The application of Fugui Zhitong recipe in the preparation of MSC-CM for relieving knee osteoarthritis oral enteric-coated preparation and the oral enteric-coated medicine

Abstract

This invention belongs to the field of pharmaceutical engineering technology, specifically relating to the application of Fugui Zhitong Fang in the preparation of MSC-CM oral enteric-coated formulations for relieving knee osteoarthritis and its oral enteric-coated drug. The oral enteric-coated formulation of MSC-CM for relieving knee osteoarthritis comprises: Fugui Zhitong Fang extract, MSC-CM lyophilized powder, enteric coating or enteric capsule shell; the Fugui Zhitong Fang extract is mixed with the MSC-CM lyophilized powder and filled into enteric capsule shells or made into enteric-coated microcapsules; wherein, the Fugui Zhitong Fang is composed of the following raw materials in parts by weight: 10-20 parts of light aconite root, 15-25 parts of cinnamon twig, 10-60 parts of astragalus root, 10-25 parts of ginger, 20-30 parts of atractylodes rhizome, 15-25 parts of saposhnikovia root, 10-20 parts of notopterygium root, 15-25 parts of eucommia bark, 10-20 parts of achyranthes root, 5-15 parts of prepared licorice root, 10-20 parts of red peony root, and 15-25 parts of anemarrhena rhizome. This formulation solves the problem of MSC-CM active factors being easily degraded by gastric acid and digestive enzymes and having low bioavailability when taken orally, and achieves synergistic effects of MSC-CM and traditional Chinese medicine in the treatment of knee osteoarthritis.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical engineering technology, specifically relating to the application of Fugui Zhitong Fang in the preparation of oral enteric-coated formulations for relieving knee osteoarthritis in MSC-CM and its oral enteric-coated drug. Background Technology

[0002] Knee osteoarthritis (KOA) is a chronic joint disease characterized by degenerative changes in articular cartilage, synovitis, and subchondral bone remodeling. Clinical manifestations include knee pain, swelling, and limited range of motion; in severe cases, it can lead to joint deformity and loss of function. It is estimated that there are approximately 355 million KOA patients worldwide, with about 60% of people over 55 years of age showing signs of knee osteoarthritis on X-rays. Furthermore, the incidence rate is increasing annually due to population aging.

[0003] Current clinical treatment strategies for KOA primarily include basic therapies, such as exercise rehabilitation, physical therapy, use of nonsteroidal anti-inflammatory drugs (NSAIDs), acetaminophen, intra-articular injections of hyaluronic acid and corticosteroids, and joint replacement surgery. However, these methods are mostly limited to symptom relief, are difficult to reverse or stop disease progression, and have various limitations such as gastrointestinal adverse reactions, cartilage damage risks, and prosthesis loosening.

[0004] Mesenchymal stem cells (MSCs) can regulate the intra-articular inflammatory microenvironment, inhibit cartilage matrix degradation, and promote cartilage repair by secreting anti-inflammatory factors (such as IL-10 and TGF-β), growth factors (such as VEGF and FGF), and extracellular vesicles through paracrine mechanisms. However, direct intra-articular injection of MSCs faces many challenges: short cell retention time in the joint cavity, low survival rate, functional decline under inflammatory conditions, and the need for professional physician operation, leading to poor patient compliance. More importantly, live cell preparations require strict cold chain transportation and liquid nitrogen storage, making clinical promotion difficult.

[0005] Mesenchymal stem cell-conditioned medium (MSC-CM) contains all the active substances secreted by MSCs, including growth factors, cytokines, and exosomes. MSC-CM has similar therapeutic effects to MSCs but without the cell-related risks of tumorigenesis and immune rejection. It can also be formulated as a lyophilized powder for easy storage and transportation. However, the active factors in MSC-CM (such as TGF-β and IL-10) are mostly proteins or peptides, which are easily degraded by gastric acid and digestive enzymes after oral administration, resulting in extremely low bioavailability. Currently, there are no reports of oral MSC-CM formulations for the treatment of knee osteoarthritis (KOA).

[0006] In the long history of traditional Chinese medicine development in China, the application of Chinese herbal medicines has become increasingly widespread. Traditional Chinese medicine believes that KOA belongs to arthralgia syndrome and bone arthralgia, and the most common pathogenesis is obstruction of yang deficiency, cold dampness. The Fuguizhitong formula is derived from the modified version of Guizhi Shaoyao Zhimu Decoction in Synopsis of the Golden Chamber, and is composed of 12 Chinese herbal medicines including Rhizoma Zingiberis Lateralis Praeparatum, Ramulus Cinnamomi, Radix Astragali, Rhizoma Atractylodis Macrocephalae, Radix Saposhnikoviae, Rhizoma seu Radix Notopterygii, Cortex Eucommiae, Radix Achyranthis Bidentatae, etc., with the effects of warming yang and dispelling cold, removing dampness and relieving pain. Clinical studies have shown that the pain improvement rate of this formula in the treatment of KOA is 76%, the joint function improvement rate is 66%, and there is no toxicity to the liver and kidneys. However, traditional oral administration has deficiencies such as low bioavailability, slow onset, and the need for long-term medication. Summary of the Invention

[0007] To solve the deficiencies of the existing technology, the present invention provides the application of the Fuguizhitong formula in the preparation of an oral enteric-coated preparation for MSC-CM to relieve knee osteoarthritis and its oral enteric-coated drug. This oral enteric-coated preparation improves the low oral bioavailability of active factors, the slow onset of traditional Chinese medicines, and the difficulty in achieving synergistic effects in the treatment of knee osteoarthritis. It not only utilizes the medicinal effects of the Chinese herbal medicines in the Fuguizhitong formula in relieving knee osteoarthritis, but also uses the natural polysaccharides in the extract of the Fuguizhitong formula as a protective agent for MSC-CM active factors, solving the problems of easy degradation of MSC-CM active factors by gastric acid and digestive enzymes and low bioavailability, and achieving synergistic effects between MSC-CM and traditional Chinese medicines in the treatment of knee osteoarthritis.

[0008] To achieve the above object, the present invention provides the following technical solutions: Provide the application of the Fuguizhitong formula in the preparation of an oral enteric-coated preparation for MSC-CM to relieve knee osteoarthritis, and the oral enteric-coated preparation for MSC-CM to relieve knee osteoarthritis includes: extract of the Fuguizhitong formula, freeze-dried powder of MSC-CM, enteric coating or enteric capsule shell; The extract of the Fuguizhitong formula and the freeze-dried powder of MSC-CM are mixed and filled into enteric capsule shells or made into enteric-coated pellets; Among them, the Fuguizhitong formula is composed of the following raw materials in parts by weight: 10-20 parts of Rhizoma Zingiberis Lateralis Praeparatum, 15-25 parts of Ramulus Cinnamomi, 10-60 parts of Radix Astragali, 10-25 parts of Rhizoma Zingiberis Recens, 20-30 parts of Rhizoma Atractylodis Macrocephalae, 15-25 parts of Radix Saposhnikoviae, 10-20 parts of Rhizoma seu Radix Notopterygii, 15-25 parts of Cortex Eucommiae, 10-20 parts of Radix Achyranthis Bidentatae, 5-15 parts of Radix Glycyrrhizae Preparata, 10-20 parts of Radix Paeoniae Rubra, 15-25 parts of Rhizoma Anemarrhenae.

[0009] The above approach combines the Fu-Gui Analgesic Formula, which has warming, dispersing, dampness-removing, and pain-relieving effects, with MSC-CM, which is rich in anti-inflammatory and repairing factors. The polysaccharides from Astragalus membranaceus, Atractylodes macrocephala, and Eucommia ulmoides in the Fu-Gui Analgesic Formula can form a hydrated gel layer in the acidic environment of the stomach, encapsulating MSC-CM microspheres. This physically blocks the degradation of MSC-CM by gastric acid and digestive enzymes, while simultaneously exerting its own therapeutic effect. This preparation balances therapeutic efficacy with local inflammation suppression, solving the problem of insufficient efficacy in single-component treatment models and achieving the therapeutic effect of MSC-CM.

[0010] Furthermore, the oral enteric-coated formulation is prepared by the following steps: The preparation of the extract of the Fugui Zhitong Formula includes: A1. Weigh out each Chinese herbal medicine component according to the formula and mix them; A2. Soak in water for 30-60 minutes, decoct and extract 2-3 times, 1-2 hours each time, and combine the filtrates; A3. Concentrate the filtrate to a relative density of 1.10~1.20, add ethanol to make the alcohol content reach 60~70%, and let it stand to precipitate; A4. Take the supernatant to recover the ethanol, concentrate and dry to obtain the extract of Fugui Zhitong Fang; The preparation of the oral enteric-coated formulation comprises: B1. Mix the extract of Fugui Zhitong Fang with MSC-CM freeze-dried powder at a weight ratio of 10~20:1 until uniform; B2. Add excipients for the self-microemulsion delivery system and granulate; B3. Fill into enteric-coated capsules or prepare enteric-coated microcapsules to obtain the oral enteric-coated preparation.

[0011] The above scheme achieves efficient integration and stable delivery of drug components. The water extraction-ethanol precipitation method for preparing the Fugui Zhitong Fang extract effectively enriches polysaccharide active ingredients and removes impurities. The relative density of the resulting extract is controlled between 1.10 and 1.20, which can be determined using a hydrometer. Ethanol precipitation removes large molecular impurities such as proteins and tannins, improving the purity of the formulation. Mixing the Fugui Zhitong Fang extract with MSC-CM lyophilized powder at a ratio of 10-20:1 ensures both the overall encapsulation and conditioning effect of the traditional Chinese medicine on the body and maintains the bioactive concentration of MSC-CM after release into the intestine. The introduction of a microemulsion delivery system promotes the dissolution of lipid-soluble components, improving absorption efficiency. Enteric treatment through filling or coating ensures that the drug is not released in the stomach and is released within 2 hours under intestinal fluid pH 6.8 conditions. The release rate is determined according to the in vitro release determination method in the General Chapter IV of the 2020 edition of the Chinese Pharmacopoeia.

[0012] Furthermore, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, or adipose-derived mesenchymal stem cells.

[0013] The above approach enables the acquisition of highly active and controllable-source MSC-CM cells with a strong secretory function, as umbilical cord, bone marrow, and adipose-derived mesenchymal stem cells all possess stable paracrine capabilities, continuously releasing functional factors such as TGF-β, IL-10, and VEGF under serum-free culture conditions. Umbilical cord mesenchymal stem cells exhibit strong proliferative capacity and low immunogenicity, making them suitable for large-scale production; bone marrow mesenchymal stem cells have a well-established research foundation and clearly defined functions; and adipose-derived mesenchymal stem cells are readily available and abundant. All three can serve as reliable cell sources for preparing conditioned media, ensuring batch-to-batch consistency of formulations.

[0014] Further, the preparation of the MSC-CM lyophilized powder includes: Revive frozen mesenchymal stem cells and passage them to the 3rd to 5th generation. Discard the culture medium and add serum-free culture medium to continue culturing for 48 h to 72 h. Collect the culture supernatant, filter it through a filter membrane, and obtain MSC-CM; Add 6%~9% trehalose, 2%~4% mannitol, and 0.5%~2% PVP, and freeze-dry to obtain MSC-CM lyophilized powder.

[0015] The above method significantly improves the lyophilization survival rate and storage stability of protein-active factors in MSCs-CM, as cells at passage 35 are in their optimal physiological state and exhibit vigorous secretory function. Serum-free culture avoids exogenous animal protein contamination, and a culture time of 48-72 hours allows for the accumulation of sufficient concentrations of exosomes and soluble factors. Filtering the supernatant through a membrane removes cell debris and microorganisms, ensuring the safety of the formulation. The addition of trehalose as a vitreous protectant, mannitol as a structural support, and PVP as an ice crystal inhibitor constitutes a composite lyophilization system, resulting in high retention rates of TGF-β activity and IL-10. The detection method is ELISA.

[0016] Furthermore, the self-microemulsion delivery system comprises medium-chain triglycerides, Tween 80, and polyethylene glycol 400.

[0017] The above approach enhances the dispersibility and transmembrane transport capacity of poorly soluble components. Medium-chain triglycerides, as an oil phase, can dissolve lipophilic active substances; Tween 80, as a nonionic surfactant, reduces interfacial tension; and polyethylene glycol 400, as a co-solvent, promotes spontaneous microemulsion formation. The synergistic effect of these three components rapidly forms microemulsion droplets with a particle size of less than 100 nm in the gastrointestinal tract, improving apparent drug solubility and lymphatic uptake efficiency.

[0018] Furthermore, the serum-free culture medium is α-MEM serum-free culture medium.

[0019] Through the above-mentioned solution, efficient secretion of mesenchymal stem cells under animal component-free conditions can be achieved. Since α-MEM contains abundant amino acids, vitamins, and trace elements, it supports cell metabolism and protein synthesis. Its serum-free formulation avoids batch differences and pathogen risks brought by fetal bovine serum, which is beneficial to the clinical transformation and quality control of subsequent preparations. This medium has strong adaptability and is widely used in the amplification and secretion induction of various mesenchymal stem cells, ensuring the consistency and functionality of the MSC-CM components.

[0020] Further, in step B2, the granulation step includes: placing each component in an autoclave, introducing supercritical CO2 until the pressure reaches 12 MP - 18 MPa and the temperature reaches 35°C - 45°C, stirring to form a slurry, and then rapidly releasing the pressure to obtain porous microspheres.

[0021] Through the above-mentioned solution, porous drug-loaded microspheres with a high specific surface area and good disintegration characteristics can be prepared. Because under supercritical CO2 conditions, CO2 penetrates into the interior of the material, and when the pressure is suddenly released, the gas rapidly escapes, forming a uniform microporous structure. This process is completed at a low temperature, avoiding the inactivation of thermosensitive components.

[0022] Further, the freeze-drying is carried out under the protection of nitrogen or argon, and the oxygen concentration is lower than 0.5%.

[0023] Through the above-mentioned solution, the impact of oxidative stress on protein activity can be minimized. Because freeze-drying and grinding are carried out in an inert gas atmosphere, oxygen can be effectively isolated to prevent oxidation denaturation or aggregation of sensitive proteins such as TGF-β and IL-10. The grinding process is carried out in a closed inert environment, avoiding the risk of dust explosion, while maintaining the particle uniformity, and the particle size distribution can be detected by a laser particle size analyzer.

[0024] Provide an oral enteric-coated drug for relieving knee osteoarthritis by combining Fufang Zhitong Prescription and MSC-CM, including the above-mentioned Fufang Zhitong Prescription in the preparation of an oral enteric-coated preparation for relieving knee osteoarthritis with MSC-CM.

[0025] Through the above-mentioned solution, an oral therapeutic product convenient for patients to use at home can be provided. This drug integrates the advantages of traditional Chinese medicine compound and stem cell secretory products, without the need for injection operation, significantly improving the treatment compliance. The preparation exists in the form of enteric-coated capsules or enteric-coated pellets, which can pass through the stomach intact and be released at a fixed point in the alkaline environment of the small intestine. Its content contains a two-component system with synergistic effects: Fufang Zhitong Prescription improves the yang deficiency and cold-dampness constitution, and MSC-CM directly inhibits the inflammatory reaction in the joint cavity.

[0026] Further, the drug is administered orally.

[0027] Through the above solution, a non-invasive and highly compliant treatment mode can be achieved. Since the oral administration method does not require professional medical staff to operate, patients can take the medicine by themselves in a home environment, greatly expanding the applicable scenarios. Compared with the live cell therapy that requires intra-articular injection, this preparation avoids the puncture risk and the possibility of infection.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention uses naturally occurring polysaccharide components in the extract of Fuguizhitong Decoction, such as astragalus polysaccharide, atractylodes polysaccharide, and eucommia polysaccharide, as the protection carriers for MSC-CM quick-frozen dry powder. In an acidic gastric environment, the above polysaccharide components can form a hydrated gel layer in situ, physically encapsulating MSC-CM microspheres, effectively isolating the degradation effects of gastric acid and digestive enzymes, and Fuguizhitong Decoction can also play a role in relieving knee osteoarthritis.

[0029] (2) The present invention has established a triple protection of traditional Chinese medicine polysaccharide gel layer, enteric coating, and freeze-drying protectant for MSC-CM, significantly reducing the destruction rate of MSC-CM active factors in simulated gastric juice, and then effectively improving the cumulative release rate in the intestinal environment, overcoming the problem of low bioavailability of traditional protein and polypeptide drugs by oral administration.

[0030] (3) Fuguizhitong Decoction regulates the overall body state and improves the pathological constitution of yang deficiency, cold and dampness in patients with knee osteoarthritis; MSC-CM rapidly inhibits local joint inflammation through anti-inflammatory factors such as TGF-β and IL-10. It is expected to be significantly superior to the单独使用附桂止痛方或单独使用MSC-CM in the KOA animal model.

[0031] (4) The present invention uses the freeze-dried powder of the conditioned medium of mesenchymal stem cells instead of a live cell preparation, fundamentally avoiding the potential risks of tumorigenesis, immune rejection reaction, and pathogenic microorganism contamination brought by live cells. Compared with the mesenchymal stem cell therapy in the prior art that must adopt an injection route, the present invention prepares MSC-CM into an oral enteric-coated preparation, and patients can take it by themselves at home without the operation of professional physicians, significantly improving the treatment compliance and clinical accessibility. Brief Description of the Drawings

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the embodiments required for the description of the embodiments or the prior art. Example 1

[0033] It should be noted that there is an unclear expression "单独使用附桂止痛方或单独使用MSC-CM" in item (3) of the original text, which may need to be further clarified for an accurate translation. Here it is translated as literally as possible.The embodiments of the present application provide an application of the Fuguizhitong formula in the preparation of an oral enteric-coated preparation for MSC-CM to relieve knee osteoarthritis. The oral enteric-coated preparation comprises an extract of the Fuguizhitong formula, freeze-dried powder of MSC-CM, enteric coating or enteric capsule shell; the extract of the Fuguizhitong formula is mixed with the freeze-dried powder of MSC-CM and filled into an enteric capsule shell or made into enteric-coated pellets; wherein, the Fuguizhitong formula is composed of the following raw materials in parts by weight: 10-20 parts of prepared common monkshood root, 15-25 parts of cassia twig, 10-60 parts of astragalus membranaceus, 10-25 parts of fresh ginger, 20-30 parts of atractylodes macrocephala, 15-25 parts of ledebouriella seseloides, 10-20 parts of notopterygium incisum, 15-25 parts of eucommia ulmoides, 10-20 parts of achyranthes bidentata, 5-15 parts of roasted licorice root, 10-20 parts of red peony root, 15-25 parts of anemarrhena asphodeloides. Compared with the problem in the prior art that the bioavailability of MSC-CM is extremely low due to its easy degradation by gastric acid and digestive enzymes after oral administration, the present invention combines the freeze-dried powder of MSC-CM with the extract of the Fuguizhitong formula and combines with an enteric-coated preparation, realizing effective protection of the active factors of MSC-CM, significantly improving its release rate and stability in the intestine, and solving the worldwide problem of oral administration of protein polypeptide drugs. Step 1: Weigh each traditional Chinese medicine component according to the formula, including 15 g of prepared common monkshood root, 20 g of cassia twig, 15 g of astragalus membranaceus, 15 g of fresh ginger, 25 g of atractylodes macrocephala, 20 g of ledebouriella seseloides, 15 g of notopterygium incisum, 20 g of eucommia ulmoides, 15 g of achyranthes bidentata, 10 g of roasted licorice root, 15 g of red peony root, 20 g of anemarrhena asphodeloides, and mix them evenly. <​​​​​​​​​​​​​​​​​​Step 8: Mix the extract of Fugui Zhitong Fang with MSC-CM lyophilized powder at a weight ratio of 15:1 until homogeneous. Step 9: Add the self-microemulsion delivery system excipients composed of medium-chain triglycerides, Tween 80, and polyethylene glycol 400, and granulate.

[0041] Step 9: Fill the granules into enteric-coated capsule shells to obtain the oral enteric-coated preparation.

[0042] Example 2 The application provided in this application also includes the preparation of the oral enteric-coated formulation through the following steps. The preparation of the Fugui Zhitong Fang extract includes: weighing each herbal component according to the formula and mixing them; soaking in water for 30-60 minutes, decocting and extracting 2-3 times, each time for 1-2 hours, and combining the filtrates; concentrating the filtrate to a relative density of 1.10-1.20, adding ethanol to achieve an alcohol content of 60-70%, allowing it to settle; recovering the ethanol from the supernatant, concentrating and drying to obtain the Fugui Zhitong Fang extract; The preparation of the oral enteric-coated formulation includes: mixing the Fugui Zhitong Fang extract with MSC-CM lyophilized powder at a weight ratio of 10-20:1; adding excipients from a self-microemulsion delivery system and granulating; filling into enteric-coated capsules or preparing enteric-coated microcapsules to obtain the oral enteric-coated formulation. This method has a clear process, is highly operable, can achieve mass production, and ensures the stability and synergistic effect of each component, which is conducive to industrial promotion.

[0043] Example 3 The applications provided in this application also include the mesenchymal stem cells being umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, or adipose-derived mesenchymal stem cells.

[0044] MSC-CM prepared from the above-mentioned mesenchymal stem cells is rich in various anti-inflammatory and repair-related factors such as TGF-β, IL-10, and VEGF, and has good biological activity. Moreover, it is widely available and has low ethical risks, making it suitable for large-scale drug development.

[0045] Example 4 The application provided in this application also includes the process of preparing the MSC-CM lyophilized powder.

[0046] Resuscitate frozen mesenchymal stem cells and passage them to passage 3-5. Discard the culture medium and add serum-free culture medium to continue culturing for 48-72 h. Collect the culture supernatant, filter it through a filter membrane to obtain MSC-CM. Add 6%-9% trehalose, 2%-4% mannitol and 0.5%-2% PVP, and freeze-dry to obtain MSC-CM lyophilized powder.

[0047] This process avoids the risk of heterologous protein contamination introduced by serum, improving the safety of the preparation; at the same time, the optimized lyophilization protection system significantly increases the retention rate of key active factors such as TGF-β and IL-10, ensuring the efficacy basis after oral administration.

[0048] Example 5 The application provided by the embodiment of the present application further includes that the self-microemulsion delivery system contains medium-chain triglycerides, Tween 80 and polyethylene glycol 400. This self-microemulsion system can rapidly form microemulsions in the gastrointestinal tract, promoting drug dispersion and transmembrane transport, improving the absorption efficiency of MSC-CM and traditional Chinese medicine extracts, and enhancing the overall bioavailability.

[0049] Example 6 The application provided by the embodiment of the present application further includes that the serum-free medium is α-MEM serum-free medium. This medium has clear components and high batch stability, can effectively support the secretion function of mesenchymal stem cells, and at the same time avoid the risks of immunogenicity and pathogen contamination brought by animal-derived components.

[0050] Example 7 The granulation steps provided by the embodiment of the present application include: placing each component in an autoclave, introducing supercritical CO2 until the pressure is 12 MP18 MPa and the temperature is 35℃45℃, stirring to form a slurry and then rapidly depressurizing to obtain porous microspheres. This supercritical CO2 granulation technology does not require high temperature and organic solvents, is green and environmentally friendly, and the obtained porous microspheres have a high specific surface area and good fluidity, which is beneficial for subsequent filling and intestinal release.

[0051] Example 8 The application provided by the embodiment of the present application further includes that the freeze-drying is carried out under the protection of nitrogen or argon and grinding, and the oxygen concentration is lower than 0.5%. The protection of inert gas can effectively prevent the oxidation and inactivation of active factors, maintain the long-term stability of the MSC-CM freeze-dried powder, and extend the product shelf life.

[0052] ]>Example 9 The embodiment of the present application also provides an oral enteric-coated drug for relieving knee osteoarthritis by combining Fufang Zhitong Prescription and MSC-CM. This drug contains the oral enteric-coated preparation prepared by the application described in any one of the foregoing embodiments and can be administered by oral administration. This drug combines traditional Chinese medicine theory with modern stem cell secretion product technology, plays the role of Fufang Zhitong Prescription in overall regulating the yang deficiency, cold and damp state of the body, and at the same time uses MSC-CM to rapidly inhibit local inflammatory reactions, realizing a synergistic treatment mode of overall conditioning and targeted repair, which is significantly superior to the treatment effect of single components.

[0053] Example 10 The oral enteric-coated drugs provided in this application are administered orally. Patients can take them themselves without the need for professional medical personnel, greatly improving treatment adherence and clinical accessibility, and are suitable for long-term management of chronic diseases.

[0054] To further illustrate the efficacy of the oral enteric-coated formulation of the present invention, the following experiments were conducted: Experimental Example 1 Experimental materials The extract of Fugui Zhitong Fang was prepared according to the method in Example 1.

[0055] Astragalus polysaccharide standard (purity ≥90%) Atractylodes macrocephala polysaccharide standard (purity ≥85%) Eucommia ulmoides polysaccharide standard (purity ≥85%) Simulated gastric juice: 0.1 mol / L HCl, pH 1.2, containing 3.2 g / L pepsin. Rheometers (such as TA Instruments Discovery HR-3) Scanning electron microscope, SEM, Hitachi SU8010 Dynamic light scattering instrument, DLS, Malvern Zetasizer Nano ZS (1) Viscosity measurement Step 1: Prepare a solution of Fugui Zhitong Fang extract with a concentration of 100 mg / mL according to Example 1, and dissolve it in simulated gastric juice.

[0056] Step 2: Incubate in a 37℃ water bath and take samples at 0, 0.5, 1 and 2 hours.

[0057] Step 3: Use a rheometer to measure the viscosity, with the shear rate set to 0.1-100 s⁻¹ and the temperature at 37℃.

[0058] Step 4: Record the apparent viscosity, focusing on the viscosity at low shear rates (0.1 s⁻¹).

[0059] (2) Control group setup: Blank control: Simulated gastric juice Single polysaccharide control: Astragalus polysaccharide, Atractylodes macrocephala polysaccharide, and Eucommia ulmoides polysaccharide, 100 mg / mL each. Flavor-deficient control: Extract of Fu Gui Zhi Tong Fang (a traditional Chinese medicine formula) with Astragalus membranaceus, Atractylodes macrocephala, and Eucommia ulmoides removed.

[0060] (3) Observation of gel microstructure Step 1: Take the sample after 2 hours of incubation and freeze it quickly with liquid nitrogen.

[0061] Step 2: Freeze-dry at -50℃ for 48 hours.

[0062] Step 3: Perform gold sputtering treatment, with a thickness of approximately 10 nm.

[0063] Step 4: Observe using a scanning electron microscope with an accelerating voltage of 5 kV and magnifications of 5000×, 10000×, and 20000×.

[0064] (4) Measurement of gel layer thickness Step 1: Take the sample after incubation for 2 hours and dilute it to an appropriate concentration.

[0065] Step 2: Use a dynamic light scattering instrument to detect the scattering angle at 173° and the temperature at 37°C.

[0066] Step 3: Record the hydrodynamic diameter (Dh) to reflect the size of the gel particles / microregions.

[0067] This shows that the natural polysaccharides in the extract of Fugui Zhitong Fang can form a hydrated gel layer in the gastric acid environment.

[0068] The final conclusion is: The 2-hour viscosity of the extract of Fugui Zhitong Fang was >1000 mPa·s (0.1 s⁻¹), which was determined by being significantly higher than that of the single polysaccharide and tasteless control.

[0069] The viscosity of a single polysaccharide at 2 hours is 300-600 mPa·s, which is lower than that of the whole extract.

[0070] The viscosity of the odorless control at 2 hours was <100 mPa·s, and the criterion was to prove that Atractylodes macrocephala, Astragalus membranaceus, and Eucommia ulmoides were key ingredients.

[0071] SEM gel pore size: 5-50 nm, the criterion is that it is smaller than the size of pepsin molecules.

[0072] DLS hydrodynamic diameter: expected value 100-500 nm, the criterion is to reflect the size of the gel microregion.

[0073] Experiment Example 2 Experimental materials MSC-CM lyophilized powder (prepared according to the method in Example 1); TGF-β ELISA kit, R&D Systems DY240; IL-10 ELISA kit, R&D Systems DY417; It simulates gastric juice with a pH of 1.2 and contains pepsin. Simulated intestinal fluid, with a pH of 6.8, containing trypsin; Enteric-coated capsule shell, coated with hydroxypropyl methylcellulose phthalate (HPMCP).

[0074] Experimental Groups Group 1: MSC-CM lyophilized powder was directly exposed to simulated gastric fluid; Group 2: MSC-CM and single Astragalus polysaccharide; Group 3: MSC-CM and the whole extract of Fugui Zhitong Fang (a traditional Chinese medicine formula); Group 4: MSC-CM and enteric-coated capsule shell, without polysaccharides; Group 5: MSC-CM and the whole extract of Fugui Zhitong Fang and enteric-coated capsule shell; Group 6: Untreated MSC-CM lyophilized powder.

[0075] Experimental methods Simulated gastric fluid exposure experiment Step 1: Add 10 mL of simulated gastric fluid to each group of samples and place them in a 37°C water bath.

[0076] Step 2: Incubate with shaking at 100 rpm for 2 hours.

[0077] Step 3: Immediately after sampling, adjust the pH to 7.0 using NaHCO3.

[0078] Step 4: Use an ELISA kit to detect TGF-β and IL-10 activity.

[0079] Simulated intestinal fluid release experiment Step 1: After 2 hours of exposure to gastric fluid, the sample is transferred to simulated intestinal fluid.

[0080] Step 2: Shake at 100 rpm for 2 hours in a 37°C water bath.

[0081] Step 3: Take samples at 0, 0.5, 1, and 2 hours.

[0082] Step 4: Use ELISA to detect the released active factors and calculate the cumulative release rate.

[0083] Calculation of activity retention rate Activity retention rate (%) = (Active concentration in experimental group / Active concentration in untreated control group) × 100% Experimental results: Group 1: TGF-β retention rate <10%, IL-10 retention rate <15%.

[0084] Group 2: TGF-β retention rate 35-50%, IL-10 retention rate 40-55%, and cumulative release rate of intestinal fluid over 2 hours 60-70%.

[0085] Group 3: TGF-β retention rate 55-70%, IL-10 retention rate 60-75%, and cumulative release rate of intestinal fluid over 2 hours 70-80%.

[0086] Group 4: TGF-β retention rate 60-75%, IL-10 retention rate 65-80%, and cumulative release rate of intestinal fluid over 2 hours 75-85%.

[0087] Group 5: TGF-β retention rate >85%, IL-10 retention rate >90%, and cumulative release rate of intestinal fluid over 2 hours >85%.

[0088] Group 6: TGF-β retention rate 100%, IL-10 retention rate 100%.

[0089] This demonstrates the synergistic protective effect of the natural polysaccharide gel layer and enteric coating on the active factors of MSC-CM.

[0090] Experimental Example 3 laboratory animals SPF-grade male SD rats, weighing 200-250 g, 8 weeks old Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animal License Number: SCXK (Beijing) 2021-0006 Modeling methods Papain was administered via intra-articular injection. Step 1: Anesthetize rats by intraperitoneal injection of 10% chloral hydrate (3 mL / kg).

[0091] Step 2: With the right hind limb knee joint flexed at 90°, insert the needle below the patella on the lateral side to locate the joint cavity.

[0092] Step 3: Inject 0.1 mL of modeling agent (4% papain and 0.03 mol / L cysteine).

[0093] Step 4: Administer the injection once on days 1, 4, and 7, for a total of 3 injections.

[0094] Step 5: On day 14, the success of the modeling is determined by the following criteria: knee joint diameter increase >20% and Lequesne score >4.

[0095] Experimental Groups Normal control group: 10 animals, no modeling, administered physiological saline by gavage. Model control group: 10 animals, model established, administered physiological saline by gavage. Example 1 group: 10 animals, modeled and administered the formulation of Example 1 (100 mg / kg) by gavage. Example 2 group: 10 animals, modeled and administered Example 2 formulation (100 mg / kg) by gavage. Example 3 group: 10 animals, modeled and administered the formulation of Example 3 (100 mg / kg) by gavage. Comparative Example 1: 10 animals, model established, administered via gavage with Comparative Example 1 preparation (Chinese medicine only). Comparative Example 2: 10 animals, modeled and administered Comparative Example 2 formulation (MSC-CM only) by gavage. Comparative Example 3: 10 animals, model established, administered via gavage with Comparative Example 3 formulation (non-enteric-coated). Comparative Example 4: 10 animals, model established, administered via gavage with the Comparative Example 4 formulation (different lyophilization protectant). Comparative Example 5: 10 animals, modeled and administered the Comparative Example 5 preparation (lacking Atractylodes macrocephala and Eucommia ulmoides) by gavage. Positive drug group: 10 animals, modeled and administered celecoxib (10 mg / kg) by gavage.

[0096] Dosing regimen Dosage schedule: Begin on day 1 after successful model establishment, and continue for 4 weeks. Route of administration: Gavage Dosage volume: 10 mL / kg Dosage frequency: once daily detection indicators Pain behavioral testing Mechanical pain threshold: The Von Frey fiber method was used, and the detection time points were before drug administration and 1, 2, 3 and 4 weeks after drug administration.

[0097] Thermal pain threshold: The hot plate method was used, and the detection time points were before drug administration and 1, 2, 3 and 4 weeks after drug administration.

[0098] Weight-bearing imbalance: The bipedal balance analgesia device was used to measure the imbalance before drug administration and at 2 and 4 weeks after drug administration.

[0099] Joint function assessment Lequesne Index: A behavioral score was used, with time points including before administration and 2 and 4 weeks after administration.

[0100] Joint range of motion: The knee flexion and extension angles were measured using a goniometer at 4 weeks after administration.

[0101] Serum biochemical indicators Four weeks after administration, the following indicators were detected using ELISA: IL-10, TGF-β, TNF-α, IL-1β, and MMP-13.

[0102] Histopathological examination Mankin score for articular cartilage: HE staining, toluidine blue staining, and safranin O-fast green staining are used. The score range is 0-14 points, with lower scores being better.

[0103] Cartilage thickness measurement: Comparison with a normal control group was performed using image analysis software.

[0104] Chondrocyte count: counted under high magnification and compared with the normal control group.

[0105] Synovial inflammation score: HE staining was used, with a score range of 0-3 points.

[0106] The results are as follows: Mechanical pain threshold (g): normal control group 25±3, model control group 8±2, Example 1 group 22±3, comparative example 1 group 15±2, comparative example 2 group 12±2*.

[0107] Lequesne index: normal control group 1±0.5, model control group 9±1.5, example group 1 2.5±0.8, comparative example group 1 5±1, comparative example group 2 6±1.2*.

[0108] Mankin score: Normal control group 1±0.3, model control group 10±1.5, Example 1 group 2.5±0.6, Comparative example 1 group 5±0.8, Comparative example 2 group 6±1*.

[0109] Serum IL-10 (pg / mL): normal control group 45±5, model control group 18±3, Example 1 group 62±7, comparative example 1 group 38±5, comparative example 2 group 28±4*.

[0110] Serum TGF-β (pg / mL): Normal control group 38±4, model control group 15±3, Example 1 group 58±6, Comparative example 1 group 32±4, Comparative example 2 group 25±3*.

[0111] It is evident that the oral enteric-coated formulation of this invention is effective in treating knee osteoarthritis.

[0112] Experiment Example 4 experiment: Animals: SD rats, half male and half female Quantity: 20 per group Dosing duration: 13 weeks (equivalent to 3 months in clinical trials) Dosage settings: low dose (50 mg / kg), medium dose (100 mg / kg), high dose (200 mg / kg) detection indicators General condition: weight, food intake, water intake, mental state, activity level.

[0113] Hematology: WBC, RBC, HGB, PLT, NEUT, LYMPH.

[0114] Blood biochemistry: ALT, AST, BUN, Cr, TP, ALB, GLU, TG.

[0115] Organ coefficient: The ratio of the weight of the heart, liver, spleen, lungs, kidneys, and brain to body weight.

[0116] Histopathology: HE staining of the heart, liver, spleen, lungs, kidneys, stomach, and duodenum.

[0117] Special toxicity: Aconitine residues were detected by LC-MS / MS.

[0118] Safety assessment criteria Aconitine residue: The acceptable standard is <0.01% (Pharmacopoeia standard).

[0119] Blood biochemical indicators: The passing standard is a difference of <20% compared with the normal control group.

[0120] Organ coefficient: The passing standard is a difference of <15% compared with the normal control group.

[0121] Histopathology: The passing standard is no obvious pathological changes.

[0122] This demonstrates the long-term safety of the oral enteric-coated formulation of the present invention.

[0123] Experimental Example 5 Experimental methods Instruments and reagents Use a smart dissolution tester (such as RC806ADK) with simulated gastric fluid (0.1 mol / L HCl, pH 1.2, 500 mL) and simulated intestinal fluid (pH 6.8 phosphate buffer, 500 mL) as the media.

[0124] Operating steps Gastric juice stage: The medium was simulated gastric juice, the time was 2 hours, the rotation speed was 100 rpm, and the temperature was 37±0.5℃.

[0125] Intestinal fluid stage: The medium was simulated intestinal fluid, the time was 2 hours, the rotation speed was 100 rpm, and the temperature was 37±0.5℃.

[0126] Sampling and Testing At 0.5h, 1h, and 2h in the gastric juice stage, 5 mL of samples were taken at each time to detect the activities of TGF-β and IL-10.

[0127] Intestinal fluid phases of 0.5h, 1h, and 2h: 5 mL samples were taken at each time to detect the activities of TGF-β and IL-10.

[0128] Qualification Standard Gastric juice TGF-β release over 2 hours: <10% Gastric juice IL-10 release over 2 hours: <10% Cumulative TGF-β release from intestinal fluid over 2 hours: >80% Cumulative IL-10 release from intestinal fluid over 2 hours: >85%.

[0129] This indicates that the oral enteric-coated formulation of the present invention has a high release rate.

[0130] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The use of Fugui Zhitong Decoction in the preparation of MSC-CM oral enteric-coated preparations for relieving knee osteoarthritis, characterized in that, The oral enteric-coated preparation of MSC-CM for relieving knee osteoarthritis comprises: the extract of Fuguizhitong formula, freeze-dried MSC-CM, enteric coating or enteric capsule shell; The extract of Fuguizhitong formula is mixed with the freeze-dried MSC-CM and filled into an enteric capsule shell or made into enteric-coated pellets; Among them, the Fuguizhitong formula is composed of the following raw materials in parts by weight: prepared rhizoma typhonii 10-20 parts, cassia twig 15-25 parts, astragalus membranaceus 10-60 parts, ginger 10-25 parts, atractylodes macrocephala 20-30 parts, ledebouriella seseloides 15-25 parts, notopterygium incisum 10-20 parts, eucommia ulmoides 15-25 parts, achyranthes bidentata 10-20 parts, roasted licorice root 5-15 parts, red peony root 10-20 parts, anemarrhena asphodeloides 15-25 parts.

2. The application of the Fugui Zhitong formula according to claim 1 in the preparation of an oral enteric-coated formulation of MSC-CM for relieving knee osteoarthritis, characterized in that, The extract of Fuguizhitong formula and the freeze-dried MSC-CM are in a weight ratio of 10-20:

1.

3. The application of the Fugui Zhitong formula according to claim 2 in the preparation of an oral enteric-coated formulation of MSC-CM for relieving knee osteoarthritis, characterized in that, The freeze-dried MSC-CM contains mesenchymal stem cells, and the mesenchymal stem cells are umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells.

4. The application of the Fugui Zhitong formula according to claim 1 in the preparation of an oral enteric-coated formulation of MSC-CM for relieving knee osteoarthritis, characterized in that, The oral enteric-coated preparation is prepared by the following steps, including: Preparing the extract of Fuguizhitong formula, which includes: A1. Weigh each traditional Chinese medicine component according to the formula and mix them; A2. Soak in water for 30-60 minutes, decoct and extract 2-3 times, each time for 1-2 hours, and combine the filtrates; A3. Concentrate the filtrate to a relative density of 1.10-1.20, add ethanol to make the alcohol content reach 60-70%, and let it stand for precipitation; A4. Take the supernatant, recover ethanol, concentrate and dry to obtain the extract of Fuguizhitong formula; Preparing the oral enteric-coated preparation, which includes: B1. Mix the extract of Fuguizhitong formula and the freeze-dried MSC-CM evenly; B2. Add self-microemulsion delivery system excipients. The extract of Fuguizhitong formula and the freeze-dried MSC-CM are filled into an enteric capsule shell in the form of the content, or jointly made into enteric-coated pellets to obtain the oral enteric-coated preparation.

5. The application of the Fugui Zhitong formula according to any one of claims 1 to 4 in the preparation of an oral enteric-coated formulation of MSC-CM for relieving knee osteoarthritis, characterized in that, Preparing the freeze-dried MSC-CM, which includes: Resuscitate cryopreserved mesenchymal stem cells and passage culture to the 3rd-5th generation; Discard the culture medium, add serum-free medium and continue to culture for 48-72 hours; Collect the culture supernatant, filter it through a filter membrane to obtain MSC-CM; Add 6%-9% trehalose, 2%-4% mannitol, 0.5%-2% PVP, and freeze-dry to obtain the freeze-dried MSC-CM.

6. The application of the Fugui Zhitong formula according to claim 2 in the preparation of an oral enteric-coated formulation of MSC-CM for relieving knee osteoarthritis, characterized in that, The self-microemulsion delivery system contains medium-chain triglycerides, tween 80 and polyethylene glycol 400.

7. The application of the Fugui Zhitong formula according to claim 5 in the preparation of an oral enteric-coated formulation of MSC-CM for relieving knee osteoarthritis, characterized in that, The serum-free medium is α-MEM serum-free medium.

8. The application of the Fugui Zhitong formula according to claim 4 in the preparation of an oral enteric-coated formulation of MSC-CM for relieving knee osteoarthritis, characterized in that, In step B2, the granulation step includes: placing each component in an autoclave, introducing supercritical CO2 to a pressure of 12 MPa-18 MPa and a temperature of 35°C-45°C, stirring to form a slurry and then depressurizing to obtain porous microspheres.

9. The application of the Fugui Zhitong formula according to claim 5 in the preparation of an oral enteric-coated formulation of MSC-CM for relieving knee osteoarthritis, characterized in that, The freeze-drying is carried out under the protection of nitrogen or argon for freeze-drying and grinding, and the oxygen concentration is lower than 0.5%.

10. A combination of Fuzi and Guizhi analgesic formula and MSC-CM for relieving knee osteoarthritis, characterized in that, [[ID= The extract of the Fuguizhitong formula is mixed with the MSC-CM freeze-dried powder and filled into enteric-coated capsule shells or made into enteric-coated pellets; Among them, the Fuguizhitong formula is composed of the following raw materials in parts by weight: prepared common monkshood 10-20 parts, cassia twig 15-25 parts, astragalus membranaceus 10-20 parts, ginger 10-20 parts, atractylodes macrocephala 20-30 parts, ledebouriella divaricata 15-25 parts, notopterygium incisum 10-20 parts, eucommia ulmoides 15-25 parts, achyranthes bidentata 10-20 parts, roasted licorice root 5-15 parts, red peony root 10-20 parts, anemarrhena asphodeloides 15-25 parts.