Ros-responsive synbiotic microgel and preparation method and application thereof

Abstract

This invention discloses a ROS-responsive synbiotic microgel, its preparation method, and its application, belonging to the field of biomedical technology. The preparation method includes the following steps: first, PBAP and CDI are reacted in a solvent; then, the product is reacted with CDPs and DMAP in a solvent to obtain PCDPs; KGM dispersion and EPTAC solution are mixed and reacted to obtain QKGM; PCDPs, QKGM solution, sodium alginate solution, and AKK bacteria are mixed to prepare a mixed solution; finally, the mixed solution is added dropwise to calcium chloride solution to obtain the final product. The ROS-responsive synbiotic microgel obtained by this invention has advantages such as uniform and controllable particle size, high encapsulation efficiency, and structural stability. It can effectively resist the degradation and destruction of AKK bacteria and PCDPs by the gastrointestinal environment, accurately achieve colon-targeted release, significantly improve the delivery efficiency and bioavailability of active ingredients, and achieve effective targeted treatment for ovarian insufficiency.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a ROS-responsive synbiotic microgel, its preparation method, and its application. Background Technology

[0002] In recent years, the incidence of premature ovarian insufficiency (POI) has been on the rise, and the age of onset is becoming increasingly younger. It not only seriously affects female fertility but also poses multiple threats to long-term health, including mental health, bones, and cardiovascular health. Cistanche deserticola polysaccharides (CDPs) possess good biocompatibility and immunomodulatory activity, and show potential application value in regulating gut microbiota and improving POI-related pathological conditions. Furthermore, they are beneficial for Akkermansia (…). Akkermansiamuciniphila The identification of AKK bacteria as a prebiotic. AKK bacteria has also been shown to be closely related to POI improvement, and constructing a synbiotic system by combining probiotics and prebiotics provides a new direction for non-invasive treatment of POI.

[0003] However, existing oral delivery systems for POIs still face significant bottlenecks: AKK bacteria, as obligate anaerobic bacteria, are extremely sensitive to gastric acid, bile salts, and reactive oxygen species (ROS), resulting in very low survival rates during gastrointestinal transport; large-molecule polysaccharides such as CDPs have poor bioavailability after oral administration, making them difficult to absorb and exert their effects directly; simultaneously, current oral delivery systems for POIs generally lack a dedicated mechanism to simultaneously protect probiotics and prebiotics and achieve colon-targeted intelligent responsive release, leading to easy degradation of active ingredients or ineffective delivery to the site of action, severely limiting the practical application of synbiotic strategies in POI treatment. Currently, there are no reports on technologies that construct ROS-responsive oral synbiotic microgel systems using CDPs-modified materials and AKK bacteria for colon-targeted POI treatment. Summary of the Invention

[0004] To address the aforementioned technical problems, the present invention aims to provide a ROS-responsive synbiotic microgel, its preparation method, and its application. This addresses the common problem that existing oral delivery systems for POIs generally lack a dedicated mechanism that can simultaneously protect probiotics and prebiotics and achieve colon-targeted intelligent responsive release, leading to the easy degradation of active ingredients or their inability to be effectively delivered to the site of action. This severely restricts the practical application effect of synbiotic strategies in POI treatment.

[0005] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides a method for preparing ROS-responsive synbiotic microgels, comprising the following steps: S1. Preparation of modified plant-derived polysaccharides: First, 4-(hydroxymethyl)phenylboronic acid pinacol ester and N,N'-carbonyldiimidazole were reacted in a solvent with stirring to obtain activated 4-(hydroxymethyl)phenylboronic acid pinacol ester; then, plant-derived polysaccharide compounds, 4-dimethylaminopyridine and activated 4-(hydroxymethyl)phenylboronic acid pinacol ester were reacted in a solvent with stirring again to obtain modified plant-derived polysaccharide; S2. Preparation of quaternized konjac glucomannan: The konjac glucomannan dispersion was mixed with a 2,3-epoxypropyltrimethylammonium chloride solution and reacted to obtain quaternized konjac glucomannan; S3, Preparation of microgels First, the modified plant-derived polysaccharide obtained from S1, the quaternized konjac glucomannan solution obtained from S2, the sodium alginate solution, and the fungi were mixed and stirred to obtain a mixed solution. Then, the mixed solution was dropped into the calcium chloride solution to prepare ROS-responsive synbiotic microgels. Plant-derived polysaccharide compounds include at least one of the following: Cistanche deserticola polysaccharide, Lycium barbarum polysaccharide, Angelica sinensis polysaccharide, Dendrobium nobile polysaccharide, Ginseng polysaccharide, Polygonatum sibiricum polysaccharide, Rehmannia glutinosa polysaccharide, Ophiopogon japonicus polysaccharide, Quinoa polysaccharide, Lonicera japonica polysaccharide, Bletilla striata polysaccharide, Rosa rugosa polysaccharide, Pueraria lobata polysaccharide, Chestnut polysaccharide, Morinda officinalis oligosaccharide, Rhodiola rosea polysaccharide, Grifola frondosa polysaccharide, and Astragalus membranaceus polysaccharide; The bacteria mentioned include Akkermansia, Lactobacillus green tea oxidase GTB1, and others. Roseburia inulinivorans and Bifidobacterium longum At least one of them.

[0006] The beneficial effects of this invention are as follows: By performing ROS-responsive functionalization modification and quaternization cationic modification on Cistanche deserticola polysaccharide and konjac glucomannan respectively, this invention obtained structurally well-defined modified Cistanche deserticola polysaccharide and quaternized konjac glucomannan. Furthermore, ROS-responsive synbiotic microgels were successfully prepared by loading AKK bacteria, which can effectively resist the degradation and damage of AKK bacteria and PCDPs by the gastrointestinal environment, precisely achieving colon-targeted release. This solves the problem that existing POI oral delivery systems cannot simultaneously protect prebiotics and probiotics, achieving targeted intelligent release, thus limiting their practical application in POI treatment. This invention significantly improves the delivery efficiency and bioavailability of active ingredients in POI oral delivery systems, achieving effective targeted treatment for ovarian insufficiency.

[0007] Furthermore, the plant-derived polysaccharide compounds are any one of the following: Cistanche deserticola polysaccharide, Lycium barbarum polysaccharide, Angelica sinensis polysaccharide, Dendrobium nobile polysaccharide, Ginseng polysaccharide, Polygonatum sibiricum polysaccharide, Rehmannia glutinosa polysaccharide, Ophiopogon japonicus polysaccharide, Quinoa polysaccharide, Lonicera japonica polysaccharide, Bletilla striata polysaccharide, Rosa rugosa polysaccharide, Pueraria lobata polysaccharide, Chestnut polysaccharide, Morinda officinalis oligosaccharide, Rhodiola rosea polysaccharide, Grifola frondosa polysaccharide, and Astragalus membranaceus polysaccharide.

[0008] Preferably, the plant-derived polysaccharide compound is Cistanche deserticola polysaccharide.

[0009] Furthermore, the bacteria included Akkermansia, Lactobacillus green tea oxidase GTB1, and others. Roseburia inulinivorans and Bifidobacterium longum Any one of them.

[0010] Preferably, the fungus is Akkermania.

[0011] Furthermore, the mass ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester and N,N'-carbonyldiimidazole in S1 is (0.5-2):(1-1.5); the mass ratio of plant-derived polysaccharide, 4-dimethylaminopyridine and activated 4-(hydroxymethyl)phenylboronic acid pinacol ester is (0.5-1):(0.3-0.5):(0.5-2).

[0012] Preferably, the mass ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester and N,N'-carbonyldiimidazole in S1 is 1:1.384; the mass ratio of plant-derived polysaccharide, 4-dimethylaminopyridine and activated 4-(hydroxymethyl)phenylboronic acid pinacol ester is 0.8:0.466:1.

[0013] Furthermore, the solvent for the reaction system of 4-(hydroxymethyl)phenylboronic acid pinacol ester and N,N'-carbonyldiimidazole in S1 is anhydrous dichloromethane.

[0014] Furthermore, the mass-to-volume ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester and anhydrous dichloromethane in S1 is 0.5-2 g: 20-40 mL.

[0015] Preferably, the mass-to-volume ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester and anhydrous dichloromethane in S1 is 1 g: 30 mL.

[0016] Furthermore, the stirring reaction time in S1 is 3-5 h, and the stirring reaction time again is 50-100 h.

[0017] Preferably, the stirring reaction time in S1 is 4 h, and the stirring reaction time again is 72 h.

[0018] Furthermore, the product of the stirred reaction of 4-(hydroxymethyl)phenylboronic acid pinacol ester and N,N'-carbonyldiimidazole in S1 was purified by the following method to obtain activated 4-(hydroxymethyl)phenylboronic acid pinacol ester: The mixture obtained from the reaction was diluted with dichloromethane, washed with double-distilled water, and the aqueous layer was discarded. The organic phase was then washed with saturated sodium chloride solution, and the sodium chloride layer was discarded. The mixture was then dried in anhydrous magnesium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure. Finally, the mixture was concentrated to obtain a pure white solid, namely activated 4-(hydroxymethyl)phenylboronic acid pinacol ester.

[0019] Furthermore, the solvent in the reaction system of plant-derived polysaccharide compounds, 4-dimethylaminopyridine, and activated 4-(hydroxymethyl)phenylboronic acid pinacol ester in S1 is DMSO.

[0020] Furthermore, the mass-to-volume ratio of plant-derived polysaccharides and DMSO in S1 is 0.5-1 g: 10-20 mL.

[0021] Furthermore, in S1, plant-derived polysaccharide compounds are first dissolved in DMSO, then 4-dimethylaminopyridine is added and stirred, and finally activated 4-(hydroxymethyl)phenylboronic acid pinacol ester is added for mixing and reaction.

[0022] Furthermore, the stirring time is 30-90 minutes.

[0023] Preferably, the stirring time is 60 minutes.

[0024] Furthermore, in S1, the product from the re-stirred reaction was dialyzed and freeze-dried to obtain modified plant-derived polysaccharides.

[0025] Furthermore, the dialysis time is 50-100 h, and the molecular weight cutoff is 3000-4000 Da.

[0026] Preferably, the dialysis time is 72 h and the molecular weight cutoff is 3500 Da.

[0027] Furthermore, the volume ratio of konjac glucomannan dispersion to 2,3-epoxypropyltrimethylammonium chloride solution in S2 is (50-100):(60-100), the concentration of konjac glucomannan dispersion is 0.08-0.16 g / mL, and the concentration of 2,3-epoxypropyltrimethylammonium chloride solution is 30 wt%-40 wt%.

[0028] Preferably, the concentration of the 2,3-epoxypropyltrimethylammonium chloride solution in S2 is 37 wt%.

[0029] Furthermore, the temperature of the mixed reaction in S2 is 70-100℃, and the time is 10-20 h.

[0030] Preferably, the temperature of the mixing reaction in S2 is 80°C and the time is 14 h.

[0031] Furthermore, the system status was observed every 2 hours during the mixed reaction. If solution evaporation occurred, a small amount of isopropanol could be added to maintain volume stability.

[0032] Furthermore, after the mixing reaction in S2 is completed, purification is performed using the following method: After the reaction was completed, the mixture was filtered under reduced pressure through a Buchner funnel and washed with an aqueous isopropanol solution to obtain a crude product. The crude product was then dissolved in an appropriate amount of deionized water, dialyzed, and dried under vacuum to obtain purified quaternized konjac glucomannan.

[0033] Furthermore, the dialysis time is 30-60 h, and the selected molecular weight cutoff is 8000-14000 Da.

[0034] Furthermore, the dialysis product can be concentrated, including the following steps: The dialysis solution was mixed with acetone solution, and the mixture was allowed to stand or incubated with stirring to precipitate. The precipitate was then filtered under reduced pressure and dried under vacuum to obtain purified quaternized konjac glucomannan.

[0035] Furthermore, the volume ratio of the dialysis solution to the acetone solution is 1:(3-4).

[0036] Furthermore, the acetone solution is pre-cooled to -30 to -10°C.

[0037] Preferably, the acetone solution is pre-cooled to -20°C.

[0038] Furthermore, the incubation time, whether static or agitated, is 2-12 hours.

[0039] Furthermore, the concentration of modified plant-derived polysaccharides in the mixed solution in S3 was 100-400 mg / mL; the mass concentration of sodium alginate in the mixed solution was 0.1%-0.5%; the mass concentration of quaternized konjac glucomannan in the mixed solution was 0.3%-0.8%; and the concentration of fungi in the mixed solution was 3×10⁻⁶. 6 -1×10 7 CFU / mL; the mass concentration of calcium chloride solution is 1%-10%.

[0040] Preferably, the concentration of the modified plant-derived polysaccharide in the mixed solution in S3 is 200 mg / mL; the mass concentration of sodium alginate in the mixed solution is 0.3%; the mass concentration of quaternized konjac glucomannan in the mixed solution is 0.5%; and the mass concentration of calcium chloride solution is 3%.

[0041] Furthermore, during mixing, sodium alginate and quaternized konjac glucomannan are first mixed evenly in a solvent, then modified plant-derived polysaccharides are added, and finally fungi are added.

[0042] Furthermore, the mixing conditions are: 3-5℃, anaerobic stirring for 30-60 min; gas microfluidic technology is used during dripping to control the gas flow rate at 3-10 L / min.

[0043] Preferably, the mixing conditions are: 4°C, anaerobic stirring for 45 min; and the gas flow rate is controlled at 5 L / min during dripping.

[0044] Furthermore, the gases used in gas microfluidics technology include nitrogen or compressed air and other inert gases.

[0045] Furthermore, after collecting the microspheres, store them at 4°C for 0-30 days before use.

[0046] Preferably, the microspheres are stored at 4°C for 7 days after collection before use.

[0047] In a second aspect, the present invention provides a ROS-responsive synbiotic microgel, which is prepared by the above-described preparation method.

[0048] A third aspect of the present invention provides the use of the above-mentioned ROS-responsive synbiotic microgel in the preparation of a medicament for colon-targeted therapy of ovarian insufficiency.

[0049] In a fourth aspect, the present invention provides a medicament for colon-targeted treatment of ovarian insufficiency, wherein the active component comprises the aforementioned ROS-responsive synbiotic microgel.

[0050] The present invention has the following beneficial effects: This invention utilizes ROS-responsive functionalization modification of Cistanche deserticola polysaccharide and quaternized cationic modification of konjac glucomannan to obtain PCDPs and QKGM derivatives with well-defined structures and stable performance. ROS-responsive synbiotic microgels AKK@PCDPs / QKGM are then prepared using gas microfluidic technology. These microgels possess advantages such as uniform and controllable particle size, high encapsulation efficiency, and structural stability. They can effectively resist degradation and damage to AKK bacteria and PCDPs by the gastrointestinal environment, precisely achieve colon-targeted release, significantly improve the delivery efficiency and bioavailability of active ingredients, and provide effective targeted treatment for ovarian insufficiency. Attached Figure Description

[0051] Figure 1 Flowchart for the preparation of ROS-responsive synbiotic microgels; Figure 2 Fourier transform infrared spectra of PCDPs and CDPs; Figure 3 The hydrogen nuclear magnetic resonance spectra of PCDPs and CDPs in d6-DMSO; Figure 4 The Fourier transform infrared spectra of QKGM and KGM are shown below. Figure 5 The hydrogen nuclear magnetic resonance spectra of QKGM and KGM in deuterated water; Figure 6 Micrograph of AKK@PCDPs / QKGM gel microspheres (10×); Figure 7 The particle size distribution of AKK@PCDPs / QKGM gel microspheres is shown. Figure 8 Gross and microscopic images of AKK@PCDPs / QKGM gel microspheres stored in physiological saline for 30 days; Figure 9 Microscopic images of AKK@PCDPs / QKGM gel microspheres after treatment with SGF, SIF, and SCF; Figure 10 The live and dead staining survival rate and relative growth rate of AKK bacteria and AKK@PCDPs / QKGM-encapsulated bacteria are shown in Figure 1. Where A represents the survival rate and B represents the relative growth rate. Figure 11 Figure 1 shows the activity characterization of AKK bacteria and AKK@PCDPs / QKGM encapsulated bacteria in a simulated gastrointestinal tract. Figure 12 This is a dynamic monitoring chart of the body weight of mice in each group over 28 days during animal experiments. Figure 13 The images show a comparison of organ indices and ovarian tissue size in mice from different groups during animal experiments. In the image, A represents organ indices and B represents size comparisons. Figure 14 The diagram shows the estrous cycle representation of mice in each group during the animal experiment. A represents the control group, B represents the model group, C represents the CDPs intervention group, and D represents the AKK@PCDPs / QKG intervention group. Detailed Implementation

[0052] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0053] Example 1: A method for preparing ROS-responsive synbiotic microgels (preparation flowchart shown below) Figure 1 (As shown), including the following steps: S1. Preparation of modified Cistanche polysaccharides (PCDPs) by self-assembly of Cistanche polysaccharides CDI activation of S101 and 4-(hydroxymethyl)phenylboronic acid pinacol ester First, 1 g of 4-(hydroxymethyl)phenylboronic acid pinacol ester (PBAP) was dried under vacuum at 50 °C for 2 h and then completely dissolved in 30 mL of anhydrous dichloromethane (DCM). Then, 1.384 g of N,N'-carbonyldiimidazole (CDI) was added and the mixture was stirred for 4 h. The resulting mixture was then diluted with dichloromethane, washed three times with 20 mL of double-distilled water, and the aqueous layer was discarded. Next, the organic phase was washed with 20 mL of saturated sodium chloride solution, and the sodium chloride layer was discarded. Finally, the mixture was dried in anhydrous magnesium sulfate, filtered under reduced pressure, and the solvent was removed by rotary evaporation under reduced pressure after filtration. After concentration, a pure white solid was obtained, which is the activated 4-(hydroxymethyl)phenylboronic acid pinacol ester (CDI-PBAP).

[0054] Preparation of S102 and PCDPs 800 mg of Cistanche deserticola polysaccharides (CDPs) were placed in a 50 mL round-bottom flask, dissolved in 16 mL of anhydrous DMSO, and then 0.466 g of 4-dimethylaminopyridine (DMAP) was added. The mixture was stirred for 1 h, and then 1 g of CDI-PBAP was added. The mixture was stirred for 72 h, and finally dialyzed for 72 h (molecular weight cutoff 3500 Da). The mixture was then dried under vacuum at -80 °C overnight to obtain PDCPs.

[0055] Preparation of S2, Quaternized Konjac Glucomannan (QKGM) Dispersion and pretreatment of S201 and konjac glucomannan (KGM) Weigh 8.00 g KGM into a 250 mL beaker, slowly add 72 mL isopropanol, turn on the water bath and heat to 60°C, and maintain this temperature while stirring for 1 h.

[0056] S202, Quaternization reaction After stirring for 1 h, while maintaining the stirring state, add 80 mL of 37 wt% 2,3-epoxypropyltrimethylammonium chloride (EPTAC) solution slowly dropwise and continue stirring for 10 min; then adjust the water bath temperature to 80℃ and react continuously for 14 h (observe the state of the system every 2 h during the reaction; if the solution evaporates, a small amount of isopropanol can be added to maintain volume stability).

[0057] S203, purification After the reaction was completed, the mixture was filtered under reduced pressure through a Buchner funnel and washed eight times with an 80 wt% isopropanol aqueous solution to obtain a crude product. The washed crude product was then completely dissolved in deionized water to obtain a QKGM aqueous solution. Finally, a dialysis bag with a molecular weight cutoff of 10,000 Da was selected for dialysis for 48 h, followed by vacuum drying to obtain QKGM.

[0058] S3, Preparation of Gel Microspheres Preparation of S301 and AKK bacteria AKK bacterial suspension (Beina Biotechnology, BNCC341917) was poured into Columbia blood agar ready-to-use plates (each plate is about 200 L; for culturing anaerobic bacteria, the culture medium should be placed in an anaerobic environment for 24 h in advance to remove oxygen), spread evenly, and then transferred to anaerobic culture conditions for cultivation.

[0059] S302, Preparation of Mixed Solution First, mix a 0.3% sodium alginate solution with a 0.5% QKGM solution until homogeneous. Then, add PCDPs and adjust the concentration to 200 mg / mL. Next, add AKK bacteria to achieve a concentration of 5 × 10⁻⁶. 6 The solution was stirred at approximately CFU / mL at 4°C under anaerobic conditions for 45 min to obtain a mixed solution.

[0060] S303, Microsphere Molding Using nitrogen as the gas phase component, the gas flow rate was controlled at 5 L / min. The mixed solution was dripped into a 3% calcium chloride solution for full cross-linking using gas microfluidics. The solution was then rinsed three times with physiological saline to obtain ROS-responsive synbiotic microgels, which were stored at 4℃.

[0061] Example 2: A method for preparing ROS-responsive synbiotic microgels includes the following steps: S1. Preparation of modified Cistanche polysaccharides (PCDPs) by self-assembly of Cistanche polysaccharides CDI activation of S101 and 4-(hydroxymethyl)phenylboronic acid pinacol ester First, 1 g of 4-(hydroxymethyl)phenylboronic acid pinacol ester (PBAP) was dried under vacuum at 50 °C for 2 h and then completely dissolved in 40 mL of anhydrous dichloromethane (DCM). Then, 1.384 g of N,N'-carbonyldiimidazole (CDI) was added and the mixture was stirred for 5 h. The resulting mixture was then diluted with dichloromethane, washed three times with 20 mL of double-distilled water, and the aqueous layer was discarded. Next, the organic phase was washed with 20 mL of saturated sodium chloride solution, and the sodium chloride layer was discarded. Finally, the mixture was dried in anhydrous magnesium sulfate, filtered under reduced pressure, and the solvent was removed by rotary evaporation under reduced pressure after filtration. After concentration, a pure white solid was obtained, which is the activated 4-(hydroxymethyl)phenylboronic acid pinacol ester (CDI-PBAP).

[0062] Preparation of S102 and PCDPs 800 mg of Cistanche deserticola polysaccharides (CDPs) were placed in a 50 mL round-bottom flask, dissolved in 20 mL of anhydrous DMSO, and then 0.466 g of 4-dimethylaminopyridine (DMAP) was added. The mixture was stirred for 1 h, and then 1 g of CDI-PBAP was added. The mixture was stirred for 72 h, and finally dialyzed for 72 h (molecular weight cutoff 3500 Da). The mixture was then dried under vacuum at -80 °C overnight to obtain PDCPs.

[0063] Preparation of S2, Quaternized Konjac Glucomannan (QKGM) Dispersion and pretreatment of S201 and konjac glucomannan (KGM) Weigh 8.00 g KGM into a 250 mL beaker, slowly add 80 mL isopropanol, turn on the water bath and heat to 60°C, and maintain this temperature while stirring for 1 h.

[0064] S202, Quaternization reaction After stirring for 1 h, while maintaining the stirring state, add 80 mL of 37 wt% 2,3-epoxypropyltrimethylammonium chloride (EPTAC) solution slowly dropwise and continue stirring for 10 min; then adjust the water bath temperature to 80℃ and react continuously for 12 h (observe the state of the system every 2 h during the reaction; if the solution evaporates, a small amount of isopropanol can be added to maintain volume stability).

[0065] S203, purification After the reaction was completed, the mixture was filtered under reduced pressure through a Buchner funnel and washed eight times with an 80 wt% isopropanol aqueous solution to obtain a crude product. The washed crude product was then completely dissolved in deionized water to obtain a QKGM aqueous solution. Finally, a dialysis bag with a molecular weight cutoff of 14000 Da was selected for dialysis for 48 h, followed by vacuum drying to obtain QKGM.

[0066] S3, Preparation of Gel Microspheres Preparation of S301 and AKK bacteria The AKK bacterial culture was poured into ready-to-use Columbia blood agar plates (each plate is about 200 L; for culturing anaerobic bacteria, the culture medium should be placed in an anaerobic environment for 24 h in advance to remove oxygen), spread evenly, and then transferred to anaerobic culture conditions for further cultivation.

[0067] S302, Preparation of Mixed Solution First, mix a 0.1% sodium alginate solution with a 0.3% QKGM solution until homogeneous. Then, add PCDPs and adjust the concentration to 300 mg / mL. Next, add AKK bacteria to achieve a concentration of 7 × 10⁻⁶. 6 The solution was stirred at approximately CFU / mL at 4°C under anaerobic conditions for 45 min to obtain a mixed solution.

[0068] S303, Microsphere Molding Compressed air was used as the gas phase component, and the gas flow rate was controlled at 10 L / min. The mixed solution was dripped into a 3% calcium chloride solution by gas microfluidics to achieve full cross-linking. The solution was washed three times with physiological saline to obtain ROS-responsive synbiotic microgels, which were then stored at 4℃.

[0069] Example 3: A method for preparing ROS-responsive synbiotic microgels includes the following steps: S1. Preparation of modified Cistanche polysaccharides (PCDPs) by self-assembly of Cistanche polysaccharides CDI activation of S101 and 4-(hydroxymethyl)phenylboronic acid pinacol ester First, 1 g of 4-(hydroxymethyl)phenylboronic acid pinacol ester (PBAP) was dried under vacuum at 50 °C for 2 h and then completely dissolved in 20 mL of anhydrous dichloromethane (DCM). Then, 1.384 g of N,N'-carbonyldiimidazole (CDI) was added and the mixture was stirred for 4 h. The resulting mixture was then diluted with dichloromethane, washed three times with 20 mL of double-distilled water, and the aqueous layer was discarded. Next, the organic phase was washed with 20 mL of saturated sodium chloride solution, and the sodium chloride layer was discarded. Finally, the mixture was dried in anhydrous magnesium sulfate, filtered under reduced pressure, and the solvent was removed by rotary evaporation under reduced pressure after filtration. After concentration, a pure white solid was obtained, which is the activated 4-(hydroxymethyl)phenylboronic acid pinacol ester (CDI-PBAP).

[0070] Preparation of S102 and PCDPs 800 mg of Cistanche deserticola polysaccharides (CDPs) were placed in a 50 mL round-bottom flask, dissolved in 10 mL of anhydrous DMSO, followed by the addition of 0.466 g of 4-dimethylaminopyridine (DMAP). The mixture was stirred for 1 h, then 1 g of CDI-PBAP was added. The mixture was stirred for 72 h, dialyzed for 72 h (molecular weight cutoff 3500 Da), and then dried under vacuum at -80 °C overnight to obtain PDCPs.

[0071] Preparation of S2, Quaternized Konjac Glucomannan (QKGM) Dispersion and pretreatment of S201 and konjac glucomannan (KGM) Weigh 8.00 g KGM into a 250 mL beaker, slowly add 60 mL isopropanol, turn on the water bath to heat to 60°C, and maintain this temperature while stirring for 1 h.

[0072] S202, Quaternization reaction After stirring for 1 h, while maintaining the stirring state, add 80 mL of 37 wt% 2,3-epoxypropyltrimethylammonium chloride (EPTAC) solution slowly dropwise and continue stirring for 10 min; then adjust the water bath temperature to 80℃ and react continuously for 14 h (observe the state of the system every 2 h during the reaction; if the solution evaporates, a small amount of isopropanol can be added to maintain volume stability).

[0073] S203, purification After the reaction was completed, the mixture was filtered under reduced pressure through a Buchner funnel and washed eight times with an 80 wt% isopropanol aqueous solution to obtain a crude product. The washed crude product was then completely dissolved in deionized water to obtain a QKGM aqueous solution. Finally, a dialysis bag with a molecular weight cutoff of 8000 Da was selected for dialysis for 48 h, followed by vacuum drying to obtain QKGM.

[0074] S3, Preparation of Gel Microspheres Preparation of S301 and AKK bacteria The AKK bacterial culture was poured into ready-to-use Columbia blood agar plates (each plate is about 200 L; for culturing anaerobic bacteria, the culture medium should be placed in an anaerobic environment for 24 h in advance to remove oxygen), spread evenly, and then transferred to anaerobic culture conditions for further cultivation.

[0075] S302, Preparation of Mixed Solution First, mix a 0.5% sodium alginate solution with a 0.8% QKGM solution until homogeneous. Then, add PCDPs and adjust the concentration to 400 mg / mL. Next, add AKK bacteria to achieve a concentration of 9 × 10⁻⁶. 6 The solution was stirred at approximately CFU / mL at 4°C under anaerobic conditions for 45 min to obtain a mixed solution.

[0076] S303, Microsphere Molding Using nitrogen as the gas phase component, the gas flow rate was controlled at 3 L / min. The mixed solution was dripped into a 3% calcium chloride solution using gas microfluidics technology to achieve full cross-linking. The solution was then rinsed three times with physiological saline to obtain ROS-responsive synbiotic microgels, which were stored at 4℃.

[0077] Experimental Example 1: Basic Performance Characterization (1) The PDPs and QKGM prepared in Example 1 were characterized by proton nuclear magnetic resonance spectroscopy and Fourier transform infrared spectroscopy. The experimental results are as follows: Figures 2-5 As shown.

[0078] Figure 2 Infrared results showed that PCDPs were at 1617 cm⁻¹ -1 1578 cm -1 1500 cm-1 A distinct absorption peak appears at [location], corresponding to the skeletal vibrations of the benzene ring in the 4-hydroxyPBA molecule. This difference directly proves that the benzene ring structure of PBA has been successfully grafted onto Cistanche deserticola polysaccharide. Figure 3 The 1H NMR results showed that PCDPs exhibited new absorption peaks at 7.77 ppm and 7.33 ppm, which correspond to the benzene ring in the PBA molecule, proving that the grafting was successful.

[0079] Figure 4 Infrared results showed that QKGM was at 1480 cm⁻¹ -1 The appearance of a new weak absorption peak at the point corresponds to the CH bond vibration of the methyl group in the quaternary ammonium salt group, proving that the grafting was successful. Figure 5 The 1H NMR spectrum results showed that the strong absorption peak of QKGM at 3.22 ppm was the methyl proton signal of the quaternary ammonium ion, proving that the grafting was successful.

[0080] (2) The ROS-responsive synbiotic microgel (AKK@PCDPs / QKGM) prepared in Example 1 was characterized by microscopic examination and particle size distribution. The experimental results are as follows: Figure 6 and Figure 7 As shown.

[0081] Figure 6 Medium-sized micrographs (10×) show that the ROS-responsive synbiotic microgels prepared in Example 1 of this invention have a uniform and stable structure. Figure 7 The particle size distribution diagram shows that the microspheres prepared in Example 1 of this invention have a particle size concentrated in the range of 150-200 μm.

[0082] (3) The storage stability of the ROS-responsive synbiotic microgel prepared in Example 1 was characterized. The experimental results are as follows: Figure 8 As shown.

[0083] Figure 8 The results showed that the microspheres maintained good stability and uniformity after being stored in physiological saline for 30 days, indicating that the ROS-responsive synbiotic microgel prepared in Example 1 of this invention has excellent storage stability.

[0084] Experimental Example 2: Colonic Targeting and Activity of AKK Bacteria (1) The ROS-responsive synbiotic microgels prepared in Example 1 were treated with simulated gastric juice (SGF), simulated small intestinal juice (SIF), and simulated colonic juice (SCF) for 1 h, 2 h, and 2 h, respectively. The experimental results are as follows: Figure 9 As shown.

[0085] Figure 9The results showed that in simulated gastric fluid, the gel microspheres shrank due to the strongly acidic environment, and their surface became denser; in simulated small intestinal fluid, the microspheres began to swell, and pores appeared on their surface; in simulated colonic fluid, the action of enzymes further accelerated the degradation of the microspheres, and obvious structural disintegration or rupture was observed. These results indicate that the ROS-responsive synbiotic microgel prepared in Example 1 of this invention can achieve colon-targeting.

[0086] (2) Using AKK bacteria cultured in the same batch as those encapsulated in AKK@PCDPs / QKGM as a control group, the survival rate, relative growth rate, and activity of the AKK@PCDPs / QKGM encapsulated bacteria in simulated gastric / small intestinal / colonic fluids were characterized. The experimental results are as follows: Figure 10 and Figure 11 As shown.

[0087] Figure 10 The results showed that the survival rate of the bacteria encapsulated in AKK@PCDPs / QKGM was comparable to that of AKK bacteria, and the relative growth rate of the bacteria encapsulated in AKK@PCDPs / QKGM on plate culture was significantly better than that of AKK bacteria. Figure 11 The results showed that, compared to AKK bacteria, the AKK@PCDPs / QKGM-encapsulated bacteria exhibited good activity in the gastrointestinal tract. These results demonstrate that the present invention, through the preparation of AKK@PCDPs / QKGM, overcomes the problem of extremely low survival rate of AKK bacteria during gastrointestinal transport.

[0088] Experimental Case 3: Characterization of POI Treatment Efficacy (1) Modeling and grouping Grouping: The experimental group was divided into POI model group (CTX), normal mouse control group (Ctrl), CDPs intervention group (CDPs) and AKK@PCDPs / QKGM intervention group (AKK@PCDPs / QKGM), with 10 mice in each group. The mice were treated for 8 weeks from the time of preventive administration to the time of death.

[0089] Among them, the CDPs intervention group and the AKK@PCDPs / QKGM intervention group were first acclimatized for 2 weeks, and then given preventive medication for 2 weeks. They were given CDPs and AKK@PCDPs / QKGM at a dose of 200 mg / kg daily, respectively. Then, the model was established. On the first day of modeling, cyclophosphamide was injected intraperitoneally at a dose of 120 mg / kg. For the next 13 days, cyclophosphamide was injected intraperitoneally at a dose of 8 mg / kg daily. The medication was continued during the modeling period until 6 weeks. Ctrl group: After 2 weeks of adaptive feeding, normal saline was administered by gavage for a total of 8 weeks; CTX group: After 2 weeks of adaptive feeding, they were given saline by gavage for 2 weeks, and then modeling was performed. On the first day of modeling, 120 mg / kg of cyclophosphamide was injected intraperitoneally, followed by 8 mg / kg of cyclophosphamide injected intraperitoneally daily for the next 13 days. Saline by gavage was continued throughout the modeling period until 6 weeks.

[0090] (2) Characteristic indicators The four groups of mice were dynamically monitored and their weight was measured daily for 28 days. After 28 days, the ovarian tissue of the mice was weighed separately and the organ index was calculated. The estrous cycle of the four groups of mice was characterized.

[0091] (3) Experimental results Experimental results are as follows Figures 12-14 As shown.

[0092] Figure 12 The results showed that the body weight of mice in the CTX model group was significantly lower than that of mice in the normal Ctrl group, proving that chemotherapy drugs caused clear damage to the mice. Meanwhile, the body weight of mice in the CDPs intervention group and the AKK@PCDPs / QKGM intervention group was higher than that of the model group. Among them, the final body weight of the AKK@PCDPs / QKGM group was basically restored to the level of the normal group.

[0093] Figure 13 The results showed that the ovarian index of mice in the CTX model group was significantly reduced, and the ovarian index showed an upward trend after intervention with CDPs and AKK@PCDPs / QKGM.

[0094] Figure 14 The results showed that the estrous cycle of mice in the CTX model group was disordered. After intervention with CDPs and AKK@PCDPs / QKGM, the estrous cycle of mice in the CDPs treatment group recovered to a certain extent, and the estrous cycle of AKK@PCDPs / QKGM basically recovered. Obvious estrous cycles and other periodic regularities could be observed.

[0095] 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 principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing ROS-responsive synbiotic microgels, characterized in that, Includes the following steps: S1. Preparation of modified plant-derived polysaccharides: First, 4-(hydroxymethyl)phenylboronic acid pinacol ester and N,N'-carbonyldiimidazole were reacted in a solvent with stirring to obtain activated 4-(hydroxymethyl)phenylboronic acid pinacol ester; then, plant-derived polysaccharide compounds, 4-dimethylaminopyridine and activated 4-(hydroxymethyl)phenylboronic acid pinacol ester were reacted in a solvent with stirring again to obtain modified plant-derived polysaccharide; S2, Preparation of quaternized konjac glucomannan: The konjac glucomannan dispersion was mixed with a 2,3-epoxypropyltrimethylammonium chloride solution and reacted to obtain quaternized konjac glucomannan; S3, Preparation of microgels First, the modified plant-derived polysaccharide obtained in S1, the quaternized konjac glucomannan obtained in S2, sodium alginate and fungi were mixed and stirred in a solvent to obtain a mixed solution. Then, the mixed solution was dropped into a calcium chloride solution to prepare ROS-responsive synbiotic microgel. The plant-derived polysaccharide compounds include at least one of the following: Cistanche deserticola polysaccharide, Lycium barbarum polysaccharide, Angelica sinensis polysaccharide, Dendrobium nobile polysaccharide, Ginseng polysaccharide, Polygonatum sibiricum polysaccharide, Rehmannia glutinosa polysaccharide, Ophiopogon japonicus polysaccharide, Quinoa polysaccharide, Lonicera japonica polysaccharide, Bletilla striata polysaccharide, Rosa rugosa polysaccharide, Pueraria lobata polysaccharide, Chestnut polysaccharide, Morinda officinalis oligosaccharide, Rhodiola rosea polysaccharide, Grifola frondosa polysaccharide, and Astragalus membranaceus polysaccharide; The bacteria mentioned include Akkermansia, Lactobacillus green tea oxidase GTB1, and others. Roseburia inulinivorans and Bifidobacterium longum At least one of them.

2. The method for preparing ROS-responsive synbiotic microgels according to claim 1, characterized in that, The mass ratio of 4-(hydroxymethyl)phenylboronic acid pinacol ester and N,N'-carbonyldiimidazole in S1 is (0.5-2):(1-1.5); the mass ratio of plant-derived polysaccharide compound, 4-dimethylaminopyridine and activated 4-(hydroxymethyl)phenylboronic acid pinacol ester is (0.5-1):(0.3-0.5):(0.5-2).

3. The method for preparing ROS-responsive synbiotic microgels according to claim 1, characterized in that, The stirring reaction time in S1 is 3-5 h, and the stirring reaction time again is 50-100 h.

4. The method for preparing ROS-responsive synbiotic microgels according to claim 1, characterized in that, The volume ratio of the konjac glucomannan dispersion to the 2,3-epoxypropyltrimethylammonium chloride solution in S2 is (50-100):(60-100), the concentration of the konjac glucomannan dispersion is 0.08-0.16 g / mL, and the concentration of the 2,3-epoxypropyltrimethylammonium chloride solution is 30 wt%-40 wt%.

5. The method for preparing ROS-responsive synbiotic microgels according to claim 1, characterized in that, The mixing reaction in S2 is carried out at a temperature of 70-100℃ for 10-20 hours.

6. The method for preparing ROS-responsive synbiotic microgels according to claim 1, characterized in that, The concentration of the modified plant-derived polysaccharide in the mixed solution in S3 is 100-400 mg / mL; the mass concentration of sodium alginate in the mixed solution is 0.1%-0.5%; the mass concentration of quaternized konjac glucomannan in the mixed solution is 0.3%-0.8%; and the concentration of fungi in the mixed solution is 3×10⁻⁶. 6 -1×10 7 CFU / mL; the mass concentration of calcium chloride solution is 1%-10%.

7. The method for preparing ROS-responsive synbiotic microgels according to claim 1, characterized in that, The mixing conditions are: 3-5℃, anaerobic stirring for 30-60 min; gas microfluidics is used during dripping to control the gas flow rate at 3-10 L / min.

8. A ROS-responsive synbiotic microgel, characterized in that, It is prepared by the preparation method according to any one of claims 1-7.

9. The use of the ROS-responsive synbiotic microgel of claim 8 in the preparation of a medicament for colon-targeted therapy of ovarian insufficiency.

10. A drug for colon-targeted therapy of ovarian insufficiency, characterized in that, The active component includes the ROS-responsive synbiotic microgel as described in claim 8.

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