Application of gardenia extract in preparation of medicine for treating rheumatoid arthritis complicated with pulmonary interstitial lesion

Abstract

The invention discloses application of a gardenia extract in preparation of a medicine for treating rheumatoid arthritis complicated with pulmonary interstitial lesion, and belongs to the technical field of new medical application of plant extracts. The preparation method comprises the following steps: by taking gardenia as a raw material, performing reflux extraction with 70% ethanol, performing vacuum concentration to obtain an extract, performing HPD-600 macroporous resin column chromatography, eluting with 35% v / v ethanol, recovering a solvent, and drying to obtain the gardenia extract. Animal experiments show that when the extract is orally taken according to 25-50 mg / kg / day to an RA-ILD model mouse infected and induced by type II collagen and Pg, the arthritis score can be remarkably reduced, synovial hyperplasia, inflammation infiltration and cartilage / bone destruction can be relieved, pulmonary interstitial collagen deposition can be reduced, and the levels of serum TNF-alpha, IL-1beta and IL-6 can be remarkably reduced. The extract is derived from medicinal and edible plants, is simple in preparation process and good in safety, and can be used as a candidate medicine for treating rheumatoid arthritis combined with pulmonary interstitial lesion.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of new medical uses of plant extracts, and particularly relates to application of gardenia extract in preparation of a medicine for treating rheumatoid arthritis combined with interstitial lung disease. BACKGROUND

[0002] Rheumatoid arthritis (RA) is a chronic immune disease involving the whole body, and the global incidence rate is close to 1%. In addition to common clinical symptoms such as joint involvement, RA is often accompanied by various complications. Interstitial lung disease (ILD) is one of the most common complications of RA, which is manifested as persistent dry cough, restrictive ventilatory dysfunction, dyspnea, and ground glass diffuse lesions on imaging. More than 40% of RA patients are accompanied by ILD symptoms. In addition, ILD is also the main cause of the decreased survival rate of RA patients, and the mortality rate of advanced ILD is even as high as more than 20%. At present, there is no recognized effective treatment drug and treatment method for RA-ILD in clinical practice. Although glucocorticoids and immunosuppressive agents can relieve symptoms for a short time, they cannot completely cure the disease and will cause many adverse reactions. In addition, traditional anti-rheumatic drugs for treating RA may induce pulmonary infection and drug-related lung disease, further causing the aggravation of ILD symptoms. Therefore, the development of new drugs for the co-treatment of RA-ILD is an important issue that needs to be explored in the field of RA.

[0003] Gardenia is the dried mature fruit of Gardenia jasminoides Ellis of Rubiaceae, which is cold in nature and bitter in taste. It is the first batch of medicinal and edible resources approved by the Ministry of Health of China, and has the effects of protecting liver, promoting bile secretion, lowering blood pressure, calming, stopping bleeding and detumescence. It is commonly used in clinical treatment of jaundice hepatitis, sprains, hypertension, diabetes and the like in traditional Chinese medicine. SUMMARY

[0004] In order to solve the above technical problems, the application provides application of gardenia extract in preparation of a medicine for treating rheumatoid arthritis combined with interstitial lung disease, which provides a new single plant source candidate drug for the co-treatment of RA-ILD.

[0005] The application provides application of gardenia extract in preparation of a medicine for treating rheumatoid arthritis combined with interstitial lung disease.

[0006] The medicine is an oral preparation, and is selected from tablets, capsules, granules, oral liquids and dripping pills.

[0007] The medicine takes the gardenia extract as the only active component.

[0008] The medicine also contains a pharmaceutically acceptable carrier, diluent or excipient, but does not contain other traditional Chinese medicines or chemical medicines having the activity of treating rheumatoid arthritis combined with interstitial lung disease.

[0009] The interstitial lung disease is interstitial pneumonia secondary to rheumatoid arthritis, usual interstitial pneumonia or diffuse alveolar damage.

[0010] The application also provides a preparation method of the Gardenia extract, comprising the following steps:

[0011] S1, taking Gardenia jasminoides Ellis dried mature fruits as raw materials, crushing, refluxing extraction with 6-10 times the amount of 60-80% low-carbon alcohol for 1-3 hours each time; combining the filtrate and concentrating under reduced pressure to obtain Gardenia extract;

[0012] S2, dispersing the Gardenia extract in water and then loading onto a HPD-600 type macroporous adsorption resin column, eluting with 6-10 times the column volume of water and 2-4 times the column volume of 35% v / v ethanol in sequence, collecting the 35% v / v ethanol elution part, concentrating under reduced pressure and drying to obtain the Gardenia extract.

[0013] The low-carbon alcohol is selected from methanol, ethanol, propanol or isopropanol.

[0014] The Gardenia extract is orally administered at a dose of 25-50 mg / kg body weight / day, which can significantly reduce the arthritis score of the RA-ILD model mice induced by type II collagen combined with Pg infection, inhibit synovial hyperplasia, inflammatory cell infiltration and cartilage / bone erosion, and reduce lung interstitial collagen deposition and serum TNF-α, IL-1β and IL-6 levels.

[0015] Compared with the prior art, the application has the following beneficial effects:

[0016] The Gardenia 35% ethanol elution part of the application shows a clear double intervention effect in the RA-ILD model mice: oral administration of 25-50 mg / kg can significantly reduce the arthritis score, reduce synovial cell hyperplasia, inflammatory infiltration and cartilage / bone destruction; at the same time, reduce lung interstitial collagen deposition and improve alveolar structure disorder. The synchronous decrease of serum TNF-α, IL-1β and IL-6 levels indicates that systemic inflammation is inhibited. The extract is derived from Gardenia jasminoides Ellis, which is both a medicine and food, has a simple preparation process, a clear dose range, good safety and accessibility, and provides a new single plant source candidate drug for long-term treatment of RA-ILD. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed for the embodiment description are briefly introduced as follows.

[0018] Figure 1 Total ion chromatogram of gardenia extract.

[0019] Figure 2 Effects of gardenia extract on arthritis score and joint histopathology of mice, wherein a is foot swelling, b is arthritis score, c is knee HE staining, d is knee histopathology score, e is ankle HE staining, and f is ankle histopathology score.

[0020] Figure 3 Effects of gardenia extract on lung histopathology and collagen deposition of mice, wherein a is lung HE staining, b is lung histopathology score, c is lung Masson staining, and d is lung collagen area proportion.

[0021] Figure 4 Effects of gardenia extract on serum inflammatory factors of mice, wherein a is serum IL-1β level, b is serum IL-6 level, and c is serum TNF-α level. DETAILED DESCRIPTION

[0022] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments.

[0023] Embodiment 1

[0024] Extraction method of gardenia extract

[0025] Take 1010 g of gardenia medicinal materials, crush into fine powder (50 mesh sieve), use 8 times the amount of 70 v / v% ethanol to reflux extract 2 times, each for 2 h, combine the filtrate, and concentrate under reduced pressure to no alcohol taste, to obtain gardenia extract 242 g. Take gardenia extract and add water to form a suspension, and pass through a HPD-600 macroporous resin column (the amount of resin is 12 times the weight of the extract, and the adsorption flow rate is 3 BV / h). First, use 8 times the column volume of water to elute, then use 3 times the column volume of 35 v / v% ethanol to elute, collect the 35 v / v% ethanol elution part, concentrate under reduced pressure to no alcohol taste, and freeze-dry to obtain gardenia extract 68.89 g.

[0026] Chromatographic conditions

[0027] ACE Excel 1.7 Super C18 column (2.1 mm x 50 mm, 1.7 um), mobile phase 0.1% formic acid water (A) - acetonitrile (B), gradient elution (0-10 min, 5%-15% B; 10-12 min, 15%-21% B; 12-15 min, 21% B; 15-18 min, 21%-23% B; 18-20 min, 23%-50% B; 20-30 min, 50%-95% B), flow rate 0.3 mL·min -1 ; injection volume 3 μL; column temperature 40℃. Ion source: electrospray ion source (ESI), parent ion mass scan range: m / z 100-1000, secondary ion fragment mass scan range: m / z 50-1000, IDA setting peaks with response value exceeding 100 cps are preferentially subjected to secondary mass spectrum scanning, dynamic background deduction (DBS) is turned on, and other parameters and parameter settings are as follows: positive ion source spray voltage is 4500V, ion source temperature is 500℃, de-clustering voltage (DP) is 100V, collision energy (CE) is 45eV, and collision energy expansion (CES) is 15eV. The atomizing gas is N2, auxiliary gas 1 (GS1) is 50PSI, auxiliary gas 2 is 50PSI, and curtain gas (CUR) is 40PSI.

[0028] The ion current diagram is shown in Figure 1 21 components were identified by comparison with reference substances, retention time, mass spectrum cracking rules, and literature information, and the results are shown in Table 1.

[0029] Table 1 Identification of chemical components of gardenia extract

[0030]

[0031]

[0032] Example 2 Animal experiment

[0033] In this embodiment, the experimental data was statistically analyzed by using GraphPad Prism 9.5 software, and all data were expressed as mean ± standard deviation (x ± s). One-way ANOVA method was used for single factor comparison among multiple groups, and two-way ANOVA method was used for double factor comparison among multiple groups. P<0.05 was determined to be a significant difference.

[0034] 1. Animal grouping, modeling and drug administration:

[0035] Forty DBA / 1 mice which had been adaptive feeding for 7 days were randomly divided into normal group (Control group), model group (RA-ILD group), gardenia extract low-dose group (G-L group), gardenia extract high-dose group (G-H group) according to body weight, 10 mice in each group. The RA-ILD group, G-L group and G-H group were constructed into the mouse disease model of rheumatoid arthritis combined with pulmonary interstitial fibrosis by the way of immunization with type II collagen combined with Porphyromonas gingivalis (Pg) infection. The immune level of bovine type II collagen was dissolved in 0.05 mol / L acetic acid, and then mixed with Freund's complete adjuvant to prepare water-in-oil emulsion by using high-speed stirrer under ice bath condition, and finally the mass concentration of type II collagen was adjusted to 1 mg / mL. After the mice were anesthetized with isoflurane, emulsion injection was performed: on the 14th day of the experiment, the mice were subcutaneously injected with 200 μL emulsion at the tail root for the first immunization. On the 35th day, the mice were subcutaneously injected with the same dose of emulsion at multiple points on the back for the second immunization. During the whole modeling period, Pg strain inoculation was performed every two days for 56 days. The normal group of mice was injected with the same amount of normal saline. From the second immunization, the G-L group and the G-H group were given 25 mg / kg and 50 mg / kg extract solution for gavage, once a day, for 21 days. The normal group and the model group were given the same amount of normal saline.

[0036] 2. Arthritis score:

[0037] The arthritis score of each group of mice was performed, and the specific scoring sites included 4 ankle and wrist joints, 4 foot segments, and 12 finger and toe joints of each mouse: the scoring standard of the ankle or wrist joint of each limb was as follows: 0 points indicated no swelling; 1 point indicated mild swelling; 2 points indicated moderate swelling; 3 points indicated severe swelling; and 4 points indicated severe swelling and no weight-bearing ability. The scoring standard of the middle foot or forepaw was the same as that of the ankle and wrist joint. The scoring standard of the 3 joints of the outermost 4 fingers was as follows: 0 points indicated no swelling; 1 point indicated swelling. The highest score of each limb was 20 points, and the highest score of each mouse was 80 points.

[0038] Effect of gardenia extract on arthritis score of mice: the normal group had good mental state, normal diet, and free activity, and no abnormal symptoms such as joint redness and swelling. Compared with the normal group, the RA-ILD group had poor mental state, decreased appetite, and significantly reduced activity, and the joint parts were significantly red and swollen, the toes were deformed, the motor function was limited, and the arthritis score was significantly higher than that of the normal group. Compared with the model group, the general state of the mice in the low-dose and high-dose gardenia extract groups was significantly improved, and the joint score was significantly reduced (P<0.01), which was manifested as increased appetite and activity, and joint redness subsided, and the results are shown in Figure 2 (a-b).

[0039] 3. HE staining to observe the pathological changes of synovial tissue in knee and ankle joints:

[0040] The synovial tissue of knee and ankle joints of mice was stripped with forceps and scissors, and the sample was placed in a 4% paraformaldehyde solution and fixed at room temperature. Sequentially dehydrated with gradient ethanol and transparentized with xylene. The tissue was immersed in a paraffin cylinder for waxing, embedded with liquid paraffin, and sectioned after cooling and solidification (4 μm thick). Oven baked at 60°C for 1 hour. Rehydrated, stained with hematoxylin for 5 minutes, washed with water; differentiated with 1% hydrochloric acid alcohol, washed with water; blued in ammonia water for 1 minute and washed again; stained with eosin for 1 minute. After washing off the floating color with distilled water, sequentially immerse in 95% and 100% ethanol and xylene to dehydrate and transparentize, mount with neutral resin, and observe the pathological changes of synovial tissue in knee and ankle joints under a microscope. Each group of sections was scored for synovial inflammation, pannus, cartilage erosion, and bone destruction: 0, almost no lesions; 1, slight lesions; 2, mild lesions; 3, obvious lesions; 4, severe lesions.

[0041] Effect of gardenia extract on synovial tissue of knee and ankle joints of mice:

[0042] The HE staining results showed that the synovial tissue structure of the knee and ankle joints of the normal group of mice was complete, the synoviocyte layer was normal without hyperplasia, the synovial surface was smooth, there was no inflammatory cell infiltration, and there was no effusion in the joint cavity. The synovial tissue of the RA-ILD group showed obvious pathological changes compared with the normal group, the synoviocytes significantly hyperplasia, the synovial layer thickness increased, a large number of inflammatory cells infiltrated, effusion was visible in the joint cavity, synovial blood vessels were dilated and congested, regional pannus formation occurred, and cartilage and bone erosion and destruction were observed, indicating that the model was successfully constructed. Compared with the model group, the pathological damage of the knee and ankle joints of mice in the low and high dose groups of gardenia extract was significantly improved, the synovial hyperplasia and joint cavity effusion were reduced, the inflammatory infiltration was significantly improved, and the erosion and destruction of cartilage and bone were significantly improved. This indicates that gardenia extract can significantly alleviate the tissue damage of knee and ankle joints of RA-ILD mice, effectively inhibit synovial hyperplasia, improve inflammatory cell infiltration, reduce joint cavity effusion and blood vessel dilation and congestion, and relieve cartilage and bone erosion and destruction. The results are shown in Figure 2 (c-f).

[0043] 4. HE staining and Masson staining to observe the pathological morphology of lung tissue:

[0044] The lung tissue was placed in a sterile culture dish, washed with pre-cooled physiological saline, and the external characteristics were observed. The lung lobes were separated, and the left lung lobe was fixed with 4% paraformaldehyde. After fixation, the tissue was sequentially dehydrated in different concentrations of ethanol, transparentized with xylene, and embedded in paraffin for sectioning. After deparaffinization in xylene and rehydration in ethanol, the sections were soaked in distilled water for 2 min. After staining with hematoxylin for 5 min, they were rinsed. After treatment with differentiation solution for about 2 min, they were washed twice with water. After staining with eosin for 3-3.5 min, they were dehydrated in ethanol until transparent, mounted with neutral balsam, and observed under a microscope for pathological changes in the lung tissue. Paraffin sections were stained with Masson. After deparaffinization and water washing of the paraffin sections, the cell nuclei were stained with hematoxylin for 5-10 min, washed with water, stained with Masson's brilliant acid fuchsin for 5-10 min, soaked in 2% acetic acid, differentiated with 1% phosphoric acid, stained with aniline blue or light green for 5 min, soaked in 0.2% acetic acid, and sequentially immersed in 95% ethanol, anhydrous ethanol, and xylene for transparency. After mounting with neutral balsam and air-drying, the collagen fibers in the lung tissue were observed.

[0045] Effects of gardenia extract on lung tissue lesions and collagen deposition in mice:

[0046] HE staining showed that the lung tissue structure of the normal group of mice was normal, the alveoli were complete, the alveolar walls were thin and clear, the cells in the interstitium were arranged normally, and there was no inflammatory cell infiltration. Compared with the normal group, the lung tissue of the model group of mice showed significant pathological damage, the alveolar structure was disordered, part of the alveolar wall was thickened, and there was obvious inflammatory infiltration. Compared with the model group, the inflammatory infiltration in the lung tissue of the low and high dose groups of gardenia extract was significantly improved Figure 3 a-b). Masson results showed that, compared with the normal group, the collagen fiber distribution in the lung tissue of the model group of mice was disordered, and the interstitial collagen fiber deposition area significantly increased. Compared with the model group, the interstitial collagen fiber deposition area in the low and high dose groups of gardenia extract was significantly reduced. The results are shown in Figure 3 (c-d).

[0047] 5. Determination of serum inflammatory factor levels

[0048] According to the operation method specified in the kit instructions, the levels of TNF-α, IL-6, and IL-1β in the serum were determined, and the readings were taken with an enzyme marker.

[0049] Effects of gardenia extract on serum inflammatory factors in mice:

[0050] The levels of IL-1β, IL-6, and TNF-α in the serum of the model group were significantly higher than those in the normal group. The high dose group of gardenia extract could significantly reduce the levels of the above inflammatory factors (P<0.01), and the low dose group could significantly inhibit the levels of IL-1β and TNF-α (P<0.05), indicating that gardenia extract had good anti-inflammatory effect. The results are shown in Figure 4 (a-c).

[0051] In summary, the 35% ethanol elution fraction of Gardenia jasminoides Ellis showed a dual intervention effect in the RA-ILD model mice: oral administration of 25-50 mg / kg significantly reduced the arthritis score, alleviated synovial cell proliferation, inflammatory infiltration and cartilage / bone destruction; at the same time, reduced lung interstitial collagen deposition and improved alveolar structure disorder. The levels of serum TNF-α, IL-1β and IL-6 were also decreased synchronously, suggesting that systemic inflammation was inhibited. The extract is derived from Gardenia jasminoides Ellis, which is both a medicinal and edible plant. The preparation process is simple, the dosage range is clear, and it has good safety and accessibility. It provides a new single plant source candidate drug for the long-term treatment of RA-ILD.

[0052] The preferred embodiments of the application disclosed above are only used to help explain the application. The preferred embodiments do not describe all the details, nor limit the application to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of the specification.

Claims

1. Application of Gardenia extract in the preparation of drugs for treating rheumatoid arthritis complicated with interstitial lung disease.

2. The application of the gardenia extract according to claim 1 in the preparation of a drug for treating rheumatoid arthritis complicated with interstitial lung disease, characterized in that, The drug is an oral preparation, selected from tablets, capsules, granules, oral liquids, or pills.

3. The application of the gardenia extract according to claim 2 in the preparation of a drug for treating rheumatoid arthritis complicated with interstitial lung disease, characterized in that, The drug uses the gardenia extract as its sole active ingredient.

4. The application of the gardenia extract according to claim 3 in the preparation of a drug for treating rheumatoid arthritis complicated with interstitial lung disease, characterized in that, The drug also contains pharmaceutically acceptable carriers, diluents or excipients, but does not contain other traditional Chinese medicines or chemical drugs with activity in treating rheumatoid arthritis with interstitial lung disease.

5. The application of the gardenia extract according to claim 4 in the preparation of a drug for treating rheumatoid arthritis complicated with interstitial lung disease, characterized in that, The interstitial lung disease refers to interstitial pneumonia secondary to rheumatoid arthritis, common interstitial pneumonia, or diffuse alveolar damage.

6. The method for preparing gardenia extract according to any one of claims 1-5, characterized in that, Includes the following steps: S1. Using dried, mature gardenia fruit as raw material, crush it and extract it by reflux with 6–10 times the volume of low-carbon alcohol with a volume fraction of 60–80% for 1–3 times, each time for 1–3 hours; combine the filtrates and concentrate under reduced pressure to obtain gardenia extract. S2. After dispersing the gardenia extract in water, the sample was loaded onto an HPD-600 macroporous adsorption resin column and eluted sequentially with 6–10 column volumes of water and 2–4 column volumes of 35% v / v ethanol. The 35% v / v ethanol eluent was collected, concentrated under reduced pressure, and dried to obtain the gardenia extract.

7. The method for preparing gardenia extract according to claim 6, characterized in that, The lower alcohol is selected from methanol, ethanol, propanol or isopropanol.

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