Lindera glauca extract, preparation method thereof and application of lindera glauca extract in preparation of medicine for preventing and / or treating rheumatoid arthritis

Abstract

The invention discloses a lindera glauca extract, a preparation method thereof and application of the lindera glauca extract in preparation of drugs for preventing and / or treating rheumatoid arthritis. The research finds that the lindera glauca extract can obviously reverse arthritic rat plantar part swelling and toe bending, relieve ankle joint injury, relieve synovial hyperplasia and inflammatory cell infiltration, inhibit joint fusion, relieve joint gap stenosis, relieve articular surface fusion tendency, increase bone mineral density and bone trabecula number, reduce bone trabecula separation degree and improve joint fusion. The lindera glauca extract can be used for relieving rheumatoid arthritis by protecting the inherent structure of joint tissues and improving inflammation, so that a novel natural medicine choice is provided for preventing and / or treating rheumatoid arthritis.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical compounds, and in particular, to a Lindera glauca extract, a preparation method thereof, and an application thereof in the preparation of drugs for preventing and / or treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic, symmetric, polyarticular inflammatory disease mainly characterized by immune dysfunction-related chronic autoimmune disease, involving hands, feet and joints throughout the body, with a very high disability rate, seriously threatening human health. Currently, the commonly used drugs in clinical practice are non-steroidal anti-inflammatory drugs, glucocorticoid drugs, disease-modifying anti-rheumatic drugs and biological agents. Although these drugs have good efficacy, as drugs used for a long time or in large doses, they will still produce a series of adverse reactions and tolerances, such as serious gastrointestinal damage, liver, kidney and lung damage, cardiovascular toxicity risk, osteoporosis, increased risk of infection, etc.; secondly, biological agents are expensive and difficult for ordinary families to afford. Therefore, in order to solve the adverse reactions and tolerances of drugs, the development of new anti-rheumatoid arthritis drugs has always been a hot topic of concern for pharmacologists.

[0003] It is clearly recorded in the "Newly Revised Materia Medica" of the Tang Dynasty, "Compendium of Chinese Ethnic Medicines", "Chinese Herbal Medicine Dictionary" and literature that Lindera glauca is warm in nature, pungent in taste, and belongs to the liver and stomach meridians, with the effects of expelling wind and dredging collaterals, promoting diuresis and detumescence, regulating qi and activating blood circulation. It is often used clinically to prevent and treat RA and is a kind of ethnic medicine. "Fujian Folk Herbs" records that Lindera glauca root is combined with pig's feet, soaked in yellow rice wine, and decocted with appropriate amount of water, which can treat rheumatic paralysis; "Anhui Chinese Herbal Medicine" also records that 15 g of Lindera glauca root and 15 g of Polygonum cuspidatum, 9 g of Chaenomeles speciosa, soaked in white wine for one week, and taken orally morning and evening can treat joint pain. Experimental studies and previous related authorized patents of this research group (ZL201910373012.1 and ZL202110308494.X) have shown that Lindera glauca and its active ingredients have good anti-inflammatory activities, but there is no public report on their effects in the treatment of rheumatoid arthritis. Summary of the Invention

[0004] The purpose of the present invention is to solve the deficiencies of the above-mentioned background art, provide a Lindera glauca extract, a preparation method thereof, and an application thereof in the preparation of drugs for preventing and / or treating rheumatoid arthritis, develop a new application of Lindera glauca extract in preventing and / or treating rheumatoid arthritis, and provide a new type of natural drug option for preventing and / or treating rheumatoid arthritis.

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] In a first aspect, the present invention provides a Litsea cubeba extract, wherein the Litsea cubeba is Litsea cubeba of the genus Litsea in the Lauraceae family, and the Litsea cubeba extract contains main active ingredients of azulenoids, naphthalenes, and [6,7]-membered ring skeleton sesquiterpenes.

[0007] The main active ingredients of the azulenoids include the following compounds 1, 2, 4, 5, and 8;

[0008] The active ingredients of the naphthalenes include the following compounds 3, 7, and 9;

[0009] The active ingredients of the [6,7]-membered ring skeleton sesquiterpenes include the following compound 6;

[0010]

[0011] The names of the above compounds 1-9 are: rupestonic N(1), aciphyllic acid(2), (5α,7αH)-2-oxoeudesma-3,11(13)-dien-12-oic acid(3), rupestonic acid(4), (-)-(4S,7S,10S)-2-oxo-guaia-1(5),11(13)-dien-12-oic acid(5), artemisia ketonic acid(6), rupestonic acid I(7), pseudoguaianelactone A(8), rupestonic acid H(9).

[0012] Preferably, the mass percentage content of the active ingredients of the azulenoids in the Litsea cubeba extract is 0.05% - 5%, the mass percentage content of the active ingredients of the naphthalenes is 0.169% - 5%, and the mass percentage content of the active ingredients of the [6,7]-membered ring skeleton sesquiterpenes is 0.007% - 5%. More preferably, the mass percentage content of the active ingredients of the azulenoids is 2.25% - 5%.

[0013] Preferably, the mass percentage content of each compound in the Litsea cubeba extract is as follows:

[0014] Compound 1 ≥ 0.69%, Compound 2 ≥ 0.98%, Compound 3 ≥ 0.07%, Compound 4 ≥ 0.54%, Compound 5 ≥ 0.01%, Compound 6 ≥ 0.007%, Compound 7 ≥ 0.09%, Compound 8 ≥ 0.03%, Compound 9 ≥ 0.009%.

[0015] In a second aspect, the present invention provides a preparation method of the Litsea cubeba extract as described above, which includes:

[0016] The roots of Litsea cubeba are extracted with lower-carbon alcohols, and the extract is concentrated to obtain a concentrated extract. The concentrated extract is suspended in water and extracted with petroleum ether to obtain a petroleum ether extract, and the petroleum ether extract is concentrated under vacuum to obtain the Litsea cubeba extract.

[0017] Preferably, the lower-carbon alcohol is a methanol solution or an ethanol solution with a volume fraction of 70%-100%.

[0018] More preferably, the lower-carbon alcohol is methanol or ethanol with a volume fraction of 100%, that is, pure methanol or pure ethanol.

[0019] Preferably, the extraction temperature is 10°C to 40°C, and the number of extraction times is 3 to 5 times;

[0020] More preferably, the extraction temperature is 25°C and the number of extraction times is 3 times.

[0021] In a third aspect, the present invention provides a composition for preventing and / or treating rheumatoid arthritis, which comprises the Litsea cubeba extract as described above.

[0022] Preferably, it further comprises pharmaceutically acceptable pharmaceutical excipients, and the pharmaceutical excipients can be selected from microcrystalline cellulose, powdered sugar, starch, dextrin, sterilized single-distilled water or physiological saline, etc.

[0023] In a fourth aspect, the present invention provides an application of the Litsea cubeba extract in the preparation of a drug for preventing and / or treating rheumatoid arthritis.

[0024] Preferably, the prevention and / or treatment of rheumatoid arthritis includes reversing the swelling and bending of the arthritis site, reducing the damage to the ankle joint, relieving synovial hyperplasia and inflammatory cell infiltration, inhibiting joint fusion, reducing joint space stenosis, relieving the tendency of joint surface fusion, increasing bone density and the number of trabeculae, reducing the trabecular separation degree, and intervening in the release of inflammatory factors.

[0025] The present invention also provides an application of the Litsea cubeba extract in the preparation of a drug that can down-regulate TNF-α, IL-6 and IL-1β in the serum of rheumatoid arthritis and up-regulate the inflammatory factor IL-10.

[0026] In the drug, the Litsea cubeba extract is used as the only active ingredient, and the rest are pharmaceutically acceptable pharmaceutical excipients. The pharmaceutical excipients can be selected from microcrystalline cellulose, powdered sugar, starch, dextrin, sterilized single-distilled water or physiological saline, etc. The dosage forms that the drug can be made into include granules, pills, capsules, tablets, powders, ointments, oral liquids, syrups, etc. The dosage forms of the drug of the present invention can be various, as long as they can enable the active ingredient to effectively reach the mammalian body.

[0027] Fifth aspect, the subject to which the drug for preventing and / or treating rheumatoid arthritis of the present invention is applied is a human or an animal. In the present invention, "animal" refers to any animal that benefits from the improvement or reduction of age-related loss or other decline of the visual system of the animal, including humans, avian animals, bovine animals, canine animals, equine animals, feline animals, hicrine animals, lupine animals, murine animals, ovine animals, and porcine animals, and preferably domestic animals. Among them, domestic animals include, for example, dogs, cats, birds, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, pleasure horses, dairy cows, goats, sheep, donkeys, pigs, and more exotic species fed by humans.

[0028] Preferably, when the subject is a rat, the effective dose of the Lindera glauca extract is 50 - 150 mg / kg. When the subject is a human, the human dosage can be converted according to the dosing standards in the pharmaceutical field.

[0029] The present invention has the following beneficial effects:

[0030] The present invention discovers that the Lindera glauca extract can significantly reverse the swelling and bending at the arthritis site, reduce bone and joint damage, relieve synovial hyperplasia and inflammatory cell infiltration, inhibit joint fusion, reduce joint space stenosis, relieve the tendency of joint surface fusion, increase bone density and the number of trabeculae, reduce the trabecular separation degree, and intervene in the release of inflammatory factors. This indicates that the Lindera glauca extract can relieve rheumatoid arthritis by protecting the inherent structure of joint tissues and improving inflammation, has the potential for clinical application in the treatment of rheumatoid arthritis, can be used to prepare anti-rheumatoid arthritis drugs, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0032] Figure 1 It is the total ion current chromatogram and extracted ion current chromatogram of the Lindera glauca extract in the embodiment of the present invention;

[0033] Figure 2 It is the structural formulas of 9 reference compounds in the embodiment of the present invention;

[0034] Figure 3 It is the extracted ion current chromatograms of 9 reference compounds in the embodiment of the present invention;

[0035] Figure 4 This is the test result diagram of each group intervened with Lindera glauca extract in the rats with rheumatoid arthritis model in the embodiments of the present invention (A. Control; B. Model; C. MTX; D. TSS-L; E. TSS-M; F. TSS-H);

[0036] Figure 5 This is the disease activity index of each group of rats in the embodiments of the present invention (All the results are represented as means±SD(n=6). ### P<0.001, ## P<0.01 versus the control group.*P<0.05,**P<0.01,***P<0.001 versus the model group.);

[0037] Figure 6 This is the Micro-CT result of the ankle joints of each group of rats in the embodiments of the present invention (A. Control; B. Model; C. MTX; D. TSS-L; E. TSS-M; F. TSS-H);

[0038] Figure 7 This is the HE staining result of the ankle joints of each group of rats in the embodiments of the present invention (A. Control; B. Model; C. MTX; D. TSS-L; E. TSS-M; F. TSS-H);

[0039] Figure 8 This is the change of inflammatory factors in the sera of each group of rats in the embodiments of the present invention (The results are represented as means±SD(n=6). ### P<0.001 versus the control group.*P<0.05,**P<0.01,***P<0.001 versus the model group.) Detailed implementation manners

[0040] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0041] The present invention provides a Lindera glauca extract, whose main components are azulenoids, naphthalenes and [6,7]-membered ring skeleton sesquiterpenes. Representative components thereof include rupestonic N (1), aciphyllic acid (2), (5α,7αH)-2-oxoeudesma-3,11(13)-dien-12-oic acid (3), rupestonic acid (4), (-)-(4S,7S,10S)-2-oxo-guaia-1(5),11(13)-dien-12-oic acid (5), artemisia ketonic acid (6), rupestonic acid I (7), pseudoguaianelactone A (8), rupestonic acid H (9). The structures of the above representative components are shown as follows:

[0042]

[0043] In this application, it has been experimentally confirmed that the Lindera glauca extract derived from the plant Lindera glauca has significant effects of reversing swelling and bending at the arthritis site, reducing bone and joint damage, alleviating synovial hyperplasia and inflammatory cell infiltration, inhibiting joint fusion, reducing joint space stenosis, alleviating the tendency of joint surface fusion, increasing bone density and the number of trabeculae, decreasing the trabecular separation degree, and intervening in the release of inflammatory factors. This indicates that the Lindera glauca extract can relieve rheumatoid arthritis by protecting the inherent structure of joint tissues and improving inflammation, and has the potential for clinical application in the treatment of rheumatoid arthritis, and can be used to prepare anti-rheumatoid arthritis drugs.

[0044] Therefore, this application also provides an anti-rheumatoid arthritis composition, which includes the above-mentioned Lindera glauca extract. In an alternative embodiment, the composition further includes pharmaceutically acceptable excipients. The above-mentioned "pharmaceutically acceptable" components are substances that are suitable for humans and / or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit / risk ratio.

[0045] Furthermore, the present invention provides the use of the Lindera glauca extract described in the foregoing embodiment in the preparation of anti-rheumatoid arthritis drugs.

[0046] Furthermore, the present invention provides the use of the Lindera glauca extract described in the foregoing embodiment in the preparation of drugs that can down-regulate TNF-α, IL-6 and IL-1β in the serum of arthritis and up-regulate the production of IL-10.

[0047] In addition, this application also provides a preparation method of the above-mentioned Lindera glauca extract only as an example, which includes:

[0048] S1. Source of Lindera glauca root medicinal material:

[0049] It is the root of Lindera glauca, a plant of the genus Lindera in the Lauraceae family. It is dried and crushed. A smaller particle size is beneficial to improving the extraction effect and enabling the extraction of active ingredients to a greater extent.

[0050] Specifically, the crushing method is not limited, and existing crushing methods can be used, all within the protection scope of the present invention.

[0051] S2. Extraction:

[0052] The Lindera glauca root powder is soaked and extracted 2 - 4 times at 10°C - 40°C (25°C in this example) with a lower-carbon alcohol, and each soaking lasts for 36 - 48 hours. The extract is concentrated to obtain a concentrated extract. The concentrated extract is suspended in water and extracted with petroleum ether at 10°C - 40°C (25°C in this example) until it becomes colorless. The petroleum ether extract is vacuum-concentrated to obtain the Lindera glauca extract.

[0053] In some embodiments, the extraction solvent (lower-carbon alcohol) is methanol or ethanol. The inventor found that using methanol or ethanol as the extraction solvent can significantly improve the extraction effect and the extraction efficiency is significantly higher.

[0054] Preferably, the extraction solvent is anhydrous ethanol to avoid the introduction of water during the extraction process.

[0055] S3. Solvent recovery:

[0056] After the extraction and extraction are completed, the extraction agent and extraction solvent are recovered, and the samples are collected. After drying to a constant weight, the extract and extractant are obtained. The method of recovering the extraction agent and extraction solvent is not limited, and existing methods for recovering organic solvents in the recovery system can be used.

[0057] Experimental example 1

[0058] Quantitative analysis was carried out on the obtained Lindera glauca extract. HPLC-MS was used for elution detection to carry out quantitative analysis on the characteristic chemical components in the Lindera glauca extract. The specific steps are as follows:

[0059] (1) Instruments

[0060] The ultra-high performance liquid system includes an LC-20AD high performance liquid chromatograph, a SIL-20AC automatic sampler, an SPD-M40 detector, a CTO-20AC column oven (Shimadzu Corporation, Japan); an API 4000 triple quadrupole mass spectrometer (AB SCIEX Corporation, USA); an Analyst 1.6 workstation, MultiQuant TM3.0 data processing software, PeakView 2.0 data processing software; SQP QUINTIX125D-1CN type one in one hundred thousandth analytical balance (Sartorius Instruments Company, Germany).

[0061] (2) Reagents

[0062] Methanol, acetonitrile (chromatographic grade, Merck Company, Germany), ultrapure water (Milli- Pure water machine, Merck Company, Germany), formic acid (chromatographic grade, Fisher Company, USA).

[0063] (3) Reference substances

[0064] Reference substances rupestonic N(1), aciphyllic acid(2), (5α,7αH)-2-oxoeudesma-3,11(13)-dien-12-oic acid(3), rupestonic acid(4), (-)-(4S,7S,10S)-2-oxo-guaia-1(5),11(13)-dien-12-oic acid(5), rupestonic acid I(7), pseudoguaianelactone A(8), rupestonic acid H(9) are sesquiterpene monomers isolated, purified and identified by our research group from Lindera glauca (Siebold) Blume previously (attached Figure 2 ), and their mass fractions are all greater than 90.0%.

[0065] (4) Preparation of test solution

[0066] Weigh 32.75 mg of the extract, place it in a 5 mL volumetric flask, add chromatographic methanol, and ultrasonically treat until completely dissolved. Take it out, let it cool, weigh it, make up the difference with chromatographic methanol and shake well; take 1.0 mL of the above solution, place it in a 10 mL volumetric flask, dilute it to the mark with chromatographic methanol, shake well, and filter through a 0.22 μm microporous filter membrane to obtain the test solution.

[0067] (5) Preparation of reference solution

[0068] Weigh the reference substances separately and place them in 1.0 mL volumetric flasks, add chromatographic methanol to dissolve and dilute to the mark to prepare single reference substance stock solutions with a mass concentration of 1.0 mg / mL. Then, accurately pipette appropriate amounts of the above stock solutions in sequence and place them in 10 mL volumetric flasks, dilute to the mark with chromatographic methanol to obtain a mixed solution reference substance stock solution with mass concentrations of 14.0, 8.0, 49.1, 56.0, 64.0, 6.2, 5.8, 4.0, and 4.8 μg / mL for compounds 1-9 respectively.

[0069] Mass spectrometry conditions: AB SCIEX API 4000 triple quadrupole mass spectrometer, equipped with an ESI ion source, data was collected in the negative ion and selected reaction monitoring scan modes. The optimized ion pairs and collision energy parameters for 9 analytes are shown in Table 1. The total ion current chromatogram and extracted ion current chromatogram are shown in Appendix Figure 1 and Appendix Figure 3 .

[0070] Table 1 Optimized ion pairs and collision energy parameters for 9 analytes

[0071]

[0072] (5) Chromatographic conditions

[0073] Waters Atlantis HPLC T3 C18 (50 mm×2.1 mm, 1.7 μm) chromatographic column, column temperature 40 °C; mobile phase A is 0.1% formic acid methanol solution, B is 0.1% formic acid aqueous solution; gradient elution: 0 - 15 min, 38% - 88% A. Flow rate 0.2 mL / min; sample chamber temperature 4 °C; injection volume 2 μL.

[0074] (6) Results of content determination

[0075] The content of each compound was determined according to the above method, and the results are shown in Table 2 below. The contents of rupestonic N(1), aciphyllic acid(2), (5α,7αH)-2-oxoeudesma-3,11(13)-dien-12-oic acid(3), rupestonic acid(4), (-)-(4S,7S,10S)-2

[0076] -oxo-guaia-1(5),11(13)-dien-12-oic acid(5), rupestonic acid I(7), pseudoguaianelactone A(8), rupestonic acid H(9) are 0.69%, 0.98%, 0.07%, 0.54%, 0.01%, 0.007%, 0.09%, 0.03%, 0.009% respectively.

[0077] Table 2 Linear range, regression equation, correlation coefficient, minimum detection limit and minimum quantitation limit of 9 analytes

[0078]

[0079] It should be noted that the 9 compound reference substances measured in the experimental examples of the present invention are the 9 compounds that can be obtained under limited conditions, and do not represent that the Litsea cubeba extract only contains these 9 compounds. Through the analysis of the applicant, it can be known that the main chemical components contained in the Litsea cubeba extract provided in this application are azulenoids, naphthalenes, and [6,7]-membered ring skeleton sesquiterpenoid structures. The 9 compounds also belong to these 3 major types, and the general formulas of their parent nucleus structures are shown in Formulas I to III. It has the above-mentioned anti-rheumatoid arthritis effect. It should be understood that the conventional substituents of the compound skeletons contained in the extract of this application also have the same or similar effects. The substituents of the compounds contained in the Litsea cubeba extract include, but are not limited to, Formulas I to III. Among them, the basic carbon skeletons are shown, and the substituents can be selected from OCH3, OH, substituted or unsubstituted alkyl groups, substituted or unsubstituted alkynyl groups, carbonyl groups, etc., respectively.

[0080]

[0081] An embodiment of the present invention provides a Litsea cubeba extract prepared by the above preparation method. The main active ingredients in the extract are azulenoids, naphthalenes, and [6,7]-membered ring skeleton sesquiterpenoid compounds, which have very good market promotion value.

[0082] An embodiment of the present invention provides a Litsea cubeba composition, which includes the above Litsea cubeba extract and pharmaceutically acceptable pharmaceutical excipients, and the specific types of the excipients are not limited.

[0083] The Litsea cubeba extract or Litsea cubeba composition provided by the embodiments of the present invention can be used in the preparation of drugs for preventing and treating rheumatoid arthritis. Since the contents of azulenoids, naphthalenes, and [6,7]-membered ring skeleton sesquiterpenoids in the Litsea cubeba extract are higher, the preventive and therapeutic effects can be further improved.

[0084] In some embodiments, the prevention and treatment of rheumatoid arthritis specifically include symptoms such as joint swelling and redness, joint ankylosis, limited mobility, uneven joint surface fusion, joint space stenosis, joint structure damage, reduced joint bone density and trabecular bone number, increased trabecular bone separation, synovial hyperplasia, inflammatory cell infiltration, and disorder of inflammatory factors in serum. Any one of the above symptoms is suitable for using the Lindera glauca extract or Lindera glauca composition in the embodiments of the present invention. The inventors found that the Lindera glauca extract or Lindera glauca composition provided in the embodiments of the present invention can relieve the swelling and bending of the arthritis site, reduce bone and joint damage, relieve synovial hyperplasia and inflammatory cell infiltration, inhibit joint fusion, reduce joint space stenosis, relieve the trend of joint surface fusion, increase bone density and trabecular bone number, reduce trabecular bone separation, down-regulate the inflammatory factors TNF-α, IL-6, and IL-1β in the serum of arthritic rats, and up-regulate IL-10, thereby achieving the effect of preventing and treating rheumatoid arthritis. The Lindera glauca extract or Lindera glauca composition provided in the embodiments of the present invention has good clinical application potential for preventing or treating rheumatoid arthritis.

[0085] It should be added that the Lindera glauca extract or Lindera glauca composition provided in the embodiments of the present invention can also be used in the preparation of drugs that inhibit the down-regulation of inflammatory factors TNF-α, IL-6, and IL-1β and up-regulate the release of IL-10.

[0086] It should be noted that the inventors induced and constructed an overall animal model of rheumatoid arthritis (CIA) using bovine type II collagen and incomplete Freund's adjuvant (IFA). The model rats showed symptoms such as significant weight loss, dull body hair, listlessness, reduced daily activities, swelling of the plantar part of the foot, toe bending, limited mobility, increased thymus index, uneven ankle joint surface, joint space stenosis, a tendency of joint surface fusion, severe joint structure damage, reduced bone density and trabecular bone number, increased trabecular bone separation, synovial hyperplasia, and inflammatory cell infiltration. TNF-α, IL-6, and IL-1β in the serum were significantly increased compared with the normal group, and IL-10 was significantly decreased compared with the normal group. After administration of the Lindera glauca extract, the above symptoms of the model rats were significantly relieved. The relevant results indicate that the Lindera glauca extract has a preventive and therapeutic effect on rheumatoid arthritis, which is conducive to the further development and utilization of the Lindera glauca extract, and at the same time provides a new natural drug option for the prevention and treatment of rheumatoid arthritis.

[0087] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0088] Example 1:

[0089] I. Experimental method

[0090] (1) Establishment of CIA animal model

[0091] Dissolve a certain amount of bovine type II collagen in 0.1 mol / L acetic acid to make the concentration 1 g / L, and then mix it with an equal volume of IFA solution to prepare a mixed solution. Stir until the mixture is fully emulsified (when dropped on the water surface, it aggregates into drops and does not disperse within 120 min). After anesthetizing the rats with 1% sodium pentobarbital, inject the above emulsifier into the intradermal of the rat's plantar, tail root and back at a dose of 0.75 mL / kg, and gently press the injection site to avoid the outflow of the injection solution. Fourteen days after the primary immunization injection, inject the emulsifier again at a dose of 0.5 mL / kg to boost the immunity.

[0092] (2) Judgment of successful model establishment

[0093] On the 21st day of the primary immunization, score according to the AI scoring rules in Table 3 below. If the AI score ≥ 4 points, it is determined that the model is successfully established:

[0094] Table 3 Rat arthritis pathology scoring criteria

[0095]

[0096] (3) Animal grouping and drug administration

[0097] Divide the rats into a normal group (Control), a model group (Model), a positive drug group (methotrexate MTX, 800 μg / kg / d), and a Lindera aggregata extract administration group (low TSS-L 0.5 g / kg / d, medium TSS-M 1.0 g / kg / d, high TSS-H 1.5 g / kg / d), with 10 rats in each group. Administer the drugs for a total of 35 days. Before animal treatment, fast the animals but do not restrict water intake.

[0098] (4) Monitoring of conventional RA pharmacodynamic indicators

[0099] To comprehensively evaluate the overall drug effect, carefully record the conditions of the rats' body hair, activities, food intake, defecation volume, etc.; after weighing on the 21st day of the primary immunization, weigh, measure the toe volume and thickness every 7 days, and perform an AI score on the arthritis.

[0100] (5) Imaging detection of ankles and toes

[0101] Perform Micro-CT imaging detection on the rats. After sampling, take pictures of the toe and ankle parts to evaluate the degree of cartilage erosion and observe the conditions of bone dissolution and osteophyte formation.

[0102] (6) Spleen index analysis: Calculate according to the following formula.

[0103] Spleen index = spleen weight / rat body weight

[0104] (7) Pathological section analysis

[0105] The ankle joints of rats were stained with H&E. After removing the skin of the joint bone tissue, it was first fixed in 4% paraformaldehyde for 5 days, and then decalcified in EDTA decalcifying solution for 3 weeks. The decalcifying solution was changed daily until the bone tissue could be easily penetrated by a needle. After rinsing with running water for 24 h, gradient dehydration with alcohol was carried out. After dehydration, permeabilization was performed, and then embedded in paraffin and sectioned. The sections were dewaxed and stained with hematoxylin-eosin. The proliferation of synoviocytes, connective tissue hyperplasia, cartilage invasion, pannus formation and other characteristics were observed under a microscope, and relevant images were collected.

[0106] (8) Detection of serum biochemical indexes

[0107] The secretion of inflammatory cytokines is a major feature of the pathogenesis of RA. The ELISA method was used to detect the intervention of the extract on the release of TNF-α, IL-6, IL-1β, and IL-10 inflammatory factors in the peripheral blood serum of rats.

[0108] II. Experimental results

[0109] (1) Observation of rat signs and arthritis index scoring

[0110] The results of this study showed that compared with the normal group of rats, the body weight of the model rats was significantly reduced, the body hair was less shiny, the spirit was listless, and the daily activities were reduced. Observing the appearance of the rat's feet (attached Figure 4 ), the plantar part of the model group was swollen, the toes were bent, and the activity was limited. After administration of the extract, the swelling and activity were significantly reversed, and these indicators were characterized by the arthritis index score. Analyzing the arthritis index scores of rats in each experimental group, after the intervention of the extract, the arthritis index scores decreased significantly. Among them, the high-dose extract group was particularly obvious. Observing the changes in the body weight of rats in each group during the experiment (attached Figure 5 ), the body weight of the model rats was significantly lower than that of the normal group (P<0.001). After administration of the medium-dose and high-dose extracts, the body weight of the rats increased significantly compared with the model group, and the difference was statistically significant (P<0.05). During the experiment, the plantar volume of the model rats became larger and the plantar thickness became thicker. Compared with the normal group (attached Figure 5 ), the difference was statistically significant (P<0.001). After administration of the high-dose extract group, the plantar volume was significantly smaller and the plantar thickness was narrower, and the difference was statistically significant (plantar volume P<0.01, plantar thickness P<0.001). Compared with the normal group, the thymus index of the model rats was significantly increased (P<0.01). After administration of the medium-dose and high-dose extracts, it was significantly reversed compared with the model group, and the difference was statistically significant (medium dose P<0.05, high dose P<0.01).

[0111] Based on the above experimental results, it was shown that the high-dose extract of terpenoids could effectively alleviate the severity of RA.

[0112] (2) Imaging results of ankles and toes

[0113] Micro-CT imaging results showed that, compared with the normal group of rats, the ankle joint surface of the rats in the CIA model group was less uniform, the joint space was narrow, there was a tendency of joint surface fusion, the joint structure was severely damaged, the bone density and the number of trabeculae were decreased, and the trabecular separation was increased; although the ankle joints of the rats in the other groups had varying degrees of injury, compared with the model group, the extract could significantly reduce the ankle joint injury, inhibit joint injury and fusion, relieve the narrowing of the joint space, alleviate the tendency of joint surface fusion, increase the bone density and the number of trabeculae, and decrease the trabecular separation (attached Figure 6 )

[0114] (3) Analysis of rat ankle joint sections

[0115] Pathological observation of the ankle joint tissues of rats in different groups after H&E staining is shown in the attachment Figure 7 . The results showed that, compared with the normal group, there were obvious synovial hyperplasia and inflammatory cell infiltration in the ankle joints of the rats in the model group; after the intervention of the extract, the synovial hyperplasia and inflammatory cell infiltration in the ankle joints of the model rats were effectively alleviated, especially in the high-dose extract group, and the pathological structure was significantly improved. This indicates that the extract can significantly relieve the pathological changes of the ankle joints of RA rats and protect the inherent structure of the ankle joints.

[0116] (4) Release of inflammatory factors in serum

[0117] The inflammatory factors TNF-α, IL-6 and IL-1β in the serum of the CIA model group were significantly increased compared with the normal group, and IL-10 was significantly decreased compared with the normal group. After the intervention of the extract, TNF-α, IL-6 and IL-1β in the serum of the model rats could be down-regulated, and IL-10 could be up-regulated, and it was dose-dependent, and the difference was statistically significant (p < 0.01) (attached Figure 8 ). This suggests that the extract can improve rheumatoid arthritis by regulating the secretion of inflammatory factors.

[0118] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Litsea cubeba extract, characterized in that, The Lindera glauca extract contains the main active ingredients of azulenoids, naphthalenes and [6,7]-membered ring skeleton sesquiterpenes. The main active ingredients of the azulenoids include the following compounds 1, 2, 4, 5, 8. The naphthalene active ingredients include the following compounds 3, 7, 9. The [6,7]-membered ring skeleton sesquiterpene active ingredients include the following compound 6.

2. The Litsea cubeba extract according to claim 1, wherein In the Lindera glauca extract, the mass percentage content of the azulenoid active ingredients is 0.05% - 5%, the mass percentage content of the naphthalene active ingredients is 0.169% - 5%, and the mass percentage content of the [6,7]-membered ring skeleton sesquiterpene active ingredients is 0.007% - 5%.

3. The Litsea cubeba extract according to claim 1, wherein The mass percentage content of each compound in the Lindera glauca extract is as follows: Compound 1 ≥ 0.69%, Compound 2 ≥ 0.98%, Compound 3 ≥ 0.07%, Compound 4 ≥ 0.54%, Compound 5 ≥ 0.01%, Compound 6 ≥ 0.007%, Compound 7 ≥ 0.09%, Compound 8 ≥ 0.03%, Compound 9 ≥ 0.009%.

4. A preparation method of the Litsea cubeba extract as described in claim 1, characterized in that, It includes: Powder of Lindera glauca root is extracted with lower-carbon alcohol, the extract is concentrated to obtain a concentrated extract, the concentrated extract is suspended in water and extracted with petroleum ether to obtain a petroleum ether extract, and the petroleum ether extract is vacuum concentrated to obtain the Lindera glauca extract.

5. A method for preparing the Litsea cubeba extract as described in claim 4, characterized in that, The lower-carbon alcohol is a methanol solution or an ethanol solution with a volume fraction of 70% - 100%.

6. A composition for preventing and / or treating rheumatoid arthritis, characterized in that, It includes the Lindera glauca extract as described in Claim 1.

7. The composition for preventing and / or treating rheumatoid arthritis according to claim 6, characterized in that, It also includes pharmaceutically acceptable pharmaceutical excipients.

8. Use of the Lindera glauca extract as described in Claim 1 in the preparation of a drug for preventing and / or treating rheumatoid arthritis.

9. Use of the Lindera glauca extract as claimed in claim 8 in the preparation of a medicament for preventing and / or treating rheumatoid arthritis, characterized in that, The prevention and / or treatment of rheumatoid arthritis includes reversing the swelling and bending of the arthritis site, reducing the damage of the ankle joint, relieving synovial hyperplasia and inflammatory cell infiltration, inhibiting joint fusion, reducing joint space stenosis, relieving the tendency of joint surface fusion, increasing bone density and the number of trabeculae, reducing the trabecular separation degree, and intervening in the release of inflammatory factors.

10. Use of the Lindera glauca extract as described in Claim 1 in the preparation of a drug that can down-regulate TNF-α, IL-6 and IL-1β in the serum of rheumatoid arthritis and up-regulate the inflammatory factor IL-10.

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