Application of caragana sinica diels stilbene glucoside part in preparation of anti-rheumatoid arthritis medicine

Abstract

The invention discloses an application of a caragana sinica stilbene glucoside part in preparation of an anti-rheumatoid arthritis medicine, and belongs to the field of biological medicines. The invention mainly relates to a preparation method of an anti-rheumatoid arthritis medicine, which comprises the following steps: taking caragana sinica rehd powder, carrying out heating reflux extraction through a solvent, combining extracting solutions, concentrating to be dry, and further drying to obtain a caragana sinica rehd extract; preparing the caragana sinica rehd extract into a dispersion liquid, adsorbing and eluting water-soluble impurities and compound impurities through an adsorbent, performing vacuum concentration on the eluent, and finally preparing the concentrated solution into freeze-dried powder to obtain the caragana sinica rehd stilbene glucoside part. Compared with the prior art, the caragana sinica diels stilbene glucoside part prepared by the preparation method disclosed by the invention has a remarkable effect of resisting rheumatoid arthritis; the foot swelling degree of AIA model rats can be obviously reduced; the bone destruction degree of AIA model rats can be obviously reduced; meanwhile, the content of inflammatory factors IL-1beta, IL-6 and TNF-alpha in serum of model rats is remarkably reduced, and the polypeptide has the potential of being developed into new anti-rheumatoid arthritis drugs.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to an application of a stilbene glycoside fraction of gentiana styraciflua in the preparation of an anti-rheumatoid arthritis drug. Background Art

[0002] Rheumatoid arthritis (RA) is an autoimmune disease with symmetrical, multiple joint inflammation as the main clinical manifestation. It recurs repeatedly. If it is not cured for a long time, the affected joints may begin to deform, even stiffen or deform, leading to joint disability and loss of work and life ability. Although the current RA treatment drugs can alleviate the RA condition to a certain extent, they have many adverse reactions and a long treatment cycle. Many cases cannot persist for a sufficient course of treatment. The development of new drugs for the treatment of RA, or the formulation of a combination drug regimen with high efficacy and few toxic and side effects, is still the ideal goal pursued by the medical community. In other words, whether the existing clinical Chinese medicine preparations can be developed or improved so that they can achieve the same or even surpass the clinical efficacy of existing anti-rheumatic drugs for improving the condition, and at the same time have few toxic and side effects, especially for patients with refractory RA inflammation who are ineffective with existing therapeutic drugs, is the focus and expectation of rheumatologists of traditional Chinese medicine and Western medicine. Therefore, it is urgent to develop new, efficient and safe RA treatment drugs. The root of Caragana sinica (Buc'hoz) Rehder is a plant of the Leguminosae family. The drug was first recorded in the ancient books "Supplement to Compendium of Materia Medica" and "Compendium of Materia Medica for Famine Relief". The "National Compendium of Chinese Herbal Medicines" records that it can dispel wind and dampness, promote blood circulation and regulate menstruation, and is mainly used to treat rheumatic joint pain, traumatic injuries, hypertension, etc. It is one of the commonly used anti-rheumatic drugs. In clinical practice, the root of brocade can be used to treat RA with definite efficacy and high safety. Brocade root shows the characteristics and advantages of definite efficacy and high safety in the treatment of RA, and has good development potential. Brocade root contains a variety of compounds, including flavonoids, diphenylethylene, phenylpropanoids, alkaloids, etc. Modern pharmacological studies have shown that brocade root has multiple biological activities such as anti-inflammatory, antioxidant, antibacterial, antiviral, and neuroprotective. Due to the complexity of the chemical composition and biological effects of brocade root, its anti-RA mechanism and material basis have not yet been scientifically elucidated. Summary of the invention

[0003] In view of the deficiencies of the prior art, the present invention proposes an application of a stilbene glycoside fraction of gentiana root in the preparation of an anti-rheumatoid arthritis drug.

[0004] The purpose of the present invention can be achieved through the following technical solutions:

[0005] The first aspect of the present invention relates to a method for preparing an anti-rheumatoid arthritis drug, comprising the following steps:

[0006] Taking brocade root powder, performing reflux extraction with a solvent, combining the extracts, concentrating to dryness, and further drying to obtain brocade root extract;

[0007] The brocade root extract is made into a dispersion liquid, adsorbed by an adsorbent, water-soluble impurities and compound impurities are eluted, the eluate is concentrated under reduced pressure, and finally the concentrate is made into a freeze-dried powder to obtain the brocade root diphenyl glycoside fraction.

[0008] Optionally, the solvent is ethanol.

[0009] Optionally, the eluent used for the elution includes water and ethanol solution.

[0010] Optionally, the mass ratio of the solvent to the siskinensis root powder is 11:1.

[0011] The second aspect of the present invention relates to an anti-rheumatoid arthritis drug, comprising the genistin stilbene glycoside fraction prepared by the above method.

[0012] Optionally, the stilbene glycoside fraction of the root of gentian contains α-glucose, Carasiphenol C, Carasinol B, Carasinol D and Kobophenol A.

[0013] The third aspect of the present invention relates to the use of the stilbene glycoside fraction obtained by the above method in the preparation of anti-rheumatoid arthritis drugs.

[0014] A fourth aspect of the present invention relates to a method for constructing a rat model, comprising the following steps:

[0015] Several healthy rats were taken as the normal group;

[0016] Several rats were injected with complete Freund's adjuvant to induce adjuvant arthritis as the model group;

[0017] Several rats were randomly selected from the model group and fed with methotrexate 1 mg / kg / 3 days to form the MTX group;

[0018] Several rats were randomly selected from the model group and fed with 50 mg / kg / day of stilbene glycosides from the root of bronchodil, forming the stilbene glycosides group;

[0019] The arthritis index scores were regularly performed for the normal group, model group, MTX group, and stilbene glycoside group; the severity of arthritis was graded according to the arthritis index scores;

[0020] After the observation and recording were completed, the rats were sacrificed and histological examination was performed by CT scanning to observe bone erosion;

[0021] Rat serum was used to measure inflammatory cytokines.

[0022] Optionally, the inflammatory cytokines include at least one of TNF-α, IL-6, and IL-1β.

[0023] Beneficial effects of the present invention:

[0024] The stilbene glycosides prepared by the present invention have significant anti-rheumatoid arthritis effects; can significantly reduce the degree of foot swelling in AIA model rats; can significantly reduce the degree of bone destruction in AIA model rats; and at the same time significantly reduce the contents of inflammatory factors IL-1β, IL-6 and TNF-α in the serum of model rats, and has the potential to be developed into a new anti-rheumatoid arthritis drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The present invention will be further described below in conjunction with the accompanying drawings.

[0026] Figure 1 Comparison of foot swelling in AIA rats of each group in Example 1 of the present application (n=6), where compared with the normal group, ***p<0.001; compared with the model group, ##p<0.01. Blank group (Control), model group (Model), methotrexate group (MTX), stilbene glycoside group (EBYXG).

[0027] Figure 2 The bone destruction of rats in each group in Example 2 of the present application: blank group (Control), model group (Model), methotrexate group (MTX), and stilbene glycoside group (EBYXG).

[0028] Figure 3 Comparison of serum inflammatory factors in each group in Example 3 of the present application (n=6), where compared with the normal group, ###p<0.001; compared with the model group, **p<0.01, ***p<0.001. Blank group (Control), model group (Model), methotrexate group (MTX), stilbene glycoside group (EBYXG). DETAILED DESCRIPTION

[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1 Preparation of stilbene glycosides from genisthen

[0031] Weigh 10 kg of brocade root powder, crush it according to the conventional method, put it into an extraction tank, and reflux extract it 3 times with 70% ethanol (solvent) by volume concentration. The amount of solvent used each time is 11 times (i.e. 110L) of the brocade root raw drug. The extraction time for each time is 2 hours. After the extracts are combined, they are concentrated to dryness and further dried to obtain the brocade root total extract.

[0032] (2) 0.2 kg of the extract was dispersed in 20% ethanol water, and the dispersion was applied to a macroporous resin, and then eluted with water to remove water-soluble impurities. The compounds adsorbed on the macroporous resin were then eluted with a 60% ethanol solution. The eluate was then concentrated under reduced pressure, and finally the concentrate was made into a lyophilized powder to obtain the stilbene glycoside fraction of the root of gentian.

[0033] 2 Determination of the content of diphenylethylene in the root of gentian

[0034] (1) Preparation of standard solutions: Accurately weigh 3 mg of α-glucose, Carasiphenol C, Carasinol D, Carasinol B, and Kobophenol A standard, add 1 mL of DMSO, and vortex to mix to obtain the stock solutions of each standard. Then take an appropriate volume of each stock solution of the standard and add methanol to dilute it to a 100 ug / mL mixed stock solution of the standard for later use.

[0035] (2) Preparation of Gentiana stilbene glycoside test solution Accurately weigh 10 mg of Gentiana stilbene glycoside fraction, add 1 mL of DMSO, and vortex to mix to obtain stilbene glycoside test solution. (3) Preparation of standard curve The mixed standard stock solution was diluted with 50% methanol water to a series of standard solutions with concentrations of 10, 20, 50, 100, 200, 500, 1000, 2500, 5000 ng / mL, and then the compounds in the standard and Gentiana stilbene glycoside solution were detected by mass spectrometry. The standard curve was drawn with the peak area as the ordinate and the concentration as the abscissa, and the regression equation was established. The content of Gentiana stilbene glycoside was determined and calculated using the standard curve. The results showed that Gentiana stilbene glycoside fraction contained α-glucose, Carasiphenol C, Carasinol D, Kobophenol A, and Carasinol B, among which the content of Kobophenol A was greater than 40%.

[0036] Example 2 Therapeutic effect of stilbene glycoside fraction of genistin on adjuvant arthritis rats

[0037] 1. Experimental Materials

[0038] Male 160-180 g SD rats. Centrifuge (Eppendorf, Germany), incomplete Freund's adjuvant (SIGMA, USA), TNF-α ELISA kit (Ruixin Biotechnology, batch number: RX302058R), IL-6 ELISA kit (Ruixin Biotechnology, batch number: RX302856R), IL-1β ELISA kit (Ruixin Biotechnology, batch number: RX302869R)

[0039] 2. Experimental Modeling and Drug Administration

[0040] Adjuvant arthritis (AIA) was induced in rats by injection of complete Freund's adjuvant. In brief, 100 μL (0.5 mg / mL) of mineral oil containing heat-killed Mycobacterium tuberculosis was injected into the tail base of SD rats (100-120 g). After modeling, the rats were randomly divided into 4 groups (n=6), including a normal group, a model group, a MTX group (1 mg / kg / 3 days), and a stilbene glycoside group (50 mg / kg / day). Arthritis index scores were performed every three days. The severity of arthritis was graded using clinical scores according to traditional methods. On the 27th day after modeling, the rats were killed and histological examinations were performed by CT scanning (SkyScan 1176, Bruker, Belgium) to observe bone erosion. Rat serum was used to determine inflammatory cytokines, including TNF-α, IL-6, and IL-1β.

[0041] 3. Statistical Analysis

[0042] Data are expressed as mean ± standard deviation (SD). Statistical analysis was performed by one-way analysis of variance (ANOVA) using GraphPad software (Prism 9, San Diego, CA, USA). P ≤ 0.05 was considered statistically significant.

[0043] 4. Experimental Results

[0044] (1) Rat foot swelling score

[0045] The rat foot swelling score is an external objective indicator for evaluating the AIA model. The rat foot swelling scoring rule is: 0 points = normal, no swelling; 1 point = mild swelling of local joints or toes; 2 points = mild swelling of toe joints and toes or ankle joints; 3 points = all paws below the ankle joint are swollen, or the ankle joint is severely swollen; 4 points = severe swelling from the ankle joint to the metatarsal bone, even deformed.

[0046] The experimental results show that ( Figure 1), on the 9th day after membrane formation, the toe swelling of the model group rats was significantly different from that of the normal group, and the stilbene glycosides of gentian root (50mg / kg / day) and the positive drug methotrexate (1mg / kg / 3 days) both had a good inhibitory effect on the toe swelling of the model rats. The results showed that the stilbene glycosides of gentian root could significantly reduce the toe swelling of AIA model rats.

[0047] (2) Visualization of bone destruction by micro-CT

[0048] The rat's hind limbs were placed in micro-CT and the bone damage of the rats could be visualized through X-ray scanning, making it easier to judge. The experimental results showed that ( Figure 2 ), compared with normal rats, the three-dimensional micro-CT imaging data of the joints of AIA rats showed that the joint gap of AIA rats was narrowed and there was severe bone erosion. The treatment with stilbene glycosides of gentian root (50 mg / kg / day) and the positive drug methotrexate (1 mg / kg / 3 days) both showed significant protective effects on the joints and improved bone erosion.

[0049] (3) Comparison of serum TNF-α, IL-1β, and IL-6 levels in rats

[0050] The experimental results show that ( Figure 3 ), compared with the normal group, the levels of IL-1β, IL-6 and TNF-α in the model group were significantly increased (P<0.001). Both the stilbene glycoside fraction of gentian root (50mg / kg / day) and the positive drug methotrexate (1mg / kg / 3 days) have a good inhibitory effect on the serum inflammatory factor levels of AIA rats. In view of the above efficacy experimental results, the stilbene glycoside fraction of gentian root has significant anti-rheumatoid arthritis activity, and can therefore be used to prepare anti-rheumatoid arthritis and other related immune and inflammatory disease drugs, health care drugs or foods.

[0051] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0052] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for preparing an anti-rheumatoid arthritis drug, characterized in that: The following steps are involved: Taking the brocade root powder, performing heating reflux extraction with a solvent, combining the extracts, concentrating to dryness, and further drying to obtain the brocade root extract; The brocade root extract is made into a dispersion liquid, adsorbed by an adsorbent, water-soluble impurities and compound impurities are eluted, the eluate is concentrated under reduced pressure, and finally the concentrate is made into a freeze-dried powder to obtain the brocade root diphenyl glycoside fraction.

2. The method for preparing the anti-rheumatoid arthritis drug according to claim 1, characterized in that: The solvent is ethanol.

3. The method for preparing the anti-rheumatoid arthritis drug according to claim 1, characterized in that: The eluent used in the elution includes water and ethanol solution.

4. The method for preparing the anti-rheumatoid arthritis drug according to claim 1, characterized in that: The mass ratio of the solvent to the goldenrod root powder is 11:

1.

5. An anti-rheumatoid arthritis drug comprising the stilbene glycoside fraction obtained by the method according to any one of claims 1 to 4.

6. The anti-rheumatoid arthritis drug according to claim 5, characterized in that: The stilbene glycoside part of the genisthenol contains α-glucose, carasiphenol C, carasinol D and kobophenol A.

7. Use of the stilbene glycoside fraction obtained by the method according to any one of claims 1 to 4 in the preparation of anti-rheumatoid arthritis drugs.

8. A method for constructing a mouse model, characterized in that: The following steps are involved: Several healthy rats were taken as the normal group; Several rats were injected with complete Freund's adjuvant to induce adjuvant arthritis as the model group; Several rats were randomly selected from the model group and fed with methotrexate 1 mg / kg / 3 days to form the MTX group; Several rats were randomly selected from the model group and fed with 50 mg / kg / day of stilbene glycosides from the root of bronchodil, forming the stilbene glycosides group; The arthritis index scores were regularly performed for the normal group, model group, MTX group, and stilbene glycoside group; the severity of arthritis was graded according to the arthritis index scores; After the observation and recording were completed, the rats were sacrificed and histological examination was performed by CT scanning to observe bone erosion; Rat serum was used to measure inflammatory cytokines.

9. The method for constructing a rat model according to claim 8, characterized in that: The inflammatory cytokines include at least one of TNF-α, IL-6 and IL-1β.

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