Tripterine sulfated metabolite and application thereof in preparation of medicine for resisting rheumatoid arthritis

Abstract

The invention provides a tripterine sulfated metabolite and application thereof in preparation of a medicine for resisting rheumatoid arthritis, and belongs to the technical field of biological medicine. The tripterine sulfated metabolite is 10-sulfated tripterine which is formed by connecting a HSO3 <-> to the 10-position carbon of the tripterine. Compared with the tripterine, the 10-sulfonated tripterine has no obvious difference in anti-rheumatoid arthritis pharmacological activity, however, the hepatotoxicity in vivo and the cytotoxicity in vitro of the 10-sulfonated tripterine are obviously reduced. The tripterine sulfated metabolite is efficient and low in toxicity, and a wide application prospect is developed for treatment of rheumatoid arthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a celastrol sulfonated metabolite with anti-rheumatoid arthritis activity and significantly reduced toxicity, and its application. Background Art

[0002] Rheumatoid arthritis, as a typical autoimmune disease, is mainly characterized by erosive arthritis, and its root cause lies in synovitis. In the initial stage of the disease, the joints of patients often show symptoms such as morning stiffness, swelling, and pain. As the disease progresses, it may ultimately lead to joint deformity and then loss of normal function. The collagen-induced arthritis (CIA) model is a classic animal model commonly used in the study of rheumatoid arthritis. This model is widely used to study the pathogenesis of rheumatoid arthritis, the disease process, and the efficacy evaluation of new drugs.

[0003] Celastrol (CEL) is a pentacyclic triterpenoid compound extracted from the roots of the plant Tripterygium wilfordii, and has various pharmacological activities such as anti-rheumatoid arthritis, anti-tumor, and anti-obesity. However, celastrol can also cause serious organ toxicity to the liver, heart, and kidneys, especially significant hepatotoxicity, which greatly hinders the wide clinical application and promotion of celastrol. Therefore, how to effectively reduce its toxicity while maintaining or enhancing the pharmacological activity of celastrol has become a technical bottleneck that needs to be overcome urgently, and is of great significance for expanding its clinical application.

[0004] C-sulfonation metabolism is a rare biological metabolic process, in which a sulfur atom with a lone pair of electrons attacks the β-carbon atom of an α,β-unsaturated carbonyl compound, and then a sulfonated metabolite is generated.

[0005] The present invention provides a C-sulfonated metabolite of celastrol, and comprehensively evaluates its toxicity and anti-rheumatoid arthritis activity. Summary of the Invention

[0006] Aiming at the problems existing in the above-mentioned prior art, in order to solve the problem of effectively reducing the toxicity of celastrol while maintaining or enhancing its pharmacological activity, the purpose of the present invention is to design and provide a highly efficient and low-toxic celastrol sulfonated metabolite with anti-rheumatoid arthritis, its preparation method, and application.

[0007] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides a tripterine sulfonated metabolite, and the tripterine sulfonated metabolite is 10-sulfonated tripterine; that is, an HSO3 is connected to the 10th carbon of tripterine - , forming 10-sulfonated tripterine.

[0009] The chemical formula of the 10-sulfonated tripterine is shown as the following formula (Ⅰ):

[0010]

[0011] On the second hand, the present invention provides a preparation method of the tripterine sulfonated metabolite as described above, comprising the following steps:

[0012] Under nitrogen protection, tripterine is added to methanol, stirred and dissolved, then sodium bisulfite or potassium bisulfite is added, dissolved in water, stirred at room temperature, dried, dissolved in methanol, centrifuged, and the supernatant is taken and dried to obtain the tripterine sulfonated metabolite.

[0013] In the described preparation method, the mass ratio of tripterine to sodium bisulfite or potassium bisulfite is 50-200:10-50;

[0014] The time of stirring at room temperature is 6-24 h;

[0015] The conditions of centrifugation are: rotation speed 5000-10000 rpm, time 5-20 min.

[0016] On the third hand, the present invention provides an application of the tripterine sulfonated metabolite or its derivative as described above in the preparation of a product for treating rheumatoid arthritis.

[0017] On the fourth hand, the present invention provides an application of the tripterine sulfonated metabolite or its derivative as described above in the preparation of a product for treating anti-tumor or anti-obesity.

[0018] On the fifth hand, the present invention provides an application of the tripterine sulfonated metabolite or its derivative as described above as a low-toxicity efficacy enhancer of a tripterine product.

[0019] On the sixth hand, the present invention provides a product for treating rheumatoid arthritis and / or anti-tumor and / or anti-obesity, comprising the tripterine sulfonated metabolite as described above and a pharmaceutically acceptable excipient.

[0020] The described product for treating rheumatoid arthritis and / or anti-tumor and / or anti-obesity, wherein the pharmaceutically acceptable excipients are at least one of solvent, binder, diluent, wetting agent, lubricant, emulsifier, thickening agent, excipient, suspending agent, disintegrant, filler, preservative, pH regulator, osmotic pressure regulator, surfactant, coating material, antioxidant, bacteriostatic agent or buffer.

[0021] The described product for treating rheumatoid arthritis and / or anti-tumor and / or anti-obesity, wherein the dosage form of the product is at least one of suspension, granule, capsule, powder, tablet, emulsion, solution, dripping pill, injection, oral preparation, suppository, enema, aerosol, patch or drop.

[0022] The described product for treating rheumatoid arthritis and / or anti-tumor and / or anti-obesity, wherein the administration method of the product is at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, nebulization administration or transdermal administration.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention first discovers that the sulfonated metabolite of celastrol is 10-sulfonate celastrol (CELS). Compared with celastrol, the anti-rheumatoid arthritis activity of 10-sulfonate celastrol has no significant difference, but the in vivo liver toxicity and in vitro hepatocyte toxicity are greatly reduced. The sulfonated metabolite of celastrol described in the present invention has the potential to be prepared into a new anti-rheumatoid arthritis drug, providing experimental data support for further clinical use and promotion. Description of the Drawings

[0025] Figure 1 For the identification of the sulfonated metabolite of celastrol (CELS), wherein, A. High-resolution mass spectrometry identification of the sulfonated metabolite of celastrol: secondary mass spectrometry diagrams of the prototype and the metabolite; B. 1H NMR spectra of celastrol and the sulfonated metabolite; C. C-position sulfonation metabolic process of celastrol;

[0026] Figure 2 For the comparison of CELS and CEL liver injury, wherein, A. Survival curve of mice; B. ALT level in plasma; C. AST level in plasma; D. Histological score map of mouse liver; E. H&E staining map of mouse liver and statistical chart of the area of liver necrosis region (original magnification 200 times);

[0027] Figure 3Comparison of cytotoxicity of CEL and CELS, including: A. Cell viability of L02 cells treated with CEL; B. Cytotoxicity of L02 cells treated with CELS; C. Calcein AM / PI staining of L02 cells treated with CEL; D. Calcein AM / PI staining of L02 cells treated with CELS;

[0028] Figure 4 Figure 2 is a graph showing the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels in L02 cells treated with CEL and CELS, where A. MMP level in L02 cells treated with CEL; B. MMP level in L02 cells treated with CELS; C. ROS level in L02 cells treated with CEL; D. ROS level in L02 cells treated with CELS;

[0029] Figure 5 Comparison of the efficacy of CEL and CELS against rheumatoid arthritis, including: A. Photographs of mouse hind paws; B. Mouse foot thickness; C. RA score; D. Body weight; E. Mouse plasma IL-6 level; F. Mouse plasma TNF-α level; G. Mouse paw histological score; H. Mouse paw HE staining. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0031] 1. Drugs and reagents: The standard product of tripterygium wilfordii (purity>99%) was purchased from Chengdu Pufeide Biotechnology Co., Ltd.; 10-sulfonated tripterygium wilfordii was synthesized in the laboratory (purity>98%); methotrexate was purchased from Shanghai Shanghai Pharmaceuticals Xinyi Pharmaceutical Co., Ltd.; bovine type II collagen, complete Freund's adjuvant, and incomplete Freund's adjuvant were purchased from Guangzhou Baben Biotechnology Co., Ltd. Sodium carboxymethyl cellulose (CMC-Na) was purchased from Sigma-Aldrich; sodium heparin was purchased from Dalian Meilun Biotechnology Co., Ltd.; DMEM culture medium was purchased from Gibco, USA; FBS serum was purchased from ZETA LIFE, USA; alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were purchased from Nanjing Jiancheng Bioengineering Institute; mitochondrial membrane potential kit and reactive oxygen species kit were purchased from Shanghai Biyuntian Biotechnology Co., Ltd.

[0032] 2. Instrumentation: The ultra-high resolution triple quadrupole mass spectrometer is from Thermo Fisher Scientific, USA; the 600 MHz nuclear magnetic resonance spectrometer is from Bruker (Beijing) Technology Co., Ltd.; the test tube vortex mixer is IKAMS3basic / MS3digital from Germany; the ultrapure water system is from Millipore, USA; the surgical instruments and syringes are from Ningbo Newzhon Biotechnology Co., Ltd.; the pipette is from Eppendorf, Germany; the -80 °C ultra-low temperature freezer is from Haier, China; the microplate reader is from Tecan, Switzerland.

[0033] 3. Statistical analysis: The experimental data in this invention are presented as Mean ± Standard Error of the Mean (Mean ± SEM). The Tukey method was used for the homogeneity of variance test. The significance criterion was α = 0.05, and a P value < 0.05 indicated a statistically significant difference.

[0034] Example 1: Preparation and Structural Identification of 10-Sulfo-celastrol

[0035] (1) Identification of Celastrol Sulfonation Metabolites by High-Resolution Mass Spectrometry

[0036] The chemical characteristics of celastrol sulfonation metabolites in mice were analyzed by a UHPLC-Orbitrap Fusion-HRMS system composed of a Vanquish UHPLC system (Thermo Fisher Scientific, USA) and an Orbitrap Fusion mass spectrometer system (Thermo Fisher Scientific, USA). Chromatographic separation experiments were carried out on a Hypersil Gold C18 column (100 × 2.1 mm, 1.9 μm, Thermo Scientific, USA). The mobile phase consisted of water (A, 0.1% formic acid) and acetonitrile (B), and the gradient elution was as follows: 0 - 1 min, 5 - 40% B; 1 - 2 min, 40 - 80% B; 2 - 6 min, 80% B; 6 - 8 min, 80 - 90% B; 8 - 9 min, 90 - 95% B; 9 - 10 min, 95% B. The flow rate was 0.3 mL / min. When the eluate was ionized by nano-spray (EasySpray ion source, Thermo Scientific) at a spray voltage of 3.5 kV and 2.5 kV in the negative ion mode and introduced into the mass spectrometer through a heated ion transfer tube (350 °C). The detection scan of small molecules in the m / z range of 100 - 1000 was carried out at a resolution of 60,000, and the AGC target for Orbitrap detection was 4.0 × 105 with a maximum injection time of 100 ms. The results are as Figure 1As shown in A, in the negative ion mode, the parent ion of the protriptophenol red prototype is 449.2690, and the main metabolite is 531.2429. Different from protriptophenol red, a fragment ion of 79.9568 (HSO3 - ) is produced by the metabolite, indicating that sulfonation metabolism of protriptophenol red has occurred.

[0037] (2) Preparation of sulfonated metabolite of protriptophenol red:

[0038] Under nitrogen protection, protriptophenol red (100 mg, 0.22 mmol) was added to 5 mL of methanol. After stirring and dissolving, sodium bisulfite (27 mg, 0.26 mmol) was added and dissolved in 2 mL of water. The mixture was stirred at room temperature for 12 - 24 h and then dried. The dried powder was dissolved in 10 mL of methanol, centrifuged at 8000 rpm for 10 min, and the supernatant was freeze-dried to obtain the product. Figure 1 In C, it is the C-position sulfonation metabolic process of protriptophenol red.

[0039] (3) NMR identification of 10-sulfonated protriptophenol red

[0040] The synthesized sulfonated metabolite of protriptophenol red was analyzed by 1H NMR spectroscopy. 1 The 1H NMR spectrum was detected on a BRUKER AVIII 400 MHz (Bruker Co.), with tetramethylsilane (TMS) as the internal standard. Chemical shift (δ) is in ppm and coupling constant (J) is in Hz. The detection results are as shown in Figure 1 B. It was analyzed that the sulfonated metabolite of protriptophenol red was identified as 10-sulfonated protriptophenol red, and the results are as follows:

[0041] CEL: 11H NMR (400 MHz, DMSO) δ 12.14 (s, 1H), 8.75 (s, 1H), 7.09 (d, J = 6.4 Hz, 1H), 6.40 (s, 1H), 6.37 (d, J = 6.5 Hz, 1H), 2.33 (d, J = 15.1 Hz, 1H), 2.22 (d, J = 9.4 Hz, 1H), 2.10 (s, 3H), 2.02 (s, 1H), 1.97 (d, J = 13.5 Hz, 1H), 1.89–1.80 (m, 1H), 1.69 (d, J = 8.5 Hz, 2H), 1.63 (d, J = 15.9 Hz, 2H), 1.58 (s, 1H), 1.56 (s, 2H), 1.44 (d, J = 14.4 Hz, 1H), 1.39 (s, 3H), 1.32 (d, J = 10.5 Hz, 1H), 1.23 (s, 3H), 1.12 (s, 3H), 1.08 (s, 3H), 0.89 (d, J = 13.4 Hz, 1H), 0.64 (s, 3H).

[0042] CELS: 1 1H NMR (400 MHz, DMSO) δ 11.99 (s, 1H), 8.83 (s, 1H), 7.66 (s, 1H), 6.59 (s, 1H), 5.81 (d, J = 6.1 Hz, 1H), 4.49 (d, J = 6.0 Hz, 1H), 2.33 (d, J = 14.7 Hz, 1H), 2.21 (s, 3H), 2.03 (d, J = 11.4 Hz, 1H), 1.97 (d, J = 11.4 Hz, 2H), 1.82 (s, 1H), 1.63 (s, 3H), 1.59 (s, 2H), 1.56 (d, J = 10.4 Hz, 2H), 1.49 (s, 2H), 1.48 (s, 2H), 1.41 (d, J = 13.0 Hz, 1H), 1.30 (d, J = 11.7 Hz, 1H), 1.19 (s, 3H), 1.10 (s, 3H), 1.06 (s, 3H), 0.86 (d, J = 10.3 Hz, 1H), 0.60 (s, 3H).

[0043] Example 2: Safety Study of 10-Sulfonated Tripterine

[0044] In this Example 2, the toxicity comparison study of 10-sulfonated tripterine prepared in Example 1 and tripterine was further carried out in C57 mice and normal human liver cells L02.

[0045] 1. Experimental Animals

[0046] SPF-grade male C57 mice, 6 - 8 weeks old, weighing 20 - 22 g.

[0047] 2. Drug Preparation

[0048] (1) Preparation of 0.5% CMC-Na solution: Accurately weigh 0.50 mg of CMC-Na powder, add it to 100 mL of distilled water and dissolve it. Vortex to mix evenly to prepare 0.5% CMC-Na solution, and store it at room temperature.

[0049] (2) Preparation of celastrol (CEL) solution: Accurately weigh 60.00 mg of CEL powder, add 2% DMSO + 98% solution containing 0.5% CMC-Na, and dissolve it by vortexing and ultrasonic treatment to prepare a CEL solution with a concentration of 6 mg / mL, and store it at 4°C for later use.

[0050] (3) Preparation of 10-sulfonated celastrol (CELS) solution: Accurately weigh 60.00 mg of CELS powder, add 2% DMSO + 98% solution containing 0.5% CMC-Na, and dissolve it by vortexing and ultrasonic treatment to prepare a CELS solution with a concentration of 6 mg / mL, and store it at 4°C for later use.

[0051] 3. Grouping and Administration

[0052] Experimental animals: Healthy male C57 mice were randomly divided into Control, CEL, and CELS groups according to body weight, with 6 mice in each group. Mice in the CEL group were orally administered celastrol (60 mg / kg), and mice in the CELS group were orally administered 10-sulfonated celastrol (60 mg / kg) once a day for 7 consecutive days. Mice in the Control group were given an equal volume of 0.5% CMC-Na solution.

[0053] 4. Collection and Detection of Plasma and Tissue Samples

[0054] (1) Sample collection: After continuously administering drugs to C57 mice for 7 days, all mice were euthanized, 500 μL of blood was collected from the eyeballs into heparin tubes, centrifuged at 5000 rpm at 4°C for 10 min, and the supernatant was taken into EP tubes and stored at -80°C for later use. Immediately separate the liver tissue, and fix a part of the liver tissue in 4% paraformaldehyde and store it at room temperature.

[0055] (2) Histopathological examination of liver tissue: Take part of the liver tissue fixed in 4% paraformaldehyde, embed the tissue in paraffin according to the conventional histopathological method, cut it into 4-μm thin sections to make slides. Subsequently, stain with hematoxylin and eosin (H&E staining), and observe the changes of liver tissue under a 200-fold microscope for each sample section for histopathological analysis.

[0056] (3) Biochemical index detection: Strictly follow the operating instructions of the kit to detect the levels of ALT and AST in mouse plasma. The levels of ALT and AST in mouse plasma are used as biochemical indicators, supplemented by H&E staining and the inflammatory infiltration and tissue pathological status of mouse liver to evaluate the severity of mouse liver injury.

[0057] The results are as follows Figure 2 As shown in the figure, at a dosage of 60 mg / kg, the mortality rate of mice in the CEL group reached 70%, while no mortality was observed in the CELS group. Compared with the Control group, the plasma ALT and AST in the CEL group were significantly increased, while the plasma ALT and AST in the CELS group were significantly lower than those in the CEL group. H&E staining results showed that multiple tissue necrosis, nuclear dissolution, increased cytoplasmic eosinophilia, and a small amount of inflammatory cell infiltration occurred in the liver of CEL mice; compared with the CEL model group, the necrosis area in the liver of mice given CELS 60 mg / kg was significantly reduced.

[0058] 5. Cell culture

[0059] Normal human liver cells L02 were cultured in DMEM complete medium containing 10% FBS and 100U / mL penicillin-streptomycin double antibody solution. The cells were cultured in a cell culture incubator at 5% CO2 and 37°C, and when they grew to 70%-80% confluence, they were digested and passaged with 0.25% trypsin.

[0060] 6. Detection of cytotoxicity indicators

[0061] (1) CCK8 assay for cell viability: When cells grew to 70%-80% confluence, they were digested with 0.25% trypsin for 2 min, centrifuged, mixed with 1 mL of whole culture medium, counted, and diluted to 1×10 5 / mL, the cells were inoculated in a 96-well plate, 100 μL per well, and the 96-well plate was incubated in a 5% CO2, 37°C incubator for 4-6 hours. Different concentrations of CEL (0, 1, 2, 4, 6, 8, 10 μM) / CELS (0, 1, 2, 4, 6, 8, 10 μM) were applied to the cells, and 5 replicate wells were set for each concentration. After incubation in a 5% CO2, 37°C incubator for 24 hours, a culture medium containing 10% CCK-8 was prepared and added in the form of liquid replacement, 100 μL per well, and the absorbance was measured at 450 nm after 1-2 hours to calculate the cell activity ( Figure 3 ).

[0062] (2) Cell viability and cytotoxicity assay: 2×10 5Cells at a density of

[0063] (3) Mitochondrial membrane potential (MMP) of cells: The drug treatment was the same as that for cell viability and cytotoxicity assays. After the drug treatment, the culture medium was aspirated, and the cells were washed once with PBS. Then, 500 μL of cell culture medium was added, followed by 1 mL of JC-1 staining working solution. The mixture was thoroughly vortexed and incubated in a 5% CO2, 37 °C incubator in the dark for 20 min. After incubation, the supernatant was aspirated, and the cells were washed twice with JC-1 staining buffer. Then, 1 mL of cell culture medium (which may contain serum and phenol red) was added, and the staining effect was observed under a fluorescence microscope (when the mitochondrial membrane potential is high, JC-1 can produce red fluorescence; when the mitochondrial membrane potential is low, JC-1 shows green fluorescence). The entire process had to be carried out in the dark. The results were as Figure 3 shown. CEL inhibited the viability of L02 cells in a concentration-dependent manner, with an IC 50 of 1.847 μM; CELS only showed a decrease in cell viability at 6 μM, with an IC 50 of 6.975 μM, which was significantly higher than that of the CEL group.

[0064] (4) Reactive oxygen species (ROS) in cells: The drug treatment was the same as that for cell viability and cytotoxicity assays. DCFH-DA (a ROS indicator) was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μM. After the drug treatment, the culture medium was aspirated, and the cells were washed once with PBS. Then, 500 μL of the diluted DCFH-DA was added, and the cells were incubated in a 5% CO2, 37 °C incubator in the dark for 30 min. The cells were washed three times with serum-free cell culture medium to thoroughly remove the DCFH-DA that had not entered the cells. Then, 500 μL of Hoechst solution was added, and the cells were incubated in the dark for 10 min. The cells were washed three times with serum-free cell culture medium, and the staining effect was observed under a fluorescence microscope (ROS in the cells can oxidize non-fluorescent DCFH to produce fluorescent DCF, generating green fluorescence, and Hoechst was used to localize the cell nucleus, producing blue fluorescence). The entire process had to be carried out in the dark. The results were as Figure 4As shown, when the drug concentration of the CEL group was 2 μM, the mitochondrial membrane potential of the cells decreased, and the intracellular reactive oxygen species (ROS) increased, showing a dose-dependent relationship. When the drug concentration in the CELS group was 2 μM and 4 μM, there was no difference in the mitochondrial membrane potential and ROS compared with the Control group. When the drug concentration was 8 μM, the mitochondrial membrane potential of the cells decreased and the intracellular reactive oxygen species increased.

[0065] In summary, compared with celastrol, the hepatotoxicity of 10-sulfonated celastrol was significantly reduced.

[0066] Example 3: Effects of 10-sulfonated celastrol on rheumatoid arthritis

[0067] 1. Experimental animals

[0068] SPF-grade male DBA / 1J mice, 6 - 8 weeks old, weighing 20 - 22 g.

[0069] 2. Drug preparation

[0070] (1) Preparation of celastrol (CEL) solution: Accurately weigh 6 mg of CEL powder, add 30 mL of 0.5% CMC-Na solution, vortex and ultrasonically dissolve to prepare a 0.2 mg / mL CEL solution, and store it at 4 °C for later use.

[0071] (2) Preparation of 10-sulfonated celastrol (CELS) solution: Accurately weigh 6 mg of the CELS powder prepared in Example 1, add 30 mL of 0.5% CMC-Na solution, vortex and ultrasonically dissolve to prepare a 0.2 mg / mL CELS solution, and store it at 4 °C for later use.

[0072] (3) Preparation of methotrexate (MTX) solution: Accurately weigh 6.00 mg of MTX, add 30 mL of normal saline, vortex and ultrasonically dissolve to prepare a 0.2 mg / mL MTX solution, and store it at 4 °C for later use.

[0073] 3. Induction of rheumatoid arthritis model

[0074] Primary immunization: Mix bovine type II collagen (CⅡ) and complete Freund's adjuvant (CFA) in a volume ratio of CⅡ:CFA = 1:1. The concentration of CFA is the same as that of CⅡ, which is 2 mg / mL. Inject 100 μL of the emulsion intradermally at the base of the tail on day 0 using a syringe. Booster immunization: Perform booster immunization on day 21 after primary immunization. Emulsify bovine type II collagen (2 mg / mL) with an equal volume of incomplete Freund's adjuvant (IFA), and inject 100 μL subcutaneously at two points at a certain distance from the base of the tail. Model evaluation: During the model establishment period, perform arthritis scoring, monitor the thickness and morphology of the paws and symptoms, and measure the levels of IL-6 and TNF-α in the plasma to evaluate whether the model is successfully established.

[0075] 4. Grouping and Administration

[0076] Forty-six male C57 mice were randomly divided into five groups according to body weight. The specific grouping and intervention plans are as follows:

[0077] Control group, Collagen-induced arthritis (CIA) group, Collagen-induced arthritis + Celastrol (CIA+CEL) group, Collagen-induced arthritis + 10-sulfonated Celastrol (CIA+CELS) group, Collagen-induced arthritis + Methotrexate (CIA+MTX) group. There were 6 mice in each of the Control and CIA groups, and they were given an equal dose of normal saline by oral gavage; there were 8 mice in each of the CIA+CEL, CIA+CELS, and CIA+MTX groups, and they were given the corresponding therapeutic drugs at a dose of 2 mg / kg by oral gavage. Administration started on the 28th day, once a day for 28 consecutive days. During the modeling and administration period, the foot thickness, arthritis score, and body weight of the mice were recorded.

[0078] 5. Collection and Detection of Plasma and Tissue Samples

[0079] (1) Sample collection: After 28 consecutive days of administration, all mice were euthanized, and 500 μL of blood was collected from the eye socket. The blood was centrifuged at 8000 rpm at 4 °C for 5 min, and the supernatant was aspirated into an EP tube and stored at -80 °C for later use. The liver tissue was separated and stored in a -80 °C refrigerator. A part of the liver tissue and the hind paws of the mice were fixed in 4% paraformaldehyde and stored at room temperature.

[0080] (2) Histopathological examination

[0081] Take some liver tissue and mouse hind paws fixed in 4% paraformaldehyde. According to the conventional histopathological method, the tissue was embedded in paraffin, cut into 4-μm thin sections, and made into slides. Subsequently, hematoxylin and eosin staining (H&E staining) was performed. Each sample slide was observed under a 200-fold microscope to observe the changes in liver tissue, and histopathological analysis was carried out.

[0082] (3) Detection of biochemical indexes

[0083] Strictly in accordance with the operating instructions of the kit, the levels of IL-6 and TNF-α in the plasma of mice were detected.

[0084] The levels of IL-6 and TNF-α in the plasma of mice were used as biochemical indexes, supplemented by H&E staining and the status of the mouse feet to evaluate the severity of mouse arthritis and the degree of foot swelling, and to investigate the anti-rheumatoid arthritis effects of CEL and CELS.

[0085] The results are as Figure 5 shown Figure 5A-D showed that compared with the Control group, the foot thickness of mice in the CIA model group was significantly increased, the arthritis score was elevated, and the body weight was significantly decreased. Compared with the CIA model group, oral administration of the positive drug MTX 2 mg / kg to CIA model mice could improve the severity of arthritis in mice, specifically manifested as a significant decrease in foot thickness, a decrease in arthritis score, and an improvement in body weight. Meanwhile, on the basis of the CIA model group, oral administration of CEL and CELS 2 mg / kg for 4 weeks had a better effect on improving rheumatoid arthritis in mice than the positive drug.

[0086] Figure 5 The results of E, F, and G showed that compared with the Control group, the levels of TNF-α and IL-6 in the plasma of the CIA model group were significantly increased. After administration of the positive drug MTX 2 mg / kg, CEL, and CELS 2 mg / kg respectively, the inflammation in CIA mice could be improved, and the plasma TNF-α and IL-6 were significantly decreased.

[0087] Figure 5 The H&E staining results of H showed that in the foot tissues of mice in the CIA model group, there were multiple synovial hyperplasias, inflammatory cell infiltration in some cartilage, synovitis and pannus formation in joints, destruction of articular cartilage and bone tissues in the ankle joint and / or toe joints, and adhesion between bone tissues and connective tissues and other arthritis lesions; compared with the CIA model group, administration of the positive drug MTX 2 mg / kg, CEL, and CELS 2 mg / kg respectively could significantly improve the inflammatory infiltration in the feet of CIA mice.

[0088] In summary, it shows that both celastrol and 10-sulfonated celastrol can significantly improve rheumatoid arthritis.

[0089] The above specific embodiments have described the present invention in detail, but the present invention is not limited to the above embodiments. Within the knowledge scope of those of ordinary skill in the art to which the present invention pertains, various changes can be made without departing from the gist of the present invention. In addition, without conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

Claims

1. A tripterine sulfonated metabolite, characterized in that, The tripterine sulfonation metabolite is 10-sulfonated tripterine; The chemical formula of the 10-sulfonated tripterine is shown as the following formula (Ⅰ):

2. The preparation method of a tripterine sulfonation metabolite according to claim 1, wherein It includes the following steps: Under nitrogen protection, tripterine is added to methanol. After stirring and dissolving, sodium bisulfite or potassium bisulfite is added and dissolved in water. Stir at room temperature, dry, dissolve in methanol, take the supernatant after centrifugation, and dry to obtain the tripterine sulfonation metabolite.

3. The preparation method according to claim 2, characterized in that, The mass ratio of the tripterine, sodium bisulfite or potassium bisulfite is 50-200:10-50; The time of stirring at room temperature is 6-24 h; The conditions of the centrifugation are: rotation speed 5000-10000 rpm, time 5-20 min.

4. Use of a tripterine sulfonation metabolite or its derivative as claimed in claim 1 in the preparation of a product for treating rheumatoid arthritis.

5. Use of a tripterine sulfonation metabolite or its derivative as claimed in claim 1 in the preparation of a product for treating tumors or obesity.

6. Use of a tripterine sulfonation metabolite or its derivative as claimed in claim 1 as a low-toxicity efficacy enhancer for tripterine products.

7. A product for treating rheumatoid arthritis and / or anti-tumor and / or anti-obesity, characterized in that, It contains the tripterine sulfonation metabolite as claimed in claim 1 and a pharmaceutically acceptable excipient.

8. A product for treating rheumatoid arthritis and / or anti-tumor and / or anti-obesity as claimed in claim 7, characterized in that, The pharmaceutically acceptable excipient is at least one of a solvent, a binder, a diluent, a wetting agent, a lubricant, an emulsifier, a thickener, a shaping agent, a suspending agent, a disintegrant, a filler, a preservative, a pH regulator, an osmotic pressure regulator, a surfactant, a coating material, an antioxidant, a bacteriostatic agent or a buffer.

9. A product for treating rheumatoid arthritis and / or anti-tumor and / or anti-obesity as claimed in claim 7, characterized in that The dosage form of the product is at least one of a suspension, a granule, a capsule, a powder, a tablet, an emulsion, a solution, a dropping pill, an injection, an oral preparation, a suppository, an enema, an aerosol, a patch or a drop.

10. A product for treating rheumatoid arthritis and / or anti-tumor and / or anti-obesity as claimed in claim 7, characterized in that, The administration method of the product is at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, aerosol administration or transdermal administration.

Images