CN102759598B

CN102759598B - Quality detection method for 29-componnet stagnation dissipation powder as Tibetan medicinal composition and preparations thereof

Info

Publication number: CN102759598B
Application number: CN201210272243.1A
Authority: CN
Inventors: 孙泰俊; 孙绪丁; 刘玉芹; 任松鹏; 魏永义
Original assignee: Shandong Arura Pharmaceutical Research & Development Co Ltd
Current assignee: JINHE TIBETAN MEDICINE CO., LTD.
Priority date: 2012-07-31
Filing date: 2012-07-31
Publication date: 2014-10-15
Anticipated expiration: 2032-07-31
Also published as: CN102759598A

Abstract

The invention discloses a quality detection method for 29-componnet stagnation dissipation powder as a Tibetan medicinal composition and preparations thereof. IN the quality detection method, thin layer chromatography is used to carry out qualitative identification on frankincense, piperine and myristica fragrans in the 29-componnet stagnation dissipation powder, high performance liquid chromatography (HPLC) is used to carry out limit test on aconitine and costustoot aristolochic acid in the 29-componnet stagnation dissipation powder, and simultaneously, the high performance liquid chromatography is used to carry out quantitative detection on aloe-emodin, chrysophanol, rhein, emodin, emodin monomethyl ether and hydroxysafflor yellow A in the 29-componnet stagnation dissipation powder. According to the invention, the thin-layer identification method for the frankincense, the piperine and the myristica fragrans, the limit test method for the aconitine and the costustoot aristolochic acid, and the content determination method for the aloe-emodin, the chrysophanol, the rhein, the emodin, the emodin monomethyl ether and the hydroxysafflor yellow A are developed, so that the quality standard of a product is increased, and drug safety and effectiveness are guaranteed while the 29-componnet stagnation dissipation powder and the preparations thereof are used for treating diseases.

Description

A kind of detection method of Tibetan medicinal composition 29 taste energy oral preparation for eliminating

Technical field

The present invention relates to the quality determining method of a kind of Tibetan medicinal composition and preparation thereof, particularly a kind of Tibetan medicinal composition 29 tastes can dissipate and the quality determining method of preparation.

Background technology

29 tastes can dissipate and be recorded in the clinical reading notes > of < < Tibetan medicine >, good the arranging of author's sea urchin roe paste is the famous Tibetan medicine in 13rd century, he sums up through clinical practice for many years, and on the basis of forefathers' prescription, concocting out 29 tastes can dissipate.Because of its good efficacy to hysteromyoma and ovarian cyst, by successive dynasties Tibetan medicine scholar, praised highly.29 tastes can dissipate and be comprised of 29 taste medicines such as Tibet inula root, gypsum rubrum (forging), myrobalan, hot millet, alkali flower, nutmeg, the Bi roots of grass, rheum officinale, Aconitum Szechenyianum Gay, safflower, banksia rose birthwort, Semen seu folium abelmoschi moschatis, have dispelling cold ruffian, help digestion, the effect of liver-regulating and kidney-nourishing.Be mainly used in accumulation of food in the stomach and intes tine due to indigestion, stomach and intestine hepatalgia, ephrosis, enteropathy, poisoning, hepatopathy, hysteropathy, yellow water spills into sering, and stomach and intestine ruffian is sick, and courage ruffian is sick, the ruffian knurl that chill causes etc.

29 tastes can dissipate and record in the national drug standards, standard number: WS3-BC-0140-95.In side, Aconitum Szechenyianum Gay contains aconitine, has higher pharmacologically active and medical value, also has stronger toxicity simultaneously, although toxic component obviously reduces after concocting, can not guarantee medicinal security; In side, banksia rose birthwort is containing aristolochic acid, and aristolochic acid can cause the bad reactions such as kidney damage.In proper mass standard, do not differentiate and assay item, the aristolochic acid that the aconitine Aconitum Szechenyianum Gay not being contained, banksia rose birthwort contain carries out limit test, makes said preparation have potential safety hazard.Meanwhile, said preparation do not differentiate and assay its effective constituent yet, thereby can not guarantee effective use of said preparation.Document and patent also have no can the dissipate report of quality testing aspect of relevant 29 tastes.Therefore, safe and effective for medication in order to guarantee patient, proper mass standard urgently needs to improve.

Summary of the invention

For the deficiencies in the prior art, the object of this invention is to provide that a kind of Tibetan medicinal composition 29 tastes can dissipate and the quality determining method of preparation.

Summary of the invention

The invention provides that a kind of Tibetan medicinal composition 29 tastes can dissipate and the limit test method of preparation mesaconitine, the limit test method of aristolochic acid A, frankincense, pipering, myristic TLC Identification are provided simultaneously, the high performance liquid chromatography content assaying method of aloe-emodin, Chrysophanol, Rhein, archen, Physcion, hydroxyl radical carthamin yellow carthamus A, make said preparation quality testing more accurately, reliably, make said preparation in treatment disease, guaranteed the safe and effective of medication.

Term explanation:

29 tastes can dissipate, and are the nomenclature of drugs that national standard for traditional Chinese medicines compilation (Chinese patent drug provincial standard rising national standard part) is recorded.

29 tastes can dissipate and preparation, comprise can dissipate Tibetan medicinal composition and with the 29 tastes preparation prepared by bulk drug formula that can dissipate of 29 tastes.

Gypsum rubrum (forging), radish (charcoal), shellfish tooth (charcoal), lime (system), vulture excrement (stir-fry): the routine techniques term that is Chinese medicine (Tibetan medicine), " forging " in bracket, " charcoal ", " system ", " stir-fry " etc. are all concocted according to the inner method of the Tibetan medicine concocted specification > > of < < Qinghai Province (Qinghai Province food and medicine Surveillance Authority compiles, 2010 years versions that the Qinghai People's Press publishes).

Technical scheme of the present invention is as follows:

A kind of bulk drug consists of Tibet inula root 25 weight portions, gypsum rubrum (forging) 125 weight portions, myrobalan's 75 weight portions, hot millet 25 weight portions, alkali is spent 125 weight portions, nutmeg 25 weight portions, the Bi roots of grass 25 weight portions, cassia seed 25 weight portions, Amomum cardamomum 25 weight portions, the rhizome of davallia 25 weight portions, pepper 25 weight portions, radish (charcoal) 2 weight portions, tsaoko 25 weight portions, natural salt 25 weight portions, asafoetide 2 weight portions, sal ammoniac 25 weight portions, kaempferia galamga 25 weight portions, shellfish tooth (charcoal) 25 weight portions, rheum officinale 100 weight portions, wide muscle rattan 13 weight portions, safflower 25 weight portions, Aconitum Szechenyianum Gay 15 weight portions, lime (system) 25 weight portions, vulture excrement (stir-fry) 40 weight portions, banksia rose birthwort 25 weight portions, Semen seu folium abelmoschi moschati 25 weight portions, HALITUM PURPUREUM 25 weight portions, frankincense 25 weight portions, Tibetan medicinal composition 29 tastes that slag is tamed and dociled cream 25 weight portions can dissipate and the quality determining method of preparation, the method comprises one or more in following discriminating and/or inspection and/or content assaying method:

Differentiate:

A. the discriminating of frankincense

Get that Tibetan medicinal composition 29 tastes can dissipate or its preparation powder 2 ~ 4g, the 20 ~ 30ml that adds diethyl ether, ultrasonic processing 15min, filters, and filtrate volatilizes, and residue adds methyl alcohol 2ml to be made to dissolve, as need testing solution; Get frankincense control medicinal material 0.5g, the 30ml that adds diethyl ether, ultrasonic processing 15min, filters, and filtrate volatilizes, and residue adds methyl alcohol 2ml to be made to dissolve, in contrast medicinal material solution; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, dimethylbenzene-ethyl acetate that the volume parts ratio of take is 8:1 ~ 2 is developping agent, launch, take out, dry; Spray is with quality volume portion rate 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;

B. the discriminating of pipering

Get that Tibetan medicinal composition 29 tastes can dissipate or its preparation powder 2 ~ 4g, add ethyl acetate 20 ~ 30ml, ultrasonic processing 30min, filters, and filtrate is concentrated into 2ml, as need testing solution; Get pipering appropriate, add methyl alcohol and make every 1ml containing the reference substance solution of 0.5mg; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, cyclohexane-ethyl acetate that the volume parts ratio of take is 3:1 ~ 3 is developping agent, launch, take out, dry; Put under ultraviolet lamp 365nm and inspect; In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

C. myristic discriminating

Get that Tibetan medicinal composition 29 tastes can dissipate or its preparation powder 4 ~ 6g, put in 250ml round-bottomed flask, add water 100ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil, in volatile oil determination apparatus, start to add a small amount of water and 1ml ethyl acetate, after having decocted, collect ethyl acetate part, as need testing solution; Get nutmeg 1g, put in 100ml round-bottomed flask, add water 50ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil, in volatile oil determination apparatus, start to add a small amount of water and 1ml ethyl acetate, after having decocted, collect ethyl acetate part, in contrast medicinal material solution; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, dimethylbenzene-ethyl acetate that the volume parts ratio of take is 20:0.1 ~ 0.5 is developping agent, launch, take out, dry, the long-pending portion rate of sprinkler body is 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;

Check:

A. the limit test of aconitine

According to high performance liquid chromatography (appendix VI D of < < Chinese Pharmacopoeia > > version in 2010), measure;

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; The volume parts of take is mobile phase than methanol-water-triethylamine-methylene chloride of 65 ~ 75:25 ~ 35:0.2:5; Detection wavelength is 235nm, and number of theoretical plate calculates and should be not less than 2000 by aconitine peak;

The preparation of reference substance solution: get aconitine reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 10 μ g, obtain;

The preparation of need testing solution: get that Tibetan medicinal composition 29 tastes can dissipate or its preparation powder 10 ~ 15g, accurately weighed, put in the conical flask of tool plug, add ammonia solution 10ml, precision adds absolute ethyl alcohol 80～120ml, close plug, weighed weight, after cold soaking 1h, add hot reflux 1h, let cool, more weighed weight, with absolute ethyl alcohol, supply the weight of less loss, shake up, filter; Precision measures subsequent filtrate 50ml, and 25 ℃ ~ 40 ℃ decompression and solvent recoveries are to dry, and residue precision adds dilute hydrochloric acid solution 20ml to dissolve, and places 30min in ice bath, filters; With ammoniacal liquor, adjust pH to 10, filtrate is transferred in separating funnel, add ethyl acetate extraction 5 times, each 20ml, combined ethyl acetate liquid, 25 ℃ ~ 40 ℃ decompression and solvent recoveries are to dry, and residue precision adds methyl alcohol 2ml to dissolve, and filters, and gets subsequent filtrate, obtains;

Determination method: precision is drawn reference substance solution and each 10 ~ 20 μ l of need testing solution respectively, and injection liquid chromatography, measures, and obtains;

The present invention's 29 tastes can dissipate or its preparation contains Aconitum Szechenyianum Gay with aconitine (C ₃₄h ₄₇nO ₁₁) meter, must not be higher than 17 μ g/g.

B. the limit test of aristolochic acid A

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; The volume parts of take is mobile phase than the methyl alcohol-volume parts of 60～70:30～40 than 1% glacial acetic acid aqueous solution; Detection wavelength is 315nm; Number of theoretical plate calculates and should be not less than 3000 by banksia rose aristolochic acid peak;

The preparation of reference substance solution: get aristolochic acid A reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 20 μ g, obtain;

The preparation of need testing solution: get that Tibetan medicinal composition 29 tastes can dissipate or its preparation powder 6 ~ 10g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 40 ~ 60ml, close plug, weighed weight, ultrasonic 20 ~ 40 minutes, let cool, weighed weight again, with methyl alcohol, supply the weight of less loss, shake up, filter, get subsequent filtrate 25ml, low temperature is concentrated into dry, it is that 0.5% sodium hydroxide solution 20ml dissolves it completely and is transferred in separating funnel that residue adds volume parts ratio, by extracted with diethyl ether 2 times, each 20ml, after extraction, alkali lye service property (quality) mark 3% salt acid for adjusting pH is to 2-3, by extracted with diethyl ether 5 times, each 20ml, merge ether extraction liquid, volatilize, with methyl alcohol, dissolve and be transferred in 5ml volumetric flask, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain,

Determination method: draw respectively each 10 ~ 20 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains.

The present invention's 29 tastes can dissipate or its preparation contains banksia rose birthwort with aristolochic acid A (C ₁₇h ₁₁nO ₇) meter, must not be higher than 30 μ g/g.

Assay:

A. the assay of aloe-emodin, Chrysophanol, Rhein, archen, Physcion

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; The methyl alcohol that the volume parts ratio of take is 80 ~ 90:10 ~ 20-volume parts is mobile phase than the phosphate aqueous solution as 0.1%; Detection wavelength is 430nm.Number of theoretical plate calculates and should be not less than 3000 by archen peak;

The preparation of reference substance solution: it is appropriate that precision takes aloe-emodin reference substance, Rhein reference substance, archen reference substance, Chrysophanol reference substance, Physcion reference substance, add methyl alcohol and make respectively every 1ml containing aloe-emodin reference substance, Rhein reference substance, archen reference substance, each 80 μ g of Chrysophanol reference substance, the solution of Physcion 40 μ g, precision measures each 2ml of above-mentioned reference substance solution respectively, mix, obtain; Be containing aloe-emodin, Rhein, archen, each 16 μ g of Chrysophanol, containing Physcion 8 μ g in every 1ml;

The preparation of need testing solution: get that Tibetan medicinal composition 29 tastes can dissipate or its preparation powder 1 ~ 3g, accurately weighed, put in the conical flask of tool plug, precision adds methyl alcohol 20 ~ 30ml, close plug, weighed weight, ultrasonic 20 ~ 40min, let cool, with methyl alcohol, supply the weight of less loss, shake up, filter, precision measures gets subsequent filtrate 5ml, put in flask, fling to solvent, adding percent by volume is 8% aqueous hydrochloric acid solution 10ml, ultrasonic 2 minutes, add again methenyl choloride 10ml, add hot reflux 1 hour, let cool, put in separating funnel, divide and get methenyl choloride layer, acid solution is extracted 3 times with methenyl choloride again, each 10ml, merge methenyl choloride liquid, recovered under reduced pressure solution is to dry, residue adds methyl alcohol to be made to dissolve, be transferred in 10ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain,

Determination method: precision is drawn reference substance solution and each 5 ~ 10 μ l of need testing solution respectively, and injection liquid chromatography, measures, and obtains.

The present invention's 29 tastes can dissipate or its preparation contains rheum officinale with aloe-emodin (C ₁₅h ₁₀o ₅), Rhein (C ₁₅h ₈o ₆), archen (C ₁₅h ₁₀o ₅), Chrysophanol (C ₁₅h ₁₀o ₄), Physcion (C ₁₆h ₁₂o ₅) total content meter, must not be less than 1.20mg/g.

B. the assay of hydroxyl radical carthamin yellow carthamus A

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take volume parts than the methyl alcohol of 20～30:70～80-volume parts ratio as 1% glacial acetic acid aqueous solution be mobile phase; Detection wavelength is 403nm; Number of theoretical plate calculates and should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak;

The preparation of reference substance solution: get hydroxyl radical carthamin yellow carthamus A reference substance appropriate, accurately weighed, put in brown bottle, add volume parts than being that 25% methanol aqueous solution is made every 1ml containing the solution of 50 μ g, obtain;

The preparation of need testing solution: get that Tibetan medicinal composition 29 tastes can dissipate or its preparation powder 3 ~ 5g, accurately weighed, put in tool plug conical flask, it is 25% methanol aqueous solution 20 ~ 30ml that precision adds volume parts ratio, close plug, weighed weight, ultrasonic 30 ~ 50 minutes, let cool, by volume parts, than being the weight that 25% methanol aqueous solution is supplied less loss, shake up, filter, get subsequent filtrate, obtain;

Determination method: draw respectively each 5 ~ 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains.

The present invention's 29 tastes can dissipate or its preparation in hydroxyl radical carthamin yellow carthamus A (C ₂₇h ₃₀o ₁₅) content must not be less than 0.20mg/g.

Described preparation refers to gets Tibetan medicinal composition 29 tastes bulk drug Tibet inula root 25 weight portions that can dissipate, gypsum rubrum (forging) 125 weight portions, myrobalan's 75 weight portions, hot millet 25 weight portions, alkali is spent 125 weight portions, nutmeg 25 weight portions, the Bi roots of grass 25 weight portions, cassia seed 25 weight portions, Amomum cardamomum 25 weight portions, the rhizome of davallia 25 weight portions, pepper 25 weight portions, radish (charcoal) 2 weight portions, tsaoko 25 weight portions, natural salt 25 weight portions, asafoetide 2 weight portions, sal ammoniac 25 weight portions, kaempferia galamga 25 weight portions, shellfish tooth (charcoal) 25 weight portions, rheum officinale 100 weight portions, wide muscle rattan 13 weight portions, safflower 25 weight portions, Aconitum Szechenyianum Gay 15 weight portions, lime (system) 25 weight portions, vulture excrement (stir-fry) 40 weight portions, banksia rose birthwort 25 weight portions, Semen seu folium abelmoschi moschati 25 weight portions, HALITUM PURPUREUM 25 weight portions, frankincense 25 weight portions, slag is tamed and dociled cream 25 weight portions, and technique, adds conventional auxiliary material to be prepared into clinical acceptable any formulation routinely, comprises micropill, dripping pill, tablet, capsule, particle or dispersing tablet.

Preferred a kind of bulk drug consists of Tibet inula root 25g, gypsum rubrum (forging) 125g, myrobalan 75g, hot millet 25g, alkali flower 125g, nutmeg 25g, Bi roots of grass 25g, cassia seed 25g, Amomum cardamomum 25g, rhizome of davallia 25g, pepper 25g, radish (charcoal) 2g, tsaoko 25g, natural salt 25g, asafoetide 2g, sal ammoniac 25g, kaempferia galamga 25g, shellfish tooth (charcoal) 25g, rheum officinale 100g, wide muscle rattan 13g, safflower 25g, Aconitum Szechenyianum Gay 15g, lime (system) 25g, vulture excrement (stir-fry) 40g, banksia rose birthwort 25g, Semen seu folium abelmoschi moschati 25g, HALITUM PURPUREUM 25g, frankincense 25g, Tibetan medicinal composition 29 tastes that slag is tamed and dociled cream 25g can dissipate and the quality determining method of preparation, the method comprises one or more in following discriminating and/or inspection and/or content assaying method:

Differentiate:

A. the discriminating of frankincense

Get the Tibetan medicinal composition 29 tastes powder 3g that can dissipate, the 25ml that adds diethyl ether, ultrasonic processing 15min, filters, and filtrate volatilizes, and residue adds methyl alcohol 2ml to be made to dissolve, as need testing solution; Get frankincense control medicinal material 0.5g, the 30ml that adds diethyl ether, ultrasonic processing 15min, filters, and filtrate volatilizes, and residue adds methyl alcohol 2ml to be made to dissolve, in contrast medicinal material solution; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the dimethylbenzene-ethyl acetate as 8:1.5, launch, take out, dry; Spray is with 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.

B. the discriminating of pipering

Get the Tibetan medicinal composition 29 tastes powder 3g that can dissipate, add ethyl acetate 25ml, ultrasonic processing 30min, filters, and filtrate is concentrated into 2ml, as need testing solution; Get pipering appropriate, add methyl alcohol and make every 1ml containing the reference substance solution of 0.5mg; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the cyclohexane-ethyl acetate as 3:2, launch, take out, dry; Put under ultraviolet lamp 365nm and inspect; In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

C. myristic discriminating

Get the Tibetan medicinal composition 29 tastes powder 5g that can dissipate, put in 250ml round-bottomed flask, add water 100ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil, in volatile oil determination apparatus, start to add a small amount of water and 1ml ethyl acetate, after decoction completes, collect ethyl acetate part, as need testing solution; Get nutmeg 1g, put in 100ml round-bottomed flask, add water 50ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil, in volatile oil determination apparatus, start to add a small amount of water and 1ml ethyl acetate, after having decocted, collect ethyl acetate part, in contrast medicinal material solution; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the dimethylbenzene-ethyl acetate as 20:0.3, launch, take out, dry, the long-pending portion rate of sprinkler body is 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.

Check:

A. the limit test of aconitine

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; The volume parts of take is mobile phase than methanol-water-triethylamine-methylene chloride of 68:32:0.2:5; Detection wavelength is 235nm, and number of theoretical plate calculates and should be not less than 2000 by aconitine peak;

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes powder 12g that can dissipate, accurately weighed, put in the conical flask of tool plug, add ammonia solution 10ml, precision adds absolute ethyl alcohol 100ml, close plug, weighed weight, after cold soaking 1h, add hot reflux 1h, let cool, more weighed weight, with absolute ethyl alcohol, supply the weight of less loss, shake up, filter; Precision measures subsequent filtrate 50ml, and 25 ~ 40 ℃ of decompression and solvent recoveries are to dry, and residue precision adds dilute hydrochloric acid solution 20ml to dissolve, and places 30min in ice bath, filters; With ammoniacal liquor, adjust pH to 10, filtrate is transferred in separating funnel, add ethyl acetate extraction 5 times, each 20ml, combined ethyl acetate liquid, 25 ~ 40 ℃ of decompression and solvent recoveries are to dry, and residue precision adds methyl alcohol 2ml to dissolve, and filters, and gets subsequent filtrate, obtains;

Determination method: precision is drawn reference substance solution and each 20 μ l of need testing solution respectively, and injection liquid chromatography, measures, and obtains;

B. the limit test of aristolochic acid A

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; The volume parts of take is mobile phase than methyl alcohol-volume parts of 66:34 than 1% glacial acetic acid aqueous solution; Detection wavelength is 315nm; Number of theoretical plate calculates and should be not less than 3000 by banksia rose aristolochic acid peak;

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes powder 8g that can dissipate, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic 30 minutes, let cool, weighed weight again, with methyl alcohol, supply the weight of less loss, shake up, filter, get subsequent filtrate 25ml, low temperature is concentrated into dry, it is that 0.5% sodium hydroxide solution 20ml dissolves it completely and is transferred in separating funnel that residue adds volume parts ratio, by extracted with diethyl ether 2 times, each 20ml, after extraction, alkali lye service property (quality) mark 3% salt acid for adjusting pH is to 2-3, by extracted with diethyl ether 5 times, each 20ml merges ether extraction liquid, volatilize, with methyl alcohol, dissolve and be transferred in 5ml volumetric flask, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain,

Determination method: draw respectively each 20 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains;

Assay:

A. the assay of aloe-emodin, Chrysophanol, Rhein, archen, Physcion

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; The volume parts of take is mobile phase than the methyl alcohol as 86:14-volume parts than the phosphate aqueous solution as 0.1%; Detection wavelength is 430nm.Number of theoretical plate calculates and should be not less than 3000 by archen peak;

The preparation of reference substance solution: it is appropriate that precision takes aloe-emodin reference substance, Rhein reference substance, archen reference substance, Chrysophanol reference substance, Physcion reference substance, add methyl alcohol and make respectively every 1ml containing aloe-emodin reference substance, Rhein reference substance, archen reference substance, each 80 μ g of Chrysophanol reference substance, the solution of Physcion 40 μ g, precision measures each 2ml of above-mentioned reference substance solution respectively, mix, obtain, in every 1ml, containing aloe-emodin, Rhein, archen, each 16 μ g of Chrysophanol, contain Physcion 8 μ g;

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes powder 2g that can dissipate, accurately weighed, put in the conical flask of tool plug, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic 30min, let cool, with methyl alcohol, supply the weight of less loss, shake up, filter, precision measures gets subsequent filtrate 5ml, put in flask, fling to solvent, adding percent by volume is 8% aqueous hydrochloric acid solution 10ml, ultrasonic 2 minutes, add again methenyl choloride 10ml, add hot reflux 1 hour, let cool, put in separating funnel, divide and get methenyl choloride layer, acid solution is extracted 3 times with methenyl choloride again, each 10ml, merge methenyl choloride liquid, recovered under reduced pressure solution is to dry, residue adds methyl alcohol to be made to dissolve, be transferred in 10ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain,

Determination method: precision is drawn reference substance solution and each 10 μ l of need testing solution respectively, and injection liquid chromatography, measures, and obtains.

The present invention's 29 tastes can dissipate or its preparation in aloe-emodin (C ₁₅h ₁₀o ₅), Rhein (C ₁₅h ₈o ₆), archen (C ₁₅h ₁₀o ₅), Chrysophanol (C ₁₅h ₁₀o ₄), Physcion (C ₁₆h ₁₂o ₅) total content must not be less than 1.20mg/g.

B. the assay of hydroxyl radical carthamin yellow carthamus A

Chromatographic condition and system suitability: take octadecylsilane chemically bonded silica as filling agent; Take volume parts than the methyl alcohol of 24:76-volume parts ratio as 1% glacial acetic acid aqueous solution be mobile phase; Detection wavelength is 403nm; Number of theoretical plate calculates and should be not less than 3000 by hydroxyl radical carthamin yellow carthamus A peak;

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes powder 4g that can dissipate, accurately weighed, put in tool plug conical flask, it is 25% methanol aqueous solution 25ml that precision adds volume parts ratio, close plug, weighed weight, ultrasonic 40 minutes, let cool, by volume parts, than being the weight that 25% methanol aqueous solution is supplied less loss, shake up, filter, get subsequent filtrate, obtain;

Determination method: draw respectively each 10 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains.

The unit corresponding relation of the weight portion described in this instructions and parts by volume is g/ml or kg/l.

The invention discloses that a kind of Tibetan medicinal composition 29 tastes can dissipate and the quality determining method of preparation, during use thin-layered chromatography can dissipate to 29 tastes, frankincense, pipering, nutmeg have carried out qualitative discriminating, use high performance liquid chromatography (HPLC method) to carry out limit test to can dissipate mesaconitine, banksia rose aristolochic acid of 29 tastes, adopt simultaneously high performance liquid chromatography (HPLC method) to 29 tastes, can dissipate in aloe-emodin, Chrysophanol, Rhein, archen, Physcion, hydroxyl radical carthamin yellow carthamus A carried out quantitative detection.The inventive method has been set up frankincense, pipering, myristic thin-layer identification method, the limit test method of aconitine, banksia rose aristolochic acid, the content assaying method of aloe-emodin, Chrysophanol, Rhein, archen, Physcion, hydroxyl radical carthamin yellow carthamus A, improved target level of product quality, make said preparation in treatment disease, guaranteed the safe and effective of medication.Simultaneously this law also can be used for other preparations that Tibetan medicinal composition 29 tastes can dissipate, as 29 tastes particle, 29 tastes ball, the 29 tastes sheet etc. that can disappear that can disappear that can disappear.

Following experimental example and embodiment are used for further illustrating but are not limited to the present invention.

Experimental example 1: thin layer identification experiment

Tibetan medicinal composition 29 tastes can dissipate and by Qinghai gold, scold Tibetan medicine medicine company incorporated company and provide.

A. the discriminating of frankincense

Get the Tibetan medicinal composition 29 tastes powder 3g that can dissipate, the 25ml that adds diethyl ether, ultrasonic processing 15min, filters, and filtrate volatilizes, and residue adds methyl alcohol 2ml to be made to dissolve, as need testing solution; Get frankincense control medicinal material 0.5g, the 30ml that adds diethyl ether, ultrasonic processing 15min, filters, and filtrate volatilizes, and residue adds methyl alcohol 2ml to be made to dissolve, in contrast medicinal material solution; Get in the negative control sample of prescription ratio preparation hypogalactia perfume (or spice), according to need testing solution preparation method with legal system for negative control sample solution; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is respectively developping agent than the dimethylbenzene-ethyl acetate as (8:1), (8:1.5), (8:2), launch, take out, dry.Spray is with 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear.

Result: in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, negative control is noiseless.In volume parts, than being under dimethylbenzene-ethyl acetate developping agent condition of 8:1 ~ 2, all there is good expansion effect.Wherein, volume parts is more best than the dimethylbenzene-ethyl acetate developping agent expansion effect for 8:1.5.Result shows, this discrimination method specificity is strong, can be used as 29 tastes can dissipate in the thin-layer identification method of frankincense.

B. the discriminating of pipering

Get the Tibetan medicinal composition 29 tastes powder 3g that can dissipate, add ethyl acetate 25ml, ultrasonic processing 30min, filters, and filtrate is concentrated into 2ml, as need testing solution; Get pipering appropriate, add methyl alcohol and make every 1ml containing the reference substance solution of 0.5mg; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is respectively developping agent than the cyclohexane-ethyl acetate as (3:1), (3:2), (1:1), launch, take out, dry.Put under ultraviolet lamp (365nm) and inspect.

Result: in test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.In volume parts, than being under cyclohexane-ethyl acetate developping agent condition of 3:1 ~ 3, all there is good expansion effect.Wherein, volume parts is more best than the cyclohexane-ethyl acetate developping agent expansion effect for 3:2.Result shows, this discrimination method chromatogram spot colour developing is clear, and degree of separation is good, can be used as 29 tastes can dissipate in the thin-layer identification method of pipering.

C. myristic discriminating

Get the Tibetan medicinal composition 29 tastes powder 5g that can dissipate, put in 250ml round-bottomed flask, add water 100ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil (starting to add a small amount of water and 1ml ethyl acetate in volatile oil determination apparatus), after decoction completes, collect ethyl acetate part, as need testing solution; Get nutmeg 1g, put in 100ml round-bottomed flask, add water 50ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil (starting to add a small amount of water and 1ml ethyl acetate in volatile oil determination apparatus), after having decocted, collect ethyl acetate part, in contrast medicinal material solution; Get in prescription ratio preparation and lack myristic negative control sample, according to need testing solution preparation method with legal system for negative control sample solution; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned three kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is respectively developping agent than the dimethylbenzene-ethyl acetate as (20:0.1), (20:0.3), (20:0.5), launch, take out, dry, the long-pending portion rate of sprinkler body is 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.

Result: in test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color, negative control is noiseless.In volume parts, than being under dimethylbenzene-ethyl acetate developping agent condition of 20:0.1 ~ 0.5, all there is good expansion effect.Wherein, volume parts is more best than the dimethylbenzene-ethyl acetate developping agent expansion effect for 20:0.3.Result shows, this discrimination method specificity is strong, can be used as 29 tastes can dissipate in myristic thin-layer identification method.

Experimental example 2: the limit test experiment of aconitine

1. instrument, reagent and test sample

Instrument: L-2100 type Hitachi high performance liquid chromatograph; AuW220D Shimadzu electronic analytical balance.

Reference substance: aconitine reference substance (Nat'l Pharmaceutical & Biological Products Control Institute) lot number: MUST-11031101.

Sample: 29 tastes can dissipate (Qinghai gold is scolded Tibetan medicine medicine company incorporated company and provided), and lot number is respectively: 20110815,20110816,20110817.

2. detect the selection of wavelength

Get aconitine reference substance solution, in 190 ~ 400nm wavelength coverage, scan, aconitine has absorption maximum at 235nm wavelength place, therefore be detection wavelength according to the selected 235nm of ultraviolet absorpting spectrum.

3. mobile phase is selected

Research discovery, the volume parts of take is mobile phase than methanol-water-triethylamine of 65 ~ 75:25 ~ 35:0.2:5-methylene chloride system, aconitine all can reach good chromatographic resolution effect.Wherein, take the volume parts ratio of methanol-water-triethylamine-methylene chloride optimum as 68:32:0.2:5 as mobile phase.

4. system suitability

Under above-mentioned chromatographic condition, precision is drawn reference substance solution, each 20 μ l of need testing solution respectively, and injection liquid chromatography, records chromatogram.Result shows, the degree of separation that test sample chromatogram mesaconitine is adjacent chromatographic peak is all greater than 1.5, good separating effect.

5. reference substance solution preparation

Get aconitine reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 10 μ g, obtain.

6. need testing solution preparation

The investigation of 6.1 extraction times

By method under check item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 3 parts, add respectively hot reflux 0.5 hour, 1 hour, 1.5 hours.The content of every gram of medicine mesaconitine of take is determined extraction time as index.The results are shown in Table 1.

Table 1 extraction time investigation test findings

Result shows, reflux 1 hour and the content of every gram of medicine mesaconitine of 1.5 hours gained basic identical, therefore select reflux extracting time, be 1 hour.

6.2 extract the investigation of solvent

By method under check item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 3 parts, by methyl alcohol, volume parts, than 80% ethanol water, absolute ethyl alcohol, extract respectively.The content of every gram of medicine mesaconitine of take is that index is determined and extracts solvent.The results are shown in Table 2.

Table 2 extracts solvent and investigates test findings

Result shows, methyl alcohol, volume parts are substantially more suitable than 80% ethanol water, three kinds of content that solvent is measured of absolute ethyl alcohol, but the test sample solvent extracting with absolute ethyl alcohol, impurity peaks is minimum, therefore adopt absolute ethyl alcohol for extracting solvent.

The investigation of 6.3 extracting method

By method under check item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 3 parts, Fen other Zhen Oscillating, ultrasonic, refluxing extraction 1 hour.The content of every gram of medicine mesaconitine of take is determined extracting method as index.The results are shown in Table 3.

Table 3 extracting method is investigated test findings

Result shows, the measured Content of Aconitine of refluxing extraction is the highest, therefore adopt, refluxes as extracting method.

7. the preparation of typical curve and the investigation of linear relationship

Precision measures aconitine reference substance stock solution solution (20.1 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml, put respectively in 10ml volumetric flask, methyl alcohol is diluted to scale, shake up, each accurate sample introduction 20 μ l, with peak area (A), reference substance concentration (C) is carried out to linear regression, obtain the regression equation of aconitine: A=12953C+249.14, related coefficient: R=0.9996.Result shows, aconitine is within the scope of 2.01 μ g/ml ~ 20.10 μ g/ml, and the peak area of aconitine (A) is good with reference substance concentration (C) linear relationship.The results are shown in Table 4.

Table 4 aconitine linear relationship is investigated result

8. precision test

The accurate aconitine reference substance solution 20 μ l that draw, injection liquid chromatography, each METHOD FOR CONTINUOUS DETERMINATION 6 times, records peak area and calculates relative standard deviation.Result shows, instrument precision is good.The results are shown in Table 5.

Table 5 aconitine Precision test result

9. quantitative limit

Accurate absorption in aconitine contrast solution (10.06 μ g/ml) 1ml to 25ml volumetric flask, dilutes and is settled to scale with methyl alcohol, shakes up, accurate 20 μ l, the injection liquid chromatography drawn.The signal to noise ratio (S/N ratio) of result aconitine chromatographic peak is about 10.Result shows: when aconitine concentration is 0.4024 μ g/ml, signal to noise ratio (S/N ratio) is about 10, and aconitine is quantitatively limited to 8.05ng.

10. replica test

Get with a collection of 29 tastes can dissipate sample (product batch number: 20110815) 12g, accurately weighed, totally 6 parts, by the method under the preparation of need testing solution, prepare need testing solution, precision is drawn 20 μ l, injection liquid chromatography, the content of calculation sample mesaconitine respectively.Result shows, this analytical approach repeatability is good.The results are shown in Table 6.

Table 6 aconitine replica test result

11. recovery tests

Precision takes with a collection of 29 tastes sample (product batch number: 20110815) 6 parts, each precision adds aconitine alkali reference substance, measures its content, calculate recovery rate that can dissipate.Result shows, this assay method measurement result is accurate.The results are shown in Table 7.

Table 7 aconitine recovery test result

12. sample determinations

Get Tibetan medicinal composition 29 tastes and can dissipate three batches, measure the also content of calculation sample mesaconitine.The results are shown in Table 8.

Table 8 sample size measurement result

Experimental example 3: the limit test experiment of aristolochic acid

1. instrument, reagent and test sample

Reference substance: aristolochic acid A reference substance (Nat'l Pharmaceutical & Biological Products Control Institute) lot number: 110746-200806.

2. detect the selection of wavelength

Get aristolochic acid A reference substance solution, in 190 ~ 400nm wavelength coverage, scan, aristolochic acid A has absorption maximum at 315nm wavelength place, therefore be detection wavelength according to the selected 315nm of ultraviolet absorpting spectrum.

3. mobile phase is selected

Research discovery, the volume parts of take is mobile phase than the methyl alcohol-volume parts of 60～70:30～40 than 1% glacial acetic acid aqueous solution, aristolochic acid A all can reach good chromatographic resolution effect.Wherein, take methyl alcohol-volume parts more optimum as 66:34 as mobile phase than the volume parts ratio of 1% glacial acetic acid aqueous solution.

4. system suitability

Under above-mentioned chromatographic condition, precision is drawn reference substance solution, each 20 μ l of need testing solution respectively, and injection liquid chromatography, records chromatogram.Result shows, the degree of separation that in test sample chromatogram, aristolochic acid A is adjacent chromatographic peak is all greater than 1.5, good separating effect.

5. reference substance solution preparation

Get aristolochic acid A reference substance appropriate, accurately weighed, add methyl alcohol and make every 1ml containing the solution of 20 μ g, obtain.

6. need testing solution preparation

The investigation of 6.1 extraction times

By method under check item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 3 parts, ultrasonic extraction is 20 minutes, 30 minutes, 40 minutes respectively.The content of aristolochic acid A in every gram of medicine of take is determined extraction time as index.The results are shown in Table 9.

Table 9 extraction time investigation test findings

Result shows, when ultrasonic 30 minutes and 40 minutes, in every gram of medicine of gained, the content of aristolochic acid A is basic identical, therefore select ultrasonic extraction time, is 30 minutes.

6.2 extract the investigation of solvent

By method under check item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 3 parts, with methyl alcohol, absolute ethyl alcohol, extract respectively.The content of aristolochic acid A of take in every gram of medicine is that index is determined and extracted solvent.The results are shown in Table 10.

Table 10 extracts solvent and investigates test findings

Result shows, the measured Determination of Aristolochic Acid A of methyl alcohol, apparently higher than absolute ethyl alcohol, is to extract solvent therefore select methyl alcohol.

The investigation of 6.3 extracting method

By method under check item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 3 parts, Fen other Zhen Oscillating, ultrasonic, refluxing extraction 30 minutes.The content of aristolochic acid A in every gram of medicine of take is determined extracting method as index.The results are shown in Table 11.

Table 11 extracting method is investigated test findings

Result shows, the measured Content of Aconitine of ultrasonic and refluxing extraction is basically identical, considers that ultrasonic extraction is easy and simple to handle, therefore select ultrasonic, is extracting method.

Precision measures aristolochic acid A reference substance stock solution solution (40.6 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml, put respectively in 10ml volumetric flask, methyl alcohol is diluted to scale, shake up, each accurate sample introduction 20 μ l, with peak area (A), reference substance concentration (C) is carried out to linear regression, obtain the regression equation of aristolochic acid A: A=17077C+5543.3, related coefficient: R=0.9998.Result shows, aristolochic acid A is within the scope of 4.06 μ g/ml ~ 40.60 μ g/ml, and the peak area of aristolochic acid A (A) is good with reference substance concentration (C) linear relationship.The results are shown in Table 12.

Table 12 aristolochic acid A linear relationship is investigated result

8. precision test

The accurate aristolochic acid A reference substance solution 20 μ l that draw, injection liquid chromatography, each METHOD FOR CONTINUOUS DETERMINATION 6 times, records peak area and calculates relative standard deviation.Result shows, instrument precision is good.The results are shown in Table 13.

Table 13 aristolochic acid A Precision test result

9. quantitative limit

Accurate absorption in aristolochic acid A contrast solution (17.68 μ g/ml) 1ml to 50ml volumetric flask, dilutes and is settled to scale with methyl alcohol, shakes up, accurate 20 μ l, the injection liquid chromatography drawn.The signal to noise ratio (S/N ratio) of result aristolochic acid A chromatographic peak is about 10.Result shows: when aristolochic acid A concentration is 0.3536 μ g/ml, signal to noise ratio (S/N ratio) is about 10, and aristolochic acid A is quantitatively limited to 7.07ng.

10. replica test

Get with a collection of 29 tastes can dissipate sample (product batch number: 20110815) 8g, accurately weighed, totally 6 parts, by the method under the preparation of need testing solution, prepare need testing solution, precision is drawn 20 μ l, injection liquid chromatography, the content of aristolochic acid A in calculation sample respectively.Result shows, this analytical approach repeatability is good.The results are shown in Table 14.

Table 14 aristolochic acid A replica test result

11. recovery tests

Precision takes with a collection of 29 tastes sample (product batch number: 20110815) 6 parts, each precision adds aristolochic acid A reference substance, measures its content, calculate recovery rate that can dissipate.Result shows, this assay method measurement result is accurate.The results are shown in Table 15.

Table 15 aristolochic acid A recovery test result

12. sample determinations

Get Tibetan medicinal composition 29 tastes and can dissipate three batches, the content of aristolochic acid A in mensuration calculation sample.The results are shown in Table 16.

Table 16 sample size measurement result

Experimental example 4: the assay experiment of aloe-emodin, Chrysophanol, Rhein, archen, Physcion

1. instrument, reagent and test sample

Instrument: L-2100 type Hitachi high performance liquid chromatograph; Shimadzu AuW220D electronic balance.

Reference substance: aloe-emodin reference substance (lot number: 110795-201007), Rhein reference substance (lot number: 110757-200206), archen reference substance (110756-200100), Chrysophanol reference substance (110796-201017), Physcion reference substance (110758-201013).

2. detect the selection of wavelength

Get the mixing reference substance solution of aloe-emodin, Rhein, archen, Chrysophanol, Physcion, in 190 ~ 500nm wavelength coverage, scan, mixing has absorption maximum to impinging upon 430nm wavelength place, therefore according to absorbing the selected 430nm of collection of illustrative plates for detecting wavelength.

3. mobile phase is selected

Research finds, it is 0.1% phosphate aqueous solution during as mobile phase that the volume parts of take compares than the methyl alcohol of 80 ~ 90:10 ~ 20-volume parts, and aloe-emodin, Rhein, archen, Chrysophanol, Physcion all can reach good chromatographic resolution effect.Methyl alcohol-the volume parts of wherein take is more optimum as 86:14 as mobile phase than the volume parts ratio of the phosphate aqueous solution as 0.1%.

4. system suitability

Under above-mentioned chromatographic condition, precision is drawn reference substance solution, each 10 μ l of need testing solution respectively, and injection liquid chromatography, records chromatogram.Result shows, the degree of separation that in test sample chromatogram, aloe-emodin, Rhein, archen, Chrysophanol, Physcion are adjacent chromatographic peak is all greater than 1.5.

5. mix reference substance solution preparation

It is appropriate that precision takes aloe-emodin reference substance, Rhein reference substance, archen reference substance, Chrysophanol reference substance, Physcion reference substance, add methyl alcohol and make respectively every 1ml containing aloe-emodin reference substance, Rhein reference substance, archen reference substance, each 80 μ g of Chrysophanol reference substance, the solution of Physcion 40 μ g, precision measures each 2ml of above-mentioned reference substance solution respectively, mix, obtain, be containing aloe-emodin, Rhein, archen, each 16 μ g of Chrysophanol, containing Physcion 8 μ g in every 1ml.

6. need testing solution preparation

The investigation of 6.1 extracting method

By method under assay item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 3 parts, ultrasonic, backflow, Zhen Oscillating processes 30 minutes respectively.Take aloe-emodin in every gram of medicine, Rhein, archen, Chrysophanol, five kinds of anthraquinone component total contents of Physcion determines extracting method as index.The results are shown in Table 17.

Table 17 extracting method is investigated test findings

Result shows, in ultrasonic and every gram of medicine of refluxing extraction gained, the total content of anthraquinone component is basically identical, considers that ultrasonic extraction is easy, easy to operate, therefore select extracting method, is ultrasonic extraction.

6.2 extract the investigation of solvent

By method under assay item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 2 parts, precision adds methyl alcohol, each 25ml of ethanol respectively.Take aloe-emodin in every gram of medicine, Rhein, archen, Chrysophanol, five kinds of anthraquinone component total contents of Physcion is that index is determined and extracted solvent.The results are shown in Table 18.

Table 18 extracts solvent and investigates test findings

Result shows, methyl alcohol is measured anthraquinone component total content apparently higher than ethanol, therefore select, to extract solvent be methyl alcohol.

The investigation of 6.3 extraction times

By method under assay item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 3 parts, ultrasonic processing is 20 minutes, 30 minutes, 40 minutes respectively.Take aloe-emodin in every gram of medicine, Rhein, archen, Chrysophanol, five kinds of anthraquinone component total contents of Physcion determines extraction time as index.The results are shown in Table 19.

Table 19 extraction time investigation test findings chemical drug

Result shows, in ultrasonic extraction 30 minutes and every gram of medicine of 40 minutes gained, the total content of anthraquinone component is basically identical, therefore select extraction time, is 30 minutes.

Precision measures mixes reference substance stock solution solution (aloe-emodin 32.6 μ g/ml, Rhein 33.8mg/ml, archen 33.2mg/ml, each 32.5 μ g/ml of Chrysophanol, containing Physcion 17.2/ml) 1ml, 3ml, 5ml, 8ml, 10ml, put respectively in 10ml volumetric flask, methyl alcohol is diluted to scale, shake up, each accurate sample introduction 10 μ l, with peak area (A), reference substance concentration (C) is carried out to linear regression, the regression equation that obtains aloe-emodin is: A=8966.4C+364.89, related coefficient: R=0.9995; The regression equation of Rhein is: A=10015C+120.03, related coefficient: R=0.9996; The regression equation of archen is: A=9087.1C+326.52, related coefficient: R=0.9995; The regression equation of Chrysophanol is: A=13338C+534.91, related coefficient: R=0.9998; The regression equation of Physcion is: A=5033.8C+68.985, related coefficient: R=0.9997.Result shows, aloe-emodin is within the scope of 3.26 μ g/ml ~ 32.60 μ g/ml, and the peak area of aloe-emodin (A) is good with reference substance concentration (C) linear relationship; Rhein is within the scope of 3.38 μ g/ml ~ 33.80 μ g/ml, and the peak area of Rhein (A) is good with reference substance concentration (C) linear relationship; Archen is within the scope of 3.32 μ g/ml ~ 33.20 μ g/ml, and the peak area of archen (A) is good with reference substance concentration (C) linear relationship; Chrysophanol is within the scope of 3.25 μ g/ml ~ 32.50 μ g/ml, and the peak area of Chrysophanol (A) is good with reference substance concentration (C) linear relationship; Physcion is within the scope of 1.72 μ g/ml ~ 17.20 μ g/ml, and the peak area of Physcion (A) is good with reference substance concentration (C) linear relationship.The results are shown in Table 20, table 21, table 22, table 23, table 24.

Table 20 aloe-emodin linear relationship is investigated result

Table 21 Rhein linear relationship is investigated result

Table 22 archen linear relationship is investigated result

Table 23 Chrysophanol linear relationship is investigated result

Table 24 Physcion linear relationship is investigated result

8. precision test

Accurate absorption mixed reference substance solution 10 μ l, injection liquid chromatography, and each METHOD FOR CONTINUOUS DETERMINATION 6 times, records peak area and calculates relative standard deviation.Result shows, instrument precision is good.The results are shown in Table 25.

Table 25 mixes contrast Precision test result

9. stability test

After prepared by need testing solution, the accurate 10 μ l that draw, injection liquid chromatography, records peak area, measures once every 2 hours later, investigates 8 hours, records peak area and calculates relative standard deviation.Result shows, in need testing solution, aloe-emodin, Rhein, archen, Chrysophanol, Physcion measurement result in 8 hours is stable.The results are shown in Table 26.

Table 26 mixes contrast stability test result

10. replica test

Get with a collection of 29 tastes sample (product batch number: 20110815) 2g that can dissipate, accurately weighed, totally 6 parts, by the method under the preparation of need testing solution, prepare need testing solution, precision is drawn 10 μ l respectively, injection liquid chromatography, aloe-emodin, Rhein, archen, Chrysophanol, five kinds of anthraquinone component total contents of Physcion in calculation sample.Result shows, this analytical approach repeatability is good.The results are shown in Table 27.

Table 27 anthraquinone component total content replica test result

11. recovery tests

Precision takes with a collection of 29 tastes sample (product batch number: 20110815) 6 parts, each precision adds aloe-emodin, Rhein, archen, Chrysophanol, five kinds of anthraquinone component reference substances of Physcion, measures its content, calculate recovery rate that can dissipate.Result shows, this assay method measurement result is accurate.The results are shown in Table 28.

Table 28 anthraquinone component recovery test result

12. sample determinations

Get Tibetan medicinal composition 29 tastes and can dissipate three batches, measure and calculate the total content of aloe-emodin, Rhein, archen, Chrysophanol, five kinds of anthraquinone components of Physcion.The results are shown in Table 29.

Table 29 sample size measurement result

Experimental example 5: the assay experiment of hydroxyl radical carthamin yellow carthamus A

1. instrument, reagent and test sample

Reference substance: hydroxyl radical carthamin yellow carthamus A reference substance (Chinese pharmaceutical biological product is identified institute), lot number: 111637-200905.

2. detect the selection of wavelength

Get hydroxyl radical carthamin yellow carthamus A reference substance solution, in 190 ~ 500nm wavelength coverage, scan, hydroxyl radical carthamin yellow carthamus A has absorption maximum at 403nm wavelength place, therefore be detection wavelength according to the selected 403nm of ultraviolet absorpting spectrum.

3. mobile phase is selected

Research finds, take volume parts than the methyl alcohol of 20～30:70～80-volume parts ratio during as 1% glacial acetic acid aqueous solution as mobile phase, and hydroxyl radical carthamin yellow carthamus A all can reach good chromatographic resolution effect.Methyl alcohol-the volume parts of wherein take is more optimum as 24:76 as mobile phase than the volume parts ratio as 1% glacial acetic acid aqueous solution.

4. system suitability

Under above-mentioned chromatographic condition, precision is drawn reference substance solution, each 10 μ l of need testing solution respectively, and injection liquid chromatography, records chromatogram.Result shows, the degree of separation that in reference substance, test sample chromatogram, hydroxyl radical carthamin yellow carthamus A is adjacent chromatographic peak is all greater than 1.5.

5. reference substance preparation

Get hydroxyl safflower anthocyanidin A reference substance appropriate, accurately weighed, put in brown bottle, add volume parts and make every 1ml containing the solution of 50 μ g than 25% methanol aqueous solution, obtain.

6. test sample preparation

The investigation of 6.1 extracting method

By method under assay item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 3 parts, ultrasonic, backflow, Zhen Oscillating processes 40 minutes respectively.The content of hydroxyl radical carthamin yellow carthamus A in every gram of medicine of take is determined extracting method as index.The results are shown in Table 30.

Table 30 extracting method is investigated test findings

Result shows, in every gram of medicine of ultrasonic extraction gained, the content of hydroxyl radical carthamin yellow carthamus A is the highest, therefore select extracting method, is ultrasonic extraction.

6.2 extract the investigation of solvent

By method under assay item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 3 parts, precision adds water, volume parts than 25% methanol aqueous solution, each 25ml of methyl alcohol respectively.The content of hydroxyl radical carthamin yellow carthamus A of take in every gram of medicine is that index is determined and extracted solvent.The results are shown in Table 31.

Table 31 extracts solvent and investigates test findings

Result shows, the hydroxyl radical carthamin yellow carthamus A content that water, volume parts are surveyed than 25% methanol aqueous solution and methyl alcohol is basically identical, owing to using water as, extracts solvent filter difficulty, and pure methyl alcohol toxicity is large, therefore select, to extract solvent be that volume parts is than 25% methanol aqueous solution.

The investigation of 6.3 extraction times

By method under assay item, need testing solution is detected.By the method under the preparation of need testing solution, prepare 3 parts, ultrasonic processing is 30 minutes, 40 minutes, 50 minutes respectively.The content of hydroxyl radical carthamin yellow carthamus A in every gram of medicine of take is determined extraction time as index.The results are shown in Table 32.

Table 32 extraction time investigation test findings

Result shows, in ultrasonic extraction 40 minutes and every gram of medicine of 50 minutes gained, the content of hydroxyl radical carthamin yellow carthamus A is basically identical, therefore select extraction time, is 40 minutes.

Precision measures hydroxyl radical carthamin yellow carthamus A reference substance stock solution solution (hydroxyl radical carthamin yellow carthamus A content is 107.8 μ g/ml) 1ml, 3ml, 5ml, 8ml, 10ml, put respectively in 10ml volumetric flask, volume parts is diluted to scale than 25% methanol aqueous solution, shake up, each accurate sample introduction 10 μ l, with peak area (A), reference substance concentration (C) is carried out to linear regression, obtain hydroxyl radical carthamin yellow carthamus A regression equation: A=6605.5C+454.39, related coefficient: R=0.9998.Result shows, hydroxyl radical carthamin yellow carthamus A is within the scope of 10.78 μ g/ml ~ 107.80 μ g/ml, and the peak area of hydroxyl radical carthamin yellow carthamus A (A) is good with reference substance concentration (C) linear relationship.The results are shown in Table 33.

Table 33 hydroxyl radical carthamin yellow carthamus A linear relationship is investigated result

8. precision test

The accurate hydroxyl radical carthamin yellow carthamus A reference substance solution 10 μ l that draw, injection liquid chromatography, each METHOD FOR CONTINUOUS DETERMINATION 6 times, records peak area and calculates relative standard deviation.Result shows, instrument precision is good.The results are shown in Table 34.

Table 34 hydroxyl radical carthamin yellow carthamus A Precision test result

9. stability test

After prepared by need testing solution, the accurate 10 μ l that draw, injection liquid chromatography, records peak area, measures once every 2 hours later, investigates 8 hours, records peak area and calculates relative standard deviation.Result shows, in need testing solution, hydroxyl radical carthamin yellow carthamus A measurement result in 8 hours is stable.The results are shown in Table 35.

Table 35 hydroxyl radical carthamin yellow carthamus A stability test result

10. replica test

Get with a collection of 29 tastes can dissipate sample (product batch number: 20110815) 4g, accurately weighed, totally 6 parts, by the method under the preparation of need testing solution, prepare need testing solution, precision is drawn 10 μ l, injection liquid chromatography, the content of hydroxyl radical carthamin yellow carthamus A in calculation sample respectively.Result shows, this analytical approach repeatability is good.The results are shown in Table 36.

Table 36 hydroxyl radical carthamin yellow carthamus A replica test result

11. recovery tests

Precision takes with a collection of 29 tastes sample (product batch number: 20110815) 6 parts, each precision adds hydroxyl radical carthamin yellow carthamus A reference substance, measures its content, calculate recovery rate that can dissipate.Result shows, this assay method measurement result is accurate.The results are shown in Table 37.

Table 37 hydroxyl radical carthamin yellow carthamus A recovery test result

12. sample determinations

Get Tibetan medicinal composition 29 tastes and can dissipate three batches, measure and calculate hydroxyl radical carthamin yellow carthamus A content.The results are shown in Table 38.

Table 38 sample size measurement result

Following embodiment all can realize the effect described in above-mentioned experimental example.

Embodiment

Below in conjunction with embodiment, the present invention is done to detailed elaboration, but be not limited to these concrete embodiment recording.Tibetan medicinal composition 29 tastes that embodiment 1 detects can be dissipated as Qinghai gold and scold Tibetan medicine medicine company incorporated company and produce and to sell.

The quality determining method that 1: two 19 taste of embodiment can dissipate

Differentiate:

A. the discriminating of frankincense

B. the discriminating of pipering

C. myristic discriminating

Check:

A. the limit test of aconitine

This product contains Aconitum Szechenyianum Gay with aconitine (C ₃₄h ₄₇nO ₁₁) meter, must not be higher than 17 μ g/g.

B. the limit test of aristolochic acid A

This product contains banksia rose birthwort with aristolochic acid A (C ₁₇h ₁₁nO ₇) meter, must not be higher than 30 μ g/g.

Assay:

A. the assay of aloe-emodin, Chrysophanol, Rhein, archen, Physcion

This product aloe-emodin (C ₁₅h ₁₀o ₅), Rhein (C ₁₅h ₈o ₆), archen (C ₁₅h ₁₀o ₅), Chrysophanol (C ₁₅h ₁₀o ₄), Physcion (C ₁₆h ₁₂o ₅) total content must not be less than 1.20mg/g.

B. the assay of hydroxyl radical carthamin yellow carthamus A

This product hydroxyl radical carthamin yellow carthamus A (C ₂₇h ₃₀o ₁₅) content must not be less than 0.20mg/g.

2: two 19 tastes of embodiment ball quality determining method that can disappear

Differentiate:

A. the discriminating of frankincense

Get the Tibetan medicinal composition 29 tastes ball powder 3g that can disappear, the 25ml that adds diethyl ether, ultrasonic processing 15min, filters, and filtrate volatilizes, and residue adds methyl alcohol 2ml to be made to dissolve, as need testing solution; Get frankincense control medicinal material 0.5g, the 30ml that adds diethyl ether, ultrasonic processing 15min, filters, and filtrate volatilizes, and residue adds methyl alcohol 2ml to be made to dissolve, in contrast medicinal material solution; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the dimethylbenzene-ethyl acetate as 8:1.5, launch, take out, dry.Spray is with 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.

B. the discriminating of pipering

Get the Tibetan medicinal composition 29 tastes ball powder 3g that can disappear, add ethyl acetate 25ml, ultrasonic processing 30min, filters, and filtrate is concentrated into 2ml, as need testing solution; Get pipering appropriate, add methyl alcohol and make every 1ml containing the reference substance solution of 0.5mg; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the cyclohexane-ethyl acetate as 3:2, launch, take out, dry.Put under ultraviolet lamp (365nm) and inspect.In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

C. myristic discriminating

Get the Tibetan medicinal composition 29 tastes ball powder 5g that can disappear, put in 250ml round-bottomed flask, add water 100ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil (starting to add a small amount of water and 1ml ethyl acetate in volatile oil determination apparatus), after decoction completes, collect ethyl acetate part, as need testing solution; Get nutmeg 1g, put in 100ml round-bottomed flask, add water 50ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil (starting to add a small amount of water and 1ml ethyl acetate in volatile oil determination apparatus), after having decocted, collect ethyl acetate part, in contrast medicinal material solution; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the dimethylbenzene-ethyl acetate as 20:0.3, launch, take out, dry, the long-pending portion rate of sprinkler body is 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.

Check:

A. the limit test of aconitine

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes ball powder 12g that can disappear, accurately weighed, put in the conical flask of tool plug, add ammonia solution 10ml, precision adds absolute ethyl alcohol 100ml, close plug, weighed weight, after cold soaking 1h, add hot reflux 1h, let cool, more weighed weight, with absolute ethyl alcohol, supply the weight of less loss, shake up, filter; Precision measures subsequent filtrate 50ml, and 25 ~ 40 ℃ of decompression and solvent recoveries are to dry, and residue precision adds dilute hydrochloric acid solution 20ml to dissolve, and places 30min in ice bath, filters; With ammoniacal liquor, adjust pH to 10, filtrate is transferred in separating funnel, add ethyl acetate extraction 5 times, each 20ml, combined ethyl acetate liquid, 25 ~ 40 ℃ of decompression and solvent recoveries are to dry, and residue precision adds methyl alcohol 2ml to dissolve, and filters, and gets subsequent filtrate, obtains;

Determination method: precision is drawn reference substance solution and each 20 μ l of need testing solution respectively, and injection liquid chromatography, measures, and obtains.

B. the limit test of aristolochic acid A

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes ball powder 8g that can disappear, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic 30 minutes, let cool, weighed weight again, with methyl alcohol, supply the weight of less loss, shake up, filter, get subsequent filtrate 25ml, low temperature is concentrated into dry, it is that 0.5% sodium hydroxide solution 20ml dissolves it completely and is transferred in separating funnel that residue adds volume parts ratio, by extracted with diethyl ether 2 times, each 20ml, after extraction, alkali lye service property (quality) mark 3% salt acid for adjusting pH is to 2-3, by extracted with diethyl ether 5 times, each 20ml merges ether extraction liquid, volatilize, with methyl alcohol, dissolve and be transferred in 5ml volumetric flask, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain,

Determination method: draw respectively each 20 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains.

Assay:

A. the assay of aloe-emodin, Rhein, archen, Chrysophanol, Physcion

The preparation of reference substance solution: it is appropriate that precision takes aloe-emodin reference substance, Rhein reference substance, archen reference substance, Chrysophanol reference substance, Physcion reference substance, add methyl alcohol and make respectively every 1ml containing aloe-emodin reference substance, Rhein reference substance, archen reference substance, each 80 μ g of Chrysophanol reference substance, the solution of Physcion 40 μ g, precision measures each 2ml of above-mentioned reference substance solution respectively, mix, obtain (being containing aloe-emodin, Rhein, archen, each 16 μ g of Chrysophanol, containing Physcion 8 μ g in every 1ml);

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes ball powder 2g that can disappear, accurately weighed, put in the conical flask of tool plug, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic 30min, let cool, with methyl alcohol, supply the weight of less loss, shake up, filter, precision measures gets subsequent filtrate 5ml, put in flask, fling to solvent, adding percent by volume is 8% aqueous hydrochloric acid solution 10ml, ultrasonic 2 minutes, add again methenyl choloride 10ml, add hot reflux 1 hour, let cool, put in separating funnel, divide and get methenyl choloride layer, acid solution is extracted 3 times with methenyl choloride again, each 10ml, merge methenyl choloride liquid, recovered under reduced pressure solution is to dry, residue adds methyl alcohol to be made to dissolve, be transferred in 10ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain,

B. the assay of hydroxyl radical carthamin yellow carthamus A

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes ball powder 4g that can disappear, accurately weighed, put in tool plug conical flask, it is 25% methanol aqueous solution 25ml that precision adds volume parts ratio, close plug, weighed weight, ultrasonic 40 minutes, let cool, by volume parts, than being the weight that 25% methanol aqueous solution is supplied less loss, shake up, filter, get subsequent filtrate, obtain;

The described Tibetan medicinal composition 29 tastes ball that can disappear refers to the 29 tastes bulk drug formula Tibet inula root 25g that can dissipate, gypsum rubrum (forging) 125g, myrobalan 75g, hot millet 25g, alkali flower 125g, nutmeg 25g, Bi roots of grass 25g, cassia seed 25g, Amomum cardamomum 25g, rhizome of davallia 25g, pepper 25g, radish (charcoal) 2g, tsaoko 25g, natural salt 25g, asafoetide 2g, sal ammoniac 25g, kaempferia galamga 25g, shellfish tooth (charcoal) 25g, rheum officinale 100g, wide muscle rattan 13g, safflower 25g, Aconitum Szechenyianum Gay 15g, lime (system) 25g, vulture excrement (stir-fry) 40g, banksia rose birthwort 25g, Semen seu folium abelmoschi moschati 25g, HALITUM PURPUREUM 25g, frankincense 25g, slag is tamed and dociled cream 25g, add conventional auxiliary material, the pill of preparing according to common process.

3: two 19 tastes of embodiment tablet quality detection method that can disappear

Differentiate:

A. the discriminating of frankincense

Get the Tibetan medicinal composition 29 tastes sheet powder 3g that can disappear, the 25ml that adds diethyl ether, ultrasonic processing 15min, filters, and filtrate volatilizes, and residue adds methyl alcohol 2ml to be made to dissolve, as need testing solution; Get frankincense control medicinal material 0.5g, the 30ml that adds diethyl ether, ultrasonic processing 15min, filters, and filtrate volatilizes, and residue adds methyl alcohol 2ml to be made to dissolve, in contrast medicinal material solution; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the dimethylbenzene-ethyl acetate as 8:1.5, launch, take out, dry.Spray is with mass volume ratio 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.

B. the discriminating of pipering

Get the Tibetan medicinal composition 29 tastes sheet powder 3g that can disappear, add ethyl acetate 25ml, ultrasonic processing 30min, filters, and filtrate is concentrated into 2ml, as need testing solution; Get pipering appropriate, add methyl alcohol and make every 1ml containing the reference substance solution of 0.5mg; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the cyclohexane-ethyl acetate as 3:2, launch, take out, dry.Put under ultraviolet lamp (365nm) and inspect.In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color.

C. myristic discriminating

Get the Tibetan medicinal composition 29 tastes sheet powder 5g that can disappear, put in 250ml round-bottomed flask, add water 100ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil (starting to add a small amount of water and 1ml ethyl acetate in volatile oil determination apparatus), after decoction completes, collect ethyl acetate part, as need testing solution; Get nutmeg 1g, put in 100ml round-bottomed flask, add water 50ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil (starting to add a small amount of water and 1ml ethyl acetate in volatile oil determination apparatus), after having decocted, collect ethyl acetate part, in contrast medicinal material solution; According to thin-layered chromatography (appendix VI B of < < Chinese Pharmacopoeia > > version in 2010), test, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the dimethylbenzene-ethyl acetate as 20:0.3, launch, take out, dry, the long-pending portion rate of sprinkler body is 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear.In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color.

Check:

A. the limit test of aconitine

Chromatographic condition and system suitability be take octadecylsilane chemically bonded silica as filling agent; The volume parts of take is mobile phase than methanol-water-triethylamine-methylene chloride of 68:32:0.2:5; Detection wavelength is 235nm, and number of theoretical plate calculates and should be not less than 2000 by aconitine peak;

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes sheet powder 12g that can disappear, accurately weighed, put in the conical flask of tool plug, add ammonia solution 10ml, precision adds absolute ethyl alcohol 100ml, close plug, weighed weight, after cold soaking 1h, add hot reflux 1h, let cool, more weighed weight, with absolute ethyl alcohol, supply the weight of less loss, shake up, filter; Precision measures subsequent filtrate 50ml, and 25 ~ 40 ℃ of decompression and solvent recoveries are to dry, and residue precision adds dilute hydrochloric acid solution 20ml to dissolve, and places 30min in ice bath, filters; With ammoniacal liquor, adjust pH to 10, filtrate is transferred in separating funnel, add ethyl acetate extraction 5 times, each 20ml, combined ethyl acetate liquid, 25 ~ 40 ℃ of decompression and solvent recoveries are to dry, and residue precision adds methyl alcohol 2ml to dissolve, and filters, and gets subsequent filtrate, obtains;

B. the limit test of aristolochic acid A

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes sheet powder 8g that can disappear, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 50ml, close plug, weighed weight, ultrasonic 30 minutes, let cool, weighed weight again, with methyl alcohol, supply the weight of less loss, shake up, filter, get subsequent filtrate 25ml, low temperature is concentrated into dry, it is that 0.5% sodium hydroxide solution 20ml dissolves it completely and is transferred in separating funnel that residue adds volume parts ratio, by extracted with diethyl ether 2 times, each 20ml, after extraction, alkali lye service property (quality) mark 3% salt acid for adjusting pH is to 2-3, by extracted with diethyl ether 5 times, each 20ml merges ether extraction liquid, volatilize, with methyl alcohol, dissolve and be transferred in 5ml volumetric flask, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain,

Assay:

A. the assay of aloe-emodin, Chrysophanol, Rhein, archen, Physcion

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes sheet powder 2g that can disappear, accurately weighed, put in the conical flask of tool plug, precision adds methyl alcohol 25ml, close plug, weighed weight, ultrasonic 30min, let cool, with methyl alcohol, supply the weight of less loss, shake up, filter, precision measures gets subsequent filtrate 5ml, put in flask, fling to solvent, adding percent by volume is 8% aqueous hydrochloric acid solution 10ml, ultrasonic 2 minutes, add again methenyl choloride 10ml, add hot reflux 1 hour, let cool, put in separating funnel, divide and get methenyl choloride layer, acid solution is extracted 3 times with methenyl choloride again, each 10ml, merge methenyl choloride liquid, recovered under reduced pressure solution is to dry, residue adds methyl alcohol to be made to dissolve, be transferred in 10ml measuring bottle, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain,

B. the assay of hydroxyl radical carthamin yellow carthamus A

The preparation of need testing solution: get the Tibetan medicinal composition 29 tastes sheet powder 4g that can disappear, accurately weighed, put in tool plug conical flask, it is 25% methanol aqueous solution 25ml that precision adds volume parts ratio, close plug, weighed weight, ultrasonic 40 minutes, let cool, by volume parts, than being the weight that 25% methanol aqueous solution is supplied less loss, shake up, filter, get subsequent filtrate, obtain;

The described Tibetan medicinal composition 29 tastes sheet that can disappear refers to the 29 tastes bulk drug formula Tibet inula root 25g that can dissipate, gypsum rubrum (forging) 125g, myrobalan 75g, hot millet 25g, alkali flower 125g, nutmeg 25g, Bi roots of grass 25g, cassia seed 25g, Amomum cardamomum 25g, rhizome of davallia 25g, pepper 25g, radish (charcoal) 2g, tsaoko 25g, natural salt 25g, asafoetide 2g, sal ammoniac 25g, kaempferia galamga 25g, shellfish tooth (charcoal) 25g, rheum officinale 100g, wide muscle rattan 13g, safflower 25g, Aconitum Szechenyianum Gay 15g, lime (system) 25g, vulture excrement (stir-fry) 40g, banksia rose birthwort 25g, Semen seu folium abelmoschi moschati 25g, HALITUM PURPUREUM 25g, frankincense 25g, slag is tamed and dociled cream 25g, add conventional auxiliary material, the tablet of preparing according to common process.

Claims

1. a bulk drug consists of Tibet inula root 25 weight portions, forge gypsum rubrum 125 weight portions, myrobalan's 75 weight portions, hot millet 25 weight portions, alkali is spent 125 weight portions, nutmeg 25 weight portions, the Bi roots of grass 25 weight portions, cassia seed 25 weight portions, Amomum cardamomum 25 weight portions, the rhizome of davallia 25 weight portions, pepper 25 weight portions, radish charcoal 2 weight portions, tsaoko 25 weight portions, natural salt 25 weight portions, asafoetide 2 weight portions, sal ammoniac 25 weight portions, kaempferia galamga 25 weight portions, shellfish tooth charcoal 25 weight portions, rheum officinale 100 weight portions, wide muscle rattan 13 weight portions, safflower 25 weight portions, Aconitum Szechenyianum Gay 15 weight portions, lime 25 weight portions processed, fry vulture excrement 40 weight portions, banksia rose birthwort 25 weight portions, Semen seu folium abelmoschi moschati 25 weight portions, HALITUM PURPUREUM 25 weight portions, frankincense 25 weight portions, slag is tamed and dociled the detection method of the Tibetan medicinal composition 29 taste energy oral preparation for eliminating of cream 25 weight portions, the method comprises following discriminating and inspection method:

A. the discriminating of pipering

Get Tibetan medicinal composition 29 taste energy oral preparation for eliminating powder 2～4g, add ethyl acetate 20～30ml, ultrasonic processing 30min, filters, and filtrate is concentrated into 2ml, as need testing solution; Get pipering appropriate, add methyl alcohol and make every 1ml containing the reference substance solution of 0.5mg; Thin-layered chromatography test according to an appendix VI B of < < Chinese Pharmacopoeia > > version in 2010, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the cyclohexane-ethyl acetate as 3:1～2, launch, take out, dry; Put under ultraviolet lamp 365nm and inspect; In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

B. myristic discriminating

Get Tibetan medicinal composition 29 taste energy oral preparation for eliminating powder 4～6g, put in 250ml round-bottomed flask, add water 100ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil, in volatile oil determination apparatus, start to add a small amount of water and 1ml ethyl acetate, after having decocted, collect ethyl acetate part, as need testing solution; Get nutmeg 1g, put in 100ml round-bottomed flask, add water 50ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil, in volatile oil determination apparatus, start to add a small amount of water and 1ml ethyl acetate, after having decocted, collect ethyl acetate part, in contrast medicinal material solution; Thin-layered chromatography test according to an appendix VI B of < < Chinese Pharmacopoeia > > version in 2010, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the dimethylbenzene-ethyl acetate as 20:0.1～0.5, launch, take out, dry, the long-pending portion rate of sprinkler body is 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;

C. the limit test of aristolochic acid A

High effective liquid chromatography for measuring according to an appendix VI D of < < Chinese Pharmacopoeia > > version in 2010;

The preparation of need testing solution: get Tibetan medicinal composition 29 taste energy oral preparation for eliminating powder 6～10g, accurately weighed, put in tool plug conical flask, precision adds methyl alcohol 40～60ml, close plug, weighed weight, ultrasonic 20～40 minutes, let cool, weighed weight again, with methyl alcohol, supply the weight of less loss, shake up, filter, get subsequent filtrate 25ml, low temperature is concentrated into dry, it is that 0.5% sodium hydroxide solution 20ml dissolves it completely and is transferred in separating funnel that residue adds volume parts ratio, by extracted with diethyl ether 2 times, each 20ml, after extraction, alkali lye service property (quality) mark 3% salt acid for adjusting pH is to 2-3, by extracted with diethyl ether 5 times, each 20ml merges ether extraction liquid, volatilize, with methyl alcohol, dissolve and be transferred in 5ml volumetric flask, add methyl alcohol to scale, shake up, filter, get subsequent filtrate, obtain,

Determination method: draw respectively each 10～20 μ l of reference substance solution and need testing solution, injection liquid chromatography, measures, and obtains.

2. the detection method of a kind of Tibetan medicinal composition 29 taste energy oral preparation for eliminating as claimed in claim 1, it is characterized in that described preparation refers to gets the Tibetan medicinal composition 29 tastes bulk drug that can dissipate, technique, adds conventional auxiliary material to be prepared into clinical acceptable any formulation routinely.

3. a kind of bulk drug as claimed in claim 1 or 2 consists of Tibet inula root 25g, forge gypsum rubrum 125g, myrobalan 75g, hot millet 25g, alkali flower 125g, nutmeg 25g, Bi roots of grass 25g, cassia seed 25g, Amomum cardamomum 25g, rhizome of davallia 25g, pepper 25g, radish charcoal 2g, tsaoko 25g, natural salt 25g, asafoetide 2g, sal ammoniac 25g, kaempferia galamga 25g, shellfish tooth charcoal 25g, rheum officinale 100g, wide muscle rattan 13g, safflower 25g, Aconitum Szechenyianum Gay 15g, lime 25g processed, fry vulture excrement 40g, banksia rose birthwort 25g, Semen seu folium abelmoschi moschati 25g, HALITUM PURPUREUM 25g, frankincense 25g, slag is tamed and dociled the detection method of the Tibetan medicinal composition 29 taste energy oral preparation for eliminating of cream 25g, the method comprises following discriminating and inspection method:

A. the discriminating of pipering

Get the Tibetan medicinal composition 29 tastes powder 3g that can dissipate, add ethyl acetate 25ml, ultrasonic processing 30min, filters, and filtrate is concentrated into 2ml, as need testing solution; Get pipering appropriate, add methyl alcohol and make every 1ml containing the reference substance solution of 0.5mg; Thin-layered chromatography test according to an appendix VI B of < < Chinese Pharmacopoeia > > version in 2010, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the cyclohexane-ethyl acetate as 3:2, launch, take out, dry; Put under ultraviolet lamp 365nm and inspect; In test sample chromatogram, with the corresponding position of reference substance chromatogram on, the spot of aobvious same color;

B. myristic discriminating

Get the Tibetan medicinal composition 29 tastes powder 5g that can dissipate, put in 250ml round-bottomed flask, add water 100ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil, in volatile oil determination apparatus, start to add a small amount of water and 1ml ethyl acetate, after decoction completes, collect ethyl acetate part, as need testing solution; Get nutmeg 1g, put in 100ml round-bottomed flask, add water 50ml, decoct 1h, with volatile oil determination apparatus, collect volatile oil, in volatile oil determination apparatus, start to add a small amount of water and 1ml ethyl acetate, after having decocted, collect ethyl acetate part, in contrast medicinal material solution; Thin-layered chromatography test according to an appendix VI B of < < Chinese Pharmacopoeia > > version in 2010, draw each 5 μ l of above-mentioned two kinds of solution, put respectively on same silica gel g thin-layer plate, the volume parts of take is developping agent than the dimethylbenzene-ethyl acetate as 20:0.3, launch, take out, dry, the long-pending portion rate of sprinkler body is 1% vanillic aldehyde ethanol solution of sulfuric acid, and 105 ℃ to be heated to spot colour developing clear; In test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, the spot of aobvious same color;

C. the limit test of aristolochic acid A