CN114689774B

CN114689774B - Preparation process and quality control method of standard decoction of radix angelicae sinensis blood replenishing decoction

Info

Publication number: CN114689774B
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: 2023-10-03
Anticipated expiration: 2040-12-30
Also published as: CN114689774A

Abstract

The invention discloses a preparation process and a quality control method of standard decoction of Chinese angelica and blood replenishing decoction, which establishes a preparation process of taking wine-washed Chinese angelica and astragalus decoction pieces as formula raw materials, takes the contents of astragaloside IV, calycosin glucoside and ferulic acid as investigation indexes in combination with the ointment yield, determines the selection of each parameter condition in the decoction process, has stable and reliable process operation, is reasonable and feasible, establishes 3 ingredient content indexes of the standard decoction of Chinese angelica and blood replenishing decoction, namely ferulic acid, calycosin glucoside and astragaloside IV, and a detection method of 2 characteristic patterns combined by HPLC-DAD and HPLC-DAD-ELSD, further realizes comprehensive quality reflection and control of the standard decoction, and ensures the stability and reliability of the preparation process of the standard decoction of Chinese angelica and blood replenishing decoction.

Description

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

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 Chinese angelica blood replenishing decoction.

Background

The ancient classical prescription is the treasure of Chinese traditional medicine prescription, is the summary of clinical practice experience of the traditional Chinese medicine, and condenses the intelligence of the traditional Chinese medicine for thousands of years. The Chinese medicine preparation is clear at the same time, and the Chinese medicine compound preparation which meets the national regulation and is derived from the ancient classical prescription can be produced, and only non-clinical safety research data can be provided when the application of medicine approval document is carried out. The classical prescription preparation meeting the requirements is applied to the market, and only the study data of pharmacy and non-clinical safety and the study data of report-free pharmacodynamics and clinical test can be provided. The basic work of classical prescription research is therefore of paramount importance.

According to the related guidance comments of the requirements (manuscript of comments) of the reporting data of the ancient classical Chinese herbal medicine compound preparation and the requirements (manuscript of comments) of the reporting data of the substance standard of the ancient classical Chinese herbal medicine compound preparation, which are issued by the national drug administration, in 2019, the compound preparation of the classical Chinese herbal medicine must conform to the requirements of the substance standard, and the substance standard must be strictly referred to the ancient Chinese herbal medicine, including the basic source, the production place, the collection period, the primary processing of the production place and the quality requirements of the medicinal material, the processing, the process and quality requirements of decoction pieces, the decoction process, the dosage and the like. Therefore, the substance reference research (standard decoction stage) of the classical prescription plays a decisive role in the safety and effectiveness of the compound preparation of the classical prescription.

The Chinese angelica blood-replenishing decoction is a classical prescription of traditional Chinese medicine, is originally carried in Jinli Yuan and is used for treating Yang Fu fever due to blood deficiency, and has the effects of tonifying qi and promoting blood production. Modern medical research finds that it has the functions of improving blood system, regulating immunity, protecting liver, resisting oxidation and the like, and can also be used for postoperative conditioning of various cancer patients. The medicine has wide applicable crowd, large base number, long administration time and larger medicine development market space. The Chinese angelica blood-replenishing decoction is from Lidong's theory of internal and external injury distinguished by "Chinese angelica blood-replenishing decoction is used for treating myofever, dryness-heat, thirsty inducing drink, conjunctival congestion and redness, and the day and night are not in charge. Its pulse is large and weak, and its repeated pressing is all the more absent. "Neijing" is: blood deficiency due to pulse deficiency. And (3) cloud: the fever due to blood deficiency is manifested as white tiger, but the pulse is not long enough to identify the ear, and the white tiger Shang Bi is taken by mistake Dead. The disease is caused by hunger and fatigue; astragalus root, chinese angelica root (wine washing) and two kinds of money; upper partThe mouth is taken as one, two water are taken, decocted to one, deslagged, taken warm and eaten before hollow food. "

However, at present, the Chinese medicine preparation "Chinese angelica blood-replenishing oral liquid" related to Chinese angelica blood-replenishing decoction, which is carried out in the Chinese pharmacopoeia, is 132g of Chinese angelica and 330g of astragalus, and is prepared by decocting Chinese angelica with astragalus after water distillation, extracting with ethanol, and has far different wine-washed Chinese angelica and decoction processes from the requirements of reporting materials (claimation manuscript) of the substance standard of the Chinese medicine compound preparation of the ancient classical prescription, and the requirements of the national Chinese medicine administration issuing the catalogue (first batch) of the ancient classical prescription. Meanwhile, in the related preparations of the Chinese angelica blood-replenishing decoction sold in the market, for example, chinese angelica blood-replenishing pills are also described as Chinese angelica, and specific use processed products are not clear. On the other hand, although the country has a table guiding principle (soliciting opinion manuscripts), only "astragalus mongholicus one two, angelica sinensis (wine washing) two money is described; upper partThe mouth is taken as one hand, two water is taken as the other hand, the mouth is decocted to one hand, deslagging, warm taking and before eating, the specific preparation process parameters are not clarified, and the application and operation of the actual preparation process of each unit are difficult.

Therefore, the related preparation process and quality control of the standard decoction of the angelica sinensis blood enriching decoction based on the national policy requirements are deficient in research report, and the research on the preparation process and quality control of the current standard decoction of the angelica sinensis blood enriching decoction should be established for further guiding the later work of the prescription granule preparation.

Ma Guhua in the research on preparation process and quality control of Angelica sinensis blood-replenishing decoction based on combination of effective component detection and physicochemical characterization, chinese herbal medicine, volume 43, 3 rd year, 3 months, 482-486, according to the components and drug effects of prescription drugs, the index components Astragaloside IV of Astragalus membranaceus, the index components Ferulic acid of Angelica sinensis, and total saponins and total polysaccharides in the prescription are selected as evaluation indexes of water decoction extraction process, wherein the total polysaccharides are measured by phenol-sulfuric acid method, the total saponins are measured by ultraviolet absorption method, the ferulic acid is measured by HPLC method, the Astragaloside IV is measured by HPLC-ELSD method, and the extraction process of Angelica sinensis blood-replenishing decoction is determined as follows: weighing 12g of Chinese angelica, 60g of astragalus, adding 12 times of water, and extracting for 3 times each time for 1.0 hour.

Pan Zihao the contents of ferulic acid, calycosin glucoside and astragaloside in the decoction of Chinese angelica are simultaneously measured by an HPLC-DAD/ELSD method in the decoction of Chinese angelica at the same time, and the contents of the ferulic acid, the calycosin glucoside and the astragaloside in the decoction of Chinese angelica at the same time are simultaneously measured by the HPLC-DAD/ELSD method in the decoction of Chinese angelica at the same time in the 3 rd stage, 547-550 of volume 27 of the Chinese medicine of the ' Zhen Guo ' in 2016 ' and examined methodologically.

In the patent with publication number of CN11155162A, the invention discloses a method for simultaneously measuring 18 compounds of different structural types in Chinese angelica blood soup based on ACQUITY UPLC-class+Xevo TX-XS, and particularly, according to the structures and specific properties of saponins, flavonoids and volatile oil components in Chinese angelica blood soup, suitable chromatographic conditions and mass spectrum conditions are screened to realize simultaneous detection of 18 compounds.

In view of the above, we found that the following problems exist in the prior art in the process of quality control of the decoction for tonifying blood:

(1) The angelica sinensis blood-replenishing soup recorded in the prior art does not define specific processed products of the angelica sinensis in the formula, and further defines a content measuring method of the angelica sinensis blood-replenishing soup in the wine washing angelica sinensis especially important in view of the difference of index components and drug effects of the angelica sinensis and the wine washing angelica sinensis.

(2) Patent CN11155162a establishes a method for simultaneously measuring 18 compounds in the decoction of angelica sinensis for replenishing blood, but the equipment input cost is high, and the conditions of chromatography and mass spectrum for screening 18 compounds are strict, so that the detection accuracy is difficult to consider.

Disclosure of Invention

The invention aims to provide a preparation process of standard decoction of Chinese angelica blood replenishing decoction by taking wine-washed Chinese angelica and astragalus decoction pieces as formula raw materials, wherein the preparation process takes the contents of astragaloside IV, calycosin glucoside and ferulic acid as investigation indexes and combines the ointment yield of the contents of the astragaloside IV, calycosin glucoside and ferulic acid to determine the selection of various parameter conditions in the decoction process, and the process operation is stable, reliable, reasonable and feasible.

The invention also provides a quality control method of the standard decoction of the Chinese angelica blood-replenishing decoction in order to realize the stability and reliability of the preparation process of the standard decoction of the Chinese angelica blood-replenishing decoction.

The invention is realized by the following technical scheme:

a preparation process of standard decoction of radix Angelicae sinensis decoction for replenishing blood comprises mixing 1 part of radix Angelicae sinensis washed with wine and 5 parts of radix astragali decoction pieces, adding 600ml of water, soaking for 30min, boiling with strong fire, decocting with slow fire for 40min, filtering while hot, and cooling and drying to obtain lyophilized powder of standard decoction of radix Angelicae sinensis decoction for replenishing blood.

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

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

Reference solution a: respectively taking ferulic acid, calycosin glucoside, senkyunolide I and ligustilide, adding methanol to prepare solutions containing 50-100 μg per 1ml,

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

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

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

In the step B, the gradient stripping 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 astragalus decoction pieces in the standard decoction of the angelica sinensis blood replenishing decoction prepared by the method, and comprises the following steps:

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

reference solution: adding methanol into formononetin, calycosin glucoside, astragaloside I, astragaloside II, iso-astragaloside I and ferulic acid to obtain 50-100 μg solutions per 1ml,

test solution II: adding water 20ml into 1g of lyophilized powder of standard decoction of radix Angelicae sinensis decoction for replenishing blood, performing ultrasonic treatment for 30 min, shaking, and filtering to obtain subsequent filtrate;

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

C. and (3) taking the characteristic spectrum of the reference object solution B as a reference spectrum, selecting a common peak from the characteristic spectrum of the test object solution II, and constructing the characteristic spectrum of astragalus decoction pieces in the standard decoction of the angelica sinensis blood replenishing decoction.

In the step B, the gradient stripping 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 replenishing decoction prepared by the method by adopting a high performance liquid chromatography method, and comprises the following steps of:

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

ferulic acid control solution: taking a proper amount of ferulic acid reference substance, adding 70% methanol to prepare solutions containing 12 μg ferulic acid per 1mL,

test solution III: taking 0.1g of freeze-dried powder of standard decoction of the angelica sinensis decoction for replenishing blood, adding 10ml of 70% methanol, sealing, weighing, performing ultrasonic extraction for 30 minutes, cooling, weighing again, supplementing the reduced weight with 10% methanol, shaking uniformly, standing, filtering the supernatant, and taking the subsequent filtrate to obtain the Chinese angelica decoction;

B. taking 10 mu l of ferulic acid reference substance solution and 10 mu l of sample solution III respectively, injecting into a high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as filler, and acetonitrile-0.085% phosphoric acid solution 17:83 is a mobile phase, the detection wavelength is 316nm, and the measurement is carried out at the column temperature of 35 ℃ to obtain the content of ferulic acid in the standard decoction of the angelica sinensis blood replenishing decoction, which is more than or equal to 0.02 percent.

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

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

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

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

B. respectively taking 10 mu l of calycosin glucoside reference substance solution and 10 mu l of test substance 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, performing gradient elution, and measuring at an inspection wavelength of 260nm to obtain the content of calycosin glucoside in the standard decoction of the Chinese angelica blood-replenishing decoction, wherein the content of calycosin glucoside in the standard decoction is more than or equal to 0.02%.

In the step B, the gradient stripping 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%.

The method also comprises the step of measuring the content of astragaloside IV in the standard decoction of the angelica sinensis blood replenishing decoction prepared by the method by adopting a high performance liquid chromatography method, and comprises the following steps of:

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

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

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

B. taking 5 μl and 10 μl of astragaloside IV reference solution and 20 μl of test solution VI respectively, and injecting into high performance liquid chromatograph with octadecylsilane chemically bonded silica as filler, acetonitrile-water 32:68 is a mobile phase, and the content of astragaloside in the standard decoction of the angelica sinensis blood replenishing decoction is more than or equal to 0.05 percent.

Compared with the prior art, the invention has the following advantages:

(1) The invention develops the preparation process research of the standard decoction of the Chinese angelica blood-replenishing decoction based on national policy guidance (wine-washed Chinese angelica and astragalus decoction pieces are taken as prescription raw materials), adopts the contents of astragaloside IV, calycosin glucoside and ferulic acid and the plaster 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 decoction of the Chinese angelica blood-replenishing decoction, effectively controls the quality of the standard decoction of the Chinese angelica blood-replenishing decoction, and provides more stable and accurate data for the application of the subsequent standard decoction of the Chinese angelica blood-replenishing decoction in the automatic traditional Chinese medicine dispensing machine.

(2) The invention also provides a method for controlling the quality of the standard decoction of the Chinese angelica blood-replenishing decoction by adopting the characteristic spectrum, which is constructed and obtained under specific detection conditions by taking the active ingredients of the Chinese angelica subjected to wine washing, namely ferulic acid, calycosin glucoside, senkyunolide I and ligustilide as reference substances, so that the quality control of the Chinese angelica subjected to wine washing in the standard decoction of the Chinese angelica blood-replenishing decoction can be rapidly and efficiently realized.

(3) According to the invention, the characteristic spectrum of astragalus membranaceus decoction pieces in the standard decoction of the Chinese angelica blood-replenishing decoction is constructed by taking the effective components of the astragalus membranaceus decoction pieces, namely, 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 astragalus membranaceus decoction pieces in the standard decoction of the Chinese angelica blood-replenishing decoction can be rapidly and efficiently realized.

(4) The method overcomes the defects that the prior method only measures the content to reflect the quality control in the industrial production process, combines the comparison of common peaks in the characteristic spectrum, has the characteristics of strong specificity, high stability and good repeatability, can comprehensively, quickly and efficiently identify decoction pieces in the standard decoction of the Chinese angelica blood enriching decoction, is applied to the quality control of the standard decoction of the Chinese angelica blood enriching decoction, and has the advantages of simple operation, stability, reliability, high precision, strong specificity and good separation degree.

In summary, the invention provides the preparation process of the standard decoction of the angelica blood replenishing decoction, which uses the wine-washed angelica as the angelica feeding decoction piece, for the first time, and also provides a quality control method for comprehensively reflecting the magnitude transfer relation of the standard decoction of the angelica blood replenishing decoction by various content measurement and characteristic spectrum methods, thereby realizing the whole process reflection from medicinal materials-decoction pieces-standard decoction, better meeting the requirements of the original prescription and ensuring the medication accuracy.

Drawings

FIG. 1 is a schematic diagram of crushing of wine-washed angelica sinensis and astragalus membranaceus decoction pieces.

Fig. 2 is a comparison characteristic map of wine-washed angelica in standard decoction of angelica sinensis blood-replenishing decoction.

Fig. 3 is a characteristic map of standard decoction of radix Angelicae sinensis decoction for replenishing blood.

Fig. 4 is a reference characteristic spectrum (DAD) of astragalus decoction pieces in standard decoction of the angelica sinensis decoction for replenishing blood.

Fig. 5 is a comparison characteristic map (ELSD) of astragalus decoction pieces in standard decoction of the angelica sinensis blood replenishing decoction.

Fig. 6 is a specific investigation map (DAD) of astragalus decoction pieces in standard decoction of the angelica sinensis blood replenishing decoction.

Fig. 7 is a specific investigation map (ELSD) of astragalus decoction pieces in standard decoction of the angelica sinensis blood replenishing decoction.

Fig. 8 is a characteristic spectrum (DAD) of standard decoction of radix Angelicae sinensis decoction for replenishing blood.

Fig. 9 is a characteristic spectrum (ELSD) of standard decoction of the decoction of Angelica sinensis.

Fig. 10 is a chart for investigating the specificity of ferulic acid in standard decoction of radix Angelicae sinensis decoction for replenishing blood.

Fig. 11 is a standard graph of ferulic acid in standard decoction of radix Angelicae sinensis decoction for replenishing blood.

Fig. 12 is a chart for investigating specificity of calycosin glucoside in standard decoction of radix Angelicae sinensis decoction for replenishing blood.

FIG. 13 is a graph showing standard profiles of calycosin glucoside in standard decoction of Angelica sinensis blood replenishing decoction.

Fig. 14 is a diagram for investigating the specificity of astragaloside IV in standard decoction of radix Angelicae sinensis decoction.

Fig. 15 is a graph of astragaloside IV standard in standard decoction of radix Angelicae sinensis decoction for replenishing blood.

Fig. 16 is a control map of standard decoction of medicinal materials, decoction pieces and Chinese angelica blood replenishing decoction.

Detailed Description

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

Example 1:

the embodiment provides a preparation process of standard decoction of Chinese angelica blood replenishing decoction.

The preparation method comprises the steps of taking wine-washed angelica sinensis and astragalus membranaceus decoction pieces as raw materials of a formula, respectively pulverizing into powder, and sieving with a 10-mesh sieve. Sieving, washing radix Angelicae sinensis 8g and radix astragali 40g with wine, soaking in 600ml water for 30min, boiling with strong fire, decocting with slow fire for 40min, filtering (200 mesh sieve), cooling to room temperature, lyophilizing, and packaging to obtain lyophilized powder of standard decoction of GUIXUE decoction.

Example 2:

the embodiment is a study on the preparation process of the standard decoction of the Gui Xuexue decoction.

1. Decoction piece selection

1.1 processing of Astragalus membranaceus decoction pieces

Reference to the processing of astragalus membranaceus decoction pieces in the 2015 edition of Chinese pharmacopoeia: removing impurities, separating, cleaning, moistening, slicing, and drying. The specific parameters are as follows: taking astragalus medicinal material, removing impurities, cleaning, moistening for about 12 hours by a moistening method, cutting into 2-4 mm thick slices, and drying at 60-70 ℃.

1.2 processing Angelica sinensis with wine

The wine washing is a traditional Chinese medicine wine preparation method used in the past, and is generally considered that the medicine can partially permeate into the tissues after the wine washing, thereby playing roles of slowing, enhancing and the like. Wine washing is firstly found in the peel and clear wine washing of rhubarb in Shang Han Dynasty Zhang Zhongjing, is used in decoction for regulating stomach and qi, and is considered as "washing with wine into yangming channel". Meanwhile, by combining with the internal and external injury confusion theory of the decoction of Chinese angelica, the Chinese angelica is considered as 'blood nourishing and middle conservation' by the Lidong palisade of doctors, and the Chinese angelica can be changed into 'through' after soaking (washing) with wine, so that the Chinese angelica can enrich blood without stagnation. The efficacy of the decoction pieces of Chinese angelica, wine Chinese angelica, in modern times is that the Chinese angelica is prepared into the solar meridian, and the two are greatly different.

Modern processing specifications describe that wine angelica is prepared by taking clean angelica slices and stir-frying the clean angelica slices according to a wine-stir-frying method (Tongsu 0213) (Chinese pharmacopoeia 2020 edition). Wine-washed angelica sinensis is described in different local processing specifications respectively: 1) Spraying yellow wine on radix Angelicae sinensis according to wine parching method (appendix I), stirring, sucking completely, and sun drying or low temperature drying. 100kg of Chinese angelica, 15kg of yellow wine (the processing standard 2008 edition of Shanghai Chinese medicine); 2) And taking angelica pedunculata or slices, adding 5kg of white spirit into 50 kg of the angelica pedunculata or slices, spraying, uniformly stirring, soaking and sucking, absorbing for about 2-3 hours, cutting or cutting into round or oblique slices, and airing (the processing standard 1986 edition of Chinese medicinal decoction pieces in Yunnan province).

In combination with literature examination and processing research, the processing specific parameters of the wine-washed angelica are as follows: the Chinese angelica medicine is prepared with Chinese angelica root as main material, and through sprinkling wine (yellow wine), moistening, low temperature drying and other steps, and the yellow wine is 100g for every 100g Chinese angelica root.

Meanwhile, an HPLC-DAD characteristic spectrum method is adopted to distinguish the three processed decoction pieces of the Chinese angelica subjected to wine washing from the Chinese angelica and the Chinese angelica, so that the accuracy of medication is ensured (see the characteristic spectrum, the construction method and the identification method of the Chinese angelica and the decoction pieces thereof recorded in the published patent 202010382210.7).

1.3 decoction piece decoction and dispersion treatment

The Chinese angelica blood-replenishing decoction is prepared by crushing medicinal material preparations according to the requirements of the ancient classical name prescription directory (first batch) issued by the national Chinese medicine administration, wherein the medicinal material preparations are required to be crushed by adopting a powder-boiling formulation, and the particle size of the medicinal material preparations is required to be determined in order to ensure the quality consistency of standard decoction of the Chinese angelica blood-replenishing decoction. The medicinal particles of the decoction powder generally have 4 specifications of coarse powder, fine powder and fine powder. Chen Shilin et al (traditional Chinese medicine accurate decoction pieces; world science and technology-modernization of traditional Chinese medicine; 2016,18 (09): 1430-1440) and Xing Dan et al (from the technical Specification of decoction of traditional Chinese medicine; J.S. of Taiping Huimin, 2012,40 (11): 73-75.) after study, considered that "coarse powder" was equivalent to the present coarse powder, and was sieved (10 mesh); the coarse powder is equal to coarse powder and is sieved by a No. two sieve (24 meshes); the "powder" is approximately between the coarse powder and the fine powder, and is sieved by a No. three sieve (50 meshes); the fine powder corresponds to middle powder and is sieved by a No. four sieve (65 meshes). To further confirm the decoction piece specifications, the following observations were carried out:

Taking 20g of wine-washed angelica and 100g of astragalus decoction pieces, respectively pulverizing into powder and sieving through a pharmacopoeia sieve. According to the requirements of the original recipe of the angelica blood-replenishing decoction, 8g of angelica and 40g of astragalus are respectively taken and washed by wine after sieving, 600ml of water is added, soaking is carried out for 30 minutes, boiling is carried out for 30 minutes by strong fire, filtering is carried out when the mixture is hot (200-mesh sieve), volume is measured after cooling, and the extract rate of decoction liquid and the contents of astragaloside IV, calycosin glucoside and ferulic acid 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 Chinese pharmacopoeia 2015. The results are shown in Table 1 below.

TABLE 1 investigation of decoction pieces of different particle sizes

Remarks: comprehensive evaluation calculation method = 25% of ointment yield%25% + of astragaloside content%25% + of calycosin glucoside%25% + of ferulic acid%25%

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

2. Investigation of decoction time

The original recipe of the Chinese angelica decoction for replenishing blood is recorded as "two decoction pieces are decocted to one decoction piece". The time for decoction is not clear in the original prescription, and the time for decoction to one cup is specially examined in order to keep the same with the original prescription. Respectively taking 3 parts of crushed and compatible Chinese angelica blood-replenishing decoction (8.0 g of Chinese angelica washed by wine and 40.0g of astragalus), respectively adding 600ml of water, soaking for 30min, covering with strong fire, boiling, then turning into slow fire, and respectively measuring the time for decocting 200ml of 3 parts of Chinese angelica blood-replenishing decoction. The results are shown in Table 2 below.

Table 2 investigation of decoction time of the Danggui Buxue decoction

Batch of	Water addition amount ml	Decocting for 20min with volume ml	Decocting for 30min with volume ml	Decocting for 40min with volume ml
					1	600	485	355	300
2	600	480	345	301
					3	600	460	330	300

According to the above experiments, in combination with the requirements of the state traditional Chinese medicine administration on the notice of the management regulations of the traditional Chinese medicine decocting room of the printing medical institution: the tonic medicine is boiled with strong fire and then decocted with slow fire for about 40-60 min. The decoction time of the angelica sinensis decoction for replenishing blood is determined to be 40 minutes.

3. Filtering and drying

3.1 filtration

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

3.2 drying

The quality standard of the Chinese angelica decoction for replenishing blood corresponds to the drying of a real object according to the requirements of standard decoction in the technical requirements of quality control and standard establishment of Chinese medicine prescription granule (request opinion manuscript) and the medical institution Chinese medicine decoction room management standard, the Chinese angelica decoction for replenishing blood is recommended to be prepared by adopting a freeze drying method, the consistency of the quality of the Chinese angelica decoction and the original decoction can be ensured, the stability of the quality of the corresponding real object is ensured, meanwhile, the corresponding real object is ensured to be easy to dissolve, and auxiliary materials are not added.

Thus, the corresponding entities of the study used a freeze-drying process, the freeze-drying process parameters of which are shown in Table 3 below.

Table 3 lyophilization process

3.3 powder collecting and storing

And (3) rapidly collecting powder under the environment of the temperature of 10-26 ℃ and the relative humidity of 40-60%. Storing the standard decoction lyophilized powder in a dryer, and placing in a shade and dry place for use.

In summary, the standard decoction of the angelica sinensis decoction for replenishing blood is prepared by the following steps:

taking crushed (sieved) wine-washed angelica sinensis and astragalus decoction pieces, and mixing according to a proportion of 1:5, adding 600ml of water, soaking for 30min, boiling with strong fire, decocting with slow fire for 40min, filtering (200 mesh sieve) while hot, cooling to room temperature, freeze-drying, and packaging to obtain lyophilized powder of standard decoction of GUIXUE decoction.

According to the preparation process, 13 batches of standard decoction of the angelica sinensis blood replenishing decoction are prepared. The lot numbers are respectively: DGBXBT01, DGBXBT02, DGBXBT03, DGBXBT04, DGBXBT05, DGBXBT06, DGBXBT07, DGBXBT08, DGBXBT09, DGBXBT10, DGBXBT11, DGBXBT12, and DGBXBT13.

Example 3:

the embodiment relates to a quality control method of standard decoction of Chinese angelica blood replenishing decoction.

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

1.1 laboratory instruments and materials (see laid-open patent 202010382210.7)

1.2 construction of a characteristic map

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

Test article solution I: taking 0.2g of freeze-dried powder of standard decoction of the angelica sinensis decoction for enriching blood, precisely weighing, placing into a conical flask, precisely adding 10ml of 70% methanol, performing ultrasonic treatment for 30 minutes, shaking uniformly, filtering, and taking subsequent filtrate to obtain the Chinese angelica decoction.

10 μl of each of the reference solution A and the sample solution I was precisely sucked, and the sample solution I was injected into a high performance liquid chromatograph, with octadecylsilane chemically bonded silica as a filler (column length: 250mm, inner diameter: 4.6mm, particle size: 5 μm), acetonitrile as mobile phase A, and 0.1% phosphoric acid solution as mobile phase B, and subjected to gradient elution according to the following Table 4, and the column temperature: 30℃and flow rate: 1.0ml per minute were measured at a detection wavelength: 290nm to obtain a corresponding characteristic spectrum.

TABLE 4 gradient desquamation

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

The characteristic spectrum of the reference object solution A is taken as a reference spectrum, a common peak is selected from the characteristic spectrum of the test object solution I, and the characteristic spectrum of the wine-washed angelica in the standard decoction of the angelica blood replenishing decoction is constructed, and the reference graph is shown in figure 2 (peak 1: calycosin glucoside; peak 2 (S): ferulic acid; peak 4: senkyunolide I; peak 7: ligustilide).

The sample chromatograph should show 7 characteristic peaks, wherein the peak corresponding to the ferulic acid reference substance is S peak. The relative retention time of each characteristic peak and the S peak was calculated to be within + -10% of the prescribed value. The specified value is: 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 methodology investigation

In order to fully and comprehensively reflect the whole appearance of the components contained in the Chinese angelica standard decoction for replenishing blood, the characteristic spectrum of the wine-washed Chinese angelica part of the standard decoction for replenishing blood is examined and verified, and the results are shown in figure 3 (DGBXBT 01, DGBXBT02, DGBXBT03, DGBXBT04, DGBXBT05, DGBXBT06, DGBXBT07, DGBXBT08, DGBXBT09, DGBXBT10, DGBXBT11, DGBXBT12, DGBXBT13, and tables 5 and 6 in sequence from bottom to top in the figure. (reference is made to the preparation method of the sample of the characteristic spectrum of the decoction pieces of Angelica sinensis and the chromatographic conditions of the decoction pieces of Angelica sinensis described in the publication 202010382210.7)

TABLE 5 liquid phase of 13 batch standard decoction and retention time

Table 6 methodological results RSD% summary criteria-relative retention time

2. Construction of characteristic spectrum of astragalus decoction pieces in standard decoction of angelica sinensis blood replenishing decoction

2.1 laboratory apparatus and materials

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

an electronic balance: ME204E/02, XP26 (Metrele Tolyduo instruments Co., ltd.);

ultrapure water machine: cell type 1810A (Shanghai mueller scientific instruments limited);

ultrasonic cleaner: KQ5200DB model (600W, 40KHz; kunshan ultrasonic instruments Co., ltd.);

Column chromatography: agilent TC-C18 (2) 5um 250X 4.6mm, etc.

Acetonitrile and formic acid are chromatographic purity, water is ultrapure water, and the rest reagents are analytical purity.

Formononetin (Sichuan Vickers biotechnology Co., ltd., lot number: wkq18112302, content 98%);

calycosin glucoside (China food and drug inspection institute, batch No. 111920-201606, content of 97.6%);

astragalus saponin I (Sichuan Uygur biotechnology Co., ltd., lot number: wkq18041805, content 98%);

astragalus saponin II (Sichuan Uygur biotechnology Co., ltd., lot number: wkq18041712, content 98%);

isofacial saponin I (Beijing century Oryza Biotechnology Co., ltd., lot number: 18050406, content of 98%);

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

2.2 construction of a feature map

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

test solution II: taking 1g of freeze-dried powder of standard decoction of the angelica sinensis decoction for replenishing blood, precisely weighing, placing in a conical flask, precisely adding 20ml of water, performing ultrasonic treatment for 30 minutes (power 600W, frequency 40 kHz), shaking uniformly, filtering, and taking subsequent filtrate to obtain the traditional Chinese medicine decoction;

Precisely sucking 10 μl of reference solution B and sample solution II, injecting into high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as filler (column length is 250mm, inner diameter is 4.6mm, granularity is 5 μm), acetonitrile as mobile phase A,0.02% formic acid solution as mobile phase B, gradient eluting according to the following table 7, detecting with ultraviolet detector and evaporative light scattering at column temperature of 25deg.C and flow rate of 1.0ml per minute, detecting wavelength of 254nm with ultraviolet detector drift tube temperature of 90deg.C and carrier gas flow rate of 1.8L/ml per minute, and obtaining corresponding characteristic map.

TABLE 7 gradient desquamation

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

Taking the characteristic spectrum of the reference object solution B as a reference spectrum, selecting a common peak from the characteristic spectrum of the test object solution II, and constructing the characteristic spectrum of astragalus decoction pieces in the standard decoction of the angelica sinensis blood replenishing decoction, wherein the characteristic spectrum is shown in a figure 4 (peak 1 (S): calycosin glucoside; peak 3: ferulic acid; peak 6: formononetin), a figure 5 (peak 1 (S): calycosin glucoside; peak 2: ferulic acid; peak 4: formononetin; peak 7: astragaloside II; peak 8: astragaloside I; peak 9: iso-astragaloside I).

The chromatogram (ultraviolet detection) of the test sample should show 9 characteristic peaks, wherein the peak corresponding to the calycosin glucoside reference substance is S peak. The relative retention time of each characteristic peak and the S peak was calculated to be within + -10% of the prescribed value. The specified value is: 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 of the test sample (evaporative light scattering detection) 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 and the S peak was calculated to be within + -10% of the prescribed value. The specified value is: 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 methodology investigation

The method for researching the characteristic spectrum of the astragalus part of the standard decoction of angelica for enriching blood comprises the steps of specialization, precision, repeatability, stability, intermediate precision and durability.

2.3.1 Properties

Preparation of negative control solution: the astragalus root standard decoction negative control solution is prepared according to the above-mentioned established conditions.

Preparation of test solution: preparing a sample solution of the characteristic spectrum of the standard decoction of the angelica sinensis blood replenishing decoction according to the above proposed conditions. The results are shown in fig. 6 and 7.

2.3.2 precision, repeatability, stability, intermediate precision and durability

The freeze-dried powder of standard decoction of the angelica sinensis decoction for replenishing blood is taken, a test solution is prepared according to the above-mentioned preparation method, and the precision, repeatability, stability, intermediate precision and durability are respectively examined, and the RSD values of the indexes are shown in the following tables 8 and 9, and the results show that the RSD values meet the requirements.

TABLE 8 DAD-methodological results RSD% summary relative retention time

TABLE 9 ELSD-methodology results RSD% summary relative retention time

2.3.3 verification investigation

By adopting the method, the characteristic spectrum analysis is carried out on 13 batches of samples, 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 in order from bottom to top), fig. 9 (DGBXBT 01, DGBXBT02, DGBXBT03, DGBXBT04, DGBXBT05, DGBXBT06, DGBXBT07, DGBXBT08, DGBXBT09, DGBXBT10, DGBXBT11, DGBXBT12, DGBXBT13 in order from bottom to top), table 10, table 11 below.

TABLE 10 batch 13 standard decoction phase to retention time DAD

TABLE 11 batch 13 standard decoction phase versus retention time ELSD

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

3. Determination of ferulic acid content in standard decoction of angelica sinensis blood replenishing decoction

3.1 laboratory apparatus and materials

Agilent 1260 type high performance liquid chromatograph;

an electronic balance: ME204E, XP260 (Metrele Tourette instruments Co., ltd.);

ultrapure water machine: cell type 1810A (Chongqing mol water treatment Co., ltd.);

constant temperature water bath: DK-98-II (Teste instruments Co., tianjin);

numerical control ultrasonic cleaner: KQ5200DB model (600W, 40KHz; kunshan ultrasonic instruments Co., ltd.);

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

methanol (analytically pure), acetonitrile (chromatographically pure), phosphoric acid (chromatographically pure), ultrapure water;

ferulic acid (Chinese food and drug inspection institute, batch No. 110773-201614, content of 99%).

3.2 determination of ferulic acid content

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

test solution III: taking 0.1g of freeze-dried powder of standard decoction of the angelica sinensis blood replenishing decoction, precisely weighing, placing in a conical bottle with a plug, precisely adding 10ml of 70% methanol, sealing, weighing, ultrasonically extracting for 30 minutes, cooling, weighing again, supplementing the weight of loss with 10% methanol, shaking uniformly, standing, filtering the supernatant, and taking a subsequent filtrate to obtain the Chinese angelica blood replenishing decoction;

precisely sucking 10 μl of ferulic acid reference solution and 10 μl of sample solution III, injecting into high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as filler, acetonitrile-0.085% phosphoric acid solution (17:83) as mobile phase, measuring at column temperature of 35deg.C with detection wavelength of 316nm, and measuring to obtain standard decoction of radix Angelicae sinensis decoction with ferulic acid content of more than or equal to 0.02%.

3.3 methodology investigation

3.3.1 investigation of specificity

The ferulic acid control solution, the test sample solution and the negative control solution were prepared according to the above-described development method, and the results are 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

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

3.3.3 repeatability investigation

Taking the same sample (DGBXBT 12), precisely weighing 6 parts, preparing a sample solution by the same operator according to a formulated method, and calculating the content of ferulic acid in the 6 parts of sample. The RSD value of the ferulic acid content is 0.63%, and the method has good repeatability.

3.3.4 linear relationship investigation

Taking a proper amount of ferulic acid reference substance, and preparing a 0.04654mg/mL reference substance mother solution by 70% methanol. Respectively diluting into control solutions with concentrations of 0.02327mg/mL, 0.0174525mg/mL, 0.00872625mg/mL, 0.004363125mg/mL and 0.0021815625mg/mL, injecting the solutions into a liquid chromatograph, analyzing to obtain peak areas, and drawing response curves with sample injection concentrations (X, mug) as abscissa and peak areas (Y) as ordinate. The results are shown in Table 12 below and FIG. 11.

TABLE 12 analysis of ferulic acid standard curve

Sample injection amount (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 results showed that the ferulic acid standard curve was y= 4.765x-2.649, r ² = 0.9996. The sample injection concentration is in the range of 21.815625-465.4 mug, and the linear relation is good.

3.3.5 stability experiments

The same sample solution (batch number: DGBXBT 12) is taken, the ferulic acid content is measured 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 established experimental conditions, the same sample solution (DGBXBT 12) is taken and respectively adopts different chromatographic columns (Agilent 1;Agilent 2;Kromaisl 1), different instruments (Agilent 1260, waters2695 and Shimadzu LC-20AD high performance liquid chromatograph), different personnel (A, B) are used for examining 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 sample recovery rate

About 0.1g of a sample (batch number: DGBXBT12, ferulic acid content 0.0.049%) with known content is taken, 6 parts are taken, a certain amount of ferulic acid reference substance (purity 99.0%) is precisely weighed, the preparation and measurement of the sample solution are carried out according to a planned method, and recovery rates are calculated and are respectively 90.46%, 91.27%, 93.23%, 91.33%, 94.12% and 90.77%, which indicate that the method is good in accuracy.

3.3.8 verification investigation

13 batches of Chinese angelica blood-replenishing standard soup are used as test samples, test sample solutions are prepared according to a planned method, the peak areas are recorded, the content of ferulic acid is calculated, and the results are shown in Table 13.

TABLE 13 results of the ferulic acid content of the Danggui blood-replenishing standard decoction

Sequence number	Standard decoction lot 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

From the table, it can be seen that ferulic acid (C) is contained in 13 batches of standard decoction of radix Angelicae sinensis for replenishing blood ₁₀ H ₁₀ O ₄ ) The content of (C) is in the range of "0.029% -0.052%", the average value is 0.037%, and the range of + -30% of the average value is "0.026% -0.048".

Determining the limit of measurement of the ferulic acid content (C ₁₀ H ₁₀ O ₄ ) Not less than 0.02%.

4. Determination of content of calycosin glucoside in standard decoction of Chinese angelica blood-replenishing decoction

4.1 laboratory apparatus and materials

High performance liquid chromatograph: agilent 1260 type high performance liquid chromatograph, shimadzu LC-20AD type high performance liquid chromatograph, waters e2695 type high performance liquid chromatograph;

an electronic balance: ME204E/02, MS205DU, XP26 (Metrele Tolyduo instruments Co., ltd.);

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

acetonitrile and formic acid are chromatographic purity, water is ultrapure water, and the rest reagents are analytical purity;

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

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 μg calycosin glucoside per 1mL,

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

precisely sucking the reference solution and the sample solution V of calycosin glucoside 10 μl respectively, injecting into high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A,0.2% formic acid solution as mobile phase B, gradient eluting according to the following table 14, measuring at 260nm inspection wavelength, and measuring to obtain the content of calycosin glucoside in standard decoction of radix Angelicae sinensis blood replenishing decoction not less than 0.02%.

TABLE 14 gradient desquamation

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

4.3 methodology investigation

4.3.1 investigation of specificity

Test solutions and negative-deficiency control solutions were prepared according to a predetermined method, 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 investigation

The control solution is continuously sampled for 6 times, the peak area of the calycosin glucoside is recorded, and the RSD value of the peak area is 0.19%, which indicates that the instrument precision is good.

4.3.3 linear relationship investigation

Taking a proper amount of calycosin glucoside reference substance, and preparing a 111.16640 mug/mL reference substance mother solution by using methanol. Preparing solutions with the following concentrations, respectively sucking 10 μl of each solution, injecting into a liquid chromatograph, analyzing to obtain peak areas, and drawing response curves by taking sample injection amounts (X, ng) as abscissa and peak areas (Y) as ordinate. The results are shown in Table 15 below and FIG. 13.

TABLE 15 results of Calycosin glucoside Standard Curve analysis

The results show that: calycosin glucoside standard curve y= 2.7826x-10.841, r ² =0.9999. The sample injection amount is in the range of 111.1664-1111.6640 ng, and the linear relation is good.

4.3.4 stability test

The same sample solution (batch number: DGBXBT 12) is taken, and the chromatographic peak areas of the calycosin glucoside are measured at 0,2,4,6, 12 and 24 hours respectively, wherein the RSD value of the peak area is 1.24%, and the sample solution has better stability within 24 hours.

4.3.5 repeatability experiments

0.5g of the same sample (batch number: DGBXBT 12) is taken, 6 parts of the sample are precisely weighed, the same operator prepares a sample solution according to a planned method, and the content of the calycosin glucoside of the 6 parts of the sample is calculated, wherein the content RSD% is 2.32%, which indicates that the method has good repeatability.

4.3.6 intermediate precision

According to the established experimental conditions, the same sample solution (DGBXBT 12) is taken and respectively adopts different chromatographic columns (Agilent 1;Agilent 2;Kromaisl 1), different instruments (Agilent 1260, waters2695 and Shimadzu LC-20AD high performance liquid chromatograph) are adopted, different personnel (A, B) are used for examining at different times (I and II), and the RSD of the content of the calycosin glucoside is respectively 3.21%, 2.14% and 5.97%.

4.3.7 sample recovery rate

About 0.5g of a test sample (batch number: DGBXBT 12) with known content is taken, 6 parts are taken, a certain amount of calycosin glucoside reference substance (purity is 97.6%) is precisely weighed, a certain amount of calycosin glucoside reference substance is precisely added respectively, the preparation and measurement of the test sample solution are carried out according to a planned method, the recovery rates are calculated and respectively 92.27%, 93.61%, 94.65%, 95.04%, 100.23% and 100.07%, and the recovery rate RSD is 6.13%, which indicates that the method is good in accuracy.

4.3.8 verification investigation

13 batches of standard decoction freeze-dried powder are used as test samples, test sample solutions are prepared according to a planned method, the peak areas are recorded, and the content of calycosin glucoside is calculated, so that the results are shown in the table 16 below.

Table 16 results of measurement of the content of lyophilized powder in 13 batches of standard decoction

Lot 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 the actual measurement range of the calycosin glucoside in the standard decoction is as follows: 0.027 to 0.096 percent,the average content was 0.065%. The content of the product is 70% -130% of the average value measured by measuring, and the content is expressed as the content of calycosin glucoside (C ₂₂ H ₂₂ NO ₁₀ )0.046～0.085％。

From the above data, calycosin glucoside (C ₂₂ H ₂₂ NO ₁₀ ) The measurement limit of (2) is not less than 0.02%.

5. Determination of astragaloside IV content in standard decoction of Chinese angelica blood-replenishing 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;

Chromatographic column: agilent ZORBAX Eclipse plus 5 μm 250×4.6mm, etc.;

acetonitrile is chromatographic purity, water is ultrapure water, and the rest reagents are analytical purity;

astragaloside IV (Chinese food and drug inspection institute, batch number: 110781-201717, content of 96.9%);

5.1 laboratory apparatus and materials

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

test solution vi: taking 1.0g of freeze-dried powder of standard decoction of the angelica sinensis blood replenishing decoction, precisely weighing, placing into a conical bottle with a plug, adding 50mL of 80% methanol containing 4% of concentrated ammonia test solution, sealing, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the weight of the loss with 80% methanol containing 4% of concentrated ammonia test solution, shaking uniformly, filtering, precisely measuring 25mL of continuous filtrate, evaporating to dryness, dissolving residues with 80% methanol, transferring to a 5mL measuring flask, adding 80% methanol to scale, shaking uniformly, filtering, and taking the continuous filtrate to obtain the traditional Chinese medicine decoction;

precisely sucking 5 μl and 10 μl of astragaloside IV reference solution, 20 μl of test solution VI, and injecting into high performance liquid chromatograph with octadecylsilane chemically bonded silica as filler, acetonitrile-water 32:68 is mobile phase, the evaporation light scattering detector is used for measurement, and the external standard two-point method logarithmic equation is used for calculation, so that the content of astragaloside in the standard decoction of the angelica sinensis blood replenishing decoction is more than or equal to 0.05%.

5.2 determination of Astragaloside IV content

5.3 investigation of extraction modes

5.3.1 extraction method investigation I

In combination with the content measurement of astragaloside IV, the following three extraction modes are used for extraction and measurement of the content of astragaloside IV:

(1) Extraction mode 1-1: weighing about 2g of test sample (batch number: DGBXBT 14), precisely weighing, placing into a Soxhlet extractor, adding 40ml of methanol, cold soaking overnight, adding appropriate amount of methanol, heating and refluxing for 4 hours, recovering solvent from the extract, concentrating to dryness, adding 10ml of water into the residue, slightly heating to dissolve, shaking and extracting with water saturated n-butanol for 4 times, 40ml each time, mixing n-butanol solutions, fully washing with ammonia solution for 2 times, discarding 40ml each time, evaporating ammonia solution and n-butanol solution, adding 5ml of water into the residue to dissolve, cooling, 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 the residue with methanol, transferring to a 5ml measuring flask, adding methanol to scale, and shaking to obtain the final product.

(2) Extraction mode 1-2: weighing about 2g of test sample (batch number: DGBXBT 14), precisely weighing, adding 10ml of water into the residue, performing ultrasonic treatment for 30min, shaking and extracting with water saturated n-butanol for 4 times, 40ml each time, combining n-butanol solutions, washing with ammonia test solution for 2 times, 40ml each time, discarding ammonia solution, evaporating n-butanol solution to dryness, adding 5ml of water into the residue to dissolve the residue, cooling, passing through D101 type macroporous adsorbent resin column (inner diameter of 1.5cm, column height of 12 cm), 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 the residue with methanol, transferring to 5ml measuring flask, adding methanol to scale, shaking to obtain the final product.

(3) Extraction modes 1-3: weighing about 0.5g of a sample (batch number: DGBXBT 14), precisely adding 80% methanol solution containing 4% of concentrated ammonia solution (4 ml of concentrated ammonia solution is taken, 80% methanol is added to 100ml, shaking up) into 50ml, sealing, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the lost weight with 80% methanol solution containing 4% of concentrated ammonia solution, shaking up, filtering, precisely measuring 25ml of a subsequent filtrate, evaporating to dryness, dissolving residues with 80% methanol, transferring into a 5ml measuring flask, adding 80% methanol to scale, shaking up, filtering, and taking the subsequent filtrate to obtain the final product.

The three extraction modes were analyzed, see table 17 below.

TABLE 17 analytical results for different extraction modes I

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

5.3.2 extraction method investigation II

The extraction mode is optimally compared on the basis of the above steps:

(1) Weighing about 1.0g of a sample (batch number: DGBXBT 14), placing the sample into a conical flask with a plug, precisely adding 50mL of 80% methanol containing 4% of concentrated ammonia test solution (4 mL of concentrated ammonia test solution is taken, 80% methanol is added to 100mL, shaking up), sealing, weighing, heating and refluxing for 1 hour, cooling, weighing again, supplementing the lost weight with 80% methanol containing 4% of concentrated ammonia test solution, shaking up, filtering, precisely measuring 25mL of a continuous 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 continuous filtrate to obtain the product.

(2) Weighing about 1.0g of a test sample (batch number: DGBXBT 14), placing the test sample into a conical flask with a plug, precisely adding 50mL of 80% methanol containing 4% of concentrated ammonia test solution (4 mL of concentrated ammonia test solution is taken, 80% methanol is added to 100mL and shaking up), sealing, weighing, carrying out ultrasonic treatment for 1 hour, cooling, weighing again, supplementing the lost weight with 80% methanol containing 4% of concentrated ammonia test solution, shaking up, filtering, precisely measuring 25mL of a continuous filtrate, evaporating to dryness, dissolving residues with 80% methanol, transferring to a 5mL measuring flask, adding 80% methanol to a scale, shaking up, filtering, and taking the continuous filtrate to obtain the product.

The two extraction modes were analyzed as 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 reflux extraction mode is selected comprehensively.

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

5.4 methodology investigation

5.4.1 specificity experiments

Test solutions and negative-deficiency control solutions were prepared according to a predetermined method, 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

The control solution is continuously sampled for 6 times, the peak area of astragaloside IV is recorded, and the RSD value is 2.41%, which shows that the method has good instrument precision.

5.4.3 Linear investigation

And (3) taking a proper amount of astragaloside IV reference substances, respectively preparing the solutions with the following concentrations, respectively sucking 20 mu l of the solutions, injecting the solutions into a liquid chromatograph, analyzing to obtain peak areas, and drawing a response curve by taking LOG sample injection quantity (X) as an abscissa and LOG peak area (Y) as an ordinate. The results are shown in Table 19 and FIG. 15 below.

TABLE 19 Astragaloside IV standard curve analysis results

Sample concentration (μg/ml)	98.6442	1972884	493.221	986.442	1233.0525	1972.884
							LOG sample injection amount (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: astragaloside IV standard curve is y= 1.4654x-2.6365, R ² =0.9965. The sample injection concentration is in the range of 98.6442-1972.8840 mug/mL, and the linear relation is good.

5.4.4 stability investigation

The same sample solution (batch number: DGBXBT 12) is taken, and the peak areas RSD of astragaloside IV are 3.55% respectively measured at 0,2,6,8, 12 and 24 hours, so that the sample solution has better stability within 24 hours.

5.4.5 repeatability investigation

1.0g of the same sample (batch number: DGBXBT 12) is taken, 6 parts of the sample are precisely weighed, the same operator prepares a sample solution according to a planned method, and the content RSD of the astragaloside IV of the 6 parts of the sample is 4.08 percent.

5.4.6 intermediate precision

According to the established experimental conditions, the same sample solution (DGBXBT 12) is taken and respectively adopts different chromatographic columns (Agilent 1;Agilent 2;Kromaisl 1), different instruments (Agilent 1260 and Shimadzu LC-20AD high performance liquid chromatograph), different persons (A, B) are used for examining at different times (I and II), and the RSD of the content of the calycosin glucoside is 6.92%, 9.87% and 9.91% respectively.

5.4.7 sample recovery rate

About 0.5g of a test sample (batch number: DGBXBT 12) with known content is taken, 6 parts are taken, a certain amount of astragaloside IV reference substance (purity is 96.9%) is precisely weighed, a certain amount of astragaloside IV reference substance is precisely added, the preparation and measurement of the test sample solution are carried out according to a planned method, and the recovery rate is calculated, wherein the recovery rate is 90.82%, 91.12%, 93.65%, 96.53%, 98.68% and 93.00%, and the average value is 91.85%, so that the method is good in accuracy.

5.4.8 verification investigation

13 batches of standard decoction freeze-dried powder are used as test samples, test sample solutions are prepared according to a planned method, the peak area is recorded, the astragaloside IV content is calculated, and the result is shown in the table 20 below.

Table 20 results of measurement of the content of lyophilized powder in 13 batches of standard decoction

The result shows that the actual measurement range of astragaloside IV in the standard decoction is as follows: 0.109-0.229%, the average content is 0.161%. The content of the product is 70-130% of the average value measured by the content measurement, and the content is limited by the content of astragaloside IV (C ₄₁ H ₆₈ NO ₁₄ )0.113～0.209％。

From the above data, astragaloside IV (C) ₂₂ H ₂₂ NO ₁₀ ) The measurement limit of (2) 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 medicinal materials-decoction pieces-standard decoction of the Chinese angelica subjected to wine washing.

By adopting the established standard decoction characteristic spectrum method of the angelica sinensis blood replenishing decoction, the rapid distinction between the wine-washed angelica sinensis decoction pieces in the angelica sinensis blood replenishing decoction from medicinal materials to decoction pieces in the standard decoction process can be realized, and the influence on the safety and effectiveness of the prescription due to the fact that other decoction pieces which do not meet the requirements of the original prescription of the classical prescription are taken as medicines is avoided.

In the method for constructing the characteristic spectrum, referring to the following figure 16 (peak 1 calycosin glucoside, peak 2 (S) ferulic acid, peak 4 senkyunolide I and peak 7 ligustilide), the characteristic spectrum of the corresponding real object of the angelica sinensis blood replenishing decoction can be seen to have 7 characteristic peaks by comparing the single decoction with the combined decoction characteristic spectrum of the wine-washed angelica sinensis and astragalus decoction pieces, wherein the peaks 1, 3 and 5 can find the corresponding peaks in the astragalus medicinal materials, decoction pieces and standard decoction. 2. The peaks 4 and 7 are respectively the peaks 4, 5 and 8 of the wine-washed Chinese angelica decoction pieces and the wine-washed Chinese angelica standard decoction pieces. The No. 6 peak is a characteristic peak generated after the astragalus and the wine-washed angelica decoction pieces are decocted together, which indicates that the Chinese medicine is decocted together to generate new substances. In the quantity value transmission process of the angelica medicinal material and the wine-washed angelica decoction pieces, the research shows that after the angelica medicinal material is subjected to wine washing, the characteristic spectrum of the angelica medicinal material is more than 1 characteristic peak (wine-washed angelica peak 1) from yellow wine.

(2) And determining relevant parameters of preparation process of the standard decoction of the angelica sinensis blood replenishing decoction based on national policy guidance.

Under the condition that the original party has no parameters of the preparation process, combining ancient books and related examination and experimental investigation, the detailed preparation parameters are established: taking crushed (sieved) wine-washed angelica sinensis and astragalus decoction pieces, and mixing according to a proportion of 1:5, adding 400ml of water, soaking for 30min, boiling with strong fire, decocting with slow fire for 40min, filtering while hot, cooling to room temperature, lyophilizing, and packaging.

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

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

On the basis of the contents of effective combination of efficacy, effective components and the like, 3 component content indexes of the standard decoction of the angelica sinensis blood enriching decoction, namely ferulic acid, calycosin glucoside and astragaloside IV are established, and a detection method of 2 characteristic patterns used by HPLC-DAD and HPLC-DAD-ELSD is further realized, so that the comprehensive quality reflection and control of the standard decoction are further realized, solid inositol is laid for later manufacturing of a blood enriching decoction preparation with better curative effect and more obvious curative effect, and a certain reference is provided for the development of other classical prescriptions.

The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent variation, etc. of the above embodiment according to the technical matter of the present invention fall within the scope of the present invention.

Claims

1. A quality control method of standard decoction of radix angelicae sinensis blood replenishing decoction is characterized by comprising the following steps: the method comprises the steps of constructing a characteristic spectrum of the wine-washed angelica in the standard decoction of the Chinese angelica decoction for enriching blood, and comprises the following steps:

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

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

The gradient elution satisfies 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%;

2. The quality control method according to claim 1, characterized in that: the method also comprises the construction of the characteristic spectrum of astragalus decoction pieces in standard decoction of the angelica sinensis blood replenishing decoction, and comprises the following steps:

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

reference solution: taking formononetin, calycosin glucoside, astragaloside I, astragaloside II, iso-astragaloside I and ferulic acid respectively, adding methanol to prepare solutions with 50-100 mug of each 1ml,

test solution II: adding water 20ml into 1g of lyophilized powder of standard decoction of radix Angelicae sinensis decoction for replenishing blood, performing ultrasonic treatment for 30min, shaking, and filtering to obtain subsequent filtrate;

B. respectively taking 10 μl of reference solution B and sample solution II, injecting into high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A,0.02% formic acid solution as mobile phase B, gradient eluting, detecting with ultraviolet detector and evaporative light scattering at 25deg.C and flow rate of 1.0ml per minute, detecting wavelength of 254nm with ultraviolet detector and drift tube temperature of 90 deg.C and carrier gas flow rate of 1.8ml per minute to obtain corresponding characteristic map,

The gradient elution satisfies 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%;

3. The quality control method according to claim 1, characterized in that: the method also comprises the steps of measuring the ferulic acid content in the standard decoction of the Chinese angelica and blood replenishing decoction by adopting a high performance liquid chromatography method, and comprises the following steps of:

ferulic acid control solution: taking a proper amount of ferulic acid reference substance, adding 70% methanol to prepare a solution containing 12 μg ferulic acid per 1mL,

4. The quality control method according to claim 1, characterized in that: the method also comprises the steps of measuring the content of calycosin glucoside in the standard decoction of the Chinese angelica and blood replenishing decoction by adopting a high performance liquid chromatography method, and comprises the following steps of:

calycosin glucoside control solution: taking a proper amount of calycosin glucoside reference substance, adding methanol to prepare a solution containing 50 μg calycosin glucoside per 1mL,

B. respectively taking 10 mu l of calycosin glucoside reference substance solution and 10 mu l of test substance solution V, injecting into a high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A,0.2% formic acid solution as mobile phase B, performing gradient elution, measuring at an inspection wavelength of 260nm to obtain the content of calycosin glucoside in the standard decoction of radix Angelicae sinensis blood replenishing decoction not less than 0.02%,

Gradient elution satisfies 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%.

5. The quality control method according to claim 1, characterized in that: the method also comprises the steps of measuring the content of astragaloside IV in the standard decoction of the Chinese angelica and blood replenishing decoction by adopting a high performance liquid chromatography method, and comprises the following steps of:

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

6. The quality control method according to any one of claims 1 to 5, characterized in that: the preparation process of the standard decoction of the angelica sinensis blood replenishing decoction comprises the following steps: mixing 1 part of wine-washed angelica and 5 parts of astragalus decoction pieces according to parts by weight, adding 600ml of water, soaking for 30min, covering with strong fire, boiling, changing into slow fire, decocting for 40min, and filtering while the mixture is hot.