CN110585404B

CN110585404B - Detection method of traditional Chinese medicine composition

Info

Publication number: CN110585404B
Application number: CN201810606184.4A
Authority: CN
Inventors: 李友林
Original assignee: China Japan Friendship Hospital
Current assignee: China Japan Friendship Hospital
Priority date: 2018-06-13
Filing date: 2018-06-13
Publication date: 2021-06-25
Anticipated expiration: 2038-06-13
Also published as: CN110585404A

Abstract

The invention provides a detection method of a traditional Chinese medicine composition preparation, which is a high performance liquid chromatography and specifically comprises the following steps: measuring conditions, namely taking octadecylsilane bonds and silica gel as fillers; using acetonitrile-water solution (30-35): (65-70) is a mobile phase; preparing reference solution by dissolving astragaloside IV reference in organic solvent to obtain reference solution; preparing a test solution, namely extracting the traditional Chinese medicine composition preparation with an organic solvent to obtain the test solution; the determination method comprises respectively sucking the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

Description

Detection method of traditional Chinese medicine composition

Technical Field

The invention relates to a detection method of a traditional Chinese medicine composition, in particular to a detection method of a traditional Chinese medicine composition for relieving cough and asthma, and belongs to the field of medicines.

Background

Asthma frequently occurs at night and in the early morning, is easily induced when people catch cold and smell pungent smells (smoke, perfume, paint, dust, pets, pollen and the like), is an inflammatory reaction involving eosinophils, T lymphocytes, mast cells and the like, increases airway reactivity, further causes airway expiration limitation, finally causes symptoms such as cough, expectoration, asthma, breathlessness, wheezing in throat and the like, can be relieved after being treated by a tracheal expansion medicament, and belongs to the category of 'asthma' in the traditional Chinese medicine.

According to WHO estimates, patients with global asthma are as high as 2.75 million people. The incidence and the fatality rate of asthma are in an increasing trend in recent 10 years, and more than 18 thousands of people die of asthma every year in the world. Nearly 500 million american children have been diagnosed with asthma as reported by the american centers for disease prevention and control (CDC). Despite active research and prevention, the asthma-related mortality rate of children in the united states has increased by 78% during 1982-1993. The incidence of asthma is not only related to regional differences, but also has a great relationship with economic status, nutrition, customs and the like, and the incidence of asthma in developed countries is higher than that in underdeveloped countries.

According to epidemiological data of Shanghai, Beijing, Shenyang and Guangdong of the world asthma conference in 2000, the incidence rate of the adult asthma in China is 0.7% -1.5%, and that of children is 0.11% -2.03%, namely 1500-2000 ten thousand patients are in China, and the number of the patients is more than that of young people and children. At present, the study of scholars at home and abroad on asthma is gradually increased, but the detailed understanding of pathogenesis of asthma is still difficult, and no medicine with obvious curative effect is found in the aspect of treatment. Adrenocortical hormone is still the first choice drug for controlling the tracheitis and preventing the recurrent attacks of the diseases at present, but has larger adverse reactions. Therefore, the research on asthma treatment methods from the aspect of Chinese medicine, the improvement of clinical curative effect and the reduction of repeated attacks of diseases become the research directions of numerous Chinese medical researchers at home and abroad, and have important significance.

The Chinese medicine dialectical treatment progress of cough variant asthma (Liangqi et Jun) discloses a treatment method related to cough variant asthma in Chinese medicine, a plurality of classical and self-prepared formulas, wherein for the formula for treating dusk cough, the formula comprises Yupingfeng powder, FUGUIZHONG pill and JINKUISHENQI pill, and the formula comprises radix astragali, Atractylodis rhizoma, radix Codonopsis, radix rehmanniae Preparata, rhizoma Dioscoreae, radix Aconiti lateralis Preparata, Zingiberis rhizoma, Glycyrrhrizae radix, fructus Chebulae, fructus Schisandrae chinensis, etc.; modified Yupingfeng powder and SHASHEN Mai Tang are selected for midnight cough, and the materials are selected from radix astragali, radix Asteris, flos Farfarae, radix Adenophorae, radix Ophiopogonis, radix asparagi, rhizoma Polygonati Odorati, fructus Schisandrae chinensis, etc.; for liver qi stagnation, qi reversal, yin deficiency and blood dryness, it is indicated for Huang Qin, Hou Po, xing ren, Zhe Bei mu and chai Hu. CN103495130A discloses a Chinese medicinal composition for treating asthma, which comprises thirty-three kinds of herbs including pinellia tuber, bitter orange, ledebouriella root, root of ballon flower, schisandra fruit, sweet wormwood, bupleurum root, astragalus root, baikal skullcap root, aster, poria cocos, dried orange peel, root of herbaceous peony, magnolia bark, dried ginger, rhubarb horsetails, cassia twig, dahurian angelica root, etc., although the curative effect is obvious (the total effective rate is 94%), the formula is huge, and the cost is high.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a traditional Chinese medicine composition with refined formula and remarkable effect of relieving cough and asthma and a preparation method thereof.

As one aspect of the present invention, the present invention provides a cough and asthma relieving traditional Chinese medicine composition, which is prepared from the following raw material medicines: astragalus root, cassia twig, dried ginger, aster, magnolia bark, dark plum, schisandra fruit, myrobalan, anemarrhena rhizome and liquorice.

In a specific embodiment, the traditional Chinese medicine composition is prepared from the following raw material medicines: 7-42 parts of astragalus membranaceus, 4-25 parts of cassia twig, 4-25 parts of dried ginger, 5-30 parts of radix asteris, 4-25 parts of mangnolia officinalis, 6-34 parts of dark plum, 4-25 parts of schisandra chinensis, 2-15 parts of myrobalan, 2-15 parts of rhizoma anemarrhenae and 2-15 parts of liquorice.

In a preferred embodiment, the traditional Chinese medicine composition is prepared from the following raw material medicines: 10-30 parts of astragalus membranaceus, 5-18 parts of cassia twig, 5-18 parts of dried ginger, 7-22 parts of radix asteris, 5-18 parts of mangnolia officinalis, 8-25 parts of dark plum, 5-18 parts of schisandra chinensis, 3-12 parts of myrobalan, 3-12 parts of rhizoma anemarrhenae and 3-12 parts of liquorice.

In a further preferred embodiment, the traditional Chinese medicine composition is prepared from the following raw material medicines: 15-22 parts of astragalus membranaceus, 6-12 parts of cassia twig, 6-12 parts of dried ginger, 9-15 parts of radix asteris, 6-12 parts of mangnolia officinalis, 12-18 parts of dark plum, 6-12 parts of schisandra chinensis, 4-9 parts of myrobalan, 4-9 parts of rhizoma anemarrhenae and 4-9 parts of liquorice.

In the most preferred embodiment, the Chinese medicinal composition is prepared from the following raw material medicaments: 18 parts of astragalus, 9 parts of cassia twig, 9 parts of dried ginger, 12 parts of aster, 9 parts of mangnolia officinalis, 15 parts of dark plum, 9 parts of schisandra chinensis, 6 parts of myrobalan, 6 parts of rhizoma anemarrhenae and 6 parts of liquorice;

or, the traditional Chinese medicine composition is prepared from the following raw material medicines: 14 parts of astragalus, 11 parts of cassia twig, 7 parts of dried ginger, 14 parts of aster, 7 parts of mangnolia officinalis, 17 parts of dark plum, 7 parts of schisandra chinensis, 8 parts of myrobalan, 5 parts of rhizoma anemarrhenae and 8 parts of liquorice;

or, the traditional Chinese medicine composition is prepared from the following raw material medicines: 21 parts of astragalus membranaceus, 7 parts of cassia twig, 11 parts of dried ginger, 10 parts of radix asteris, 11 parts of mangnolia officinalis, 13 parts of dark plum fruit, 11 parts of schisandra chinensis, 5 parts of myrobalan, 8 parts of rhizoma anemarrhenae and 5 parts of liquorice.

In a preferred embodiment, the astragalus is raw astragalus; the licorice is raw licorice.

The traditional Chinese medicine composition can be a composition formed by crushing and mixing raw material medicines, can also be an extract obtained by mixing or extracting the raw material medicines independently, or an effective part obtained by further refining and purifying the extract, and can also be a conventional preparation form prepared by adding pharmaceutically acceptable auxiliary materials.

Wherein the extraction method comprises decocting extraction, reflux extraction, immersion extraction, ultrasonic extraction, percolation extraction, microwave extraction, etc.; the purification method comprises water extraction and alcohol precipitation, alkali dissolution and acid precipitation and various column chromatography purification methods, such as a macroporous resin column, a silica gel column, a reverse phase column and the like; the conventional dosage forms include but are not limited to injections, capsules, tablets, granules, gels, sustained-release agents, oral liquids, dropping pills or nano preparations; the pharmaceutically acceptable auxiliary materials comprise: fillers, disintegrants, lubricants, suspending agents, binders, sweeteners, flavoring agents, preservatives, bases, and the like. The filler comprises: starch, pregelatinized starch, lactose, mannitol, chitin, microcrystalline cellulose, sucrose, etc.; the disintegrating agent comprises: starch, pregelatinized starch, microcrystalline cellulose, sodium carboxymethyl starch, crospolyvinylpyrrolidone, low-substituted hydroxypropylcellulose, croscarmellose sodium, etc.; the lubricant comprises: magnesium stearate, sodium lauryl sulfate, talc, silica, and the like; the suspending agent comprises: polyvinylpyrrolidone, microcrystalline cellulose, sucrose, agar, hydroxypropyl methylcellulose, and the like; the binder includes starch slurry, polyvinylpyrrolidone, hydroxypropyl methylcellulose, etc.

The traditional Chinese medicine composition can be fed in the form of an extract besides being fed in the form of raw medicines, so that the invention further discloses a traditional Chinese medicine composition which is prepared from the following raw materials: 7-42 parts of astragalus extract, 4-25 parts of cassia twig extract, 4-25 parts of dried ginger extract, 5-30 parts of aster extract, 4-25 parts of mangnolia officinalis extract, 6-34 parts of dark plum extract, 4-25 parts of schisandra extract, 2-15 parts of myrobalan extract, 2-15 parts of rhizoma anemarrhenae extract and 2-15 parts of liquorice extract.

In a preferred embodiment, the Chinese medicinal composition is prepared from the following raw materials: 10-30 parts of astragalus extract, 5-18 parts of cassia twig extract, 5-18 parts of dried ginger extract, 7-22 parts of aster extract, 5-18 parts of mangnolia officinalis extract, 8-25 parts of dark plum extract, 5-18 parts of schisandra extract, 3-12 parts of myrobalan extract, 3-12 parts of rhizoma anemarrhenae extract and 3-12 parts of liquorice extract.

In a further preferred embodiment, the Chinese medicinal composition is prepared from the following raw materials: 15-22 parts of astragalus extract, 6-12 parts of cassia twig extract, 6-12 parts of dried ginger extract, 9-15 parts of aster extract, 6-12 parts of mangnolia officinalis extract, 12-18 parts of dark plum extract, 6-12 parts of schisandra extract, 4-9 parts of myrobalan extract, 4-9 parts of rhizoma anemarrhenae extract and 4-9 parts of liquorice extract.

In the most preferred embodiment, the Chinese medicinal composition is prepared from the following raw materials: 18 parts of astragalus extract, 9 parts of cassia twig extract, 9 parts of dried ginger extract, 12 parts of aster extract, 9 parts of mangnolia officinalis extract, 15 parts of dark plum extract, 9 parts of schisandra extract, 6 parts of myrobalan extract, 6 parts of rhizoma anemarrhenae extract and 6 parts of liquorice extract;

or, the traditional Chinese medicine composition is prepared from the following raw material medicines: 14 parts of astragalus extract, 11 parts of cassia twig extract, 7 parts of dried ginger extract, 14 parts of aster extract, 7 parts of mangnolia officinalis extract, 17 parts of dark plum extract, 7 parts of schisandra extract, 8 parts of myrobalan extract, 5 parts of rhizoma anemarrhenae extract and 8 parts of liquorice extract;

or, the traditional Chinese medicine composition is prepared from the following raw material medicines: 21 parts of astragalus extract, 7 parts of cassia twig extract, 11 parts of dried ginger extract, 10 parts of aster extract, 11 parts of mangnolia officinalis extract, 13 parts of dark plum extract, 11 parts of schisandra extract, 5 parts of myrobalan extract, 8 parts of rhizoma anemarrhenae extract and 5 parts of liquorice extract.

The extracts are respectively aqueous extracts or organic solvent extracts of the raw material medicines, and the extraction method comprises conventional decoction extraction, reflux extraction, ultrasonic extraction, percolation extraction and the like.

As a second aspect of the invention, the invention provides application of the traditional Chinese medicine composition in preparing a medicine for relieving cough and asthma.

As a third aspect of the present invention, the present invention provides a preparation method of the traditional Chinese medicine composition, which comprises the following raw material medicines: 7-42 parts of astragalus membranaceus, 4-25 parts of cassia twig, 4-25 parts of dried ginger, 5-30 parts of radix asteris, 4-25 parts of mangnolia officinalis, 6-34 parts of dark plum, 4-25 parts of schisandra chinensis, 2-15 parts of myrobalan, 2-15 parts of rhizoma anemarrhenae and 2-15 parts of liquorice; the raw materials are directly crushed and then mixed, or extracted by a conventional extraction method independently or together. Wherein the extraction method comprises decoction extraction, reflux extraction, immersion extraction, ultrasonic extraction, percolation extraction, microwave extraction and the like.

In a specific embodiment, the preparation method comprises the following steps:

step a, extracting volatile oil from cassia twig and dried ginger to obtain volatile oil and water extract;

extracting the eight ingredients of astragalus, aster, magnolia officinalis, dark plum, schisandra, myrobalan, rhizoma anemarrhenae and liquorice singly or together by adopting a conventional solvent;

and c, combining the extracting solutions obtained in the steps a and b, and adding the volatile oil obtained in the step a to obtain the traditional Chinese medicine composition.

Wherein, the steam distillation method is preferably adopted to extract the volatile oil in the step a; the volatile oil is preferably coated with BETA-cyclodextrin; the ratio of the volatile oil to the betacyclodextrin is 1: (5-10); preferably 1: (6-9); most preferably 1: 8; the inclusion temperature is 30-50 ℃, and the inclusion time is 1-3 hours;

the conventional solvent in the step b comprises water, methanol and ethanol; preferably by water extraction; the extraction time is 1-2 hours, and the extraction times are 1-3 times;

and c, combining the extracting solutions, and then concentrating, precipitating with ethanol and drying, namely concentrating the extracting solution to obtain clear paste with the relative density of 1.10-1.15 (60 ℃), adding ethanol until the ethanol content is 50% -70%, recovering ethanol, concentrating the ethanol solution to obtain thick paste, and drying at the temperature below 70 ℃.

The traditional Chinese medicine composition can be further added with conventional auxiliary materials to prepare oral formulations, preferably granules. Therefore, the invention further provides a preparation method of the traditional Chinese medicine composition granules, which comprises the following steps: taking the traditional Chinese medicine composition, and mixing with an excipient according to the ratio of (1-2): 1, granulating by a wet method, and drying to obtain granules.

In a specific embodiment, the excipient is selected from any one or more of dextrin, lactose and soluble starch, and is preferably dextrin; the wetting agent is 85-95% ethanol solution, preferably 90% ethanol solution. The ratio of the composition extract powder to the excipient is preferably 1.5: 1.

In a specific embodiment, the ambient relative humidity during the granulation process is below 72%.

As a fourth aspect of the present invention, the present invention provides a detection method of the Chinese medicinal composition preparation, wherein the method specifically comprises the following steps:

measuring conditions, namely taking octadecylsilane bonds and silica gel as fillers; using acetonitrile-water solution (30-35): (65-70) is a mobile phase;

preparing reference solution by dissolving astragaloside IV reference in organic solvent to obtain reference solution;

preparing a test solution, namely extracting the traditional Chinese medicine composition preparation with an organic solvent to obtain the test solution;

the determination method comprises respectively sucking the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

In a specific embodiment, the organic solvent is selected from any one of methanol, acetone, n-butanol, ethyl acetate, petroleum ether, and chloroform; the extraction method is selected from ultrasonic extraction, reflux extraction, cold leaching extraction, percolation extraction, microwave extraction and other methods.

In a preferred embodiment, the sample solution is prepared by ultrasonic extraction with methanol; further preferably, the method for preparing the test solution comprises: extracting the Chinese medicinal composition preparation with methanol under ultrasound, recovering solvent from the extractive solution, concentrating to dry, dissolving the residue in water, extracting with water saturated n-butanol under shaking, washing the n-butanol extractive solution with ammonia test solution, discarding the ammonia test solution, evaporating the washed n-butanol solution, and dissolving the residue with methanol.

In a preferred embodiment, the mobile phase is an acetonitrile-water solution (32: 68).

The detection method of the invention also comprises the following identification of any one or more of A-D:

identification A

Preparation of a test solution: extracting the Chinese medicinal composition preparation with organic solvent to obtain test solution;

preparing a reference medicinal material solution: extracting radix astragali reference material with organic solvent to obtain reference material solution;

and (3) detection: respectively sucking a test solution and a reference medicinal material solution, dropping the test solution and the reference medicinal material solution on the same silica gel plate, and mixing the solutions with chloroform-methanol-water (10-15): (5-9): (1-3) developing with a developing agent, developing with a 10% sulfuric acid ethanol solution, heating at 105 ℃ until the spots are clearly developed, and inspecting in sunlight;

identification B

preparation of control solutions: dissolving shionone control in organic solvent to obtain control solution;

and (3) detection: respectively sucking a test solution and a reference solution, dropping the test solution and the reference solution on the same silica gel plate, and adding petroleum ether-ethyl acetate (5-10): (0.5-2) developing with 10% sulfuric acid ethanol solution, heating at 105 deg.C until the color of spots is clear, and inspecting under sunlight and ultraviolet lamp (365 nm);

identification C

Preparing a test solution: extracting the Chinese medicinal composition preparation with organic solvent to obtain test solution;

preparing a reference medicinal material solution: extracting fructus Schisandrae chinensis control material with organic solvent to obtain control solution;

preparation of a reference solution: dissolving schisandrin A reference substance in organic solvent to obtain reference substance solution;

and (3) detection: respectively sucking a test solution, a reference medicinal material solution and a reference medicinal material solution, dropping the solutions on the same silica gel plate, and mixing the solutions with petroleum ether-ethyl formate-formic acid (12-18): (4-8): (0.5-2) developing with developing agent, and inspecting under an ultraviolet lamp (254 nm);

authentication D

preparing a reference medicinal material solution: extracting mume fructus reference material with organic solvent to obtain reference material solution;

preparation of a reference solution: dissolving ursolic acid reference substance in organic solvent to obtain reference substance solution;

and (3) detection: respectively sucking a test solution, a reference medicinal material solution and a reference medicinal material solution, dropping the solutions on the same silica gel plate, and mixing the solutions with cyclohexane-trichloromethane-ethyl acetate-formic acid (18-22): (4-6): (6-10): (0.05-0.1) as developing agent, developing with 10% sulfuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed.

In a preferred embodiment, the identification a test solution is prepared by the following steps: extracting the Chinese medicinal composition preparation with methanol under ultrasound, recovering solvent from filtrate, concentrating to dry, dissolving residue in water, extracting with water saturated n-butanol under shaking, washing n-butanol extractive solution with ammonia test solution, discarding ammonia test solution, evaporating washed n-butanol solution, and dissolving residue with methanol.

In a preferred embodiment, the identification D test solution is prepared by: taking the Chinese medicinal composition preparation, adding methanol, performing ultrasonic extraction, evaporating the extracting solution to dryness, dissolving in water, extracting with diethyl ether, evaporating the diethyl ether solution to dryness, soaking with petroleum ether, removing the petroleum ether solution, and dissolving the residue with anhydrous ethanol to obtain the final product.

The raw materials are collected in the 'Chinese pharmacopoeia' 2015 edition, and the processing method is the 'pharmacopoeia' recording method.

In the formula of the invention, the astragalus root is sweet and warm, enters lung and spleen channels, invigorates qi and strengthens exterior, and strengthens spleen and lung; cassia twig, pungent, sweet and warm, enters heart, lung and bladder meridians, warms and unblocks meridians and collaterals, and helps yang and regulate qi; the two herbs are indicated for warming yang, invigorating spleen and tonifying lung, and are used as monarch herbs. Aster, bitter and warm, entering lung meridian, warming lung, descending qi, dissolving phlegm and relieving cough; magnolia bark, cortex magnoliae officinalis, bitter and pungent, warm, enters spleen, stomach, lung and large intestine channels, eliminates dampness and phlegm, purges qi and removes fullness, and is good at removing dampness and stagnancy to injure the middle-jiao, phlegm and fluid retention and dyspnea and cough; dried ginger, pungent and hot in flavor, enters spleen, stomach, kidney, heart and lung channels, warms middle-jiao to dispel cold, returns yang to activate pulse, eliminates dampness and eliminates phlegm; cortex Magnoliae officinalis and adjuvant materials, radix Asteris, cortex Magnoliae officinalis and adjuvants, can be used for improving lung function; the three medicines help the monarch medicine to earth up, benefit lung and reduce adverse qi of lung to be used as ministerial medicine. Dark plum, sour, astringent and neutral, enters liver, spleen, lung and large intestine channels, astringes lung and produces body fluid, and has special effects of lung deficiency, chronic cough, deficiency heat and thirst quenching; fructus Schisandrae chinensis, sour, sweet, warm, enter lung, heart, kidney channel, astringe, invigorate qi, promote fluid production, tonify kidney, tranquilize heart, it is good for chronic cough, asthma of deficiency type, body fluid impairment thirst, shortness of breath and pulse, internal heat, diabetes, palpitation insomnia; myrobalan, bitter, sour, astringent and flat, enters lung and large intestine channels, astringes lung, lowers fire and relieves sore throat, and is used for treating lung deficiency, cough, chronic cough, pharyngalgia and hoarseness; the three herbs can astringe lung to relieve cough, and prevent acridity and warmth; rhizoma anemarrhenae, bitter, sweet and cold, enters lung, stomach and kidney channels, clears heat and purges fire, promotes fluid production and moistens dryness, and prevents the generation of stagnated heat due to deficiency of both lung and spleen; the four medicines are used as adjuvant medicines for astringing lung, relieving cough and asthma, and dispelling stagnation of heat. The liquorice is sweet and neutral, and has the functions of tonifying heart, lung, spleen and stomach channels, invigorating spleen and replenishing qi, clearing away heat and toxic materials, eliminating phlegm and stopping cough, and harmonizing the drugs of the specification, and is used as a guiding drug. The whole formula combines the principle of 'warm-moistening, pungent-golden and banking up root' and combines the functions of warming yang and strengthening spleen, relieving cough and relieving asthma by dispelling cold and warming and resolving yang, and treating diseases and seeking root causes.

Pharmacodynamic researches show that the traditional Chinese medicine composition can relieve cough symptoms, reduce airway resistance and relieve pathological damage of lung tissues, and the action mechanism of the traditional Chinese medicine composition is probably related to adjusting imbalance of Th1/Th2/Th17 cytokines and inhibiting NF-kB signal pathways and 5-LOX/LTs/CysLTR signal pathways.

Clinical researches show that the total clinical effective rate of the traditional Chinese medicine composition for treating cough and asthma is 96.7%, the control rate is 83.3%, and the total clinical effective rate is remarkably higher than 66.7% and 41.7% of that of a control group (Shuflumetom and Shulii). In addition, compared with the traditional Chinese medicine composition for treating cough and asthma disclosed by the prior art (CN103495130A), the traditional Chinese medicine composition disclosed by the invention has a simple formula and a more remarkable effect (the total effective rate of treating cough and asthma of the traditional Chinese medicine composition is 96.7%, the control rate is 83.3%, and the total effective rate of the traditional Chinese medicine composition in the prior art is 94%, and the control rate is 68%).

Detailed Description

Example 1 Chinese medicinal composition decoction

The formula is as follows: 18g of astragalus, 9g of cassia twig, 9g of dried ginger, 12g of radix asteris, 9g of magnolia officinalis, 15g of dark plum fruit, 9g of schisandra chinensis, 6g of myrobalan, 6g of rhizoma anemarrhenae and 6g of liquorice;

the preparation method comprises the following steps: the raw materials are taken according to the proportion and decocted and extracted by water to obtain the traditional Chinese medicine composition decoction.

Example 2 Chinese medicinal composition decoction

The formula is as follows: 14g of astragalus, 11g of cassia twig, 7g of dried ginger, 14g of radix asteris, 7g of mangnolia officinalis, 17g of dark plum, 7g of schisandra chinensis, 8g of myrobalan, 5g of rhizoma anemarrhenae and 8g of liquorice;

Example 3 Chinese medicinal composition decoction

The formula is as follows: 21g of astragalus, 7g of cassia twig, 11g of dried ginger, 10g of aster, 11g of magnolia officinalis, 13g of dark plum, 11g of schisandra chinensis, 5g of myrobalan, 8g of rhizoma anemarrhenae and 5g of liquorice;

Example 4 Chinese medicinal composition granules

the preparation method comprises the following steps:

adding 8 times of water into ramulus Cinnamomi and Zingiberis rhizoma (wherein Zingiberis rhizoma is crushed into small pieces of about 0.7cm 3), extracting volatile oil for 11h, collecting volatile oil, clathrating with BETA-cyclodextrin at 50 deg.C for 2h, wherein the ratio of oil (mL) to BETA-cyclodextrin (g) is 1:8, filtering and drying to obtain an inclusion compound for later use; filtering the water solution obtained by extracting the volatile oil for later use;

adding 10 times of water into the other eight medicines such as astragalus, rhizoma anemarrhenae and the like, decocting for three times, each time for 1.5 hours, combining decoction liquids, filtering, combining with the water liquid obtained by extracting the volatile oil, concentrating to obtain clear paste with the relative density of 1.10-1.15 (60 ℃), adding ethanol to ensure that the alcohol content reaches 60%, stirring, standing overnight, filtering, recovering ethanol and concentrating into thick paste, drying under reduced pressure below 70 ℃, crushing into fine powder, and uniformly mixing with the beta-cyclodextrin inclusion compound to obtain extract powder;

mixing the composition extract powder and dextrin at a ratio of 1.5:1, wet granulating, taking 90% ethanol as wetting agent, controlling relative humidity of environment below 72%, granulating, drying, and packaging to obtain the final product granule.

The research of the invention finds that the relative humidity of the environment is strictly controlled to be below 72% in the granulating process, and if the relative humidity is higher than 72%, the materials absorb moisture and the granulating is difficult.

Example 5 Chinese medicinal composition granule

the preparation method is the same as example 4.

Example 6 Chinese medicinal composition granule

the preparation method is the same as example 4.

Example 7 Chinese medicinal composition granule

the preparation method comprises the following steps:

adding 6 times of water into ramulus Cinnamomi and Zingiberis rhizoma (wherein Zingiberis rhizoma is crushed into small pieces of about 0.7cm 3), extracting volatile oil for 18h, collecting volatile oil, clathrating with BETA-cyclodextrin at 30 deg.C for 1h, wherein the ratio of oil (mL) to BETA-cyclodextrin (g) is 1: 6, filtering and drying to obtain an inclusion compound for later use; filtering the water solution obtained by extracting the volatile oil for later use;

adding 8 times of water into the other eight medicines such as astragalus, rhizoma anemarrhenae and the like, decocting for three times, each time for 1 hour, combining decoction, filtering, combining with the water solution obtained by extracting the volatile oil, concentrating to obtain clear paste with the relative density of 1.10-1.15 (60 ℃), adding ethanol to ensure that the alcohol content reaches 70%, stirring, standing overnight, filtering, recovering ethanol and concentrating into thick paste, drying under reduced pressure below 70 ℃, crushing into fine powder, and uniformly mixing with the beta-cyclodextrin inclusion compound to obtain extract powder;

mixing the composition extract powder and soluble starch at a ratio of 1:1, wet granulating, taking 95% ethanol as wetting agent, controlling relative humidity of environment below 72%, granulating, drying, and packaging to obtain the final product granule.

Example 8 Chinese medicinal composition granule

The formula is as follows: 20g of astragalus, 8g of cassia twig, 10g of dried ginger, 11g of aster, 10g of magnolia officinalis, 14g of dark plum, 10g of schisandra chinensis, 4g of myrobalan, 7g of rhizoma anemarrhenae and 4g of liquorice;

the preparation method is the same as example 7.

Example 9 Chinese medicinal composition granule

The formula is as follows: 11g of astragalus, 17g of cassia twig, 6g of dried ginger, 20g of aster, 6g of magnolia officinalis, 22g of dark plum, 6g of schisandra chinensis, 11g of myrobalan, 3g of rhizoma anemarrhenae and 11g of liquorice;

the preparation method is the same as example 7.

Example 10 Chinese medicinal composition granule

the preparation method comprises the following steps:

adding 12 times of water into ramulus Cinnamomi and Zingiberis rhizoma (wherein Zingiberis rhizoma is crushed into small pieces of about 0.7cm 3), extracting volatile oil for 14h, collecting volatile oil, clathrating with BETA-cyclodextrin at 40 deg.C for 3h, wherein the ratio of oil (mL) to BETA-cyclodextrin (g) is 1: 10, filtering and drying to obtain an inclusion compound for later use; filtering the water solution obtained by extracting the volatile oil for later use;

adding 12 times of water into the other eight medicines such as astragalus, rhizoma anemarrhenae and the like, decocting for three times, each time for 2 hours, combining decoction, filtering, combining with the water liquid obtained by extracting the volatile oil, concentrating to obtain clear paste with the relative density of 1.10-1.15 (60 ℃), adding ethanol to ensure that the alcohol content reaches 50%, stirring, standing overnight, filtering, recovering ethanol and concentrating into thick paste, drying under reduced pressure below 70 ℃, crushing into fine powder, and uniformly mixing with the beta-cyclodextrin inclusion compound to obtain extract powder;

mixing the composition extract powder and lactose at a ratio of 1.5:1, wet granulating, taking 85% ethanol as wetting agent, controlling relative humidity below 72%, granulating, drying, and packaging to obtain the final product.

EXAMPLE 11 Chinese medicinal composition granule

The formula is as follows: 16g of astragalus, 10g of cassia twig, 8g of dried ginger, 13g of radix asteris, 8g of mangnolia officinalis, 16g of dark plum, 8g of schisandra chinensis, 7g of myrobalan, 4g of rhizoma anemarrhenae and 9g of liquorice;

the preparation method is the same as that of example 10.

EXAMPLE 12 Chinese medicinal composition granule

The formula is as follows: 28g of astragalus, 6g of cassia twig, 16g of dried ginger, 8g of aster, 16g of magnolia officinalis, 10g of dark plum, 16g of schisandra chinensis, 3g of myrobalan, 11g of rhizoma anemarrhenae and 3g of liquorice;

the preparation method is the same as that of example 10.

Example 13 Chinese medicinal composition decoction

The formula is as follows: 12g of astragalus, 16g of cassia twig, 7g of dried ginger, 18g of radix asteris, 5g of magnolia officinalis, 20g of dark plum, 5g of schisandra chinensis, 10g of myrobalan, 4g of rhizoma anemarrhenae and 10g of liquorice;

Example 14 Chinese medicinal composition decoction

The formula is as follows: 26g of astragalus, 5g of cassia twig, 14g of dried ginger, 7g of aster, 15g of magnolia officinalis, 11g of dark plum, 14g of schisandra chinensis, 4g of myrobalan, 10g of rhizoma anemarrhenae and 4g of liquorice;

Example 15 Chinese medicinal composition decoction

The formula is as follows: 8g of astragalus, 22g of cassia twig, 5g of dried ginger, 28g of aster, 5g of magnolia officinalis, 32g of dark plum, 4g of schisandra chinensis, 14g of myrobalan, 3g of rhizoma anemarrhenae and 14g of liquorice;

EXAMPLE 16 Chinese medicinal composition tablet

The formula is as follows: 38g of astragalus, 5g of cassia twig, 23g of dried ginger, 6g of aster, 24g of magnolia officinalis, 7g of dark plum, 23g of schisandra chinensis, 2g of myrobalan, 14g of rhizoma anemarrhenae and 2g of liquorice;

the preparation method comprises the following steps:

adding 10 times of water into ramulus Cinnamomi and Zingiberis rhizoma (wherein Zingiberis rhizoma is crushed into small pieces of about 0.7cm 3), extracting volatile oil for 11h, collecting volatile oil, clathrating with BETA-cyclodextrin at 50 deg.C for 2h, wherein the ratio of oil (mL) to BETA-cyclodextrin (g) is 1:8, filtering and drying to obtain an inclusion compound for later use; filtering the water solution obtained by extracting the volatile oil for later use;

adding the composition extract powder into conventional tablet adjuvants, and making into tablet.

EXAMPLE 17 Chinese medicinal composition tablet

The formula is as follows: 9g of astragalus, 20g of cassia twig, 4g of dried ginger, 25g of aster, 4g of magnolia officinalis, 29g of dark plum, 6g of schisandra chinensis, 13g of myrobalan, 2g of rhizoma anemarrhenae and 13g of liquorice;

the preparation method is the same as example 16.

EXAMPLE 18 Chinese medicinal composition tablet

The formula is as follows: 34g of astragalus, 4g of cassia twig, 20g of dried ginger, 5g of aster, 22g of magnolia officinalis, 6g of dark plum, 21g of schisandra chinensis, 3g of myrobalan, 13g of rhizoma anemarrhenae and 2g of liquorice;

the preparation method is the same as example 16.

Example 19

The formula is as follows: 18g of astragalus extract, 9g of cassia twig extract, 9g of dried ginger extract, 12g of aster extract, 9g of mangnolia officinalis extract, 15g of dark plum extract, 9g of schisandra extract, 6g of myrobalan extract, 6g of rhizoma anemarrhenae extract and 6g of liquorice extract.

Example 20

The formula is as follows: 14g of astragalus extract, 11g of cassia twig extract, 7g of dried ginger extract, 14g of aster extract, 7g of mangnolia officinalis extract, 17g of dark plum extract, 7g of schisandra extract, 8g of myrobalan extract, 5g of rhizoma anemarrhenae extract and 8g of liquorice extract.

Example 21

The formula is as follows: 21g of astragalus extract, 7g of cassia twig extract, 11g of dried ginger extract, 10g of aster extract, 11g of mangnolia officinalis extract, 13g of dark plum extract, 11g of schisandra extract, 5g of myrobalan extract, 8g of rhizoma anemarrhenae extract and 5g of liquorice extract.

Example 22

The formula is as follows: 20g of astragalus extract, 8g of cassia twig extract, 10g of dried ginger extract, 11g of aster extract, 10g of mangnolia officinalis extract, 14g of dark plum extract, 10g of schisandra extract, 4g of myrobalan extract, 7g of rhizoma anemarrhenae extract and 4g of liquorice extract.

Example 23

The formula is as follows: 11g of astragalus extract, 17g of cassia twig extract, 6g of dried ginger extract, 20g of aster extract, 6g of mangnolia officinalis extract, 22g of dark plum extract, 6g of schisandra extract, 11g of myrobalan extract, 3g of rhizoma anemarrhenae extract and 11g of liquorice extract.

The extract of cinnamomi ramulus and the extract of zingiber officinale roscoe described in examples 19 to 23 above are volatile oil extracts of cinnamomi ramulus and zingiber officinale roscoe, respectively; the astragalus extract, the aster extract, the magnolia bark extract, the dark plum extract, the schisandra extract, the myrobalan extract, the anemarrhena rhizome extract and the liquorice extract are respectively prepared by water extraction and alcohol precipitation processes of astragalus, aster, magnolia bark, dark plum, schisandra chinensis, myrobalan, anemarrhena rhizome and liquorice, and the alcohol precipitation concentration is 60%.

EXAMPLE 24 clinical Effect test

1 source of cases

All 120 cases were from outpatients and inpatients during the period of 2009-2012 3 months in the department of lung disease, department of respiration and department of internal medicine of traditional Chinese medicine, respectively, in the friendly hospital of the middle-aged.

2 case selection

The case diagnosis was included as criteria:

(1) no obvious inducement for continuous cough or repeated attack for more than 1 month; (2) the aggravation of the disease can be induced by sports, cold air, upper respiratory tract infection and the like; (3) histamine or acetylcholine challenge test positive; (4) antibiotics and antitussives are both ineffective, and treatment with bronchial spasmolytic or corticosteroid drugs is effective; (5) personal allergy history or family allergy history, and allergen positive; (6) physical examination shows no positive signs, chest radiograph is normal, lung ventilation function is normal, no abnormality is found in examination of the department of five sense organs, and the patients have no history of esophageal reflux and chronic bronchitis and are not treated by steroid hormone 1 month before the test.

3 grouping method

The selected patients were divided into 60 cases of treatment group and 60 cases of control group according to the principle of random grouping.

General data of 4 cases

Of all 120 CVA patients, 85 were outpatient and 35 were inpatient. 53 men and 67 women; the age is 18 years minimum, 70 years maximum, and 45.26 +/-15.28 years average; the disease course of cough is 1 month at the shortest, 6 years at the longest, and the average disease course is 3.56 +/-1.02 months.

5 methods of treatment

5.1 therapeutic Agents

The granules prepared in example 4 are taken in a treatment group;

the control group was treated with a combination of suflunomide plus sulpiride (salmeterol ticasone at 50ug/250 ug).

5.2 application method and treatment course

The granules prepared in example 4 were orally administered to the treatment groups 2 times a day in the morning and evening. The control group was given 200mg of suflunomide twice daily, sulpiride, 1 inhalation each time, twice daily. 3 weeks is 1 course of treatment, and 1 course of treatment is observed.

5.3 Observation indicators and methods

5.3.1 cough symptom Scale:

mild (+): intermittent cough, no effect on normal life. Moderate (++) -is between mild and severe cough. Severe (+++) -frequent or paroxysmal coughing during the day and night, affecting work and sleep.

5.3.2 airway reactivity assay (measurement of air responsiveness), also known as bronchial challenge test (Broeal promotion test BPT):

the respiratory resistance (Rrs) is well correlated with the airway resistance (Raw) measured by a plethysmograph, and has indexes for evaluating airway responsiveness by showing airway sensitivity and airway responsiveness. We used an inhaled Methacholine (MCH) challenge test with increasing concentrations, with a gas-channel reactivity assay for both groups of patients before treatment, 4 weeks after treatment, respectively. The indexes mainly comprise a minimum response threshold (Dmin) and a conductivity decline slope (SGrs), wherein the Dmin can reflect airway sensitivity, and the SGrs reflects airway reactivity.

5.4 efficacy assessment method

5.4.1 therapeutic Effect determination method

The cough graduation standard is mild (+): intermittent cough does not influence normal life and work; moderate (++) -between mild and severe cough; severe (+++) -frequent or paroxysmal coughing during the day and night, affecting work and sleep.

5.4.2 therapeutic efficacy assessment criteria (self-simulation)

And (3) clinical control: cough symptoms were significantly reduced or eliminated, and airway reactivity was measured negative. The effect is shown: cough symptoms turned from (+++) to (+) or from (++) to (-); airway reactivity assay Dmin ↓, sGrs ↓. Effective cough symptoms were either shifted from (+++) to (++) or from (++) to (+), and airway reactivity was measured Dmin ≠ sGrs ↓. And (4) invalidation: after the medicine is taken, cough symptoms are not improved or converted into asthma, and Dmin and sGrs values measured by airway reactivity are not changed.

5.5 therapeutic results

5.5.1 reduction of cough symptoms, resolution and night time of rest comparison

The cough relieving, the cough eliminating and the night stopping time of the treatment group are obviously shorter than those of the control group, and the treatment group is obviously better than that of the control group through statistical treatment with significant difference (P < 0.01). See table 1.

TABLE 1 two groups of cough relieving medicineComparison of changes

Group of	N	Time to cough relief	Time to resolve cough	Time to arrest cough at night
					Treatment group	60	4.5±1.5^**	7.2±1.3^**	6.3±1.8^**
Control group	60	8.5±1.6	13.5±1.8	14.2±4.4

Note: p <0.05 p <0.01 in comparison to control group

5.5.2 comparison of the overall efficacy of the two groups

And judging according to the curative effect evaluation standard. The total effective rate calculation formula is as follows: clinical control + significant effect + significant (example)/number of samples in group × 100%. And (3) calculating the result: the control rate and the total effective rate of the treatment group are respectively 83.3 percent and 96.7 percent; the control group has a control rate and a total effective rate of 41.7 percent and 66.7 percent respectively. The two groups of control rates have significant difference (P < 0.01); the comparison of the overall curative effects between the two groups also has obvious difference (P <0.01), which shows that the curative effect of the treatment group is obviously superior to that of the control group. See Table 2

TABLE 2 comparison of clinical overall efficacy between groups

Note: p <0.01 in comparison to control group

Example 25 examination of preparation method of granules

The invention adopts wet granulation

1 selection of wetting Agents

A common wetting agent for wet granulation is ethanol, so ethanol was chosen as the wetting agent and the effect of different ethanol concentrations on the granulation was examined.

2 investigation of the type of auxiliary Material

The moisture absorption investigation result of the mixed extract powder and the auxiliary materials shows that the moisture absorption of the extract powder can be reduced by the dextrin, the lactose and the soluble starch, so that the wet granulation auxiliary materials are further preferably selected by taking the particle forming rate, the soft material property, the granulation difficulty and the like as investigation indexes.

Mixing the extract powder with dextrin, lactose and soluble starch according to the weight ratio of 3: 2, mixing, spraying 95% ethanol, granulating, and taking the soft material property, the granule forming rate, the granulation difficulty, the granule color, the fine powder, the bonding condition and the like as evaluation indexes, and the results are shown in table 9.

The granule molding ratio%.

Qualified granules: referring to the general requirements of the preparation under the item of granules in the section of 'Chinese pharmacopoeia' (appendix I C) of the 2015 edition, the granules which can pass through the No. one sieve and can not pass through the No. five sieve are collected.

Preparing composition extract powder:

adding 10 times of water into the other eight medicines such as the astragalus root, the rhizoma anemarrhenae and the like, decocting for three times, each time lasting for 1.5 hours, merging decoction, filtering, merging with the water liquid obtained by extracting the volatile oil, concentrating to obtain clear paste with the relative density of 1.10-1.15 (60 ℃), adding ethanol to ensure that the alcohol content reaches 60%, stirring, standing overnight, filtering, recovering ethanol and concentrating into thick paste, drying under reduced pressure below 70 ℃, crushing into fine powder, and uniformly mixing with the beta-cyclodextrin inclusion compound to obtain extract powder.

TABLE 3 investigation of the types of adjuvants

The results show that the three auxiliary materials can form granules and are easy to granulate, but the forming rate of the granules is highest when dextrin is added, and the dextrin is low in price, good in stability and good in fluidity compared with lactose and soluble starch, so that the dextrin is selected as the auxiliary material.

3 investigating the dosage of auxiliary materials and the concentration of wetting agent ethanol

Respectively mixing an appropriate amount of extract powder and dextrin according to the weight ratio of 2: 1. 1.5:1, 1:1, respectively spraying appropriate amount of ethanol with concentration of 85%, 90% and 95% to granulate, and the granulation condition results are shown in table 4.

TABLE 4 granule formation Process optimization test results

From the results, it is understood that the soft material is relatively loose, the fine powder is relatively large, and the molding rate is not high when the ethanol concentration is 95%, the molding rate is improved when the ethanol concentration is 90%, and the soft material is likely to form hard lumps and is difficult to sieve when the ethanol concentration is 85%. The forming rate and the granulating difficulty degree of the granules are found by comparing the forming rate and the granulating difficulty degree of the granules when the proportion of the auxiliary materials to the auxiliary materials is different, and the granulating is easier and the forming rate is higher when the proportion of the auxiliary materials to the auxiliary materials is 1.5: 1. Therefore, 90% ethanol is selected as a wetting agent, and the medicine-auxiliary ratio is 1.5:1 for granulation.

EXAMPLE 26 measurement of the content of the granule of the Chinese medicinal composition

1 establishment of chromatographic conditions

Chromatographic conditions are as follows: agilent 1260 high performance liquid chromatograph, evaporative light scattering detector Agilent380-ELSD, Agilent Zorbax SB-C18 chromatographic column (4.6mm × 250mm, 5 μm), acetonitrile-water (32:68) as mobile phase; column temperature 30 ℃, volume flow 1.0mL/min, sample injection amount 20 μ L, ELSD atomization temperature: 85 ℃, evaporator temperature 85 ℃, carrier gas: high purity nitrogen, gas flow rate: 1.00 SLM.

2 preparation and measurement of solutions

(1) Preparation of control solutions

Taking a proper amount of astragaloside IV reference substance, precisely weighing, and adding methanol to obtain solution containing 0.2945mg astragaloside IV per 1 mL.

(2) Preparation of test solution

A reflux method: taking 6g of granules, precisely weighing, placing in a conical flask with a plug, adding 80mL of methanol, carrying out cold soaking overnight, carrying out reflux extraction for 2h, cooling, filtering, concentrating the filtrate to dryness, adding 40mL of water to dissolve the residue, shaking and extracting with water-saturated n-butyl alcohol for 4 times, 40mL each time, combining the n-butyl alcohol solutions, washing with ammonia test solution for 2 times, 50mL each time, discarding the ammonia solution, evaporating the n-butyl alcohol solution to dryness, adding methanol to dissolve the residue, fixing the volume to a 5mL volumetric flask, and shaking up to obtain the product.

An ultrasonic method: taking 6g of granules, precisely weighing, placing in a conical flask with a plug, adding 80mL of methanol, ultrasonically extracting for 30min, cooling, filtering, and performing the other steps by the same reflux method.

Respectively sucking 20 μ L of each solution, injecting into a high performance liquid chromatograph, recording peak area, and calculating astragaloside content, wherein the result is shown in Table 5.

TABLE 5 Astragaloside extraction method investigation

The result shows that the average value of the astragaloside content in the ultrasonic extraction is 0.1333mg/g, which is obviously better than 0.966mg/g in the reflux extraction, so the ultrasonic extraction method is selected to extract the particles.

(3) Preparation of negative test solution

Preparing a blank sample without astragalus into a negative test sample solution.

The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution, the test solution and the negative test solution, injecting into a liquid chromatograph, and measuring by the method.

3 methodology examination

(1) Specificity experiments

Precisely sucking 10 μ L of the reference solution, the negative sample solution and the sample solution, respectively, injecting into a liquid chromatograph, and measuring.

The results show that the negative sample has no chromatographic peak within the same retention time as the astragaloside IV control, so that the negative sample is considered to have no interference.

(2) Investigation of linear relationships

Preparing reference solution, and diluting to standard solutions with concentrations of 0.0974mg/g, 0.1461mg/g, 0.1948mg/g, 0.2922mg/g, 0.3896mg/g, and 0.487 mg/g. Respectively and precisely sucking 10 mul, injecting into a high performance liquid chromatograph, measuring, and drawing a standard curve to obtain a regression equation: 1.6141x +5.1654, R²When the concentration was changed to 0.9992, it was found that astragaloside IV was in a good linear relationship between 0.0974mg/mL and 0.4870 mg/mL.

(3) Precision test

Accurately sucking 20 μ L of paeoniflorin control solution, continuously injecting sample for 6 times, and measuring peak area of the control, the result is shown in Table 6.

Table 6 precision test results (n ═ 6)

The results show that: the relative standard deviation RSD < 3%, indicating good precision.

(4) Stability test

Taking the Chinese medicinal composition granules (grinding), accurately weighing 5g, preparing a test solution according to the method, and respectively carrying out sample injection analysis 0, 2, 4, 6, 8, 10, 11 and 24 hours after preparation, wherein the results are shown in Table 7.

TABLE 7 stability test results

The results show that: the relative standard deviation RSD of the astragaloside content measured within 24 hours is less than 3 percent, which indicates that the test solution has good stability within 24 hours.

(5) Reproducibility test

Taking the same batch of Chinese medicinal composition granule samples, precisely weighing 5g, preparing 6 parts of test solution according to the method, measuring peak area, and calculating astragaloside content.

Table 8 results of reproducibility test (n ═ 6)

The results show that: the content average value of paeoniflorin in the test sample is measured to be 0.2567mg/g, and the RSD is less than 3 percent, which indicates that the reproducibility is good.

Example 27

Identification A

Collecting 5g of the product granule, grinding, adding 60ml of methanol, performing ultrasonic treatment for 30min, filtering, evaporating the filtrate on a water bath, dissolving the residue with 40ml of water, extracting with water saturated n-butanol for 4 times, 40ml each time, mixing n-butanol solutions, washing with ammonia solution twice, 50ml each time, discarding the ammonia solution, evaporating the n-butanol solution, and dissolving the residue with 5ml of methanol to obtain a sample solution. And preparing 4g of astragaloside IV reference medicinal material into reference medicinal material solution by the same method. Performing thin layer chromatography (general rule 0502) test, sucking 10 μ l of each of the test solution and the control solution, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13: 7: 2) lower layer solution as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. In the chromatogram of the test solution, the same brown spots appear in the sunlight at the corresponding positions of the chromatogram of the control solution.

Identification B

Taking the granules, adding 25ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, volatilizing the filtrate, and adding ethyl acetate lml into residues to dissolve the residues to obtain a test solution. Separately, a shionone control substance was added with ethyl acetate to prepare a solution containing lmg per lml as a control solution. Performing thin layer chromatography (general rule 0502) test, sucking 3 μ l of the above two solutions, respectively dropping on the same silica gel G thin layer plate, spreading with petroleum ether (60-90 deg.C) -ethyl acetate (9:1) as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, heating at 105 deg.C until the color development of spots is clear, and respectively inspecting under sunlight and ultraviolet lamp (365 nm). In the chromatogram of the test solution, spots or fluorescent spots of the same color appear at the corresponding positions of the chromatogram of the control solution.

Identification C

Taking 2g of the product particles, grinding, adding 25mL of trichloromethane, carrying out ultrasonic treatment for 30 minutes, filtering, drying the filtrate by distillation on a water bath kettle, and adding 1mL of dichloromethane into residues to dissolve the residues to obtain a test solution. Taking 1g of fructus Schisandrae chinensis as reference material, and making into reference material solution by the same method. Adding methanol into Schizandrol A control to obtain solution containing 0.5mg per 1mL as control solution. Performing thin layer chromatography (general rule 0502) test by sucking 10 μ L of test solution and control solution, and spotting on the same silica gel GF₂₅₄On the thin layer plate, petroleum ether (30 to E)60 ℃) ethyl formate-formic acid (15: 6: 1) the upper solution is a developing agent, is developed and taken out, and is dried under an ultraviolet lamp (254nm) for inspection. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to those on the chromatogram of the control solution.

Authentication D

Taking the granules of the product, adding 30ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating filtrate to dryness, adding 20ml of water into residues for dissolving, adding diethyl ether for shaking and extracting for 2 times, 20ml each time, combining diethyl ether solutions, evaporating to dryness, soaking residues in petroleum ether (30-60 ℃) for 2 times, 15ml each time (soaking for about 2 minutes), pouring off the petroleum ether, and adding 2ml of absolute ethyl alcohol into the residues for dissolving to obtain a sample solution. Taking another 5g of mume fructus as reference material, and making into reference material solution by the same method. Then adding anhydrous ethanol into ursolic acid control to obtain solution containing 0.5mg per lml as control solution. Performing thin layer chromatography (general rule 0502) test, sucking 1-2 μ l of the above three solutions, respectively dropping on the same silica gel G thin layer plate, developing with cyclohexane-chloroform-ethyl acetate-formic acid (20:5:8:0.1) as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clear. Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.

Claims

1. A detection method of a traditional Chinese medicine composition preparation is characterized by being a high performance liquid chromatography, and specifically comprising the following steps:

the determination method comprises respectively sucking reference solution and sample solution, injecting into liquid chromatograph, and determining;

the traditional Chinese medicine composition preparation is prepared from the following raw material medicines: 7-42 parts of astragalus membranaceus, 4-25 parts of cassia twig, 4-25 parts of dried ginger, 5-30 parts of radix asteris, 4-25 parts of mangnolia officinalis, 6-34 parts of dark plum, 4-25 parts of schisandra chinensis, 2-15 parts of myrobalan, 2-15 parts of rhizoma anemarrhenae and 2-15 parts of liquorice.

2. The detection method according to claim 1, wherein the organic solvent is selected from any one of methanol, acetone, n-butanol, ethyl acetate, petroleum ether, and chloroform; the extraction method is selected from ultrasonic extraction, reflux extraction, cold leaching extraction, percolation extraction and microwave extraction.

3. The assay of claim 1, wherein the test solution is prepared by a method comprising: extracting the Chinese medicinal composition preparation with methanol under ultrasound, recovering solvent from the extractive solution, concentrating to dry, dissolving the residue in water, extracting with water saturated n-butanol under shaking, washing the n-butanol extractive solution with ammonia test solution, discarding the ammonia test solution, evaporating the washed n-butanol solution, and dissolving the residue with methanol.

4. The detection method according to claim 1, wherein the mobile phase is 32:68 acetonitrile-water solution.

5. The assay of any one of claims 1 to 4 further comprising identifying any one or more of A-D as follows:

identification A

identification B

and (3) detection: respectively sucking a test solution and a reference solution, dropping the test solution and the reference solution on the same silica gel plate, and adding petroleum ether-ethyl acetate (5-10): (0.5-2) developing with a developing agent, developing with a 10% sulfuric acid ethanol solution, heating at 105 ℃ until the color of spots is clearly developed, and respectively inspecting under sunlight and 365nm ultraviolet lamps;

identification C

and (3) detection: respectively sucking a test solution, a reference medicinal material solution and a reference medicinal material solution, dropping the solutions on the same silica gel plate, and mixing the solutions with petroleum ether-ethyl formate-formic acid (12-18): (4-8): (0.5-2) developing with a developing agent, and inspecting under an ultraviolet lamp of 254 nm;

authentication D

6. The detection method according to claim 5, wherein the organic solvent is selected from any one of methanol, acetone, n-butanol, ethyl acetate, petroleum ether, and chloroform; the extraction method is selected from ultrasonic extraction, reflux extraction, cold leaching extraction, percolation extraction and microwave extraction.

7. The assay of claim 5 wherein the identification a test solution is prepared by: extracting the Chinese medicinal composition preparation with methanol under ultrasound, recovering solvent from filtrate, concentrating to dry, dissolving residue in water, extracting with water saturated n-butanol under shaking, washing n-butanol extractive solution with ammonia test solution, discarding ammonia test solution, evaporating washed n-butanol solution, and dissolving residue with methanol.

8. The assay of claim 5 wherein the assay D test solution is prepared by: taking the Chinese medicinal composition preparation, adding methanol, performing ultrasonic extraction, evaporating the extracting solution to dryness, dissolving in water, extracting with diethyl ether, evaporating the diethyl ether solution to dryness, soaking with petroleum ether, removing the petroleum ether solution, and dissolving the residue with anhydrous ethanol to obtain the final product.

9. The assay method of any one of claims 1 to 4, wherein the preparation of the Chinese medicinal composition is prepared by a method comprising:

and c, combining the extracting solutions obtained in the step a and the step b, adding the volatile oil obtained in the step a, and preparing a preparation.