CN103432217B - External medicine composition for treating rheumatoid arthritis, preparation method and application of external medicine composition - Google Patents

Abstract

The invention provides an external medicine composition for treating rheumatoid arthritis, which is an external preparation prepared by the following active ingredients in parts by weight: 15-25 parts of gentiana macrophylla, 15-25 parts of caulis sinomenii, 15-25 parts of radix paeoniae alba, 15-25 parts of Chinese angelica and 10-20 parts of tripterygium wilfordii. The invention further provides a preparation method and an application of the external medicine composition. The external medicine composition is used for treating the rheumatoid arthritis, is obvious and controllable in efficacy, and simple and convenient to use, and provides a new clinical administration choice.

Description

A kind of externally-applied medicinal composition for the treatment of rheumatoid arthritis and its production and use

Technical field

The present invention relates to a kind of externally-applied medicinal composition for the treatment of rheumatoid arthritis.

Background technology

Rheumatoid arthritis (Rheumatoid Arthritis, RA) being a kind of autoimmune disease, is the chronic inflammatory disease being principal character with joint synovitis disease and getting involved with multisystem, is apt to occur in the Minor articulus such as hands, wrist, foot, recurrent exerbation, is symmetric.Pathogenesis is not yet completely clear and definite at present, and infection and autoimmune response are the key factors of morbidity and state of an illness migration, and the factor such as endocrine, h and E adds its susceptibility.RA is 0.3% ~ 1% at global sickness rate, and domestic is 0.32% ~ 0.36%, and each age cohort all can be fallen ill.Main manifestations is the symmetric multi-joint chronic nonspecific inflammation of surrounding, morbidity has joint red and swollen heat pain and dysfunction in early days, ankylosis deformity in various degree can be there is late period, simultaneously with the atrophy of bone and bones flesh, and very easily disable, disability rate about 20% in onset 1 year, up to 60% in 10 years.Harmful effect is brought, the serious quality of life affecting patient to the physiology of patient, psychology.Therefore, the research and development for RA medicine are significant.

In recent years, China RA patient numbers is more than 4,000,000, and medicine conventional clinically has NSAID (non-steroidal anti-inflammatory drug), slow-acting drug, glucocorticoid etc., but its therapeutic effect is not good enough.NSAID (non-steroidal anti-inflammatory drug) is the mainstay classification of RA medicine always, clinical conventional have indomethacin, aspirin, diclofenac, ibuprofen etc., such preparation is mainly through suppressing the activity of Cycloxygenase (COX), reduce the release of the inflammatory mediator such as prostaglandin, thromboxane that arachidonic acid metabolic generates, thus improve inflammatory symptom, there is certain curative effect to the releasing etc. of pain, but the pathogenesis of RA can not be changed, and have certain gastrointestinal reaction; Slow effect antirheumatic is considered to the possibility of symptom management progress, act on the different immunizing compositions in the RA course of disease, conventional medicine has methotrexate, ciclosporin, penicillamine and golden preparation etc., this type of medicine often acts on immunocyte, the symptom of RA can be improved preferably, prevent the destruction in joint, but make it apply because price is higher to be restricted; Glucocorticoid medicine rapidly, significantly can improve rheumatoid arthritis symptom, but often has rebound phenomena after drug withdrawal, and can produce the untoward reaction such as osteoporosis, hypertension, obesity along with the prolongation of the course for the treatment of.

Clinical expert is thought, desirable resisting rheumatoid disease medicine should have 1. definite anti-inflammatory and antalgic curative effect; 2. gastrointestinal damage is not caused; 3. rational medical expense.Therefore, clinically safe, effective, economic RA medicine is needed.

Chinese medicine has unique advantage in RA treatment, RA belongs to the category of " arthromyodynia " in motherland's medical science, just detailed record is done to it as far back as ancient Chinese, as elaboration " wet three gas of wind and cold are mixed extremely; combined into numbness; its migratory BI-syndrome caused mainly by pathogenic wind, BI-syndrome caused mainly by pathogenic cold being called painful BI, and dampness victor is damp arthralgia " just detailed in " Plain Questions arthromyodynia opinion "; Zhang Zihe also proposes in " Confucian's Duties to Their Parents " " numbness is sick is source with damp and hot, and wind and cold is double, and three gas are combined into numbness ".The traditional Chinese medical science is on the basis of its organic conception and determination for the treatment of based on pathogenesis obtained through differentiation of symptoms and signs, sum up in treatment and dispel the wind, cold expelling, dehumidifying, heat clearing away and blood circulation promoting and blood stasis dispelling, relax through therapeutic rules such as dredging collateral, especially be classical with " inner disease outer treat " theory, medicine is directly contacted with lesions position, to reach the object playing rapidly curative effect of medication, the rapid recovery misery of patient, the Qing Dynasty well-known doctor Xu Hui Xi think " with patch it, its gas inaccessible, the property of medicine is made to enter its space between skin and muscles from pore, stimulate the menstrual flow and pass through network, or carry it, or attack and fall apart it, outstanding strong than taking medicine, this extremely wonderful method also ", Qing Dynasty's Wu Shi machine is shown in " Li Yue Pianwen, Rhymed Discourse for Topical Remedies " also " reason of external treatment and the reason for the treatment of by oral administration of medicines, different way and reach the same goal " record.

In addition, compared with chemical medicine, Chinese medicine has the action character of multicomponent, multipath, Mutiple Targets, acts on more comprehensive, and meanwhile, the form of external transdermal administration, the first pass effect, the toxic and side effects that avoid liver are less.Therefore, Chinese medicine external treatment RA has unique advantage.

Gel ointment is a kind of Novel external patch, and original name is cataplasma, is commonly called as hydrogel, is, after extract, decoction pieces and chemicals and suitable hydrophilic matrix being mixed, to coat the outer emplastrum that material of mounting is made.Because this dosage form is substrate with water-soluble high-molecular material, better with the affinity of skin, enhance the hydration of skin and be conducive to the absorption of skin.Circumventing first pass effect and the gastrointestinal tract destruction of liver except having traditional external preparation, maintaining except the feature such as constant temperature, lasting blood drug level, this dosage form also has the advantages such as drug loading is large, bioavailability is high, good permeability, skin irritation are little.Therefore, this dosage form is suitable for that the RA state of an illness is very long, patient needs the features such as long-term prescription.

Summary of the invention

Technical scheme of the present invention there is provided a kind of externally-applied medicinal composition for the treatment of rheumatoid arthritis, and another technical scheme of the present invention there is provided preparation method and the purposes of this pharmaceutical composition.

The invention provides a kind of externally-applied medicinal composition for the treatment of rheumatoid arthritis, it is the external preparation be prepared from by the crude drug of following weight proportioning:

Radix Gentianae Macrophyllae 15-25 part, Caulis Sinomenii 15-25 part, Radix Paeoniae Alba 15-25 part, Radix Angelicae Sinensis 15-25 part, Radix Tripterygii Wilfordii 10-20 part.

Further preferably, it is the external preparation be prepared from by the crude drug of following weight proportioning:

Radix Gentianae Macrophyllae 20 parts, Caulis Sinomenii 20 parts, the Radix Paeoniae Alba 20 parts, Radix Angelicae Sinensis 20 parts, Radix Tripterygii Wilfordii 15 parts.

Pharmaceutical composition of the present invention is active component by the water of Radix Gentianae Macrophyllae, Caulis Sinomenii, the Radix Paeoniae Alba, Radix Angelicae Sinensis, Radix Tripterygii Wilfordii or extractive with organic solvent, adds the external preparation that pharmaceutically acceptable adjuvant or adjuvant composition are prepared from.

Wherein, described external preparation is emplastrum, tincture, liniment, gel, plaster, ointment; Wherein, described emplastrum is rubber-emplastrum, gel ointment, patch.

Wherein, described unguentum is gel ointment, and wherein, every sheet contains Radix Gentianae Macrophyllae with gentiopicrin (C ₁₆h ₂₀o ₉) must not count and be less than 51.16mg.

Wherein, described gel ointment is prepared from by the stock and adjunct of following weight proportioning:

Radix Gentianae Macrophyllae 318.5-591.5 part, Caulis Sinomenii 318.5-591.5 part, Radix Paeoniae Alba 318.5-591.5 part, Radix Angelicae Sinensis 318.5-591.5 part, Radix Tripterygii Wilfordii 238-442 part, adhesive 46.2-85.8 part, excipient 17.5-32.5 part, cross-linking agent 1.26-2.34 part, cross-linking regulator 1.96-3.64 part, wetting agent 87.5-162.5 part, penetration enhancers 22.4-41.6 part.

Wherein, described adhesive is selected from one or more the mixing in sodium polyacrylate, polyvinyl alcohol, carboxymethyl cellulose class, gelatin; Described excipient is selected from one or more the mixing in carbomer, Kaolin, titanium dioxide, calcium carbonate, zinc oxide, Pulvis Talci; Described wetting agent is selected from propylene glycol, glycerol, Oleum Ricini; It is aluminum salt that described cross-linking agent is selected from cross-linking agent, as the mixing of one or more in aluminum glycinate, aluminium hydroxide, aluminum chloride, dihydroxyaluminum aminoacetate; Described cross-linking regulator is selected from citric acid, tartaric acid; Described penetration enhancers is selected from one or more the mixing in azone, propylene glycol, Borneolum Syntheticum.

Wherein, described gel ointment is prepared from by the raw material of following weight proportioning:

Radix Gentianae Macrophyllae 455 parts, Caulis Sinomenii 455 parts, the Radix Paeoniae Alba 455 parts, Radix Angelicae Sinensis 455 parts, Radix Tripterygii Wilfordii 340 parts, sodium polyacrylate 66 parts, carbomer 25 parts, aluminum glycinate 1.8 parts, citric acid 2.8 parts, glycerol 100 parts, Oleum Ricini 25 parts, Borneolum Syntheticum 32 parts.

Present invention also offers and a kind ofly prepare described externally-applied medicinal composition, it comprises the steps:

A, take the crude drug of weight proportion;

B, Radix Angelicae Sinensis extract volatile oil, and extracting solution is for subsequent use;

C, Radix Gentianae Macrophyllae, Caulis Sinomenii, the Radix Paeoniae Alba, Radix Tripterygii Wilfordii add water or ethanol extraction, add the Radix Angelicae Sinensis extracting solution of b step, concentrated;

D, extractum step c concentrated mix with the Radix Angelicae Sinensis volatile oil of b step, add the acceptable adjuvant of pharmacy or are complementaryly prepared into pharmaceutically conventional external preparation.

Further preferably, the method that the Radix Angelicae Sinensis described in b step extracts volatile oil is steam distillation or supercritical fluid extraction; Described ethanol is 50% ethanol.

Present invention also offers the purposes of this externally-applied medicinal composition in the medicine of preparation treatment rheumatoid arthritis.

Being monarch with Radix Gentianae Macrophyllae in medicine material of the present invention, arrogating to oneself Eradicates rheumatism, relaxing muscles and tendons network, clearind deficient heat, is the key medicine for the treatment of arthromyodynia; Caulis Sinomenii is minister, helps Radix Gentianae Macrophyllae wind-damp dispelling, and it has effect of the meridian dredging, diuresis, makes heresy have outlet; Pure with pungent loose product, probably consumption impairment of QI Tianjin, therefore use Radix Paeoniae Alba nourishing blood to suppress the hyperactive liver, pain relieving in slow, yin fluid astringing receives antiperspirant; Be equipped with Radix Angelicae Sinensis, it has enriches blood, invigorates blood circulation, menstruction regulating and pain relieving, moisturizes effect of laxation, and blood is lived the loose and not impairment of YIN blood of wind; Assistant is dispeled the wind with Radix Tripterygii Wilfordii, is detoxified, parasite killing.All medicines coordinate, and play expelling wind and clearing away heat altogether, the effect of blood nourishing acupuncture-stimulating.

Externally-applied medicinal composition of the present invention is used for the treatment of rheumatoid arthritis, and drug effect is obvious, controlled, and easy to use, provides a kind of new medication selection for clinical.

Accompanying drawing explanation

Fig. 1 improved Franz diffusing cells method structure chart

Fig. 2 gentiopicrin reference substance HPLC collection of illustrative plates

Fig. 3 blank skin receiver media HPLC collection of illustrative plates

The HPLC collection of illustrative plates of Fig. 4 drug gel unguentum of the present invention receiver media

Detailed description of the invention

Embodiment 1 medicine material medicine of the present invention extracts

Raw material prescription: Radix Gentianae Macrophyllae 20g Caulis Sinomenii 20g Radix Paeoniae Alba 20g Radix Angelicae Sinensis 20g Radix Tripterygii Wilfordii 15g

Radix Angelicae Sinensis adopts extraction by steam distillation volatile oil, and pulverizing medicinal materials is 10 orders, adds the water of 10 times amount, soaks 3h, extracts 8h, collect volatile oil respectively and extracting solution for subsequent use.Radix Gentianae Macrophyllae, Caulis Sinomenii, Radix Paeoniae Alba Radix Tripterygii Wilfordii 4 taste medicine, soak 1h, uses 50% alcohol heating reflux three times of 9 times amount, 8 times amount, 8 times amount respectively, each 1 hour.Extracting solution reclaims ethanol to without alcohol taste, merges concentrated with the mother solution after volatile oil is drawn.

The preparation of embodiment 2 drug gel unguentum of the present invention

Raw material: Radix Gentianae Macrophyllae 455g, Caulis Sinomenii 455g, Radix Paeoniae Alba 455g, Radix Angelicae Sinensis 455g, Radix Tripterygii Wilfordii 340g, sodium polyacrylate 66g, carbomer 25g, aluminum glycinate 1.8g, citric acid 2.8g, glycerol 100g, Oleum Ricini 25g, Borneolum Syntheticum 32g, water 1000g; Amount to 100 to paste.

Method for making: above five kinds of Chinese medicine, Radix Angelicae Sinensis pulverized 10 mesh sieves, add 10 times of water gagings, soak 3h, steam distillation distillation 8h, respectively collect volatile oil and extracting solution for subsequent use, all the other 4 taste medicines soak 1h, use 50% alcohol heating reflux three times of 9 times amount, 8 times amount, 8 times amount respectively, each 1 hour, extracting solution reclaims ethanol to without alcohol taste, merge that to be concentrated into relative density be 1.25 with the mother solution after volatile oil is drawn ~

1.30(50 DEG C) extractum, then volatile oil to be added, stirs, obtain total extractum.Take the sodium polyacrylate of recipe quantity, after being uniformly dispersed with glycerol, add suitable quantity of water, swelling evenly as A phase; Carbomer add suitable quantity of water swelling after mix homogeneously with above-mentioned total extractum, then will add wherein with the finely dispersed aluminum glycinate of Oleum Ricini, stir as B phase; Borneolum Syntheticum is as C phase; Aqueous citric acid solution is as D phase; After B and C two-phase mixtures is even, then add A phase mix homogeneously, after finally mixing with D phase, 60 DEG C,

Refining and 20min under 100r/min condition, adjustments coating thickness is 2.5mm, is applied on non-woven fabrics, and 60 DEG C of oven dry, are cut into 7cm × 10cm size, and common 100 lose money in a business invention drug gel unguentum.

[inspection] paste containing amount measures the second method (" Chinese Pharmacopoeia " version in 2010 annex I I) according to emplastrum paste containing amount and measures, every 100cm ²should be 11.83 ~ 14.45g.

Plastic property measures according to gel ointment plastic property test (" Chinese Pharmacopoeia " version in 2010 annex I I), should conform with the regulations.

Adhesion gets this product 3, and removing lid lining, makes non-overlapping copies, place more than 2 hours in room temperature.Be placed in and become the swash plate of 30 ° central with horizontal plane, get No. 10 steel balls, measure according to emplastrum adhesive force algoscopy (" Chinese Pharmacopoeia " version in 2010 annex Ⅻ first method), should conform with the regulations.

[assay] measures according to high performance liquid chromatography (" Chinese Pharmacopoeia " version in 2010 annex VI D).

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filler; Methanol-water solution (gradient elution, 0 ~ 20min, 25 ~ 35%A, 20 ~ 25min, 35 ~ 95%A) is mobile phase; Determined wavelength is 254nm.Number of theoretical plate calculates should be not less than 3000 by gentiopicrin peak.

The preparation precision of reference substance solution takes gentiopicrin reference substance in right amount, makes the solution of every 1ml containing 0.5mg, to obtain final product with methanol.

This product 1 is got in the preparation of need testing solution, and removing lid lining, is cut into small pieces, get about 2.5g, accurately weighed, put in tool plug conical flask, precision adds 70% methanol 50ml, soaks 20min, weighed weight, supersound extraction 30min, taking-up lets cool, and supplies weight, shake up filtration with corresponding solvent, get subsequent filtrate, to obtain final product.

Algoscopy precision draws reference substance solution and each 5 μ l of need testing solution, injects high performance liquid chromatograph, measures, to obtain final product.

The every sheet of this product contains Radix Gentianae Macrophyllae with gentiopicrin (C ₁₆h ₂₀o ₉) must not count and be less than 51.16mg.

Embodiment 3 medicine material technological parameter of the present invention screening test

1. the Study on extraction of Extracting Oil of Radix Angelica Sinensis

1.1 instruments and reagent

SB500 gram of type pulverizer (Shanghai Guang Sha Trade Co., Ltd.); Volatile oil extractor (Shanghai instrument manufacturing factory); YP202N electronics Libra (Shanghai Precision Scientific Apparatus Co., Ltd); Water is deionized water.

1.2 method and result

1.2.1 the extraction of Radix Angelicae Sinensis volatile oil

With reference to version " Chinese Pharmacopoeia " (one) annex X D in 2010, get Radix Angelicae Sinensis 100g, put in round-bottomed flask, add zeolite number and several times water gaging, connect volatile oil extractor and condensing tube, soak certain hour, slowly add thermic to boil, and stop heating after keeping micro-a few hours of boiling, leave standstill more than 2h, read reservoir volume, calculate volatile oil is drawn rate.

Volatile oil is drawn rate (ml/100g)=volatile oil volume/quality of medicinal material × 100%

1.2.2 the experiment of single factor of Radix Angelicae Sinensis volatile oil extraction process

1.2.2.1 distillation time is on the impact of volatile oil is drawn rate

According to method under " 1.2.1 " item, take angelica sinensis 5 parts, every part of 100g, all add the water of 10 times amount, after soaking 3h under room temperature, connect volatile oil extractor and condensing reflux pipe, respectively vapor distillation 2,4,6,8,10h extracts volatile oil, the extraction ratio of calculating volatile oil, the results are shown in Table 1.

Table 1 distillation time affects result to volatile oil is drawn rate

Distillation time (h)	2	4	6	8	10
						Volatile oil is drawn rate (%)	0.21	0.28	0.37	0.45	0.46

As shown in Table 1, when distillation time reaches 8h, Radix Angelicae Sinensis volatile oil extraction ratio substantially no longer increases along with the prolongation of distillation time.

1.2.2.2 soak time is on the impact of volatile oil is drawn rate

According to method under " 1.2.1 " item, take angelica sinensis 5 parts, every part of 100g, all add the water of 10 times amount, under room temperature, soak 0 respectively, 1,3,5, after 7h, connect volatile oil extractor and condensing reflux pipe, vapor distillation 8h extracts volatile oil, calculates the extraction ratio of volatile oil, the results are shown in Table 2.

Table 2 soak time affects result to volatile oil is drawn rate

As shown in Table 2, soak time reaches 3h, and the extraction ratio of Radix Angelicae Sinensis volatile oil no longer increases along with the prolongation of soak time.

1.2.2.3 amount of water is on the impact of volatile oil is drawn rate

According to method under " 1.2.1 " item, take angelica sinensis 5 parts, every part of 100g, add 6,8,10,12,14 times of water gagings respectively, after soaking 3h under room temperature, connect volatile oil extractor and condensing reflux pipe, vapor distillation 8h extracts volatile oil, calculates the extraction ratio of volatile oil, the results are shown in Table 3.

Table 3 amount of water affects result to volatile oil is drawn rate

As shown in Table 3, when amount of water arrives 10 times, the extraction ratio of Radix Angelicae Sinensis volatile oil no longer increases along with amount of water and increases.

1.2.2.4 degree of grinding is on the impact of volatile oil is drawn rate

According to method under " 1.2.1 " item, take angelica sinensis, degree of grinding is 10 object angelica sinensis, degree of grinding is each 1 part of 24 object angelica sinensis 100g, add 10 times of water gagings respectively, after soaking 3h under room temperature, connect volatile oil extractor and condensing reflux pipe, vapor distillation 8h extracts volatile oil, calculates the extraction ratio of volatile oil, the results are shown in Table 4.

Table 4 degree of grinding affects result to volatile oil is drawn rate

Table 4 shows, when degree of grinding is 10 order, volatile oil is drawn rate is the highest.

1.2.3 orthogonal experiment

As can be seen from the experiment of single factor result of 1.2.2 Radix Angelicae Sinensis volatile oil extraction process, the extraction ratio impact of soak time on volatile oil is less, and therefore this research adopts L ₉(3 ⁴) orthogonal design, with volatile oil is drawn rate for index, with degree of grinding (A), distillation time (B), amount of water (C) for investigation factor, optimize Radix Angelicae Sinensis volatile oil extraction process, factor level table is in table 5.

Take Radix Angelicae Sinensis totally 9 parts, every part of 100g, by orthogonal test table, result and Nogata analysis in table 6, variance analysis is in table 7.

Table 5 factor level table

Table 6 orthogonal design, result of the test and Nogata analysis

The analysis of variance table of table 7 volatile oil is drawn rate

Note: F _0.05(2,2)=19, F _0.01namely (2,2)=99, * have significant difference

Size from table 7 extreme difference R: R _a>R _c>R _bthe influence degree of each factor to Radix Angelicae Sinensis volatile oil extraction process is followed successively by A>C>B, namely degree of grinding > adds water multiple > distillation time, wherein variance analysis shows that degree of grinding is larger on the impact of volatile oil is drawn rate, and difference has statistical significance (P<0.05), amount of water and distillation time are on its impact not quite.The optimum extraction process of volatile oil is: A ₂b ₂c ₂, being degree of grinding is 10 orders, adds the water of 10 times amount, soaks 3 hours, extracts 8 hours.

Get Radix Angelicae Sinensis totally 3 parts, every part of 100g, according to the A that above-mentioned screening obtains ₂b ₂c ₂technique carries out orthogonal proving test research, and it is 1.15% that the volatile oil is drawn rate that result obtains is respectively 0.51%, 0.50%, 0.50%, RSD.Show that optimizing the Extraction Process of Volatile Oil obtained stablizes feasible, repeated good.

1.3 brief summary

Through the optimization of extraction process, the optimum extraction process of Radix Angelicae Sinensis volatile oil is degree of grinding 10 order, adds 10 times of water gagings, soaks 3 hours, extracts 8 hours.

2. the Study on extraction of Radix Gentianae Macrophyllae, Caulis Sinomenii, the Radix Paeoniae Alba, Radix Tripterygii Wilfordii four Chinese medicine

2.1 instruments and reagent

2.1.1 instrument

Shimadzu 1200 Ultra Performance Liquid Chromatography instrument; 1200SL work station; Balance (Japanese Shimadzu, AEG-45SM/AM220); Waster XTerra C18 chromatographic column (250 × 4.6mm, 5 μm).

2.1.2 reagent

Gentiopicrin reference substance (Nat'l Pharmaceutical & Biological Products Control Institute, lot number 110770-20071); Methanol is chromatographically pure, and all the other reagent are analytical pure; Water is redistilled water.

2.2 experimental techniques and result

2.2.1 the investigation of concentration of alcohol

Taking Radix Gentianae Macrophyllae, Caulis Sinomenii, the Radix Paeoniae Alba, Radix Tripterygii Wilfordii four Chinese medicine material according to prescription ratio, fixing soak time is 1h, adding corresponding solvent amount is 10 times amount, extraction time 3 times and each 1h, merging filtrate, standardize solution.With gentiopicroside in different morphological and paste-forming rate for evaluation index, concentration of alcohol is investigated.

2.2.1.1 Determination of Gentiopicroside

Chromatographic condition: chromatographic column: Waster XTerra C18(250 × 4.6mm, 5 μm); Mobile phase: methanol-water solution (gradient elution, 0 ~ 20min, 25 ~ 35%A, 20 ~ 25min, 35 ~ 95%A); Flow velocity: 1mL/min; Column temperature: 25 DEG C; Determined wavelength 254nm; Number of theoretical plate calculates should be not less than 3000 by gentiopicrin peak.

The preparation of need testing solution: accurate absorption extracting solution is appropriate, and water-bath volatilizes, and residue dissolve with methanol is transferred in 25ml volumetric flask, and supersound process 30min, is cooled to room temperature, by methanol constant volume to scale, as need testing solution.

The preparation of reference substance solution: precision takes reference substance 0.00527g and puts in 10ml measuring bottle, adds methanol to scale, shakes up, and obtains the gentiopicrin reference substance solution of 0.527mg/ml.

The mensuration of test sample: accurate absorption gentiopicrin reference substance solution and each 5 μ l of need testing solution, injects high performance liquid chromatograph, measures, to obtain final product.

2.2.1.2 the mensuration of paste-forming rate

Precision pipettes extracting solution 50ml in evaporating dish, after water-bath volatilizes, then dries to constant weight in 105 DEG C of baking ovens, takes out to put in exsiccator to cool 30min, weighs rapidly, calculates yield of extract.

2.2.1.3 experimental result

This research adopts Synthetic weighted mark method result, wherein gentiopicroside in different morphological, paste-forming rate to account for that weight coefficient is respectively be 0.8,0.2, i.e. total score Y=Y1/42.39*80+Y2/20.43*20, the results are shown in Table 8.

The investigation result that table 8 concentration of alcohol affects extraction process

From table 8, concentration of alcohol is 50% ~ 70% time, and overall merit score is higher than 85 points, and the gentiopicroside in different morphological now in Radix Gentianae Macrophyllae is higher, when concentration of alcohol higher than 70% time, gentiopicroside in different morphological obviously declines.Consider concentration of alcohol and extraction time, add the reciprocal effect of the factors such as solvent amount, extraction time, therefore the follow-up orthogonal design that will the basis investigated at this adopt determines best concentration of alcohol further.

2.2.2 the investigation of pick up

Take Radix Gentianae Macrophyllae, Caulis Sinomenii, the Radix Paeoniae Alba, Radix Tripterygii Wilfordii four Chinese medicine material 75g altogether according to prescription ratio, add 50%, 60%, 70% ethanol 500ml respectively, and observe the Wet Out of medical material, after result 1h, medical material all runs through, leach whole unabsorbed solvent, calculate pick up, the results are shown in Table 9.

The investigation of table 9 pick up

As shown in Table 9, pick up is about 1 times of quality of medicinal material, should add the solvent of a times amount before therefore extracting, and soaks 1h.

2.2.3 orthogonal design is to the optimization of extraction process

According to bibliographical information and trial test result, select to affect the principal element concentration of alcohol of ethanol extraction, add amount of alcohol, extraction time, extraction time be factor, select L ₉(3) ⁴orthogonal array is tested.Gentiopicroside in different morphological and paste-forming rate is selected to be that index is evaluated, screening optimum extraction process.The design of orthogonal array gauge outfit is in table 10.

Table 10 experimental factor water-glass

Experimental result

Adopt Synthetic weighted mark method, shared by gentiopicroside in different morphological, paste-forming rate, weight coefficient is respectively 0.8,0.2, i.e. total score Y=Y1/39.65*80+Y2/19.83*20, experimental result and Nogata analysis in table 11, variance analysis are in table 12.

Table 11 positive quadraturing design test result and Nogata analytical table

Table 12 analysis of variance table

Note: * has significant difference, * * has pole significant difference

From Nogata analysis and variance analysis, for gentiopicrin index, the size of each factor impact is R _c>R _b>R _a>R _d, for paste-forming rate index, R _c>R _a>R _d>R _b, overall merit, R _c>R _b>R _a>R _d, i.e. extraction time > solvent amount > solvent concentration > extraction time, C factor all has significant difference to three indexs, A, B, D factor there was no significant difference.Consider from cost-saving angle, therefore select A ₁(50% ethanol), B ₁(8 times amount), C ₃(3 times), D ₁(1h), optimizing optimised process is A ₁b ₁c ₃d ₁, in conjunction with pick up, optimum extraction process is and soaks 1h, uses 50% alcohol heating reflux three times of 9 times amount, 8 times amount, 8 times amount respectively, each 1 hour.

2.2.3.4 process certification

Take Radix Gentianae Macrophyllae, Caulis Sinomenii, the Radix Paeoniae Alba, Radix Tripterygii Wilfordii four Chinese medicine material three parts according to prescription ratio, every part of 75g, checking research is carried out to the above-mentioned extraction process determined, measures gentiopicroside in different morphological and paste-forming rate respectively, the results are shown in Table 13.

Table 13 extraction process demonstration test result

As seen from Table 13, the extraction process of screening is stablized feasible, reproducible.

The optimised process of this prescription is, Radix Angelicae Sinensis adopts extraction by steam distillation volatile oil, and pulverizing medicinal materials is 10 orders, adds the water of 10 times amount, soaks 3h, extracts 8h, collect volatile oil respectively and extracting solution for subsequent use.Radix Gentianae Macrophyllae, Caulis Sinomenii, Radix Paeoniae Alba Radix Tripterygii Wilfordii 4 taste medicine, soak 1h, uses 50% alcohol heating reflux three times of 9 times amount, 8 times amount, 8 times amount respectively, each 1 hour.Extracting solution reclaims ethanol to without alcohol taste, merges that to be concentrated into relative density be 1.25 ~ 1.30 with the mother solution after volatile oil is drawn.

Embodiment 4 inventive gel unguentum Study on Forming

One, substrate prescription screening

1. instrument and reagent

1.1 instrument

Gel ointment coating machine (self-control); JJ-1 reinforcement motor stirrer (Fuhua Instrument Ltd. of Jintan City); DGG-9240 type electric heating constant-temperature blowing drying box (Shanghai gloomy reliable test Instrument Ltd.); BS224S (ten thousand/, Beijing Sai Duolisi scientific instrument company limited).

1.2 reagent

Sodium polyacrylate (Zhong Sheng bio tech ltd, Guangdong); Carbomer (Langfang Ju Tong Chemical Co., Ltd., lot number: 20120528); Aluminum glycinate (Shanghai Sen Lei Chemical Co., Ltd.); Polyvinyl alcohol (Chengdu Ke Long chemical reagent factory); Sodium carboxymethyl cellulose (Chengdu Ke Long chemical reagent factory, lot number: 20110909); Polyvinylpyrrolidone (Chengdu Ke Long chemical reagent factory, lot number: 20110823); Zinc oxide (Chengdu Ke Long chemical reagent factory, lot number: 20111122); Gelatin (Tianjin great Mao chemical reagent factory, lot number: 20090728); Oleum Ricini Chengdu Ke Long chemical reagent factory, lot number: 20090815); Glycerol (Chongqing Chuan Dong Chemical Co., Ltd., lot number: 20110901); Citric acid (Chongqing Chuan Dong Chemical Co., Ltd.); Tartaric acid (Chongqing chemical reagent factory, lot number: 901112); Kaolin (Fengxian, Shanghai City Feng Cheng chemical reagent work).

2. method and result

2.1 substrate initial options

The substrate of gel cream is primarily of compositions such as adhesive, excipient, cross-linking agent, cross-linking regulator, wetting agents.Each component distinct, the effect in substrate is also different.This research, in conjunction with document, carries out preliminary screening to substrate conventional over nearly 5 years.

2.1.1 adhesive

Adhesive is the main matter that gel ointment mastic produces stickiness.Adhesive has cross-linking type and non-crosslinked type, cross-linking type as sodium polyacrylate, polyvinyl alcohol, carboxymethyl cellulose class, non-crosslinked type based on animal glue, as gelatin etc.The gel ointment poor stability that non-crosslinked type adhesive is prepared, is easily subject to the impact of temperature, and time as too high in summer temperature, cream thin-skinnedization likely occurs that cream hydrorrhea goes out, and therefore this research selects emphatically cross-linking type adhesive when prescription screening.

2.1.2 excipient

Conventional inorganic excipients Kaolin, calcium carbonate, zinc oxide, Pulvis Talci etc.In recent years, the high-molecular organic materials such as carbomer are introduced in the substrate of gel ointment as excipient, make the character of gel ointment have certain change.

2.1.3 wetting agent

In gel ointment substrate, water content is higher, therefore needs to select wetting agent to prevent water loss.Wetting agent conventional at present has propylene glycol, glycerol etc.

2.1.4 cross-linking agent

The high volence metal ion of cross-linking agent and macromolecular compound such as sodium polyacrylate are cross-linked, and form network structure, thus improve the cohesive strength of substrate, Ca in theory ²⁺, Zn ²⁺, Al ³⁺deng high volence metal ion, all there is crosslinked action, but the most frequently used cross-linking agent is aluminum salt, as aluminum glycinate, aluminium hydroxide, aluminum chloride, an Al ³⁺three carboxyls can be cross-linked.

2.1.5 cross-linking regulator

Cross-linking regulator is the key substance that catalysis regulates cross-linking agent and macromolecular compound crosslinking rate, crosslinking degree, conventional cross-linking regulator mainly organic acid as citric acid, tartaric acid etc.

2.2 each dissolving of component addition sequence and the designs of addition sequence

2.2.1 the dissolving of sodium polyacrylate (PAAS)

Be dispersed in inside a small amount of glycerol by PAAS, get appropriate water, add under rapid stirring, and stir rapidly, leave standstill swelling, the mastic bubble obtained is less, clear.

2.2.2 the dissolving of gelatin, sodium carboxymethyl cellulose

Gelatin, sodium carboxymethyl cellulose are directly scattered in aqueous solution, heating in water bath makes swelling, and finally obtain colloidal sol homogeneous, stickiness is larger.

2.2.3 the dissolving of polyvinyl alcohol

Getting appropriate polyvinyl alcohol adds in appropriate water, and 60 DEG C of water-baths are dissolved, and can dissolve completely in 30min.

2.2.4 the dissolving of carbomer

By carbomer dispersion in suitable quantity of water, rapid stirring, swellingly spends the night, and must clarify uniform substrate.

2.2.5 the process of Kaolin, titanium dioxide and feed postition

Kaolin, titanium dioxide are excipient, generally added dissolved gelatin, in poly-vinyl alcohol solution, then to mix mutually with other.

2.2.6 citric acid, tartaric acid, AlCl ₃, dihydroxyaluminum aminoacetate etc. process

By AlCl ₃or dihydroxyaluminum aminoacetate is dispersed in a small amount of glycerol, is uniformly dispersed, and is added in extractum and stirs, and mixs homogeneously with other, finally add citric acid or aqueous tartaric acid solution mix homogeneously, can slow down crosslinking time to a certain extent.

The selection of 2.3 mixings time, temperature, speed

Mixing time is generally at about 20 ~ 60min, and overlong time, easily brings a large amount of bubbles on the one hand, and the shearing force continued on the other hand can be destroyed the hydrogen bond of mastic formation and stickiness is declined, and the time is too short, then mastic not easily mixes.Temperature generally should not higher than 80 DEG C, and temperature is high, and mastic deformation is very fast, easy mix homogeneously, but the stickiness of mastic can be made to decline ^[26].Mixing speed also can not be ignored the impact of the physical behavior of mastic, excessive velocities, also can give in mastic and bring a lot of bubble, and mastic stickiness also can be caused to decline due to the reason of shearing force; Speed is too slow, and mastic can not be made equally to stir ^[27].

Through trial test research, this gel ointment with blender (speed is about 100r/min) 60 DEG C stir 20 minutes comparatively suitable, the mastic uniformity of preparation, coating are better.

2.4 ocular estimate standards of grading

2.4.1 mastic uniformity

Mastic evenly, without graininess micelle, without mixing, speckle is chosen as 5 ~ 4 points; Granule micelle is little, and assorted speckle is seldom chosen as 3 ~ 2 points; Mastic is uneven, graininess micelle is larger or be morely chosen as 1 ~ 0 point.

2.4.2 film residual

Get the gel ointment of molding, the sample of the wide 4cm of clip, long 5cm, with an iron block pressure 20min, open lining cap rock, the amount remained on polyethylene film is film residual quantity, and standards of grading are 5 points: <0.050g; 4 points: 0.050 ~ 0.149g; 3 points: 0.150 ~ 0.249g; 2 points: 0.250 ~ 0.349g; 1 point: 0.350 ~ 0.449g; 0 point: >0.450g.

2.4.3 stretchability

Coating easily completes, and is chosen as 5 points; Coating can complete, and is chosen as 4 ~ 3 points; Be coated with more difficult completing, be chosen as 2 ~ 1 points; Coating effort, cannot complete, be chosen as 0 point.

2.4.4 degree of oozing out

After gel ointment rubber cement is coated with while hot, the non-woven fabrics back side is chosen as 5 points without the person of oozing out; Ooze out area to account for the gross area 1 ~ 20% and be chosen as 4 points; 21 ~ 40% are chosen as 3 points; 41 ~ 60% are chosen as 2 points; 61 ~ 80% are chosen as 1 point; >80% person is chosen as 0 point.

2.4.5 skin tracing ability

Formed gel unguentum is affixed on wrist portion, firmly gets rid of 5 times, get rid of the 5th time person that do not come off and be chosen as 5 points, get rid of the 5th time person of coming off and be chosen as 4 points, get rid of the 4th time person of coming off and be chosen as 3 points, by that analogy, get rid of the 1st time person of coming off, be chosen as 0 point.

2.5 substrate prescription screening result

According to the character of each component and the effect in gel ointment, in conjunction with literature method, carry out the research of preliminary experiment prescription, formulation study is as shown in table 14,15:

The research of table 14 drug gel paste substrate of the present invention prescription Preliminary screening

Note: A item is adhesive, B item is excipient, C item is cross-linking agent, D item is cross-linking regulator, E item is that compound recipe extracts extractum

Table 15 drug gel paste substrate of the present invention prescription Preliminary screening result of study

The display of above result, every evaluation indexes such as the mastic uniformity of prescription 2 and prescription 6, film residual, stretchability, degree of oozing out and skin tracing ability are more suitable, and the composition of two prescriptions is comparatively similar, and prescription 6 is adjuvant PVA than prescription more than 2; Therefore on the basis of prescription 6, Box-Behken is adopted to design further, the consumption of each adjuvant in optimization formulation.

Two, Box-Behken design optimization formulation study

1. instrument and reagent: the same

2. method and result

2.1 evaluation index

First externally applied transdermal absorption preparation will meet and be a bit sticky, and admittedly could be affixed on skin and discharge medicine.Evaluation at present for gel ointment stickiness has first viscous force, cohesion, holds viscous force etc., and the evaluation of each mechanical index all needs the instrument and equipment of being correlated with, and this studies the experimentally existing condition in room, selects just viscous force to be paper examines index.

In order to the statistical analysis of data is convenient in experiment, the assay method reference literature of first viscous force ^[28]and American Pressure Sensitive Tape association PSTC-6 method, adopt spin plane stop method, namely spin rolls down from a smooth bevel, through the gel cream surface of horizontal positioned, measure the rolling distance of spin in gel cream face, judge the stickiness size of gel cream, rolling distance is less, and stickiness is larger.Each sample determination 5 times, with mean value calculation.Through preliminary experiment screening, be advisable with 30 °, inclination angle in inclined-plane, spin is with No. 10 beads, and diameter is 7.144mm, and weight is that 1.50g is advisable.

2.2Box-Behken design

Select PAAS(X larger on the impact of gel ointment stickiness in prescription ₁), carbomer (X ₂), PVA(X ₃), aluminum glycinate (X ₄), citric acid (X ₅) be factor, first viscous force is evaluation index, and experimental level is set to-1,0,1, and factor level table is shown in table 16.

Table 16Box-Behken design factor water-glass

According to the preparation method of fixed gel ointment substrate, according to the arrangement of table 16Box-Behken design factor water-glass, take the consumption of each adjuvant, prepare sample, measure the first viscous force of each sample, result is shown in table 17.

Table 17Box-Behken design measures just viscous force result

2.3 date processing

To above-mentioned experimental result, Design-Expert8.0 software is adopted to adopt the different models fitting process such as Linear, 2FI, Quadratic, Cubic, with P value in confidence interval in models fitting equation and model coefficient R to data ²for criterion selects suitable model, shown in table 18.

Table 18Design-Expert software data analysis result

P=0.0079<0.01 in Linear models fitting equation, has pole significant difference, but model coefficient R ²=0.3146, model is not remarkable; P=0.2133>0.05 in 2FI models fitting equation, there was no significant difference, and model coefficient R ²=0.4106, model is not remarkable; Quadratic models fitting equation, P=0.0014<0.01, has pole significant difference, model coefficient R ²=0.7436, models fitting is not ideal enough; Cubic models fitting equation P=0.0011<0.01, has pole significant difference, and R ²=0.9619, model-fitting degree is high, model ideal.Therefore Cubic models fitting is carried out to data, arrange certain P value level, rejected by the item of P>0.05, analysis result is in table 19, and the fit equation after simplification is:

Y=10.67-1.75X ₁+0.049X ₂+0.22X ₃+2.09X ₄-0.65X ₅+0.24X ₁X ₂-1.28X ₁X ₄+0.85X ₂X ₄-1.56X ₃X ₄+1.21X ₃X ₅-1.43X ₁ ²-2.59X ₂ ²-2.22X ₃ ²-1.14X ₄ ²+1.41X ₁X ₂ ²-2.32X ₂ ²X ₄-1.92X ₃ ²X ₅，（P<0.0001，R ²=0.8496）

The variance analysis of table 19Cubic models fitting

As seen from Table 19, X ₁, X ₄pole significance impact is had, X on the stickiness of gel ointment ₁x ₄, X ₃x ₄between have significant reciprocal action, X ₃x ₅between also have faint reciprocal action.

On the basis in optimum value region, in conjunction with the solution that saving adjuvant, less cost cost and Design-Expert8.0 software provide, optimize drug gel unguentum optimum substrate of the present invention: PAAS1.11g, carbomer 1.29g, aluminum glycinate 0.09g, citric acid 0.14g.

2.4 optimization formulation checkings

In order to the reliability of verification model, 3 batches of drug gel paste substrate prescription checkings of the present invention have been carried out in this research on the basis of the optimum prescription of prediction, and calculate the error between measured value and predictive value, result is shown in table 20.

Table 20 predictive value compares with experiment value

Table 20 shows, the error of measured value and predictive value is within 5%, and model is reliable.

Three, the screening of transdermal penetration enhancer

1. experimental apparatus, material and animal

1.1 instrument

Agilent 1200 high performance liquid chromatograph, 1200SL work station, DAD detector, binary gradient pump; Waster XTerra C ₁₈chromatographic column (4.6 × 250mm, 5 μm); Electronic balance (Japanese Shimadzu, AEG-45SM/AM220); TK-20A type percutaneous dispersion test instrument (the triumphant scientific and technological trade Co., Ltd of Shanghai armour); Gel ointment coating machine (self-control).

1.2 reagent

With the reagent in this chapter first segment 1.2.

1.3 animal

SPF level Kunming mouse, male, body weight 20 ~ 25g, is provided by Chongqing Institute of Chinese Medicine Institute of Botany.

2. the foundation of experimental technique

The preparation of 2.1 different transdermal penetration enhancer drug gel unguentum of the present invention

Take the sodium polyacrylate of recipe quantity, after being uniformly dispersed with glycerol, add suitable quantity of water, swelling evenly as A phase; Carbomer add suitable quantity of water swelling after mix homogeneously with the total extractum of a certain amount of medicaments compound extract of the present invention, then will add wherein with the finely dispersed aluminum glycinate of Oleum Ricini, stir as B phase; The transdermal penetration enhancer of amount of calculation is as C phase; Aqueous citric acid solution is as D phase; By B and C two-phase mixtures evenly after, then add A phase mix homogeneously, finally mix with D phase, 60 DEG C, refine and 20min under 100r/min condition, be applied on non-woven fabrics, oven dry, cuts out, obtains the drug gel unguentum of the present invention containing different transdermal penetration enhancer.

The preparation of 2.2 transdermal experiment skin models

Adopt 8%Na ₂s solution sloughs the hair of animal corresponding site, by sacrifice of animal after 24h, cut skin, be laid on glass plate, horny layer is downward, carefully rejects subcutaneous fat and is adhered tissue, then repeatedly rinse well with normal saline with single back of the body blade and cotton swab, be cut into suitable size, put in normal saline and save backup in 4 DEG C.Take out during test, the dermal depot time is no more than 3 days.Before each experiment, check zoodermic integrity, any breakage must not be had.

2.3 transdermal diffusion experiment devices

Transdermal absorption disperser is the Franz diffusion cell of improvement, and it is involuted by upper and lower two tubular glass tubings, and the skin be sandwiched between glass is divided into upper and lower two Room.Upper room is diffuser casing, and lower room is receiving chamber, connects a probe tube at the right part of acceptable solution, and for sample introduction, sampling and eliminating bubble, structural representation refers to Fig. 1.Reception tank volume is 18.5ml, and effective diffusion area is 2.92cm ², heating in water bath, temperature precise hard_drawn tuhes, at 32 ± 0.5 DEG C, tests maintenance 32 DEG C of transdermal penetration conditions.

2.4 body outer osmotic experimental techniques

Be fixed between the supply pool of diffusion cell and acceptance pool by the skin handled well, corium side contacts with receiving liquid, and emptying bubble, stratum corneum side is to supply pool.Supply pool adds test sample extracting solution or gel ointment sample, and acceptance pool adds normal saline.Magnetic agitation speed is about 200r/min, and bath temperature is 32 DEG C.Respectively at 2,4,8,12,24 hours, from acceptance pool, take out receiver media 2ml, in acceptance pool, add the fresh receiver media of equivalent simultaneously.The receiver media taken out, after 0.45 μm of filtering with microporous membrane, is analyzed for HPLC sample introduction.

2.5 transdermal experiment date processing

Processed by high performance liquid chromatography data measured, try to achieve unit are accumulation infiltration capacity (Qn), cumulative release percentage rate (Q), percutaneous rate (J), enhancing rate (ER) and lag time (T _lAG).

Qn: unit are cumulative release amount

Qn=(V _alwaysc _n+ Σ C _n-1v _get)/A

In formula: C _nit is the drug level (μ g/ml) that the n-th sample point records

C _n-1it is the drug level (μ g/ml) that (n-1)th sample point records

A is infiltrating area (cm ²)

V _alwaysfor the volume (ml) of diffusion cell receiving chamber

V _getfor sampling amount (ml)

∑ C _n-1v _getby sampling the Cumulate Sum of loss dose

Due in this experiment, V _always=18.5ml, V _get=2.0ml, A=2.92cm ²therefore above formula can be abbreviated as:

Qn=（C _n×18.5+ΣC _n-1×2）/2.92

Q: accumulative through percentage rate=unit are gentiopicrin transit dose/unit are cataplasma gentiopicroside in different morphological * 100%

J: be percutaneous rate, return time of penetration t with unit are Percutaneous permeability Qn, the side of obtaining returns journey, and this equation slope is percutaneous rate;

ER: be enhancing rate, weighs Percutaneous absorption enhancer to the size of the transdermal penetration effect of medicine;

ER=Je/Jo=Ke/Ko；

Je: be the percutaneous rate of medicine after application penetrating agent;

Jo: be the percutaneous rate that medicine is intrinsic;

Ke: be the slope of percutaneous penetration of drugs kinetic curve straight line portion after application penetrating agent, i.e. infiltration coefficient;

Ko: be percutaneous penetration of drugs kinetic curve straight line portion slope;

T _lAGh (): lag time, carry out linear regression with the straight line portion of unit are Percutaneous permeability Qn to time of penetration t mapping curve, the intercept in X-axis is lag time.

3. experimental result

The selection of 3.1 test temperatures

In order to physiological disposition that is virtually reality like reality, Ligustrazine hydrochloride test must be in temperature constant state.At present, general vitro permeation assay selective temperature is 32 DEG C or 37 DEG C ^{[29 ~ 30]}, this research considers that 32 DEG C closer to body surface temperature, and the too high corruption accelerating skin of temperature, and therefore this experimental selection 32 DEG C is experimental temperature.

The selection of 3.2 process of the test rotor rotating speeds

In transdermal penetration process of the test, drugs through skin spreads in accepting medium, static diffusion layer is there is in the interface of skin and receiver media, thus the concentration difference created in receiver media, in order to eliminate static diffusion layer, certain magnetic agitation rotor must be selected to stir, interface is disappeared, thus simulation human body sink conditions.Preliminary experiment compares rotor 50r/min, 100r/min, 200r/min, 300r/min, rotor speed, when 200r/min, can make homogeneous media in reception tank flow stably, not have whirlpool to produce, therefore, the rotating speed of rotor is selected to be 200r/min in test.

The foundation of 3.3 transdermal test in vitro analytical methods

3.3.1 the determination of chromatographic condition

Chromatographic column: Waster XTerra C ₁₈chromatographic column (4.6 × 250mm, 5 μm); Mobile phase: methanol-water, gradient elution, 0 ~ 20min, 25 ~ 35%A, 20 ~ 25min, 35 ~ 95%A; Flow velocity: 1ml/min, determined wavelength 254nm.Component under above-mentioned chromatographic condition in gentiopicrin and skin and other component all can reach baseline separation, and negative noiseless, as shown in Figure 2, Figure 3, Figure 4.

3.3.2 the investigation of linear relationship

Precision takes the gentiopicrin reference substance 11.38mg being dried to constant weight, puts in 50ml measuring bottle, adds dissolve with methanol and be settled to scale, shaking up.Respectively precision pipette 0.2,1.0,3.0,6.0,9.0,10.0ml, by methanol constant volume to 10ml, make the gentiopicrin reference substance solution that concentration is respectively 4.552,22.76,68.28,136.56,204.84,227.6 μ g/ml, according to the chromatographic condition sample introduction 100 μ l in above-mentioned 3.3.1, measure gentiopicrin peak area.With gentiopicrin reference substance solution concentration (μ g/ml) for abscissa, take peak area as vertical coordinate, drawing standard curve, obtains equation of linear regression y=85.683x-1.990, r=1.Result shows that gentiopicrin concentration is good in 4.552 ~ 227.6 μ g/ml scope internal linear relations.

The selection of 3.4 receiver medias

Get drug gel unguentum of the present invention (not containing penetration enhancers), with mouse web portion isolated skin for object of study, respectively with distilled water, normal saline for receiver media, to investigate in drug gel unguentum of the present invention gentiopicrin through the transdermal penetration situation of mouse web portion isolated skin in different accepting medium, unit of account area cumulative release amount (Qn, μ g/cm ²), percutaneous rate (J), total release percentage (Q) and lag time (T _lAG), the results are shown in Table 21,22.

Table 21 gentiopicrin is external unit are accumulation infiltration capacity (n=3) in different medium

The transdermal test in vitro permeability parameters of table 22 gentiopicrin in different medium

From table 22, compare with normal saline, gentiopicrin less, accumulative percent penetration lag time in distilled water is higher, percutaneous rate is higher, and therefore, this research take distilled water as receiver media.

Gentiopicrin in 3.5 drug gel unguentum of the present invention is through the research of the Transdermal absorption of different isolated skin

Get drug gel unguentum of the present invention (not containing penetration enhancers), respectively with the abdominal part of mice, skin of back for object of study, take distilled water as accepting medium, investigate the release behavior of gentiopicrin in drug gel unguentum of the present invention, unit of account area cumulative release amount (Qn, μ g/cm ²), percutaneous rate (J), total release percentage (Q) and lag time (T _lAG), the results are shown in Table 23,24.

Table 23 gentiopicrin is external unit are accumulation infiltration capacity (n=3) in different animals skin

The body outer osmotic parameter of table 24 gentiopicrin in different animals skin

From table 24, compare with mouse back skin, gentiopicrin less, accumulative percent penetration lag time in mouse part skin is higher, percutaneous rate is higher, therefore, this research take mouse part skin as object of study, carries out the screening of drug gel unguentum penetrating agent of the present invention.

3.6 different transdermal penetration enhancer are on the impact of gentiopicrin permeation behavior in drug gel unguentum of the present invention

3.6.1 variable concentrations azone is on the impact of permeation behavior

Get the drug gel unguentum of the present invention containing 1%, 3%, 5%, 7% azone respectively, with the skin of abdomen of mice for object of study, take distilled water as accepting medium, investigate the release behavior of gentiopicrin in drug gel unguentum of the present invention, unit of account area cumulative release amount (Qn, μ g/cm ²), percutaneous rate (J), total release percentage (Q), enhancing rate (ER) and lag time (T _lAG), the results are shown in Table 25,26.

Table 25 azone is on the impact (n=3) of gentiopicrin external unit are accumulation infiltration capacity

Table 26 variable concentrations azone is to gentiopicrin transdermal test in vitro permeability parameters in drug gel unguentum of the present invention

Variable concentrations azone shows the result of calculation of gentiopicrin transdermal test in vitro permeability parameters in drug gel unguentum of the present invention, the anatonosis effect of azone to gentiopicrin transdermal test in vitro infiltration capacity has certain dose-effect relationship, when azone concentration reaches a certain amount of, play inhibitory action on the contrary, when concentration is 3%, the short effect of oozing is best.

3.6.2 variable concentrations propylene glycol is on the impact of permeation behavior

Get the drug gel unguentum of the present invention containing 5%, 7%, 9%, 10% propylene glycol respectively, with the skin of abdomen of mice for object of study, take distilled water as accepting medium, investigate the release behavior of gentiopicrin in drug gel unguentum of the present invention, unit of account area cumulative release amount (Qn, μ g/cm ²), percutaneous rate (J), total release percentage (Q), enhancing rate (ER) and lag time (T _lAG), the results are shown in Table 27, table 28.

Table 27 propylene glycol is on the impact (n=3) of gentiopicrin external unit are accumulation infiltration capacity

Table 28 variable concentrations propylene glycol is to gentiopicrin transdermal test in vitro permeability parameters in drug gel unguentum of the present invention

Variable concentrations propylene glycol shows the result of calculation of gentiopicrin transdermal test in vitro permeability parameters in drug gel unguentum of the present invention, the anatonosis effect of propylene glycol to gentiopicrin transdermal test in vitro infiltration capacity is certain linear relationship, when PG concentration reaches 9%, Q24 and percutaneous rate no longer increase.

3.6.3 variable concentrations Borneolum Syntheticum is on the impact of permeation behavior

Get the drug gel unguentum of the present invention containing 1%, 2%, 4%, 6% Borneolum Syntheticum respectively, with the skin of abdomen of mice for object of study, take distilled water as accepting medium, investigate the release behavior of gentiopicrin in drug gel unguentum of the present invention, unit of account area cumulative release amount (Qn, μ g/cm ²), percutaneous rate (J), total release percentage (Q), enhancing rate (ER) and lag time (T _lAG), the results are shown in Table 29,30.

Table 29 Borneolum Syntheticum is on the impact (n=3) of gentiopicrin external unit are accumulation infiltration capacity

Table 30 variable concentrations Borneolum Syntheticum is to gentiopicrin transdermal test in vitro permeability parameters in drug gel unguentum of the present invention

Variable concentrations Borneolum Syntheticum shows the result of calculation of gentiopicrin transdermal test in vitro permeability parameters in drug gel unguentum of the present invention, compared with 0% Borneolum Syntheticum, adding the short effect of oozing of gentiopicrin of Borneolum Syntheticum is remarkable, enhancing rate ER is its 4 ~ 5 times, when wherein concentration is 1% ~ 2%, Q24 and percutaneous rate are more or less the same, and when concentration is greater than 2%, short effect of oozing reduces on the contrary.

3.7 containing the drug gel unguentum ocular estimate result of the present invention of different penetration enhancers

According to the ocular estimate standard in 2.2, outward appearance scoring is carried out to the drug gel unguentum of the present invention containing different penetrating agent, the results are shown in Table 31.

Table 31 is containing the drug gel unguentum ocular estimate result of the present invention of different penetration enhancers

As can be seen from above-mentioned evaluation result, penetration enhancers add mastic uniformity, film residual, stretchability change are little; Azone increases along with the increase adding percentage ratio is oozed out, propylene glycol to add degree of oozing out comparatively large, and adding of Borneolum Syntheticum does not affect degree of oozing out; Each penetrating agent is all along with the increase skin tracing ability adding percentage ratio reduces.

3.8 containing the drug gel unguentum comprehensive evaluation result of the present invention of different penetration enhancers

This research is with ocular estimate, first viscous force, percutaneous rate for evaluation index, and adopt Synthetic weighted mark method, carry out overall merit to each gel ointment, weight coefficient shared by it is respectively 0.3,0.3,0.4, and evaluation result is in table 32.

Table 32 is containing the drug gel unguentum comprehensive evaluation result of the present invention of different penetration enhancers

Note: total score=(ocular estimate score/outer light evaluates score maximum) × 0.3 × 100+(just viscous force minima/first viscous force value) × 0.3 × 100+(percutaneous rate/percutaneous rate maximum) × 0.4 × 100

Table 32 shows, with 2% Borneolum Syntheticum for penetrating agent is little on the impact of the presentation quality of drug gel unguentum of the present invention, first viscous force, and increase significantly for accumulative permeability, therefore this research selection 2% Borneolum Syntheticum is the penetrating agent of drug gel unguentum of the present invention.

The establishment of embodiment 5 medicine moulding process of the present invention prescription and technique are amplified

1. preparations shaping technology preparation is established

The establishment of 1.1 extractum water content

Drug gel unguentum of the present invention is used as medicine with Chinese medicine extract extractum, and early-stage Study finds that the water content of extractum has larger impact for the molding of preparation and stability, and the too high meeting of water content causes the rotten cream of preparation, stickiness to reduce, and storage is easily mouldy.Therefore, production strictly should control the content of moisture in extractum.The relative density of extractum, through test of many times, controls 1.25 ~ 1.30 by this research, to make drug gel unguentum preparations shaping process stabilizing of the present invention.

The establishment of 1.2 prescription substrate drug loading

The drug loading of gel ointment substrate also affects the performance of preparation, and drug loading is excessive, easily occurs leaking cream, skin tracing ability is poor, skin residual quantity is large.Drug loading is too small, needs larger administration area because unit are content of dispersion is little, causes the waste of substrate.This research is on the substrate bases optimized, and investigate the drug loading of drug gel unguentum of the present invention, result is shown in table 33.

Table 33 drug loading is on the impact of drug gel unguentum moulding process of the present invention

Table 33 shows, it is more suitable to add 6g extractum in the substrate often optimized, and prepared drug gel unguentum of the present invention is homogeneous, fine and smooth, and bubble is less, and skin tracing ability is good, therefore adds 6g extractum by the substrate often optimized.

1.3 bubble removing methods

Due to the restriction of test apparatus equipment, the research of room temperature placement, ultrasonic bubble removing has only been carried out in this research, all do not realize satisfactory results, but the production of bubble can be reduced when finding to stir in experimentation along single direction, BT/S-I type gel ointment coating machine is adopted to carry out prolonging platen press coating during coating, because of the squeezing action of coating process, part bubble can be eliminated.

The establishment of 1.4 coating duration

Coating duration has larger impact for the production of drug gel unguentum of the present invention, after finding in test that mastic has prepared 6 hours, mastic starts to be cross-linked, elasticity strengthens, now be coated with and easily occur block and make cream face uneven, therefore coating should complete in 0 ~ 6 hour after prepared by mastic.

The establishment of 1.5 coating thickness and dosage

The mastic that this research is prepared according to every recipe quantity, with reference to the specification of commercially available gel ointment, compares through repetition test, think coating time thickness select 2.5mm(wherein to comprise embossing film and the 100g/m of 30c ²non-woven fabrics), area 7cm × 10cm is more suitable.

The establishment of 1.6 conditions of cure

Early-stage Study finds that time of gel ointment molding after fixing and speed and temperature are closely related.The drug gel unguentum of the present invention that preparation molding is got in this research is put in room temperature respectively and dries and the drying in oven of different temperatures, and after hardening time and solidification, the performance of mastic is shown in table 34.

The different condition of cure of table 34 is on the impact of drug gel unguentum performance of the present invention

Table 34 shows, drug gel unguentum room temperature of the present invention is placed and dried and 40 DEG C, 60 DEG C, 80 DEG C oven dry, and hardening time used reduces successively.Except 80 DEG C of bake out temperatures are higher, make outside mastic shrinkage, stickiness reduction, room temperature is placed to dry and is dried paste properties without obvious difference with 40 DEG C, 60 DEG C.Consider, select 60 DEG C to dry as curing mode is comparatively suitable.

2. preparation process amplifies research

2.1 technique amplification test prescription compositions

Radix Gentianae Macrophyllae 455g, Caulis Sinomenii 455g, Radix Paeoniae Alba 455g, Radix Angelicae Sinensis 455g, Radix Tripterygii Wilfordii 340g, sodium polyacrylate 66g, carbomer 25g, aluminum glycinate 1.8g, citric acid 2.8g, glycerol 100g, Oleum Ricini 25g, Borneolum Syntheticum 32g, water 1000g, amount to 100 and paste.

2.2 method for making

Above five kinds of Chinese medicine, Radix Angelicae Sinensis pulverized 10 mesh sieves, added 10 times of water gagings, soak 3h, steam distillation distillation 8h, respectively collect volatile oil and extracting solution for subsequent use, all the other 4 taste medicines soak 1h, use 50% alcohol heating reflux three times of 9 times amount, 8 times amount, 8 times amount respectively, each 1 hour, extracting solution reclaims ethanol to without alcohol taste, merge that to be concentrated into relative density be 1.25 ~ 1.30(50 DEG C with the mother solution after volatile oil is drawn) extractum, again volatile oil is added, stir, obtain total extractum.Take the sodium polyacrylate of recipe quantity, after being uniformly dispersed with glycerol, add suitable quantity of water, swelling evenly as A phase; Carbomer add suitable quantity of water swelling after mix homogeneously with above-mentioned total extractum, then will add wherein with the finely dispersed aluminum glycinate of Oleum Ricini, stir as B phase; Borneolum Syntheticum is as C phase; Aqueous citric acid solution is as D phase; After B and C two-phase mixtures is even, then add A phase mix homogeneously, after finally mixing with D phase, 60 DEG C, refining and 20min under 100r/min condition, adjustment coating thickness is 2.5mm, is applied on non-woven fabrics, 60 DEG C of oven dry, are cut into 7cm × 10cm size, altogether 100 to lose money in a business invention drug gel unguentum.

Beneficial effect of the present invention is proved below by way of concrete pharmacodynamics test.

1. test apparatus, animal and reagent

1.1 instrument

YLS-6A intelligence hot-plate instrument (Shandong Academy of Medical Sciences's equipment station); Card punch (8mm); Timer; BS224S electronic balance (ten thousand/, Beijing Sai Duolisi scientific instrument company limited).

1.2 animal

Kunming mouse, body weight 18 ~ 22g, by Chongqing Institute of Chinese Medicine, institute of lab animals provides.1.3 reagent

Sodium sulfide (Chongqing Mao Ye chemical reagent company limited, lot number 20080506); Glacial acetic acid (Chongqing Chuan Dong Chemical Group company limited chemical reagent factory, lot number 20110301); Dimethylbenzene (Chengdu Ke Long chemical reagent factory, lot number 20091225); SHANGSHI ZHITONG GAO (hygienic material limited company of alliance is revised in Jiaozhuo, lot number 20110203).

2. the preparation of pharmacological evaluation need testing solution

The preparation (extraction process by water) of test sample: take the 5 taste decoction pieces such as Radix Gentianae Macrophyllae by recipe quantity, with 8 times amount soak by water three times, each 1h, filters.Be concentrated into the medicinal liquid containing crude drug 1.0g/ml, obtain final product.

The preparation (alcohol extraction process) of test sample: take the 5 taste decoction pieces such as Radix Gentianae Macrophyllae by recipe quantity, with 8 times amount 50% alcohol reflux three times, each 1h, filters.Decompression recycling ethanol, to without alcohol taste, is concentrated into the medicinal liquid of 1.0g crude drug/ml, obtains final product.

The preparation (volatile oil+extraction process by water) of test sample: get recipe quantity Radix Angelicae Sinensis and pulverized 10 mesh sieves, add the water of 10 times amount, soak 3h, extraction by steam distillation volatile oil 8h, respectively collect volatile oil and extracting solution for subsequent use.Radix Gentianae Macrophyllae etc. 4 taste medicine, with 8 times amount soak by water three times, each 1h, filters, merges above-mentioned filtrate, be concentrated into the medicinal liquid of 1.0g crude drug/ml, then add Radix Angelicae Sinensis volatile oil, to obtain final product.

The preparation (volatile oil+alcohol extraction process) of test sample: get recipe quantity Radix Angelicae Sinensis and pulverized 10 mesh sieves, add the water of 10 times amount, soak 3h, extraction by steam distillation volatile oil 8h, respectively collect volatile oil and extracting solution for subsequent use.Radix Gentianae Macrophyllae etc. 4 taste medicine, with 8 times amount 50% alcohol reflux three times, each 1h, filters, and decompression recycling ethanol, to without alcohol taste, merges above-mentioned filtrate, is concentrated into the medicinal liquid of 1.0g crude drug/ml, then adds Radix Angelicae Sinensis volatile oil, to obtain final product.

Test example 1 hot plate method in mice analgesic test

First adopt intelligent hot-plate instrument under the condition of 55 ± 0.5 DEG C, measure the normal pain threshold of every mice respectively before administration.Be put on intelligent hot-plate instrument to occurring licking the time of metapedes as pain threshold (second) using mice, METHOD FOR CONTINUOUS DETERMINATION 2 times, midfeather 30min, and using the meansigma methods of twice mensuration as this mice administration before normal pain threshold, as appointed more than 60s so without licking rear foot phenomenon, then in 60s.Pain threshold is less than 5 seconds or be greater than 30 seconds, leaper gives it up.

Get the qualified female mice of pain threshold 60, be divided into 6 groups at random by its normal pain threshold size, often organize 10, be respectively: 1. blank group: give distilled water; 2. water extraction group: give water extraction test sample; 3. alcohol extraction group: give alcohol extraction test sample; 4. volatile oil+water extraction group: give volatile oil+water extraction test sample; 5. volatile oil+alcohol extraction group: give volatile oil+alcohol extraction test sample; 6. positive drug group: give SHANGSHI ZHITONG GAO.Mice 8%Na ₂3cm × 2cm size is lost hair or feathers in S solution nape portion, and after 24h, apply ointment or force in epilating area, and volume of applying ointment is 0.28ml/20g body weight.Every day 1 time, for three days on end, apply ointment 30 in the 4th day last, 60, be measured in the same method each mice pain threshold after 90min.The results are shown in Table 35.

Table 35 different process route is to the analgesic activity (hot plate method) of mice

Note: compare with blank group, * P<0.05, * * P<0.01

From table 35, each extraction process route is improved the effect of pain threshold of mice, and wherein, volatile oil+alcohol extraction process and positive drug have significant analgesic activity to hot plate method is pain caused.In addition, table 35 also demonstrates, and volatile oil part has good analgesic effect.

Test example 2 mouse writhing method analgesic test

Get mice 60, be divided into 6 groups at random, often organize 10, male and female half and half, grouping and administration are with under the item of hot plate method in mice analgesic test.Each group of mice is after last administration 1h, every mouse peritoneal injection 0.3ml concentration is the glacial acetic acid solution of 0.7%, and there is number of times and writhing time first of writhing response (i.e. mouse web portion indent, stretch hind leg, buttocks is raised) in observed and recorded mice immediately in 20min.Calculate analgesia suppression ratio, analgesia suppression ratio (%)=[(matched group writhing number of times-administration group writhing number of times)/matched group writhing number of times] × 100%.The results are shown in Table 36.

Table 36 different process route is to the analgesic activity (writhing method) of mice

Note: compare with blank group, * P<0.05, * * P<0.01

From table 36, after each extraction process and SHANGSHI ZHITONG GAO administration, all there is remarkable analgesic activity, can the incubation period of significant prolongation mice generation writhing response and the number of times of remarkable minimizing writhing response.

Test example 3 mice dimethylbenzene auricle causes scorching test

Get mice 60, be divided into 6 groups at random, often organize 10, male and female half and half, grouping and administration are with under the item of hot plate method in mice analgesic test.Each group of mice, after last administration 30min, draws 20 μ l dimethylbenzene with liquid-transfering gun and is spread evenly across mouse right ear exterior feature and causes inflammation.To cause sacrifice after scorching 1h, cut left and right sides auricle simultaneously, lay circular auricle at the position that two auricles are identical respectively with the card punch that diameter is 8mm, accurately weighed two auricle weight.The difference (mg) of left auricle weight is deducted as inflammation swelling using auris dextra sheet weight.Calculate inflammation suppression ratio, inflammation suppression ratio (%)=[(matched group swelling-administration group swelling)/matched group swelling] × 100%.The results are shown in Table 37.

The impact of table 37 different process route xylol induced mice auricle inflammation swelling

Note: compare with blank group, * P<0.05, * * P<0.01

From table 37, except blank group, all the other each group all has antiinflammatory action, wherein, volatile oil+water extraction group, volatile oil+alcohol extraction group and the swelling of positive group xylol induced mice auricle inflammation all have significant inhibitory action, also show that volatile oil has good antiinflammatory action simultaneously.

In sum, we adopt volatile oil+alcohol extraction process to have significant analgesia and antiinflammatory action, meanwhile, also show that volatile oil has the effect of antalgic and inflammation relieving.

Claims (12)

1. treat an externally-applied medicinal composition for rheumatoid arthritis, it is characterized in that: it is the external preparation be prepared from by the crude drug of following weight proportioning:

Radix Gentianae Macrophyllae 15-25 part, Caulis Sinomenii 15-25 part, Radix Paeoniae Alba 15-25 part, Radix Angelicae Sinensis 15-25 part and Radix Tripterygii Wilfordii 10-20 part.

2. externally-applied medicinal composition according to claim 1, is characterized in that: it is the external preparation be prepared from by the crude drug of following weight proportioning:

Radix Gentianae Macrophyllae 20 parts, Caulis Sinomenii 20 parts, the Radix Paeoniae Alba 20 parts, Radix Angelicae Sinensis 20 parts, Radix Tripterygii Wilfordii 15 parts.

3. externally-applied medicinal composition according to claim 1 and 2, it is characterized in that: it is active component by the water of Radix Gentianae Macrophyllae, Caulis Sinomenii, the Radix Paeoniae Alba, Radix Angelicae Sinensis, Radix Tripterygii Wilfordii or extractive with organic solvent, add the external preparation that pharmaceutically acceptable adjuvant or adjuvant composition are prepared from.

4. externally-applied medicinal composition according to claim 3, is characterized in that: described external preparation is emplastrum, tincture, liniment, gel.

5. externally-applied medicinal composition according to claim 4, is characterized in that: described emplastrum is rubber-emplastrum, gel ointment, patch.

6. externally-applied medicinal composition according to claim 3, is characterized in that: described external preparation is ointment.

7. externally-applied medicinal composition according to claim 5, is characterized in that: in described gel ointment, every sheet contains Radix Gentianae Macrophyllae with gentiopicrin (C ₁₆h ₂₀o ₉) must not count and be less than 51.16mg.

8. treat an external-use gel unguentum for rheumatoid arthritis, it is characterized in that: be prepared from by the stock and adjunct of following weight proportioning:

Radix Gentianae Macrophyllae 318.5-591.5 part, Caulis Sinomenii 318.5-591.5 part, Radix Paeoniae Alba 318.5-591.5 part, Radix Angelicae Sinensis 318.5-591.5 part, Radix Tripterygii Wilfordii 238-442 part, adhesive 46.2-85.8 part, excipient 17.5-32.5 part, cross-linking agent 1.26-2.34 part, cross-linking regulator 1.96-3.64 part, wetting agent 87.5-162.5 part, penetration enhancers 22.4-41.6 part

Wherein, described adhesive is selected from one or more the mixing in sodium polyacrylate, polyvinyl alcohol, carboxymethyl cellulose class, gelatin; Described excipient is selected from one or more the mixing in carbomer, Kaolin, titanium dioxide, calcium carbonate, zinc oxide, Pulvis Talci; Described wetting agent is selected from propylene glycol, glycerol, Oleum Ricini; It is aluminum salt that described cross-linking agent is selected from cross-linking agent, one or more the mixing in aluminum glycinate, aluminium hydroxide, aluminum chloride, dihydroxyaluminum aminoacetate; Described cross-linking regulator is selected from citric acid, tartaric acid; Described penetration enhancers is selected from one or more the mixing in azone, propylene glycol, Borneolum Syntheticum.

9. external-use gel unguentum according to claim 8, is characterized in that: described gel ointment is prepared from by the raw material of following weight proportioning:

Radix Gentianae Macrophyllae 455 parts, Caulis Sinomenii 455 parts, the Radix Paeoniae Alba 455 parts, Radix Angelicae Sinensis 455 parts, Radix Tripterygii Wilfordii 340 parts, sodium polyacrylate 66 parts, carbomer 25 parts, aluminum glycinate 1.8 parts, citric acid 2.8 parts, glycerol 100 parts, Oleum Ricini 25 parts, Borneolum Syntheticum 32 parts.

10. prepare a method for the externally-applied medicinal composition described in claim 1-4 any one, it comprises the steps:

A, take the crude drug of weight proportion;

B, Radix Angelicae Sinensis extract volatile oil, and extracting solution is for subsequent use;

C, Radix Gentianae Macrophyllae, Caulis Sinomenii, the Radix Paeoniae Alba, Radix Tripterygii Wilfordii add water or ethanol extraction, add the Radix Angelicae Sinensis extracting solution of b step, concentrated;

D, extractum step c concentrated mix with the Radix Angelicae Sinensis volatile oil of b step, add the acceptable adjuvant of pharmacy or are complementaryly prepared into pharmaceutically conventional external preparation.

The preparation method of 11. externally-applied medicinal compositions according to claim 10, is characterized in that: the method that the Radix Angelicae Sinensis described in b step extracts volatile oil is steam distillation or supercritical fluid extraction; Described ethanol is 50% ethanol.

The purposes of externally-applied medicinal composition described in 12. claim 1-4 any one in the medicine of preparation treatment rheumatoid arthritis.