Preparation method and application of chlorogenic acid carbon quantum dot microsphere capsule

Abstract

The invention discloses a preparation method and application of a chlorogenic acid carbon quantum dot microsphere capsule, a natural active product chlorogenic acid (CHA) in coffee is used as a raw material, and chlorogenic acid carbon quantum dots (CHA CDs) with anti-inflammatory and antioxidant activity are synthesized through a hydration method; then, the chlorogenic acid carbon quantum dots (CHACDs) are packaged with chitosan, and meanwhile, the chitosan on the outer layer of the capsule is modified with the DNA aptamer, so that the chlorogenic acid carbon quantum dot microsphere capsule Apt-CS (at) CHA CDs are obtained. The chlorogenic acid carbon quantum dot microsphere capsule disclosed by the invention not only has anti-inflammatory and antioxidant activity, but also can regulate intestinal flora, enhance intestinal barrier function and immune conditions; moreover, the microsphere capsule can be retained in the intestinal tract for a long time, so that the microsphere capsule can resist inflammation and remove high-level reactive oxygen species (ROS), can be retained in the intestinal tract for a long time and play a role in regulating intestinal flora, finally, the disease development of IBS is comprehensively relieved, and the microsphere capsule has a very good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to a preparation method and application of chlorogenic acid carbon quantum dot microsphere capsules. Background Art

[0002] Irritable bowel syndrome (IBS) is an extremely common, chronic, non-organic disorder of the digestive system. IBS patients may experience low-grade intestinal inflammation, a mild inflammatory response that does not cause significant tissue damage but may impair normal intestinal function. Inflammatory stimulation leads to macrophage activation and degranulation, releasing a variety of bioactive substances that affect intestinal motility and sensation, potentially contributing to the underlying symptoms of IBS. Some IBS patients exhibit immune cell infiltration into the intestinal mucosa, including increased numbers of lymphocytes and macrophages within the epithelium and in the lamina propria. Furthermore, during the inflammatory response, inflammatory cells become activated and aggregate to damaged areas, producing a range of inflammatory mediators and cytokines. These mediators and cytokines can induce more inflammatory cells to reach the damaged area, exacerbating the inflammatory response and triggering oxidative stress. The disparity between excessive reactive oxygen species (ROS) and weak endogenous defenses can lead to damage to cellular structure and molecules such as lipids, proteins, and DNA, ultimately exacerbating IBS. Current treatments for IBS include lifestyle and dietary interventions, antiperistaltic medications, probiotics, antispasmodics, and antidepressants. Currently, there is still a lack of a comprehensive IBS treatment plan to continuously relieve the multiple symptoms of IBS. Summary of the Invention

[0003] In view of the above-mentioned deficiencies in the prior art, the object of the present invention is to provide a preparation method and application of chlorogenic acid carbon quantum dot microsphere capsules, so as to solve the problem that the prior art lacks a complete comprehensive treatment method for IBS and a lack of drugs that can continuously relieve multiple symptoms of IBS.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] A method for preparing chlorogenic acid carbon quantum dot microsphere capsules, the specific steps are as follows:

[0006] Step 1: Preparation of chlorogenic acid carbon quantum dots:

[0007] Dissolving chlorogenic acid in water, heating at 200° C. to 250° C. for 1 to 3 hours, and cooling to room temperature to obtain a mixture, filtering to obtain a brown transparent solution, and purifying by dialysis to obtain CHA CDs; wherein the concentration of chlorogenic acid in water is 5 to 10 mg / mL;

[0008] Step 2: Preparation of Apt-CS:

[0009] The aptamer is dissolved in water and heated to 90°C to 100°C, cooled to room temperature, and then cooled at 0°C for at least 40 seconds to obtain an aptamer solution. Then, chitosan is mixed with the aptamer solution and incubated at 30°C to 40°C for at least 12 hours. The reaction product is purified to obtain Apt-CS. The mass volume ratio of the aptamer to water is (1-10) mg: (1-10) μL; the mass volume ratio of the chitosan to the aptamer solution is (1-10) mg: (1-10) μL.

[0010] Step 3: Preparation of Apt-CS@CHACDs:

[0011] The Apt-CS prepared in step 2 was dissolved in a PBS buffer solution, and then the CHA CDs solution prepared in step 1 was added dropwise to obtain chlorogenic acid carbon quantum dot microsphere capsules Apt-CS@CHA CDs through electrostatic interaction; wherein the mass ratio of Apt-CS to CHACDs was (1-10):(1-10), and the pH value of the PBS buffer solution was 7-8.

[0012] Preferably, the sequence of the aptamer is as follows:

[0013] GGTGGGTTTGGCTGGTACTTAGGGCGTCGTCCC.

[0014] Preferably, in step 1, the reaction is carried out at 230° C. for 2 h.

[0015] Preferably, in step 2, the aptamer is dissolved in water and then heated to 95°C.

[0016] Preferably, in step 2, the chitosan and aptamer solution are incubated at 35° C. to 37° C. for 12 h to 24 h.

[0017] The present invention also provides an application of chlorogenic acid carbon quantum dot microsphere capsules. The microsphere capsules prepared by the above preparation method are used to prepare a drug for treating irritable bowel syndrome.

[0018] The present invention provides a pharmaceutical composition comprising the microsphere capsule prepared by the above preparation method and a pharmaceutically acceptable carrier.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention uses the natural active product chlorogenic acid (CHA) as raw material and synthesizes chlorogenic acid carbon quantum dots (CHACDs) with anti-inflammatory and antioxidant activities through hydration method; then, chitosan is used to encapsulate the chlorogenic acid carbon quantum dots (CHACDs), and DNA aptamers are modified on the chitosan layer of the capsule to obtain chlorogenic acid carbon quantum dot microsphere capsules Apt-CS@CHACDs.

[0021] 2. The present invention unexpectedly discovered that the chlorogenic acid carbon quantum dot microsphere capsules prepared by the preparation method not only have anti-inflammatory and antioxidant activities, but can also regulate the intestinal flora, enhance the intestinal barrier function and immune conditions; and the microsphere capsules can be retained in the intestine for a long time, so that the microsphere capsules can remain in the intestine for a long time while anti-inflammatory and clearing high levels of reactive oxygen species (ROS) and play a role in regulating the intestinal flora, ultimately comprehensively alleviating the progression of IBS, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the molecular weight distribution curve of Apt-CS.

[0023] Figure 2 Preparation and characterization of Apt-CS@CHACDs; Figure 2 A is the transmission electron microscopy (TEM) image and particle size distribution histogram of CHACDs; Figure 2 B is the X-ray photoelectron spectroscopy (XPS) graph; Figure 2 C is the hydrogen nuclear magnetic resonance (1H-NMR) spectrum; Figure 2 D is the TEM image and particle size distribution histogram of Apt-CS@CHA CDs; Figure 2 E is the XPS spectrum of Apt-CS@CHACDs; Figure 2 F is the zeta potential; Figure 2 G is the element distribution pattern of carbon (C), nitrogen (N), oxygen (O) and phosphorus (P) in Apt-CS@CHACDs.

[0024] Figure 3 is the C1s spectrum of CHACDs.

[0025] Figure 4 is the O1s spectrum of CHACDs.

[0026] Figure 5 N1s spectrum of Apt-CS@CHACDs.

[0027] Figure 6 is the C1s spectra of Apt-CS@CHACD.

[0028] Figure 7 This is the O1s spectrum of Apt-CS@CHACD.

[0029] Figure 8 For the in vitro therapeutic effect of IBS; Figure 8 A is the dose-dependent scavenging effect of free CHA, CHA CDs, and Apt-CS@CHA CDs on DPPH free radicals; Figure 8 B is the scavenging effect of free CHA, CHACDs and Apt-CS@CHA CDs on ABTS free radicals; Figure 8 C is the scavenging effect of free CHA, CHACDs and Apt-CS@CHACDs on PTIO free radicals; Figure 8 D~8F are the ROS levels in Caco-2 cells after different treatments: Figure 8 D is the intracellular ROS fluorescence image induced by Rosup; Figure 8 E is the result of flow cytometry; Figure 8 F is the statistical analysis of ROS levels; Figure 8 Figures G, 8H, and 8I are graphs showing the mRNA expression levels of (G) TNF-α, (H) IL-6, and (I) IL-1β in Caco-2 cells detected by real-time fluorescence quantitative PCR (drug concentrations were converted to 100 μg / mL CHA, n=5, *p<0.05, **p<0.01, ***p<0.001, ns, no significant difference).

[0030] Figure 9 Effects of free CHA, CHACDs, and Apt-CS@CHACDs at different concentrations (6.25-200 μg / mL) on RAW 264.7 cells (n=6)

[0031] Figure 10 Effects of free CHA, CHA CDs, and Apt-CS@CHA CDs at different concentrations (6.25-200 μg / mL) on Caco-2 cell viability. (n=6)

[0032] Figure 11 Statistical analysis of fluorescence of DHE images.

[0033] Figure 12 Retention, distribution and penetration in the body; Figure 12 A is the fluorescence imaging of IBS model mice at 0, 4, 8, 12, 16, and 24 hours after oral administration of CS-FITC@CHA CDs and Apt-CS-FITC@CHACDs; Figure 12B is the histogram analysis of the fluorescence signal intensity of CS-FITC@CHA CDs and Apt-CS-FITC@CHA CDs at 0, 4, 8, 12, 16, and 24 h; Figure 12 C is the half-life of the two nanoparticles CS-FITC@CHACDs and Apt-CS-FITC@CHA CDs in vivo; Figure 12 D is the fluorescence imaging of the gastrointestinal tract at 0, 8, and 16 hours after oral administration; Figure 12 E is the corresponding fluorescence signal intensity analysis graph.

[0034] Figure 13 The body weight change curves of IBS mice in different treatment groups (n=5)

[0035] Figure 14 is the in vivo efficacy of LBL in the treatment of IBS; Figure 14 A is a representative image of the collected intestine; Figure 14 B is colorectal length (n=5); Figure 14 C-14E ​​is the serum levels of IL-6, IL-1β, and TNF-α (n=5); Figure 14 F is a representative image of immunohistochemistry for IL-6, IL-1β, and TNF-α in the colon; Figure 14 G-14I is the mean integrated optical density (IOD) of IL-6, IL-1β, and TNF-α quantitatively analyzed by Image J (n=5). (*p<0.05, **p<0.01, ***p<0.001, ns no significant difference)

[0036] Figure 15 The improvement effect of the intestinal barrier after administration of Apt-CS@CHACDs; Figure 15 A is a representative immunofluorescence staining image of occludin-1 (ZO-1) in colon tissue; Figure 15 B is a representative immunofluorescence staining image of occludin; Figure 15 C is a representative immunofluorescence staining image of reactive oxygen species (ROS); Figure 15 D-15F is the fluorescence intensity of ZO-1, occludin and ROS measured by ImageJ (n=5). (*p<0.05, **p<0.01, ***p<0.001, ns no significant difference)

[0037] Figure 16 Representative photos of colon tissue stained with Alcian blue.

[0038] Figure 17 The regulatory effect of Apt-CS@CHACDs on intestinal microbiota; Figure 17A is the Chao1 index; Figure 17 B is the Ace index; Figure 17 C is the number of observed species (Sob); Figure 17 D is the inverse-Simpson index (Inverse-Simpson) and principal component analysis (PCA); Figure 17 E is the heat map of the top ten abundant taxa at the family level; Figure 17 F is the LEfSe histogram, and bacterial taxa with LDA scores > 3.5 were labeled as biomarkers; Figure 17 G is a Circos diagram showing the distribution ratio of dominant bacteria among different groups. (*p<0.05, **p<0.01, ***p<0.001, ns, no significant difference)

[0039] Figure 18 The figure shows the process of the preparation method of the present invention and the working principle diagram of the microsphere capsule. DETAILED DESCRIPTION

[0040] The present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments derived by persons of ordinary skill in the art based on the present invention are within the scope of protection of the present invention.

[0041] Unless otherwise indicated in specific cases, the numerical ranges listed herein include the upper and lower limits, and all integers and fractions within the range, and are not limited to the specific values ​​listed when defining the range.

[0042] 1. Preparation method of chlorogenic acid carbon quantum dot microsphere capsules

[0043] Step 1: Preparation of chlorogenic acid carbon quantum dots:

[0044] Dissolving chlorogenic acid in water, heating at 200° C. to 250° C. for 1 to 3 hours, and cooling to room temperature to obtain a mixture, filtering to obtain a brown transparent solution, and purifying by dialysis to obtain CHA CDs; wherein the concentration of chlorogenic acid in water is 5 to 10 mg / mL;

[0045] Step 2: Preparation of Apt-CS:

[0046] The aptamer is dissolved in water and heated to 90°C to 100°C, cooled to room temperature, and then cooled at 0°C for at least 40 seconds to obtain an aptamer solution. Then, chitosan is mixed with the aptamer solution and incubated at 30°C to 40°C for at least 12 hours. The reaction product is purified to obtain Apt-CS. The mass volume ratio of the aptamer to water is (1-10) mg: (1-10) μL; the mass volume ratio of the chitosan to the aptamer solution is (1-10) mg: (1-10) μL.

[0047] Step 3: Preparation of Apt-CS@CHACDs:

[0048] The Apt-CS prepared in step 2 was dissolved in a PBS buffer solution, and then the CHA CDs solution prepared in step 1 was added dropwise to obtain chlorogenic acid carbon quantum dot microsphere capsules Apt-CS@CHA CDs through electrostatic interaction; wherein the mass ratio of Apt-CS to CHACDs was (1-10):(1-10), and the pH value of the PBS buffer solution was 7-8.

[0049] While researching IBS-related therapeutic agents, the present inventors discovered that chlorogenic acid (CHA), a polyphenolic compound found in plants such as honeysuckle and Eucommia ulmoides, possesses anti-inflammatory, antioxidant, and neuroprotective properties. CHA can improve anxiety, depression, and PTSD-like symptoms, regulate the intestinal microbiome, and increase the abundance of bacteria that produce short-chain fatty acids (SCFAs). CHA treatment can increase the relative abundance of Bifidobacterium and the expression of the intestinal tight junction (TJ) proteins occludin and zonula occludens-1 (ZO-1), protecting the intestinal barrier and reducing inflammatory responses. However, how can chlorogenic acid be ensured to enter the intestine in a sustained and stable manner? Further research by the present inventors led them to consider formulating chlorogenic acid into chlorogenic acid carbon quantum dots. The CDs have multiple functional groups on their surface, including ketocarbonyl, hydroxyl, and carboxyl groups, which mimic different enzyme catalytic active sites to provide excellent quantum yield and bioavailability. While natural product-derived CDs retain their biological activity, the structural characteristics, physicochemical properties, and biological functions of the CDs vary depending on the natural product. Based on this, the present invention further discovered that chlorogenic acid CHA, a natural active product in coffee, was used as a raw material to synthesize chlorogenic acid carbon quantum dots (CHACDs) with anti-inflammatory and antioxidant activities by a hydrothermal method. This retains the biological activity of chlorogenic acid, and the obtained chlorogenic acid carbon quantum dots can also be better encapsulated in chitosan microsphere capsules. However, if the microsphere capsules are only such, the retention time in the intestine is not long, and it is difficult to play a sustained therapeutic role. To this end, the present invention discovered a DNA aptamer (ssDNA) that can specifically recognize and bind to melatonin, and it can be successfully modified on the chitosan (CS) on the outer layer of the capsule through electrostatic interaction, so that the prepared microsphere capsule can be retained in the intestine for a longer period of time. Finally, the microsphere capsule has the "trinity" therapeutic function of anti-inflammatory, scavenging high levels of ROS, and regulating intestinal flora, aiming to comprehensively alleviate the progression of IBS and provide a useful reference for the future treatment of IBS.

[0050] In some embodiments of the present invention, the chlorogenic acid is derived from an active substance extracted from coffee, and the active substance also contains at least caffeic acid or quinic acid. In actual use, the active substance extracted from coffee can also be used instead of chlorogenic acid, and the coffee active extract is dissolved in water to ensure that the concentration of the chlorogenic acid in the water reaches 5 to 10 mg / mL. Therefore, when chlorogenic acid is dissolved in water, the concentration of chlorogenic acid in water can be 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL or 10 mg / mL, or in a numerical range consisting of any two of the above specific values ​​as endpoints. It should be understood that in the embodiment, any of the above ranges can be combined with any other ranges.

[0051] In some embodiments of the present invention, in step 1, chlorogenic acid dissolved in water is heated to 200°C to 250°C and reacted for 1 hour to 3 hours to ensure that chlorogenic acid can form a sufficient amount of chlorogenic acid carbon quantum dots. A temperature lower than this will result in incomplete reaction and difficulty in obtaining the desired carbon quantum dots; however, the temperature should not be too high, as it will damage the already formed chlorogenic acid carbon quantum dots. A reaction time that is too short will result in insufficient carbon quantum dots being generated and poor therapeutic effect; a reaction time that is too long will lead to the formation of other byproducts, which will also affect the therapeutic effect. Therefore, the heating temperature in step 1 can be 200°C, 210°C, 220°C, 230°C, 240°C or 250°C, or a range consisting of any two of the above specific values ​​as endpoints; the reaction time in step 1 can be 1 hour, 2 hours or 3 hours, or a range consisting of any two of the above specific values ​​as endpoints; it should be understood that in the embodiments, any of the above ranges can be combined with any other ranges.

[0052] In some embodiments of the present invention, a DNA aptamer (ssDNA) has been discovered that specifically recognizes and binds to melatonin. The aptamer sequence is as follows: GGTGGGTTTGGCTGGTACTTAGGGCGTCGTCCC. This aptamer is not only uniquely capable of specifically recognizing and binding melatonin in the intestine, but also, more importantly, is successfully electrostatically modified onto the chitosan (CS) outer layer of the capsule, enabling the resulting microsphere capsule to remain in the intestine for a longer period of time.

[0053] In some embodiments of the present invention, in step 2, the aptamer is dissolved in water and heated to 90°C to 100°C for at least 5 minutes. The solution is then cooled to room temperature and then placed at 0°C for at least 40 seconds to obtain an aptamer solution. This treatment ensures that any misfolding in the aptamer structure is eliminated. Chitosan is then mixed with the aptamer solution and incubated at 30°C to 40°C for at least 12 hours, preferably at 37°C. The reaction product is then purified to obtain Apt-CS. Wherein, in step 2, the heating temperature after the aptamer is dissolved in water can be 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C or 100°C, or a numerical range consisting of any two of the above specific values ​​as endpoints; the incubation temperature can be 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, or a numerical range consisting of any two of the above specific values ​​as endpoints; the mass volume ratio of the aptamer to water can be 1 mg:1 μL, 1 mg:2 μL, 1 mg:3 μL, 1 mg:4 μL, 1 mg:5 μL, 1 mg:6 μL, 1 mg:7 μL, 1 mg:8 μL, 1 mg:9 μL, 2 mg:7 μL, 4 mg:9 μL, 7 mg:10 μL, 1 0mg:1μL or 1mg:10μL, or in the numerical range consisting of any two of the above specific values ​​as endpoints; the mass volume ratio of chitosan to aptamer solution can be 1mg:1μL, 1mg:2μL, 1mg:3μL, 1mg:4μL, 1mg:5μL, 1mg:6μL, 1mg:7μL, 1mg:8μL, 1mg:9μL, 2mg:1μL, 2mg:3μL, 2mg:5μL, 2mg:7μL, 2mg:9μL, 3mg:1μL, 3mg:2μL, 4mg:9μL, 7mg:10μL, 10mg:1μL or 1mg:10μL, or in the numerical range consisting of any two of the above specific values ​​as endpoints; it should be understood that, in the embodiment, any of the above ranges can be combined with any other ranges.

[0054] 2. Application of a chlorogenic acid carbon quantum dot microsphere capsule

[0055] The microsphere capsules prepared by the above-described preparation method are used to prepare a medicament for treating irritable bowel syndrome. The medicament is a pharmaceutical composition comprising the microsphere capsules prepared by the above-described preparation method and a pharmaceutically acceptable carrier. Examples include, but are not limited to, solid dosage forms and liquid dosage forms. Solid dosage forms include tablets, capsules, powders, and granules. Liquid dosage forms include solutions, syrups, emulsions, and suspensions.

[0056] 3. Examples and Comparative Examples

[0057] 1. Reagents used

[0058] Chlorogenic acid (CHA) and chitosan (CS) were purchased from Shanghai Mecoxlin Technology Co., Ltd. (Shanghai, China).

[0059] The aptamer (Apt) was commissioned to be synthesized by BGI.

[0060] 2′-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS), 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH), and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl radical (PTIO) were purchased from Shanghai Mecoxlin Technology Co., Ltd. (Shanghai, China).

[0061] CCK-8 was purchased from Shengbio Biotechnology Co., Ltd. (Shanghai, China).

[0062] Fetal bovine serum (FBS), penicillin-streptomycin, Dulbecco's modified Eagle's medium (DMEM), and RPMI 1640 medium were purchased from Thermo Fisher Scientific (Beijing, China).

[0063] 4% paraformaldehyde was purchased from Beijing Langbo Technology Co., Ltd. (Beijing, China).

[0064] 2. Preparation of Examples

[0065] Example 1

[0066] Step 1: Preparation of CHACDs

[0067] 150 mg of chlorogenic acid was dissolved in 30 mL of deionized water and transferred to a 25 mL Teflon-lined autoclave and heated at 230°C for 2 h. After cooling to room temperature, the resulting mixture was filtered through a 0.02 μm microporous membrane to yield a brown, transparent solution. Finally, CHACDs were recovered by dialysis (Mw = 1 kDa) for 8 h.

[0068] Step 2: Preparation of Apt-CS

[0069] The aptamer (4 mg) was heated in a 95°C water bath for 5 min and then cooled at room temperature for 30 min. The aptamer solution was further cooled on ice for 40 s to remove misfolding in its structure. 200 mg of CS was incubated with 200 μL of aptamer solution (containing 4 mg of aptamer) in a 37°C water bath overnight. The aptamer-modified NHS active ester reacted with the amino group of CS to form an amide bond, thereby obtaining Apt-CS. The reaction solution was then dialyzed against deionized water and freeze-dried to obtain the Apt-CS material, and the molecular weight of the obtained material was determined by gel permeation chromatography ( Figure 1 ).

[0070] Step 3: Preparation of Apt-CS@CHACDs

[0071] Apt-CS was dissolved in a PBS solution with a pH of 7.4, and then CHACDs solution was added dropwise (the mass ratio of the two was 10:1). Apt-CS@CHACDs was obtained through the electrostatic interaction between the two.

[0072] Comparative Example 1

[0073] The method was adjusted based on Example 1, except that only step 1 was performed, and the product prepared was CHACDs.

[0074] Comparative Example 2

[0075] Adjustments were made based on Example 1, except that no aptamer Apt was added in Comparative Example 2. Other steps were exactly the same as in Example 1, and the product prepared was CS@CHACDs.

[0076] 3. Statistical analysis

[0077] All experiments were performed using a randomized, blinded method. Statistical analysis was performed using Prism (Graphpad 9), and data are presented as mean ± standard deviation (SD). Statistical significance was determined at P < 0.05 by one-way analysis of variance with Tukey's / Dunn's multiple comparison test.

[0078] 4. Characterization of Apt-CS@CHACDs

[0079] Taking the product prepared in Example 1 as an example, CHACDs were dissolved in DMSO-d 6 Then transfer 600 μL of the solution into an NMR tube. 1 H-NMR spectra were obtained on an AVANCE NEO 400 spectrometer (Bruker, Switzerland). The hydrodynamic size and zeta potential of CHACDs and Apt-CS@CHACDs were determined using a Zetasizer Nano ZS 90 instrument (Malvern, UK). The morphology of CHACDs and Apt-CS@CHACDs, as well as the elemental distribution of Apt-CS@CHACDs, were observed using a Talos F200S transmission electron microscope (Thermo Fisher, Czech Republic).

[0080] Chlorogenic acid CHA is mainly found in coffee. Therefore, inspired by the coffee roasting process, the present invention synthesized CHACDs by hydrothermal treatment of CHA. DLS analysis showed that the average particle size distribution of CHACDs was 50.34±3.71nm, and under transmission electron microscopy (TEM) observation, they were mainly well-dispersed quasi-spherical in shape ( Figure 2 A). Subsequently, X-ray photoelectron spectroscopy (XPS) and nuclear magnetic resonance H spectroscopy ( 1 H-NMR) was used to characterize the elemental composition and main groups of CHACDs. XPS of CHACDs showed two obvious characteristic peaks at 532.91 and 284.81 eV, which represent O1s and C1s respectively. This data shows that CHACDs mainly contain two elements, C and O, with a molar ratio of 67.43:32.57 ( Figure 2 B). In the HR C1s spectrum ( Figure 3 ), the binding energies of 284.8 and 288.89 eV are assigned to C=C / CCC, and NC=O bonds, respectively. In the HR O1s spectrum ( Figure 4 ), the binding energies of 531.3 and 532.5 eV are attributed to C=O and C-OH / COC. 1 H-NMR showed a number of characteristic peaks from 7.54 to 7.01 ppm, which were attributed to the aromatic rings of the CHA original substance in CHACDs ( Figure 2 C). Then, the present invention utilizes electrostatic interaction to allow CS modified by ssDNA aptamer (Apt) to encapsulate CHACDs to obtain nanocapsules (Apt-CS@CHACDs). First, the successful preparation of Apt-CS@CHACDs was studied by TEM. Figure 2 As shown in D, Apt-CS@CHACDs are well-dispersed quasi-spherical in shape with an average diameter of 739.7±203.6nm. XPS was then used to further analyze the changes in the elements of Apt-CS@CHACDs. From the results, there are three obvious peaks at 284.8401.29 and 532.6eV in the XPS of Apt-CS@CHACDs, representing C1s, N1s, and O1s, respectively. In the HR N1s spectrum ( Figure 5 ), the binding energy at 400.1eV is attributed to the amino group N, indicating the presence of amino groups. In the HR C1s spectrum ( Figure 6 ), the binding energies of 288.6eV and 287.9 ​​are attributed to the NC=O and C=N / C=O bonds. In the HR O1s spectrum ( Figure 7), the binding energy of 532.5eV is attributed to C-OH / C=OC. And because CS mainly exhibits positive charge in aqueous solution, the potential of CHACDs after being wrapped by Apt-CS changes from the original -24.2±1.01mV to 56.1±4.38mV, and the potential has been significantly reversed. These data show that Apt-CS successfully encapsulates CHACDs in it. In order to further study the successful preparation of Apt-CS@CHACDs, the present invention carried out elemental analysis of nanocapsules. From the results ( Figure 2 G) The Apt-CS@CHACDs surface shows a uniform distribution of O, N, and P elements. The presence of P clearly indicates the successful Apt bonding. Although XPS analysis did not reveal the presence of P, this may be due to its low content. The observation of nanocapsules plays a key role in supplementing the P element's presence. Combined with these characterization results, the successful preparation of Apt-CS@CHACDs is fully demonstrated.

[0081] 5. Verification of free radical scavenging effect

[0082] (1) ABTS scavenging activity

[0083] ABTS was prepared by mixing 2.45 mM K2S2O8 and 2 mM ABTS in phosphate buffered saline (PBS, 25 mM, pH = 7.4). + Solution and store in the dark for 12 h. Dilute ABTS + The absorbance of the solution was measured at 734 nm and was 0.750 ± 0.025. + Different concentrations of free CHA, CHACDs, and Apt-CS@CHACDs solutions (0.1 mL, 0-100 μg / mL) were added. After the reaction, the absorbance was measured. Distilled water was used as a control.

[0084] ABTS scavenging rate (%) = (1-Abs Sample / Abs Control )×100%

[0085] Abs Sample : Samples and ABTS + Absorbance value of the solution after reaction; Abs Control :ABTS + The absorbance value of the solution before reaction.

[0086] (2) DPPH scavenging activity

[0087] DPPH solution (3 mL, 0.1 mol / L) was added to various concentrations of free CHA, CHACDs, and Apt-CS@CHACDs solutions (1 mL, 0–100 μg / mL). The reaction was carried out at room temperature in the dark for 30 min. Anhydrous ethanol was used as a control.

[0088] DPPH scavenging rate (%) = (1-Abs Sample / Abs Control )×100%

[0089] Abs Sample : absorbance value after the sample reacts with DPPH solution; Abs Control : Absorbance value of DPPH solution before reaction.

[0090] (3) PTIO scavenging activity

[0091] PTIO solution (3 mL, 0.1 mol / L) was added to various concentrations of free CHA, CHACDs, and Apt-CS@CHACDs solutions (1 mL, 0–100 μg / mL). The reaction was carried out at room temperature in the dark for 30 min. Anhydrous ethanol was used as a control.

[0092] PTIO scavenging rate(%)=(1-Abs Sample / Abs Control )×100%

[0093] Abs Sample : absorbance value after the sample reacts with PTIO solution; Abs Control : Absorbance value of PTIO solution before reaction.

[0094] (4) Elimination of intracellular reactive oxygen species (ROS)

[0095] RAW264.7 cells were first seeded, treated with free CHA, CHACDs, and Apt-CS@CHACDs solution (CHA: 100 μg / mL), and then cultured with DHE (10 μM). Finally, the cells were observed by fluorescence microscopy and measured by ImageJ.

[0096] RAW 264.7 cells were treated with an equal volume of 600 μM H₂O₂ to induce cell stress and incubated for 2 h. Cells were then treated with three CHA preparations at 20 μg / mL concentrations and incubated with DHE. Cells were harvested and quantified by FCM analysis.

[0097] (5) Cleaning effect

[0098] Excellent ROS scavenging ability is the basis of antioxidant and anti-inflammatory therapy. The IBS therapeutic potential of Apt-CS@CHACDs was evaluated by testing its antioxidant capacity. Specifically, the broad-spectrum antioxidant activity of Apt-CS@CHACDs against 1,1-diphenyl-2-trinitrophenylhydrazine radical (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS), and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO) was systematically examined ( Figure 8 A-8C). ABTS, DPPH, and PTIO are commonly used reagents for antioxidant evaluation. Apt-CS@CHA CDs exhibited a significant concentration-dependent scavenging activity against these three free radicals. At a concentration of 100 μg / mL, Apt-CS@CHACDs achieved scavenging rates of 91.14%, 99.59%, and 100.01% for ABTS, DPPH, and PTIO, respectively. However, at the same concentration, free CHA exhibited significantly lower scavenging rates for ABTS, DPPH, and PTIO than Apt-CS@CHACDs. Antioxidant results demonstrated that Apt-CS@CHACDs exhibited broad-spectrum antioxidant properties and a higher free radical scavenging efficiency than free CHA. These results confirm that the Apt-CS@CHA CDs prepared in this invention possess potent antioxidant activity and are effective in scavenging ROS.

[0099] 6. Inflammatory gene expression detection

[0100] To evaluate the expression of three cholesterol efflux receptor genes, IL-6, IL-1β, and TNF-α, Raw 264.7 cells were incubated with ox-LDL. Cells were treated with free CHA, CHACDs, and Apt-CS@CHACDs solution (CHA: 100 μg / mL) for 24 hours, and mRNA expression was then extracted and measured. The primer pairs are listed below:

[0101] β-Actin:Forward Primer:5'-GTGAAGGTGACAGCAGTCGGTT-3'

[0102] Reverse Primer:5'-GAAGTGGGGTGCTTTTAGGA-3';

[0103] IL-6:Forward Primer:5'-GTTGCCTTCTTGGGACTGATG-3'

[0104] Reverse Primer:5'-TTGGGAGTGGTATCCTCTGTGA-3';

[0105] INF-α:Forward Primer:5'-CACCACGCTCTTCTGTCTACTG-3'

[0106] Reverse Primer:5'-GGTCTGGGCCATAGAACTGA-3';

[0107] IL-1β:Forward Primer:5'-TTGAAGTTGACGGACCCCA-3'

[0108] Reverse Primer:5'-GAGTGATACTGCCTGCCTGAAG-3';

[0109] Before in vitro and in vivo biological evaluations, it is necessary to determine the biocompatibility of nanomedicines. The cell compatibility of Apt-CS@CHACDs was studied using mouse macrophage cell line RAW 264.7 cells and human cloned colon adenocarcinoma cells (Caco-2). Figure 9-10 As shown in the figure, after incubating cells with different concentrations of Free CHA, CHACDs, or Apt-CS@CHACDs for 24 hours, Free CHA, CHACDs, or Apt-CS@CHACDs showed no obvious cytotoxicity against RAW 264.7 and Caco-2 cells. Even at an equivalent drug concentration of up to 100 μg / mL, the cells maintained good cell viability. These results indicate that Apt-CS@CHACDs has satisfactory biocompatibility.

[0110] In addition, ROS has important biological functions in organisms. At appropriate levels, ROS regulates cell signaling, apoptosis, and immune responses, among others. However, excessive production of ROS exceeding endogenous antioxidant capacity will lead to adverse oxidative cell damage, further leading to cell dysfunction and inflammatory responses. Based on the good antioxidant activity of Apt-CS@CHACDs, the present invention also studied the ROS scavenging ability of Apt-CS@CHACDs in cells. The Caco-2 cells in the model group were treated with lipopolysaccharide (LPS) for 12 hours, while Caco-2 cells cultured alone in culture medium served as normal controls. The production of intracellular ROS was monitored by the ROS fluorescent probe dihydroethidium (DHE). As Figure 8As shown in D, Caco-2 cells in the model group showed higher ROS accumulation. In contrast, Caco-2 treated with equal doses of Free CHA, CHA CDs, or Apt-CS@CHACDs for 12 h showed significantly weakened FL signals, especially in the Apt-CS@CHA CDs-treated group, where only negligible ROS were detected, similar to the normal control group. Semi-quantitative statistics of the red fluorescence of each group of images ( Figure 11 ), showing that the red fluorescence intensity of the Apt-CS@CHACDs group was only 0.16 times that of the control group. Further quantitative analysis of the effective scavenging of ROS by the Apt-CS@CHA CDs prepared by the present invention by flow cytometry showed that Apt-CS@CHACDs can effectively scavenge ROS in cells, restoring the ROS level in cells to a normal range ( Figure 8 E-8F).

[0111] IBS is often accompanied by inflammatory reactions. Inflammatory cells are activated and aggregated to damaged areas, producing inflammatory mediators and cytokines, inducing more inflammatory cells to arrive and exacerbate the inflammatory response, triggering oxidative stress. Excessive ROS and weak endogenous defense capabilities cause damage to cell structures and molecules such as lipids, proteins, and DNA, ultimately worsening IBS. To this end, after treatment with Free CHA, CHACDs, or Apt-CS@CHACDs, the reduction of inflammation was observed by quantifying the mRNA expression of three inflammatory factors in Caco-2. From the results ( Figure 8 G-8I), all three treatment groups effectively suppressed the expression of TNF-α, IL-6, and IL-1β. In particular, the Apt-CS@CHA CDs group showed a more pronounced suppression of IL-1β mRNA expression compared to FreeCHA and CHA CDs. This may be attributed to the enhanced ROS scavenging ability of Apt-CS@CHACDs. These results suggest that Apt-CS@CHACDs can effectively scavenge highly expressed ROS within cells, thereby inhibiting further exacerbation of the inflammatory response.

[0112] 7. Distribution in the body

[0113] First, an IBS model was established by daily gavage of senna leaf decoction (0.6 g / kg) plus restraint stress (starting 1 hour after gavage and lasting 1 hour). Mice were randomly divided into two groups of five: one group received Apt-CS-FITC@CHACDs by gavage, while the other group received CS-FITC@CHACDs. Abdominal fluorescence in the mice was then monitored at specific time points, and fluorescence values ​​were calculated. Simultaneously, mice were sacrificed at specific time points, and intestinal tissue and various organs were removed for fluorescence imaging to observe the distribution of the nanocapsules in vivo and to calculate fluorescence values.

[0114] Targeting inflamed intestinal tissue is crucial for oral IBS treatment. Observing the absorption of the target intestinal tissue can directly reflect the delivery status and targeting ability. Therefore, CS-FITC@CHACDs and Apt-CS-FITC@CHACDs are used to evaluate the effect of Apt-modified nanocapsules on administration to specific sites in inflamed intestinal tissue. Images were acquired using the in vivo imaging system IVIS. The changes in the fluorescence intensity of the mice reflected the retention of FITC in the body, and the fluorescence of each organ reflected the distribution of FITC. The present invention observed that more fluorescence signals were seen in the colitis tissues of Apt-CS-FITC@CHACDs-treated mice compared with those treated with CS-FITC@CHA CDs. Although the fluorescence intensity in the mice of the two treatment groups decreased to varying degrees over time, there was still an obvious fluorescence signal in the mice of the Apt-CS-FITC@CHA CDs-treated group at 16 hours. Even at 24 hours, some fluorescence signals were still observed in the Apt-CS-FITC@CHA CDs group ( Figure 12 A). By statistically analyzing the fluorescence in each group of mice, it was found that the fluorescence signal was consistent with the observed fluorescence signal ( Figure 12 B). The fluorescence half-life of Apt-CS-FITC@CHA CDs (16±0.55h) was much longer than that of CS-FITC@CHA CDs (12±0.25h) ( Figure 12 C) These results demonstrate that Apt-CS-FITC@CHA CDs can effectively deliver and release CDs to sites of colonic inflammation. Apt-CS-FITC@CHA CDs can then preferentially adhere to the inflamed mucosa by interacting with melatonin, which is highly expressed on the surface of the inflamed mucosa, further enhancing the targeting of CDs to the inflammatory site.

[0115] By further detecting the fluorescence in the intestine, the distribution of FIRC in the body can be clearly reflected. Figure 12As shown in Figure D-12E, the fluorescence of Apt-CS-FITC@CHACDs initially accumulated in the colon and was significantly higher than that of CS-FITC@CHACDs. After 8 hours, fluorescence from Apt-CS-FITC@CHACDs and CS-FITC@CHACDs was primarily present in the small intestine and colon, while only a small amount of fluorescence was observed in the intestine from Apt-CS-FITC@CHACDs. After 16 hours, the substantial fluorescence from CS-FITC@CHACDs had completely disappeared, leaving fluorescence from Apt-CS-FITC@CHACDs primarily in the colon. These results demonstrate that Apt-CS-FITC@CHACDs protects CHACDs from gastrointestinal destruction and specifically reaches inflamed intestinal tissue. No fluorescence was observed in other organs throughout the entire process, confirming the excellent colon targeting properties of Apt-CS-FITC@CHACDs.

[0116] 8. Treatment of IBS

[0117] IBS mice were randomly divided into five groups (5 animals each): control, free CHA, CHACDs, CS@CHACDs, and Apt-CS@CHACDs. Drugs were administered via oral gavage every other day for 10 days. The control group received 0.5 ml of normal saline by oral gavage every two days for 10 days.

[0118] (1) Histopathological analysis

[0119] Colon tissue was harvested and colon length was measured. All tissue samples were fixed in 4% paraformaldehyde and embedded in paraffin, then cut into 4 μm sections. Hematoxylin and eosin (H&E) staining was used to assess pathological progression. Alcian blue staining was used to detect mucus accumulation and identify goblet cells in the colon. For immunofluorescence staining, colon sections were treated with ZO-1 or anti-occludin at 4°C, then rinsed and incubated with Cy5.5-conjugated secondary antibodies and counterstained with 4',6-diamidino-2-phenylindole. For immunohistochemistry, colon tissue sections were incubated with anti-IL-6, IL-1β, TNF-α antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Tissue sections were stained with dihydroacetylcholine (DHE) to determine ROS levels.

[0120] (2) Intestinal flora sequencing

[0121] Cecal contents were collected, and the fecal 16S rRNA gene (V3-V4338F-806R) region was sequenced using the Illumina Miseq platform (Majorbio, CHN) according to the manufacturer's protocol. Principal coordinates analysis (PCoA) and NMDS of the microbiota were performed based on Bray-Curtis similarity.

[0122] (3) Treatment effect

[0123] After the mice were acclimated for 1 week, IBS model mice were established by daily gavage of senna leaf decoction (0.6 g / kg) and restraint stress (starting 1 hour after gavage and lasting 1 hour) for 2 weeks. The weight changes of mice in each group were recorded during the experiment. A sharp decrease in weight was observed in the control group of IBS-D model mice, while the treated group showed significant improvement in physical function ( Figure 13 The length of the colorectum was measured after treatment. The Apt-CS@CHACDs treatment group showed a significant protective effect on the reduction of colorectal length in the IBS-D model group mice ( Figure 14 A-14B). In addition, the therapeutic effect of Apt-CS@CHACDs on IBS-D was evaluated in terms of inflammation and intestinal barrier integrity. ELISA was used to measure serum levels of proinflammatory cytokines (including IL-1β, TNF-α, and IL-6). Compared with free CHA, CHACDs significantly reduced the levels of these inflammatory factors, and Apt-CS@CHACDs exerted a better inhibitory effect on inflammation than CHACDs ( Figure 14 C-14E). After Apt-CS@CHA CDs treatment, the expression levels of inflammatory factors detected by immunohistochemistry showed a decrease in colon inflammation. Figure 14 As shown in F, compared with the DSS-treated group, the Apt-CS@CHACDs-treated group significantly reduced the expression of IL-6, IL-1β, and TNF-α. In addition, by statistically analyzing the optical density (IOD) values ​​of the expression of each inflammatory factor, it was found that ( Figure 14 G-14I), Apt-CS@CHACDs was able to effectively inhibit inflammatory expression by about 80%, compared with the control group. Specifically, the Apt-CS@CHACDs-treated group was able to effectively inhibit inflammatory expression, indicating that Apt-CS@CHA CDs has an effective anti-inflammatory effect.

[0124] Intestinal permeability is a functional characteristic of the intestinal barrier. Dysfunction of the intestinal barrier can lead to increased intestinal permeability and intestinal inflammatory diseases. Therefore, the expression levels of cytoplasmic tight adhesion protein 1 (ZO-1) and occludin were detected as indicators of tight junction integrity. The IBS-D control group showed a significant decrease in this indicator, however, Apt-CS@CHACDs could reverse this trend (e.g., Figure 15 A, 15B, 15D, 15E). Epithelial goblet cells produce mucus, a key barrier in the intestine, and IBS-D, as an inflammatory disease of the intestine, is also characterized by the depletion of goblet cells. Therefore, goblet cell density was assessed by Alcian blue staining. Consistent with the results of ZO-1 and occludin, the number of goblet cells was significantly reduced in the control group, but the destructive effect was significantly attenuated after administration of Apt-CS@CHA CDs ( Figure 16 ). In addition, the excessive ROS produced by the inflammatory tissue will further aggravate the inflammatory response, so the effective removal of ROS can synergistically achieve the relief of inflammation. Here, the present invention uses DHE to stain the inflammatory tissue site for ROS, and it can be seen from the fluorescence images of each group ( Figure 15 C, 15F). Compared to the control group, Apt-CS@CHACDs administration effectively scavenged overexpressed ROS in tissue sites, which further led to tissue damage and inflammation. Colonic histological sections stained with hematoxylin and eosin revealed a well-preserved, integrated tissue architecture in healthy control mice. Despite the presence of intestinal villi rupture in the IBS-D model group, no significant damage was observed in Apt-CS@CHACDs-treated mice. Instead, alterations in colonic epithelial integrity were prevented.

[0125] Intestinal flora imbalance is believed to play a key role in IBS-D. Therefore, the present invention also analyzed the effect of Apt-CS@CHACDs on the structure of intestinal flora. By using Qiime2 to analyze amplicon sequence variation (ASV), multiple diversity indicators were calculated. The results showed that the α diversity index (including Chao 1, Ace, Sob and inverse Simpson index) of mice treated with Apt-CS@CHACDs increased significantly, indicating that Apt-CS@CHACDs can positively regulate the structure of intestinal flora ( Figure 17 A-17D).

[0126] The detailed changes in intestinal microorganisms were analyzed and visualized by heat maps, showing the average relative abundance of the 10 most abundant bacterial groups at the family level ( Figure 17E) and corresponding statistical analysis was performed. The results showed that Apt-CS@CHACDs treatment significantly increased the abundance of Muribaculaceae, norank_o_Clostridia_UCG-014, Prevotellaceae_UCG-001, Ruminococcaceae, Turicibacter, and Eubacterium, while decreasing the abundance of Staphylococcaceae, Helicobacter, Escherichia-Shigella, and Clostridium_cocleatum. These bacterial communities can affect the host by producing short-chain fatty acids, converting primary bile acids into secondary bile acids, and enhancing resistance to colonization by intestinal pathogens. Prevotellaceae_UCG-001 and norank_o_Clostridia_UCG-014 are considered beneficial to intestinal inflammation and are negatively correlated with inflammation. These results further confirm that Apt-CS@CHACDs can positively regulate the structure of the intestinal microbial community.

[0127] Subsequently, the biomarkers that were significant at the genus level were identified by linear discriminant analysis effect size (LEfSe) analysis. Figure 17 F), the drug intervention group was mainly characterized by beneficial bacteria. Muribaculaceae can participate in food digestion and nutrient absorption, regulate the immune system, and is considered to be beneficial bacteria. The Circos diagram shows the proportion of Muribaculaceae in each group. The Apt-CS@CHACDs group had the highest proportion (72%) ( Figure 17 G). Taken together, these results highlight that Apt-CS@CHACDs can significantly restore the imbalanced intestinal microbiota in IBS-D by increasing beneficial bacteria and reducing pathogenic bacteria.

[0128] Figure 18 The following diagram illustrates the process flow and the mechanism of action of the microsphere capsules described in this invention. Apt-CS@CHACDs were successfully prepared and demonstrated excellent antioxidant and anti-inflammatory properties in vitro. They effectively scavenged ROS and inhibited the expression of inflammatory factors. In vivo experiments demonstrated that Apt-CS@CHACDs had excellent colon targeting and sustained retention in inflamed intestinal tissue. They significantly improved symptoms in IBS mice, reduced inflammation, restored intestinal barrier function, and regulated the intestinal flora.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the technical solutions. Those skilled in the art should understand that modifications or equivalent replacements of the technical solutions of the present invention that do not depart from the purpose and scope of the technical solutions of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing chlorogenic acid carbon quantum dot microsphere capsules, characterized in that: The specific steps are as follows: Step 1: Preparation of chlorogenic acid carbon quantum dots: Dissolving chlorogenic acid in water, heating at 200° C. to 250° C. for 1 to 3 hours, and cooling to room temperature to obtain a mixture, filtering to obtain a brown transparent solution, and purifying by dialysis to obtain CHA CDs; wherein the concentration of chlorogenic acid in water is 5 to 10 mg / mL; Step 2: Preparation of Apt-CS: The aptamer is dissolved in water and heated to 90°C to 100°C, cooled to room temperature, and then cooled at 0°C for at least 40 seconds to obtain an aptamer solution. Then, chitosan is mixed with the aptamer solution and incubated at 30°C to 40°C for at least 12 hours. The reaction product is purified to obtain Apt-CS. The mass volume ratio of the aptamer to water is (1-10) mg: (1-10) μL; the mass volume ratio of the chitosan to the aptamer solution is (1-10) mg: (1-10) μL. Step 3: Preparation of Apt-CS@CHACDs: The Apt-CS prepared in step 2 was dissolved in a PBS buffer solution, and then the CHACDs solution prepared in step 1 was added dropwise to obtain chlorogenic acid carbon quantum dot microsphere capsules Apt-CS@CHA CDs through electrostatic action; wherein the mass ratio of Apt-CS and CHACDs was (1-10):(1-10), and the pH value of the PBS buffer solution was 7-8.

2. The preparation method according to claim 1, characterized in that The sequence of the aptamer is as follows: GGTGGGTTTGGCTGGTACTTAGGGCGTCGTCCC.

3. The preparation method according to claim 1, characterized in that: In step 1, the reaction was carried out at 230°C for 2 hours.

4. The preparation method according to claim 1, characterized in that In step 2, the aptamer is dissolved in water and then heated to 95°C.

5. The preparation method according to claim 1, characterized in that: In step 2, the chitosan and aptamer solution are incubated at 35° C. to 37° C. for 12 h to 24 h.

6. An application of chlorogenic acid carbon quantum dot microsphere capsules, characterized in that: The microsphere capsules prepared by the preparation method according to any one of claims 1 to 5 are used to prepare drugs for treating irritable bowel syndrome.

7. A pharmaceutical composition, characterized in that The invention comprises a microsphere capsule prepared by the preparation method according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

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