Preparation method of eight-treasure decoction nano-emulsion temperature-sensitive hydrogel for treating osteoporosis

Abstract

A preparation method of an eight-treasure decoction nano-emulsion temperature-sensitive hydrogel for treating osteoporosis comprises the following steps: S1, taking eight-treasure decoction raw medicinal materials according to a prescription amount, mixing two extracting solutions, concentrating, and drying the concentrated solution to obtain a final product eight-treasure decoction dry extract; s2, precisely weighing an emulsifier in a penicillin bottle, adding a proper amount of the eight-treasure soup extract, and continuously adding pure water, so that the emulsifier and the eight-treasure soup extract are fully dissolved to prepare a water phase; precisely weighing angelica sinensis oil to serve as an oil phase, slowly adding the water phase into the oil phase, carrying out vortex uniform mixing, and carrying out ultrasonic treatment in an ultrasonic cell crusher, so as to obtain the eight-treasure decoction nano-emulsion; s3, precisely weighing P188, P407 and bletilla striata polysaccharide respectively, adding pure water, and uniformly stirring to obtain a mixed solution; and transferring the eight-treasure decoction nano-emulsion in the step S2, adding the eight-treasure decoction nano-emulsion into the mixed solution, and uniformly stirring to obtain the nano-emulsion temperature-sensitive hydrogel. The prepared eight-treasure decoction nanoemulsion temperature-sensitive gel has good temperature sensitivity, the preparation method is simple, the process is stable, repeatability is good, and safety and reliability are achieved.

Description

Technical Field

[0001] This invention relates to the field of nanoemulsion gel technology, and more particularly to a method for preparing a Bazhentang nanoemulsion thermosensitive hydrogel for the treatment of osteoporosis. Background Technology

[0002] Bazhen Decoction is often used as an adjunct therapy in the clinical treatment of osteoporosis, in combination with conventional anti-osteoporosis drugs. Liu Yunbing analyzed the effect of modified Bazhen Decoction combined with zoledronic acid on postoperative rehabilitation of patients with osteoporotic vertebral compression fracture (OVCF). The results showed that postoperative treatment with modified Bazhen Decoction and zoledronic acid in OVCF patients could improve bone density, relieve postoperative pain, and accelerate the recovery of various indicators such as lumbar spine function, with a low incidence of adverse reactions, demonstrating high safety and effectiveness. Liu Jianjun et al. treated children with nonunion fractures using modified Bazhen Decoction. In a comparative study with a control group treated with Traumatology Bone-Setting Tablets, they found that modified Bazhen Decoction could increase serum BMP-7 and VEGF levels, thereby promoting fracture healing and achieving satisfactory results. For elderly patients with osteoporosis, Bazhen Decoction can improve digestive and absorptive functions through its "spleen and kidney tonifying" effect, promoting the absorption and utilization of calcium and nutrients, thereby enhancing the effect of basic treatment. It is worth noting that traditional Bazhen Decoction formulations have problems such as low bioavailability, short duration of action, and inconvenient administration. Modifying the dosage form of Bazhen Decoction into a nanoemulsion thermosensitive gel system is expected to solve these problems and significantly enhance its clinical application value.

[0003] Nanoemulsions (also known as microemulsion systems), as colloidal dispersion systems, can be classified into three basic forms based on their structural characteristics: oil-in-water (O / W), water-in-oil (W / O), and bicontinuous phase structure. This system consists of oil and water phases, surfactants, and co-emulsifiers. The dispersed phase particle size is controlled within the 1-100 nm range, exhibiting thermodynamic stability and a transparent to semi-transparent state. The O / W type is characterized by oil droplets dispersed in a continuous aqueous phase, the W / O type consists of aqueous microparticles encapsulated in an oil matrix, and the bicontinuous type displays a unique spatial configuration of interwoven oil and water phases. Nanoemulsions can deliver drugs through multiple pathways, including transdermal, mucosal absorption, and systemic administration, exhibiting superior carrier performance, especially in special drug delivery scenarios such as the nasal cavity and ocular mucosa. Their nanoscale droplet structure not only enhances drug solubility but also allows for precise control of lipophilic or hydrophilic properties through surface modification, thereby optimizing targeted delivery to different tissues and organs.

[0004] As an innovative formulation technology, nanoemulsion systems achieve multiple pharmacodynamic optimizations through unique physicochemical properties. Their nanoscale drug-loading structures not only ensure drug stability control but also significantly improve bioavailability and precise targeted delivery capabilities through pharmacokinetic optimization. This delivery platform, integrating targeted modification and sustained-release / controlled-release, demonstrates enormous application potential in the field of novel formulation development. However, pure nanoemulsion formulations still suffer from problems such as rapid in vivo clearance and the need for frequent dosing. Combining nanoemulsions with thermosensitive gel technology can further optimize drug release kinetics and achieve long-term therapeutic effects, which is precisely the design intention of the Bazhen Tang nanoemulsion thermosensitive gel system.

[0005] Thermosensitive hydrogels are a class of smart polymer materials that undergo reversible sol-gel phase transitions in response to changes in ambient temperature, and have attracted widespread attention in the biomedical field in recent years. These materials exhibit significant changes in rheological properties near their critical phase transition temperatures (LCST or UCST), making them ideal for smart drug delivery systems and tissue engineering scaffolds. Thermosensitive hydrogels are mainly divided into two categories: gels with a low critical solution temperature (LCST) and gels with a high critical solution temperature (UCST). LCST-type gels dissolve at low temperatures, and phase separation occurs and a gel forms when the temperature rises to the LCST. Typical examples include poly(N-isopropylacrylamide) (PNIPAAm) and its derivatives, methylcellulose, and poloxamer. UCST-type gels, on the other hand, dissolve at high temperatures and form a gel upon cooling, such as the polyacrylamide / polyacrylic acid interpenetrating network system. This temperature responsiveness mainly stems from changes in the hydrophilic and hydrophobic balance within the polymer chains; temperature changes lead to a reorganization of intramolecular and intermolecular interactions. The most widely used type of thermosensitive gel in the medical field is LCST. Its phase transition temperature can be precisely controlled by adjusting the polymer composition and concentration. It is usually set below room temperature and above body temperature (25-37℃) so that gelation is triggered by body temperature after injection or application.

[0006] Zhang Chunyan et al. constructed a temperature-sensitive microemulsion gel delivery system, using ethyl oleate as the oil phase matrix, combined with polysorbate 80 surfactant and anhydrous ethanol as a co-emulsifier, and incorporating poloxamer 188 and 407 to form a temperature-sensitive structure. They studied the microstructural characteristics and physical stability of this formulation and compared its transdermal performance with commercially available products. The results showed that the novel gel had a faster transdermal rate than traditional creams, and the amount of drug accumulated in the skin was five times that of commercially available formulations. Experiments confirmed that this innovative formulation can rapidly penetrate the stratum corneum to form a subcutaneous drug reservoir, significantly enhancing antibacterial efficacy by increasing local drug concentration.

[0007] Deng Peng developed a triptolide-based thermosensitive gel system based on the solubilizing properties of nanoemulsions. A temperature-responsive system was constructed using poloxamer 407 and 188 matrices, and the phase transition temperature was optimized using response surface methodology. A pseudo-ternary phase diagram was established to screen the ratios of the oil phase, surfactant, and co-emulsifier. The nanoemulsion particle size was controlled using a central group design method. Systematic evaluation showed that the formulation possesses sustained-release properties and broad-spectrum antibacterial activity. Animal experiments confirmed that it has a prolonged ocular retention time and significantly reduced irritation. In an LPS-induced stromal keratitis model, the gel's anti-inflammatory effect showed a dose-response relationship. The results indicate that this preparation process is feasible and demonstrates advantages of low irritation and high delivery efficiency in ophthalmic formulations.

[0008] Thermosensitive nanogel delivery systems have attracted significant attention in the field of novel formulations due to their intelligent controlled release and precise targeting characteristics. This technology achieves long-term sustained drug release through phase transition regulation, improving bioavailability while simultaneously reducing systemic toxicity, and has important translational value in the field of precision medicine. Summary of the Invention

[0009] To address the problems mentioned in the background section, this invention provides a method for preparing a thermosensitive hydrogel containing Bazhentang nanoemulsion for treating osteoporosis.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A method for preparing a thermosensitive hydrogel based on Bazhen Tang nanoemulsion for treating osteoporosis includes the following steps:

[0012] S1. Take the prescribed amount of the original medicinal materials of Bazhen Decoction, soak them in 10 times the amount of water for 2 hours, decoct for 30 minutes, filter out the extract, add 8 times the amount of water for the second time, continue to decoct for 30 minutes, filter out the extract again, combine the two extracts and concentrate them, dry the concentrate to obtain the final product Bazhen Decoction dry extract.

[0013] S2. Accurately weigh the emulsifier into a vial, add an appropriate amount of Bazhentang extract, and continue to add pure water to fully dissolve the emulsifier and Bazhentang extract to form an aqueous phase; accurately weigh the Angelica sinensis oil as the oil phase, slowly add the aqueous phase to the oil phase, vortex mix for 10 minutes, and then place it in an ultrasonic cell disruptor (240-420W, 5s working, 5s intermittent) for 6-8 minutes to obtain Bazhentang nanoemulsion.

[0014] S3. Accurately weigh poloxamer 188 (P188), poloxamer 407 (P407), and Bletilla striata polysaccharide, place them in a 25 mL beaker, add pure water, and stir well to obtain a mixed solution. Accurately transfer the Bazhen Tang nanoemulsion from S2 to the above mixed solution, stir well, and place it in a refrigerator at 3-5℃ for 20-24 h to swell, thus obtaining the nanoemulsion thermosensitive hydrogel.

[0015] Preferably, the emulsifier in S2 is tea saponin; the amount of emulsifier added in S2 is 0.6%-1.2% of pure water.

[0016] Preferably, the mass ratio of emulsifier to Bazhentang extract in S2 is (30-60):(2-10).

[0017] Preferably, the amount of Angelica oil added in S2 is 0.5%-3% of pure water.

[0018] Preferably, the mass ratio of poloxamer 188, poloxamer 407 and Bletilla striata polysaccharide in S3 is (4-4.5):(18-18.5):1.

[0019] Preferably, the volume ratio of pure water to Bazhentang nanoemulsion in S3 is (2-2.2):(5-5.5).

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] The thermosensitive gel of Bazhentang nanoemulsion prepared by this invention has a uniform appearance and good fluidity at room temperature. It rapidly undergoes a phase transition to form a semi-solid gel near body temperature (32-37℃), with the gelation time controlled within 60-90 seconds. This reflects the temperature response characteristics of the thermosensitive material (poloxam). This characteristic can ensure that the drug is accurately retained and slowly released at the target site, reducing the risk of systemic exposure.

[0022] The nanoemulsion system exhibited good stability: the encapsulation efficiency was 104.3% (paeoniflorin component), attributed to the synergistic effect of the thermosensitive material and the amphiphilic structure of the nanoemulsion; the pH value remained stable within the range of 5-6, compatible with the physiological environment of the skin, avoiding local irritation; the erosion test showed that the gel eroded 100% within 2 hours under simulated physiological conditions. The results indicate that the Bazhentang nanoemulsion gel is superior to traditional dosage forms in terms of quality controllability, functionality, and application potential. The Bazhentang nanoemulsion thermosensitive gel prepared by this invention has good temperature sensitivity, a simple preparation method, stable process, good repeatability, and is safe and reliable. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The appearance of the Bazhentang nanoemulsion thermosensitive hydrogel prepared in Example 1 of the present invention at 4°C (A) and 37°C (B);

[0025] Figure 2 The stability of the Bazhentang nanoemulsion thermosensitive hydrogel prepared in Example 1 of this invention;

[0026] Figure 3 Centrifugal stability of the Bazhentang nanoemulsion thermosensitive hydrogel prepared in Example 1 of this invention;

[0027] Figure 4 The gelation time of the Bazhentang nanoemulsion thermosensitive hydrogel prepared in Example 1 of this invention was determined;

[0028] Figure 5 The erosion rate of the Bazhentang nanoemulsion thermosensitive hydrogel prepared in Example 1 of this invention;

[0029] Figure 6 The amount of gel remaining during the dissolution process of the Bazhentang nanoemulsion thermosensitive hydrogel prepared in Example 1 of this invention;

[0030] Figure 7 The pH of the Bazhen Tang nanoemulsion thermosensitive hydrogel prepared in Example 1 of this invention after 7 days;

[0031] Figure 8 This is the standard curve for paeoniflorin. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the raw materials used in this invention are all from commercially available conventional products.

[0034] Tea saponins were purchased from Shanghai Yuanye Biotechnology Co., Ltd., BR, 65%;

[0035] Angelica oil was purchased from Hubei Nuona Technology Co., Ltd., 99% purity;

[0036] Poloxamer 188 was purchased from Beijing Solarbio Technology Co., Ltd.

[0037] Poloxamer 407 was purchased from Beijing Solarbio Technology Co., Ltd.

[0038] Bletilla striata polysaccharide, Shaanxi Baichuan Biotechnology Co., Ltd. / 99%.

[0039] Example 1

[0040] A method for preparing a thermosensitive hydrogel based on Bazhen Tang nanoemulsion for treating osteoporosis includes the following steps:

[0041] S1. Take 13.2g of the original medicinal materials of Bazhen Decoction according to the prescription. Soak in 10 times the amount of water for 2 hours, decoct for 30 minutes, and filter out the extract. Add 8 times the amount of water for the second time, continue to decoct for 30 minutes, filter out the extract again, combine the two extracts and concentrate them. Dry the concentrate to obtain the final product, Bazhen Decoction dry extract.

[0042] S2. Accurately weigh 0.045g of emulsifier into a vial, add 6mg of Bazhentang extract, and continue to add 5mL of pure water to fully dissolve the emulsifier and Bazhentang extract to form an aqueous phase; accurately weigh 0.05g of Angelica sinensis oil as the oil phase, slowly add the aqueous phase to the oil phase, vortex mix for 10min, and then place it in an ultrasonic cell disruptor (300W, 5s working, 5s intermittent) for 7min to obtain Bazhentang nanoemulsion;

[0043] S3. Accurately weigh 0.44g of poloxamer 188, 1.82g of poloxamer 407, and 0.10g of Bletilla striata polysaccharide, place them in a 25mL beaker, add 2.2mL of pure water, and stir well to obtain a mixed solution. Accurately transfer 5.5mL of the Bazhen Tang nanoemulsion from S2 to the above mixed solution, stir well, and place in a 4℃ refrigerator for swelling for 24h to obtain the nanoemulsion thermosensitive hydrogel.

[0044] Example 2

[0045] A method for preparing a thermosensitive hydrogel based on Bazhen Tang nanoemulsion for treating osteoporosis includes the following steps:

[0046] S1. Take 13g of the original medicinal materials of Bazhen Decoction according to the prescription. Soak in 10 times the amount of water for 2 hours, decoct for 30 minutes, filter out the extract. Add 8 times the amount of water for the second time, continue to decoct for 30 minutes, filter out the extract again, combine the two extracts and concentrate them. Dry the concentrate to obtain the final product Bazhen Decoction dry extract.

[0047] S2. Accurately weigh 0.05g of emulsifier into a vial, add 2mg of Bazhentang extract, and continue to add 5mL of pure water to fully dissolve the emulsifier and Bazhentang extract to form an aqueous phase; accurately weigh 0.1g of Angelica sinensis oil as the oil phase, slowly add the aqueous phase to the oil phase, vortex mix for 10min, and then place it in an ultrasonic cell disruptor (300W, 5s working, 5s intermittent) for 7min to obtain Bazhentang nanoemulsion;

[0048] S3. Accurately weigh 0.4g of poloxamer 188, 1.8g of poloxamer 407, and 0.10g of Bletilla striata polysaccharide, place them in a 25mL beaker, add 2.2mL of pure water, and stir well to obtain a mixed solution. Accurately transfer 5.5mL of the Bazhen Tang nanoemulsion from S2 to the above mixed solution, stir well, and place in a 4℃ refrigerator for swelling for 24h to obtain the nanoemulsion thermosensitive hydrogel.

[0049] Example 3

[0050] A method for preparing a thermosensitive hydrogel based on Bazhen Tang nanoemulsion for treating osteoporosis includes the following steps:

[0051] S1. Take 13.5g of the original medicinal materials of Bazhen Decoction according to the prescription. Soak in 10 times the amount of water for 2 hours, decoct for 30 minutes, filter out the extract. Add 8 times the amount of water for the second time, continue to decoct for 30 minutes, filter out the extract again, combine the two extracts and concentrate them. Dry the concentrate to obtain the final product Bazhen Decoction dry extract.

[0052] S2. Accurately weigh 0.045g of emulsifier into a vial, add 8mg of Bazhentang extract, and continue to add 5mL of pure water to fully dissolve the emulsifier and Bazhentang extract to form an aqueous phase; accurately weigh 0.05g of Angelica sinensis oil as the oil phase, slowly add the aqueous phase to the oil phase, vortex mix for 10min, and then place it in an ultrasonic cell disruptor (300W, 5s working, 5s intermittent) for 7min to obtain Bazhentang nanoemulsion;

[0053] S3. Accurately weigh 0.44g of poloxamer 188, 1.82g of poloxamer 407, and 0.10g of Bletilla striata polysaccharide, place them in a 25mL beaker, add 2.2mL of pure water, and stir well to obtain a mixed solution. Accurately transfer 5.5mL of the Bazhen Tang nanoemulsion from S2 to the above mixed solution, stir well, and place in a 4℃ refrigerator for swelling for 24h to obtain the nanoemulsion thermosensitive hydrogel.

[0054] Example 4

[0055] A method for preparing a thermosensitive hydrogel based on Bazhen Tang nanoemulsion for treating osteoporosis includes the following steps:

[0056] S1. Take 13.2g of the original medicinal materials of Bazhen Decoction according to the prescription. Soak in 10 times the amount of water for 2 hours, decoct for 30 minutes, and filter out the extract. Add 8 times the amount of water for the second time, continue to decoct for 30 minutes, filter out the extract again, combine the two extracts and concentrate them. Dry the concentrate to obtain the final product, Bazhen Decoction dry extract.

[0057] S2. Accurately weigh 0.045g of emulsifier into a vial, add 4mg of Bazhentang extract, and continue to add 5mL of pure water to fully dissolve the emulsifier and Bazhentang extract to form an aqueous phase; accurately weigh 0.05g of Angelica sinensis oil as the oil phase, slowly add the aqueous phase to the oil phase, vortex mix for 10min, and then place it in an ultrasonic cell disruptor (240W, 5s working, 5s intermittent) for 6min to obtain Bazhentang nanoemulsion;

[0058] S3. Accurately weigh 0.45g of poloxamer 188, 1.85g of poloxamer 407, and 0.10g of Bletilla striata polysaccharide, place them in a 25mL beaker, add 2.2mL of pure water, and stir well to obtain a mixed solution. Accurately transfer 5.5mL of the Bazhen Tang nanoemulsion from S2 to the above mixed solution, stir well, and place it in a 3℃ refrigerator for 24h to swell, thus obtaining the nanoemulsion thermosensitive hydrogel.

[0059] Example 5

[0060] A method for preparing a thermosensitive hydrogel based on Bazhen Tang nanoemulsion for treating osteoporosis includes the following steps:

[0061] S1. Take 13.2g of the original medicinal materials of Bazhen Decoction according to the prescription. Soak in 10 times the amount of water for 2 hours, decoct for 30 minutes, and filter out the extract. Add 8 times the amount of water for the second time, continue to decoct for 30 minutes, filter out the extract again, combine the two extracts and concentrate them. Dry the concentrate to obtain the final product, Bazhen Decoction dry extract.

[0062] S2. Accurately weigh 0.045g of emulsifier into a vial, add 10mg of Bazhentang extract, and continue to add 5mL of pure water to fully dissolve the emulsifier and Bazhentang extract to form an aqueous phase; accurately weigh 0.05g of Angelica sinensis oil as the oil phase, slowly add the aqueous phase to the oil phase, vortex mix for 10min, and then place it in an ultrasonic cell disruptor (400W, 5s working, 5s intermittent) for 8min to obtain Bazhentang nanoemulsion;

[0063] S3. Accurately weigh 0.43g of poloxamer 188, 1.81g of poloxamer 407, and 0.10g of Bletilla striata polysaccharide, place them in a 25mL beaker, add 2.2mL of pure water, and stir well to obtain a mixed solution. Accurately transfer 5.5mL of the Bazhen Tang nanoemulsion from S2 to the above mixed solution, stir well, and place it in a 5℃ refrigerator for 20h to swell, thus obtaining the nanoemulsion thermosensitive hydrogel.

[0064] Example 6

[0065] A method for preparing a thermosensitive hydrogel based on Bazhen Tang nanoemulsion for treating osteoporosis includes the following steps:

[0066] S1. Take 13g of the original medicinal materials of Bazhen Decoction according to the prescription. Soak in 10 times the amount of water for 2 hours, decoct for 30 minutes, filter out the extract. Add 8 times the amount of water for the second time, continue to decoct for 30 minutes, filter out the extract again, combine the two extracts and concentrate them. Dry the concentrate to obtain the final product Bazhen Decoction dry extract.

[0067] S2. Accurately weigh 0.06g of emulsifier into a vial, add 4mg of Bazhentang extract, and continue to add 5mL of pure water to fully dissolve the emulsifier and Bazhentang extract to form an aqueous phase; accurately weigh 0.1g of Angelica sinensis oil as the oil phase, slowly add the aqueous phase to the oil phase, vortex mix for 10min, and then place it in an ultrasonic cell disruptor (360W, 5s working, 5s intermittent) for 6min to obtain Bazhentang nanoemulsion;

[0068] S3. Accurately weigh 0.4g of poloxamer 188, 1.8g of poloxamer 407, and 0.10g of Bletilla striata polysaccharide, place them in a 25mL beaker, add 2.2mL of pure water, and stir well to obtain a mixed solution. Accurately transfer 5.5mL of the Bazhen Tang nanoemulsion from S2 to the above mixed solution, stir well, and place in a 4℃ refrigerator for swelling for 24h to obtain the nanoemulsion thermosensitive hydrogel.

[0069] Example 7

[0070] A method for preparing a thermosensitive hydrogel based on Bazhen Tang nanoemulsion for treating osteoporosis includes the following steps:

[0071] S1. Take 13g of the original medicinal materials of Bazhen Decoction according to the prescription. Soak in 10 times the amount of water for 2 hours, decoct for 30 minutes, filter out the extract. Add 8 times the amount of water for the second time, continue to decoct for 30 minutes, filter out the extract again, combine the two extracts and concentrate them. Dry the concentrate to obtain the final product Bazhen Decoction dry extract.

[0072] S2. Accurately weigh 0.05g of emulsifier into a vial, add 6mg of Bazhentang extract, and continue to add 5mL of pure water to fully dissolve the emulsifier and Bazhentang extract to form an aqueous phase; accurately weigh 0.1g of Angelica sinensis oil as the oil phase, slowly add the aqueous phase to the oil phase, vortex mix for 10min, and then place it in an ultrasonic cell disruptor (380W, 5s working, 5s intermittent) for 8min to obtain Bazhentang nanoemulsion;

[0073] S3. Accurately weigh 0.4g of poloxamer 188, 1.8g of poloxamer 407, and 0.10g of Bletilla striata polysaccharide, place them in a 25mL beaker, add 2.2mL of pure water, and stir well to obtain a mixed solution. Accurately transfer 5.5mL of the Bazhen Tang nanoemulsion from S2 to the above mixed solution, stir well, and place in a 4℃ refrigerator for swelling for 24h to obtain the nanoemulsion thermosensitive hydrogel.

[0074] Example 8

[0075] A method for preparing a thermosensitive hydrogel based on Bazhen Tang nanoemulsion for treating osteoporosis includes the following steps:

[0076] S1. Take 13.5g of the original medicinal materials of Bazhen Decoction according to the prescription. Soak in 10 times the amount of water for 2 hours, decoct for 30 minutes, filter out the extract. Add 8 times the amount of water for the second time, continue to decoct for 30 minutes, filter out the extract again, combine the two extracts and concentrate them. Dry the concentrate to obtain the final product Bazhen Decoction dry extract.

[0077] S2. Accurately weigh 0.05g of emulsifier into a vial, add 6mg of Bazhentang extract, and continue to add 5mL of pure water to fully dissolve the emulsifier and Bazhentang extract to form an aqueous phase; accurately weigh 0.05g of Angelica sinensis oil as the oil phase, slowly add the aqueous phase to the oil phase, vortex mix for 10min, and then place it in an ultrasonic cell disruptor (300W, 5s working, 5s intermittent) for 7min to obtain Bazhentang nanoemulsion;

[0078] S3. Accurately weigh 0.44g of poloxamer 188, 1.82g of poloxamer 407, and 0.10g of Bletilla striata polysaccharide, place them in a 25mL beaker, add 2.2mL of pure water, and stir well to obtain a mixed solution. Accurately transfer 5.5mL of the Bazhen Tang nanoemulsion from S2 to the above mixed solution, stir well, and place it in a 3℃ refrigerator for 20h to swell, thus obtaining the nanoemulsion thermosensitive hydrogel.

[0079] Example 9

[0080] A method for preparing a thermosensitive hydrogel based on Bazhen Tang nanoemulsion for treating osteoporosis includes the following steps:

[0081] S1. Take 13.5g of the original medicinal materials of Bazhen Decoction according to the prescription. Soak in 10 times the amount of water for 2 hours, decoct for 30 minutes, filter out the extract. Add 8 times the amount of water for the second time, continue to decoct for 30 minutes, filter out the extract again, combine the two extracts and concentrate them. Dry the concentrate to obtain the final product Bazhen Decoction dry extract.

[0082] S2. Accurately weigh 0.03g of emulsifier into a vial, add 6mg of Bazhentang extract, and continue to add 5mL of pure water to fully dissolve the emulsifier and Bazhentang extract to form an aqueous phase; accurately weigh 0.05g of Angelica sinensis oil as the oil phase, slowly add the aqueous phase to the oil phase, vortex mix for 10min, and then place it in an ultrasonic cell disruptor (320W, 5s working, 5s intermittent) for 7min to obtain Bazhentang nanoemulsion;

[0083] S3. Accurately weigh 0.45g of poloxamer 188, 1.85g of poloxamer 407, and 0.10g of Bletilla striata polysaccharide, place them in a 25mL beaker, add 2.2mL of pure water, and stir well to obtain a mixed solution. Accurately transfer 5.5mL of the Bazhen Tang nanoemulsion from S2 to the above mixed solution, stir well, and place it in a 5℃ refrigerator for 24h to swell, thus obtaining the nanoemulsion thermosensitive hydrogel.

[0084] Experimental methods:

[0085] 1. Appearance and properties of Bazhentang nanoemulsion thermosensitive gel

[0086] The appearance of the Bazhentang nanoemulsion gel in Example 1 was observed at 4 and 37°C.

[0087] The results are as follows Figure 1 As shown: The appearance of the nanoemulsion thermosensitive hydrogel at 4℃ and 37℃ is as follows. Figure 1 As shown, the nanoemulsion thermosensitive hydrogel is a yellow solution with fluidity at 4℃ (A); and a yellow semi-solid with no fluidity at 37℃ (B), indicating that the nanoemulsion gel has good temperature sensitivity.

[0088] 2. Stability of Bazhentang Nanoemulsion Thermosensitive Gel

[0089] Room temperature stability: After storing the prepared Bazhentang nanoemulsion thermosensitive gel at 4℃, room temperature, and 37℃ for 1 day, 5 days, and 10 days, observe the appearance for any obvious changes, and whether there are any phenomena such as layering or precipitation.

[0090] Centrifugal stability: The Bazhentang nanoemulsion thermosensitive gel prepared in Example 1 was centrifuged at 4°C and 37°C for 20 min (4000 r / min) respectively, and the appearance was observed to see if there were any changes, or if there were any flocculent or layered phenomena.

[0091] The thermosensitive nanoemulsion gel of Bazhentang (a traditional Chinese medicine formula) remained uniform and transparent after storage at 4℃, room temperature, and 37℃ for 1 day, 5 days, and 10 days, respectively. No layering or precipitation was observed, indicating good stability of the nanoemulsion gel at these temperatures. Figure 2 As shown.

[0092] The prepared Bazhentang nanoemulsion thermosensitive gel was centrifuged at 4℃ and 37℃ for 20 min (4000 r / min). No flocculent or layered phenomena were observed. Furthermore, the temperature values ​​(T) before and after centrifugation were (31.9±0.1)℃ and (32.5±0.3)℃, respectively, showing no significant change. This indicates that the nanoemulsion thermosensitive gel has good centrifugal stability. Figure 3 As shown.

[0093] 3. Determination of gelation time of Bazhentang nanoemulsion thermosensitive gel

[0094] Precisely transfer 5 mL of Bazhen Tang nanoemulsion thermosensitive gel into a 10 mL vial. Heat in a 37℃ constant temperature water bath, ensuring the gel-containing portion of the vial is completely submerged. Remove the vial every 10 seconds to observe the gel state until the vial is inverted for 15 seconds and the gel stops flowing. Record the time t, which is the gelation time (6 parallel determinations). Results are as follows: Figure 4As shown.

[0095] 4. Erosion rate of Bazhentang nanoemulsion thermosensitive gel

[0096] The gel erosion rate indicates the rate at which the gel dissolves without detaching from the site of action.

[0097] The gel erosion rate was determined according to the method of Zhou Feilong et al. Michael CW, Kevin C, Ellen E. Curcumin, a turmeric extract, for oral lichen planus: A systematic review. [J]. Oral diseases, 2019, 25(3):720-7.

[0098] Before the experiment, the weight of the vial was recorded as M0. 1 mL of sample was transferred to the vial, and the total weight (M1) was measured. The vial was then placed in a 36°C water bath for 1 minute to equilibrate. Then, 3 mL of 36°C PBS buffer was slowly added to the vial. The vial was then placed in a 36°C water bath and shaken (50 rpm). At different time points (20 min, 40 min, 60 min, 80 min, 100 min, 120 min), all the PBS buffer on top of the gel was removed. The weight of the vial and gel at these times was recorded as Mt (t = 20 min, 40 min, 60 min, 80 min, 100 min, 120 min), and 3 mL of 36°C PBS buffer was added again. The erosion rate of the gel at a given time point was calculated using the following formula: Erosion rate = (M1 - Mt) / (M1 - M0) × 100%.

[0099] The erosion rate results of the gel are shown in Table 1. Figure 5 , Figure 6 As shown, all samples were completely dissolved (100%) within 100 minutes. The dissolution rate was time-dependent: significant differences were observed in the first 60 minutes (y1 was the fastest, reaching 77.78% at 60 minutes, while y3 was the slowest at 60%), but they tended to synchronize in the later stages (y1: 88.89% at 80 minutes, y2: 87.5%, y3: 80%). The batch-to-batch differences were mainly reflected in the initial dissolution stage, which may be due to differences in nanoemulsion particle size or cross-linking degree, but the final dissolution behavior was consistent, indicating that the gel system has stable overall release performance.

[0100] Table 1. Erosion rate of Bazhentang nanoemulsion gel

[0101]

[0102]

[0103] 5. pH of Bazhentang Nanoemulsion Thermosensitive Gel

[0104] Take an appropriate amount of the prepared Bazhentang nanoemulsion temperature-sensitive gel and measure its pH value using a pH meter at 4℃ and room temperature, repeating the measurement three times. Monitor the pH stability for seven consecutive days.

[0105] pH stability results are as follows Figure 7 As shown, the pH of the Bazhentang nanoemulsion thermosensitive gel is not significantly different between 4℃ and room temperature, and is basically stable between 5 and 5.5.

[0106] 6. Construction of the paeoniflorin standard curve

[0107] Because the composition of Bazhen Decoction extract is complex, the amount of paeoniflorin, one of the main components, was selected and determined by ultraviolet spectrophotometry to determine the drug loading in Bazhen Decoction gel.

[0108] Accurately weigh 10.5 mg of paeoniflorin standard and dilute to 50 ml in a volumetric flask with 50% ethanol to prepare a reference standard with a concentration of 0.21 mg / ml. Accurately remove 0.2 ml, 0.3 ml, 0.35 ml, 0.4 ml, and 0.5 ml of the reference standard solution into five 10 ml volumetric flasks, respectively, and dilute to the mark with 50% ethanol. Using these as references, measure the absorbance at 230 nm using a UV spectrophotometer. Establish a standard curve with A as the ordinate (y) and paeoniflorin concentration as the abscissa (x).

[0109] The regression equation for paeoniflorin is y = 74.81x - 0.1089, with a correlation coefficient r = 0.9994. (From...) Figure 8 It can be seen that the paeoniflorin standard has a good linear relationship in the concentration range of 0.0042 to 0.0105 mg / mL.

[0110] 7. Stability of the sample solution

[0111] Dissolve 0.05g of Bazhentang nanoemulsion thermosensitive gel in 50% ethanol, filter through a 0.45µm microporous membrane, and bring the volume to 25ml to obtain the sample solution. Take an appropriate amount of the sample solution in a test tube and measure the absorbance at 0h, 2h, 4h, 8h, and 12h. The stability of the sample solution can be determined by observing the absorbance at different time points.

[0112] As shown in Table 2, the RSD value of the stability test was 0.24%, which proves that the test solution has good stability within 12 hours.

[0113] Table 2 Stability test results

[0114]

[0115] 8. Precision test

[0116] Following the method for establishing a standard curve, three paeoniflorin solutions with different mass concentrations of 0.0063, 0.0074, and 0.0084 mg / ml were prepared. The absorbance of each concentration was measured at a maximum wavelength of 230 nm using an ultraviolet spectrophotometer. Each concentration was measured six times to determine the intraday precision.

[0117] As shown in Table 3, the RSD values ​​of the three concentrations (0.0063 mg / ml, 0.0074 mg / ml, and 0.0084 mg / ml) are 0.300%, 0.156%, and 0.089%, respectively. Since the RSD values ​​are all less than 1%, the intraday precision of the solution is good.

[0118] Table 3 Precision Experiment Results

[0119]

[0120] 9. Recovery rate experiment

[0121] Three paeoniflorin solutions with different mass concentrations of 0.0063, 0.0074, and 0.0084 mg / ml were prepared. The absorbance of each concentration was measured at the maximum absorption wavelength of 230 nm. Each concentration in each group was measured three times, and the average value was taken. The measured absorbance was substituted into the known paeoniflorin standard curve to calculate the concentration of the paeoniflorin solution. The recovery rate was calculated (recovery rate = calculated concentration / known concentration × 100%), and the relative standard deviation (RSD%) was calculated.

[0122] As shown in Table 4, the recoveries of the three concentrations (0.0063 mg / ml, 0.0074 mg / ml, and 0.0084 mg / ml) were 98.48%, 100.76%, and 101.51%, respectively, with RSD values ​​of 0.27%, 0.34%, and 0.32%, respectively. The recoveries were between 98% and 102%, and the RSD values ​​were within 2%, which proves that the method has good recovery and the determination results are relatively accurate.

[0123] Table 4 Recovery rates of paeoniflorin at different concentrations

[0124]

[0125] 10. Encapsulation rate

[0126] 0.05 g of nanoemulsion gel was dissolved in ethanol, centrifuged at 4000 r / min, and the supernatant was collected. The absorbance was measured at the maximum wavelength of 230 nm. The amount of paeoniflorin encapsulated in the nanoemulsion gel, M1, was determined by substituting the absorbance into the standard curve. (Encapsulation rate = M1 / M0 × 100%, where M0 is the content of paeoniflorin in the nanoemulsion gel, mg / g). The encapsulation rate of this Bazhen Decoction nanoemulsion thermosensitive gel was 104.3%.

[0127] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a thermosensitive hydrogel containing Bazhen Tang nanoemulsion for treating osteoporosis, characterized in that, Includes the following steps: S1. Take the original medicinal materials of Bazhen Tang, soak them in water for 2 hours for the first time, decoct for 30 minutes, filter out the extract, add water for the second time, continue to decoct for 30 minutes, filter out the extract again, combine the two extracts and concentrate them, dry the concentrate to obtain the final product Bazhen Tang dry extract. S2. Accurately weigh the emulsifier into a vial, add an appropriate amount of Bazhentang extract, and continue to add pure water to fully dissolve the emulsifier and Bazhentang extract to form an aqueous phase. Precisely weigh out Angelica sinensis oil as the oil phase, slowly add the aqueous phase to the oil phase, vortex mix well, and then sonicate in an ultrasonic cell disruptor to obtain Bazhentang nanoemulsion. S3. Place poloxamer 188, poloxamer 407 and Bletilla striata polysaccharide in beakers, add pure water, stir well to prepare a mixed solution, accurately transfer the Bazhentang nanoemulsion from S2, add it to the above mixed solution, stir well, and then refrigerate in a refrigerator to swell, thus obtaining the nanoemulsion thermosensitive hydrogel.

2. The method for preparing the Bazhen Tang nanoemulsion thermosensitive hydrogel for treating osteoporosis according to claim 1, characterized in that, The amount of water added in the first step of S1 is ten times that of the original medicinal materials for Bazhen Tang. The amount of water added the second time is eight times the amount of the original medicinal materials for Bazhen Tang.

3. The method for preparing a Bazhen Tang nanoemulsion thermosensitive hydrogel for treating osteoporosis according to claim 1, characterized in that, The emulsifier in S2 is tea saponin; The amount of emulsifier added in S2 is 0.6%-1.2% of pure water.

4. The method for preparing a Bazhen Tang nanoemulsion thermosensitive hydrogel for treating osteoporosis according to claim 1, characterized in that, The mass ratio of emulsifier to Bazhentang extract in S2 is (30-60):(2-10).

5. The method for preparing a Bazhen Tang nanoemulsion thermosensitive hydrogel for treating osteoporosis according to claim 1, characterized in that, The amount of Angelica oil added in S2 is 0.5%-3% of pure water.

6. The method for preparing a Bazhen Tang nanoemulsion thermosensitive hydrogel for treating osteoporosis according to claim 1, characterized in that, The ultrasonic cell disruptor in S2 has a power of 240-420W and an ultrasonic time of 6-8 minutes.

7. The method for preparing a Bazhen Tang nanoemulsion thermosensitive hydrogel for treating osteoporosis according to claim 1, characterized in that, In S2, the ultrasound operates for 5 seconds, followed by a 5-second interval.

8. The method for preparing a Bazhen Tang nanoemulsion thermosensitive hydrogel for treating osteoporosis according to claim 1, characterized in that, The mass ratio of poloxamer 188, poloxamer 407 and Bletilla striata polysaccharide in S3 is (4-4.5):(18-18.5):

1.

9. The method for preparing a Bazhen Tang nanoemulsion thermosensitive hydrogel for treating osteoporosis according to claim 1, characterized in that, The volume ratio of pure water to Bazhentang nanoemulsion in S3 is (2-2.2):(5-5.5).

10. The method for preparing a thermosensitive hydrogel of Bazhen Tang nanoemulsion for treating osteoporosis according to claim 1, characterized in that, The refrigerator temperature in S3 is 3-5℃, and the swelling time is 20-24h.

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