CN109030699B

CN109030699B - Sugar-free cold cough relieving granule quality detection method

Info

Publication number: CN109030699B
Application number: CN201810666560.9A
Authority: CN
Inventors: 穆滨
Original assignee: HARBIN KANGLONG PHARMACEUTICAL CO Ltd
Current assignee: HARBIN KANGLONG PHARMACEUTICAL CO Ltd
Priority date: 2018-06-26
Filing date: 2018-06-26
Publication date: 2020-09-18
Anticipated expiration: 2038-06-26
Also published as: CN109030699A

Abstract

The invention relates to a quality control method of a traditional Chinese medicine preparation, and particularly discloses a quality detection method of sugar-free cold and cough granules. The quality detection method of the invention has good reproducibility, strong specificity and good durability of thin-layer chromatography detection, improves the quality control of the medicine, ensures that the qualitative and quantitative detection of the medicine is more accurate, and the quality is more easily controlled.

Description

Sugar-free cold cough relieving granule quality detection method

Technical Field

The invention relates to a quality control method of a traditional Chinese medicine preparation, in particular to a quality detection method of sugar-free cold and cough relieving granules.

Background

The sugar-free cold and cough relieving granule comprises the following main components in percentage by weight:

the preparation method comprises the following steps: decocting the above nine materials except Mentholum and the other eight materials such as bupleuri radix with water twice each for 4 hr, filtering the decoction, mixing the filtrates, concentrating to appropriate amount, spray drying the concentrated solution to obtain fine powder, adding sucralose, dextrin and Mentholum (encapsulated with appropriate amount of BETA-cyclodextrin), mixing, and dry granulating to obtain 300g (without sucrose).

The sugar-free cold and cough relieving granule is mainly used for clearing heat, relieving exterior syndrome, relieving cough and reducing sputum. Can be used for treating common cold caused by affection of exogenous wind-heat, with symptoms of fever, aversion to wind, headache, nasal obstruction, sore throat, cough, and general malaise.

The sugar-free cold and cough relieving granule is used as a Chinese patent medicine preparation, and the drug effect of the granule is the result of the combined action of a plurality of active ingredients. The existing quality standard does not comprehensively measure the effective components of the sugar-free cold and cough granules, has the limitation of one, and cannot comprehensively reflect the quality level of the sugar-free cold and cough granules.

At present, few patents are disclosed about the quality detection method of the cold and cough relieving granules. Chinese patent application 201010011610.3 discloses a detection method of a traditional Chinese medicine preparation for treating infantile common cold, which establishes a thin-layer chromatography identification method of folium isatidis, radix bupleuri, radix scutellariae, platycodon grandiflorum and liquorice and a baicalin content determination method, wherein the thin-layer identification of radix bupleuri is as follows: taking 5g of radix bupleuri as a reference medicinal material, adding 100ml of 50% ethanol, heating and refluxing for 1 hour, filtering, concentrating the filtrate on a water bath to about 20ml, cooling, preparing the rest according to a preparation method of a sample solution under the folium isatidis identification item, and adding 2ml of ethanol into residues to dissolve the residues to serve as a reference medicinal material solution; performing thin-layer chromatography test, taking 2 μ l of control medicinal material solution and 8 μ l of folium Isatidis sample solution under identification, respectively dropping on the same silica gel G thin-layer plate with sodium carboxymethylcellulose as binder, developing with chloroform-methanol-water as developing agent at a ratio of 8:2:0.2, taking out, air drying, spraying with 5% phosphomolybdic acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to those in the chromatogram of the reference solution; the patent application more effectively controls the quality of the product and ensures the clinical curative effect. Due to the difference of the components of the traditional Chinese medicine preparation for treating the infantile common cold, the detection method may have larger difference.

Chinese patent application 201610259721.3 discloses a method for simultaneously detecting baicalin, baicalein and wogonin in cold and cough syrup, which sequentially comprises the following steps: 1) measuring 1ml of the product, placing the product in a 20ml volumetric flask, and adding the mixture in a volume ratio of 1:1, dissolving methanol and acetonitrile, evaporating the solvent to dryness, fixing the volume to 20ml of scale mark by using the acetonitrile, uniformly mixing and filtering a membrane, and putting the filtrate into an ultra-high performance liquid chromatograph for detection; 2) the parameters for the analysis by the ultra performance liquid chromatography are set as follows: the analytical column ACEExcel2C18-PFP has the specification of 4.6 mu m multiplied by 250mm and 5 mu m, the flow rate is 0.8ml min < -1 >, the column temperature is 30 ℃, the detection wavelength is 276nm, and the sample injection amount is 2 mu L; the mobile phase A is acetonitrile, and the mobile phase B is 0.1% phosphoric acid solution; gradient elution procedure: 0-2.5 min, 5% of A and 95% of B; 2.5-5.1min, 30-70% of A and 70-30% of B; 5.1-12min, 90% A, 10% B; equilibrate for 1min at 2% A, 98% B. The application can simultaneously and rapidly detect the contents of baicalin, baicalein and wogonin in the syrup for treating cold and cough.

In the prior art, no comprehensive quality control method reflects the quality condition of the cold and cough relieving granules, so that the production process and the product quality of the cold and cough relieving granules cannot be effectively controlled, and the curative effect of the medicine cannot be better ensured. Therefore, the establishment of the quality detection of the sugar-free cold and cough relieving granules has great significance.

Disclosure of Invention

The invention aims to provide a quality detection method of sugar-free cold cough granules, which can comprehensively and accurately analyze active components of a medicament, is beneficial to quality control of active ingredients of a product and ensures the clinical curative effect of the product.

The purpose of the invention is realized by the following technical scheme: a quality detection method of sugar-free granule for treating common cold and cough comprises identifying Mentholum, herba Artemisiae Annuae and bupleuri radix,

(1) identification of menthol:

preparation of a test solution: taking 10-20g of the product, grinding, placing in a round-bottom flask, adding 20-60mL of water, sealing, performing ultrasonic treatment, and soaking; adding 140 mL of water, connecting with a volatile oil tester, adding 30-60mL of water from the upper end of the tester, overflowing the water to the flask, adding ethyl acetate, connecting with a reflux condenser tube, slowly heating to boil, keeping slightly boiling for 0.5-1.5 hours, stopping heating, separating an ethyl acetate layer, rapidly filtering by using a funnel paved with anhydrous sodium sulfate, washing a volatile oil extractor and a filter by using a small amount of ethyl acetate, placing the filtrate in a 10mL measuring flask, diluting to a scale by using ethyl acetate, and shaking uniformly to obtain a sample solution;

preparation of control solutions: taking a menthol control, adding ethyl acetate to prepare a solution containing 2mg in each 1mL as a control solution;

preparation of negative control solution: taking other medicinal materials except menthol according to the prescription respectively to prepare a negative control sample without menthol, and preparing a negative control sample solution without menthol according to the preparation method of the test sample solution;

thin-layer chromatography test: pipette 5. mu.l of each of the two solutions, spot on the same silica gel G thin layer plate, and mix with cyclohexane: developing ethyl acetate (12-17): (3-6) as developing agent, taking out, air drying, spraying 2-4% vanillin sulfuric acid solution, and heating at 105 deg.C until the color of spots is clear; in the chromatogram of the test solution, the main spots with the same color appear at the corresponding positions of the chromatogram of the reference solution;

(2) identification of sweet wormwood herb:

preparation of a test solution: grinding 2-5g of the product, adding water 30-50ml, heating under reflux, filtering, extracting the filtrate with petroleum ether under shaking, collecting aqueous layer solution 10ml, extracting with ethyl acetate under shaking, mixing the ethyl acetate solutions, recovering solvent to dryness, and dissolving the residue with ethanol 2ml to obtain sample solution;

preparation of control solutions: adding 15ml of water into lg of herba Artemisiae Annuae control material, heating and refluxing for 1 hr, filtering, and preparing the filtrate into control material solution by the same method;

preparation of negative control solution: taking the other medicinal materials except the sweet wormwood according to the prescription respectively to prepare a negative control sample without the sweet wormwood, and preparing a negative control sample solution without the sweet wormwood according to the preparation method of the test sample solution;

thin-layer chromatography test: sucking 1 mul of each of the two solutions, respectively dropping on the same silica gel G thin layer plate, adding petroleum ether: ethyl acetate ═ (5-8): (4-6) developing with developing agent, taking out, air drying, and inspecting under ultraviolet light; in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution;

(3) identification of bupleurum:

preparation of a test solution: taking 3-8g of the product, adding water, heating and refluxing for 1h, filtering, extracting the filtrate with petroleum ether under shaking, taking the aqueous layer solution, extracting with water-saturated n-butanol under shaking for 1-3 times, combining the n-butanol solution, washing with sodium hydroxide solution, discarding the washing solution, washing with n-butanol-saturated water, discarding the washing solution, recovering solvent from the n-butanol solution until dry, and dissolving the residue with 1ml of methanol to obtain a sample solution;

preparation of control solutions: adding 15ml of water into 1g of radix bupleuri control medicinal material to obtain control medicinal material solution;

preparation of negative control solution: taking the other medicinal materials except for radix bupleuri according to the prescription respectively to prepare a negative control sample without radix bupleuri, and preparing a negative control sample solution without radix bupleuri according to the preparation method of the test sample solution;

thin-layer chromatography test: pipetting 5 mul of each of the two solutions, dropping the solution on the same silica gel G thin layer plate, adding chloroform: methanol: developing with water 8:2:0.2 as developing agent, taking out, air drying, spraying 1% p-dimethylaminobenzaldehyde sulfuric acid ethanol solution, heating at 105 deg.C until the spot color is clear, and respectively inspecting under sunlight and 365nm ultraviolet light; in the chromatogram of the test solution, at least two main spots with the same color appear in the corresponding positions of the chromatogram of the reference solution under sunlight; at least two main spots of fluorescence with the same color appear under ultraviolet light.

Further, the sugar-free cold cough granule comprises the following components: bupleurum l00g, lonicera confusa 75g, kudzuvine root 100g, sweet wormwood 75g, weeping forsythia 75g, baikal skullcap root 75g, balloonflower root 50g, bitter apricot seed 50g, menthol 0.15 g;

the preparation method comprises the following steps: decocting the above nine materials except Mentholum and the other eight materials such as bupleuri radix with water twice each for 4 hr, filtering the decoction, mixing the filtrates, concentrating to appropriate amount, spray drying the concentrated solution to obtain fine powder, adding sucralose, dextrin and Mentholum, mixing, and dry granulating to obtain 300g sucrose-free granule.

Further, in the (1) identification of menthol, preparation of a test solution: taking 10-20g of the product, grinding, placing in a round bottom flask, adding 30-50mL of water, sealing, performing ultrasonic treatment for 2-4min, and soaking for 20-30 min; continuously adding 150-180mL of water, connecting a volatile oil tester, adding 30-60mL of water from the upper end of the tester, overflowing the water to the flask, adding 2-7mL of ethyl acetate, connecting a reflux condenser tube, slowly heating to boil, keeping slightly boiling for 0.5-1.5 hours, stopping heating, separating an ethyl acetate layer, rapidly filtering by using a funnel paved with anhydrous sodium sulfate, washing a volatile oil extractor and a filter by using a small amount of ethyl acetate, placing the filtrate in a 10-mL measuring flask, diluting to a scale by using ethyl acetate, and shaking uniformly to serve as a test solution;

further, in the (1) identification of menthol, the preparation of the test article: taking 15g of the product, grinding, placing in a round bottom flask, adding 40mL of water, sealing, performing ultrasonic treatment for 3min, and soaking for 25 min; adding 160mL of water, connecting with a volatile oil tester, adding 40mL of water from the upper end of the tester again, overflowing the water to the flask, adding 5mL of ethyl acetate, connecting with a reflux condenser tube, slowly heating to boil and keeping slightly boiling for 1.0 h, stopping heating, separating ethyl acetate layer, rapidly filtering with a funnel paved with anhydrous sodium sulfate, washing the volatile oil extractor and filter with a small amount of ethyl acetate, placing the filtrate in a 10mL measuring flask, diluting with ethyl acetate to scale, and shaking up to obtain a sample solution.

Further, in the identification of (1) menthol, a thin layer chromatography test: pipette 5. mu.l of each of the two solutions, spot on the same silica gel G thin layer plate, and mix with cyclohexane: developing with ethyl acetate 14:6 as developing agent, taking out, air drying, spraying 4% vanillin-sulfuric acid solution, and heating at 105 deg.C until the spots are clearly developed; the main spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution.

Further, in the (2) identification of the sweet wormwood herb, the preparation of a test sample comprises the following steps: grinding 2-5g of the product, adding 30-50ml of water, heating and refluxing for 1h, filtering, extracting the filtrate with petroleum ether under shaking for 1-3 times (20-40 ml each time), extracting the aqueous layer solution with 10ml of water, extracting with ethyl acetate under shaking for 1-5 times (10 ml each time), mixing the ethyl acetate solutions, recovering solvent to dryness, and dissolving the residue with 2ml of ethanol to obtain a sample solution.

Further, in the (2) identification of the sweet wormwood herb, the preparation of a test sample comprises the following steps: collecting 3g of the product, grinding, adding 40ml of water, heating and refluxing for 1h, filtering, extracting the filtrate with petroleum ether under shaking for 2 times (30 ml each time), collecting aqueous layer solution 10ml, extracting with ethyl acetate under shaking for 2 times (10 ml each time), mixing ethyl acetate solutions, recovering solvent to dry, and dissolving the residue with 2ml of ethanol to obtain a sample solution.

Further, in the identification of the sweet wormwood herb (2), a thin layer chromatography test is as follows: sucking 1 mul of each of the two solutions, respectively dropping on the same silica gel G thin layer plate, adding petroleum ether: ethyl acetate ═ 6: 4.5 is developing agent, developing, taking out, airing, and inspecting under 365nm ultraviolet light; in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.

Further, in the (3) identification of bupleurum, the preparation of a test solution: taking 3-8g of the product, adding 20-40ml of water, heating and refluxing for 1h, filtering, extracting the filtrate with petroleum ether under shaking for 1-3 times, 15ml each time, taking 10ml of aqueous layer solution, extracting with water-saturated n-butyl alcohol under shaking for 1-3 times, 20ml each time, combining n-butyl alcohol solutions, washing with 40ml of 1-3% sodium hydroxide solution, discarding the washing solution, washing with 40ml of water saturated with n-butyl alcohol, discarding the washing solution, recovering solvent from the n-butyl alcohol solution until dry, and dissolving the residue with 1ml of methanol to obtain a sample solution.

Further, in the (3) identification of bupleurum, the preparation of a test solution: taking 6g of the product, adding 30ml of water, heating and refluxing for 1h, filtering, extracting the filtrate with petroleum ether for 2 times and 15ml each time, taking 10ml of aqueous layer solution, extracting with water saturated n-butyl alcohol for 2 times and 20ml each time, combining the n-butyl alcohol solutions, washing with 40ml of 1% sodium hydroxide solution, discarding the washing solution, washing with 40ml of water saturated with n-butyl alcohol, discarding the washing solution, recovering the solvent from the n-butyl alcohol solution until the n-butyl alcohol solution is dry, and adding 1ml of methanol to dissolve the residue to obtain a sample solution.

Further, in the identification of bupleurum root in the step (3), in the preparation process of the test sample solution, the solvent of the n-butanol solution is recovered until the sample is dried, the sample is added into the mixed solution of hydrochloric acid and acetic acid for soaking, and then the sample is added with 1ml of methanol for dissolution after freeze drying to be used as the test sample solution. The inventor finds that in the research process, in the preparation process of the bupleurum test sample solution, the detection limit of bupleurum can be greatly reduced by soaking a sample in a mixed solution of hydrochloric acid and acetic acid and then freeze-drying the sample.

Further, the mass fraction of the hydrochloric acid is 3%, and the mass ratio of the hydrochloric acid to acetic acid is 1: 1.

Further, the quality detection method of the sugar-free cold and cough relieving granules also comprises an identification method of forsythia, kudzu root and lonicera confusa:

(4) the identification method of the forsythia suspense comprises the following steps:

preparation of a test solution: grinding 3g of the product, adding 100ml of water, heating and refluxing for 30 minutes, cooling, filtering, adjusting the pH value of the filtrate to 2 by using hydrochloric acid solution, shaking and extracting by using diethyl ether for 2 times, 30ml of each time, combining diethyl ether extracting solutions, volatilizing, and adding ethanol lml into residues to dissolve the residues to obtain a test solution;

preparation of control solutions: collecting control material of fructus forsythiae lg, adding water 50ml, and preparing control material solution by the same method;

and (3) testing according to thin-layer chromatography: sucking 10 mul of test solution and 5 mul of reference solution, respectively dropping on the same silica gel G thin layer plate, adding chloroform: ethyl acetate: developing with methanol 20:0.5:0.5 as developing agent, taking out, air drying, spraying 10% sulphuric acid ethanol solution, heating at 105 deg.C until the spots are clearly developed; in the chromatogram of the test solution, main spots with the same color appear at the corresponding positions of the chromatogram of the reference solution;

(5) the identification method of the kudzuvine root comprises the following steps:

preparation of a test solution: grinding 3g of the product, adding 30ml of methanol, performing ultrasonic treatment, filtering, evaporating the filtrate to dryness, and dissolving the residue in 3ml of methanol to obtain a sample solution;

preparation of control solutions: taking puerarin reference substance, adding methanol to prepare solution containing lmg per l ml as reference substance solution;

and (3) testing according to thin-layer chromatography: sucking the two solutions each at 2 μ 1, and dropping on the same silica gel GF₂₅₄On thin layer plates, with chloroform: developing with methanol 3:1 as developing agent, taking out, air drying, and inspecting with ultraviolet lamp. Spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution;

(6) the identification method of the lonicera confusa comprises the following steps:

preparation of a test solution: taking 3g of the product, grinding, adding ethyl acetate, carrying out ultrasonic treatment for 2 times (30 ml each time), discarding an ethyl acetate solution, adding 0.25ml of lmol/L hydrochloric acid solution into residues, adding ethyl acetate, carrying out ultrasonic treatment for 2 times (20 ml each time), combining the ethyl acetate solutions, evaporating to dryness, and adding ethanol lml into the residues to dissolve the residues to obtain a sample solution;

preparation of control solutions: adding methanol into chlorogenic acid reference substance to obtain lmg solution per l ml as reference substance solution;

and (3) testing according to thin-layer chromatography: sucking the two solutions 1-2 mu 1 respectively, and spotting on the same polyamide film to form strips, adding toluene: ethyl acetate: formic acid: glacial acetic acid: taking the upper solution with water as a developing agent, developing, taking out, drying, and placing under an ultraviolet lamp for inspection; in the chromatogram of the test solution, fluorescent stripes with the same color appear at the corresponding positions of the chromatogram of the reference solution.

Furthermore, in the puerarin identification, the ultrasonic treatment time is 10-15min, and the ultraviolet wavelength of the ultraviolet lamp is 254 nm; in the identification of the lonicera confusa, the ultrasonic treatment time is 5-8min, and the ultraviolet wavelength of the ultraviolet lamp is 365 nm.

Further, the quality detection method of the sugar-free cold and cough granules also comprises the content determination of baicalin, puerarin and chlorogenic acid,

wherein, the baicalin content determination method is determined by high performance liquid chromatography:

chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; methanol, water and phosphoric acid are taken as mobile phases, wherein the ratio of the methanol to the water to the phosphoric acid is 50:50: 0.2; the detection wavelength is 280 nm; the number of theoretical plates is not less than 3000 calculated according to baicalin peak;

preparation of control solutions: taking appropriate amount of baicalin as reference substance, precisely weighing, and adding methanol to obtain solution containing 60 μ g per lml;

preparation of a test solution: taking the product with different filling amount, grinding, taking about lg or 3g, precisely weighing, placing in a conical flask with a plug, precisely adding 25ml of 70% ethanol, sealing the plug, weighing, ultrasonically treating, cooling, weighing again, supplementing the lost weight with 70% ethanol, shaking up, filtering, precisely weighing 10ml of subsequent filtrate, placing in a 25ml measuring flask, adding 70% ethanol to scale, shaking up, filtering, and taking the subsequent filtrate;

the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into a liquid chromatograph, and measuring;

the puerarin content determination method is determined by high performance liquid chromatography:

chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; taking methanol and water as a mobile phase, wherein the ratio of the methanol to the water is 27: 73; the detection wavelength is 250 nm; the number of theoretical plates is not less than 4000 calculated according to puerarin peak;

preparation of control solutions: taking a proper amount of puerarin reference substance, precisely weighing, and adding methanol to prepare a solution containing 40 μ g of puerarin per 1 ml;

preparation of a test solution: grinding the product with different contents, precisely weighing 0.3g, placing in a 50ml measuring flask, adding appropriate amount of 30% methanol, ultrasonic treating, cooling, adding 30% methanol to desired volume, shaking, and filtering;

the content of chlorogenic acid is determined by high performance liquid chromatography:

chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile and 0.4 percent phosphoric acid solution which are 13:87 are taken as mobile phases; the detection wavelength is 327 nm; the number of theoretical plates is not less than 1000 calculated according to the peak of chlorogenic acid;

preparation of control solutions: precisely weighing 12.5mg chlorogenic acid reference substance, placing in 25ml brown measuring flask, adding methanol to dilute to scale, precisely weighing 2ml chlorogenic acid reference substance, placing in 25ml brown measuring flask, adding water to dilute to scale, and making into solution containing 40 μ g per 1ml to obtain the final product;

preparation of a test solution: grinding the product with different contents, precisely weighing 0.6g, placing in a 50ml measuring flask, adding appropriate amount of 50% methanol, ultrasonic treating, cooling, adding 50% methanol to desired volume, shaking, and filtering;

the determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.

In the present invention, the component ratios of the developing solvent are volume ratios unless otherwise specified.

The detection method of the sugar-free cold cough granules disclosed by the invention is used for identifying the menthol, the sweet wormwood herb, the radix bupleuri, the fructus forsythiae, the radix puerariae and the lonicera confusa by using the thin-layer chromatography, has the advantages of good reproducibility, strong specificity and good durability, and is used for determining the contents of the baicalin, the puerarin and the chlorogenic acid in the sugar-free cold cough granules by using the high performance liquid chromatography. The method realizes the comprehensive characterization of each component of the sugar-free cold and cough granules, avoids the one-sidedness of judging the whole quality of the sugar-free cold and cough granules only by measuring a plurality of components, improves the quality control of the medicine, ensures that the qualitative and quantitative detection of the medicine is more accurate, and the quality is more easily controlled.

Drawings

FIG. 1 thin layer chromatography of menthol; FIG. 2 reproducibility of thin layer chromatography of menthol; FIG. 3 menthol specificity test; FIG. 4(a) different room temperature durability tests of menthol; FIG. 4(b) durability test of menthol at different relative humidity; FIG. 5 thin layer chromatography detection limits for menthol; FIG. 6(a) effect of different developing agents on thin layer chromatography of menthol; FIG. 6(b) effect of different test sample preparation methods on thin layer chromatography of menthol; FIG. 7 Artemisia annua thin layer chromatography; FIG. 8 Artemisia annua specificity test; FIG. 9(a) durability test of Artemisia annua at different room temperatures; FIG. 9(b) durability test of Artemisia annua with different relative humidity; FIG. 10 is a graph showing the detection limit of thin layer chromatography of Artemisia annua; FIG. 11 effect of different treatments on thin layer chromatography of Artemisia annua; FIG. 12 thin layer chromatography of Bupleurum chinense Mill; FIG. 13A bupleuri radix specificity test; FIG. 14(a) tolerance test of Bupleurum chinense to different room temperatures; FIG. 14(b) different relative humidity tolerance tests of bupleuri radix; FIG. 15 the detection limit of bupleuri radix.

Detailed Description

The invention is further illustrated by the following examples. These examples are for illustrative purposes only and do not limit the scope and spirit of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

The sugar-free cold and cough relieving granule is prepared from the following components in parts by weight: bupleurum l00g, lonicera confusa 75g, kudzuvine root 100g, sweet wormwood 75g, weeping forsythia 75g, baikal skullcap root 75g, balloonflower root 50g, bitter apricot seed 50g and menthol 0.15 g; the preparation method comprises the following steps: decocting the above nine materials except Mentholum and the other eight materials such as bupleuri radix with water twice each for 4 hr, filtering the decoction, mixing the filtrates, concentrating to appropriate amount, spray drying the concentrated solution to obtain fine powder, adding sucralose, dextrin and Mentholum (encapsulated with appropriate amount of beta-cyclodextrin), mixing, and dry granulating to obtain 300g (without sucrose).

Example 1

1.1 identification of menthol:

preparation of a test solution: taking 15g of the product, grinding, placing in a round bottom flask, adding 40mL of water, sealing, performing ultrasonic treatment for 3min, and soaking for 25 min; adding 160mL of water continuously, connecting a volatile oil tester, adding 40mL of water from the upper end of the tester again, overflowing the water to a flask, adding 5mL of ethyl acetate, connecting a reflux condenser tube, slowly heating to boil and keeping slightly boiling for 1.0 hour, stopping heating, separating an ethyl acetate layer, rapidly filtering by using a funnel paved with anhydrous sodium sulfate, washing a volatile oil extractor and a filter by using a small amount of ethyl acetate, placing the filtrate in a 10mL measuring flask, diluting to a scale by using the ethyl acetate, and shaking up to obtain a sample solution;

thin-layer chromatography test: pipette 5. mu.l of each of the two solutions, spot on the same silica gel G thin layer plate, and mix with cyclohexane: developing with ethyl acetate 14:6 as developing agent, taking out, air drying, spraying 4% vanillin-sulfuric acid solution, and heating at 105 deg.C until the spots are clearly developed;

the results are shown in FIG. 1, where 1-6 are negative control solution, test solution, control solution, and test solution, respectively, at high temperature for 10 days; it can be seen that the main spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution; negative no spots.

1.2 reproducibility

The results of preparing two test solutions according to the test solution preparation method of 1.1 are shown in FIG. 2, where 1-5 are menthol control solution (10. mu.l), test solution (5. mu.l), menthol control solution (2. mu.l) and test solution (2. mu.l), respectively. It can be seen that the main spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatogram of the reference solution, which indicates the reproducibility of the method.

1.3 specificity test

1 sample of sugar-free type after alteration (batch No. 160902), 1 sample before alteration (batch No. 1601001) and a menthol-negative sample were treated in accordance with the method described in 1.1 and measured.

The results are shown in FIG. 3, where 1-5 are respectively the sample (lot number: 1601001), sample (lot number: 160902), sample solution (lot number: 160902), menthol control, and negative sample;

it can be seen that, in the sample chromatogram before and after the change, the main spots of the same color appear at the positions corresponding to the control chromatogram; in the chromatogram of the negative sample, no spot appears at the position corresponding to the chromatogram of the control; negatives do not interfere with the assay, and the method is well-defined.

1.4 durability test

Different room temperatures: respectively sucking 5 μ l of each of the sugar-free sample after modification (lot number: 160902) solution, the sample before modification (lot number: 1601001) solution, and the menthol control solution under the above specificity test items, respectively dropping on the same silica gel G thin layer plate, developing at room temperature different from that under the specificity test items (specificity room temperature is 22 deg.C, and the test room temperature is 20 deg.C), with cyclohexane-ethyl acetate (14:6) as developing agent, taking out, air drying, spraying 4% vanillin sulfuric acid solution, and heating at 105 deg.C until spots clearly develop.

As shown in FIG. 4(a), 1 to 6 are respectively a negative sample, a test sample (lot No. 1601001), a test sample (lot No. 160902), a menthol control, a test sample (lot No. 1601001), and a test sample (lot No. 160902); it can be seen that the sample chromatogram before and after the change showed the same color of the main spot at the position corresponding to the control chromatogram.

Different relative humidities: respectively sucking 5 μ l of each of the sugar-free sample (lot number: 160902) solution after modification, the sample (lot number: 1601001) solution before modification, and the menthol control solution under the above-mentioned specificity test items, respectively dropping on the same silica gel G thin layer plate, developing with cyclohexane-ethyl acetate (14:6) as developing agent under the condition of relative humidity (specificity test relative humidity is 28%, the test is that 27.5% sulfuric acid solution is placed on one side of double-layer developing tank, and relative humidity is 72%) different from that under the specificity test items, taking out, air drying, spraying with 4% vanillin sulfuric acid solution, and heating at 105 deg.C until the spots are clearly developed.

The results are shown in FIG. 4(b), where 1 to 6 are respectively a negative sample, a test sample (lot No. 1601001), a test sample (lot No. 1601001), a menthol control, a test sample (lot No. 160902), and a test sample (lot No. 160902); it can be seen that the sample chromatogram before and after the change showed the same color of the main spot at the position corresponding to the control chromatogram.

In conclusion, the durability of the method is good.

1.5 detection Limit

Taking menthol control solution, diluting with ethyl acetate to different concentrations (0.475mg/ml, 0.1583mg/ml), respectively dropping on the same silica gel G thin layer plate, taking cyclohexane-ethyl acetate (17:3) as developing agent, developing, taking out, air drying, spraying with 5% vanillin sulfuric acid solution, and heating at 105 deg.C until the spots are clearly developed. The results are shown in FIG. 5, where 1-5 are menthol control (0.475 mg/ml: 1. mu.l), menthol control (0.475 mg/ml: 0.5. mu.l), menthol control (0.1583 mg/ml: 2. mu.l), menthol control (0.1583 mg/ml: 1. mu.l), and menthol control (0.1583 mg/ml: 0.5. mu.l), respectively; it can be seen that the detection limit for determining menthol is: 0.1583 μ g.

Comparative example 1.1

The developer in the measurement method of 1.1 of example 1 was replaced, and the developer was adjusted to: toluene-ethyl acetate (19: 1). The results of the same assay are shown in FIG. 6(a), wherein 1-3 are negative, test solution, and menthol control solution, respectively; it can be seen that only the standard sample showed clear spots, and the test sample showed no spots. This developer is shown to be less effective.

Comparative example 1.2

The method for preparing the sample solution and the control solution in the measurement method of 1.1 in example 1 was changed, and the other methods were adjusted to: test solution: taking 3g of the product (sugar-free type, batch number: 160902), grinding, adding 25ml of petroleum ether (60-90 deg.C), performing ultrasonic treatment for 5 minutes, filtering, and volatilizing the filtrate to about 1ml at room temperature to obtain a sample solution. Negative: taking a proper amount (equivalent to one bag) of sample without menthol, grinding, adding 25mL of petroleum ether (60-90 ℃), performing ultrasonic treatment for 5 minutes, filtering, and volatilizing the filtrate to about 1mL at room temperature to obtain a negative sample solution. Control solution: taking Mentholum control, adding petroleum ether (60-90 deg.C) to make into solution containing 2mg per 1ml as control solution. The results are shown in FIG. 6(b), where 1-5 are negative, menthol control, small test sample solution, and small test sample solution, respectively; as can be seen, only the control shows fuzzy spots, and the others do not show spots, which indicates that the method has poor effect.

Example 2

2.1 identification of sweet wormwood:

preparation of a test solution: grinding 3g of the product, adding 40ml of water, heating and refluxing for 1h, filtering, extracting the filtrate with petroleum ether under shaking for 2 times (30 ml each time), extracting the aqueous layer solution with 10ml of water, extracting with ethyl acetate under shaking for 2 times (10 ml each time), mixing the ethyl acetate solutions, recovering solvent to dryness, and dissolving the residue with 2ml of ethanol to obtain a sample solution;

thin-layer chromatography test: sucking 1 mul of each of the two solutions, respectively dropping on the same silica gel G thin layer plate, adding petroleum ether: ethyl acetate ═ 6: 4.5 is developing agent, developing, taking out, airing, and inspecting under 365nm ultraviolet light; the results are shown in FIG. 7, where 1-6 are respectively the test sample (lot number: 160901; 2. mu.l), the test sample (lot number: 160902; 2. mu.l), the test sample (lot number: 160903; 2. mu.l), the Artemisia apiacea reference drug (2. mu.l), the test sample (lot number: 1601001; 2. mu.l), and the test sample (lot number: 1601002; 2. mu.l); in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution;

2.2 specificity test

Taking a sugar-containing sample (batch number: 1601001), a sugar-free sample (batch number: 160902), a negative sample and a sweet wormwood herb control medicinal material, respectively preparing a sugar-containing sample solution, a sugar-free sample solution, a negative sample solution and a sweet wormwood herb control medicinal material solution according to the above method, and determining according to the above method, wherein the results are shown in figure 8, and 1-7 are respectively a negative sample (no lonicera confusa and no sweet wormwood herb), a negative sample (no sweet wormwood herb), a test sample 3 (batch number: 1601001), a test sample 4 (batch number: 160902), a sweet wormwood herb control medicinal material 5, a test sample 6 (batch number: 1601001) and a test sample 7 (batch number: 160902); as can be seen, the method has better specificity without negative interference of the sweet wormwood herb and the lonicera confusa in the determination.

2.3 durability test

Different room temperatures: respectively sucking 1 μ l of each of the solution of the sugar-free sample after modification (lot number: 160902), the solution of the sample before modification (lot number: 1601001), and the solution of the herba Artemisiae Annuae control drug under the above specificity test item, respectively dropping on the same silica gel G thin layer plate, developing at room temperature different from that under the specificity test item (specificity room temperature is 22 deg.C, and the test room temperature is 20 deg.C) with petroleum ether (60-90 deg.C) -ethyl acetate (6: 4.5) as developing agent, taking out, air drying, and inspecting under ultraviolet light (365 mn). The results are shown in FIG. 9(a), wherein 1-4 are 1 negative sample, 2 test sample (lot number: 1601001), 3 herba Artemisiae Annuae control drug, 4 test sample (lot number: 160902), respectively; it can be seen that the sample chromatogram before and after the change shows fluorescence spots of the same color at the positions corresponding to the control chromatogram.

Different relative humidities: respectively sucking 1 μ l of each of the modified sugar-free sample (lot number: 160902) solution, 1 lot of the pre-modified sample (lot number: 1601001) solution, and the herba Artemisiae Annuae control solution under the above-mentioned specificity test items, respectively dropping on the same silica gel G thin layer plate, developing with petroleum ether (60-90 deg.C) -ethyl acetate (6: 4.5) as developing agent under the condition of different relative humidity (30% of specificity test, 72% of relative humidity) in a double-layer developing tank, taking out, air drying, and inspecting under ultraviolet light (365 nm). The results are shown in FIG. 9(b), where 1-4 are 1 test sample (lot number: 1601001), 2 herba Artemisiae Annuae control drug, 3 test sample (lot number: 160902), and 4 negative samples, respectively; it can be seen that the sample chromatogram before and after the change shows the main fluorescence spots of the same color at the corresponding positions of the control chromatogram.

In conclusion, the method has good durability.

2.4 detection Limit

The herba Artemisiae Annuae control medicinal material solution is diluted with ethanol to different concentrations in series for detection, and the results are shown in FIG. 10, wherein 1-7 are 1 herba Artemisiae Annuae control medicinal material (1 mg/ml; 0.5 μ l), 2 herba Artemisiae Annuae control medicinal material (0.33 mg/ml; 2 μ l), 3 herba Artemisiae Annuae control medicinal material (0.33 mg/ml; 1 μ l), 4 herba Artemisiae Annuae control medicinal material (0.33 mg/ml; 0.5 μ l), 5 herba Artemisiae Annuae control medicinal material (0.165 mg/ml; 0.5 μ l), 6 herba Artemisiae Annuae control medicinal material (0.33 mg/ml; 5 μ l), 7 herba Artemisiae Annuae control medicinal material (0.165 mg/ml; 2 μ l); it can be seen that the detection limit of the artemisia apiacea reference medicinal material is as follows: 0.33. mu.g.

Comparative example 2

Preparing a test solution: the preparation method of the test solution is preliminarily formulated according to the literature as follows: sampling 10g or 3g (sugar-free type), adding water 30ml, heating to dissolve completely, cooling, extracting with petroleum ether (60-90 deg.C) under shaking for 2 times (30 ml each time), collecting aqueous layer solution 10ml, extracting with ethyl acetate under shaking for 2 times (10 ml each time), mixing ethyl acetate solutions, recovering solvent to dryness, and dissolving the residue with ethanol 2ml to obtain sample solution.

Taking a negative sample of the sweet wormwood: 1/10 the medicinal materials in the prescription are prepared into dry paste powder without sweet wormwood, the menthol clathrate and the auxiliary materials are added according to the sugar-free prescription, and the mixture is uniformly mixed, and the negative sample is prepared into the negative sample solution by the same method.

Control solution: taking 1g of sweet wormwood herb as a reference medicinal material, adding 15ml of water, heating and refluxing for 1 hour, and filtering. The filtrate is prepared into control medicinal solution by the same method.

And (3) determination: sucking 10 μ l of each of the above two solutions, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-ethyl acetate (8: 2) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm).

The results are shown in FIG. 11, and 1-4 are 1 negative sample (without flos Lonicerae and herba Artemisiae Annuae) (1. mu.l), 2 herba Artemisiae Annuae control drug (1. mu.l), 3 test sugar-free small sample (1. mu.l), and 4 test sample (lot number: 1601001) (1. mu.l), respectively; as can be seen, in the chromatogram of the test solution, at the position corresponding to the chromatogram of the control solution, the fluorescence spots with the same color are displayed, but the tailing phenomenon still exists, but the spots are visible. No corresponding fluorescent spots appeared in negatives.

Example 3

3.1 identification of Bupleurum:

preparation of a test solution: taking 6g of the product, adding 30ml of water, heating and refluxing for 1h, filtering, extracting the filtrate with petroleum ether for 2 times, 15ml each time, taking 10ml of aqueous layer solution, extracting with water-saturated n-butanol for 2 times, 20ml each time, combining n-butanol solutions, washing with 40ml of 1% sodium hydroxide solution, discarding the washing solution, washing with 40ml of water saturated with n-butanol, discarding the washing solution, recovering solvent from the n-butanol solution to dryness, and dissolving the residue with 1ml of methanol to obtain a sample solution;

thin-layer chromatography test: pipetting 5 mul of each of the two solutions, dropping the solution on the same silica gel G thin layer plate, adding chloroform: methanol: developing with water 8:2:0.2 as developing agent, taking out, air drying, spraying 1% p-dimethylaminobenzaldehyde sulfuric acid ethanol solution, heating at 105 deg.C until the spot color is clear, and respectively inspecting under sunlight and 365nm ultraviolet light;

the results are shown in FIG. 12 (left: under sunlight, right: under ultraviolet light), where 1-7 are 1 sample (lot number: 1601001), 2 Bupleurum negative, 3 sample (lot number: 1601002), 4 sample (lot number: 160901), 5 sample (lot number: 160902), 6 sample (lot number: 160903), and 7 Bupleurum reference drug, respectively; wherein the sugar-containing granules for treating cold and cough comprise two batches of samples (batch numbers: 1601001, 1601002) before modification, and the sugar-free granules for treating cold and cough comprise 3 batches of samples (batch numbers: 160901, 160902, 160903) after modification; it can be seen that in the chromatogram of the test solution, at least two main spots with the same color appear in the sunlight at the corresponding positions of the chromatogram of the reference solution; at least two main spots of fluorescence with the same color appear under ultraviolet light.

3.2 specificity test

The sugar-free samples (lot number: 160902) after 1 lot of alteration, the samples (lot number: 1601001) before 1 lot of alteration, and the bupleuri radix-negative samples were collected and measured in the same manner as described above. The results are shown in FIG. 13 (left: under sunlight, right: under ultraviolet light), where 1-6 are 1 bupleuri radix negative, 2 bupleuri radix control, 3 bupleuri radix control, 4 test sample (lot: 160902), 5 test sample (lot: 1601001), and 6 test sample (lot: 160902), respectively; it can be seen that in the chromatogram of the test solution, at least two main spots with the same color appear in the sunlight at the corresponding positions of the chromatogram of the reference solution; at least two main spots of fluorescence with the same color appear under ultraviolet light. No spots appear in negatives, and the specificity is strong.

3.3 durability test

Different room temperatures: respectively sucking 1 μ l of each of the modified sugar-free sample (lot number: 160902) solution, 1 lot of the pre-modified sample (lot number: 1601001) solution, and the bupleuri radix control solution under the above specificity test items, respectively dropping on the same silica gel G thin layer plate, developing at room temperature different from that under the specificity test items (specificity room temperature is 22 deg.C, and the test room temperature is 20 deg.C), taking out, air drying, spraying 1% p-dimethylaminobenzaldehyde sulfuric acid ethanol solution, heating at 105 deg.C until the spots are clear, and respectively inspecting under sunlight and ultraviolet lamp (365 nm). The results are shown in FIG. 14(a), where 1-4 are 1 Bupleurum root negative, 2 test samples (lot number: 160902), 3 Bupleurum root reference drug, 4 test samples (lot number: 1601001); it can be seen that at least two main spots with the same color appear in the sample chromatogram before and after the change at the positions corresponding to the reference medicinal material chromatogram in the sunlight; at least two main spots of fluorescence with the same color appear under ultraviolet light.

Different relative humidities: respectively sucking 1 μ l of each of the sugar-free sample after modification (lot number: 160902) solution, the sample before modification (lot number: 1601001) solution, and the bupleuri radix control solution under the above-mentioned specificity test items, respectively dropping on the same silica gel G thin layer plate, spreading with chloroform-methanol-water (8:2:0.2) as developing agent under the condition of relative humidity different from that under the specificity test items (30% of specificity test relative humidity, the test is that 27.5% sulfuric acid solution is placed on one side of a double-layer developing tank, the relative humidity is 72%), taking out, drying, spraying 1% p-dimethylaminobenzaldehyde sulfuric acid ethanol solution, heating at 105 deg.C until the spots are clear, and respectively inspecting under sunlight and ultraviolet lamp (365 nm). The results are shown in FIG. 14(b), where 1 to 4 are: 1 Bupleurum negative, 2 test samples (batch number: 160902), 3 test samples (batch number: 1601001), 4 Bupleurum reference medicinal materials; it can be seen that at least two main spots with the same color appear in the sample chromatogram before and after the change at the positions corresponding to the reference medicinal material chromatogram in the sunlight; at least two main spots of fluorescence with the same color appear under ultraviolet light.

In conclusion, the method has good durability.

3.4 detection Limit

Diluting bupleuri radix reference medicinal material solution with methanol to different concentrations, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (8:2:0.2) as developing agent, taking out, air drying, spraying 1% p-dimethylaminobenzaldehyde sulfuric acid ethanol solution, heating at 105 deg.C until the spots are clear, and respectively inspecting under sunlight and ultraviolet lamp (365 nm). The results are shown in FIG. 15 (left: under sunlight and right: under ultraviolet light), 1-9 are 1 bupleuri radix reference medicinal material (0.2 mg/ml; 3ul), 2 bupleuri radix reference medicinal material (0.2 mg/ml; 2.5ul), 3 bupleuri radix reference medicinal material (0.2 mg/ml; 1.5ul), 4 bupleuri radix reference medicinal material (0.2 mg/ml; 2ul), 5 bupleuri radix reference medicinal material (0.2 mg/ml; 1ul), 6 bupleuri radix reference medicinal material (0.2 mg/ml; 0.5ul), 7 bupleuri radix reference medicinal material (0.04 mg/ml; 3ul), 8 bupleuri radix reference medicinal material (0.04 mg/ml; 1ul), 9 bupleuri radix reference medicinal material (0.04 mg/ml; 0.5 ul); according to the map, the detection limit is 0.2 ug.

Example 4

The difference from example 3 is that, in the preparation process of the sample solution, the sample from which the solvent was recovered from the n-butanol solution until it was dried was added to a mixed solution of hydrochloric acid and acetic acid (wherein the mass fraction of hydrochloric acid was 3% and the mass ratio of hydrochloric acid to acetic acid was 1:1), and the mixture was immersed for 0.5 hour, freeze-dried, and then dissolved in 1ml of methanol to prepare the sample solution. The detection limit was 0.04ug as measured in example 3.

Example 5

Identification of menthol:

preparation of a test solution: taking 20g of the product, grinding, placing in a round bottom flask, adding 50mL of water, sealing, performing ultrasonic treatment for 4min, and soaking for 20 min; continuously adding 180mL of water, connecting a volatile oil tester, adding 30mL of water from the upper end of the tester again, overflowing the water to a flask, adding 6mL of ethyl acetate, connecting a reflux condenser tube, slowly heating to boil and keeping slightly boiling for 1.4 hours, stopping heating, separating an ethyl acetate layer, rapidly filtering by using a funnel paved with anhydrous sodium sulfate, washing a volatile oil extractor and a filter by using a small amount of ethyl acetate, placing the filtrate in a 10mL measuring flask, diluting to a scale by using the ethyl acetate, and shaking up to obtain a sample solution;

thin-layer chromatography test: pipette 5. mu.l of each of the two solutions, spot on the same silica gel G thin layer plate, and mix with cyclohexane: developing with ethyl acetate 14:6 as developing agent, taking out, air drying, spraying 4% vanillin-sulfuric acid solution, and heating at 105 deg.C until the spots are clearly developed; in the chromatogram of the test solution, the main spots with the same color appear at the corresponding positions of the chromatogram of the reference solution; negative no spots.

Identification of sweet wormwood herb:

preparation of a test solution: grinding 2g of the product, adding 30ml of water, heating and refluxing for 1h, filtering, extracting the filtrate with petroleum ether under shaking for 3 times (20 ml each time), extracting the aqueous layer solution with 10ml of water, extracting with ethyl acetate under shaking for 4 times (10 ml each time), mixing the ethyl acetate solutions, recovering solvent to dryness, and dissolving the residue with 2ml of ethanol to obtain a sample solution;

preparation of negative control solution: taking the other medicinal materials except the sweet wormwood according to the prescription respectively to prepare a negative control sample without the sweet wormwood, and preparing a negative control sample solution without the sweet wormwood according to the preparation method of the test sample solution; in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.

Identification of bupleurum:

preparation of a test solution: taking 8g of the product, adding 40ml of water, heating and refluxing for 1h, filtering, extracting the filtrate with petroleum ether under shaking for 1 time and 15ml each time, taking 10ml of aqueous layer solution, extracting with water saturated n-butanol under shaking for 3 times and 20ml each time, combining n-butanol solutions, washing with 40ml of 3% sodium hydroxide solution, discarding the washing solution, washing with 40ml of water saturated with n-butanol, discarding the washing solution, recovering solvent from the n-butanol solution to dryness, and dissolving the residue with 1ml of methanol to obtain a sample solution;

Example 6

The identification method of the forsythia suspense comprises the following steps:

the identification method of the kudzuvine root comprises the following steps:

preparation of a test solution: grinding 3g of the product, adding 30ml of methanol, performing ultrasonic treatment for 10min, filtering, evaporating the filtrate, and dissolving the residue in 3ml of methanol to obtain a sample solution;

and (3) testing according to thin-layer chromatography: sucking the two solutions each at 2 μ 1, and dropping on the same silica gel GF₂₅₄On thin layer plates, with chloroform: developing with methanol 3:1 as developing agent, taking out, air drying, and inspecting with ultraviolet lamp (254 nm). Spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution;

the identification method of the lonicera confusa comprises the following steps:

preparation of a test solution: taking 3g of the product, grinding, adding ethyl acetate, carrying out ultrasonic treatment for 2 times, carrying out ultrasonic treatment for 5min, removing an ethyl acetate solution 30ml each time, adding 0.25ml of lmol/L hydrochloric acid solution into residues, adding ethyl acetate, carrying out ultrasonic treatment for 2 times, combining the ethyl acetate solutions 20ml each time, drying by distillation, adding ethanol lml into the residues to dissolve the residues to obtain a sample solution;

and (3) testing according to thin-layer chromatography: sucking the two solutions 1-2 mu 1 respectively, and spotting on the same polyamide film to form strips, adding toluene: ethyl acetate: formic acid: glacial acetic acid: developing with water as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm); in the chromatogram of the test solution, fluorescent stripes with the same color appear at the corresponding positions of the chromatogram of the reference solution.

Example 7

The baicalin content determination method adopts high performance liquid chromatography for determination:

The detection method is obviously superior to a comparative example, which shows that the detection method has the advantages of close connection of all links, reasonable collocation and no defect, and the detection method formed by the specific combination generates obvious synergistic effect.

The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims

1. A quality detection method of sugar-free granule for treating common cold and cough comprises identification method of Mentholum, herba Artemisiae Annuae and bupleuri radix,

(1) identification of menthol:

thin-layer chromatography test: pipette 5. mu.l of each of the three solutions, spot on the same silica gel G thin layer plate, and mix with cyclohexane: developing ethyl acetate (12-17): (3-6) as developing agent, taking out, air drying, spraying 2-4% vanillin sulfuric acid solution, and heating at 105 deg.C until the color of spots is clear; in the chromatogram of the test solution, the main spots with the same color appear at the corresponding positions of the chromatogram of the reference solution;

(2) identification of sweet wormwood herb:

thin-layer chromatography test: sucking 1 mul of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, adding petroleum ether: ethyl acetate ═ (5-8): (4-6) developing with developing agent, taking out, air drying, and inspecting under ultraviolet light; in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution;

(3) identification of bupleurum:

thin-layer chromatography test: pipetting 5 mul of each of the three solutions, dropping the solution on the same silica gel G thin layer plate, adding chloroform: methanol: developing with water 8:2:0.2 as developing agent, taking out, air drying, spraying 1% p-dimethylaminobenzaldehyde sulfuric acid ethanol solution, heating at 105 deg.C until the color of the spot is clear, and inspecting under sunlight and 365nm ultraviolet light respectively; in the chromatogram of the test solution, at least two main spots with the same color appear in the corresponding positions of the chromatogram of the reference solution under sunlight; at least two fluorescent main spots with the same color appear under ultraviolet light;

the quality detection method of the sugar-free cold and cough granules also comprises the content determination of baicalin, puerarin and chlorogenic acid,

2. The quality detection method of claim 1, wherein the sugar-free cold cough granule has a composition comprising: bupleurum l00g, lonicera confusa 75g, kudzuvine root 100g, sweet wormwood 75g, weeping forsythia 75g, baikal skullcap root 75g, balloonflower root 50g, bitter apricot seed 50g, menthol 0.15 g;

3. The quality inspection method according to claim 1, wherein in the step (1), the preparation of the test solution: taking 10-20g of the product, grinding, placing in a round bottom flask, adding 30-50mL of water, sealing, performing ultrasonic treatment for 2-4min, and soaking for 20-30 min; adding 150 mL of water, connecting with a volatile oil tester, adding 30-60mL of water from the upper end of the tester, overflowing the water to the flask, adding 2-7mL of ethyl acetate, connecting with a reflux condenser tube, slowly heating to boil, keeping slightly boiling for 0.5-1.5 hours, stopping heating, separating an ethyl acetate layer, rapidly filtering by using a funnel paved with anhydrous sodium sulfate, washing a volatile oil extractor and a filter by using a small amount of ethyl acetate, placing the filtrate in a 10mL measuring flask, diluting to a scale by using ethyl acetate, and shaking uniformly to obtain a test solution.

4. The quality inspection method according to claim 1, wherein in the (1), the thin layer chromatography test: pipette 5. mu.l of each of the three solutions, spot on the same silica gel G thin layer plate, and mix with cyclohexane: developing with ethyl acetate 14:6 as developing agent, taking out, air drying, spraying 4% vanillin-sulfuric acid solution, and heating at 105 deg.C until the spots are clearly developed; the main spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution.

5. The quality inspection method according to claim 1, wherein in the step (2), the preparation of the test solution: grinding 2-5g of the product, adding 30-50ml of water, heating and refluxing for 1h, filtering, extracting the filtrate with petroleum ether under shaking for 1-3 times (20-40 ml each time), extracting the aqueous layer solution with 10ml of water, extracting with ethyl acetate under shaking for 1-5 times (10 ml each time), mixing the ethyl acetate solutions, recovering solvent to dryness, and dissolving the residue with 2ml of ethanol to obtain a sample solution.

6. The quality inspection method according to claim 1, wherein in the (2), the thin layer chromatography test: sucking 1 mul of each of the three solutions, respectively dropping on the same silica gel G thin layer plate, adding petroleum ether: ethyl acetate ═ 6: 4.5 is developing agent, developing, taking out, airing, and inspecting under 365nm ultraviolet light; in the chromatogram of the test solution, fluorescent spots with the same color appear at the corresponding positions of the chromatogram of the reference solution.

7. The quality inspection method according to claim 1, wherein in the step (3), the preparation of the test solution: taking 3-8g of the product, adding 20-40ml of water, heating and refluxing for 1h, filtering, extracting the filtrate with petroleum ether under shaking for 1-3 times, 15ml each time, taking 10ml of aqueous layer solution, extracting with water-saturated n-butyl alcohol under shaking for 1-3 times, 20ml each time, combining n-butyl alcohol solutions, washing with 40ml of 1-3% sodium hydroxide solution, discarding the washing solution, washing with 40ml of water saturated with n-butyl alcohol, discarding the washing solution, recovering solvent from the n-butyl alcohol solution until dry, and dissolving the residue with 1ml of methanol to obtain a sample solution.

8. The quality detection method of claim 1, wherein the quality detection method of the sugar-free cold and cough granule further comprises the identification methods of fructus forsythiae, radix puerariae and lonicera confusa:

and (3) testing according to thin-layer chromatography: sucking the two solutions each at 2 μ 1, and dropping on the same silica gel GF₂₅₄On thin layer plates, with chloroform: developing with methanol 3:1 as developing agent, taking out, air drying, and inspecting with ultraviolet lamp; spots with the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference solution;

9. The quality detection method according to claim 8, wherein in the identification method of radix Puerariae, the ultrasonic treatment time is 10-15min, and the ultraviolet light wavelength of the ultraviolet light is 254 nm; in the identification method of lonicera confusa, the ultrasonic treatment time is 5-8min, and the ultraviolet wavelength of the ultraviolet lamp is 365 nm.