CN115236225B

CN115236225B - Method for constructing characteristic spectrum of standard decoction of Chinese honeylocust spine traditional Chinese medicine decoction pieces

Info

Publication number: CN115236225B
Application number: CN202210822278.1A
Authority: CN
Inventors: 李振雨; 梁月仪; 位翠杰; 刘晓霞; 叶瑞平; 何民友; 陈向东; 孙冬梅
Original assignee: Guangdong Yifang Pharmaceutical Co Ltd
Current assignee: Guangdong Yifang Pharmaceutical Co Ltd
Priority date: 2022-07-13
Filing date: 2022-07-13
Publication date: 2023-09-29
Anticipated expiration: 2042-07-13
Also published as: CN115236225A

Abstract

The invention relates to the technical field of medicine analysis, in particular to a method for constructing a characteristic map of a standard decoction of Chinese honeylocust spine traditional Chinese medicine decoction pieces. The construction method comprises the following steps: preparing a sample solution; performing ultra-high performance liquid chromatography measurement on the sample solution; the chromatographic conditions of the ultra-high performance liquid chromatography include: the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises acetonitrile and methanol, the mobile phase B comprises aqueous solution of formic acid, and the elution mode is gradient elution. The UPLC characteristic spectrum of the standard decoction of the spina gleditsiae traditional Chinese medicine decoction pieces constructed by the invention can be used for representing not only flavone and phenolic acid components in the standard decoction of the spina gleditsiae traditional Chinese medicine decoction pieces, but also coumarin components (scopolamine and scopoletin) with better water solubility. And the separation degree among characteristic peaks of each component is good.

Description

Method for constructing characteristic spectrum of standard decoction of Chinese honeylocust spine traditional Chinese medicine decoction pieces

Technical Field

The invention relates to the technical field of medicine analysis, in particular to a method for constructing a characteristic map of a standard decoction of Chinese honeylocust spine traditional Chinese medicine decoction pieces.

Background

Spina Gleditsiae is also called spina Gleditsiae, and is the thorn of the tree of Leguminosae deciduous tree. Spina Gleditsiae contains flavonoids, phenols, amino acids, coumarin, etc., and has repercussive, toxic materials removing, pus expelling, and parasite killing effects. The quality standard of spina gleditsiae is not studied much, and only the characteristics, microscopic identification and thin-layer identification are included under the quality standard item of spina gleditsiae medicinal materials and decoction pieces in Chinese pharmacopoeia of 2020 edition, the quality standard is still imperfect.

The standard decoction of the traditional Chinese medicine decoction pieces is prepared by referring to a modern extraction method and a standard process based on the traditional Chinese medicine theory and clinical application, can be used for standardizing clinical medication as a standard substance and a standard system, standardizes the novel decoction piece form which is widely used in clinic at present and comprises prescription particles, and ensures the medication accuracy and the dosage consistency. Therefore, by adopting the modern analysis technology, the quality standard of the standard decoction of the traditional Chinese medicine decoction pieces is established, and important references can be provided for the establishment of the quality standards of the corresponding formula particles, the classical prescription and other modern traditional Chinese medicine preparations.

The chemical components of the traditional Chinese medicine are a multi-component complex system, the traditional chemical quality control mode is difficult to evaluate the internal quality of the traditional Chinese medicine, the types and the amounts of the chemical components contained in the traditional Chinese medicine and the preparation thereof can be comprehensively reflected due to the integral control concept, the medicine quality is integrally described and evaluated, and the traditional Chinese medicine quality control method is increasingly applied to the fields of chemical components and quality control research of the traditional Chinese medicine.

However, the basic research of the spina gleditsiae is weak, the chemical components are complex, the content is low, and the research difficulty is high. And the research on spina gleditsiae mainly focuses on the research on flavone and phenolic acid components of spina gleditsiae on medicinal materials and formula particles, and the research on other components is less. For example, there are studies on establishing HPLC fingerprint of spina gleditsiae formula particles by using an HPLC method, and identifying 9 common peaks, but the identified common peaks are not clearly identified, and the separation effect of each characteristic peak is poor. In addition, the HPLC method is adopted to establish the fingerprint of the spina gleditsiae medicinal material, 29 common peaks are identified, wherein 5 chromatographic peaks are respectively identified as flavonoid and phenolic acid, the fingerprint peaks are smaller, and the overall separation effect is poor.

Disclosure of Invention

Based on the above, the invention provides a method for constructing the characteristic spectrum of the standard decoction of the Chinese honeylocust spine traditional Chinese medicine decoction pieces. The technical scheme is as follows:

a construction method of a characteristic spectrum of a standard decoction of Chinese honeylocust spine traditional Chinese medicine decoction pieces comprises the following steps:

mixing standard decoction of Chinese medicinal decoction pieces of spina Gleditsiae with extraction solvent, extracting, dissolving the obtained extract, and preparing test solution;

performing ultra-high performance liquid chromatography measurement on the sample solution;

the chromatographic conditions of the ultra-high performance liquid chromatography include:

the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises acetonitrile and methanol, the mobile phase B comprises aqueous solution of formic acid, and the elution mode is gradient elution;

the gradient elution specifically comprises the following steps:

0-11 min, wherein the volume fraction of the mobile phase A is increased from 5% to 15%, and the volume fraction of the mobile phase B is reduced from 95% to 85%;

11-26 min, wherein the volume fraction of the mobile phase A is increased from 15% to 18%, and the volume fraction of the mobile phase B is reduced from 85% to 82%;

26-45 min, wherein the volume fraction of the mobile phase A is increased from 18% to 22%, and the volume fraction of the mobile phase B is reduced from 82% to 78%;

45-50 min, wherein the volume fraction of the mobile phase A is increased from 22% to 90%, and the volume fraction of the mobile phase B is reduced from 78% to 10%;

50-55 min, wherein the volume fraction of the mobile phase A is kept at 90%, and the volume fraction of the mobile phase B is kept at 10%.

In some embodiments, the volume ratio of acetonitrile to methanol is 1 (0.5-2). Preferably, the volume ratio of acetonitrile to methanol is 1:1.

In some embodiments, the formic acid is present in the aqueous solution at a volume fraction of 0.02 to 0.2%. Preferably, the volume fraction of formic acid in the aqueous solution of formic acid is 0.1%.

In some embodiments, the chromatographic conditions of the ultra performance liquid chromatography further comprise: the stationary phase is a chromatographic column with octadecylsilane chemically bonded silica as filler.

Alternative chromatographic columns include, but are not limited to: chromatographic columns of Thermo acclaim C18 (2.1 mm. Times.150 mm,2.2 μm), waters HSS T3C 18 (2.1 mm. Times.150 mm,1.8 μm), agilent SB C18 (2.1 mm. Times.150 mm,1.8 μm), shimadzu C18 (2.1 mm. Times.150 mm,1.9 μm)

In some embodiments, the chromatographic conditions of the ultra performance liquid chromatography further comprise: the flow rate is 0.34 mL-0.36 mL per minute, and/or the column temperature is 40-44 ℃, and/or the detection wavelength is 320-360nm. Preferably, the flow rate is 0.35mL per minute, and/or the column temperature is 42 ℃, and/or the detection wavelength is 340nm.

In some embodiments, the extraction solvent is methanol, an aqueous solution of methanol, ethanol, or an aqueous solution of ethanol. Alternatively, the volume fraction of methanol in the aqueous solution of methanol may be 20% to 80%, and the volume fraction of ethanol in the aqueous solution of ethanol may be 20% to 80%.

In some embodiments, the extraction process is an ultrasonic process or a heat reflow process.

Optionally, the extraction treatment is ultrasonic treatment, and the time of ultrasonic treatment is not less than 25min. Preferably, the time of the ultrasonic treatment is not less than 30 minutes.

Optionally, the extraction treatment is ultrasonic treatment, and the dosage of the extraction solvent is as follows: each 0.5g of the spina gleditsiae traditional Chinese medicine decoction piece standard decoction corresponds to 10-20 mL of the extraction solvent. Preferably, each 0.5g of the spina gleditsiae traditional Chinese medicine decoction piece standard decoction corresponds to 14-16 mL of the extraction solvent.

Optionally, the extraction treatment is ultrasonic treatment, the power of the ultrasonic treatment is 400-600W, and the frequency is 30-50 kHz.

Optionally, the extraction treatment is a heat reflux treatment, the temperature of the heat reflux treatment is 70-80 ℃, and the time is not less than 25min, preferably not less than 30min.

It will be appreciated that after the extraction process, the supernatant is the extract, and the extract is dried to obtain the extract.

It will be appreciated that the solvent in which the resulting extract is dissolved may be water and that the step of washing may also be included after the resulting extract is dissolved.

In some embodiments, the number of extraction treatments is not less than 2. Preferably, the number of extraction treatments is not less than 3.

Optionally, the extractant is n-butanol.

It will be appreciated that after the extraction process, the n-butanol solution is the extract, and the extract is dried to obtain the extract.

It will be appreciated that the solvent in which the extract is dissolved may be methanol.

Compared with the traditional scheme, the invention has the following beneficial effects:

the UPLC characteristic spectrum of the standard decoction of the spina gleditsiae, constructed by the invention, can simultaneously characterize as many characteristic components of the standard decoction of the spina gleditsiae as possible, for example, modern pharmacological research shows that coumarin components represented by scopolamine are substance bases with anticancer, cancer inhibiting, bacteriostasis and other activities of spina gleditsiae. And the separation degree among characteristic peaks of each component is good. The UPLC characteristic spectrum of the standard decoction of the spina gleditsiae decoction pieces constructed by the invention not only provides a basis for establishing the quality standard of the standard decoction of the spina gleditsiae decoction pieces, but also provides an important reference for establishing the quality standard of modern traditional Chinese medicine preparations such as spina gleditsiae formula particles, classical prescriptions thereof and the like.

Drawings

FIG. 1 is a full-wavelength scanning 3D map of a standard decoction feature map of spina gleditsiae decoction pieces;

FIG. 2 is a comparison chromatogram of different detection wavelengths of the standard decoction feature spectrum of spina Gleditsiae decoction pieces;

FIG. 3 is a comparison chromatogram of standard decoction feature of spina Gleditsiae decoction pieces with different chromatographic columns;

FIG. 4 is a graph showing the characteristic spectrum of standard decoction of spina Gleditsiae in different mobile phases;

FIG. 5 is a graph showing the comparison of different extraction times of the standard decoction feature of spina Gleditsiae;

FIG. 6 is a comparison chromatogram of different ultrasonic extraction times of the standard decoction feature of spina Gleditsiae decoction pieces;

FIG. 7 is a graph showing the comparison of the characteristic spectrum of the standard decoction of spina gleditsiae with different amounts of ultrasonic extraction solvent;

FIG. 8 is a comparison chromatogram of different extraction modes of the standard decoction feature spectrum of spina gleditsiae decoction pieces;

FIG. 9 is a comparison chromatogram of different extraction solvents of the standard decoction feature of spina Gleditsiae decoction pieces;

FIG. 10 is a pattern common to the characteristic spectrum of the standard decoction pieces of 18 batches of spina Gleditsiae;

FIG. 11 is a graph of the characteristic of standard decoction of spina Gleditsiae;

FIG. 12 is a characteristic map of spina Gleditsiae as a control;

FIG. 13 is a total ion flow chart and ultraviolet absorption chromatogram of a standard decoction sample solution of spina Gleditsiae;

FIG. 14 is a graph showing the characteristic peaks of standard decoction of spina Gleditsiae.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

1 instrument and reagents

Instrument: rotary evaporator (RE-3000A, shanghai Rong Biochemical instruments Co., ltd.), ceramic health care kettle (3L, shenzhen King Co., ltd.), circulating water vacuum pump (SHZ-D class, ind.), low temperature cooling liquid circulating water pump (DLSB-5/20, santa Classification, chengzhou, great wall Co., ltd.), vacuum freeze dryer (TRL-0.5, dalian Shuangrui technology Co., ltd.), electrothermal constant temperature blast drying oven (DHG-9626A, shanghai Seisakusho laboratory equipment Co., ltd.), thermo ultra high performance liquid chromatograph (Vanquish, simer Feishan technology Co., ltd.); agilent ultra-high performance liquid chromatograph (model 1290, agilent inc.); a Thermo Acclaim C18 (2.1 mm. Times.150 mm,2.2 μm) column; an Agilent SB C18 (2.1 mm. Times.150 mm,1.8 μm) column; waters HSS T3C 18 (2.1 mm. Times.150 mm,1.8 μm) column; shimadzu C18 (2.1 mm. Times.150 mm,1.9 μm) column. One hundredth electronic analytical balance (JJ 600, a well-established market double jetty test instrumentation factory); one ten-thousandth electronic analytical balance (ME 204E, mertrer-tolidox); parts per million of electronic analytical balance (XP 26, mertrer-tolidol); numerical control ultrasonic cleaner (KQ-500 DE, kunshan ultrasonic instruments Co., ltd.); thermostatic water bath (HWS 28, shanghai-Heng technology Co., ltd.); ultrapure water systems (Milli-Q Direct, merck Co., ltd.).

2 reagent

2.1 preparation of spina Gleditsiae decoction pieces

The Chinese honeylocust spine decoction pieces are prepared according to the processing rules of Chinese pharmacopoeia 2020 edition honeylocust spine. The specific processing method comprises the following steps: removing impurities from spina Gleditsiae, cutting into suitable segments, placing in a cleaning basket, rapidly cleaning, covering with clean wet towel, moistening, cutting into thick pieces with specification of 2-4 mm, drying at 50deg.C for 4-5 hr, taking out, and cooling.

2.2 preparation of standard decoction of spina Gleditsiae decoction pieces

Decocting 200g of spina Gleditsiae in water twice, adding 10 times of water for the first time, soaking for 30 minutes, boiling with strong fire (500W), keeping slight boiling with slow fire (200W) for 30 minutes, filtering with 350 mesh screen, and rapidly cooling with cold water; adding 8 times of water for the second time, boiling with strong fire (500W), keeping micro-boiling with slow fire (200W) for 25min, filtering with 350 mesh sieve, rapidly cooling with cold water, and mixing the two filtrates. Transferring the decoction into a 2000mL round bottom flask, and concentrating at low temperature under reduced pressure by adopting a rotary evaporator (temperature: 65 ℃ C.; vacuum degree: minus 0.08 to minus 0.1 MPa) to 100mL; under the magnetic stirring, subpackaging into 10mL penicillin bottles, wherein the subpackaging volume of each bottle is 2mL, transferring into a vacuum freeze dryer for freeze-drying after the subpackaging is finished, taking out, and rolling an aluminum cover to obtain standard decoction freeze-dried powder, wherein the batch number of the standard decoction freeze-dried powder prepared from 18 batches of spina gleditsiae decoction pieces at different producing places is shown in table 1.

TABLE 1

2.3 preparation of reference solutions

Taking about 1.5g of spina gleditsiae reference medicine, placing the spina gleditsiae reference medicine into a conical flask with a plug, adding 50mL of water, heating and refluxing for 30 minutes, centrifuging, evaporating to dryness, adding 15mL of methanol into residues, performing ultrasonic treatment (power 500W, frequency 40 kHz) for 30 minutes, centrifuging, taking supernatant, evaporating to dryness, adding 10mL of water into residues to dissolve, adding 10mL of ammonia water for washing, transferring to a separating funnel, extracting with n-butanol for 3 times by shaking, merging n-butanol liquid each time by 20mL, evaporating to dryness, adding methanol into residues to dissolve, transferring to a 5mL measuring flask, adding methanol to scale, shaking uniformly, filtering, and taking a subsequent filtrate to obtain a reference medicine solution.

Accurately weighing a proper amount of taxifolin reference substance, placing into a 25mL measuring flask, and adding methanol to obtain reference substance solution containing taxifolin 40 μg per 1 mL. Obtaining the douglas fir element reference substance solution.

2.4 preparation of sample solutions

Taking a proper amount of standard decoction pieces of spina gleditsiae (batch GT 1611181), grinding, taking about 0.5g, precisely weighing, placing into a conical flask, precisely adding 15mL of methanol, performing ultrasonic treatment (power 500W, frequency 40 kHZ) for 30 minutes, centrifuging, taking supernatant, evaporating to dryness, adding 10mL of water into residues to dissolve, adding 10mL of ammonia water for washing, transferring into a separating funnel, shaking and extracting 3 times with n-butanol, 20mL each time, merging n-butanol liquid, evaporating to dryness, adding methanol into residues to dissolve, transferring into a 5mL measuring flask, adding methanol to scale, shaking uniformly, filtering, and taking subsequent filtrate to obtain a sample solution.

3 chromatographic conditions

3.1 selection of detection wavelength

Taking the sample solution under the item "2.4", respectively selecting 210nm, 254nm and 340nm as detection wavelengths of the sample solution, and performing full-wave scanning under other chromatographic conditions as follows: a Thermo acclaim C18 (2.1 mm. Times.150 mm,2.2 μm) column was selected, acetonitrile-methanol (1:1, v/v) as mobile phase A, and 0.1% formic acid aqueous solution as mobile phase B, and gradient elution was performed as specified in Table 2; the flow rate was 0.35mL per minute; column temperature was 42 ℃; the sample loading was 2. Mu.L. And (3) sample injection measurement, namely recording the absorption spectrum of the sample solution in the range of 210 nm-400 nm, and the result is shown in fig. 1 and 2.

The results show that: the detection wavelength is 340nm, chromatographic peak information is more and the base line is more stable, so 340nm is selected as the detection wavelength of the characteristic spectrum.

3.2 selection of chromatographic columns

The sample solutions under the "2.4" item were used as columns for Waters HSS T3C 18 (2.1 mm. Times.150 mm,1.8 μm), shimadzu C18 (2.1 mm. Times.150 mm,1.9 μm) and Thermo acclaim C18 (2.1 mm. Times.150 mm,2.2 μm), respectively, and the other chromatographic conditions were as follows: acetonitrile-methanol (1:1, v/v) as mobile phase A, and formic acid aqueous solution with volume fraction of 0.1% as mobile phase B, gradient elution was performed as specified in Table 2; the flow rate was 0.35mL per minute; column temperature was 42 ℃; the sample injection amount is 2 mu L; the detection wavelength was 340nm. Sample injection measurement is carried out, a chromatogram of the sample solution is recorded, and the result is shown in fig. 3.

The results show that: when Thermo acllim (2.1 mm. Times.150 mm,2.2 μm) was used, the peak type was good and the degree of separation was high. Thus, thermo acclaim (2.1 mm. Times.150 mm,2.2 μm) was chosen as the chromatographic column.

3.3 selection of mobile phases

Taking the sample solution under the item "2.4", and taking a formic acid aqueous solution with the volume fraction of 0.1% and a phosphoric acid aqueous solution with the volume fraction of 0.1% as fluidity B respectively, wherein other chromatographic conditions are as follows: a Thermo acclaim C18 (2.1 mm. Times.150 mm,2.2 μm) column was selected and gradient eluted with acetonitrile-methanol (1:1, v/v) as mobile phase A as specified in Table 2; the flow rate was 0.35mL per minute; column temperature was 42 ℃; the sample injection amount is 2 mu L; the detection wavelength was 340nm. Sample injection measurement is carried out, a chromatogram of the sample solution is recorded, and the result is shown in fig. 4.

The results show that: when formic acid is used, the main characteristic chromatographic peak type is better. Thus, an aqueous formic acid solution having a volume fraction of 0.1% was selected as mobile phase B.

3.4 confirmation of chromatographic conditions

In summary, the chromatographic conditions of the standard decoction feature patterns of the spina gleditsiae decoction pieces are confirmed as follows: a Thermo acclaim C18 (2.1 mm. Times.150 mm,2.2 μm) column was selected, acetonitrile-methanol (1:1, v/v) as mobile phase A, and 0.1% formic acid aqueous solution as mobile phase B, and gradient elution was performed as specified in Table 2; the flow rate was 0.35mL per minute; column temperature was 42 ℃; the sample injection amount is 2 mu L; the detection wavelength was 340nm.

TABLE 2

4 investigation of sample solution preparation method

4.1 investigation of extraction times

Taking a proper amount of standard decoction pieces of Chinese honeylocust (GT 1611181), grinding, taking about 0.5g, precisely weighing, referring to the preparation method of the sample solution under the item "2.4", respectively extracting for 1 time, 2 times, 3 times and 4 times, comparing the influence of different extraction times on the total peak area/sample weighing of the characteristic peaks of the standard decoction of Chinese honeylocust spine by the total peak area/sample weighing of 8 characteristic peaks which are temporarily determined, and selecting the optimal extraction times.

Specifically: after preparing the test solution, the sample was taken and assayed according to the chromatographic conditions identified under item "3.4". Recording the chromatograms and the peak areas of the characteristic peaks, and calculating the value of total peak area/sample weighing, wherein the chromatograms are shown in figure 5, and the inspection results of the extraction times of the standard decoction characteristic maps of the spina gleditsiae decoction pieces are shown in table 3.

TABLE 3 Table 3

The results show that: comparing the values of total peak area/sample weighing of characteristic peaks of the characteristic patterns of the sample solution processed by different extraction times, the fact that the values of total peak area/sample weighing of 8 characteristic peaks are larger when the extraction is adopted for 3 times and 4 times, no obvious difference exists between the two, the operation is simple and convenient, the reagent is saved, and the extraction times are selected to be 3 times.

4.2 ultrasonic extraction time investigation

Taking a proper amount of standard decoction pieces of Chinese honeylocust (GT 1611181), grinding, taking about 0.5g, precisely weighing, referring to the preparation method of the sample solution under the item "2.4", respectively carrying out ultrasonic extraction for 15 minutes, 30 minutes and 45 minutes, comparing the influence of different ultrasonic extraction time on the total peak area/sample weighing of the characteristic peaks of the standard decoction pieces of Chinese honeylocust by temporarily determining the total peak area/sample weighing of 8 characteristic peaks, and selecting the optimal ultrasonic extraction time.

Specifically: after preparing the test solution, the sample was taken and assayed according to the chromatographic conditions identified under item "3.4". Recording the chromatograms and the peak areas of the characteristic peaks, and calculating the value of total peak area/sample weighing, wherein the chromatograms are shown in figure 6, and the ultrasonic extraction time investigation results of the standard decoction characteristic maps of the spina gleditsiae decoction pieces are shown in table 4.

TABLE 4 Table 4

The results showed that when extracted ultrasonically for 30 minutes, the "total peak area/sample size" value of 8 characteristic peaks was large, indicating that 30 minutes was sufficient for extraction. Thus, the time of sonication was chosen to be 30 minutes.

4.3 investigation of the amount of solvent used for ultrasonic extraction

Taking a proper amount of standard decoction of Chinese honeylocust decoction pieces (GT 1611181), grinding, taking about 0.5g, precisely weighing, referring to the preparation method of the sample solution under the item "2.4", respectively adding 10mL, 15mL and 20mL of extraction solvent (methanol), comparing the influence of different ultrasonic extraction solvent dosages on the total peak area/sample amount of the characteristic peaks of the Chinese honeylocust spine standard decoction characteristic spectrum by temporarily determining the total peak area/sample amount of 8 characteristic peaks, and selecting the optimal ultrasonic extraction solvent dosage.

Specifically: after preparing the test solution, the sample was taken and assayed according to the chromatographic conditions identified under item "3.4". Recording the chromatograms and the peak areas of the characteristic peaks, and calculating the value of total peak area/sample weighing, wherein the chromatograms are shown in figure 7, and the ultrasonic extraction solvent dosage investigation results of the standard decoction characteristic maps of the spina gleditsiae decoction pieces are shown in table 5.

TABLE 5

The results show that under the condition of using three ultrasonic extraction solvents, the peak type and the separation effect of each characteristic peak are not obviously different, wherein when the using amount of the ultrasonic extraction solvents is 15mL, the value of total peak area/sample weighing of each characteristic peak is larger. Therefore, the amount of ultrasonic extraction solvent was selected to be 15mL.

4.4 investigation of extraction method

Taking a proper amount of standard decoction of spina gleditsiae (GT 1704004), grinding, taking about 0.5g, precisely weighing, referring to the preparation method of the sample solution under the item "2.4", respectively adopting two extraction modes of ultrasonic extraction and heating reflux for treatment, wherein the ultrasonic extraction (power 500W, frequency 40 kHZ) is carried out for 30 minutes, and the heating reflux (water bath temperature is 70-80 ℃) is carried out for 30 minutes. The optimal extraction mode is selected by comparing the influence of different extraction modes on the total peak area/sample weighing of the characteristic peaks of the characteristic spectrum of the spina gleditsiae standard decoction by the temporarily determined total peak area/sample weighing of 8 characteristic peaks.

Specifically: after preparing the test solution, the sample was taken and assayed according to the chromatographic conditions identified under item "3.4". Recording the chromatograms and the peak areas of the characteristic peaks, calculating the total peak area/sample weighing value, wherein the chromatograms are shown in figure 8, and the inspection results of the standard decoction characteristic patterns of the spina gleditsiae decoction pieces are shown in table 6.

TABLE 6

The results show that the peak type, the separation effect and the value of the total peak area/sample amount of each characteristic peak are not obviously different by adopting two different extraction modes, and the ultrasonic treatment is selected by considering the simplicity and convenience of operation.

4.5 investigation of extraction solvent

Taking a proper amount of standard decoction of spina gleditsiae (GT 1704004), grinding, taking about 0.5g, precisely weighing, and referring to the preparation method of the sample solution under the item "2.4", wherein methanol, a 70% methanol aqueous solution by volume fraction, a 50% methanol aqueous solution by volume fraction, ethanol and a 50% ethanol aqueous solution by volume fraction are respectively used as extraction solvents. The influence of different extraction solvents on the total peak area/sample weighing of the characteristic peaks of the spina gleditsiae standard decoction characteristic spectrum is compared through the temporarily determined total peak area/sample weighing value of 8 characteristic peaks, and the optimal extraction solvent is selected.

Specifically: after preparing the test solution, the sample was taken and assayed according to the chromatographic conditions identified under item "3.4". Recording the chromatograms and the peak areas of the characteristic peaks, and calculating the value of total peak area/sample weighing, wherein the chromatograms are shown in figure 9, and the extraction solvent investigation results of the standard decoction characteristic maps of the spina gleditsiae decoction pieces are shown in table 7.

TABLE 7

The results show that the peak types and the separation effects of the characteristic peaks are not obviously different by adopting different extraction solvents, wherein methanol is used as the extraction solvent, and the total peak area/sample weighing amount of 8 characteristic peaks is maximum. Therefore, methanol was selected as the extraction solvent.

4.6 confirmation of sample solution preparation method

In summary, the preparation method of the sample solution of the standard decoction feature map of the spina gleditsiae decoction pieces is confirmed as follows: taking a proper amount of the product, grinding, precisely weighing 0.5g, placing into a conical flask, precisely adding 15mL of methanol, performing ultrasonic treatment (with the power of 500W and the frequency of 40 kHZ) for 30 minutes, centrifuging, taking supernatant, evaporating to dryness, adding 10mL of water into residues to dissolve, adding 10mL of ammonia water for washing, transferring to a separating funnel, shaking and extracting 3 times with n-butanol, 20mL each time, merging n-butanol liquid, evaporating to dryness, adding methanol into residues to dissolve, transferring to a 5mL measuring flask, adding methanol to a scale, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the product.

5 confirmation of characteristic peaks

Taking 18 batches of spina gleditsiae decoction piece standard decoction freeze-dried powder samples, preparing a sample solution according to the preparation method of the sample solution confirmed under the item "4.6", precisely sucking 2 mu L of each of the sample solution and the reference solution respectively under the item "2.3", and carrying out sample injection measurement according to the chromatographic conditions confirmed under the item "3.4". The characteristic patterns of 18 batches of standard decoction of spina gleditsiae are identified by using Chinese medicine chromatographic fingerprint similarity evaluation software, 8 common peaks with known components, better peak type and separation degree and higher purity are selected as characteristic peaks of the standard decoction of spina gleditsiae, as shown in figure 10, the taxifolin chromatographic peaks with relatively central retention time and easily obtained reference substances are used as reference peaks S, the relative retention time of each characteristic peak and S peak is calculated, the relative retention time average value of each characteristic peak of the characteristic patterns of 18 batches of standard decoction of spina gleditsiae is calculated, and the characteristic peaks are positioned by using the relative retention time average value of each characteristic peak.

Analyzing the characteristic spectrum of the standard decoction pieces of 18 batches of spina gleditsiae, taking the peak corresponding to the taxifolin reference peak as an S peak, and calculating the relative retention time of the peak 1, the peak 2, the peak 3, the peak 5, the peak 6, the peak 7, the peak 8 and the S peak, wherein the relative retention time is within +/-10% of a specified value, and the specified value is: 0.34 (Peak 1), 0.50 (Peak 2), 0.86 (Peak 3), 1.11 (Peak 5), 1.28 (Peak 6), 1.42 (Peak 7), 1.56 (Peak 8).

6, drawing up a characteristic map

Matching the UPLC characteristic spectrum of 18 batches of spina gleditsiae decoction pieces by using a traditional Chinese medicine chromatographic fingerprint similarity evaluation system, generating a comparison spectrum according to an average method, and establishing a spina gleditsiae decoction piece standard decoction comparison characteristic spectrum which has 8 characteristic peaks in total and corresponds to 8 characteristic peaks in a spina gleditsiae comparison medicinal material reference chromatograph (the spina gleditsiae comparison medicinal material characteristic spectrum is shown in figure 12). Calculating the relative retention time of peak 1, peak 2, peak 3, peak 5, peak 6, peak 7, peak 8 and S peak by taking the peak corresponding to the taxifolin reference peak as the S peak, wherein the relative retention time is within +/-10% of a specified value, and the specified value is: 0.34 (Peak 1), 0.50 (Peak 2), 0.86 (Peak 3), 1.11 (Peak 5), 1.28 (Peak 6), 1.42 (Peak 7), 1.56 (Peak 8).

High resolution mass spectrometry validation of 7 characteristic peaks

7.1 conditions for ultra high Performance liquid chromatography Mass Spectrometry

The chromatographic conditions of the ultra-high performance liquid chromatography were as described in item "3.4".

The mass spectrum conditions are shown in table 8.

TABLE 8

7.2 preparation of sample solutions

Taking standard decoction of spina gleditsiae decoction pieces (GT 1702118), and preparing a sample solution according to the preparation method of the sample solution confirmed under the item of 4.6.

7.3 test sample measurement

2 mu L of the sample solution is precisely sucked, injected into an ultra-high performance liquid chromatography-mass spectrometer, and detected by adopting the ultra-high performance liquid chromatography conditions and the mass spectrometry conditions, and the total ion flow diagram and the ultraviolet absorption chromatogram of the sample solution are shown in figure 13.

7.4 analysis of results

Through mass spectrum accurate molecular weight and fragment ion comparison analysis and matching with a standard database of the Siemens femzVault, 6 components of protocatechuic acid (peak 1), scopolamine (peak 2), scopoletin (peak 3), taxifolin (peak 4), vitexin (peak 6) and isovitexin (peak 7) in a standard decoction characteristic map of the spina gleditsiae decoction piece are confirmed, and compound information is shown in table 9.

TABLE 9

8 identification of reference substance with characteristic peak

8.1 chromatographic conditions were as under "3.4".

8.2 preparation of control solution

1.049mg of protocatechuic aldehyde reference substance, 2.159mg of scopoletin reference substance, 2.142mg of scopoletin, 1.023mg of taxifolin, 1.124mg of vitexin and 1.205mg of isovitexin are precisely weighed, placed in 25mL measuring flask respectively, and methanol is added to prepare reference substance solutions containing 41.792 mug of protocatechuic aldehyde, 84.633 mug of scopoletin, 85.423 mug of scopoletin, 40.470 mug of taxifolin, 42.667 mug of vitexin and 47.236 mug of isovitexin.

8.3 preparation of sample solutions

8.4 measurement

2 mu l of each of the control solution and the sample solution is precisely sucked and measured by ultra-high performance liquid chromatography, and the chromatogram is shown in figure 14.

The results show that the chromatogram of the test solution shows the same chromatographic peak at the retention time corresponding to the chromatogram of the control, thereby confirming that characteristic peak 1 is catechin, characteristic peak 2 is scopoletin, characteristic peak 3 is scopoletin, characteristic peak 4 is taxifolin, characteristic peak 6 is vitexin, and characteristic peak 7 is isovitexin.

9 methodological verification

9.1 precision investigation

Taking standard decoction of spina Gleditsiae (batch number: GT 1710002), grinding, taking about 0.5g, precisely weighing, preparing sample solution according to the sample solution preparation method confirmed under item "4.6", sampling and measuring according to the chromatographic condition confirmed under item "3.4", repeating sampling for 6 times, and analyzing. The relative retention time and relative peak area of each characteristic peak and S peak were calculated using douglas fir peak as reference peak S, and RSD values were calculated, and the results are shown in tables 10 and 11. Wherein, table 10 is the precision investigation result (relative retention time) of the standard decoction feature spectrum of the spina gleditsiae decoction pieces, and table 11 is the precision investigation result (relative peak area) of the standard decoction feature spectrum of the spina gleditsiae decoction pieces.

Table 10

TABLE 11

The result shows that the same sample solution is continuously sampled for 6 times, the taxifolin chromatographic peak is taken as a reference peak S, the relative retention time RSD value of each characteristic peak and the S peak is within the range of 0.13-0.79 percent, the relative peak area RSD value is within the range of 1.34-2.70 percent, and the relative retention time RSD value is less than 3.0 percent, which indicates that the instrument precision is good.

9.2 repeatability investigation

Taking standard decoction of spina Gleditsiae (batch number: GT 1710002), grinding, taking about 0.5g, precisely weighing, preparing 6 parts of sample solution according to the sample solution preparation method confirmed under the item "4.6", and sampling and measuring according to the chromatographic condition confirmed under the item "3.4". The relative retention time and relative peak area of each characteristic peak and S peak were calculated using douglas fir peak as reference peak S, and RSD values were calculated, and the results are shown in tables 12 and 13. Wherein, table 12 is the result of the repeated investigation of the characteristic spectrum of the standard decoction of the spina gleditsiae decoction pieces (relative retention time), and table 13 is the result of the repeated investigation of the characteristic spectrum of the standard decoction of the spina gleditsiae decoction pieces (relative peak area).

Table 12

/>

TABLE 13

The result shows that the same sample is repeatedly measured for 6 times, the taxifolin chromatographic peak is taken as a reference peak S, the relative retention time RSD value of each characteristic peak and the S peak is in the range of 0.01-0.03 percent, the relative peak area RSD value is in the range of 2.42-4.94 percent, and the relative retention time RSD value is less than 5.0 percent, which shows that the method has good repeatability.

9.3 stability investigation

Taking standard decoction of spina Gleditsiae (batch number: GT 1710002), grinding, taking about 0.5g, precisely weighing, preparing sample solution according to the sample solution preparation method confirmed under item "4.6", and respectively sampling and measuring at 0,2,4,8, 12 and 24 hours according to the chromatographic conditions confirmed under item "3.4". The relative retention time and relative peak area of each characteristic peak and S peak were calculated using douglas fir peak as reference peak S, and RSD values were calculated, and the results are shown in tables 14 and 15. Wherein, table 14 is the stability inspection result (relative retention time) of the standard decoction feature spectrum of the spina gleditsiae decoction pieces, and table 15 is the stability inspection result (relative peak area) of the standard decoction feature spectrum of the spina gleditsiae decoction pieces.

TABLE 14

TABLE 15

The results show that the same sample solution is analyzed in 0,2,4,8, 12 and 24 hours respectively, the taxifolin peak is taken as a reference peak S, the relative retention time RSD value of each characteristic peak and the S peak is in the range of 0.01-0.05%, the relative peak area RSD value is in the range of 0.33-0.65%, and the relative stability of the sample solution is good in 24 hours.

9.4 intermediate precision investigation

Other analysts in the project group operate under different dates and different chromatographs, take a proper amount of standard decoction pieces of gleditsia sinensis lam (batch number: GT 1710002), grind, take about 0.5g, precisely measure, prepare 6 parts of sample solution according to the sample solution preparation method confirmed under the item of '4.6', sample and measure according to the chromatographic conditions confirmed under the item of '3.4', calculate the relative retention time and relative peak area of each characteristic peak and S peak by taking the taxifolin peak as a reference peak S, calculate RSD value, and see the results in tables 16 and 17. Wherein, table 16 is the intermediate precision investigation result (relative retention time) of the standard decoction feature spectrum of the spina gleditsiae decoction pieces, and table 17 is the intermediate precision investigation result (relative peak area) of the standard decoction feature spectrum of the spina gleditsiae decoction pieces.

Table 16

TABLE 17

The results show that the relative retention time RSD value of each characteristic peak and S peak is in the range of 0.01-0.04%, and the relative peak area RSD value is in the range of 1.74-4.86% and is less than 5.0% by operating on different instruments at different times by different analysts and repeating the measurement for 6 times on the same batch of samples. The RSD values of the relative retention time and the repeatability test 6 data are in the range of 0.05% -0.55%, the relative peak area and the RSD values of the repeatability test 6 data are in the range of 4.59% -7.34%, which shows that the relative retention time of each characteristic peak is good in intermediate precision on different instruments, and the relative peak area RSD is larger, so that the relative peak area is not stipulated at all.

9.5 durability inspection

9.5.1 durability inspection of different chromatographic columns

Taking standard decoction of spina Gleditsiae (batch number: GT 1710002), grinding, taking about 0.5g, precisely weighing, preparing sample solution according to the sample solution preparation method confirmed under item "4.6", preparing sample solution, wherein the chromatographic column is a Thermo Acclaim C18 chromatographic column (2.1 mm×150mm,2.2 μm), a Waters HSS T3C 18 (2.1 mm×150mm,1.8 μm), an Agilent SB C18 chromatographic column (2.1 mm×150mm,1.8 μm) respectively, and other chromatographic conditions are measured by sample injection according to the chromatographic conditions confirmed under item "3.4". The relative retention time and relative peak area of each characteristic peak and the S peak are calculated by taking taxifolin as a reference peak S, and the RSD value is calculated. The results are shown in Table 18 and Table 19. Wherein, table 18 is the results (relative retention time) of investigation of the durability of the different chromatographic columns of the standard decoction feature spectrum of the spina gleditsiae decoction pieces, and table 19 is the results (relative peak area) of investigation of the durability of the different chromatographic columns of the standard decoction feature spectrum of the spina gleditsiae decoction pieces.

TABLE 18

TABLE 19

The results show that under different chromatographic column brands, the relative retention time RSD value of each characteristic peak and S peak is in the range of 1.15% -3.53%, the relative peak area RSD value is in the range of 1.20% -4.8%, and both are smaller than 5%, which indicates that the influence of the chromatographic column of different brands on the relative retention time and relative peak area of each characteristic peak is smaller, and the durability of different chromatographic columns is good.

9.5.2 investigation of durability at different column temperatures

Taking standard decoction of spina Gleditsiae (batch number: GT 1710002), grinding, taking about 0.5g, precisely weighing, preparing sample solution according to the sample solution preparation method confirmed under item "4.6", preparing sample solution, and performing sample injection measurement according to chromatographic conditions confirmed under item "3.4" except column temperature of 40 ℃, 42 ℃ and 44 ℃ respectively. The relative retention time and relative peak area of each characteristic peak and the S peak are calculated by taking taxifolin as a reference peak S, and the RSD value is calculated. The results are shown in Table 20 and Table 21. Wherein, table 20 is the results (relative retention time) of investigation of different column temperatures of the standard decoction feature spectrum of the spina gleditsiae decoction pieces, and table 21 is the results (relative peak area) of investigation of different column temperatures of the standard decoction feature spectrum of the spina gleditsiae decoction pieces.

Table 20

Table 21

The results show that at different column temperatures, the relative retention time RSD value of each characteristic peak and the S peak is in the range of 0.22% -1.76%, the relative peak area RSD value is less than 3%, and the relative peak area RSD value is in the range of 1.68% -5.31%, and is less than 8.0%, which shows that when the column temperature is +/-2 ℃, the change of the column temperature has less influence on the relative retention time of each characteristic peak and has a certain influence on the relative peak area of each characteristic peak.

Investigation of durability of 9.5.3 different flow rates

Taking standard decoction of spina gleditsiae decoction pieces (batch number: GT 1710002), grinding, taking about 0.5g, precisely weighing, preparing a sample solution according to the sample solution preparation method confirmed under the item "4.6", and carrying out sample injection measurement on other chromatographic conditions according to the chromatographic conditions confirmed under the item "3.4" except for the flow rates of 0.34mL/min, 0.35mL/min and 0.36mL/min respectively. The relative retention time and relative peak area of each characteristic peak and the S peak are calculated by taking taxifolin as a reference peak S, and the RSD value is calculated. The results are shown in Table 22 and Table 23. Wherein, table 22 is the results (relative retention time) of investigation of the different chromatographic column spectra of the standard decoction feature spectrum of the spina gleditsiae decoction pieces, and table 23 is the results (relative peak area) of investigation of the different chromatographic column spectra of the standard decoction feature spectrum of the spina gleditsiae decoction pieces.

Table 22

Table 23

The results show that at different flow rates, the relative retention time RSD value of each characteristic peak and the S peak is in the range of 0.03% -0.66%, and the relative peak area RSD value is in the range of 1.93% -2.74%, which shows that when the flow rate is +/-0.01 mL/min, the change of the flow rate has less influence on the relative retention time and the relative peak area.

According to the method, by combining an ultra-high performance liquid chromatography-mass spectrometry (UPLC MS/MS), the constructed UPLC characteristic spectrum of the standard decoction of the spina gleditsiae decoction pieces is characterized by simultaneously characterizing flavone, phenolic acid and coumarin components in the standard decoction of the spina gleditsiae decoction pieces, the characteristic spectrum method has better separation degree of each characteristic peak, and better method precision, repeatability and durability, can reflect the chemical component characteristics of the standard decoction of the spina gleditsiae decoction pieces more comprehensively, and provides an important reference for establishing the quality standard of the standard decoction of the spina gleditsiae decoction pieces.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. The method for constructing the characteristic spectrum of the standard decoction of the Chinese honeylocust spine traditional Chinese medicine decoction pieces is characterized by comprising the following steps:

mixing standard decoction of Chinese medicinal decoction pieces of spina Gleditsiae with extraction solvent, extracting, dissolving the obtained extract, and preparing test solution by dissolving the obtained extract, wherein the extraction solvent is methanol, aqueous solution of methanol, ethanol or 50% ethanol aqueous solution;

the mobile phase comprises a mobile phase A and a mobile phase B, wherein the mobile phase A comprises acetonitrile and methanol, the volume ratio of the acetonitrile to the methanol is 1:1, the mobile phase B comprises an aqueous solution of formic acid, the volume fraction of the formic acid in the aqueous solution of the formic acid is 0.02-0.2%, and the elution mode is gradient elution;

the gradient elution specifically comprises the following steps:

50-55 min, wherein the volume fraction of the mobile phase A is kept at 90%, and the volume fraction of the mobile phase B is kept at 10%;

the chromatographic conditions of the ultra performance liquid chromatography further include: the stationary phase is a chromatographic column with octadecylsilane chemically bonded silica as filler.

2. The method for constructing a characteristic spectrum of a standard decoction of Chinese honeylocust spine decoction pieces of Chinese medicine according to claim 1, wherein the chromatographic conditions of the ultra performance liquid chromatography further comprise: the flow rate is 0.34-0.36 mL/min, and/or the column temperature is 40-44 ℃, and/or the detection wavelength is 320-360nm.

3. The method for constructing a characteristic spectrum of a standard decoction of Chinese honeylocust spine decoction pieces according to claim 2, wherein the flow rate is 0.35mL per minute, and/or the column temperature is 42 ℃, and/or the detection wavelength is 340nm.

4. The method for constructing a characteristic spectrum of a standard decoction of Chinese honeylocust spine decoction pieces of Chinese traditional medicine according to any one of claims 1-2, wherein the extraction treatment is ultrasonic treatment or heating reflux treatment.

5. The method for constructing a characteristic spectrum of a standard decoction of Chinese honeylocust spine decoction pieces of Chinese medicine of claim 4, wherein the ultrasonic treatment time is not less than 25min.

6. The method for constructing a characteristic spectrum of a standard decoction of Chinese honeylocust spine decoction pieces of Chinese medicine according to claim 4, wherein the extraction treatment is ultrasonic treatment, and the dosage of the extraction solvent is as follows: every 0.5g of the spina gleditsiae traditional Chinese medicine decoction piece standard decoction corresponds to 10-20 mL of the extraction solvent.

7. The method for constructing a characteristic spectrum of a standard decoction of Chinese honeylocust spine decoction pieces of Chinese medicine according to any one of claims 1-2, 5 and 6, wherein the number of times of extraction treatment is not less than 2.