Pharmaceutical composition, pharmaceutical preparation containing pharmaceutical composition and application

Abstract

The invention relates to the field of medicine, in particular to a pharmaceutical composition, a pharmaceutical preparation containing the pharmaceutical composition and application, the pharmaceutical composition comprises ligustrazine nanoparticles and a tendon relaxing and collateral activating compound composition, the ligustrazine nanoparticles comprise a carrier and ligustrazine loaded in the carrier, the ligustrazine nanoparticles and the tendon-relaxing and collateral-activating compound composition are combined for use, so that a synergistic effect is achieved, and the treatment effect on the OA is remarkably improved. In addition, combined use of the two shows remarkable curative effects in the aspects of relieving synovitis, repairing chondrocyte apoptosis and relieving pain-related biological indexes, which is of great significance in improving the life quality of OA patients.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a pharmaceutical composition and a pharmaceutical preparation and application thereof. Background Art

[0002] Osteoarthritis (OA) belongs to the category of "bi syndrome" in traditional Chinese medicine. It is a chronic disease caused by wind, cold, dampness and other factors, with joint pain, swelling and dysfunction as the main manifestations. According to statistics from the World Health Organization, there are about 350 million confirmed cases of OA in the world, of which nearly 60% are elderly people. With the aging of the population, its prevalence is increasing year by year. As an important part of traditional Chinese medicine, traditional Chinese medicine has unique advantages in the treatment of OA. Scientific prescription composition and preparation form are the key content of the modernization application and development of traditional Chinese medicine.

[0003] The inventors of the present application have formulated a compound composition for relaxing muscles and activating collaterals based on many years of clinical experience and basic research, based on the theory of traditional Chinese medicine and modern pharmacological research. Previous studies have shown that the compound composition for relaxing muscles and activating collaterals has a significant effect on regulating the proliferation, apoptosis and migration of OA synovial fibroblasts (fibroblast-like synoviocytes, FLSs) and inhibiting the release of inflammatory factors (see Chinese patent document CN111920849A).

[0004] Chuanxiong is the main ingredient of the Shujin Huoluo prescription. It has the effects of promoting blood circulation, removing blood stasis, promoting qi and relieving pain. In addition to being used in the treatment of cardiovascular and gynecological diseases, it is also widely used in the treatment of joint diseases (see the literature Guan Yongmei, Jiang Cheng, Zang Zhenzhong, et al. Research progress on the chemical composition, pharmacological effects and clinical application of Chuanxiong volatile oil [J]. Chinese Traditional Patent Medicine, 2024, 46(03): 873-880. Liang Qi, Zhang Laibin, Lv Jieli. Research progress on the chemical composition and pharmacological effects of Chuanxiong [J]. Journal of Xinxiang Medical College, 2024, 41(03): 275-285.). Ligustrazine (TMP) is the main active ingredient of Ligustrazine Rhizome and is often selected as the main quality control standard for compound preparations containing Ligustrazine Rhizome (see the literature Wang Wei. Analysis of the method for determining the content of Ligustrazine in compound Ligustrazine capsules by HPLC [J]. Journal of Clinical Rational Drug Use, 2012, 5(23):123.). Modern pharmacological studies have found that ligustrazine has analgesic, microcirculation-improving and immunomodulatory effects. It can inhibit the release of various inflammatory factors such as IL-1β and TNF-α, and has an inhibitory effect on the TLR4 / MyD88 / NF-κB inflammatory pathway in innate immunity (see Zhao Junli, Cheng Ju, Chen Jianjun, et al. Research progress on the pharmacological activity of ligustrazine derivatives [J]. West China Journal of Pharmaceutical Sciences, 2023, 38(03): 340-344. Tang Yan, Zhang Li, Liu Haihong. Ligustrazine inhibits TLR4 / MyD88 / NF-κB signaling pathway to reduce inflammatory response in rats with endometriosis [J]. Journal of Immunology, 2023, 39(06): 469-477.), but TMP has the effects of vasodilation, sedation and analgesia, microcirculation improvement, and immunity enhancement. The previous research of the research group also found that ligustrazine can significantly reduce the levels of NO and prostaglandin E2 in osteoarthritis synovial fluid, and has a certain effect on reducing the inflammation of osteoarthritis (see the literature Xie Pingjin, et al. Ligustrazine intervenes in the expression of type II collagen fiber α1 gene, vascular endothelial growth factor mRNA and miR20b in cartilage of rats with early knee osteoarthritis [J]. Chinese Journal of Tissue Engineering Research, 2018, 22(12): 1846-1851.). In order to improve the therapeutic effect of ligustrazine on osteoarthritis and improve the quality of life of OA patients, it is necessary to develop a new drug composition and drug preparation for the treatment of OA. Summary of the invention

[0005] Therefore, the purpose of the present invention is to improve the therapeutic effect of ligustrazine on osteoarthritis, thereby providing a pharmaceutical composition and a pharmaceutical preparation and application containing the pharmaceutical composition.

[0006] To this end, the present invention provides a pharmaceutical composition comprising ligustrazine nanoparticles and a muscle-soothing and collateral-activating compound composition, wherein the ligustrazine nanoparticles comprise a carrier and ligustrazine loaded in the carrier, and the muscle-soothing and collateral-activating compound composition comprises the following raw materials in parts by weight: 20-40 parts of ligustrazine, 20-40 parts of Sinomenium sinensis, 10-30 parts of Notopterygium wilfordii, 10-35 parts of Angelica dahurica, 5-25 parts of Sappan wood, 10-30 parts of Artemisia argyi, 5-20 parts of Angelica sinensis, 5-20 parts of Carthamus tinctorius, 5-20 parts of frankincense, 5-20 parts of myrrh, 5-20 parts of Asarum, 5-25 parts of Cyathula capitata, 5-25 parts of Drynaria fortunei, 5-25 parts of Herba Lycopodii, 5-25 parts of Herba Polygoni Multiflori, and 5-25 parts of Achyranthes bidentata.

[0007] Furthermore, in the pharmaceutical composition, the ratio of the mass of ligustrazine provided by the ligustrazine nanoparticles to the mass of ligustrazine provided by the muscle-soothing and collateral-activating compound composition is 1:0.1-50.

[0008] Furthermore, the content of ligustrazine in the muscle-relaxing and collateral-activating compound composition is 0.1-10 μg / mg; and / or, the content of ligustrazine in the ligustrazine nanoparticles is 25-500 μg / mg; and / or, the content of ligustrazine in the pharmaceutical composition is 500-1500 μg / mL.

[0009] Further, the carrier comprises exosomes, liposomes or polymeric carrier materials;

[0010] Preferably, the raw materials for preparing the liposome include phospholipids and cholesterol; more preferably, the phospholipids include phosphatidylcholine, 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC), 1,2-dilauroyl-sn-glyceryl-3-phosphoethanolamine (DLPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2- One or more of dilauroyl-sn-glyceryl-3-phosphoethanolamine (DLPE), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), 1,2-dipalmitoyl-rac-glyceromethoxypolyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glyceryl-3-methylpolyoxyethylene (DSG-PEG), and 1,2-dimyristoyl-rac-glyceryl-3-methylpolyoxyethylene (DMG-PEG).

[0011] Further, the polymer carrier material includes one or more of polylactic acid (PLA), polylactic acid-co-glycolic acid (PLGA), chitosan, polycaprolactone (PCL), polyethylene glycol (PEG) modified materials, sodium alginate, gelatin, polymethyl methacrylate, and silica nanoparticles. Among them, the polyethylene glycol modified materials include PEG-PLA, PEG-PCL, PEG-PLGA and other materials.

[0012] Preferably, the exosomes are derived from peripheral blood, urine, saliva, cerebrospinal fluid, mesenchymal stem cells or milk.

[0013] The present invention also provides a method for preparing any of the above-mentioned pharmaceutical compositions, which is prepared by mixing a liquid containing ligustrazine nanoparticles with a liquid containing the muscle-relaxing and collateral-activating compound composition.

[0014] Optionally, the concentration of ligustrazine in the liquid containing ligustrazine nanoparticles is 500-3000 μg / mL;

[0015] Optionally, the concentration of ligustrazine in the liquid containing the muscle-relaxing and collateral-activating compound composition is 100-2000 μg / mL;

[0016] Optionally, the volume ratio of the liquid containing ligustrazine nanoparticles to the liquid containing the muscle-relaxing and collateral-activating compound composition is 1:20 to 20:1.

[0017] Furthermore, the preparation method of the muscle-relaxing and collateral-activating compound composition comprises weighing chuanxiong, sinomenium, notopterygium, angelica, sappan wood, wormwood, angelica, safflower, frankincense, myrrh, asarum, chuanduan, drynaria, smilax glabra, radix polygoni multiflori, and rhizoma spatholobi according to selected weight proportions, mixing and extracting according to conventional extraction methods or extracting respectively according to conventional extraction methods and then mixing to obtain the compound composition;

[0018] Optionally, the conventional extraction method includes one or more of maceration extraction, decoction extraction, reflux extraction, percolation extraction, ultrasonic extraction and steam distillation;

[0019] Optionally, after the conventional extraction method is used for extraction, the method further includes a step of refining and purifying the obtained extract; the refining and purification treatment includes one or more of water extraction and alcohol precipitation, extraction, silica gel chromatography column separation and macroporous resin column separation.

[0020] Furthermore, the preparation method of the muscle-relaxing and collateral-activating compound composition comprises the following steps:

[0021] S1, water extraction step: extract each API with water for 1-5 times, and then concentrate to obtain a water extract, wherein the mass of water used for each water extraction is more than 8 times the mass of the API; the time for each water extraction is greater than or equal to 1.5 hours; the water extraction temperature is 80-100°C;

[0022] S2, alcohol precipitation step: the water extract is mixed with 70-90% alcohol solution, and the alcohol extract is collected after alcohol precipitation, wherein the volume of the alcohol solution is more than 5 times the volume of the water extract obtained after concentration; the alcohol precipitation time is greater than or equal to 48 hours.

[0023] Preferably, the alcohol solution is one or more of ethanol solution and methanol solution.

[0024] Preferably, in the water extraction step, the number of water extractions is 1.5-2.5 times, the mass of water used in each water extraction is 10-12 times the mass of the raw material drug, and the time for each water extraction is 2-2.5 hours. In the alcohol precipitation step, 80%-88% ethanol solution is used for alcohol precipitation, the volume of the ethanol solution is 5-6 times the volume of the concentrated water extract, and the alcohol precipitation time is 48-60 hours.

[0025] Preferably, both step S1 and step S2 include a concentration step. The volume of the alcohol solution is 5-6 times the volume of the concentrated water extract. Optionally, step S1 is concentrated to obtain a water extract with a relative density of 1.05-1.15, and step S2 is concentrated to obtain a refined solution with a relative density of 1.16-1.25.

[0026] Furthermore, the preparation method of the ligustrazine nanoparticles includes preparing a blank carrier, and using the blank carrier and a solution containing ligustrazine as raw materials to carry out drug loading according to a conventional drug loading method, to obtain;

[0027] Optionally, the mass ratio of the blank carrier to ligustrazine is 1 to 10:1;

[0028] Optionally, the concentration of the solution containing ligustrazine is 0.01-50 mg / mL;

[0029] Optionally, the solvent of the solution containing ligustrazine is one or more of methanol, ethanol, acetone, glycerol, petroleum ether, acetonitrile, chloroform, water, saline, PBS, DMSO, PEG, propylene glycol, sodium acetate buffer, dichloromethane, n-butanol, isopropanol, tetrahydrofuran, ether, dimethylformamide (DMF), dimethylacetamide (DMAc), toluene, and cyclohexane;

[0030] Optionally, the conventional drug loading method includes one or more of ultrasound method, electroporation method, solvent volatilization method, double emulsification method, nanoprecipitation method, spray drying method, freeze drying method, ion gel method, self-assembly method, high pressure homogenization method, and hot melt extrusion method;

[0031] Optionally, after the conventional drug loading method is used for drug loading, the step of purifying the obtained mixed solution is also included; the purification treatment method includes one or more of centrifugation, ultrafiltration, dialysis, gel filtration, chromatography, dialysis combined with freeze-drying, precipitation-resuspension, magnetic separation, membrane filtration, and washing-centrifugation.

[0032] The present invention also provides a pharmaceutical preparation, comprising any of the above-mentioned pharmaceutical compositions or the pharmaceutical compositions prepared by any of the above-mentioned preparation methods, and optionally one or more pharmaceutically acceptable carriers;

[0033] Optionally, the pharmaceutical preparation is a gel, cream, ointment, patch, emulsion or suspension;

[0034] Optionally, the pharmaceutically acceptable carrier is selected from at least one or more of a pharmaceutically acceptable solvent, a water-soluble matrix, an oil-soluble matrix, a penetration enhancer, a humectant, a solubilizer, an antioxidant, a preservative, a pH adjuster, an emulsifier, and a thickener.

[0035] For example, but not limited to, one or more of stearic acid, glyceryl monostearate, paraffin, liquid paraffin, white vaseline, lanolin, cetyl alcohol, stearyl alcohol, Span series, beeswax, animal and plant oils and fats, glycerin, sorbitol, polyethylene glycol, Tween series, sodium lauryl sulfate, dimethyl sulfoxide, triethanolamine, sodium carboxymethyl cellulose, sodium hyaluronate, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, polyvinyl acid, carbomer, xanthan gum, and carboxymethyl cellulose.

[0036] Use of any of the above-mentioned pharmaceutical compositions or the pharmaceutical compositions prepared by any of the above-mentioned preparation methods or the pharmaceutical preparations in the preparation of drugs for preventing or treating bone and joint diseases.

[0037] Furthermore, the bone and joint disease is at least one of osteoarthritis, traumatic arthritis, rheumatoid arthritis, gouty arthritis, synovitis, bursitis, cervical spondylosis, lumbar spondylosis, scapulohumeral periarthritis and bone hyperplasia.

[0038] The technical solution of the present invention has the following advantages:

[0039] 1. A pharmaceutical composition provided by the present invention comprises ligustrazine nanoparticles and a muscle-soothing and activating compound composition, wherein the ligustrazine nanoparticles comprise a carrier and ligustrazine loaded in the carrier, and the muscle-soothing and activating compound composition comprises the following raw materials in parts by weight: 20-40 parts of ligustrazine, 20-40 parts of Sinomenium, 10-30 parts of Notopterygium, 10-35 parts of Angelica dahurica, 5-25 parts of Sappanwood, 10-30 parts of Artemisia argyi, 5-20 parts of Angelica sinensis, 5-20 parts of Carthamus tinctorius, 5-20 parts of Olibanum, 5-20 parts of Myrrh, 5-20 parts of Asarum, 5-25 parts of Chuanduan, 5-25 parts of Drynariae, 5-25 parts of Herba Lycopodii, 5-25 parts of Herba Polygoni Multiflori, and 5-25 parts of Herba Cynoglossi. By using ligustrazine nanoparticles and the muscle-soothing and activating compound composition in combination, a synergistic effect is exerted, and the therapeutic effect on OA is significantly improved. In addition, the combined use of the two showed significant therapeutic effects in reducing synovial inflammation, repairing chondrocyte apoptosis, and alleviating pain-related biological indicators, which is of great significance for improving the quality of life of OA patients.

[0040] 2. A pharmaceutical composition provided by the present invention, wherein the carrier includes exosomes, liposomes or polymeric carrier materials, preferably exosomes. Exosomes are nanoscale carriers with good biocompatibility and targeting, which can carry a variety of bioactive molecules, fuse with target cells through cell membranes, and deliver the encapsulated Chinese medicine monomers directly and accurately to the lesion site, which can effectively protect the drug components from degradation in the body environment, improve the stability of the drug, and reduce the side effects on healthy tissues. More preferably, when mesenchymal stem cell exosomes are used as carriers, TMP is encapsulated in mesenchymal stem cell exosome carriers to form nanoscale drug particles, which can better improve the therapeutic effect on OA. In addition, using mesenchymal stem cell exosomes as carriers can also increase the drug loading and encapsulation rate of ligustrazine nanoparticles.

[0041] 3. The pharmaceutical preparation provided by the present invention can be prepared into various dosage forms according to practical applications, and can be used externally, such as but not limited to various dosage forms such as gel, cream, ointment, plaster, emulsion or suspension. The pharmaceutical preparation used in this study can be used as a massage medium, combining standardized massage techniques with modern technology to form a set of efficient drug delivery systems. During the massage process, the synergistic effect of mechanical stimulation and drug delivery is fully utilized, so that the drug can penetrate into the affected area more effectively, increase the local drug concentration, enhance the therapeutic effect, and provide an innovative solution for the drug delivery of massage therapy. In order to comprehensively evaluate the therapeutic effect of this "traditional and modern" combination of standardized treatment regimens on OA, considering the important role of articular cartilage and synovium in the pathological process of OA, the inventor of the present application has improved the application of "relaxing muscles and activating collaterals" massage by comparing the therapeutic effects of each group and further analyzing the molecular mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 Single factor optimization results of water extraction process; ****P<0.00001, ***P<0.0001, **P<0.01, *P<0.05, ns P>0.05;

[0044] Figure 2 Single factor optimization results of alcohol precipitation process; ****P<0.00001, ***P<0.0001, **P<0.01, *P<0.05, ns P>0.05;

[0045] Figure 3-5 This is the result diagram of the response surface analysis method (Box-Behnken) for water extraction process optimization, where: Figure 3 The water extraction time and water addition amount, Figure 4 is the number of water extractions and the amount of water added, Figure 5 The number of water extractions and the time of water extraction;

[0046] Figure 6-8 This is the Box-Behnken result diagram for alcohol precipitation process optimization; Figure 6 is the amount of ethanol and the concentration of ethanol, Figure 7 is the alcohol precipitation time and ethanol concentration, Figure 8 is the alcohol precipitation time and ethanol dosage;

[0047] Fig. 9 This is a specificity chromatogram; from top to bottom are the reference solution; the test solution; the blank solution;

[0048] Fig.10 is the standard curve of TMP reference;

[0049] Fig.11 The results of the regulation of the Shujin Huoluo compound on FLS apoptosis (A: high concentration group, B: medium concentration group, C: low concentration group, D: conventional extraction group, E: blank group);

[0050] Fig.12 The results of the regulation of the release of NO and IL-1β by the Shujin Huoluo compound, ****P<0.00001, the left figure is the NO level, and the right figure is the IL-1β level. Vertical axis: Concentration;

[0051] Fig.13 The particle size results of the blank exosome solution (EXO before drug loading); the horizontal and vertical scales Size represent the size, and Concentration (particles / ml) is translated as the particle concentration (particles / mL);

[0052] Fig.14 The particle size results of drug-loaded exosomes (EXO after drug loading); the horizontal and vertical scales Size represent the size, and Concentration (particles / ml) is translated as the particle concentration (particles / mL);

[0053] Fig.15 The electron microscopy results of EXO before drug loading (100000×);

[0054] Fig.16 The electron microscopy results of EXO after drug loading (100000×);

[0055] Fig.17 Western blot results of EXO before and after drug loading;

[0056] Fig.18 It is the potential result of EXO before drug loading; the horizontal and vertical scales Zeta Potential are translated as Zeta potential; the vertical scale Frequency is translated as frequency;

[0057] Fig.19 It is the potential result of EXO after drug loading; the horizontal and vertical scales Zeta Potential are translated as Zeta potential; the vertical scale Frequency is translated as frequency;

[0058] Fig. 20 HE staining of synovium; A. Normal group; B. Model group; C. Simple massage group; D. Compound massage group; E. Combined massage group; Scale bar = 200μm / 100μm;

[0059] Fig.21 HE staining of cartilage; A. Normal group; B. Model group; C. Simple massage group; D. Compound massage group; E. Combined massage group; Scale bar = 200μm / 100μm;

[0060] Fig. 22 TUNEL staining of cartilage; A. Normal group; B. Model group; C. Simple massage group; D. Compound massage group; E. Combined massage group; Scale bar = 100 μm;

[0061] Fig.23The WB detection chart of OA-related protein expression levels, A. normal group; B. model group; C. simple massage group; D. compound massage group; E. combined massage group; ****P<0.00001, ***P<0.0001, **P<0.01, *P<0.05; Relative protein level is translated as relative protein level;

[0062] Fig.24 is the release amount of TNF-α and MMP-13; ****P<0.00001; Vertical axis: Concentration. DETAILED DESCRIPTION

[0063] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0064] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0065] 1. Materials

[0066] 1.1 Chinese medicine The decoction pieces used in the Shujinhuoluo prescription in this study include: Chuanxiong, Qingfengteng, Qianghuo, Angelica dahurica, Sappan wood, Artemisia argyi, Angelica sinensis, Carthamus tinctorius, Frankincense, Myrrh, Asarum, Chuanduan, Drynaria fortunei, Semen armeniacae, Herba Polygoni multiflori and Achyranthes bidentata, which were purchased from the Chinese medicine pharmacy of the China-Japan Friendship Hospital and passed the quality inspection and met the standards of the Pharmacopoeia of the People's Republic of China.

[0067] 1.2 Reagents and instruments

[0068] 1.2.1 Reagents Ethanol, methanol and acetonitrile (AR grade, analytical grade) were purchased from Chengdu Jinshan Chemical Reagent Co., Ltd., and ligustrazine standard was provided by the Chinese Institute for the Authentication of Pharmaceutical and Biological Products.

[0069] 1.2.2 Instruments High-speed multifunctional pulverizer (LFP-800A): Life Flagship Store; Digital display water bath constant temperature oscillator (HZ-9212S): Jintan Jerel Electric Co., Ltd.; Rotary evaporator (RE52AA): Shanghai Yarong Biochemical Instrument Factory; Electronic balance (JA3003): Shanghai Sunny Hengping Scientific Instrument Co., Ltd.; Liquid chromatograph (Agilent 6890N 5975): Agilent Technologies Co., Ltd., USA.

[0070] 1.3 Cells

[0071] The mesenchymal stem cells (MSC) cell lines used in this study were donated by the Pathology Laboratory of the Clinical Trial Institute of the China-Japan Friendship Hospital.

[0072] Example 1-5 Pharmaceutical Composition

[0073] Examples 1-5 provide a series of pharmaceutical compositions and preparation methods thereof, all of which include ligustrazine nanoparticles and a muscle-soothing compound composition. The pharmaceutical composition is prepared by mixing a liquid containing ligustrazine nanoparticles with a liquid containing a muscle-soothing compound composition. The liquid containing ligustrazine nanoparticles and the liquid containing the muscle-soothing compound composition used in each embodiment have the same composition and preparation method, the only difference being that the volume ratio of the two mixed is different, resulting in a different total content of ligustrazine in the pharmaceutical composition, and in the pharmaceutical composition, the ratio of the mass of ligustrazine provided by the drug-loaded exosomes to the mass of ligustrazine provided by the muscle-soothing compound composition (TMP1:TMP2) is different, as shown in Table 1 for details.

[0074] The preparation method of the pharmaceutical composition is as follows: a liquid containing ligustrazine nanoparticles (referred to as liquid 1) and a liquid containing the muscle-relaxing and collateral-activating compound composition (referred to as liquid 2) are mixed according to the volume ratio in Table 1 to obtain the pharmaceutical composition.

[0075] The raw materials of the Shujin Huoluo compound composition are as follows: 30g of Chuanxiong, 30g of Qingfengteng, 20g of Qianghuo, 20g of Baizhi, 15g of Sappanwood, 20g of Artemisia argyi, 10g of Angelica sinensis, 10g of Carthamus tinctorius, 10g of Frankincense, 10g of Myrrh, 10g of Asarum, 15g of Chuanduan, 15g of Drynariae, 15g of Herba Lycopodii, 15g of Herba Polygoni Multiflori, and 15g of Achyranthes bidentata. The ligustrazine nanoparticles include exosomes and ligustrazine loaded in the exosomes.

[0076] The preparation method of the muscle-relaxing and collateral-activating compound composition is as follows:

[0077] Weigh chuanxiong, sinomenium, notopterygium, angelica, sappan wood, wormwood, angelica, safflower, frankincense, myrrh, asarum, chuanduan, drynaria, smilax china, radix polygoni multiflori and rhizoma cyrtonemae according to the above weight, wash and mix the raw materials except frankincense and myrrh, add water and boil twice, add water 10 times the weight of the raw materials for the first time, soak for 1 hour, boil for 2 hours, add water 10 times the weight of the raw materials for the second time, boil for 2 hours; add frankincense and myrrh when boiling for 1.5 hours for the second time, and continue boiling for 0.5 hours, and combine the decoctions; filter and set aside, Pour into a rotary evaporator, concentrate under reduced pressure to obtain an extract with a relative density of 1.1, add 80% ethanol solution in an amount that is 5 times the volume of the extract (5 times the volume means that the volume of the ethanol solution is 5 times the volume of the concentrated extract), mix well and let stand for 48 hours, collect the supernatant, pour into a rotary evaporator, and concentrate under reduced pressure to obtain a compound refined solution with a relative density of 1.2 (that is, the mass of the compound extract in each milliliter of refined solution is 1200 mg), which is recorded as a liquid containing the muscle-relaxing and collateral-activating compound composition.

[0078] Test: 1 mL of the compound refined liquid was dried to obtain a Chinese medicine compound extract, and 30 mg of the Chinese medicine compound extract was taken as a test sample. After HPLC detection in Experimental Example 2, the content of ligustrazine in the Chinese medicine compound extract (or the dried compound refined liquid) was 0.8187 μg / mg. After calculation, the content of ligustrazine in the compound refined liquid was 982.44 μg / mL.

[0079] The preparation method of ligustrazine nanoparticles (drug-loaded exosomes) is as follows:

[0080] Preparation of blank exosomes: Mesenchymal stem cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and maintained in a constant temperature incubator at 37°C and 5% carbon dioxide (CO2). The cell supernatant was removed and placed on ice to melt, and then centrifuged at 500xg, 5min, 4°C to collect the supernatant; then transferred to a new 50ml centrifuge tube, 2000xg, 30min, 4°C to collect the supernatant; centrifuged at 10000xg for 60min, collected the supernatant, filtered with a 0.22um sterile filter, added to an ultra-high speed centrifuge tube, and centrifuged at 120000xg for 70min at 4°C. After centrifugation, the supernatant was carefully removed, the precipitate was dissolved with sterile PBS, and the resuspension was collected and recorded as the blank exosome solution for subsequent experiments. The concentration of exosomes contained in the blank exosome solution tested in Experimental Example 2 was 6.01846 μg / μL.

[0081] Preparation of drug-loaded exosomes: Take 20 mg of ligustrazine and add 1 ml of methanol to dissolve it to obtain a ligustrazine solution with a ligustrazine concentration of 20 μg / μl. According to the mass ratio of exosomes contained in the blank exosome solution to ligustrazine contained in the ligustrazine solution of 2:1, measure the blank exosome solution and ligustrazine solution (wherein the mass of ligustrazine in the ligustrazine solution is 800 μg), mix them into 1 tube, and place them on ice. Ultrasound on ice: Use the Biosafer 650-92 ultrasonic instrument provided by Saifei (China) Co., Ltd., set the power ratio to 20%, turn on for 30s and then turn off for 3 minutes as 1 cycle, and perform 6 cycles in total. After ultrasonic treatment, incubate the solution at 37°C for 60 minutes to restore the exosome membrane. Ultrafiltration (100KD) treatment was performed, and the upper liquid was collected to obtain a drug-loaded exosome solution (volume 184μL), which was recorded as a liquid containing ligustrazine nanoparticles. The HPLC test of Experimental Example 2 measured the concentration of ligustrazine in the drug-loaded exosome solution: 1374.88μg / mL. The BCA quantitative test in Experimental Example 2 showed that the concentration of exosomes contained in the drug-loaded exosome solution was 4.34267μg / μL.

[0082] Table 1 Recipe

[0083]

[0084] Example 6 Pharmaceutical Composition

[0085] This embodiment provides a pharmaceutical composition, which is basically the same as Example 1, except that the raw materials of the compound composition for relaxing muscles and activating collaterals are different. The raw materials in this embodiment are composed as follows: 20g of Chuanxiong, 40g of Sinomenium windflower, 10g of Notopterygium wilfordii, 35g of Angelica dahurica, 5g of Sappan wood, 30g of Artemisia argyi, 20g of Angelica sinensis, 5g of Carthamus tinctorius, 20g of frankincense, 20g of Myrrh, 5g of Asarum, 5g of Chuanduan, 25g of Drynaria fortunei, 5g of Herba Lycopodii, 25g of Herba Polygoni Multiflori, and 25g of Achyranthes bidentata.

[0086] The preparation method of the pharmaceutical composition of this embodiment is the same as that of Example 1.

[0087] Example 7 Pharmaceutical Composition

[0088] This embodiment provides a pharmaceutical composition, which is basically the same as Example 1, except that the raw materials of the compound composition for relaxing muscles and activating collaterals are different. The raw materials in this embodiment are composed as follows: 40g of Chuanxiong, 20g of Sinomenium windflower, 30g of Notopterygium wilfordii, 10g of Angelica dahurica, 25g of Sappan wood, 10g of Artemisia argyi, 5g of Angelica sinensis, 20g of Carthamus tinctorius, 5g of frankincense, 20g of Myrrh, 5g of Asarum, 25g of Chuanduan, 5g of Drynaria fortunei, 25g of Herba Lycopodii, 5g of Herba Polygoni Multiflori, and 20g of Achyranthes bidentata.

[0089] The preparation method of the pharmaceutical composition of this embodiment is the same as that of Example 1.

[0090] Example 8 Pharmaceutical Composition

[0091] The pharmaceutical composition of this embodiment includes ligustrazine nanoparticles and a muscle-soothing and activating compound composition. The raw material composition of the muscle-soothing and activating compound composition and the preparation method of the pharmaceutical composition are the same as those in Example 1, except that the preparation method of the ligustrazine nanoparticles is different.

[0092] The preparation method of ligustrazine nanoparticles in this embodiment is as follows:

[0093] Weigh 0.1g of lecithin and 0.02g of cholesterol respectively, mix them, add 2mL of chloroform to dissolve them, and remove the chloroform by rotary evaporation to form a lipid film. Add 1mL of distilled water, incubate in a 60℃ water bath for 20min, and homogenize with a high-speed dispersing homogenizer to make 1mL of blank liposomes. Dissolve 20mg of ligustrazine in 1ml of methanol to obtain a ligustrazine solution with a ligustrazine concentration of 20μg / μl. Take 1mL of ligustrazine solution and add it to 1mL of blank liposomes, and homogenize with a high-speed dispersing homogenizer to obtain drug-loaded liposomes.

[0094] Example 9 Ointment

[0095] The ointment of this embodiment is prepared as follows: 90 g of the pharmaceutical composition prepared according to the preparation method of Example 1, 10 g of triethanolamine, and 40 g of carbomer 940 are mixed, stirred evenly, distilled water is added to 300 mL, and heated in a water bath at 80° C. with stirring for later use; 40 g of stearic acid glyceryl and 30 g of vaseline are melted together at 80° C., white mineral oil is added to 100 mL, and the above-mentioned materials are added for later use, and finally 10 g of Sy-45 composite preservative is added, and the mixture is stirred and mixed until it is in a creamy state to obtain the ointment.

[0096] Example 10 Hydrogel

[0097] The hydrogel of this example includes 30 g of the pharmaceutical composition prepared in Example 1, and also includes the following carriers: 1 g of carbomer, 1.35 g of triethanolamine, 60 g of water, 5 g of glycerol and 2.65 g of ethanol.

[0098] Preparation method: Dissolve carbomer in water and stir until completely dissolved. Add glycerol and ethanol and continue to stir evenly. Use triethanolamine to adjust the pH value to neutral (about 6.8-7.2). While stirring, add the pharmaceutical composition prepared in Example 1 and stir evenly to obtain a hydrogel for use.

[0099] Example 11 Emulsion

[0100] The emulsion of this embodiment includes 30 g of the pharmaceutical composition prepared in Example 1, and also includes the following carriers: 10 g of vaseline, 5 g of stearic acid, 5 g of white mineral oil, 1.5 g of Tween 80, 40 g of water, 5 g of glycerol, and 0.1 g of vitamin E.

[0101] Preparation method: Mix vaseline, stearic acid, white mineral oil, and Tween 80, heat to 70°C, and stir evenly to obtain an oil phase. Mix water, glycerin, and vitamin E, heat to the same temperature (70°C), and stir evenly to obtain an aqueous phase. Slowly add the aqueous phase to the oil phase, and keep stirring until the emulsification is complete for 20 minutes. Cool to about 40°C, add the pharmaceutical composition prepared in Example 1, and stir evenly to obtain an emulsion.

[0102] Experimental Example 1

[0103] 1. Preliminary screening of extraction process and alcohol precipitation process by single factor experiment

[0104] (1) Single factor experiment of water extraction process

[0105] Weigh 30g of Chuanxiong, 30g of Qingfeng Caulis, 20g of Qianghuo, 20g of Angelica dahurica, 15g of Sappanwood, 20g of Artemisia argyi, 10g of Angelica sinensis, 10g of Carthamus tinctorius, 10g of Olibanum, 10g of Myrrh, 10g of Asarum, 15g of Chuanduan, 15g of Drynariae Rhizoma, 15g of Lycopodiella ciliatae, 15g of Herba Polygoni Multiflori, and 15g of Achyranthes bidentata, and after washing, mix the raw materials except for Olibanum and Myrrh, add water and decoct twice, add 10 times the mass of water of the raw materials for the first time, soak for 1h, decoct for 2h, add 10 times the mass of water of the raw materials for the second time, decoct for 2h; add Olibanum and Myrrh when the second decoction is 1.5h, continue to decoct for 0.5h, combine the decoctions; filter for standby use, pour into a rotary evaporator, and concentrate under reduced pressure to obtain an extract with a relative density of 1.1 (abbreviated as "SHCE"). Based on this method, each parameter in the water extraction process is investigated through a single factor experiment.

[0106] The water extraction of SHC was carried out by changing the amount of water added for decoction, the water extraction time for decoction and the number of water extractions respectively, and the extract was prepared by the above method. The only difference is that in the water extraction process, the mass of water added for decoction is adjusted to 4, 6, 8, 10 or 12 times the weight of the raw material drug respectively; other conditions remain unchanged; or, the water extraction time level is adjusted to 0.5, 1, 1.5, 2 or 2.5h; other conditions remain unchanged; or, the number of water extractions level is adjusted to 1, 2, 3, 4 or 5 times; other conditions remain unchanged. Frankincense and myrrh are added when the decoction is left for 0.5h, and the decoction time after adding frankincense and myrrh is continued for 0.5h.

[0107] The experiment was repeated three times for each condition, and the SHCE yield was calculated.

[0108] The calculation formula is as follows: SHCE yield (%) = (SHCE weight / raw material weight) × 100%, wherein the SHCE weight refers to the weight of the extract obtained after concentration, and the raw material mass in the water extraction process refers to the weight of the medicinal materials used, and the unit is gram (g).

[0109] (2) Single factor experiment of alcohol precipitation process

[0110] Weigh 30g of Chuanxiong, 30g of Sinomenium, 20g of Notopterygium, 20g of Angelica dahurica, 15g of Sappanwood, 20g of Artemisia argyi, 10g of Angelica sinensis, 10g of Carthamus tinctorius, 10g of frankincense, 10g of myrrh, 10g of Asarum, 15g of Chuanduan, 15g of Drynariae, 15g of Herba Lycopodiellae, 15g of Herba Polygoni Multiflori, and 15g of Achyranthes bidentata, and after washing, mix the raw materials except frankincense and myrrh with water 10 times the mass of the raw materials, soak for 1h, and boil twice with water, adding 10 times the mass of the raw materials each time, and boil for 2h each time; the second time When the decoction is 1.5 hours, frankincense and myrrh are added and the decoction is continued for 0.5 hours, and the decoction is combined; filtered for use, poured into a rotary evaporator, and concentrated under reduced pressure to obtain an extract with a relative density of 1.1, and an ethanol solution with a volume percentage of 80% accounting for 5 times the volume of the extract (5 times the volume means that the volume of the ethanol solution is 5 times the volume of the concentrated extract), mixed evenly and allowed to stand for 48 hours, the supernatant is collected, poured into a rotary evaporator, and concentrated under reduced pressure to obtain a compound refined solution with a relative density of 1.2 (abbreviated as "SHCE"). Based on this method, each parameter in the alcohol precipitation process is investigated through a single factor experiment.

[0111] The alcohol precipitation of SHC was carried out by changing the three factors of ethanol concentration, ethanol volume and alcohol precipitation time respectively, and the compound refined liquid was prepared by the above method. The only difference is that the ethanol concentration level in the alcohol precipitation process is adjusted to 35%, 50%, 65%, 80% or 95%; other conditions remain unchanged; or, the volume level of ethanol is adjusted to 2, 3, 4, 5 or 6 times the volume of the extract; other conditions remain unchanged; or, the alcohol precipitation time level is adjusted to 12, 24, 36, 48 or 60h; other conditions remain unchanged.

[0112] The experiment was repeated three times for each condition, and the SHCE yield was calculated.

[0113] The calculation formula is as follows: SHCE yield (%) = (SHCE weight / raw material weight) × 100%, wherein the SHCE weight refers to the weight of the composite refined liquid obtained after alcohol precipitation.

[0114] (3) Experimental results

[0115] Results Figure 1As shown in the results, the SHCE yield increased first and then decreased with the increase of water addition, water extraction time and water extraction times. When the water addition was 10 times, the SHCE yield was the highest, and the difference was statistically significant compared with 4, 6, and 8 times of water addition (P < 0.05); when the water extraction time was 2h, the SHCE yield was the highest, and the difference was statistically significant compared with 0.5, 1, and 1.5h (P < 0.05); when the water extraction times were 2 times, the SHCE yield was the highest, and the difference was statistically significant compared with other groups (P < 0.05). When the water addition was 12 times or the extraction time was 2.5h, the SHCE yield was slightly lower than the optimal conditions, but the difference was not significant (P > 0.05).

[0116] The results showed that the SHCE yield increased first and then decreased with the increase of ethanol concentration, ethanol dosage and alcohol precipitation time. When the ethanol concentration was 80%, the SHCE yield was the highest, and the difference was statistically significant compared with 35%, 50%, and 65% (P < 0.05); when the ethanol dosage was 5 times, the SHCE yield was the highest, and the difference was statistically significant compared with 2, 3, and 4 times (P < 0.05); when the alcohol precipitation time was 48h, the SHCE yield was the highest, and the difference was statistically significant compared with 12, 24, and 36h (P < 0.05). When the ethanol dosage was 6 times or the alcohol precipitation time was 60h, the SHCE yield was slightly lower than the optimal conditions, but the difference was not significant (P > 0.05). Results are shown in Figure 2 .

[0117] 2. Box-Behnken response surface experiment to optimize the extraction process

[0118] (1) Experimental design

[0119] According to the Box-Behnken star-point response surface design principle in Design Expert 13.0 software, combined with the above SHC water extraction and alcohol precipitation single-factor test results, the amount of water added (A), water extraction time (B) and number of water extractions (C) were used as water extraction process investigation factors, and the ethanol concentration (A), ethanol dosage (B) and alcohol precipitation time (C) were used as alcohol precipitation process investigation factors. -1, 0, 1 were used to represent the low, medium and high levels respectively, and the SHCE yield (R) was used as the response surface value. A three-factor three-level response surface optimization experiment was carried out. The experimental factors and level designs are shown in Tables 2 and 3.

[0120] Table 2. Level table of factors in response surface analysis of water extraction process

[0121]

[0122] Table 3. Level table of factors in response surface analysis of alcohol precipitation process

[0123]

[0124]

[0125] (2) Response surface optimization experiment of water extraction process

[0126] 1) Water extraction regression model and variance analysis

[0127] The response surface experimental design and response values ​​(Table 4) were subjected to regression analysis using Design Expert 13.0 software. After regression fitting of each factor, the ternary quadratic regression equation of SHCE yield with respect to water addition amount (A), water extraction time (B) and water extraction times (C) was obtained: Y = 30.22 + 0.6250A + 1.19B + 1.02C - 0.0200AB + 0.0550AC + 0.0125BC - 4.13A 2 -2.38B 2 -3.26C 2 .

[0128] Table 4 Experimental design and response values ​​of response surface analysis of water extraction process

[0129]

[0130] The results of variance analysis and significance test of the regression equation are shown in Table 5. The results show that when the water-extractable SHCE content is used as the response value, the model P value is 0.0002, indicating that the model is significant, and the regression equation has a good fit and is statistically significant. The P value of the model lack of fit term is 0.7033, which is greater than 0.05, indicating that the unknown factors have little interference with the test results. The model can significantly fit the effects of water addition (A), water extraction time (B) and water extraction times (C) on the water-extractable SHCE yield without lack of fit. It can replace the actual test points to reflect the actual situation and perform result analysis and prediction. Determine the model coefficient R 2 The influence of each factor on the SHCE extraction rate is as follows: water extraction time (B) > water extraction times (C) > water addition amount (A). The interaction terms AB, AC, and BC are not significant factors in the model. The quadratic term A of water addition is 2 , the quadratic term of water extraction time B 2 and the quadratic term C of the number of water extractions 2 It has a significant effect on the SHCE yield.

[0131] Table 5 Variance analysis of water extraction process regression equation

[0132]

[0133] 2) Analysis of water lift contour lines and surface plots

[0134] The response surface contours and surface plots obtained by using the three-variable quadratic regression fitting analysis of the Box-Behnken test data of SHC water extraction using Design Expert 13.0 software are shown in Figure 3-5 , this group of graphs can intuitively reflect the effects of various water extraction factors and their interactions on the SHCE yield of SHC water extraction. The results show that the contour maps of the interaction terms of the water addition amount, water extraction time, and water extraction times are close to circles, and the interactions are not significant. This result is consistent with the results in Table 5. After optimization and prediction, the optimal process parameters for SHC water extraction are: 10.15 times the water addition amount, 2.12h water extraction time, and 2.16 water extraction times. Under this condition, the predicted SHCE yield of SHC water extraction is 30.47%, which is close to the highest value in the single factor experiment.

[0135] (3) Response surface optimization experiment of alcohol precipitation process

[0136] 1) Alcohol precipitation regression model and variance analysis

[0137] The response surface experimental design and response values ​​(Table 6) were subjected to regression analysis using Design Expert 13.0 software. After regression fitting of each factor, the ternary quadratic regression equation of SHCE yield with respect to water addition amount (A), water extraction time (B) and water extraction times (C) was obtained: Y = 23.88 + 0.8525A + 0.9363B + 0.8838C - 0.0950AB - 1.01AC + 0.0675BC - 6.92A 2 -2.62B 2 -1.73C 2 .

[0138] Table 6 Response surface analysis experimental design and response values ​​of alcohol precipitation process

[0139]

[0140]

[0141] The results of variance analysis and significance test of the regression equation are shown in Table 7. The results show that when the water-extractable SHCE content is used as the response value, the model P value is less than 0.0001, indicating that the model is extremely significant, and the regression equation has a good fit and is statistically significant. The P value of the model lack of fit term is 0.8383, which is greater than 0.05, indicating that the unknown factors have little interference with the test results. The model can significantly fit the effects of ethanol concentration (A), ethanol dosage (B) and alcohol precipitation time (C) on the water-extractable SHCE yield without lack of fit. It can replace the actual test point to reflect the actual situation and perform result analysis and prediction. Determine the model coefficient R 2The influence of each factor on the SHCE extraction rate is in the following order: ethanol dosage (B) > alcohol precipitation time (C) > ethanol concentration (A). The interaction terms AB, AC, and BC are not significant factors in the model. The quadratic term A of ethanol concentration is 2 , the quadratic term B of ethanol usage 2 and the quadratic term C of the alcohol precipitation time 2 It has a significant effect on the SHCE yield.

[0142] Table 7 Analysis of variance of regression equation for alcohol precipitation process

[0143]

[0144] 2) Analysis of alcohol precipitation contour lines and surface plots

[0145] The response surface contours and surface plots obtained by using the Design Expert 13.0 software to perform a three-variable quadratic regression fitting analysis on the Box-Behnken test data of SHC alcohol precipitation are shown in Figure 6-8 , this group of graphs can intuitively reflect the effects of various factors of alcohol precipitation and their interactions on the SHCE yield of SHC alcohol precipitation. The results show that the contour maps of the interaction terms of alcohol precipitation time and ethanol dosage are close to circles, and the interaction is not significant. This result is consistent with the results in Table 7; the contour maps of ethanol concentration and alcohol precipitation time or ethanol dosage are elliptical, and the slope of the surface map is slightly steep, indicating that there is a certain interaction between them, but combined with the results in Table 7, there is no significant difference in their interaction. After optimization and prediction, the optimal process parameters for SHC alcohol precipitation are: ethanol concentration 87.97%, ethanol dosage 5.19 times, and alcohol precipitation 49.32h. Under this condition, the predicted SHCE yield of SHC alcohol precipitation is 22.49%, which is close to the highest value in the single factor experiment.

[0146] Experimental Example 2 Determination of related parameters of the muscle-relaxing and collateral-activating compound composition and ligustrazine nanoparticles

[0147] 1. Method for determining the content of ligustrazine in the compound composition for relaxing muscles and activating collaterals

[0148] (1) Preparation of reference solution

[0149] Accurately weigh 80 mg of ligustrazine reference substance and add 2 mL of acetonitrile to make a mass concentration of 0.04 mg mL -1 Reference stock solution.

[0150] (2) Preparation of test solution

[0151] Accurately weigh 30 mg of the test sample, add 2 mL of acetonitrile and shake to dissolve for 30 min at 5000 r / min. -1Centrifuge for 20 minutes, take the supernatant and filter it through a 0.22 μm microporous filter membrane into a sample injection bottle for testing.

[0152] (3) Detection

[0153] High performance liquid chromatography was used for detection. The chromatographic conditions were as follows: chromatographic column: Diamonsil C18 (250 nm × 4.6 mm, 5 μm); mobile phase: acetonitrile-water (volume ratio 30:70); detection wavelength: 295 nm; injection volume: 10 μL; flow rate: 1 mL·min -1 , column temperature: 30℃.

[0154] (4) Methodological validation

[0155] 1) Specificity test

[0156] Under the above chromatographic column conditions, 10 μL of the prepared TMP reference solution, the Shujin Huoluo test solution and the blank solution (acetonitrile) were accurately aspirated and injected into the HPLC for determination. The results showed that the chromatographic peaks displayed by the Shujin Huoluo test solution matched the main peaks in the reference solution, confirming that the test sample contained TMP components; the chromatogram of the blank solution did not show chromatographic peaks similar to those of the reference solution, excluding interference from other components, indicating that this HPLC test has good specificity and the results are reliable. Results are shown in Fig. 9 .

[0157] 2) Linear relationship investigation

[0158] Accurately pipette the TMP reference stock solution and prepare reference solutions of different mass concentrations with acetonitrile-water (30:70) to make the TMP concentrations of 1 μg mL -1 , 2μg·mL -1 , 4 μg·mL -1 , 6μg·mL -1 , 8μg·mL -1 , 10μg·mL -1 , 20 μg·mL -1 , 40 μg·mL -1 . Accurately pipette 10 μL of each of the above concentration solutions into the HPLC, sample and measure under the chromatographic conditions of item (3), and perform linear regression with the peak area corresponding to the mass concentration of the reference substance. With the mass concentration of the reference substance as the horizontal axis (X) and the peak area RSD as (Y), the regression equation of TMP is Y=26.535X+2.2955(R 2 =0.9998), 1-40 μg·mL -1 The linear relationship is good within the range. Fig.10 .

[0159] 3) Precision test

[0160] Accurately weigh the Chinese herbal compound extract prepared in Example 1 and prepare the test solution (6 μg mL -1 ), under the above chromatographic conditions, 10 μL was injected into the HPLC, and the injection was repeated 6 times to measure the relative standard deviation (RSD) of the TMP peak area.

[0161] The calculation formula is as follows: RSD (%) = (SD / Mean) × 100, wherein SD refers to standard deviation (SD), and Mean refers to mean value.

[0162] Through continuous measurement of TMP peak area, the relative standard deviation (RSD) was 1%, which met the acceptance criteria (RSD ≤ 2%), indicating that the instrument used in the test had high precision and stable performance during the measurement process, ensuring the reliability of the experimental data.

[0163] 4) Repeatability test

[0164] Accurately weigh the same batch of Chinese herbal compound extracts prepared in Example 1, and prepare 6 test solutions (6 μg mL -1 ), and the TMP peak area RSD was determined under the above chromatographic conditions.

[0165] The repeatability test showed that the RSD of the peak area of ​​TMP content was 3%, which was within the acceptable range (usually RSD≤5%), indicating that the standardized operating procedure of this method had good repeatability.

[0166] 5) Stability test

[0167] Accurately weigh the Chinese herbal compound extract prepared in Example 1, prepare the same test solution according to the above method, and inject the sample at 0, 2, 4, 8, 12, and 24 h under the above chromatographic conditions to determine the TMP peak area RSD.

[0168] In the stability test, the RSD of the peak area of ​​TMP content was 2.82%, indicating that the sample had good stability within 24 hours.

[0169] 6) Sample recovery test

[0170] Accurately weigh 6 portions of the same batch of known contents of the Chinese herbal compound extract prepared in Example 1, about 0.5 g per portion, and add 6 μg mL -1 The TMP reference solution was prepared according to the above method, and the test solution was prepared. 10 μL of each sample was injected for measurement under the above chromatographic conditions, and the sample recovery rate was calculated.

[0171] In the sample recovery test, the average sample recovery of TMP was 99.76% and the RSD was 2.15%. Both parameters were better than the industry standard (the sample recovery should be between 95-105% and the RSD should be ≤5%). The sample recovery close to 100% indicates that the method used has high accuracy in quantitative analysis and is not affected by factors such as matrix effects. The lower RSD value further confirms the reliability and repeatability of the method.

[0172] 7) Determination of TMP content

[0173] Accurately weigh 3 batches of the Chinese herbal compound extract prepared in Example 1, 30 mg per batch, in parallel, prepare the test solution according to the above method, inject 10 μL of each sample for measurement under the above chromatographic conditions, record the TMP peak area RSD, and substitute it into the linear formula to calculate the TMP content.

[0174] Through the above precision, repeatability, stability and sample recovery tests, we established a reliable analytical method. Using this method, we determined the content of TMP in the sample to be 0.8187μg / mg, which provides a reference for further quality control research.

[0175] 2. Determination of the content of ligustrazine in ligustrazine nanoparticles

[0176] (1) Preparation of reference solution

[0177] Accurately weigh 80 mg of ligustrazine reference substance and add 2 mL of acetonitrile to make a mass concentration of 0.04 mg mL -1 Reference stock solution.

[0178] (2) Preparation of test solution

[0179] The liquid containing ligustrazine nanoparticles was accurately weighed, diluted 20 times with PBS, and filtered through a 0.22 μm microporous filter membrane into a sample injection bottle for testing.

[0180] (3) Detection

[0181] The chromatographic conditions were as follows: chromatographic column: Welchrom Vantage C18 (250 nm × 4.6 mm, 5 μm); mobile phase: acetonitrile: 0.02% phosphoric acid water = 30:70 (V:V); detection wavelength: 292 nm; injection volume: 10 μL; flow rate: 1 mL min -1 , column temperature: 30℃.

[0182] 3. BCA quantitative test

[0183] The BCA protein concentration kit (Servicebio, G2026) was used to determine the content of exosomes in the blank exosome solution and drug-loaded exosomes according to the method in the kit instructions.

[0184] 4. Drug loading and encapsulation efficiency

[0185] (1) Calculation of drug loading and encapsulation efficiency of drug-loaded exosomes

[0186] According to the ligustrazine concentration measured by the above-mentioned high-performance liquid chromatography and the total volume of exosomes, the mass of the drug encapsulated in the carrier (referred to as the drug loading amount) was calculated according to the following formula, the encapsulation rate was calculated according to the drug loading amount and the total mass of ligustrazine added, and the content of ligustrazine in the drug-loaded exosomes was calculated according to the drug loading amount, the concentration of exosomes in the drug-loaded exosomes, and the total volume of the exosomes.

[0187] Drug loading amount = ligustrazine concentration in drug-loaded exosomes × total volume of exosomes. Encapsulation efficiency = (drug loading amount / total mass of ligustrazine added) × 100%. Content of ligustrazine in drug-loaded exosomes = drug loading amount / (drug loading amount + concentration of exosomes measured by BSA × total volume of exosomes).

[0188] It was calculated that the drug loading amount of the drug-loaded exosomes prepared in Example 1 was 253.28 μg, the content of ligustrazine in the drug-loaded exosomes was 240.47 μg / mg, and the encapsulation efficiency was 31.66%.

[0189] (2) Calculation of drug loading and encapsulation efficiency of drug-loaded liposomes

[0190] Take 200 μl of drug-loaded liposomes and put them into a dialysis bag. Use 20 mL of phosphate buffered saline (PBS) as the dialysis medium and dialyze for 24 hours. Replace the dialysate outside the dialysis bag every 4 hours, and replace the dialysate 6 times in total, with a total dialysate volume of 120 mL. Take 1 mL of the dialysate, filter it through a 0.22 μm microporous filter membrane into a sample injection bottle for testing. Test the TMP content in the dialysate under the chromatographic conditions under Item 2 (3) of this experimental example, calculate the amount of free TMP, and calculate the drug loading and encapsulation efficiency according to the following formula.

[0191] The mass of encapsulated drug = the mass of total TMP added to the drug-loaded liposomes - the mass of free TMP.

[0192] The drug loading capacity of the drug-loaded liposomes μg / mg=(mass of encapsulated TMP) / (mass of encapsulated TMP+mass of carrier).

[0193] The encapsulation efficiency of drug-loaded liposomes = the mass of encapsulated TMP / the mass of total TMP added to the drug-loaded liposomes*100%.

[0194] It was calculated that the mass of the drug encapsulated in the drug-loaded liposomes prepared in Example 8 was 4.21 mg, the mass of the free drug was 15.79 mg (distributed in 2 mL of the drug-loaded liposome solution), the drug loading was 30.07 μg / mg, and the encapsulation efficiency was 21.05%.

[0195] 5. Particle size, morphology, Zeta potential and marker protein level test of ligustrazine nanoparticles

[0196] The particle size, morphology, potential and marker protein level of the blank exosome solution (EXO before drug loading) and the drug-loaded exosomes (EXO after drug loading) in Example 1 were tested. The particle size test method is as follows: take the drug-loaded exosomes, dilute them with sterile PBS, filter them with a 0.22μm filter, and detect them on a particle size detector. The Zeta potential of each group of samples was tested three times, and the average value was taken. Figure 13-14 and 18-19 and Table 8.

[0197] Table 8 Particle size and potential

[0198]

[0199] The morphology was tested using an electron microscope. Fig.15 and 16 As shown, the morphology of exosomes before and after drug loading is similar, which is disc-shaped or cup-shaped, which is consistent with the characteristics of exosomes.

[0200] Western blot was used to detect the levels of exosome marker proteins. Fig.17 The results showed that they all contained characteristic exosome proteins: CD9 protein, TSG101, and HSP70 protein.

[0201] Experimental Example 3

[0202] 1. Animal information and drug testing

[0203] Thirty 8-week-old male Wistar rats weighing approximately 300 g (± 20 g) were selected for this study.

[0204] Test drug of the compound massage group: a muscle-soothing compound composition was prepared according to Examples 1-5, and the same mass of the muscle-soothing compound composition was used to replace the drug composition prepared in Example 1. An ointment was prepared using the muscle-soothing compound composition as the active pharmaceutical ingredient using the method of Example 9, hereinafter referred to as muscle-soothing ointment.

[0205] Test drug for combined treatment massage group: an ointment was prepared according to the method of Example 9, hereinafter referred to as compound ointment.

[0206] 2. Experimental grouping and sample preparation

[0207] In this study, rats were randomly divided into 5 groups: normal group, model group, simple massage group, compound massage group and combined massage group. There were 6 rats in each group. The normal group did not receive any treatment, and the other groups were induced with anterior cruciate ligament transection (ACLT) to induce knee OA model (induction method reference: Huan Wang, Jun Shu, Chengfei Zhang, Yang Wang, Rongxing Shi, Fan Yang, Xuezhang Tang. Extracellular Vesicle-Mediated miR-150-3p Delivery in Joint Homeostasis: A Potential Treatment for Osteoarthritis [J]. Cells, 2022, 11 (17): 2766.).

[0208] The model group did not need treatment; the simple massage group received massage manipulation treatment; the combined massage group received 1g of compound ointment as a medium combined with massage manipulation treatment each time, and the massage manipulation treatment was the same as that of the simple massage group; the compound massage group received 1g of Shujin Huoluo ointment as a medium combined with massage manipulation treatment each time, and the massage manipulation treatment was the same as that of the simple massage group. The simple massage group, combined massage group, and compound massage group were all treated twice a week for 4 weeks. After all treatments, the articular cartilage and synovial tissues and samples of the rats in each group were collected for subsequent analysis.

[0209] The massage operation plan for the simple massage group, combined massage group and compound massage group was based on the team's characteristic "relaxing muscles and activating collaterals" massage method, formulated according to actual conditions and preliminary experiments, and in accordance with the distribution of acupoints in "Experimental Acupuncture" (edited by Guo Yi, published by China Traditional Chinese Medicine Press, 1st edition). The specific massage treatment steps were as follows: the rats were placed in a lateral decubitus position with their forelimbs fixed, and the thumb and index finger of the operator were used to apply relative force to loosen the modeling side knee joint and surrounding, meridian muscles, fascia and other soft tissues using gentle kneading and grasping methods, focusing on the knee patella, patellar ligament, medial and lateral collateral ligaments, quadriceps and other parts (the key treatment acupoints were: bilateral Dubi, Heding, Weizhong, Yanglingquan, Liangqiu, Zusanli), with an average treatment time of about 1 min for each acupoint, and the above-mentioned loosening time was 10 min; at the same time, passive activities such as knee flexion, extension and rotation were coordinated, and the treatment time was about 2 min; finally, the rat's knee joint was rubbed with the thumb to end the treatment.

[0210] 3. HE staining for histological observation

[0211] Articular cartilage and synovial samples were fixed in 4% paraformaldehyde for 24 hours, and articular cartilage was further decalcified with EDTA for 14 days. Dehydrated with gradient ethanol (70%, 80%, 90%, 95%, 100%) for 60 minutes each, transparentized with xylene for 20 minutes each, embedded in paraffin, and cut into 4 μm thick sections. Dewaxed with xylene (10 minutes per step, 2 steps), rehydrated with gradient ethanol (100%, 95%, 90%, 80%, 70%) for 5 minutes each. Stained with hematoxylin for 8 minutes, differentiated with 1% hydrochloric acid alcohol for 10 seconds, washed with running water for 5 minutes to blue, and stained with eosin for 2 minutes. Dehydrated with gradient ethanol, transparentized with xylene (3 minutes per step). Finally, the sections were sealed with neutral gum and observed and photographed under a microscope.

[0212] 4. TUNEL staining to observe chondrocyte apoptosis

[0213] Staining was performed according to the instructions of the TUNEL kit. Incubate the sections with proteinase K working solution (20 μg / mL) at room temperature for 15 min, then wash 3 times with PBS for 5 min each time. Add 50 μL of TUNEL reaction mixture and incubate at 37°C for 60 min in the dark. After incubation, wash 3 times with PBS for 5 min each time. DAPI was used to stain nucleic acids at room temperature in the dark for 5 min, then washed 3 times with PBS for 5 min each time. Finally, the sections were sealed with anti-fluorescence quenching sealing agent. Chondrocyte apoptosis was observed using a fluorescence microscope.

[0214] 5. WB detection of OA-related protein expression

[0215] The synovial samples were lysed on ice for 30 minutes, centrifuged at 12000rpm for 10 minutes, and the supernatant was collected and the protein concentration was determined using a BCA kit. Equal amounts of protein samples were taken, denatured at 95℃ for 5 minutes, separated by gel electrophoresis, and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder for 1 hour, incubated with specific primary antibodies at 4℃ overnight, incubated with secondary antibodies for 1 hour, treated with ECL, and the signal was detected in a chemiluminescence imaging system. The grayscale value of the bands was analyzed to evaluate the protein expression level.

[0216] 6. Statistics

[0217] The experimental data were statistically analyzed using SPSS 22.0 software. All data were expressed as mean ± standard deviation (Mean ± SD). One-way analysis of variance (ANOVA) was used for comparison between groups, and P < 0.05 was considered statistically significant.

[0218] 7. Results

[0219] (1) Histological observation of synovium

[0220] The synovial tissue structure of the normal group was normal, the lining layer was intact, and 1-2 layers of synovial cells were neatly arranged, without obvious hyperplasia and angiogenesis. The synovial tissue of the model group showed significant hyperplasia, the lining layer was significantly thickened, the number of synovial cells increased significantly, the arrangement was chaotic, the cell fibrosis was severe, the entire synovial tissue showed hyaline degeneration, and was accompanied by obvious angiogenesis. The degree of synovial hyperplasia in all treatment groups was alleviated to varying degrees. Compared with the model group, the number of synovial cells, the degree of disordered arrangement, the thickness of the lining layer, and the angiogenesis were all alleviated. Among them, the combined treatment massage group showed the most significant improvement effect, followed by the compound massage group. Although the effect of the simple massage group was not as good as that of the compound massage group and the combined treatment massage group, it was also significantly improved compared with the model group. Results are shown in Fig. 20 .

[0221] (2) Histological observation of cartilage

[0222] In the normal group, the cartilage tissue structure was intact, the chondrocytes were neatly arranged, the matrix was uniform, the tide line was neat, and the deep subchondral bone and the base of the calcified layer were well embedded. In contrast, the cartilage tissue in the model group was significantly degraded, the chondrocytes were unclearly stratified, the number was significantly reduced, the arrangement was disordered, the matrix showed obvious cracks and signs of degradation, and a large amount of fibrous components replaced the normal chondrocytes, showing obvious ossification changes. Cartilage degeneration was alleviated in all treatment groups. Compared with the model group, the number of chondrocytes in the treatment group increased, the arrangement gradually returned to neatness, and the matrix structure was also repaired. Among them, the improvement effect of the combined treatment massage group was the most significant, which was manifested in the repair of the number and arrangement of chondrocytes, the tide line was relatively complete, and the matrix structure was significantly improved. The compound massage group was second. Although the effect of the simple massage group was not as good as the first two groups, it was also significantly improved compared with the model group. Results are shown in Fig.21 .

[0223] (3) Chondrocyte apoptosis detection

[0224] In the normal group, the apoptosis rate of chondrocytes was low, and TUNEL staining showed only a small number of positive cells with weak fluorescence intensity. In contrast, chondrocyte apoptosis in the model group increased significantly, the number of TUNEL-positive cells increased significantly, and the fluorescence intensity was strong. The apoptosis rate of chondrocytes in all treatment groups decreased, and the number and fluorescence intensity of TUNEL-positive cells decreased significantly. Compared with the model group, the number of apoptotic cells in the treatment group decreased, and the fluorescence intensity weakened, indicating that the apoptosis was alleviated. Among them, the combined treatment massage group showed a more significant anti-apoptosis effect, with a small number of TUNEL-positive cells, weaker fluorescence intensity, and a significantly reduced chondrocyte apoptosis rate. The improvements on chondrocyte apoptosis in the compound massage group and the simple massage group decreased in turn. Results are shown in Fig. 22 .

[0225] (4) Determination of OA-related protein expression levels

[0226] WB results showed that Cox-2, IL-1β, TNF-α, VEGF and ADAMTS-5 were expressed in synovial tissue, and the protein bands were clearly visible at their respective molecular weights. The expression levels of these proteins were low in the normal group. In the model group, the expression of Cox-2 increased significantly, marking inflammation and pain; the expression of IL-1β and TNF-α increased significantly, indicating an enhanced inflammatory response; the expression of VEGF increased, indicating active angiogenesis; the expression of ADAMTS-5 increased, reflecting increased cartilage degradation. In each treatment group, the expression levels of these proteins decreased, among which the protein expression level in the combined treatment massage group decreased most significantly; the protein expression levels of the compound massage group and the simple massage group decreased successively, but the effect was not as good as that of the combined treatment massage group. Overall, each treatment group significantly alleviated inflammation, pain, angiogenesis and cartilage degradation, especially the combined treatment massage group. Results are shown in Fig.23 .

[0227] Experimental Example 4: Mechanism of action of the compound composition for relaxing muscles and activating collaterals on OA

[0228] 1. Preparation of test drug solution

[0229] Solution of the compound composition for relaxing tendons and activating collaterals: according to the weight of the compound composition for relaxing tendons and activating collaterals in Example 1, weigh Chuanxiong, Qingfengteng, Qianghuo, Angelica dahurica, Sappan wood, Artemisia argyi, Angelica sinensis, Carthamus tinctorius, frankincense, myrrh, Asarum, Chuanduan, Drynaria fortunei, Herba Lycopodii, Herba Polygoni Multiflori and Achyranthes bidentata, wash and mix the raw materials except frankincense and myrrh, add water and boil twice, add 10 times the weight of the raw materials for the first time, soak for 1 hour, boil for 2 hours, add 10 times the weight of the raw materials for the second time, boil for 2 hours; when the second time is boiled for 1.5 hours Add frankincense and myrrh and continue to decoct for 0.5h, combine the decoctions; filter for later use, pour into a rotary evaporator, and concentrate under reduced pressure to obtain an extract with a relative density of 1.1, add 80% ethanol solution (5 times the volume of the extract) (5 times the volume means that the volume of the ethanol solution is 5 times the volume of the extract), mix well and let stand for 48h, collect the supernatant, pour into a rotary evaporator, and concentrate under reduced pressure to obtain a compound refined solution with a relative density of 1.2, and dilute with culture medium to obtain 400μg·mL -1 , 200 μg·mL -1 , 100 μg·mL -1 of dilution.

[0230] The test drug solution of the conventional extraction group: according to the weight of the Shujin Huoluo compound composition in Example 1, Chuanxiong, Qingfengteng, Qianghuo, Angelica dahurica, Sappan wood, Artemisia argyi, Angelica sinensis, Carthamus tinctorius, Frankincense, Myrrh, Asarum, Chuanduan, Drynaria fortunei, Lysimachia striata, Herba peonyii and Achyranthes bidentata were weighed, and the raw materials except frankincense and myrrh were mixed and boiled twice after washing. The first time, 10 times the weight of the raw materials were added with water, soaked for 1 hour, and boiled for 2 hours. The second time, 10 times the weight of the raw materials were added with water, and boiled for 2 hours; when the second decoction was 1.5 hours, frankincense and myrrh were added and continued to be boiled for 0.5 hours, and the decoctions were combined; filtered for use, poured into a rotary evaporator, and concentrated under reduced pressure to obtain an extract with a relative density of 1.1. Dilute with culture medium to obtain a concentration of 400 μg·mL -1 of aqueous extract.

[0231] 2. Isolation and culture of FLS

[0232] The synovial tissue obtained during joint replacement was cleaned and minced under sterile conditions and treated with a digestion solution containing collagenase II in a 37° cell culture incubator for 4 h. After digestion, the incompletely digested tissue was removed by filtering through a 100-mesh filter. Subsequently, the mixture was heated at 1000 r / min. -1 The cells were centrifuged for 10 min and resuspended in DMEM F12 medium containing 10% FBS to obtain FLS. [4] The FLS in the logarithmic growth phase were cultured at 2×10 4 pc·mL -1 The cells were seeded at a density of 100 μL in a 24-well culture plate, and each well was filled with 500 μL of cell suspension for subsequent experimental studies.

[0233] 3. FCM detection of cell apoptosis

[0234] The patients were randomly divided into five groups: a high concentration group (400 μg mL -1 ), medium concentration group of the Shujin Huoluo compound composition (200 μg·mL -1 ), low concentration group of Shujin Huoluo compound composition (100 μg·mL -1 ), conventional extraction group (400 μg mL -1 1 mL of the corresponding solution was added to each well and the mixture was heated at 1000 r / min for 24 h. -1The cells were collected by centrifugation at a speed of 5 min, washed with PBS and resuspended into 100 μL cell suspension, 5 μL of FITC-labeled Annexin V and 5 μL of propidium iodide (PI) were added to each sample for staining, and incubated at room temperature in the dark for 15 min. 400 μL of binding buffer was added to each sample, and then apoptosis was detected and analyzed using a flow cytometer to evaluate the effects of different concentrations of the Shujin Huoluo compound composition on apoptosis, and the effect advantages of the preferred extraction method compared with conventional extraction were evaluated.

[0235] 4. ELISA method to detect NO and IL-1β release

[0236] The release of inflammatory mediators NO and IL-1β in FLS induced by LPS was detected by ELISA method, and the groups were divided into the following groups: high concentration group of Shujin Huoluo compound composition (LPS 1 μg mL -1 +800μg·mL -1 ), medium concentration group of the Shujin Huoluo compound composition (LPS 1 μg·mL -1 +400μg·mL -1 ), low concentration group of the Shujin Huoluo compound composition (LPS 1 μg mL -1 +200μg·mL -1 ), conventional extraction group (LPS 1 μg mL -1 +400μg·mL -1 water extract), model group (LPS 1 μg mL -1 ), blank group (cell culture medium). After 24 hours of treatment, the supernatant was collected and the release of NO and IL-1β was analyzed according to the operating instructions provided by the kit manufacturer.

[0237] 5. Statistical analysis

[0238] The data were collected as mean ± SD. SPSS27.0, GraphPad Prism 9 and Design Expert 13.0 software were used for statistical analysis. One-way ANOVA was used for inter-group comparison. The results were considered statistically significant when P < 0.05.

[0239] 6. Experimental results

[0240] (1) Regulation of FLS apoptosis by muscle-soothing and collateral-activating preparations

[0241] Table 9 Regulation of the Shujin Huoluo compound composition on FLS apoptosis (%)

[0242]

[0243] *Compared with the blank group, P<0.05; △ Compared with the conventional extraction group, P < 0.05

[0244] Compared with the blank group, the conventional extraction group and the Shujin Huoluo compound combination of each concentration group induced FLS early apoptosis and late apoptosis to varying degrees, and the total apoptosis rate also increased (P<0.05). Among them, the high concentration group had the most significant effect on FLS apoptosis induction, and the late apoptosis rate and total apoptosis rate of each concentration group of the Shujin Huoluo compound combination were higher than those of the conventional extraction group (P<0.05). The results are shown in Table 9. Fig.11 .

[0245] (2) Regulation of NO and IL-1β release by the combination of the Shujin Huoluo compound

[0246] Table 10 Regulation of NO and IL-1β release by the Shujin Huoluo compound composition

[0247]

[0248] *Compared with the blank group, P<0.05; △ Compared with the conventional extraction group, P < 0.05

[0249] After LPS induction, the release of inflammatory factors NO and IL-1β in the model group was significantly higher than that in the blank group (P<0.05), indicating that the synovial inflammation model has been established; compared with the model group, the release of NO and IL-1β in the conventional extraction group and the low, medium and high concentration groups of the Shujin Huoluo compound combination was reduced, and the difference was statistically significant (P<0.05). Among them, the high concentration group of the Shujin Huoluo compound combination had the most obvious inhibitory effect on the release of NO and IL-1β, and the inhibitory effect of each concentration group of the Shujin Huoluo compound combination on the release of inflammatory mediators was higher than that of the conventional extraction group (P<0.05). The results are shown in Table 10. Fig.12 .

[0250] Experimental Example 5

[0251] 1. Medication

[0252] (1) Preparation of the test drug solution used in the compound group: The compound refined solution prepared in step (1) of Example 1 was added to the culture medium for dilution, and lipopolysaccharide (LPS) was added to obtain a compound drug solution with an LPS concentration of 1 μg / mL and a ligustrazine concentration of 654.96 μg / mL.

[0253] (2) Preparation of the test drug solution used in the TMP encapsulation group: The drug-loaded exosomes prepared in step (2) of Example 1 were diluted in the culture medium, and lipopolysaccharide (LPS) was added to obtain a drug-loaded exosome solution with an LPS concentration of 1 μg / mL and a ligustrazine concentration of 458.29 μg / mL.

[0254] (3) Preparation of the test drug solution used in the combined treatment group: The compound refined solution prepared in step (1) of Example 1 and the drug-loaded exosomes prepared in step (2) of Example 1 (the volume ratio of drug-loaded exosomes to compound refined solution was 1:2) were taken respectively, diluted with culture medium, and lipopolysaccharide (LPS) was added to obtain a combined drug solution with an LPS concentration of 1 μg / mL and a ligustrazine concentration of 1113.25 μg / mL.

[0255] 2. Isolation and culture of FLS

[0256] The synovial tissue obtained during joint replacement was cleaned and minced under sterile conditions and treated with a digestion solution containing collagenase II in a 37° cell culture incubator for 4 h. After digestion, the incompletely digested tissue was removed by filtering through a 100-mesh filter. Subsequently, the mixture was heated at 1000 r / min. -1 The cells were centrifuged for 10 min and resuspended in DMEM F12 medium containing 10% FBS to obtain FLS. [4] The FLS in the logarithmic growth phase were cultured at 2×10 4 pc·mL -1 The cells were seeded at a density of 1000 μL in a 24-well culture plate, and each well was filled with 1000 μL of cell suspension for subsequent experimental studies.

[0257] 3. FCM detection of cell apoptosis

[0258] The cells were randomly grouped as follows:

[0259] TMP encapsulation group: 500 μL of drug-loaded exosome solution containing 1 μg / mL LPS and 458.29 μg / mL ligustrazine was added to each well;

[0260] Compound group (LPS+SHCE): Add compound drug solution with LPS concentration of 1 μg / mL and ligustrazine concentration of 654.96 μg / mL, 500 μL per well;

[0261] Combined treatment group: Add a combined drug solution with an LPS concentration of 1 μg / mL and a ligustrazine concentration of 1113.25 μg / mL, 500 μL per well.

[0262] Model group (LPS): add cell culture medium containing 1 μg / mL LPS, 500 μL per well.

[0263] Blank group (cell culture medium): add cell culture medium, 500 μL per well.

[0264] After 24 h of treatment, the supernatant was collected and analyzed for NO and IL-1β release according to the instructions provided by the kit manufacturer.

[0265] 4. Results

[0266] After LPS induction, the release of inflammatory factors TNF-α and MMP-13 in the model group was significantly higher than that in the blank group (P<0.05), indicating that the synovial inflammation model had been established; compared with the model group, the release of TNF-α and MMP-13 in the TMP encapsulation group, the compound group, and the combined treatment group was reduced, and the difference was statistically significant (P<0.05). Among them, the combined treatment group had the most obvious inhibitory effect on the release of TNF-α and MMP-13, and the effect was greater than the sum of the compound group and the TMP encapsulation group. The results are shown in Table 11. Fig.24 .

[0267] In OA research, TNF-α and MMP-13 are two key representative indicators. TNF-α, as a major proinflammatory factor, drives synovial inflammation and exacerbates local inflammatory responses by activating pathways such as NF-κB; MMP-13 is the main enzyme that degrades cartilage matrix (especially type II collagen), directly leading to cartilage destruction. Both play a core role in the pathological progression of OA, so detecting their release can effectively evaluate the anti-inflammatory and cartilage protective effects of drugs. Through the LPS-induced FLS model, the quantitative detection of TNF-α and MMP-13 using ELISA technology can reveal the regulatory effects of drugs on synovial inflammation and cartilage degradation, thereby providing an important reference for OA treatment.

[0268] Table 11 TNF-α and MMP-13 release

[0269]

[0270] *Compared with the blank group, P<0.05; △ Compared with the model group, P<0.05

[0271] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A pharmaceutical composition, characterized in that The invention comprises ligustrazine nanoparticles and a muscle-relaxing and collateral-activating compound composition. The ligustrazine nanoparticles comprise a carrier and ligustrazine loaded in the carrier. The muscle-relaxing and collateral-activating compound composition comprises the following raw materials in parts by weight: 20-40 parts of ligustrazine, 20-40 parts of sinomenium wilfordii, 10-30 parts of notopterygium wilfordii, 10-35 parts of angelica dahurica, 5-25 parts of sappan wood, 10-30 parts of argyi leaf, 5-20 parts of angelica sinensis, 5-20 parts of safflower, 5-20 parts of frankincense, 5-20 parts of myrrh, 5-20 parts of asarum, 5-25 parts of chuanxiong, 5-25 parts of drynaria, 5-25 parts of schizonepeta tenuifolia, 5-25 parts of schizonepeta radix, and 5-25 parts of achyranthes bidentata.

2. The pharmaceutical composition according to claim 1, characterized in that In the pharmaceutical composition, the ratio of the mass of ligustrazine provided by the ligustrazine nanoparticles to the mass of ligustrazine provided by the muscle-soothing and collateral-activating compound composition is 1:0.1-50.

3. The pharmaceutical composition according to claim 1 or 2, characterized in that The content of ligustrazine in the muscle-relaxing and collateral-activating compound composition is 0.1-10 μg / mg; and / or, the content of ligustrazine in the ligustrazine nanoparticles is 25-500 μg / mg; and / or, the content of ligustrazine in the pharmaceutical composition is 500-1500 μg / mL.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that The carrier includes exosomes, liposomes or polymeric carrier materials; Preferably, the raw materials for preparing the liposome include phospholipids and cholesterol; more preferably, the phospholipids include phosphatidylcholine, 1,2-distearoyl-sn-glyceryl-3-phosphocholine (DSPC), 1,2-dilauroyl-sn-glyceryl-3-phosphoethanolamine (DLPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2- One or more of dilauroyl-sn-glyceryl-3-phosphoethanolamine (DLPE), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (DSPE-PEG), 1,2-dipalmitoyl-rac-glyceromethoxypolyethylene glycol (DPG-PEG), 1,2-distearoyl-rac-glyceryl-3-methylpolyoxyethylene (DSG-PEG), and 1,2-dimyristoyl-rac-glyceryl-3-methylpolyoxyethylene (DMG-PEG); Preferably, the exosomes are derived from peripheral blood, urine, saliva, cerebrospinal fluid, mesenchymal stem cells or milk; Preferably, the polymer carrier material includes one or more of polylactic acid, polylactic acid-glycolic acid copolymer, chitosan, polycaprolactone, polyethylene glycol modified material, sodium alginate, gelatin, polymethyl methacrylate, and silica nanoparticles.

5. A method for preparing the pharmaceutical composition according to any one of claims 1 to 4, characterized in that: The mixture is prepared by mixing a liquid containing ligustrazine nanoparticles with a liquid containing a muscle-relaxing and collateral-activating compound composition; Optionally, the concentration of ligustrazine in the liquid containing ligustrazine nanoparticles is 500-3000 μg / mL; Optionally, the concentration of ligustrazine in the liquid containing the muscle-relaxing and collateral-activating compound composition is 100-2000 μg / mL; Optionally, the volume ratio of the liquid containing ligustrazine nanoparticles to the liquid containing the muscle-relaxing and collateral-activating compound composition is 1:20 to 20:

1.

6. The method for preparing the pharmaceutical composition according to claim 5, characterized in that: The preparation method of the muscle-relaxing and collateral-activating compound composition comprises weighing chuanxiong, sinomenium, notopterygium, angelica, sappan wood, wormwood, angelica, safflower, frankincense, myrrh, asarum, chuanduan, drynaria, smilax glabra, radix polygoni multiflori, and rhizoma spatholobi according to selected weight proportions, mixing and extracting according to conventional extraction methods or extracting respectively according to conventional extraction methods and then mixing to obtain the compound composition; Optionally, the conventional extraction method includes one or more of maceration extraction, decoction extraction, reflux extraction, percolation extraction, ultrasonic extraction and steam distillation; Optionally, after the conventional extraction method is used for extraction, the method further includes a step of refining and purifying the obtained extract; the refining and purification treatment includes one or more of water extraction and alcohol precipitation, extraction, silica gel chromatography column separation and macroporous resin column separation.

7. The pharmaceutical composition according to claim 5 or 6, characterized in that The preparation method of the ligustrazine nanoparticles comprises preparing a blank carrier, using the blank carrier and a solution containing ligustrazine as raw materials to carry out drug loading according to a conventional drug loading method, and obtaining the nanoparticles; Optionally, the mass ratio of the blank carrier to ligustrazine is 1 to 10:1; Optionally, the concentration of the solution containing ligustrazine is 0.01-50 mg / mL; Optionally, the solvent of the solution containing ligustrazine is one or more of methanol, ethanol, acetone, glycerol, petroleum ether, acetonitrile, chloroform, water, saline, PBS, DMSO, PEG, propylene glycol, sodium acetate buffer, dichloromethane, n-butanol, isopropanol, tetrahydrofuran, ether, dimethylformamide (DMF), dimethylacetamide (DMAc), toluene, and cyclohexane; Optionally, the conventional drug loading method includes one or more of ultrasound method, electroporation method, solvent volatilization method, double emulsification method, nanoprecipitation method, spray drying method, freeze drying method, ion gel method, self-assembly method, high pressure homogenization method, and hot melt extrusion method; Optionally, after the conventional drug loading method is used for drug loading, the step of purifying the obtained mixed solution is also included; the purification treatment method includes one or more of centrifugation, ultrafiltration, dialysis, gel filtration, chromatography, dialysis combined with freeze-drying, precipitation-resuspension, magnetic separation, membrane filtration, and washing-centrifugation.

8. A pharmaceutical preparation, characterized in that A pharmaceutical composition comprising any one of claims 1 to 4 or a pharmaceutical composition prepared by the preparation method of any one of claims 5 to 7, and optionally one or more pharmaceutically acceptable carriers; Optionally, the pharmaceutical preparation is a gel, cream, ointment, patch, emulsion or suspension; Optionally, the pharmaceutically acceptable carrier is selected from at least one or more of a pharmaceutically acceptable solvent, a water-soluble matrix, an oil-soluble matrix, a penetration enhancer, a humectant, a solubilizer, an antioxidant, a preservative, a pH adjuster, an emulsifier, and a thickener.

9. Use of the pharmaceutical composition according to any one of claims 1 to 4, or the pharmaceutical composition prepared by the preparation method according to any one of claims 5 to 7, or the pharmaceutical preparation according to claim 8 in the preparation of drugs for preventing or treating bone and joint diseases.

10. The use according to claim 9, characterized in that: The bone and joint disease is at least one of osteoarthritis, traumatic arthritis, rheumatoid arthritis, gouty arthritis, synovitis, bursitis, cervical spondylosis, lumbar spondylosis, scapulohumeral periarthritis and bone hyperplasia.

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