Method for preparing phloretin solid dispersion by applying supercritical CO2 anti-solvent technology

Abstract

The invention discloses a method for preparing a phloretin solid dispersion by applying a supercritical CO2 anti-solvent technology, which comprises the following steps: dissolving phloretin and a carrier in an organic solvent to prepare a phloretin-carrier mixed solution; adjusting the temperature in the crystallization kettle to a set value, introducing CO2 into the crystallization kettle, and pressurizing to a set value; keeping the temperature and the pressure constant, adjusting the flow rate of CO2, and pumping the phloretin-carrier solution into a crystallization kettle at a certain volume flow rate; after sample introduction is finished, CO2 continues to be introduced for a period of time, then pressure relief is conducted, and the phloretin solid dispersion in the crystallization kettle is collected. According to the method provided by the invention, the phloretin solid dispersion with obviously improved dissolution performance can be prepared, so that the bioavailability of an indissolvable drug is improved, and the problem of organic solvent residue generally existing in the traditional technology can be solved.

Description

Technical Field

[0001] The invention relates to a method for preparing phloretin solid dispersion by using supercritical CO2 antisolvent technology. Background Art

[0002] Phloretin's chemical name is 2,4,6-trihydroxy-3-(4-hydroxyphenyl)propiophenone, which is a natural dihydrochalcone flavonoid, mainly found in the peel or root bark of fruits and vegetables or plants such as strawberries, apples, cherries, crabapple leaves, and pears. Phloretin has pharmacological activities such as anti-diabetes, anti-osteoporosis, antioxidant, protection of the nervous system, anti-atherosclerosis, and antibacterial. In addition, phloretin has many beauty effects such as whitening and moisturizing, and anti-aging, and has broad application prospects in the fields of medicine, cosmetics, and food. However, the low solubility of phloretin (26.51μg / mL), poor stability, and low bioavailability of 8.68% limit the drug application of phloretin. Therefore, improving the water solubility and bioavailability of phloretin is the difficulty and focus of phloretin research and development.

[0003] Solid dispersions can improve the water solubility and bioavailability of BCS class II drugs by enhancing wettability, reducing particle size, high porosity and amorphous state. Its preparation methods include melting method, solvent evaporation method, freeze drying method, spray drying method, supercritical fluid technology, etc. Traditional solid dispersion preparation techniques, including spray drying, grinding, solvent evaporation and freeze drying, have limitations, such as high temperature, many operation steps, difficulty in controlling the particle size and particle size distribution, poor reproducibility and high solvent residue. Some studies have reported the use of solvent method to prepare phloretin solid dispersions to improve the water solubility of the drug; in addition, some researchers have used spray drying technology to prepare phloretin solid dispersions to improve the dissolution rate and stability of the poorly soluble drug; however, the spray drying and freeze drying methods have high preparation costs, the crystal form is difficult to reproduce, and a large amount of solvent is consumed and the solvent is not easy to completely remove; the melting method is not suitable for drugs or carriers that are unstable at high temperatures of melting or evaporation; the solvent method has relatively high requirements for process parameters and low reproducibility; the grinding method cannot determine whether the mixture is uniform, requires a large proportion of carrier materials, and is only suitable for the preparation of small doses of drugs. It is urgent to develop a more efficient method for preparing phloretin solid dispersions. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for preparing phloretin solid dispersion using supercritical CO2 antisolvent technology.

[0005] Technical solution: The present invention discloses a method for preparing a phloretin solid dispersion using supercritical CO2 antisolvent technology, comprising the following steps:

[0006] (1) dissolving phloretin and a carrier in an organic solvent to prepare a phloretin-carrier mixed solution;

[0007] (2) adjusting the temperature in the crystallization kettle, passing CO2 into the crystallization kettle, and pressurizing it to a set value;

[0008] (3) adjusting the CO2 flow rate to pump the phloretin-carrier solution into the crystallization kettle;

[0009] (4) After the injection is completed, CO2 is continuously introduced and the pressure is released to collect the phloretin solid dispersion in the crystallization kettle.

[0010] Wherein, in step (1), the organic solvent is a mixed solvent of ethanol and acetone, and the volume ratio of ethanol to acetone is 1:1 to 2, preferably 1:1.

[0011] Wherein, in step (1), the drug-carrying ratio of phloretin to carrier is 1:2-3, preferably 1:3.

[0012] Wherein, in step (1), the carrier is polyvinyl pyrrolidone K30 (PVP K30).

[0013] Wherein, in step (1), the mass concentration of phloretin is 2-7 mg / mL.

[0014] Wherein, in step (2), the temperature is 37-62°C.

[0015] Wherein, in step (2), the pressure is 9 to 19 MPa.

[0016] Wherein, in step (3), the CO2 flow rate is 3.5±0.2L / min.

[0017] Wherein, in step (3), the volume flow rate of the phloretin-carrier solution pumped in is 0.6 to 1.6 mL / min.

[0018] Wherein, in step (4), the time for continuing to introduce CO2 is 60 to 80 minutes.

[0019] The phloretin solid dispersion prepared by the above method is fluffy and has a solubility of 81.70%.

[0020] Principle of the invention: The method for preparing phloretin solid dispersion by supercritical CO2 antisolvent technology of the present invention, the phloretin solid dispersion prepared by supercritical CO2 antisolvent technology is fluffy and flocculent, which greatly provides the dissolution performance of solid dispersion, significantly improves the solubility of phloretin, and improves its bioavailability. And the present invention uses supercritical carbon dioxide as antisolvent, reduces the cost in preparation production, is green and environmentally friendly, has no organic solvent residue, mild conditions, and is suitable for thermosensitive drugs and carriers. In addition, the recovery rate of the phloretin solid dispersion of the present invention is closely related to the drug loading and process conditions, and the pressure factor has the most significant effect on the result, and the temperature has a small effect. The present invention prepares a solid dispersion with high yield, high drug loading, more concentrated particle size, and smaller particle size by optimizing the effects of different process parameters such as solvent, drug-to-carrier ratio, temperature and pressure on the recovery rate and drug loading, and it has been verified that the process conditions have high reproducibility.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) the phloretin solid dispersion prepared by the method of the present invention has significantly improved dissolution performance compared with the phloretin raw material drug; at the 180-min time point, the solubility of the raw material drug is 13.60%, while that of the solid dispersion prepared by the supercritical process is 81.70%; (2) the solubility of phloretin is significantly improved, the bioavailability of the poorly soluble drug is increased, and the problem of organic solvent residues commonly existing in traditional technologies can be overcome, providing a reference for the further clinical development of phloretin. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The schematic diagram of the experimental equipment structure, where 1 is a CO2 gas cylinder, 2 is a low-temperature constant temperature bath, 3 is a CO2 pump, 4 is a CO2 preheater, 5 is a crystallization kettle, and 6 is a high-pressure infusion pump;

[0023] Figure 2 This is the relationship diagram of the effect of crystallization temperature on the recovery rate and drug loading of phloretin solid dispersion;

[0024] Figure 3 This is the relationship diagram of the effect of solution volume flow rate on the recovery rate and drug loading of phloretin solid dispersion;

[0025] Figure 4 This is the relationship diagram of the effect of crystallization pressure on the recovery rate and drug loading of phloretin solid dispersion;

[0026] Figure 5 This is the relationship diagram of the effect of drug mass concentration on the recovery rate and drug loading of phloretin solid dispersion;

[0027] Figure 6 SEM images of phloretin API, PVP K30, a physical mixture of phloretin and PVP K30, and a phloretin solid dispersion prepared in Example 3 of the present invention;

[0028] Figure 7 XRD patterns of phloretin API, PVP K30, a physical mixture of phloretin and PVP K30, and a phloretin solid dispersion prepared in Example 3 of the present invention;

[0029] Figure 8 FTIR images of phloretin API, PVP K30, a physical mixture of phloretin and PVP K30, and a phloretin solid dispersion prepared in Example 3 of the present invention;

[0030] Fig. 9 The figure is a comparison chart of the 180 min in vitro dissolution curves of the phloretin bulk drug and the phloretin solid dispersion prepared by the present invention. DETAILED DESCRIPTION

[0031] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0032] 1. Experimental Materials and Instruments

[0033] The experimental materials are shown in Table 1, and the experimental instruments are shown in Table 2.

[0034] Table 1. Experimental materials

[0035]

[0036] Table 2. Experimental instruments

[0037]

[0038] Example 1

[0039] The process flow chart of supercritical CO2 antisolvent method is shown in Figure 1 , the operation steps are as follows:

[0040] (1) Check the air tightness of the entire device, open the low-temperature thermostat and set it to 4°C, set the temperature of the crystallization kettle, and turn on the heating switch;

[0041] (2) Heat to the set temperature, turn on the CO2 cylinder, air compressor pump and equipment pump, and slowly adjust the pressure indication to pressurize to the set value;

[0042] (3) After the pressure reading stabilizes, unscrew the CO2 outlet valve, and control the CO2 flow rate by fine-tuning the rotor flowmeter, and pump the prepared solution into the crystallization kettle through a high-pressure infusion pump;

[0043] (4) After the injection is completed, continue to introduce CO2 for a period of time, turn off the gas cylinder, air compressor pump and equipment pump, reduce the pressure to 4.5PMa, open the CO2 exhaust valve until the pressure reading is 0, and open the crystallization kettle to collect the sample.

[0044] Example 2

[0045] Single factor experiment: Effect of carrier type on the recovery rate of phloretin solid dispersion

[0046] Under the same conditions, hydroxypropyl methylcellulose, zein, soy lecithin, PVP / VA64 and PVP K30 were used as carriers respectively. The solid dispersion prepared with PVP K30 as the carrier was fluffy in shape and had a high yield, the sample prepared with PVP / VA64 as the carrier was not fluffy, and the samples prepared with the other carriers were viscous and had a low yield, so PVP K30 was selected as the carrier.

[0047] Single factor experiment: Effect of solvent type on the recovery rate of phloretin solid dispersion

[0048] Under the same conditions, methanol, acetone, acetone: dichloromethane = 1:1, ethanol: acetone = 1:1, ethanol: acetone = 1:2, ethanol: acetone = 1:3, ethanol: acetone = 2:3 were used as solvents (all volume ratios), and methanol, acetone, acetone: dichloromethane = 1:1, ethanol: acetone = 1:3 were used as solvents, but no molded samples could be prepared. The samples prepared with ethanol: acetone = 1:1, ethanol: acetone = 1:2, and ethanol: acetone = 2:3 were fluffy, and the recovery rates were 84.87%, 76.09%, and 78.03%, respectively. Therefore, the solvent selected was ethanol: acetone = 1:1-2, preferably 1:1.

[0049] Single factor experiment: Effect of drug-loading ratio on the recovery rate of phloretin solid dispersion

[0050] Under the same conditions, the effects of drug-loading ratios of 1:1, 1:2, 1:3, and 1:4 on sample recovery were investigated, and the recovery rates were 63.47%, 75.49%, 84.87%, and 68.51%, respectively. When the drug-loading ratio was 1:3, the recovery rate was the highest, so the drug-loading ratio was selected to be 1:2-3, preferably 1:3.

[0051] Single factor experiment: Effect of crystallization temperature on the recovery rate and drug loading of phloretin solid dispersion

[0052] Under the same conditions, the effects of temperatures of 37, 42, 47, 52, 57, and 62°C on the recovery rate and drug loading of phloretin solid dispersion were investigated. The results are as follows: Figure 2 As shown in the figure, as the temperature increases, the sample recovery increases first and then decreases. It may be that the increase in temperature promotes the mass transfer effect, which increases the recovery rate and drug loading. However, when the temperature rises to a certain value, the gaseous properties of the supercritical fluid play a leading role, and the solubility of the drug and carrier decreases, resulting in a decrease in the recovery rate and drug loading. After comprehensive consideration, the temperature range is determined to be 47-57°C.

[0053] Single factor experiment: Effect of solution volume flow rate on recovery rate and drug loading of phloretin solid dispersion

[0054] Under the same conditions, the effects of solution volume flow rates of 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 mL / min on the recovery rate and drug loading of phloretin solid dispersion were investigated. The results are as follows: Figure 3 As shown. As the volume flow rate of the solution increases, the drug loading of the sample shows a downward trend, which may be due to the shortened contact time between the supercritical fluid and the drug, the weakened mass transfer effect, and the reduced drug loading. The recovery first increases and then decreases, which may be due to the fact that within the lower rate range, the drug and the carrier can be quickly precipitated. When the rate is too high, part of the drug and the carrier are taken out of the pipeline, resulting in a decrease in the recovery rate. Considering all factors, the solution volume flow rate range is selected to be 0.8-1.2mL / min.

[0055] Single factor experiment: Effect of crystallization pressure on the recovery rate and drug loading of phloretin solid dispersion

[0056] Under the same conditions, the effects of pressures of 9, 11, 13, 15, 17, and 19 MPa on the recovery rate and drug loading of phloretin solid dispersion were investigated. The results are as follows: Figure 4 As shown in the figure, as the pressure increases, the recovery rate and drug loading of the sample first increase and then decrease. This may be because the density of the supercritical fluid increases with the increase in pressure, and the solubility of the drug and the carrier also increases, resulting in an increase in the recovery rate and drug loading. When the pressure reaches a certain critical value, the drug is not precipitated in time and is brought into the pipeline, resulting in a decrease in the recovery rate and drug loading. Taking all factors into consideration, the pressure range is selected to be 13-17MPa.

[0057] Single factor experiment: Effect of drug concentration on recovery rate and drug loading of phloretin solid dispersion

[0058] Under the same conditions, the effects of drug concentrations of 2, 3, 4, 5, 6, and 7 mg / mL on the recovery rate and drug loading of phloretin solid dispersion were investigated. The results are as follows: Figure 5 As shown in the figure, as the drug mass concentration increases, the sample recovery increases first and then decreases. This may be because the increase in drug concentration causes the drug to diffuse rapidly, quickly reaching a supersaturated state in the supercritical fluid, increasing the recovery rate and drug loading. When the concentration reaches a certain value, some drugs and carriers cannot be precipitated, causing the recovery rate and drug loading to decrease. Taking all factors into consideration, the drug mass concentration range is determined to be 4-6 mg / mL.

[0059] Example 3

[0060] Orthogonal test to optimize process parameters

[0061] Orthogonal experimental design and results:

[0062] The comprehensive scoring method was adopted. According to the influence size, the weight of recovery rate was 0.6, the weight of drug loading was 0.4, and the comprehensive score was calculated according to the formula comprehensive score = recovery rate * 0.6 + drug loading / theoretical drug loading * 0.4. The effects of four factors, namely crystallization temperature (A), crystallization pressure (B), drug mass concentration (C), and solution volume flow rate (D), on recovery rate and drug loading were investigated. Table 3 is the orthogonal test factor level table, Table 4 is the orthogonal test design and results, and Table 5 is the variance analysis results.

[0063] Table 3. Factor level table

[0064]

[0065] Table 4. Orthogonal experimental design and results

[0066]

[0067] Table 5. Results of variance analysis

[0068]

[0069] Analysis of orthogonal test results:

[0070] From the range analysis in Table 4, it can be seen that the influence of each factor on the recovery rate and drug loading of phloretin solid dispersion is B>D>C>A, that is, crystallization pressure>solution volume flow>solution mass concentration>crystallization temperature. The optimal process condition combination is A1B2C2D1, that is, the crystallization temperature is 47℃, the crystallization pressure is 15MPa, the solution mass concentration is 5mg / mL, and the solution volume flow is 0.8mL / min.

[0071] From the range analysis, we can see that factor A has the least impact on the comprehensive results. Therefore, factor A is used as the error factor to perform variance analysis on the results. From the variance analysis in Table 5, we can see that the significant P values ​​of factor B and factor D are both less than 0.05. According to the test standard of the significant level a=0.05, factors B and D have significant impacts on the results, while factors A and C have little impact on the results. According to the P value, the impact size is B>D>C, which is consistent with the results of the range analysis.

[0072] Example 4

[0073] Preparation of phloretin solid dispersion using optimal process conditions

[0074] The phloretin solid dispersion was prepared by supercritical CO2 antisolvent method, and the steps are as follows:

[0075] (1) dissolving phloretin and a carrier in an organic solvent to prepare a phloretin-carrier mixed solution;

[0076] (2) adjusting the temperature in the crystallization kettle to a set value, passing CO2 into the crystallization kettle, and pressurizing it to a set value;

[0077] (3) maintaining constant temperature and pressure, adjusting the CO2 flow rate, and pumping the phloretin-carrier solution into the crystallization kettle at a certain volume flow rate;

[0078] (4) After the injection is completed, CO2 is continuously introduced for a period of time and then the pressure is released to collect the phloretin solid dispersion in the crystallization kettle.

[0079] Among them, the carrier is PVP K30, the drug-carrier ratio is 1:3, the drug mass concentration is 5 mg / mL, the organic solvent is a mixed solvent of ethanol:acetone = 1:1, the temperature is 47°C, the pressure is 15 MPa, the CO2 flow rate is (3.5±0.2) L / min, the volume flow rate is 0.8 mL / min, and the CO2 is continued to be introduced for 1 h.

[0080] Optimal process validation:

[0081] The optimal process combination conditions A1B2C2D1 were verified, and the results of five batches of experiments are shown in Table 6. The relative standard deviations (RSDs) of the recovery rate and drug loading of phloretin solid dispersion were 1.81% and 1.13%, respectively, both less than 2%, indicating that the process of preparing phloretin solid dispersion by supercritical CO2 antisolvent method is stable and feasible.

[0082] Table 6. Optimal process verification

[0083]

[0084] Characterization and analysis of phloretin solid dispersion

[0085] SEM analysis: Figure 6 The SEM images of phloretin API, PVP K30, physical mixture of phloretin and PVP K30, and phloretin solid dispersion are shown in order. It can be seen from the figure that the API has a rod-like morphology, PVP K30 has a concave spherical shape, the physical mixture has not changed the morphology of the two, and the phloretin solid dispersion prepared by the supercritical process has a regular spherical shape and is evenly distributed.

[0086] XRD analysis: X-ray diffraction analysis was performed on the phloretin API, PVP K30, physical mixture and solid dispersion. The results are as follows: Figure 7 As shown in the figure, the physical mixture basically includes all the diffraction peaks of the raw material drug, but the absorption intensity becomes weaker. The phloretin solid dispersion prepared by the supercritical process has no obvious diffraction peaks, and the diffraction peaks in the raw material drug basically disappear, indicating that there is no drug crystal in the solid dispersion, and the drug exists in an amorphous state.

[0087] FTIR analysis: Figure 8These are infrared spectra of phloretin API, PVP K30, physical mixture of phloretin and PVP K30, and phloretin solid dispersion. It can be seen from the figure that the characteristic peak of the physical mixture is just a simple superposition of the API and the carrier, and the characteristic peak of the solid dispersion is offset compared to the API, indicating that the phloretin solid dispersion prepared by the supercritical process has undergone corresponding changes.

[0088] In vitro dissolution test

[0089] According to the first basket method of the dissolution determination in the 2020 edition of the Chinese Pharmacopoeia, 900 mL of phosphate buffer solution pH 6.8 was used as the dissolution medium. At 37°C and 100 r / min, 10 mL of samples were taken at 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, and 180 min, and an equal amount of medium was added at the same time. After the solution was filtered through a 0.22 μm water filter membrane, the absorbance was measured at a wavelength of 288, and the concentration was substituted into the standard curve to calculate the cumulative dissolution according to the formula. The dissolution curve is as follows: Fig. 9 As can be seen from the figure, the phloretin API showed a slow release trend, with a dissolution rate of less than 15% at 180 minutes. The phloretin solid dispersion prepared by the supercritical process had a faster release rate, with a cumulative release rate of about 82% at 180 minutes, which is 6 times the dissolution rate of the API. It can be seen that the solid dispersion prepared by the supercritical process significantly improved the dissolution performance of phloretin.

[0090] The above experimental results show that the phloretin solid dispersion prepared by supercritical technology has a more uniform distribution, smaller particle size, and significantly improved dissolution performance, thereby improving the bioavailability of the poorly soluble drug and overcoming the problem of organic solvent residues that is common in traditional technologies.

Claims

1. A method for preparing a phloretin solid dispersion using supercritical CO2 antisolvent technology, characterized in that: The method comprises the following steps: (1) dissolving phloretin and a carrier in an organic solvent to prepare a phloretin-carrier mixed solution; (2) adjusting the temperature in the crystallization kettle, passing CO2 into the crystallization kettle, and pressurizing it to a set value; (3) adjusting the CO2 flow rate to pump the phloretin-carrier solution into the crystallization kettle; (4) After the injection is completed, CO2 is continuously introduced and the pressure is released to collect the phloretin solid dispersion in the crystallization kettle.

2. The method according to claim 1, characterized in that In step (1), the organic solvent is a mixed solvent of ethanol and acetone.

3. The method according to claim 1, characterized in that In step (1), the carrier is polyvinyl pyrrolidone K30.

4. The method according to claim 1, characterized in that In step (1), the mass concentration of phloretin is 2-7 mg / mL.

5. The method according to claim 1, characterized in that In step (2), the temperature is 37-62°C.

6. The method according to claim 1, characterized in that In step (2), the pressure is 9 to 19 MPa.

7. The method according to claim 1, characterized in that In step (3), the CO2 flow rate is 3.5±0.2 L / min.

8. The method according to claim 1, characterized in that In step (3), the volume flow rate of the phloretin-carrier solution pumped in is 0.6 to 1.6 mL / min.

9. The method according to claim 1, characterized in that: In step (4), the time for continuing to introduce CO2 is 60 to 80 minutes.

10. A phloretin solid dispersion prepared by the method according to any one of claims 1 to 9, characterized in that: The phloretin solid dispersion is fluffy.

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