An external traditional Chinese medicine preparation for removing blood stasis and reducing swelling and a preparation method thereof

Abstract

The application discloses a kind of external use traditional Chinese medicine preparation for removing blood stasis and reducing swelling and a preparation method thereof, and belongs to the technical field of traditional Chinese medicine.The preparation is made of the following raw medicinal materials: Fengxian Tougucao, Qiyelian, Gushugupu, San Duiye Danshen, Niuma Teng, Bai Niuchan, Hantao Leaf, Huosuo Ma, Yeshangzhu and Yuejihua.The ten kinds of medicines are matched with precision, and jointly play the functions of removing wind and dampness, reducing swelling and relieving pain, promoting blood circulation to remove blood stasis and tonifying kidney and strengthening bone, to form a unique prescription with special curative effect.The preparation has remarkable curative effect in inhibiting inflammation, improving joint function and protecting chondrocytes, and is especially suitable for osteoarthritis, which provides a theoretical basis for improving patient symptoms and delaying disease progression.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, specifically to a topical traditional Chinese medicine preparation for removing blood stasis and reducing swelling, and its preparation method. Background Technology

[0002] Knee osteoarthritis (KOA) is a chronic degenerative joint disease characterized by degeneration and destruction of articular cartilage and bone hyperplasia. Clinically, it mainly manifests as progressively worsening joint pain and limited mobility. Its incidence gradually increases with age, and in severe cases, it can lead to permanent disability, seriously affecting the patient's physical and mental health. Because articular cartilage lacks regenerative capacity, the disease often progresses continuously. Current treatments primarily focus on symptom relief, but long-term oral nonsteroidal anti-inflammatory drugs (NSAIDs) pose a risk of gastrointestinal damage and cannot reverse cartilage damage. End-stage patients often require joint replacement surgery.

[0003] Traditional Chinese medicine (TCM) treatment for knee osteoarthritis (KOA) possesses the characteristics of "multi-target and holistic regulation," showing significant advantages in analgesia, anti-inflammation, delaying cartilage degeneration, and improving microcirculation, enabling comprehensive conditioning. Clinically, graded and personalized treatment should be provided according to the patient's condition to alleviate symptoms and maintain good joint function. To maximize efficacy and shorten the recovery process, effective external TCM therapies are essential. TCM external therapies, through holistic diagnosis, are characterized by definite efficacy and high safety in treating KOA. However, several problems remain in clinical implementation: the clinical practice and theoretical system of TCM external therapies are still immature, related research lacks rigorous experimental design, and there is limited exploration of the mechanism of action. Therefore, it is necessary to continuously improve the rigorous efficacy evaluation system of TCM external therapies to provide a scientific and objective basis for further exploration and innovation, aiming to provide more ideas and methods for the clinical diagnosis and care of knee osteoarthritis using TCM. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a topical traditional Chinese medicine preparation for removing blood stasis and reducing swelling, and its preparation method.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] On the one hand, a topical Chinese medicine preparation for removing blood stasis and reducing swelling, wherein the Chinese medicine preparation is made from the following Chinese herbal medicines in parts by weight: 10-40 parts of Impatiens balsamina, 8-25 parts of Achyranthes bidentata, 10-45 parts of Dryopteris crassirhizoma, 5-25 parts of Salvia miltiorrhiza, 5-25 parts of Achyranthes bidentata, 1-18 parts of Ophiopogon japonicus, 1-10 parts of Prunus persica leaf, 1-18 parts of Hedyotis diffusa, 1-20 parts of Phyllanthus urinaria, and 1-10 parts of Rosa rugosa.

[0009] Furthermore, the traditional Chinese medicine preparation is made from the following parts by weight of Chinese herbs as raw materials: 15-30 parts of Impatiens balsamina, 12-20 parts of Achyranthes bidentata, 12-30 parts of Dryopteris crassirhizoma, 8-20 parts of Salvia miltiorrhiza, 8-20 parts of Achyranthes bidentata, 3-12 parts of Ox Gallbladder, 3-8 parts of Prunus persica leaf, 3-12 parts of Hedyotis diffusa, 3-12 parts of Phyllanthus urinaria, and 3-8 parts of Rosa rugosa.

[0010] Furthermore, the traditional Chinese medicine preparation is made from the following parts by weight of Chinese herbs: 20 parts of Impatiens balsamina, 15 parts of Achyranthes bidentata, 15 parts of Eucommia ulmoides, 10 parts of Salvia miltiorrhiza trifoliata, 10 parts of Achyranthes bidentata, 5 parts of Ophiopogon japonicus, 5 parts of Prunus persica leaf, 5 parts of Hemp seed, 5 parts of Phyllanthus urinaria, and 5 parts of Rosa rugosa.

[0011] On the other hand, the present invention provides the application of a topical Chinese medicine preparation with the effect of removing blood stasis and reducing swelling in the preparation of a drug for treating or improving osteoarthritis, wherein the topical Chinese medicine preparation is a Chinese medicine tincture product.

[0012] Furthermore, the method for preparing the traditional Chinese medicine tincture for treating or improving osteoarthritis is carried out according to the following steps:

[0013] (1) Take the above-mentioned Chinese herbal medicine raw materials, clean and dry them, and then coarsely crush each herb to obtain various herb coarse powders for later use;

[0014] (2) Take the coarse powder of Chinese medicinal materials from step (1) and 75% alcohol, add them to a light-proof wide-mouth glass bottle in a ratio of 1:5 to 1:8, seal the container, store for one month, filter out the liquid, and repackage it in a ball bottle, spray bottle or sealed glass bottle for later use.

[0015] The herbal tincture of this invention is prepared from Chinese herbal medicines. The sources, efficacy, and indications of the Chinese herbal medicines are described below:

[0016] Impatiens balsamina L., a plant belonging to the genus Impatiens in the family Balsamicaceae, is a type of herb. It has a bitter and pungent taste, and is neutral in nature; it enters the liver and kidney meridians. It has the effects of dispelling wind and dampness, promoting blood circulation, and relieving pain. It is mainly used to treat rheumatic pain and traumatic injuries.

[0017] Seven-leaf arboricola: The root or stem and leaves of *Heptapleurum arboricola* Hayata, a plant belonging to the genus *Heptapleurum* in the family Araliaceae. It has a pungent and slightly bitter taste and is warm in nature. It possesses the effects of dispelling wind and relieving pain, promoting blood circulation and reducing swelling. It is commonly used for rheumatic pain, headache, toothache, abdominal pain, dysmenorrhea, postpartum abdominal pain, traumatic swelling and pain, fractures, and sores.

[0018] Drynaria Rhizome: It is the dried rhizome of Drynaria fortunei (Kunze) J.Sm., a plant of the genus Drynaria in the family Polypodiaceae. It has the effects of treating injuries and relieving pain, tonifying the kidney and strengthening bones. It is mainly used for treating falls and contusions, tendon and bone fractures, kidney deficiency low back pain, flaccidity of muscles and bones, tinnitus and deafness, loose teeth, alopecia areata, and vitiligo.

[0019] Salvia miltiorrhiza var. trijugata: It is a perennial herb of the genus Salvia in the family Lamiaceae. It tastes bitter and slightly sweet, is warm in nature, and belongs to the liver, heart, and kidney meridians. It has the effects of promoting blood circulation and regulating menstruation, removing blood stasis and generating new blood, tonifying the kidney and calming the mind. It is mainly used for treating irregular menstruation, dysmenorrhea, kidney deficiency low back pain, and traumatic injuries.

[0020] Evergreen Mucuna Stem: It is the stem of Mucuna sempervirens Hemsl., a plant of the family Fabaceae. It tastes sweet and slightly bitter, is warm in nature, and belongs to the liver and stomach meridians. It has the effects of promoting blood circulation and regulating menstruation, tonifying blood and relaxing tendons. It is commonly used for irregular menstruation, dysmenorrhea, amenorrhea, postpartum blood deficiency, anemia, rheumatic arthralgia, numbness of the limbs, and traumatic injuries.

[0021] Inula Root: It is the whole herb of Inula cappa (Buch.-Ham.) DC. It has the effects of expelling wind, promoting diuresis, regulating qi, and resolving stagnation. It is used to treat rheumatic joint pain, chest oppression, malaria, dysentery, diarrhea, postpartum cold, hepatitis, hemorrhoids, and scabies.

[0022] Schefflera kwangsiensis Stem: It is the dried stem branch or stem branch with leaves of Schefflera kwangsiensis Merr. ex Li, a plant of the family Araliaceae. It tastes slightly bitter and astringent, is warm in nature, and belongs to the liver and stomach meridians. It has the effects of expelling wind and relieving pain, relaxing tendons and activating collaterals. It is mainly used for rheumatic arthralgia, low back and leg pain, headache, toothache, and traumatic pain.

[0023] Helicteres hirsuta Root: It is the dried root of Helicteres hirsuta Lour., with a mild taste of bitterness and a neutral nature. Its root can be collected throughout the year, washed, sliced, dried, and used as medicine. It has the effect of regulating qi and relieving pain, and is mainly used for chronic gastritis and gastric ulcer.

[0024] Helwingia Leaf or Fruit: It is the leaf or fruit of Helwingia japonica (Thunb.) Dietr., Helwingia himalaica Hook. f. et Thoms. ex C. B. Clarke, and Helwingia chinensis Batal. of the family Cornaceae. It tastes bitter and pungent, is neutral in nature, and belongs to the lung and heart meridians. It has the effects of expelling wind and dampness, promoting blood circulation and detoxifying. It is used for cold and cough, rheumatic arthralgia, stomachache, dysentery, hematochezia, irregular menstruation, traumatic swelling and stasis, fracture, carbuncle and furuncle, and snake bite.

[0025] Rose: The dried flower of *Rosa chinensis* Jacq., a plant belonging to the genus *Rosa* in the family Rosaceae. It has a sweet taste and warm nature, and enters the liver meridian. It has the effects of promoting blood circulation and regulating menstruation, soothing the liver and relieving depression. It is mainly used to treat qi stagnation and blood stasis, irregular menstruation, dysmenorrhea, amenorrhea, and chest and rib pain.

[0026] (III) Beneficial Effects

[0027] The core pathogenesis of knee osteoarthritis lies in the obstruction of meridians by pathogenic factors, leading to poor circulation of qi and blood. The principal ingredient in this formula is *Impatiens balsamina*, which dispels wind and dampness, invigorates blood, and relieves pain. Its pungent and dispersing properties open the pores, guiding the medicinal effects directly to the deepest parts of the affected area, penetrating the bones and tendons. The secondary ingredients are *Symplocos rubra*, *Prunus persica* leaves, and *Bletilla striata* gall. *Symplocos rubra* is specifically effective in dispelling wind and relieving pain; *Prunus persica* leaves are good at relaxing muscles and tendons; and *Bletilla striata* gall is effective in dispelling wind and dampness. These three herbs work synergistically to eliminate lingering wind, cold, and dampness in the meridians and joints, collectively acting as the main force in relieving pain. The adjuvant herbs are administered in three ways: First, a combination to invigorate blood and regulate qi, using *Salvia miltiorrhiza* (three-leaf salvia) to invigorate blood, remove blood stasis, promote metabolism, and calm the mind and benefit the kidneys; *Achyranthes bidentata* (codonopsis) is particularly good at relaxing muscles and invigorating blood, assisting *Salvia miltiorrhiza* in clearing stagnation and enhancing the circulation of new blood; *Rosa chinensis* (rose) invigorates blood and relieves depression and regulates qi, the combined effects of these blood-invigorating herbs are specifically for treating "swelling due to blood stasis" in arthralgia, which is localized swelling, hardening, bluish-purple discoloration, and stinging pain caused by "blood outside the meridians." Once the stagnation is dispersed, the meridians become unobstructed, the swelling subsides, and new blood is generated, which is the fundamental treatment of removing blood stasis to reduce swelling; *Hypericum perforatum* (firethorn) promotes qi circulation, relieves pain, and opens the muscles and pores, alleviating numbness and difficulty in flexion and extension of the limbs. The four herbs work together to regulate qi and blood, nourishing the muscles and bones and making the joints smooth. Secondly, it serves a cooling and balancing function. The bitter and pungent taste of the *Dryopteris crassirhizoma* leaf clears heat and swelling caused by stagnation, while also counteracting the warming and dispersing effects of *Achyranthes bidentata*, *Rosa rugosa*, and *Prunus persica* leaves. This ensures that the entire formula, while warming and dispersing, avoids the risk of internal heat generation or damage to the collaterals. By slightly adjusting the proportions of *Achyranthes bidentata*, *Prunus persica* leaves, and *Rosa rugosa* in clinical practice, it can address the complex course of osteoarthritis, characterized by "mixed cold and heat, and alternating acute and chronic symptoms," making the entire formula suitable for various types of swelling and pain. Thirdly, it contains *Dryopteris crassirhizoma*, which tonifies the kidneys, strengthens bones, invigorates blood, and dispels wind. Among a large group of herbs that attack pathogens and clear the collaterals, it uniquely possesses a tonifying effect, combining dispersing with tonifying, ensuring that the elimination of pathogens does not harm the body's vital energy. It reduces swelling and improves joint mobility, while also nourishing degenerated tendons and bones, delaying joint aging. The entire formula works synergistically to dissipate swelling and restore bone and tendon suppleness, making it an excellent method for external treatment of arthralgia.

[0028] The wonder of this external treatment lies in its adherence to the principle of "removing blood stasis and reducing swelling." Firstly, it penetrates the affected area, leveraging the power of Impatiens balsamina and Clematis armandii, whose "penetrating" nature is crucial. This allows the medicinal power to break through the skin barrier, reaching directly the bones, tendons, and veins, and aiding in the transdermal absorption of other medications, thus paving the way for the dissipation of blood stasis and swelling. Secondly, the assistant herbs work together to expel pathogenic factors, dispersing stagnation, wind, cold, and dampness. With the removal of stagnation, the meridians are unblocked, and with unblocked meridians, swelling subsides—this is the key to removing blood stasis and reducing swelling. Thirdly, it employs a combination of warming and dispersing herbs to attack pathogenic factors and cooling herbs to prevent complications. This not only resolves local redness and swelling caused by blood stasis but also allows for slight adjustments to the proportions of herbs like Ox Gallbladder according to the specific symptoms. This adapts to the complex course of osteoarthritis, characterized by "mixed cold and heat, and alternating acute attacks and chronic remissions," making the entire formula applicable to various types of swelling and pain. The addition of bone-strengthening herbs enhances the body's ability to dispel pathogens, relieve pain, promote blood circulation, and unblock meridians, while also providing nourishment. This allows the body to reduce swelling and remove blood stasis without harming its vital energy. Furthermore, the combination of tendon-relaxing herbs such as *Achyranthes bidentata* and *Prunus persica* leaves creates a synergy between "softening tendons" and "aligning bones," ultimately achieving the effects of eliminating swelling and blood stasis, nourishing tendons and bones, and making joints smooth and flexible.

[0029] This traditional Chinese medicine preparation possesses the effects of dispelling wind and dampness, clearing the meridians and relieving pain, promoting blood circulation and removing blood stasis, and tonifying the kidneys and strengthening bones. Through in vitro pharmacodynamic screening, the optimal formulation and dosage form were determined. This preparation can significantly downregulate inflammatory factors and protect the cartilage microenvironment. Further intervention in animal models of knee osteoarthritis showed that it can relieve pain, stiffness, and swelling, improve joint function, inhibit inflammation, activate chondrocytes, and promote matrix synthesis and repair, demonstrating significant efficacy and providing patients with a better treatment option. Attached Figure Description

[0030] Figure 1 The levels of inflammatory markers IL-6 (A) and TNF-α (B) and the protein expression levels of Col II (C) and MMP13 (D) in cells of each group were compared with those of the control group. * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01; compared with the topical Chinese medicine preparation group, & P<0.05, && P<0.01.

[0031] Figure 2 The levels of inflammatory markers IL-6 (A), IL-1β (B), and TNF-α (C) in the serum of rats in each group, as well as the Krenn (D) and Mankin (E) histopathological scores of rat tissue, were compared with those of the control group. * P<0.05, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01; compared with the topical Chinese medicine preparation group, & P<0.05,&& P<0.01. Detailed Implementation

[0032] 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.

[0033] Example 1

[0034] A topical Chinese medicine preparation for removing blood stasis and reducing swelling is made from the following Chinese herbal raw materials in the indicated weights: 40g of Impatiens balsamina, 25g of Achyranthes bidentata, 45g of Dryopteris crassirhizoma, 25g of Salvia miltiorrhizae, 25g of Achyranthes bidentata, 18g of Ox Gallbladder, 10g of Prunus persica leaf, 18g of Hedyotis diffusa, 20g of Phyllanthus urinaria, and 10g of Rosa rugosa.

[0035] The preparation method of traditional Chinese medicine tincture is as follows:

[0036] (1) Take the above-mentioned Chinese herbal medicine raw materials, clean and dry them, and then coarsely crush each herb to obtain various herb coarse powders for later use;

[0037] (2) Take the coarse powder of Chinese medicinal materials from step (1) and 75% alcohol, add them to a light-proof wide-mouth glass bottle in a ratio of 1:6, seal the container, store for one month, filter out the liquid, and repackage it in a ball bottle, spray bottle or sealed glass bottle for later use.

[0038] Example 2

[0039] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is made from the following herbal raw materials by weight: 10g of Impatiens balsamina, 8g of Achyranthes bidentata, 10g of Eucommia ulmoides, 5g of Salvia miltiorrhiza var. trifoliata, 5g of Achyranthes bidentata, 1g of Ox Gallbladder, 1g of Prunus persica leaf, 1g of Hemp seed, 1g of Phyllanthus urinaria, and 1g of Rosa rugosa.

[0040] Example 3

[0041] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is made from the following Chinese herbal raw materials by weight: 40g of Impatiens balsamina, 25g of Achyranthes bidentata, 45g of Dryopteris crassirhizoma, 25g of Salvia miltiorrhizae, 25g of Achyranthes bidentata, 18g of Ox Gallbladder, 10g of Prunus persica leaf, 18g of Hemp seed, 20g of Phyllanthus urinaria, and 10g of Rosa rugosa.

[0042] Example 4

[0043] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is made from the following Chinese herbal raw materials by weight: 15g of Impatiens balsamina, 12g of Achyranthes bidentata, 12g of Eucommia ulmoides, 8g of Salvia miltiorrhiza var. trifoliata, 8g of Achyranthes bidentata, 3g of Ox Gallbladder, 3g of Prunus persica leaf, 3g of Hemp seed, 3g of Phyllanthus urinaria, and 3g of Rosa rugosa.

[0044] Example 5

[0045] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is made from the following Chinese herbal raw materials by weight: 30g of Impatiens balsamina, 20g of Achyranthes bidentata, 30g of Eucommia ulmoides, 20g of Salvia miltiorrhiza var. trifoliata, 20g of Achyranthes bidentata, 12g of Ox Gallbladder, 8g of Prunus persica leaf, 12g of Hedyotis diffusa, 12g of Phyllanthus urinaria, and 8g of Rosa rugosa.

[0046] Example 6

[0047] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is made from the following Chinese herbal raw materials by weight: 35g of Impatiens balsamina, 22g of Achyranthes bidentata, 38g of Dryopteris crassirhizoma, 23g of Salvia miltiorrhizae, 23g of Achyranthes bidentata, 16g of Ox Gallbladder, 9g of Prunus persica leaf, 16g of Hedyotis diffusa, 16g of Phyllanthus urinaria, and 9g of Rosa rugosa.

[0048] Example 7

[0049] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is made from the following Chinese herbal raw materials by weight: 13g of Impatiens balsamina, 10g of Achyranthes bidentata, 35g of Eucommia ulmoides, 6g of Salvia miltiorrhiza var. trifoliata, 6g of Achyranthes bidentata, 14g of Ox Gallbladder, 2g of Prunus persica leaf, 10g of Hedyotis diffusa, 10g of Phyllanthus urinaria, and 2g of Rosa rugosa.

[0050] Example 8

[0051] The difference between this embodiment and Embodiment 1 is that the external Chinese medicine preparation is made from the following Chinese herbal raw materials by weight: 20g of Impatiens balsamina, 15g of Achyranthes bidentata, 15g of Eucommia ulmoides, 10g of Salvia miltiorrhiza var. trifoliata, 10g of Achyranthes bidentata, 5g of Ox Gallbladder, 5g of Prunus persica leaf, 5g of Hemp seed, 5g of Phyllanthus urinaria, and 5g of Rosa rugosa.

[0052] Experiment 1

[0053] 1 Experimental Methods

[0054] 1.1 Experimental Cells

[0055] Human articular chondrocytes were purchased from Wuhan Pronosei Life Science Co., Ltd., catalog number CP-H096.

[0056] 1.2 Experimental Drugs

[0057] Traditional Chinese medicine preparation: 20g of Impatiens balsamina, 15g of Achyranthes bidentata, 15g of Dryopteris crassirhizoma, 10g of Salvia miltiorrhiza var. sanguisorba officinalis, 10g of Achyranthes bidentata, 5g of Ophiopogon japonicus, 5g of Prunus persica leaf, 5g of Hedyotis diffusa, 5g of Phyllanthus urinaria, and 5g of Rosa rugosa.

[0058] Preparation 2: 15g of Heptaphyllum japonicum, 15g of Dryopteris crassirhizoma, 10g of Salvia miltiorrhizae, 10g of Achyranthes bidentata, 5g of Ophiopogon japonicus, 5g of Prunus persica leaves, 5g of Hemp seed, 5g of Phyllanthus urinaria, and 5g of Rosa rugosa.

[0059] Preparation 3: 20g of Impatiens balsamina, 15g of Luffa cylindrica, 15g of Drynaria fortunei, 10g of Salvia miltiorrhiza var. salviae, 10g of Achyranthes bidentata, 5g of Ox gall, 5g of Prunus persica leaf, 5g of Hemp seed, 5g of Phyllanthus urinaria, and 5g of Rosa rugosa.

[0060] Preparation method of decoction: Weigh each raw material according to the above weight ratio, clean and select them, add 8 times the amount of purified water of the total amount of medicinal materials, and soak for 30 minutes; first bring to a boil over high heat, then reduce to low heat and maintain a gentle boil, and decoct twice; combine the two decoctions, filter through a 200-mesh sieve, collect the filtrate, and discard the dregs; combine the filtrates and concentrate under reduced pressure until the raw drug content is 4g / mL to obtain the original Chinese medicine solution of each group.

[0061] Preparation of drug-containing serum: Twelve female SD rats, 6-8 weeks old, were randomly divided into four groups: blank control group, traditional Chinese medicine preparation group, preparation group 2, and preparation group 3. Except for the blank control group, the other groups were administered the corresponding drug at 20 g / kg / day by gavage. The blank control group was administered the same volume of physiological saline by gavage once a day for 8 days. Within 2 hours after the last administration, blood was collected from the anesthetized animals, serum was separated, and inactivated and labeled at 56℃ for later use.

[0062] 1.3 Experimental grouping and modeling methods

[0063] Control group: Logarithmic growth phase cells were pretreated with culture medium containing 10% blank rat serum for 2 hours, and then cultured in normal culture medium for 24 hours.

[0064] Model group: Logarithmic growth phase cells were pretreated with culture medium containing 10% blank rat serum for 2 h, and then cultured with 10 ng / ml IL-1β for 24 h.

[0065] Traditional Chinese medicine preparation (ZYZJ): Take cells in the logarithmic growth phase, pre-treat them with a culture medium containing 10% serum from the traditional Chinese medicine preparation for 2 hours, and then add 10 ng / ml IL-1β and culture for 24 hours.

[0066] Formulation 2 (ZJ2): Take logarithmic growth phase cells, pretreat them with culture medium containing 10% Formulation 2 serum for 2 hours, and then add 10 ng / ml IL-1β and culture for 24 hours.

[0067] Formulation 3 (ZJ3): Take cells in the logarithmic growth phase, pretreat them for 2 hours with a culture medium containing 10% Formulation 3 serum, and then add 10 ng / ml IL-1β and culture for 24 hours.

[0068] 1.4 Detection Indicators

[0069] 1.4.1 Levels of inflammatory factors IL-6 and TNF-α in cell supernatant

[0070] Cells were processed according to the method described in "1.3" above. After processing, the cell supernatant was collected and centrifuged. The supernatant was then used for detection according to the kit instructions.

[0071] 1.4.2 Western blot analysis of Col II and MMP13 protein expression levels

[0072] Cells were treated according to the method described in "1.3" above. After treatment, cell pellets were collected, lysed, and proteins were extracted for Western blotting. Finally, Image J was used for semi-quantitative analysis.

[0073] 1.5 Statistical Analysis

[0074] Data were processed using GraphPad Prism 10 software. ANOVA was used for comparisons between groups. P < 0.05 indicated significant differences, and P < 0.01 indicated highly significant differences.

[0075] 2. Experimental Results

[0076] 2.1 Levels of inflammatory factors IL-6 and TNF-α in each group

[0077] The results showed that, compared with the blank group, the inflammatory markers in the cell supernatant of the model group were significantly increased (P<0.01); compared with the model group, the inflammatory markers in the cell supernatant of each drug administration group decreased to varying degrees, among which the traditional Chinese medicine preparation group had the best anti-inflammatory effect, thus inhibiting the catabolism of chondrocytes.

[0078] 2.2 Expression levels of Col II and MMP13 proteins, which are related to cartilage degradation in each group

[0079] Osteoarthritis is an age-related disease characterized by degenerative changes in articular cartilage, with the core pathology being the progressive loss of cartilage matrix. Col II is a key component in maintaining the structural integrity of the extracellular matrix (ECM), while MMP13 is a key enzyme mediating ECM degradation, specifically degrading Col II and other matrix components, and exacerbating cartilage destruction through a cascade activation effect. Results showed that compared to the control group, the expression level of MMP13 protein in the model group was significantly increased, while the expression level of Col II protein was significantly decreased (P<0.05). Compared to the model group, the expression level of Col II protein in the cells of all treatment groups recovered, and the expression level of MMP13 protein decreased to varying degrees. The improvement effect in the traditional Chinese medicine preparation group showed a significant difference (P<0.05), indicating that this preparation can effectively balance the synthesis and catabolism of chondrocytes.

[0080] 3. Discussion

[0081] Chondrocytes are the only cell type in articular cartilage, maintaining cartilage homeostasis by producing extracellular matrix (ECM). Therefore, regulating chondrocyte metabolism is crucial for maintaining cartilage homeostasis and preventing and delaying osteoarthritis. Treatment with the traditional Chinese medicine preparation of this invention, after inducing chondrocytes to release large amounts of inflammatory factors and mediators using IL-1β, effectively inhibited the inflammatory response, increased Col II expression, enhanced chondrocyte anabolic metabolic activity, and consequently inhibited MMP13 protein expression, thereby preventing cartilage degeneration.

[0082] Experiment 2

[0083] 1 Experimental Methods

[0084] 1.1 Laboratory Animals

[0085] Healthy adult female SD rats, SPF grade, weighing approximately 200-220g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed in separate cages with free access to water, at a relative humidity of 22-24℃ and 30-70%, under 12-hour light / dark cycles.

[0086] 1.2 Experimental Drugs

[0087] External application of traditional Chinese medicine preparation (ZYZJ): Prepared according to the formula and preparation method described in Example 8 above.

[0088] Yunnan Baiyao Group (YNBY): Yunnan Baiyao Tincture was selected and purchased from Yunnan Baiyao Group Co., Ltd.

[0089] Tongbi Tincture (TB): 50 g each of raw frankincense, raw myrrh, raw aconite, centipede, raw pinellia, raw arisaema, asarum, long pepper, raw aconite, camphor, scorpion, and borneol; 100 g each of white mustard seed and raw rhubarb; and prepared according to the preparation method of external Chinese medicine tincture.

[0090] 1.3 Modeling Methods and Grouping

[0091] Eight SD rats were randomly selected as the control group. The remaining rats were anesthetized, and after skin preparation and disinfection, the skin on the medial side of the right posterior knee joint was longitudinally incised. The quadriceps tendon was longitudinally incised along the outer edge of the patella to expose the anterior cruciate ligament. The anterior cruciate ligament was cut with a sharp blade to cause knee instability. The layers were sutured together, and the rats were given antibiotic intervention for three days.

[0092] Seven days after modeling, the animals were randomly divided into four groups: the model group, the topical Chinese medicine preparation group, the Yunnan Baiyao group, and the Tongbi tincture group, with eight animals in each group. The model group received bilateral application of the corresponding volume of tincture solvent to both knee joints; the topical Chinese medicine preparation group received 0.1 ml of the Chinese medicine preparation to both knee joints; the Yunnan Baiyao group received 0.1 ml of Yunnan Baiyao tincture to both knee joints; and the Tongbi tincture group received 0.1 ml of Tongbi tincture to both knee joints, twice daily for eight weeks.

[0093] 1.4 Detection Indicators

[0094] 1.4.1 Behavioral observations of each group

[0095] After the administration was completed, the mental state, activity level, gait, range of motion, and degree of knee joint swelling of the rats in each group were observed, and the Lequesne MG behavioral scores were performed on the rats.

[0096] This score is often used to assess the degree of pain, swelling, and limitation of movement in knee joint diseases.

[0097] 1.4.2 Serum levels of inflammatory factors IL-6, IL-1β, and TNF-α

[0098] After the rats were administered the drug, blood was collected, serum was separated, and the tests were performed according to the instructions of the kit.

[0099] 1.4.3 Morphological observation of knee joint tissues

[0100] After fixation for 48 hours, rat knee joint cartilage tissue specimens were decalcified, stained with hematoxylin and eosin and bone green, and semi-quantitative analysis was performed on the tissue sections. Krenn synovitis score and Mankin cartilage tissue score were also used. Krenn score: enlargement of the inner synovial cell layer (0-3 points); density of intrinsic cells (0-3 points); inflammatory infiltration (0-3 points); Mankin score: cartilage structure (0-6 points); chondrocytes (0-3 points); cartilage matrix staining (0-4 points); tidal line integrity (0-1 point).

[0101] 1.5 Statistical Analysis

[0102] Data were processed using GraphPad Prism 10 software. ANOVA was used for comparisons between groups. P < 0.05 indicated significant differences, and P < 0.01 indicated highly significant differences.

[0103] 2. Experimental Results

[0104] 2.1 Lequesne MG behavioral scores of rats in each group

[0105] After the drug intervention ended, no animals showed signs of death, mental abnormalities, or obvious allergic symptoms (such as redness, swelling, and rashes) at the joint sites during the observation period. The Lequesne MG behavioral scores were assessed in the rats. Results showed that the scores of the model group rats were all above 6 points, significantly higher than the control group. Compared with the model group, the scores of all drug-treated groups were significantly lower; and the score reduction was more significant in the topical Chinese medicine preparation group compared to other drug-treated groups. This suggests that the topical Chinese medicine preparation can effectively relieve knee pain and improve knee joint function.

[0106] Table 1. Lequesne MG behavioral scores of rats in each group

[0107]

[0108] 2.2 Serum levels of inflammatory factors IL-6, IL-1β, and TNF-α

[0109] In knee osteoarthritis, cartilage damage releases inflammatory mediators such as IL-6, IL-1β, and TNF-α. These mediators not only stimulate nerve endings, leading to increased pain, but also accelerate the degradation of cartilage matrix. Results showed that, compared to the control group, the serum levels of IL-6, IL-1β, and TNF-α were significantly increased in the model group. Compared to the model group, the levels of inflammatory factors in each treatment group decreased to varying degrees, with the topical traditional Chinese medicine preparation group showing the most significant effect.

[0110] 2.3 Morphological observation of knee joint tissue

[0111] The Krenn synovitis score and Mankin cartilage tissue score were determined based on the staining results. The results showed that the model group scores were significantly higher than the control group (P<0.01). After drug intervention, the pathological damage to the cartilage tissue of each group of animals improved to varying degrees. Among them, the external application of traditional Chinese medicine preparations effectively repaired cartilage damage and improved the pathological damage (P<0.01).

[0112] 3. Discussion

[0113] This traditional Chinese medicine preparation possesses the effects of dispelling wind and dampness, clearing the meridians and relieving pain, promoting blood circulation and removing blood stasis, and tonifying the kidneys and strengthening bones. Preliminary efficacy screening was conducted through in vitro experiments, and the most scientifically formulated and effective combination was selected for in vivo efficacy studies. The optimal dosage form was chosen based on the principle of enhancing the efficacy of the preparation. In vitro results show that this preparation can significantly downregulate inflammatory factor levels and exert potential effects such as protecting the cartilage microenvironment. Based on the holistic concept and syndrome differentiation and treatment characteristics of traditional Chinese medicine external application, it was used in animal models of knee osteoarthritis for intervention. The results showed that this invention can effectively relieve symptoms such as joint pain and stiffness, improve joint function and mobility, eliminate joint swelling and effusion, inhibit inflammatory responses, activate chondrocyte activity, and promote cartilage matrix synthesis and tissue repair. This external traditional Chinese medicine preparation provides patients with a better treatment option.

[0114] 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 removing blood stasis and reducing swelling, characterized in that, The external Chinese medicine preparation is made from the following Chinese herbal raw materials in parts by weight: 10-40 parts of Impatiens balsamina, 8-25 parts of Achyranthes bidentata, 10-45 parts of Eucommia ulmoides, 5-25 parts of Salvia miltiorrhiza, 5-25 parts of Achyranthes bidentata, 1-18 parts of Ox Gallbladder, 1-10 parts of Prunus persica leaf, 1-18 parts of Hedyotis diffusa, 1-20 parts of Phyllanthus urinaria, and 1-10 parts of Rosa rugosa.

2. The external application traditional Chinese medicine preparation according to claim 1, characterized in that, The external Chinese medicine preparation is made from the following herbal raw materials in parts by weight: 15-30 parts of Impatiens balsamina, 12-20 parts of Achyranthes bidentata, 12-30 parts of Dryopteris crassirhizoma, 8-20 parts of Salvia miltiorrhiza, 8-20 parts of Achyranthes bidentata, 3-12 parts of Ox Gallbladder, 3-8 parts of Prunus persica leaf, 3-12 parts of Hedyotis diffusa, 3-12 parts of Phyllanthus urinaria, and 3-8 parts of Rosa rugosa.

3. The external application traditional Chinese medicine preparation according to claim 1, characterized in that, The external Chinese medicine preparation is made from the following herbal raw materials in the indicated weight proportions: 20 parts of Impatiens balsamina, 15 parts of Achyranthes bidentata, 15 parts of Eucommia ulmoides, 10 parts of Salvia miltiorrhiza var. trifoliata, 10 parts of Achyranthes bidentata, 5 parts of Ophiopogon japonicus, 5 parts of Prunus persica leaf, 5 parts of Hedyotis diffusa, 5 parts of Phyllanthus urinaria, and 5 parts of Rosa rugosa.

4. The use of a topical traditional Chinese medicine preparation with the effect of removing blood stasis and reducing swelling as described in any one of claims 1 to 3 in the preparation of a drug for treating or improving osteoarthritis, characterized in that, The aforementioned external Chinese medicine preparation is a tincture product made from the above-mentioned weight proportions of Chinese herbs as raw materials, supplemented with pharmaceutically acceptable excipients.

5. The application of the topical traditional Chinese medicine preparation with the effect of removing blood stasis and reducing swelling as described in claim 4 in the preparation of a drug for treating or improving osteoarthritis, characterized in that... The herbal tincture product for treating or improving osteoarthritis is prepared according to the following steps: (1) Take the above-mentioned Chinese herbal medicine raw materials, clean and dry them, and then coarsely crush each herb to obtain various herb coarse powders for later use; (2) Take the coarse powder of Chinese medicinal materials from step (1) and 75% alcohol, add them to a light-proof wide-mouth glass bottle in a ratio of 1:5 to 1:8, seal the container, store for one month, filter out the liquid, and repackage it in a ball bottle, spray bottle or sealed glass bottle for later use.

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