CN113624854A

CN113624854A - Quality control method of Qinqiu powder and its preparation

Info

Publication number: CN113624854A
Application number: CN202010389132.3A
Authority: CN
Inventors: 黄广伟; 孙仁弟; 杨素德; 马云飞; 杨瑞花; 张媛媛; 保红云; 韦悦; 高志泓; 张玥莉
Original assignee: Shanghai Lvguyuan Biomedicine Co ltd
Current assignee: Shanghai Lvguyuan Biomedicine Co ltd
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2021-11-09
Anticipated expiration: 2040-05-09
Also published as: CN113624854B

Abstract

The invention provides quality control methods of Qinqiu powder and a preparation thereof. Specifically, the invention provides a quality control method of effective parts of a traditional Chinese medicine, wherein the traditional Chinese medicine comprises the following medicinal materials: scutellariae radix, fructus Perillae, caulis Perillae, radix Sophorae Flavescentis and Saviae Miltiorrhizae radix. The quality control of the Qinsu powder and the preparation thereof is carried out by controlling the percentage content of the representative active ingredients in the effective parts and/or utilizing the similarity of fingerprint spectrums, thereby achieving accurate and comprehensive quality control.

Description

Quality control method of Qinqiu powder and its preparation

Technical Field

The invention relates to the field of medicines, in particular to a quality control method of Qinsu powder and a preparation thereof.

Background

The Chinese medicine composition comprising five Chinese medicinal materials of scutellaria root, perilla seed, perilla stem, flavescent sophora root and salvia root has several functions of clearing away heat, relieving pain and diminishing inflammation. In the formula, the scutellaria is a monarch drug, the perilla (seed and stem) and the sophora flavescens are ministerial drugs, and the salvia is an assistant drug. The extract (Qinsu powder) obtained by extracting, separating and purifying the traditional Chinese medicinal materials in the formula has the advantages of less impurities, low side effect and the like, and can be further conveniently prepared into various preparations (such as Qinsu capsules) convenient to take.

However, the traditional Chinese medicine extract, especially the traditional Chinese medicine extract of various medicinal materials, has very complex components and much interference of impurities, and brings great difficulty to true and false identification, component analysis, pharmacological research and quality control of the traditional Chinese medicine extract. And the differences of the producing areas, the growth years, the medicinal parts and the like of the same Chinese medicinal materials can influence the medicines.

Therefore, the quality control method capable of accurately and comprehensively controlling the Qinsu powder and the preparation thereof is urgently needed in the field.

Disclosure of Invention

The invention aims to provide accurate and comprehensive Qinsu powder and a preparation quality control method thereof.

The invention aims to provide a Qinsu powder, a fingerprint construction method of a Qinsu powder preparation and a quality control method based on the Qinsu powder preparation.

The invention also aims to provide a combination of active ingredients of a plurality of traditional Chinese medicine monomers in reasonable proportion, a pharmaceutical composition and application thereof.

The invention provides a quality control method of effective parts of traditional Chinese medicine,

wherein the traditional Chinese medicine comprises the following medicinal materials: scutellariae radix, fructus Perillae, caulis Perillae, radix Sophorae Flavescentis and Saviae Miltiorrhizae radix;

the method comprises the following steps:

(a) determining the weight percentage of active ingredients in the effective parts of the traditional Chinese medicine, based on the dry weight of the effective parts of the traditional Chinese medicine, wherein the active ingredients comprise the following group A: baicalin, salvianolic acid B, rosmarinic acid and matrine;

(b) judging whether the content of the active ingredients meets the following standard content range according to the measurement result in the step (a):

and (4) when the content of each active ingredient meets the corresponding standard content range, the active ingredient is qualified, otherwise, the active ingredient is unqualified.

In another preferred embodiment, the quality control method further includes the steps of:

(c) and (C) when the determination result in the step (b) is unqualified and the closest end value C0 of the content C1 of the corresponding active ingredient exceeding the standard content range in the Chinese medicinal effective part and the corresponding standard content range meets | C1-C0|/C0 is less than or equal to 10%, adjusting the composition of the Chinese medicinal effective part to be qualified.

In another preferred embodiment, the adjustment of the composition of the effective part of the traditional Chinese medicine refers to the adjustment of one or more of the following parameters of one or more of the medicinal materials or components: source of the medicinal material, amount of the medicinal material, extraction method, active ingredient, or combination thereof.

In another preferred embodiment, | C1-C0|/C0 is 5% or less, more preferably 3% or less, 2% or less or 1% or less in step (C).

In another preferred embodiment, the step (b) further comprises determining whether the sum of the four ingredients (A1) - (A4) meets the following standard content range:

(A1) the sum of the contents of the four components (A4) is 26-36 wt%.

In another preferred embodiment, the method further comprises determining one or more of the following active ingredients in group B and determining whether they meet the following respective standard content ranges:

in another preferred embodiment, the method further comprises determining one or more of the following group C active ingredients and determining whether they meet the following respective standard content ranges:

in another preferred embodiment, the Chinese medicinal herbs are scutellaria baicalensis, perilla seeds, perilla stems, sophora flavescens and salvia miltiorrhiza.

In another preferred embodiment, the traditional Chinese medicine comprises the following components:

400 portions of perilla seed;

the perilla stem is 400-600 parts by weight;

600 and 800 parts of kuh-seng;

600 and 800 parts of scutellaria baicalensis; and

the salvia miltiorrhiza is 1000-1400 parts by weight.

In another preferred example, the effective part of the traditional Chinese medicine is an extract of the traditional Chinese medicine or a preparation form of the extract.

In another preferred example, the Chinese medicine extract is obtained by extracting the Chinese medicine with a mixed solvent of water and an organic solvent.

In another preferred embodiment, the organic solvent in the mixed solvent is selected from the group consisting of: a C1-C6 lower alcohol, acetonitrile, or a combination thereof.

In another preferred embodiment, the Chinese medicinal extract is obtained by extracting each medicinal material respectively and then combining the medicinal materials or is obtained by combining the medicinal materials and then extracting the medicinal materials.

In another preferred embodiment, step (a) includes the steps of:

(a1) preparation of control solutions: precisely weighing reference substances of each active component, diluting with a solvent, fixing the volume, and filtering with a microporous filter membrane to obtain a reference substance mixed solution;

(a2) preparation of test sample solution: precisely weighing the effective part of the traditional Chinese medicine, diluting with a solvent, fixing the volume, and filtering with a microporous filter membrane to obtain a sample solution;

(a3) loading the reference solution and the test sample solution to a high performance liquid chromatography for detection,

wherein the high performance liquid chromatography detection comprises:

(a3-1) when the active ingredient detected is not an alkaloid, the liquid phase conditions are characterized by:

(i) the chromatographic column is an octadecyl bonding silica gel column;

(ii) the mobile phase is acetonitrile (A) and 0.04-0.06 wt% phosphoric acid water solution (B); and

(iii) gradient elution;

(a3-2) when the active ingredient to be detected is an alkaloid, the liquid phase condition has the following characteristics:

(i) the chromatographic column is an amino bonded silica gel column;

(ii) the mobile phase is acetonitrile-absolute ethyl alcohol-2.5-3.5 wt% phosphoric acid water solution;

(iii) the elution procedure is isocratic elution;

(a4) and (b) calculating the weight percentage of each active ingredient in the test sample according to the chromatogram obtained in the step (a3) by the dry weight of the effective parts of the traditional Chinese medicine.

In another preferred example, in the step (a3-1), the mobile phase is acetonitrile (A) and 0.05 wt% phosphoric acid aqueous solution (B).

In another preferred example, in the step (a3-2), the mobile phase is acetonitrile-absolute ethyl alcohol-2.8-3.2 wt% phosphoric acid aqueous solution, preferably acetonitrile-absolute ethyl alcohol-3 wt% phosphoric acid aqueous solution.

In another preferred example, in step (a3-1), the gradient elution comprises a continuous process in which% is a volume percentage relative to the total flow rate:

in another preferred embodiment, the isocratic elution is acetonitrile: anhydrous ethanol: the volume ratio of the phosphoric acid aqueous solution is as follows: 75-85:8-15: 6-12; preferably 75-85:8-12: 6-10.

In another preferred embodiment, the isocratic elution is acetonitrile: anhydrous ethanol: the volume ratio of the 3 wt% phosphoric acid aqueous solution is 81: 11: 8.

in another preferred embodiment, the alkaloid is selected from one or more of the following group: matrine, oxymatrine, sophocarpine, and oxysophoridine.

In another preferred example, in the step (a1), the control solutions include 5 to 10 control solutions with gradient concentration.

In the step (a3-2), the method for pretreating the test sample solution includes the steps of:

preparation of alkaloid test sample solution: precisely weighing the effective parts of the traditional Chinese medicines, diluting with concentrated ammonia water and dichloromethane, and fixing the volume to obtain a diluent; precisely measuring the diluent, evaporating to obtain residue, dissolving the residue with solvent, diluting to desired volume, and filtering with microporous membrane to obtain alkaloid sample solution

In another preferred embodiment, in step (a4), the content of each active ingredient is calculated using an external standard method and/or an external standard point method.

In another preferred example, in step (a4), the peak area of each active ingredient is determined by its maximum ultraviolet absorption wavelength.

In another preferred embodiment, in step (a1), the solvent is selected from the group consisting of: C1-C6 lower alcohol, acetonitrile, or their mixed solvent with water.

In another preferred embodiment, the volume of water in the mixed solvent is 5 to 40%, preferably 10 to 25%, based on the total volume of the mixed solvent.

In another preferred embodiment, the concentrated ammonia water is 15-30 wt% ammonia water, preferably 20-28 wt%, and more preferably 25-28 wt%.

In another preferred embodiment, the column length of the chromatographic column is 20-25 cm.

In another preferred embodiment, the packing particle size of the chromatographic column is 5-10 μm.

In another preferred embodiment, the column diameter of the chromatographic column is 4mm to 6mm, preferably 4.6 mm.

In another preferred embodiment, the column temperature of the elution is 10 to 40 ℃, more preferably 20 to 35 ℃.

In another preferred embodiment, the sample is introduced in an amount of 10. mu.L to 50. mu.L, more preferably, 10. mu.L to 20. mu.L.

In another preferred embodiment, in the step (a3-1), the number of theoretical plates of the column is not less than 1500, preferably 2000, 2500 or 3000, calculated as baicalin.

In another preferred embodiment, in step (a3-2), the column has a theoretical plate number of not less than 1500, preferably 2000, 2500 or 3000, calculated as matrine.

In another preferred example, the elution procedure further comprises an equilibration process of 5-20min before and/or after, wherein the equilibration process elution procedure is as follows: acetonitrile 5% and phosphoric acid water solution 95%.

In another preferred embodiment, the total flow rate of the eluted mobile phase is 0.8-1.2mL/min, preferably 0.8-1.0 mL/min.

In another preferred embodiment, the UV detection wavelength of the HPLC is 250-300nm, preferably 260-280nm, more preferably 270-275 nm.

In another preferred embodiment, the formulation is selected from the group consisting of: liquid formulations (suspensions, solutions), solid formulations, or combinations thereof.

In another preferred embodiment, the liquid formulation is selected from the group consisting of: oral liquid, injection, tincture, or combination thereof.

In another preferred embodiment, the solid formulation is selected from the group consisting of: tablets, capsules, pills, powders, granules, or combinations thereof.

The second aspect of the invention provides a method for constructing the fingerprint of the effective part of the traditional Chinese medicine,

the fingerprint map construction method comprises the following steps:

(I) preparation of test sample solution: precisely weighing the effective part of the traditional Chinese medicine, diluting with a solvent, fixing the volume, and filtering with a microporous filter membrane to obtain a sample solution;

(II) loading the control solution and the test sample solution to high performance liquid chromatography and separating,

wherein the separation conditions of the high performance liquid chromatography have the following characteristics:

(i) the chromatographic column is an octadecyl bonding silica gel column;

(ii) the mobile phase is acetonitrile (A)0.04-0.06 wt% phosphoric acid water solution (B);

(iii) gradient elution;

(iv) the ultraviolet detection wavelength is 250-300 nm; and

and (III) analyzing and comparing the chromatograms obtained in the step (II) to obtain an HPLC sample fingerprint and a reference fingerprint which are formed by common characteristic peaks of the chromatograms.

In another preferred embodiment, in step (III), the common characteristic peak is generated by using a multipoint calibration method, and the control fingerprint is generated by using an average value method.

In another preferred example, the method further comprises the steps of:

(IV) applying a control solution to the HPLC and determining the active ingredient represented by each peak by comparing the retention time of the control with the peaks in the fingerprint.

In another preferred embodiment, in step (IV), the preparation method of the control solution comprises:

preparation of control solutions: precisely weighing one or more reference substances, fixing the volume of the solvent, and filtering with a microporous filter membrane to obtain a reference substance solution containing one or more reference substances.

In another preferred embodiment, in step (IV), the control is selected from the group consisting of: rosmarinic acid, baicalin, trifolium pterocarpus santalin, oroxylin-7-O-beta-D-glucuronide, salvianolic acid, wogonoside, salvianolic acid, luteolin, baicalein, wogonin, kushenketone, cryptotanshinone, and tanshinone IIA, or a combination thereof.

In another preferred example, the elution procedure of the gradient elution comprises the following successive procedure, wherein% is the volume percentage relative to the total flow rate:

time/min acetonitrile (A)% phosphoric acid aqueous solution (B)%

In another preferred example, the method further comprises the steps of:

(E) constructing a fingerprint of alkaloid of the effective parts of the traditional Chinese medicine;

the construction of the alkaloid fingerprint comprises the following sub-steps:

(E1) preparation of alkaloid test sample solution: precisely weighing the effective parts of the traditional Chinese medicines, diluting with concentrated ammonia water and chloromethane, and fixing the volume to obtain a diluent; precisely measuring the diluent, evaporating to obtain residue, dissolving the residue with a solvent, fixing the volume, and filtering with a microporous filter membrane to obtain an alkaloid test sample solution;

(E2) loading the alkaloid reference substance solution and the alkaloid test sample solution to a high performance liquid chromatography and separating,

(i) the chromatographic column is an amino bonded silica gel column;

(iii) the elution procedure was isocratic:

(iv) the ultraviolet detection wavelength is 200-230 nm; and

(E4) and (4) analyzing and comparing the chromatograms obtained in the step (E3) to obtain an HPLC sample alkaloid fingerprint and a control alkaloid fingerprint which are formed by common characteristic peaks of the chromatograms.

In another preferred embodiment, in step (E4), the common characteristic peak is generated by using a multipoint correction method, and the control fingerprint is generated by using an average value method.

In another preferred example, the method further comprises the steps of:

(E4) loading the alkaloid control solution onto the high performance liquid chromatography, and comparing the retention time of the control with the peak in the fingerprint, thereby determining the active ingredient represented by each peak.

In another preferred example, in the step (E4), the method for preparing the alkaloid control solution comprises: preparation of alkaloid control solutions: precisely weighing one or more alkaloid reference substances: fixing the volume of the solvent, and filtering with microporous membrane to obtain alkaloid reference substance solution containing one or more reference substances.

In another preferred embodiment, the alkaloid is selected from the group consisting of: matrine, oxymatrine, sophocarpine, and oxysophoridine.

In another preferred example, in the step (E3), the mobile phase is acetonitrile-absolute ethyl alcohol-2.8-3.2 wt% phosphoric acid aqueous solution, preferably acetonitrile-absolute ethyl alcohol-3 wt% phosphoric acid aqueous solution.

In another preferred embodiment, the elution conditions for the isocratic elution are acetonitrile: anhydrous ethanol: the volume ratio of the 3 wt% phosphoric acid aqueous solution is 81: 11: 8.

in another preferred embodiment, the method further comprises one or more of the following steps:

(F) similarity evaluation is carried out on each fingerprint by using similarity software;

(G) and continuously injecting the same sample solution for 3-6 times, and evaluating the precision, reproducibility and/or stability of the method according to the obtained fingerprint.

In a third aspect of the invention, a method for controlling the quality of an effective part of a traditional Chinese medicine is provided,

the method comprises the following steps:

(Z1) constructing a fingerprint of the effective part of the Chinese medicine using the method according to the second aspect of the present invention;

(Z2) performing similarity evaluation on the fingerprints by using similarity software, wherein the similarity between the sample fingerprint and the comparison fingerprint is not less than 0.95.

In another preferred example, the quality control method further comprises quality control of alkaloids:

(Z4) constructing an alkaloid fingerprint of the effective part of the traditional Chinese medicine;

(Z5) similarity evaluation is performed on the fingerprint by using similarity software, and the similarity between the sample alkaloid fingerprint and the control alkaloid fingerprint is not less than 0.95.

In another preferred embodiment, the similarity is 0.96 or more, preferably 0.97 or more, 0.98 or more, or 0.99 or more.

In another preferred example, the method further comprises the steps of:

and continuously feeding the same sample solution for 3-6 times, and evaluating the precision, reproducibility and/or stability of the method according to the obtained fingerprint, wherein the RSD of each common peak area ratio is less than or equal to 3%.

In another preferred embodiment, the relative retention time of each common peak is within ± 2min, preferably ± 1min, of the mean relative retention time.

In a fourth aspect of the invention, there is provided a combination of active ingredients, said combination comprising the following active ingredients of group a:

in another preferred embodiment, the combination further comprises one or more active ingredients from group B:

in another preferred embodiment, the combination further comprises one or more active ingredients from group C below:

in another preferred embodiment, the baicalin is 15.5-19.5 parts by weight.

In another preferred embodiment, the amount of wogonoside is 3.3 to 4.0 parts by weight.

In another preferred embodiment, the baicalein is 0.6 to 1.3 parts by weight.

In another preferred embodiment, the wogonin is 0.3 to 0.6 parts by weight.

In another preferred embodiment, the oroxylin-7-O-beta-D-glucuronide accounts for 1.9-3.2 parts by weight.

In another preferred embodiment, the salvianolic acid B is 9.7-14.0 parts by weight.

In another preferable example, the tanshinone IIA is 0.3-0.5 weight part.

In another preferred embodiment, the matrine is 1.7-2.4 parts by weight.

In another preferred embodiment, the amount of the trifoliosid is 1.2 to 1.9 parts by weight.

In another preferred embodiment, the kurarinone is 0.4-0.9 weight parts.

In another preferred example, the luteolin is 0.1-0.3 part by weight.

In another preferred embodiment, the rosmarinic acid is 0.4-0.6 part by weight.

In another preferred embodiment, the salvianolic acid A is 0.1-0.4 weight parts.

In another preferred embodiment, the cryptotanshinone is 0.2-0.4 weight part.

In another preferred embodiment, the oxymatrine is 0.6 to 0.9 parts by weight.

In another preferred embodiment, the sophocarpine is 0.6-0.8 weight parts.

In another preferred embodiment, the sophoridine oxide is 0.4-0.7 parts by weight.

In another preferred embodiment, the combination comprises:

in a fifth aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a combination of active ingredients according to the fourth aspect of the invention; and a pharmaceutically acceptable carrier.

In another preferred embodiment, the composition comprises the following a species:

in another preferred embodiment, the composition further comprises one or more of the following group B:

in another preferred embodiment, the composition further comprises one or more of the following group C:

in another preferred embodiment, the sum of the contents of the four components (A1) - (A4) is 26-36 wt%.

In another preferred embodiment, the baicalin accounts for 15.5-19.5 wt%.

In another preferred embodiment, the weight of wogonoside is 3.3-4.0 wt%.

In another preferred embodiment, the oroxylin-7-O-beta-D-glucuronide accounts for 1.9-3.2 wt%. In another preferred embodiment, the salvianolic acid B is 9.7-14.0 wt%.

In another preferred embodiment, the tanshinone IIA is 0.3-0.5 wt%.

In another preferred embodiment, the matrine is 1.7-2.4 wt%.

In another preferred embodiment, the baicalein is 0.6-1.3 wt%.

In another preferred embodiment, the wogonin is 0.3 to 0.6 wt%.

In another preferred embodiment, the amount of the trifolioside is 1.2-1.9 wt%.

In another preferred embodiment, the content of the kurarinone is 0.4-0.9 wt%.

In another preferred embodiment, the luteolin is 0.1-0.3 wt%.

In another preferred embodiment, the rosmarinic acid is 0.4 to 0.6 wt%.

In another preferred embodiment, the salvianolic acid A is 0.1-0.4 wt%.

In another preferred embodiment, the cryptotanshinone is 0.2-0.4 wt%.

In another preferred embodiment, the oxymatrine is 0.6-0.9 wt%.

In another preferred embodiment, the sophocarpine is 0.6-0.8 wt%.

In another preferred embodiment, the sophoridine oxide is 0.4-0.7 wt%.

In another preferred embodiment, the composition comprises:

wherein the percentages are based on the total weight of the composition.

In another preferred embodiment, in the pharmaceutical composition, each active ingredient is chemically synthesized.

In another preferred embodiment, each active ingredient is derived from extracts of scutellaria baicalensis, perilla seeds, perilla stems, sophora flavescens and salvia miltiorrhiza, and the sum of the active ingredients accounts for more than 80 wt%, preferably more than 90 wt%, and more preferably more than 95 wt% of the extracts.

In a sixth aspect of the invention there is provided the use of a combination of active ingredients according to the fourth aspect of the invention or a pharmaceutical composition according to the fifth aspect of the invention in the manufacture of a medicament for: (a) treating or preventing cough after cold; and/or (b) antibacterial and/or treatment of bacterial infections; (c) preventing and/or treating bronchitis; (d) clearing heat; (e) anti-inflammatory; (f) inhibition of capillary permeability; (g) relieving pain; (h) relieving cough; and/or (i) expectorant.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows HPLC sample fingerprints and reference fingerprints (R) of 9 batches of Qinsu capsules; wherein: 10: rosmarinic acid, 11: baicalin, 13: trifoliosid, 16: oroxylin-7-O-beta-D-glucuronide, 17: salvianolic acid B, 18: wogonoside, 19: salvianolic acid a, 20: luteolin, 21: baicalein, 23: wogonin, 25: kurarinone, 28: cryptotanshinone, 33: tanshinone IIA.

FIG. 2 shows the HPLC fingerprint similarity calculation results of 9 batches of Qinsu capsules.

FIG. 3 shows HPLC sample alkaloid fingerprint and control alkaloid fingerprint (R) of alkaloid components of 9 Qinsu capsules; wherein 1: sophocarpine, 2: matrine, 3: oxysophoridine, 4: oxymatrine.

FIG. 4 shows the HPLC fingerprint similarity calculation results for the alkaloid components of 9 Qinsu capsules.

FIG. 5A is a liquid chromatogram of acetonitrile-0.2% formic acid as the mobile phase; FIG. 5B is a liquid chromatogram of a mobile phase of acetonitrile-0.05% phosphoric acid water.

Detailed Description

The inventor provides an extract consisting of five traditional Chinese medicines of scutellaria baicalensis, perilla seeds, perilla stems, radix sophorae flavescentis and salvia miltiorrhiza and a quality control method of a preparation prepared from the extract through extensive and intensive research and a large number of screening and tests. The invention also provides a Qinsu powder fingerprint construction method and a quality control method based on the Qinsu powder fingerprint, the obtained fingerprint can mark more than 30 common peaks, and the product quality can be accurately and comprehensively controlled. The present invention has been completed based on this finding.

Term(s) for

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

The term "room temperature" as used herein means a temperature of 4-40 ℃, preferably 25 ± 5 ℃.

As used herein, the term "C1-C6 lower alcohol", "C1-C4 lower alcohol" or "C1-C3 lower alcohol" refers to an alcohol having 1-6, 1-4 or 1-3 carbon atoms, such as methanol, ethanol, n-propanol, isopropanol, n-butanol, or the like.

Chinese medicinal raw material

The invention provides traditional Chinese medicine raw medicinal materials, which comprise (but are not limited to) one or more medicinal materials selected from the following groups: perilla seed, perilla stem, radix sophorae flavescentis, scutellaria baicalensis and salvia miltiorrhiza.

400 portions of perilla seed;

the perilla stem is 400-600 parts by weight;

600 and 800 parts of kuh-seng;

600 and 800 parts of scutellaria baicalensis; and

the salvia miltiorrhiza is 1000-1400 parts by weight.

In the invention, the main raw medicinal materials have the following properties and main effects:

fructus Perillae is dried mature fruit of Perilla frutescens of Labiatae, and has effects of benefiting five internal organs, descending qi, relieving cholera, emesis, regurgitation, and tonifying consumptive disease.

CAULIS Perillae is named as CAULIS PERILLAE, and is dried stem of Perilla Perillarietescens (L.) Britt. of Labiatae, and CAULIS Perillae has effects of regulating qi-flowing, and treating gastric cavity pain, belch and emesis.

RADIX Sophorae Flavescentis is named RADIX Sophorae FLAVESCENTIS, and is dried root of Sophora flavescens ait of Leguminosae, and has effects in clearing away heat, eliminating dampness, killing parasite, and promoting urination.

The Salvia miltiorrhiza, named as RADIX SALVIAE MILTIORRHIZAE, is dried root and rhizome of Salvia miltiorrhiza bge of Labiatae, has effects of removing blood stasis, promoting blood circulation, and can be used for treating menoxenia, amenorrhea, and dysmenorrhea.

RADIX Scutellariae, also known as RADIX Scutellariae, is the dried root of Scutellaria baicalensis Georgi of Labiatae, and has effects in clearing away heat and toxic materials, eliminating dampness, purging pathogenic fire, stopping bleeding, and preventing miscarriage.

Effective part of traditional Chinese medicine

In the invention, the effective parts of the traditional Chinese medicine comprise the extract (also called Qinlusu powder) of the traditional Chinese medicine and the preparation form of the extract.

The Chinese medicinal raw materials can be mixed and then effective components are extracted by a proper method to obtain an extract; in addition, each raw material can also be respectively extracted (for example, the same or different extraction or processing methods are respectively adopted) to obtain effective components (namely, extracts of the medicinal materials), and the extracts are obtained after combination. It should be understood that the active ingredients obtained by extraction may exist in various forms, such as single or mixed pure active ingredient powder obtained by extracting and purifying raw material herbs, or extract obtained by extracting and concentrating raw material herbs with solvent (the active ingredients exist in the extract), or other forms.

The Chinese medicine effective part can also exist in the form of preparation. Such as preparing the extract and a pharmaceutically acceptable carrier into any suitable preparation. Commonly used formulations include, but are not limited to: liquid preparations, such as suspensions, solutions; more specifically, oral liquid, injection, aerosol, etc.; solid preparations, for example, tablets, capsules, granules, pills, powders, and the like.

In the present invention, when the effective components of the traditional Chinese medicine exist in the form of a preparation, the content of active ingredients in the effective components of the traditional Chinese medicine is based on the dry weight of the extract added to the preparation (i.e., the carrier is not included in the total weight).

In the present invention, the extraction method of the effective part of the Chinese medicine is not particularly limited, and a Chinese medicine extraction method commonly used in the field can be adopted. Preferably, the extract meets the quality control requirements of the present invention.

Preferably, the traditional Chinese medicine extract is obtained by extracting the traditional Chinese medicine with a mixed solvent of water and an organic solvent. For example, the mixed solvent is an alcohol-water mixed solution or a mixed solvent of acetonitrile and water, preferably ethanol-water.

Preferably, the extraction method of scutellaria baicalensis comprises the following steps: taking a scutellaria baicalensis medicinal material, adding 6-10 times of 65-75% ethanol water, heating, refluxing and extracting for 2-4 times, wherein each time lasts for 1-4 hours, filtering, recovering ethanol from filtrate under reduced pressure, merging and concentrating the filtrate into an extract with the relative density of 1.05-1.20 (50-60 ℃), and drying under reduced pressure to obtain a dry extract A.

Preferably, the method for extracting sophora flavescens comprises the following steps: taking a lightyellow sophora root medicinal material, adding 45-55% ethanol water which is 8-12 times the amount of the lightyellow sophora root medicinal material, heating, refluxing and extracting for 2-4 times, wherein each time lasts for 1-4 hours, filtering, recovering ethanol from filtrate under reduced pressure, and concentrating to obtain a concentrated solution with the relative density of 1.02-1.12 (50-60 ℃); diluting the concentrated solution with purified water to obtain a dispersion liquid (containing 0.05kg/L of crude drug), passing the dispersion liquid through a neutral alumina column, eluting with 90-95% ethanol water in an amount which is 6-10 times that of the dispersion liquid, collecting eluent, recovering ethanol from the eluent under reduced pressure, concentrating the ethanol to obtain an extract with the relative density of 1.05-1.20 (50-60 ℃), and drying under reduced pressure to obtain a dry extract B.

Preferably, the method for extracting salvia miltiorrhiza comprises the following steps: taking a salvia miltiorrhiza medicinal material, adding 8-12 times of 85-90% ethanol water, heating, refluxing and extracting for 2-4 times, each time for 1-4 hours, filtering, recovering ethanol from filtrate under reduced pressure, merging and concentrating the filtrate to a concentrated solution with the relative density of 1.05-1.15 (50-60 ℃), adding purified water into the concentrated solution to dilute the concentrated solution to form a dispersion liquid (containing 0.05kg/L of crude drug), taking the dispersion liquid to pass through a weak-polarity or non-polarity macroporous resin column, eluting with 6-10 times of 90-95% ethanol water, collecting eluent, recovering ethanol from the eluent under reduced pressure, merging and concentrating the eluent to an extract with the relative density of 1.05-1.20 (50-60 ℃), and drying under reduced pressure to obtain a dry extract C.

Preferably, the extraction method of perilla seed and/or perilla stem comprises the following steps: taking perilla seeds and/or perilla stems, adding 8-12 times of 75-85% ethanol water, heating, refluxing and extracting for 2-4 times, each time for 1-4 hours, filtering, recovering ethanol from filtrate under reduced pressure, mixing, concentrating to obtain an extract with the relative density of 1.05-1.20 (50-60 ℃), and drying under reduced pressure to obtain a dry extract D.

Preferably, the traditional Chinese medicine effective parts are obtained by mixing the dry extracts A, B, C and D, and are preferably completely mixed according to the amount of the extracted paste.

In another preferred embodiment, the effective component of the traditional Chinese medicine is obtained by extracting the mixture of the medicinal materials, and specifically, the extraction method may include the steps of:

(1) mixing the traditional Chinese medicine raw materials, and extracting with an alcohol-water solution to obtain an extracting solution;

(2) purifying the extracting solution by using a macroporous adsorption resin column to obtain the extract.

In another preferred embodiment, in the step (1), the alcohol content of the alcohol aqueous solution is 10-90% (v/v), preferably 20-80% (v/v), more preferably 30-70% (v/v), and most preferably 40-60% (v/v).

In another preferred embodiment, in the step (1), the alcohol is a C1-C6 lower alcohol, preferably a C1-C4 lower alcohol, more preferably a C1-C3 lower alcohol. Typically, the alcohol is ethanol.

In another preferred example, in the step (2), the macroporous resin is a weak polar or non-polar macroporous adsorption resin. Typically, in the step (2), the macroporous resin is styrene type weak polar macroporous adsorption resin.

Construction method of fingerprint

The traditional Chinese medicine fingerprint spectrum refers to a spectrum which is obtained by adopting a certain analysis means after the traditional Chinese medicine is properly processed and can identify the common peak of the characteristics of the traditional Chinese medicine. The traditional Chinese medicine fingerprint has the basic characteristics of integrity. The information reflected is comprehensive, emphasizing the relatively stable proportion, arrangement order and mutual implication of a plurality of components (common peaks). The authenticity of the traditional Chinese medicine can be effectively identified through the characteristics of the fingerprint. The quality of the traditional Chinese medicine can be effectively controlled and the relative stability of the quality of the traditional Chinese medicine can be ensured by setting the area and the proportion of the main characteristic peak of the fingerprint.

Therefore, the fingerprint of the semi-finished product (such as extract) of the traditional Chinese medicine and the preparation thereof can identify the authenticity of the quality and prove the consistency and stability of the quality among product batches.

The invention provides a method for constructing the fingerprint of the effective part of the traditional Chinese medicine, wherein the method comprises a method for constructing the fingerprint of flavonoid components, polyphenol components and diterpenoid quinone components and a method for constructing alkaloid components.

Preferably, the fingerprint construction method of the flavonoid component, the polyphenol component and the diterpene quinone component comprises the following steps:

(i) the chromatographic column is an octadecyl bonding silica gel column;

(iii) gradient elution;

(iv) the ultraviolet detection wavelength is 250-300 nm; and

In the present invention, the "octadecyl silica gel column" is also referred to as a C18 column, and refers to a reverse phase chromatography column using octadecyl silica gel as a packing. Preferably, the number of theoretical plates of the chromatographic column calculated by baicalin is not less than 1500, preferably not less than 2000, not less than 2500 or not less than 3000.

More preferably, when separating the flavonoid component, the polyphenol component and the diterpene quinone component, the mobile phase is acetonitrile (a) and 0.05 wt% phosphoric acid aqueous solution (B).

More preferably, the gradient elution comprises a continuous process wherein% is the volume percentage relative to the total flow:

generally, when chromatographic peaks are obtained by high performance liquid chromatography, analytical comparison of spectra and data processing methods are known to those skilled in the art, and data processing can be performed, for example, according to the "similarity evaluation system for chromatography fingerprints of chinese medicine" (2012 edition).

Preferably, the alkaloid fingerprint map is constructed by the following sub-steps:

(i) the chromatographic column is an amino bonded silica gel column;

(iii) the elution procedure was isocratic:

(iv) the ultraviolet detection wavelength is 200-230 nm; and

In the present invention, the "amino-bonded silica gel column" refers to a column packed with amino-bonded silica gel. The method of the present invention may use various amino-bonded silica gel columns commonly used in the art, such as a bonded phase obtained by bonding 3-aminopropyltriethoxysilane (3-aminopropyltriethoxysilane), -Si- (CH2)3NH2, etc., to silica gel particles. Preferably, the chromatographic column has a theoretical plate number, calculated as matrine, of not less than 1500, preferably not less than 2000, not less than 2500 or not less than 3000.

Preferably, when the alkaloid component is separated, the mobile phase is acetonitrile-absolute ethyl alcohol-2.8-3.2 wt% phosphoric acid aqueous solution, preferably acetonitrile-absolute ethyl alcohol-3 wt% phosphoric acid aqueous solution.

Preferably, the volume ratio of the mobile phase is acetonitrile: anhydrous ethanol: phosphoric acid water solution 75-85:8-15: 6-12; preferably 75-85:8-12: 6-10.

Quality control method

The invention also provides a quality control method of the effective parts of the traditional Chinese medicine, wherein the method comprises a similar quality control method obtained by analyzing the fingerprint and/or an active ingredient content quality control method.

Specifically, the quality control method for the content of the active ingredients comprises the following steps:

In general, the content of the active ingredient may be obtained by any suitable quantitative detection method in the art, for example, the quantitative detection methods that may be used include, but are not limited to: high performance liquid chromatography, mass spectrometry, liquid chromatography-mass spectrometry, etc.

Preferably, the content of the active ingredient is obtained by detecting with a high performance liquid chromatograph and a diode array detector, for example, by using the above fingerprint spectrum construction method. When a certain active ingredient is quantified, it is preferable to use the absorption peak at its maximum ultraviolet absorption (. lamda.max. + -. 5nm) as a basis for quantification.

In general, the methods of quantification and data analysis of the above-described detection methods are known to those skilled in the art. Generally, peak assignment and active ingredient content can be determined by a control. For example, commonly used quantification methods include, but are not limited to: a standard curve method (external standard method, internal standard method), an external standard one-point method, or a combination thereof.

The quality control method further comprises the following steps: (c) and (C) when the determination result in the step (b) is unqualified and the closest end value C0 of the content C1 of the corresponding active ingredient exceeding the standard content range in the Chinese medicinal effective part and the corresponding standard content range meets | C1-C0|/C0 is less than or equal to 10%, adjusting the composition of the Chinese medicinal effective part to be qualified.

Specifically, the similarity-based quality control method includes the steps of:

the method comprises the following steps:

(Z1) constructing the fingerprint of the effective part of the traditional Chinese medicine by using the method;

Preferably, the fingerprint comprises fingerprints and/or alkaloids fingerprints of the flavonoid components, the polyphenol components and the diterpenoid quinones.

Experiments prove that the method has good reproducibility and stability, the sample similarity result can be more than or equal to 0.99, and the similarity can be more than or equal to 0.95 in the application process, so that the quality control requirement can be met.

More preferably, the two quality control methods of the similarity can be combined to achieve comprehensive and accurate quality control.

Combination of active ingredients, pharmaceutical compositions and uses thereof

The invention also provides a combination of active ingredients: the combination comprises the following active ingredients of group a: (A1) 14.5 to 20.5 weight portions of baicalin; (A2) 9-15 parts of salvianolic acid B; (A3) 0.3-0.7 parts by weight of rosmarinic acid; and (A4) 1.5-2.8 parts by weight of matrine.

In another preferred embodiment, the content of other substances from scutellaria, perilla stem, sophora flavescens and salvia miltiorrhiza in the combination of the active ingredients is less than 20 wt%, less than 10% or 5%.

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a combination of active ingredients as described above; and a pharmaceutically acceptable carrier.

The combinations and pharmaceutical compositions of active ingredients of the present invention are useful for: (a) treating or preventing cough after cold; and/or (b) antibacterial and/or treatment of bacterial infections; (c) preventing and/or treating bronchitis; (d) clearing heat; (e) anti-inflammatory; (f) inhibition of capillary permeability; (g) relieving pain; (h) relieving cough; and/or (i) expectorant. See, e.g., chinese applications 201911033104.1, 201410229647.1, the entire contents of which are incorporated herein by reference.

As used herein, the term "therapeutically effective amount" refers to any amount of a drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as manifested by a decrease in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic phases of the disease, or the prevention of a disorder or disability resulting from the disease. The "therapeutically effective dose" of the drug of the present invention also includes a "prophylactically effective dose", which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or suffering from a recurrence of a disease, inhibits the development or recurrence of the disease. Typically, the pharmaceutical composition contains 1-2000mg of the active ingredient combination/dose according to the invention, more preferably 10-500mg of the active ingredient combination/dose according to the invention. Preferably, said "dose" is a capsule or tablet.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and may be any dosage form suitable for mammals. Preferably, the dosage form may comprise tablets, capsules, granules, pills, powders, oral liquids, buccal tablets, or aerosols. The preferred composition is a solid composition from the standpoint of ease of preparation, administration or administration. Oral administration is preferred.

The composition of the present invention may be added with various conventional carriers and/or adjuvants required for preparing different dosage forms, such as filler (e.g. starch, cellulose, dextrin), disintegrant (sodium carboxymethyl starch), lubricant (magnesium stearate), solvent (purified water), cosolvent (ethanol), and coating material. Can be prepared into any common dosage form such as tablet, capsule, granule, capsule, pill, and powder by conventional method.

As used herein, the term "pharmaceutically acceptable carrier" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), buffers, chelating agents, thickeners, pH adjusters, transdermal enhancers, colorants, flavors, stabilizers, antioxidants, preservatives, bacteriostats, pyrogen-free water, etc.

The main advantages of the invention include:

1. the invention provides a quality control method for accurately and comprehensively controlling the quality of active ingredients of Qinsu powder and a preparation thereof.

2. The invention also provides a liquid phase fingerprint construction method of the Qinsu powder and the preparation thereof, wherein the liquid phase separation method has good separation effect and more peak number, can efficiently separate the components of the Qinsu powder, and can obtain more accurate and comprehensive results.

3. The liquid phase fingerprint spectrum of the invention has good reproducibility and high stability, and can be used for comprehensively analyzing and identifying the Qinsu powder and the preparation thereof.

4. The quality control method of the Qin Su powder and the preparation thereof based on the liquid phase fingerprint spectrum can control the product quality on the whole.

5. The invention also provides an active ingredient combination and a pharmaceutical composition, wherein the pharmaceutical composition can replace traditional Chinese medicine extracts to treat diseases, and reduce side effects caused by impurities in the extracts.

The invention is further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Instrument for measuring the position of a moving object

High performance liquid chromatograph: agilent 1260, (fitted with quaternary pump, autosampler, column oven, diode array detector), Agilent ChemStation.

Reagent

Example 1

1.1, prescription

1.2 methods of pharmaceutical preparation and formulation

Taking a scutellaria baicalensis medicinal material, adding 8 times (weight ratio) of 70% ethanol water, heating, refluxing and extracting for 2 times, each time for 2 hours, filtering, recovering ethanol from filtrate under reduced pressure, merging and concentrating the filtrate into an extract with the relative density of 1.05-1.20 (50-60 ℃), and drying under reduced pressure (-0.07-0.1 MPa, less than or equal to 60 ℃) to obtain a dry extract A.

Taking a lightyellow sophora root medicinal material, adding 10 times of 50% ethanol water, heating and refluxing for 2 times, extracting for 2 hours each time, filtering, recovering ethanol from filtrate under reduced pressure, and concentrating to obtain a concentrated solution with the relative density of 1.02-1.12 (50-60 ℃). Diluting the concentrated solution with purified water to obtain a dispersion liquid (containing 0.05kg/L of crude drug), passing the dispersion liquid through a neutral alumina column, eluting with 8 times of 95% ethanol, collecting eluent, recovering ethanol from the eluent under reduced pressure, merging and concentrating the ethanol into an extract with the relative density of 1.05-1.20 (50-60 ℃), and drying under reduced pressure (-0.07-0.1 MPa, less than or equal to 60 ℃) to obtain a dry extract B.

Taking a salvia miltiorrhiza medicinal material, adding 10 times of 90% ethanol water, heating, refluxing and extracting for 2 times, each time for 2 hours, filtering, recovering ethanol from filtrate under reduced pressure, merging and concentrating the filtrate to a concentrated solution with the relative density of 1.05-1.15 (50-60 ℃), adding purified water into the concentrated solution to dilute the concentrated solution to a dispersion solution (containing 0.05kg/L of crude drug), taking the dispersion solution to pass through an AB-8 resin column, eluting with 8 times of 95% ethanol water, collecting eluent, recovering ethanol from the eluent under reduced pressure, merging and concentrating the eluent to an extract with the relative density of 1.05-1.20 (50-60 ℃), and drying under reduced pressure (-0.07-0.1 MPa, at most 60 ℃) to obtain a dry extract C.

Taking perilla seeds and perilla stems, adding 10 times of 80% ethanol water, heating, refluxing and extracting for 2 times, each time for 2 hours, filtering, recovering ethanol from filtrate under reduced pressure, merging and concentrating the ethanol into extract with the relative density of 1.05-1.20 (50-60 ℃), drying under reduced pressure (-0.07-0.1 MPa, less than or equal to 60 ℃) to obtain dry extract D.

Pulverizing the dry extract A, B, C, D, sieving with 60 mesh sieve, and mixing to obtain QINSU powder.

And (3) capsule preparation: mixing the dry extract powder with appropriate amount of starch, wet granulating with 95% ethanol as binder, drying, adding appropriate amount of silica gel micropowder, grading, mixing, and filling to obtain 1000 granules.

Example 2

HPLC fingerprint and similarity analysis

2.1 construction method of HPLC fingerprint of Qinqiu capsule

2.1.1 preparation of test solutions

Grinding the content of Qinsu capsule, precisely weighing about 50mg, placing into 25ml brown measuring flask, adding 75% methanol 20ml, ultrasonic treating for 10min (power 250kw, frequency 50KHz), cooling to room temperature, adding 75% methanol to scale, shaking, filtering with 0.45 μm microporous membrane, and collecting the filtrate.

2.1.2 chromatographic conditions

A Waters Xbridge Shield RP18 column (4.6 mm. times.250 mm, 5 μm) packed with octadecyl-bonded silica gel; mobile phase: acetonitrile (A) -0.05% phosphoric acid water (B) gradient elution, the gradient conditions are shown in Table 1; detection wavelength: 270 nm; flow rate: 0.8 ml/min; column temperature: 30 ℃; operating time: 105 minutes (run 10 minutes at the starting concentration after the end of a single run to equilibrate the column); sample introduction amount: 10 μ L.

TABLE 1 HPLC gradient elution conditions

2.2 fingerprint and similarity analysis

The evaluation of the measurement results of 9 batches of Qinsu capsules is carried out by a traditional Chinese medicine chromatography fingerprint similarity evaluation system (2012 edition), a Qinsu powder fingerprint common mode is generated by adopting a multipoint correction method, and a Qinsu powder control fingerprint (R) is generated by adopting an average value calculation method.

The obtained fingerprint is shown in figure 1, and it can be seen that the HPLC fingerprint of the Qin Su capsule calibrates 33 common peaks, and the resolution between the peaks is very high, which suggests that more accurate and comprehensive results can be obtained when the chromatogram obtained by the method of the present invention is used for data analysis.

Representative peaks are shown in table 2:

TABLE 2 information table of common peaks of Qinsu capsules (batch No. 200101)

In table 2, 13 components in the HPLC fingerprint of the qin su capsule are identified by the control of the control, which are: baicalin, wogonoside, baicalein, wogonin, oroxylin A-7-O-beta-D-glucuronide, trifolio red sandalwood glycoside, kushenketone, luteolin, salvianolic acid B, rosmarinic acid, salvianolic acid A, tanshinone IIA and cryptotanshinone.

The above common mode is taken as a standard, the overall similarity evaluation is carried out on 9 batches of samples, the calculation result is shown in figure 2, and the results show that the similarities of S1-S9 samples and the comparison fingerprint (R) are respectively 0.999, 0.997, 0.998, 0.999, and 0.999, respectively >0.990 (compared with the comparison fingerprint, the similarity >0.95 can meet the quality control requirement), which shows that the fingerprint method established by the invention has high sensitivity, good repeatability, reliable result and stable quality of the detected Qinsu capsules when applied to the quality control of the Qinsu capsules.

Example 3

Quantitative analysis

3.1 methodological validation

3.1.1 chromatographic conditions

Waters Xbridge Shield RP18 column (4.6 mm. times.250 mm, 5 μm); mobile phase: acetonitrile (B) -0.05% phosphoric acid water (C) gradient elution, the gradient conditions are shown in Table 1; detection wavelength: 270nm (baicalin, wogonoside, baicalein, wogonin, cryptotanshinone, tanshinone IIA), 310nm (salvianolic acid B), and 330nm (rosmarinic acid); flow rate: 0.8 mL/min; column temperature: 30 ℃; operating time: 105 minutes (run 10 minutes at the starting concentration after the end of a single run to equilibrate the column).

3.1.2 solution preparation

Preparation of control solutions: taking appropriate amount of baicalin, wogonoside, baicalein, wogonin, salvianolic acid B, rosmarinic acid, cryptotanshinone, and tanshinone IIA reference substances, precisely weighing, and making into standard mother liquor containing 0.75mg, 0.16mg, 0.04mg, 0.02mg, 0.32mg, 0.03mg, 0.01 and 0.01mg per 1mL with methanol.

Preparation of a test solution: taking the product with different filling amount, grinding, taking about 50mg, precisely weighing, placing in a 25mL brown measuring flask, adding 20mL of 75% methanol, performing ultrasonic treatment for 10 minutes (power 250kw, frequency 50KHz), cooling to room temperature, adding 75% methanol to scale, shaking, filtering with 0.45 μm microporous membrane, and taking the subsequent filtrate.

3.1.3 Linear relationship investigation

Precisely sucking appropriate amount of control mother liquor, sequentially diluting with methanol to obtain mixed control solutions 1-5 (shown in Table 3), respectively measuring by HPLC, injecting 20 μ L of sample, and showing standard curve data of each index component in Table 3.

Taking the concentration (X) of the reference substance as a horizontal coordinate and the peak area (Y) as a vertical coordinate, and performing linear regression to obtain a standard curve equation which respectively comprises the following components: baicalin: y 81.404x +96.908(r 1), wogonoside: y 96.778x +61.506(r 1), baicalein: y 128.14x-32.67(r 1), wogonin: y 153.54x +10.49(r 1), salvianolic acid B: y 31.158x-4.2264(r 0.9999), rosmarinic acid: y-74.693 x +3.3339 (r-1), cryptotanshinone: y 121.75x +6.5767(r 1), tanshinone iia: y 140.9x +6.6845(r 0.9999). The results show that baicalin, wogonin, baicalein, wogonin, salvianolic acid B, rosmarinic acid, cryptotanshinone and tanshinone IIA respectively have good linear relationship in the test range.

TABLE 3 Standard Curve data of index components

3.1.4 precision test

Precision in the day: mixing baicalin, wogonoside, baicalein, wogonin, salvianolic acid B, rosmarinic acid, cryptotanshinone, and tanshinone IIA with high, medium, and low concentrations to obtain a reference solution (shown in Table 4), repeating the sample injection for 6 times, each sample injection for 20 μ L, and calculating peak area RSD. Calculated baicalin, wogonoside, baicalein, wogonin, salvianolic acid B, rosmarinic acid, cryptotanshinone and tanshinone IIA RSD are all less than 3%, which shows that the precision in the day is good.

Precision in the daytime: the above control solution was injected 1 time a day, 20 μ L each time, and measured for three consecutive days to calculate the peak area RSD. The results are shown in table 5, where the RSD for each control was calculated to be less than 3%, indicating good precision between days of the sample.

TABLE 4 results of in-day precision measurement (n ═ 6)

TABLE 5 results of day precision measurement (n ═ 3)

3.1.5 stability test

The test solution of Qinsu capsule (batch No. 190601) was injected into each sample at a volume of 10 μ L for 0, 2, 4, 8, 16, 24 and 48h, respectively, and then measured. The results are shown in Table 6, and the RSDs of the peak areas of baicalin, wogonoside, baicalein, wogonin, salvianolic acid B, rosmarinic acid, cryptotanshinone and tanshinone IIA are respectively 0.13%, 2.43%, 1.33%, 0.12%, 0.71%, 1.14%, 0.49% and 0.63%, which indicates that the test solution is in a stable state within 48 hours.

TABLE 6 measurement results (peak area) of component stability for each index

3.1.6 repeatability test

Weighing 6 parts of Diqin Su capsule (lot number: 190601) 50mg each, preparing according to the preparation method of test solution, measuring, and injecting 10 μ L. The results are shown in table 7, and the contents of baicalin, wogonoside, baicalein, wogonin, salvianolic acid B, rosmarinic acid, cryptotanshinone and tanshinone IIA are respectively 185.03, 35.97, 9.83, 4.14, 96.81, 6.87, 3.09 and 4.28 mg/g; RSD is 0.43%, 2.58%, 0.27%, 0.30%, 1.25%, 2.03%, 0.26% and 0.96% respectively, which indicates that the method has good repeatability.

TABLE 7 repeatability test results (content mg/g) of each index component

3.1.7 sample recovery test

Precisely weighing about 25mg (lot number: 190601) of QINSU Capsule, precisely weighing, respectively weighing appropriate amount of baicalin, wogonoside, baicalein, wogonin, salvianolic acid B, rosmarinic acid, cryptotanshinone, and tanshinone IIA reference substance, precisely weighing, dissolving in 75% methanol, and making into mixed reference substance solutions with concentrations of 2.0369, 0.4157, 0.1603, 0.0534, 1.0024, 0.1089, 0.0379, and 0.0494 mg/mL. Adding mixed reference substance solutions with different volumes into 50%, 100% and 150%, respectively, preparing according to the preparation method of the sample solution, injecting 10 μ L, measuring content, and calculating sample recovery rate and average sample recovery rate. The results are shown in table 8, the average recovery rates of baicalin, wogonoside, baicalein, wogonin, salvianolic acid B, rosmarinic acid, cryptotanshinone and tanshinone iia in the qinsu capsule are respectively 99.65%, 99.71%, 103.31%, 99.65%, 100.04%, 99.60%, 99.17% and 101.45%, and the RSD is respectively 0.37%, 1.20%, 0.75%, 1.41%, 2.26%, 0.98%, 1.44% and 2.26%, which indicates that the recovery rate is good.

TABLE 8 sample application recovery test (n ═ 9)

The quantitative determination results are summarized in table 16, wherein baicalin, wogonoside, baicalein, wogonin, salvianolic acid B, rosmarinic acid, cryptotanshinone and tanshinone iia are calculated by an external standard curve method, and the contents of oroxylin-7-O- β -D-glucuronide, trifolio pterocarcinoside, kurarinone, luteolin and salvianolic acid a are calculated by an external standard one-point method, wherein the percentage content of the active ingredients is calculated by the dry weight of the extract (scutellaria baicalensis).

In conclusion, the HPLC content determination method established by the invention has the advantages of accuracy, good reproducibility, high precision and strong specificity, and is suitable for content determination of flavonoid components, polyphenol components and diterpene quinine components in the Qinsu powder and the preparation thereof.

Example 4

HPLC fingerprint spectrum of alkaloid component

4.1 preparation of test solutions

Taking the content of the Qinsu capsule, grinding, taking about 0.1g, precisely weighing, placing in a conical flask with a plug, adding 0.5ml of concentrated ammonia test solution (25-28 wt% concentrated ammonia water), precisely adding 25ml of dichloromethane, shaking uniformly, sealing the plug, weighing, ultrasonically treating (with the power of 250W and the frequency of 50kHz) for 10 minutes, cooling, weighing again, complementing the lost weight with dichloromethane, shaking uniformly, filtering, precisely weighing 10ml of subsequent filtrate, evaporating the filtrate to dryness, dissolving residues by adding a proper amount of absolute ethyl alcohol, transferring to a 10ml measuring flask, adding the absolute ethyl alcohol to the scale, shaking uniformly, and filtering through a 0.45 mu m microporous filter membrane to obtain the medicine.

4.2 chromatographic conditions

Agilent ZORBAX NH₂(4.6 mm. times.250 mm, 5 μm, filler amino-bonded silica gel); acetonitrile-absolute ethyl alcohol-3% phosphoric acid solution (81: 11: 8) is used as a mobile phase; detection wavelength: 210 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃; operating time: 35min, sample size 10. mu.L.

4.3. Fingerprint and similarity analysis

The same fingerprint generation method and similarity analysis method as in example 2 were used, and the results are shown in fig. 3 and 4.

Representative peaks are shown in table 9:

TABLE 9 common Peak Table of Qinsu capsules (batch No. 190601)

The results show that the similarity of the S1-S9 samples and the reference products is respectively 0.997, 0.991, 0.995, 1.000, 0.997, 0.992, 0.998 and 0.998, which are all more than 0.990; the reference substance is compared with 4 components and totally identified as follows: matrine, oxymatrine, sophocarpine, and oxysophoridine.

Example 5

Quantitative analysis of alkaloid components

5.1 methodological validation

5.1.1 preparation of control solutions

Taking appropriate amount of matrine, sophoridine and oxymatrine as reference substances, precisely weighing, and making into standard mother liquor containing 1.2mg, 1.3mg and 1.1mg per 1mL respectively with acetonitrile-anhydrous ethanol (80:20) mixed solution.

5.1.2 preparation of test solutions

Taking the product with different loading amounts, grinding, taking about 0.1g, precisely weighing, placing in a conical flask with a plug, adding 0.5ml of concentrated ammonia test solution, precisely adding 25ml of dichloromethane, shaking uniformly, sealing the plug, weighing, ultrasonically treating (power 250W, frequency 50kHz) for 10min, cooling, weighing again, supplementing the lost weight with dichloromethane, shaking uniformly, filtering, precisely weighing 10ml of subsequent filtrate, evaporating the filtrate to dryness, dissolving the residue with appropriate amount of anhydrous ethanol, transferring to a 10ml measuring flask, adding anhydrous ethanol to scale, shaking uniformly, and filtering with a 0.45 mu m microporous membrane to obtain the final product.

5.1.3 chromatographic conditions and System Adaptation

Amino-bonded silica gel as filler [ Agilent ZORBAX NH ]₂](ii) a Acetonitrile-absolute ethyl alcohol-3% phosphoric acid solution (81: 11: 8) is used as a mobile phase; detection wavelength: 210 nm; flow rate: 1.0 mL/min; column temperature: 30 ℃; operating time: and (5) 35 min.

5.1.4 Linear relationship investigation

Precisely sucking a proper amount of the stock solution of the reference substance, sequentially diluting with acetonitrile-absolute ethanol (80:20) mixed solution to obtain mixed reference substance solutions 1-6 shown in Table 10, respectively measuring, and injecting 10 μ L of the sample.

Taking the concentration (X) of the reference substance as a horizontal coordinate and the peak area (Y) as a vertical coordinate, and performing linear regression to obtain a standard curve equation which respectively comprises the following components: matrine: y 14927x +26.36(r 1), sophoridine: y 12293x +5.20(r 0.9999), oxymatrine: y 15380x +10.40(r 0.9998). The data of the standard curve of each index component are shown in the table 10, and the result shows that the matrine, the sophoridine and the oxymatrine respectively have good linear relation in the measured range.

TABLE 10 Standard Curve data of index components

5.1.5 precision test

As shown in Table 11, the reference solutions of matrine, sophoridine and oxymatrine with high, medium and low concentrations were sampled 6 times, 10 μ L each time, and the peak area RSD was calculated.

Injecting sample for 1 time every day, injecting 10 mu L each time, measuring for three consecutive days, and calculating peak area RSD.

TABLE 11 results of in-day precision measurement (n ═ 6)

Table 12 results of day precision measurement (n ═ 3)

As is clear from tables 11 and 12, the method for measuring an alkaloid of the present invention has excellent daily precision and daytime precision.

5.1.6 stability test

Sample solutions of QINSU Capsule (lot: 190601) were injected into each sample solution at 0, 4, 8, 16, 24 and 48 hr, respectively, and 10 μ L of the sample solution was injected.

The results are shown in table 13, and the RSDs of the peak areas of matrine, sophoridine and oxymatrine were measured to be 1.0%, 2.7% and 2.6%, respectively, which indicates that the test solution was in a stable state within 48 hours, and the content of each index component did not change much.

TABLE 13 results of stability determination (peak area)

5.1.7 repeatability test

The test solutions were prepared by accurately weighing 6 parts of Geranium strictipes capsules (lot No. 0050101-one 190601), 0.1g each, and measuring 10. mu.L each.

As shown in Table 14, the matrine, sophoridine and oxymatrine contents were determined to be 21.31, 5.48 and 6.36mg/g, respectively; RSD is 0.5%, 2.2% and 2.3%, respectively, which indicates that the method has good repeatability.

TABLE 14 repeatability test results (content mg/g)

5.1.8 sample recovery test

Taking 0.05g of Qinsu capsule (batch number: 0050101-190601, matrine, sophoridine and oxymatrine respectively with content of 21.31, 5.48 and 6.36mg/g), precisely weighing, taking appropriate amount of matrine, sophoridine and oxymatrine respectively, precisely weighing, dissolving with methanol, and making into mixed control solution with concentration of 0.5263, 0.1339 and 0.1435 mg/mL. Adding mixed reference substance solutions with different concentrations according to 50%, 100% and 150%, respectively, preparing according to the preparation method of the sample solution, injecting 10 μ L, measuring content, and calculating sample recovery rate and average sample recovery rate.

As a result, as shown in Table 15, the average recovery rate of total alkaloids in Qinsu capsule was 96.34%, and the RSD was 0.67%.

TABLE 15 sample application recovery test (n ═ 9)

The quantitative determination results are summarized in table 16, wherein the content of matrine and oxymatrine is calculated by an external standard curve method; the sophocarpine and oxysophoridine are calculated by external standard one-point method, wherein the percentage of the active ingredients is calculated by dry weight of the extract (Qin Su powder).

In conclusion, the HPLC content determination method established by the invention has the advantages of accuracy, good reproducibility, high precision and strong specificity, and is suitable for content determination of alkaloid components in the Qinsu powder and the preparation thereof.

Proved by experiments, the Qinsu capsules of each batch tested in the embodiment have good drug effects in the aspects of treating or preventing cough after cold, resisting bacteria and/or treating bacterial infection, preventing and/or treating bronchitis, relieving fever, resisting inflammation, inhibiting capillary permeability, easing pain, relieving cough and eliminating phlegm, and meet the drug requirement.

Comparative example 1

Essentially the same as example 2 except that the mobile phase was acetonitrile-0.2% formic acid water.

The results are shown in fig. 5, when acetonitrile-0.2% formic acid water mobile phase system is used to carry out gradient elution on the sample solution, the phenolic acid component is greatly influenced by the mobile phase system, and the phenomenon that retention time is unstable or the separation degree is poor often occurs (for example, the separation degree of the salvianolic acid B as the index component in fig. 5A is only 1.4, and does not reach baseline separation, and the separation degree of salvianolic acid B as the index component in fig. 5B is 1.6-1.5, and achieves baseline separation); and after an acetonitrile-phosphoric acid water mobile phase system is adopted, index components obtain better separation degree, peak shape and stable retention time.

Discussion of the related Art

The invention combines HPLC-DAD content analysis and traditional Chinese medicine fingerprint spectrum technology to successfully establish the HPLC fingerprint spectrum of the extract of the traditional Chinese medicine formula containing perilla fruit, perilla stem, radix sophorae flavescentis, scutellaria baicalensis and salvia miltiorrhiza and the preparation thereof, can comprehensively evaluate various components in the medicine such as flavonoids, polyphenols, diterpene quinines and alkaloids simultaneously, has high stability and stable and comprehensive result, can quantitatively analyze the main active ingredients by using the obtained liquid phase spectrum, and is very suitable for the overall quality control of the scutellaria baicalensis and the preparation thereof.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A quality control method of effective parts of traditional Chinese medicine,

the method comprises the following steps:

2. The quality control method according to claim 1, wherein the quality control method further comprises the steps of:

3. The quality control method according to claim 1, wherein the step (a) comprises the steps of:

wherein the high performance liquid chromatography detection comprises:

(i) the chromatographic column is an octadecyl bonding silica gel column;

(iii) gradient elution;

(i) the chromatographic column is an amino bonded silica gel column;

(iii) the elution procedure is isocratic elution;

4. The quality control method according to claim 3, wherein in the step (a3-1), the gradient elution includes a continuous process in which% is a volume percentage with respect to a total flow rate:

5. a method for constructing a fingerprint of an effective part of a traditional Chinese medicine,

the fingerprint map construction method comprises the following steps:

(i) the chromatographic column is an octadecyl bonding silica gel column;

(iii) gradient elution;

(iv) the ultraviolet detection wavelength is 250-300 nm; and

6. The fingerprint construction method according to claim 5, wherein the elution procedure of the gradient elution comprises the following successive procedures, wherein% is the volume percentage relative to the total flow rate:

7. a quality control method of effective parts of traditional Chinese medicine,

the method comprises the following steps:

(Z1) constructing a fingerprint of the effective part of the chinese medicine using the method of claim 5;

8. A combination of active ingredients, characterized in that the combination comprises the following active ingredients of group a:

9. a pharmaceutical composition comprising a therapeutically effective amount of a combination of active ingredients according to claim 8; and a pharmaceutically acceptable carrier.

10. Use of a combination of active ingredients according to claim 8 or a pharmaceutical composition according to claim 9 for the preparation of a medicament for: (a) treating or preventing cough after cold; and/or (b) antibacterial and/or treatment of bacterial infections; (c) preventing and/or treating bronchitis; (d) clearing heat; (e) anti-inflammatory; (f) inhibition of capillary permeability; (g) relieving pain; (h) relieving cough; and/or (i) expectorant.