CN114689774A

CN114689774A - Preparation process and quality control method of standard decoction of radix Angelicae sinensis decoction for replenishing blood

Info

Publication number: CN114689774A
Application number: CN202011605399.8A
Authority: CN
Inventors: 周厚成; 胡昌江; 黄宇; 姜艳娇; 仰莲
Original assignee: Sichuan New Green Pharmaceutical Technology Development Co ltd
Current assignee: Sichuan New Green Pharmaceutical Technology Development Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2022-07-01
Anticipated expiration: 2040-12-30
Also published as: CN114689774B

Abstract

The invention discloses a preparation process of standard decoction of angelica sinensis blood-enriching decoction and a quality control method thereof, wherein a preparation process taking radix angelicae sinensis and radix astragali decoction pieces washed with wine as formula raw materials is established, the contents of astragaloside, calycosin glucoside and ferulic acid are taken in combination with the paste yield as investigation indexes, the selection of each parameter condition in the decoction process is determined, the process operation is stable, reliable, reasonable and feasible, meanwhile, the content indexes of 3 components of ferulic acid, calycosin glucoside and astragaloside in the standard decoction of the angelica sinensis blood-enriching decoction and the detection method of HPLC-DAD and HPLC-DAD-ELSD combined 2 characteristic maps are established, the comprehensive reflection and control of the quality of the standard decoction are further realized, and the stability and reliability of the preparation process of the standard decoction of the angelica sinensis blood-enriching decoction are ensured.

Description

Preparation process and quality control method of standard decoction of radix Angelicae sinensis decoction for replenishing blood

Technical Field

The invention belongs to the field of traditional Chinese medicine preparations and detection, and particularly relates to a preparation process and a quality control method of standard decoction of angelica sinensis blood-enriching decoction.

Background

The ancient classical prescription is a treasure of Chinese traditional medicine prescription, is a summary of clinical practice experience of doctors of all generations, and gathers thousands of years of wisdom of Chinese traditional medicine people. The Chinese medicine law is clear at the same time, and the Chinese medicine compound preparation which meets the national regulation and is derived from the ancient classical famous prescription is produced, so that only non-clinical safety research data can be provided when a medicine approval document number is applied. The simplified registration approval management regulation of the ancient classical famous prescription traditional Chinese medicine compound preparation released in 2018 is proposed again, the classical famous prescription preparation meeting the requirements is applied to be listed on the market, and only pharmaceutical and non-clinical safety research data can be provided, and pharmacodynamic research and clinical test data are not reported. Therefore, the basic work of classical famous research is very important.

According to the related guidance suggestions of the declaration data requirement (request for comments) of the ancient classical famous-party Chinese herbal compound preparation and the declaration data requirement (request for comments) of the ancient classical famous-party Chinese herbal compound preparation on the basis of the substance, which are published by the State administration of drug supervision and management in 2019, the compound preparation of the classical famous-party must comply with the requirement on the basis of the substance, and the substance standard must strictly refer to the ancient prescription, including the requirements on the basis of medicinal materials, the production place, the harvesting period, the initial processing of the production place and the quality of the medicinal materials, the processing, the process and the quality of decoction pieces, the decocting process, the dosage and the like. Therefore, the development of the material benchmark research (standard decoction stage) of the classical famous prescription plays a decisive role in the safety and the effectiveness of the compound preparation of the ancient classical famous prescription.

The Dang Gui Buxue Tang is a classic and famous prescription of traditional Chinese medicine, which was recorded in "differentiation and confusion of internal and external injuries" of jin-Li Dongyuan, and has the efficacies of tonifying qi and generating blood, etc., and is mainly used for treating heat syndrome due to blood deficiency and yang floating. Modern medical research finds that the traditional Chinese medicine composition has the effects of improving the function of a blood system, regulating immunity, protecting the liver, resisting oxidation and the like, and can also be used for postoperative conditioning of various cancer patients. Wide applicable population, large base number, long taking time and larger medicine development market space. The Dang Gui Bu Xue Tang comes from Li Dongyuan's "Wai Shang Bian Huo Lun", which is originally for treating myoheat, dryness-heat, thirst with water, conjunctival congestion and flushing, and lingering day and night. It is surging and deficient in pulse, and forcefully pressing all without. The "internal classic" is in the name: the pulse is deficient and the blood is deficient. And cloud: fever due to blood deficiency with white appearanceTiger, being unable to feel comfortable, is identified by taking the decoction of Baihu by mistake. The disease is caused by hunger and sleepy labor; astragalus root, radix astragali, angelica sinensis (washed with wine) and two coins; upper piece

The mouth is taken as one piece, two pieces of water are taken, one piece is decocted, dregs are removed, the medicine is taken warmly, and the medicine is taken before eating. "

However, the present preparation, angelica blood-enriching oral liquid, which is related to angelica blood-enriching decoction and is collected in the chinese pharmacopoeia, has a prescription composition of 132g of angelica and 330g of astragalus, and the preparation method adopts the steps of adding water into angelica for distillation, then decocting the angelica with the astragalus, and extracting with ethanol, which is far different from the requirements of declaration data (survey of comments) of the ancient classic famous Chinese medicinal compound preparation material standard, the requirements of wine-washing angelica and the decoction process of the ancient classic famous Chinese medicinal catalog (first batch) published by the national traditional Chinese medicine administration. Meanwhile, in the relevant preparations of angelica blood-enriching soup sold in the market, for example, the angelica blood-enriching pill is also described as angelica, and the specific processed product is not clear. On the other hand, although the state has developed a guiding principle (solicited for comments), the description is only "astragalus root is one or two, and angelica (washed with wine) is two money; upper piece

The mouth is taken as one piece, two pieces are taken in water, one piece is decocted, dregs are removed, the mouth is taken warmly, and the mouth is taken before eating, so that the specific preparation process parameters are not determined, and the actual preparation process application operation of each unit is difficult.

Therefore, the research reports on the relevant preparation process and quality control of the standard decoction of the angelica blood-enriching decoction based on the national policy requirements are deficient, and the research on the preparation process and quality control of the current standard decoction of the angelica blood-enriching decoction should be established for further guiding the later work of the formula particle preparation.

In the 'Chinese herbal medicine'

volume

43, 3 rd month 2012 in 3 rd period, 482 plus 486, according to the components and the drug effects of the prescription drugs, the index components of astragalus astragaloside, the index line components ferulic acid of angelica, and the total saponins and total polysaccharides in the prescription are selected as evaluation indexes of the water decoction extraction process, wherein the amount of the total polysaccharides is determined by adopting a phenol-sulfuric acid method, the amount of the total saponins is determined by adopting an ultraviolet absorption method, the amount of the ferulic acid is determined by adopting an HPLC method, the amount of the astragaloside is determined by adopting an HPLC-ELSD method, and the extraction process of the angelica blood-enriching decoction is determined as follows: weighing 12g of angelica and 60g of astragalus, adding 12 times of water, and extracting for 3 times, wherein each time lasts for 1.0 hour.

The contents of ferulic acid, calycosin glucoside and astragaloside IV in the Chinese angelica blood replenishing decoction are simultaneously measured by adopting an HPLC-DAD/ELSD method of Panegor et al in 'the Chinese angelica blood replenishing decoction for simultaneously measuring three index components' No. 3, 547-550 of 2016, the contents of ferulic acid, calycosin glucoside and astragaloside IV in the Chinese angelica blood replenishing decoction are simultaneously measured by adopting an HPLC-DAD/ELSD method, and methodology is investigated.

The invention discloses a method for simultaneously determining 18 compounds with different structure types in angelica sinensis blood-enriching soup, which is disclosed in an invention patent with the publication number of CN11155162A and the name of the method for detecting the quality of the angelica sinensis blood-enriching soup, and particularly discloses a method for simultaneously detecting 18 compounds in the angelica sinensis blood-enriching soup by screening appropriate chromatographic conditions and mass spectrum conditions according to the structures and the property specificities of saponins, flavonoids and volatile oil components in the angelica sinensis blood-enriching soup.

In view of the above situation, we find that the following problems still exist in the prior art in the research process of quality control of the angelica blood-enriching decoction:

(1) the angelica sinensis blood-enriching soup recorded in the prior art does not specify the specific processed product of angelica sinensis in the formula, and in view of the difference of index components and medicinal effects of angelica sinensis and alcohol-washed angelica sinensis, a method for further specifying the content of the alcohol-washed angelica sinensis in the angelica sinensis blood-enriching soup is particularly important.

(2) Although the patent CN11155162A establishes a method for simultaneously measuring 18 compounds in the angelica sinensis blood-enriching decoction, the equipment investment cost is high, the conditions of chromatography and mass spectrum for screening 18 compounds are harsh, and the detection accuracy is difficult to be considered at the same time.

Disclosure of Invention

The invention aims to provide a preparation process of standard angelica sinensis blood-enriching decoction liquid by taking alcohol-washed angelica sinensis and astragalus membranaceus decoction pieces as formula raw materials.

In order to realize the stability and reliability of the preparation process of the standard angelica blood-enriching decoction, the invention also provides a quality control method of the standard angelica blood-enriching decoction.

The invention is realized by the following technical scheme:

a preparation process of standard decoction of angelica sinensis blood-enriching soup comprises the steps of mixing 1 part of wine-washed angelica sinensis and 5 parts of astragalus membranaceus decoction pieces in parts by weight, adding 600ml of water, soaking for 30min, adding a cover, boiling with strong fire, turning to slow fire, decocting for 40min, filtering while hot, cooling and drying to obtain freeze-dried powder of the standard decoction of the angelica sinensis blood-enriching soup.

A quality control method of standard decoction of radix Angelicae sinensis decoction for tonifying blood comprises constructing characteristic spectrum of wine-washed radix Angelicae sinensis in the standard decoction of radix Angelicae sinensis decoction for tonifying blood prepared by the above method, and comprises the following steps:

A. preparing reference solution A and test solution I respectively,

reference solution a: respectively taking ferulic acid, calycosin glucoside, senkyunolide I and ligustilide, adding methanol to prepare solutions of 50-100 μ g per 1ml,

test solution I: taking 0.2g of lyophilized powder of standard decoction of radix Angelicae sinensis decoction for replenishing blood, adding 10ml of 70% methanol, performing ultrasonic treatment for 30 minutes, shaking up, filtering, and taking the subsequent filtrate to obtain the final product;

B. respectively taking 10 mu l of reference substance solution A and sample solution I, injecting into a high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A, and 0.1% phosphoric acid solution as a mobile phase B, performing gradient elution, and measuring at a column temperature of 30 ℃, a flow rate of 1.0ml per minute and a detection wavelength of 290nm to obtain corresponding characteristic maps;

C. and (3) taking the characteristic spectrum of the reference substance solution A as a reference spectrum, selecting common peaks from the characteristic spectrum of the test solution I, and constructing the characteristic spectrum of the wine-washed angelica in the standard decoction of the angelica blood-enriching decoction.

In the step B, gradient elution meets the following conditions:

0-30 min, mobile phase A: 10% → 30%, mobile phase B: 90% → 70%;

30-45 min, mobile phase A: 30% → 80%, mobile phase B: 70% → 20%;

45-60 min, mobile phase A: 80% → 95%, mobile phase B: 20% → 5%.

The method also comprises the construction of the characteristic spectrum of the astragalus decoction pieces in the standard decoction of the angelica blood-enriching decoction prepared by the method, and the steps are as follows:

A. respectively preparing a reference substance solution B and a test solution II,

reference solution: respectively adding formononetin, calycosin glucoside, astragaloside I, astragaloside II, isoastragaloside I and ferulic acid into methanol to obtain solutions each containing 50-100 μ g per 1ml,

sample solution ii: taking 1g of lyophilized powder of standard decoction of radix Angelicae sinensis decoction for replenishing blood, adding 20ml of water, performing ultrasonic treatment for 30 minutes, shaking up, filtering, and taking subsequent filtrate;

B. respectively taking 10 mu L of reference substance solution B and sample solution II, injecting into a high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A, and 0.02% formic acid solution as a mobile phase B, performing gradient elution, detecting by using an ultraviolet detector and evaporative light scattering at a column temperature of 25 ℃ and a flow rate of 1.0ml per minute, wherein the detection wavelength of the ultraviolet detector is 254nm, the temperature of a drift tube of the evaporative light scattering detector is 90 ℃, and the flow rate of carrier gas is 1.8L/ml per minute, and obtaining corresponding characteristic maps;

C. and (4) taking the characteristic map of the reference substance solution B as a reference map, selecting common peaks from the characteristic maps of the test solution II, and constructing the characteristic map of the astragalus decoction pieces in the standard decoction of the angelica blood-enriching decoction.

In the step B, gradient elution meets the following conditions:

0-30 min, mobile phase A: 20% → 45%, mobile phase B: 80% → 55%;

30-40 min, mobile phase A: 45% → 80%, mobile phase B: 55% → 20%.

The method also comprises the step of measuring the content of ferulic acid in the standard decoction of the angelica sinensis blood-enriching decoction prepared by the method by adopting a high performance liquid chromatography, and the steps are as follows:

A. respectively preparing ferulic acid reference solution and test solution III,

ferulic acid control solution: taking appropriate amount of ferulic acid reference substance, adding 70% methanol to obtain solutions containing 12 μ g ferulic acid per 1mL,

test solution iii: taking 0.1g of lyophilized powder of standard decoction of radix Angelicae sinensis blood replenishing decoction, adding 10ml of 70% methanol, sealing, weighing, ultrasonically extracting for 30min, cooling, weighing again, supplementing the reduced weight with 10% methanol, shaking, standing, collecting supernatant, filtering, and collecting filtrate;

B. respectively taking 10 mu l of each ferulic acid reference solution and test solution III, injecting into a high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as a filler, and taking acetonitrile-0.085% phosphoric acid solution 17: 83 is mobile phase, the detection wavelength is 316nm, the column temperature is 35 deg.C, and the content of ferulic acid in standard decoction of radix Angelicae sinensis decoction for replenishing blood is not less than 0.02%.

The method also comprises the step of measuring the content of calycosin glucoside in the standard decoction of the angelica sinensis blood-enriching decoction prepared by the method by adopting a high performance liquid chromatography, and the steps are as follows:

A. respectively preparing a calycosin glucoside reference solution and a test solution V,

calycosin glucoside control solution: taking appropriate amount of calycosin glucoside reference, adding methanol to obtain solutions containing 50 μ g calycosin glucoside per 1mL respectively,

test solution v: taking 0.5g of freeze-dried powder of standard decoction of the angelica blood-enriching decoction, adding 10mL of methanol, sealing, weighing, performing ultrasonic treatment at the frequency of 40kHz and the power of 600W for 30 minutes, cooling, weighing again, supplementing the lost weight with the methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the Chinese angelica blood-enriching decoction;

B. respectively taking 10 mul of each of the calycosin glucoside reference solution and the test solution V, injecting into a high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A, and 0.2% formic acid solution as a mobile phase B for gradient elution, and measuring at an inspection wavelength of 260nm to obtain the standard decoction of the angelica sinensis hematinic soup, wherein the content of calycosin glucoside in the standard decoction is not less than 0.02%.

In the step B, gradient elution meets the following conditions:

0-20 min, mobile phase A: 20% → 40%, mobile phase B: 80% → 60%;

20-30 min, mobile phase A: 40%, mobile phase B: 60 percent.

Also comprises the step of measuring the content of astragaloside in the standard decoction of the angelica sinensis blood-enriching decoction prepared by the method by adopting a high performance liquid chromatography, and the steps are as follows:

A. preparing astragaloside IV reference substance solution and test solution VI,

astragaloside IV control solution: taking appropriate amount of astragaloside IV reference substance, adding methanol to obtain solutions containing astragaloside IV 0.5mg per 1mL respectively,

test solution vi: taking 1.0g of freeze-dried powder of standard decoction of the angelica blood-enriching decoction, adding 50mL of 80% methanol containing 4% concentrated ammonia solution, sealing, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the reduced weight with 80% methanol containing 4% concentrated ammonia solution, shaking up, filtering, precisely taking 25mL of subsequent filtrate, evaporating to dryness, dissolving the residue with 80% methanol, transferring to a 5mL measuring flask, adding 80% methanol to scale, shaking up, filtering, and taking subsequent filtrate;

B. respectively taking 5 mul and 10 mul of astragaloside IV reference substance solution and 20 mul of test substance solution VI, injecting into a high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as a filler, and mixing acetonitrile-water 32: 68 is mobile phase, measuring with evaporative light scattering detector to obtain standard decoction of radix Angelicae sinensis decoction for replenishing blood with astragaloside IV content of 0.05% or more.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the invention develops the research on the preparation process of the standard angelica sinensis blood-enriching decoction based on the national policy guidance (angelica sinensis and astragalus decoction pieces are used as formula raw materials) for the first time, adopts the contents of astragaloside IV, calycosin glucoside and ferulic acid and the cream yield thereof as main investigation indexes, determines related parameters involved in the decoction process, lays a foundation for the reliability and stability of the preparation process, can also be used for quality control of the preparation process of the standard angelica sinensis blood-enriching decoction, effectively controls the quality of the standard angelica sinensis blood-enriching decoction, and provides more stable and accurate data for the application of the standard angelica sinensis blood-enriching decoction in an automatic traditional Chinese medicine preparation machine.

(2) The invention also provides a method for controlling the quality of the standard decoction of the angelica sinensis blood-enriching decoction by adopting the characteristic map, wherein the characteristic map of the wine-washing angelica sinensis in the standard decoction of the angelica sinensis blood-enriching decoction is constructed by selecting the active ingredients of ferulic acid, calycosin glucoside, senkyunolide I and ligustilide of the wine-washing angelica sinensis as reference substances under specific detection conditions, so that the quality control of the wine-washing angelica sinensis in the standard decoction of the angelica sinensis blood-enriching decoction can be quickly and efficiently realized.

(3) According to the invention, the characteristic spectrum of the radix astragali decoction pieces in the standard decoction of the angelica blood-enriching decoction is constructed and obtained by selecting the active ingredients of the radix astragali decoction pieces, namely the formononetin, calycosin glucoside, astragaloside I, astragaloside II, isoastragaloside I and ferulic acid as reference substances under specific detection conditions, so that the quality control of the radix astragali decoction pieces in the standard decoction of the angelica blood-enriching decoction can be quickly and efficiently realized.

(4) The method overcomes the defects that the quality control in the industrial production process is reflected by measuring the content of the angelica sinensis blood-enriching decoction only, has the characteristics of strong specificity, high stability and good repeatability by combining the comparison of common peaks in a characteristic map, can comprehensively, quickly and efficiently realize the identification of decoction pieces in the standard angelica sinensis blood-enriching decoction, is applied to the quality control of the standard angelica sinensis blood-enriching decoction, and has the advantages of simple operation, stability, reliability, high precision, strong specificity and good separation degree.

In conclusion, the invention provides a preparation process of the standard angelica sinensis blood-enriching decoction used by using the alcohol washed angelica sinensis as the angelica sinensis feeding decoction piece for the first time, and also provides a quality control method for comprehensively reflecting the quantity value transmission relationship of the standard angelica sinensis blood-enriching decoction by using various content measurement and characteristic spectrum methods, so that the reflection from the whole process of medicinal materials-decoction pieces-standard decoction is realized, the requirements of the original prescription are better met, and the accuracy of medication is ensured.

Drawings

FIG. 1 is a schematic diagram of the pulverization of radix Angelicae sinensis and radix astragali decoction pieces washed with wine.

FIG. 2 is a control feature spectrum of alcohol-washed Angelica sinensis in standard decoction of Angelica sinensis decoction for tonifying blood.

FIG. 3 is a characteristic spectrum of standard decoction of DANGGUIBUXUE decoction.

FIG. 4 is a control characteristic spectrum (DAD) of radix astragali decoction pieces in standard decoction of radix Angelicae sinensis decoction for tonifying blood.

FIG. 5 is a reference characteristic spectrum (ELSD) of radix astragali decoction pieces in standard decoction of radix Angelicae sinensis decoction for tonifying blood.

FIG. 6 is a investigation atlas (DAD) of specificity of radix astragali decoction pieces in standard decoction of DANGGUIBUXUE decoction.

FIG. 7 is an ELSD (ELSD) map of radix astragali decoction pieces in standard decoction of DANGGUIBUXUE decoction.

FIG. 8 is a characteristic spectrum (DAD) of standard decoction of DANGGUIBUXUE decoction.

FIG. 9 is a characteristic spectrum (ELSD) of standard decoction of DANGGUIBUXUE decoction.

FIG. 10 is a ferulic acid specificity study map of standard decoction of radix Angelicae sinensis decoction for tonifying blood.

FIG. 11 is a standard ferulic acid curve chart of standard decoction of DANGGUIBUXUE decoction.

FIG. 12 is a map of the specificity of calycosin glucoside in the standard decoction of DANGGUIBUXUE decoction.

FIG. 13 is a graph showing the standard of calycosin glucoside in the standard decoction of DANGGUIBUXUE decoction.

FIG. 14 is a map of Astragaloside IV specificity in standard decoction of radix Angelicae sinensis decoction for tonifying blood.

FIG. 15 is a standard curve diagram of astragaloside IV in standard decoction of DANGGUIBUXUE decoction.

FIG. 16 is a control chart of the standard decoction of herbs, decoction pieces and Dang Gui Buxue Tang.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

Example 1:

the embodiment provides a preparation process of a standard decoction of angelica sinensis blood-enriching decoction.

The Chinese angelica and astragalus root decoction pieces washed with wine are taken as formula raw materials, and are respectively ground into powder and sieved by a sieve with 10 meshes. Washing 8g of radix Angelicae sinensis and 40g of radix astragali with wine after sieving, adding 600ml of water, soaking for 30min, adding a cover, boiling with strong fire, turning to slow fire, decocting for 40min, filtering while hot (200 mesh), cooling to room temperature, freeze drying, and packaging to obtain lyophilized powder of standard decoction of radix Angelicae sinensis decoction for replenishing blood.

Example 2:

this example is a study of the preparation process of standard decoction of Dang Gui Buxue Tang.

1. Selection of decoction pieces

1.1 processing radix astragali decoction pieces

According to the processing of astragalus mongholicus decoction pieces in the 2015 edition of Chinese pharmacopoeia: removing impurities, separating into different sizes, cleaning, moistening, slicing into thick pieces, and drying. The specific parameters are as follows: taking the astragalus root, removing impurities, cleaning, moistening for about 12 hours by adopting a closed moistening method, cutting into pieces with the thickness of 2-4 mm, and drying at the temperature of 60-70 ℃.

1.2 processing radix Angelicae sinensis with wine

Wine washing is a method for preparing traditional Chinese medicine wine which is used throughout the past, and generally, the medicine can partially permeate into tissues of the medicine after being washed by the wine to play the roles of delaying, enhancing the effect and the like. The wine washing is firstly seen in the rhubarb of Shang Zhong Jing Shang Han Lun (Shang Han treatise on Cold-induced diseases) of the Han Dynasty, is used in the stomach regulating and qi supporting decoction, and is considered to enter Yangming channel. Meanwhile, by combining the 'internal and external injury theory' of the blood-enriching decoction of Chinese angelica, the Hospital Lidongyuan considers that the body of the Chinese angelica is 'blood-nourishing and middle-guard', and the 'middle-guard' of the Chinese angelica can be changed into 'dredging away' after the Chinese angelica is soaked in wine (washed by wine), so that the Chinese angelica can enrich the blood without stagnation. The modern common angelica decoction pieces-wine angelica have the effect of 'making wine into the solar meridian', and the two have great difference.

The modern processing specifications describe that the alcohol-processed angelica is prepared by taking clean angelica slices, and frying the angelica slices dry by an alcohol-processing method (generally 0213) (2020 version of Chinese pharmacopoeia). The processing specifications of wine-washed Chinese angelica are respectively described in different places: 1) mixing radix Angelicae sinensis, parching with wine (appendix I), spraying yellow wine, stirring, drying in the sun or at low temperature. Every 100kg of angelica is processed with 15kg of yellow wine (2008 edition of Chinese medicine processing standard in Shanghai city); 2) adding Chinese liquor 5kg per 50 kg, spraying, stirring, soaking for 2-3 hr, cutting or chopping into round or oblique pieces, and air drying (1986 edition).

Combining literature examination and processing research, the specific processing parameters of wine-washed Chinese angelica are as follows: the Chinese angelica wine is prepared by taking Chinese angelica as a raw material, uniformly stirring the Chinese angelica with wine (yellow wine), moistening the mixture, drying the mixture at a low temperature and processing the mixture, wherein 100g of yellow wine is taken for every 100g of Chinese angelica.

Meanwhile, HPLC-DAD characteristic spectrum method is adopted to distinguish processed decoction pieces of radix Angelicae sinensis processed with wine, radix Angelicae sinensis processed with wine and radix Angelicae sinensis processed with wine, so as to ensure accuracy of medication (see characteristic spectrum, construction method and identification method of radix Angelicae sinensis and its decoction pieces recorded in patent publication 202010382210.7).

1.3 decoction piece decoction treatment

The angelica sinensis blood-enriching soup adopts a boiling preparation formulation according to the requirements of ' ancient classic famous-cube catalogue ' (first batch) ' issued by the State administration of traditional Chinese medicine, the boiling preparation formulation requires crushing of processed medicinal materials, and the particle size of the processed medicinal materials needs to be determined in order to ensure the quality consistency of standard decoction of the angelica sinensis blood-enriching soup. The boiling powder medicinal particles generally have 4 specifications of coarse powder, powder and fine powder. Chen Lin et al (Chinese medicine accurate decoction pieces. world science and technology-Chinese medicine modernization, 2016,18(09):1430-1440) and Chen Dan (Chinese medicine decoction technical specification from Taiping Huimin and Ju Fang. J. Chinese clinician, 2012,40(11):73-75.) after research, consider that "coarse powder" is equivalent to the coarsest powder nowadays, and pass through a No. one sieve (10 meshes); the coarse powder is equivalent to coarse powder and is sieved by a second sieve (24 meshes); the powder is between the coarse powder and the fine powder and is sieved by a third sieve (50 meshes); the "fine powder" is equivalent to the medium powder, and is sieved by a No. four sieve (65 meshes). To further confirm the tablet size, the following observations were made:

taking 4 parts of 20g of Chinese angelica washed with wine and 100g of astragalus decoction pieces respectively, pulverizing the Chinese angelica and the astragalus decoction pieces respectively, and sieving the powder by a pharmacopoeia sieve. According to the requirements of the original prescription of the angelica blood-replenishing decoction, 8g of sieved angelica sinensis and 40g of astragalus membranaceus are respectively washed with wine, 600ml of water is added, the angelica sinensis and the astragalus membranaceus are soaked for 30min, the mixture is boiled with strong fire and then decocted for 30min, the mixture is filtered while the mixture is hot (with a 200-mesh sieve), the volume of the mixture is measured after the mixture is cooled, and the paste yield and the contents of astragaloside, calycosin glucoside and ferulic acid of the decoction under different crushing particle sizes are respectively calculated. The content determination method of astragaloside IV, calycosin glucoside and ferulic acid refers to the content determination method of radix Angelicae sinensis and radix astragali decoction pieces in 2015 edition of Chinese pharmacopoeia. The results are shown in Table 1 below.

TABLE 1 investigation of decoction pieces of different particle sizes

Remarking: the overall evaluation calculation method:% cream yield 25% + astragaloside content 25% + calycosin glucoside 25% + ferulic acid 25%

According to the comprehensive evaluation index, the crushed particle size of the angelica sinensis and astragalus membranaceus decoction pieces washed with the wine is finally determined to be coarse powder (a sieve), the powder is in accordance with the dosage form in the original prescription as shown in figure 1, and the cream yield and the contents of astragaloside, calycosin glucoside and ferulic acid are suitable.

2. Observation of decoction time

The original prescription of the angelica blood-enriching decoction records that two people take water and one person takes the decoction. The decoction time is not clear in the original formula, and the time taken for decocting to one piece is specially considered to be consistent with the original formula. Respectively taking 3 parts of the crushed and compatible angelica sinensis blood-enriching decoction (8.0 g of wine-washed angelica sinensis and 40.0g of astragalus membranaceus), respectively adding 600ml of water, soaking for 30min, adding a cover, boiling with strong fire, then turning to slow fire, and respectively measuring the time for decocting 3 parts of angelica sinensis blood-enriching decoction with 200ml of water decoction. The results are shown in Table 2 below.

TABLE 2 study of decoction time of Angelica sinensis decoction for tonifying blood

Batches of	Amount of added water ml	Decocting for 20min, and volume ml	Decocting for 30min and volume ml	Decocting for 40min and volume ml
					1	600	485	355	300
2	600	480	345	301
					3	600	460	330	300

According to the above experiment, the following requirements are met in the 'Notification of the State administration of traditional Chinese medicine administration about the administration regulations of traditional Chinese medicine decoction rooms of the issuing medical institution': the nourishing medicine is boiled by strong fire and then is slowly decocted by slow fire for about 40 to 60 minutes. The decoction time of the angelica sinensis blood-enriching decoction is determined to be 40 min.

3. Filtering and drying

3.1 filtration

The standard decoction of the angelica blood-enriching decoction is filtered by a 200-mesh screen by adopting a solid-liquid separation mode, and the filtrate is cooled to room temperature.

3.2 drying

Drying of substance-based corresponding real objects of the angelica blood-enriching decoction is recommended to be prepared by adopting a freeze-drying method according to technical requirements (survey suggestions) formulated by quality control and standards of Chinese medicinal formula particles and requirements of standard decoction in management standards of Chinese medicinal decoction rooms in medical institutions, so that the consistency of the quality of the Chinese medicinal decoction and the quality of the corresponding real objects can be ensured, the stability of the quality of the corresponding real objects is ensured, and the corresponding real objects are easy to dissolve and are free of auxiliary materials.

Therefore, the corresponding material of this study was freeze-dried with the freeze-drying process parameters shown in table 3 below.

TABLE 3 Freeze drying Process

3.3 collecting and storing the powder

And rapidly collecting the powder in an environment with the temperature of 10-26 ℃ and the relative humidity of 40-60%. Storing standard decoction lyophilized powder in a dryer, and placing in a cool and dry place for use.

In summary, the preparation process of the standard decoction of the angelica blood-enriching decoction is as follows:

taking the crushed Chinese angelica and astragalus root decoction pieces (screened by a first sieve) which are washed by wine, and mixing the Chinese angelica and the astragalus root decoction pieces according to the proportion of 1: and 5, adding 600ml of water, soaking for 30min, adding a cover, boiling with strong fire, then turning to slow fire, decocting for 40min, filtering while hot (200-mesh sieve), cooling to room temperature, freeze-drying, and subpackaging to obtain the freeze-dried powder of the standard decoction of the angelica blood-enriching decoction.

According to the preparation process, 13 batches of standard angelica sinensis blood-enriching decoction are prepared. The batch numbers are respectively: DGBXBT01, DGBXBT02, DGBXBT03, DGBXBT04, DGBXBT05, DGBXBT06, DGBXBT07, DGBXBT08, DGBXBT09, DGBXBT10, DGBXBT11, DGBXBT12 and DGBXBT 13.

Example 3:

the embodiment relates to a quality control method of standard angelica blood-enriching decoction.

1. Construction of characteristic spectrum of wine-washed angelica in standard decoction of angelica blood-enriching decoction

1.1 Experimental instruments and materials (see published patent 202010382210.7)

1.2 construction of feature maps

Reference solution a: precisely weighing ferulic acid, calycosin glucoside, senkyunolide I and ligustilide respectively, and adding methanol to obtain solutions each containing 50-100 μ g per 1 ml.

Test solution I: precisely weighing 0.2g of lyophilized powder of standard decoction of radix Angelicae sinensis decoction for replenishing blood, placing in a conical flask, precisely adding 10ml of 70% methanol, performing ultrasonic treatment for 30min, shaking, filtering, and collecting filtrate.

Precisely sucking 10 μ l of each of the reference solution A and the sample solution I, injecting into a high performance liquid chromatograph, performing gradient elution according to the following table 4 by using octadecylsilane chemically bonded silica as a filler (the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 μm), acetonitrile as a mobile phase A, and a 0.1% phosphoric acid solution as a mobile phase B, and measuring at a column temperature of 30 ℃, a flow rate of 1.0ml per minute, and a detection wavelength of 290nm to obtain corresponding characteristic maps.

TABLE 4 gradient elution

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0～30	10→30	90→70
30～45	30→80	70→20
			45～60	80→95	20→5

Taking the characteristic spectrum of the reference substance solution A as a reference spectrum, selecting common peaks from the characteristic spectra of the test solution I, and constructing the characteristic spectrum of wine-washed angelica in the standard decoction of the angelica blood-enriching decoction, as shown in figure 2 (peak 1: calycosin glucoside; peak 2 (S): ferulic acid; peak 4: senkyunolide I; peak 7: ligustilide).

7 characteristic peaks are presented in the chromatogram of the test sample, wherein the peak corresponding to the ferulic acid reference is an S peak. The relative retention time of each characteristic peak to the S peak is calculated and should be within ± 10% of the specified value. The specified values are: 0.914 (peak 1), 1.000 (peak 2S), 1.252 (peak 3), 1.369 (peak 4), 1.729 (peak 5), 2.108 (peak 6), 2.461 (peak 7).

1.3 methodological investigation

In order to fully and comprehensively reflect the overall appearance of the components contained in the standard angelica blood-enriching decoction, the feature maps of the portions of the wine-washed angelica in the standard angelica blood-enriching decoction are examined and verified, and the results are shown in fig. 3 (in the figure, from bottom to top, DGBXBT01, DGBXBT02, DGBXBT03, DGBXBT04, DGBXBT05, DGBXBT06, DGBXBT07, DGBXBT08, DGBXBT09, DGBXBT10, DGBXBT11, DGBXBT12 and DGBXBT13), and tables 5 and 6. (refer to the characteristic chromatogram test sample preparation method, chromatogram condition, etc. of radix Angelicae sinensis medicinal material and radix Angelicae sinensis decoction pieces washed with wine described in patent publication 202010382210.7)

TABLE 513 relative residence time of standard batches of decoction

TABLE 6 methodological results RSD% summary criteria-relative retention time for each project

2. Construction of characteristic spectrum of radix astragali decoction pieces in standard decoction of radix Angelicae sinensis decoction for tonifying blood

2.1 laboratory instruments and materials

High performance liquid chromatograph: agilent 1260 type high performance liquid chromatograph (Agilent evaporable light 1260ELSD), Shimadzu 20AD type high performance liquid chromatograph (Shimadzu ELSD-LTII type evaporable light);

an electronic balance: ME204E/02, XP26 (Mettler-Tollido instruments, Inc.);

an ultra-pure water machine: cell type 1810A (Shanghai Mohler scientific instruments, Inc.);

an ultrasonic cleaner: model KQ5200DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);

agilent TC-C18(2)5um 250X 4.6mm and the like.

Acetonitrile and formic acid are chromatographically pure, water is ultrapure water, and other reagents are analytically pure.

Formononetin (Vickci Biotech Co., Ltd., Sichuan province, lot number wkq18112302, content 98%);

calycosin glucoside (China institute for testing food and drug, batch No.: 111920-201606, content is 97.6%);

astragaloside I (Vickqi Biotech Co., Ltd., Sichuan province, lot number wkq18041805, content in 98%);

astragaloside II (Vickqi Biotech Co., Ltd., Sichuan, lot number wkq18041712, content 98%);

isoastragaloside I (Beijing century Olympic Biotechnology Co., Ltd., lot number: 18050406, content 98%);

13 batches of angelica sinensis blood-enriching decoction standard decoction freeze-dried powder.

2.2 construction of feature maps

Reference solution: precisely weighing formononetin, calycosin glucoside, astragaloside I, astragaloside II, isoastragaloside I and ferulic acid respectively, adding methanol to obtain solutions each containing 50-100 μ g per 1ml,

sample solution ii: precisely weighing 1g lyophilized powder of standard decoction of radix Angelicae sinensis decoction for replenishing blood, placing in a conical flask, precisely adding 20ml water, performing ultrasonic treatment for 30min (power 600W, frequency 40kHz), shaking, filtering, and collecting filtrate;

precisely absorbing 10 μ L of each of the reference solution B and the sample solution II, injecting into a high performance liquid chromatograph, performing gradient elution according to the following table 7 by using octadecylsilane chemically bonded silica as a filler (the column length is 250mm, the inner diameter is 4.6mm, and the particle size is 5 μm), acetonitrile as a mobile phase A, and a 0.02% formic acid solution as a mobile phase B, detecting by using an ultraviolet detector and evaporative light scattering at a column temperature of 25 ℃ and a flow rate of 1.0ml per minute, wherein the detection wavelength of the ultraviolet detector is 254nm, the temperature of a drift tube of the evaporative light scattering detector is 90 ℃, and the flow rate of a carrier gas is 1.8L/ml per minute, and obtaining corresponding characteristic maps.

TABLE 7 gradient elution

Time (minutes)	Mobile phase A (%)	Mobile phase B (%)
			0～30	20→45	80→55
30～40	45→80	55→20

Taking the characteristic map of the reference substance solution B as a reference map, selecting a common peak from the characteristic maps of the test solution II to construct the characteristic maps of the radix astragali decoction pieces in the standard decoction of the angelica sinensis blood-enriching decoction, referring to fig. 4 (peak 1 (S): calycosin glucoside, peak 3: ferulic acid, peak 6: formononetin), and fig. 5 (peak 1 (S): calycosin glucoside, peak 2: ferulic acid, peak 4: formononetin, peak 7: astragaloside II, peak 8: astragaloside I, and peak 9: isoastragaloside I).

The chromatogram (ultraviolet detection) of the test sample should present 9 characteristic peaks, wherein the peak corresponding to the calycosin glucoside reference substance is S peak. The relative retention time of each characteristic peak to the S peak is calculated and should be within ± 10% of the specified value. The specified values are: 1.000(S peak), 1.104 (peak 2), 1.308 (peak 3), 1.437 (peak 4), 1.796 (peak 5), 1.834 (peak 6), 1.942 (peak 7), 2.329 (peak 8), 2.365 (peak 9).

The chromatogram (evaporative light scattering detection) of the test sample should show 10 characteristic peaks, wherein the peak corresponding to the calycosin glucoside reference is S peak. The relative retention time of each characteristic peak to the S peak is calculated and should be within ± 10% of the specified value. The specified values are: 1.000(S peak), 1.303 (peak 2), 1.432 (peak 3), 1.824 (peak 4), 2.066 (peak 5), 2.313 (peak 6), 3.688 (peak 7), 4.323 (peak 8), 4.438 (peak 9), 4.542 (peak 10).

2.3 methodological investigation

The methodology of the radix astragali characteristic spectrum of the standard radix Angelicae sinensis blood-enriching decoction is examined, including specificity, precision, repeatability, stability, intermediate precision and durability.

2.3.1 specificity

Preparation of negative control solution: preparing the control solution lacking the standard decoction of radix astragali for yin deficiency according to the conditions set out above.

Preparation of a test solution: preparing a test solution for the characteristic spectrum of the standard decoction of the angelica blood-enriching decoction according to the conditions. See fig. 6 and 7 for results.

2.3.2 precision, repeatability, stability, intermediate precision and durability

The lyophilized powder of standard decoction of radix Angelicae sinensis decoction for replenishing blood is prepared into test solution by the above method, and precision, repeatability, stability, intermediate precision and durability are respectively examined, and RSD values of each index are shown in tables 8 and 9 below, and the results show that RSD values all meet the requirements.

TABLE 8 DAD-methodology the results RSD% for each project summarized relative retention time

TABLE 9 ELSD-methodological results RSD% summary of relative retention times

2.3.3 validation Studies

By adopting the method, the 13 batches of samples are subjected to characteristic spectrum analysis, and the relative retention time and the relative peak area are calculated. See fig. 8 (DGBXBT 01, DGBXBT02, DGBXBT03, DGBXBT04, DGBXBT05, DGBXBT06, DGBXBT07, DGBXBT08, DGBXBT09, DGBXBT10, DGBXBT11, DGBXBT12, DGBXBT13, fig. 9 (DGBXBT 01, DGBXBT02, DGBXBT03, DGBXBT04, DGBXBT05, DGBXBT06, bxbt07, DGBXBT08, DGBXBT09, DGBXBT10, DGBXBT11, DGBXBT12, bxbt13, from bottom to top) and table 10, table 11.

TABLE 1013 Standard batches of relative residence time of decoction DAD

TABLE 1113 relative retention time of standard decoction in batches

According to the characteristic spectrum verification data of 13 batches of standard decoction of the angelica blood-enriching decoction, 9 peaks with better repeatability are selected by an ultraviolet detector and 10 peaks with better repeatability are selected by an evaporative light scattering detector as the characteristic peaks of the standard decoction of the angelica blood-enriching decoction according to the principle that the relative retention time is stable, and samples of each batch can be detected and the peaks are relatively higher.

3. Determination of ferulic acid content in standard decoction of angelica blood-enriching decoction

3.1 Experimental instruments and materials

Agilent model 1260 hplc;

an electronic balance: ME204E, XP260 (mettler-toledo instruments ltd);

an ultra-pure water machine: cell type 1810A (Chongqing Moore Water treatment Co., Ltd.);

a constant-temperature water bath kettle: DK-98-II (Tensted instruments, Tianjin);

numerical control ultrasonic cleaner: model KQ5200DB (600W, 40 KHz; ultrasonic instruments, Inc. of Kunshan);

and (3) chromatographic column: agilent SB-C18 (250X 4.6mm, 5 μm), etc.;

methanol (analytical grade), acetonitrile (chromatographic grade), phosphoric acid (chromatographic grade), ultrapure water;

ferulic acid (China institute for food and drug assay, batch No. 110773) -201614, 99% content).

3.2 determination of Ferulic acid content

Ferulic acid control solution: taking a proper amount of ferulic acid reference substance, precisely weighing, placing in a brown measuring flask, adding 70% methanol to obtain solutions containing 12 μ g ferulic acid per 1mL,

test solution iii: taking 0.1g of freeze-dried powder of standard decoction of the angelica blood-enriching decoction, precisely weighing, placing in a conical flask with a plug, precisely adding 10ml of 70% methanol, sealing the plug, weighing, ultrasonically extracting for 30 minutes, cooling, weighing again, supplementing the lost weight with 10% methanol, shaking up, standing, taking supernatant, filtering, and taking subsequent filtrate;

precisely absorbing 10 mu l of each of the ferulic acid reference solution and the test solution III, injecting into a high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as a filler, taking acetonitrile-0.085% phosphoric acid solution (17: 83) as a mobile phase, measuring at a detection wavelength of 316nm and a column temperature of 35 ℃, and measuring to obtain the content of ferulic acid in the standard decoction of the angelica blood-enriching decoction of more than or equal to 0.02%.

3.3 methodological inspection

3.3.1 specialization examination

Ferulic acid reference solution, test solution and negative reference solution were prepared according to the above-mentioned preparation method, and the results were shown in FIG. 10. The result shows that the negative solution chromatogram has no interference to the measurement of the peak to be measured, and the method has good specificity.

3.3.2 precision investigation

And continuously feeding the ferulic acid reference substance solution for 6 times, recording the peak area of the ferulic acid, calculating the RSD value, and obtaining the result that the RSD% is 0.11, which indicates that the injection precision of the instrument is good.

3.3.3 repeatability test

Precisely weighing 6 parts of the same test sample (DGBXBT12), preparing a test sample solution by the same operator according to a proposed method, and calculating the content of ferulic acid in the 6 parts of test sample. The content RSD value of the ferulic acid is 0.63 percent, and the method has good repeatability.

3.3.4 Linear relationship investigation

Taking a proper amount of ferulic acid reference substance, and preparing into 0.04654mg/mL reference substance mother liquor with 70% methanol. The solutions were diluted to 0.02327mg/mL, 0.0174525mg/mL, 0.00872625mg/mL, 0.004363125mg/mL and 0.0021815625mg/mL, respectively, and the solutions were injected into a liquid chromatograph, and analyzed to obtain peak areas, and response curves were plotted with the injection concentration (X, μ g) as abscissa and the peak area (Y) as ordinate. The results are shown in Table 12 below and FIG. 11.

TABLE 12 Ferulic acid Standard Curve analysis results

Sample size (X, mug)	21.815625	43.63125	87.2625	174.525	232.700	465.4
							Peak area (Y)	106.39305	205.98497	419.46851	835.72577	1076.53442	2225.87256

The result shows that the standard curve of ferulic acid is that y is 4.765x-2.649, R²0.9996. The linear relation is good when the injection concentration is 21.815625-465.4 mug.

3.3.5 stability test

The content of ferulic acid is measured in the same sample solution (batch number: DGBXBT12) at 0, 2,4, 8, 12 and 24 hours respectively, and the RSD value is 2.39%, which shows that the method has good stability.

3.3.6 intermediate precision

According to the proposed experimental conditions, the same sample solution (DGBXBT12) is respectively taken and inspected by different chromatographic columns (Agilent 1; Agilent 2; Kromasil 1) and different instruments (Agilent 1260, Waters2695 and Shimadzu LC-20AD high performance liquid chromatograph), and different personnel (A, B) examine at different times (I and II), and the RSD of the ferulic acid content is respectively 1.66%, 6.95% and 6.75%.

3.3.7 recovery rate of sample

Taking about 0.1g of a test sample (batch number: DGBXBT12, ferulic acid content 0.0.049%) with known content, precisely weighing 6 parts in total, precisely adding a certain amount of ferulic acid reference substance (purity 99.0%) respectively, preparing and measuring a test sample solution according to a proposed method, and calculating the recovery rates to be 90.46%, 91.27%, 93.23%, 91.33%, 94.12% and 90.77% respectively, thereby indicating that the method has good accuracy.

3.3.8 verification survey

13 batches of angelica sinensis blood-enriching standard soup are used as a test sample, a test sample solution is prepared and measured according to a formulated method, peak areas are recorded, and the content of ferulic acid is calculated, and the results are shown in a table 13.

TABLE 1313 blood-enriching Chinese angelica decoction ferulic acid content results

Serial number	Standard decoction batch number	Content (%)
			1	DGBXBT01	0.030
2	DGBXBT02	0.030
			3	DGBXBT03	0.032
4	DGBXBT04	0.029
			5	DGBXBT05	0.029
6	DGBXBT06	0.042
			7	DGBXBT07	0.044
8	DGBXBT08	0.027
			9	DGBXBT09	0.040
10	DGBXBT10	0.041
			11	DGBXBT11	0.052
12	DGBXBT12	0.050
			13	DGBXBT13	0.035

As shown in the table, ferulic acid (C) in 13 Chinese angelica standard blood-enriching decoction₁₀H₁₀O₄) The content range of the (B) is 0.029% -0.052% ", the average value is 0.037%, and the range of +/-30% of the average value is 0.026% -0.048%.

Integrating the actual measurement range of ferulic acid to determine ferulic acid content determination limit (C)₁₀H₁₀O₄) Not less than 0.02%.

4. Determination of calycosin glucoside content in standard decoction of Angelica sinensis decoction for tonifying blood

4.1 Experimental instruments and materials

High performance liquid chromatograph: agilent 1260 type HPLC, Shimadzu LC-20AD type HPLC, and Waters e2695 type HPLC;

an electronic balance: ME204E/02, MS205DU, XP26 (Mettler-Tollido instruments, Inc.);

a chromatographic column: agilent ZORBAX Eclipse plus 5 μm 250X 4.6mm, etc.;

acetonitrile and formic acid are chromatographically pure, water is ultrapure water, and other reagents are analytically pure;

calycosin glucoside (Chinese institute for testing and testing food and drug; batch No. 111920-201606, content is 97.6%);

13 batches of Chinese angelica blood-enriching decoction freeze-dried powder.

4.2 determination of Calycosin glucoside content

Calycosin glucoside control solution: taking appropriate amount of calycosin glucoside reference substance, precisely weighing, adding methanol to obtain solutions containing 50 μ g of calycosin glucoside per 1mL,

test solution v: taking 0.5g of freeze-dried powder of standard decoction of the angelica blood-enriching decoction, precisely weighing, placing in a conical flask with a plug, adding 10mL of methanol, sealing the plug, weighing, carrying out ultrasonic treatment at the power of 600W and the frequency of 40kHz for 30 minutes, cooling, weighing again, supplementing the weight loss by using methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the Chinese angelica blood-enriching decoction;

precisely sucking 10 mu l of each of the calycosin glucoside reference solution and the test solution V, injecting the solution into a high performance liquid chromatograph, performing gradient elution according to the following table 14 by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and 0.2% formic acid solution as a mobile phase B, and determining at an inspection wavelength of 260nm to obtain a standard decoction of the angelica sinensis hematinic decoction, wherein the content of calycosin glucoside in the standard decoction is not less than 0.02%.

TABLE 14 gradient elution

Time (minutes)	Mobile phase A (%)	Mobile phase (B)
			0～20	20～40	80～60
20～30	40	60

4.3 methodological inspection

4.3.1 specialization examination

The test solution and the negative control solution were prepared according to the proposed method and tested, and the results are shown in FIG. 12. The result shows that the negative solution chromatogram has no interference to the measurement of the peak to be measured, and the method has good specificity.

4.3.2 precision review

And continuously injecting the reference substance solution for 6 times, recording the area of the peak of calycosin glucoside, and indicating that the instrument precision is good, wherein the RSD value of the peak area is 0.19%.

4.3.3 Linear relationship investigation

Taking a proper amount of calycosin glucoside reference substance, and preparing 111.16640 μ g/mL reference substance mother liquor with methanol. Solutions with the following concentrations are prepared respectively, 10 mu l of each solution is sucked and injected into a liquid chromatograph, the peak area is obtained through analysis, and a response curve is drawn by taking the sample volume (X, ng) as a horizontal coordinate and the peak area (Y) as a vertical coordinate. The results are shown in Table 15 below and FIG. 13.

TABLE 15 results of analysis of Calycosin glucoside Standard curves

The results show that: the standard curve of calycosin glucoside is y-2.7826 x-10.841, R²0.9999. The linear relation is good when the sample injection amount is in the range of 111.1664-1111.6640 ng.

4.3.4 stability test

The chromatographic peak area of calycosin glucoside in the same test solution (batch number: DGBXBT12) is measured at 0, 2,4, 6, 12 and 24 hours, the RSD value of the peak area is 1.24%, and the test solution has good stability within 24 hours.

4.3.5 repeatability experiments

Taking 0.5g of the same test sample (batch number: DGBXBT12), precisely weighing 6 parts, preparing a test sample solution by the same operator according to a proposed method, calculating the content of calycosin glucoside of the 6 parts of test sample, wherein the content of RSD is 2.32 percent, and the method has good repeatability.

4.3.6 intermediate precision

According to the proposed experimental conditions, the same sample solution (DGBXBT12) is respectively taken and inspected by different chromatographic columns (Agilent 1; Agilent 2; Kromasil 1) and different instruments (Agilent 1260, Waters2695 and Shimadzu LC-20AD high performance liquid chromatograph), and different persons (A, B) examine at different times (I and II), and the RSD of the content of calycosin glucoside is respectively 3.21%, 2.14% and 5.97%.

4.3.7 recovery rate of sample

Taking about 0.5g of a test sample (batch number: DGBXBT12) with a known content, taking 6 parts in total, precisely weighing, precisely adding a certain amount of calycosin glucoside reference substance (purity is 97.6%) respectively, preparing and measuring a test sample solution according to a proposed method, calculating recovery rates which are 92.27%, 93.61%, 94.65%, 95.04%, 100.23% and 100.07% respectively, and the RSD recovery rate is 6.13%, which indicates that the method has good accuracy.

4.3.8 verification of surveys

Taking 13 batches of standard decoction freeze-dried powder as a test sample, preparing a test sample solution according to a proposed method, measuring, recording peak area, calculating the content of calycosin glucoside, and obtaining the result shown in the following table 16.

TABLE 1613 measurement of lyophilized powder content of standard decoction

Batch number	Calycosin glucoside content (%)
		DGBXBT01	0.088
DGBXBT02	0.096
		DGBXBT03	0.040
DGBXBT04	0.059
		DGBXBT05	0.057
DGBXBT06	0.068
		DGBXBT07	0.066
DGBXBT08	0.063
		DGBXBT09	0.027
DGBXBT10	0.064
		DGBXBT11	0.066
DGBXBT12	0.055
		DGBXBT13	0.075

The results show that calycosin-dextran in the standard decoctionThe measured range of glucoside is: 0.027-0.096%, and the average content is 0.065%. The content of the product is limited to calycosin-containing glucoside (C) 70% -130% of the average value₂₂H₂₂NO₁₀)0.046～0.085％。

Combining the above data, determining calycosin glucoside (C)₂₂H₂₂NO₁₀) The determination limit of (A) is not less than 0.02%.

5. Determination of astragaloside content in standard decoction of angelica blood-enriching decoction

High performance liquid chromatograph: agilent 1260 type high performance liquid chromatograph, Agilent evaporative light scattering detector, Shimadzu LC-20AD type high performance liquid chromatograph;

a chromatographic column: agilent ZORBAX Eclipse plus 5 μm 250X 4.6mm, etc.;

acetonitrile is chromatographically pure, water is ultrapure water, and other reagents are analytically pure;

astragaloside (China institute for testing and testing food and drug; batch No. 110781-containing 201717, content is 96.9%);

13 batches of Chinese angelica blood-enriching decoction freeze-dried powder.

5.1 Experimental instruments and materials

Astragaloside IV control solution: taking appropriate amount of astragaloside IV reference substance, precisely weighing, adding methanol to obtain solutions containing astragaloside IV 0.5mg per 1mL respectively,

test solution vi: taking 1.0g of freeze-dried powder of standard decoction of the angelica blood-enriching soup, precisely weighing, placing in a conical flask with a plug, adding 50mL of 80% methanol containing 4% concentrated ammonia test solution, sealing the plug, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the lost weight with 80% methanol containing 4% concentrated ammonia test solution, shaking up, filtering, precisely weighing 25mL of subsequent filtrate, evaporating to dryness, dissolving residues with 80% methanol, transferring to a 5mL measuring flask, adding 80% methanol to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the Chinese medicinal preparation;

precisely sucking 5 mul and 10 mul of astragaloside IV reference substance solution and 20 mul of test substance solution VI, injecting into a high performance liquid chromatograph, using octadecylsilane chemically bonded silica as a filler, and mixing acetonitrile-water 32: 68 is mobile phase, measuring by an evaporative light scattering detector, and calculating by an external standard two-point method logarithmic equation to obtain the standard decoction of radix Angelicae sinensis hematinic decoction containing astragaloside IV more than or equal to 0.05%.

5.2 determination of Astragalus glycoside content

5.3 examination of extraction methods

5.3.1 extraction Studies I

Combining the content determination of the decoction piece astragaloside IV, extracting and determining the astragaloside IV content by the following three extraction modes:

(1) extraction mode 1-1: weighing about 2g of a sample (lot number: DGBXBT14), precisely weighing, placing in a Soxhlet extractor, adding 40ml of methanol, cold soaking overnight, adding an appropriate amount of methanol, heating and refluxing for 4 hours, recovering a solvent from an extracting solution, concentrating to dryness, adding 10ml of water into residues, slightly heating to dissolve the residues, shaking and extracting with water-saturated n-butanol for 4 times, 40ml each time, combining n-butanol solutions, fully washing with an ammonia test solution for 2 times, 40ml each time, discarding the ammonia solution, evaporating the n-butanol solution to dryness, adding 5ml of water into the residues to dissolve, cooling, passing through a D101 type macroporous adsorption resin column (the inner diameter is 1.5cm, the column height is 12cm), eluting with 50ml of water, discarding a water solution, eluting with 30ml of 40% ethanol, discarding an eluent, eluting with 80ml of 70% ethanol, collecting the eluent, evaporating to dryness, adding methanol into residues to dissolve, transferring to a 5ml volumetric flask, adding methanol to the scale, and shaking uniformly to obtain the reagent.

(2) Extraction mode 1-2: weighing about 2g of sample (lot number: DGBXBT14), precisely weighing, adding 10ml of water into residue, performing ultrasonic treatment for 30min, extracting with water-saturated n-butanol under shaking for 4 times, 40ml each time, mixing n-butanol solutions, washing with ammonia sample solution for 2 times, 40ml each time, discarding ammonia solution, evaporating n-butanol solution to dryness, adding 5ml of water into residue to dissolve, cooling, passing through D101 type macroporous adsorbent resin column (inner diameter of 1.5cm, column height of 12cm), eluting with 50ml of water, discarding water solution, eluting with 30ml of 40% ethanol, discarding eluate, eluting with 80ml of 70% ethanol, collecting eluate, evaporating to dryness, dissolving residue with methanol, transferring into 5ml volumetric flask, adding methanol to scale, and shaking uniformly to obtain the final product.

(3) Extraction mode 1-3: weighing about 0.5g of a sample (batch number: DGBXBT14), precisely adding 50ml of 80% methanol solution (4 ml of concentrated ammonia solution, 80% methanol to 100ml, shaking up) containing 4% concentrated ammonia solution, sealing, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the weight loss by 80% methanol solution containing 4% concentrated ammonia solution, shaking up, filtering, precisely taking 25ml of subsequent filtrate, evaporating to dryness, dissolving residues by 80% methanol, transferring to a 5ml measuring flask, adding 80% methanol to scale, shaking up, filtering, and taking subsequent filtrate.

The above three extraction methods were analyzed and are shown in Table 17 below.

TABLE 17 analysis results of different extraction methods I

The result shows that the extraction mode 3 has the highest content in the three extraction modes, and the operation mode is simpler and more convenient than the first two extraction modes, so the extraction mode 3 is selected by comprehensive consideration.

5.3.2 examination of extraction mode II

And optimizing and comparing the extraction modes on the basis:

(1) weighing about 1.0g of a sample (batch number: DGBXBT14), placing the sample in a conical flask with a plug, precisely adding 50mL of 80% methanol (taking 4mL of concentrated ammonia test solution, adding 80% methanol to 100mL, shaking up) containing 4% concentrated ammonia test solution, sealing the plug, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the reduced weight with 80% methanol containing 4% concentrated ammonia test solution, shaking up, filtering, precisely taking 25mL of subsequent filtrate, evaporating to dryness, dissolving residues with 80% methanol, transferring to a 5mL volumetric flask, adding 80% methanol to scale, shaking up, filtering, and taking subsequent filtrate to obtain the final product.

(2) Weighing about 1.0g of a sample (batch number: DGBXBT14), placing the sample in a conical flask with a plug, precisely adding 50mL of 80% methanol (taking 4mL of concentrated ammonia test solution, adding 80% methanol to 100mL, shaking up) containing 4% concentrated ammonia test solution, sealing the plug, weighing, performing ultrasonic treatment for 1 hour, cooling, weighing again, supplementing the weight loss with 80% methanol containing 4% concentrated ammonia test solution, shaking up, filtering, precisely taking 25mL of subsequent filtrate, evaporating to dryness, dissolving residues with 80% methanol, transferring to a 5mL measuring flask, adding 80% methanol to scale, shaking up, filtering, and taking subsequent filtrate.

The above two extraction methods were analyzed and are shown in Table 18 below.

TABLE 18 analysis results of different extraction modes II

The result shows that the reflux extraction content of the extraction mode is higher, and the extraction mode is selected as reflux by comprehensive consideration.

The preparation method of the final test sample comprises the following steps: taking 1.0g of a sample, placing the sample in a conical flask with a plug, precisely adding 50mL of 80% methanol containing 4% concentrated ammonia solution, sealing the plug, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the reduced weight with 80% methanol containing 4% concentrated ammonia solution, shaking up, filtering, precisely measuring 25mL of subsequent filtrate, evaporating to dryness, dissolving residues with a solvent, transferring to a 5mL measuring flask, adding the solvent to scale, shaking up, filtering, and taking the subsequent filtrate.

5.4 methodological examination

5.4.1 specificity experiments

The test solution and the negative control solution were prepared according to the proposed method and tested, and the results are shown in FIG. 14. The result shows that the negative solution chromatogram has no interference to the measurement of the peak to be measured, and the method has good specificity.

5.4.2 precision investigation

And continuously injecting the reference substance solution for 6 times, recording the peak area of the astragaloside IV, and recording the RSD value of 2.41 percent, which shows that the precision of the instrument of the method is good.

5.4.3 Linear investigation

Taking proper amount of astragaloside IV reference substance, respectively preparing the following concentration solutions, respectively sucking 20 μ l of each solution, injecting into a liquid chromatograph, analyzing to obtain peak area, and drawing a response curve by taking LOG sample amount (X) as a horizontal coordinate and LOG peak area (Y) as a vertical coordinate. The results are shown in Table 19 below and FIG. 15.

TABLE 19 Standard Curve analysis of astragaloside IV

Sample concentration (mu g/ml)	98.6442	1972884	493.221	986.442	1233.0525	1972.884
							LOG sample size (X)	3.2951	3.5961	3.9941	4.2951	4.392	4.5961
LOG Peak area (Y)	2.1419	2.6591	3.2701	3.6824	3.797	4.0466

The results show that: the astragaloside standard curve is that y is 1.4654x-2.6365, R²0.9965. The linear relation is good when the injection concentration is 98.6442-1972.8840 mu g/mL.

5.4.4 stability Studies

The same test solution (batch number: DGBXBT12) is taken and respectively tested for 0 hour, 2 hours, 6 hours, 8 hours, 12 hours and 24 hours, the peak area RSD of the astragaloside IV is 3.55 percent, and the test solution has better stability within 24 hours.

5.4.5 repeatability test

1.0g of the same test sample (batch number: DGBXBT12) is precisely weighed to obtain 6 parts, the same operator prepares test sample solution according to a proposed method, the content RSD of the astragaloside of the 6 parts of test sample is 4.08 percent, and the method has good repeatability.

5.4.6 intermediate precision

According to the proposed experimental conditions, the same sample solution (DGBXBT12) is respectively taken and inspected by different chromatographic columns (Agilent 1; Agilent 2; Kromasil 1) and different instruments (Agilent 1260 and Shimadzu LC-20AD high performance liquid chromatograph), and different persons (A, B) examine at different times (I and II), and the RSD of the content of calycosin glucoside is respectively 6.92%, 9.87% and 9.91%.

5.4.7 recovery rate of sample

Taking about 0.5g of a test sample (batch number: DGBXBT12) with a known content, taking 6 parts in total, precisely weighing, precisely adding a certain amount of astragaloside control (with the purity of 96.9%) respectively, preparing and measuring a test sample solution according to a proposed method, and calculating the recovery rates, wherein the recovery rates are 90.82%, 91.12%, 93.65%, 96.53%, 98.68% and 93.00%, and the average value is 91.85%, which indicates that the method has good accuracy.

5.4.8 verification of surveys

Taking 13 batches of standard decoction freeze-dried powder as a test sample, preparing a test sample solution according to a proposed method, measuring, recording peak area, calculating astragaloside content, and obtaining the result shown in the following table 20.

TABLE 2013 measurement results of lyophilized powder content of standard decoction

The result shows that the actual measurement range of the astragaloside in the standard decoction is as follows: 0.109-0.229%, and the average content is 0.161%. The product contains astragaloside IV (C) 70-130% of the average value of the content measurement₄₁H₆₈NO₁₄)0.113～0.209％。

Combining the above data to determine astragaloside IV (C)₂₂H₂₂NO₁₀) The determination limit of (A) is not less than 0.05%.

Based on the above, the invention also has the following advantages and effects:

(1) can realize the characteristic spectrum reflected by the whole process of the medicinal material-decoction piece-standard decoction of the wine-washed angelica.

By adopting the method of the established characteristic spectrum of the standard decoction of the angelica sinensis blood-enriching decoction, the angelica sinensis decoction pieces washed with wine in the angelica sinensis blood-enriching decoction can be quickly distinguished from the medicinal materials to the decoction pieces in the standard decoction process, and the influence on the safety and the effectiveness of the prescription caused by the fact that other decoction pieces which do not meet the requirements of the original prescription of the classical famous prescription are used as the medicine is avoided.

In the method for constructing the characteristic map, referring to fig. 16 (peak 1 calycosin glucoside, peak 2(S) ferulic acid, peak 4 senkyunolide i and peak 7 ligustilide), the characteristic map of the single decoction and the combined decoction of angelica sinensis and astragalus membranaceus washed with wine can be compared, so that the characteristic map of the corresponding real object of the angelica sinensis blood-enriching decoction mainly has 7 characteristic peaks, wherein the

peaks

1, 3 and 5 can be found in the astragalus membranaceus medicinal material, the decoction pieces and the standard decoction. 2.

Peaks

4 and 7 are

peaks

4, 5 and 8 of the decoction pieces and standard decoction of radix Angelicae sinensis. The peak No. 6 is a characteristic peak generated after the astragalus and the Chinese angelica decoction pieces are decocted together, which indicates that new substances are generated during the combined decoction of the traditional Chinese medicines. In the quantity value transmission process of the angelica medicinal material and the angelica decoction pieces washed with wine, the research finds that the characteristic spectrum of the angelica medicinal material is more than 1 characteristic peak (angelica peak 1 washed with wine) from yellow wine after the angelica medicinal material is washed with wine.

(2) Determining relevant parameters of the preparation process of the standard decoction of the angelica sinensis blood-enriching decoction based on the national policy guidance.

Under the condition that the original recipe has no parameters of a preparation process, by combining ancient books, related examination certificates and experimental investigation, detailed preparation parameters are established: taking the crushed Chinese angelica and astragalus root decoction pieces which are washed with wine (sieved by a first sieve) according to the proportion of 1: 5, adding 400ml of water, soaking for 30min, boiling with strong fire, then turning to slow fire, decocting for 40min, filtering while hot, cooling to room temperature, freeze-drying, and packaging to obtain the final product.

The standard decoction established by the method has stable quality corresponding to real objects (namely freeze-dried powder) and can better reflect the comprehensive components of the angelica blood-enriching decoction.

(3) The quality of standard decoction of the angelica blood-enriching decoction is comprehensively controlled by various contents and characteristic spectrum methods.

On the basis of effectively combining the contents of efficacy and active ingredients, 3 ingredient content indexes of ferulic acid, calycosin glucoside and astragaloside IV in the standard decoction of the angelica blood-enriching decoction and a detection method of 2 characteristic maps combining HPLC-DAD and HPLC-DAD-ELSD are established, so that the quality of the standard decoction is comprehensively reflected and controlled, the zhuangguan inositol is established for later-stage preparation of the better blood-enriching decoction with more obvious curative effect, and a certain reference is provided for the development of other classical formulas.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiments according to the technical spirit of the present invention are included in the scope of the present invention.

Claims

1. A preparation process of standard decoction of angelica sinensis blood enriching soup is characterized by comprising the following steps: mixing 1 part of Chinese angelica with 5 parts of astragalus decoction pieces according to the parts by weight, adding 600ml of water, soaking for 30min, adding a cover to boil with strong fire, turning to slow fire, decocting for 40min, filtering while hot, cooling and drying to obtain the freeze-dried powder of the Chinese angelica blood-enriching decoction standard decoction.

2. A quality control method of standard decoction of angelica sinensis blood-enriching decoction is characterized in that: comprises the construction of the characteristic map of the wine-washed angelica in the standard decoction of the angelica blood-enriching decoction prepared by the method of claim 1, and comprises the following steps:

A. preparing reference solution A and test solution I respectively,

test solution I: taking 0.2g of freeze-dried powder of standard decoction of the angelica blood-enriching decoction, adding 10ml of 70% methanol, carrying out ultrasonic treatment for 30 minutes, shaking up, filtering, and taking subsequent filtrate to obtain the Chinese angelica blood-enriching decoction;

3. The quality control method of standard decoction of angelica sinensis blood-enriching soup according to claim 3, which is characterized in that: in the step B, gradient elution meets the following conditions:

0-30 min, mobile phase A: 10% → 30%, mobile phase B: 90% → 70%;

30-45 min, mobile phase A: 30% → 80%, mobile phase B: 70% → 20%;

45-60 min, mobile phase A: 80% → 95%, mobile phase B: 20% → 5%.

4. The quality control method of standard decoction of angelica sinensis blood-enriching decoction according to claim 2, which is characterized in that: the method also comprises the construction of the characteristic spectrum of the astragalus decoction pieces in the standard decoction of the angelica sinensis blood-enriching decoction prepared by the method of claim 1, and the steps are as follows:

A. preparing reference solution B and test solution II,

C. and (4) taking the characteristic map of the reference substance solution B as a reference map, selecting common peaks from the characteristic maps of the test solution II, and constructing the characteristic map of the astragalus decoction pieces in the standard decoction of the angelica sinensis blood-enriching decoction.

5. The quality control method of standard decoction of Angelica sinensis blood-enriching decoction according to claim 4, which is characterized in that: in the step B, gradient elution meets the following conditions:

0-30 min, mobile phase A: 20% → 45%, mobile phase B: 80% → 55%;

30-40 min, mobile phase A: 45% → 80%, mobile phase B: 55% → 20%.

6. The quality control method of standard decoction of Angelica sinensis blood-enriching decoction according to claim 4, which is characterized in that: further comprises measuring the content of ferulic acid in the standard decoction of the angelica sinensis blood-enriching decoction prepared by the method of claim 1 by adopting a high performance liquid chromatography, which comprises the following steps:

B. respectively taking 10 mu l of each of the ferulic acid reference solution and the test solution III, injecting the ferulic acid reference solution and the test solution III into a high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as a filler, and mixing the ferulic acid reference solution and the test solution III to obtain a mixture, wherein the mixture comprises 17 percent of acetonitrile-0.085% phosphoric acid solution: 83 is mobile phase, the detection wavelength is 316nm, the column temperature is 35 deg.C, and the content of ferulic acid in standard decoction of radix Angelicae sinensis decoction for replenishing blood is not less than 0.02%.

7. The quality control method of standard decoction of Angelica sinensis blood-enriching decoction according to claim 4, which is characterized in that: further comprising the step of measuring the content of calycosin glucoside in the standard decoction of the angelica sinensis hematinic decoction prepared by the method of claim 1 by using high performance liquid chromatography, which comprises the following steps:

8. The quality control method of standard decoction of angelica sinensis blood-enriching decoction according to claim 7, which is characterized in that: in the step B, gradient elution meets the following conditions:

0-20 min, mobile phase A: 20% → 40%, mobile phase B: 80% → 60%;

20-30 min, mobile phase A: 40%, mobile phase B: 60 percent.

9. The quality control method of standard decoction of Angelica sinensis blood-enriching decoction according to claim 4, which is characterized in that: further comprises measuring the content of astragaloside in the standard decoction of the angelica sinensis blood-enriching decoction prepared by the method of claim 1 by adopting a high performance liquid chromatography, and the steps are as follows:

test solution vi: taking 1.0g of freeze-dried powder of standard decoction of angelica sinensis blood-enriching soup, adding 50mL of 80% methanol containing 4% concentrated ammonia solution, sealing, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the lost weight with 80% methanol containing 4% concentrated ammonia solution, shaking up, filtering, precisely taking 25mL of subsequent filtrate, evaporating to dryness, dissolving the residue with 80% methanol, transferring to a 5mL measuring flask, adding 80% methanol to scale, shaking up, filtering, and taking subsequent filtrate to obtain the Chinese medicinal preparation;