Construction of HPLC-ELSD (High Performance Liquid Chromatography-Evaporative Light Scattering Detector) characteristic chromatogram of standard decoction of garden balsam

Abstract

According to the method, a high performance liquid chromatograph and an evaporative light scattering detector are used for detection, the establishment method of the HPLC-ELSD characteristic chromatogram of the garden balsam seed decoction piece standard decoction is provided, 10 characteristic peaks are shown in the characteristic chromatogram established through the method, the characteristic peak information of a sample can be fully reflected, the chemical component characteristics of the garden balsam seed standard decoction are shown, and the method is suitable for large-scale popularization and application. A relatively comprehensive and effective evaluation method can be provided for quality control of the standard garden balsam seed decoction piece decoction; furthermore, the adulterant standard decoction losing medicinal material characters can be identified through the established HPLC-ELSD characteristic chromatogram, and a basis is provided for formulating the quality standard of the garden balsam seed decoction piece standard decoction; in addition, the construction method provided by the invention also has the advantages of rapidness, stability, good repeatability and strong specificity.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine identification and drug analysis, and specifically relates to the construction of an HPLC-ELSD characteristic spectrum of a standard decoction of Jijizi decoction pieces and its identification application. Background Art

[0002] Standard decoctions of Chinese herbal medicines are single-flavor decoctions of Chinese herbal medicines prepared by standardized processes under the guidance of traditional Chinese medicine theory and based on clinical application, with reference to modern extraction methods. Standard decoctions of Chinese herbal medicines are the reference for quality control of related Chinese medicine products and are the key hub connecting Chinese herbal medicines and modern Chinese medicine preparations. Therefore, the research on the quality standards of standard decoctions of Chinese herbal medicines can lay the foundation for the formulation of formula granules derived from decoctions and other Chinese medicine finished products.

[0003] Impatiens balsamina L. is the dried mature seed of Impatiens balsamina L., a plant of the Impatiens family. It is harvested in summer and autumn when the fruit is about to mature, dried in the sun, and the peel and impurities are removed. It tastes pungent, bitter, and warm in nature. It enters the spleen and liver meridians, and has the effects of breaking blood and promoting qi, dredge menstruation and relieving pain. It is used for chest and flank stabbing pain, chest pain, dysmenorrhea, amenorrhea, symptom scars, rheumatic shoulder and arm pain, and swelling and pain from falls. Studies have shown that the main chemical components of Impatiens balsamina include fatty oils, diterpenes, flavonoids, triterpenoid saponins, sterols, polypeptide compounds and proteins. It has the effects of promoting skin penetration, anti-fertility, anti-tumor, anti-cancer, and antibacterial. Among them, flavonoids and triterpenoid saponins are the material basis of the main pharmacological effects of Impatiens balsamina. The Impatiens balsamina medicinal material item of the "Chinese Pharmacopoeia" (2020 edition) lacks fingerprint / characteristic map items, and it is difficult to control its quality as a whole, so the quality standards need to be further improved.

[0004] Fingerprint / characteristic spectrum can comprehensively reflect the types and quantities of chemical components contained in traditional Chinese medicine and its preparations due to its overall control concept, and can identify the differences between medicinal samples and related counterfeits and confusion products, so as to comprehensively evaluate the quality of traditional Chinese medicine. Due to the weak research on the material basis of schisandra chinensis, there are very few studies on its fingerprint / characteristic spectrum. The separation effect of each chromatographic peak of the fingerprint / characteristic spectrum of schisandra chinensis established in the report is very poor, only one type of compound (flavonoids) is concentrated, and the number of identified compounds is very small. There is no report on the research related to its decoction standard decoction.

[0005] In the relevant literature reports and the catalog of 130 commonly used Chinese medicine counterfeits and confusion products, it is mentioned that Perilla seed, Chrysanthemum seed, Wangjiang South seed, Radish seed, Astragalus seed, and Melia seed are similar to the properties of Jijizi and are confused products of Jijizi. In addition, due to the loss of medicinal properties, the standard decoction of Jijizi and related preparations cannot be identified by traditional medicinal identification methods such as properties and smell, and then the different confusion products are distinguished, which seriously affects the safety of its medication based on the difference in efficacy. Therefore, this study established the characteristic spectrum of the standard decoction of Jijizi decoction pieces, and simultaneously characterized as many of the main chemical components (flavonoids and saponins) in the standard decoction of Jijizi as possible, which can effectively qualitatively identify related confusion products. In addition, the establishment of this characteristic spectrum not only provides a basis for the establishment of the quality standard of the standard decoction of Jijizi decoction pieces, but also provides an important reference for the establishment of the quality standard of Jijizi formula granules and related Chinese medicine preparations. Summary of the invention

[0006] The purpose of the present invention is to provide a method for establishing an HPLC-ELSD characteristic spectrum of a standard decoction of Jijizi medicinal slices, and to identify characteristic components. The method is rapid, stable and highly specific. The established HPLC-ELSD characteristic spectrum can be used to identify standard decoctions of confused products that have lost their medicinal properties, thereby providing a more comprehensive and effective evaluation method for the quality control of standard decoctions of Jijizi medicinal slices.

[0007] The first aspect of the present invention provides a method for establishing a HPLC-ELSD characteristic spectrum of a standard decoction of Jijizi decoction pieces, comprising the following steps:

[0008] Take one or more of the reference substances of impatiens tetraol saponin K, impatiens tetraol saponin A, impatiens tetraol saponin G, impatiens tetraol saponin L, impatiens tetraol saponin B, and quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside to prepare a reference substance solution;

[0009] Prepare the test solution from the standard decoction of Jijizi decoction pieces;

[0010] The reference solution and the test solution are respectively detected by high performance liquid chromatography-evaporative light scattering detection method;

[0011] The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method include: using acetonitrile as mobile phase A and water as mobile phase B for gradient elution;

[0012] The procedure of the gradient elution includes:

[0013] From 0 to 25 min, the volume percentage of the mobile phase A increased from 16% to 32%, and the volume percentage of the mobile phase B decreased from 84% to 68%;

[0014] 25min~30min, the volume percentage of the mobile phase A is 32%, and the volume percentage of the mobile phase B is 68%;

[0015] The detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 1 to 3; a carrier gas flow rate of 2.5 L / min to 3.5 L / min; and a temperature of 110° C. to 120° C.

[0016] In some embodiments of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: using an Agilent Poroshell 120EC-C18 chromatographic column.

[0017] In some embodiments of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a flow rate of 0.8 mL / min to 1.2 mL / min.

[0018] In some embodiments of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a column temperature of 33°C to 37°C.

[0019] In some embodiments of the present invention, methanol is used as a dissolving reagent to prepare the reference solution; the concentration of the impatiens tetraol saponin K reference solution is 280 μg / mL to 320 μg / mL; the concentration of the impatiens tetraol saponin A reference solution is 260 μg / mL to 300 μg / mL; the concentration of the impatiens tetraol saponin G reference solution is 270 μg / mL to 310 μg / mL; the concentration of the impatiens tetraol saponin L reference solution is 290 μg / mL to 330 μg / mL; the concentration of the impatiens tetraol saponin B reference solution is 270 μg / mL to 310 μg / mL; the concentration of the quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside reference solution is 140 μg / mL to 180 μg / mL.

[0020] In some embodiments of the present invention, the step of preparing the test solution includes: mixing the standard decoction of Jijizi decoction pieces with a first alcohol-containing aqueous solution having a volume fraction of 50% to 80% for extraction, concentrating, dissolving the residue, filtering, and obtaining a filtrate;

[0021] The alcohol in the first alcohol-containing aqueous solution is selected from methanol or ethanol;

[0022] The extraction method is ultrasonic extraction or heating reflux extraction, and the conditions of ultrasonic extraction include: power of 200W to 300W, frequency of 35kHz to 45kHz, and time of 30min to 60min.

[0023] In some embodiments of the present invention, in the step of preparing the test solution, the amount of ethanol solution corresponding to every 0.1 g of the standard decoction of the Aconitum carmichaelii is 15 mL to 25 mL.

[0024] It can be understood that in the present invention, the test sample of the standard decoction of Jijizi medicinal pieces is in the form of freeze-dried powder.

[0025] It can be understood that before the extraction solvent is used for extraction, the step of grinding the standard decoction sample of the Jijizi decoction piece to prepare the powder sample of the standard decoction sample of the Jijizi decoction piece is also included. Furthermore, the standard decoction sample of the Jijizi decoction piece in the "the amount of ethanol solution corresponding to each 0.1g of the standard decoction sample of the Jijizi decoction piece is 15mL to 25mL" refers to the powder sample of the standard decoction sample of the Jijizi decoction piece.

[0026] In some embodiments of the present invention, 10 characteristic peaks are presented in the characteristic spectrum, and the peak corresponding to the reference peak of the impatiens tetraol saponin K reference substance is the S peak. The relative retention times of peaks 1 to 3, peaks 5 to 8, and peak 10 with the S peak are calculated, and the relative retention times are within ±10% of the specified values, and the specified values ​​are: peak 1 is 0.21, peak 2 is 0.78, peak 3 is 0.89, peak 5 is 1.04, peak 6 is 1.07, peak 7 is 1.14, peak 8 is 1.24, and peak 10 is 1.59.

[0027] In some embodiments of the present invention, the establishment method further comprises the step of performing high-resolution mass spectrometric identification of characteristic peaks in the characteristic spectrum in conjunction with mass spectrometry;

[0028] The mass spectrometry conditions include:

[0029] HESI ion source parameters include: sheath gas flow rate of 30arb~40arb, auxiliary gas flow rate of 8arb~12arb, spray voltage of 3.5kV~4.0kV, S-lens voltage of 45V~55V, heating temperature of 320℃~380℃, and capillary temperature of 320℃~380℃;

[0030] The mass spectrometry scanning parameters include: the scanning mode is positive ion mode and / or negative ion mode, the scanning range is 120m / z to 1200m / z, the normalized collision energy is 40V, and the mass spectrum type is a peak shape diagram.

[0031] The second aspect of the present invention also provides a method for identifying a standard decoction of Jijizi decoction pieces, comprising the following steps:

[0032] Take the test product to prepare a test product solution;

[0033] The test solution is detected by high performance liquid chromatography-evaporative light scattering detection, and the obtained chromatogram of the test product is compared with the characteristic spectrum obtained by the method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of the Jijizi decoction pieces;

[0034] The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method include: using acetonitrile as mobile phase A and water as mobile phase B for gradient elution;

[0035] The procedure of the gradient elution includes:

[0036] From 0 to 25 min, the volume percentage of the mobile phase A increased from 16% to 32%, and the volume percentage of the mobile phase B decreased from 84% to 68%;

[0037] 25min~30min, the volume percentage of the mobile phase A is 32%, and the volume percentage of the mobile phase B is 68%;

[0038] The detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 1 to 3; a carrier gas flow rate of 2.5 L / min to 3.5 L / min; and a temperature of 110° C. to 120° C.

[0039] In some embodiments of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: using an Agilent Poroshell 120EC-C18 chromatographic column.

[0040] In some embodiments of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a flow rate of 0.8 mL / min to 1.2 mL / min.

[0041] In some embodiments of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a column temperature of 33°C to 37°C.

[0042] In some embodiments of the identification method of the present invention, the step of preparing the test solution comprises:

[0043] The sample to be tested is mixed with a second alcohol-containing aqueous solution having a volume fraction of 50% to 80% to perform extraction, concentrated, the residue is dissolved, filtered, and the filtrate is collected;

[0044] The alcohol in the second alcohol-containing aqueous solution is selected from methanol or ethanol;

[0045] The extraction method is ultrasonic extraction or heating reflux extraction, and the conditions of ultrasonic extraction include: power of 200W to 300W, frequency of 35kHz to 45kHz, and time of 30min to 60min.

[0046] In some embodiments of the identification method of the present invention, in the step of preparing the test product solution, the amount of ethanol solution corresponding to each 0.1 g of the test product is 15 mL to 25 mL.

[0047] In some embodiments of the identification method of the present invention, the test product includes at least one of Perilla seed, Chrysanthemum chinense seed, Rhizoma Cibotii flavescentis seed, Radish seed, Astragalus complanatus seed and Malva ovata seed.

[0048] Compared with traditional technologies, the present invention has the following beneficial effects: the present invention utilizes high performance liquid chromatography-evaporative light scattering detection to detect, and provides a method for establishing an HPLC-ELSD characteristic spectrum of a standard decoction of Jijizi herbal pieces by selecting appropriate conditions. The method is rapid, stable and highly specific, and can identify standard decoctions of confused products that have lost their medicinal properties through the established HPLC-ELSD characteristic spectrum, thereby providing a more comprehensive and effective evaluation method for the quality control of standard decoctions of Jijizi herbal pieces. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the chromatogram of the standard decoction of Jijizi under different chromatographic conditions;

[0050] Figure 2 The chromatograms of different chromatographic columns for the standard decoction of Jijizi;

[0051] Figure 3 This is a survey diagram of different extraction solvents for the characteristic spectrum of the standard decoction of Jijizi;

[0052] Figure 4 This is a survey diagram of different extraction methods for the characteristic spectrum of the standard decoction of Jijizi;

[0053] Figure 5 This is a graph showing the characteristic spectrum of the standard decoction of Jijizi at different extraction times;

[0054] Figure 6 This is a graph for investigating the specificity of the characteristic spectrum of the standard decoction of Jijizi;

[0055] Figure 7 This is an overlay of characteristic spectra of 21 batches of standard decoctions of Jijizi;

[0056] Figure 8 This is the characteristic spectrum of the reference medicinal material of Aconitum; among them, Peak 1: Quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside; Peak 2: Impatiens tetraol saponin B; Peak 4: Impatiens tetraol saponin K; Peak 8: Impatiens tetraol saponin G; Peak 9: Impatiens tetraol saponin A; Peak 10: Impatiens tetraol saponin L;

[0057] Fig. 9This is the reference characteristic spectrum of the standard decoction of Jijizi; among them, Peak 1: Quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside; Peak 2: Impatiens tetraol saponin B; Peak 4: Impatiens tetraol saponin K; Peak 8: Impatiens tetraol saponin G; Peak 9: Impatiens tetraol saponin A; Peak 10: Impatiens tetraol saponin L;

[0058] Fig.10 This is the total ion flow chart and ultraviolet absorption chromatogram of the test solution of the standard decoction of Jijizi decoction pieces;

[0059] Fig.11 The results were confirmed by the reference substance for the characteristic spectrum of the standard decoction of Jijizi;

[0060] Fig.12 This is a comparison chart of the characteristic spectra of standard decoctions of Perilla seeds, Chrysanthemum seeds and Radix Aconiti Lateralis;

[0061] Fig.13 This is a comparison chart of the characteristic spectra of the standard decoctions of Radish Seed and Aconitum;

[0062] Fig.14 This is a comparison chart of the characteristic spectra of standard decoctions of Fructus Lysimachiae and Fructus Aconitum;

[0063] Fig.15 This is a comparison chart of the characteristic spectra of the standard decoctions of Astragalus and Aconitum;

[0064] Fig.16 This is a comparison chart of the characteristic spectra of the standard decoctions of Wangjiangnan and Jijizi. DETAILED DESCRIPTION

[0065] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0067] The complexity and diversity of the chemical components of traditional Chinese medicine are the basis for its efficacy, and are also the difficulty and key point in evaluating the quality of medicinal materials. Fingerprints / characteristics can completely and systematically characterize the similarity characteristics of the main chemical components in medicinal samples; chemical pattern recognition can identify the differences between medicinal samples to comprehensively evaluate the quality of traditional Chinese medicine. According to the survey, due to the weak research on the material basis of acute sputum, there are few studies on the fingerprints / characteristics of acute sputum, so the research on fingerprints / characteristics is more difficult. At present, the research on the fingerprint / characteristic spectrum of the licorice root mainly focuses on medicinal materials. For example, Xu Yan et al. established the fingerprint spectrum of the medicinal materials of the licorice root in the study of the HPLC fingerprint spectrum of the licorice root. Moreover, the separation effect of the chromatographic peaks of the licorice root fingerprint / characteristic spectrum established in the report is poor. Most of the studies only involve one type of compound (flavonoids or saponins), such as Tan Lina, Study on saponin components of the licorice root [D]; Pei Hui et al., HPLC-ELSD method for determining the contents of four balsam pear tetraglycosides in the licorice root [J]; Wang Wei et al., Preliminary study on the HPLC fingerprint spectrum of flavonoid components in the licorice root [J]. It is difficult to fully reflect the intrinsic quality of the licorice root.

[0068] As a reference for the quality control of related Chinese medicine products, the standard decoction of Chinese herbal medicine pieces is the key hub connecting Chinese herbal medicine pieces and modern Chinese medicine preparations. Due to the loss of medicinal material properties, the standard decoction of Jinjizi pieces cannot be identified by traditional medicinal material identification methods such as properties and smell, and then different confusion products can be distinguished, which seriously affects its medication safety based on the difference in efficacy. Therefore, this study established a characteristic spectrum of the standard decoction of Jinjizi pieces, and simultaneously characterized as many of the main chemical components (flavonoids and saponins) in the standard decoction of Jinjizi as possible, which can effectively qualitatively identify related confusion products. In addition, the establishment of this characteristic spectrum not only provides a basis for the establishment of the quality standard of the standard decoction of Jinjizi pieces, but also provides an important reference for the establishment of the quality standard of Jinjizi formula granules and related Chinese medicine preparations.

[0069] In the present invention, the standard decoction of the Jijizi decoction pieces can be prepared by the following method:

[0070] 1. Preparation of Jijizi decoction pieces

[0071] Since there is no processing item for decoction pieces under the item of Jijizi in the Chinese Pharmacopoeia (2020 edition), the processing method is as follows: take Jijizi medicinal materials and pick out impurities and spoiled products. According to the principle of "every shell must be crushed and every seed must be broken" under the item "Preparation of decoctions for research and characterization of standard decoctions" in the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules", Jijizi decoction pieces are crushed.

[0072] 2. Preparation of standard decoction of Jijizi slices

[0073] Take 100g of Radix Achyranthis Bidentatae decoction piece, add water and decoct twice, decoct for the first time and add 8 times of water gagings, soak for 30 minutes, high heat (500W) boils and then slow fire (200W) keeps slightly boiling for 30 minutes, decocting liquid is filtered while hot with 350 mesh screens, and filtrate is cooled with cold water rapidly. Decoction for the second time adds 6 times of water gagings, high heat (500W) boils and then slow fire (200W) keeps slightly boiling for 25 minutes, decocting liquid is filtered while hot with 350 mesh screens, and filtrate is cooled with cold water rapidly, and merges twice of filtrate. Filtrate is transferred in 2000ml round-bottomed flask, and reduced pressure low temperature is concentrated to 100ml, and vacuum freeze drying obtains standard decoction freeze-dried powder.

[0074] The standard decoction of the Jijizi herbal medicine pieces in the embodiment of the present invention can be prepared by the above preparation method. It can be understood that it is not limited to this and one or more steps can be adjusted on the basis of the above preparation method.

[0075] The first aspect of the present invention provides a method for establishing a HPLC-ELSD characteristic spectrum of a standard decoction of Jijizi decoction pieces, comprising the following steps:

[0076] Take one or more of the reference substances of impatiens tetraol saponin K, impatiens tetraol saponin A, impatiens tetraol saponin G, impatiens tetraol saponin L, impatiens tetraol saponin B, and quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside to prepare a reference substance solution;

[0077] Prepare the test solution from the standard decoction of Jijizi decoction pieces;

[0078] The reference solution and the test solution are respectively detected by high performance liquid chromatography-evaporative light scattering detection method;

[0079] The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method include: using acetonitrile as mobile phase A and water as mobile phase B for gradient elution;

[0080] The procedure of the gradient elution includes:

[0081] From 0 to 25 min, the volume percentage of the mobile phase A increased from 16% to 32%, and the volume percentage of the mobile phase B decreased from 84% to 68%;

[0082] 25min~30min, the volume percentage of the mobile phase A is 32%, and the volume percentage of the mobile phase B is 68%;

[0083] The detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 1 to 3; a carrier gas flow rate of 2.5 L / min to 3.5 L / min; and a temperature of 110° C. to 120° C.

[0084] Preferably, the detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 2; a carrier gas flow rate of 3.0 L / min; and a temperature of 110°C.

[0085] In some examples of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: using an Agilent Poroshell 120EC-C18 chromatographic column.

[0086] Preferably, the specifications of the chromatographic column are: column length of 150 mm, inner diameter of 4.6 mm, and filler particle size of 2.7 μm.

[0087] In some examples of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a flow rate of 0.8 mL / min to 1.2 mL / min.

[0088] It can be understood that in the present invention, the flow rate is, for example, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, and preferably, the flow rate is 1.0 mL / min.

[0089] In some examples of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method further include: a column temperature of 30°C to 37°C.

[0090] It can be understood that in the present invention, the column temperature is, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. Preferably, the column temperature is 35°C.

[0091] In some examples of the present invention, methanol is used as a dissolving reagent to prepare the reference solution; the concentration of the impatiens tetraol saponin K reference solution is 280μg / mL to 320μg / mL; the concentration of the impatiens tetraol saponin A reference solution is 260μg / mL to 300μg / mL; the concentration of the impatiens tetraol saponin G reference solution is 270μg / mL to 310μg / mL; the concentration of the impatiens tetraol saponin L reference solution is 290μg / mL to 330μg / mL; the concentration of the impatiens tetraol saponin B reference solution is 270μg / mL to 310μg / mL; the concentration of the quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside reference solution is 140μg / mL to 180μg / mL.

[0092] In some examples of the present invention, the step of preparing the test solution includes: mixing the standard decoction of Jijizi decoction pieces with a first alcohol-containing aqueous solution having a volume fraction of 50% to 80% for extraction, concentrating, dissolving the residue, filtering, and taking the filtrate;

[0093] The alcohol in the first alcohol-containing aqueous solution is selected from methanol or ethanol;

[0094] The extraction method is ultrasonic extraction or heating reflux extraction, and the conditions of ultrasonic extraction include: power of 200W to 300W, frequency of 35kHz to 45kHz, and time of 30min to 60min.

[0095] It can be understood that the volume percentage of the first alcohol-containing aqueous solution includes but is not limited to 50%, 60%, 70%, 80%; preferably, it is 50% ethanol aqueous solution.

[0096] It can be understood that the dissolving agent for dissolving the residue is consistent with the extraction solvent used in the extraction process.

[0097] It can be understood that in the present invention, the power of the ultrasonic extraction is, for example, 200W, 220W, 250W, 280W, and 300W, the frequency of the ultrasonic extraction is, for example, 35kHz, 36kHz, 38kHz, 40kHz, 42kHz, and 45kHz, and the time of the ultrasonic extraction is, for example, 30min, 40min, 45min, 48min, 50min, 55min, and 60min. Preferably, the conditions of the ultrasonic extraction include: the power is 250W, the frequency is 40kHz, and the time is 45min.

[0098] In some examples of the present invention, in the step of preparing the test solution, the amount of ethanol solution corresponding to every 0.1 g of the standard decoction of Jijizi herbal medicine sample is 15 mL to 25 mL.

[0099] It can be understood that the amount of ethanol solution corresponding to each 0.1g of the standard decoction test sample of the Jijizi herbal medicine slices is 15mL, 18mL, 20mL, 22mL, or 25mL. Preferably, the amount of ethanol solution corresponding to each 0.1g of the standard decoction test sample of the Jijizi herbal medicine slices is 20mL.

[0100] In some examples of the present invention, 10 characteristic peaks are presented in the characteristic spectrum, and the peak corresponding to the reference peak of the impatiens tetraol saponin K reference substance is the S peak. The relative retention times of peaks 1 to 3, peaks 5 to 8, and peak 10 and the S peak are calculated, and the relative retention times are within ±10% of the specified values, and the specified values ​​are: peak 1 is 0.21, peak 2 is 0.78, peak 3 is 0.89, peak 5 is 1.04, peak 6 is 1.07, peak 7 is 1.14, peak 8 is 1.24, and peak 10 is 1.59.

[0101] In some examples of the present invention, the establishment method further comprises the step of performing high-resolution mass spectrometric identification of characteristic peaks in the characteristic spectrum in conjunction with mass spectrometry;

[0102] The mass spectrometry conditions include:

[0103] HESI ion source parameters include: sheath gas flow rate of 30arb~40arb, auxiliary gas flow rate of 8arb~12arb, spray voltage of 3.5kV~4.0kV, S-lens voltage of 45V~55V, heating temperature of 320℃~380℃, and capillary temperature of 320℃~380℃;

[0104] The mass spectrometry scanning parameters include: the scanning mode is positive ion mode and / or negative ion mode, the scanning range is 120m / z to 1200m / z, the normalized collision energy is 40V, and the mass spectrum type is a peak shape diagram.

[0105] It can be understood that in the present invention, the sheath gas flow rate is, for example, 30arb, 32arb, 35arb, 38arb, 40arb, the auxiliary gas flow rate is, for example, 8arb, 9arb, 10arb, 11arb, 12arb, the spray voltage is, for example, 3.5kV, 3.6kV, 3.8kV, 4.0kV, the S-lens voltage is, for example, 45V, 48V, 50V, 52V, 55V, the heating temperature is, for example, 320℃, 350℃, 360℃, 380℃, and the capillary temperature is, for example, 320℃, 350℃, 360℃, 380℃.

[0106] Preferably, the sheath gas flow rate is 35 arb, the auxiliary gas flow rate is 10 arb, the spray voltage is 3.8 kV, the S-lens voltage is 50 V, the heating temperature is 350°C, and the capillary temperature is 350°C.

[0107] The second aspect of the present invention also provides a method for identifying a standard decoction of Jijizi decoction pieces, comprising the following steps:

[0108] Take the test product to prepare a test product solution;

[0109] The test solution is detected by high performance liquid chromatography-evaporative light scattering detection, and the obtained chromatogram of the test product is compared with the characteristic spectrum obtained by the method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of the Jijizi decoction pieces;

[0110] The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method include: using acetonitrile as mobile phase A and water as mobile phase B for gradient elution;

[0111] The procedure of the gradient elution includes:

[0112] From 0 to 25 min, the volume percentage of the mobile phase A increased from 16% to 32%, and the volume percentage of the mobile phase B decreased from 84% to 68%;

[0113] 25min~30min, the volume percentage of the mobile phase A is 32%, and the volume percentage of the mobile phase B is 68%;

[0114] The detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 1 to 3; a carrier gas flow rate of 2.5 L / min to 3.5 L / min; and a temperature of 110° C. to 120° C.

[0115] Preferably, the detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 2; a carrier gas flow rate of 3.0 L / min; and a temperature of 110°C.

[0116] In some examples of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method also include: using an Agilent Poroshell 120EC-C18 chromatographic column.

[0117] Preferably, the specifications of the chromatographic column are: column length of 150 mm, inner diameter of 4.6 mm, and filler particle size of 2.7 μm.

[0118] In some examples of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method also include: a flow rate of 0.8 mL / min to 1.2 mL / min.

[0119] It can be understood that in the present invention, the flow rate is, for example, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, and preferably, the flow rate is 1.0 mL / min.

[0120] In some examples of the identification method of the present invention, the chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method also include: a column temperature of 33°C to 37°C.

[0121] It can be understood that in the present invention, the column temperature is, for example, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, or 37°C. Preferably, the column temperature is 35°C.

[0122] In some examples of the identification method of the present invention, the step of preparing the test solution includes:

[0123] The sample to be tested is mixed with a second alcohol-containing aqueous solution having a volume fraction of 50% to 80% to perform extraction, concentrated, the residue is dissolved, filtered, and the filtrate is collected;

[0124] The alcohol in the second alcohol-containing aqueous solution is selected from methanol or ethanol;

[0125] The extraction method is ultrasonic extraction or heating reflux extraction, and the conditions of ultrasonic extraction include: power of 200W to 300W, frequency of 35kHz to 45kHz, and time of 30min to 60min.

[0126] It can be understood that the volume percentage of the second alcohol-containing aqueous solution includes but is not limited to 50%, 60%, 70%, 80%; preferably, it is 50% ethanol aqueous solution.

[0127] It can be understood that in the present invention, the power of the ultrasonic extraction is, for example, 200W, 220W, 250W, 280W, and 300W, the frequency of the ultrasonic extraction is, for example, 35kHz, 36kHz, 38kHz, 40kHz, 42kHz, and 45kHz, and the time of the ultrasonic extraction is, for example, 30min, 40min, 45min, 48min, 50min, 55min, and 60min. Preferably, the conditions of the ultrasonic extraction include: the power is 250W, the frequency is 40kHz, and the time is 45min.

[0128] In some embodiments of the identification method of the present invention, in the step of preparing the test product solution, the amount of the extraction solvent corresponding to every 0.1 g of the test product is 15 mL to 25 mL.

[0129] It can be understood that the amount of ethanol solution corresponding to 0.1g of the test product is 15mL, 18mL, 20mL, 22mL, or 25mL. Preferably, the amount of ethanol solution corresponding to 0.1g of the test product is 20mL.

[0130] In some embodiments of the identification method of the present invention, the test product includes at least one of Perilla seed, Chrysanthemum chinense seed, Rhizoma Cibotii flavescentis seed, Radish seed, Astragalus complanatus seed and Malva ovata seed.

[0131] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are preferably referred to the guidance provided in the present invention, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0132] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0133] Example 1

[0134] A method for establishing HPLC-ELSD characteristic spectrum of standard decoction of Jijizi decoction pieces

[0135] 1. Instruments, reagents and test drugs

[0136] Instruments: Waters ultra-high performance liquid chromatograph (H-Class PLus, Waters Technology Co., Ltd.), Agilent ultra-high performance liquid chromatograph (1290, Agilent Technology Co., Ltd.), Poroshell 120EC-C18 (4.6 mm × 150 mm, 2.7 μm, No. JS-320, JS-319, JS-321) chromatographic columns, Waters Xbridge C18 (4.6 mm × 150 mm, 5 μm) chromatographic columns, Waters Xselect HSS T3 (4.6 mm × 150 mm, 5 μm) chromatographic columns, evaporative light scattering detector (ELSD6000, Alltech Chrom), 1% electronic analytical balance (JJ600, Changshu Shuangjie Testing Instrument Factory), 1 / 10,000 electronic analytical balance (ME204E, Mettler-Toledo), 1 / 100,000 electronic analytical balance (ABT220-5DM, Guangzhou Dexiang Technology Co., Ltd.), 1 / 1,000,000 electronic analytical balance (XP26, Mettler-Toledo), CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.), electric constant temperature water bath (HWS28, Shanghai Yiheng Technology Co., Ltd.), ultrapure water system (Milli-Q Direct, Merck AG).

[0137] Reagents: Methanol (Xilong Scientific Co., Ltd.) and ethanol (Xilong Scientific Co., Ltd.) were of analytical grade; acetonitrile (Merck AG) was of chromatographic grade; and water was ultrapure water (self-made in the laboratory).

[0138] Test drugs: Impatiens tetraol saponin K (batch number: 112005-201902, content: 91.2%, China Food and Drug Inspection Institute), Impatiens tetraol saponin A (batch number: 112009-202102, content: 95.4%, China Food and Drug Inspection Institute), Impatiens tetraol saponin G (batch number: 18020704, content: 99.47%, Chengdu Pufeide Biotechnology Co., Ltd.), Impatiens tetraol saponin B (batch number: 060032-201906, content: ≥98%, Guangzhou Langou Biotechnology Co., Ltd. Technology Co., Ltd.), impatiens tetraol saponin L (batch number: 060033-201906, content: ≥98%, Guangzhou Langou Biotechnology Co., Ltd.), quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside (content: ≥98%, Sichuan Weikeqi Biotechnology Co., Ltd.), Aconitum carmichaelii reference medicinal materials (batch number: 121606-201602, China Food and Drug Inspection Institutes); 21 batches of freeze-dried powders of standard decoctions prepared from Aconitum carmichaelii slices from different origins.

[0139] 2. Preparation of Jijizi Decoction Pieces

[0140] Since there is no processing item for decoction pieces under the item of Jijizi in the Chinese Pharmacopoeia (2020 edition), the processing method is as follows: take Jijizi medicinal materials and pick out impurities and spoiled products. According to the principle of "every shell must be crushed and every seed must be broken" under the item "Preparation of decoctions for research and characterization of standard decoctions" in the "Technical Requirements for Quality Control and Standardization of Chinese Medicine Formula Granules", Jijizi decoction pieces are crushed.

[0141] 3. Preparation of standard decoction of Jijizi slices

[0142] Take 100g of the Radix Achyranthis Bidentatae slices, add water and decoct twice, add 8 times of water for the first decoction, soak for 30 minutes, boil over high heat (500W) and keep a slight boil for 30 minutes with a slow fire (200W), filter the decoction with a 350-mesh sieve while hot, and cool the filtrate with cold water rapidly. Add 6 times of water for the second decoction, boil over high heat (500W) and keep a slight boil for 25 minutes with a slow fire (200W), filter the decoction with a 350-mesh sieve while hot, and cool the filtrate with cold water rapidly, and merge the two filtrates. The filtrate is transferred to a 2000ml round-bottomed flask, concentrated to 100ml under reduced pressure and freeze-dried in vacuum to obtain a standard decoction freeze-dried powder. 21 batches of standard decoction freeze-dried powders prepared from Radix Achyranthis Bidentatae slices from different origins are shown in Table 1.

[0143] Table 1 Origin information of 21 batches of freeze-dried powder and medicinal materials of Jijizi standard decoction

[0144]

[0145] 4. Chromatographic conditions and preparation of test solution

[0146] 4.1 Chromatographic conditions

[0147] An Agilent Poroshell 120EC-C18 (4.6×150 mm, 2.7 μm) chromatographic column was used, with acetonitrile as mobile phase A and water as mobile phase B, and gradient elution was performed according to the provisions in Table 2 below; the flow rate was 1.0 mL per minute; the column temperature was 35°C; the evaporative light scattering detector detection: the gain was 2; the carrier gas flow rate was 3.0 L / min; and the temperature was 110°C.

[0148] Table 2 Gradient elution table

[0149]

[0150] 4.2 Preparation of reference solution

[0151] Accurately weigh 7.860 mg of impatiens tetraol saponin K reference substance, put it into a 25 ml volumetric flask, add methanol to make a solution containing 299.963 μg of impatiens tetraol saponin K per 1 ml, and obtain the impatiens tetraol saponin K reference substance solution.

[0152] Accurately weigh 7.755 mg of impatiens tetraol saponin A reference substance, place it in a 25 ml volumetric flask, add methanol to make a solution containing 286.733 μg of impatiens tetraol saponin A per 1 ml, to obtain the impatiens tetraol saponin A reference substance solution.

[0153] Accurately weigh 7.280 mg of impatiens tetraol saponin G reference substance, place it in a 25 ml volumetric flask, add methanol to make a solution containing 289.657 μg of impatiens tetraol saponin G per 1 ml, to obtain the impatiens tetraol saponin G reference substance solution.

[0154] Accurately weigh 7.674 mg of the Impatiens tetraol saponin L reference substance, place it in a 25 ml volumetric flask, add methanol to make a solution containing 310.841 μg of Impatiens tetraol saponin L per 1 ml, to obtain the Impatiens tetraol saponin L reference substance solution.

[0155] Accurately weigh 7.463 mg of impatiens tetraol saponin B reference substance, place it in a 25 ml volumetric flask, add methanol to make a solution containing 292.574 μg of impatiens tetraol saponin B per 1 ml, to obtain the impatiens tetraol saponin B reference substance solution.

[0156] Accurately weigh 1.628 mg of quercetin-3-O-[α-L-rhamnose-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside reference substance, place it in a 10 ml volumetric flask, add methanol to make a solution containing 159.544 μg of quercetin-3-O-[α-L-rhamnose-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside per 1 ml, and obtain the quercetin-3-O-[α-L-rhamnose-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside reference substance solution.

[0157] 4.3 Preparation of control medicinal material solution

[0158] Take about 1g of the quick-acting reference medicinal material, accurately weigh it, put it in a stoppered conical flask, add 50mL of water, heat and reflux for 30 minutes, cool, filter, take the filtrate, evaporate to dryness, add 50ml of 50% ethanol to the residue, weigh the weight, heat and reflux for 45 minutes, cool, weigh again, make up the lost weight with 50% ethanol, centrifuge, accurately measure 25ml of the supernatant, evaporate to dryness in a water bath, add 50% ethanol to the residue to dissolve it, transfer it to a 5ml volumetric flask, add 50% ethanol to the scale, shake well, filter, and take the filtrate as the reference solution of the reference medicinal material.

[0159] 4.4 Preparation of test solution

[0160] Take an appropriate amount of this product, grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 20mL of 50% ethanol, weigh it, and treat it with ultrasound (power 250W, frequency 40kHz) for 45 minutes. Let it cool, weigh it again, make up the lost weight with 50% ethanol, centrifuge it, accurately measure 10ml of the supernatant, evaporate it in a water bath, dissolve the residue in 50% ethanol, transfer it to a 5mL volumetric flask, add 50% ethanol to the scale, shake it well, filter it, and take the filtrate to obtain it.

[0161] 4.5 Determination method

[0162] Accurately pipette 5 μL of the test solution, reference solution and reference medicinal material solution respectively, inject into the liquid chromatograph for determination.

[0163] 5. Optimization of chromatographic conditions

[0164] 5.1 Optimization of elution gradient

[0165] Take an appropriate amount of the standard decoction of Jijizi (No.: BT1), grind it into powder, take about 0.1g, and prepare the test solution according to the method under "4.4". Except for the different elution gradients, all other analyses are carried out according to the provisions under "4.1". The results show that by comparing the chromatograms of different gradient elutions ( Figure 1), it can be found that when gradient 3 is selected, the baseline of the chromatogram is stable and the separation effect of each chromatographic peak is good, so gradient 3 is selected as the final elution gradient.

[0166] Table 3 Gradient 1

[0167]

[0168] Table 4 Gradient 2

[0169]

[0170] Table 5 Gradient 3

[0171]

[0172] 5.2 Column selection

[0173] Take an appropriate amount of the standard decoction of Acute Zi (No.: BT1), grind it into powder, take about 0.1g, and prepare the test solution according to the method under "4.4". Except for using different chromatographic columns, all other analyses were carried out according to the provisions under "4.1". The results showed that when the Agilent Poroshell 120EC-C18 (4.6×150mm, 2.7μm) chromatographic column was selected for elution, the response values ​​of each characteristic peak were large, the baseline was stable, the interference was small, and the peak shape and separation effect of each chromatographic peak were good. Therefore, the Agilent Poroshell120EC-C18 (4.6×150mm, 2.7μm) chromatographic column was selected as the best chromatographic column ( Figure 2 ).

[0174] 6 Investigation of the preparation method of test solution

[0175] 6.1 Investigation of extraction solvent

[0176] Take appropriate amount of the same batch of standard decoction of Jijizi (No.: BT1), grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of methanol, 80% methanol, 50% methanol, ethanol, 80% ethanol, and 50% ethanol, weigh it, and treat it with ultrasound (power 250W, frequency 40kHz) for 45 minutes. Let it cool, weigh it again, make up the lost weight with the corresponding solvent, centrifuge it, accurately measure 10ml of the supernatant, evaporate it in a water bath, dissolve the residue in the corresponding solvent, transfer it to a 5ml volumetric flask, add the corresponding solvent to the scale, shake it well, filter it, and take the filtrate. The determination was carried out according to the chromatographic conditions specified in "4.1". The results showed that the characteristic spectra had obvious differences when different extraction solvents were used. When 50% ethanol was used as the extraction solvent, the chromatographic peak shape was better and the extraction rate was higher. Therefore, 50% ethanol was used as the extraction solvent ( Figure 3 ).

[0177] 6.2 Investigation of extraction methods

[0178] Take appropriate amount of the same batch of standard decoction of Jijizi (No.: BT1), grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical bottle, accurately add 20ml of 50% ethanol, weigh it, treat it with ultrasound (power 250W, frequency 40kHz) for 45 minutes, reflux it for 45 minutes, let it cool, weigh it again, make up the lost weight with 50% ethanol, centrifuge it, accurately measure 10ml of the supernatant, evaporate it in a water bath, dissolve the residue in 50% ethanol, transfer it to a 5ml volumetric bottle, add 50% ethanol to the scale, shake it well, filter it, and take the filtrate. Determine it according to the chromatographic conditions specified in "4.1". The results show that ( Figure 4 ), using different extraction methods, there was no significant difference in the peak shape and separation effect of each characteristic peak. Considering the simplicity of operation, ultrasonic extraction was determined as the extraction method.

[0179] 6.3 Extraction time investigation

[0180] Take appropriate amount of the same batch of Jijizi standard decoction (No.: BT1), grind it into powder, take about 0.1g, weigh it accurately, put it in a stoppered conical flask, accurately add 20ml of 50% ethanol, weigh it, and treat it with ultrasound (power 250W, frequency 40kHz) for 30 minutes, 45 minutes, and 60 minutes respectively. Let it cool, weigh it again, make up the lost weight with 50% ethanol, centrifuge it, accurately measure 10ml of the supernatant, evaporate it in a water bath, dissolve the residue in 50% ethanol, transfer it to a 5ml volumetric flask, add 50% ethanol to the scale, shake it well, filter it, and take the filtrate. Determine it according to the chromatographic conditions specified under "4.1". The results showed that different extraction times did not have a significant effect on the characteristic spectrum of the Jijizi standard decoction. To ensure complete extraction, the ultrasonic extraction time was selected as 45 minutes ( Figure 5 ).

[0181] 7 Methodological considerations

[0182] 7.1 Specificity Investigation

[0183] Take an appropriate amount of the standard decoction of Jijizi (No.: BT1), grind it into powder, take about 0.1g, prepare the test solution according to the method under "4.4", accurately pipette 5μl of the test solution, the reference solution under "4.2" and the blank solvent, and analyze according to the chromatographic conditions under "4.1". The results show that the chromatogram of the test sample has the same chromatographic peak at the corresponding retention time of the chromatogram of the reference sample, and the blank solvent has no interference, indicating that the method has good specificity ( Figure 6 ).

[0184] 7.2 Precision investigation

[0185] Take an appropriate amount of the standard decoction of Acute Radix (No.: BT1), grind it into powder, take about 0.1g, prepare the test solution according to the method under "4.4", and repeat the injection 6 times according to the chromatographic conditions under "4.1". Take the tetraol saponin K of Impatiens as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The relative retention time RSD value of the 9 common peaks is in the range of 0.01% to 0.21%, and the relative peak area RSD value is in the range of 0.41% to 2.73%, indicating that the instrument has good precision.

[0186] 7.3 Repeatability Study

[0187] Take an appropriate amount of the standard decoction of Acute Herba (No.: BT1), grind it into powder, take about 0.1g, prepare 6 test solutions according to the method under "4.4", and inject and measure according to the chromatographic conditions under "4.1". Take the tetraol saponin K of Impatiens as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The relative retention time RSD value of the 9 common peaks is in the range of 0.02% to 0.30%, and the relative peak area RSD value is in the range of 1.29% to 2.88%, indicating that the method has good repeatability.

[0188] 7.4 Stability Study

[0189] Take an appropriate amount of the standard decoction of Acute Herba (No.: BT1), grind it into powder, take about 0.1g, prepare the test solution according to the method under "4.4", and inject and measure at 0, 2, 4, 6, 8, 10, 12, and 24 hours according to the chromatographic conditions under "4.1". Take the tetraol saponin K of Impatiens as the reference peak S, calculate the relative retention time and relative peak area of ​​each characteristic peak and the S peak, and calculate the RSD value. The relative retention time RSD value of the 9 common peaks is in the range of 0.02% to 0.53%, and the relative peak area RSD value is in the range of 2.03% to 2.90%, indicating that the test solution is relatively stable within 24 hours.

[0190] 8 Establishment of characteristic spectrum of standard decoction of acute

[0191] 8.1 Results of Characteristic Spectrum Determination of Standard Decoction of Acute Zi

[0192] Take 21 batches of standard decoction of Jijizi, according to the chromatographic conditions under "4.1" and the preparation method of the test solution determined under "4.4", take impatiens tetraol saponin K as the reference peak S, calculate the relative retention time of each characteristic peak and the S peak, and calculate the average relative retention time of each characteristic peak to be 0.21 (peak 1), 0.78 (peak 2), 0.89 (peak 3), 1.04 (peak 5), 1.07 (peak 6), 1.14 (peak 7), 1.24 (peak 8), 1.59 (peak 10). The characteristic spectra of 21 batches of standard decoction of Jijizi are shown in Figure 7 shown.

[0193] 8.2 Establishment of the common pattern of characteristic spectra of standard decoctions of Acute

[0194] Take the reference medicinal material of Jijizi (batch number: 121606-201602, China Food and Drug Inspection Institute), prepare the reference medicinal material reference solution according to the method under "4.3", prepare the test sample solution of 21 batches of Jijizi standard decoction according to the method under "4.4", and inject and measure according to the chromatographic conditions under "4.1" to obtain the characteristic spectrum of Jijizi reference medicinal material ( Figure 8 ); the Chinese medicine chromatographic fingerprint similarity evaluation software was used to generate a reference spectrum by the average method, and the reference characteristic spectrum of the standard decoction of Jijizi was established ( Fig. 9 ).

[0195] It is determined that 10 characteristic peaks should be present in the chromatogram of the test sample, and the retention times of the 10 characteristic peaks in the chromatogram of the reference medicinal material should correspond to each other, among which peak 4 should correspond to the retention times of the corresponding reference material peaks. The peak corresponding to the peak of the reference material of impatiens tetraol saponin K is the S peak, and the relative retention times of peaks 1 to 3, 5 to 8, and 10 to the S peak are calculated. The relative retention times should be within the range of ±10% of the specified values, and the specified values ​​are: 0.21 (peak 1), 0.78 (peak 2), 0.89 (peak 3), 1.04 (peak 5), 1.07 (peak 6), 1.14 (peak 7), 1.24 (peak 8), and 1.59 (peak 10).

[0196] 9 High-resolution mass spectrometry identification of characteristic peaks

[0197] 9.1 HPLC-MS Conditions

[0198] Liquid chromatography conditions: Poroshell 120EC-C18 column (150 mm×4.6 mm, 2.7 μm) was used; acetonitrile was used as mobile phase A, water was used as mobile phase B, and gradient elution was performed according to the provisions in Table 6; the flow rate was 1.0 ml per minute; the column temperature was 35° C.; and detection was performed by evaporative light scattering detector.

[0199] Table 6 Gradient elution table

[0200]

[0201] The mass spectrometry conditions are shown in Table 7:

[0202] Table 7 Mass spectrometry parameters

[0203]

[0204] 9.2 Preparation of test solution

[0205] Same as the preparation of test solution under “4.4”.

[0206] 9.3 Determination

[0207] Accurately pipette 5 μL of the test solution and inject it into the liquid chromatography-mass spectrometer. Use the above liquid chromatography conditions and mass spectrometry conditions to detect the test solution. The total ion flow diagram and ultraviolet absorption chromatogram of the test solution are shown in Fig.10 .

[0208] 9.4 Results Analysis

[0209] By comparing the mass spectrometry molecular weight and fragment ions, matching with the Thermo Fisher Scientific mzVault standard database, identification and reference confirmation, the six characteristic peaks in the characteristic spectrum of the Jijizi decoction were determined to be: quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside (peak 1), impatiens tetraol saponin B (peak 2), impatiens tetraol saponin K (peak 4), impatiens tetraol saponin G (peak 8), impatiens tetraol saponin A (peak 9) and impatiens tetraol saponin A (peak 10). The compound information is shown in Table 8, and the reference confirmation results are shown in Fig.11 .

[0210] Table 8 Mass spectrometry identification results of compounds in the standard decoction of Jijizi decoction pieces

[0211]

[0212] Example 2

[0213] Identification of the standard decoction of Jijizi and its different adulterants

[0214] It is mentioned in relevant literature reports and the catalog of 130 commonly used counterfeit and mixed products of Chinese medicine that Perilla seed, Chrysanthemum chrysanthemum seed, Amomum villosum seed, Radish seed, Astragalus membranaceus seed and Melaleuca alternifolia seed are similar to Rhizoma Cibotii in properties and are mixed products of Rhizoma Cibotii.

[0215] The method for preparing the standard decoction of Jinjizi was used to prepare the standard decoction of Perilla seed, Chrysanthemum seed, Ganoderma lucidum, Radish seed, Astragalus seed and Malus seed. The method for preparing the standard decoction of Jinjizi and the characteristic spectrum detection method were used to determine the characteristic spectrum of the standard decoction of Perilla seed, Chrysanthemum seed, Ganoderma lucidum, Radish seed, Astragalus seed and Malus seed, and compared with the characteristic spectrum of the standard decoction of Jinjizi. The results are as follows: Figure 12 to Figure 16 shown.

[0216] The results showed that the characteristic spectra of the standard decoctions of Perilla seed, Chrysanthemum seed, Amomum villosum seed, Radish seed, Astragalus seed, and Melaleuca seed were significantly different from those of the standard decoction of Rhizoma Cimicifugae. The characteristic spectra of the standard decoctions of Perilla seed, Chrysanthemum seed, Amomum villosum seed, and Melaleuca seed did not have the 10 characteristic peaks in the characteristic spectra of the standard decoction of Rhizoma Cimicifugae. The characteristic spectra of the standard decoction of Radish seed only contained characteristic peak 2 with very low response, and peak 11 was exclusive to Radish seed. Although the characteristic spectra of the standard decoction of Astragalus villosum contained characteristic peaks 1, 2, and 8, the response was lower than that of Rhizoma Cimicifugae, and peaks 11 to 13 were characteristic peaks exclusive to Rhizoma Cimicifugae. In summary, the established characteristic spectrum method can effectively distinguish various confused products of Rhizoma Cimicifugae that have lost their medicinal properties.

[0217] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0218] The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in detail, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to interpret the contents of the claims.

Claims

1. A method for establishing a HPLC-ELSD characteristic spectrum of a standard decoction of Radix Aconitum, characterized in that: The steps include: Take one or more of the reference substances of impatiens tetraol saponin K, impatiens tetraol saponin A, impatiens tetraol saponin G, impatiens tetraol saponin L, impatiens tetraol saponin B, and quercetin-3-O-[α-L-rhamnosyl-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside to prepare a reference substance solution; Prepare the test solution from the standard decoction of Jijizi decoction pieces; The reference solution and the test solution are respectively detected by high performance liquid chromatography-evaporative light scattering detection method; The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method include: using acetonitrile as mobile phase A and water as mobile phase B for gradient elution; The procedure of the gradient elution includes: From 0 to 25 min, the volume percentage of the mobile phase A increased from 16% to 32%, and the volume percentage of the mobile phase B decreased from 84% to 68%; 25min~30min, the volume percentage of the mobile phase A is 32%, and the volume percentage of the mobile phase B is 68%; The detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 1 to 3; a carrier gas flow rate of 2.5 L / min to 3.5 L / min; and a temperature of 110° C. to 120° C.

2. The method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of the Jijizi decoction pieces according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method also include one or more of the following technical features: (1) Using Agilent Poroshell 120EC-C18 column; (2) Flow rate: 0.8 mL / min to 1.2 mL / min; (3) The column temperature is 33℃~37℃.

3. The method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of the Jijizi decoction pieces according to claim 1, characterized in that: Methanol is used as a dissolving agent to prepare the reference solution; the concentration of the impatiens tetraol saponin K reference solution is 280 μg / mL to 320 μg / mL; The concentration of the impatiens tetraol saponin A reference solution is 260 μg / mL to 300 μg / mL; The concentration of the Impatiens tetraol saponin G reference solution is 270 μg / mL to 310 μg / mL; The concentration of the Impatiens tetraol saponin L reference solution is 290 μg / mL to 330 μg / mL; The concentration of the impatiens tetraol saponin B reference solution is 270 μg / mL to 310 μg / mL; The concentration of the quercetin-3-O-[α-L-rhamnose-(1→2)-β-D-glucosyl]-5-O-β-D-glucoside reference solution is 140 μg / mL to 180 μg / mL.

4. The method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of the Jijizi decoction pieces according to claim 1, characterized in that: The steps of preparing the test solution include: mixing the standard decoction of Jijizi slices with a first alcohol-containing aqueous solution with a volume fraction of 50% to 80% to perform extraction, concentrating, dissolving the residue, filtering, and taking the filtrate; The alcohol in the first alcohol-containing aqueous solution is selected from methanol or ethanol; The extraction method is ultrasonic extraction or heating reflux extraction, and the conditions of ultrasonic extraction include: power of 200W to 300W, frequency of 35kHz to 45kHz, and time of 30min to 60min.

5. The method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of the Jijizi decoction pieces according to claim 4, characterized in that, In the step of preparing the test solution, the amount of ethanol solution corresponding to every 0.1 g of the standard decoction of the Aconitum carmichaelii slices is 15 mL to 25 mL.

6. The method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of Jijizi decoction pieces according to any one of claims 1 to 5, characterized in that: There are 10 characteristic peaks in the characteristic spectrum, and the peak corresponding to the peak of the reference substance of impatiens tetraol saponin K is the S peak. The relative retention times of peaks 1 to 3, peaks 5 to 8, peak 10 and the S peak are calculated, and the relative retention times are within ±10% of the specified values, and the specified values ​​are: peak 1 is 0.21, peak 2 is 0.78, peak 3 is 0.89, peak 5 is 1.04, peak 6 is 1.07, peak 7 is 1.14, peak 8 is 1.24 and peak 10 is 1.

59.

7. The method for establishing the HPLC-ELSD characteristic spectrum of the standard decoction of Jijizi decoction pieces according to any one of claims 1 to 5, characterized in that: The establishment method also includes the step of performing high-resolution mass spectrometry identification on the characteristic peaks in the characteristic spectrum by combined mass spectrometry; The mass spectrometry conditions include: HESI ion source parameters include: sheath gas flow rate of 30arb~40arb, auxiliary gas flow rate of 8arb~12arb, spray voltage of 3.5kV~4.0kV, S-lens voltage of 45V~55V, heating temperature of 320℃~380℃, and capillary temperature of 320℃~380℃; The mass spectrometry scanning parameters include: the scanning mode is positive ion mode and / or negative ion mode, the scanning range is 120m / z to 1200m / z, the normalized collision energy is 40V, and the mass spectrum type is a peak shape diagram.

8. A method for identifying a standard decoction of Jijizi decoction pieces, characterized in that: The steps include: Take the test product to prepare a test product solution; The test solution is tested by high performance liquid chromatography-evaporative light scattering detection, and the obtained chromatogram of the test product is compared with the characteristic spectrum obtained by the method established in any one of claims 1 to 7; The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method include: using acetonitrile as mobile phase A and water as mobile phase B for gradient elution; The procedure of the gradient elution includes: From 0 to 25 min, the volume percentage of the mobile phase A increased from 16% to 32%, and the volume percentage of the mobile phase B decreased from 84% to 68%; 25min~30min, the volume percentage of the mobile phase A is 32%, and the volume percentage of the mobile phase B is 68%; The detection conditions of the high performance liquid chromatography-evaporative light scattering detection method include: a gain of 1 to 3; a carrier gas flow rate of 2.5 L / min to 3.5 L / min; and a temperature of 110° C. to 120° C.

9. The identification method of the standard decoction of Jijizi decoction pieces according to claim 8 is characterized in that: The chromatographic conditions of the high performance liquid chromatography-evaporative light scattering detection method also include one or more of the following technical features: (1) Using Agilent Poroshell 120EC-C18 column; (2) Flow rate: 0.8 mL / min to 1.2 mL / min; (3) The column temperature is 33℃~37℃.

10. The identification method of the standard decoction of Jijizi decoction pieces according to claim 8, characterized in that: The steps of preparing the test solution include: The sample to be tested is mixed with a second alcohol-containing aqueous solution having a volume fraction of 50% to 80% to perform extraction, concentrated, the residue is dissolved, filtered, and the filtrate is collected; The alcohol in the second alcohol-containing aqueous solution is selected from methanol or ethanol; The extraction method is ultrasonic extraction or heating reflux extraction, and the conditions of ultrasonic extraction include: power of 200W to 300W, frequency of 35kHz to 45kHz, and time of 30min to 60min.

11. The identification method of the standard decoction of Jijizi decoction pieces according to claim 9 is characterized in that: In the step of preparing the test product solution, the amount of ethanol solution corresponding to 0.1 g of the test product is 15 mL to 25 mL.

12. The identification method of the standard decoction of Jijizi decoction pieces according to any one of claims 8 to 11, characterized in that: The sample to be tested includes at least one of Perilla seed, Chrysanthemum chinense seed, Rhizoma Cibotii flavescentis seed, Radish seed, Astragalus complanatus seed and Malva ovata seed.

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