CN114159435B

CN114159435B - Application of Fuziling in preparing medicine for treating arthritis

Info

Publication number: CN114159435B
Application number: CN202111649116.4A
Authority: CN
Inventors: 朱丽君; 刘中秋; 李小翠; 倪家栋; 许仪红; 傅愉; 区晓君
Original assignee: Guangzhou University of Traditional Chinese Medicine
Current assignee: Guangzhou University of Traditional Chinese Medicine
Priority date: 2021-12-30
Filing date: 2021-12-30
Publication date: 2023-06-16
Anticipated expiration: 2041-12-30
Also published as: CN114159435A

Abstract

The invention discloses the application of aconitine in preparing the medicine for treating arthritis, and it can also be applied in preparing the medicine for treating arthritis and its anti-inflammatory, analgesic and antipyretic activity, the invention verifies that aconitine has obvious therapeutic effect to rheumatoid arthritis through rat experiment, it can obviously reduce MMP3 concentration in serum, and its therapeutic effect is obviously better than indometacin, and has no side effect; experiments prove that the medicine has the characteristics of obvious anti-inflammatory and analgesic activities, and the like, has the advantages of high permeability, good absorption, high oral bioavailability and the like, has good pharmaceutical prospect, and can enrich the treatment means for rheumatoid arthritis.

Description

Application of Fuziling in preparing medicine for treating arthritis

Technical Field

The invention relates to the technical field of medical biology, in particular to application of a traditional Chinese medicine active ingredient, namely application of aconite root in preparation of a medicine for treating arthritis.

Background

Rheumatoid arthritis (Rheumatoid arthritis, RA) is a chronic, inflammatory, systemic autoimmune disease. The clinical characteristics are that the joints are symmetrical swelling, morning stiffness, local joint pain, dysfunction, systemic light or moderate fever and the like. The RA disability rate is high, and the erosion of multi-joint cartilage and bone can occur in time when the treatment is not performed, so that the life quality of patients is seriously affected. The etiology and pathogenesis of RA are not completely defined, and the current treatment of RA is mainly to control symptoms and delay illness. The western medicine treatment RA mainly comprises non-steroidal anti-inflammatory drugs, glucocorticoids, immunosuppressants and the like, so that adverse reactions can not be ignored while the disease is delayed. In recent years, scholars at home and abroad have conducted extensive and intensive studies on the aspect of treating RA by using vegetable drugs, and found that a plurality of natural medicinal components such as tripterygium glycosides, total glucosides of paeony, thaumatin and the like have better RA treatment effect. Therefore, the development of safe and efficient RA treatment drugs from plant drugs has broad prospect.

Fuziline (FE) is C-19 aconite alkaloid extracted and separated from traditional Chinese medicine aconite, the research on FE at home and abroad is mainly focused on the aspects of extraction separation and structural identification, the research on pharmacological aspects is very little, the research on the pharmacological activity and application of FE at home and abroad is very little, the activity and application of FE in resisting arrhythmia, heart failure and pain are seen at present, and the FE has the effects of tonifying heart, dilating blood vessels, reducing blood pressure and the like in vitro. However, research reports of FE for treating rheumatoid arthritis are not yet seen.

Disclosure of Invention

Therefore, based on the background, the invention provides the application of aconite (CAS number: 80665-72-1) in preparing the medicine for treating arthritis, and a new means can be provided for treating rheumatoid arthritis through the new application research thereof.

The technical scheme of the invention is as follows:

one of the invention is that: the application of aconite root in preparing medicine for treating arthritis; the aconite root medicine has the following structural formula:

further, the arthritis is rheumatoid arthritis.

Further, the dosage form of the medicine is a conventional dosage form for clinically treating arthritis.

Further, the dosage form of the medicine can be an oral preparation or an injection preparation or an external preparation.

The invention also provides an anti-rheumatoid arthritis medicament, which comprises an effective amount of FE and pharmaceutically acceptable auxiliary agents.

The anti-rheumatoid arthritis medicament can be an oral preparation or an injection preparation or an external preparation, wherein the oral preparation comprises, but is not limited to, tablets, capsules, granules and the like.

The invention also provides application of the FE in preparing medicines with anti-inflammatory, analgesic and antipyretic activities.

By adopting the technical scheme, the beneficial effects are as follows:

the invention verifies through rat experiments that FE has obvious curative effect on rheumatoid arthritis, can obviously reduce the concentration of arthritis marker matrix metalloproteinase-3 (Matrix metalloproteinase, MMP 3) in serum, and has obvious curative effect superior to indometacin; experiments prove that the medicine has the characteristics of obvious anti-inflammatory, analgesic and pyrolytic activities, and the like, has the advantages of high permeability, good absorption, high oral bioavailability and the like, has good pharmaceutical prospect, and can enrich the treatment means for rheumatoid arthritis.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a photograph of a right hind paw lesion of a rat in an experiment of FE versus RA for example 1 of the present invention;

wherein Normal (fig. 1A), model (fig. 1B), positive (fig. 1C), high-dose FE (4 mg/kg FE) (fig. 1D), and low-dose FE (2 mg/kg FE) (fig. 1E);

fig. 2 shows joint and foot swelling rate scores on

days

9, 12, 15, 18 and 21 for normal, model, positive, high and low dose FE groups of rats in the FE versus RA treatment trial of example 1 of the present invention;

fig. 3 is a graph showing the joint swelling score on

days

9, 12, 15, 18 and 21 of rats in the normal, model, positive, high and low dose FE groups of the treatment experiment of FE versus RA according to example 1 of the present invention;

fig. 4 shows the arthritis index scores on

days

9, 12, 15, 18 and 21 for rats in the normal, model, positive, high and low dose FE groups of the treatment experiment with RA for FE of example 1 of the present invention;

fig. 5 shows the whole body scores of rats in the normal, model, positive, high-dose and low-dose FE groups on

days

9, 12, 15, 18 and 21 in the FE-RA treatment experiment of example 1 of the present invention;

fig. 6 shows weight changes on

days

9, 12, 15, 18 and 21 in normal, model, positive, high and low dose FE groups of rats in the FE versus RA treatment experiment of example 1 of the present invention;

FIG. 7 shows the concentration of matrix metalloproteinase 3 (MMP 3) in the serum of rats in the normal, model, positive, high and low dose FE groups of the treatment experiment of RA with FE of example 1 of the present invention;

FIG. 8 shows the foot swelling rate of the mice according to example 2 of the present invention after injection of lambda carrageenan for 4 hours in the right hind foot of the mice after 30min of gastric normal Saline (Saline), 50mg/kg diclofenac sodium (Positive), 15mg/kg FE and 7.5mg/kg FE, respectively;

FIG. 9 shows the number of pain twists after intraperitoneal injection of 1% acetic acid after 30min of gastric lavage Saline (Saline), 50mg/kg diclofenac sodium (Positive), 15mg/kg FE and 7.5mg/kg FE, respectively, in the mice of example 2 of the present invention;

FIG. 10 shows the thermosensitive threshold of mice in example 2 of the present invention, which were recorded by liquid crystal display after the mice had developed behaviors such as leg lifting, beating, licking, etc., after the mice were respectively perfused with physiological Saline (Saline), 50mg/kg diclofenac sodium (Positive), 15mg/kg FE, and 7.5mg/kg FE for 30min, and then given with IR55 at the right hind paw;

FIG. 11 is a graph showing the blood concentration versus time after oral administration of 15mg/kg FE (FIG. 11A) and intravenous injection of 1.5mg/kg FE (FIG. 11B) in the mice of example 3 of the present invention.

FIG. 12 shows pharmacokinetic parameters of mice of example 3 of the present invention after oral administration of 15mg/kg FE and intravenous injection of 1.5mg/kg FE, respectively:

wherein FIG. 12A is the area under the blood concentration versus time curve (AUC _0-t )；

FIG. 12B is an average residence time (MRT) _0-t )；

FIG. 12C is half-life (T _1/2 )；

FIG. 12D is an apparent distribution volume (V _d )；

FIG. 12E is Clearance (CL);

FIG. 13 shows the absorption transport characteristics of FE in Caco-2 monolayer cell model according to example 3 of the present invention:

FIG. 13A is the effect of efflux transporters P-gp inhibitor (Verapeil), BCRP inhibitor (KO 143) and MRP2 inhibitor (MK 571) on BL-side transport when FE is transported from the AP-BL side;

FIG. 13B is the effect of 3 efflux transporter inhibitors on AP side transport capacity when FE is transported from BL-AP side;

FIG. 13C is the apparent permeability coefficient (P) of 3 efflux transporter inhibitors to FE _app ) Is a function of (1);

fig. 13D is the effect of 3 efflux transporter inhibitors on the efflux rate of FE.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: treatment experiment of RA with FE

In this example, experiments on treatment of rat adjuvant arthritis with Fuziling (FE) were performed to investigate the effect of FE on treatment of rheumatoid arthritis.

30 male SD rats of 120+ -20 g were used, and 6 were randomly divided into 5 groups, namely, a normal group, a model group, an indomethacin group (positive drug), a 4mg/kg FE group (high dose FE group) and a 2mg/kg FE group (low dose FE group). After the rats were fixed, the hairs near the tail root were shaved off, and the rats were sterilized by wiping with 75% alcohol, and 0.1mL of physiological saline was subcutaneously injected at about 1cm of the tail root of the normal group of rats, and the other group of rats was injected with the same volume of Freund's complete adjuvant. Rats started to develop disease on day 9 after injection of Freund's complete adjuvant. From day 9, rats were perfused with 20% beta-cyclodextrin, 2mg/kg indomethacin, 4mg/kg FE and 2mg/kg FE, respectively, daily in a volume of 10mL/kg for 14 days in the normal, model, indomethacin, 4mg/kg FE (high dose FE) and 2mg/kg FE (low dose FE) groups. In the experiment, 2mg/kg of indomethacin, 4mg/kg of FE and 2mg/kg of FE are prepared by 20% beta-cyclodextrin. After onset of the disease from day 9 rats, the foot volume and body weight of the rats were measured and recorded every 3 days while the joint swelling number, arthritis score and systemic score of the rats were calculated.

(1) Calculation of foot swelling rate in rats:

joint swelling number 1 wrist ankle joint (or ankle joint) and 5 finger (toe) joints per paw, up to 24 joints per rat.

(2) The arthritis index scoring criteria were:

normally, marking 0 point;

erythema and slight swelling of the ankle joint were noted for 1 minute;

erythema and slight swelling of ankle to toe or metacarpal joints, recorded 2 minutes;

erythema and moderate swelling of ankle to toe or metacarpal joints, recorded 3 minutes;

erythema and severe swelling occurred from ankle to toe, recorded 4 minutes.

(3) The whole body scoring criteria were:

the ears have no nodule and redness symptoms, and record 0 points;

one ear nodule and redness symptoms, recorded 1 minute;

two ear nodules and redness symptoms, recorded 2 points;

the nose has no connective tissue swelling, and the score is 0;

obvious connective tissue swelling, recorded 1 minute;

the tail has no nodule, and the score is 0;

if there are nodules, record 1 minute;

the full paw has no swelling, and the score is 0;

one forefoot paw is swollen, 1 minute;

swelling of the two forefoot paws, recorded 2 minutes;

hind paw is free from swelling, and the score is 0;

one hind paw is swollen, 1 minute;

swelling of the two hind paws, recorded 2 minutes; each rat was rated at most 8 points.

After the experiment is finished, fixing the rats, and taking right hind foot pictures of all groups of rats; rats were then anesthetized, collected by abdominal aorta, and after the blood sample was allowed to stand at room temperature for 2 hours, serum was isolated by centrifugation at 3000rpm for 10 minutes, and the concentration of arthritis marker matrix metalloproteinase-3 (Matrix metalloproteinase-3, MMP 3) in serum of each rat was measured according to the operating instructions of the Elisa kit. The results obtained in this experiment are shown in fig. 1 to 7. The results of fig. 2-7 are expressed in terms of mean ± standard deviation, the data were analyzed using a Shapiro-Wilk test for normal distribution, an independent sample t-test for normal distribution and variance alignment for the two sets of mean-compared data, and a Satterthwaite corrected t-test for normal distribution and variance alignment. In addition, the data were analyzed using a non-parametric Wilcoxon rank sum test when the data did not fit the normal distribution. P <0.05 indicates a statistical difference. In the figure "#" indicates a statistical difference between the model group and the normal group, and "×" indicates a statistical difference between the drug-treated group and the model group (n=6).

From the analysis of the results of fig. 1 to 7, rats began to develop disease on day 9 of modeling. Model group rats showed obvious joint swelling, redness and deformation (fig. 1B) compared to normal group rats (fig. 1A); from day 9, the model group rats had an increased rate of foot swelling score (fig. 2), joint swelling number score (fig. 3), arthritis index score (fig. 4) and systemic score (fig. 5) over the normal group; and from day 12 on, the model group rats had a significant increase in the rate of foot swelling (fig. 2), the number of joint swelling (fig. 3), the arthritis index (fig. 4) and the whole body (fig. 5) scores (P < 0.05) over the normal group. Meanwhile, from day 9, the body weight of the model group rats began to decrease (fig. 6), and from day 12, the body weight of the model group rats was significantly lower than that of the normal group rats (P < 0.05). In addition, the concentration of MMP3 (arthritis marker) in the serum of rats in the model group was also significantly increased over that in the normal group (fig. 7, p < 0.05). The results show that the rat adjuvant arthritis model in the embodiment of the invention is successfully established.

And as is evident from fig. 1 to 7, FE has a remarkable effect of treating rat adjuvant arthritis. Compared with the model group, after the rats in the high-dose FE group and the high-dose FE group are treated by FE, the joint swelling, redness and deformation of the rats are obviously improved, and the improvement degree is equivalent to that of the indomethacin treatment group (figure 1); both FE and indomethacin treatments reduced the rate of foot swelling score in rats compared to the model group (fig. 2), and from day 12, treatment with the high dose (4 mg/kg) FE group significantly reduced the rate of foot swelling in rats (P < 0.05), and treatment with the low dose (2 mg/kg) FE also significantly reduced the rate of foot swelling in rats (P < 0.05) on day 21; compared to the model group, both FE and indomethacin treatments reduced the joint swelling score of rats (fig. 3), and both FE and indomethacin treatments at high/low doses from day 12 significantly reduced the joint swelling score of rats (P < 0.05), with FE treatments at high/low doses having a greater effect on the reduction of the joint swelling score of adjuvanted arthritic rats than indomethacin (fig. 3); compared with the model group, the treatment of FE and indomethacin can reduce the arthritis index score of rats (figure 4), the treatment of high-dose (4 mg/kg) FE from the 12 th day can obviously reduce the arthritis index score of adjuvant arthritis rats (P < 0.05), the reduction effect is stronger than that of indomethacin, and the treatment of low-dose (2 mg/kg) FE can obviously reduce the arthritis index score of adjuvant arthritis rats on the 12 th, 15 th and 18 th days (figure 4, P < 0.05); compared with the model group, the high/low dose of FE and indomethacin treatment can remarkably reduce the whole body score of the rat with adjuvant arthritis (figure 5, P < 0.05), and the effect of the high/low dose of FE treatment on reducing the whole body score of the rat is stronger than that of indomethacin; compared to the model group, both high/low doses of FE and indomethacin treatment restored body weight of the adjuvanted arthritic rats (fig. 6), and high doses (4 mg/kg) of FE treatment from day 12 were able to significantly restore body weight of rats (P < 0.05), while their restoring effect on rat body weight was stronger than indomethacin (fig. 6); compared to the model group, both high/low dose FE and indomethacin treatments significantly reduced the concentration of MMP3 in serum of the adjuvanted arthritic rats (fig. 7, p < 0.05), and the high/low dose FE treatment reduced MMP3 in serum of the adjuvanted arthritic rats more strongly than indomethacin. In conclusion, the effect of the FE on treating RA is remarkable, so that the joint swelling, redness and deformation of the adjuvant arthritis rat can be remarkably improved, the foot swelling rate score, the joint swelling number score, the arthritis index score and the whole body score of the rat can be remarkably reduced, the weight of the rat can be remarkably recovered, and in addition, the concentration of MMP3 in serum of the adjuvant arthritis rat can be remarkably reduced; based on the experimental results of this example, it can be seen that FE has a better therapeutic effect on RA than the non-steroidal anti-inflammatory drug indomethacin.

Example 2: pharmacological and pharmacodynamic related experiments of FE for treating RA

RA is clinically characterized by joint inflammation and swelling, localized joint pain, and associated systemic mild or moderate fever. Thus, anti-inflammatory, analgesic and antipyretic are key to alleviating RA symptoms, effectively eliminating joint swelling, alleviating pain and restoring body temperature. The pharmacological activities of anti-inflammatory, analgesic and antipyretic activities of FE are verified through experiments.

(1) Anti-inflammatory Activity of FE

This example uses a classical carrageenan-induced murine paw tumefaction inflammation model to examine the anti-inflammatory activity of FE. Male 8-week-old C57 mice were selected 24, randomly divided into 4 groups of 6, and the right hind foot thickness of each mouse was measured with vernier calipers. After the 4 groups of mice were perfused with physiological saline, 50mg/kg of diclofenac sodium (positive drug), 15mg/kg of FE and 7.5mg/kg of FE for 30min, respectively, 20. Mu.L of lambda carrageenan was subcutaneously injected into the right foot plantar of each group of mice to cause inflammation. After 4h of inflammation, the right foot thickness of each mouse was measured with a vernier caliper. The foot swelling rate of each mouse was calculated using the following formula:

the results of the anti-inflammatory experiments of FE are shown in fig. 8, the results are expressed as mean ± standard deviation, the data are analyzed using independent sample t-test, and the symbol "x" indicates P <0.05, i.e. there is a significant difference between the drug-treated group and the physiological saline group (n=6). As shown in FIG. 8, 50mg/kg of diclofenac sodium and 15 and 7.5mg/kg of FE significantly reduced the rate of foot swelling in mice by 58.95%, 34.87% and 35.21% (P < 0.05), respectively, compared to the saline group. The lambda carrageenan inflammatory model is a classical anti-inflammatory drug screening model, and experimental results show that FE has remarkable anti-inflammatory activity. The remarkable anti-inflammatory activity of FE is probably one of the pharmacodynamic bases of RA treatment, and can remarkably inhibit the arthritis of RA patients.

(2) Analgesic effect of FE

The invention adopts a classical acetic acid induced mouse pain torsion model to examine the analgesic activity of FE. Male 8-week-old C57 mice were randomized into 4 groups of 6. After 4 groups of mice are respectively infused with normal saline, 50mg/kg of diclofenac sodium (positive medicine), 15mg/kg of FE and 7.5mg/kg of FE for 30min according to the volume of 10mL/kg, 1% acetic acid solution is injected intraperitoneally according to the volume of 10mL/kg, and the times of pain and torsion of the mice are recorded after 5min, and the total observation is 15min. The pain inhibition rate of each group of medicines is calculated by the number of times of twisting the body, and the calculation formula is as follows:

the results of the analgesic experiments of FE are shown in fig. 9, the results are expressed as mean ± standard deviation, the data are analyzed using independent sample t-test, and the symbol "×" indicates P <0.05, i.e. there is a significant difference between the drug-treated group and the saline group (n=6). Compared with the normal saline group, 50mg/kg of diclofenac sodium, 15mg/kg of FE and 7.5mg/kg of FE can obviously reduce the pain torsion times (P < 0.05) of mice, and the pain inhibition rates are 45.44%, 54.54% and 58.18%, respectively. The acetic acid induced mouse pain torsion model is a classical analgesic screening model, and experimental results show that FE has remarkable analgesic effect and analgesic activity of the FE is stronger than that of a non-steroidal anti-inflammatory drug sodium diclofenac. Thus, analgesic activity may be one of the pharmacodynamic bases of FE treatment for RA, significantly alleviating the symptoms of joint pain in RA patients.

(3) Antipyretic activity of FE

In the embodiment, the heat-relieving activity of FE is examined by adopting a classical infrared thermosensitive experimental model. Male 8-week-old C57 mice were randomized into 4 groups of 6. After the group 4 mice were respectively perfused with physiological saline, 50mg/kg of diclofenac sodium (positive drug), 15mg/kg of FE and 7.5mg/kg of FE for 30min in a volume of 10mL/kg, infrared heat radiation was given to the right hind legs of the mice, and the infrared light intensity was IR55. And recording the thermosensitive threshold of the mouse displayed by the liquid crystal display after the behaviors of leg lifting, flapping, foot adding and the like of the mouse occur. To avoid scalding the mice, the mice without leg lifting reaction still have the detection stopped after 25s, and the threshold value is calculated according to 25 s.

The results of the fever test of FE are shown in fig. 10, the results are expressed as mean ± standard deviation, the data are analyzed using independent sample t-test, and the symbol "×" indicates P <0.05, i.e. there is a significant difference between the drug-treated group and the physiological saline group (n=6). Compared with the physiological saline group, 50mg/kg of diclofenac sodium, 15mg/kg of FE and 7.5mg/kg of FE can obviously increase the thermosensitive threshold of mice by 188.86%, 197.83% and 143.21% (P < 0.05), respectively. The infrared thermosensitive experiment is a commonly used antipyretic screening model, and the experimental result shows that FE has remarkable antipyretic effect. Therefore, the antipyretic activity of FE is probably one of the drug efficacy bases of RA treatment, and can remarkably restore the body temperature of RA patients.

Example 3: oral availability and absorption transport characteristics of FE

In this example, the oral bioavailability and absorption and transport characteristics of FE were experimentally examined to preliminarily evaluate the drug properties.

(1) Oral bioavailability of FE

Male 8-week-old C57 mice were randomized into 2 groups of 5. After 15mg/kg FE and 1.5mg/kg FE were infused intravenously, 2 groups of mice were bled from the tail vein at

time points

0, 3, 7, 10, 15, 20, 30, 50, 80, 120, 240, 360 and 480 min. After blood sample is centrifugally separated to obtain plasma, the plasma is treated by organic solvent to precipitate protein, 2 mu L of the plasma is taken to enter a UHPLC-MS/MS system to quantitatively detect the concentration of FE in the plasma, a blood concentration-time curve is drawn, pharmacokinetic parameters are calculated, and the oral bioavailability is calculated according to the following formula:

oral bioavailability (100%) = (AUC _p.o ×Dose _i.v )/(AUC _i.v ×Dose _p.o )×100％

(2) Absorption transport characteristics of FE

The Caco-2 cells in the logarithmic growth phase were diluted with DMEM complete medium containing 10% fetal calf serum, 1% diabody and 1% nonessential amino acids and inoculated into 6-well Transwell plates to give 50 ten thousand cells/well, and cultured in an incubator at 37℃and 5% CO2 for 21 days. After cells grow into stable and compact cell tissues with a monolayer, the cells are changed once a day during the cell culture period, and after the cells grow into stable and compact cell tissues with HBSS buffer solution at 37 ℃ for 3 times, transepithelial cell resistance is measured, and when the resistance value is more than 420 omega/cm < 2 >, the cells form a compact and complete cell monolayer, and the cell can be used for performing a transmembrane transport experiment. After the resistance measurement is completed, 2mL of HBSS buffer solution is added to two sides of each hole, and the mixture is placed on a shaking table at 37 ℃ for incubation for 30min, so that the cells release nutrient substances absorbed in the culture process. Drug transport process from AP side to BL side: 2mL of HBSS solution containing 2. Mu.M FE was added from the AP side, and an equal volume of blank HBSS solution was added to the BL side. Samples of 0.5mL were taken on both sides at 30, 60, 90 and 120min after dosing, while 0.5mL of HBSS solution containing 2. Mu.M FE was added on the AP side and an equal volume of blank HBSS solution was added on the BL side, each group of samples was in parallel 3. The drug transfer from BL side to AP side was identical to the AP side to BL side transfer experiment operation, i.e., drug was added to BL side and blank HBSS solution was added to AP side.

Inhibitor experiments: when the drug is transported from the AP-BL, adding a mixed HBSS solution containing 2 mu M FE and an inhibitor from the AP side, and simultaneously adding a blank HBSS solution from the B side; when the drug is transported from BL-AP, the drug is added to BL side, and the HBSS solution containing the inhibitor is added to A side, and the rest steps are the same. 100 mu L of acetonitrile solution containing an internal standard is added into a sample, and after uniform mixing and centrifugation, 2 mu L of supernatant is taken and subjected to UHPLC-MS/MS analysis to detect the concentration of FE.

The experimental results are shown in fig. 11-13, wherein the results are expressed in terms of mean ± standard deviation, the data are analyzed using independent sample t-test, and the symbol "x" represents P<0.05, i.e. there was a significant difference between the different inhibitor groups and the control group (n=6). The in vivo pharmacokinetic experiment result shows that FE is absorbed rapidly in mice, the blood concentration reaches a peak within 10min (figure 11A), and the second peak is reached within 100-300 min, and the calculated oral bioavailability is 16.06%. In vitro cell transport experiments, apparent permeability coefficient (P _app ) Mainly at 0.78 x 10 ^-5 cm/s to 1.73 x 10 ^-5 In cm/s range (FIG. 13C), indicating good absorption of FE on cells, possibly into and out of cells mainly by passive diffusion; in addition, when inhibitors of efflux transporters P-gp and BCRP were added, the efflux rate of FE on Caco-2 cells was significantly reduced (FIG. 13D, P)<0.05 Indicating that Efflux transporters P-gp and BCRP are both involved in the Efflux of FE, but the rate of FE Efflux is very low (Effect ratio<1.22). In conclusion, the FE has good oral absorption, low excretion rate, higher bioavailability and better drug property, and can be applied to preparing the medicine for treating RA.

Through the experiments of examples 1 to 3, it is obvious that FE can significantly treat and improve symptoms of the adjuvant arthritis rats, and the treatment effect of FE is superior to that of non-steroidal anti-inflammatory drug indomethacin; through a pharmacodynamic basic experiment, the FE has very excellent anti-inflammatory activity, analgesic activity and antipyretic activity, and the excellent anti-inflammatory, analgesic and antipyretic activity can be used for purposefully relieving and improving the symptoms of arthritis; through an oral bioavailability experiment and an in vitro absorption and transport experiment, the FE has the advantages of good oral absorption, low excretion rate, high bioavailability and better drug property.

The invention and its embodiments have been described above with no limitation, and the actual construction is not limited to the embodiments of the invention as shown in the drawings. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present invention.

Claims

1. The application of the aconite root medicine in preparing the medicine for treating the rheumatoid arthritis is characterized in that the aconite root medicine has the following structural formula:

（Ⅰ）

the only active component of the medicine is aconite root extract.

2. The use of aconite root in preparing medicine for treating rheumatoid arthritis according to claim 1, wherein the dosage form of the medicine is a conventional dosage form for treating arthritis clinically.

3. The use of aconite root in preparing medicine for treating rheumatoid arthritis according to claim 2, wherein the dosage form of the medicine is oral preparation or injection preparation or external preparation.

4. The use of aconite root in the preparation of a medicament for the treatment of rheumatoid arthritis according to claim 2, wherein the medicament further comprises pharmaceutically acceptable adjuvants.