CN114935612A

CN114935612A - Method for constructing body pain stasis-removing decoction characteristic spectrum

Info

Publication number: CN114935612A
Application number: CN202210498780.1A
Authority: CN
Inventors: 刘艳梅; 朱德全; 蔡盛康; 彭致铖; 刘金金; 陶晨璐; 范倩; 陈向东; 孙冬梅; 罗文汇; 魏梅; 叶梅霞
Original assignee: Guangdong Yifang Pharmaceutical Co Ltd
Current assignee: Guangdong Yifang Pharmaceutical Co Ltd
Priority date: 2022-05-09
Filing date: 2022-05-09
Publication date: 2022-08-23
Anticipated expiration: 2042-05-09
Also published as: CN114935612B

Abstract

The invention discloses a method for constructing a body pain stasis-removing decoction characteristic spectrum, and relates to the technical field of traditional Chinese medicine quality analysis and detection. The method specifically comprises the following steps: dissolving or extracting tryptophan, loganin, gentiopicrin, hydroxysafflor yellow A, ferulic acid, liquiritin, apioside liquiritin, glycyrrhizic acid, and Notopterygii rhizoma alcohol with solvent to obtain reference solution; extracting the body pain stasis-removing decoction preparation with an extraction solvent to obtain a test solution; and (3) testing by adopting a liquid chromatograph, specifically, carrying out gradient elution by taking acetonitrile as a mobile phase A and a phosphoric acid aqueous solution as a mobile phase B, and constructing a characteristic map of the body pain stasis removing decoction. The method can provide a data basis for the quality control of the body pain stasis removing soup, and effectively ensure the stability and controllability of the quality of the body pain stasis removing soup product.

Description

Construction method of body pain stasis-removing decoction characteristic spectrum

Technical Field

The invention relates to the technical field of traditional Chinese medicine quality analysis and detection, in particular to a construction method of a body pain stasis-removing decoction characteristic spectrum.

Background

The body pain stasis-removing soup is prepared from the lower volume of the clinical and clinical evidence works of various departments of traditional Chinese medicine written by the Wang Qing and ren of famous Chinese medical scientists of the Qing Dynasty, and the original text is recorded as follows: general name of arthralgia is arthralgia syndrome, which is all the symptoms of shoulder pain, arm pain, lumbago, leg pain or general body pain. It is obvious that the disease is affected by wind-cold, and the disease is not cured by warm-heat dispersing medicine; when damp-heat is known, damp-heat-clearing herbs are used to reduce pathogenic fire. Long-term muscle wasting is due to yin deficiency, which is not effective when yin-nourishing herbs are used. So far, the cloud: the disease is in the skin and pulse, so the disease is easy to do; if the disease is in the muscles and bones, the effect is difficult to be obtained. There is pain in the skin due to wind, cold, dampness and heat. Entering the trachea, pain will flow away; entering blood vessels, pain does not move. For example, weakness is caused by disease, but not by deficiency. … … the ancient recipe is too many to be effective for the treatment of pain and stasis. The Wangqingren considers that the existing treatment of the arthralgia syndrome is mostly treated from etiology and pathogenesis, namely wind cold, damp heat and yin deficiency, the heat is radiated when the patient is on a smaller side, the dampness is removed, the fire is reduced or the deficiency is nourished and supplemented, and the medicine is mostly used at one end, and the medicine is mostly used to the ineffective end, so the pain-relieving and stasis-removing decoction for the body is self-prepared. The efficacy of the recipe is recorded by ancient medical records as activating blood and promoting qi, removing blood stasis and dredging collaterals, and relieving pain, and is mainly used for treating the syndrome of blood stasis and blood obstruction in channels and collaterals, such as shoulder pain, arm pain, lumbago, leg pain, or general pain, like acupuncture, which is not cured for a long time. The body pain stasis-removing decoction is widely applied to treating various orthopedic diseases such as lumbar intervertebral disc protrusion, knee osteoarthritis, rheumatism cold arthralgia, fracture and postoperative complications, acute lumbar sprain and the like, and is also applied to diseases such as thrombophlebitis, soft tissue injury, malignant tumor bone metastasis pain, diabetic complications, neuritis and the like.

In recent years, in order to clarify the material basis of the body pain stasis-eliminating decoction for treating diseases and guarantee the effectiveness and safety of clinical medication, many scholars study the chemical components and active ingredients in the body pain stasis-eliminating decoction in the aspects of pharmacology, pharmacodynamics, pharmacokinetics and the like, and continuously improve the quality standard thereof, thereby laying the foundation for the development of the classical famous prescription. At present, the research on the body pain stasis-removing decoction mainly focuses on the research aspect of pharmacology, and the research on the material basis, the extraction process, the content determination of multi-index components and the characteristic spectrum of the decoction is less, and the systematic property is lacked. And the research on how to measure the consistency between the mass production preparation and the quality of the traditional decoction is less.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for constructing a body pain stasis-removing soup characteristic spectrum, which can provide a data basis for the control of the mass production quality of the body pain stasis-removing soup and ensure the stability and controllability of the quality of a body pain stasis-removing soup product.

In order to solve the technical problems, the invention provides a method for constructing a body pain stasis-removing decoction characteristic spectrum, which comprises the following steps:

(1) dissolving or extracting tryptophan, loganin, gentiopicrin, hydroxysafflor yellow A, ferulic acid, liquiritin, apioside liquiritin, glycyrrhizic acid, and Notopterygii rhizoma alcohol with solvent to obtain reference solution;

(2) extracting the body pain stasis-removing decoction preparation with an extraction solvent to obtain a test solution;

(3) and injecting preset amounts of reference substance solution and test solution into a liquid chromatograph, wherein the liquid chromatograph is subjected to gradient elution by using octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and phosphoric acid aqueous solution as a mobile phase B to construct a characteristic map of the body pain stasis-removing soup.

As a modification of the above technical means, in the step (3), gradient elution is carried out according to the following procedure:

0-5 min, wherein the mobile phase A is 6% and the mobile phase B is 94%;

5-12 min, the mobile phase A is 6% → 10%, and the mobile phase B is 94% → 90%;

12-22 min, wherein the mobile phase A is 10% → 12%, and the mobile phase B is 90% → 88%;

22-24 min, wherein the mobile phase A is 12% → 16%, and the mobile phase B is 88% → 84%;

24-36 min, wherein the mobile phase A is 16% → 22%, and the mobile phase B is 84% → 78%;

36-46 min, wherein the mobile phase A is 22% → 32%, and the mobile phase B is 78% → 68%;

46-50 min, wherein the content of the mobile phase A is 32% → 50%, and the content of the mobile phase B is 68% → 50%;

50-58 min, wherein the mobile phase A is 50% → 65%, and the mobile phase B is 50% → 35%;

58-68 min, the mobile phase A is 65% → 78%, and the mobile phase B is 35% → 22%.

As an improvement of the technical scheme, in the step (3), 0.5-1.5 μ L of each of the reference solution and the sample solution is respectively absorbed and injected into a liquid chromatograph for detection, wherein the liquid chromatograph takes octadecylsilane chemically bonded silica as a filling agent, the column length of the liquid chromatograph is 150mm, the inner diameter of the liquid chromatograph is 2.1mm, the particle size of the liquid chromatograph is 1.6 μm, and the column temperature is 25-35 ℃; the liquid chromatograph takes acetonitrile as a mobile phase A, takes 0.1-0.2% phosphoric acid aqueous solution as a mobile phase B, has a flow rate of 0.25-0.35 mL/min, and has a detection wavelength of 220-254 nm.

As an improvement of the technical scheme, in the method for constructing the body pain and stasis removing soup characteristic map, in the step (3), 1 μ L of each of a reference solution and a test solution is respectively absorbed and injected into a liquid chromatograph for detection, wherein the liquid chromatograph takes octadecylsilane chemically bonded silica as a filling agent, the column length of the liquid chromatograph is 150mm, the inner diameter of the liquid chromatograph is 2.1mm, the particle size of the liquid chromatograph is 1.6 μm, and the column temperature is 30 ℃; the liquid chromatograph takes acetonitrile as a mobile phase A, takes 0.2% phosphoric acid water solution as a mobile phase B, has the flow rate of 0.3mL/min, and has the detection wavelength of 235 nm.

As an improvement of the above technical solution, in the step (1), the preparation method of the reference solution comprises: tryptophan, loganin, gentiopicrin, hydroxysafflor yellow A, ferulic acid, liquiritin, apioside liquiritin, glycyrrhizic acid and notopterygium alcohol are respectively weighed, and methanol is added to prepare a mixed solution containing 15 mu g of tryptophan, 40 mu g of loganin, 60 mu g of gentiopicrin, 60 mu g of hydroxysafflor yellow A, 20 mu g of ferulic acid, 30 mu g of liquiritin, 70 mu g of apioside, 40 mu g of glycyrrhizic acid and 20 mu g of notopterygium alcohol per 1mL, so as to obtain a reference substance solution.

As an improvement of the technical scheme, in the step (2), the extraction solvent is 50-100% of methanol, the extraction time is 15-45 min, and the extraction mode is ultrasonic extraction and/or reflux extraction.

As an improvement of the technical scheme, the step (2) comprises the following steps:

taking 0.2-0.5 g of the shentongyu decoction freeze-dried powder, placing the powder into a conical flask with a plug, adding 25-30 mL of 70% methanol, weighing, carrying out ultrasonic treatment for 30 minutes, taking out, cooling, weighing again, complementing the lost weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the test solution.

As an improvement of the technical scheme, the characteristic spectrum of the body pain stasis removing decoction comprises 15 characteristic peaks; wherein, peak 1 is a loganin acid peak, peak 2 is a tryptophan peak, peak 4 is a gentiopicroside peak, peak 5 is a hydroxysafflor yellow A peak, peak 7 is a ferulic acid peak, peak 8 is a liquiritin peak, peak 9 is a liquiritin peak in apiary pond, peak 13 is a glycyrrhizic acid peak, and peak 14 is a notopterygium alcohol peak;

relative retention times for peaks 7 as reference S peaks are: peak 1 was 0.321, peak 2 was 0.372, peak 3 was 0.624, peak 4 was 0.662, peak 5 was 0.774, peak 6 was 0.941, peak 7 was 1.000, peak 8 was 1.277, peak 9 was 1.333, peak 10 was 1.510, peak 11 was 1.616, peak 12 was 1.765, peak 13 was 2.443, peak 14 was 2.610, and peak 15 was 2.733.

As an improvement of the above technical solution, the method further comprises:

(4) and (3) identifying the characteristic peak chemical components of the characteristic spectrum of the body pain stasis removing decoction by using a UPLC-MS identification method.

As an improvement of the technical scheme, the establishment method of the UPLC-MS identification method comprises the following steps:

(1) dissolving neochlorogenic acid, L-tryptophan, loganin, D-amygdalin, swertiamarin, hydroxysafflor yellow A, cryptochlorogenic acid, ferulic acid, liquiritin, gentiopicrin, swertiamarin, liquiritigenin, rutin, astragalin, naringenin, apioside liquiritin, senkyunolide I, glycyrrhizic acid, ammonium glycyrrhizinate, glycyrrhetinic acid and ginsenoside Ro in 70% methanol to obtain a first UPLC-MS control solution; dissolving reference substances including umbelliferone, decursin, imperatorin, isoimperatorin, ligustilide, notopterygium alcohol and alpha-cyperone in methanol to obtain a second UPLC-MS reference substance solution;

(2) extracting the body pain stasis-removing decoction preparation with an extraction solvent to obtain a UPLC-MS test sample solution;

(3) injecting the first UPLC-MS reference substance solution, the second UPLC-MS reference substance solution and the UPLC-MS test solution into an ultra-high performance liquid chromatography-quadrupole-electrostatic orbit trap-high resolution mass spectrometer, and establishing chromatographic conditions and mass spectrum conditions of the UPLC-MS identification method;

(4) calculating the element composition and molecular formula of the unknown compound by using a workstation; and calculating and matching the retention time of the chromatographic peak, the excimer ion peak and the characteristic MS/MS fragment ion peak information with a database, and identifying the chemical components of the characteristic peak of the characteristic spectrum.

As an improvement of the technical scheme, in the UPLC-MS identification method, the preparation method of the UPLC-MS test sample comprises the following steps: taking 0.2-0.5 g of the pain-relieving stasis-removing decoction freeze-dried powder, placing the powder into a conical flask with a plug, adding 25-30 mL of 70% methanol, weighing, carrying out ultrasonic treatment for 15-45 minutes, centrifuging for 5 minutes at 8000r/min, taking supernate, filtering with a 0.22 mu m filter membrane, and collecting filtrate.

As an improvement of the technical scheme, in the UPLC-MS identification method, the chromatographic conditions are as follows: the chromatographic column adopts an octadecylsilane chemically bonded silica chromatographic column, the length of the chromatographic column is 150mm, the inner diameter of the chromatographic column is 2.1mm, the particle size of the chromatographic column is 1.8 mu m, acetonitrile is used as a mobile phase A, 0.05-0.1% formic acid aqueous solution is used as a mobile phase B, and the column temperature is 25-35 ℃; the flow rate is 0.28-0.32 mL/min; the detection wavelength is 210-250 nm, and the sample injection amount is 0.5-1.5 mu L; gradient elution was performed according to the following procedure:

0-3 min, wherein the mobile phase A is 0% and the mobile phase B is 100%;

3-7 min, wherein the content of mobile phase A is from 0% → 4%, and the content of mobile phase B is from 100% → 96%;

7-15 min, wherein the content of mobile phase A is from 4% → 10%, and the content of mobile phase B is from 96% → 90%;

15-25 min, wherein the mobile phase A is 10% → 12%, and the mobile phase B is 90% → 88%;

25-30 min, wherein the mobile phase A is 12% → 15%, and the mobile phase B is 88% → 85%;

30-40 min, wherein the mobile phase A is 15% → 22%, and the mobile phase B is 85% → 78%;

40-50 min, the mobile phase A is 22% → 32%, and the mobile phase B is 78% → 68%;

50-55 min, wherein the mobile phase A is 32% → 50%, and the mobile phase B is 68% → 50%;

55-60 min, the mobile phase A is 50% → 65%, and the mobile phase B is 50% → 35%;

60-70 min, the mobile phase A is 65% → 78%, and the mobile phase B is 35% → 22%.

As an improvement of the above technical solution, in the UPLC-MS identification method, the mass spectrometry conditions are: the ESI ion source is in a positive and negative ion monitoring mode; the cracking voltage is 3.80kV, the auxiliary gas flow is 10mL/min, the temperature of the ion transmission tube is 320 ℃, the temperature of the auxiliary gas is 350 ℃, and the temperature of the ion transmission tube is sweptThe drawing mode is Full MS/dd-MS ² With Full MS resolution of 70000, dd-MS ² Resolution is 17500; the mass scanning range m/z is 100-1500; in the MS/MS mode, the collision energy in the positive and negative ion modes is 20eV and 40eV respectively, and the mass accuracy is corrected by taking leucine enkephalin as an internal standard.

As an improvement of the technical scheme, the body pain stasis removing soup comprises the following components in parts by weight:

3.73 parts of gentiana macrophylla, 7.46 parts of ligusticum wallichii, 11.19 parts of peach kernel, 11.19 parts of safflower, 7.46 parts of liquorice, 3.73 parts of notopterygium root, 7.46 parts of myrrh, 11.19 parts of angelica, 7.46 parts of vinegar trogopterus dung, 3.73 parts of rhizoma cyperi, 11.19 parts of achyranthes bidentata and 7.46 parts of earthworm.

As an improvement of the technical scheme, the preparation method of the body pain stasis removing decoction comprises the following steps:

taking gentiana macrophylla, ligusticum wallichii, peach kernel, safflower, liquorice, notopterygium root, myrrh, angelica, vinegar trogopterus dung, rhizoma cyperi, achyranthes and earthworm, adding 6-10 times of water of the total weight of the raw materials, soaking for 30-60 min, boiling with strong fire, keeping slight boiling for 20-40 min with slow fire, and filtering; adding water which is 5-8 times of the total weight of the raw material medicines into the obtained filter residue, decocting for 20-30 min, and filtering; mixing the two decoctions.

taking gentiana macrophylla, ligusticum wallichii, peach kernel, safflower, liquorice, notopterygium root, myrrh, angelica sinensis, vinegar trogopterus dung, rhizoma cyperi, achyranthes and earthworm, adding water which is 6-8 times of the total weight of the raw materials, and slightly boiling with slow fire until the filtrate is 3-4 times of the weight of the raw materials.

The implementation of the invention has the following beneficial effects:

the invention establishes a body pain stasis-removing soup characteristic map. The UPLC-MS is adopted to confirm basic research of related substances, 80 chemical components are identified, 9 characteristic peaks are calibrated through a reference substance, the 9 characteristic peaks belong to 9 medicinal flavors, the characteristics of the chemical components of the body pain stasis removing decoction are fully displayed, the characteristic peak information content is rich, the method is stable, accurate and reliable, and the quality monitoring of the characteristic components of a plurality of medicinal flavors can be realized.

Drawings

FIG. 1 is a characteristic spectrum of the pain-relieving and stasis-removing decoction of the invention measured by gradient 1;

FIG. 2 is a characteristic diagram of the pain-relieving and stasis-removing decoction of the invention measured by gradient 2;

FIG. 3 is a characteristic diagram of the pain-relieving and stasis-removing decoction of the invention measured by gradient 3;

FIG. 4 is a characteristic spectrum of the pain-relieving stasis-removing decoction of the invention measured by a Waters CORTECS T3 column;

FIG. 5 is a characteristic spectrum of the pain-relieving stasis-removing decoction of the invention measured by a Waters BEH C18 column;

FIG. 6 is a characteristic spectrum of the pain-relieving and stasis-removing decoction for body pain of the present invention measured by a Waters HSS T3 column;

FIG. 7 is a characteristic spectrum of the pain-relieving and stasis-removing decoction of the present invention measured by different wavelengths;

FIG. 8 is a characteristic diagram of the body pain stasis-eliminating decoction of the present invention measured with acetonitrile-0.1% acetic acid as the mobile phase;

FIG. 9 is a characteristic diagram of the pain-relieving and stasis-removing decoction of the invention measured by using acetonitrile-0.1% formic acid as a mobile phase;

FIG. 10 is a characteristic diagram of the pain-relieving and stasis-removing decoction of the present invention measured using acetonitrile-0.1% phosphoric acid as the mobile phase;

FIG. 11 is a characteristic diagram of the pain-relieving and stasis-removing decoction of the invention measured by acetonitrile-0.2% phosphoric acid as a mobile phase;

FIG. 12 is a characteristic diagram of the body pain stasis-removing decoction of the present invention measured by different flow rates;

FIG. 13 is a characteristic diagram of the pain-relieving and stasis-removing decoction of the present invention measured by different column temperatures;

FIG. 14 is a characteristic map of a body pain stasis-eliminating decoction sample, a gentiana macrophylla negative sample and a gentiana macrophylla single medicine in the research of the body pain stasis-eliminating decoction characteristic map specificity of the invention; wherein, peak 1 is a loganin acid peak, peak 2 is a tryptophan peak, peak 4 is a gentiopicroside peak, peak 5 is a hydroxysafflor yellow A peak, peak 7 is a ferulic acid peak, peak 8 is a liquiritin peak, peak 9 is an apiose liquiritin peak, peak 13 is a glycyrrhizic acid peak, and peak 14 is a notopterygium alcohol peak;

FIG. 15 is a characteristic spectrum of a body pain stasis-eliminating decoction test sample, a licorice negative sample and a licorice single medicine in the body pain stasis-eliminating decoction characteristic spectrum specificity investigation of the invention; wherein, peak 1 is a loganin acid peak, peak 2 is a tryptophan peak, peak 4 is a gentiopicroside peak, peak 5 is a hydroxysafflor yellow A peak, peak 7 is a ferulic acid peak, peak 8 is a liquiritin peak, peak 9 is an apiose liquiritin peak, peak 13 is a glycyrrhizic acid peak, and peak 14 is a notopterygium alcohol peak;

FIG. 16 is a characteristic diagram of a body pain stasis-removing decoction test sample, a safflower negative sample and a safflower single medicine in the body pain stasis-removing decoction characteristic diagram special attribute investigation of the invention; wherein, peak 1 is a loganin acid peak, peak 2 is a tryptophan peak, peak 4 is a gentiopicroside peak, peak 5 is a hydroxysafflor yellow A peak, peak 7 is a ferulic acid peak, peak 8 is a liquiritin peak, peak 9 is an apiose liquiritin peak, peak 13 is a glycyrrhizic acid peak, and peak 14 is a notopterygium alcohol peak;

FIG. 17 is a characteristic spectrum of a body pain stasis-removing decoction sample, a vinegar trogopterus dung negative sample and a vinegar trogopterus dung single medicine in the body pain stasis-removing decoction characteristic spectrum special attribute investigation of the invention; wherein, peak 1 is a loganin acid peak, peak 2 is a tryptophan peak, peak 4 is a gentiopicroside peak, peak 5 is a hydroxysafflor yellow A peak, peak 7 is a ferulic acid peak, peak 8 is a liquiritin peak, peak 9 is an apiose liquiritin peak, peak 13 is a glycyrrhizic acid peak, and peak 14 is a notopterygium alcohol peak;

FIG. 18 is a characteristic diagram of a body pain stasis-eliminating decoction test sample, a peach kernel negative sample and a peach kernel single medicine in the body pain stasis-eliminating decoction characteristic diagram special attribute investigation of the invention; wherein, peak 1 is a loganin acid peak, peak 2 is a tryptophan peak, peak 4 is a gentiopicroside peak, peak 5 is a hydroxysafflor yellow A peak, peak 7 is a ferulic acid peak, peak 8 is a liquiritin peak, peak 9 is an apiose liquiritin peak, peak 13 is a glycyrrhizic acid peak, and peak 14 is a notopterygium alcohol peak;

FIG. 19 is a characteristic diagram of a body pain stasis-eliminating decoction sample, a rhizoma Ligustici Chuanxiong negative sample and a rhizoma Ligustici Chuanxiong single medicine in the body pain stasis-eliminating decoction characteristic diagram special attribute investigation of the invention; wherein, the peak 1 is a loganin peak, the peak 2 is a tryptophan peak, the peak 4 is a gentiopicroside peak, the peak 5 is a hydroxysafflor yellow A peak, the peak 7 is a ferulic acid peak, the peak 8 is a liquiritin peak, the peak 9 is an apiose liquiritin peak, the peak 13 is a glycyrrhizic acid peak, and the peak 14 is a notopterygium alcohol peak;

FIG. 20 is a characteristic diagram of a body pain stasis-eliminating decoction sample, an angelica sinensis negative sample and an angelica sinensis single medicine in the body pain stasis-eliminating decoction characteristic diagram special attribute investigation of the invention; wherein, peak 1 is a loganin acid peak, peak 2 is a tryptophan peak, peak 4 is a gentiopicroside peak, peak 5 is a hydroxysafflor yellow A peak, peak 7 is a ferulic acid peak, peak 8 is a liquiritin peak, peak 9 is an apiose liquiritin peak, peak 13 is a glycyrrhizic acid peak, and peak 14 is a notopterygium alcohol peak;

FIG. 21 is a characteristic diagram of a body pain stasis-removing decoction test sample, a Notopterygium incisum negative sample and a Notopterygium incisum single medicine in the body pain stasis-removing decoction characteristic diagram special attribute investigation of the invention; wherein, the peak 1 is a loganin peak, the peak 2 is a tryptophan peak, the peak 4 is a gentiopicroside peak, the peak 5 is a hydroxysafflor yellow A peak, the peak 7 is a ferulic acid peak, the peak 8 is a liquiritin peak, the peak 9 is an apiose liquiritin peak, the peak 13 is a glycyrrhizic acid peak, and the peak 14 is a notopterygium alcohol peak;

FIG. 22 is a characteristic spectrum of a body pain stasis-removing decoction sample, a myrrh negative sample and a myrrh single medicine in the body pain stasis-removing decoction special attribute investigation of the invention; wherein, the peak 1 is a loganin peak, the peak 2 is a tryptophan peak, the peak 4 is a gentiopicroside peak, the peak 5 is a hydroxysafflor yellow A peak, the peak 7 is a ferulic acid peak, the peak 8 is a liquiritin peak, the peak 9 is an apiose liquiritin peak, the peak 13 is a glycyrrhizic acid peak, and the peak 14 is a notopterygium alcohol peak;

FIG. 23 is a characteristic diagram of a body pain stasis-eliminating decoction test sample, a rhizoma Cyperi negative sample and rhizoma Cyperi single medicine in the body pain stasis-eliminating decoction characteristic diagram specificity investigation of the invention; wherein, the peak 1 is a loganin peak, the peak 2 is a tryptophan peak, the peak 4 is a gentiopicroside peak, the peak 5 is a hydroxysafflor yellow A peak, the peak 7 is a ferulic acid peak, the peak 8 is a liquiritin peak, the peak 9 is an apiose liquiritin peak, the peak 13 is a glycyrrhizic acid peak, and the peak 14 is a notopterygium alcohol peak;

FIG. 24 is a characteristic spectrum of a body pain stasis-removing decoction sample, a achyranthes negative sample and a achyranthes single medicine in the investigation of the body pain stasis-removing decoction characteristic spectrum specificity of the invention; wherein, the peak 1 is a loganin peak, the peak 2 is a tryptophan peak, the peak 4 is a gentiopicroside peak, the peak 5 is a hydroxysafflor yellow A peak, the peak 7 is a ferulic acid peak, the peak 8 is a liquiritin peak, the peak 9 is an apiose liquiritin peak, the peak 13 is a glycyrrhizic acid peak, and the peak 14 is a notopterygium alcohol peak;

FIG. 25 is a characteristic spectrum of the body pain stasis-removing decoction in the examination of the body pain stasis-removing decoction characteristic spectrum specificity, earthworm negative sample and earthworm single medicine; wherein, the peak 1 is a loganine acid peak, the peak 2 is a tryptophan peak, the peak 4 is a gentiopicroside peak, the peak 5 is a hydroxysafflor yellow A peak, the peak 7 is a ferulic acid peak, the peak 8 is a liquiritin peak, the peak 9 is an apiose liquiritin peak, the peak 13 is a glycyrrhizic acid peak, and the peak 14 is a notopterygium alcohol peak;

FIG. 26 is a feature map overlay of 15 batches of pain relieving stasis removing soup samples; wherein, peak 1 is a loganin acid peak, peak 2 is a tryptophan peak, peak 4 is a gentiopicroside peak, peak 5 is a hydroxysafflor yellow A peak, peak 7 is a ferulic acid peak, peak 8 is a liquiritin peak, peak 9 is an apiose liquiritin peak, peak 13 is a glycyrrhizic acid peak, and peak 14 is a notopterygium alcohol peak;

FIG. 27 is a control feature spectrum of the pain-relieving stasis-removing decoction of the present invention; wherein, peak 1 is a loganin acid peak, peak 2 is a tryptophan peak, peak 4 is a gentiopicroside peak, peak 5 is a hydroxysafflor yellow A peak, peak 7 is a ferulic acid peak, peak 8 is a liquiritin peak, peak 9 is an apiose liquiritin peak, peak 13 is a glycyrrhizic acid peak, and peak 14 is a notopterygium alcohol peak;

FIG. 28 is total ion flux mass spectrum and ultraviolet liquid chromatogram (ESI) of test solution of SHENTONGZHUYU decoction ⁺ )；

FIG. 29 is a total ion current mass spectrum and ultraviolet liquid chromatogram (ESI) of a body pain stasis-removing decoction sample solution ^- )。

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and detailed description.

The inventor carries out various aspects of archaeological examinations based on ancient book literature, and the determined body pain stasis-removing decoction is as follows: 3.73g of gentiana macrophylla, 7.46g of ligusticum wallichii, 11.19g of peach kernel, 11.19g of safflower, 7.46g of liquorice, 3.73g of notopterygium root, 7.46g of myrrh, 11.19g of angelica, 7.46g of vinegar trogopterus dung, 3.73g of rhizoma cyperi, 11.19g of achyranthes bidentata and 7.46g of earthworm.

Further, based on the ancient books and literature examination, the preparation method of the traditional decoction of the body pain stasis-removing decoction is determined as follows: putting 3.73g of large-leaved gentian, 7.46g of ligusticum wallichii, 11.19g of peach kernel, 11.19g of safflower, 7.46g of liquorice, 3.73g of notopterygium root, 7.46g of myrrh, 11.19g of angelica, 7.46g of trogopterus dung with vinegar, 3.73g of rhizoma cyperi, 11.19g of achyranthes bidentata and 7.46g of earthworm in a 2L decocting earthenware pot, adding 8 times of water into the first decoction, soaking for 45 minutes, heating to boil with strong fire, slightly boiling with slow fire for 30 minutes, and filtering; adding 6 times of water into the second decoction, decocting for 25 minutes, and combining the two decoction filtrates to obtain the body pain-relieving and stasis-removing decoction. Further, the bodily pain stasis removing soup samples adopted in the invention are the bodily pain stasis removing soup freeze-dried powder, and specifically, the bodily pain stasis removing soup decoction is freeze-dried in a vacuum freeze-drying machine and taken out to obtain the bodily pain stasis removing soup freeze-dried powder.

Further, in another embodiment of the present invention, the preparation method of the traditional decoction of the body pain stasis removing decoction comprises: 3.73g of gentiana macrophylla, 7.46g of ligusticum wallichii, 11.19g of peach kernel, 11.19g of safflower, 7.46g of liquorice, 3.73g of notopterygium root, 7.46g of myrrh, 11.19g of angelica, 7.46g of vinegar trogopterus dung, 3.73g of rhizoma cyperi, 11.19g of achyranthes bidentata and 7.46g of earthworm are taken and placed in a 2L decocting earthen pot, 600mL of water is added into the 2L decocting earthen pot, and the mixture is kept to be slightly boiled by slow fire and decocted to 300mL, so that the body pain and stasis removing decoction is obtained. Further, the body pain stasis removing soup samples adopted in the invention are body pain stasis removing soup freeze-dried powder, and specifically, the body pain stasis removing soup is freeze-dried in a vacuum freeze-drying machine and taken out to obtain the body pain stasis removing soup freeze-dried powder.

In order to comprehensively reflect the quality information of the body pain stasis-removing soup and realize comprehensive and effective control of the quality of a body pain stasis-removing soup product, the invention provides a construction method of a body pain stasis-removing soup characteristic map, which is explained in detail as follows:

instrument and reagent

The instrument comprises the following steps: waters ultra-high performance liquid chromatograph (H-Class, waterltd), Thermo ultra-high performance liquid chromatograph (Vanquish, seemer femtoll technologies (china)), Agilent 1290 high performance liquid chromatograph (Agilent 1290, Agilent ltd), Waters high performance liquid chromatograph (Arc, waterltd), ten thousandth balance (ME204E, mettler-tolimo), millionth of dail (XP26, mettler-tolimo), digital controlled ultrasonic cleaner (KQ500D, kunshan instruments ltd), constant temperature water bath (HWS28, shanghai constant technology ltd), ultra-pure water system (Milli-Q Direct, merck technologies ltd).

Reagent: ethanol (west longa science, ltd) and methanol (west longa science, ltd) were both analytically pure; the liquid phase is prepared from formic acid (Mimi European chemical reagent Co., Ltd., Tianjin), glacial acetic acid (Mimi European chemical reagent Co., Ltd., Tianjin), acetonitrile (Merck Co., Ltd.), methanol (Merck Co., Ltd.) by HPLC chromatography, and water (prepared by laboratory) by ultrapure water.

Reagent testing: loganin acid reference substance (batch No. 111865-202005, content: 97.5%, China institute for food and drug identification); a tryptophan control (batch No. 140686-201904, content: 99.9%, China institute for food and drug identification); a gentiopicroside reference substance (batch number: 110770-201918, content: 97.1%, China institute for food and drug identification); a hydroxysafflor yellow A reference substance (batch number: 111637-201810, content 93.1%, China institute for food and drug identification); ferulic acid reference (batch No. 110773-201915, content: 99.4%, China institute for food and drug identification); a liquiritin reference substance (batch number: 111610 and 201908, content: 95.0%, China institute for food and drug identification); apioside liquiritin reference substance (batch number: 110730-; ammonium glycyrrhizinate reference (batch No. 110731 and 202021, content: 96.2%, China institute for food and drug identification); senkyunolide I reference substance (batch number: DSTDY000901, content: 99.45%, Chengdu le Meitian medicine science and technology Co., Ltd.); notopterygium incisium alcohol reference substance (batch number: 111820-201705, content: 99.9%, China institute for food and drug identification); the pain-relieving and stasis-removing decoction samples (batch numbers: S1-S15) are all from Guangdong one-party pharmaceutical Co., Ltd.).

Second, method for constructing body pain and stasis removing soup characteristic spectrum

2.1 preparation of chromatographic conditions and reference solutions and test solutions

2.1.1 chromatographic conditions

Chromatographic conditions are as follows: octadecylsilane chemically bonded silica was used as a filler (Waters CORTECS T3, column length 150mm, inner diameter 2.1mm, particle diameter 1.6 μm); acetonitrile was used as mobile phase a, 0.2 vol% phosphoric acid was used as mobile phase B, and gradient elution was performed as specified in table 1; the flow rate is 0.3mL per minute; the column temperature is 30 ℃; the detection wavelength was 235 nm. The number of theoretical plates is not less than 10000 calculated according to gentiopicrin peak.

TABLE 1 gradient elution table for characteristic spectrum of pain-relieving and stasis-removing decoction

2.1.2 preparation of reference solutions

Preparation of reference solutions: accurately weighing tryptophan reference substance, loganin reference substance, gentiopicrin reference substance, hydroxysafflor yellow A reference substance, ferulic acid reference substance, liquiritin reference substance, apioside liquiritin reference substance, glycyrrhizic acid reference substance, and Notopterygii rhizoma alcohol reference substance, accurately weighing, adding methanol to obtain mixed solution containing tryptophan 15 μ g, loganin 40 μ g, gentiopicrin 60 μ g, hydroxysafflor yellow A60 μ g, ferulic acid 20 μ g, liquiritin 30 μ g, apioside 70 μ g, glycyrrhizic acid 40 μ g, and Notopterygii rhizoma alcohol 20 μ g per 1mL, and obtaining reference substance solution.

2.1.3 method for preparing test solution

Taking 0.5g of the pain-relieving stasis-removing decoction freeze-dried powder, precisely weighing, placing in a conical flask with a plug, adding 25mL of 70 vol% methanol, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with 70 vol% methanol, shaking up, filtering, and taking a subsequent filtrate to obtain a test solution.

2.1.4 assay

Precisely sucking 1 μ L of reference solution and sample solution respectively, injecting into liquid chromatograph, and measuring.

Wherein, the test sample characteristic spectrum should present 15 characteristic peaks, and 9 peaks should respectively correspond to the retention time of the corresponding reference sample peak. Specifically, peak 1 is a loganin acid peak, peak 2 is a tryptophan peak, peak 4 is a gentiopicroside peak, peak 5 is a hydroxysafflor yellow a peak, peak 7 is a ferulic acid peak, peak 8 is a liquiritin peak, peak 9 is an apiose liquiritin peak, peak 13 is a glycyrrhizic acid peak, and peak 14 is a notopterygium alcohol peak. The peak corresponding to the ferulic acid control peak was selected as the reference S peak, and the relative retention time of each characteristic peak and the S peak was calculated to be within ± 5% of the specified value, the specified value of the characteristic peak being 0.323 peak 2 to 0.373 peak 3 to 0.625, peak 4 to 0.662, peak 5 to 0.774, peak 6 to 0.941, peak 7 to 1.000, peak 8 to 1.276, peak 9 to 1.332, peak 10 to 1.616, peak 11 to 1.765, peak 12 to 2.442, peak 13 to 2.609, and peak 14 to 2.732.

2.2 determination of chromatographic conditions

2.2.1 elution gradient investigation

Gradient 1: a Waters CORTECS T3 column (2.1X 150mm, 1.6 μm) was used as a chromatographic column; acetonitrile is taken as a mobile phase A, 0.2 vol% phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the table 2; the detection wavelength was 235nm, the column temperature was 30 ℃, the flow rate was 0.3mL per minute, and the sample size was 1. mu.L, as shown in FIG. 1.

Table 2 gradient elution table 1

Gradient 2: using a Waters CORTECS T3 column (2.1 × 150mm, 1.6 μm) as a chromatographic column; acetonitrile was used as mobile phase a, 0.2 vol% phosphoric acid was used as mobile phase B, and gradient elution was performed according to table 3; the detection wavelength was 235nm, the column temperature was 30 ℃, the flow rate was 0.3mL per minute, and the sample size was 1. mu.L, as shown in FIG. 2.

Table 3 gradient elution table 2

Gradient 3: a Waters CORTECS T3 column (2.1X 150mm, 1.6 μm) was used as a chromatographic column; gradient elution was performed according to the gradient (gradient 3) in table 1 using acetonitrile as mobile phase a and 0.2 vol% phosphoric acid as mobile phase B; the detection wavelength was 235nm, the column temperature was 30 ℃, the flow rate was 0.3mL per minute, and the sample size was 1. mu.L, as shown in FIG. 3.

Comparing 3 different elution gradients, more chromatographic peaks are obtained by the gradient 1 and the gradient 2, but chromatographic peaks with higher response values are not separated, the base line is not stable, chromatographic peaks at the middle polar part are stacked, the separation degree is poor, and the base line is seriously drifted. By adopting the gradient 3, the separation degree of each chromatographic peak is better, the chromatographic peak information is more comprehensive, more medicinal flavor information can be contained, and the chromatographic peak with higher response value and the nearby miscellaneous peak are separated from each other by a base line. In summary, gradient 3 was chosen as the optimal chromatographic elution condition.

2.2.2 chromatographic column inspection

The method is characterized by comprising the following steps of (1) investigating ultrahigh chromatographic columns of different manufacturers, wherein the method comprises the following steps: waters CORTECS T3 column (2.1X 150mm, 1.6 μm); waters HSS T3 (2.1X 150mm, 1.8 μm) column; waters BEH C18 (2.1X 150mm, 1.7 μm) chromatography column; gradient elution was performed as specified in table 1 using acetonitrile as mobile phase a and 0.2 vol% phosphoric acid aqueous solution as mobile phase B; the flow rate is 0.3mL per minute; the column temperature is 30 ℃; the sample injection amount is 1 mu L; the detection wavelength was 235 nm. The results are shown in FIGS. 4 to 6.

The results show that the chromatographic columns of three different manufacturers have different separation effects on each chromatographic peak, and the chromatographic column of Waters CORTECS T3 has better separation effect and better peak type on each chromatographic peak. Thus, Waters CORTECS T3 (2.1X 150mm, 1.6 μm) was chosen as analytical column.

2.2.3 absorption wavelength investigation

Investigating different absorption wavelengths; the absorption wavelengths were 220nm, 235nm, 254nm, 274nm and 316nm, respectively. A Waters CORTECS T3(2.1 mm. times.150 mm, 1.6 μm) column was used; acetonitrile is taken as a mobile phase A, 0.2% phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the specification in the table 1; the flow rate is 0.3mL per minute; the sample injection amount is 1 mu L; the results are shown in FIG. 7.

By comparing the 5 detection wavelength chromatograms, the method can find that when 220nm is selected as the detection wavelength, the number of chromatographic peaks is large, but the response value of the whole chromatographic peak is low, and after the retention time is 40 minutes, the base line is not stable; when the wavelengths of 254nm, 274nm and 316nm are selected for detection, liquiritin and ferulic acid have maximum absorption, the response values of the rest chromatographic peaks become low, and part of chromatographic peaks disappear; when the wavelength of 235nm is selected, the overall response value is high, the number of chromatographic peaks is the largest, and the baseline is stable. In order to reflect characteristic peaks of the medicinal materials as much as possible, ensure response values of the various spectral peaks and ensure stable baseline, the detection wavelength is selected to be 235nm by combining the consideration.

2.2.4 Mobile phase inspection

Investigating the type and concentration of the buffer solution (mobile phase B) in the mobile phase, and selecting a 0.1 vol% acetic acid solution, a 0.1 vol% formic acid solution, a 0.1 vol% phosphoric acid solution and a 0.2 vol% phosphoric acid solution respectively; a Waters CORTECS T3(2.1 mm. times.150 mm, 1.6 μm) column was used; acetonitrile is taken as a mobile phase A, and gradient elution is carried out according to the specification in the table 1; the flow rate was 0.3mL per minute; the detection wavelength is 235 nm; the column temperature is 30 ℃; the sample size was 1. mu.L, and the results are shown in FIGS. 8 to 11.

Comparing three chromatograms of 0.1 vol% phosphoric acid solution, 0.1 vol% acetic acid solution and 0.1 vol% formic acid solution, finding that 0.1 vol% acetic acid and 0.1 vol% formic acid solution are mobile phases, the base line has uneven drift, the base line is concave after 45 minutes, and the number of chromatographic peaks is small; if acetonitrile-phosphoric acid is used as a mobile phase, chromatogram information is rich, chromatographic peak separation degree is good, a base line is stable, the peak appearance time is proper, and the separation effect of 0.2% phosphoric acid on polar components with early peak appearance time is better than that of 0.1 vol% phosphoric acid. In summary, 0.2 vol% phosphoric acid solution was used as buffer (mobile phase B).

2.2.5 flow Rate investigation

Flow phase flow rates are examined and are respectively 0.25mL, 0.30mL and 0.35mL per minute; a Waters CORTECS T3 (2.1X 150mm, 1.6 μm) column was used; gradient elution was performed as specified in table 1 using acetonitrile as mobile phase a and 0.2 vol% phosphoric acid as mobile phase B; the column temperature is 30 ℃; the sample injection amount is 1 mu L; the detection wavelength was 235 nm. The results are shown in FIG. 12.

As can be seen, when the flow rate is 0.3mL/min, the separation effect of each chromatographic peak is better, the peak pattern is better, the fluctuation of the flow rate up and down by 0.05mL/min can be accepted, and the flow rate durability of the method is good.

2.2.6 investigation of column temperature

Examining the column temperature, wherein the column temperature is 25 ℃, 30 ℃, 35 ℃ and 40 ℃ respectively; a Waters CORTECS T3 (2.1X 150mm, 1.6 μm) column was used; acetonitrile is taken as a mobile phase A, 0.2 percent phosphoric acid is taken as a mobile phase B, and gradient elution is carried out according to the specification in the table 1; the flow rate is 0.3mL per minute; the sample injection amount is 1 mu L; the detection wavelength was 235 nm. The results are shown in FIG. 13.

As can be seen from the figure, when the column temperature is 30 ℃, the separation effect of each chromatographic peak is good, the peak type is good, the fluctuation of the flow rate up and down at 5 ℃ is acceptable, and the durability of the column temperature of the method is good.

2.2.7 chromatographic Condition determination

From the above experiment, the chromatographic conditions were determined as follows: octadecylsilane chemically bonded silica was used as a filler (Waters CORTECS T3, column length 150mm, inner diameter 2.1mm, particle diameter 1.6 μm); gradient elution was performed as specified in table 1 using acetonitrile as mobile phase a and 0.2 vol% phosphoric acid aqueous solution as mobile phase B; the flow rate is 0.3mL per minute; the column temperature is 30 ℃; the detection wavelength is 235nm, and the number of theoretical plates is not less than 10000 calculated according to gentiopicroside peak.

2.3 examination of the preparation method of the test solution

2.3.1 examination of extraction solvent

The influence of different solvents on the characteristic spectrum of the pain relieving stasis removing decoction is examined, 100 vol% methanol, 70 vol% methanol and 50 vol% methanol are respectively used as extraction solvents, the influence of different extraction solvents on the characteristic spectrum of the pain relieving stasis removing decoction is compared by observing the peak types and the separation degrees of 15 characteristic peaks in the pain relieving stasis removing decoction, calculating the total peak area/sample weighing amount of the 15 characteristic peaks, and selecting the optimal extraction solvent.

Specifically, a proper amount of the pain-relieving stasis-removing decoction freeze-dried powder is taken, ground, about 0.5g of the fine-weighing decoction is taken, three groups of the fine-weighing decoction are parallelly arranged, two parts of each group are placed in a conical flask with a plug, 25mL of the extraction solvent is precisely added, the weight is weighed, ultrasonic treatment (power 250W and frequency 40kHz) is carried out for 30 minutes, the mixture is taken out, cooled, weighed again, the lost weight is complemented with the corresponding extraction solvent, the mixture is shaken uniformly, filtered, and a subsequent filtrate is taken and is subjected to sample injection analysis under the chromatographic condition of 2.1.1, and the result is shown in table 4.

TABLE 4 examination result table of body pain stasis-removing decoction feature spectrum extraction solvent

The results show that: when 100 vol% methanol, 70 vol% methanol and 50 vol% methanol are used as extraction solvents, the extraction effects of all characteristic peaks are similar, and the peak types and the separation effects of all characteristic peaks have no obvious difference, wherein when 70 vol% methanol is used as the extraction solvent, the total peak area/sample weighing amount of 15 characteristic peaks is the largest, and the theoretical plate number of each characteristic peak is more optimal, so 70 vol% methanol is selected as the extraction solvent.

2.3.2 examination of the extraction method

The influence of different extraction modes on the characteristic spectrum of the body pain stasis-removing decoction is investigated, two extraction modes of ultrasonic and reflux are respectively investigated, the peak types and the separation degrees of 15 characteristic peaks are observed, the total peak area/sample weighing amount of the 15 characteristic peaks is calculated, and the influence of different extraction modes on the characteristic spectrum of the body pain stasis-removing decoction is compared.

Specifically, a proper amount of the pain-relieving stasis-removing decoction freeze-dried powder is taken, ground, about 0.5g of the pain-relieving stasis-removing decoction freeze-dried powder is precisely weighed, two groups of the powder are arranged in parallel, two parts of each group are placed in a conical flask with a plug, 25mL of 70 vol% methanol is precisely added, the weight is weighed, ultrasonic treatment (power 250W and frequency 40kHz) is respectively carried out for 30 minutes, heating reflux is carried out for 30 minutes, the mixture is taken out, cooled and weighed again, the weight loss is complemented with 70 vol% methanol, shaking up is carried out, filtering is carried out, a subsequent filtrate is taken, sample injection analysis is carried out according to the chromatographic condition under 2.1.1, and the result is shown in Table 5.

TABLE 5 evaluation result table of characteristic spectrum extraction method of pain-relieving and stasis-removing decoction

The results show that: by adopting different extraction modes, the peak types and the separation effects of all characteristic peaks are not obviously different, the difference of the total peak area/sample weighing amount is not large, and ultrasonic treatment is selected in consideration of simplicity and convenience of an ultrasonic treatment method.

2.3.3 extraction time study

Examining the influence of different extraction times on the body pain stasis-removing decoction characteristic spectrum, and selecting and examining three different extraction times of 15min, 30min and 45 min.

Specifically, a proper amount of the shentongxuezhuyu decoction freeze-dried powder is taken, ground, about 0.5g of the freeze-dried powder is precisely weighed, three groups of the freeze-dried powder are parallelly arranged, each group of the freeze-dried powder is placed in a conical flask with a plug, 25mL of 70 vol% methanol is precisely added, the weight is weighed, ultrasonic treatment (power of 250W and frequency of 40kHz) is respectively carried out for 15min, 30min and 45min, the mixture is taken out, cooled, weighed again, the weight loss is complemented with 70 vol% methanol, shaken up, filtered, a subsequent filtrate is taken, sample injection analysis is carried out according to 2.1.1 chromatographic conditions, and the result is shown in table 6.

TABLE 6 evaluation result table of body pain stasis-removing decoction feature spectrum extraction time

The results show that: different extraction times are adopted, the total peak area/sample weighing amount of 15 characteristic peaks has no obvious difference, and the RSD value of the total peak area/sample weighing amount of three extraction times is 0.98%; in order to ensure the durability and complete extraction of the method, the ultrasonic extraction time is selected to be 30 min.

2.3.4 determination of method for preparing test solution

According to the experimental result, the pretreatment method of the body pain stasis removing decoction characteristic spectrum sample is determined as follows:

taking 0.5g of the shentongxueyu decoction freeze-dried powder, accurately weighing, placing the powder in a conical flask with a plug, adding 25mL of 70 vol% methanol, sealing the plug, weighing, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, taking out, cooling, weighing again, complementing the weight loss with 70 vol% methanol, shaking up, and filtering to obtain a test solution.

2.4 methodological validation

2.4.1 specialization examination

And respectively taking the pain-relieving stasis-removing decoction lacking each medicine, and preparing according to the preparation method of the test solution to obtain a negative sample solution lacking each medicine.

Taking appropriate amount of radix Gentianae Macrophyllae, rhizoma Ligustici Chuanxiong, semen Persicae, Carthami flos, Glycyrrhrizae radix, Notopterygii rhizoma, Myrrha, radix Angelicae sinensis, vinegar Oletum Trogopterori, rhizoma Cyperi, Achyranthis radix, and Lumbricus control medicinal materials, respectively, decocting according to the decoction method of body pain and blood stasis dispelling decoction to obtain single reference medicinal material solution, and taking appropriate amount to prepare reference material solution of each medicinal material according to the preparation method of test sample.

Injecting 1 μ L of each of the test solution, the negative sample solution lacking each medicinal material, and the reference solution of single control medicinal material into a liquid chromatograph, and analyzing by sample injection under chromatographic conditions of 2.1.1 bar, with the results shown in FIGS. 14-25.

As can be seen from fig. 14 to 25: peak 1 (loganin) and Peak 4 (gentiopicroside) are characteristic components of radix Gentianae Marcrophyllae; peak 2 (tryptophan), Peak 8 (liquiritin), Peak 9 (apioside liquiritin), Peak 12, Peak 13 (glycyrrhizic acid) are characteristic components of Glycyrrhrizae radix; peak 3, Peak 5 (hydroxysafflor yellow A), Peak 11 are characteristic components of safflower, Peak 6 is characteristic component of trogopterus dung and peach kernel, Peak 7 (ferulic acid) and Peak 10 are characteristic components of Ligusticum wallichii, Peak 7 (ferulic acid) is characteristic component of Angelica sinensis, Peak 7 (ferulic acid), Peak 10, Peak 11 and Peak 14 (notopterygium alcohol) are characteristic components of Notopterygium incisum, Peak 15 is characteristic component of myrrh; wherein peak 7 is common component of rhizoma Ligustici Chuanxiong, radix Angelicae sinensis, and Notopterygii rhizoma, peak 10 is common component of radix Angelicae sinensis and Notopterygii rhizoma, and peak 11 is common component of Notopterygii rhizoma and Carthami flos; the main components of earthworm, nutgrass galingale rhizome and twotooth achyranthes root have low response under the chromatographic condition, have poor chromatographic peak separation effect and are easily influenced by background, and no characteristic peak belongs to the medicinal materials.

In addition, as can be seen from fig. 14 to 25, the chromatogram of the test sample has the same chromatographic peak at the corresponding retention time of the chromatogram of the single drug control drug, and the negative sample without the single drug has no interference, which indicates that the specificity of the method is good.

2.4.2 precision investigation

Taking a sample solution of the same batch of the body pain stasis-removing decoction freeze-dried powder preparation, repeatedly injecting the sample for 6 times according to the determined chromatographic condition (subsection 2.1.1), taking a peak (peak 7) corresponding to the peak of the ferulic acid reference substance as an S peak, calculating the relative retention time and the relative peak area of characteristic peaks 1-15 and the S peak, and calculating the RSD value of the sample solution. The results are shown in tables 7 to 8.

TABLE 7 evaluation result table for body pain stasis-removing decoction feature spectrum precision (relative retention time)

TABLE 8 evaluation result table (relative peak area) for body pain stasis-removing decoction feature spectrum precision

The result shows that the relative retention time RSD of each characteristic peak and the S peak is in the range of 0.06% -0.20%, the relative peak area RSD is in the range of 0.25% -16.69%, the relative peak area RSD is more than 3% by taking the ferulic acid (peak 7) chromatographic peak as a reference peak S, the relative peak area range is recommended not to be specified, and the instrument precision is good because the relative retention time of each chromatographic peak is less than 3.0%.

2.4.3 repeatability Studies

6 parts of the same batch of the shentong Zhuyu decoction freeze-dried powder are taken to prepare a test solution, the test solution is measured under the chromatographic condition of 2.1.1 sections, the peak (peak 7) corresponding to the peak of the ferulic acid reference substance is the S peak, the relative retention time and the relative peak area of the characteristic peaks 1-15 and the S peak are calculated, and the RSD value is calculated. The results are shown in tables 9 to 10.

TABLE 9 repeatability test results of characteristic spectra of the decoction for treating body pain and dissipating blood stasis (relative retention time)

TABLE 10 repeatability survey results of characteristic spectra of the decoction for treating body pain and dissipating blood stasis (relative peak area)

The result shows that the method has good repeatability as the relative retention time RSD of each characteristic peak and the S peak is in the range of 0.02-0.63%, the relative peak area RSD is in the range of 0.34-7.04%, and the relative peak area RSD is more than 3% by taking the ferulic acid (peak 7) chromatographic peak as a reference peak S, and the relative retention time RSD of each chromatographic peak is less than 3.0% without specifying the relative peak area range.

2.4.4 stability Studies

Taking a test solution of the same batch of the body pain stasis-removing decoction freeze-dried powder, analyzing the test solution at the chromatographic conditions of 2.1.1 bar in 0, 4, 8, 12, 16 and 25 hours respectively, taking a peak (peak 7) corresponding to the ferulic acid reference peak as an S peak, calculating the relative retention time and the relative peak area of characteristic peaks 1-15 and the S peak, and calculating the RSD value of the test solution. The results are shown in tables 11 to 12.

TABLE 11 stability test results of body pain-eliminating stasis decoction (relative retention time)

TABLE 12 stability survey results of body pain-eliminating stasis decoction characteristics (relative peak area)

The results show that the relative retention time RSD of each characteristic peak and the S peak is in the range of 0.08-1.31%, the relative peak area RSD is in the range of 0.63-5.32%, and the relative peak area RSD is more than 3% by taking the ferulic acid (peak 7) chromatographic peak as a reference peak S, the relative peak area range is not recommended to be specified, and the relative retention time of each chromatographic peak is less than 3.0%, which indicates that the sample solution is relatively stable within 25 h.

2.4.5 intermediate precision investigation

Operating on different instruments at different time by different analysts, taking the same test solution of the pain-eliminating stasis-removing decoction freeze-dried powder of the same batch, and determining according to chromatographic conditions of 2.1.1 bar; the peak (peak 7) corresponding to the ferulic acid control peak was the S peak, and the relative retention time and relative peak area of characteristic peaks 1 to 15 and the S peak were calculated to calculate the RSD value thereof. The results are shown in tables 13 to 14.

TABLE 13 recipe intermediate precision test results (relative retention time) of decoction for removing blood stasis and relieving pain

TABLE 14 recipe intermediate precision test results (relative peak area) of the decoction for treating body pain and dissipating blood stasis

The results show that the relative retention time RSD of each characteristic peak and the S peak is in the range of 0.12-8.98%, the relative peak area RSD is in the range of 2.40-85.70%, the relative peak area RSD value is more than 5.0% by taking the ferulic acid (peak 7) chromatographic peak as a reference peak S, the relative peak area range is not recommended to be specified, the relative retention time of the peak 1 and the peak 2 is more than 5%, the remaining characteristic peaks are less than 5%, the relative retention time of other chromatographic peaks is less than 3.0%, and the middle precision of each characteristic peak is good.

2.5 determination of samples from different batches and determination of common peaks

Respectively taking 15 batches of body pain stasis removing soup reference sample small samples (numbers S1-S15), preparing a test solution according to a preparation method determined under 2.1.3, precisely absorbing the test solution, performing sample injection measurement according to chromatographic conditions under 2.1.1, calculating the relative retention time and the relative peak area of each characteristic peak and an S peak according to a characteristic map superposed graph 26 of the 15 batches of body pain stasis removing soup samples by taking ferulic acid (peak 7) as a reference peak S, and calculating the relative retention time and the relative peak area of each characteristic peak and the S peak in tables 15-18.

TABLE 1515 decoction for relieving pain and removing blood stasis with common peak relative retention time table (Peak 1-Peak 8)

TABLE 1615 SANTONGZHUYU decoction feature chromatogram common peak relative retention time table (Peak 9-Peak 15)

TABLE 1715 decoction for relieving pain and removing blood stasis with common peak relative peak area table (Peak 1-Peak 8)

TABLE 1815 lot of general pain dispelling stasis decoction feature map common peak relative peak area table (Peak 9-Peak 15)

And determining the standard of the characteristic spectrum of the body pain stasis-removing decoction by referring to the result of the characteristic spectrum as follows: the chromatogram of the test sample should show 15 characteristic peaks, wherein 9 peaks correspond to the retention time of the corresponding control peak, specifically, peak 1 is loganin acid peak, peak 2 is tryptophan peak, peak 4 is gentiopicrin peak, peak 5 is hydroxysafflor yellow A peak, peak 7 is ferulic acid peak, peak 8 is liquiritin peak, peak 9 is liquiritin peak, peak 13 is glycyrrhizic acid peak, and peak 14 is notopterygium alcohol peak. The relative retention time of each peak should be within ± 5% of the specified values: 0.321 (peak 1), 0.372 (peak 2), 0.624 (peak 3), 0.662 (peak 4), 0.774 (peak 5), 0.941 (peak 6), 1.277 (peak 8), 1.333 (peak 9), 1.510 (peak 10), 1.616 (peak 11), 1.765 (peak 12), 2.443 (peak 13), 2.610 (peak 14), 2.733 (peak 15).

In addition, the RSD value of the common peak of the 15 batches of the body pain stasis removing soup is in the range of 0.05-0.52%, and the RSD value of the relative peak area is in the range of 14.21-126.22%. Therefore, the general pain stasis-removing decoction prepared from different batches of medicinal materials in different producing areas has stable common peak relative retention time, and has larger difference of relative peak areas, so that the components are stable in types and have larger content difference.

UPLC-MS identification method of body pain and stasis removing decoction

3.1 preparation of chromatographic conditions and reference solutions, test solutions

3.1.1 chromatographic conditions

Chromatographic conditions are as follows: octadecylsilane chemically bonded silica was used as a filler (Waters HSS T3, column length 150mm, inner diameter 2.1mm, particle diameter 1.8 μm); acetonitrile was used as mobile phase a, 0.1% formic acid was used as mobile phase B, and gradient elution was performed as specified in table 19; the flow rate is 0.3mL per minute; the column temperature is 30 ℃; the detection wavelength was 235 nm. The number of theoretical plates is not less than 10000 calculated according to gentiopicroside peak.

TABLE 19 gradient elution Table

3.1.2 Mass Spectrometry conditions

Mass spectrum conditions: HESI ion source, positive and negative ion monitoring mode; the cracking voltage is 3.80 kV; the auxiliary air flow is 10 mL/min; the temperature of the ion transmission tube is 320 ℃; the temperature of the auxiliary gas is 350 ℃; scanning mode Full MS/dd-MS ² Full MS resolution 70000, dd-MS ² Resolution 17500; the mass scanning range m/z is 100-1500. In the MS/MS mode, the collision energy in the positive and negative ion modes is 20eV and 40eV respectively, and the mass accuracy is corrected by taking leucine enkephalin as an internal standard.

3.1.3 preparation of reference solutions

Precisely weighing reference substances of neochlorogenic acid, L-tryptophan, loganin, D-amygdalin, swertiamarin, hydroxysafflor yellow A, cryptochlorogenic acid, ferulic acid, liquiritin, gentiopicrin, swertiamarin, liquiritin, rutin, astragalin, naringenin, apioside liquiritin, senkyunolide I, glycyrrhizic acid, ammonium glycyrrhizinate, glycyrrhetinic acid and ginsenoside Ro, placing into a 20mL measuring flask, adding 70% methanol to prepare a solution containing neochlorogenic acid 30 μ g, L-tryptophan 15 μ g, loganin 40 μ g, D-amygdalin 40 μ g, swertiamarin 30 μ g, hydroxysafflor yellow A60 μ g, cryptochlorogenic acid 30 μ g, ferulic acid 10 μ g, liquiritin 30 μ g, gentiamarin 60 μ g, swertiamarin 30 μ g, liquiritigenin 20 μ g, rutin 10 μ g, astragalin 10 μ g, Dissolving a mixed solution of 10 mug naringenin, 70 mug apiose liquiritin, 10 mug senkyunolide I, 40 mug glycyrrhizic acid, 40 mug ammonium glycyrrhizinate, 30 mug glycyrrhetinic acid and 30 mug ginsenoside Ro to a constant volume, and shaking up to obtain a first UPLC-MS reference substance solution; accurately weighing appropriate amount of reference substances including umbelliferone, decursin, imperatorin, isoimperatorin, ligustilide, notopterygium alcohol and alpha-cyperone, placing into a 20mL measuring flask, adding methanol to prepare a mixed solution containing 80 μ g of umbelliferone, 20 μ g of decursin, 40 μ g of imperatorin, 40 μ g of isoimperatorin, 20 μ g of ligustilide, 20 μ g of notopterygium alcohol and 40 μ g of alpha-cyperone in each 1mL, dissolving, fixing the volume to scale, and shaking uniformly to obtain a second UPLC-MS reference substance solution; storing in a refrigerator at 4 deg.C, and filtering with 0.22 μm microporous membrane before use.

3.1.4 method for preparing test solution

Taking 0.5g of the shentongyuyu decoction freeze-dried powder, precisely weighing, placing in a conical flask with a plug, adding 25mL of 70% methanol, carrying out ultrasonic treatment (power 250W and frequency 40kHz) for 30 minutes, centrifuging at 8000r/min for 5 minutes, taking supernate, filtering with a 0.22 mu m filter membrane, and collecting filtrate to obtain the UPLC-MS test solution.

3.1.5 assay

Precisely absorbing 1 mu L of each of the first UPLC-MS reference substance solution, the second UPLC-MS reference substance solution and the UPLC-MS test solution, injecting into an ultra-high performance liquid chromatography-quadrupole-electrostatic orbit trap-high resolution mass spectrometer, and measuring to obtain the final product.

3.2 results of the analysis

According to the literature reports, TCMSP database and reference substance, the chemical components in the sample are identified by combining the chromatographic retention behavior, molecular weight information and mass spectrum cracking rule, and the results are shown in table 20, fig. 28 and fig. 29. In conclusion, UPLC-Q-active Orbitrap MS is adopted to carry out chromatographic separation, mass spectrum data acquisition and structural identification of inherent components on various components in the body pain stasis removing soup, so as to identify 80 components such as organic acids, nucleosides, iridoids, flavonoids, saponins, coumarins and the like, and provide basis and premise for further researching the drug effect substance basis of the body pain stasis removing soup.

TABLE 20 identification of pain-relieving and stasis-removing decoction UPLC-MS

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims

1. A construction method of a body pain stasis-removing decoction characteristic spectrum is characterized by comprising the following steps:

2. The method for constructing body pain stasis-removing decoction characteristic map as claimed in claim 1, wherein in the step (3), gradient elution is carried out according to the following procedure:

0-5 min, wherein the mobile phase A is 6% and the mobile phase B is 94%;

22-24 min, wherein the content of the mobile phase A is 12% → 16%, and the content of the mobile phase B is 88% → 84%;

24-36 min, wherein the content of mobile phase A is 16% → 22%, and the content of mobile phase B is 84% → 78%;

46-50 min, wherein the mobile phase A is 32% → 50%, and the mobile phase B is 68% → 50%;

50-58 min, the mobile phase A is 50% → 65%, and the mobile phase B is 50% → 35%;

3. The method for constructing a body pain stasis-removing soup characteristic map according to claim 1, wherein in the step (3), 0.5 to 1.5 μ L of each of the reference solution and the test solution is respectively sucked and injected into a liquid chromatograph for detection, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler, and has a column length of 150mm, an inner diameter of 2.1mm, a particle size of 1.6 μm and a column temperature of 25 to 35 ℃; the liquid chromatograph takes acetonitrile as a mobile phase A, takes 0.1-0.2% phosphoric acid aqueous solution as a mobile phase B, has a flow rate of 0.25-0.35 mL/min, and has a detection wavelength of 220-254 nm.

4. The method for constructing the characteristic spectrum of jingtongzhuyu decoction according to claim 1 or 3, wherein in the step (3), 1 μ L of each of the reference solution and the sample solution is respectively absorbed and injected into a liquid chromatograph for detection, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler, and has a column length of 150mm, an inner diameter of 2.1mm, a particle size of 1.6 μm, and a column temperature of 30 ℃; the liquid chromatograph takes acetonitrile as a mobile phase A, takes 0.2% phosphoric acid aqueous solution as a mobile phase B, and has the flow rate of 0.3mL/min and the detection wavelength of 235 nm.

5. The method for constructing the body pain stasis removing soup characteristic map as claimed in claim 1, wherein in the step (1), the reference substance solution is prepared by the following steps: tryptophan, loganin, gentiopicrin, hydroxysafflor yellow A, ferulic acid, liquiritin, apioside liquiritin, glycyrrhizic acid and notopterygium alcohol are respectively weighed, and methanol is added to prepare a mixed solution containing 15 mu g of tryptophan, 40 mu g of loganin, 60 mu g of gentiopicrin, 60 mu g of hydroxysafflor yellow A, 20 mu g of ferulic acid, 30 mu g of liquiritin, 70 mu g of apioside, 40 mu g of glycyrrhizic acid and 20 mu g of notopterygium alcohol per 1mL, so as to obtain a reference substance solution.

6. The method for constructing the body pain stasis removing soup characteristic map as claimed in claim 1, wherein in the step (2), the extraction solvent is 50-100% methanol, the extraction time is 15-45 min, and the extraction mode is ultrasonic extraction and/or reflux extraction.

7. The method for constructing the body pain stasis removing soup characteristic map as claimed in claim 1 or 6, wherein the step (2) comprises the following steps:

taking 0.2-0.5 g of the pain-relieving stasis-removing decoction freeze-dried powder, placing the powder into a conical flask with a plug, adding 25-30 mL of 70% methanol, weighing, carrying out ultrasonic treatment for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with 70% methanol, shaking up, filtering, and taking the subsequent filtrate to obtain the test solution.

8. The method for constructing the body pain stasis removing soup characteristic spectrum according to claim 1, wherein the body pain stasis removing soup characteristic spectrum comprises 15 characteristic peaks; wherein, peak 1 is a loganin acid peak, peak 2 is a tryptophan peak, peak 4 is a gentiopicroside peak, peak 5 is a hydroxysafflor yellow A peak, peak 7 is a ferulic acid peak, peak 8 is a liquiritin peak, peak 9 is a liquiritin peak in apiary pond, peak 13 is a glycyrrhizic acid peak, and peak 14 is a notopterygium alcohol peak;

relative retention time of each peak with peak 7 as reference S peak: peak 1 was 0.321, peak 2 was 0.372, peak 3 was 0.624, peak 4 was 0.662, peak 5 was 0.774, peak 6 was 0.941, peak 7 was 1.000, peak 8 was 1.277, peak 9 was 1.333, peak 10 was 1.510, peak 11 was 1.616, peak 12 was 1.765, peak 13 was 2.443, peak 14 was 2.610, peak 15 was 2.733.

9. The method for constructing the body pain stasis-removing decoction characteristic map according to claim 1, further comprising the following steps:

10. The method for constructing the feature map of the decoction for eliminating body pain according to claim 9, wherein the method for identifying UPLC-MS is as follows:

11. The method for constructing a feature map of the body pain stasis-removing decoction according to claim 10, wherein in the UPLC-MS identification method, the UPLC-MS sample is prepared by the following steps: taking 0.2-0.5 g of the pain-relieving stasis-removing decoction freeze-dried powder, placing the powder into a conical flask with a plug, adding 25-30 mL of 70% methanol, weighing, carrying out ultrasonic treatment for 15-45 minutes, centrifuging for 5 minutes at 8000r/min, taking supernate, filtering with a 0.22 mu m filter membrane, and collecting filtrate.

12. The method for constructing the feature map of the pain-relieving and stasis-removing decoction according to claim 10, wherein in the UPLC-MS identification method, the chromatographic conditions are as follows: the chromatographic column adopts an octadecylsilane chemically bonded silica chromatographic column, the length of the chromatographic column is 150mm, the inner diameter of the chromatographic column is 2.1mm, the particle size of the chromatographic column is 1.8 mu m, acetonitrile is used as a mobile phase A, 0.05-0.1% formic acid aqueous solution is used as a mobile phase B, and the column temperature is 25-35 ℃; the flow rate is 0.28-0.32 mL/min; the detection wavelength is 210-250 nm, and the sample injection amount is 0.5-1.5 mu L; gradient elution was performed according to the following procedure:

0-3 min, wherein the mobile phase A is 0% and the mobile phase B is 100%;

7-15 min, wherein the content of the mobile phase A is from 4% → 10%, and the content of the mobile phase B is from 96% → 90%;

25-30 min, wherein the content of the mobile phase A is 12% → 15%, and the content of the mobile phase B is 88% → 85%;

30-40 min, wherein the content of the mobile phase A is 15% → 22%, and the content of the mobile phase B is 85% → 78%;

50-55 min, wherein the content of the mobile phase A is 32% → 50%, and the content of the mobile phase B is 68% → 50%;

60-70 min, 65% → 78% for mobile phase A and 35% → 22% for mobile phase B.

13. The method for constructing the body pain stasis-removing soup characteristic map as claimed in claim 10, wherein in the UPLC-MS identification method, the mass spectrum conditions are as follows: the ESI ion source is in a positive and negative ion monitoring mode; the cracking voltage is 3.80kV, the auxiliary gas flow is 10mL/min, the temperature of the ion transmission tube is 320 ℃, the auxiliary gas temperature is 350 ℃, and the scanning mode is Full MS/dd-MS ² With Full MS resolution of 70000, dd-MS ² Resolution is 17500; the mass scanning range m/z is 100-1500; in the MS/MS mode, the collision energy in the positive and negative ion modes is 20eV and 40eV respectively, and the mass accuracy is corrected by taking leucine enkephalin as an internal standard.

14. The method for constructing the body pain stasis-removing soup characteristic spectrum according to claim 1, wherein the body pain stasis-removing soup is composed of the following components in parts by weight:

15. The method for constructing the body pain stasis-removing decoction characteristic map according to claim 1, wherein the body pain stasis-removing decoction is prepared by the following steps:

taking gentiana macrophylla, ligusticum wallichii, peach kernel, safflower, liquorice, notopterygium root, myrrh, angelica sinensis, vinegar trogopterus dung, rhizoma cyperi, achyranthes and earth worm, adding water which is 6-10 times of the total weight of the raw materials, soaking for 30-60 min, boiling with strong fire, keeping slight boiling with slow fire for 20-40 min, and filtering; adding water which is 5-8 times of the total weight of the raw material medicines into the obtained filter residue, decocting for 20-30 min, and filtering; mixing the two decoctions.

16. The method for constructing the body pain stasis-removing decoction characteristic map according to claim 1, wherein the body pain stasis-removing decoction is prepared by the following steps:

taking gentiana macrophylla, ligusticum wallichii, peach kernel, safflower, liquorice, notopterygium root, myrrh, angelica, vinegar trogopterus dung, rhizoma cyperi, achyranthes and earthworm, adding 6-8 times of water of the total weight of the raw materials, and slightly boiling with slow fire until the filtrate is 3-4 times of the weight of the raw materials.