CN109288907B - Yandan capsule, preparation method, detection method and new application thereof - Google Patents

Abstract

The invention relates to the field of traditional Chinese medicines, in particular to a delavay capsule, a preparation method and a detection method thereof and new application thereof. The Yandan capsule is prepared from the following medicinal raw materials in parts by weight: 4-6 parts of salvia miltiorrhiza, 4-6 parts of corydalis tuber processed with vinegar, 2-4 parts of trogopterus dung, 4-6 parts of snakegourd fruit, 2-4 parts of frankincense processed with vinegar, 2-4 parts of white paeony root, 2-4 parts of fructus aurantii and 2-4 parts of radix bupleuri. The invention provides a method for simultaneously measuring the content of active ingredients of a delavay capsule by adopting a high performance liquid chromatography, and also provides a new application of the delavay capsule in preparing medicines for treating pulmonary heart disease.

Description

Yandan capsule, preparation method, detection method and new application thereof

Technical Field

The invention relates to the field of traditional Chinese medicines, in particular to a delavay capsule, a preparation method and a detection method thereof and new application thereof.

Background

Pulmonary heart disease (pulmonary heart disease for short) is mainly caused by pulmonary hypertension due to bronchial-pulmonary tissue or pulmonary artery vasculopathy. According to the onset and duration of disease, it can be divided into acute and chronic types. Clinically, the disease is slow in development, and is mainly characterized by progressive lung and heart failure and other organ damage except various symptoms and signs of the original lung and chest diseases.

In the prior art, some traditional Chinese medicine compositions for treating pulmonary heart disease are disclosed, for example:

the patent name: an application of a Chinese medicinal composition in preparing medicine for treating pulmonary heart disease is disclosed in application number: 201810677806.2, filing date: 2018-06-27, which discloses an application of a traditional Chinese medicine composition in preparing a medicine for treating pulmonary heart disease, wherein the traditional Chinese medicine composition is prepared from the following raw material medicines in parts by weight: 5-15 parts of antelope horn or 50-150 parts of goat horn, 10-60 parts of fritillaria ussuriensis, 15-60 parts of rheum officinale, 7-30 parts of scutellaria baicalensis, 7-30 parts of lapis chlorite, 10-50 parts of gypsum, 5-20 parts of artificial bezoar and 15-60 parts of liquorice.

The patent name: a traditional Chinese medicine composition for treating chronic pulmonary heart disease in stationary phase is disclosed in application number: 201410857156.1, filing date: 2014-12-29, which discloses a traditional Chinese medicine composition for treating chronic stable-phase pulmonary heart disease, comprising monkshood, cassia twig, cinnamon, herba epimedii, ginger, radix astragali, semen lepidii, earthworm, angelica, poria cocos, schisandra chinensis, bighead atractylodes rhizome, dried orange peel, salvia miltiorrhiza and fructus psoraleae.

The patent name: a Chinese medicinal composition for treating chronic pulmonary heart disease and its preparation method, application number: 201711259044.6, filing date: 2017-12-02, which discloses a traditional Chinese medicine composition for treating chronic pulmonary heart disease and a preparation method thereof, wherein the traditional Chinese medicine composition is prepared from the following traditional Chinese medicine raw materials in parts by weight: 6-9 parts of ephedra, 3-6 parts of asarum, 10-15 parts of schizonepeta, 10-20 parts of divaricate saposhnikovia root, 10-20 parts of dried ginger, 10-25 parts of schisandra chinensis, 20-30 parts of honeysuckle, 10-15 parts of fructus forsythiae, 20-30 parts of gypsum, 10-15 parts of scutellaria baicalensis, 10-15 parts of cortex mori radicis, 10-20 parts of reed rhizome, 10-15 parts of semen coicis, 8-15 parts of semen benincasae, 10-20 parts of almond, 15-20 parts of radix scrophulariae, 10-15 parts of caulis bambusae in taeniam and 6-10 parts of liquorice.

The patent name: a traditional Chinese medicine for treating chronic pulmonary heart disease, application No.: 201110397612.5, filing date: 2011-12-02, which discloses a traditional Chinese medicine for treating chronic pulmonary heart disease. The traditional Chinese medicine comprises the following raw materials in parts by weight: 10-15 parts of ginseng, 10-15 parts of radix ophiopogonis, 10-15 parts of schisandra chinensis, 10-15 parts of bighead atractylodes rhizome, 10-15 parts of sappan wood, 5-8 parts of cassia twig, 10-15 parts of astragalus membranaceus, 10-15 parts of salvia miltiorrhiza, 10-15 parts of radix aconiti carmichaeli, 8-12 parts of gecko, 10-15 parts of epimedium, 10-15 parts of Chinese yam, 10-15 parts of bitter apricot seed, 10-15 parts of fructus psoraleae, 10-15 parts of semen lepidii, 10-15 parts of radix stemonae, 10-15 parts of poria cocos, 10-15 parts of peach kernel, 10-15 parts of flos farae, 5-8 parts of honey-fried licorice root, 10-15 parts of earthworm and 10-15 parts of.

The patent name: a Chinese medicinal composition for treating chronic pulmonary heart disease and its preparation method, application number: 201510026679.6, filing date: 2015-01-15, which discloses a traditional Chinese medicine composition for treating chronic pulmonary heart disease, which is prepared from ephedra, cassia twig, dried ginger, asarum, pinellia ternate, schisandra chinensis, white peony root, blackberry lily, semen lepidii, astragalus mongholicus, angelica sinensis, rhodiola rosea, gecko, agilawood, peach kernel, safflower carthamus, ligusticum wallichii, salvia miltiorrhiza, spina gleditsiae, codonopsis pilosula, fried bighead atractylodes rhizome, poria cocos and honey-fried licorice root according to a certain weight ratio.

Although some medicines for treating the pulmonary heart disease exist in the prior art, the effect is not ideal, the invention provides the traditional Chinese medicine composition, the medicine can effectively treat the pulmonary heart disease, the curative effect is exact, and a brand new choice is provided for treating the pulmonary heart disease.

Research on Wangyilong of salt city traditional Chinese medicine institute, Yuanjuan of salt city medicine inspection institute, Zhahaixin of Min Hospital department of medicine, Jianhu county, Liuyun of Ji City Hospital pharmaceutical institute and the like establishes a method for measuring contents of protocatechualdehyde, paeoniflorin and hesperidin in Yandan capsules by an HPLC method, the method adopts a high performance liquid chromatography method to simultaneously measure contents of protocatechualdehyde, paeoniflorin and hesperidin in Yandan capsules, and Waters CAPCELL PAK C is adopted to measure the contents of protocatechualdehyde, paeoniflorin and hesperidin in Yandan capsules₁₈Chromatographic column (150mm × 4.6mm,5 μm), mobile phase acetonitrile-0.1% phosphoric acid, test wavelength 230nm, column temperature 25 deg.C. The results show that protocatechualdehyde, paeoniflorin and hesperidin respectively have good linear relations in the ranges of 0.007728-0.4508 mu g, 0.06462-4.308 mu g and 0.06984-3.492 mu g, and the average recovery rates are 99.00%, 98.92% and 98.65%. The method is simple, accurate, and has good repeatability, and can be used as quality control method of YANDAN Capsule. See: journal of liberty military medicine 2015 12 th page 104-108.

The danshensu contained in the salvia miltiorrhiza in the delavay capsule, the tetrahydropalmatine contained in the rhizoma corydalis processed with vinegar and the chrysoeriol contained in the trichosanthes kirilowii maxim are all important active ingredients for treating the pulmonary heart disease in the delavay capsule, so that the applicant carries out repeated experimental research on the essential active ingredients, and provides and establishes a method for simultaneously detecting the contents of the danshensu, the tetrahydropalmatine and the chrysoeriol by adopting an HPLC (high performance liquid chromatography).

Disclosure of Invention

Based on the technical problems in the background art, the invention provides a delavay capsule, a preparation method and a detection method thereof and new application.

The invention aims to provide a delavay capsule and a preparation method thereof.

Another object of the present invention is to provide a method for detecting Yandan capsules.

The invention also provides a new pharmaceutical application of the Yandan capsule.

The purpose of the invention is realized by the following modes:

the delavay pill capsule is prepared from the following medicinal raw materials in parts by weight: 4-6 parts of salvia miltiorrhiza, 4-6 parts of corydalis tuber processed with vinegar, 2-4 parts of trogopterus dung, 4-6 parts of snakegourd fruit, 2-4 parts of frankincense processed with vinegar, 2-4 parts of white paeony root, 2-4 parts of fructus aurantii and 2-4 parts of radix bupleuri.

The delavay pill capsule is preferably prepared from the following medicinal raw materials in parts by weight: 5 parts of salvia miltiorrhiza, 4.8 parts of corydalis tuber processed with vinegar, 3 parts of trogopterus dung, 4.8 parts of snakegourd fruit, 3 parts of frankincense processed with vinegar, 3 parts of white paeony root, 3 parts of fructus aurantii and 3 parts of radix bupleuri.

The preparation method of the Yandan capsule comprises the following medicinal raw materials in parts by weight: 4-6 parts of salvia miltiorrhiza, 4-6 parts of corydalis tuber processed with vinegar, 2-4 parts of trogopterus dung, 4-6 parts of snakegourd fruit, 2-4 parts of frankincense processed with vinegar, 2-4 parts of white paeony root, 2-4 parts of fructus aurantii and 2-4 parts of radix bupleuri; the preparation method comprises the following steps:

(1) mixing vinegar processed Olibanum, fructus Aurantii, and bupleuri radix, pulverizing into fine powder, and keeping;

(2) taking the salvia miltiorrhiza, adding 1-3 times of 80-90% ethanol, performing reflux extraction for 1-3 times, each time for 1-2 hours, combining extracting solutions, filtering, recovering ethanol from filtrate until no ethanol smell exists, obtaining clear paste, and reserving for later use, wherein the obtained dregs are reserved for later use;

(3) mixing snakegourd fruit, rhizoma corydalis processed with vinegar, trogopterus dung and radix paeoniae alba with the dregs obtained in the step (2), adding water for decocting for 1-3 times, adding 8-12 times of water for decocting for 1-3 h each time, mixing decoctions, filtering, concentrating the filtrate into an extract, and reserving for later use;

(4) and (3) taking the fine powder obtained in the step (1), the clear paste obtained in the step (2) and the extract obtained in the step (3), mixing uniformly, drying at low temperature, crushing into fine powder, sieving and encapsulating to obtain the capsule.

The preparation method of the delavay pills comprises the following steps of: 5 parts of salvia miltiorrhiza, 4.8 parts of corydalis tuber processed with vinegar, 3 parts of trogopterus dung, 4.8 parts of snakegourd fruit, 3 parts of frankincense processed with vinegar, 3 parts of white paeony root, 3 parts of fructus aurantii and 3 parts of radix bupleuri; the preparation method is preferably as follows:

(1) mixing vinegar processed Olibanum, fructus Aurantii, and bupleuri radix, pulverizing into fine powder, and keeping;

(2) extracting Saviae Miltiorrhizae radix with 2 times of 85% ethanol under reflux for 2 times (each time for 1.5 hr), mixing extractive solutions, filtering, recovering ethanol from filtrate until there is no ethanol smell to obtain fluid extract, and reserving the residue;

(3) mixing fructus Trichosanthis, rhizoma corydalis processed with vinegar, Oletum Trogopterori, and radix Paeoniae alba with the residue obtained in step (2), decocting in water for 2 times, each time adding 10 times of water, decocting for 2 hr, mixing decoctions, filtering, concentrating the filtrate to obtain extract, and keeping;

(4) and (3) taking the fine powder obtained in the step (1), the clear paste obtained in the step (2) and the extract obtained in the step (3), mixing uniformly, drying at low temperature, crushing into fine powder, sieving and encapsulating to obtain the capsule.

A detection method of a Yandan capsule is characterized in that the Yandan capsule is prepared from the following medicinal raw materials in parts by weight: 5 parts of salvia miltiorrhiza, 4.8 parts of corydalis tuber processed with vinegar, 3 parts of trogopterus dung, 4.8 parts of snakegourd fruit, 3 parts of frankincense processed with vinegar, 3 parts of white paeony root, 3 parts of fructus aurantii and 3 parts of radix bupleuri; the Yandan capsule is prepared by the following steps:

(1) mixing vinegar processed Olibanum, fructus Aurantii, and bupleuri radix, pulverizing into fine powder, and keeping;

(2) extracting Saviae Miltiorrhizae radix with 2 times of 85% ethanol under reflux for 2 times (each time for 1.5 hr), mixing extractive solutions, filtering, recovering ethanol from filtrate until there is no ethanol smell to obtain fluid extract, and reserving the residue;

(3) mixing fructus Trichosanthis, rhizoma corydalis processed with vinegar, Oletum Trogopterori, and radix Paeoniae alba with the residue obtained in step (2), decocting in water for 2 times, each time adding 10 times of water, decocting for 2 hr, mixing decoctions, filtering, concentrating the filtrate to obtain extract, and keeping;

(4) mixing the fine powder obtained in step (1), the fluid extract obtained in step (2) and the extract obtained in step (3), drying at low temperature, pulverizing into fine powder, sieving, and encapsulating to obtain capsule;

the contents of danshensu, tetrahydropalmatine and chrysoeriol in the delavay salviae miltiorrhizae capsule by adopting the high performance liquid chromatography comprise the following steps:

(1) chromatographic conditions are as follows: a chromatographic column: c₁₈Column, mobile phase: acetonitrile-0.15 mol.L^-1Sodium dihydrogen phosphate aqueous solution, gradient elution, elution order is as follows: from the beginning to 5min, acetonitrile was maintained at 15%, 0.15 mol.L^-1The sodium dihydrogen phosphate aqueous solution was kept at 85%; from 6min to 15min, the acetonitrile linearly increased from 15% to 25%, 0.15 mol.L^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 85% to 75%; from 16min to 25min, the acetonitrile linearly increased from 25% to 30%, 0.15 mol.L^-1The sodium dihydrogen phosphate aqueous solution linearly decreased from 75% to 70%; from 26min to 35min, the acetonitrile linearly increased from 30% to 45%, 0.15 mol.L^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 70% to 55%; detection wavelength: the detection wavelength of 0-15 min is 285nm, and the method is used for detecting the tanshinol; the detection wavelength of 16-25 min is 305nm, and the detection is used for detecting tetrahydropalmatine; the detection wavelength of 26-35 min is 328nm, and the detection wavelength is used for detecting chrysoeriol; flow rate: 0.5 to 1.5 mL/min^-1(ii) a Column temperature: 30-35 ℃; sample introduction amount: 5-20 mu L;

(2) preparation of control solutions: accurately weighing danshensu and danyan respectivelyAdding appropriate amount of each of the reference substances of the huosoxisu and the chrysoeriol into methanol to prepare the reference substances with the mass concentration of 150.0-250.0 mu g/mL^-1，100.0～200.0μg·mL^-1，250.0～350.0μg·mL^-1The control solution of (4);

(3) preparation of a test solution: taking a capsule content sample, precisely weighing 4.00-6.00 g, placing the sample in a 50mL conical flask with a plug, adding 30-50 mL of methanol, weighing the mass after sealing, refluxing and extracting for 0.5-1.5 h, cooling, supplementing the loss reduction mass with methanol, filtering with a 0.45 mu m microporous membrane, and taking a subsequent filtrate to obtain a test solution;

(4) and (3) determination: precisely sucking 5-20 μ L of each of the reference solution and the sample solution, and injecting into a high performance liquid chromatograph for measurement.

The detection method of the Yandan capsule adopts the contents of danshensu, tetrahydropalmatine and chrysoeriol in the Yandan capsule of the high performance liquid chromatography, and the preferable steps are as follows:

(1) chromatographic conditions are as follows: a chromatographic column: wondasil C₁₈Chromatographic column, specification: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-0.15 mol.L^-1Sodium dihydrogen phosphate aqueous solution, gradient elution, elution order is as follows: from the beginning to 5min, acetonitrile was maintained at 15%, 0.15 mol.L^-1The sodium dihydrogen phosphate aqueous solution was kept at 85%; from 6min to 15min, the acetonitrile linearly increased from 15% to 25%, 0.15 mol.L^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 85% to 75%; from 16min to 25min, the acetonitrile linearly increased from 25% to 30%, 0.15 mol.L^-1The sodium dihydrogen phosphate aqueous solution linearly decreased from 75% to 70%; from 26min to 35min, the acetonitrile linearly increased from 30% to 45%, 0.15 mol.L^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 70% to 55%; detection wavelength: the detection wavelength of 0-15 min is 285nm, and the method is used for detecting the tanshinol; the detection wavelength of 16-25 min is 305nm, and the detection is used for detecting tetrahydropalmatine; the detection wavelength of 26-35 min is 328nm, and the detection wavelength is used for detecting chrysoeriol; flow rate: 1.0 mL/min^-1(ii) a Column temperature: at 32 ℃; sample introduction amount: 10 mu L of the solution;

(2) preparation of control solutions: accurately weighing danshensu and corydalis B respectivelyAdding appropriate amount of reference substances including phytin and chrysoeriol into methanol to obtain reference substances with mass concentration of 200.0 μ g/mL^-1，150.0μg·mL^-1，300.0μg·mL^-1The control solution of (4);

(3) preparation of a test solution: taking a capsule content sample, precisely weighing 5.00g, placing in a 50mL conical flask with a plug, adding 50mL of methanol, weighing the quality after sealing, refluxing and extracting for 1h, cooling, supplementing the loss mass with methanol, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain a sample solution;

(4) and (3) determination: precisely sucking 10 μ L of each of the reference solution and the sample solution, and injecting into high performance liquid chromatograph for measurement.

An application of a delavay pill capsule in preparing a medicament for treating pulmonary heart disease is disclosed, wherein the delavay pill capsule is prepared from the following medicinal raw materials in parts by weight: 5 parts of salvia miltiorrhiza, 4.8 parts of corydalis tuber processed with vinegar, 3 parts of trogopterus dung, 4.8 parts of snakegourd fruit, 3 parts of frankincense processed with vinegar, 3 parts of white paeony root, 3 parts of fructus aurantii and 3 parts of radix bupleuri; the Yandan capsule is prepared by the following steps:

(1) mixing vinegar processed Olibanum, fructus Aurantii, and bupleuri radix, pulverizing into fine powder, and keeping;

(2) extracting Saviae Miltiorrhizae radix with 2 times of 85% ethanol under reflux for 2 times (each time for 1.5 hr), mixing extractive solutions, filtering, recovering ethanol from filtrate until there is no ethanol smell to obtain fluid extract, and reserving the residue;

(3) mixing fructus Trichosanthis, rhizoma corydalis processed with vinegar, Oletum Trogopterori, and radix Paeoniae alba with the residue obtained in step (2), decocting in water for 2 times, each time adding 10 times of water, decocting for 2 hr, mixing decoctions, filtering, concentrating the filtrate to obtain extract, and keeping;

(4) mixing the fine powder obtained in step (1), the fluid extract obtained in step (2) and the extract obtained in step (3), drying at low temperature, pulverizing into fine powder, sieving, and encapsulating to obtain capsule;

the contents of danshensu, tetrahydropalmatine and chrysoeriol in the delavay salviae miltiorrhizae capsule by adopting the high performance liquid chromatography comprise the following steps:

(1) chromatographic conditions are as follows: a chromatographic column: wondasil C₁₈Chromatographic column, specification: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-0.15 mol.L^-1Sodium dihydrogen phosphate aqueous solution, gradient elution, elution order is as follows: from the beginning to 5min, acetonitrile was maintained at 15%, 0.15 mol.L^-1The sodium dihydrogen phosphate aqueous solution was kept at 85%; from 6min to 15min, the acetonitrile linearly increased from 15% to 25%, 0.15 mol.L^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 85% to 75%; from 16min to 25min, the acetonitrile linearly increased from 25% to 30%, 0.15 mol.L^-1The sodium dihydrogen phosphate aqueous solution linearly decreased from 75% to 70%; from 26min to 35min, the acetonitrile linearly increased from 30% to 45%, 0.15 mol.L^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 70% to 55%; detection wavelength: the detection wavelength of 0-15 min is 285nm, and the method is used for detecting the tanshinol; the detection wavelength of 16-25 min is 305nm, and the detection is used for detecting tetrahydropalmatine; the detection wavelength of 26-35 min is 328nm, and the detection wavelength is used for detecting chrysoeriol; flow rate: 1.0 mL/min^-1(ii) a Column temperature: at 32 ℃; sample introduction amount: 10 mu L of the solution;

(2) preparation of control solutions: accurately weighing appropriate amount of danshensu, tetrahydropalmatine and chrysoeriol reference substances, respectively, adding methanol to obtain reference substances with mass concentration of 200.0 μ g/mL^-1，150.0μg·mL^-1，300.0μg·mL^-1The control solution of (4);

(3) preparation of a test solution: taking a capsule content sample, precisely weighing 5.00g, placing in a 50mL conical flask with a plug, adding 50mL of methanol, weighing the quality after sealing, refluxing and extracting for 1h, cooling, supplementing the loss mass with methanol, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain a sample solution;

(4) and (3) determination: precisely sucking 10 μ L of each of the reference solution and the sample solution, and injecting into high performance liquid chromatograph for measurement.

An application of a delavay pills in preparing a medicine for treating coronary heart disease is disclosed, wherein the delavay pills are prepared from the following medicinal raw materials in parts by weight: 5 parts of salvia miltiorrhiza, 4.8 parts of corydalis tuber processed with vinegar, 3 parts of trogopterus dung, 4.8 parts of snakegourd fruit, 3 parts of frankincense processed with vinegar, 3 parts of white paeony root, 3 parts of fructus aurantii and 3 parts of radix bupleuri; the Yandan capsule is prepared by the following steps:

(1) mixing vinegar processed Olibanum, fructus Aurantii, and bupleuri radix, pulverizing into fine powder, and keeping;

(2) extracting Saviae Miltiorrhizae radix with 2 times of 85% ethanol under reflux for 2 times (each time for 1.5 hr), mixing extractive solutions, filtering, recovering ethanol from filtrate until there is no ethanol smell to obtain fluid extract, and reserving the residue;

(3) mixing fructus Trichosanthis, rhizoma corydalis processed with vinegar, Oletum Trogopterori, and radix Paeoniae alba with the residue obtained in step (2), decocting in water for 2 times, each time adding 10 times of water, decocting for 2 hr, mixing decoctions, filtering, concentrating the filtrate to obtain extract, and keeping;

(4) mixing the fine powder obtained in step (1), the fluid extract obtained in step (2) and the extract obtained in step (3), drying at low temperature, pulverizing into fine powder, sieving, and encapsulating to obtain capsule;

the contents of danshensu, tetrahydropalmatine and chrysoeriol in the delavay salviae miltiorrhizae capsule by adopting the high performance liquid chromatography comprise the following steps:

(1) chromatographic conditions are as follows: a chromatographic column: wondasil C₁₈Chromatographic column, specification: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-0.15 mol.L^-1Sodium dihydrogen phosphate aqueous solution, gradient elution, elution order is as follows: from the beginning to 5min, acetonitrile was maintained at 15%, 0.15 mol.L^-1The sodium dihydrogen phosphate aqueous solution was kept at 85%; from 6min to 15min, the acetonitrile linearly increased from 15% to 25%, 0.15 mol.L^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 85% to 75%; from 16min to 25min, the acetonitrile linearly increased from 25% to 30%, 0.15 mol.L^-1The sodium dihydrogen phosphate aqueous solution linearly decreased from 75% to 70%; from 26min to 35min, the acetonitrile linearly increased from 30% to 45%, 0.15 mol.L^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 70% to 55%; detection wavelength: the detection wavelength of 0-15 min is 285nm, and the method is used for detecting the tanshinol; the detection wavelength of 16-25 min is 305nm, and the detection is used for detecting tetrahydropalmatine; the detection wavelength of 26-35 min is 328nm, and the detection wavelength is used for detecting chrysoeriol; flow rate: 1.0 mL/min^-1(ii) a Column temperature: at 32 ℃; sample introduction amount: 10 mu L of the solution;

(2) preparation of control solutions: accurately weighing appropriate amount of danshensu, tetrahydropalmatine and chrysoeriol reference substances, respectively, adding methanol to obtain reference substances with mass concentration of 200.0 μ g/mL^-1，150.0μg·mL^-1，300.0μg·mL^-1The control solution of (4);

(3) preparation of a test solution: taking a capsule content sample, precisely weighing 5.00g, placing in a 50mL conical flask with a plug, adding 50mL of methanol, weighing the quality after sealing, refluxing and extracting for 1h, cooling, supplementing the loss mass with methanol, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain a sample solution;

(4) and (3) determination: precisely sucking 10 μ L of each of the reference solution and the sample solution, and injecting into high performance liquid chromatograph for measurement.

The following experimental studies are used to verify the technical content and technical effects of the technical solutions of the present invention, but do not limit the scope of the present invention.

Experiment one: drug screening research for treating experimental pulmonary heart disease of rat

1 materials and methods

1.1 Experimental animals

SD rat, male, weight 180 ~ 220g, purchased from Anhui Chinese medicine university experimental animals center provides, animal production license number: SCXK (Anhui) 2012 and 2013. SPF grade. Animals were kept under standard conditions with 12h alternating light and shade at 22 + -2 deg.C and were allowed free access to drinking water. The experiment was started after 1 week of acclimatization of the animals in the above environment.

1.2 drugs and reagents

1.2.1 Experimental drugs

(1) Test drug Y1: prepared by Tianlongyi Safan pharmaceutical Co., Ltd, and the prescription and preparation method are as follows:

prescription: 125g of salvia miltiorrhiza, 120g of rhizoma corydalis processed with vinegar, 75g of trogopterus dung, 120g of snakegourd fruit, 75g of frankincense processed with vinegar, 75g of white paeony root, 75g of fructus aurantii and 75g of radix bupleuri.

The preparation method comprises the following steps: adding 2000mL of 55% ethanol into the salvia miltiorrhiza, the rhizoma corydalis processed with vinegar, the trogopterus dung, the snakegourd fruit, the frankincense processed with vinegar, the white paeony root, the bitter orange and the radix bupleuri, performing reflux extraction for 2 times, wherein each time lasts for 1.5h, combining extracting solutions, filtering, recovering ethanol from filtrate until no ethanol smell exists, obtaining clear paste, drying at low temperature, and crushing into fine powder to obtain the tested medicine Y1.

(2) Test drug Y2: prepared by Tianlongyi Safan pharmaceutical Co., Ltd, and the prescription and preparation method are as follows:

prescription: 125g of salvia miltiorrhiza, 120g of rhizoma corydalis processed with vinegar, 75g of trogopterus dung, 120g of snakegourd fruit, 75g of frankincense processed with vinegar, 75g of white paeony root, 75g of fructus aurantii and 75g of radix bupleuri.

The preparation method comprises the following steps: adding 2000mL of 95% ethanol into the salvia miltiorrhiza, the corydalis tuber processed with vinegar, the trogopterus dung, the snakegourd fruit, the frankincense processed with vinegar, the white paeony root, the bitter orange and the radix bupleuri, performing reflux extraction for 2 times, 1.5h each time, combining extracting solutions, filtering, recovering ethanol from filtrate until no alcohol smell exists, obtaining clear paste, drying at low temperature, and crushing into fine powder to obtain the tested medicine Y2.

(3) Test drug Y3: prepared by Tianlongyi Safan pharmaceutical Co., Ltd, and the prescription and preparation method are as follows:

prescription: 125g of salvia miltiorrhiza, 120g of rhizoma corydalis processed with vinegar, 75g of trogopterus dung, 120g of snakegourd fruit, 75g of frankincense processed with vinegar, 75g of white paeony root, 75g of fructus aurantii and 75g of radix bupleuri.

The preparation method comprises the following steps: firstly, mixing vinegar-processed frankincense, bitter orange and radix bupleuri, crushing into fine powder and reserving for later use;

② taking the salvia miltiorrhiza, adding 250mL of 85 percent ethanol, reflux extracting for 2 times, each time for 1.5h, merging the extracting solutions, filtering, recovering ethanol from the filtrate until no ethanol smell exists, obtaining clear paste, reserving for later use, and reserving the obtained dregs for later use;

thirdly, mixing the snakegourd fruit, the corydalis tuber processed by vinegar, the trogopterus dung and the white paeony root with the dregs obtained in the step (2), adding water for decocting for 2 times, adding 10 times of water for decocting for 2 hours each time, mixing the decoctions, filtering, concentrating the filtrate into an extract, and reserving for later use;

and fourthly, taking the fine powder obtained in the step (1), the clear paste obtained in the step (2) and the extract obtained in the step (3), mixing uniformly, drying at low temperature, and crushing into fine powder to obtain the tested medicine Y3.

(4) Test drug Y4: prepared by Tianlongyi Safan pharmaceutical Co., Ltd, and the prescription and preparation method are as follows:

prescription: 125g of salvia miltiorrhiza, 120g of rhizoma corydalis processed with vinegar, 75g of trogopterus dung, 120g of snakegourd fruit, 75g of frankincense processed with vinegar, 75g of white paeony root, 75g of fructus aurantii and 75g of radix bupleuri.

The preparation method comprises the following steps: decocting Saviae Miltiorrhizae radix, rhizoma corydalis processed with vinegar, Oletum Trogopterori, fructus Trichosanthis, Olibanum processed with vinegar, radix Paeoniae alba, fructus Aurantii, and bupleuri radix with 10 times of water for 2 times, each time for 2 hr, mixing decoctions, filtering, concentrating the filtrate to obtain extract, drying at low temperature, and pulverizing into fine powder to obtain tested drug Y4.

(5) Lung-power cough capsule: manufactured by Guizhou Jianxing pharmaceutical industry Co., Ltd, approved document No.: national standard of medicine Z20025240, specification: each pill is 0.3 g.

(6) Captopril tablets: manufactured by Shanghai Shigui pharmaceutical Co., Ltd, of China, and approved by the following text: the Chinese medicine standard character H31022986, the product specification: 12.5 mg.

1.2.2 Experimental reagents

(1) Monocrotaline: produced by Shanghai Huatu medical science and technology development GmbH;

(2) WULAI sugar, produced by Beijing Soilebao science and technology Limited, 500 g/bottle;

(3) 0.9% sodium chloride injection: manufactured by Anhui double-crane pharmaceutical industry, approved by the company with limited liability, the letter number: the Chinese medicine standard is H34023607, 500 mL/bottle;

(4) an endothelin detection kit produced by kainoku reagent (shanghai) ltd;

(5) an NO detection kit produced by Eimei technologies, Inc.

1.3 instruments

An electronic analytical balance (manufactured by sydow sedolis, germany, purchased from yokoku instruments ltd); an automatic biochemical analyzer, manufactured by Beijing Jiuqiang Biotechnology GmbH.

1.4 methods

80 SD rats with body mass of 180-220 g, half male and half female. The test results are divided into 8 groups according to body mass and gender, and each group comprises 10 test medicines Y1, Y2, Y3, Y4, cough due to lung force, captopril, a model group and a normal control group.

1.5 Molding

Except for normal control groups, rats of each group are injected with 50mg/5mL/Kg of 1% monocrotaline aqueous solution at one time into the abdominal cavity for molding. Gavage administration was started 1 week after molding, 1 time a day, and the test drugs Y1 group: test drug Y1 was administered at the following dose: 5.0g crude drug/Kg; test drug Y2 group: test drug Y2 was administered at the following dose: 5.0g crude drug/Kg; test drug Y3 group: test drug Y3 was administered at the following dose: 5.0g crude drug/Kg; test drug Y4 group: test drug Y4 was administered at the following dose: 5.0g crude drug/Kg; lung power cough group: administering a lung cough capsule, wherein the administration dose is as follows: 5.0g crude drug/Kg; captopril group: captopril was administered at the dose: 10 mg/Kg; the administration volume is 10mL/Kg, and normal control group and model group are infused with physiological saline with equal volume for continuous administration for 10 days.

After the administration by gavage for 1 hour on day 10, blood was collected from the orbit. Separating serum, and detecting the content of nitric oxide NO; separating plasma, and detecting the content of endothelin 1 (ET-1).

The rats were sacrificed and the rat hearts were dissected and the Right Ventricle (RV) and Left Ventricle (LV) + ventricular septum (S) were weighed separately and the right heart hypertrophy index was calculated.

Right heart hypertrophy index is right ventricle/(left ventricle + interventricular septum) × 100%.

1.6 inspection measurement

The rat lung is taken and put into 5 percent formaldehyde solution for fixation, paraffin embedding, slicing to 4 mu m, HE staining and observation under a light microscope.

1.6.1 detection of pulmonary artery area, pulmonary artery vessel wall area

Firstly, observing pulmonary arteries in rat lung tissues of HE stained sections under a low power microscope (100X), selecting 5 pulmonary arteries with similar cross sections as measurement targets, measuring the areas of blood vessels and blood vessel walls of the cross sections of the 5 pulmonary arteries by adopting an image analysis system under a medium power microscope (200X), and calculating the sum of the areas of the cross sections and the area of the blood vessel walls.

1.6.2 detection of pulmonary artery lumen area

The area of the pulmonary artery lumen is equal to the sum of the cross section area and the blood vessel wall area

1.6.3 calculating the absolute value of the area of the blood vessel wall of the pulmonary artery and the absolute value of the area of the blood vessel cavity

The absolute value of the area of the vessel wall is the area of the vessel wall/the area of the artery;

lumen area absolute value is lumen area/artery area.

1.7 statistical methods

Statistical treatment is carried out by SPSS20.0 statistical software, and the difference is statistically significant when P is less than 0.05 by using t test.

2 results of the experiment

The experimental result shows that compared with a normal control group, the content of ET-1 in the blood of the rat in the model group is increased, the content of NO is reduced, and the difference has statistical significance (P is less than 0.05), which indicates that the rat in the model group forms ET-1/NO imbalance of the pulmonary heart disease model, the vasoconstriction factor ET-1 is increased, the vasodilation factor NO is reduced, and the life modeling is successful.

Compared with the model group, the tested medicine Y3 group and the captopril group can reduce the level of the ET-1 in the blood plasma, and the difference has statistical significance (P is less than 0.05); the tested drug Y1 group, the tested drug Y2 group, the tested drug Y4 group and the lung cough group can also reduce the level of the plasma ET-1, but the reduction range is small, and the difference has no statistical significance (P is more than 0.05). See table 1 for details.

Compared with the model group, the tested medicine Y3 group and the captopril group can increase the level of serum NO, and the difference has statistical significance (P is less than 0.05); the tested medicine Y1 group, the tested medicine Y2 group, the tested medicine Y4 group and the lung strength cough group can also increase the level of serum NO, but the increase range is small, and the difference has NO statistical significance (P is more than 0.05). See table 1 for details.

The experimental result shows that the tested medicines Y3 and captopril can improve ET-1/NO imbalance of the pulmonary heart disease model by reducing ET-1 level and increasing NO level.

The experimental result shows that compared with the control group, the right heart hypertrophy index of the model group rats is increased, and the difference has statistical significance (P is less than 0.05).

Compared with the model group, the tested medicines Y3 group and captopril group can obviously reduce the right heart hypertrophy index, and the difference has statistical significance (P is less than 0.05); the tested drug Y1 group, the tested drug Y2 group, the tested drug Y4 group and the lung power cough group can also reduce the right heart hypertrophy index, but the reduction range is small, and the difference has no statistical significance (P is more than 0.05). See table 1 for details.

TABLE 1 Effect on ET-1, NO, Right Heart hypertrophy index in rats with cor pulmonale

Note: comparison with the control group:^#p is less than 0.05; comparison with model groups:^*P＜0.05。

histopathological statistics show that: compared with the model group, the tested drug Y3 group, the lung cough group and the captopril group can obviously reduce the thickness of the pulmonary artery blood vessel wall and increase the blood vessel cavity area, and the difference has statistical significance (P is less than 0.05); the tested medicine Y1 group, the tested medicine Y2 group and the tested medicine Y4 group can also reduce the area of the pulmonary artery blood vessel wall to different degrees, thin the pulmonary artery blood vessel wall and increase the blood vessel cavity area, but the difference has no statistical significance (P is more than 0.05). The results are shown in Table 2.

TABLE 2 influence on the absolute value of the vessel wall area and the vessel lumen area of the pulmonary artery in rats

Note: comparison with the control group:^#p is less than 0.05; comparison with model groups:^*P＜0.05。

the pathological micrographs are shown in the accompanying fig. 1 to 8 of the specification.

4 conclusion

The experimental result shows that the tested drug Y3 can obviously improve the imbalance (P is less than 0.05) of vasoconstriction factor endothelin/vasodilator nitric oxide in the blood of a rat model of pulmonary heart disease caused by monocrotaline, and has the tendency of reducing the right heart hypertrophy index RV/(LV + S). The pathological histological result shows that the tested drug Y3 reduces the thickness of the pulmonary artery blood vessel wall to different degrees and increases the area of the blood vessel cavity, which indicates that the tested drug Y3 can reduce the blood vessel hyperplasia of the pulmonary artery. The tested medicine Y3 can be used for treating cor pulmonale by relieving ET-1/NO imbalance, and relieving right heart hypertrophy and pulmonary vascular hyperplasia reconstruction.

Experiment two: HPLC method for determining content of tanshinol, tetrahydropalmatine and chrysoeriol in capsule

The applicant finds that danshensu contained in salvia miltiorrhiza, tetrahydropalmatine contained in rhizoma corydalis processed with vinegar and chrysoeriol contained in trichosanthes kirilowii maxim in the delavay pill capsule provided by the invention are important active ingredients for treating pulmonary heart disease, so that the applicant carries out repeated experimental research on the danshensu, the tetrahydropalmatine and the chrysoeriol contained in the dela pill capsule, proposes and establishes a method for simultaneously detecting the contents of the danshensu, the tetrahydropalmatine and the chrysoeriol by using an HPLC (high performance liquid chromatography) method, and provides experimental basis for the detection of the dela pill capsule.

1 Instrument and reagent

1.1 instruments

Model U-3000 Shimadzu high performance liquid chromatograph (model 6000 ELSD detector, Samorfei, USA, available from Synbiotic State Industriage instruments Co., Ltd.); an electronic analytical balance (manufactured by sydow sedolis, germany, purchased from yokoku instruments ltd); model KH-2200DB numerically controlled ultrasonic cleaner (manufactured by taiwan aste instruments & ltd., available from yokoku instruments & ltd.).

1.2 reagent

Danshensu reference substance (purchased from Nantong Henghua biological products, Inc. and having a purity of 99.8%), tetrahydropalmatine reference substance (purchased from Jiangsu Zhirun science and technology, Inc. and having a purity of 99.2%), chrysoeriol reference substance (purchased from Jiangsu Zhirun science and technology, Inc. and having a purity of 99.6%); acetonitrile is chromatographically pure (purchased from Zhirun science and technology Limited, Jiangsu); the water is ultrapure water; the other reagents are analytically pure (purchased from Jiangsu Zhirun science and technology Co., Ltd.);

yandan capsules: 125g of salvia miltiorrhiza, 120g of rhizoma corydalis processed with vinegar, 75g of trogopterus dung, 120g of snakegourd fruit, 75g of frankincense processed with vinegar, 75g of white paeony root, 75g of fructus aurantii, 75g of radix bupleuri and a delavay capsule are prepared by the following steps:

(1) mixing vinegar processed Olibanum, fructus Aurantii, and bupleuri radix, pulverizing into fine powder, and keeping;

(2) adding 250mL of 85% ethanol into Saviae Miltiorrhizae radix, reflux-extracting for 2 times, each time for 1.5 hr, mixing extractive solutions, filtering, recovering ethanol from filtrate until there is no ethanol smell, to obtain fluid extract, and reserving the obtained residue;

(3) mixing fructus Trichosanthis, rhizoma corydalis processed with vinegar, Oletum Trogopterori, and radix Paeoniae alba with the residue obtained in step (2), decocting in water for 2 times, each time adding 10 times of water, decocting for 2 hr, mixing decoctions, filtering, concentrating the filtrate to obtain extract, and keeping;

(4) and (3) taking the fine powder obtained in the step (1), the clear paste obtained in the step (2) and the extract obtained in the step (3), mixing uniformly, drying at low temperature, crushing into fine powder, sieving and encapsulating to obtain the capsule.

2 methods and results

2.1 chromatographic conditions

A chromatographic column: wondasil C₁₈A chromatographic column (specification: 4.6 mm. times.250 mm,5 μm); mobile phase: acetonitrile-0.15 mol.L^-1Sodium dihydrogen phosphate in water, gradient elution, in the following order, table 3:

TABLE 3 mobile phase gradient elution order Table

Detection wavelength: the detection wavelength of 0-15 min is 285nm, and the method is used for detecting the tanshinol; the detection wavelength of 16-25 min is 305nm, and the detection is used for detecting tetrahydropalmatine; the detection wavelength of 26-35 min is 328nm, and the detection wavelength is used for detecting chrysoeriol; flow rate: 1.0 mL/min^-1(ii) a Column temperature: at 32 ℃; sample introduction amount: 10 μ L.

2.2 preparation of the solution

2.2.1 preparation of control solutions:

accurately weighing appropriate amount of danshensu, tetrahydropalmatine and chrysoeriol reference substances, respectively, adding methanol to obtain reference substances with mass concentration of 200.0 μ g/mL^-1，150.0μg·mL^-1，300.0μg·mL^-1The control solution of (4).

2.2.2 preparation of test solutions:

taking a capsule content sample, precisely weighing 5.00g, placing in a 50mL conical flask with a plug, adding 50mL of methanol, weighing the quality after sealing, refluxing and extracting for 1h, cooling, supplementing the loss of quality with methanol, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain a sample solution.

2.2.3 preparation of negative sample solutions:

according to the proportion and the preparation process of the Yandan capsule prescription, a negative sample without salvia miltiorrhiza, a negative sample without corydalis ambigua processed with vinegar and a negative sample without trichosanthes kirilowii are respectively prepared, and then according to the method provided by the invention, a negative sample solution without salvia miltiorrhiza, a negative sample solution without corydalis ambigua processed with vinegar and a negative sample solution without trichosanthes kirilowii are respectively prepared.

2.3 specialization examination

Taking 10 mu L of each of the mixed reference solution, the test solution and the negative sample solution, measuring according to the chromatographic conditions provided by the invention, and recording a chromatogram, which is shown in the attached figure 9, the attached figure 10, the attached figure 11, the attached figure 12 and the attached figure 13 of the specification. The result shows that the chromatogram of the test sample has the same chromatographic peak at the corresponding position on the chromatogram of the reference sample, and the negative sample has no interference.

2.4 Linear relationship investigation

Precisely sucking 1mL, 2mL, 4mL, 8mL, and 16mL of the reference solution, respectively, injecting into a high performance liquid chromatograph, measuring according to the chromatographic conditions provided by the invention, and recording the peak area. Respectively using the injection concentrations (X, mu g. mL) of danshensu, tetrahydropalmatine and chrysoeriol^-1) Taking the peak area (Y) as the ordinate to perform linear regression to obtain the following regression equation:

the regression equation of danshensu is 35.125X +5.268(r is 0.9997);

the regression equation of tetrahydropalmatine is 32.185X-1.254 (r is 0.9995);

the regression equation for chrysoeriol is 98.254X +8.124(r 0.9994).

The result shows that the content of danshensu is 0.256-25.128 mug.mL^-1Within the range of 0.256-25.128 mug/mL of tetrahydropalmatine^-1Within the range of 0.256 to 25.128 [ mu ] g/mL of chrysoeriol^-1Within the range, the content and the peak area show a good linear relationship.

2.5 precision test

Precisely sucking 10 μ L of the reference solution, continuously sampling for 6 times according to the chromatographic conditions provided by the invention, and recording the peak area. The results showed that the RSDs of the peak areas of danshensu, tetrahydropalmatine and chrysoeriol were 0.95%, 0.85% and 0.76%, respectively (n-6), indicating good precision.

2.6 stability test

Taking the same sample solution, respectively standing for 0, 1, 2, 4, 8 and 16h, then carrying out sample injection determination according to the chromatographic conditions provided by the invention, and recording the peak area. The results show that the peak areas RSD of the danshensu, tetrahydropalmatine and chrysoeriol are respectively 1.26%, 1.12% and 1.28% (n is 6), which indicates that the stability of the test solution is better within 16 h.

2.7 repeatability test

Taking the same batch of Yandan capsules, preparing 6 parts of test solution in parallel according to the method provided by the invention, carrying out sample injection measurement according to the chromatographic conditions provided by the invention, recording the peak area and calculating the contents of danshensu, tetrahydropalmatine and chrysoeriol. As a result, the average contents of the 3 components were 0.285, 0.195 and 0.150mg g, respectively^-1RSD were 1.12%, 1.26%, and 0.85% (n ═ 6), respectively, indicating that the method was good in reproducibility.

2.8 sample recovery test

Taking 6 parts of the measured content of the delavay capsule sample powder, each part is about 1g, precisely weighing, precisely adding 3 parts of high, medium and low control solutions respectively, preparing a sample adding test solution according to the method provided by the invention, measuring according to the chromatographic conditions provided by the invention, recording the peak area and calculating the recovery rate of each component. The results showed that the average recovery of danshensu was 99.84% (RSD 1.12%), the average recovery of tetrahydropalmatine was 100.83% (RSD 1.06%) and the average recovery of chrysoeriol was 100.74% (RSD 0.96%), as detailed in table 4.

TABLE 4 sample recovery test (n ═ 6)

2.9 assay of samples

Taking 3 batches of dela dane capsules, preparing a test solution according to the method provided by the invention, determining according to the chromatographic conditions provided by the invention, recording peak areas, calculating the content of each component according to the obtained standard curve equation provided by the invention, and obtaining the result shown in table 5.

TABLE 5 measurement of sample content (mg. g)^-1，n＝3)

Discussion of 3

3.1 selection of the Mobile phase

The test examined acetonitrile-0.25% glacial acetic acid solution, acetonitrile-0.15 mol.L ^-13 different mobile phases of sodium dihydrogen phosphate aqueous solution and methanol-water, and the test result shows that acetonitrile-0.15 mol.L is used^-1When the sodium dihydrogen phosphate aqueous solution system is used as a mobile phase, the separation effect of each chromatographic peak is good, so the mobile phase system is adopted in the detection method.

3.2 selection of wavelength

Setting different detection wavelengths according to different time periods: the detection wavelength of 0-15 min is 285nm, and the method is used for detecting the tanshinol; the detection wavelength of 16-25 min is 305nm, and the detection is used for detecting tetrahydropalmatine; the detection wavelength of 26-35 min is 328nm, and the method is used for detecting chrysoeriol. When a certain fixed detection wavelength is adopted, other components cannot obtain maximum absorption, and the separation degree is good, so that the wavelength switching method is adopted to detect the components at the maximum absorption wavelength of each component, and the detection effect is good.

4 conclusion

The research establishes a method for measuring the contents of danshensu, tetrahydropalmatine and chrysoeriol 3 in the Yandan capsules, and the method is stable and feasible, has high specificity, high precision, good repeatability, small interference of other components and high recovery rate, and can be used for detecting the Yandan capsules.

Description of the drawings:

FIG. 1 is a photomicrograph of lung histology of a placebo run

FIG. 2 is a microscopic picture of lung organization pathology in model group

FIG. 3 is a photomicrograph of lung tissue pathology of the Y1 group tested

FIG. 4 is a photomicrograph of lung tissue pathology of the Y2 group tested

FIG. 5 is a photomicrograph of lung tissue pathology of the Y3 group tested

FIG. 6 is a photomicrograph of lung tissue pathology of the Y4 group tested

FIG. 7 is a microscopic picture of lung organization pathology in the coughing lung group

FIG. 8 is a photomicrograph of lung histology of the captopril group

Fig. 9 is a high performance liquid chromatogram of a control solution, wherein: the No. 1 peak is a danshensu chromatographic peak, the No. 2 peak is a tetrahydropalmatine chromatographic peak, and the No. 3 peak is a chrysoeriol chromatographic peak.

FIG. 10 is a high performance liquid chromatogram of a test solution, wherein: the No. 1 peak is a danshensu chromatographic peak, the No. 2 peak is a tetrahydropalmatine chromatographic peak, and the No. 3 peak is a chrysoeriol chromatographic peak.

FIG. 11 is a high performance liquid chromatogram of a negative sample solution without Angelica sinensis, wherein: the peak 2 is tetrahydropalmatine chromatographic peak, and the peak 3 is chrysoeriol chromatographic peak.

FIG. 12 is a high performance liquid chromatogram of a negative sample solution without Taxus chinensis, wherein: the No. 1 peak is the danshensu chromatographic peak, and the No. 3 peak is the chrysoeriol chromatographic peak.

FIG. 13 is a high performance liquid chromatogram of a negative sample solution without cinnamon twig, wherein: the No. 1 peak is tanshinol chromatographic peak, and the No. 2 peak is tetrahydropalmatine chromatographic peak.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples.

Example 1:

prescription: 125g of salvia miltiorrhiza, 120g of rhizoma corydalis processed with vinegar, 75g of trogopterus dung, 120g of snakegourd fruit, 75g of frankincense processed with vinegar, 75g of white paeony root, 75g of fructus aurantii and 75g of radix bupleuri.

The preparation method comprises the following steps:

(1) mixing vinegar processed Olibanum, fructus Aurantii, and bupleuri radix, pulverizing into fine powder, and keeping;

(2) adding 250mL of 85% ethanol into Saviae Miltiorrhizae radix, reflux-extracting for 2 times, each time for 1.5 hr, mixing extractive solutions, filtering, recovering ethanol from filtrate until there is no ethanol smell, to obtain fluid extract, and reserving the obtained residue;

(3) mixing fructus Trichosanthis, rhizoma corydalis processed with vinegar, Oletum Trogopterori, and radix Paeoniae alba with the residue obtained in step (2), decocting in water for 2 times, each time adding 10 times of water, decocting for 2 hr, mixing decoctions, filtering, concentrating the filtrate to obtain extract, and keeping;

(4) and (3) taking the fine powder obtained in the step (1), the clear paste obtained in the step (2) and the extract obtained in the step (3), mixing uniformly, drying at low temperature, crushing into fine powder, sieving and encapsulating to obtain the capsule.

The contents of danshensu, tetrahydropalmatine and chrysoeriol in the delavay salviae miltiorrhizae capsule by adopting the high performance liquid chromatography comprise the following steps:

(1) chromatographic conditions are as follows: a chromatographic column: wondasil C₁₈Chromatographic column, specification: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-0.15 mol.L^-1Sodium dihydrogen phosphate aqueous solution, gradient elution, elution order is as follows: from the beginning to 5min, acetonitrile was maintained at 15%, 0.15 mol.L^-1The sodium dihydrogen phosphate aqueous solution was kept at 85%; from 6min to 15min, the acetonitrile linearly increased from 15% to 25%, 0.15 mol.L^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 85% to 75%; from 16min to 25min, the acetonitrile linearly increased from 25% to 30%, 0.15 mol.L^-1The sodium dihydrogen phosphate aqueous solution linearly decreased from 75% to 70%; from 26min to 35min, the acetonitrile linearly increased from 30% to 45%, 0.15 mol.L^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 70% to 55%; detection ofWavelength: the detection wavelength of 0-15 min is 285nm, and the method is used for detecting the tanshinol; the detection wavelength of 16-25 min is 305nm, and the detection is used for detecting tetrahydropalmatine; the detection wavelength of 26-35 min is 328nm, and the detection wavelength is used for detecting chrysoeriol; flow rate: 1.0 mL/min^-1(ii) a Column temperature: at 32 ℃; sample introduction amount: 10 mu L of the solution;

(2) preparation of control solutions: accurately weighing appropriate amount of danshensu, tetrahydropalmatine and chrysoeriol reference substances, respectively, adding methanol to obtain reference substances with mass concentration of 200.0 μ g/mL^-1，150.0μg·mL^-1，300.0μg·mL^-1The control solution of (4);

(3) preparation of a test solution: taking a capsule content sample, precisely weighing 5.00g, placing in a 50mL conical flask with a plug, adding 50mL of methanol, weighing the quality after sealing, refluxing and extracting for 1h, cooling, supplementing the loss mass with methanol, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain a sample solution;

(4) and (3) determination: precisely sucking 10 μ L of each of the reference solution and the sample solution, and injecting into a high performance liquid chromatograph for determination;

(5) and (3) measuring results: each gram of the delavay salviae miltiorrhizae capsule contains 0.286mg of danshensu, 0.195mg of tetrahydropalmatine and 0.151mg of chrysoeriol.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (2)

1. A detection method of a Yandan capsule is characterized in that the Yandan capsule is prepared from the following medicinal raw materials in parts by weight: 5 parts of salvia miltiorrhiza, 4.8 parts of corydalis tuber processed with vinegar, 3 parts of trogopterus dung, 4.8 parts of snakegourd fruit, 3 parts of frankincense processed with vinegar, 3 parts of white paeony root, 3 parts of fructus aurantii and 3 parts of radix bupleuri; the Yandan capsule is prepared by the following steps:

(1) mixing vinegar processed Olibanum, fructus Aurantii, and bupleuri radix, pulverizing into fine powder, and keeping;

(2) extracting Saviae Miltiorrhizae radix with 2 times of 85% ethanol under reflux for 2 times (each time for 1.5 hr), mixing extractive solutions, filtering, recovering ethanol from filtrate until there is no ethanol smell to obtain fluid extract, and reserving the residue;

(3) mixing fructus Trichosanthis, rhizoma corydalis processed with vinegar, Oletum Trogopterori, and radix Paeoniae alba with the residue obtained in step (2), decocting in water for 2 times, each time adding 10 times of water, decocting for 2 hr, mixing decoctions, filtering, concentrating the filtrate to obtain extract, and keeping;

(4) mixing the fine powder obtained in step (1), the fluid extract obtained in step (2) and the extract obtained in step (3), drying at low temperature, pulverizing into fine powder, sieving, and encapsulating to obtain capsule;

the method is characterized in that the contents of danshensu, tetrahydropalmatine and chrysoeriol in the delavay capsule are detected by adopting a high performance liquid chromatography, and the method comprises the following steps:

(1) chromatographic conditions are as follows: a chromatographic column: c₁₈Chromatographic column, specification: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-0.15 mol.L^-1Sodium dihydrogen phosphate aqueous solution, gradient elution, elution order is as follows: acetonitrile is maintained at 15% from start to 5min, 0.15mol^-1The sodium dihydrogen phosphate aqueous solution was kept at 85%; from 6min to 15min, acetonitrile linearly rose from 15% to 25%, 0.15mol^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 85% to 75%; from 16min to 25min, acetonitrile linearly rose from 25% to 30%, 0.15mol^-1The sodium dihydrogen phosphate aqueous solution linearly decreased from 75% to 70%; from 26min to 35min, acetonitrile linearly rose from 30% to 45%, 0.15mol^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 70% to 55%; detection wavelength: the detection wavelength of 0-15 min is 285nm, and the method is used for detecting the tanshinol; the detection wavelength of 16-25 min is 305nm, and the detection is used for detecting tetrahydropalmatine; the detection wavelength of 26-35 min is 328nm, and the detection wavelength is used for detecting chrysoeriol; flow rate: 0.5 to 1.5 mL/min^-1(ii) a Column temperature: 30-35 ℃; sample introduction amount: 5-20 mu L;

(2) preparation of control solutions: precisely weighing appropriate amounts of danshensu, tetrahydropalmatine and chrysoeriol reference substances respectively,adding methanol to prepare the mixture with the mass concentration of 150.0-250.0 mu g/mL^-1，100.0～200.0μg·mL^-1，250.0～350.0μg·mL^-1The control solution of (4);

(3) preparation of a test solution: taking a capsule content sample, precisely weighing 4.00-6.00 g, placing the sample in a 50mL conical flask with a plug, adding 30-50 mL of methanol, weighing the mass after sealing, refluxing and extracting for 0.5-1.5 h, cooling, supplementing the loss reduction mass with methanol, filtering with a 0.45 mu m microporous membrane, and taking a subsequent filtrate to obtain a test solution;

(4) and (3) determination: precisely sucking 5-20 μ L of each of the reference solution and the sample solution, and injecting into a high performance liquid chromatograph for measurement.

2. The method for detecting the delavay salviae miltiorrhizae capsules as claimed in claim 1, wherein the contents of danshensu, tetrahydropalmatine and chrysoeriol in the delavay salviae miltiorrhizae capsules are detected by high performance liquid chromatography, and the method comprises the following steps:

(1) chromatographic conditions are as follows: a chromatographic column: wondasil C₁₈Chromatographic column, specification: 4.6mm × 250mm, 5 μm; mobile phase: acetonitrile-0.15 mol.L^-1Sodium dihydrogen phosphate aqueous solution, gradient elution, elution order is as follows: acetonitrile is maintained at 15% from start to 5min, 0.15mol^-1The sodium dihydrogen phosphate aqueous solution was kept at 85%; from 6min to 15min, acetonitrile linearly rose from 15% to 25%, 0.15mol^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 85% to 75%; from 16min to 25min, acetonitrile linearly rose from 25% to 30%, 0.15mol^-1The sodium dihydrogen phosphate aqueous solution linearly decreased from 75% to 70%; from 26min to 35min, acetonitrile linearly rose from 30% to 45%, 0.15mol^-1The linear reduction of the sodium dihydrogen phosphate aqueous solution from 70% to 55%; detection wavelength: the detection wavelength of 0-15 min is 285nm, and the method is used for detecting the tanshinol; the detection wavelength of 16-25 min is 305nm, and the detection is used for detecting tetrahydropalmatine; the detection wavelength of 26-35 min is 328nm, and the detection wavelength is used for detecting chrysoeriol; flow rate: 1.0 mL/min^-1(ii) a Column temperature: at 32 ℃; sample introduction amount: 10 mu L of the solution;

(2) preparation of control solutions: respectively precisely weighingAppropriate amount of danshensu, tetrahydropalmatine and chrysoeriol reference substances, and methanol to obtain final product with mass concentration of 200.0 μ g/mL^-1，150.0μg·mL^-1，300.0μg·mL^-1The control solution of (4);

(3) preparation of a test solution: taking a capsule content sample, precisely weighing 5.00g, placing in a 50mL conical flask with a plug, adding 50mL of methanol, weighing the quality after sealing, refluxing and extracting for 1h, cooling, supplementing the loss mass with methanol, filtering with a 0.45-micrometer microporous membrane, and taking the subsequent filtrate to obtain a sample solution;

(4) and (3) determination: precisely sucking 10 μ L of each of the reference solution and the sample solution, and injecting into high performance liquid chromatograph for measurement.

Images