CN113304163B

CN113304163B - Application of trifolium pterocarpus santalin in treating arthritis

Info

Publication number: CN113304163B
Application number: CN202110641583.6A
Authority: CN
Inventors: 孔祥英; 苏晓慧; 林娜
Original assignee: Institute of Materia Medica of CAMS
Current assignee: Institute of Materia Medica of CAMS
Priority date: 2021-06-09
Filing date: 2021-06-09
Publication date: 2022-05-20
Anticipated expiration: 2041-06-09
Also published as: CN113304163A

Abstract

The invention provides the application of the trifoliate red sandalwood glycoside or the plant containing the trifoliate red sandalwood glycoside or the extract of the trifoliate red sandalwood glycoside in treating arthritis, and compared with the existing medicine for treating arthritis, the trifoliate red sandalwood glycoside has the advantages of excellent effect and low cost, and has the potential of being combined with other medicines. The technical scheme of the invention expands the selectivity of the existing medicines for treating RA, and provides more medicine choices for the effective treatment of the RA; the clinical treatment cost of RA patients is further reduced, and the application range of the trifolium pterocarpus santalin is expanded.

Description

Application of trifolium pterocarpus santalin in treating arthritis

Technical Field

The invention relates to the technical field of medicines, in particular to application of trifolirhizin or plants containing trifolialin or extracts of trifolialin in preparation of medicines for treating arthritis.

Background

Rheumatoid Arthritis (RA) is a world-refractory, autoimmune disease characterized by progressive erosion of cartilage and bone by chronic synovitis, ultimately leading to destruction of bone and joints. The disease is better in young and middle-aged 30-50 years, the two-year disability rate of untreated patients is reported to be about 50%, the three-year disability rate is reported to be 70%, and the disease becomes one of the main causes of the loss of labor force and disability of people in China. Recent guidelines and treatment strategies for RA in the American College of Rheumatology (ACR) emphasize the use of disease-modifying antirheumatic drugs (DMARDs) in the early stages of confirmed diagnosis of RA, with the ultimate goal of preventing and controlling joint destruction. The european antirheumatic alliance (EULAR) in 2017 has a first suggestion for the therapeutic renewal of RA: the comprehensive consideration of the individual cost, the medical cost and the social cost of the patient is increased, and a reasonable individualized treatment scheme is made. It can be seen that pharmacogenomics is a real problem that we must face in the clinical application of drugs. Although the biological agents on the market have played an important role in the treatment of RA in recent years, their high price limits their widespread use. Therefore, the search for an anti-RA medicament with definite target, obvious curative effect, economy, cheapness and independent property in the traditional Chinese medicine becomes an urgent problem to be solved.

Joint synovitis, pannus formation, cartilage damage and bone destruction are important pathological links in the progress of the disease course of RA, and particularly, bone erosion is the main pathological feature of RA and is the final cause of disability of patients. It is generally accepted that once bone is destroyed, it means that its pathological changes enter an irreversible phase. Therefore, delaying or even blocking bone destruction becomes a key node for RA treatment, and is also the ultimate goal of RA treatment. The search for attempts to block RA bone destruction based on this has also attracted considerable attention from researchers in the medical community.

Trifolium pterocarpus santalin, also called trifolialin, can exist in subprostrate sophora, radix sophorae flavescentis, folium mori, sophora alopecuroides and other plants, belongs to flavonoid compounds, and has obvious activities of resisting inflammation, resisting tumor, resisting oxidation, resisting bacteria, reducing asthma, inhibiting melanin formation and the like. In recent years, attention has been paid to the wide range of biological activities, for example: CN106727653A discloses the application of trifoliate red sandalwood glycoside as hypoglycemic medicine and medicine for treating diabetic nephropathy. CN111202740A discloses the application of trifoliate red sandalwood glycoside in the preparation of antidepressant drugs. CN105497293A discloses the use of pharmaceutical compositions comprising trifoliosid in the treatment of cerebral palsy. CN104784666A discloses a traditional Chinese medicine ointment containing subprostrate sophora and the like, which is used for orthopedic nursing. On one hand, the traditional Chinese medicine ointment has more active ingredients, and does not mention whether the subprostrate sophora can play a role in treatment alone, and on the other hand, the traditional Chinese medicine ointment has no comparability in orthopedic nursing and arthritis treatment. Therefore, reports of the trifolioside in the aspect of treating arthritis are not disclosed in the prior art. At present, no satisfactory therapy for RA exists, and the research on the trifolium pterocarpus santalin is insufficient, so that the invention is also provided.

Disclosure of Invention

The application provides application of trifolioloid pterocarpan glycoside in treating arthritis, in particular, the treatment effect of trifolioloid pterocarpan glycoside on bovine type II collagen-induced mouse and rat arthritis (CIA) models is researched through an intraperitoneal injection administration method, and the treatment effect of trifolioloid pterocarpan glycoside on CIA mice and rats is evaluated through comparing ankle joint swelling degrees and pathological changes of various groups of animals and detecting bone destruction conditions through Micro-CT. In addition, the influence of the trifoliosid on the differentiation and activation of osteoclasts is observed through a mouse bone marrow macrophage osteoclast differentiation model induced by RANKL, and a key way for treating RA by the trifoliosid is discussed. The effect of the trifoliolate pterocarpan glycoside on the expression level of the proinflammatory cytokines and an RANKL-induced in-vitro osteoclast differentiation model are observed through an in-vitro synoviocyte inflammation model, the differentiation and activation level of osteoclasts are detected, the influence of the trifoliolate pterocarpan glycoside on the function of the osteoclasts is evaluated, and therefore the treatment action mechanism of the trifoliolate pterocarpan glycoside on RA is clarified.

The chronic synovitis in RA causes erosion damage to bones and cartilages and further causes bone damage to the whole body, which is currently recognized as an important mechanism in the pathogenesis of RA, and the trilobatin reduces synovitis and then bone damage to local joints and the whole body, has the treatment effect equivalent to that of a positive control drug methotrexate, and part of indexes are superior to that of methotrexate, so that the trilobatin has clinical value in RA treatment. Specifically, the method comprises the following steps:

in a first aspect of the invention, the invention provides an application of trifoliarioside or pharmaceutically acceptable salt, hydrate, solvate, enantiomer, stereoisomer or tautomer thereof in preparing a medicament for preventing and/or treating arthritis.

In a second aspect of the present invention, there is provided a use of trifoliosid or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, stereoisomer or tautomer thereof for the treatment and/or prevention of arthritis.

Preferably, the trifoliosid can be obtained commercially or extracted from plants. The plant is selected from radix Sophorae Tonkinensis, radix Sophorae Flavescentis, folium Mori or herba Sophorae Alopecuroidis.

In a third aspect of the present invention, there is provided a use of a plant or an extract thereof in the preparation of a medicament for preventing and/or treating arthritis, wherein the plant is selected from the group consisting of radix sophorae tonkinensis, radix sophorae flavescentis, folium mori, and sophora alopecuroides.

In a fourth aspect of the present invention, there is provided a use of a plant or an extract thereof selected from the group consisting of radix sophorae tonkinensis, radix sophorae flavescentis, folium mori, and sophora alopecuroides for preventing and/or treating arthritis.

Preferably, the extract of the plant contains pterocarpus santalin. Further preferably, the trifoliosid contains 0.01% to 99.9% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of trifoliosid.

The arthritis includes but is not limited to osteoarthritis, rheumatoid arthritis, infectious arthritis, gouty arthritis, metabolic arthritis or childhood idiopathic arthritis.

In one embodiment of the invention, the arthritis is selected from chronic synovitis, erosive arthritis, or rheumatoid arthritis.

In a fifth aspect, the invention provides an application of the trifolium pterocarpus indicus glycoside in preparing a medicament for relieving the joint swelling degree, prolonging the attack time of arthritis, inhibiting the destruction degree of knee joints, reducing pannus formation, inhibiting osteoclast differentiation, reducing the actin ring formation proportion or reducing the expression level of inflammatory factors.

The sixth aspect of the invention provides an application of trifolium pterocarpus santaline in alleviating joint swelling degree, prolonging the attack time of arthritis, inhibiting the destruction degree of knee joints, reducing pannus formation, inhibiting osteoclast differentiation, reducing the actin ring formation ratio or reducing the expression level of inflammatory factors.

Preferably, the medicament also comprises pharmaceutically acceptable auxiliary materials. Further preferably, the adjuvant is selected from a carrier, a diluent, a binder, a lubricant or a wetting agent.

In one embodiment of the present invention, the adjuvant is selected from starch, lactose, microcrystalline cellulose, dextrin, hydroxypropyl cellulose, crospovidone, pregelatinized starch, talc, magnesium stearate, aerosil, sodium lauryl sulfate, tween 80, hydrogenated vegetable oil, or the like.

In a seventh aspect of the present invention, there is provided a medicament for treating and/or preventing arthritis, the medicament comprising trifolio pterocarpan glycoside or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, stereoisomer or tautomer thereof, or comprising a plant or an extract thereof, the plant being selected from the group consisting of subprostrate sophora, sophora flavescens, mulberry leaves and sophora alopecuroides.

The arthritis is selected from one or the combination of more than two of osteoarthritis, rheumatoid arthritis, infectious arthritis, gouty arthritis, metabolic arthritis or infant idiopathic arthritis.

The medicine also comprises pharmaceutically acceptable auxiliary materials. Further preferably, the adjuvant is selected from a carrier, a diluent, a binder, a lubricant or a wetting agent.

Preferably, the content of the trifoliosid or its pharmaceutically acceptable salt, hydrate, solvate, enantiomer, stereoisomer or tautomer in the drug is 0.01-99.9% (preferably 1-99%) by mass, such as 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%.

Preferably, the content of the plant or its extract in the medicine is 0.01-99.9% (preferably 1-99%) by mass, such as 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%.

Preferably, the mode of administration of the drug is suitable for gastrointestinal or parenteral administration.

In one embodiment of the present invention, the drug is administered by a method selected from oral administration, intestinal administration, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, transdermal administration, subconjunctival administration, intra-ocular administration, orbital administration, retrobulbar administration, retinal administration, choroidal administration, intrathecal injection, and the like.

Preferably, the medicament is a freeze-dried preparation, an injection, a tablet, a granule, a pill, a capsule, an injection (such as an injection or a freeze-dried powder for injection), an inhalant, a buccal tablet, a suppository, an emulsion, a microemulsion, a submicron emulsion, a nanoparticle, a gel, a powder, a suspoemulsion, a cream, a jelly, a spray and the like, and is prepared by adopting a conventional preparation method in the field. The administration dose varies depending on the subject to be administered and the disease stage, and local administration is selected as the standard.

Preferably, the medicament also comprises other active ingredients, and the other active ingredients can be medicaments for treating arthritis (nonsteroidal anti-inflammatory medicaments such as ibuprofen, naproxen and the like, antirheumatic medicaments such as methotrexate, hydroxychloroquine and the like, biological response modifiers such as tumor necrosis factor inhibitors or etanercept, infliximab and the like, corticosteroids such as prednisone, cortisone and the like, uric acid reducing medicaments such as purine alcohol, benzbromarone, febuxostat and the like) and the triafolacin or plants containing the triafol or extracts thereof. The other active ingredients can also be drugs for alleviating side effects in the process of treating arthritis. The other active ingredient may also be a medicament for treating complications arising from arthritis. The other active ingredient may also be an analgesic (e.g. acetaminophen or an opioid such as tramadol, oxycodone or hydrocodone).

Preferably, the medicament is suitable for use in a human or non-human mammal. The non-human mammal is selected from mouse, rat, monkey, dog, pig, cow or sheep, etc.

In an eighth aspect of the present invention, there is provided a method for preparing a medicament for treating and/or preventing arthritis, the method comprising: the active ingredient is the trifoliate red sandalwood glycoside or the pharmaceutically acceptable salt, hydrate, solvate, enantiomer, stereoisomer or tautomer thereof, or comprises plants or extracts thereof, and the plants are selected from subprostrate sophora, sophora flavescens, mulberry leaves or sophora alopecuroides.

In a ninth aspect of the present invention, there is provided a method for treating and/or preventing arthritis, said method comprising administering a therapeutically effective amount of trifoliolium pterocarpus santalin or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, stereoisomer or tautomer thereof, or alternatively, administering a therapeutically effective amount of the above plant or extract thereof, said plant being selected from the group consisting of subprostrate sophora, sophora flavescens, mulberry leaves and sophora alopecuroides, or administering a therapeutically effective amount of the above drug.

In a tenth aspect of the present invention, there is provided a method for reducing the degree of joint swelling, prolonging the time of onset of arthritis, inhibiting the degree of knee joint destruction, reducing pannus formation, inhibiting osteoclast differentiation, reducing the proportion of actin ring formation, or reducing the level of expression of inflammatory factors, the method comprising administering a therapeutically effective amount of trifoliosirin or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, stereoisomer, or tautomer thereof, or administering a therapeutically effective amount of the above plant or extract thereof, the plant being selected from the group consisting of subprostrate sophora, sophora flavescens, mulberry leaves, or sophora alopecuroides, or administering a therapeutically effective amount of the above drug.

The "extract" of the present invention can be extracted by any conventional method for extracting Chinese herbs in the prior art, such as decoction, percolation, distillation or reflux method.

The trifolium pterocarpus santaline belongs to flavonoid active ingredients, and the molecular formula is C₂₂H₂₂O₁₀Molecular weight 446.404, having the formula:

the term "pharmaceutically acceptable" as used herein means that the biological activity and properties of the active substance are neither significantly stimulating the organism nor inhibiting the administered product. The "pharmaceutically acceptable auxiliary materials" include, but are not limited to, carriers, diluents, binders, lubricants, wetting agents. The term "pharmaceutically acceptable salt" refers to a salt prepared from a pharmaceutically acceptable non-toxic acid or base, wherein the acid or base includes an inorganic acid or base or an organic acid or base. The inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid, hydroiodic acid or sulfuric acid. The inorganic alkali is selected from calcium, magnesium, lithium, sodium, zinc, aluminum or potassium. The organic acid is selected from formic acid, glycolic acid, propionic acid, acetic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, maleic acid, glutamic acid, benzoic acid, stearic acid, alginic acid, benzenesulfonic acid, glucuronic acid, pamoic acid or galacturonic acid. The organic base is selected from diethanolamine, choline, procaine, lysine or 1, 2-ethylenediamine.

"treating" as used herein refers to slowing, interrupting, arresting, controlling, stopping, alleviating, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve complete elimination of all disease-related signs, symptoms, conditions, or disorders.

"prevention" as referred to herein means suppression of symptoms or delay of all actions of a particular symptom stress by administration of a product as described herein.

An "effective amount" as referred to herein, refers to an amount or dose of a composition of the present invention which provides the desired treatment or prevention after administration to a subject or organ to which it is applied, in single or multiple doses.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1: expression of trifoliosid (Trif) on clinical symptoms of the joints of CIA mice (red swelling deformity).

FIG. 2: effect of trifolioside (Trif) on the arthritis score in CIA mice.

FIG. 3: effect of trifolioside (Trif) on the incidence of arthritis in CIA mice.

FIG. 4: influence of triafol triallata (Trif) on three-dimensional models of the left hind limb knee joint of CIA mice knee joints.

FIG. 5: effect of trifolioside (Trif) on bone destruction of knee joints in CIA mice.

FIG. 6: effects of trifolioside (Trif) on histopathology of CIA mice and statistics (x 200).

FIG. 7: effect of trifolioside (Trif) on body weight of CIA mice.

FIG. 8: influence of Trifolium Lenticularis Triprocedure (Trif) on the visceral-brain index of CIA mice.

FIG. 9: effect of trifolioside (Trif) on hepatotoxicity in CIA model mice.

FIG. 10: alleviation effect of trifolioside (Trif) on CIA rat arthritis score.

FIG. 11: influence of Tripterocarpus santalin (Trif) on micro-CT of hind limb knee joints of CIA rats.

FIG. 12: influence of Tripterocarpus santalin (Trif) on micro-CT of hind ankle joints of CIA rats.

FIG. 13: statistical analysis of Trifolium Lenticularis glycoside (Trif) for bone destruction in the knee joint of CIA rats.

FIG. 14: effect of trifolioside (Trif) on body weight in CIA rats.

FIG. 15: influence of Trifolium Millettiae Fortunei glycoside (Trif) on the organ index of CIA rats.

FIG. 16: trifolium pterocarpus santalin (Trif) has no obvious influence on the activity of RA-FLS cells.

FIG. 17: tripterocarpus santalin (Trif) can obviously inhibit the TNF-alpha induced mRNA expression of RA-FLS inflammatory factors.

FIG. 18: effect of trifolioside (Trif) on osteoclast differentiation and activation (x 200).

FIG. 19: effect of trifolioside (Trif) on osteoclast activation (x 200).

FIG. 20: effect of trifoliosid (Trif) on mRNA expression of osteoclast differentiation-associated genes.

FIG. 21: effect of trifolialin (Trif) on osteoclast differentiation-associated protein expression (x 1000).

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: effect of Trifolium Millettiae Spectabilis glycosides on CIA mice

1. Animal molding: healthy DBA/1J mice, male, SPF level, 8-10 weeks old, adaptive breeding for 7d, modeling: on day 0 (primary immunization), 100. mu.L/mouse of bovine type II collagen and complete Freund's adjuvant was injected intradermally into the root of the tail of the mouse, and on day 21 (booster immunization), 100. mu.L/mouse of bovine type II collagen and complete Freund's adjuvant was injected into the same site. The emulsifier is prepared by adding 2mg/mL bovine type II collagen into equal volume of Freund's adjuvant, emulsifying with homogenizer while dripping, rotating for 3min, stopping for 1min until the emulsifier is dripped into water and will not diffuse, and the whole process is carried out in ice bath (the emulsifier is prepared immediately before use).

2. Grouping and administration: randomly dividing DBA/1J mice into a normal group, a model group, a methotrexate group (0.5mg/kg/3d, MTX), a low dose (5mg/kg/d, Trif) of trifoliosid and a high dose (10mg/kg/d, Trif) of trifoliosid, wherein 6-8 mice are respectively administrated in a stomach irrigation way, and the administration volume is 0.2ml/10 g; the trifolium pterocarpus santaline group is administrated by intraperitoneal injection, and the administration volume is 0.1ml/10 g; the normal group and the model group were injected with an equal volume of saline i.p. each day, and the administration volume was 0.1ml/10 g.

3. Material taking and pesticide effect index detection: after the administration, blood is taken from eyeballs of the mice, and blood plasma of the mice is collected and stored at minus 80 ℃ for subsequent detection; weighing heart, liver, spleen, lung, thymus and brain tissue; the right ankle joints of each group of mice after administration are additionally taken, peripheral soft tissues are removed, the mice are fixed for 48 hours in 4 percent paraformaldehyde, and then the mice are placed in 10 percent EDTA decalcification solution for decalcification (the joint bones can be completely decalcified by a 1mL syringe needle). The remaining (2 forelimbs and 1 left hind limb) were stored in a-80 ℃ refrigerator.

(1) After successful modeling of body weight and arthritis index, body weight and arthritis scores were recorded every 3 days. Arthritis scoring criteria: 0 is normal, 1 is mild redness and swelling of the ankle joint, 2 is mild redness and swelling of the ankle joint to the metatarsal or metacarpal bone joint, 3 is moderate redness and swelling of the ankle joint to the metatarsophalangeal or metacarpophalangeal joint, and 4 is severe redness and swelling of the ankle joint to the toe or finger.

(2) Index of the zang-brain: at 28d, materials were drawn and heart, liver, spleen, lung, thymus and brain tissues were excised. The index of each organ was calculated according to the following formula: the visceral brain index (%) — the organ mass (g)/brain tissue (g) × 100%.

(3) Micro-CT scanning: carrying out Micro-CT scanning on animal joint tissues by adopting a GE Healthcare RS0800604-0063Micro-CT system, wherein the scanning time of each time is 72min, and the resolution is 27 mu m; the results were reconstructed three-dimensionally and data analyzed using GEHC Microviw software.

(4) And (3) dyeing the knee joint HE: paraffin sections were routinely subjected to HE staining. The photographed pictures were statistically analyzed using the following scoring criteria, and the final score for each mouse was the sum of the following four points, which were 12 points in total: (ii) degree of inflammatory cell infiltration (percentage): 0: none; 1: mild (1-10%); 2: medium (11-50%); 3: Severe (51-100%). ② synovial membrane hyperplasia degree: 0, none; 1: light (5-10 layers); 2: medium (11-20 layers); 3: severe (>20 layers). Degree of pannus formation: 0: none; 1, light; 2, moderate; 3, the weight is high. Degree of bone and cartilage destruction (percentage): 0: none; 1: mild (1-10%); 2: medium (11-50%); 3: Severe (51-100%).

(5) And (3) liver tissue HE staining: paraffin sections were routinely subjected to HE staining. The general toxicity of trifolioside to the liver was evaluated.

4. Results of the experiment

In the experiment, the collagen-induced arthritis mouse classical model (CIA mouse model) is established, the low-treatment and high-treatment effects of the trifoliosid are given, and the pharmacodynamic action of the trifoliosid on the CIA mouse model is evaluated. As a result, it was found that:

(1) the trifoliosid can reduce the joint red swelling expression of a CIA model mouse: at 1W after secondary immunization, the feet and claws of the CIA mice are swollen, and daily activities are reduced; at 2W after secondary immunization, the feet and claws of the CIA mice reach the peak swelling, the swelling of the feet and the ankles is obvious, and partial joints are malformed. Compared with the model group, the mice had significantly reduced joint swelling after trifolialin (Trif) intervention, and the reduction was greater than that of the positive Methotrexate (MTX) group, as shown in FIG. 1.

(2) Effect of trifolioside on arthritis score in CIA model mice: compared with a normal control group, the arthritis score of the mice in the CIA model group is obviously increased (P < 0.01); compared with the model control group, each dose group of the Trif and the MTX group can obviously reduce the arthritis score (P <0.01) of the mouse in the CIA model, and the Trif low dose group (5mg/kg) has the best effect of inhibiting the joint swelling degree, which is shown in figure 2.

(3) The trifoliosid can obviously inhibit the incidence rate of arthritis of a mouse in a CIA model and delay the incidence time of the arthritis of the mouse: mice developed disease on day 3 after the second immunization, and the incidence of disease gradually increased with time, and the incidence of disease in the model group reached 100% on day 9 after the second immunization. The MTX group started to develop on day 3, and the low and high Trif dose groups started to develop on day 6 after the second immunization, but the incidence rate is lower than that of the model group. And low dose groups of Trif had lower morbidity than high dose groups, see figure 3.

(4) The trifolium pterocarpus santalin can obviously inhibit the destruction of knee joints and ankle joints of mice of a CIA model (Micro-CT): Micro-CT scanning and 3D reconstruction are respectively carried out on knee joints and ankle joints of all groups of CIA mice, and bone destruction degree is quantitatively evaluated by measuring BMC (bone mineral content), Bone Mineral Density (BMD), tissue bone mineral content (TMC), Tissue Mineral Density (TMD), bone body integral number (BV/TV), bone trabecular number (Tb.N), bone trabecular thickness (Tb.Th), bone surface area volume ratio (BS/BV), bone trabecular separation degree (Tb.Sp) and the like of the knee joints of the CIA mice. As a result, the joint appearance of the normal group of mice is complete, the surface is smooth, no bursa becomes collapsed, and the bone tissue is compact. The model group mice have incomplete joint appearance, uneven surface, obvious capsular collapse and sparse bone tissues. Compared with the Control group, the Bone Mineral Content (BMC), the bone density (BMD), the tissue bone mineral content (TMC), the tissue bone density (TMD), the bone body integral number (BV/TV) and the bone trabecular thickness (Tb.Th) of the Model group CIA mice are all obviously reduced (P is less than 0.01), the bone surface area-to-volume ratio (BS/BV), the bone trabecular number (Tb.N) and the bone trabecular separation degree (Tb.Sp) are obviously increased (P is less than 0.01), and Trif5mg/kg has a better bone destruction inhibition effect than MTX and is better than Trif 10mg/kg (see figures 4-5).

(5) The trifolioside can improve the histopathological changes of the knee joints of the CIA mice: CIA mouse joint tissue section is subjected to HE staining, and synovial membranes and cartilage and bone pathological states of the mouse knee joint tissue section are observed under a light microscope. It can be observed that the normal group of mice has obvious joint tissue gaps, no hyperplasia of synovium and complete and smooth bone surface. The model group mice exhibited massive inflammatory cell infiltration, synovial hyperplasia, pannus formation in the joint space, accompanied by cartilage erosion and bone destruction (P < 0.01). After the Trif is administrated, inflammatory joint histopathology of CIA mice is improved to different degrees, wherein Trif5mg/kg can obviously reduce inflammatory infiltration, pannus formation, cartilage destruction and bone destruction joint pathology micro-integration, and the difference has statistical significance compared with a model group (P <0.01) (see figure 6).

(6) The trifolium pterocarpus santalin has no obvious influence on the weight change of a CIA model mouse: mice in the CIA model group lost significantly (P <0.01) compared to the normal control group. No significant change in body weight was seen for each of the Trif dose groups and the MTX group compared to the model control group, see fig. 7.

(7) The trifolium pterocarpus santalin has no obvious influence on the visceral brain index of a CIA model mouse: compared with a normal control group, the spleen-brain index of the model group is remarkably increased (P <0.05), and other visceral brain indexes are not remarkably changed; except that the low dose group of the Trif group can significantly improve the pulmonary brain index, the index of each other brain is not significantly different from the model group (P >0.05), which is shown in figure 8.

(8) The trifolium pterocarpus santalin has no obvious influence on the liver histopathology of a mouse model CIA: the HE pathological section shows that the liver cell morphology of the mice in the normal group under the light microscope is normal, the liver lobule structure is complete, the junction area is normal, and the liver blood sinus structure is clear. The model group can show that liver cells in partial visual fields are hypertrophied, the intercellular space is reduced, and liver blood sinuses are reduced; each dose group of the Trif was not significantly different from the model group, as shown in fig. 9. The experimental results show that the dose of the trifoliate red sandalwood glycoside used in the experiment has no obvious hepatotoxicity to the CIA model mouse.

Example 2: effect of Trifolium Millettiae Spectabilis glycosides on CIA rats

1. CIA rat modeling: healthy SD rats, male, SPF grade, 160-170 g, molding: on day 0 (primary immunization) rats, 200. mu.L/rat of bovine type II collagen and complete Freund's adjuvant in the skin were injected into the roots of the rats, and on day 7 (booster immunization) 100. mu.L/rat of bovine type II collagen and incomplete Freund's adjuvant in the same sites. The emulsifier is prepared by adding 2mg/mL bovine type II collagen into equal volume of Freund's adjuvant completely or incompletely, emulsifying with homogenizer while dripping, stopping for 1min every 3min until the emulsifier is not dispersed in water, and performing the whole process in ice bath (the emulsifier is prepared when used).

2. Grouping and administration: SD rats were randomly divided into a normal group, a model group, a trilobe pterocarpin low dose group (1.75mg/kg/d, Trif-L), a trilobe pterocarpin medium dose group (3.5mg/kg/d, Trif-M), a trilobe pterocarpin high dose group (7mg/kg/d, Trif-H), and a methotrexate group (0.6mg/kg/3d), with 6-7 rats per group. The administration is started from the 7 th day and is continuously carried out to the 30 th day, wherein each administration group is subjected to intraperitoneal injection of the trifoliate red sandalwood glycoside or methotrexate with the corresponding dose every day, the administration volume is 0.5ml/100g, and the normal group and the model group are subjected to intraperitoneal injection of the same volume of normal saline every day.

3. Material taking and drug effect index detection: after the administration is finished, taking materials from a 30 th rhizoma gastrodiae drunk rat, wherein the materials are as follows: collecting blood serum of each group of rats after taking blood from abdominal aorta, and storing at-80 ℃ for subsequent detection; weighing heart, liver, spleen, lung, thymus and brain tissue; taking the right hind limb, removing peripheral soft tissues, fixing in 4% paraformaldehyde for 48h, and decalcifying in 10% EDTA decalcifying liquid (the joint bone can be completely decalcified by penetrating a 1mL syringe needle); the remaining three limbs were frozen.

(1) Body weight, arthritis index: after successful modeling, body weight and arthritis scores were recorded every 3 days. Arthritis scoring criteria: 0 is normal, 1 is slight swelling of the ankle joint, 2 is slight swelling of the ankle joint to the metatarsal or metacarpal joint, 3 is moderate swelling of the ankle joint to the metatarsophalangeal or metacarpophalangeal joint, and 4 is severe swelling of the ankle joint to the toes or fingers.

(2) Micro-CT scanning: carrying out micro-CT scanning after the right hind limb of the rat is fixed, and observing the protective effect of the pterocarpus santalin on the joint of the CIA rat

(3) Index of the zang-brain: heart, liver, spleen, lung, thymus and brain tissue were collected. The index of each organ was calculated according to the following formula: heart index (%) ═ heart (g)/brain tissue (g) × 100%; liver index (%) ═ liver (g)/brain tissue (g) × 100%; spleen index (%) ═ spleen (g)/brain tissue (g) × 100%; lung index (%) ═ lung (g)/brain tissue (g) × 100%; kidney index (%) ═ kidney (g)/brain tissue (g) × 100%.

4. Results of the experiment

In the experiment, the collagen-induced arthritis rat classical model (CIA rat model) is established, and the low, medium and high treatments of the trifoliosid are given, so that the pharmacodynamic action of the trifoliosid on the CIA rat model is evaluated. As a result, it was found that:

(1) the trifolium pterocarpus santalin obviously reduces the CIA rat arthritis score: after 12 days after the first immunization, the score of the model group rats is obviously increased; the doses of the trifolium pterocarpus santalin can reduce the arthritis score of a CIA rat to different degrees; by day 21, high doses of trifoliosid (Trif-H) significantly reduced the rat arthritis score P <0.01, comparable to the positive control, Methotrexate (MTX), as shown in FIG. 10.

(2) Effect of trifolioside on destruction of articular bone in CIA rats: compared with the normal group, the joint bone of the rat in the model group is obviously damaged; compared with the model group, the pterocarpus santalinus glycoside with different doses has a certain improvement trend on the bone joint destruction of the CIA rats, but has no statistical difference. See fig. 11-13.

(3) Effect of trifolioside on body weight in CIA rats: the rats in each group showed a steady increase in body weight over the time of the experiment. Compared with the normal group, after the model is made, the body weight of the CIA rats is gradually reduced and is obviously lower than that of the normal group, and the dosage groups of the trifoliosid have no significant influence on the body weight of the CIA rats, as shown in figure 14.

(4) Influence of trifolioside on the organs of CIA rats: after the CIA model is established, the index of each organ of the rat is reduced, the reduction of the center, the liver and the kidney of the rat is most obvious, and the establishment of the CIA model has certain influence on the state of the rat; compared with the model group, the indexes of the organs of rats in each dose group of the trifolialin have no obvious change, which indicates that the trifolialin has no obvious toxic effect on the heart, the liver, the spleen, the lung, the kidney and the thymus of the CIA rats, and is shown in figure 15.

Example 3: effect of Trifolium Lepidii glycoside on TNF-induced expression of human fibroblast-like synovial cell inflammatory factor

1. Human fibroblast-like synoviocytes (RA-FLS) cell culture: resuscitating RA-FLS, addingDMEM medium containing 15% serum and 1% penicillin-streptomycin, at 37 deg.C and 5% CO₂Cultured in an incubator. Changing the liquid every 2-3d for 1 time, and carrying out subculture when the cell density reaches more than 80%. Subsequent experiments were performed using RA-FLS from generations 4-8.

2. Cell activity assay: taking cells in logarithmic growth phase, adjusting the density of the complete culture medium to 5 multiplied by 10⁴One/ml, inoculate 100. mu.l in a 96-well plate, set up a blank separately, and add an equal volume of complete medium. After the cells are shaped by adherence, 0, 2.5, 5, 10, 20, 40 and 80 mu M of trifoliolate pterocarpan glycoside is added, 20 mu l of MTS is added into each hole after 24h of culture, the obtained product is placed in an incubator for incubation for 2h, an enzyme-labeling instrument is used for measuring the absorbance (OD) of each hole at 490nm wavelength, three multiple holes are arranged in each group, and the test is repeated for 3 times.

3. Grouping and administration: taking cells in logarithmic growth phase, adjusting the density of complete medium to 1 × 10⁵One/ml, 2ml in 6-well plates were inoculated. The grouping is as follows: control, TNF-alpha + different concentrations of Trifolium Lepidioides (10, 20, 40. mu.M). The control group is not added with any intervention factors, 5ng/ml of TNF-a cultured cells are given to the TNF-alpha group for 12h, and TNF-alpha 5ng/ml and trilobatin with different concentrations are added to the other groups.

4. RT-qPCR detection of the expression of RA-FLS cytokines IL-1 beta and IL-6: after stem prognosis of each group of cells, total RNA of the cells is extracted by using Trizol reagent, and then the purity and concentration of the RNA are detected by using an ultraviolet spectrophotometer. And carrying out reverse transcription according to a reverse transcription kit to obtain cDNA, and detecting the expression levels of IL-1 beta, IL-6 and GAPDH genes by adopting an RT-qPCR instrument. The results are expressed as the ratio of the target gene to GAPDH, using GAPDH as an internal reference.

5. Results

(1) The pterocarpus santalinus glycoside has no obvious influence on the activity of RA-FLS cells: after incubation of trilobatin (5-160 μ M) with different concentrations and RA-FLS for 24h, cell activity was detected, and it was found that trilobatin within this concentration range had no significant effect on RA-FLS cell viability, as shown in FIG. 16.

(2) The trifolium pterocarpus santalin can obviously inhibit the expression of RA-FLS inflammatory factors induced by TNF-alpha: the test detects the expression of the mRNA of the inflammatory cytokines IL-1 beta and IL-6 by constructing a model for inducing the inflammation of human fibroblast-like synovial cells by TNF-alpha and giving the pterocarpus santalin with different concentrations. As a result, it was found that: the pterocarpus santalin with different concentrations can reduce the expression level of mRNA of RA-FLS cell inflammatory factors IL-1 beta and IL-6 induced by TNF-alpha, as shown in figure 17.

Example 4: effect of Trifolium Millettiae Volubilis glycoside on in vitro osteoclast differentiation

1. Osteoclast-induced differentiation: killing a mouse by a neck-off method, aseptically separating the femur and the tibia of the bilateral hind limb, repeatedly purging a marrow cavity by using a 5mL injector filled with a serum-free alpha-MEM culture medium until the marrow cavity becomes white, adding an erythrocyte lysate, uniformly mixing, standing for 2min, centrifuging for 5min at 1500r/min, and removing a supernatant; the cells were counted in a complete medium resuspension, and finally diluted with medium containing 20ng/mL M-CSF and seeded in 24-well plates at 1 mL/well and a density of 2.5X 10/well⁵one/mL. RANKL is induced after 72 hours of culture, the induction concentration is 50ng/mL, and the liquid is changed every other day.

2. TRAP staining for osteoclast differentiation: trif (administration dose of 10, 20, 40. mu. mol. L) was administered simultaneously with the induction culture of cell RANKL^-1) After 5d, taking out the slide with the cells, washing with PBS for 3 times, fixing with 4% formaldehyde at room temperature for 10min, and washing with deionized water for 3 times; adding an incubation solution with naphthol AS-BI phosphate AS a substrate, placing in a dark box at 37 ℃ for incubation for 15min, washing with PBS for 3 times, counterstaining with hematoxylin for 30s, washing with PBS for 3 times, observing under an inverted microscope, and counting the number of TRAP positive cells (the number of nuclei is ≧ 3, and osteoclasts).

3. Actin ring staining for osteoclast activation function: trif (administration dose of 10, 20, 40. mu. mol. L) was administered simultaneously with RANKL induction culture^-1) After 5d, taking out the slide with the cells, washing with PBS for 3 times, fixing with 4% formaldehyde at room temperature for 10min, and washing with deionized water for 3 times; adding 0.1% triton, clearing for 5min, incubating with TRITC-phalloidin labeling solution in a wet box at room temperature for 30min, washing with PBS for 3 times, adding Hoechst dye solution, incubating for 15min, washing with PBS for 3 times, and observing formation of osteoclast actin ring under a fluorescence inverted microscope. Thoroughly washed, observed and photographed, and the actin ring formation ratio, i.e., myokinetic, is calculatedThe number of cells forming the protein loop was divided by the total number of cells.

4. Osteoclast bone resorption was observed by staining bone fragments with toluidine blue: at 2X 10⁴The cells were inoculated in a 96-well plate containing bovine bone fragments at a density of one ml, and Trif (

administration dose

10, 20, 40. mu. mol. L) was administered simultaneously with the induction culture of RANKL cells^-1) After 9 days, the bone slices are placed in the liquid for ultrasonic washing for 5min multiplied by 3 times, then the bone slices are dehydrated step by step in gradient alcohol (80%, 90%, 95%, 100% I and 100% II), 1% toluidine blue is dyed, the bone slices are observed under a light mirror, and the area percentage of the bone pits on the whole bone slices is counted by adopting Image Pro Plus software, namely the area occupied by the bone absorption pits is divided by the total area of the bone slices.

5. Detecting the expression of the osteoclast differentiation related gene by a Real-time fluorescent quantitative PCR (Real-time PCR) experiment: trif (administration dose of 10, 20, 40. mu. mol. L) was administered simultaneously with RANKL induction culture^-1) After 5d, total mRNA of the cells is extracted by a Trizol method, and the RNA concentration is detected by a nucleic acid quantifier. Then, RNA is reversely transcribed into cDNA according to the instruction of a reverse transcription kit, and Real-Time fluorescence quantitative PCR detection is carried out by using SYBR Premix Ex Taq II kit (Perfect read-Time, TaKaRa, Japan). PCR amplification procedure: pre-denaturation at 94 deg.C for 5 min; followed by 94 ℃, 15s and 60 ℃, 30s, respectively, for 40 cycles, in accordance with 2-^ΔΔAnd calculating the expression of the osteolytic associated factor gene relative to the internal reference by CT.

6. Detecting the expression of osteoclast differentiation related genes by an immunofluorescence staining experiment: trif (administration dose of 10, 20, 40. mu. mol. L) was administered simultaneously with RANKL induction culture^-1) After 5d, fixing 4% formaldehyde at room temperature for 10min, perforating 0.1% Ttiton-100 cells for 15min, washing with PBS, adding primary antibody with corresponding concentration, and incubating overnight in a dark place at 4 ℃; washing with PBS for 3 times the next day, adding secondary antibody, incubating for 1h in dark, and washing with PBS for 3 times; dyeing rhodamine-phalloidin in dark for 60 minutes, and washing with PBS for 3 times; and dyeing with DAPI for 5 minutes, washing with PBS for 3 times, absorbing excessive water, sealing with fluorescent sealing agent, and observing and photographing under a laser scanning confocal microscope.

7. Results

In the experiment, the influence of the trifoliolate pterocarpus santalin on the in vitro osteoclast differentiation is evaluated by establishing an in vitro osteoclast differentiation model and giving three doses of low, medium and high trifoliolate pterocarpus santalin. As a result, it was found that:

(1) trif inhibits osteoclast differentiation formation: after mouse BMMs are induced by M-CSF and RANKL, a large number of TRAP positive multinucleated cells (nucleus is more than or equal to 3) can be observed, the volume of the TRAP positive multinucleated cells is obviously increased compared with that of common mononuclear cells, obvious wine red can be observed in cytoplasm, the boundary of an osteoclast cell membrane is not integral, and the periphery of the mouse BMMs can be seen in false foot extension. Compared with a normal group, the number of osteoclast TRAP positive cells in an RANKL induction group is obviously increased; the dose-dependent inhibition of osteoclast formation was compared to RANKL group for each administration group, see fig. 18.

(2) Trif decreases osteoclast activation function: the intracellular excitant protein microfilament (F-actin) is one of the main components of a pseudopodosoma and a cytoskeleton, and the F-actin in cells shows green fluorescence after being dyed by rhodamine-phalloidin; DAPI binds to DNA and the nucleus fluoresces blue after cell staining. After the mouse BMMs are subjected to induction culture for 5 days, rhodamine-phalloidin and DAPI staining are carried out, the staining is carried out by a fluorescence microscope, after RANKL induction, obvious actin ring formation can be seen, and a large number of cell nucleuses in cells are observed; compared with the RANKL group, the rate of actin ring formation was significantly reduced in the Trif-administered group and was dose-dependent, as shown in fig. 19.

(3) Effect of Trif on osteoclast differentiation-related factors: and (3) carrying out qPCR detection on the mouse BMMs after RANKL induction culture for 5 days. The results show that the expressions of specific genes TRAP, MMP-9, NFATc1 and CtsK mRNA of osteoclast differentiation are obviously improved compared with a blank group; compared with the RANKL group, the mRNA expression of MMP-9, TRAP, NFATc1, CtsK, Intergrin-beta 3 and the like of each administration group is obviously reduced, and the figure 20 shows. Immunofluorescence detection results also show that TRAP, MMP-9, NFATc1 and CTSK proteins in the induction group are expressed in large quantity in osteoclasts, the expression trend is consistent with mRNA, and after the Trif stem is given, the expression of TRAP, MMP-9, NFATc1 and CTSK proteins is inhibited in a dose-dependent mode in each administration group, and the figure 21 shows that the expression of TRAP, MMP-9, NFATc1 and CTSK proteins is inhibited.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. Use of trifoliosid or a pharmaceutically acceptable salt thereof as the sole active ingredient in the manufacture of a medicament for the prevention and/or treatment of arthritis selected from osteoarthritis or rheumatoid arthritis.

2. The use of claim 1, wherein the medicament further comprises a pharmaceutically acceptable excipient.

3. Use according to claim 2, wherein the excipient is selected from carriers, diluents, binders, lubricants or wetting agents.