A traditional Chinese medicine composition for treating chronic musculoskeletal diseases, its preparation method and its application

Abstract

This invention belongs to the field of traditional Chinese medicine technology, and relates to a traditional Chinese medicine composition for treating chronic musculoskeletal diseases, its preparation method, and its application. The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 4-50 parts of *Drynaria fortunei*, 3-30 parts of *Cibotium barometz*, 1-20 parts of *Ligustrum lucidum*, 1-20 parts of *Spatholobus suberectus*, 1-20 parts of *Pyrola rotundifolia*, and 1-20 parts of *Achyranthes bidentata*. This invention uses *Drynaria fortunei*, *Cibotium barometz*, *Ligustrum lucidum*, *Spatholobus suberectus*, *Pyrola rotundifolia*, and *Achyranthes bidentata* in combination, with synergistic effects of the principal, assistant, and adjuvant herbs, to tonify the kidneys and strengthen bones, nourish blood and promote blood circulation, and unblock meridians and relieve pain. It can improve bone density, regulate bone metabolism, increase muscle strength, relieve bone and joint pain, improve spinal joint function, and effectively treat chronic musculoskeletal diseases such as osteoporosis and degenerative osteoarthritis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of traditional Chinese medicine, and relates to a traditional Chinese medicine composition for treating chronic muscle and bone diseases, a preparation method and application thereof. BACKGROUND

[0002] Chronic muscle and bone disease is a kind of chronic degenerative disease of bone, joint, muscle and tendon, or chronic injury, which causes imbalance of muscle and bone around the spine and bone joints, and causes systemic or local pain, swelling, numbness, muscle atrophy, limited activity and other symptoms. Chronic muscle and bone disease has a long course, and most patients are middle-aged and elderly people. Liver and kidney deficiency, deficiency of vital qi, cause emptying of bone collaterals, deficiency of qi and blood, and inability to nourish muscle and bone. At the same time, invasion of external pathogens, such as wind, cold and dampness, phlegm and blood stasis, causes obstruction of bone collaterals, and interference of bone collaterals by pathological products such as external pathogens and phlegm and blood stasis, which also blocks the transmission of nutrients such as qi and blood, and affects the growth and repair of bone. Muscle and tendon are connected with bone, and bone supports muscle and tendon. Muscle and bone are connected, and they depend on each other and maintain the dynamic balance between muscle and bone. Abnormal bone will inevitably lead to abnormal muscle, and cause muscle and bone disease. Therefore, the present inventors refine the bone collateral theory according to years of clinical experience, and believe that the pathological properties of chronic muscle and bone disease are deficiency in origin and excess in superficiality, and deficiency and excess are mixed. Deficiency in origin refers to liver and kidney deficiency or deficiency of qi and blood, and emptying of bone collaterals. Excess in superficiality refers to invasion of external pathogens or endogenous phlegm and blood stasis, and obstruction of bone collaterals. Emptying of bone collaterals causes obstruction of bone collaterals due to weakness of qi and blood. On the contrary, obstruction of bone collaterals causes poor circulation of qi and blood, which further aggravates emptying of bone collaterals. Emptying and obstruction of bone collaterals influence each other, and deficiency and excess are mixed. Therefore, emptying and obstruction of bone collaterals are the key links of chronic muscle and bone disease.

[0003] According to the pathogenesis of emptying and obstruction of bone collaterals and the pathological characteristics of deficiency in origin and excess in superficiality and deficiency and excess being mixed, the treatment mainly adopts the method of dredging collaterals. The specific treatment methods can be divided into two categories: supporting healthy qi and dredging collaterals, and eliminating evil and dredging collaterals. The treatment methods commonly used in supporting healthy qi and dredging collaterals include tonifying qi and blood and dredging collaterals, and tonifying kidney and filling essence and dredging collaterals. For patients with deficiency of qi and blood, the method of tonifying qi and blood and dredging collaterals is used. For patients with kidney deficiency and essence deficiency, the method of tonifying kidney and filling essence and dredging collaterals is used. The treatment methods commonly used in eliminating evil and dredging collaterals include removing blood stasis and phlegm and dredging collaterals, dispelling cold and dredging collaterals, and eliminating wind and dampness and dredging collaterals.

[0004] The existing internal treatment method for treating chronic muscle and bone disease, especially for joint pain, is mainly to tonify liver and kidney and strengthen muscle and bone. The prior art CN103263560A discloses a traditional Chinese medicine composition for tonifying kidney and dredging collaterals, which is composed of Fructus Psoraleae, Radix Cyathulae, Ramulus Loranth, Radix Drynariae, Radix Paeoniae Alba, Rhizoma Chuanxiong, Caulis Spatholobi and Rhizoma Dioscoreae Nipponicae.

[0005] CN107638465A also discloses a traditional Chinese medicine composition for treating joint pain, consisting of Rehmannia glutinosa (processed), Drynaria fortunei, Angelica sinensis, Psoralea corylifolia, Cynanchum paniculatum, Eupolyphaga sinensis, Bombyx mori, Cibotium barometz, Scolopendra subspinipes, Buthus martensii, Vespa nidus, Dipsacus asper, Pheretima aspergillum, Zaocys dhumnades, Corydalis yanhusuo, Pyrola rotundifolia, Eucommia ulmoides, Epimedium brevicornu, Clematis chinensis, Geranium wilfordii, Taxillus chinensis, Spatholobus suberectus, Humulus scandens, Rehmannia glutinosa (raw), Polygonum cuspidatum, and Ligustrum lucidum. This formula contains many ingredients, which presents challenges. Firstly, the quality requirements for the raw materials are high, and their availability cannot be guaranteed. Secondly, its effects are slow, requiring long-term use. Many patients either cannot adhere to long-term use or experience adverse reactions such as abdominal distension, abdominal pain, loss of appetite, constipation, and heatiness after prolonged use, making it difficult to fundamentally treat osteoarthritis. Discontinuing the medication can easily lead to disease recurrence and increased pain. Furthermore, achieving industrial-scale manufacturing in the pharmaceutical field presents significant operational difficulties.

[0006] Therefore, the treatment of chronic musculoskeletal diseases with traditional Chinese medicine still requires a combination of Chinese herbal medicines that can target the core pathogenesis, use simplified drugs, have precise dosage ratios, be highly effective and potent, and be easy to produce and transform, so as to achieve more significant and efficient therapeutic effects. Summary of the Invention

[0007] Based on the aforementioned background technology, this invention provides a novel self-formulated prescription based on the "bone network theory," targeting the core pathogenesis of "bone network obstruction" in chronic musculoskeletal diseases. It employs treatment methods such as tonifying the kidney and unblocking the network, nourishing blood and unblocking the network, and activating blood and unblocking the network. The prescription is formulated with *Drynaria fortunei*, *Cibotium barometz*, *Ligustrum lucidum*, *Spatholobus suberectus*, *Pyrola rotundifolia*, and *Achyranthes bidentata* or *Achyranthes bidentata*, with synergistic effects of the principal, assistant, and adjuvant herbs. It possesses multiple effects of tonifying the kidney and strengthening bones, nourishing blood and promoting blood circulation, and unblocking the network and relieving pain. It has excellent therapeutic effects on osteoporosis and osteoarthritis. The targeted nature of this invention, which "corrects imbalances with imbalances," provides a scientific basis for the precise treatment of chronic musculoskeletal diseases.

[0008] The preparation method and application of the above-mentioned traditional Chinese medicine composition for treating chronic musculoskeletal diseases are also provided.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A traditional Chinese medicine composition for treating chronic musculoskeletal diseases, comprising 4-50 parts of Drynaria fortunei, 3-30 parts of Cibotium barometz, 1-20 parts of Ligustrum lucidum, 1-20 parts of Spatholobus suberectus, 1-20 parts of Pyrola rotundifolia, and 1-20 parts of Achyranthes bidentata.

[0011] Preferably, the ingredients are 8-20 parts of Drynaria fortunei, 5-18 parts of Cibotium barometz, 6-15 parts of Ligustrum lucidum, 6-15 parts of Spatholobus suberectus, 6-15 parts of Pyrola rotundifolia, and 6-15 parts of Achyranthes bidentata.

[0012] Preferably, the ingredients are 15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Spatholobus suberectus, 10 parts of Pyrola rotundifolia, and 10 parts of Achyranthes bidentata.

[0013] More preferably, the Sichuan Achyranthes bidentata can be replaced with Achyranthes bidentata. The herbal composition may also include 3-5 parts of Epimedium, 3-5 parts of Cinnamomum cassia, and 3-5 parts of Angelica pubescens; or 1-3 parts of Rehmannia glutinosa and 1-3 parts of Cornus officinalis; or 1-3 parts of Corydalis yanhusuo, 1-3 parts of Lindera strychnifolia, and 1-3 parts of Pheretima aspergillum.

[0014] A second objective of this invention is to provide a method for preparing the traditional Chinese medicine composition, comprising the following steps:

[0015] S1. Mix all the raw materials of the composition, add water and boil twice, according to the amount of raw materials, the amount of water added in the first boil is more than the amount of water added in the second boil;

[0016] S2. Combine the filtrates obtained from the two decoctions and perform two vacuum concentration processes to obtain a clear extract and an extract.

[0017] S3. Mix the dried extract, clear extract and excipients to obtain the final product.

[0018] Preferably, in step S1, the first time 10 to 15 times the amount of water is added and decocted for 1.0 to 2.5 hours, and the second time 8 to 12 times the amount of water is added and decocted for 1.0 to 2.5 hours.

[0019] Preferably, in step S1, the first time 11 times the amount of water is added and decocted for 1.5 hours, and the second time 9 times the amount of water is added and decocted for 1.5 hours.

[0020] Preferably, in step S2, the concentration includes primary concentration and secondary concentration, and the concentration temperature is 60-80°C.

[0021] Preferably, the relative density of the clear extract is 1.15-1.25, and the relative density of the concentrated extract is 1.30-1.35.

[0022] Preferably, the excipients include a wetting agent and a filler, wherein the filler is dextrin and lactose, and the mixture is granulated, sieved, and blended to obtain a traditional Chinese medicine granule preparation.

[0023] Preferably, the ratio of the dry extract to the filler is 1:0.6-1.0, wherein the preferred ratio is dry extract:lactose:dextrin = 6.25:2.5:1.25.

[0024] A third objective of this invention is to provide a pharmaceutical preparation whose active ingredient includes the above-mentioned traditional Chinese medicine composition, or a traditional Chinese medicine composition prepared according to the above-mentioned preparation method.

[0025] Preferably, the pharmaceutical preparation can be formulated as a mixture, tincture, injection, tablet, powder, oral liquid, capsule, granule, ointment, powder, emulsion, drop pill, pill, lozenge, gel, suppository, or aerosol. Suitable drug carriers in the art can be selected for different dosage forms. The drug carrier used can be solid, liquid, or gaseous. Examples of solid carriers include lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. Examples of liquid carriers include syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0026] A fourth objective of this invention is to provide the above-mentioned traditional Chinese medicine composition, or the traditional Chinese medicine composition prepared according to the above-mentioned preparation method, or the above-mentioned pharmaceutical preparation for use in the preparation of a drug for treating osteoarthritis.

[0027] Preferably, the drug is used in the preparation of a drug for treating osteoarthritis by reducing knee joint swelling in rats, increasing weight-bearing capacity of rat hind limbs, and alleviating lower limb pain.

[0028] Preferably, the drug is used in the preparation of a drug for treating osteoarthritis by increasing the number of trabeculae, the bone volume fraction, and the trabeculae thickness.

[0029] Preferably, the drug is used in the preparation of a drug for treating osteoarthritis by reducing the trabecular pattern factor, structural pattern index, trabecular separation, total porosity, and the ratio of bone surface area to bone volume.

[0030] Preferably, the drug is used in the preparation of a drug for treating osteoarthritis by reducing the levels of TNF-α, IL-1β, IL-6 and IL-8.

[0031] Another object of the present invention is to provide the above-mentioned traditional Chinese medicine composition, or the traditional Chinese medicine composition prepared according to the above-mentioned preparation method, or the above-mentioned pharmaceutical preparation in the preparation of a drug for treating osteoporosis.

[0032] Preferably, the drug is used in the preparation of a drug for treating osteoporosis by increasing bone density, regulating bone metabolism, and increasing muscle strength.

[0033] The traditional Chinese medicine composition (hereinafter referred to as "TCM composition") for treating chronic musculoskeletal diseases described in this invention is based on the bone and collaterals theory of Professor Li Yanmin, a renowned TCM doctor in Shaanxi Province. It was invented by Professor Yang Feng, a National Young Qihuang Scholar, and his orthopedic team, based on years of clinical experience in treating chronic musculoskeletal diseases. The TCM composition adheres to the industrialization concept of concise and effective formulation, and is based on the actual use of hospital-prepared formulations. Ultimately, six TCM herbs were selected, each with its own specific function, to achieve the following explanation of the formula.

[0034] Drynaria fortunei: It has the effects of tonifying the kidney and strengthening bones, promoting blood circulation and relieving pain; it is bitter and warm in nature, and enters the liver and kidney meridians; it is used for kidney deficiency and lower back pain, traumatic injuries, and weakness of muscles and bones; as the principal herb, it has the effects of strengthening muscles and bones and promoting blood circulation.

[0035] Cibotium barometz: tonifies the liver and kidneys, strengthens the lower back and knees, dispels wind and dampness. It is bitter, sweet, and warm in nature, and enters the liver and kidney meridians. Used for soreness and weakness of the lower back and knees, and rheumatic pain; it also assists Drynaria fortunei in tonifying the liver and kidneys, while dispelling wind and dampness.

[0036] Privet fruit: Nourishes the liver and kidneys, improves eyesight and darkens hair, clears deficiency heat; sweet and cool in nature, enters the liver and kidney meridians. Used for lower back and knee weakness, dizziness and blurred vision due to liver and kidney yin deficiency. Nourishes yin to balance the warming properties of the formula, nourishes both the liver and kidneys, and prevents dryness and heat from damaging yin; it is used as an assistant herb along with Cibotium barometz.

[0037] Pyrola rotundifolia: dispels wind and dampness, strengthens tendons and bones, stops bleeding. It is sweet, bitter, and warm in nature, and enters the liver and kidney meridians. It is used for rheumatic pain and kidney deficiency-related lower back pain; as an adjuvant herb, it works synergistically with Cibotium barometz to dispel wind and dampness and enhance the liver and kidney tonifying effects.

[0038] Chicken Blood Vine: Promotes blood circulation and replenishes blood, relaxes muscles and tendons, and is bitter, sweet, and warm in nature. It enters the liver and kidney meridians. It is used for numbness of limbs and joint pain due to blood deficiency and blood stasis. It promotes blood circulation and replenishes blood, and guides other herbs into the collaterals, serving as a guiding herb.

[0039] Achyranthes bidentata or Achyranthes bidentata: It tonifies the liver and kidneys, strengthens tendons and bones, benefits joints, removes blood stasis and unblocks meridians, and guides blood downwards. Its properties are bitter, sweet, sour and neutral, and it enters the liver and kidney meridians. It guides other herbs downwards to the waist and knees, enhancing the targeted effect of the formula. It is used together with Spatholobus suberectus as an adjuvant.

[0040] The present invention is mainly based on the "Bone Meridian Theory". Aiming at the core pathogenesis of "obstruction of bone meridian" in chronic bone and tendon diseases, treatment methods such as tonifying the kidney and dredging the meridian, nourishing blood and dredging the meridian, promoting blood circulation and dredging the meridian are adopted. Among them, Drynaria rhizome is bitter and warm in nature, and belongs to the liver and kidney meridians. It has the functions of tonifying the kidney and strengthening bones, promoting blood circulation and relieving pain. According to the specific syndrome types, the compatibility is flexibly adjusted to form a clear composition of monarch, minister, assistant and envoy: Drynaria rhizome tonifies the kidney and strengthens bones, promotes blood circulation and relieves pain as the monarch drug; Cibotium barometz is bitter, sweet and warm in nature, with the effects of tonifying the liver and kidney, strengthening the waist and knees, and dispelling wind-dampness. "Shennong Ben Cao Jing" records that it "is mainly used for strong waist and back, relaxation of joints, arthralgia due to wind-cold-dampness, and knee pain", and "Hai Yao Ben Cao" also says that it "treats wind, warms and tonifies the waist and feet... strengthens bones and muscles", with the effect of warming and tonifying the kidney yang to strengthen the root; Glossy privet fruit is sweet, bitter and slightly cold, nourishing the yin of the liver and kidney. "Compendium of Materia Medica" says that it "strengthens yin, strengthens the waist and knees, and improves eyesight", forming a pattern of tonifying both yin and yang with the monarch drug to prevent yin damage caused by warm and dryness; Pyrola calliantha is bitter and flat. "Compendium of Materia Medica of Yunnan" records that it "treats bone and muscle pain, flaccidity due to phlegm-fire, and numbness of hands and feet", with the functions of dispelling wind-dampness, strengthening bones and muscles, promoting blood circulation and relieving pain; Millettia reticulata is bitter, sweet and warm in nature, and can promote blood circulation and nourish blood, relax tendons and activate collaterals. These two herbs assist the monarch and minister drugs to enhance the effects of promoting blood circulation and dredging collaterals, expelling wind and removing dampness; Cyathula officinalis or Achyranthes bidentata guide all the herbs to descend to the waist and knees, enhancing the targeting of the prescription, and are used as envoy drugs together; The compatibility of the whole prescription is exquisite, with both tonifying and dredging: warming yang without being too dry and fierce, nourishing yin without being greasy and stagnant, using qi-tonifying and blood-activating simultaneously, and jointly achieving the effects of warming the kidney and nourishing the liver, tonifying qi and activating blood circulation, expelling wind and removing dampness, and strengthening bones and muscles. It is mainly used to treat symptoms such as soreness and weakness of the waist and knees, joint pain, and limited flexion and extension caused by deficiency of the liver and kidney and blood stasis. The whole prescription takes "dredging collaterals" as the core treatment method, achieving the purpose of dredging collaterals and relieving pain for the main symptoms of "pain" and "numbness" in chronic bone and tendon diseases such as osteoporosis and osteoarticular degenerative diseases. It is particularly suitable for chronic bone and tendon diseases belonging to the syndrome of deficiency of the liver and kidney and obstruction of blood stasis.

[0041] Combined with modern pharmacological research, it is further explained as follows: Drynaria rhizome, Cibotium barometz, and Glossy privet fruit improve bone density by regulating the osteoblast-osteoclast balance, and improve kidney deficiency-type osteoporosis. Among them, the flavonoid components contained in Drynaria rhizome can promote osteoblast differentiation and bone matrix mineralization, shorten the fracture healing period, and at the same time reduce bone resorption by inhibiting the RANKL / OPG signaling pathway, induce the differentiation of bone marrow mesenchymal stem cells into osteoblasts, and enhance bone repair ability. The flavonoid components in Pyrola calliantha and Millettia reticulata can inhibit the NF-κB inflammatory pathway, reduce joint synovial inflammation, and form an anti-inflammatory and blood-activating mechanism; Cibotium barometz and Pyrola calliantha improve pain and dysfunction caused by "blood stasis" by inhibiting platelet aggregation and reducing blood viscosity.

[0042] Compared with the prior art, the beneficial effects of the present invention are:

[0043] 1. The traditional Chinese medicine composition for treating chronic musculoskeletal diseases described in this invention has the following characteristics: 1) Simultaneous tonification of the liver and kidneys, addressing both the root cause and symptoms: Drynaria fortunei, Cibotium barometz, and Ligustrum lucidum tonify the liver and kidneys and strengthen muscles and bones to treat the root cause; Pyrola rotundifolia and Spatholobus suberectus work together to dispel wind and dampness, invigorate blood and unblock collaterals, thus treating the symptoms; 2) Combination of warming and cooling properties: Drynaria fortunei and Cibotium barometz are warming, while Ligustrum lucidum is cooling, preventing dryness and heat from damaging yin; 3) Combination of attack and tonification: The combination of liver and kidney tonifying herbs with blood-activating and collateral-unblocking herbs achieves a combination of attack and tonification, tonifying without stagnation.

[0044] 2. This invention, through a comparative study of single-herb deficiency groups and complete formula groups, verifies the overall efficacy of the traditional Chinese medicine composition. The absence of a single herb weakens the therapeutic effect, while the complete formula can exert the effects of multiple components and multiple targets, achieving the best therapeutic effect on knee osteoarthritis. Based on this, six groups with different dosage ratios of the complete formula were designed for research. The results showed that all groups had therapeutic effects, but the differences were significant. Group 6 (15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Spatholobus suberectus, 10 parts of Pyrola rotundifolia, and 10 parts of Achyranthes bidentata) was superior to other groups in relieving joint swelling, increasing hind limb weight-bearing, repairing bone microstructure, and reducing the level of pro-inflammatory factors. This result fully confirms that the dosage ratio of Group 6 can maximize the synergistic effects of the principal herb in tonifying the kidney and strengthening bones, the assistant herb in assisting tonification, the adjuvant herb in unblocking the meridians and relieving pain, and the guiding herb in promoting blood circulation and guiding the meridians, providing a scientific basis for precise clinical medication.

[0045] 3. The preferred dosage form of the herbal composition of this invention is granules, which has the advantages of being easy to carry and absorb, and is especially suitable for the effective treatment and conditioning of chronic musculoskeletal diseases. Combined with the extraction and molding processes of the granules described in this invention, it has beneficial technical effects such as high enrichment rate of effective ingredients and simple and feasible processes. The beneficial effects of the process have been verified successively through "small-scale," "pilot-scale," and "large-scale production." Specifically: 1) In the "small-scale" stage: key process conditions such as water extraction, concentration, drying, and molding in the preparation of the granules of this invention were systematically screened under laboratory conditions. The content of protocatechuic acid and naringin was used as dual indicator components for evaluation, and the process combination and parameter range of "decoction extraction + vacuum concentration" were determined. 2) In the "pilot-scale" stage: a pilot-scale study was conducted at a ratio of 1:5000 to further verify and optimize each process parameter. The granule specification is 10g / bag, the yield of extract and clear extract can reach 85-115%, the yield of granules reaches 95-105%, and the material balance rate reaches 95-100%, showing significant advantages of high production yield and low process cost. 3) Large-scale production stage: Referring to the equipment and parameters of the pilot test, and in combination with the GMP operation requirements of large-scale production, the naringin content in the principal drug Drynaria fortunei is used as the quality test indicator to carry out more than 3 batches of process optimization and industrialization determination.

[0046] 4. This invention successfully established an animal model of osteoarthritis by injecting collagenase into the joint cavity of rats, fully revealing the pharmacological effects of the traditional Chinese medicine composition. The results showed that it significantly reduced the inflammatory response in the model rats and effectively improved the symptoms of knee osteoarthritis (KOA), confirming its therapeutic effect on osteoarthritis. Simultaneously, this invention, combining animal model construction and clinical human drug use, systematically elucidated the mechanism of action of this traditional Chinese medicine composition in treating osteoporosis and osteoarthritis, providing innovative theoretical support and reliable medication basis for the clinical diagnosis and treatment of chronic musculoskeletal diseases such as osteoporosis and osteoarthritis. Attached Figure Description

[0047] Figure 1 A process flow diagram for the preparation of traditional Chinese medicine compositions;

[0048] Figure 2 The changes in knee joint diameter in rats were investigated in response to different prescriptions.

[0049] Figure 3 The weight-bearing capacity of the lower limbs of rats in different groups corresponding to different prescriptions;

[0050] Figure 4 Micro-CT analysis results of subchondral bone microstructure morphology parameters for different prescriptions: (A) Bone volume fraction: BV / TV; (B) Bone surface area to bone volume ratio: BS / BV; (C) Trabecular thickness: Tb.Th; (D) Bone surface area to tissue volume ratio: Tb.Sp; (E) Number of trabeculae: Tb.N; (F) Structural pattern index: SMI; (G) Po(tot): Total porosity; (H) Bone volume: BV; (I) Trabecular pattern factor: Tb.pf;

[0051] Figure 5 Serum TNF-α, IL-1β, IL-6, and IL-8 levels in rats of different prescriptions were measured.

[0052] Figure 6 The changes in knee joint diameter in rats of different formulation ratios were investigated.

[0053] Figure 7 The weight-bearing capacity of the lower limbs of rats in different groups corresponding to different formulation ratios;

[0054] Figure 8 Micro-CT analysis results of the morphological parameters of subchondral bone microstructure for different ratios: (A) Bone volume fraction: BV / TV; (B) Bone surface area to bone volume ratio: BS / BV; (C) Trabecular thickness: Tb.Th; (D) Bone surface area to tissue volume ratio: Tb.Sp; (E) Number of trabeculae: Tb.N; (F) Structural pattern index: SMI; (G) Po(tot): Total porosity; (H) Bone volume: BV; (I) Trabecular pattern factor: Tb.pf;

[0055] Figure 9 The serum TNF-α, IL-1β, IL-6, and IL-8 levels in each group of rats were determined by different formulation ratios.

[0056] Figure 10 The changes in knee joint diameter in rats of different doses were investigated.

[0057] Figure 11 The weight-bearing capacity of the lower limbs of rats in different dose groups;

[0058] Figure 12 Micro-CT images of subchondral bone in rats at different doses: (A) Blank group; (B) Model group; (C) Diacerein group (9.00 mg / kg); (D) GLT (2.25 mL / kg) group; (E) GLT (4.50 mL / kg); (F) GLT (9.00 mL / kg);

[0059] Figure 13 Micro-CT analysis results of subchondral bone microstructure morphology parameters for different doses in each group; (A) Bone volume fraction: BV / TV; (B) Bone surface area to bone volume ratio: BS / BV; (C) Trabecular thickness: Tb.Th; (D) Bone surface area to tissue volume ratio: BS / TV; (E) Number of trabeculae: Tb.N; (F) Structural pattern index: SMI; (G) Po(tot): Total porosity; (H) Bone volume: BV; (I) Trabecular pattern factor: Tb.pf; Note: Compared with the blank group, # P<0.05, ## P<0.01; compared with the model group, *P<0.05, **P<0.01;

[0060] Figure 14 HE staining results of subchondral bone for different doses in each group; (A) Blank group; (B) Model group; (C) GLT (2.25 mL / kg) group; (D) GLT (4.50 mL / kg); (E) GLT (9.00 mL / kg); (F) Double crude Resine group (9.00 mg / kg);

[0061] Figure 15 The results of safranin-fast green staining for different doses are shown below: (A) Blank group; (B) Model group; (C) GLT (2.25 mL / kg) group; (D) GLT (4.50 mL / kg); (E) GLT (9.00 mL / kg); (F) Double crude resveratrol group (9.00 mg / kg);

[0062] Figure 16Serum TNF-α, IL-1β, IL-6, and IL-8 levels in rats of different doses; Note: Compared with the control group, * P<0.05, ** P<0.01, compared with the model group, # P<0.05, ## P<0.01. Detailed Implementation

[0063] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments, but the embodiments of the present invention are not limited thereto. Other methods for preparing the compositions of the present invention, with some conventional modifications to the reaction conditions according to the present invention, are considered to be within the scope of the present invention.

[0064] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0065] Techniques, methods, and equipment known to those skilled in the art need not be discussed in detail, but where appropriate, such techniques, methods, and equipment should be considered part of the specification.

[0066] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

[0067] This invention combines modern pharmacological research and clinical experience of renowned doctors to summarize the fundamental treatment principle for chronic musculoskeletal diseases, which is to tonify the kidneys and strengthen bones, nourish blood and promote blood circulation, and unblock the meridians and relieve pain. The formula is composed of the following ingredients: 4-50 parts of Drynaria fortunei, 3-30 parts of Cibotium barometz, 1-20 parts of Ligustrum lucidum, 1-20 parts of Spatholobus suberectus, 1-20 parts of Pyrola rotundifolia, and 1-20 parts of Achyranthes bidentata.

[0068] Depending on the characteristics of different symptoms, other herbs can be added to the above formula as appropriate, such as 3-5 parts of Epimedium, 3-5 parts of Cinnamon Twig, and 3-5 parts of Angelica pubescens; or 1-3 parts of Rehmannia glutinosa and 1-3 parts of Cornus officinalis; or 1-3 parts of Corydalis yanhusuo, 1-3 parts of Lindera strychnifolia, and 1-3 parts of Pheretima aspergillum.

[0069] In the early stages of the experiment, key process conditions such as water extraction, concentration, drying, and molding in the preparation of traditional Chinese medicine granules were systematically screened under laboratory conditions. A preliminary process combination of "two decoction extractions + two vacuum concentrations" was determined. Subsequently, an animal model of osteoarthritis was constructed, and the therapeutic mechanism was theoretically studied through animal experiments. Based on the initial laboratory tests and theoretical research, pilot-scale production was further expanded, and small-scale clinical trials were conducted. Clinically, it was found to be suitable for chronic musculoskeletal diseases with kidney deficiency and blood stasis syndrome, characterized by neck, shoulder, waist, and leg pain, swelling, and limited mobility. It was particularly effective for osteoporosis and osteoarthritis, corresponding to those diagnosed with kidney deficiency and blood stasis syndrome in traditional Chinese medicine (symptoms include soreness and pain in the waist and knees, difficulty in joint flexion and extension, swelling and numbness of limbs, limited mobility, dark tongue or ecchymosis, thin coating, and deep, thready or wiry pulse). The combined effects of the various herbs—tonifying the kidney and strengthening bones, nourishing blood and promoting blood circulation, and unblocking the meridians and relieving pain—are demonstrated.

[0070] Example 1

[0071] This embodiment provides a traditional Chinese medicine composition for treating chronic musculoskeletal diseases. The raw materials are composed of the following components by weight: 4 parts of Drynaria fortunei, 3 parts of Cibotium barometz, 1 part of Ligustrum lucidum, 1 part of Spatholobus suberectus, 1 part of Pyrola rotundifolia, and 1 part of Achyranthes bidentata.

[0072] Example 2

[0073] This embodiment provides a traditional Chinese medicine composition for treating chronic musculoskeletal diseases. The raw materials are composed of the following components by weight: 8 parts of Drynaria fortunei, 5 parts of Cibotium barometz, 6 parts of Ligustrum lucidum, 6 parts of Spatholobus suberectus, 6 parts of Pyrola rotundifolia, and 6 parts of Achyranthes bidentata.

[0074] Example 3

[0075] This embodiment provides a traditional Chinese medicine composition for treating chronic musculoskeletal diseases. The raw materials are composed of the following components by weight: 15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Spatholobus suberectus, 10 parts of Pyrola rotundifolia, and 10 parts of Achyranthes bidentata.

[0076] Example 4

[0077] This embodiment provides a traditional Chinese medicine composition for treating chronic musculoskeletal diseases. The raw materials are composed of the following components by weight: 30 parts of Drynaria fortunei, 24 parts of Cibotium barometz, 20 parts of Ligustrum lucidum, 20 parts of Spatholobus suberectus, 20 parts of Pyrola rotundifolia, and 20 parts of Achyranthes bidentata.

[0078] Example 5

[0079] This embodiment provides a traditional Chinese medicine composition for treating chronic musculoskeletal diseases. The raw materials are composed of the following components by weight: 20 parts of Drynaria fortunei, 18 parts of Cibotium barometz, 15 parts of Ligustrum lucidum, 15 parts of Spatholobus suberectus, 15 parts of Pyrola rotundifolia, and 15 parts of Achyranthes bidentata.

[0080] Example 6

[0081] This embodiment provides a traditional Chinese medicine composition for treating chronic musculoskeletal diseases. The raw materials are composed of the following components by weight: 40 parts of Drynaria fortunei, 30 parts of Cibotium barometz, 20 parts of Ligustrum lucidum, 20 parts of Spatholobus suberectus, 20 parts of Pyrola rotundifolia, and 20 parts of Achyranthes bidentata.

[0082] Example 7

[0083] Replace Achyranthes bidentata in Example 3 with Achyranthes bidentata.

[0084] To further adapt to different symptoms, other medicinal ingredients can be added to the above formula.

[0085] Example 8

[0086] This embodiment provides a traditional Chinese medicine composition for treating chronic musculoskeletal diseases. The raw materials are composed of the following components by weight: 15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Spatholobus suberectus, 10 parts of Pyrola rotundifolia, 10 parts of Achyranthes bidentata, 5 parts of Epimedium brevicornu, 5 parts of Cinnamomum cassia, and 5 parts of Angelica pubescens. It is used for cold and dampness syndrome.

[0087] Example 9

[0088] This embodiment provides a traditional Chinese medicine composition for treating chronic musculoskeletal diseases. The raw materials are composed of the following components by weight: 15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Spatholobus suberectus, 10 parts of Pyrola rotundifolia, 10 parts of Achyranthes bidentata, 2 parts of Rehmannia glutinosa, and 2 parts of Cornus officinalis. It is used for symptoms of yin deficiency.

[0089] Example 10

[0090] This embodiment provides a traditional Chinese medicine composition for treating chronic musculoskeletal diseases. The raw materials, by weight, consist of the following components: 15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Spatholobus suberectus, 10 parts of Pyrola rotundifolia, 10 parts of Achyranthes bidentata, 2 parts of Corydalis yanhusuo, 2 parts of Lindera strychnifolia, and 2 parts of Pheretima aspergillum. It is used for patients with significant pain.

[0091] The preparation process and therapeutic effects of the traditional Chinese medicine composition of this invention will be further verified below in conjunction with the research process.

[0092] I. Screening of preparation process conditions in the pilot-scale stage

[0093] Using the medicinal materials in Example 3, 15g of Drynaria fortunei, 12g of Cibotium barometz, 10g of Ligustrum lucidum, 10g of Spatholobus suberectus, 10g of Pyrola rotundifolia, and 10g of Achyranthes bidentata were selected to screen the preparation process conditions.

[0094] The specific experimental procedure is as follows:

[0095] 1.1 Investigation of water extraction process

[0096] 1.1.1 Water Absorption Rate Test

[0097] Take 67g of the medicinal slices prescribed in this invention, add a certain amount of water, and soak until the medicinal material is fully absorbed (soaking for about 4 hours). Filter, weigh the slices after water absorption, and calculate the water absorption rate, as shown in Table 1. Table 1 shows that the water absorption rate of the medicinal slices is 143.51%, meaning that approximately 1.5 times more water should be added initially during extraction to compensate for the water absorption rate.

[0098]

[0099] Table 1. Results of water absorption rate test of medicinal materials (n=3)

[0100]

[0101] 1.1.2 Method for determining the dry paste yield

[0102] Take an appropriate amount of concentrated drug solution from single-factor and orthogonal studies, add it to a constant-weight and numbered evaporating dish, evaporate it to dryness in a water bath, place it in an oven to dry, set the oven temperature to 105℃, dry for 3 hours, then place it in a desiccator to cool for 30 minutes, weigh it quickly and record the value, and calculate the dry extract rate.

[0103] 1.1.3 Determination of protocatechuic acid and naringin content by HPLC

[0104] ①Chromatographic conditions

[0105] An Agilent 5TC-C18(2) 250×4.6mm, 5μm column was used. The mobile phase was 0.4% phosphoric acid solution (A): acetonitrile (B); 0-30min, 5%-10% B; 30-50min, 10%-25% B; 55-65min, 25% B; 65-70min, 5%-10% B; flow rate 1mL / min; PAD detector; detection wavelength 270nm; injection volume 10μL; column temperature 30℃; time 70min.

[0106] ② Preparation of reference solution

[0107] Take approximately 1.0 mg of protocatechuic acid and naringin reference standards into a 25 mL volumetric flask, dissolve in methanol, and dilute to the mark to prepare reference stock solutions with concentrations of 40 μg / mL. Accurately measure 1.5 mL to 10 mL of the protocatechuic acid and naringin reference stock solutions into the flasks, add 60% methanol, and dilute to the mark. The concentrations of protocatechuic acid and naringin in the mixed standard are 0.0044 mg / mL and 0.02144 mg / mL, respectively. Filter through a 0.22 μm microporous membrane and collect the filtrate.

[0108] ③ Preparation of test solution

[0109] Accurately measure 20 mL of the extract obtained from each orthogonal experiment into a stoppered conical flask, add 30 mL of 60% methanol, weigh the flask, sonicate for 30 min, cool, replenish the lost mass with 60% methanol, filter through a 0.22 μm microporous membrane, and collect the filtrate.

[0110] Accurately pipette the water extract sample for the orthogonal experiment, prepare the test solution according to the above method, inject the sample for determination, and repeat the determination twice for each sample. Calculate the content of protocatechuic acid and naringin in the sample based on the linear regression equation.

[0111] 1.1.4 Orthogonal Experimental Design and Results

[0112] The water decoction extraction process of the traditional Chinese medicine composition is as follows: Mix *Drynaria fortunei*, *Cibotium barometz*, *Ligustrum lucidum*, *Spatholobus suberectus*, *Pyrola rotundifolia*, and *Achyranthes bidentata*, then add water and decoct twice. For the first decoction, add 10-12 times the amount of water and decoct for 1.5-2.5 hours; for the second decoction, add 8-12 times the amount of water and decoct for 1.5-2.5 hours. In this experiment, the amount of water added (A), extraction time (B), and number of extractions (C) are orthogonal factors. An orthogonal experiment was designed with three levels for each factor. The orthogonal experiment factor level table is shown in Table 2.

[0113] Table 2. Factor Level Table for Orthogonal Experiment

[0114]

[0115] The dry extract rate, protocatechuic acid, and naringin content were used as evaluation indicators in the orthogonal experiment. The comprehensive score (comprehensive score = dry extract rate / maximum dry extract rate × 0.2 + protocatechuic acid content / highest protocatechuic acid content × 0.4 + naringin content / highest naringin content × 0.4) was used for evaluation. The results are shown in Tables 3 and 4.

[0116] Table 3 Results of the orthogonal experiment

[0117]

[0118] Analysis of the experimental results in Table 3 of the orthogonal experiment shows that factor C has the largest range, followed by factors B and A. Therefore, the order of influence of each factor on this process can be considered as C>A>B. Analysis of the influence of each factor level on the yield of the extracted dry extract (K value) shows that for factor A, K2>K3>K1; for factor B, K3>K1>K2; and for factor C, K2>K3>K1. Based on these results, the water decoction extraction process conditions are determined to be A2B1C2. A significant variance analysis was performed on each factor, and the results are shown in Table 4.

[0119] Table 4. Results of Variance Analysis of Orthogonal Experiments

[0120]

[0121] Table 4 shows that, taking the blank as the error, factor C has a highly significant impact on the dry extract rate and the comprehensive score of protocatechuic acid and naringin content of the water-decocted extract, while factors A and B have a smaller impact. Based on the intuitive analysis in Table 4, and considering the pharmacological and chemical characteristics of the drugs in the formula and the actual situation of the water-decocting process of the original decoction, as well as the efficiency and cost in actual production, the water-decocted extraction process conditions are determined to be two decoctions: the first decoction is prepared by adding 11.5 times the amount of water and decocting for 90 minutes, and the second decoction is prepared by adding 10 times the amount of water and decocting for 90 minutes.

[0122] 1.1.5 Validation of water extraction process

[0123] To verify the stability and feasibility of the optimal extraction process parameters determined by the orthogonal experiment, the medicinal materials in the prescription amount of Example 4 were extracted according to the optimal process parameters, and the extraction was repeated 3 times. The dry extract rate and the contents of protocatechuic acid and naringin were determined, and the results are shown in Table 5.

[0124] Table 5. Results of the three verifications

[0125]

[0126] The results show that the process described in this invention is stable, reliable, and has good repeatability.

[0127] 1.2 Investigation of Concentration Process

[0128] The experiment further investigated the effects of two different concentration methods, atmospheric pressure concentration and vacuum concentration, on the dry extract yield, protocatechuic acid, and naringin content.

[0129] According to the weight proportions of each drug in Example 3, each medicinal slice was accurately weighed, totaling 67g. Extraction was performed according to the established process, and the filtrate was divided into two parallel portions. One portion was concentrated under normal pressure at 100℃. The other portion was concentrated under reduced pressure at a temperature below 70℃. Concentration was carried out until the relative density reached 1.20–1.25 (60℃). The dry extract yield, protocatechuic acid content, and naringin content were determined, and the results are shown in Table 6.

[0130] Table 6 Results of Concentration Process Investigation

[0131]

[0132] The results above show that the concentration method has little effect on the dry extract yield and the content of protocatechuic acid and naringin. However, the atmospheric pressure concentration process is more time-consuming and not conducive to improving production efficiency. Multi-effect evaporators are also commonly used in industrial production to concentrate pharmaceutical solutions. Further experiments were conducted under pilot-scale production conditions, where the aqueous decoction was concentrated in a double-effect energy-saving concentration tank according to the standard operating procedure (SOP). The steam pressure did not exceed 0.06 MPa. Temperature control was maintained at 60–80℃ for both Effect I and Effect II, with a vacuum degree of 0.025–0.045 MPa for Effect I and 0.06–0.08 MPa for Effect II. Concentration was carried out below 80℃ to a thick paste with a relative density of 1.20–1.25 (60℃), which was then used in the subsequent drying process.

[0133] 1.3. Investigation of Drying Process

[0134] The above-mentioned water decoction extract was concentrated to obtain an extract, which was then placed in microwave drying, hot air drying and vacuum drying machines to investigate the effects of the three different methods on the content of protocatechuic acid and naringin. The results are shown in Table 7 below.

[0135] Table 7 Results of Drying Process Investigation

[0136]

[0137]

[0138] The results above show that microwave drying has little effect on the content of protocatechuic acid and naringin, and takes less time. Therefore, microwave drying was adopted as the drying method in this study.

[0139] 1.4. Molding process investigation

[0140] After drying, dry extract powder was prepared. The results of the powder property investigation showed that the extract powder was highly hygroscopic and could not be directly used with wetting agents with high moisture content. After experimental screening, 75% ethanol was selected as the wetting agent to prevent excessive moisture from causing soft material to clump together and become too hard to granulate.

[0141] Further investigation was conducted into the types and amounts of excipients used in granule preparation. Granules were prepared using starch, dextrin, and sucrose as fillers, respectively. It was found that starch-based granules were relatively loose, while sucrose easily agglomerated into lumps during granulation. Therefore, dextrin was selected as the filler. The results of the excipient dosage investigation showed that a ratio of dry extract to dextrin of 1:0.6–1.2 yielded the best granule results. The results are shown in Table 8.

[0142] Table 8. Investigation of Molding Process

[0143]

[0144] Through laboratory investigations of the water extraction, concentration, drying, and molding processes, the optimal conditions for each process under laboratory conditions were finally optimized as follows: first addition of 11.5 times the amount of water, decoction for 90 minutes; second addition of 10 times the amount of water, decoction for 90 minutes; concentration at below 70℃ to a relative density of 1.20–1.25 (60℃); microwave drying; dextrin as the filler, with a dry extract to dextrin ratio of 1:0.6–1.2.

[0145] II. Pilot-scale study

[0146] Based on the aforementioned laboratory research, and according to the weight ratio of each drug in Example 3, a pilot-scale production study was conducted at a ratio of 1:5000 to further optimize process parameters to meet the needs of mass production.

[0147] 2.1 Process Parameters

[0148] Table 9. Decoction Process Parameters

[0149]

[0150]

[0151] Table 10 Concentration Process Parameters

[0152]

[0153] Table 11 Drying process parameters

[0154]

[0155]

[0156] Table 12 Granulation process conditions

[0157]

[0158] 2.2 Research Results

[0159] In the pilot-scale study, the preparation process of the traditional Chinese medicine composition was as follows: Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Spatholobus suberectus, Pyrola rotundifolia, and Achyranthes bidentata were mixed and decocted twice with water. The first decoction was 1.5 hours with 11 times the amount of water, and the second decoction was 1.5 hours with 9 times the amount of water. The filtrates from the two decoctions were combined and subjected to two vacuum concentration processes, including first-effect and second-effect concentration, at a temperature of 60–80℃, to obtain a clear extract and an extract. The relative density of the clear extract was 1.15–1.25 (60℃), and the relative density of the extract was 1.30–1.35 (60℃). The extract was dried and pulverized to obtain a dry extract powder.

[0160] III. Preparation of Pharmaceutical Formulations

[0161] The dry extract powder, excipients, and flavoring agents are mixed and granulated. The excipients include wetting agents and fillers, with dextrin and lactose as fillers; the wetting agent is a clear extract; and the flavoring agent is steviol glycosides. The ratio of dry extract to filler is 1:0.6-1.0, with the preferred ratio being dry extract:lactose:dextrin = 6.25:2.5:1.25. The mixture is then granulated, sieved, and finally mixed to obtain the traditional Chinese medicine granule preparation.

[0162] The produced granules are packaged in 10g / bag specifications. Actual test data shows that: the yield of both extract and clear extract can reach 85-115% (calculated as (extract amount / theoretical amount) × 100%); the yield of traditional Chinese medicine granules can reach 95-105% (calculated as (number of small bags + sample amount) / planned amount) × 100%); and the material balance rate can reach 95-100% (calculated as (weight of small bags + sample amount + waste amount + packaging material loss + remaining packaging material - packaging material usage) / mixed granule input amount) × 100%). This demonstrates significant advantages in high production yield and low process cost. Furthermore, the traditional Chinese medicine granules prepared by this process have significant advantages over other traditional dosage forms in the treatment of osteoporosis and osteoarthritis, including convenient use and clear efficacy.

[0163] The compositions of the present invention can also be formulated into other dosage forms, such as mixtures, tinctures, injections, tablets, powders, oral liquids, capsules, granules, ointments, powders, emulsions, drop pills, pills, lozenges, gels, suppositories, or aerosols, by adding other excipients or pharmaceutically acceptable carriers according to conventional processes.

[0164] To further investigate the efficacy of the traditional Chinese medicine composition of this invention in treating chronic musculoskeletal diseases such as osteoporosis and degenerative osteoarthritis, a rat model of knee osteoarthritis was constructed. The combination, compatibility, and dosage of the traditional Chinese medicine composition of this invention were screened using behavioral indicators, joint pathological examinations, and biochemical index detection. The aim was to elucidate the therapeutic effects of different dosage combinations on knee osteoarthritis and the correlation between drug-dosage-efficacy, ultimately screening out the optimal combination for treating knee osteoarthritis and clarifying the synergistic mechanism of each herb. This will provide experimental evidence for optimizing prescriptions, developing precise medication regimens, and improving efficacy in clinical practice.

[0165] IV. Verification of the therapeutic effects of different prescriptions

[0166] A group lacking single herbs such as *Drynaria fortunei*, *Cibotium barometz*, *Ligustrum lucidum*, *Pyrola rotundifolia*, and *Achyranthes bidentata* was set up and compared with the group containing the whole formula. The therapeutic effects of different herbal combinations on knee osteoarthritis were analyzed by combining behavioral indicators, joint pathological examinations, and biochemical index tests. The specific experimental procedure is as follows:

[0167] 4.1 Instruments and Materials

[0168] 4.1.1. Instruments

[0169] Sterile insulin syringe (Shanghai Kantele Enterprise Development Co., Ltd.); Bipedal balance algometer (IITC Life Science, 123019-600); Microplate reader (Thermo scientific, 51119670DP); Micro-CT (SKYSCAN-1276, 19G17070); High-speed low-temperature tissue grinder (Wuhan Servier Biotechnology Co., Ltd., KZ-III-F).

[0170] 4.1.2. Materials

[0171] The traditional Chinese medicine composition herbs were provided by the hospital. Collagenase (Sigma-Aldrich (Shanghai) Trading Co., Ltd., V900892); Diacerein capsules (TRB Pharma S.A. 21830); Rat interleukin-1β (IL-1β) kit, rat interleukin-6 (IL-6) kit, rat tumor necrosis factor (TNF-α) kit, rat interleukin-8 (IL-8) kit, all of which were purchased from Shaanxi Liming Kant Biotechnology Co., Ltd.).

[0172] 4.1.3. Experimental animals

[0173] 90 male SD rats (150-200 g), provided by Chengdu Dashuo Experimental Animal Co., Ltd., license number SCXK (Sichuan 2020-0030). They were housed in an environment with a temperature of 22-24°C and a relative humidity of 50%-60%, and maintained under a light / dark cycle of 12 h / 12 h, with free access to standard diet and water. After 2 weeks of adaptive feeding, they were fasted for 12 h before the experiment and allowed free access to water. The experimental animals in this study were approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine.

[0174] 4.2. Methods and results

[0175] 4.2.1. In vivo activity evaluation method

[0176] 4.2.1.1. Animal experiments and sample collection

[0177] Ninety rats were acclimatized for one week and then randomly divided into nine groups according to body weight stratification: control group, model group, double-coarse renin group, group lacking *Drynaria fortunei*, group lacking *Cibotium barometz*, group lacking *Ligustrum lucidum*, group lacking *Pyrola rotundifolia*, group lacking *Achyranthes bidentata*, and complete formula. Except for the control group, the rats in the other groups were anesthetized with freshly prepared 10% sodium pentobarbital (0.35 mL / 100 g). The right knee joint was disinfected three times with povidone-iodine, and then a 1 mL syringe was used to puncture the right knee joint. After no blood return was observed upon aspiration, 50 μL of prepared collagenase solution (500 U dissolved in physiological saline, freshly prepared) was injected into the joint cavity. After needle withdrawal, the wound was cleaned with povidone-iodine swabs and covered with an injection dressing. The control group rats were injected with 50 μL of physiological saline at the same location and time point. No other special treatment was given postoperatively. The above procedure was repeated on day 4.

[0178] After successful model replication, rats in each group underwent a 30-day gavage intervention, once daily, starting the following day. Following successful model replication, rats were randomly divided into the following groups based on body weight: control group; model group; positive control group; group lacking *Drynaria fortunei*; group lacking *Cibotium barometz*; group lacking *Ligustrum lucidum*; group lacking *Pyrola rotundifolia*; group lacking *Achyranthes bidentata*; and a complete treatment group. The dosage of double-crude renin was 9 mg / kg. The dosage for other groups was 4.5 mg / kg. Administration was performed via gavage for 30 days, once daily. The model group and control group received an equal volume of physiological saline orally via gavage.

[0179] After the administration of the drug, the rats in each group were weighed, fasted for 12 hours but allowed free access to water, anesthetized with 1 mL / kg of 3% sodium pentobarbital, and blood was collected from the abdominal aorta. The serum was collected by centrifugation and stored at -80℃ for later use. Tibial plateau articular cartilage was collected from each group for later use.

[0180] 4.2.1.2 Behavioral Indicators Assessment

[0181] Knee joint circumference measurement: During the experiment, the diameter of the right knee joint of rats was measured using an electronic digital vernier caliper on day 21 before drug administration, day 7 before drug administration, day 7 after drug administration, and day 28 after drug administration. The changes in knee joint diameter of rats in each group were observed and compared.

[0182] Bipedal balance test: During the experiment, on days 7, 14, 30, 45, and 60 after the initial modeling, the weight-bearing capacity of the rats' hind limbs was measured using a bipedal balance analgesia device, and changes in each group were observed. The bipedal balance analgesia device was used to detect the force on the affected limbs of KOA model rats. Before the experiment, the instrument was calibrated, with no load set to 0 for both sides and calibrated at 500g for both sides. The time was set to 5s. The rats were placed upright in the transparent organic glass grid of the bipedal balance analgesia device, with both hind feet placed stably in the center of the pedal, head and body upright, and stationary. The pressure value within 5s in the rat's resting state was recorded, with each measurement at 5-minute intervals, for a total of 3 measurements. The average value was taken and calculated using the formula: (Right foot / Weight-bearing capacity of both feet) × 100%. The baseline value was 50%. When KOA symptoms appeared on one side, the weight-bearing ratio of the affected side was <50%.

[0183] 4.2.1.3 Joint pathological examination

[0184] Micro-CT Bone Microenvironment Analysis: After drug administration, rats in each group were weighed, fasted for 12 hours but allowed to drink water, and anesthetized with 1 mL / kg of 3% sodium pentobarbital. Tibial plateau articular cartilage was harvested from each group and the articular cartilage microenvironment was analyzed by Micro-CT.

[0185] 4.2.1.4 Biochemical Indicator Detection

[0186] Elisa serum TNF-α, IL-1β, IL-6, and IL-8 levels: After administration, rats in each group were weighed, fasted for 12 hours but allowed free access to water, anesthetized with 1 mL / kg of 3% sodium pentobarbital, blood was collected from the abdominal aorta, the serum was collected by centrifugation, and stored at -80℃ for later use. The levels of TNF-α, IL-1β, IL-6, and IL-8 in the serum were measured strictly according to the kit instructions.

[0187] 4.2.2 Results of in vivo activity evaluation

[0188] 4.2.2.1 Behavioral Indicators Assessment

[0189] The results of measuring the diameter of the rat knee joint using vernier calipers are as follows: Figure 2 As shown in Table 13: In the control group, the knee joint diameter remained at a normal level on days 21 before administration, day 7, and days 7 and 28 after administration, with no significant swelling. In the model group, the knee joint diameter was significantly higher than that in the control group at all time points (P<0.01), and the swelling remained significant and showed no trend of relief. After administration, the positive group showed the best improvement, with a significant decrease in knee joint diameter on day 7 compared to before administration, exhibiting the largest reduction. On day 28, there was no difference between the model group and the control group (P>0.05). The complete formula group showed the second best effect, with a smaller reduction in diameter on day 7 than the positive group but significantly greater than other drug-deficient groups. On day 28, there was no difference between the model group and the control group (P>0.05). The groups lacking Cibotium barometz, Ligustrum lucidum, Pyrola rotundifolia, and Achyranthes bidentata showed less improvement than the complete formula group. Although there was no difference between the model group and the control group on day 28 (P>0.05), their overall effect was inferior to that of the complete formula group. The group lacking *Drynaria fortunei* showed the worst effect, although it was better than the model group. The diameter reduction was the smallest on day 7, and although there was no difference from the control group on day 28 (P>0.05), the degree of improvement was still weaker than other treatment groups. In summary, the complete herbal composition of this invention can effectively relieve knee joint swelling. Although the effect is slightly inferior to the positive control group, it is superior to all groups lacking the corresponding herbs. The absence of *Drynaria fortunei* significantly weakens the swelling-reducing effect, verifying the importance of the synergistic effect of each herb.

[0190] Table 13 Comparison of knee joint diameter in rats of different prescriptions ( mm, n=9)

[0191]

[0192] Note: Compared with the control group, #p<0.05, ##p<0.01.

[0193] The weight-bearing ratio of rats' hind limbs was measured using a bipedal balance analgesia device to assess pain intensity. The results are shown in Table 14 and... Figure 3 The results showed that the weight-bearing ratio of the affected limb in the control group remained at a normal level at all time points after modeling (days 7, 14, 30, 45, and 60). The weight-bearing ratio in the model group was significantly lower than that in the control group at all time points (P<0.01), and there was no obvious trend of improvement. On days 7, 14, and 30 after modeling, the weight-bearing ratios in the positive group, the complete formula group, and each drug-deficient group were significantly lower than those in the control group (P<0.01), and there was no significant difference from the model group (P>0.05). On day 45, the weight-bearing ratio of the positive group increased the most, significantly higher than that of the model group (P<0.01); the increase in the complete formula group was the second largest, significantly higher than that of each drug-deficient group (P<0.01); the increases in the groups lacking Cibotium barometz, Ligustrum lucidum, Pyrola rotundifolia, and Achyranthes bidentata were similar, all higher than those lacking Drynaria fortunei; although the Drynaria fortunei group increased significantly compared to the model group (P<0.01), the increase was the smallest, and the effect was the worst among the drug-treated groups. On day 60, there was no difference between the positive control group and the control group (P>0.05); there was no difference between the complete formula group and the control group (P>0.05), but the control group was slightly lower than the positive control group; other drug-deficient groups reached the level of the control group, while the group lacking *Drynaria fortunei* was still slightly lower than the other drug-treated groups. In summary, the herbal compound granules can significantly increase hind limb weight-bearing to alleviate pain, with the positive control group showing the best effect, followed by the complete formula group, which was superior to all drug-deficient groups. The absence of *Drynaria fortunei* significantly weakens the therapeutic effect.

[0194] Table 14 Comparison of weight-bearing capacity of rats in different prescription groups ( mm, n=9)

[0195]

[0196] Note: Compared with the model group, *p<0.05, **p<0.01.

[0197] 4.2.2.2 Joint Pathological Examination

[0198] Key indicators of rat bone microstructure were detected using Micro-CT, and the results are shown in Table 15 and 16. Figure 4The results showed that in the blank group, bone volume (BV), bone volume fraction (BV / TV), and trabecular thickness (Tb.Th) were all within normal levels, while the ratio of bone surface area to bone volume (BS / BV), trabecular pattern factor (Tb.Pf), structural pattern index (SMI), total porosity (Po(tot)), and trabecular separation (Tb.Sp) were all within normal ranges. Compared with the blank group, the model group showed significantly reduced BV, BV / TV, and Tb.Th (P<0.05), and significantly increased other indicators (P<0.015), indicating severe damage to bone microstructure. After drug administration, the positive group showed the best improvement, with all indicators significantly improved compared to the model group (P<0.05) and approaching the levels of the blank group. The full-formula group showed the second best effect, with significantly better reductions in BS / BV, Tb.Pf, SMI, Po(tot), and Tb.Sp, and significantly better increases in BV / TV and Tb.Th than the drug-free groups (P<0.05), but slightly worse than the positive group. The groups lacking Cibotium barometz, Ligustrum lucidum, Pyrola rotundifolia, and Achyranthes bidentata showed similar improvement, all superior to the group lacking Drynaria fortunei. Although the Drynaria fortunei group showed significant improvement compared to the model group (P<0.05), its optimization level was the lowest, making it the worst performing group. There were no significant differences in trabecular bone number (Tb.N) and bone volume (BV) between the model group and the various treatment groups (P>0.05). In conclusion, the herbal composition granules can improve bone microstructure, with the positive control group showing the best results, followed by the complete formula group, which is superior to all the groups lacking specific herbs.

[0199] Table 15. Micro-CT analysis results of subchondral bone microstructure morphological parameters (n=6). )

[0200]

[0201] 4.2.2.3 Biochemical index detection

[0202] The levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) in rat serum were measured to assess the anti-inflammatory effect. The results are as follows: Figure 5The results showed that the levels of all factors in the blank group remained at the normal baseline, with no abnormal increases. In the model group, the expression levels of the aforementioned factors were significantly increased (P<0.01), indicating a severe inflammatory response. After administration, the positive control group showed the best anti-inflammatory effect, with all factor levels significantly lower than the model group (P<0.01), and the largest decrease, with indicators closest to the blank group. The whole-formula group showed the second best effect, with all factor levels significantly lower than the model group (P<0.01), the decrease being smaller than the positive control group but significantly greater than the groups lacking any specific drug (P<0.01). The levels of all factors in the groups lacking Cibotium barometz, Ligustrum lucidum, Epimedium brevicornu, and Achyranthes bidentata were significantly lower than the model group (P<0.01), with similar improvement rates, and all were better than the group lacking Drynaria fortunei. Although the levels of all factors in the Drynaria fortunei group were significantly lower than the model group (P<0.01), indicating a better anti-inflammatory effect than the model group, the decrease was the smallest among all administered groups, indicating the worst effect. In summary, the Chinese herbal medicine granules can significantly reduce the level of pro-inflammatory cytokines in the serum of osteoarthritis rats and exert an anti-inflammatory effect. The positive control group showed the best effect, followed by the whole formula group, which was better than each group lacking the medicine. The absence of Drynaria fortunei significantly weakened the overall anti-inflammatory effect, which verifies the importance of the synergistic effect of each herb.

[0203] Through the comparative study of the single-herb deficiency groups (lacking Drynaria fortunei, Cibotium barometz, Ligustrum lucidum, Pyrola rotundifolia, and Achyranthes bidentata) and the complete formula group, the complete formula group, with Drynaria fortunei as the principal herb, Cibotium barometz and Ligustrum lucidum as assistant herbs, Pyrola rotundifolia as adjuvant herbs, and Spatholobus suberectus and Achyranthes bidentata as guiding herbs, showed the best efficacy in improving joint swelling, relieving pain, repairing bone microstructure, and reducing pro-inflammatory cytokine levels, significantly outperforming the single-herb deficiency groups. This result verifies the synergistic effect of Drynaria fortunei as the principal herb tonifying the kidney and strengthening bones, assistant herbs assisting in tonification, adjuvant herbs clearing the channels and relieving pain, and guiding herbs invigorating blood and directing the flow of qi. Each herb performs its specific function while working together to target the pathogenesis and exert a comprehensive therapeutic effect, while the absence of single herbs weakens the overall efficacy to varying degrees. Therefore, the comprehensive analysis indicates that the complete formula of this invention should be selected for formulation, as it not only reflects the overall advantages of traditional Chinese medicine formulation but also achieves the best therapeutic effect on knee osteoarthritis through multi-component and multi-target action, providing a scientific basis for optimizing prescriptions and improving efficacy in clinical practice.

[0204] V. Verification of the therapeutic effects of different formulations

[0205] To further explore the relationship between different dosage ratios and efficacy, six specific dosage combinations were designed for the six herbs in the formula. By comparing the differences in behavioral indicators, joint pathology examinations, and biochemical indicators among the groups, the aim was to screen out the dosage ratio with the optimal efficacy.

[0206] Table 16 Different dosage combinations of the 6 herbs in the whole formula

[0207]

[0208] 5.1 Instruments and Materials

[0209] 5.1.1. Instruments

[0210] Sterile insulin syringe (Shanghai Kantele Enterprise Development Co., Ltd.); Bipedal balance algometer (IITC Life Science, 123019-600); Microplate reader (Thermo scientific, 51119670DP); Micro-CT (SKYSCAN-1276, 19G17070); High-speed low-temperature tissue grinder (Wuhan Sevier Biotechnology Co., Ltd., KZ-III-F).

[0211] 5.1.2. Materials

[0212] The medicinal materials of the traditional Chinese medicine composition were provided by Shaanxi University of Chinese Medicine. Collagenase (Sigma-Aldrich (Shanghai) Trading Co., Ltd., V900892); Diacerein capsules (TRB Pharma S.A. 21830); Rat interleukin 1β (IL-1β) kit, rat interleukin 6 (IL-6) kit, rat tumor necrosis factor (TNF-α) kit, rat interleukin (IL-8) kit, all of which were purchased from Shaanxi Liming Kant Biotechnology Co., Ltd.

[0213] 5.1.3. Experimental animals

[0214] 90 male SD rats (150-200 g), provided by Chengdu Dashuo Experimental Animal Co., Ltd., license number SCXK (Sichuan 2020-0030). They were housed in an environment with a temperature of 22-24 °C and a relative humidity of 50%-60%, and were freely provided with standard diet and water. After 2 weeks of adaptive feeding, they were fasted for 12 h before the experiment and had free access to water. The experimental animals in this study were approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine.

[0215] 5.2. Methods and results

[0216] 5.2.1. In vivo activity evaluation method

[0217] 5.2.1.1. Animal experiments and sample collection

[0218] Ninety rats were acclimatized for one week and then randomly divided into nine groups according to body weight stratification: control group, model group, group 1, group 2, group 3, group 4, group 5, and group 6. Except for the control group, the rats in the other groups were anesthetized with freshly prepared 10% sodium pentobarbital (0.35 mL / 100 g). The right knee joint was disinfected three times with povidone-iodine, and then a 1 mL syringe was used to puncture the right knee joint. After no blood return was observed upon aspiration, 50 μL of prepared collagenase solution (500 U dissolved in physiological saline, freshly prepared) was injected into the joint cavity. After needle withdrawal, the wound was cleaned and massaged with povidone-iodine swabs, and then covered with an injection dressing. The control group rats were injected with 50 μL of physiological saline at the same location and time point. No other special treatment was given postoperatively. The above procedure was repeated on day 4.

[0219] After successful model replication, rats in each group underwent a 30-day gavage intervention, once daily, starting the following day. Following successful model replication, rats were randomly divided into stratified groups based on body weight: control group, model group, group 1, group 2, group 3, group 4, group 5, and group 6. The dosage of diclofenac sodium was 9 mg / kg. The dosage for other groups was 4.5 mg / kg. Gavage administration was administered once daily for 30 days, while the model group and control group received an equal volume of physiological saline orally.

[0220] After the administration of the drug, the rats in each group were weighed, fasted for 12 hours but allowed free access to water, anesthetized with 1 mL / kg of 3% sodium pentobarbital, and blood was collected from the abdominal aorta. The serum was collected by centrifugation and stored at -80℃ for later use. Tibial plateau articular cartilage was collected from each group for later use.

[0221] 5.2.1.2 Behavioral Indicators Assessment

[0222] Knee joint circumference measurement: During the experiment, the diameter of the right knee joint of rats was measured using an electronic digital vernier caliper on day 21 before drug administration, day 7 before drug administration, day 7 after drug administration, and day 28 after drug administration. The changes in knee joint diameter of rats in each group were observed and compared.

[0223] Bipedal balance test: During the experiment, on days 7, 14, 30, 45, and 60 after the initial modeling, the weight-bearing capacity of the rats' hind limbs was measured using a bipedal balance analgesia device, and changes in each group were observed. The bipedal balance analgesia device was used to detect the force on the affected limbs of KOA model rats. Before the experiment, the instrument was calibrated, with no load set to 0 for both sides and calibrated at 500g for both sides. The time was set to 5s. The rats were placed upright in the transparent organic glass grid of the bipedal balance analgesia device, with both hind feet placed stably in the center of the pedal, head and body upright, and stationary. The pressure value within 5s was recorded in the rat's resting state, with each measurement taken at 5-minute intervals, for a total of 3 measurements. The average value was calculated using the formula: (Right foot / Weight-bearing capacity of both feet) * 100%. The baseline value was 50%. When KOA symptoms appeared on one side, the weight-bearing ratio of the affected side was <50%.

[0224] 5.2.1.3 Joint Pathological Examination

[0225] Micro-CT Bone Microenvironment Analysis: After drug administration, rats in each group were weighed, fasted for 12 hours but allowed to drink water, and anesthetized with 1 mL / kg of 3% sodium pentobarbital. Tibial plateau articular cartilage was harvested from each group and the articular cartilage microenvironment was analyzed by Micro-CT.

[0226] 5.2.1.4 Biochemical Indicator Detection

[0227] Elisa serum TNF-α, IL-1β, IL-6, and IL-8 levels: After administration, rats in each group were weighed, fasted for 12 hours but allowed free access to water, anesthetized with 1 mL / kg of 3% sodium pentobarbital, blood was collected from the abdominal aorta, the serum was collected by centrifugation, and stored at -80℃ for later use. The levels of TNF-α, IL-1β, IL-6, and IL-8 in the serum were measured strictly according to the kit instructions.

[0228] 5.2.2 Results of in vivo activity evaluation

[0229] 5.2.2.1 Behavioral Indicators Assessment

[0230] The results of rat knee joint diameter measurements are shown in Table 17 and Figure 6 As shown: In the control group, the diameter remained normal at all time points, with no swelling. In the model group, the diameter was significantly higher than that in the control group at all time points (P<0.01), and the swelling persisted without relief. The positive group showed the best improvement, with the largest diameter reduction on day 7 after administration, and no difference from the control group on day 28 (P>0.05). Group 6 was the second best, with a smaller diameter reduction on day 7 than the positive group but greater than other dosage groups, and no difference from the control group on day 28 (P>0.05). Groups 2 and 3 showed less improvement than group 6, and although there was no difference from the control group on day 28, the overall effect was slightly inferior. Groups 1, 4, and 5 showed even smaller improvement and were less effective than groups 2 and 3. In conclusion, all dosage groups of the Bone-Strengthening Formula could relieve swelling, with group 6 showing the best effect (slightly inferior to the positive group), verifying the importance of dosage optimization in enhancing efficacy and providing a basis for determining the optimal dosage.

[0231] Table 17 Comparison of knee joint diameter in rats with different formulation ratios ( mm, n=9)

[0232]

[0233] Note: Compared with the control group, #p<0.05, ##p<0.01.

[0234] The weight-bearing ratio of rats' hind limbs was measured using a bipedal balance analgesia device to assess pain levels. The results are shown in Table 18 and... Figure 7As shown: In the blank group, the weight-bearing ratio of the affected limb remained at normal levels at all time points after modeling (days 7, 14, 30, 45, and 60). In the model group, the weight-bearing ratio was significantly lower than that of the blank group at all time points (P<0.01), and there was no trend of improvement. On days 7, 14, and 30 after modeling, the weight-bearing ratios of the positive group and groups 1 to 6 were significantly lower than those of the blank group (P<0.01), and there was no significant difference compared to the model group (P>0.05). On day 45, the positive group showed the largest increase in weight-bearing ratio, significantly higher than the model group (P<0.01); group 6 showed the second largest increase, significantly higher than the other dose groups (P<0.01); the increases in groups 1, 2, 3, 4, and 5 were similar, all lower than group 6; all dose groups were significantly higher than the model group (P<0.01). On day 60, there was no difference between the positive control group and the control group (P>0.05); group 6 showed no difference between the positive control group and the control group (P>0.05), but was slightly lower than the positive control group; groups 1, 2, 3, 4, and 5 were significantly higher than the model group, but all were lower than group 6. In summary, all dosage groups of the Bone-Strengthening Formula could alleviate pain, with group 6 showing the best effect (slightly less than the positive control group), demonstrating the enhancing effect of dosage optimization on efficacy.

[0235] Table 18 Comparison of weight-bearing capacity of rats with different formulation ratios in each group ( mm, n=9)

[0236]

[0237]

[0238] Note: Compared with the model group, *p<0.05, **p<0.01.

[0239] 5.2.2.2 Joint Pathological Examination

[0240] Key indicators of rat bone microstructure were detected using Micro-CT, and the results are shown in Table 19 and 10. Figure 8As shown: In the blank group, bone volume (BV), bone volume fraction (BV / TV), and trabecular thickness (Tb.Th) were all within normal levels, while the ratio of bone surface area to bone volume (BS / BV), trabecular pattern factor (Tb.Pf), structural pattern index (SMI), total porosity (Po(tot)), and trabecular separation (Tb.Sp) were all within normal ranges. Compared with the blank group, the model group showed significantly reduced BV, BV / TV, and Tb.Th (P<0.05), and significantly increased other indicators (P<0.01), indicating severe damage to bone microstructure. After administration, the positive group showed the best improvement, with all indicators significantly improved compared to the model group (P<0.05) and approaching the levels of the blank group. Group 6 showed the second best effect, with significantly better reductions in BS / BV, Tb.Pf, SMI, Po(tot), and Tb.Sp, and significantly better increases in BV / TV and Tb.Th than other dose groups (P<0.05), but slightly worse than the positive group. Groups 2, 3, 4, and 5 showed similar improvement, all superior to group 1. Although group 1 showed significant improvement compared to the model group (P<0.05), it had the lowest degree of optimization across all indicators, making it the worst performing group. There were no significant differences in trabecular bone number (Tb.N) and bone volume (BV) between the model group and each dose group (P>0.05). In conclusion, all dose groups of the Bone-Strengthening Formula improved bone microstructure damage, with group 6 showing the best effect (slightly inferior to the positive control group), validating the importance of dosage optimization in enhancing bone structure repair.

[0241] Table 19. Micro-CT analysis results of subchondral bone microstructure morphology parameters in different ratios (n=6). )

[0242]

[0243]

[0244] 5.2.2.3 Biochemical Indicator Detection

[0245] The levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) in rat serum were measured to assess the anti-inflammatory effect. The results are as follows: Figure 9As shown: In the blank group, the levels of all factors remained at the normal baseline, with no abnormal increases. In the model group, the protein expression levels of the above factors were significantly increased (P<0.01), indicating a severe inflammatory response. After administration, the positive group showed the best anti-inflammatory effect, with the levels of all factors significantly lower than the model group (P<0.01), and the largest decrease, with the indicators closest to the blank group. Group 6 showed the second best effect, with the levels of all factors significantly lower than the model group (P<0.01), although the decrease was smaller than that in the positive group, but significantly greater than that in groups 1 to 5 (P<0.01). The levels of all factors in groups 1 to 5 were significantly lower than those in the model group (P<0.01), indicating that their anti-inflammatory effect was better than that in the model group, but the improvement was similar and all smaller than that in group 6, making them relatively less effective among all administered groups. In conclusion, Guluotong granules can significantly reduce the levels of pro-inflammatory cytokines in the serum of rats with osteoarthritis, thus exerting an anti-inflammatory effect. The positive control group showed the best effect, followed by group 6, which was significantly better than groups 1 to 5. Although groups 1 to 5 were better than the model group, their effects were not as good as other drug-treated groups. This verifies the importance of dose optimization in enhancing the anti-inflammatory effect and provides a basis for determining the optimal dose.

[0246] Six groups with different dosage ratios were designed based on the above-mentioned traditional Chinese medicine composition (Drynaria fortunei as the principal ingredient, Cibotium barometz and Ligustrum lucidum as the assistant ingredients, Pyrola rotundifolia as the adjuvant ingredient, Spatholobus suberectus and Achyranthes bidentata as the guiding ingredients). Through behavioral, joint pathological and biochemical index detection, the results showed that all groups could improve joint swelling, pain, cartilage degeneration and inflammatory response, but there were significant differences in the effects: Group 6 (15 parts of Drynaria fortunei, 12 parts of Cibotium barometz, 10 parts of Ligustrum lucidum, 10 parts of Spatholobus suberectus, 10 parts of Pyrola rotundifolia, and 10 parts of Achyranthes bidentata) was significantly better than Groups 1 to 5 in terms of relieving joint swelling, improving hind limb weight-bearing capacity, repairing bone microstructure integrity and reducing the level of pro-inflammatory cytokines (TNF-α, IL-1β, etc.). Although it was slightly inferior to the positive control group, it showed the most balanced multi-target synergistic effect. This result also verifies that the dosage ratio of Group 6 maximizes the synergistic effects of the principal herb in tonifying the kidneys and strengthening bones, the assistant herb in assisting tonification, the adjuvant herb in unblocking meridians and relieving pain, and the guiding herb invigorating blood and guiding the medicine through the meridians. Its dosage setting avoids the weaker efficacy caused by insufficient dosage of some herbs in Groups 1 and 3, and also overcomes the impact of imbalanced dosages of the principal or assistant herbs in Groups 2 and 5 on the overall efficacy. Therefore, the dosage ratio of Group 6 should be selected for the entire formula of this invention, providing a scientific basis for precise clinical medication and maximizing efficacy.

[0247] VI. Verification of treatment efficacy without dosage

[0248] To further investigate the efficacy of the traditional Chinese medicine composition of this invention in treating chronic musculoskeletal diseases such as osteoporosis and degenerative osteoarthritis, a rat model of knee osteoarthritis was constructed to further confirm the efficacy and mechanism of the traditional Chinese medicine granule preparation prepared in the above-mentioned "pilot-scale study" in treating rats with knee osteoarthritis, thus providing a theoretical basis for the treatment of knee osteoarthritis.

[0249] 6.1 Instruments and Materials

[0250] 6.1.1. Instruments

[0251] Sterile insulin syringe (Shanghai Kantele Enterprise Development Co., Ltd.); Bipedal balance algometer (IITC Life Science, 123019 - 600); Microplate reader (Thermo scientific, 51119670DP); Micro-CT (SKYSCAN-1276, 19G17070); Inverted optical microscope (Nikon, Japan, Nikon Eclipse E100); Imaging system (Nikon, Japan, Nikon DS-U3); High-speed low-temperature tissue grinder (Wuhan Sevier Biotechnology Co., Ltd., KZ-III-F).

[0252] 6.1.2. Materials

[0253] The traditional Chinese medicine composition granules (hereinafter referred to as traditional Chinese medicine composition or granules) were prepared with the above optimal ratio. Collagenase (Sigma-Aldrich (Shanghai) Trading Co., Ltd., V900892); Diacerein capsules (TRB Pharma S.A. 21830); Rat interleukin-1β (IL-1β) kit, rat interleukin-6 (IL-6) kit, rat tumor necrosis factor (TNF-α) kit, rat interleukin (IL-8) kit, all of which were purchased from Shaanxi Liming Kant Biotechnology Co., Ltd.; HE staining solution set (Chengdu Lilai Biotechnology Co., Ltd.); Safranin fast green staining solution set (Chengdu Lilai Biotechnology Co., Ltd.).

[0254] 6.1.3. Experimental animals

[0255] 96 male SD rats (150 - 200 g), provided by Chengdu Dashuo Experimental Animal Co., Ltd., license number SCXK (Sichuan 2020 - 0030). They were housed in an environment with a temperature of 22 - 24 °C and a relative humidity of 50% - 60%, and were freely provided with standard diet and water. After 2 weeks of adaptive feeding, they were fasted for 12 h before the experiment and had free access to water. The experimental animals in this study were approved by the Animal Ethics Committee of Shaanxi University of Chinese Medicine.

[0256] 6.2. Methods and results

[0257] 6.2.1. In vivo activity evaluation method

[0258] 6.2.1.1. Animal experiments and sample collection

[0259] Ninety rats were acclimatized for one week and then randomly divided into six groups according to body weight stratification: control group, model group, double-coarse renin group, low-dose group, medium-dose group, and high-dose group of traditional Chinese medicine granules. Except for the control group, the rats in the other groups were anesthetized with freshly prepared 10% sodium pentobarbital (0.35 mL / 100 g). The right knee joint was disinfected three times with povidone-iodine, and then a 1 mL syringe was used to puncture the right knee joint. After no blood return was observed upon aspiration, 50 μL of prepared collagenase solution (500 U dissolved in physiological saline, freshly prepared) was injected into the joint cavity. After needle withdrawal, the wound was cleaned with povidone-iodine swabs and covered with an injection dressing. The control group rats were injected with 50 μL of physiological saline at the same location and time point. No other special treatment was given postoperatively. The above procedure was repeated on day 4.

[0260] After successful model replication, rats in each group underwent a 30-day gavage intervention, once daily, starting the following day. Following successful model replication, rats were randomly stratified by body weight and divided into: ① Model group; ② Low-dose group of traditional Chinese medicine granules (2.25 mL / kg); ③ Medium-dose group of traditional Chinese medicine granules (4.5 mL / kg); ④ High-dose group of traditional Chinese medicine granules (9 mL / kg); ⑤ Blank control group; sham-operated group served as a blank control. ⑥ Positive group: The dosage of double crude resine was 9 mg / kg. The clinical dosage of the traditional Chinese medicine granules in this invention is 8 g × 2 times / day. Administration was performed via gavage for 30 days, once daily. The model group and control group received an equal volume of physiological saline orally via gavage. The model group and blank group received an equal volume of physiological saline orally via gavage, while the dosage of double crude resine in the double crude resine group was 9.00 mg / kg.

[0261] After the administration of the drug, the rats in each group were weighed, fasted for 12 hours but allowed free access to water, anesthetized with 1 mL / kg of 3% sodium pentobarbital, and blood was collected from the abdominal aorta. The serum was collected by centrifugation and stored at -80℃ for later use. Tibial plateau articular cartilage was collected from each group for later use.

[0262] 6.2.1.2 Behavioral Indicator Assessment

[0263] Knee joint circumference measurement: During the experiment, the diameter of the right knee joint of rats was measured using an electronic digital vernier caliper on day 21 before drug administration, day 7 before drug administration, day 7 after drug administration, and day 28 after drug administration. The changes in knee joint diameter of rats in each group were observed and compared.

[0264] Bipedal balance test: During the experiment, on days 7, 14, 30, 45, and 60 after the initial modeling, the weight-bearing capacity of the rats' hind limbs was measured using a bipedal balance analgesia device, and changes in each group were observed. The bipedal balance analgesia device was used to detect the force on the affected limbs of KOA model rats. Before the experiment, the instrument was calibrated, with no load set to 0 for both sides and calibrated at 500g for both sides. The time was set to 5s. The rats were placed upright in the transparent organic glass grid of the bipedal balance analgesia device, with both hind feet placed stably in the center of the pedal, head and body upright, and stationary. The pressure value within 5s was recorded in the rat's resting state. Measurements were taken 3 times at 5-minute intervals, and the average value was calculated using the formula: (Right foot / Weight-bearing capacity of both feet) * 100%. The baseline value was 50%. When KOA symptoms appeared on one side, the weight-bearing ratio on the affected side was <50%.

[0265] 6.2.1.3 Joint pathological examination

[0266] Micro-CT Bone Microenvironment Analysis: After drug administration, rats in each group were weighed, fasted for 12 hours but allowed to drink water, and anesthetized with 1 mL / kg of 3% sodium pentobarbital. Tibial plateau articular cartilage was harvested from each group and the articular cartilage microenvironment was analyzed by Micro-CT.

[0267] HE staining: Tibial plateau articular cartilage from each group was taken, trimmed to approximately 0.5cm × 0.5cm × 0.5cm, fixed with 4% paraformaldehyde, and subjected to routine series of dehydration and decalcification. The cartilage was then embedded in paraffin, sectioned, dewaxed, rinsed with PBS, and stained with HE. The morphology and structure of the articular cartilage in each group were observed under an optical microscope.

[0268] Safranin-Fixed Green Staining: After fixation, the tissue was removed and placed in a prepared 10% EDTA decalcification solution. The decalcification effect was observed every 3 days, and the solution was replaced with fresh decalcification solution until decalcification was complete. The fixed tissue was then dehydrated using an automated dehydrator (dehydration time: 75% ethanol 2h, 85% ethanol 1h, 95% ethanol 1h, 100% ethanol I 20min, 100% ethanol II 20min, 100% ethanol III 20min, 100% ethanol IV 20min, clearing agent I 25min, clearing agent II 30min, paraffin I 30min, paraffin II 1h, paraffin III 1h), embedded, and sectioned as follows:

[0269] ① Dewaxing the sections to water, the specific steps are as follows: Dewaxing solution I for 30 min, dewaxing solution II for 30 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% alcohol for 5 min, 85% alcohol for 5 min, 75% alcohol for 5 min, and then rinsing with running tap water for 5 min.

[0270] ② Stain with freshly prepared Weigert staining solution for 3-5 minutes, then rinse with water;

[0271] ③ Differentiate in acidic solution for 15 seconds, then wash with distilled water for 10 minutes;

[0272] ④ Immerse in Fast Green staining solution for 5 minutes;

[0273] ⑤ Quickly wash the sections with a weak acid solution for 10-15 seconds;

[0274] ⑥ Immerse in safranin staining solution for 5 minutes;

[0275] ⑦ Dehydrate with graded alcohol, clear with a transparent agent, and seal with neutral resin.

[0276] 6.2.1.4 Biochemical Indicator Detection

[0277] Elisa serum TNF-α, IL-1β, IL-6, and IL-8 levels: After administration, rats in each group were weighed, fasted for 12 hours but allowed free access to water, anesthetized with 1 mL / kg of 3% sodium pentobarbital, blood was collected from the abdominal aorta, the serum was collected by centrifugation, and stored at -80℃ for later use. The levels of TNF-α, IL-1β, IL-6, and IL-8 in the serum were measured strictly according to the kit instructions.

[0278] 6.2.2 Results of in vivo activity evaluation

[0279] 6.2.2.1 Behavioral Indicators Assessment

[0280] The diameter of the rat knee joint was measured using vernier calipers (see Table 20 and 20). Figure 10 The study found that, compared with the control group, the knee joint diameter of rats in all groups was significantly increased on days 21 and 7 before administration (P<0.01). On day 7 after administration, compared with the control group, the knee joint diameter of rats in the model group was significantly increased (P<0.01), while the knee joint diameter of rats in other groups decreased compared with before administration, but was still significantly increased compared with the control group (P<0.01). On day 28 after administration, compared with the control group, the knee joint diameter of rats in the model group was significantly increased (P<0.01), while there was no significant difference in knee joint diameter among other groups (P>0.05). These results indicate that the traditional Chinese medicine granule preparation can significantly reduce the degree of knee joint swelling in rats. Measurement of the knee joint diameter in rats demonstrates that the traditional Chinese medicine granule preparation can alleviate knee joint swelling in rats.

[0281] Table 20 Comparison of knee joint diameter in rats of different dosage groups ( mm, n=10)

[0282]

[0283] Note: Compared with the control group, #p<0.05, ##p<0.01.

[0284] Compare the changes in weight-bearing on the right leg of rats in different groups at the same time point (see Table 21 and 2023). Figure 11On days 7, 14, and 30 after modeling, compared with the control group, the weight-bearing ratio of the affected limb in other groups was significantly reduced (P<0.01). Compared with the model group, there was no significant difference in weight-bearing ratio between the treatment groups and the model group (P>0.05). On days 45 and 60, the weight-bearing ratio of the positive group and each treatment group of the traditional Chinese medicine granules was significantly increased compared with the model group (P<0.01). On day 60, there was no significant difference between the positive group and each treatment group of the traditional Chinese medicine granules and the control group (P>0.05). The results indicate that the traditional Chinese medicine granules can significantly increase the weight-bearing ratio of the hind limbs of rats and alleviate hind limb pain. The weight-bearing ratio of the hind limbs of rats measured by a bipedal balance pain tester can reflect the pain symptoms of the hind limbs of rats, indicating that the traditional Chinese medicine granules can alleviate the pain of the hind limbs of rats.

[0285] Table 21 Comparison of weight-bearing capacity of rats in different dose groups ( ) mm, n=10)

[0286]

[0287]

[0288] Note: Compared with the model group, *p<0.05, **p<0.01.

[0289] 6.2.2.2 Joint Pathological Examination

[0290] Micro-CT observation results: Compared with the blank group, the model group rats showed a significant decrease in the number of trabeculae, irregular arrangement, sparse distribution, and thinner thickness, indicating a change in trabecular bone structure; compared with the model group, the number of trabeculae in each group treated with traditional Chinese medicine granules increased, the arrangement became more orderly, the distribution became denser, and the thickness increased. Figure 12 Compared with the control group, the model group showed significantly decreased bone volume (BV), bone volume fraction (BV / TV), and trabecular thickness (Tb.Th) (P<0.05). However, the ratio of bone surface area to bone volume (BS / BV), trabecular pattern factor (Tb.Pf), structural pattern index (SMI), total porosity (Po(tot)), and trabecular separation (Tb.Sp) were significantly increased (P<0.015). In contrast, the traditional Chinese medicine granule preparations showed significantly decreased trabecular pattern factor (Tb.Pf), structural pattern index (SMI), trabecular separation (Tb.Sp), total porosity (Po(tot)), and ratio of bone surface area to bone volume (BS / BV) (P<0.05). Meanwhile, bone volume fraction (BV / TV) and trabecular thickness (Tb.Th) were significantly increased (P<0.05). There were no significant differences in trabecular bone number (Tb.N) and bone volume (BV) between the model group and each of the compound Eucommia ulmoides treatment groups (P>0.05). Figure 13 AI, Table 22).

[0291] Table 22 Micro-CT analysis results of subchondral bone microstructure morphological parameters in different dose groups (n=6, )

[0292]

[0293] Note: Compared with the blank group, # P<0.05, ## P < 0.01. Compared with the model group, * P<0.05, ** P<0.01.

[0294] Histopathological changes in the subchondral bone of KOA rats were observed using HE staining. In the control group, the joint structure was intact, the cartilage layer was intact, and the contrast between bone and cartilage tissue was clear. In the model group, the joint structure was disrupted, the cartilage was severely damaged, and inflammatory cells showed significant infiltration. Compared with the model group, the traditional Chinese medicine granule preparation group and the diacerein group showed reduced inflammatory cell infiltration, improved cartilage thickness, and significantly improved joint structure. (See...) Figure 14 (AF) In safranin-fast green staining, safranin dye binds to cartilage and turns it red, while fast green dye binds to bone tissue and appears green or blue, which contrasts sharply with the red cartilage, thus distinguishing cartilage tissue from bone tissue.

[0295] As shown in the figures, the joint structures of rats in the blank control group were intact and smooth, with uniform matrix staining. After staining, the cartilage showed a clear color distribution, regular chondrocyte arrangement, and a clear contrast between bone and cartilage tissues, with intact tide lines. In the model group, the joint structures of rats were damaged, with some cartilage defects, cartilage loss of staining, loss of cartilage matrix, and no change in color hierarchy. The contrast between bone and cartilage tissues was blurred. In contrast, the joint structures of rats in all groups treated with the traditional Chinese medicine granules were clear, the cartilage showed slightly deeper safranin staining, still retained layers, the cell arrangement was slightly more regular, and the contrast between bone and cartilage tissues was more obvious. Figure 15 AF).

[0296] 6.2.2.3 Biochemical Indicator Detection

[0297] In rats with osteoarthritis, the levels of TNF-α, IL-1β, IL-6, and IL-8 were elevated. We measured the levels of pro-inflammatory cytokines in rat serum. The results showed that the protein expression of TNF-α, IL-1β, IL-6, and IL-8 was significantly increased in the model group (P<0.01). However, the traditional Chinese medicine granule preparation could reduce the levels of TNF-α, IL-1β, IL-6, and IL-8 (P<0.01). Figure 16 The results suggest that traditional Chinese medicine granules have a significant anti-inflammatory effect on rats with osteoarthritis.

[0298] VII. Clinical Trial Research

[0299] To further verify the clinical efficacy of the herbal granule preparation of this invention, this study employed a multicenter, randomized, controlled scientific research method, enrolling 120 patients with osteoporosis of the kidney deficiency and blood stasis type for a systematic clinical study. The aim was to objectively evaluate the efficacy and safety of this preparation in clinical application based on rigorous evidence-based medicine. The 120 patients were randomly divided into two groups: Experimental group: Calcium carbonate D3 (each tablet containing 600mg calcium / vitamin D3), 1 tablet, orally, once daily; and the herbal granule preparation of this invention (each 10g sachet), 1 sachet, orally, twice daily. Control group: Calcium carbonate D3 (each tablet containing 600mg calcium / vitamin D3), 1 tablet, orally, once daily. Treatment lasted 6 months, with a follow-up of 1 year.

[0300] The results showed that after treatment, the VAS pain score, bone metabolism marker levels (P1NP, CTX), and TCM syndrome scores of both groups were significantly lower than before treatment, and the treatment group showed a significantly higher decrease than the control group (P<0.05). After treatment, the levels of BMD, LH, E2, FSH, and ECOS quality of life scores of both groups were significantly higher than before treatment, and the treatment group showed a significantly higher increase than the control group (P<0.05). No adverse reactions were observed. This indicates that the granule formulation of the present invention can effectively improve bone mineral density, regulate bone metabolism, improve TCM syndromes, increase muscle strength, improve balance, prevent falls, and improve quality of life, and is both effective and safe.

[0301] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features, all of which should be included within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for treating chronic rheumatism, characterized in that, The traditional Chinese medicine composition is composed of rhizoma drynariae 4-50 parts, dracaena 3-30 parts, ligustrum lucidum aiton 1-20 parts, caulis spatholobi 1-20 parts, pyrola 1-20 parts, and radix cyathulae 1-20 parts.

2. The traditional Chinese medicine composition for treating chronic rheumatism according to claim 1, characterized in that, The traditional Chinese medicine composition is composed of rhizoma drynariae 4-50 parts, dracaena 3-30 parts, ligustrum lucidum aiton 1-20 parts, caulis spatholobi 1-20 parts, pyrola 1-20 parts, and radix cyathulae 1-20 parts. 3.The traditional Chinese medicine composition for treating chronic rheumatism according to claim 2, characterized in that, The traditional Chinese medicine composition is composed of rhizoma drynariae 4-50 parts, dracaena 3-30 parts, ligustrum lucidum aiton 1-20 parts, caulis spatholobi 1-20 parts, pyrola 1-20 parts, and radix cyathulae 1-20 parts.

4. The traditional Chinese medicine composition for treating chronic rheumatism according to any one of claims 1 to 3, characterized in that, The radix cyathulae is replaced by radix cyathulae.

5. A method for preparing the traditional Chinese medicine composition according to claim 4, characterized in that, The method comprises the following steps: S1, mixing the raw materials of the composition, decocting twice with water, and adding more water in the first time than in the second time according to the amount of the raw materials; S2, combining the filtrates obtained by decocting twice to perform vacuum concentration twice to obtain clear paste and extract paste; S3, mixing the dried extract paste, clear paste and excipient to obtain the traditional Chinese medicine granule preparation.

6. The production method according to claim 5, wherein In S1, 10-15 times of water is added for decocting 1.0-2.5 hours in the first time, and 8-12 times of water is added for decocting 1.0-2.5 hours in the second time.

7. The production method according to claim 6, wherein In S1, 11 times of water is added for decocting 1.5 hours in the first time, and 9 times of water is added for decocting 1.5 hours in the second time.

8. The preparation method according to claim 5, characterized in that, In S2, the concentration comprises one-effect concentration and two-effect concentration, and the concentration temperature is 60-80 ℃.

9. The preparation method according to claim 5, characterized in that, The relative density of the clear paste is 1.15-1.25, and the relative density of the extract paste is 1.30-1.

35.

10. The method of claim 5, wherein, In S3, the excipient comprises wetting agent and filler, the filler is dextrin and lactose, and the granule is obtained by whole granulation, sieving and total mixing.

11. The method of claim 10, wherein, The ratio of the dried extract paste to the filler is 1:0.6-1.0, wherein the ratio of the dried extract paste, lactose and dextrin is 6.25:2.5:1.

25.

12. A pharmaceutical preparation, characterized in that, The effective component is composed of the traditional Chinese medicine composition of claim 4, or the traditional Chinese medicine composition prepared by the preparation method of any one of claims 6-10.

13. The pharmaceutical preparation according to claim 12, characterized in that The pharmaceutical preparation further comprises pharmaceutically acceptable carrier; and the dosage form of the pharmaceutical preparation is mixture, tincture, tablet, powder, oral liquid, capsule, granule, powder, emulsion or pill.

14. The pharmaceutical preparation according to claim 13, characterized in that The tablet is buccal tablet, and the pill is drop pill.

15. The traditional Chinese medicine composition of claim 4, or the traditional Chinese medicine composition prepared by the preparation method of claim 5, or the pharmaceutical preparation of claim 12, is used for preparing medicine for treating osteoarthritis.

16. The traditional Chinese medicine composition of claim 4, or the traditional Chinese medicine composition prepared by the preparation method of claim 5, or the pharmaceutical preparation of claim 12, is used for preparing medicine for treating osteoporosis.

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