Honokiol nanoparticle temperature-sensitive gel as well as preparation method and application thereof

Abstract

The invention discloses a temperature-sensitive gel which is prepared from the following raw materials in percentage by mass volume: 0.5-1.5% of a mixture of lentinan and chitosan, 10-20% of poloxamer 407 and the balance of an aqueous solution with the pH value of 7-7.5. The temperature-sensitive gel disclosed by the invention is in a liquid state at room temperature, is rapidly gelatinized to form semi-solid gel at the mammary gland temperature of 36.5 DEG C, and has high retention property and a good sustained and controlled release effect. The medicine is specifically administered to the mammary gland in an injection manner, so that the carried medicine can be adhered to the mucous membrane of the mammary gland, directly reaches the disease site and is not easy to flow out, and the utilization rate of the medicine is improved. When the temperature-sensitive gel is used for carrying honokiol nanoparticles, in-vitro tests show that the temperature-sensitive gel has a better bacteriostatic effect than free honokiol and honokiol nanoparticles, has a more remarkable curative effect when being applied to prevention and treatment of animal mastitis, and has actual popularization and application values.

Description

Technical Field

[0001] The invention relates to a honokiol nanoparticle thermosensitive gel and a preparation method and application thereof, belonging to the field of pharmaceutical preparations. Background Art

[0002] Animal mastitis is an inflammation of the mammary glands of animals, which is mainly manifested by redness, swelling, heat and pain in the udder and significant changes in milk. It is a complex disease that causes the most serious economic losses to the animal husbandry industry, especially the dairy industry, and no complete solution has been proposed so far.

[0003] At present, mastitis is mainly treated with antibiotics, and vaccines are also used to prevent mastitis, such as vaccines to prevent E. coli mastitis. However, due to the inconsistency of pathogenic E. coli strains and the high probability of genetic variation, it is difficult to effectively control E. coli mastitis using a single strain of vaccine. Antibiotic treatment is prone to drug resistance, which is transmitted to humans through the food chain and affects the health of the public, so it is not easy to use as a common drug.

[0004] Honokiol is a natural bisphenol compound extracted from the Chinese herbal medicine Magnolia officinalis, which has multiple biological activities, including anti-inflammatory, antibacterial, antioxidant, and anti-tumor effects. Patent CN118453692A discloses that honokiol can be used for the prevention and treatment of bovine mastitis. However, honokiol is poorly soluble in water and has poor stability. After oral administration, it is less absorbed and has low bioavailability after absorption. It is necessary to provide a dosage form of honokiol for local mammary administration to improve the efficacy of honokiol in treating animal mastitis. Summary of the invention

[0005] To achieve the above object, the present invention provides a thermosensitive gel, which is prepared from the following raw materials in mass volume percentage: Lentinan and carboxymethyl chitosan mixture 0.5-1.5%, poloxamer 407 10-20%, and the rest is aqueous solution with pH 7-7.5; The mass ratio of lentinan to carboxymethyl chitosan is 1-3:1-3.

[0006] Furthermore, it is prepared from the following raw materials in mass volume percentage: Lentinan and carboxymethyl chitosan mixture 1%, poloxamer 407 16%, and the rest is aqueous solution with pH 7-7.5; Furthermore, the mass ratio of Lentinan to carboxymethyl chitosan is 1:1.

[0007] The present invention also provides a use of the aforementioned thermosensitive gel as a drug matrix for local administration.

[0008] Furthermore, the drug matrix is ​​a matrix for local administration to the mammary gland.

[0009] The invention also provides a medicine for preventing and / or treating animal mastitis, which is a thermosensitive gel loaded with honokiol nanoparticles.

[0010] Furthermore, it is prepared from the following raw materials in mass volume percentage: Lentinan and carboxymethyl chitosan mixture 0.5-1.5%, poloxamer 407 10-20%, and the rest is honokiol nanoparticle solution; The mass ratio of Lentinan to carboxymethyl chitosan is 1-3:1-3; The honokiol nanoparticle solution is obtained by homogenizing a PBS buffer solution containing whey protein and an ethanol solution containing honokiol, removing the ethanol and filtering to obtain the nanoparticle solution.

[0011] The present invention also provides a method for preparing the aforementioned medicine, comprising the following steps: 1) Take whey protein, add PBS sustained-release solution and ultrasonically dissolve it to obtain whey protein solution; take magnolia officinalis, add anhydrous ethanol to dissolve it, and obtain magnolia officinalis ethanol solution; 2) taking the honokiol ethanol solution obtained in step 1), dropping it into the whey protein solution obtained in step 1), removing the ethanol and then homogenizing, filtering, and taking the filtrate to obtain the honokiol nanoparticle solution; 3) Take a mixture of Lentinan and carboxymethyl chitosan, add the honokiol nanoparticle solution obtained in step 1) according to the ratio to dissolve, then add poloxamer 407, stir and disperse evenly, and completely swell until there are no bubbles.

[0012] Furthermore, in step 1), the pH value of the PBS buffer is 7.0-7.5, the concentration of the whey protein solution is 4 mg / mL; the ethanol is anhydrous ethanol, and the concentration of the honokiol ethanol solution is 4 mg / mL; and / or, the volume ratio of the honokiol ethanol solution obtained in step 2) to the whey protein solution is 1:10; the dripping speed is 1 ml / min; or, the homogenization is ultrasonic homogenization; or, the filtration is filtering with a 0.22 μm microporous filter membrane; And / or, the step 3) of completely swelling until there are no bubbles is to stand in a 4° C. environment until there are complete swelling and no bubbles.

[0013] Finally, the present invention provides a use of the aforementioned drug in the preparation of a drug for preventing and / or preventing animal mastitis; the mastitis includes Escherichia coli mastitis.

[0014] The "drugs" mentioned in the present invention include drugs for human use and drugs for animals, with veterinary drugs being the most suitable.

[0015] The "mass volume percentage" mentioned in the present invention refers to the percentage of the mass g of the solute in the solution to the volume ml of the solution.

[0016] The thermosensitive gel of the present invention is liquid at room temperature, and rapidly gels to form a semisolid gel at a mammary gland temperature of 36.5°C, has high retention, and has a good sustained-release effect. The drug is administered to the mammary gland in a targeted manner by injection, so that the drug carried by the gel can adhere to the mammary gland mucosa, directly reach the diseased site, and is not easy to flow out, thereby improving the utilization rate of the drug. The thermosensitive gel of the present invention is used to carry honokiol nanoparticles, and in vitro tests show that the gel has a better antibacterial effect than free honokiol and honokiol nanoparticles, and is applied to the prevention and treatment of animal mastitis, especially Escherichia coli mastitis, with more significant therapeutic effects, and has practical promotion and application value.

[0017] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0018] The above contents of the present invention are further described in detail below through specific implementation methods in the form of examples. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Effects on mouse mammary gland histopathology (HE staining, n=3, scale bar 20 μm); Figure 2 IL-1β, IL-6, and TNF-α contents in mammary gland tissues of mice in each group (x±s, n=6); Figure 3 MPO content in mammary gland tissue of mice in each group (x±s, n=6); Figure 4 Effect of P407 dosage on gelation temperature Figure 5 HK-NS-Gel needle-passing property; Figure 6 SEM image of HK-NS-Gel; Figure 7 HK-NS-Gel rheology diagram; Figure 8 In vitro release profiles of nanoparticle solution and HK-NS-Gel; Fig. 9 DPPH free radical scavenging ability; Fig.10Antibacterial ability of Oxford cup antibacterial method (A. blank control group, B. HK (1 mg / mL) group, C. HWN (1 mg / mL) group, D. HK-NS-Gel group, E. blank gel group) DETAILED DESCRIPTION

[0020] The raw materials, reagents and equipment used in the specific implementation of the present invention are all purchased from the market, wherein Lentinan is purchased from Shanghai Yuanye Biotechnology Co., Ltd., with product specifications: UV detection content 40%.

[0021] Example 1 Preparation of Honokiol Nanoparticle Thermosensitive Gel 1) Take 40 mg of whey protein, add 10 mL of PBS buffer (pH 7.4) and ultrasonically dissolve to obtain a whey protein solution; take 4 mg of honokiol, add 1 mL of anhydrous ethanol to dissolve to obtain a honokiol ethanol solution; stir the honokiol ethanol solution and drip it into the whey protein solution at a rate of 1 ml / min, remove the ethanol and ultrasonically homogenize, then filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain a honokiol nanoparticle solution; 2) Mix 75 mg of Lentinan and 75 mg of carboxymethyl chitosan, add 10 ml of the honokiol nanoparticle solution obtained in step 1) to dissolve, then add poloxamer 407 to a concentration of 16% (g / ml), stir and disperse evenly, and finally completely swell in a 4°C environment to obtain the product.

[0022] Example 2 Preparation of Honokiol Nanoparticle Thermosensitive Gel 1) Take 40 mg of whey protein, add 10 mL of PBS buffer (pH 7.4) and ultrasonically dissolve to obtain a whey protein solution; take 4 mg of honokiol, add 1 mL of anhydrous ethanol to dissolve to obtain a honokiol ethanol solution; stir the honokiol ethanol solution and drip it into the whey protein solution at a rate of 1 ml / min, remove the ethanol and ultrasonically homogenize, then filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain a honokiol nanoparticle solution; 2) Mix 75 mg of Lentinan and 75 mg of carboxymethyl chitosan, add 10 ml of the honokiol nanoparticle solution obtained in step 1) to dissolve, then add poloxamer 407 to a concentration of 10% (g / ml), stir and disperse evenly, and finally completely swell in a 4°C environment to obtain the product.

[0023] Example 3 Preparation of Honokiol Nanoparticle Thermosensitive Gel 1) Take 40 mg of whey protein, add 10 mL of PBS buffer (pH 7.4) and ultrasonically dissolve to obtain a whey protein solution; take 4 mg of honokiol, add 1 mL of anhydrous ethanol to dissolve to obtain a honokiol ethanol solution; stir the honokiol ethanol solution and drip it into the whey protein solution at a rate of 1 ml / min, remove the ethanol and ultrasonically homogenize, then filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain a honokiol nanoparticle solution; 2) Mix 25 mg of Lentinan and 25 mg of carboxymethyl chitosan, add 10 ml of the honokiol nanoparticle solution obtained in step 1) to dissolve, then add poloxamer 407 to a concentration of 20% (g / ml), stir and disperse evenly, and finally completely swell in a 4°C environment to obtain the product.

[0024] The beneficial effects of the present invention are specifically described below through test examples.

[0025] Test Example 1 Study on the efficacy of honokiol and its preparations 1. Methods 1. Study on the efficacy of honokiol against LPS mastitis 1.1 Grouping and modeling Sixty female ICR mice and 20 male ICR mice were housed in a ratio of 3:1 after one week of adaptive feeding. They were provided with sufficient water and feed. The pregnancy status was determined by checking the mating plug and recording the daily body weight. All mice were kept in an indoor environment with a temperature of 5±0.5℃, a relative humidity of 55±5% and a 12-h light cycle. During the experiment, all mice had free access to food and water. Female mice were housed in single cages after pregnancy. The lactating mice 5-7 days after delivery were randomly divided into a blank group, a model group, a low-dose honokiol group (20 mg / kg), a medium-dose honokiol group (40 mg / kg), a high-dose honokiol group (60 mg / kg), and a positive control group (dexamethasone 5 mg / kg), with 10 mice in each group. Honokiol was intraperitoneally injected into each group for three days. The blank control group and the lipopolysaccharide (LPS) model group were intraperitoneally injected with an equal volume of normal saline, and the positive control group was intraperitoneally injected with dexamethasone. One hour after the last administration, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital, and 50 μl of LPS at a concentration of 0.2 mg / mL was injected into the mammary ducts of both nipples using a microinjection needle. 12 hours after modeling, the administration was repeated, and the mice were killed 24 hours later.

[0026] Drug treatment (1) LPS stock solution Weigh 0.2 mg LPS and accurately add 1 mL of physiological saline with a pipette to prepare a concentration of 0.2 mg / mL. Use a pipette to blow repeatedly to dissolve it completely, and prepare it fresh each time.

[0027] (2) Positive drugs Take 1 mL of dexamethasone (DEX) injection (5 mg) and accurately add 9 mL of normal saline using a pipette to dilute to 10 mL, with a concentration of 0.5 mg / mL.

[0028] (3) Honokiol suspension Low-dose group: accurately weigh 2 mg of honokiol and add it to 1 mL of saline with a pipette to prepare a concentration of 2 mg / mL. Medium-dose group: accurately weigh 4 mg of honokiol and add it to 1 mL of saline with a pipette to prepare a concentration of 4 mg / mL. High-dose group: accurately weigh 6 mg of honokiol and add it to 1 mL of saline with a pipette to prepare a concentration of 6 mg / mL.

[0029] Animal tissue sample processing and collection All mice in the six groups were killed after 24 h, and the skin was cut open along the midline of the abdomen to expose the fourth pair of mammary glands. The mammary gland lesions were observed and the fourth pair of mammary gland tissues were peeled off. One part was rinsed with ice PBS, wrapped in tin foil, and frozen in a -80℃ ultra-low temperature refrigerator for ELISA experiments; the other part was fixed in 4% paraformaldehyde solution and kept at room temperature for mammary gland HE staining and immunohistochemistry experiments.

[0030] Observation of breast tissue pathological changes by staining The mammary tissues were fixed with paraformaldehyde, dehydrated, paraffin-embedded, and sliced, and then HE-stained. The morphological manifestations of the mammary tissue structure, mucosa, goblet cells, villus arrangement, and inflammatory cell infiltration of the mice were observed under an optical microscope and photographed.

[0031] Detection of serum inflammatory factors The mammary gland tissue of each group of mice was taken, weighed, added with an appropriate amount of PBS, homogenized, centrifuged, and the supernatant was collected. The levels of IL-1β, IL-6, and TNF-α were detected according to the instructions of the ELISA kit.

[0032] Detection of serum inflammatory factors The mammary gland tissue of each group of mice was taken, weighed, added with an appropriate amount of PBS, homogenized, centrifuged, and the supernatant was collected. The MPO level was detected according to the instructions of the ELISA kit.

[0033] Statistical analysis SPSS 25.0 statistical software was used for analysis, and one-way analysis of variance was used for comparison among the groups. P The difference was statistically significant when the value was <0.05.

[0034] Preparation and characterization of in situ thermosensitive hydrogel loaded with honokiol nanoparticles 2.1 Preparation method of honokiol nanoparticles Honokiol nanoparticles (HWN) were prepared by the desolvation method reported by Zhou et al. Weigh 40 mg whey protein (WPI) and dissolve it in 10 mL ultrapure water by ultrasonication. After complete dissolution, place it in the refrigerator overnight to fully hydrate, which is the antisolvent phase; 4 mg honokiol (HK) was fully dissolved in 1 mL anhydrous ethanol, which was the solvent phase. While slowly stirring, the HK ethanol solution was slowly dripped into the WPI solution at a rate of 1 ml / min using a syringe. After constant temperature magnetic stirring until the ethanol was completely removed, it was homogenized on a probe ultrasonic instrument (power 900 W, amplitude 10%, on 2 s, off 3 s, cycle 2 min), and finally passed through a 0.22 μm microporous filter membrane to separate free HK, and the HWN solution was obtained. Blank nanoparticles without HK were prepared in the same way.

[0035] Design of honokiol nanoparticle thermosensitive hydrogel (HK-NS-Gel) First, lentinan (LNT) and carboxymethyl chitosan (CS) were mixed and fully dissolved in the honokiol nanoparticle solution to produce physical crosslinking such as polyelectrolyte and non-covalent bonding, and then combined with the thermosensitive material poloxamer 407. Lentinan, carboxymethyl chitosan and poloxamer 407 were physically crosslinked by hydrogen bonds between each other to finally form a blank thermosensitive gel, and finally the HK-NS-Gel nanoparticle in-situ gel was prepared. This method is not only simple and easy to operate, but also avoids the use of toxic crosslinking agents such as glutaraldehyde.

[0036] Forming Under gentle stirring, dissolve LNT and CS powder in pure water at a ratio of 1:1 and stir until completely dissolved. Dissolve the investigated 16% P407 in the above solution and completely swell it in an ice bath at 4°C to prepare a blank gel solution. Replace pure water with HWN solution, and similarly obtain HK-NS-Gel, which is stored in a refrigerator at 4°C to remove bubbles and set aside. 2.4 Determination of gelation temperature The gelation temperature of thermosensitive gel was determined by the "test tube inversion method". Measure 2 mL of thermosensitive gel solution and place it in a 5 mL stoppered glass test tube. Place the stoppered test tube in a 100 mL beaker. The water bath liquid level in the beaker is about 2 cm higher than the liquid level in the test tube. Leave it for about 5 minutes until the solution in the test tube is consistent with the water bath temperature. Place a mercury thermometer in the beaker and keep the mercury ball of the thermometer consistent with the liquid level in the stoppered test tube. Place the system in a constant temperature water bath, keep the water bath in the beaker heated to 50 °C at 1~2 °C / min, and quickly tilt the test tube every 5 seconds to observe whether the liquid in the test tube flows. If the liquid does not flow within 5 seconds, it is considered to be completely gelled. The temperature recorded at this time is the gelation temperature of the thermosensitive gel.

[0037] Determination of gel time Place 2 mL of the gel solution to be tested into a vial, preheat it to 25°C to make the internal temperature of the solution uniform, then place it in a 36.5°C constant temperature water bath, and immediately start timing with a stopwatch. When the solution stops flowing completely, record the time. Repeat 3 times, and the average value is the time required for phase transition, i.e., the gelation time.

[0038] Study on the preparation process of Take a certain amount of Lentinan, carboxymethyl chitosan, and poloxamer 407, put them in a 10 mL vial, mix them evenly, add the prescribed amount of HWN, stir to disperse evenly, and refrigerate at 4°C for 24 h to obtain HK-NS-Gel. The prepared gel was placed at room temperature, and after the temperature was balanced, its morphological properties were observed, and its gelation temperature and gelation time were determined. Since the mammary gland is about 36.5°C, in order to achieve the purpose of rapid gelation at the administration site, there must be a certain temperature difference between the gelation temperature and the mammary gland temperature. The physiological temperature of the mammary gland was used as the investigation index. The optimal ratio of Lentinan, carboxymethyl chitosan, and the amount of poloxamer 407 (w / v) on the gelation temperature were investigated by single factor to determine the concentration of the nanoparticle thermosensitive hydrogel. Each group of experiments was repeated 3 times.

[0039] Single factor study on the ratio of lentinan and carboxymethyl chitosan in gel formation The concentration of poloxamer 407 was fixed at 20%, the amount of nanoparticle solution added was 5 mL, and different ratios of lentinan and carboxymethyl chitosan (3:1, 2:1:1, 1:2, 1:3) were added to screen the optimal ratio for gel formation.

[0040] Single factor study on the dosage of P407 (w / v) The ratio of Lentinan and carboxymethyl chitosan was fixed at 1:1, the amount of nanoparticle solution added was 5 mL, and different concentrations of Poloxamer 407 (12%, 16%, 20%) were added. The gelation temperature and gelation time were determined according to the methods under "2.4 Determination of gelation temperature" and "2.5 Determination of gelation time" to screen the concentration of Poloxamer 407 suitable for mammary administration.

[0041] Table 1 Single factor study of the formula composition of each group

[0042] Note: P407 is poloxamer 407, LNT:CS is lentinan:chitosan 2.9 HK-NS-Gel morphology 2.9.1 Study on the acupuncture properties of HK-NS-Gel At room temperature, the prepared nanoemulsion gel samples were injected with a syringe connected to a 1mL needle to examine their needle permeability.

[0043] Determination of value The pH meter was calibrated with a standard solution having a pH value of 6.8, and the pH value of each group of HK-NS-Gel was measured with the pH meter after calibration.

[0044] Particle size potential determination The electrodes were inserted directly into the sample in the beaker. Zeta potential analysis of the gel was performed using a Zetasizer Nano.

[0045] Morphological observation HK-NS-Gel obtained by optimizing the best preparation process was placed in a -80℃ refrigerator for 24 h, and then freeze-dried in a vacuum freeze dryer at -40℃, 9 Pa for 48 h to make freeze-dried powder. Take an appropriate amount of HK-NS-Gel powder sample and stick it directly on the conductive glue. Use Oxford Quorum SC7620 sputtering coater to spray gold for 45 s at 10 mA. Then use a scanning electron microscope to photograph the surface morphology of the sample. The accelerating voltage is 3 kV during the morphology photography. Electronic scanning is performed at 100 times and 400 times magnification to collect the microstructure of HK-NS-Gel and observe the microstructure morphology and pore size of the gel surface.

[0046] Rheological analysis of The viscosity of HK-NS-Gel obtained by the optimal preparation process was measured by rheometer. The specific method is to take 2 mL of HK-NS-Gel, use dynamic viscosity test (rotation mode), and measure the change of its viscosity with temperature at a constant shear rate in the temperature range of 0~100℃ and the constant shear rate of 200 s -1 , the heating rate is 2℃ / min.

[0047] In vitro release studies In vitro release was performed in phosphate buffer solution (PBS) at pH = 7.4, and 0.5% Tween 80 was added as the release medium. 3 ml of HWN and HK-NS-Gel were added to the dialysis bag (molecular weight 3000), and the packed dialysis bag was immersed in 30 ml of release medium. The dialysis was maintained at 37 ° C with continuous stirring at 100 rpm to simulate the environment of the cow's mammary gland. At the specified time intervals (0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 h), 1 mL of the sample was taken and 1 mL of fresh PBS was added. The concentration of HK in the sample was determined by high performance liquid chromatography, and then the cumulative release curve was drawn using Graphpad prism (version 7.0). The cumulative release of HK was determined by the following formula: Cumulative release of HK (%) = Rt / L × 100, where L and Rt represent the initial loading and cumulative release of HK at time t, respectively.

[0048] Study on the in vitro activity of honokiol nanoparticle thermosensitive gel 3.1 Antioxidant activity of HK-NS-Gel The free radical scavenging ability of RFX-CS nanogel was determined by 2,2-diphenyl-2-picrylhydrazide (DPPH). Briefly, the DPPH stock solution was diluted to 0.1 mmol / L with anhydrous ethanol, and 2 mL of 0.5, 1, 2, 4, 8, 16, and 32 mg / mL mass concentrations of HK, HWN, HK-NS-Gel, and blank Gel were respectively added to 2 mL of DPPH solution (0.1 mmol / L), mixed thoroughly, and allowed to stand at room temperature in the dark for 30 minutes. The absorbance (A1) was measured at 517 nm using a UV-visible spectrophotometer. The absorbance A0 was measured by replacing the sample solution with 2 mL of anhydrous ethanol, and the absorbance A2 was measured by replacing the DPPH solution with 2 mL of anhydrous ethanol. Vc was used as a positive control. Each sample and control at each mass concentration were repeated 3 times, and the average value was taken.

[0049] The scavenging capacity was calculated as follows: DPPH free radical scavenging capacity = (Ac-As / Ac) × 100 (9) where Ac is the control absorbance and As is the sample absorbance.

[0050] Inhibitory activity against E. coli 3.2.1 Oxford cup antibacterial method Activate E. coli and prepare bacterial suspension. Calculate the concentration of bacterial suspension using a cell counting plate and calibrate to 10 6 CFU / mL for later use. Under sterile conditions, a plate was made with sterilized culture medium. After cooling and solidification, 200 μL of bacterial suspension was added respectively, and evenly coated with a sterile applicator, cooled and dried for later use. HK group (1 mg / mL), HWN group (1 mg / mL), HK-NS-Gel group (1 mg / mL), blank Gel group were set up, and deionized water was used as the blank control group. Three sterile Oxford cups were evenly placed in the prepared plate, and 100 μL of the corresponding group solution was added to each cup. Each group was paralleled three times and placed in a constant temperature incubator at 37 °C for 24 h. The diameter of the inhibition zone was measured and recorded.

[0051] Antimicrobial MIC value determination With reference to Zhou Huizhen et al. and with slight modifications according to the experiment, the minimum inhibitory concentration (MIC) of HK-NS-Gel against clinically isolated Escherichia coli was determined by the test tube two-fold dilution method. The Oxford cup antibacterial method was used, and the method was the same as 3.2.1 Oxford cup antibacterial method. Gel (1 mg / mL) was prepared and two-fold diluted. 100 μL of gel solution containing different concentrations was added to the Oxford cups respectively, and placed in a constant temperature incubator at 37°C for 24 h to determine the diameter of the inhibition zone.

[0052] 2. Results 1. Study results of the efficacy of honokiol against LPS mastitis 1.1 Effects on pathological changes in mouse mammary gland tissue The morphology of mouse mammary gland tissue was observed. Figure 1 As shown in Group A, it can be found that the mammary gland tissue of mice in the normal group was healthy, without redness, swelling or congestion. The mammary glands of mice in the LPS group were severely congested and swollen, while the redness, swelling and congestion of the mammary glands of mice in the DEX and HK (20, 40, 60 mg / kg) groups were improved. The mammary gland tissue sections of mice were stained with HE and observed, as shown in Figure 2 Figure 1 As shown in Group B, the mammary gland tissue of the blank control group appeared normal, while the mammary gland tissue of the mice in the LPS group showed obvious inflammatory changes, with disordered alveolar structure, congestive edema of the alveolar cavity, thickening of the alveolar wall, and infiltration of neutrophils and macrophages in the mammary alveoli, ducts and connective tissues. The DEX and HK (20, 40, 60 mg / kg) administration groups significantly alleviated the histopathological changes of the mammary gland caused by LPS, among which the high-dose HK group had the most obvious effect.

[0053] Effects on the levels of inflammatory factors in mouse mammary gland tissue Compared with the normal group, the levels of IL-1β and TNF-α in the mammary gland tissue of the model group mice increased significantly ( P <0.0001), IL-6 level was significantly increased ( P <0.001). Compared with the model group, the levels of IL-1β and TNF-α in the mammary gland tissue of DEX mice were significantly reduced ( P <0.0001), IL-6 level was significantly decreased ( P <0.01); the IL-1β levels in the low-dose, medium-dose and high-dose groups of honokiol were significantly decreased ( P <0.001), IL-6 level was significantly decreased ( P <0.01), and TNF-α level decreased significantly ( P <0.0001). The TNF-α level in the high-dose honokiol group was significantly lower than that in the low-dose honokiol group ( P<0.01), but there was no significant difference with the medium dose group. The results showed that honokiol can effectively inhibit the secretion of pro-inflammatory factors, and the effect is enhanced in a dose-dependent manner. Figure 2 .

[0054] Effects on MPO levels in mouse mammary tissue Compared with the normal group, the MPO level in the mammary gland tissue of the model group mice increased significantly ( P <0.0001). Compared with the model group, the MPO level in the mammary gland tissue of DEX mice was significantly reduced ( P <0.0001); the MPO levels in the low-dose, medium-dose and high-dose groups of honokiol were significantly reduced ( P <0.0001). The MPO level in the high-dose honokiol group was lower than that in the low-dose honokiol group ( P <0.05), but there was no significant difference with the medium dose group. The results showed that honokiol can effectively alleviate the degree of inflammatory infiltration, and the effect is enhanced in a dose-dependent manner. Figure 3 .

[0055] Preparation and characterization of in situ thermosensitive hydrogel loaded with honokiol nanoparticles 2.1 Single factor investigation results of HK-NS-Gel 2.1.1 Determination of the gelling ratio of Lentinan and carboxymethyl chitosan The results of the investigation on the gel formation of Lentinan and carboxymethyl chitosan in different ratios are shown in Table 2. As the ratio changes, precipitation gradually occurs. Only when the ratio of Lentinan and carboxymethyl chitosan is 1:1 can a sol of qualified quality be formed.

[0056] Table 2 Investigation of the gelation ratio of Lentinan and chitosan (, n = 3)

[0057] 2.1.2 Determination of the dosage of P407 (w / v) The results of the study on the effect of different poloxamer 407 concentrations on the gelation temperature of HK-NS-Gel are as follows: Figure 4 As shown in the figure, with the increase of poloxamer 407 concentration, the gelation temperature decreases continuously, showing a concentration-dependent manner. At the same time, with the increase of poloxamer 407 concentration, the gelation time also shortens. When the concentration of poloxamer 407 is 16%, the gelation temperature is closest to the mammary gland temperature.

[0058] In summary, the preparation process of HK-NS-Gel under the optimal prescription is: 1) Preparation of Honokiol Nanoparticles (HWN) ① Take whey protein, add PBS buffer (pH 7.4) and dissolve by ultrasonic to obtain 4 mg / mL whey protein solution; ②Take honokiol and dissolve it in anhydrous ethanol to obtain a 4 mg / mL honokiol ethanol solution; ③ Take the honokiol ethanol solution in step ②, and drip it into the whey protein solution in step ① at a rate of 1 ml / min at a volume ratio of 1:10, remove the ethanol and then homogenize by ultrasonication, and then filter with a 0.22 μm microporous filter membrane, and take the filtrate to obtain the honokiol nanoparticle solution; 2) Preparation of honokiol nanoparticle thermosensitive gel HK-NS-Gel Take Lentinan, carboxymethyl chitosan and the honokiol nanoparticle solution obtained in step 1), mix and dissolve them in a mass volume ratio of 50 mg:50 mg:10 ml, then add poloxamer 407 to make the concentration of 16% (g / ml), stir and disperse evenly, finally swell completely in a 4°C environment, and remove bubbles to obtain the product.

[0059] The characterization results 2.2.1 Study on the acupuncture properties of HK-NS-Gel At room temperature, the prepared HK-NS-Gel was injected with a syringe connected to a 1 ml needle. The results showed that HK-NS-Gel had good needle permeability and was suitable for local injection ( Figure 5 ).

[0060] pH The pH value of the optimal formula HK-NS-Gel was measured using a pH acidity meter. The results showed that the pH value of HK-NS-Gel was around 6.5, which was a weakly acidic substance and consistent with the mastitis environment. At the same time, the pH value of the nanoparticle solution was measured and found to be 6.4, proving that the gel preparation process had no significant effect on its pH value.

[0061] Table 3 pH values ​​of HK-NS-Gel and HWN at 4℃ (, n = 3)

[0062] 2.2.3 Surface morphology of HK-NS-Gel The surface microstructure and pore size of HK-NS-Gel were observed by SEM at different magnifications (100x and 400x). It was found that HK-NS-Gel showed a loose lamellar grid structure with many circular holes and a uniform internal structure ( Figure 6 ).

[0063] Rheological analysis of As a local breast injection, HK-NS-Gel needs to undergo a sol-gel phase transition at body temperature after being injected into breast tissue. Rheometers are usually used to test the temperature-dependent viscosity change to study the rheological behavior of thermosensitive gels. Figure 7 The study showed that the formation temperature of HK-NS-Gel is between 34℃ and 38℃, and HK-NS-Gel is liquid-like at 4 and 25℃, indicating that the gel can be accurately and quantitatively injected into the breast at room temperature, with strong spreadability and uniform administration. When it reaches the gelation temperature, it quickly forms a semi-solid gel with high retention, which can further improve the bioavailability of the drug.

[0064] In vitro release results Depend on Figure 8 It can be seen that HWN releases magnolol rapidly within 12 h, and releases slowly from 12 to 48 h. However, the continuous release of HK in HK-NS-Gel within the first 12 h was only about 22%, and HK was continuously released within 48 h, with a cumulative release rate of more than 40%. Compared with HWN solution, HK-NS-Gel has a good sustained-release effect.

[0065] Results of in vitro activity study of honokiol nanoparticle thermosensitive gel 3.1 Antioxidant activity of HK-NS-Gel Results Fig. 9 HWN and HK-NS-Gel have a certain ability to scavenge DPPH free radicals. In the mass concentration range of 0-1 mg / mL, the scavenging ability of HWN and HK-NS-Gel on DPPH free radicals increases with the increase of mass concentration. The scavenging rate of HK-NS-Gel can reach 90.98%, which is much higher than that of free drugs, but lower than that of VC. This proves that the introduction of LNT and CMC gel materials greatly enhances the anti-E. coli effect of HK and plays a synergistic role.

[0066] Inhibitory activity against E. coli 3.2.1 Oxford cup diffusion method to measure the antibacterial activity of nanoparticle gel The results are as follows Fig.10 As shown, the diameter of the inhibition zone of the HK group (1 mg / mL) was (1.55±0.01) cm, the diameter of the inhibition zone of the HWN group (1 mg / mL) was (1.79±0.01) cm, the diameter of the inhibition zone of the HK-NS-Gel group (1 mg / mL) was (2.22±0.01) cm, the diameter of the inhibition zone of the blank Gel group was (1.21±0.01) cm, and the diameter of the inhibition zone of the blank control group was (0.5±0.01) cm, indicating that the HWN group and the HK-NS-Gel group had significant antibacterial effects compared with free HK, and the effect of the HK-NS-Gel group was significantly enhanced.

[0067] Determination of antibacterial MIC value of nanoparticle gel HK-NS-Gel (1 mg / mL) was prepared and diluted twice. 100 μL of HK-NS-Gel solution containing different concentrations was added to Oxford cups respectively, and then placed in a constant temperature incubator at 37 °C for 24 h, and the diameter of the inhibition zone was measured. The results showed that the diameter of the inhibition zone was positively correlated with the mass concentration of HK-NS-Gel, and the measured MIC concentration was 0.125 mg / mL.

[0068] Escherichia coli is an important environmental microorganism that causes animal mastitis. Compared with free honokiol, honokiol nanoparticle gel has a more significant inhibitory effect on Escherichia coli, indicating that honokiol nanoparticle gel has outstanding advantages in the treatment of Escherichia coli mastitis in animals.

[0069] In summary, the thermosensitive gel of the present invention is liquid at room temperature, and rapidly gels to form a semisolid gel at a mammary gland temperature of 36.5°C, has high retention, and has a good sustained-release effect. The drug is administered to the mammary gland in a targeted manner by injection, so that the drug carried by it can adhere to the mammary gland mucosa, directly reach the diseased site, and is not easy to flow out, thereby improving the utilization rate of the drug. The thermosensitive gel of the present invention is used to carry honokiol nanoparticles. In vitro tests show that it has a better antibacterial effect than free honokiol and honokiol nanoparticles. It is applied to the prevention and treatment of animal mastitis, especially Escherichia coli mastitis, and has a more significant therapeutic effect, and has practical promotion and application value.

Claims

1. A thermosensitive gel, characterized in that: It is prepared from the following raw materials in mass volume percentage: Lentinan and carboxymethyl chitosan mixture 0.5-1.5%, poloxamer 407 10-20%, and the rest is aqueous solution with pH 7-7.5; The mass ratio of lentinan to carboxymethyl chitosan is 1-3:1-3.

2. The thermosensitive gel according to claim 1, characterized in that: It is prepared from the following raw materials in mass volume percentage: Lentinan and carboxymethyl chitosan mixture 1%, poloxamer 407 16%, and the rest are aqueous solutions with a pH value of 7-7.

5.

3. The thermosensitive gel according to claim 1 or 2, characterized in that: The mass ratio of lentinan to carboxymethyl chitosan is 1:

1.

4. Use of the thermosensitive gel according to any one of claims 1 to 3 as a drug matrix for topical administration.

5. The use according to claim 4, characterized in that: The drug matrix is ​​a mammary gland topical drug administration matrix.

6. A drug for preventing and / or treating animal mastitis, characterized in that: It is a thermosensitive gel loaded with honokiol nanoparticles; The thermosensitive gel is the thermosensitive gel according to any one of claims 1 to 3.

7. The drug according to claim 6, characterized in that: It is prepared from the following raw materials in mass volume percentage: Lentinan and carboxymethyl chitosan mixture 0.5-1.5%, poloxamer 407 10-20%, and the rest is honokiol nanoparticle solution; The mass ratio of Lentinan to carboxymethyl chitosan is 1-3:1-3; The honokiol nanoparticle solution is obtained by homogenizing a PBS buffer solution containing whey protein and an ethanol solution containing honokiol, removing the ethanol and filtering to obtain the nanoparticle solution.

8. A method for preparing the drug according to claim 6 or 7, characterized in that: The steps include: 1) Take whey protein, add PBS buffer and ultrasonically dissolve it to obtain a whey protein solution; take magnolia officinalis, add ethanol to dissolve it, and obtain a magnolia officinalis ethanol solution; 2) taking the honokiol ethanol solution obtained in step 1), dropping it into the whey protein solution obtained in step 1), removing the ethanol and then homogenizing, filtering, and taking the filtrate to obtain the honokiol nanoparticle solution; 3) Take a mixture of Lentinan and carboxymethyl chitosan, add the honokiol nanoparticle solution obtained in step 1) according to the ratio to dissolve, then add poloxamer 407, stir and disperse evenly, and completely swell until there are no bubbles.

9. The preparation method according to claim 8, characterized in that: Step 1) the pH value of the PBS buffer is 7.0-7.5, the concentration of the whey protein solution is 4 mg / mL; the ethanol is anhydrous ethanol, and the concentration of the honokiol ethanol solution is 4 mg / mL; and / or, the volume ratio of the honokiol ethanol solution obtained in step 2) to the whey protein solution is 1:10; or, the dripping speed is 1 ml / min; or, the homogenization is ultrasonic homogenization; and the filtration is performed by filtering with a 0.22 μm microporous membrane; And / or, the step 3) of completely swelling until there are no bubbles is to stand in a 4° C. environment until there are complete swelling and no bubbles.

10. Use of the drug according to claim 6 or 7 in the preparation of a drug for preventing and / or preventing animal mastitis, characterized in that: The mastitis includes Escherichia coli mastitis.

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