A microfluidic preparation method of nano-apigenin

Abstract

The present invention discloses a method for preparing nano-apigenin by a microfluidic field. Apigenin is dissolved in an organic solvent to obtain a homogeneous solution A; a surfactant is dissolved in deionized water to obtain a homogeneous solution B; the homogeneous solution A is used as the solvent phase, and the homogeneous solution B is used as the antisolvent phase, and they are simultaneously pumped into a microfluidic mixer for full reaction; the obtained nano-apigenin suspension is collected, the precipitate is separated by centrifugation and washed with water, and then an aqueous solution containing a freeze-drying protectant is added to completely dissolve the apigenin precipitate to re-obtain an apigenin suspension; preferably, the obtained apigenin suspension is freeze-dried to obtain the product. The present invention adopts a microfluidic device to strengthen the mixing process of the solvent and the antisolvent, uses a surfactant as a dispersant to regulate the transport efficiency and supersaturation of apigenin drug molecules, realizes the preparation of nano-apigenin, has simple operation, fast reaction speed and high safety.

Description

Technical Field

[0001] The present invention belongs to the field of micro-chemical engineering, and particularly relates to a method for preparing nano-apigenin by a microfluidic field method. Background Art

[0002] Apigenin is a dietary flavonoid that widely exists in various plants such as fruits, vegetables, beans, and tea. It is recognized as having various biological activities and values such as anti-inflammatory, anti-tumor, antioxidant, anti-anxiety, immunomodulatory, and cardiovascular protection, and has good market application prospects.

[0003] The solubility of apigenin is particularly poor. It is almost insoluble in water and has a very low solubility in organic solvents, resulting in low bioavailability. Solubilization methods for poorly soluble drugs include drug nano-sizing, solid dispersions, liposomes, inclusion complexes, self-emulsifying drug delivery systems, polymer nanoparticles, etc. However, these nano-preparation strategies usually have problems such as energy waste, low production efficiency, and high production costs due to the need for a large amount of carrier materials or large-scale instrument equipment. Summary of the Invention

[0004] Object of the Invention: To solve the problems of low solubility and low utilization rate of existing nano-apigenin. The present invention provides a method for preparing nano-apigenin by a microfluidic reaction technology, which is efficient, safe, environmentally friendly and can effectively reduce energy loss during the reaction process.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing nano-apigenin by a microfluidic field method, comprising the following steps:

[0007] (1) Dissolve apigenin in an organic solvent to obtain a homogeneous solution A;

[0008] (2) Dissolve a surfactant in deionized water to obtain a homogeneous solution B;

[0009] (3) Use the homogeneous solution A as the solvent phase and the homogeneous solution B as the antisolvent phase, and simultaneously pump them into a microfluidic mixer for full reaction;

[0010] (4) Collect the apigenin nano-suspension obtained from the reaction in step (3), centrifuge to separate the precipitate and wash it with water, then add an aqueous solution containing a lyophilization protectant to completely dissolve the apigenin precipitate, and re-obtain an apigenin suspension;

[0011] (5) Freeze-dry the apigenin suspension obtained in step (4) to obtain the product.

[0012] Specifically, in step (1), the organic solvent is selected from any one of dimethyl sulfoxide, ethanol, and benzene, preferably dimethyl sulfoxide; the concentration of apigenin in the homogeneous solution A is 5-30 mg / mL, preferably 10 mg / mL.

[0013] Specifically, in step (2), the surfactant is selected from any one or a mixture of two or more of sodium dodecylbenzenesulfonate, cetyltrimethylammonium bromide, and dodecyldimethylbetaine, preferably a mixture of cetyltrimethylammonium bromide and dodecyldimethylbetaine; in the homogeneous solution B, the mass concentration of the surfactant is 0.1%-1.0%.

[0014] Specifically, in step (3), the pumping rate of the homogeneous solution A is 0.01-1.5 mL / min, preferably 0.125 mL / min; the pumping rate of the homogeneous solution B is 2.5-50 mL / min, preferably 2.5 mL / min.

[0015] Specifically, in step (3), the flow rate ratio of the solvent phase to the antisolvent phase in the microfluidic mixer is 1:1-1:20, preferably 1:5.

[0016] Preferably, in step (3), the volume of the microfluidic mixer is 15-25 mL, the reaction temperature is 0-30 °C, and the reaction residence time is 4-6 min.

[0017] Specifically, in step (3), the homogeneous solution A and the homogeneous solution B are respectively injected into the microfluidic mixer by a precision syringe pump; a corresponding pressure gauge is provided at the outlet of the precision syringe pump;

[0018] The microfluidic mixer adopts any one of a T-shaped mixer, a Y-shaped mixer, a serpentine micro mixer, or an ellipsoidal internal member micro mixer; a corresponding pressure gauge is provided at the reaction liquid outlet of the microfluidic mixer.

[0019] Preferably, in step (4), the centrifugation speed is controlled at 10000-12500 r / min, and the centrifugation time is 10-15 min.

[0020] Preferably, in step (4), the solution containing the lyoprotectant includes, but is not limited to, any one of mannitol solution, glucose solution, and sucrose solution; the concentration of the apigenin precipitate in the solution containing the lyoprotectant is 5-20 mg / mL.

[0021] Further, in step (10), before freeze-drying, pre-freeze at -40 °C for 2-3 h, and then freeze-dry at -15-0 °C for 2-3 days.

[0022] Beneficial effects:

[0023] (1) The present invention uses a microfluidic device to strengthen the mixing process of the solvent and the anti-solvent. Using a surfactant as a dispersant, it regulates the transport efficiency and supersaturation of apigenin drug molecules to achieve the preparation of nano-apigenin. The operation is simple, the reaction speed is fast, and the safety is high.

[0024] (2) The present invention uses a microfluidic device to explore the molecular transport process of apigenin in the solvent-anti-solvent, and examines the effects of the flow rate ratio, flow rate, raw material drug concentration, surfactant, etc. on the particle size, providing a new idea for the preparation of nano-apigenin by microfluidic technology.

[0025] (3) The present invention discovers for the first time that compared with the use of a single surfactant, the use of a mixed surfactant can have a significant impact on the reduction of the particle size. In particular, the combined use of cetyltrimethylammonium bromide and dodecyldimethylbetaine can significantly reduce the particle size of apigenin particles, showing unexpected technical effects. Description of the Drawings

[0026] The following further describes the present invention in detail in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0027] Figure 1 is a schematic diagram of the preparation of nano-apigenin using the microfluidic device adopted by the present invention.

[0028] Figure 2 is the particle size distribution curve of the nano-apigenin prepared in Examples 1-6.

[0029] Figure 3 is the scanning electron microscope photograph of the nano-apigenin prepared in Examples 1-3.

[0030] Figure 4 is the particle size distribution curve of the nano-apigenin prepared in Examples 1 and 7-9. Detailed Description of the Invention

[0031] According to the following embodiments, the present invention can be better understood.

[0032] As Figure 1 shown, the microfluidic device adopted by the present invention includes 2 precision syringe pumps, 3 pressure gauges, 1 microfluidic mixer, 1 sample collection device, and several connecting components. Among them, the microfluidic mixer uses an ellipsoidal inner microreactor, referring to ZL2021114510508, with a volume of 15 mL.

[0033] Example 1:

[0034] Dissolve 1 g of apigenin in an appropriate amount of dimethyl sulfoxide solvent to obtain a homogeneous solution A with a concentration of 20 mg / mL; dissolve the surfactant sodium dodecylbenzenesulfonate (SDBS) in deionized water to obtain a homogeneous solution B with a concentration of 0.1 wt%. Use two syringes with a specification of 50 mL each to aspirate the prepared solution A and solution B respectively. Fix the syringes on a precision syringe pump. Under the condition of 25 °C, start the precision syringe pump and pump the solutions into the ellipsoidal internal component microreactor at a flow rate ratio of A:B = 1:20 (the flow rate of homogeneous solution A is 1 mL / min, and the flow rate of homogeneous solution B is 20 mL / min). After reacting for 5 min, collect the apigenin nano-suspension at the outlet, centrifuge it at 12,500 r / min for 10 min, wash the precipitate with water. Then take 0.2 g of the precipitate and add it to 10 mL of a mannitol solution containing a freeze-drying protectant to completely dissolve the apigenin precipitate and obtain a new apigenin suspension. First, pre-freeze it in a -40 °C ultra-low temperature storage box for 2 h, and then put it into a freeze-dryer and freeze-dry it at -4 °C for 64 h. Detect its particle size using a laser particle size analyzer.

[0035] Example 2:

[0036] Dissolve 1 g of apigenin in an appropriate amount of dimethyl sulfoxide solvent to obtain a homogeneous solution A with a concentration of 20 mg / mL; dissolve the surfactant dodecyldimethylbetaine (BS-12) in deionized water to obtain a homogeneous solution B with a concentration of 0.1 wt%. Use two syringes with a specification of 50 mL each to aspirate the prepared solution A and solution B respectively. Fix the syringes on a precision syringe pump. Under the condition of 25 °C, start the precision syringe pump and pump the solutions into the ellipsoidal internal component microreactor at a flow rate ratio of A:B = 1:20 (the flow rate of homogeneous solution A is 1 mL / min, and the flow rate of homogeneous solution B is 20 mL / min). After reacting for 5 min, collect the apigenin nano-suspension at the outlet, centrifuge it at 12,500 r / min for 10 min using a centrifuge, wash the precipitate with water. Then take 0.2 g of the precipitate and add it to 10 mL of a mannitol solution containing a freeze-drying protectant to completely dissolve the apigenin precipitate and obtain a new apigenin suspension. First, pre-freeze it in a -40 °C ultra-low temperature storage box for 2 h, and then put it into a freeze-dryer and freeze-dry it at -4 °C for 64 h. Detect its particle size using a laser particle size analyzer.

[0037] Example 3:

[0038] Dissolve 1 g of apigenin in an appropriate amount of dimethyl sulfoxide solvent to obtain a homogeneous solution A with a concentration of 20 mg / mL; dissolve the surfactant cetyltrimethylammonium bromide (CTAB) in deionized water to obtain a homogeneous solution B with a concentration of 0.1 wt%. Use two syringes with a specification of 50 mL each to aspirate the prepared solution A and solution B respectively. Fix the syringes on a precision syringe pump. Under the condition of 25 °C, start the precision syringe pump and pump them into the ellipsoidal internal component microreactor according to a flow rate ratio of A:B = 1:20 (the flow rate of the homogeneous solution A is 1 mL / min, and the flow rate of the homogeneous solution B is 20 mL / min). After reacting for 5 min, collect the apigenin nano-suspension at the outlet, centrifuge it at 12,500 r / min for 10 min, wash the precipitate with water. Then take 0.2 g of the precipitate and add it to a 10 mL mannitol solution containing a freeze-drying protectant to completely dissolve the apigenin precipitate. After re-obtaining the apigenin suspension, pre-freeze it in a -40 °C ultra-low temperature storage box for 2 h, and then put it into a freeze-dryer to freeze-dry at -4 °C for 64 h. Detect its particle size with a laser particle size analyzer.

[0039] Example 4:

[0040] Dissolve 1 g of apigenin in an appropriate amount of dimethyl sulfoxide solvent to obtain a homogeneous solution A; dissolve the mixed surfactant dodecyldimethylbetaine / sodium hexadecylbenzenesulfonate (BS-12 / SDBS) in deionized water to obtain a homogeneous solution B with a mixed surfactant concentration of 0.4% (the concentration of dodecyl betaine is 0.2 wt%, and the concentration of sodium hexadecylbenzenesulfonate is 0.2 wt%). Use two syringes with a specification of 50 mL each to aspirate the prepared solution A and solution B respectively. Fix the syringes on a syringe pump. Under the condition of 25 °C, start the precision syringe pump and pump them into the ellipsoidal internal component microreactor according to a flow rate ratio of A:B = 1:20 (the flow rate of the homogeneous solution A is 1 mL / min, and the flow rate of the homogeneous solution B is 20 mL / min). After reacting for 5 min, collect the apigenin nano-suspension at the outlet, centrifuge it at 12,500 r / min, wash the precipitate with water. Then take 0.2 g of the precipitate and add it to a 10 mL mannitol solution containing a freeze-drying protectant to completely dissolve the apigenin precipitate. After re-obtaining the apigenin suspension, pre-freeze it in a -40 °C ultra-low temperature storage box for 2 h, and then put it into a freeze-dryer to freeze-dry at -4 °C for 64 h. Detect its particle size with a laser particle size analyzer.

[0041] Example 5:

[0042] Dissolve 1 g of apigenin in an appropriate amount of dimethyl sulfoxide solvent to obtain a homogeneous solution A with a concentration of 20 mg / mL; dissolve the mixed surfactant dodecyldimethylbetaine / cetyltrimethylammonium bromide (BS-12 / CTAB) in deionized water to obtain a homogeneous solution B with a mixed surfactant concentration of 0.4 wt% (the concentration of dodecyl betaine is 0.2 wt%, and the concentration of cetyltrimethylammonium bromide is 0.2 wt%); use two syringes with a specification of 50 mL each to suck up the prepared solutions A and B respectively, fix the syringes on an injection pump, and at 25 °C, start the injection pump to deliver to the ellipsoidal internal component microreactor according to a flow rate ratio of A:B = 1:20 (the flow rate of the homogeneous solution A is 1 mL / min, and the flow rate of the homogeneous solution B is 20 mL / min). After reacting for 5 min, collect the apigenin nano-suspension at the outlet, centrifuge at 12500 r / min for 10 min, wash the precipitate with water, then take 0.2 g of the precipitate, add it to a mannitol solution containing 10 mL of freeze-drying protectant to completely dissolve the apigenin precipitate, and after re-obtaining the apigenin suspension, pre-freeze it in a -40 °C ultra-low temperature storage box for 2 h, and then put it into a freeze-dryer to freeze-dry at -4 °C for 64 h, and detect its particle size through a laser particle size analyzer.

[0043] Example 6:

[0044] Dissolve 1 g of apigenin in an appropriate amount of dimethyl sulfoxide solvent to obtain a homogeneous solution A with a concentration of 20 mg / mL; dissolve the mixed surfactant sodium dodecylbenzenesulfonate / cetyltrimethylammonium bromide (SDBS / CTAB) in deionized water to obtain a homogeneous solution B with a mixed surfactant concentration of 0.4 wt% (the concentration of sodium dodecylbenzenesulfonate is 0.2 wt%, and the concentration of cetyltrimethylammonium bromide is 0.2 wt%); use two syringes with a specification of 50 mL each to suck up the prepared solutions A and B respectively, fix the syringes on an injection pump, and at 25 °C, start the injection pump to deliver to the ellipsoidal internal component microreactor according to a flow rate ratio of A:B = 1:20 (the flow rate of the homogeneous solution A is 1 mL / min, and the flow rate of the homogeneous solution B is 20 mL / min). After reacting for 5 min, collect the apigenin nano-suspension at the outlet, centrifuge at 12500 r / min for 10 min, wash the precipitate with water, then take 0.2 g of the precipitate, add it to a mannitol solution containing 10 mL of freeze-drying protectant to completely dissolve the apigenin precipitate, and after re-obtaining the apigenin suspension, pre-freeze it in a -40 °C ultra-low temperature storage box for 2 h, and then put it into a freeze-dryer to freeze-dry at -4 °C for 64 h, and detect its particle size through a laser particle size analyzer.

[0045] Example 7:

[0046] Dissolve 1 g of apigenin in an appropriate amount of dimethyl sulfoxide solvent to obtain a homogeneous solution A with a concentration of 20 mg / mL; dissolve the surfactant sodium dodecylbenzenesulfonate (SDBS) in deionized water to obtain a homogeneous solution B with a concentration of 0.1 wt%. Use two syringes with a specification of 50 mL each to suck the prepared solutions A and B respectively, fix the syringes on an injection pump, and at 25 °C, start the injection pump to transport them to an ellipsoidal internal member microreactor according to a flow rate ratio of A:B = 1:10 (the flow rate of homogeneous solution A is 1 mL / min, and the flow rate of homogeneous solution B is 10 mL / min). After reacting for 5 min, collect the apigenin nano-suspension at the outlet, centrifuge it at 12500 r / min for 10 min, wash the precipitate with water, then take 0.2 g of the precipitate and add it to a mannitol solution containing 10 mL of freeze-drying protectant to completely dissolve the apigenin precipitate. After re-obtaining the apigenin suspension, pre-freeze it in a -40 °C ultra-low temperature storage box for 2 h, and then put it into a freeze-dryer for freeze-drying at -4 °C for 64 h. Detect its particle size through a laser particle size analyzer.

[0047] Example 8:

[0048] Dissolve 1 g of apigenin in an appropriate amount of dimethyl sulfoxide solvent to obtain a homogeneous solution A with a concentration of 20 mg / mL; dissolve the surfactant sodium dodecylbenzenesulfonate (SDBS) in deionized water to obtain a homogeneous solution B with a concentration of 0.1 wt%. Use two syringes with a specification of 50 mL each to suck the prepared solutions A and B respectively, fix the syringes on an injection pump, and at 25 °C, start the injection pump to transport them to an ellipsoidal internal member microreactor according to a flow rate ratio of A:B = 1:5 (the flow rate of homogeneous solution A is 2 mL / min, and the flow rate of homogeneous solution B is 10 mL / min). After reacting for 5 min, collect the apigenin nano-suspension at the outlet, centrifuge it at 12500 r / min for 10 min, wash the precipitate with water, then take 0.2 g of the precipitate and add it to a mannitol solution containing 10 mL of freeze-drying protectant to completely dissolve the apigenin precipitate. After re-obtaining the apigenin suspension, pre-freeze it in a -40 °C ultra-low temperature storage box for 2 h, and then put it into a freeze-dryer for freeze-drying at -4 °C for 64 h. Detect its particle size through a laser particle size analyzer.

[0049] Example 9:

[0050] Dissolve 1 g of apigenin in an appropriate amount of dimethyl sulfoxide solvent to obtain a homogeneous solution A with a concentration of 20 mg / mL; dissolve the surfactant sodium dodecylbenzenesulfonate (SDBS) in deionized water to obtain a homogeneous solution B with a concentration of 0.1 wt%. Use two syringes with a specification of 50 mL each to suck the prepared solutions A and B respectively, fix the syringes on the syringe pump, and start the syringe pump to transport them to the ellipsoidal internal component microreactor at 25 °C according to the flow rate ratio of A:B = 1:2 (the flow rate of the homogeneous solution A is 5 mL / min, and the flow rate of the homogeneous solution B is 10 mL / min). After reacting for 5 min, collect the apigenin nano-suspension at the outlet, centrifuge it at 12500 r / min for 10 min, wash the precipitate with water, then take 0.2 g of the precipitate and add it to a 10 mL mannitol solution containing a freeze-drying protectant to completely dissolve the apigenin precipitate. After re-obtaining the apigenin suspension, pre-freeze it in a -40 °C ultra-low temperature storage box for 2 h, and then put it into a freeze-dryer for freeze-drying at -4 °C for 64 h. Detect its particle size with a laser particle size analyzer.

[0051] Table 1 shows the particle size distribution of nano-apigenin obtained in Examples 1 to 9. It can be seen from the table that the particle sizes of the single surfactant SDBS at D 10 , D 50 , D 90 are all smaller than those of the single surfactants CTAB and BS-12, which are 97.50 nm, 129.77 nm, and 297.43 nm respectively. In addition, the particle sizes of the surfactants D 10 , D 50 , D 90 of the CTAB / BS-12 combination are all smaller than those of the other 5 combinations, which are 91.28 nm, 114.05 nm, and 177.15 nm respectively, and its PDI is also the narrowest. As the flow rate ratio increases from 1:20 to 1:2, the particle size of the apigenin particles gradually becomes larger. Among them, the flow rate ratios of 1:20 and 1:5 have smaller particle sizes and narrower particle size distributions than 1:10.

[0052] Table 1

[0053]

[0054] , Figure 2 is the particle size distribution curve of nano-apigenin prepared in Examples 1 - 6. It can be seen that the particle sizes of CTAB / BS-12 are concentrated in the range of 90 - 180 nm, and the particle size distribution is relatively narrow; the particle sizes of SDBS / CTAB are larger, mainly distributed in the range of 300 - 1000 nm; CTAB, SDBS, and SDBS / BS-12 show bimodal distributions, with a small part of larger particle sizes, which increases the PDI. The results show that the surfactant CTAB / BS-12 has the best effect of inhibiting the growth and aggregation between apigenin particles.

[0055] Figure 3 It is the scanning electron microscope photograph of the nano-apigenin prepared in Examples 1-3. It can be seen that: the CTAB and BS-12 nanoparticles are flaky, and the SDBS has relatively good dispersibility and can form spherical nanoparticles with uniform size and smaller particle size.

[0056] Figure 4 It is the particle size distribution curve of the nano-apigenin prepared in Example 1 and Examples 7-9. It can be seen that: when the flow rate increases from 0.125 mL / min to 1 mL / min, the particle size of the apigenin particles gradually decreases. The D10, D50, and D90 at the flow rate of 1 mL / min are the smallest and are 1 / 2 of those at the flow rate of 0.25 mL / min. The results show that: the larger the flow rate, the smaller the particle size. When the flow rate is 1 mL / min, its particle size is small and the particle size distribution is narrow.

[0057] The present invention provides an idea and method for the preparation method of nano-apigenin in a microfluidic field. There are many methods and ways to specifically implement this technical solution. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be realized by the prior art.

Claims

1. A microfluidic preparation method of nano-apigenin, characterized in that, It includes the following steps: (1) Dissolve apigenin in an organic solvent to obtain a homogeneous solution A; (2) Dissolve a surfactant in deionized water to obtain a homogeneous solution B; (3) Use the homogeneous solution A as the solvent phase and the homogeneous solution B as the antisolvent phase, and simultaneously pump them into a microfluidic mixer for full reaction; (4) Collect the apigenin nano-suspension obtained from the reaction in step (3), centrifuge to separate the precipitate and wash it with water, then add an aqueous solution containing a freeze-drying protectant to completely dissolve the apigenin precipitate to re-obtain an apigenin suspension; (5) Perform freeze-drying on the apigenin suspension obtained in step (4) to obtain the product; In step (1), the organic solvent is dimethyl sulfoxide; the concentration of apigenin in the homogeneous solution A is 20 mg / mL; In step (2), the surfactant is a mixture of cetyltrimethylammonium bromide and dodecyldimethylbetaine; in the homogeneous solution B, the mass concentration of the surfactant is 0.1% - 1.0%; In step (3), the pumping rate of the homogeneous solution A is 1 mL / min, and the pumping rate of the homogeneous solution B is 20 mL / min; In step (3), the flow rate ratio of the solvent phase to the antisolvent phase in the microfluidic mixer is 1:20; In step (3), the volume of the microfluidic mixer is 15 - 25 mL, the reaction temperature is 0 - 30 °C, and the reaction residence time is 4 - 6 min.

2. The microfluidic preparation method of nanoapigenin according to claim 1, characterized in that, In step (3), the homogeneous solution A and the homogeneous solution B are respectively injected into the microfluidic mixer using a precision syringe pump; a corresponding pressure gauge is provided at the outlet of the precision syringe pump; The microfluidic mixer uses any one of a T-shaped mixer, a Y-shaped mixer, a serpentine micro-mixer, or an ellipsoidal internal component micro-mixer; a corresponding pressure gauge is provided at the reaction liquid outlet of the microfluidic mixer.

3. The microfluidic preparation method of nano-apigenin according to claim 1, characterized in that, In step (4), the centrifugation speed is controlled at 10000 - 12500 r / min, and the centrifugation time is 10 - 15 min.

4. The microfluidic preparation method of nanoapigenin according to claim 1, characterized in that In step (4), the solution containing the freeze-drying protectant is any one of a mannitol solution, a glucose solution, or a sucrose solution; the concentration of the apigenin precipitate in the solution containing the freeze-drying protectant is 5 - 20 mg / mL.

5. The microfluidic preparation method of nano-apigenin according to claim 1, characterized in that, In step (10), before freeze-drying, pre-freeze at -40 °C for 2 - 3 h, and then perform freeze-drying at -15 - 0 °C for 2 - 3 days.

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