Screening method and quantitative analysis method of processing degree marker of traditional Chinese medicine decoction pieces, and control method of processing degree of traditional Chinese medicine decoction pieces

Abstract

The present application relates to a screening method for processing degree markers of traditional Chinese medicine decoction pieces, which combines metabolomics and chemometrics methods to analyze the relationship between metabolites and processing degree, find differential markers from processing metabolite information, and further screen processing degree markers, thereby establishing a fast and reliable method for discovering processing degree markers. A quantitative analysis method for processing degree markers of traditional Chinese medicine decoction pieces is also established, which adopts high performance liquid chromatography-ultraviolet quantitative analysis method, and is verified through linearity, detection limit and quantitative limit, repeatability, precision, recovery rate and stability analysis to meet the quantitative analysis requirements of processing markers. A control method for the processing degree of traditional Chinese medicine decoction pieces is also provided, which controls the processing degree by using the quantitative limit of the screened processing degree markers of traditional Chinese medicine decoction pieces. The present application also relates to a screening method for general processing degree markers, which screens 9-HODE and 13-HODE as general processing degree markers due to their strong universality.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine processing, in particular to a screening method of a processing degree marker of traditional Chinese medicine decoction pieces, a quantitative analysis method of a processing marker of traditional Chinese medicine decoction pieces and a control method of the processing degree of traditional Chinese medicine decoction pieces. BACKGROUND

[0002] Traditional Chinese medicine processing is a process of transforming Chinese herbal medicines into traditional Chinese medicine decoction pieces, and its purposes include reducing toxicity and increasing efficacy, transforming or moderating drug properties, facilitating the decoction of effective components, etc. The processing methods of traditional Chinese medicine include frying, baking, steaming, boiling, calcining and baking, and some of them can be processed with or without auxiliary materials, most of which need to go through a heating process.

[0003] Since traditional Chinese medicine decoction pieces are medicines that need to be directly used in clinical practice of traditional Chinese medicine or production of preparations, the quality of the medicines needs to be guaranteed and the quality consistency between different batches needs to be ensured. In the Ming Dynasty, Chen Jiamou recorded in Bencao Mengxun that "in the production of medicines, the key is to be moderate, if not, the efficacy will be difficult to obtain, and if too much, the odor will be lost", which briefly summarizes the important influence of the processing degree on the quality of decoction pieces. However, most of the traditional Chinese medicine decoction pieces lack clear processing degree markers, and the processing endpoint can only be determined by observing their appearance and color.

[0004] Therefore, it is necessary to find effective processing degree markers of traditional Chinese medicine decoction pieces for the quality control of traditional Chinese medicine decoction pieces. SUMMARY

[0005] Therefore, it is necessary to find effective processing degree markers of traditional Chinese medicine decoction pieces for the quality control of traditional Chinese medicine decoction pieces.

[0006] The first aspect of the present application provides a screening method of a processing degree marker of traditional Chinese medicine decoction pieces, comprising the following steps:

[0007] According to the set category of Chinese herbal medicines, the set category of processing methods and the set category of processing parameters, the processing parameters of the set category are adjusted to obtain processing products with different processing degrees;

[0008] The test sample is detected to obtain a detection result, wherein the test sample comprises the processing products with different processing degrees and corresponding raw products;

[0009] The detection result is analyzed by a metabolomics method to obtain metabolite information of the processing products with different processing degrees;

[0010] The detection result is analyzed by a chemometrics method to obtain difference information between the processing products with different processing degrees;

[0011] The difference markers with positive or negative correlation with the processing degree are screened.

[0012] identifying the differential markers and establishing a quantitative analysis method for the differential markers;

[0013] According to real world samples and self-made samples, the quantitative analysis method is used to verify the differential markers, and the processing degree markers of the set category of Chinese herbal medicines are screened out;

[0014] Optionally, the following step is further included: universal verification is performed on the screened processing degree markers of the set category of Chinese herbal medicines.

[0015] In some embodiments of the present application, the set category of Chinese herbal medicines is any one of Gansui, Yiyiren, Badigtian, Yansuo, Shenglu; and / or,

[0016] The set category of processing methods is any one of frying method, roasting method, steaming method, boiling method, baking method, and baking method; and / or,

[0017] The set category of processing parameters is at least one of processing time, processing temperature, and auxiliary material amount; and / or,

[0018] The processed products with different processing degrees are any one of vinegar Gansui, vinegar Yansuo, vinegar Shenglu, bran-fried Yiyiren, salt Badigtian.

[0019] In some embodiments of the present application, the metabolomics method is at least one of the following methods: high performance liquid chromatography method, gas chromatography method, high performance liquid chromatography method combined with mass spectrometry, and gas chromatography method combined with mass spectrometry;

[0020] Preferably, the test sample is detected by the high performance liquid chromatography method combined with mass spectrometry, and the detection conditions of the high performance liquid chromatography method combined with mass spectrometry are as follows: a high performance liquid chromatography system; a chromatographic column: Agilent Zorbaxplus C8, 150 mm*2.1 mm, 1.8 μm; a mobile phase: acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B, and the gradient program of the mobile phase is as follows: 0 / 8 / 10 / 20 / 30 / 45 / 50 min; 53 / 65 / 65 / 80 / 90 / 100 / 100% acetonitrile; after 50 min, the initial mobile phase is changed to balance for 0-10 min, and the total running time is 50-60 min; the flow rate is 0.20-0.50 mL / min; the column temperature is 25-35 °C; the injection volume is 5 μL; a linear ion trap orbitrap combined mass spectrometry detector is used for detection in a positive ion mode; the source spray voltage is 3-5 kV, the capillary temperature is 300-400 °C, the source heating temperature is 250-350 °C, the sheath gas flow rate is 30%-40%, the auxiliary gas flow rate is 1%-10%, the split ratio is 1:1, in a full scan mode, the mass spectrometry acquisition range is set to 200 m / z-1500 m / z, the resolution is set to 20000-40000, MS 4 the collision energy is set to 20-40%, and the resolution is set to 7000-8000.

[0021] In some embodiments of the present application, the chemometric method comprises using SIMCA 14.1 software and GraphPad 9.0 software; and the analysis by the chemometric method comprises the following steps:

[0022] The principal component analysis method is used to determine the difference information between the different processed products with different processing degrees;

[0023] The orthogonal partial least squares discriminant analysis is used to determine the difference information between the processed product with the lowest processing degree and the processed product with the highest processing degree;

[0024] The VIP plot analysis is used to screen out the differential metabolites with a VIP value > 7;

[0025] The single factor variance analysis is used to screen out the differential metabolites with a P value < 0.05 and a mass spectrometry response value positively or negatively correlated with the processing degree.

[0026] In some embodiments of the present application, the quantitative analysis method is at least one of the following methods: a high performance liquid chromatography-ultraviolet quantitative analysis method, a high performance liquid chromatography-evaporative light scattering detector quantitative analysis method, and a high performance liquid chromatography-electrospray detector quantitative analysis method.

[0027] Preferably, the real world sample and the self-made sample are detected by the high performance liquid chromatography-ultraviolet quantitative analysis method, and the detection conditions of the high performance liquid chromatography-ultraviolet quantitative analysis method are as follows: a chromatographic column is Acquity UPLC BEH C18, 100 mm*2.1 mm, 1.7 μm; a mobile phase is acetonitrile as a mobile phase A and 0.1% phosphoric acid aqueous solution as a mobile phase B, a mobile phase gradient program is 1 / 20 / 21 / 27 min, 44 / 44 / 100 / 100% acetonitrile, a flow rate is 0.35 mL / min, a column temperature is 30 DEG C, a sample injection volume is 1 μL, and an ultraviolet detection wavelength is 230 nm.

[0028] In some embodiments of the present application, the real world sample comprises commercially available raw Kansu and commercially available vinegar Kansu.

[0029] The self-made sample comprises vinegar Kansu prepared by adjusting at least one of a processing time, a processing temperature and an amount of vinegar.

[0030] In some embodiments of the present application, the processing degree marker is at least one of the following compounds: octadecadienoic acid metabolite, octadecatrienoic acid metabolite.

[0031] The octadecadienoic acid metabolite is preferably octadecadienoic acid oxidative metabolite, more preferably at least one of hydroxyoctadecadienoic acid and oxooctadecadienoic acid, and further preferably at least one of the following compounds: 9-hydroxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, 15-hydroxyoctadecadienoic acid, 9-oxooctadecadienoic acid and 13-oxooctadecadienoic acid; and / or,

[0032] The octadecatrienoic acid metabolite is preferably octadecatrienoic acid oxidative metabolite, more preferably hydroxyoctadecatrienoic acid, and further preferably at least one of the following compounds: 9-hydroxyoctadecatrienoic acid and 13-hydroxyoctadecatrienoic acid.

[0033] The second aspect of the present application provides a quantitative analysis method of a traditional Chinese medicine processing marker, comprising the following steps:

[0034] A test sample containing a processing marker is provided, and the test sample containing the processing marker is extracted by using an extraction reagent to prepare a test sample to be tested;

[0035] A control sample containing the processing marker is provided, and the control sample containing the processing marker is extracted by using the extraction reagent to prepare a control sample to be tested; the processing marker is octadecadienoic acid metabolite or / and octadecatrienoic acid metabolite;

[0036] The test sample to be tested is detected by high performance liquid chromatography-ultraviolet detection to obtain a test sample chromatogram.

[0037] performing high performance liquid chromatography-ultraviolet detection on the sample to be tested to obtain a chromatogram of the sample;

[0038] comparing the chromatogram of the sample with the chromatogram of the control to determine the content of the processing marker.

[0039] In some embodiments of the present application, the extraction solvent is any one of the following solvents or any combination thereof: methanol, a methanol / water mixed solvent, ethanol, an ethanol / water mixed solvent, ethyl acetate, chloroform, dichloromethane, n-hexane, and diethyl ether.

[0040] In some embodiments of the present application, in the high performance liquid chromatography-ultraviolet detection, a reversed-phase octadecyl-bonded phase column or an octyl-bonded phase column is used for high performance liquid chromatography separation, and / or an ultraviolet detector is used.

[0041] In some embodiments of the present application, the detection conditions of the high performance liquid chromatography-ultraviolet detection are as follows: a chromatographic column: Acquity UPLC BEH C18, 100 mm x 2.1 mm, 1.7 μm; a mobile phase: acetonitrile as mobile phase A and 0.1% phosphoric acid aqueous solution as mobile phase B, a mobile phase gradient program: 1 / 20 / 21 / 27 min, 44 / 44 / 100 / 100% acetonitrile; a flow rate: 0.3-0.4 mL / min; a column temperature: 25-35°C; an injection volume: 1 μL; and an ultraviolet detection wavelength: 220-240 nm.

[0042] A third aspect of the present application provides a method for controlling the processing degree of traditional Chinese medicine decoction pieces, comprising the following steps: providing raw products of a set category of Chinese herbal medicines, selecting a set category of processing methods, processing using the set category of processing methods, preparing processed products of the set category of Chinese herbal medicines, controlling the content of a processing degree marker in the processed products of the set category of Chinese herbal medicines to be not more than a quantitative limit, and the quantitative limit being formulated according to the content of the processing degree marker in real-world samples.

[0043] In some embodiments of the present application, the set category of Chinese herbal medicines is Gansui, the set category of processing methods is vinegar processing, the processing degree marker is 9-hydroxyoctadecadienoic acid or / and 13-hydroxyoctadecadienoic acid, and the quantitative limit is a mass content of 0.0011%; and / or,

[0044] the set category of Chinese herbal medicines is Yiyiren, the set category of processing methods is stir-frying, and the processing degree marker is 9-hydroxyoctadecadienoic acid or / and 13-hydroxyoctadecadienoic acid; and / or,

[0045] The set category of Chinese herbal medicine is Fuzi, the set category of processing method is vinegar processing, and the processing degree marker is 9-hydroxyoctadecadienoic acid or / and 13-hydroxyoctadecadienoic acid; and / or,

[0046] The set category of Chinese herbal medicine is Shanglu, the set category of processing method is vinegar processing, and the processing degree marker is 9-hydroxyoctadecadienoic acid or / and 13-hydroxyoctadecadienoic acid; and / or,

[0047] The set category of Chinese herbal medicine is Bajitian, the set category of processing method is steaming, and the processing degree marker is 9-hydroxyoctadecadienoic acid or / and 13-hydroxyoctadecadienoic acid.

[0048] In some embodiments of the present application, the processing degree marker is 9-hydroxyoctadecadienoic acid or / and 13-hydroxyoctadecadienoic acid which is negatively correlated with the processing degree.

[0049] The screening method of the processing degree marker of the traditional Chinese medicine decoction piece of the present application can be used to analyze the differences between traditional Chinese medicine decoction pieces with different processing degrees, find the difference markers from the metabolites of the traditional Chinese medicine decoction pieces, and then screen the processing degree markers, and a fast and reliable method for finding effective processing degree markers is established. In the method, the use of metabolomics combined with chemometrics is beneficial to find the difference markers of biological metabolism changes caused by specific interference (such as the change of processing parameters). At present, most of the non-targeted metabolomics researches are carried out between Chinese herbal medicines and their corresponding processed traditional Chinese medicine decoction pieces, and the present application uses it to study and screen the processing degree markers related to processing parameters such as processing temperature, processing time and auxiliary material amount.

[0050] The processing degree marker obtained by the screening method of the processing degree marker of the traditional Chinese medicine decoction piece provided by the present application can clearly indicate the heating processing process of Chinese herbal medicine; according to the screened processing degree marker, the quantitative limit (which can be upper limit or lower limit) of the processing marker in the processed traditional Chinese medicine decoction piece is formulated, which can realize quality control in the processing of traditional Chinese medicine, and is beneficial to improve the quality consistency of traditional Chinese medicine decoction pieces.

[0051] Most of the Chinese herbal pieces lack clear processing degree markers, and the processing endpoint can only be determined by observing the appearance and color. It is necessary to find the processing degree markers of Chinese herbal pieces. The processing markers, such as octadecadienoic acid oxidation metabolites, determined by the method can clearly indicate the heating processing of Euphorbia L. Chinese herbal medicines, and facilitate the quality control of related processed products / Chinese herbal pieces. Some processing metabolites including but not limited to 9-HODE and 13-HODE are screened as processing degree markers, and have good universality and can be used as processing degree markers of various Chinese herbal pieces. For Chinese herbal medicines (such as Gansui) using 9-HODE or / and 13-HODE as processing degree markers, after the limit of quantification in Chinese herbal pieces (such as vinegar Gansui) processed by a specific processing method is determined, the processing degree of Chinese herbal medicines can be controlled, and the quality control of Chinese herbal pieces can be realized, which provides an effective guide for the quality control of Chinese herbal pieces.

[0052] The present application not only discloses the feasibility of processing degree markers, but also provides a method for screening processing degree markers, which provides a valuable research idea for future processing degree marker searching. By screening the markers, new objective indicators can be provided for the quality control of different kinds of Chinese herbal medicines / Chinese herbal pieces.

[0053] Gansui is a Chinese herbal piece with reduced toxicity by processing, which is derived from the dried root of Euphorbia kansui T.N.Liou ex T.P.Wang and is used for treating edema, ascites and asthma. According to clinical practice, vinegar processing is a common method to reduce the toxicity of Gansui and alleviate its drastic purgative effect. Other Chinese medicines such as Phytolacca acinosa Roxb. or Phytolacca americana L. dried roots can also reduce toxicity by vinegar processing. Corydalis yanhusuo W.T.Wang dried tubers have enhanced analgesic effect after vinegar processing. In addition, some Chinese herbal pieces are processed in other ways, such as Coix lacryma-jobi L.var mayuen (Roman.) Stapf dried mature seed kernels, which are fried with bran to enhance the spleen- strengthening effect; Morinda officinalis How dried roots are steamed with salt to enhance the kidney- tonifying effect. At present, there is almost no marker to control the processing degree of the above-mentioned Chinese herbal pieces. 9-HODE and 13-HODE screened by the method of the present application can be used as processing degree markers of vinegar Gansui, vinegar Corydalis yanhusuo, vinegar Phytolacca acinosa Roxb., bran-fried Coix lacryma-jobi L.var mayuen (Roman.) Stapf and salt Morinda officinalis How, and are effective universal processing markers. Since 9-HODE and 13-HODE have the characteristic that their contents decrease after heating, they have the potential to be used as processing degree markers of more kinds of Chinese herbal medicines.

[0054] This invention also provides a method for controlling the degree of processing of traditional Chinese medicine (TCM). Based on the processing degree markers screened by this invention, quantitative limits for these markers in TCM decoction pieces are set, thereby controlling the quality of the TCM decoction pieces by controlling the degree of processing. For example, this invention provides a quality control method using 9-HODE and / or 13-HODE as processing degree markers. By controlling the content of these processing degree markers during the vinegar-processing of Euphorbia kansui, the quality control of vinegar-processed Euphorbia kansui TCM decoction pieces is achieved. More specifically, for example, a quantitative limit (e.g., 0.0011%) is set as the upper limit, using 120% of the average mass content of 9-HODE and / or 13-HODE in commercially available vinegar-processed Euphorbia kansui. This limit is used as the content index of the processing degree marker to control the degree of processing, resulting in vinegar-processed Euphorbia kansui TCM decoction pieces that meet the processing requirements. Attached Figure Description

[0055] Figure 1 This is a representative total ion chromatogram of vinegar-processed Euphorbia kansui prepared by two different frying times (5 min and 15 min) in one embodiment of the present invention; the black chromatographic peak numbers in the figure are consistent with the identified metabolite numbers, and the gray chromatographic peak numbers 14 and 50 are candidate markers of the degree of processing screened in this application.

[0056] Figure 2 This is a chemometric analysis chart for identifying potential markers of processing degree in one embodiment of the present invention. (A) PCA score charts of raw Euphorbia kansui and Euphorbia kansui processed with vinegar for different times; (B) OPLS-DA score charts of Euphorbia kansui processed with vinegar for 5 min and 15 min; (C) VIP score charts of various metabolic characteristics, VIP value > 7, number of characteristics = 35, t in the chart. R 6.37_m / z296.2350n、t R 30.52_m / z and 590.4907n correspond to two metabolic features used for screening candidate biomarkers; (D) shows an S-line plot with some identified metabolites, where the lines are numbered according to... Figure 1 The peak numbers in the text are consistent; (E) as the vinegar roasting time increases, the characteristic t R The mass spectrometry response at 6.37 m / z and 296.2350 nm gradually decreased; (F) with the extension of vinegar roasting time, the characteristic t R The mass spectrometry response at 30.52 m / z and 590.4907 n gradually increases.

[0057] Figure 3is the standard substance comparison and quantitative analysis method of potential processing markers in one embodiment of the present application.(A) Secondary mass spectrometry data of 13-HODE and 9-HODE in standard substance and test sample; (B) Chemical structural formula of 13-HODE and 9-HODE identified in the embodiment (both hydroxyl and diene bond positions are not adjustable); (C) High performance liquid chromatography-ultraviolet chromatogram of mixed standard substance (13-HODE and 9-HODE), raw Kansui root sample and vinegar Kansui root sample.

[0058] Figure 4 is a chart of the verification analysis of potential processing markers using real-world samples and laboratory self-made samples in one embodiment of the present application.(A) Content analysis of 13-HODE and 9-HODE in commercially available raw Kansui root and commercially available vinegar Kansui root; (B) Effect of frying temperature on the content of 13-HODE and 9-HODE in fried products (frying time is 8 min, and the amount of rice vinegar is 20% (w / w) of the mass of raw product); (C) Effect of the amount of rice vinegar on the content of 13-HODE and 9-HODE in fried products (frying temperature is 150℃, and frying time varies with the amount of vinegar to ensure drying).

[0059] Figure 5 is the high performance liquid chromatography-ultraviolet chromatogram of different kinds of traditional Chinese medicine decoction pieces in one embodiment of the present application.(A) Fried coix seed; (B) salt baijiatian; (C) vinegar corydalis; (D) vinegar phytolacca; (E) wine angelica; (F) salt alismatis; (G) vinegar cyperus; (H) vinegar daphne.

[0060] Figure 6 is the change of the content of 13-HODE and 9-HODE in different traditional Chinese medicine decoction pieces in their respective processing processes in one embodiment of the present application (the amount of auxiliary materials meets the relevant requirements of Chinese Pharmacopoeia).(A) Fried coix seed (250℃); (B) salt baijiatian; (C) vinegar corydalis (150℃); (D) vinegar phytolacca (150℃); (E) wine angelica (100℃); (F) salt alismatis (100℃); (G) vinegar cyperus (150℃); (H) vinegar daphne (150℃).

[0061] Figure 7 is a summary of the use of 13-HODE and 9-HODE as processing markers of different processing methods in one embodiment of the present application, wherein 13-HODE and 9-HODE can be used as processing markers of traditional Chinese medicine decoction pieces marked with a check mark (). DETAILED DESCRIPTION

[0062] For the purpose of promoting an understanding of the application, the application will be described in further detail below with reference to the drawings and some preferred embodiments or specific examples of the application. It should be understood that the application can be carried out in many different forms and should not be considered limited to the embodiments and specific examples described herein. Instead, these embodiments and examples are provided so that this disclosure will be thorough and complete, and fully convey the scope of the application to those skilled in the art. Also, it should be understood that the application can be carried out in many different forms and should not be considered limited to the embodiments and specific examples described herein, as this application can be practiced with many modifications, alterations, and variations within the scope of the present application, which should be considered to fall within the scope of the application. In addition, throughout this application the use of "or" means "and / or" unless strictly stated otherwise. Furthermore, in the following description, numerous specific details are set forth in order to provide a thorough understanding of the application. However, it will be recognized by one skilled in the art, that the application can be practiced without one or more of these specific details. In other instances, well known structures and functions have not been described in detail in order to avoid obscuring the application.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing embodiments and examples only and is not intended to be limiting of the application.

[0064] Terminology

[0065] Unless otherwise indicated or unless contradicted by context, terms or phrases used herein have the following meanings:

[0066] The selection scope of the terms "and / or", "or / and", "and / or" used herein includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, which includes any two relevant listed items, any more relevant listed items, or all relevant listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in this application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B, and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (i.e., the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C, and D, i.e., includes the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (i.e., the technical solution connected by "logical and").

[0067] In the present application, if there are multiple technical features in one technical solution, and these features have preferences and examples respectively, the preferences and / or examples of different features can be combined in any suitable manner.

[0068] As used herein, "combinations thereof", "any combination thereof", "any combination manner thereof" and the like include all suitable combination manners of any two or more of the listed items.

[0069] As used herein, "suitable", "suitable manner", "any suitable manner" and the like are subject to the ability to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the intended technical effects of the present application.

[0070] As used herein, "preferred" is only to describe the implementation manner or embodiment with better effect, and it should be understood that it does not constitute a limitation on the protection scope of the present application.

[0071] In the present application, "optionally", "optional" means optional, that is, either "yes" or "no" of the two parallel schemes. If there are multiple "optionally" in one technical solution, and there is no contradiction or mutual restriction relationship, each "optionally" is independent.

[0072] In the present application, in the terms "first aspect", "second aspect", "third aspect", "fourth aspect" and the like, the terms "first", "second", "third", "fourth" and the like are only for description purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implying the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration and description, and should be understood as not constituting a closed limitation on the quantity.

[0073] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features. For example, for "the real world sample includes commercially available raw gansuon and commercially available vinegar gansuon", it includes "the real world sample is commercially available raw gansuon and commercially available vinegar gansuon".

[0074] Traditional Chinese medicine processing: It is the process of transforming Chinese herbal medicine into traditional Chinese medicine decoction pieces, and its purposes include reducing toxicity and increasing efficacy, changing or moderating drug properties, facilitating the decoction of effective components, etc.

[0075] Processing method: It refers to the method of transforming Chinese herbal medicine into traditional Chinese medicine decoction pieces, which can or can not add auxiliary materials, including but not limited to frying, baking, steaming, boiling, calcining, and baking.

[0076] Chinese medicine processed product: a substance obtained by processing Chinese herbal medicine. The processing degree of the processed product described in the present application is not specifically limited. The Chinese medicine processed product is one of Chinese herbal pieces. In the present application, unless defined otherwise, "Chinese medicine processed product" and "processed product" have the same meaning and can be used interchangeably.

[0077] Chinese medicine raw product: a Chinese herbal piece without heating processing. In the present application, unless defined otherwise, "Chinese medicine raw product" and "raw product" have the same meaning and can be used interchangeably.

[0078] Chinese medicine piece processing degree marker: in the present application, processing degree marker and Chinese medicine piece processing degree marker have the same meaning and can be used interchangeably. The Chinese medicine piece processing degree marker refers to an endogenous chemical component in Chinese herbal medicine, which has a positive or negative correlation between the content change in the processing process and the processing degree of Chinese medicine pieces, and can be used as a marker substance reflecting the processing degree of Chinese medicine pieces for the control of the processing process of Chinese medicine.

[0079] Difference marker: refers to a characteristic differential component that can reflect characteristic information difference. The difference marker of the processing process refers to a differential component that can reflect the characteristic difference of the processing process, which can be used as a candidate for the processing process marker.

[0080] Test sample: refers to a sample to be tested.

[0081] Standard substance: refers to a standard substance in the national drug standard for identification, inspection, content determination, impurity and related substance inspection, etc.

[0082] Reference sample: a sample used for comparison with the test sample. The standard substance is a typical reference sample.

[0083] Metabolite: refers to a substance produced or consumed by an organism through a metabolic process, which can reflect the degree of metabolism according to the content change of the metabolite. Unless otherwise specified, in the present application, metabolite mainly refers to endogenous chemical components in Chinese medicine raw products. For example, 13-HODE, 9-HODE, 15-HODE, 9-OODE, 13-OODE, etc. are metabolites in Chinese medicine raw products.

[0084] Metabolite characteristics: refers to characteristics derived from mass spectrometry data processing software. Typical examples include the retention time and m / z value of ions, which represent potential metabolite ions.

[0085] Frying method: after cleaning or cutting the medicine, put it in a preheated frying container, add auxiliary materials or not, then heat with different fire and continuously stir-fry or rotate to reach a certain degree of processing method.

[0086] Stir-frying with accessories: the method of frying the cleaned or cut medicinal materials with solid accessories. According to the different accessories, it can include but not limited to bran frying, rice frying, earth frying, sand frying, clam powder frying, talc powder frying, etc.

[0087] Baking method: the method of taking the cleaned medicinal materials and mixing them with liquid accessories, and then putting them into a container and heating them. According to the different liquid accessories, it can include but not limited to vinegar baking, wine baking, honey baking, ginger juice baking, salt baking, oil baking, medicinal juice baking, etc.

[0088] Steaming method: the method of putting the cleaned medicinal materials into a steaming container with or without accessories and heating them with water vapor or waterless heating to a certain degree. The accessories can be salt. Without accessories, it is clear steaming.

[0089] Boiling method: the method of putting the cleaned medicinal materials into a container with or without accessories (the solid accessories need to be crushed first), and boiling them with water. Without accessories, it is clear boiling. According to the different accessories, it can include but not limited to vinegar boiling, medicinal juice boiling, bean curd boiling, etc.

[0090] Scalding method: the method of putting the medicinal materials into boiling water for a short time, taking them out, and separating the seed coat.

[0091] Roasting method: the purpose of roasting is to remove part of the volatile oil and irritating components in the medicinal materials, so as to reduce the side effects, or to moderate the medicinal properties and enhance the curative effect. It includes but not limited to flour roasting, paper roasting, wheat bran roasting, talc powder roasting, etc.

[0092] Metabolomics method: the science about the whole body and its change rule of endogenous metabolites in organisms. Non-targeted metabolomics based on liquid chromatography-high resolution mass spectrometry is a problem-oriented technology, which has been widely used in the study of metabolic changes in biological systems.

[0093] Chemometrics method: in the present invention, the chemometrics method can be used for statistical analysis of metabolite characteristics, including but not limited to: using principal component analysis (PCA) method to view the difference between different processed products; using orthogonal partial least squares discriminant analysis (OPLS-DA) to view the difference between the lowest and highest processed products; comparing the contribution of group difference through VIP diagram, and screening out the components with large contribution to group difference; through one-way ANOVA, screening out the chemical components with obvious group difference and the characteristic response value (such as mass spectrometry response value) representing the content, which is positively or negatively correlated with the processing degree. The PCA method, OPLS-DA method, VIP analysis, one-way ANOVA are known to those skilled in the art of chemometrics. The chemometrics analysis method used in the present invention can be carried out by analysis software such as SIMCA 14.1 software, GraphPad 9.0 software, etc.

[0094] In the present invention, when referring to a numerical interval (i.e. a numerical range), unless otherwise specified, both endpoints of the numerical interval are included. Unless otherwise specified, an optional numerical distribution within the numerical interval is considered to be continuous and includes both numerical endpoints (i.e. the minimum and maximum values) of the numerical range, as well as every numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, both endpoints of the numerical range are included, as well as every integer between the two endpoints. Furthermore, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise indicated, ranges disclosed herein are to be understood to include any and all sub-ranges subsumed therein. Temperature parameters in the present invention, unless otherwise specified, allow for constant temperature treatment as well as variations within a temperature interval. It is to be understood that the constant temperature treatment allows for fluctuations within the accuracy of the instrument control.

[0095] In the present invention, percentage content, unless otherwise specified, refers to volume percentage for gas-gas mixtures, mass percentage wt% for solid-solid mixtures, volume percentage % (v / v) for liquid-liquid mixtures, and mass percentage wt% or solid-liquid percentage % (w / v) for solid-liquid mixtures.

[0096] In the present invention, % (w / w) and wt% both refer to weight percentage.

[0097] The first aspect of the present invention provides a screening method for traditional Chinese medicine processing markers, comprising the following steps:

[0098] S100: According to the set category of Chinese herbal medicine, the set category of processing method and the set category of processing parameter, adjust the set category of processing parameter, obtain the processing products with different processing degrees;

[0099] S110: Detect the test sample to obtain the detection result, wherein the test sample includes the processing products with different processing degrees and the corresponding raw products;

[0100] S120: Analyze the detection result obtained in step S110 by using a metabolomics method combined with a chemometrics method, screen out the differential markers with a positive or negative correlation between the characteristic response representing the content and the processing degree, as potential processing markers, including the following processes:

[0101] Analyzing the detection result by using a metabolomics method;

[0102] Analyzing the detection result by using a chemometrics method;

[0103] The differential markers with characteristic response values representing content positively or negatively correlated with the processing degree are screened, preferably, differential markers with mass spectrometry response positively or negatively correlated with the processing degree are screened;

[0104] S130: identifying the differential markers and establishing a quantitative analysis method for the differential markers;

[0105] S140: verifying the differential markers according to real-world samples and self-made samples using the quantitative analysis method, and screening the processing degree markers of the set category of Chinese herbal medicines;

[0106] Optionally, S150: verifying the universality of the screened processing markers of the set category of Chinese herbal medicines.

[0107] The screening method combines metabolomics and chemometrics to analyze the differences between Chinese herbal pieces with different processing degrees, can find differential markers from Chinese herbal piece metabolites, and further screen processing degree markers, and establishes a fast and reliable method for finding effective processing degree markers. In the screening method, metabolomics combined with chemometrics is beneficial to find differential markers caused by specific interference (such as changes in processing parameters).

[0108] In some embodiments of the present application, the processing markers are selected from endogenous chemical components in Chinese herbal pieces. The same component can produce metabolites with different molecular formulas, and metabolites with the same molecular formula can also have different isomers. It should be noted that although a certain molecular formula may theoretically have a very large number of isomers, the component that can be generated by a specific processing method can be used as a processing marker of the present application, and therefore, through the limitation of raw materials and processing methods, the range of isomers involved can be considered clear and definite.

[0109] In some embodiments of the present application, the processing degree marker is an octadecadienoic acid oxidation metabolite and / or other lipid compounds. The octadecadienoic acid oxidation metabolite specifically includes, for example, hydroxyl-substituted octadecadienoic acid, oxo-octadecadienoic acid, and the like.

[0110] In some embodiments of the present application, the processing marker is at least one of the following compounds: octadecadienoic acid metabolite, octadecatrienoic acid metabolite. The olefin bond can have different positions to form different isomers.

[0111] In some embodiments of the present application, the octadecadienoic acid metabolite is an octadecadienoic acid oxidation metabolite (which can be independently preferred), and / or the octadecatrienoic acid metabolite is an octadecatrienoic acid oxidation metabolite (which can be independently preferred).

[0112] In some embodiments of the present application, the octadecadienoic acid metabolite is preferably an oxidized octadecadienoic acid metabolite, more preferably at least one of hydroxyoctadecadienoic acid (HODE), oxo-octadecadienoic acid (OOD E), and more preferably at least one of 9-hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), 15-hydroxyoctadecadienoic acid (15-HODE), 9-oxo-octadecadienoic acid (9-OODE), 13-oxo-octadecadienoic acid (13-OODE), and the like.

[0113] In some embodiments of the present application, the octadecatrienoic acid metabolite is preferably an oxidized octadecatrienoic acid metabolite, more preferably hydroxyoctadecatrienoic acid (HOTE), and further preferably at least one of 9-hydroxyoctadecatrienoic acid (9-HOTE), 13-hydroxyoctadecatrienoic acid (13-HOTE), and the like.

[0114] In some embodiments of the present application, the processing marker is at least one of hydroxyoctadecadienoic acid (HODE), oxo-octadecadienoic acid (OOD E), hydroxyoctadecatrienoic acid (HOTE), and the like; the aforementioned listed compounds include various isomers thereof, such as HODE isomers can include 9-hydroxyoctadecadienoic acid (9-HODE), 13-hydroxyoctadecadienoic acid (13-HODE), 15-hydroxyoctadecadienoic acid (15-HODE), and the like; OOD E isomers can include 9-oxo-octadecadienoic acid (9-OODE), 13-oxo-octadecadienoic acid (13-OODE), and the like; and HOTE isomers can include 9-hydroxyoctadecatrienoic acid (9-HOTE), 13-hydroxyoctadecatrienoic acid (13-HOTE), and the like.

[0115] In some embodiments of the present application, the processing marker is at least one of 9-HODE, 13-HODE, 15-HODE, 9-OODE, 13-OODE, 9-HOTE, 13-HOTE, and the like.

[0116] In some embodiments of the present application, the processing marker is at least one of 9-HODE, 13-HODE, and other isomers of HODE. The other isomers of HODE are isomers that can be generated in the processing process, and do not exceed the set of isomers that exist in theory.

[0117] In some preferred embodiments of the present application, the processing marker is 9-HODE or / and 13-HODE.

[0118] In some preferred embodiments of the present application, the structures of 9-HODE and 13-HODE are shown as follows, respectively.

[0119]

[0120] S100: Obtain processed products of different processing degrees.

[0121] In some embodiments of the present application, the set category of Chinese herbal medicine is the genus of Jatropha.

[0122] In some embodiments of the present application, the different processing degrees of processed products include at least three different processing degrees of processed products. For example, 3, 4, 5, 6, 7, or more different processing degrees. The different processing degrees can be caused by single factors or multiple factors. Preferably, at least one single factor corresponds to at least 3 different processing degrees. More preferably, each single factor corresponds to at least 3 different processing degrees.

[0123] In some embodiments of the present application, the set category of Chinese herbal medicine is any one of Kansui, Yiyiren, Danggui, Yansu, and Heilu.

[0124] In some embodiments of the present application, the set category of processing method is any one of stir-frying, baking, steaming, boiling, calcining, and baking.

[0125] In some embodiments of the present application, the set category of processing parameter is at least one of processing time, processing temperature, and amount of auxiliary material.

[0126] In some embodiments of the present application, the different processing degrees of processed products are any one of vinegar Kansui, vinegar Yansu, vinegar Heilu, bran-stir-fried Yiyiren, and salt Danggui.

[0127] In some embodiments of the present application, the different processing degrees of processed products are vinegar-baked processed products, which are processed by adjusting at least one of processing time, processing temperature, and amount of vinegar.

[0128] In some preferred embodiments of the present application, the vinegar-processed product is any one of vinegar Euphorbia kansui, vinegar Corydalis, or vinegar Phytolacca.

[0129] In some preferred embodiments of the present application, the different processing degrees of the processed product are vinegar Euphorbia kansui processed for different processing times. For example, 5 min, 10 min, 15 min. The different processing degrees of vinegar Euphorbia kansui can be obtained by selecting different processing times according to the method recorded in Chinese Pharmacopoeia (2020 edition). Please refer to section 1.2 of Example 1.

[0130] S110: Detect the test samples (including processed products of different processing degrees).

[0131] The test sample is detected, including preparing the test sample into a test sample to be detected.

[0132] The detection method of the test sample to be detected includes but is not limited to liquid detection and solid detection.

[0133] The preparation method of the test sample to be detected can include the following steps: extracting the test sample with an extraction solvent to prepare the test sample to be detected. For example, see Example 2.

[0134] In some embodiments of the present application, the test sample to be detected is a solution of the test sample.

[0135] In some embodiments of the present application, the preparation method of the test sample to be detected includes the following steps: taking a certain amount of sieved raw powder, accurately weighing, adding an extraction solvent, ultrasonic treatment, vacuum filtration, washing the residue; combining the filtrate; evaporating the filtrate to dryness, dissolving the residue with methanol, adding methanol to a certain volume, shaking well, and obtaining it.

[0136] In some preferred embodiments of the present application, the preparation method of the test sample to be detected includes the following steps: taking 0.50 g of Euphorbia kansui powder (passed through a No. 4 sieve), accurately weighing, placing it in a conical flask with a stopper, adding 25 mL of ethyl acetate, tightly stoppering, ultrasonic treatment (power 100 W, frequency 37 kHz) for 30 min, vacuum filtration, washing the residue; combining the filtrate; evaporating the filtrate to dryness, dissolving the residue with methanol, transferring it to a 5 mL volumetric flask, adding methanol to the mark, shaking well, and obtaining it.

[0137] In some embodiments of the present application, the detection of the different processing degrees of the processed product is selected from one of the following modes: based on high performance liquid chromatography method, based on gas chromatography method, based on high performance liquid chromatography method combined with mass spectrometry, based on gas chromatography method combined with mass spectrometry, etc.

[0138] In some preferred embodiments of the present application, the different processed products of different processing degrees are detected based on a high performance liquid chromatography method combined with mass spectrometry. The extraction solvent used for preparing the test sample for detection can be, but is not limited to, any one of the following solvents or any combination thereof: methanol, methanol / water mixed solvent, ethanol, ethanol / water mixed solvent, ethyl acetate, chloroform, dichloromethane, n-hexane, and diethyl ether, etc.

[0139] In some preferred embodiments of the present application, the detection conditions of the high performance liquid chromatography method combined with mass spectrometry are as follows: acetonitrile is used as mobile phase A, and formic acid aqueous solution (independently preferably 0.1% formic acid aqueous solution) is used as mobile phase B, the flow phase gradient program is: 0 / 8 / 10 / 20 / 30 / 45 / 50 min; 53 / 65 / 65 / 80 / 90 / 100 / 100% acetonitrile; after 50 min, the initial mobile phase is changed to balance for 0-10 min, and the total running time is 50-60 min; the flow rate is 0.20-0.50 mL / min (for example, 0.2 mL / min, 0.25 mL / min, 0.3 mL / min, 0.35 mL / min, 0.4 mL / min, 0.45 mL / min, 0.5 mL / min); the column temperature is 25-35°C (for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C); the linear ion trap orbitrap combined mass spectrometry detector is used for detection, in positive ion mode; the source spray voltage is 3-5 kV (for example, 3 kV, 3.5 kV, 4 kV, 4.5 kV, 5 kV), the capillary temperature is 300-400°C (for example, 250°C, 260°C, 280°C, 300°C, 320°C, 350°C), the source heating temperature is 250-350°C (for example, 250°C, 260°C, 270°C, 280°C, 290°C, 300°C, 310°C, 320°C, 330°C, 340°C, 350°C), the sheath gas flow rate is 30%-40% (for example, 30%, 32%, 34%, 35%, 36%, 38%, 40%), the auxiliary gas flow rate is 1%-10% (for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%), the split ratio is 1:1, in full scan mode, the mass spectrometry acquisition range is set to 200 m / z-1500 m / z, the resolution is set to 20000-40000 (for example, 20000, 25000, 30000, 35000, 40000), MS 4 The collision energy is set to 20%-40% (for example, 20%, 25%, 30%, 35%, 40%), and the resolution is set to 7000-8000 (for example, 7000, 7500, 8000). The preferences and / or examples of the above-mentioned features can be combined in any suitable manner.

[0140] In some more preferred embodiments of the present application, the detection condition of the high performance liquid chromatography method combined with mass spectrometry is as follows: a high performance liquid chromatography system (e.g., an Acquity Ultimate 3000 high performance liquid chromatography system); a chromatographic column: Agilent Zorbax plus C8 (150 mm x 2.1 mm, 1.8 μm); mobile phase: acetonitrile as mobile phase A, formic acid aqueous solution (independently preferably 0.1% formic acid aqueous solution) as mobile phase B, and the mobile phase gradient program is as follows: 0 / 8 / 10 / 20 / 30 / 45 / 50 min; 53 / 65 / 65 / 80 / 90 / 100 / 100% acetonitrile; after 50 min, change to the initial mobile phase for equilibration for 4 min to 10 min (for example, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min); the total running time is 54 min to 60 min; the flow rate is 0.20 to 0.50 mL / min (independently preferably 0.35 mL / min); the column temperature is 25 to 35 °C (independently preferably 30 °C); the injection volume is 1 to 10 μL (preferably 5 μL); a linear ion trap orbitrap combined mass spectrometry (e.g., an LTQ-Orbitrap Velos Pro MS system) detector is used for detection in positive ion mode; the source spray voltage is 3 to 5 kV (independently preferably 4 kV), the capillary temperature is 300 to 400 °C (independently preferably 350 °C), the source heating temperature is 250 to 350 °C (independently preferably 200 °C), the sheath gas flow rate is 30% to 40% (independently preferably 35%), the auxiliary gas flow rate is 1% to 10% (independently preferably 5%), the split ratio is 1:1, in Full scan mode, the mass spectrometry acquisition range is set to 200 m / z to 1500 m / z, the resolution is set to 20000 to 40000 (independently preferably 30000), MS 4 The collision energy is set to 20% to 40% (independently preferably 30%), and the resolution is set to 7000 to 8000 (independently preferably 7500). The preferences and / or examples of the above-mentioned features can be combined in any suitable manner.

[0141] S120: Analyze the detection results obtained in S110 by using metabolomics methods combined with chemometrics methods, screen out differential markers with characteristic responses (such as mass spectrometry responses) positively or negatively correlated with processing degree as potential processing markers. S130: Establish a quantitative analysis method for the differential markers. Time / min

[0142] In some embodiments of the present application, the metabolomics method is based on the detection results of high performance liquid chromatography-high resolution mass spectrometry, thereby obtaining the metabolite information of the detected sample.

[0143] In some embodiments of the present application, the detection results obtained in step S110 include mass spectrometry data. The original mass spectrometry data are first analyzed qualitatively by Xcalibur 2.1 software. In one specific embodiment, 52 compounds are identified by m / z value and fragment spectrum comparison.

[0144] In some embodiments of the present application, the chemometric method is analyzed by using SIMCA 14.1 software and GraphPad 9.0 software. It should be understood that the chemometric method of the present application can also be realized by using other software.

[0145] In some embodiments of the present application, the analysis by using the chemometric method includes steps S121, S122 and S123.

[0146] S121: Differences between the different processed products of different processing degrees are viewed by using principal component analysis.

[0147] In some embodiments of the present application, the original mass spectrometry data are processed by Progenesis QI 2.3 software (Waters, Milford, USA); in one specific embodiment, a data matrix of 12373 metabolite characteristics is generated.

[0148] In some embodiments of the present application, the metabolite characteristic data matrix is imported into SIMCA 14.1 (Umetrics, Sweden) for multivariate analysis.

[0149] In some embodiments of the present application, after principal component analysis, the processed products of different processing degrees can be well separated according to the principal component analysis results; for example, raw Kansui Root and four groups of vinegar Kansui Root with different frying times (5 min, 10 min and 15 min) can be well separated.

[0150] S122: Differences between the processed product with the lowest processing degree and the processed product with the highest processing degree are viewed by using orthogonal partial least squares discriminant analysis.

[0151] The viewing range can be the different processed products of different processing degrees or part of the processed products; however, generally, the orthogonal partial least squares discriminant analysis is used to determine the difference information between the processed product with the lowest processing degree and the processed product with the highest processing degree among the different processed products of different processing degrees.

[0152] In some embodiments of the present application, for the vinegar Kansui Root samples with different processing times, the samples with the lowest and highest processing degrees are vinegar Kansui Root with frying times of 5 min and 15 min, respectively.

[0153] S123: screening out the processing metabolites with obvious group difference and the characteristic response value (such as mass spectrometry response value) representing content positively or negatively correlated with the degree of processing as potential processing degree markers, including the following processes:

[0154] VIP figure analysis is adopted to screen out metabolites with large contribution to group difference (preferably meeting VIP value > 7);

[0155] Single factor variance analysis is adopted to screen out metabolites with obvious group difference (preferably meeting P value < 0.05) and the characteristic response value (such as mass spectrometry response value) representing content positively or negatively correlated with the degree of processing.

[0156] In some embodiments of the present application, the VIP limit value is set to 7.0, and 35 metabolic characteristics are obtained.

[0157] In some embodiments of the present application, in the S-line figure, the metabolic characteristics are arranged according to the retention time and labeled according to the identified components, and the changes of the metabolic characteristics in the processing process can be observed.

[0158] In some embodiments of the present application, single factor variance analysis (ANOVA) is adopted to analyze the mass spectrometry response values of the 35 metabolic characteristics in the four groups (raw Kansui root and vinegar Kansui roots with different frying times) respectively to test the group difference.

[0159] In some embodiments of the present application, two characteristics with gradually decreasing mass spectrometry response values with the vinegar frying time are screened out, characteristic t R 6.37_m / z 296.2350n and characteristic t R 4.83_m / z 294.2197n, which are identified as 13-HODE isomers and 13-HOTE isomers respectively; wherein, characteristic 6.37_296.2350n is derived from QI software, 6.37 represents the retention time, 296.2350 represents the m / z of the characteristic ion, and n represents that the characteristic ion is the neutral ion derived from QI software. Characteristics with gradually increasing mass spectrometry response values are also screened out, characteristic t R 30.52_m / z 590.4907n, which is identified as DG (16:0 / 0:0 / 18:3n3) isomer.

[0160] When screening potential processing markers in S120 step, components with higher mass spectrometry response value and smaller P value are preferred.

[0161] When screening potential processing degree markers in S120 step, components with higher mass spectrometry response value and smaller P value are preferred.

[0162] In some embodiments of the present application, the 13-HODE isomers with higher mass spectrometry response values and smaller P values are selected as potential processing markers.

[0163] In some embodiments of the present application, the screening method of the traditional Chinese medicine processing marker further comprises the following steps in the S120 step: identifying or determining the chemical composition of the differential marker so as to determine what the differential marker is. For example, the process of identifying the differential marker in Example 2 is confirmed to be the chemical composition of 13-HODE isomer, 13-HOTE isomer, and DG(16:0 / 0:0 / 18:3n3) isomer. The mass spectrometry of the test sample and the standard sample can be compared, and other methods for identifying unknown metabolites used by those skilled in the art can also be used.

[0164] Mobile phase composition

[0165] In some embodiments of the present application, the quantitative analysis method of the differential marker comprises: establishing a high performance liquid chromatography-ultraviolet quantitative analysis method, and verifying the high performance liquid chromatography-ultraviolet quantitative analysis method. In some preferred embodiments of the present application, the verification comprises at least one of the following analyses (one of the preferred ways comprises all of the following analyses): linearity analysis, limit of detection and limit of quantification analysis, repeatability analysis, precision analysis, recovery rate analysis, and stability analysis. After verification, the high performance liquid chromatography-ultraviolet quantitative analysis method provided by the present application meets the quantitative analysis requirements of the processing marker, and can refer to Example 3.

[0166] In some embodiments of the present application, the potential processing marker (also referred to as the candidate differential marker) is identified as 9-HODE and 13-HODE by comparison with the standard sample.

[0167] In some embodiments of the present application, the quantitative analysis method of the S130 step adopts a high performance liquid chromatography-ultraviolet method for analysis.

[0168] In some embodiments of the present application, in the step S130, the sample weighing amount and the extraction solvent volume are 2 g and 100 mL respectively, and the remaining parameters can be in the same manner as in the S110 step. The step S130 further comprises preparing standard sample solutions of 13-HODE and 9-HODE as control samples to be tested.

[0169] In some embodiments of the present application, in the step S130, an Acquity UPLC H-Class high performance liquid chromatography system is used; an Acquity UPLC BEH C18(100mm×2.1mm, 1.7μm) chromatographic column is used; the column temperature is 30℃; and the injection volume is 1μL.

[0170] In some embodiments of the present application, in step S130, the mobile phase is acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and the mobile phase gradient program is:

[0171] 44% acetonitrile 44% acetonitrile 0 100% acetonitrile 20 100% acetonitrile 21 S140: According to real-world samples and self-made samples, verify the differential markers by using the quantitative analysis method, and screen out processing degree markers of the set category of Chinese herbal medicines. 27 Optional S150 step: Verify the processing markers of the set category of Chinese herbal medicines screened out for universality.

[0172] The mobile phase gradient program in the above table is recorded as 1 / 20 / 21 / 27 min, 44 / 44 / 100 / 100% acetonitrile.

[0173] In some embodiments of the present application, in step S130, a UV detector or other type of detector is used, and the detection wavelength is 220-240 nm, and in some specific examples, 230 nm.

[0174] In some embodiments of the present application, in step S130, the high-performance liquid chromatography-ultraviolet quantitative analysis method used is verified to meet the quantitative analysis requirements through linear, limit of detection and limit of quantification, repeatability, precision, recovery rate and stability methodological investigations. The limit of quantification can reach 0.5 ng.

[0175] Figure 1 Figure 2

[0176] In some embodiments of the present application, the set category of Chinese herbal medicine is Kansui, and the real-world sample includes commercially available raw Kansui and commercially available vinegar Kansui.

[0177] In some embodiments of the present application, the set category of Chinese herbal medicine is Kansui, and the self-made sample includes vinegar Kansui made by adjusting at least one processing parameter of processing time, processing temperature, and amount of vinegar.

[0178] In some embodiments of the present application, the real-world sample is represented by commercially available products. As shown in Example 4.

[0179] In some embodiments of the present application, the self-made sample is a laboratory self-made sample.

[0180] In some embodiments of the present application, the self-made sample is, for example, the self-made sample used in Examples 1-5.

[0181] In some embodiments of the present application, the real-world sample includes commercially available raw Kansui and commercially available vinegar Kansui.

[0182] In some embodiments of the present application, the self-made sample includes vinegar Kansui made by adjusting at least one processing parameter of processing time, processing temperature, and amount of vinegar.

[0183] In some embodiments of the present application, the real-world sample is commercially available raw Kansui and commercially available vinegar Kansui.

[0184] In some embodiments of the present application, the real-world sample is market circulating raw Kansu and vinegar Kansu.

[0185] In some embodiments of the present application, the self-made sample is vinegar Kansu prepared by adjusting at least one of processing time, processing temperature, and amount of vinegar.

[0186] In some embodiments of the present application, the laboratory self-made sample is vinegar Kansu prepared by using different processing temperatures or / and different amounts of vinegar.

[0187] In some embodiments of the present application, in step S140, the content of 13-HODE in the market raw Kansu and the market vinegar Kansu is 12.6-20.59 ppm and 5.76-12.98 ppm, respectively. The content of 9-HODE in the market raw Kansu and the market vinegar Kansu is 12.45-21.52 ppm and 6.07-13.34 ppm, respectively.

[0188] In some embodiments of the present application, by T test, the content of 9-HODE in the market raw Kansu and the market vinegar Kansu has significant difference, and the content of 13-HODE in the two also has significant difference. It can be seen that 9-HODE and 13-HODE can be used as processing degree markers to effectively identify different processing degrees, thereby effectively analyzing the effect of processing on Chinese herbal medicine materials.

[0189] In some embodiments of the present application, in step S140, the upper limit of the average content of the market vinegar Kansu is 120%, and the upper limit of 13-HODE and 9-HODE is set to 0.0011%. The content of 13-HODE and 9-HODE in most of the market vinegar Kansu meets the processing requirements.

[0190] In some embodiments of the present application, in step S140, in the vinegar Kansu prepared by different processing temperatures, the content of 9-HODE and 13-HODE is negatively correlated with the processing degree; different amounts of vinegar have no significant effect on the content of 9-HODE and 13-HODE in the processed product.

[0191] Figure 2 Figure 2

[0192] The processing markers of the set type of Chinese herbal medicines screened in step S140 are applied to other types of traditional Chinese medicine decoction pieces or / and the set type of Chinese herbal medicines obtained by other processing methods, to verify whether the processing markers are still effective processing markers for other types of traditional Chinese medicine decoction pieces or other processing methods, and whether they can still effectively identify different processing degrees. The method in step S140 can be used for verification.

[0193] In some embodiments of the present invention, other processing methods include stir-frying, roasting, steaming, and / or other types of Chinese medicinal slices (including but not limited to vinegar-processed Corydalis, vinegar-processed Phytolacca acinosa, wheat bran-fried Coix lacryma-jobi, salt-processed Morinda officinalis, wine-processed Angelica sinensis, salt-processed Alisma plantago-aquatica, vinegar-processed Cyperus rotundus, and vinegar-processed Daphne genkwa).

[0194] In some embodiments of the present invention, the contents of 9-HODE and 13-HODE in the processed Chinese medicine products prepared by methods such as stir-frying, roasting, and steaming are negatively correlated with the degree of processing.

[0195] In some embodiments of the present invention, the contents of 9-HODE and 13-HODE in vinegar-processed Corydalis yanhusuo, vinegar-processed Phytolacca acinosa, bran-fried Coix lacryma-jobi, salt-processed Morinda officinalis, and some other types of Chinese medicinal herbs are negatively correlated with the degree of processing.

[0196] According to the results of several embodiments of the present invention, it was found that although the contents of 13-HODE and 9-HODE in the decoction pieces are low, they are heat-sensitive and negatively correlated with the degree of processing, and can be used as general processing markers for most Chinese herbal medicine decoction pieces.

[0197] The 9-HODE and 13-HODE selected by this invention have strong versatility as markers of the degree of processing.

[0198] In some embodiments of the present invention, the method for screening markers of the degree of processing of traditional Chinese medicine decoction pieces includes the following steps:

[0199] The detection results were analyzed using metabolomics methods to obtain information on metabolites of processed products at different processing levels (i.e., the types of processed metabolites are at least two).

[0200] The test results were analyzed using chemometric methods to obtain information on the differences between the processed products with different degrees of processing.

[0201] Based on the information on metabolites of processed products with different processing degrees and the difference information, differential markers whose content is positively or negatively correlated with the processing degree are screened from the metabolites of processed products with different processing degrees.

[0202] Identify the differential markers and establish quantitative analysis methods for the differential markers;

[0203] Based on real-world samples and self-made samples, the quantitative analysis method was used to verify the differential markers and screen out the processing degree markers for the specified types of Chinese herbal medicines.

[0204] A second aspect of the present invention provides a method for quantitative analysis of biomarkers in traditional Chinese medicine processing, comprising the following steps:

[0205] Obtaining a test sample containing the processing marker, extracting the test sample containing the processing marker by using an extraction reagent to prepare a test sample to be tested;

[0206] Obtaining a control sample containing the processing marker, extracting the control sample containing the processing marker by using the extraction reagent to prepare a control sample to be tested;

[0207] Performing high performance liquid chromatography-ultraviolet detection on the test sample to be tested to obtain a test sample chromatogram;

[0208] Performing high performance liquid chromatography-ultraviolet detection on the control sample to be tested to obtain a control sample chromatogram;

[0209] Comparing the test sample chromatogram and the control sample chromatogram to determine the content of the processing marker;

[0210] The processing marker is screened according to the method of the first aspect of the present application.

[0211] The quantitative analysis method provided by the second aspect of the present application is a high performance liquid chromatography-ultraviolet quantitative analysis method.

[0212] The quantitative analysis method part of the S130 step of the first aspect of the present application, the listed implementation example, the preferred embodiment mode, the specific embodiment, and the preferred embodiment are also applicable to the quantitative analysis method of the second aspect of the present application.

[0213] When performing high performance liquid chromatography-ultraviolet detection on the test sample to be tested, the preparation method of the test sample to be tested can refer to Embodiment 3.

[0214] In some embodiments of the present application, the extraction solvent is any one of the following solvents or any combination thereof: methanol, methanol / water mixed solvent, ethanol, ethanol / water mixed solvent, ethyl acetate, chloroform, dichloromethane, n-hexane, and diethyl ether.

[0215] In some embodiments of the present application, in the high performance liquid chromatography-ultraviolet detection, a reversed-phase octadecyl bonded phase chromatographic column or an octyl bonded phase chromatographic column is used for high performance liquid chromatography separation, and / or an ultraviolet detector is used.

[0216] In some preferred embodiments of the present application, the detection conditions of the high performance liquid chromatography-ultraviolet detection are as follows: acetonitrile is used as mobile phase A, and phosphoric acid aqueous solution (preferably 0.1% phosphoric acid aqueous solution) is used as mobile phase B, the elution mode is gradient elution, and further preferably, the ultraviolet detection wavelength is 220nm-240nm (more specifically, such as 230nm).

[0217] In some embodiments of the present application, the detection conditions of the high performance liquid chromatography-ultraviolet detection are as follows: a chromatographic column: Acquity UPLC BEH C18 (100 mm x 2.1 mm, 1.7 μm); a mobile phase: a mobile phase A is acetonitrile, a mobile phase B is a phosphoric acid aqueous solution (preferably 0.1% phosphoric acid aqueous solution), a mobile phase gradient program is: 1 / 20 / 21 / 27 min, 44 / 44 / 100 / 100 acetonitrile%; a flow rate is 0.3-0.4 mL / min; a column temperature is 25-35°C; further preferably, an injection volume is 1 μL, and an ultraviolet detection wavelength is 220-240 nm. In some preferred embodiments of the present application, the flow rate is 0.35 mL / min; and / or, the column temperature is 30°C; and / or, the injection volume is 1 μL; and / or, the ultraviolet detection wavelength is 220-240 nm (more preferably 230 nm).

[0218] In some more preferred embodiments of the present application, the detection conditions of the high performance liquid chromatography-ultraviolet detection are as follows: a chromatographic column: Acquity UPLC BEH C18 (100 mm x 2.1 mm, 1.7 μm); a mobile phase: a mobile phase A is acetonitrile, a mobile phase B is a phosphoric acid aqueous solution (preferably 0.1% phosphoric acid aqueous solution), a mobile phase gradient program is: 1 / 20 / 21 / 27 min, 44 / 44 / 100 / 100 acetonitrile%; a flow rate is 0.35 mL / min; a column temperature is 30°C; an injection volume is 1 μL; and an ultraviolet detection wavelength is 230 nm.

[0219] The third aspect of the present application provides a method for controlling the processing degree of traditional Chinese medicine, comprising the following steps: providing raw products of a set category of Chinese herbal medicines, selecting a set category of processing methods, determining processing markers of the set category of Chinese herbal medicines, processing by using the set category of processing methods, preparing processed products of the set category of Chinese herbal medicines, and controlling the content of the processing markers in the processed products of the set category of Chinese herbal medicines to be no more than a quantitative limit.

[0220] The processing markers of the set category of Chinese herbal medicines can be determined according to the method of the first aspect of the present application.

[0221] The quantitative limit can be an upper limit or a lower limit, and the selection basis is related to the correlation direction of the processing marker and the processing degree. For example, 9-HODE and 13-HODE are negatively correlated with the processing degree, and the quantitative limit thereof in the traditional Chinese medicine decoction pieces is set as an upper limit, that is, the content thereof should not exceed the quantitative limit at the end of processing.

[0222] In some embodiments of the present application, the quantitative limit is set according to the content of the processing marker in real-world samples. For example, the quantitative limit is 120% of the average mass content of the processing marker in commercially available Chinese herbal pieces. For example, the upper limit of 9-HODE or / and 13HODE in vinegar Gansui Chinese herbal pieces can be set to 0.0011% of the mass content, which can be referred to Example 4.

[0223] In some embodiments of the present application, the set category of Chinese herbal medicine is Gansui, the set category of processing method is vinegar roasting method, and the processing marker is 9-HODE or / and 13HODE. In some more specific embodiments, the quantitative limit is the upper limit and is 0.0011% of the mass content.

[0224] In some embodiments of the present application, the set category of Chinese herbal medicine is Yiyiren, the set category of processing method is stir-frying, and the processing marker is 9-HODE or / and 13-HODE. The corresponding Chinese herbal piece is stir-fried Yiyiren, and more specifically, for example, bran-stir-fried Yiyiren.

[0225] In some embodiments of the present application, the set category of Chinese herbal medicine is Yansuo, the set category of processing method is vinegar roasting method, and the processing marker is 9-HODE or / and 13-HODE. The corresponding Chinese herbal piece is vinegar Yansuo.

[0226] In some embodiments of the present application, the set category of Chinese herbal medicine is Shanglu, the set category of processing method is vinegar roasting method, and the processing marker is 9-HODE or / and 13-HODE. The corresponding Chinese herbal piece is vinegar Shanglu.

[0227] In some embodiments of the present application, the set category of Chinese herbal medicine is Bajitian, the set category of processing method is steaming, and the processing marker is 9-HODE or / and 13-HODE. The corresponding Chinese herbal piece is steamed salt Bajitian.

[0228] In some embodiments of the present application, the processing marker is 9-HODE or / and 13-HODE, which is negatively correlated with the degree of processing.

[0229] Based on the screening method of the processing marker of the first aspect, the present application can also establish a general screening method of processing markers. A kind of Chinese herbal medicine can be selected first, and the processing marker is screened according to the method, and then it is applied or verified in other categories of Chinese herbal medicine, and it is confirmed whether it is the common processing marker of different categories of Chinese herbal medicine. Different processing markers can also be screened for different categories of Chinese herbal medicine, and then further screening the common processing marker of different categories of Chinese herbal medicine after comparison and analysis.

[0230] The fourth aspect of the present application provides a screening method of a universal processing marker, comprising the following steps:

[0231] selecting one kind of Chinese herbal medicine, and screening a processing marker PM according to the screening method of the first aspect;

[0232] verifying the processing marker PM for other kinds of Chinese herbal medicines;

[0233] When the processing marker PM is a common processing marker of at least three different kinds of Chinese herbal medicines, the processing marker PM is a screened universal processing marker.

[0234] The fifth aspect of the present application provides a screening method of the universal processing marker, comprising the following steps:

[0235] selecting at least three different kinds of Chinese herbal medicines, and screening respective processing markers PMi according to the screening method of the first aspect;

[0236] screening a common processing marker of at least three different kinds of Chinese herbal medicines as a screened universal processing marker.

[0237] In some embodiments of the present application, the screened processing marker is a common processing marker of at least three kinds of Chinese herbal medicines, which indicates that the processing marker has good universality.

[0238] The following are specific embodiments.

[0239] In the following specific embodiments, the experimental parameters not written in the embodiments are preferably referred to the guidance given in the present application, and can also be referred to the Chinese Pharmacopoeia (2020 edition), experimental manuals in the art or other experimental methods known in the art, or to the experimental conditions recommended by the manufacturers.

[0240] The raw materials and reagents involved in the following specific embodiments can be obtained from the market, or can be easily obtained or prepared by those skilled in the art.

[0241] In the following specific embodiments, if the matrix is not written for the mixed solvent used for preparing the test sample (including the test sample of the test product and the test sample of the standard product) of the present application, the matrix is water; if the unit is not written when referring to the percentage content of the components, it refers to the volume percentage % (v / v). For example, 0.1% formic acid solution refers to 0.1% (v / v) formic acid aqueous solution.

[0242] Example 1. Sample source

[0243] 1.1. Metabolomics research samples, commercially available and laboratory self-made verification samples

[0244] Table 1 below is a list of metabolomics research samples used in the examples, commercially available and laboratory-made validation samples.

[0245] Table 1 Sources of commercially available samples used in the examples

[0246]

[0247] 1.2. Different sample preparation methods

[0248] The sources of the raw products in this Part 1.2 are shown in Table 1.

[0249] Vinegar-processed Echinocystis rhizome: 20 batches of samples were prepared by vinegar- processing. According to the processing method recorded in the Chinese Pharmacopoeia (2020 edition), the raw Echinocystis rhizome (No. 01 raw Echinocystis rhizome, purchased from Beijing Huamai Pharmaceutical Co., Ltd. of Sinopharm Group) was divided into 20 parts, and each part was mixed with vinegar (the amount of vinegar was 20% of the mass of the raw Echinocystis rhizome) and then steamed. After steaming, the Echinocystis rhizome was put into a special stir-frying container and then taken out after stir-frying. The stir-frying temperature was controlled at 180-220°C, and the stir-frying time was 5 min (5 batches of parallel experiments), 10 min (10 batches of parallel experiments), and 15 min (5 batches of parallel experiments), respectively. According to the difference in stir-frying time, the different batches were also marked as vinegar-processed for 5 min, 10 min, and 15 min.

[0250] Fried Coicis Semen with Bran: According to the processing method recorded in the Chinese Pharmacopoeia (2020 edition), a flat-bottomed frying pan was heated until smoke was generated when bran was added (250°C), and then Coicis Semen was immediately added and stirred rapidly. After frying, the bran was removed, and the Coicis Semen was cooled. The frying time was 2 min, 4 min, and 5 min, respectively.

[0251] Salt-processed Morindae Radicis: According to the processing method recorded in the Chinese Pharmacopoeia (2020 edition), the raw Morindae Radicis was mixed with salt water and steamed, and then placed in a steaming container and heated with steam to a specified degree. The wood core was removed while hot, cut into segments, and dried.

[0252] Vinegar-processed Corydalis: According to the processing method recorded in the Chinese Pharmacopoeia (2020 edition), the raw Corydalis was mixed with vinegar and steamed, and then placed in a flat-bottomed frying pan and taken out after frying. The frying temperature was controlled at 150°C, and the frying time was 6 min, 12 min, and 18 min, respectively.

[0253] Vinegar-processed Polygoni Multiflori Radix: According to the processing method recorded in the Chinese Pharmacopoeia (2020 edition), the raw Polygoni Multiflori Radix was mixed with vinegar and steamed, and then placed in a flat-bottomed frying pan and taken out after frying. The frying temperature was controlled at 150°C, and the frying time was 6 min, 8 min, 12 min, and 15 min, respectively.

[0254] Rhubarb: according to the processing method recorded in the Chinese Pharmacopoeia (2020 edition), add yellow wine to Angelica tablet and mix evenly, soak until transparent (1-2 hours), place in a frying machine, fry dry with a low fire, take out and cool down; the frying temperature is controlled at 80-120℃, and the wine roasting time is 5min (2 batches of parallel experiments), 10min (2 batches of parallel experiments), and 15min (2 batches of parallel experiments) respectively.

[0255] Salted Alisma: according to the processing method recorded in the Chinese Pharmacopoeia (2020 edition), add salt water to Alisma tablet and mix evenly, soak until transparent, place in a frying machine, fry dry with a low fire, take out and cool down; the frying temperature is controlled at 80-120℃, and the salt roasting time is 5min (2 batches of parallel experiments), 10min (2 batches of parallel experiments), and 15min (2 batches of parallel experiments) respectively.

[0256] Vinegar Cyperus: according to the processing method recorded in the Chinese Pharmacopoeia (2020 edition), take clean Cyperus, add vinegar and mix evenly, soak until transparent, place in a flat-bottom frying pan, fry and take out; the frying temperature is controlled at 150℃, and the frying time is 6min, 11min and 16min respectively.

[0257] Vinegar Daphne: according to the processing method recorded in the Chinese Pharmacopoeia (2020 edition), take clean Daphne, add vinegar and mix evenly, soak until transparent, place in a flat-bottom frying pan, fry and take out; the frying temperature is controlled at 150℃, and the frying time is 10min, 14min and 18min respectively.

[0258] When the above traditional Chinese medicine decoction pieces are processed, auxiliary materials are added, and the amount of auxiliary materials added is in accordance with the relevant provisions in the Chinese Pharmacopoeia (2020 edition).

[0259] Example 2. Screening of potential processing markers by non-targeted metabolomics combined with chemometrics (taking vinegar Euphorbia as an example).

[0260] Select No.01 raw Euphorbia and vinegar Euphorbia with different vinegar roasting times (5+10+5 batches) in 1.1. of Example 1 as test samples, prepare Euphorbia powder by powdering, mixing, and passing through a No.4 sieve, and use it to prepare test sample to be tested (test sample in a state that can be directly sampled and detected).

[0261] 2.1. High performance liquid chromatography-mass spectrometry detection

[0262] Prepare the test sample to be tested: take 0.50g of Euphorbia powder (pass through a No.4 sieve), accurately weigh, place in a conical flask with a plug, add 25mL of ethyl acetate (as an extraction solvent), tightly plug, ultrasonic treatment (power 100W, frequency 37kHz) for 30min, filter under reduced pressure, and wash the residue; combine the filtrate; evaporate the filtrate to dryness, dissolve the residue with methanol, transfer to a 5mL volumetric flask, add methanol to the mark, shake well, and the obtained solution is the test sample to be tested.

[0263] The total ion chromatogram, i.e. the original mass spectrum data, was obtained by detecting the test sample by high performance liquid chromatography-mass spectrometry. The No. 01 Gansui samples in 1.1. part of Example 1 and vinegar Gansui samples (5+10+5 batches) with different vinegar frying times were detected by high performance liquid chromatography-mass spectrometry.

[0264] The detection conditions of high performance liquid chromatography-mass spectrometry were as follows: Acquity Ultimate 3000 high performance liquid chromatography system; chromatographic column: Agilent Zorbax plus C8 (150 mm x 2.1 mm, 1.8 μm); mobile phase: acetonitrile as mobile phase A, 0.1% formic acid aqueous solution as mobile phase B, mobile phase gradient program: 0 / 8 / 10 / 20 / 30 / 45 / 50 min; 53 / 65 / 65 / 80 / 90 / 100 / 100% acetonitrile; after 50 min, change to the initial mobile phase for balance for 4 min; total running time is 54 min; flow rate 0.35 mL / min; column temperature 30℃; injection volume 5 μL; linear ion trap orbitrap combination mass spectrometry (LTQ-Orbitrap Velos Pro MS system) detector detection, positive ion mode; source spray voltage 4 kV, capillary temperature 350℃, source heating temperature 200℃, sheath gas flow rate 35%, auxiliary gas flow rate 5%, split ratio 1:1, in Full scan mode, mass spectrometry acquisition range is set to 200 m / z-1500 m / z, resolution is set to 30000, MS 4 Collision energy is set to 30%, resolution is set to 7500.

[0265] 2.2. Metabolite information of the sample was obtained by non-targeted metabolomics analysis

[0266] The original mass spectrum data was analyzed qualitatively by Xcalibur 2.1 software, and the metabolites were identified. The m / z value (mass-to-charge ratio) and fragment spectrum were matched with the relevant information in the reference and online database for identification, and the online database included Metlin database (http: / / metlin.scripps.edu / ), human metabolome database (http: / / www.hmdb.ca / ) and Scifinder-n (https: / / scifinder-n.cas.org / ).

[0267] Taking the vinegar Gansui samples with two different frying times as examples, Figure 2is the total ion chromatogram of two different roasting time (5 min and 15 min) prepared in Example 1 of vinegar Euphorbia. Different peak values form different metabolic characteristics (also known as metabolite characteristics), and different metabolic characteristics correspond to corresponding metabolites, from which candidate markers (as potential processing markers) can be screened. A total of 52 compounds were identified, including 31 diterpenoids, 8 triterpenoids, 4 fatty acids, 5 lipids and 4 other compounds. The black chromatographic peak numbers (1, 2, 3, 6, 10, 12, 16, 19, 20, 22, 23, 25, 28, 29, 30, 31, 33, 37, 39, 42, 48, 51, 52) in the figure are consistent with the identified metabolite numbers, and the gray chromatographic peak numbers 14 and 50 correspond to potential processing degree markers.

[0268] 2.3. Finding candidate processing degree markers by chemometrics

[0269] The raw mass spectrometry data was processed by Progenesis QI 2.3 software (Waters, Milford, USA). Feature detection was performed by QI software, and the peak values of the total ion chromatogram of different samples were selected, then the data matrix of the metabolite characteristics was generated and imported into SIMCA 14.1 (Umetrics, Sweden) for multivariate analysis. In order to observe the overall chemical changes between raw Euphorbia and vinegar Euphorbia, principal component analysis (PCA) was performed. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to discriminate between the two groups with large differences in processing degree. The VIP score plot and S line plot were used to find candidate markers. By performing one-way analysis of variance on the characteristic mass spectrometry response values obtained by the mass spectrometry detector, it was determined whether there were significant differences between groups (P value < 0.05), and then the characteristics closely related to the processing parameters (in this example, vinegar roasting time) were screened as the basis for screening potential processing markers. In this example, for vinegar Euphorbia treated with different vinegar roasting times, metabolite characteristics that change in content with vinegar roasting time were screened, and the corresponding difference markers were used as potential processing degree markers.

[0270] For the raw mass spectrometry data of raw Euphorbia and vinegar Euphorbia with different vinegar roasting times (5+10+5 batches) in Example 1.1, a total of 12373 features were detected by QI software. Figure 2 is the result of chemometrics analysis for finding potential processing markers taking Euphorbia as an example.

[0271] Figure 2A is the PCA score plot of Kansui Radix and vinegar Kansui Radix (5+10+5 batches) in Example 1, Part 1.1, with vinegar processing time of 5 min, 10 min and 15 min, respectively. Among them, the three coordinate axes t[1], t[2], t[3] represent three different principal component axes. From the figure, it can be seen that the four groups of Kansui Radix and vinegar Kansui Radix with different vinegar processing time (5 min, 10 min and 15 min) can be well separated, which indicates that vinegar processing can cause time-dependent chemical transformation. These chemical transformations reflect the changes in components during processing. Figure 2 As can be seen from A, the four groups of Kansui Radix and vinegar Kansui Radix with different vinegar processing time (5 min, 10 min and 15 min) can be well separated, which indicates that vinegar processing can cause time-dependent chemical transformation. These chemical transformations reflect the changes in components during processing.

[0272] Figure 2 B is the OPLS-DA score plot of vinegar Kansui Radix with vinegar processing time of 5 min and 15 min in Example 1. According to the OPLS-DA score plot, the two groups of vinegar Kansui Radix with different vinegar processing time can be well separated, which indicates that the difference in processing degree can cause chemical transformation. Figure 1 B can study metabolic differences and reflect the differences between processed products with large differences in processing degree.

[0273] Figure 1 C is the VIP score plot of vinegar Kansui Radix with vinegar processing time of 5 min and 15 min in Example 1. The VIP threshold value is set to 7.0, and 35 metabolic characteristics are obtained, Figure 2 C reflects the VIP values of the 35 metabolic characteristics. Figure 2 In C, the characteristic t R 6.37_m / z 296.2350n indicates that the chromatographic retention time of the metabolic characteristic is 6.37, and the m / z value of the metabolic characteristic is 296.2350, corresponding to Figure 1 Characteristic peak number 14 in C, characteristic t R 30.52_m / z 590.4907n indicates that the chromatographic retention time of the metabolic characteristic is 30.52, and the m / z value of the metabolic characteristic is 590.4907, corresponding to Figure 2 Characteristic peak number 50 in C; in addition, n indicates that the metabolic characteristic is a neutral ion derived by QI software. From Figure 1 As can be seen from C, the metabolic characteristic has a high VIP score and a small error, indicating that the difference between groups is large and the fluctuation within groups is small.

[0274] Figure 2 D is the S line plot of vinegar Kansui Radix with vinegar processing time of 5 min and 15 min in Example 1, in which the line numbers of some identified metabolites are marked. In the figure, the horizontal axis is the retention time of chromatographic detection, the left vertical axis p(ctr)[1] represents the difference degree (covariance) of the measured variable in the discrimination model, and the right vertical axis represents the reliability (correlation) of the measured variable in the discrimination model. In the figure, the line numbers of some identified metabolites are marked 3, 10, 12, 14, 16, 19, 20, 22, 23, 31, 33, 37, 42, 48, 50, 51, which are the same as the line numbers of some identified metabolites in Figure 2The peaks in the data have the same numbering and correspond to the same metabolite components. Figure 2 In the S-curve plot of D, metabolite characteristics are arranged according to retention time and according to Figure 2 By labeling the components identified in the process, we can observe the degree of contribution and reliability of the components to the differences during the processing.

[0275] One-way ANOVA was used to analyze the variance of the samples. Figure 2 The mass spectrometry response values ​​of 35 metabolic features in C were analyzed across four groups (raw Euphorbia kansui and Euphorbia kansui processed with vinegar for 5 min, 10 min, and 15 min) to examine inter-group differences. Two features whose mass spectrometry response values ​​gradually decreased with vinegar processing time were identified and identified as 13-hydroxyoctadecadienoic acid (13-HODE) isomers and 13-hydroxyoctadecadienoic acid (13-HOTE) isomers, respectively, by matching m / z values ​​(mass-to-charge ratio) and fragment spectra with relevant information in the Human Metabolomics Database (http: / / www.hmdb.ca / ). A feature whose mass spectrometry response values ​​gradually increased with prolonged processing time was also identified and identified as the DG (16:0 / 0:0 / 18:3n3) isomer by matching m / z values ​​(mass-to-charge ratio) and fragment spectra with relevant information in the Human Metabolomics Database (http: / / www.hmdb.ca / ). Figure 3 E is a feature t R The mass spectrometry response values ​​of 6.37 m / z and 296.2350 n in four groups of samples: raw Euphorbia kansui (EK) and vinegar-processed Euphorbia kansui (VEK) at 5 min, 10 min, and 15 min, are shown. Figure 3 As can be seen from E, with the extension of vinegar roasting time, characteristic t R The response at 6.37 m / z 296.2350 n gradually decreased, indicating that the content of the candidate marker 13-HODE decreased with the extension of vinegar-roasting time during the processing, i.e., a negative correlation. Figure 3 F is a feature t R The characteristic mass spectrometry response values ​​of 30.52 m / z and 590.4907 n in four groups of samples—raw Euphorbia kansui (EK) and vinegar-processed Euphorbia kansui (VEK) at 5 min, 10 min, and 15 min—showed changes from... Figure 4 As can be seen from F, with the extension of vinegar roasting time, characteristic t R The gradually increasing response at 30.52 m / z and 590.4907n indicates a positive correlation between the content of the candidate biomarker DG(16:0 / 0:0 / 18:3n3) and the vinegar-roasting time during processing. Figure 4 E and Figure 4The P value obtained by ANOVA analysis is also marked in F. Metabolites with high mass spectrometry response value and small P value are preferred as potential processing markers. In this embodiment, among 13-HODE isomers, 13-HOTE isomers and DG (16:0 / 0:0 / 18:3n3) isomers, the 13-HODE isomer with higher mass spectrometry response value and smaller P value is more preferred as a potential processing marker.

[0276] The identification method of the compound corresponding to the characteristic is to match the m / z value (mass-to-charge ratio) and fragment spectrum with the relevant information in the online database for identification.

[0277] The identification process of the 13-HODE isomer is as follows: match the m / z value (mass-to-charge ratio) and fragment spectrum with the relevant information in the human metabolome database (http: / / www.hmdb.ca / ) for identification.

[0278] The identification process of the 13-HODT isomer is as follows: match the m / z value (mass-to-charge ratio) and fragment spectrum with the relevant information in the human metabolome database (http: / / www.hmdb.ca / ) for identification.

[0279] The identification process of the DG (16:0 / 0:0 / 18:3n3) isomer is as follows: match the m / z value (mass-to-charge ratio) and fragment spectrum with the relevant information in the human metabolome database (http: / / www.hmdb.ca / ) for identification.

[0280] Example 3. Establishment of high performance liquid chromatography-ultraviolet quantitative analysis method (taking vinegar gansu as an example).

[0281] A high performance liquid chromatography-ultraviolet quantitative analysis method was established to analyze the potential processing markers.

[0282] 3.1. Sample preparation and high performance liquid chromatography-ultraviolet detection

[0283] Prepare the test sample: take 2 g of the test sample, use ethyl acetate as the extraction solvent, the volume is 100 mL, and the rest of the parameters are the same as those of the test sample preparation in the high performance liquid chromatography-mass spectrometry detection part of Example 2.1. Take gansu as an example, the samples used as test samples include: commercially available raw gansu and vinegar gansu (6 batches each).

[0284] Prepare the standard sample: purchase 13-HODE and 9-HODE standard samples, dilute them to a certain concentration with methanol solvent to prepare the corresponding standard sample. Here, the standard sample includes both single-component standard samples (i.e., corresponding to one potential processing marker) and mixed standard samples (i.e., at least two potential processing markers).

[0285] The test sample and the standard sample were subjected to high performance liquid chromatography-ultraviolet detection to obtain corresponding chromatograms.

[0286] High performance liquid chromatography-ultraviolet detection conditions: Acquity UPLC H-Class high performance liquid chromatography system; chromatographic column: Acquity UPLC BEH C18 (100 mm x 2.1 mm, 1.7 μm); mobile phase: acetonitrile as mobile phase A, 0.1% phosphoric acid aqueous solution as mobile phase B, mobile phase gradient program: 1 / 20 / 21 / 27 min; 44 / 44 / 100 / 100% acetonitrile; flow rate 0.35 mL / min; column temperature 30°C; injection volume 1 μL; ultraviolet detection wavelength 230 nm.

[0287] 3.2. Secondary mass spectrometry analysis and high performance liquid chromatography-ultraviolet detection (taking 13-HODE and 9-HODE as examples)

[0288] The secondary mass spectrometry data of the potential processing markers 13-HODE and 9-HODE in the test sample and the standard sample were compared. It was found that the secondary mass spectrometry data of 13-HODE and 9-HODE in the test sample and the standard sample were completely matched (A), and the chemical structural formulas of the two potential processing degree markers are shown in Figure 4 B. Figure 4

[0289] According to the chromatogram of high performance liquid chromatography-ultraviolet detection, the retention time of the processing degree markers in the test sample and the standard sample was compared. It was found that the chromatographic peaks of 13-HODE and 9-HODE corresponded in retention time in the chromatogram of raw Euphorbia kansui, the chromatogram of Euphorbia kansui vinegar and the chromatogram of 13-HODE / 9-HODE mixed standard (C). Figure 5

[0290] 3.3. Validation of high performance liquid chromatography-ultraviolet quantitative analysis method (taking 13-HODE and 9-HODE as examples)

[0291] The quantitative analysis method was verified through linear, detection limit and quantitative limit, repeatability, precision, recovery rate and stability and other methodological investigations, and the results are shown in Tables 1-5. If not specified, the sample weight is 2 g, and the sample used is commercially available Euphorbia kansui vinegar 2.

[0292] Table 1 is the linear regression equation, linear range, quantitative limit and detection limit of the analytes 13-HODE and 9-HODE. Among them, y represents the peak area of the chromatographic peak, x represents the concentration of the substance, R 2 represents the correlation coefficient, LOQ represents the quantitative limit, and LOD represents the detection limit. The closer R 2 is to 1, the better the linearity. According to the data in Table 1, the linearity is good. ​​

[0293] Table 2 is repeatability analysis. Different sample weights are used for high performance liquid chromatography-ultraviolet detection. Low-1, 2, 3 are three parallel samples with low sample weight (about 1 g), Medium-1, 2, 3 are three parallel samples with medium sample weight (about 2 g), and High-1, 2, 3 are three parallel samples with high sample weight (about 3 g). The peak area is the peak area corresponding to the potential marker in the high performance liquid chromatography-ultraviolet chromatogram. The content is the ten-thousandth (ppm) content of the potential marker in the sample. The average value in the table is the average relative content. RSD represents the relative standard deviation. According to the RSD in Table 2, it can be reflected that the repeatability is good.

[0294] Table 3 is inter-day precision analysis. The sample weight is 2 g. The samples prepared on three consecutive days are detected and analyzed. The meanings of peak area, content and RSD are the same as above. The average value is the average relative content. According to the RSD in Table 3, it can be reflected that the inter-day precision is good.

[0295] Table 4 is recovery rate analysis. Three different levels of standard addition are used, and the sample weight is 2 g. The measured amount represents the material content of the mixed test sample of the sample and the standard sample, the original amount represents the theoretical material content of the sample without adding the standard sample, and the added amount represents the actual standard sample addition. The calculation method of recovery rate (%) is (measured amount-original amount) / added amount x 100. The average value is the average recovery rate. From Table 4, it can be seen that the recovery rate meets the requirements of quantitative analysis.

[0296] Table 5 is stability analysis. The standard sample and the test sample are respectively placed at 4℃ for 0h, 2h, 4h, 5h, 8h, 12h, 18h, 24h, and then injected for high performance liquid chromatography-ultraviolet detection, and the peak area of the potential marker in the chromatogram of the standard sample and the test sample is counted. From the RSD data in Table 5, it can be seen that it has good stability.

[0297] According to the analysis results in Tables 1-5, it can be seen that the methodological investigation results are good, and the high performance liquid chromatography-ultraviolet quantitative analysis method can be used for the routine quantitative analysis of 13-HODE and 9-HODE.

[0298] Table 1. Linear regression equation, linear range, limit of quantification and limit of detection of analyte

[0299]

[0300] Table 2. Repeatability

[0301]

[0302] Table 3. Inter-day precision

[0303]

[0304]

[0305] Table 4. Recovery

[0306]

[0307] Table 5. Stability

[0308]

[0309] Example 4. Validation of real world samples and lab-made samples (using Kansu Vinegar as an example).

[0310] 4.1. Analysis of real world samples

[0311] Real world samples used commercially available Chinese herbal pieces.

[0312] To validate the processing markers, commercially available raw and Kansu Vinegar were detected and analyzed using the established HPLC-UV quantitative analysis method. The commercially available raw Kansu Vinear included the six commercially available raw Kansu Vinear listed in Table 1; the commercially available Kansu Vinegar included the six commercially available Kansu Vinear listed in Table 1.

[0313] After detection and quantitative analysis, the content of 13-HODE in the six commercially available raw Kansu Vinear and the six commercially available Kansu Vinear was 12.6 ppm-20.59 ppm and 5.76 ppm-12.98 ppm, respectively. The content of 9-HODE in the commercially available raw Kansu Vinear and Kansu Vinear was 12.45 ppm-21.52 ppm and 6.07 ppm-13.34 ppm, respectively. T-test was performed, and the content of 13-HODE and 9-HODE was significantly different between the commercially available raw Kansu Vinear and Kansu Vinear. Figure 5 A, P value of 13-HODE content was 0.001, and P value of 9-HODE content was 0.0015.

[0314] To avoid insufficient processing, it is recommended to set the upper limit of 13-HODE and 9-HODE at 120% of the average mass content in commercially available Kansu Vinear, which is equivalent to an upper limit of 0.0011% for the mass content of 13-HODE and 9-HODE. Therefore, it is recommended to use the point when the mass content of 13-HODE and 9-HODE is not more than 0.0011% as the end point of roasting.

[0315] After verification analysis, the contents of 13-HODE and 9-HODE in most of the commercially available vinegar Echinopsis alba meet the above requirements. Among them, the mass contents of 13-HODE in the six commercially available vinegar Echinopsis alba in Table 1 are 7.46 ppm, 6.49 ppm, 5.76 ppm, 8.99 ppm, 10.94 ppm, and 12.98 ppm, respectively; and the mass contents of 9-HODE in the six commercially available vinegar Echinopsis alba in Table 1 are 7.68 ppm, 6.84 ppm, 6.07 ppm, 9.95 ppm, 11.09 ppm, and 13.34 ppm, respectively.

[0316] 4.2. Laboratory-made samples

[0317] The response of potential processing degree markers to other processing parameters was investigated. The vinegar Echinopsis alba samples were prepared by using different roasting temperatures (120°C, 150°C, 180°C, roasting time 8 min, using rice vinegar, and the amount of vinegar was 20% (w / w) of the mass of the raw product), different amounts of vinegar (using rice vinegar, the amount of vinegar (w / w) was 10%, 20%, and 30% of the mass of the raw product, respectively, and the roasting time was changed with the amount of vinegar to ensure that it was roasted to dryness, and the roasting time was 4 min, 8 min, and 12 min, respectively, and the roasting temperature was 150°C), and the rest of the processing parameters were consistent with those in Example 1, Part 1.2. The effects of roasting temperature and the amount of vinegar on the contents of 13-HODE and 9-HODE in the processed products were studied, and the results are shown in Tables Figure 6 B and Figure 6 C.

[0318] From Figure 6 B, it can be seen that the contents of 13-HODE and 9-HODE gradually decrease with the increase of roasting temperature (roasting time 8 min, and the amount of rice vinegar is 20% (w / w) of the mass of the raw product). According to the requirements of the Chinese Pharmacopoeia, the amount of rice vinegar is 20% of the mass of the sample to be processed, which theoretically does not affect the contents of 13-HODE and 9-HODE. However, in actual operation, different roasting times were used for samples with different amounts of vinegar to ensure that they were roasted to dryness, resulting in a gradual decrease in the contents of 13-HODE and 9-HODE with the increase of the amount of vinegar (as shown in Figure 6 C).

[0319] Therefore, the roasting temperature and the roasting time both have an effect on the contents of 13-HODE and 9-HODE in the processed products, which means that these two compounds can be used as processing markers to monitor the processing production process, thereby effectively controlling the quality of the processed products.

[0320] Example 5. Investigation of the universality of 13-HODE and 9-HODE as processing markers.

[0321] To investigate the general applicability of 13-HODE and 9-HODE as processing markers.

[0322] For several commonly used processing methods (including adding adjuvant stir-frying, baking and steaming), several representative traditional Chinese medicine decoction pieces were prepared in the laboratory. The processing was carried out by the method of 1.2 in Example 1, and the amount of adjuvant met the relevant requirements of Chinese Pharmacopoeia. The representative traditional Chinese medicine decoction pieces included bran-fried coix seed, salt baji-ten, vinegar rhizoma corydalis, vinegar phytolacca, wine angelica, salt alismatis, vinegar rhizoma zedoariae, vinegar genkwa, which were processed by the following methods respectively: bran-fried coix seed was processed by adding adjuvant stir-frying (smoking temperature about 250℃); salt baji-ten was processed by adding salt steaming; vinegar rhizoma corydalis was processed by vinegar baking (frying temperature 150℃); vinegar phytolacca was processed by vinegar baking (frying temperature 150℃); wine angelica was processed by wine baking (frying temperature 100℃); salt alismatis was processed by salt baking (frying temperature 100℃); vinegar rhizoma zedoariae was processed by vinegar baking (frying temperature 150℃); vinegar genkwa was processed by vinegar baking (frying temperature 150℃).

[0323] The traditional Chinese medicine decoction pieces obtained by processing were subjected to high performance liquid chromatography-ultraviolet detection and quantitative analysis. The detection conditions of high performance liquid chromatography-ultraviolet were referred to Example 3, and the results are shown in Figure 6 From the detection results of Figure 6 , it can be seen that the above-mentioned several traditional Chinese medicine decoction pieces obtained by different processing methods all contain 13-HODE and 9-HODE, and the characteristic peaks are obvious. According to the peak area, the content of the two compounds can be quantitatively analyzed.

[0324] According to the high performance liquid chromatography-ultraviolet chromatogram, the content of 13-HODE and 9-HODE in different types of traditional Chinese medicine decoction pieces can be analyzed with the change of the corresponding processing parameters, and the results are shown in Figure 6 Figure 6 A corresponds to bran-fried coix seed obtained by different frying time, Figure 6 B corresponds to salt baji-ten obtained by adding salt steaming, Figure 6 C corresponds to vinegar rhizoma corydalis obtained by different frying time, Figure 7 D corresponds to vinegar phytolacca obtained by different frying time, Figure 7 E corresponds to wine angelica obtained by different wine baking time, Figure 7 F corresponds to salt alismatis obtained by different salt baking time, ​ G corresponds to vinegar rhizoma zedoariae obtained by different frying time, ​ H corresponds to vinegar genkwa obtained by different frying time. From ​ , it can be seen that with the progress of the corresponding processing, the content of 13-HODE and 9-HODE in A, B, C, D four graphs gradually decreases; while in E, F, G, H four graphs, the content of 13-HODE and 9-HODE has no significant change.

[0325] ​The use of 13-HODE and 9-HODE as markers for the three processing methods is summarized in Table 1 ​ During processing, the contents of 13-HODE and 9-HODE in the Chinese medicinal pieces marked with a check mark (V) decreased with processing time, and 13-HODE and 9-HODE can be used as markers for the processing of the corresponding Chinese medicinal pieces, ​ The roasted Yiyiren was processed by frying (with solid adjuvants), Gansui, Yansu and Shenglu were processed by vinegar-frying (belonging to liquid adjuvants), and Badijitian was processed by steaming (with salt). The Chinese medicinal pieces marked with a cross (X) did not show significant changes in the contents of 13-HODE and 9-HODE before and after processing. From ​ It can be seen from the above that 13-HODE and 9-HODE have strong universality as markers for processing.

[0326] Through the foregoing qualitative and quantitative analysis, it was found that the contents of 13-HODE and 9-HODE decreased with processing time during the high-temperature adjuvant frying and steaming processes of Chinese medicinal pieces, indicating that they can be used as markers for processing; and the contents of 13-HODE and 9-HODE did not change significantly during the low-temperature wine-frying and salt-frying processes, indicating that they are not suitable for use as markers for processing. For vinegar-frying, which is not specified in the Pharmacopoeia, the contents of 13-HODE and 9-HODE in more than half of the Chinese medicinal pieces decreased with processing time (suitable for use as markers for processing). Therefore, 13-HODE and 9-HODE have strong universality as markers for processing. Whether 13-HODE and 9-HODE can be used as markers for processing for other different Chinese medicinal pieces and / or different processing methods still needs to be verified.

[0327] In general, although the contents of 13-HODE and 9-HODE in the pieces are low, they are heat-sensitive and negatively correlated with the degree of processing, and can be used as universal markers for the degree of processing of most Chinese medicinal pieces.

[0328] The technical features of the above-described embodiments and examples can be combined in any suitable manner. To make the description concise, not all possible combinations of the technical features in the above-described embodiments and examples are described, but as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure.

[0329] The above embodiments only express several implementation manners and embodiments of the present application, facilitate to understand the technical scheme of the present application concretely and in detail, but cannot be understood as the limitation of the patent protection scope of the present application. It should be pointed out that, for the ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which all belong to the protection scope of the present application. It should be understood that, the technical scheme obtained by the skilled in the art through logical analysis, reasoning or limited test on the basis of the technical scheme provided by the present application, all belong to the protection scope of the appended claims of the present application. Therefore, the protection scope of the present application patent should be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Use of 9-hydroxyoctadecadienoic acid as a processing degree marker in the quality control of the processing process of traditional Chinese medicines, characterized in that, The processing process of the traditional Chinese medicine is a heating processing process, and the 9-hydroxyoctadecadienoic acid is used to adjust the processing parameter in the processing process. The processed product with different processing degrees is any one of vinegar Euphorbia kansui and vinegar Corydalis. The processing parameter of the vinegar Euphorbia kansui includes at least one of processing time and processing temperature. The processing parameter of the vinegar Corydalis is processing time.

2. Use according to claim 1, characterized in that, The processing degree marker further includes 13-hydroxyoctadecadienoic acid.

3. A method for controlling the processing degree of traditional Chinese medicine decoction pieces, characterized in that, The method comprises the following steps: The raw product of a set category of Chinese herbal medicines is provided, a set category of processing methods is selected, the set category of processing methods is used for processing, the processed product of the set category of Chinese herbal medicines is prepared, and the content of the processing degree marker in the processed product of the set category of Chinese herbal medicines is controlled to be not more than a quantitative limit, wherein the quantitative limit is determined according to the content of the processing degree marker in the commercially available raw product and processed product. The processed product with different processing degrees is any one of vinegar Euphorbia kansui and vinegar Corydalis; the set category of processing parameters is at least one of processing time and processing temperature; the processing parameter of the vinegar Euphorbia kansui includes at least one of processing time and processing temperature; the processing parameter of the vinegar Corydalis is processing time; and the processing degree marker is 9-hydroxyoctadecadienoic acid.

4. The method for controlling the processing degree of traditional Chinese medicine decoction pieces according to claim 3, characterized in that, The set category of Chinese herbal medicines is Euphorbia kansui, the set category of processing methods is vinegar roasting method, the processing degree marker is 9-hydroxyoctadecadienoic acid or 9-hydroxyoctadecadienoic acid and 13-hydroxyoctadecadienoic acid; and the quantitative limit refers to that the mass content of 9-hydroxyoctadecadienoic acid is 0.0011%, or the mass content of 9-hydroxyoctadecadienoic acid and 13-hydroxyoctadecadienoic acid is 0.0011% respectively.

5. The method for controlling the processing degree of traditional Chinese medicine decoction pieces according to claim 3, characterized in that, The set category of Chinese herbal medicines is Corydalis, the set category of processing methods is vinegar roasting method, and the processing degree marker is 9-hydroxyoctadecadienoic acid or 9-hydroxyoctadecadienoic acid and 13-hydroxyoctadecadienoic acid.

6. The method for controlling the processing degree of traditional Chinese medicine decoction pieces according to claim 3, characterized in that, The processing degree marker is negatively correlated with the processing degree.

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