Traditional Chinese medicine monomer composition for treating rheumatoid arthritis and application

Abstract

The invention provides a traditional Chinese medicine monomer composition for treating rheumatoid arthritis and application, and belongs to the technical field of traditional Chinese medicine preparations. The traditional Chinese medicine monomer composition is prepared from triptolide and medipterocarpin. In the implementation process, it is accidentally found that the triptolide and the mediodine have a synergistic effect when combined use of low concentration is used for inhibiting osteoclast differentiation, and due to the combined effect disclosed by the research result, the triptolide and the mediodine have the application prospect of being developed into the medicine for treating rheumatoid arthritis in the form of the composition.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine preparations, and particularly relates to a traditional Chinese medicine monomer composition for treating rheumatoid arthritis and an application thereof. Background Art

[0002] Rheumatoid arthritis (RA) affects approximately 0.5% to 1% of the global population and is characterized by inflammation and bone destruction in the affected joints, ultimately leading to joint deformity and loss of function. Excessive bone resorption caused by osteoclasts (OCs) is the direct cause of cortical bone destruction and damage in RA patients. With the continuous development of disease-modifying antirheumatic drugs (DMARDs), significant success has been achieved in preventing and alleviating symptoms in RA patients. However, these drugs often fail to reverse existing joint destruction, and even with potent anti-inflammatory treatment strategies, such as blocking TNF-α or IL-6R, there are only limited signs of bone erosion repair. Therefore, it is necessary to optimize the treatment options for RA bone destruction.

[0003] Chinese patent CN 110638955 A discloses a Chinese medicine composition for treating arthralgia, and its raw materials include Notopterygium wilfordii, Gentiana macrophylla, Saposhnikovia divaricata, Pueraria root, Bupleurum root, Cyperus rotundus, Angelica sinensis, Red Peony root, Chuanxiong, Astragalus, Codonopsis pilosula, Atractylodes macrocephala, Coix seed, Atractylodes macrocephala, Tangerine peel, Pinellia tuber, Carthamus tinctorius, Semen safflower, Luffa, Cinnamomum cassia, Achyranthes bidentata and Dipsacus asper. Among them, the herbal medicine is widely available, with low cost, easy to make, and remarkable therapeutic effect; the prescription does not contain medicinal materials with large toxic and side effects such as Tripterygium wilfordii and Semen strychni, and long-term use has little stimulation to the gastrointestinal tract; starting from the two aspects of replenishing qi and blood and promoting blood circulation and removing blood stasis, it can improve human immunity and prevent bone destruction. It has unique curative effect for rheumatoid arthritis, compression fracture, periarthritis of shoulder, and limb pain after stroke, which belong to qi deficiency and blood stasis type or liver and kidney deficiency type according to syndrome differentiation, and no obvious adverse reactions are observed, so it is suitable for promotion and application.

[0004] The existing technology increases the large side effects of Tripterygium wilfordii. However, studies have found that triptolide (Tp), one of the main active ingredients in Tripterygium wilfordii, can inhibit OC differentiation to relieve RA. Although the therapeutic window of triptolide is very narrow and it can cause toxicity at slightly higher doses, its combination with other drugs can reduce the dosage, improve the efficacy, and thus reduce side effects.

[0005] Chinese patent CN103735649A discloses an application of particles in the preparation of drugs for treating bone and joint chondrocyte destruction and inflammation. The particles are obtained according to the following preparation method: extracting and concentrating Saussurea involucrata, Cistanche deserticola, Clematis chinensis, Herba Epimedii and Millettia reticulata with alcohol, obtaining a thick paste, extracting volatile oil from Atractylodes macrocephala, extracting, concentrating and precipitating crude polysaccharides from Anazi root, Morus alba, Herba Lycopodii, Herba Polygoni Multiflori and alcohol-extracted drug residues and drug residues after volatile oil extraction from Atractylodes macrocephala, encapsulating the volatile oil to obtain an inclusion compound, mixing the thick paste and the crude polysaccharide, adding auxiliary materials, granulating and granulating, and adding the inclusion compound to obtain the particles. The particles of the invention have low toxicity, have good effects on bone and joint chondrocyte destruction and inflammation, and have a certain analgesic effect, and can be used to treat bone and joint chondrocyte destruction and inflammatory diseases.

[0006] The prior art uses Millettia reticulata as one of the drug ingredients, but direct extraction of traditional Chinese medicine for use as medicine has certain toxic side effects. Therefore, in recent years, combined use of drugs to act on multiple targets to enhance pharmacological activity and reduce toxic side effects has become a new strategy. Med is one of the flavonoid active ingredients derived from the traditional Chinese medicine Millettia reticulata. It has anti-inflammatory, anti-osteoclastogenesis, and anti-angiogenesis effects. It can be combined with triptolide to relieve and treat rheumatoid arthritis, and can enhance pharmacological activity and reduce toxic side effects. There is no report of related research in the prior art. Summary of the invention

[0007] Based on the deficiencies in the prior art, the present invention aims to provide a Chinese medicine monomer composition and application for treating rheumatoid arthritis. During the implementation process, it was unexpectedly found that triptolide and medipterocarbone have a synergistic effect when used in combination at a lower concentration to inhibit osteoclast differentiation. The combined effect revealed by the research results of the present invention makes triptolide and medipterocarbone have an application prospect of being developed into a drug for treating rheumatoid arthritis in the form of a composition.

[0008] In order to achieve the above object, the present invention adopts the following technical solution: The invention provides a traditional Chinese medicine monomer composition for treating rheumatoid arthritis. The traditional Chinese medicine monomer composition consists of triptolide and meditartin.

[0009] The molar concentration ratio of triptolide and medipterostilbene is 1:500-2000.

[0010] Further preferably, the molar concentration ratio of triptolide to medipterostilbene is 1:1000.

[0011] The concentration of the triptolide is 5-20 nM, and the concentration of the medipatorhinol is 5-20 μM.

[0012] Preferably, the concentration of triptolide is 10-20 nM, and the concentration of meditartin is 10-20 μM.

[0013] As some preferred embodiments, the concentration of triptolide is 10 nM, and the concentration of medipatrin is 10 μM; Or, the concentration of triptolide is 10 nM, and the concentration of medipatorhinol is 20 μM; Or, the concentration of triptolide is 20 nM, and the concentration of medipatrin is 5 μM; Or, the concentration of triptolide is 20 nM, and the concentration of medipatorhinol is 10 μM; Or, the concentration of triptolide is 20 nM, and the concentration of meditartin is 20 μM.

[0014] The present invention also provides the use of the above-mentioned Chinese medicine monomer composition in preparing a medicine for treating rheumatoid arthritis.

[0015] A medicine for treating rheumatoid arthritis, comprising the above-mentioned Chinese medicine monomer composition and pharmaceutically acceptable excipients.

[0016] "Pharmaceutically acceptable excipients" refer to excipients and additives used in the production of drugs and the preparation of prescriptions. They refer to substances that have been reasonably evaluated for safety and are included in drug preparations in addition to active ingredients. The same pharmaceutical excipient can be used in drug preparations for different routes of administration and has different effects and uses. The pharmaceutical excipients added to the drugs provided by the present invention can play the role of excipient, carrier or stability improvement. In addition, they also have important functions such as solubilization, solubilization or sustained release.

[0017] Typical but non-limiting excipients include: one or more of solvents, solubilizers, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adherents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids or release retardants.

[0018] Fillers include: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; disintegrants include: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, etc.; lubricants include: magnesium stearate, sodium lauryl sulfate, talc, silicon dioxide, etc.; suspending agents include: polyvinyl pyrrolidone, Microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, etc.; binders include: starch slurry, polyvinyl pyrrolidone, hydroxypropyl methylcellulose, etc.; sweeteners include: saccharin sodium, aspartame, sucrose, sodium cyclamate, glycyrrhetinic acid, etc.; flavoring agents include: sweeteners and various flavors; preservatives include: parabens, benzoic acid, sodium benzoate, sorbic acid and its salts, benzalkonium bromide, chloroethidine acetate, eucalyptus oil, etc.; matrices include: PEG6000, PEG4000, insect wax, etc.

[0019] The pharmaceutical dosage forms of the present invention include tablets, capsules, solutions, granules, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, creams, sprays, patches, sustained-release preparations, controlled-release preparations or targeted preparations.

[0020] Preferably, the pharmaceutical dosage form is a tablet Pharmaceutical excipients used in the preparation of tablets include but are not limited to: (1) Fillers or diluents, which are used to increase the weight or volume of tablets to facilitate tableting, including starch, powdered sugar, dextrin, lactose, compressible starch, microcrystalline cellulose, inorganic salts, and mannitol; (2) Wetting agents or binders. Wetting agents refer to excipients that are non-viscous but can induce the viscosity of soft-material drugs, thereby achieving the purpose of granulation and tableting. They include distilled water and ethanol. Binders refer to excipients that give viscosity to non-viscous or insufficiently viscous materials, thereby agglomerating the materials into granules. They include starch slurry, methyl cellulose, hydroxypropyl cellulose, hypromellose, sodium carboxymethyl cellulose, ethyl cellulose, povidone, gelatin, polyethylene glycol, 50%-70% sucrose solution, and sodium alginate solution. (3) Disintegrants refer to excipients that promote the rapid breakup of tablets into fine particles in gastrointestinal fluid, including dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, and cross-linked polyvinylpyrrolidone; (4) Lubricant, which is a general term for glidants, anti-adherents and lubricants (in a narrow sense). glidants refer to auxiliary materials that reduce the friction between particles, thereby improving the fluidity of powders and reducing weight differences; anti-adherents refer to auxiliary materials that prevent materials from adhering to the surface of the punch and die during tableting to ensure smooth tableting operations and a smooth tablet surface; lubricants (in a narrow sense) refer to auxiliary materials that reduce the friction between the tablet and the die wall during tableting and ejection to ensure uniform stress distribution during tableting and prevent tablet cracking; lubricants include: magnesium stearate, micronized silica gel, Talc, hydrogenated vegetable oil, polyethylene glycols such as PEG4000, PEG6000, sodium lauryl sulfate.

[0021] The auxiliary materials may also contain colorants and flavoring agents; colorants refer to auxiliary materials that give the drug color, including pharmaceutical or food grade pigments, including chlorophyll, caramel, amaranth, lemon yellow, carmine, indigo, and their content in the pharmaceutical composition does not exceed 0.05%. Flavoring agents refer to pharmaceutical auxiliary materials used in drugs to improve or shield the bad smell and taste of drugs, making it difficult for patients to perceive the strong bitter taste of drugs (or other peculiar smells such as spicy, irritating, etc.), including sucrose, citrus glycosides, saccharin sodium, protein sugar, sorbitol, aspartame, acesulfame-K.

[0022] The preparation method of the above-mentioned medicine of the present invention is not strictly limited, and it can be made into an injection form, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Medicines such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.

[0023] Dosage levels of the active compounds in the medicaments contemplated by the present invention will depend on the activity of the particular compound, the route of administration, the severity of the disease being treated and the condition and previous medical history of the patient being treated.

[0024] The administration routes of the drug of the present invention include, but are not limited to, the following: administration via the gastrointestinal tract, such as oral administration; and administration without the gastrointestinal tract, such as intramuscular injection, intravenous injection or transdermal administration.

[0025] Compared with the prior art, the present invention has the following beneficial effects: During the implementation of the present invention, it was unexpectedly discovered that triptolide and medipterostilbene have a synergistic effect when used in combination at lower concentrations to inhibit osteoclast differentiation. The combined effect revealed by the research results of the present invention makes triptolide and medipterostilbene have the application prospect of being developed into a drug for treating rheumatoid arthritis in the form of a combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The combination of triptolide and medipatin inhibits the generation and differentiation of osteoclasts; Figure symbols: A is TRAP staining for osteoclast formation, scale bar 200 μm; B is the quantification of the number of osteoclasts in each group; C is the CI value of the drug inhibition rate on TRAP positive cells; Compared with the model group, ***p<0.001, **p<0.01, *p<0.05; compared with the combination group, ###p<0.001, ##p<0.01, #p<0.05.

[0027] Figure 2 The combination of triptolide and meditartin inhibits the resorption function of osteoclasts; Figure symbols: A is the formation of F-actin ring of osteoclasts by phalloidin staining, scale bar 100 μm; B is the area ratio of F-actin ring in each group; C is the representative scanning electron microscopy image of bone resorption pits in bone slices, scale bar 21 mm; D is the area ratio of bone resorption pits.

[0028] Compared with the model group, ***p<0.001, **p<0.01, *p<0.05; compared with the combination group, ###p<0.001, ##p<0.01, #p<0.05.

[0029] Figure 3 Effects of the combination of triptolide and meditartin on osteoclastogenesis, differentiation, and absorption-related mRNA; Figure symbols: A is the effect on Nfatc1 mRNA; B is the effect on C-fos mRNA; C is the effect on Atp6v0d2 mRNA; D is the effect on Dc-stamp mRNA; E is the effect on Ctsk mRNA. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and carefully in combination with preferred embodiments below, but the protection scope of the present invention is not limited to the following specific embodiments. Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0031] Among them: triptolide (CAS No. 38748-32-2) and medipterostilbene (CAS No. 73340-41-7).

[0032] The invention discloses a Chinese medicine monomer composition for treating rheumatoid arthritis. The Chinese medicine monomer composition consists of triptolide and medipterocarpin; the molar concentration ratio of the triptolide and medipterocarpin is 1:500-2000.

[0033] Further preferably, the molar concentration ratio of triptolide to medipterostilbene is 1:1000.

[0034] Preferably, the concentration of triptolide is 10-20 nM, and the concentration of meditartin is 10-20 μM.

[0035] As some preferred embodiments, the concentration of triptolide is 10 nM, and the concentration of medipatrin is 10 μM; Or, the concentration of triptolide is 10 nM, and the concentration of medipatorhinol is 20 μM; Or, the concentration of triptolide is 20 nM, and the concentration of medipatrin is 5 μM; Or, the concentration of triptolide is 20 nM, and the concentration of medipatorhinol is 10 μM; Or, the concentration of triptolide is 20 nM, and the concentration of meditartin is 20 μM.

[0036] When triptolide and medipterostilbene are used in combination at the above lower concentrations to inhibit osteoclast differentiation, the combined effect revealed by the research results of the present invention enables triptolide and medipterostilbene to be used in the form of a composition to be developed into a drug for treating rheumatoid arthritis.

[0037] Example 1: Effect of Chinese medicine monomer composition on primary osteoclast differentiation Primary monocytes from the bone marrow of C57 mice were extracted and inoculated into 96-well plates. Different concentrations of drugs (as shown in Table 1) were added for 4-6 days. The prepared TRAP working solution was added and incubated for 1 hour. The number of osteoclasts in each group was calculated. The results are shown in Tables 1 and Figure 1 .

[0038] Table 1 Analysis of synergistic effects of triptolide and medipterostilbene

[0039] According to Table 1 and Figure 1It can be seen that triptolide or medipterostilbene alone can inhibit RANKL-induced osteoclastogenesis and reduce the number of osteoclasts (OC) in a dose-dependent manner. It is worth noting that the combination of 10 nM Tp and 10μM Med, 10 nM Tp and 20μM Med, 20 nM Tp and 5μMMed, 20 nM Tp and 10μM Med, and 20 nM Tp and 20μM Med had the best effect on reducing the number of osteoclasts (OC), with synergistic combination index (CI) values ​​less than 1.

[0040] Example 2: Effect of Chinese medicine monomer composition on primary osteoclast bone resorption Primary monocytes were extracted from the bone marrow of C57 mice, inoculated into 96-well plates, and treated with drugs of different concentrations (as shown in Table 1) for 4-6 days. The prepared phalloidin working solution was added and the F-actin ring area of ​​osteoclasts in each group was calculated. The results are shown in Table 1. Figure 2 .

[0041] according to Figure 2 The results showed that the use of triptolide or medipterostilbene alone could inhibit the F-actin ring of osteoclasts induced by receptor activator of nuclear factor κ-B ligand (RANKL) and reduce the area of ​​F-actin ring in a dose-dependent manner. It is worth noting that the inhibition of the F-actin ring area of ​​osteoclasts in the combination group of triptolide and medipterostilbene was statistically different from that in the single-use group. The bone slice results showed that the combined group had a more obvious inhibitory effect on bone resorption than the single-use group.

[0042] Example 3: Effects of Chinese medicine monomer composition on osteoclast differentiation and absorption-related mRNA according to Figure 1 and 2 Results: 10 nM triptolide and 10 μM medipterostilbene were selected as the combined drug group, osteoclast RNA was extracted, and RT-qPCR was used to detect the effects of Nfatc1, C-fos, Atp6v0d2, Dc-stamp, and Ctsk mRNA. The results were as follows Figure 3 .

[0043] The specific groups are: Normal group: macrophage colony-stimulating factor (M-CSF) 25 ng / mL; Model group: macrophage colony-stimulating factor (M-CSF) 25 ng / mL, receptor activator of nuclear factor κ-B ligand (RANKL) 50 ng / mL; Triptolide group: macrophage colony-stimulating factor (M-CSF) 25 ng / mL, receptor activator of nuclear factor κ-B ligand (RANKL) 50 ng / mL, triptolide 10 nM; Medipterocarb group: macrophage colony-stimulating factor (M-CSF) 25 ng / mL, receptor activator of nuclear factor κ-B ligand (RANKL) 50 ng / mL, and Medipterocarb 10 μM; Chinese medicine monomer composition group: macrophage colony stimulating factor (M-CSF) 25ng / mL, receptor activator of nuclear factor κ-B ligand (RANKL) 50ng / mL, triptolide 10nM, and meditartin 10μM.

[0044] according to Figure 3 The results showed that triptolide and medipterostilbene had an inhibitory effect on Nfatc1, C-fos, Atp6v0d2, Dc-stamp, and Ctsk mRNA in osteoclasts when used alone or in combination, and the inhibitory effect of the Chinese medicine monomer combination on Nfatc1, Atp6v0d2, Dc-stamp, and CtskmRNA was significantly better than that of the single drug group.

[0045] The above studies show that triptolide and medipterostilbene have a synergistic effect when used together at lower concentrations to inhibit osteoclast differentiation. The combined effect makes triptolide and medipterostilbene have application prospects in the form of a combination to be developed into a drug for the treatment of rheumatoid arthritis.

[0046] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A Chinese medicine monomer composition for treating rheumatoid arthritis, characterized in that: The traditional Chinese medicine monomer composition consists of triptolide and meditartin.

2. The Chinese medicine monomer composition according to claim 1, characterized in that: The molar ratio of triptolide to mediterranean is 1:500-2000.

3. The Chinese medicine monomer composition according to claim 2, characterized in that: The molar ratio of triptolide to medipterostilbene is 1:1000.

4. The Chinese medicine monomer composition according to claim 1, characterized in that: The concentration of the triptolide is 5-20 nM, and the concentration of the mediterranean is 5-20 μM.

5. The Chinese medicine monomer composition according to claim 4, characterized in that: The concentration of the triptolide is 5-20 nM, and the concentration of the mediterranean is 5-20 μM.

6. The Chinese medicine monomer composition according to claim 5, characterized in that: The concentration of the triptolide is 10-20 nM, and the concentration of the mediterranean is 10-20 μM.

7. The Chinese medicine monomer composition according to claim 5, characterized in that: The concentration of triptolide is 10 nM, and the concentration of medipatrin is 10 μM; Or, the concentration of triptolide is 10 nM, and the concentration of medipatorhinol is 20 μM; Or, the concentration of triptolide is 20 nM, and the concentration of medipatrin is 5 μM; Or, the concentration of triptolide is 20 nM, and the concentration of medipatorhinol is 10 μM; Or, the concentration of triptolide is 20 nM, and the concentration of meditartin is 20 μM.

8. Use of the Chinese medicine monomer composition according to any one of claims 1 to 7 in the preparation of a medicine for treating rheumatoid arthritis.

9. A drug for treating rheumatoid arthritis, characterized in that: The medicine comprises the Chinese medicine monomer composition according to any one of claims 1 to 7 and pharmaceutically acceptable excipients.

10. The drug according to claim 9, characterized in that: The auxiliary materials are one or more of solvents, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids or release retardants.

Images