CN115837063B

CN115837063B - Preparation and detection methods of lung force cough pharmaceutical composition

Info

Publication number: CN115837063B
Application number: CN202211690239.7A
Authority: CN
Inventors: 孙小鹦; 廖萍; 廖伟; 廖忠
Original assignee: GUIZHOU JIANXING PHARMACEUTICAL CO Ltd
Current assignee: GUIZHOU JIANXING PHARMACEUTICAL CO Ltd
Priority date: 2022-12-27
Filing date: 2022-12-27
Publication date: 2023-12-08
Anticipated expiration: 2042-12-27
Also published as: CN115837063A

Abstract

The invention relates to a preparation and detection method of a lung force cough pharmaceutical composition, which comprises the steps of crushing and separately extracting baikal skullcap root, radix peucedani and radix stemonae in a prescription, mixing and decocting gentian carthami, chinese phoenix tree root, oldenlandia diffusa and red tube medicine in water, and finally adding auxiliary materials into the obtained extract to prepare a preparation. The preparation method is simple and easy to implement, has low energy consumption and high production benefit, not only improves the content of the effective components, but also avoids the damage of the effective components in the medicine; on the basis of the original standard, thin-layer identification of the baical skullcap root is revised, thin-layer identification of the safflower gentian and oldenlandia diffusa medicinal materials is newly established, and the content measurement and control project of the baicalin is revised again. The method is simple and accurate, and the quality of the lung force cough capsule can be effectively controlled by the standard through the examination of the sample.

Description

Preparation and detection methods of lung force cough pharmaceutical composition

Technical Field

The invention belongs to the technical field of traditional Chinese medicine pharmacy, and particularly relates to a preparation and detection method of a lung force cough pharmaceutical composition.

Background

The lung force cough capsule is a product of Guizhou Jianxing pharmaceutical industry Co., ltd, and has the effects of relieving cough and asthma, clearing heat and detoxicating, guiding qi downward and eliminating phlegm. It can be used for treating cough, asthma, excessive phlegm, dyspnea, acute and chronic bronchitis, and emphysema.

The lung force cough capsule is received in WS-10217 (ZD-0217) -2002, and the original detection items in the standard are: a trait; thin-layer identification of radix scutellariae, thin-layer identification of radix peucedani and thin-layer identification of radix stemonae; and (5) measuring the content of baicalin.

[ PREPARATION ] radix Scutellariae 173g, radix Peucedani 167g, radix Stemonae 160g, radix Gentianae carthami 150g, radix Firmianae 130g, herba Hedyotidis Diffusae 120g, and radix Ginseng Rubra 96g; the preparation method comprises the steps of taking 30g of radix stemonae, crushing the radix stemonae into fine powder for later use, decocting the rest of radix stemonae and the rest of radix scutellariae and the like with water for two times, wherein the first time is 2 hours, the second time is 1.5 hours, merging decoction, filtering, concentrating filtrate into clear paste with the relative density of 1.17-1.20 (80 ℃), adding the fine powder, mixing uniformly, drying, crushing and encapsulating.

Chinese invention CN201910165098.9 capsule extract for pulmonary cough, its use and quality control method, applicant, guizhou Jianxing pharmaceutical industry limited, the disclosed preparation method is as follows: the lung-force cough capsule extract is characterized by being prepared by the following steps: taking radix Scutellariae, radix Peucedani, radix Stemonae, radix Gentianae, radix Firmianae, radix Rhodiolae, and herba Hedyotidis Diffusae according to the prescription; pulverizing Scutellariae radix, and sieving with 16mm sieve plate; and then taking part of radix stemonae, crushing the part of radix stemonae into fine powder with the particle size smaller than 80 meshes for standby, adding boiling water into the rest six materials for decoction twice, adding boiling water which is 8 times of the weight of the medicinal materials for decoction for 2 hours for the first time, adding boiling water which is 6 times of the weight of the medicinal materials for decoction for 1.5 hours for the second time, merging the decoctions, filtering, and deeply shrinking the filtrate to obtain the clear paste with the relative density of 1.17-1.20 (80 ℃).

The prior art has the following defects: (1) The yield of the water decoction extract is not ideal enough, the content of active ingredients is low, and the baicalin conversion rate is low; (2) In the formula, the medicinal materials such as stemona root and the like mainly contain alkaloid effective components, the alkaloid property is unstable, and the structure is easy to change under the condition of heating and decocting, so that the effective components are damaged, and the content of the effective components is reduced; (3) In the detection project, thin-layer identification spots of the radix scutellariae are blurred, the reproducibility is poor, and the judgment of the detection result is affected; the content of baicalin is measured, the stability is poor, and the condition of poor separation degree of adjacent peaks can occur; (4) In the detection project, thin-layer chromatography identification inspection is not carried out on the gentiana carthami and oldenlandia diffusa.

In order to solve the problems, the quality of the product is effectively improved, the quality standard of the lung force cough capsule is improved, and the preparation process of the lung force cough source is improved and the quality detection and research of the lung force cough source are simultaneously carried out. The preparation method and the detection method of the lung force cough pharmaceutical composition are established by taking main active ingredients in the prescription as evaluation indexes, examining the controllability of the prescription process and quality and combining with the standard of the original lung force cough capsule for deep research and development. The preparation method is simple, has high production benefit, not only improves the content of effective components and the conversion rate of baicalin, but also avoids the destruction of main alkaloid components of medicinal materials such as radix Stemonae. The detection method improves thin layer identification and content measurement of Scutellariae radix in original standard, increases thin layer identification of Carthami flos radix and herba Hedyotidis Diffusae in FEILITE Capsule, can effectively control product quality, and further improves product quality standard.

Disclosure of Invention

The invention aims to provide a preparation method of a lung force cough pharmaceutical composition.

The invention also aims to provide a detection method of the lung force cough pharmaceutical composition.

The invention relates to a preparation method of a lung force cough pharmaceutical composition, which comprises 173g of radix scutellariae, 167g of radix peucedani, 160g of radix stemonae, 150g of safflower and gentian, 130g of Chinese parasol root, 120g of oldenlandia diffusa and 96g of red tube medicine, and the preparation method comprises the following steps:

(1) Preparation of fine powder of radix Scutellariae, radix Peucedani and radix Stemonae: pulverizing Scutellariae radix, radix Peucedani, and radix Stemonae respectively, and sieving with 80-120 mesh sieve;

(2) Preparation of traditional Chinese medicine extract:

(1) preparation of the radix Scutellariae extract: soaking the fine powder of the scutellaria baicalensis obtained in the step (1) in 50-90% ethanol solution with the mass-volume ratio of 1:8-12 and preheated to 30 ℃ for 60min, performing ultrasonic extraction for 30-60min, filtering at the extraction temperature of 30-60 ℃, recovering ethanol from the filtrate, concentrating under reduced pressure to obtain an extract with the relative density of 1.20-1.30 at 50 ℃, and then drying under reduced pressure at the vacuum degree of 0.8-0.9mpa and 60 ℃ to obtain a dry extract of the scutellaria baicalensis;

(2) preparation of Peucedanum root extract: soaking the radix Peucedani Hu Xifen obtained in the step (1) in 50-90% ethanol solution with the mass-volume ratio of 1:8-12 and preheated to 30 ℃ for 60min, ultrasonically extracting for 30-60min, filtering at the extraction temperature of 30-60 ℃, recovering ethanol from the filtrate, concentrating under reduced pressure to obtain extract with the relative density of 1.20-1.30 at 50 ℃, and drying the extract under reduced pressure at the vacuum degree of 0.8-0.9mpa and 60 ℃ to obtain radix Peucedani extract;

(3) Preparation of Stemonae radix extract: soaking the radix stemonae fine powder obtained in the step (1) in 50-90% ethanol solution with the mass volume ratio of 1:8-12 and preheated to 30 ℃ for 60min, performing ultrasonic extraction for 30-60min, filtering at the extraction temperature of 30-60 ℃, recovering ethanol from the filtrate, concentrating under reduced pressure to obtain extract with the relative density of 1.20-1.30 at 50 ℃, and drying the extract under reduced pressure at the vacuum degree of 0.8-0.9mpa and 60 ℃ to obtain a dry extract;

(4) preparation of extracts of radix Gentianae, radix Firmianae, herba Hedyotidis Diffusae and radix Rhodiolae: mixing four medicinal materials of gentian carthami, chinese phoenix tree root, oldenlandia diffusa and red tube, adding water which is 8-12 times of the total amount of the four medicinal materials, heating and decocting for two times, wherein the first time of decoction is 1.5-2.5 hours, the second time of decoction is 1.0-2.0 hours, merging the two decoctions, filtering, concentrating the filtrate under reduced pressure to an extract with the relative density of 1.07-1.10 at 50 ℃, and drying the extract under reduced pressure at the vacuum degree of 0.8-0.9mpa and the temperature of 60 ℃ to obtain a dry extract of the four medicinal materials;

(3) Preparation of the capsules: pulverizing the above extracts, adding starch 10-20% of the total amount of the extracts, mixing, granulating with 85-95% ethanol, sieving with 20-35 mesh sieve, drying at 40-60deg.C, and encapsulating.

Further, the preparation method of the invention comprises the following steps:

(1) Preparation of fine powder of radix Scutellariae, radix Peucedani and radix Stemonae: pulverizing Scutellariae radix, radix Peucedani, and radix Stemonae respectively, and sieving with 100 mesh sieve;

(2) Preparation of traditional Chinese medicine extract:

(1) preparation of the radix Scutellariae extract: soaking the fine powder of the scutellaria baicalensis obtained in the step (1) in 70% ethanol solution with the mass-volume ratio of 1:10 and the preheating temperature of 30 ℃ for 60min, carrying out ultrasonic extraction for 45min, filtering at the extraction temperature of 60 ℃, recovering ethanol from the filtrate, concentrating the filtrate under reduced pressure to obtain an extract with the relative density of 1.20-1.30 at the temperature of 50 ℃, and then drying the extract under reduced pressure at the vacuum degree of 0.8-0.9mpa and the temperature of 60 ℃ to obtain a dry extract of the scutellaria baicalensis;

(2) preparation of Peucedanum root extract: soaking Hu Xifen obtained in the step (1) in 70% ethanol solution with a mass-volume ratio of 1:8 and preheated to 30deg.C for 60min, ultrasonically extracting for 30min at 60deg.C, filtering, recovering ethanol from the filtrate, concentrating under reduced pressure to obtain extract with relative density of 1.20-1.30 at 50deg.C, and oven drying under reduced pressure at 60 deg.C under vacuum degree of 0.8-0.9mpa to obtain radix Peucedani extract;

(3) preparation of Stemonae radix extract: soaking the radix stemonae fine powder obtained in the step (1) in 70% ethanol solution with the mass-volume ratio of 1:10 and preheated to 30 ℃ for 60min, carrying out ultrasonic treatment for 45min, extracting at the temperature of 45 ℃, filtering, recovering ethanol from the filtrate, concentrating under reduced pressure to obtain extract with the relative density of 1.20-1.30 at the temperature of 50 ℃, and drying the extract under reduced pressure at the temperature of 60 ℃ under the vacuum degree of 0.8-0.9mpa to obtain a dry extract;

(4) Preparation of extracts of radix Gentianae, radix Firmianae, herba Hedyotidis Diffusae and radix Rhodiolae: mixing four medicinal materials of safflower gentian, chinese phoenix tree root, oldenlandia diffusa and red tube, adding water 10 times of the total amount of the four medicinal materials, heating and decocting for two times, wherein the first time is 2 hours, the second time is 1.5 hours, merging the two decoctions, filtering, concentrating the filtrate under reduced pressure to an extract with the relative density of 1.07-1.10 at 50 ℃, and drying the extract under reduced pressure at the vacuum degree of 0.8-0.9mpa and the temperature of 60 ℃ to obtain a dry extract, wherein the dry extract is a mixed extract of the four medicinal materials;

(3) Preparation of the capsules: pulverizing the above extracts, adding starch 20% of the total amount of the extracts, mixing, granulating with 90% ethanol, sieving with 20-35 mesh sieve, drying at 50deg.C, and encapsulating.

The detection method of the lung force cough pharmaceutical composition provided by the invention comprises the following steps: thin-layer identification of radix Scutellariae, radix Gentianae and herba Hedyotidis Diffusae in the medicine, and determination of baicalin content in the medicine.

The thin-layer identification method of the medicinal materials of the baical skullcap root, the safflower, the gentian and the oldenlandia diffusa comprises the following steps:

(1) Thin layer identification of radix scutellariae medicinal material: taking 1.0-3.0g of sample, adding 40-60mL of 60-80% methanol, heating and refluxing for 0.5-1.5h, filtering, evaporating filtrate to dryness, adding 20-30mL of water into residues to dissolve, adjusting pH value to 2.0-2.5 with 2.5-3.5% hydrochloric acid solution, shaking and extracting for 1-3 times with ethyl acetate, each time of 25-35mL, mixing the extracting solutions, evaporating to dryness, and adding 5mL of methanol into residues to dissolve, thereby obtaining a sample solution; adding methanol into baicalin reference substance to obtain 1mg solution per 1mL as reference substance solution; sucking 4-6 mu L of each of the 2 solutions, and respectively spotting on the same silica gel G thin layer plate according to the proportion of 8-12:1-2:0.5:1-2, developing n-butanol-acetic acid-acetone-water as developing agent, taking out, air drying, spraying 3-10% acid ferric trichloride ethanol solution for developing, and making spots of the same color appear on the positions corresponding to the control material chromatogram and the control material chromatogram in the sample chromatogram;

(2) Thin layer identification of gentian medicinal material of safflower: taking 2.0-4.0g of sample, adding 20-30mL of 50-70% methanol, heating and refluxing for 0.5-1.5h, filtering, evaporating filtrate to dryness, and adding 1mL of methanol into residue to dissolve the residue to obtain a sample solution; additionally, 0.5g of safflower gentian is used as a reference medicine, and the prepared reference medicine solution is prepared by the same method; adding methanol into mangiferin reference substance to prepare a solution containing 1mg per 1mL as reference substance solution; sucking 4-6 mu L of each of the 3 solutions, and respectively spotting on the same silica gel G254 thin layer plate according to the proportion of 11-12:1-1.5:1-2, developing, taking out, and air drying, wherein spots with the same color appear on the positions corresponding to the control medicine chromatogram and the control medicine chromatogram in the sample chromatogram;

(3) Thin layer identification of oldenlandia diffusa medicinal material: taking 3.0-5.0 g of sample, adding 50-70ml of 60-70% methanol, heating and refluxing for 0.5-1.5h, filtering, evaporating filtrate to dryness, and adding 5ml of methanol into residues to dissolve to obtain a sample solution; taking oleanolic acid reference substance, adding methanol to prepare a solution containing 1mg per 1ml, and taking the solution as a reference solution; sucking 3-5 mu L of each of the two solutions, and spotting the two solutions on the same silica gel G thin layer plate according to the proportion of 24-26:6-8:3-5:0.1 petroleum ether (30-60 ℃) and dimethylbenzene-ethanol-glacial acetic acid are taken as developing agents, and are developed, taken out, dried and sprayed with 5-10% sulfuric acid ethanol solution for developing color, and the color spectrum of the sample to be tested shows spots with the same color on the corresponding position of the reference.

Further, the thin layer identification method of the medicinal materials of the baical skullcap root, the safflower, the gentian and the oldenlandia diffusa in the medicament comprises the following steps:

(1) Thin layer identification of radix scutellariae medicinal material: taking 2.0g of a sample, adding 50mL of 70% methanol, heating and refluxing for 1h, filtering, evaporating the filtrate to dryness, adding 25mL of water into the residue to dissolve, adjusting the pH value to 2.2 with 3% hydrochloric acid solution, extracting for 2 times with 30mL of ethyl acetate under shaking, combining the extracting solutions, evaporating to dryness, and adding 5mL of methanol into the residue to dissolve to obtain a sample solution; adding methanol into baicalin reference substance to obtain 1mg solution per 1mL as reference substance solution; the 2 solutions were each drawn 5 μl and spotted onto the same silica gel G thin layer plate in a ratio of 10:2:0.5:2, developing n-butanol-acetic acid-acetone-water as developing agent, taking out, air drying, spraying 5% acid ferric trichloride ethanol solution for developing color, and in the chromatogram of the sample, showing spots with the same color at the positions corresponding to the chromatogram of the reference substance;

(2) Thin layer identification of gentian medicinal material of safflower: taking 3.0g of sample, adding 25mL of 60% methanol, heating and refluxing for 1h, filtering, evaporating filtrate to dryness, and adding 1mL of methanol into residues to dissolve the residues to obtain a sample solution; additionally, 0.5g of safflower gentian is used as a reference medicine, and the prepared reference medicine solution is prepared by the same method; adding methanol into mangiferin reference substance to prepare a solution containing 1mg per 1mL as reference substance solution; 5 mu L of each of the 3 solutions is sucked and respectively spotted on the same silica gel G254 thin layer plate, and the proportion is 12:1.5:1, developing with chloroform-acetone-water as developing agent, taking out, air drying, and making spots of the same color appear on the positions corresponding to the control medicine chromatogram and the control medicine chromatogram in the sample chromatogram;

(3) Thin layer identification of oldenlandia diffusa medicinal material: taking 4.0g of sample, adding 60ml of 70% methanol, heating and refluxing for 1h, filtering, evaporating filtrate to dryness, and dissolving residues with 5ml of methanol to obtain a sample solution; taking oleanolic acid reference substance, adding methanol to prepare a solution containing 1mg per 1ml, and taking the solution as a reference solution; the two solutions were each drawn 4 μl and spotted on the same silica gel G thin layer plate in a ratio of 25:7:4:0.1 petroleum ether (30-60 ℃) and dimethylbenzene-ethanol-glacial acetic acid are taken as developing agents, and are developed, taken out, dried and sprayed with 10% sulfuric acid ethanol solution for developing color, and spots with the same color appear on the corresponding positions of the sample chromatograph.

The method for measuring the content of baicalin in the medicine comprises the following steps:

(1) Preparation of test solution: taking 0.1-0.4g of sample, precisely weighing, placing in a 50ml measuring flask, adding appropriate amount of 60-80% ethanol, performing ultrasonic treatment for 15-45min, cooling, adding 60-80% ethanol to dilute to scale, shaking, filtering, and collecting the subsequent filtrate;

(2) Preparation of a control solution: precisely weighing appropriate amount of baicalin reference substance, and adding methanol to obtain solution containing 70ug of baicalin per 1 mL;

(3) Chromatographic conditions and system suitability test: the chromatographic column is CORTECS UPLC T3.8 um; the column temperature is 25-30 ℃; acetonitrile solution containing 0.01-0.02% glacial acetic acid is used as a mobile phase A, and 0.05-0.15% glacial acetic acid water solution is used as a mobile phase B for gradient elution; the detection wavelength is 275nm; the flow rate is 0.5-0.9mL/min, and the theoretical plate number is not lower than 3500 according to baicalin peak calculation; the gradient elution specifically comprises the following steps:

0-12min, mobile phase A is 20% -36%; 12-20min, wherein the mobile phase A is 36% -48%; 20-30min, wherein the mobile phase A is 48% -57%; 30-45min, wherein the mobile phase A is 57% -48%; 45-55min, wherein the mobile phase A is 48% -24%;

(4) Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.

Further, the method for measuring the content of baicalin in the medicine comprises the following steps:

(1) Preparation of test solution: accurately weighing 0.2g of sample, placing in 50ml measuring flask, adding appropriate amount of 70% ethanol, ultrasonic treating for 30min, cooling, diluting with 70% ethanol to scale, shaking, filtering, and collecting filtrate;

(3) Chromatographic conditions and system suitability test: the chromatographic column is CORTECS UPLC T3.8 um; the column temperature is 30 ℃; acetonitrile solution containing 0.01% glacial acetic acid is taken as a mobile phase A, and 0.10% glacial acetic acid water solution is taken as a mobile phase B for gradient elution; the detection wavelength is 275nm; the flow rate is 0.7mL/min, and the theoretical plate number is not lower than 3500 according to baicalin peak calculation; the gradient elution specifically comprises the following steps:

The ethanol in the step (1) of the invention can be any one of acetonitrile and methanol.

The ultrasonic conditions in the step (1) are as follows: the power is 250-400W, and the frequency is 20-40 kHz.

Further, the condition of the ultrasound in the step (1) is as follows: the power is 300W and the frequency is 25kHz.

The beneficial effects of the invention are as follows:

1. compared with the traditional process, the invention has the remarkable advantages that the extraction and preparation technology is adopted:

(1) The extraction efficiency is high: the invention adopts an ultrasonic extraction mode, can promote the wall breaking or deformation of plant cell tissues, and ensures that the extraction of the effective components of the traditional Chinese medicine is more complete; the ultrasonic extraction process of baicalin, peucedanum root and radix stemonae medicinal materials is subjected to orthogonal experimental investigation, the concentration of the obtained extraction solvent is the maximum influencing factor, the extraction solvent has significance, the best effect can be achieved by taking 70% ethanol as the extraction solvent, and the extraction rate of the main medicinal materials such as baicalin, peucedanum root and radix stemonae is obviously improved by more than 20-40% compared with the traditional process.

(2) Avoid destroying the effective components of the baical skullcap root, the peucedanum root and the stemona root medicinal materials in the prescription: according to the invention, the radix scutellariae, the radix peucedani and the radix stemonae are subjected to ultrasonic extraction, and through the examination of an orthogonal test, the optimal temperature of the ultrasonic extraction is 45+/-15 ℃, and the ultrasonic extraction temperature is lower than that of the traditional water decoction reflux and other extraction temperatures, so that the ultrasonic extraction method has a protective effect on the active ingredients in the radix stemonae and other medicinal materials which are unstable when encountering heat and are easy to hydrolyze or oxidize, avoids the destruction of the active ingredients of the medicinal materials under severe conditions such as long-time heating and decoction, and simultaneously greatly saves energy and reduces consumption.

2. By adopting the method, the average content of the baicalin in the active ingredient of the extract is 21.39mg/g, which is higher than the average content of the baicalin in the prior art by 13.07mg/g; the measured transfer rate of baicalin is 81.06%, the transfer rate of procyanidin is 75.31%, and the transfer rate of radix stemonae sessilifolia is 85.47%; comparative example experiment the transfer rate of baicalin measured by the traditional process of water decoction was 60.13%, the transfer rate of procyanidine was 54.39%, and the transfer rate of stemonine was 51.24%. The transfer rate of the medicinal materials is far higher than that of the prior art, the content of main effective components is obviously improved, and the transfer rate of the main components of the medicinal materials is obviously improved. Because the extract preparation is adopted, the content of the active ingredients of the traditional Chinese medicinal materials in the preparation is improved, and the dosage of the medicine is reduced.

3. Compared with the prior art, the thin-layer identification of the baical skullcap root medicinal material is improved, the main spots are clear, the durability is good, and the problems of fuzzy spots and poor reproducibility of the prior art method are solved; on the basis of the original quality detection project, the thin-layer identification of the gentian and the oldenlandia diffusa in the lung force cough capsule is added, so that the quality standard is improved, and the quality is better controlled. The method of the invention examines the extraction mode, the extraction solvent, the extraction times, the sample application amount of the sample and the developing agent by examining the sample preparation and the thin layer chromatography conditions, and finally establishes a thin layer chromatography identification method; and the feasibility and the reliability of the method are further verified through tests on the silica gel G thin layer plates of different manufacturers, different unfolding temperatures, different relative humidity and other influencing factors.

4. Compared with the prior art, the method improves the baicalin content determination method, performs investigation experiments on mobile phase, gradient, detection wavelength, extraction solvent, extraction mode and extraction time, establishes high performance liquid chromatography, and inspects by methodology: (1) Through linear relation investigation, a linear equation is obtained and is Y=6E+06x-12369, and R=0.9998, which shows that the linear relation between 0.2754 mug and 1.3770 mug is good in baicalin content measurement; (2) The RSD is 2.59% through the investigation of the precision test of the instrument, and the result shows that the precision of the instrument is good; (3) The repeatability test and investigation show that the RSD is 2.12%, and the result shows that the repeatability is good; (4) The stability test is examined to obtain RSD of 1.83%, and the result shows that the stability of the sample solution is good within 12 hours; (5) And (3) obtaining RSD of 1.35% through recovery rate test and investigation, and the result shows that the RSD meets the requirements. The method is accurate, stable and reliable.

Detailed Description

The present invention will be further understood by those skilled in the art by reference to the following examples, which are included herein by way of illustration and not limitation, and various changes and modifications may be made by those skilled in the art without departing from the spirit of the invention.

Example 1 preparation method one

Prescription: 173g of radix scutellariae, 167g of radix peucedani, 160g of radix stemonae, 150g of gentian carthami, 130g of Chinese phoenix tree root, 120g of oldenlandia diffusa and 96g of red tube medicine.

The preparation method comprises the following steps: (1) preparation of fine powder of radix scutellariae, radix peucedani and radix stemonae medicinal materials: pulverizing Scutellariae radix, radix Peucedani, and radix Stemonae respectively, and sieving with 100 mesh sieve;

(2) Preparation of traditional Chinese medicine extract:

(3) Preparation of the capsules: pulverizing the above extracts, adding starch 20% of the total amount of the extracts, mixing, granulating with 90% ethanol, sieving with 20 mesh sieve, drying at 50deg.C, and encapsulating.

Example 2 preparation method two:

The preparation method comprises the following steps: (1) preparation of fine powder of radix scutellariae, radix peucedani and radix stemonae medicinal materials: pulverizing Scutellariae radix, radix Peucedani, and radix Stemonae respectively, and sieving with 120 mesh sieve;

(2) Preparation of traditional Chinese medicine extract:

(1) preparation of the radix Scutellariae extract: soaking the fine powder of the scutellaria baicalensis obtained in the step (1) in 90% ethanol solution with the mass-volume ratio of 1:12 and the preheating temperature of 30 ℃ for 60min, carrying out ultrasonic extraction for 60min, filtering at the extraction temperature of 60 ℃, recovering ethanol from the filtrate, concentrating the filtrate under reduced pressure to obtain an extract with the relative density of 1.20-1.30 at the temperature of 50 ℃, and then drying the extract under reduced pressure at the vacuum degree of 0.8-0.9mpa and the temperature of 60 ℃ to obtain a dry extract of the scutellaria baicalensis;

(2) preparation of Peucedanum root extract: soaking Hu Xifen obtained in the step (1) in 90% ethanol solution with a mass-volume ratio of 1:12 and preheated to 30deg.C for 60min, ultrasonically extracting for 60min, filtering at 60deg.C, recovering ethanol from the filtrate, concentrating under reduced pressure to obtain extract with relative density of 1.20-1.30 at 50deg.C, and oven drying under reduced pressure at vacuum degree of 0.8-0.9Mpa at 60deg.C to obtain radix Peucedani extract;

(3) Preparation of Stemonae radix extract: soaking the radix stemonae fine powder obtained in the step (1) in 90% ethanol solution with the mass-volume ratio of 1:12 and preheated to 30 ℃ for 60min, performing ultrasonic treatment for 60min, extracting at 60 ℃, filtering, recovering ethanol from the filtrate, concentrating under reduced pressure to obtain extract with relative density of 1.20-1.30 at 50 ℃, and drying the extract under reduced pressure at 60 ℃ under vacuum degree of 0.8-0.9mpa to obtain radix stemonae extract;

(4) preparation of extracts of radix Gentianae, radix Firmianae, herba Hedyotidis Diffusae and radix Rhodiolae: mixing four medicinal materials of safflower gentian, chinese phoenix tree root, oldenlandia diffusa and red tube, adding water which is 12 times of the total amount of the four medicinal materials, heating and decocting for two times, wherein the first time is 2.5 hours, the second time is 2.0 hours, merging the two decoctions, filtering, concentrating the filtrate under reduced pressure to an extract with the relative density of 1.07-1.10 at 50 ℃, and drying the extract under reduced pressure at 60 ℃ under the vacuum degree of 0.8-0.9mpa to obtain a dry extract, wherein the dry extract is prepared from the four medicinal materials;

(3) Preparation of the capsules: pulverizing the above extracts, adding starch 15% of the total amount of the extracts, mixing, granulating with 95% ethanol, sieving with 35 mesh sieve, drying at 60deg.C, and making into capsule.

Example 3 preparation method three:

The preparation method comprises the following steps: (1) preparation of fine powder of radix scutellariae, radix peucedani and radix stemonae medicinal materials: pulverizing Scutellariae radix, radix Peucedani, and radix Stemonae respectively, and sieving with 80 mesh sieve;

(2) Preparation of traditional Chinese medicine extract:

(1) preparation of the radix Scutellariae extract: soaking the fine powder of the scutellaria baicalensis obtained in the step (1) in 50% ethanol solution with the mass-volume ratio of 1:8 and the preheating temperature of 30 ℃ for 60min, carrying out ultrasonic extraction for 30min, filtering at the extraction temperature of 30 ℃, recovering ethanol from the filtrate, concentrating the filtrate under reduced pressure to obtain an extract with the relative density of 1.20-1.30 at the temperature of 50 ℃, and then drying the extract under reduced pressure at the vacuum degree of 0.8-0.9mpa and the temperature of 60 ℃ to obtain a dry extract of the scutellaria baicalensis;

(2) preparation of Peucedanum root extract: soaking Hu Xifen obtained in the step (1) in 50% ethanol solution with a mass-volume ratio of 1:8 and preheated to 30deg.C for 60min, ultrasonically extracting for 30min at 30deg.C, filtering, recovering ethanol from the filtrate, concentrating under reduced pressure to obtain extract with relative density of 1.20-1.30 at 50deg.C, and drying the extract under reduced pressure at vacuum degree of 0.8-0.9Mpa and 60deg.C to obtain radix Peucedani extract;

(3) Preparation of Stemonae radix extract: soaking the radix stemonae fine powder obtained in the step (1) in 50% ethanol solution with the mass-volume ratio of 1:8 and the preheating temperature of 30 ℃ for 60min, carrying out ultrasonic treatment for 30min, extracting at the temperature of 30 ℃, filtering, recovering ethanol from the filtrate, concentrating under reduced pressure to obtain extract with the relative density of 1.20-1.30 at the temperature of 50 ℃, and drying the extract under reduced pressure at the vacuum degree of 0.8-0.9mpa and the temperature of 60 ℃ to obtain a radix stemonae extract;

(4) preparation of extracts of radix Gentianae, radix Firmianae, herba Hedyotidis Diffusae and radix Rhodiolae: mixing four medicinal materials of gentian carthami, chinese phoenix tree root, oldenlandia diffusa and red tube, adding water which is 8 times of the total amount of the four medicinal materials, heating and decocting for two times, wherein the first time is 1.5 hours, the second time is 1.0 hour, merging the two decoctions, filtering, concentrating the filtrate under reduced pressure to an extract with the relative density of 1.07-1.10 at 50 ℃, and drying the extract under reduced pressure at 60 ℃ under the vacuum degree of 0.8-0.9mpa to obtain a dry extract;

(3) Preparation of the capsules: pulverizing the above extracts, adding starch 10% of the total amount of the extracts, mixing, granulating with 85% ethanol, sieving with 20 mesh sieve, drying at 40deg.C, and making into capsule.

The products prepared in examples 1-3 were tested by the thin layer identification method or the assay method of any one of examples 4-9.

Example 4 thin layer authentication

(1) Thin layer identification of radix scutellariae medicinal material: taking 2.0g of a sample, adding 50mL of 70% methanol, heating and refluxing for 1h, filtering, evaporating the filtrate to dryness, adding 25mL of water into the residue to dissolve, adjusting the pH value to 2.2 with 3% hydrochloric acid solution, extracting for 2 times with 30mL of ethyl acetate under shaking, combining the extracting solutions, evaporating to dryness, and adding 5mL of methanol into the residue to dissolve to obtain a sample solution; adding methanol into baicalin reference substance to obtain 1mg solution per 1mL as reference substance solution; the 2 solutions were each drawn 5 μl and spotted onto the same silica gel G thin layer plate in a ratio of 10:2:0.5:2, developing n-butanol-acetic acid-acetone-water as developing agent, taking out, air drying, spraying 5% acid ferric trichloride ethanol solution for developing color, and in the sample chromatogram, showing the same color spots on the positions corresponding to the control medicinal material chromatogram and the control chromatogram;

(3) Thin layer identification of oldenlandia diffusa medicinal material: taking 4.0g of sample, adding 60ml of 70% methanol, heating and refluxing for 1h, filtering, evaporating filtrate to dryness, and dissolving residues with 5ml of methanol to obtain a sample solution; taking oleanolic acid reference substance, adding methanol to prepare a solution containing 1mg per 1ml, and taking the solution as a reference solution; the two solutions were each drawn 4 μl and spotted on the same silica gel G thin layer plate in a ratio of 25:7:4:0.1 petroleum ether (30-60 ℃) and dimethylbenzene-ethanol-glacial acetic acid are taken as developing agents, and are developed, taken out, dried and sprayed with 10% sulfuric acid ethanol solution for developing color, and spots with the same color appear on the corresponding positions of the sample chromatogram and the reference chromatogram.

Example 5 thin layer authentication

(1) Thin layer identification of radix scutellariae medicinal material: taking 1.0g of a sample, adding 40mL of 60% methanol, heating and refluxing for 0.5h, filtering, evaporating the filtrate, adding 20mL of water into the residue to dissolve, adjusting the pH value to 2.0 with 2.5% hydrochloric acid solution, shaking and extracting for 1 time with ethyl acetate, wherein the dosage is 25mL, extracting solution, evaporating to dryness, and adding 5mL of methanol into the residue to dissolve to obtain a sample solution; adding methanol into baicalin reference substance to obtain 1mg solution per 1mL as reference substance solution; the 2 solutions were each drawn 4 μl and spotted onto the same silica gel G thin layer plate in a ratio of 8:1:0.5:1, developing n-butanol-acetic acid-acetone-water as developing agent, taking out, air drying, spraying 3% acid ferric trichloride ethanol solution for developing color, and in the chromatogram of the sample, showing spots with the same color at the positions corresponding to the chromatogram of the reference substance;

(2) Thin layer identification of gentian medicinal material of safflower: taking 2.0g of sample, adding 20mL of 50% methanol, heating and refluxing for 0.5h, filtering, evaporating filtrate to dryness, and adding 1mL of methanol into residues to dissolve the residues to obtain a sample solution; additionally, 0.5g of safflower gentian is used as a reference medicine, and the prepared reference medicine solution is prepared by the same method; adding methanol into mangiferin reference substance to prepare a solution containing 1mg per 1mL as reference substance solution; the 3 solutions were each aspirated at 4 μl and spotted onto the same silica gel G254 thin layer plate in a ratio of 11:1:1, developing with chloroform-acetone-water as developing agent, taking out, air drying, and making spots of the same color appear on the positions corresponding to the control medicine chromatogram and the control medicine chromatogram in the sample chromatogram;

(3) Thin layer identification of oldenlandia diffusa medicinal material: taking 3.0g of sample, adding 50ml of 60-70% methanol, heating and refluxing for 0.5h, filtering, evaporating filtrate to dryness, and dissolving residues with 5ml of methanol to obtain a sample solution; taking oleanolic acid reference substance, adding methanol to prepare a solution containing 1mg per 1ml, and taking the solution as a reference solution; the two solutions were each drawn 3 μl and spotted on the same silica gel G thin layer plate in a ratio of 24:6:3:0.1 petroleum ether (30-60 ℃) and dimethylbenzene-ethanol-glacial acetic acid are taken as developing agents, and are developed, taken out, dried and sprayed with 5-10% sulfuric acid ethanol solution for developing color, and spots with the same color appear on the corresponding positions of the sample chromatogram and the reference chromatogram.

Example 6 thin layer authentication

(1) Thin layer identification of radix scutellariae medicinal material: 3.0g of a sample is added with 60mL of 80% methanol, heated and refluxed for 1.5h, filtered, evaporated to dryness, and the residue is added with 30mL of water to dissolve, the pH value is adjusted to 2.5 by 3.5% hydrochloric acid solution, the mixture is extracted for 3 times by shaking with 35mL each time, the extracting solutions are combined and evaporated to dryness, and the residue is added with 5mL of methanol to dissolve to be used as a sample solution; adding methanol into baicalin reference substance to obtain 1mg solution per 1mL as reference substance solution; sucking 4-6 mu L of each of the 2 solutions, and respectively spotting on the same silica gel G thin layer plate according to the proportion of 12:2:0.5:2, developing n-butanol-acetic acid-acetone-water as developing agent, taking out, air drying, spraying 10% acid ferric trichloride ethanol solution for developing color, and in the chromatogram of the sample, showing spots with the same color at the positions corresponding to the chromatogram of the reference substance;

(2) Thin layer identification of gentian medicinal material of safflower: taking 4.0g of a sample, adding 30mL of 70% methanol, heating and refluxing for 1.5h, filtering, evaporating the filtrate to dryness, and adding 1mL of methanol into the residue to dissolve the residue to obtain a sample solution; additionally, 0.5g of safflower gentian is used as a reference medicine, and the prepared reference medicine solution is prepared by the same method; adding methanol into mangiferin reference substance to prepare a solution containing 1mg per 1mL as reference substance solution; 6 mu L of each of the 3 solutions is sucked and respectively spotted on the same silica gel G254 thin layer plate, and the proportion is 12:1.5:2, developing with chloroform-acetone-water as developing agent, taking out, air drying, and making spots of the same color appear on the positions corresponding to the control medicine chromatogram and the control medicine chromatogram in the sample chromatogram;

(3) Thin layer identification of oldenlandia diffusa medicinal material: taking 5.0g of sample, adding 70ml of 70% methanol, heating and refluxing for 1.5h, filtering, evaporating filtrate to dryness, and dissolving residues with 5ml of methanol to obtain a sample solution; taking oleanolic acid reference substance, adding methanol to prepare a solution containing 1mg per 1ml, and taking the solution as a reference solution; the two solutions were each drawn 5 μl and spotted on the same silica gel G thin layer plate in a ratio of 26:8:5:0.1 petroleum ether (30-60 ℃) and dimethylbenzene-ethanol-glacial acetic acid are taken as developing agents, and are developed, taken out, dried and sprayed with 10% sulfuric acid ethanol solution for developing color, and spots with the same color appear on the corresponding positions of the sample chromatogram and the reference chromatogram.

Example 7 content determination method one

(1) Preparation of test solution: accurately weighing 0.2g of sample, placing in a 50ml measuring flask, adding appropriate amount of 70% ethanol, performing ultrasonic treatment for 30 min, cooling, adding 70% ethanol to dilute to scale, shaking, filtering, and collecting filtrate;

At 0min, mobile phase A was 20% and mobile phase B was 80%;12min, 36% mobile phase A and 64% mobile phase B; 20min, 48% mobile phase A and 52% mobile phase B; 30min, mobile phase A57%, mobile phase B43%; 45min, 48% mobile phase A and 52% mobile phase B; 55min, mobile phase A24% and mobile phase B76%;

Example 8 method for measuring content

(1) Preparation of test solution: accurately weighing 0.1g of sample, placing in 50ml measuring flask, adding appropriate amount of 60% ethanol, ultrasonic treating for 15min, cooling, diluting with 60% ethanol to scale, shaking, filtering, and collecting filtrate;

(3) Chromatographic conditions and system suitability test: the chromatographic column is CORTECS UPLC T3.8 um; the column temperature is 30 ℃; acetonitrile solution containing 0.01% glacial acetic acid is taken as a mobile phase A, and 0.05% glacial acetic acid water solution is taken as a mobile phase B for gradient elution; the detection wavelength is 275nm; the flow rate is 0.5mL/min, and the theoretical plate number is not lower than 3500 according to baicalin peak calculation; the gradient elution specifically comprises the following steps:

0min, mobile phase A20% and mobile phase B80%; 12min, 36% mobile phase A and 64% mobile phase B; 20min, 48% mobile phase A and 52% mobile phase B; 30min, mobile phase A57%, mobile phase B43%; 45min, 48% mobile phase A and 52% mobile phase B; 55min, mobile phase A24% and mobile phase B76%;

Example 9 content determination method three

(1) Preparation of test solution: accurately weighing 0.3g of sample, placing in 50ml measuring flask, adding appropriate amount of 80% ethanol, ultrasonic treating for 45min, cooling, diluting with 80% ethanol to scale, shaking, filtering, and collecting filtrate;

(3) Chromatographic conditions and system suitability test: the chromatographic column is CORTECS UPLC T3.8 um; the column temperature is 30 ℃; acetonitrile solution containing 0.015% glacial acetic acid is used as a mobile phase A, and 0.15% glacial acetic acid water solution is used as a mobile phase B for gradient elution; the detection wavelength is 275nm; the flow rate is 0.9mL/min, and the theoretical plate number is not lower than 3500 according to baicalin peak calculation; the gradient elution specifically comprises the following steps:

To further illustrate the beneficial effects of the present invention, the applicant conducted a series of experimental studies of the preparation method and provided the following experimental data:

1. research and investigation of preparation process

1.1 design of preparation Process route

The formula comprises the following components: 173g of radix scutellariae, 167g of radix peucedani, 160g of radix stemonae, 150g of gentian carthami, 130g of Chinese phoenix tree root, 120g of oldenlandia diffusa and 96g of red tube medicine.

The original preparation process adopts a method of simple water decoction, filtration and concentration into extractum in the traditional process, the process is backward, the time consumption is long, the problems of lower extraction rate of main effective components and more impurities exist, the clinical application of the product is seriously influenced, and the medicinal materials such as stemona root and the like in the recipe mainly contain alkaloid effective components, the alkaloid property is unstable, and the structure is easy to change under the condition of heating, so the traditional process needs to be improved. The selection and improvement method is that three main medicinal materials of baical skullcap root, whiteflower hogfennel root and radix stemonae are singly extracted by ultrasonic.

The specific method comprises the following steps: pulverizing Scutellariae radix, radix Peucedani, and radix Stemonae, soaking in ethanol, ultrasonic extracting, concentrating under reduced pressure, and drying to obtain dry extract; decocting the rest materials in water, concentrating, drying under reduced pressure to obtain dry extract, pulverizing, adding starch, mixing, granulating with ethanol, sieving, drying, and making into capsule. The extraction process of the lung force cough capsule is finally determined through pre-test and orthogonal design test by referring to a large amount of data and combining the analysis, wherein the transfer rate and the content of main effective components of baicalin, procyanidine and tuberose are used as evaluation indexes.

1.2 ultrasonic extraction Pre-test of Baikal skullcap root, peucedanum root and Stemonae radix

Pulverizing Scutellariae radix, radix Peucedani, and radix Stemonae according to the prescription ratio, soaking in 70% ethanol, ultrasonic extracting, respectively measuring the content of index components of each medicine in ethanol ultrasonic extractive solution, concentrating under reduced pressure to obtain extract with relative density of 1.20-1.30 (50deg.C), and oven drying under reduced pressure at 60 deg.C under vacuum degree of 0.8-0.9mpa to obtain dry extract. The content measurement method of each index component comprises the following steps:

1.2.1 method for determining baicalin content according to High Performance Liquid Chromatography (HPLC) method of "Chinese pharmacopoeia" 2020 edition (general rule 0512)

Chromatographic conditions and system applicability test chromatographic column is CORTECS UPLC T3.8 um; mobile phase: methanol-water-phosphoric acid (55:45:0.2); detection wavelength: 280nm; column temperature: 30 ℃; flow rate: 1.0 ml.min-1; sample injection amount: 5 μl.

Preparation of control solution A proper amount of baicalin control solution is obtained by drying under reduced pressure at 60deg.C for 4 hr, precisely weighing, and adding methanol to obtain solution containing 60 μg per 1 ml.

Preparation of sample solution about 0.3g of sample solution, precisely weighing, adding 40ml of 70% ethanol, heating and refluxing for 3 hours, cooling, filtering, placing filtrate into a 100ml measuring flask, washing container and residue with a small amount of 70% ethanol, filtering the washing solution into the same measuring flask, adding 70% ethanol to scale, and shaking. Precisely measuring 1ml, placing into a 10ml measuring flask, adding methanol to scale, and shaking.

1.2.2.2 method for measuring content of Violet peucedanum praeruptorum according to high performance liquid chromatography, chinese pharmacopoeia 2020 edition (general rule 0512)

Chromatographic conditions and system applicability test chromatographic column is CORTECS UPLC T3.8 um; mobile phase: methanol-water (40:60); detection wavelength: 334nm; column temperature: 30 ℃; flow rate: 1.0 ml/min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Sample injection amount: 5 μl.

Preparation of control solution A proper amount of decursin control is precisely weighed, and methanol is added to prepare 50 mug solution per 1 ml.

Preparation of sample solution about 0.5g, precisely weighing, placing into conical flask with plug, precisely adding 25ml of methanol, weighing, soaking for 1 hr, ultrasonic treating (power 100W, frequency 40 kHz) for 20 min, cooling, weighing again, supplementing the reduced weight with methanol, shaking, filtering, precisely measuring 1ml of filtrate, placing into 10ml measuring flask, adding methanol to scale, and shaking.

1.2.3 method for measuring the content of Stemona tuberosa alkali reference to the literature name "determination of alkaloid content in Stemona tuberosa 4 in different producing regions of Guangxi" determination of cough-relieving Activity comparison

Chromatographic conditions and system applicability test chromatographic column is CORTECS UPLC T3.8 um; mobile phase: 0.05% ammonia (A) -acetonitrile (B), gradient elution (0-2 min,40% B;2-30min,40% -65% B); detection wavelength: 210nm; column temperature: 40 ℃; flow rate: 1.0 ml/min ^-1 The method comprises the steps of carrying out a first treatment on the surface of the Sample injection amount: 20 μl.

Preparation of reference solution A proper amount of the reference solution of stemonine is precisely weighed, and methanol is added to prepare a solution with 400 mug/ml per 1 ml.

Preparation of sample solution 1.0g of sample solution, precisely weighing, placing in a 20ml measuring flask, adding appropriate amount of 70% methanol, soaking at room temperature for 30min, and ultrasonic extracting (250W, 25 kHz) for 1 hr. Centrifuging for 10min, and filtering with 0.22 μm microporous membrane.

1.2.4 results of the transfer Rate measurement

The test results are shown in Table 1.

TABLE 1 results of transfer Rate determination for ultrasonic extraction

1.3 ultrasonic extraction investigation

1.3.1 selection of orthogonality factors and levels

Under certain conditions, the 1.3.1.1 ethanol concentration takes ethanol as an extraction solvent, the extraction amount of the glycosides is increased along with the increase of the water content, and the extraction amount of the glycosides in the ethanol with more water content is larger. However, when the concentration of ethanol is less than 50%, the increase of the water content in the ethanol extraction solvent does not increase the extraction amount of glycosides, but increases the viscosity of the extract, increases pigment interference, and increases the complexity of post-treatment before application of the extract. In summary, 50%, 70%, 90% ethanol was chosen as the factor and level of the orthogonal test investigation.

1.3.1.2 different material-solution ratio and different extraction effect. The material solution ratio is too small, so that the ultrasonic extraction effect is reduced; the material solution ratio is too large, so that the solvent is wasted, and the concentration treatment difficulty of the later-stage extracting solution is increased. Comprehensive analysis, selecting a material solution ratio of 1: 8. 1: 10. 1:12 as orthogonal test factor level.

1.3.1.3 the ultrasonic extraction has the characteristics of short time and high efficiency. The ultrasonic wave can break cell walls and cell membranes of the traditional Chinese medicinal materials through cavitation effect, mechanical effect, thermal effect and the like, so that the intracellular speed of the solvent penetrating into the medicinal materials is increased, or the diffusion of chemical components in cells to the outside of the cells is accelerated, and the extraction rate is improved. Taking extraction time of 30, 45 and 60min as investigation factors and levels of orthogonal tests according to the specific conditions of the tests.

1.3.1.4 extraction temperature the temperature of extraction is one of the key factors affecting extraction. Too low a temperature may affect dissolution of the active ingredient in the extraction solvent; too high a temperature may destroy the thermosensitive components and the solvent may be seriously volatilized. Comprehensively considering 30, 45 and 60 ℃ as investigation factors and levels of orthogonal tests.

1.3.2 radix Scutellariae orthogonal experiments

The orthogonal experimental factor level table is designed by taking the concentration (A) of ethanol in an extraction solvent, the ratio (B) of materials to solution, the extraction time (C) and the ultrasonic temperature (D) as factors, wherein each factor comprises 3 levels, and L is selected ₉ (3 ⁴ ) Orthogonal table the orthogonal test was designed, the level of each factor is shown in table 2, the results of the orthogonal test are shown in table 3, and the analysis of variance is shown in table 4.

TABLE 2 level of orthogonal test factors

TABLE 3 results of orthogonal test table

TABLE 4 analysis of variance of extraction results

Visual analysis from Table 3 shows that the influence sequence of each factor on the content is baicalin A > D > B > C, and the optimal extraction process A ₂ B ₃ C ₃ D ₃ As can be seen from table 4, the effect of a on the extraction was significant, and neither the effect of B, C nor the effect of D was significant, i.e., the maximum factor affecting the extraction rate was ethanol concentration a. Comprehensively considering cost and benefit factors, determining the optimized extraction process as A ₂ B ₂ C ₂ D ₃ Namely adding 10 times of 70% ethanol for extraction for 45min at 60 ℃.

1.3.3 Pre-Hu Zhengjiao test

The orthogonal experimental factor level table is designed by taking the concentration (A) of ethanol in an extraction solvent, the ratio (B) of materials to solution, the extraction time (C) and the ultrasonic temperature (D) as factors, wherein each factor comprises 3 levels, and L is selected ₉ (3 ⁴ ) Orthogonal table the orthogonal test was designed, the level of each factor is shown in table 5, the results of the orthogonal test are shown in table 6, and the analysis of variance is shown in table 7.

TABLE 5 level of orthogonal test factors

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TABLE 6 results of orthogonal test table

TABLE 7 analysis of variance of extraction results

Visual analysis is carried out on the principle of Table 6, the influence sequence of each factor on the content is that the decursin A is more than D is more than B is more than C, and the optimal extraction process A is adopted ₂ B ₁ C ₂ D ₃ As shown in table 7, the effect of a on the extraction was significant, and the effects of B, C and D were not significant, i.e., the maximum factor affecting the extraction rate was ethanol concentration a. Comprehensively considering cost and benefit factors, determining the optimized extraction process as A ₂ B ₁ C ₁ D ₃ Namely adding 8 times of 70% ethanol for extraction for 30min at 60 ℃.

1.3.4 Stemona orthometric test

The orthogonal experimental factor level table is designed by taking the concentration (A) of ethanol in an extraction solvent, the ratio (B) of materials to solution, the extraction time (C) and the ultrasonic temperature (D) as factors, wherein each factor comprises 3 levels, and L is selected ₉ (3 ⁴ ) Orthogonal table the orthogonal test was designed, the level of each factor is shown in table 8, the results of the orthogonal test are shown in table 9, and the analysis of variance is shown in table 10.

TABLE 8 level of orthogonal test factors

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TABLE 9 results of orthogonal test table

TABLE 10 analysis of variance of extraction results

Visual analysis from Table 9 shows that the order of influence of each factor on the content is that the stemonine A > B > D > C, and the optimal extraction process A ₂ B ₃ C ₃ D ₃ As can be seen from Table 10, A has a significant effect on the extraction effect, B, C and the effect of D factorThe response is not obvious, namely the maximum factor influencing the extraction rate is the concentration A of ethanol. Comprehensively considering cost and benefit factors, determining the optimized extraction process as A ₂ B ₂ C ₂ D ₂ Namely adding 10 times of 70% ethanol for extraction for 45min at 45 ℃.

1.4 final preparation method

Pulverizing Scutellariae radix, radix Peucedani, and radix Stemonae respectively, sieving, soaking in 70% ethanol solution preheated to 30deg.C for one hr, ultrasonic treating, filtering, recovering ethanol from filtrate, concentrating under reduced pressure to obtain extract with relative density of 1.20-1.30 at 50deg.C, and oven drying under reduced pressure at 60deg.C under vacuum degree of 0.8-0.9mpa to obtain Scutellariae radix extract, radix Peucedani extract, and radix Stemonae extract; mixing the rest four medicinal materials of radix Gentianae, radix Firmianae, herba Hedyotidis Diffusae and radix Rhodiolae, adding 10 times of water, decocting twice, decocting for 2 hr for the first time and 1.5 hr for the second time, mixing the decoctions, filtering, concentrating the filtrate under reduced pressure to obtain extract with relative density of 1.07-1.10 at 50deg.C, and oven drying under reduced pressure at 60 deg.C under vacuum degree of 0.8-0.9mpa to obtain dry extract; and finally, crushing the obtained extract, adding starch accounting for 20% of the total amount of the extract, uniformly mixing, granulating with 90% ethanol, sieving with a 20-mesh sieve, granulating, drying at 50 ℃, and encapsulating after drying.

2. Screening of thin-layer identification method of herbal materials of radix scutellariae, radix gentianae carthami and oldenlandia in lung-force cough pharmaceutical composition

2.1 thin-layer chromatography identification of Scutellaria baicalensis Georgi medicinal material in Lung force cough Capsule

2.1.1 method sources: reference to the original standard of the lung-force cough capsule

Taking 1g of the content of the product, adding 20ml of methanol, carrying out ultrasonic treatment for 20 minutes, filtering, evaporating the filtrate to dryness, and adding 5ml of methanol into the residue to dissolve the residue to obtain a sample solution. And adding methanol into appropriate amount of baicalin reference substance to obtain 1mg solution per 1ml, and taking the solution as reference substance solution. According to thin layer chromatography (appendix VI B of China pharmacopoeia 2000 edition), sucking 5-10 μl of each of the above two solutions, respectively spotting on the same silica gel G thin layer plate, pre-saturating with n-butanol-glacial acetic acid-water (7:1:2) as developing agent for 20 min, spreading, taking out, air drying, and spraying 5% ferric trichloride ethanol solution. Spots of the same color appear in the sample chromatogram at positions corresponding to those of the control chromatogram.

Results: thin-layer identification of radix Scutellariae has small and blurred main spots, tailing phenomenon, small spot Rf value and poor reproducibility.

According to the thin layer result detected by the reference method, the problems, the combination property, the extraction solvent, the extraction mode, the choice of the developing agent, the proportion of the developing agent and the like in the thin layer identification method of the baical skullcap root are examined.

2.1.2 thin-layer identification method of baical skullcap root medicinal material in capsule for treating lung force cough after optimization

According to the thin layer result under the item 2.1.1, in combination with the physicochemical properties of the baical skullcap root medicinal material, the water extract is required to be adjusted to be acidic by adding dilute hydrochloric acid or sulfuric acid, and then flavone components are extracted, and the tentative thin layer identification method is as follows:

thin layer identification of radix scutellariae medicinal material: taking 2.0g of lung force cough fluid extract, adding 50mL of 70% methanol, heating and refluxing for 1h, filtering, evaporating the filtrate to dryness, adding 25mL of water into the residue to dissolve, adjusting the pH value to 2.2 with 3% dilute hydrochloric acid, extracting with ethyl acetate for 2 times by shaking for 30mL each time, mixing the extracting solutions, evaporating to dryness, and adding 5mL of methanol into the residue to dissolve to obtain a sample solution; adding methanol into baicalin reference substance to obtain 1mg solution per 1mL as reference substance solution; sucking 3-8 mu L of each of the 2 solutions, and respectively spotting on the same silica gel G thin layer plate according to the proportion of 10:2:0.5:2, developing n-butanol-acetic acid-acetone-water as developing agent, taking out, air drying, spraying 5% acid ferric trichloride ethanol solution for developing color, and in the sample chromatogram, showing the same color spots on the positions corresponding to the control medicinal material chromatogram and the control chromatogram;

2.1.2.1 extraction mode selection

Method 1: shaking and extracting for 60min.

Method 2: ultrasonic extraction is carried out for 60min.

Method 3: extracting under reflux for 60min.

The samples treated in the above three extraction modes were subjected to thin layer detection according to the method under item "2.1.2", respectively, and the results are shown in Table 11.

Table 11 screening table of extraction method

As a result, as shown in Table 11, the sample treated by "method 3" was clear in spots, satisfactory in resolution, moderate in Rf value and good in reproducibility, so that "heat reflux extraction" was the preferred extraction method.

2.1.2.2 selection of sample extraction solvent

Method 1: sample 2g was taken and extracted with 50ml of diethyl ether under reflux with heating for 60min.

Method 2: 2g of the sample was taken, and extracted with 50ml of methanol under reflux with heating for 60min.

Method 3: sample 2g was taken and extracted with 70% methanol 50ml under reflux with heating for 60min.

Method 4: sample 2g was taken and extracted with 50ml of 50% methanol under reflux with heating for 60min.

Method 5: 2g of the product is taken, 50ml of ethyl acetate is used for heating reflux extraction for 60min.

The above five samples treated with the extraction solvents were subjected to thin layer detection according to the method under item "2.1.2", respectively, and the results are shown in Table 12.

TABLE 12 screening table of sample extraction solvents

As a result, as shown in Table 12, the sample treated by "method 3" was clear in spots, satisfactory in resolution, moderate in Rf value and good in reproducibility, so that "70% methanol" was the preferred extraction solvent.

2.1.2.3 selection of the number of extractions

Method 1: 2g of the sample was taken, and extracted with 50ml of 70% methanol under reflux with heating for 1 time each for 30min.

Method 2: 5g of the sample is taken, 50ml of 70% methanol is used, and the mixture is extracted by heating and refluxing for 2 times for 30min each time.

Method 3: 2g of the sample was taken, and extracted with 50ml of 70% methanol under reflux with heating for 3 times each for 30min.

Method 4: 2g of the sample was taken, and extracted with 50ml of 70% methanol under reflux with heating for 4 times each for 30min.

The above four samples treated with the number of extraction times were subjected to thin layer detection according to the tentative identification method described under item "2.1.2", respectively, and the results are shown in Table 13.

TABLE 13 screening Table of the number of extractions

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Results: as is clear from Table 13, the samples developed in methods 1, 2 and 3 were clear in spots, free from interference, satisfactory in resolution, moderate in Rf value and good in reproducibility, but in view of cost and efficiency, the optimal number of heat reflux extractions was selected to be 1.

2.1.2.4 selection of sample application amount of test sample

The test solutions were aspirated at 2. Mu.L, 5. Mu.L and 10. Mu.L, respectively, and the thin layer test was performed according to the tentative identification method described under item "2.1.2", and the results are shown in Table 14.

TABLE 14 sample application amount screening Table for test samples

As a result, it was found from Table 14 that the sample amount was 5. Mu.L, the spot was satisfactory, the Rf value was moderate, and the reproducibility was good, and therefore, the optimal sample amount of the sample was 5. Mu.L.

2.1.2.5 developer selection

Suitable developing solvents are selected according to the nature of the compounds and the principle of "similar miscibility".

Method 1: chloroform-methanol (8:2) is used as developing agent;

method 2: methanol-ethyl acetate-acetone (7:2:1) is taken as a developing agent;

method 3: n-butanol-ethyl acetate-acetone-water (7:1:1:1) is used as a developing agent;

method 4: n-butanol-acetic acid-acetone-water (10:2:0.5:2) is used as developing agent;

the above four developing agents were developed, and the results of the thin layer test were shown in Table 15, respectively, according to the tentative identification method described in item "2.1.2".

TABLE 15 developer screening Table

As a result, as shown in Table 15, the sample developed in method 4 was clear in spots, satisfactory in separation degree and good in reproducibility, and therefore, the optimal developing agent was selected to be n-butanol-acetic acid-acetone-water (10:2:0.5:2).

2.1.3 methodological verification

Proved by methodology experiments, the method examines the specificity, reproducibility and durability, and has the thin layer effect under the conditions of different humidity and different temperature, and as a result, the thin layer spots are clear, the separation meets the requirements, the Rf value is moderate, and the verification experiments show that the repeatability is good and the durability is good.

2.2 thin-layer chromatography identification of safflower gentian in Lung force cough capsule

2.2.1 method Source references the standard of the safflower gentian medicine of the edition 2020 of Chinese pharmacopoeia

0.5g of sample is taken, 10ml of methanol is added, ultrasonic treatment is carried out for 15 minutes, and filtration is carried out, and the filtrate is taken as a sample solution. A medicinal solution prepared from 0.5g of radix Gentianae is prepared. And adding methanol into mangiferin reference substance to prepare a solution containing 1mg per 1ml, wherein the solution is used as reference substance solution. According to a thin layer chromatography (general rule 0502) test, 5 μl of each of the above three solutions is sucked and respectively spotted on the same silica gel GF254 thin layer plate, and developed with ethyl acetate-methanol-water (10:2:1) as developing agent, taken out, dried, and inspected under an ultraviolet lamp (254 nm). In the sample chromatogram, spots with the same color appear at the positions corresponding to the control chromatogram and the control chromatogram.

Results: thin layer identification of radix Gentianae in Carthami flos shows that major speckle is not obvious, and Rf value of speckle is small.

According to the thin layer result detected by the reference method, the problems, the combination property, the extraction solvent, the extraction mode, the choice of the developing agent, the proportion of the developing agent and the like in the thin layer identification method of the gentian carthami.

2.2.2 method for identifying thin layer of safflower gentian in capsule for treating cough caused by lung force after optimization

According to the thin layer result under the item 2.2.1, combining the physicochemical properties of gentian medicinal material of safflower, the tentative thin layer identification method is as follows:

Thin layer identification of gentian medicinal material of safflower: taking 3.0g of lung force cough fluid extract, adding 25mL of 60% methanol, heating and refluxing for 0.5h, filtering, evaporating filtrate to dryness, and adding 1mL of methanol into residues to dissolve the residues to obtain a sample solution; additionally, 0.5g of safflower gentian is used as a reference medicine, and the prepared reference medicine solution is prepared by the same method; adding methanol into mangiferin reference substance to prepare a solution containing 1mg per 1mL as reference substance solution; 5 mu L of each of the 3 solutions is sucked and respectively spotted on the same silica gel G254 thin layer plate, and the proportion is 12:1.5:1, developing with chloroform-acetone-water as developing agent, taking out, air drying, and making spots of the same color appear on the positions corresponding to the control medicine chromatogram and the control medicine chromatogram in the sample chromatogram;

2.2.2.1 extraction mode selection

Method 1: shaking and extracting for 30min.

Method 2: ultrasonic extraction is carried out for 30min.

Method 3: extracting under reflux for 30min.

The samples treated in the above three extraction modes were subjected to thin layer detection according to the method under item "2.2.2", respectively, and the results are shown in Table 16.

Table 16 screening table of extraction method

As a result, as shown in Table 16, the sample treated by "method 3" was clear in spots, satisfactory in resolution, moderate in Rf value and good in reproducibility, so that "heat reflux extraction" was the preferred extraction method.

2.2.2.2 selection of sample extraction solvent

Method 1: sample 3g was taken and extracted with 25ml of ethyl acetate under reflux with heating for 30min.

Method 2: 3g of the sample was taken, and extracted with 25ml of methanol under reflux with heating for 30min.

Method 3: sample 3g was taken and extracted with 25ml of 60% methanol under reflux with heating for 30min.

Method 4: sample 3g was taken and extracted with 50% methanol 25ml under reflux with heating for 30min.

The above four samples treated with the extraction solvent were subjected to thin layer detection according to the method under item "2.2.2", respectively, and the results are shown in Table 17.

TABLE 17 screening Table of sample extraction solvents

As a result, as shown in Table 17, the sample treated by "method 3" was clear in spots, satisfactory in resolution, moderate in Rf value and good in reproducibility, so that "60% methanol" was the preferred extraction solvent.

2.2.2.3 selection of the number of extractions

Method 1: 3g of the sample was taken, and 50ml of 60% methanol was used for reflux extraction with heating for 1 time each for 30min.

Method 2: 3g of the sample was taken, and 50ml of 60% methanol was used for reflux extraction with heating for 2 times each for 30min.

Method 3: 3g of the sample was taken, and extracted with 50ml of 60% methanol under reflux with heating for 3 times each for 30min.

The three samples treated with the above extraction times were subjected to thin layer detection according to the tentative identification method described under item "2.2.2", respectively, and the results are shown in Table 18.

Table 18 screening table of the number of extractions

As a result, as shown in Table 18, the samples treated by the various methods were clear in spots, satisfactory in resolution, moderate in Rf value and good in reproducibility, and therefore, the heat reflux extraction was preferable for 1 time.

2.2.2.4 sample application amount selection

The sample solutions of 2. Mu.L, 5. Mu.L and 10. Mu.L were aspirated, and the thin layer test was performed according to the tentative identification method described under item "2.2.2", and the results are shown in Table 19.

TABLE 19 sample application amount screening Table for test substances

As a result, as shown in Table 19, the amount of spotting was 5. Mu.L, spots were satisfactory, the Rf value was moderate, and reproducibility was good, so that the optimal amount of spotting of the sample was 5. Mu.L.

2.2.2.5 developer selection

Method 1: chloroform-water (8:2) as developing agent;

method 2: chloroform-ethyl acetate-acetic acid (8:2:1) is used as developing agent;

method 3: chloroform-acetone-acetic acid (10:2:2) is used as developing agent;

method 4: chloroform-acetone-water (12:1.5:1) was used as developing solvent; chloroform-acetone-water

The above four developing agents were developed, and the results of the thin layer test were shown in Table 20, respectively, according to the tentative identification method described in item "2.2.2".

TABLE 20 developer screening Table

As a result, as shown in Table 20, the sample developed in method 4 was clear in spots, satisfactory in separation degree and good in reproducibility, and therefore, the optimal developing agent was selected to be n-butanol-acetic acid-acetone-water (12:1.5:1).

2.2.3 methodological validation

2.3 thin-layer chromatography identification of Hedyotis diffusa medicinal material in Lung-force cough capsule

2.3.1 method Source reference literature name "Hedyotis Diffusa quality Standard study"

1g of a sample was taken, 15ml of methanol was added thereto, the mixture was sonicated for 30 minutes, filtered, the filtrate was evaporated to dryness, and 5ml of methanol was added to the residue to dissolve the residue, thereby obtaining a sample solution. And preparing a control medicinal material solution by the same method as the control medicinal material. According to thin layer chromatography (four parts 0502 of 2015 edition of Chinese pharmacopoeia), the two solutions are absorbed by 4-8 mu L of each solution and respectively spotted on the same silica gel G thin layer plate, and the solution is spread, taken out, dried and sprayed with 10% sulfuric acid ethanol solution, and heated at 105 ℃ until spots are clearly displayed by using ethyl acetate-methanol-water (25:5:0.5) as a spreading agent.

Results: the thin-layer identification of the oldenlandia diffusa herbal material has smaller main spots, is not obvious, has smaller spot Rf value and has poor reproducibility.

According to the thin layer result detected by the reference method, the problems, the combination property, the extraction solvent, the extraction mode, the choice of the developing agent, the proportion of the developing agent and the like in the oldenlandia diffusa thin layer identification method are examined.

2.3.2 method for identifying herba Hedyotidis Diffusae herbal material thin layer in capsule for treating pulmonary cough after optimization

According to the thin layer result under the item "2.3.1", combining the physicochemical properties of the oldenlandia diffusa medicinal material, the tentative thin layer identification method is as follows:

thin layer identification of oldenlandia diffusa medicinal material: taking 4.0g of a sample, adding 60mL of 70% methanol, heating and refluxing for 1h, filtering, evaporating the filtrate to dryness, and adding 5mL of methanol into the residue to dissolve the residue to obtain a sample solution; taking oleanolic acid reference substance, adding methanol to prepare a solution containing 1mg per 1mL, and taking the solution as reference substance solution; sucking 4-8 mu L of each of the 2 solutions, and respectively spotting on the same silica gel G thin layer plate according to the proportion of 25:7:4: petroleum ether (30-60 ℃) of 0.1-dimethylbenzene-ethanol-glacial acetic acid is taken as developing agent, developed, taken out, dried and sprayed with 10% sulfuric acid ethanol solution for developing color.

2.3.2.1 extraction mode selection

Method 1: shaking and extracting for 1h.

Method 2: ultrasonic extraction is carried out for 1h.

Method 3: extracting under reflux for 1h.

The samples treated in the above three extraction modes were subjected to thin layer detection according to the method under item "2.3.2", respectively, and the results are shown in Table 21.

Table 21 screening table of extraction method

As a result, as shown in Table 21, the sample treated by "method 3" was clear in spots, satisfactory in resolution, moderate in Rf value and good in reproducibility, so that "heat reflux extraction" was the preferred extraction method.

2.3.2.2 selection of sample extraction solvent

Method 1: 4g of the sample was taken, and 50ml of methanol was used for extraction under reflux with heating for 1 hour.

Method 2: 4g of the sample was taken and extracted with 50ml of 70% methanol under reflux with heating for 1 hour.

Method 3: 4g of the sample was taken and extracted with 50ml of 50% methanol under reflux with heating for 1h.

Method 4: 4g of the product is taken, 50ml of ethyl acetate is used for heating reflux extraction for 1h.

The above four samples treated with the extraction solvent were subjected to thin layer detection according to the method under item "2.3.2", respectively, and the results are shown in Table 22.

TABLE 22 screening Table of sample extraction solvents

As a result, as shown in Table 22, the sample treated by "method 2" was clear in spots, satisfactory in resolution, moderate in Rf value and good in reproducibility, so that "70% methanol" was the preferred extraction solvent.

2.3.2.3 selection of the number of extractions

Method 1: 5g of the sample was taken, and 50ml of 70% methanol was used for reflux extraction with heating for 1 time each for 1 hour.

Method 2: 5g of the sample was taken, and 50ml of 70% methanol was used for reflux extraction with heating for 2 times each for 1 hour.

Method 3: 5g of the sample was taken, and 50ml of 70% methanol was used for reflux extraction with heating for 3 times each for 1 hour.

The three samples treated with the above extraction times were subjected to thin layer detection according to the tentative identification method described under item "2.3.2", respectively, and the results are shown in Table 23.

Table 23 screening table of the number of extraction times

As a result, as shown in Table 23, the samples treated by "method 1 or 2" were clear in spots, satisfactory in resolution, moderate in Rf value and good in reproducibility, and therefore, the number of extraction times was preferably 1.

2.3.2.4 sample application amount selection

The test solutions were aspirated at 2. Mu.L, 4. Mu.L and 8. Mu.L, respectively, and the thin layer test was performed according to the tentative identification method described under item "2.3.2", and the results are shown in Table 24.

TABLE 24 sample application amount screening Table for test substances

As a result, as shown in Table 24, the amount of spotting was 4. Mu.L, the spots were satisfactory, the Rf value was moderate, and the reproducibility was good, so that the optimal amount of spotting of the sample was 4. Mu.L.

2.3.2.5 selection of the developing Agents

Method 1: chloroform-methanol (7:3) is used as developing agent;

method 2: ethyl acetate-methanol-water (7:2:1) is taken as a developing agent;

method 3: cyclohexane-xylene-ethanol-glacial acetic acid (20:10:1:0.2) is used as a developing agent;

method 4: petroleum ether (30-60 ℃) xylene-ethanol-glacial acetic acid (25:7:4:0.1) is used as a developing agent;

the above four developing agents were developed, and the results of the thin layer test were shown in Table 25, respectively, according to the tentative identification method described in item "2.3.2".

TABLE 25 developer screening Table

As a result, it was found from Table 25 that the sample developed in method 4 was clear in spots, satisfactory in resolution and good in reproducibility, and therefore, the optimal developing agent was petroleum ether (30-60 ℃ C.) and xylene-ethanol-glacial acetic acid (25:7:4:0.1).

2.3.3 methodological validation

3. Method for measuring baicalin content for investigation

3.1 method Source

Firstly, the content of baicalin in WS-10217 (ZD-0217) -2002 standard lung force cough capsule is measured according to high performance liquid chromatography (appendix VI D of the 2000 edition of Chinese pharmacopoeia) by referring to the content measuring method of baicalin.

Octadecylsilane chemically bonded silica is used as a filler for chromatographic conditions and system applicability tests; methanol-water-phosphoric acid (55:45:0.2) as mobile phase; the detection wavelength is 280nm; the column temperature is 25 ℃; the flow rate was 1.0mL/min. The theoretical plate number is calculated by baicalin peak and should not be lower than 2000.

Preparation of control solution A proper quantity of baicalin control is precisely weighed, and methanol is added to prepare solution with 70 mug per 1 ml.

Preparation of sample 0.2g of sample solution, precisely weighing, placing in 50ml measuring flask, adding appropriate amount of 70% ethanol, ultrasonic treating (power 300W, frequency 25 kHz) for 30 min, cooling, adding 70% ethanol, diluting to scale, shaking, filtering, and collecting filtrate.

Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.

Results: the problems of poor peak separation degree, poor peak stability, poor accuracy and the like are found, and the problems are examined for sample treatment, chromatographic conditions, extraction solvents and the like, specifically as follows:

3.1.1 selection of mobile phases and gradients

The optimization method comprises the following steps: the chromatographic column is CORTECS UPLC T3.8 um; the column temperature is 30 ℃; acetonitrile solution containing 0.01% glacial acetic acid is taken as a mobile phase A, and 0.10% glacial acetic acid water solution is taken as a mobile phase B for gradient elution; the detection wavelength is 280nm; the flow rate is 0.7mL/min, and the theoretical plate number is not lower than 3500 according to baicalin peak calculation;

TABLE 26 gradient elution TABLE

Results: the method has good peak shape and peak purity, but poor stability, and the conditions of poor separation degree of adjacent peaks appear respectively.

And the optimization method II comprises the following steps: the chromatographic column is CORTECS UPLC T3.8 um; the column temperature is 30 ℃; acetonitrile solution containing 0.01% glacial acetic acid is taken as a mobile phase A, and 0.1% glacial acetic acid water solution is taken as a mobile phase B for gradient elution; the detection wavelength is 280nm; the flow rate is 0.7mL/min, and the theoretical plate number is not lower than 3500 according to baicalin peak calculation;

TABLE 27 gradient elution TABLE

Results: the peak separation degree, peak shape and peak purity are all good, and the stability is good. Therefore, "optimization method two" is preferred as the best solution for the content detection chromatographic conditions.

3.1.2 determination of detection wavelength

The best solution to select wavelengths tentatively under item "3.1.1":

0.1g of sample is precisely weighed, placed in a 50ml measuring flask, added with a proper amount of 70% ethanol, subjected to ultrasonic treatment (power 300W, frequency 25 kHz) for 30 minutes, cooled, diluted to a scale by adding 70% ethanol, shaken uniformly, filtered, and the subsequent filtrate is taken to obtain the medicine. The absorption spectrum in the 190-400nm range is recorded.

Results: the baicalin has maximum absorption at about 275nm, and has high peak response and stable baseline, so 275nm is selected as the wavelength for detecting the baicalin content.

3.1.3 extraction solvent investigation

Determining chromatographic conditions under the condition of 3.1.2, taking 5 parts of the prepared sample, each part of the sample is about 0.2g, precisely weighing, placing the sample into a 50ml volumetric flask, respectively adding proper amounts of 70% methanol, ethanol, 80% ethanol, 70% ethanol and 60% ethanol, performing ultrasonic treatment (power 300W, frequency 25 KHz) for 0.5h, cooling, adding various diluting solvents to scale, shaking uniformly, filtering, and taking subsequent filtrate to obtain the product. The results are shown in Table 28 below:

TABLE 28 screening of extraction solvents

Extraction solvent	70% methanol	Methanol	80% ethanol	70% ethanol	60% ethanol
						Baicalin content (mg/g)	17.3	16.8	19.2	21.6	20.4

From the results, it was found that 70% ethanol was preferable as the extraction solvent because baicalin content was high when 70% ethanol was used as the extraction solvent.

3.1.4 extraction method investigation

Determining chromatographic conditions under the condition of '3.1.2', taking 3 parts of the product, each part of the product is about 0.2g, precisely weighing, placing one part of the product into a 50ml measuring flask, adding a proper amount of 75% ethanol, performing ultrasonic treatment (power 300W, frequency 25 kHZ) for 30 minutes, cooling, adding 75% ethanol diluent to a scale, shaking uniformly, filtering, and taking subsequent filtrate to obtain the product. And (3) placing the other part of the mixture into a conical flask, precisely adding 50ml of 75% ethanol, weighing, heating and refluxing for 1 hour, cooling, adding 75% ethanol to compensate for weight loss, shaking uniformly, filtering, and taking a subsequent filtrate to obtain the finished product. The results are shown in Table 29:

Table 29 extraction of baicalin content by different methods

Extraction method	Shaking machine	Heating and refluxing	Ultrasonic wave
				Baicalin content (mg/g)	16.1	19.3	21.4

As is clear from the results, when ultrasonic extraction is used, baicalin content is the highest, so ultrasonic is preferable as the extraction method.

3.1.5 investigation of extraction time

Determining chromatographic conditions under the condition of '3.1.2', taking 3 parts of the product, each part of the product is about 0.2g, precisely weighing, placing the product into a 50ml measuring flask, adding a proper amount of 75% ethanol, respectively carrying out ultrasonic treatment (power 300W, frequency 25 kHZ) for 20 minutes, 30 minutes and 40 minutes, cooling, respectively adding 75% ethanol diluent to scales, shaking uniformly, filtering, and taking subsequent filtrate, wherein the result is shown in the table 30:

TABLE 30 baicalin content at different extraction times

Extraction method	20min	30min	40min
				Baicalin content (mg/g)	20.5	21.4	21.8

As shown by the results, the ultrasonic extraction is carried out for 30min and 40min, the content of baicalin is high, the baicalin content is almost unchanged, and the ultrasonic time is preferably 30min due to the consideration of cost factors and efficiency.

3.1.6 method conclusion

Through the above investigation experiments, the preferred method for determining the baicalin content is as follows:

chromatographic conditions and system applicability test chromatographic column is CORTECS UPLC T3.8 um; the column temperature is 30 ℃; acetonitrile solution containing 0.01% glacial acetic acid is taken as a mobile phase A, and 0.1% glacial acetic acid water solution is taken as a mobile phase B for gradient elution; the detection wavelength is 275nm; the flow rate is 0.7mL/min, and the theoretical plate number is not lower than 3500 calculated according to baicalin peak.

3.2 methodological validation investigation

3.2.1 sample preparation and methods

3.2.2 linear relationship investigation

4, 8, 12, 16 and 20 mu l of baicalin reference solution (68.85 mu g/ml) is precisely sucked, chromatography is recorded, peak area is taken as an abscissa, sample injection quantity (mu g) is taken as an ordinate to be plotted, and a linear equation of Y=6E+06 x-12369 and R=0.9998 is obtained, so that the linear relation between 0.2754 mu g and 1.3770 mu g of baicalin content measurement is good. See table 31.

Table 31 linear relation investigation of baicalin

3.2.3 precision investigation

The baicalin reference substance solution is prepared by the method under the item 3.2.1 and is continuously injected for 6 times, the precision of the instrument is inspected, the RSD is calculated, and the RSD is 2.59%. The results show that the precision is good. See table 32.

Meter 32 precision test

3.2.4 repeatability investigation

6 lung force cough samples are respectively weighed, sample test solutions are respectively prepared according to the test solution preparation method under the item of 3.2.1, the content is measured according to the established chromatographic condition, the precision of the method is examined, and the RSD is calculated, so that the RSD is 2.12%. The results showed good reproducibility. See table 33.

Table 33 repeatability test

3.2.5 stability investigation

Preparing a sample solution and a baicalin reference substance solution according to the method under the item 3.2.1, measuring the content by using a set chromatographic condition, performing sample injection analysis at 0, 2, 6, 8 and 12 hours, examining the stability of the method, and calculating RSD, wherein the result RSD is 1.83%. The results show that the component to be measured is stable within 12 hours. See table 34.

Table 34 stability test

3.2.6 recovery investigation

6 lung force cough samples are respectively weighed precisely, a proper amount of corresponding baicalin reference substance solution is added, the content is measured under the established chromatographic condition, and the RSD is calculated, so that the average recovery rate is 96.80.16%, and the RSD is 1.35%. Meets the requirements. See table 35.

Table 35 recovery test

3.2.7 sample content determination

Taking 5 batches of lung force cough samples, preparing test liquid according to the method under the item 3.2.1, measuring the content and calculating. As a result, the baicalin contents of the three pilot samples were 21.25mg/g, 20.76mg/g, 21.18mg/g, 21.09mg/g and 21.39mg/g, respectively. The method of the invention is stable and feasible. See table 36.

Table 36 sample content data table

3.2.8 conclusion

In conclusion, the method has the advantages of good peak shape, qualified separation degree and peak purity, high accuracy, good stability and simple and quick operation.

Comparative example 1

The standard of execution of the lung force cough capsule issued by the national drug administration is received in WS-10217 (ZD-0217) -2002, and the preparation method is disclosed as follows: 173g of radix scutellariae, 167g of radix peucedani, 160g of radix stemonae, 150g of gentian carthami, 130g of Chinese phoenix tree root, 120 oldenlandia diffusa and 96g of red tube medicine, taking 30g of radix stemonae, crushing into fine powder for standby, decocting the rest of radix stemonae and the rest of radix scutellariae and the like with water twice, 2 hours for the first time and 1.5 hours for the second time, merging decoctions, filtering, concentrating filtrate into clear paste with the relative density of 1.17-1.20 (80 ℃), adding the fine powder, mixing evenly, drying, crushing and encapsulating.

According to the original loading standard preparation process, the transfer rate of baicalin is measured to be 60.13%, the transfer rate of procyanidine is measured to be 54.39%, and the transfer rate of stemona sessilifolia is measured to be 51.24%.

According to the original loading standard, three test sample solutions are prepared, and the content of baicalin which is the main active ingredient in the lung-force cough capsule is measured as follows: 13.29mg/g, 12.87mg/g, 13.06mg/g.

In conclusion, the transfer rate of the main active ingredient baicalin in the preparation method can reach more than 80%, which is higher than that of the traditional process contrast test by more than twenty percent; the transfer rate of the decursin is more than 75%, which is more than twenty percent higher than that of the traditional process contrast test; the transfer rate of the main active ingredient in the stemona root medicinal material to the stemona root can reach more than 85 percent, and is nearly thirty percent higher than that of the traditional process; the average content 21.39mg/g of baicalin is higher than that of the comparative test 13.07mg/g, and the content of the effective component of the baicalin of the composition prepared by the preparation method of the lung force cough pharmaceutical composition is higher than that of the composition prepared by the prior art, and the damage of the water decoction process to the effective component in the heat-sensitive medicinal material is avoided. By the preparation method of the lung force cough pharmaceutical composition, the content of active ingredients in the pharmaceutical composition is obviously improved, the extraction rate of the composition extract is obviously improved, and the quality of the medicine is obviously improved.

While the invention has been described in detail in the foregoing general description, embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims

1. The preparation method of the lung force cough pharmaceutical composition comprises 173g of radix scutellariae, 167g of radix peucedani, 160g of radix stemonae, 150g of safflower gentian, 130g of Chinese phoenix tree root, 120g of oldenlandia diffusa and 96g of red tube medicine, and is characterized by comprising the following steps:

(2) Preparation of traditional Chinese medicine extract:

2. A method of testing a pharmaceutical composition prepared by the method of claim 1, comprising: thin-layer identification of baicalin in the medicine, namely thin-layer identification of the baicalin in the medicine comprises the following specific steps of:

(3) Thin layer identification of oldenlandia diffusa medicinal material: taking 4.0g of sample, adding 60ml of 70% methanol, heating and refluxing for 1h, filtering, evaporating filtrate to dryness, and dissolving residues with 5ml of methanol to obtain a sample solution; taking oleanolic acid reference substance, adding methanol to prepare a solution containing 1mg per 1ml, and taking the solution as a reference solution; the two solutions were each drawn 4 μl and spotted on the same silica gel G thin layer plate in a ratio of 25:7:4:0.1, using 30-60 ℃ petroleum ether-dimethylbenzene-ethanol-glacial acetic acid as developing agent, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution for developing color, and displaying spots with the same color on the corresponding position of the sample chromatogram and the reference chromatogram;

(4) The content determination method of baicalin comprises the following steps:

1) Preparation of test solution: accurately weighing 0.2g of sample, placing in 50ml measuring flask, adding appropriate amount of 70% ethanol, ultrasonic treating for 30min, cooling, diluting with 70% ethanol to scale, shaking, filtering, and collecting filtrate;

2) Preparation of a control solution: precisely weighing appropriate amount of baicalin reference substance, and adding methanol to obtain solution containing 70 μg of baicalin per 1 mL;

3) Chromatographic conditions and system suitability test: the chromatographic column is CORTECS UPLC T3.8 um; the column temperature is 30 ℃; acetonitrile solution containing 0.01% glacial acetic acid is taken as a mobile phase A, and 0.10% glacial acetic acid water solution is taken as a mobile phase B for gradient elution; the detection wavelength is 275nm; the flow rate is 0.7mL/min, and the theoretical plate number is not lower than 3500 according to baicalin peak calculation; the gradient elution specifically comprises the following steps:

4) Assay: respectively precisely sucking 10 μl of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement.

3. The method according to claim 2, wherein the ethanol in step (1) is either acetonitrile or methanol.

4. The method according to claim 2, wherein the condition of the ultrasound in step (1) is: the power is 300W and the frequency is 25kHz.