Curcumin complex, method of preparation and use and pharmaceutical composition thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a curcumin compound, a preparation method and use and a pharmaceutical composition thereof. The curcumin compound (Cur-VE-Zn) is prepared, wherein the addition of vitamin E or a derivative thereof can improve the solubility of the curcumin-Zn compound and reduce the toxicity of the curcumin-Zn compound, so that the Cur-VE-Zn has low cytotoxicity and good biological safety; the curcumin compound of the application can play a role in treating radiation-induced oral mucositis by eliminating active oxygen, reducing inflammatory response, promoting mucosal repair and reducing DNA damage. The application also prepares a temperature-sensitive hydrogel with the curcumin compound as a main component, improves the drug utilization rate, realizes the targeted delivery of the curcumin compound in the oral mucosa, and further exhibits better therapeutic effect, thereby providing a new strategy for the treatment of radiation-induced oral mucositis and having good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a curcumin complex, its preparation method and uses, and its pharmaceutical composition. Background Technology

[0002] Head and neck cancers (HNC) are the sixth most common malignant tumors worldwide, accounting for 5-10% of all cancers. In recent years, the incidence and mortality rates of head and neck malignancies in my country have been rising, especially in South China. Radiotherapy, a routine treatment for HNC, causes radiation oral mucositis (RIOM) in up to 90% of cases, posing a significant challenge to both doctors and patients during cancer treatment. RIOM can disrupt the integrity of the oral mucosa, accompanied by many complex mucosal and submucosal changes, often causing oral pain, ulcers, anorexia, dehydration, dysphagia, insufficient nutrient intake, and general malaise. Furthermore, oral mucositis, especially ulcerative oral mucositis, increases the incidence, mortality, and cost of cancer treatment. Studies have found that two weeks after conventional fractionated radiotherapy (2 Gy / day), when the cumulative radiation dose reaches 10-20 Gy, over 90% of patients develop clinical symptoms (grade 1-2), manifesting as dry mouth, halitosis, mucosal redness, bleeding, erosion, and a white pseudomembrane covering the mucosa. By the fifth week of radiotherapy, when the cumulative radiation dose reaches 50-60 Gy, 30-60% of patients develop severe ROM (grade 3-4). At this stage, patients are unable to eat normally and often require opioid peptide analgesics to relieve pain caused by ulcerative mucositis. Severe ROM can also lead to systemic infections and delays, cessation, or dose reduction of radiotherapy and chemotherapy, thus adversely affecting the patient's prognosis, impacting cancer treatment efficacy, and the patient's quality of life. Therefore, the prevention and treatment of radiation-induced oral mucositis are of paramount importance for ensuring the implementation and effectiveness of radiotherapy for head and neck cancer patients, as well as improving their prognosis and quality of life. This is also a challenging problem that urgently needs to be addressed in clinical practice.

[0003] The five stages of radiation-induced oral mucositis (RIOM) work together to ultimately lead to adverse reactions such as pain, dry mouth, ulcers, and difficulty swallowing. Blocking or reducing any one of these stages could potentially slow the progression of RIOM or reduce its severity. Currently, clinical treatments for RIOM are based on the five-stage theory, focusing on adjusting radiation dose, anti-oxidation, anti-inflammation, and antibacterial processes. These mainly include the following categories: (1) routine oral care; (2) intensity-modulated radiotherapy (IMRT); (3) anti-inflammatory drugs; (4) growth factors and cytokines; (5) anesthetics and analgesics; (6) photobiological regulation; (7) antibacterial agents; and (8) natural drugs. Although many studies have been conducted, the actual prevention and treatment effects of various interventions are not ideal, and there are varying degrees of side effects. The prevention and treatment of RIOM should be based on safety and effectiveness, and be more economical and feasible to improve patient compliance, reduce treatment costs, and improve the quality of life of patients during treatment. With the arrival of an aging society, the number of cancer patients is increasing, and the demand for radiotherapy is also increasing. Therefore, in-depth research and development of effective and safe new strategies for the prevention and treatment of radiotherapy are of great significance for improving patients' clinical symptoms, prognosis and quality of life, and optimizing the overall treatment effect of cancer.

[0004] Curcumin is inexpensive, readily available, and non-toxic, and has been approved as a natural food additive by the World Health Organization / Food and Drug Administration (WHO / FDA). Curcumin also possesses excellent antioxidant, anti-inflammatory, antiviral, antibacterial, and wound-healing properties, and has been proven to have good therapeutic effects on cardiovascular diseases, respiratory diseases, metabolic diseases, and neurodegenerative diseases, making it an ideal treatment for recurrent acute myelopathy (RIOM). However, curcumin's low solubility, poor stability, rapid metabolism, and low absorption rate result in poor bioavailability, thus affecting its practical clinical application. Studies have found that modifying the structure of curcumin can improve its physiological efficacy, solubility, stability, and safety. Curcumin complexes with metal ions to form curcumin-metal complexes, among which curcumin-Zn is formed with zinc ions. 2+ The complex not only amplifies the antioxidant, anti-inflammatory, and antibacterial properties of curcumin itself, but also improves its stability and solubility. Furthermore, zinc itself plays a positive role in maintaining oral health; it can reduce the production of free radicals, inhibit inflammatory mediators, and promote wound healing. Zinc supplementation can effectively combat oral diseases such as gingivitis, periodontitis, and halitosis. Multiple clinical studies have suggested that zinc supplementation may reduce the severity and duration of mucositis during chemotherapy in cancer patients. However, the cytotoxicity of the curcumin-Zn complex is increased compared to curcumin monomers, inhibiting cell viability. Vitamin E, as a natural antioxidant, exerts its antioxidant effect by scavenging free radicals and blocking lipid peroxidation chain reactions, thereby maintaining the biological activity of cell membranes.

[0005] Existing clinical treatment strategies and other drugs and natural products have limited effectiveness in treating RIOM, and there is currently no effective means to treat RIOM. Therefore, there is an urgent need to find a new curcumin complex with higher stability and safety, so as to provide a new option for the treatment of RIOM. Summary of the Invention

[0006] To address the above-mentioned problems, the present invention provides a curcumin complex, its preparation method, its uses, and its pharmaceutical composition.

[0007] A curcumin complex, which is made from the following raw materials in parts by weight:

[0008] Curcumin-Vitamin E complex 4-4.8 parts

[0009] Zinc source 2-2.4 parts;

[0010] The curcumin-vitamin E complex is made from the following raw materials in parts by weight:

[0011] 470-590 parts of vitamin E or its derivatives

[0012] 370-450 parts of dehydrating agent

[0013] Curcumin 330-410 parts

[0014] Catalyst 55-67 parts.

[0015] Preferably, it is made from raw materials comprising the following parts by weight:

[0016] 4.4 parts of curcumin and vitamin E complex

[0017] 2.2 parts zinc source;

[0018] The curcumin-vitamin E complex is made from the following raw materials in parts by weight:

[0019] 530.78 parts of vitamin E or its derivatives

[0020] 412.66 parts of dehydrating agent

[0021] Curcumin 368.38 parts

[0022] Catalyst 61.09 parts.

[0023] Preferably, the vitamin E derivative is selected from α-tocopherol succinate;

[0024] And / or, the dehydrating agent is selected from dicyclohexyldiimide;

[0025] And / or, the catalyst is selected from 4-dimethylaminopyridine;

[0026] And / or, the zinc source is selected from zinc acetate dihydrate.

[0027] The present invention also provides a method for preparing the above-mentioned curcumin complex, comprising the following steps:

[0028] Step 1: React vitamin E or its derivatives with a dehydrating agent to obtain mixture 1;

[0029] Step 2: React curcumin with a catalyst to obtain mixture 2;

[0030] Step 3: React mixture 1 with mixture 2 to obtain curcumin-VE complex;

[0031] Step 4: React the curcumin-VE complex with a zinc source to obtain the final product.

[0032] Preferably, in step 1, the solvent for the reaction is dichloromethane;

[0033] And / or, in step 1, the reaction temperature is 20-30℃, and the reaction time is 30-60 min;

[0034] And / or, in step 2, the solvent for the reaction is dichloromethane;

[0035] And / or, in step 3, the reaction temperature is 3-5℃ and the reaction time is 6-8h;

[0036] And / or, in step 4, the solvent for the reaction is ethanol;

[0037] And / or, in step 4, the reaction temperature is 50-70℃ and the reaction time is 1-2h.

[0038] The present invention also provides the use of the above-mentioned curcumin complex in the preparation of medicaments for the prevention and / or treatment of oral mucositis.

[0039] Preferably, the oral mucositis is radiation-induced oral mucositis.

[0040] The present invention also provides a pharmaceutical composition, which is a formulation prepared by adding pharmaceutically acceptable excipients to the above-mentioned curcumin complex as the active ingredient.

[0041] Preferably, the formulation is at least one of nano-formulation, cream, gel, and paste.

[0042] Preferably, the pharmaceutical composition is made from raw materials comprising the following parts by weight:

[0043] 15-25 parts of curcumin complex

[0044] 100-110 parts of surfactant

[0045] 20-30 parts of matrix material

[0046] 1240-1260 parts of gelling agent;

[0047] The surfactant is selected from Tween 80;

[0048] And / or, the matrix material is selected from chitosan;

[0049] And / or, the gelling agent is selected from at least one of poloxamer F127 and poloxamer F68.

[0050] This invention prepares a curcumin complex (Cur-VE-Zn), wherein the addition of vitamin E or its derivatives improves the solubility and reduces the toxicity of the curcumin-Zn complex, resulting in low cytotoxicity and good biocompatibility of Cur-VE-Zn. The curcumin complex of this invention exerts its therapeutic effect on radiation-induced oral mucositis by scavenging reactive oxygen species, reducing inflammatory responses, promoting mucosal repair, and reducing DNA damage. This invention also prepares a temperature-sensitive hydrogel with the curcumin complex as the main component, improving drug utilization and enabling targeted delivery of the curcumin complex to the oral mucosa, thereby exhibiting better therapeutic effects and providing a new strategy for the treatment of RIOM (recurrent irritation of oral mucosa), with promising application prospects.

[0051] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0052] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0053] Figure 1 Infrared spectra of curcumin, D-α-tocopherol succinate, curcumin-VE, curcumin-Zn, and curcumin-VE-Zn complex.

[0054] Figure 2 XPS full spectrum of curcumin-VE-Zn complex.

[0055] Figure 3 High-resolution spectrum of Zn 2p of curcumin-VE-Zn complex.

[0056] Figure 4 The effects of different treatment groups on the cell viability of HaCaT cells.

[0057] Figure 5 The cell scratch healing rate was calculated for each group at 12h and 24h.

[0058] Figure 6 The effects of different treatment groups on HaCaT cell migration.

[0059] Figure 7 The effects of different treatment groups on the expression level of γ-H2AX in the nucleus of HaCaT cells at 6h, 12h, and 24h.

[0060] Figure 8 The effects of different treatment groups on ROS levels in HaCaT cells.

[0061] Figure 9 To investigate the effects of different treatment groups on the expression levels of inflammatory factors in HaCaT cells.

[0062] Figure 10 This refers to the sol-gel reaction of a gel.

[0063] Figure 11 The curve showing the temperature change of G' (storage modulus) / G" (loss modulus) for curcumin-VE-Zn gel.

[0064] Figure 12 This is a scanning electron microscope image of curcumin-VE-Zn gel after lyophilization.

[0065] Figure 13 The swelling ratio of curcumin-VE-Zn gel.

[0066] Figure 14 The degradation rate of curcumin-VE-Zn gel.

[0067] Figure 15 The adhesion strength of curcumin-VE-Zn gel.

[0068] Figure 16 The change in body weight of mice in different treatment groups after X-ray irradiation.

[0069] Figure 17 The changes in the area of ​​tongue ulcers and mucositis in mice under different treatment groups.

[0070] Figure 18 HE staining of tongue tissue from mice in different treatment groups.

[0071] Figure 19 The expression levels of inflammatory factors in the tongue tissue of mice in different treatment groups.

[0072] Figure 20 The antioxidant levels of the tongue tissue of mice in different treatment groups.

[0073] Figure 21The expression levels of γ-H2AX in the tongue tissue of mice in different treatment groups. Detailed Implementation

[0074] In the following examples and experimental cases, reagents and raw materials not specifically described are all commercially available products.

[0075] Curcumin (Beijing Solarbio Science & Technology Co., Ltd.); α-Tocopherol Succinate (Shanghai Yuanye Biotechnology Co., Ltd.); Dicyclohexyldiimide, 4-Dimethylaminopyridine (Shanghai Aladdin Biochemical Technology Co., Ltd.); Zinc acetate dihydrate (Shanghai Maclean Biochemical Technology Co., Ltd.); Poloxamer F127 (Shanghai Yuanye Biotechnology Co., Ltd., Item No.: S30692), Poloxamer F68 (Shanghai Yuanye Biotechnology Co., Ltd., Item No.: S30691); Chitosan (Shanghai Maclean Biochemical Technology Co., Ltd., Item No.: C766420).

[0076] Example 1: Curcumin complex (Cur-VE-Zn) and its preparation method

[0077] Step 1: Preparation of Cur-VE

[0078] 1 mmol of α-tocopherol succinate (VE, 530.78 mg) and 2 mmol of dicyclohexyldiimide (412.66 mg) were weighed and added to 45 mL of dichloromethane. The mixture was stirred for 30 min at room temperature under nitrogen to obtain mixture 1. 1 mmol of curcumin (Cur, 368.38 mg) and 0.5 mmol of 4-dimethylaminopyridine (61.09 mg) were weighed and dissolved in 5 mL of dichloromethane. The solution was then slowly added dropwise to mixture 1. The mixture was reacted at 4 °C under nitrogen protection for 7 h. After filtration, the filtrate was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 3:1) and then rotary evaporated to obtain Cur-VE dry powder.

[0079] Step 2: Preparation of curcumin complex (Cur-VE-Zn)

[0080] 4.4 g of dried Cur-VE powder and 2.2 g of zinc acetate dihydrate were added to 100 mL of anhydrous ethanol and completely dissolved under nitrogen. The mixture was then mixed at 60 °C and reacted under nitrogen for 1.5 h to form a stable precipitate before the reaction was terminated. The Cur-VE-Zn complex was obtained after vacuum drying.

[0081] Example 2: Hydrogel loaded with curcumin complex (Cur-VE-Zn@gel) and its synthesis method

[0082] 100 μL of Tween80 (108 mg) was ultrasonically dispersed in 4650 μL of deionized water. 20 mg of the Cur-VE-Zn complex was dissolved in 250 μL of dimethyl sulfoxide and slowly added dropwise to the deionized water to prepare the Cur-VE-Zn complex formulation. 28.6 μL of glacial acetic acid was added dropwise to the complex formulation, and the volume was adjusted to 5 mL to achieve a glacial acetic acid concentration of 0.1 mol / L. 0.025 g of chitosan was dissolved in the above formulation and magnetically stirred at room temperature for 1 h to obtain a 0.5% chitosan solution. Poloxamer F127 (1 g) and F68 (0.25 g) in a mass ratio of 20:5 were dissolved in the chitosan solution and allowed to swell completely at 4 °C for 24 h to obtain a clear, viscous Cur-VE-Zn gel (Cur-VE-Zn@gel).

[0083] The following comparative examples provide the control samples used in the experiment.

[0084] Comparative Example 1: Curcumin-Zn (Cur-Zn)

[0085] Curcumin-Zn was prepared according to the method in step 2 of Example 1, except that curcumin was reacted with zinc acetate dihydrate.

[0086] Comparative Example 2: Curcumin-VE (Cur-VE)

[0087] Curcumin-VE was prepared according to the method in step 1 of Example 1.

[0088] Comparative Example 3 Cur @gel

[0089] The synthesis method is the same as in Example 2, except that the drug loaded is different. The drug loaded in this comparative example is curcumin.

[0090] The technical solution of the present invention will be further illustrated by the following experiments.

[0091] Characterization of Curcumin Complex (Cur-VE-Zn) in Experiment Example 1

[0092] I. Experimental Methods

[0093] Cur-VE-Zn was analyzed using Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy.

[0094] II. Experimental Results

[0095] like Figure 1 As shown, the Cur-VE complex was measured at 1755 and 2926 cm⁻¹, respectively. -1The presence of characteristic -COO-R and -CH3 peaks belonging to D-α-tocopherol succinate at the position indicates that the Cur-VE complex was successfully synthesized; the presence of these peaks with Zn... 2+ After coordination, the original bending and stretching vibration peaks of curcumin decreased, most of the characteristic peaks disappeared, and they were located at 1628 / 1598 cm⁻¹. -1 The C=O and C=C conjugate stretching vibration peaks at Zn exhibit a redshift, a characteristic observed in Cur-Zn and Cur-VE-Zn complexes but not in VE and Cur-VE complexes, indicating that Zn... 2+ Successfully chelated with curcumin and Cur-VE complex to form Cur-Zn and Cur-VE-Zn complex.

[0096] like Figure 2 As shown, the XPS full spectrum reveals characteristic peaks of binding energies for C 1s (284.78 eV), O 1s (531.06 eV), and Zn 2p (1022.72 eV), indicating the presence of elements such as C, O, and Zn in the complex.

[0097] like Figure 3 As shown, the high-resolution spectrum of Zn 2p reveals two spin orbitals of Zn 2p: Zn 2p³ / 2 (1022.72 eV) and Zn 2p¹ / ² (1045.66 eV), indicating that Zn 2+ It exists within the complex and may have accepted a lone pair of electrons from the O in the ligand Cue, thus remaining stable in an oxidized state. These results indicate the successful construction of the curcumin complex (Cur-VE-Zn).

[0098] Experiment Example 2: Effect of curcumin complex on radiation resistance of HaCaT cells after irradiation

[0099] I. Experimental Methods

[0100] 1. Experimental materials, drugs and reagents

[0101] Human immortalized keratinocytes (HaCaT) (China Center for Type Culture Collection, CCTCC); DMEM high-glucose medium, FBS (BDBIO); penicillin antibody (Beyotime Biotechnology Co., Ltd.); CCK-8 kit (Dojindo, Tongren Chemical); trypsin-EDTA solution (NewSemi Biotechnology); crystal violet (Beijing Solarbio Science & Technology Co., Ltd.); γ-H2AX monoclonal antibody (ZhengNeng Biotechnology Co., Ltd.); iFluor™ 488 Conjugated Goat anti-rabbit IgG polyclonal Antibody (Hangzhou Huaan Biotechnology Co., Ltd.); ROS detection kit (APE&BIO); IL-1β, IL-6, TNF-α detection kit (Wuhan Elairite Biotechnology Co., Ltd.).

[0102] 2. Method

[0103] (1) Cytotoxicity test: HaCaT cells were administered at a rate of 4 × 10⁻⁶ cells / year. 3 The cells were seeded at a concentration of / ml in 96-well plates. The concentration gradients of curcumin (Cur), curcumin-Zn complex (Cur-Zn), curcumin-VE complex (Cur-VE), and Cur-VE-Zn complex (Cur-VE-Zn) were set to 0, 10, 20, 40, 80, 160, and 200 μM. The cells were co-cultured with the cells for 24 h and 48 h. After washing with DMEM, 10 μL of CCK-8 solution was added and the cells were reacted at 37℃ for 1-2 h. The absorbance value at 450 nm was then measured to determine the cell viability.

[0104] (2) Cell scratch assay: HaCaT cells were seeded in 6-well plates (2×10⁻⁶ cells / wells). 5 After incubation for 24 h in 10 μM curcumin and curcumin-VE-Zn complex, cells were irradiated with X-rays (160 kV, 20 mA, total dose 20 Gy, dose rate 1.903 Gy / min). Cells were then co-cultured with 10 μM curcumin and curcumin-VE-Zn complex for 24 h. Cells were then scratched with the tip of a 1 mL pipette and cultured in DMEM medium for 24 h. During this period, the width of the scratched area was observed at 0 h, 12 h, and 24 h using an inverted microscope, and quantitative analysis was performed using Image-J.

[0105] (3) Transwell assay: HaCaT cells were seeded in 24-well plates (3 × 10⁻⁶ cells per well). 4 After incubation in 10 μM curcumin ( / ml) for 24 h, the cells were irradiated with X-rays and co-cultured with 10 μM curcumin and curcumin-VE-Zn complex for 24 h. After digestion and centrifugation, the cells were resuspended in DMEM to obtain a cell suspension, counted using a cell counter, and the cell suspension concentration was adjusted to 1×10⁻⁶. 5 / ml. A 24-well plate was divided into upper and lower sections using Transwell cell culture chambers. 200 μL of cell suspension was added to the upper chamber, and 500 μL of culture medium (DMEM + 15% FBS) was added to the lower chamber. After 24 h, cells were fixed with 4% paraformaldehyde, incubated with crystal violet for 20 min, washed with PBS, and observed under an inverted microscope to monitor cell migration and record the number of migrating cells.

[0106] (4) γ-H2AX expression: HaCaT cells were seeded in 96-well plates (4×10⁻⁶ cells / wells). 3 After incubation for 24 h in 10 μM curcumin and curcumin-VE-Zn complex, the cells were irradiated with X-rays and co-cultured for 24 h with 10 μM curcumin and curcumin-VE-Zn complex. The cells were then fixed with 4% paraformaldehyde, incubated with 0.5% Triton-X100 (10 min) at room temperature, blocked with blocking solution at room temperature for 1 h, and then incubated sequentially with γ-H2AX monoclonal antibody and iFluor™ 488 Conjugated Goat anti-rabbit IgG polyclonal antibody. Fluorescence images were captured and quantitatively analyzed using a high-throughput multi-parameter cell dynamics analysis system.

[0107] (5) ROS level determination: HaCaT cells were seeded in 6-well plates (2×10⁻⁶ cells / wells). 5 After incubation in 10 μM curcumin and curcumin-VE-Zn complex for 24 h, the cells were irradiated with X-rays and co-cultured for 24 h with 10 μM curcumin and curcumin-VE-Zn complex. After 24 h of culture, the cells were digested, centrifuged, washed, and then DCFH-DA dilution buffer was added. The cells were incubated at 37°C in the dark for 30 min, and the ROS level was detected by flow cytometry.

[0108] (6) Inflammatory factor levels: HaCaT cells were seeded in 6-well plates (2×10⁻⁶ cells / well). 5 After incubation in 10 μM curcumin and curcumin-VE-Zn complex for 24 h, the levels of IL-1β, IL-6, and TNF-α were detected by ELISA kit, and the absorbance value was measured at 450 nm.

[0109] II. Experimental Results

[0110] like Figure 4As shown, incubation with 10 μM curcumin for 24 h had no significant effect on HaCaT cell viability, but incubation with 20–200 μM curcumin for 24 h significantly inhibited cell viability, and the inhibition rate further increased after 48 h of incubation. Incubation with the curcumin-Zn complex (Cur-Zn group) resulted in increased cytotoxicity and inhibited cell viability compared to curcumin monomer, indicating that the curcumin-Zn complex can increase the cytotoxicity of curcumin. Incubation with different concentrations of curcumin-VE complex and curcumin-VE-Zn complex significantly reduced the inhibitory effect on HaCaT cell viability compared to curcumin monomer. The curcumin-VE-Zn complex group showed higher cell viability than the curcumin-VE complex group, indicating that both curcumin-VE complex and curcumin-VE-Zn complex reduced the cytotoxicity of free curcumin, but the curcumin-VE-Zn complex showed a more significant effect in reducing curcumin toxicity. Cytotoxicity tests showed that the addition of vitamin E reduced the cytotoxicity of the curcumin-VE complex, and the target product curcumin-VE-Zn complex had better biocompatibility compared to the intermediate curcumin-Zn complex and the curcumin-VE complex.

[0111] like Figure 5 As shown, the trends of cell scratch healing rates were consistent across groups at 12h and 24h. Compared to the normal group, radiation inhibited cell migration, but curcumin did not improve cell migration under X-ray exposure; instead, it increased the inhibitory effect of radiation on HaCaT cell migration. However, the curcumin-VE-Zn complex (Cur-VE-Zn) repaired the migration ability of radiation-affected HaCaT cells, significantly increasing the scratch healing rate, indicating that the curcumin-VE-Zn complex has a better effect on promoting cell scratch healing.

[0112] like Figure 6 As shown, curcumin improved radiation-induced cell migration, but the difference was not significant (P>0.05), while the number of HaCaT cells that migrated significantly increased under curcumin-VE-Zn complex incubation (P<0.01), indicating that the curcumin-VE-Zn complex is more effective in promoting cell migration.

[0113] like Figure 7As shown, compared with the control group, radiation induced the formation of γ-H2AX lesions. The γ-H2AX levels in the curcumin group (X-ray + Cur group) were lower than those in the radiation group (X-ray group) at 6h and 12h, but were higher in the curcumin group after 24h of intervention, suggesting that the effect of curcumin in alleviating radiation-induced DNA damage is unstable and may exacerbate DNA damage at 24h. The γ-H2AX content in the nuclei of HaCaT cells in the curcumin-VE-Zn complex group (X-ray + Cur-VE-Zn group) was significantly lower than that in the radiation group at 6h, 12h, and 24h, suggesting that the curcumin-VE-Zn complex does not cause DNA damage to cells and can reverse X-ray-induced DNA damage.

[0114] like Figure 8 The HaCaT cells treated with the radiation showed that they produced a large amount of ROS, while the ROS in the irradiated cells treated with curcumin and curcumin-VE-Zn complex were largely cleared. The curcumin-VE-Zn complex showed a more significant ROS clearing ability than curcumin.

[0115] like Figure 9 As shown, radiation increased the expression of inflammatory factors in HaCaT cells. The curcumin group (X-ray+Cur group) decreased the expression of IL-6 and IL-1β, but increased the expression of TNF-α. The curcumin-VE-Zn complex (X-ray+Cur-VE-Zn group) decreased the expression of IL-6, IL-1β, and TNF-α, indicating that both can inhibit radiation-induced inflammatory damage to some extent.

[0116] The above results indicate that the Cur-VE-Zn complex shows potential in treating radiation-induced oral mucositis by scavenging reactive oxygen species, reducing inflammation, promoting mucosal repair, and minimizing DNA damage.

[0117] Example 3: Characterization of the thermosensitive hydrogel loaded with curcumin complex (Cur-VE-Zn@gel)

[0118] I. Experimental Methods

[0119] 1. Sol-gel reaction of thermosensitive gel: The synthesized gel was placed in environments of 4℃ and 37℃ respectively, and the gel flow was observed.

[0120] 2. Rheological testing of the gel: The sol of the temperature-sensitive gel was subjected to rheological analysis using a rheometer to evaluate the gelation temperature of the gel.

[0121] 3. The prepared curcumin-VE-Zn gel (Cur-VE-Zn@gel) was placed in liquid nitrogen for rapid freezing and then vacuum freeze-dried to obtain a freeze-dried sample. After sputtering with gold, its internal structure and microstructure were observed by scanning electron microscopy.

[0122] 4. Swelling Test: Accurately weigh 100 mg (W0) of the freeze-dried blank gel and curcumin-VE-Zn gel, place them in 20 mL of phosphate buffered saline (PBS) (pH=7.4), and immerse them at 37℃. At different time points, blot the surface moisture of the gel with filter paper and weigh (WS) until the gel sample reaches swelling equilibrium and the mass no longer changes. Swelling rate calculation formula: Swelling rate = (WS – W0) / W0.

[0123] 5. Degradability Test: Accurately weigh 100 mg (W0) of the freeze-dried blank gel and curcumin-VE-Zn gel, place them in 20 mL of PBS (pH=7.4) solution, and incubate them in a constant temperature shaker (37℃, 100 r / min). Remove the hydrogels every 12 hours, freeze-dry them under vacuum, and record the weight (WS). Degradation rate calculation formula: Degradation rate = (W0 – WS) / W0.

[0124] 6. Adhesion Test: Simulating a moist oral environment, the adhesion of the blank gel and curcumin-VE-Zn gel was determined using an overlap shear test. Fresh pigskin was rigorously cleaned of surface hair and grease, cut into 60 mm × 15 mm pieces, and soaked in artificial saliva for 1 hour. 100 μL of the blank gel and curcumin-VE-Zn gel were evenly applied to the surfaces of two pigskin pieces, each with a coating area of ​​15 mm × 15 mm. After stabilization at 37°C for 1 hour, the adhesion properties were measured using a universal testing machine. The tensile speed was 5 mm / min, and the adhesion strength was expressed as the maximum tensile force (in N) divided by the overlap area of ​​the two pigskin pieces (in m²).

[0125] II. Experimental Results

[0126] like Figure 10 As shown, the sol-gel reaction of the gel is demonstrated. It is a liquid gel at 4°C and a solid gel when the temperature is raised to 37°C. This property enables the gel to be sprayed onto the wound during interventional treatment.

[0127] like Figure 11As shown, when the temperature is below 30.61℃, the G' (storage modulus) of the curcumin-VE-Zn gel is lower than the G" (loss modulus). However, as the temperature increases, under certain frequencies and strain values, both G' and G" increase with increasing temperature. But when the temperature reaches 30.61℃, G' begins to be higher than G", at which point the gel undergoes a sol-gel reaction, changing from a liquid sol to a solid gel. This indicates that the curcumin-VE-Zn gel is thermosensitive, and it can form a gel when the temperature rises to 30.61℃. That is, under oral temperature conditions, the curcumin-VE-Zn gel can adhere to the oral mucosa in a solid gel state.

[0128] like Figure 12 As shown, the internal microstructure of curcumin-VE-Zn gel after freeze-drying exhibits a porous network structure with pore sizes mainly ranging from 5 to 20 μm. This structure facilitates the entry and exit of small molecule drugs, water, and nutrients, which is beneficial for wound healing.

[0129] like Figure 13 As shown, the blank gel rapidly absorbed water within 4 hours, with a swelling rate exceeding 300%, and then reached swelling equilibrium. The results indicate that the blank gel has good swelling properties and can appropriately absorb and store water, while the loading of curcumin-VE-Zn complex has no significant effect on the swelling properties of the gel (P>0.05).

[0130] like Figure 14 As shown, the degradation rates of both gels exceeded 95% after 48 h, indicating that the addition of the curcumin-VE-Zn complex had no significant effect on the gel degradation rate (P>0.05).

[0131] like Figure 15 As shown, in a simulated moist oral environment, the adhesion strength of the blank gel was 13.3 kPa, and that of the curcumin-VE-Zn gel was 12.748 kPa. There was no statistically significant difference (P>0.05). Moreover, the adhesion strength of both gels was higher than that of common dressings (0-5 kPa), indicating that the gel can adhere stably in the oral environment and prolong the time of drug action on the wound.

[0132] The above results indicate that the Cur-VE-Zn gel is temperature-responsive and possesses good swelling, degradation, adhesion, and drug delivery capabilities.

[0133] Experiment Example 4: The therapeutic effect of a thermosensitive hydrogel loaded with curcumin complex (Cur-VE-Zn@gel) on RIOM mice

[0134] I. Experimental Methods

[0135] 1. Laboratory animals and grouping

[0136] Fifty 8-week-old male BALB / c mice weighing 21–23 g were purchased from Sichuan Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK (Sichuan) 2023-0040. Mice were randomly divided into four groups (n=10 per group): a normal group (blank), an X-ray irradiation group (X-ray group), a blank gel group (X-ray + gel group), a curcumin gel group (X-ray + Cur@gel group), and a curcumin-VE-Zn gel group (X-ray + Cur-VE-Zn@gel group). Mice were housed at the Animal Experiment Center of West China School of Basic Medical Sciences and Forensic Medicine, Sichuan University, under an ambient temperature of 20–26℃ and a relative humidity of 40%–70%. They were acclimatized for 7 days with free access to water and food. The animal experiments were approved by the Laboratory Animal Ethics Committee of West China Hospital, Sichuan University, ethics registration number: 20240618001.

[0137] 2. Establishment of the RIOM animal model

[0138] The RIOM model was established using a single high-dose radiation injection. After weighing, mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital (3.5 mL / kg), fixed in a supine position in a lead box with the head and neck exposed, and the rest of the body covered with a 5 mm thick lead plate to isolate ionizing radiation. The mice were then placed in an X-RAY small animal precision irradiation instrument for local radiotherapy of the head and neck. The radiation conditions were: voltage 160 kV, current 20 mA, total radiation dose 25 Gy, and dose rate 1.903 Gy / min. The control group was only anesthetized and did not receive radiation.

[0139] 3. Administration method

[0140] The day of radiation was recorded as day 0. Treatment intervention was initiated on day 1 post-radiation and continued for 7 consecutive days. The control group and X-ray irradiation group received standard feed and water, with no other treatment. The control gel group, curcumin gel group, and curcumin-VE-Zn gel group received gel intervention once daily at 10:00 AM and 6:00 PM, in addition to standard feed and water. The gel intervention methods for each group are as follows:

[0141] Blank gel group: 100 μL of blank gel was taken and thoroughly applied to the oral cavity of mice with a sterile cotton swab. After fasting and water restriction for 1 h, mice were allowed to drink water and eat freely.

[0142] Curcumin gel group: 100 μL of curcumin gel was applied to the oral cavity of mice using a sterile cotton swab. After fasting and water restriction for 1 h, mice were allowed to drink water and eat freely.

[0143] Curcumin-VE-Zn gel group: 100 μL of curcumin-VE-Zn gel was applied thoroughly to the oral cavity of mice using a sterile cotton swab. After fasting and water restriction for 1 h, mice were allowed to drink water and eat freely.

[0144] On day 8, mice were euthanized by intraperitoneal anesthesia and cervical dislocation, and the data were collected.

[0145] 4. General condition and weight of mice: The general condition of mice was observed at the same time on the day of radiation and every day after radiation, and the weight of mice was recorded.

[0146] 5. Measurement of ulcer area in mice: After mice were euthanized by cervical dislocation, their tongue tissue was immediately taken. The surface of the tongue tissue was cleaned with 1% acetic acid solution using a cotton swab, and 1% toluidine blue staining solution was evenly applied and left to stand for 5 minutes. Excess staining solution was thoroughly washed off with 1% acetic acid solution. After taking pictures, Image-J analysis was performed to determine the ratio of ulcer area, mucosal inflammation area to the total area of ​​tongue tissue.

[0147] 6. Histopathological observation: After toluidine blue staining, the tongue tissue was washed twice with PBS and fixed in 4% paraformaldehyde for 24-48 h. The tongue tissue was divided into two parts along the sagittal plane, dehydrated, embedded in paraffin, sectioned, stained with hematoxylin and eosin, dehydrated and mounted, observed and photographed under a microscope.

[0148] 7. Determination of Inflammatory Factor Levels in Mouse Tongue Tissue: ELISA was used to determine the levels of tumor necrosis factor-α (TNF-α), interleukin IL-1β, and IL-6 in tongue tissue. The minced tissue was mixed with pre-cooled PBS at a weight-to-volume ratio of 1:9, homogenized thoroughly in a tissue homogenizer, and centrifuged at 500×g for 10 min at 4°C to obtain a 10% homogenate supernatant. The supernatant was then used to determine the levels of each inflammatory factor according to the ELISA detection procedure.

[0149] 8. Determination of antioxidant levels in mouse tongue tissue: The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) content in mouse tongue tissue were measured. The shredded tissue was added to physiological saline at a weight-to-volume ratio of 1:9, homogenized thoroughly in a tissue homogenizer, centrifuged at 400×g for 10 min, and the supernatant was collected. The determination was performed strictly following the operating procedures in the instruction manual.

[0150] 9. Distribution and Expression of γ-H2AX in Mouse Tongue Tissue: Mouse tongue tissue was fixed, collected, dehydrated, embedded, sectioned, dewaxed, and antigen-retrieved. Endogenous peroxidase was blocked, and bovine serum albumin was added for blocking at room temperature for 20 min. The corresponding primary antibody (Nrf2 1:200) was diluted and incubated overnight at 4°C. After overnight incubation, the tissue was warmed, washed with PBS, and incubated with secondary antibody at 37°C for 30 min. After washing with PBS, diaminobenzidine was used for staining, and hematoxylin was used to counterstain the cell nuclei. After dehydration and mounting, the tissue was observed under a microscope. Three fields of view were randomly selected from each section for photography, and the number of positive cells was analyzed using Image-J. The phosphorylated histone H2AX (γ-H2AX) monoclonal antibody was purchased from Chengdu Zhengneng Biotechnology Co., Ltd.

[0151] II. Experimental Results

[0152] like Figure 16 As shown, the body weight of mice in all groups decreased sharply after X-ray irradiation. However, by day 5, the weight loss of mice in the curcumin gel group (X-ray + Cur@gel group) and the curcumin-VE-Zn gel group (X-ray + Cur-VE-Zn@gel group) began to slow down, with the curcumin-VE-Zn gel group showing the most significant reduction in body weight. On day 7 after irradiation, the total weight change in the X-ray irradiation group (-6.76g) was significantly greater than that in the normal group (-1.76g), while the total weight change in the curcumin-VE-Zn gel group (-4.35g) was lower than that in the X-ray irradiation group (-6.76g), the blank gel group (-6.12g), and the curcumin gel group (-5.62g), indicating that the curcumin-VE-Zn gel had the best effect in inhibiting radiation-induced weight loss in mice.

[0153] like Figure 17 As shown, the relative area of ​​tongue ulcers in mice increased significantly after X-ray irradiation. The blank gel, curcumin gel, and curcumin-VE-Zn gel all reduced ulcer occurrence to some extent, while almost no ulcers were observed in the curcumin-VE-Zn gel group. Similar to the trend of ulceration, the curcumin-VE-Zn gel most significantly reduced the severity of radiation-induced oral mucositis.

[0154] like Figure 18 As shown, the normal mouse tongue tissue had an intact epithelial structure, neatly arranged cells, and no inflammatory cell infiltration in the epithelium and lamina propria. In the X-ray irradiation group and the blank gel group, the integrity of the epithelial structure of the mouse tongue tissue disappeared, most of the keratinized epidermis was shed, the cells were arranged in a disordered manner, and a large number of inflammatory cells were infiltrated in the lamina propria. The curcumin gel group and the curcumin-VE-Zn gel group could reduce the loss of epithelial structure and reduce inflammatory cell infiltration. However, compared with the curcumin gel group, the curcumin-VE-Zn gel group had more basal cells, more neatly arranged cells, reduced inflammatory infiltration, and was morphologically closer to the normal mouse tongue tissue.

[0155] like Figure 19 As shown, the levels of TNF-α, IL-1β, and IL-6 were significantly increased in the X-ray irradiation group, while the blank gel group did not reduce the expression of these factors. In the curcumin gel group and the curcumin-VE-Zn gel group, the levels of TNF-α, IL-1β, and IL-6 in the curcumin-VE-Zn gel group were significantly lower than those in the curcumin gel group.

[0156] like Figure 20As shown, the activities of the three most representative antioxidant enzymes, SOD, CAT, and GSH-Px, in the curcumin-VE-Zn gel group were significantly higher than those in the X-ray irradiation group, the blank gel group, and the curcumin gel group, while the level of lipid peroxidation product MDA was significantly lower than that in the other groups.

[0157] like Figure 21 As shown, the percentage of epithelial cells expressing γ-H2AX positive was as high as 100% in the X-ray irradiation group and the blank gel group. The proportion of γ-H2AX positive cells decreased in the curcumin gel group and the curcumin-VE-Zn gel group, with the curcumin-VE-Zn gel group showing the lowest γ-H2AX expression. This indicates that in the animal model, the curcumin-VE-Zn gel has the strongest ability to resist DNA damage after X-ray irradiation.

[0158] The above results indicate that curcumin-VE-Zn gel can help improve radiation-induced weight loss, oral mucosal ulcers, oxidative damage, inflammatory damage, and DNA damage in mice, and promote the treatment of radiation-induced oral mucositis.

[0159] In summary, this invention prepares a curcumin complex (Cur-VE-Zn), wherein the addition of vitamin E or its derivatives improves the solubility and reduces the toxicity of the curcumin-Zn complex, resulting in low cytotoxicity and good biocompatibility of Cur-VE-Zn. The curcumin complex of this invention exerts its therapeutic effect on radiation-induced oral mucositis by scavenging reactive oxygen species, reducing inflammatory responses, promoting mucosal repair, and reducing DNA damage. This invention also prepares a thermosensitive hydrogel using the curcumin complex as the main component, improving drug utilization and enabling targeted delivery of the curcumin complex to the oral mucosa, thereby exhibiting better therapeutic effects and providing a new strategy for the treatment of RIOM with promising application prospects.

Claims

1. A curcumin complex Cur-VE-Zn, characterized in that, It is made of raw materials in the following weight parts: Curcumin-vitamin E complex 4-4.8 parts, Zinc source 2-2.4 parts; The curcumin-vitamin E complex is made of raw materials in the following weight parts: Vitamin E or α-tocopherol succinate 470-590 parts, Dehydrating agent 370-450 parts, Curcumin 330-410 parts, Catalyst 55-67 parts; The dehydrating agent is selected from dicyclohexyl diimine; the catalyst is selected from 4-dimethylamino pyridine; and the zinc source is selected from zinc acetate dihydrate.

2. The curcumin complex Cur-VE-Zn according to claim 1, characterized by: It is made of raw materials in the following weight parts: Curcumin-vitamin E complex 4.4 parts, Zinc source 2.2 parts; The curcumin-vitamin E complex is made of raw materials in the following weight parts: Vitamin E or α-tocopherol succinate 530.78 parts, Dehydrating agent 412.66 parts, Curcumin 368.38 parts, Catalyst 61.09 parts.

3. A process for the preparation of the curcumin complex Cur-VE-Zn according to claim 1 or 2, characterized in that: It comprises the following steps: Step 1, vitamin E or α-tocopherol succinate is reacted with a dehydrating agent to obtain mixture 1; Step 2, curcumin is reacted with a catalyst to obtain mixture 2; Step 3, mixture 1 is reacted with mixture 2 to obtain a curcumin-VE complex; Step 4, the curcumin-VE complex is reacted with a zinc source, and the curcumin complex Cur-VE-Zn is obtained.

4. The method of claim 3, wherein: In step 1, the solvent for the reaction is dichloromethane; And / or, in step 1, the temperature for the reaction is 20-30℃, and the reaction time is 30-60 min; And / or, in step 2, the solvent for the reaction is dichloromethane; And / or, in step 3, the temperature for the reaction is 3-5℃, and the reaction time is 6-8h; And / or, in step 4, the solvent for the reaction is ethanol; And / or, in step 4, the temperature for the reaction is 50-70℃, and the reaction time is 1-2h.

5. Use of the curcumin complex of claim 1 or 2 in the preparation of a medicament for preventing and / or treating radiation-induced oral mucositis.

6. A pharmaceutical composition, characterized by: It is a preparation prepared by taking the curcumin complex Cur-VE-Zn of claim 1 or 2 as an active ingredient and adding pharmaceutically acceptable adjuvants.

7. The pharmaceutical composition according to claim 6, characterized in that: The preparation is at least one of a nano-preparation, a cream, a gel, and a gum.

8. The pharmaceutical composition according to claim 6, characterized by: The pharmaceutical composition is made of raw materials in the following weight parts: Curcumin complex Cur-VE-Zn 15-25 parts, Surfactant 100-110 parts, Matrix material 20-30 parts, Gel forming agent 1240-1260 parts; The surfactant is selected from Tween 80; And / or, the matrix material is selected from chitosan; And / or, the gel forming agent is selected from at least one of poloxamer F127 and poloxamer F68.

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