CN102895348B

CN102895348B - Pharmaceutical compositions for treating rheumatic joint disease, preparation method thereof and use thereof

Info

Publication number: CN102895348B
Application number: CN201210443908.0A
Authority: CN
Inventors: 徐世军
Original assignee: Chengdu University of Traditional Chinese Medicine
Current assignee: Chengdu University of Traditional Chinese Medicine
Priority date: 2012-11-08
Filing date: 2012-11-08
Publication date: 2014-07-30
Anticipated expiration: 2032-11-08
Also published as: CN102895348A

Abstract

The invention provides pharmaceutical compositions for treating rheumatic joint disease, a preparation method thereof and application thereof. The pharmaceutical compositions mainly consist of the following raw materials in parts by weight: 10-70 parts of silky ant, 10-50 parts of astragalus mongholicus, 5-40 parts of pawpaw, 5-40 parts of salted aconite root and 2-20 parts of pseudo-ginseng. The invention further provides a preparation method of the pharmaceutical compositions and the use of the pharmaceutical compositions. The experiment improves that the pharmaceutical compositions provided by the invention have the effects of deficiency tonifying, consolidating basis, eliminating evil and smoothening numbness, not only can be used for effectively treating and relieving the arthralgia, the morning stiffness, the numbness and the bad flexibility caused by the rheumatism and the rheumatoid arthritis, but also can be used for preventing, treating and relieving the kidney function harm caused by the rheumatism, have the functions for treating the pathological changes such as arthrocele, pain, inhibited bending and stretching and the like, and can be particularly used for the rheumatic arthritis, the chronic infectious arthritis, the joint synovitis and the rheumatoid renal damage.

Description

A kind of pharmaceutical composition and preparation method and purposes for the treatment of rheumatic joint disease

Technical field

The present invention relates to a kind of pharmaceutical composition, particularly relate to a kind of pharmaceutical composition being formed by crude drug Formica fusca, Radix Aconiti Lateralis Preparata, the Radix Astragali, Fructus Chaenomelis and Radix Notoginseng, belong to technical field of Chinese medicines.

Background technology

Rheumatoid arthritis (Rheumatoid Arthritis, RA) belongs to " arthromyodynia " category in motherland's medical science, and sickness rate is high, falls ill in the majority with the person between twenty and fifty of 25～55 years old, and women falls ill higher than male, and men and women's ratio is 1:4, and the whole world has more than one hundred million patients.According to statistics, the external prevalence of RA is 1～2%, and indivedual areas are up to 5%; China's sickness rate is about 0.74%, existing 1,500 ten thousand people left and right patients; Primary disease disability rate is higher, and the patient with rheumatoid arthritis of in time diagnosis and treatment 50%, the three year disability rate that disables for 2 years does not reach 70%.And the patient who has suffered from rheumatoid arthritis, average life shortens 10～15 years.From course of disease length, discuss, the disability rate approximately 20% of falling ill in 1 year, more than 2 years can reach 40% left and right, heavy damage work capacity, very big to human health risk, cause very big burden to society, family, have serious impact also to patient's physical and mental health and quality of life, therefore have the title of " not dead cancer ".

The exact mechanism of RA is not very clear and definite, but modern study thinks that RA is that a kind of a large amount of T cellular infiltration is the autoimmune disease that main chronic synovitis is feature at present, and crucial immunological abnormality comprises abnormal activation, the especially CD of the imbalance of T cell subsets and T cell ₄ ⁺t cell has participated in exciting of RA and has continued.RA patient's pathological study result shows, it is main feature that patient's synovial membrane be take synovial cell's tumor sample propagation, blood vessel hyperplasia and inflammatory cell infiltration, and in the inflammatory cell infiltrating with T cell especially CD ₄ ⁺t cell subsets is main, in addition, and CD in RA patient T cell subsets ₄ ⁺t cell proportion and clonal expansion ability all change to some extent, prompting T lymphocyte, especially CD ₄ ⁺t has extremely important status in RA occurs and develops.T cell gathers and activates in the synovial membrane of inflammation, by secretion of gamma-IFN, contacts with the direct of IL-17 and cell-cell, in RA inflammatory reaction, has brought into play important function, has promoted RA bone to damage in addition by TNF the drawing together inducible factor of being correlated with.RA patient is owing to existing CD ₄ ⁺the increase of T cell quantity or CD ₈ ⁺the decline of T quantity, makes CD ₄ ⁺t/CD ₈ ⁺the rising that compares of T, relatively hyperfunction weaken relative with Ts cell function of Th cell function, thus cause that B cell function is hyperfunction, immunoglobulin expression is out of control.It is believed that in the past RA seldom involves kidney, even if the common renal amyloidosis of renal damage or the membranous nephropathy being caused by golden preparation or penicillamine occur and treat relevant interstitial nephritis with on-steroidal AID (NSAIDs).Just because of the deficiency in understanding, so that formed for a long time the wrong views that RA itself seldom causes nephropathy.Day by day deep along with the extensive use of kidney Biopsy and immunology research in recent years, people recognize that RA not only can cause renal damage gradually, and infringement is 100%; Research finds that RA renal damage is mainly mesangial proliferative nephritis (RA-MsPGN), accounts for 30.1%～75.0% (being also reported as 25%-50%) of RA constitutional renal damage.

RA there is no specific short at present, and clinical treatment is mainly anti symptom treatment.World rheumatism alliance is divided into two large classes by existing antirheumatic, improve the antirheumatic of symptom and the antirheumatic of control disease, the former is still current clinical common medicine, so far without a kind of antirheumatic of real control disease, clinical normal drug combination is to reaching the object of controlling disease, but with regard at present obtained in the recent period, with regard to late result, result all can not be satisfactory; Although carried out in recent years Biotherapeutics, eventually because of expensive, Orally active is poor, can not in shortcomings such as target tissues, limit its application by selectively acting; Gene therapy seems can overcome in theory the part defect of Biotherapeutics, but in the clinical research of more than 300, the world, 3000 many cases are the patient of receptor gene treatment, does not have an example clearly to show clinical improvements.Existing clinical application is as salicylic acid and other NSAID (non-steroidal anti-inflammatory drug), antimalarial, penicillamine, Tripterygium Preparations, golden preparation, 17-hydroxy-11-dehydrocorticosterone even, and its therapeutic effect is all undesirable, and side effect is large.The traditional Chinese medical science has accumulated rich experience in the clinical practice of several thousand, quickening along with the surging of back to nature tide and modernization of Chinese medicine step, seek Chinese medicine means and medicine and become the emphasis of global concern and the focus of research, especially be accompanied by the understanding of RA target organ damage is deepened and paid attention to, (dosage is little in developmental research " three is little ", toxicity is little, side effect is little) triple effect is (efficiently, quick-acting, long-acting) five convenience (take medicine, produce, transportation, carry, storage), both there is good anti rheumatism action, main target organ (the lung again RA being damaged, kidney, bone etc.) medicine that has a protective effect becomes the common focus of being concerned about of Chinese and western medicine.

Summary of the invention

First object of the present invention is to provide a kind of pharmaceutical composition for the treatment of rheumatic joint disease;

Second object of the present invention is to provide the preparation method of this pharmaceutical composition;

The 3rd object of the present invention is to provide this pharmaceutical composition in preparation treatment and alleviates the application in rheumatic arthropathy and rheumatic renal damage medicine.

The object of the invention is to be achieved through the following technical solutions:

Treat a pharmaceutical composition for rheumatic joint disease, the crude drug of this pharmaceutical composition consists of:

Formica fusca 10-70 part, Radix Astragali 10-50 part, Fructus Chaenomelis 5-40 part, Radix Aconiti Lateralis Preparata 5-40 part, Radix Notoginseng 2-20 part;

Further, its crude drug consists of:

Formica fusca 15-60 part, Radix Astragali 15-45 part, Fructus Chaenomelis 10-35 part, Radix Aconiti Lateralis Preparata 10-30 part, Radix Notoginseng 3-15 part;

Further, its crude drug consists of:

Formica fusca 15-50 part, Radix Astragali 15-40 part, Fructus Chaenomelis 10-30 part, Radix Aconiti Lateralis Preparata 10-30 part, Radix Notoginseng 3-15 part;

Further, its crude drug consists of:

Formica fusca 20-40 part, Radix Astragali 18-30 part, Fructus Chaenomelis 12-20 part, Radix Aconiti Lateralis Preparata 10-20 part, Radix Notoginseng 4-10 part;

Further, its crude drug consists of:

Formica fusca 25-40 part, Radix Astragali 18-30 part, Fructus Chaenomelis 12-20 part, Radix Aconiti Lateralis Preparata 10-20 part, Radix Notoginseng 5-10 part;

Further, its crude drug consists of:

30 parts of Formica fuscas, 20 parts of the Radixs Astragali, 15 parts of Fructus Chaenomeliss, 12 parts of Radix Aconiti Lateralis Preparata, 6 parts of Radix Notoginseng;

Or, 25 parts of Formica fuscas, 30 parts of the Radixs Astragali, 12 parts of Fructus Chaenomeliss, 20 parts of Radix Aconiti Lateralis Preparata, 5 parts of Radix Notoginseng;

Or, 40 parts of Formica fuscas, 18 parts of the Radixs Astragali, 20 parts of Fructus Chaenomeliss, 10 parts of Radix Aconiti Lateralis Preparata, 10 parts of Radix Notoginseng.

Pharmaceutical composition of the present invention can be prepared as follows:

Get in proportion five tastes crude drug, with water or with the organic solvent extraction that water dissolves each other, be prepared from;

Or, get in proportion five tastes crude drug, wherein Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata merging obtain extract with water or with the organic solvent extraction that water dissolves each other; Radix Notoginseng is pulverized, by Radix Notoginseng powder join above-mentioned extract obtained in, mix homogeneously forms.

Further, the described organic solvent dissolving each other with water is selected from any one in methanol, ethanol, acetone; Further be preferably the ethanol that concentration is 30%-80%;

Extracting method used comprises any one mode decocting in extraction, reflux, extract,, immersion extraction, supersound extraction or percolation extraction, or the combination of Different Extraction Method.

Further, above-mentioned preparation method Raw medicine also can be purified, refining after extracting, as crossed macroporous resin column, and further preparation process is routinely made the acceptable any conventional dosage form of pharmaceutics, comprises granule, tablet, capsule, drop pill, oral liquid, suspension, emulsion, injection.

Pharmaceutical composition of the present invention also can adopt: get in proportion five tastes crude drug crude drug and directly pulverize, be mixed clinical acceptable dosage form;

Described pulverizing is preferably: after micronizing, make the super fine below particle diameter 100 μ m; Further preferably be prepared as the super fine below 80 μ m; Further preferably be prepared as the super fine below 70 μ m; Further preferably be prepared as the super fine below 60 μ m; Further preferably be prepared as the super fine below 50 μ m; Further preferably be prepared as the super fine below 40 μ m; Further preferably be prepared as the super fine below 30 μ m; Further preferably be prepared as the super fine below 20 μ m; Further preferably be prepared as the super fine below 10 μ m; Further preferably be prepared as the super fine below 8 μ m;

Further, before described micronizing, crude drug is first ground into coarse powder (crossing 24 mesh sieves), and makes moisture lower than 4% through dried.

For above-mentioned dosage form can be realized, need when these dosage forms of preparation, add the acceptable adjuvant of pharmacy, such as: filler, disintegrating agent, lubricant, suspending agent, binding agent, sweeting agent, correctives, antiseptic, substrate etc.Filler comprises: starch, pregelatinized Starch, lactose, mannitol, chitin, microcrystalline Cellulose, sucrose etc.; Disintegrating agent comprises: starch, pregelatinized Starch, microcrystalline Cellulose, carboxymethyl starch sodium, crospolyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, cross-linking sodium carboxymethyl cellulose etc.; Lubricant comprises: magnesium stearate, sodium lauryl sulphate, Pulvis Talci, silicon dioxide etc.; Suspending agent comprises: polyvinylpyrrolidone, microcrystalline Cellulose, sucrose, agar, hydroxypropyl emthylcellulose etc.; Binding agent comprises, starch slurry, polyvinylpyrrolidone, hydroxypropyl emthylcellulose etc.; Sweeting agent comprises: saccharin sodium, Aspartane, sucrose, cyclamate, enoxolone etc.; Correctives comprises: sweeting agent and various essence; Antiseptic comprises: parabens, benzoic acid, sodium benzoate, sorbic acid and its esters, benzalkonium bromide, acetic acid chloroethene are determined, Folium eucalypti globueli (Eucalyptus globulus Labill.) wet goods; Substrate comprises: PEG6000, PEG4000, insect wax etc.For making above-mentioned dosage form can realize pharmacy of Chinese materia medica, need add acceptable other adjuvant of pharmacy (the green booth < of model < pharmacy of Chinese materia medica > >, the adjuvant that in Shanghai Science Press December in 1997 the 1st edition, each dosage form is recorded) during these dosage forms in preparation.

The pharmaceutical composition for the treatment of rheumatic joint disease of the present invention is except the form feeding intake with Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata and Radix Notoginseng crude drug, can also adopt the form feeding intake with Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata and Radix Notoginseng extract (effective site), therefore the present invention further discloses the pharmaceutical composition for the treatment of rheumatic joint disease:

Treat a pharmaceutical composition for rheumatic joint disease, the raw material of this pharmaceutical composition consists of:

Formica fusca extract 10-70 part, Radix Astragali extract 10-50 part, Fructus Chaenomelis extract 5-40 part, Radix Aconiti Lateralis Preparata extract 5-40 part, Radix Notoginseng extract 2-20 part;

Further, its raw material consists of:

Formica fusca extract 20-50 part, Radix Astragali extract 15-40 part, Fructus Chaenomelis extract 10-30 part, Radix Aconiti Lateralis Preparata extract 10-30 part, Radix Notoginseng extract 3-15 part;

Further, its raw material consists of:

Formica fusca extract 25-40 part, Radix Astragali extract 18-30 part, Fructus Chaenomelis extract 12-20 part, Radix Aconiti Lateralis Preparata extract 10-20 part, Radix Notoginseng extract 5-10 part;

Further, its raw material consists of:

30 parts of Formica fusca extracts, 20 parts of Radix Astragali extracts, 15 parts of Fructus Chaenomelis extract, 12 parts of Radix Aconiti Lateralis Preparata extracts, 6 parts of Radix Notoginseng extracts;

Or, 25 parts of Formica fusca extracts, 30 parts of Radix Astragali extracts, 12 parts of Fructus Chaenomelis extract, 20 parts of Radix Aconiti Lateralis Preparata extracts, 5 parts of Radix Notoginseng extracts;

Or, 40 parts of Formica fusca extracts, 18 parts of Radix Astragali extracts, 20 parts of Fructus Chaenomelis extract, 10 parts of Radix Aconiti Lateralis Preparata extracts, 10 parts of Radix Notoginseng extracts.

Formica fusca extract of the present invention, Radix Astragali extract, Fructus Chaenomelis extract, Radix Aconiti Lateralis Preparata extract, Radix Notoginseng extract are respectively the water extract of Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata, Radix Notoginseng or the extractive with organic solvent dissolving each other with water; Or the refining thing further obtaining through refining purification process after the organic solvent extraction dissolving each other through water or with water for Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata, Radix Notoginseng, as crossed macroporous resin column.

Further, the described organic solvent dissolving each other with water is selected from any one in methanol, ethanol, acetone; The ethanol that more preferably concentration is 30%-80%;

Extracting method used can be any one mode decocting in extraction, reflux, extract,, immersion extraction, supersound extraction or percolation extraction, or the combination of Different Extraction Method.

Pharmaceutical composition of the present invention can also be used with other drug compatibility, and described other drug is for the medicine in relevant or similar field, as rheumatic, antirheumatic, immunoregulation medicament, analgesic drug product etc.

Pharmaceutical composition of the present invention can be used for treatment and alleviates rheumatic arthropathy, and described rheumatic arthropathy is rheumatic arthritis, rheumatoid arthritis, articular synovitis and rheumatic renal damage.

Formica fusca of the present invention is that < < Guangxi Chinese crude drug standard > > second (1996) records, and is the dry body of Formicidae animal bitooth multi-ant Polyrhachis dives Smith; The described Radix Astragali, Radix Aconiti Lateralis Preparata, Fructus Chaenomelis, Radix Notoginseng are 2010 editions Chinese medicine or its processed products that < < Chinese Pharmacopoeia > > First records.

Ingredients of the present invention is prescription under traditional Chinese medical science QI and blood essence dynamic circulation theoretical direction, through the clinical basis of repeatedly verifying for many years and optimizing, forms.This theory think gas, blood, smart three under physiological status mutual promotion, mutually transform; Under pathological state, influence each other, jointly cause a disease; Rheumatoid arthritis be the process that is broken and rebuilds due to extraneous inducement effect therapeutic method to keep the adverse QI flowing downwards hemospermia balance sysmte, build the multiple pathological product that in equilibrium process, QI and blood essence produces mutually cementing, influencing each other is to cause the main cause that disease is complicated and treatment is thorny again; The basic pathogenesis of RA is that QI and blood essence is all empty, and the dynamic equilibrium of QI and blood essence is rebuild on low-level, expectorant stasis of blood water cementing being at a stop in joint tissue of wet, and the normal circulation of obstruction QI and blood essence, causes vicious cycle.In prescription, with Formica fusca, Radix Aconiti Lateralis Preparata QI invigorating hemospermia, rouse oneself vigour, promote QI and blood essence blood circulation to transform to physiological status, be aided with its medicine and reach treating both the principal and secondary aspects of a disease, the effect for the treatment of must aim at the pathogenesis of disease; The circulation of qi promoting of Radix Astragali QI invigorating, reaches qi-supplementing, blood-engendering, and gas promoting the circulation of blood is moving, thereby drives QI and blood fine balance to recover to physiological level; The medicine blood-supplementing blood-nourishings such as Radix Notoginseng, thus reach the logical object of mending of essence and blood; Formica fusca is worm medicine, and benefaction, has promoting blood circulation to remove obstruction in the collateral, and the effect of stasis-dispelling and pain-killing is aided with the effect of invigorating blood circulation of Radix Notoginseng, reaches promoting tissue regeneration by removing blood stasis, promotes the object of joint function recovery, and Fructus Chaenomelis relaxing muscles and tendons and activating QI and blood in the collateral, for clinical cardinal symptom; In this prescription, Radix Aconiti Lateralis Preparata is pungent salty flat, and Gan Wen, returns spleen kidney channel, gets its dispelling cold and removing dampness, the justice of warming YANG dredging collateral; The salty kidney of returning, salty energy softening the hard mass, the joint caused cellulose sample precipitation of softening RA, according to modern medical theory, Radix Aconiti Lateralis Preparata can improve again body Na+ concentration, changes osmotic pressure in body, by permeability, is dewatered and is reached the effect of elimination inflammatory exudate.The medicines such as Formica fusca, tepor, micro-salty, sour sweet, enters liver,spleen,kidney warp, dispelling wind dredging collateral, nourishing muscle and tendon blood stasis dispelling, anti-inflammatory analgesic.Sour in taste acting on the liver, liver governing tendons, the logical bone of acid, thus can easing joint movement, softening distortion joint, prevents or alleviates joint distortion, salty flavor entering the kidney, the kidney generating marrow and dominating bone, sweet flavour enters the spleen, spleen is source of generating QI and blood, thus can nourishing muscle and tendon, bone growth promoting is mended marrow.All medicines share, and amount to invigorating deficiency and tonifying vital QI, and the effect of eliminating evil blood stasis dispelling promotes QI and blood essence physiological equilibrium's recovery, reaches the object for the treatment of.

In an embodiment of the present invention, provide the pharmaceutical composition of the present invention of different proportion for the experimentation of anti-model of adjuvant arthritis in rats, thereby drawn the ratio range of the comparatively preferred crude drug of crude drug compositions of the present invention.

In other embodiment of the present invention, provided the extract of the more excellent proportioning of primary raw material medicine of the present invention for the pharmacological evaluation of anti-dimethylbenzene mice ear, Ovum Gallus domesticus album rat paw edema, thermostimulation pain, chemical stimulation pain, adjuvant-induced arthritis, rheumatic renal damage (RA-MsPGN), proved that pharmaceutical composition of the present invention can be used for rheumatic arthritis, rheumatoid arthritis, rheumatic MsPGN.

In following experimental example, laboratory animal used is clearly not dated, is: KM mice, body weight 20 ± 2g, SD rat, body weight 200 ± 20g, by Sichuan Academy of Medical Sciences institute of lab animals, provide the animal quality certification number: real moving pipe matter SCXK (river) 2004-16 in river.Experimental site is three grades of laboratorys of Chengdu University of Traditional Chinese Medicine of State Administration of Traditional Chinese Medicine herbal pharmacology, numbering: TCM2032043.Animal, all at room temperature 22-24 ℃, is raised under light and shade cycle 12h/12h condition, freely drinks water and ingests.

The research of the different compatible composition antalgic and inflammation relieving of experimental example 1 proportioning ratio

1 material

1.1 medicines and reagent

Formica fusca, Radix Aconiti Lateralis Preparata, Radix Notoginseng, the Radix Astragali, Fructus Chaenomelis, purchased from the large pharmacy of Tongrentang, are identified by the Ma Yuntong of Chengdu University of Traditional Chinese Medicine.Tramadol hydrochloride is produced (lot number: 0406245) by Beijing Sihuan Medicine Science and Technology Co., Ltd; Dexamethasone acetate injection is produced (lot number: 66040099) by Xinan Pharmaceutical Co., Ltd..

1.2 laboratory animal

KM kind white mice, clean level, male and female dual-purpose, body weight (20 ± 2) g; By Sichuan Academy of Medical Sciences institute of lab animals, provided, the laboratory animal quality certification number is: SCXK(river) 2004-16.

2 methods and result

2.1, the setting of Experimental agents proportioning ratio

Table 1 Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata and Radix Notoginseng proportioning ratio (g)

Experiment number	Formica fusca	The Radix Astragali	Fructus Chaenomelis	Radix Aconiti Lateralis Preparata	Radix Notoginseng
						1	10	10	5	5	2
2	20	15	10	8	4
						3	30	20	15	12	6
4	40	25	20	15	8
						5	50	30	25	20	10

By table 1, get respectively the crude drug of recipe quantity, Formica fusca, Radix Aconiti Lateralis Preparata, the Radix Astragali, Fructus Chaenomelis and Radix Notoginseng powder are broken into coarse powder, and adding distil water soaked after 30 minutes, added 6 times of water gagings to decoct three times, and each 1 hour, filter, get 4 ℃ of Refrigerator stores of filtrate standby.

2.2 impact---70 of writhing method mices on pain reaction, male and female half and half, by being divided at random 7 groups after body weight layering, i.e. blank group, positive controls (tramadol group) and 5 dosage ratio groups, 10 every group, every day, 1 continuous gastric infusion was 3 days.The results are shown in Table 2.

The impact of the different composition of prescription of table 2 on mouse writhing reaction

Note: with the comparison of blank group, ^*p < 0.05; ^*p < 0.01; ^{* *}p < 0.001

As can be seen from Table 2, the prescription of proportion compatibility all can make mice occur that the prolongation of latency of writhing response and writhing number of times reduce within the scope of the present invention, relatively there is significant statistical significance (P < 0.05 or P < 0.01 or P < 0.001) with blank group, the suppression ratio of mouse writhing number of times is all greater than to 60%, but Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata and Radix Notoginseng proportioning ratio be 30,20,15,12 and 6g time effect the most remarkable.2.3 70 of mice caused by dimethylbenzene xylene ear swelling test mices, entirely male, by being divided at random 7 groups after body weight layering, i.e. blank group, positive controls (dexamethasone acetate) and and 5 dosage ratio groups, 10 every group.Except positive controls is in test intraperitoneal injection on the same day, the continuous gastric infusion of all the other every treated animals 3 days.The results are shown in Table 3.

The impact of the different composition of prescription of table 3 on mice auricle swelling

Group	N(only)	Dosage (g/kg)	Ear swell poor (mg)	Suppression ratio (%)
					Blank group	10	Equal-volume	14.4±1.6	?
Positive controls	10	0.005	4.3±1.1 ^***	70.14
					1	10	10.95	7.2±2.7 ^*	50.00
2	10	10.95	8.5±2.7 ^*	40.97
					3	10	10.95	6.8±2.2 ^***	52.78
4	10	10.95	7.3±1.8 ^**	49.31
					5	10	10.95	9.3±1.7 ^*	35.42

As can be seen from Table 3, the mice ear that the prescription xylol of different proportion compatibilities causes has obvious inhibitory action, relatively has obvious statistical significance (P < 0.05) with blank group.And Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata and Radix Notoginseng proportioning ratio be 30,20,15,12 and 6g time effect the most remarkable.

3 conclusions

By antiinflammatory and analgesia research, result shows that Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata and Radix Notoginseng proportioning ratio are 30,20,15,12 and acts on antiinflammatory during 6g and analgesic activity is all the most remarkable.

The experimental study of experimental example 2 drug particles analgesic and anti-inflammatory effects of the present invention

1 material

1.1 medicines and reagent

Exempt from arthralgia eliminating granule (abbreviation granule), by embodiment 1 method preparation; Tramadol hydrochloride is produced (lot number: 0406245) by Beijing Sihuan Medicine Science and Technology Co., Ltd; Dexamethasone acetate injection is produced (lot number: 66040099) by Xinan Pharmaceutical Co., Ltd.; Glucosidorum Tripterygll Totorum is produced (lot number: 20040503) by Xieli Pharmaceutical Co., Ltd., Hunan; Luohe City the second pharmaceutical factory in cortisone acetate Pian You Henan Province produces (lot number: 2,004,021 3).The fresh configuration of normal saline before use of all medicines, gastric infusion, 1 times/day.Freund's complete adjuvant (FCA) (Sigma company product).

1.2 laboratory animal

KM kind white mice, clean level, male and female dual-purpose, body weight (20 ± 2) g; Healthy SD rat, male, clean level, body weight (200 ± 20) g; By Sichuan Academy of Medical Sciences institute of lab animals, provide, the laboratory animal quality certification number is: SCXK(river) 2004-16.

2 methods and result

2.1 analgesic activity

2.1.1 granule is on 50 of impact-hot plate method mices of pain reaction, entirely female, by being divided at random 5 groups after body weight layering, it is the groups of grains (32.4,16.2,8.1g crude drug in whole/kg) of blank group, positive controls and high, medium and low 3 dosage, every group 10, every day, 1 continuous gastric infusion was 3 days.The results are shown in Table 4.

The impact of table 4 granule on hot plate method latency of pain response

As can be seen from Table 4, after administration, in 2h, all can there is the incubation period that pain is reacted by significant prolongation mice in granule and tramadol hydrochloride, relatively have significant statistical significance (P < 0.05 or P < 0.01) with blank group, after administration, 4～6h is also improved the trend of latency of pain response.

2.1.2 50 of the impact of granule on pain reaction---writhing method mices, male and female half and half, grouping and the same 2.1.1 of administration, the results are shown in Table 5.

The impact of table 5 Granules on Mouse writhing response

As can be seen from Table 5, granule can make mice occur that the prolongation of latency of writhing response and writhing number of times reduce, relatively there is significant statistical significance (P < 0.05 or P < 0.01 or P < 0.001) with blank group, the suppression ratio of mouse writhing number of times is all greater than to 50%.

2.2 antiinflammatory action

2.2.1 mice caused by dimethylbenzene xylene ear swelling test mice is 50, complete male, by being divided at random 5 groups after body weight layering, i.e. blank group, the groups of grains of positive controls and high, medium and low 3 dosage (32.4,16.2,8.1g crude drug in whole/kg), 10 every group.Except positive controls is in test intraperitoneal injection on the same day, the continuous gastric infusion of all the other every treated animals 3 days.The results are shown in Table 6.

The impact of table 6 Granules on Mouse auricle edema

Group	N(only)	Dosage (g/kg)	Ear swell poor (mg)	Suppression ratio (%)
					Blank group	10	Equal-volume	14.8±1.8	?
Dexamethasone group	10	0.005	6.3±1.7 ^***	57.4
					Granule high dose group	10	32.4	12.1±2.1 ^*	1?8.2
Dosage group in granule	10	16.2	8.5±2.7 ^***	42.6
					Granule low dose group	10	8.1	1?1.9±2.17 ^*	19.6

As can be seen from Table 6, the mice ear that granule xylol causes has obvious inhibitory action, relatively has obvious statistical significance (P < 0.05) with blank group.

2.2.2 acetic acid causes the hyperfunction experiment grouping of mouse peritoneal capillary permeability and the same 2.2.1 of administration, the results are shown in Table 7.

The impact of table 7 ant Granules on Mouse abdominal cavity capillary permeability

Group	N(only)	Dosage (g/kg)	OD value
				Blank group	10	Equal-volume	1.5±.50
Dexamethasone	10	0.005	0.7±0.6 ^***
				Granule high dose group	10	32.4	1.0±0.4 ^**
Dosage group in granule	10	16.2	0.8±0.3 ^***
				Granule low dose group	10	8.1	0.7±0.4 ^***

As can be seen from Table 7, mouse peritoneal capillary permeability that granule causes glacial acetic acid is hyperfunction has obvious inhibitory action, relatively has significant statistical significance (P < 0.01 or P < 0.001) with blank group.

2.2.3 50 of healthy male SD rats, body weight 1 80～220g are got in the experiment that Ovum Gallus domesticus album causes rat paw edema.Be divided at random 5 groups, it is blank group, the groups of grains of positive controls and high, medium and low 3 dosage (25.2,12.6,6.3g crude drug in whole/kg), except positive controls is in test intraperitoneal injection on the same day, the continuous gastric infusion of all the other every treated animals 5 days.The results are shown in Table 8

The impact of table 8 granules in rats Ovum Gallus domesticus album foot swelling

() represents foot swelling inhibition percentage

As can be seen from Table 8, the rat paw edema that granule causes Ovum Gallus domesticus album has obvious inhibitory action, relatively has significant statistical significance (P < 0.05 or P < 0.01) with blank group.

2.2.4 the impact on chronic inflammation model-mice granuloma induced by implantation of cotton pellets

Get 50 of healthy male mices, by body weight, be divided at random 5 groups, the same 2.2.1 that divides into groups, successive administration 7 days, outside positive group intraperitoneal injection every day, all the other are gavage, the results are shown in Table 9.

The impact of table 9 Granules on Mouse granuloma induced by implantation of cotton pellets

Group	N(only)	Dosage (g/kg)	Granulation dry weight (mg)	Granulation index
					Blank group	10	Equal-volume	1?0.6±2.6	4.7±1.1
Dexamethasone group	10	0.005	4.9±1.3 ^***	2.3±0.6 ^***
					Granule high dose group	10	32.4	7.5±0.9 ^***	3.4±0.5 ^***
Dosage group in granule	10	16.2	7.7±0.8 ^***	3.4±0.5 ^***
					Granule low dose group	10	8.1	7.0±0.8 ^***	3.1±0.5 ^***

As can be seen from Table 9, granule is to the significant inhibitory action of being formed with of granulation, and granulation dry weight and granulation index and blank group more all have extremely significant statistical significance (P < 0.001).

2.2.5 the inhibitory action to adjuvant-induced arthritis

Get 50 of rats, by body weight, be divided at random 5 groups: (1) matched group; (2) granule prevention group; (3) granule therapy group; (4) cortisone prevention group; (5) cortisone treatment group, 10 every group.Rat under etherization, with FCA0.1ml, be injected in right back foot pad, injection adjuvant started administration the same day is prevention group, the 8th day starts administration is treatment group, and be administered once every day, granule 12.6g/kgd, cortisone 25mg/kgd, matched group gavages isopyknic normal saline, continuous 21 days, within the 22nd day, kills and cuts open animal.Injection adjuvant before and after 3 hours and fix the date and respectively survey the sufficient sole of the foot volume in left and right once every one, is swelling with the parapodum sole of the foot in giving the difference of volume before and after adjuvant, and observes Mus ear erythema, afterbody tuberosity etc. and occur situation.

Preventative and the therapeutic administration of granule, all can obviously suppress Earlier period of inflammation reaction and the swelling once again after 12 days of injection site, can suppress again the foot swelling that another hind leg delayed hypersensitivity causes, action intensity is similar to cortisone, the results are shown in Table 10.Each administration group rat ear's erythema and afterbody tuberosity and normal saline group more obviously alleviate.

The impact of table 10 granule on adjuvant arthritis rats pedal swelling

Note: with the comparison of normal saline group, ^*p < 0.05; ^*p < 0.01; ^{* *}p < 0.001

As can be seen from Table 10, granule is preventative all has inhibitory action significantly with therapeutic administration to adjuvant arthritis rats constitutional and Secondary cases foot swelling, more all has statistical significance (P < 0.05 or P < 0.01 or P < 0.001) significantly with blank group.

3. result

This experiment confirms that by hot plate method and the experiment of acetic acid twisting method granule has good analgesic activity; By mice dimethylbenzene ear swelling, glacial acetic acid abdominal cavity capillary permeability, resist into confirming that with rat Ovum Gallus domesticus album foot swelling experiment granule all has good antagonism to acute exudative inflammation; By mice granuloma induced by implantation of cotton pellets, experimental results show that granule also has good antagonism to chronic inflammatory disease; By adjuvant-induced arthritis, tested and confirmed that granule has significant antagonism to immunologic injury inflammation, result of study prompting granule has significant analgesia and antiinflammatory action.

The impact of experimental example 3 drug particles of the present invention on adjuvant arthritis rats synovial tissue pathomorphology

1, material

1.1 medicines and reagent

Exempt from arthralgia eliminating granule (abbreviation granule), by embodiment 1 method preparation;

Glucosidorum Tripterygll Totorum is produced (lot number: 20040503) by Xieli Pharmaceutical Co., Ltd., Hunan;

Freund's complete adjuvant (FCA) (Sigma company product);

1.2 key instrument

Microscope BH-2, Olympus;

1.3 laboratory animal

Healthy SD rat, SPF level, male, body weight (200 ± 20) g, is provided by Sichuan Academy of Medical Sciences institute of lab animals, and the laboratory animal quality certification number is: SCXK(river) 2004-16.

2, method

2.1 group technology

60 SD rats are divided into 6 groups after by body weight layering at random, be dosage group (middle dosage group) and granule low dose group (low dose group) in Normal group (blank group), model control group (model group), Radix Tripterygii Wilfordii positive controls (positive group), granule high dose group (high dose group), granule, every group 10, gastric infusion once a day.Except normal group, respectively organize rat all by modeling method modeling.

2.2 modeling method

With Freund's complete adjuvant, in the subcutaneous inserting needle of the right back toes of modeling Mus to ankle joint, injection Freund adjuvant 0.1ml/ only induces arthritic generation.

2.3 medication

The high, medium and low dosage group of granule gavages that medicinal liquid is respectively 25.2,12.6g and 6.3 crude drug in whole/(kgd); Radix Tripterygii Wilfordii group gavages medicinal liquid 5mg/ (kgd), and normal group and model group gavage isopyknic normal saline, and modeling starts gastric infusion on the 14th day, until experiment finishes.

The drawing materials and prepare of 2.4 specimen

After testing last administration in the 32nd day, rat fasting was put to death rat after 24 hours, on aseptic working platform, take off right ankle joint, 1 0% neutral formalin articular cavity perfusion is fixedly after 6-8 hour, separated synovial tissue, conventional film-making, HE dyeing, marks to cell infiltration, synovial cell proliferation and synovial membrane proliferation of fibrous tissue, by 5 grades of point systems of lymphocyte of adjurant arthritis rat pathology ^[2](cell infiltration, 0: without inflammatory cell infiltration; 1:1～5/HP; 2:6～10/HP; 3:11～15/HP; 4: form microabscess.Synovial cell proliferation degree: 0: without infiltrating; 1: swelling hypertrophy (many); 2: increase connected; 3: increase bilayer; More than 4:3 layer.Synovial membrane proliferation of fibrous tissue degree: 0: without hypertrophy; 1: hypertrophy accounts for 1/3HP; 2: hypertrophy accounts for 1/2HP; 3: hypertrophy accounts for 1HP; 4: hypertrophy accounts for >1HP), the morphologic change of tissues observed under light microscopic.

3 results

Adjuvant Arthritis Model in Rats synovium of joint cell infiltration, hypertrophy are obvious, and especially hypertrophy is remarkable, and the pathological change of each administration group synovium of joint of granule is light compared with model group.The results are shown in Table 11

Table 11 synovium of joint pathological evaluation (HE dyeing, scoring method)

Group	N (only)	Dosage (g/kg)	Cell infiltration	Synovial cell proliferation	Synovial membrane fibrous tissue increases
						Normal group	1?0	Equal-volume	0.30±48 ^***	0.20±0.42 ^***	0.1?0±0.32 ^***
Model group	8	Equal-volume	2.88±0.99	2.25±1.04	2.13±0.99
						Radix Tripterygii Wilfordii group	9	0.005	0.78±0.67 ^***	1.22±0.67 ^**	1.22±0.44 ^**
High dose group	9	25.2	1.44±0.88 ^***	1.44±0.88 ^*	1.33±0.50 ^**
						Middle dosage group	9	12.6	1.11±0.60 ^***	1.33±0.50 ^**	1.22±0.44 ^**
Low dose group	9	6.3	2.00±1.00 ^*	1.56±0.53 ^*	1.44±0.53 ^*

Note: with model group comparison, ^*p < 0.05; ^*p < 0.01; ^{* *}p < 0.001

Experimental example 4 drug particles of the present invention is on the impact on adjuvant arthritis rats T cell subsets

1 material

1.1 medicines and reagent

Exempt from arthralgia eliminating granule (abbreviation granule), by embodiment 1 method preparation; Glucosidorum Tripterygll Totorum is produced (lot number: 20040503) by Xieli Pharmaceutical Co., Ltd., Hunan; The fresh configuration of normal saline before use of all medicines, gastric infusion, 1 time/d; Freund's complete adjuvant (FCA) (Sigma company product); The Mus FITC-CD of the Chinese People's Anti-Japanese Military and Political College ₃/ PE-CD ₄two mark monoclonal antibodies, the Mus FITC-CD of the Chinese People's Anti-Japanese Military and Political College ₃/ PE-CD ₈two mark monoclonal antibodies, hemolysin lysing solution (BD company product).

1.2 key instrument

BECTON DICKINSON FACSCAN flow cytometer;

XH-B type vortex mixer, Jiangyan City healthy medical apparatus company limited;

CENTRIFVGE MODEU 0412-7 centrifuge, Shanghai Surgical Operation Equipment Factory;

1.3 laboratory animal

Healthy SD rat, SPF level, male, body weight (200 ± 20) g, is provided by Sichuan Academy of Medical Sciences institute of lab animals, and the laboratory animal quality certification number is: SCXK(river) 2004-16.All animals are all tested after the animal housing of constant temperature and humidity raises 1 week.

2 methods

2.1 group technology

40 SD rats are divided into 5 groups after by body weight layering at random, be Normal group (normal group), model control group (model group), Radix Tripterygii Wilfordii positive controls (Radix Tripterygii Wilfordii group), granule high dose group (high dose group), granule low dose group (low dose group), every group 8, dosage is in Table 9.Except normal group, respectively organize rat all by modeling method modeling.

2.2 modeling method

2.3 medication

Granule high dose group gavages medicinal liquid 12.6g crude drug in whole/(kgd), and granule low dose group gavages medicinal liquid 6.3g crude drug in whole/(kgd); Radix Tripterygii Wilfordii group gavages medicinal liquid 5mg/ (kgd), and normal group and model group gavage isopyknic normal saline, and modeling starts gastric infusion on the 14th day, until experiment finishes.

The drawing materials and prepare of 2.4 specimen

Test after last administration in the 32nd day rat fasting 24 hours, femoral artery sacrificed by exsanguination.With heparinization test tube (100 μ/ml), collect blood.Get anticoagulation 100 μ l and add after mensuration pipe, add corresponding antibodies 10 μ l, whirlpool mixes, room temperature lucifuge is hatched 30min, add again hemolysin 1ml, mix, hatch 10min, add PBS washing 1000rpm/min, each 5min, continuous three times, adjustment cell number is 106/ml, upper machine, collects 30000 cells and analyzes with cellquest software; Negative control pipe removes the corresponding antibodies adding and is respectively FITC-IgG1 and PE-IgG2, all the other time-and-motion study pipes.

3 results

Table 12 is respectively organized the comparison that rat peripheral blood T cells subgroup positive cell changes

Note: with model group comparison, ^*p < 0.05 ^*p < 0.01

As shown in Table 12, model group rat CD ₄ ⁺t cell compares not statistically significant (P > 0.05), CD with blank group ₈ ⁺t cell presents significantly low (P < 0.05), CD ₄ ⁺/ CD ₈ ⁺ratio extremely significantly increases (P < 0.01), and Radix Tripterygii Wilfordii group, high dose group and low dose group all have significant regulating action to disorderly T cell subsets.

Granule has good regulating action to the disorder of T cell subsets, and its mechanism may be in the positive selection at thymus at T cell, the specific raising of granule CD ₃ ⁺cD ₈ ⁺two positive cells are combined with thymic cortex surface epithelial cell mhc class i molecule, make CD ₈ ⁺the generation of cell increases, thereby the Th/Ts balance of imbalance is recovered, thus performance therapeutical effect, and this may be one of approach of granule performance therapeutical effect.

The impact of experimental example 5 medicine super fine of the present invention on RA-MsPGN Renal Function in Rats

1 material

1.1 Experimental agents

Super fine (abbreviation micropowder), by embodiment 2 method preparations; Tripterygium wilfordii Polyglycosidium Tablets is produced (lot number: 20040503) by Xieli Pharmaceutical Co., Ltd., Hunan.The fresh configuration of normal saline before use of all medicines, gastric infusion, 1 times/day.

1.2 reagent and instrument

Freund's complete adjuvant (FCA), incomplete Freund's adjuvant (FIA), LPS (E.Coli O111:B4) and bovine serum albumin (N-BSA), be U.S. Sigma company; Holland prestige figure biochemical instruments.

1.3 laboratory animal

Healthy SD rat, male, SPF level, body weight (200 ± 20) g; By Sichuan Academy of Medical Sciences institute of lab animals, provided, the laboratory animal quality certification number is: SCXK(river) 2004-16.

2 experimental techniques

2.1 reconstruction animal models [Xu Shijun, Shen Yingjun, Eurasian dragon, etc.The impact of the attached super fine of ant on RA-MsPGN rat model renal function.China's combination of Chinese and Western medicine nephropathy magazine, 2007; 8 (7): 391～393]

(1) immunity in advance: other group rat except Normal group adds 3mg bovine serum albumin (N-BSA) in the right sufficient lift hemostasis complete Freund's adjuvant 0.1ml of experimental mouse, after this respectively at strengthening 1 time at 1 weekend, 2 weekends, the incomplete Freund's adjuvant of the subcutaneous branch injection in back simultaneously 0.1ml, includes 3mgBSA; 3 weekends, continuous 4 the injection BSA in abdominal cavity, interval 1h, injected dose is respectively every 0.5mg, 1.0mg, 1.5mg, 3.0mg; Strengthen morning next day 1 time (2.0mg/ only).

(2) formally immunity: other group rat N-BSA tail vein injection and abdominal cavity multiple spot subcutaneous injection except Normal group hocket every other day, and tail vein injection dosage, from every 0.5mg, increases 0.5mg, till 2.5mg at every turn.Lumbar injection amount is 1 times of tail vein injection amount.2 weeks rear tail vein injection escherichia coli endotoxin 200ng/ of immunity only.Formal immunity totally 7 weeks.

(3) model evaluation: from modeling the 4th week, choose at random two model group rats per weekend and do synovium of joint and Pathological inspection, whether successful with evaluation model.

2.2 grouping and administrations:

73 rats are divided into 6 groups at random, and blank group (blank group) is 13,20 of model control group (model group); Positive controls (Radix Tripterygii Wilfordii group), the high, medium and low dosage group of micropowder (high, medium and low dosage group), every group each 10, the free diet of animal, lower minute cage of equal conditions fed.After modeling 3 weeks, gastric infusion once a day, blank group and model group give equal-volume normal saline; Positive group gives Radix Tripterygii Wilfordii suspension 5mg/kg; The medicinal liquid of high, medium and low dosage group difference 25.2,12.6 and 6.3g crude drug in whole/kg, to 10 weekends.

2.3 draw materials and specimen preparation

After last administration, water is can't help in fasting, adopts metabolism of rat cage to collect twenty-four-hour urine liquid, adopts biuret method to measure urine protein quantitation; Femoral artery sacrificed by exsanguination rat, collects blood, and separation of serum is measured serum urea nitrogen and creatinine; Asepticly win left kidney, 10% formaldehyde is fixed, and paraffin embedding is cut 2～3um thin slice, HE dyeing, om observation.

3 statistical method

Measurement data adopts one factor analysis of variance, relatively adopts t check between group; Ranked data adopt Ridit to analyze.

4 results

4.1 animal models:

Animal injection complete Freund's adjuvant right sufficient obvious tumefaction next day, adds the peak that weighs day by day, then alleviates gradually, after the 10th day, occurs swelling for the second time, the 12nd day offside hind leg, the swelling of forelimb secondary, and the 3rd week there is " rheumatism tuberosity " in afterbody; Within the 4th week, pathomorphology inspection synovium of joint is shown in that typical adjuvant-induced arthritis changes, and kidney mesentery is slightly bred simultaneously, to the 6th week remarkable hypertrophy of kidney mesentery, occurs the pathological change of typical mesentery appreciation nephritis.This model modeling factor and pathogenic process meet the feature of RA mesentery appreciation nephritis substantially, pathological change also proves that rat not only has typical arthritic feature, the feature simultaneously with MsPGN, illustrates this model modeling success, can be for the research of RA mesentery appreciation nephritis.

The impact of 4.2 micropowders on Rat 24 h urine protein quantitation, creatinine (BUN) and blood urea nitrogen (Crea), the results are shown in Table 13.

The impact of table 13 super fine on rat urine protein quantification, creatinine and blood urea nitrogen

Note: with model group comparison, ^*p < 0.05; ^*p < 0.01, ^{* *}p < 0.001

The impact of 4.3 micropowders on rat mesangial cell in vitro, extracellular matrix pathological change

The observation of rat kidney pathological change adopts Wang Shi [Wang Jun, Zheng Jiaxin, Sun Ruitao.The pathology effects of the former capsule of kidney to MsPGN rat model.China's combination of Chinese and Western medicine nephropathy magazine, the grade scale of 2002,3 (3) 6:346-347..That is: (1)+slight: each mesangial region is containing 3 nucleus; Extracellular matrix slightly increases, and capillary lumen is not squeezed and is open shape, and hypertrophy mesentery width is no more than the diameter of blood capillary.(2) ++ moderate: each mesangial region is containing 4 nucleus; Extracellular matrix moderate increases, and be less than 50% Capillary loops and be squeezed, tube chamber mild stenosis, the mesentery width of hypertrophy surpasses the diameter of blood capillary, and capillary lumen presents the extruding phenomenon that weight does not wait.(3) +++ severe: each mesangial region is containing 5 or more nucleus; Extracellular matrix severe increases, and the capillary loops more than 50% is squeezed, on the basis that the mesentery of tube chamber severe stenosis or inaccessible hypertrophy distributes in diffusivity finger-like, being block assembles, extracellular matrix showed increased, the position of assembling in lumps hypertrophy, capillary structure is destroyed blood vessel and is disappeared.Concrete data are in Table 14.

The impact of table 14 micropowder on rat mesangial cell in vitro, extracellular matrix pathological change

Note: the comparison of pathology degree, with model group comparison, ^*p < 0.05; ^*p < 0.01

5. conclusion

Result of study shows, model group compared with normal group twenty-four-hour urine amount, urine protein quantitation and serum creatinine be significantly rising (p < 0.05 or p < 0.001) all, blood urea nitrogen is without significant change (P > 0.05), and each dosage group of the attached super fine of Tripterygium wilfordii Polyglycosidium Tablets and ant all can make twenty-four-hour urine amount, urine protein quantitation and serum creatinine, and it significantly reduces (p < 0.05 or p < 0.01); Each dosage group of the attached super fine of ant and Tripterygium wilfordii Polyglycosidium Tablets group be zero difference (P > 0.05) relatively; Match with pathology testing result, illustrate that super fine has good protective effect to the renal function injury due to RA, to the hypertrophy of mesangial cell and extracellular matrix, also have good inhibition to do.

The impact of experimental example 6 drug powder of the present invention on adjuvant arthritis rats foot swelling and T cell subsets

1. material is in method:

1.1 trial drugs: super fine, by embodiment 2 method preparations.Hydrocortisone injection, Xinan Pharmaceutical Co., Ltd.'s product, lot number: 66040099.

1.2, experimental animal: SD rat, body weight 1 80～220g, is SPF level, and the animal quality certification number is provided by Sichuan Academy of Medical Sciences institute of lab animals: real moving pipe matter SCXK (river) 2004-16 in river.Experiment is carried out at Chengdu University of Traditional Chinese Medicine's herbal pharmacology laboratory (three grades of herbal pharmacology laboratorys of State Administration of Traditional Chinese Medicine, certificate number is TCM-03.043).

1.3, reagent and instrument:

1.3.1 reagent: complete Freund's adjuvant, Sigma company product; The two mark of the Mus FITC-CD3/PE-CD4 of Chinese People's Anti-Japanese Military and Political College monoclonal antibody, the two mark of the Mus FITC-CD3/PE-CD8 of Chinese People's Anti-Japanese Military and Political College monoclonal antibody, hemolysin lysing solution (U.S. company BD).

13.2 key instruments: Becton Dickinson Facscan flow cytometer (U.S. Block Scientific company).

1.4, method:

1.4.1, grouping and administration: 60 SD rats are divided into 6 groups at random by body weight, be dosage group, super fine low dose group in Normal group (normal group), model control group (model group), cortisone matched group (cortisone group), super fine high dose group, super fine, 10 every group.Except normal group, respectively organize rat all by modeling method modeling.Modeling started administration, the corresponding medicinal liquid 10mlkg of each administration treated animal ig the same day ^-1, normal group and model group ig10 mlkg ^-1normal saline, every day 1 time, successive administration 21 days, grouping and dosage in Table 15.

1.4.2, model preparation: in the subcutaneous inserting needle of the right back toes of modeling Mus to ankle joint, injection Freund adjuvant 0.1ml/ only induces arthritic generation with Freund's complete adjuvant (FCA).

1.4.3, measuring:

1) impact on adjuvant arthritis rats foot volume: before experiment, the right back ankle joint of each Mus is marked, before injection FCA, after 3h and injection, respectively survey at regular intervals the sufficient sole of the foot volume in left and right once, the difference that gives adjuvant front and back volume with the parapodum sole of the foot is swelling, the results are shown in Table 12

2) impact of human peripheral blood T cell subgroup: rat fasting 24h after last administration, femoral artery sacrificed by exsanguination.Every group choose at random 8 for detection of.With heparinization test tube (100Uml ^-1) collection blood.Get anticoagulation 100 μ l and add after mensuration pipe, add corresponding antibodies 10 μ l, whirlpool mixes, and room temperature lucifuge is hatched 30min, then adds hemolysin 1ml, mixes, and hatches 10min, adds PBS washing 1 * 10 ³rpmmin ^-1, each 5min, continuous three times, adjusting cell number is 10 ⁶cellml ^-1, upper machine, collects 3 * 10 with Cellquest software ⁴individual cell is also analyzed; Negative control pipe is except the corresponding antibodies adding is respectively FITC-IgG1 and PE-IgG2, and all the other operations are with measuring pipe.The results are shown in Table 13

1.4.4, statistical procedures: application SPSS15.0 statistical software, data represent with X ± S, each is organized data and is normal distribution and carries out one factor analysis of variance (one-wayANOVA), nonnormal distribution is carried out K-W check.

2. result

2.1 impacts on adjuvant arthritis rats foot volume:

The results are shown in Table 15.From table, with model group comparison, the positive group of cortisone after modeling 1,3h, and 3,5,8,12,15, the foot swelling value of 18d is all starkly lower than model group (P < 0.01), high, medium and low three the dosage groups of super fine after modeling 1,3h, and 3,5,8,12,15, the foot swelling value of 18d and model group significantly reduce (P < 0.05 or P < 0.01), shows good antiphlogistic effects.

The impact of table 15 super fine on adjuvant arthritis rats pedal swelling

Note: with model control group comparison, ^*p < 0.05; ^*p < 0.01

The impact of 2.2 human peripheral blood T cell subgroups:

The results are shown in Table 16.

The comparison of table 16 super fine t lymphocyte subsets of peripheral blood impact

Note: with model group comparison, ^*p < 0.05; ^*p < 0.01

From table, with model group comparison, high, medium and low three the dosage groups of super fine can significantly reduce CD ₄ ⁺/ CD ₈ ⁺ratio (P < 0.05 or P < 0.01); High, the middle dosage group of super fine can significantly reduce CD ₄ ⁺% value (P < 0.01), low dose group performance is not obvious; Super fine shows good antiphlogistic effects.

3. conclusion

This experimental result shows, each dosage of super fine all has and alleviates preferably rat model foot swelling and reduce CD ₄ ⁺t value.

Experimental example 7 drug particles of the present invention and the comparative study of super fine anti-inflammatory and analgesic effect

1 material

1.1 trial drug super fines (abbreviation micropowder), by embodiment 2 method preparations; Exempt from arthralgia eliminating granule (abbreviation granule), by embodiment 1 method preparation.The dosage that in test, the crude drug in whole consumption of two kinds of dosage forms is clinical identical multiple (1/12 that micropowder is granule).Tramadol hydrochloride is produced (lot number: 0406245) by Beijing Sihuan Medicine Science and Technology Co., Ltd; Dexamethasone acetate injection is produced (lot number: 66040099) by Xinan Pharmaceutical Co., Ltd.; Tripterygium wilfordii Polyglycosidium Tablets is produced (lot number: 20040503) by Xieli Pharmaceutical Co., Ltd., Hunan.All medicines are used the fresh configuration of normal saline, gastric infusion, 1 times/day before use.

1.2 animal KM kind white mice, male and female dual-purpose, body weight (20 ± 2) g; SD rat, SPF level, male, body weight (200 ± 20) g; By Sichuan Academy of Medical Sciences institute of lab animals, provide, the laboratory animal quality certification number is: SOCK (river) 2004-16.

2 methods and result

2.1 xylol cause 80 of the male mices of impact of mice auricle swelling, by body weight stratified random, are divided into 8 groups.High, medium and low 3 dosage of groups of grains are respectively 32.4,16.2,8.1 crude drug in whole/kg, and high, medium and low 3 dosage of micropowder group are respectively 2.7,1.35,0.675g crude drug in whole/kg.Except Dexamethasone group is in test intraperitoneal injection on the same day, all the other every treated animal gastric infusions, every day 1 time, for three days on end.1h after last administration, is evenly applied to the wide two sides of mouse right ear by 2Oral dimethylbenzene and causes inflammation, left ear in contrast, cause scorching after 30min put to death animal, with the card punch of diameter 8mrn, take off left and right auricle and weigh.Using left and right auricle weight difference as swelling, calculate swelling inhibition percentage, the results are shown in Table 17.

The impact of two kinds of dosage forms of table 17 on mice auricle swelling

Group	Mus number (only)	Dosage (g/kg)	The ear method of double differences (mg)	Suppression ratio (%)
					Matched group	10	?	14.8±1.8	?
Dexamethasone	10	0.005	6.32±1.7 ^**	57.4
					Granule high dose group	10	32.40	12.12±2.1 ^*	1?8.2
Dosage group in granule	10	16.20	8.47±2.7 ^**	42.6
					Granule low dose group	10	8.1?0	11.85±2.17 ^*	19.6
Micropowder high dose group	10	2.70	1?1.43±3.6 ^**	23.0
					Dosage group in micropowder	10	1.35	9.98±3.3 ^**	32.4
Micropowder low dose group	10	0.675	11.52±3.9 ^**	22.3

With matched group comparison ^*p < 0.05, ^*p < 0.01 (lower same)

The demonstration of table 17 result, the granule of 3 dosage and micropowder xylol cause mice ear all obvious inhibitory action, and under clinical identical multiple consumption, two kinds of dosage forms compare not statistically significant.

2.2 Dichlorodiphenyl Acetates cause mouse peritoneal capillary permeability hyperfunction affect grouping and administration with 2.1,1h after last administration, mouse tail vein injection 1% azovan blue 0.1M1/10g body weight, lumbar injection 0.6% acetum 0.2ml/ is only simultaneously.After 20min, put to death mice, with 5ml distilled water flushing abdominal cavity, collect flushing liquor, centrifugal, get supernatant in spectrophotometer 590nm colorimetric, with the permeability of absorbance (OD) value judgement mouse peritoneal blood capillary.The results are shown in Table 18.

The impact that two kinds of dosage forms of table 18 are hyperfunction on mouse peritoneal capillary permeability

Group	Mus number (only)	Dosage (g/kg)	OD value
				Matched group	1?0	?	1.45±0.50
Dexamethasone	1?0	0.05	0.66±0.6 ^**
				Granule high dose group	1?0	32.40	0.96±0.4 ^**
Dosage group in granule	1?0	16.20	0.83±0.3 ^**
				Granule low dose group	1?0	8.10	0.74±0.4 ^**
Micropowder high dose group	1?0	2.70	0.52±0.1 ^**
				Dosage group in micropowder	1?0	1.35	0.39±0.2 ^**
Micropowder low dose group	1?0	0.675	0.54±0.2 ^**

Table 18 result shows, the granule of 3 dosage and super fine Dichlorodiphenyl Acetate cause that mouse peritoneal capillary permeability is hyperfunction all an extremely significant inhibitory action, two kinds of dosage forms not statistically significants relatively under identical multiple consumption.

2.3 on the foot swelling of rat Ovum Gallus domesticus album affect 60 of rats, by body weight stratified random, be divided into 6 groups.Except dexamethasone is in test intraperitoneal injection on the same day.All the other every treated animal gastric infusions, every day 1 time, continuous 5 days.Before experiment, at the right back ankle joint of each Mus, mark, with sufficient volume measuring apparatus, measure twice of each Mus foot volume.Average as normal foot volume.30min after last administration, every subcutaneous inserting needle of Rat Right metapedes sole of the foot portion is near subcutaneous injection lO～X ankle joint, the clear solution 0.1ml of Fresh Egg causes inflammation, respectively at cause scorching after 30,60,120,240,360min measures and causes scorching sufficient volume, calculate each rat and cause the right back sufficient sole of the foot volume-variation value in scorching front and back, the anti-inflammatory effect that represents medicine with paw swelling and the swollen inhibition percentage of foot, the results are shown in Table 19.

The impact of two kinds of dosage forms of table 19 on the foot swelling of rat Ovum Gallus domesticus album

() interior data are that foot swelling suppresses percentage

The demonstration of table 19 result, the granule of high and low dose and micropowder all have extremely significant inhibitory action to the rat paw edema due to Ovum Gallus domesticus album. and under clinical identical multiple consumption, two kinds of dosage forms compare not statistically significant.

2.4 groupings of the impact on mice granuloma induced by implantation of cotton pellets are with 2.1.The same day was implanted sterilizing cotton balls (10mg ± 0.5mg) in mouse armpit subcutaneous operation in administration, and gastric infusion is 7 days continuously, Dexamethasone group intraperitoneal injection.After 7 days, put to death mice.Peel off granulation tissue, in 60 ℃ of baking ovens, dry, weigh, calculate granuloma and weigh and granulation index, the results are shown in Table 20.The impact of two kinds of dosage forms of table 20 on mice granuloma induced by implantation of cotton pellets

Group	Mus number (only)	Dosage (g/kg)	Granulation dry weight (mg)	Granuloma index
					Matched group	10	?	10.60±2.6	4.70±1.1
Dexamethasone	10	0.005	4.92±1.3 ^**	2.26±0.6 ^**
					Granule high dose group	10	32.40	7.52±0.9 ^**	3.42±0.5 ^**
Dosage group in granule	10	16.20	7.65±0.8 ^**	3.44±0.5 ^**
					Granule low dose group	10	8.1?0	6.99±0.8 ^**	3.14±0.5 ^**
Micropowder high dose group	10	2.70	8.30±1.3 ^**	3.62±0.5 ^**
					Dosage group in micropowder	10	1.35	9.30±1.5 ^**	3.88±0.7 ^*
Micropowder low dose group	10	0.675	8.80±1.8 ^**	4.03±1.0 ^*

The demonstration of table 20 result, the granule of 3 dosage and micropowder all have significant inhibitory action to the formation of granulation, and under clinical identical multiple consumption, two kinds of dosage forms compare not statistically significant.

2.5 impacts on hot plate method test mice pain reaction regulate hot plate dolorimeter temperature, constant in 55 ± 0.5 ℃, the female mice of take is put and in people's dolorimeter, starts to clock to occurring licking metapedes required time (5) as pain week value. screen pain threshold 80 of the mices of asking of 5～30s, by body weight stratified random, be divided into 8 groups, matched group and each administration group are 10 mices, measure respectively every group every the wealthy value of mice pain 2 times, every minor tick 5min, averages as normal pain threshold (being the front pain threshold of administration).Then be subject to reagent or same volume normal saline 0.1ml/10g.Every day 1 time, for three days on end, after last administration 30,60,120,240,360min measure respectively organize mice bitterly the value of closing as administration after gate of a village value bitterly, pain threshold is greater than 60s person in 60s.The results are shown in Table 21.

The impact of two kinds of dosage forms of table 21 on hot plate method test mice pain reaction

The demonstration of table 21 result, the micropowder of 3 dosage, granule and tramadol hydrochloride all can significantly improve pain threshold after administration, and under clinical same amount multiple, two kinds of dosage forms compare not statistically significant.

2.6 Dichlorodiphenyl Acetate writhing method mice pains reactions affect 80 male and female half and half of mice, grouping and administration be with 2.1, the normal saline 0.1ml/l0g of gastric infusion merit same volume, after administration 30min, lumbar injection 0.6% glacial acetic acid solution 0.2ml/ is only.Writhing response number of times in 1 5min after observation injection acetic acid, and calculate analgesia rate, the results are shown in Table 22.

The impact of two kinds of dosage form Dichlorodiphenyl Acetate writhing method mice pain reactions of table 22

The demonstration of table 22 result, the micropowder of 3 dosage and granule can make mice occur that the prolongation of latency of writhing response and writhing number of times reduce, and under clinical identical multiple consumption, two kinds of dosage forms compare not statistically significant.

3 results

Result of study shows, under clinical identical multiple dosage, granule consumption is 12 times of micropowder, but the antalgic and inflammation relieving effect of two kinds of dosage forms is substantially similar.Prompting is under same or analogous therapeutic effect, and the consumption of micropowder is only 1/12 of granule.

The specific embodiment

Embodiment 1

Prescription crude drug forms: Formica fusca 3000g, Radix Astragali 2000g, Fructus Chaenomelis 1500g, Radix Aconiti Lateralis Preparata 1200g, Radix Notoginseng 600g

Get the crude drug of recipe quantity, Formica fusca, Radix Aconiti Lateralis Preparata, the Radix Astragali and papaya powder are broken into coarse powder, and adding distil water soaked after 30 minutes, added 6 times of water gagings to decoct three times, and each 1 hour, filter, filtrate was concentrated into relative density 1.07(60 ℃) clear paste, standby; Separately get the Radix Notoginseng of recipe quantity, pulverize, cross 120 mesh sieves, obtain Radix Notoginseng impalpable powder, Radix Notoginseng impalpable powder is mixed homogeneously with the clear paste of said extracted gained, add in pharmacy acceptable adjuvant according to the routine techniques of this area preparation, granulation agent.

Every gram of granule of the present invention is equivalent to 6 grams of crude drug.

Embodiment 2

Get the crude drug of the present invention of recipe quantity, Formica fusca, Radix Aconiti Lateralis Preparata, the Radix Astragali, Radix Notoginseng and Fructus Chaenomelis are pulverized, and cross 24 mesh sieves, obtain raw material coarse powder; Raw material coarse powder is placed in to the drying in oven 5h of 60 ℃, takes out, guarantee that pan feeding moisture should be lower than 4%; Coarse powder after drying is carried out to micronizing, and grinding time is 20min, obtains particle diameter and is about 8 μ m super fines.

Every gram of super fine of the present invention is equivalent to 1 gram of crude drug.

Embodiment 3

Prescription crude drug forms: Formica fusca 2500g, Radix Astragali 3000g, Fructus Chaenomelis 1200g, Radix Aconiti Lateralis Preparata 2000, Radix Notoginseng 500g

Get the crude drug of the present invention of recipe quantity, Formica fusca, Radix Aconiti Lateralis Preparata, the Radix Astragali, Radix Notoginseng and Fructus Chaenomelis are pulverized, and cross 24 mesh sieves, obtain raw material coarse powder, add the alcohol reflux three times of 10 times of amounts 70%, and each 1.5 hours, filter, merging filtrate, reclaims ethanol to without alcohol taste; Filtrate, by macroporous resin, is first used 5 times of water gaging eluting, and eluent is abandoned or adopted, then with adding 70% ethanol elution, collects eluent, and decompression recycling ethanol is extremely without alcohol taste; Add again acceptable adjuvant in pharmacy to make tablet according to the routine techniques of this area preparation.

Embodiment 4

Prescription crude drug forms: Formica fusca 4000g, Radix Astragali 1800g, Fructus Chaenomelis 2000g, Radix Aconiti Lateralis Preparata 1200g, Radix Notoginseng 1000g

Get the crude drug of the present invention of recipe quantity, Formica fusca, Radix Aconiti Lateralis Preparata, the Radix Astragali and Fructus Chaenomelis were soaked after 30 minutes, add 6 times of water gagings to decoct three times, each 1 hour, filter, filtrate is concentrated into relative density 1.07(60 ℃) clear paste, spraying is dry, and (inlet temperature is decided to be: 170～1 80 ℃, leaving air temp is decided to be: 90～1 00 ℃), collect dry extract powder; Radix Notoginseng is pulverized, and crosses 120 mesh sieves, obtains Radix Notoginseng impalpable powder, mixs homogeneously, then add acceptable adjuvant in pharmacy to make capsule according to the routine techniques of this area preparation with above-mentioned dry extract powder.

Embodiment 5

Prescription crude drug forms: Formica fusca 2000g, Radix Astragali 1500g, Fructus Chaenomelis 1000g, Radix Aconiti Lateralis Preparata 800g, Radix Notoginseng 400g;

Get recipe quantity crude drug, Formica fusca, Radix Aconiti Lateralis Preparata, the Radix Astragali, Radix Notoginseng and Fructus Chaenomelis are pulverized, cross 24 mesh sieves, obtain raw material coarse powder, add 8 times of amount methanol supersound extraction three times, front twice each 40min, 20min, filters merging filtrate for the third time, filtrate decompression is concentrated into relative density 1.1 5(60 ℃) clear paste, standby; Extracting resulting clear paste adds acceptable adjuvant in pharmacy to make drop pill according to the routine techniques of this area preparation again.

Embodiment 6

Prescription crude drug forms: Formica fusca 6000g, Radix Astragali 2000g, Fructus Chaenomelis 3000g, Radix Aconiti Lateralis Preparata 1000g, Radix Notoginseng 1500g;

Get the crude drug of the present invention of recipe quantity, Formica fusca, Radix Aconiti Lateralis Preparata, the Radix Astragali and Fructus Chaenomelis add 8 times of water gaging dipping 6h, percolation 24h, flow velocity 2L/h.Collect percolate, centrifugal, upper macroporous adsorptive resins, first wash with water, then use 70% ethanol elution, collect ethanol elution, filtrate is concentrated into relative density 1.07(60 ℃) clear paste, spraying dry (inlet temperature is decided to be: 170～1 80 ℃, leaving air temp is decided to be: 90～1 00 ℃), collects dry extract powder; Radix Notoginseng is pulverized, and crosses 120 mesh sieves, obtains Radix Notoginseng impalpable powder, mixs homogeneously, then add in pharmacy acceptable adjuvant according to the routine techniques granulation agent of this area preparation with above-mentioned dry extract powder.

Embodiment 7

Prescription crude drug forms: Formica fusca 2000g, Radix Astragali 4000g, Fructus Chaenomelis 1000g, Radix Aconiti Lateralis Preparata 3000g, Radix Notoginseng 500g;

Get recipe quantity crude drug, Formica fusca, Radix Aconiti Lateralis Preparata, the Radix Astragali, Radix Notoginseng and Fructus Chaenomelis are pulverized, and cross 24 mesh sieves, obtain raw material coarse powder, add 12 times of water gaging reflux, extract, three times, each 1.5h, filter, merging filtrate, adds acceptable adjuvant in pharmacy to make oral liquid according to the routine techniques of this area preparation.

Embodiment 8

Prescription raw material forms: Formica fusca extract 6000g, Radix Astragali extract 2000g, Fructus Chaenomelis extract 3000g, Radix Aconiti Lateralis Preparata extract 1000g, Radix Notoginseng extract 1500g;

Described Formica fusca extract, Radix Astragali extract, Fructus Chaenomelis extract, Radix Aconiti Lateralis Preparata extract, Radix Notoginseng extract are respectively the extract that Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata, Radix Notoginseng prepare through 60% alcohol reflux.Said extracted thing is pulverized as fine powder, mixed, add conventional adjuvant according to the routine techniques granulation agent of this area preparation.

Embodiment 9

Prescription raw material forms: Formica fusca extract 3000g, Radix Astragali extract 2000g, Fructus Chaenomelis extract 1500g, Radix Aconiti Lateralis Preparata extract 1200g, Radix Notoginseng extract 600g;

Described Formica fusca extract, Radix Astragali extract, Fructus Chaenomelis extract, Radix Aconiti Lateralis Preparata extract, Radix Notoginseng extract are respectively the extract that Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata, Radix Notoginseng prepare through acetone reflux, extract.Said extracted thing is pulverized as fine powder, mixed, add conventional adjuvant to make powder according to the routine techniques of this area preparation.

Embodiment 10

Prescription raw material forms: Formica fusca extract 2500g, Radix Astragali extract 3000g, Fructus Chaenomelis extract 1200g, Radix Aconiti Lateralis Preparata extract 2000, Radix Notoginseng extract 500g;

Described Formica fusca extract, Radix Astragali extract, Fructus Chaenomelis extract, Radix Aconiti Lateralis Preparata extract, Radix Notoginseng extract are respectively Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata, Radix Notoginseng and through decocting, boil the extract preparing.Said extracted thing is pulverized as fine powder, mixed, add conventional adjuvant to make tablet according to the routine techniques of this area preparation.

Claims

1. treat a pharmaceutical composition for rheumatic joint disease, it is characterized in that the crude drug of described pharmaceutical composition consists of:

Formica fusca 30g, Radix Astragali 20g, Fructus Chaenomelis 15g, Radix Aconiti Lateralis Preparata 12g, Radix Notoginseng 6g.

2. the preparation method of pharmaceutical composition as claimed in claim 1, is characterized in that the method comprises:

A, get five tastes crude drug in proportion, with water or the organic solvent extraction that dissolves each other with water; Or, get in proportion five tastes crude drug, wherein Formica fusca, the Radix Astragali, Fructus Chaenomelis, Radix Aconiti Lateralis Preparata merge and obtain extract with water or with the organic solvent extraction that water dissolves each other, Radix Notoginseng is pulverized, by Radix Notoginseng powder join above-mentioned extract obtained in, mix; Or get in proportion five tastes crude drug and directly pulverize, mix;

B, make clinical acceptable dosage form.

3. preparation method as claimed in claim 2, is characterized in that the described organic solvent dissolving each other with water is any one or two kinds in methanol, ethanol or acetone; Described extraction is any one mode or its combination decocting in extraction, reflux, extract,, immersion extraction, supersound extraction or percolation extraction.

4. preparation method as claimed in claim 2, is characterized in that, described five tastes crude drug is directly pulverized as micronizing, and before micronizing, crude drug is first pulverized, and crosses 24 mesh sieves and obtains crude drug coarse powder, and make moisture lower than 4% through dried.

5. the application of pharmaceutical composition as claimed in claim 1 in preparation treatment and alleviation rheumatic arthropathy or rheumatic renal damage medicine.

6. application as claimed in claim 5, is characterized in that described rheumatic arthropathy is rheumatic arthritis, rheumatoid arthritis or articular synovitis.