An external traditional Chinese medicine preparation for arthritis and a preparation method thereof

Abstract

The application provides a kind of external use traditional Chinese medicine preparation for arthritis and its preparation method, it is related to traditional Chinese medicine pharmaceutical technical field, the external use traditional Chinese medicine preparation is composed of ten traditional Chinese medicines such as radix paeoniae alba, equus asinus, radix rubi, radix curdlans, caesalpinia sappan, rhizoma dipsaci, honeycomb, salix matsudana koidz, selaginella, honeyed licorice.The preparation of the application has significant anti-inflammatory effect, can inhibit the secretion of key proinflammatory factors such as IL-6, IL-1 beta of MH7A cell induced by TNF-alpha, effectively reverse the inflammatory activation state of synoviocyte;In addition, the preparation of the application can reduce the swelling rate of knee joint of KOA model rat, improve gait and paw pressure score, reduce local inflammatory edema of joint;At the same time, the preparation can down-regulate the expression level of TNF-alpha, IL-6, IL-1 beta in serum, reduce the content of MMP-3 and COMP, block the inflammatory cascade amplification reaction from the source, inhibit the excessive degradation of cartilage matrix, delay the progression of cartilage degeneration.The traditional Chinese medicine preparation of the application has good drug safety, provides a safe and effective new choice for the treatment of arthritis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine pharmaceutical technology, specifically to a topical traditional Chinese medicine preparation for arthritis and its preparation method. Background Technology

[0002] Arthritis is a chronic degenerative joint disease characterized by progressive degeneration of articular cartilage, sclerosis and remodeling of subchondral bone, osteophyte formation at joint margins, and synovitis. Clinically, it is mainly manifested by joint pain, swelling, morning stiffness, difficulty in flexion and extension, and limited range of motion. The disease is protracted and recurrent, and in the terminal stage, it can lead to joint deformity and complete loss of function. It is one of the leading causes of limb disability in middle-aged and elderly people.

[0003] Modern medicine believes that the occurrence and development of arthritis are related to multiple factors, including mechanical stress injury, inflammatory factor mediation, cartilage matrix metabolic imbalance, chondrocyte apoptosis, and oxidative stress. Its pathogenesis is complex, and there is currently no cure. Clinical treatment is generally divided into three categories: non-pharmacological intervention, pharmacological treatment, and surgical intervention. In terms of pharmacological treatment, non-steroidal anti-inflammatory drugs (NSAIDs) are currently the most widely used drugs, with representative drugs including ibuprofen, naproxen, and celecoxib, playing an important role in clinical practice. However, long-term use of NSAIDs has significant limitations: gastrointestinal adverse reactions are prominent, manifesting as nausea, vomiting, abdominal pain, and diarrhea; in severe cases, it can lead to gastrointestinal ulcers, perforation, and even bleeding. Some NSAIDs can also damage liver and kidney function and increase the risk of adverse cardiovascular events. More importantly, NSAIDs can only relieve clinical symptoms and cannot delay or reverse the degenerative changes in articular cartilage. While intra-articular injections of sodium hyaluronate or corticosteroids can improve symptoms in the short term, the effects are limited in duration, and repeated injections may increase the risk of joint infection and even accelerate cartilage damage. Joint replacement surgery, as an end-stage treatment, can resolve functional impairments in some patients, but it is highly invasive, expensive, and some patients still experience unsatisfactory joint function recovery post-surgery.

[0004] Traditional Chinese medicine (TCM) categorizes arthritis under the terms "Bi syndrome," "bone Bi," and "knee Bi," believing its pathogenesis to be rooted in deficiency of vital energy (Qi) and exacerbated by external pathogenic factors. Blood stasis, phlegm stagnation, and Qi stagnation are considered important pathological products, which intertwine and obstruct the meridians. Prolonged illness penetrates these meridians, ultimately leading to joint pain, stiffness, deformity, and even loss of function. TCM treatment of arthritis adheres to a holistic approach and the principle of syndrome differentiation and treatment, possessing unique advantages in multi-component, multi-target, and multi-pathway intervention. It exerts therapeutic effects through multiple pathways, including anti-inflammatory analgesia, immunomodulation, inhibition of chondrocyte apoptosis, and promotion of cartilage matrix repair. Modern pharmacological research further confirms that various Chinese herbs and their active ingredients can regulate inflammation and cartilage metabolism-related signaling pathways at the molecular level, effectively delaying the pathological progression of arthritis. However, existing traditional Chinese medicine (TCM) preparations for treating arthritis still have significant shortcomings: most formulas cover only a single pathogenesis, focusing primarily on tonifying the liver and kidneys and dispelling wind and cold, lacking sufficient intervention in the core pathogenesis of phlegm-blood stasis, muscle and bone stagnation, and meridian obstruction in mid-to-late stage patients. They also offer limited relief for patients with joint stiffness and deformities, nerve entrapment pain, and severe limitation of movement, failing to effectively slow disease progression. Therefore, there is an urgent need to develop a well-formulated, comprehensive, effective, and safe arthritis treatment. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a topical traditional Chinese medicine preparation for arthritis and its preparation method.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a topical traditional Chinese medicine preparation for arthritis, which is composed of the following raw materials in parts by weight: 10-30 parts of white peony root, 5-25 parts of *Zou Ma Tai* (a type of herb), 5-25 parts of *Tou Gu Cao* (another herb), 10-30 parts of *Chuan Po Shi* (another herb), 3-20 parts of sappanwood, 3-20 parts of *Xu Die* (another herb), 3-15 parts of processed bee honeycomb, 3-20 parts of *Han Liu Ye* (another herb), 3-20 parts of *Juan Bai* (another herb), and 3-20 parts of processed licorice root.

[0010] Preferably, the external Chinese medicine preparation is composed of the following raw materials in parts by weight: 15-25 parts of white peony root, 10-20 parts of horse fetus, 10-20 parts of clematis root, 15-25 parts of stone-cutting stone, 8-16 parts of sappanwood, 5-15 parts of dipsacus root, 3-12 parts of processed bee honeycomb, 5-15 parts of willow leaf, 8-16 parts of selaginella, and 5-15 parts of processed licorice root.

[0011] Preferably, the external Chinese medicine preparation is composed of the following raw materials in parts by weight: 20 parts of white peony root, 15 parts of horse fetus, 15 parts of clematis root, 20 parts of stone-cutting stone, 12 parts of sappanwood, 10 parts of dipsacus root, 8 parts of processed bee honeycomb, 10 parts of willow leaf, 12 parts of selaginella, and 10 parts of processed licorice root.

[0012] This invention provides a method for preparing the aforementioned topical Chinese medicine preparation. The preparation is carried out according to conventional Chinese medicine manufacturing methods, with the addition of excipients, and is prepared into a topical dosage form, including ointments, patches, liniments, powders, tinctures, and sprays.

[0013] Preferably, the preparation of the traditional Chinese medicine spray includes the following steps:

[0014] (1) Raw material preparation: Weigh each raw medicinal material according to the above weight proportions, clean and select them, and then grind them into coarse powder for later use;

[0015] (2) Extraction and concentration: Add 8-10 times the amount of purified water to the above crude powder, soak for 30-60 minutes, then decoct twice, each time for 1.5-2 hours. Combine the two decoctions, filter and concentrate under reduced pressure to a fluid extract with a relative density of 1.10-1.20 (measured at 60℃).

[0016] (3) Alcohol precipitation purification: Add 95% ethanol to the fluid extract obtained in step (2) to make the alcohol content reach 60%-70%, let stand for 12-24 hours, filter, and recover the ethanol until there is no alcohol taste to obtain the purified extract.

[0017] (4) Add 40-70 parts of glycerol and 20-50 parts of propylene glycol to the purified extract obtained in step (3) and stir until completely dissolved; take 5-20 parts of Tween-80, dissolve it in water and slowly add it to the above drug solution, and stir evenly; then add 5-20 parts of azone, and dissolve 0.1-1.0 parts of ethylparaben in an appropriate amount of 70-80℃ hot water and let it cool before adding; add purified water to make up to 1000 parts, stir continuously for 15-20 min until homogeneous, filter through a 0.22 μm microporous membrane, and fill into a spray bottle to obtain the product.

[0018] This invention provides the application of the aforementioned topical traditional Chinese medicine preparation in the preparation of a drug for treating arthritis.

[0019] The properties, flavors, meridian tropisms, and pharmacological effects of each herb in the herbal preparation of this invention are as follows:

[0020] White peony root: The dried root of Paeonia lactiflora Pall., a plant in the Ranunculaceae family. It tastes bitter and sour, and is slightly cold in nature. It enters the liver and spleen meridians. It has the effects of nourishing blood and regulating menstruation, astringing yin and stopping sweating, softening the liver and relieving pain, and calming liver yang. It is used for blood deficiency and chlorosis, irregular menstruation, spontaneous sweating, night sweats, hypochondriac pain, abdominal pain, limb spasms, headache, and dizziness.

[0021] Rhizome of *Ardisia gigantifolia* Stapf, a plant in the Myrsinaceae family. It has a bitter and slightly pungent taste, and is warm in nature. It enters the liver, spleen, and kidney meridians. It has the effects of dispelling wind and dampness, strengthening muscles and bones, and promoting blood circulation and removing blood stasis. It is used for rheumatic pain in muscles and bones, traumatic injuries, postpartum blood stasis, carbuncles, and ulcers.

[0022] *Speranskia tuberculata* (Bunge) Baill., a plant belonging to the genus *Speranskia* in the family Euphorbiaceae. It has a pungent and bitter taste, and is warm in nature. It enters the liver and kidney meridians. It has the effects of dispelling wind, eliminating dampness, relaxing muscles and tendons, promoting blood circulation, and relieving pain. It is used for rheumatic pain, muscle and bone contractures, cold-damp beriberi, and sores and boils.

[0023] Piercing Stone: The root of *Maclura cohinchinensis* (Lour.) Kudo et Masam. and *M. tricuspidata* Carr., both belonging to the Moraceae family. It has a slightly bitter taste and cool properties. It possesses the effects of relieving cough and phlegm, dispelling wind and dampness, and dispersing blood stasis and relieving pain. It is used for pulmonary tuberculosis, jaundice-type hepatitis, hepatosplenomegaly, gastric and duodenal ulcers, and rheumatic lumbago; externally, it is used to treat fractures and traumatic injuries.

[0024] Sappanwood: The dried heartwood of *Caesalpinia sappan* L., a plant in the legume family. It has a sweet and salty taste, and is neutral in nature. It enters the heart, liver, and spleen meridians. It has the effects of promoting blood circulation, removing blood stasis, reducing swelling, and relieving pain. It is used for traumatic injuries, fractures, muscle injuries, swelling and pain due to blood stasis, amenorrhea, dysmenorrhea, postpartum blood stasis, chest and abdominal pain, and carbuncles and boils.

[0025] Dipsacus asperoides root: The dried root of *Dipsacus asperoides* Wall. ex Henry, a plant in the Dipsacaceae family. It tastes bitter and pungent, and is slightly warm in nature. It enters the liver and kidney meridians. It has the effects of tonifying the liver and kidneys, strengthening tendons and bones, healing fractures, and stopping metrorrhagia. It is used for liver and kidney deficiency, soreness and weakness of the lower back and knees, rheumatic pain, injuries from falls, tendon injuries and fractures, metrorrhagia, and threatened abortion. Wine-processed Dipsacus asperoides is mostly used for rheumatic pain, injuries from falls, and tendon injuries and fractures. Salt-processed Dipsacus asperoides is mostly used for soreness and weakness of the lower back and knees.

[0026] Roasted bee nest: A processed product of the nests of the fruit wasp *Polistes olivaceus* (DeGeer), the Japanese long-legged wasp *Polistes jokahamae* Radoszkowski, or the parapolybia varia (Fabricius). It has a sweet taste and neutral properties. It enters the stomach meridian. It has the effects of attacking toxins and killing parasites, dispelling wind and relieving pain. It is used for sores and carbuncles, mastitis, scrofula, stubborn skin diseases, tinea pedis, toothache, and rheumatic pain.

[0027] Salix matsudana leaves: The tender leaves, branches, or bark of Salix matsudana Koidz., a plant in the Salicaceae family. They are bitter and cold in nature. They have the effects of clearing heat and dampness, dispelling wind and relieving pain. They are used for jaundice, acute cystitis, dysuria, arthritis, impetigo, sores, and toothache.

[0028] Selaginella tamariscina (Beauv.) Spring or Selaginella pulvinata (Hook. et Grev.) Maxim., belonging to the Selaginellaceae family. It has a pungent taste and neutral properties. It enters the liver and heart meridians. It has the effects of promoting blood circulation and regulating menstruation. It is used for amenorrhea, dysmenorrhea, abdominal masses, and injuries from falls. Carbonized Selaginella tamariscina can stop bleeding. It is used for hematemesis, metrorrhagia, hematochezia, and rectal prolapse.

[0029] Prepared licorice root: A processed product of the dried roots and rhizomes of *Glycyrrhiza uralensis* Fisch., *Glycyrrhiza inflata* Bat., or *Glycyrrhiza glabra* L. (all belonging to the Fabaceae family). It has a sweet taste and neutral properties. It enters the heart, lung, spleen, and stomach meridians. It tonifies the spleen and stomach, replenishes qi, and restores the pulse. It is used for spleen and stomach weakness, fatigue, palpitations, and irregular pulse.

[0030] (III) Beneficial Effects

[0031] This invention provides a topical traditional Chinese medicine preparation for arthritis and its preparation method. The topical traditional Chinese medicine preparation consists of ten traditional Chinese medicines: white peony root, *Zou Ma Tai* (a type of herb), *Tou Gu Cao* (another herb), *Chuan Po Shi* (a type of herb), *Su Mu* (a type of herb), *Xu Dipsacus* (a type of herb), *Zhi Feng Fang* (a type of herb), *Han Liu Ye* (a type of herb), *Juan Bai* (a type of herb), and *Zhi Gan Cao* (a type of herb). White peony root and *Zou Ma Tai* are the principal herbs in the formula, one nourishing and the other clearing, directly targeting the core pathogenesis of arthritis. White peony root is bitter and sour, entering the liver and spleen meridians, and its function is to nourish blood, soften the liver, and relieve pain. The sourness can astringe yin and soften tendons, while the sweetness can relieve pain, perfectly aligning with the principle of "treating wind by first treating blood, for when blood flows smoothly, wind will naturally subside." *Zou Ma Tai* is bitter and pungent, entering the liver and kidney meridians, and its function is to dispel wind and dampness, invigorate blood, relieve pain, and strengthen tendons and bones. It can both dispel wind, cold, and dampness externally and clear stagnant meridians internally, while also addressing the root cause of arthritis by tonifying the liver and kidneys and strengthening tendons and bones. The combination of these two herbs, one gentle and one firm, one nourishing and one unblocking, works synergistically to soothe the liver, relieve pain, dispel wind and dampness, and strengthen tendons and bones, directly addressing the core pathogenesis of arthritis. The assistant herbs, *Clematis chinensis*, *Scutellaria baicalensis*, and *Caesalpinia sappan*, specifically target the superficial symptoms, assisting the principal herbs in enhancing their ability to relieve pain and alleviate numbness. *Clematis chinensis* is pungent and sweet, warm in nature, and readily penetrates to the tendons and bones, dispelling wind and dampness, relaxing muscles and tendons, promoting blood circulation, and relieving pain. It guides the medicine directly to the affected area, enhancing the principal herbs' ability to unblock meridians and relieve pain. *Scutellaria baicalensis* is slightly bitter and cool in nature, dispelling wind and dampness, dispersing blood stasis, and relieving pain. It helps the principal herbs to unblock meridians, dissipate stagnation, and clear the meridians of chronic arthritis, enhancing the overall blood-activating and pain-relieving effects of the formula. *Caesalpinia sappan* is sweet and salty, neutral in nature, entering the blood aspect of the heart, liver, and spleen meridians. It effectively activates blood circulation, removes blood stasis, reduces swelling, and relieves pain, specifically entering the blood to disperse stagnation, reduce swelling, and relieve pain, assisting the principal herbs in enhancing their blood-activating and pain-relieving effects. When used together, the three herbs work synergistically: *Clematis chinensis* dispels wind and dampness to unblock the meridians; *Smilax china* dispels wind and dampness to unblock the channels; and *Sappanwood* invigorates blood and removes blood stasis to relieve pain. Together, they assist the principal herbs in dispelling wind and dampness, invigorating blood and unblocking the meridians, thus relieving the symptoms of joint pain. *Dipsacus asper*, *Bee honeycomb*, *Salix matsudana* leaves, and *Selaginella tamariscina* are added as adjuvants, combining tonification and purgation. *Dipsacus asper* excels at tonifying the liver and kidneys, strengthening tendons and bones, and healing fractures, assisting the principal herbs in tonifying the liver and kidneys to strengthen the foundation and strengthening tendons and bones to treat the symptoms. *Bee honeycomb* dispels wind and relieves pain, attacks toxins and disperses nodules, and clears the meridians. Its penetrating nature allows it to penetrate deep into the joints, dispelling pathogens, clearing the meridians, dispersing nodules, and relieving pain, assisting the principal and adjuvant herbs in overcoming stubborn pain. *Salix matsudana* leaves clear heat and dampness, dispel wind and relieve pain, addressing the concurrent symptoms of prolonged pain and stagnation leading to heat, clearing damp-heat, detoxifying, and reducing swelling, thus helping the entire formula clear damp-heat from the joints. *Selaginella tamariscina* invigorates blood, unblocks the meridians, and removes blood stasis, targeting the symptoms of blood stasis obstruction, assisting the principal and adjuvant herbs in enhancing the power of invigorating blood and unblocking the meridians. The four herbs work together to both strengthen the body's resistance and eliminate pathogenic factors, dispelling wind and dampness without harming the body's vital energy, and invigorating blood circulation without depleting yin and blood. This achieves a holistic effect, addressing both the root cause and the symptoms. Prepared licorice root is used as the guiding herb to harmonize the entire formula and direct its flow to the affected area. Prepared licorice root is sweet and neutral in nature, capable of tonifying the spleen and replenishing qi, relieving spasms and pain, and harmonizing the effects of the other herbs. Combined with the principal herb, white peony root, its sweet and sour properties nourish yin, soften tendons and relieve spasms, enhancing the analgesic effect. It also harmonizes the formula's properties, balancing the cold and warm, tonifying and purging, and dispersing and astringent effects, ensuring that each herb performs its function without conflict, promoting circulation without being harsh, and tonifying without causing stagnation. Furthermore, it guides the herbs to their respective meridians, harmonizing the herbs to reach the affected area directly, achieving a comprehensive therapeutic effect.

[0032] In vitro experiments have verified that the traditional Chinese medicine preparation of this invention has no significant toxic effect on human rheumatoid synovial cells MH7A at a concentration of 200 μg / mL, and has good drug safety. At the same time, the preparation can significantly inhibit the secretion of key pro-inflammatory factors such as IL-6 and IL-1β induced by TNF-α in MH7A cells, and effectively reverse the inflammatory activation state of synovial cells.

[0033] This invention's traditional Chinese medicine preparation can reduce knee joint swelling in KOA model rats induced by the modified Hulth method, significantly improve gait and claw pressure scores, rapidly alleviate local inflammatory edema in the joint, and restore the affected limb's normal weight-bearing walking ability. Simultaneously, this preparation can significantly downregulate the expression levels of key pro-inflammatory factors such as TNF-α, IL-6, and IL-1β in serum, and concurrently reduce the serum levels of key cartilage degradation enzyme MMP-3 and cartilage damage-specific marker COMP. It blocks the inflammatory cascade amplification reaction at its source, prevents continuous inflammatory infiltration of the synovium and cartilage tissue, directly inhibits excessive degradation of the cartilage matrix, and delays the cartilage degeneration process, achieving a triple progressive effect of "reducing swelling, anti-inflammation, and protecting cartilage," providing a safe and effective new option for the clinical treatment of arthritis. Attached Figure Description

[0034] Figure 1 The knee joint swelling rate and joint function of rats in each group were compared; among them, compared with the Sham group, # P<0.05, ## P<0.01; compared with the KOA group, * P<0.05, ** P<0.01.

[0035] Figure 2 The levels of serum inflammatory factors and cartilage metabolic markers in rats from different groups were compared; among them, compared with the Sham group, # P<0.05, ## P<0.01; compared with the KOA group, * P<0.05, ** P<0.01. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Example 1

[0038] A topical Chinese medicine preparation for arthritis, the topical Chinese medicine preparation being composed of the following raw materials in parts by weight: 20 parts of white peony root, 15 parts of horsetail, 15 parts of clematis root, 20 parts of stonewort, 12 parts of sappanwood, 10 parts of dipsacus root, 8 parts of processed bee honeycomb, 10 parts of willow leaf, 12 parts of selaginella, and 10 parts of processed licorice root.

[0039] The external Chinese medicine preparation is prepared according to conventional Chinese medicine manufacturing methods, using the above-mentioned weight proportions of Chinese medicinal materials as raw materials and excipients, into an external dosage form, which includes ointments, patches, liniments, powders, tinctures, and sprays.

[0040] The preparation method of the external application Chinese medicine spray includes the following steps:

[0041] (1) Raw material preparation: Weigh each raw medicinal material according to the above weight proportions, clean and select them, and then grind them into coarse powder for later use;

[0042] (2) Extraction and concentration: Add 8-10 times the amount of purified water to the above crude powder, soak for 30-60 minutes, then decoct twice, each time for 1.5-2 hours. Combine the two decoctions, filter and concentrate under reduced pressure to a fluid extract with a relative density of 1.10-1.20 (measured at 60℃).

[0043] (3) Alcohol precipitation purification: Add 95% ethanol to the fluid extract obtained in step (2) to make the alcohol content reach 60%-70%, let stand for 12-24 hours, filter, and recover the ethanol until there is no alcohol taste to obtain the purified extract.

[0044] (4) Add 50 parts of glycerol and 30 parts of propylene glycol to the purified extract obtained in step (3) and stir until completely dissolved; take 5 parts of Tween-80, dissolve it in water and slowly add it to the above drug solution, and stir evenly; then add 10 parts of azone, and dissolve 0.5 parts of ethylparaben in an appropriate amount of 70-80℃ hot water and let it cool before adding; add purified water to make up to 1000 parts, stir continuously for 15-20 min until homogeneous, filter through a 0.22 μm microporous membrane, and fill into a spray bottle to obtain the product.

[0045] Example 2

[0046] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is composed of the following raw materials in parts by weight: 10 parts of white peony root, 5 parts of horse fetus, 5 parts of clematis root, 10 parts of stone-cutting stone, 3 parts of sappanwood, 3 parts of dipsacus root, 3 parts of roasted bee honeycomb, 3 parts of willow leaf, 3 parts of selaginella, and 3 parts of roasted licorice root.

[0047] Example 3

[0048] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is composed of the following raw materials in parts by weight: 30 parts of white peony root, 25 parts of horse fetus, 25 parts of clematis root, 30 parts of stone-cutting stone, 20 parts of sappanwood, 20 parts of dipsacus root, 15 parts of processed bee honeycomb, 20 parts of willow leaf, 20 parts of selaginella, and 20 parts of processed licorice root.

[0049] Example 4

[0050] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is composed of the following raw materials in parts by weight: 15 parts of white peony root, 10 parts of horse fetus, 10 parts of clematis root, 15 parts of stone-cutting stone, 8 parts of sappanwood, 5 parts of teasel root, 3 parts of processed bee honeycomb, 5 parts of willow leaf, 8 parts of selaginella, and 5 parts of processed licorice root.

[0051] Example 5

[0052] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is composed of the following raw materials in parts by weight: 25 parts of white peony root, 20 parts of horse fetus, 20 parts of clematis root, 25 parts of stone-cutting stone, 16 parts of sappanwood, 15 parts of dipsacus root, 12 parts of processed bee honeycomb, 15 parts of willow leaf, 16 parts of selaginella, and 15 parts of processed licorice root.

[0053] Example 6

[0054] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is composed of the following raw materials in parts by weight: 12 parts of white peony root, 8 parts of horse fetus, 7 parts of clematis root, 12 parts of stone-cutting stone, 6 parts of sappanwood, 4 parts of teasel root, 3 parts of processed bee honeycomb, 4 parts of willow leaf, 7 parts of selaginella, and 4 parts of processed licorice root.

[0055] Example 7

[0056] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is composed of the following raw materials in parts by weight: 28 parts of white peony root, 22 parts of horse fetus, 23 parts of clematis root, 27 parts of stone-cutting stone, 18 parts of sappanwood, 17 parts of dipsacus root, 13 parts of processed bee honeycomb, 18 parts of willow leaf, 18 parts of selaginella, and 17 parts of processed licorice root.

[0057] Experimental Example 1

[0058] 1. Materials and Methods

[0059] 1.1 Cell lines

[0060] The human rheumatoid arthritis fibroblast synovial cell line MH7A was purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0061] 1.2 Test Drug

[0062] The formulation composition of each group of test drugs is shown in Table 1. Drug preparation method: Weigh the medicinal materials according to the formulation proportions for each group, add 8 times the volume of purified water and soak for 30 minutes, then decoct twice, 1.5 hours each time. Combine the two decoctions and concentrate to a crude drug concentration of 1 g / mL. Filter through a 0.22 μm microporous membrane for sterilization and store at 4℃ for later use. Dilute to the required concentration with RPMI-1640 complete culture medium before use.

[0063] Table 1. Composition of the test drug prescriptions in each group

[0064] Traditional Chinese medicine preparations White peony root 20g, *Zouma fetus* 15g, *Tougucao* 15g, *Chuanposhi* 20g, *Caesalpinia sappan* 12g, *Xuduan* 10g, *Zhifengfang* 8g, *Hanliuye* 10g, *Juepai* 12g, *Zhigancao* 10g. Group 1 of test substances White peony root 20g, calamus 15g, clematis root 15g, clematis root 20g, sappanwood 12g, dipsacus root 10g, roasted bee honeycomb 8g, roasted licorice root 10g. Group 2 of test substances 15g of Clematis armandii, 20g of Scutellaria baicalensis, 12g of Sappanwood, 10g of Dipsacus asper, 8g of processed bee honeycomb, 10g of Salix matsudana, 12g of Selaginella tamariscina, and 10g of processed licorice root. Group 3 of test substances White peony root 20g, horse fetus 15g, sappanwood 12g, teasel root 10g, roasted bee honeycomb 8g, willow leaf 10g, selaginella tamariscina 12g, roasted licorice root 10g. Group 4 of test substances White peony root 20g, *Zoumatai* 15g, *Tougucao* 15g, *Chuanposhi* 20g, *Hanliuye* 10g, *Juanbai* 12g, *Zhigancao* 10g.

[0065] 1.3 Methods

[0066] 1.3.1 Cell Culture

[0067] MH7A cells were cultured in RPMI-1640 complete medium containing 10% FBS and 1% penicillin antibiotics at 37°C in a 5% CO2 incubator. When the cells reached 80%-90% confluence, they were passaged using 0.25% trypsin, and cells in the logarithmic growth phase were used for experiments.

[0068] 1.3.2 Experimental grouping and drug administration

[0069] The experiment included a blank control group, a model control group, a traditional Chinese medicine preparation group, and test substance groups 1-4, with three replicates for each group. MH7A cells in logarithmic growth phase were harvested and cultured at a density of 4 × 10⁴ cells / well. 4 Cells were seeded at a density of 10 cells / well in 96-well plates and cultured for 24 hours until adherence. Except for the blank control group, all other groups were induced to establish a cell inflammation model by adding recombinant human TNF-α at a final concentration of 10 ng / mL. After 24 hours of stimulation, each drug-treated group was replaced with 100 μL of complete culture medium containing the corresponding drug (final concentration of 200 μg / mL), while the blank control group and the model control group were given an equal volume of complete culture medium. After culturing for another 24 hours, the cell supernatant of each group was collected.

[0070] 1.4 Detection Indicators

[0071] 1.4.1 Cell Viability

[0072] The effect of each test drug on TNF-α-induced MH7A cell viability was detected using the CCK-8 assay. After drug administration and culture, 10 μL of CCK-8 solution was added to each well, and incubation was continued for 2 h. The absorbance at 450 nm was measured using a microplate reader, and the relative cell viability of each group was calculated based on the blank control group.

[0073] 1.4.2 Levels of inflammatory cytokine secretion

[0074] Collect cell culture supernatant from each group and strictly follow the ELISA kit instructions to detect the levels of IL-6 and IL-1β in the cell supernatant of each group.

[0075] 1.5 Statistical Analysis

[0076] Data analysis was performed using SPSS 26.0 statistical software. Quantitative data were expressed as mean ± standard deviation (Mean ± SD), and one-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0077] 2 Results

[0078] 2.1 Cell viability

[0079] The cell survival rates of each group are shown in Table 2. Compared with the blank control group, there was no significant difference in cell survival rates between the model control group and each drug-treated group (P>0.05), indicating that each tested drug had no significant toxicity to MH7A cells at a concentration of 200 μg / mL and the drug was safe.

[0080] Table 2 Comparison of cell survival rates in each group (%)

[0081] Blank control group 100.00±0.00 Model control group 98.79±6.64 Traditional Chinese medicine preparations 102.09±5.48 Group 1 of test substances 97.60±3.02 Group 2 of test substances 99.67±4.95 Group 3 of test substances 100.64±5.21 Group 4 of test substances 96.61±4.14

[0082] 2.2 Inflammatory factor levels

[0083] The levels of inflammatory factors in the cell supernatant of each group are shown in Table 3. Compared with the blank control group, the levels of IL-6 and IL-1β in the cell supernatant of the model control group were significantly increased (P<0.01). Compared with the model control group, the levels of IL-6 and IL-1β in the traditional Chinese medicine preparation group were significantly decreased (P<0.01). The levels of IL-6 and IL-1β in test substance groups 1-4 were also significantly decreased (P<0.05), but the decrease was smaller than that in the traditional Chinese medicine preparation group.

[0084] Table 3 Comparison of inflammatory factor levels in cell supernatant of each group (pg / mL)

[0085] Blank control group 124.48±10.51 50.69±5.94 Model control group <![CDATA[532.26±14.80 ## ]]> <![CDATA[142.72±6.61 ## ]]> Traditional Chinese medicine preparations <![CDATA[167.40±12.24 ** ]]> <![CDATA[68.48±3.96 ** ]]> Group 1 of test substances <![CDATA[240.12±17.47 ** ]]> <![CDATA[89.51±4.03 ** ]]> Group 2 of test substances <![CDATA[429.27±14.99 ** ]]> <![CDATA[129.13±7.16 * ]]> Group 3 of test substances <![CDATA[389.14±18.45 ** ]]> <![CDATA[103.74±5.68 ** ]]> Group 4 of test substances <![CDATA[310.60±13.83 ** ]]> <![CDATA[92.81±3.40 ** ]]>

[0086] Note: Compared with the blank control group ## P<0.01; compared with the model control group, * P<0.05, ** P<0.01.

[0087] 3. Conclusion

[0088] This study used a TNF-α-induced MH7A cell inflammation model as a vector to systematically evaluate the in vitro anti-inflammatory activity and safety of the traditional Chinese medicine preparation of this invention. The results showed that the traditional Chinese medicine preparation of this invention had no significant toxic effect on cells at a concentration of 200 μg / mL, demonstrating good drug safety. Simultaneously, the traditional Chinese medicine preparation of this invention significantly inhibited the secretion of key pro-inflammatory factors such as IL-6 and IL-1β induced by TNF-α in MH7A cells, effectively reversing the inflammatory activation state of synovial cells, providing experimental evidence for the clinical treatment of arthritis. Furthermore, further analysis revealed that the anti-inflammatory effect of the traditional Chinese medicine preparation of this invention is the result of the synergistic effect of multiple medicinal materials; the absence of any one of these materials weakened the overall anti-inflammatory effect, reflecting the scientific and holistic characteristics of the "principal, assistant, adjuvant, and guide" principle of traditional Chinese medicine formulation.

[0089] Experimental Example 2

[0090] 1. Materials and Methods

[0091] 1.1 Laboratory Animals

[0092] Sixty healthy SPF-grade SD rats, half male and half female, aged 6-8 weeks and weighing 200±20 g, were purchased from Henan Spekes Biotechnology Co., Ltd. The rats were housed in an environment with a temperature of 22±2℃, humidity of 50±10%, and a 12h light / 12h dark cycle, with free access to water and food, and their bedding was changed daily. Experiments began after one week of acclimatization.

[0093] 1.2 Preparation of the test drug solution

[0094] The traditional Chinese medicine preparation of this invention is prepared according to the prescription ratio (each part by weight is 1g) and preparation method described in Example 1.

[0095] Positive control 1, Tianhe Zhuifeng Ointment, manufacturer: Guilin Huarun Tianhe Pharmaceutical Co., Ltd., main ingredients: raw aconite, raw Sichuan aconite, ephedra, asarum, notopterygium, angelica pubescens, clematis, safflower, frankincense, myrrh, Guangxi dragon's blood, cinnamon oil, borneol, menthol, etc., national drug approval number: Z45021872.

[0096] Positive control 2, diclofenac sodium ointment, manufacturer: Beijing Novartis Pharmaceutical Co., Ltd., national drug approval number H20020176, specification: 15g:0.15g (as C 14 H 10 (Calculated using Cl2NNaO2).

[0097] 1.3 Methods

[0098] 1.3.1 Experimental Grouping and KOA Model Construction

[0099] Rats were randomly divided into 6 groups using a random number table: sham operation group (Sham group), model group (KOA group), traditional Chinese medicine preparation group (ZYZJ), positive control group 1 (TianHe) and positive control group 2 (Diclofenac group), with 10 rats in each group.

[0100] A modified Hulth method was used to establish a knee osteoarthritis model. Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (30 mg / kg), fixed in a supine position, and the right hind limb knee joint was prepared and disinfected with povidone-iodine. A longitudinal incision of approximately 1.5 cm was made on the medial side of the knee joint, and the subcutaneous tissue and fascia were dissected layer by layer to expose the knee joint capsule. The joint capsule was opened, the patella was dislocated laterally, the knee joint was flexed, the anterior cruciate ligament was exposed, and the anterior cruciate ligament was transected under direct vision, followed by subtotal resection of the medial meniscus. After confirming the anterior cruciate ligament rupture by the drawer test, the joint cavity was irrigated with saline, and the joint capsule, fascia, and skin were sutured layer by layer. Postoperatively, penicillin 80,000 U / day was administered intramuscularly for 3 consecutive days to prevent infection. In the Sham group, only the joint capsule was opened and then sutured layer by layer, without transecting the ligament or resecting the meniscus. Postoperatively, all rats were housed individually and allowed free movement. The rats' mental state, diet, and local joint condition were observed daily.

[0101] 1.3.2 Administration

[0102] On day 7 after modeling, the circumference of the right knee joint of the rats was measured and gait scores were performed. Rats in the modeling group showed significant knee swelling and limping, indicating successful modeling, and drug intervention was initiated. All rats in each group received topical medication on the right knee joint. Before administration, the hair around the right knee joint was shaved, exposing approximately 2×2 cm of skin. The specific protocols for each group were as follows: the herbal preparation of this invention was administered as a spray, two puffs each time, followed by natural cooling; the TianHe group received topical application, one TianHe Zhuifeng plaster (cut to approximately 2×2 cm) each time; the Diclofenac group received topical application, 0.1 g of diclofenac ointment each time. Administered once daily for 4 consecutive weeks.

[0103] 1.4 Observation Indicators

[0104] 1.4.1 Joint swelling

[0105] Before modeling and after drug administration, the circumference of the widest part of the patella of the right knee joint of rats was measured using electronic vernier calipers, and the knee joint swelling rate was calculated.

[0106] Swelling rate = (Circumference after drug administration - Circumference before modeling) / Circumference before modeling × 100%

[0107] 1.4.2 Joint Function

[0108] After drug administration, gait score and paw pressure weight-bearing score were used to assess the knee joint function of rats.

[0109] Gait score (0-3 points): 0 points, normal walking, no limping; 1 point, mild limping, the affected limb touches the ground slightly, and the stride is slightly small; 2 points, moderate limping, the affected limb bears some weight, and walking is obviously sluggish; 3 points, severe limping, the affected limb cannot bear weight at all, and walking requires lifting the foot.

[0110] Claw pressure rating (0-3 points): The hind paw landing shape is observed through mirror reflection. 0 points: the whole palm is on the ground and the force is evenly distributed; 1 point: part of the paw is on the ground and the force is slightly reduced; 2 points: only the paw tip is on the ground and the force is significantly reduced; 3 points: completely suspended in the air and not on the ground.

[0111] 1.4.3 Detection of serum inflammatory factors and cartilage metabolic markers

[0112] After the behavioral tests, all rats were deeply anesthetized by intraperitoneal injection of 3% sodium pentobarbital (50 mg / kg), and blood was collected from the abdominal aorta. After standing at room temperature for 30 min, the blood was centrifuged at 3000 r / min for 15 min at 4℃, and the supernatant serum was separated. The serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), matrix metalloproteinase-3 (MMP-3), and chondroitin oligomeric matrix protein (COMP) were detected by enzyme-linked immunosorbent assay (ELISA).

[0113] 1.4.4 Statistical Analysis

[0114] Data analysis was performed using GraphPad Prism 8.0 statistical software. Quantitative data were expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups, and LSD-t tests were used for pairwise comparisons between groups. A p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered statistically significant.

[0115] 2 Results

[0116] 2.1 Comparison of knee joint swelling in rats of different groups

[0117] Depend on Figure 1 It can be seen that, compared with the Sham group, the swelling rate of the knee joint of rats in the KOA group was significantly increased, and the difference was statistically significant (P<0.01); compared with the KOA group, the swelling rate of each treatment group was significantly reduced (P<0.05, P<0.01), among which the swelling rate of the ZYZJ group was lower than that of the TianHe group and slightly higher than that of the Diclofenac group, but the difference was not statistically significant (P>0.05), indicating that the traditional Chinese medicine preparation of the present invention can effectively alleviate the joint swelling symptoms of rats with knee arthritis.

[0118] 2.2 Comparison of joint function scores among different groups of rats

[0119] Depend on Figure 1 It can be seen that, compared with the Sham group, the gait score and claw pressure score of the KOA group rats were significantly increased (P<0.01), and the rats showed obvious lameness and weight-bearing impairment. Compared with the KOA group, the gait score and claw pressure score of each treatment group were significantly decreased (P<0.05, P<0.01), among which the ZYZJ group showed a greater degree of improvement than the TianHe group, indicating that the traditional Chinese medicine preparation of the present invention can improve the joint activity and weight-bearing function of KOA rats.

[0120] 2.3 Comparison of serum inflammatory factors and cartilage metabolic markers in different groups of rats

[0121] Depend on Figure 2 It was found that, compared with the Sham group, the serum levels of TNF-α, IL-6, and IL-1β in the KOA group were significantly increased (P<0.01), and the levels of MMP-3 and COMP were also significantly increased (P<0.01), indicating that the KOA model rats had a significant inflammatory response and cartilage matrix degradation. Compared with the KOA group, the serum levels of TNF-α, IL-6, IL-1β, MMP-3, and COMP in each treatment group were significantly decreased (P<0.01), with the ZYZJ group showing a greater degree of reduction than the TianHe group. These results indicate that the traditional Chinese medicine preparation of this invention can effectively inhibit joint inflammation and cartilage degeneration by downregulating the expression of pro-inflammatory cytokines and cartilage-degrading enzymes.

[0122] 3. Conclusion

[0123] The results of this study showed that the herbal preparation of this invention significantly reduced the knee joint swelling rate in rats, and significantly improved gait and claw pressure scores. Serological tests showed that the levels of TNF-α, IL-6, IL-1β, MMP-3, and COMP were significantly reduced in all treatment groups. These results indicate that the herbal preparation of this invention has a significant therapeutic effect on the modified Hulth method-induced KOA model rats, effectively reducing joint swelling, improving joint function, inhibiting the expression of inflammatory factors, and delaying cartilage degeneration.

[0124] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A topical traditional Chinese medicine preparation for arthritis, characterized in that, The external Chinese medicine preparation is composed of the following raw materials in parts by weight: 10-30 parts of white peony root, 5-25 parts of horse fetus, 5-25 parts of clematis root, 10-30 parts of stone-cutting stone, 3-20 parts of sappanwood, 3-20 parts of dipsacus root, 3-15 parts of processed bee honeycomb, 3-20 parts of willow leaf, 3-20 parts of selaginella, and 3-20 parts of processed licorice root.

2. The external application traditional Chinese medicine preparation according to claim 1, characterized in that, The external Chinese medicine preparation is composed of the following raw materials in parts by weight: 15-25 parts of white peony root, 10-20 parts of horse fetus, 10-20 parts of clematis root, 15-25 parts of stone-cutting stone, 8-16 parts of sappanwood, 5-15 parts of teasel root, 3-12 parts of processed bee honeycomb, 5-15 parts of willow leaf, 8-16 parts of selaginella, and 5-15 parts of processed licorice root.

3. The external application traditional Chinese medicine preparation according to claim 1, characterized in that, The external Chinese medicine preparation is composed of the following raw materials in parts by weight: 20 parts of white peony root, 15 parts of horse fetus, 15 parts of clematis root, 20 parts of stone-cutting stone, 12 parts of sappanwood, 10 parts of dipsacus root, 8 parts of processed bee honeycomb, 10 parts of willow leaf, 12 parts of selaginella, and 10 parts of processed licorice root.

4. The method for preparing the external application traditional Chinese medicine preparation according to any one of claims 1 to 3, characterized in that, The external Chinese medicine preparations are prepared into external dosage forms by adding excipients according to conventional Chinese medicine manufacturing methods. The external dosage forms include ointments, patches, liniments, powders, tinctures, and sprays.

5. The preparation method according to claim 4, characterized in that, The preparation of the traditional Chinese medicine spray includes the following steps: (1) Raw material preparation: Weigh each raw medicinal material according to the above weight proportions, clean and select them, and then grind them into coarse powder for later use; (2) Extraction and concentration: Add 8-10 times the amount of purified water to the above crude powder, soak for 30-60 minutes, then decoct twice, each time for 1.5-2 hours. Combine the two decoctions, filter and concentrate under reduced pressure to a fluid extract with a relative density of 1.10-1.

20. (3) Alcohol precipitation purification: Add 95% ethanol to the fluid extract obtained in step (2) to make the alcohol content reach 60%-70%, let stand for 12-24 hours, filter, and recover the ethanol until there is no alcohol taste to obtain the purified extract. (4) Add 40-70 parts of glycerol and 20-50 parts of propylene glycol to the purified extract obtained in step (3) and stir until completely dissolved; take 5-20 parts of Tween-80, dissolve it in water and slowly add it to the above drug solution, and stir evenly; then add 5-20 parts of azone, and dissolve 0.1-1.0 parts of ethylparaben in an appropriate amount of 70-80℃ hot water and let it cool before adding; add purified water to make up to 1000 parts, stir continuously for 15-20 min until homogeneous, filter through a 0.22 μm microporous membrane, and fill into a spray bottle to obtain the product.

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