CN103816172A - Application of eleutheroside E in preparation of rheumatoid arthritis treating medicine - Google Patents

Abstract

The invention relates to a novel application of eleutheroside E, in particular to an application of the eleutheroside E in preparation of a rheumatoid arthritis treatment medicine and medicine composition for treating the rheumatoid arthritis. The medicine composition for treating the rheumatoid arthritis can be prepared into tablets, capsules, a dissolved medicine, drops, granules, an ointment or an injection through the eleutheroside E with purity higher than 98% (mass percentage) added with universal auxiliary medical components. Pharmacology experiment results show that the eleutheroside E can remarkably improve the joint swelling condition of the rheumatoid arthritis, reduce the arthritis index, reverse pathologic change of arthritis, has no critical defects of clinically common medicines, and has the function of the rheumatoid arthritis resistance with high efficiency and low toxicity. Therefore, the eleutheroside E can be used for preparing the medicine for treating the rheumatoid arthritis. The invention provides a novel medicine source for treating the rheumatoid arthritis.

Description

The application of eleutheroside E in preparation treatment medicine for treating rheumatoid arthritis

Technical field

The present invention relates to medical technical field, is the application of eleutheroside E in preparation treatment medicine for treating rheumatoid arthritis.

Background technology

Rheumatoid arthritis (Rheumatoid arthritis, RA) is that a kind of joint around involved is main multisystem inflammatory autoimmune disease, and the cause of disease is not bright.Rheumatoid arthritis is modal struvite joint disease clinically, has a strong impact on patient's function status, quality of life and life expectancy, because of its chronic feature, related complication and long-term dysfunction, society and economy is caused to tremendous influence.To the treatment of rheumatoid arthritis, take Drug therapy as master, (common medicine is as NSAID (non-steroidal anti-inflammatory drug) at present, new listing biological preparation is as TNF-a inhibitor), but neither one medicine can really be controlled the development of the state of an illness completely, and there is serious side effect, therefore urgently research and develop novel resisting rheumatoid arthritis medicine.Natural drug is the important rarity of China, because its wide material sources, composition enrich, untoward reaction is few, is developed to new drug and is easy to the feature that human body is accepted, and more and more receives domestic and international researcher's concern.The novelty for the treatment of RA is found in trial at present from natural drug, effectively active pharmaceutical substance has become the focus of research.

Eleutheroside E (Eleutheroside E) is colourless acicular crystal in dilute methanol, and fusing point 269-271 ℃ is the main effective ingredient of Chinese medicine Radix Et Caulis Acanthopanacis Senticosi, and this composition all has existence in the positions such as the root of Radix Et Caulis Acanthopanacis Senticosi, stem, leaf, flower.It dissolves in methanol or ethanol, and all unstable to soda acid, structural formula is as follows.

Eleutheroside E is as a kind of known monomers compound, although have definite molecular formula and structural formula, but the activity of its research is not high, considerably less about the pharmacological action report of eleutheroside E at present, its pharmaceutical research report is mainly and reduces blood glucose, anti-central fatigue, improves the myocardiac mechanics being caused by pituitrin and change, reduce the effect of myocardial infarction incidence rate, the clinical control cardiovascular and cerebrovascular disease aspect that is mainly used in.So far, as monomer, the application in preparation treatment medicine for treating rheumatoid arthritis yet there are no research report to eleutheroside E.

Summary of the invention

The technical problem that the present invention will solve first aspect is to provide the new purposes of eleutheroside E, i.e. the application of eleutheroside E in preparation treatment medicine for treating rheumatoid arthritis.

For addressing the above problem, the technical scheme that first aspect present invention provides is: the application of eleutheroside E in preparation treatment medicine for treating rheumatoid arthritis compositions.Described pharmaceutical composition comprises inertia, the excipient that nontoxic, pharmacology is suitable.

Second aspect present invention provides a kind of pharmaceutical composition for the treatment of rheumatoid arthritis, and said composition contains the eleutheroside E active component for the treatment of effective dose.

Preferably, described pharmaceutical composition also comprises medicine one or more inertia, nontoxic, excipient that pharmacology is suitable.

Preferably, described excipient is carrier, solvent, emulsifying agent, dispersant, wetting agent, binding agent, stabilizing agent, coloring agent, spice.

Preferably, described pharmaceutical composition is tablet, capsule, electuary, drop, lyophilized products, granule, ointment or injection.

In technique scheme, described treatment medicine for treating rheumatoid arthritis compositions can be added general medicinal auxiliary element by the above eleutheroside E of purity 98% (mass percent), makes tablet, capsule, electuary, drop, lyophilized products, granule, ointment or injection.

Can whole body and/or work partly according to pharmaceutical composition of the present invention, for this reason, can in suitable mode, for example, take by the approach of mouth, parenteral, lung or nose, the form of taking that can be suitable for these route of administration according to compositions of the present invention is taken.

Being suitable for oral is to work and promptly and/or discharge the form of medication of pharmaceutical composition of the present invention with improved procedure according to prior art level, and comprise with crystallization and/or unbodied and/or dissolved form, for example tablet is (without coating or cated tablet, it for example has the gastric juice of resistance or delayed dissolved or undissolved coating, discharge according to compositions of the present invention), rapid broken tablet in mouth, or diaphragm, film/lyophilized products, capsule (for example hard or soft capsule), coated tablet, granule, piller, powder, Emulsion, suspension, aerosol or solution according to pharmaceutical composition of the present invention.

Parenteral admistration can avoid absorption step (for example intravenous, intra-arterial, intracardiac, in spinal column or in waist or intraarticular) or comprise that absorption (for example intramuscular, subcutaneous, Intradermal, percutaneous or intraperitoneal) carries out simultaneously.Be suitable for the suitable form of taking of parenteral admistration method in particular for injecting and injecting with solution suspension, Emulsion, the preparation of lyophilized products or sterilized powder form.

What be suitable for another route of administration is for example medicament forms for sucking, and for example powder sucks seven or nebulizer, or the medicament forms of can nose taking, typing drop, solution or spray.

Can be converted into the described form of taking according to pharmaceutical composition of the present invention.This can be in a manner known way by carrying out with mixed with excipients inertia, nontoxic, that pharmacology is suitable.These excipient comprise carrier (for example microcrystalline Cellulose, lactose, mannitol especially, starch), solvent (for example liquid macrogol), emulsifying agent and dispersant or wetting agent (for example sodium lauryl sulphate, oleic acid polyoxy sorbitan esters, propylene glycol), binding agent (for example polyvinylpyrrolidone), synthesize and natural polymer (for example albumin), stabilizing agent (for example antioxidant, as ascorbic acid), coloring agent (for example inorganic pigment, as iron oxides) and masking property spice and abnormal smells from the patient.

The effective dosage of eleutheroside E can change according to administering mode, patient's age body weight, coincident with severity degree of condition and other relevant factor, and when oral administration, recommended dose is 20-70mg/ time, every day 1-2 time; Parenteral recommended dose is 10-30mg/ time, every day 1 time.

Technical scheme of the present invention is: set up rheumatoid arthritis mice (CIA) model by collagen-induced method, select first eleutheroside E as medicine, study it learns index and arthropathology change impact on CIA mice serum related immune, observe the preventive and therapeutic effect of eleutheroside E to rheumatoid arthritis mice (CIA) animal model, provide experimental basis thereby prevent and treat rheumatoid arthritis for eleutheroside E.

The invention has the advantages that: (1) provides eleutheroside E to have the purposes of Effective Anti rheumatoid arthritis: significantly mitigation symptoms, as arthroncus, arthritis index, and reverse joint pathology damage, reduce anti-CII antibody horizontal, " treating both the principal and secondary aspects of a disease ".Overcome the defect that prior art " is cured the symptoms, not the disease " as NSAID (non-steroidal anti-inflammatory drug).

(2) eleutheroside E provided by the invention has the purposes of Effective Anti rheumatoid arthritis, there is potential great economy effect: current global rheumatoid arthritis treatment medicine market is huge: have data to show, patient with rheumatoid arthritis in life branched go out expense be approximately 61000-122000 dollar.Preparation prepared by eleutheroside E or its pharmaceutically acceptable carrier has the application prospect for the treatment of medicine for treating rheumatoid arthritis, develop according to national original new drug drug approval law procedure, be expected to become country's 1 class high-efficiency low-toxicity treatment rheumatoid arthritis new drug with the independent intellectual property of China, industrialization prospect is clear and definite.

Accompanying drawing explanation

Below in conjunction with drawings and Examples, the invention will be further described:

Figure 1A is each group of CIA mice rear solid end picture substantially; Figure 1B is the impact of eleutheroside E on the scoring of CIA mice joint; Fig. 1 C is the impact of eleutheroside E on CIA Mouse Weight; Fig. 1 D is the impact of eleutheroside E on CIA mice claw thickness;

Fig. 2 A is each group of mice rear solid end mammography picture; Fig. 2 B is each group of mice rear solid end micro-CT picture; Fig. 2 C is the impact of eleutheroside E on CIA mice bone destruction scoring;

Fig. 3 A is that each group of mice joint part pathological tissue changes picture; Fig. 3 B is the impact of eleutheroside E on the scoring of CIA mice arthropathology;

Fig. 4 is the impact of eleutheroside E on anti-C II antibody horizontal in Mice Body.

Wherein, Vehicle is excipient; The positive contrast medicine of TG; EE is eleutheroside E; EE-15 is 15mg/kg gavage group; EE-30 is 30mg/kg gavage group, and EE-60 is 60mg/kg gavage group.

The specific embodiment

Below in conjunction with accompanying drawing, the specific embodiment of the invention is described.

One, material

1. laboratory animal

DBA/1J mice, 30, male, 8 week age, body weight 18～25g, clean level, is provided by Shanghai Slac Experimental Animal Co., Ltd..Laboratory animal production licence number (SCXK (Shanghai) 2012-0002).20 ± 3.2 ℃ of duration of test receptacle temperature, relative humidity is 65～75%, freely ingests and drinks water.

2, medicine and reagent:

2.1 medicine

Eleutheroside E, white powder, content is >=98%, and 4 ℃ of Refrigerator stores are provided by Sigma-Aldrich (Louis, MO, USA) company.Facing the used time adds 1% sodium carboxymethyl cellulose (CMC-Na) and makes suspension, matching while using; Tripterygium wilfordii Polyglycosidium Tablets, Xieli Pharmaceutical Co., Ltd., Hunan produces, the accurate word of traditional Chinese medicines: Z43020138.

2.2 reagent

Cattle II Collagen Type VI, complete Freund's adjuvant and incomplete Freund's adjuvant, Chondrex company of the U.S. produces; Hyclone, Gibco company produces, lot number: 948573; RPMI-1640 cultivates powder, and Gibco company produces, lot number: 984716; THP-1 cell line, is so kind as to give by University Of Suzhou's hematopathy institute; Penicillin (1000U/ml), streptomycin (10mg/ml), produces lot number: 20121025 by Beijing Solarbio company; Horseradish peroxidase sheep anti-mouse antibody, is produced lot number: 130318 by Abgent company.

2.3 instrument

Electronic balance scale, EL104 type, Shanghai Yue Ping scientific instrument company limited produces; Slide gauge (HAMILTION); 3K15 type tabletop refrigerated centrifuge, German Sigma company produces; CO2 incubator, electrical machinery of Japanese sanyo Co., Ltd. manufactures; Sunrise microplate reader, is produced by Tecan company of Switzerland; FACSCanto II flow cytometer, U.S. company BD production; DK-8AD type electric heating constant temperature tank, Shanghai Yi Heng Science and Technology Ltd. produces.

Two, experimental technique

1, modeling and administration

30 of DBA/1J mices, male, weigh, numbering, is divided into 5 groups at random, i.e. CIA model group, eleutheroside E low (15mg/kg), in (30mg/kg) and high (60mg/kg) dosage group and positive control drug Radix Tripterygii Wilfordii (TG15mg/kg) group, 6 every group.The mice of model group and each treatment group is all induced rheumatoid arthritis morbidity.Concrete operation step is as follows: by cattle II Collagen Type VI acetic acid solution (2mg/ml) and Freund's complete adjuvant (containing 5mg/ml BCG) mixed in equal amounts emulsifying, form Emulsion.In one glass of clear water, splash into an Emulsion, emulsion droplet maintains the original state, and that does not disperse is considered to qualified stability.Every experiment mice back is got 4-6 point and is carried out subcutaneous injection, 200 μ l altogether.After three weeks, with incomplete Freund's adjuvant and cattle II Collagen Type VI acetic acid solution (2mg/ml) mixed in equal amounts emulsifying, every mouse tail root is got 3-5 point subcutaneous injection, altogether 100 μ l.Each treated animal starts from every morning per os gastric infusion 1 time, successive administration 35 days in 2 second days of exempting from.Every other day mice is carried out to joint scoring, weighs and surveys rear solid end thickness once.

2, collection of specimens

The processing in joint: when experiment finishes, respectively organize mice and all pluck eyeball through eye socket and get blood, and get the rear solid end (comprising rear solid end and joint part) of mice.Respectively rear solid end is placed in to 10% paraformaldehyde and fixedly exceedes 24 hours, wherein a rear solid end is for pathological study, and another rear solid end is for imaging observation (molybdenum target and microCT).

The preparation of serum: respectively organize mice and pluck eyeball through eye socket and get after blood, 4 ℃ are spent the night, the centrifugal 20min of 4000r/min, carefully gets supernatant and is placed in the centrifuge tube of 0.5ml.Serum-20 that prepare ℃ Refrigerator store, to measure the level of anti-II Collagen Type VI antibody in serum.

3, joint scoring

Mouse arthritis disease and scoring are as follows: 0: without obviously changing; 1: one toe head obvious tumefaction and rubescent; 2: the swelling of slight swelling and the erythema of limbs or two toe heads; There is the erythema of obvious swelling and limbs in 3: three toe heads; 4: swelling and rubescent, even joint stiffness appear in whole joint.

4, pathological study:

The rear solid end (rear solid end and joint part) of mice is fixed with 10% formaldehyde, then within two days, carries out decalcification through 10% EDTA solution soaking, by paraffin embedding, and tissue slice 5 μ m, rear row HE dyeing.According to organizing the change of microcosmic as neutrophilic granulocyte in the infiltration of synovial membrane and periarticular tissue and monocytic infiltration (inflammation), the pannus (pannus) of boundary zone, the damage (loss of proteoglycan, the death of chondrocyte and the destruction of collagen stroma) of osteoclasia and cartilage is marked.Carry out overall scoring according to the parameter in sufficient pawl, knee joint and joint.Be divided into five grades: 0: without changing; 1: the change of minimum degree; 2: slightly change; 3: medium change; 4: obviously change; 5: severe changes.

5, the mensuration of serumanti-typeⅡcollagenantibody

With coated 96 orifice plates of cattle II Collagen Type VI (10 μ g/ml), put 4 ℃ and spend the night.Then use PBST(phosphate tween buffer) to clean three times, every hole adds 200ul sealer (0.01mol/L PBS and 5% skim milk), hatches 2 hours for 37 ℃.Serum is carried out to 1:32000 dilution with sample buffer (0.01M PBST, 5% skim milk), (100 μ l) and at 37 ℃ are hatched 1 hour to join every hole.After cleaning 3 times, in every hole, add 100 μ l horseradish peroxidase sheep anti-mouse antibodies (1:2000), hatch 1 hour for 37 ℃.Clean 3 times again, add tmb substrate solution, place 10min colour developing under room temperature, then every hole adds the sulphuric acid color development stopping of 50ul2M.Under 450nm wavelength, read the OD value in every hole by automatic microplate reader.

6, imaging evaluation

The rear solid end of mice is carried out to x-ray mammography, mark according to destruction of joint and bone erosion degree: 0: not damage; 1: find that there is slight osteoclasia at a place; 2: moderate change, has 2-4 point to destroy at a place; 3: have 2-4 point significantly to destroy in many places; 4: the destruction that joint part is serious; 5: joint part is completely destroyed.

7, micro-CT(micro-CT) assessment

The mice rear solid end fixing with formaldehyde is placed on to the scanning bed enterprising line scanning of micro-CT, has scanned and carried out three-dimensional reconstruction with NRecon software afterwards, then with CT analysis software, image is further processed.

8, statistical method

Adopt SPSS16.0 statistical software processing system, experimental data with

represent, statistical analysis adopts Mann – Whitney U check, and P<0.05 has statistical significance.

Three, result of implementation

The impact of embodiment 1 eleutheroside E on mice joint, body weight and sufficient volume

Experiment mice carries out gavage treatment with eleutheroside E, add up its joint scoring, body weight and rear solid end thickness, result shows: model group mice rear solid end has obvious redness and joint stiffness, and the mice lesion degree of eleutheroside E and TG treatment group obviously reduces (Figure 1A).Compared with model group, 15mg/kg TG treatment group, 30mg/kg and the scoring of 60mg/kg eleutheroside E treatment group small mouse joint and the degree losing weight obviously reduce (P<0.01).The mice joint scoring of 15mg/kg eleutheroside E treatment group also reduces compared with model group with the degree losing weight, but not remarkable (P>0.05) of significant difference is shown in Figure 1B and 1C.The thickness of 30mg/kg and 60mg/kg eleutheroside E treatment group small mouse rear solid end obviously declines after exempting from one for 42 days to 56 days, but 15mg/kg eleutheroside E treatment group does not demonstrate obvious therapeutic effect, sees Fig. 1 D.

Embodiment 2 eleutheroside Es are treated the impact on joint bone destruction

The rear solid end of mice is carried out to molybdenum target X-ray plain film and micro-CT inspection, molybdenum palladium X-ray film display model group mice rear solid end joint is obviously destroyed, the hypertrophy of displacement and irregular bone, and eleutheroside E (30mg/kg, 60mg/kg) and TG(15mg/kg) the bone destruction degree for the treatment of group small mouse obviously lower, see Fig. 2 A.Correspondingly eleutheroside E (30mg/kg, 60mg/kg) and TG(15mg/kg) the also obviously reduction (P<0.01) of score of the bone destruction degree for the treatment of group small mouse, be shown in Fig. 2 C.And the mice of 15mg/kg eleutheroside E treatment group is compared with model group, do not show obvious improvement (P>0.05).The rear solid end of mice is scanned with micro-CT, find that equally model group mice has obvious destruction of joint, eleutheroside E (30mg/kg, 60mg/kg) and TG(15mg/kg) treatment group small mouse joint and osteoclasia degree obviously reduce, and see Fig. 2 B.

The impact of embodiment 3 eleutheroside Es on synovial tissue of joint

When experiment finishes, get mice rear solid end and ankle joint, through the parallel HE dyeing of paraffin section, under light microscopic, observed result shows: in the mice synovial membrane of CIA model group, have obvious cell infiltration, pannus formation, cartilage destruction and bone erosion, see Fig. 3 A.Eleutheroside E (30mg/kg, 60mg/kg) and TG(15mg/kg) treatment group small mouse cell infiltration degree alleviate, pannus quantity obviously reduces, also significantly suppressed (P<0.001) of the degree of cartilage destruction and bone erosion, and eleutheroside E 15mg/kg treatment group is compared with model group, changes also not obvious (P>0.05) and see Fig. 3 B.

The impact of embodiment 4 eleutheroside E antagonism II Collagen Type VI antibody horizontals

Get a serum of exempting from mice after 42 days and carry out the detection of anti-II Collagen Type VI antibody horizontal, interpretation of result is found: in model group mice serum, anti-II Collagen Type VI antibody horizontal is very high, and in eleutheroside E (30mg/kg, 60mg/kg) and TG(15mg/kg) treatment group, anti-II Collagen Type VI antibody horizontal obviously reduces (P<0.01).See Fig. 4.

Result of implementation brief summary

The continuous gavage of 1.CIA mice gave eleutheroside E treatment after 35 days, and eleutheroside E (30mg/kg and 60mg/kg) can reduce the order of severity and the weight loss degree of CIA mice rear solid end swelling significantly.

2. molybdenum target X-ray plain film and micro-CT check and show, successive administration is after 35 days, and eleutheroside E (30mg/kg and 60mg/kg) can improve the degree of destruction of joint significantly.

3. histopathology observed result shows, after eleutheroside E treatment, the erosion of mice joint part cell infiltration, pannus formation, cartilage destruction and bone obviously alleviates.

4. after eleutheroside E treatment, in model Mice Body, anti-II Collagen Type VI antibody horizontal obviously reduces.

In sum, eleutheroside E corrodes and has certain preventive and therapeutic effect cell infiltration, bone destruction, articular cartilage in CIA mouse arthritis.

The present invention can further illustrate by experimental example below.

Prepare the embodiment 1 of medicament:

Get eleutheroside E monomeric compound 20g, add medical starch 280g, the two fully mixes, and makes 1000 capsules, and every heavy 0.3g, containing eleutheroside E 20mg.

Prepare the embodiment 2 of medicament:

Get eleutheroside E monomeric compound 100g, add medical starch 200g, the two fully mixes, and makes 1000, and every heavy 0.3g, containing eleutheroside E 100mg.

Prepare the embodiment 3 of medicament:

Get eleutheroside E monomeric compound 20g, add 1,2-PD 100m1, fully dissolve, add sterilized water for injection and be diluted to 1000m1, after mixing, divide and install in 1000 ampullas, every lml, containing eleutheroside E 20mg.

More than show and described ultimate principle of the present invention, principal character and advantage of the present invention.The technical staff of the industry should understand; the present invention is not subject to the restriction of above-mentioned example; that in above-mentioned example and description, describes just illustrates principle of the present invention; the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and these changes and improvements all fall in the claimed scope of the invention.The claimed scope of the present invention is defined by appending claims and equivalent thereof.

Claims (7)

1. the application of eleutheroside E in preparation treatment medicine for treating rheumatoid arthritis compositions.

2. purposes according to claim 1, is characterized in that, described pharmaceutical composition comprises inertia, the excipient that nontoxic, pharmacology is suitable.

3. purposes according to claim 1, is characterized in that, described pharmaceutical composition is tablet, capsule, electuary, drop, lyophilized products, granule, ointment or injection.

4. a treatment medicine for treating rheumatoid arthritis compositions, is characterized in that, said composition contains the eleutheroside E active component for the treatment of effective dose.

5. pharmaceutical composition according to claim 4, is characterized in that, it also comprises one or more inertia, the excipient that nontoxic, pharmacology is suitable.

6. pharmaceutical composition according to claim 5, is characterized in that, described excipient is carrier, solvent, emulsifying agent, dispersant, wetting agent, binding agent, stabilizing agent, coloring agent, spice.

7. pharmaceutical composition according to claim 4, is characterized in that, described pharmaceutical composition is tablet, capsule, electuary, drop, lyophilized products, granule, ointment or injection.

Images