CN102908519B

CN102908519B - A kind ofly treat the medicine of influenza and the preparation method of preparation thereof and quality determining method

Info

Publication number: CN102908519B
Application number: CN201210361460.8A
Authority: CN
Inventors: 何国军
Original assignee: TONGYITANG PHARMACEUTICAL CO Ltd
Current assignee: Tongyitang Pharmaceutical Co., Ltd.
Priority date: 2012-09-26
Filing date: 2012-09-26
Publication date: 2015-11-25
Anticipated expiration: 2032-09-26
Also published as: CN102908519A

Abstract

The present invention relates to a kind of medicine for the treatment of influenza, it is characterized in that by the preparation method of the blue or green roc preparation of Chinese medicine preparation-nine taste of form improvement, its technical scheme is optimized in extraction process and screens, adopt modern new equipment, new technique, new method technically, be applicable to industrialized great production; Quality standard research improves on the basis of primary standard, and newly formulated TLC distinguish, content assaying method and ester alkaloid limit examine method, target level of product quality advanced person is controlled, ensure that clinical efficacy and drug safety.

Description

A kind ofly treat the medicine of influenza and the preparation method of preparation thereof and quality determining method

Technical field

The invention belongs to field of traditional Chinese, relate to a kind of Chinese medicine, particularly a kind ofly treat the medicine of influenza and the preparation method of preparation thereof and quality determining method.

Background technology

Influenza (Influenza) is called for short influenza, is a kind of Acute respiratory infectious disease caused by influenza virus, and infectiousness is strong, and sickness rate is high, easily causes outbreak of epidemic or is very popular.It is mainly through propagating containing the virulent spittle, interpersonal contact or also can propagate with the contact of contaminated article.Typical clinical characters is the anxious high heat, significantly weak of rising, muscular soreness of whole body, and have a stuffy nose, the upper mucositis symptom of breathing such as watery nasal discharge and sneeze is relatively light.Autumn and winter season is occurred frequently.Primary disease has self limiting, but causes death at the severe complication such as infant, old people and the easy Complicating Pneumonia In Patients of patient that there is cardiopulmonary underlying diseases.Data shows according to the relevent statistics, and China about has the people of 75% to suffer from least every year once to catch a cold every year.Influenza not only has influence on the live and work of people, for old people, infant, anemia of pregnant woman and some have the people of other diseases, influenza is a kind of very dangerous disease, and the complication occurred with influenza even can produce life and threaten.Because flu episode is rapid, complicated symptoms is various, thus do not have a kind of medicine to address all of these issues so far.Doctor trained in Western medicine adopts antipyretic analgesic usually, neuraminidase inhibitor, M2 ion channel blocking agents, antiviral agents etc., but the feature that the side effect of Western medicine ubiquity is large, and very easily produce drug resistance, while treatment influenza, also reduce body immunity to a certain extent, fundamentally can not reach the object of healing.Chinese medicine think flu be due to ailment said due to cold or exposure take advantage of human body to drive evil scarce capacity time, invasion and attack lung defend caused by fur.The most common with wind and cold, wind heat two kinds of diseases clinically, in addition, the administration in season, wet, pathogenic dryness also can random thoughts and be disease.Therefore Chinese traditional treatment influenza, adopts dialectical treatmert usually, from organic conception, with multipath, multi-level, conditioner body immunity function, prevent pathologic progress, and herbal toxic effect is little, shows good advantages for development.Therefore develop safe and effective, taking convenience, the flu medicine that fundamentally can reach therapeutic purposes is one and significantly works.

In view of the kind " the blue or green roc of nine tastes falls apart " recorded in " Drug Standard of Ministry of Public Health of the Peoples Republic of China Tibetan medicine " first, its reasonable recipe, Rhizoma Rhodiolae kirilowii, list loud, high-pitched sound Radix aconiti szechenyiani (seedling), Herba pterocephali clearing away heat to alleviate pain in side, except pestilence pestilence; Radix Solms-Laubachiae pulcherrimatis clearing away lung-heat; Clear " dragon " blood complication of Radix Pecteilis susannae; Radix Inulae, Benzoinum, Herba Oxytropis falcatae clearing away heat and alleviating pain eliminating phlegm and stopping cough; Fructus Chebulae's coordinating the actions of various ingredients in a prescription, heat-clearing and toxic substances removing, invigorating the spleen and replenishing QI.All medicines 5 plays sharp lung, antiinflammatory, the merit of cough-relieving mutually altogether, and to plague diseases, the fever that influenza causes, pulmonary's pain, pneumonia, throat such as to swell and ache at the successful.Therefore we have carried out further research and development to this kind.

The prescription that the blue or green roc of nine tastes falls apart is: Radix aconiti szechenyiani (seedling) 50g Fructus Chebulae (enucleation) 50g Radix Inulae 50g Benzoinum 27.5g Herba pterocephali 10g Rhizoma Rhodiolae kirilowii 47.5g Radix Pecteilis susannae 47.5g Radix Solms-Laubachiae pulcherrimatis 47.5g Herba Oxytropis falcatae 50g.Traditional preparation method is: above nine tastes, are ground into fine powder, sieve, and mixing, to obtain final product.The company producing the blue or green roc of nine tastes loose has 3, is respectively Tibet Tibetan Medicine Plant, Tibet hanuman Pharmaceutical Co., Ltd, Fromlingzhi, tibet Yu Tuo Tibetan medicine Co., Ltd.Patent search result, does not retrieve Patents; Open source literature result for retrieval, document HPLC measures the blue or green roc of tibetan traditional medicine nine taste and falls apart that (Pan Guoqing, HPLC measure the blue or green roc of tibetan traditional medicine nine taste and to fall apart the content of middle gallic acid for the content of middle gallic acid, Chinese patent medicine, the 2006,28th volume 9 phase, 1391-1392), the content assaying method of gallic acid is disclosed; Document HPLC method measures the content of Mongolia patent drug nine taste blue or green roc ball mesaconitine, discloses the content assaying method of aconitine.

Existing what fall apart that the preparation of medicine adopts about the blue or green rocs of nine tastes is traditional method, and crude drug un-extracted is directly pulverized and is used as medicine, and effective ingredient release slowly, badly influences absorbing of effective ingredient.The existing quality standard loose about the blue or green rocs of nine tastes, without differentiating and assay item, the HPLC detection method of the rarely seen gallic acid of disclosed quality determining method document and aconitine, therefore the blue or green rocs of the nine tastes quality standard that falls apart has much room for improvement.

Summary of the invention

The object of the invention is reform in extraction process, adopt modern new equipment, new technique, new method technically, formulated the production technology of reasonable science, be applicable to industrialized great production.As adopted modern extraction purification technology, prescription medical material being extracted, accelerating the stripping of effective ingredient; And for example to containing volatile oil medical material, extract volatile oil, adopt beta-cyclodextrin inclusion compound technology to carry out enclose, effectively can reduce the loss of finished product volatile oil in storage process, and mask the bad smell of volatile oil, improve mouthfeel when finished product is taken; For another example in preparation technique, adopt one-step palletizing, have and avoid heat time heating time in conventional formulation long, the deficiency that effective ingredient destroys, and substantially reduce the production cycle, efficiently advanced.

Another object of the present invention is to the weak point for former nine tastes blue or green roc powder type, carry out rational form improvement, consider that former powder instructions of taking is with mixing in water for oral taking, but easily eke out a living when taking, not easily swallow, in addition, this product is every packed 10g, once take 1g, take inconvenience, and dosage accurately should not control.Be that the modern formulations such as granule, capsule, tablet, drop pill, micropill, dispersible tablet can give full play to granule and absorb fast, rapidly effective by its form improvement, take and easy to carry, the feature that mouthfeel is good.For this prescription adds novel form again, adapt to extensive patients to the demand of different dosage form.

Another object of the present invention has carried out detailed deep quality standard research to preparation, and the basis of primary standard is improved.Do not formulate discriminating and assay under former powder item, this product, in development process, has all been carried out more deep Study on Identification to ingredients in prescription, has been established the thin-layer identification method of Fructus Chebulae, Radix Pecteilis susannae, Radix Inulae, Rhizoma Rhodiolae kirilowii.Method is all simple and feasible, and characteristic spots is obvious, and specificity is strong.Principal agent in Benzoinum, the Herba pterocephali side of being, its principle active component is respectively cinnamic acid, oleanolic acid and ursolic acid, adopt high performance liquid chromatography, measure the content of cinnamic acid, oleanolic acid and the ursolic acid in this product, result shows that method is simple and feasible, there is good accuracy and precision, effectively can control this quality, ensure that clinical efficacy.Radix aconiti szechenyiani (seedling) is aconitum plant, containing ester alkaloid class toxic component, adopts high performance liquid chromatography, controls the aconitine in this product, hypaconitine, mesaconitine limitation simultaneously, ensure that the clinical drug safety of this product.

Technical scheme of the present invention is as follows:

Treat the medicine of influenza and the preparation method of preparation thereof and a quality determining method, the crude drug of the medicine of described treatment influenza consists of: the blue or green rocs of former nine tastes fall apart prescription composition.Preparation method comprises the following steps:

(1) get Radix Inulae by crude drug composition and ratio, add water 6-12 times of weight, adopts steam distillation, extracts volatile oil 1-6h, collects volatile oil, and medicinal liquid filters, and obtains extracting solution A ' and medicinal residues A;

Add in the beta cyclodextrin aqueous solution of percent weight in volume 3-6% by collecting the volatile oil obtained, the envelope-bulk to weight ratio of volatile oil and beta cyclodextrin is 1ml:3-6g, under stirring condition, keep temperature 40-60 DEG C, stir 2-8h, 0 DEG C of-4 DEG C of refrigerated overnight, sucking filtration, get precipitation, 40-60 DEG C of vacuum drying, obtains volatile oil clathrate compound B;

(2) Radix aconiti szechenyiani (seedling) is got by crude drug composition and ratio, add water 6-14 times of weight, reflux, extract, 3-6h, be incorporated to the medicinal residues A after step (1) extraction volatile oil, Fructus Chebulae's (enucleation) is added again by crude drug composition and ratio, Benzoinum, Herba pterocephali, Rhizoma Rhodiolae kirilowii, Radix Pecteilis susannae, Radix Solms-Laubachiae pulcherrimatis, Herba Oxytropis falcatae, the reflux, extract, that adds water 1-4 time, the amount at every turn added water is 8-12 times of described 9 taste medical material gross weights, each extraction 1-4h, filter, filtrate merges, extract the extracting solution A ' after volatile oil with step (1) to merge, concentrating under reduced pressure, be concentrated into the fluid extract C that relative density under 25 DEG C of conditions is 1.08-1.12, use Boiling Fuel Injection Spray one-step palletizing, obtain extract D.

(3) the extract D that volatile oil clathrate compound B step (1) obtained and step (2) obtain, mixing, obtain the blue or green roc extract of nine tastes, select customary adjuvant, preparation process makes the dosage forms such as granule, capsule, tablet, drop pill, micropill, dispersible tablet routinely, obtains the blue or green roc preparation of nine tastes.

preferred preparation method is:

(1) get Radix Inulae by crude drug composition and ratio, add water 8 times of weight, adopts steam distillation, extracts volatile oil 4h, collects volatile oil, and medicinal liquid filters, and obtains extracting solution A ' and medicinal residues A;

Add in the beta cyclodextrin aqueous solution of percent weight in volume 4% by collecting the volatile oil obtained, the envelope-bulk to weight ratio of volatile oil and beta cyclodextrin is 1ml:4g, under stirring condition, keep temperature 50 C, stir 6h, 0 DEG C of-4 DEG C of refrigerated overnight, sucking filtration, get precipitation, 50 DEG C of vacuum dryings, obtain volatile oil clathrate compound B;

(2) Radix aconiti szechenyiani (seedling) is got by crude drug composition and ratio, add water 10 times of weight, reflux, extract, 4h, be incorporated to the medicinal residues A after step (1) extraction volatile oil, Fructus Chebulae's (enucleation) is added again by crude drug composition and ratio, Benzoinum, Herba pterocephali, Rhizoma Rhodiolae kirilowii, Radix Pecteilis susannae, Radix Solms-Laubachiae pulcherrimatis, Herba Oxytropis falcatae, the reflux, extract, that adds water 2 times, the amount that first time adds water is 12 times of described 9 taste medical material gross weights, extract 2h, filter, the amount that medicinal residues add water again is 10 times of described 9 taste medical material gross weights, extract 2h, filter, filtrate merges, extract the extracting solution A ' after volatile oil with step (1) to merge, concentrating under reduced pressure, be concentrated into the fluid extract C that relative density under 25 DEG C of conditions is 1.10, use Boiling Fuel Injection Spray one-step palletizing, obtain extract D.

(3) the extract D that volatile oil clathrate compound B step (1) obtained and step (2) obtain, mixing, obtain the blue or green roc extract of nine tastes, select customary adjuvant, preparation process makes tablet routinely, obtains the blue or green roc sheet of nine tastes.

The quality determining method of the blue or green roc preparations of nine tastes of the present invention comprises following discriminating and/or one or more in content assaying method:

differentiate:

the discriminating of A, Fructus Chebulae:get nine tastes blue or green roc preparation 3-5g, add dehydrated alcohol 25-50ml, supersound process 20-30min, filter paper evenly adds kieselguhr 1.0g, filter, filtrate is concentrated into 2ml, as need testing solution; Separately get Fructus Chebulae's control medicinal material 1-2g, add dehydrated alcohol 25-50ml, supersound process 20-30min, filter paper evenly adds kieselguhr 1.0g, filter, filtrate is concentrated into 2ml, in contrast medical material solution; Separately get gallic acid reference substance, add methanol and make the solution of every 1ml containing 1mg, product solution in contrast; Test according to thin layer chromatography (Pharmacopoeia of the People's Republic of China version in 2010 annex VIB), draw above-mentioned solution each 5-10 μ l, put respectively on same silica gel g thin-layer plate, with volume parts ratio for the dichloromethane-methyl acetate-formic acid of 3-9:2-6:0.5-1.5 is for developing solvent, launch, take out, dry, spray is 1% ferric chloride alcoholic solution with quality volume portion rate, 105 DEG C to be heated to spot development clear, in test sample chromatograph, on the position corresponding to reference substance and control medicinal material chromatograph, the speckle of aobvious same color;

b. the discriminating of Radix Pecteilis susannae:get nine tastes blue or green roc preparation 2-5g, add methanol 20-50ml, supersound process 10-40min, filter, filter evaporate to dryness, the residue 25ml that adds water makes dissolving, with ethyl acetate extraction 1-3 time, each 25ml, merge ethyl acetate liquid, evaporate to dryness, residue adds methanol 1ml and dissolves, as need testing solution.Separately get Radix Pecteilis susannae control medicinal material 1g, add methanol 30ml, supersound process 30min, filter, filter evaporate to dryness, the residue 25ml that adds water makes dissolving, with ethyl acetate extraction 3 times, each 25ml, merge ethyl acetate liquid, evaporate to dryness, residue adds methanol 1ml and dissolves, medical material solution in contrast.Test according to thin layer chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ L of above-mentioned two kinds of solution, point is on same silica gel g thin-layer plate, with normal hexane-ethyl acetate (9:1) for developing solvent, launch, take out, dry, spray, with volume ratio 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear, inspects under putting ultra-violet lamp (365nm).In test sample chromatograph, on the position corresponding to control medicinal material chromatograph, the fluorescence speckle of aobvious same color.

the discriminating of Radix Inulae:get nine tastes blue or green roc preparation 2-5g, add boiling range 30-60 DEG C of petroleum ether 10-40ml, supersound process 10-30min, filter, filtrate is lower than evaporate to dryness under 60 DEG C of conditions, and residue adds ethyl acetate 1ml and dissolves, as need testing solution.Separately get Lignum Santali Albi control medicinal material 0.5g, add boiling range 30-60 DEG C of petroleum ether 30ml, supersound process 30min, filter, filtrate is lower than evaporate to dryness under 60 DEG C of conditions, and residue adds ethyl acetate 1ml and dissolves, medical material solution in contrast.According to thin layer chromatography (Chinese Pharmacopoeia version in 2010 annex VI B) test, draw each 10 μ L of above-mentioned two kinds of solution, put on same silica gel g thin-layer plate, with chloroform-Ethyl formate-methanol (5:4:0.4) for developing solvent, launch, take out, dry.Spray is with mass volume ratio 1% vanillin-sulfuric acid solution, and 105 DEG C to be heated to spot development clear.In test sample chromatograph, on the position corresponding to control medicinal material chromatograph, the fluorescence speckle of aobvious same color.

the discriminating of Rhizoma Rhodiolae kirilowii:get nine tastes blue or green roc preparation 3-6g, add n-butanol extraction 30-50ml, supersound process 20-40min, filter, n-butyl alcohol liquid evaporate to dryness, residue adds methanol 5ml and dissolves, be added in neutral alumina column (100-200 order, 2.5g, internal diameter is 1.0cm) on, with methanol 30ml eluting, collect meoh eluate, evaporate to dryness, residue adds methanol 5ml makes dissolving, as need testing solution; Separately get Rhizoma Rhodiolae kirilowii control medicinal material 1g, add n-butanol extraction 30-50ml, supersound process 20-40min, filter, n-butyl alcohol liquid evaporate to dryness, residue adds methanol 5ml and dissolves, be added in neutral alumina column (100-200 order, 2.5g, internal diameter is 1.0cm) on, with methanol 30ml eluting, collect meoh eluate, evaporate to dryness, residue adds methanol 5ml makes dissolving, medical material solution in contrast.Test according to thin layer chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ L of above-mentioned two kinds of solution, put respectively on same silica GF254 lamellae, with chloroform-Ethyl formate-methanol (5:4:1.5) for developing solvent, launch, take out, dry, inspect under putting ultra-violet lamp (254nm), in test sample chromatograph, the speckle of aobvious same color on the position corresponding to reference substance chromatograph.

assay:

benzoic assay:measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VID).

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filler; With methanol-0.8% glacial acetic acid (35:65) for mobile phase; Determined wavelength is 270nm.Number of theoretical plate calculates should be not less than 3000 by cinnamic acid peak.

The preparation of reference substance solution gets cinnamic acid reference substance in right amount, accurately weighed, puts in brown volumetric flask, adds methanol and makes the solution of every 1ml containing 50 μ g, to obtain final product.

After the preparation nine taste blue or green roc preparation porphyrize of need testing solution, get powder 3-5g, accurately weighed, put in tool plug conical flask, precision adds methanol 40-60ml, close plug, weighed weight, supersound process 20-40min, let cool, weighed weight again, the weight of less loss is supplied with methanol, shake up, filter, get subsequent filtrate 25ml, be concentrated into dry, it is that 0.5% potassium hydroxide solution 20ml makes dissolving that residue adds volume parts ratio, by extracted with diethyl ether 2 times, each 20ml, discard ether solution, alkaline solution regulates pH4 with mass fraction hydrochloric acid again, by extracted with diethyl ether 4 times, each 20ml, merge ether solution, volatilize, be transferred in the brown volumetric flask of 5ml with dissolve with methanol, add methanol to scale, shake up, filter, get subsequent filtrate, obtain,

Algoscopy is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product.

the assay of Herba pterocephali:measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VID).

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filler; With methanol-0.6% glacial acetic acid (85:15) for mobile phase; Evaporative light scattering detector detects, drift tube temperature 85 DEG C, nitrogen pressure 48ps.

Oleanolic acid reference substance is got in the preparation of reference substance solution and ursolic acid reference substance is in right amount each, accurately weighed respectively, adds methanol and makes every 1ml respectively containing the solution of 0.5mg, to obtain final product.

After the preparation nine taste blue or green roc preparation porphyrize of need testing solution, get powder 3-5g, accurately weighed, put in tool plug conical flask, precision adds methanol 40-60ml, close plug, weighed weight, supersound process 20-40min, lets cool, weighed weight again, supply the weight of less loss with methanol, shake up, filter, get subsequent filtrate, to obtain final product;

ester alkaloid determination limit:measure according to high performance liquid chromatography (Chinese Pharmacopoeia version in 2010 annex VI D).

Chromatographic condition and system suitability acetonitrile-oxolane (5:3) are mobile phase A, and with 0.1mol/L Spirit of Mindererus. for Mobile phase B, the regulation according to the form below 1 carries out gradient elution, and determined wavelength is 235nm.Number of theoretical plate calculates should be not less than 2500 by mesaconitine peak.

Table 1 gradient elution mobile phase

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	15	85
60	35	65

Aconitine reference substance is got in the preparation of reference substance solution, hypaconitine reference substance, mesaconitine reference substance are appropriate, accurately weighed, the mixed solution adding hydrochloric acid and methanol volume ratio 1:100 makes the mixed solution of every 1ml containing aconitine, hypaconitine, each 50ug of mesaconitine, to obtain final product.

The preparation nine taste blue or green roc preparation porphyrize of need testing solution, gets powder 10g, accurately weighed, put in the conical flask of tool plug, precision adds the mixed solution 50ml of hydrochloric acid and methanol volume ratio 1:100, weighed weight, supersound process 60min, let cool, weighed weight, supply the weight of less loss with the mixed solution of hydrochloric acid and methanol volume ratio 1:100, shake up, filter, get subsequent filtrate, to obtain final product.

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

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

The prescription weight that the blue or green roc of nine tastes of the present invention falls apart is as follows: Radix aconiti szechenyiani (seedling) 50g Fructus Chebulae (enucleation) 50g Radix Inulae 50g Benzoinum 27.5g Herba pterocephali 10g Rhizoma Rhodiolae kirilowii 47.5g Radix Pecteilis susannae 47.5g Radix Solms-Laubachiae pulcherrimatis 47.5g Herba Oxytropis falcatae 50g.

Experimental example 1-5 and embodiment 1-4 raw material medicines in portions by weight proportioning are the loose prescription weight proportion of the blue or green roc of nine tastes.

experimental example 1: volatile oil bag and on stability of volatile oil impact test

Volatile oil is extracted by the method for embodiment 1, and enclose, prepare the blue or green roc sheet of nine tastes simultaneously; The preparation method that the blue or green roc of nine tastes recorded by background technology falls apart is prepared the blue or green rocs of nine tastes and is fallen apart; Identical conditions (temperature 40 DEG C ± 2 DEG C, humidity 75% ± 5%) transfer set to 0,1,2,3,6 month, detect volatile oil content, the results are shown in Table 1.

Table 1 beta-cyclodextrin inclusion compound is to the investigation result of stability of volatile oil

Conclusion: after volatile oil beta-cyclodextrin inclusion compound of the present invention, substantially increases volatile oil stability in the formulation.

experimental example 2: Different Extraction Method compares

A. water extraction:

Add in the beta cyclodextrin aqueous solution of percent weight in volume 3% by collecting the volatile oil obtained, the envelope-bulk to weight ratio of volatile oil and beta cyclodextrin is 1ml:3g, under stirring condition, keep temperature 50 C, stir 6h, 0 DEG C of-4 DEG C of refrigerated overnight, sucking filtration, get precipitation, 50 DEG C of vacuum dryings, obtain volatile oil clathrate compound B;

(2) Radix aconiti szechenyiani (seedling) is got by crude drug composition and ratio, add water 10 times of weight, reflux, extract, 4h, be incorporated to the medicinal residues A after step (1) extraction volatile oil, Fructus Chebulae's (enucleation) is added again by crude drug composition and ratio, Benzoinum, Herba pterocephali, Rhizoma Rhodiolae kirilowii, Radix Pecteilis susannae, Radix Solms-Laubachiae pulcherrimatis, Herba Oxytropis falcatae, the reflux, extract, that adds water 2 times, the amount at every turn added water is 12 times of described 9 taste medical material gross weights, each extraction 2h, filter, filtrate merges, extract the extracting solution A ' after volatile oil with step (1) to merge, concentrating under reduced pressure, be concentrated into the fluid extract C that relative density under 25 DEG C of conditions is 1.08-1.12,

B.60% ethanol extraction:

(2) Radix aconiti szechenyiani (seedling) is got by crude drug composition and ratio, add 60% ethanol, 10 times of weight, reflux, extract, 4h, be incorporated to the medicinal residues A after step (1) extraction volatile oil, Fructus Chebulae's (enucleation) is added again by crude drug composition and ratio, Benzoinum, Herba pterocephali, Rhizoma Rhodiolae kirilowii, Radix Pecteilis susannae, Radix Solms-Laubachiae pulcherrimatis, Herba Oxytropis falcatae, add 60% alcohol reflux 2 times, the amount at every turn adding 60% ethanol is 12 times of described 9 taste medical material gross weights, each extraction 2h, filter, filtrate merges, extract the extracting solution A ' after volatile oil with step (1) to merge, concentrating under reduced pressure, be concentrated into the fluid extract C that relative density under 25 DEG C of conditions is 1.08-1.12,

C.30% ethanol extraction:

(2) Radix aconiti szechenyiani (seedling) is got by crude drug composition and ratio, add 30% ethanol, 10 times of weight, reflux, extract, 4h, be incorporated to the medicinal residues A after step (1) extraction volatile oil, Fructus Chebulae's (enucleation) is added again by crude drug composition and ratio, Benzoinum, Herba pterocephali, Rhizoma Rhodiolae kirilowii, Radix Pecteilis susannae, Radix Solms-Laubachiae pulcherrimatis, Herba Oxytropis falcatae, add 30% alcohol reflux 2 times, the amount at every turn adding 30% ethanol is 12 times of described 9 taste medical material gross weights, each extraction 2h, filter, filtrate merges, extract the extracting solution A ' after volatile oil with step (1) to merge, concentrating under reduced pressure, be concentrated into the fluid extract C that relative density under 25 DEG C of conditions is 1.08-1.12,

Get Different Extraction Method gained fluid extract C, measure active constituent content, calculate the rate of transform, result is as follows:

Table 2 Different Extraction Method effective ingredient rate of transform result

Extracting method	The gallic acid rate of transform (%)	Oleanolic acid and the total rate of transform of ursolic acid (%)	The cinnamic acid rate of transform (%)
				Water extraction	88	68	86
30% ethanol extraction	86	70	85
				60% ethanol extraction	75	75	73

Result shows, water extraction is compared with 30% ethanol and 60% ethanol extraction, the rate of transform zero difference of oleanolic acid and the total rate of transform of ursolic acid, water extraction is compared with 30% ethanol extraction, both rates of transform zero difference of gallic acid, cinnamic acid, and all higher than 60% ethanol extraction, consider production cost and production safety, determine to adopt water extraction.

experimental example 3:the preferred amount of water of orthogonal test, extraction time and extraction time

Adopt orthogonal experiment, selected amount of water, extraction time and extraction time, as three factors investigated, respectively get three levels, and design sees the following form 2:

Table 3 water extraction orthogonal test table designs

* note: the amount of water in laboratory and extraction time are all determined by extraction time, and amount of water and extraction time carry out from front to back successively.

Test method: take Radix aconiti szechenyiani (seedling) 50g Fructus Chebulae (enucleation) 50g Radix Inulae 50g Benzoinum 27.5g Herba pterocephali 10g Rhizoma Rhodiolae kirilowii 47.5g Radix Pecteilis susannae 47.5g Radix Solms-Laubachiae pulcherrimatis 47.5g Herba Oxytropis falcatae 50g totally 9 taste medical materials, get 9 parts altogether, enter process test research by orthogonal table order, result is as following table 4:

Table 4L9(3 ⁴) orthogonal test designs table and result

Tested number	A (amount of water)	B (extraction time)	C (extraction time)	D (blank)	Oleanolic acid and ursolic acid total amount (mg)
						1	1	1	1	1	47.5
2	1	2	2	2	59.4
						3	1	3	3	3	60.2
4	2	1	2	3	67.4
						5	2	2	3	1	62.1
6	2	3	1	2	53.9
						7	3	1	3	2	53.6
8	3	2	1	3	48.4
						9	3	3	2	1	52．0
K1	55.700	56.167	49.933	53.867	——
						K2	61.133	56.633	59.600	55.633	——
K3	51.333	55.367	58.633	58.667	——
						R	9.800	1.267	9.667	4.800	——

As can be seen from Table 4, each factor effect primary and secondary is A>C>B, and optimal processing parameter is A ₂b ₂c ₂.Take reflux, extract, 2 times, add water 12 times amount for the first time, extracts 2h, and add water 10 times amount for the second time, extracts 2h.

Amplify demonstration test through three batches, result confirms that selected technique is optimised process.

experimental example 4,different decocting time is on the impact of toxic component

Radix aconiti szechenyiani (seedling) medical material contains aconitine toxic component, aconitine can be destroyed through long decoction, the different decocting time of this experiment investigation is on the impact of Content of Aconitine, get Radix aconiti szechenyiani (seedling) 50g, put in 1000ml round-bottomed flask, add 500ml soak by water, decoct 6h continuously, from 2h, start sampling and measuring Content of Aconitine, the results are shown in Table 5.

The different decocting time of table 5 is on the impact of Content of Aconitine

Table 5 result shows, the content of aconitine is along with the increase of extraction time, and its content reduces gradually, prompting extraction time the destruction of longer aconitine more, its toxicity also can be less.Therefore determine, aconitine first singly carries 4h, and then carries altogether with other medical materials.

experimental example 5: the selection of different granulating process

Prepare fluid extract C by the method for embodiment 1, take dextrin as adjuvant, consumption is 60% of finished particle, adopts one-step palletizing and wet granulation respectively, prepares granule, and gained granule measures the loss of effective ingredient.

The different method of granulating of table 6 is on the impact of effective ingredient

Granulating process	Cinnamic acid loss rate (%)	Oleanolic acid and ursolic acid total loss rate (%)
			One-step palletizing	1.2	2.0
Wet granulation	15.8	8.5

Result shows, one-step palletizing, a few free of losses of effective ingredient, and wet granulation effective ingredient, and the loss of esp meat cinnamic acid is comparatively serious, therefore determines to adopt one-step palletizing.

Gained granule and volatile oil clathrate compound mix, and add proper auxiliary materials further, can be made into the preparations such as granule, capsule, tablet.

experimental example 6: the selection of different dosage form

The nine taste medical materials that blue or green for former nine tastes roc falls apart in prescription are prepared according to the following steps

(3) the extract D that volatile oil clathrate compound B step (1) obtained and step (2) obtain, mixing, obtains the blue or green roc extract of nine tastes.

Get the blue or green roc extract of above-mentioned steps (3) gained nine taste, add the adjuvants such as suitable disintegrating agent, filler, lubricant, prepare different dosage form.

The yellow extract of table 7 nine taste Zhu prepares different dosage form result

Illustrate: granule, sheet, capsule, drop pill, buccal tablet, oral liquid, micropill, dispersible tablet are qualified, refer to meet Chinese Pharmacopoeia version in 2010 annex c, d, l, k, d, j, k, to the relevant regulations of granule, sheet, capsule, drop pill, buccal tablet, oral liquid, micropill, dispersible tablet under D item.

Result shows, the blue or green roc extract of nine tastes of the present invention, except being suitable for preparing except granule, tablet, capsule, is also suitable for being prepared into drop pill, micropill, dispersible tablet, buccal tablet and oral liquid etc.

experimental example 7: effective ingredient dissolution test

By tablet prepared by the granule that blue or green for former nine tastes roc falls apart and prepared by the embodiment of the present invention 1, embodiment 2, capsule prepared by embodiment 3 carries out effective ingredient dissolution test respectively.

Table 8 effective ingredient dissolution results

Result shows, granule prepared by the embodiment of the present invention 1, the tablet of embodiment 2 preparation, and the dissolution of capsule prepared by embodiment 3 falls apart apparently higher than the blue or green rocs of former nine tastes.

embodiment 1: the nine taste medical materials that blue or green for former nine tastes roc falls apart in prescription are prepared granule according to the following steps:

(2) Radix aconiti szechenyiani (seedling) is got by crude drug composition and ratio, add water 10 times of weight, reflux, extract, 4h, be incorporated to the medicinal residues A after step (1) extraction volatile oil, Fructus Chebulae's (enucleation) is added again by crude drug composition and ratio, Benzoinum, Herba pterocephali, Rhizoma Rhodiolae kirilowii, Radix Pecteilis susannae, Radix Solms-Laubachiae pulcherrimatis, Herba Oxytropis falcatae, the reflux, extract, that adds water 2 times, the amount that first time adds water is 12 times of described 9 taste medical material gross weights, extract 2h, filter, the amount that medicinal residues add water again is 10 times of described 9 taste medical material gross weights, extract 2h, filter, filtrate merges, extract the extracting solution A ' after volatile oil with step (1) to merge, concentrating under reduced pressure, be concentrated into the fluid extract C that relative density under 25 DEG C of conditions is 1.10, with sucrose, dextrin and step (1) volatile oil clathrate compound B do bed material, use Boiling Fuel Injection Spray one-step palletizing, dry, granulate, packaging, obtain the blue or green roc granule of nine tastes of the present invention.

embodiment 2: the nine taste medical materials that blue or green for former nine tastes roc falls apart in prescription are prepared tablet according to the following steps:

(1) get Radix Inulae by crude drug composition and ratio, add water 6 times of weight, adopts steam distillation, extracts volatile oil 6h, collects volatile oil, and medicinal liquid filters, and obtains extracting solution A ' and medicinal residues A;

Add in the beta cyclodextrin aqueous solution of percent weight in volume 6% by collecting the volatile oil obtained, the envelope-bulk to weight ratio of volatile oil and beta cyclodextrin is 1ml:6g, under stirring condition, keep temperature 40 DEG C, stir 2h, 0 DEG C of-4 DEG C of refrigerated overnight, sucking filtration, get precipitation, 40 DEG C of vacuum dryings, obtain volatile oil clathrate compound B;

(2) Radix aconiti szechenyiani (seedling) is got by crude drug composition and ratio, add water 14 times of weight, reflux, extract, 6h, be incorporated to the medicinal residues A after step (1) extraction volatile oil, Fructus Chebulae's (enucleation) is added again by crude drug composition and ratio, Benzoinum, Herba pterocephali, Rhizoma Rhodiolae kirilowii, Radix Pecteilis susannae, Radix Solms-Laubachiae pulcherrimatis, Herba Oxytropis falcatae, the reflux, extract, that adds water 2 times, the amount that first time adds water is 14 times of described 9 taste medical material gross weights, extract 1h, filter, the amount that medicinal residues add water again is 8 times of described 9 taste medical material gross weights, extract 3h, filter, filtrate merges, extract the extracting solution A ' after volatile oil with step (1) to merge, concentrating under reduced pressure, be concentrated into the fluid extract C that relative density under 25 DEG C of conditions is 1.20, use Boiling Fuel Injection Spray one-step palletizing, obtain extract D.

(3) the extract D that volatile oil clathrate compound B step (1) obtained and step (2) obtain, mixing, obtains the blue or green roc extract of nine tastes, selects starch, microcrystalline Cellulose, carboxymethyl starch sodium, magnesium stearate to be adjuvant, preparation process makes tablet routinely, obtains the blue or green roc sheet of nine tastes.

embodiment 3: the nine taste medical materials that blue or green for former nine tastes roc falls apart in prescription are prepared capsule according to the following steps:

(1) get Radix Inulae by crude drug composition and ratio, add water 14 times of weight, adopts steam distillation, extracts volatile oil 2h, collects volatile oil, and medicinal liquid filters, and obtains extracting solution A ' and medicinal residues A;

Add in the beta cyclodextrin aqueous solution of percent weight in volume 3% by collecting the volatile oil obtained, the envelope-bulk to weight ratio of volatile oil and beta cyclodextrin is 1ml:3g, under stirring condition, keep temperature 60 C, stir 6h, 0 DEG C of-4 DEG C of refrigerated overnight, sucking filtration, get precipitation, 60 DEG C of vacuum dryings, obtain volatile oil clathrate compound B;

(2) Radix aconiti szechenyiani (seedling) is got by crude drug composition and ratio, add water 6 times of weight, reflux, extract, 3h, be incorporated to the medicinal residues A after step (1) extraction volatile oil, Fructus Chebulae's (enucleation) is added again by crude drug composition and ratio, Benzoinum, Herba pterocephali, Rhizoma Rhodiolae kirilowii, Radix Pecteilis susannae, Radix Solms-Laubachiae pulcherrimatis, Herba Oxytropis falcatae, the reflux, extract, that adds water 2 times, the amount that first time adds water is 10 times of described 9 taste medical material gross weights, extract 2h, filter, the amount that medicinal residues add water again is 8 times of described 9 taste medical material gross weights, extract 1h, filter, the amount that medicinal residues add water again is 6 times of described 9 taste medical material gross weights, extract 1h, filter, filtrate merges, extract the extracting solution A ' after volatile oil with step (1) to merge, concentrating under reduced pressure, be concentrated into the fluid extract C that relative density under 25 DEG C of conditions is 1.05, use Boiling Fuel Injection Spray one-step palletizing, obtain extract D.

(3) the extract D that volatile oil clathrate compound B step (1) obtained and step (2) obtain, mixing, obtains the blue or green roc extract of nine tastes, selects starch, microcrystalline Cellulose, magnesium stearate to be adjuvant, preparation process makes capsule routinely, obtains the blue or green roc capsule of nine tastes.

embodiment 4: the nine taste medical materials that blue or green for former nine tastes roc falls apart in prescription are prepared other preparations according to the following steps:

(1) get Radix Inulae by crude drug composition and ratio, add water 8 times of weight, adopts steam distillation, extracts volatile oil 5h, collects volatile oil, and medicinal liquid filters, and obtains extracting solution A ' and medicinal residues A;

Add in the beta cyclodextrin aqueous solution of percent weight in volume 4% by collecting the volatile oil obtained, the envelope-bulk to weight ratio of volatile oil and beta cyclodextrin is 1ml:4g, under stirring condition, keep temperature 50 C, stir 8h, 0 DEG C of-4 DEG C of refrigerated overnight, sucking filtration, get precipitation, 50 DEG C of vacuum dryings, obtain volatile oil clathrate compound B;

(2) Radix aconiti szechenyiani (seedling) is got by crude drug composition and ratio, add water 10 times of weight, reflux, extract, 6h, be incorporated to the medicinal residues A after step (1) extraction volatile oil, Fructus Chebulae's (enucleation) is added again by crude drug composition and ratio, Benzoinum, Herba pterocephali, Rhizoma Rhodiolae kirilowii, Radix Pecteilis susannae, Radix Solms-Laubachiae pulcherrimatis, Herba Oxytropis falcatae, the reflux, extract, that adds water 2 times, the amount that first time adds water is 10 times of described 9 taste medical material gross weights, extract 2h, filter, the amount that medicinal residues add water again is 10 times of described 9 taste medical material gross weights, extract 3h, filter, filtrate merges, extract the extracting solution A ' after volatile oil with step (1) to merge, concentrating under reduced pressure, be concentrated into the fluid extract C that relative density under 25 DEG C of conditions is 1.10, use Boiling Fuel Injection Spray one-step palletizing, obtain extract D.

(3) the extract D that volatile oil clathrate compound B step (1) obtained and step (2) obtain, mixing, obtain the blue or green roc extract of nine tastes, select customary adjuvant, preparation process makes drop pill, micropill, dispersible tablet etc. routinely, obtains the preparations such as the blue or green roc drop pill of nine tastes, the blue or green roc micropill of nine tastes, the blue or green roc dispersible tablet of nine tastes.

embodiment 5: nine taste blue or green roc granular mass detection method

The present invention has all carried out deep discriminating and assay research to the ingredients in prescription, and establish the thin-layer identification method of Fructus Chebulae, Radix Pecteilis susannae, Radix Inulae, Rhizoma Rhodiolae kirilowii, the ester alkaloid limit examine method of the HPLC content assaying method of Benzoinum, Herba pterocephali and Radix aconiti szechenyiani (seedling).The present embodiment specimen in use is embodiment 1 granule.

differentiate:

the discriminating of A, Fructus Chebulae:get nine tastes blue or green roc granule 3g, add dehydrated alcohol 25ml, supersound process 20min, filter paper evenly adds kieselguhr 1.0g, filter, filtrate is concentrated into 2ml, as need testing solution; Separately get Fructus Chebulae's control medicinal material 1-2g, add dehydrated alcohol 25ml, supersound process 20min, filter paper evenly adds kieselguhr 1.0g, filter, filtrate is concentrated into 2ml, in contrast medical material solution; Separately get gallic acid reference substance, add methanol and make the solution of every 1ml containing 1mg, product solution in contrast; Test according to thin layer chromatography (Pharmacopoeia of the People's Republic of China version in 2010 annex VIB), draw each 10 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with volume parts ratio for the dichloromethane-methyl acetate-formic acid of 3:6:0.5 is for developing solvent, launch, take out, dry, spray is 1% ferric chloride alcoholic solution with quality volume portion rate, 105 DEG C to be heated to spot development clear, in test sample chromatograph, on the position corresponding to reference substance and control medicinal material chromatograph, the speckle of aobvious same color;

b. the discriminating of Radix Pecteilis susannae:get nine tastes blue or green roc granule 5g, add methanol 50ml, supersound process 40min, filter, filter evaporate to dryness, the residue 25ml that adds water makes dissolving, and with ethyl acetate extraction 3 times, each 25ml, merge ethyl acetate liquid, evaporate to dryness, residue adds methanol 1ml and dissolves, as need testing solution.Separately get Radix Pecteilis susannae control medicinal material 1g, add methanol 30ml, supersound process 30min, filter, filter evaporate to dryness, the residue 25ml that adds water makes dissolving, with ethyl acetate extraction 3 times, each 25ml, merge ethyl acetate liquid, evaporate to dryness, residue adds methanol 1ml and dissolves, medical material solution in contrast.Test according to thin layer chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ L of above-mentioned two kinds of solution, point is on same silica gel g thin-layer plate, with normal hexane-ethyl acetate (9:1) for developing solvent, launch, take out, dry, spray, with volume ratio 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear, inspects under putting ultra-violet lamp (365nm).In test sample chromatograph, on the position corresponding to control medicinal material chromatograph, the fluorescence speckle of aobvious same color.

the discriminating of Radix Inulae:get nine tastes blue or green roc granule 5g, add boiling range 30-60 DEG C of petroleum ether 40ml, supersound process 30min, filter, filtrate is lower than evaporate to dryness under 60 DEG C of conditions, and residue adds ethyl acetate 1ml and dissolves, as need testing solution.Separately get Lignum Santali Albi control medicinal material 0.5g, add boiling range 30-60 DEG C of petroleum ether 30ml, supersound process 30min, filter, filtrate is lower than evaporate to dryness under 60 DEG C of conditions, and residue adds ethyl acetate 1ml and dissolves, medical material solution in contrast.According to thin layer chromatography (Chinese Pharmacopoeia version in 2010 annex VI B) test, draw each 10 μ L of above-mentioned two kinds of solution, put on same silica gel g thin-layer plate, with chloroform-Ethyl formate-methanol (5:4:0.4) for developing solvent, launch, take out, dry.Spray is with mass volume ratio 1% vanillin-sulfuric acid solution, and 105 DEG C to be heated to spot development clear.In test sample chromatograph, on the position corresponding to control medicinal material chromatograph, the fluorescence speckle of aobvious same color.

the discriminating of Rhizoma Rhodiolae kirilowii:get nine tastes blue or green roc granule 6g, add n-butanol extraction 50ml, supersound process 40min, filter, n-butyl alcohol liquid evaporate to dryness, residue adds methanol 5ml and dissolves, be added in neutral alumina column (100-200 order, 2.5g, internal diameter is 1.0cm) on, with methanol 30ml eluting, collect meoh eluate, evaporate to dryness, residue adds methanol 5ml makes dissolving, as need testing solution; Separately get Rhizoma Rhodiolae kirilowii control medicinal material 1g, add n-butanol extraction 50ml, supersound process 40min, filter, n-butyl alcohol liquid evaporate to dryness, residue adds methanol 5ml and dissolves, be added in neutral alumina column (100-200 order, 2.5g, internal diameter is 1.0cm) on, with methanol 30ml eluting, collect meoh eluate, evaporate to dryness, residue adds methanol 5ml makes dissolving, medical material solution in contrast.Test according to thin layer chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ L of above-mentioned two kinds of solution, put respectively on same silica GF254 lamellae, with chloroform-Ethyl formate-methanol (5:4:1.5) for developing solvent, launch, take out, dry, inspect under putting ultra-violet lamp (254nm), in test sample chromatograph, the speckle of aobvious same color on the position corresponding to reference substance chromatograph.

assay:

After the preparation nine taste blue or green roc granule porphyrize of need testing solution, get powder 5g, accurately weighed, put in tool plug conical flask, precision adds methanol 60ml, close plug, weighed weight, supersound process 40min, let cool, weighed weight again, the weight of less loss is supplied with methanol, shake up, filter, get subsequent filtrate 25ml, be concentrated into dry, it is that 0.5% potassium hydroxide solution 20ml makes dissolving that residue adds volume parts ratio, by extracted with diethyl ether 2 times, each 20ml, discard ether solution, alkaline solution regulates pH4 with mass fraction hydrochloric acid again, by extracted with diethyl ether 4 times, each 20ml, merge ether solution, volatilize, be transferred in the brown volumetric flask of 5ml with dissolve with methanol, add methanol to scale, shake up, filter, get subsequent filtrate, obtain,

After the preparation nine taste blue or green roc preparation porphyrize of need testing solution, get powder 5g, accurately weighed, put in tool plug conical flask, precision adds methanol 60ml, close plug, weighed weight, supersound process 40min, lets cool, more weighed weight, supply the weight of less loss with methanol, shake up, filter, get subsequent filtrate, to obtain final product;

Table 1 gradient elution mobile phase

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	15	85
60	35	65

Aconitine reference substance is got in the preparation of reference substance solution, hypaconitine reference substance, mesaconitine reference substance are appropriate, accurately weighed, the mixed solution adding hydrochloric acid and methanol volume ratio 1:100 makes the mixed solution of every 1ml containing aconitine, hypaconitine, each 50 μ g of mesaconitine, to obtain final product.

embodiment 6: nine taste blue or green roc tablet quality detection method

The present invention has all carried out deep discriminating and assay research to the ingredients in prescription, and establish the thin-layer identification method of Fructus Chebulae, Radix Pecteilis susannae, Radix Inulae, Rhizoma Rhodiolae kirilowii, the ester alkaloid limit examine method of the HPLC content assaying method of Benzoinum, Herba pterocephali and Radix aconiti szechenyiani (seedling).The present embodiment specimen in use is the blue or green roc sheet of embodiment 2 nine taste.

differentiate:

the discriminating of A, Fructus Chebulae:get nine tastes blue or green roc sheet 5g, add dehydrated alcohol 50ml, supersound process 30min, filter paper evenly adds kieselguhr 1.0g, filter, filtrate is concentrated into 2ml, as need testing solution; Separately get Fructus Chebulae's control medicinal material 2g, add dehydrated alcohol 50ml, supersound process 30min, filter paper evenly adds kieselguhr 1.0g, filter, filtrate is concentrated into 2ml, in contrast medical material solution; Separately get gallic acid reference substance, add methanol and make the solution of every 1ml containing 1mg, product solution in contrast; Test according to thin layer chromatography (Pharmacopoeia of the People's Republic of China version in 2010 annex VIB), draw each 5 μ l of above-mentioned solution, put respectively on same silica gel g thin-layer plate, with volume parts ratio for the dichloromethane-methyl acetate-formic acid of 9:2:1.5 is for developing solvent, launch, take out, dry, spray is 1% ferric chloride alcoholic solution with quality volume portion rate, 105 DEG C to be heated to spot development clear, in test sample chromatograph, on the position corresponding to reference substance and control medicinal material chromatograph, the speckle of aobvious same color;

b. the discriminating of Radix Pecteilis susannae:get nine tastes blue or green roc preparation 2g, add methanol 20ml, supersound process 10-40min, filter, filter evaporate to dryness, the residue 25ml that adds water makes dissolving, and with ethyl acetate extraction 1 time, each 25ml, merge ethyl acetate liquid, evaporate to dryness, residue adds methanol 1ml and dissolves, as need testing solution.Separately get Radix Pecteilis susannae control medicinal material 1g, add methanol 30ml, supersound process 30min, filter, filter evaporate to dryness, the residue 25ml that adds water makes dissolving, with ethyl acetate extraction 1 time, each 25ml, merge ethyl acetate liquid, evaporate to dryness, residue adds methanol 1ml and dissolves, medical material solution in contrast.Test according to thin layer chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ L of above-mentioned two kinds of solution, point is on same silica gel g thin-layer plate, with normal hexane-ethyl acetate (9:1) for developing solvent, launch, take out, dry, spray, with volume ratio 10% ethanol solution of sulfuric acid, is heated to spot development at 105 DEG C clear, inspects under putting ultra-violet lamp (365nm).In test sample chromatograph, on the position corresponding to control medicinal material chromatograph, the fluorescence speckle of aobvious same color.

the discriminating of Radix Inulae:get nine tastes blue or green roc preparation 2g, add boiling range 30-60 DEG C of petroleum ether 10ml, supersound process 10min, filter, filtrate is lower than evaporate to dryness under 60 DEG C of conditions, and residue adds ethyl acetate 1ml and dissolves, as need testing solution.Separately get Lignum Santali Albi control medicinal material 0.5g, add boiling range 30-60 DEG C of petroleum ether 30ml, supersound process 30min, filter, filtrate is lower than evaporate to dryness under 60 DEG C of conditions, and residue adds ethyl acetate 1ml and dissolves, medical material solution in contrast.According to thin layer chromatography (Chinese Pharmacopoeia version in 2010 annex VI B) test, draw each 10 μ L of above-mentioned two kinds of solution, put on same silica gel g thin-layer plate, with chloroform-Ethyl formate-methanol (5:4:0.4) for developing solvent, launch, take out, dry.Spray is with mass volume ratio 1% vanillin-sulfuric acid solution, and 105 DEG C to be heated to spot development clear.In test sample chromatograph, on the position corresponding to control medicinal material chromatograph, the fluorescence speckle of aobvious same color.

the discriminating of Rhizoma Rhodiolae kirilowii:get nine tastes blue or green roc preparation 3g, add n-butanol extraction 30ml, supersound process 20min, filter, n-butyl alcohol liquid evaporate to dryness, residue adds methanol 5ml and dissolves, be added in neutral alumina column (100-200 order, 2.5g, internal diameter is 1.0cm) on, with methanol 30ml eluting, collect meoh eluate, evaporate to dryness, residue adds methanol 5ml makes dissolving, as need testing solution; Separately get Rhizoma Rhodiolae kirilowii control medicinal material 1g, add n-butanol extraction 30ml, supersound process 20min, filter, n-butyl alcohol liquid evaporate to dryness, residue adds methanol 5ml and dissolves, be added in neutral alumina column (100-200 order, 2.5g, internal diameter is 1.0cm) on, with methanol 30ml eluting, collect meoh eluate, evaporate to dryness, residue adds methanol 5ml makes dissolving, medical material solution in contrast.Test according to thin layer chromatography (Chinese Pharmacopoeia version in 2010 annex VI B), draw each 10 μ L of above-mentioned two kinds of solution, put respectively on same silica GF254 lamellae, with chloroform-Ethyl formate-methanol (5:4:1.5) for developing solvent, launch, take out, dry, inspect under putting ultra-violet lamp (254nm), in test sample chromatograph, the speckle of aobvious same color on the position corresponding to reference substance chromatograph.

assay:

After the preparation nine taste blue or green roc preparation porphyrize of need testing solution, get powder 3g, accurately weighed, put in tool plug conical flask, precision adds methanol 40ml, close plug, weighed weight, supersound process 20min, let cool, weighed weight again, the weight of less loss is supplied with methanol, shake up, filter, get subsequent filtrate 25ml, be concentrated into dry, it is that 0.5% potassium hydroxide solution 20ml makes dissolving that residue adds volume parts ratio, by extracted with diethyl ether 2 times, each 20ml, discard ether solution, alkaline solution regulates pH4 with mass fraction hydrochloric acid again, by extracted with diethyl ether 4 times, each 20ml, merge ether solution, volatilize, be transferred in the brown volumetric flask of 5ml with dissolve with methanol, add methanol to scale, shake up, filter, get subsequent filtrate, obtain,

After the preparation nine taste blue or green roc preparation porphyrize of need testing solution, get powder 3g, accurately weighed, put in tool plug conical flask, precision adds methanol 40ml, close plug, weighed weight, supersound process 20min, lets cool, more weighed weight, supply the weight of less loss with methanol, shake up, filter, get subsequent filtrate, to obtain final product;

Table 1 gradient elution mobile phase

Time (min)	Mobile phase A (%)	Mobile phase B (%)
			0	15	85
60	35	65

Claims

1. treat a preparation method for flu pharmaceutical, comprise the following steps:

(1) get Radix Inulae by the blue or green rocs of nine tastes prescription proportioning of faling apart, add water 6-12 times of weight, adopts steam distillation, extracts volatile oil 1-6h, collects volatile oil, and medicinal liquid filters, and obtains extracting solution A ' and medicinal residues A;

(2) Radix aconiti szechenyiani seedling is got by the blue or green rocs of nine tastes prescription proportioning of faling apart, add water 6-14 times of weight, reflux, extract, 3-6h, be incorporated to the medicinal residues A after step (1) extraction volatile oil, the Fructus Chebulae of enucleation is added again by the blue or green rocs of nine tastes prescription proportioning of faling apart, Benzoinum, Herba pterocephali, Rhizoma Rhodiolae kirilowii, Radix Pecteilis susannae, Radix Solms-Laubachiae pulcherrimatis, Herba Oxytropis falcatae, the reflux, extract, that adds water 1-4 time, the amount at every turn added water is 8-12 times of described 9 taste medical material gross weights, each extraction 1-4h, filter, filtrate merges, extract the extracting solution A ' after volatile oil with step (1) to merge, concentrating under reduced pressure, be concentrated into the fluid extract C that relative density under 25 DEG C of conditions is 1.08-1.12, use Boiling Fuel Injection Spray one-step palletizing, obtain extract D,

(3) the extract D that volatile oil clathrate compound B step (1) obtained and step (2) obtain, mixing, obtain the blue or green roc extract of nine tastes, select customary adjuvant, preparation process makes granule, capsule, tablet, drop pill, micropill routinely.

2. a kind of preparation method for the treatment of flu pharmaceutical according to claim 1, comprises the following steps:

(1) get Radix Inulae by the blue or green rocs of nine tastes prescription proportioning of faling apart, add water 8 times of weight, adopts steam distillation, extracts volatile oil 4h, collects volatile oil, and medicinal liquid filters, and obtains extracting solution A ' and medicinal residues A;

(2) Radix aconiti szechenyiani seedling is got by the blue or green rocs of nine tastes prescription proportioning of faling apart, add water 10 times of weight, reflux, extract, 4h, be incorporated to the medicinal residues A after step (1) extraction volatile oil, the Fructus Chebulae of enucleation is added again by the blue or green rocs of nine tastes prescription proportioning of faling apart, Benzoinum, Herba pterocephali, Rhizoma Rhodiolae kirilowii, Radix Pecteilis susannae, Radix Solms-Laubachiae pulcherrimatis, Herba Oxytropis falcatae, the reflux, extract, that adds water 2 times, the amount that first time adds water is 12 times of described 9 taste medical material gross weights, extract 2h, filter, the amount that medicinal residues add water again is 10 times of described 9 taste medical material gross weights, extract 2h, filter, filtrate merges, extract the extracting solution A ' after volatile oil with step (1) to merge, concentrating under reduced pressure, be concentrated into the fluid extract C that relative density under 25 DEG C of conditions is 1.10, use Boiling Fuel Injection Spray one-step palletizing, obtain extract D,

(3) the extract D that volatile oil clathrate compound B step (1) obtained and step (2) obtain, mixing, obtain the blue or green roc extract of nine tastes, select customary adjuvant, preparation process makes tablet routinely.

3. a kind of preparation method for the treatment of flu pharmaceutical according to claim 1 or 2, and to differentiate as follows and/or one or more in assay method combine:

differentiate:

the discriminating of A, Fructus Chebulae:get nine tastes blue or green roc preparation 3-5g, add dehydrated alcohol 25-50ml, supersound process 20-30min, filter paper evenly adds kieselguhr 1.0g, filter, filtrate is concentrated into 2ml, as need testing solution; Separately get Fructus Chebulae's control medicinal material 1-2g, add dehydrated alcohol 25-50ml, supersound process 20-30min, filter paper evenly adds kieselguhr 1.0g, filter, filtrate is concentrated into 2ml, in contrast medical material solution; Separately get gallic acid reference substance, add methanol and make the solution of every 1ml containing 1mg, product solution in contrast; According to the annex VIB thin layer chromatography test of Chinese Pharmacopoeia version in 2010, draw above-mentioned solution each 5-10 μ l, put respectively on same silica gel g thin-layer plate, with volume parts ratio for the dichloromethane-methyl acetate-formic acid of 3-9:2-6:0.5-1.5 is for developing solvent, launch, take out, dry, spray is 1% ferric chloride alcoholic solution with quality volume portion rate, 105 DEG C to be heated to spot development clear, in test sample chromatograph, on the position corresponding to reference substance and control medicinal material chromatograph, the speckle of aobvious same color;

the discriminating of B, Radix Pecteilis susannae:get nine tastes blue or green roc preparation 2-5g, add methanol 20-50ml, supersound process 10-40min, filter, filter evaporate to dryness, the residue 25ml that adds water makes dissolving, with ethyl acetate extraction 1-3 time, each 25ml, merge ethyl acetate liquid, evaporate to dryness, residue adds methanol 1ml and dissolves, as need testing solution; Separately get Radix Pecteilis susannae control medicinal material 1g, add methanol 30ml, supersound process 30min, filter, filter evaporate to dryness, the residue 25ml that adds water makes dissolving, with ethyl acetate extraction 3 times, each 25ml, merge ethyl acetate liquid, evaporate to dryness, residue adds methanol 1ml and dissolves, medical material solution in contrast; According to the annex VI B thin layer chromatography test of Chinese Pharmacopoeia version in 2010, draw each 10 μ L of above-mentioned two kinds of solution, put on same silica gel g thin-layer plate, with the normal hexane-ethyl acetate of 9:1 for developing solvent, launch, take out, dry, spray is with volume ratio 10% ethanol solution of sulfuric acid, be heated to spot development at 105 DEG C clear, inspect under putting 365nm ultra-violet lamp, in test sample chromatograph, on the position corresponding to control medicinal material chromatograph, the fluorescence speckle of aobvious same color;

the discriminating of C, Rhizoma Rhodiolae kirilowii:get nine tastes blue or green roc preparation 3-6g, add n-butanol extraction 30-50ml, supersound process 20-40min, filter, n-butyl alcohol liquid evaporate to dryness, residue adds methanol 5ml and dissolves, be added in 100-200 order, 2.5g, internal diameter is on the neutral alumina column of 1.0cm, with methanol 30ml eluting, collect meoh eluate, evaporate to dryness, residue adds methanol 5ml makes dissolving, as need testing solution; Separately get Rhizoma Rhodiolae kirilowii control medicinal material 1g, add n-butanol extraction 30-50ml, supersound process 20-40min, filter, n-butyl alcohol liquid evaporate to dryness, residue adds methanol 5ml and dissolves, be added in 100-200 order, 2.5g, internal diameter is on the neutral alumina column of 1.0cm, with methanol 30ml eluting, collect meoh eluate, evaporate to dryness, residue adds methanol 5ml makes dissolving, medical material solution in contrast; According to the annex VI B thin layer chromatography test of Chinese Pharmacopoeia version in 2010, draw each 10 μ L of above-mentioned two kinds of solution, put respectively on same silica GF254 lamellae, with the chloroform-Ethyl formate-methanol of 5:4:1.5 for developing solvent, launch, take out, dry, inspect under putting 254nm ultra-violet lamp, in test sample chromatograph, the speckle of aobvious same color on the position corresponding to reference substance chromatograph;

assay:

a, benzoic assay:according to Chinese Pharmacopoeia version in 2010 annex VID high effective liquid chromatography for measuring

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filler; With methanol-0.8% glacial acetic acid of 35:65 for mobile phase; Determined wavelength is 270nm; Number of theoretical plate calculates should be not less than 3000 by cinnamic acid peak;

The preparation of reference substance solution gets cinnamic acid reference substance in right amount, accurately weighed, puts in brown volumetric flask, adds methanol and makes the solution of every 1ml containing 50 μ g, to obtain final product;

Algoscopy is accurate respectively draws reference substance solution and each 10 μ l of need testing solution, injection liquid chromatography, measures, to obtain final product;

the assay of B, Herba pterocephali:according to Chinese Pharmacopoeia version in 2010 annex VID high effective liquid chromatography for measuring

Chromatographic condition and system suitability take octadecylsilane chemically bonded silica as filler; With methanol-0.6% glacial acetic acid of 85:15 for mobile phase; Evaporative light scattering detector detects, drift tube temperature 85 DEG C, nitrogen pressure 48ps;

Oleanolic acid reference substance is got in the preparation of reference substance solution and ursolic acid reference substance is in right amount each, accurately weighed respectively, adds methanol and makes every 1ml respectively containing the solution of 0.5mg, to obtain final product;

c, ester alkaloid determination limit:according to Chinese Pharmacopoeia version in 2010 annex VI D high effective liquid chromatography for measuring

Chromatographic condition and system suitability are with the acetonitrile-oxolane of 5:3 for mobile phase A, and with 0.1mol/L Spirit of Mindererus. for Mobile phase B, the regulation according to the form below 1 carries out gradient elution, and determined wavelength is 235nm; Number of theoretical plate calculates should be not less than 2500 by mesaconitine peak;

Table 1 gradient elution mobile phase

Time (min) Mobile phase A (%) Mobile phase B (%) 0 15 85 60 35 65

Aconitine reference substance is got in the preparation of reference substance solution, hypaconitine reference substance, mesaconitine reference substance are appropriate, accurately weighed, the mixed solution adding hydrochloric acid and methanol volume ratio 1:100 makes the mixed solution of every 1ml containing aconitine, hypaconitine, each 50ug of mesaconitine, to obtain final product;

The preparation nine taste blue or green roc preparation porphyrize of need testing solution, gets powder 10g, accurately weighed, put in the conical flask of tool plug, precision adds the mixed solution 50ml of hydrochloric acid and methanol volume ratio 1:100, weighed weight, supersound process 60min, let cool, weighed weight, supply the weight of less loss with the mixed solution of hydrochloric acid and methanol volume ratio 1:100, shake up, filter, get subsequent filtrate, to obtain final product;