Preparation method and application of chitosan oral microcapsule of tripterine

Abstract

The invention relates to the technical field of pharmacy and materials, in particular to a preparation method and application of chitosan oral microcapsules of tripterine, and the preparation method comprises the following steps: S100, dissolving tripterine in absolute ethyl alcohol to prepare a primary solution; s200, dissolving porous starch in the primary solution and carrying out ultrasonic dispersion; s300, carrying out rotary evaporation under the action of reduced pressure to remove ethanol so as to obtain a primary product; and S400, dissolving chitosan in an acetic acid solution to prepare a mixed solution, adding the primary precipitation product, and freeze-drying to obtain the (C-PS / tripterine). According to the invention, the oral bioavailability of the tripterine is successfully improved through the chitosan microencapsulated porous starch carrier.

Description

Technical Field

[0001] The invention relates to the field of pharmacy and material technology, and in particular to a method for preparing chitosan oral microcapsules of tripterygium wilfordii and application thereof. Background Art

[0003] Porous starch (PS) is a modified starch with pores formed within its molecular structure, providing a larger surface area and pore volume. By physically or chemically treating conventional starch, irregular or uniform pores can be formed, increasing the surface area of ​​starch particles and enhancing their ability to adsorb, store and release other substances. This makes porous starch a multifunctional material with applications in the food industry, pharmaceutical industry and environmental fields.

[0004] Chitosan is a natural polysaccharide obtained by deacetylation of chitin extracted from the shells of crustaceans. It is biocompatible and degradable. It carries a positive charge and can interact with negatively charged substances. These properties make chitosan widely used in medicine, especially in improving the absorption of oral drugs. Chitosan can enhance the permeability of the gastrointestinal mucosa, act as a carrier of nanoparticles or microparticles, encapsulate drugs into complexes, and improve the stability of sensitive drugs, thereby helping them to cross the gastrointestinal barrier and increase bioavailability.

[0005] Celastrol is a triterpenoid extracted from the root bark of the traditional Chinese medicinal plant Tripterygium hypoglaucum (Levl.) Hutch. It is a red crystal with antioxidant properties that can modulate the function of the NF-κB signaling pathway. The compound exhibits multiple biological activities, including anti-inflammatory, anti-cancer, antioxidant, and immunomodulatory effects, making it a promising candidate for the treatment of rheumatic diseases, inflammatory conditions, cancer, and obesity. However, the oral administration of celastrol faces certain limitations, mainly due to its poor stability and solubility. Its instability in vivo and its sensitivity to the gastrointestinal environment lead to its low oral bioavailability.

[0006] Therefore, those skilled in the art are committed to developing a method for preparing and applying chitosan oral microcapsules of tripterygium wilfordii, and successfully improved the oral bioavailability of tripterygium wilfordii through a porous starch carrier of chitosan microencapsulation. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a method for preparing chitosan oral microcapsules of tripterygium wilfordii and its application, and the oral bioavailability of tripterygium wilfordii is successfully improved by the porous starch carrier of chitosan microencapsulation. The technical solution of the present invention to solve the above technical problems is as follows: A method for preparing chitosan oral microcapsules of tripterygium wilfordii, comprising the following steps: S100. Dissolve tripterygium wilfordii in anhydrous ethanol to prepare a primary solution, wherein the concentration after dissolution is 1 mg / mL; S200. Dissolving porous starch (PS) in the primary solution prepared in step S100 and dispersing by ultrasonication, wherein the mass ratio of tripterygium wilfordii to porous starch is 1:20; S300. The ethanol is removed by rotary evaporation under reduced pressure to obtain a primary product, the primary product is washed with pure water, the primary product precipitate is collected by centrifugation, and the primary product precipitate is frozen and stored for later use; S400. dissolving chitosan (C) in an acetic acid solution to prepare a mixed solution, and mixing the mixed solution with the primary precipitation product prepared in step S300 at a volume-weight ratio of 1:10 (v / w) and stirring, collecting the precipitate after centrifugation to prepare a secondary product precipitate, washing the secondary product precipitate with pure water, freeze-drying it, and storing it in a frozen state; S500. Precipitate the secondary product in step S400 into capsules.

[0009] The beneficial effects of adopting the above scheme are: 1. Solved the problem of poor stability and solubility of tripterygium wilfordii; 2. The outer coating of chitosan enhances the protective effect of porous starch on tripterygium wilfordii; 3. The oral bioavailability of tripterygium wilfordii was successfully improved by using porous starch carriers microencapsulated with chitosan; 4. The bioadhesiveness and intestinal permeability of chitosan combined with the large specific surface area of ​​porous starch create an effective delivery platform for tripterygium wilfordii, which protects tripterygium wilfordii from degradation by gastric acid and promotes its release and absorption in the small intestine; 5. The dissolution of chitosan in acetic acid solution can effectively improve the solubility of chitosan and reduce the thermal damage of chitosan molecular structure. In addition, chitosan will aggregate due to the adsorption of acetate ions in the initial stage of dissolution in acetic acid solution, but as the dissolution process proceeds, a stable solution will be formed, which is conducive to the subsequent preparation of capsules. 6. Ultrasonic dispersion of porous starch is beneficial to improve the porosity of porous starch, enhance the specific surface area and improve the pore structure, which is beneficial for celastrol to adhere to the pores of porous starch, improve the adsorption capacity of porous starch for celastrol, and improve the oral bioavailability of celastrol.

[0010] Based on the above technical solution, the present invention can also be improved as follows.

[0011] Furthermore, after step S400, the following steps are further included: S410. The porous starch is dissolved in anhydrous ethanol and ultrasonically dispersed, chitosan and bio-glue are added and mixed and stirred evenly, and the anhydrous ethanol is rotary evaporated under reduced pressure to obtain a colloid; S420. The colloid prepared in step S410 is evenly spread out, and the secondary precipitate frozen in step S400 is evenly sprinkled on the spread colloid, and finally the colloid is cut into pieces and kneaded so that the colloid wraps the secondary precipitate to form pills; S430. Prepare the pills in step S420 into capsules.

[0012] The beneficial effect of adopting the above further scheme is that the pills made of tripterygium wilfordii, porous starch and chitosan are wrapped with an outer coat made of porous starch and chitosan to improve the stability of the active ingredient of tripterygium wilfordii.

[0013] Furthermore, the temperature of the frozen storage in step S300 and step S400 is -25°C to -18°C.

[0014] The beneficial effect of adopting the above further scheme is: storing the prepared product at low temperature to avoid high temperature denaturation and reduce the effectiveness of the product.

[0015] Further, in step S400, the pH value of the acetic acid solution is 5.5 to 6.5.

[0016] The beneficial effect of adopting the above further solution is that it is conducive to the rapid dissolution of chitosan.

[0017] Further, in step S400, the stirring rate of the mixed stirring is 500 rpm to 700 rpm, and the stirring time is 0.5 h to 1.5 h.

[0018] The beneficial effect of adopting the above further scheme is: various ingredients are stirred and mixed evenly.

[0019] The use of chitosan oral microcapsules of tripterygium wilfordii as described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The scanning electron microscopy (A) and surface potential (B) results of porous starch, porous starch-triptertium wilfordii, and chitosan-porous starch-triptertium wilfordii microcapsules of the present invention are shown; Figure 2 Fourier transform infrared spectra and X-ray diffraction patterns of different groups of the present invention; Figure 3 The in vitro release curves of porous starch-triptolamine and chitosan-porous starch-triptolamine microcapsules of the present invention; Figure 4 Figures 1 and 2 are fluorescence imaging analysis (A) and flow cytometry quantitative analysis (B) of the present invention; Figure 5 The in vivo and in vitro fluorescence imaging analysis diagrams of porous starch and chitosan-porous starch of the present invention; Figure 6 The graph is a concentration-time curve of tripterygium wilfordii in plasma after oral administration of different tripterygium wilfordii preparations (Example 1, Example 2, Comparative Example 1, Comparative Example 2 and Comparative Example 3) of the present invention. DETAILED DESCRIPTION

[0021] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0022] In the description of the present invention, it is necessary to understand that the terms "center", "length", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "inside", "outside", "peripheral", "circumferential" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred system or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present invention.

[0023] In the description of the present invention, “plurality” means at least two, for example, two, three, etc., unless otherwise clearly and specifically defined.

[0024] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, it can be the internal connection of two elements or the interaction relationship between two elements, unless otherwise clearly defined. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0025] A method for preparing chitosan oral microcapsules of tripterygium wilfordii, comprising the following steps: S100. Dissolve tripterygium wilfordii in anhydrous ethanol to prepare a primary solution, wherein the concentration after dissolution is 1 mg / mL; S200. Dissolving porous starch (PS) in the primary solution prepared in step S100 and dispersing by ultrasonication, wherein the mass ratio of tripterygium wilfordii to porous starch is 1:20; S300. The ethanol is removed by rotary evaporation under reduced pressure to obtain a primary product, the primary product is washed with pure water, the primary product precipitate is collected by centrifugation, and the primary product precipitate is frozen and stored for standby use, and the frozen storage temperature is -25°C to -18°C; S400. Dissolve chitosan (C) in an acetic acid solution to prepare a mixed solution, wherein the pH value of the acetic acid solution is 5.5 to 6.5, and mix and stir the mixed solution with the primary precipitation product prepared in step S300 at a volume-weight ratio of 1:10 (v / w), the stirring rate of the mixed stirring is 500 rpm to 700 rpm, the stirring time is 0.5 h to 1.5 h, collect the precipitate after centrifugation to prepare a secondary product precipitate, wash the secondary product precipitate with pure water, freeze-dry it, and store it in a frozen state, and the temperature of the frozen storage is -25°C to -18°C; S500. Precipitate the secondary product in step S400 into capsules, The present invention also provides a use of the above-mentioned tripterygium wilfordii chitosan oral microcapsule product.

[0026] Embodiment 1 Porous starch was purchased from Liaoning Biotechnology Co., Ltd. (Liaoning, China). Nile red, triptolide (purity ≥ 98%), and chitosan (deacetylation degree ≥ 90%, high viscosity > 400 mPa·s) were supplied by Adamas Life Company. Hoechst33342 and DiR were purchased from Suzhou Biotechnology Co., Ltd. (Suzhou, China). All other chemicals were of analytical grade.

[0027] Preparation of porous starch-celastrol (PS / celastrol) microspheres: S100. Dissolve tripterygium wilfordii in anhydrous ethanol (1 mg / mL); S200. mixing tripterygium wilfordii and porous starch at a mass ratio of 1:20 (m / m), and ultrasonically treating to ensure uniform dispersion; S300. Then, the ethanol was removed by rotary evaporator under reduced pressure, and the product attached to the round-bottom flask was scraped off. The product was washed with water, and the precipitate was collected by centrifugation at 5000 g for 5 minutes. This process was repeated three times to obtain porous starch-triptertium wilfordii microspheres (PS / triptertium wilfordii), which were stored at -20°C for future use; Preparation of chitosan-coated PS / celastrol microspheres (C-PS / celastrol): S400. Chitosan was dissolved in acetic acid solution (pH = 6) under stirring (600 rpm). The chitosan solution was mixed with porous starch-celastrol (PS / celastrol) microspheres at a volume-weight ratio of 1:10 (v / w) and stirred for 1 hour. The precipitate (C-PS / celastrol) was then collected by centrifugation at 5000 g for 5 minutes, washed three times with water to remove unbound chitosan, freeze-dried and stored at -20°C for use, and finally prepared into capsules. The capsules can be made of cellulose lipids or polyacrylic acid resins.

[0028] Embodiment 2 The difference between Example 2 and Example 1 is that in Example 2, after preparing tripterygium wilfordii, porous starch and chitosan into a medicine, the medicine is wrapped in an outer coat made of chitosan, porous starch and biological glue to control the release rate of tripterygium wilfordii and improve the absorption efficiency of the human body, comprising the following steps: S100. Dissolve tripterygium wilfordii in anhydrous ethanol (1 mg / mL); S200. mixing tripterygium wilfordii and porous starch at a mass ratio of 1:20 (m / m), and ultrasonically treating to ensure uniform dispersion; S300. Then, the ethanol was removed by rotary evaporator under reduced pressure, and the product attached to the round-bottom flask was scraped off. The product was washed with water, and the precipitate was collected by centrifugation at 5000 g for 5 minutes. This process was repeated three times to obtain porous starch-triptertium wilfordii microspheres (PS / triptertium wilfordii), which were stored at -20°C for future use; Preparation of chitosan-coated PS / celastrol microspheres (C-PS / celastrol): S400. Chitosan was dissolved in acetic acid solution (pH = 6) under stirring (600 rpm). The chitosan solution was mixed with porous starch-celastrol (PS / celastrol) microspheres at a volume-weight ratio of 1:10 (v / w) and stirred for 1 hour. The precipitate (C-PS / celastrol) was then collected by centrifugation at 5000 g for 5 minutes, washed three times with water to remove unbound chitosan, freeze-dried and stored at -20°C for future use; S410. The porous starch is dissolved in anhydrous ethanol and ultrasonically dispersed, and then chitosan and biological glue are added and mixed and stirred evenly, and then the anhydrous ethanol is rotary evaporated under reduced pressure to obtain a colloid. The biological glue can be honey, starch paste or syrup.

[0029] S420. The colloid prepared in step S410 is evenly spread out, and the secondary precipitate frozen in step S400 is evenly sprinkled on the spread colloid, and finally the colloid is cut into pieces and kneaded so that the colloid wraps the secondary precipitate to form pills; S500. Prepare the pills in step S420 into capsules. The capsules can be made of cellulose lipids or polyacrylic acid resins, which are stable in gastric juice and can be degraded in the intestine due to changes in pH or the action of intestinal bacteria, thereby releasing the outer coating prepared by mixing chitosan and porous starch, thereby facilitating the intestinal absorption of tripterygium wilfordii.

[0030] Comparative Example 1 The tripterygium wilfordii suspension used in this comparative example 1 is prepared by dissolving tripterygium wilfordii and a stabilizer in an organic solvent, then slowly dripping the mixture into water by ultrasonic injection, and then removing the organic solvent by a rotary evaporator to obtain the tripterygium wilfordii suspension.

[0031] Comparative Example 2 The first comparative example adopts porous starch-triptertium wilfordii. Specifically, the triptertium wilfordii is first dissolved in anhydrous ethanol, then the porous starch is added and ultrasonically mixed evenly, and then the ethanol is removed by a rotary evaporator under reduced pressure to obtain the porous starch-triptertium wilfordii.

[0032] Comparative Example 3 The difference between Comparative Example 3 and Examples 1 and 2 is that in Comparative Example 3, tripterygium wilfordii, porous starch and chitosan are mixed according to a certain proportion and then prepared into capsules, which specifically includes the following steps: S100. Dissolve tripterygium wilfordii in anhydrous ethanol (1 mg / mL); S200. mixing tripterygium wilfordii and porous starch at a mass ratio of 1:20 (m / m), and ultrasonically treating to ensure uniform dispersion; S300. Chitosan was dissolved in acetic acid solution (pH = 6) under stirring (600 rpm), and the chitosan solution was mixed with porous starch-celastrol (PS / celastrol) microspheres at a volume-weight ratio of 1:10 (v / w) and stirred for 1 h; S400. Then, the ethanol was removed by a rotary evaporator under reduced pressure, and the product attached to the round-bottom flask was scraped off. The product was washed with purified water and the precipitate was collected by centrifugation.

[0033] S500. Prepare the precipitate into capsules, which can be made of cellulose lipids or polyacrylic acid resin.

[0034] like Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 As shown, the analysis of Comparative Example 1 (triptertium wilfordii suspension), Comparative Example 2 (porous starch-triptertium wilfordii) and Example 1 (chitosan-porous starch-triptertium wilfordii) and porous starch (PS) are as follows: 1. Analysis of chitosan-porous starch-tripterygium wilfordii microcapsules 1.1 Structural characteristics analysis The freeze-dried microcapsules were sputter-coated with gold, and their cross-sectional microstructures were observed using a scanning electron microscope (TESCAN MIRA LMS, Czech Republic) at 5.0 kV. Fourier transform infrared (FTIR) spectra of the samples were recorded using a Nicoleti S20 FTIR spectrometer (USA) by KBr pellet technique in the range of 4000 to 400 cm⁻¹, with 32 scans and a resolution of 4 cm⁻¹. X-ray diffraction (XRD) patterns of the microcapsules were determined using an X-ray diffractometer (Rigaku D / MAX-2600, Japan). The zeta potential of the samples was analyzed using a Malvern laser particle size analyzer (Mastersizer 3000, Worcestershire, UK). The samples were dispersed to a concentration of 10 mg / mL to achieve an appropriate opacity factor (2% to 8%).

[0035] 1.2 Encapsulation efficiency analysis The microcapsule powder was added to 10 mL of anhydrous ethanol to prepare a suspension. The suspension was vortexed for 1 min to disperse the powder and then sonicated at 500 W power for 30 min in the dark. Finally, centrifuged at 5000 g for 10 min. 1 mL of the supernatant was placed in a high-performance liquid chromatography (HPLC) bottle for analysis. The HPLC conditions were as follows: column: Agilent ZORBAX SB-Aq (4.6x250 mm, 5 μm), mobile phase: acetonitrile-0.1% phosphoric acid aqueous solution (65:35), flow rate: 1 mL / min, column temperature: 20°C, injection volume: 15 μL, detection wavelength: 425 nm. The encapsulation efficiency (EE) was calculated based on the prepared tripterygium wilfordii standard curve. The encapsulation efficiency was calculated as follows: EE (%) = Mm / Mt × 100, where Mt represents the total amount of tripterygium wilfordii added, and Mm represents the amount of tripterygium wilfordii in the microspheres.

[0036] 1.3 In vitro release analysis Experimental cells and animals Caco-2 cells were purchased from Pronocell Life Science and Technology Co., Ltd. (Wuhan, China) and cultured in MEM medium supplemented with 20% fetal bovine serum (FBS) (Adamas Life, China) and 1% non-essential amino acids. Cells were passaged using 0.25% trypsin / 0.5 mM EDTA.

[0037] Female BALB / c mice (6–8 weeks old, 18–22 g) were housed in a specific pathogen-free (SPF) laboratory environment with a 12-h light–dark cycle, a temperature of 22–24°C, and a relative humidity of 30–50%. All animal experiments were performed in accordance with the guidelines and regulations approved by the Laboratory Animal Ethics Review Committee of a medical university in Shandong Province.

[0038] To compare the in vitro drug release properties of different starch microsphere formulations, a dialysis bag diffusion technique was used. Each formulation (2 mL) was placed in a dialysis bag with a molecular weight cutoff of 8-14 kDa. Each dialysis bag was immersed in 250 mL of release medium (PBS buffer at pH 7.4 containing 0.5% Tween 80 as a solubilizer) to maintain appropriate dissolution conditions. The medium was stirred at 100 rpm and maintained at a temperature of 37°C ± 0.5°C. At predetermined time points (2, 4, 8, 12, 18, and 24 h), 1 mL of the dispersion was removed and replaced with an equal volume of fresh medium to maintain a constant total volume. In vitro drug release was quantified using high performance liquid chromatography (HPLC), and each formulation was tested in triplicate.

[0039] 1.4 Caco-2 cell uptake analysis Caco-2 cells in logarithmic growth phase were seeded at a density of 50,000 cells per well in clear bottom culture dishes and cultured overnight. Nile red labeled microspheres were dispersed in culture medium at a concentration of 1 mg / mL and added to the wells. After 2 h of culture, the cells were washed three times with PBS, fixed with 4% formaldehyde, and stained with Hoechst 33342 to visualize the cell nuclei. Fluorescence microscopy was performed using an EVOS M5000 microscope (Thermo Fisher Scientific).

[0040] For quantitative analysis, 5 × 105 Caco-2 cells were cultured in 25 cm² plastic bottles until a monolayer was formed. The medium was removed and washed three times with sterile PBS. Nile red-labeled microspheres were added and incubated for 2 hours. After incubation, the cells were washed three times with PBS (pH 7.2), dissociated with trypsin, and the fluorescence intensity was measured by flow cytometry.

[0041] 1.5 In vivo and ex vivo fluorescence analysis DiR-labeled microspheres were dispersed in PBS at a concentration of 50 mg / mL and orally administered to mice. In vivo imaging was performed 1, 2, 4, 8, and 12 hours after administration to observe the in vivo distribution of the microspheres. After the last imaging, the mice were sacrificed and excised organs and tissues were subjected to ex vivo fluorescence imaging analysis to evaluate the biodistribution of the microspheres.

[0042] 1.6 Pharmacokinetic analysis in mice BALB / c mice were randomly divided into three groups. Mice were orally administered one of three test formulations: tripterygium wilfordii suspension (tripterygium wilfordii dispersed in 5% sodium carboxymethylcellulose solution), PS / tripterygium wilfordii, or C-PS / tripterygium wilfordii. The tripterygium wilfordii dose was 2 mg / kg. After administration, mice were sacrificed and blood samples were collected at 1, 2, 4, 8, 12, and 24 hours. Blood samples were centrifuged at 3000 rpm for 10 minutes to separate plasma.

[0043] 1.7 Statistical analysis All experiments were repeated at least three times. The results are expressed as mean ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test, and P < 0.05 was considered statistically significant.

[0044] Experimental results of porous starch, comparative example 2 and example 1: Table 1 shows the encapsulation efficiency (EE) of various porous starch microspheres. The encapsulation efficiency of all microspheres exceeded 71.8%, indicating that the porous starch microspheres have good encapsulation performance. Porous starch has a large specific surface area and porous properties, so it has a high adsorption capacity. The morphology of PS, PS / tripterygium wilfordii, and C-PS / tripterygium wilfordii was observed by scanning electron microscopy (SEM). Figure 1 As shown in A, the PS surface has uniform macropores. After loading with tripterygium wilfordii, the pore size of the porous starch becomes smaller.

[0045] sample Average particle size (μm) PDI Zeta potential (mV) Encapsulation efficiency (%) Porous starch 5.12 0.44±0.06 -44.9±0.86 / Porous starch-triptolide 6.64 0.39±0.06 -38.03±0.90 71.8±1.82 Chitosan-porous starch-tripterygium wilfordii microcapsules 6.98 0.43±0.02 14.26±1.35 68.7±2.13 Table 1. Particle size, dispersion coefficient, Zeta potential and encapsulation efficiency of each group of samples To further investigate the structure and surface charge of starch, the Zeta potential of starch dispersions was measured (Table 1 and Figure 1 B). The Zeta potential of porous starch is negative. The positive Zeta potential of chitosan dispersion is attributed to the protonation of nitrogen atoms in the amino groups on the surface of chitosan particles. Figure 1 As shown in B, C-PS / tripterygium wilfordii showed a positive Zeta potential, which was significantly higher than that of PS, indicating that chitosan successfully covered the porous starch surface during the modification process.

[0046] Table 2. Pharmacokinetic parameters of different groups sample Maximum plasma concentration (μg / L) <![CDATA[AUC 0-t ( μg / L•h)]]> Tripterygium wilfordii suspension 123.56±16.15 891.23±75.78 Porous starch-triptolide 233.02±47.46 1819.59±244.51 Chitosan-porous starch-tripterygium wilfordii microcapsules <![CDATA[486.78±81.50 **, ## ]]> <![CDATA[4668.71±637.97 ***, ## ]]> Comparative Example 3 <![CDATA[452.78±81.50 **, ## ]]> <![CDATA[4568.71±637.97 ***, ## ]]> Embodiment 2 <![CDATA[492.78±81.50 **, ## ]]> <![CDATA[4758.71±637.97 ***, ## ]]> *p<0.05, **p<0.01 and ***p<0.001 vs Celastrol suspension; # p<0.05, ## p<0.01 and ### p<0.001 vs Porous starch-triptolide 2. FTIR and XRD analysis The characteristic absorptions of porous starch are as follows: –OH stretching vibration at 3420 cm⁻¹, –CH2 stretching vibration at 2929 cm⁻¹, C–O–O stretching vibration (carbohydrate group) at 1642 cm⁻¹, C–O stretching vibration at 1020 cm⁻¹, and C–C, C–O stretching vibration at 1155 cm⁻¹. –CH2 bending vibration at 1454 and 1370 cm⁻¹, C–O–H bending vibration at 1082 cm⁻¹. For chitosan, the peak at 1082 cm⁻¹ involves C–O stretching vibration, and 1154 cm⁻¹ (asymmetric stretching vibration of C–O–C bridge, glycosidic band) is related to its polysaccharide structure. The absorption bands at 1659, 1598, and 1384 cm⁻¹ correspond to C=O stretching vibration (amide I), –NH2 bending vibration (amide II), and –CH2 bending vibration, respectively. The absorption band at 2924 cm⁻¹ is attributed to –CH bending vibration (from –NHCOCH3, –CH3). In addition, the strong and broad absorption band at 3450 cm⁻¹ is attributed to –OH stretching vibration, –NH stretching vibration, and intermolecular hydrogen bonding between polysaccharide components. Compared with starch and chitosan, characteristic absorption bands appeared in the spectrum of C-PS / tripterygium wilfordii, and the –OH stretching vibration of chitosan slightly shifted from 3450 cm⁻¹ to 3420 cm⁻¹, and the C=O stretching vibration slightly shifted from 1379 cm⁻¹ (amide I) to 1369 cm⁻¹, indicating the presence of intermolecular hydrogen bonding (O---H–O and O---H–N) at the starch-chitosan interface. In the FTIR spectrum of tripterygium wilfordii, sharp absorption bands were observed at 1450 cm⁻¹, 1740 cm⁻¹, and 1950 cm⁻¹, of which 1450 cm⁻¹ corresponds to the C=C skeleton vibration in the aromatic ring, and 1950 cm⁻¹ and 1740 cm⁻¹ correspond to the C=O stretching vibration. These three characteristic peaks disappeared in the spectra of PS / tripterygium wilfordii and C-PS / tripterygium wilfordii, confirming the successful encapsulation of tripterygium wilfordii, see Figure 2 A.

[0047] In order to compare the changes in crystallinity before and after nanoparticle preparation, we used X-ray diffraction (XRD) to determine the crystallinity of tripterygium wilfordii, PS, C-PS, PS / tripterygium wilfordii, and C-PS / tripterygium wilfordii ( Figure 2 B). PS exhibits a typical A-type crystal pattern, with diffraction peaks appearing at 15.06°, 17.04°, 18.01°, and 22.96°. Specifically, compared with tripterygium wilfordii, the diffraction peak at 13.1° is missing in PS / tripterygium wilfordii and C-PS / tripterygium wilfordii, while the diffraction peak at 22.96° is enhanced, indicating that porous starch can be used for the encapsulation of tripterygium wilfordii. In addition, no characteristic diffraction peaks of PS monomers were observed in C-PS / tripterygium wilfordii, indicating that chitosan was coated on the surface of PS / tripterygium wilfordii.

[0048] 3. In vitro release The release rates of tripterygium wilfordii from PS / tripterygium wilfordii and C-PS / tripterygium wilfordii increased rapidly from 0% to 24.9% and 15.96% in the first 60 min, respectively, and then the release rates gradually leveled off ( Figure 3 ). It is speculated that the tripterygium wilfordii molecules attached to the starch surface were released rapidly, resulting in a linear increase in the release rate. Between 60 and 300 minutes, the release rates of PS / tripterygium wilfordii and C-PS / tripterygium wilfordii increased slowly, from 24.9% to 43.0%, and from 15.96% to 38.7%, respectively. This may be due to the leakage of a small amount of tripterygium wilfordii from the gaps on the starch surface. As the release time increased, the release rate of tripterygium wilfordii gradually slowed down, probably because the concentration gradient of tripterygium wilfordii in the system decreased, thereby slowing down the release process. At this time, the release rate of tripterygium wilfordii from the modified porous starch microspheres was different. The release rate of tripterygium wilfordii from C-PS / tripterygium wilfordii was significantly lower than that from PS / tripterygium wilfordii, indicating that the outer coating of chitosan enhanced the protective effect of porous starch on tripterygium wilfordii.

[0049] 4. Cellular Uptake and In Vivo Imaging Caco-2 cells are a human colon adenocarcinoma cell line that structurally and functionally resemble differentiated intestinal epithelial cells. Figure 4 As shown in A, after 2 hours of incubation, the uptake of C-PS / Nile Red by Caco-2 cells was significantly higher than that of PS / Nile Red. Flow cytometry quantitative analysis confirmed that the uptake of C-PS / Nile Red by Caco-2 cells was approximately twice that of PS / Nile Red ( Figure 4 B). The cell surface carries a negative charge, while intestinal mucus contains a large amount of sialic acid and other substances. Therefore, after being coated with positively charged chitosan, C-PS can effectively enhance its interaction with Caco-cells.

[0050] PS was further labeled with a near-infrared dye and its in vivo distribution was analyzed after oral administration to mice. Figure 5 As shown, the fluorescence of DiR decreased rapidly, confirming that the unprotected fluorescently labeled product would be quickly cleared by the body. In contrast, C-PS / DiR still maintained a relatively high content in the digestive tract for up to 12 hours and was significantly higher than PS / DiR. This confirms that chitosan modification enhances the retention of C-PS / DiR in the intestine, providing a basis for improving drug absorption.

[0051] 5. Pharmacokinetic Analysis in Mice Figure 6 Table 2 shows the temporal pharmacokinetic profiles of tripterygium wilfordii in mouse plasma in Comparative Example 1 (tripterygium wilfordii suspension), Comparative Example 2 (porous starch-tripterygium wilfordii), Example 1 (chitosan-porous starch-tripterygium wilfordii), Example 2 and Comparative Example 3.

[0052] Oral administration of the tripterygium wilfordii suspension in Comparative Example 1 resulted in very low plasma levels, indicating limited absorption of tripterygium wilfordii in this formulation, and a higher plasma drug concentration of PS / tripterygium wilfordii in Comparative Example 2 compared to the tripterygium wilfordii suspension. However, the use of C-PS / tripterygium wilfordii in Example 1 resulted in a higher plasma drug concentration.

[0053] These results show that the bioadhesion provided by chitosan has a significant effect on the absorption of C-PS / tripterygium wilfordii. However, in Comparative Example 3, tripterygium wilfordii, porous starch and chitosan were only mixed, and the steps involved in Example 1 were not adopted. It can be seen that the time (Tmax) for the suspension of tripterygium wilfordii, PS / tripterygium wilfordii and C-PS / tripterygium wilfordii to reach the maximum concentration was about 4 hours, and there was no significant difference in time. The maximum plasma concentration (Cmax) of C-PS / tripterygium wilfordii was 486.8 μg / L, which was significantly higher than PS / tripterygium wilfordii (233.1 μg / L) and tripterygium wilfordii suspension (123.5 μg / L), and also higher than 452.8 μg / L in Comparative Example 3, which confirmed that chitosan and porous starch synergistically encapsulated tripterygium wilfordii to improve its bioavailability, and chitosan and porous starch synergistically encapsulated tripterygium wilfordii using the steps of Example 1 to significantly improve the bioavailability of tripterygium wilfordii.

[0054] In Example 2, tripterygium wilfordii is first dissolved in anhydrous ethanol, then mixed with porous starch, rotary evaporated, precipitated, and freeze-dried for later use. Chitosan is dissolved in acetic acid solution, and the freeze-dried tripterygium wilfordii and starch solution are mixed, and centrifugal drying is performed to remove the precipitate to prepare microcapsules. The microcapsules are then wrapped in an outer coat made of porous starch, chitosan and biological glue. After tripterygium wilfordii enters the human body, there will first be a relatively slow release process, which is similar to the "preheating" effect of the intestine and intestinal cells. This reaction process is mainly due to the fact that the microorganisms or cells in the intestine first decompose the outer coat made of porous starch and chitosan. After the chitosan and porous starch outer coats are decomposed, the pills formed by the mixture of chitosan, porous starch and tripterygium wilfordii are decomposed, so that the tripterygium wilfordii is quickly released, and the final release rate is significantly better than the release curves of Example 1 and Comparative Example 3.

[0055] One possible reason is that porous starch is degraded in the intestine to produce short-chain fatty acids (such as acetic acid, propionic acid and butyric acid), while chitosan is further broken down by microorganisms in the intestine to form molecules with amino and hydroxyl groups.

[0056] Short-chain fatty acids have a nourishing effect on intestinal epithelial cells and increase the activity of epithelial cells. At the same time, short-chain fatty acids can regulate the composition and metabolic activity of intestinal microorganisms. The products of porous starch and chitosan that are first decomposed in the intestine (short-chain fatty acids, amino groups, hydroxyl groups) preferentially act on the cells in the intestine, thereby pre-increasing the activity of intestinal cells.

[0057] When the chitosan and porous starch shells are decomposed, the chitosan, porous starch and tripterygium wilfordii mixed pills are exposed. At this time, the cells and microorganisms in the intestine decompose and absorb the mixed pills. During the decomposition process, the decomposition products of chitosan and porous starch (short-chain fatty acids, amino groups, and hydroxyl groups) continue to maintain the activity of intestinal cells and microorganisms, and improve the decomposition and absorption efficiency of tripterygium wilfordii. At the same time, the amino groups and hydroxyl groups produced by the decomposition of chitosan will introduce hydrophilic groups, which can improve the water solubility of tripterygium wilfordii. When the activity of epithelial cells is high, tripterygium wilfordii can penetrate quickly, which may increase its solubility and bioavailability in the body. Moreover, the number of beneficial microorganisms in the intestine increases, and the metabolic activity is strong. The introduction of transdermal peptides or other fat-soluble groups on amino groups or hydroxyl groups can improve the transdermal absorption rate of tripterygium wilfordii, making it easier to enter the blood circulation through the skin, thereby improving the metabolism and conversion of the active ingredients in tripterygium wilfordii.

[0058] Therefore, the present invention successfully improves the oral bioavailability of tripterygium wilfordii through a porous starch carrier microencapsulated by chitosan. The combination of chitosan and porous starch in microcapsules not only ensures the stability of tripterygium wilfordii, but also significantly improves its absorption efficiency in the intestine. Specifically, the bioadhesion and intestinal permeability of chitosan plus the large specific surface area of ​​porous starch create an effective tripterygium wilfordii delivery platform. The platform protects tripterygium wilfordii from degradation by gastric acid and promotes its release and absorption in the small intestine. Therefore, the porous starch carrier microencapsulated by chitosan provides a feasible method for improving the bioavailability of tripterygium wilfordii and other poorly soluble natural products, and provides new ideas and technical support for the development of functional foods, health supplements and pharmaceutical products.

[0059] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing chitosan oral microcapsules of tripterygium wilfordii, characterized in that: The following steps are involved: S100. Dissolve tripterygium wilfordii in anhydrous ethanol to prepare a primary solution, wherein the concentration after dissolution is 1 mg / mL; S200. Dissolving the porous starch in the primary solution prepared in step S100 and dispersing by ultrasonication, wherein the mass ratio of tripterygium wilfordii to the porous starch is 1:20; S300. The ethanol is removed by rotary evaporation under reduced pressure to obtain a primary product, the primary product is washed with pure water, the primary product precipitate is collected by centrifugation, and the primary product precipitate is frozen and stored for later use; S400. Dissolving chitosan in an acetic acid solution to prepare a mixed solution, and mixing the mixed solution with the primary precipitate product prepared in step S300 at a volume-weight ratio of 1:10 and stirring, collecting the precipitate after centrifugation to prepare a secondary product precipitate, washing the secondary product precipitate with pure water, freeze-drying it, and storing it frozen; S500. Precipitate the secondary product in step S400 into capsules.

2. The method for preparing chitosan oral microcapsules of tripterygium wilfordii according to claim 1, characterized in that: After step S400, the following steps are also included: S410. The porous starch is dissolved in anhydrous ethanol and ultrasonically dispersed, chitosan and bio-glue are added and mixed and stirred evenly, and the anhydrous ethanol is rotary evaporated under reduced pressure to obtain a colloid; S420. The colloid prepared in step S410 is evenly spread out, and the secondary precipitate frozen in step S400 is evenly sprinkled on the spread colloid, and finally the colloid is cut into pieces and kneaded so that the colloid wraps the secondary precipitate to form pills; S430. Prepare the pills in step S420 into capsules.

3. The method for preparing chitosan oral microcapsules of tripterygium wilfordii according to claim 1, characterized in that: The temperature of the frozen storage in step S300 and step S400 is -25°C to -18°C.

4. The method for preparing chitosan oral microcapsules of tripterygium wilfordii according to claim 1, characterized in that: In step S400, the pH value of the acetic acid solution is 5.5 to 6.

5.

5. The method for preparing chitosan oral microcapsules of tripterygium wilfordii according to claim 1, characterized in that: In step S400, the stirring rate of the mixed stirring is 500 rpm to 700 rpm, and the stirring time is 0.5 h to 1.5 h.

6. Use of the chitosan oral microcapsule of tripterygium wilfordii according to any one of claims 1 to 5 in a product.

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