Traditional Chinese medicine composition and application thereof in preparation of medicine for treating ankylosing spondylitis

Abstract

The invention discloses a traditional Chinese medicine composition and application thereof in preparation of a medicine for treating ankylosing spondylitis, and belongs to the technical field of medicines. The traditional Chinese medicine composition is prepared from the following raw materials in parts by mass: 5 to 10 parts of myrrh, 8 to 15 parts of rhizoma drynariae, 8 to 15 parts of fructus lycii, 8 to 15 parts of herba epimedii, 8 to 15 parts of radix achyranthis bidentatae, 2 to 6 parts of rhizoma galangae, 8 to 15 parts of fructus forsythiae and 10 to 15 parts of radix astragali seu hedysari. Animal experiments verify that the traditional Chinese medicine composition can remarkably inhibit inflammatory response and bone destruction by regulating T cell immune balance, has good pharmacological activity and safety for treating ankylosing spondylitis, and provides a scientific basis and an innovative application approach for research and development of new traditional Chinese medicines for treating ankylosing spondylitis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a traditional Chinese medicine composition and its application in the preparation of a medicament for treating ankylosing spondylitis. Background Technology

[0002] Ankylosing spondylitis (AS) is a chronic, progressive inflammatory rheumatic disease that primarily affects the spine and sacroiliac joints, with a significant genetic predisposition. Its clinical features include inflammatory pain in the axial and sacroiliac joints, bone destruction, joint stiffness, and pathological new bone formation. Current research suggests that the pathogenesis of AS is not fully understood, but it may be related to the interaction of genetic, immune, and environmental factors. Among these, the imbalance and dysfunction of T cell subsets play a crucial role in the occurrence and development of the disease, promoting its progression by influencing inflammatory responses, bone metabolism, and immune homeostasis.

[0003] Although there is no specific disease name for "ankylosing spondylitis" in Traditional Chinese Medicine (TCM), based on its clinical manifestations, it can be categorized under "Bi syndrome," "Great hunchback," "Spine stiffness," "Bamboo joint wind," or "Bone Bi syndrome." TCM theory considers the core pathogenesis of this disease to be "deficiency of the kidney and the Du meridian, stagnation of Qi and blood, and obstruction of the meridians," thus proposing "tonifying the kidney and unblocking the meridians" as the fundamental treatment principle. The latest clinical expert consensus indicates that common syndrome types of AS include kidney and Du meridian deficiency syndrome, damp-heat obstruction syndrome, cold-damp obstruction syndrome, liver and kidney deficiency syndrome, and blood stasis obstruction syndrome, with single or combined syndromes observed clinically. In terms of syndrome differentiation and treatment, the basic principle is "tonifying the kidney and strengthening the Du meridian to treat the root cause, clearing heat, dispersing cold, and eliminating dampness to treat the symptoms, and promoting blood circulation and removing blood stasis throughout the treatment." Existing traditional Chinese medicine formulas for treating AS are mostly based on the core principles of "tonifying the kidney, dispelling dampness, and promoting blood circulation," such as Eucommia Kidney-Tonifying Decoction, Angelica and Loranthus Decoction, and Notopterygium incisum Dampness-Relieving Decoction. However, these formulas rely heavily on experience in their formulation, and the rules of compatibility and targets of action are not systematically explained. At the same time, because traditional Chinese medicine theory is formed at the macroscopic experience level, it lacks a microscopic molecular basis and modern pharmacological verification, resulting in the unclear mechanism of action of traditional formulas at the molecular level, which makes it difficult to meet the needs of precision treatment and drug development.

[0004] In recent years, with the development of multi-omics technologies, artificial intelligence (AI), and network pharmacology, researchers have attempted to systematically explore the compatibility rules and mechanisms of action of traditional Chinese medicine (TCM) through methods such as deep learning, collaborative filtering, and graph network models. These technologies can integrate multimodal disease data, drug target information, and the properties and meridian tropism characteristics of TCM, providing new insights for the modernization of TCM. However, currently available literature on AI-predicted TCM formulas is mostly used for pharmacodynamic correlation analysis, lacking systematic validation for specific diseases and optimization of specific drug combinations. Summary of the Invention

[0005] The purpose of this invention is to provide a traditional Chinese medicine composition and its application in the preparation of a drug for treating ankylosing spondylitis, in order to solve the problems existing in the prior art. For the first time, it is proposed that a traditional Chinese medicine composition based on the theory of tonifying the kidney and unblocking the meridians can effectively relieve joint inflammation and improve joint function through the dual pathways of immune regulation and inflammation control, providing a new application direction for the treatment of related immune joint diseases by traditional Chinese medicine.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a traditional Chinese medicine composition for treating ankylosing spondylitis, the composition comprising the following raw materials in parts by weight: 5-10 parts of myrrh, 8-15 parts of Drynaria fortunei, 8-15 parts of Lycium barbarum, 8-15 parts of Epimedium brevicornu, 8-15 parts of Achyranthes bidentata, 2-6 parts of Alpinia officinarum, 8-15 parts of Forsythia suspensa, and 10-15 parts of Astragalus membranaceus.

[0007] Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight: 5 parts myrrh, 10 parts drynaria, 10 parts wolfberry, 10 parts epimedium, 10 parts achyranthes, 6 parts galangal, 10 parts forsythia, and 15 parts astragalus.

[0008] The present invention also provides the use of the aforementioned traditional Chinese medicine composition in the preparation of a medicament for treating ankylosing spondylitis.

[0009] The present invention also provides the application of the aforementioned traditional Chinese medicine composition in the preparation of drugs that regulate T cell immune balance.

[0010] The present invention also provides the use of the aforementioned traditional Chinese medicine composition in the preparation of a drug for inhibiting bone destruction.

[0011] The present invention also provides a medicine for treating ankylosing spondylitis, wherein the medicine uses the aforementioned traditional Chinese medicine composition as the sole active ingredient.

[0012] Preferably, the drug further includes pharmaceutically acceptable excipients.

[0013] Preferably, the dosage form of the drug includes oral preparations, topical preparations, or injectable preparations.

[0014] Preferably, the oral formulation includes tablets, capsules, and granules.

[0015] The present invention discloses the following technical effects: This invention provides a traditional Chinese medicine formula comprising myrrh, drynaria fortunei, wolfberry, epimedium, achyranthes bidentata, galangal, forsythia, and astragalus. This formula uses myrrh to invigorate blood circulation and unblock collaterals, and achyranthes bidentata to tonify the kidneys and strengthen bones and muscles, directly targeting the pathogenesis of AS (asthenia gravis) characterized by blood stasis obstructing collaterals and kidney deficiency leading to depletion of the Du meridian. Wolfberry nourishes yin and tonifies the kidneys, assisting achyranthes bidentata in strengthening the body's foundation and regulating immunity. Drynaria fortunei and epimedium are used as adjuvants to warm the kidneys and strengthen bones, galangal dispels cold and unblocks the meridians, forsythia clears heat and dissipates nodules, combining warming and cooling properties, and astragalus invigorates qi, promotes diuresis, strengthens the spleen, and eliminates dampness. The combined effects of these herbs integrate kidney tonification, blood invigoration, collateral unblocking, and dampness elimination into one formula, addressing both the root cause and symptoms by tonifying the liver and kidneys to regulate immunity and by improving the inflammatory microenvironment through blood invigoration, dampness elimination, and heat clearing.

[0016] The components of this traditional Chinese medicine formula are derived from natural active ingredients of Chinese herbs. The extraction process is mature and inexpensive, with low cytotoxicity. It is suitable for various formulations such as oral, topical, or injection, making it easy to promote clinical use and long-term use.

[0017] This invention, through animal experiments, clarifies that the traditional Chinese medicine formula composition can significantly inhibit inflammatory responses and bone destruction by regulating T-cell immune balance, demonstrating good pharmacological activity and safety in the treatment of ankylosing spondylitis. This provides a scientific basis and innovative application approach for the research and development of new traditional Chinese medicine drugs for ankylosing spondylitis. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 To assess hind paw swelling and spinal appearance in mice from different groups on day 28 after drug administration; Figure 2 Changes in hind paw thickness during the drug administration period in mice; Figure 3 Changes in arthritis swelling scores in mice during the drug administration period; Figure 4 HE staining results of the spine of mice in each group; Figure 5 The results of microCT scans of L5-6 mice in each group; Figure 6 To assess the proportions of CD80 and CD86 in each group of mice using flow cytometry; Figure 7 The expression of CD4 and IL17A in mice of each group was detected by immunofluorescence co-localization. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] This invention proposes the application of a traditional Chinese medicine formula with "kidney-tonifying and meridian-clearing" effects in the preparation of drugs for treating ankylosing spondylitis. This formula (ASD-A) integrates disease omics characteristics, drug target information, and traditional Chinese medicine properties and flavors using a deep learning model, and is optimized and screened through collaborative filtering and clustering algorithms. ASD-A consists of myrrh, Drynaria fortunei, Lycium barbarum, Epimedium, Achyranthes bidentata, Alpinia officinarum, Forsythia suspensa, and Astragalus membranaceus. Myrrh is the principal ingredient, promoting blood circulation and clearing the meridians, while Achyranthes bidentata tonifies the kidneys and strengthens bones and muscles, directly targeting the pathogenesis of AS characterized by blood stasis in the meridians and kidney deficiency. Lycium barbarum nourishes yin and tonifies the kidneys, assisting Achyranthes bidentata in strengthening the body and regulating immunity. Drynaria fortunei and Epimedium are used as adjuvants to warm the kidneys and strengthen bones, Alpinia officinarum dispels cold and clears the meridians, Forsythia suspensa clears heat and dissipates nodules using a combination of warming and cooling properties, and Astragalus membranaceus invigorates qi, promotes diuresis, strengthens the spleen, and eliminates dampness. The combined use of these herbs integrates kidney tonification, blood circulation promotion, meridian clearing, and dampness removal into one formula. It not only regulates the immune system by tonifying the liver and kidneys, but also improves the inflammatory microenvironment by promoting blood circulation, removing dampness, and clearing heat, thus treating both the symptoms and the root cause.

[0026] Example 1 This embodiment provides a traditional Chinese medicine formula for treating ankylosing spondylitis, which is prepared from the following raw materials: 5g of myrrh, 8g of Drynaria fortunei, 8g of wolfberry, 8g of Epimedium, 8g of Achyranthes bidentata, 2g of Alpinia officinarum, 8g of Forsythia suspensa, and 10g of Astragalus membranaceus.

[0027] Preparation method: Weigh each component raw material, soak them for 30 minutes, and decoct them according to the conventional method of decocting Chinese medicine to make a decoction with a concentration of 1g / mL of raw medicinal material. Shake well to mix and filter with a 0.22μm filter. Store at 4℃ for later use.

[0028] Example 2 This embodiment provides a traditional Chinese medicine formula for treating ankylosing spondylitis, which is prepared from the following raw materials: 5g of myrrh, 10g of Drynaria fortunei, 10g of wolfberry, 10g of Epimedium, 10g of Achyranthes bidentata, 6g of Alpinia officinarum, 10g of Forsythia suspensa, and 15g of Astragalus membranaceus.

[0029] The preparation method is the same as in Example 1.

[0030] Example 3 This embodiment provides a traditional Chinese medicine formula for treating ankylosing spondylitis, which is prepared from the following raw materials: 10g of myrrh, 15g of Drynaria fortunei, 15g of wolfberry, 15g of Epimedium, 15g of Achyranthes bidentata, 6g of Alpinia officinarum, 15g of Forsythia suspensa, and 15g of Astragalus membranaceus.

[0031] The preparation method is the same as in Example 1.

[0032] Experimental Example 1: Establishment of a mouse model of ankylosing spondylitis and intervention experiment with a traditional Chinese medicine formula for tonifying the kidney and unblocking the meridians. 1. Experimental conditions (1) Animal source: 24 SKG mice and 12 SPF-grade BALB / c male mice, 6–8 weeks old, weighing (20±3) g; (2) Breeding conditions: temperature 18~25℃, humidity 40~55%, 12h / 12h day and night alternating light, noise <60 decibels; mice are raised in groups of 5 per cage, fed with a maintenance diet containing 60% carbohydrates, 22% protein and 4.0% fat, with free access to food and water; (3) Experimental ethics: All animal experiments comply with the regulations of the Animal Experimentation Ethics Committee and have been approved with the corresponding ethical approval documents (ethics number: ZYZC202508007S).

[0033] (4) Drug preparation, ASD-A composition: Myrrh 5g, Drynaria fortunei 10g, Lycium barbarum 10g, Epimedium 10g, Achyranthes bidentata 10g, Alpinia officinarum 6g, Forsythia suspensa 10g, and Astragalus membranaceus 15g. All Chinese herbal medicine pieces were purchased from Beijing Tongrentang Pharmacy and conformed to the 2015 edition of the Pharmacopoeia of the People's Republic of China (Part I) and related provisions. A decoction of the Chinese herbs was selected, soaked for 30 minutes, and then decocted to a concentration of 1g / mL of raw herbs. The decoction was thoroughly shaken and mixed, filtered through a 0.22μm filter, and stored at 4℃ for later use.

[0034] 2. Modeling and Drug Administration (1) Modeling method: Mice in the model group were injected intraperitoneally with dextran (Curdlan, CAS 54724-00-4) at a dose of 0.3 mg / g once a week for 2 weeks; mice in the control group (BALB / c) were injected with an equal volume of physiological saline. (2) Grouping and administration: After successful modeling, the patients were randomly divided into the AS model group and the traditional Chinese medicine prescription group (ASD-A), and were administered the medicine by gavage for 4 consecutive weeks. (3) Tissue collection: After the experiment, the spinal bone tissue of the mouse and the surrounding muscle and ligament tissue were collected and flash-frozen in liquid nitrogen at -80℃.

[0035] Experimental Example 2: An Arthritis Scoring and Histological Evaluation Experiment in Ankylosing Spondylitis Mice 1. Detection Method (1) Two researchers assessed the arthritis score weekly using a double-blind method, using a 0–4 point scale (0 = normal, 4 = severe swelling and deformity), with a maximum cumulative score of 8 points for both hind paws; (2) Use vernier calipers to measure the thickness of the hind claw and record the mortality and complications; (3) After euthanasia, spinal tissue was taken, fixed with 4% paraformaldehyde, washed with PBS, and stained with HE. The total pathological score was calculated by semi-quantitative scoring of three spinal segments.

[0036] 2. Experimental Results The results are as follows Figures 1-4 As shown in the mouse model experiment, from day 14 after modeling, the AS group showed obvious joint swelling and increased thickness of the hind paw. P <0.05). After 4 weeks of continuous administration, compared with the model group, the ASD-A group mice showed a significant decrease in hind paw thickness and pathological arthritis score ( P <0.05); indicating that the traditional Chinese medicine formula can significantly improve arthritis symptoms. HE staining results showed that the vertebral bodies of mice in the normal group were intact, while the vertebral bodies of mice in the AS group were severely damaged, with varying degrees of bone loss and vertebral collapse; the bone loss of the vertebral bodies of mice in the ASD-A group was significantly improved after intervention with the traditional Chinese medicine formula.

[0037] Experiment Example 3: A micro-CT test to detect changes in bone structure in mice with ankylosing spondylitis 1. Testing conditions (1) Instrument parameters: energy 40 kV, current 500 μA, sampling time 40 ms, resolution 768×972 pixels; (2) Threshold setting: Mature bone threshold of L5-6 vertebral body and left posterior claw 550–3000 mg HA / cm 3 The threshold for new bone formation is 200–750 mg HA / cm². 3 .

[0038] 2. Experimental Results As shown in Table 1 and Figure 5 As shown, micro-CT scans revealed that in the AS group mice, the L5-L6 vertebral bodies had sparse trabeculae and a significantly reduced bone volume ratio (BV / TV). P <0.05); After intervention with the traditional Chinese medicine formula in the ASD-A group, the bone volume ratio significantly increased, the intertrabecular spacing (Tb.Sp) decreased, and the number of trabecular bones (Tb.N) increased ( P <0.05), some groups recovered trabecular bone arrangement, suggesting that the herbal formula of this invention can significantly improve bone destruction and bone microstructure damage in AS model mice. The bone remodeling index (Conn.Dn) showed no significant difference between groups ( P >0.05), but the overall trend of the intervention group was better than that of the AS group.

[0039] Table 1. MicroCT detection levels of bone formation and bone loss in mice of different groups Experimental Example 4: A Safety Experiment of ELISA for Detecting Inflammatory Factors and Serum 1. Detection Method (1) Homogenize the tissue using lysis buffer, centrifuge and collect the supernatant, and test serum IL-6, TNF-α, IL-17A, IL-23, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, blood urea nitrogen, and complete blood count [including white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), red blood cells (RBC), hemoglobin (Hb) and platelets (PLT)] according to the kit instructions; (2) Data normalization and comparison with the control group.

[0040] 2. Experimental Results As shown in Table 2, the ELISA results showed that the serum levels of IL-6, TNF-α, IL-17A, and IL-23 in the AS group mice were significantly higher than those in the control group. P<0.05). The levels of the above-mentioned inflammatory factors significantly decreased after drug intervention ( P <0.05). The results showed that the formulation of this invention could effectively inhibit the systemic inflammatory response in AS model mice, thereby alleviating immune-mediated joint inflammation. Serological tests showed that the levels of erythrocytes, leukocytes, hemoglobin, ALT, AST, creatinine, and blood urea nitrogen in each group of mice were all within the normal range. P >0.05), with no obvious liver or kidney toxicity observed, indicating that the herbal formula of this invention has good safety and tolerability.

[0041] Table 2. ELISA detection of liver and kidney function, blood routine tests, and expression levels of inflammation-related markers in mice of each group. Experimental Example 5: An experiment using flow cytometry to detect CD80 / CD86 expression and immune homeostasis in mice. 1. Detection Method (1) The proportion of lymphocytes and the expression of CD80 and CD86 were detected by flow cytometry; (2) Calculate the CD80 / CD86 ratio to assess the degree of immune activation.

[0042] 2. Experimental Results like Figure 6 As shown, flow cytometry results revealed that CD80 expression was significantly upregulated and CD86 expression was downregulated in AS model mice, leading to an increased CD80 / CD86 ratio. P <0.01) indicates excessive T cell activation. The CD80 / CD86 ratio in ASD-A mice significantly decreased after drug intervention ( P <0.05), close to the control group level, indicating that this traditional Chinese medicine formula can restore the balance of immune co-stimulatory signals and inhibit abnormal T cell activation.

[0043] Experimental Example 6: An Immunohistochemical Co-localization Detection Experiment for CD4 and IL17A Expression in Mice 1. Detection Method (1) Glass slides were incubated with CD4 mouse primary antibody and IL-17A rabbit antibody at room temperature for 1 hour, followed by incubation with AlexaFluor 488-conjugated anti-mouse antibody, Cy3-Alexa-conjugated anti-rabbit antibody, and Cy5-conjugated anti-rat antibody at room temperature for 1 hour. To avoid non-specific staining, the stained slides were treated with DAPI using an autofluorescence quenching kit. Immunofluorescence images were collected using a confocal microscope. (2) Calculate the fluorescence expression ratio of CD4 and IL17A.

[0044] 2. Experimental Results like Figure 7As shown, immunohistochemical colocalization results revealed that CD4 and IL-17A were significantly upregulated in the spinal tissue of AS model mice, with a significantly increased fluorescence expression ratio and significantly enhanced colocalization, suggesting increased Th17 cell infiltration. After ASD-A intervention, the fluorescence expression ratio was significantly decreased compared to the AS group, and the colocalization signal was significantly reduced. P <0.05), close to the control level. This indicates that the herbal formula of this invention can effectively inhibit abnormal activation of Th17 cells, thereby promoting the restoration of Th17 / Treg immune balance.

[0045] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A traditional Chinese medicine composition for treating ankylosing spondylitis, characterized in that, The traditional Chinese medicine composition comprises the following raw materials in parts by weight: myrrh 5-10 parts, drynaria 8-15 parts, wolfberry 8-15 parts, epimedium 8-15 parts, achyranthes bidentata 8-15 parts, galangal 2-6 parts, forsythia 8-15 parts, and astragalus 10-15 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition comprises the following raw materials in parts by weight: 5 parts myrrh, 10 parts drynaria, 10 parts wolfberry, 10 parts epimedium, 10 parts achyranthes, 6 parts galangal, 10 parts forsythia, and 15 parts astragalus.

3. The use of the traditional Chinese medicine composition as described in claim 1 or 2 in the preparation of a medicament for treating ankylosing spondylitis.

4. The use of the traditional Chinese medicine composition as described in claim 1 or 2 in the preparation of a drug for regulating T cell immune balance.

5. The use of the traditional Chinese medicine composition as described in claim 1 or 2 in the preparation of a drug for inhibiting bone destruction.

6. A drug for treating ankylosing spondylitis, characterized in that, The drug uses the traditional Chinese medicine composition as described in claim 1 or 2 as its sole active ingredient.

7. The drug as described in claim 6, characterized in that, The drug also includes pharmaceutically acceptable excipients.

8. The drug as described in claim 6, characterized in that, The dosage forms of the drug include oral preparations, topical preparations, or injectable preparations.

9. The medicament as described in claim 8, characterized in that, The oral preparations include tablets, capsules, and granules.

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