KR20220143266A - Orally cyclodextrin-curcumin encapsulated chitosan/alginate nanoparticles and use thereof in treating colitis - Google Patents

Abstract

The present invention relates to orally administrable nanoparticles of cyclodextrin-curcumin encapsulated chitosan/alginate and a use thereof in treating colitis, wherein curcumin (Cur) is added to ring-shaped β-cyclodextrin (CD) to form a water-soluble Cur-CD conjugate and then the Cur-CD conjugate is encapsulated by chitosan/alginate nanoparticles (CANPs). The generated Cur-CD-CANPs have unimodal size distribution and a negatively charged surface. The efficiency of encapsulation of Cur-CD-CANPs is 81.5 ± 7.4 % at an average diameter of 89.7 nm. In addition, as a result of an in vivo experiment, it is shown that oral administration of Cur-CD-CANPs has excellent therapeutic effects in DSS-induced colitis, compared to free CUR. According to a change in the body weight, a degree of bloody excrement, the length of the large intestine, and a result of histopathology, it is shown that the oral administration of Cur-CD-CANPs has excellent effects in treating DSS-induced colitis. Accordingly, the Cur-CD-CANPs of the present invention can be useful as a drug delivery system for oral administration in order to treat ulcerative colitis (UC).

Description

Cytodextrin-curcumin encapsulated chitosan/alginate oral nanoparticles and their use for treating colitis

The present invention relates to cyclodextrin-curcumin encapsulated chitosan/alginate oral nanoparticles and their use for the treatment of colitis.

Inflammatory bowel disease (IBD) is an idiopathic inflammatory bowel disease involving the ileum, rectum and large intestine that presents with clinical symptoms of diarrhea, abdominal pain and even bloody stools caused by a variety of genetic and environmental factors. Such diseases include ulcerative colitis (UC) and Crohn's disease (CD). The incidence of UC is increasing rapidly worldwide. It seriously threatens public health because it is easy to relapse and difficult to treat. Therefore, prevention and treatment of IBD is one of the major tasks of modern society.

Oral administration of drugs is recognized as the most accessible method because it has the advantageous characteristics of safety, high compliance and cost-effectiveness. Accordingly, various oral drug delivery systems (ODDSs) such as tablets, microparticles and liposomes have been developed for the treatment of UC. Among the above ODDSs, polymeric NPs are of great interest because they represent the potential to improve the aqueous solubility of hydrophobic drugs, improve the in vivo stability of drugs, and optimize the pharmacokinetic properties of drugs. . Importantly, they have the ability to specifically deliver drugs to colitis tissues through epithelial enhanced permeability and retention (eEPR) effects. This effect is known to be due to histopathological abnormalities of the inflammatory colon, which include destruction of the mucosal layer and accumulation of immune cells. However, the eEPR effect can only passively promote the accumulation of ODDS in the inflammatory colon tissue, and the low cell uptake efficiency for target cells is still a limitation in treatment.

According to a previous study, chitosan-alginate nanoparticles were found to enhance the cytotoxicity and cellular uptake of curcumin diethyl disuccinate (CDD) in breast cancer cells. More importantly, chitosan-alginate nanoparticles showed good stability, and slow release of nanoparticles resulted in higher in vitro cellular uptake in Caco-2 cells, and Caco-2, HepG2 and MDA-MB -231 cells showed improved anticancer activity. Although many chitosan-alginate nanoparticles have been shown to exhibit excellent therapeutic effects in various diseases, little has been known about their anti-inflammatory effects in dextran sodium sulfate-induced colitis. In addition, the effect of orally administered chitosan/alginate nanoparticles (CANPs) on the intestinal microflora has not been reported so far.

Chinese Patent Publication CN 107432951 A (published on Dec. 5, 2017)

An object of the present invention is an oral cyclodextrin-curcumin (cyclodextrin-curcumin; Cur-CD) complex containing encapsulated chitosan/alginate nanoparticles (CANPs) as an active ingredient with improved water solubility and bioavailability To provide a composition for

Another object of the present invention is to provide a method for preparing an oral composition having improved water solubility and bioavailability.

Another object of the present invention is to provide a composition for preventing, improving or treating ulcerative colitis (UC) comprising the oral composition as an active ingredient.

Another object of the present invention is to provide a composition for drug delivery to the large intestine comprising the oral composition as an active ingredient.

In order to achieve the above object, the present invention is a cyclodextrin-curcumin (cyclodextrin-curcumin; Cur-CD) complex encapsulated chitosan / alginate nanoparticles (chitosan / alginate nanoparticles; CANPs) water solubility and biomaterials containing as an active ingredient An oral composition with improved utilization is provided.

In addition, the present invention comprises the steps of (1) mixing cyclodextrin dissolved in an aqueous phase and curcumin dissolved in an oil phase to form a water-soluble cyclodextrin-curcumin (Cur-CD) complex; (2) mixing the formed Cur-CD complex with sodium alginate solution; (3) preparing a pre-gel state by adding a calcium chloride solution to the mixed solution of step (2); and (4) adding a chitosan solution to the pre-gel state mixture of step (3), and encapsulating the Cur-CD complex into CANPs. An oral composition with improved aqueous solubility and bioavailability A manufacturing method is provided.

In addition, the present invention provides a pharmaceutical composition for preventing or treating ulcerative colitis (UC) comprising the oral composition as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving ulcerative colitis (UC) comprising the oral composition as an active ingredient.

In addition, the present invention provides a composition for drug delivery to the large intestine comprising the oral composition as an active ingredient.

The present invention relates to cyclodextrin-curcumin encapsulated chitosan/alginate oral nanoparticles and their use for the treatment of colitis, wherein curcumin (Cur) is added to cyclic β-cyclodextrin (β-cyclodextrin; CD), After forming the water-soluble Cur-CD inclusion complex, it was encapsulated with chitosan/alginate nanoparticles (CANPs). The resulting Cur-CD-CANPs showed a monodisperse size distribution and a negative charge surface. The encapsulation efficiency of Cur-CD-CANPs was 81.5 ± 7.4% at an average diameter of 89.7 nm. In addition, as a result of in vivo experiments, oral administration of Cur-CD-CANPs showed superior therapeutic effect on DSS-induced colitis compared to free CUR. According to the results of body weight change, bloody stool, colon length and histopathology, oral administration of Cur-CD-CANPs showed excellent therapeutic effect on DSS-induced colitis. Accordingly, the Cur-CD-CANPs of the present invention can be effectively utilized as a drug delivery system for oral administration for the treatment of ulcerative colitis (UC).

1 shows the results of TEM and zeta potential analysis of CANPs.
2 shows the encapsulation rate and drug release results.
3 shows the improvement effect of orally administered Cur-CD-CANPs in DSS-induced colitis.
4 shows the experimental results through the DSS-induced animal model.
Figure 5 shows the change of the mouse intestinal flora profile by orally administered CANPs.
6 shows a schematic diagram of the production of Cur-CD-CANPs.

The present invention is a cyclodextrin-curcumin (cyclodextrin-curcumin; Cur-CD) complex encapsulated chitosan / alginate nanoparticles (chitosan / alginate nanoparticles; CANPs) containing as an active ingredient water solubility and bioavailability is improved An oral composition is provided.

Preferably, the cyclodextrin-curcumin (Cur-CD) complex has a molar ratio of cyclodextrin (CD) to curcumin (Cur) of 1:1 (v/v), and a water-soluble complex may be, but is not limited thereto.

In addition, the present invention comprises the steps of (1) mixing cyclodextrin dissolved in an aqueous phase and curcumin dissolved in an oil phase to form a water-soluble cyclodextrin-curcumin (Cur-CD) complex; (2) mixing the formed Cur-CD complex with sodium alginate solution; (3) preparing a pre-gel state by adding a calcium chloride solution to the mixed solution of step (2); and (4) adding a chitosan solution to the pre-gel state mixture of step (3), and encapsulating the Cur-CD complex into CANPs. An oral composition with improved aqueous solubility and bioavailability A manufacturing method is provided.

In addition, the present invention provides a pharmaceutical composition for preventing or treating ulcerative colitis (UC) comprising the oral composition as an active ingredient.

The pharmaceutical composition or combination formulation of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant includes an excipient, a disintegrant, a sweetener, a binder, a coating agent, a swelling agent, and a lubricant. , a solubilizing agent such as a lubricant or a flavoring agent may be used. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition including one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. For compositions formulated as liquid solutions, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one of these components. More than the components can be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostats can be added as needed. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form pills, capsules, granules or tablets.

The pharmaceutical formulation of the pharmaceutical composition of the present invention may be granules, powders, coated tablets, tablets, capsules, syrups, juices, suspensions or emulsions. The effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required for preventing or treating a disease. Therefore, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, weight, general health status, sex and diet of the patient, administration time, administration route and composition It can be adjusted according to various factors including secretion rate, duration of treatment, and drugs used at the same time. Although not limited thereto, for example, for adults, when administered once to several times a day, the composition of the present invention can be administered at a dose of 0.01 ng/kg to 10 g/kg when administered once to several times a day. .

In addition, the present invention provides a health functional food composition for preventing or improving ulcerative colitis (UC) comprising the oral composition as an active ingredient.

The health functional food composition of the present invention may further include one or more additives selected from the group consisting of organic acids, phosphates, antioxidants, lactose casein, dextrin, glucose, sugar and sorbitol. The organic acid may be, but is not limited to, citric acid, humic acid, adipic acid, lactic acid or malic acid, and the phosphate salt may be, but is not limited to, sodium phosphate, potassium phosphate, acid pyrophosphate or polyphosphate (polyphosphate), and antioxidant The agent may be, but is not limited to, a natural antioxidant such as polyphenol, catechin, alpha-tocopherol, rosemary extract, licorice extract, chitosan, tannic acid or phytic acid.

In another embodiment of the present invention, the health functional food is, in addition to the active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.) ), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the food composition according to an embodiment of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage.

According to an embodiment of the present invention, the dosage form of the health functional food is not limited thereto, but may be in the form of solid, powder, granule, tablet, capsule, liquid or beverage.

In addition, the health functional food is not limited thereto, but confectionery, sugar, ice cream products, dairy products, meat products, fish meat products, tofu or jelly products, edible oils and fats, noodles, teas, beverages, special nutritional foods, health supplements, seasoned foods , ice, ginseng products, pickled kimchi food, raisins, fruits, vegetables, dried fruits or vegetables, cut products, fruit juice, vegetable juice, mixed juices thereof, chips, noodles, processed livestock food, processed seafood, It can be used in the manufacture of foods such as milk processed foods, fermented milk foods, pulse foods, cereal foods, microbial fermented foods, confectionery baking, seasonings, meat products, acidic beverages, licorice, herbs, and the like.

In addition, the present invention provides a composition for drug delivery to the large intestine comprising the oral composition as an active ingredient.

The drug used in the present invention is a substance capable of inducing a desired biological or pharmacological effect by promoting or inhibiting physiological functions in the body of an animal or human, and is a chemical or biological substance or compound suitable for administration to an animal or human has a preventive effect on organic matter by preventing unwanted biological effects, such as preventing infection, alleviating conditions caused by disease, for example, alleviating pain or infection resulting from disease, and removing disease from organic matter It can play a role in mitigating, reducing, or completely eliminating it.

In the present invention, the composition for drug delivery may further include one or more pharmaceutically acceptable carriers, excipients or diluents in addition to the drug.

Examples of such carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In addition, the composition for drug delivery may further include a filler, an anti-aggregant, a lubricant, a wetting agent, a fragrance, an emulsifier, and a preservative.

The composition for drug delivery according to the present invention may be administered orally, and the dosage of the drug may be appropriately selected according to various factors such as the age, sex, weight and severity of the patient. In addition, the composition for drug delivery of the present invention may be administered in parallel with a known compound capable of increasing the desired effect of the drug.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are provided to more completely explain the present invention to those with average knowledge in the art, and are only illustrative of the contents of the present invention, so that the scope of the present invention is limited to the following examples not.

< Experimental example >

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Experimental Materials

Curcumin and β-cyclodextrin were purchased from Solarbio (Beijing, China). Chitosan (Mw 50,000-190,000 Da, viscosity 20-30 cP and deacetylation 75%) and sodium alginate (low viscosity, Mw 80,000-120,000 Da, ≥ viscosity 2000 cP) were synthesized by Sigma-Aldrich (St. Louis, MO, USA). ) was purchased from Dextran sodium sulfate (DSS) was purchased from MP Biomedicals (Irvine, CA, USA). Male C57BL/6 mice (8-10 weeks old, 18-22 g) were obtained from Pengyue Laboratory Animal Technology Co., Ltd. It was purchased from (Jinan, China) and bred in a sterile environment. Rhodamine B isothiocyanate (RBITC) was purchased from Macklin (Shanghai, China).

2. cyclodextrin - Curry Min ( Cyclodextrin - curcumin ; Cur-CD) production

The molar ratio of cyclodextrin to curcumin was 1:1 (v/v). Briefly, 647 mg cyclodextrin was dissolved in 120 mL water, 147 mg curcumin was dissolved in 2 mL acetone, and the oil phase was added to the aqueous solution and centrifuged at 400 r/min. The acetone was volatilized by stirring in the dark without a cover for 24 hours, followed by centrifugation at 1000 r/min for 5 minutes. The supernatant was collected and lyophilized, and aqueous Cur-CD was recovered.

3. Preparation and Measurement of Nanoparticles

As shown in Fig. 6, Cur-CD-CANPs were prepared by the ion gel method. 0.0315% (w/v) sodium alginate solution and 0.07% (w/v) chitosan were dissolved in water and 1% acetic acid solution, respectively, and stirred overnight. Then, 1 mL aqueous Cur-CD and 23.5 mL sodium alginate solution were mixed and dispersed evenly. Then, a 0.2% (w/v) 1.5 mL calcium chloride solution was dropped into the solution at a rate of 0.1 mL/min, and stirred for 30 minutes to make a pre-gel state. Finally, 2ml chitosan solution was added at a rate of 0.1mL/min, and left for 30 minutes to form a uniformly dispersed solution. After centrifugation at 12,000 r/min for 30 minutes, the supernatant was removed and washed 3 times with ultrapure water. The powder was obtained by vacuum freeze-drying. The particle size and zeta potential of the synthetic Cur-CD-CANPs were measured with a Zetasizer Nano ZS (Malvern Instrument, UK) through dynamic light scattering (DLS). Morphological analysis was performed in open field mode using transmission electron microscopy (TEM).

4. Encapsulation rate and drug release

Drug encapsulation efficiency (EE) and loading efficiency (LE) were measured by the following method. 5 mg of synthetic Cur-CD-CANPs were dispersed in 10 mL of absolute ethanol and vibrated at 37° C. at a constant temperature for 24 hours to obtain fully expanded nanoparticles. Then, the solution was ultrasonically washed for 30 minutes and centrifuged at 12000 rpm for 30 minutes. The concentration of Cur in the supernatant was measured according to a predetermined standard curve at 425 nm using an ultraviolet spectrometer. Finally, EE and LE were calculated according to the following equation.

Using a dialysis bag, the pH sensitivity of the formed nanoparticles was measured. Briefly, 5 mg of Cur-CD-CANP was dispersed in 10 mL water and transferred to a dialysis bag. Then, 100 mL of several solution buffers (pH 1.2, 6.8 and 7.4) were used as release media and mixed at 37° C. for 24 hours (100 rpm). At the same time, samples were removed at different times and the concentration of free Cur was measured at 425 nm.

The basket rotation method was used to simulate the entry of oral drugs into the human gastrointestinal tract. First, the nanoparticles were exposed to a simulated gastric acid environment (pH 1.2) for 2 h, and then transferred to a pH 6.8 buffer system to simulate the small intestine environment for an additional 2 h. Finally, it was transferred to 7.4 to simulate the colonic environment for 20 h. On the other hand, a 2 mL aliquot of the sample solution was removed at different times to detect the free Cur content, and mixed with an equivalent amount of fresh sustained-release medium. The cumulative drug release rate of the compound particles at time T was calculated according to the following equation.

5. to RBITC Fluorescence-labeling of chitosan

The synthesis of RBITC-labeled chitosan is based on the reaction between the isothiocyanate of RBITC and the primary amino acid group of chitosan. Briefly, 0.5 g chitosan was dissolved in 100 mL of 2% (v/v) acetic acid solution for 30 min via electromagnetic stirring. The pH was then adjusted to 7.5 with 1 mol/L NaOH. While stirring, 1 mL of DMSO solution containing RBITC (1 mg) was added to the chitosan solution. After reacting at 40° C. for 1 hour in the dark, the reaction was carried out at room temperature overnight. Thereafter, the solution was centrifuged at 4000 rpm for 10 minutes, and washed repeatedly with distilled water until a clear supernatant was obtained to remove free RBITC. RBITC-labeled chitosan was used to prepare RBITC-labeled Cur-CD-CANPs.

6. Animal Models and Treatments

8-week-old male C57BL/6 mice (20-22 g) were housed in a temperature- and humidity-controlled facility (25 ± 2 °C, 50 ± 5% relative humidity) with a 12-hour light-dark cycle and free access to water and food. was bred in Colitis was induced by providing mice with 3% DSS (molecular weight 36-50 kDa) instead of drinking water for 7 consecutive days. After 1 week of adaptation, mice were randomly divided into the next 5 groups of 6 mice. (1) Ctrl: negative control group and mice provided with normal drinking water; (2) DSS model group; (3) Cur-CD-treated DSS group; (4) Cur-CD-CANPs-treated DSS group; and (5) RBITC-labeled Cur-CD-CANPs-treated DSS group. Mice in the drug treatment group received an equivalent amount of Cur (50 mg/kg) in the form of a suspension via oral gavage for 7 days. The status of mice in groups 1-4 was carefully recorded and observed daily for signs of disease (weight loss, defecation status and rectal bleeding). Disease activity index (DAI) was calculated according to the percentage of daily weight loss, defecation status and mean score of bloody/occult blood. At the end of the experiment, feces from groups 1-4 were collected and stored at -80°C for further analysis. The colon and spleen tissues were quickly removed and weighed. Tissues were photographed and stored at -80°C for further analysis.

Group 5 mice were used to determine the tissue-targeting effect of CANPs in colitis. RBITC-labeled Cur-CD-CANPs (100 μl) were administered 7 days after establishment of the colitis model. To avoid background interference with food in the GIT, the diet of the mice was restricted for 6 h prior to image analysis. After 6 and 12 hours, using an in vivo 3D imaging system (PerkinElmer), the tissue and the living body of the mouse were image analyzed. All animal experiments were approved through the ethics committee of Qingdao Medical University.

7. H&E Staining and ELISA Analysis

Hematoxylin and eosin (H&E) staining was performed with Sevicepio (Wuhan, China). Colonic fragments were fixed in 4% paraformaldehyde solution for 24 hours, dehydrated, infiltrated, and embedded in paraffin. 4-μm thick sections were obtained with a slicer and placed on slides. Sections were stained with H&E. Images were obtained using a microscope at 200× and 400× magnification.

Colonic tissue was homogenized in ice-cold PBS (pH 7.4). The homogenate was centrifuged at 14000 rpm for 5 minutes at 4°C, and the supernatant was collected. The concentrations of IL-1β, IL-6 and TNF-alpha were measured according to ELISA kit instructions (Nanjing jiancheng Bioengineering Institute, Nanjing, China). Results were expressed as pg of cytokines per mg of total protein in the colon homogenate.

8. Real-time Quantitation PCR

Total RNA was isolated from tissues using the mirVana miRNA Isolation Kit (Ambion, Carlsbad, CA, USA). The primers of the present invention were synthesized by Sangong Biotech (Shanghai, China). Gene expression levels were normalized to GAPDH, and relative quantification of gene expression was performed using the 2-ΔΔCt method. All real-time PCR reactions were performed in triplicate.

9. western blot

The total protein in the colon tissue was extracted using the RIPA tissue extract, and the protein concentration was measured using a bicinchoninic acid (BCA) protein quantification kit. Western blotting analysis was performed as previously reported.

10. feces Microbial Species Analysis

Using qPCR, the microbial composition of each sample was analyzed. Total DNA of fecal microorganism samples was extracted using TIANamp Stool DNA Kit (Tiangen Biotechnology Co., Ltd., Beijing, China). The concentration of the DNA sample was measured using a NanoDrop spectrophotometer (ThermoFisher, Pittsburgh, PA, USA). RT-PCR (Applied Biosystems 7500 Real-Time PCR System, ABI Co., Ltd., Foster City, CA, U.S.A.) with Power SYBR® Green PCR Master Mix Kit (ABI Co., Ltd. Foster City, CA) The microbiome was quantified through After initiation of the reaction by activation at 95°C for 5 minutes, 40 cycles of target cDNA amplification (denaturation at 95°C for 15 seconds and extension at 60°C for 35 seconds) were performed. A standard DNA template was used to quantify the target DNA copy number. Briefly, serial 10-fold gradient dilutions of the standard products were used, and at least six non-zero standard concentrations were applied to each assay. Concentrations are expressed as log 10 copies.

11. Statistical Analysis

The present inventors performed statistical analysis using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA). Results are expressed as mean ± SEM. To measure the statistical significance between the two groups, the present inventors performed Student's t-test to calculate the associated p-value. Statistical significance between multiple groups was calculated through one-way analysis of variance (ANOVA).

< Example 1> CANPs Characterization

CANPs are potential candidates for the development of ODDSs, but their application in the treatment of colitis is rare. In the present invention, Cur is provided as a hydrophobic model drug, and a ring-form β-CD is added thereto to form a water-soluble Cur-CD inclusion complex. As shown in Figure 1A, the Cur-CD complex was dissolved in water and then encapsulated into CANPs. Then, as a result of TEM image analysis, the spherical shape of the particles prepared with a narrow size distribution and a dense structure was confirmed. The particle size was about 100-300 nm, which promoted the adhesion and migration of microspheres in vivo (Fig. 1B). As a result of dynamic light scattering (DLS) image analysis, the average particle size of the formed Cur-CD-CANPs was 462.1 nm ( FIG. 1C ). It should be noted that, due to the drying process of TEM sample processing, the particle size of TEM may be smaller than that of DLS, and the particle size measured in solution may be larger than that of TEM in the dry state. The polydispersity index (PDI) was 0.227 ± 0.17, indicating a monodisperse size distribution. The zeta potential of the synthetic Cur-CD-CANPs is -19 mV, which supports the excellent stability of the formed NPs.

< Example 2> Analysis of encapsulation efficiency, drug loading and cumulative release rate

The EE and drug loading (LC) of Cur-CD-CANPs were calculated indirectly through measurement of residual drug in solution. The EE and LC of CANPs were 88.89% and 3.49, respectively, indicating that the encapsulation ability is effective. The dialysis bag method was used to measure the permeability of Cur in three different pH media (pH 1.2, 6.8 and 7.4, respectively). As shown in Figure 2A, Cur has a maximum rate at pH 7.4. As the pH is decreased, the permeation also decreases. Because the strong electrostatic bonding between the carboxyl group of alginate and the amino group of chitosan induces shrinkage and gel formation in a low pH environment, Cur-CD-CANPs release only 15% of Cur within 24 hours at pH 1.2. This property protects Cur from GIT and aggressive gastric environment. To further confirm the protection of CANPs in GIT, Cur-CD-CANPs were reacted with a medium with a gradual change in pH to prevent drug passage through the stomach (pH 1.2), small intestine (pH 6.8) and large intestine (pH 7.4). simulated. Only 12% and 27% of Cur was released within the first 4 hours at pH 1.2 and 6.8 levels, representing pH levels in the stomach and upper small intestine, respectively. However, in the ileum (pH 7.4), more than 60% of the drug was explosively released within 24 hours (Fig. 2B). These results indicate that CANPs have therapeutic potential in pH sensitivity and colitis.

< Example 3> in vivo ( in in vivo ) drug formation distribution profile

The DSS-induced colitis mouse model is easy to establish and reproduce, and its symptoms are similar to human UC (eg, weight loss, colonic ulcers and bloody stools). Therefore, it is widely used to evaluate new drugs or carriers for the treatment of UC. To investigate the bio-distribution of CANPs in vivo , we gavaged RBITC-embedded Cur-CD-CANPs in DSS-induced colitis mice, and time-dependent passage and in vivo ( In vivo ) targeting efficiency was analyzed using a small animal in vivo 3D imaging system. After oral administration of RBITC-Cur-CD-CANPs, the fluorescence intensity of whole mice was quantitatively analyzed at 6 and 12 hours ( FIG. 3 ). After oral administration of NPs, fluorescence signals were mainly observed in GIT, and faint signals were observed in other internal organs. In addition, the percentage of RBITC amount in major GIT fragments (ie, stomach, small intestine and large intestine including caecum) was analyzed 6 and 12 hours after NPs administration. As a result of ex vivo RBITC image analysis, the signal accumulated through the digestive tract within 6 hours. At the same time, a strong signal was mainly observed at 12 h in the colon of UC mice, indicating that synthetic Cur-CD-CANPs have strong colon-targeting and retention capacity in mice with colitis ( FIG. 3 ). Results obtained from drug release in vitro (Fig. 2) and bio-distribution of NPs in GIT (Fig. 3) in vivo indicate that synthetic Cur-CD-CANPs have tremendous therapeutic potential in the treatment of colitis. indicates

< Example 4> DSS-induced colitis alleviating Cur-CD- CANPs

To evaluate the therapeutic effect of Cur-CD-CANPs in colitis, a DSS-induced acute colitis mouse model was established. The protocol for induction of colitis and treatment of Cur-CD-CANPs is shown in Figure 4A. Mice developed colitis and severe diarrhea 3 days after DSS treatment, accompanied by piling, prostrate and hypomotility. As shown in Figure 4B, mice treated with DSS showed significant weight loss by day 4, whereas Cur-CD treatment prevented weight loss until day 7. Cur-CD-CANPs significantly prevented weight loss by day 5. DSS administration induced severe inflammation, and DSS treatment significantly reduced colon length ( P < 0.001). Treatment with Cur-CD-CANPs significantly inhibited the decrease in colon length ( P < 0.01, FIGS. 4C and 4D ). Meanwhile, disease severity was scored by DAI. The DAI score of the colitis model group was significantly increased until the 6th day compared to the Cur-CD-CANPs treatment group ( P < 0.05, FIG. 4E). At day 7, both the Cur-CD-CANPs and Cur-CD groups showed a significant decrease in DAI scores ( P < 0.01). In addition, DSS induction significantly increased the weight of the spleen ( P < 0.05), and treatment with Cur-CD-CANPs significantly inhibited the increase in spleen weight ( P < 0.05, FIGS. 4F-G). These results support that treatment with Cur-CD-CANPs alleviates the symptoms of DSS-induced colitis, indicating potential therapeutic potential in UC.

< Example 5> Cur-CD-CANPs to reduce the histological damage of colon tissue in DSS-induced colitis mouse model

To further confirm the inhibitory effect of Cur-CD-CANPs on the histological damage of colon tissues in a DSS-induced colitis mouse model, the severity of colon damage and inflammation was analyzed using H&E staining (FIG. 4). As expected, DSS administration induced severe damage and inflammation in the colon tissue, which was consistent with the DAI score and colon morphology ( FIGS. 4C and 4E ). Compared with the control group, the DSS group had severe damage to the colonic mucosa; destruction of almost all intestinal glands; rapid decrease in goblet cells; Inflammatory cell invasion in the lamina propria; glandular disorders; and severe ulcers. Cur-CD treatment significantly reduced the colitis symptoms, but the therapeutic effect was not comparable to that of Cur-CD-CANPs. In the Cur-CD-CANPs group, there was no damage to the colon tissue, the intestinal glands were relatively intact, the glandular tissue was clear, the goblet cells were complete, and the colonic tissue morphology was similar to that of the control group ( FIGS. 4F and 4G ). The above results indicate that treatment with Cur-CD-CANPs reduces the inflammatory response of colonic tissue in a mouse model of DSS-induced colitis.

< Example 6> Cur-CD-CANPs Reduce Inflammation and Preserve Intestinal Integrity in a DSS-Induced Colitis Mouse Model

To verify the anti-inflammatory effect of Cur-CD-CANPs, gene and protein levels of several anti-inflammatory cytokines were detected. DSS administration significantly induced the expression of inflammatory factors ( FIGS. 5A-B ). Compared with the Cur-CD group, Cur-CD-CANPs inhibited the relative expression of genes/proteins more effectively. Upon treatment with Cur-CD-CANPs, three pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) were significantly reduced in DSS-induced colitis mice. These results support that Cur-CD-CANPs significantly reduce inflammation in the serum and colon tissues of DSS-induced colitis mice.

To further elucidate the role of Cur-CD-CANPs in the regulation of intestinal tight junctions, major tight junction proteins (ZO-1 and occludin) were analyzed in colonic tissues by Western blot analysis (FIGS. 5C-D). DSS treatment significantly reduced protein expression of ZO-1 ( P < 0.01) and occludin ( P < 0.001). On the other hand, the expression of ZO-1 ( P < 0.01) and occludin ( P < 0.01) reduced by DSS treatment was significantly reversed by treatment with Cur-CD-CANPs. Cur-CD partially improved the protein expression of ZO-1 and occludin in DSS-induced colitis, but it was not significant. These results support that treatment with Cur-CD-CANPs improves the integrity of the DSS-induced colitis-induced mouse intestinal wall. These results indicate that Cur-CD-encapsulated CANPs reduce the inflammatory response and protect the intestinal integrity of mice.

< Example 7> Intestine in mice with DSS-induced colitis dysbacteriosis The protective effect of Cur-CD-CANPs against

In order to verify the protective effect of Cur-CD-CANP against intestinal dysplasia, the composition of the intestinal flora was analyzed in the collected fecal pellet samples. The total levels of intestinal microbes, Bacteroides, Bifidobacterium , Lactobacillus, Clostridium leptum group and C. coccoides , were quantitatively analyzed by qPCR. As shown in FIG. 5 , among the four groups, there was no significant difference in the overall level of intestinal microflora. However, significant changes were observed in the structure of some intestinal flora. Bifidobacterium ( P < 0.01) and Lactobacillus ( P < 0.001) species in mice were significantly reduced following DSS treatment. Cur-CD-CANPs treatment significantly reversed the reduction in Bifidobacterium ( P < 0.05) and Lactobacillus ( P < 0.001) levels caused by DSS treatment, whereas the effect of Cur-CD treatment was not as pronounced compared to the Cur-CD-CANPs group. . Among the four groups, no significant difference was observed in the levels of Bacteroides , C. leptum and C. coccoides . Therefore, regulation of intestinal microbial composition through Cur-CD-CANPs may play a central role in alleviating DSS-induced colitis.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (6)

Cytodextrin-curcumin (cyclodextrin-curcumin; Cur-CD) complex encapsulated chitosan / alginate nanoparticles (chitosan / alginate nanoparticles; CANPs) as an active ingredient, aqueous solubility and bioavailability is improved oral composition. The method of claim 1, wherein the cyclodextrin-curcumin (Cur-CD) complex has a molar ratio of cyclodextrin (CD) to curcumin (Cur) of 1:1 (v/v), An oral composition with improved water solubility and bioavailability, characterized in that it is a water-soluble complex. (1) mixing cyclodextrin dissolved in an aqueous phase and curcumin dissolved in an oil phase to form a water-soluble cyclodextrin-curcumin (Cur-CD) complex;
(2) mixing the formed Cur-CD complex with sodium alginate solution;
(3) preparing a pre-gel state by adding a calcium chloride solution to the mixed solution of step (2); and
(4) by adding a chitosan solution to the pre-gel state mixture of step (3), and encapsulating the Cur-CD complex into CANPs. Preparation of an oral composition with improved water solubility and bioavailability Way. A pharmaceutical composition for preventing or treating ulcerative colitis (UC) comprising the oral composition of claim 1 or 2 as an active ingredient. A health functional food composition for preventing or improving ulcerative colitis (UC) comprising the oral composition of claim 1 or 2 as an active ingredient. A composition for drug delivery to the large intestine comprising the oral composition of claim 1 or 2 as an active ingredient.

Images