Traditional Chinese medicine compound temperature-sensitive slow-release hydrogel as well as preparation method and application thereof

Abstract

The invention discloses traditional Chinese medicine compound temperature-sensitive slow-release hydrogel as well as a preparation method and application thereof. Sodium alginate, gelatin and polyvinylpyrrolidone are used as raw materials to prepare microspheres, traditional Chinese medicine compound liquid medicine and the microspheres are mixed to prepare medicine carrying microspheres, and an F127-PEG200 system is introduced to prepare the traditional Chinese medicine compound temperature-sensitive slow-release hydrogel. The traditional Chinese medicine compound temperature-sensitive slow-release hydrogel has the properties of slow release, temperature sensitivity and the like, and is administrated to an affected part in a spray form, so that the administration contact is reduced, and the risk of inflammation increase of the affected part caused by the contact is reduced; the phase change temperature of the gel is closer to the skin temperature, the temperature range of the gel which is in a liquid state at normal temperature and can be used as a spray is increased, and the gel can be changed into gel to cover an affected part, so that the wound surface is protected, and the infection risk is reduced; due to the temperature-sensitive characteristic, the hydrogel is changed into liquid at the temperature lower than 31 DEG C, so that the hydrogel is easy to wash and change medicine, and the biocompatibility is good.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and specifically relates to a traditional Chinese medicine compound temperature-sensitive sustained-release hydrogel and a preparation method and application thereof. Background Art

[0002] Allergic contact dermatitis (ACD) is mainly caused by specific antigens activating the body's acquired immune system, which in turn produces a skin inflammatory reaction mediated by effector T cells. Common symptoms are local skin inflammation, edema, fever, pain and severe itching. The incidence of ACD in the population is as high as 22%, which seriously affects the patient's daily life and work. The commonly used treatment methods are ineffective, which seriously affects the patient's health and quality of life, and may even induce mental illness, increasing the burden on patients and their families. Traditional antihistamines such as loratadine and diphenhydramine have little effect and are almost ineffective, while other types of drugs often have poor efficacy and serious side effects. There is currently no specific drug for this disease. ACD is a common inflammatory skin disease, which belongs to the category of "lacquer sores" in traditional Chinese medicine. It is mentioned in Volume 35 of "Treatise on the Causes and Symptoms of Various Diseases". Traditional Chinese medicine has been used to treat "lacquer sores" for more than a thousand years, with significant efficacy and few side effects. Traditional Chinese medicine skin topical preparations have a long history of development. They are directly applied or attached to the skin surface without passing through the gastrointestinal tract, thereby reducing the risk of being damaged by gastric acid, protecting the gastrointestinal tract from irritation, and having a significant local therapeutic effect.

[0003] However, Chinese herbal medicines for external use on the skin still have many shortcomings: (1) patients need to apply them several times a day, which is inconvenient to use; (2) they are not easy to adhere to the surface of the affected area in solution state, and they quickly slide off and leave the affected area, making it difficult for them to exert their original efficacy; (3) some Chinese medicines have poor water solubility, low bioavailability, and short duration of efficacy; (4) direct contact with the affected area during administration can easily increase the risk of inflammation of the affected area.

[0004] Compared with traditional preparations, ideal temperature-sensitive sustained-release hydrogels have multiple advantages: (1) Sustained-release hydrogels have sustained-release properties, which can reduce the number of dosing times and prolong the duration of action; (2) Thermosensitive hydrogels are administered to the affected area in the form of a spray, which reduces the contact of administration and reduces the risk of increased inflammation of the affected area caused by contact; (3) Thermosensitive sustained-release hydrogels shorten the gel phase transition temperature range, so that the phase transition occurs only when it is closer to the skin temperature, increasing the temperature range in which it can be used as a spray when it is liquid at room temperature, and at the same time, it becomes a gel to cover the affected area, protecting the wound surface, reducing the risk of infection, and prolonging the duration of drug efficacy; (4) Due to its temperature-sensitive properties, the hydrogel becomes liquid at temperatures below 31°C, and can be cleaned and replaced by rinsing with sterile water at room temperature. However, it is not easy to obtain a temperature-sensitive sustained-release hydrogel with the above advantages.

[0005] Sodium alginate (SA) has fixation, displacement and adsorption capabilities and is often used as a fixed embedding carrier material. Polyvinyl pyrrolidone (PVP) has good adsorption properties and strong ability to form hydrogen bonds and complexes. However, there are no reports in the prior art on the use of sodium alginate and polyvinyl pyrrolidone as raw materials to prepare temperature-sensitive sustained-release hydrogels, and there are no reports on sprayable carrier materials for traditional Chinese medicine compound temperature-sensitive sustained-release hydrogels that can be used to improve the effect of treating ACD. Summary of the invention

[0006] The primary purpose of the present invention is to overcome the deficiencies of the existing technology and provide a method for preparing a thermosensitive sustained-release hydrogel of a traditional Chinese medicine compound.

[0007] Another object of the present invention is to provide a traditional Chinese medicine compound thermosensitive sustained-release hydrogel obtained by the above preparation method.

[0008] Another object of the present invention is to provide an application of the above-mentioned traditional Chinese medicine compound temperature-sensitive sustained-release hydrogel.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A method for preparing a thermosensitive sustained-release hydrogel of a traditional Chinese medicine compound comprises the following preparation steps:

[0011] (1) Extraction of Chinese herbal compound liquid;

[0012] (2) Preparation of SA-GEL-PVP microspheres: Sodium alginate, gelatin and water were mixed and stirred until completely dissolved; polyvinyl pyrrolidone solution was added and mixed to obtain a mixture, and the supernatant was collected by centrifugation and dried to obtain freeze-dried powder of SA-GEL-PVP microspheres;

[0013] (3) Preparation of thermosensitive sustained-release hydrogel of traditional Chinese medicine compound: the traditional Chinese medicine compound liquid obtained in step (1) and the freeze-dried powder of microspheres obtained in step (2) are mixed and stirred; F127 is added, allowed to stand overnight, and then PEG200 is added and mixed to obtain the thermosensitive sustained-release hydrogel of traditional Chinese medicine compound.

[0014] In step (1):

[0015] The extraction is preferably carried out by water extraction.

[0016] The water extraction method preferably includes the following steps: soaking the Chinese medicinal materials in water, boiling and then decocting, separating the solid and the liquid to obtain the residue and the liquid A; concentrating the liquid A to obtain the Chinese medicinal compound liquid.

[0017] In order to fully extract the effective ingredients in the Chinese medicinal materials, the water extraction method also includes the following steps: repeatedly soaking the medicinal residues with water, decocting and extracting steps to obtain liquid medicine B; combining and concentrating liquid medicine B and liquid medicine A to obtain a Chinese medicinal compound liquid medicine.

[0018] The Chinese medicinal materials preferably include Bupleurum chinense, Sophora flavescens, Dictamni cortex, Periostracum cicadae, Schizonepeta tenuifolia, Coptis chinensis, Phellodendron chinense, Saposhnikovia divaricata, Schisandra chinensis, Ebony plum, and Licorice; more preferably, the composition is as follows: 15g Bupleurum chinense, 15g Sophora flavescens, 15g Dictamni cortex, 10g Periostracum cicadae, 10g Schizonepeta tenuifolia, 10g Coptis chinensis, Phellodendron chinense, 10g Saposhnikovia divaricata, 5g Schisandra chinensis, 5g Ebony plum, and 10g Licorice.

[0019] The mass volume ratio (g:mL) of the Chinese medicinal materials to water is preferably 1:2-3.

[0020] The soaking time is preferably 30 min to 90 min, more preferably 60 min.

[0021] The time for continued decocting is preferably 10 to 30 minutes, and more preferably 20 minutes.

[0022] The concentration is preferably rotary evaporation concentration.

[0023] The concentration is preferably to concentrate the medicinal liquid to 3-5 g of raw medicinal material / mL; more preferably to concentrate the medicinal liquid to 4 g of raw medicinal material / mL.

[0024] In step (2):

[0025] The water is preferably ultrapure water.

[0026] The sodium alginate, gelatin and polyvinyl pyrrolidone are preferably mixed in a ratio of 10-15g: 5-7g: 1-3g; more preferably in a ratio of 12g: 6g: 2g.

[0027] The concentration of the polyvinyl pyrrolidone solution is preferably 0.3-0.5 g / 100 mL, and more preferably 0.4 g / 100 mL.

[0028] The amount of water is preferably calculated based on the concentration of sodium alginate in the mixture of 1 to 1.5 g / 100 mL; more preferably, the concentration of sodium alginate in the mixture is 1.2 g / 100 mL.

[0029] The stirring condition is preferably 400-600 rpm, and more preferably 500 rpm.

[0030] The centrifugal conditions are preferably centrifugal at 6000-8000 rpm for 10 min; more preferably centrifugal at 7000 rpm for 10 min.

[0031] The drying is preferably freeze-drying.

[0032] The freeze-drying conditions are preferably a vacuum degree of 20Pa, a gradual temperature increase from -45°C to -25°C, an increase of 5 to 10°C every 2 hours, and maintaining -25°C until the freeze-drying is completed. The total freeze-drying time is 24 hours.

[0033] In step (3):

[0034] The concentration of the Chinese herbal compound liquid is preferably 0.01-0.02 g raw medicinal material / mL; more preferably 0.01875 g raw medicinal material / mL.

[0035] The volume-to-mass ratio of the traditional Chinese medicine compound liquid to the microspheres is preferably 8-12 mL: 0.005-0.02 g; more preferably 10 mL: 0.01 g.

[0036] The stirring condition is preferably stirring at 800-1000 rpm for 20-28 h; more preferably stirring at 900 rpm for 24 h.

[0037] The dosage of F127 is preferably calculated based on the ratio of the Chinese medicine compound liquid to F127 of 10 mL: 1.9 g.

[0038] The dosage of PEG200 is preferably calculated based on a volume ratio of the Chinese medicine compound liquid to PEG200 of 250:1 (v / v).

[0039] A traditional Chinese medicine compound thermosensitive sustained-release hydrogel is obtained by the preparation method.

[0040] Application of the above-mentioned traditional Chinese medicine compound thermosensitive sustained-release hydrogel in the preparation of drugs for treating allergic contact dermatitis.

[0041] Compared with the prior art, the present invention has the following advantages and effects:

[0042] (1) The main components of the traditional Chinese medicine compound KMF were detected for the first time.

[0043] (2) It was confirmed for the first time that the traditional Chinese medicine compound KMF can slow down the abnormal proliferation of keratinocytes.

[0044] (3) SA-GEL-PVP was prepared from a non-microsphere form into a microsphere form, and a new SA-GEL-PVP sustained-release microsphere system that can carry traditional Chinese medicine compound was constructed.

[0045] (4) The present invention provides a new Chinese medicine dosage form, which is prepared into a temperature-sensitive type, that is, a solution at room temperature, which becomes a gel when sprayed on the skin surface, and has a sustained-release gel preparation. At the same time, the Chinese medicine compound KMF, microspheres and PEG200 reduce the hydrogen bonding force of the cross-linked gel formed by F127, thereby increasing the adaptability to the skin surface.

[0046] (5) The thermosensitive sustained-release hydrogel of the Chinese medicine compound KMF is liquid at room temperature and can be sprayed, indicating that the matrix concentration is dilute enough; it is gel-like at 32.5°C (i.e., the surface temperature of the human body), indicating that the matrix strength is strong enough; the presence of the Chinese medicine compound KMF and microspheres enables F127-PEG200 to simultaneously achieve a concentration that is dilute enough to be sprayed and a strength that is strong enough to form a gel, solving the problem of "the dilute solution has low strength", which cannot be achieved without the Chinese medicine compound KMF, microspheres, and PEG200.

[0047] (6) The traditional Chinese medicine compound KMF was prepared into a temperature-sensitive, sustained-release hydrogel for the first time. The characteristics of the temperature-sensitive hydrogel make it suitable for use in sprays. The drug is administered to the affected area in the form of a spray, which reduces the contact between the drug and the dosage form and optimizes the efficacy. The sustained-release hydrogel can reduce the number of doses and prolong the duration of action. It is also easy to store, transport, and use.

[0048] (7) The F127-PEG200 system reduces the amount of F127 used and is economical and applicable. It also reduces the viscosity of the system, making it easier to spray. The traditional Chinese medicine compound KMF, microspheres, and PEG200 make F127 liquid at 25°C-28.5°C when the F127 concentration is 19%, and solid at 29°C, reducing the amount of F127 used and the viscosity of the system, making it economical and applicable. It is liquid at room temperature, and turns into a solid after contacting the skin and adheres to the skin.

[0049] (8) The Chinese medicine compound KMF is prepared into molded Chinese medicine compound KMF microspheres, which can reduce the phase transition temperature range of the Chinese medicine compound KMF thermosensitive sustained-release hydrogel and increase its liquid range at room temperature. The human body surface temperature is 32.5°C, and the phase transition occurs at 31°C, which is closer to the skin temperature.

[0050] (9) The new system is biocompatible, indicating that it is safe for use in animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The above graph is the analysis result of the effective ingredients of the traditional Chinese medicine compound KMF; the upper graph represents the negative ion flow graph in the total ion flow graph of the traditional Chinese medicine compound KMF, and the lower graph represents the positive ion flow graph in the total ion flow graph of the traditional Chinese medicine compound KMF.

[0052] Figure 2 This is a graph showing the effect of different concentrations of LPS on the abnormal proliferation of Hacat cells.

[0053] Figure 3 This is the cytotoxicity (24h) result of the water extract of the traditional Chinese medicine compound KMF.

[0054] Figure 4 This is the cytotoxicity (48h) result of the water extract of the traditional Chinese medicine compound KMF.

[0055] Figure 5 This is a graph showing the effect of the traditional Chinese medicine compound KMF on the abnormal proliferation of keratinocytes.

[0056] Figure 6 This is the phase transition temperature result diagram of the traditional Chinese medicine compound KMF thermosensitive sustained-release hydrogel.

[0057] Figure 7 This is the transmission electron microscopy result of KMF@SA-GEL-PVP.

[0058] Figure 8 This is the biocompatibility result diagram of F127-PEG200 (KMF@SA-GEL-PVP).

[0059] Fig. 9 This is the biocompatibility result diagram of F127-PEG200.

[0060] Fig.10 This is the biocompatibility result diagram of KMF@F127-PEG200.

[0061] Fig.11 This is the biocompatibility result diagram of KMF@SA-GEL-PVP.

[0062] Fig.12 The figure shows the biocompatibility results of SA-GEL-PVP.

[0063] Fig.13 The transmission electron microscopy results of the blank microspheres prepared in Comparative Example 1 are shown; the left picture represents the blank microspheres A prepared in Comparative Example 1; and the right picture represents the blank molded microspheres B prepared in Comparative Example 1.

[0064] Fig.14 Phase transition temperature results of blank microsphere A hydrogel and blank molded microsphere B hydrogel prepared in Comparative Example 1.

[0065] Fig.15 Phase transition temperature results of product C and product D traditional Chinese medicine compound KMF thermosensitive sustained-release hydrogel prepared in comparative example 1.

[0066] Fig.16 The stability results of the drug-loaded microspheres of the KMF aqueous extract of the traditional Chinese medicine compound and the product D prepared in comparative example 1 after being placed for one month; wherein, the left figure represents the KMF aqueous extract of the traditional Chinese medicine compound without adding microspheres and F127-PEG200; the right figure represents the microspheres loaded with the KMF aqueous extract of the traditional Chinese medicine compound prepared in comparative example 1. DETAILED DESCRIPTION

[0067] The present invention will be further described below in conjunction with specific embodiments. These embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified, the conditions recommended by the manufacturer or the conventional conditions are followed. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0068] The material information involved in the present invention is as follows:

[0069] (1) Experimental instruments: CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., product model: KQ2200DE), intelligent magnetic stirrer (Guangzhou Yuhua Instrument Co., Ltd., product model: ZNCL-B 140), electronic balance (Shimadzu Corporation, Kyoto, Japan, product model: ATY124), electrostatic field orbital trap high-resolution liquid spectrometer (Thermo Fisher, Germany, model: QExactive Plus)

[0070] (2) Experimental drugs and reagents: chitosan quaternary ammonium salt (HTCC, Shanghai MacLean Biochemical Technology Co., Ltd., 850125), gelatin (Shanghai MacLean Biochemical Technology Co., Ltd., G6317-500g); polyvinyl pyrrolidone (Shanghai MacLean Biochemical Technology Co., Ltd., P816205-500g); polyethylene glycol 200 (Guangzhou Xueyou Biotechnology Co., Ltd., S30183-500g); F127 (Shanghai Aladdin Biochemical Technology Co., Ltd., P434419-50g); methanol (Tianjin Best Chemical Co., Ltd.), acetonitrile (CNW Technologies GmbH, Germany).

[0071] Example 1 Study on the analysis of effective components of the traditional Chinese medicine compound KMF

[0072] (1) Composition of the Chinese medicine compound KMF

[0073] The traditional Chinese medicine compound KMF includes 15g of Bupleurum chinense, 15g of Sophora flavescens, 15g of Dictamni cortex, 10g of Periostracum cicadae, 10g of Schizonepeta tenuifolia, 10g of Coptis chinensis, 10g of Phellodendron chinense, 10g of Saposhnikovia divaricata, 5g of Schisandra chinensis, 5g of Prunus mume, and 10g of Licorice.

[0074] (2) Preparation of KMF aqueous extract of traditional Chinese medicine compound

[0075] Weigh 214g of the traditional Chinese medicine compound KMF on a balance, add 600ml of water to cover the traditional Chinese medicine compound KMF, soak for 1 hour, perform water extraction, heat to boil, simmer for 20 minutes on low heat after boiling, filter through 4 layers of gauze, and pour out the liquid medicine; add 4 / 5 of the first amount of water, perform water extraction again, heat to boil, continue to boil for 20 minutes after boiling, filter through 4 layers of gauze, combine the filtrate, concentrate the filtrate to 100ml by rotary evaporation, and store it in a refrigerator at 4°C.

[0076] (3) Preparation of mass spectrometry samples

[0077] The concentration of the mother liquor of the Chinese herbal compound KMF water extract was 4 g of raw medicinal material / mL, and a certain amount of 70% (v / v) methanol was added for dilution to finally prepare a sample solution with a concentration of 0.05 g of raw medicinal material / mL.

[0078] (4) Mass spectrometry

[0079] The chromatographic column was ACQUITY UPLC HSS T3 Column (1.8 μm, 2.1×100 mm, Waters), and the column oven was 25°C. The mobile phase A was 0.1% formic acid water, and the mobile phase B was acetonitrile. The gradient elution (v / v) was: 0-5 min 7% B; 5-6 min 7-12% B; 6-16 min 12%-25% B; 16-23 min 25-70% B; 23-24 min 70%-90% B; 24-26 min 90% B. The injection volume was 3 μL, the liquid chromatography flow rate was 0.3 mL / min, and the detection wavelength was 280 nm.

[0080] (5) Experimental results

[0081] according to Figure 1 The active ingredients of KMF can be determined to be tryptophan, atractylenolide III, artemisinin, cimicifuga glycosides, valerophenone, 7,2′,6′-trihydroxy-5-methoxyl-dihydroflavone, meranzinhydrate, cinnamaldehyde, fumaric acid, pinocembrin, epoxybergamottin or cnidicin, 4-isopropylbenzaldehyde, isovalascoside, and hesperidin.

[0082] Example 2 Effect of the Chinese herbal compound KMF on abnormal proliferation of keratinocytes

[0083] (1) Construction of abnormal keratinocyte proliferation model

[0084] HaCat cells (purchased from China Type Culture Collection, Wuhan University, catalog number: GDC0106) were taken and counted. The cells were counted at 3.5×10 4 The cells were seeded into a 96-well plate at a concentration of 100 μL / well, and cultured with DMEM complete medium. After 24 hours, the medium was discarded, and different concentrations of lipopolysaccharide (LPS) were added and cultured for another 24 hours. The volume of LPS added to each well was 100 μL, and then the activity was measured by MTT method to determine the optimal LPS concentration. Complete medium refers to DMEM medium containing 10% calf serum or fetal bovine serum by volume.

[0085] Experimental groups: blank control group (DMEM complete medium without LPS was added), different LPS concentration groups (7.5, 15, 31.25, 62.5, 125, 250, 500, 1000 μg / mL; 5, 10, 20, 40, 80, 160, 320, 640 μg / mL; all prepared with DMEM complete medium, two batches of experiments).

[0086] Experimental results: Figure 2 As shown, human keratinocytes (Hacat cells) were treated with different concentrations of LPS for 24 hours, and the absorbance was detected by MTT method to observe cell viability. The results showed that Hacat cells treated with 7.5μg / mL and 10μg / mL LPS for 24 hours had obvious abnormal proliferation.

[0087] (2) Determination of cytotoxicity of the aqueous extract of the traditional Chinese medicine compound KMF

[0088] Take HaCat cells in the logarithmic growth phase, count them and adjust the cell number to 3.5×10 4 The cells were seeded into 96-well plates at a concentration of 100 μL / well per well, and cultured with DMEM complete medium overnight until the cells were completely attached. The complete medium was discarded, and different concentrations of complete medium containing the traditional Chinese medicine compound KMF were added at 100 μL / well. After culturing for 24 h and 48 h, the cell supernatant was aspirated, and 100 μL of 0.5% MTT solution was added to each well. After culturing for 4 h, the cell supernatant was aspirated, and 150 μL of DMSO solution was added. The plates were shaken on an oscillator for 10 min to completely dissolve the purple formazan crystals. The OD value was measured at a wavelength of 490 nm on a microplate reader to determine the safe dosage range of the traditional Chinese medicine compound KMF for HaCat cells.

[0089] Experimental groups: control group (DMEM medium without drugs), various concentration groups of the Chinese medicine compound KMF (1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, 0.015 mg / mL, 0.0075 mg / mL, all prepared with DMEM complete medium; the concentration is based on the freeze-dried powder mg / mL after the raw medicinal materials are extracted, the supernatant is taken and freeze-dried, the same below).

[0090] Experimental results: Hacat cells were treated with complete medium containing 7.5, 15, 31.25, 62.5, 125, 250, 500, and 1000 μg / mL KMF for 24 h and 48 h, respectively. Cell viability was detected by MTT assay. Complete medium containing KMF at a concentration of 1000 μg / mL or less had no obvious toxicity to Hacat cells and increased cell viability (e.g. Figure 3 and Figure 4 shown).

[0091] (3) Effect of the Chinese herbal compound KMF on abnormal proliferation of keratinocytes

[0092] Take HaCat cells, count and adjust the cell number to 3.5×10 4 The cells were seeded into 96-well plates at a concentration of 100 μL / well, and cultured in DMEM complete medium. Complete medium containing different concentrations of the traditional Chinese medicine compound KMF (1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, 0.015 mg / mL, 0.0075 mg / mL) was added. The above are the safe dosing concentrations screened in (2). The control group was replaced with a drug-free medium and cultured for 24 hours. LPS (10 μg / mL) was added according to the optimal concentration in the results of (1) and cultured for 24 hours. Then, the activity was measured by MTT method.

[0093] The experimental groups were: control group, LPS model group, and Chinese medicine compound KMF+LPS group.

[0094] Experimental results: Hacat cells were treated with complete medium containing 7.5, 15, 31.25, 62.5, 125, 250, 500, and 1000 μg / mL KMF for 24 h, and then treated with 10 μg / mL LPS for 24 h to induce abnormal proliferation of Hacat cells. Cell viability was detected by MTT method. Figure 5 As shown, KMF pretreatment reduced LPS-induced abnormal proliferation of Hacat cells, with statistical significance.

[0095] Example 3 Preparation of Traditional Chinese Medicine Compound KMF Thermosensitive Sustained Release Hydrogel

[0096] (1) Preparation of microspheres

[0097] 12 g of sodium alginate (SA) and 6 g of gelatin (GEL) were added to ultrapure water (500 mL), and the solution was continuously stirred (5000 rpm) until it was completely dissolved to obtain a mixture. Next, polyvinyl pyrrolidone (PVP) was mixed at a ratio of 2 g:500 mL of PVP and ultrapure water to obtain a PVP aqueous solution, and then the PVP aqueous solution was added to the mixture, centrifuged at 7000 rpm for 10 min, and the supernatant was taken and freeze-dried. The temperature was gradually increased from -45°C to -25°C under a vacuum degree of 20 Pa, and the temperature was increased by 5 to 10°C every 2 hours, and -25°C was maintained until the freeze-drying was completed. The total freeze-drying time was 24 hours to obtain blank microspheres (SA-GEL-PVP).

[0098] (2) Preparation of KMF thermosensitive sustained-release hydrogel

[0099] The prepared Chinese medicine compound water extract (0.01875g raw medicinal material / mL) and the prescribed amount of freeze-dried microspheres were mixed at a ratio of 10mL:0.01g, and stirred at 900rpm for 24h to obtain drug-loaded microspheres. Finally, F127 was added at a ratio of 10mL:1.9g of the Chinese medicine compound water extract and F127, and the mixture was placed overnight by cold dissolution method, and then polyethylene glycol 200 (PEG200) was added at a ratio of 10mL:0.04mL of the Chinese medicine compound water extract and polyethylene glycol 200 (PEG200) and mixed.

[0100] (3) Structural characterization of microspheres

[0101] The dried blank microspheres prepared in step (1) were added to 0.01875 g of the raw medicinal material / mL KMF aqueous extract at a ratio of 10 mL: 0.01 g, stirred at 900 rpm for 24 h, and then dripped onto a copper sheet and allowed to air dry naturally. Figure 6 As shown in Figure 2, the morphology of KMF-loaded SA-GEL-PVP microspheres (KMF@SA-GEL-PVP) was observed by transmission electron microscopy. The microspheres were slightly irregular spherical with a particle size of about 1 μm.

[0102] (4) Phase change temperature detection

[0103] Experimental method: The prepared 0.01875 g crude drug / mL Chinese medicine compound water extract was mixed with different masses of F127, F127-PEG200, and F127-HTCC, respectively, so that the final mass fraction of F127 reached 19%, 20%, 21%, 22%, 23%, 24%, and 25%, respectively. Among them, the amount of PEG200 added was calculated according to the ratio of Chinese medicine compound water extract to PEG200 of 10 mL: 0.04 mL. The concentration of HTCC in the obtained gel was 0.05 g / mL, which were recorded as KMF@F127, KMF@F127-PEG200, and KMF@F 127-HTCC; the prepared 0.01875g crude drug / mL Chinese medicine compound water extract was mixed with the prescribed amount of freeze-dried microspheres (10mL: 0.01g), and the drug-loaded microspheres were obtained by stirring at 900rpm for 24h, and different masses of F127 (the final mass fraction of F127 was 19%, 20%, 21%, 22%, 23%, 24%, and 25%) were added, and the mixture was placed overnight by the cold dissolution method, and then PEG200 was added according to the ratio of the Chinese medicine compound water extract to PEG200 of 10mL:0.04mL, and the mixture was mixed to obtain F127-PEG200 (KMF@SA-GEL-PVP). Vials containing KMF@F127, KMF@F127-PEG200, F127-PEG200 (KMF@SA-GEL-PVP), and KMF@F127-HTCC with different F127 mass fractions were placed in a water bath and set at different temperatures. The phase transition temperature was recorded when the liquid surface stopped flowing within 5 minutes at this temperature.

[0104] Experimental results: Figure 7 As shown, compared with the use of F127 alone, the addition of PEG200 or HTCC causes F127 to undergo phase transition at a lower concentration. We need the gel to undergo phase transition at 31°C. We found that after adding PEG200, the F127 concentration that undergoes phase transition at 31°C is 21%. After further adding microspheres, the F127 concentration that undergoes phase transition at 31°C is 19%, which helps to reduce the concentration of F127 and makes it easier to spray. The F127 content of F127-HTCC at the time of phase transition at 31°C is 19.5%, and the consistency is too large.

[0105] The process of preparing the gel using HTCC is the same as the process of preparing the thermosensitive sustained-release hydrogel of the traditional Chinese medicine compound KMF, and the amount of HTCC used is 0.05 g / mL.

[0106] Example 4 Biocompatibility determination of KMF thermosensitive sustained-release hydrogel of traditional Chinese medicine compound

[0107] The MTT assay was used to detect the effects of various preparations on cell viability: L929 cells in the logarithmic growth phase were cultured at 1.0×10 4The cells / well were seeded at a density of 1:1280, 1:640, 1:160, 1:80, 1:40, 1:20, 1:10 g / mL and incubated at 37°C for 24 h. The optical density of each well was measured at 450 nm using a microplate reader.

[0108] Preparations: blank microspheres (SA-GEL-PVP, prepared according to Example 3), blank hydrogel (F127-PEG200, F127 was added to pure water at a ratio of 10 mL: 1.9 g, cold dissolution method, left overnight, then PEG200 was added at a ratio of 10 mL: 0.04 mL of pure water and PEG200, and mixed.), drug-loaded microspheres (KMF@SA-GEL-PVP, prepared according to Example 3), drug-loaded gel without microspheres (KMF@F1 27-PEG200, according to the ratio of 10mL:1.9g of the traditional Chinese medicine compound KMF water extract of 0.01875g raw material / mL and F127, placed overnight, and then according to the ratio of 10mL:0.04mL of the traditional Chinese medicine compound KMF water extract of 0.01875g raw material / mL and PEG200, PEG200 was added and mixed), traditional Chinese medicine compound KMF thermosensitive sustained-release hydrogel (F127-PEG200(KMF@SA-GEL-PVP)).

[0109] Experimental results: Figure 8-12 The results showed that blank microspheres, blank hydrogel (F127-PEG200), drug-loaded microspheres (KMF@SA-GEL-PV), drug-loaded gel without microspheres (KMF@F127-PEG200), and Chinese medicine compound KMF thermosensitive sustained-release hydrogel had good safety; the F127-PEG200 (KMF@SA-GEL-PVP) extract did not affect cell viability and had good biocompatibility.

[0110] Comparative Example 1

[0111] (1) Preparation of sodium alginate / gelatin / PVP molded microspheres: 12 g of sodium alginate (SA) and 6 g of gelatin (GEL) were added to ultrapure water (500 mL), and the solution was continuously stirred (5000 rpm) until it was completely dissolved to obtain a mixture. Next, polyvinyl pyrrolidone (PVP) was mixed at a ratio of 2 g PVP to ultrapure water to obtain a PVP aqueous solution, and then the PVP aqueous solution was added to the mixture, centrifuged at 7000 rpm for 10 min, and the supernatant was taken and freeze-dried. The temperature was gradually increased from -45°C to -25°C under a vacuum degree of 20 Pa, and the temperature was increased by 5 to 10°C every 2 hours. The temperature was maintained at -25°C until the freeze-drying was completed. The total freeze-drying time was 24 hours to obtain blank microspheres (SA-GEL-PVP). The blank microspheres were dissolved at a ratio of 0.01 g microspheres to pure water to obtain product A. The product was left to stand for 24 hours without stirring. Blank microspheres were dissolved in a ratio of 0.01 g microspheres to pure water of 10 mL and stirred at 900 rpm for 24 h to obtain product B. Fig.13 As shown: Fig.13 A in the figure means no stirring. It can be seen that without stirring, it is impossible to prepare formed microspheres with microsphere morphology. Fig.13 B in the sample was stirred at 900 rpm for 24 h, and it can be seen that stable blank molded microspheres can be formed.

[0112] (2) Add product A and product B obtained in step (1) to F127 respectively, with the final mass fraction of F127 being 19%. Use cold dissolution method, place overnight, then add PEG200 in a ratio of 10 mL:0.04 mL of pure water to PEG200, mix well, and obtain F127-PEG200 (SA-GEL-PVP) (unstirred) and F127-PEG200 (SA-GEL-PVP) (stirred). The obtained gel was subjected to temperature phase change test, and the results are as follows: Fig.14 As shown: It can be seen that when F127-PEG200 is added and the F127 content is 19%, the hydrogel containing blank formed microspheres stirred at 900 rpm for 24 hours can undergo phase transition at 30°C, while the hydrogel without blank formed microspheres that has not been stirred needs to undergo phase transition at 32°C. This result shows that the preparation of blank formed microspheres reduces the gelation phase transition temperature and can further reduce the amount of F127.

[0113] (3) Prepare blank microspheres (SA-GEL-PVP) according to step (1). Dissolve the blank microspheres in 0.01875 g / mL of the Chinese medicine compound KMF water extract at a ratio of 0.01 g:10 mL of the Chinese medicine compound KMF water extract, without stirring, and place naturally for 24 hours to obtain product C. Dissolve the blank microspheres in a ratio of 0.01 g:10 mL of the Chinese medicine compound KMF water extract, stir at 900 rpm for 24 hours, and obtain product D. Product C and product D were added with F127, the final mass fraction of F127 was 19%, cold dissolution method, left overnight, and then PEG200 was added according to the ratio of 0.01875g / mL Chinese medicine compound KMF water extract and PEG200 of 10mL:0.04mL, mixed, and obtained F127-PEG200 (KMF@SA-GEL-PVP) (unstirred) and F127-PEG200 (KMF@SA-GEL-PVP) (stirred). The obtained gel was tested for temperature phase change, and the results are as follows: Fig.15 As shown: It can be seen that when the F127 content is 19%, the hydrogel containing the traditional Chinese medicine compound KMF molded microspheres stirred at 900 rpm for 24 hours undergoes a phase transition at 31°C, while the hydrogel containing the traditional Chinese medicine compound KMF but not molded microspheres without stirring undergoes a phase transition at 29°C (as shown in Figure 2). Fig.15 The hydrogel prepared into the microspheres of the traditional Chinese medicine compound has a narrowed phase transition temperature range, and the phase transition occurs when the temperature is closer to the skin surface temperature, and the liquid range is increased, and the hydrogel is liquid and sprayable at room temperature.

[0114] The Chinese medicine compound KMF water extract and F127-PEG200 (KMF@SA-GEL-PVP) (stirred) were light yellow and transparent after preparation. Fig.16 As shown: Fig.16 A in the figure shows that the aqueous extract of the traditional Chinese medicine compound KMF has flocculated substances and its transparency is reduced; Fig.16 B in the figure shows that F127-PEG200 (KMF@SA-GEL-PVP) (stirred) has almost no flocculated substances, and the higher transparency indicates that the hydrogel prepared with microspheres increases its stability.

[0115] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a thermosensitive sustained-release hydrogel of a traditional Chinese medicine compound, characterized in that The method comprises the following preparation steps: (1) Extraction of Chinese herbal compound liquid; (2) Preparation of SA-GEL-PVP microspheres: Sodium alginate, gelatin and water were mixed and stirred until completely dissolved; polyvinyl pyrrolidone solution was added and mixed to obtain a mixture, and the supernatant was collected by centrifugation and dried to obtain SA-GEL-PVP microspheres; (3) Preparation of thermosensitive sustained-release hydrogel of traditional Chinese medicine compound: the traditional Chinese medicine compound liquid obtained in step (1) and the microspheres obtained in step (2) are mixed and stirred; F127 is added, allowed to stand overnight, and then PEG200 is added and mixed to obtain the thermosensitive sustained-release hydrogel of traditional Chinese medicine compound.

2. The preparation method according to claim 1, characterized in that: In step (1): The extraction is water extraction; The Chinese medicinal materials include Bupleurum chinense, Sophora flavescens, Dictamni cortex, Periostracum cicadae, Schizonepeta tenuifolia, Coptis chinensis, Phellodendron amurense, Saposhnikovia divaricata, Schisandra chinensis, Prunus mume, and Licorice.

3. The preparation method according to claim 2, characterized in that: The water extraction method comprises the following steps: soaking the Chinese medicinal materials in water, boiling and then decocting, separating the solid and the liquid to obtain the medicinal residue and the medicinal liquid A; concentrating the medicinal liquid A to obtain the Chinese medicinal compound liquid; The composition of the Chinese medicinal materials is as follows: 15g of Bupleurum chinense, 15g of Sophora flavescens, 15g of Dictamni cortex, 10g of Periostracum cicadae, 10g of Schizonepeta tenuifolia, 10g of Coptis chinensis, 10g of Phellodendron amurense, 10g of Saposhnikovia divaricata, 5g of Schisandra chinensis, 5g of Prunus mume, and 10g of Licorice.

4. The preparation method according to claim 3, characterized in that: The water extraction method also includes the following steps: repeatedly soaking the medicinal residues with water, decocting and extracting, to obtain medicinal solution B; combining and concentrating the medicinal solution B and the medicinal solution A to obtain the Chinese medicine compound medicinal solution.

5. The preparation method according to claim 1, characterized in that: In step (2): The water is ultrapure water; The sodium alginate, gelatin and polyvinyl pyrrolidone are mixed in a ratio of 10-15g: 5-7g: 1-3g; The concentration of the polyvinyl pyrrolidone solution is 0.3-0.5 g / 100 mL; The freeze-drying conditions are as follows: a vacuum degree of 20 Pa, a gradual temperature increase from -45°C to -25°C, an increase of 5 to 10°C every 2 hours, and maintaining -25°C until the freeze-drying is completed. The total freeze-drying time is 24 hours.

6. The preparation method according to claim 1, characterized in that: In step (2): The stirring condition is 400-600 rpm; The centrifugal condition is centrifugal at 6000-8000 rpm for 10 min; The drying is freeze drying.

7. The preparation method according to claim 1, characterized in that: In step (3): The concentration of the Chinese medicine compound liquid is 0.01-0.02 g of raw medicinal material / mL; The volume mass ratio of the Chinese medicine compound liquid to the microspheres is 8-12 mL: 0.005-0.02 g; The dosage of F127 is calculated based on the ratio of the Chinese medicine compound liquid to F127 of 10 mL: 1.9 g; The dosage of PEG200 is calculated based on the volume ratio of the Chinese medicine compound liquid to PEG200 being 250:1 (v / v).

8. The preparation method according to claim 1, characterized in that: In step (3): The stirring condition is 800-1000 rpm for 20-28 hours.

9. A Chinese medicine compound thermosensitive sustained-release hydrogel, characterized in that: The method is obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the traditional Chinese medicine compound temperature-sensitive sustained-release hydrogel according to claim 9 in the preparation of a drug for treating allergic contact dermatitis.

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