CN103405487A - Chinese medicine compound effective part with effect of treating arthritis - Google Patents
Chinese medicine compound effective part with effect of treating arthritis Download PDFInfo
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Abstract
The invention discloses a Chinese medicine compound effective part with an effect of treating arthritis. The effective part is prepared via the steps as follows: 10-50 parts of frankincense and 10-50 parts of myrrh are abstracted via water or alcohol, impurities are removed via alcohol deposition, and the frankincense and the myrrh are placed on a macropore adsorption resin column, and eluted and purified with alcohol gradient with optimized concentrate. The effective part is refined and prepared via modern analytical apparatuses and pharmacological experiments section and according to Chinese medicine theories, the pathogenesis of rheumatic and rheumatoid arthritis. Shown by the experiment result, the Chinese medicine compound effective part provided by the invention has the good effects of antiphlogosis, pain easing, immunization regulation and the like, can be used for treating rheumatic and rheumatoid arthritis, proliferative arthritis, and inflammation pain diseases, is clear in ingredient, low in clinical dosage and lower in adverse reaction, and possibly can be developed as a new generation of medicine for treating the arthritis.
Description
Technical field:
The present invention relates to a kind of Chinese medicine compound effective site, be specifically related to a kind of effective ingredient in Chinese and application in preparation treatment medicine for treating rheumatoid arthritis thereof that can prevent and treat rheumatoid arthritis.
Background technology
Rheumatoid arthritis (RheumatoidArthritis, RA) is a kind of chronic systemic autoimmune disease of articular synovitis as feature of take.Synovitis is outbreak repeatedly lastingly, the destruction that can cause intraarticular cartilage and bone, and function of joint generation obstacle, even maimed.The vasculitis pathological changes is involved each organ of whole body, therefore primary disease is called again rheumatoid disease.Its cause of disease is not yet clear, and the pathological changes outside chronic, symmetry, many synovial jointss inflammation and joint of take is main clinical manifestation, belongs to the autoimmune inflammatory diseases.This disease is mainly in the little joints such as hands, wrist, foot, outbreak repeatedly, and be symmetric.Joint red and swollen heat pain and dysfunction are arranged in early days, and late period, stiff deformity in various degree can appear in joint, and, with the atrophy of bone and bones flesh, very easily disabled.From the angle of pathological change, rheumatoid arthritis is a kind of synovium of joint (later can feed through to articular cartilage, osseous tissue, articular ligament and tendon) that mainly involves, and is secondly the popularity inflammation disease of the connective tissues such as serous coat, the heart, lung and eye.The general performance of rheumatoid arthritis, except arthropathy, also has heating, fatigue and weak, pericarditis, subcutaneous nodule, pleuritis, arteritis, peripheral neuropathy etc.
The rheumatoid arthritis of broad sense, except the inflammatory disorders of joint part, also comprises the popularity pathological changes of whole body.This disease is more common in female middle-aged, and the female patient sickness rate is about 2/3 times of male, and onset peak is 45-65 year, and the prevalence of developed country is about 1% in the adult population, and China's sickness rate is about 0.37%.Although up to the present this disease still can not be effected a radical cure, early discovery rational therapy, the state of an illness can be controlled preferably and be alleviated.
Rheumatoid arthritis (RA) belongs to " arthromyodynia " category in Chinese medicine, be called again " general arthralgia ", " severe and migratory arthralgia ", " insensitive impediment ", " rheumatism involving the bone " etc.As " Yu Ji Wei Yi arthromyodynia door ", say: " numbness, the wet gas of sense wind and cold the moon is subjected to it, and for the multiple pain of disease is heavy, patient's difficulty that is easy to get is gone." opinion numbness head see " Bian Que's heart book numbness disease " say " the wind and cold dampness closes and is numbness, migratory arthralgia caused by wind pain, or the contracture of arm waist foot knee joint, two elbows lead urgency, are that the cold-evil gathers between boundary between muscles also.Numbness person, the solidifying numbness of QI and blood and not all right, be detained outside the five internal organs, and and be disease "; " Prescriptions for Succouring the Sick numbness " says " all because of body void, space between skin and muscles is empty, and the dampness of having a cold forms numbness also ".
Western medicine RA disease ubiquity price is high, toxic and side effects is large, and the drug-induced disease of supervening makes the patient be difficult to reach a desirable treatment platform, and can't effect a radical cure.Yet Chinese medicine RA's is with a long history, penetrating theory and the abundant method of preventing and treating have been accumulated.Adopt the dialectical opinion of Chinese medicine to control, good effect, side effect be little, eliminating evil effects a permanent cure, can strengthen body immunity, more and more demonstrates its advantage in the control of RA and treatment.But current compound Chinese medicinal preparation, exist flavour of a drug to form complicated mostly, and dosage is large, has certain weak points such as untoward reaction.
Summary of the invention
Goal of the invention: technical problem to be solved by this invention is to overcome the deficiencies in the prior art, the refine more of a kind of prescription proportioning is provided, proportioning is scientific and reasonable, effective ingredient is clear and definite, good effect, side effect are little, the Chinese medicine compound effective site with the effect for the treatment of class arthritis of taking convenience, and another object of the present invention is to provide its application in preparing medicament for treating arthritis.
Technical scheme: in order to realize above purpose, the technical scheme that the present invention takes is:
A kind of Chinese medicine compound effective site with treatment of arthritis effect, it is prepared by following method:
(1) get Olibanum 10-50 part, Myrrha 10-50 part, the water or the alcohol extraction that add total medical material weight 5-10 doubly to measure, merge extractive liquid, obtains concentrated solution at 20-70 ℃ of concentrating under reduced pressure;
(2) get the concentrated solution that step (1) obtains and add ethanol, make the concentration of ethanol reach 60-90%, place, precipitation, separate to obtain supernatant, concentrated, obtains concentrate;
(3) get macroporous adsorptive resins on the concentrate that step (2) obtains, ethanol gradient elution by concentration 60% to 80%, collection concentration is 60% to 80% ethanol elution, reclaim solvent, obtain the boswellic acids derivatives, Myrrha sesquiterpenoids and the diterpene-kind compound that in Chinese medicine compound effective site, include percentage by weight 20-90%.
As preferred version, Olibanum 35-45 part, Myrrha 25-35 part in above-described Chinese medicine compound effective site with treatment of arthritis effect, step (1).
The present invention is on the basis to treating at all times rheumatic, rheumatoid arthritis and pathogenesis analysis thereof, under the guidance of Chinese medical theory, determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs, in conjunction with its pathogeny and disease symptom, carry out the refine prescription, determine with Olibanum invigorate blood circulation, the loose blood of Myrrha carries out prescription, to declare logical internal organs, circulation meridians.Wherein Olibanum is arduous warm in nature, and gas perfume (or spice) is altered, and partially enters edema caused by disorder of QI and is good at regulating QI, and pain relieving power is strong.Myrrha is bitter flat, and gas is thin enters blood system partially, and is longer than dissipating blood stasis, and the broken power of rushing down is large.Two medicines share, and QI and blood is also controlled, and brings out the best in each other, and merit potentiation is grand.The effect that has promoting blood circulation to remove blood stasis, reducing swelling and alleviating pain after two flavour of a drug compatibilities, the clinical all pains of heart stomach flank limbs joint that are mainly used to, evident in efficacy.
And the present invention is according to the physicochemical property characteristics of prescription flavour of a drug, extract to compound recipe removes crudely and store essence by modern separation and purification means, according to Modern Analytical Instrument and pharmacological screening result, show, the effective site active component of gained is clearer and more definite, promoting blood circulation to remove blood stasis, reducing swelling and alleviating pain, antiinflammatory action are strong, and untoward reaction is lower.
As preferred version, in above-described Chinese medicine compound effective site with treatment of arthritis effect, step (1), extracting solvent alcohol is the ethanol of concentration 10-80%, and extracting method is reflux, extract, or soxhlet extraction, and extraction time is 1-3 time.
As preferred version, above-described Chinese medicine compound effective site with treatment of arthritis effect, the macroporous adsorbent resin described in step (3) is D101, D102 or AB-8 type macroporous adsorbent resin.The present invention is by adsorbing the screening with the purification ability to opposed polarity and different model resin, determine that model is D101, absorption with macroporous adsorbent resin and the purification effect of D102 or AB-8 type are better, the present invention has simultaneously carried out preferably the eluting power of 5% to 95% different concentration ethanol, with total eluate yield and boswellic acids, sesquiterpenoids, the content of Diterpenes is as index, determine that concentration is that the eluate total content of 30% to 90% ethanol is higher, boswellic acids, sesquiterpenoids, the Diterpenes active constituent content is high, and impurity is few, therefore the ethanol of determining concentration 30% to 90% is the gradient elution solvent, as further preferred version, the ethanol elution of concentration 60% to 80%, the boswellic acids obtained, sesquiterpenoids, the Diterpenes active constituent content is the highest, activity is the strongest.Therefore, the present invention collects the ethanol elution of concentration 60% to 80%.
As preferred version, above-described Chinese medicine compound effective site with treatment of arthritis effect, through HPLC/HPLC-MS, analyze, the boswellic acids derivatives that includes respectively percentage by weight 10%-60% in the Chinese medicine compound effective site that step (3) obtains, the sesquiterpenoids of 1%-30%, the diterpene-kind compound of 1%-40%.
As preferred version, step of the present invention adds ethanol in (2), make the concentration of ethanol reach 75-85%, place precipitation, can precipitate and remove a large amount of large macromolecular compounds of polarity in extracting solution, as polysaccharide, resin, protein etc., be conducive to follow-up separation purifying technique, improve purification efficiency.
The application of Chinese medicine compound effective site in preparing medicament for treating arthritis with treatment of arthritis effect of the present invention.
As preferred version, arthritis of the present invention is rheumatic arthritis, rheumatoid arthritis or proliferative arthritis.
The application of Chinese medicine compound effective site in preparing medicament for treating arthritis with treatment of arthritis effect of the present invention, make Chinese medicine compound effective site and pharmaceutically acceptable carrier the medicine of soft capsule, capsule, granule, spray, injection, microcapsule, tablet, ointment or skin-permeable and control-released plaster dosage form.
Chinese medicine compound effective site handle when making tablet with treatment of arthritis effect provided by the invention has Chinese medicine compound effective site and carrier lactose or the corn starch for the treatment of of arthritis effect, while needing, add magnesium stearate lubricant, mix homogeneously, then tabletting is made tablet.
When the Chinese medicine compound effective site with treatment of arthritis effect provided by the invention was made capsule, handle had Chinese medicine compound effective site and carrier lactose or the corn starch mix homogeneously for the treatment of of arthritis effect, granulate, the then encapsulated capsule of making.
During the Chinese medicine compound effective site granulation agent with treatment of arthritis effect provided by the invention, the Chinese medicine compound effective site with treatment of arthritis effect and diluent lactose or corn starch mix homogeneously, granulate, drying, granulation agent.
Chinese medicine compound effective site with treatment of arthritis effect provided by the invention, when being prepared into other dosage form, prepares according to Chinese Pharmacopoeia described method of version in 2005.
Beneficial effect: compared to the prior art the Chinese medicine compound effective site with treatment of arthritis effect provided by the invention have the following advantages:
Chinese medicine compound effective site with treatment of arthritis effect provided by the invention, according to the pathogenesis of Chinese medical theory and rheumatoid arthritis, choose raw material refine prescription, two component proportionings are scientific and reasonable, and according to the character characteristics of prescription flavour of a drug chemical composition, prescription is extracted, and extract is removed crudely and store essence by modern separation and purification means, according to Modern Methods and pharmacology tracking the selection result, show, the effective site active component of gained of the present invention is clearer and more definite, has good promoting blood circulation to remove blood stasis, the effect of promoting the circulation of QI to relieve pain, consumption is few, and rapid-action, treating both the principal and secondary aspects of a disease, be difficult for recurrence, life-time service has no adverse reaction, and the Chinese medicine compound with treatment of arthritis effect provided by the invention can be prepared into the medicine of various dosage forms easily with pharmaceutically acceptable carrier, facilitate clinical taking.
The specific embodiment
According to following embodiment, the present invention may be better understood.Yet, those skilled in the art will readily understand, the described concrete material proportion of embodiment, process conditions and result thereof be only be used to the present invention is described, and should also can not limit the present invention described in detail in claims.
Embodiment 1
A kind of method with Chinese medicine compound effective site for the treatment of of arthritis effect, its preparation method is:
(1) taking in proportion 10 parts of Olibanums, 10 parts of Myrrhas, is 40 orders through being crushed to particle diameter, mixes, by water boiling and extraction 2 times, add up for the first time 10 times of water gagings of medical material and decoct 1.5h, add up for the second time 8 times of water gagings of medical material and decoct 1h, merge fried liquid twice, be concentrated into 1g crude drug/ml, obtain concentrated solution;
(2) by concentration, be that concentrated solution to the concentration of alcohol that 95% ethanol pacing rapid (1) obtains is 80%, place 12h, filtering-depositing, get supernatant, is concentrated into 5kg, obtains concentrate;
(3) get D101 macroporous adsorptive resins on the concentrate that step (2) obtains, after standing 12h, with the ethanol that pure water, concentration are 10%-95%, carry out gradient elution respectively, collect concentration 60%-80% alcohol elution, concentrate, must have the Chinese medicine compound effective site for the treatment of of arthritis.
Through HPLC/HPLC--MS, analyze, in above Chinese medicine compound effective site, contain percentage by weight and be respectively 20% boswellic acids derivatives; 15% sesquiterpenoids; 30% diterpene-kind compound.
Get the above effective site 10g prepared, add medical starch 16g, mix homogeneously, add water and make soft material, cross 20 mesh sieves and granulate, drying, granulation, incapsulate, every 0.2g, or get effective site 10g, corn starch 20g, add magnesium stearate lubricant 5g, mix homogeneously, then tabletting is made tablet.
Embodiment 2
A kind of method with Chinese medicine compound effective site for the treatment of of arthritis effect, its preparation method is:
(1) get 35 parts of Olibanums, 25 parts of Myrrhas, mix, through being crushed to particle diameter, be 40 orders, by concentration, be 60% alcohol reflux 3 times, add for the first time 8 times of amount 60% alcohol reflux 2h, add for the second time 6 times of amount 60% alcohol reflux 2h, add for the third time 6 times of amount 60% alcohol reflux 1.5h, merge extractive liquid,, obtain concentrated solution 5kg at 50-70 ℃ of concentrating under reduced pressure;
(2) get the concentrated solution that step (1) obtains and add ethanol, make the concentration of ethanol reach 90%, place precipitation, separation of supernatant, concentrated, obtain concentrate;
(3) get D102 type macroporous adsorptive resins on the concentrate that step (2) obtains, ethanol gradient elution by concentration 10% to 95%, collect the 60%-80% alcohol elution, reclaim solvent, must have the Chinese medicine compound effective site for the treatment of rheumatoid arthritis.
Through HPLC/HPLC--MS, analyze, in Chinese medicine compound effective site, contain percentage by weight and be respectively 30% boswellic acids; 5% sesquiterpenoids; 10% diterpene-kind compound.
Get the above effective site 10g prepared, add medical starch 16g, mix homogeneously, add water and make soft material, cross 20 mesh sieves and granulate, drying, granulation, incapsulate, every 0.2g, or get effective site 10g, corn starch 20g, add magnesium stearate lubricant 5g, mix homogeneously, then tabletting is made tablet.
Embodiment 3
A kind of method with Chinese medicine compound effective site for the treatment of of arthritis effect, its preparation method is:
(1) getting 50 parts of Olibanums, 50 parts of Myrrhas, mix, is 40 orders through being crushed to particle diameter, by concentration, be 80% alcohol reflux 2 times, add for the first time 80 times of amount 80% alcohol reflux 2h, add for the second time 8 times of amount 80% alcohol reflux 1.5h, merge extractive liquid,, obtain concentrated solution 7kg at 50-70 ℃ of concentrating under reduced pressure;
(2) get the concentrated solution that step (1) obtains and add ethanol, make the concentration of ethanol reach 90%, place precipitation, separation of supernatant, concentrated, obtain concentrate;
(3) get AB-8 type macroporous adsorptive resins on the concentrate that step (2) obtains, after standing 12h, with the ethanol of pure water, 10%-95%, carry out gradient elution respectively, collect the 60%-80% alcohol elution, reclaim solvent, must have the Chinese medicine compound effective site for the treatment of of arthritis.
Through HPLC/HPLC--MS, analyze, in Chinese medicine compound effective site, contain percentage by weight and be respectively 50% boswellic acids; 8% sesquiterpenoids; 15% diterpene-kind compound.
Get the above effective site 15g prepared, add medical starch 30g, mix homogeneously, add water and make soft material, crosses 20 mesh sieves and granulate, drying, granulate, the granulation agent, or get effective site 15g, corn starch 30g, add magnesium stearate lubricant 6g, mix homogeneously, and then tabletting is made tablet.
The impact of embodiment 4 on adjuvant arthritis rats
1. experiment material
Instrument Drict-Q5 ultrapure water machine (Millipore); Rotary evaporator (Nanjing Nintaus instruments used for education company limited); Electronic balance (SartorisBP211D); Microscope (model OLMPUSCKX31); Microplate reader (model 680, BIO-RAD); Microplate reader (DENLEYDRAGONWellscanMK3, Thermo); Wash plate machine (Wellwash4MK2, Thermo); Electronic balance (model JA2003N, Shanghai Precision Scientific Apparatus Co., Ltd); Centrifuge (AnkeLXJ-IIB); Constant water bath box (HH-S Jintan City Medical Instruments factory); Eddy mixer (XW-80A, the western instrument plant in Qingpu Shanghai Shanghai).
Reagent and sample F reund ' s Freund's complete adjuvant (100M8717CAS9007-81-2, sigma); Polyoxyethylene sorbitan monoleate (Shen, Shanghai space medication chemistry company limited, 95-18661); Dehydrated alcohol (the inferior chemical industry company limited of containing of Wuxi City); Rat IL-2ELISA test kit, P of Rats GE
2ELISA test kit, rat TNF α ELISA test kit (all purchased from Shanghai western Tang bio tech ltd); Indomethacin (Shanghai Xin Pasi pharmaceutical Co. Ltd, lot number: H31020148); Pentobarbital sodium (German packing, lot number: WS20080810); Sodium citrate (Nanjing Chemistry Reagent Co., Ltd., lot number: 050580052); Ultra-pure water is self-control; Sample is self-control.
The clean level of animal SD rat (Shanghai Slac Experimental Animal Co., Ltd.).The raising condition: clean level Animal Lab., room temperature 23-25 ℃, relative humidity 40%-70%, every day, suitable ventilation was 15 times.
2. experimental technique
2.1 the foundation of rat adjuvant type arthritis model
72 of SD rats, male and female half and half, body weight, at 180-220g, are surveyed the left and right metapedes sole of the foot of each Mus volume with homemade simple and easy sufficient sole of the foot measuring device, separate at random 8 as the blank group by weight, and all the other 64 are carried out modeling by literature method.Conventional method carries out disinfection to SD Rat Right metapedes sole of the foot bottom skin, and 0.1mL/ only causes inflammation with microsyringe intradermal injection Freund ' s Freund's complete adjuvant.After modeling the 13rd day, measure every sufficient sole of the foot volume in rat left and right, relatively there were significant differences with blank group for model group foot sole of the foot volume, and observe and find the red and swollen obviously prompting of sufficient sole of the foot modeling success.
2.2 experiment grouping
Experiment is divided into dosage group (FM) in Chinese medicine compound effective site high dose group (FH) that blank group, model group, positive controls (Indomethacin, IMT), the embodiment of the present invention 1 prepare, effective site low dose group (FL), effective site at random by rat weight and foot swelling degree.
2.3 the evaluation of the observation of rat general state and swollen joint expansibility
Before modeling, reach administration fore-and-aft observing rat chaeta color and luster and activity situation, the increase variation of appetite and redness and swelling of joints situation, and before modeling, after modeling after 13d, modeling after 20d, modeling after 24d, modeling after 27d, modeling 30d measure the body weight (g) of rat and the volume (mL) of the left and right sole of the foot.
Body weight: use the electronic scale weighing rat body weight; Foot sole of the foot volume: survey the volume of respectively organizing the rear sole of the foot in rat left and right with the sufficient sole of the foot measuring device of self-control, relatively respectively organize the variation of rat paw edema degree after administration.
2.4 dosage and administering mode
13d after modeling, after dividing into groups and measuring the left and right sufficient sole of the foot volume of rat and body weight, start gastric infusion.Indomethacin positive controls group (10mg/Kgd), Chinese medicine compound effective site high dose group (FH) (90.48mg/Kgd), effective site low dose group (FL) (56.12mg/Kgd), (76.92mg/Kgd) gavage 2mL of dosage group (FM) in effective site, Normal group and model group give the Tween-80 of equivalent 2/1000ths, and administration is 17 days altogether.
2.5 the sampling and processing of rat plasma, tissue samples
With 10% chloral hydrate intraperitoneal injection of anesthesia rat, dorsal position is fixed, carotid artery is got blood, with 3.8% sodium citrate (blood and anticoagulant volume ratio are 9:1) anticoagulant, collect whole blood in the centrifuge tube of 10mL, with low speed centrifuge with the centrifugal 10min of 3000r/min rotating speed, draw supernatant ,-20 ℃ of lower stored frozen, detect NO, MDA, PGE in blood plasma
2, IL-2, TNF
α.Cut simultaneously the peeling of the right back foot swelling foot sole of the foot, pick a bone, soak (tissue and normal saline volume ratio are 1:9) with normal saline, the centrifugal 10min of 3000r/min, draw supernatant, and-20 ℃ of freezing preservations detect NO, MDA, PGE in tissue fluid
2, IL-2, TNF
α.
2.6 the assay method of BCA protein concentration in rat tissue
The BCA determination of protein concentration test kit of the western Tang in Shanghai biological production is adopted in experiment, and assay method is as follows:
1) quantity per sample, add 1 volume BCA reagent B(50:1 by 50 volume BCA reagent A) the appropriate BCA working solution of preparation, fully mix.BCA working solution room temperature was stablized in 24 hours.
2) complete soluble protein standard substance, get 25 μ L and be diluted to 100 μ L with 0.9%NaCl or PBS, and making final concentration is 0.5mg/mL.
3) standard substance are added in the standard substance hole of 96 orifice plates by 0,1,2,4,8,12,16,20 μ L, add the standard substance diluent and supply 20 μ L.
4) add the proper volume sample aerial to the sample of 96 orifice plates, add the standard substance diluent to 20 microlitres.
5) each hole adds 200 μ LBCA working solutions, places 30 minutes for 37 ℃.
6) measuring wavelength is 562nm, and the wavelength between 540-595nm also can be accepted.According to standard curve, calculate protein concentration.
2.7NO assay method
Press the operation of nitric oxide (Nitricoxide, NO) test kit (enzyme process) description, measure the burst size of NO.The NO chemical property is active, and internal metabolism transfers NO2-and NO3-very soon to, and NO2-is further converted to NO3-, and this law utilizes the nitrate reductase enzyme spcificity that NO3-is reduced to NO2-, by the colour developing depth, measures the height of its concentration.
Get above-mentioned gained blood plasma, organize supernatant, each sample is respectively got eight pipes, and every pipe 100 μ L, as measuring pipe, get 100 μ L100 μ mol/LKNO
3As standard pipe totally eight pipes, the distilled water of getting 100 μ L is as blank tube totally eight pipes, and every pipe adds 400 μ L mix reagents (nitrate reductase), mix gently, 37 ℃ of water-baths are after 1 hour, and each manages reagent adding 3 successively, reagent 4, fully vortex mixes 30 seconds, standing 40 minutes of room temperature, the centrifuging and taking supernatant, add developer reaction 60min, 540nm surveys and respectively manages the OD value, is calculated as follows:
(1) computing formula of serum NO content:
(2) computing formula of NO content in tissue:
2.8 the assay method of malonaldehyde (MDA)
The operational approach of test kit description is measured in experiment according to malonaldehyde (MDA), measure the burst size of MDA.MDA test kit test philosophy: the malonaldehyde in the lipid peroxide catabolite (MDA) can with thiobarbituricacidα-(TBA) condensation, form red product, at the 532nm place, maximum absorption band is arranged.
Press test kit explanation configuration reagent, get above-mentioned gained serum, organize supernatant, each specimen is got eight pipes, every pipe 50 μ L, blank tube and standard pipe are respectively got eight pipes, every pipe 50 μ L, every pipe adds respectively mix reagent 1000 μ L, with the vortex vortex mixer, mix, the test tube mouth is tightened with preservative film, with syringe needle thorn one aperture, 95 ℃ of water-baths (or uncap and boil with pot) 40 minutes, after taking out, flowing water is cooling, 3500-4000r/min then, centrifugal 10 minutes.Get supernatant 200 μ L in 96 orifice plates, the 532nm place surveys the absorbance of each pipe, calculates by following formula:
(1) computing formula of MDA content in serum:
(2) computing formula of NO content in tissue:
2.9 the assay method of interleukin-2 (IL-2), PGE2, TNF α
Double antibodies sandwich ABC-ELISA method is adopted in experiment, with Chinese People's Anti-Japanese Military and Political College's Mus IL-2 monoclonal antibody, be coated on ELISA Plate, IL-2 in standard substance and sample is combined with monoclonal antibody, adds biotinylation to obtain the Mus IL-2 of the Chinese People's Anti-Japanese Military and Political College, forms immune complex and connects onboard, the streptavidin of horseradish peroxidase-labeled is combined with biotin, add the substrate working solution aobvious blue, finally add stop buffer sulphuric acid, at the 450nm place, survey the OD value, IL-2 concentration is directly proportional to the OD value, by the drawing standard curve, obtains the concentration of IL-2 in specimen.PGE
2, TNF
αThe detection principle of content is similar to the detection principle of IL-2.
Requirement configuration standard product, 10 * sample diluent, cleaning mixture by test kit.Take out 96 hole ELISA Plate, every hole adds standard substance and testing sample 100 μ L successively, Sptting plate is fully mixed to rearmounted 37 ℃ and hatched 120 minutes.With cleaning mixture, Sptting plate is fully washed 4-6 time, to print on filter paper dry after, in every hole, add first antibody working solution 100 μ L, Sptting plate is fully mixed to latter 37 ℃ hatched 60 minutes, wash plate, print dry rear every hole and add substrate working solution 100 μ L, Sptting plate is put to dark place reaction 15 minutes.Every hole adds the stop buffer of 100 μ L to mix again, in 30 minutes, at the 450nm place, surveys light absorption value by microplate reader.
All OD values are gone and are calculated again after all should reducing blank value.Take standard substance 2000,1000,500,250,125,62.5,31.2,0pg/mL is abscissa, and the OD value is vertical coordinate, maps on graph paper, draws standard curve.The OD value is found corresponding IL-2, PGE on this curve chart per sample
2, TNF
αContent.
3 experimental results
3.1 cause after inflammation the observation and comparison of respectively organizing the rat ordinary circumstance
3.1.1 respectively organize the variation of rat general state after causing inflammation
The rats in normal control group food-intake is many, and the mental status is good, glossy by hair, flexibly movable, occurs without abnormal state, and along with the prolongation of feeding time, the bodily form is gradually long, and body weight increases obviously.The model group rat, female appetite reduces more obvious, loses weight obviously, listless, and hair is withered lackluster, and activity reduces, later stage heavy burden difficulty, obstacle appears in joint motion; Male rat appetite has minimizing but is not obvious, loses weight also not obvious, listless, and the few gloss of hair, activity also reduces, but it is obvious to be not so good as female rats.The high, medium and low dosage group of Chinese medicine compound effective site provided by the invention Hair of Rats, the mental status, body weight and joint movement disorder all take an evident turn for the better than model group.
3.1.2 cause after inflammation the variation of respectively organizing rat different time body weight
Before administration, respectively organize rat body weight and model group weight ratio, normal group body weight and model group body weight have significant difference (P≤0.05), all the other each groups and the equal not statistically significant of model group comparing difference (P>0.05).
3.2 cause after inflammation the impact of respectively organizing the right pedal swelling degree of rat different time
Cause after inflammation the impact of respectively organizing the right pedal swelling degree of rat different time, table 1 is as can be known, before modeling, normal group, administration group Rat Right foot sole of the foot volume all with model group there was no significant difference (P>0.05); 13d after modeling, normal group and model group relatively have utmost point significant difference (P<0.001), all the other each group and model group relatively there are no significant difference (P>0.05); 20d after modeling, normal group, IMT group, medicine group and model group relatively have utmost point significant difference (P<0.001), high, the middle dosage group of Chinese medicine compound effective site and model group relatively have significant differences (P<0.01), and low dose group and model group relatively have significant difference (P<0.05); 24d after modeling, high, the middle dosage group of Chinese medicine compound effective site and model group relatively have significant differences (P<0.01), and all the other each groups more all have significant difference (P<0.01) with model group; After modeling, 27d, 30d respectively organize Rat Right foot sole of the foot volume and the more even utmost point significant difference of model group (P<0.01), and Chinese medicine compound effective site provided by the invention has demonstrated good detumescence effect.
The variation of table 1 Rat Right pedal swelling degree
Annotate: with model group, compare, * P<0.05 means to have significant difference, and * * P<0.01 means to have significant differences, and * * * P<0.001 means to have utmost point significant difference.
3.3 the impact on NO, MDA, IL-2, PGE2, TNF α in each group rat plasma
As shown in Table 2, the high, medium and low dosage group of Chinese medicine compound effective site of the present invention can significantly suppress NO level in rat plasma, with model group, utmost point significant difference (P<0.001) is arranged relatively; In normal rats blood plasma, MDA content and model group relatively have highly significant difference (P<0.01), and ITM group, the high, medium and low dosage group of Chinese medicine compound effective site and model group relatively have highly significant difference (P<0.01); IL-2, PGE in IMT group, the high, medium and low dosage group of Chinese medicine compound effective site rat plasma
2, TNF
αLevel, more all have utmost point significant difference (P<0.001) with model group.Result shows that the Chinese medicine compound effective site that the present invention prepares has NO, MAD, IL-2, PGE in significant inhibition adjuvant arthritis rats blood plasma
2, TNF
αLevel, have good antiphlogistic effects.
The impact of table 2 on NO, MDA, IL-2, PGE2, TNF alpha content in rat plasma
Annotate: with model group, compare, * P<0.05 means to have significant difference, and * * P<0.01 means to have significant differences, and * * * P<0.001 means to have utmost point significant difference.
3.4 the impact on NO, MDA, IL-2, PGE2, TNF α in Rat Right pedal swelling tissue
As shown in Table 3, NO, PGE in IMT group, the high, medium and low dosage group of Chinese medicine compound effective site of the present invention Rat Right pedal swelling tissue
2, TNF
αContent and model group utmost point significant difference (P<0.001) is more all arranged, experimental result shows, Chinese medicine compound effective site provided by the invention can significantly suppress NO, MDA in the pedal swelling tissue, IL-2, PGE2, TNF alpha levels, has demonstrated good antiphlogistic effects.
The impact of table 3 on NO, MDA, IL-2, PGE2, TNF alpha content in Rat Right pedal swelling tissue
(annotate: with model group, compare, * P<0.05 means to have significant difference, and * * P<0.01 means to have significant differences, and * * * P<0.001 means to have utmost point significant difference.）
Embodiment 5 antioxidant activity evaluations
1. experiment material
Instrument 680BIO-RAD microplate reader, JA2003N electronic balance (Shanghai Precision Scientific Apparatus Co., Ltd), Drict-Q5 ultrapure water machine (Millipore), the liquid-transfering gun of all size.
Reagent and sample dehydrated alcohol (the inferior chemical industry company limited of containing of Wuxi City), DPPH(sigma company), vitamin C (Chemical Reagent Co., Ltd., Sinopharm Group), the ultra-pure water self-control, sample is self-control.
2. experimental technique
Accurately take DPPH and be made into 6.5 * 10 with dehydrated alcohol
-4The storing solution of mol/L, the refrigerator 30min that keeps in Dark Place can be used.Vitamin C (positive control) dissolves with distilled water, each effective-site anhydrous alcohol solution of variable concentrations before and after Olibanum-Myrrha compatibility.The effective section of the Chinese medicine compound high concentration (F01) respectively the 100 μ L embodiment of the present invention 1 prepared, middle concentration (F02) and low concentration (F03) are added in 96 orifice plates, then add 100 μ LDPPH solution (final concentration is 80 μ M), rock evenly, lucifuge reaction 30min, measure the OD value at the 517nm place.According to following formula, calculate free radical scavenging activity:
Clearance rate=[1-(A
1-A
2)/A
0] * 100%
In formula, A
0For the absorbance after solvent and DPPH effect; A
1For the absorbance after tested material and DPPH effect; A
2For not adding the absorbance of DPPH.
The calculating of half suppression ratio: half suppression ratio (IC
50) refer to the concentration of required antioxidant when clearance rate is 5O%, according to the clearance rate of variable concentrations antioxidant, obtain as curve.
3. experimental result
Different solubility per sample is dissolved in different solvents by sample, measures according to the method described above the clearance rate of each sample to DPPH, the results are shown in Table 4.As shown in Table 4, the embodiment of the present invention 1 Chinese medicine compound effective site high dose group free radical scavenging is the strongest, IC
50Be 67.11 μ g/mL; In, low dose group IC
50Be respectively 111.55 μ g/mL, 93.53 μ g/mL; Show that effective site prepared by the present invention has stronger antioxidant activity.
Table 4 is removed DPPH free radical half inhibitory action (n=4)
Embodiment 6 mice hot plate induced pain experiments
1. experimental technique
Female ICR mice, 50, body weight 22～26 grams, be divided at random model control group, positive drug group (dolantin), dosage group (FM) in the Chinese medicine compound effective site high dose group (FH) that the embodiment of the present invention 1 prepares, effective site low dose group (FL), effective site, 10 every group.With the hot plate stimulus method, estimate the analgesic effect of Chinese medicine compound effective site.Mice first uses hot plate dolorimeter device (55.5 ℃) to measure respectively the normal pain threshold of every mice.Administration every day 1 time, continuous 2 days, after the last administration 30 minutes, 1 hour, 2 hours, 4 hours, measured threshold of pain time, relatively group difference in 10 hours.
2. experimental result
The Chinese medicine compound effective site group that the embodiment of the present invention 1 prepares stimulates the induced mice threshold of pain time, than the front threshold of pain of the administration time, the prolongation effect is arranged to hot plate after administration, and obvious analgesic activity was arranged still after 10 hours, with the blank group, compare, can significantly improve the mice threshold of pain time.Concrete experimental result such as table 5.
The analgesic activity (n=10) of table 5 Chinese medicine compound effective site to the mice hot-plate
With comparison before administration, * * P<0.01, * P<0.05; With the normal saline group, compare △ △ P<0.01, △ P<0.05.
Experimental result by above table 5 shows, Chinese medicine compound effective site provided by the invention has good analgesic activity, can improve pain threshold, and the persistent period is longer, can bring into play good anti-inflammatory and antalgic effect when the treatment of arthritis disease.
Above embodiment only is explanation technical conceive of the present invention and characteristics; its purpose is to allow the person skilled in the art understand content of the present invention and implemented; can not limit the scope of the invention with this; all equivalences that spirit is done according to the present invention change or modify, and all should be encompassed in protection scope of the present invention.
Claims (8)
1. the Chinese medicine compound effective site with treatment of arthritis effect, is characterized in that, it is prepared by following method:
(1) get Olibanum 10-50 part, Myrrha 10-50 part, the water or the alcohol extraction that add total medical material weight 5-10 doubly to measure, merge extractive liquid, obtains concentrated solution at 20-70 ℃ of concentrating under reduced pressure;
(2) get the concentrated solution that step (1) obtains and add ethanol, make the concentration of ethanol reach 60-90%, place, precipitation, separate to obtain supernatant, concentrated, obtains concentrate;
(3) get macroporous adsorptive resins on the concentrate that step (2) obtains, ethanol gradient elution by concentration 30% to 90%, collection concentration is 60% to 80% ethanol elution, reclaim solvent, obtain the boswellic acids derivatives, Myrrha sesquiterpenoids and the diterpene-kind compound that in Chinese medicine compound effective site, include percentage by weight 20-90%.
2. the Chinese medicine compound effective site with treatment of arthritis effect according to claim 1, is characterized in that, Olibanum 35-45 part, Myrrha 25-35 part in step (1).
3. the Chinese medicine compound effective site with treatment of arthritis effect according to claim 1, is characterized in that, in step (1), extracting solvent alcohol is the ethanol of concentration 10-80%, and extracting method is reflux, extract,, and extraction time is 1-3 time.
4. the Chinese medicine compound effective site with treatment of arthritis effect according to claim 1, is characterized in that, the macroporous adsorbent resin described in step (3) is D101, D102 or AB-8 type macroporous adsorbent resin.
5. the Chinese medicine compound effective site with treatment of arthritis effect according to claim 4, it is characterized in that, the boswellic acids derivatives that includes percentage by weight 10%-60% in the Chinese medicine compound effective site that step (3) obtains, the sesquiterpenoids of 1%-30%, the diterpene-kind compound of 1%-40%.
6. the described application of Chinese medicine compound effective site in preparing medicament for treating arthritis with treatment of arthritis effect of claim 1 to 5 any one.
7. the application of Chinese medicine compound effective site in preparing medicament for treating arthritis with treatment of arthritis effect according to claim 6, is characterized in that, described arthritis is rheumatic arthritis, rheumatoid arthritis or proliferative arthritis.
8. the application of Chinese medicine compound effective site in preparing medicament for treating arthritis with treatment of arthritis effect according to claim 7, its feature is in Chinese medicine compound effective site and pharmaceutically acceptable carrier being made to the medicine of soft capsule, capsule, granule, spray, injection, microcapsule, tablet, ointment or skin-permeable and control-released plaster dosage form.
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| CN108785357A (en) * | 2018-09-18 | 2018-11-13 | 南京中医药大学 | A kind of masticinic acid and myrrh terpene compatible composition and its preparation method and application |
| CN110101768A (en) * | 2019-06-17 | 2019-08-09 | 郭珈宜 | A kind of black plaster formula that treating osteoarthropathy and its preparation process |
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