by CCK-8 method. Figure 8 As shown, Figure 8 A and Figure 8 B are the cell viability of PT and HP-β-CD in Caco-2 cells. The cell viability of the two drugs was in the concentration range of 1-10 μg·mL -1 The survival rate reached over 80% within the range of 10 μg·mL. Therefore, the maximum concentration of the drug to be added in subsequent experiments was determined to be 10 μg·mL -1 The high, medium and low concentrations were set at 10, 5 and 1 μg·mL, respectively. -1 .

[0167] Figure 8 C, D, E, and F are the cell survival rates of four different transporter inhibitors in Caco-2 cells. Except for EDTA, the other three drugs were 1-25 μg mL-1 The survival rate reached more than 80% within the range of 15 μg·mL. -1 EDTA at 5 μg·mL -1 When the cell viability is greater than 80%, the dosage concentration is 5 μg·mL -1 .

[0168] (2) Validation of the Caco-2 monolayer cell model

[0169] Establish Caco-2 monolayer cell model: Caco-2 cells in logarithmic growth phase were plated at 2×10 5 The cells were inoculated into 12-well Transwell plates and cultured for 21 days, with the medium changed every two days during the first week and every day thereafter.

[0170] ①Integrity assessment

[0171] Trans-epithelial electrical resistance (TEER) is related to the integrity of tight junctions between cells. Therefore, the integrity of the monolayer cell model is determined by measuring the trans-epithelial electrical resistance of the monolayer cell. During measurement, the short electrode of the resistance meter is inserted into the Transwell chamber, and the long electrode is inserted into the outer chamber. The resistance value is calculated according to formula (2).

[0172] TEER=(TEERc-TEERb)×S (2)

[0173] Where TEER is the final resistance of the cell; TEERc is the resistance of the experimental group; TEERb is the resistance of the blank group; S is the membrane area of ​​the 12-well Transwell chamber (cm 2 ).

[0174] ② Transmittance evaluation

[0175] Sodium fluorescein is a paracellular transporter and is commonly used to detect the integrity and density of cell monolayers. A sodium fluorescein solution of a certain concentration was prepared. When the cells were cultured for 21 days, the prepared sodium fluorescein solution was used to calculate the transmembrane flux from the AP side to the BL side. The permeability of sodium fluorescein in the experimental well, P, was calculated according to formula (3). app Less than 1.0×10 -6 cms -1 This indicates that a complete single cell layer has been formed with good density and can be used for transport experiments.

[0176]

[0177] Where dQ / dt is the permeation rate per unit time; A is the surface area of ​​the polycarbonate membrane, which is 1.12 (cm 2 ); C0 is the initial concentration of the drug (μg·mL -1 ).

[0178] The resistance value and fluorescent yellow transmittance of the established Caco-2 monolayer cell model were verified as follows: Figure 9 As shown in Table 10, taking one of the plates as an example, it was found that after 21 days of culture, the cells were well intact and tightly connected. As shown in Table 10, the fluorescent yellow transmittance P of all Transwell plates was app All of them are less than 1.0×10 -6 cm·s -1 , indicating that the cells are tightly connected and well differentiated, and can be used for transport experiments.

[0179] Table 10

[0180]

[0181] [Example 7]

[0182] (1) Effects of different concentrations of PT and HP-β-CD on transmembrane function in Caco-2 cells

[0183] Based on the cytotoxicity results of PT and PT-HP-β-CD, the high, medium and low concentrations of the drug were determined. On the basis of the successful preparation of the Caco-2 monolayer cell model, different concentrations of PT and PT-HP-β-CD were added to the AP side of the Transwell plate, and 1.5 mL of Hank's was added to the BL side. The plates were then cultured in an incubator at 37°C and 5% CO2. Finally, samples were taken from the BL side of the Transwell plate after 30, 60, 90 and 120 min of culture, and the samples were centrifuged at 13000 rpm for 5 min. The sample concentration was then detected in a high performance liquid chromatography (HPLC) column. Finally, the transmembrane permeability of the drug, P, was calculated according to formula (3): app .

[0184] (2) Effects of different transport directions on the transmembrane transport of PT and PT-HP-β-CD in Caco-2 cells

[0185] Different concentrations of PT and PT-HP-β-CD were added to the BL side of the Transwell plate, and 1.5 mL of Hank's was added to the AP side. The plates were then cultured in an incubator at 37°C and 5% CO2. Samples were taken from the AP side of the Transwell plate after 30, 60, 90, and 120 min of culture. The samples were centrifuged at 13,000 rpm for 5 min, and then the sample concentrations were determined by high performance liquid chromatography. The transmembrane permeability of the drug, P, was calculated according to formula (3). app .

[0186] Caco-2 cell model app The values ​​are shown in Table 11. The results showed that the AP-BL of PT was 1, 5 and 10 μg·mL -1 P app The values ​​were (4.793±0.917)×10 -7 、(5.017±0.203)×10 -7 and (5.350±0.316)×10 - 7 cm·s -1 , BL-AP's P app The values ​​were (7.407±0.555)×10 -7 、(8.123±0.567)×10 -7 、(8.983±0.161)×10 -7 cm·s -1 The ER values ​​of PT at different concentrations were 1.545, 1.619, and 1.679, respectively. The ER value of PT was greater than 1.5, which may be involved in efflux transport and active transport.

[0187] The AP-BL of PT-HP-β-CD was 1, 5 and 10 μg·mL -1 P app The values ​​were (5.595±0.901)×10 -6 、(2.040±0.125)×10 -6 and (1.983±0.180)×10 -6 cm·s -1 , BL-AP's P app The values ​​were (2.053±0.372)×10 -6 、(0.813±0.063)×10 -6 and (0.825±0.046)×10 -6 cm·s -1The ER values ​​of PT-HP-β-CD at different concentrations were 0.366, 0.398 and 0.416 respectively. In summary, due to the addition of HP-β-CD, the P app The values ​​were higher than those of PT app value.

[0188] Table 11

[0189]

[0190] (3) Effects of different temperatures on transmembrane transport of PT and PT-HP-β-CD in Caco-2 cells

[0191] PT and PT-HP-β-CD of medium concentration among high, medium and low concentrations were added to the AP side of the Transwell plate, 1.5 mL of Hank's was added to the BL side, and three replica wells were prepared. The plates were cultured at 37°C and 4°C, respectively. Finally, samples were taken from the BL side of the Transwell plate after 30, 60, 90 and 120 min of culture, and the samples were centrifuged at 13000 rpm for 5 min. The sample concentration was then detected in HPLC, and the transmembrane permeability P of the drug was calculated according to formula (3): app .

[0192] The transmembrane permeability P of PT and PT-HP-β-CD at different temperatures from the AP-BL side in the Caco-2 monolayer cell model was calculated. app ,like Figure 10 As shown in the figure, the permeability of PT at 4℃ decreased significantly compared with that at 37℃. Its transmembrane transport is affected by temperature, that is, it is energy-dependent. Therefore, it is speculated that PT may have active transport. However, PT-HP-β-CD at 37℃ is lower than that at 4℃. app The difference in values ​​is small, although there are obvious differences, but the P app The values ​​were higher than those of PT. Although there was a certain degree of active transport, it was not obvious compared with pure PT. It may be that the addition of HP-β-CD changed the transport mode.

[0193] (4) Effects of different transporter inhibitors on transmembrane transport of PT and PT-HP-β-CD in Caco-2 cells

[0194] The four drugs at medium concentrations were added to the AP side of the Transwell plate, and 1.5 mL of Hank's was added to the BL side. The concentration of the inhibitor added was determined based on the cytotoxicity results of the three transporter inhibitors. The plates were then placed in an incubator at 37°C and 5% CO2. Samples were taken from the BL side of the Transwell plate after 30, 60, 90, and 120 min of incubation, and the transmembrane permeability of the drugs, P, was calculated according to formula (3). app .

[0195] like Figure 11 As shown in A, in the Caco-2 monolayer cell model, the P of PT increased with the addition of different efflux protein inhibitors. app The value of PT was changed; the addition of indomethacin and verapamil increased the P value of PT. app The value of PT increased significantly, which indicates that PT may be the substrate of MRP2 and P-gp protein, which has a significant efflux effect on PT. However, the addition of reserpine increased the P value of PT. app The values ​​did not change significantly, indicating that PT was not affected by the efflux of BCRP protein in Caco-2 cells;

[0196] After PT was included by HP-β-CD, its efflux function was changed, such as Figure 11 As shown in B, it can be seen that after adding different efflux protein inhibitors, the P app The values ​​of PT and HP-β-CD did not change significantly. In other words, the PT was prevented from being expelled by efflux proteins after HP-β-CD was used to encapsulate PT. app The values ​​of both of them changed significantly, which indicated that both of them passed through the monolayer cell model through the intercellular gap, so their P app The values ​​increased.

[0197] (5) In vivo one-way intestinal perfusion study of PT and PT-HP-β-CD

[0198] Use a small amount of distilled water to dissolve and prepare KR nutrient solution (NaCl 7.80g, NaHCO3 1.37g, KCl 0.35g, NaH2PO4 0.32g, MgCl2 0.02g, CaCl2 0.37g, glucose 1.40g). After CaCl2 is dissolved, add it dropwise. Glucose is added during the experiment. After dissolution, use ultrapure water to make up to 1L and store it in a refrigerator at 4℃ for later use.

[0199] Accurately weigh PT and PT-HP-β-CD, dissolve them in KR solution, and prepare a concentration of 50 μg mL -1The drug-containing perfusate was stored at 4°C for future use. Protein inhibitors were added to the prepared 50 μg mL -1 Just add it to the drug-containing perfusion fluid.

[0200] Preheat the perfusion fluid in a 37°C water bath to fill the tubing with the perfusion fluid, and balance the perfusion system to make the drug concentrations at the inlet and outlet equal.

[0201] Rats were fasted for 24 h, with free access to water, and anesthetized with isoflurane. The ileum and colon were selected, and a small opening was made at each end of the intestinal segment. A catheter was inserted and 37°C saline was slowly flushed into the intestine before the perfusion fluid was connected. The residual saline in the intestinal cavity was then emptied and the intestinal fluid was purged at a rate of 0.2 mL min. -1 The flow rate was perfused and timed, starting after approximately 30 minutes of intestinal equilibration. Each experiment consisted of 15 minutes, during which samples were collected. The sample solution was centrifuged at 13,000 rpm for 10 minutes, and the supernatant was collected for HPLC determination of PT concentration. The rats were sacrificed, and the length and radius of the intestinal segments were measured to calculate the surface area of ​​the perfused intestinal segments. The absorption rate constants (Ka) of PT and PT-HP-β-CD in different intestinal segments were calculated according to formula (4).

[0202]

[0203] Where Ka is the absorption rate constant; C in and C out : Mass concentration of intestinal inlet and outlet perfusate (μg·mL -1 ); l and r 2 : length (cm) and cross-sectional radius (cm) of the perfused intestinal segment; Q: perfusion rate (mL·min -1 );Q in and Q out : The mass of the test tube containing the perfusion solution and the mass of the test tube after perfusion (g).

[0204] Different protein inhibitors (verapamil hydrochloride, reserpine, and indomethacin) and EDTA (EDTA) were added to the perfusate of PT and PT-HP-β-CD to investigate their effects on intestinal absorption. The absorption rate constant, Ka, of the drug in the ileum was calculated using the formula.

[0205] The results of PT and PT-HP-β-CD in ileum and colon in vivo unidirectional intestinal perfusion are as follows Figure 12As shown, the Ka values ​​of PT in the ileum were 0.102 ± 0.022, and those of PT-HP-β-CD were 0.204 ± 0.040, indicating a significant difference between PT-HP-β-CD and PT. In the colon, the Ka values ​​of PT were 0.055 ± 0.007, and those of PT-HP-β-CD were 0.081 ± 0.009, indicating a significant difference between PT-HP-β-CD and PT. In summary, the Ka values ​​of PT-HP-β-CD were greater than those of the API, PT, indicating that the addition of HP-β-CD increased the permeability of PT in the ileum and colon.

[0206] like Figure 13 As shown in the figure, the Ka values ​​calculated by intestinal perfusion show that after the addition of indomethacin, the Ka value of the raw material PT in the ileum increases significantly, indicating that after inhibiting the MRP2 protein, MRP2 loses its ability to excrete PT, resulting in increased ileal permeability of PT. The Ka value of PT-HP-β-CD after the addition of indomethacin is not significantly different from that without indomethacin, indicating that after HP-β-CD inclusion, MRP2 protein has no significant effect on its excretion, indicating that HP-β-CD inclusion can prevent PT from being excreted by MRP2.

[0207] After the addition of verapamil hydrochloride, the Ka value of the API PT in the ileum increased significantly, indicating that the addition of verapamil hydrochloride inhibited P-gp protein in the ileum, significantly increasing the Ka value of PT, consistent with the results of Caco-2 transmembrane transport. The Ka value of PT-HP-β-CD after the addition of verapamil hydrochloride was not significantly different from that without verapamil hydrochloride, indicating that the inclusion of HP-β-CD prevented the efflux of P-gp protein.

[0208] After adding reserpine, the Ka of the raw material PT in the ileum becomes higher, with obvious differences, which means that the addition of reserpine significantly improves the absorption of PT in the ileum. Compared with the situation in Caco-2, reserpine does not increase P app The Ka value of PT-HP-β-CD with reserpine was not significantly different from that without reserpine, indicating that inclusion of HP-β-CD prevented the efflux of BCRP protein.

[0209] The above cell experiments and animal experiments verified that after PT was included in HP-β-CD, its permeability changed, and PT-HP-β-CD had better permeability than pure PT.

[0210] [Example 8]

[0211] {Study on Antioxidant Capacity}

[0212] (1) Ability to scavenge free radicals

[0213] DPPH method: Accurately weigh 1 mg of PT, an equal amount of PT in PT-HP-β-CD, and an equal amount of HP-β-CD and dissolve them in 10 mL of ultrapure water. Mix thoroughly and centrifuge to collect the filtrate. Add equal volumes of aqueous solutions of PT, PT-HP-β-CD, and HP-β-CD to the DPPH reagent and store in the dark for 30 min. Observe the color change.

[0214] ABTS method: prepare 7mmol·L -1 ABTS solution and 140mmol·L -1 Potassium persulfate solution was mixed in proportion to form ABTS free radical stock solution, and allowed to stand in the dark for 16 h; before use, it was diluted with 80% anhydrous ethanol at 30°C to an ABTS working solution with an absorbance of 0.700±0.200.

[0215] Accurately weigh 1 mg of PT, an equal amount of PT-HP-β-CD, and an equal amount of HP-β-CD, and dissolve them in 10 mL of ultrapure water. Mix thoroughly, centrifuge, and collect the filtrate. Add equal volumes of PT, PT-HP-β-CD, and HP-β-CD aqueous solutions to the ABTS working solution, protect from light for 30 min, and observe the color change.

[0216] like Figure 14 As shown, after 30 minutes of storage in the dark, the color of the HP-β-CD group remained unchanged from that of the ABTS and DPPH reagents, indicating that it has no antioxidant activity. Although the color of the PT group also changed significantly, it did not completely discolor the reagents. However, PT-HP-β-CD completely discolored both reagents, indicating that HP-β-CD, after incorporating PT, has a more effective free radical scavenging effect. This is because HP-β-CD, after incorporating PT, greatly increases its water solubility, thereby significantly enhancing its antioxidant capacity compared to pure PT.

[0217] (2) Reducing ability determination

[0218] Using potassium ferricyanide method, Na2HPO4 and KH2PO4 were used to prepare 0.2 mol·L -1 , pH = 6.6 buffer, use this buffer to prepare samples of different concentrations, and then add 2.5mL10g·L -1 Potassium ferrocyanide, react in a 50℃ water bath for 30 min, then add 100g·L -12.5 mL of trichloroacetic acid was added to 2.5 mL of the supernatant after standing, and 2.5 mL of deionized water and 0.5 mL of 1 g·L - 1 FeCl3, the absorbance value was measured at a wavelength of 700nm.

[0219] like Figure 15 As shown in the figure, after HP-β-CD encapsulation of PT, the reducing performance of PT-HP-β-CD is significantly stronger than that of the original drug at all concentrations, and HP-β-CD remains unchanged, indicating that it has no reducing properties. This is because the hydrogen bonding interaction between PT and HP-β-CD significantly enhances the reducing ability of PT-HP-β-CD.

[0220] As can be seen from the above, the present invention forms an inclusion complex by including PT with HP-β-CD, which greatly improves the water solubility of phloretin, thereby improving the free radical scavenging ability of phloretin. At the same time, due to the action of the inclusion complex, the reducing ability of phloretin is greatly improved, thereby improving the antioxidant performance of phloretin.

[0221] While the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Persons skilled in the art will readily appreciate that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. Application of hydroxypropyl-β-cyclodextrin as an ingredient to enhance antioxidant capacity in the preparation of phloretin preparations.

2. The use according to claim 1, characterized in that Phloretin is encapsulated in the cavity of hydroxypropyl-β-cyclodextrin and cooperates with hydroxypropyl-β-cyclodextrin to form an inclusion complex, and the antioxidant property of phloretin is improved by the formed inclusion complex.

3. A phloretin preparation, characterized in that The preparation contains phloretin (PT) and hydroxypropyl-β-cyclodextrin (HP-β-CD), wherein the phloretin is located in the cavity of the hydroxypropyl-β-cyclodextrin, and the phloretin and the hydroxypropyl-β-cyclodextrin cooperate to form an inclusion compound, thereby improving the antioxidant performance of the phloretin through the formed inclusion compound.

4. The phloretin preparation according to claim 3, characterized in that Phloretin and hydroxypropyl-β-cyclodextrin form the inclusion complex through hydrogen bonding interaction.

5. The phloretin preparation according to claim 3, characterized in that In the inclusion compound, the aromatic ring of phloretin is located at one end of the narrow side of the hydroxypropyl-β-cyclodextrin cavity.

6. The phloretin preparation according to claim 3, characterized in that In the inclusion compound, the molar ratio of phloretin to hydroxypropyl-β-cyclodextrin is 1:

1.

7. A method for preparing the phloretin preparation according to any one of claims 3 to 6, characterized in that: The following steps are involved: dissolving hydroxypropyl-β-cyclodextrin in ultrapure water to form a hydroxypropyl-β-cyclodextrin solution; dissolving phloretin in anhydrous ethanol to form a phloretin solution; The phloretin solution is added to the hydroxypropyl-β-cyclodextrin solution, and the mixture is stirred to react, so that the phloretin enters the cavity of the hydroxypropyl-β-cyclodextrin, and the phloretin and the hydroxypropyl-β-cyclodextrin cooperate to form an inclusion complex; After the reaction, the mixture was cooled to room temperature and refrigerated in a 4°C refrigerator for 24 h, and then the solvent was removed by rotary evaporation and vacuum dried. The free phloretin was then washed to remove the free phloretin, dried, and sieved to obtain phloretin-hydroxypropyl-β-cyclodextrin inclusion complex (PT-CDs) powder.

8. The method for preparing the phloretin preparation according to claim 7, wherein The mass ratio of phloretin to hydroxypropyl-β-cyclodextrin is 1:(4-12).

9. The method for preparing the phloretin preparation according to claim 7, wherein The phloretin solution is added to the hydroxypropyl-β-cyclodextrin solution, and the stirring reaction conditions include: stirring at a temperature of 35 to 55° C. and a rotation speed of 250 to 550 rpm for 90 to 150 minutes.

10. The method for preparing the phloretin preparation according to claim 7, wherein The conditions for removing the solvent by rotary evaporation include: rotary evaporation at a temperature of 60 to 80° C. and a rotation speed of 80 to 120 rpm.

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