CN108815162A - Benzoyl aconine compatibility Paeoniflorin is preparing the purposes in treating rheumatoid arthritis drug - Google Patents

Benzoyl aconine compatibility Paeoniflorin is preparing the purposes in treating rheumatoid arthritis drug Download PDF

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CN108815162A
CN108815162A CN201810973543.XA CN201810973543A CN108815162A CN 108815162 A CN108815162 A CN 108815162A CN 201810973543 A CN201810973543 A CN 201810973543A CN 108815162 A CN108815162 A CN 108815162A
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drug
paeoniflorin
compatibility
rheumatoid arthritis
benzoyl aconine
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李晋奇
顾平
王佳凤
童荣生
李�杰
何丹
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Sichuan Provincial Peoples Hospital
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    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

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Abstract

The invention belongs to field of medicine invention, specifically disclose benzoyl aconine compatibility Paeoniflorin and are preparing the purposes in treating rheumatoid arthritis drug and its drug of preparation, disclose benzoyl aconine compatibility Paeoniflorin, can be by inhibiting serum PG E2Generation, adjust serum cytokines (IL-1 β, TNF-α and VEGF), inhibit IgG synthesis and secretion, and/or regulate and control the expression of STAT1 and STAT3 in JAK-STAT signal path, to play anti-inflammatory, the formation for analgesia, mitigating synovium of joint hyperplasia, inhibiting pannus, the infiltration for reducing inflammatory cell, reduce osteoclasia and/or reduction ankle-joint pathological change collaboration multiple-effect function.The efficient significant therapeutic effect of multiaction target spot, low toxicity is played to rheumatoid arthritis, it increases patient with rheumatoid arthritis cure rate, reduce a possibility that types of medicines and dosage, it is used for using in preparation treatment medicine for treating rheumatoid arthritis, there is prominent marked improvement meaning.

Description

Benzoyl aconine compatibility Paeoniflorin is in preparing treating rheumatoid arthritis drug Purposes
Technical field
The present invention relates to field of medicine invention, and in particular to benzoyl aconine compatibility Paeoniflorin is closed in preparation rheumatoid Save the purposes in scorching therapeutic agent.
Background technique
It is main that rheumatoid arthritis (rheumatoid arthritis, RA), which is with aggressivity, symmetry panarthritis, Chronic, the systemic autoimmune disease of clinical manifestation.Global incidence is about 0.24%, disability rate with higher.RA Pathogenesis is still indefinite, and main pathological characters are that synovial tissue's hyperplasia, inflammatory cell infiltration and pannus are formed, and are gone out The destruction of existing cartilage and bone can finally cause joint deformity and function to lose.The treatment of RA at present uses drug mainly to alleviate and close Symptom is saved, the state of an illness, Saving cortilage function etc. are delayed.Primary treatment drug includes non-steroidal anti-inflammatory drugs (NSAIDs), sugared cortical hormone Element improves state of an illness antirheumatic drug (DMARDs), biological agent TNF-α inhibitor and IL-1 antagonist etc., but its toxic side effect compared with Greatly, the compliance of patient is seriously affected.
Benzoyl aconine (benzoylaconine, BAC), molecular formula C32H45NO10, it is monoester alkaloid, it is main It is present in the Chinese medicines such as aconiti preparata,radix, radix aconiti agrestis and monkshood, is the main hydrolysate of aconitine, toxicity drops significantly compared with aconitine It is low.Primary Study finds that benzoyl aconine has anti-inflammatory effect, and effect is better than Benzoylmesaconitine and benzoyl Secondary aconine, can lower adjuvant-induced arthritis (Adjuvant arthritis, AA) rat primary and secondary foot is swollen It is swollen.
Paeoniflorin (paeoniflorin, PAE) is the main active of Radix Paeoniae Alba, molecular formula C23H28O11, there is solution Convulsion, analgesic, it is anti-inflammatory, adjust the effects of immune and neuroprotection, to the curative for effect of rheumatoid arthritis.
Summary of the invention
Present invention aims at overcome medication toxic side effect existing for existing treating rheumatoid arthritis drug big, medication The strong disadvantage of compliance discloses a kind of rheumatoid arthritis using benzoyl aconine compatibility Paeoniflorin as effective component Therapeutic agent and its preparing the purposes in antirheumatic.Through inventor accidentally experimental studies have found that：Benzene Less toxic efficient, multiaction target spot collaboration can be played to rheumatoid arthritis after formyl aconine and Paeoniflorin compatibility to increase Strong therapeutic effect uses as treating rheumatoid arthritis medicine preparation, can play significant remarkable result.
To achieve the above object, following technical solution provided by the invention：
Benzoyl aconine compatibility Paeoniflorin is preparing the purposes in treating rheumatoid arthritis drug.
Wherein, through inventor accidentally experimental studies have found that：Select benzoyl aconine compound and two kinds of Paeoniflorin changes After closing object combination drug, anti-inflammatory, the formation for analgesia, mitigating synovium of joint hyperplasia, inhibiting pannus, reduction inflammatory can be passed through The infiltration of cell reduces osteoclasia and/or ankle-joint pathology multiple action mechanism, plays collaboration enhancing to rheumatoid arthritis Significant therapeutic effect.For existing treating rheumatoid arthritis drug, it is efficient to play multiaction target spot, low toxicity Significant therapeutic effect increases patient with rheumatoid arthritis cure rate, reduces types of medicines and dosage, further to open Sending out the effective therapeutic agent of rheumatoid arthritis has marked improvement meaning.
Preferably, the combination drug ratio between benzoyl aconine and Paeoniflorin is 0.1-2.0：24.It is highly preferred that Combination drug ratio between benzoyl aconine and Paeoniflorin is：0.5-1.5：24.More preferably, benzoyl aconite is former Combination drug ratio between alkali and Paeoniflorin is：1.5：24.
Further, benzoyl aconine and Paeoniflorin extract to obtain from crude drug respectively.Wherein, benzoyl aconite is former Alkali is to extract to obtain from monkshood, radix aconiti agrestis and/or monkshood crude drug.Paeoniflorin is extracted from Radix Paeoniae Alba and/or radix paeoniae rubra It arrives.
It studies and finds through inventor's further experiment：By benzoyl aconine and the combination drug of Paeoniflorin between the two Limited proportion is in 0.1-2.0：24, more preferably it is limited to 0.5-1.5：24, further preferably 1.5：When in 24 ranges, the two compatibility association It is more significant with effect, it is more significant to be used to prepare effect in treating rheumatoid arthritis drug.
Further, the drug is to have anti-inflammatory, the formation for analgesia, mitigating synovium of joint hyperplasia, inhibiting pannus, reduce The infiltration of inflammatory cell, the drug for reducing osteoclasia and/or reducing ankle-joint pathological change collaboration multiple-effect function.
Further, the drug is especially by inhibition serum PG E2Generation, adjust serum cytokines, inhibit IgG Synthesis and secretion, and/or in regulation JAK-STAT signal path STAT1 and STAT3 expression to inhibiting synovial cell different The drug of collaboration multiple-effect function is realized in normal secretion and hyperplasia.Wherein, the adjusting serum cytokines are IL-1 β, TNF-α And/or VEGF.
A kind for the treatment of rheumatoid arthritis drug contains benzoyl aconine and Paeoniflorin as effective component.
Wherein, through inventor accidentally experimental studies have found that：Select benzoyl aconine compound and two kinds of Paeoniflorin changes After closing object combination drug, anti-inflammatory, the formation for analgesia, mitigating synovium of joint hyperplasia, inhibiting pannus, reduction inflammatory can be passed through The infiltration of cell reduces osteoclasia and/or ankle-joint pathology multiple action mechanism, plays collaboration enhancing to rheumatoid arthritis Significant therapeutic effect.For existing treating rheumatoid arthritis drug, it is efficient to play multiaction target spot, low toxicity Significant therapeutic effect increases patient with rheumatoid arthritis cure rate, reduces types of medicines and dosage, further to open Sending out the effective therapeutic agent of rheumatoid arthritis has marked improvement meaning.
Preferably, the combination drug ratio between benzoyl aconine and Paeoniflorin is 0.1-2.0：24.It is highly preferred that Combination drug ratio between benzoyl aconine and Paeoniflorin is：0.5-1.5：24.More preferably, benzoyl aconite is former Combination drug ratio between alkali and Paeoniflorin is：1.5：24.
It studies and finds through inventor's further experiment：By benzoyl aconine and the combination drug of Paeoniflorin between the two Limited proportion is in 0.1-2.0：24, more preferably it is limited to 0.5-1.5：24, further preferably 1.5：When in 24 ranges, the two compatibility association It is more significant with effect, it is more significant to be used to prepare effect in treating rheumatoid arthritis drug.
Further, the drug further includes pharmaceutical acceptable other compositions, including receivable auxiliary material and/or excipient Deng.Further, capsule, tablet, microcapsule formulation, oral agents and/or injection are preferably made oral agents or injection.
It is shown through experimental result of the present invention：After benzoyl aconine and the treatment of Paeoniflorin compatibility group, can significantly it subtract The sufficient pawl swelling and AI of light CIA rat, effectively mitigate the synovium of joint hyperplasia of CIA rat, the formation of pannus and inflammatory cell Infiltration, reduce osteoclasia and joint pathology scoring, to CIA rat have good therapeutic effect.Its mechanism of action may be led It will be with inhibition serum PG E2Generation, adjust serum cytokines (IL-1 β, TNF-α and VEGF), inhibit the synthesis of IgG with point It secretes, regulates and controls the expression of STAT1 and STAT3 in JAK-STAT signal path, inhibit the secretion and hyperplasia phase of synovial cell's exception It closes.
Thus illustrate, benzoyl aconine compatibility Paeoniflorin can by anti-inflammatory, analgesia, mitigate synovium of joint hyperplasia, The formation for inhibiting pannus, the infiltration for reducing inflammatory cell, reduction osteoclasia and/or the collaboration enhancing of ankle-joint pathological change are made With, uses as treating rheumatoid arthritis medicine preparation, rheumatoid arthritis can be played collaboration enhance it is significant Therapeutic effect.
Detailed description of the invention：
Fig. 1 shows influence of the benzoyl aconine compatibility Paeoniflorin to CIA rat foot claw swelling；
In figure：A1：Blank control group group；B1：Model control group；C1：Tripterygium glycosides group；F1：Compatibility group (before administration)； A2：Blank control group group；B2：Model control group；C2：Tripterygium glycosides group；F2：Compatibility group (at the end of administration).
Influence Fig. 2 shows benzoyl aconine compatibility Paeoniflorin to CIA rat ankle joint pathological change；
In figure：A1：Blank control group；B1：Model control group；C1：Tripterygium glycosides group F1:Compatibility group (H.E × 4)； A2：Blank control group；B2：Model control group；C2：Tripterygium glycosides group；F2：Compatibility group (H.E × 10).
Specific embodiment
Below with reference to test example and specific embodiment, the present invention is described in further detail.But this should not be understood It is all to be belonged to based on the technology that the content of present invention is realized for the scope of the above subject matter of the present invention is limited to the following embodiments The scope of the present invention.
The following embodiments of the present invention are assessed by selecting SD rat to establish Collagen-induced Arthritis (CIA) rat model Drug action of the benzoyl aconine compatibility Paeoniflorin to CIA rat, while being exempted from by detection serum cytokines, serum The indexs such as epidemic disease globulin (immunoglobulin G IgG), inquire into its mechanism of action, for RA treatment provide new scheme and Theoretical basis.Specific embodiment operation is as follows：
Embodiment 1
Benzoyl aconine compatibility Paeoniflorin studies the drug action of Collagen-induced Arthritis (CIA)
(1) experimental animal
SD rat, male, weight (140 ± 10) g, by People's Hospital, Sichuan Prov. of Sichuan Academy of Medical Sciences experimental animal Research institute provides, production licence number：SCXK (river) 2013-15.
It is carried out after Sichuan Academy of Medical Sciences's Experimental Animal Center adaptable fed 5 days before all experimental rat experiments real It tests.Feeding environment：22 DEG C~23 DEG C of temperature, humidity 30%~50%；Method for breeding：By a group difference sub-cage rearing, 5, every cage, Free diet, drinking-water.
(2) experimental drug and drug solution preparing
(2.1) experimental drug
Benzoyl aconine, Chengdu Man Site Biotechnology Co., Ltd and Chengdu Inst. of Biology, Chinese Academy of Sciences grind System, purity：99.96% (HPLC), lot number：MUST-16012815；
Paeoniflorin, Chengdu Man Site Biotechnology Co., Ltd and Chengdu Inst. of Biology, Chinese Academy of Sciences develop, purity： 99.30% (HPLC), lot number：MUST-16041901；
Tripterygium wilfordii Polyglycosidium Tablets, Hunan Qianjin Xieli Pharmaceutical Co., Ltd., specification 10mg/ piece 100, authentication code：Traditional Chinese medicines Quasi- word group Z43020138, batch number：20160704.
(2.2) drug solution preparing is tested
Benzoyl aconine compatibility Paeoniflorin high dose compatibility group (hereinafter referred to as compatibility group)：Benzoyl aconine and The dosage of Paeoniflorin is respectively 1.5mgkg-1·d-1And 24mgkg-1·d-1, when experiment is dissolved in distilled water respectively, prepares It is spare at the medical fluid containing benzoyl aconine 0.03% and Paeoniflorin 0.48%.
Tripterygium glycosides group：Dosage is 8mgkg-1·d-1, when experiment is dissolved in distilled water, is configured to more containing tripterygium wilfordii The medical fluid of glycosides 0.08% is spare.
(3) major experimental reagent
II Collagen Type VI solution (2mgml-1) of ox is produced, lot number by Chondrex company of the U.S.：SLBR1681V；
Complete Freund's adjuvant (CFA), is produced, lot number by Sigma Co., USA：160346；
Formic acid, Chengdu Ke Long chemical reagent factory, lot number：2013123001；
Haematoxylin dye liquor and eosin stain, Beisuo Biological Technology Co., Ltd., Zhuhai, lot number：716123.
Specific experiment operates：
1, the foundation and grouping of CIA model
It SPF grades male SD rat 70, after weight (140 ± 10) g, adaptable fed 5d, randomly selects 10 and is only used as just Normal control group, remaining carries out the modeling of collagen induced arthritis model.Specific method is：
By the acetum (II 2.0mgml-1 of C containing ox) of II Collagen Type VI of ox (type II collagen, C II) and equal bodies Product complete Freund's adjuvant (complete Freund ' s adjuvant, CFA) mixes completely, fully emulsified, and emulsion is with its drop Being added on indiffusion in water is degree, is finally configured to the emulsion of II 1.0mgml-1 of C containing ox.Take emulsion by every 0.2mL Initial immunity (d0) is carried out in intradermal point of back, root of the tail portion 4 points of injection (back two o'clock, root of the tail portion two o'clock), initial immunity the Booster immunization again according to the above method after 7d (d7), in the 14th day (d14) carry out arthritis index (arthritis index, AI it) scores.Every rat highest scoring is 16 points, is induced successfully using at least 1 foot pawl AI >=2 points of persons of scoring as arthritis. Modeling experimental result is as follows：
CIA rat model, after the emulsion of intracutaneous injection C containing ox II, injection site festers, general 7 days left sides The right side can form a scab healing.The 7th day (d7) after initial immunity, it is seen that redness occurs in a little small toe joint of rat portions metapedes, again After immune, rat hindleg pawl swelling is gradually aggravated, and the time that swelling occurs in front foot pawl is later than metapedes pawl, and the 14th day (d14) when can See that most of rat foot claw is obviously red and swollen, AI scoring is carried out to CIA, apparent arthritic symptom, modeling occur in 53 rats Success rate is 88.33%.
Finally, random screening in the successful CIA rat of above-mentioned modeling is gone out successful model 30, it is randomly divided into CIA model Group (10), tripterygium glycosides group (positive controls) (10), benzoyl aconine compatibility Paeoniflorin group (compatibility group) (10).
2, it is administered
The day initial immunity the 14th (d14) starts gastric infusion, and dosage selects the compatibility dosage screened in first part, I.e.
Benzoyl aconine compatibility Paeoniflorin group (compatibility group), wherein benzoyl aconine group dosage is 1.5mg·kg-1·d-1, Paeoniflorin dosage is 24mgkg-1·d-1；
Tripterygium glycosides group, dosage 8mgkg-1·d-1。
Specifically dosage regimen is：Blank control group and model control group stomach-filling distilled water, other each groups are dense by each group drug Degree carries out stomach-filling after preparing, wherein blank control group, model control group, every group of tripterygium glycosides group according to 10mlkg-1Agent Amount carries out stomach-filling, benzoyl aconine compatibility Paeoniflorin group benzoyl aconine medical fluid and Paeoniflorin medical fluid respectively according to 5ml·kg-1Dosage carry out stomach-filling, 1 time a day, continuous 4 weeks.
3, benzoyl aconine compatibility Paeoniflorin is to Collagen-induced Arthritis (CIA) sufficient pawl swelling degree therapeutic effect
The measurement of rat foot claw swelling：It is every after every 7d (d7, d14) and treatment after (d0), initial immunity before initial immunity 4d (d18, d22, d26, d30, d34, d38, d42) once, with vernier caliper measures its thickness in right hind foot of rat fixed position Degree.
Said determination result is subjected to statistical procedures again：Measurement data is indicated with x ± s, counts soft with SPSS 17.0 Part is for statistical analysis using one-way analysis of variance, and P < 0.05 is that difference is statistically significant.
Testing result：See Table 1 for details and table 2 for each group right hind foot of rat sufficient pawl swelling degree measurement result, and the sufficient pawl in administration front and back is swollen It is swollen to see Fig. 1.
1 each group right hind foot of rat swelling situation of change (x ± s) of table
Note：The * P compared with blank control group<0.05, * * P<0.01；The #P compared with model control group<0.05, ##P<0.01.
2 each group right hind foot of rat swelling situation of change (x ± s) of table
Note：Compared with blank control group*P<0.05,**P<0.01；Compared with model control group#P<0.05,##P<0.01。
It can be seen that：There was no significant difference (P > 0.05) for (d0) rat foot claw swelling before initial immunity for each group rat. 14d (d14) after initial immunity, relative to blank control group rat foot claw obvious tumefaction, difference has each CIA group before treating Statistical significance (P < 0.01), there was no significant difference (P > 0.05) for each CIA group mouse sufficient pawl swelling degree.Treat 20 days (d34) Afterwards, compared with model control group, sufficient pawl swelling degree is significantly reduced for tripterygium glycosides group and compatibility group, and difference has statistical significance (P < 0.05).After administration 24 days (d38), compared with model control group, sufficient pawl swelling degree is significant for tripterygium glycosides group, compatibility group Lower, difference has statistical significance (P < 0.01 or P < 0.05).
4, benzoyl aconine compatibility Paeoniflorin scores to Collagen-induced Arthritis (CIA) arthritis index (AI) It influences
Arthritis index (AI) scoring measurement：
In 14d after initial immunity (d14), carried out every 4d (d18, d22, d26, d30, d34, d38, d42) primary AI observation scoring.
Said determination result is subjected to statistical procedures：Measurement data is indicated with x ± s, with 17.0 statistical software of SPSS For statistical analysis using one-way analysis of variance, P < 0.05 is that difference is statistically significant.
Testing result：See Table 3 for details and table 4 shown in.
3 each group CIA rat arthritis index score situation of change (x ± s) of table
Note：The * P compared with blank control group<0.05, * * P<0.01；The #P compared with model control group<0.05, ##P<0.01.
4 each group CIA rat arthritis index score situation of change (x ± s) of table
Note：The * P compared with blank control group<0.05, * * P<0.01；The #P compared with model control group<0.05, ##P<0.01.
According to known to above-mentioned experimental result：The 14th day (d14) after initial immunity, each CIA group is relative to blank pair before treating It is significantly increased according to a group rat AI, difference has statistical significance (P < 0.01), each CIA group rat AI there was no significant difference (P > 0.05).After treatment 24 days (d34), tripterygium glycosides group and compatibility group are compared with model control group, and AI is significantly reduced, difference With statistical significance (P < 0.01 or P < 0.05).After treatment 28 days (d42), tripterygium glycosides group, compatibility group and model pair Compare according to group, AI significantly lowers, and difference has statistical significance (P < 0.01 or P < 0.05).
5, benzoyl aconine compatibility Paeoniflorin is to the influence to CIA rat ankle joint pathological change
Joint histopathology observation：
After experiment, rat is put to death, cuts right metapedes ankle-joint, separated after skin and muscle with 10% neutral buffered good fortune The fixed 48h of your Malin's solution, is then put into decalcification 3d~5d in 10% formic acid formalin for ankle-joint sample, carries out conventional Dehydration, paraffin embedding, slice (5 μm) and Hematoxylin-eosin (Hematoxylin-eosin, HE) dyeing, optical microscopy are seen The synovial tissue's hyperplasia, cell infiltration, pannus for examining rat ankle joint are formed and articular cartilage and bone destruction etc. are sick Reason changes, and scores the above lesion degree, it is without exception be 0 point, be slightly 1 point, be slightly 2 points, moderate be 3 points and Severe is 4 points.Each ankle-joint pathological score highest 12 is divided.
Said determination result is subjected to statistical procedures：Measurement data is indicated with x ± s, with 17.0 statistical software of SPSS For statistical analysis using one-way analysis of variance, P < 0.05 is that difference is statistically significant.
Testing result：As shown in table 5 and Fig. 2.
The influence (x ± s) that 5 benzoyl aconine compatibility Paeoniflorin of table scores to CIA rat ankle joint pathological change
Note：The * P compared with blank control group<0.05, * * P<0.01；The #P compared with model control group<0.05, ##P<0.01.
Above-mentioned testing result is shown：Blank control group synovial membrane is thin and complete, is made of 1~2 layer of synovial cell, has no tissue Oedema, cell infiltration and blood vessel hyperplasia, cartilage surface is smooth, has no that osteoclasia, joint structure are complete.
Synovial tissue's hyperplasia is obvious in model control group articular cavity, forms multilayer (up to 10~15 layers) stratum synoviale, arrangement Disorder, part synovial membrane missing, and with the blood vessel dilatation of synovial membrane undertissue, quantity increases, and pannus is formed；Synovial tissue's hyperplasia And go deep into articular cavity in villiform, there are a large amount of cell infiltrations in articular cavity, articular hyaline cartilage and bone tissue occur broken Bad, local fibrosis and stick to each other, joint structure is unclear, and compared with blank control group, ankle-joint pathological score is significantly risen Height, difference have statistical significance (P<0.01).
Tripterygium glycosides group synovial cell layer number is substantially at or slightly above normal level, cell infiltration and pannus base This disappearance, accidental cartilage surface is coarse or a little bone destruction, and compared with model control group, pathological change significantly mitigates, pathology Scoring significantly reduces, and difference has statistical significance (P<0.01).
Compatibility group synovial cell proliferation degree is substantially reduced, it is seen that 3~6 layers, a small amount of cell infiltration and blood vessel hyperplasia close It is slightly coarse to save cartilage, has no cartilage and bone destruction substantially, compared with model group, pathological score is significantly reduced, and difference has Statistical significance (P<0.01).
In summary known to test result：After benzoyl aconine and the treatment of Paeoniflorin compatibility group, can significantly it subtract The sufficient pawl swelling and AI of light CIA rat.Pathological section is the results show that the synovium of joint that compatibility group effectively mitigates CIA rat increases Raw, the formation of pannus and the infiltration of inflammatory cell reduce osteoclasia and joint pathology scoring, have to CIA rat good Therapeutic effect.Thus illustrate, benzoyl aconine compatibility Paeoniflorin can be increased by anti-inflammatory, analgesia, mitigation synovium of joint Formation that is raw, inhibiting pannus, the infiltration for reducing inflammatory cell reduce osteoclasia and/or the collaboration enhancing of ankle-joint pathological change Effect, plays synergistic therapeutic effect to rheumatoid arthritis.
Embodiment 2
The study on mechanism of benzoyl aconine compatibility Paeoniflorin treatment Collagen-induced Arthritis
(1) experimental animal, (2) experimental drug and drug solution preparing, it is same as Example 1；
(3) major experimental reagent
II Collagen Type VI solution (2mgml-1) of ox is produced, lot number by Chondrex company of the U.S.：SLBR1681V；
Complete Freund's adjuvant (CFA), is produced, lot number by Sigma Co., USA：160346；
Rat interleukin-11 β (IL-1 β) ELISA kit, by Wuhan Yi Lai Rett Biotechnology Co., Ltd (Elabscience) it produces, lot number：AK0016DEC23031；
Rat tumor necrosin & (TNF-α) ELISA kit, is produced, lot number by Wuhan Elabscience： AK0016DEC23032
Rat VEGF ELISA kit, is produced by Xin Bosheng Biotechnology Co., Ltd, lot number R161221-103a
Rat immunoglobulin G (IgG) ELISA kit, is produced, lot number by Wuhan Elabscience： AK0017JAN12020
Rat prostate element E2 (PGE2) ELISA kit, is produced, lot number by Wuhan Elabscience AK0017JAN12021
Haematoxylin dye liquor and eosin stain, Beisuo Biological Technology Co., Ltd., Zhuhai, lot number：Lot number：716123；
STAT1 (primary antibody), SAB company, lot number：5807；
STAT3 (primary antibody), SAB company, lot number：6D22；
Anti-Rabbit IgG (secondary antibody), SAB company, lot number 8715；
DAB color developing agent, Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge, lot number：K166622C
Specific experiment operates：
1, the foundation and grouping of CIA model
With embodiment 1
2, it is administered
With embodiment 1
3, experiment materials
After administration, according to rat body weight, by 3mlkg-1Dosage give 10% chloral hydrate anesthesia rat, open Disposable blood taking needle is used after abdomen, acquires rat aorta blood in additive-free vacuum blood collection tube, the blood sample of acquisition is put Set 30min at room temperature, 3000rmin-1It is centrifuged 5min, separates serum, -20 DEG C save backup；Right metapedes ankle-joint is cut, It is fixed after separation skin and muscle with 10% neutral buffered formalin solution spare；Left back ankle synovium of joint is taken, with 3% penta The fixation of dialdehyde solution is spare.
4, influence of the benzoyl aconine compatibility Paeoniflorin to CIA rat blood serum PGE2
Serum inflammatory factors of senile PGE2The measurement of content：Rat blood serum is taken, using enzyme linked immunosorbent assay (enzyme Linked immunosorbent assay, ELISA) method detection serum in PGE2Content, according to kit specification into Row detection.
Detection data is indicated with x ± s, carries out statistical using one-way analysis of variance with 17.0 statistical software of SPSS Analysis, P < 0.05 are that difference is statistically significant.
Benzoyl aconine compatibility Paeoniflorin is to CIA rat blood serum PGE2Influence, detection statistics result see Table 6 for details institute Show：
Influence (x ± s) of the 6 benzoyl aconine compatibility Paeoniflorin of table to CIA rat blood serum PGE2
Note：The * P compared with blank control group<0.05, * * P<0.01；The #P compared with model control group<0.05, ##P<0.01.
It is shown according to upper table statistical data：Model control group rat blood serum PGE2It is significantly increased relative to blank control group, Difference has statistical significance (P<0.01), wherein tripterygium glycosides group and compatibility group rat blood serum PGE2Relative to model pair It is remarkably decreased according to group, difference has statistical significance (P<0.05).
5, influence of the benzoyl aconine compatibility Paeoniflorin to CIA rat blood serum cell factor
Serum cytokines measurement：Rat blood serum is taken, using IL-1 β, TNF-α and blood vessel in ELISA method detection serum The content of endothelial growth factors (vascular endothelial growth factor, VEGF), according to the explanation of kit Book is detected.
Detection data is indicated with x ± s, carries out statistical using one-way analysis of variance with 17.0 statistical software of SPSS Analysis, P < 0.05 are that difference is statistically significant.
Detection statistics result is as shown in Table 7 below：
Influence (x ± s) of the 7 benzoyl aconine compatibility Paeoniflorin of table to CIA rat blood serum cell factor
Note：The * P compared with blank group<0.05, * * P<0.01；The #P compared with model control group<0.05, ##P<0.01.
It is shown according to upper table statistical data：Model control group rat blood serum IL-1 β is significantly increased relative to blank control group, Difference has statistical significance (P < 0.01)；Each treatment group's rat blood serum IL-1 β is remarkably decreased relative to model control group, Difference has statistical significance (P < 0.01 or P < 0.05).
Model control group rat blood serum TNF-α is significantly increased relative to blank control group, and difference has statistical significance (P < 0.05)；Each treatment group's rat blood serum TNF-α is remarkably decreased group relative to model, and difference has statistical significance (P < 0.01 or P < 0.05).
Model control group rat blood serum VEGF is significantly increased relative to blank control group, and difference has statistical significance (P < 0.01)；Tripterygium glycosides group and compatibility group rat blood serum VEGF are significantly reduced relative to model control group, and difference has statistics It learns meaning (P < 0.01).
6, influence of the benzoyl aconine compatibility Paeoniflorin to rheumatoid factor IgG in CIA rat blood serum
Rheumatoid factor IgG level measures in serum：Rat blood serum is taken, is contained using IgG in ELISA method detection serum Amount, is detected according to the specification of kit.
Detection data is indicated with x ± s, carries out statistical using one-way analysis of variance with 17.0 statistical software of SPSS Analysis, P < 0.05 are that difference is statistically significant.
Detection statistics result is as shown in Table 8 below：
Influence (x ± s) of the 8 benzoyl aconine compatibility Paeoniflorin of table to IgG in CIA rat blood serum
Note：The * P compared with blank control group<0.05, * * P<0.01；The #P compared with model control group<0.05, ##P<0.01.
It is shown according to upper table statistical data：Model control group serum IgG is significantly increased relative to blank control group, poor It is different that there is meter to learn meaning (P < 0.01)；Tripterygium glycosides group and compatibility group serum IgG are significant relative to model control group It reduces, difference has statistical significance (P < 0.05).
It can be seen that：Benzoyl aconine compatibility Paeoniflorin
7, the influence that benzoyl aconine compatibility Paeoniflorin expresses CIA rat articular synovial tissue STAT1 and STAT3
Immunohistochemistry detects synovial tissue of joint's signal transduction and activating transcription factor (signal transducers and Activators of transcription 1, STAT1) and STAT3 expression：
After aquation after (5 μm) of synovium of joint paraffin section dewaxings, STAT1 (1 is first added dropwise respectively:And STAT3 (1 100): 100) then Anti-Rabbit IgG (1 is added dropwise in monoclonal antibody:1000), DAB color developing agent color development at room temperature, haematoxylin are redyed, With microscopic observation after neutral gum mounting.Immunohistochemistry is to there is clear brown yellow granule in cytoplasm and/or nucleus For positive cell, this experiment carries out sxemiquantitative scoring, specific methods of marking according to the ratio and staining power of positive cell For：First scored according to the ratio of positive cell, wherein 0 point of inaction, 1%~25% is 1 point, and 26%~50% is 2 points, 56%~75% is 3 points, 76%~100% is 4 points；It scores further according to staining power, wherein no positive reacting cells are 0 point, Light yellow is 1 point, and brown color is 2 points, and sepia is 3 points, and finally two parts score is multiplied, obtains the general comment of this sample Point.
Detection data is indicated with x ± s, carries out statistical using one-way analysis of variance with 17.0 statistical software of SPSS Analysis, P < 0.05 are that difference is statistically significant.
Detection statistics result is as shown in Table 9 below：
9 rat ankle joint synovial membrane Immunohistochemical detection of table scores (x ± s)
Note：The * P compared with blank control group<0.05, * * P<0.01；The #P compared with model control group<0.05, ##P<0.01.
Each group rat ankle joint synovial tissue's Immunohistochemical detection is the results show that model control group lymphocyte of adjurant arthritis rat STAT1 and STAT3 positive expression is dramatically increased relative to blank control group, and difference has statistical significance (P < 0.01)；Respectively Treatment group's lymphocyte of adjurant arthritis rat STAT1 and STAT3 positive expression is on a declining curve relative to model control group, and wherein tripterygium wilfordii is more Glycosides group, compatibility group lymphocyte of adjurant arthritis rat STAT1 and STAT3 positive expression are remarkably decreased relative to model control group, and difference has statistics It learns meaning (P < 0.01 or P < 0.05).
In summary known to test result：Benzoyl aconine compatibility Paeoniflorin can increase benzoyl aconine, Chinese herbaceous peony Medicine glycosides treat rheumatoid arthritis drug action, mechanism of action may mainly with inhibit serum PG E2Generation, adjust blood Clear cell factor (IL-1 β, TNF-α and VEGF), inhibits the synthesis and secretion of IgG, regulates and controls in JAK-STAT signal path The expression of STAT1 and STAT3 inhibits the secretion of synovial cell's exception related to hyperplasia.
3 Medications Example of embodiment
Benzoyl aconine and Paeoniflorin are taken, according to usage ratio 0.1-2.0：24 (unit mgkg-1·d-1) dissolution Afterwards, then be optionally added into pharmaceutically acceptable other compositions, including auxiliary material, excipient etc., it is made according to existing pharmaceutical technology Capsule, tablet, microcapsule formulation, oral agents and/or injection are preferably made oral agents or injection, so that a type be made Rheumathritis therapeutic agent.

Claims (10)

1. benzoyl aconine compatibility Paeoniflorin is preparing the purposes in treating rheumatoid arthritis drug.
2. purposes according to claim 1, which is characterized in that the combination drug between benzoyl aconine and Paeoniflorin Amount is than being 0.1-2.0：24.
3. purposes according to claim 1, which is characterized in that benzoyl aconine and Paeoniflorin are respectively from crude drug Extraction obtains.
4. purposes according to claim 1, which is characterized in that the drug is to have anti-inflammatory, analgesia, mitigate synovium of joint Hyperplasia, the infiltration for reducing inflammatory cell, reduces osteoclasia and/or reduces ankle-joint pathological change association the formation for inhibiting pannus With the drug of multiple-effect function.
5. purposes according to claim 1, which is characterized in that the drug is especially by inhibition serum PG E2Generation, STAT1 and STAT3 in the synthesis and secretion for adjusting serum cytokines, inhibiting IgG, and/or regulation JAK-STAT signal path Expression to inhibit the secretion and hyperplasia of synovial cell's exception, realize the drug for cooperateing with multiple-effect function.
6. purposes according to claim 1, which is characterized in that the adjusting serum cytokines be IL-1 β, TNF-α and/ Or VEGF.
7. a kind for the treatment of rheumatoid arthritis drug, which is characterized in that contain benzoyl aconine and Paeoniflorin as drug effect Ingredient.
8. drug according to claim 7, which is characterized in that the combination drug amount between benzoyl aconine and Paeoniflorin Than for 0.1-2.0：24 .
9. drug according to claim 7, which is characterized in that further include medically acceptable other compositions.
10. drug according to claim 7, which is characterized in that the pharmaceutical dosage form is capsule, tablet, microcapsule formulation, mouth Take agent and/or injection.
CN201810973543.XA 2018-08-24 2018-08-24 Benzoyl aconine compatibility Paeoniflorin is preparing the purposes in treating rheumatoid arthritis drug Pending CN108815162A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102038699A (en) * 2009-10-19 2011-05-04 周亚伟 Novel application of paeoniflorin to inflammation resistance

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102038699A (en) * 2009-10-19 2011-05-04 周亚伟 Novel application of paeoniflorin to inflammation resistance

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YUN-YUN GUAN等: "Suppressive effects of Wang-Bi Tablet on adjuvant-induced arthritis in rats via NF-κB and STAT3 signaling pathways", 《INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE》 *
顾平: "苯甲酰乌头原碱配伍芍药苷治疗类风湿关节炎的药效学及作用机制研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 *

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