Electrostatic spinning microsphere of ellagic acid and urolithin and preparation method thereof

Abstract

The invention relates to the technical field of drug delivery systems, and provides electrostatic spinning microspheres of ellagic acid and urolithin and a preparation method of the electrostatic spinning microspheres. According to the preparation method disclosed by the invention, the nano-microspheres prepared from alginate and chitosan are successfully prepared by utilizing an electrostatic spinning technology, and ellagic acid and metabolin thereof, namely urolithin, are effectively encapsulated, so that a guarantee is provided for efficient delivery of medicines. The prepared electrostatic spinning microspheres are uniform in particle size distribution, the packaging efficiency reaches 60-88%, and FTIR and TGA prove that the medicine is successfully embedded and has good thermal stability. Compared with free drugs, the drug-loaded microspheres still show excellent antioxidant activity, and the DPPH and ABTS removal activity before and after EA and Uro C are encapsulated is the highest. An in-vitro digestion experiment shows that compared with a free drug, the drug loss of the microsphere in stomach and intestine stages is reduced by 32%-69%, the drug can be protected to smoothly reach the colon to play a role, and the bioavailability of the drug is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug delivery systems, and in particular to an electrospun microsphere of ellagic acid and urolithin and a preparation method thereof. Background Art

[0002] Ellagic acid (EA) is a common polyphenol in plants such as pomegranates, raspberries, strawberries, and walnuts, and is also an intestinal metabolite of soluble ellagitannins. In addition to its general anti-inflammatory and antioxidant characteristics, it also has anti-cancer potential and cardiovascular protection ability, and can metabolize different types of urolithins depending on the intestinal flora, such as urolithin A (Uro A), urolithin B (UroB), urolithin C (Uro C), and isourolithin A (Iso A), etc. These urolithins will in turn regulate the intestinal flora, showing the effect of reducing obesity and metabolic complications caused by obesity. However, the bioavailability of EA is extremely low, which greatly limits the exertion of its medicinal value, but its metabolite urolithin can greatly enhance its utilization and better exert its medicinal value. The metabolic type of urolithin is dependent on the intestinal flora and there are individual differences. Research shows that type A metabolizers (UM-A) can metabolize to generate Uro A, type 0 metabolizers (UM-0) cannot convert EA into urolithin, and type B metabolizers (UM-B) can metabolize not only Uro A but also Uro B and Iso A.

[0003] In order to analyze the effects of EA and urolithin in the colon, it is first necessary to improve their instability in the gastrointestinal tract and reduce their loss during the process of reaching the colon. Therefore, it is necessary to design a suitable drug delivery system. Common drug delivery systems include nanotechnology delivery, liposome delivery, biomass delivery such as proteins and polysaccharides, and microcapsule technology delivery, etc. However, the current drug delivery systems have poor stability, are difficult to resist the destruction of gastric acid, and cannot achieve colon-targeted delivery. Summary of the Invention

[0004] In view of this, the present invention provides an electrospun microsphere of ellagic acid and urolithin and a preparation method thereof. The electrospun microsphere provided by the present invention has good stability, high encapsulation efficiency, has good tolerance in the human gastrointestinal environment, can achieve colon-targeted delivery, and greatly improves the bioavailability of the drug.

[0005] In order to achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0006] A preparation method of an electrospun microsphere of ellagic acid and urolithin, comprising the following steps:

[0007] Mix sodium alginate, an active ingredient, and water to obtain a spinning solution; the active ingredient includes one or more of ellagic acid and urolithin;

[0008] The spinning solution is injected into a calcium chloride solution by electrospinning for solidification to obtain solidified microspheres;

[0009] The solidified microspheres are mixed with a chitosan solution for coating to obtain the electrospun microspheres of ellagic acid and urolithin.

[0010] Preferably, the urolithin includes one or more of urolithin A, urolithin B, urolithin C, and isourolithin A.

[0011] Preferably, the concentration of sodium alginate in the spinning solution is 1-2 wt%, and the content of the active ingredient is 0.5-2 mg / mL.

[0012] Preferably, the mixing of sodium alginate, the active ingredient, and water includes: dissolving sodium alginate in water to obtain a sodium alginate solution, and then mixing the sodium alginate solution and the active ingredient; the temperature of the dissolution is 35-45°C, and the temperature of the mixing of the alginate and the active ingredient is 35-45°C.

[0013] Preferably, the electrospinning method includes: fixing the spinning solution on the injection pump of an electrospinning machine, and vertically injecting the spinning solution into the calcium chloride solution through a syringe.

[0014] Preferably, the injection voltage is 12-16 kV.

[0015] Preferably, the concentration of the calcium chloride solution is 2-4 wt%, and the solidification time is 1-2 h.

[0016] Preferably, the chitosan solution is a VC solution of chitosan, the concentration of chitosan in the chitosan solution is 0.5-2 wt%, and the coating time is 12-24 h.

[0017] The present invention also provides electrospun microspheres of ellagic acid and urolithin prepared by the preparation method described in the above solution, including calcium alginate microspheres, the active ingredient encapsulated in the calcium alginate microspheres, and chitosan coated on the surface of the calcium alginate microspheres; the active ingredient includes one or more of ellagic acid and urolithin.

[0018] Preferably, the average particle size of the electrospun microspheres ＜ 100 μm.

[0019] The present invention provides a method for preparing electrospun microspheres of ellagic acid and urolithin, comprising the following steps: mixing sodium alginate, an active ingredient, and water to obtain a spinning solution; the active ingredient includes one or more of ellagic acid and urolithin; injecting the spinning solution into a calcium chloride solution by electrospinning for curing to obtain cured microspheres; mixing the cured microspheres and a chitosan solution for coating to obtain the electrospun microspheres of ellagic acid and urolithin. The present invention utilizes electrospinning technology to successfully prepare nanoscale electrospun microspheres made of alginate and chitosan, effectively encapsulating ellagic acid and its metabolite urolithin, providing guarantee for the efficient delivery of drugs. The electrospun microspheres prepared by the present invention have high encapsulation efficiency and good stability, and have good tolerance in the human gastrointestinal environment, can protect the drug to pass through the gastrointestinal tract smoothly and reach the colon to play a role, greatly improving the bioavailability of the drug, providing a reliable way to supplement the required urolithin in the human body, and providing an important theoretical basis for the treatment of colon health and the application of electrospun microspheres in the field of drug delivery.

[0020] The results of the examples show that the electrospun microspheres prepared by the present invention have a uniform particle size distribution, small particles, and high encapsulation efficiency (60 - 88%). FTIR and TGA analyses confirm that the drug is stably embedded in the microspheres through hydrogen bonding and electrostatic interactions, and the thermal stability is significantly better than that of the blank microspheres, indicating that the microspheres encapsulating the drug have good tolerance in the human gastrointestinal environment. Compared with the free drug, the drug-loaded microspheres still exhibit excellent antioxidant activity, and the DPPH and ABTS scavenging activities of EA and Uro C are the highest before and after encapsulation. The in vitro digestion simulation experiment shows that the microspheres significantly reduce the loss of the drug during gastrointestinal digestion (reducing by 32% - 69%), which means that the microspheres can protect the drug to pass through the gastrointestinal tract smoothly and reach the colon to play a role, greatly improving the bioavailability of the drug, and providing a reliable way to supplement the required urolithin in the human body. Description of the Drawings

[0021] Figure 1 Average particle size and encapsulation efficiency of electrospun microspheres encapsulating EA with different concentrations of SA, where a is the average particle size and b is the encapsulation efficiency;

[0022] Figure 2 Average particle size and encapsulation efficiency of 1% SA microspheres with different compositions, where a is the encapsulation efficiency and b is the average particle size;

[0023] Figure 3Surface morphologies of different electrospun microspheres, where a is 1% SA-MP, b is 1% SA-MP@EA, c is 1.5% SA-MP, d is 1.5% SA-MP@EA, e is 2% SA-MP, f is 2% SA-MP@EA, g is 1% SA-MP@Uro A, h is 1% SA-MP@Uro B, i is 1% SA-MP@Uro C, j is 1% SA-MP@Iso A, and the scale bars of a - j are all 50 μm;

[0024] Figure 4 FTIR spectra of different electrospun microspheres;

[0025] Figure 5 TGA-DTG curves of different electrospun microspheres, where a is the TGA curve and b is the DTG curve;

[0026] Figure 6 TGA-DTG curves of SA microspheres with different concentrations, where a is the TGA curve and b is the DTG curve;

[0027] Figure 7 Antioxidant activity test results of different samples, where a is the DPPH scavenging activity and b is the ABTS scavenging activity;

[0028] Figure 8 Simulated digestion curves of different samples, where a is EA and MP@EA, b is Uro A and MP@Uro A, c is Uro B and MP@Uro B, d is Uro C and MP@Uro C, e is Iso A and MP@Iso A;

[0029] Figure 9 Optical microscopy images of microspheres encapsulating different drugs after gastrointestinal digestion, where a, b, and c are the original SEM image, SEM image after gastric digestion, and SEM image after intestinal digestion of MP@EA microspheres, respectively, d, e, and f are the original SEM image, SEM image after gastric digestion, and SEM image after intestinal digestion of MP@Uro A microspheres, respectively, g, h, and i are the original SEM image, SEM image after gastric digestion, and SEM image after intestinal digestion of MP@Uro B microspheres, respectively, j, k, and l are the original SEM image, SEM image after gastric digestion, and SEM image after intestinal digestion of MP@Uro C microspheres, respectively, and m, n, and o are the original SEM image, SEM image after gastric digestion, and SEM image after intestinal digestion of MP@Iso A microspheres, respectively. Detailed implementation methods

[0030] The present invention provides a preparation method of electrospun microspheres of ellagic acid and urolithin, comprising the following steps:

[0031] Mix sodium alginate, active ingredient(s), and water to obtain a spinning solution; the active ingredient(s) includes one or more of ellagic acid and urolithins.

[0032] Use electrospinning to inject the spinning solution into a calcium chloride solution for curing to obtain cured microspheres.

[0033] Mix the cured microspheres and a chitosan solution for coating to obtain the electrospun microspheres of ellagic acid and urolithins.

[0034] In the present invention, sodium alginate (SA), active ingredient(s), and water are mixed to obtain a spinning solution. In the present invention, the active ingredient(s) includes one or more of ellagic acid (EA) and urolithins; the urolithins preferably include one or more of urolithin A (Uro A), urolithin B (Uro B), urolithin C (Uro C), and isourolithin A (Iso A).

[0035] In the present invention, the concentration of sodium alginate in the spinning solution is preferably 1 - 2 wt%, specifically it can be 1 wt%, 1.5 wt%, or 2 wt%, more preferably 1 wt%, and the content of the active ingredient is preferably 0.5 - 2 mg / mL, more preferably 1 mg / mL; the mixing of sodium alginate, active ingredient(s), and water preferably includes: dissolving sodium alginate in water to obtain a sodium alginate solution, and then mixing the sodium alginate solution and the active ingredient; the temperature of the dissolution is preferably 35 - 45 °C, more preferably 40 - 45 °C, the dissolution is preferably carried out under stirring conditions, and the rotation speed of the stirring is preferably 300 rpm; the temperature for mixing sodium alginate and the active ingredient is preferably 35 - 45 °C, more preferably 40 °C.

[0036] After obtaining the spinning solution, the present invention uses electrospinning to inject the spinning solution into a calcium chloride solution for curing to obtain cured microspheres. In the present invention, the electrospinning preferably includes: fixing the spinning solution on the injection pump of an electrospinning machine, and vertically injecting the spinning solution into the calcium chloride solution through a syringe; the specification of the syringe is 28G - 32G (inner diameter is 0.18 mm - 0.11 mm), preferably 30G, and the injection voltage is preferably 12 - 16 kV; using electrospinning to inject the spinning solution into the calcium chloride solution in the present invention is beneficial to obtaining microspheres with uniform and small particle sizes, and the microspheres with small particle sizes can be better dispersed in digestive juices such as the gastrointestinal tract and will not settle rapidly due to gravity.

[0037] In the present invention, the concentration of the calcium chloride solution is preferably 2 - 4 wt%, the curing time is preferably 1 - 2 h, and the curing can be carried out at room temperature; after the spinning solution is injected into the calcium chloride solution, sodium alginate reacts with calcium chloride to form calcium alginate microspheres and encapsulate the active ingredient inside. After curing is completed, it is preferred to separate and collect the obtained cured microspheres.

[0038] After obtaining the cured microspheres, the present invention mixes the cured microspheres with a chitosan solution for coating to obtain the electrospun microspheres of ellagic acid and urolithin. In the present invention, the chitosan solution is a VC solution of chitosan. The concentration of chitosan in the chitosan solution is preferably 0.5-2 wt%, more preferably 1 wt%; the content of VC (vitamin C) in the VC solution of chitosan is preferably 2 wt%. In the present invention, chitosan is more easily soluble in an acidic environment (pH < 6.5), and VC, as an acidic substance, can improve the solubility of chitosan and enable it to form a uniform solution at a lower pH. The coating time is preferably 12-24 h; the coating is preferably carried out under stirring conditions; the coating can be carried out at room temperature. After the coating is completed, the present invention preferably separates and freeze-dries the obtained electrospun microspheres, and the present invention has no special requirements for the specific conditions of the freeze-drying. The present invention first prepares cured microspheres and then coats the cured microspheres in a chitosan solution, which is beneficial to enhancing the electrostatic interaction between alginate and chitosan and enabling chitosan to coat on the surface of calcium alginate microspheres. The present invention first prepares calcium alginate cured microspheres by electrospinning and then coats chitosan on the surface of the calcium alginate cured microspheres, which can enable chitosan to coat on the outermost layer of the microspheres. Chitosan is a cationic compound, which can increase the adhesion of the microspheres. Moreover, when the microspheres are in an acidic condition, the outer layer of chitosan will be preferentially dissolved, avoiding the dissolution of calcium alginate, thereby protecting the drug to reach the colon, achieving colon-targeted delivery, and improving the bioavailability.

[0039] The present invention also provides electrospun microspheres of ellagic acid and urolithin prepared by the preparation method described in the above solution, including calcium alginate microspheres, active ingredients encapsulated in the calcium alginate microspheres, and chitosan coated on the surface of the calcium alginate microspheres; the active ingredients include one or more of ellagic acid and urolithin. In the present invention, according to the different types of active ingredients, the electrospun microspheres include one or more of ellagic acid microspheres (MP@EA), urolithin A microspheres (Uro A@EA), urolithin B microspheres (Uro B@EA), urolithin C microspheres (Uro C@EA), and isourolithin A microspheres (Iso A@EA).

[0040] In the present invention, the average particle size of the electrospun microspheres is preferably < 100 μm. In the specific embodiments of the present invention, when the concentration of sodium alginate in the spinning solution is 1 wt%, the particle size of the electrospun microspheres is preferably < 95 μm, and the average particle size of ellagic acid microspheres (MP@EA), urolithin A microspheres (Uro A@EA), urolithin B microspheres (Uro B@EA), and urolithin C microspheres (Uro C@EA) is < 80 μm, and the average particle size of isourolithin A microspheres (Iso A@EA) is 94 μm.

[0041] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0042] The reagents and instruments required in the following examples are shown in Table 1 and Table 2.

[0043] Table 1 Experimental drugs and reagents

[0044] Name Purity Specification Manufacturer Sodium Alginate AR Shanghai Yuanye Bio-Technology Co., Ltd. Calcium Chloride AR Shanghai Yuanye Bio-Technology Co., Ltd. Chitosan AR Shanghai Aladdin Biochemical Technology Co., Ltd. Folin-Ciocalteu Reagent AR Shanghai Yuanye Bio-Technology Co., Ltd. Sodium Carbonate AR Shanghai Yuanye Bio-Technology Co., Ltd. DPPH HPLC≥97% Shanghai Yuanye Bio-Technology Co., Ltd. ABTS HPLC≥98% Shanghai Yuanye Bio-Technology Co., Ltd. KBr SP Shanghai Macklin Biochemical Co., Ltd. Ellagic Acid (EA) HPLC≥97% Hubei Ruike Chemical Co., Ltd. Urolithin A (Uro A) HPLC≥98% Shanghai Jizhi Biochemical Technology Co., Ltd. Urolithin B (Uro B) HPLC≥97% Shanghai Yuanye Biotechnology Co., Ltd. Urolithin C (Uro C) HPLC≥98% Shanghai Jizhi Biochemical Technology Co., Ltd. Iso Urolithin A (Iso A) HPLC≥98% Shanghai Jizhi Biochemical Technology Co., Ltd.

[0045] Table 2 Experimental instruments

[0046] Name Model Manufacturer Electrospinning Machine E20 Foshan Lepton Precision Measurement and Control Technology Co., Ltd. Magnetic Stirrer DF-101Z Xi'an Taikang Biotechnology Co., Ltd. Scanning Electron Microscope TM4000 Hitachi High-Technologies Corporation Microplate Reader ReadMax 1500 Shanghai Shanspec Biotechnology Co., Ltd. Fourier Transform Infrared Spectrometer Nicolet 5700 Thermo Fisher Scientific Thermogravimetric Analyzer TGA / DSC 3+ METTLER TOLEDO Optical Microscope SK160 Macrotech Industrial Group Co., Ltd. Analytical Balance FA1204B Shanghai Jingke Tianmei Scientific Instrument Co., Ltd. Freeze Dryer FD-1A-50 Shanghai Xinweng Scientific Instrument Co., Ltd.

[0047] Example 1 Preparation of electrospun microspheres

[0048] First, prepare a sodium alginate (SA) solution. Add sodium alginate to water at 45 °C and 300 rpm, stir with a magnetic stirrer, adjust the temperature to 40 °C after dissolution, add EA, and make a spinning solution after uniform dispersion; the concentrations of sodium alginate in the spinning solution are 1%, 1.5%, and 2% respectively, and the concentration of EA is 1 mg / mL. Fix the above solution on the syringe pump of an electrospinning machine (E20, Foshan Lepton Precision Measurement and Control Technology Co., Ltd., China), and vertically inject it into a 4 wt% CaCl2 solution at a voltage of 16 kV through a syringe (specification 28G). After curing for 1 h, add the obtained cured microspheres to a VC solution containing 1 wt% chitosan (CS) and stir overnight to obtain electrospun microspheres encapsulating EA, denoted as MP@EA (denoted as 1% SA-MP@EA, 1.5% SA-MP@EA, 3% SA-MP@EA respectively according to the different SA concentrations in the spinning solution).

[0049] Encapsulate Uro A, Uro B, Uro C, and Iso A respectively according to the above method, and the obtained electrospun microspheres are denoted as MP@Uro A, MP@Uro B, MP@Uro C, and MP@Iso A respectively. The concentration of sodium alginate in the spinning solution used for encapsulating Uro A, Uro B, Uro C, and Iso A is 1%.

[0050] Meanwhile, prepare blank microspheres according to the above method, omitting the addition of EA, and keeping other conditions the same (denoted as 1% SA-MP, 1.5% SA-MP, 3% SA-MP respectively according to the different SA concentrations in the spinning solution).

[0051] Among the electrospun microspheres obtained by the above method, according to the different concentrations of sodium alginate, they are respectively denoted as 1% SA microspheres, 1.5% SA microspheres, and 2% SA microspheres.

[0052] The microspheres prepared in Example 1 were used for subsequent experiments. All subsequent experiments were repeated three times, and the values were expressed as the mean (three independent measurements) ± SD (standard deviation). IBM SPSS Statistics 22.0 was used for data analysis. One-way analysis of variance (ANOVA) was used to test the significance of differences between independent variables. p < 0.05 indicates that the data is statistically significant.

[0053] Characterization and testing of the microspheres in Example 2

[0054] 1. Particle size test and encapsulation efficiency test

[0055] Particle size test: The freeze-dried electrospun microspheres were evenly dispersed on a glass slide, and images were taken using a scanning electron microscope SEM (TM4000, Hitachi High-Technologies Corporation, Japan). Subsequently, more than 300 microspheres were randomly measured using Nano Measure software to analyze the size distribution and average particle size of the microspheres.

[0056] Encapsulation efficiency test: The drug quantification method was to scan the full wavelength using an enzyme-linked immunosorbent assay reader (ReadMax 1500, Shanghai Flash Spectrum Biotechnology Co., Ltd., China). After determining the maximum absorption wavelength, the absorbance of different concentrations of the drug was measured to plot a quantitative standard curve. The calculation method of the encapsulation efficiency (EE) is shown in Equation I:

[0057] EE (%) = (M0 - C1V1) / M0 × 100% Equation I.

[0058] In Equation I, M0 (unit: μg) is the total drug mass, C1 (unit: μg / mL) is the drug concentration remaining in the CaCl2 receiving solution, and V1 (unit: mL) is the total volume of the CaCl2 receiving solution.

[0059] Figure 1 are the average particle size and encapsulation efficiency of electrospun microspheres encapsulating EA with different concentrations of SA, where a is the average particle size and b is the encapsulation efficiency. Figure 2 are the average particle size and encapsulation efficiency of different 1% SA microspheres, where a is the encapsulation efficiency and b is the average particle size; Figures 1 - 2 Different letters in indicate the differences between groups (p < 0.05).

[0060] According to Figure 1As can be seen from Fig. a, with the increase in SA concentration, the average particle size of the blank microspheres increased from 70 μm to 129 μm. The results showed that the particle size was concentration-dependent on the SA dose. At the same SA concentration, the average particle size of MP@EA with 1% SA was 18% larger than that of the blank MP, indicating the successful encapsulation of EA. To ensure that the microspheres could be better dispersed in digestive fluids such as the gastrointestinal tract and would not settle rapidly due to gravity, the formulation with the smallest particle size (i.e., the 1% SA formulation) was selected for the subsequent preparation of microspheres. According to Figure 1 As can be seen from Fig. b, at different SA concentrations, there were no significant differences in the EE of different microspheres, all of which were above 80%. Combining with the particle size results, 1% SA was selected for the subsequent experiments.

[0061] Figure 2 The average particle size and encapsulation efficiency of electrospun microspheres encapsulating different drugs (EA, Uro A, Uro B, Uro C, and Iso A) at a SA concentration of 1% were investigated. MP was the blank microsphere of the same formulation with a particle size of 70 μm. According to Figure 2 As can be seen from Fig. a, the EE of the hydrogel microspheres encapsulating Uro A, Uro B, Uro C, and Iso A were all above 60%. According to Figure 2 As can be seen from Fig. b, the particle size of the microspheres increased after encapsulating the drugs. The average particle sizes of MP@Uro A, MP@Uro B, and MP@Uro C were all below 80 μm at a 1% SA concentration, while MP@Iso A was relatively large, reaching 94 μm. This may be related to its structure. The amino group of chitosan and the carboxyl group of sodium alginate form a polyelectrolyte complex through electrostatic interaction. The phenolic hydroxyl group at a unique position of Iso A may form hydrogen bonds with the amino group in the chitosan molecule and the carboxyl group in the sodium alginate molecule. This hydrogen bond interaction can change the molecular conformation and aggregation state of the polymer, affecting their solubility and dispersibility in solution, resulting in a relatively large particle size of the formed microspheres.

[0062] 2. Observation of Microspheres by Scanning Electron Microscope

[0063] The surface morphology of the electrospun microspheres was evaluated by SEM (TM4000, Hitachi High-Technologies Corporation, Japan). The freeze-dried electrospun microspheres were attached to a conductive tape and photographed under vacuum, and the acceleration voltage was set at 15 kV.

[0064] Figure 3Surface morphologies of different electrospun microspheres, where a is 1% SA-MP, b is 1% SA-MP@EA, c is 1.5% SA-MP, d is 1.5% SA-MP@EA, e is 2% SA-MP, f is 2% SA-MP@EA, g is 1% SA-MP@Uro A, h is 1% SA-MP@Uro B, i is 1% SA-MP@Uro C, and j is 1% SA-MP@Iso A.

[0065] Figure 3 The results of a-f in [reference] show that different concentrations of SA have no significant effect on the morphology of microspheres. There are differences in the morphology of microspheres before and after encapsulating EA. The surface of microspheres without encapsulating EA is relatively smooth, and a large number of wrinkles appear on the surface of microspheres after encapsulating EA. This may be because multiple phenolic hydroxyl groups in the EA molecule can form hydrogen bonds with carboxyl groups in sodium alginate. This hydrogen bond interaction enhances the tight binding of EA and sodium alginate. At the same time, the phenolic hydroxyl groups in EA bind to the hydroxyl and amino groups of chitosan through hydrogen bond interactions, increasing the stability of the system, indicating the successful encapsulation of EA. Figure 3 g-j in [reference] are electrospun microspheres of 1% SA encapsulating Uro A, Uro B, Uro C, and Iso A. Compared with Figure 3 the blank control of a in [reference], different drugs show wrinkles to varying degrees. The above results all confirm the feasibility of encapsulation.

[0066] 3. Fourier Transform Infrared Spectroscopy

[0067] The Fourier Transform Infrared Spectrometer (Nicolet 5700, Thermo Fisher Scientific, USA) was used to study the structure of the samples, scanning the samples from 4000 to 500 cm -1 The test samples were SA, CS, EA, Uro A, Uro B, Uro C, Iso A, microspheres after encapsulating 1% SA, and blank microspheres MP prepared under 1% SA.

[0068] Figure 4 is the FTIR spectrum of different electrospun microspheres (1% SA).

[0069] Figure 4 In [reference], the characteristic absorption peaks of the EA spectrum at 3560 cm -1 , 3480 cm -1 , and 3150 cm -1 represent four interacting hydroxyl groups, corresponding to the polyphenol structure. The absorption peaks at 1621 cm -1 and 1506 cm -1 are attributed to the stretching vibration of C=C-C. The broad peak between 3000 and 3700 cm -1 in the CS spectrum is the stretching vibration of O-H, and at 2878 cm-1 and 2917 cm -1 、1654 cm -1 、1154 cm -1 and 1031 cm -1 The peaks appearing at are the stretching vibrations of C-H in CH2OH, the characteristic absorption peak of type I amide group (C-N-H), and the characteristic absorption peak of ether group (C-O-C), respectively. The spectral diagram of SA shows characteristic absorption peaks at 3435 cm -1 、2930 cm -1 、1613 and 1417 cm -1 、1029 cm -1 respectively corresponding to the stretching vibration of hydroxyl O-H, the stretching vibration of C-H, the asymmetric and symmetric stretching vibrations of COO-, and the stretching vibration of C-O-C, among which the C-O-C stretching vibration corresponds to the sugar structure. And at 1508 cm in the MP@EA spectral diagram -1 is the characteristic peak of EA, and the blank microspheres (MP) do not contain this peak, which indicates the successful encapsulation of EA. In the spectral diagram of MP@EA, some characteristic peaks of EA may overlap with the peaks of calcium alginate and chitosan at similar wavenumber positions, making the spectrum difficult to identify. The inconsistency between the spectral diagram of the complex and the spectral diagrams of single substances also indirectly proves the success of encapsulation.

[0070] Figure 4 In the spectral diagram of Uro A, the characteristic absorption peaks at 3335 cm -1 and 3148 cm -1 、1700 cm -1 、1613 cm -1 、1534 cm -1 are the stretching vibrations of O-H, C=O, -COO, and C=C respectively. The spectral diagram of Uro B shows characteristic absorption peaks at 3295 cm -1 、2917 cm -1 、1698 cm -1 、1467 cm -1 、1116 cm -1 、944 cm -1 corresponding to the stretching vibrations of phenolic hydroxyl, C-H group, carbonyl, C-C, N-H, and C-O respectively. The absorption peak at 3366 cm in the spectral diagram of Uro C -1 is the stretching vibration of -OH, and the absorption peak at 1710 cm -1 is the stretching vibration of carbonyl (C=O). The absorption peak at 3307 cm in the spectral diagram of Iso A -1 is the stretching vibration of -OH, and at 1689 cm -1The absorption peak at [[]] is the stretching vibration of the carbonyl group (C=O). Among them, the characteristic absorption peaks of four drugs at [[]] represent the characteristic absorption peaks of the benzene ring. However, the absorption peak intensity of the benzene ring in the spectrograms of MP@Uro A, MP@UroB, MP@Uro C, and MP@Iso A at [[]] weakens, and the characteristic absorption peak of the phenolic hydroxyl group does not appear either, indicating that these functional groups are encapsulated in the calcium alginate and chitosan microspheres and shielded, which confirms the stability of the microsphere-encapsulated drugs. Uro A, Uro B, Uro C, and Iso A are all successfully encapsulated in the microspheres. -1 The characteristic absorption peaks that appear at [[]] represent the characteristic absorption peaks of the benzene ring. For MP@Uro A, MP@UroB, MP@Uro C, and MP@Iso A spectrograms, the absorption peak intensity of the benzene ring at [[]] weakens, and the characteristic absorption peak of the phenolic hydroxyl group does not appear either, indicating that these functional groups are encapsulated in the calcium alginate and chitosan microspheres and shielded, which confirms the stability of the microsphere-encapsulated drugs. Uro A, Uro B, Uro C, and Iso A are all successfully encapsulated in the microspheres. -1 The absorption peak intensity of the benzene ring at [[]] weakens, and the characteristic absorption peak of the phenolic hydroxyl group does not appear either, indicating that these functional groups are encapsulated in the calcium alginate and chitosan microspheres and shielded, which confirms the stability of the microsphere-encapsulated drugs. Uro A, Uro B, Uro C, and Iso A are all successfully encapsulated in the microspheres.

[0071] 4. Thermogravimetric analysis

[0072] The thermal stability of the samples was tested by TGA (TGA / DSC 3+, Mettler Toledo, Switzerland) from 30 °C to 800 °C, with a heating rate of 20 °C / min under nitrogen. The tested samples were CS, SA, EA, Uro A, Uro B, UroC, Iso A, microspheres encapsulated with 1% SA, MP@EA microspheres encapsulated at different SA concentrations, and blank microspheres MP.

[0073] Figure 5 Figure [[]] shows the TGA-DTG curves of different electrospun microspheres (1% SA), where a is the TGA curve and b is the DTG curve ( Figure 5 MP in is 1% SA-MP). The TGA-DTG curves of microspheres with different SA concentrations are shown in Figure 6 , where a is the TGA curve and b is the DTG curve.

[0074] According to Figure 5 It can be seen that the first stage of weight loss of EA is below 388 °C ( Figure 5 a in ), and the DTG curve shows ( Figure 5 b in ), the maximum weight loss rate temperature T m is 479 °C, and the weight loss rate in this stage is 13%, mainly due to the loss of water and other volatile components. The second stage of weight loss is in the range of 388 - 561 °C, and the weight loss rate is 38%, which is related to the decomposition and degradation of polymer molecular chains. The third stage of weight loss is in the range of 561 - 800 °C, and the weight loss rate is 21%. Among them, the maximum weight loss rate temperatures T m (and the weight loss rate in the current stage) of EA, CS, and SA are 479 °C (32%), 309 °C (28%), and 249 °C (31%) respectively, indicating that EA has the strongest thermal stability. For the TGA-DTG curves of microspheres with different SA concentrations ( Figure 6) Among them, the T of 1.0% SA-MP, 1.0% SA-MP@EA, 1.5% SA-MP, 1.5% SA-MP@EA, 2.0% SA-MP, and 2.0% SA-MP@EA m (and the weight loss rate at the current stage) were 246 °C (54%), 261 °C (40%), 235 °C (42%), 252 °C (44%), 224 °C (40%), and 246 °C (52%) respectively. The results showed that the higher the sodium alginate content, the lower the thermal stability of the microspheres. And compared with the empty microspheres, encapsulating EA improved the heat resistance of the microspheres.

[0075] The first stage of Uro A weight loss was below 268 °C ( Figure 5 in a)), the DTG curve showed ( Figure 5 in b)), T m was 388 °C. The second stage of weight loss was in the range of 268 - 410 °C, and the weight loss rate was 88%. This may be because the substituents on the benzene ring fell off and further decomposed, and the breaking and recombination of multiple chemical bonds occurred simultaneously, resulting in a sharp decrease in weight. The third stage of weight loss was at 410 - 800 °C, and the weight loss rate was 5%. The T of Uro B m was 325 °C, and the weight loss rate at this stage was 91%. The T of Uro C m was 416 °C, and the weight loss rate at this stage was 76%. The T of Iso A m was 366 °C, and the weight loss rate at this stage was 39%. Due to the different positions of phenolic hydroxyl groups in several samples, the thermal stabilities were also different. The heat resistance of Uro B had no significant difference from that of CS. In addition, the thermal stabilities of other several samples were better than those of CS and SA. The thermal stabilities of the microspheres encapsulating these samples were between 240 - 243 °C, slightly increased compared with the blank control MP, showing better thermal stability.

[0076] Antioxidant activity of the microspheres in Example 3

[0077] 1. DPPH scavenging activity

[0078] Test the scavenging activity of the test drugs and microspheres on 1,1-diphenyl-2-picrylhydrazyl (DPPH). Evaluate the antioxidant properties between the drugs before encapsulation and between the samples after encapsulation respectively by the DPPH scavenging rate. The concentration of the free drug is 50 μg / mL. The microspheres (containing the same mass of the drug) are ground into powder and then formulated into an equal-concentration solution (the solvent is ethanol in both cases). The reaction solution is composed of 2 mL of DPPH and 2 mL of the sample solution. After reacting in the dark for 40 min, measure the absorbance value A1 at 517 nm; mix 2 mL of DPPH with 2 mL of the solvent and then measure the absorbance value A2; mix 2 mL of 95% ethanol with 2 mL of the sample and then measure the absorbance value A3. The calculation method of the DPPH radical scavenging activity is shown in Equation II:

[0079] DPPH scavenging rate (%) = [1 - (A1 - A3) / A2] × 100% Equation II.

[0080] 2. ABTS scavenging activity

[0081] Test the scavenging activity of the test drugs and microspheres on 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The sample concentration is the same as that in the DPPH scavenging activity test. Mix 1 mL of the sample solution with 3 mL of the ABTS reaction solution, react in the dark for 6 min, and then measure the absorbance at 734 nm, denoted as A4. Mix 1 mL of 70% ethanol with 3 mL of the ABTS reaction solution as a control, and its absorbance value is A5. The calculation method of the ABTS scavenging activity is shown in Equation III:

[0082] ABTS scavenging rate (%) = (A5 - A4) / A5 × 100% Equation III.

[0083] Figure 7 are the test results of the antioxidant activities of different samples, where a is the DPPH scavenging activity and b is the ABTS scavenging activity. Different letters (a - d, A - B) indicate significant differences in the antioxidant activities of different samples, p < 0.05.

[0084] According to Figure 7 it can be seen that among the several free drugs, Uro C and EA have the highest DPPH scavenging activity. At a concentration of only 50 μg / mL, the scavenging rate can reach about 50%. Followed by Uro A, with a scavenging rate of 40%. Immediately followed by Uro A, the EC 50 value is 0.027 μmol / L. The DPPH scavenging activity trend of the microspheres is generally consistent with that of the free drugs ( Figure 7In a), the activity is slightly higher than that of the free drug. After encapsulation with EA, the scavenging activity increased by 3%, and after encapsulation with Uro C, the activity increased by 2%. This indicates that the antioxidant activity of the drug increases after encapsulation in microspheres. The ABTS scavenging activities of different drugs and their microsphere-encapsulated forms are as shown in Figure 7 Figure b). It is generally consistent with the DPPH scavenging activity. Among the free drugs, Uro C and EA have relatively high ABTS scavenging activities, reaching more than 68%. Followed by Uro B and Iso A. The activity of MP@Uro C has no significant difference from that of Uro C, and the activity of MP@EA is 2% higher than that of EA. Generally speaking, the encapsulated microspheres still have good antioxidant activity, and the urolithin series drugs after encapsulation are expected to replace EA and be directly supplemented into the human body.

[0085] Example 4 In vitro digestion simulation experiment

[0086] The release of free drugs and microsphere-encapsulated drugs was detected by simulating in vitro digestion.

[0087] Oral digestion: The free drug was uniformly dispersed in 12 mL of deionized (DI) water at a concentration of 4 mg / mL, and the microspheres encapsulating the same amount of drug were also dispersed in an equal volume of deionized water. The pH of the solution was adjusted to 7.0 using 1 mol / L HCl, 10 mg of α-amylase was added, and it was continuously shaken and incubated at 37 °C and 100 rpm for 2 min. The supernatant sample was collected to detect the drug content in the simulated oral environment.

[0088] Gastric digestion: After oral digestion, the pH of the solution was adjusted to 1.0 using 1 mol / L HCl, 20 mg of pepsin was added, and it was continuously shaken and incubated at 37 °C and 100 rpm for 2 h. The supernatant sample was taken every 30 min to detect the drug content in the simulated gastric environment.

[0089] Intestinal digestion: After gastric digestion, the pH of the remaining digestive fluid was adjusted to 7.0 using 1 mol / L NaOH solution, 100 mg of pancreatin was added, and it was continuously shaken and incubated at 37 °C and 100 rpm for 4 h. The supernatant sample was taken every 1 h to detect the drug content in the intestinal environment.

[0090] Figure 8 Figure shows the simulated digestion curves of different samples, where a is EA and MP@EA, b is Uro A and MP@Uro A, c is Uro B and MP@Uro B, d is Uro C and MP@Uro C, e is Iso A and MP@Iso A, and the microspheres used are all 1% SA microspheres.

[0091] The in vitro digestion process is divided into three stages, namely oral, gastric, and intestinal digestion processes. The purpose of digestion simulation is to detect whether the microspheres encapsulating the drug can protect the drug during the digestion process. During the digestion process, there was no significant difference in the effect of SA concentration on the digestion rate of EA. Microspheres with 1% SA were selected for testing in the experiment.

[0092] Figure 8 The results in [reference] showed that about 30% of the EA group was consumed during gastric digestion. After entering the intestinal digestion stage, the digestion rate of EA increased significantly, reaching more than 80%. Compared with the digestion of the EA group, the final digestion amount of the microsphere group decreased by about 85%. The microsphere group significantly reduced the EA loss in each stage, indicating that the microspheres played a protective role on EA and prevented it from being decomposed during the initial digestion process (oral, gastric, intestinal). Similarly, the digestion curves of Uro A, Uro B, Uro C, Iso A and their microspheres showed a consistent trend ([reference] Figure 8 b - e in). A large amount of free samples were digested after passing through the oral cavity, stomach, and intestine. After being encapsulated by microspheres, their digestion rates were significantly reduced, by 86%, 67%, 84%, and 74% respectively. The results showed that the samples encapsulated by microspheres could protect the drug from being digested during the process of passing through the oral cavity, stomach, and intestine, so that it could reach the colon to exert its efficacy. Research has shown that EA cannot be directly absorbed in the human body. It is usually metabolized into different types of urolithins and then absorbed by the human body. However, different constitutions may have different metabolic conditions and metabolize different types of urolithins, and some may not even metabolize urolithins. Therefore, preparing a series of urolithin microspheres can supplement high - activity urolithins symptomatically, which is beneficial to improving the health level of the human body.

[0093] An optical microscope (SK160, MacAudie Industrial Group Co., Ltd., China) was selected to analyze the morphology of microspheres after gastric and intestinal digestion. After simulating gastric digestion for 2 h and intestinal digestion for 4 h, some drug - loaded microspheres were respectively aspirated and observed under the microscope.

[0094] Figure 9 are the optical microscopy images of microspheres encapsulating different drugs after gastrointestinal digestion. Among them, a, b, and c are the original SEM images, SEM images after gastric digestion, and SEM images after intestinal digestion of MP@EA microspheres respectively. d, e, and f are the original SEM images, SEM images after gastric digestion, and SEM images after intestinal digestion of MP@Uro A microspheres respectively. Among them, g, h, and i are the original SEM images, SEM images after gastric digestion, and SEM images after intestinal digestion of MP@Uro B microspheres respectively. j, k, and l are the original SEM images, SEM images after gastric digestion, and SEM images after intestinal digestion of MP@Uro C microspheres respectively. m, n, and o are the original SEM images, SEM images after gastric digestion, and SEM images after intestinal digestion of MP@Iso A microspheres respectively.Figure 9 The results in Figure 8 show that there are no significant changes in the microscopic images of the five microspheres after gastrointestinal digestion. This may be because only a small amount of the drug on the surface is released into the digestive fluid, and gastrointestinal digestion is not sufficient to significantly damage the structure of the microspheres, which corresponds to the digestion curve (

[0095] ). Therefore, there is no obvious change on the surface of the microspheres. The results show that the microspheres play a good role in protecting the drug during gastrointestinal digestion.

[0096] The above are only the preferred embodiments 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.

Claims

1. A preparation method of electrospun microspheres of ellagic acid and urolithin, characterized in that, It includes the following steps: Mix sodium alginate, active ingredient and water to obtain a spinning solution; the active ingredient includes one or more of ellagic acid and urolithin; Inject the spinning solution into a calcium chloride solution by electrospinning for curing to obtain cured microspheres; Mix the cured microspheres and a chitosan solution for coating to obtain the electrospun microspheres of ellagic acid and urolithin.

2. The preparation method according to claim 1, wherein The urolithin includes one or more of urolithin A, urolithin B, urolithin C and isourolithin A.

3. The preparation method according to claim 1, wherein The concentration of sodium alginate in the spinning solution is 1-2 wt%, and the content of the active ingredient is 0.5-2 mg / mL.

4. The preparation method according to claim 1, wherein The mixing of sodium alginate, active ingredient and water includes: dissolving sodium alginate in water to obtain a sodium alginate solution, and then mixing the sodium alginate solution and the active ingredient; the temperature of the dissolution is 35-45 °C, and the temperature of the mixing of the alginate and the active ingredient is 35-45 °C.

5. The preparation method according to claim 1, characterized in that, The electrospinning method includes: fixing the spinning solution on the injection pump of an electrospinning machine, and vertically injecting the spinning solution into the calcium chloride solution through a syringe.

6. The preparation method according to claim 1 or 5, characterized in that, The injection voltage is 12-16 kV.

7. The preparation method according to claim 1, characterized in that, The concentration of the calcium chloride solution is 2-4 wt%, and the curing time is 1-2 h.

8. The preparation method according to claim 1, wherein, The chitosan solution is a VC solution of chitosan, the concentration of chitosan in the chitosan solution is 0.5-2 wt%, and the coating time is 12-24 h.

9. The electrospun microspheres of ellagic acid and urolithin prepared by the preparation method according to any one of claims 1 to 8, characterized in that, It includes calcium alginate microspheres, an active ingredient encapsulated in the calcium alginate microspheres, and chitosan coated on the surface of the calcium alginate microspheres; the active ingredient includes one or more of ellagic acid and urolithin.

10. The electrospun microspheres of ellagic acid and urolithin according to claim 9, characterized in that, The average particle size of the electrospun microspheres ＜ 100 μm.

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