Chondroitin sulfate modified scutellarin nano-composite as well as preparation method and application thereof

Abstract

The invention discloses a chondroitin sulfate modified scutellarin nano-composite as well as a preparation method and application thereof, and belongs to the field of medical biological materials. The chondroitin sulfate modified scutellarin nano-composite disclosed by the invention has good oxidation resistance and can be used for removing various active oxygen and active nitrogen free radicals; the colon-targeting liposome has good targeting ability, can target M1 macrophages at a colon part, effectively reduces adverse reactions, and improves bioavailability; the water solubility is good, and the cell compatibility and the blood compatibility are good. According to the preparation method of the chondroitin sulfate modified scutellarin nano-composite, esterification reaction is adopted, carboxyl of chondroitin sulfate and hydroxyl of scutellarin are utilized to form ester compounds through catalysis, operation is easy, raw material sources are wide, reaction conditions are mild, and the problems of high temperature, toxic solvents and the like are solved.

Description

Technical Field

[0001] The present invention belongs to the field of medical biomaterials, in particular to a chondroitin sulfate-modified scutellarin nanocomposite, a preparation method and an application thereof. Background Art

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory disease of the intestine. Lesions are primarily confined to the colorectal mucosa and are characterized by continuous, diffuse inflammatory changes. Its primary clinical manifestations include diarrhea, abdominal pain, and bloody stools. Patients often experience weight loss, fatigue, and recurrent episodes. The pathogenesis of UC is complex, with current research suggesting it is the result of an intertwined network of genetic, environmental, immune system, and intestinal barrier dysfunction.

[0003] In recent years, the incidence of IBD has risen significantly. Although commonly used clinical treatments, such as aminosalicylate, immunomodulatory drugs, corticosteroids, and antibiotics, have certain efficacy in treating IBD, they are still accompanied by various adverse reactions and poor prognosis. According to statistics, approximately 40% of IBD patients will try complementary or alternative therapies while receiving traditional drug treatment. While the widespread use of biological drugs has improved the clinical treatment remission rate of ulcerative colitis to a certain extent, achieving a clinical cure for UC remains a challenge. The long-term cure rate has not increased significantly, and the high price of biological drugs has undoubtedly put heavy economic pressure on the public health system. Therefore, the development of drugs that are both in line with my country's national conditions and have the ability to effectively treat UC is of vital importance for improving the quality of life of UC patients and reducing the national medical burden. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings of existing traditional medicines and provide a chondroitin sulfate-modified scutellarin nanocomposite, a preparation method and an application thereof.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] Dissolve 92.6 mg of chondroitin sulfate in 15 mL of deionized water, add 115 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 73.3 mg of 4-dimethylaminopyridine (DMAP) as catalyst, and stir to activate for one hour.

[0007] 2) Dissolve 92.4 mg of (2S,3S,4S,5R,6S)-6-((5,6-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-4H-chromen-7-yl)oxy)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxylic acid (scutellarin) in 20 mL of dimethyl sulfoxide and add to the reaction mixture in 1) with stirring for 12 hours.

[0008] 3) The reaction solution in 2) was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed with deionized water for 3 days to remove unreacted scutellarin, dimethyl sulfoxide, and catalyst to obtain a pure chondroitin sulfate-scutellarin nanocomplex solution.

[0009] 4) The solution in the dialysis bag in 3) is placed in a vacuum freeze dryer for freeze drying to obtain a chondroitin sulfate-scutellarin nanocomplex product.

[0010] Compared with the prior art, the present invention has the following beneficial effects:

[0011] The chondroitin sulfate-modified scutellarin nanocomplex of the present invention has good antioxidant properties and can scavenge a variety of reactive oxygen and reactive nitrogen free radicals; has good targeting ability and can target M1 macrophages in the colon, effectively reducing adverse reactions and improving bioavailability; has good water solubility, cell compatibility and blood compatibility.

[0012] The preparation method of the chondroitin sulfate-modified scutellarin nanocomplex of the present invention adopts an esterification reaction, utilizing the carboxyl group of chondroitin sulfate and the hydroxyl group of scutellarin to form an ester compound through catalysis. The method has simple operation, a wide source of raw materials, mild reaction conditions, and avoids problems such as high temperature and toxic solvents.

[0013] The chondroitin sulfate-modified scutellarin nanocomposite of the present invention is added into a hydrogel as a medicine for treating ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the nuclear magnetic resonance spectrum of the chondroitin sulfate scutellarin nanocomplex of Example 1;

[0015] Figure 2 This is the infrared absorption spectrum of the chondroitin sulfate scutellarin nanocomplex of Example 1;

[0016] Figure 3 This is a transmission electron microscope image of the chondroitin sulfate scutellarin nanocomplex of Example 1;

[0017] Figure 4The ability of the chondroitin sulfate-scutellarin nanocomplex of Example 1 to scavenge reactive oxygen species and reactive nitrogen species at different concentrations, wherein the reactive nitrogen species include DPPH· and ABTS + ·, reactive oxygen species include O2 - ·and PTIO·;

[0018] Figure 5 This is the hemolysis test result of the chondroitin sulfate scutellarin nanocomplex of Example 1;

[0019] Figure 6 This is the fluorescence staining result of the cytotoxicity of the chondroitin sulfate scutellarin nanocomplex to Caco2 cells in Example 1;

[0020] Figure 7 These are H&E and Masson's trichrome staining images of colon tissue pathology sections. DETAILED DESCRIPTION

[0021] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0022] It should be noted that the terms "first", "second", etc. in the description and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that the numbers used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0023] Scutellarin is a flavonoid compound extracted from the entire herb of Erigeron breviscapus. It possesses multiple pharmacological effects and a wide range of clinical applications. It is commonly used in clinical treatments for ischemic cardiovascular and cerebrovascular diseases to assist in vasodilation and reduce blood viscosity. Furthermore, scutellarin exhibits significant anti-inflammatory, antioxidant, and anti-tumor biological activities. It can inhibit the release of inflammatory factors and reduce inflammatory responses, while scavenging free radicals, protecting cells from oxidative damage, and inhibiting the proliferation and invasion of cancer cells. It has broad application value and potential in the medical field.

[0024] Chondroitin sulfate is a naturally occurring endogenous polysaccharide widely distributed in the extracellular matrix and cell surfaces of animal tissues. Its excellent biocompatibility and water solubility make it a promising material for biomedical applications. Its structure contains a rich array of reactive groups, such as carboxyl and hydroxyl groups, which can be covalently coupled to small molecule drugs for the delivery of hydrophobic drugs. Furthermore, chondroitin sulfate can target the CD44 receptor, which is highly expressed in the colon, making it an ideal nanoparticle delivery vehicle.

[0025] The present invention is described in further detail below with reference to the accompanying drawings:

[0026] Example 1

[0027] 92.6 mg of chondroitin sulfate was dissolved in 20 mL of deionized water, and 115 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 73.3 mg of 4-dimethylaminopyridine (DMAP) were added as catalysts, and the mixture was stirred and activated for one hour. 92.4 mg of scutellarin was dissolved in 20 mL of dimethyl sulfoxide and added to the reaction solution, and the mixture was stirred and reacted for 12 hours. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed against deionized water for 3 days to remove unreacted scutellarin, dimethyl sulfoxide, and catalyst, thereby obtaining a pure chondroitin sulfate-scutellarin nanocomplex solution. The solution in the dialysis bag was placed in a vacuum freeze dryer for freeze drying to obtain the chondroitin sulfate-scutellarin nanocomplex product.

[0028] The chondroitin sulfate used in Example 1 was purchased from MacLean Biotechnology Co., Ltd. with a purity of 95%, and scutellarin was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd. with a purity of 97%.

[0029] The chondroitin sulfate scutellarin nanocomplex of Example 1, chondroitin sulfate and scutellarin were added to potassium bromide, ground and pressed into tablets, and infrared absorption spectra of the three were obtained by Fourier transform infrared spectrometer. Figure 1 As shown in the figure, characteristic absorption peaks of chondroitin sulfate and scutellarin appeared in the chondroitin sulfate-scutellarin nanocomplex, verifying its structure.

[0030] The chondroitin sulfate scutellarin nanocomplex and chondroitin sulfate of Example 1 were dissolved in heavy water and the hydrogen spectra of the two were obtained by nuclear magnetic resonance spectroscopy at 400 MHz. Figure 2 As shown, the methyl peak of chondroitin sulfate and the benzene ring peak of scutellarin appeared in the chondroitin sulfate-scutellarin nanocomplex, verifying its structure.

[0031] The chondroitin sulfate scutellarin nanocomplex of Example 1 was dissolved in ultrapure water and sonicated, and the solution was dripped onto a copper mesh. After drying, the morphology of the nanocomplex was observed using a transmission electron microscope. Figure 3 As shown, transmission electron microscopy shows that the chondroitin sulfate-scutellarin nanocomplex has a uniform spherical structure.

[0032] The chondroitin sulfate scutellarin nanocomposite sample prepared in Example 1 was prepared into a gradient concentration solution. + · reacts with the sample to determine the nitrogen free radical scavenging ability. The superoxide anion free radical and PTIO· scavenging ability is evaluated by the riboflavin-methionine light irradiation method and reaction with PTIO·. Figure 4 As shown, the chondroitin sulfate-scutellarin nanocomplex can scavenge a variety of oxygen and nitrogen free radicals in a concentration-dependent manner.

[0033] Biosafety experiments

[0034] 1) Hemolysis test to determine the blood compatibility of the chondroitin sulfate-scutellarin nanocomplex in Example 1. Fresh mouse blood was collected, centrifuged at 1500 rpm for 10 min, the supernatant was discarded, washed with physiological saline, and centrifuged again, and repeated until the supernatant was clear. Finally, red blood cells were taken to prepare a 2% red blood cell suspension. Then 500 μL of the red blood cell suspension was incubated with different concentrations of chondroitin sulfate-scutellarin nanocomplex solutions at 37°C for 2 h, and the OD value was measured after centrifugation. Figure 5 As shown, the chondroitin sulfate-scutellarin nanocomplex has excellent blood compatibility.

[0035] 2) Live-dead cell staining assay to determine the cytocompatibility of the chondroitin sulfate / scutellarin nanocomplex in Example 1. Caco2 cells were seeded into confocal microplates. After the cells attached, the culture medium was replaced with medium containing different drug concentrations. Following the same culture conditions, staining solution was added after 1-2 days of culture and incubation continued for 0.5 hours. The culture was terminated, the culture supernatant was aspirated, and the cells were washed with PBS before observation under a laser confocal microscope. Figure 6 The results showed that the chondroitin sulfate-scutellarin nanocomplex had excellent cell compatibility.

[0036] Evaluation of therapeutic effects in a mouse colitis model

[0037] 1) C57BL / 6 mice were used as experimental animals. The normal group was fed with mouse chow and drinking water for 8 days, while the remaining groups were given free access to a 2% DSS solution for 8 days to induce an ulcerative colitis model. On the second day, mice were treated with a chondroitin sulfate / scutellarin nanocomplex. This group served as the experimental group, while a control group was given 5-aminosalicylic acid (5-ASA) for 7 consecutive days.

[0038] 2) On day 8, colon tissues of mice in each group were collected for pathological sectioning and H&E and Masson trichrome staining, see Figure 7 It can be observed that the model group had obvious inflammatory cell infiltration and tissue mucosal damage in the colon, while the pathological damage such as inflammatory cell infiltration and mucosal damage in the treatment group was improved, which proved that the chondroitin sulfate-modified scutellarin nanocomplex has a protective effect on colitis and can significantly improve the pathological damage caused by ulcerative colitis.

[0039] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A chondroitin sulfate-modified scutellarin nanocomposite, characterized in that: It is a nanocomplex formed by esterification reaction of chondroitin sulfate and scutellarin.

2. The chondroitin sulfate-modified scutellarin nanocomplex according to claim 1, characterized in that: The chondroitin sulfate-modified scutellarin nanocomplex presents a uniform spherical structure.

3. The chondroitin sulfate-modified scutellarin nanocomplex according to claim 1, characterized in that: The molar ratio of scutellarin to chondroitin sulfate is 1:

3.

4. The chondroitin sulfate-modified scutellarin nanocomplex according to claim 1, characterized in that: The molecular weight of the chondroitin sulfate is 70,000; the molecular weight of the scutellarin is 462.

5. A method for preparing a chondroitin sulfate-modified scutellarin nanocomposite, characterized in that: 1) dissolving chondroitin sulfate in deionized water to obtain a chondroitin sulfate aqueous solution; dissolving scutellarin in dimethyl sulfoxide to obtain a scutellarin solution; 2) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 4-dimethylaminopyridine (DMAP) as catalysts to the chondroitin sulfate aqueous solution. After stirring and activating for one hour, add scutellarin solution and stir for 12 hours. 3) The reaction solution was placed in a dialysis bag, dialyzed with deionized water for 3 days, and then freeze-dried to obtain a chondroitin sulfate-scutellarin nanocomplex product.

6. The method for preparing the chondroitin sulfate-modified scutellarin nanocomplex according to claim 5, characterized in that: In step 1), the concentration of the chondroitin sulfate aqueous solution is 6.17 mg / mL; the concentration of the scutellarin solution is 4.62 mg / mL.

7. The method for preparing the chondroitin sulfate-modified scutellarin nanocomplex according to claim 5, characterized in that: In step 2), the molar ratio of chondroitin sulfate to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 4-dimethylaminopyridine (DMAP) is 1:3:

3.

8. The method for preparing the chondroitin sulfate-modified scutellarin nanocomplex according to claim 5, characterized in that: The molecular weight cut-off of the dialysis bag in step 3) is 3500 Da.

9. The method for preparing the chondroitin sulfate-modified scutellarin nanocomplex according to claim 5, characterized in that: The molecular weight of the chondroitin sulfate is 70,000; the molecular weight of the scutellarin is 462.

10. A use of the chondroitin sulfate-modified scutellarin nanocomplex according to any one of claims 1 to 4, characterized in that: It is used as an oral medication to treat ulcerative colitis.

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