CN114515301B

CN114515301B - Semen astragali Complanati standard decoction, and preparation method and detection method thereof

Info

Publication number: CN114515301B
Application number: CN202011301371.5A
Authority: CN
Inventors: 谢隼; 娄涛涛; 安悦; 李慧馨; 孙宜春; 卿勇军
Original assignee: Guoyaojituan Tongjitang Guizhou Pharmaceutical Co ltd; Guangdong Yifang Pharmaceutical Co Ltd; Jiangyin Tianjiang Pharmaceutical Co Ltd
Current assignee: Guoyaojituan Tongjitang Guizhou Pharmaceutical Co ltd; Guangdong Yifang Pharmaceutical Co Ltd; Jiangyin Tianjiang Pharmaceutical Co Ltd
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2024-03-29
Anticipated expiration: 2040-11-19
Also published as: CN114515301A

Abstract

The invention relates to a semen astragali complanati standard decoction, a preparation method and a detection method. The invention provides a semen astragali complanati standard decoction, which contains: myricetin-3-O-beta-D-glucopyranoside, kaempferol-3-O- [ beta-D-xylopyranose- (1- > 2) ] -beta-D-glucopyranoside, kaempferol-3-O-beta-D-glucopyranoside and flastem milkvetch seed glycoside. The semen astragali complanati standard decoction provided by the invention can be used as a quality standard, and provides a reference for controlling the quality of semen astragali complanati decoction and formula granules.

Description

Semen astragali Complanati standard decoction, and preparation method and detection method thereof

Technical Field

The invention relates to the field of modernization of traditional Chinese medicines, in particular to a semen astragali complanati standard decoction, a preparation method and a detection method thereof.

Background

The semen astragali Complanati standard decoction is water extract of seed of Astragalus membranaceus (Astragalus com-planatus R.Br.) of Leguminosae. Semen astragali Complanati is dry mature seed of Astragalus membranaceus (Astvagalus complanatus R.Br.) bge. Of Leguminosae, which is a traditional Chinese medicine in China, and is mainly produced in regions of Shaanxi, shanxi, hebei, inner Mongolia, etc., and has been used as medicine in the traditional Chinese medicine in the names of semen astragali Complanati, sha Yuanbai fructus Tribuli, fructus Atriplicis Sibiricae, semen astragali Complanati, xia Huangcao, etc. Semen astragali Complanati mainly contains flavonoids, triterpenes, amino acids, microelements, etc., and has effects of resisting oxidation, resisting tumor, protecting liver, regulating blood lipid, inhibiting platelet aggregation, lowering blood pressure, resisting fibrosis, improving blood rheology index, etc.

The standard decoction is also called as standard decoction, is a traditional medicine form widely used clinically, and is prepared by standardized decoction according to a clinical decoction method, solid-liquid separation, proper concentration preparation or drying by a proper method according to the theory of traditional Chinese medicine, and is used as a standard reference for measuring whether the traditional Chinese medicine formula particles are basically consistent with the clinical decoction.

The standard decoction is a bridge connecting traditional Chinese medicine decoction pieces and modern Chinese medicine preparations, provides a reference for controlling the quality of Chinese medicine end products, provides a reference for marking different administration forms of Chinese medicines, ensuring uniformity of quality and consistency of curative effect, and provides a reference for evaluating consistency of quality of products of different manufacturers, so that the establishment of the quality standard of the Chinese medicine standard decoction provides a basis for the establishment of the quality standard of all end products of decoction pieces derived from decoction pieces.

Disclosure of Invention

Therefore, the technical problem solved by the invention is to provide the semen astragali complanati standard decoction.

On the other hand, the invention also provides a detection method of the semen astragali complanati standard decoction component so as to realize the standardization of quality control and standard research of semen astragali complanati formula particles.

The invention provides a semen astragali complanati standard decoction, which contains:

myricetin-3-O-beta-D-glucopyranoside,

kaempferol-3-O- [ beta-D-xylopyranose- (1- > 2) ] -beta-D-glucopyranoside,

kaempferol-3-O-beta-D-glucoside and its preparation

Semen astragali Complanati glycoside.

Preferably, the semen astragali complanati standard decoction further comprises:

Myricetin-3-O- [ beta-D-xylopyranose- (1- & gt 2) ] -beta-D-glucopyranoside,

Semen astragali Complanati myricetin,

rhamnocitrin-3-O- [ beta-D-apiose- (1- & gt 2) ] -beta-D-glucoside,

rhamnocitrin-3-O- [ beta-D-apiose- (1 '. Fwdarw.2 ') -6 ' -acetyl ] -beta-D-glucoside, acetyl-complanatoside, kaempferol-3-O-beta-D-arabinoside, and,

rhamnocitrin-3-O- [5 ' -O-feruloyl-beta-D-apiose (1 ' -2 ') ] -beta-D-glucopyranoside,

Or, one or more than two of rhamnocitrin-3-O- [5 ' -O-p-coumaroyl-beta-D-apiose- (1 '. Fwdarw.2 ') ] -beta-D-glucopyranoside.

The invention provides a preparation method of a semen astragali Complanati standard decoction, which comprises the following steps:

(1) Decocting semen astragali Complanati with water, and filtering to obtain filtrate;

(2) Concentrating and drying the filtrate in the step (1), and then freeze-drying, wherein the freeze-drying is divided into three stages: a. pre-freezing: the pre-freezing temperature is-50 ℃ to-45 ℃; b. primary drying: the drying temperature is between 20 ℃ below zero and 0 ℃; c. and (3) secondary drying: the drying temperature is 5-25 ℃, and the semen astragali complanati standard decoction is obtained.

The invention also provides a method for detecting the semen astragali complanati glycoside, which comprises the following steps:

(1) Preparation of a control solution:

adding ethanol into semen astragali Complanati glycoside reference substance to obtain solution;

(2) Preparation of test solution:

extracting semen astragali Complanati standard decoction with solvent;

(3) Ultra high performance liquid chromatography

Sucking the reference substance solution and the sample solution, injecting into an ultra-high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A, water phase as mobile phase B, and detecting at a ratio of A-B of 22:78 to obtain the content of semen astragali Complanati glycoside.

Preferably, in the step (2), the solvent is selected from one of water, 60% ethanol, 75% ethanol, absolute ethanol, 60% methanol, 75% methanol, and methanol, preferably, the solvent is methanol;

preferably, in step (2), the extraction is one of ultrasonic extraction or dissolution extraction,

preferably, the extraction is carried out using ultrasound extraction,

preferably, the extraction time is 10-45min,

preferably, the extraction time is 30min;

preferably, in step (3), the flow rates of mobile phase A and mobile phase B are in the range of 0.9-1.1ml/min,

preferably, the flow rate of mobile phase A and mobile phase B is 1.0ml/min,

Preferably, in step (3), the aqueous phase is selected from water, 0.1% aqueous phosphoric acid or 0.05% aqueous phosphoric acid, preferably 0.1% aqueous phosphoric acid.

The invention also provides a detection method of the characteristic spectrum of the semen astragali complanati standard decoction, which comprises the following steps:

(1) Preparation of reference solution:

respectively weighing semen astragali Complanati glycoside reference substances, and adding ethanol to obtain solutions;

(2) Preparation of test solution:

extracting semen astragali Complanati standard decoction with solvent;

(3) Ultra high performance liquid chromatography

Sucking the reference substance solution and the sample solution, injecting into an ultra-high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as filler, acetonitrile as mobile phase A, and water phase as mobile phase B, and performing gradient elution to obtain the characteristic map of semen astragali Complanati standard decoction.

Preferably, in step (2), the solvent is selected from one of water, 95% ethanol, 75% ethanol, diluted ethanol, methanol, 75% methanol, 50% methanol, preferably, the solvent is 75% methanol,

preferably, in step (2), the extraction is one of reflux extraction, ultrasonic extraction or dissolution extraction,

preferably, the extraction is carried out using ultrasound extraction,

Preferably, the extraction time is 10-45min,

preferably, the extraction time is 15min.

Preferably, in step (3), the flow rates of mobile phase A and mobile phase B are in the range of 0.1-0.5ml/min,

preferably, the flow rate of the mobile phase A and the mobile phase B is 03ml/min,

preferably, the gradient elution procedure is:

0-9min, the volume percentage of the mobile phase A is 25-27.5%, and the volume percentage of the mobile phase B is 75-72.5%;

9-17min, the volume percentage of the mobile phase A is 27.5-28%, and the volume percentage of the mobile phase B is 72.5-72%;

17-18min, 28-29% of mobile phase A and 72-71% of mobile phase B;

18-35min, the volume percentage of the mobile phase A is 29-38%, and the volume percentage of the mobile phase B is 71-62%;

35-55min, the volume percentage of the mobile phase A is 38-53%, and the volume percentage of the mobile phase B is 62-47%;

55-55.1min, the volume percentage of the mobile phase A is 53-25%, and the volume percentage of the mobile phase B is 47-75%;

55.1-58min, the volume percentage of the mobile phase A is 25-25%, and the volume percentage of the mobile phase B is 75-75%.

Preferably, the column temperature in step (3) is 32-38 ℃, preferably the column temperature is 35 ℃.

Preferably, the characteristic spectrum has no less than 10 characteristic peaks, the complanatoside corresponding to the reference substance is an S peak, and the relative retention time of each characteristic peak and the S peak is calculated, wherein the relative retention time is within + -10% of a specified value, and the specified value contains 0.212, 0.243, 0.312, 0.570, 0.628, 0.777, 0.964, 1.103 and 1.128.

In one embodiment of the present invention, the characteristic spectrum has no less than 10 characteristic peaks, the peak 8 (complanatoside) corresponding to the reference is the S peak, and the relative retention time of each characteristic peak and the S peak is calculated to be within ±10% of a predetermined value, and the predetermined value contains 0.212 (peak 1), 0.243 (peak 2), 0.312 (peak 3), 0.570 (peak 4), 0.628 (peak 5), 0.777 (peak 6), 0.964 (peak 7), 1.103 (peak 9), and 1.128 (peak 10).

Preferably, the characteristic peak is not less than 12, more preferably, the characteristic peak is not less than 15.

The beneficial effects obtained by the invention are as follows:

the characteristic spectrum method established by the invention adopts ultra-high phase liquid chromatography, has the characteristics of simplicity, convenience, stability, high precision, good reproducibility and the like, and the obtained standard decoction fingerprint has multiple peaks, good peak pattern, easy identification, accuracy and reliability.

Drawings

FIG. 1 shows the effect of different extraction solvents on the content of astragali semen glycoside;

FIG. 2 shows a survey HPLC profile of different extraction modes;

FIG. 3 shows a survey HPLC profile for different extraction times;

FIG. 4 shows a specific investigation of the content measurement of the standard decoction of semen astragali Complanati;

FIG. 5 shows a standard graph of astragali semen astragali Complanati glycoside;

FIG. 6 shows a HPLC chart for different column durability studies;

FIG. 7A is a diagram showing a durability inspection HPLC at a column temperature of 25 ℃;

FIG. 7B is a diagram showing a durability inspection HPLC at a column temperature of 30 ℃;

FIG. 7C is a HPLC chart showing a durability test with a column temperature of 35 ℃;

FIG. 8A is a HPLC chart showing a durability study with a flow rate of 0.9 ml/min;

FIG. 8B is a chart showing a durability review HPLC at a flow rate of 1.0 ml/min;

FIG. 8C is a HPLC chart showing a durability study with a flow rate of 1.1 ml/min;

FIG. 9A shows a UPLC spectrum of methanol-0.1% phosphoric acid water as mobile phase system;

FIG. 9B shows a UPLC spectrum of acetonitrile-0.1% phosphoric acid water as mobile phase system;

FIG. 10A shows the effect of methanol-water on peak profile for mobile phase;

FIG. 10B shows the effect of methanol-0.1% phosphoric acid in the mobile phase on peak profile;

FIG. 10C shows the effect of acetonitrile-0.1% formic acid water as mobile phase on peak profile;

FIG. 10D shows the effect of acetonitrile-0.1% acetic acid water as mobile phase on peak profile;

FIG. 11A shows a UPLC spectrum of water as the extraction solvent investigation;

FIG. 11B shows a UPLC spectrum of 95% ethanol as extraction solvent;

FIG. 11C shows a UPLC spectrum of 75% ethanol as extraction solvent investigation;

FIG. 11D shows a UPLC spectrum of a dilute ethanol as an extraction solvent investigation;

FIG. 11E shows a UPLC spectrum of methanol as an extraction solvent investigation;

FIG. 11F shows a UPLC spectrum of 75% methanol as extraction solvent;

FIG. 11G shows a UPLC spectrum for 50% methanol as extraction solvent investigation;

FIG. 12 shows the extraction efficiency versus peak area/sample size for different extraction solvents;

FIG. 13A shows a UPLC spectrum examined by ultrasonic extraction;

FIG. 13B shows a UPLC spectrum examined by a reflux extraction method;

FIG. 14 shows the extraction efficiency versus (peak area/sample size) for different extraction modes;

FIG. 15A shows a UPLC spectrum examined by ultrasonic extraction for 15 min;

FIG. 15B shows a UPLC spectrum examined by ultrasonic extraction for 30 min;

FIG. 15C shows a UPLC spectrum examined by ultrasonic extraction for 45 min;

FIG. 16 is a graph showing the extraction efficiency versus time (peak area/sample size);

fig. 17 shows a specific investigation of a characteristic spectrum of a standard decoction of semen astragali complanati;

FIG. 18 shows the overall investigation of the characteristic spectrum of the standard decoction of semen astragali Complanati;

FIG. 19 shows an intermediate precision comparison (Sieimer's fly);

FIG. 20A shows a UPLC spectrum for an Agilent C18 chromatographic column;

FIG. 20B shows a Dikma C18 column looking at the UPLC spectrum;

FIG. 20C shows ACQUITY UPLCC18 chromatographic column to examine UPLC spectrum;

FIG. 21A shows a UPLC spectrum for a column temperature of 25 ℃;

FIG. 21B shows a UPLC spectrum for a column temperature of 30deg.C;

FIG. 21C shows a UPLC spectrum for a column temperature of 35 ℃;

FIG. 22A shows a UPLC spectrum for a flow rate of 0.25 ml/min;

FIG. 22B shows a UPLC spectrum for a flow rate of 0.30 ml/min;

FIG. 22C shows a UPLC spectrum for a flow rate of 0.35 ml/min;

FIG. 23 is a characteristic map of 17 batches of semen astragali Complanati standard decoction;

FIG. 24 is a diagram showing LC-MS mass spectrum of semen astragali Complanati standard decoction;

FIG. 24A is a diagram showing a negative ion mode BPI mass spectrum;

FIG. 24B shows a 266nm ultraviolet chromatogram;

FIG. 25 shows a DAD spectrum of peak 1;

FIG. 26 shows an MS/MS mass spectrum of peak 1;

FIG. 27 shows a DAD spectrum of peak 2;

FIG. 28 shows the MS/MS mass spectrum of peak 2;

FIG. 29 shows a DAD spectrum of peak 3;

FIG. 30 shows the MS/MS mass spectrum of peak 3;

FIG. 31 shows the DAD spectrum of peak 4;

FIG. 32 shows the MS/MS mass spectrum of peak 4;

FIG. 33 shows the DAD spectrum of peak 5;

FIG. 34 shows the MS/MS mass spectrum of peak 5;

FIG. 35 shows the DAD spectrum of peak 6;

FIG. 36 shows the MS/MS mass spectrum of peak 6;

FIG. 37 shows the DAD spectrum of peak 7;

FIG. 38 is a MS/MS mass spectrum of peak 7;

FIG. 39 shows the DAD spectrum of peak 9;

FIG. 40 shows the MS/MS mass spectrum of peak 9;

FIG. 41 shows the DAD spectrum of peak 10;

FIG. 42 shows the MS/MS mass spectrum of peak 10;

FIG. 43 shows the DAD spectrum of peak 11;

FIG. 44 shows an MS/MS mass spectrum of peak 11;

FIG. 45 shows the DAD spectrum of peak 12;

FIG. 46 is a MS/MS mass spectrum of peak 12;

FIG. 47 is a further validated UPLC plot of Peak 2, peak 3, peak 5, peak 8;

FIG. 48 shows an LC-MS mass spectrum of a standard decoction of semen astragali Complanati;

fig. 49 shows a reference map of the standard decoction of semen astragali Complanati.

Detailed Description

The term "diluted ethanol" is prepared by adopting an annex XVB test solution in the first part of Chinese pharmacopoeia, namely 529ml of ethanol is taken, and water is added for dilution to 1000ml, thus obtaining the diluted ethanol. The liquid contains C at 20deg.C ₂ H ₅ OH should be 49.5% -50.5% (ml/ml).

The manufacturers of the raw materials used in this example are described below, wherein the chemicals are not identified as chemically pure grades of conventional reagents. The information on the raw materials used in the examples and the experimental equipment are shown in tables 1 and 2, respectively.

Table 1 information on the raw materials used in the examples

Raw materials	Purity/lot number	Manufacturer' s
			Semen astragali complanati decoction pieces	201801	Fengchengxiang town in Weinan city of Shaanxi province
Semen astragali Complanati glycoside reference substance	111803-201403	China Institute for food and drug control

Table 2 Experimental apparatus used in the examples

Example 1

1. Preparation method of semen astragali Complanati standard decoction

Decocting semen astragali Complanati decoction pieces 100g in ceramic pot with water twice, adding 8 times of water for the first time, soaking for 30 min, boiling with strong fire (500W), keeping boiling with slow fire (200W) for 60 min, filtering the decoction with 200 mesh sieve, and cooling the filtrate with cold water. Adding 6 times of water for the second time, boiling with strong fire, keeping boiling with slow fire for 50 min, filtering the decoction with 200 mesh sieve, cooling the filtrate with cold water, and mixing the decoctions. Transferring the decoction into a 2000ml round bottom flask, concentrating under reduced pressure and low temperature (temperature: 65deg.C; vacuum degree: -0.080-0.090 MPa) to 200ml extract by rotary evaporator; under magnetic stirring, packaging into 10ml brown penicillin bottles, wherein each bottle has a packaging volume of 1.0ml, half-plugging, transferring into a vacuum freeze dryer for freeze-drying after packaging, performing primary drying after pre-freezing for 240min at-45 ℃ and maintaining for 420min at-30 ℃ and maintaining for 120min at-10 ℃ and 180min at 0 ℃, then performing analytical drying after maintaining for 120min at 5 ℃ and maintaining for 120min at 15 ℃ and 180min at 25 ℃, taking out, and rolling an aluminum cover to obtain the semen astragali Complanati standard decoction.

The calculation formula of the paste yield of the standard decoction of the semen astragali complanati is as follows:

calculated, the paste yield of the standard decoction of the semen astragali complanati is 13.8%.

2. Establishment of method for measuring and analyzing content of semen astragali complanati glycoside in semen astragali complanati standard decoction

(1) Preparation of control solution

Precisely weighing semen astragali Complanati glycoside reference substance 15mg, placing into 100ml volumetric flask, and adding 60% ethanol to obtain mother liquor containing 0.15mg per 1 ml. And precisely transferring 1mL of mother solution, placing into a 10mL volumetric flask, and adding 60% ethanol to a constant volume to prepare a control solution containing 15 mug of flastem milkvetch seed glycoside per 1 mL.

(2) Preparation of test solutions

Grinding appropriate amount of semen astragali Complanati standard decoction prepared in step 1, precisely weighing 0.15g, placing into conical flask with plug, precisely adding 25ml of solvent, sealing, weighing, extracting for a period of time, cooling, weighing again, supplementing with organic solvent for weight loss, shaking, filtering, and collecting filtrate.

Octadecylsilane chemically bonded silica is used as filler, water-acetonitrile is used as mobile phase, 10 μl of each of the reference solution and the sample solution is precisely sucked, and the mixture is injected into a liquid chromatograph for measurement. The detection wavelength is 266nm, and the theoretical plate number is not lower than 4000 calculated according to the peak of semen astragali Complanati glycoside.

2.1 examining experimental conditions in the detection method:

A. investigation of the extraction solvent

Taking a proper amount of standard decoction of semen astragali complanati, grinding, taking about 0.15g, precisely weighing, parallel 7 groups, 2 parts of each group, placing into a conical flask with a plug, precisely adding 25ml of water, 60% ethanol, 75% ethanol, absolute ethanol, 60% methanol, 75% methanol and methanol respectively, weighing, performing ultrasonic treatment (power is 250W and frequency is 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with corresponding solvent, shaking uniformly, filtering, and taking the subsequent filtrate. Precisely sucking 10 μl, injecting into liquid chromatograph, and measuring. The experimental results are shown in Table 3 and FIG. 1.

TABLE 3 determination of the content of Astragaloside in different extraction solvents of semen astragali Complanati standard decoction

As can be seen from Table 3 and FIG. 1, the extraction rate of the flastem milkvetch seed glycoside in the standard decoction of flastem milkvetch seed by methanol is higher by comparing the content of the flastem milkvetch seed glycoside in the 7 extraction solvent samples, so that the methanol is selected as the extraction solvent for measuring the content of the flastem milkvetch seed glycoside in the standard decoction of flastem milkvetch seed.

B. Investigation of the extraction method

Taking a proper amount of standard decoction of semen astragali complanati, grinding, taking about 0.15g, precisely weighing, paralleling 2 groups, placing 2 parts of each group into a conical bottle with a plug, precisely adding 25ml of methanol, weighing, directly dissolving respectively, heating, ultrasonically refluxing for 30 minutes, taking out, cooling, weighing again, supplementing the weight loss with methanol, shaking uniformly, filtering, and taking the subsequent filtrate. Precisely sucking 10 μl, injecting into liquid chromatograph, and measuring. The experimental results are shown in table 4 and fig. 2.

TABLE 4 determination of the content of Astragaloside in different extraction methods of semen astragali Complanati standard decoction

As can be seen from table 4 and fig. 2, the content of the methanol ultrasonic extraction under the investigation of the extraction solvent is known, the content of the ultrasonic reflux extraction is higher, and the ultrasonic operation is convenient to consider, so that the ultrasonic reflux is selected as the extraction mode.

C. Investigation of extraction time

Taking a proper amount of standard decoction of semen astragali Complanati, grinding, taking about 0.15g, precisely weighing, parallel 4 groups, 2 parts each, placing into a conical flask with a plug, precisely adding 25ml of methanol, weighing, respectively performing ultrasonic treatment (power 250W, frequency 40 kHz) for 15min, 30min, 45min and 60min, taking out, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the filtrate. Precisely sucking 10 μl, injecting into liquid chromatograph, and measuring. The experimental results are shown in Table 5 and FIG. 3.

TABLE 5 determination of the content of Astragaloside in different extraction times of semen astragali Complanati standard decoction

As can be seen from Table 5 and FIG. 3, by comparing the contents of the Asteraside at different ultrasonic times, it was found that the ultrasonic time had less influence on the content of the Asteraside, and the ultrasonic time was selected to be 30 minutes in consideration of convenience of operation.

Determination of the method for preparing a sample solution

According to the sample pretreatment investigation experimental result, the preparation method of the test sample can be determined as follows:

taking a proper amount of standard decoction of semen astragali Complanati, grinding, taking about 0.15g, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of methanol, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and collecting the subsequent filtrate.

2.2 examination of methodology of detection methods

a. Investigation of specificity

Precisely sucking 10 μl of semen astragali Complanati standard decoction sample solution, semen astragali Complanati glycoside reference substance solution and blank solvent, and injecting into liquid chromatograph, and measuring under the above chromatographic conditions. The results are shown in FIG. 4 and Table 6.

TABLE 6 chromatographic peak system adaptation parameters

As can be seen from fig. 4 and table 6, there is no chromatographic peak in the blank solvent chromatography at the retention time corresponding to the complanatoside, which indicates that the solvent has no interference to the determination of complanatoside, and the content of complanatoside in the standard decoction of complanatoside determined by the method has specificity.

b. Linear investigation

1, 4, 8, 12, 16 and 20 mu l of the flatstem milkvetch seed glycoside reference substance solution (14.816 mu g/ml) are respectively sucked precisely, injected into a liquid chromatograph, measured according to the chromatographic conditions, and a standard curve is drawn by taking a peak area integral value as an ordinate and the flatstem milkvetch seed glycoside injection amount (mu g) as an abscissa, and the results are shown in the following table 7 and fig. 5.

TABLE 7 Linear investigation of the results of Asteraside

As shown in fig. 5 and table 7, the obtained linear regression equation is: y= 33314x-5518.7, correlation coefficient R ² =1, indicating that the concentration of complanatoside is linearly related to peak area in the range of 0.014816 μg to 0.29632 μg.

c. Precision investigation

Precisely sucking the sample solution of semen astragali Complanati standard decoction, repeating sample injection for 6 times, and sampling volume of 10 μl. The peak area RSD was calculated and the measurement results are shown in table 8.

Table 8 results of precision investigation

As can be seen from table 8, the content of the flastem milkvetch seed glycoside in the 6 parallel test sample solutions is stable, RSD% = 0.788%, and the repeatability is good, which indicates that the instrument precision is good.

d. Stability investigation

Sample solutions of the standard decoction of the semen astragali Complanati are precisely sucked, sample injection is carried out at 0,2,4,6,8, 10 and 12 hours respectively according to the chromatographic conditions, the sample injection volume is 10 μl, the peak area of the semen astragali Complanati glycoside in the sample solutions is measured, the peak area RSD is calculated, and the measurement results are shown in Table 9.

Table 9 stability investigation results

As shown in the experimental results of Table 9, the peak area RSD of the test sample within 12 hours was 2.65%. RSD was 1.88% within 6 hours, indicating that the test sample was stable for less than 6 hours.

e. Intermediate precision investigation

The other analysts of the project group operate under different dates and different chromatographs, about 0.15g of the same batch of semen astragali complanati standard decoction is taken, precisely weighed, 6 parts are parallel, test sample solutions are prepared, and the results are shown in table 10 according to the chromatographic conditions.

Table 10 results of intermediate precision investigation

The experimental results in table 10 show that different analysts operate on different dates and with different chromatographs, the content result RSD of the semen astragali complanati glycoside is less than 2%, and the intermediate precision of the analytical method is good.

f. Durability inspection

(1) Investigation of different chromatographic columns

The effect of three chromatographic columns of Inertsil ODS-HL, YMC-Triar C18 and Ultimate XB-C18 on the content of the semen astragali Complanati glycoside in the semen astragali Complanati standard decoction was compared.

The sample solution of semen astragali Complanati standard decoction is measured according to the above chromatographic conditions, and the experimental results are shown in FIG. 6 and Table 11.

TABLE 11 different column System Adaptation parameters

As can be seen from FIGS. 6 and 11, the separation degree was 1.38 when the method was used with the column Inertsil ODS-HL, and the symmetry factor was 1.34 when the method was used with the column Ultimate XB-C18, and the adaptability test of the two column systems was poor. The measured RSD value of the content is larger. Experimental results show that the method has certain selectivity to the chromatographic column.

(2) Investigation by different chromatographs

According to the existing equipment in the laboratory, a Waters chromatograph and an Agilent chromatograph are selected, and the influence of the two chromatographs on the determination of the content of the semen astragali complanati glycoside in the semen astragali complanati standard decoction is compared.

The sample solution of semen astragali Complanati standard decoction was measured according to the above chromatographic conditions, and the experimental results are shown in Table 12.

Table 12 different chromatograph system adaptation parameters

The experimental results in table 12 show that the analytical method has better durability for different chromatographs. The chromatograph changes can meet the system adaptability requirements.

(3) Investigation of different column temperatures

Comparing the effect of different Wen Shayuan sub-standard decoction of semen astragali Complanati glycoside at 25deg.C, 30deg.C and 35deg.C.

The standard decoction of semen astragali Complanati, the test solution, were measured according to the above chromatographic conditions, and the experimental results are shown in FIGS. 7A-7C and Table 13.

Table 13 different column temperature system adaptation parameters

As shown in FIGS. 7A to 7C and Table 13, the degree of separation at 25℃was 1.35, less than 1.5, and was not satisfactory. The target peak and incomplete separation at 35℃and the system adaptability tests at 25℃and 35℃were poor, so the content RSD was large, indicating that the method was selective for column temperature, which was 30 ℃.

(4) Investigation of different flow rates

The effect of different flow rates at 0.9ml/min, 1.0ml/min and 1.1ml/min on the content of the semen astragali Complanati glycoside in the semen astragali Complanati standard decoction was compared.

Taking semen astragali Complanati standard decoction sample solution, and performing experiments according to the above chromatographic conditions to obtain experimental results shown in figures 8A-8C and Table 14.

Table 14 different flow rate system adaptation parameters

As shown in the experimental results of FIGS. 8A to 8C and Table 14, the RSD of the content measurement of the astragali complanatoside was large at different flow rates, which indicates that the method was selective to the flow rate, and the method prescribes the flow rate to be 1.0ml/min.

3. Characteristic spectrum analysis of semen astragali Complanati standard decoction

3.1 optimization of the conditions of the detection method

(1) Preparation of reference solutions

(2) Preparation of test solutions

Taking a proper amount of standard decoction of semen astragali Complanati, grinding, taking about 0.15g, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of methanol, weighing, performing ultrasonic treatment (power 250W, frequency 40 kHz) for 30 minutes, cooling, weighing again, supplementing the weight loss with methanol, shaking, filtering, and taking filtrate.

Octadecylsilane chemically bonded silica is used as a filler; eluting with acetonitrile-0.1% phosphoric acid water (22:78) as mobile phase; the detection wavelength was 266nm. The theoretical plate number should be not less than 4000 as calculated by the peak of semen astragali Complanati glycoside.

The following examined experimental conditions in the detection method:

i. selection of mobile phase and gradient

(1) Large gradient investigation a is the organic phase and B is the aqueous phase. The gradients are shown in table 15.

TABLE 15

A is an organic phase and B is an aqueous phase. The effect of phase A being methanol or acetonitrile, phase B being water, 0.1% aqueous phosphoric acid, 0.1% aqueous formic acid or 0.1% aqueous acetic acid on the peaks was examined.

As can be seen from fig. 9A and 9B, the eluting ability of acetonitrile-0.1% phosphoric acid water is stronger than that of methanol-0.1% phosphoric acid water, but in the later stage condition investigation, the separation effect of some chromatographic peaks is not ideal due to the adoption of acetonitrile-0.1% phosphoric acid water, the separation effect of methanol-0.1% phosphoric acid water is improved, and the use of methanol is more economical and cost-effective than that of acetonitrile, so that the condition investigation is carried out by selecting methanol-0.1% phosphoric acid water, and various acids used in the mobile phase are further investigated in the later stage.

(2) The gradient was adjusted and the acid addition and no acid addition were examined. A is an organic phase and B is an acid-containing aqueous phase. The gradients are shown in table 16. The experimental results are shown in FIGS. 10A-10D.

Table 16

/>

As can be seen from FIGS. 10A to 10D, the acid addition was better than the acid addition, but the peak pattern was the best when the mobile phase was acetonitrile-0.1% phosphoric acid aqueous system, so acetonitrile-0.1% phosphoric acid aqueous system was selected for elution.

Therefore, the following chromatographic conditions were obtained:

octadecylsilane chemically bonded silica is used as filler (column length is 150mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile as mobile phase a and 0.1% phosphoric acid aqueous solution as mobile phase B, and gradient elution was performed as specified in table 17 below; the column temperature is 35 ℃; the flow rate is 0.3ml/min; the detection wavelength was 266nm. The theoretical plate number should be not less than 4000 calculated on the basis of the semen astragali Complanati glycoside.

TABLE 17 gradient elution procedure

Examination of pretreatment method of sample solution

The extraction solvent, extraction time and extraction mode of the semen astragali Complanati standard decoction are examined, and the sample pretreatment method of the semen astragali Complanati standard decoction characteristic spectrum is determined.

a. Investigation of the extraction solvent

The experiment respectively examines the influence of different extraction solvents on the characteristic spectrum of the standard decoction of the semen astragali complanati, selects water, ethanol, 75% ethanol, diluted ethanol, methanol, 75% methanol and 50% methanol as the extraction solvents, and compares the characteristic spectrum results of the different extraction solvents through the total peak area/sample weighing amount of 15 chromatographic peaks which are temporarily identified and the chromatogram.

Taking a proper amount of standard decoction of semen astragali Complanati, grinding, taking about 0.14g, precisely weighing, placing into conical flask with plug, precisely adding 25ml of water, ethanol, 75% ethanol, diluted ethanol, methanol, 75% methanol and 50% methanol respectively, weighing, performing ultrasonic treatment (power 500W, frequency 40 kHz) for 30min, cooling, supplementing the weight of loss with corresponding solvent, shaking, filtering, and collecting filtrate. 20. Mu.l of each sample solution was precisely aspirated, and the sample solution was injected into a liquid chromatograph, and the peak area was recorded by measuring the sample solution under the above-mentioned chromatographic conditions. The results are shown in FIGS. 11A-11G and 12.

As can be seen from fig. 11A to 11G and fig. 12, the chromatographic peak information is more and the extraction efficiency is higher when the extraction solvent is 75% methanol.

b. Investigation of the extraction method

The experiment respectively examines the influence of different extraction modes on the characteristic spectrum of the standard decoction of the semen astragali complanati, selects ultrasonic and reflux as the extraction modes, and compares the characteristic spectrum results of different extraction modes through the total peak area/sample weighing amount of 15 chromatographic peaks which are temporarily identified and the chromatogram.

Taking a proper amount of standard decoction of semen astragali Complanati, grinding, taking about 0.14g, precisely weighing, placing into conical flask with plug, precisely adding 25ml of 75% methanol, respectively, weighing, respectively treating with ultrasound and reflux for 30min, cooling, supplementing the lost weight with 75% methanol, shaking, filtering, and collecting filtrate. 20. Mu.l of each sample solution was precisely aspirated, and the sample solution was injected into a liquid chromatograph, and the peak area was recorded by measuring the sample solution under the above-mentioned chromatographic conditions. The results are shown in FIGS. 13A, 13B and 14.

The experimental results of fig. 13A, 13B and 14 show that the ultrasonic extraction is not quite different from the reflux extraction, and the ultrasonic extraction is selected in consideration of the simplicity of the actual operation.

c. Investigation of extraction time

The experiment respectively examines the influence of different extraction times on the characteristic spectrum of the standard decoction of the semen astragali complanati, selects and examines three different extraction times of 15min, 30min and 45min, and compares the characteristic spectrum results of different extraction times through the total peak area/sample weighing amount of 15 chromatographic peaks which are temporarily identified and the chromatogram.

Grinding semen astragali Complanati standard decoction, precisely weighing about 0.14g, placing into conical flask with plug, precisely adding 75% methanol 25ml, weighing, ultrasonic treating (power 500W, frequency 40 kHz) for 15min, 30min, 45min, cooling, respectively adding 75% methanol to compensate for the weight loss, shaking, filtering, and collecting the filtrate. 20. Mu.l of each sample solution was precisely aspirated, and the sample solution was injected into a liquid chromatograph, and the peak area was recorded by measuring the sample solution under the above-mentioned chromatographic conditions. The results are shown in FIGS. 15A-15C and 16.

As can be seen from FIGS. 15A to 15C and FIG. 16, the areas of the chromatographic peaks are not greatly different by using ultrasonic treatment for 15min, 30min and 45 min.

In summary, the pretreatment method of the semen astragali complanati standard decoction feature map sample can be determined as follows:

Dissolving semen astragali Complanati standard decoction in 75% methanol, ultrasonic treating for 15min, cooling, adding 75% methanol to the weight of the decoction, shaking, filtering, and collecting the filtrate for measuring characteristic map.

3.2 examination of methodology of detection methods

a. Investigation of specificity

Precisely sucking 1 μl of each of the sample solution, reference solution and blank solvent of semen astragali Complanati standard decoction, and measuring with liquid chromatograph. The results are shown in FIG. 17.

As can be seen from FIG. 17, the analytical method can correctly detect the complanatoside without being interfered by the extraction solvent.

b. Integrity inspection

Precisely sucking semen astragali Complanati standard decoction sample solution, injecting into liquid chromatograph, measuring under the above chromatographic conditions, maintaining the same gradient, eluting for one time under the mobile phase proportion of gradient end point, and analyzing characteristic spectrum. The results are shown in FIG. 18.

The results in FIG. 18 show that the chromatographic conditions have no obvious chromatographic peak after being prolonged by one time, which indicates that the chromatographic conditions basically meet the principle of maximum information.

c. Precision investigation

Sample solutions of the standard decoction of semen astragali Complanati are taken in parallel 6 parts, and the sample volume is 1 μl. The relative retention time and relative peak area were calculated using the astragali complanatoside peak as a reference peak, and the experimental results are shown in tables 18 and 19.

Table 18 semen astragali Complanati standard decoction feature map precision results (relative retention time)

Table 19 precision results (relative peak area) of the characteristic spectrum of semen astragali Complanati standard decoction

The experimental results in tables 18 and 19 show that the relative retention time of each chromatographic peak and the RSD of the relative peak area are less than 3.0%, and the precision of the instrument is good.

d. Stability investigation

Sample solutions of the standard decoction of semen astragali Complanati are taken and respectively injected at 0,2,4,8, 12 and 24 hours, and the injection volume is 1 μl. The relative retention time and relative peak area were calculated using the astragali complanatoside peak as a reference peak, and the results are shown in tables 20 and 21.

Table 20 stability results (relative retention time) of the characteristic spectrum of the semen astragali Complanati standard decoction

Table 21 stability results (relative Peak area) of the characteristic spectrum of the standard decoction of semen astragali Complanati

/>

The results of the experiments in tables 20 and 21 show that the relative retention time of the chromatographic peak and the RSD of the relative peak area are <5.0%, indicating that the sample solution is relatively stable.

e. Repeatability investigation

About 0.14g of semen astragali Complanati standard decoction is taken, precisely weighed, 6 parts are parallel to each other, sample solutions to be tested are prepared respectively, and 1 μl of sample solutions are injected respectively. The relative retention time and relative peak area were calculated using the astragali complanatoside peak as a reference peak, and the experimental results are shown in tables 22 and 23.

Table 22 repeated results (relative retention time) of the characteristic spectrum of the standard decoction of semen astragali Complanati

/>

Table 23 repeated results (relative peak area) of the characteristic spectrum of the standard decoction of semen astragali Complanati

/>

The results of the experiments in tables 22 and 23 show that the RSD of the relative retention time and the relative peak area of each chromatographic peak is less than 3.0%, which indicates that the repeatability of the method is good.

f. Intermediate precision investigation

Other analysts in the project group operate under different dates and different chromatographs (Sieimer's flight), about 0.14g of semen astragali Complanati standard decoction is taken, precisely weighed, 6 parts are parallel, sample solutions are respectively prepared, and 1 μl of sample solutions are respectively injected. The relative retention time and relative peak area were calculated using the astragali complanatoside peak as a reference peak, and the experimental results are shown in tables 24 and 25 and fig. 19.

Table 24 intermediate precision results (relative retention time) of the characteristic spectrum of semen astragali Complanati standard decoction

/>

Table 25 intermediate precision results (relative peak area) of the characteristic spectrum of semen astragali Complanati standard decoction

/>

As shown by the experimental results in Table 24, table 25 and FIG. 19, different analysts operate on different dates and with different chromatographs, and the relative retention time RSD is less than 3.0%, which indicates that the method has good intermediate precision.

g. Durability inspection

(1) Investigation of different chromatographic columns

3 chromatographic columns were examined, respectively: column 1-Agilent C18 (2.1 x 150mm,1.8 μm); column 2-Dikma C18 (2.1 x 150mm,1.8 μm); chromatographic column 3-ACQUITY UPLC C18 (2.1 mm. Times.150 mm,1.7 μm), the effect of three chromatographic columns on the durability of the characteristic spectrum of the standard decoction of semen astragali Complanati was examined.

About 0.14g of semen astragali Complanati standard decoction is taken, precisely weighed, prepared into test solution, and 1 μl of each sample is introduced. The experimental results are shown in FIGS. 20A-20C.

The experimental results of fig. 20A-20C show that the chromatographic column 1-Agilent C18 (2.1 x 150mm,1.8 μm) is adopted for elution, and the chromatographic peak-to-peak type is better, so that the method is suggested to adopt the ultra-high performance liquid chromatographic column with the model of Agilent C18 (2.1 x 150mm,1.8 μm).

(2) Investigation of different column temperatures

Comparing different column temperatures, respectively: the durability of the characteristic spectrum of the standard decoction of semen astragali Complanati at 25 ℃, 30 ℃ and 35 ℃.

About 0.14g of semen astragali Complanati standard decoction is taken, precisely weighed, prepared into test solution, and 1 μl of each sample is injected. The experimental results are shown in FIGS. 21A-21C.

The results of the experiments in FIGS. 21A to 21C show that the column temperature has a large influence on the peak condition, and the separation effect is optimal when the column temperature is 35 ℃, so that the measurement is carried out at 35 ℃ by using the column temperature.

(3) Investigation of different flow rates

Comparing the different flow rates, respectively: the durability of the characteristic spectrum of the semen astragali complanati standard decoction is affected by 0.25ml/min, 0.30ml/min and 0.35 ml/min.

About 0.14g of semen astragali Complanati standard decoction is taken, precisely weighed, prepared into test solution, and 1 μl of each sample is introduced. Experimental results fig. 22A-22C.

The experimental results of FIGS. 22A-22C show that the flow rate has a larger influence on the peak-out condition, and the peak type is optimal when the flow rate is 0.3 ml/min. Therefore, it is recommended to use 0.3ml/min as the measurement flow rate.

Example 2

1. Preparation of semen astragali Complanati standard decoction

2. Semen astragali Complanati glycoside content determination and accuracy experiment

(1) Determination of semen astragali Complanati glycoside content

About 0.15g of semen astragali complanati standard decoction is taken, precisely weighed, 6 parts are weighed in parallel, put into a conical flask with a plug, precisely added with 25ml of methanol, weighed, subjected to ultrasonic treatment (power 250W and frequency 40 kHz) for 30 minutes, cooled, weighed again, complemented with methanol to reduce the weight, shaken uniformly, filtered, and the subsequent filtrate is taken, thus obtaining 6 parts of sample solution. YMC-Triart C18 column (4.6X250 mm,5 μm); acetonitrile-0.1% phosphoric acid aqueous solution (22:78) is taken as a mobile phase; the detection wavelength is 266nm, the sample injection amount is 10 mu l, the flow rate is 1.0ml/min, the column temperature is 30 ℃, 10 mu l of sample solution is precisely sucked each time, the content of the flastem milkvetch seed glycoside in the sample solution is measured, the RSD is calculated, and the measurement result is shown in Table 26.

Table 26 results of repeatability investigation

The experimental result shows that the repeatability of the analysis method is good.

(2) Accuracy experiment

Taking 0.075g of sample with known content of the semen astragali complanati glycoside, precisely weighing the content of the semen astragali complanati glycoside to be 0.487wt%, adding 2.380ml of semen astragali complanati glycoside reference substance solution (0.148 mg/ml) respectively, preparing 6 parts of sample solutions according to the preparation method of the sample solutions, respectively injecting 10 μl, measuring peak area of the semen astragali complanati glycoside, calculating the content, and calculating recovery rate and RSD according to the following formula, wherein the result is shown in the table 27 below.

Table 27 accuracy test

The experimental result shows that the recovery rate of the astragali complanatoside is 102.663 percent, and the recovery rate limit is 92-105 percent when the content of the component to be detected in the sample is 0.1-1.0 percent according to the verification and guidance principle of the drug quality standard analysis method of four parts of Chinese pharmacopoeia 2015, which indicates that the recovery rate is good.

Example 3

1. Preparation of semen astragali Complanati standard decoction

Taking 17 parts of semen astragali complanati decoction pieces, each 100g, placing into an electric ceramic pot, adding water for decoction twice, adding 8 times of water for the first time, soaking for 30 minutes, boiling with strong fire (500W), keeping micro-boiling with slow fire (200W) for 60 minutes, filtering the decoction with a 200-mesh screen while the decoction is hot, and rapidly cooling the filtrate with cold water. Adding 6 times of water for the second time, boiling with strong fire, keeping boiling with slow fire for 50 min, filtering the decoction with 200 mesh sieve, cooling the filtrate with cold water, and mixing the decoctions. Transferring the decoction into a 2000ml round bottom flask, concentrating under reduced pressure and low temperature (temperature: 65deg.C; vacuum degree: -0.080-0.090 MPa) to 200ml extract by rotary evaporator; under magnetic stirring, packaging into 10ml brown penicillin bottles, wherein each bottle has a packaging volume of 1.0ml, half-plugging, transferring into a vacuum freeze dryer for freeze-drying after packaging, performing primary drying after pre-freezing for 240min at-45 ℃ and maintaining for 420min at-30 ℃ and maintaining for 120min at-10 ℃ and 180min at 0 ℃, then performing analytical drying after maintaining for 120min at 5 ℃ and maintaining for 120min at 15 ℃ and 180min at 25 ℃, taking out, and rolling an aluminum cover to obtain the semen astragali Complanati standard decoction.

2. Characteristic spectrum measurement of semen astragali Complanati standard decoction

Taking a proper amount of 17 batches of semen astragali complanati standard decoction prepared in the first embodiment, grinding, respectively taking about 0.14g, precisely weighing, placing into a conical flask with a plug, precisely adding 25ml of 75% methanol, weighing, performing ultrasonic treatment for 15min, performing ultrasonic power of 500W and frequency of 40kHz, cooling, supplementing the lost weight with 75% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to measure a characteristic map under the following conditions.

Octadecylsilane chemically bonded silica is used as filler (column length is 150mm, inner diameter is 2.1mm, and particle diameter is 1.7 μm); acetonitrile as mobile phase a and 0.1% phosphoric acid aqueous solution as mobile phase B, and gradient elution was performed as specified in table 4; the column temperature is 35 ℃; the flow rate is 0.3ml/min; the detection wavelength was 266nm. The theoretical plate number should be not less than 4000 calculated on the basis of the semen astragali Complanati glycoside. The measured characteristic spectrum is shown in fig. 23. The measured relative retention times and relative peak areas of 17 batches of semen astragali Complanati standard decoction characteristic patterns are shown in tables 28 and 29.

As can be seen from FIG. 23, the total number of peaks in the characteristic spectrum of 17 batches of semen astragali Complanati standard decoction is 10, the peak 8 corresponding to the semen astragali Complanati glycoside peak is S peak, and the relative retention time of each characteristic peak and S peak is within + -10% of the specified value. By analysis, the common peaks that can be determined are: 0.212 (Peak 1), 0.243 (Peak 2), 0.312 (Peak 3), 0.570 (Peak 4), 0.628 (Peak 5), 0.777 (Peak 6), 0.964 (Peak 7), semen astragali Complanati (Peak S), 1.103 (Peak 9), 1.128 (Peak 10).

Table 28 relative retention time of characteristic spectrum of 17 batches of semen astragali Complanati standard decoction

Table 29 17 characteristic spectrum of the standard decoction of semen astragali Complanati with respect to peak area

/>

Example 4

1. Preparation of semen astragali Complanati standard decoction

2. Characteristic peak assignment

1. LC-MS analysis conditions

(1) UPLC condition:

waters ACQUITY UPLC chromatograph; chromatographic column: agilent SB-C18 column (2.1 x 150mm,1.8 μm); mobile phase system: methanol (a): 0.1% formic acid water (B); elution was performed according to the gradient procedure of table 30; flow rate: 0.25mL/min; detection wavelengths 270, 266, 210nm; column temperature: 30 ℃; sample injection amount: 3 μL;0.8 μl (control), gradient program elution; flow rate: 0.4mL/min; detection wavelengths 270, 254, 210nm; column temperature: 30 ℃; sample injection amount: 2.5. Mu.l (sample), 0.2. Mu.l (control).

TABLE 30 gradient elution procedure

(2) Mass spectrometry conditions: the method comprises the steps of (1) detecting anions of an ESI ion source by a Waters Xevo G2-XS QTOF mass spectrometer; source voltage: 2.5kV, N2 flow rate: 800L/h, the collision gas is nitrogen; the capillary temperature is 400 ℃; taper hole gas flow rate: 100L/h; air source temperature: 120 ℃; adopting a full scanning mode, wherein the molecular weight scanning range is 50-1500; collision induced dissociation voltage: 6V (low energy), 30-60V (high energy);

(3) sample treatment: taking the semen astragali complanati standard soup freeze-dried powder prepared in the step 1, precisely weighing about 0.20g, placing into a conical flask with a plug, precisely adding 25ml of 75% methanol, weighing, performing ultrasonic treatment for 15min, cooling, shaking uniformly, filtering, and taking the subsequent filtrate to pass through a 0.22 mu m filter membrane.

(4) Control treatment: the semen astragali Complanati glycoside reference substance solution (concentration 15.6 μg/mL) is prepared by dissolving appropriate amount of astragalin (batch number DST171212-001, product of Du De Si biological Co., ltd., purity not less than 98%) and myricetin-3-O-beta-D-glucoside (batch number DST181109-272, product of Du Si biological Co., ltd., purity not less than 98%) with 50% methanol.

2. LC-MS analysis results

2.1LC-MS Mass Spectrometry Profile

The LC-MS mass spectrum of the obtained semen astragali Complanati standard decoction is shown in FIG. 24, wherein FIG. 24A shows negative ion mode BPI mass spectrum, and FIG. 24B shows 266nm ultraviolet chromatogram.

From the 266nm ultraviolet chromatogram, the LC-MS mass spectrum chromatogram peak 1-12 is suspected to be consistent with the characteristic fingerprint spectrum No. 1-12 peak. The ultraviolet spectrogram and the mass spectrogram of the 12 chromatographic peaks are analyzed as follows.

DAD uv plot (200-400 nm) for each chromatographic peak: fig. 25, 27, 29, 31, 33, 35, 37, 39, 41, 43 and 45 are DAD uv absorbance comparisons of characteristic peaks 1-7, 9-12 on two different instruments and methods.

2.2 identification of the main chromatographic peak at 266nm of uv:

2.21 Peak 1

The retention time of peak 1 was 10.99min: the DAD spectrum showed maximum absorption wavelengths of 254 and 354nm, suggesting a flavonoid component. The mass spectrum shows that M/z 611.12565 is an excimer ion peak, M/z 461.07255 is an ion peak for removing one molecule of xylose [ M-132-18], M/z 316.02224 is an aglycone excimer ion peak formed by removing one molecule of xylose and one part of glucose [ M-H-132-162], and myricetin glycoside is suggested, so that the peak is identified as myricetin-3-O- [ beta-D-xylopyranose- (1- & gt 2) ] -beta-D-glucopyranoside. FIG. 25 shows the DAD spectrum of peak 1. FIG. 26 shows the MS/MS mass spectrum of peak 1.

2.2.2 Peak 2

The retention time of peak 2 was 16.04min, and the DAD spectrum showed maximum absorption wavelengths of 254 and 354nm, suggesting a flavonoid component. The mass spectrum shows that M/z 479.08262 is an excimer ion peak, M/z 316.02206 is an aglycone excimer ion peak formed by removing one part of glucose [ M-H-162], which is suggested to be myricetin glycoside, and therefore, the peak is identified as myricetin-3-O-beta-D-glucopyranoside or myricetin-3-O-beta-D-galactopyranoside. FIG. 27 shows the DAD spectrum of peak 2. FIG. 28 shows the MS/MS mass spectrum of peak 2.

2.2.3 Peak 3

The retention time of peak 3 was 27.16min and the dad profile showed maximum absorption wavelengths of 265 and 346nm, suggesting a flavonoid component. The mass spectrum shows that M/z 579.13581 is an excimer ion peak, M/z 429.08282 is an ion peak for removing one molecule of xylose [ M-132-18], and M/z 284.03252 is an aglycone excimer ion peak formed by removing one molecule of xylose and one part of glucose [ M-H-132-162], which is suggested to be kaempferol glucoside, so that the peak is identified as kaempferol-3-O- [ beta-D-xylopyranose- (1- > 2) ] -beta-D-glucopyranoside. FIG. 29 shows the DAD spectrum of peak 3. FIG. 30 shows a mass spectrum of peak 3 MS/MS.

2.2.4 Peak 4

The retention time of peak 4 was 29.09min and the dad profile showed maximum absorption wavelengths of 265 and 346nm, suggesting a flavonoid component. The mass spectrum shows that M/z 493.09818 is an excimer ion peak, M/z 330.03806 is an aglycone ion peak for removing one molecule of glucose [ M-162], and M/z 315.01432 is an ion peak formed by removing one molecule of methyl from aglycone, so the peak is identified as the semen astragali complanati myricetin. FIG. 31 shows the DAD spectrum of peak 4. FIG. 32 shows the MS/MS mass spectrum of peak 4.

2.2.5 Peak 5

The retention time of peak 5 was 35.26min and the dad profile showed maximum absorption wavelengths of 264 and 343nm, suggesting a flavonoid component. The mass spectrum shows that M/z 447.09264 is an excimer ion peak, M/z 284.03239 is an aglycone ion peak for removing one molecule of glucose [ M-162], and the peak is identified as kaempferol-3-O-beta-D-glucoside. FIG. 33 shows the DAD spectrum of peak 5. FIG. 34 shows the MS/MS mass spectrum of peak 5.

2.2.6 Peak 6

The retention time of peak 6 was 36.29min and the DAD spectrum showed maximum absorption wavelengths of 263 and 350nm, suggesting a flavonoid component. The mass spectrum shows that M/z 417.08232 is an excimer ion peak, M/z 284.03282 is an aglycone ion peak for removing one molecule of arabinose [ M-132], and the peak is identified as kaempferol-3-O-beta-D-arabinoside. FIG. 35 shows the DAD spectrum of peak 6. FIG. 36 shows the MS/MS mass spectrum of peak 6.

2.2.7 Peak 7

The retention time of peak 7 was 36.95min and the dad profile showed maximum absorption wavelengths of 266 and 333nm, suggesting a flavonoid component. The mass spectrum shows that M/z 593.15156 is an excimer ion peak, M/z 461.10988 is an ion peak for removing one molecule of arabinose [ M-132], 298.04824 is an aglycone excimer ion peak for removing one molecule of xylose and one molecule of glucose [ M-H-132-162], and therefore the component is determined to be rhamnocitrin glycoside, and the component is identified as rhamnocitrin-3-O- [ beta-D-apiose- (1- > 2) ] -beta-D-glucoside. FIG. 37 shows the DAD spectrum of peak 7. FIG. 38 shows the MS/MS mass spectrum of peak 7.

2.2.8 Peak 8

The retention time of peak 8 was 38.49min, which was determined to be astragali complanatoside by comparison with the control.

2.2.9 Peak 9

The retention time of peak 9 was 41.39min and the dad profile showed maximum absorption wavelengths of 266 and 321nm, suggesting a flavonoid component. The mass spectrum shows that m/z 593.15156 is an excimer ion peak, m/z 298.04842 is rhamnogenin, the peak is also rhamnoside, the molecular weight of the rhamnoside is 42 more than that of peak 7, acetylation is indicated, and m/z 593.15333 is an ion peak for removing one molecule of acetyl, so that the component is identified as rhamnogenin-3-O- [ beta-D-apigenin sugar- (1 ' -2 ') -6 ' -acetyl ] -beta-D-glucoside. FIG. 39 shows the DAD spectrum of peak 9. FIG. 40 shows the MS/MS mass spectrum of peak 9.

2.2.10 Peak 10

The retention time of peak 10 was 42.65min and the dad profile showed maximum absorption wavelengths of 265 and 333nm, suggesting a flavonoid component. The mass spectrum shows that M/z 665.17244 is an excimer ion peak, M/z 299.05557 is rhamnogenin, the peak is also rhamnoside, the molecular weight of the rhamnoside is 42 more than that of the flastem milkvetch seed glycoside, acetylation is indicated, 503.11901 is an ion peak for removing one molecule of glucose [ M-162], and M/z 461.10840 is an ion peak for removing one molecule of acetyl, so that the component is identified as acetyl flastem milkvetch seed glycoside. FIG. 41 shows the DAD spectrum of peak 10. FIG. 42 shows the MS/MS mass spectrum of peak 10.

2.2.11 Peak 11

The retention time of peak 11 was 47.76min and the DAD spectrum showed maximum absorption wavelengths of 268 and 329nm, suggesting a flavonoid component. The mass spectrum shows that M/z 769.19919 is an excimer ion peak, M/z 299.05557 is rhamnogenin, the peak is also rhamnogenin glucoside, M/z193.05082 is a ferulic acid excimer ion peak, the structure contains a molecule of feruloyl, 593.15173 is an ion peak for removing a molecule of feruloyl [ M-176], 575.14110 is an ion peak for removing a molecule of feruloyl [ M-H-194], 443.09878 is an ion peak formed by removing a molecule of feruloyl and a molecule of apigenin, and on the basis, an excimer ion peak M/z298.04833 for removing a molecule of glucose to form aglycone is removed. Thus, the component was identified as rhamnocitrin-3-O- [5 '-O-feruloyl-beta-D-apiose furanose- (1'. Fwdarw.2 ") ] -beta-D-glucopyranoside. FIG. 43 shows the DAD spectrum of peak 11. FIG. 44 shows the MS/MS mass spectrum of peak 11.

2.2.12 Peak 12

The retention time of peak 12 was 48.89min and the DAD spectrum showed maximum absorption wavelengths of 267 and 311nm, suggesting a flavonoid component. The mass spectrum shows that m/z 739.18871 is an excimer ion peak, m/z 298.04848 is rhamnogenin, the peak is also rhamnogenin glycoside, m/z167.05096 indicates that one molecule of coumaroyl is contained in the molecule, 593.15216 is an ion peak formed by removing one molecule of coumaroyl, 575.14154 is an ion peak formed by removing one molecule of coumaric acid, on the basis, one molecule of apiose is removed to form an ion m/z 461.10961, and one molecule of glucose is removed to form an aglycone excimer ion peak m/z 298.04848. Thus, the component was identified as rhamnocitrin-3-O- [5 '"-O-p-coumaroyl- β -D-apiose- (1'" -2 ") ] - β -D-glucopyranoside, fig. 45 shows the DAD profile of peak 12. FIG. 46 shows the MS/MS mass spectrum of peak 12.

Further validation of Peak 2, peak 3, peak 5, peak 8

For further verification, peak 2 (myricetin-3-O-beta-D-glucopyranoside), peak 3 (kaempferol-3-O- [ beta-D-xylopyranose- (1.fwdarw.2) ] -beta-D-glucopyranoside), peak 5 (kaempferol-3-O-beta-D-glucopyranoside), peak 8 (flastem milkvetch seed glycoside) were again identified using the conditions of the standard decoction profile of flastem milkvetch seed, and the isolated peaks 2, 3, 5, 8 were consistent in retention time, as shown in FIG. 47.

As indicated in fig. 48:

the retention time of peak 1, which is myricetin-3-O- [ beta-D-xylopyranose- (1.fwdarw.2) ] -beta-D-glucopyranoside, was 10.99 min;

the retention time of peak 2, which was myricetin-3-O-beta-D-glucopyranoside, was 16.04 min;

the retention time of peak 3, which was kaempferol-3-O- [ beta-D-xylopyranose- (1.fwdarw.2) ] -beta-D-glucopyranoside, was 23.16 min;

the retention time of peak 4, which is semen astragali Complanati myricetin, was 29.09 min;

the retention time of peak 5, which is kaempferol-3-O-beta-D-glucoside, was 35.26 min;

the retention time of peak 6, which was kaempferol-3-O- β -D-arabinoside, was 36.28 min;

the retention time of peak 7, which is rhamnocitrin-3-O- [ beta-D-apiose- (1→2) ] -beta-D-glucoside, was 36.95 min;

The retention time of peak 8, which is complanatoside, was 38.49 min;

the retention time of peak 9, which is rhamnocitrin-3-O- [ beta-D-apiose- (1' "-2") -6 "-acetyl ] -beta-D-glucoside, was 41.39 min;

the retention time of peak 10, which is acetyl flatstem milkvetch seed glycoside, was 42.65 min;

the retention time of peak 11, which is rhamnocitrin-3-O- [5 '"-O-feruloyl- β -D-apiose- (1'" -2 ") ] - β -D-glucopyranoside, was 47.76 min;

the retention time of peak 12, which was rhamnocitrin-3-O- [5 '"-O-p-coumaroyl- β -D-apiose- (1'" -2 ") ] - β -D-glucopyranoside, was 48.89 min.

The above description is not intended to limit the invention in any way, but is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. A method for detecting the content of flastem milkvetch seed glycoside in a flastem milkvetch seed standard decoction is characterized by comprising the following steps:

(1) Preparation of a control solution:

(2) Preparation of test solution:

Adding methanol solvent into semen astragali Complanati standard decoction for ultrasonic extraction; the preparation method of the semen astragali complanati standard decoction comprises the following steps: (1) Decocting semen astragali Complanati with water, and filtering to obtain filtrate; (2) Concentrating and drying the filtrate in the step (1), and then freeze-drying, wherein the freeze-drying is divided into three stages: a. pre-freezing: the pre-freezing temperature is-50 ℃ to-45 ℃; b. primary drying: the drying temperature is between 20 ℃ below zero and 0 ℃; c. and (3) secondary drying: drying at 5-25 deg.c to obtain standard decoction of semen astragali Complanati;

(3) Ultra-high performance liquid chromatography analysis:

sucking the reference substance solution and the sample solution, injecting into an ultra-high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as filler, taking acetonitrile as mobile phase A with detection wavelength of 266nm, taking water phase as mobile phase B, and detecting with A-B ratio of 22:78 to obtain the content of the semen astragali Complanati glycoside.

2. The method according to claim 1, wherein in the step (2), the extraction time is 10 to 45 minutes.

3. The content detecting method according to claim 1, wherein in the step (2), the extraction time is 30min.

4. The content detection method according to claim 1, wherein in the step (3), the flow rate of the mobile phase a and the mobile phase B is 1.0ml/min.

5. The content detection method according to claim 2, wherein in the step (3), the flow rate of the mobile phase a and the mobile phase B is 1.0ml/min.

6. The method according to claim 1, wherein in the step (3), the aqueous phase is selected from water, a 0.1% aqueous phosphoric acid solution or a 0.05% aqueous phosphoric acid solution.

7. The method according to claim 2, wherein in the step (3), the aqueous phase is selected from water, a 0.1% aqueous phosphoric acid solution or a 0.05% aqueous phosphoric acid solution.

8. The method according to claim 4, wherein in the step (3), the aqueous phase is selected from water, a 0.1% aqueous phosphoric acid solution or a 0.05% aqueous phosphoric acid solution.

9. The method according to claim 1, wherein in the step (3), the aqueous phase is a 0.1% phosphoric acid aqueous solution.

10. The method for detecting the characteristic spectrum of the standard decoction of semen astragali complanati is characterized by comprising the following steps of:

(1) Preparation of reference solution:

(2) Preparation of test solution:

adding 75% methanol into semen astragali Complanati standard decoction for ultrasonic extraction; the preparation method of the semen astragali complanati standard decoction comprises the following steps: (1) Decocting semen astragali Complanati with water, and filtering to obtain filtrate; (2) Concentrating and drying the filtrate in the step (1), and then freeze-drying, wherein the freeze-drying is divided into three stages: a. pre-freezing: the pre-freezing temperature is-50 ℃ to-45 ℃; b. primary drying: the drying temperature is between 20 ℃ below zero and 0 ℃; c. and (3) secondary drying: drying at 5-25 deg.c to obtain standard decoction of semen astragali Complanati;

(3) Ultra-high performance liquid chromatography analysis:

sucking the reference substance solution and the sample solution, injecting into an ultra-high performance liquid chromatograph, taking octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A, and water phase as a mobile phase B, and performing gradient elution to obtain a characteristic map of the semen astragali Complanati standard decoction;

in the step (3), the gradient elution procedure is as follows:

17-18min, 28-29% of mobile phase A and 72-71% of mobile phase B;

11. The method according to claim 10, wherein in the step (2), the extraction time is 10 to 45 minutes.

12. The method according to claim 11, wherein in the step (2), the extraction time is 15min.

13. The method according to claim 10, wherein in the step (3), the flow rate of the mobile phase a and the mobile phase B is 0.3ml/min.

14. The method according to claim 10, wherein in step (3), the aqueous phase is selected from water, 0.1% aqueous phosphoric acid solution or 0.05% aqueous phosphoric acid solution.

15. The method according to claim 11, wherein in step (3), the aqueous phase is selected from water, 0.1% aqueous phosphoric acid solution or 0.05% aqueous phosphoric acid solution.

16. The method according to claim 13, wherein in step (3), the aqueous phase is selected from water, 0.1% aqueous phosphoric acid solution or 0.05% aqueous phosphoric acid solution.

17. The method according to claim 10, wherein in step (3), the aqueous phase is a 0.1% aqueous phosphoric acid solution.

18. The method according to claim 10, wherein the column temperature in step (3) is 32 to 38 ℃.

19. The method according to claim 11, wherein the column temperature in step (3) is 32 to 38 ℃.

20. The method according to claim 13, wherein the column temperature in step (3) is 32 to 38 ℃.

21. The method according to claim 14, wherein in step (3), the column temperature is 35 ℃.

22. The method according to any one of claims 10 to 21, wherein the characteristic spectrum has at least 10 characteristic peaks, the reference substance is computable to obtain S peaks, and the relative retention time of each characteristic peak and S peak is calculated to be within ±10% of a predetermined value, and the predetermined value contains 0.212, 0.243, 0.312, 0.570, 0.628, 0.777, 0.964, 1.103, and 1.128.