A chlorogenic acid thermosensitive sol, a preparation method and application thereof, and a chlorogenic acid gel and application thereof

Abstract

The application belongs to the technical field of pharmaceutical preparations, and particularly relates to a chlorogenic acid temperature-sensitive sol, a preparation method and application thereof, a chlorogenic acid gel and application thereof. The chlorogenic acid temperature-sensitive sol provided by the application combines chlorogenic acid with anti-inflammatory activity and a temperature-sensitive material for the first time, and constructs a temperature-responsive in-situ gel delivery system. The chlorogenic acid temperature-sensitive sol is in a flowable sol state at room temperature, and is convenient for administration in a liquid state through a syringe or an enema device. When contacting a human body cavity mucosa, the temperature-sensitive material triggers sol-gel phase transition through intermolecular hydrophobic interaction, forms a semi-solid three-dimensional network gel, and is closely adhered to the mucosa surface, so that the drug is positioned and enriched in a lesion area and is released for a long time, the symptoms of ulcerative colitis are relieved, the disease activity index is reduced, the colon length shortening and the pathological damage of colon tissue are improved, the technical defects such as uneven distribution of traditional suppositories and short retention time of enema liquid are overcome, and the chlorogenic acid temperature-sensitive sol has clinical application potential.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology, specifically relating to a chlorogenic acid thermosensitive sol and its preparation method and application, and a chlorogenic acid gel and its application. Background Technology

[0002] Ulcerative colitis is an inflammatory bowel disease characterized by chronic inflammation of the colonic and rectal mucosa. Clinical manifestations include bloody stools, diarrhea, and tenesmus; approximately 15% of patients progress to severe stage at initial diagnosis. Long-term inflammation can lead to damage to the intestinal mucosal barrier and an increased risk of colon cancer. Current treatment options primarily consist of aminosalicylic acid preparations, glucocorticoids, and biologics, but significant technical limitations remain. These include side effects such as immunosuppression and osteoporosis caused by systemic administration, and insufficient local drug concentrations due to poor mucosal adhesion and short retention time of traditional rectal preparations.

[0003] Chlorogenic acid (CGA), a natural polyphenol compound, is widely found in plants such as coffee, honeysuckle, and stevia. It possesses multiple effects, including anti-inflammatory, antioxidant, immunomodulatory, and intestinal barrier repair properties, showing significant potential in the treatment of ulcerative colitis. However, the clinical application of chlorogenic acid is limited by its physicochemical defects, such as low oral bioavailability, poor water solubility, and insufficient gastrointestinal stability. Conventional formulations struggle to achieve effective drug concentrations in the rectal lesion area, and existing rectal suppositories / enemas are easily washed away by physiological fluids due to insufficient mucosal adhesion.

[0004] The chemical structural formula of chlorogenic acid is as follows:

[0005]

[0006] In recent years, in-situ thermosensitive gel technology has provided an innovative direction for optimizing local rectal delivery systems. These formulations are in a flowable liquid state at room temperature, facilitating wide distribution through enema administration. Upon temperature triggering (e.g., the rectal environment of 36–37°C), they rapidly undergo a sol-gel phase transition, forming a semi-solid gel network, thereby achieving precise targeted drug delivery, effectively preventing drug leakage and reducing operational difficulty. Compared to traditional suppositories or enema solutions, thermosensitive gels adhere tightly to the mucosal surface, significantly prolonging retention time and supporting sustained drug release, which is highly compatible with the long-acting effect required for the treatment of ulcerative colitis. However, existing thermosensitive gels still suffer from a drawback: insufficient adhesive strength to resist the erosion caused by the dynamic physiological environment of the rectum, leading to a shortened effective drug retention time. Summary of the Invention

[0007] The purpose of this invention is to provide a chlorogenic acid thermosensitive sol, its preparation method and application, and a chlorogenic acid gel and its application.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a chlorogenic acid thermosensitive sol, comprising the following components in weight percentage:

[0010] Chlorogenic acid 0.5-2%, thermosensitive material 15-25%, temperature regulator 1-8%, bioadhesive 0.1-1.0%, and the balance being a pH buffer solution;

[0011] The pH value of the chlorogenic acid thermosensitive sol is 4.0 to 5.0.

[0012] Preferably, the temperature-sensitive material includes at least one of poloxamer 407, poly(N-isopropylacrylamide), and chitosan.

[0013] Preferably, the temperature regulator includes at least one of poloxamer 188, polyethylene glycol 4000, and polyethylene glycol 6000.

[0014] Preferably, the bioadhesive comprises at least one of sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, dextran, β-cyclodextrin, and carbomer.

[0015] Preferably, the pH buffer solution is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system;

[0016] The pH value of the pH buffer solution is 6.0 to 8.0.

[0017] Preferably, the chlorogenic acid thermosensitive sol further includes an antioxidant, which includes at least one of sodium bisulfite, sodium metabisulfite, L-cysteine, and vitamin C;

[0018] The antioxidant in the chlorogenic acid thermosensitive sol has a mass percentage of 0.05-0.3%.

[0019] This invention also provides a method for preparing the chlorogenic acid thermosensitive sol described in the above technical solution, comprising the following steps:

[0020] Chlorogenic acid and a portion of pH buffer solution were mixed to obtain the first mixture;

[0021] The temperature-sensitive material, temperature regulator, bioadhesive, and remaining pH buffer solution are mixed a second time to obtain a second mixture.

[0022] The first mixture and the second mixture are mixed together and swollen to obtain the chlorogenic acid thermosensitive sol.

[0023] Preferably, the swelling temperature is 4±1℃ and the time is 22 to 26 hours.

[0024] The present invention also provides a chlorogenic acid gel, which is obtained by gelation of a sol, wherein the sol is the chlorogenic acid thermosensitive sol described in the above technical solution or the chlorogenic acid thermosensitive sol prepared by the preparation method described in the above technical solution;

[0025] The gelation temperature is 28–36°C.

[0026] The present invention also provides the application of the chlorogenic acid thermosensitive sol described in the above technical solution, the chlorogenic acid thermosensitive sol prepared by the preparation method described in the above technical solution, and the chlorogenic acid gel described in the above technical solution in the preparation of drugs for treating ulcerative colitis.

[0027] This invention provides a chlorogenic acid thermosensitive sol, comprising the following components in weight percentage: 0.5-2% chlorogenic acid, 15-25% thermosensitive material, 1-8% temperature regulator, 0.1-1.0% bioadhesive, and the balance being a pH buffer solution; the pH value of the chlorogenic acid thermosensitive sol is 4.0-5.0.

[0028] The chlorogenic acid thermosensitive sol provided by this invention is the first to combine chlorogenic acid, which has anti-inflammatory activity, with a thermosensitive material to construct a temperature-responsive in-situ gel delivery system. The chlorogenic acid thermosensitive sol provided by this invention is a flowable sol at room temperature, facilitating drug administration in liquid form via syringe or enema device. When it comes into contact with the mucosa of human cavities, the thermosensitive material triggers a sol-gel phase transition through intermolecular hydrophobic interactions, forming a semi-solid three-dimensional network gel that adheres tightly to the mucosal surface. This achieves localized enrichment and long-lasting sustained release of the drug in the lesion area, alleviating symptoms of ulcerative colitis, reducing the disease activity index, and improving shortened colon length and pathological damage to colonic tissue. This overcomes the technical shortcomings of traditional suppositories, such as uneven distribution and short retention time of enema fluid, and has potential for clinical application. Attached Figure Description

[0029] Figure 1 The images show the physical samples of the chlorogenic acid thermosensitive sol and chlorogenic acid gel obtained in Example 1 of this invention.

[0030] Figure 2 This is a scanning electron microscope image of the chlorogenic acid thermosensitive sol obtained in Example 1;

[0031] Figure 3 The rheological scanning curves of the chlorogenic acid thermosensitive sol obtained in Example 1 are shown. In the figure, A is the curve of storage modulus and loss modulus as a function of temperature, and B is the curve of composite viscosity as a function of temperature.

[0032] Figure 4 The in vitro dissolution and release curves of the chlorogenic acid thermosensitive sol obtained in Example 1 are shown.

[0033] Figure 5To evaluate the retention time of chlorogenic acid thermosensitive sol based on fluorescence tracer (Example 1) in mouse rectum, where A is the in vivo real-time fluorescence imaging of fluorescently labeled chlorogenic acid and chlorogenic acid thermosensitive gel, B is the fluorescence intensity-time dynamics curve of chlorogenic acid and chlorogenic acid thermosensitive gel, and C is the ex vivo fluorescence imaging of colon after 24 h.

[0034] Figure 6 This study systematically evaluates the therapeutic effect of chlorogenic acid thermosensitive sol obtained in Example 1 on DSS-induced ulcerative colitis in mice. In the figures, A is a schematic diagram of the in vivo experimental design, B is the curve of mouse body weight change over time, C is the curve of disease activity index (DAI) change over time, D is a comparison of colon tissue morphology on day 35, E is the quantitative analysis of colon length in each group, F is HE staining of colon tissue, and G is the pathological score of colon tissue. Detailed Implementation

[0035] This invention provides a chlorogenic acid thermosensitive sol, comprising the following components in weight percentage:

[0036] Chlorogenic acid 0.5-2%, thermosensitive material 15-25%, temperature regulator 1-8%, bioadhesive 0.1-1.0%, and the balance being a pH buffer solution;

[0037] The pH value of the chlorogenic acid thermosensitive sol is 4.0 to 5.0.

[0038] The chlorogenic acid thermosensitive sol provided by the present invention comprises 0.5% to 2% chlorogenic acid, specifically 0.5%, 1.0%, 1.5%, and 2.0%.

[0039] The chlorogenic acid thermosensitive sol provided by this invention comprises 15-25% thermosensitive material by weight percentage, specifically 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, and 25%. In this invention, the thermosensitive material preferably includes at least one of poloxamer 407, poly(N-isopropylacrylamide), and chitosan. In this invention, the thermosensitive material can rapidly change from a liquid state to a solid gel upon body temperature triggering, solving the problems of rapid drug release and short residence time in traditional rectal preparations. By limiting the content of the thermosensitive material to 15-25%, both fluidity during injection and the formation of a stable gel in vivo are ensured, thereby achieving long-term sustained drug release. Too low a content of the thermosensitive material will result in insufficient gel strength and excessively rapid drug release; too high a content will result in excessive viscosity, making injection difficult and affecting efficacy.

[0040] The chlorogenic acid thermosensitive sol provided by this invention comprises 1-8% temperature regulator by weight percentage, specifically 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%. In this invention, the temperature regulator preferably includes at least one of poloxamer 188, polyethylene glycol 4000, and polyethylene glycol 6000. In this invention, the temperature regulator can further precisely control the phase transition temperature to better adapt to physiological environmental requirements, ensuring the sol remains liquid during storage and transportation and rapidly triggers gelation at body temperature. By limiting the content to 1-8%, the stability and injectability of the sol can be maintained, while excessive viscosity due to over-addition can be avoided. When the temperature regulator content is below 1%, the phase transition temperature fluctuates too much; when it is above 8%, the sol has poor flowability and cannot achieve precise temperature-sensitive response.

[0041] The chlorogenic acid thermosensitive sol provided by this invention comprises 0.1-1.0% (by weight) of a bioadhesive, specifically 0.1%, 0.2%, 0.3%, 0.4%, 0.45%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, and 1.0%. In this invention, the bioadhesive preferably comprises at least one of sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, dextran, β-cyclodextrin, and carbomer. In this invention, the bioadhesive can bind to the mucous membrane through intermolecular forces, significantly enhancing the adhesion of the gel in human cavities and solving the problem of drug loss caused by short retention time in traditional formulations. By limiting the content to 0.1-1.0%, both sol adhesion and retention time can be ensured, while excessive addition can avoid excessively high viscosity or hindered drug release. When the content of bio-adhesive is below 0.1%, the adhesion effect is significantly insufficient, while when it is above 1.0%, the flowability and uniformity of the sol will decrease significantly.

[0042] The chlorogenic acid thermosensitive sol provided by this invention comprises, by weight percentage, the remainder being a pH buffer solution. In this invention, the pH buffer solution is preferably a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (PBS buffer); the pH value of the pH buffer solution is preferably 6.0–8.0.

[0043] In this invention, the chlorogenic acid thermosensitive sol preferably includes an antioxidant, which preferably includes at least one selected from sodium bisulfite, sodium metabisulfite, L-cysteine, and vitamin C. In this invention, the antioxidant in the chlorogenic acid thermosensitive sol is preferably 0.05-0.3% by mass, specifically 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, and 0.3%. In this invention, the antioxidant can effectively inhibit the oxidative degradation of chlorogenic acid, ensuring the chemical stability of the formulation during storage and use by scavenging free radicals or blocking oxidation chain reactions, and avoiding reduced efficacy due to oxidative inactivation of the active pharmaceutical ingredient. Limiting the antioxidant content to 0.05-0.3% strictly adheres to the regulations of the Chinese Pharmacopoeia regarding the safety and effectiveness of antioxidants, significantly extending the shelf life of the sol while avoiding biosafety issues caused by excessive addition. When the antioxidant content is below 0.05%, the antioxidant effect is insufficient; when it is above 0.3%, it may cause local irritation.

[0044] In this invention, the pH value of the chlorogenic acid thermosensitive sol is 4.0–5.0. During the dissolution process of the chlorogenic acid, its acidic groups release protons, which neutralize the alkaline components in the pH buffer solution, causing the final sol's pH value to self-adjust to 4.0–5.0. This acidic environment significantly inhibits the oxidative degradation of chlorogenic acid by maintaining its protonated state.

[0045] This invention also provides a method for preparing the chlorogenic acid thermosensitive sol described in the above technical solution, comprising the following steps:

[0046] Chlorogenic acid and a portion of pH buffer solution were mixed to obtain the first mixture;

[0047] The temperature-sensitive material, temperature regulator, bioadhesive, and remaining pH buffer solution are mixed a second time to obtain a second mixture.

[0048] The first mixture and the second mixture are mixed together and swollen to obtain the chlorogenic acid thermosensitive sol.

[0049] In this invention, the first mixing is preferably performed under ultrasonic conditions, with the ultrasonic temperature preferably at 30°C. In this invention, the mass ratio of the partial pH buffer solution to the remaining pH buffer solution is preferably 1:1 to 1:5. In this invention, the swelling temperature is preferably 4±1°C, and the time is preferably 22 to 26 hours. In this invention, the third mixing and swelling are preferably performed under light-protected conditions. In this invention, after swelling, the resulting system is preferably stirred to form a homogeneous and transparent sol. In this invention, the chlorogenic acid thermosensitive sol is preferably stored under low-temperature and light-protected conditions.

[0050] In this invention, when the chlorogenic acid thermosensitive sol also includes an antioxidant, the antioxidant is preferably added in the second mixture.

[0051] The present invention also provides a chlorogenic acid gel, which is obtained by gelation of a sol, wherein the sol is the chlorogenic acid thermosensitive sol described in the above technical solution or the chlorogenic acid thermosensitive sol prepared by the preparation method described in the above technical solution;

[0052] The gelation temperature is 28–36°C.

[0053] The present invention also provides the application of the chlorogenic acid thermosensitive sol described in the above technical solution, the chlorogenic acid thermosensitive sol prepared by the preparation method described in the above technical solution, and the chlorogenic acid gel described in the above technical solution in the preparation of drugs for treating ulcerative colitis.

[0054] Unless otherwise specified, the materials and equipment used in this invention are all commercially available products in the field.

[0055] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0056] Examples 1-16

[0057] The formulations for Examples 1 to 16 are shown in Table 1;

[0058] Table 1 Formulations of Examples

[0059]

[0060]

[0061] Preparation method:

[0062] Chlorogenic acid was dissolved in a partial sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (pH 7.4) and sonicated at 30°C until completely dissolved to obtain the first mixture. Poloxamer 407, poloxamer 188, and sodium carboxymethyl cellulose were dispersed in the remaining sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system (added in this step if sodium bisulfite is present) to obtain the second mixture. The mass ratio of the partial buffer system to the remaining buffer system was 1:1.

[0063] Mix the first and second mixtures, transfer them to a light-proof container, let them stand at 4°C for 24 hours to allow them to swell, stir until homogeneous to form a semi-transparent sol, and store at low temperature in the dark for later use.

[0064] Performance testing

[0065] Test Example 1

[0066] The sol obtained in the examples was gelled; the gelation temperature (T) was adjusted. gel ) to be measured;

[0067] The sol-gel phase transition temperature was determined using the rotor method. 1 mL of sol was injected into a 5 mL EP tube, which was vertically fixed in the center of a thermostatic magnetic stirrer. The PTFE rotor was positioned at the center of the liquid surface. The initial temperature was set to 20 °C, and the temperature was increased at a rate of 0.5 °C / min. The rotor's status was monitored in real time. When the rotor completely stopped rotating, the temperature at this point was recorded as the gelation temperature T. gel The gelation temperature of the chlorogenic acid thermosensitive sol prepared in the above examples was measured, and the results are shown in Table 2; among them, the actual images of the sol obtained in Example 1 and the gel obtained after gelation are shown in the figure. Figure 1 As shown;

[0068] Table 2 shows the gel temperatures of the sols obtained in the examples.

[0069]

[0070]

[0071] Thermosensitive sols need to remain liquid at room temperature for easy administration. The phase transition temperature is usually designed to be 1-2°C lower than the physiological rectal temperature (36-37°C) to compensate for local temperature fluctuations during administration. As shown in the table above, in order to achieve a suitable gelation temperature, the optimal mass percentage of poloxamer 407 in the chlorogenic acid thermosensitive sol is 19%, the optimal mass percentage of poloxamer 188 in the chlorogenic acid thermosensitive sol is 6%, and the optimal mass percentage of sodium carboxymethyl cellulose in the chlorogenic acid thermosensitive sol is 0.3%.

[0072] Comparing Examples 1 and 15-16, the addition of sodium bisulfite had almost no effect on the gelation temperature and pH of the chlorogenic acid thermosensitive sol.

[0073] Test Example 2

[0074] The sol prepared in Example 1 was cured at 37°C and then subjected to liquid nitrogen brittle fracture. After vacuum freeze-drying for 24 hours, a gold coating was sputtered using an ion sputtering apparatus, and the cross-sectional morphology was observed using a scanning electron microscope. Figure 2 The image shown is a scanning electron microscope image of the chlorogenic acid thermosensitive sol obtained in Example 1. It can be seen that the gel has a three-dimensional porous network structure, indicating that it is suitable for use as a drug carrier.

[0075] Test Example 3

[0076] The sol from Example 1 was loaded onto a 40 mm stainless steel parallel plate of a rotational rheometer. After equilibration at 10°C for 5 min, the temperature was increased to 40°C at a rate of 2°C / min. The storage modulus (G'), loss modulus (G”), and composite viscosity (η*) were recorded in oscillation mode (frequency 1 Hz, strain 1%).

[0077] Figure 3 The rheological scanning chromatograms of the chlorogenic acid thermosensitive sol obtained in Example 1 are shown below. In Figure A, storage modulus and loss modulus change with temperature; in Figure B, composite viscosity changes with temperature. Figure 3 As shown, the intersection temperature of G' and G” is 31℃, and the mutation point of η* is also 31℃. This temperature is the gelation temperature T. gel .

[0078] Test Example 4

[0079] Accurately measure 1.0 mL of the sol obtained in Example 1 and inject it into a pre-weighed 5 mL centrifuge tube (empty tube mass W1). Incubate in a 37°C water bath for 10 min to form a gel. Wipe the outer wall dry and weigh (W). D Add 1 mL of preheated simulated colon fluid and place in a constant-temperature shaker (100 rpm) at 37 ± 0.5 °C. Pour off all liquid at 15, 30, 45, 60, 90, 120, 180, and 240 min and weigh the residual gel + centrifuge tube (W). Add 1 mL of the same temperature medium after each sampling. The erosion rate is calculated as (W). D -W) / (W D -W1)×100%; after the decanted medium was filtered through a 0.22μm filter membrane, the concentration of chlorogenic acid was determined by high performance liquid chromatography.

[0080] Figure 4 The in vitro dissolution and release curves of the chlorogenic acid thermosensitive sol obtained in Example 1 are shown below. Figure 4 As shown, the dissolution rate reached 94.96% at 240 min, and the chlorogenic acid release curve was y = 86.49684(1-exponential). -0.00834t This conforms to the classic Higuchi sustained-release model (R...). 2 =0.9968), indicating that it has a certain sustained-release effect.

[0081] Test Example 5

[0082] The effect of pH on the stability of chlorogenic acid thermosensitive sol was tested.

[0083] Two sets of chlorogenic acid rectal in-situ thermosensitive sols were prepared:

[0084] Group with pH=4.0: Following the formulation of Example 1, conventional PBS buffer solution (pH=7.4) was used to self-regulate the pH of the final sol to 4.0 through the acidic release of chlorogenic acid;

[0085] pH=6.0 group: Following the formulation of Example 1, a modified phosphate buffer (950 μL of conventional PBS buffer solution with 50 μL of 1.0 M Na2HPO4 solution added) was used to self-regulate the pH of the final sol to 6.0 through the acidic release of chlorogenic acid;

[0086] The two sols were stored at 4℃, and the gelation temperature, drug content, and appearance were measured at 0d, 5d, and 10d. The results are shown in Table 3 below.

[0087] Table 3 Effect of pH value on the stability of chlorogenic acid thermosensitive sol

[0088]

[0089] The comparison shows that the acidic condition of pH=4.0 can effectively maintain the protonated state of chlorogenic acid, inhibit chlorogenic acid oxidation, and achieve a degradation rate of ≤0.21% and a gelation temperature fluctuation of ≤0.1℃, which is significantly better than pH=6.0. This confirms the protective effect of the acidic buffer system on drug stability. Therefore, pH=4.0 was selected as the optimal embodiment of the present invention.

[0090] Test Example 6

[0091] Accelerated stability test of antioxidants;

[0092] The stability data (chlorogenic acid content in the gel) of the chlorogenic acid thermosensitive sols of Examples 1 and 15-16 after being placed at high temperature (60°C) for 5 days are shown in Table 4.

[0093] Table 4. Stability test results of chlorogenic acid thermosensitive sol.

[0094]

[0095] Compared with Examples 1 and 15-16, the stability of chlorogenic acid was significantly improved, indicating that sodium bisulfite can significantly inhibit its oxidative degradation and improve the stability of the gel.

[0096] Test Example 7

[0097] Evaluation of the rectal retention time of chlorogenic acid thermosensitive sol;

[0098] Six 6-8 week old SPF-grade male C57BL / 6 mice were selected and randomly divided into two groups (n=3) after fasting for 24 hours. One group received rectal administration of a chlorogenic acid solution (CGA) containing 60 μg / 100 μL indocyanine green fluorescent labeling, while the other group received chlorogenic acid thermosensitive sol (CGA-Gel). Rectal fluorescence signals were dynamically monitored at 0h, 4h, 8h, 10h, 12h, and 24h after administration using a small animal in vivo imaging system. 4.4 The software was used to analyze the normalized fluorescence intensity of the region of interest (ROI). At the experimental endpoint (24 h), mice were dissected and colon tissue was separated for fluorescence verification.

[0099] Figure 5 To evaluate the retention time of chlorogenic acid thermosensitive sol (Example 1) based on fluorescence tracer in mouse rectum, A is the in vivo real-time fluorescence imaging of fluorescently labeled chlorogenic acid and chlorogenic acid thermosensitive gel, B is the fluorescence intensity-time dynamics curve of chlorogenic acid and chlorogenic acid thermosensitive gel, and C is the ex vivo fluorescence imaging of the colon after 24 h. The specific results are shown in Table 5.

[0100] Table 5. Fluorescence intensity changes of chlorogenic acid thermosensitive gel and chlorogenic acid solution over time.

[0101] 0h 4h 8h 10h 12h 24h CGA 1.8065 3.1270 5.7390 5.1765 6.0695 2.4840 CGA-Gel 1.5508 12.6075 10.2537 10.1160 9.9473 6.2698

[0102] Note: The units of values ​​in the table are ×10. 10 Units: p / sec / cm 2 / sr.

[0103] like Figure 5 As shown in Table 5, the fluorescence intensity in the rectum of the CGA-Gel group was higher than that of the CGA group, confirming that the thermosensitive gel can prolong the drug retention time in the rectum to more than 24 hours. Its sustained-release properties were verified by both in vivo imaging and in vitro tissue fluorescence signals.

[0104] Test Example 8

[0105] Verification of the therapeutic effect of chlorogenic acid thermosensitive sol on ulcerative colitis;

[0106] Thirty 6-8 week old SPF-grade male C57BL / 6 mice were randomly divided into 6 groups (n=5): blank control group (Control), DSS model group (DSS), DSS+5-aminosalicylic acid group (5-ASA), DSS+chlorogenic acid solution group (CGA), DSS+blank thermosensitive gel matrix group (Vehicle), and DSS+chlorogenic acid thermosensitive sol group (CGA-Gel).

[0107] Except for the control group, which had free access to sterilized water, the other groups alternated between drinking a 1% (wt / vol) sodium dextran sulfate (DSS, Mw 40000) aqueous solution and sterilized water for 7-day cycles, for a total of 5 cycles. During the experiment, the mice's body weight, fecal characteristics, and fecal occult blood tests were monitored daily, and the severity of the model was quantified using the Disease Activity Index (DAI) scoring system. The DAI score is determined according to the following criteria: Weight loss score: 0-1% weight loss is 0 points, 1-5% is 1 point, 6-10% is 2 points, 11-20% is 3 points, and greater than 20% is 4 points; Stool consistency score: normal is 0 points, loose but formed is 1 point, loose is 2 points, very loose and moist is 3 points, diarrhea, liquid and adhering to the anus is 4 points; Fecal occult blood score: negative is 0 points, weakly positive is 1 point, positive or strongly positive is 2 points, bloody stool is 3 points, and bloody stool is 4 points; The total score is the sum of the scores of the above three parts.

[0108] Starting on day 8, each treatment group received 100 mg / kg of the drug (200 μL / mouse) via rectal instillation every other day. The Control and DSS groups received an equal volume of phosphate-buffered saline. A total of 14 administrations were administered. Mice were sacrificed 24 hours after the last administration. Colonic tissue was completely dissected and its length measured. After fixation with 4% paraformaldehyde, paraffin sections were prepared and stained using hematoxylin and eosin (H&E) staining. Histopathological scoring was performed based on the degree of mucosal epithelial surface loss, crypt structure integrity, inflammatory cell infiltration and edema, and goblet cell depletion.

[0109] Figure 6 This is a systematic evaluation of the therapeutic effect of the chlorogenic acid thermosensitive sol obtained in Example 1 on DSS-induced ulcerative colitis in mice. In the figures, A is a schematic diagram of the in vivo experimental design; B is the curve of mouse body weight change over time; C is the curve of disease activity index (DAI) change over time; D is a comparison of colonic tissue morphology on day 35; E is the quantitative analysis of colon length in each group; F is HE staining of colonic tissue; and G is the pathological score of colonic tissue. Figure 6 As shown, the drug-treated groups alleviated weight loss, loose stools, and rectal bleeding caused by DSS, thereby reducing the DAI score. Among them, the CGA-Gel group had the greatest weight gain and the lowest DAI score. In addition, the colon length decreased by 33.4%, 19%, 17.9%, 26.3%, and 14.6% in the DSS, 5-ASA, CGA, Vehicle, and CGA-Gel groups, respectively. All drug-treated groups significantly improved the colon length shortening caused by DSS, and the improvement rate was CGA-Gel>CGA>5-ASA. There was no significant difference between the Vehicle and DSS groups, indicating that the gel matrix itself has no therapeutic effect.

[0110] Further examination of colonic tissue pathological damage using HE staining and scoring revealed that the colonic tissue of mice in the Control group showed an intact and continuous colonic wall, normal and orderly glandular structure, abundant goblet cells, and no inflammatory cell infiltration. However, mice in the DSS group showed severe damage to the colonic epithelial barrier, disordered tissue structure, lack of goblet cells, disappearance of crypts, and extensive inflammatory cell infiltration. These pathological phenomena were improved to varying degrees in the drug-treated groups, with the CGA-Gel group showing the lowest histopathological score and the best improvement. In conclusion, CGA-Gel demonstrates the best therapeutic effect for ulcerative colitis.

[0111] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A chlorogenic acid thermosensitive sol, characterized in that, The components are those with the following mass percentages: The solution contains 1% chlorogenic acid, 19% thermosensitive material, 6% temperature regulator, 0.3% bioadhesive, and the balance being a pH buffer solution. The temperature-sensitive material is poloxamer 407; The temperature regulator is poloxamer 188; The bioadhesive is sodium carboxymethyl cellulose; The pH value of the chlorogenic acid thermosensitive sol is 4.08; The preparation method of the chlorogenic acid thermosensitive sol includes the following steps: Chlorogenic acid and a portion of pH buffer solution were mixed to obtain the first mixture; The temperature-sensitive material, temperature regulator, bioadhesive, and remaining pH buffer solution are mixed a second time to obtain a second mixture. The first mixture and the second mixture are mixed together and swollen to obtain the chlorogenic acid thermosensitive sol.

2. The chlorogenic acid thermosensitive sol according to claim 1, characterized in that, The pH buffer solution is a sodium dihydrogen phosphate-disodium hydrogen phosphate buffer system; The pH value of the pH buffer solution is 6.0~8.

0.

3. The method for preparing the chlorogenic acid thermosensitive sol according to claim 1 or 2, characterized in that, The steps are as follows: Chlorogenic acid and a portion of pH buffer solution were mixed to obtain the first mixture; The temperature-sensitive material, temperature regulator, bioadhesive, and remaining pH buffer solution are mixed a second time to obtain a second mixture. The first mixture and the second mixture are mixed together and swollen to obtain the chlorogenic acid thermosensitive sol.

4. The preparation method according to claim 3, characterized in that, The swelling temperature is 4±1℃, and the time is 22~26h.

5. A chlorogenic acid gel, obtained by gelation of a sol, characterized in that, The sol is the chlorogenic acid thermosensitive sol according to claim 1 or 2, or the chlorogenic acid thermosensitive sol prepared by the preparation method according to claim 3 or 4. The gelation temperature is 28~36℃.

6. The use of the chlorogenic acid thermosensitive sol according to claim 1 or 2, the chlorogenic acid thermosensitive sol prepared by the preparation method according to claim 3 or 4, and the chlorogenic acid gel according to claim 5 in the preparation of a drug for treating ulcerative colitis.

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