Preparation method and detection method of angelica sinensis blood-enriching pharmaceutical composition

文档序号：648480 发布日期：2021-05-14 浏览：2次中文

阅读说明：本技术 一种当归补血药物组合物的制备方法及其检测方法 (Preparation method and detection method of angelica sinensis blood-enriching pharmaceutical composition ) 是由喻懋国杨志华屠国丽郁华军甯欢欢李会林付登林于 2020-12-21 设计创作，主要内容包括：本发明涉及一种当归补血药物组合物的制备方法及其检测方法,进一步优化了当归补血药物组合物的工艺,以出膏率、黄芪甲苷和阿魏酸提取总量等为指标,对物质基准对应实物制备工艺中的煎煮时间、滤过、浓缩及干燥等相关参数进行研究,摸索提取时间,浓缩方式等最佳工艺,进一步提高了阿魏酸、黄芪甲苷的含量以及产品的出膏率。通过对当归补血药物组合物检测方法进行摸索,确定了最佳的检测方法,更好的控制了产品的质量。(The invention relates to a preparation method and a detection method of a Chinese angelica blood-enriching pharmaceutical composition, which further optimizes the process of the Chinese angelica blood-enriching pharmaceutical composition, takes the extraction rate, the total extraction amount of astragaloside and ferulic acid and the like as indexes, researches related parameters such as decoction time, filtration, concentration, drying and the like in a physical preparation process corresponding to a material standard, and searches for the optimal processes such as extraction time, concentration mode and the like, thereby further improving the contents of ferulic acid and astragaloside and the extraction rate of products. The best detection method is determined by searching the detection method of the angelica blood-enriching pharmaceutical composition, and the quality of the product is better controlled.)

1. A preparation method of a Chinese angelica blood-enriching pharmaceutical composition is characterized by comprising the following steps: placing 300-500 parts of astragalus and 70-90 parts of angelica in an electronic decoction pot, adding 4000-6000 ml of water, soaking for 20-60 min, covering and decocting, boiling with strong fire, changing to small fire for decocting and keeping slightly boiling, decocting for 40-80 min, filtering with 200-mesh filter cloth while hot, concentrating the filtrate to 100-400 ml under vacuum reduced pressure at the temperature of 45-75 ℃, freeze-drying the concentrated solution at the temperature of-40-minus 80 ℃ and the vacuum degree of less than 300Pa, and collecting dry paste powder, or adding pharmaceutically acceptable auxiliary materials into the dry paste powder to prepare a pharmaceutically acceptable dosage form.

2. The preparation method according to claim 1, wherein the preparation method specifically comprises: placing 400 parts of astragalus and 80 parts of angelica in an electronic decoction pot, adding 4000ml of water, soaking for 40min, covering and decocting, boiling with strong fire, changing to small fire for decocting and keeping slightly boiling, decocting for 60min, filtering with 200-mesh filter cloth while hot, concentrating the filtrate to 200ml under vacuum and reduced pressure at 55-65 ℃, freeze-drying the concentrated solution at the temperature of-50 to-70 ℃ and the vacuum degree of less than 300Pa, and collecting dry extract powder, or adding pharmaceutically acceptable auxiliary materials after collecting the dry extract powder to prepare a pharmaceutically acceptable preparation.

3. The preparation method according to any one of claims 1-2, wherein the pharmaceutically acceptable preparation is a solid preparation or a liquid preparation, and the solid preparation is a granule, a capsule, a tablet, a pill, a powder, a lyophilized powder for injection; the liquid preparation is injection preparation and oral liquid.

4. The detection method of the angelica sinensis blood-enriching pharmaceutical composition is characterized by being used for detecting a product prepared by the preparation method of any one of claims 1-2 or the preparation method of claim 3, and comprises the following specific detection methods:

the characters are as follows: the product is light yellow powder; light smell, slightly sweet taste;

the identification method of the astragalus membranaceus thin layer comprises the following steps:

taking 1.0-3.0 g of the product, adding 15-35 mL of water to dissolve the product, carrying out ultrasonic treatment for 20-40 minutes, filtering, shaking and extracting filtrate for 2-4 times by using water saturated n-butyl alcohol, 15-35 mL each time, combining n-butyl alcohol extracting solutions, washing for 2-4 times by using ammonia test solution, 20-40 mL each time, taking n-butyl alcohol solution to evaporate to dryness, and adding methanol lmL to dissolve residues to obtain a test solution; taking 1-2 g of astragalus decoction pieces, adding 40-60 ml of water, decocting for 20-40 minutes, filtering, and preparing a control medicinal solution by the same method; adding methanol to a proper amount of astragaloside IV reference substance to obtain a solution containing 2mg per 1ml as reference substance solution; performing thin-layer chromatography test according to 0502 thin-layer chromatography of general guidelines of the four departments of the national pharmacopoeia 2015 edition, sucking 10 μ l of each of a test solution, a reference medicinal material solution and a reference solution, respectively dropping the solution on the same silica gel G thin-layer plate, taking out a lower-layer solution of trichloromethane-methanol-water as a developing agent according to a ratio of 13:7:2, drying the solution, spraying 10% sulfuric acid ethanol solution, heating the solution at 105 ℃ until spots are clearly developed, and inspecting the spots under 365nm ultraviolet lamp; spots with the same color appear at the corresponding positions of the chromatograms of the reference medicinal material and the reference substance;

the angelica sinensis thin-layer identification method comprises the following steps:

taking 1.0-3.0 g of the product, adding 40-60 ml of 0.5-1.5% sodium bicarbonate solution, carrying out ultrasonic treatment for 5-15 minutes, centrifuging, taking the supernatant, adjusting the pH value of the supernatant to 2-3 with dilute hydrochloric acid, shaking and extracting with diethyl ether for 2-4 times, 15-25 ml each time, combining the diethyl ether solutions, volatilizing, adding methanol lml into the residue to dissolve the residue to obtain a sample solution; taking 1.0-3.0 g of the angelica sinensis reference medicinal material, adding 40-60 ml of water, decocting for 20-40 minutes, filtering, and preparing the filtrate into a reference medicinal material solution by the same method; adding methanol into ferulic acid control to obtain lmg solution per lml as control solution; according to the thin-layer chromatography test of 0502 of the four ministry of the Ministry of the China pharmacopoeia 2015, 10 mul of reference solution, reference solution and test solution are respectively absorbed and respectively spotted on the same silica gel G thin-layer plate according to the proportion of 4: 1: 1: 0.1 cyclohexane-dichloromethane-ethyl acetate-formic acid as developing agent, developing, taking out, drying, and inspecting under 365nm ultraviolet lamp; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution;

the method for checking the moisture comprises the following steps: the water content is measured to be not more than 12.0 percent according to the 0832 second method of the general rule of the four departments of the version 2015 of Chinese pharmacopoeia;

the method for inspecting the extract comprises: 2g of the product is precisely weighed and measured by a hot dipping method under the item of 2201 alcohol-soluble extract measuring method according to the general rules of the four departments of 2015, namely Chinese pharmacopoeia, ethanol is used as a solvent, and the content of the alcohol-soluble extract is 30.0-55.0 percent;

fingerprint detection: the determination is carried out according to the general rules 0512 of four departments of the year 2015 of high performance liquid chromatography (Chinese pharmacopoeia).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification; detecting a wavelength ofColumn temperatureThe number of theoretical plates is not less than 5000 calculated according to calycosin glucoside peak; gradient elution is specified as:

time (min) acetonitrile (A) 0.1% phosphoric acid (B)

0 20 80

60 40 60

Preparation of reference solution A proper amount of calycosin glucoside reference substance and a proper amount of ferulic acid reference substance are precisely weighed and addedPreparing methanol into mixed solution containing calycosin glucoside 200 μ g and ferulic acid 100 μ g per 1 ml;

preparation of test solution precision weighing articleBy usingMethanolUltrasoundFiltering, recovering solvent from the filtrate under reduced pressure to near dryness, dissolving the residue in 20ml of water, and extracting with water saturated n-butanol25ml each time, combine the n-butanol solutions and useExtracting with ammonia solution for 1 time, discarding ammonia solution, and collecting n-butyl alcoholEvaporating the alcohol layer solution, dissolving the residue with 2ml water, subjecting to column chromatography with 10ml D101 macroporous adsorbent resin, and purifying with waterEluting with 95% ethanolEluting, recovering solvent from 95% ethanol eluate under reduced pressure to near dryness, dissolving 50% methanol, transferring to 10ml volumetric flask, fixing volume to scale, shaking, and filtering to obtain the final product;

the determination method comprises precisely sucking reference solution and sample solution 10 μ l each, injecting into liquid chromatograph, determining, and recording chromatogram;

the sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 10 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatogram fingerprint similarity evaluation system, the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90, and the reference fingerprint is shown in figure 1.

The content is measured according to 0512 of the general rules of four departments of 2015 edition of Chinese pharmacopoeia by high performance liquid chromatography;

determination of Astragalus root content

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; in a ratio of 35: 65 taking acetonitrile-water as a mobile phase; detecting with evaporative light scattering detector, wherein the number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak;

preparing reference solution by accurately weighing appropriate amount of astragaloside IV reference, and adding methanol to obtain solution containing 0.4mg per lml;

preparation of test solutionAccurately weighing, and accurately adding waterWeighing, and ultrasonic treating with power of 720W and frequency of 40kHzWeighing, adding water to make up for the lost weight, shaking, filtering, precisely taking 25ml of the filtrate, and extracting with water saturated n-butanol under shaking25ml each time, mixing n-butanol extractive solutions, and washing with ammonia solutionOne at a timeDiscarding the ammonia test solution washing solution, separating n-butanol solution, evaporating to dryness, dissolving the residue with methanol, transferring to 5ml measuring flask, adding methanol to dilute to scale, and shaking;

the determination method comprises precisely sucking 10 μ L and 20 μ L of reference solution and 20 μ L of test solution, subjecting to liquid chromatograph, determining, and calculating with external standard two-point method logarithmic equation;

the content of the astragalus root in each g of the product is 0.40-2.3 mg calculated as astragaloside IV according to dry products;

determination of Angelica sinensis content

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; in a ratio of 17:83 acetonitrile-0.085% phosphoric acid solution as mobile phase; detecting a wavelength ofThe column temperature is 35 ℃, and the number of theoretical plates is not less than 5000 according to the peak of ferulic acid;

preparing a reference substance solution, precisely weighing a proper amount of ferulic acid reference substance, placing the ferulic acid reference substance into a brown measuring flask, and adding 60-80% methanol to prepare a solution containing 12 mu g of ferulic acid per 1 mL;

preparing a test solution, namely precisely weighing 1.5-3.5 g of powder of the test solution, adding water to dissolve the powder, transferring the powder into a 50ml volumetric flask, adding water to fix the volume to a scale, weighing 10ml of aqueous solution into a 25ml volumetric flask, precisely weighing, and adding methanol to fix the volume to the scale to obtain the test solution;

the determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and determining;

the content of the angelica sinensis in each g of the product is 0.001-0.817 mg calculated by ferulic acid based on a dry product.

5. The detection method according to claim 4, wherein the radix astragali thin layer identification method comprises the following steps: dissolving 1.5g of the product in 25mL of water, performing ultrasonic treatment for 30 minutes, filtering, extracting the filtrate with 25mL of water-saturated n-butanol under shaking for 2 times, mixing the n-butanol extractive solutions, washing with ammonia test solution for 2 times, 30mL each time, collecting n-butanol extractive solution, evaporating to dry, and dissolving the residue with lmL of methanol to obtain test solution; decocting radix astragali decoction pieces 1.5g in water 50ml for 30min, filtering, and making into control medicinal solution by the same method; adding methanol to a proper amount of astragaloside IV reference substance to obtain a solution containing 2mg per 1ml as reference substance solution; according to a thin-layer chromatography test of 0502 of the general guidelines of the four departments of the 'Chinese pharmacopoeia' 2015 edition, 10 mul of each of a test solution, a reference medicinal material solution and a reference solution is respectively spotted on the same silica gel G thin-layer plate, a lower layer solution of trichloromethane-methanol-water in a ratio of 13:7:2 is taken as a developing agent, the lower layer solution is developed, taken out and dried, 10% sulfuric acid ethanol solution is sprayed, the spots are heated at 105 ℃ until the color development is clear, the spots are inspected under a 365nm ultraviolet lamp, and the spots with the same color are displayed on the positions corresponding to the chromatograms of the reference medicinal material and the reference.

6. The detection method according to claim 4, wherein the angelica sinensis thin layer identification method comprises the following steps:

taking 1.5g of the product, adding 50ml of 1% sodium bicarbonate solution, carrying out ultrasonic treatment for 10 minutes, centrifuging, taking the upper liquid, adjusting the pH value of the upper liquid to 2-3 by using dilute hydrochloric acid, shaking and extracting by using ether for 2 times, wherein 20ml of the upper liquid is extracted each time, combining the ether solutions, volatilizing the ether solutions, and adding methanol lml into residues to dissolve the residues to obtain a sample solution; adding water 50ml into radix Angelicae sinensis decoction pieces 1.5g, decocting for 30min, filtering, and making into control medicinal solution by the same method; and then taking ferulic acid reference substance, adding methanol to prepare a solution containing lmg per lml, taking the solution as a reference substance solution, performing thin-layer chromatography test according to 0502 general guidelines of the four ministry of the national pharmacopoeia 2015 edition, sucking 10 mu l of each of the test solution, the reference medicinal material solution and the reference substance solution, respectively dropping the solution on the same silica gel G thin-layer plate according to the proportion of 4: 1: 1: 0.1 cyclohexane-dichloromethane-ethyl acetate-formic acid as developing agent, developing, taking out, air drying, inspecting under 365nm ultraviolet lamp, and displaying the same color spot on the corresponding position of the reference medicinal material chromatogram and the reference substance chromatogram.

7. The detection method according to claim 4, wherein the fingerprint detection method comprises the following steps:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the following specification; the detection wavelength is 208-230 nm, and the column temperature is 22-28 ℃; the number of theoretical plates is not less than 5000 calculated according to calycosin glucoside peak; gradient elution is specified as:

time (min) acetonitrile (A) 0.1% phosphoric acid (B)

0 20 80

60 40 60

Preparing reference solution by precisely weighing appropriate amount of calycosin glucoside reference and ferulic acid reference, and adding 70% methanol to obtain mixed solution containing 200 μ g of calycosin glucoside and 100 μ g of ferulic acid per 1 ml;

preparation of test solutionPrecisely weigh and decideMethanolUltrasound Filtering, recovering solvent from the filtrate under reduced pressure to near dryness, dissolving the residue in 20ml of water, and extracting with water saturated n-butanol25ml each time, combine the n-butanol solutions and useExtracting the ammonia test solution for 1 time, discarding the ammonia test solution, volatilizing the n-butanol layer solution, dissolving the residue with 2ml of water, performing column chromatography with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, then eluting with 100ml of 95% ethanol, recovering the solvent from 95% ethanol eluate under reduced pressure until the eluate is nearly dry, dissolving 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to obtain the final product;

the determination method comprises precisely sucking reference solution and sample solution 10 μ l each, injecting into liquid chromatograph, determining, and recording chromatogram;

the sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 10 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90. The reference fingerprint is shown in FIG. 1.

8. The detection method according to claim 7, wherein the fingerprint detection method comprises:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the following specification; the detection wavelength is 210nm, and the column temperature is 25 ℃; the number of theoretical plates is not less than 5000 calculated according to calycosin glucoside peak; gradient elution is specified as:

time (min) acetonitrile (A) 0.1% phosphoric acid (B)

0 20 80

60 40 60

preparing a sample solution, precisely weighing about 2.5g of the product, performing ultrasonic treatment with 50ml of 50% methanol for 45min, filtering, recovering the solvent from the filtrate under reduced pressure till the sample is nearly dry, dissolving the residue in 20ml of water, extracting with water-saturated n-butyl alcohol for 2 times, 25ml each time, mixing the n-butyl alcohol solutions, extracting with 30ml of ammonia test solution for 1 time, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residue in 2ml of water, performing column chromatography with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, then eluting with 100ml of 95% ethanol, recovering the solvent from 95% ethanol eluent under reduced pressure till the sample is nearly dry, dissolving the 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to obtain the product;

the determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram.

The sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 10 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatogram fingerprint similarity evaluation system, the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90, and the reference fingerprint is shown in figure 1.

9. The detection method according to claim 4, wherein the radix astragali content determination method comprises:

measuring according to high performance liquid chromatography 'Chinese pharmacopoeia' 2015 edition general rules of four parts 0512;

chromatographic conditions and system applicability test octadecylsilane chemically bonded silica is used as a filler; in a ratio of 35: 65 taking acetonitrile-water as a mobile phase; detecting with evaporative light scattering detector, wherein the number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak;

preparing reference solution by accurately weighing appropriate amount of astragaloside IV reference, and adding methanol to obtain solution containing 0.4mg per lml;

preparing a test solution, namely taking 5.0g of the product, precisely weighing, precisely adding 50ml of water, weighing, carrying out ultrasonic treatment at the power of 720W and the frequency of 40kHz for 45 minutes, then weighing, supplementing the weight loss with water, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, shaking and extracting with water-saturated n-butyl alcohol for 4 times, 25ml each time, combining n-butyl alcohol extract, washing with an ammonia test solution for 3 times, 30ml each time, discarding an ammonia test solution washing solution, taking out the n-butyl alcohol solution to dry, dissolving residues with methanol, transferring the residues into a 5ml measuring flask, adding methanol to dilute to a scale, and shaking up to obtain the test solution;

the determination method comprises precisely sucking 10 μ L and 20 μ L of reference solution and 20 μ L of test solution, subjecting to liquid chromatograph, and calculating with external standard two-point method logarithmic equation;

the content of the astragalus root in each g of the product is 0.40-2.3 mg calculated as astragaloside IV according to dry products.

10. The detection method according to claim 4, wherein the angelica content determination method comprises: measuring by high performance liquid chromatography (0512 in the four-department general regulation of 2015 pharmacopoeia);

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; in a ratio of 17:83 acetonitrile-0.085% phosphoric acid solution as mobile phase; the detection wavelength is 316 nm; the column temperature is 35 ℃; the number of theoretical plates is not less than 5000 according to the ferulic acid peak;

preparing a reference substance solution, precisely weighing a proper amount of ferulic acid reference substance, placing into a brown measuring flask, and adding 70% methanol to prepare a solution containing 12 μ g per 1 mL;

preparing a test solution, precisely weighing 2.5g of the powder, adding water to dissolve the powder, transferring the powder into a 50ml volumetric flask, adding water to a constant volume to scale, weighing 10ml of the aqueous solution into a 25ml volumetric flask, precisely weighing, and adding methanol to a constant volume to scale to obtain the test solution;

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

The content of the angelica sinensis in each g of the product is 0.001-0.817 mg calculated by ferulic acid based on a dry product.

Technical Field

The invention relates to the field of pharmacy and drug detection, in particular to a preparation method and a detection method of a Chinese angelica blood-enriching drug composition.

Background

The Dang Gui Bu Tang is prepared from Dang Gui and Huang Qi, which are recorded in Jingyuan Lidonyuan Lun (treatise on differentiation of internal and external injury). Radix astragali and radix astragali, radix Angelicae sinensis and fructus forsythiae (washed with wine) and the mouth of the first piece are taken as the first piece. Decocting two pieces of water to obtain a decoction, removing residues, and taking the decoction warm or empty before eating. The method is different from the modern people in decocting methods and measurement, the data unit in the preparation method and the use method in the angelica blood-enriching decoction is converted from the modern data unit according to the historic measurement scale and the evolution simple table of Chinese historic standard, and the angelica blood-enriching decoction is prepared according to the method recorded in the classic famous prescription catalog and the classic famous prescription substance standard by combining the health department and the national traditional Chinese medicine administration 'management standard of the traditional Chinese medicine decoction room of the medical institution' (the national traditional Chinese medicine issue [ 2009 ] 3). The Chinese era simple form records that one or two of the golden period is equal to 40g at present, one money is equal to 4g at present, and one is equal to 200-300 ml at present, so that the formula comprises 40g of astragalus root and 8g of angelica, 400-600 ml of water is added, and the mixture is decocted to 200-300 ml. The angelica sinensis blood-enriching soup has the efficacy of enriching blood and tonifying qi, is mainly used for treating floating heat syndrome of deficiency yang, and can also be used for treating symptoms of fever, headache and the like caused by blood deficiency during menstrual period and postpartum period of women and the symptoms of chronic but unhealed hemorrhoid and ulcer.

In order to further optimize the process of the angelica sinensis blood-enriching pharmaceutical composition, the extraction rate, the total extraction amount of astragaloside and ferulic acid and the like are taken as indexes, relevant parameters such as decoction time, filtration, concentration, drying and the like in the preparation process of material-based corresponding real objects are researched, and the content of ferulic acid and astragaloside and the extraction rate of products are further improved by searching the optimal processes such as extraction time, concentration mode and the like. The best detection method is determined by searching the detection method of the angelica blood-enriching pharmaceutical composition, and the quality of the product is better controlled.

Disclosure of Invention

The invention aims to provide a preparation method of a Chinese angelica blood-enriching pharmaceutical composition.

The invention also aims to provide a detection method of the angelica sinensis blood-enriching pharmaceutical composition.

The preparation method of the angelica sinensis blood-enriching pharmaceutical composition specifically comprises the following steps: taking 300-500 parts of astragalus and 70-90 parts of angelica, placing the astragalus and 70-90 parts of angelica in a 9L electronic decoction pot, adding 4000-6000 ml of water, soaking for 20-60 min, covering the pot for decoction, boiling with strong fire, changing to small fire for decoction, keeping the boiling slightly, decocting for 40-80 min, filtering with 200-mesh filter cloth while hot, concentrating the filtrate to 100-400 ml under the conditions of vacuum pressure reduction and 45-75 ℃, freeze-drying the concentrated solution at the temperature of-40 to-80 ℃ and the vacuum degree of less than 300Pa, and collecting dry paste powder, or adding pharmaceutically acceptable auxiliary materials into the dry paste powder to prepare a pharmaceutically acceptable preparation formulation.

Preferably, the preparation method of the angelica sinensis blood-enriching pharmaceutical composition provided by the invention specifically comprises the following steps: taking 400 parts of astragalus and 80 parts of angelica, placing the astragalus and 80 parts of angelica in a 9-electron decoction pot, adding 4000ml of water, soaking for 40min, covering and decocting, boiling with strong fire, changing to small fire for decocting and keeping slightly boiling, decocting for 60min, filtering with 200-mesh filter cloth while hot, concentrating the filtrate to 200ml under the condition of vacuum reduced pressure and at the temperature of 55-65 ℃, freeze-drying the concentrated solution at the temperature of-50 to-70 ℃ and the vacuum degree of less than 300Pa, and collecting dry paste powder, or adding pharmaceutically acceptable auxiliary materials into the dry paste powder to prepare a pharmaceutically acceptable preparation.

The pharmaceutically acceptable preparation is a solid preparation or a liquid preparation, and the solid preparation is granules, capsules, tablets, pills, powder or freeze-dried powder injection; the liquid preparation is injection preparation and oral liquid.

The auxiliary materials are not limited and can be accepted in pharmacy.

The vacuum decompression concentration is preferably performed in the vacuum degree of 0.04MPa to 0.08 MPa.

The detection method comprises the following steps:

the characteristics are as follows: the product is light yellow powder; light smell, slightly sweet taste;

the identification method of the astragalus membranaceus thin layer comprises the following steps:

the angelica sinensis thin-layer identification method comprises the following steps:

taking 1.0-3.0 g of the product, adding 40-60 ml of 0.5-1.5% sodium bicarbonate solution, carrying out ultrasonic treatment for 5-15 minutes, centrifuging, taking the upper clear solution, adjusting the pH value of the upper clear solution to 2-3 by using dilute hydrochloric acid, shaking and extracting by using ether for 2-4 times, combining ether solutions by 15-25 ml each time, volatilizing, adding methanol lml into residues to dissolve, and taking the residues as a test solution; taking 1.0-3.0 g of the angelica sinensis reference medicinal material, adding 40-60 ml of water, decocting for 20-40 minutes, filtering, and preparing the filtrate into a reference medicinal material solution by the same method; adding methanol into ferulic acid control to obtain lmg solution per lml as control solution; according to the thin-layer chromatography test of 0502 of the four ministry of the Ministry of the China pharmacopoeia 2015, 10 mul of reference solution and 10 mul of reference solution are respectively spotted on the same silica gel G thin-layer plate according to the proportion of 4: 1: 1: 0.1 cyclohexane-dichloromethane-ethyl acetate-formic acid as developing agent, developing, taking out, drying, and inspecting under 365nm ultraviolet lamp; spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution;

fingerprint detection: the determination is carried out according to the general rules 0512 of four departments of the year 2015 of high performance liquid chromatography (Chinese pharmacopoeia).

time (min) acetonitrile (A) 0.1% phosphoric acid (B)

0 20 80

60 40 60

preparation of test solution precision weighing articleBy usingMethanolUltrasoundFiltering, recovering solvent from the filtrate under reduced pressure to near dryness, dissolving the residue in 20ml of water, and extracting with water saturated n-butanol25ml each time, combine the n-butanol solutions and useExtracting with ammonia solution for 1 time, discarding ammonia solution, evaporating n-butanol layer solution, dissolving residue with 2ml water, purifying with column chromatography containing 10ml D101 macroporous adsorbent resin, and purifying with waterEluting with 95% ethanolEluting, recovering solvent from 95% ethanol eluate under reduced pressure to near dryness, dissolving 50% methanol, transferring to 10ml volumetric flask, fixing volume to scale, shaking, and filtering to obtain the final product;

the determination method comprises precisely sucking reference solution and sample solution 10 μ l each, injecting into liquid chromatograph, determining, and recording chromatogram;

the sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 10 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90. The reference fingerprint is shown in FIG. 1.

Measuring the content according to high performance liquid chromatography 'Chinese pharmacopoeia' 2015 edition general rules of four parts 0512;

and (3) measuring the content of the astragalus:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; in a ratio of 35: 65 taking acetonitrile-water as a mobile phase; detecting with evaporative light scattering detector, wherein the number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak;

preparing reference solution by accurately weighing appropriate amount of astragaloside IV reference, and adding methanol to obtain solution containing 0.4mg per lml;

the product contains astragaloside IV (C) per g of radix astragali₄₁H₆₈O₁₄) Calculated, 0.40-2.3 mg;

determination of Angelica sinensis content

the determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, and determining;

the product contains ferulic acid (C) per g of radix Angelicae sinensis₁₀H₁₀O₄) The amount of the active ingredient is 0.001-0.817 mg.

Preferably, the astragalus membranaceus thin layer identification method provided by the invention comprises the following steps: dissolving 1.5g of the product in 25mL of water, performing ultrasonic treatment for 30 minutes, filtering, extracting the filtrate with 25mL of water-saturated n-butanol under shaking for 2 times, mixing the n-butanol extractive solutions, washing with ammonia test solution for 2 times, 30mL each time, collecting n-butanol extractive solution, evaporating to dry, and dissolving the residue with lmL of methanol to obtain test solution; decocting radix astragali decoction pieces 1.5g in water 50ml for 30min, filtering, and making into control medicinal solution by the same method; adding methanol to a proper amount of astragaloside IV reference substance to obtain a solution containing 2mg per 1ml as reference substance solution; according to a thin-layer chromatography test of 0502 of the general guidelines of the four departments of the 'Chinese pharmacopoeia' 2015 edition, 10 mul of each of a test solution, a reference medicinal material solution and a reference solution is respectively spotted on the same silica gel G thin-layer plate, a lower layer solution of trichloromethane-methanol-water in a ratio of 13:7:2 is taken as a developing agent, the lower layer solution is developed, taken out and dried, 10% sulfuric acid ethanol solution is sprayed, the spots are heated at 105 ℃ until the color development is clear, the spots are inspected under a 365nm ultraviolet lamp, and the spots with the same color are displayed on the positions corresponding to the chromatograms of the reference medicinal material and the reference.

Preferably, the angelica sinensis thin layer identification method of the invention comprises the following steps:

taking 1.5g of the product, adding 50ml of 1% sodium bicarbonate solution, carrying out ultrasonic treatment for 10 minutes, centrifuging, taking the upper liquid, adjusting the pH value of the upper liquid to 2-3 by using dilute hydrochloric acid, shaking and extracting by using ether for 2 times, combining the ether solution by 20ml each time, volatilizing, adding methanol lml into residues for dissolving to obtain a sample solution; adding water 50ml into radix Angelicae sinensis decoction pieces 1.5g, decocting for 30min, filtering, and making into control medicinal solution by the same method; and then taking ferulic acid reference substance, adding methanol to prepare a solution containing lmg per lml, taking the solution as a reference substance solution, performing thin-layer chromatography test according to 0502 general guidelines of the four ministry of the national pharmacopoeia 2015 edition, sucking 10 mu l of each of the test solution, the reference medicinal material solution and the reference substance solution, respectively dropping the solution on the same silica gel G thin-layer plate according to the proportion of 4: 1: 1: 0.1 cyclohexane-dichloromethane-ethyl acetate-formic acid as developing agent, developing, taking out, air drying, inspecting under 365nm ultraviolet lamp, and displaying the same color spot on the corresponding position of the reference medicinal material chromatogram and the reference substance chromatogram.

Preferably, the fingerprint detection method of the invention comprises the following steps:

time (min) acetonitrile (A) 0.1% phosphoric acid (B)

0 20 80

60 40 60

preparation of test solutionPrecisely weigh and decideMethanolUltrasound Filtering, recovering solvent from the filtrate under reduced pressure to near dryness, dissolving the residue in 20ml of water, and extracting with water saturated n-butanol25ml each time, combine the n-butanol solutions and useExtracting with ammonia solution for 1 time, discarding ammonia solution, evaporating n-butanol layer solution, dissolving residue with 2ml water, subjecting to column chromatography with 10ml D101 macroporous adsorbent resin, eluting with 50ml water, eluting with 100ml 95% ethanol, washing with 95% ethanolRemoving liquid, recovering solvent under reduced pressure to near dryness, dissolving with 50% methanol, transferring to 10ml volumetric flask, fixing volume to scale, shaking, and filtering to obtain the final product;

the determination method comprises precisely sucking reference solution and sample solution 10 μ l each, injecting into liquid chromatograph, determining, and recording chromatogram;

the sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 10 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90. The reference fingerprint is shown in FIG. 1.

Further preferably, the fingerprint detection method of the present invention comprises:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the following specification; the detection wavelength is 210nm, and the column temperature is 25 ℃; the number of theoretical plates is not less than 5000 calculated according to calycosin glucoside peak; gradient elution is specified as:

time (min) acetonitrile (A) 0.1% phosphoric acid (B)

0 20 80

60 40 60

the determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram.

The sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 10 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90. The reference fingerprint is shown in FIG. 1.

Preferably, the method for measuring the content of astragalus comprises the following steps:

measuring according to high performance liquid chromatography 'Chinese pharmacopoeia' 2015 edition general rules of four parts 0512;

preparing reference solution by accurately weighing appropriate amount of astragaloside IV reference, and adding methanol to obtain solution containing 0.4mg per lml;

the product contains astragaloside IV (C) per g of radix astragali₄₁H₆₈O₁₄) The amount of the active ingredient is 0.40-2.3 mg.

Preferably, the method for measuring the content of the angelica comprises the following steps: measuring by high performance liquid chromatography (0512 in the four-department general regulation of 2015 pharmacopoeia);

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

The product contains ferulic acid (C) per g of radix Angelicae sinensis₁₀H₁₀O₄) The amount of the active ingredient is 0.001-0.817 mg.

Has the advantages that:

compared with the prior art, the invention has the following beneficial effects:

1. the process of the Chinese angelica blood-enriching pharmaceutical composition is further optimized, the extraction rate, the total extraction amount and the content of astragaloside and ferulic acid are taken as indexes, relevant parameters such as decoction time, filtration, concentration, drying and the like in the preparation process of a substance reference corresponding substance are researched, and the optimal processes such as extraction time, concentration mode and the like are searched, so that the contents of ferulic acid and astragaloside and the extraction rate of the product are further improved. The best detection method is determined by searching the detection method of the angelica blood-enriching pharmaceutical composition, and the quality of the product is better controlled.

2. The invention determines that the water adding amount in the preparation method is preferably 4000-6000 ml, the optimal water adding amount is 4000ml, the highest content of ferulic acid in the extracting solution reaches 0.018mg/ml, and the highest content of astragaloside IV in the extracting solution reaches 0.035 mg/g; the highest transfer rate content of ferulic acid reaches 67.17%, the highest transfer rate content of astragaloside reaches 11.14%, and the highest solid content reaches 0.053 g/mL.

3. The invention adopts the cream yield, the total amount of ferulic acid and astragaloside IV as indexes and inspects the heating modes of open fire and an electronic decocting pot. The ancient decocting mode is changed, the heating mode of the electronic decocting pot is determined, and the heating efficiency is controlled through power, so that the effect of controlling firepower is more stable.

4. According to the invention, a decocting time investigation test is carried out according to a decocting time factor table, the preferable decocting time is 30-60 min, the optimal decocting time is 60min, the highest content of ferulic acid in an extracting solution reaches 0.021mg/ml, and the highest content of astragaloside IV in the extracting solution reaches 0.031 mg/ml; the highest transfer rate content of ferulic acid reaches 71.36%, the highest transfer rate content of astragaloside IV reaches 10.81%, and the highest solid content reaches 0.052 g/mL.

5. According to the invention, the content and transfer rate of astragaloside IV and ferulic acid are taken as indexes, the concentration method is considered, the concentration mode is determined to preferentially select vacuum reduced pressure concentration, the vacuum degree is preferably in the range of 0.04 MPa-0.08 MPa, the content of ferulic acid in the concentrated solution is up to 0.072mg/ml, and the content of astragaloside IV in the concentrated solution is up to 0.272 mg/ml; the highest transfer rate content of ferulic acid reaches 46.87%, the highest transfer rate content of astragaloside reaches 41.31%, and the highest solid content reaches 0.407 g/mL.

6. According to the invention, through investigation, the optimal concentration is carried out at the temperature of 45-60 ℃, the highest ferulic acid content of the concentrated solution reaches 0.071mg/ml, and the highest astragaloside content of the concentrated solution reaches 0.272 mg/ml; the highest transfer rate content of ferulic acid reaches 39.99%, the highest transfer rate content of astragaloside reaches 34.90%, and the highest solid content reaches 0.412 g/mL.

7. According to the invention, a filtration process is adopted for investigation, and the extracting solution is subjected to freeze drying, so that the freeze-dried powder is not foamed and melted, and the dry paste powder is loose in texture.

8. According to the invention, the average value of the volume of the intermediate is 2500ml through three batches of process verification, and is close to 2000ml of the original text examination endpoint, so that the determination of the process decocting time is reasonable.

9. According to the invention, the data of the extracting solution and the dry paste powder are basically parallel by three batches of verification analysis on the total amount of ferulic acid and astragaloside IV, and the material transfer loss in the process is small; three batches verify that the dry paste powder has no obvious difference by analyzing extracts and water.

10. The invention analyzes from the fingerprint, peaks 1, 3, 4, 5, 6, 7, 8, 9 and 10 are chromatographic peaks of radix astragali, and peak 2 is chromatographic peak of radix Angelicae sinensis. The peak No. 1 is temporarily taken as a reference peak to calculate the relative retention time, and the result shows that the common peak in the chromatogram of the medicinal materials, the decoction pieces, the extracting solution, the concentrated solution and the dry paste powder has the relative retention time RSD less than 2.0 percent, so that the determined substance standard preparation process is stable and feasible.

11. According to the determination process, the daily prescription amount is adopted for feeding, each group of experiments obtains a batch of Chinese angelica blood-enriching soup substance standard, 15 batches of substance standards correspond to objects, the 15 batches of substance standards have the paste yield, the determination range is 10.69-14.16%, the average value is 12.1%, and the substance standard paste yield is determined to be 7.00-25.00%.

12. The invention carries out a groping experiment of thin-layer chromatography on astragalus to determine the optimal sampling point, extraction solvent, developing solvent and the like, and tests prove that the developed spots at the corresponding positions are clear and visible without trailing. Through durability investigation of low-temperature, high-temperature, normal-temperature, low-humidity and high-humidity environments and different silica gel G thin-layer plates, the durability of the identification condition of the astragalus membranaceus thin layer is good under different influence factors. In the chromatogram of the test sample based on different batches of substances, spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance.

13. The invention carries out thin-layer groping experiment on angelica by ferulic acid chemical components, determines the optimal amount of sample, extraction solvent, developing solvent and the like, and tests prove that the developed spots at corresponding positions are clear and visible without trailing. Through durability investigation of low-temperature, high-temperature, normal-temperature, low-humidity and high-humidity environments and different silica gel G thin-layer plates, the durability of the identification condition of the astragalus membranaceus thin layer is good under different influence factors. In the chromatogram of the test sample based on different batches of substances, spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance.

14. The invention determines the detection method of the fingerprint by researching the fingerprint, and has good spectrum separation degree and few impurity peaks. Through methodology investigation, comparison of a blank solvent spectrum and a chromatographic peak of a test solution shows that the blank is free of interference, and the instrument has good precision, stability and repeatability.

15. The invention has good linear relation, and good precision, stability and repeatability of the instrument by carrying out methodology investigation on the content measurement of the astragalus.

16. The invention has good linear relation, instrument precision, stability and repeatability through methodology investigation of angelica content measurement.

Drawings

FIG. 1 shows a control fingerprint (10 common peaks with a peak 1: Calycosin glucoside 3: Ferulic acid).

FIG. 2 shows comparison of fingerprint spectra of radix Angelicae sinensis decoction for nourishing blood by reference process (S1: first batch of extractive solution; S2: second batch of extractive solution; S3: third batch of extractive solution; S4: first batch of concentrated solution; S5: second batch of concentrated solution; S6: third batch of concentrated solution; S7: first batch of dried paste; S8: second batch of dried paste; S9: third batch of dried paste; S10: radix Angelicae sinensis decoction pieces; S11: radix astragali decoction pieces; S12: reference substance.

FIG. 3 shows that different batches correspond to physical (lyophilized) powder properties.

FIG. 4 a Qingdao marine silica gel G thin layer plate (specification: 10 × 10 cm); temperature: humidity at 22.8 ℃: 74 percent; 1. 5. mu.l of a test solution (lot: DGBXT-2018051507D); 2. test solution 8. mu.l (batch: DGBXT-2018051507D); 3. 10. mu.l of a test solution (lot: DGBXT-2018051507D); 4. 10 μ l of Astragalus membranaceus decoction pieces solution (batch number: HQ-LF-2017110801Y); 5. 10 μ l of Mongolian radix astragali decoction pieces solution (batch number: HQ-GY-2017110801Y); 6. 10 μ l of astragaloside IV control (batch No. 110781-; 7. mu.l of astragalus-deficient negative sample solution (batch number: DGBXT-HQ-20180511-D01).

FIG. 5 is a diagram of the identification of Angelica sinensis (1. 5. mu.l of test solution (lot: DGBXT-2018051507D), 2. 8. mu.l of test solution (lot: DGBXT-2018051507D), 3. 10. mu.l of test solution (lot: DGBXT-2018051507D), 4. 10. mu.l of ferulic acid control solution (lot: 110773 and 201313, China food and drug testing institute), 5. 10. mu.l of Angelica sinensis decoction piece solution (lot: DG-LT-2017112901Y), 6. 5. mu.l of Angelica sinensis deficiency negative sample (Mongolian milkvetch root) solution (lot: DGBXT-DG-20180816-D01), and 7. 5. mu.l of Angelica sinensis deficiency negative sample (membranous milkvetch root) solution (lot: DGBXT-20180816-D02).

FIG. 6 is a first finger-print chromatogram (first search method for chromatographic conditions).

FIG. 7 is a fingerprint chromatogram (second search method for chromatographic conditions).

FIG. 8 is a chromatogram of a fingerprint (third search method for chromatographic conditions).

FIG. 9 is a fingerprint chromatogram (fourth method for searching for chromatographic conditions).

FIG. 10 is a fingerprint chromatogram (method for searching for chromatographic conditions, step five).

FIG. 11 is a fingerprint chromatogram (sixth method for searching for chromatographic conditions).

FIG. 12 is a fingerprint chromatogram (seventh search method for chromatographic conditions).

FIG. 13 is a fingerprint chromatogram (eighth search method for chromatographic conditions).

FIG. 14 shows an assignment chromatogram of decoction pieces of finger print (S1: blank; S2: decoction pieces of Astragalus membranaceus; S3: decoction pieces of Angelica sinensis; S4: reference substance; S5: reference substance).

FIG. 15 examines the chromatograms in their entirety.

Figure 16 fingerprint accuracy chromatogram.

FIG. 17 chromatogram for fingerprint stability assay (S1: DGBXT-2018050801D-0 h; S2: DGBXT-2018050801D-3 h; S3: DGBXT-2018050801D-6 h; S4: DGBXT-2018050801D-9 h; S5: DGBXT-2018050801D-12 h; S6: DGBXT-2018050801D-24 h).

FIG. 18 chromatogram for repeated fingerprint determination (S1: DGBXT-2018050801D-1; S2: DGBXT-2018050801D-2; S3: DGBXT-2018050801D-3; S4: DGBXT-2018050801D-4; S5: DGBXT-2018050801D-5; S6: DGBXT-2018050801D-6).

FIG. 19 shows the consensus patterns of the reference fingerprints of different batches of substances (R control spectrum; S1. DGBXT-2018050801D; S2. DGBXT-2018050802D; S3. DGBXT-2018050803D; S4. DGBXT-2018051504D; S5. DGBXT-2018051505D; S6. DGBXT-2018051506D; S7. DGBXT-2018051507D; S8. DGBXT-2018051508D; S9. DGBXT-2018051509D; S10. DGBXT-2018052110D; S11. DGBXT-2018052111D; S12. DGBXT-2018052112D; S13. DGBXT-2018052113D; S14. DGBXT-2018052114D; S15. DGBXT-2018052115D).

FIG. 20 specializes in chromatograms.

Detailed Description

The technical solution of the present invention will be further specifically described below by way of specific examples.

Example 1 preparation of a Chinese angelica hematinic composition

Placing 400g of astragalus and 80g of angelica in an electronic decoction pot, adding 4000ml of water, soaking for 40min, covering for decoction, boiling with strong fire, changing to small fire for decoction and keeping slightly boiling, decocting for 60min, filtering while hot through 200-mesh filter cloth, concentrating the filtrate to 200ml under vacuum and reduced pressure at 55-65 ℃, freeze-drying the concentrated solution at the temperature of-50 to-70 ℃ and the vacuum degree of less than 300Pa, and collecting dry extract powder, or adding soluble starch (the ratio of the soluble starch to the dry extract is 1: 1-1: 4) after collecting the dry extract powder, uniformly mixing, and preparing into granules.

Example 2 preparation of a Chinese angelica hematinic composition

Placing 400g of astragalus and 80g of angelica in an electronic decoction pot, adding 4000ml of water, soaking for 40min, covering for decoction, boiling with strong fire, changing to small fire for decoction and keeping slightly boiling, decocting for 60min, filtering while hot through 200-mesh filter cloth, concentrating the filtrate to 200ml under vacuum and reduced pressure at the temperature of 55-65 ℃, freeze-drying the concentrated solution at the temperature of-50 to-70 ℃ and the vacuum degree of less than 300Pa, and collecting dry extract powder, or adding soluble starch (the ratio of the soluble starch to the dry extract is 1: 1-1: 4) after collecting the dry extract powder, uniformly mixing, and filling into capsules to obtain the capsules.

Example 3 preparation of a Chinese angelica hematinic composition

Example 4 preparation of a Chinese angelica hematinic composition

Placing 400g of astragalus and 80g of angelica in an electronic decoction pot, adding 4000ml of water, soaking for 40min, covering for decoction, boiling with strong fire, changing to small fire for decoction and keeping slightly boiling, decocting for 60min, filtering while hot through 200-mesh filter cloth, concentrating the filtrate to 200ml under vacuum and reduced pressure at 55-65 ℃, freeze-drying the concentrated solution at the temperature of-50 to-70 ℃ and the vacuum degree of less than 300Pa, and collecting dry extract powder, or collecting the dry extract powder, adding soluble starch (the ratio of the soluble starch to the dry extract is 1: 1-1: 4), uniformly mixing, drying and preparing pills.

Example 5 preparation of a Chinese angelica hematinic composition

Placing 400g of astragalus and 80g of angelica in an electronic decoction pot, adding 4000ml of water, soaking for 40min, covering and decocting, boiling with strong fire, changing to small fire for decocting and keeping slightly boiling, decocting for 60min, filtering while hot through 200-mesh filter cloth, concentrating the filtrate to 200ml under vacuum and reduced pressure at 55-65 ℃, freeze-drying the concentrated solution at the temperature of-50 to-70 ℃ and the vacuum degree of less than 300Pa, and collecting dry extract powder, or adding 30 times of water for injection after collecting the dry extract powder, mixing uniformly, filtering and sterilizing to obtain the injection.

Example 6 preparation of a Chinese angelica hematinic composition

300g of astragalus and 90g of angelica are put into an electronic decoction pot, 3000ml of water is added, the mixture is soaked for 20min, a cover is added for decoction, the mixture is boiled by strong fire, the mixture is changed into small fire for decoction, the decoction is kept slightly boiling, the decoction is carried out for 40min, 200-mesh filter cloth is filtered when the mixture is hot, the filtrate is concentrated to 100ml under the conditions of vacuum pressure reduction and temperature of 45 ℃, the concentrated solution is freeze-dried under the conditions that the temperature is between 40 ℃ below zero and 80 ℃ and the vacuum degree is less than 300Pa, and dry extract powder is collected, or the dry extract powder is collected, then soluble starch (the ratio of the soluble starch to the dry extract is 1: 1-1: 4).

Example 7 preparation of a Chinese angelica hematinic composition

Example 8 preparation of a Chinese angelica hematinic composition

300g of astragalus and 90g of angelica are put into an electronic decoction pot, 3000ml of water is added, the mixture is soaked for 20min, a cover is added for decoction, the mixture is boiled by strong fire, the mixture is changed into small fire for decoction, the decoction is kept slightly boiling, the decoction is carried out for 40min, 200-mesh filter cloth is filtered when the mixture is hot, the filtrate is concentrated to 100ml under the conditions of vacuum pressure reduction and temperature of 45 ℃, the concentrated solution is freeze-dried under the conditions that the temperature is between 40 ℃ below zero and 80 ℃ and the vacuum degree is less than 300Pa, and dry extract powder is collected, or the dry extract powder is collected and then added with soluble starch (the ratio of the soluble starch to the dry extract is 1: 1-1.

Example 9 preparation of a Chinese angelica hematinic composition

300g of astragalus and 90g of angelica are put into an electronic decoction pot, 3000ml of water is added, the mixture is soaked for 20min, a cover is added for decoction, the mixture is boiled by strong fire, the mixture is changed into small fire for decoction, the decoction is kept slightly boiling, the decoction is carried out for 40min, 200-mesh filter cloth is filtered when the mixture is hot, the filtrate is concentrated to 100ml under the conditions of vacuum pressure reduction and temperature of 45 ℃, the concentrated solution is freeze-dried under the conditions that the temperature is between 40 ℃ below zero and 80 ℃ and the vacuum degree is less than 300Pa, and dry extract powder is collected, or the dry extract powder is collected and then added with soluble starch (the ratio of the soluble starch to the dry extract is 1: 1-1.

Example 10 preparation of a Chinese angelica hematinic composition

300g of astragalus and 90g of angelica are put into an electronic decoction pot, 3000ml of water is added, the mixture is soaked for 20min, a cover is added for decoction, the mixture is boiled by strong fire, the mixture is changed into small fire for decoction, the decoction is kept slightly boiling, the decoction is carried out for 40min, 200-mesh filter cloth is filtered when the mixture is hot, the filtrate is concentrated to 100ml under the conditions of vacuum pressure reduction and temperature of 45 ℃, the concentrated solution is freeze-dried under the conditions that the temperature is between 40 ℃ below zero and 80 ℃ and the vacuum degree is less than 300Pa, dry paste powder is collected, and the injection is obtained or 30 times of water for injection is added, mixed evenly, filtered.

Example 11 preparation of a Chinese angelica hematinic composition

500g of astragalus and 70g of angelica are put into an electronic decoction pot, 5000ml of water is added, the mixture is soaked for 60min, a cover is added for decoction, the mixture is boiled by strong fire, the mixture is changed into small fire for decoction, the decoction is kept slightly boiling, the decoction is carried out for 80 min, 200-mesh filter cloth is filtered when the mixture is hot, the filtrate is concentrated to 400ml under the conditions of vacuum pressure reduction and 75 ℃, the concentrated solution is freeze-dried under the conditions that the temperature is between 40 ℃ below zero and 80 ℃ and the vacuum degree is less than 300Pa, and dry extract powder is collected, or the dry extract powder is collected, then soluble starch (the ratio of the soluble starch to the dry extract is 1: 1-1: 4) is added.

Example 12 preparation of a Chinese angelica hematinic composition

Example 13 preparation of a Chinese angelica hematinic composition

500g of astragalus and 70g of angelica are put into an electronic decoction pot, 5000ml of water is added, the mixture is soaked for 60min, a cover is added for decoction, the mixture is boiled by strong fire, the mixture is changed into small fire for decoction, the decoction is kept slightly boiling, the decoction is carried out for 80 min, 200-mesh filter cloth is filtered when the mixture is hot, the filtrate is concentrated to 400ml under the conditions of vacuum pressure reduction and 75 ℃, the concentrated solution is freeze-dried under the conditions that the temperature is between 40 ℃ below zero and 80 ℃ and the vacuum degree is less than 300Pa, and dry extract powder is collected, or the dry extract powder is collected and then added with soluble starch (the ratio of the soluble starch to the dry extract is 1: 1-1: 4).

EXAMPLE 14 preparation of Angelica sinensis hematinic composition

500g of astragalus and 70g of angelica are put into an electronic decoction pot, 5000ml of water is added, the mixture is soaked for 60min, a cover is added for decoction, the mixture is boiled by strong fire, the mixture is changed into small fire for decoction, the decoction is kept slightly boiling, the decoction is carried out for 80 min, 200-mesh filter cloth is filtered when the mixture is hot, the filtrate is concentrated to 400ml under the conditions of vacuum pressure reduction and 75 ℃, the concentrated solution is freeze-dried under the conditions that the temperature is between 40 ℃ below zero and 80 ℃ and the vacuum degree is less than 300Pa, and dry extract powder is collected, or the dry extract powder is collected and then added with soluble starch (the ratio of the soluble starch to the dry extract is 1: 1-1: 4.

Example 15 preparation of a Chinese angelica hematinic composition

500g of astragalus and 70g of angelica are put into an electronic decoction pot, 5000ml of water is added, the mixture is soaked for 60min, a cover is added for decoction, the mixture is boiled by strong fire, the mixture is changed into small fire for decoction, the decoction is kept slightly boiling, the decoction is carried out for 80 min, 200-mesh filter cloth is filtered when the mixture is hot, the filtrate is concentrated to 400ml under the conditions of vacuum pressure reduction and 75 ℃, the concentrated solution is freeze-dried under the conditions that the temperature is between 40 ℃ below zero and 80 ℃ below zero and the vacuum degree is less than 300Pa, and dry extract powder is collected, or 30 times of water for injection is added, mixed evenly, filtered and sterilized, so.

Examples 1 to 15 were tested by any of the following test methods

Example 16

[ PROPERTIES ] the product is light yellow powder; light smell, slightly sweet taste.

[ IDENTIFICATION ] collecting 1.5g of the product, adding 25mL of water to dissolve, ultrasonic treating for 30min, filtering, shaking and extracting the filtrate with water saturated n-butanol for 2 times, 25mL each time, mixing the n-butanol extractive solutions, washing with ammonia test solution for 2 times, 30mL each time, collecting the n-butanol extractive solution, evaporating to dryness, and dissolving the residue with methanol lmL to obtain a test solution. And adding water 50ml into astragalus root decoction pieces 1.5g, decocting for 30min, filtering, and preparing a control medicinal solution by the same method. Adding methanol to a proper amount of astragaloside IV reference substance to obtain a solution containing 2mg per 1ml, and using as reference substance solution. Performing thin layer chromatography (0502 of the four ministerial rules of the four parts of the book of the Chinese pharmacopoeia 2015), sucking 10 μ l of each of the four solutions, dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) lower layer solution as developing agent, taking out, air drying, spraying with 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under ultraviolet lamp (365 nm).

(2) Taking 1.5g of the product, adding 50ml of 1% sodium bicarbonate solution, carrying out ultrasonic treatment for 10 minutes, centrifuging, taking the upper liquid, adjusting the pH value of the upper liquid to 2-3 by using dilute hydrochloric acid, shaking and extracting by using ether for 2 times, combining 20ml of ether solution each time, volatilizing, adding methanol lml into residues to dissolve, and taking the residues as a test solution. And adding water 50ml into 1.5g of the angelica control medicinal material, decocting for 30 minutes, filtering, and preparing the filtrate into a control medicinal material solution by the same method. Then, the ferulic acid control is taken and added with methanol to prepare a solution containing lmg per lml as a control solution. Performing thin layer chromatography (0502 of the four ministerial rules of the republic of China pharmacopoeia 2015), collecting 10 μ l of each of the reference solution, the reference solution and the sample solution, dropping on the same silica gel G thin layer plate, developing with cyclohexane-dichloromethane-ethyl acetate-formic acid (4: 1: 1: 0.1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.

[ EXAMINATION ] the water content should not exceed 12.0% (0832 second method in the fourth division of the design reside in the pharmacopoeia of China 2015).

[ EXTRACT ] about 2g of the product is precisely weighed, and is measured by hot dipping method under alcohol soluble extract measuring method (2201 in the four ministry of the pharmacopoeia of China 2015), ethanol is used as solvent, and the content should be 30.0-55.0%.

[ fingerprinting ] was measured by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 210nm, and the column temperature is 25 ℃. The number of theoretical plates is not less than 5000 calculated according to calycosin glucoside peak.

Preparation of reference solution A proper amount of calycosin glucoside reference and a proper amount of ferulic acid reference are precisely weighed, and 70% methanol is added to prepare a mixed solution containing 200 μ g of calycosin glucoside and 100 μ g of ferulic acid per 1 ml.

Preparing a sample solution, precisely weighing about 2.5g of the product, performing ultrasonic treatment with 50ml of 50% methanol for 45min, filtering, recovering the solvent from the filtrate under reduced pressure until the solvent is nearly dry, dissolving the residue in 20ml of water, extracting with water-saturated n-butyl alcohol for 2 times, 25ml each time, mixing the n-butyl alcohol solutions, extracting with 30ml of ammonia test solution for 1 time, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residue in 2ml of water, performing column chromatography with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, then eluting with 100ml of 95% ethanol, recovering the solvent from 95% ethanol eluent under reduced pressure until the solvent is nearly dry, dissolving the 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to obtain the product.

The determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram.

The sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 10 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90. The reference fingerprint is shown in FIG. 1.

[ CONTENT DETERMINATION ] radix astragali is determined by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-water (35: 65) is used as a mobile phase; detection by an evaporative light scattering detector. The number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak.

Preparation of control solution A proper amount of astragaloside IV control is precisely weighed, and added with methanol to obtain solution containing 0.4mg per lml.

Preparing a test solution, precisely weighing 5.0g of the product, precisely adding 50ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 45 minutes, weighing again, supplementing the weight loss with water, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, shaking up and extracting with water-saturated n-butyl alcohol for 4 times, 25ml each time, combining n-butyl alcohol extract, washing with ammonia test solution for 3 times, 30ml each time, discarding ammonia test solution washing liquor, separating n-butyl alcohol solution, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product.

The determination method comprises precisely sucking 10 μ L and 20 μ L of reference solution and 20 μ L of test solution, subjecting to liquid chromatograph, determining, and calculating with external standard two-point method logarithmic equation.

The product contains astragaloside IV (C) per g of radix astragali₄₁H₆₈O₁₄) The amount of the active ingredient is 0.40-2.3 mg.

Radix Angelicae sinensis was measured by high performance liquid chromatography (0512 in the four-department general regulation of 2015 pharmacopoeia).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.085% phosphoric acid solution (17:83) is used as a mobile phase; the detection wavelength is 316 nm; the column temperature was 35 ℃. The number of theoretical plates is not less than 5000 calculated according to ferulic acid peak.

Preparation of reference substance solution A proper amount of ferulic acid reference substance is precisely weighed, placed in a brown measuring flask, and added with 70% methanol to prepare a solution containing 12 μ g per 1 mL.

Preparing a test solution, precisely weighing 2.5g of the powder, adding a proper amount of water to dissolve the powder, transferring the powder into a 50ml volumetric flask, adding water to fix the volume to a scale, weighing 10ml of the aqueous solution into a 25ml volumetric flask, precisely weighing, and adding methanol to fix the volume to the scale to obtain the test solution.

The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

The product contains ferulic acid (C) per g of radix Angelicae sinensis₁₀H₁₀O₄) The amount of the active ingredient is 0.001-0.817 mg.

[ PACKAGE ] vacuum packaging bag is sealed.

[ STORAGE ] the seeds are stored in a cool and dry place.

Example 17

[ PROPERTIES ] the product is light yellow powder; light smell, slightly sweet taste.

[ IDENTIFICATION ] collecting 1.0g of the product, adding 15mL of water to dissolve, ultrasonic treating for 20min, filtering, shaking and extracting the filtrate with water saturated n-butanol for 2 times (15 mL each time), mixing the n-butanol extractive solutions, washing with ammonia test solution for 2 times (20 mL each time), collecting the n-butanol extractive solution, evaporating to dryness, and dissolving the residue with methanol lmL to obtain a test solution. And adding water 40ml into astragalus root decoction pieces 1.0g, decocting for 20min, filtering, and preparing a control medicinal solution by the same method. Adding methanol to a proper amount of astragaloside IV reference substance to obtain a solution containing 2mg per 1ml, and using as reference substance solution. Performing thin layer chromatography (0502 of the four ministerial rules of the four parts of the book of the Chinese pharmacopoeia 2015), sucking 10 μ l of each of the four solutions, dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) lower layer solution as developing agent, taking out, air drying, spraying with 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under ultraviolet lamp (365 nm).

(2) Taking 1.0g of the product, adding 40ml of 0.5% sodium bicarbonate solution, carrying out ultrasonic treatment for 5 minutes, centrifuging, taking the upper liquid, adjusting the pH value of the upper liquid to 2-3 by using dilute hydrochloric acid, shaking and extracting by using ether for 2 times, combining ether solutions by 15ml each time, volatilizing, adding methanol lml into residues for dissolving to obtain a sample solution. And adding water 40ml into 1.0g of angelica sinensis control medicinal material, decocting for 20 minutes, filtering, and preparing the filtrate into a control medicinal material solution by the same method. Then, the ferulic acid control is taken and added with methanol to prepare a solution containing lmg per lml as a control solution. Performing thin layer chromatography (0502 of the four ministerial rules of the republic of China pharmacopoeia 2015), collecting 10 μ l of each of the reference solution, the reference solution and the sample solution, dropping on the same silica gel G thin layer plate, developing with cyclohexane-dichloromethane-ethyl acetate-formic acid (4: 1: 1: 0.1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.

[ EXAMINATION ] the water content should not exceed 12.0% (0832 second method in the fourth division of the design reside in the pharmacopoeia of China 2015).

[ fingerprinting ] was measured by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength was 203nm and the column temperature was 20 ℃. The number of theoretical plates is not less than 5000 calculated according to calycosin glucoside peak.

Preparation of reference solution A proper amount of calycosin glucoside reference and a proper amount of ferulic acid reference are precisely weighed, and 50% methanol is added to prepare a mixed solution containing 200 μ g of calycosin glucoside and 100 μ g of ferulic acid per 1 ml.

Preparing a sample solution, precisely weighing about 2.0g of the product, performing ultrasonic treatment for 30min by using 40ml of 40% methanol, filtering, recovering the solvent from the filtrate under reduced pressure until the solvent is nearly dry, dissolving the residue in 20ml of water, extracting the residue with water-saturated n-butyl alcohol for 2 times, 25ml each time, mixing the n-butyl alcohol solutions, extracting the solution with 20ml of ammonia test solution for 1 time, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residue in 2ml of water, performing column chromatography by using 10ml of D101 macroporous adsorption resin, eluting by using 40ml of water, then eluting by using 80ml of 95% ethanol, recovering the solvent from 95% ethanol eluent under reduced pressure until the solvent is nearly dry, dissolving the 50% methanol, transferring the dissolved methanol into a 10ml bottle, fixing the volume to the scale, shaking uniformly, and.

The determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram.

The sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 10 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90. The reference fingerprint is shown in FIG. 1.

[ CONTENT DETERMINATION ] radix astragali is determined by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China).

Preparation of control solution A proper amount of astragaloside IV control is precisely weighed, and added with methanol to obtain solution containing 0.4mg per lml.

Preparing a test solution, precisely weighing 4.0g of the product, precisely adding 40ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 30 minutes, weighing again, supplementing the weight loss with water, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, shaking up and extracting with water-saturated n-butanol for 2 times (20 ml each time), combining the n-butanol extract, washing with an ammonia test solution for 2 times (20 ml each time), discarding the ammonia test solution washing liquid, separating the n-butanol solution, evaporating to dryness, dissolving the residue with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product.

The product contains astragaloside IV (C) per g of radix astragali₄₁H₆₈O₁₄) The amount of the active ingredient is 0.40-2.3 mg.

Radix Angelicae sinensis was measured by high performance liquid chromatography (0512 in the four-department general regulation of 2015 pharmacopoeia).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.085% phosphoric acid solution (17:83) is used as a mobile phase; the detection wavelength is 280 nm; the column temperature was 35 ℃. The number of theoretical plates is not less than 5000 calculated according to ferulic acid peak.

Preparing a test solution, precisely weighing 1.5g of the powder, adding a proper amount of water to dissolve the powder, transferring the powder into a 50ml volumetric flask, adding water to fix the volume to a scale, weighing 10ml of the aqueous solution into a 25ml volumetric flask, precisely weighing the aqueous solution, and adding methanol to fix the volume to the scale to obtain the test solution.

The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

The product contains ferulic acid (C) per g of radix Angelicae sinensis₁₀H₁₀O₄) The amount of the active ingredient is 0.001-0.817 mg.

[ PACKAGE ] vacuum packaging bag is sealed.

[ STORAGE ] the seeds are stored in a cool and dry place.

Example 18

[ PROPERTIES ] the product is light yellow powder; light smell, slightly sweet taste.

[ IDENTIFICATION ] collecting 3.0g of this product, adding 35mL of water to dissolve, ultrasonic treating for 40min, filtering, shaking and extracting the filtrate with water saturated n-butanol for 4 times, 35mL each time, mixing the n-butanol extractive solutions, washing with ammonia test solution for 4 times, 40mL each time, collecting the n-butanol solution, evaporating to dryness, and dissolving the residue with methanol lmL to obtain a test solution. And adding water 40ml into astragalus root decoction pieces 1.0g, decocting for 20min, filtering, and preparing a control medicinal solution by the same method. Adding methanol to a proper amount of astragaloside IV reference substance to obtain a solution containing 2mg per 1ml, and using as reference substance solution. Performing thin layer chromatography (0502 of the four ministerial rules of the four parts of the book of the Chinese pharmacopoeia 2015), sucking 10 μ l of each of the four solutions, dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) lower layer solution as developing agent, taking out, air drying, spraying with 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under ultraviolet lamp (365 nm).

(2) Taking 3.0g of the product, adding 60ml of 1.5% sodium bicarbonate solution, carrying out ultrasonic treatment for 15 minutes, centrifuging, taking the upper liquid, adjusting the pH value of the upper liquid to 2-3 by using dilute hydrochloric acid, shaking and extracting by using ether for 4 times, combining 25ml of ether solution each time, volatilizing, adding methanol lml into residues to dissolve, and taking the residues as a sample solution. And adding water 40ml into 1.0g of angelica sinensis control medicinal material, decocting for 20 minutes, filtering, and preparing the filtrate into a control medicinal material solution by the same method. Then, the ferulic acid control is taken and added with methanol to prepare a solution containing lmg per lml as a control solution. Performing thin layer chromatography (0502 of the four ministerial rules of the republic of China pharmacopoeia 2015), collecting 10 μ l of each of the reference solution, the reference solution and the sample solution, dropping on the same silica gel G thin layer plate, developing with cyclohexane-dichloromethane-ethyl acetate-formic acid (4: 1: 1: 0.1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). Spots of the same color appear on the chromatogram of the test solution at the positions corresponding to the chromatograms of the control solution and the reference solution.

[ EXAMINATION ] the water content should not exceed 12.0% (0832 second method in the fourth division of the design reside in the pharmacopoeia of China 2015).

[ fingerprinting ] was measured by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength was 203nm and the column temperature was 30 ℃. The number of theoretical plates is not less than 5000 calculated according to calycosin glucoside peak.

Preparation of reference solution A proper amount of calycosin glucoside reference and a proper amount of ferulic acid reference are precisely weighed, and 90% methanol is added to prepare a mixed solution containing 200 μ g of calycosin glucoside and 100 μ g of ferulic acid per 1 ml.

Preparing a test solution, precisely weighing about 3.0g of the product, performing ultrasonic treatment for 60min by using 40ml of 60% methanol, filtering, recovering the solvent from the filtrate under reduced pressure until the solution is nearly dry, dissolving the residue in 20ml of water, extracting the residue with water-saturated n-butyl alcohol for 2 times, 25ml each time, mixing the n-butyl alcohol solutions, extracting the solution with 40ml of ammonia test solution for 1 time, discarding the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residue in 2ml of water, performing column chromatography by using 10ml of D101 macroporous adsorption resin, eluting with 60ml of water, then eluting with 120ml of 95% ethanol, recovering the solvent from 95% ethanol eluent under reduced pressure until the solution is nearly dry, dissolving the 50% methanol, transferring the dissolved methanol into a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering to.

The determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram.

The sample fingerprint should present a chromatographic peak with the same retention time as the reference substance chromatographic peak, the sample chromatogram should be basically consistent with the reference fingerprint, there are 10 corresponding common peaks, the similarity is calculated by the common peaks according to the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity between the sample fingerprint and the reference fingerprint should not be lower than 0.90. The reference fingerprint is shown in FIG. 1.

[ CONTENT DETERMINATION ] radix astragali is determined by high performance liquid chromatography (0512 in the four Ministry of communications in the 2015 pharmacopoeia of China).

Preparation of control solution A proper amount of astragaloside IV control is precisely weighed, and added with methanol to obtain solution containing 0.4mg per lml.

Preparing a test solution, precisely weighing 6.0g of the product, precisely adding 60ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 30 minutes, weighing again, supplementing the weight loss with water, shaking up, filtering, precisely weighing 25ml of a subsequent filtrate, shaking up and extracting with water-saturated n-butanol for 4 times (40 ml each time), combining the n-butanol extract, washing with an ammonia test solution for 4 times (40 ml each time), discarding the ammonia test solution washing liquid, separating the n-butanol solution, evaporating to dryness, dissolving the residue with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product.

The product contains astragaloside IV (C) per g of radix astragali₄₁H₆₈O₁₄) The amount of the active ingredient is 0.40-2.3 mg.

Radix Angelicae sinensis was measured by high performance liquid chromatography (0512 in the four-department general regulation of 2015 pharmacopoeia).

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.085% phosphoric acid solution (17:83) is used as a mobile phase; the detection wavelength is 280 nm; the column temperature was 35 ℃. The number of theoretical plates is not less than 5000 calculated according to ferulic acid peak.

Preparation of reference substance solution A proper amount of ferulic acid reference substance is precisely weighed, placed in a brown measuring flask, and added with 80% methanol to prepare a solution containing 12 μ g per 1 mL.

Preparing a test solution, precisely weighing 3.5g of the powder, adding a proper amount of water to dissolve the powder, transferring the powder into a 50ml volumetric flask, adding water to fix the volume to a scale, weighing 10ml of the aqueous solution into a 25ml volumetric flask, precisely weighing the aqueous solution, and adding methanol to fix the volume to the scale to obtain the test solution.

The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

The product contains ferulic acid (C) per g of radix Angelicae sinensis₁₀H₁₀O₄) The amount of the active ingredient is 0.001-0.817 mg.

[ PACKAGE ] vacuum packaging bag is sealed. [ STORAGE ] the seeds are stored in a cool and dry place.

In order to verify the feasibility and the effectiveness of the invention, the inventor conducts a series of tests on the process and the detection method, and extracts the following:

1. classical famous-square substance benchmark study

1.1 Process description and flow sheet

1.1.1 preparation Process and parameters of substance reference substance

1.1.1.1 prescription

40g of astragalus root and 8g of angelica

1.1.1.2 preparation

Taking decoction pieces of 10 times of prescription amount, placing in a 9L electronic decoction pot, adding 4000ml of water, heating and decocting with open fire, boiling with strong fire, decocting with slow fire for 60 minutes, filtering with 200-mesh filter cloth while hot under normal pressure, concentrating under reduced pressure to 200ml, freeze drying, vacuum packaging to obtain corresponding substance, and storing in a cool and dry place.

1.1.3 procedure

1.1.3.1 times of decoction and dosage per decoction

According to the record of original texts, the decocting times are one time, and the decocting feeding amount is the daily prescription amount. In order to keep consistency with the ancient book recording process, the decoction times are determined as one time; because the daily prescription dosage is less and is inconvenient to study, the dosage of each decoction is enlarged to 10 times of the daily prescription dosage, 400g of astragalus and 80g of angelica.

1.1.3.2 decoction piece pretreatment

The prescription of the angelica sinensis blood-enriching decoction comprises two medicines, namely astragalus mongholicus and angelica sinensis, and the astragalus mongholicus decoction pieces are 2-4 mm thick pieces, can be directly fed, and do not need decoction piece pretreatment. The specification of the angelica sinensis decoction pieces is 2-4 mm thick pieces, and the angelica sinensis is quickly washed in yellow wine and filtered, so that the angelica sinensis decoction pieces are suitable for decoction without pretreatment.

1.1.3.3 decocting Process

(1) Investigation of heating mode

Taking the total extraction amount and the cream yield of ferulic acid and astragaloside IV in the extracting solution as investigation indexes, and carrying out single-factor comparison test research on two heating modes of open fire and an electronic decoction pot. The difference between the two heating modes is small from the comprehensive analysis of the decocting time, the paste yield and the total index components, and the decocting method of the Chinese angelica blood-enriching soup is selected as the decocting method of the electronic decocting pot because the electronic decocting pot is convenient to decoct.

(2) Observation of decoction time

Because the decoction end point in the prescription original text is the volume of the extracting solution, the operation is inconvenient in the decoction process by taking the volume of the extracting solution as the decoction end point, and the fixed heating mode is adopted, the research is carried out on the decoction time as the decoction end point instead of the volume of the extracting solution.

1.1.3.4 investigation of filtration, concentration and drying Processes

The extracting solution is inspected by a normal pressure filtration process, and the clarity of the liquid medicine is taken as an index. As a result: the extract is filtered by a nylon filter cloth with 200 meshes while the extract is hot, and the filtrate is clear and has no impurities. Determining the filtering mode as follows: filtering with 200 mesh filter cloth under normal pressure. Since the volume of the extract is about 2000ml, the freeze-drying is inconvenient, so the concentration process is considered. The concentration method, the concentration vacuum degree and the concentration temperature are respectively considered, and the total extraction amount of the ferulic acid and the astragaloside in the concentrated solution is taken as an investigation index. As a result: vacuum concentration is adopted, the vacuum degree is in the range of 0.06-0.08 Mpa, and the total extraction amount of active ingredients of astragaloside IV and ferulic acid is higher when the concentration temperature is 50-65 ℃. After the concentrated solution is subjected to freeze drying, loose dry paste powder can be collected, and the freeze drying mode is feasible.

1.1.3.5 packaging

Vacuum sealing bag for inner packing material, and sealing;

1.1.3.6 storage

Storing in a shady and cool warehouse.

1.1.4 basis for determination of Process parameters

According to the methods described in the classical name list: radix astragali and radix astragali, radix Angelicae sinensis and fructus forsythiae (washed with wine) and the mouth of the first piece are taken as the first piece. Decocting two pieces of water to obtain a decoction, removing residues, and taking the decoction warm or empty before eating. The research adopts an original text decoction method, adopts an electronic decoction pot (the capacity is 9L) for decoction, takes the extraction rate, the total extraction amount of ferulic acid and astragaloside IV and a fingerprint spectrum as indexes, and researches relevant parameters of the processes such as pretreatment, a heating mode, decoction time, filtration, concentration, drying and the like of substance-based decoction pieces so as to determine the substance-based process of the angelica blood-enriching decoction.

1.1.4.1 pretreatment

The angelica blood-enriching decoction prescription contains two medicines, namely astragalus and angelica, which are in decoction piece specifications and are suitable for decoction without pretreatment.

1.1.4.2 decocting process

The process research researches two heating modes of open fire and an electronic decoction pot, the results are comprehensively analyzed from the decoction time, the paste yield, the total amount of ferulic acid and astragaloside IV, the difference of the two heating modes is small, the electronic decoction pot is convenient to decoct, and therefore the heating mode is selected as the electronic decoction pot. And (3) researching the decoction time instead of the volume of the extracting solution as a decoction endpoint, and determining the standard decoction time of the angelica blood-enriching decoction material to be 60 minutes.

1.1.4.3 filtration, concentration, drying Process

Determining solid-liquid separation to be 200 meshes, concentrating under vacuum at vacuum degree of 0.06-0.08 Mpa and concentration temperature of 50-65 deg.C to obtain active ingredients with high total extraction amount of astragaloside IV and ferulic acid. After the concentrated solution is subjected to freeze drying (the freeze drying temperature is between 50 ℃ below zero and 70 ℃ below zero and the vacuum degree is less than 300Pa), loose dry paste powder can be collected, and the feasible freeze drying mode is determined.

1.1.4.4 Process verification procedure

Sampling three batches of medicinal materials, decoction pieces, extracting solution and dry paste powder of the corresponding batch based on the substance standard, determining the content of index components and a fingerprint, analyzing from the total amount of ferulic acid and astragaloside IV, verifying that the data of the extracting solution and the dry paste powder are basically parallel by three batches, and verifying that the substance transmission loss in the technical process is small, wherein the chromatographic profiles of the medicinal materials, the decoction pieces, the extracting solution and the dry paste powder have common peaks and the relative retention time RSD is less than 2.0 percent by analyzing from the fingerprint.

1.1.4.5 packaging

Vacuum sealing bag for inner packing material, and sealing;

1.1.4.6 storage

Storing in a shady and cool warehouse.

The standard process for determining the substances of the angelica blood-enriching decoction comprises the following steps: placing 400g of astragalus and 80g of angelica in a 9L electronic decoction pot, adding 4000ml of water, heating by open fire, covering and decocting, boiling by strong fire, changing to small fire for decocting and keeping slightly boiling, decocting for 60 minutes, decocting to 2000ml, filtering by 200-mesh filter cloth while hot, concentrating, freeze-drying (the freeze-drying temperature is-50 to-70 ℃, and the vacuum degree is less than 300Pa), and collecting dry paste powder.

1.1.5 Main devices and models are shown in Table 1

TABLE 1 Main Equipment and model

1.2 study of the Process

Decocting according to the method recorded in the list of classical famous prescriptions, in combination with the Ministry of health and the administration of Chinese medicine and drug administration of the State administration of traditional Chinese medicine and drug administration of medical institutions (the State administration of traditional Chinese medicine [ 2009 ] 3). Decocting in an electronic decocting pot, concentrating the extract, freeze-drying to obtain dry extract, and studying the decoction time, filtration, concentration and drying parameters in the preparation process of the substance-based corresponding substance to determine the substance-based process of the radix Angelicae sinensis decoction for replenishing blood by using the extract yield, the total amount of astragaloside IV and ferulic acid, and fingerprint as indexes.

8.2.1 pretreatment

According to the ancient book original texts and examination results, decoction pieces used in the prescription are prepared from medicinal materials according to corresponding processing standards, wherein the astragalus decoction pieces are 2-4 mm thick pieces, the angelica decoction pieces are 2-4 mm thick pieces, and the decoction pieces are quickly washed in yellow wine, filtered and directly fed without pretreatment of the decoction pieces. See table 2.

TABLE 2 decoction piece information for research

1.2.2 decoction

1.2.2.1 Experimental examination of Water absorption

The medicinal part of the astragalus root is the root part, and the medicinal root part of the angelica is very easy to absorb water, so the water absorption test of the decoction pieces is carried out.

The experimental method comprises the following steps: taking 40g of astragalus decoction pieces and 8g of angelica decoction pieces, uniformly mixing, adding 400ml of water, filtering at regular time, weighing and recording the weight of the filtered medicinal materials, and calculating the water absorption weight of the medicinal materials at different times. The results are shown in Table 3.

Table 3 water absorption test investigation table

And (4) conclusion: from the above tests, the soaking time of the mixed medicinal materials is 40min, the water absorption weight of the mixed medicinal materials tends to be stable, and the water absorption amount is about 2.68 times of the weight of the medicinal materials, so the extracting and soaking time of the angelica blood-enriching decoction medicinal materials is determined to be 40 min.

1.2.2.2 investigation of water addition

The decoction of the prescription of the blood-enriching decoction requires two pieces of water, one piece of water is decocted until one piece of water is decocted, and the decoction is obtained after the residue is removed, wherein the amount of the one piece of water in the prescription is equivalent to 200ml to 300ml of the current amount according to relevant literature examination, so the water addition amount is investigated. Magnifying the prescription ten times, collecting radix astragali decoction pieces (HQ-HX-2017110801Y)400g and radix Angelicae sinensis decoction pieces (DG-MX-2017110801Y)80g, respectively, and performing investigation according to the decoction volume factor table, as shown in Table 4

TABLE 4 extraction of volume factor table

The values of all factors are the contents and transfer rates of ferulic acid and astragaloside IV; the solid content was used as an index for investigation, and the detailed results are shown in Table 5.

TABLE 5 summary of volume factor content

And (4) conclusion: according to the examination of the volume of the added water, the difference between the content of astragaloside and ferulic acid and the difference between the content of transfer is not obvious, the difference between the solid content is not obvious, but the decoction time of the experimental group with the added water of 4000ml is shorter, so the reference decoction added water of the angelica blood-enriching decoction is 4000 ml.

8.2.2.3 examination of heating modes

The ancient decocting mode is open fire decocting, an electric stove is used as the open fire heating mode, meanwhile, the heating effect of an electronic decocting pot is more stable under the condition that the heating mode of the electronic decocting pot controls the heating efficiency through power, so that the open fire and the heating mode of the electronic decocting pot are considered by adopting the paste yield, the total amount of ferulic acid and astragaloside as indexes.

Weighing 4 parts of ten-day prescription amount decoction pieces (astragalus lot number HQ-LX-2017110801Y: 400g, angelica lot number DG-MX-2017110801Y: 80g), adding 4000ml of water, respectively placing in a 9L electronic decocting pot and a casserole, covering and decocting, decocting with strong fire until boiling, decocting with slow fire until 2000ml, filtering the extract with a 200-mesh sieve, and recording the volume of the extract; decocting with open fire and electronic decocting pot (2200W for strong fire and 400W for slow fire) to obtain two extractive solutions, measuring ferulic acid and astragaloside IV content and extraction rate of the extractive solution according to index component measurement method, and examining influence of heating modes of open fire and electronic decocting pot (2200W for strong fire and 400W for slow fire) on index component.

The method for measuring the cream yield of the intermediate (extract liquid) comprises the following steps: precisely measuring 20ml of the extractive solution, placing in an evaporating dish W1 dried to constant weight, evaporating to dryness in water bath, drying at 105 deg.C to constant weight, cooling in a desiccator for 30min, and precisely weighing W2 rapidly, as shown in Table 6.

TABLE 6 examination of heating methods

The test result shows that the content determination index has no significant difference in total amount due to two heating modes of open fire and an electronic decoction pot, and the electronic decoction pot is selected for decoction.

1.2.2.4 determination of the decoction time

The classic famous prescription is decocted according to the traditional Chinese medicine, a container is preferably a marmite decocting pot or a ceramic pot, an electronic decocting pot is adopted for decoction, the angelica blood-replenishing decoction in the classic famous prescription is decocted to one piece by using two pieces of water, and dregs are removed, so that the angelica blood-replenishing decoction is decocted once, and the time required for decocting to one piece is investigated. Taking 400g of radix astragali decoction pieces (HQ-LX-2017110801Y) and 80g of radix Angelicae sinensis decoction pieces (DG-MX-2017110801Y), respectively, and performing decoction time investigation test according to the decoction time factor table, wherein the results are shown in Table 7.

TABLE 7 decoction time factor Table

And (4) conclusion: as can be seen from the data in the above table, the decoction time is approximately half of the addition amount when the decoction time is 60min, so the decoction extraction time of the classic famous prescription substance standard of the Angelica sinensis blood-enriching decoction is determined to be 60 min. The measurement of the content of each factor and the data of the transfer rate are shown in Table 8:

TABLE 8 summary of the determination of the content of the decoction time factors

And (4) conclusion: as can be seen from the data in the table above, when the decoction time is 60min, the content difference of ferulic acid and astragaloside IV is not large, the transfer rate has no significant difference, the solid content is higher when the decoction is 60min, so the standard preparation decoction time of the angelica blood-enriching decoction is determined to be 60 min.

1.2.2.5 summary of the decoction Process

In summary, the standard decocting process of the tentative angelica blood-enriching decoction comprises the following steps: taking 400g of astragalus and 80g of angelica. Adding 4000ml water, placing in a 9L electronic decocting pot, covering, decocting with strong fire until boiling, and decocting with slow fire for 60 min.

1.2.3 filtration, concentration and drying

1.2.3.1 filtration Process Studies

Respectively taking the decoction time under the investigation item, decocting for 45min, decocting for 60min, respectively, filtering the hot extractive solutions with 200 and 300 mesh filter cloth, and observing the difference between the two filtrates.

The results show that: both the two kinds of filter materials are easy to filter, and the clarity difference of the filtrate is not large by observing the state of the filtered liquid medicine, so 200-mesh filter cloth is selected for filtering.

1.2.3.2 concentration Process Studies

1.2.3.2.1 examination of concentration method

As can be seen from the material standard preparation results, the extract is about 2000ml, concentration treatment is required, the concentration method used comprises two methods of normal pressure concentration and reduced pressure concentration, the concentration method is considered to determine the concentration method by taking the content and transfer rate of astragaloside and ferulic acid as indexes.

The experimental method comprises the following steps: taking 400g of astragalus decoction pieces (batch number HQ-LX-2017110801Y) and 80g of angelica decoction pieces (batch number DG-MX-2017110801Y), preparing the angelica blood-enriching soup according to a decoction process, filtering an extracting solution through 200-mesh filter cloth, averagely dividing into 2 parts, wherein each part is 1000ml, concentrating to about 200ml to obtain the angelica blood-enriching soup, and respectively concentrating under normal pressure and reduced pressure, wherein the concentration results are shown in Table 9.

TABLE 9 examination results of concentration method

And (4) conclusion: the concentration under normal pressure and the concentration under vacuum have no obvious difference on the content and the transfer rate of the astragaloside ferulic acid, but the vacuum reduced pressure concentration time is shorter than the concentration time, so the concentration method selects vacuum reduced pressure concentration.

1.2.3.2.2 concentration vacuum examination

According to the investigation experiment of the 8.2.3.2.1 concentration method, the concentration method is determined to be reduced pressure concentration, and the vacuum degree is further examined.

The experimental method comprises the following steps: 400g of astragalus decoction pieces (batch number HQ-LX-2017110801Y) and 80g of angelica decoction pieces (batch number DG-MX-2017110801Y) are taken, the angelica blood-enriching soup is prepared according to a decoction process, the liquid medicine is averagely divided into 3 parts, the temperature is fixed, a reduced pressure rotary steaming concentration method is adopted, the concentration conditions under different vacuum conditions are examined, and the results are shown in Table 10.

TABLE 10 examination of the degree of vacuum of concentration

And (4) conclusion: the vacuum degree has no obvious influence on the content and the transfer rate of the astragaloside and the ferulic acid, but the steam pressure is increased along with the increase of the vacuum degree, and the vacuum degree is concentrated within the range of 0.04MPa to 0.08MPa by combining the practical production and the comprehensive consideration of time saving and consumption reduction.

1.2.3.2.3 examination of concentration temperature

According to 1.2.3.2.2 and 1.2.3.2.3 concentration methods and vacuum degree test, the concentration method is determined to be reduced pressure concentration, and the concentration temperature is further examined.

The experimental method comprises the following steps: 400g of astragalus decoction pieces (batch number HQ-LX-2017110801Y) and 80g of angelica decoction pieces (batch number DG-MX-2017110801Y) are taken, the angelica blood-enriching soup is prepared according to the decoction process, the liquid medicine is averagely divided into 4 parts, and the liquid medicine is respectively concentrated under different temperature conditions, and the results are shown in Table 11.

TABLE 11 examination of the concentration temperature

The results show that: the temperature is between 45 ℃ and 60 ℃, the contents and transfer rates of the astragaloside and the ferulic acid are not obviously influenced, and the concentration is carried out at the temperature of between 45 ℃ and 60 ℃ by combining the production practice and the comprehensive consideration of time saving and consumption reduction.

In conclusion, the following results are obtained: the concentration process adopts a method of decompression concentration to carry out concentration, and the process parameters are determined as the concentration temperature of 45-60 ℃ and the vacuum degree of 0.04-0.06 MPa.

1.2.3.2.4 summary of concentration Process

In conclusion, the concentration process of the angelica sinensis blood enriching soup is vacuum concentration at 0.04-0.08MPa, and the concentration temperature is 45-66 ℃.

1.2.3.3 study of drying Process

And (3) taking the filtration process to observe the drying condition, and freeze-drying the extract (the freeze-drying temperature is-45 to-60 ℃, and the vacuum degree is less than 300 Pa). The results are shown in Table 12.

TABLE 12 results of reference process study of substances

In summary, the following steps: the preparation process of the provisional Chinese angelica blood-enriching soup substance standard comprises the following steps: placing 400g of radix astragali and 80g of radix Angelicae sinensis in a 9L casserole, heating in an electronic decocting pot, adding 4000ml of water, covering, decocting, boiling with strong fire, decocting with slow fire, keeping slightly boiling, decocting for 60min, filtering with 200 mesh filter cloth, concentrating the extractive solution to about 200ml, freeze drying, and collecting dry extract powder.

1.2.4 Key Process Steps and intermediates

1.2.4.1 Process validation sample preparation

Weighing 3 parts of decoction pieces (astragalus root 400g batch number: HQ-LX-2017110801Y and angelica 80g batch number: DG-LT-2017112901Y) with the daily prescription amount of 10 times, adding 4000ml of water, placing the mixture into a 9L electronic decoction pot, soaking for 40min, covering and decocting, decocting with strong fire until the mixture is boiled, decocting with slow fire for 60min, decocting to about 2000ml, filtering with 200-mesh filter cloth while hot, reserving 250ml of extract, concentrating the rest filtrate (vacuum decompression, concentration at 55-65 ℃) to about 200ml, and freeze-drying. (the cold trap temperature is between 50 ℃ below zero and 70 ℃ below zero, the vacuum degree is less than 300Pa), dry paste powder is collected, and the paste yield is calculated. Sampling, measuring the water content, extract, ferulic acid and astragaloside IV content in the dry extract powder, and respectively calculating the content transfer rate in the process. And preparing single-ingredient decoction pieces and medicinal material extracting solution according to the process, and performing fingerprint spectrum determination.

1.2.4.2 Process validation sample determination

The results are shown in tables 13 to 14, FIG. 2.

TABLE 13 summary of standard process verification data of Angelica sinensis decoction for tonifying blood

TABLE 14 basic process verification of Chinese angelica decoction for tonifying blood and relative retention time results

1.2.4.3 Process determination

From the analysis of the decoction volume, the average value of the volume of the intermediate is 2500ml which is verified by the three batches of processes and is close to 2000ml of the original text examination end point, and the determination of the process decoction time is reasonable.

Three batches of verification result shows that the data of the extracting solution and the dry paste powder are basically parallel and the material transmission loss is small in the technical process by analyzing the total amount of the ferulic acid and the astragaloside IV; three batches verify that the dry paste powder has no obvious difference by analyzing extracts and water.

When the fingerprint is analyzed, peaks 1, 3, 4, 5, 6, 7, 8, 9 and 10 are chromatographic peaks of radix astragali, and peak 2 is chromatographic peak of radix Angelicae sinensis. The peak No. 1 is temporarily taken as a reference peak to calculate the relative retention time, and the result shows that the common peak in the chromatogram of the medicinal materials, the decoction pieces, the extracting solution, the concentrated solution and the dry paste powder has the relative retention time RSD less than 2.0 percent, so that the determined substance standard preparation process is stable and feasible.

In summary, the material reference process was determined to be: placing 400g of astragalus and 80g of angelica in a 9L electronic decoction pot, adding 4000ml of water, soaking for 40min, covering the pot for decoction, boiling with strong fire, changing to small fire for decoction and keeping slightly boiling, decocting for 60min, filtering with 200-mesh filter cloth while hot, concentrating the filtrate (vacuum decompression, vacuum degree of 0.04-0.08MPa, temperature of 55-65 ℃) to about 200ml, freeze-drying (freeze-drying temperature is-50-70 ℃ and vacuum degree of less than 300Pa), and collecting dry paste powder to obtain the Chinese medicinal composition.

1.2.4.4 preparation of Multi-batch Material basis

At present, decoction pieces are collected on the principle of 5 producing areas and not less than 15 batches; astragalus is collected from Gansu province Longxi county, Gansu province Huating county river western county, Gansu province Weiyuan county lotus peak town, Hebei Bao City and inner Mongolia Guyang county, 3 batches of extracts are obtained in each production area, and the extracts meet the regulations; in the prescription, 5 Chinese angelica in 5 counties such as Weiyuan county, Min county, Wushan county, Tongwei county and Lin moreover 27950 county of Gansu province are collected from Chinese angelica, 3 batches of extracts are obtained in each production place, and the extracts meet the specification; the decoction pieces of 15 batches of each herb were randomly sorted to prepare 15 batches of the material basis. The results of the 15 batches of decoction pieces sorted and combined are shown in Table 15.

TABLE 15 summary of material basis feeds for different batches

Note: numbered 1-15, namely 15 combination modes

According to the determination process, daily prescription amount is adopted for feeding, a batch of Chinese angelica blood-enriching soup substance standard is obtained in each group of experiments, 15 batches of substance standards correspond to real objects, the 15 batches of substance standard paste yield determination results are shown in the following table, the determination range is 10.69-14.16%, the average value is 12.1%, and the substance standard paste yield determination range is 7.00-25.00%. The results are shown in Table 16.

TABLE 16 Multi-batch substance reference batch information

Study of detection method

1.3.3.2 validation of analytical methods

The analysis methodology verification of the item listed in the detection method is carried out according to the relevant guiding principle in the current edition of Chinese pharmacopoeia, and the methodology verification data is as follows.

1.3.3.2.1 trait

The actual properties of 15 batches of the corresponding real objects (dry paste powder) are observed and recorded, and the results are shown in a table 17 and a figure 3.

TABLE 17 Properties

And (4) conclusion: temporarily setting the product to be light yellow powder according to the actual observation condition of 15 batches of corresponding object (dry paste powder) samples; light smell, slightly sweet taste.

1.3.3.2.2 authentication

The product is prepared from two traditional Chinese medicines, is a compound preparation prepared by a water decoction process, adopts a thin-layer identification method with strong specificity, rapidness, simplicity and good reproducibility, and carries out identification test research on main characteristic components of Chinese angelica and astragalus in the prescription. The results are summarized as follows:

astragalus root

The radix astragali and Glycyrrhrizae radix contain saponins and flavonoids as main chemical components, and also contain polysaccharide. Experiments were performed on Astragalus membranaceus according to the above chemical composition by thin layer chromatography as follows.

a method of establishing thin layer chromatography

The method comprises the following steps: refer to thin layer identification method under item of 'radix astragali granule' in the first part of 'Chinese pharmacopoeia' 2015 edition for groping

Taking 1.5g of the product and negative samples without astragalus root respectively, taking 1.5g of the product powder, adding 25mL of water, carrying out ultrasonic treatment for 30 minutes, filtering, shaking and extracting the filtrate for 2 times with 25mL of water saturated n-butyl alcohol each time, combining n-butyl alcohol extracts, washing with ammonia test solution for 2 times, 30mL each time, taking the n-butyl alcohol solution to evaporate to dryness, and adding methanol lmL into residues to dissolve the residues to obtain a test solution. And adding water 50ml into astragalus root decoction pieces 1.5g, decocting for 30min, filtering, and preparing a control medicinal solution by the same method. Adding methanol to a proper amount of astragaloside IV reference substance to obtain a solution containing 2mg per 1ml, and using as reference substance solution. Performing thin layer chromatography (0502 of the four ministerial rules of the republic of China pharmacopoeia 2015), sucking 5 μ l of each of the four solutions, dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) lower layer solution as developing agent, taking out, air drying, spraying with 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under ultraviolet lamp (365 nm).

And (4) analyzing results: in the chromatogram of the test sample, the same spots are present at the corresponding positions of the chromatogram of the astragaloside IV and the radix astragali, and the negative is free of interference. The method is feasible by using astragaloside IV as a reference substance and using the radix astragali decoction piece solution as a reference substance and inspecting under ultraviolet light (365nm), wherein the spots are lighter than the test solution, and the sample amount of the reference substance and the radix astragali decoction piece solution is further searched for in order to make the spots more clear.

The second method comprises the following steps: sample amount searching

Sucking sample solution (DGBXT-2018051507D) prepared by the first method, the sample application amount of astragaloside IV reference substance solution, the astragalus mongholicus decoction piece solution and the negative sample is not changed to 10 mul, the sample application amount of the sample is changed to 5 mul, 8 mul and 10 mul, respectively dropping the sample solution, the sample solution and the negative sample on the same silica gel G thin layer plate, taking a lower layer solution of chloroform-methanol-water (13:7:2) as a developing agent, developing, taking out, airing, spraying with 10% sulfuric acid ethanol solution, and heating at 105 ℃ until spots are clearly developed. Spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; the film was observed under an ultraviolet lamp (365 nm). See fig. 4.

The result shows that the sample application amount of the astragaloside control solution and the astragalus mongholicus decoction piece is 10 mul, the sample application amount of the test solution is 5 mul, 8 mul and 10 mul, the color spots at corresponding positions are clear and visible, and no tailing phenomenon occurs, so that the concentration of the astragaloside control solution is determined to be 10 mul of sample application amount of 2mg/ml, the sample application amount of the astragalus mongholicus decoction piece solution is 10 mul of 1.5g/ml, and the sample application amount of the test solution is 10 mul. Comprehensive analysis, confirmation of thin layer identification method: dissolving 1.5g of the product in 25mL of water, performing ultrasonic treatment for 30 minutes, filtering, extracting the filtrate with 25mL of water-saturated n-butanol under shaking for 2 times, mixing the n-butanol extractive solutions, washing with ammonia test solution for 2 times, 30mL each time, collecting n-butanol extractive solution, evaporating to dryness, and dissolving the residue with lmL of methanol to obtain test solution. And adding water 50ml into astragalus root decoction pieces 1.5g, decocting for 30min, filtering, and preparing a control medicinal solution by the same method. Adding methanol to a proper amount of astragaloside IV reference substance to obtain a solution containing 2mg per 1ml, and using as reference substance solution. Performing thin layer chromatography (0502 of the four ministerial rules of the republic of China pharmacopoeia 2015), sucking 5 μ l of each of the four solutions, dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) lower layer solution as developing agent, taking out, air drying, spraying with 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under ultraviolet lamp (365 nm).

b durability examination

And (3) carrying out a durability test according to the determined method, and inspecting influence factors such as thin-layer plates (Qingdao ocean chemical plants) of the self-made silica gel G plate and the machine-made silica gel G plate, different development high temperature and high humidity (temperature 42 ℃ and humidity 75%), relative low temperature and low humidity (temperature 4 ℃ and humidity 45%) and the like.

And (4) conclusion: through durability investigation of low-temperature, high-temperature, normal-temperature, low-humidity and high-humidity environments and different silica gel G thin-layer plates, the durability of the identification condition of the astragalus membranaceus thin layer is good under different influence factors. The thin layer method is hereby incorporated into the text.

c method determination

Collecting 1.5g of the product, adding 25mL of water, performing ultrasonic treatment for 30min, filtering, extracting the filtrate with water saturated n-butanol under shaking for 2 times, each time 25mL, mixing n-butanol extractive solutions, washing with ammonia test solution for 2 times, each time 30mL, collecting n-butanol solution, evaporating to dryness, and dissolving the residue with methanol lmL to obtain test solution. And adding water 50ml into 1.5g of astragalus control medicinal material, decocting for 30 minutes, filtering, and preparing the filtrate into a control medicinal material solution by the same method. Adding methanol into astragaloside IV control to obtain solution containing 2mg of astragaloside IV per lmL as control solution. Performing thin layer chromatography (0502 of the four ministerial general rules of the design reside in the Chinese pharmacopoeia 2015), sucking 10 μ l of each of the control solution, and the sample solution, respectively dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) lower layer solution as developing agent, taking out, air drying, spraying with 10% sulphuric acid ethanol solution, and heating at 105 deg.C until the spots are clearly developed. Spots of the same color appear in the chromatogram of the test solution at the positions corresponding to the chromatograms of the reference medicinal material and the reference solution; the fluorescent spots with the same color are observed under an ultraviolet lamp (365 nm).

d thin layer identification of corresponding real object (dry paste powder) in different batches

Weighing 1.5g of corresponding substance (dry extract powder) of different batches, and making into sample solution by the same method. And (5) performing thin-layer identification.

And (4) conclusion: in the chromatogram of the test sample based on different batches of substances, spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance.

② Chinese angelica root

The angelica mainly contains benzene compounds such as ferulic acid, ligustilide and the like. The volatile oil is the main active component of angelica and mainly contains ester components such as ligustilide, etc., and the ligustilide is extracted and dried to be less in proportion in dry paste powder, so the chemical component of ferulic acid is used for carrying out the following thin-layer grope experiment on angelica.

a method of establishing thin layer chromatography

The method comprises the following steps: refer to the thin-layer identification method under the term "Chinese angelica" in the section of the "Chinese pharmacopoeia" 2015 edition

Collecting 1.5g of the product and negative sample of radix Angelicae sinensis, adding 1% sodium bicarbonate solution 50ml, ultrasonic treating for 10 min, centrifuging, collecting the upper clear solution, and adjusting pH with dilute hydrochloric acid toExtracting with diethyl ether under shaking for 2 times, mixing 20ml each time, volatilizing, and dissolving the residue with methanol lml to obtain test solution. And adding 50ml of water into 1.5g of angelica decoction pieces, decocting for 30 minutes, filtering, and preparing a control medicinal solution by the same method. Adding methanol into ferulic acid control to obtain lmg solution per lml, performing thin layer chromatography (0502 in 2015 th four parts of the pharmacopoeia of China)The four solutions each having a volume of 10. mu.L were spotted on the same silica gel G thin layer plate, developed with cyclohexane-dichloromethane-ethyl acetate-formic acid (4: 1: 1: 0.1) as developing agent, taken out, air-dried, and inspected under an ultraviolet lamp (365 nm).

And (4) analyzing results: in the chromatogram of the test sample, the same spots are present at the positions corresponding to the chromatograms of ferulic acid and radix Angelicae sinensis, and the negative is free of interference. Taking ferulic acid as a reference substance and radix Angelicae sinensis decoction piece solution as a reference substance, and inspecting under ultraviolet light (365nm), the method is feasible, but the spots of the reference substance and radix Angelicae sinensis decoction piece solution are lighter than those of the test solution, so the next step is to grope the sample amount of the reference substance and radix Angelicae sinensis decoction piece solution.

The second method comprises the following steps: sample amount searching

Sucking the three sample solutions prepared by the first method, increasing the sample application amount of the ferulic acid reference solution and the Chinese angelica decoction piece solution to 10 mu l, changing the sample application amount of the negative sample to 5 mu l, changing the sample application amount of the sample to 5 mu l, 8 mu l and 10 mu l, respectively applying the sample solution to the same silica gel G thin layer plate, developing by using cyclohexane-dichloromethane-ethyl acetate-formic acid (4: 1: 1: 0.1) as a developing agent, taking out, airing, and inspecting under an ultraviolet lamp (365 nm). See fig. 5.

The results show that the sample application amount of the ferulic acid control solution and the Chinese angelica decoction piece is 10 mul, the sample application amount of the test solution is 5 mul, 8 mul and 10 mul, the color spots at corresponding positions are clear and visible, and no tailing phenomenon occurs, so that the concentration of the ferulic acid control solution is determined to be lml solution containing lmg, the sample application amount is 10 mul, the sample application amount of the Chinese angelica piece solution is 1.5g/ml sample application amount is 10 mul, and the sample application amount of the test solution is 10 mul.

Confirmation thin layer identification method: 1.5g each of the product and the negative sample without radix Angelicae sinensis, adding 1% sodium bicarbonate solution 50ml, ultrasonic treating for 10 min, centrifuging, collecting the upper clear solution, adjusting pH with dilute hydrochloric acid toExtracting with diethyl ether under shaking for 2 times, mixing 20ml each time, volatilizing, and dissolving the residue with methanol lml to obtain test solution. Decocting 1.5g radix Angelicae sinensis decoction pieces in 50ml water for 30min, filtering, and making into control medicinal solution by the same method. Taking ferulic acid reference substance, adding methanol to obtain solution containing lmg per lml, taking as reference substance solution, performing thin layer chromatography (0502 in the four departments of the national pharmacopoeia 2015), sucking 10 μ L of the above four solutions, dropping on the same silica gel G thin layer plate, developing with cyclohexane-dichloromethane-ethyl acetate-formic acid (4: 1: 1: 0.1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm).

b durability examination

And (4) conclusion: through durability investigation of low-temperature, high-temperature, normal-temperature, low-humidity and high-humidity environments and different silica gel G thin-layer plates, the durability of the angelica sinensis thin-layer identification condition is good under different influence factors. The thin layer method is hereby incorporated into the text.

c method determination

Collecting 1.5g of the product, adding 1% sodium bicarbonate solution 50ml, ultrasonic treating for 10 min, centrifuging, collecting the upper clear solution, and adjusting pH with dilute hydrochloric acid toExtracting with diethyl ether under shaking for 2 times, mixing 20ml each time, volatilizing, and dissolving the residue with methanol lml to obtain test solution. And adding water 50ml into 1.5g of the angelica control medicinal material, decocting for 30 minutes, filtering, and preparing the filtrate into a control medicinal material solution by the same method. Then, the ferulic acid control is taken and added with methanol to prepare a solution containing lmg per lml as a control solution. Performing thin layer chromatography (0502 of the four ministerial rules of the republic of China pharmacopoeia 2015), sucking 10 μ l of control solution and 10 μ l of control solution, respectively dropping 10 μ l of test solution on the same silica gel G thin layer plate, developing with cyclohexane-dichloromethane-ethyl acetate-formic acid (4: 1: 1: 0.1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm). The chromatogram of the test solution shows the same color at the corresponding positions of the chromatogram of the reference solution and the chromatogram of the reference solutionSpots of color.

d thin layer identification of corresponding real object (dry paste powder) in different batches

Weighing 1.5g of corresponding substance (dry extract powder) of different batches, and making into sample solution by the same method. And (5) performing thin-layer identification.

1.3.3.2.3 examination

(ii) water content

The reference moisture content of the material was measured in 15 lots according to a moisture measurement method (the second method 0832, general rule of four parts of the edition of "Chinese pharmacopoeia" 2015), and the results are shown in Table 18.

TABLE 18 moisture test results Table

The results show that: the 15 batches of the material have the reference moisture range of 5.56-14.54 percent, the average moisture of 9.40 percent and the moisture of the tentative product is not more than 12.0 percent.

(4) Extract of plant

TABLE 19 leachate test results Table

The results show that: the range of 15 material standard extracts prepared from different batches of decoction pieces is 27.47-57.33%, and the average extract is 42.15%. Considering various influence factors and the like in the actual production process, the content of the alcohol-soluble extract of the product is temporarily set to be 30.0-55.0%.

1.3.3.2.4 fingerprint map

Due to the simple qualitative identification and quantitative analysis of index components, the quality of the reference quality of the substance is difficult to reflect. As a standard reference for measuring whether a substance reference corresponding real object is basically consistent with a substance reference, the quality of the standard reference should be enhanced by specificity identification and multi-component and overall quality control. Therefore, the quality control based on material quality control emphasizes the overall quality control mainly based on fingerprint.

Instrument and reagent

Liquid chromatograph: ultimibe 3000, Agilent 1260 II in Sammerfoil;

a chromatographic column: GL Sciences LncAQ-C18(5μm，250×4.6mm)；AQ-C18(5 μm, 250X 4.6mm), etc.

An electronic balance: aohaos instrument (Changzhou) Limited ScottSE (hundredth)

Shanghai Yueping scientific instruments Co., Ltd FA1104 (one in ten thousand)

Japan Shimadzu science and technology Co., Ltd AUW1220D (one hundred thousand)

Acetonitrile is chromatographically pure: TEDIA production; methanol is chromatographically pure: TEDIA production; phosphoric acid is chromatographically pure, and Aladdin is produced; methanol was analytically pure: hongsheng fine chemical company, Heizhou city; water is ultrapure water, Onhancon technologies, Inc.

Ferulic acid control (batch No. 110773-201313, purity: 99.6%), calycosin glucoside control (batch No. 111920-201505, purity: 97.1%), purchased from China institute of food and drug testing.

② investigation of chromatographic conditions

The method comprises the following steps: referring to the method of 'high performance liquid characteristic spectrum comparison of different preparations of angelica blood-enriching decoction',

chromatographic conditions and System suitability test GL Sciences LncAQ-C18(5 μm, 250X 4.6mm) as a chromatographic column; methanol was used as mobile phase a, and 0.4% formic acid solution B was used for gradient elution as specified in table 20; the flow rate is 1ml/min, and the column temperature is 30 ℃; the detection wavelength was 280 nm.

TABLE 20 mobile phase procedure

Preparation of reference solution A proper amount of calycosin glucoside reference and a proper amount of ferulic acid reference are precisely weighed, and methanol is added to prepare a mixed solution containing 200 μ g of calycosin glucoside and 100 μ g of ferulic acid per 1 ml.

Preparation of test solution 1.0g of this product (lot number: DGBXT-2018050801D) is precisely weighed, 1g of dried powder of Angelica sinensis decoction for tonifying blood is weighed, 1.2ml of methanol is added into a 25ml volumetric flask, water is added to the scale, and the solution is filtered to obtain the test solution.

The measurement method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, measuring, and recording chromatogram (see figure 6).

And (4) analyzing results: the method is known to have poor separation effect from a map.

The second method comprises the following steps: reference method for researching HPLC fingerprint spectrum of angelica sinensis blood enriching decoction (I)

Chromatographic conditions and System suitability test GL Sciences LncAQ-C18(5 μm, 250X 4.6mm) as a chromatographic column; methanol was used as mobile phase a, and 0.2% glacial acetic acid solution B was subjected to gradient elution as specified in table 19; flow rate of 1ml/min, columnThe temperature is 25 ℃; the detection wavelength was 254 nm.

TABLE 21 mobile phase procedure

Preparing a sample solution, taking 2.0g of the product (lot number: DGBXT-2018050801D), adding 25ml of water for dissolving, extracting for 3 times by using petroleum ether (60-90 ℃) and 20ml each time, discarding the petroleum ether, extracting water liquid by using water saturated n-butyl alcohol for 4 times and 30ml each time, combining the n-butyl alcohol, washing for 3 times by using the water saturated n-butyl alcohol and 25ml each time, collecting the n-butyl alcohol liquid, evaporating to dryness, adding methanol into residues for dissolving, and fixing the volume to 5ml to obtain the sample solution.

The determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram, as shown in figure 7.

And (4) analyzing results: the method is known to have poor separation effect from a map.

The third method comprises the following steps: reference method for HPLC fingerprint preliminary analysis of radix astragali in n-butanol part of radix Angelicae sinensis decoction for replenishing blood

Chromatographic conditions and System suitability test GL Sciences LncAQ-C18(5 μm, 250X 4.6mm) as a chromatographic column; acetonitrile was used as mobile phase a, and aqueous solution B was subjected to gradient elution as specified in table 22; the flow rate is 1ml/min, and the column temperature is 30 ℃; the detection wavelength was 203 nm.

TABLE 22 mobile phase procedure

Preparing sample solution by dissolving 2.0g of product (lot number: DGBXT-2018050801D) in 50ml of water, extracting with ethyl acetate for 3 times (50 ml each time), extracting water solution with n-butanol for 3 times (50 ml each time), mixing n-butanol solutions, washing with n-butanol saturated water for 2 times (30 ml each time), evaporating n-butanol solution to dryness, dissolving residue with methanol, and diluting to 5ml to obtain sample solution.

And (4) analyzing results: the method is known to have poor separation effect from a map.

The method four comprises the following steps: reference method for HPLC fingerprint chromatogram research of saponin components of angelica blood-enriching decoction

Chromatographic conditions and System suitability test GL Sciences LncAQ-C18(5 μm, 250X 4.6mm) as a chromatographic column; acetonitrile was used as mobile phase a, and aqueous solution B was subjected to gradient elution as specified in table 23; the flow rate is 1ml/min, and the column temperature is 30 ℃; the detection wavelength was 203 nm.

TABLE 23 procedure for mobile phase

Preparing sample solution by dissolving 2.0g of product (lot number: DGBXT-2018050801D) in 50ml of water, extracting with diethyl ether for 3 times (40 ml each time), discarding the diethyl ether solution, extracting the water solution with water saturated n-butanol for 4 times (40 ml each time), mixing the n-butanol solutions, washing with ammonia water for 2 times (40 ml each time), evaporating the n-butanol solution to dryness, dissolving the residue with 5ml of methanol, eluting with 100ml of 40% methanol on a neutral alumina column (100 mesh, 200 mesh, 5g, 1.0cm inner diameter), collecting the eluate, evaporating to dryness, dissolving the residue with 5ml of water, passing through D101 type macroporous adsorbent resin column (inner diameter 1.0cm, length 10cm), eluting with 50ml water, discarding water solution, eluting with 30ml 40% ethanol, discarding eluate, eluting with 80ml 70% ethanol, collecting eluate, evaporating, dissolving residue with methanol, and diluting to 5ml to obtain sample solution.

And (4) analyzing results: the method is known to have poor separation effect from a map.

The method five comprises the following steps: reference method for HPLC fingerprint spectrum research of total glycosides of angelica sinensis blood-enriching decoction

Chromatographic conditions and System suitability test GL Sciences LncAQ-C18(5 μm, 250X 4.6mm) as a chromatographic column; acetonitrile was used as mobile phase a, and aqueous solution B was subjected to gradient elution as specified in table 24; the flow rate is 1ml/min, and the column temperature is 30 ℃; the detection wavelength was 203 nm.

TABLE 24 mobile phase procedure

Preparing a sample solution, taking 2.0g of the product (lot number: DGBXT-2018050801D), adding 50ml of absolute ethanol, carrying out ultrasonic treatment for 30min, extracting with water saturated n-butanol for 3 times, 40ml each time, combining n-butanol solutions, passing through a D101 type macroporous adsorption resin column (with the inner diameter of 1.0cm and the length of 10cm), eluting with 50ml of water, discarding water solution, eluting with 30ml of 40% ethanol, discarding eluent, eluting with 80ml of 70% ethanol, collecting eluent, evaporating to dryness, dissolving residues with methanol, and fixing the volume to 5ml to obtain the sample solution.

And (4) analyzing results: the method is known to have poor separation effect from a map.

The method six: reference method for HPLC fingerprint spectrum and liquid chromatogram-mass spectrum combined analysis of angelica sinensis medicinal material

Chromatographic conditions and System suitability test GL Sciences LncAQ-C18(5 μm, 250X 4.6mm) as a chromatographic column; methanol was used as mobile phase a, and 0.05% formic acid solution B was used for gradient elution as specified in table 25; the flow rate is 1ml/min, and the column temperature is 30 ℃; the detection wavelength was 203 nm.

TABLE 25 mobile phase procedure

Preparation of test solution 2.0g of the product (lot number: DGBXT-2018050801D) is precisely weighed, placed in a conical flask, precisely added with 25ml of methanol-formic acid (95:5), shaken uniformly, sealed, weighed, ultrasonically extracted for 60min, cooled, weighed again, replenished with methanol-formic acid (95:5), shaken uniformly, stood, supernatant is taken, filtered, and a subsequent filtrate is taken to obtain the test solution.

And (4) analyzing results: the method is known to have poor separation effect from a map.

The method comprises the following steps: reference method for HPLC-UV fingerprint spectrum research of astragalus mongholicus

Chromatographic conditions and System suitability test GL Sciences LncAQ-C18(5 μm, 250X 4.6mm) as a chromatographic column; acetonitrile was used as the mobile phase a, and 0.1% phosphoric acid solution B was used for gradient elution as specified in table 26; the flow rate is 1ml/min, and the column temperature is 210 ℃; the detection wavelength was 210 nm.

TABLE 26 mobile phase procedure

Preparing a test solution, taking 2.5g of the product (batch number: DGBXT-2018050801D), carrying out ultrasonic treatment for 45min by using 50ml of 50% methanol, filtering, recovering the solvent from the filtrate under reduced pressure until the filtrate is nearly dry, dissolving the residue by using 2ml of water, carrying out column chromatography by using 10ml of D101 macroporous adsorption resin, eluting by using 50ml of water, then eluting by using 100ml of 95% ethanol, recovering the solvent from the 95% ethanol eluent under reduced pressure until the filtrate is nearly dry, dissolving the 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to a scale, shaking up, and filtering to obtain the test solution.

And (4) analyzing results: the separation effect of the method is better according to the atlas, so that the angelica blood-enriching decoction 'classic famous prescription substance standard' liquid adopts the method to carry out further investigation, verification and research on the fingerprint map.

The method eight: reference method for HPLC-UV fingerprint spectrum research of astragalus mongholicus

Chromatographic conditions and System suitability test GL Sciences LncAQ-C18(5 μm, 250X 4.6mm) as a chromatographic column; acetonitrile was used as mobile phase a, and 0.1% phosphoric acid solution B was used to perform gradient elution as specified in table 27; the flow rate is 1ml/min, and the column temperature is 25 ℃; the detection wavelength was 210 nm.

TABLE 27 mobile phase procedure

Preparation of test solution

Taking 2.5g of the product (lot number: DGBXT-2018050801D), precisely weighing, performing ultrasonic treatment for 45min by using 50% methanol 50ml, filtering, recovering the solvent from the filtrate under reduced pressure till the solvent is nearly dry, dissolving the residue in 20ml of water, extracting for 2 times by using water saturated n-butyl alcohol 25ml each time, combining the n-butyl alcohol solution, extracting for 1 time by using 30ml of ammonia test solution, removing the ammonia test solution, volatilizing the n-butyl alcohol layer solution, dissolving the residue in 2ml of water, performing column chromatography by using 10ml of D101 macroporous adsorption resin, eluting by using 50ml of water, then eluting by using 100ml of 95% ethanol, recovering the solvent from 95% ethanol eluent under reduced pressure till the solvent is nearly dry, dissolving the 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to scale, shaking uniformly, and filtering to obtain the product.

And (4) analyzing results: according to the atlas, the method has fewer impurity peaks than the seventh method, so that the angelica sinensis blood-enriching decoction 'classic famous prescription substance standard' is subjected to fingerprint image research by adopting the method.

Selection of peak attribution and reference object

a attribution of herb flavor

According to the substance standard preparation method, freeze-dried powder of single medicinal material and freeze-dried powder of negative sample lacking single medicinal material are prepared, the sample solution is prepared according to the determined preparation method of the sample solution of fingerprint spectrum, the determination is carried out according to the determined chromatographic conditions, and the chromatographic peak in the fingerprint spectrum of the freeze-dried powder belongs to which medicinal slices, as shown in figure 14 and table 28.

TABLE 28 attribution list of herbs and flavors

And (4) experimental conclusion: the substance standard chromatographic peak is mainly the characteristic peak of angelica and astragalus, and the blank of the mobile phase is free from interference.

b selection of reference

The calycosin glucoside has moderate retention time of chromatographic peak, high response value and baseline separation, so that the calycosin glucoside is selected as a reference peak of a reference substance, the calycosin glucoside is marked as a peak S, and the calycosin glucoside reference substance is used as a reference substance.

Preliminary determination of method

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile was used as mobile phase a, 0.1% phosphoric acid solution was used as mobile phase B, and gradient elution was performed as specified in table 29; the detection wavelength was 210nm, the flow rate was 1ml per minute, and the column temperature was 25 ℃. The number of theoretical plates is not less than 5000 calculated according to calycosin glucoside peak.

TABLE 29 gradient elution Specification

Preparation of reference solution A proper amount of calycosin glucoside reference and a proper amount of ferulic acid reference are precisely weighed, and methanol is added to prepare a mixed solution containing 50 mu g of calycosin glucoside and 20 mu g of ferulic acid per 1ml, so as to obtain the reference solution.

The determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram.

(iii) methodology investigation

a specificity and integrity test

In order to investigate whether the blank solvent has interference, the blank solvent has no interference. Meanwhile, the integrity of the test sample is also considered, and the result basically meets the principle of maximum information quantity, which is shown in figure 15.

And (4) analyzing results: and (3) the blank is found to be free of interference through comparison of a blank solvent spectrum and a chromatographic peak of the test solution.

b precision test

Taking 2.5g of this product (lot number: DGBXT-2018050801D), precisely weighing, carrying out continuous sample injection for 6 times according to the preparation and measurement items of the test solution, calculating the relative retention time and relative peak area, and obtaining the specific measurement results of the relative retention time and relative peak area shown in tables 30-31, FIG. 16

TABLE 29 calculation of fingerprint precision vs. retention time

TABLE 30 summary of relative peak areas of fingerprint precision

The results show that: the relative retention time difference is small, the RSD is less than 2.0 percent, and the precision of the instrument is good. The relative peak area RSD is less than 10.0%, which indicates that the precision of the instrument is good.

c stability test

2.5g of the product (lot number: LGZGT-B-180707-D04) is precisely weighed, the sample is respectively placed for 0, 3, 6, 9, 12 and 24 hours for sample injection and determination after preparation according to the preparation and determination items of the test solution in the text, the relative retention time and the relative peak area are calculated, and the specific determination results of the relative retention time and the relative peak area are shown in the following tables 31 to 32, and figure 17.

TABLE 31 calculation of fingerprint stability versus retention time

TABLE 32 summary of stability of finger prints versus peak area

The result shows that the relative retention time difference is not large, and the RSD is less than 2.0 percent; relative peak area RSD < 10.0%. Indicating that the stability in the test solution is good within 24 hours.

d repeatability test

About 2.5g (total 6 parts) of this product (lot: LGZGT-B-180707-D04) was weighed out precisely and subjected to the following procedures in the text of test solution preparation and measurement. The relative retention time and relative peak area were calculated, and the specific measurement results of the relative retention time and relative peak area are 33 to 34, as shown in FIG. 18.

TABLE 33 calculation of fingerprint repeatability versus retention time

TABLE 34 summary of relative peak areas of repeatability of finger prints

The result shows that the relative retention time difference is not large, and the RSD is less than 2.0 percent; the relative peak area RSD is less than 10.0%, which shows that the repeatability of the method is good.

Fingerprint pattern determination method confirmation

Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 210nm, and the column temperature is 25 ℃. The number of theoretical plates is not less than 5000 calculated according to calycosin glucoside peak.

TABLE 35 gradient elution Table

Preparation of reference solution A proper amount of calycosin glucoside reference and a proper amount of ferulic acid reference are precisely weighed, and methanol is added to prepare a mixed solution containing 50 mu g of calycosin glucoside and 20 mu g of ferulic acid per 1ml, so as to obtain the reference solution.

Preparation of test solution about 2.5g of the product is taken, precisely weighed, ultrasonically treated for 45min by 50ml of 50% methanol, filtered, the solvent is recovered by reducing pressure of the filtrate until the solution is nearly dry, 20ml of water is added into the residue for dissolving, water saturated n-butyl alcohol is used for extracting for 2 times, 25ml of the solution is used for each time, the n-butyl alcohol solution is combined, 30ml of ammonia test solution is used for extracting for 1 time, the ammonia test solution is discarded, the n-butyl alcohol layer solution is volatilized, the residue is dissolved by 2ml of water, the mixture is subjected to column chromatography with 10ml of D101 macroporous adsorption resin, 50ml of water is used for eluting, then 100ml of 95% ethanol is used for eluting, the solvent is recovered by reducing pressure of 95% ethanol eluent until the solution is nearly dry, 50% methanol is dissolved and transferred into a 10ml volumetric flask, the volume.

The determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram

Fifthly, establishing the fingerprint

a calibration of common peaks

Measuring chromatographic peaks of 15 batches of samples by adopting a method under the text (fingerprint), analyzing the obtained fingerprint, selecting chromatographic peaks with good stability and proper response value in the 15 batches of sample fingerprints as common peaks, and calibrating 10 common peaks in total. The results are shown in FIG. 19.

b establishing a control map

The following comparison fingerprint R is generated by adopting the software system 2012 issued by the State pharmacopoeia Committee for evaluating the similarity of traditional Chinese medicine chromatography fingerprints and correcting 10 marked common peaks according to the measurement result of 15 batches of substance standard fingerprints. The results are shown in FIG. 1.

c calculation of similarity

The similarity between 15 batches of samples and the reference fingerprint is calculated by adopting a traditional Chinese medicine chromatogram fingerprint similarity evaluation software system and using common peaks, and the result is shown in Table 36

Table 3615 batch similarity evaluation results

R contrast fingerprint S1.DGBXT-2018050801D S2.DGBXT-2018050802D S3.DGBXT-2018050803D S4.

DGBXT-2018051504D S5.DGBXT-2018051505D S6.DGBXT-2018051506D S7.

DGBXT-2018051507D S8.DGBXT-2018051508D S9.DGBXT-2018051509D S10.

DGBXT-2018052110D S11.DGBXT-2018052111D S12.DGBXT-2018052112D S13.

DGBXT-2018052113D S14.DGBXT-2018052114D S15.DGBXT-2018052115D。

The fingerprint of the corresponding real object (lyophilized powder) of the 15 batches of angelica sinensis blood-enriching soup generates a comparison fingerprint R, so that the similarity of the corresponding real object (lyophilized powder) of the 15 batches is more than 0.85, and the similarity of the corresponding real object (lyophilized powder) of different batches is good. The similarity between the fingerprint of the temporary sample and the reference fingerprint should not be less than 0.850.

1.3.3.2.5 content determination

And comprehensively determining content measurement indexes by combining the prescription efficacy indication and the positions of the prescription medicines in the prescription. As the process research determines that water is used as a solvent, the angelica blood-enriching soup has more water-soluble components on the basis of the material, wherein the monarch drug astragalus contains flavonoids, saponins and the like as the main components, the astragaloside content can be determined, and the ministerial drug angelica contains organic acid, volatile oil and the like as the main components, so the ferulic acid content can be determined to comprehensively evaluate the material basis quality.

Astragalus root

The measurement is carried out by high performance liquid chromatography (0512 in the four-department general regulation of the 2015 edition in China pharmacopoeia).

a instruments and reagents

Liquid chromatograph: shimadzu 2040C, saimer flyaway UltiMate 3000;

a chromatographic column: GL Sciences LncAQ-C18(5μm，250×4.6mm)；WelchAQ-C18(5 μm, 250X 4.6mm), etc.

Shanghai Yueping scientific instruments Co., Ltd FA1104 (one in ten thousand)

Japan Shimadzu science and technology Co., Ltd AUW1220D (one hundred thousand)

Astragaloside IV reference substance (batch number: 110781-.

Preliminary determination of content determination method

According to the content determination of the astragalus root granules in the Chinese pharmacopoeia

Taking 5.0g of the product, accurately weighing, accurately adding 50ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 45 minutes, weighing again, supplementing the weight loss with water, shaking up, filtering, accurately weighing 25ml of subsequent filtrate, shaking up and extracting with water-saturated n-butyl alcohol for 4 times, 25ml each time, combining n-butyl alcohol extract, washing with ammonia test solution for 3 times, 30ml each time, discarding ammonia test solution washing liquor, separating n-butyl alcohol solution, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product. Octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-water (35: 65) is used as a mobile phase; detection by an evaporative light scattering detector. The number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak.

Chromatographic conditions and system applicability test with octadecylsilane chemically bonded silica as filler and acetonitrile-water (35: 65) as mobile phase; detection by an evaporative light scattering detector. The number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak.

Preparation of control solution A proper amount of astragaloside IV control is precisely weighed, and added with methanol to obtain solution containing 0.4mg per lml.

Preparing a test solution, precisely weighing 5.0g of the product, precisely adding 50ml of water, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 30 minutes, weighing again, supplementing the weight loss with water, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, shaking up and extracting with water-saturated n-butyl alcohol for 4 times, 25ml each time, combining n-butyl alcohol extract, washing with ammonia test solution for 3 times, 30ml each time, discarding ammonia test solution washing liquor, separating n-butyl alcohol solution, evaporating to dryness, dissolving residues with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the product.

e methodology examination

a) Investigation of linear relationships

Accurately weighing 15.33071mg of astragaloside control (batch number: 110781-.

The regression equation: 467084x-340818

Correlation coefficient: r-0.9989

The result shows that the astragaloside IV has good linearity in the range of 0.02757-0.22055 mu g.

TABLE 37 Linear relationship of Astragaloside IV

b) Precision of the instrument

Taking astragaloside IV reference substance solution, and continuously injecting sample for 6 times according to text determination method, wherein the determination result is shown in Table 38.

TABLE 38 Instrument precision test results (n ═ 6)

The results show that: RSD is less than 1%, and the precision of the instrument is good.

c) Stability test

5g of the product (lot: DGBXT-2018050801D) is weighed, and the product is prepared and tested according to the procedures of determining the preparation and testing of the test solution, and is respectively placed for 0, 2, 4, 8, 12 and 24 hours after preparation for sample injection and testing, and the testing results are shown in the following table 39.

TABLE 39 stability test results

d) Repeatability test

The experimenter (A) takes about 5g (6 parts in total) of the product (batch number: DGBXT-2018050801D), precisely weighs the product, and operates according to the text sample solution preparation and measurement items. The results are shown in Table 40.

Table 40 repeatability test results (n ═ 6)

The result shows that RSD is less than 3.0%, which indicates that the repeatability of the method is good.

e) Sample application recovery test

Accurately weighing 15.33071mg of astragaloside IV reference substance (batch number: 110786-one 201616, purity 97.4%), placing in a 25ml measuring flask, adding appropriate amount of methanol to dissolve and dilute to scale, and shaking to obtain reference substance solution with concentration of 0.5973 mg/ml. Taking a substance reference sample, namely 2.263mg/g of astragaloside content, precisely weighing 2.5g of astragaloside, adding sample and recovering 6 parts of astragaloside, precisely adding an astragaloside reference substance solution (concentration: 0.5973mg/ml)8.5ml, and the sample solution was prepared according to the method for preparing the test solution and the results were determined according to the method shown in Table 41.

TABLE 41 astragaloside IV accuracy test results (n ═ 6)

As a result, the average recovery rate was 100.79%, and the RSD was < 3.0%, indicating that the method is accurate.

f determination method confirmation

The research result tests show that the method for measuring the content of astragaloside in the real object (freeze-dried powder) corresponding to the angelica blood-enriching soup has strong specificity, the stability, the repeatability, the accuracy and the like all accord with the regulations, and the durability of the chromatographic condition is good, so that the liquid chromatographic condition can be used for measuring the content of astragaloside in the real object (freeze-dried powder) corresponding to the angelica blood-enriching soup, and the specific measuring conditions are as follows:

chromatographic conditions and system applicability test octadecylsilane chemically bonded silica is used as a filler; acetonitrile-water (35: 65) is used as a mobile phase; detection by an evaporative light scattering detector. The number of theoretical plates is not less than 4000 calculated according to astragaloside IV peak.

Preparation of control solution A proper amount of astragaloside IV control is precisely weighed, and added with methanol to obtain solution containing 0.4mg per lml.

The determination method comprises precisely sucking 10 μ L and 20 μ L of reference solution and 20 μ L of test solution, subjecting to liquid chromatograph, and calculating with external standard two-point method.

g multiple batches of corresponding substance (freeze-dried powder) content determination

The astragaloside content in 15 batches of the corresponding real object (lyophilized powder) was determined according to the procedures of preparation and determination of test solution, and the results are shown in Table 42.

TABLE 4215 corresponding Astragaloside content in real object (lyophilized powder)

The results show that: 15 batches of corresponding real object (lyophilized powder) astragaloside with the range of 1.040-1.903 mg/g and the average content of 1.359mg/g, and considering various influence factors and the like in the actual production process, the product is tentatively calculated according to the dry product, and each 1g astragaloside (C) is contained₄₁H₆₈O₁₄) Should be 0.40-2.3 mg (. + -. 3 times SD).

② Chinese angelica root

The measurement is carried out by high performance liquid chromatography (0512 in the four-department general regulation of the 2015 edition in China pharmacopoeia).

a instruments and reagents

Liquid chromatograph: ultimibe 3000, Agilent 1260 II in Sammerfoil;

a chromatographic column: GL Sciences LncAQ-C18(5μm，250×4.6mm)；WelchAQ-C18(5 μm, 250X 4.6mm), etc.

An electronic balance: aohaos instrument (Changzhou) Limited ScottSE (hundredth)

Shanghai Yueping scientific instruments Co., Ltd FA1104 (one in ten thousand)

Japan Shimadzu science and technology Co., Ltd AUW1220D (one hundred thousand)

Ferulic acid reference (batch No. 110773-201313, purity: 99.6%) was purchased from China food and drug testing institute.

b selection of chromatographic conditions

a) Selection of detection wavelength

Taking a proper amount of ferulic acid reference substance, precisely weighing, adding methanol to prepare a solution containing 12 mug per 1ml, and scanning at the wavelength of 190-400 nm, so that the ferulic acid has a maximum absorption peak at 316nm and has no impurity interference, and 316nm is selected as the detection wavelength.

b) Selection of mobile phase

The method comprises the following steps: content determination of Chinese angelica according to Chinese pharmacopoeia

Weighing 2.5g of the powder, precisely weighing, adding appropriate amount of water to dissolve, transferring to a 50ml volumetric flask, adding water to a constant volume to scale, weighing 10ml of the water solution in a 25ml volumetric flask, precisely weighing, and adding methanol to a constant volume to scale to obtain the test solution. Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.085% phosphoric acid solution (17:83) is used as a mobile phase; the detection wavelength is 316 mn; the column temperature was 35 ℃.

The second method comprises the following steps: content determination of gynecological menstruation regulating tablet according to Chinese pharmacopoeia

Weighing 2.5g of the powder, precisely weighing, adding appropriate amount of water to dissolve, transferring to a 50ml volumetric flask, adding water to a constant volume to scale, weighing 10ml of the water solution in a 25ml volumetric flask, precisely weighing, and adding methanol to a constant volume to scale to obtain the test solution. Octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; the wavelength was 320nm as measured with acetonitrile-0.3% phosphoric acid solution (18.5:81.5) as the mobile phase.

As a result: by comparison, the resolution of ferulic acid achieved in the method one. Octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile-0.085% phosphoric acid solution (17:83) is used as a mobile phase; the detection wavelength is 316 mn; the column temperature was 35 ℃.

c examination of preparation method of test solution

a) Examination of extraction methods

Taking about 2.5g (total 3 groups) of the product (batch number: DGBXT-2018050801D), precisely weighing, adding a proper amount of water to dissolve, transferring to a 50ml volumetric flask, adding water to fix the volume to a scale, respectively carrying out ultrasonic treatment (power 500W, frequency 40kHz) for 30 minutes, refluxing and extracting for 30 minutes, directly dissolving, precisely weighing 10ml of aqueous solution in a 25ml volumetric flask, and adding methanol to fix the volume to the scale to obtain the product. The results are shown in Table 43.

Table 43 results of examination of extraction mode

The results show that the extraction rate difference is not large in different extraction modes, and the dissolution treatment is selected in consideration of convenient operation.

b) Examination of extraction vehicle

Precisely weighing about 2.5g (total 3 groups) of the product (lot number: DGBXT-2018050801D), precisely weighing 2.5g of the product powder, adding appropriate amount of water to dissolve, transferring to a 50ml volumetric flask, adding water to a constant volume to scale, precisely weighing 10ml of the aqueous solution in a 25ml volumetric flask, and respectively adding water and methanol to a constant volume to scale. Precisely sucking 10 μ l of the subsequent filtrate, injecting into a liquid chromatograph, measuring, and calculating to obtain the final product. The results are given in Table 44 below.

Table 44 results of the extraction vehicle study

The results show that: the different extraction solvents are not very different, but the methanol treatment content is slightly higher, so methanol is selected as the extraction solvent.

Preliminary determination of the method of determining the content

Chromatographic conditions and system applicability test by using octadecylsilane chemically bonded silica as filler and acetonitrile-0.085% phosphoric acid solution (17:83) as mobile phase; the detection wavelength is 316 nm; the column temperature was 35 ℃. The number of theoretical plates is not less than 5000 calculated according to ferulic acid peak.

Preparation of control solution A proper amount of ferulic acid control was precisely weighed, placed in a brown measuring flask, and 70% methanol was added to obtain a solution containing 12 μ g per 1mL

Preparing test solution by precisely weighing 2.5g of the powder, dissolving with appropriate amount of water, transferring into 50ml volumetric flask, adding water to desired volume to desired scale, weighing 10ml of water solution into 25ml volumetric flask, precisely weighing, and adding methanol to desired volume to desired scale to obtain test solution

The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

e methodology examination

a) Specificity test

Ferulic acid is an index component of angelica in the angelica blood-enriching decoction, in order to investigate whether other medicinal materials interfere the determination of ferulic acid, the medicinal materials are weighed according to the prescription proportion to prepare a negative sample (batch number: DGBXT-2018050801D) by the same method, a solution sample of the negative sample lacking the angelica is prepared according to the treatment method of a test sample, and a chromatogram is recorded. The result shows that the chromatographic peak is present in the negative chromatogram in the retention time corresponding to the ferulic acid, the sample peak area is 2.941, the negative peak area is 0.200, and the calculation shows that the negative peak area is 3.4% and less than 5.0% of the sample peak area, so that the determination of the ferulic acid negative interfering acid in the angelica blood-enriching soup without interference by neglecting the astragalus dry paste is specific to the determination of the ferulic acid in the product by the method. (see FIG. 20)

The result shows that the color peak of the retention time corresponding to the ferulic acid in the negative chromatogram has certain interference, but the interference is within 5 percent and meets the requirement, so the specificity of determining the content of the ferulic acid in the product is good.

b) Investigation of linear relationships

Precisely weighing 13.84mg of ferulic acid reference substance (batch number: 110773-201313, purity: 99.6%), putting into a 100ml measuring flask, adding a proper amount of 70% methanol to dissolve, adding 70% methanol to dilute to scale, and shaking up to obtain 0.1378464mg/ml ferulic acid reference substance storage solution; precisely measuring 10ml of the control stock solution, placing the control stock solution in a 100ml measuring flask, diluting the control stock solution to the scale with 70% methanol, shaking up, and preparing into a control solution with the concentration of 13.78464 mu g/ml. Precisely sucking control solution with concentration of 13.78464 μ g/ml, 2 μ l, 4 μ l, 8 μ l, 12 μ l, 16 μ l, 18 μ l, and 20 μ l, injecting into liquid chromatograph, and measuring by high performance liquid chromatography (0512 in the fourth Proc. 2015 pharmacopoeia). The sample amount (μ g) was taken as the abscissa and the peak area as the ordinate, and a standard curve was drawn. The results are given in Table 45 below.

The regression equation: y 57.909x +0.0238

Correlation coefficient: r is 0.9999

The result shows that the ferulic acid has good linearity in the range of 0.02757-0.27569 mu g

TABLE 45 Linear relationship of ferulic acid

The ferulic acid reference solution was taken, and the sample injection was performed continuously for 6 times according to the text determination method, and the determination results are shown in Table 46.

Table 46 instrument precision test results (n ═ 6)

The results show that: RSD is less than 1%, and the precision of the instrument is good.

e) Stability test

The product (lot: DGBXT-2018050801D) is weighed to about 2.5 m, and is processed according to the items of preparation and determination of the test solution, and is respectively placed for 0, 2, 4, 8, 12 and 24 hours after preparation for injection determination, and the determination results are shown in Table 47 below.

TABLE 47 stability test results

f) Repeatability test

The experimenter (A) takes about 2.5g (total 6 parts) of the product (batch number: DGBXT-2018050801D), precisely weighs the product, and operates according to the text sample solution preparation and determination items. The results are shown in Table 48 below.

Table 48 repeatability test results (n ═ 6)

The result shows that RSD is less than 3.0%, which indicates that the repeatability of the method is good.

g) Sample application recovery test

10.09548mg of ferulic acid reference substance (batch number: 110773-201313, purity 99.6%) is precisely weighed, placed in a 10ml measuring flask, added with a proper amount of methanol to dissolve and dilute to scale, and shaken evenly to obtain a reference substance solution with the concentration of 0.9833 mg/ml. Taking a substance reference sample with ferulic acid content of 0.6293mg/g), precisely weighing 1.5g of ferulic acid in total 6 parts by adding sample and recovering, precisely adding astragaloside IV reference solution (concentration: 0.9833mg/ml)1ml, and the sample solution was prepared according to the method for preparing the test solution and the results were determined according to the method shown in Table 49.

TABLE 49 ferulic acid accuracy test results (n ═ 6)

As a result, the average recovery rate is 99.14%, and the RSD is less than 3.0%, which shows that the method has good accuracy.

f determination method confirmation

The research result tests show that the method for measuring the content of the ferulic acid in the substance (freeze-dried powder) corresponding to the angelica blood-enriching soup has strong specificity, and the stability, the repeatability, the accuracy and the like all accord with the regulations, so the liquid chromatography condition can be used for measuring the content of the ferulic acid in the substance (freeze-dried powder) corresponding to the angelica blood-enriching soup, and the specific measurement conditions are as follows:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile-0.085% phosphoric acid solution (17:83) is used as a mobile phase; the detection wavelength is 316 nm; the column temperature was 35 ℃. The number of theoretical plates should not be less than 7000 calculated from the ferulic acid peak.

The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

g multiple batches of corresponding substance (freeze-dried powder) content determination

The contents of ferulic acid in 15 batches of corresponding substances (lyophilized powder) were determined according to the procedures of preparation and determination of test solution, and the results are shown in Table 50.

TABLE 5015 Ferulic acid content in corresponding substance (lyophilized powder) of batch

The results show that: the range of 15 batches of corresponding substance (freeze-dried powder) ferulic acid is 0.296-0.660 mg/g, the average content is 0.409mg/g, simultaneously various influence factors and the like in the actual production process are considered, the product is tentatively calculated according to the dry product, and every 1g of Chinese angelica blood-enriching soup substance contains ferulic acid (C)₁₀H₁₀O₄) Should be 0.001-0.817 mg (. + -. 3SD value).

1.3.4 Mass analysis of corresponding entities

1.3.4.1 preparation of corresponding entities

Preparing 15 batches of qualified decoction pieces in a prescription according to a determined process, which is detailed in 'data 8.2 process research', preparing 15 batches of substance references in parallel, wherein the dosage of each batch is 10 times daily prescription amount and is about 480g, and specific preparation results are shown in a table 51

TABLE 5115 batch Material benchmark corresponding batch number

1.3.4.2 corresponding substance determination method

(1) And (3) measuring the cream yield: the standard paste yield of different batches of substances is detected according to the determination method of the invention.

(2) And (3) moisture determination: according to the second method of 0832 moisture determination in accordance with the general rule of the four departments of the edition of the Chinese pharmacopoeia 2015, about 2g of a sample is taken and spread in a flat weighing bottle which is dried to constant weight, the thickness is not more than 5mm, the loosened sample is not more than 10mm, the sample is precisely weighed, a bottle cover is opened to be dried for 5 hours at 105 ℃, the bottle cover is covered, the sample is moved into a drier to be cooled for 30 minutes, the sample is precisely weighed, the sample is dried for 1 hour at the temperature, cooled and weighed until the difference between two successive weighing is not more than 5 mg. The water content (%) in the test article was calculated from the weight loss.

(3) And (3) extract determination: the alcohol-soluble hot-dipping method is determined according to a rule 2201 of China pharmacopoeia (general regulations of China pharmacopoeia) 2015: taking a proper amount of the product, grinding, precisely weighing about 2g, placing in a 100ml conical flask, precisely adding 100ml of ethanol, sealing, weighing, standing for 1 hour, performing extract measurement by adopting a hot dipping method, precisely weighing 25ml of filtrate, placing in an evaporation dish dried to constant weight, drying by distillation on a water bath, drying at 105 ℃ for 3 hours, placing in a dryer for cooling for 30 minutes, and rapidly precisely weighing. The content (%) of the alcohol-soluble extract in the test sample was calculated.

(4) Fingerprint spectrum determination: the method for measuring the medicinal materials, the decoction pieces, the intermediates and the corresponding real objects is detailed in a data 6.3.4.1 quality standard text, 10 common peaks are marked according to the measurement result of 15 batches of substance reference fingerprints, a reference fingerprint is generated, and the similarity is calculated. The preparation method of the medicinal materials, the decoction pieces and the intermediate samples comprises the following steps:

a astragalus root medicinal solution: weighing 2.5g of radix astragali powder, precisely weighing with 50ml of 50% methanol, ultrasonically extracting for 45min, filtering, recovering the solvent from the filtrate under reduced pressure until the solvent is nearly dry, dissolving the residue with 20ml of water, extracting with water-saturated n-butanol for 2 times, 25ml each time, mixing the n-butanol solutions, extracting with 30ml of ammonia test solution once, discarding the ammonia test solution layer, evaporating the n-butanol layer to dryness, dissolving the residue with 2ml of water, performing column chromatography with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, discarding the eluent, eluting with 100ml of 95% ethanol, recovering the solvent from the 95% ethanol eluent under reduced pressure until the solvent is nearly dry, dissolving the residue with 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to scale, shaking up, and filtering with a 0.45 mu m filter membrane to obtain the Chinese medicinal composition.

b, astragalus membranaceus decoction piece solution: weighing 2.5g of radix astragali decoction piece powder, precisely weighing with 50% methanol 50ml, ultrasonically extracting for 45min, filtering, recovering solvent from filtrate under reduced pressure till the solution is nearly dry, dissolving residue with 20ml of water, extracting with water saturated n-butanol for 2 times, 25ml each time, mixing n-butanol solutions, extracting with 30ml of ammonia test solution once, discarding ammonia test solution layer, evaporating n-butanol layer to dryness, dissolving residue with 2ml of water, subjecting to column chromatography with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, discarding eluent, eluting with 100ml of 95% ethanol, recovering solvent from 95% ethanol eluent under reduced pressure till the solution is nearly dry, dissolving residue with 50% methanol, transferring to 10ml volumetric flask, fixing volume to scale, shaking up, and filtering with 0.45 μm filter membrane to obtain the Chinese medicinal composition.

c, Chinese angelica medicinal material solution: weighing 2.5g of angelica medicinal material powder, precisely weighing with 50ml of 50% methanol, carrying out ultrasonic extraction for 45min, filtering, recovering a solvent from a filtrate under reduced pressure till the filtrate is nearly dry, dissolving the residue in 20ml of water, extracting with water-saturated n-butyl alcohol for 2 times, 25ml each time, mixing the n-butyl alcohol solutions, extracting with 30ml of ammonia test solution once, discarding an ammonia test solution layer, evaporating the n-butyl alcohol layer to dryness, dissolving the residue in 2ml of water, carrying out column chromatography by using 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, discarding an eluent, eluting with 100ml of 95% ethanol, recovering the solvent from the 95% ethanol eluent under reduced pressure till the residue is nearly dry, dissolving the residue in 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to scale, shaking up, and filtering by using a 0.45 mu m filter membrane to.

d, angelica sinensis decoction piece solution: weighing 2.5g of angelica decoction piece powder, precisely weighing with 50ml of 50% methanol, ultrasonically extracting for 45min, filtering, recovering the solvent from the filtrate under reduced pressure until the solution is nearly dry, dissolving the residue in 20ml of water, extracting with water-saturated n-butyl alcohol for 2 times, 25ml each time, mixing the n-butyl alcohol solutions, extracting with 30ml of ammonia test solution once, discarding the ammonia test solution layer, evaporating the n-butyl alcohol layer to dryness, dissolving the residue in 2ml of water, performing column chromatography with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, discarding the eluent, then eluting with 100ml of 95% ethanol, recovering the solvent from the 95% ethanol eluent under reduced pressure until the solution is nearly dry, dissolving the residue in 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to the scale, shaking up, and filtering with a 0.45 mu m filter membrane to obtain the Chinese angelica.

e intermediate (extract): precisely measuring 25ml of standard extract of angelica blood-enriching soup material, precisely measuring with 50ml of 50% methanol, carrying out ultrasonic extraction for 45min, filtering, recovering the solvent from the filtrate under reduced pressure until the extract is nearly dry, dissolving the residue in 20ml of water, extracting with water-saturated n-butyl alcohol for 2 times, 25ml each time, mixing the n-butyl alcohol solutions, extracting with 30ml of ammonia test solution once, removing the ammonia test solution layer, evaporating the n-butyl alcohol layer to dryness, dissolving the residue in 2ml of water, carrying out column chromatography with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, removing the eluent, eluting with 100ml of 95% ethanol, recovering the solvent from the 95% ethanol eluent under reduced pressure until the extract is nearly dry, dissolving the residue in 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to scale, shaking up, and filtering with a 0.45 mu m filter membrane to.

Intermediate (concentrate): precisely measuring 5ml of angelica blood-enriching soup material reference concentrated solution, precisely measuring with 50ml of 50% methanol, carrying out ultrasonic extraction for 45min, filtering, recovering the solvent from the filtrate under reduced pressure until the solution is nearly dry, dissolving the residue in 20ml of water, extracting with water-saturated n-butyl alcohol for 2 times, 25ml each time, mixing the n-butyl alcohol solutions, extracting with 30ml of ammonia test solution once, discarding the ammonia test solution layer, evaporating the n-butyl alcohol layer to dryness, dissolving the residue in 2ml of water, carrying out column chromatography with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, discarding the eluent, then eluting with 100ml of 95% ethanol, recovering the solvent from the 95% ethanol eluent under reduced pressure until the solution is nearly dry, dissolving the residue in 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to scale, shaking up, and filtering with a 0.45 mu m filter membrane to.

g dry paste powder: precisely measuring 2.5g of standard dry extract powder of the angelica blood-enriching soup substance, precisely measuring with 50ml of 50% methanol, ultrasonically extracting for 45min, filtering, recovering the solvent from the filtrate under reduced pressure until the solution is nearly dry, dissolving the residue in 20ml of water, extracting with 25ml of water-saturated n-butyl alcohol for 2 times, mixing the n-butyl alcohol solutions, extracting with 30ml of ammonia test solution once, discarding the ammonia test solution layer, evaporating the n-butyl alcohol layer to dryness, dissolving the residue in 2ml of water, performing column chromatography with 10ml of D101 macroporous adsorption resin, eluting with 50ml of water, discarding the eluent, then eluting with 100ml of 95% ethanol, recovering the solvent from the 95% ethanol eluent under reduced pressure until the solution is nearly dry, dissolving the residue in 50% methanol, transferring to a 10ml volumetric flask, fixing the volume to scale, shaking up, and filtering with a 0.45 mu m filter membrane to obtain.

(5) Content determination: the determination is carried out according to the method for determining the corresponding substance of the ferulic acid astragaloside IV. The preparation method of the intermediate sample comprises the following steps:

1) extract liquid

Measuring ferulic acid content

Extracting solution: precisely measuring 10ml of the extractive solution, placing in a 25ml measuring flask, adding methanol to dilute to scale, shaking, and filtering.

② determination of astragaloside IV content

Extracting solution: precisely measuring 50ml of the extract, weighing, carrying out ultrasonic treatment (power of 720W and frequency of 40kHz) for 30 minutes, weighing again, supplementing the lost weight with water, shaking up, filtering, precisely measuring 25ml of the subsequent filtrate, shaking up and extracting for 4 times with water-saturated n-butyl alcohol, 25ml each time, combining n-butyl alcohol extract, washing for 3 times with ammonia test solution, 30ml each time, discarding ammonia test solution washing liquid, separating n-butyl alcohol solution, evaporating to dryness, dissolving the residue with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the final product.

2) Liquid medicine

Measuring ferulic acid content

Concentrating the solution: precisely measuring 2ml of the extractive solution, placing in a 25ml measuring flask, adding methanol to dilute to scale, shaking, and filtering.

② determination of astragaloside IV content

Concentrating the solution: precisely measuring 10ml of concentrated solution, placing in a 50ml measuring flask, adding water to dilute to scale, weighing, ultrasonically treating (power of 720W and frequency of 40kHz) for 30 minutes, weighing again, supplementing the lost weight with water, shaking up, filtering, precisely weighing 25ml of subsequent filtrate, shaking up and extracting with water saturated n-butanol for 4 times, 25ml each time, combining n-butanol extractive solutions, washing with ammonia test solution for 3 times, 30ml each time, discarding ammonia test solution washing solution, separating n-butanol solution, evaporating to dryness, dissolving residue with methanol, transferring to a 5ml measuring flask, adding methanol to dilute to scale, and shaking up to obtain the final product.

1.3.4.3 measurement results

The results are shown in summary 52

TABLE 52 reference measurement results for different batches of substances

1.3.4.4 determination of object key quality attribute range

(1) Rate of paste discharge

The standard cream yield range of 15 batches of materials is 13.39-22.25%, and the average cream yield is 17.83%, which are 10.67-25.0% in the 3SD floating range; the +/-30% floating range is 12.48-23.18%. The range of the temporary cream yield is 10.00-25.00%.

(2) Moisture content

The standard moisture range of 15 batches of the material is 5.56-11.80%, the average moisture is 9.04%, and the tentative moisture is not more than 12.0%.

(3) Extract of plant

The range of 15 batches of material standard extract is 27.47-57.33%, the average extract is 42.15%, and the range of tentative extract is 30.0-55.0%.

(4) Finger print

15 batches of angelica sinensis blood-enriching soup substance reference fingerprints generate comparison fingerprints, 15 batches of substance reference similarity is larger than 0.850 in the next comparison, the substance reference similarity between different batches is good, and the provisional fingerprint similarity limit is larger than or equal to 0.850.

(5) Determination of content

The range of 15 batches of substance-based ferulic acid is 0.296-0.660 mg/g, the average content is 0.409mg/g, and the ranges are +/-30% and 3 SD; referring to + -30% and 3SD floating range, considering various influence factors in actual production process, etc., tentatively calculating the product according to dry product, and adding ferulic acid (C) to every 1g radix Angelicae sinensis₁₀H₁₀ O₄) The amount of the active ingredient is 0.001-0.817 mg.

The range of 15 batches of material standard astragaloside IV is 1.040-1.903 mg/g, the average content is 1.359mg/g, the astragaloside IV is within 3SD floating range, considering various influence factors in the actual production process, the product is tentatively calculated according to the dry product, and each 1g of astragaloside IV (C)₄₁H₆₈O₁₄) The amount of the active ingredient is 0.40-2.3 mg.

1.4.1 detection method of radix Angelicae sinensis blood-tonifying medicinal composition

1.4.1.1 prescription

The recipe is in the book of internal and external injury theory of Lidonyuan, gold Yuan. The original prescription name is Dang Gui Buxue Tang, and the detailed study is shown in the data of prescription examination and historical welfare.

1.4.1.2 method for making same

Decocting according to the method described in the list of classical name prescriptions. Decocting in an electronic decoction pot (capacity of 9L), and researching related parameters by using extract yield, total extraction amount of ferulic acid and astragaloside IV and fingerprint spectrum as indexes, such as pretreatment of material-based decoction pieces, heating mode, decocting time, filtering and drying process, etc., thereby determining the material-based process of the Chinese angelica blood-enriching decoction. According to the determined process, 10 times daily prescription is adopted for feeding, two parallel samples are designed in each group of experiment, each parallel sample comprises 10 parts of daily prescription, extracting solutions are mixed, concentrated and then subjected to plate freeze drying. 15 batches of substance-based corresponding real objects are obtained, and the result shows that the substance-based preparation process is stable and feasible. The standard preparation method of the angelica blood-enriching decoction comprises the following steps: 400g of astragalus root and 80g of angelica, adding 4000ml of water into the two medicines, soaking for 40min, decocting for 60min in an electronic decoction pot, filtering, concentrating, and freeze-drying to obtain the traditional Chinese medicine.

1.4.1.3 trait

Temporarily setting the product as light yellow powder according to the actual observation condition of a plurality of batches of samples; light smell, slightly sweet taste.

1.4.1.4 identification

The product is prepared from two traditional Chinese medicines, is a compound preparation prepared by a water decoction process, adopts a thin-layer identification method with strong specificity, rapidness, simplicity and good reproducibility, carries out identification test research on main characteristic components of astragalus and angelica in the prescription, and confirms that the astragalus and the angelica are brought into quality standard.

(1) Thin layer identification of astragalus

The radix astragali contains saponins and flavonoids as main chemical components, and also contains polysaccharide. Performing thin layer chromatography experiments on radix astragali according to the above chemical components to determine identification method

Dissolving 1.5g of the product in 25mL of water, performing ultrasonic treatment for 30 minutes, filtering, extracting the filtrate with 25mL of water-saturated n-butanol under shaking for 2 times, mixing the n-butanol extractive solutions, washing with ammonia test solution for 2 times, 30mL each time, collecting n-butanol extractive solution, evaporating to dryness, and dissolving the residue with lmL of methanol to obtain test solution. And adding water 50ml into astragalus root decoction pieces 1.5g, decocting for 30min, filtering, and preparing a control medicinal solution by the same method. Adding methanol to a proper amount of astragaloside IV reference substance to obtain a solution containing 2mg per 1ml, and using as reference substance solution. Performing thin layer chromatography (0502 of the four ministerial rules of the four parts of the book of the Chinese pharmacopoeia 2015), sucking 10 μ l of each of the four solutions, dropping on the same silica gel G thin layer plate, developing with chloroform-methanol-water (13:7:2) lower layer solution as developing agent, taking out, air drying, spraying with 10% ethanol sulfate solution, heating at 105 deg.C until the spots are clearly developed, and inspecting under ultraviolet lamp (365 nm).

Durability of the self-made silica gel G thin layer plate manufactured in low-temperature, normal-temperature and high-temperature environments and by a machine-made silica gel G thin layer plate is inspected, and the thin layer identification condition is good in durability under different influence factors. Spots of the same color appear on the chromatogram of the test solution corresponding to the real object (lyophilized powder) in different batches at the positions corresponding to the chromatogram of the reference solution. The thin layer method is hereby incorporated into the text.

(2) Thin layer identification of Chinese angelica

The angelica mainly contains benzene compounds such as ferulic acid, ligustilide and the like. The volatile oil is the main active component of angelica, mainly contains ester components such as ligustilide, etc., after ligustilide is extracted and dried, the ratio of the ligustilide in dry paste powder is not large, the angelica is subjected to the following thin-layer grope experiment by deliberately using the chemical components of ferulic acid, and the following method is determined.

Collecting 1.5g of the product, adding 1% sodium bicarbonate solution 50ml, ultrasonic treating for 10 min, centrifuging, collecting the upper clear solution, and adjusting pH with dilute hydrochloric acid toExtracting with diethyl ether under shaking for 2 times, mixing 20ml each time, volatilizing, and dissolving the residue with methanol lml to obtain test solution. And adding 50ml of water into 1.5g of angelica decoction pieces, decocting for 30 minutes, filtering, and preparing a control medicinal solution by the same method. Taking ferulic acid reference substance, adding methanol to obtain solution containing lmg per lml, taking as reference substance solution, performing thin layer chromatography (0502 in the four departments of the national pharmacopoeia 2015), sucking 10 μ L of the above four solutions, dropping on the same silica gel G thin layer plate, developing with cyclohexane-dichloromethane-ethyl acetate-formic acid (4: 1: 1: 0.1) as developing agent, taking out, air drying, and inspecting under ultraviolet lamp (365 nm).

Durability of the self-made silica gel G thin layer plate in low-temperature, normal-temperature and high-temperature environments and a machine-made silica gel G thin layer plate is inspected, and the durability of the angelica sinensis thin layer identification condition is good under different influence factors. In the chromatogram of the test sample based on different batches of substances, spots with the same color appear at the corresponding positions of the chromatogram of the reference medicinal material and the chromatogram of the reference substance. The thin layer method is hereby incorporated into the text.

1.4.1.5 examination

(1) Moisture content

According to a water content measuring method (a second method 0832 in the four ministry of general rules of China pharmacopoeia 2015), the standard water content of different batches is measured, the standard water content of 15 batches of materials is 5.56-14.54%, the average water content is 9.40%, and the water content of a provisional product is not more than 12.0%.

1.4.1.6 extract

Most of chemical components or major components in the Chinese medicinal materials can be dissolved out in water or alcohol, and then extract measurement is carried out. Because the standard of the angelica sinensis blood-enriching soup is water extraction, and the significance of establishing extract measurement by selecting water as a solvent is not great, and the internal quality is difficult to reflect, the solvent is selected, the solubility of the major components is considered, and ethanol is selected for extract measurement so as to control the quality of the standard of the substance. The content of extract in 15 batches of classical famous prescription material standards is measured according to an extract measuring method (2201 alcohol-soluble hot dipping method in the four ministry of communications in the national pharmacopoeia 2015), the extract range is 27.47-57.33%, and the average extract is 42.15%. Considering various influence factors and the like in the actual production process, the content of the alcohol-soluble extract of the product is temporarily set to be 30.0-55.0%. .

1.4.1.7 finger print

The method is searched, and the selection of drug flavor attribution and reference substances is carried out on each spectrum peak in the substance standard fingerprint (the calycosin glucoside with moderate retention time and high response value which achieves baseline separation is selected as the reference substance), and the fingerprint determination method is preliminarily determined. The methodological verification is carried out on the preliminarily determined fingerprint spectrum method, the method has good specificity, the blank solvent has no interference, and simultaneously the principle of the maximum information content is basically satisfied, the precision, the repeatability and the stability (within 24 hours) of the method accord with the regulations (the relative retention time RSD of each spectrum peak is less than 2 percent), and the common peak in the chromatogram is sharp in shape, symmetrical and good in separation degree, so the method is determined as the fingerprint spectrum determination method and listed in the standard text:

octadecylsilane chemically bonded silica is used as a filler in chromatographic conditions and system applicability tests; acetonitrile is taken as a mobile phase A, 0.1 percent phosphoric acid solution is taken as a mobile phase B, and gradient elution is carried out according to the specification in the following table; the detection wavelength is 210nm, and the column temperature is 25 ℃. The number of theoretical plates is not less than 5000 calculated according to calycosin glucoside peak.

TABLE 53 gradient elution Table

Preparing sample solution by collecting 2.5g, precisely weighing, subjecting to 50ml of 50% methanol ultrasonic treatment for 45min, filtering, recovering solvent from filtrate under reduced pressure to near dryness, dissolving residue in 20ml of water, extracting with water saturated n-butanol for 2 times (25 ml each time), mixing n-butanol solutions, extracting with 30ml of ammonia solution for 1 time, discarding ammonia solution, volatilizing n-butanol layer solution, dissolving residue in 2ml of water, subjecting to column chromatography with 10ml of D101 macroporous adsorbent resin, eluting with 50ml of water, eluting with 100ml of 95% ethanol, recovering solvent from 95% ethanol eluate under reduced pressure to near dryness, dissolving 50% methanol, transferring to 10ml volumetric flask, fixing volume to desired scale, shaking, and filtering to obtain the final product

The determination method comprises precisely sucking 10 μ l of reference solution and sample solution respectively, injecting into liquid chromatograph, determining, and recording chromatogram.

And (3) measuring 15 batches of substance references according to the determination method, analyzing the obtained fingerprints, selecting chromatographic peaks with better stability and proper response value in the 15 batches of substance reference fingerprints as common peaks, and calibrating 10 common peaks in total. The method comprises the steps of automatically matching 1 batch of angelica sinensis blood-enriching soup substance standard with a physical object (dry paste powder) HPLC chromatogram peak by adopting 'traditional Chinese medicine chromatogram fingerprint similarity evaluation software system 2012 edition' issued by the State pharmacopoeia Committee to form a common pattern diagram, and establishing a comparison spectrum. The similarity of 15 batches of samples and the comparison fingerprint is calculated by using the common peak and is more than 0.90, so that the fingerprint is selected as the evaluation standard of the angelica blood-enriching soup substance standard, and the similarity of the product and the angelica blood-enriching soup substance standard comparison fingerprint is tentatively determined to be not less than 0.90.

1.4.1.8 content determination

(1) Radix astragali

And (4) groping the astragaloside determination method to preliminarily determine the content determination method. The methodology of the preliminary determination method is verified, the method has good specificity, the astragalus root-lacking negative sample has no interference, the purity of the target peak is qualified, and the astragaloside has good linearity within the range of 1.1946-11.946 mug. And the method has the advantages of precision (RSD < 2.0%), repeatability (RSD < 3.0%), stability (RSD < 3.0% in 24 hours), average recovery rate of 100.79%, RSD of 2.09% meeting the specification, and good durability of chromatographic conditions, so the liquid chromatographic conditions can be used for measuring the content of astragaloside in the corresponding real object (freeze-dried powder) of the angelica blood-enriching soup, and the specific measuring conditions are as follows:

Preparation of control solution A proper amount of astragaloside IV control is precisely weighed, and added with methanol to obtain solution containing 0.4mg per lml.

15 batches of corresponding real object (lyophilized powder) astragaloside with the range of 1.040-1.903 mg/g and the average content of 1.359mg/g, and considering various influence factors and the like in the actual production process, the product is tentatively calculated according to the dry product, and each 1g astragaloside (C) is contained₄₁H₆₈O₁₄) Should be 0.40-2.3 mg (. + -. 3 times SD).

(2) Radix Angelicae sinensis

And (4) groping the ferulic acid determination method and primarily determining the content determination method. The methodology of the preliminary determination method is verified, the specificity of the method is good, the interference of the angelica-deficient negative sample is not obvious, the purity of the target peak is qualified, and the linearity of the ferulic acid is good within the range of 0.02757-0.27569 mug. And the method has the advantages of precision (RSD less than 2.0%), repeatability (RSD less than 3.0%), stability (RSD less than 3.0% in 24 hours), accuracy average recovery rate of 99.71%, RSD 0.86% meeting the specification, and good durability of chromatographic conditions, so the liquid chromatographic conditions can be used for measuring the content of ferulic acid in a corresponding real object (freeze-dried powder) of the angelica blood-enriching soup, and the specific measuring conditions are as follows:

The determination method comprises precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and determining.

The range of 15 batches of corresponding substance (freeze-dried powder) ferulic acid is 0.296-0.660 mg/g, the average content is 0.409mg/g, simultaneously various influence factors and the like in the actual production process are considered, the product is tentatively calculated according to the dry product, and every 1g of Chinese angelica blood-enriching soup substance contains ferulic acid (C)₁₀H₁₀O₄) Should be 0.001-0.817 mg (. + -. 3 SD).

While the invention has been described in detail in the foregoing by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that certain changes and modifications may be made therein based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

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