CN114935612B - Construction method of body pain and stasis dispelling decoction characteristic spectrum - Google Patents

Construction method of body pain and stasis dispelling decoction characteristic spectrum Download PDF

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CN114935612B
CN114935612B CN202210498780.1A CN202210498780A CN114935612B CN 114935612 B CN114935612 B CN 114935612B CN 202210498780 A CN202210498780 A CN 202210498780A CN 114935612 B CN114935612 B CN 114935612B
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mobile phase
decoction
relieving
body pain
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CN114935612A (en
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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
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Guangdong Yifang Pharmaceutical Co Ltd
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Abstract

The invention discloses a construction method of a body pain and 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: respectively taking tryptophan, loganin acid, gentiopicroside, hydroxysafflor yellow A, ferulic acid, glycyrrhizin, apioside, glycyrrhizic acid and notopterygium alcohol reference substances, adding solvent for dissolving or extracting, and preparing reference substance solution; extracting decoction with solvent to obtain sample solution; and (3) testing by adopting a liquid chromatograph, specifically, performing gradient elution by taking acetonitrile as a mobile phase A and phosphoric acid aqueous solution as a mobile phase B, and constructing a characteristic map of the body pain and blood stasis removing decoction. The method can provide a data basis for the quality control of the body pain-relieving and blood stasis-removing soup, and effectively ensures the stability and controllability of the quality of the body pain-relieving and blood stasis-removing soup product.

Description

Construction method of body pain and stasis dispelling decoction characteristic spectrum
Technical Field
The invention relates to the technical field of traditional Chinese medicine quality analysis and detection, in particular to a method for constructing a body pain and stasis removing decoction characteristic map.
Background
The body pain-relieving and stasis-removing decoction is prepared from the books of the medical science clinical evidence of various kinds of books of Qing dynasty and famous traditional Chinese medicine King of Qing dynasty, and the original text is recorded as follows: "pain in shoulder, arm, back, leg, or whole body" is known as arthralgia. It is known that wind-cold is not healed by warm-heat dispersing herbs; it is known that damp-heat is reactive with damp-excreting and fire-reducing herbs. For long-term emaciation of muscles, the theory of yin deficiency is that yin-nourishing herbs are not effective. So far, the cloud: the disease is in the skin and pulse, which is easy to be done; the disease is in tendons and bones, and the excessive disease is difficult to take effect. Pain is caused by cold, damp and heat entering the skin. Is inserted into trachea, and pain must flow away; it is introduced into blood vessel and pain does not move. As such, deficiency is caused by disease, and is not caused by deficiency. … … Gu Fang Poduo, for instance, gu Fang is not effective, and Shen Tong Zhu Yu Tang is used for treating body pain. The Wangqing conception considers that the previous arthralgia is mainly treated by pathogenic factors, namely wind cold, damp heat and yin deficiency, the paranoid warm heat is dispersed, dampness is removed, fire is reduced, or yin is nourished, one end of the decoction is used, and the other end is used for treating the deficiency, so that the decoction is suitable for treating body pain and removing blood stasis, the dosage of the decoction is recorded, the original prescription is composed of twelve medicines such as gentiana macrophylla, ligusticum wallichii, peach kernel, safflower, liquorice, notopterygium root, myrrh, angelica, vinegar trogopterus dung, nutgrass galingale rhizome, achyranthes bidentata, peach kernel, achyranthes bidentata, earthworm and the like, and the principal medicines such as safflower, peach kernel, ligusticum wallichii and angelica can both activate blood and remove stasis, the principal medicines such as notopterygium root and gentiana macrophylla are good at dispelling wind and removing dampness, the trogopterus dung, myrrh, the nutgrass galingale rhizome, the qi and blood circulation of the drug and stopping pain are good at dredging the channels and collaterals to facilitate the joints, and the liquorice regulates the medicines. The recipe is recorded as blood circulation promoting, qi circulation promoting, blood stasis removing, meridian obstruction dredging, arthralgia relieving, and meridian obstruction syndrome such as shoulder pain, arm pain, lumbago, leg pain, or pain of the whole body, such as acupuncture, which is not healed for a long time. The body pain and blood stasis dispelling decoction is widely applied to treating various bone traumatology diseases such as lumbar disc herniation, knee joint osteoarthritis, rheumatic cold joint pain, fracture, postoperative complications, acute lumbar sprain and the like, and simultaneously is also applied to diseases such as thrombophlebitis, soft tissue injury, malignant tumor bone metastasis pain, diabetes complications, neuritis and the like.
In recent years, in order to clarify the material standard of the body pain and blood stasis removing decoction for treating diseases, ensure the effectiveness and safety of clinical medication, many students conduct pharmacological, pharmacodynamic and pharmacokinetic researches on chemical components and effective components in the body pain and blood stasis removing decoction, and continuously perfect the quality standard of the decoction, thereby laying a foundation for the development of classical prescriptions. At present, the research on decoction for relieving body pain and removing blood stasis is mainly focused on pharmacological research, and the research on the material basis, extraction process, multi-index component content measurement and characteristic spectrum of the decoction is less and lacks systemicity. And less research is conducted on how to measure the consistency of mass-produced preparations and traditional decoction quality.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a construction method of a characteristic map of the body pain and blood stasis removing decoction, which can provide a data base for mass production quality control of the body pain and blood stasis removing decoction and ensure stability and controllability of the quality of the body pain and blood stasis removing decoction product.
In order to solve the technical problems, the invention provides a method for constructing a body pain-relieving stasis-removing decoction characteristic map, which comprises the following steps:
(1) Respectively taking tryptophan, loganin acid, gentiopicroside, hydroxysafflor yellow A, ferulic acid, glycyrrhizin, apioside, glycyrrhizic acid and notopterygium alcohol reference substances, adding solvent for dissolving or extracting, and preparing reference substance solution;
(2) Extracting decoction with solvent to obtain sample solution;
(3) Taking a preset amount of reference substance solution and sample solution, and injecting into a liquid chromatograph, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler, acetonitrile as a mobile phase A and phosphoric acid aqueous solution as a mobile phase B for gradient elution, so as to construct a characteristic map of the body pain and blood stasis removing decoction.
As an improvement of the above technical scheme, in the step (3), gradient elution is performed according to the following procedure:
0-5 min, mobile phase A is 6%, mobile phase B is 94%;
5-12 min, mobile phase A from 6% to 10%, mobile phase B from 94% to 90%;
12-22 min, mobile phase A from 10% to 12%, mobile phase B from 90% to 88%;
22-24 min, mobile phase A from 12% to 16%, mobile phase B from 88% to 84%;
24-36 min, mobile phase A from 16% to 22%, mobile phase B from 84% to 78%;
36-46 min, mobile phase A from 22% to 32%, mobile phase B from 78% to 68%;
46-50 min, mobile phase A from 32% to 50%, mobile phase B from 68% to 50%;
50-58 min, mobile phase A from 50% to 65%, mobile phase B from 50% to 35%;
58-68 min, mobile phase A from 65% to 78% and mobile phase B from 35% to 22%.
As an improvement of the technical scheme, in the step (3), respectively sucking 0.5-1.5 mu L of a reference object solution and 0.5 mu L of a test object solution, and injecting the reference object solution and the test object solution into a liquid chromatograph for detection, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica as a filler, 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 mu m, and the column temperature of the liquid chromatograph 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 the flow rate of 0.25-0.35 mL/min and the detection wavelength of 220-254 nm.
In the method for constructing the characteristic spectrum of the decoction for relieving body pain and removing blood stasis, in the improvement of the technical scheme, in the step (3), 1 mu L of each of a reference substance solution and a test sample solution is respectively absorbed, and is injected into a liquid chromatograph for detection, wherein the liquid chromatograph takes octadecylsilane chemically bonded silica gel as a filler, 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 mu m, and the column temperature of the liquid chromatograph is 30 ℃; the liquid chromatograph takes acetonitrile as a mobile phase A, 0.2% phosphoric acid aqueous solution as a mobile phase B, the flow rate is 0.3mL/min, and the detection wavelength is 235nm.
As an improvement of the above technical solution, in step (1), the preparation method of the reference solution is as follows: the method comprises the steps of respectively weighing tryptophan, loganin acid, gentiopicroside, hydroxysafflor yellow A, ferulic acid, glycyrrhizin, apioside, glycyrrhizic acid and notopterygium alcohol, adding methanol to prepare a mixed solution containing 15 mug tryptophan, 40 mug loganin acid, 60 mug gentiopicroside, 60 mug hydroxysafflor yellow A, 20 mug ferulic acid, 30 mug glycyrrhizin, 70 mug apioside, 40 mug glycyrrhizic acid and 20 mug notopterygium alcohol per 1mL, and obtaining a reference solution.
As an improvement of the technical scheme, 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.
As an improvement of the above technical solution, the step (2) includes:
taking 0.2-0.5 g of body pain-relieving stasis decoction freeze-dried powder, placing the powder into a conical bottle with a plug, adding 25-30 mL of 70% methanol, weighing, performing ultrasonic treatment for 30 minutes, taking out, cooling, weighing again, supplementing the weight of loss with 70% methanol, shaking uniformly, 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 removing blood stasis decoction comprises 15 characteristic peaks; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin yellow A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
the relative retention time of each peak, with peak 7 as reference S peak, is: 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, further comprising:
(4) And (3) carrying out characteristic peak chemical component identification on the characteristic spectrum of the body pain-relieving stasis decoction by adopting a UPLC-MS identification method.
As an improvement of the technical scheme, the establishing method of the UPLC-MS identification method comprises the following steps:
(1) Dissolving neochlorogenic acid, L-tryptophan, loganin acid, D-amygdalin, swertiamarin, hydroxysafflor yellow A, cryptochlorogenic acid, ferulic acid, glycyrrhizin, gentiopicroside, swertiamarin, glycyrrhizin, rutin, astragalin, naringenin, apigenin, senkyunolide I, glycyrrhizic acid, ammonium glycyrrhizate, glycyrrhetinic acid and ginsenoside Ro in 70% methanol to obtain a first UPLC-MS reference solution; adding methanol into control substances such as umbelliferone, decursin, imperatorin, isoimperatorin, ligustilide, notopterygol and alpha-cypnone, and dissolving to obtain second UPLC-MS control solution;
(2) Extracting decoction with solvent to obtain UPLC-MS test solution;
(3) Taking a first UPLC-MS reference substance solution, a second UPLC-MS reference substance solution and a UPLC-MS test substance solution, injecting into an ultra-high performance liquid chromatography-quadrupole-electrostatic orbitrap-high resolution mass spectrometer, and establishing chromatographic conditions and mass spectrometry conditions of a UPLC-MS identification method;
(4) Calculating the unknown compound element composition and molecular formula by using a workstation; and (3) 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 spectrum characteristic peak.
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 body pain-relieving stasis decoction freeze-dried powder, placing the powder into a conical bottle with a plug, adding 25-30 mL of 70% methanol, weighing, performing ultrasonic treatment for 15-45 minutes, centrifuging for 5 minutes at 8000r/min, taking supernatant, filtering with a 0.22 mu m filter membrane, and collecting filtrate.
As an improvement of the above technical scheme, in the UPLC-MS identification method, the chromatographic conditions are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica gel chromatographic column with column length of 150mm, inner diameter of 2.1mm and particle diameter of 1.8 μm, acetonitrile as mobile phase A, 0.05-0.1% formic acid water solution as mobile phase B, and column temperature of 25-35deg.C; 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 as follows:
0-3 min, mobile phase A is 0%, mobile phase B is 100%;
3-7 min, mobile phase A from 0% -4% and mobile phase B from 100% -96%;
7-15 min, the mobile phase A is from 4% to 10%, and the mobile phase B is from 96% to 90%;
15-25 min, mobile phase A from 10% to 12%, mobile phase B from 90% to 88%;
25-30 min, mobile phase A from 12% -15%, mobile phase B from 88% -85%;
30-40 min, mobile phase A from 15% to 22%, mobile phase B from 85% to 78%;
40-50 min, mobile phase A from 22% to 32%, mobile phase B from 78% to 68%;
50-55 min, the mobile phase A is from 32% to 50%, and the mobile phase B is from 68% to 50%;
55-60 min, mobile phase A from 50% -65%, mobile phase B from 50% -35%;
60-70 min, the mobile phase A is from 65% to 78%, and the mobile phase B is from 35% to 22%.
As an improvement of the above technical scheme, in the UPLC-MS identification method, the mass spectrometry 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 air flow is 10mL/min, the temperature of the ion transmission tube is 320 ℃, the auxiliary air temperature is 350 ℃, and the scanning mode is Full MS/dd-MS 2 Full MS resolution of 70000, dd-MS 2 Resolution 17500; the mass scanning range m/z is 100-1500; in MS/MS mode, collision energy in positive and negative ion modes is 20eV and 40eV respectively, and leucine enkephalin is used as an internal standard to correct mass accuracy.
As an improvement of the technical scheme, the body pain and blood 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 and 7.46 parts of earthworm.
As an improvement of the technical scheme, the preparation method of the body pain and blood stasis removing soup comprises the following steps:
adding 6-10 times of water of the total weight of the raw materials into the large-leaf gentian root, the szechuan lovage rhizome, the peach seed, the safflower, the liquorice, the notopterygium root, the myrrh, the angelica, the vinegar trogopterus dung, the nutgrass galingale rhizome, the achyranthes root and the earthworm, soaking for 30-60 min, boiling with strong fire, keeping micro-boiling with slow fire for 20-40 min, and filtering; adding water which is 5-8 times of the total weight of the raw materials into the obtained filter residues, decocting for 20-30 min, and filtering; mixing the two decoctions to obtain the final product.
As an improvement of the technical scheme, the preparation method of the body pain and blood stasis removing soup comprises the following steps:
taking gentiana macrophylla, ligusticum wallichii, peach kernel, safflower, liquorice, notopterygium root, myrrh, angelica, vinegar trogopterus dung, nutgrass galingale rhizome, achyranthes root and earthworm, adding 6-8 times of water of the total weight of the raw materials, and boiling with slow fire until the weight of 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 characteristic spectrum of the decoction for relieving body pain and removing blood stasis. The UPLC-MS is adopted to confirm the basic research of related substances, 80 chemical components are identified, 9 characteristic peaks are marked by a reference substance, the 9 characteristic peaks are classified into 9 medicinal herbs, the characteristics of the chemical components of the body pain and blood stasis removing decoction are fully displayed, the characteristic peak information is rich, and the method is stable, accurate and reliable and can realize the quality monitoring of the characteristic components of a plurality of medicinal herbs.
Drawings
FIG. 1 is a characteristic map of the decoction for relieving body pain and removing blood stasis according to the invention when measured by a gradient 1;
FIG. 2 is a characteristic map of the decoction for relieving body pain and removing blood stasis according to the invention when the decoction is measured by using a gradient 2;
FIG. 3 is a characteristic map of the decoction for relieving body pain and removing blood stasis according to the invention, which is measured by using a gradient 3;
FIG. 4 is a characteristic map of the decoction for relieving body pain and removing blood stasis according to the invention, which is measured by using a Waters CORTECS T3 column;
FIG. 5 is a characteristic spectrum of the decoction for relieving body pain and removing blood stasis according to the invention, as measured by a Waters BEH C18 column;
FIG. 6 is a characteristic map of the decoction for relieving body pain and removing blood stasis according to the invention, which is measured by Waters HSS T3 column;
FIG. 7 is a characteristic spectrum of the decoction for relieving body pain and removing blood stasis according to the invention when different wavelengths are used for measurement;
FIG. 8 is a characteristic spectrum of the decoction for relieving body pain and removing blood stasis according to the invention, when acetonitrile-0.1% acetic acid is used as mobile phase for measurement;
FIG. 9 is a characteristic spectrum of the decoction for relieving body pain and removing blood stasis according to the present invention when acetonitrile-0.1% formic acid is used as mobile phase;
FIG. 10 is a characteristic spectrum of the decoction for relieving body pain and removing blood stasis according to the present invention when acetonitrile-0.1% phosphoric acid is used as mobile phase;
FIG. 11 is a characteristic spectrum of the decoction for relieving body pain and removing blood stasis according to the present invention when acetonitrile-0.2% phosphoric acid is used as mobile phase;
FIG. 12 is a characteristic map of the decoction for relieving body pain and removing blood stasis according to the invention when different flow measurements are used;
FIG. 13 is a characteristic map of the decoction for relieving body pain and removing blood stasis according to the invention when different column temperatures are used for measurement;
FIG. 14 is a characteristic spectrum of a single medicine of Gentiana macrophylla, a negative sample of the Gentiana macrophylla, which is a specific investigation of the pattern of the body pain-relieving and stasis-relieving Shang Tezheng of the present invention; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 15 is a characteristic spectrum of a body pain-relieving and stasis-relieving decoction sample, a licorice negative sample and a licorice single drug, which are specifically examined by a Shang Tezheng spectrum of the body pain-relieving and stasis-relieving decoction of the invention; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 16 is a characteristic spectrum of a body pain-relieving and stasis-relieving decoction sample, a safflower negative sample and safflower single drug, which is specifically studied by a Shang Tezheng spectrum of the body pain-relieving and stasis-relieving decoction of the invention; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 17 is a characteristic spectrum of a body pain-relieving and stasis-relieving Shang Tezheng map specificity investigation of a body pain-relieving and stasis-relieving decoction sample, a vinegar-trogopterus dung negative sample and a vinegar-trogopterus dung single drug; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 18 is a characteristic spectrum of a body pain-relieving and stasis-relieving decoction sample, a peach kernel negative sample and a peach kernel single drug, which are specifically studied by the graph specificity of the body pain-relieving and stasis-relieving Shang Tezheng; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 19 is a characteristic spectrum of a body pain-relieving and stasis-relieving decoction sample, a Ligusticum wallichii negative sample and a Ligusticum wallichii single drug, which are specifically examined by a Shang Tezheng spectrum of the body pain-relieving and stasis-relieving decoction of the invention; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 20 is a characteristic spectrum of a body pain-relieving and stasis-relieving decoction sample, a Chinese angelica negative sample and a Chinese angelica single drug, which are specifically examined by a Shang Tezheng spectrum of the body pain-relieving and stasis-relieving decoction of the invention; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 21 is a characteristic spectrum of a body pain-relieving and stasis-relieving decoction sample, a notopterygium root negative sample and a notopterygium root single drug, which are specifically examined by a Shang Tezheng spectrum of the body pain-relieving and stasis-relieving decoction of the invention; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 22 is a characteristic spectrum of a body pain-relieving and stasis-relieving decoction sample, a myrrh negative sample and a myrrh single drug, which are specifically examined by the pattern specificity of the body pain-relieving and stasis-relieving Shang Tezheng; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 23 is a characteristic spectrum of a body pain-relieving and stasis-relieving decoction sample, a nutgrass galingale rhizome negative sample and a nutgrass galingale rhizome single drug, which are specially studied by the body pain-relieving and stasis-relieving Shang Tezheng spectrum of the invention; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 24 is a characteristic spectrum of a body pain-relieving and stasis-relieving decoction sample, a achyranthes negative sample and achyranthes single drug, which are specially inspected by the Shang Tezheng spectrum of the body pain-relieving and stasis-relieving decoction of the invention; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 25 is a graph showing characteristics of a body pain-relieving and stasis-relieving decoction sample, a earthworm negative sample and a earthworm single drug, which are specifically examined by the graph of the body pain-relieving and stasis-relieving Shang Tezheng of the invention; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan acid peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin yellow A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 26 is a graph showing the superposition of characteristic maps of 15 batches of body pain-relieving stasis decoction samples; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 27 is a comparison feature map of the body pain-relieving stasis decoction of the present invention; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
FIG. 28 is a total ion mass spectrum and ultraviolet liquid chromatography (ESI) of a body pain relieving and blood stasis removing decoction sample solution + )；
FIG. 29 is a total ion mass spectrum and ultraviolet liquid chromatography (ESI) of a body pain relieving and blood stasis removing decoction sample solution - )。
Detailed Description
The present invention will be described in further detail with reference to the drawings and the detailed description, for the purpose of making the objects, technical solutions and advantages of the present invention more apparent.
The inventor carries out various archaeological examination based on ancient books, and the determined body pain and stasis removal Shang Fangji are 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 and 7.46g of earthworm.
Further, based on ancient books literature examination, the preparation method of the traditional decoction for determining body pain and stasis removing decoction comprises the following steps: 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 nutgrass galingale rhizome, 11.19g of achyranthes root and 7.46g of earthworm are taken, put into a 2L medicine decocting marmite, 8 times of water is added into the first decoction, the first decoction is soaked for 45 minutes, the mixture is heated and boiled by strong fire, the micro boiling is kept for 30 minutes by slow fire, and the filtration is carried out; adding 6 times of water into the second decoction, decocting for 25 minutes, and combining the two decoctions to obtain the decoction for relieving body pain and removing blood stasis. Furthermore, the body pain and blood stasis removing Shang Yangpin adopted in the invention are all body pain and blood stasis removing soup freeze-dried powder, specifically, the body pain and blood stasis removing Shang Shangji is freeze-dried in a vacuum freeze-dryer, and the body pain and blood stasis removing soup freeze-dried powder is obtained after being taken out.
Further, in another embodiment of the present invention, a method for preparing a traditional decoction of a body pain-relieving stasis 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 nutgrass galingale rhizome, 11.19g of achyranthes root and 7.46g of earthworm are put into a 2L medicinal-decocting marmite, are added with 600mL of water, and are decocted with slow fire until the volume is 300mL, thus obtaining the body pain-relieving and blood stasis-removing soup. Furthermore, the body pain and blood stasis removing Shang Yangpin adopted in the invention are all body pain and blood stasis removing soup freeze-dried powder, specifically, the body pain and blood stasis removing Shang Shangji is freeze-dried in a vacuum freeze-dryer, and the body pain and blood stasis removing soup freeze-dried powder is obtained after being taken out.
In order to comprehensively reflect the quality information of the body ache and blood stasis removing soup and realize comprehensive and effective control of the quality of the body ache and blood stasis removing soup product, the invention provides a construction method of a characteristic spectrum of the body ache and blood stasis removing soup, which is described in detail as follows:
1. instrument and reagent
Instrument: waters ultra-high performance liquid chromatograph (H-Class, wolter's Co., ltd.), thermo ultra-high performance liquid chromatograph (Vanquish, siemens Feisher technology (China Co.), agilent 1290 high performance liquid chromatograph (Agilent 1290, agilent Co., ltd.), waters high performance liquid chromatograph (Arc, wolter's Co., ltd.), one part per million balance (ME 204E, meiler-Toli Co., ltd.), one part per million (XP 26, meiler-Toli Co., ltd.), digital controlled ultrasonic cleaner (KQ 500D, kunshan ultrasonic instruments Co., ltd.), thermostatic water bath (HWS 28 type, shanghai Heng He technology Co., ltd.), ultra-pure water system (Milli-Q Direct, merck Co., ltd.).
Reagent: ethanol (limited of the scientific sciences of the ridge) and methanol (limited of the scientific sciences of the ridge) are all analytically pure; the liquid phase was HPLC chromatographic grade with formic acid (Tianjin chemical agent Co., ltd.), glacial acetic acid (Tianjin chemical agent Co., ltd.), acetonitrile (Merck Co., ltd.), methanol (Merck Co., ltd.), and water was ultra pure water (laboratory self-made).
Reagent: loganin acid reference (lot number: 111865-202005, content: 97.5%, national institute of food and drug identification); tryptophan reference (lot number: 140686-201904, content: 99.9%, national food and drug identification institute); gentiopicroside reference (lot number: 110770-201918, content: 97.1%, national food and drug identification institute); hydroxysafflor yellow A control (lot number: 111637-201810, content 93.1%, national food and drug identification institute); ferulic acid reference substance (batch number: 110773-201915, content: 99.4%, national food and drug identification institute); glycyrrhizin reference substance (lot number: 111610-201908, content: 95.0%, national food and drug identification institute); apigenin reference (batch number: 110730-201915, content: 99.8%, national food and drug identification institute); ammonium glycyrrhizate reference substance (batch number: 110731-202021, content: 96.2%, national institute of food and drug identification); senkyunolide I reference (lot number: DSTDY000901, content: 99.45%, chengdu Mei Tian medicine Co., ltd.); notopterygium alcohol reference (lot number: 111820-201705, content: 99.9%, china food and drug identification institute); all of the body aches and stasis removing Shang Yangpin (lot numbers S1 to S15) are from Guangdong party pharmaceutical Co., ltd.
2. Construction method of body pain and stasis dispelling decoction characteristic spectrum
2.1 preparation of chromatographic conditions and reference solutions, test solutions
2.1.1 chromatographic conditions
Chromatographic conditions: octadecylsilane chemically bonded silica (Waters CORTECS T3, column length of 150mm, inner diameter of 2.1mm, particle diameter of 1.6 μm) was used as filler; gradient elution was performed as specified in table 1 with acetonitrile as mobile phase a and 0.2vol% phosphoric acid as mobile phase B; the flow rate was 0.3mL per minute; the column temperature is 30 ℃; the detection wavelength was 235nm. The theoretical plate number is not less than 10000 calculated according to gentiopicroside peak.
Table 1 body pain-relieving stasis Shang Tezheng atlas gradient elution table
2.1.2 method for preparing reference solution
Preparation of reference solution: respectively precisely weighing tryptophan reference substance, loganin reference substance, gentiopicroside reference substance, hydroxysafflor yellow A reference substance, ferulic acid reference substance, glycyrrhizin reference substance, apiose glycyrrhizin reference substance, glycyrrhizic acid reference substance, and notopterygium alcohol reference substance, precisely weighing, adding methanol to prepare mixed solution containing tryptophan 15 μg, loganin 40 μg, gentiopicroside 60 μg, hydroxysafflor yellow A60 μg, ferulic acid 20 μg, glycyrrhizin 30 μg, apiose glycyrrhizin 70 μg, glycyrrhizic acid 40 μg, and notopterygium alcohol 20 μg per 1mL, and obtaining reference substance solution.
2.1.3 preparation method of sample solution
Taking 0.5g of body pain-relieving stasis decoction freeze-dried powder, precisely weighing, placing into a conical bottle with a plug, adding 25mL of 70vol% methanol, performing ultrasonic treatment (power is 250W, frequency is 40 kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the weight of the decoction with 70vol% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain a sample solution.
2.1.4 assays
Precisely sucking 1 μl of each of the reference solution and the sample solution, and measuring with a liquid chromatograph.
Wherein, 15 characteristic peaks should be presented in the characteristic spectrum of the test sample, and 9 peaks should correspond to the retention time of the corresponding reference sample peak respectively. Specifically, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin a peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak. The peak corresponding to the peak of the ferulic acid reference substance is selected as a reference S peak, and the relative retention time of each characteristic peak and the S peak is calculated, wherein the relative retention time is within +/-5% of a specified value, the specified value of the characteristic peak is 0.323, the specified value of the peak 2 is 0.373, the peak 3 is 0.625, the peak 4 is 0.662, the peak 5 is 0.774, the peak 6 is 0.941, the peak 7 is 1.000, the peak 8 is 1.276, the peak 9 is 1.332, the peak 10 is 1.616, the peak 11 is 1.765, the peak 12 is 2.442, the peak 13 is 2.609 and the peak 14 is 2.732.
2.2 determination of chromatographic conditions
2.2.1 elution gradient investigation
Gradient 1: a Waters CORTECS T3 column (2.1X105 mm,1.6 μm) was used as the column; gradient elution was performed according to table 2 with acetonitrile as mobile phase a and 0.2vol% phosphoric acid as mobile phase B; the detection wavelength is 235nm, the column temperature is 30 ℃, the flow rate is 0.3mL per minute, the sample injection amount is 1 mu L, and the result is shown in FIG. 1.
TABLE 2 gradient elution TABLE 1
Gradient 2: a Waters CORTECS T3 column (2.1X105 mm,1.6 μm) was used as the column; gradient elution was performed according to table 3 with acetonitrile as mobile phase a and 0.2vol% phosphoric acid as mobile phase B; the detection wavelength is 235nm, the column temperature is 30 ℃, the flow rate is 0.3mL per minute, the sample injection amount is 1 mu L, and the result is shown in FIG. 2.
TABLE 3 gradient elution TABLE 2
Gradient 3: a Waters CORTECS T3 column (2.1X105 mm,1.6 μm) was used as the column; gradient elution was performed according to the gradient of table 1 (gradient 3) using acetonitrile as mobile phase a and 0.2vol% phosphoric acid as mobile phase B; the detection wavelength is 235nm, the column temperature is 30 ℃, the flow rate is 0.3mL per minute, the sample injection amount is 1 mu L, and the result is shown in FIG. 3.
Comparing 3 different elution gradients, gradient 1 and gradient 2 have more chromatographic peaks, but the chromatographic peak with higher response value is not separated, the base line is unstable, the chromatographic peak at the middle polarity part is piled up, the separation degree is poor, and the base line drift is serious. The gradient 3 is adopted, the separation degree of each chromatographic peak is better, the chromatographic peak information is more comprehensive, more medicinal flavor information can be included, and the chromatographic peak with higher response value and nearby impurity peaks reach the baseline separation. In summary, gradient 3 was chosen as the optimal chromatographic elution condition.
2.2.2 column chromatography investigation
Investigation of ultra-high chromatographic columns of different manufacturers includes: waters CORTECS T3 column (2.1X105 mm,1.6 μm); waters HSS T3 (2.1X105 mm,1.8 μm) column; waters BEH C18 (2.1X105 mm,1.7 μm) column; gradient elution was performed as specified in table 1 with acetonitrile as mobile phase a and 0.2vol% phosphoric acid aqueous solution as mobile phase B; the flow rate was 0.3mL per minute; the column temperature is 30 ℃; the sample injection amount is 1 mu L; the detection wavelength was 235nm. 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 various chromatographic peaks, and the Waters CORTECS T3 chromatographic column has better separation effect on various chromatographic peaks and better peak type. Therefore, waters CORTECS T3 (2.1X105 mm,1.6 μm) was chosen as analytical column.
2.2.3 absorption wavelength investigation
Examining 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 as mobile phase a and 0.2% phosphoric acid as mobile phase B, and performing gradient elution according to the specifications in table 1; the flow rate was 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 number of chromatographic peaks is more when 220nm is selected as the detection wavelength, but the response value of the integral chromatographic peak is lower, and the base line is unstable after the retention time is 40 minutes; when 254nm, 274nm and 316nm detection wavelengths are selected, the glycyrrhizin and ferulic acid have maximum absorption, the response values of other chromatographic peaks become low, and partial chromatographic peaks disappear; when 235nm wavelength is selected, the overall response value is higher, the number of chromatographic peaks is the largest, and the base line is stable. By combining the above considerations, the detection wavelength is selected to be 235nm in order to embody the characteristic peak of each medicinal material as much as possible, ensure the response value of each chromatographic peak and ensure the baseline to be stable.
2.2.4 mobile phase investigation
The type and concentration of the buffer solution (mobile phase B) in the mobile phase were examined, and 0.1vol% acetic acid solution, 0.1vol% formic acid solution, 0.1vol% phosphoric acid solution, and 0.2vol% phosphoric acid solution were used, respectively; a Waters CORTECS T3 (2.1 mm. Times.150 mm,1.6 μm) column was used; acetonitrile as mobile phase a, gradient elution was performed as specified in table 1; the flow rate was 0.3mL per minute; the detection wavelength is 235nm; the column temperature is 30 ℃; the sample injection amount was 1. Mu.L, and the results are shown in FIGS. 8 to 11.
Comparing three chromatograms of the 0.1vol% phosphoric acid solution, the 0.1vol% acetic acid solution and the 0.1vol% formic acid solution, the baseline drift is uneven, the baseline is concave after 45 minutes, and the number of chromatographic peaks is small; if acetonitrile-phosphoric acid is used as a mobile phase, the chromatogram information is rich, the chromatographic peak separation degree is good, the base line is stable, the peak-forming time is proper, and the separation effect of 0.2% phosphoric acid on polar components with early peak-forming time is better than that of 0.1vol% phosphoric acid. To sum up, a 0.2vol% phosphoric acid solution was used as buffer (mobile phase B).
2.2.5 flow Rate investigation
Examining the flow rate of the mobile phase, wherein the flow rates are respectively 0.25mL, 0.30mL and 0.35mL per minute; a Waters CORTECS T3 (2.1X105 mm,1.6 μm) column was used; gradient elution was performed as specified in table 1 with acetonitrile as mobile phase a and 0.2vol% phosphoric acid as mobile phase B; the column temperature is 30 ℃; the sample injection amount is 1 mu L; the detection wavelength was 235nm. The results are shown in FIG. 12.
As can be seen from the graph, when the flow rate is 0.3 mL/min, the separation effect of each chromatographic peak is good, the peak type is good, the flow rate can be accepted when the flow rate floats up and down by 0.05mL/min, and the flow rate durability of the method is good.
2.2.6 investigation of column temperature
Examining column temperatures, wherein the column temperatures are 25 ℃, 30 ℃, 35 ℃ and 40 ℃ respectively; a Waters CORTECS T3 (2.1X105 mm,1.6 μm) column was used; acetonitrile as mobile phase a and 0.2% phosphoric acid as mobile phase B, and performing gradient elution according to the specifications in table 1; the flow rate was 0.3mL per minute; the sample injection amount is 1 mu L; the detection wavelength was 235nm. 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 better, the peak type is better, the flow speed can be accepted when the column temperature floats up and down by 5 ℃, and the durability of the column temperature is good.
2.2.7 chromatographic condition determination
According to the above experiments, the chromatographic conditions were determined as follows: octadecylsilane chemically bonded silica (Waters CORTECS T3, column length of 150mm, inner diameter of 2.1mm, particle diameter of 1.6 μm) was used as filler; gradient elution was performed as specified in table 1 with acetonitrile as mobile phase a and 0.2vol% phosphoric acid aqueous solution as mobile phase B; the flow rate was 0.3mL per minute; the column temperature is 30 ℃; the detection wavelength is 235nm, and the theoretical plate number is not lower than 10000 calculated according to gentiopicroside peak.
2.3 examination of the preparation method of the sample solution
2.3.1 extraction solvent investigation
The influence of different solvents on the characteristic spectrum of the body pain-relieving and blood stasis-relieving decoction is examined, 100vol% of methanol, 70vol% of methanol and 50vol% of methanol are respectively used as extraction solvents, the peak areas/sample weighing amounts of 15 characteristic peaks in the body pain-relieving and blood stasis-relieving decoction are calculated by observing the peak and separation degree of the 15 characteristic peaks, the influence of different extraction solvents on the characteristic spectrum of the body pain-relieving and blood stasis-relieving decoction is compared, and the optimal extraction solvent is selected.
Specifically, taking a proper amount of freeze-dried powder of the decoction for relieving body pain and removing blood stasis, grinding, taking about 0.5g, precisely weighing, and combining three groups of two parts, placing into a conical flask with a plug, precisely adding 25mL of the extraction solvent, weighing, performing ultrasonic treatment (with the power of 250W and the frequency of 40 kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with the corresponding extraction solvent, shaking uniformly, filtering, taking the subsequent filtrate, and performing sample injection analysis according to chromatographic conditions under the condition of 2.1.1, wherein the result is shown in Table 4.
Table 4 results table of results of solvent investigation of decoction for relieving body pain and removing blood stasis characteristic spectrum extraction
The results show that: the extraction effect of each characteristic peak is similar when 100vol% methanol, 70vol% methanol and 50vol% methanol are used as the extraction solvent, and the peak type and separation effect of each characteristic peak are not obviously different, wherein when 70vol% methanol is used as the extraction solvent, the total peak area/sample weighing amount of 15 characteristic peaks is maximum, and the theoretical plate number of each characteristic peak is better, so 70vol% methanol is selected as the extraction solvent.
2.3.2 investigation of extraction modes
The influence of different extraction modes on the body pain and blood stasis removing decoction feature map is examined, two extraction modes of ultrasound and reflux are respectively examined, the peak type and the separation degree of 15 feature peaks are observed, the total peak area/sample weighing amount of the 15 feature peaks is calculated, and the influence of different extraction modes on the body pain and blood stasis removing decoction feature map is compared.
Specifically, taking a proper amount of freeze-dried powder of the body pain-relieving and stasis-removing decoction, grinding, taking about 0.5g, precisely weighing, two groups of two parts in parallel, placing into a conical flask with a plug, precisely adding 25mL of 70vol% methanol, weighing, respectively carrying out ultrasonic treatment (with the power of 250W and the frequency of 40 kHz) for 30 minutes, heating and refluxing for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with 70vol% methanol, shaking uniformly, filtering, taking the subsequent filtrate, and carrying out sample injection analysis according to chromatographic conditions under the condition of 2.1.1, wherein the result is shown in Table 5.
TABLE 5 body pain-relieving stasis decoction feature pattern extraction method investigation result table
The results show that: the method adopts different extraction modes, the peak types and separation effects of the characteristic peaks are not obviously different, the difference of total peak area/sample weighing is not large, and the ultrasonic treatment method is considered to be simpler and more convenient, so that the ultrasonic treatment is selected.
2.3.3 extraction time investigation
And (3) observing the influence of different extraction times on the characteristic spectrum of the body pain-relieving and stasis-relieving decoction, and selecting three different extraction times of 15min, 30min and 45 min.
Specifically, taking a proper amount of freeze-dried powder of the body pain-relieving and stasis-removing decoction, grinding, taking about 0.5g, precisely weighing, and combining three groups of two parts, placing into a conical flask with a plug, precisely adding 25mL of 70vol% methanol, weighing, respectively performing ultrasonic treatment (with the power of 250W and the frequency of 40 kHz) for 15min, 30min and 45min, taking out, cooling, weighing again, supplementing the lost weight with 70vol% methanol, shaking uniformly, filtering, taking the subsequent filtrate, and performing sample injection analysis according to chromatographic conditions under the condition of 2.1.1, wherein the result is shown in Table 6.
TABLE 6 results table for investigating extraction time of decoction for relieving body pain and removing blood stasis
The results show that: the total peak area/sample weighing of 15 characteristic peaks is not obviously different by adopting different extraction times, and the RSD value of the total peak area/sample weighing of the three extraction times is 0.98%; in order to ensure the durability and the extraction completion of the method, the ultrasonic extraction time is selected to be 30min.
2.3.4 determination of the method for preparing the sample solution
According to the experimental results, the pretreatment method of the body pain and blood stasis removing decoction characteristic spectrum sample is determined as follows:
taking 0.5g of body pain-relieving stasis decoction freeze-dried powder, precisely weighing, placing into a conical bottle with a plug, adding 25mL of 70vol% methanol, sealing, weighing, performing ultrasonic treatment (power is 250W, frequency is 40 kHz) for 30 minutes, taking out, cooling, weighing again, supplementing the lost weight with 70vol% methanol, shaking uniformly, and filtering to obtain a sample solution.
2.4 methodological verification
2.4.1 specificity investigation
Taking the body pain and blood stasis removing soup of each medicine, and preparing according to the preparation method of the sample solution to obtain the negative sample solution of each medicine.
The preparation method comprises the steps of respectively taking a proper amount of control medicinal materials of gentiana macrophylla, ligusticum wallichii, peach kernel, safflower, liquorice, notopterygium root, myrrh, angelica, vinegar trogopterus dung, rhizoma cyperi, achyranthes root and earthworm, preparing the control medicinal materials into single medicinal materials according to a body pain stasis-removing decoction method, and taking a proper amount of single control medicinal material reference solution of each medicinal material according to a sample preparation method.
1 mu L of each of the sample solution, the negative sample solution without each medicinal material and the single reference solution with each medicinal material is injected into a liquid chromatograph, and sample injection analysis is performed according to the chromatographic conditions of 2.1.1 sections, and the results are shown in fig. 14-25.
As can be seen from fig. 14 to 25: peak 1 (loganin acid) 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 Carthami flos, peak 6 is characteristic component of Oletum Trogopterori Vinegar and semen Persicae, peak 7 (Ferulic acid), peak 10 is characteristic component of rhizoma Ligustici Chuanxiong, peak 7 (Ferulic acid) is characteristic component of radix Angelicae sinensis, peak 7 (Ferulic acid), peak 10, peak 11, peak 14 (Notopterygii rhizoma alcohol) is characteristic component of Myrrha; 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 achyranthes root have low response under the chromatographic condition, the chromatographic peak separation effect is poor, the main components are easily affected by the 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 sample has the same chromatographic peak at the corresponding retention time as the chromatogram of the single drug control drug, and the negative sample lacking the single drug has no interference, thus demonstrating that the method has good specificity.
2.4.2 precision investigation
Taking the sample solution of the freeze-dried powder preparation of the body pain-relieving and blood stasis-relieving decoction in the same batch, repeatedly sampling for 6 times according to the determined chromatographic condition (2.1.1 sections), taking the 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 the characteristic peaks 1-15 and the S peak, and calculating the RSD value. The results are shown in tables 7 to 8.
Table 7 body pain-relieving stasis Shang Tezheng atlas precision investigation result table (relative retention time)
Table 8 body pain-relieving stasis Shang Tezheng map precision investigation result table (relative peak area)
The result shows that the relative retention time RSD of each characteristic peak and 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%, and the relative peak area RSD is more than 3%, the relative peak area range is not recommended, and the relative retention time of each chromatographic peak is less than 3.0%, so that the instrument precision is good.
2.4.3 repeatability investigation
Taking 6 parts of freeze-dried powder of the soup for removing body pain and blood stasis in the same batch, preparing a test solution, determining the peak (peak 7) corresponding to the peak of the ferulic acid reference substance as an S peak according to the chromatographic condition of section 2.1.1, calculating the relative retention time and relative peak area of the characteristic peaks 1-15 and the S peak, and calculating the RSD value. The results are shown in tables 9 to 10.
Table 9 results table of the repeated investigation of the pattern of body pain and stasis dispelling Shang Tezheng (relative retention time)
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Table 10 pain-relieving stasis Shang Tezheng map repeatability investigation result table (relative peak area)
The results showed that the relative retention time RSD of each characteristic peak and S peak was in the range of 0.02% to 0.63%, the relative peak area RSD was in the range of 0.34% to 7.04%, and the relative peak area RSD was more than 3%, with the ferulic acid (peak 7) chromatographic peak as the reference peak S, and it was suggested that the relative peak area range was not specified, since the relative retention time of each chromatographic peak was less than 3.0%, indicating that the reproducibility of the method was good.
2.4.4 stability investigation
Taking the sample solution of the freeze-dried powder of the soup for removing body pain and blood stasis in the same batch, analyzing at 0, 4, 8, 12, 16 and 25 hours according to the chromatographic condition of 2.1.1 sections, respectively, taking the 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 the characteristic peaks 1 to 15 and the S peak, and calculating the RSD value. The results are shown in tables 11 to 12.
Table 11 results table (relative retention time) for investigating stability of decoction for relieving body pain and removing blood stasis
Table 12 results table (relative peak area) for investigating stability of decoction for relieving body pain and removing blood stasis
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The results showed that the relative retention time RSD of each characteristic peak and S peak was in the range of 0.08% -1.31%, the relative peak area RSD was in the range of 0.63% -5.32%, and the relative peak area RSD was more than 3%, with the ferulic acid (peak 7) chromatographic peak as the reference peak, and the relative retention time of each chromatographic peak was less than 3.0%, indicating that the sample solution was relatively stable in 25 hours.
2.4.5 intermediate precision investigation
Different analysts operate on different instruments at different times, and sample solutions of the freeze-dried powder of the soup for removing the stasis of the same batch of body pain are taken and measured according to chromatographic conditions of 2.1.1 sections; the peak corresponding to the peak of the ferulic acid control (peak 7) was S-peak, and the relative retention time and relative peak area of the characteristic peaks 1 to 15 and S-peak were calculated to calculate the RSD value. The results are shown in tables 13 to 14.
Table 13 results of intermediate precision test (relative retention time) of decoction for relieving pain and removing blood stasis
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Exterior 14 decoction for relieving pain of body and removing blood stasis characteristic spectrum middle precision test result (relative peak area)
The results show that the relative retention time RSD of each characteristic peak and 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%, the relative peak area range is not recommended, the relative retention time of peak 1 and bee 2 is more than 5%, the other characteristic peaks are less than 5%, the relative retention time of other chromatographic peaks is less than 3.0%, and the intermediate precision of each characteristic peak is good.
2.5 determination of samples of different batches and determination of the Co-peak
15 small samples (numbered S1-S15) of the sample Shang Jizhun for relieving the body pain and removing the blood stasis are respectively taken, a sample solution is prepared according to a preparation method determined under the condition of 2.1.3, the sample solution is precisely sucked and measured according to the chromatographic condition under the condition of 2.1.1, the superimposed graph of the characteristic graph Shang Yangpin for relieving the body pain and removing the blood stasis is shown as 26, the ferulic acid (peak 7) is used as a reference peak S, and the relative retention time and the relative peak area of each characteristic peak and the S peak are shown as tables 15-18.
TABLE 15 general pain-relieving stasis decoction feature patterns sharing peak-to-hold timetable (Peak 1-Peak 8)
Table 16 15 decoction for relieving pain of body and removing blood stasis characteristic spectra sharing peak-to-hold time table (peak 9-15)
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Table 17 Table 15 batch decoction for relieving pain of the body and removing blood stasis characteristic spectrum sharing the peak-to-peak area table (Peak 1-Peak 8)
TABLE 18 15 general pain-relieving stasis decoction feature atlas sharing the peak-to-peak area Table (Peak 9-Peak 15)
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And (3) referring to the characteristic spectrum results, determining the characteristic spectrum standard of the body pain and blood stasis removing decoction as follows: the chromatogram of the sample should show 15 characteristic peaks, wherein 9 peaks respectively correspond to the retention time of corresponding reference peaks, specifically, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin yellow A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin glycyrrhizin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak. The relative retention time of each peak should be within + -5% of the specified value: 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 relative retention time RSD value of the common peak of 15 batches of body pain-relieving and stasis-relieving soup is in the range of 0.05-0.52%, and the relative peak area RSD value is in the range of 14.21-126.22%. Therefore, the common peak of the body pain and blood stasis removing decoction prepared from different batches of medicinal materials in different producing places has stable relative retention time, and the relative peak area difference is larger, so that the components of the decoction have stable types and larger content difference.
3. UPLC-MS identification method of body pain and stasis removing decoction
3.1 preparation of chromatographic conditions and control solution, test solution
3.1.1 chromatographic conditions
Chromatographic conditions: octadecylsilane chemically bonded silica is used as filler (Waters HSS T3, column length of 150mm, inner diameter of 2.1mm, particle diameter of 1.8 μm); gradient elution was performed as specified in table 19 with acetonitrile as mobile phase a and 0.1% formic acid as mobile phase B; the flow rate was 0.3mL per minute; the column temperature is 30 ℃; the detection wavelength was 235nm. The theoretical plate number is not less than 10000 calculated according to gentiopicroside peak.
TABLE 19 gradient elution table
3.1.2 Mass Spectrometry conditions
Mass spectrometry conditions: HESI ion source, positive and negative ion monitoring mode; the cracking voltage is 3.80kV; the auxiliary air flow is 10mL/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 MS/MS mode, collision energy in positive and negative ion modes is 20eV and 40eV respectively, and leucine enkephalin is used as an internal standard to correct mass accuracy.
3.1.3 preparation method of control solution
Precisely weighing appropriate amounts of reference substances such as neochlorogenic acid, L-tryptophan, loganin acid, D-amygdalin, swertiamarin, hydroxysafflor yellow A, cryptochlorogenic acid, ferulic acid, glycyrrhizin, gentiopicroside, swertiamarin, glycyrrhizin, astragalin, naringenin, apigenin, senkyunolide I, glycyrrhizic acid, ammonium glycyrrhizate, glycyrrhetinic acid and ginsenoside Ro, placing into a 20mL measuring flask, adding 70% methanol to prepare a solution containing 30 μg of neochlorogenic acid, 15 μg of L-tryptophan, 40 μg of loganin acid, 40 μg of D-amygdalin, 30 μg of swertiamarin, 60 μg of hydroxysafflor yellow A, 30 μg of cryptochlorogenic acid, 10 μg of ferulic acid, 30 μg of glycyrrhizin, 60 μg of gentiopicroside, 30 μg of swertiamarin, 20 μg of glycyrrhizin, 10 μg of astragalin, 10 μg of naringin, 70 μg of naringin, 10 μg of glycyrrhizin, 40 μg of glycyrrhizin, and 40 μg of glycyrrhizic acid, and 40 μg of shaking to obtain a solution of the reference substances such as a liquid, and mixing the solution with equal volume of the reference substances such as 40 μg of the liquid; precisely weighing the control substances of umbelliferone, imperatorin, isoimperatorin, ligustilide, notopterygium alcohol and alpha-cypnone in a 20mL measuring flask, adding methanol to prepare a mixed solution containing 80 mug of umbelliferone, 20 mug of imperatorin, 40 mug of isoimperatorin, 20 mug of ligustilide, 20 mug of notopterygium alcohol and 40 mug of alpha-cypnone in each 1mL of mixed solution, dissolving, fixing the volume to a scale, and shaking uniformly to obtain a second UPLC-MS control solution; stored in a refrigerator at 4deg.C, and filtered with a 0.22 μm microporous filter membrane before use.
3.1.4 preparation method of sample solution
Taking 0.5g of body pain-relieving stasis decoction freeze-dried powder, precisely weighing, placing into a conical bottle with a plug, adding 25mL of 70% methanol, performing ultrasonic treatment (power is 250W, frequency is 40 kHz) for 30 minutes, centrifuging for 5 minutes at 8000r/min, taking supernatant, filtering with a 0.22 mu m filter membrane, and collecting filtrate to obtain a UPLC-MS test sample solution.
3.1.5 assays
Precisely absorbing 1 μl of the first UPLC-MS reference substance solution, the second UPLC-MS reference substance solution and UPLC-MS sample solution respectively, injecting into an ultra-high performance liquid chromatography-quadrupole-electrostatic orbitrap-high resolution mass spectrometer, and measuring.
3.2 analysis results
According to literature reports, TCMSP databases and reference substances, the chemical components in the TCMSP databases are identified by combining chromatographic retention behavior, molecular weight information and mass spectrum cracking rules, and the results are shown in Table 20, FIG. 28 and FIG. 29. In summary, UPLC-Q-Exactive Orbitrap MS is adopted to carry out chromatographic separation, mass spectrum data acquisition and structure identification of inherent components on various components in the decoction for relieving body pain and removing blood stasis, and 80 components such as organic acid, nucleosides, iridoids, flavonoids, saponins, coumarin and the like are identified, so that basis and precondition are provided for further researching the medicinal effect substance basis of the decoction for relieving body pain and removing blood stasis.
Table 20 decoction UPLC-MS component identification result for relieving pain of body and removing blood stasis
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While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that changes and modifications may be made without departing from the principles of the invention, such changes and modifications are also intended to be within the scope of the invention.

Claims (14)

1. The method for constructing the body pain stasis-removing decoction characteristic spectrum is characterized by comprising the following steps of:
(1) Respectively taking tryptophan, loganin acid, gentiopicroside, hydroxysafflor yellow A, ferulic acid, glycyrrhizin, apioside, glycyrrhizic acid and notopterygium alcohol reference substances, and adding methanol for dissolving or extracting to obtain reference substance solution;
(2) Taking a body pain removing and stasis removing decoction, adding 70vol% methanol for extraction, and preparing a test solution;
(3) Respectively sucking 0.5-1.5 mu L of reference substance solution and 0.5 mu L of sample solution, and injecting the solutions into a liquid chromatograph for detection, wherein the liquid chromatograph uses octadecylsilane chemically bonded silica gel as a filler, the column length is 150mm, the inner diameter is 2.1mm, the particle size is 1.6 mu m, and the column temperature is 25-35 ℃; the liquid chromatograph takes acetonitrile as a mobile phase A, 0.2% phosphoric acid aqueous solution as a mobile phase B, the flow rate is 0.25-0.35 mL/min, and the detection wavelength is 235nm;
Wherein the gradient elution is performed according to the following procedure:
0-5 min, mobile phase A is 6%, mobile phase B is 94%;
5-12 min, mobile phase A from 6% to 10%, mobile phase B from 94% to 90%;
12-22 min, mobile phase A from 10% to 12%, mobile phase B from 90% to 88%;
22-24 min, mobile phase A from 12% to 16%, mobile phase B from 88% to 84%;
24-36 min, mobile phase A from 16% to 22%, mobile phase B from 84% to 78%;
36-46 min, mobile phase A from 22% to 32%, mobile phase B from 78% to 68%;
46-50 min, mobile phase A from 32% to 50%, mobile phase B from 68% to 50%;
50-58 min, mobile phase A from 50% to 65%, mobile phase B from 50% to 35%;
58-68 min, mobile phase A from 65% to 78% and mobile phase B from 35% to 22%.
2. The method for constructing a body pain and blood stasis removing decoction feature map according to claim 1, wherein in the method for constructing a body pain and blood stasis removing decoction feature map, in the step (3), 1 μl of each of a reference solution and a test solution is sucked and injected into a liquid chromatograph for detection, wherein the column temperature is 30 ℃; the flow rate was 0.3mL/min and the detection wavelength was 235nm.
3. The method for constructing a body pain-relieving stasis decoction feature map according to claim 1, wherein in the step (1), the reference solution is prepared by the following steps: the method comprises the steps of respectively weighing tryptophan, loganin acid, gentiopicroside, hydroxysafflor yellow A, ferulic acid, glycyrrhizin, apioside, glycyrrhizic acid and notopterygium alcohol, adding methanol to prepare a mixed solution containing 15 mug tryptophan, 40 mug loganin acid, 60 mug gentiopicroside, 60 mug hydroxysafflor yellow A, 20 mug ferulic acid, 30 mug glycyrrhizin, 70 mug apioside, 40 mug glycyrrhizic acid and 20 mug notopterygium alcohol per 1mL, and obtaining a reference solution.
4. The method for constructing a body pain and blood stasis removing decoction feature map according to claim 1, wherein in the step (2), the extraction time is 15-45 min, and the extraction mode is ultrasonic extraction and/or reflux extraction.
5. The method for constructing a body pain-relieving stasis decoction feature map according to claim 1 or 4, wherein the step (2) comprises:
taking 0.2-0.5 g of body pain-relieving stasis decoction freeze-dried powder, placing the powder into a conical bottle with a plug, adding 25-30 mL of 70% methanol, weighing, performing ultrasonic treatment for 30 minutes, taking out, cooling, weighing again, supplementing the weight of loss with 70% methanol, shaking uniformly, filtering, and taking the subsequent filtrate to obtain the test solution.
6. The method for constructing a body pain-relieving stasis decoction feature map according to claim 1, wherein the body pain-relieving stasis decoction feature map comprises 15 feature peaks; wherein, peak 1 is horse Qian Gansuan peak, peak 2 is tryptophan peak, peak 4 is gentiopicroside peak, peak 5 is hydroxy carthamin yellow A peak, peak 7 is ferulic acid peak, peak 8 is glycyrrhizin peak, peak 9 is apigenin peak, peak 13 is glycyrrhizin peak, and peak 14 is notopterygium alcohol peak;
the relative retention time of each peak, with peak 7 as reference S peak, is: 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.
7. The method for constructing a body pain-relieving stasis decoction feature map according to claim 1, further comprising:
(4) And (3) carrying out characteristic peak chemical component identification on the characteristic spectrum of the body pain-relieving stasis decoction by adopting a UPLC-MS identification method.
8. The method for constructing a body pain-relieving stasis decoction feature map according to claim 7, wherein the method for constructing a UPLC-MS identification method is as follows:
(1) Dissolving neochlorogenic acid, L-tryptophan, loganin acid, D-amygdalin, swertiamarin, hydroxysafflor yellow A, cryptochlorogenic acid, ferulic acid, glycyrrhizin, gentiopicroside, swertiamarin, glycyrrhizin, rutin, astragalin, naringenin, apigenin, senkyunolide I, glycyrrhizic acid, ammonium glycyrrhizate, glycyrrhetinic acid and ginsenoside Ro in 70% methanol to obtain a first UPLC-MS reference solution; adding methanol into control substances such as umbelliferone, decursin, imperatorin, isoimperatorin, ligustilide, notopterygol and alpha-cypnone, and dissolving to obtain second UPLC-MS control solution;
(2) Extracting decoction with solvent to obtain UPLC-MS test solution;
(3) Taking a first UPLC-MS reference substance solution, a second UPLC-MS reference substance solution and a UPLC-MS test substance solution, injecting into an ultra-high performance liquid chromatography-quadrupole-electrostatic orbitrap-high resolution mass spectrometer, and establishing chromatographic conditions and mass spectrometry conditions of a UPLC-MS identification method;
(4) Calculating the unknown compound element composition and molecular formula by using a workstation; and (3) 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 spectrum characteristic peak.
9. The method for constructing a body pain-relieving stasis decoction feature map according to claim 8, wherein in a UPLC-MS identification method, the preparation method of the UPLC-MS test sample is as follows: taking 0.2-0.5 g of body pain-relieving stasis decoction freeze-dried powder, placing the powder into a conical bottle with a plug, adding 25-30 mL of 70% methanol, weighing, performing ultrasonic treatment for 15-45 minutes, centrifuging for 5 minutes at 8000r/min, taking supernatant, filtering with a 0.22 mu m filter membrane, and collecting filtrate.
10. The method for constructing a body pain and blood stasis removing decoction feature map according to claim 8, wherein in the UPLC-MS identification method, the chromatographic conditions are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica gel chromatographic column with column length of 150mm, inner diameter of 2.1mm and particle diameter of 1.8 μm, acetonitrile as mobile phase A, 0.05-0.1% formic acid water solution as mobile phase B, and column temperature of 25-35deg.C; 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 as follows:
0-3 min, mobile phase A is 0%, mobile phase B is 100%;
3-7 min, mobile phase A from 0% -4% and mobile phase B from 100% -96%;
7-15 min, the mobile phase A is from 4% to 10%, and the mobile phase B is from 96% to 90%;
15-25 min, mobile phase A from 10% to 12%, mobile phase B from 90% to 88%;
25-30 min, mobile phase A from 12% -15%, mobile phase B from 88% -85%;
30-40 min, mobile phase A from 15% to 22%, mobile phase B from 85% to 78%;
40-50 min, mobile phase A from 22% to 32%, mobile phase B from 78% to 68%;
50-55 min, the mobile phase A is from 32% to 50%, and the mobile phase B is from 68% to 50%;
55-60 min, mobile phase A from 50% -65%, mobile phase B from 50% -35%;
60-70 min, the mobile phase A is from 65% to 78%, and the mobile phase B is from 35% to 22%.
11. The method for constructing a body pain-relieving stasis decoction feature map according to claim 8, 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 air flow is 10mL/min, the temperature of the ion transmission tube is 320 ℃, the auxiliary air temperature is 350 ℃, and the scanning mode is FullMS/dd-MS 2 FullMS resolution is 70000, dd-MS 2 Resolution 17500; the mass scanning range m/z is 100-1500; in MS/MS mode, collision energy in positive and negative ion modes is 20eV and 40eV respectively, and leucine enkephalin is used as an internal standard to correct mass accuracy.
12. The method for constructing a characteristic spectrum of body pain and blood stasis removing soup as claimed in claim 1, wherein the body pain and blood 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 and 7.46 parts of earthworm.
13. The method for constructing a characteristic spectrum of a body pain-relieving and stasis-relieving decoction according to claim 1, wherein the method for preparing the body pain-relieving and stasis-relieving decoction is as follows:
adding 6-10 times of water of the total weight of the raw materials into the large-leaf gentian root, the szechuan lovage rhizome, the peach seed, the safflower, the liquorice, the notopterygium root, the myrrh, the angelica, the vinegar trogopterus dung, the nutgrass galingale rhizome, the achyranthes root and the earthworm, soaking for 30-60 min, boiling with strong fire, keeping micro-boiling with slow fire for 20-40 min, and filtering; adding water which is 5-8 times of the total weight of the raw materials into the obtained filter residues, decocting for 20-30 min, and filtering; mixing the two decoctions to obtain the final product.
14. The method for constructing a characteristic spectrum of a body pain-relieving and stasis-relieving decoction according to claim 1, wherein the method for preparing the body pain-relieving and stasis-relieving decoction is as follows:
Taking gentiana macrophylla, ligusticum wallichii, peach kernel, safflower, liquorice, notopterygium root, myrrh, angelica, vinegar trogopterus dung, nutgrass galingale rhizome, achyranthes root and earthworm, adding 6-8 times of water of the total weight of the raw materials, and boiling with slow fire until the weight of filtrate is 3-4 times of the weight of the raw materials.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
CN108578541A (en) * 2018-07-06 2018-09-28 海王（湖北）中医药研究总院有限公司 A kind of Chinese medical extract that treating rheumatism and cardiovascular and cerebrovascular disease, preparation method and applications

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Publication number Priority date Publication date Assignee Title
CN108578541A (en) * 2018-07-06 2018-09-28 海王（湖北）中医药研究总院有限公司 A kind of Chinese medical extract that treating rheumatism and cardiovascular and cerebrovascular disease, preparation method and applications

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王琳 等.经典名方身痛逐瘀汤物质基准UPLC指纹图谱建立及其5种成分含量测定.《中国中药杂志》.2022,第47卷(第47期),334-342. *

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