CN112190684A

CN112190684A - A Chinese medicinal composition for treating intractable cough and asthma, and its preparation method and quality control method

Info

Publication number: CN112190684A
Application number: CN202011224365.4A
Authority: CN
Inventors: 邹纯朴; 王庆其; 张彤; 丁越; 李孝刚; 梁尚华; 李婷; 王少墨; 裘世轲
Original assignee: Shanghai University of Traditional Chinese Medicine
Current assignee: Shanghai University of Traditional Chinese Medicine
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2021-01-08
Anticipated expiration: 2040-11-05
Also published as: CN112190684B

Abstract

The present invention relates to a Chinese medicine composition for curing intractable cough and asthma, its preparation process and quality control method. The traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 10-13 parts of raw ephedra, 12-16 parts of scutellaria baicalensis, 9-13 parts of dried ginger, 3-10 parts of asarum, 9-12 parts of gentian, 10-15 parts of roasted myrobalan and 6-13 parts of raw liquorice. The invention proves that the weight proportion relation of the raw material medicines of the traditional Chinese medicine composition has obvious influence on the curative effect of the traditional Chinese medicine composition on the intractable cough and asthma, and the traditional Chinese medicine composition should be prepared according to the weight proportion strictly. The invention further optimizes the preparation process of the traditional Chinese medicine composition particles, establishes a fingerprint detection method of the traditional Chinese medicine composition particles and a method for simultaneously detecting the contents of multiple components in a traditional Chinese medicine composition sample, and provides a systematic research basis for the reasonable development of the compound preparation of the traditional Chinese medicine composition.

Description

A Chinese medicinal composition for treating intractable cough and asthma, and its preparation method and quality control method

Technical Field

The invention relates to the field of traditional Chinese medicines, in particular to a traditional Chinese medicine composition for treating intractable cough and asthma, a preparation process and a quality control method thereof.

Background

Intractable cough and asthma are the common term for intractable cough and asthma syndrome, and are clinically common chronic respiratory diseases, which can occur at all ages. Clinically, the medicine is mainly characterized by wheezing and breathing, suffocation such as obstruction, cough and expectoration, cyanosis of lips and nails, abdominal fullness and distention, lingering course of disease and long-term difficult healing. The modern medicine is mainly diagnosed with cough variant asthma, allergic asthma, chronic obstructive pulmonary disease and the like.

The intractable cough and asthma belongs to the categories of ' cough ', ' cough and asthma ', ' asthma ' and phlegm asthma ' in the traditional Chinese medicine, and the symptoms and the etiology and pathogenesis of the disease are recorded in a plurality of chapters in the ' internal classic ' more than two thousand years ago. The traditional Chinese medicine theory considers that the generation of the intractable cough is caused by factors such as exogenous pathogenic factors, fatigue, diet and the like because the persistent phlegm is accumulated in the lung. Thereby causing the disorder of the lung qi dispersing and descending and the blockage of the air passage by the phlegm stagnation. The main cause of this disease is phlegm in the body, and the generation of phlegm is the result of spleen, lung and kidney failing to distribute, transport and transform body fluids. Body fluids stagnated in the body gradually congeals into phlegm, which is hidden in the lung and becomes an objective root of disease, and cough and asthma can be caused by sudden change of climate, emotional disorder, improper diet or fatigue. With the rapid development of urbanization and industrialization, the living environment and climate conditions are greatly changed, and the incidence and mortality of cough and asthma show a continuously rising trend in the global scope. The prevalence of global cough and asthma has been particularly prominent in recent years, increasing at a dramatic rate of 20% to 50% every 10 years. As a threat to human health, many countries attach more importance to the treatment of cough and asthma, and scholars at home and abroad also make more intensive studies on cough and asthma, but the pathogenesis of the cough and asthma is still unclear at present. In recent years, inhaled glucocorticoids and beta 2 receptor agonists are common medicines for treating cough and asthma, but the medicines have obvious adverse reactions and drug resistance and are not suitable for long-term administration. The traditional Chinese medicine has a long history of treating cough and asthma, has better curative effect in clinical aspect, some Chinese patent medicines are already on the market, but effective Chinese patent medicines are lacked for treating intractable cough and asthma.

The Qinhima prescription is an proved prescription of professor of Shanghai medical university and famous old traditional Chinese medicine fur Peiran by the applicant, is evolved from Xiaoqinglong decoction, and specifically consists of ephedra, asarum, gentian, scutellaria, dried ginger, myrobalan and liquorice. The prescription is clinically used for many years, has good treatment effect on cough and asthma, and has little toxic and side effect. The proportion relationship of the dosage of each raw material medicine of the formula is not disclosed so far, the dosage of the medicine directly determines the potency of the medicine, and the change of the dosage proportion can change the compatibility relationship of the prescription, so that the proportion relationship of the dosage of each raw material medicine needs to be determined to improve the curative effect.

In addition, the formula of qin ma is always applied clinically in the form of decoction, but in consideration of effectiveness and convenience of clinical treatment, further research on extraction and preparation process and quality control method is needed to stably produce solid preparation with large drug-loading rate, safety and high efficiency, which is suitable for long-term convenient administration on a large scale, so as to promote clinical application of qin ma formula.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine composition for treating intractable cough, a preparation process of granules of the traditional Chinese medicine composition and a quality control method of the traditional Chinese medicine composition, aiming at the defects in the prior art.

In a first aspect, the invention provides a traditional Chinese medicine composition for treating intractable cough, which is prepared from the following raw material medicines in parts by weight: 10-13 parts of raw ephedra, 12-16 parts of scutellaria baicalensis, 9-13 parts of dried ginger, 3-10 parts of asarum, 9-12 parts of gentian, 10-15 parts of roasted myrobalan and 6-13 parts of raw liquorice.

In a second aspect, the invention provides a preparation process of Qinuman granules, which comprises the following steps: weighing the following medicinal materials in parts by weight: 10-13 parts of raw ephedra, 12-16 parts of scutellaria baicalensis, 9-13 parts of dried ginger, 3-10 parts of asarum, 9-12 parts of gentian, 10-15 parts of stewed myrobalan and 6-13 parts of raw liquorice, adding 10 times of water into the six medicinal materials except the scutellaria baicalensis, soaking for 30 minutes, adding the scutellaria baicalensis before decoction, decocting for 2 times, 1 hour each time, combining decoction, filtering, and concentrating the filtrate to the density of 1.08-1.18g/ml (measured thermally), thereby obtaining an aqueous extract concentrated solution.

Preferably, the method further comprises the following steps: vacuum drying and crushing the water extraction concentrated solution at 60 ℃ to obtain extract powder; adding a proper amount of dextrin into the obtained extract powder, uniformly mixing, and taking 55% ethanol as a binder to prepare the Qinma granules.

In a third aspect, the invention provides a method for establishing a qinlan granule fingerprint spectrum, which comprises the following steps:

(A) preparation of mixed control solution: mixing gallic acid, ephedrine hydrochloride, pseudoephedrine hydrochloride, liquiritin, baicalin, wogonoside, baicalein, and wogonin with methanol to obtain mixed reference solution;

(B) preparing a test solution: weighing Scutellariae radix and folium Sesami granules, adding methanol, performing ultrasonic treatment, cooling, and making into sample solution;

(C) and respectively carrying out high performance liquid chromatography analysis on the mixed reference substance solution and the test solution, wherein the chromatographic conditions are as follows: c₁₈The chromatographic column uses acetonitrile as a mobile phase A and 0.3% phosphoric acid water (containing 0.2% triethylamine) as a mobile phase B, and the elution procedure is as follows:

preferably, the chromatographic conditions further comprise: the flow rate is 0.4 ml/min; the column temperature was 25 ℃; the detection wavelength was 210 nm.

In a fourth aspect, the invention provides a quality control method for a Qinma granule sample, which comprises the following steps:

(A) establishing a standard fingerprint spectrum of the radix scutellariae and semen cannabis granules according to the establishing method of the radix scutellariae and semen cannabis granule fingerprint spectrum;

(B) detecting the qin ma particle sample to be detected according to any one of the establishment methods of the qin ma particle fingerprint spectrum, and establishing the fingerprint spectrum of the qin ma particle sample to be detected;

(C) comparing the fingerprint of the radix scutellariae barbatae granule sample to be detected with the standard fingerprint of the radix scutellariae barbatae granule, and evaluating the quality of the radix scutellariae barbatae granule sample according to the chromatographic peak parameters in the fingerprint.

Preferably, in step (C), it is determined whether the fingerprint of the sample of the sisal particles to be detected contains 18 peaks whose relative retention time does not deviate more than ± 10% from 0.058(1), 0.103(2), 0.119(3), 0.436(4), 0.516(5), 0.566(6), 0.591(7), 0.602(8), 0.625(9), 0.672(10), 0.714(11), 0.874(12), 1.000(S), 1.074(14), 1.130(15), 1.168(16), 1.279(17) and 1.538 (18).

In a fifth aspect, the invention provides a method for simultaneously determining the contents of multiple components in a formula sample of radix scutellariae, and simultaneously determining gallic acid, ephedrine hydrochloride, pseudoephedrine hydrochloride, gentiopicrin, baicalin, wogonoside,Performing high performance liquid chromatography analysis on the scutellarin square sample according to the contents of scutellarin, wogonin, 6-gingerol, ammonium glycyrrhizinate, sesamin and asaricin, wherein the chromatographic conditions are as follows: c₁₈The chromatographic column uses acetonitrile as a mobile phase A and 0.3% phosphoric acid water (containing 0.2% triethylamine) as a mobile phase B, and the elution procedure is as follows:

preferably, the chromatographic conditions further comprise: the flow rate is 0.4 ml/min; the column temperature was 25 ℃; the detection wavelength is 210nm (gallic acid, ephedrine hydrochloride and pseudoephedrine hydrochloride), 249nm (ammonium glycyrrhizinate), 271nm (gentiopicroside), 275nm (wogonin and baicalein), 278nm (baicalin and wogonin), 280nm (6-gingerol), 287nm (sesamin and asaricin).

In a sixth aspect, the present invention provides a quality control method for a Qinwan square sample, comprising the step of measuring the contents of the components in the Qinwan square sample according to any one of the above-mentioned methods for simultaneously measuring the contents of the components in the Qinwan square sample.

The invention has the advantages that:

1. the invention discloses the weight ratio of each raw material medicine in the formula of Qinmen for the first time. In the formula, the ephedra is a monarch drug, and has the effects of inducing sweat, dispelling cold, ventilating lung and relieving asthma; the gentian and the scutellaria are bitter and cold, clear phlegm and heat and lower lung qi, while the asarum and the dried ginger are pungent and warm, warm the lung and resolve retention, and the two groups supplement each other, and the four medicines are all ministerial medicines; the myrobalan is an adjuvant drug, astringes lung and relieves cough, and the ephedra herb is pungent and disperses exterior syndrome relieving, so that the efficacy of relieving asthma and relieving cough is enhanced, and lung qi is not lost; the liquorice can regulate the medicine property, clear away heat and toxic material, eliminate phlegm and relieve cough. The medicines are combined to play the efficacies of freeing lung and relieving asthma, purging fire and removing toxin, clearing phlegm and heat and descending lung qi. Experiments show that the weight ratio of the raw material medicines in the formula has obvious influence on the curative effect, so that the prescription should strictly follow the proportional relation of the dosage of the raw material medicines in the formula in order to obtain the optimal curative effect.

2. The invention optimizes the preparation process of the radix scutellariae barbatae granules, adopts baicalin and ephedrine hydrochloride as indexes, determines the process of not soaking radix scutellariae barbatae, and determines the optimal extraction preparation process of the radix scutellariae barbatae granules by comparing the extraction efficiency of different extraction processes of the radix scutellariae barbatae formulas on the baicalin and the ephedrine hydrochloride. The extraction process can ensure that the transfer rate of baicalin is not less than 80%, the transfer rate of ephedrine hydrochloride is not less than 50%, and the repeatability is good. The invention also determines that the transfer rate of each index component is reduced more due to the purification treatment, so the alcohol precipitation treatment is not selected. And further screening the types of the formed auxiliary materials and the types of the adhesives. Small experiments and pilot plant studies prove that the process can ensure that the transfer rate of baicalin is not less than 40 percent, the transfer rate of ephedrine hydrochloride is not less than 50 percent, the content of baicalin in each package of the radix scutellariae granules is not less than 250mg, the content of ephedrine hydrochloride is not less than 20mg, the process has good stability, and the forming rate is more than 80 percent.

3. The invention establishes the fingerprint of the Qinma granules and evaluates the variety, the transmission rule and the content limit of the main components of the inner chemical substances of the Qinma granules on the whole. The fingerprint detection method has excellent precision, repeatability and stability, and can be used for quality control of radix Scutellariae granules.

4. The invention establishes a content determination method for simultaneously determining 12 components in the Qinmen prescription, the method has good linear relation, excellent precision, repeatability and stability and high recovery rate, can be used for quality control of water extraction concentrated solution, dry extract and particle samples in the Qinmen particle preparation process, and can further guide the optimization of the preparation production process by comparing the content change conditions of the components in each sample through content determination.

Drawings

FIG. 1: the lung organization pathology (HE, × 200) was determined for each group of rats.

FIG. 2: the chromatogram map of ephedrine hydrochloride. A. Test sample, control sample, and negative control sample (1, ephedrine hydrochloride).

FIG. 3: baicalin specificity chromatogram. A. Test sample, control sample, and negative control sample (1. baicalin).

FIG. 4: a flow chart of pilot plant test process of Qinma granules.

FIG. 5: HPLC chromatogram of Qinwan prescription. A. Mixing standard, B. radix Scutellariae granules (1. gallic acid, 2. ephedrine hydrochloride, 3. pseudoephedrine hydrochloride, 11. liquiritin, 13. baicalin, 16. wogonoside, 17. baicalein, 18. wogonoside).

FIG. 6: standard fingerprint spectrum of Qinhua granules.

FIG. 7: and 10 batches of Qinhua granule fingerprint spectra are calculated.

FIG. 8: a multi-component content determination chromatogram of the twelve components. (1) Gallic acid, (2) ephedrine hydrochloride, (3) pseudoephedrine hydrochloride, (4) gentiopicrin, (5) baicalin, (6) wogonin, (7) baicalein, (8) wogonin, (9) 6-gingerol, (10) ammonium glycyrrhizinate, (11) sesamin, and (12) asaricin.

Detailed Description

The following detailed description of the present invention will be made with reference to the accompanying drawings. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products which can be purchased in the market.

Example 1A Chinese medicinal composition for treating intractable cough and asthma of the present invention

10 parts of raw ephedra, 12 parts of scutellaria baicalensis, 10 parts of dried ginger, 3 parts of asarum, 10 parts of gentian, 10 parts of roasted myrobalan and 10 parts of raw liquorice, and decocting by a conventional method.

Example 2A Chinese medicinal composition for treating intractable cough and asthma of the present invention

12 parts of raw ephedra herb, 15 parts of baical skullcap root, 9 parts of dried ginger, 3 parts of manchurian wildginger, 9 parts of gentian, 12 parts of roasted myrobalan and 6 parts of raw liquoric root by weight through a conventional method.

Example 3 the present invention relates to a Chinese medicinal composition for treating intractable cough and asthma

12 parts of raw ephedra, 16 parts of scutellaria, 12 parts of dried ginger, 3 parts of asarum, 9 parts of gentian, 12 parts of roasted myrobalan and 13 parts of raw liquorice, and decocting by a conventional method.

Example 4 Chinese medicinal composition for treating intractable cough and asthma of the invention

13 parts of raw ephedra, 12 parts of scutellaria baicalensis, 13 parts of dried ginger, 10 parts of asarum, 12 parts of gentian, 10 parts of roasted myrobalan and 6 parts of raw liquorice, and decocting by a conventional method.

Example 5 the present invention provides a Chinese medicinal composition for treating intractable cough and asthma

10 parts of raw ephedra herb, 16 parts of baical skullcap root, 9 parts of dried ginger, 3 parts of manchurian wildginger, 9 parts of gentian, 15 parts of roasted myrobalan and 13 parts of raw liquoric root by weight through a conventional method.

Example 6 Chinese medicinal composition for treating intractable cough and asthma of the present invention

13 parts of raw ephedra, 16 parts of scutellaria, 13 parts of dried ginger, 10 parts of asarum, 10 parts of gentian, 13 parts of roasted myrobalan and 13 parts of raw liquorice, and decocting by a conventional method.

Example 7 Experimental study on Effect of the Chinese medicinal composition on airway remodeling of chronic obstructive pulmonary disease model rat

The experiment proves that the traditional Chinese medicine composition has the effects of resisting inflammation, relieving asthma, reconstructing airways and the like in the aspect of animal experiments by constructing a Chronic Obstructive Pulmonary Disease (COPD) rat model.

1 materials and methods

1.1 materials

1.1.1 animals

70 healthy male Wistar rats with SPF grade, age of 10-12 weeks and body mass of 180-220 g are purchased from Shanghai Sphall (Bikeka) laboratory animals company. Animal certification number: 2008001655632.

1.1.2 drugs and reagents

The radix scutellariae and ephedra formula (13 g of raw ephedra herb, 16g of baical skullcap root, 13g of dried ginger, 10g of manchurian wildginger, 10g of gentian, 13g of roasted myrobalan and 13g of raw liquoric root) and the medicine extract powder are provided by the traditional Chinese medicine engineering laboratory of the school, and each plaster is equivalent to 6.5g of extract. Six monarch drugs decoction (ginseng 9g, poria cocos wolf 9g, white atractylodes rhizome 9g, honey-fried licorice root 6g, dried orange peel 9g, pinellia tuber 12g, ginger 2 tablets, jujube 3) are all Chinese medicinal formula granules, and are purchased from Jiangyin Tianjiang pharmaceutical industry Co.

Aminophylline (batch No. B141104), Shanxi Yunpeng pharmaceuticals, Inc.; lipopolysaccharide, Sigma company, usa; pepsin, shanghai weio biotechnology ltd; rat ELISA kit, shanghai weiao biotechnology ltd; rabbit anti-rat MMP-9, TIMP-1, Strobile GmbH, Wuhan doctor; diazepam injection (batch No. H31021864), Shanghai Asahi Kazai pharmaceutical Co., Ltd; cigarette (Huangguoshu brand, tar content 11mg), Guizhou cigarette Co., Ltd.

1.1.3 instruments

A wooden semitransparent smoking box (52cm multiplied by 40cm multiplied by 28cm) which is made by self; optical microscope (NIKON YS 100 type), japan NIKON corporation; biological function experiment analysis system (BY210 type), Gendtis Hispanics science and technology Limited; noninvasive respiratory function (WBP), Buxco corporation, usa; microplate reader (MK3), Thermo corporation, usa; image analysis software Image-Pro Plus 6.0, Media Cybernetics, USA.

1.2 grouping, Molding and administration

The experimental rats were randomly divided into 10 normal groups and 60 model groups. The normal group had free diet and water without intervention. Model building reference literature (Guo Guizhou, Song Wen Long, Ganzume, etc.. study of mechanism for intervention of tonifying qi prescription in chronic obstructive pulmonary disease rat airway remodeling based on MMP-9/TIMP-1 imbalance [ J ]. Chinese medicine J, 2016, 16(11):4678-4680.) model building. The specific method comprises the following steps: on day 1 of molding, 1. mu.l of a dithiol chloride solution (decomposed to sulfur dioxide in the presence of air) was injected into the trachea with a microinjector. On the 2 nd and 15 th days after sulfur dioxide inhalation, the model-built rats were anesthetized with 3% pentobarbital sodium (40. mu.g/g) by intraperitoneal injection. Fixing the rat in a supine position on a fixing plate of the rat, using a self-made probe lamp injector, probing the injector into the glottis part of the rat, finding the trachea part, injecting 50 mu l of solution (200 mu g of lipopolysaccharide and 200 mu g of pepsin prepared by PBS) into the trachea, and after injection, the rat is obviously choked and fixed by rapid rotation, so that the lipopolysaccharide and the pepsin can be uniformly distributed on two lungs. Starting on the 3 rd day (3-29) of sulfur dioxide inhalation, rats are placed in a self-made semitransparent smoking box, 15 rats are placed each time, 7 cigarettes are used, and smoking is carried out for 0.5h, 1 time every morning and continuously for 1 month.

The rats in the model group are randomly divided into a model group, a Qinmao prescription (low and medium dosage), a six-monarch decoction group (traditional Chinese medicine control group) and an aminophylline group (western medicine control group), and each group contains 10 rats. The model group was administered distilled water daily for intragastric administration. The Qinmao prescription is given at 1.17g/kg (clinical times), 0.58g/kg (clinical dosage) and 0.29g/kg (clinical half dosage) according to the body surface area folding algorithm of human and rat. The dosage of the six-monarch decoction is 4.9g/kg (clinical dosage), and the dosage of the aminophylline group is 45 mug/g (clinical dosage). The administration was performed by gavage for 3 weeks. The normal group was not intervened.

1.3 method of obtaining materials

And (3) carrying out 25% urethane anesthesia, collecting abdominal aorta blood, naturally coagulating at room temperature for 10-20 min, centrifuging for 20min after blood is layered, and collecting supernatant at 3500 r/min. After centrifugation, rat serum was dispensed and stored at-20 ℃ for further use. Exposing the two lungs, injecting 4% formaldehyde 8ml into the two lungs to make the two lungs full, and fixing in 4% formaldehyde for later use.

1.4 detection index and method

1.4.1 general status of rats

The body mass, hair, cough, diet, mental state, etc. of the rats were observed.

1.4.2 measurement of respiratory function in non-invasive rats

Before the rat measures respiration, 1.4 mu l/g diazepam is injected into the abdominal cavity, and after the rat is quiet, the rat is placed in a self-made closed container, and a noninvasive respiration function tester is adopted to obtain various parameters: respiratory rate (Hz), respiratory area (ml/s)²) Peak expiratory flow (ml/s), peak inspiratory flow (ml/s).

1.4.3 pathological section of rat Lung tissue

Rat lung tissues are taken for dehydration, xylene is transparent, paraffin immersion and embedding are carried out, slicing is carried out, hematoxylin-eosin (HE) staining is carried out, and pathological changes are observed under a 20-time microscope. The inflammatory parenchyma of lung tissues is changed by dark blue in trachea and alveolus under a 10-fold microscope by adopting Image-Pro software, the percentage of the inflammatory parenchyma in a lung section is obtained, and the thickness of the bronchiole and the thickness of smooth muscle of 5 visual fields in the section are compared at the same time.

1.4.4 immunohistochemical staining of Lung tissue and detection of MMP-9 and TIMP-1

Before immunohistochemistry, each section was dewaxed to water, and the expression of MMP-9 and TIMP-1 on the lung tissue sections was measured by the streptavidin-peroxidase method (SABC method). SABC incubation, DAB color development, positive response is the appearance of tan or yellow in lung tissue. The negative control used PBS instead of primary antibody, and the rest steps were the same. And (5) reading under a microscope and shooting. Optionally, 5 fields per sheet were selected, and a positive response was a tan or yellow color in the lung tissue. Cumulative optical density (IOD) was collected per sheet using Image-Pro Image analysis software. The expression of MMP-9 and TIMP-1 proteins is represented by the accumulated optical density, and the higher the accumulated optical density is, the higher the expression of the proteins is, and the stronger the positive reaction is.

1.4.5 TGF-. beta.1 content

Rat serum is taken, the content of TGF-beta 1 in the serum is measured by adopting an ELISA method, and the operation is strictly carried out according to the kit instruction.

1.5 statistical methods

Statistical data were analyzed using SPSS 21.0 software. Measure data to

And (4) showing. The single-factor variance analysis is adopted to analyze the relevant data, and the LSD method is adopted to compare every two groups. The difference is statistically significant when P is less than 0.05.

2 results

2.1 general State

When smoked for 1 week, the rats have obvious cough, shortness of breath, asthma, moist nose, fluffy and yellow fur, lassitude, pricking and reduced activity. Compared with the normal group, the animal quality of the model group is obviously reduced (P is less than 0.05). See table 1.

TABLE 1 comparison of the changes in body mass of rats in the model

Note: p < 0.05 compared to normal group

2.2 Effect on non-invasive rat respiratory function related indices

After 3 weeks of treatment, the differences in the model rat respiratory rate, respiratory area, peak expiratory flow and peak inspiratory flow were statistically significant (P < 0.05) compared to the normal group. Compared with the model group, the respiratory frequency of the low, medium and high dose group of the radix scutellariae and ephedra is obviously reduced (P is less than 0.05); the group suction area of the Qinmao prescription is increased in low, medium and high dose groups and aminophylline group (P is less than 0.05); the peak expiratory flow and inspiratory flow of the Qinmen prescription middle and high dose groups and the six monarch decoction group are obviously increased (P is less than 0.05). See table 2.

TABLE 2 changes in respiratory function of rats in groups after 3 weeks of treatment

Note: p < 0.05 compared to normal group; compared with the model group, # P < 0.05

2.3 pathological morphology of Lung tissue and trachea of rat and related indexes

The normal group had less inflammatory cell infiltration, normal alveolar structure, and no expansion. The pulmonary alveoli of the model group are expanded, the pulmonary alveoli are fused into a larger sac cavity, the intimal fibers of interstitial pulmonary arterioles are thickened, the bronchial wall is thickened, and inflammatory cells are infiltrated. The tracheae of the low and medium dose group of the formula of Qinmao still have inflammatory cell infiltration phenomenon, and the alveolar structure still has damage. The inflammatory cell infiltration of the high dose Qinmao group is obviously reduced. The lung tissue bronchial lumen and alveolar cavity inflammatory cell infiltration of the rats in the Liujunzi Tang group is obvious, the alveolar structure is damaged, and the small bronchus can be seen to change the inflammation. The aminophylline group still has inflammatory cell infiltration, the damage of alveolar structure is reduced, and alveolar wall is thickened. See fig. 1.

The percentage of lung section inflammatory consolidation was significantly higher in the model group compared to the normal group (P < 0.05). Compared with the model group, the lung section inflammatory excess change percentage of the Qinmian prescription medium and high dose groups and the aminophylline group is obviously reduced (P is less than 0.05). Compared with the Qinma prescription low dose group and the Liujunzi decoction group, the Qinma prescription high dose group has the advantage that the lung slice inflammatory excess change percentage is obviously reduced (P is less than 0.05). See table 3.

The model group showed significantly higher bronchial wall thickness and smooth muscle thickness (P < 0.05) compared to the normal group. Compared with the model group, the bronchial wall thickness and the smooth muscle thickness of the Qinming prescription medium/high dose group and the aminophylline group are obviously reduced (P is less than 0.05). Compared with the Qinma prescription low dose group and the Liujunzi decoction group, the Qinma prescription high dose group has obviously reduced bronchial wall thickness and smooth muscle thickness (P is less than 0.05). See table 3.

TABLE 3 comparison of percent lung section inflammatory consolidation, bronchial wall thickness, smooth muscle thickness in rats 3 weeks after treatment

Note: p < 0.05 compared to normal group; compared with the model group, # P < 0.05; compared with the low dose group of the formula of the radix scutellariae and the ephedra, the delta P is less than 0.05; compared with the six monarch drug decoction group,

2.4 Effect on serum TGF-. beta.1 content

Compared with the normal group, the model group has higher serum TGF-beta 1 content (P is less than 0.05). Compared with the model group, the TGF-beta 1 content of the serum of the Qinmao prescription, the high dose group and the aminophylline group is obviously reduced (P is less than 0.05). See table 4.

TABLE 4 comparison of TGF-. beta.1 content in serum of rats in each group

2.5 immunohistochemical staining of Lung tissue and MMP-9 and TIMP-1 expression

Compared with the normal group, the model group MMP-9 and TIMP-1 cumulative optical density (IOD) are obviously increased (P is less than 0.05). Compared with the model group, the accumulated optical density of MMP-9 and TIMP-1 in the Qinmian prescription, the high dose group and the aminophylline group is obviously reduced (P is less than 0.05). Compared with the Qinma prescription low dose group and the Liujunzi decoction group, the IOD of MMP-9 and TIMP in the Qinma prescription high dose group is obviously reduced (P is less than 0.05). See table 5.

TABLE 5 IOD and MMP-9/TIMP-1 comparison of rat lung tissues MMP-9 and TIMP-1 in each group

2.6 results of correlation analysis

The IOD of TIMP-1 is inversely related to peak expiratory flow (r ═ 0.315), and MMP-9/TIMP-1 is positively related to respiratory area (r ═ 0.314). Bronchial thickness is positively correlated with the IOD of TGF- β 1, MMP-9 (r ═ 0.354, r ═ 0.356, r ═ 0.357).

EXAMPLE 8 preparation of Qinhua granules

1 index component measuring method

1.1 method for measuring extract yield

Precisely transferring 20ml of the extracting solution, placing the extracting solution in an evaporating dish which is dried to constant weight, evaporating the extracting solution by evaporation in a water bath, drying the extracting solution to constant weight at 60 ℃, weighing the extracting solution, and calculating the yield of the extract.

1.2 determination method of ephedrine hydrochloride content

1.2.1 chromatographic conditions

The column was a polar Ether-linked Phenyl-bonded silica gel column (Ultimate Phenyl-Ether, 4.6 × 300mm, 5 μm, yuxu scientific (shanghai) ltd); acetonitrile is used as a mobile phase A, 0.3% triethylamine water (containing 0.02mol/L potassium dihydrogen phosphate and being adjusted to pH 3.0 by phosphoric acid) is used as a mobile phase B, the ratio of the mobile phase A to the mobile phase B is 2:98, and the elution is carried out for 30 minutes at equal speed; the detection wavelength is 210nm, the sample injection amount is 2 mul, the column temperature is 30 ℃, and the flow rate is 1.0 ml/min. Under the condition, the ephedrine hydrochloride is completely separated from other components (R is more than or equal to 1.5), and the number of theoretical plates is not less than 3000 calculated according to the ephedrine hydrochloride peak.

1.2.2 preparation of control solutions

Taking appropriate amount of ephedrine hydrochloride reference, precisely weighing, and preparing into 1ml solution containing 0.42mg with methanol.

1.2.3 preparation of test solutions

Weighing each decoction piece according to the prescription amount (120 g of raw ephedra herb, 150g of baical skullcap root, 90g of dried ginger, 30g of manchurian wildginger, 90g of gentian, 120g of stewed myrobalan and 60g of raw liquoric root, the same below), adding 8 times of water, soaking for 0.5 hour, decocting for 2 times, each time for 1 hour, combining the decoctions, filtering and concentrating to 1g of crude drug/ml, thus obtaining the concentrated solution. Precisely transferring 1ml of the concentrated solution, putting into a 20ml volumetric flask, adding 50% methanol to constant volume, and shaking up to obtain the product.

1.2.4 preparation of negative control solution

Weighing each decoction piece (except herba Ephedrae) according to the prescription amount, adding 8 times of water, soaking for 0.5h, decocting for 2 times, 1h each time, filtering, mixing the filtrates, and concentrating to 1g crude drug/ml to obtain concentrated solution. Precisely transferring 1ml of the concentrated solution, putting into a 20ml volumetric flask, adding 50% methanol to constant volume, and shaking up to obtain the product.

1.2.5 specialization examination

The above-mentioned control, test sample and negative control solution were aspirated, and respectively injected into HPLC, and analyzed under the chromatographic conditions of "1.2.1", as shown in FIG. 2. The result shows that the negative control solution with the ephedra left in the formula of radix scutellariae has no absorption peak at the corresponding position in the chromatogram of the ephedrine hydrochloride control, which indicates that other medicines in the formula have no negative interference on the determination of ephedrine hydrochloride in the formula of radix scutellariae and ephedra.

1.3 method for measuring baicalin content

1.3.1 chromatographic conditions

The chromatographic column is C₁₈Liquid chromatography column (Platisil ODS, 4.6X 250mm, 5 μm, Shanghai Dima analytical technique Co., Ltd.); methanol is taken as a mobile phase A, 0.3 percent phosphoric acid water is taken as a mobile phase B, the proportion of the mobile phase is that methanol and the 0.3 percent phosphoric acid water are 50:50, and the elution is carried out for 20 minutes at equal intervals; the detection wavelength is 280nm, the sample injection amount is 10 mul, the column temperature is 30 ℃, and the flow rate is 1.0 ml/min. Under the condition, baicalin and other components are completely separated (R is more than or equal to 1.5), and the theoretical plate number is not less than 2500 calculated according to baicalin peak.

1.3.2 preparation of control solutions

Taking appropriate amount of baicalin reference substance, precisely weighing, and preparing into 1ml solution containing 0.5mg with methanol.

1.3.3 preparation of test solutions

Weighing each decoction piece according to the prescription amount, adding 8 times of water, soaking for 0.5 hour, decocting for 2 times, 1 hour each time, mixing decoctions, filtering, and concentrating to 1g crude drug/ml to obtain concentrated solution. Precisely transferring 1ml of the concentrated solution, adding 50% methanol into a 20ml volumetric flask to constant volume, shaking up, precisely transferring 1ml of the concentrated solution, adding 50% methanol into a 20ml volumetric flask to constant volume, and shaking up to obtain the product.

1.3.4 preparation of baicalin negative control solution

Weighing each decoction piece (except for radix Scutellariae) according to the prescription, adding 8 times of water, soaking for 0.5 hour, decocting for 2 times, each time for 1 hour, mixing decoctions, filtering, and concentrating to 1g crude drug/ml to obtain concentrated solution. Precisely transferring 1ml of the concentrated solution, adding 50% methanol into a 20ml volumetric flask to constant volume, shaking up, precisely transferring 1ml of the concentrated solution, adding 50% methanol into a 20ml volumetric flask to constant volume, and shaking up to obtain the product.

1.3.5 specialization examination

The above-mentioned control, test sample and negative control solution were aspirated, and the samples were respectively injected into HPLC, and analyzed under the chromatographic conditions of "1.3.1", as shown in FIG. 3. The result shows that the negative control solution of the scutellarin lacking in the formula has no absorption peak at the corresponding position in the chromatogram of the scutellarin control product, and the other medicines in the formula have no negative interference on the determination of the scutellarin in the formula.

2 investigation of extraction mode of Qinlian prescription

2.1 preparation of sample solutions

(1) Concentrate sample solution: weighing each decoction piece according to the prescription amount, adding 8 times of water, soaking for 0.5 hour, decocting for 2 times, each time for 1 hour, filtering, combining filtrates, and concentrating to 1g crude drug/ml to obtain the final product.

(2) Alcohol extraction of sample solution: weighing each decoction piece according to the prescription amount, adding 8 times of 75% ethanol, soaking for 0.5 hour, refluxing for 2 times, each time for 1 hour, filtering, combining filtrates, and concentrating under reduced pressure to 1g crude drug/ml to obtain the final product.

2.2 measurement of target component content

TABLE 6 results of content measurement of samples obtained by two extraction methods (n. 3)

The results show that the transfer rates of ephedrine hydrochloride and baicalin in the 75% ethanol reflux extraction solution are slightly higher than those of water decoction, but no significant difference exists, and the problem is to select water extraction as the extraction mode of the scutellaria baicalensis prescription considering that the pharmacodynamic experiment results show that the pharmacological effects of the scutellaria baicalensis prescription ethanol extract and the water extract are not significantly different and the actual production cost.

2.3 Single factor test

As the main component baicalin in the scutellaria baicalensis is easy to decompose in cold water, whether the scutellaria baicalensis is soaked or not and the extraction frequency are examined by adopting a single-factor test method before decoction.

2.3.1 investigation of Process for whether or not Scutellaria baicalensis Georgi is soaked

Weighing each decoction piece according to the prescription amount, adding 8 times of water, soaking for 0.5 hour, decocting for 2 times, each time for 1 hour, filtering, combining filtrates, and concentrating to 1g crude drug/ml to obtain Scutellariae radix soaking concentrated solution; weighing decoction pieces according to the prescription, adding 8 times of water, soaking the decoction pieces of the other medicinal materials except for the scutellaria baicalensis for 0.5 hour, adding the scutellaria baicalensis before decoction, decocting for 2 times, 1 hour each time, filtering, combining filtrates, and concentrating to 1g crude drug/ml to obtain a scutellaria baicalensis non-soaking concentrated solution. The results are shown in Table 7.

TABLE 7 investigation of the process of soaking Scutellaria baicalensis (n ═ 3)

And (4) conclusion: whether to soak scutellaria before decocting has obvious influence on the transfer rate of baicalin, so that a method that scutellaria is not soaked before decocting is adopted subsequently.

2.3.2 examination of the number of extractions

Weighing each decoction piece according to the prescription amount, adding 8 times of water, soaking the decoction pieces of the other medicinal materials except the scutellaria baicalensis for 0.5 hour, adding the scutellaria baicalensis before decoction, respectively decocting for 1 time, 2 times and 3 times (two parts in parallel) for 1 hour each time, filtering, combining the filtrates, and concentrating to 1g crude drug/ml to obtain the traditional Chinese medicine. The results are shown in Table 8.

TABLE 8 results of the number of times of decoction (n 2)

And (4) conclusion: the transfer rate of each index component, the extract yield and the production cost are comprehensively considered, and the effect of decocting 2 times in water is considered to be better.

2.4 orthogonal test optimized extraction process

2.4.1 orthogonal Experimental design and results

According to the result of preliminary experiment, the soaking time, the extraction time and the solvent amount which have great influence on the water extraction effect are taken as investigation factors of orthogonal experiment, and L is designed₉(3⁴) The results of the orthogonal test are shown in Table 9.

TABLE 9 orthogonal experiment factor horizon

Weighing 9 parts of each decoction piece in parallel according to the prescription amount, and weighing according to L₉(3⁴) Arranging orthogonal table, adding corresponding amount of water, soaking the rest Chinese medicinal decoction pieces except Scutellariae radix under corresponding conditions, adding Scutellariae radix before decocting, and processing into corresponding stripsExtracting, filtering, mixing filtrates, and concentrating to 1g crude drug/ml to obtain each sample solution. Then measuring the transfer rate and extract yield of baicalin and ephedrine hydrochloride in the water extract. The results are shown in tables 10 and 11.

TABLE 10L₉(3⁴) Visual analysis result of orthogonal test

Note: the weight of baicalin transfer rate is 40%, the weight of ephedrine hydrochloride is 40%, and the weight of extract yield is 20%

TABLE 11 results of ANOVA

Note: f_0.05(2,2)＝19

As can be seen from the visual analysis results of Table 10, the evaluation index is used as the standard, and the primary and secondary relationship of the three factors is C>A>B, amount of instant solution>Soaking time>And (4) extracting time. As can be seen from the results of the ANOVA in Table 11, the three factors have no significant influence on the extraction effect. The optimal extraction process is A according to the visual analysis and variance analysis results and the actual production conditions₂B₁C₃Namely 10 times of water, soaking for 0.5 hour and decocting for 1 hour.

2.4.2 extraction Process verification test

Weighing each decoction piece with the prescription amount of 10 times, extracting according to the optimal optimized process (10 times of water, soaking for 0.5 hour, decocting for 1 hour, extracting for 2 times), paralleling three batches, and determining the transfer rate of ephedrine hydrochloride and baicalin in the extract and the extract yield, the results are shown in Table 12.

Table 12 extraction process verification results (n ═ 3)

The test results show that the transfer rates and the extract yields of ephedrine hydrochloride and baicalin in the three batches of samples are basically consistent, which shows that the extraction process has good repeatability.

3 study of purification Process

Because the extraction process adopts a water decoction method, the extract yield is higher, and the ethanol precipitation method is supposed to be adopted to purify the water extract in view of reducing the oral dosage and ensuring the curative effect of the preparation.

Weighing each decoction piece according to the prescription amount of 10 times, adding 10 times of water, soaking the decoction pieces except for scutellaria baicalensis for 0.5 hour, adding the scutellaria baicalensis decoction pieces before decoction, decocting for 2 times, 1 hour each time, filtering, combining filtrates, concentrating to 1g crude drug/ml to serve as a concentrated solution sample solution, taking 50ml of the concentrated solution sample solution, adding ethanol with corresponding volume respectively to ensure that the alcohol content in the sample solution respectively reaches 0%, 30%, 50%, 60%, 70% and 80%, precipitating for 12 hours at room temperature, filtering, concentrating the filtrate at 60 ℃ under reduced pressure to 50ml, and determining the transfer rate and extract yield of ephedrine hydrochloride and baicalin in the alcohol precipitation sample solution, wherein the results are shown in Table 13.

Table 13 alcohol precipitation concentration measurement results (n ═ 3)

Test results show that although the extract yield of a sample subjected to alcohol precipitation is effectively reduced, the transfer rate of each index component is reduced more, and the transfer rate of each index component in subsequent pilot plant production is considered to be further reduced, so that the alcohol precipitation is not selected.

4 study of Molding Process

The common preparation methods of the granules include dry granulation, one-step granulation, wet granulation and the like. The extract powder obtained by the method is sensitive to humidity and temperature, and is easy to generate the phenomenon of sticking and connecting blocks. The preliminary experiments show that dry granulation is adopted, the required auxiliary material dosage is large, and the dissolubility of finished product particles is poor; and wet granulation is adopted, so that agglomeration is easy to occur in the soft material preparation process, and granulation is difficult, so that the Qinma granules are prepared by adopting a one-step granulation method which is simple to operate and easy to generate industrially.

4.1 investigation of the types of auxiliary materials

The common adjuvants for granule include dextrin, soluble starch and maltodextrin. The results of preliminary experiments prove that the granules prepared from the soluble starch have poor forming effect, and the granules prepared from the dextrin and the maltodextrin have no obvious difference. Dextrin is selected as an auxiliary material because of low price and better water solubility.

4.2 Adhesives species investigation

Through preliminary experiments, 35% ethanol, 55% ethanol, 75% ethanol and 20% PVP-K30 ethanol solution are respectively used as the adhesive, the extract powder and dextrin are uniformly mixed in equal proportion, one-step granulation is carried out, the type of the adhesive is investigated by taking the forming rate and the granulation condition as evaluation indexes, and the result is shown in Table 14.

TABLE 14 adhesive type selection

The result shows that when the 35 percent ethanol is used as the adhesive, the adhesive has excessive water content and is not easy to volatilize, so that the substrate is agglomerated and is easy to collapse, and the molding rate of the prepared particles is low; when 55% ethanol is used as the adhesive, the fluidization condition is good, the particle size is moderate, and the agglomeration phenomenon is avoided; when 75% ethanol solution is used as a bonding agent, the fluidization condition is good, but the obtained particles are fine and fragile, and the particle size is unqualified; when the 20% PVP-K30 ethanol solution is used as a binder, the granule forming rate is high, but the granule forming rate can react with materials, and the aqueous solution of the granule generates floccules, so that the dissolubility is not qualified. So 55% ethanol is selected as the binder.

Study of the 5 th laboratory

Weighing 120g of raw ephedra, 150g of scutellaria baicalensis, 90g of dried ginger, 30g of asarum, 90g of gentian, 120g of roasted myrobalan and 60g of raw liquorice according to the proportion of the prescription, adding 10 times of water for soaking for 30 minutes, adding scutellaria baicalensis before decocting, decocting for 2 times, 1 hour each time, combining decoctions, filtering, and concentrating the filtrate to the density of 1.08-1.18g/ml (thermal test) to obtain a water extraction concentrated solution. Vacuum drying at 60 deg.C, and pulverizing to obtain extract powder. Adding appropriate amount of dextrin into the obtained extract powder, mixing, and granulating with 55% ethanol as binder in an experimental one-step granulator.

5.1 bench test results

According to the optimized preparation process, three batches of small samples of the Qinma granules are prepared, and the results are shown in tables 15 and 16.

TABLE 15 bench test results

TABLE 16 results of transfer rate

And (4) conclusion: the transfer rates of baicalin and ephedrine hydrochloride in the concentrated solution, the extract powder and the granules are all more than 50 percent, and the finished product rate of the preparation is all more than 80 percent, which proves that the preparation process is stable and feasible.

5.2 one-step granulation Process Studies

According to the early-stage preliminary experiment results, the parameters of the one-step granulating machine in the granulating process are properly adjusted to obtain specific parameter conditions of the one-step granulating process, which are shown in table 17.

TABLE 17 one-step granulation Process

In conclusion, the one-step granulation is carried out under the condition, the prepared granules have consistent sizes, high forming rate and short preparation time, and can be used for pilot scale-up tests.

6 pilot study

6.1 Pilot plant test Process

Weighing 4.2kg of raw ephedra herb, 5.25kg of scutellaria baicalensis, 3.15kg of dried ginger, 1.05kg of asarum, 3.15kg of gentian, 4.2kg of stewed myrobalan and 2.1kg of raw liquorice according to the proportion of the prescription, adding 10 times of water for soaking for 0.5 hour, adding the scutellaria baicalensis before decocting, decocting for 2 times, 1 hour each time, filtering, combining filtrates, and concentrating under reduced pressure until the density is 1.08-1.18g/ml (measured thermally), thereby obtaining an aqueous extract concentrated solution. Vacuum drying at 60 deg.C, and pulverizing to obtain extract powder. Adding appropriate amount of dextrin into the obtained extract powder, mixing, and granulating with 55% ethanol as binder. Granulating and packaging to obtain QINMA granule with a packaging amount of 10g per bag, and the process flow chart is shown in figure 4.

6.2 Pilot test results

Three batches of the sisal particles were prepared according to the method of "6.1", and the results are shown in tables 18 and 19.

TABLE 18 results of pilot plant test

Table 19 pilot plant test results of the preparation process

And (4) conclusion: the transfer rates of scutelloside and ephedrine hydrochloride in the concentrated solution and the extract powder of the three batches of pilot samples are both more than 50 percent, and the finished product rate of the preparation is both more than 80 percent, which proves that the process is stable and feasible.

Example 9 determination of the content of baicalin and ephedrine hydrochloride in Qinma granules

1 determination of baicalin content

1.1 chromatographic conditions

1.2 preparation method of test solution of Qinma granule

Taking about 0.2g of the Qinma granules prepared by the best process in the embodiment 8, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 75% methanol respectively, weighing, carrying out ultrasonic treatment (power 300W, frequency 25kHz) for 20 minutes, cooling, supplementing weight, carrying out high-speed centrifugation (15000rmp, 10min), and taking supernatant to obtain the product.

1.3 preparation of control solutions

Precisely weighing a proper amount of baicalin reference substance solution, placing the solution into a 25ml volumetric flask, adding 75% methanol to a constant volume, and preparing reference substance solution containing 325.4 mu g of baicalin in each 1 ml.

1.4 measurement results of Qinma granule sample

Taking three batches of Qinma granule samples to prepare a test solution according to the method under the item '1.2', respectively sucking 10 mu L of each of a high-concentration (325.4 mu g/ml) reference solution, a low-concentration (3.254 mu g/ml) reference solution and the test solution, injecting into HPLC, calculating the content of baicalin in the samples, calculating the transfer rate, and finding the result in a table 20.

TABLE 20 determination of baicalin in Qinma granule (n ═ 3)

The results in Table 20 show that the amount of baicalin contained in each pack of granules is 272.32-294.04 mg, and the content of baicalin in the Qinmen granules is initially set to be not less than 250mg per pack.

2 determination of ephedrine hydrochloride content

2.1 chromatographic conditions

A chromatographic column: mercury Ether-phenyl5 μm, 4.6X 250mm (Guangzhou Philomen scientific instruments, Inc.); acetonitrile is used as a mobile phase A: 0.3% triethylamine (containing 0.02 mol/potassium dihydrogen phosphate, pH 3 adjusted with phosphoric acid) in water as mobile phase B (2:98), and isocratic elution for 25 min; the detection wavelength is 210nm, the sample injection amount is 2 muL, the column temperature is 25 ℃, and the flow rate is 1.0 ml/min. Under the condition, the ephedrine hydrochloride can be completely separated from other components (R is more than or equal to 1.5), and the number of theoretical plates is not less than 2500 calculated according to the ephedrine hydrochloride peak.

2.2 preparation of the sample solution of Qinma granule

Weighing about 0.5g of radix Scutellariae and folium Sesami, accurately weighing, placing into conical flask with plug, accurately adding 10ml of 50% methanol, weighing, ultrasonic processing (power 300W, frequency 25kHz) for 30 min, cooling, and supplementing weight.

2.3 preparation of control solutions

Precisely weighing appropriate amount of ephedrine hydrochloride reference solution, placing in 25ml volumetric flask, adding 50% methanol to constant volume, and preparing into 1258 μ g ephedrine hydrochloride reference solution per 1 ml.

2.4 results of sample measurement

Taking three batches of Qinma granule samples to prepare a sample solution according to the method under the item '2.2', respectively sucking 2 mu L of each of a high-concentration (1258 mu g/ml) reference solution, a low-concentration (25.16 mu g/ml) reference solution and the sample solution, injecting into HPLC, calculating the content of ephedrine hydrochloride in the samples, calculating the transfer rate, and finding the result in Table 21.

TABLE 21 determination of ephedrine hydrochloride in Qinma granule (n ═ 3)

The results in Table 21 show that the ephedrine hydrochloride transfer rate in three batches of Qin Ma granule samples is greater than 50%, the ephedrine hydrochloride content in each granule is 21.69-23.88 mg, and the ephedrine hydrochloride content in the Qin Ma granule is not less than 18 mg.

Example 10 establishment of fingerprint of Qinhua granule

1 chromatographic conditions

Agilent ZORBAX RRHD SB-C18 column (2.1 mm. times.50 mm, 1.8 μm); a pre-pillar 1290 definition In-Line Filter; acetonitrile is taken as a mobile phase A, 0.3 percent phosphoric acid water (containing 0.2 percent triethylamine) is taken as a mobile phase B, and gradient elution is carried out according to the specification of a table 22; the flow rate is 0.4 ml/min; the sample amount is 2 mul; the column temperature was 25 ℃; the detection wavelength was 210 nm.

TABLE 22 radix Scutellariae, fructus Cannabis, and radix Scutellariae fingerprint mobile phase

2 chromatographic Condition optimization

Different mobile phase conditions were compared: a. acetonitrile is taken as a mobile phase A, and water is taken as a mobile phase B; b. acetonitrile is used as a mobile phase A, 0.1% phosphoric acid water is used as a mobile phase B, and the pH value is 1.99; c. acetonitrile is taken as a mobile phase A, 0.1% formic acid water is taken as a mobile phase B, and the pH value is 2.38; d. acetonitrile is used as a mobile phase A, 0.2% phosphoric acid water (containing 0.2% triethylamine) is used as a mobile phase B, and the pH value is 2.43; e. the results of the separation of the compounds in the formula of Qinhima with acetonitrile as the mobile phase A and 0.3% phosphoric acid water (containing 0.2% triethylamine) as the mobile phase B at pH2.10 show that the chromatographic peaks can achieve baseline separation and the best separation effect with acetonitrile as the mobile phase A and 0.3% phosphoric acid water (containing 0.2% triethylamine) as the mobile phase B at pH 2.10.

3 preparation of Mixed control solution

Taking appropriate amount of gallic acid, ephedrine hydrochloride, pseudoephedrine hydrochloride, liquiritin, baicalin, wogonin, baicalein, and wogonin reference substances, precisely weighing, and adding methanol to obtain mixed reference substance solution containing gallic acid 40 μ g, ephedrine hydrochloride 33 μ g, pseudoephedrine hydrochloride 33 μ g, liquiritin 24 μ g, baicalin 60 μ g, wogonin 30 μ g, baicalein 30 μ g, and wogonin 30 μ g per 1 ml.

4 preparation of test solution

Taking about 0.5g of the Qinma granules prepared by the best process in the embodiment 8, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of 50% methanol, weighing, carrying out ultrasonic treatment (power 300W and frequency 25kHz) for 30 minutes, cooling and then supplementing weight to obtain the Qinma granules.

5 selection and Structure determination of common peaks

By comparing the fingerprint spectrums of the concentrated solution sample, the dry extract sample and the particle sample, the peak emergence time, the peak area and the separation degree are used as indexes, and 18 common peaks are determined. The total area of the common peaks is calculated to be more than 80 percent of the total peak area. By comparison with a control, the structure of 8 common peaks was determined, peak 1 being gallic acid, peak 2 being ephedrine hydrochloride, peak 3 being pseudoephedrine hydrochloride, peak 11 being liquiritin, peak 13 being baicalin, peak 16 being wogonoside, peak 17 being baicalein, and peak 18 being wogonoside. Wherein, the baicalin content is the highest, the separation degree is good, the retention time is proper, so the baicalin content is selected as a reference peak. See fig. 5.

6 comparison fingerprint map generation

Preparing 10 batches of radix Scutellariae granule test sample according to the preparation method of the test sample solution under item "3", measuring according to the chromatographic condition under item "1", guiding the above 10 batches of radix Scutellariae granule fingerprints into AIA file, introducing into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and simulating the control fingerprint of radix Scutellariae granule. See fig. 6.

7 methodology examination

7.1 fingerprint similarity evaluation mode

7.1.1 precision test

Preparing the sample according to the preparation method of the sample solution under item "3", continuously injecting sample for 6 times under the chromatographic condition under item "1", and recording the UPLC chromatogram. And similarity is calculated by adopting traditional Chinese medicine chromatogram fingerprint similarity evaluation system software, and the result is shown in a table 23.

TABLE 23 calculation of similarity of Qinlanfang fingerprint precision test

The result shows that the similarity of 6 fingerprints is 1.000, which indicates that the precision of the instrument is better.

7.1.2 repeatability test

Preparing 6 parts of sample solution in parallel according to the preparation method of the sample solution under the item '3', carrying out sample injection measurement according to the chromatographic condition under the item '1', and recording the UPLC chromatogram. And similarity is calculated by adopting traditional Chinese medicine chromatogram fingerprint similarity evaluation system software, and the result is shown in a table 24.

TABLE 24 calculation results of similarity of Qinlian prescription fingerprint repeatability tests

The result shows that the similarity of 6 fingerprints is 1.000, which indicates that the repeatability of the method is better.

7.1.3 stability test

Preparing the sample according to the preparation method of the sample solution under item "3", performing sample injection measurement under the chromatographic condition under item "1" for 0, 2, 4, 6, 8, 10, 12 and 24h, and recording the UPLC chromatogram. And similarity is calculated by adopting traditional Chinese medicine chromatogram fingerprint similarity evaluation system software, and the result is shown in a table 25.

TABLE 25 calculation of similarity of Qinlanfang fingerprint stability test

The result shows that the similarity of 8 fingerprint spectrums is 1.000, which indicates that the stability of the test solution is good within 24 hours.

7.2 evaluation mode of common peaks of fingerprint

7.2.1 precision test

The sample is prepared according to the preparation method of the sample solution under the item '3', the sample is continuously injected for 6 times under the chromatographic condition under the item '1', the baicalin chromatographic peak (No. 13 peak) is taken as a reference peak, the relative retention time of each characteristic peak and the average value and RSD value of the relative peak area in the liquid chromatogram are calculated, and the results are shown in a table 26, a table 27 and a table 28.

TABLE 26 common peak relative retention time of precision test of Qinlima prescription

TABLE 27 common peak relative peak area in precision test of Qinlima prescription

TABLE 28 percentage of total area of common peaks to total peak area (%)

The result shows that the total peak area of 18 common peaks in the liquid chromatogram of the Qinmen sample is more than 79%. The RSD of the relative retention time of each common peak is less than 0.3 percent, and the RSD of the relative peak area is less than 2.2 percent, which indicates that the precision of the instrument is better.

7.2.2 repeatability test

6 parts of test solution are prepared in parallel according to the preparation method of the test solution under the item '3', the sample injection determination is carried out according to the chromatographic condition under the item '1', the baicalin chromatographic peak (peak No. 13) is taken as a reference peak, the relative retention time of each characteristic peak and the average value and RSD value of the relative peak area in the liquid chromatogram are calculated, and the results are shown in a table 29, a table 30 and a table 31.

TABLE 29 common peak relative retention time for Qinlima's repeatability tests

TABLE 30 relative peak area of common peak in Qinlima's repeatability test

TABLE 31 percentage of total area of common peaks to total peak area (%)

The result shows that the area of 18 common peak peaks in the liquid chromatogram of the Qinmen sample accounts for more than 80% of the total peak area. The RSD of the relative retention time of each common peak is less than 0.4%, and the RSD of the relative peak area is less than 10.1%, which indicates that the method has good repeatability.

7.2.3 stability test

Preparing the sample according to the preparation method of the sample solution under the item '3', carrying out sample injection measurement under the chromatographic condition under the item '1' for 0, 2, 4, 8, 12 and 24 hours, taking the baicalin chromatographic peak (the No. 13 peak) as a reference peak, and calculating the relative retention time of each characteristic peak in the liquid chromatogram, the average value of the relative peak area and the RSD value, wherein the results are shown in tables 32, 33 and 34.

TABLE 32 common peak relative retention time of stability test of Qinlima formula

TABLE 33 relative peak area of common peak in stability test of Qinwan prescription

TABLE 34 percentage of total area of common peaks to total peak area (%)

The result shows that the total peak area of 18 common peaks in the liquid chromatogram of the Qinmen sample is more than 79%. The RSD of the relative retention time of each common peak is less than 0.5 percent, and the RSD of the relative peak area is less than 6.5 percent, which indicates that the test solution has good stability within 24 hours.

8 calculation of Qinlun granule fingerprint

Fingerprint spectrum research of 10 batches of Qinlian granules is carried out by adopting the Qinlian granule fingerprint spectrum technology, and the similarity of the Qinlian granule fingerprint spectrum is calculated.

8.1 fingerprint similarity evaluation mode

Preparing the sample according to the preparation method of the sample solution under the item '3', and recording the UPLC chromatogram according to the chromatographic condition under the item '1'. And similarity is calculated by adopting traditional Chinese medicine chromatogram fingerprint similarity evaluation system software, and the result is shown in figure 7 and table 35.

Table 3510 batches of Qinlanfang granule sample fingerprint similarity calculation results

The result shows that the similarity range of the liquid chromatogram fingerprints of 10 batches of radix scutellariae barbatae particles is between 0.964 and 1, and the similarity of the liquid chromatogram fingerprints of the radix scutellariae barbatae particles is preliminarily determined to be more than 0.95.

8.2 evaluation mode of common peaks of fingerprint

Preparing the sample according to the preparation method of the sample solution under item "3", performing sample injection measurement under the chromatographic condition under item "1", taking baicalin chromatographic peak (peak 13) as a reference peak, and calculating the average value of the relative retention time of each characteristic peak and RSD value in the liquid chromatogram, wherein the results are shown in tables 36 and 37.

TABLE 36 common peak relative retention time of the Qin hemp square samples

TABLE 37 percentage of total area of common peaks to total peak area (%)

The results show that the liquid chromatography fingerprints of 10 batches of the qinma particles have 18 common chromatographic peaks, the average relative retention time is 0.058(1), 0.103(2), 0.119(3), 0.436(4), 0.516(5), 0.566(6), 0.591(7), 0.602(8), 0.625(9), 0.672(10), 0.714(11), 0.874(12), 1.000(S), 1.074(14), 1.130(15), 1.168(16), 1.279(17) and 1.538(18), and the relative deviation is less than +/-10%. The ratio of the total area of the common peaks to the total peak area is not less than 80%.

Example 11 Studies on the content determination of 10 chemical components in Qinma granules

Various chemical components in the formula of Qinhma have pharmacological effects, for example, ephedrine hydrochloride and pseudoephedrine hydrochloride are proved to be capable of relieving bronchial smooth muscle and have anti-inflammatory and antiviral effects. The Scutellariae radix total flavone has the effect of relieving pulmonary fibrosis. In addition, monoammonium glycyrrhizinate, gallic acid, liquiritin, 6-gingerol, sesamin and asaricin are reported to have antipyretic and anti-inflammatory effects. Therefore, the method for simultaneously measuring the contents of 12 components in 7 medicinal materials of the formula of Qinmao is established, can be used for measuring the contents of a water extract concentrated solution, a dry extract and a particle sample in the preparation process of Qinmao particles, and compares the content change conditions of the components in each sample.

1 chromatographic conditions

Agilent ZORBAX RRHD SB-C18 column (2.1 mm. times.50 mm, 1.8 μm); a pre-pillar 1290 definition In-Line Filter; acetonitrile is taken as a mobile phase A, 0.3 percent phosphoric acid water (containing 0.2 percent triethylamine) is taken as a mobile phase B, and gradient elution is carried out according to the specification of a table 38; the flow rate is 0.4 ml/min; the sample amount is 2 mul; the column temperature was 25 ℃; the detection wavelength is 210nm (gallic acid, ephedrine hydrochloride and pseudoephedrine hydrochloride), 249nm (ammonium glycyrrhizinate), 271nm (gentiopicroside), 275nm (wogonin and baicalein), 278nm (baicalin and wogonin), 280nm (6-gingerol), 287nm (sesamin and asaricin).

TABLE 38 determination of multicomponent content of radix Scutellariae and fructus Cannabis mobile phase

2 optimization of chromatographic conditions

3 preparation of control solutions

Taking 2.15mg of gallic acid, 385 mu g of ephedrine hydrochloride, 397 mu g of pseudoephedrine hydrochloride, 340 mu g of gentiopicroside, 5.59mg of baicalin, 2.02mg of wogonoside, 279.5 mu g of baicalein, 249.5 mu g of wogonin, 213 mu g of 6-gingerol, 251 mu g of ammonium glycyrrhizinate, 287 mu g of sesamin and 243.5 mu g of asaricin, precisely weighing, placing in a 10ml volumetric flask, adding methanol to fix the volume to prepare the product with the mass concentration of 215 mu g/ml of gallic acid, 38.5 mu g/ml of ephedrine hydrochloride, 39.7 mu g/ml of pseudoephedrine hydrochloride, 34 mu g/ml of gentiopicroside, 559 mu g/ml of baicalin, 202 mu g/ml of wogonoside, 27.95 mu g/ml of baicalein, 24.95 mu g/ml of wogonin, 21.3 mu g/ml of 6-gingerol, 21.1 mu g/ml of ammonium glycyrrhizin and 28.7 mu g/ml of sesamin, a solution of asaricin 24.35. mu.g/ml was used as a mother liquor.

4 preparation of test solution

Taking 0.5g of the Qinma granules prepared according to the best process of the embodiment 8, precisely weighing, putting into a 20ml volumetric flask, adding 50% methanol, ultrasonically dissolving, fixing the volume and shaking up to obtain the medicine.

5 methodological investigation

The established method for simultaneously measuring the contents of 12 components in the Qinmao prescription is verified by methodology, and mainly verified by linear relation (lowest detection limit and lowest quantification limit), precision (precision within day and during day), repeatability, stability and sample adding and recycling tests.

5.1 systematic Adaptation test

Precisely absorbing the mixed standard solution under the chromatographic condition of the item 1, and injecting the mixed standard solution into an UPLC liquid chromatograph. The chromatogram of the mixture of 12 components is shown in FIG. 8.

5.2 Linear relationship and repeatability test

Taking the mother liquor under the item '3', gradually diluting the mother liquor with a 50% methanol solution, and preparing standard substance solutions with different concentrations. Mu.l of the control solution of each concentration was pipetted into UPLC, and a standard curve was drawn with the mixed standard concentration (. mu.g/ml) as abscissa and the peak area as ordinate. All components showed good linearity (r > 0.9995) over the range of the assay. The lowest detection limit and lowest quantitation limit were located with signal-to-noise ratios (S/N) of 3 and 10, respectively. To examine the reproducibility of the method, test solutions were prepared according to the method under item "3", 6 portions were treated in parallel, and measured according to the chromatographic conditions under item "1". The results are shown in Table 39.

TABLE 39 Linear relationship and repeatability test results

5.3 precision test

Precisely absorbing 2 mul of mixed standard solution with high, medium and low concentrations respectively, injecting into UPLC, measuring according to chromatographic condition under item 1, and continuously repeating sample injection for 6 times for 3 days. As a result, the intra-day precision RSD was less than 3.0%, and the inter-day precision RSD was less than 3.0%, as shown in Table 40.

TABLE 40 precision test results

5.4 sample application recovery and stability test

Taking methanol ultrasonic sample liquid with known content, respectively adding reference substances with equivalent sample component content of 80%, 100% and 120%, and performing sample adding recovery test on 3 parts in parallel; the same sample solution was taken and left at room temperature for 0, 2, 4, 6, 8, 10, 12, 24h for stability examination, and the results are shown in Table 41, measured according to the chromatographic conditions under item "1".

TABLE 41 sample recovery and stability test results

6 sample determination

The sample solution was prepared according to the method under item "4", and the contents of the respective components in the Qinhua granules were measured according to the chromatographic conditions under item "1", and the results are shown in Table 42.

TABLE 42 measurement results of contents of respective components in the sample

As can be seen from the table above, the method can effectively determine the content of 12 components in the Qinmao square granules, and meanwhile, the method can also be used for detecting the transfer rate of different components in the Qinmao square preparation process and observing the influence of the extraction method on the extraction rate of different chemical components in the Qinmao square.

Monitoring of 7 Qinwan preparation process

The best process of example 8 was used to prepare the Qinma granules, the contents of 12 ingredients such as gallic acid in the concentrated solution sample, the dry extract sample and the granule sample during the preparation of the Qinma formula were measured, and the transfer rates of the ingredients were calculated, and the results are shown in Table 43.

TABLE 43 measurement results of sample content

The results show that in the experiment, due to the fact that most of samples are low in sesamin and asarum lignan content, other 10 components can be accurately quantified, and the results show that the dry part of the water extract of the formula of the scutellaria baicalensis is reduced, the transfer rate is 81.11% -89.32%, wherein the transfer rate of gallic acid, wogonin and ammonium glycyrrhetate is more than 85%, and the transfer rate of other components is 79.53% -84.69%. Meanwhile, most chemical components are lost after granulation, and the loss of raw materials in the granulation process is mainly considered. Therefore, the method for measuring the content of 12 components in the formula of Qinmen can be effectively used for quality monitoring in the preparation process of the formula of Qinmen.

The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and additions can be made without departing from the method of the present invention, and these modifications and additions should also be regarded as the protection scope of the present invention.

Claims

1. The traditional Chinese medicine composition for treating intractable cough is characterized by being prepared from the following raw material medicines in parts by weight: 10-13 parts of raw ephedra, 12-16 parts of scutellaria baicalensis, 9-13 parts of dried ginger, 3-10 parts of asarum, 9-12 parts of gentian, 10-15 parts of roasted myrobalan and 6-13 parts of raw liquorice.

2. A preparation process of Qinma granules is characterized by comprising the following steps: weighing the following medicinal materials in parts by weight: 10-13 parts of raw ephedra, 12-16 parts of scutellaria baicalensis, 9-13 parts of dried ginger, 3-10 parts of asarum, 9-12 parts of gentian, 10-15 parts of stewed myrobalan and 6-13 parts of raw liquorice, adding 10 times of water into the six medicinal materials except the scutellaria baicalensis, soaking for 30 minutes, adding the scutellaria baicalensis before decoction, decocting for 2 times, 1 hour each time, combining decoction, filtering, and concentrating the filtrate to the density of 1.08-1.18g/ml (measured thermally), thereby obtaining an aqueous extract concentrated solution.

3. The process for preparing Qinwan granules according to claim 2, characterized by further comprising the following steps: vacuum drying and crushing the water extraction concentrated solution at 60 ℃ to obtain extract powder; adding a proper amount of dextrin into the obtained extract powder, uniformly mixing, and taking 55% ethanol as a binder to prepare the Qinma granules.

4. A method for establishing a Qinma granule fingerprint spectrum is characterized by comprising the following steps:

5. the method for establishing the qinma granule fingerprint spectrum according to claim 4, wherein the chromatographic conditions further comprise: the flow rate is 0.4 ml/min; the column temperature was 25 ℃; the detection wavelength was 210 nm.

6. A quality control method of a radix scutellariae and ramie particle sample is characterized by comprising the following steps:

(A) establishing a standard fingerprint of the Qinma granules according to the establishing method of the Qinma granule fingerprint of the claim 4 or 5;

(B) detecting a scutellaria baicalensis and semen cannabis granule sample to be detected according to the establishment method of the scutellaria baicalensis and semen cannabis granule fingerprint spectrum of claim 4 or 5, and establishing the fingerprint spectrum of the scutellaria baicalensis and semen cannabis granule sample to be detected;

7. The quality control method for a Qin hemp particle sample according to claim 6, characterized in that in step (C), it is determined whether the fingerprint of the Qin hemp particle sample to be detected contains 18 chromatographic peaks having a relative retention time not more than ± 10% deviation from 0.058(1), 0.103(2), 0.119(3), 0.436(4), 0.516(5), 0.566(6), 0.591(7), 0.602(8), 0.625(9), 0.672(10), 0.714(11), 0.874(12), 1.000(S), 1.074(14), 1.130(15), 1.168(16), 1.279(17) and 1.538 (18).

8. A method for simultaneously determining the contents of multiple components in a formula sample of radix scutellariae is characterized in that the contents of gallic acid, ephedrine hydrochloride, pseudoephedrine hydrochloride, gentiopicrin, baicalin, wogonoside, baicalein, wogonin, 6-gingerol, ammonium glycyrrhizinate, sesamin and asafetida are simultaneously determined, the formula sample of radix scutellariae is subjected to high performance liquid chromatography analysis, and the chromatographic conditions are as follows: c₁₈The chromatographic column uses acetonitrile as a mobile phase A and 0.3% phosphoric acid water (containing 0.2% triethylamine) as a mobile phase B, and the elution procedure is as follows:

9. the method for simultaneously determining the contents of multiple components in a Qinwang sample according to claim 8, wherein the chromatographic conditions further comprise: the flow rate is 0.4 ml/min; the column temperature was 25 ℃; the detection wavelength is 210nm (gallic acid, ephedrine hydrochloride and pseudoephedrine hydrochloride), 249nm (ammonium glycyrrhizinate), 271nm (gentiopicroside), 275nm (wogonin and baicalein), 278nm (baicalin and wogonin), 280nm (6-gingerol), 287nm (sesamin and asaricin).

10. A quality control method of a Qinhua sample, which is characterized by comprising the step of measuring the contents of the components in the Qinhua sample according to the method for simultaneously measuring the contents of the components in the Qinhua sample as claimed in claim 8 or 9.