Quercetin nanogel for treating animal kidney injury and preparation method of quercetin nanogel

Abstract

The invention discloses a quercetin nanogel for treating animal kidney injury. The quercetin nanogel is prepared from the following raw materials in percentage by mass: 0.2-3% of quercetin; 0.1 to 4% of 4-formylphenylboronic acid; 0.5 to 5% of carboxymethyl chitosan; 1-10% of absolute ethyl alcohol; and the balance of sterile water. The quercetin nanogel with an imine borate bond is formed through 4-formylphenylboronic acid and carboxymethyl chitosan on the basis of a one-pot method for the first time and is used for treating animal kidney injury. According to the quercetin nanogel disclosed by the invention, the oxidation resistance of the quercetin is enhanced; the targeting effect of quercetin in renal injury is improved; and the clinical curative effect on animal kidney injury is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary drug preparation, and particularly relates to a quercetin nanogel for treating animal kidney damage and a preparation method thereof. Background Art

[0002] Animal renal injury is a common disease in veterinary clinics. It is characterized by metabolic disorders and oliguria or anuria, which greatly threatens the healthy growth of animals. Studies have shown that renal injury is often accompanied by oxidative stress. When animals suffer from renal injury, the level of antioxidant stress in the body decreases, and the concentration of lipid peroxidation products increases, resulting in a large accumulation of oxygen free radicals in the body, which makes renal injury more severe through oxidative stress.

[0003] As a flavonol compound, quercetin has multiple biological activities such as antioxidant and anti-inflammatory, which are closely related to its antioxidant effect. Its antioxidant effects mainly include direct scavenging of reactive oxygen free radicals (ROS), integrating metal ions, and inhibiting LDL and DNA oxidative damage. Because quercetin is rich in phenolic hydroxyl groups, it can protect cells and tissues from the attack of electrophilic reagents and oxygen free radicals, thereby effectively scavenging oxygen free radicals (ROS). Although quercetin has antioxidant effects, it lacks targeted release in the body, resulting in weak antioxidant effects in the body, which limits its clinical application. Summary of the invention

[0004] In order to enhance the antioxidant capacity and targeting effect of quercetin and improve the therapeutic effect on animal renal injury, the present invention uses quercetin as a material, and forms a quercetin nanogel with an imide borate ester bond through 4-formylphenylboronic acid and carboxymethyl chitosan, which is used to treat animal renal injury. The antioxidant capacity of quercetin is enhanced, and the targeting effect and clinical efficacy of quercetin in renal injury are improved.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A quercetin nanogel for treating animal kidney damage is prepared from the following raw materials in percentage by mass: 0.2-3% quercetin; 0.1-4% 4-formylphenylboronic acid; 0.5-5% carboxymethyl chitosan; 1-10% ethanol; and the balance is water.

[0007] Preferably, the invention is prepared from the following raw materials in percentage by mass: 0.5-2% quercetin; 0.5-2% 4-formylphenylboronic acid; 1-4% carboxymethyl chitosan; 3-8% ethanol; and the balance is water.

[0008] Preferably, the invention is prepared from the following raw materials in percentage by mass: 0.5-1% quercetin; 0.25-1% 4-formylphenylboronic acid; 1-2% carboxymethyl chitosan; 3-5% ethanol; and the balance is water.

[0009] More preferably, it is made of the following raw materials in percentage by mass: 1% quercetin; 0.5% 4-formylphenylboronic acid; 1% carboxymethyl chitosan; 5% ethanol; and the balance is water.

[0010] Most preferably, the ethanol is anhydrous ethanol and the water is sterile water.

[0011] The preparation method of the quercetin nanogel comprises the following steps:

[0012] (1) adding the quercetin into ethanol and stirring to dissolve to obtain solution 1;

[0013] (2) adding the 4-formylphenylboronic acid into water and stirring to dissolve it to obtain solution 2;

[0014] (3) adding the carboxymethyl chitosan into water and stirring to dissolve to obtain solution 3;

[0015] (4) At the same time, solution 1 and solution 2 are slowly added dropwise to solution 3, heated at 75-85° C. (0.5-2 h), and stirred to form a nanogel, thereby obtaining quercetin nanogel.

[0016] The beneficial effects of the present invention are:

[0017] The present invention forms a quercetin nanogel with an iminoborate bond based on a one-pot method for the first time; the iminoborate bond can be targeted for cleavage under oxidative conditions, thereby releasing the drug in a targeted manner, thereby improving the targeted effect of quercetin in renal injury, enhancing the antioxidant capacity of quercetin, and improving the clinical efficacy of quercetin on animal renal injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings required for use are briefly introduced below.

[0019] Figure 1 is a schematic diagram of the preparation of quercetin nanogel and the treatment of kidney injury in Example 4;

[0020] Figure 2 is a transmission electron microscopic diagram of the quercetin nanogel in Example 4;

[0021] Figure 3 is a schematic diagram of the particle size distribution of quercetin nanogel in Example 4;

[0022] Figure 4 is a schematic diagram of the Zeta potential distribution of the quercetin nanogel in Example 4;

[0023] Figure 5 Schematic diagram of ·OH removal rate of quercetin bulk drug solution and quercetin nanogel in Example 4;

[0024] Figure 6 is a schematic diagram of the in vitro release of quercetin nanogel in simulated oxidation conditions in Example 4;

[0025] Figure 7 This is a schematic diagram of B-mode ultrasound examination of the mouse renal injury model in the negative control group of the treatment experiment;

[0026] Figure 8 This is a schematic diagram of B-mode ultrasound examination of the mouse renal injury model in the positive control group of the treatment experiment;

[0027] Fig. 9 This is a schematic diagram of B-mode ultrasound examination of the mouse renal injury model treated with quercetin API solution in the treatment trial;

[0028] Fig.10 This is a schematic diagram of B-mode ultrasound examination of the quercetin nanogel in the treatment of the mouse renal injury model in the treatment trial;

[0029] Fig.11 This is a schematic diagram of the tissue sections of the mouse renal injury model in the negative control group of the treatment experiment;

[0030] Fig.12 This is a schematic diagram of the tissue sections of the mouse kidney injury model in the positive control group of the treatment experiment;

[0031] Fig.13 This is a schematic diagram of tissue sections of the infected mouse renal injury model treated with quercetin API in the treatment trial;

[0032] Fig.14 This is a schematic diagram of tissue sections of the mouse renal injury model treated with quercetin nanogel in the therapeutic trial. DETAILED DESCRIPTION

[0033] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0034] Example 1

[0035] A quercetin nanogel for treating animal kidney damage is composed of the following raw materials in percentage by mass: 2% quercetin; 1% 4-formylphenylboronic acid; 2% carboxymethyl chitosan; 10% anhydrous ethanol; and the balance is sterile water. The preparation method specifically comprises the following steps:

[0036] (1) adding the quercetin into anhydrous ethanol, stirring and dissolving to obtain solution 1;

[0037] (2) adding the 4-formylphenylboronic acid to sterile water, stirring and dissolving, to obtain solution 2;

[0038] (3) adding the carboxymethyl chitosan into sterile water and stirring to dissolve to obtain solution 3;

[0039] (4) At the same time, solution 1 and solution 2 were slowly added dropwise to solution 3, heated at 80° C. for 1 h, and stirred to form a nanogel, thereby obtaining quercetin nanogel.

[0040] Example 2

[0041] A quercetin nanogel for treating animal kidney damage is composed of the following raw materials in percentage by mass: 1.5% quercetin; 0.75% 4-formylphenylboronic acid; 1.5% carboxymethyl chitosan; 7.5% anhydrous ethanol; and the balance is sterile water. The preparation method is as in Example 1.

[0042] Example 3

[0043] A quercetin nanogel for treating animal kidney damage is composed of the following raw materials in percentage by mass: 2% quercetin; 2% 4-formylphenylboronic acid; 4% carboxymethyl chitosan; 8% anhydrous ethanol; and the balance is sterile water. The preparation method is as described in Example 1.

[0044] Example 4

[0045] A quercetin nanogel for treating animal kidney damage is composed of the following raw materials in percentage by mass: 1% quercetin; 0.5% 4-formylphenylboronic acid; 1% carboxymethyl chitosan; 5% anhydrous ethanol; and the balance is sterile water. The preparation method is as in Example 1.

[0046] Example 5

[0047] A quercetin nanogel for treating animal kidney damage is composed of the following raw materials in percentage by mass: 0.5% quercetin; 0.5% 4-formylphenylboronic acid; 1% carboxymethyl chitosan; 3% anhydrous ethanol; and the balance is sterile water. The preparation method is as in Example 1.

[0048] Example 6

[0049] A quercetin nanogel for treating animal kidney damage is composed of the following raw materials in percentage by mass: 1% quercetin; 0.25% 4-formylphenylboronic acid; 1% carboxymethyl chitosan; 5% anhydrous ethanol; and the balance is sterile water. The preparation method is as in Example 1.

[0050] Example 7

[0051] A quercetin nanogel for treating animal kidney damage is composed of the following raw materials in percentage by mass: 1% quercetin; 1% 4-formylphenylboronic acid; 1% carboxymethyl chitosan; 3% anhydrous ethanol; and the balance is sterile water. The preparation method is as in Example 1.

[0052] Treatment trials:

[0053] 1. Take an appropriate amount of the nanogels in Examples 1-7 and observe whether the prepared nanogels have uniform color and whether they form a gel. Among them, Example 1 has uneven color and does not form a gel; Examples 2-3 have uniform color but do not form a gel; the quercetin nanogels prepared in Examples 4-7 have good uniformity and can form a stable gel, which can be used as a therapeutic test agent for the subsequent mouse renal injury model.

[0054] 2. Morphological observation: After the quercetin nanogel obtained in Example 4 was diluted 100 times, its microscopic morphology was observed by transmission electron microscopy. The results showed that the prepared quercetin nanogel was light yellow with uniform color, and no floccules or precipitation were found. Under transmission electron microscopy, the quercetin nanogel was spherical with uniform size and uniform distribution, as shown in FIG. Figure 2 shown.

[0055] 3. Determination of Zeta potential and particle size: After the quercetin nanogel prepared in Example 4 was diluted 100 times, its Zeta potential and particle size were measured by Malvern nano laser particle size analyzer, and the average value was obtained by repeating 3 times. The results showed that the average particle size and Zeta potential of the quercetin nanogel were 66.5±4.8nm and 25.7±1.9mV, respectively. Figure 3 and Figure 4 shown.

[0056] 3. Determination of ·OH clearance rate: Reaction system: 0.50 mL of 9 mmol / L FeSO4 solution, 0.50 mL of 9 mmol / L salicylic acid ethanol solution and 2.50 mL of quercetin bulk drug solution or quercetin nanogel prepared in Example 4. After mixing evenly, add 0.5 mL of 8.8 mmol / L H2O2 and place in a 37 ° C water bath for 15 minutes. After taking out and cooling to room temperature, measure its absorbance at 510 nm, and calculate its ·OH clearance rate. The ·OH clearance rates of quercetin bulk drug solution and quercetin nanogel are 34.3% ± 1.8% and 88.3% ± 0.9%, respectively. Figure 5 As shown, the quercetin nanogel prepared in Example 4 enhances the antioxidant capacity of quercetin.

[0057] 4. In vitro release: The quercetin nanogel prepared in Example 4 was placed in a medium containing H2O2 or not containing H2O2, and the release rate of quercetin was calculated at different time points. The results showed that in the medium containing H2O2, 92.1%±4.0% of quercetin was released at 4h; in the medium without H2O2, only 56.9%±3.8% of quercetin was released at 4h, and only 64.0%±4.0% of quercetin was released at 24h. Figure 6 As shown, the quercetin nanogel prepared in Example 4 can improve the targeting effect of quercetin under oxidative conditions through the imide boronate bond.

[0058] 7. Therapeutic test of mouse renal injury model:

[0059] 1. Materials and Methods

[0060] 1.1 Drugs: Quercetin bulk drug solution (1%), quercetin nanogel (1%).

[0061] 1.2 Experimental Animals Twenty-four clean grade (ICR) Kunming mice were purchased from the Animal Experiment Station of Tarim University.

[0062] 1.3 Instruments: laser nanoparticle size analyzer, heat-collecting constant temperature magnetic stirrer, heating magnetic stirrer, freeze dryer, variable vacuum ultra-high resolution field emission scanning electron microscope, UV-visible spectrophotometer, nanoparticle size and Zeta potential analyzer, full digital color veterinary ultrasound diagnostic system.

[0063] 1.4 Test methods

[0064] 1.4.1 Establishment of mouse renal injury model

[0065] Twenty-four mice weighing 20±0.3g were randomly divided into four groups (① negative control group, ② positive control group, ③ quercetin API solution treatment group, ④ quercetin nanogel treatment group), with 6 mice in each group. The mice in group 1 were not infected (negative control group). Each mouse in groups 2-4 was inoculated with adenine suspension and infected for 7 consecutive days to construct a mouse renal injury model. The clinical manifestations of mice with renal injury were decreased drinking water and difficulty urinating; B-ultrasound examination results showed that the edges of the mouse's renal capsule were unclear and rough, the morphology and structure changed significantly, and the kidney diameter became larger; through tissue section observation, it was found that the mouse's renal tubules and glomeruli had severe pathological changes, glomerular atrophy, tubular cavity enlargement, and disordered tissue structure. The mice in the negative control group drank water normally, urinated normally, and no obvious clinical symptoms appeared; B-ultrasound examination results showed that the mouse's renal capsule was clear and intact, and the contrast between the cortex and medulla was clear; through tissue section observation, it was found that the mouse's renal tubules and glomeruli did not undergo pathological changes and the structure was intact. Therefore, it was concluded that the mouse renal injury model was successfully established.

[0066] 1.4.2 Treatment Experiments

[0067] Group 1 was not infected and no treatment was performed (negative control group). Group 2 infected mice and did not perform any treatment (positive control group). Group 3 treated the mouse renal injury model with quercetin bulk drug solution. Group 3 treated the mouse renal injury model with quercetin nanogel. The treatment was continued for 14 days, and the clinical symptoms of the mice were observed and recorded. On the 14th day, the kidneys of each group of mice were examined by B-mode ultrasound, and the kidneys of each group of mice were collected for tissue section (HE staining) observation.

[0068] 1.4.3 Result determination

[0069] Determination of clinical symptoms: If after treatment, the infected mice drink water normally, urinate normally, and show no obvious clinical symptoms, the treatment is considered successful from a clinical perspective.

[0070] B-mode ultrasound examination judgment: The results of B-mode ultrasound examination showed that the renal capsule of the mice was clear and intact, and the contrast between the cortex and medulla was clear and distinct. The treatment was judged to be successful from the perspective of B-mode ultrasound examination.

[0071] Tissue section judgment: Tissue section observation revealed that the renal tubules and glomeruli of the mice did not undergo pathological changes and their structures were intact, so the treatment was judged to be successful from the perspective of tissue sections.

[0072] 1.5 Test results

[0073] The mice in the negative control group drank water and urinated normally, and showed no obvious clinical symptoms. The results of B-mode ultrasound examination showed that the renal capsule of the mice was clear and intact, and the contrast between the cortex and medulla was clear. Figure 7 ; Through tissue section observation, it was found that the renal tubules and glomeruli of mice did not undergo pathological changes and their structures were intact, such as Fig.11 Compared with the negative control group, the mice in the positive control group showed clinical symptoms such as decreased water intake and difficulty urinating. The results of B-mode ultrasound examination showed that the edges of the mice's kidney capsules were unclear and rough, the morphology and structure changed significantly, and the kidney diameter became larger. Figure 8 ; Through tissue section observation, it was found that the renal tubules and glomeruli of mice had serious pathological changes, glomerular atrophy, enlarged tubular cavities, and disordered tissue structure, such as Fig.12 .

[0074] After the mouse renal injury model was treated with quercetin API solution and quercetin nanogel, drinking water returned to normal, urination was normal, and no obvious clinical symptoms appeared, which was considered a clinical success. The results of B-mode ultrasound examination showed that the renal capsule of the mouse was clear and intact, and the contrast between the cortex and medulla was clear, which was considered a B-mode ultrasound examination success, respectively. Fig. 9 and Fig.10As shown in the figure, the observation of tissue sections showed that the renal tubules and glomeruli of the mice did not undergo pathological changes and their structures were intact. The treatment was successful based on the perspective of tissue sections, as shown in the figure. Fig.13 and Fig.14 shown.

[0075] The cure rate of group 3 was 33.3%, and the cure rate of group 4 was 83.3%, which indicated that quercetin nanogel could improve the clinical efficacy of quercetin on animal renal injury.

[0076] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A quercetin nanogel for treating animal kidney damage, characterized in that: The invention is prepared from the following raw materials in percentage by mass: 0.2-3% of quercetin; 0.1-4% of 4-formylphenylboronic acid; 0.5-5% of carboxymethyl chitosan; 1-10% of ethanol; and the balance is water.

2. The quercetin nanogel according to claim 1, characterized in that The invention is prepared from the following raw materials in percentage by mass: 0.5-2% of quercetin; 0.5-2% of 4-formylphenylboronic acid; 1-4% of carboxymethyl chitosan; 3-8% of ethanol; and the balance is water.

3. The quercetin nanogel according to claim 1, characterized in that The invention is prepared from the following raw materials in percentage by mass: 0.5-1% of quercetin; 0.25-1% of 4-formylphenylboronic acid; 1-2% of carboxymethyl chitosan; 3-5% of ethanol; and the balance is water.

4. The quercetin nanogel according to claim 1, characterized in that The invention is prepared from the following raw materials in percentage by mass: 1% of quercetin; 0.5% of 4-formylphenylboronic acid; 1% of carboxymethyl chitosan; 5% of ethanol; and the balance of water.

5. The quercetin nanogel according to claim 1, characterized in that: The ethanol is anhydrous ethanol, and the water is sterile water.

6. The method for preparing the quercetin nanogel according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) adding the quercetin into ethanol and stirring to dissolve to obtain solution 1; (2) adding the 4-formylphenylboronic acid into water and stirring to dissolve it to obtain solution 2; (3) adding the carboxymethyl chitosan into water and stirring to dissolve to obtain solution 3; (4) At the same time, solution 1 and solution 2 are slowly added dropwise to solution 3, heated, and stirred to form a nanogel, thereby obtaining quercetin nanogel.

7. The preparation method according to claim 6, characterized in that: The heating is performed at 75-85° C. for 0.5-2 h.

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