CN111568895A

CN111568895A - Anti-arthritis pharmaceutical composition

Info

Publication number: CN111568895A
Application number: CN202010299994.7A
Authority: CN
Inventors: 易涛; 陈绮蕾; 陈虎彪; 祝琳; 江滔; 吴亮; 叶桦珍
Original assignee: Hong Kong Baptist University HKBU
Current assignee: Hong Kong Baptist University HKBU
Priority date: 2020-04-16
Filing date: 2020-04-16
Publication date: 2020-08-25
Anticipated expiration: 2040-04-16
Also published as: CN111568895B

Abstract

The invention discloses an anti-arthritis pharmaceutical composition. The invention relates to a method for preparing scopoletin and syringin by mixing scopoletin and syringin in a mass ratio of 1: 1, the medicine composition has good heart safety, COX-2 inhibition in vivo and in vitro and anti-arthritis effect in vivo; the composition has certain synergistic effect in safety and therapeutic activity. The invention has certain significance for improving the condition that the chemosynthesis medicine in the existing anti-arthritis medicine generally has cardiotoxicity.

Description

Anti-arthritis pharmaceutical composition

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an anti-arthritis pharmaceutical composition.

Background

Arthritis is a chronic joint disease, which can cause progressive joint injury and systemic complications, causing a heavy social and economic burden. The most common arthritis includes Rheumatoid Arthritis (RA) and Osteoarthritis (OA). RA is an autoimmune disease, the joints of which are attacked by the human autoimmune system, with a global prevalence of about 1%, and can occur at any age. OA is a degenerative joint disease caused by cartilage wear, with about 10% of the population over the age of 60 affected, ranking the fifth of all forms of disability worldwide. Common symptoms of RA and OA include joint swelling due to soft tissue inflammation, and pain during or after exercise, especially morning stiffness and loss of mobility of the joint. RA and OA have complex etiology and involve genetic, environmental, biological and other factors. These factors may collectively alter the threshold of immune activation or dysregulation, thereby accelerating the development of joint inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most commonly used drugs for the treatment of RA and OA. For RA, the american college of rheumatology (american college of rheumatology) recommends treatment with NSAIDs first to reduce joint swelling and pain. For OA, NSAIDs are widely taken by 65% of patients to relieve symptoms. NSAIDs act by inhibiting cyclooxygenase (COX-1 and/or COX-2) and thereby reduce the synthesis of inflammatory mediators such as prostaglandins and thromboxanes. In order to avoid adverse reactions such as gastrointestinal bleeding caused by COX-1 inhibition, NSAIDs which selectively inhibit COX-2 should be developed. However, not only does this COX-2 selective inhibitor have no significant therapeutic effect on joint damage, but it also increases the risk to the cardiovascular system of the patient. Currently, most synthetic COX-2 selective inhibitors (e.g., rofecoxib, valdecoxib, etc.) are being withdrawn from the market by the U.S. food and drug administration, the European Union, and other multinational governments.

Research shows that the toxic side effect of the existing synthetic COX-2 selective inhibitor on the cardiovascular system may be related to the chemical structure of the inhibitor. However, attempts to develop COX-2 inhibitors by chemical synthesis or modification have met with little success. Compared with chemical synthesis, the natural product compounds (especially active ingredients in medicinal plants) have remarkable structural diversity and long-term clinical safety and effectiveness, are proved to be important sources of new drugs or new drug lead compounds, and can be used as the starting point of screening natural COX-2 selective inhibitors. Medicinal plants contain a variety of active ingredients which may act in whole or in concert to maximize therapeutic efficacy or reduce toxicity.

Disclosure of Invention

The invention aims to provide an anti-arthritis pharmaceutical composition which has lower cost, higher safety and more selectivity on COX-2 inhibition, aiming at the defect of the prior art that NSAIDs selectively inhibiting COX-2 have toxic and side effects on the heart. The pharmaceutical composition can remarkably relieve arthritis-related symptoms, and has better effect than the pharmacological effect of a single compound; the preparation method saves cost compared with the medicine for relieving arthritis-related symptoms.

Through a series of literature arrangement, microscopic identification, chemical analysis, pharmacological activity and in vivo metabolism and separation researches, the applicant finds that Saussurea lanuginosa (Saussurea laniceps hand-Mazz.) has strong anti-inflammatory and analgesic effects as a rare medicinal material commonly used in minority regions in China, and the effects are exerted through a natural COX-2 selective inhibitor contained in the Saussurea lanuginosa, wherein the main components are scopoletin and syringin. The invention utilizes the optimal proportion combination of the two compounds to improve the treatment effect on arthritis and simultaneously reduce adverse reactions.

The anti-arthritis pharmaceutical composition is characterized by containing effective amounts of scopoletin and syringin. Preferably, the mass ratio of the scopoletin to the syringin is 1: 1.

the invention discloses a method for preparing scopoletin and syringin by using a mass ratio of 1: 1, the heart safety and the anti-arthritis effect of the prepared pharmaceutical composition. The pharmaceutical composition has good heart safety, in vivo and in vitro COX-2 selective inhibition, and in vivo anti-arthritis effects; the composition has certain synergistic effect in safety and therapeutic activity. The invention has certain significance for improving the current situation that the chemical synthetic drugs in the anti-arthritis drugs generally have cardiotoxicity.

The technical problems solved in the invention include:

(1) NSAIDs which selectively inhibit COX-2 are used as the main medicaments for treating arthritis at present, have obvious cardiovascular toxic and side effects, and are easy to increase the risks of patients suffering from myocardial infarction and forming thrombus. The invention utilizes natural compounds, aims to develop safe pharmaceutical compositions with COX-2 selective inhibition, and has heart safety which is obviously higher than that of celecoxib in vivo and in vitro (figures 1 and 4).

(2) There is currently no systematic study of cardiac safety for the two natural compounds (scopoletin and syringin) and their combinations in different ratios encompassed by the present invention, and pharmacological studies on anti-arthritis in vivo models are not complete. The invention discloses the heart safety of scopoletin and syringin combined in different proportions (figures 1 and 4), selective COX-2 inhibition (figures 3, 9 and 14) and pharmacological effects for treating arthritis (figures 5 to 12; figures 13 to 16) by applying various cells and different rat arthritis models, and summarizes the optimal combination proportion (1: 1) of scopoletin and syringin to be used as a safe and effective anti-arthritis medicinal composition.

(3) For some complex diseases, the drug combination therapy can improve the treatment effect and reduce the toxic and side effects compared with single drug treatment. At present, no research on the treatment of arthritis by the combined medication of scopoletin and syringin exists. The invention discloses that scopoletin and syringin have synergistic effect in vivo and in vitro, can selectively inhibit COX-2 level and relieve serial symptoms of arthritis.

The invention has the following beneficial effects:

1. scopoletin and syringin were mixed at a ratio of 1: 1 proportion combination, and has higher in-vivo and in-vitro heart safety. The cardiac safety of the pharmaceutical composition is significantly higher than that of NSAIDs which selectively inhibit COX-2 and are represented by celecoxib. The pharmaceutical composition has no significant influence on the survival rate of rat myocardial cells H9c2 within the concentration range of 0-200 mu M; in contrast, celecoxib had a significant toxic effect on H9c2 cells at concentrations of 50 μ M and above (fig. 1). Furthermore, after 6 weeks of continuous oral administration, the combination was effective in reducing the cardiomyocyte cross-sectional area and reducing the percentage of myocardial fibrosis compared to celecoxib, thereby avoiding the cardiotoxic side effects of NSAIDs, which typically selectively inhibit COX-2 (fig. 4).

2. Scopoletin and syringin were mixed at a ratio of 1: the combination of the proportion 1 has obvious synergistic effect, selectively inhibits COX-2 expression level, and is an excellent COX-2 selective inhibitor. The in vitro COX-2 selective inhibitory effect (LPS-stimulated RAW264.7 cell model) of this pharmaceutical composition was significantly greater than that of the two single compounds and the other combination ratios, and the effect was directly proportional to the concentration (FIG. 3). This combination ratio (1: 1) also has significant in vivo selective COX-2 inhibition: both in the adjuvant arthritis rat model (FIG. 9) and in the pre-Crohn's disease-induced OA rat model (FIG. 14), joint synovial COX-2 expression levels were significantly reduced, while COX-1 expression levels were unaffected or even elevated.

3. Scopoletin and syringin were mixed at a ratio of 1: 1 proportion, obviously relieves the series symptoms of RA and OA of rats and has certain synergistic action. In rat adjuvant arthritis RA model, the pharmaceutical composition has effects of relieving clinical symptoms (figure 5-8), regulating immune response (figure 9-10), and improving ankle joint pathological manifestations (figure 11-12). In the OA model induced by anterior cross-ligament resection, the pharmaceutical composition can significantly restore the body weight of rats (FIG. 13), selectively inhibit the COX-2 in the knee synovium (FIG. 14), and improve the pathological manifestations of the knee joint (especially, restore the thickness of hyaline cartilage at the distal femur and proximal tibia, with a significantly stronger restoration effect than that of two single compounds; FIGS. 15-16).

Drawings

FIG. 1 shows that scopoletin and syringin in different proportions have higher in vitro cardiac safety. The data are shown as the mean value plus or minus the standard deviation, and n is more than or equal to 3; compared to the blank group: ns^aNo significant difference exists; a, p<0.01；***，p<0.001；****；p<0.0001。

FIG. 2 shows that scopoletin and syringin in different proportions have higher in vitro anti-inflammatory safety. The data are shown as the mean value plus or minus the standard deviation, and n is more than or equal to 3; compared to the model group (LPS-stimulated RAW264.7 cells): ns^bNo significant difference exists; #, p<0.01；###，p<0.001；####，p<0.0001。

FIG. 3 shows that different ratios of scopoletin and syringin affect the expression of COX-1 and COX-2 in RAW264.7 cells stimulated by LPS. Scopoletin and syringin are synthesized in the following formula (A) 1: 0. (B) 0: 1 and (C) 1: 1. 1: 5. 1: 9 ratio mixing, (D) comparison shows 1: the ratio of 1 has the strongest selective inhibition effect on COX-2 and is in direct proportion to the concentration; data are shown as mean ± standard deviation, n is 4; compared to the blank group: ns_aNo significant difference exists; a, p<0.05；**，p<0.01；***，p<0.001；****，p<0.0001; compared to the model set: ns^bNo significant difference exists; #, p<0.05；##，p<0.01；###，p<0.001；####，p<0.0001。

FIG. 4 shows that scopoletin and syringin in different proportions have higher in vivo cardiac safety. Different ratios of scopoletin and syringin affected rats (a) cardiac index (n ═ 6 rats), (B) cardiomyocyte cross-sectional area (n ═ 45, each group measured 15 cells on 3 sections from different rats), (C) percent myocardial fibrosis (n ═ 3 sections from different rats), and (D) left ventricular myocardial histopathological manifestations (solid arrow: necrotic cardiomyocytes are wavy; dashed arrow: fibrotic tissue); data are presented as mean ± standard deviation of the mean; compared to the blank group: ns_aNo significant difference exists; a, p<0.05；***，p<0.001；****，p<0.0001; compared to the celecoxib group: # #, p<0.001；####，p<0.0001；L，50mg/kg/d；M，100mg/kg/d；H，200mg/kg/d。

FIG. 5 shows that scopoletin and syringin in different proportions affect the rate of change of body weight in adjuvant arthritis rats. Data are shown as mean ± standard deviation of mean, n ═ 6 rats; compared to the blank group: ns^aNo significant difference exists; a, p<0.05；***，p<0.001；****，p<0.0001; compared to the model set: ns^bNo significant difference exists; #, p<0.05；##，p<0.01；###，p<0.001；####，p<0.0001。

FIG. 6 shows that scopoletin and syringin in different ratios affect the volume of swelling in hind paw of adjuvant arthritis rats. Data are shown as mean ± standard deviation of mean, n ═ 6 rats, mean of two hind paws per rat; compared to the blank group: ns^aNo significant difference exists; a, p<0.0001; compared to the model set: ns^bNo significant difference exists; #, p<0.05；###，p<0.001；####，p<0.0001。

FIG. 7 shows that scopoletin and syringin in different proportions affect the rate of inhibition of swelling of the feet after adjuvant arthritis. n-6 rats, each rat counting the average of two hind paws.

FIG. 8 shows rat clinical scores of different ratios of scopoletin and syringin affecting adjuvant arthritis. Data are shown as mean ± standard deviation of mean, n ═ 6 rats, and each rat counted the total of two hind paws and tails; compared to the blank group: ns^aNo significant difference exists; a, p<0.0001; compared to the model set: ns^bNo significant difference exists; #, p<0.05；##,p<0.01；###,p<0.001；####,p<0.0001。

FIG. 9 shows that scopoletin and syringin in different ratios affect the ankle synovial membrane COX-1/COX-2 protein expression levels of adjuvant arthritis rats 21 days after molding. The data are displayed by the standard deviation of the mean value plus or minus the mean value, and n is more than or equal to 3; compared with the control group: p < 0.05; p < 0.01; p < 0.001; p < 0.0001; compared to the model set: # # #, p < 0.0001.

FIG. 10 is a graph of the effect of scopoletin and syringin on the level of ankle synovial inflammatory factor in adjuvant arthritis rats 21 days after molding. The data are displayed by the standard deviation of the mean value plus or minus the mean value, and n is more than or equal to 3; compared with the control group: p < 0.01; compared to the model set: #, p < 0.05; #, p < 0.01.

FIG. 11 shows the effect of different ratios of scopoletin to syringinThe adjuvant arthritis rats showed hind paw X-ray imaging analysis 21 days after model building. Data are shown as mean ± standard deviation of mean, n ═ 3 rats, each rat counting the mean of the left and right hind paw; compared with the control group: a, p<0.0001; compared to the model set: ns^bNo significant difference exists; #, p<0.05；##,p<0.01；###,p<0.001；####，p<0.0001; s, soft tissue swelling; j, narrowing the joint cavity; b, bone erosion; p, periosteal hyperplasia; o, osteophyte.

FIG. 12 shows that scopoletin and syringin at different ratios affect the histological appearance of ankle joints of rats with adjuvant arthritis 21 days after molding. Data are shown as mean ± standard deviation of mean, n ═ 3 rats, each rat counting the mean of the left and right hind paw; compared with the control group: a, p<0.0001; compared to the model set: ns^bNo significant difference exists; #, p<0.05；##,p<0.01；###,p<0.001；####,p<0.0001; tib, tibia; tal, talus; nav, scaphoid; cal, calcaneus; sy, synovial hyperplasia; i, cell infiltration; c, cartilage erosion; pa, pannus formation.

FIG. 13 is the rate of change of body weight of OA rats induced by pre-Crohn's disease with varying ratios of scopoletin to syringin. Data are shown as mean ± standard deviation of mean, n ═ 6 rats; compared to the sham group: ns_aNo significant difference exists; a, p<0.05；**，p<0.01；***，p<0.001；****，p<0.0001; compared to the model set: ns^bNo significant difference exists; #, p<0.05；##，p<0.01；###，p<0.001；####，p<0.0001。

FIG. 14 shows the levels of expression of gonadal COX-1/COX-2 protein in OA rats induced by pre-Crohn's ligament resection effected by scopoletin and syringin at different ratios. Data are shown as mean ± standard deviation of mean, n ═ 6 rats; compared to the sham group: a, p<0.001；****，p<0.0001; compared to the model set: ns^bNo significant difference exists; #, p<0.01；###，p<0.001；####，p<0.0001。

FIG. 15 shows the pre-intercross ligament excision induced OA rat knee joint groups affected by scopoletin and syringin in different proportionsAnd (4) performing histological manifestation. Data are shown as mean ± standard deviation of mean, n ═ 3 rats, each rat counting the mean of the left and right hind paw; compared to the sham group: a, p<0.0001; compared to the model set: ns^bNo significant difference exists; #, p<0.05；##，p<0.01；###，p<0.001；####，p<0.0001; dotted arrow, synovial hyperplasia; solid arrows, cartilage vertical fissures extend to transitional layers; dotted line, incomplete tide line; arrowhead, chondrocyte death; HC, hyaline cartilage; CC, calcified cartilage.

FIG. 16 is a graph of the effect of varying proportions of scopoletin and syringin on pre-chiasmatomy-induced cartilage thickness in the knee joints of OA rats. Data are shown as mean ± standard deviation of mean, n is 6; compared to the sham group: a, p<0.0001; compared to the model set: ns^bNo significant difference exists; #, p<0.05；##，p<0.01；###，p<0.001；####，p<0.0001。

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Cardiac safety and anti-arthritis evaluations were performed using a series of in vivo and in vitro tests in the following examples. The principle of adopting in vivo and in vitro test models comprises the following steps:

(1) h9c2 rat cardiomyocytes were used for in vitro cardiac safety studies because they have a similar and stable in vitro drug response to the in situ heart;

(2) RAW264.7 mouse macrophages stimulated by Lipopolysaccharide (LPS) were used for in vitro anti-inflammatory studies because, as a recognized model of inflammatory cells, cellular activity and cellular COX-2 expression levels are indicators of drug toxicity and anti-inflammatory effects, respectively;

(3) according to observation of rat hearts after administration, because the main side effect of the COX-2 selective inhibitor on the market at present is cardiovascular diseases mainly caused by myocardial infarction, pathological cardiac remodeling can be caused, and particularly, the size, the shape and the function of myocardial tissues of a left ventricle are clinically changed;

(4) rat adjuvant arthritis acts as a Rheumatoid Arthritis (RA) model because in this model, injection of complete freund's adjuvant results in a series of immune responses common to human RA, including polyarthritis, cartilage and bone destruction, and intra-articular lymphocyte infiltration;

(5) rat anterior cruciate ligament resection-induced Osteoarthritis (OA) serves as a model of OA because in this model, rupture of the anterior cruciate ligament leads to joint instability, which in turn leads to post-traumatic OA, which is characterized by joint cartilage degradation, subchondral bone sclerosis and synovial inflammation.

In vitro cell tests, the scopoletin and the syringin are combined and prepared into test medicines according to the quantitative proportion of different substances: 1: 0. 0: 1. 1: 1. 1: 5. 1: 9. in an in vivo animal test, the mass ratio of the scopoletin to the syringin is as follows: 1: 0. 0: 1. 1: 1.

example 1: formulation of pharmaceutical compositions (for in vitro studies)

1.9216g of scopoletin is taken, 5mL of dimethyl sulfoxide (DMSO) is added, and the mixture is mixed uniformly for standby use to be used as a mother solution (2M) of scopoletin. Taking 3.7237g of syringin, adding 5mL of DMSO, and mixing uniformly for later use to serve as a mother solution (2M) of the syringin. Scopoletin and syringin in quantitative ratios of different substances prepared for in vitro studies:

(1)1：0

diluting scopoletin mother liquor with DMEM medium to 12.5, 25, 50, 100, 150 and 200 mu M;

(2)0：1

diluting syringin mother liquor with DMEM medium to 12.5, 25, 50, 100, 150, 200 μ M;

(3)1：1

the scopoletin and syringin mother liquor is prepared by mixing the following raw materials in a proportion of 1: 1 proportion to obtain a mixture, and diluting the solute concentration of the mixture to 12.5, 25, 50, 100, 150 and 200 mu M by using a culture medium;

(4)1：5

the scopoletin and syringin mother liquor is prepared by mixing the following raw materials in a proportion of 1: 5 to obtain a mixture, and diluting the solute concentration of the mixture to 12.5, 25, 50, 100, 150 and 200 mu M by using the culture medium;

(5)1：9

the scopoletin and syringin mother liquor is prepared by mixing the following raw materials in a proportion of 1: 9 proportion to obtain a mixture, and diluting the solute concentration of the mixture to 12.5, 25, 50, 100, 150 and 200 mu M by using the culture medium.

Example 2: formulation of pharmaceutical compositions (for in vivo studies)

Different mass ratios of scopoletin to syringin were made for in vivo studies:

(1)1：0

taking 10g of scopoletin, adding 50mL of 5% CMC-Na, and diluting into 200mg/mL solution;

(2)0：1

taking 10g of syringin, adding 50mL of 5% CMC-Na, and diluting into 200mg/mL solution;

(3)1：1

taking 5g of scopoletin and syringin respectively, adding 50mL of 5% CMC-Na, and diluting to 200mg/mL solution.

Example 3: in vitro myocardial safety test for pharmaceutical compositions

The cardiomyocytes H9c2 were plated in 96-well plates (5000 cells/well) and 24 hours later, the cells grew well, and different ratios of scopoletin and syringin (1: 0, 0: 1, 1: 1, 1: 5, 1: 9; concentrations of 12.5, 25, 50, 100, 200. mu.M)/celecoxib (12.5, 25, 50, 100, 200. mu.M) were added and incubated for 24 hours. The culture medium is used as a negative control, the celecoxib is used as a positive control, the cell survival rate is detected by an MTT method, and the cytotoxic effect of the pharmaceutical composition is further evaluated.

30mg of MTT powder was dissolved in 6mL of Phosphate Buffered Saline (PBS) in the dark, 10. mu.L of the MTT powder was added to each well, and the mixture was incubated in an incubator for 2 hours in the dark. The mixture was then centrifuged (1000rpm,10min), the supernatant carefully aspirated, 100. mu.L DMSO was added to each well, and the mixture was shaken on a shaker for 10min to dissolve the crystals thoroughly. The absorbance of each well was measured at OD 570nm (630nm calibration) of an enzyme linked immunosorbent assay.

Results of in vitro myocardial safety evaluation:

the MTT results are shown in figure 1, and the scopoletin and syringin with different proportions have no obvious cytotoxic effect on H9c2 cells within the range of the tested drug concentration, and the toxicity is obviously lower than that of celecoxib.

Example 4: in vitro anti-inflammatory safety test for pharmaceutical compositions

Macrophage RAW264.7 was inoculated in 96-well plates (5000/well), LPS (0.1. mu.g/mL) was added for stimulation after the cells were confluent, and scopoletin and syringin (1: 0, 0: 1, 1: 1, 1: 5, 1: 9; concentration of each ratio 12.5, 25, 50, 100, 200. mu.M)/celecoxib (12.5, 25, 50, 100, 200. mu.M) were added at 24 hours later for incubation. The culture medium is used as a negative control, the celecoxib is used as a positive control, the cell survival rate is detected by an MTT method, and the cytotoxic effect of the pharmaceutical composition is further evaluated.

30mg of MTT powder was dissolved in 6ml of PBS in the absence of light, 10. mu.L of the MTT powder was added to each well, and the mixture was cultured in an incubator for 2 hours in the absence of light. The mixture was then centrifuged (1000rpm,10min), the supernatant carefully aspirated, 100. mu.L of DMMSO was added to each well, and the mixture was shaken on a shaker for 10min at low speed to dissolve the crystals sufficiently. The absorbance of each well was measured at OD 570nm (630nm calibration) of an enzyme linked immunosorbent assay.

In vitro anti-inflammatory safety evaluation results:

the MTT results are shown in figure 2, and the scopoletin and syringin with different proportions have no obvious cytotoxic effect on RAW264.7 cells induced by lipopolysaccharide within the range of the measured drug concentration, and the toxicity is obviously lower than that of celecoxib.

Example 5: in vitro COX-2 selective inhibition assay for pharmaceutical compositions

Macrophage RAW264.7 was seeded in 6-well plates (2 × 10)⁴Perwell), after 24 hours the cells were confluent, LPS (0.1. mu.g/mL) was added, scopoletin and syringin (1: 0, 0: 1, 1: 1, 1: 5, 1: 9; each ratio 12.5, 25, 50, 100, 200. mu.M)/celecoxib (10. mu.M) were added at different ratios, incubated for 24 hours, the cells were collected, β -actin was used as an internal standard, and intracellular COX-1 and COX-2 expression was detected by the WesternBlot method, the medium was used as a negative control, the LPS group as a model group, and the celecoxib group as a positive control.

In vitro COX-2 selective inhibition evaluation results:

western Blot results are shown in FIG. 3, with scopoletin and syringin in a ratio of 1: 1 proportion, the selective inhibition effect on COX-2 is strongest, the effect and the concentration are in a direct proportion relation, and the proportion combination obviously enhances the COX-2 selective inhibition effect of a single compound.

Example 6: in vivo anti-inflammatory safety test for pharmaceutical compositions

66 three-month male SD rats of 400 g. + -.20 g were randomly assigned to 11 groups: (1) blank control groups (5% CMC-Na; 1mL/d), (2) celecoxib (10mg/kg/d), (3) - (5) scopoletin (50, 100, 200mg/kg/d), (6) - (8) syringin (50, 100, 200mg/kg/d), (9) - (11) scopoletin and syringin were mixed at a ratio of 1: mixing at a ratio of 1 (50, 100, 200 mg/kg/d). Gavage was performed daily for 42 days. 5% CMC-Na was used as a negative control (blank group) and celecoxib was used as a positive control.

After 42 days, the rats were sacrificed and the rat hearts were collected, washed and weighed, heart index ═ heart weight/rat body weight. Taking the myocardial tissue of the left ventricle as a tissue slice, observing the shape of the myocardial cells, recording the cross section area of the myocardial cells and calculating the percent of myocardial fibrosis.

Rat heart safety evaluation results:

the heart index and histopathological results are shown in fig. 4, and the measured cardiotoxicity of the combination of scopoletin and syringin in each proportion is significantly lower than that of celecoxib.

Example 7: mitigation effect of pharmaceutical compositions on RA

Two month male SD rats of 72 were taken at 280g ± 20g and randomly divided into 12 groups: (1) blank control group (5% CMC-Na; 1mL/d), (2) rat adjuvant arthritis model group (5% CMC-Na; 1mL/d), (3) celecoxib (10mg/kg/d), (4) to (6) scopoletin (50, 100, 200mg/kg/d), (7) to (9) syringin (50, 100, 200mg/kg/d), (10) to (12) scopoletin and syringin were mixed at a ratio of 1: mixing at a ratio of 1 (50, 100, 200 mg/kg/d). Gavage was performed daily for 21 days. The rats without model formation are used as negative control (blank group) by intragastric administration of 5% CMC-Na every day, the model formation rats are used as model group by intragastric administration of 5% CMC-Na every day, and the model formation rats are used as positive control by intragastric administration of celecoxib every day.

And (3) performing arthritis molding on day 0: 0.1mL of complete Freund's adjuvant was injected subcutaneously at the base of the tail.

Weight changes were recorded every 2 days with a rate of weight change of (new weight-original weight)/original weight 100%.

The volume of the left and right hind paws is measured by a toe swelling measuring instrument every 3 days, and the inhibition rate of each group of medicaments on the hind paw swelling is calculated, wherein the inhibition rate is (the average swelling volume of the hind paw after the model group-the average swelling volume of the hind paw after the medicament group)/the average swelling volume of the hind paw after the model group is 100%).

The left and right hind soles and tail were scored for clinical inflammation every 3 days. Grading standard: hind feet: 0, no red and no swelling, 1, small or large joints with light swelling and/or redness, 2, small or large joints with moderate swelling and/or redness, 3, large joints with severe red swelling, and small joints with moderate swelling and/or redness, 4, large joints with very severe red swelling, and small joints with severe swelling and/or redness; tail part: 0-no inflammatory presentation, 1-slight swelling of the injection site and surrounding tissues, 2-approximately 25% of the tail inflamed or ulcerated, and 3-greater than 25% of the tail inflamed or ulcerated.

After 21 days:

photographing and recording the inflammation conditions of the soles and the tails of the rats in each group.

The rats in each group were sacrificed and the ankle synovium was collected. Synovial tissue samples were washed with PBS (0.01M, pH 7.4) to remove residual blood or impurities on the tissue surface. The tissue mass was weighed, and after recording, minced and placed in an EP tube. The tissue 1: 9(w/v) Pre-cooled PBS (protease inhibitor added just before use) was added to the homogenate, which was placed on ice or in an ice bath. And (3) sucking the homogenate liquid into a centrifuge tube, centrifuging for 5-10 min at the temperature of 4 ℃ by 5000 Xg, taking the supernatant, and preserving at the temperature of minus 20 ℃ or minus 80 ℃ to avoid repeated freeze thawing. The expression of COX-1/COX-2 protein in the supernatant of the homogenate was measured by Western Blot method using beta-actin as an internal standard. The levels of IL-2 and IL-10, inflammatory factors of the homogenate supernatants, were measured by ELISA.

Taking the hind paw for X-ray imaging analysis. The comprehensive conditions of soft tissue swelling, narrowing of joint cavity, bone erosion and periosteum hyperplasia are evaluated: normal, light, moderate, and severe 0, 2, and severe 3.

The ankle joint was taken and examined as a tissue section. Synovial hyperplasia, cellular infiltration, cartilage erosion, pannus formation were evaluated separately:

(1) synovial hyperplasia

0 is normal; 1, slight hyperplasia, 2-4 layers of reactive synovial cells; 2-moderate hyperplasia, 4 or more layers of reactive synovial cells, increased mitotic activity, mild or no infiltration of synovial cells into adjacent bone and connective tissue; 3-severe hyperplasia, characterized by infiltration and elimination of the joint space and adjacent cartilage, bone and connective tissue.

(2) Cell infiltration

0 is normal; 1, a small amount of local infiltration; 2, extensive local infiltration; 3, it is widely infiltrated, agglomerated and invaded the joint capsule.

(3) Cartilage erosion

0 is normal; 1 ═ localized superficial cartilage degeneration; 2 ═ localized deep cartilage degeneration; multiple sites of extensive deep cartilage degeneration.

(4) Formation of pannus

0 is normal; pannus formation in no more than two locations; 2, the pannus is formed at no more than four parts, and the articular surface infiltrates or is flat and overgrown; more than four sites of pannus formation or two sites of extensive pannus formation.

The evaluation result of the curative effect of the rat adjuvant arthritis model is as follows:

rat body weights were recorded every 2 days and the rate of body weight change results are shown in figure 5.

The volume of hind paw, volume of swelling results every 3 days are shown in figure 6; the inhibition rate of each group of drugs on sole swelling was calculated, and the results are shown in fig. 7.

Clinical inflammation scores were performed every 3 days for the left and right hind paw and tail, and the results are shown in fig. 8.

After 21 days:

each group of rats was sacrificed and hind paw ankle synovium was collected. The results of measurement of COX-1/COX-2 protein expression in the ankle synovia by the Western Blot method are shown in FIG. 9. The results of measurement of IL-6 and IL-10 levels in the synovial membrane of the hindfoot ankle by ELISA are shown in FIG. 10.

The hind paw was taken and analyzed by X-ray imaging, the results are shown in figure 11.

The ankle joint was taken and observed as a tissue section, and the results are shown in FIG. 12.

The test results show that the scopoletin and syringin are mixed in a ratio of 1: 1 proportion, has stronger improving effect on the adjuvant arthritis of rats, and is mainly shown in relieving clinical symptoms (weight loss, hind foot swelling and tail inflammation), regulating ankle synovial membrane immune response (selectively inhibiting COX-2, improving the level of an anti-inflammatory factor IL-10, reducing the level of a proinflammatory factor IL-6), and improving ankle joint pathological manifestations (synovial membrane hyperplasia, lymphocyte infiltration, cartilage erosion, pannus formation and the like).

Example 8: OA-relieving effect of pharmaceutical composition

Three month male SD rats of 400 g. + -. 20g were taken 72 and randomly divided into 12 groups: (1) blank control group (5% CMC-Na; 1mL/d), (2) osteoarthritis model group induced by rat anterior cruciate ligament resection (sham group) (5% CMC-Na; 1mL/d), (3) celecoxib (10mg/kg/d), (4) - (6) scopoletin (50, 100, 200mg/kg/d), (7) - (9) syringin (50, 100, 200mg/kg/d), (10) - (12) scopoletin and syringin were mixed at a ratio of 1: mixing at a ratio of 1 (50, 100, 200 mg/kg/d). Gavage was performed daily for 42 days. The rats in the sham operation group were gavaged with 5% CMC-Na every day as negative control, the molded rats were gavaged with 5% CMC-Na every day as model group, and the molded rats were gavaged with celecoxib every day as positive control.

Antegrade transection of the ligament on day 21 for both hind paws.

After 42 days, each group of rats was sacrificed and the gonadal synovium was collected. Synovial tissue samples were washed with PBS (0.01M, pH 7.4) to remove residual blood or impurities on the tissue surface. The tissue mass was weighed, and after recording, minced and placed in an EP tube. The tissue 1: 9(w/v) Pre-cooled PBS (protease inhibitor added just before use) was added to the homogenate, which was placed on ice or in an ice bath. And (3) sucking the homogenate liquid into a centrifuge tube, centrifuging for 5-10 min at the temperature of 4 ℃ by 5000 Xg, taking the supernatant, and preserving at the temperature of minus 20 ℃ or minus 80 ℃ to avoid repeated freeze thawing. The expression of COX-1/COX-2 protein in the supernatant of the homogenate was measured by Western Blot method using beta-actin as an internal standard.

Taking the knee joint, and observing a tissue section: performing histopathology (Mankin) scoring of the articular cartilage; the thickness of the calcified cartilage and hyaline cartilage was recorded.

Evaluation results of therapeutic effect of rat anterior cross ligament resection-induced OA model:

rat body weights were recorded every 2 days and the rate of body weight change results are shown in figure 13.

After 42 days:

each group of rats was sacrificed and the gonadal synovium was collected. The expression of COX-1/COX-2 proteins in the knee synovial membrane was measured by Western Blot (results are shown in FIG. 14).

The knee joint was taken and observed as a tissue section, and the results are shown in FIG. 15. The thickness of the calcified cartilage and hyaline cartilage of the knee joint was recorded and the results are shown in figure 16. Scopoletin and syringin were mixed at a ratio of 1: 1 ratio mixing, particularly effective in restoring hyaline cartilage thickness in the knee joint; this effect was directly correlated with dose and significantly enhanced the recovery effect of the single compound.

The test results show that the scopoletin and syringin are mixed in a ratio of 1: 1 proportion, has stronger improving effect on OA induced by major anterior cruciate ligament resection, is mainly expressed in weight loss caused by recovery surgery and inflammation, selectively inhibits COX-2 level in knee synovial membrane, and improves pathological manifestations (cartilage tissue structure, cartilage thickness and the like) of knee joint.

In view of the above results of cell and animal experiments, the amounts of scopoletin and syringin are 1: 1 proportion has higher cell safety, COX-2 selective inhibition effect and obvious anti-RA and OA effects.

Claims

1. An anti-arthritis pharmaceutical composition is characterized by comprising effective amounts of scopoletin and syringin.

2. The pharmaceutical composition of claim 1, wherein the weight ratio of scopoletin to syringin is 1: 1.