Use of tetrahydroharman in the preparation of a medicament for the treatment of rheumatoid arthritis

Abstract

The present application belongs to the technical field of medicine, and particularly relates to the use of tetrahydrojatrorrhizine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating rheumatoid arthritis. Experimental results show that tetrahydrojatrorrhizine can effectively alleviate the foot swelling level of CIA rats, reduce the arthritis score, and inhibit the progression of rheumatoid arthritis. Blood routine analysis shows that the administration of tetrahydrojatrorrhizine reduces the number of immune cells in the blood and weakens the immune level of the body. In addition, the experimental results also show that tetrahydrojatrorrhizine can inhibit synovial hyperplasia and relieve joint damage. Therefore, tetrahydrojatrorrhizine has the prospect of being developed into an oral medicament for treating rheumatoid arthritis, provides more treatment options for clinically treating rheumatoid arthritis, and has important social and economic values.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of tetrahydropyridine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a common inflammatory autoimmune disease. Epidemiological studies show that the incidence of RA is 0.5% to 1%, and the incidence in women is about three times that in men. RA is characterized by persistent synovitis, systemic inflammation, and continuous production of autoantibodies. It is a chronic autoimmune disease with a protracted course. Most patients experience recurrent symmetrical pain, swelling, and dysfunction in the small joints of the hands and feet, accompanied by morning stiffness. Its pathological features are mainly inflammatory cell infiltration and synovial cell proliferation, affecting multiple joints throughout the body, impacting the patient's normal life, and in severe cases, leading to joint deformities and disability.

[0003] Contrary to popular belief, rheumatoid arthritis (RA) and osteoarthritis are two diseases with significant differences in their manifestations and treatments. Osteoarthritis is a non-infectious, chronic, and progressive disease characterized by degenerative changes in the joints, with the knee joint being the most commonly affected. Its causative factors are related to age, weight, trauma, and the intensity of physical activity. Rheumatoid arthritis, on the other hand, is a systemic autoimmune disease primarily characterized by synovitis, manifesting mainly as synovial hyperplasia and joint damage in the fingers, toes, wrists, and ankles. Its causative factors are not yet fully understood. Synovial cell proliferation is an important cellular process in the development of rheumatoid arthritis, eroding cartilage and leading to bone damage, while the degeneration of joints in osteoarthritis is less related to synovial cells.

[0004] The goal of RA treatment is to control inflammation while preserving joint structure and function, preventing structural damage, and restoring normal bodily functions to maximize quality of life. Currently, clinical treatment primarily uses glucocorticoids, anti-inflammatory drugs, and disease-modifying antirheumatic drugs (DMARDs) to treat RA. Furthermore, in the context of precision medicine, biologics and targeted therapies will be future directions for RA treatment. However, RA is a chronic disease requiring long-term medication to control its progression. Long-term, high-dose administration can lead to gastrointestinal reactions and damage to the liver, kidneys, and blood systems. Therefore, finding novel drugs with higher efficacy and lower side effects remains a key direction for the future prevention and treatment of RA.

[0005] Natural product monomers are monomeric components extracted and isolated from natural products. They may possess a variety of pharmacological activities, such as antioxidant, antitumor, antibacterial, and anti-inflammatory effects. Due to their single component and minimal side effects, they are valuable resources for new drug development. Tetrahydrocorypalmine is an isoquinoline alkaloid derived from traditional Chinese medicines such as *Xia Tian Wu*, *Huang Lian*, and *Yan Hu Suo*. Its molecular formula is C1. 20 H 23 NO4, its chemical structural formula is shown below:

[0006]

[0007] Chinese patent CN 111184719A discloses a dopamine receptor antagonist and its uses. The dopamine receptor antagonist is D-tetrahydrodramine and its pharmaceutically acceptable salts or precursor compounds. This dopamine receptor antagonist can antagonize the calcium ion influx caused by dopamine receptor activation. Animal experiments have demonstrated that D-tetrahydrodramine, as a dopamine receptor antagonist, has good analgesic effects on acute pain, inflammatory pain, and bone cancer pain. Simultaneously, D-tetrahydrodramine also has anti-inflammatory, antiviral, and antiarrhythmic effects. Furthermore, Chinese patent CN117379379A discloses the role of nanoparticles containing tetrahydrodramine in the treatment of osteoarthritis.

[0008] Currently, there are no reports on the use of tetrahydrocannabinoids in the treatment of rheumatoid arthritis. Summary of the Invention

[0009] The purpose of this invention is to address the current shortage of highly effective, safe, and inexpensive oral medications of natural origin for the treatment of rheumatoid arthritis available for clinical use. This invention aims to achieve in-depth development and utilization of natural products using modern pharmaceutical research methods, combined with extensive pharmacodynamic experiments, to provide the use of tetrahydroraconazole in the preparation of drugs for the treatment of rheumatoid arthritis.

[0010] Specifically, the present invention is achieved through the following technical solutions:

[0011] This invention provides the use of tetrahydrodraconine or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating rheumatoid arthritis.

[0012] Alternatively, in the above-described uses, the tetrahydroraffinate is L-tetrahydroraffinate or D-tetrahydroraffinate.

[0013] Preferably, the pharmaceutically acceptable salt is selected from hydrochloride salts.

[0014] As an alternative, in the above-mentioned uses, the tetrahydrophane alkaloid inhibits the progression of rheumatoid arthritis, reduces the number of immune cells in the blood, weakens the body's immune level, inhibits synovial hyperplasia, and relieves joint damage.

[0015] Alternatively, in the above-described uses, the drug comprises a therapeutically effective amount of tetrahydrodraconine and a pharmaceutically acceptable carrier.

[0016] Alternatively, in the above-described uses, the medicine may also contain other natural products commonly used in the clinical treatment of rheumatoid arthritis.

[0017] Alternatively, in the above-described uses, the active ingredient in the drug consists of tetrahydrodrae and other natural products.

[0018] Alternatively, in the above uses, the other natural products are selected from one or more of the following: oridonin, tripterygin, resveratrol, perillyl alcohol, curcumin, decalinine, sophora root pine olein, punicin, gentianic acid, or naringin.

[0019] Preferably, the other natural product is sophora root phenoxyline.

[0020] Alternatively, in the above-mentioned uses, the weight ratio of the tetrahydroraconazole to the other natural products is 1:10 to 10:1.

[0021] Preferably, the weight ratio of the tetrahydroraconazole to the other natural products is 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0022] Alternatively, in the above-mentioned uses, when tetrahydroraconazole is used in combination with other natural products, the dosage forms of the former and the latter can be the same or different. Furthermore, the former and the latter can be administered simultaneously or separately.

[0023] Alternatively, in the above-described uses, the drug may be in oral dosage form.

[0024] Alternatively, in the above-described uses, the oral dosage form is selected from oral liquids, tablets, powders, capsules, or granules.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) Combining my country’s advantages in natural product research, this invention has discovered a new use of the natural product tetrahydrodrynine in the treatment of rheumatoid arthritis for the first time.

[0027] (2) Experimental results showed that tetrahydroraconazole could effectively alleviate paw edema in CIA rats, reduce arthritis scores, and inhibit the progression of rheumatoid arthritis. Blood routine analysis showed that administration of tetrahydroraconazole reduced the number of immune cells in the blood and weakened the body's immune level. In addition, experimental results also showed that tetrahydroraconazole could inhibit synovial hyperplasia and alleviate joint damage.

[0028] (3) Tetrahydropyridine has the potential to be developed into an oral drug for the treatment of rheumatoid arthritis, providing more treatment options for the clinical treatment of rheumatoid arthritis, and has important social and economic benefits.

[0029] (4) In addition, the present invention has also found that the combination of tetrahydropyrrolizine with certain natural products (in particular, sophora root pine ... Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 Changes in paw volume of rats' hind paws during drug administration. *, p < 0.5; **, p < 0.01, compared with the CIA group.

[0032] Figure 2 Changes in the thickness of the hind paw of rats during drug administration. *, p < 0.5; **, p < 0.01, compared with the CIA group.

[0033] Figure 3 Changes in arthritis scores in rats during drug administration. *, p < 0.5; **, p < 0.01, compared with the CIA group.

[0034] Figure 4 Micro-CT analysis of rat ankle joints (bone injury assessment) after drug administration.

[0035] Figure 5 HE staining analysis of rat ankle joints after drug administration (evaluation of synovial hyperplasia).

[0036] Figure 6 Safranin-Fix-Green staining analysis of rat ankle joints after drug administration (evaluation of cartilage damage).

[0037] Figure 7Immunohistochemical analysis of S100A4 markers (synovial fibroblast markers) in the ankle joint of rats after drug administration.

[0038] Figure 8 Immunohistochemical analysis of Vimentin in the ankle joint of rats after drug administration (analysis of synovial fibroblast markers).

[0039] Figure 9 Tetrahydropyridine inhibits the migration of synovial fibroblasts.

[0040] Figure 10 Statistical graph of tetrahydrocannabinoids inhibiting synovial fibroblast migration. **, p<0.01, compared with the 0-dose group.

[0041] Figure 11 MTT assay for the inhibition of synovial fibroblast proliferation by tetrahydrocannabinoids. **, p < 0.01, compared with the 0-dose group.

[0042] Figure 12 Edu staining analysis of tetrahydrocannabinoids inhibiting synovial fibroblast proliferation.

[0043] Figure 13 Statistical analysis of edu staining on the inhibitory effect of tetrahydrocannabinoids on synovial fibroblast proliferation. *, p < 0.5; **, p < 0.01, compared with the 0-dose group. Detailed Implementation

[0044] The inventors employed modern pharmaceutical research methods to deeply develop and utilize natural products. Through extensive screening, they discovered for the first time that oral tetrahydrocannabinoids can effectively treat rheumatoid arthritis. Based on this, the present invention was completed.

[0045] The preferred dosage form of the drug of the present invention is an oral dosage form.

[0046] The oral dosage form is a capsule, tablet, granule, or oral liquid. Tablets or capsules are preferred.

[0047] The pharmaceutically acceptable carrier refers to a conventional drug carrier in the field of pharmaceutical formulations, selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, wetting agents, pigments, flavoring agents, solvents, and surfactants.

[0048] The fillers described in this invention include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; the lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, poloxamer, etc.; the binders include, but are not limited to, water, ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidone, etc.; the disintegrants include, but are not limited to, starch effervescent mixtures, i.e., sodium bicarbonate and citric acid, tartaric acid, low-substituted hydroxypropyl cellulose, etc.; the suspending agents include, but are not limited to, polysaccharides such as farnesian gum, agar, alginic acid, cellulose ether, and carboxymethyl chitosan, etc.; and the solvents include, but are not limited to, water, balanced salt solutions, etc.

[0049] The drug can be formulated into various solid oral dosage forms, liquid oral dosage forms, etc. Pharmaceutically acceptable solid oral dosage forms include: ordinary tablets, dispersible tablets, enteric-coated tablets, granules, capsules, pellets, powders, etc., while liquid oral dosage forms include oral liquids, emulsions, etc.

[0050] The above dosage forms can be prepared using conventional processes in the pharmaceutical formulation field.

[0051] "Tetrahydroraphrine" can be extracted and isolated from plants containing the active ingredient, such as *Ipomoea aquatica*, using conventional natural product extraction methods in the field, or it can be purchased from commercially available products.

[0052] In the pharmaceutical applications described herein, the timing, frequency, and duration of administration of tetrahydrocannabinoids should be determined based on the specific diagnostic results of the condition, which is within the technical scope of those skilled in the art.

[0053] Applying treatment regimens for rats to humans allows for the conversion of the effective human dose of all drugs to the effective rat dose, which is easily achievable by those skilled in the art.

[0054] To better understand the essence of the present invention, the following detailed description section uses pharmacodynamic experiments and their results to further illustrate the novel use of tetrahydrodraconine in the pharmaceutical field for the treatment of rheumatoid arthritis.

[0055] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0056] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0057] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0058] In the following embodiments, for the purpose of saving costs, this application and another patent application filed by the inventor on the same day for corydalis alkaloids share the normal control group, model group, positive drug control group and some natural product monomers for combined use.

[0059] Example 1: In vivo efficacy study of tetrahydrodraconine in a collagen-induced rheumatoid arthritis animal model

[0060] 1.1 Experimental Methods:

[0061] Rheumatoid arthritis is an autoimmune disease primarily characterized by synovitis, with pathological manifestations including enhanced immunity, synovial hyperplasia, and bone destruction. A collagen-induced rat arthritis model (CIA model) can be used to simulate rheumatoid arthritis.

[0062] SD rats, 180-200g, were obtained from the Department of Laboratory Animal Science, Peking University School of Medicine. A total of 75 rats, 8 weeks old, male, SPF grade, were divided into 5 groups.

[0063] Animal groups: normal control group, 15 animals / group; CIA model group, 15 animals / group; positive drug group (methotrexate, 1 mg / kg / 2d), 15 animals / group; drug administration group (L-tetrahydropyrrolizine, 5 mg / kg / d, 20 mg / kg / d), divided into 2 groups, 15 animals / group in each group.

[0064] Administration method: Gavage.

[0065] Procedure: Animals were first divided into 7 groups according to the grouping plan and were acclimatized for 6 days to adapt to the environment. The animal room was kept in a 12-hour light and 12-hour dark cycle. During this period, they were allowed free food and water. The experiment used bovine type II collagen injection at the base of the tail to create a model. The specific method is as follows: (1) Bovine type II collagen solution: Bovine type II collagen was dissolved in an acetic acid aqueous solution with pH=3 and the concentration was 2mg / mL. The solution was shaken at 4℃ for 2 hours. (2) Emulsion I: 2mg / mL bovine type II collagen solution was mixed with complete Freund's adjuvant in a 1:1 ratio and vortexed. (3) Emulsion II: 2mg / mL bovine type II collagen solution was mixed with incomplete Freund's adjuvant in a 1:1 ratio and vortexed. After anesthetizing the SD rats with an anesthesia machine, 0.1mL of Emulsion I was subcutaneously injected into the base of the tail of the model group and the drug administration group. Seven days later, the survival status of the rats was observed. If no abnormalities were found, the SD rats were anesthetized again using an anesthesia machine, and 0.1 mL of emulsion II was subcutaneously injected into the tail base of both the model group and the treatment group. Seven days later, the model was successfully established (the signs of successful arthritis model establishment: significant swelling of the hind leg toes, inability to bend the hind leg joints, and crawling gait). Grouped drug administration then began.

[0066] Animals in the treatment group were administered the drug via gavage, while the control and model groups were given only an equal volume of 0.5% CMC-Na solution. During the treatment period, paw swelling levels, foot thickness, and arthritis scores were monitored. Four weeks after administration, the animals were anesthetized, and micro-CT scans of the hind limbs were performed. Blood was collected from the orbital fossa, and the animals were euthanized.

[0067] Animal handling and subsequent analysis: After anesthesia, blood was collected from the orbital cavity, serum was separated, and the animals were euthanized. Organs such as the spleen, thymus, and hind limbs were harvested. Three animals from each group were randomly selected for paraffin embedding. The ankle joints were harvested for HE staining, Safranin-Fix-Green staining, and immunohistochemical analysis of S100A4 and vimentin.

[0068] Experimental data representation: Foot swelling level (foot volume), foot thickness, and arthritis score records are shown in [link to relevant documentation]. Figures 1 to 3 The mean ± standard deviation of each group's data is given at each time point. Anticoagulation was detected using a complete blood count analyzer, and the results are reported in Table 1. Statistical analysis of the experimental data was performed using GraphPad-Prism 6.0 software. The method used was a one-way ANOVA t-test to compare whether there were significant differences in the means of each column; p < 0.05 was considered significant.

[0069] 1.2 Experimental Results:

[0070] The results show (see) Figures 1 to 3In CIA rats, paw volume, paw thickness, and arthritis scores were significantly higher than in the normal group, and these values ​​did not decrease during the administration period. Tetrahydrocannabinoids (CP) effectively alleviated paw swelling in CIA rats, reduced arthritis scores, and inhibited the progression of rheumatoid arthritis. The high-dose group showed better efficacy than the positive control group.

[0071] Blood routine analysis showed that administration of tetrahydrocannabinoids reduced the number of immune cells (neutrophils, lymphocytes) in the blood (see Table 1), weakening the body's immune level.

[0072] Table 1: Results of routine blood analysis in various experimental animals after administration of tetrahydrocannabinoids

[0073]

[0074]

[0075] CT analysis showed reduced bone damage (see...) Figure 4 HE staining showed that synovial hyperplasia was inhibited (see...). Figure 5 Safranin-Fix-Green staining showed that it alleviated cartilage damage in CIA rats (see...). Figure 6 Immunohistochemistry with S100A4 and vimentin showed that synovial fibroblast proliferation was inhibited (see S100A4 and vimentin immunohistochemistry). Figures 7 to 8 ).

[0076] Example 2: In vitro efficacy study of tetrahydrocannabinoids in primary synovial fibroblasts 2.1 Experimental methods:

[0077] The cells were derived from primary synovial fibroblasts of rats with rheumatoid arthritis.

[0078] Primary rat synovial fibroblast culture: Synovial tissue from the hind foot of rats was digested with type II collagenase at 37℃ for 2 hours. The supernatant was filtered through a filter and centrifuged at 1000×g for 2 minutes to collect synovial fibroblasts. The cells were cultured in DMEM complete medium (DMEM high glucose medium supplemented with a mixture of 10% fetal bovine serum and 1% penicillin and streptomycin) under the following conditions: 37℃, 5% CO2 and 95% humidity.

[0079] (1) MTT experiment

[0080] Synovial fibroblasts were seeded in 48-well plates at a density of 2 × 10⁶ cells / well. 4After 12 hours of growth in an incubator, control and treatment groups were randomly assigned to four wells. The control group received no treatment, while the treatment groups were treated with tetrahydroraffinin at concentrations of 1.5, 3, and 6 μM. After 24 hours of tetrahydroraffinin treatment, the original culture medium in the 48-well plates was carefully aspirated, and DMEM high-glucose medium containing 50 μg / mL MTT was added. The plates were incubated at 37°C, 5% CO2, and 95% humidity in the dark for 4 hours. The culture medium in the 48-well plates was carefully aspirated, and 350 μL of DMSO was added to dissolve the purple crystals. The absorbance of the samples was measured using a multi-mode microplate reader at a wavelength of 570 nm. Cell viability was calculated as (absorbance of treatment group - blank) / (absorbance of control group - blank) * 100%.

[0081] (2) Wound healing experiment

[0082] Synovial fibroblasts were seeded in 12-well plates at a density of 5 × 10⁶ cells / well. 5 After the cells reached confluence in each well, uniform wounds were made using a sterile 200 μL pipette tip. After washing twice with PBS, control and treatment groups were randomly assigned. The control group received no treatment, while the treatment groups received tetrahydrophagocytic alkaloid treatment at concentrations of 1.5, 3, and 6 μM. After 24 h of tetrahydrophagocytic alkaloid treatment, the culture medium in the 12-well plates was discarded, and the cells were fixed with 4% paraformaldehyde solution for 30 min. Images were captured using a Zeiss microscope, and ImageJ software was used to analyze relative migration rate. Relative migration rate = (initial wound area - wound area after healing) / initial wound area * 100%.

[0083] (3) Edu experiment

[0084] Synovial fibroblasts were seeded in 24-well plates at a density of 4 × 10⁶ cells / well. 4After 12 hours of growth in an incubator, control and treatment groups were randomly assigned to each well, with three replicates per well. The control group received no treatment, while the treatment groups received tetrahydroraffinate at concentrations of 1.5, 3, and 6 μM. After 4 hours of culture, 10 μM EdU solution preheated to 37°C was added. After 20 hours of incubation, the culture medium in the 24-well plate was carefully aspirated, and cells were fixed with 4% paraformaldehyde solution at room temperature for 40 minutes. After washing the cells twice with PBS, wells were punched with 0.5% Triton X-100 PBS solution at room temperature for 40 minutes. After washing the cells twice with PBS, 400 μL / well Click reaction solution (a mixture of 430 μL Click Reaction Buffer, 20 μL CuSO4, 1 μL Alexa Fluor 488, and 50 μL Click Additive Solution) was added under light-protected conditions, and the reaction was carried out at room temperature under light-protected conditions for 30 minutes. Discard the Click reaction solution, wash three times with pre-cooled PBS, add 400 μL of Hoechst 33342 diluted 1:1000 with PBS to each well, incubate at room temperature in the dark for 10 min, wash thoroughly with PBS, and image using a Zeiss fluorescence microscope. ImageJ software was used to analyze the number of EDU-positive cells. The excitation / emission wavelengths for the Alexa Fluor 488 were 495 nm / 520 nm, and for Hoechst 33342, they were 360 ​​nm / 450 nm. EDU-positive cell rate = (Number of 488-positive cells / Number of nuclei) * 100%

[0085] The experimental data were statistically analyzed using GraphPad-Prism 6.0 software. The method used was a one-way ANOVA t-test to compare whether there was a significant difference in the mean of each column. p < 0.05 was considered to be a significant difference.

[0086] 2.2 Experimental Results:

[0087] Wound healing experiments show (see...) Figures 9 to 10 The wound healing rate in the drug-treated group was lower than that in the untreated group, indicating that CP inhibits synovial fibroblast migration. MTT assay (see...) Figure 11 ), edu proliferation experiment (see Figures 12 to 13 The number of edu-positive cells in the drug-treated group was significantly lower than that in the untreated group, indicating that CP inhibits the proliferation of synovial fibroblasts. CP can alleviate rheumatoid arthritis by inhibiting the proliferation of synovial fibroblasts.

[0088] Example 3: In vitro efficacy study of tetrahydropyrrolizine in combination with other natural products in primary synovial fibroblasts.

[0089] In this embodiment, the inventors also preliminarily investigated the effects of using tetrahydroraconazole in combination with other natural product monomers that may have therapeutic effects on rheumatoid arthritis (oridone, tripterygium lactone, resveratrol, perillyl alcohol, curcumin, decalinine, sophorabolic acid, punicin, gentianic acid, naringin).

[0090] 3.1 Experimental Methods:

[0091] The cells were derived from primary synovial fibroblasts of rats with rheumatoid arthritis.

[0092] Primary rat synovial fibroblast culture: Synovial tissue from the hind foot of rats was digested with type II collagenase at 37°C for 2 h. The supernatant was filtered through a filter and centrifuged at 1000×g for 2 min to collect synovial fibroblasts. The cells were cultured in DMEM complete medium (DMEM high glucose medium supplemented with a mixture of 10% fetal bovine serum and 1% penicillin and streptomycin) under the following conditions: 37°C, 5% CO2 and 95% humidity.

[0093] MTT Experiment

[0094] Synovial fibroblasts were seeded in 48-well plates at a density of 2 × 10⁶ cells / well. 4 After 12 hours of growth in an incubator, control and drug-treated groups were randomly assigned to four wells. The control group received no treatment, while the drug-treated groups received the various test natural product monomers at a concentration of 3 μM. After 24 hours of drug treatment, the original culture medium in the 48-well plate was carefully aspirated, and DMEM high-glucose medium containing 50 μg / mL MTT was added. The plate was incubated at 37°C, 5% CO2, and 95% humidity in the dark for 4 hours. The culture medium in the 48-well plate was carefully aspirated, and 350 μL of DMSO was added to dissolve the purple crystals. The absorbance of the samples was detected using a multi-mode microplate reader at a wavelength of 570 nm. Cell viability was calculated as: (Absorbance of drug-treated group - Blank) / (Absorbance of control group - Blank) * 100%.

[0095] The experimental data were statistically analyzed using GraphPad-Prism 6.0 software. The method used was a one-way ANOVA t-test to compare whether there was a significant difference in the mean of each column. p < 0.05 was considered to be a significant difference.

[0096] The synergy index is determined using the Jin Zhengjun q-value method, and the q-value is obtained by the following formula:

[0097] q = P A+B / (P A +P B -P A ×P BIn the formula, P A P B and P A+B The treatment improvement rates are categorized into drug A group, drug B group, and the combination of the two drugs, respectively. q < 1 indicates an antagonistic effect after the two drugs are used together; q > 1 indicates a synergistic effect after the two drugs are used together; and q = 1 indicates an additive effect after the two drugs are used together.

[0098] 3.2 Experimental Results:

[0099] The experimental results are shown in Table 2 below.

[0100] Table 2. Survival rate of synovial fibroblasts in each drug group (%)

[0101]

[0102]

[0103] Experimental results show that although natural product monomers such as oridonin and tripterygium lactone may inhibit the proliferation of rat synovial fibroblasts to varying degrees when used alone, the combination of these natural product monomers with tetrahydroraconazole may produce three results: antagonistic, additive, and synergistic effects. It is evident that the effect of combining tetrahydroraconazole with other natural products on rat synovial fibroblasts is difficult to predict.

[0104] As shown in Table 2, the combined use of tetrahydropyrrolizine and sophora root pine tinctoria may have a synergistic effect in the treatment of rheumatoid arthritis.

[0105] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of a combination of tetrahydroharman and sophorubin in the manufacture of a medicament for the treatment of rheumatoid arthritis, characterized in that: The weight ratio of the tetrahydrooxymatrine to the sophoridine pheffinol is 3:

1.

2. Use according to claim 1, characterized in that: The tetrahydrooxymatrine is L-tetrahydrooxymatrine or D-tetrahydrooxymatrine.

3. Use according to claim 1, characterized in that: The tetrahydrooxymatrine inhibits rheumatoid arthritis progression, reduces the number of immune cells in the blood, and weakens the immune level of the body. The tetrahydrooxymatrine inhibits synovial hyperplasia and relieves joint damage.

4. Use according to claim 1, characterized in that: The medicine comprises a therapeutically effective amount of tetrahydrooxymatrine and a pharmaceutically acceptable carrier.

5. Use according to claim 1, characterized in that: The medicine is in an oral dosage form.

6. Use according to claim 5, characterized in that: The oral dosage form is selected from an oral liquid, a tablet, a powder, a capsule, or a granule.

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