CN117169411A

CN117169411A - Method for detecting loquat lung-heat-clearing drink

Info

Publication number: CN117169411A
Application number: CN202210594855.6A
Authority: CN
Inventors: 李艳英; 黄能听; 康伟; 张璞
Original assignee: Beijing Increase Innovative Drug Co ltd
Current assignee: Beijing Increase Innovative Drug Co ltd
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2023-12-05

Abstract

The invention relates to a method for detecting loquat lung-heat-clearing drink. The detection method establishes thin-layer identification of 6 medicinal herbs of loquat leaf, white mulberry root-bark, coptis root, amur corktree bark, ginseng and liquoric root in the classical prescription loquat lung-heat clearing drink and other dosage forms, has good reproducibility in different production places and different batches, can fully detect, can rapidly carry out qualitative and quantitative detection on unknown samples, and effectively controls the quality of the loquat lung-heat clearing drink.

Description

Method for detecting loquat lung-heat-clearing drink

Technical Field

The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting loquat lung-heat-clearing drink.

Background

The loquat lung-heat-clearing drink is from qing Wu Qian, yizong jin Jian (medical and religious gold authentication), and the prescription comprises: ginseng three-part, loquat leaf two-part (brush hair removal and honey processing), licorice three-part (raw), coptis root one-part, mulberry bark two-part (good for fresh), and phellodendron bark one-part. The loquat lung-heat clearing decoction has the efficacy of clearing lung-heat and is mainly used for treating lung wind and wind thorns. The symptoms are facial pimples, red, swelling and pain, broken powder juice or bits, etc.

The Chinese medicine administration in 2018 issues a 'ancient classical name prescription directory (first batch)' in 4 months, the loquat lung-heat-clearing drink is 94 th prescription, and decoction pieces processing methods, prescription doses, decoction modes and administration methods exist in a 'ancient classical name prescription key information table (7 first prescriptions)'. The loquat lung-heat-clearing drink is still widely applied to the past, has definite curative effect, has high development and application values, and is a popular field for current research.

The existing literature data of UPLC fingerprint and multi-index component content analysis of classical famous prescription loquat lung-heat clearing drink discloses a preparation method of a substance standard, a fingerprint and a content determination method.

However, the preparation of the substance standard in the document lacks detailed parameters, such as whether the medicinal materials of different places of origin can cause the substance standard sample standard to be difficult to unify.

In addition, the document does not consider the coincidence of the preparation process and quality standard (chemical component composition, content limit and the like) with the traditional decoction in the production of the compound preparation, and can not ensure that the preparation method of the ancient traditional classical prescription can become the equivalent preparation method of the modern traditional Chinese medicine compound preparation.

Further, the quality control method of the substance standard studied in this document is not necessarily applicable to formulation studies, and the characterization of ginseng is lacking in the quality standard, and the quality of ginseng in the formulation and the content transferred to the formulation cannot be detected and controlled.

Finally, the fingerprint detection method with the standard quality is long in time (50 min), high in detection cost, and has the advantages that the wavelength is required to be changed in the detection process, and the method is not verified after being enlarged.

In conclusion, a quality standard method for rapidly, comprehensively and directly reacting loquat lung-heat-clearing drink samples is lacking.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides a method for detecting loquat lung-heat clearing drink, which comprises the following steps:

1) The thin-layer chromatography detection is used for qualitatively judging 6 ingredients in the loquat lung-heat-clearing drink sample to be detected, namely ginseng, loquat leaf, licorice, coptis chinensis, white mulberry root-bark and amur corktree bark:

step a, ginseng qualitative detection: adding water into a loquat lung-heat-clearing drink sample to be detected, heating to dissolve the loquat lung-heat-clearing drink sample, extracting by shaking with water saturated n-butanol for at least 1 time, combining n-butanol liquid, and extracting by shaking with a sodium hydroxide solution with the mass fraction of 0.1% -1% for at least 1 time; washing the n-butanol solution with n-butanol saturated water with pH value of 3-4 for at least 1 time, discarding the water washing solution, washing with n-butanol saturated water for at least 1 time, discarding the water washing solution, evaporating the n-butanol solution to dryness, and dissolving the residue with methanol or ethanol to obtain a sample solution; decocting ginseng reference medicinal materials in water for 10-60 min, filtering, and preparing the filtrate into reference medicinal material solution according to the identical operation of the preparation of the sample solution; adding methanol or ethanol into ginsenoside Rb1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance and ginsenoside Rg1 reference substance to obtain mixed solution containing 0.1-1.0 mg per 1ml as reference substance solution; according to a thin-layer chromatography test, absorbing a reference substance solution, a reference medicinal material solution and a test substance solution, respectively spotting on the same silica gel G thin-layer plate, spreading with a lower layer solution placed below 30 ℃ of chloroform-methanol-water with a volume ratio of 13:7:2 as a spreading agent, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating until spots develop clearly, and observing whether spots with the same color are displayed on positions corresponding to the reference medicinal material chromatogram and the reference substance chromatogram in the test substance chromatogram;

Step b, detecting honey loquat She Dingxing: taking a loquat lung-heat-clearing drink sample to be detected, adding methanol or ethanol for ultrasonic treatment, filtering, and concentrating the filtrate to be used as a sample solution; decocting folium Eriobotryae control with water for 10-60 min, filtering, evaporating filtrate to dryness, and preparing the residue into control medicinal solution according to the same preparation method as the sample solution; adding ethanol into ursolic acid and/or oleanolic acid reference substances to prepare solutions with the concentration of 0.1-1.0 mg per 1ml respectively as reference substance solutions; according to a thin layer chromatography test, sucking the sample solution, the reference medicinal material solution and the reference substance solution to be respectively spotted on the same silica gel G thin layer plate, developing by taking a mixed solution of cyclohexane-chloroform-ethyl acetate-glacial acetic acid with the volume ratio of 10:15:8:0.5 as a developing agent, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating until spots are clear, and inspecting under a 365nm ultraviolet lamp; observing whether spots with the same color are displayed on the positions corresponding to the control medicine chromatogram and the reference substance chromatogram in the sample chromatogram;

step c, qualitative detection of liquorice: taking a loquat lung-heat-clearing drink sample to be detected, adding methanol or ethanol, carrying out ultrasonic treatment for 10-60 minutes, filtering, and concentrating the filtrate to be used as a sample solution; adding methanol or ethanol into the glycyrrhizin reference substance to prepare a solution containing 0.1-1.0 mg per 1ml as reference substance solution; according to a thin-layer chromatography test, respectively sucking a sample solution and a reference substance solution to be tested on the same silica gel G thin-layer plate, taking ethyl acetate-formic acid-glacial acetic acid-water with the volume ratio of 15:1:1:2 as developing agents, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating until spots develop clearly, inspecting under a 365nm ultraviolet lamp, and observing whether spots with the same color are displayed on the positions corresponding to the reference substance chromatogram in the sample chromatogram; or,

Adding water into a loquat lung-heat-clearing drink sample to be detected, heating to dissolve, extracting with water saturated n-butanol for at least 1 time, merging n-butanol solutions, extracting with 0.5% sodium hydroxide solution for at least 1 time, merging sodium hydroxide solutions, adjusting the pH value of the sodium hydroxide solution to 3-4 with hydrochloric acid, extracting with ethyl acetate for at least 1 time, merging ethyl acetate solutions, evaporating to dryness, and adding methanol or ethanol into residues to dissolve to be used as a sample solution; decocting Glycyrrhrizae radix control medicine in water for 10-60 min, filtering, and preparing filtrate into control medicine solution according to the same preparation method of the sample solution; adding methanol into the glycyrrhizin reference substance to prepare a solution containing 0.1-1.0 mg per 1 ml; according to a thin-layer chromatography test, sucking a sample solution, a reference drug solution and a reference substance solution to be respectively spotted on the same silica gel G thin-layer plate, spreading the solution at the lower layer below 30 ℃ with the volume ratio of 15:7:2 chloroform-methanol-water as a developing agent, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating until the spot color is clear, inspecting under a 365nm ultraviolet lamp, and observing whether spots with the same color are displayed on positions corresponding to the reference drug chromatogram and the reference substance chromatogram in the sample chromatogram;

Step d, qualitative detection of cortex phellodendri and rhizoma coptidis simultaneously; taking a loquat lung-heat-clearing drink sample to be detected, adding methanol or ethanol, carrying out ultrasonic treatment for 10-60 minutes, filtering, and taking filtrate, adding methanol or ethanol, and diluting to obtain a sample solution; decocting cortex Phellodendri reference medicinal material and Coptidis rhizoma reference medicinal material with water for 10-60 min, filtering, evaporating filtrate, and making residue into reference medicinal material solution according to the same preparation method as the sample solution; adding methanol into berberine hydrochloride reference substance to prepare a solution containing 0.1-1.0 mg per 1ml as reference substance solution; according to a thin layer chromatography test, sucking the solutions to be respectively spotted on the same silica gel G plate, taking cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine with a volume ratio of 3:3.5:1.5:0.5:1 as a developing agent, placing the developing agents in a developing cylinder with a presaturated concentrated ammonia test solution for 5-50 minutes for developing, taking out, airing, and placing the developing agents under a 365nm ultraviolet lamp for inspection; observing whether spots with the same color are displayed on the positions corresponding to the control medicine chromatogram and the control substance chromatogram in the sample chromatogram;

step e, qualitative detection of the white mulberry root-bark: adding water into a loquat lung-heat-clearing drink sample to be detected, performing ultrasonic treatment to dissolve, centrifuging, taking supernatant, shaking and extracting with chloroform for at least 1 time, mixing the extracting solutions, washing with 0.1-2.0% sodium hydroxide solution for at least 1 time, discarding washing solution, evaporating chloroform solution to dryness, and adding chloroform into residues to dissolve to serve as a sample solution; adding water into the cortex mori reference medicinal material, performing ultrasonic treatment for 10-60 minutes, and preparing a reference medicinal material solution according to the same preparation method of the sample solution; according to the thin layer chromatography test, the two solutions are respectively spotted on the same silica gel G thin layer plate, and the volume ratio is 2:1: developing with 2 petroleum ether-toluene-chloroform as developing agent, taking out, air drying, spraying 5% vanillin sulfuric acid solution, and heating until the color of spots is clear; observing whether spots with the same color are displayed on the positions corresponding to the control medicine chromatogram in the sample chromatogram;

2) The quantitative detection of phellodendrine, an active ingredient in phellodendron, comprises the following steps:

2a) Preparation of a control solution: taking a proper amount of phellodendrine hydrochloride reference substance, precisely weighing, and adding methanol to prepare a solution with a known concentration of 10-30 mug per 1 ml;

2b) Preparation of test solution: grinding a loquat lung-heat-clearing drink sample to be detected, precisely weighing 0.1-1 g, placing the loquat lung-heat-clearing drink sample into a conical bottle with a plug, precisely adding 15-35 ml of methanol, or 15-35 ml of 70% methanol water solution, or 15-35 ml of acetonitrile-0.1% phosphoric acid solution, sealing, weighing, performing ultrasonic treatment for 15-120 minutes, or heating and refluxing for 15-120 minutes, or shaking and extracting for 15-120 minutes, taking out, cooling, weighing again, supplementing the reduced weight with methanol, shaking uniformly, filtering, and taking filtrate to obtain the loquat lung-heat-clearing drink;

2c) Precisely sucking 5-15 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement; the chromatographic conditions are: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.1% phosphoric acid solution is used as a mobile phase; the detection wavelength is 284nm; the theoretical plate number is not lower than 6000 according to peak of phellodendrine hydrochloride;

3) The quantitative detection of berberine, epiberberine, berberine and palmatine as active components in cortex Phellodendri and Coptidis rhizoma comprises the following steps:

3a) The chromatographic conditions used were: octadecylsilane chemically bonded silica is used as a filler; acetonitrile is taken as a mobile phase A, 8-12 mmol/L formic acid-0.08-0.15% ammonium formate solution is taken as a mobile phase B, and gradient elution is carried out as follows; the detection wavelength is 280nm;

3b) Preparation of a control solution: taking a proper amount of berberine hydrochloride reference substance, epiberberine hydrochloride reference substance, berberine hydrochloride reference substance and palmatine hydrochloride reference substance, precisely weighing, and adding methanol to prepare a mixed solution containing 110-130 mug of berberine hydrochloride, 55-75 mug of epiberberine, 450-650 mug of berberine and 100-120 mug of palmatine hydrochloride in each 1 ml;

3c) Taking 0.1-1.0 g of loquat lung-heat clearing drink sample to be detected, precisely weighing, placing the loquat lung-heat clearing drink sample into a conical bottle with a plug, precisely adding methanol, or methanol aqueous solution with the volume fraction of 40-60%, or methanol aqueous solution with the volume fraction of 61-80%, 15-40 ml, sealing, weighing, performing ultrasonic treatment for 15-120 minutes, or reflux extraction for 15-120 minutes, or shaking extraction for 15-120 minutes, taking out, cooling, weighing again, supplementing the reduced weight with methanol, shaking uniformly, centrifuging, and taking supernatant to obtain a sample solution; precisely sucking 0.5-2 μl of each of the control solution and the sample solution, and injecting into an ultra-high performance liquid chromatograph for detection;

4) High performance liquid chromatography qualitative detection: the preparation process of the loquat lung-heat clearing drink sample to be detected is the same as that in the step 3), except that the extraction solvent is methanol or methanol water solution with the volume fraction of 1-80%;

the chromatographic conditions used for the detection were: octadecylsilane chemically bonded silica is used as a filler; acetonitrile is taken as a mobile phase A, 8-12 mmol/L formic acid-0.08-0.15% ammonium formate solution is taken as a mobile phase B; the detection wavelength is 280nm; the gradient elution conditions were:

and calculating the similarity between the obtained chromatogram and a comparison fingerprint spectrum shown in fig. 9 by adopting a traditional Chinese medicine chromatographic fingerprint similarity evaluation software system, wherein the similarity between the obtained chromatogram and the comparison fingerprint spectrum is more than 90%.

According to the embodiment of the invention, the loquat lung-heat clearing drink to be detected comprises a decoction prepared by an ancient classical prescription or a solid (such as granules) prepared by a current technology, or other liquid dosage forms and the like.

According to the embodiment of the invention, the water amount for dissolving the loquat lung-heat clearing drink sample to be detected in the step a by adding water is not particularly required, and the sample can be dissolved and can be detected by thin-layer chromatography, for example, 1g of the sample is added with 5-50 ml of water, for example, 25ml of water.

According to an embodiment of the invention, the amount of water-saturated n-butanol in step a is 1g of the sample plus 5 to 50ml of water-saturated n-butanol, for example 25ml of water-saturated n-butanol.

According to an embodiment of the invention, said at least 1 more times in step a means 1 to 5 times, for example 2 to 3 times.

According to an embodiment of the invention, there is no particular requirement for the amount of methanol or ethanol used in step b, and the sample can be dissolved and thin layer chromatography detection can be achieved, for example using 1g of sample plus 10 to 100ml of methanol or ethanol, for example 10 to 50ml of methanol or ethanol.

According to an embodiment of the invention, there is no particular requirement for the amount of methanol or ethanol used in step c, and the sample can be dissolved and thin layer chromatography detection can be achieved, for example using 1g of sample plus 10 to 100ml of methanol or ethanol, for example 10 to 50ml of methanol or ethanol.

According to the embodiment of the invention, the water amount for dissolving the loquat lung-heat clearing drink sample to be detected in the step c by adding water is not particularly required, and the sample can be dissolved and can be detected by thin-layer chromatography, for example, 1g of the sample is added with 5-50 ml of water, for example, 25ml of water.

According to an embodiment of the invention, said at least 1 more times in step c means 1 to 5 times, for example 2 to 3 times.

According to an embodiment of the invention, there is no particular requirement for the amount of methanol or ethanol used in step d, and the sample can be dissolved and thin layer chromatography detection can be achieved, for example using 1g of sample plus 10 to 100ml of methanol or ethanol, for example 10 to 50ml of methanol or ethanol.

According to an embodiment of the invention, said at least 1 more times in step e means 1 to 5 times, for example 2 to 3 times.

According to the embodiment of the invention, the water amount for dissolving the loquat lung-heat clearing drink sample to be detected in the step e by adding water is not particularly required, and the sample can be dissolved and can be detected by thin-layer chromatography, for example, 1g of the sample is added with 5-50 ml of water, for example, 25ml of water.

According to an embodiment of the present invention, the thin layer chromatography in the thin layer chromatography detection in step 1) is performed according to the method described in the fourth edition of the general rule 0502 of the chinese pharmacopoeia 2020.

According to an embodiment of the present invention, the preparation of the control solution of step 2 a) may be performed in the following manner: taking a proper amount of phellodendrine hydrochloride reference substance, precisely weighing, and adding methanol to prepare a solution containing 20 mug per 1 ml;

according to the embodiment of the invention, 0.3-0.8 g of loquat lung-heat clearing drink sample to be detected is taken in the preparation of the test solution in the step 2 b), and is precisely weighed, placed in a conical bottle with a plug, and precisely added with 20-30 ml of methanol, or 20-30 ml of 70% methanol aqueous solution with volume fraction, or 20-30 ml of acetonitrile-0.1% phosphoric acid solution, and then subjected to subsequent operation.

According to an embodiment of the invention, the power of the ultrasound in step 2 b) is 400-600W and the frequency is 30-500 kHz.

According to an embodiment of the invention, in step 2 b) acetonitrile-0.1% phosphoric acid solution (0.2 g per 100ml sodium dodecyl sulphate).

According to an embodiment of the invention, the ultrasonic treatment in step 2 b) is carried out for 15 to 45 minutes, or heated under reflux for 15 to 45 minutes, or shaking extraction for 15 to 45 minutes, for example for 30 minutes, or heated under reflux for 30 minutes, or shaking extraction for 30 minutes.

According to an embodiment of the invention, the chromatography column in step 3 a) is of model ACQUCITY UPLC BEH Shield RP, column length 100mm, inner diameter 2.1mm and particle size 1.7 μm.

According to an embodiment of the invention, the preparation process of the control solution of 3 b) is: taking a proper amount of berberine hydrochloride reference substance, epiberberine reference substance, berberine hydrochloride reference substance and palmatine hydrochloride reference substance, precisely weighing, and adding methanol to prepare a mixed solution containing 120 mug of berberine hydrochloride, 65 mug of epiberberine, 550 mug of berberine and 110 mug of palmatine hydrochloride per 1 ml.

According to an embodiment of the invention, the ultrasonic treatment in step 3 c) is carried out for 15 to 45 minutes, or heated under reflux for 15 to 45 minutes, or shaking extraction for 15 to 45 minutes, for example for 30 minutes, or heated under reflux for 30 minutes, or shaking extraction for 30 minutes.

According to an embodiment of the invention, step 3 c) employs the following operations: taking 0.5g of loquat lung-heat clearing drink sample to be detected, precisely weighing, placing the sample into a conical bottle with a plug, precisely adding methanol, or a 50% methanol aqueous solution with a volume fraction of 25ml, sealing, weighing, performing ultrasonic treatment for 15-120 minutes, or reflux extraction for 15-120 minutes, or shaking extraction for 15-120 minutes, taking out, cooling, weighing again, supplementing the reduced weight with methanol, shaking uniformly, centrifuging, and taking supernatant to obtain a sample solution; respectively precisely sucking 1 μl of the reference substance solution and 1 μl of the sample solution, and detecting by ultra high performance liquid chromatograph.

According to an embodiment of the invention, the chromatographic column in step 4) is model ACQUCITY UPLC BEH Shield RP, column length 100mm, inner diameter 2.1mm and particle size 1.7 μm.

According to an embodiment of the present invention, the fingerprint in step 5) is shown in fig. 9, wherein 11 sets of common peaks are shown as follows:

。

advantageous effects

1. The detection method establishes thin-layer identification of 6 medicinal herbs of loquat leaf, white mulberry root-bark, coptis root, amur corktree bark, ginseng and liquoric root in the classical prescription loquat lung-heat clearing drink and other dosage forms, has good reproducibility in different production places and different batches, can fully detect, can rapidly carry out qualitative and quantitative detection on unknown samples, and effectively controls the quality of the loquat lung-heat clearing drink.

2. The fingerprint spectrum measuring method established by the invention is consistent with the content measuring method of coptis chinensis and phellodendron barks (sample preparation, chromatographic conditions and the like), and enhances the specificity identification and the multi-component and overall quality control. The fingerprint spectrum is established to control the whole quality, and the running time is 21 minutes, so that the energy consumption is effectively reduced, and the project cost is reduced.

3. The method establishes quantitative research of 5 chemical components of coptis chinensis and phellodendron bark in total, has good precision, stability and repeatability, comprehensively and effectively detects the standard of loquat lung-heat clearing drink substances and the content of the preparation, provides a detailed and reliable research basis for subsequent process research, quality standard establishment and stability investigation, and is beneficial to future production and sale of loquat lung-heat clearing drink.

Drawings

FIG. 1-1 is the chromatogram of comparative example 4, and FIG. 1-2 is the chromatogram of example 14.

FIG. 2 is a chromatogram of example 11.

Fig. 3 is a 3D view of example 16.

FIGS. 4-8 are chromatograms of samples of example 16 analyzed at 254nm, 270nm, 280nm, 300nm, 330nm, respectively.

Fig. 9 is a chromatogram of a control fingerprint.

Fig. 10 is a comparison chart of the fingerprint of example 18.

Detailed Description

The technical scheme of the invention will be further described in detail below with reference to specific embodiments. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.

Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.

The preparation method of the product in the following examples and comparative examples comprises the following steps: taking 1.12g of ginseng (broken), 7.46g of loquat leaf, 1.12g of licorice, 3.73g of coptis (broken), 7.46g of white mulberry root-bark and 3.73g of amur corktree bark, adding 300ml of water, boiling with strong fire, decocting with slow fire for 30 minutes, filtering with 200-mesh filter cloth while the mixture is hot at normal pressure, freeze-drying, and crushing to obtain corresponding substances. And (5) sealing.

Example 1

Dissolving 1g of the product in 25ml of water under heating, extracting with water saturated n-butanol under shaking for 2 times (25 ml each time), mixing n-butanol solutions, extracting with 0.5% sodium hydroxide solution under shaking for 2 times (25 ml each time). Washing the n-butanol solution with n-butanol saturated water with pH value of 3-4 for 2 times, each time 25ml, discarding the water washing solution, washing with n-butanol saturated water for 3 times, each time 25ml, discarding the water washing solution, evaporating the n-butanol solution to dryness, and dissolving the residue with 1ml methanol to obtain the test sample solution. Decocting 0.5g of ginseng reference medicinal material with 30ml of water for 30 min, filtering, and preparing filtrate into reference medicinal material solution according to the same operation as the sample solution. And adding methanol into ginsenoside Rb1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance and ginsenoside Rg1 reference substance to obtain mixed solution containing 0.5mg of each 1ml of the above materials as reference solution. According to thin layer chromatography (rule 0502 of four parts of Chinese pharmacopoeia 2020 edition), sucking 1 μl of each of the control solution and 2 μl of the test solution, respectively spotting on the same silica gel G thin layer plate, spreading with lower layer solution of chloroform-methanol-water (volume ratio of 13:7:2) below 10deg.C as developing agent, taking out, air drying, spraying 10% ethanol sulfate solution, and heating until spot color is clear.

Results: spots of the same color appear in the sample chromatogram at the positions corresponding to the control chromatogram and the control chromatogram. Therefore, the method can rapidly and qualitatively detect the ginseng in the product.

Comparative example 1

Taking 1g of the product, adding 10ml of chloroform, carrying out ultrasonic treatment for 30 minutes, discarding the chloroform solution, volatilizing the solvent from the dregs, adding 15ml of water saturated n-butanol, carrying out ultrasonic treatment for 30 minutes, filtering, adding 30ml of ammonia test solution into the filtrate, shaking uniformly, standing for layering, taking the upper layer solution, evaporating the upper layer solution, and adding 1ml of methanol into the residue to dissolve the residue to be used as a test solution. Decocting Ginseng radix reference material 1g with water 30ml for 30 min, filtering, evaporating filtrate, and making the residue into reference material solution by the same preparation method as above test sample. And adding methanol into ginsenoside Rb1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance and ginsenoside Rg1 reference substance to obtain mixed solution containing 0.5mg of each 1ml as reference substance solution. According to thin layer chromatography (four-part rule 0502 of Chinese pharmacopoeia 2020 edition), absorbing 2 μl of each of the control solution and the control medicinal solution, and 4 μl of the test solution, respectively spotting on the same silica gel G thin layer plate, spreading with lower layer solution of chloroform-methanol-water (volume ratio of 13:7:2) below 10deg.C as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, and heating until spot color is clear.

Results: the color band interference in the sample chromatograph is serious, and whether the sample contains ginseng cannot be determined.

Example 2

Taking 1g of the product, adding 30ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, taking 5ml of filtrate, and concentrating to 1ml as a sample solution. And (3) adding 50ml of water into 1g of the loquat leaf reference medicinal material, decocting for 30 minutes, filtering, evaporating the filtrate to dryness, and preparing the residue into a reference medicinal material solution according to the same preparation method of the sample solution. And adding ethanol into ursolic acid and oleanolic acid reference substance to obtain solutions containing 0.1mg of each 1ml of the reference substance solution. According to thin layer chromatography (four-part rule 0502 of Chinese pharmacopoeia 2020 edition), 5 μl of the sample solution, 8 μl of the control medicinal material solution and 1 μl of the control medicinal material solution are sucked and respectively spotted on the same silica gel G thin layer plate, and the mixed solution of cyclohexane-chloroform-ethyl acetate-glacial acetic acid (volume ratio of 10:15:8:0.5) is used as developing agent to develop, taken out, dried in the air, sprayed with 10% sulfuric acid ethanol solution, heated at 105deg.C until spots are clear, and then placed under ultraviolet lamp (365 nm) for inspection.

Results: the sample chromatogram has the same spots on the corresponding positions of the reference material chromatogram and the reference material chromatogram. Therefore, the method can rapidly and qualitatively detect the loquat leaf in the product.

Example 3

Taking 1g of the product, adding 30ml of ethanol, carrying out ultrasonic treatment for 30 minutes, filtering, evaporating the filtrate to dryness, and adding 1ml of ethanol into residues to dissolve the residues to obtain a sample solution. Decocting 1g of folium Eriobotryae control with 50ml of water for 30 min, filtering, evaporating filtrate, and making the residue into control medicinal solution according to the same preparation method as the test solution. And adding ethanol into ursolic acid as reference substance to obtain solution containing 0.1mg per 1ml of ursolic acid as reference substance solution. According to thin layer chromatography (four-part rule 0502 of Chinese pharmacopoeia 2020 edition), the sample solution 4 μl, the control medicinal material solution 3 μl and the control solution 1 μl are sucked and respectively spotted on the same silica gel G thin layer plate, and the mixed solution of cyclohexane-chloroform-ethyl acetate-glacial acetic acid (volume ratio of 10:15:8:0.5) is used as developing agent to develop, taken out, dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105deg.C until the spot color is clear, and then observed under ultraviolet lamp (365 nm).

Results: the sample chromatogram shows fluorescence main spots with the same color at the corresponding positions of the control chromatogram and the control chromatogram. Therefore, the method can rapidly and qualitatively detect the loquat leaf in the product.

Example 4

Taking 1g of the product, adding 25ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, taking 5ml of filtrate, and concentrating to 1ml to obtain a sample solution. And adding methanol into the control of liquiritin (which is the main active ingredient in Glycyrrhrizae radix) to obtain a solution containing 0.3mg per 1ml as control solution. According to thin layer chromatography (four-part rule 0502 of Chinese pharmacopoeia 2020 edition), sucking 2 μl of each of the sample solution and the reference solution, respectively spotting on the same silica gel G thin layer plate, spreading with ethyl acetate-formic acid-glacial acetic acid-water (volume ratio of 15:1:1:2) as developing agent, taking out, air drying, spraying 10% sulfuric acid ethanol solution, heating at 105deg.C until the color of spots is clear, and placing under ultraviolet lamp (365 nm) for inspection.

Results: the spots in the sample chromatogram are identical to those in the control chromatogram. Therefore, the method can rapidly and qualitatively detect the liquorice in the product.

Example 5

Adding 25ml of water into 1g of the product, heating to dissolve, extracting with water saturated n-butanol for 2 times by shaking, extracting with 25ml of water each time, combining n-butanol solutions, extracting with 0.5% sodium hydroxide solution for 2 times by shaking, extracting with 25ml of water each time, combining sodium hydroxide solutions, adjusting the pH value of the sodium hydroxide solution to 3-4 by hydrochloric acid, extracting with ethyl acetate for 2 times by shaking, extracting with 25ml of ethyl acetate each time, combining ethyl acetate solutions, evaporating to dryness, and dissolving residues with 3ml of methanol to obtain a sample solution. Decocting Glycyrrhrizae radix control material 0.25g with water 30ml for 30 min, filtering, and making filtrate into control material solution according to the same preparation method as the test sample solution. Adding methanol into the glycyrrhizin reference substance to obtain solution containing 0.3mg per 1 ml. According to thin layer chromatography (four-part rule 0502 of Chinese pharmacopoeia 2020 edition), 1 μl of each of the sample solution, the control medicinal solution and the control solution is absorbed, respectively spotted on the same silica gel G thin layer plate, and the lower layer solution placed below 10deg.C of chloroform-methanol-water (volume ratio of 15:7:2) is used as developing agent to spread, taken out, air dried, sprayed with 10% sulfuric acid ethanol solution, heated at 105deg.C until the spot color is clear, and observed under ultraviolet lamp (365 nm).

Results: the sample chromatogram shows the same color of fluorescent spots on the corresponding positions of the control chromatogram and the control chromatogram. Therefore, the method can rapidly and qualitatively detect the liquorice in the product.

Example 6

Taking 1g of the product, adding 25ml of methanol, carrying out ultrasonic treatment for 30 minutes, filtering, and taking 1ml of filtrate, adding methanol to dilute to 10ml to obtain a sample solution. And (2) taking 0.1g of phellodendron bark reference medicinal material and 0.25g of coptis root reference medicinal material, respectively adding 50ml of water, decocting for 30 minutes, filtering, evaporating filtrate to dryness, and preparing residues into a reference medicinal material solution according to the same preparation method of the sample solution. And adding methanol into berberine hydrochloride (which is the common effective component of golden thread and cortex Phellodendri) as reference substance to obtain solution containing 0.1mg per 1ml of the reference substance. According to thin layer chromatography (rule 0502 of four parts of the 2020 edition of Chinese pharmacopoeia), 1 μl of each solution is sucked and respectively spotted on the same silica gel G plate, cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine (volume ratio of 3:3.5:1:1.5:0.5:1) is used as developing agent, and the solution is placed in a developing cylinder of a concentrated ammonia solution presaturation 20 minutes for developing, taken out, dried and placed under an ultraviolet lamp (365 nm) for inspection.

Experiment: the sample chromatogram shows the same color of fluorescent spots on the corresponding positions of the control chromatogram and the control chromatogram. Therefore, the method can simultaneously and rapidly and qualitatively detect the phellodendron bark and the coptis chinensis in the product.

Example 7

Taking 1g of the product, adding 25ml of water, carrying out ultrasonic treatment to dissolve, centrifuging, taking supernatant, shaking and extracting for 2 times by using chloroform, 30ml each time, combining extracting solutions, washing for 2 times by using a sodium hydroxide solution with the mass fraction of 1%, 20ml each time, discarding washing solution, evaporating the trichloromethane solution to dryness, and adding 1ml of chloroform into residues to dissolve the residues to serve as a sample solution. And adding 30ml of water into 2g of the cortex mori reference medicinal material, performing ultrasonic treatment for 30 minutes, and preparing a reference medicinal material solution according to the same preparation method of the sample solution. According to a thin-layer chromatography (four-part rule 0502 of Chinese pharmacopoeia 2020 edition), 5 mu l of each of the two solutions is absorbed and respectively spotted on the same silica gel G thin-layer plate, petroleum ether (60-90 ℃) and toluene-chloroform (volume ratio of 2:1:2) are used as developing agents to develop, the mixture is taken out, dried in the air, sprayed with 5% vanillin sulfuric acid solution, and heated at 105 ℃ until the spots develop clearly.

Results: the main spots of the same color appear in the sample chromatogram at the positions corresponding to the control chromatogram. Therefore, the method can simultaneously and rapidly and qualitatively detect the white mulberry root-bark in the product.

Comparative example 3

Taking 1g of the product, adding 20ml of saturated sodium carbonate solution, carrying out ultrasonic treatment for 20 minutes, filtering, adding dilute hydrochloric acid into the filtrate to regulate the pH value to 1-2, standing for 30 minutes, filtering, extracting the filtrate with ethyl acetate for 2 times by shaking, mixing ethyl acetate solutions each time by 10ml, evaporating to dryness, and adding 2ml of methanol into residues to dissolve the residues to serve as a sample solution. Decocting 2g of cortex Mori reference medicinal material in 50ml of water for 30 min, filtering, evaporating filtrate, and making the residue into reference medicinal material solution according to the same preparation method as the sample solution. And adding methanol into the Mortiered-A reference substance to obtain a solution containing 0.4mg per 1ml of Mortiered-A reference substance solution. According to thin layer chromatography (rule 0502 of four parts of Chinese medicine dictionary 2020 edition), 3 μl of each of the above three solutions is absorbed, respectively spotted on the same polyamide film, and developed with acetic acid as developing agent for about 10cm, taken out, air dried, and inspected under ultraviolet lamp (365 nm).

Results: the sample chromatogram has no same spot corresponding to the control materials and the control chromatogram, and no detection of mulberroside A, and the sample color band is heavy, the separation is poor, and the spot is unclear.

In the qualitative detection of loquat lung-heat-clearing drink, the inventor discovers that not all extraction reagents or thin-layer chromatography can realize the qualitative detection of the sample. The inventors have unexpectedly found that the above method of the present invention can rapidly identify the taste of a drug in a prescription.

Examples 8-10 below provide quantitative determination methods of phellodendrine, respectively, using chromatographic conditions of:

octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.1% phosphoric acid solution (0.2 g of sodium dodecyl sulfonate per 100 ml) (32:68) was used as mobile phase; the detection wavelength was 284nm. The theoretical plate number is not less than 6000 according to peak of phellodendrine hydrochloride.

Preparation of control solution A proper amount of phellodendrine hydrochloride control is taken, precisely weighed, and methanol is added to prepare a solution containing 35 mug per 1 ml.

The method comprises the following steps:

example 8: investigation of extraction solvent

Because the product is a liquid or solid preparation, the product needs to be prepared into a liquid sample which can be detected by liquid chromatography before the quantitative detection of the active ingredient phellodendrine (the active ingredient of phellodendron). Thus, properly, the solvent in which the active ingredient is eluted is critical for subsequent detection. This example examined the effect of different vehicles.

About 1g (total 3 groups) of the product is precisely weighed and placed in a conical bottle with a plug, methanol, 70% methanol aqueous solution with volume fraction and acetonitrile-0.1% phosphoric acid solution (0.2 g containing sodium dodecyl sulfonate per 100 ml) (32:68) are precisely added, 25ml of each product is sealed, the product is weighed and subjected to ultrasonic treatment (power 500W and frequency 40 kHz) for 30 minutes, the product is taken out and cooled, the product is then weighed and cooled, methanol, 70% methanol aqueous solution with volume fraction and acetonitrile-0.1% phosphoric acid solution (0.2 g containing sodium dodecyl sulfonate per 100 ml) (32:68) are respectively used for supplementing the lost weight, the product is uniformly shaken and filtered, and filtrate is obtained, thus obtaining the sample solution. Respectively precisely sucking the reference substance solution and 10 μl of the sample solution prepared in this example, and injecting into a liquid chromatograph for detection. The test results of the phellodendrine content in the test sample solution are shown in the following table 1:

TABLE 1

Extracting solvent	Phellodendrine content (mg/g)
		Methanol	1.216
70% methanol	1.263
		Mobile phase	1.238

The results show that the contents of phellodendrine extracted by three extraction solvents of methanol, 70% methanol aqueous solution and mobile phase have no obvious difference. Therefore, all three extraction solvents can realize the extraction detection of the sample. In view of the convenience of operation, methanol is selected as the extraction solvent.

Example 9: investigation of extraction modes

As described above, the solvent extraction method is also critical for accurate quantitative detection of cortex Phellodendri. The extraction mode of the solvent is examined as follows:

about 1g (total 3 groups) of the product is taken, precisely weighed, placed in a conical bottle with a plug, precisely added with 25ml of methanol, sealed, weighed by weight, respectively extracted by adopting a heating reflux extraction mode, a shaking extraction mode and an ultrasonic treatment mode (with the power of 500W and the frequency of 40 kHz) for 30 minutes, taken out, cooled, weighed by weight again, complemented by the lost weight by the methanol, shaken evenly, filtered, and taken out of filtrate to obtain the sample solution. Respectively precisely sucking the reference substance solution and 10 mu l of the sample solution prepared in the embodiment, and injecting into a liquid chromatograph for detection. The test results of the phellodendrine content in the test sample solution are shown in the following table 2:

TABLE 2

Extraction mode	Phellodendrine content (mg/g)
		Ultrasonic wave	1.216
Reflow process	1.135
		Shaking machine	1.116

As shown by test results, the extraction mode of ultrasonic treatment has higher phellodendrine content than other modes, so the preferred extraction mode is ultrasonic extraction.

Example 10: investigation of extraction time

As described above, the extraction time of solvent is also critical for accurate quantitative detection of cortex Phellodendri. The extraction time of the solvent was examined as follows:

About 1g (total 3 groups) of the product is precisely weighed, placed in a conical bottle with a plug, precisely added with 25ml of methanol, sealed, weighed by weight, respectively subjected to ultrasonic treatment (power 500W, frequency 40 kHz) for 15 minutes, 30 minutes and 45 minutes, taken out, cooled, weighed by weight again, complemented by the lost weight by methanol, shaken uniformly, filtered, and filtered to obtain filtrate, thus obtaining the solution of the sample.

Respectively precisely sucking the reference substance solution and 10 μl of the sample solution prepared in this example, and injecting into a liquid chromatograph for detection. The test results of the phellodendrine content in the test sample solution are shown in the following table 3:

TABLE 3 Table 3

Extraction time	Phellodendrine content (mg/g)
		15	1.131
30	1.216
		45	1.133

As shown by the test results, the extraction time of 30 minutes is preferably 30 minutes because the phellodendrine content is higher than the extraction time of 15 minutes and 45 minutes.

Examples 11-13 below provide quantitative detection of berberine, epiberberine, berberine, palmatine (these 4 are active ingredients in coptis and phellodendron), respectively, using chromatographic conditions of:

octadecylsilane chemically bonded silica is used as filler (chromatographic column model ACQUCITY UPLC BEH Shield RP, column length of 100mm, inner diameter of 2.1mm, and particle diameter of 1.7 μm); acetonitrile was used as mobile phase A, 10mmol/L ammonium formate-0.1% formic acid solution was used as mobile phase B, and gradient elution was performed as specified in Table 4 below; the detection wavelength was 280nm.

TABLE 4 Table 4

Preparation of reference solution A mixed solution containing berberine hydrochloride 120 μg, berberine 65 μg, berberine 550 μg and palmatine hydrochloride 110 μg per 1ml is prepared by precisely weighing the reference solution of berberine hydrochloride, epiberberine, berberine hydrochloride and palmatine hydrochloride.

The method comprises the following steps:

EXAMPLE 11 investigation of extraction vehicle

As described above, the solvent extraction method is also critical for detecting Coptidis rhizoma and cortex Phellodendri with a certain amount. Taking coptisine, epiberberine, berberine and palmatine as investigation objects, and investigating the influence of a solvent:

about 0.5g (total 3 groups) of the product is taken, precisely weighed, placed in a conical bottle with a plug, respectively precisely added with methanol, 50% methanol aqueous solution with volume fraction and 25ml methanol aqueous solution with volume fraction of 70%, sealed, weighed, ultrasonically treated for 30 minutes, taken out, cooled, weighed again, and the weight loss is complemented by methanol, shaken uniformly, centrifuged (the rotating speed is 13000 rpm) for 10 minutes, and the supernatant is taken to obtain the solution of the sample. 1 μl of each of the control solution and the sample solution prepared in the above examples was precisely sucked, and the samples were injected into an ultra-high performance liquid chromatograph for detection, and the results of the measurement of the content of each active ingredient in the sample solution are shown in Table 5 below.

TABLE 5

The experimental results show that the content of coptisine, epiberberine, berberine and palmatine in the samples obtained by extracting with methanol is slightly higher than that of 50% of methanol and 70% of methanol. But the three can realize the effective extraction and detection of coptisine, epiberberine, berberine and palmatine.

Example 12 investigation of extraction modes

About 0.5g (total 3 groups) of the product is taken, precisely weighed, placed in a conical bottle with a plug, respectively and precisely added with 25ml of methanol, sealed, weighed, respectively subjected to ultrasonic treatment, reflux extraction and shaking extraction for 30 minutes, taken out, cooled, weighed again, complemented with the reduced weight by methanol, shaken uniformly, centrifuged (the rotating speed is 13000 rpm) for 10 minutes, and the supernatant is taken to obtain the sample solution. 1 μl of each of the control solution and the sample solution prepared in the above example was precisely sucked, and the samples were injected into an ultra-high performance liquid chromatograph for detection, and the results of the measurement of the content of each active ingredient in the sample solution are shown in Table 6 below.

TABLE 6

The experimental results show that the three extraction modes of ultrasonic, reflux and shaking have no obvious difference; can realize the effective extraction and detection of coptisine, epiberberine, berberine and palmatine.

Example 13 investigation of extraction time

About 0.5g (total 3 groups) of the product is taken, precisely weighed, placed in a conical bottle with a plug, respectively and precisely added with 25ml of methanol, sealed, weighed, respectively subjected to ultrasonic treatment for 15 minutes, 30 minutes and 45 minutes, taken out, cooled, weighed again, complemented with methanol for the weight loss, shaken uniformly, centrifuged (the rotating speed is 13000 rpm) for 10 minutes, and the supernatant is taken to obtain the sample solution.

1 μl of each of the control solution and the sample solution prepared in the above examples was precisely sucked, and the samples were injected into an ultra-high performance liquid chromatograph for detection, and the results of the measurement of the content of each active ingredient in the sample solution are shown in Table 7 below.

TABLE 7

From the above experimental results, it is clear that there is no obvious difference in the extraction of berberine, epiberberine, berberine and palmatine with different times. Can realize the effective extraction and detection of coptisine, epiberberine, berberine and palmatine.

Therefore, the sample extraction method and the high-efficiency chromatographic method can accurately and effectively monitor the content condition of coptis chinensis and phellodendron barks in the method, thereby confirming whether the loquat lung-heat-clearing drink sample is qualified or not.

Comparative example 4: preliminary investigation of mobile phase gradient of fingerprint

Chromatographic conditions: octadecylsilane chemically bonded silica is used as filler (column length is 100mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile is taken as a mobile phase A, 0.1% formic acid solution is taken as a mobile phase B, and the flow rate is 0.4ml per minute; the detection wavelength was 280nm, and elution was performed according to the gradient shown in Table 8 below.

TABLE 8

Preparation of test solution: taking a proper amount of the product, grinding, taking about 0.5g, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of methanol, sealing, weighing, performing ultrasonic treatment (power 500W and frequency 40 kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with methanol, shaking uniformly, centrifuging (rotating at 13000 rpm) for 10 minutes, and taking supernatant.

Assay: 1 μl of the sample solution was precisely aspirated, injected into the chromatograph, measured, and the chromatogram was recorded, and the results are shown in FIG. 1-1.

Example 14

On the basis of comparative example 4, acetonitrile was used as mobile phase A, 10mmol/L formic acid-0.1% ammonium formate solution was used as mobile phase B, and the elution gradient was changed to the gradient shown in Table 9 below, with the other conditions unchanged. The results are shown in FIGS. 1-2.

TABLE 9

Observing the patterns obtained in comparative example 4 and example 14, the chromatographic peaks of comparative example 4 are concentrated at 0-11 min, and the information amount is less after 11 min; the separation effect of the chromatographic peak of example 14 was significantly better than that of comparative example 4, and the chromatographic peak was uniformly distributed and the information amount was large, so that the elution gradient was determined as shown in the above table 9.

In addition, when the mobile phase B is 10mmol/L ammonium formate-0.08% formic acid solution or 10mmol/L ammonium formate-0.15% formic acid solution, the fingerprint with rich information and high separation degree can be obtained.

Example 15

The test sample solutions were examined for 50% methanol, 70% methanol and methanol as solvents, and the other chromatographic conditions and the measurement method were the same as in example 14. The results are shown in FIG. 2.

The results are shown in FIG. 2.

Analysis of results: according to the graph, the investigation of three solvents of 50% methanol, 70% methanol and methanol has no obvious difference, so that the content measurement result is combined, and the methanol is selected as an extraction solvent.

Example 16

The test sample of the solvent extracted under the 'example 14' is methanol, and is scanned by a diode array detector at the wavelength of 190-400 nm, and is analyzed by a 3D view (see figure 3), and the result shows that the chromatographic peak is in the wavelength range of 250-330 nm, and the information amount is large. Wavelengths 254nm, 270nm, 280nm, 300nm, 330nm were selected for analysis (see FIGS. 4-8).

After comparing the chromatograms, the peak response value of each chromatogram is moderate, the peak information amount is large, and the base line is stable under the wavelength of 280nm. The detection wavelength was therefore chosen to be 280nm.

Example 17 determination of finger print

According to the chromatographic conditions of example 14, methanol is used as an extraction solvent, 15 batches (30 parts) of samples are measured, the obtained fingerprints are analyzed, chromatographic peaks with good stability and proper response value in 15 batches (30 parts) of samples are selected as common peaks, and 11 common peaks are calibrated in total. Fingerprint judgment result: the chromatographic peak with the same retention time as the chromatographic peak of the reference substance should be displayed in the fingerprint of the test sample, the chromatographic spectrum of the test sample should be basically consistent with the reference fingerprint, and has 11 corresponding common peaks, and the similarity is calculated according to the similarity evaluation system of the traditional Chinese medicine chromatographic fingerprint by the common peaks, and the similarity between the fingerprint of the test sample and the reference fingerprint should not be lower than 0.90.

The obtained chromatogram, i.e. the fingerprint, is shown in FIG. 9, and as can be seen from FIG. 9, the obtained fingerprint has 11 common peaks, and the peak assignment and attribution conditions are shown in Table 10 below.

Table 10

The berberine chromatographic peak has moderate retention time and higher response value, and achieves baseline separation, so berberine is selected as reference peak of reference substance, and berberine is marked as peak S, and berberine reference substance is used as reference substance.

The fingerprint spectrum measuring method established by the method is consistent with the content measuring method of coptis chinensis and phellodendron barks (sample preparation, chromatographic conditions and the like), 11 common peaks are marked, belonging to 5 medicinal flavors, and the specificity identification and multi-component and overall quality control are enhanced. The fingerprint spectrum is established to control the whole quality, and the running time is 21 minutes, so that the energy consumption is effectively reduced, and the project cost is reduced.

The method establishes thin-layer identification of 6 medicinal herbs of loquat leaf, white mulberry root-bark, coptis root, phellodendron bark, ginseng and liquorice in the classical prescription loquat lung-heat clearing drink and other dosage forms. The repeatability of detection results of different production places and different batches is good, and the standard of loquat lung-heat clearing drink substances and the standard of preparations are fully detected and effectively controlled. Meanwhile, a method for detecting the content of 5 chemical components of coptis chinensis and phellodendron bark is established, the content of loquat lung-heat-clearing drink substance standard and preparation is comprehensively and effectively detected, the content range is determined through multi-batch substance standard and preparation prepared from different batches of medicinal materials in different production places, and the foundation and standard are provided for subsequent amplification and mass production. Is favorable for the future production and sales of loquat lung-heat-clearing drink. The fingerprint spectrum is established to control the whole quality of the method, so that the operation time is short and the energy consumption is low.

Example 18

The sample preparation process of this example was: weighing 1.87kg of ginseng (broken), 12.43kg of loquat leaf, 1.87kg of liquorice, 6.22kg of coptis (broken), 12.43kg of white mulberry root-bark and 6.22kg of phellodendron bark, adding 41.04kg of water, decocting for 1 hour, filtering, concentrating the filtrate, drying, sieving with a 80-mesh sieve, adding a proper amount of aspartame and maltodextrin, uniformly mixing, granulating into 10kg of granules, and packaging. A total of 3 samples were prepared.

The sample is detected according to the method of the embodiment of the invention, the thin layer identification can detect corresponding spots, and the content detection and fingerprint detection results are shown in the following table:

from the results, the invention provides a method for comprehensively, accurately, quickly and intuitively detecting the loquat lung-heat clearing drink substance standard and the compound preparation.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of protection of the present invention.

Claims

1. The method for detecting the loquat lung-heat clearing drink is characterized by comprising the following steps of:

1) The thin-layer chromatography detection is used for qualitatively judging 6 ingredients in the loquat lung-heat-clearing drink sample to be detected, namely ginseng, loquat leaf, liquorice, coptis chinensis, white mulberry root-bark and amur corktree bark:

Step a, ginseng qualitative detection: adding water into a loquat lung-heat-clearing drink sample to be detected, heating to dissolve the loquat lung-heat-clearing drink sample, extracting by shaking with water saturated n-butanol for at least 1 time, combining n-butanol liquid, and extracting by shaking with a sodium hydroxide solution with the mass fraction of 0.1% -1% for at least 1 time; washing the n-butanol solution with n-butanol saturated water with pH value of 3-4 for at least 1 time, discarding the water washing solution, washing with n-butanol saturated water for at least 1 time, discarding the water washing solution, evaporating the n-butanol solution to dryness, and dissolving the residue with methanol or ethanol to obtain a sample solution; decocting ginseng reference medicinal materials in water for 10-60 min, filtering, and preparing the filtrate into reference medicinal material solution according to the identical operation of the preparation of the sample solution; adding methanol or ethanol into ginsenoside Rb1 reference substance, ginsenoside Re reference substance, ginsenoside Rf reference substance and ginsenoside Rg1 reference substance to obtain mixed solution containing 0.1-1.0 mg per 1ml as reference substance solution; according to a thin-layer chromatography test, absorbing a reference substance solution, a reference medicinal material solution and a test sample solution, respectively spotting on the same silica gel G thin-layer plate, spreading with a lower layer solution placed below 30 ℃ of chloroform-methanol-water with a volume ratio of 13:7:2 as a developing agent, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating until spot color development is clear, and observing whether spots with the same color are displayed on positions corresponding to the reference medicinal material chromatogram and the reference substance chromatogram in the test sample chromatogram;

Step b, detecting honey loquat She Dingxing: taking a loquat lung-heat-clearing drink sample to be detected, adding methanol or ethanol for ultrasonic treatment, filtering, and concentrating the filtrate to be used as a sample solution; decocting folium Eriobotryae control with water for 10-60 min, filtering, evaporating filtrate to dryness, and preparing the residue into control medicinal solution according to the same preparation method as the sample solution; adding ethanol into ursolic acid and/or oleanolic acid reference substances to prepare solutions with the concentration of 0.1-1.0 mg per 1ml respectively as reference substance solutions; according to a thin layer chromatography test, sucking the sample solution, the reference medicinal material solution and the reference substance solution to be respectively spotted on the same silica gel G thin layer plate, developing by taking a mixed solution of cyclohexane-chloroform-ethyl acetate-glacial acetic acid with the volume ratio of 10:15:8:0.5 as a developing agent, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating until spots are clear, and inspecting under a 365nm ultraviolet lamp; observing whether spots with the same color are displayed on the positions corresponding to the control medicine chromatogram and the control substance chromatogram in the sample chromatogram;

step c, qualitative detection of liquorice: taking a loquat lung-heat-clearing drink sample to be detected, adding methanol or ethanol, carrying out ultrasonic treatment for 10-60 minutes, filtering, and concentrating the filtrate to be used as a sample solution; adding methanol or ethanol into the glycyrrhizin reference substance to prepare a solution containing 0.1-1.0 mg per 1ml as reference substance solution; according to a thin-layer chromatography test, sucking a sample solution and a reference substance solution to be respectively spotted on the same silica gel G thin-layer plate, taking ethyl acetate-formic acid-glacial acetic acid-water with a volume ratio of 15:1:1:2 as developing agents, developing, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating until spots develop clearly, inspecting under a 365nm ultraviolet lamp, and observing whether spots with the same color are displayed on positions corresponding to the reference substance chromatogram in the sample chromatogram; or,

Adding water into a loquat lung-heat-clearing drink sample to be detected, heating to dissolve, extracting with water saturated n-butanol for at least 1 time, combining n-butanol solutions, extracting with 0.5% sodium hydroxide solution for at least 1 time, combining sodium hydroxide solutions, adjusting the pH value of the sodium hydroxide solution to 3-4 with hydrochloric acid, extracting with ethyl acetate for at least 1 time, combining ethyl acetate solutions, evaporating to dryness, and adding methanol or ethanol into residues to dissolve the residues to be used as a sample solution; decocting Glycyrrhrizae radix control medicine in water for 10-60 min, filtering, and preparing filtrate into control medicine solution according to the same preparation method of the sample solution; adding methanol into the glycyrrhizin reference substance to prepare a solution containing 0.1-1.0 mg per 1 ml; according to a thin-layer chromatography test, sucking a sample solution, a reference medicinal material solution and a reference substance solution to be respectively spotted on the same silica gel G thin-layer plate, spreading the solution at the lower layer below 30 ℃ with the volume ratio of 15:7:2 chloroform-methanol-water as a developing agent, taking out, airing, spraying 10% sulfuric acid ethanol solution, heating until spots develop clearly, inspecting under a 365nm ultraviolet lamp, and observing whether spots with the same color are displayed on positions corresponding to the reference medicinal material chromatogram and the reference substance chromatogram in the sample chromatogram;

Step d, qualitative detection of cortex phellodendri and rhizoma coptidis simultaneously; taking a loquat lung-heat-clearing drink sample to be detected, adding methanol or ethanol, carrying out ultrasonic treatment for 10-60 minutes, filtering, and taking filtrate, adding methanol or ethanol, and diluting to obtain a sample solution; decocting cortex Phellodendri reference medicinal material and Coptidis rhizoma reference medicinal material with water for 10-60 min, filtering, evaporating filtrate, and making the residue into reference medicinal material solution according to the same preparation method as the sample solution; adding methanol into berberine hydrochloride reference substance to prepare a solution containing 0.1-1.0 mg per 1ml as reference substance solution; according to a thin layer chromatography test, sucking the solutions to be respectively spotted on the same silica gel G plate, taking cyclohexane-ethyl acetate-isopropanol-methanol-water-triethylamine with a volume ratio of 3:3.5:1.5:0.5:1 as a developing agent, placing the developing agents in a developing cylinder with a presaturated concentrated ammonia test solution for 5-50 minutes for developing, taking out, airing, and placing the developing agents under a 365nm ultraviolet lamp for inspection; observing whether spots with the same color are displayed on the positions corresponding to the control medicine chromatogram and the control substance chromatogram in the sample chromatogram;

step e, qualitative detection of the white mulberry root-bark: adding water into a loquat lung-heat-clearing drink sample to be detected, performing ultrasonic treatment to dissolve, centrifuging, taking supernatant, shaking and extracting for at least 1 time by using chloroform, mixing the extracting solutions, washing for at least 1 time by using a sodium hydroxide solution with the mass fraction of 0.1-2.0%, discarding washing solution, evaporating the chloroform solution to dryness, and adding chloroform into residues to dissolve to serve as a sample solution; adding water into the cortex mori reference medicinal material, performing ultrasonic treatment for 10-60 minutes, and preparing a reference medicinal material solution according to the same preparation method of the sample solution; according to a thin layer chromatography test, the two solutions are respectively absorbed on the same silica gel G thin layer plate according to the volume ratio of 2:1: developing with 2 petroleum ether-toluene-chloroform as developing agent, taking out, air drying, spraying 5% vanillin sulfuric acid solution, and heating until the color of spots is clear; observing whether spots with the same color are displayed on the positions corresponding to the control medicine chromatogram in the sample chromatogram;

2b) Preparation of test solution: grinding a loquat lung-heat-clearing drink sample to be detected, precisely weighing 0.1-1 g, placing the loquat lung-heat-clearing drink sample into a conical bottle with a plug, precisely adding 15-35 ml of methanol, or 15-35 ml of 70% methanol water solution, or 15-35 ml of acetonitrile-0.1% phosphoric acid solution, sealing, weighing, performing ultrasonic treatment for 15-120 minutes, or heating and refluxing for 15-120 minutes, or shaking and extracting for 15-120 minutes, taking out, cooling, weighing again, supplementing the reduced weight with methanol, shaking uniformly, and filtering to obtain filtrate;

2c) Precisely sucking 5-15 μl of each of the reference solution and the sample solution, and injecting into a liquid chromatograph for measurement; the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filler; acetonitrile-0.1% phosphoric acid solution is used as a mobile phase; the detection wavelength is 284nm; the theoretical plate number is not lower than 6000 according to peak of phellodendrine hydrochloride;

3c) Taking 0.1-1.0 g of loquat lung-heat clearing drink sample to be detected, precisely weighing, placing the loquat lung-heat clearing drink sample into a conical bottle with a plug, precisely adding methanol, or methanol aqueous solution with the volume fraction of 40-60% or methanol aqueous solution with the volume fraction of 61-80% by 15-40 ml, sealing, weighing, ultrasonically treating for 15-120 minutes, or reflux-extracting for 15-120 minutes, or shaking and extracting for 15-120 minutes, taking out, cooling, weighing again, supplementing the reduced weight with methanol, shaking uniformly, centrifuging, and taking supernatant to obtain a sample solution; precisely sucking 0.5-2 μl of each of the control solution and the sample solution, and injecting into an ultra-high performance liquid chromatograph for detection;

and calculating the similarity between the obtained chromatogram and the control fingerprint shown in fig. 9 by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation software system, wherein the similarity between the obtained chromatogram and the control fingerprint is more than 90%.

2. The method according to claim 1, wherein the loquat lung-heat clearing drink to be detected comprises a decoction prepared by an ancient classical prescription or a solid preparation prepared by a modern technology.

3. The method according to claim 1 or 2, wherein the water amount of the loquat lung-heat clearing drink sample to be detected in the step a is 1g sample added with 5-50 ml water;

preferably, the amount of water-saturated n-butanol in step a is 1g of sample plus 5 to 50ml of water-saturated n-butanol.

Preferably, the at least 1 more times in step a means 1 to 5 times.

4. A method according to any one of claims 1-3, characterized in that 1g of sample is used in step b with 10-100 ml of methanol or ethanol.

5. The method according to any one of claims 1 to 4, wherein the amount of methanol or ethanol used in step c is from 1g of sample plus 10 to 100ml of methanol or ethanol.

Preferably, the water amount of the loquat lung-heat clearing drink sample to be detected in the step c, which is dissolved by adding water, is 1g of sample and 5-50 ml of water.

Preferably, said at least 1 more times in step c means 1 to 5 times.

Preferably, the amount of methanol or ethanol used in step d is 1g of sample plus 10 to 100ml of methanol or ethanol.

Preferably, said at least 1 more times in step e means 1 to 5 times.

Preferably, the water amount of the loquat lung-heat clearing drink sample to be detected in the step e, which is dissolved by adding water, is 1g of sample and 5-50 ml of water.

6. The method according to any one of claims 1 to 5, wherein the thin layer chromatography in the thin layer chromatography detection in step 1) is performed according to the method described in the fourth edition of the general rule 0502 of the chinese pharmacopoeia 2020.

7. The method according to any one of claims 1 to 6, wherein the power of the ultrasound in step 2 b) is 400 to 600W and the frequency is 30 to 500kHz.

Preferably, the mobile phase in step 2 b) is acetonitrile-0.1% phosphoric acid solution (0.2 g per 100ml sodium dodecyl sulfonate).

Preferably, in step 2 b) the ultrasonic treatment is carried out for 15-45 minutes, or the reflux is carried out for 15-45 minutes, or the extraction is carried out for 15-45 minutes by shaking.

8. The method according to any one of claims 1 to 7, wherein the chromatographic column in step 3 a) is of model ACQUCITY UPLC BEH Shield RP, column length 100mm, inner diameter 2.1mm and particle size 1.7 μm.

Preferably, in step 3 c) the ultrasonic treatment is carried out for 15-45 minutes, or the reflux is carried out for 15-45 minutes, or the extraction is carried out for 15-45 minutes by shaking.

9. The method according to any one of claims 1 to 8, wherein the chromatographic column in step 4) is of model ACQUCITY UPLC BEH Shield RP, column length 100mm, inner diameter 2.1mm and particle size 1.7 μm.

10. The method according to any one of claims 1 to 9, wherein the fingerprint in step 4) is as shown in fig. 9, wherein 11 sets of common peaks are as follows:

。