Use of total iridoid glycosides in paeonia suffruticosa andrews in preparation of medicine for reducing side effects of methotrexate

Abstract

The application discloses a use of total iridoid glycosides in radix morindae officinalis in preparation of a medicine for reducing side effects of methotrexate, and the total iridoid glycosides in radix morindae officinalis are effective traditional Chinese medicine components prepared by extraction and separation from radix morindae officinalis medicinal materials. The total iridoid glycosides in radix morindae officinalis and main component crystal larkspur glycoside are combined with methotrexate to treat rheumatoid arthritis rats, and the animal experiment proves that the combined administration can effectively treat inflammation of the rheumatoid arthritis rats, and can inhibit bone loss, especially can effectively reduce side effects caused by methotrexate, reduce damage caused by methotrexate to the organism, make the application more safe and effective, and provide a new treatment strategy for rheumatoid arthritis.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically the pharmaceutical use of a combination of total iridoid glycosides extracted from the traditional Chinese herbal medicine Morinda officinalis, which have anti-inflammatory and anti-rheumatoid arthritis properties, and methotrexate, a first-line drug for the treatment of rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by joint destruction and persistent synovitis. Its main pathological features are multi-joint involvement, persistent and recurrent synovitis, and a lingering and difficult-to-cure condition. Ultimately, it leads to the destruction of cartilage and bone within the joints, making fractures more likely and severely affecting the patient's quality of life.

[0003] Methotrexate (MTX) is widely recognized as the first-line disease-modifying antirheumatic drug (DMARD) and a fundamental component of combination therapy for rheumatoid arthritis (RA). Both the 2015 American College of Rheumatology and the 2016 European League Against Rheumatism guidelines designate MTX as the first-line drug for initial RA treatment. For newly diagnosed RA, MTX can rapidly control disease progression. However, long-term use of MTX can cause serious adverse reactions such as gastrointestinal discomfort, bone marrow suppression, liver damage, pulmonary fibrosis, nephrotoxicity, and dermatitis. It has been reported that 20-70% of patients experience gastrointestinal side effects; and 70% of patients experience hepatotoxicity during the first 2-4 years of MTX treatment. Studies show that in RA patients who do not respond well to MTX, combination therapy with other drugs can better control disease activity and help improve joint function. In recent years, with the development of traditional Chinese medicine, MTX is often used in combination with other drugs to treat RA. Therefore, seeking drugs to reduce the toxic side effects of MTX is of great significance for its widespread clinical application.

[0004] Morinda officinalis How., a plant in the Rubiaceae family, is the dried root of the plant. It is warm in nature, pungent and sweet in taste, and enters the liver and kidney meridians. It tonifies kidney yang, strengthens tendons and bones, and dispels wind and dampness. It is used for impotence, seminal emission, infertility due to cold uterus, irregular menstruation, lower abdominal pain due to cold, rheumatic pain, and weakness of tendons and bones. Pharmacological experiments have found that total iridoid glycosides (MOIG) from Morinda officinalis have significant anti-inflammatory and anti-RA effects. Summary of the Invention

[0005] The purpose of this invention is to address the toxic side effects of methotrexate in the treatment of rheumatoid arthritis (RA) by providing a traditional Chinese medicine extract that can reduce the toxic side effects of methotrexate for combined treatment of RA.

[0006] The specific technical solution adopted in this invention is as follows:

[0007] The use of total iridoid glycosides from Morinda officinalis in the preparation of a drug to reduce the toxic side effects of methotrexate, wherein the total iridoid glycosides from Morinda officinalis are used in combination with methotrexate to reduce the toxic side effects of methotrexate.

[0008] Furthermore, the total iridoid glycosides of Morinda officinalis, when used in combination with methotrexate, can reduce the toxic side effects of methotrexate in the preparation of an anti-rheumatoid arthritis drug.

[0009] The total iridoid glycosides of Morinda officinalis of the present invention, combined with methotrexate, can treat RA, especially RA patients who do not respond well to MTX treatment. When used in combination with other drugs, it can better control disease activity and help improve joint function.

[0010] The use of a crystallized glycoside in the preparation of a drug that reduces the toxic side effects of methotrexate, wherein the crystallized glycoside is used in combination with methotrexate to reduce the toxic side effects of methotrexate.

[0011] Furthermore, the combination of crystallized glycoside and methotrexate in the preparation of an anti-rheumatoid arthritis drug reduces the toxic side effects of methotrexate.

[0012] The beneficial effects of this invention are: the total iridoid glycosides of Morinda officinalis and its main component, crystal blue glycoside, combined with methotrexate can treat RA, while reducing the toxic side effects caused by methotrexate, providing a new treatment strategy for RA. Attached Figure Description

[0013] Figure 1 These are synovial tissue sections from CIA rats (n = 10, × 1 mm); in the image, green arrows indicate bone loss; yellow arrows indicate synovial hyperplasia; blue arrows indicate inflammatory cell infiltration; and red arrows indicate pannus.

[0014] Figure 2 These are bone tissue sections from CIA rats (n = 10, × 1 mm); in the image, purple arrows indicate articular cartilage, collagen, and bone collagen; black arrows indicate muscle fibers and cellulose.

[0015] Figure 3 These are liver tissue sections from CIA rats (n = 10, × 200 μm); in the image, black arrows indicate hepatocyte swelling, blue arrows indicate nodule formation, and red arrows indicate inflammatory cells;

[0016] Figure 4 These are gastric tissue sections from CIA rats (n = 10, × 200 μm); in the image, red arrows indicate inflammatory cells;

[0017] Figure 5 These are lung tissue sections from CIA rats (n = 10, × 200 μm); in the image, red arrows indicate inflammatory cells; blue arrows indicate fibrosis.

[0018] Figure 6 These are pathological sections of liver tissue from mice with acute liver injury. Detailed Implementation

[0019] This invention utilizes a rat model of type II collagen-induced arthritis (CIA) and a rat model of MTX-induced acute liver injury to observe the effects of MOIG and its main component, crystallized blue glycoside, in alleviating the side effects of MTX, particularly its protective effect against liver injury, aiming to achieve a more effective and safer treatment for RA. The effects of this invention are further illustrated below with specific embodiments and accompanying drawings.

[0020] Example 1: Preparation of total iridoid glycosides from Morinda officinalis

[0021] The preparation method of total iridoid glycosides from Morinda officinalis can be found in published patent CN201710902667.4, and is briefly described below:

[0022] The pulverized Morinda officinalis herb was cold-soaked in 30-95 vol% ethanol according to conventional methods, percolated to obtain an extract, concentrated, filtered, and the filtrate was adsorbed through a macroporous resin. The macroporous resin was a polystyrene-type porous adsorption resin with styrene as the crosslinking material, such as D101, HPD-722, HPD400, XDA-1, AB-8 type macroporous resins, etc. Impurities were removed by elution with water or less than 5 vol% aqueous ethanol, followed by elution with 10%-70 vol% aqueous ethanol. The ethanol eluent was collected, concentrated to dryness under reduced pressure, and the total iridoid glycosides of Morinda officinalis of this invention were obtained, with a yield of more than 2% of the crude drug. HPLC analysis showed that it contained 38.6% cynomolgus glycoside and 23.6% deacetylated cynomolgus acid.

[0023] Example 2: Effects of total iridoid glycosides from Morinda officinalis on the efficacy and toxic side effects of methotrexate in the treatment of rheumatoid arthritis 1.1 Experimental materials and methods

[0024] 1.1.1 Laboratory Animals

[0025] Wistar clean-grade rats, male (170±10)g, were purchased from Shanghai Xipubikai Experimental Animal Co., Ltd.

[0026] 1.1.2 Reagents

[0027] Methotrexate (batch number 036150201) was purchased from Shanghai Xinyi Pharmaceutical Co., Ltd.; bovine type II collagen (CII, batch number 180055) was purchased from Chondrex, Inc., USA; Freund's incomplete adjuvant (batch number SLBQ2284V) was purchased from Sigma-Aldrich, Inc., USA; HE staining reagent and Masson staining solution kit were purchased from Wuhan Google Biotechnology Co., Ltd.; ELISA kits were purchased from Nanjing Jiancheng Biotechnology Institute and Hangzhou Lianke Biotechnology Co., Ltd.; other reagents and materials (analytical grade and chemically pure) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0028] 1.1.3 Experimental Apparatus

[0029] TGL-16M high-speed refrigerated centrifuge, Aida Corporation; ELx800 continuous wavelength microplate reader, Berten Instruments, Inc.; 13050655 electronic display caliper, Shanghai Shenhan Measuring Instruments Co., Ltd.; JA1003 precision electronic balance, Shanghai Hengping Scientific Instruments Co., Ltd.; JB-P5 embedding machine, Wuhan Junjie Electronics Co., Ltd.; RM2016 pathological slide machine, Leica Instruments Co., Ltd.; BX61VS Olympus microscope, OLYMPUS Corporation.

[0030] 1.1.4 Preparation of the medicinal solution

[0031] A 2 mg / mL solution of type II collagen acetic acid was added dropwise to cold Freund's incomplete adjuvant at a ratio of 1:1 (v / v). The mixture was stirred continuously in an ice bath until the two liquids were completely emulsified. The emulsion was obtained when the solution did not disperse after 30 seconds when dropped into water.

[0032] Take MTX tablets and prepare solutions with concentrations of 0.025, 0.05, and 0.075 mg / mL using 0.5 wt% CMC-Na.

[0033] Accurately weigh MOIG powder and prepare a solution with a concentration of 5 mg / mL using 0.5% CMC-Na.

[0034] 1.2 Experimental Methods

[0035] Rats were randomly divided into 7 groups according to body weight: normal group, model group, MTX group (0.5 mg / kg), MOIG group (50 mg / kg), and low, medium, and high dose groups of MOIG combined with MTX (MOIG-50 mg / kg-MTX-0.25, 0.5, and 0.75 mg / kg), with 10 rats in each group. Except for the normal group, all other groups received an intradermal injection of 0.2 mL of the emulsion prepared in section 1.1.4 at a distance of 2 cm from the tail base. A booster injection was administered at the same site using the same method 21 days later. Starting on day 28, all groups were administered the drug by gavage at a dose of 1 mL / 100 g. The MTX group was administered the drug twice a week, while the other groups were administered it six times a week. The normal and model groups were each given an equal amount of 0.5 wt% CMC-Na. Rats' body weight and paw edema were measured weekly. Six weeks later, the rats were anesthetized with 3 wt% sodium pentobarbital, blood was collected from the abdominal aorta, serum was separated, and the thymus and spleen were quickly dissected and weighed to determine the spleen and thymus indices.

[0036] 1.2.1 Serum biochemical index determination

[0037] Serum levels of IL-1β, IL-6, OCN, OPG, CTX-I, RANKL, TRAP, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were measured using an ELISA kit, strictly following the instructions for use of the ELISA kit.

[0038] 1.2.2 Tissue Sections

[0039] The tibia and femur of rats were harvested, and excess soft tissue, muscles, and ligaments were removed to obtain fresh bone tissue. After decalcification, dehydration, embedding, and sectioning, the tissue was stained with HE and Masson staining solutions and observed under a microscope.

[0040] The liver, lungs, stomach, and other organs of rats were stored in 4 wt% paraformaldehyde at room temperature. Tissue sections of the stomach, liver, and lungs were prepared, stained with hematoxylin and eosin (HE), and their pathological features were observed.

[0041] 1.3 Statistical Analysis

[0042] Mean ± standard deviation The analysis process involves first performing a normality test. If the data conforms to a normal distribution, a homogeneity of variance test is performed. If it also conforms, a t-test is conducted, with P < 0.05 considered statistically significant. For data that does not conform to a normal distribution or homogeneity of variance, a rank-sum test is used. SPSS 21.0 software was used for analysis.

[0043] 1.4 Experimental Results

[0044] 1.4.1 Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on body weight in CIA rats

[0045] As shown in Table 1, compared with the normal group, the body weight of the CIA rats in the model group was significantly reduced (P<0.05), and the body weight of the CIA rats was lowest at 41 days. The body weight of the MTX group was significantly reduced in the later stage of drug administration, while the body weight of rats treated with iridoid glycosides and combined therapy was slowly increased (P<0.05).

[0046] Table 1. Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on body weight in CIA rats (n=10)

[0047]

[0048] Compared with the normal group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.05

[0049] 1.4.2 Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on thymus and spleen indices in CIA rats

[0050] As shown in Table 2, compared with the normal group, the spleen index of CIA rats in the model group was significantly increased, while the spleen index decreased after drug administration, but the difference was not statistically significant. Compared with the normal group, the thymus index of CIA rats in the model group was significantly decreased (P<0.05). Both methotrexate and iridoid glycosides significantly increased the thymus index of CIA rats (P<0.01, P<0.05), while the combined drug administration group increased the thymus index of CIA rats, but the difference was not statistically significant.

[0051] Table 2. Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on spleen and thymus indices in CIA rats (n=10)

[0052]

[0053] Compared with the normal group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.05.

[0054] 1.4.3 Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on paw edema in CIA rats

[0055] As shown in Table 3, compared with the normal group, the paw swelling of the model group CIA rats was significantly increased (P<0.01). Compared with the model group CIA rats, the paw swelling of the MTX group was significantly reduced from day 55 (P<0.05, P<0.01). After the administration was completed, except for the low-dose combined administration which showed no significant difference, all other treatment groups significantly reduced the paw swelling of CIA rats (P<0.05, P<0.01).

[0056] Table 3. Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on paw edema in CIA rats (n=10)

[0057]

[0058] Compared with the normal group, #P<0.05, ##P<0.01; compared with the model group, *P<0.05, **P<0.05

[0059] 1.4.4 Synovial tissue sections - HE staining

[0060] like Figure 1 As shown, the model group of CIA rats exhibited severe synovial hyperplasia of the knee joint, with synovial margin rupture, extensive inflammatory cell infiltration in the synovial tissue, and severe destruction of articular cartilage. Compared with the model group of CIA rats, synovial hyperplasia was reduced, cartilage surface destruction was alleviated, and subchondral bone destruction was essentially unaffected in all groups after drug treatment. These results indicate that iridoid glycosides combined with methotrexate can effectively alleviate synovial inflammation in CIA rats, thereby reducing bone destruction and protecting the knee joint.

[0061] 1.4.5 Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on serum inflammatory factors in CIA rats

[0062] As shown in Table 4, the serum levels of IL-1β and IL-6 in the model group CIA rats were significantly increased. After 49 days of treatment, the serum levels of IL-1β and IL-6 in CIA rats were significantly improved, but there was no statistical significance (P>0.05).

[0063] Table 4. Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on serum inflammatory factors in CIA rats (n=10)

[0064]

[0065] 1.4.6 Bone tissue sections - Masson staining

[0066] Depend on Figure 2 As shown, the joint tissues of normal rats were rich in cartilage collagen (stained blue), and contained abundant muscle fibers, cellulose, and erythrocytes (stained red), as well as abundant trabeculae and bone matrix. In the model group of CIA rats, the joint tissues showed less cartilage collagen and trabeculae destruction. Rats treated with methotrexate and iridoid glycosides showed increased cartilage collagen and reduced bone destruction. Compared to rats treated with methotrexate alone, the combined use of methotrexate and iridoid glycosides significantly increased cartilage collagen and significantly alleviated bone destruction, indicating that iridoid glycosides can enhance the protective effect of methotrexate against bone destruction in CIA model rats.

[0067] 1.4.7 Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on serum bone formation and bone metabolism markers in CIA rats

[0068] As shown in Table 5, there were no significant changes in serum OCN and CTX-I levels in the model group and each treatment group. Compared with the normal group, the serum TRAP activity of the CIA rats in the model group was significantly increased (P<0.01), and the OPG / RANKL ratio was significantly decreased (P<0.01). Compared with the CIA rats in the model group, the serum TRAP activity of the iridoid glycosides and the combined administration of the two was significantly decreased (P<0.05, P<0.01), and the OPG / RANKL ratio was significantly increased (P<0.05, P<0.01). Compared with the MTX group, the serum TRAP activity of the combined administration group was significantly decreased, and the OPG / RANKL ratio was significantly increased.

[0069] Table 5. Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on serum bone biochemical parameters in CIA rats (n=10)

[0070]

[0071]

[0072] Compared with the normal group, ##P<0.01; compared with the model group, *P<0.05, **P<0.05

[0073] 1.4.8 Liver tissue sections - HE staining

[0074] like Figure 3 It was found that, compared with normal rats, the liver tissue of the model group CIA rats showed mild fibrosis and inflammatory cell infiltration. Compared with the model group CIA rats, the inflammatory cell infiltration was significantly increased, hepatocytes swelled, and focal centrilobular necrosis and fibrosis were severe after methotrexate administration. The liver tissue fibrosis and inflammatory cell infiltration of the iridoid glycoside group were significantly reduced. Compared with the MTX group, the liver tissue fibrosis and inflammatory cell infiltration of the combined iridoid glycoside and methotrexate group were significantly reduced.

[0075] 1.4.9 Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on serum liver function indicators in CIA rats

[0076] As shown in Table 6, compared with the normal group, the serum ALT levels in the model group and the MTX group were significantly increased (P<0.01), and the AST levels were also increased, but there was no statistical difference. Compared with the model group CIA rats, the serum ALT and AST levels in the MTX group were increased, but there was no statistical difference. The serum ALT level in the MOIG group was significantly decreased (P<0.01), and the AST level was also decreased, but there was no statistical difference. Compared with the MTX group, the serum ALT level in the iridoid glycoside combined with methotrexate group was significantly decreased (P<0.01), and the serum AST level in the iridoid glycoside combined with low-dose methotrexate group was significantly decreased (P<0.05). The serum AST levels in the other groups were decreased, but there was no statistical difference.

[0077] Table 6. Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on serum liver function indicators in CIA rats (n=10)

[0078]

[0079] Compared with the normal group, ##P<0.01; compared with the model group, *P<0.05, **P<0.05; compared with the MTX group, ▲ P<0.05, ▲▲ P<0.01

[0080] 1.4.10 Histopathological evaluation of gastric tissue

[0081] like Figure 4 Compared with the normal group, the gastric mucosa of the CIA model group rats showed inflammatory cell infiltration; compared with the CIA model group, the gastric mucosa of the MTX group rats showed significantly increased inflammatory cell infiltration, microvascular congestion, altered gastric parietal cell structure, and reduced number of cells; the gastric inflammatory cells of the MOIG group rats were significantly reduced; compared with the MTX group rats, the gastric mucosa inflammation of the rats in the combined administration group of iridoid glycosides and methotrexate was significantly reduced, microvascular congestion was reduced, and the gastric parietal cell structure was normal.

[0082] 1.4.11 Pathological assessment of lung tissue

[0083] like Figure 5 Compared with the normal group, the model group of CIA rats showed mild inflammatory cell infiltration and alveolar wall thickening in the lung tissue. Compared with the model group of CIA rats, the MTX group showed significantly increased inflammatory cell infiltration and alveolar wall thickening in the lung tissue, and severe parenchymal fibrosis in the lungs. The MOIG group showed significantly reduced inflammatory cell infiltration and alveolar wall thinning in the lung tissue. Compared with the MTX group, the group treated with iridoid glycosides and methotrexate showed significantly reduced inflammatory cells, alveolar wall thinning, and significantly reduced pulmonary fibrosis.

[0084] This invention employs a method of administering iridoid glycosides in combination with different doses of methotrexate. The effects of reducing the adverse reactions induced by methotrexate were evaluated by detecting serum biochemical indicators, bone tissue, and observing histopathological sections of liver, lung, and stomach tissues in CIA rats. The results show that iridoid glycosides combined with methotrexate are effective in treating RA, while simultaneously reducing the side effects caused by methotrexate, thus mitigating its toxicity. This invention lays the research foundation for developing a novel drug for treating RA by combining methotrexate and iridoid glycosides.

[0085] Example 3: The effect of total iridoid glycosides from Morinda officinalis in alleviating methotrexate-induced liver injury in the treatment of rheumatoid arthritis (RA).

[0086] 2.1 Experimental Materials and Methods

[0087] 2.1.1 Laboratory Animals

[0088] Wistar clean-grade rats, male (170±10)g, were purchased from Shanghai Xipubikai Experimental Animal Co., Ltd.

[0089] 2.1.2 Reagents

[0090] Methotrexate (batch number 036150201) was purchased from Shanghai Xinyi Pharmaceutical Co., Ltd.; bovine type II collagen (CII, batch number 180055) was purchased from Chondrex, Inc., USA; Freund's incomplete adjuvant (batch number SLBQ2284V) was purchased from Sigma-Aldrich, Inc., USA; ELISA kits and biochemical kits were purchased from Nanjing Jiancheng Bioengineering Institute; other reagents and materials (analytical grade and chemically pure) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0091] 2.1.3 Experimental Apparatus

[0092] TGL-16M high-speed refrigerated centrifuge, Aida Corporation; ELx800 continuous wavelength microplate reader, Berten Instruments, Inc.; 13050655 electronic display caliper, Shanghai Shenhan Measuring Instruments Co., Ltd.; JA1003 precision electronic balance, Shanghai Hengping Scientific Instruments Co., Ltd.

[0093] 2.2 Experimental Methods

[0094] Rats were randomly divided into 7 groups according to body weight: normal group, model group, MTX group (1 mg / kg), MOIG group (100 mg / kg), and low- and high-dose MTX combined with MOIG (MTX-1 mg / kg-MOIG-50, 100 mg / kg), with 10 rats in each group. Except for the normal group, the other groups of rats were injected intradermally with 0.2 mL of the emulsion prepared in 1.1.4 at 2 cm from the tail base. After 21 days, a booster injection was given at the same site using the same method. Starting from day 28, each group was administered 1 mL / 100 g of the drug by gavage. MTX was administered once a week, while the other groups were administered 6 times a week. The normal and model groups were given an equal amount of 0.5 wt% CMC-Na. After 6 weeks, the rats were anesthetized with 3 wt% sodium pentobarbital, and blood was collected from the abdominal aorta to separate the serum.

[0095] 2.2.1 Serum biochemical index determination

[0096] Serum AST and ALT levels were measured using an ELISA kit, strictly following the instructions for use of the ELISA kit.

[0097] 2.2.2 Liver tissue biochemical indicators

[0098] The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione reductase (GSH), and catalase (CAT) in liver tissue homogenates were determined using a biochemical reagent kit, strictly following the instructions of the biochemical reagent kit.

[0099] 2.3 Statistical Analysis

[0100] Mean ± standard deviation The analysis process involves first performing a normality test. If the data conforms to a normal distribution, a homogeneity of variance test is performed. If it also conforms, a t-test is conducted, with P < 0.05 considered statistically significant. For data that does not conform to a normal distribution or homogeneity of variance, a rank-sum test is used. SPSS 21.0 software was used for analysis.

[0101] 2.4 Experimental Results

[0102] 2.4.1 Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on liver function indicators in CIA rats

[0103] As shown in Table 7, compared with the normal group, the serum AST levels in the model group and the MTX group were significantly increased (P<0.001, P<0.05); compared with the model group, the serum AST level in the MOIG group was significantly decreased (P<0.05); compared with the MTX group, the serum AST level in the iridoid glycoside combined with methotrexate group was decreased, but there was no statistically significant difference. Compared with the normal group, the serum ALT level in the model group was increased, but there was no significant difference; and there were no statistically significant differences in ALT levels among the various treatment groups.

[0104] Table 7. Effects of total iridoid glycosides from Morinda officinalis on liver function enzyme activities in methotrexate-treated CIA rats (n=10)

[0105]

[0106] Note: Compared with the normal group, ## P < 0.001; *P < 0.05 compared to the model group.

[0107] 2.4.2 Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on the activity of antioxidant enzymes in liver tissue of CIA rats

[0108] As shown in Table 8, compared with the normal group, the MDA level in the liver tissue of rats in the model group and MTX group was significantly increased (P<0.05, P<0.01), and the GSH enzyme activity was significantly decreased (P<0.05, P<0.01). The SOD and CAT activities in rats in the MTX group were significantly decreased (P<0.01), while there were no significant changes in rats in the model group. Compared with the model group, there were no significant differences in the enzyme activities of MDA, GSH, SOD, and CAT in the liver tissue of rats in the MOIG group. Compared with the MTX group, the MDA level in the liver tissue of rats in the low- and high-dose groups of iridoid glycosides combined with the treatment was significantly decreased (P<0.01), and the SOD, CAT, and GSH enzyme activities were significantly increased (P<0.005, P<0.01), indicating that iridoid glycosides have a significant ameliorative effect on the oxidative damage to rat liver tissue caused by methotrexate treatment for RA.

[0109] Table 8. Effects of total iridoid glycosides from Morinda officinalis on oxidative damage in liver tissue of methotrexate-treated CIA rats (n=10)

[0110]

[0111] Note: Compared with the normal group, #P<0.05, ##P<0.01; compared with the model group, **P<0.05; compared with the MTX group, ▲ P<0.05, ▲▲ P<0.01

[0112] 2.4.3 Effects of total iridoid glycosides from Morinda officinalis combined with methotrexate on liver inflammation in CIA rats

[0113] As shown in Table 9, compared with the normal group, IL-1β levels in the liver tissue of rats in the model group and MTX group were significantly increased (P < 0.001), IL-6 levels in the liver tissue of the MTX group were significantly increased (P < 0.001), while IL-6 levels in the model group were increased, but the difference was not statistically significant. Compared with the model group, IL-6 levels in the liver tissue of rats in the MOIG group were decreased, but the difference was not statistically significant. Compared with the MTX group, IL-6 levels in the liver tissue of rats in the low- and high-dose groups of iridoid glycosides combined with the treatment were significantly decreased (P < 0.05), while IL-1β showed no significant change. The results indicate that iridoid glycosides can alleviate liver inflammation induced by methotrexate treatment for rheumatoid arthritis (RA).

[0114] Table 9. Effects of total iridoid glycosides from Morinda officinalis on liver inflammation in methotrexate-treated CIA rats (n=10)

[0115]

[0116]

[0117] Note: Compared with the normal group, #P<0.05, ##P<0.01; compared with the MTX group, ▲ P<0.05

[0118] This invention employs a method of administering methotrexate in combination with different doses of iridoid glycosides. By detecting liver function indicators, liver tissue inflammatory factors, and the activity of antioxidant enzymes in CIA rats, the study further evaluated the effect of iridoid glycosides on reducing the adverse liver reactions induced by methotrexate. The results show that the combination of iridoid glycosides and methotrexate reduces the elevation of liver function enzymes induced by long-term methotrexate administration and improves liver tissue inflammation and oxidative damage.

[0119] Example 4: Protective effect of crystal blue glycoside, the main component of total iridoid glycosides from Morinda officinalis, against methotrexate-induced acute liver injury.

[0120] 3.1 Experimental Materials and Methods

[0121] 3.1.1 Laboratory Animals

[0122] Thirty male BALB / c mice aged 6–8 weeks, weighing 16–24 g, were purchased from Shanghai Silex Company.

[0123] 3.1.2 Reagents

[0124] Methotrexate (batch number T1485) was purchased from Shanghai Taoshu Biotechnology Co., Ltd.; folic acid tablets (National Drug Approval Number H10970079) were purchased from Beijing Silian Pharmaceutical Co., Ltd.; biochemical reagent kits were purchased from Nanjing Jiancheng Bioengineering Institute; eosin staining solution (G1001) and hematoxylin staining solution (GB1004) were purchased from Wuhan Google Biotechnology Co., Ltd.; modified Oil Red O staining kit (G1261) was purchased from Beijing Solarbio Technology Co., Ltd.

[0125] 3.1.3 Experimental Apparatus

[0126] TGL-16M high-speed refrigerated centrifuge, Aida Corporation; ELx800 continuous wavelength microplate reader, Bertek Instruments, Inc.; JA1003 precision electronic balance, Shanghai Hengping Scientific Instruments Co., Ltd.

[0127] 3.2 Experimental Methods

[0128] Thirty BALB / c mice were randomly divided into five groups: a normal group, an MTX group (20 mg / kg), low- and high-dose MTX combined with monoclonal antibody (MON) groups (MTX-20 mg / kg-MON-40, 80 mg / kg), and an MTX combined with folic acid group (MTX-20 mg / kg-FA-0.1 mg / kg), with six mice in each group. Mice were administered the medication by gavage for 10 days. The normal and MTX groups were given physiological saline. One hour after the end of gavage on day 10, 20 mg / kg of MTX was injected intraperitoneally. On day 11, the mice were sacrificed by cervical spine manipulation, and their livers were harvested, weighed, and liver indexes calculated.

[0129] 3.2.1 Measurement of liver function indicators and oxidative indicators

[0130] The contents of liver function indicators AST and ALT, and the contents of oxidation indicators GSH, SOD and MDA enzymes in liver tissue homogenate were determined using a biochemical reagent kit, strictly following the instructions of the biochemical reagent kit.

[0131] 3.2.2 Liver tissue sections

[0132] Mouse liver tissue was fixed in 4 wt% paraformaldehyde for at least 24 h, embedded and sectioned, stained with HE and Oil Red O, and observed under a Zeiss upright microscope.

[0133] 3.3 Statistical Analysis

[0134] Mean ± standard deviation The analysis process involves first performing a normality test. If the data conforms to a normal distribution, a homogeneity of variance test is performed. If it also conforms, a t-test is conducted, with P < 0.05 considered statistically significant. For data that does not conform to a normal distribution or homogeneity of variance, a rank-sum test is used. SPSS 26.0 software was used for analysis.

[0135] 3.4 Experimental Results

[0136] 3.4.1 Effects of crystallized blue glycoside on liver index in mice with methotrexate-induced acute liver injury

[0137] As shown in Table 10, the liver index of mice in the MTX group was increased compared with the normal group. Compared with the MTX group, the liver index of mice in the low- and high-dose groups of methotrexate combined with crystallizing glycoside was significantly reduced (P<0.05), indicating that crystallizing glycoside can alleviate methotrexate-induced acute liver injury.

[0138] Table 10 Effects of crystallized blue on liver index in mice with methotrexate-induced acute liver injury (n=6)

[0139]

[0140] Note: *P<0.05 compared to the MTX group.

[0141] 3.4.2 Effects of crystallized blue glycosides on pathological morphological changes and lipid accumulation in liver tissue of mice with methotrexate-induced acute liver injury

[0142] like Figure 6 As shown, compared with the normal group, the MTX group exhibited disordered hepatocyte arrangement (400×, 100×) and partial loss of hepatic cord structure under the microscope. Small vacuoles were occasionally observed in the cytoplasm, and some hepatocytes showed foamy degeneration. Mild inflammatory cell infiltration was observed near the central vein. Some centrilobular hepatocytes showed condensed eosinophilic cytoplasm and slightly angular outlines, suggesting apoptosis. A significant amount of lipid droplets accumulated between cells (200×).

[0143] Microscopic examination of the MTX combined with folic acid group revealed mild perichondral fibrosis accompanied by bile duct hyperplasia, with a significant reduction in lipid droplet accumulation. Microscopic examination of the methotrexate combined with crystal blue glycoside low and high dose groups revealed disordered hepatic cord arrangement, a small amount of inflammatory cell aggregation around the bile ducts, occasional polynuclearization and heterogeneous nuclei in periportal hepatocytes, and reduced intercellular lipid accumulation in the high dose group.

[0144] 3.4.3 Effects of crystallized blue glycoside on liver function enzymes in mice with methotrexate-induced acute liver injury

[0145] As shown in Table 11, compared with the normal group, the serum AST level of mice in the MTX group was significantly increased (P<0.01). Compared with the MTX group, the serum AST level of mice in the MTX combined with high-dose crystallizing glycoside and folic acid groups was decreased, but there was no statistical difference. The serum AST level of the low-dose crystallizing glycoside group was significantly decreased (P<0.01). Compared with the normal group, the serum ALT level of mice in the MTX group was significantly increased (P<0.05). Compared with the MTX group, the MTX combined with crystallizing glycoside and folic acid groups significantly reduced the serum ALT level of mice (P<0.05). The results indicate that crystallizing glycoside can alleviate methotrexate-induced liver injury.

[0146] Table 11 Effects of crystallized blue on liver function enzymes in mice with methotrexate-induced acute liver injury (n=6)

[0147]

[0148] Note: Compared with the MTX group, *P<0.05, **P<0.01

[0149] 3.4.4 Effects of crystallizing glycosides on the activity of antioxidant enzymes in liver tissue of mice with methotrexate-induced acute liver injury

[0150] As shown in Table 12, compared with the normal group, the MTX group showed significantly increased MDA levels in liver tissue (P<0.01), significantly decreased SOD activity (P<0.05), and decreased GSH levels, but without significant differences. Compared with the MTX group, the low- and high-dose crystallizing glycoside groups showed significantly increased GSH levels in liver tissue (P<0.05, P<0.01), and the high-dose group showed significantly increased SOD activity (P<0.05), while the low-dose group showed no significant difference. The folic acid group had no significant effect on GSH and SOD. The high-dose crystallizing glycoside and folic acid groups significantly reduced MDA levels (P<0.05, P<0.01), but the low-dose group had no significant effect. These results indicate that crystallizing glycoside has a significant ameliorative effect on oxidative damage induced by methotrexate treatment.

[0151] Table 3. Effects of MON on the activity of antioxidant enzymes in liver tissue of mice with acute liver injury caused by MTX (n=6)

[0152]

[0153] Note: Compared with the MTX group, *P<0.05, **P<0.01

[0154] This invention uses crystallized blue glycosides to prevent methotrexate-induced acute liver injury as a model. The study evaluated the effect of crystallized blue glycosides in alleviating methotrexate-induced acute liver injury in mice by detecting liver function indicators, the activity of antioxidant enzymes in liver tissue, and examining liver tissue pathological sections. The results showed that crystallized blue glycosides can reduce the elevation of liver function enzymes in mice with methotrexate-induced acute liver injury, improve oxidative damage in liver tissue, and reduce the accumulation of lipid droplets in liver tissue. This invention further lays the research foundation for the combined development of methotrexate with total iridoid glycosides of Morinda officinalis or its main component, crystallized blue glycosides, into a novel drug for the treatment of rheumatoid arthritis (RA).

[0155] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The use of total iridoid glycosides from Morinda officinalis in combination with methotrexate in the preparation of an antirheumatoid arthritis drug, characterized in that, The total iridoid glycosides of Morinda officinalis, when used in combination with methotrexate, reduce the liver-damaging toxicity of methotrexate. The total iridoid glycosides of Morinda officinalis are prepared by the following method: The pulverized Morinda officinalis herb was cold-soaked in 30-95 vol% ethanol, and the extract was obtained by percolation. The extract was concentrated and filtered. The filtrate was adsorbed through a macroporous resin, which is a polystyrene-type porous adsorption resin with styrene as the crosslinking material. Impurities were removed by elution with water or less than 5 vol% aqueous ethanol. Then, it was eluted with 10%-70 vol% aqueous ethanol. The ethanol eluent was collected and concentrated to dryness under reduced pressure.

2. The use according to claim 1, characterized in that, The total iridoid glycosides of Morinda officinalis are crystallized glycosides.

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