Silybin lecithin compound nano-particles as well as preparation method and application thereof

Abstract

The invention discloses a silybin lecithin compound nanoparticle and a preparation method and application thereof, the preparation method comprises the following steps: completely dissolving lecithin and silybin in an organic solvent to prepare an internal phase solution; dissolving a freeze-drying protective agent and a stabilizer in deionized water to prepare an external phase solution; the two-phase solution is mixed in a micro-fluidic device in a coaxial confocal form, and the silybin lecithin carried by the inner-phase organic solvent is spontaneously assembled to form nano-scale particles in the process of diffusing the inner-phase organic solvent to the outer-phase solution; the silybin and lecithin compound nano-particles have good dispersibility and stability, the particle size is 150-500 nm, the particle size dispersity is 0.1-0.3, the system composition is simple, the biocompatibility is good, lecithin serves as a carrier, nano-scale release of silybin in the intestinal tract can be achieved, the solubility of indissolvable drugs is increased, and the bioavailability of the silybin is improved. Wide application prospects are realized in the aspect of promoting medicine absorption and utilization.

Description

Technical Field

[0001] The invention relates to silybin lecithin complex nanoparticles, and also relates to a preparation method and application of the complex nanoparticles. Background Art

[0002] Silibinin (SIB) is a flavonoid lignin compound extracted and separated from the fruit of the Asteraceae plant Silybum marianum. It is also known as 2,3-dihydro-3-(4-hydroxy-3-methoxyphenyl)-2-hydroxymethyl-6-(3,5,7-trihydroxy-4-oxobenzopyran-2-yl)benzodioxane. It is a mixture of silibinin A and silibinin B in approximately equal molar ratios. Its molecular formula is C 25 H 22 O 10 , molecular weight is 482.436. The morphological characteristics are light yellow powder, odorless, slightly bitter, hygroscopic, insoluble in water and chloroform, easily soluble in alkaline aqueous solution, slightly soluble in methanol and ethanol, soluble in organic solvents such as acetone and ethyl acetate. The structural formulas of silybin A (left) and silybin B (right) are as follows:

[0003]

[0004] Current studies have shown that silybin has an inhibitory effect on lipoxygenase and peroxidase, and has the ability to resist oxidation, lipid peroxidation, fibrosis, maintain cell membrane stability and regulate liver regeneration. It is mainly used in the treatment of acute and chronic hepatitis, fatty liver, cirrhosis, alcohol-induced liver damage, metabolic toxic liver damage, etc. Recent studies have shown that silybin also has the effects of lowering blood lipids, protecting the myocardium, and anti-diabetes. In addition, its excellent performance in anti-cancer has also attracted more and more attention from domestic and foreign researchers. Due to its extremely low water solubility, silybin has poor oral absorption and extremely low bioavailability. In order to solve the problems of poor stability, poor water solubility, and low bioavailability of silybin, current research is mainly focused on making it salt, cocrystal or prepared into emulsions, polymer nanoparticles, etc. Emulsions and polymer nanoparticles have the defects of easy aggregation, easy demulsification, and poor solution stability. Currently, the silibinin preparations on the market at home and abroad are mainly silibinin capsules, silibinin meglumine tablets, etc. Reducing the particle size of silibinin can further improve the release and absorption efficiency of the raw material.

[0005] Phospholipid complexes are relatively stable compounds or complexes formed by drugs and phospholipid molecules through charge migration. The preparation method is simple and the cost is low. In recent years, their application in pharmaceutical preparations has gradually increased. The methods commonly used to prepare phospholipid complexes into nano-scale particles are high-pressure homogenization, direct instillation, and thin film hydration. These preparation methods often require a large amount of organic solvents, have complicated steps, and high energy consumption. The prepared particles are not uniform, and it is difficult to control the particle size, which is not conducive to achieving quality control of the production of phospholipid complex nanoparticles.

[0006] Microfluidic technology provides a continuous production platform for controllable and repeatable preparation of nanomedicines. Its advantages lie in the ability to digitally simulate flow processes, design channel structures, and timely adjust process parameters. It can achieve one-step formation of nanoparticles. Compared with conventional methods, the nanoparticles prepared are uniform and of controllable size, which is of great significance for the stable release of drugs, maintaining stable blood drug concentrations, and improving absorption efficiency. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to provide a silybin phosphatidylcholine complex nanoparticles, and also provide a preparation method and application of the above-mentioned complex nanoparticles.

[0008] Technical solution: The present invention discloses a method for preparing silybin phosphatidylcholine complex nanoparticles, comprising the following steps:

[0009] (a) dissolving lecithin and silybin in an organic solvent to prepare an internal phase solution;

[0010] (b) dissolving a lyophilization protectant and a stabilizer in water to prepare an external phase solution;

[0011] (c) using microfluidics technology, dispersing the complex formed in the internal phase solution in the external phase solvent for co-precipitation to obtain a silybin lecithin complex nanodispersion;

[0012] (d) The silybin phosphatidylcholine complex nano-dispersion solution is subjected to rotary evaporation and then freeze-dried to obtain silybin phosphatidylcholine complex nano-particles.

[0013] Wherein, in step (a), the drug-lipid ratio of silybin to lecithin is 2:1-1:2 (w / w), the concentration of silybin is 1-15 mg / ml, and the mass concentration of lecithin is 1-15 mg / ml.

[0014] Wherein, in step (a), the organic solvent includes methanol, ethanol or acetone, and the lecithin is soybean lecithin or egg yolk lecithin.

[0015] Wherein, in step (b), the stabilizer includes sodium dodecyl sulfate (SDS), Tween 80, polyvinyl pyrrolidone (PVP-K30), hydroxypropyl methylcellulose (HPMC) or poloxamer 188, and the mass concentration of the stabilizer in the external phase solution is 0.1% to 0.5%.

[0016] Wherein, in step (b), the lyophilization protective agent includes sucrose or D mannitol, and the mass concentration of the lyophilization protective agent in the external phase solution is 1% to 5%.

[0017] Wherein, in step (c), the microfluidic technology, the total flow rate of the inner phase solution and the outer phase solution is 100-500 μL / min, and the flow rate ratio of the inner phase solution to the outer phase solution is 1:1-1:20.

[0018] Among them, the microfluidic technology adopts a device composed of an inner phase capillary nested in a stainless steel tube, a dispensing needle, a square glass tube, and a receiving capillary glass tube nested in the stainless steel tube, which are connected in sequence, and a glass slide is provided at the bottom; wherein the dispensing needle is vertically fixed at the intersection of the inner phase capillary and the square glass tube, and the stainless steel tube extends out and fixes both sides of the glass slide.

[0019] Specifically, the assembly of the microfluidic device is as follows: the square glass tube is fixed in the center of the glass slide by epoxy resin glue, the injection capillary and the collection capillary are inserted into the square glass tube from both ends, and the injection needle is vertically placed at the interface between the injection capillary and the square tube; two iron tubes are respectively inserted into the injection capillary and the outflow capillary, and fixed to the edge of the glass slide and extended half the length, used to connect the polytetrafluoroethylene hose to prevent the capillary from breaking. The internal and external phase solutions flow into the microfluidic device through the polytetrafluoroethylene hose under the push of the injection pump; the outflow port is guided into the receiving bottle through the polytetrafluoroethylene hose.

[0020] Among them, the tip diameter of the injection capillary glass tube is 50-150μm. The tip is obtained by stretching the capillary with a needle puller, and then the tip is polished to the target size using 2000 mesh sandpaper to obtain the inner phase capillary; the injection needle is an 18G dispensing needle, and the lower end of the needle has two pre-processed grooves for fixing the capillary and the square tube.

[0021] The present invention also discloses silybin lecithin complex nanoparticles prepared by the above-mentioned preparation method, with a particle size range of 150-500nm and a polydispersity index range of 0.1-0.3; the nanoparticles have a small particle size and good polydispersity, and can effectively improve the water solubility and gastrointestinal release and absorption capacity of silybin.

[0022] The silybin phosphatidylcholine complex nanoparticles can also be used in the preparation of silybin oral medicines; the processed and prepared solid preparations have good long-term stability and can be adapted to long-term storage and transportation.

[0023] Principle of the invention: The silybin-lecithin complex of the present invention is a relatively stable compound or complex formed by the drug and phospholipid molecules through charge migration. Silybin and lecithin are closely connected in an organic solvent through the van der Waals force generated by the polar parts. Then, the complex dissolved in the organic solvent is quickly and evenly dispersed into an aqueous phase containing a stabilizer and a freeze-drying protective agent by a microfluidic coprecipitation method. As the aqueous phase environment improves, the long-chain molecules of the lecithin hydrophobic ends in the complex are polymerized and bent to form a spherical structure that wraps the raw material drug. At the same time, the high molecular weight stabilizer present in the aqueous phase is adsorbed in the spherical structure, which not only prevents electrostatic adsorption between charged particles, but also can net the complex sphere to prevent the leakage of the raw material drug, thereby improving the recombination rate of the complex particles.

[0024] Specifically, the flow focusing principle is adopted, and an injection pump is used to pass the internal and external phase solvents into the microfluidic device. The two solvents meet at a certain flow rate ratio in the microfluidic device, and the internal and external phase solutions are quickly mixed in the device. The internal phase solution is squeezed into a thin stream by the external phase solution at a higher flow rate, thereby increasing the contact area between the internal phase solvent and the external phase solvent. After the silybin lecithin complex in the internal phase solvent contacts the water phase, it is dispersed and precipitated into nano-scale particles as it flows and mixes, and flows out of the device as the solvent flows. A polytetrafluoroethylene hose is used to introduce the mixed solution into a receiving bottle, and the silybin lecithin complex suspended nanoparticles are obtained. The silybin lecithin complex precipitation rate can be adjusted by using the flow focusing principle of the microfluidic device.

[0025] The preparation method of the present invention is simple and low in cost. The drug forming the complex can improve the total dissolution rate of the drug in the aqueous solution by utilizing the hydrophilicity of the phospholipid molecules. After being taken through the long-chain lipid port of the phospholipid molecules, microemulsions are spontaneously formed in the gastrointestinal tract through bile salt and peristalsis, releasing the prototype drug, which is beneficial to the aggregation and release of the drug in the intestine. At the same time, lecithin is an important component of the cell membrane, which is beneficial to the transmembrane absorption of the drug, and thus the bioavailability can be significantly improved. Through the operation of microfluidic flow focusing and coprecipitation, the complex can be directly prepared into nanometer-sized particles, increasing the specific surface area of ​​the drug, which is more conducive to promoting drug dissolution and absorption by the human body.

[0026] Therefore, the present invention uses microfluidic technology to prepare silybin phosphatidylcholine complex nanoparticles. The silybin phosphatidylcholine complex dissolved in an organic solvent is added to an aqueous phase through a microfluidic coprecipitation method to directly form precipitated particles. At the same time, a stabilizer and a lyophilization protectant in the aqueous phase will maintain the suspension of the precipitated particles to prevent aggregation, thereby maintaining the particle size and forming lyophilized particles. The obtained nanoparticles have good long-term stability.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The silybin phosphatidylcholine complex of the present invention has the characteristics of small particle size, high dispersibility, good stability, etc., and can significantly improve the dispersibility of the poorly soluble drug silybin in the aqueous solution system. The freeze-dried product has a fast dispersion speed and good stability after reconstitution; the entire system is green and harmless, has good biocompatibility, and has broad application prospects; (2) The raw materials of the preparation method of the present invention are simple and easy to obtain, and the cost is low; the microfluidic fluid has its unique microscale characteristics, which can break through the limitations of conventional methods, and the product particle size can be controlled by adjusting the flow rate, flow rate ratio, capillary caliber and other methods; (3) The preparation conditions of the flow focusing capillary glass microfluidic device are mild, which will not affect the formation of silybin phosphatidylcholine complex nanoparticles. The preparation process is continuous, the operation is convenient and simple, and large-scale production can be achieved through high throughput and other methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the microfluidic device, including inner phase capillary glass tube-1; receiving capillary glass tube-2; square glass tube-3; glass slide-4; dispensing needle-5; stainless steel tube-6;

[0029] Figure 2 Process flow diagram for preparing nanoparticles for microfluidics;

[0030] Figure 3 This is a graph showing the effect of different solvents and different flow rate ratios on the particle size of the prepared nanoparticles;

[0031] Figure 4 The result diagram of the complexation rate of lecithin complex prepared by different solvents;

[0032] Figure 5 The figure shows the effect of stabilizer on the particle size, monodispersity and recombination rate of prepared nanoparticles, where (a) shows the particle size and monodispersity results, and (b) shows the recombination rate results;

[0033] Figure 6 The results of the effect of the internal phase solvent concentration on the particle size, monodispersity and recombination rate of the prepared nanoparticles are shown in Figure 1, where (a) is the particle size and monodispersity results, and (b) is the recombination rate result;

[0034] Figure 7 The results of the drug-lipid ratio on the particle size, monodispersity and composite rate of the prepared nanoparticles are shown in Figure 1, where (a) is the particle size and monodispersity results, and (b) is the composite rate result;

[0035] Figure 8 This is the result diagram of the effect of the stirring time of the internal phase solution on the recombination rate;

[0036] Fig. 9 This is the particle size and potential distribution diagram of silybin phosphatidylcholine complex nanoparticles;

[0037] Fig.10 This is a transmission electron microscopy image of silybin phosphatidylcholine complex nanoparticles;

[0038] Fig.11 Comparison of Fourier transform infrared spectra of silybin lecithin complex nanoparticles, physical mixture, PVP-K30, D-mannitol, and silybin API;

[0039] Fig.12 This is the in vitro release result of silybin phosphatidylcholine complex nanoparticles. DETAILED DESCRIPTION

[0040] The technical scheme of the present invention is further described below in conjunction with the embodiments. The test materials used in the embodiments can be purchased through conventional channels. The experimental schemes without specifying specific conditions in the embodiments are all carried out under normal temperature and pressure (25°C, 101.325kPa).

[0041] Magnetic stirrer (IKARET basic, Germany);

[0042] Inverted microscope (ICX41, Ningbo Sunny Optical Instrument Co., Ltd.);

[0043] High-speed camera (Hefei Fuhuang Junda Hi-Tech Information Technology Co., Ltd. 2F04M);

[0044] Syringe pump (TYD01-01-CE, Baoding Leifu Fluid Technology Co., Ltd.);

[0045] Laser particle size analyzer (Anton Paar Litesizer 500, Austria);

[0046] Transmission electron microscope (Hitachi HT7800, Japan);

[0047] Needle pulling instrument (Japanese NarishigePC-100);

[0048] UV-visible spectrophotometer (Thermo Fisher GENESYS, USA) TM 180 UV-Vis);

[0049] Fourier transform infrared spectrometer (Bruker Tensor 27, Germany).

[0050] Example 1

[0051] Capillary glass microfluidic device construction:

[0052] like Figure 1As shown, the injection capillary and the collection capillary are cylindrical glass capillaries with an inner diameter of 0.35 mm and an outer diameter of 0.98 mm. Using a needle puller, the capillary glass tube is heated and melted from the middle and stretched to obtain a tip. Using 2000-mesh sandpaper, the tip of the capillary glass tube is polished to a diameter of 50-150 μm for the injection capillary; the collection capillary is to use sandpaper to polish the broken end of the capillary glass tube to a smooth surface to obtain a 350 μm caliber outflow capillary with smooth ends. The capillary polished to the target size is placed in an ethanol solution for ultrasonic cleaning several times until the capillary glass tube is transparent and free of dirt and glass chips under a microscope. Select a square capillary with an inner diameter of 1.02 mm and an outer diameter of 1.5 mm, use epoxy resin glue to fix the square capillary in the center of the glass slide, place the injection capillary and the collection capillary cones opposite to the central axis, insert the square capillary from both sides, observe under a microscope, and adjust the distance between the injection capillary and the collection capillary to 100 μm; embed a stainless steel tube in the part of the capillary extending from the glass slide at the left and right ends, so that the stainless steel tube extends out of the edge of the glass slide and is fixed with AB glue; select an 18G dispensing needle and fix it vertically at the interface between the internal phase capillary and the square capillary as the internal phase injection port; use AB glue to seal the stainless steel tube, capillary, square tube, dispensing needle and other joints to ensure that the device does not leak. Leave it overnight for 24 hours until the epoxy resin glue is completely cured. At this point, the capillary glass microfluidic device is prepared, where the a end is the internal phase flow inlet, the b end is the external phase flow inlet, and the c end is the sample flow outlet.

[0053] Example 2

[0054] Effects of total flux and FRR on particle size and PDI of silybin-phosphatidylcholine complex nanoparticles

[0055] like Figure 2 As shown, it is a process flow chart of preparing nanoparticles by microfluidics. The specific preparation method is as follows:

[0056] (1) 10 mg of silybin and soybean lecithin of molar ratio were accurately weighed and dissolved in methanol, ethanol, and acetone solution at 35°C in a water bath, stirred at 150 rpm for 24 h, and then taken out and transferred to a 10 ml volumetric flask and fixed to the mark to obtain a 1 mg / ml silybin lecithin complex internal phase solution;

[0057] (2) Accurately weigh 1 g of D-mannitol and dissolve it in 100 ml of ultrapure water at room temperature. Ultrasonicate at 80 Hz for 5 min to obtain an external phase solution containing 1% D-mannitol.

[0058] (3) transferring the prepared internal phase and external phase solutions into a syringe, fixing the syringe on a syringe pump, connecting the syringe and the device with a polytetrafluoroethylene hose, adjusting the internal and external phase flow rates using the syringe pump control panel, setting the total flux of the device to 100, 200, and 300 μl / min, and setting the internal and external phase flow rate ratio to 1:1, 1:2, 1:3, 1:4, 1:5, 1:8, 1:10, and 1:15, collecting the effluent, and obtaining a silybin phosphatidylcholine complex nanoparticle suspension;

[0059] (4) After removing the organic solvent by rotary evaporation at 40° C., the mixture was freeze-dried to obtain silybin phosphatidylcholine complexes prepared with different solvents.

[0060] Particle size and particle size distribution of silybin phosphatidylcholine complex nanoparticles

[0061] The nanoparticle suspension collected above was appropriately diluted and then the particle size and PDI% of the prepared composite nanoparticles were tested using a Malvern laser particle size analyzer. The test results are shown in Figure 3 As shown, by adjusting the total flux of the internal and external phase solutions passing through the microfluidic device and the internal and external phase flow rate ratio, the particle size of the nanoparticles can be significantly adjusted. When the total flux of the device is 200 μl / min and the internal and external phase flow rate ratio is 1:8 to 1:10, the particle size is lower.

[0062] Example 3

[0063] Single factor investigation on the preparation of silybin lecithin complex to verify the effect of solvent on the preparation of silybin lecithin complex:

[0064] (1) Accurately weigh three groups of equimolar silybin and soybean lecithin, dissolve them in methanol, ethanol, and acetone solutions, respectively, in a 35°C water bath, stir them with a magnetic stirrer at 150 rpm for 24 h, take them out, and dilute them to the mark in a 10 ml volumetric flask to obtain a 1 mg / ml silybin lecithin complex internal phase solution with methanol, ethanol, and acetone as solvents;

[0065] (2) Accurately weigh 1 g of D-mannitol and dissolve it in 100 ml of ultrapure water at room temperature. Ultrasonicate at 80 Hz for 5 min to obtain an external phase solution containing 1% D-mannitol.

[0066] (3) The inner phase solution is introduced into the inlet of the microfluidic device, the outer phase solution is an aqueous solution containing 1% D-mannitol, the total flux TFR of the device is set to 200 μl / min, the inner and outer phase solution flow rate ratio FRR is set to 1:9, and the effluent is collected;

[0067] (4) Two 1 ml portions of the collected solution were removed, and the organic solvent was removed by rotary evaporation at 40° C., followed by freeze-drying to obtain silybin phosphatidylcholine complexes prepared with different solvents.

[0068] Taking advantage of the solubility difference between silybin phosphatidylcholine complex soluble in chloroform and free silybin insoluble in chloroform, one freeze-dried sample of silybin phosphatidylcholine complex was reconstituted with ethanol and fixed to the mark of 10ml volumetric flask, and the other group was reconstituted with 2ml chloroform, filtered with 0.45 microporous membrane, and the filtrate was collected. After evaporation of the filtrate, it was reconstituted with ethanol and fixed to the mark of 10ml volumetric flask. The sample obtained by fixed volume was subjected to UV quantitative test at 288nm.

[0069] Recombination rate % = extraction group / redissolution group × 100%

[0070] The results of the effect of the internal phase solution solvent on the recombination rate are as follows Figure 4 As shown, the complex rate when acetone was used as the internal phase solution was significantly higher than that of the methanol group and the ethanol group. Acetone as an aprotic solvent is more conducive to the formation of phosphatidylcholine complexes.

[0071] Example 4

[0072] Single factor investigation on the preparation of silybin lecithin complex to verify the effect of stabilizer on the complexation rate of silybin lecithin complex:

[0073] Compared with Example 3, the fixed internal phase solvent is acetone, the drug-lipid ratio is 1:1, and the mixture is placed in a magnetic stirrer and stirred at 150 rpm in a 35°C water bath for 24 hours to prepare a 1 mg / ml silybin lecithin complex internal phase solution, the stabilizer types are PVP-K30, SDS, Poloxamer188, HPMC, tween80, and a certain amount of freeze-drying protective agent and stabilizer are accurately weighed and dissolved in ultrapure water to prepare an external phase solution, wherein the stabilizer concentration is 0.2% (w / v), and the freeze-drying protective agent D-mannitol concentration is 1% (w / v). The internal and external phase solutions are passed into a microfluidic device, TFR is set to 200 μl / min, FRR is 1:9, and the effluent is collected for particle size test and composite rate test, and the test method is the same as before.

[0074] Figure 5 Figure a shows the effect of different stabilizers on the particle size, monodispersity and recombination rate of the prepared nanoparticles. The particle size of the particles prepared using the microfluidic device under each stabilizer is less than 400nm, and the PDI% is less than 30%, indicating that the stability and repeatability of the nanoparticles prepared by the microfluidic device are good.

[0075] Figure 5 Result b shows the influence of the stabilizer recombination rate. The recombination rate was significantly improved by using the non-ionic stabilizer PVP-K30. Compared with other stabilizers, PVP-K30 can form a dense protective film on the particles to prevent particle aggregation and prevent the leakage of silybin in the composite particles.

[0076] Example 5

[0077] Single factor investigation on the preparation of silybin lecithin complex, the effect of internal phase solution concentration on the preparation of silybin lecithin complex:

[0078] Compared with Example 3, the fixed internal phase solvent is acetone, the drug-lipid ratio is 1:1, the silybin concentration in the internal phase solution is set to 1 mg / ml, 2 mg / ml, 4 mg / ml, and 8 mg / ml, and placed in a magnetic stirrer at 35°C water bath and 150 rpm magnetic stirring for 24 hours to prepare different degrees of silybin lecithin complex internal phase solutions. Accurately weigh a certain amount of freeze-dried protective agent and stabilizer, dissolve in ultrapure water to prepare an external phase solution, wherein the concentration of stabilizer PVP-K30 is 0.2% (w / v), and the concentration of freeze-dried protective agent D-mannitol is 1% (w / v). The internal and external phase solutions are passed into a microfluidic device, TFR is 200 μl / min, FRR is 1:9, and the effluent is collected for particle size test and composite rate test, and the test method is the same as before.

[0079] Figure 6 The results of the particle size, monodispersity and composite rate of nanoparticles prepared by different internal phase solution concentrations are shown in the figure. Too high a concentration causes the internal phase solution to precipitate quickly when it contacts the external phase solution, which easily clogs the device. At the same time, the precipitated particles are retained in the device channel, which significantly reduces the composite rate and the standard deviation of the particle polydispersity is large. When the internal phase solution concentration is low, the composite particles form slowly, resulting in a larger particle size. The sample prepared when the internal phase solution concentration is 2mg / ml is better.

[0080] Example 6

[0081] Single factor investigation on the preparation of silybin lecithin complex, the effect of drug-lipid ratio on the preparation of silybin lecithin complex:

[0082] Compared with Example 3, the fixed internal phase solvent is acetone, the drug-lipid ratio is set to 1:0.5, 1:0.75, 1:1, 1:1.5, 1:2, and placed in a magnetic stirrer at 35°C water bath and 150rpm magnetic stirring for 24h to prepare a 2mg / ml silybin lecithin complex internal phase solution. Accurately weigh a certain amount of freeze-drying protective agent and stabilizer, dissolve in ultrapure water to prepare an external phase solution, wherein the concentration of stabilizer PVP-K30 is 0.2% (w / v), and the concentration of freeze-drying protective agent D-mannitol is 1% (w / v). The internal and external phase solutions are passed into a microfluidic device, TFR is 200μl / min, FRR is 1:9, and the effluent is collected for particle size test and composite rate test, and the test method is the same as before.

[0083] Figure 7The figure shows the effect of different internal phase preparation conditions on the encapsulation efficiency of prepared nanoparticles. When the drug-lipid ratio is in the range of 2:1 to 1:1.5, the particle size distribution of the prepared nanocomplex is in the range of 200-300nm, and the PDI% is less than 30%. When the drug-lipid ratio is 1:2, the viscosity of the internal phase solution increases, making the complex easier to aggregate and the particle size larger. The composite rate of the complex will increase if the proportion of lipids in the complex is appropriately increased. When the mass ratio of silybin to soybean lecithin is 1:1.5, the composite rate of lecithin to silybin is significantly increased, and further increasing the mass proportion of lecithin has little effect on increasing the composite rate.

[0084] Example 7

[0085] Single factor investigation on the preparation of silybin lecithin complex and the effect of internal phase solution complexation time on the preparation of silybin lecithin complex

[0086] Compared with Example 3, the fixed internal phase solvent is acetone, the drug-lipid ratio is 1:1.5, and the mixture is placed in a magnetic stirrer with a 35°C water bath and magnetic stirring at 150 rpm. The stirring time is 12 h, 24 h, 48 h, and 72 h, respectively, to prepare a 2 mg / ml silybin lecithin complex internal phase solution. A certain amount of lyophilization protectant and stabilizer are accurately weighed and dissolved in ultrapure water to prepare an external phase solution, wherein the concentration of stabilizer PVP-K30 is 0.2% (w / v), and the concentration of lyophilization protectant D-mannitol is 1% (w / v). The internal and external phase solutions are passed into a microfluidic device, with a TFR of 200 μl / min and a FRR of 1:9. The effluent is collected for particle size test and composite rate test, and the test method is the same as before.

[0087] Figure 8 To study the effect of complexation time on the complexation rate of lecithin complex, the formation of lecithin complex is mainly based on van der Waals force. Insufficient stirring may affect the formation of the complex. The results show that the complex reaction is close to completion when the complex is stirred for 24 hours, and further increasing the reaction time has little effect on the complexation rate.

[0088] like Fig. 9 As shown in the figure, the particle size of the nanocomposite obtained after the optimization of the silybin lecithin complex prepared by the microfluidic method is 221.50nm, and the PDI% is 23.48%, indicating that the complex is a nanoscale particle with uniform size and concentrated particle size distribution. The Zeta potential is -19.11mV, indicating that there is a certain electrostatic repulsion between the complexes, and the system can exist stably. Fig.10 This is a transmission electron microscopy image of the silybin lecithin complex. It can be seen that the complex is spherical and stably distributed at around 250nm.

[0089] Example 8

[0090] According to the silybin lecithin complex nanoparticle freeze-dried powder prepared in Example 7, silybin, lecithin, mannitol, and PVP-K30 were physically mixed and ground in appropriate amounts according to the proportions in the case to obtain a physical mixture powder. The complex freeze-dried powder, the physical mixture, and samples of each component were taken for spectral analysis using a Fourier infrared spectrometer, and the scanning wavenumber range was 4000 cm-1 to 400 cm-1.

[0091] The component scanning results are as follows Fig.11 As shown, the characteristic peaks in the structure of silybin API are as follows: ①3606.0 is the OH stretching vibration of phenolic hydroxyl group; 3454.8 is the OH stretching vibration of hydroxyl group; ②1636.9 is the C=O stretching vibration conjugated with benzene ring; ③1269.1 and 1082.1 are the Ar-O and RO stretching vibrations of ether bond respectively; in the structure of soybean lecithin, there is a characteristic peak ①1741.1 of C=O stretching vibration of saturated ester group; ②1235.7 of P=O stretching vibration of phospholipid and 1062.6 of POC stretching vibration; D-mannitol characteristic peak ①3288.0 is OH stretching vibration; ②1019.8 of primary alcohol CO stretching and 1081.1 of secondary alcohol CO stretching; in the structure of PVP-K30, there is a characteristic peak of C=O stretching vibration of amide group at 1654.8.

[0092] like Fig.11 As shown, the physical mixture has peaks at the aforementioned silybin phenol hydroxyl group (3605.3, 3454.7), ester group (1639.2), ether bond (1269.2, 1082.2) and ester group (1740.5) and phospholipid (1234.4, 1041.4) of lecithin, which are the superposition of carrier stabilizer, raw material drug and lecithin, indicating that simple physical mixing does not change the change in the spatial structure of the components.

[0093] The characteristic peaks of phenolic hydroxyl groups from silybin and phospholipids from soybean lecithin disappeared in the lecithin complex, presumably because the complex formed with lecithin was hidden. The C=O stretching vibration of the saturated ester group from soybean lecithin was blue-shifted, and the C=O stretching vibration conjugated with the benzene ring from silybin was red-shifted, indicating that the formation of the complex was not a simple physical mixing, and some new intermolecular forces were generated between silybin and soybean lecithin.

[0094] Example 9

[0095] Lecithin complex dissolution test:

[0096] (1) Establishment of Silybin Standard Curve

[0097] Weigh 25 mg of silybin accurately, dissolve it in anhydrous ethanol, transfer it to a 25 ml volumetric flask and make up to volume with anhydrous ethanol to obtain a 1 mg / ml silybin anhydrous ethanol stock solution. Use a pipette to transfer 2 ml of the stock solution to a 50 ml volumetric flask, and make up to volume with a phosphate buffer solution with a pH value of 6.8 and a dilute hydrochloric acid solution with a pH value of 1.2. Scan it with a UV-visible spectrophotometer in the wavelength range of 200-600 nm. The maximum absorption wavelength of silybin at both pH values ​​is 288 nm. Pipette the pH 6.8 and pH 1.2 silybin stock solutions to prepare a series of standard solutions, and measure the corresponding absorbance values ​​at a wavelength of 288 nm. Draw a standard curve with concentration as the horizontal axis and absorbance as the vertical axis. The linear regression equations at the two pH values ​​are as follows.

[0098] pH=6.8, y=0.0275x+0.0447, R2=0.999

[0099] pH=1.2, y=0.0435x-0.0138, R2=0.999

[0100] (2) In vitro release study of silybin phosphatidylcholine complex

[0101] Buffer solutions with pH values ​​of 1.2 and 6.8 were used as release media to simulate the release effect of silybin API and silybin lecithin complex in gastric and intestinal body fluids. The dissolution apparatus was set at 37°C and the stirring paddle speed was 50 rpm. Sampling and rehydration were performed at 5, 10, 15, 30, 45, 60, 90, and 120 minutes, and the dissolution at each sampling point was calculated.

[0102] Dissolution calculation formula

[0103]

[0104] Where Cn is the silybin concentration at the nth sampling point, V is the total volume of the release medium (in this experiment, V = 1L), Ci is the silybin concentration at the ith sampling point, Vi is the sampling volume at the ith sampling point (in this experiment, Vi is 5ml), and M is the mass of silybin in the sample. Release results are shown in Fig.12 The results showed that after silybin was prepared into lecithin complex, the release effect in gastric and intestinal simulated pH solutions was significantly improved, and the release effect in pH 6.8 buffer solution was better than that in pH 1.2 buffer solution.

[0105] Therefore, the silybin lecithin complex nanoparticles of the present invention have good dispersibility and stability, a particle size of 150 to 500 nm, a particle size dispersion of 0.1 to 0.3, a simple system composition, and good biocompatibility. Lecithin can be used as a carrier to achieve nanoscale release of silybin in the intestine, and has broad application prospects in increasing the solubility of poorly soluble drugs and promoting drug absorption and utilization.

Claims

1. A method for preparing silybin-phosphatidylcholine complex nanoparticles, characterized in that: The preparation method comprises the following steps: (a) dissolving lecithin and silybin in an organic solvent to prepare an internal phase solution; (b) dissolving a lyophilization protectant and a stabilizer in water to prepare an external phase solution; (c) using microfluidics technology, dispersing the complex formed in the internal phase solution in the external phase solvent for co-precipitation to obtain a silybin lecithin complex nanodispersion; (d) The silybin phosphatidylcholine complex nano-dispersion solution is subjected to rotary evaporation and then freeze-dried to obtain silybin phosphatidylcholine complex nano-particles.

2. The preparation method according to claim 1, characterized in that: In step (a), the drug-lipid ratio of silybin to lecithin is 2:1 to 1:2 (w / w).

3. The preparation method according to claim 1, characterized in that: In step (a), the organic solvent comprises methanol, ethanol or acetone, and the lecithin is soybean lecithin or egg yolk lecithin.

4. The preparation method according to claim 1, characterized in that: In step (b), the stabilizer includes sodium dodecyl sulfate, Tween 80, polyvinyl pyrrolidone, hydroxypropyl methylcellulose or poloxamer 188, and the mass concentration of the stabilizer in the external phase solution is 0.1% to 0.5%.

5. The preparation method according to claim 1, characterized in that: In step (b), the freeze-drying protective agent includes sucrose or D mannitol, and the mass concentration of the freeze-drying protective agent in the external phase solution is 1% to 5%.

6. The preparation method according to claim 1, characterized in that: In step (c), the microfluidic technology has a total flow rate of the inner phase solution and the outer phase solution of 100 to 500 μL / min, and a flow rate ratio of the inner phase solution to the outer phase solution of 1:1 to 1:

20.

7. The preparation method according to claim 1, characterized in that: In step (c), the microfluidic technology adopts a device composed of an inner phase capillary (1) nested in a stainless steel tube (6), a dispensing needle (5), a square glass tube (3), and a receiving capillary glass tube (2) nested in the stainless steel tube (6), which are connected in sequence, and a glass slide (4) is provided at the bottom.

8. The preparation method according to claim 7, characterized in that: The dispensing needle (5) is vertically fixed at the intersection of the inner phase capillary (1) and the square glass tube (3), and the stainless steel tube (6) extends out and is fixed on both sides of the glass slide (4).

9. A silybin phosphatidylcholine complex nanoparticle prepared by the preparation method according to any one of claims 1 to 8, characterized in that: The composite nanoparticles have a particle size range of 150 to 500 nm and a polydispersity index range of 0.1 to 0.

3.

10. Use of the silybin phosphatidylcholine complex nanoparticles according to claim 9 in the preparation of silybin oral medicine.

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