CN103405487B - 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 part, be specifically related to a kind of energy and prevent and treat the effective ingredient in Chinese of rheumatoid arthritis and the application in preparation treatment medicine for treating rheumatoid arthritis thereof.
Background technology
The chronic generalized autoimmune disease of rheumatoid arthritis (RheumatoidArthritis, RA) to be a kind of with articular synovitis be feature.The lasting recurrent exerbation of synovitis, can cause the destruction of intraarticular cartilage and bone, function of joint generation obstacle, even maimed.Vasculitis pathological changes involves each organ of whole body, therefore primary disease is also called rheumatoid disease.Its cause of disease is not yet clear, for main clinical manifestation, belongs to autoimmune inflammatory disease with pathological changes outside chronic, symmetry, many synovial jointss inflammation and joint.This disease is mainly in the Minor articulus such as hands, wrist, foot, recurrent exerbation, and is symmetric.Having joint red and swollen heat pain and dysfunction in early days, can there is stiff deformity in various degree in advanced joint, and with the atrophy of bone and bones flesh, very easily disables.From the angle of pathological change, rheumatoid arthritis is that one mainly involves synovium of joint (later can feed through to articular cartilage, osseous tissue, articular ligament and tendon), is secondly the popularity inflammation disease of the connective tissues such as serous coat, the heart, lung and eye.The systemic manifestation 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 female patient sickness rate is about 2/3 times of male, and onset peak is 45-65 year, and the prevalence of developed country about 1% in adult population, 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, is also called " general arthralgia ", " severe and migratory arthralgia ", " insensitive impediment ", " rheumatism involving the bone " etc.As " Yu Ji Wei Yi arthromyodynia door " is said: " numbness, the gas that sense wind and cold is wet is then cloudy by it, and for the multiple pain of disease is heavy, patient's difficulty that is easy to get is gone." opinion numbness head sees " Bian Que's heart book numbness sick " and say that " wind and cold dampness combined into numbness, migratory arthralgia caused by wind pain, or the contracture of arm waist foot knee joint, two elbows lead urgency, are that cold-evil to gather between boundary between muscles also.Numbness person, QI and blood coagulates numbness and not all right, is detained outside the five internal organs, and and is disease "; " Prescriptions for Succouring the Sick numbness " is said " all because body is empty, space between skin and muscles is empty, and 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 supervened makes patient be difficult to reach a desirable treatment platform, and cannot effect a radical cure.But Chinese medicine RA's is with a long history, have accumulated penetrating theory and abundant prevention and controls.Adopt Chinese medicine dialectical treatmert, good effect, side effect be little, eliminating evil to effect a permanent cure, can body immunity be strengthened, in control and the treatment of RA, more and more demonstrate its advantage.But current compound Chinese medicinal preparation, mostly there is flavour of a drug composition complicated, dosage is large, there is the weak points such as certain untoward reaction.
Summary of the invention
Goal of the invention: technical problem to be solved by this invention overcomes 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 part with the effect for the treatment of class arthritis of taking convenience, and another object of the present invention is to provide it and is preparing the application in medicament for treating arthritis.
Technical scheme: in order to realize above object, the technical scheme that the present invention takes is:
Have a Chinese medicine compound effective part for treatment of arthritis effect, it is prepared by following method:
(1) get Olibanum 10-50 part, Myrrha 10-50 part, add water or the alcohol extraction of total medical material weight 5-10 times amount, merge extractive liquid, obtains concentrated solution at 20-70 DEG C 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, is separated to obtain supernatant, concentrated, obtains concentrate;
(3) macroporous adsorptive resins on concentrate that step (2) obtains is got, with the ethanol gradient elution of concentration 60% to 80%, collecting concentration is the ethanol elution of 60% to 80%, recycling design, obtains in Chinese medicine compound effective part the boswellic acids derivatives, Myrrha sesquiterpenoids and the diterpene-kind compound that include percentage by weight 20-90%.
Preferably, the above-described Chinese medicine compound effective part with treatment of arthritis effect, Olibanum 35-45 part, Myrrha 25-35 part in step (1).
The present invention to treat at all times rheumatic, rheumatoid arthritis and pathogenesis thereof analyze basis on, under the guidance of Chinese medical theory, determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs, refine prescription is carried out in conjunction with its pathogeny and disease symptom, determine to invigorate blood circulation with Olibanum, the Myrrha blood that falls apart carries out prescription, to declare logical internal organs, circulation meridians.Wherein Olibanum is worked hard 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 hardship is flat, and gas is thin enters blood system partially, and is longer than dissipating blood stasis, and 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.There is after two flavour of a drug compatibilities effect of promoting blood circulation to remove blood stasis, reducing swelling and alleviating pain, be clinically mainly used to all pains of heart stomach flank limbs joint, evident in efficacy.
And the present invention is according to the physicochemical property feature of prescription flavour of a drug, the extract of compound recipe is removed crudely and store essence by modern separation and purification means, show according to Modern Analytical Instrument and pharmacological screening result, 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.
Preferably, the above-described Chinese medicine compound effective part with treatment of arthritis effect, in step (1), Extraction 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.
Preferably, the above-described Chinese medicine compound effective part 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 opposed polarity and different model resin and the screening of purification capacity, determine that model is D101, absorption with macroporous adsorbent resin and the purification effect of D102 or AB-8 type are better, simultaneously the present invention carries out preferably to 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 the ethanol of 30% to 90% is higher, boswellic acids, sesquiterpenoids, Diterpenes active constituent content is high, and impurity is few, therefore determine that the ethanol of concentration 30% to 90% is solvent gradient, as further preferred version, the ethanol elution of concentration 60% to 80%, the boswellic acids obtained, sesquiterpenoids, Diterpenes active constituent content is the highest, activity is the strongest.Therefore, the present invention collects the ethanol elution of concentration 60% to 80%.
Preferably, the above-described Chinese medicine compound effective part with treatment of arthritis effect, analyze through HPLC/HPLC-MS, the boswellic acids derivatives of percentage by weight 10%-60% is included respectively in the Chinese medicine compound effective part that step (3) obtains, the sesquiterpenoids of 1%-30%, the diterpene-kind compound of 1%-40%.
Preferably, step of the present invention adds ethanol in (2), the concentration of ethanol is made to reach 75-85%, place precipitation, can precipitate and remove the macromolecular compound that in extracting solution, a large amount of polarity is large, as polysaccharide, resin, protein etc., be conducive to follow-up separation purifying technique, improve purification efficiency.
The Chinese medicine compound effective part with treatment of arthritis effect of the present invention is preparing the application in medicament for treating arthritis.
Preferably, arthritis of the present invention is rheumatic arthritis, rheumatoid arthritis or proliferative arthritis.
The Chinese medicine compound effective part with treatment of arthritis effect of the present invention is preparing the application in medicament for treating arthritis, Chinese medicine compound effective part and pharmaceutically acceptable carrier is made the medicine of soft capsule, capsule, granule, spray, injection, microcapsule, tablet, ointment or skin-permeable and control-released plaster dosage form.
The Chinese medicine compound effective part with treatment of arthritis effect provided by the invention when making tablet having the Chinese medicine compound effective part for the treatment of of arthritis effect and carrier lactose or corn starch, magnesium stearate lubricant is added when needing, mix homogeneously, then tablet made by tabletting.
There is the Chinese medicine compound effective part for the treatment of of arthritis effect and carrier lactose or corn starch mix homogeneously, granulate when the Chinese medicine compound effective part with treatment of arthritis effect provided by the invention makes capsule, then encapsulatedly make capsule.
When the Chinese medicine compound effective part with treatment of arthritis effect provided by the invention makes granule, there is the Chinese medicine compound effective part for the treatment of of arthritis effect and diluent lactose or corn starch mix homogeneously, granulate, dry, make granule.
The Chinese medicine compound effective part with treatment of arthritis effect provided by the invention, when being prepared into other dosage form, prepares according to method described in China's coastal port.
Beneficial effect: compared to the prior art the Chinese medicine compound effective part with treatment of arthritis effect provided by the invention has the following advantages:
The Chinese medicine compound effective part with treatment of arthritis effect provided by the invention, pathogenesis according to Chinese medical theory and rheumatoid arthritis chooses raw material refine prescription, two component proportionings are scientific and reasonable, and according to the character feature 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, follow the tracks of the selection result according to Modern Methods and pharmacology to show, the effective site active component of gained of the present invention is clearer and more definite, there is good promoting blood circulation to remove blood stasis, the effect of promoting the circulation of QI to relieve pain, consumption is few, and it is rapid-action, treating both the principal and secondary aspects of a disease, not easily recur, 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 form with pharmaceutically acceptable carrier easily, facilitate clinical taking.
Detailed description of the invention
According to following embodiment, the present invention may be better understood.But those skilled in the art will readily understand, concrete material proportion, process conditions and result thereof described by embodiment only for illustration of the present invention, and should can not limit the present invention described in detail in claims yet.
Embodiment 1
Have a method for the Chinese medicine compound effective part for the treatment of of arthritis effect, its preparation method is:
(1) taking Olibanum 10 parts, Myrrha 10 parts in proportion, is 40 orders through being crushed to particle diameter, mixing, by water boiling and extraction 2 times, first time adds up medical material 10 times amount soak by water 1.5h, and second time adds up medical material 8 times amount soak by water 1h, merge twice fried liquid, be concentrated into 1g crude drug/ml, obtain concentrated solution;
(2) be concentrated solution to the concentration of alcohol that 95% ethanol pacing rapid (1) obtains by concentration be 80%, place 12h, filtering-depositing, gets supernatant, is concentrated into 5kg, obtains concentrate;
(3) D101 macroporous adsorptive resins on concentrate that step (2) obtains is got, after leaving standstill 12h, gradient elution is carried out respectively with the ethanol that pure water, concentration are 10%-95%, collect concentration 60%-80% alcohol elution, concentrated, the Chinese medicine compound effective part for the treatment of of arthritis must be had.
Analyze through HPLC/HPLC--MS, in above Chinese medicine compound effective part, be respectively the boswellic acids derivatives of 20% containing percentage by weight; The sesquiterpenoids of 15%; The diterpene-kind compound of 30%.
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, dry, make granule, incapsulate, every 0.2g, or get effective site 10g, corn starch 20g, add magnesium stearate lubricant 5g, mix homogeneously, then tablet made by tabletting.
Embodiment 2
Have a method for the Chinese medicine compound effective part for the treatment of of arthritis effect, its preparation method is:
(1) Olibanum 35 parts, Myrrha 25 parts is got, mixing, be 40 orders through being crushed to particle diameter, be 60% alcohol reflux 3 times by concentration, first time adds 8 times amount 60% alcohol reflux 2h, and second time adds 6 times amount 60% alcohol reflux 2h, and third time adds 6 times amount 60% alcohol reflux 1.5h, merge extractive liquid, obtains concentrated solution 5kg at 50-70 DEG C 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) D102 type macroporous adsorptive resins on concentrate that step (2) obtains is got, with the ethanol gradient elution of concentration 10% to 95%, collect 60%-80% alcohol elution, recycling design, the Chinese medicine compound effective part for the treatment of rheumatoid arthritis must be had.
Analyze through HPLC/HPLC--MS, in Chinese medicine compound effective part, be respectively the boswellic acids of 30% containing percentage by weight; The sesquiterpenoids of 5%; The diterpene-kind compound of 10%.
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, dry, make granule, incapsulate, every 0.2g, or get effective site 10g, corn starch 20g, add magnesium stearate lubricant 5g, mix homogeneously, then tablet made by tabletting.
Embodiment 3
Have a method for the Chinese medicine compound effective part for the treatment of of arthritis effect, its preparation method is:
(1) get Olibanum 50 parts, Myrrha 50 parts, mixing is 40 orders through being crushed to particle diameter, be 80% alcohol reflux 2 times by concentration, first time adds 80 times amount 80% alcohol reflux 2h, and second time adds 8 times amount 80% alcohol reflux 1.5h, merge extractive liquid, obtains concentrated solution 7kg at 50-70 DEG C 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) AB-8 type macroporous adsorptive resins on concentrate that step (2) obtains is got, after leaving standstill 12h, gradient elution is carried out respectively with the ethanol of pure water, 10%-95%, collect 60%-80% alcohol elution, recycling design, must have the Chinese medicine compound effective part for the treatment of of arthritis.
Analyze through HPLC/HPLC--MS, in Chinese medicine compound effective part, be respectively the boswellic acids of 50% containing percentage by weight; The sesquiterpenoids of 8%; The diterpene-kind compound of 15%.
Get the above effective site 15g prepared, add medical starch 30g, mix homogeneously, add water and make soft material, cross 20 mesh sieves and granulate, drying, granulate, makes granule, or gets effective site 15g, corn starch 30g, add magnesium stearate lubricant 6g, mix homogeneously, then tablet made by tabletting.
Embodiment 4 is on the impact of adjuvant arthritis rats
1. experiment material
Instrument Drict-Q5 ultrapure water machine (Millipore); Rotary evaporator (instruments used for education company limited of Nanjing Nintaus); Electronic balance (SartorisBP211D); Microscope (model OLMPUSCKX31); Microplate reader (model 680, BIO-RAD); Microplate reader (DENLEYDRAGONWellscanMK3, Thermo); Wash trigger (Wellwash4MK2, Thermo); Electronic balance (model JA2003N, Shanghai Precision Scientific Apparatus Co., Ltd); Centrifuge (AnkeLXJ-IIB); Constant water bath box (Medical Instruments factory of HH-S Jintan City); Eddy mixer (XW-80A, Qingpu Shanghai Hu Xi instrument plant).
Reagent and sample F reund ' s Freund's complete adjuvant (100M8717CAS9007-81-2, sigma); Polyoxyethylene sorbitan monoleate (Shanghai Shen Yu medication chemistry company limited, 95-18661); Dehydrated alcohol (Wuxi City Ya Sheng Chemical Co., Ltd.); Rat IL-2ELISA test kit, P of Rats GE
2eLISA kit, rat TNF α ELISA kit (all purchased from Xi Tang bio tech ltd, Shanghai); Indomethacin (Shanghai Xin Pasi pharmaceutical Co. Ltd, lot number: H31020148); Pentobarbital sodium (German subpackage, lot number: WS20080810); Sodium citrate (Nanjing Chemistry Reagent Co., Ltd., lot number: 050580052); Ultra-pure water is self-control; Sample is self-control.
Animal cleaning grade SD rat (Shanghai Slac Experimental Animal Co., Ltd.).Rearing conditions: cleaning grade Animal Lab., room temperature 23-25 DEG C, relative humidity 40%-70%, suitable ventilation 15 times every day.
2. experimental technique
The foundation of 2.1 rat adjuvant type arthritis models
SD rat 72, male and female half and half, body weight is at 180-220g, and survey each Mus left and right metapedes sole of the foot volume with homemade simple and easy sufficient sole of the foot measuring device, separate 8 at random as blank group by weight, all the other 64 are carried out modeling by literature method.Conventional method carries out disinfection to skin bottom the SD Rat Right metapedes sole of the foot, only carries out causing inflammation with microsyringe intradermal injection Freund ' s Freund's complete adjuvant 0.1mL/.After modeling the 13rd day, sufficient sole of the foot volume about measuring every rat, model group foot sole of the foot volume compared with blank group that there were significant differences, and observes and find that the sufficient sole of the foot red and swollen obviously prompting modeling is successful.
2.2 experiment groupings
Dosage group (FM) in the Chinese medicine compound effective part high dose group (FH) that experiment is divided into blank group, model group, positive controls (Indomethacin, IMT), the embodiment of the present invention 1 to prepare by rat weight and foot swelling degree at random, effective site low dose group (FL), effective site.
The observation of 2.3 rat general states and the evaluation of Articular swelling
Before modeling and administration fore-and-aft observing rat chaeta color and luster and activity situation, the increase change 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 electronic scale weighing rat body weight; Foot sole of the foot volume: the volume surveying the sole of the foot after about each group of rat with the sufficient sole of the foot measuring device of self-control, the change of each group rat paw edema degree after comparing administration.
2.4 dosages and administering mode
13d after modeling, divides into groups and after measuring the left and right sufficient sole of the foot volume of rat and body weight, starts gastric infusion.Dosage group (FM) (76.92mg/Kgd) gavage 2mL in indomethacin positive controls group (10mg/Kgd), Chinese medicine compound effective part high dose group (FH) (90.48mg/Kgd), effective site low dose group (FL) (56.12mg/Kgd), effective site, Normal group and model group give the Tween-80 of equivalent 2/1000ths, altogether administration 17 days.
The sampling and processing of 2.5 rat plasmas, tissue samples
With 10% chloral hydrate intraperitoneal injection of anesthesia rat, dorsal position is fixed, carotid artery gets blood, whole blood is collected in the centrifuge tube of 10mL with 3.8% sodium citrate (blood and anticoagulant volume ratio are 9:1) anticoagulant, with low speed centrifuge with the centrifugal 10min of 3000r/min rotating speed, Aspirate supernatant, stored frozen at-20 DEG C, detects NO, MDA, PGE in blood plasma
2, IL-2, TNF
α.Cut the peeling of the right back foot swelling foot sole of the foot simultaneously, pick a bone, soak (tissue and normal saline volume ratio are 1:9) with normal saline, the centrifugal 10min of 3000r/min, Aspirate supernatant ,-20 DEG C of freezen protective, detect NO, MDA, PGE in tissue fluid
2, IL-2, TNF
α.
The assay method of BCA protein concentration in 2.6 rat tissues
Experiment adopts the BCA determination of protein concentration test kit of the western Tang in Shanghai biological production, and assay method is as follows:
1) quantity per sample, adds 1 volume BCA reagent B(50:1 by 50 volume BCA reagent A) prepare appropriate BCA working solution, fully mix.BCA working solution room temperature was stablized in 24 hours.
2) complete soluble protein standard substance, get 25 μ L 0.9%NaCl or PBS and are diluted to 100 μ L, make final concentration be 0.5mg/mL.
3) standard substance are added in the standard sample wells of 96 orifice plates by 0,1,2,4,8,12,16,20 μ L, add standard dilutions and supply 20 μ L.
4) add proper volume sample aerial to the sample of 96 orifice plates, add standard dilutions to 20 microlitres.
5) each hole adds 200 μ LBCA working solutions, places 30 minutes for 37 DEG C.
6) measuring wavelength is that wavelength between 562nm, 540-595nm also can accept.Protein concentration is calculated according to standard curve.
The assay method of 2.7NO
By the operation of nitric oxide (Nitricoxide, NO) test kit (enzyme process) description, measure the burst size of NO.NO chemical property is active, and internal metabolism transfers NO2-and NO3-to very soon, and NO2-is further converted to NO3-, and this law utilizes nitrate reductase enzyme spcificity that NO3-is reduced to NO2-, is measured the height of its concentration by the colour developing depth.
Get above-mentioned gained blood plasma, tissue supernatant, eight pipes respectively got by each sample, often pipe 100 μ L, as mensuration pipe, get 100 μ L100 μm ol/LKNO
3as standard pipe totally eight pipes, get the distilled water of 100 μ L as blank tube totally eight pipes, often pipe adds 400 μ L mix reagents (nitrate reductase), mix gently, 37 DEG C of water-baths, after 1 hour, respectively manage reagent adding 3 successively, reagent 4, abundant vortex mixes 30 seconds, room temperature leaves standstill 40 minutes, centrifuging and taking supernatant, adds developer reaction 60min, 540nm surveys each pipe OD value, is calculated as follows:
(1) computing formula of serum NO content:
(2) computing formula of NO content in tissue:
The assay method of 2.8 malonaldehyde (MDA)
Experiment measures the operational approach of test kit description according to malonaldehyde (MDA), measure the burst size of MDA.MDA test kit test philosophy: the malonaldehyde (MDA) in lipid peroxide catabolite can with thiobarbituricacidα-(TBA) condensation, form red product, have maximum absorption band at 532nm place.
By test kit, configuration reagent is described, gets above-mentioned gained serum, tissue supernatant, eight pipes got by each test sample, often pipe 50 μ L, eight pipes respectively got by blank tube and standard pipe, often pipe 50 μ L, often pipe adds mix reagent 1000 μ L respectively, with the mixing of vortex vortex mixer, test tube mouth preservative film is tightened, and stings an aperture with syringe needle, 95 DEG C of water-baths (or uncap with pot boil) 40 minutes, flowing water cooling after taking out, then 3500-4000r/min, centrifugal 10 minutes.Get supernatant 200 μ L in 96 orifice plates, 532nm place surveys the absorbance of each pipe, by following formulae discovery:
(1) computing formula of MDA content in serum:
(2) computing formula of NO content in tissue:
The assay method of 2.9 interleukin-2s (IL-2), PGE2, TNF α
Experiment adopts double antibodies sandwich ABC-ELISA method, be coated in ELISA Plate with Chinese People's Anti-Japanese Military and Political College's Mus IL-2 monoclonal antibody, IL-2 in standard substance and sample is combined with monoclonal antibody, adds biotinylation and obtains Chinese People's Anti-Japanese Military and Political College Mus IL-2, forms immune complex and connects onboard, the streptavidin of horseradish peroxidase-labeled is combined with biotin, add substrate working solution aobvious blue, finally add stop buffer sulphuric acid, survey OD value at 450nm place, IL-2 concentration is directly proportional to OD value, is obtained the concentration of IL-2 in specimen by drawing standard curve.PGE
2, TNF
αthe Cleaning Principle of content is similar to the Cleaning Principle of IL-2.
By requirement configuration standard product, 10 × sample diluent, the cleaning mixture of 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 rearmounted 37 DEG C and hatches 120 minutes.With cleaning mixture, Sptting plate is fully washed 4-6 time, after print is done on filter paper, in every hole, add first antibody working solution 100 μ L, Sptting plate is fully mixed latter 37 DEG C and hatch 60 minutes, wash plate, after print is dry, every hole adds substrate working solution 100 μ L, Sptting plate is put dark place and reacts 15 minutes.Every hole adds the stop buffer mixing of 100 μ L again, surveys light absorption value in 30 minutes by microplate reader at 450nm place.
All OD values are gone after all should reducing blank value again and are calculated.With standard substance 2000,1000,500,250,125,62.5,31.2,0pg/mL for abscissa, OD value is vertical coordinate, and graph paper is mapped, and draws standard curve.OD value finds corresponding IL-2, PGE on the graph per sample
2, TNF
αcontent.
3 experimental results
3.1 cause scorching rear each observation and comparison organizing rat ordinary circumstance
3.1.1 the change of scorching rear each group rat general state is caused
Rats in normal control group food-intake is many, and the mental status is good, glossy by hair, flexibly movable, and state without exception occurs, along with the prolongation of feeding time, the bodily form is gradually long, and body weight increases obviously.Model group rats, female appetite reduces comparatively obvious, and lose weight obviously, listless, hair dries up lackluster, and activity reduces, later stage heavy burden difficulty, and obstacle appears in joint motion; Male rat appetite has and to reduce but not obvious, and lose weight also not obvious, listless, the few gloss of hair, activity also reduces, but obvious not as female rats.Chinese medicine compound effective part provided by the invention high, medium and low dosage group Hair of Rats, the mental status, body weight and joint movement disorder comparatively model group all take an evident turn for the better.
3.1.2 the change of scorching rear each group rat different time body weight is caused
Before administration, comparatively, normal group body weight and model group body weight have significant difference (P≤0.05) for each group rat body weight and model group weight ratio, and all the other are group and the equal not statistically significant of model group comparing difference (P > 0.05) respectively.
3.2 cause scorching rear each impact organizing the right pedal swelling degree of rat different time
Cause scorching after the impact of the right pedal swelling degree of each group rat different time, table 1 is 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 compares with model group pole significant difference (P < 0.001), and all the other respectively organize the difference (P > 0.05) that to compare with model group that there are no significant; 20d after modeling, normal group, IMT group, medicine group compare with model group pole significant difference (P < 0.001), high, the middle dosage group of Chinese medicine compound effective part compares with model group significant differences (P < 0.01), and low dose group compares with model group significant difference (P < 0.05); 24d after modeling, high, the middle dosage group of Chinese medicine compound effective part compares with model group significant differences (P < 0.01), and all the other are respectively organized more all has significant difference (P < 0.01) with model group; After modeling, 27d, 30d respectively organize Rat Right foot sole of the foot volume and model group more even pole significant difference (P < 0.01), and Chinese medicine compound effective part provided by the invention shows effect of well subsiding a swelling.
The change of table 1 Rat Right pedal swelling degree
Note: compare with model group, * P < 0.05 indicates significant difference, and * * P < 0.01 indicates significant differences, and * * * P < 0.001 indicates pole significant difference.
3.3 on each impact organizing NO, MDA, IL-2, PGE2, TNF α in rat plasma
As shown in Table 2, the high, medium and low dosage group of Chinese medicine compound effective part of the present invention can significantly suppress NO level in rat plasma, compares have pole significant difference (P < 0.001) with model group; In normal rats blood plasma, MDA content compares with model group highly significant difference (P < 0.01), and the high, medium and low dosage group of ITM group, Chinese medicine compound effective part compares with model group highly significant difference (P < 0.01); IL-2, PGE in IMT group, Chinese medicine compound effective part high, medium and low dosage group rat plasma
2, TNF
αlevel, more all has pole significant difference (P < 0.001) with model group.Result shows that the Chinese medicine compound effective part that the present invention prepares has NO, MAD, IL-2, PGE in significant suppression adjuvant arthritis rats blood plasma
2, TNF
αlevel, has good antiphlogistic effects.
Table 2 is on the impact of NO, MDA, IL-2, PGE2, TNF alpha content in rat plasma
Note: compare with model group, * P < 0.05 indicates significant difference, and * * P < 0.01 indicates significant differences, and * * * P < 0.001 indicates pole significant difference.
3.4 impacts on NO, MDA, IL-2, PGE2, TNF α in Rat Right pedal swelling tissue
As shown in Table 3, NO, PGE in IMT group, Chinese medicine compound effective part of the present invention high, medium and low dosage group Rat Right pedal swelling tissue
2, TNF
αcontent more all have pole significant difference (P < 0.001) with model group, experimental result shows, Chinese medicine compound effective part provided by the invention significantly can suppress NO, MDA, IL-2, PGE2, TNF alpha levels in pedal swelling tissue, shows good antiphlogistic effects.
Table 3 is on the impact of NO, MDA, IL-2, PGE2, TNF alpha content in Rat Right pedal swelling tissue
(note: compare with model group, * P < 0.05 indicates significant difference, and * * P < 0.01 indicates significant differences, and * * * P < 0.001 indicates pole significant difference.）
The evaluation of embodiment 5 antioxidant activity
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 (Wuxi City Ya Sheng Chemical Co., Ltd.), DPPH(sigma company), vitamin C (Chemical Reagent Co., Ltd., Sinopharm Group), ultra-pure water is made by oneself, and sample is self-control.
2. experimental technique
Accurately take DPPH dehydrated alcohol and be made into 6.5 × 10
-4the storing solution of mol/L, the refrigerator 30min that keeps in Dark Place can use.Vitamin C (positive control) distilled water dissolves, the anhydrous alcohol solution of each effective-site variable concentrations before and after Olibanum-Myrrha compatibility.Chinese medicine compound effective portion 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 100 μ LDPPH solution (final concentration is 80 μMs) are added, rock evenly, lucifuge reaction 30min, measures OD value at 517nm place.According to following formulae discovery 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) concentration of required antioxidant when referring to that clearance rate is 5O%, the clearance rate according to variable concentrations antioxidant is obtained as curve.
3. experimental result
Sample is dissolved in different solvents by different solubility per sample, measures each sample according to the method described above to the clearance rate of DPPH, the results are shown in Table 4.As shown in Table 4, the embodiment of the present invention 1 Chinese medicine compound effective part 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.
DPPH free radical half inhibitory action (n=4) removed by table 4
Embodiment 6 mouse hot-plate induced pain is tested
1. experimental technique
Female ICR mice, 50, body weight 22 ~ 26 grams, be divided into model control group, positive drug group (dolantin) at random, dosage group (FM) in the Chinese medicine compound effective part high dose group (FH) that the embodiment of the present invention 1 prepares, effective site low dose group (FL), effective site, often organizes 10.The analgesic effect of Chinese medicine compound effective part is evaluated with hot plate stimulus method.Mice first uses hot plate dolorimeter device (55.5 DEG C) to measure the normal pain threshold of every mice respectively.Administration every day 1 time, continuous 2 days, after last administration 30 minutes, 1 hour, 2 hours, 4 hours, measures the threshold of pain time, compares group difference for 10 hours.
2. experimental result
The Chinese medicine compound effective part group that the embodiment of the present invention 1 prepares stimulates the induced mice threshold of pain time to hot plate upon administration, and comparatively there is prolongation effect the administration front threshold of pain time, and still have obvious analgesic activity after 10 hours, compared with blank group, the mice threshold of pain time can be significantly improved.Specific experiment result is as table 5.
The analgesic activity (n=10) that table 5 Chinese medicine compound effective part is tested mouse hot-plate
Compare with before administration, * * P<0.01, * P<0.05; Compare with normal saline group, △ △ P<0.01, △ P<0.05.
Shown by the experimental result of above table 5, Chinese medicine compound effective part provided by the invention has good analgesic activity, can improve pain threshold, and the persistent period is longer, can play good anti-inflammatory and antalgic effect when treatment of arthritis disease.
Above embodiment is only for illustrating technical conceive of the present invention and feature; its object is to allow person skilled in the art understand content of the present invention and to be implemented; can not limit the scope of the invention with this; all equivalences done according to spirit of the present invention change or modify, and all should be encompassed in protection scope of the present invention.
Claims (4)
1. have a Chinese medicine compound effective part for treatment of arthritis effect, it is characterized in that, it is prepared by following method:
(1) get Olibanum 35-45 part, Myrrha 25-35 part, adding total medical material weight 5-10 times amount concentration is 10-80% alcohol reflux 1-3 time, and merge extractive liquid, obtains concentrated solution at 20-70 DEG C 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, is separated to obtain supernatant, concentrated, obtains concentrate;
(3) D101, D102 or AB-8 type macroporous adsorptive resins on concentrate that step (2) obtains is got, with the ethanol gradient elution of concentration 30% to 90%, collecting concentration is the ethanol elution of 60% to 80%, recycling design, obtain the boswellic acids derivatives including percentage by weight 10%-60% in Chinese medicine compound effective part, the sesquiterpenoids of 1%-30%, the diterpene-kind compound of 1%-40%.
2. the Chinese medicine compound effective part with treatment of arthritis effect according to claim 1 is preparing the application in medicament for treating arthritis.
3. the Chinese medicine compound effective part with treatment of arthritis effect according to claim 2 is preparing the application in medicament for treating arthritis, and it is characterized in that, described arthritis is rheumatic arthritis, rheumatoid arthritis or proliferative arthritis.
4. the Chinese medicine compound effective part with treatment of arthritis effect according to claim 3 is preparing the application in medicament for treating arthritis, it is characterized in that, Chinese medicine compound effective part and pharmaceutically acceptable carrier are made the medicine of capsule, granule, spray, injection, microcapsule, Tabules.
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| CN105997954A (en) * | 2016-06-24 | 2016-10-12 | 贵州弘康药业有限公司 | Itching-relieving and pain-relieving pollen liniment and preparation method thereof |
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| CN107158180A (en) * | 2017-05-24 | 2017-09-15 | 郭庆忠 | A kind of Chinese medicine lotion for external use for treating rheumatism bone disease |
| 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 |
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