A pharmaceutical composition, a pharmaceutical preparation comprising the same, and use thereof

Abstract

The present application relates to the field of medicine, and more particularly to a kind of pharmaceutical composition, and the pharmaceutical preparation and application comprising the pharmaceutical composition, the pharmaceutical composition includes ligustrazine nanoparticle and Shujin Huoluo compound composition, ligustrazine nanoparticle includes carrier and ligustrazine loaded in carrier, by ligustrazine nanoparticle and Shujin Huoluo compound composition combined use, play synergistic effect, significantly improve the treatment effect to OA.In addition, both combined use shows significant curative effect in reducing synovial inflammation and repairing chondrocyte apoptosis and reducing pain-related biological indicators, which is of great significance to improve the quality of life of OA patients.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and more specifically to a pharmaceutical composition, a pharmaceutical preparation comprising the pharmaceutical composition, and its application. Background Technology

[0002] Osteoarthritis (OA) falls under the category of "Bi syndrome" in Traditional Chinese Medicine (TCM). It is a chronic disease primarily characterized by joint pain, swelling, and functional impairment, caused by factors such as wind, cold, and dampness. According to statistics from the World Health Organization, there are approximately 350 million confirmed cases of OA globally, nearly 60% of whom are elderly. With the increasing aging of the population, its prevalence is showing a year-on-year upward trend. TCM, as an important component of traditional Chinese medicine, possesses unique advantages in the treatment of OA. Scientific formula composition and preparation methods are key aspects of the modern application and development of TCM.

[0003] Based on years of clinical experience and basic research, and grounded in traditional Chinese medicine theory and modern pharmacology, the inventors of this application have formulated a muscle-relaxing and blood-activating compound composition. Previous studies have shown that this muscle-relaxing and blood-activating compound composition has significant effects on regulating the proliferation, apoptosis, and migration of OA synovial fibroblasts (FLSs) and inhibiting the release of inflammatory factors (see Chinese patent document CN111920849A).

[0004] Chuanxiong is the principal herb in the Shujin Huoluo formula, which has the effects of promoting blood circulation, removing blood stasis, regulating qi, and relieving pain. In addition to its use in the treatment of cardiovascular and gynecological diseases, it is also widely used in the treatment of joint diseases (see references: Guan Yongmei, Jiang Cheng, Zang Zhenzhong, et al. Research progress on chemical composition, pharmacological effects and clinical application of volatile oil of Chuanxiong [J]. Chinese Traditional and Herbal Drugs, 2024, 46(03): 873-880. Liang Qi, Zhang Laibin, Lü Jieli. Research progress on chemical composition and pharmacological effects of Chuanxiong [J]. Journal of Xinxiang Medical College, 2024, 41(03): 275-285.). Tetramethylpyrazine (TMP) is the main active ingredient of Ligusticum chuanxiong and is often selected as the main quality control standard for compound preparations containing Ligusticum chuanxiong (see the literature Wang Wei. Analysis of the method for determining the content of tetramethylpyrazine in compound Ligusticum chuanxiong capsules by high performance liquid chromatography [J]. Journal of Rational Clinical Drug Use, 2012, 5(23):123.). Modern pharmacological studies have found that tetramethylpyrazine 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 pharmacological activity of tetramethylpyrazine derivatives [J]. West China Journal of Pharmaceutical Sciences, 2023, 38(03):340-344. Tang Yan, Zhang Li, Liu Haihong. Tetramethylpyrazine inhibits TLR4 / MyD88 / NF-κB signaling pathway to reduce inflammatory response in rats with endometriosis [J]. Journal of Immunology, 2023, 39(06):469-477.). However, TMP has vasodilatory, sedative, analgesic, microcirculation-improving, and immune-enhancing effects. Previous studies by our research group have also found that tetramethylpyrazine can significantly reduce the levels of NO and prostaglandin E2 in the synovial fluid of osteoarthritis, and has a certain effect on alleviating inflammation in osteoarthritis (see Xie Pingjin, et al. Intervention of tetramethylpyrazine on the expression of type II collagen fiber α1 gene, vascular endothelial growth factor mRNA and miR20b in 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 tetramethylpyrazine on osteoarthritis and improve the quality of life of OA patients, it is necessary to develop a new drug composition and drug formulation for the treatment of OA. Summary of the Invention

[0005] Therefore, the purpose of this invention is to improve the therapeutic effect of tetramethylpyrazine on osteoarthritis, thereby providing a pharmaceutical composition, a pharmaceutical preparation comprising the pharmaceutical composition, and its application.

[0006] Therefore, the present invention provides a pharmaceutical composition comprising ligustrazine nanoparticles and a muscle-relaxing and blood-activating compound composition. The ligustrazine nanoparticles comprise a carrier and ligustrazine loaded within the carrier. The muscle-relaxing and blood-activating compound composition comprises the following raw materials in parts by weight: 20-40 parts of Ligusticum chuanxiong, 20-40 parts of Sinomenium acutum, 10-30 parts of Notopterygium incisum, 10-35 parts of Angelica dahurica, 5-25 parts of Caesalpinia sappan, 10-30 parts of Artemisia argyi, 5-20 parts of Angelica sinensis, 5-20 parts of Carthamus tinctorius, 5-20 parts of Boswellia carterii, 5-20 parts of Commiphora myrrha, 5-20 parts of Asarum heterotropoides, 5-25 parts of Dipsacus asper, 5-25 parts of Drynaria fortunei, 5-25 parts of Lycopodium clavatum, 5-25 parts of Clematis chinensis, and 5-25 parts of Achyranthes bidentata.

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

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

[0009] Furthermore, the carrier includes exosomes, liposomes, or polymeric carrier materials;

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

[0011] Furthermore, the polymer carrier material includes one or more of the following: polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), chitosan, polycaprolactone (PCL), polyethylene glycol (PEG) modified materials, sodium alginate, gelatin, polymethyl methacrylate, and silica nanospheres. Among these, polyethylene glycol modified materials include PEG-PLA, PEG-PCL, and PEG-PLGA.

[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-described pharmaceutical compositions, wherein a liquid containing ligustrazine nanoparticles is mixed with a liquid containing a muscle-relaxing and blood-activating compound composition.

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

[0015] Optionally, the concentration of ligustrazine in the liquid containing the muscle-relaxing and blood-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 blood-activating compound composition is 1:20 to 20:1.

[0017] Furthermore, the preparation method of the muscle-relaxing and blood-activating compound composition includes weighing Chuanxiong, Qingfengteng, Qianghuo, Baizhi, Sumu, Aiye, Danggui, Honghua, Ruxiang, Moyao, Xixin, Chuanduan, Gusuibu, Shenjincao, Tougucao, and Niuxi according to the selected weight proportions, mixing them and then extracting them according to conventional extraction methods or extracting them separately according to conventional extraction methods and then mixing them to obtain the product;

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

[0019] Optionally, after extraction using the conventional extraction method, the step of purifying the obtained extract may be included; the purification process may include one or more of the following: water extraction and alcohol precipitation, extraction, silica gel column separation, and macroporous resin column separation.

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

[0021] S1. Water extraction step: After extracting each raw material with water 1-5 times, concentrate to obtain an aqueous extract. The mass of water used in each water extraction is more than 8 times the mass of the raw material; the extraction time for each water extraction is greater than or equal to 1.5 hours; and the extraction temperature is 80-100℃.

[0022] S2. Alcohol precipitation step: Mix the aqueous extract with a 70-90% alcohol solution, precipitate with alcohol, and collect the alcohol extract. The volume of the alcohol solution is more than 5 times the volume of the concentrated aqueous extract. 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 water extraction is performed 1.5-2.5 times, the mass of water used in each water extraction is 10-12 times the mass of the raw drug, and the extraction time is 2-2.5 hours. In the alcohol precipitation step, an 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, a concentration step is included after both steps S1 and S2. The volume of the alcohol solution is 5-6 times the volume of the concentrated aqueous extract. Optionally, step S1 concentrates to obtain an aqueous extract with a relative density of 1.05-1.15, and step S2 concentrates to obtain a purified solution with a relative density of 1.16-1.25.

[0026] Furthermore, the preparation method of the tetramethylpyrazine nanoparticles includes preparing a blank carrier, and loading the blank carrier and a solution containing tetramethylpyrazine into a drug using conventional drug loading methods to obtain the nanoparticles.

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

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

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

[0030] Optionally, the conventional drug loading methods include one or more of the following: ultrasonication, electroporation, solvent evaporation, dual emulsification, nanoprecipitation, spray drying, freeze drying, ion gelation, self-assembly, high-pressure homogenization, and hot melt extrusion.

[0031] Optionally, after drug loading using the conventional drug loading method, the process further includes a step of purifying the resulting mixture; the purification method includes one or more of centrifugation, ultrafiltration, dialysis, gel filtration, chromatography, dialysis combined with lyophilization, precipitation-resuspension, magnetic separation, membrane filtration, and washing-centrifugation.

[0032] The present invention also provides a pharmaceutical formulation comprising any of the pharmaceutical compositions described above or a pharmaceutical composition prepared by any of the preparation methods described above, 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 the following pharmaceutically acceptable solvents, water-soluble matrices, oil-soluble matrices, penetration enhancers, humectants, solubilizers, antioxidants, preservatives, pH adjusters, emulsifiers, and thickeners.

[0035] Examples include, but are not limited to, one or more of the following: stearic acid, glyceryl monostearate, paraffin wax, liquid paraffin, white petrolatum, lanolin, cetyl alcohol, octadecyl alcohol, Span series, beeswax, animal and vegetable oils, glycerin, sorbitol, polyethylene glycol, Tween series, sodium lauryl sulfate, dimethyl sulfoxide, triethanolamine, sodium carboxymethyl cellulose, sodium hyaluronate, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl ester, carbomer, xanthan gum, and carboxymethyl cellulose.

[0036] The use of any of the above-described pharmaceutical compositions or pharmaceutical compositions or pharmaceutical preparations prepared by any of the above-described methods in the preparation of medicaments for the prevention or treatment of osteoarthritis.

[0037] Furthermore, the bone and joint diseases mentioned are at least one of osteoarthritis, traumatic arthritis, rheumatoid arthritis, gouty arthritis, synovitis, bursitis, cervical spondylosis, lumbar spondylosis, frozen shoulder, and bone hyperplasia.

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

[0039] 1. This invention provides a pharmaceutical composition comprising ligustrazine nanoparticles and a muscle-relaxing and blood-activating compound composition. The ligustrazine nanoparticles include a carrier and ligustrazine loaded within the carrier. The muscle-relaxing and blood-activating compound composition comprises the following raw materials in parts by weight: 20-40 parts of Ligusticum chuanxiong, 20-40 parts of Sinomenium acutum, 10-30 parts of Notopterygium incisum, 10-35 parts of Angelica dahurica, 5-25 parts of Caesalpinia sappan, 10-30 parts of Artemisia argyi, 5-20 parts of Angelica sinensis, 5-20 parts of Carthamus tinctorius, 5-20 parts of Boswellia carterii, 5-20 parts of Commiphora myrrha, 5-20 parts of Asarum heterotropoides, 5-25 parts of Dipsacus asper, 5-25 parts of Drynaria fortunei, 5-25 parts of Lycopodium clavatum, 5-25 parts of Clematis chinensis, and 5-25 parts of Achyranthes bidentata. The combined use of ligustrazine nanoparticles and the muscle-relaxing and blood-activating compound composition achieves a synergistic effect, significantly improving the therapeutic effect on osteoarthritis (OA). Furthermore, the combined use of these two treatments showed significant efficacy in reducing synovial inflammation, repairing chondrocyte apoptosis, and alleviating pain-related biomarkers, which is of great significance for improving the quality of life of OA patients.

[0040] 2. The present invention provides a pharmaceutical composition in which the carrier comprises exosomes, liposomes, or polymeric carrier materials, preferably exosomes. Exosomes are nanoscale carriers with good biocompatibility and targeting capabilities, capable of carrying multiple bioactive molecules. They fuse with target cells through the cell membrane, directly and precisely delivering the encapsulated traditional Chinese medicine monomers to the lesion site. This effectively protects the drug components from degradation by the in vivo environment, improves drug stability, and reduces side effects on healthy tissues. More preferably, when mesenchymal stem cell exosomes are used as the carrier, TMP encapsulates the mesenchymal stem cell exosome carrier to form nanoscale drug particles, which can better enhance the therapeutic effect on OA. Furthermore, using mesenchymal stem cell exosomes as the carrier can also increase the drug loading and encapsulation efficiency of tetramethylpyrazine nanoparticles.

[0041] 3. The pharmaceutical preparations provided by this invention can be further formulated into various dosage forms according to actual applications. These can be used externally, for example, but not limited to, gels, creams, ointments, plasters, emulsions, or suspensions. The pharmaceutical preparations used in this study can be used as a massage medium, combining standardized massage techniques with modern technology to form a highly efficient drug delivery system. During massage, the synergistic effect of mechanical stimulation and drug delivery is fully utilized, enabling the drug to penetrate the affected area more effectively, increasing local drug concentration, and enhancing the therapeutic effect, providing an innovative solution for drug delivery in massage therapy. To comprehensively evaluate the therapeutic effect of this standardized treatment plan combining "tradition and modernity" on OA, considering the important role of articular cartilage and synovium in the pathological process of OA, the inventors of this application have improved the application of the "relaxing muscles and activating collaterals" massage method by comparing the treatment effects of each group and further analyzing the molecular mechanisms. Attached Figure Description

[0042] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0043] Figure 1 The results are the single-factor optimization results for the water extraction process; ****P < 0.00001, ***P < 0.0001, **P < 0.01, *P < 0.05, nsP > 0.05;

[0044] Figure 2 The results are the single-factor optimization results for the alcohol precipitation process; ****P < 0.00001, ***P < 0.0001, **P < 0.01, *P < 0.05, nsP > 0.05;

[0045] Figure 3-5 The results of Box-Behnken response surface methodology (RSM) for water extraction process optimization are shown in the figure. Figure 3 For water extraction time and water volume, Figure 4 The number of water extractions and the amount of water added, Figure 5 The number of water extractions and the water extraction time;

[0046] Figure 6-8 Box-Behnken results for optimizing the alcohol precipitation process; among them, Figure 6 This refers to the amount and concentration of ethanol used. Figure 7 The time for alcohol precipitation is related to the ethanol concentration. Figure 8 The time for alcohol precipitation and the amount of ethanol used;

[0047] Figure 9 This is a specific chromatogram; from top to bottom, they represent the reference solution, the test solution, and the blank solution.

[0048] Figure 10 This is the standard curve for TMP reference standard;

[0049] Figure 11 The regulatory effect of the muscle-relaxing and blood-activating compound composition on FLS apoptosis (A: high concentration group, B: medium concentration group, C: low concentration group, D: conventional extraction group, E: blank group);

[0050] Figure 12 The results of the muscle-relaxing and blood-activating compound composition on the regulation of NO and IL-1β release are shown. ****P < 0.00001. The left graph represents NO level, and the right graph represents IL-1β level. The vertical axis represents concentration.

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

[0052] Figure 14 The particle size results are for drug-loaded exosomes (EXOs after drug loading); the horizontal and vertical labels Size represents the size, and Concentration (particles / ml) is translated as particle concentration (particles / mL);

[0053] Figure 15 Electron microscopy results of EXO before drug loading (100000×);

[0054] Figure 16 Electron microscopy results of EXO after drug loading (100000×);

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

[0056] Figure 18 The potential results of EXO before drug loading; the horizontal and vertical axes are translated as Zeta Potential; the vertical axis is translated as Frequency.

[0057] Figure 19 The results show the potential of EXO after drug loading; the horizontal and vertical axes are translated as Zeta Potential and Frequency.

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

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

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

[0061] Figure 23Western blots showing the expression levels of OA-related proteins: A. Normal group; B. Model group; C. Simple massage group; D. Compound massage group; E. Combined treatment massage group; ****P<0.00001, ***P<0.0001, **P<0.01, *P<0.05; Relative protein level is translated as relative protein level.

[0062] Figure 24 The values ​​represent the release amounts of TNF-α and MMP-13; ****P < 0.00001; the vertical axis represents concentration. Detailed Implementation

[0063] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0064] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0065] 1. Materials

[0066] 1.1 Traditional Chinese Medicine The medicinal materials used in this study to promote blood circulation and relieve muscle tension include: Chuanxiong, Qingfengteng, Qianghuo, Baizhi, Sumu, Aiye, Danggui, Honghua, Ruxiang, Moyao, Xixin, Chuanduan, Gusuibu, Shenjincao, Tougucao, and Niuxi. They were purchased from the Chinese Medicine Pharmacy of China-Japan Friendship Hospital, passed 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 The reagents ethanol, methanol, and acetonitrile (AR grade, analytical grade) were all purchased from Chengdu Jinshan Chemical Reagent Co., Ltd., and the tetramethylpyrazine standard was provided by the China Institute for Drug and Biological Products.

[0069] 1.2.2 Instruments: High-speed multi-functional pulverizer (LFP-800A): Laifu flagship store; Digital display water bath constant temperature shaker (HZ-9212S): Jintan Jerell Electric Appliance Co., Ltd.; Rotary evaporator (RE52AA): Shanghai Yarong Biochemical Instrument Factory; Electronic balance (JA3003): Shanghai Shunyu Hengping Scientific Instrument Co., Ltd.; Liquid chromatograph (Agilent 6890N 5975): Agilent Technologies Inc.

[0070] 1.3 Cells

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

[0072] Examples 1-5 Pharmaceutical Compositions

[0073] Examples 1-5 provide a series of pharmaceutical compositions and their preparation methods. These pharmaceutical compositions all include tetramethylpyrazine nanoparticles and a muscle-relaxing and blood-activating compound composition. The pharmaceutical compositions are prepared by mixing a liquid containing tetramethylpyrazine nanoparticles with a liquid containing the muscle-relaxing and blood-activating compound composition. The composition and preparation method of the liquid containing tetramethylpyrazine nanoparticles and the liquid containing the muscle-relaxing and blood-activating compound composition used in each example are the same. The only difference is that the volume ratio of the two is different, resulting in different total contents of tetramethylpyrazine in the pharmaceutical composition, and different mass ratios (TMP1:TMP2) of tetramethylpyrazine provided by drug-loaded exosomes to tetramethylpyrazine provided by the muscle-relaxing and blood-activating compound composition. See Table 1 for details.

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

[0075] The raw materials of the muscle-relaxing and blood-activating compound composition are as follows: 30g of Ligusticum chuanxiong, 30g of Sinomenium acutum, 20g of Notopterygium incisum, 20g of Angelica dahurica, 15g of Caesalpinia sappan, 20g of Artemisia argyi, 10g of Angelica sinensis, 10g of Carthamus tinctorius, 10g of Boswellia carterii, 10g of Commiphora myrrha, 10g of Asarum heterotropoides, 15g of Dipsacus asper, 15g of Drynaria fortunei, 15g of Lycopodium clavatum, 15g of Clematis chinensis, and 15g of Achyranthes bidentata. The ligustrazine nanoparticles include exosomes and ligustrazine loaded within the exosomes.

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

[0077] Weigh out the following herbs according to the above weights: Ligusticum chuanxiong, Sinomenium acutum, Notopterygium incisum, Angelica dahurica, Caesalpinia sappan, Artemisia argyi, Angelica sinensis, Carthamus tinctorius, Boswellia carterii, Commiphora myrrha, Asarum heterotropoides, Dipsacus asper, Drynaria fortunei, Lycopodium clavatum, Clematis chinensis, and Achyranthes bidentata. After washing, mix all the herbs except Boswellia carterii and Commiphora myrrha and decoct twice with water. For the first decoction, add 10 times the weight of the herbs in water, soak for 1 hour, and decoct for 2 hours. For the second decoction, add 10 times the weight of the herbs in water and decoct for 2 hours. After 1.5 hours of the second decoction, add Boswellia carterii and Commiphora myrrha and continue decoction for another 0.5 hours. Combine the decoctions and filter for later use. Pour the solution into a rotary evaporator and concentrate under reduced pressure to obtain an extract with a relative density of 1.1. Add an 80% ethanol solution, which 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 it into a rotary evaporator, and concentrate under reduced pressure to obtain a compound purified solution with a relative density of 1.2 (i.e., the mass of the compound extract in each milliliter of purified solution is 1200 mg), which is denoted as the liquid containing the muscle-relaxing and blood-activating compound composition.

[0078] Test: 1 mL of the compound purified solution was dried to obtain the traditional Chinese medicine compound extract. 30 mg of the traditional Chinese medicine compound extract was used as the test sample. HPLC analysis using the method described in Example 2 showed that the content of tetramethylpyrazine in the traditional Chinese medicine compound extract (or the dried compound purified solution) was 0.8187 μg / mg. Calculations showed that the content of tetramethylpyrazine in the compound purified solution was 982.44 μg / mL.

[0079] The preparation method of tetramethylpyrazine 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). Cell supernatant was collected, thawed on ice, centrifuged at 500 x g for 5 min at 4°C, and the supernatant was collected. The supernatant was then transferred to a new 50 ml centrifuge tube, centrifuged at 2000 x g for 30 min at 4°C, and the supernatant was collected again. After centrifugation at 10000 x g for 60 min, the supernatant was collected, filtered through a 0.22 μm sterile filter, and added to an ultracentrifuge tube. The tube was centrifuged at 120000 x g for 70 min at 4°C. After centrifugation, the supernatant was carefully removed, the precipitate was dissolved in sterile PBS, and the resuspended solution was collected and designated as the blank exosome solution, ready for subsequent experiments. The concentration of exosomes in the blank exosome solution was tested in Experiment Example 2 and found to be 6.01846 μg / μL.

[0081] Preparation of drug-loaded exosomes: Dissolve 20 mg of tetramethylpyrazine in 1 ml of methanol to obtain a tetramethylpyrazine solution with a concentration of 20 μg / μl. Measure the blank exosome solution and the tetramethylpyrazine solution (containing 800 μg of tetramethylpyrazine) at a mass ratio of 2:1 (exosomes in the blank exosome solution to tetramethylpyrazine in the tetramethylpyrazine solution), mix them into one tube, and place on ice. Ultrasonication on ice: Use a Biosafer 650-92 ultrasound machine (provided by Cytec (China) Co., Ltd.), set to 20% power ratio, with a cycle of 30 seconds on and 3 minutes off, for a total of 6 cycles. After ultrasound treatment, incubate the solution at 37℃ for 60 minutes to restore the exosome membrane. The solution was subjected to ultrafiltration (100 kDa), and the supernatant was collected to obtain a drug-loaded exosome solution (volume 184 μL), denoted as the liquid containing tetramethylpyrazine nanoparticles. HPLC analysis in Example 2 showed that the concentration of tetramethylpyrazine in the drug-loaded exosome solution was 1374.88 μg / mL. BCA quantitative analysis in Example 2 showed that the concentration of exosomes in the drug-loaded exosome solution was 4.34267 μg / μL.

[0082] Table 1 Formula Table

[0083]

[0084] Example 6 Pharmaceutical Composition

[0085] This embodiment provides a pharmaceutical composition that is basically the same as that in Example 1, except that the raw materials of the muscle-relaxing and blood-activating compound composition are different. The raw material composition in this embodiment is as follows: 20g of Ligusticum chuanxiong, 40g of Sinomenium acutum, 10g of Notopterygium incisum, 35g of Angelica dahurica, 5g of Caesalpinia sappan, 30g of Artemisia argyi, 20g of Angelica sinensis, 5g of Carthamus tinctorius, 20g of Boswellia carterii, 20g of Commiphora myrrha, 5g of Asarum heterotropoides, 5g of Dipsacus asper, 25g of Drynaria fortunei, 5g of Lycopodium clavatum, 25g of Clematis chinensis, and 25g of Achyranthes bidentata.

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

[0087] Example 7 Pharmaceutical Composition

[0088] This embodiment provides a pharmaceutical composition that is basically the same as that in Example 1, except that the raw materials of the muscle-relaxing and blood-activating compound composition are different. The raw material composition in this embodiment is as follows: 40g of Ligusticum chuanxiong, 20g of Sinomenium acutum, 30g of Notopterygium incisum, 10g of Angelica dahurica, 25g of Caesalpinia sappan, 10g of Artemisia argyi, 5g of Angelica sinensis, 20g of Carthamus tinctorius, 5g of Boswellia carterii, 20g of Commiphora myrrha, 5g of Asarum heterotropoides, 25g of Dipsacus asper, 5g of Drynaria fortunei, 25g of Lycopodium clavatum, 5g of Clematis chinensis, and 20g of Achyranthes bidentata.

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

[0090] Example 8 Pharmaceutical Composition

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

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

[0093] Weigh out 0.1g of lecithin and 0.02g of cholesterol separately, 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 using a high-speed homogenizer to prepare 1mL of blank liposomes. Dissolve 20mg of tetramethylpyrazine in 1mL of methanol to obtain a tetramethylpyrazine solution with a concentration of 20μg / μl. Add 1mL of the tetramethylpyrazine solution to 1mL of blank liposomes and homogenize using a high-speed homogenizer to obtain drug-loaded liposomes.

[0094] Example 9 Ointment

[0095] The ointment of this embodiment is prepared as follows: Take 90g of the pharmaceutical composition prepared according to the method of Example 1, 10g of triethanolamine, and 40g of carbomer 940, mix them, stir evenly, add distilled water to 300mL, heat and stir in an 80°C water bath, and set aside; melt 40g of glyceryl stearate and 30g of petrolatum together at 80°C, add white mineral oil to 100mL, then add the above-mentioned prepared materials, and finally add 10g of Sy-45 compound preservative, stir and mix until it becomes an ointment, and the ointment is obtained.

[0096] Example 10 Hydrogel

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

[0098] Preparation: Dissolve carbomer in water and stir until completely dissolved. Add glycerol and ethanol, and continue stirring until homogeneous. Adjust the pH to neutral (approximately 6.8-7.2) with triethanolamine. While stirring, add the pharmaceutical composition prepared in Example 1 and stir until homogeneous to obtain a hydrogel for later use.

[0099] Example 11 Emulsion

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

[0101] Preparation: Vaseline, stearic acid, white mineral oil, and Tween 80 are mixed and heated to 70°C, stirred until homogeneous to obtain the oil phase. Water, glycerin, and vitamin E are mixed and heated to the same temperature (70°C), stirred until homogeneous to obtain the aqueous phase. The aqueous phase is slowly added to the oil phase while stirring until emulsification is complete in 20 minutes. The mixture is cooled to approximately 40°C, and the pharmaceutical composition prepared in Example 1 is added, stirred until homogeneous to obtain the emulsion.

[0102] Experimental Example 1

[0103] 1. Preliminary screening of extraction and alcohol precipitation processes using single-factor experiments.

[0104] (1) Single-factor experiments on water extraction process

[0105] Weigh out 30g of Ligusticum chuanxiong, 30g of Sinomenium acutum, 20g of Notopterygium incisum, 20g of Angelica dahurica, 15g of Caesalpinia sappan, 20g of Artemisia argyi, 10g of Angelica sinensis, 10g of Carthamus tinctorius, 10g of Boswellia carterii, 10g of Commiphora myrrha, 10g of Asarum heterotropoides, 15g of Dipsacus asper, 15g of Drynaria fortunei, 15g of Lycopodium clavatum, 15g of Clematis chinensis, and 15g of Achyranthes bidentata. After washing, mix all the raw materials except for Boswellia carterii and Commiphora myrrha and decoct twice with water. For the first decoction, add 10 times the weight of the raw materials in water, soak for 1 hour, and decoct for 2 hours. For the second decoction, add 10 times the weight of the raw materials in water and decoct for 2 hours. After 1.5 hours of the second decoction, add Boswellia carterii and Commiphora myrrha and continue to decoct for 0.5 hours. Combine the decoctions, filter and set aside. 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, single-factor experiments were conducted to investigate the parameters in the water extraction process.

[0106] SHC water extraction was performed by varying three factors: the amount of water added during decoction, the water extraction time, and the number of water extractions. The extract was prepared using the method described above. The only difference was that, in the water extraction process, the mass of water added during decoction was adjusted to 4, 6, 8, 10, or 12 times the weight of the raw material, while other conditions remained unchanged; or, the water extraction time was adjusted to 0.5, 1, 1.5, 2, or 2.5 hours, while other conditions remained unchanged; or, the number of water extractions was adjusted to 1, 2, 3, 4, or 5 times, while other conditions remained unchanged. Frankincense and myrrh were added when 0.5 hours of decoction remained, and the decoction time was continued for another 0.5 hours after adding frankincense and myrrh.

[0107] Each condition was repeated three times, and the SHCE yield was calculated.

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

[0109] (2) Single-factor experiments on alcohol precipitation process

[0110] Weigh out 30g of Ligusticum chuanxiong, 30g of Sinomenium acutum, 20g of Notopterygium incisum, 20g of Angelica dahurica, 15g of Sappanwood, 20g of Artemisia argyi, 10g of Angelica sinensis, 10g of Carthamus tinctorius, 10g of Boswellia carterii, 10g of Commiphora myrrha, 10g of Asarum heterotropoides, 15g of Dipsacus asper, 15g of Drynaria fortunei, 15g of Lycopodium clavatum, 15g of Clematis chinensis, and 15g of Achyranthes bidentata. After washing, mix all the raw materials except for Boswellia carterii and Commiphora myrrha with 10 times the weight of the raw materials in water, soak for 1 hour, then decoct twice, adding 10 times the weight of the raw materials in water each time, and decoct for 2 hours each time; the second time... Frankincense and myrrh were added after 1.5 hours of decoction, and the decoction was continued for another 0.5 hours. The decoctions were combined, filtered, and then poured into a rotary evaporator for concentration under reduced pressure to obtain an extract with a relative density of 1.1. An 80% ethanol solution (5 times the volume of the extract) was added (5 times the volume of the concentrated extract). After mixing thoroughly, the mixture was allowed to stand for 48 hours. The supernatant was collected, poured into a rotary evaporator, and concentrated under reduced pressure to obtain a compound purified solution (SHCE) with a relative density of 1.2. Based on this method, single-factor experiments were conducted to investigate the parameters in the alcohol precipitation process.

[0111] SHC was subjected to alcohol precipitation by varying the ethanol concentration, ethanol volume, and precipitation time, specifically to obtain the compound purified solution using the method described above. The only differences were: adjusting the ethanol concentration to 35%, 50%, 65%, 80%, or 95% while keeping other conditions constant; or adjusting the ethanol volume to 2, 3, 4, 5, or 6 times the volume of the extract while keeping other conditions constant; or adjusting the precipitation time to 12, 24, 36, 48, or 60 hours while keeping other conditions constant.

[0112] Each condition was repeated three times, and the SHCE yield was calculated.

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

[0114] (3) Experimental Results

[0115] See results Figure 1As shown, the SHCE yield initially increased and then decreased with increasing water volume, extraction time, and number of extractions. The highest SHCE yield was achieved when the water volume was 10 times the optimal value, showing a statistically significant difference compared to 4, 6, and 8 times the optimal value (P < 0.05). Similarly, the highest SHCE yield was achieved when the extraction time was 2 hours, showing a statistically significant difference compared to 0.5, 1, and 1.5 hours (P < 0.05). The highest SHCE yield was also achieved when the number of extractions was 2 times, showing a statistically significant difference compared to other groups (P < 0.05). At 12 times the optimal value or an extraction time of 2.5 hours, the SHCE yield was slightly lower than under the optimal conditions, but the difference was not statistically significant (P > 0.05).

[0116] The results showed that the SHCE yield first increased and then decreased with increasing ethanol concentration, ethanol dosage, and precipitation time. The highest SHCE yield was achieved at an ethanol concentration of 80%, with statistically significant differences compared to 35%, 50%, and 65% (P < 0.05). The highest SHCE yield was also achieved at a 5-fold increase in ethanol dosage, with statistically significant differences compared to 2, 3, and 4-fold increases (P < 0.05). The highest SHCE yield was achieved at a precipitation time of 48 h, with statistically significant differences compared to 12, 24, and 36 h (P < 0.05). At 6-fold ethanol dosage or a precipitation time of 60 h, the SHCE yield was slightly lower than the optimal conditions, but the difference was not statistically significant (P > 0.05). See the results below. Figure 2 .

[0117] 2. Optimization of extraction process using Box-Behnken response surface methodology

[0118] (1) Experimental Design

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

[0120] Table 2. Factor Levels in Response Surface Analysis of Water Extraction Process

[0121]

[0122] Table 3. Factor Levels 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 analysis of variance

[0127] The response surface methodology and response values ​​(Table 4) were analyzed using Design Expert 13.0 software. After regression fitting of each factor, a ternary quadratic regression equation was obtained for the SHCE yield of water extraction on the factors of water addition (A), water extraction time (B), and number of water extractions (C): 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 ​​for Water Extraction Process Response Surface Analysis

[0129]

[0130] The results of the ANOVA and significance tests on the regression equation are shown in Table 5. The results indicate that when the water-extracted SHCE content is used as the response value, the model P-value is 0.0002, indicating that the model is significant, the regression equation has a good fit, and is statistically significant. The model lack of fit P-value is 0.7033, greater than 0.05, indicating that unknown factors have little interference with the experimental results. This model can significantly fit the effects of water addition (A), water extraction time (B), and number of water extractions (C) on the yield of water-extracted SHCE, without any lack of fit. It can replace the actual experimental points to reflect the actual situation for result analysis and prediction. The model coefficient R was determined. 2 The value is 0.9687, indicating that 96.87% of the response value changes can be explained and predicted by this model, making it relatively reliable. The influence of each factor on the SHCE extraction yield is as follows: water extraction time (B) > number of water extractions (C) > water addition (A). The interaction terms AB, AC, and BC are not significant factors in the model. The quadratic term of water addition, A... 2 The quadratic term B of the water extraction time 2 The quadratic term C of the number of water extractions 2 It has a significant impact on the yield of SHCE.

[0131] Table 5. Analysis of Variance of Regression Equations for Water Extraction Process

[0132]

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

[0134] The response surface contour lines and surface plots obtained by performing ternary quadratic regression fitting analysis on the Box-Behnken experimental data of SHC water extraction using Design Expert 13.0 software are shown below. Figure 3-5 This set of figures visually reflects the influence of various factors and their interactions on the SHCE yield of SHC. The results show that the contour plots of the interaction terms between water addition, water extraction time, and the number of water extractions are all close to circles, indicating that the interactions are not significant, a result consistent with Table 5. After optimization and prediction, the optimal process parameters for SHC water extraction are: water addition of 10.15 times, water extraction time of 2.12 h, and 2.16 extractions. Under these conditions, the predicted SHCE yield of SHC is 30.47%, close to the highest value in the single-factor experiments.

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

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

[0137] The response surface methodology and response values ​​(Table 6) were analyzed using Design Expert 13.0 software. After regression fitting of each factor, a ternary quadratic regression equation was obtained for the SHCE yield of water extraction on the factors of water addition (A), water extraction time (B), and number of water extractions (C): 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. Experimental Design and Response Values ​​for Alcohol Precipitation Process Response Surface Analysis

[0139]

[0140]

[0141] The results of the ANOVA and significance tests on the regression equation are shown in Table 7. The results indicate that when the water-extracted SHCE content is used as the response value, the model P-value is less than 0.0001, indicating that the model is highly significant, the regression equation has a good fit, and is statistically significant. The model lack of fit P-value is 0.8383, greater than 0.05, indicating that unknown factors have little interference with the experimental results. This model can significantly fit the effects of ethanol concentration (A), ethanol dosage (B), and ethanol precipitation time (C) on the yield of water-extracted SHCE, without any lack of fit. It can be used to represent the actual experimental data for result analysis and prediction. The model coefficient R was determined. 2The value is 0.9806, indicating that 98.06% of the response value changes can be explained and predicted by this model, which is relatively reliable. The influence of each factor on the SHCE extraction yield is in the order of ethanol dosage (B) > ethanol 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 significant. 2 The quadratic term B of ethanol usage 2 The quadratic term C of the precipitation time 2 It has a significant impact on the yield of SHCE.

[0142] Table 7. Analysis of Variance of Regression Equations for Alcohol Precipitation Process

[0143]

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

[0145] The response surface contour lines and surface plots obtained by performing ternary quadratic regression fitting analysis on the Box-Behnken experimental data of SHC alcohol precipitation using Design Expert 13.0 software are shown below. Figure 6-8 This set of figures visually reflects the influence of various factors and their interactions on the SHCE yield of SHC alcohol precipitation. The results show that the contour plots of the pairwise interactions between alcohol precipitation time and ethanol dosage are nearly circular, indicating no significant interaction, consistent with the results in Table 7. The contour plots of ethanol concentration and alcohol precipitation time or ethanol dosage are elliptical, with a slightly steep slope, indicating some interaction, but combined with the results in Table 7, there is no significant difference in the 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 time 49.32 h. Under these conditions, the predicted SHCE yield of SHC alcohol precipitation is 22.49%, close to the highest value in the single-factor experiments.

[0146] Experimental Example 2: Determination of Relevant Parameters of Muscle-Relaxing and Collateral-Activating Compound Composition and Ligustrazine Nanoparticles

[0147] 1. Determination method of ligustrazine content in the muscle-relaxing and blood-activating compound composition

[0148] (1) Preparation of reference solution

[0149] Accurately weigh 80 mg of tetramethylpyrazine reference standard, add 2 mL of acetonitrile, and prepare a solution with a mass concentration of 0.04 mg / mL. -1 The reference standard stock solution.

[0150] (2) Preparation of the test solution

[0151] Accurately weigh 30 mg of the test sample, add 2 mL of acetonitrile, shake to dissolve for 30 min, 5000 rpm. -1Centrifuge for 20 min, then filter the supernatant through a 0.22 μm microporous membrane into a sample vial for testing.

[0152] (3) Detection

[0153] High-performance liquid chromatography (HPLC) was used for detection. The chromatographic conditions were as follows: column: Diamonsil C18 (250 nm × 4.6 mm, 5 μm); mobile phase: acetonitrile-water (30:70 v / v); 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 each of the prepared TMP reference solution, the muscle relaxant test solution, and the blank solution (acetonitrile) were accurately injected into the high-performance liquid chromatograph (HPLC) for determination. The results showed that the chromatographic peaks of the muscle relaxant test solution matched the main peaks in the reference solution, confirming the presence of TMP in the test sample. The chromatogram of the blank solution did not show similar peaks to the reference solution, excluding interference from other components, indicating that this HPLC test has good specificity and reliable results. (See attached figures). Figure 9 .

[0157] 2) Examination of linear relationships

[0158] Accurately pipette the TMP reference stock solution and prepare reference solutions of different mass concentrations with acetonitrile-water (30:70) to achieve a TMP concentration of 1 μg·mL⁻¹. -1 2 μg·mL -1 4 μg·mL -1 6 μg·mL -1 8 μg·mL -1 10 μg·mL -1 20 μg·mL -1 40 μg·mL -1 10 μL of each of the above concentration solutions was precisely pipetted into the high-performance liquid chromatograph and measured under the chromatographic conditions specified in section (3). Linear regression was performed using the peak area to correspond to the mass concentration of the reference standard. The regression equation for TMP was obtained as Y = 26.535X + 2.2955(R0 / R0). 2 =0.9998), in 1-40 μg·mL -1 The linear relationship is good within the range. See the results below. Figure 10 .

[0159] 3) Precision test

[0160] Accurately weigh the traditional Chinese medicine compound extract obtained in Example 1, and prepare the test solution (6 μg·mL) according to the above method. -1 Under the above chromatographic conditions, 10 μL was injected into the high-performance liquid chromatograph, and the injection was repeated 6 times to determine the relative standard deviation (RSD) of the TMP peak area.

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

[0162] The relative standard deviation (RSD) was 1% by continuous measurement of the TMP peak area, which meets the acceptance criteria (RSD≤2%). This indicates that the instrument used in the experiment has high precision and stable performance, ensuring the reliability of the experimental data.

[0163] 4) Repeatability test

[0164] Accurately weigh the traditional Chinese medicine compound extract obtained in Example 1 from the same batch, and prepare 6 test solutions (6 μg / mL) in parallel according to the above method. -1 Under the above chromatographic conditions, the peak area RSD of TMP was determined by injection.

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

[0166] 5) Stability test

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

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

[0169] 6) Recovery test

[0170] Accurately weigh 6 portions of the traditional Chinese medicine compound extract prepared in Example 1, each approximately 0.5g, from the same batch and containing known amounts. Add 6μg·mL⁻¹ to each portion. -1 The TMP reference solution was prepared according to the above method, and 10 μL of each solution was injected under the above chromatographic conditions to determine the recovery rate.

[0171] In the spiking recovery test, the average recovery rate of TMP was 99.76%, with an RSD of 2.15%. Both parameters are better than the industry standard (spiking recovery rate should be between 95-105%, and RSD should be ≤5%). A recovery rate close to 100% indicates high accuracy of the method in quantitative analysis, unaffected by matrix effects or other factors. The low RSD further confirms the reliability and repeatability of the method.

[0172] 7) Determination of TMP content

[0173] Accurately weigh 30 mg of the traditional Chinese medicine compound extract prepared in Example 1, and prepare test solutions in triplicate. Inject 10 μL of each solution under the above chromatographic conditions and record the peak area RSD of TMP. Substitute the RSD into the linear formula to calculate the TMP content.

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

[0175] 2. Determination of tetramethylpyrazine content in tetramethylpyrazine nanoparticles

[0176] (1) Preparation of reference solution

[0177] Accurately weigh 80 mg of tetramethylpyrazine reference standard, add 2 mL of acetonitrile, and prepare a solution with a mass concentration of 0.04 mg / mL. -1 The reference standard stock solution.

[0178] (2) Preparation of the test solution

[0179] Accurately weigh the liquid containing ligustrazine nanoparticles, dilute it 20 times with PBS, and then filter it through a 0.22μm microporous membrane into a sample vial for testing.

[0180] (3) Detection

[0181] High-performance liquid chromatography (HPLC) was used for detection. The chromatographic conditions were as follows: 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 content of exosomes in blank exosome solutions and drug-loaded exosomes was determined using the BCA protein concentration kit (Servicebio, G2026) according to the kit instructions.

[0184] 4. Drug loading and encapsulation efficiency

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

[0186] Based on the concentration of tetramethylpyrazine and the total volume of exosomes determined by the above high performance liquid chromatography method, the mass of drug encapsulated in the carrier (referred to as drug loading) is calculated according to the following formula. The encapsulation efficiency is calculated based on the drug loading and the total mass of added tetramethylpyrazine. The content of tetramethylpyrazine in the drug-loaded exosomes is calculated based on the drug loading, the concentration of exosomes in the drug-loaded exosomes, and the total volume of exosomes.

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

[0188] The drug loading capacity of the drug-loaded exosomes prepared in Example 1 was calculated to be 253.28 μg, the content of tetramethylpyrazine 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] 200 μl of drug-loaded liposomes were placed in a dialysis bag and dialyzed for 24 h using 20 mL of phosphate-buffered saline (PBS) as the dialysis medium. The dialysate outside the dialysis bag was replaced every 4 hours, for a total of 6 replacements, with a total dialysate volume of 120 mL. 1 mL of the dialysate was filtered through a 0.22 μm microporous membrane into a sample vial for analysis. The TMP content in the dialysate was tested under the chromatographic conditions described in section 2(3) of this experimental example. The amount of free TMP was calculated, and the drug loading and encapsulation efficiency were calculated using the following formulas.

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

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

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

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

[0195] 5. Measurement of particle size, morphology, zeta potential, and marker protein levels of tetramethylpyrazine nanoparticles.

[0196] The particle size, morphology, zeta potential, and marker protein levels of blank exosome solutions (EXO before drug loading) and drug-loaded exosomes (EXO after drug loading) in Example 1 were tested. The particle size analysis method was as follows: drug-loaded exosomes were diluted with sterile PBS, filtered through a 0.22 μm filter, and measured using a particle size analyzer. Zeta potential was measured three times for each sample group, and the average value was taken. (See [link to example 1]). Figure 13-14 As shown in 18-19 and Table 8.

[0197] Table 8 Particle size and potential

[0198]

[0199] The morphology was examined using electron microscopy. Results are shown in [Figure number missing]. Figure 15 and 16 As shown, the exosomes have similar morphology before and after drug loading, being 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. Results are shown below. Figure 17 As shown in the figure. The results showed that all contained characteristic exosome proteins: CD9 protein, TSG101, and HSP70 protein.

[0201] Experimental Example 3

[0202] 1. Animal Information and Drug Testing

[0203] This study used 30 male Wistar rats aged 8 weeks, weighing approximately 300g (±20g).

[0204] The test drug in the compound massage group: The muscle-relaxing and blood-activating compound composition was prepared according to Examples 1-5. The same mass of the muscle-relaxing and blood-activating compound composition was used to replace the drug composition prepared in Example 1. The ointment was prepared using the method of Example 9 with the muscle-relaxing and blood-activating compound composition as the active pharmaceutical ingredient. It is referred to as muscle-relaxing and blood-activating ointment below.

[0205] The test drug in the combined treatment massage group: an ointment 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 treatment massage group. There were 6 rats in each group. The normal group did not receive any treatment, while the other groups were induced to develop knee OA model by anterior cruciate ligament transection (ACLT) (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 required no treatment; the simple massage group received massage therapy; the combined massage therapy group received massage therapy with 1g of compound ointment as a medium each time, with the massage therapy being the same as that in the simple massage group; the compound massage therapy group received massage therapy with 1g of muscle-relaxing and blood-activating ointment as a medium each time, with the massage therapy being the same as that in the simple massage group. All three groups—simple massage, combined massage therapy, and compound massage therapy—received treatment twice a week for four weeks. After all treatments were completed, articular cartilage and synovial tissue samples were collected from each group of rats for subsequent analysis.

[0209] The massage procedures for the simple massage group, the combined massage group, and the compound massage group were based on the team's unique "relaxing muscles and tendons" massage method, formulated according to actual conditions and pre-experiments, and in accordance with the acupoint distribution 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 rat was placed in a lateral recumbent position with its forelimb fixed. The practitioner used the thumb and index finger to apply force to the model side of the knee joint and surrounding muscles, fascia, and other soft tissues with gentle kneading and grasping techniques, focusing on the patella, patellar ligament, medial and lateral collateral ligaments, quadriceps femoris muscle, etc. (the key acupoints for treatment were: bilateral Dubi, Heding, Weizhong, Yanglingquan, Liangqiu, and Zusanli). The average treatment time for each acupoint was about 1 minute, and the total treatment time for the above-mentioned loosening was 10 minutes. At the same time, passive movements such as knee flexion, extension, and rotation were performed, with a treatment time of about 2 minutes. Finally, the rat's knee joint was rubbed with the thumb to finish the treatment.

[0210] 3. Histological observation using HE staining.

[0211] Articular cartilage and synovial membrane samples were fixed in 4% paraformaldehyde for 24 hours. Articular cartilage underwent further decalcification with EDTA for 14 days. Dehydration was performed using a gradient of ethanol (70%, 80%, 90%, 95%, 100%) for 60 minutes each, followed by clearing with xylene for 20 minutes each. The samples were then embedded in paraffin and sectioned to a thickness of 4 μm. Dewaxing was then performed with xylene (10 minutes each, 2 steps), followed by rehydration with a gradient of ethanol (100%, 95%, 90%, 80%, 70%) for 5 minutes each. Hematoxylin staining was performed for 8 minutes, followed by differentiation with 1% hydrochloric acid alcohol for 10 seconds, rinsing with running water for 5 minutes for bluing, and eosin staining for 2 minutes. Dehydration was then performed using a gradient of ethanol, followed by clearing with xylene (3 minutes each step). Finally, the slides were mounted with neutral resin and observed and photographed under a microscope.

[0212] 4. TUNEL staining to observe chondrocyte apoptosis

[0213] Staining was performed according to the TUNEL kit instructions. Sections were incubated with proteinase K working solution (20 μg / mL) at room temperature for 15 min, followed by washing three times with PBS for 5 min each time. 50 μL of the TUNEL reaction mixture was added, and the sections were incubated at 37°C for 60 min in the dark. After incubation, the sections were washed three times with PBS for 5 min each time. Nucleic acids were stained with DAPI at room temperature in the dark for 5 min, followed by washing three times with PBS for 5 min each time. Finally, the sections were mounted with anti-fluorescence quenching mounting medium. Chondrocyte apoptosis was observed using a fluorescence microscope.

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

[0215] Synovial membrane samples were lysed on ice for 30 min, centrifuged at 12000 rpm for 10 min, and the supernatant was collected. Protein concentration was determined using a BCA kit. An equal volume of protein sample was denatured at 95℃ for 5 min, separated by gel electrophoresis, and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder for 1 h, incubated overnight with a specific primary antibody at 4℃, and then incubated with a secondary antibody for 1 h. The membrane was treated with ECL, and the signal was detected using a chemiluminescence imaging system. The gray values ​​of the bands were analyzed to assess protein expression levels.

[0216] 6. Data Statistics

[0217] Experimental data were statistically analyzed using SPSS 22.0 software. All data are expressed as mean ± standard deviation (Mean ± SD). One-way ANOVA was used for comparisons between groups, and p < 0.05 was considered statistically significant.

[0218] 7. Results

[0219] (1) Histological observation of synovium

[0220] The normal group showed normal synovial tissue structure, an intact lining layer, and 1-2 layers of synovial cells arranged neatly, without significant hyperplasia or angiogenesis. The model group showed significant synovial tissue hyperplasia, a markedly thickened lining layer, a substantial increase in the number of synovial cells, disordered arrangement, severe cell fibrosis, and hyaline degeneration throughout the synovial tissue, accompanied by significant angiogenesis. All treatment groups showed varying degrees of reduction in synovial hyperplasia; compared to the model group, the number of synovial cells, the degree of disordered arrangement, the thickness of the lining layer, and angiogenesis were all alleviated. Among these, the combined massage therapy group showed the most significant improvement, followed by the compound massage therapy group. While the effect of the simple massage group was not as good as the compound massage therapy group and the combined massage therapy group, it still showed significant improvement compared to the model group. Results are shown below. Figure 20 .

[0221] (2) Histological observation of cartilage

[0222] In the normal group, the cartilage tissue structure was intact, chondrocytes were neatly arranged, the matrix was uniform, the tidal line was regular, and the deep subchondral bone and calcified base were well embedded. In contrast, the model group showed significant cartilage degeneration, with unclear chondrocyte stratification, a significantly reduced number of chondrocytes, disordered arrangement, and obvious cracks and degradation signs in the matrix. A large amount of fibrous material replaced normal chondrocytes, showing obvious ossification changes. All treatment groups showed some relief from cartilage degeneration. Compared with the model group, the treatment groups showed an increase in the number of chondrocytes, a gradual restoration of neat arrangement, and some repair of the matrix structure. Among them, the combined massage therapy group showed the most significant improvement, manifested in the repair of chondrocyte number and arrangement, a more intact tidal line, and a significantly improved matrix structure. The compound massage therapy group was second best, and although the effect of the simple massage therapy group was not as good as the first two groups, it was still significantly improved compared to the model group. Results are shown in […]. Figure 21 .

[0223] (3) Detection of chondrocyte apoptosis

[0224] In the normal group, the chondrocyte apoptosis rate was low, and TUNEL staining showed only a small number of positive cells with weak fluorescence intensity. In contrast, the model group showed a significant increase in chondrocyte apoptosis, a marked increase in the number of TUNEL-positive cells, and strong fluorescence intensity. All treatment groups showed a decrease in chondrocyte apoptosis rate, with a significant reduction in both the number of TUNEL-positive cells and fluorescence intensity. Compared to the model group, the treatment groups showed a decrease in the number of apoptotic cells and a weakening of fluorescence intensity, indicating an improvement in apoptosis. The combined massage therapy group showed a more significant anti-apoptotic effect, with fewer TUNEL-positive cells, weaker fluorescence intensity, and a significantly reduced chondrocyte apoptosis rate. The improvement in chondrocyte apoptosis decreased sequentially between the compound massage group and the simple massage group. Results are shown below. Figure 22 .

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

[0226] Western blot results showed that Cox-2, IL-1β, TNF-α, VEGF, and ADAMTS-5 were all expressed in synovial tissue, with clear protein bands visible at their respective molecular weights. The expression levels of these proteins were low in the normal group. In the model group, Cox-2 expression was significantly increased, indicating inflammation and pain; IL-1β and TNF-α expression were significantly increased, suggesting enhanced inflammatory response; VEGF expression was increased, indicating active angiogenesis; and ADAMTS-5 expression was increased, reflecting accelerated cartilage degradation. In all treatment groups, the expression levels of these proteins decreased, with the most significant decrease observed in the combined massage therapy group; the protein expression levels decreased sequentially in the compound massage group and the simple massage group, but the effect was not as significant as in the combined massage therapy group. Overall, all treatment groups significantly alleviated inflammation, pain, angiogenesis, and cartilage degradation, with the combined massage therapy group showing the most outstanding effect. (See attached results). Figure 23 .

[0227] Experimental Example 4: Mechanism of Action of Muscle-Relaxing and Collateral-Activating Compound Composition on OOA

[0228] 1. Preparation of the test drug solution

[0229] Muscle-relieving and blood-activating compound solution: Weigh out the following ingredients according to the weight specified in Example 1: Ligusticum chuanxiong, Sinomenium acutum, Notopterygium incisum, Angelica dahurica, Sappanwood, Artemisia argyi, Angelica sinensis, Carthamus tinctorius, Boswellia carterii, Commiphora myrrha, Asarum heterotropoides, Dipsacus asper, Drynaria fortunei, Lycopodium clavatum, Clematis chinensis, and Achyranthes bidentata. After washing, mix all the raw materials except Boswellia carterii and Commiphora myrrha and decoct twice with water. For the first decoction, add 10 times the weight of the raw materials in water, soak for 1 hour, and decoct for 2 hours. For the second decoction, add 10 times the weight of the raw materials in water and decoct for 2 hours. After 1.5 hours of the second decoction... Add frankincense and myrrh and continue decocting for 0.5 hours. Combine the decoctions; filter and set aside. Pour into a rotary evaporator and concentrate under reduced pressure to obtain an extract with a relative density of 1.1. Add an 80% ethanol solution (5 times the volume of the extract) to the 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 purified solution with a relative density of 1.2. Dilute with culture medium to obtain 400 μg / mL. -1 200 μg·mL -1 100 μg·mL -1 The diluent.

[0230] The test drug solution for the conventional extraction group: According to the weights of the muscle-relaxing and blood-activating compound composition in Example 1, the following herbs were weighed: Ligusticum chuanxiong, Sinomenium acutum, Notopterygium incisum, Angelica dahurica, Caesalpinia sappan, Artemisia argyi, Angelica sinensis, Carthamus tinctorius, Boswellia carterii, Commiphora myrrha, Asarum heterotropoides, Dipsacus asper, Drynaria fortunei, Lycopodium clavatum, Clematis chinensis, and Achyranthes bidentata. After washing, the raw materials except for Boswellia carterii and Commiphora myrrha were mixed and decocted twice with water. For the first decoction, 10 times the weight of the raw materials were added to water, and the mixture was soaked for 1 hour and decocted for 2 hours. For the second decoction, 10 times the weight of the raw materials were added to water and decocted for 2 hours. After 1.5 hours of the second decoction, Boswellia carterii and Commiphora myrrha were added and the decoction was continued for another 0.5 hours. The decoctions were combined, filtered, and then concentrated under reduced pressure in a rotary evaporator to obtain an extract with a relative density of 1.1. This was diluted with culture medium to obtain a concentration of 400 μg / mL. -1 Aqueous extract.

[0231] 2. Isolation and culture of FLS

[0232] Synovial tissue obtained during joint replacement surgery was aseptically cleaned and minced, then treated with a digestion solution containing collagenase II in a 37°C cell culture incubator for 4 hours. After digestion, incompletely digested tissue was removed by filtering through a 100-mesh filter. Subsequently, the mixture was cultured at 1000 rpm. -1 Centrifuge for 10 min to pellet the cells, then resuspend in DMEM F12 medium containing 10% FBS to obtain FLS. [4] FLS in the logarithmic growth phase will be increased by 2×10 4 pcs·mL -1 Cells were seeded at a density of 500 μL in each well of a 24-well culture plate for subsequent experimental studies.

[0233] 3. FCM detection of cell apoptosis

[0234] The patients were randomly divided into five groups as follows: a high-concentration group of the muscle-relaxing and blood-activating compound composition (400 μg / mL). -1 ), the concentration group of the muscle-relaxing and blood-activating compound composition (200 μg·mL) -1 Low concentration group of muscle-relaxing and blood-activating compound composition (100 μg·mL) -1 ), conventional extraction group (400 μg·mL) -1 Aqueous extract and blank control group (cell culture medium) were used. 1 mL of the corresponding solution was added to each well, and after 24 h of treatment, the mixture was incubated at 1000 rpm. -1Cells were collected by centrifugation for 5 min, washed with PBS, and resuspended in 100 μL cell suspension. Each sample was stained with 5 μL of FITC-labeled Annexin V and 5 μL of Propidium Iodide (PI) and incubated at room temperature in the dark for 15 min. 400 μL of binding buffer was added to each sample, and then flow cytometry was used to detect and analyze apoptosis, evaluating the effect of different concentrations of the muscle-relaxing and blood-activating compound on apoptosis, and assessing the advantages of this optimized extraction method compared to conventional extraction.

[0235] 4. ELISA method for detecting NO and IL-1β release.

[0236] The release of inflammatory mediators NO and IL-1β by FLS induced by LPS was detected by ELISA. The groups were as follows: high concentration group of the muscle-relaxing and blood-activating compound composition (LPS 1 μg·mL). -1 +800μg·mL -1 ), the concentration group of the muscle-relaxing and blood-activating compound composition (LPS 1 μg·mL) -1 +400μg·mL -1 Low concentration group of muscle-relaxing and blood-activating compound (LPS 1 μg·mL) -1 +200μg·mL -1 ), conventional extraction group (LPS 1 μg·mL) -1 +400μg·mL -1 Aqueous extract), model group (LPS 1 μg·mL) -1 (1) Control 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 instructions provided by the kit manufacturer.

[0237] 5. Statistical Analysis

[0238] Data were collected in the form of mean ± standard deviation (Mean ± SD). Statistical analysis was performed using SPSS 27.0, GraphPad Prism 9, and Design Expert 13.0 software. One-way ANOVA was used for inter-group comparisons, and the p-value < 0.05 was considered statistically significant.

[0239] 6. Experimental Results

[0240] (1) Regulation of FLS apoptosis by muscle relaxants

[0241] Table 9. Regulation of FLS apoptosis by the muscle-relaxing and blood-activating compound composition (%)

[0242]

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

[0244] Compared with the control group, both the conventional extraction group and the various concentration groups of the muscle-relaxing and blood-activating compound induced early and late apoptosis in FLS to varying degrees, and the total apoptosis rate also increased accordingly (P<0.05). Among them, the high concentration group had the most significant apoptosis-inducing effect on FLS, and the late apoptosis rate and total apoptosis rate of each concentration group of the muscle-relaxing and blood-activating compound induced apoptosis were higher than those of the conventional extraction group (P<0.05). The results are shown in Table 9. Figure 11 .

[0245] (2) Regulation of NO and IL-1β release by the muscle-relaxing and blood-activating compound composition

[0246] Table 10. Regulation of NO and IL-1β release by the muscle-relaxing and blood-activating compound composition.

[0247]

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

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

[0250] Experimental Example 5

[0251] 1. Medicine

[0252] (1) Preparation of the test drug solution used in the compound group: The compound purified solution obtained 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 obtained in step (2) of Example 1 were added to the culture medium for dilution, 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: Take the compound purified solution obtained in step (1) of Example 1 and the drug-loaded exosomes obtained in step (2) of Example 1 (the volume ratio of drug-loaded exosomes to compound purified solution is 1:2), add culture medium to dilute, add lipopolysaccharide (LPS) 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] Synovial tissue obtained during joint replacement surgery was aseptically cleaned and minced, then treated with a digestion solution containing collagenase II in a 37°C cell culture incubator for 4 hours. After digestion, incompletely digested tissue was removed by filtering through a 100-mesh filter. Subsequently, the mixture was cultured at 1000 rpm. -1 Centrifuge for 10 min to pellet the cells, then resuspend in DMEM F12 medium containing 10% FBS to obtain FLS. [4] FLS in the logarithmic growth phase will be increased by 2×10 4 pcs·mL -1 Cells were seeded at a density of 1000 μL in each well of a 24-well culture plate for subsequent experimental studies.

[0257] 3. FCM detection of cell apoptosis

[0258] The cells were randomly grouped as follows:

[0259] TMP encapsulation group: Add 500 μL of drug-loaded exosome solution with LPS concentration of 1 μg / mL and tetramethylpyrazine concentration of 458.29 μg / mL to each well;

[0260] Compound group (LPS+SHCE): 500 μL of compound drug solution with LPS concentration of 1 μg / mL and tetramethylpyrazine concentration of 654.96 μg / mL was added to each well;

[0261] Combined treatment group: 500 μL of combined drug solution with LPS concentration of 1 μg / mL and ligustrazine concentration of 1113.25 μg / mL was added to each 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 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.

[0265] 4. Results

[0266] After LPS induction, the release levels of inflammatory factors TNF-α and MMP-13 in the model group were significantly higher than those in the control group (P<0.05), indicating that the synovial inflammation model had been established. Compared with the model group, the release levels of TNF-α and MMP-13 in the TMP encapsulation group, compound group, and combined treatment group were all reduced, and the differences were statistically significant (P<0.05). Among them, the combined treatment group had the most significant 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. Figure 24 .

[0267] In OA research, TNF-α and MMP-13 are two key representative indicators. TNF-α, as a major pro-inflammatory factor, drives synovial inflammation and exacerbates local inflammatory responses by activating pathways such as NF-κB; MMP-13 is a major enzyme that degrades cartilage matrix (especially type II collagen), directly leading to cartilage destruction. Both play a central role in the pathological progression of OA, therefore, detecting their release levels can effectively assess the anti-inflammatory and chondrogenic effects of drugs. Using an LPS-induced FLS model and ELISA technology to quantitatively detect TNF-α and MMP-13, the regulatory effects of drugs on synovial inflammation and cartilage degradation can be revealed, thus providing important reference for OA treatment.

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

[0269]

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

[0271] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A pharmaceutical composition for osteoarthritis, characterized in that, This pharmaceutical composition is made from ligustrazine nanoparticles and a muscle-relaxing and blood-activating compound. The ligustrazine nanoparticles include a carrier and ligustrazine loaded within the carrier. The muscle-relaxing and blood-activating compound is made from the following raw materials in parts by weight: 30 parts of Ligusticum chuanxiong, 30 parts of Sinomenium acutum, 20 parts of Notopterygium incisum, 20 parts of Angelica dahurica, 15 parts of Caesalpinia sappan, 20 parts of Artemisia argyi, 10 parts of Angelica sinensis, 10 parts of Carthamus tinctorius, 10 parts of Boswellia carterii, 10 parts of Commiphora myrrha, 10 parts of Asarum heterotropoides, 15 parts of Dipsacus asper, 15 parts of Drynaria fortunei, 15 parts of Lycopodium clavatum, 15 parts of Clematis chinensis, and 15 parts of Achyranthes bidentata. The carrier is an exosome derived from mesenchymal stem cells. In the pharmaceutical composition, the mass ratio of ligustrazine provided by the ligustrazine nanoparticles to the mass of ligustrazine provided by the muscle-relaxing and blood-activating compound is 1:1.

43.

2. A method for preparing the pharmaceutical composition according to claim 1, characterized in that, The liquid containing ligustrazine nanoparticles is prepared by mixing a liquid containing a muscle-relaxing and blood-activating compound composition; the volume ratio of the liquid containing ligustrazine nanoparticles to the liquid containing the muscle-relaxing and blood-activating compound composition is 1:

2.

3. The method for preparing the pharmaceutical composition according to claim 2, characterized in that, The preparation method of the liquid containing the muscle-relaxing and blood-activating compound composition includes weighing the following ingredients according to the selected weight proportions: Ligusticum chuanxiong, Sinomenium acutum, Notopterygium incisum, Angelica dahurica, Caesalpinia sappan, Artemisia argyi, Angelica sinensis, Carthamus tinctorius, Boswellia carterii, Commiphora myrrha, Asarum heterotropoides, Dipsacus asper, Drynaria fortunei, Lycopodium clavatum, Clematis chinensis, and Achyranthes bidentata. The raw materials, except for Boswellia carterii and Commiphora myrrha, are mixed and decocted twice with water. For the first decoction, 10 times the weight of the raw materials are added to water, and the mixture is soaked for 1 hour and decocted for 2 hours. For the second decoction, 10 times the weight of the raw materials are added to water and decocted for 2 hours. The second decoction lasts for 1.5 hours. Add frankincense and myrrh to the decoction and continue decocting for 0.5 hours. Combine the decoctions and filter for later use. Pour the decoction into a rotary evaporator and concentrate under reduced pressure to obtain an extract with a relative density of 1.

1. Add an 80% ethanol solution (5 times the volume of the concentrated extract) and mix well. Let stand for 48 hours. Collect the supernatant and pour it into a rotary evaporator for concentrated under reduced pressure to obtain a liquid containing the muscle-relaxing and blood-activating compound with a relative density of 1.

2.

4. The method for preparing the pharmaceutical composition according to claim 2, characterized in that, The method for preparing the tetramethylpyrazine nanoparticles includes preparing a blank carrier, and loading the blank carrier and a solution containing tetramethylpyrazine into the drug using conventional drug loading methods.

5. A pharmaceutical preparation, characterized in that, The pharmaceutical composition is prepared by the pharmaceutical composition of claim 1 or by any of the preparation methods of claims 2-4, and by a pharmaceutically acceptable carrier.

6. The pharmaceutical preparation according to claim 5, characterized in that, The pharmaceutical preparation is a gel, cream, ointment, patch, emulsion, or suspension; And / or, the pharmaceutically acceptable carrier is selected from at least one or more of the following pharmaceutically acceptable solvents, water-soluble matrices, oil-soluble matrices, penetration enhancers, humectants, solubilizers, antioxidants, preservatives, pH adjusters, emulsifiers, and thickeners.

7. The use of the pharmaceutical composition of claim 1, or the pharmaceutical composition prepared by any of the preparation methods of claims 2-4, or the pharmaceutical preparation of claim 5 or 6, in the preparation of a medicament for the prevention or treatment of osteoarthritis.

Images