Application of polygala tenuifolia or processed polygala tenuifolia fingerprint spectrum construction method in identification of processed polygala tenuifolia and crude products thereof

Abstract

The invention relates to the technical field of traditional Chinese medicine detection and analytical chemistry, in particular to application of a polygala tenuifolia or processed polygala tenuifolia fingerprint spectrum construction method to identification of processed polygala tenuifolia and crude products thereof. The fingerprint construction method provided by the invention comprises the step of analyzing by adopting ultra-high performance liquid chromatography under the chromatographic conditions that acetonitrile is taken as a mobile phase A and a formic acid aqueous solution with the volume concentration of 0.05-0.2% is taken as a mobile phase B. The fingerprint spectrum has characteristics, reproducibility and operability, and common components and differential components of the polygala tenuifolia and the processed polygala tenuifolia can be determined. The construction method can be applied to identification of the processed polygala tenuifolia and the crude polygala tenuifolia, the polygala tenuifolia and the processed polygala tenuifolia are identified according to whether the chromatographic peak exists or not, and the method is visual and accurate.

Description

Technical Field

[0001] The present application relates to the technical field of traditional Chinese medicine detection and analytical chemistry, and specifically to a method for constructing a fingerprint spectrum of Polygala tenuifolia or processed Polygala tenuifolia and its application in identifying processed Polygala tenuifolia and its raw products. Background Art

[0002] Polygala is derived from the dried roots of Polygala tenuifolia Willd. or Polygala sibirica L. of the Polygalaceae family. It is warm in nature, bitter and pungent in taste, and enters the heart, kidney and lung meridians. It has the effects of calming the mind and improving intelligence, connecting the heart and kidney, removing phlegm and reducing swelling. The processed products of Polygala included in the 2020 edition of the Chinese Pharmacopoeia include Polygala and processed Polygala. Polygala is made by removing impurities from the dried roots of Polygala, washing them slightly, moistening them, cutting them into sections and drying them. Processed Polygala is made by removing impurities from the dried roots of Polygala, adding them to the decoction of licorice, simmering them over a low heat until the soup is absorbed, and then drying them. Ancient books record that raw Polygala "makes people feel stuffy" and "hurts the throat", while processed Polygala can reduce its toxicity of "hurting the throat" and enhance its calming and intelligence-improving effects. Modern pharmacological research shows that Chinese medicine processing can change the properties of the medicine, guide the medicine to the meridians, and change the tendency of the medicine to act on the body, thus explaining the differences in toxicity and efficacy between Polygala tenuifolia and processed Polygala tenuifolia.

[0003] The pharmacopoeia records that Polygala is a cylindrical segment, with a grayish yellow to grayish brown outer skin, transverse wrinkles, a brownish yellow cross section, and a hollow center. The processed Polygala is shaped like a Polygala segment, with a yellowish brown surface. It can be seen that it is difficult to distinguish Polygala from processed Polygala from the appearance, and no other identification methods have been reported so far. In view of the differences in pharmacological activity between Polygala and processed Polygala, it is necessary to establish an identification method and quality control method for processed Polygala and its raw Polygala slices in order to ensure the safety and effectiveness of clinical medication.

[0004] The fingerprint of traditional Chinese medicine is an understanding of the overall effect of the substance group of traditional Chinese medicine. It can explain the chemical composition of traditional Chinese medicine at the overall level. It is also one of the important indicators of traditional Chinese medicine quality control. It can be used to judge the quality of medicinal materials, identify the authenticity of medicinal materials, distinguish different parts of medicinal materials, and identify raw and processed medicinal materials. By establishing the fingerprint of processed Polygala tenuifolia and its raw products and screening the specific indicators of the difference in chemical composition between the two, the two can be objectively and scientifically identified and distinguished.

[0005] Based on this, it is necessary to provide a method for constructing the fingerprint of Polygala tenuifolia and processed Polygala tenuifolia at the same time, so as to realize the scientific identification of the two. Summary of the invention

[0006] Based on this, one or more embodiments of the present application provide a fingerprint map construction method for Polygala tenuifolia or processed Polygala tenuifolia for use in identifying processed Polygala tenuifolia and its raw products. The fingerprint map obtained by the fingerprint map construction method of the present application can objectively, scientifically and comprehensively reflect the intrinsic chemical characteristic information of Polygala tenuifolia and processed Polygala tenuifolia, and serve as a basis for quality detection and identification.

[0007] The technical solution of this application includes the following contents:

[0008] Application of a fingerprint spectrum construction method of Polygala tenuifolia or processed Polygala tenuifolia in identifying processed Polygala tenuifolia and its raw products;

[0009] The fingerprint spectrum construction method of Polygala tenuifolia or processed Polygala tenuifolia comprises the following steps:

[0010] preparing a test solution, wherein the test solution comprises an extract of Polygala tenuifolia or processed Polygala tenuifolia;

[0011] The test solution is subjected to liquid chromatography analysis to establish a fingerprint of Polygala tenuifolia or processed Polygala tenuifolia;

[0012] The conditions of the liquid chromatography analysis include:

[0013] Acetonitrile is used as mobile phase A, and an aqueous solution of an acid with a volume concentration of 0.05% to 0.2% is used as mobile phase B, and gradient elution is adopted;

[0014] The procedure of the gradient elution includes:

[0015] 0min~3min, mobile phase A from 10%→13%;

[0016] 3min~10min, mobile phase A from 13%→15%;

[0017] 10min~11min, mobile phase A from 15%→18%;

[0018] 11min~16min, mobile phase A from 18%;

[0019] 16min~20min, mobile phase A from 18%→20%;

[0020] 20min~21min, mobile phase A from 20%→23%;

[0021] 21min~35min, mobile phase A from 23%;

[0022] 35min~39min, mobile phase A from 23%→27%;

[0023] 39min~43min, mobile phase A changed from 27% to 28%;

[0024] 43min~44min, mobile phase A changed from 28% to 30%;

[0025] 44min~45min, mobile phase A changed from 30% to 34%;

[0026] 45min~64min, mobile phase A from 34%→35%;

[0027] 64min~68min, mobile phase A from 35%→38%;

[0028] 68min~73min, mobile phase A changed from 38% to 40%;

[0029] 73min~73.1min, mobile phase A from 40%→90%;

[0030] 73.1min~75min, mobile phase A from 90%;

[0031] Among them, the detection wavelength is 275nm-285nm at 0min-50min, 245nm-255nm at 50min-56min, and 275nm-285nm at 56min-75min.

[0032] In some embodiments, the conditions of the liquid chromatography analysis further include one or more of the following:

[0033] (1) The chromatographic column is an octadecylsilane bonded silica gel chromatographic column;

[0034] (2) Column temperature is 38°C to 42°C;

[0035] (3) The flow rate of the mobile phase is 0.28 mL / min to 0.32 mL / min;

[0036] (4) The injection volume is 1 μL to 3 μL;

[0037] (5) The acid in the mobile phase B includes at least one of phosphoric acid and formic acid.

[0038] In some embodiments, the method for preparing the extract of Polygala tenuifolia or processed Polygala tenuifolia comprises the following steps:

[0039] Extracting Polygala tenuifolia or processed Polygala tenuifolia with an extraction solvent to obtain an extract of the Polygala tenuifolia or processed Polygala tenuifolia;

[0040] The extraction solvent is an aqueous solution of alcohol with a volume concentration of 30% to 70%;

[0041] Optionally, the alcohol comprises methanol.

[0042] In some embodiments, the weight-to-volume ratio of the Polygala tenuifolia or processed Polygala tenuifolia to the extraction solvent is 1 g: 50-100 mL.

[0043] In some embodiments, the extraction method is ultrasound;

[0044] Optionally, the power of the ultrasound is 200W to 300W, the frequency is 30kHz to 50kHz, and the time is 20min to 40min.

[0045] In some embodiments, the application further comprises the following steps:

[0046] preparing a reference solution, wherein the reference solution comprises a reference medicinal material solution and a reference substance solution;

[0047] The reference substance solution includes at least one of the following reference substances: 4-hydroxybenzoic acid, Siberian Polygala sugar A5, Siberian Polygala sugar A6, Siberian Polygala xanthone B, ferulic acid, globuloside A, Polygala Ketone III, polygala glycoside B, 3,6'-dianisopylsucrose, polygala glycoside A, 4-methoxycinnamic acid, polygala glycoside C, polygala saponin B, polygala saponin F, liquiritigenin, liquiritigenin, and glycyrrhizic acid.

[0048] In some embodiments, the method for preparing the reference solution comprises the following steps:

[0049] Mixing the reference substances, adding an organic solvent to dissolve the mixture, and preparing the reference substance solution;

[0050] The organic solvent includes methanol.

[0051] In some embodiments, the concentration of each of the reference substances in the reference substance solution is 10-100 μg / mL.

[0052] In some embodiments, the fingerprint of processed Polygala tenuifolia comprises 50 peaks, namely peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16, peak 17, peak 18, peak 19, peak 20, peak 21, peak 22, peak 23, peak 24, peak 25, peak 26, peak 27, peak 28, peak 29, peak 30, peak 31, peak 32, peak 33, peak 34, peak 35, peak 36, peak 37, peak 38, peak 39, peak 40, peak 41, peak 42, peak 43, peak 44, peak 45, peak 46, peak 47, peak 48, peak 49 and peak 50;

[0053] The fingerprint of the Polygala root lacks the peak 10, the peak 12, the peak 13, the peak 18 and the peak 36 relative to the fingerprint of the processed Polygala root;

[0054] Among them, taking peak 16 as the reference peak, the relative retention times of other peaks are as follows:

[0055] Peak 1: 0.12 ± 10%; Peak 2: 0.13 ± 10%; Peak 3: 0.18 ± 10%; Peak 4: 0.20 ± 10%; Peak 5: 0.22 ± 10%; Peak 6: 0.42 ± 10%; Peak 7: 0.45 ± 10%; Peak 8: 0.46 ± 10%; Peak 9: 0.51 ± 10%; Peak 10: 0.55 ± 10%;

[0056] Peak 11: 0.57 ± 10%; Peak 12: 0.59 ± 10%; Peak 13: 0.60 ± 10%; Peak 14: 0.62 ± 10%; Peak 15: 0.69 ± 10%; Peak 17: 1.04 ± 10%; Peak 18: 1.11 ± 10%; Peak 19: 1.18 ± 10%; Peak 20: 1.20 ± 10%;

[0057] Peak 21: 1.32 ± 10%; Peak 22: 1.42 ± 10%; Peak 23: 1.46 ± 10%; Peak 24: 1.47 ± 10%; Peak 25: 1.57 ± 10%; Peak 26: 1.61 ± 10%; Peak 27: 1.67 ± 10%; Peak 28: 1.69 ± 10%; Peak 29: 1.87 ± 10%; Peak 30: 2.02 ± 10%;

[0058] Peak 31: 2.05 ± 10%; Peak 32: 2.17 ± 10%; Peak 33: 2.25 ± 10%; Peak 34: 2.30 ± 10%; Peak 35: 2.38 ± 10%; Peak 36: 2.89 ± 10%; Peak 37: 3.17 ± 10%; Peak 38: 3.36 ± 10%; Peak 39: 3.39 ± 10%; Peak 40: 3.45 ± 10%;

[0059] Peak 41: 3.51±10%; Peak 42: 3.59±10%; Peak 43: 3.61±10%; Peak 44: 3.65±10%; Peak 45: 3.72±10%; Peak 46: 3.75±10%; Peak 47: 3.77±10%; Peak 48: 3.78±10%; Peak 49: 3.80±10%; Peak 50: 3.85±10%.

[0060] In some embodiments, the method of identifying the test product as Polygala tenuifolia or Polygala tenuifolia processed by comparing the chromatogram of the test product with the fingerprint of Polygala tenuifolia or Polygala tenuifolia processed comprises:

[0061] (1) comparing the chromatogram of the test product with the fingerprint of the processed Polygala tenuifolia, if the chromatogram of the test product has 50 characteristic peaks with the same retention times as the 50 peaks in the fingerprint of the processed Polygala tenuifolia, then identifying the test product as processed Polygala tenuifolia; and

[0062] (2) Compare the chromatogram of the test product with the fingerprint of Polygala tenuifolia. If the chromatogram of the test product has 45 characteristic peaks with the same retention times as the 45 peaks in the fingerprint of Polygala tenuifolia, and there are no chromatographic peaks at the retention times corresponding to peaks 18, 10, 36, 12 and 13, then the test product is identified as Polygala tenuifolia.

[0063] The method for constructing the fingerprint of Polygala or processed Polygala in the embodiment of the present application can comprehensively detect the components of the object to be tested by adopting appropriate chromatographic conditions according to the characteristics of the differences in the components contained in Polygala and processed Polygala, and the method is stable, precise and reliable. The fingerprint obtained by the construction method has characteristic, reproducible and operable characteristics, can provide more abundant characteristic peak information, thereby more comprehensively reflecting the quality of the sample, and providing a fast, sensitive, objective and accurate detection method for the quality of Polygala and processed Polygala.

[0064] Processed Polygala is processed from Polygala, and the traditional technology is to identify it by judging the appearance of the decoction pieces, which has certain subjectivity and ambiguity. The identification method of Polygala and processed Polygala in the embodiment of the present application is based on the construction of fingerprint spectrum, and the identification is realized by the presence or absence of chromatographic peaks, so as to scientifically and effectively identify Polygala and processed Polygala, making up for the defects of inconsistent artificial discrimination standards and insufficient accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0066] Figure 1 It is a comparison chart of the chromatographic detection charts of the test samples under different extraction solvents in Section 1.2.1 of Example 1;

[0067] Figure 2 It is a comparison chart of the chromatographic detection charts of the test products under different extraction methods in Section 1.2.2 of Example 1;

[0068] Figure 3 It is a comparison chart of the chromatographic detection chart of the test sample under different mobile phase conditions in Section 1.3.1 of Example 1;

[0069] Figure 4 This is a comparison chart of the chromatographic detection chart of the test sample under different flow rate conditions in Section 1.3.2 of Example 1;

[0070] Figure 5It is a comparison chart of the chromatographic detection chart of the test sample under different column temperature conditions in Section 1.3.3 of Example 1;

[0071] Figure 6 This is a comparison chart of the chromatographic detection charts of the test products under different column model conditions in Section 1.3.4 of Example 1;

[0072] Figure 7 It is a comparison chart of the chromatographic detection charts of the test products under different elution program conditions in Section 1.3.5 of Example 1;

[0073] Figure 8 The superimposed chromatograms of 10 batches of prepared Polygala test products in Example 1;

[0074] Fig. 9 The chromatogram of the prepared Polygala reference substance of Example 1;

[0075] Fig.10 The chromatogram of one of the reference solutions in Example 1, wherein the peaks are: Peak 3: 4-hydroxybenzoic acid, Peak 4: Siberian Polygala sugar A5, Peak 5: Siberian Polygala sugar A6, Peak 7: Siberian Polygala xanthone B, Peak 8: Ferulic acid, Peak 9: Globuloside A, Peak 14: Polygala Ketone III, peak 15: polygalaside B, peak 16: 3,6'-dianisinoylsucrose, peak 19: polygalaside A, peak 20: 4-methoxycinnamic acid, peak 24: polygalaside C, peak 44: polygalaside B, peak 50: polygalaside F;

[0076] Fig.11 is a chromatogram of one of the reference solutions in Example 1, wherein the peaks are: peak 10: liquiritin, peak 12: apigenin, peak 18: liquiritigenin, peak 36: glycyrrhizic acid;

[0077] Fig.12 The superimposed chromatograms of 10 batches of Polygala test samples in Example 1;

[0078] Fig.13 The chromatogram of the Polygala reference substance of Example 1;

[0079] Fig.14 The chromatographic detection chart of the sample to be tested in Example 2 measured according to the chromatographic conditions under item "1.3.6";

[0080] Fig.15 The cluster analysis diagram of Polygala tenuifolia and Polygala radix processed in Example 3;

[0081] Fig.16 This is the principal component analysis score graph of Polygala tenuifolia and Polygala tenuifolia processed in Example 3. DETAILED DESCRIPTION

[0082] The present application will be further described below in conjunction with the embodiments, examples and accompanying drawings. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. In addition, it should be understood that after reading the content taught in the present application, those skilled in the art may make various changes or modifications to the present application, and these equivalent forms also fall within the scope of protection of the claims attached to the present application.

[0083] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0084] the term

[0085] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0086] The term "and / or" used herein includes any one of two or more related listed items, and also includes any and all combinations of the related listed items, which include any combination of any two related listed items, any more related listed items, or all related listed items. For example, "A and / or B" includes three parallel solutions of A, B, and A+B.

[0087] Herein, "preferred", "preferable", "better", etc. are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of this application.

[0088] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0089] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0090] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0091] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, and ±0.1°C are allowed.

[0092] In the present application, weight may be μg, mg, g, kg or other mass units known in the chemical industry.

[0093] The present application provides a method for constructing a fingerprint of Polygala and processed Polygala for use in identifying processed Polygala and its raw products. The fingerprint construction method of the present application can comprehensively reflect the quality of Polygala and processed Polygala by adopting appropriate chromatographic conditions. The method is stable, with good precision and reproducibility, and provides a more objective basis for quality detection and sample identification.

[0094] Application of the fingerprint construction method of Polygala tenuifolia or processed Polygala tenuifolia of the present application in identifying processed Polygala tenuifolia and its raw products;

[0095] The method for constructing a fingerprint spectrum includes the following steps:

[0096] Providing a test solution, the test solution comprising an extract of Polygala tenuifolia or processed Polygala tenuifolia;

[0097] The test solution is subjected to liquid chromatography analysis to establish a fingerprint of Polygala tenuifolia or processed Polygala tenuifolia;

[0098] The conditions of the liquid chromatography analysis include:

[0099] Acetonitrile is used as mobile phase A, and an aqueous solution of an acid with a volume concentration of 0.05% to 0.2% is used as mobile phase B;

[0100] Gradient elution was used;

[0101] The procedure of the gradient elution includes:

[0102] From 0min to 3min, the volume percentage of the mobile phase A increases from 10% to 13%;

[0103] 3min to 10min, the volume percentage of the mobile phase A increases from 13% to 15%;

[0104] 10min-11min, the volume percentage of the mobile phase A increases from 15% to 18%;

[0105] 11min-16min, maintaining the volume percentage of the mobile phase A at 18%;

[0106] 16min-20min, the volume percentage of the mobile phase A increases from 18% to 20%;

[0107] From 20min to 21min, the volume percentage of the mobile phase A increases from 20% to 23%;

[0108] 21min-35min, maintaining the volume percentage of the mobile phase A at 23%;

[0109] From 35min to 39min, the volume percentage of the mobile phase A increased from 23% to 27%;

[0110] From 39min to 43min, the volume percentage of the mobile phase A increased from 27% to 28%;

[0111] From 43 to 44 minutes, the volume percentage of the mobile phase A increased from 28% to 30%;

[0112] 44min-45min, the volume percentage of the mobile phase A increases from 30% to 34%;

[0113] From 45min to 64min, the volume percentage of the mobile phase A increases from 34% to 35%;

[0114] From 64 to 68 minutes, the volume percentage of the mobile phase A increases from 35% to 38%;

[0115] From 68min to 73min, the volume percentage of the mobile phase A increases from 38% to 40%;

[0116] 73min-73.1min, the volume percentage of the mobile phase A increases from 40% to 90%;

[0117] 73.1min~75min, maintaining the volume percentage of the mobile phase A at 90%;

[0118] Among them, the detection wavelength is 275nm-285nm at 0min-50min, 245nm-255nm at 50min-56min, and 275nm-285nm at 56min-75min.

[0119] In some embodiments, the conditions for liquid chromatography analysis also include one or more of the following:

[0120] (1) The chromatographic column is an octadecylsilane bonded silica gel chromatographic column;

[0121] (2) Column temperature is 38°C to 42°C;

[0122] (3) The flow rate of the mobile phase is 0.28 mL / min to 0.32 mL / min;

[0123] (4) The injection volume is 1 μL to 3 μL;

[0124] (5) The acid in mobile phase B includes at least one of phosphoric acid and formic acid.

[0125] In some embodiments, the method for preparing the extract of Polygala tenuifolia or processed Polygala tenuifolia comprises the following steps:

[0126] Extracting Polygala tenuifolia or processed Polygala tenuifolia with an extraction solvent to obtain the extract of Polygala tenuifolia or processed Polygala tenuifolia; the extraction solvent is an aqueous solution of alcohol with a volume concentration of 30% to 70%; optionally, the alcohol includes methanol.

[0127] In some embodiments, the weight-to-volume ratio of the Polygala tenuifolia or processed Polygala tenuifolia to the extraction solvent is 1 g: 50-100 mL.

[0128] In some embodiments, the extraction method is ultrasound; optionally, the power of ultrasound is 200W to 300W, the frequency is 30kHz to 50kHz, and the time is 20min to 40min.

[0129] In some implementation modes, the application further comprises the following steps:

[0130] preparing a reference solution, wherein the reference solution comprises a reference medicinal material solution and a reference substance solution;

[0131] The reference substance solution includes at least one of the following reference substances: 4-hydroxybenzoic acid, Siberian Polygala sugar A5, Siberian Polygala sugar A6, Siberian Polygala xanthone B, ferulic acid, globuloside A, Polygala Ketone III, polygala glycoside B, 3,6'-dianisopylsucrose, polygala glycoside A, 4-methoxycinnamic acid, polygala glycoside C, polygala saponin B, polygala saponin F, liquiritigenin, liquiritigenin, and glycyrrhizic acid.

[0132] In some embodiments, the method for preparing the reference solution comprises the following steps:

[0133] The reference substances are mixed and dissolved in an organic solvent to prepare a reference substance solution;

[0134] Organic solvents include methanol.

[0135] In some embodiments, the concentration of each reference substance in the reference substance solution is 10-100 μg / mL.

[0136] In some embodiments, the fingerprint of processed Polygala tenuifolia comprises 50 peaks, namely peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16, peak 17, peak 18, peak 19, peak 20, peak 21, peak 22, peak 23, peak 24, peak 25, peak 26, peak 27, peak 28, peak 29, peak 30, peak 31, peak 32, peak 33, peak 34, peak 35, peak 36, peak 37, peak 38, peak 39, peak 40, peak 41, peak 42, peak 43, peak 44, peak 45, peak 46, peak 47, peak 48, peak 49 and peak 50;

[0137] The fingerprint of Polygala root lacks peaks 10, 12, 13, 18 and 36 compared with the fingerprint of processed Polygala root;

[0138] Among them, taking peak 16 as the reference peak, the relative retention times of other peaks are as follows:

[0139] Peak 1: 0.12 ± 10%; Peak 2: 0.13 ± 10%; Peak 3: 0.18 ± 10%; Peak 4: 0.20 ± 10%; Peak 5: 0.22 ± 10%; Peak 6: 0.42 ± 10%; Peak 7: 0.45 ± 10%; Peak 8: 0.46 ± 10%; Peak 9: 0.51 ± 10%; Peak 10: 0.55 ± 10%;

[0140] Peak 11: 0.57 ± 10%; Peak 12: 0.59 ± 10%; Peak 13: 0.60 ± 10%; Peak 14: 0.62 ± 10%; Peak 15: 0.69 ± 10%; Peak 17: 1.04 ± 10%; Peak 18: 1.11 ± 10%; Peak 19: 1.18 ± 10%; Peak 20: 1.20 ± 10%;

[0141] Peak 21: 1.32 ± 10%; Peak 22: 1.42 ± 10%; Peak 23: 1.46 ± 10%; Peak 24: 1.47 ± 10%; Peak 25: 1.57 ± 10%; Peak 26: 1.61 ± 10%; Peak 27: 1.67 ± 10%; Peak 28: 1.69 ± 10%; Peak 29: 1.87 ± 10%; Peak 30: 2.02 ± 10%;

[0142] Peak 31: 2.05 ± 10%; Peak 32: 2.17 ± 10%; Peak 33: 2.25 ± 10%; Peak 34: 2.30 ± 10%; Peak 35: 2.38 ± 10%; Peak 36: 2.89 ± 10%; Peak 37: 3.17 ± 10%; Peak 38: 3.36 ± 10%; Peak 39: 3.39 ± 10%; Peak 40: 3.45 ± 10%;

[0143] Peak 41: 3.51±10%; Peak 42: 3.59±10%; Peak 43: 3.61±10%; Peak 44: 3.65±10%; Peak 45: 3.72±10%; Peak 46: 3.75±10%; Peak 47: 3.77±10%; Peak 48: 3.78±10%; Peak 49: 3.80±10%; Peak 50: 3.85±10%.

[0144] The application of the fingerprint construction method of Polygala tenuifolia or processed Polygala tenuifolia in the present application in identifying processed Polygala tenuifolia and its raw products, which compares the chromatogram of the test product with the fingerprint of Polygala tenuifolia or processed Polygala tenuifolia to identify the test product as Polygala tenuifolia or processed Polygala tenuifolia, comprises:

[0145] (1) comparing the chromatogram of the test product with the fingerprint of the processed Polygala tenuifolia. If the chromatogram of the test product has 50 characteristic peaks with the same retention times as the 50 peaks in the fingerprint of the processed Polygala tenuifolia, then the test product is identified as processed Polygala tenuifolia; and

[0146] (2) Compare the chromatogram of the test product with the fingerprint of Polygala tenuifolia. If the chromatogram of the test product shows 45 characteristic peaks with the same retention times as the 45 peaks in the fingerprint of Polygala tenuifolia, and there are no chromatographic peaks at the retention times corresponding to peaks 18, 10, 36, 12 and 13, then the test product is identified as Polygala tenuifolia.

[0147] In some embodiments, the method for constructing the chromatogram of the test product is as follows: extracting the test product with an extraction solvent to prepare a test product solution;

[0148] Performing liquid chromatography analysis on the solution of the test product to generate a chromatogram of the test product;

[0149] The extraction solvent and extraction method can refer to the preparation method of the test solution mentioned above; the conditions of liquid chromatography analysis can refer to the chromatographic detection conditions in the method for constructing the fingerprint of Polygala tenuifolia or processed Polygala tenuifolia mentioned above;

[0150] Among them, peak 16 is a characteristic peak with a retention time consistent with that of the 3,6'-dicerinoylsucrose reference solution.

[0151] In some embodiments, after the sample to be tested is identified as processed Polygala tenuifolia according to method (1), in order to further confirm the quality of the slices, it is determined whether peaks 10, 12, 13, 16, 18, and 36 appearing in the chromatogram of the sample to be tested meet the following conditions:

[0152] (i) the ratio of the peak area of ​​peak 18 to peak 16 is greater than 0.04;

[0153] (ii) the ratio of the peak area of ​​peak 10 to peak 16 is greater than 0.05;

[0154] (iii) the ratio of the peak area of ​​peak 36 to peak 16 is greater than 0.03;

[0155] (iv) the ratio of the peak area of ​​peak 12 to peak 16 is greater than 0.03;

[0156] (v) the ratio of the peak area of ​​peak 13 to peak 16 is greater than 0.01;

[0157] If all the above conditions are met, the quality is judged to be excellent.

[0158] In some embodiments, in the method for identifying Polygala tenuifolia and processed Polygala tenuifolia, one or more analysis methods selected from one-way analysis of variance, cluster analysis, and principal component analysis are further used to verify the identification results.

[0159] In some embodiments, one-way ANOVA is performed using SPSS 20.0 software.

[0160] In some embodiments, cluster analysis and principal component analysis are performed using SIMCA 14.1 software.

[0161] The following are some specific embodiments.

[0162] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, auxiliary materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified. Among them:

[0163] The instruments and models are as follows:

[0164] Waters ACQUITY ultra-high performance liquid chromatography system (Waters Corporation, USA); ME204E and XP26 analytical balances (Mettler-Toledo, Switzerland); KQ-700DE digitally controlled ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); 111B 2-liang high-speed Chinese medicine pulverizer (Ruian Yongli Pharmaceutical Machinery Co., Ltd., Zhejiang Province).

[0165] The reagents are as follows:

[0166] 4-Hydroxybenzoic acid (batch number: 101149-202204, purity 100%, China Food and Drug Inspection Institute); Siberian Polygala A5 (batch number: 10096, purity 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); Siberian Polygala A6 (batch number: 9886, purity 95%, Shanghai Shidande Standard Technology Service Co., Ltd.); Siberian Polygala xanthone B (batch number: 15703, purity 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); Polygala glycoside B (batch number: 20110201, purity 98.68%, Chengdu Pufeide Biotechnology Co., Ltd.); Ferulic acid (batch number: 110773-202316, purity 99.3%, China Food and Drug Inspection Institute); Globuloside A (batch number: 11034, purity 95%, Shanghai Shidande Standard Technology Service Co., Ltd.); Polygala Ketone III (batch number: 111850-202207, purity 95.8%, China Food and Drug Inspection Institute); 3,6'-dienasinoylsucrose (batch number: 111848-202307, purity 96.7%, China Food and Drug Inspection Institute); Polygala tenuifolia glycoside A (batch number: 16614, purity 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); 4-methoxycinnamic acid (batch number: 13275, purity 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); Polygala tenuifolia glycoside C (batch number: 14003, purity 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); Polygala tenuifolia saponin B (batch number: 15710, purity 95%, Shanghai Shidande Standard Technology Service Co., Ltd.); Polygala tenuifolia saponin F (batch number: 15709, purity 95%, Shanghai Shidande Standard Technology Service Co., Ltd.); liquiritin (batch number: 111610-202209, purity 95.2%, China Food and Drug Inspection Institute); apigenin (batch number: DST230404, purity 98%, Lemeitian Pharmaceutical); glycyrrhizin (batch number: wkq21032002, purity 99.72%, Sichuan Weikeqi Biotechnology Co., Ltd.); glycyrrhizic acid (batch number: 15199, purity 98%, Shanghai Shidande Standard Technology Service Co., Ltd.); chromatographic grade methanol and acetonitrile (Merck Co., Ltd., Germany); chromatographic grade formic acid (Tianjin Komiou Chemical Reagent Co., Ltd.); water was ultrapure water (taken from the laboratory Milli-Q ultrapure water system), and the other reagents were of analytical grade.

[0167] The materials are as follows:

[0168] The 10 batches of Polygala root materials were identified as the dried roots of Polygala tenuifolia Willd. of the Polygalaceae family by Dr. Dongmei Sun, chief Chinese medicine pharmacist of Guangdong Yifang Pharmaceutical Co., Ltd. The 10 batches of Polygala root pieces and 10 batches of processed Polygala root pieces were made in the laboratory of Guangdong Yifang Pharmaceutical Co., Ltd. The specific sample information is shown in Table 1.

[0169] Table 1 Source information of Polygala and processed Polygala samples

[0170]

[0171] The following tests involve chromatographic detection, and all use the following determination method: accurately pipette 2 μL of the solution to be tested (reference solution or test solution), inject it into the ultra-high performance liquid chromatograph, and record the chromatogram.

[0172] Example 1 Construction of fingerprint

[0173] 1.1 Preparation of reference materials

[0174] Accurately weigh 4-hydroxybenzoic acid, Siberian Polygala sugar A5, Siberian Polygala sugar A6, Siberian Polygala xanthone B, ferulic acid, globuloside A, Polygala An appropriate amount of ketone III, polygala glycoside B, 3,6'-dicerinoylsucrose, polygala glycoside A, 4-methoxycinnamic acid, polygala glycoside C, polygala saponin B, and polygala saponin F reference substances were accurately weighed and methanol was added to make the mass concentrations of 14.0000μg / mL, 27.3420μg / mL, 34.5325μg / mL, 21.5600μg / mL, 21.1175μg / mL, 12.3629μg / mL, 13.5850μg / mL, 21.5550μg / mL, and 7 7.0699μg / mL, 88.1020μg / mL, 22.4420μg / mL, 27.0970μg / mL, 212.6575μg / mL, and 74.9075μg / mL of mixed reference solution I; accurately weigh appropriate amounts of liquiritigenin, liquiritigenin, and glycyrrhizic acid reference substances, and add methanol to make mixed reference solution II with mass concentrations of 19.9920μg / mL, 11.3680μg / mL, 51.4555μg / mL, and 27.3910μg / mL, respectively.

[0175] 1.2 Investigation of test sample preparation methods

[0176] 1.2.1 Investigation of extraction solvent

[0177] Take about 0.5g of the decoction powder (passed through a No. 2 sieve), use 25mL of water, methanol, 50% methanol, ethanol, and 50% ethanol as extraction solvents, ultrasonicate for 30min, and use the same pre-experimental chromatographic conditions for injection and detection. The results are as follows: Figure 1 A, B, C, D and E. The results showed that when 50% methanol was used as the extraction solvent, the chromatographic peak shape was good and the number of peaks was large, so 50% methanol was selected as the extraction solvent.

[0178] 1.2.2 Investigation of extraction methods

[0179] Take about 0.5g of the decoction powder (passed through a No. 2 sieve), use 25mL of 50% methanol as the extraction solvent, sonicate and heat under reflux for 30min, and use the same pre-experimental chromatographic conditions for injection and detection. The results are as follows: Figure 2 A and B in the sample. The results showed that the effects of ultrasonic extraction and heating reflux extraction were similar. Ultrasonic extraction was selected as the extraction method because of its simplicity.

[0180] 1.2.3 Determine the preparation method of the test product

[0181] Based on the above investigation results, the determined preparation method is as follows: take about 0.5 g of the decoction powder (passed through No. 2 sieve), accurately weigh it, put it in a stoppered conical flask, accurately add 25 mL of 50% methanol, weigh the weight, ultrasonicate (250W, 40kHz) for 30 minutes, cool, weigh the weight again, make up the lost weight with 50% methanol, shake well, filter, and take the filtrate to obtain.

[0182] 1.3 Establishing chromatographic conditions

[0183] 1.3.1 Mobile phase investigation

[0184] Different proportions of phosphoric acid and formic acid were used to prepare mobile phase B, namely: 0.05% formic acid, 0.1% formic acid, 0.1% phosphoric acid, and 0.2% formic acid. Liquid phase detection was performed separately. The results are as follows Figure 3 A, B, C and D in the.

[0185] 1.3.2 Flow rate investigation

[0186] The flow rates of 0.2 mL / min, 0.25 mL / min, and 0.3 mL / min were used for detection. The results are as follows Figure 4 A, B and C in .

[0187] 1.3.3 Column temperature investigation

[0188] The column temperatures of 20℃, 25℃, 30℃, 35℃ and 40℃ were used for testing. The results are as follows Figure 5 A, B, C, D and E in the.

[0189] 1.3.4 Chromatographic column inspection

[0190] Waters ACQUITY HSS T3 (2.1 mm × 150 mm, 1.8 μm) chromatographic column, Waters ACQUITY UPLC BEH Shield RP18 (2.1 mm × 150 mm, 1.7 μm) chromatographic column, ThermoScientific TM Syncronis TMThe results were as follows: Figure 6 A, B, C and D in the.

[0191] 1.3.5 Elution procedure investigation

[0192] The following elution procedures 1 to 6 were used for detection, and the results were as follows Figure 7 A, B, C, D, E and F in the.

[0193] Elution procedure 1:

[0194] From 0 to 13 minutes, the volume percentage of mobile phase A increased from 5% to 22%.

[0195] From 13min to 20min, the volume percentage of mobile phase A increased from 22% to 25%.

[0196] 20min~26min, the volume percentage of mobile phase A is maintained at 25%,

[0197] From 26min to 35min, the volume percentage of mobile phase A increased from 25% to 28%.

[0198] From 35min to 37min, the volume percentage of mobile phase A increased from 28% to 38%.

[0199] From 37min to 45min, the volume percentage of mobile phase A increased from 38% to 42%.

[0200] From 45min to 50min, the volume percentage of mobile phase A increased from 42% to 44%.

[0201] Elution procedure 2:

[0202] From 0 to 3 minutes, the volume percentage of mobile phase A increased from 5% to 8%.

[0203] 3min~5min, the volume percentage of mobile phase A increased from 8% to 11%,

[0204] 5min~15min, the volume percentage of mobile phase A increased from 11% to 14%,

[0205] 15min~16min, the volume percentage of mobile phase A increased from 14% to 17%,

[0206] From 16min to 24min, the volume percentage of mobile phase A increased from 17% to 22%.

[0207] 24min~27min, the volume percentage of mobile phase A is maintained at 22%,

[0208] From 27min to 35min, the volume percentage of mobile phase A increased from 22% to 28%.

[0209] From 35min to 36min, the volume percentage of mobile phase A increased from 28% to 37%.

[0210] From 36 min to 60 min, the volume percentage of mobile phase A increased from 37% to 40%.

[0211] Elution procedure 3:

[0212] From 0 to 3 minutes, the volume percentage of mobile phase A increased from 5% to 8%.

[0213] 3min~5min, the volume percentage of mobile phase A increased from 8% to 13%,

[0214] 5min~8min, the volume percentage of mobile phase A increased from 13% to 17%,

[0215] From 8min to 27min, the volume percentage of mobile phase A increased from 17% to 24%.

[0216] From 27min to 36min, the volume percentage of mobile phase A increased from 24% to 28%.

[0217] From 36min to 37min, the volume percentage of mobile phase A increased from 28% to 35%.

[0218] 37min~39min, the volume percentage of mobile phase A is maintained at 35%,

[0219] From 39min to 60min, the volume percentage of mobile phase A increased from 35% to 37%.

[0220] From 60min to 62min, the volume percentage of mobile phase A increased from 37% to 90%.

[0221] Elution procedure 4:

[0222] From 0 to 3 minutes, the volume percentage of mobile phase A increased from 4% to 8%.

[0223] From 3min to 11min, the volume percentage of mobile phase A increased from 8% to 15%.

[0224] From 11min to 15min, the volume percentage of mobile phase A increased from 15% to 19%.

[0225] From 15min to 27min, the volume percentage of mobile phase A increased from 19% to 21%.

[0226] From 27min to 33min, the volume percentage of mobile phase A increased from 21% to 24%.

[0227] From 33min to 42min, the volume percentage of mobile phase A increased from 24% to 29%.

[0228] From 42min to 43min, the volume percentage of mobile phase A increased from 29% to 33%.

[0229] From 43min to 50min, the volume percentage of mobile phase A increased from 33% to 35%.

[0230] 50min~60min, the volume percentage of mobile phase A increased from 35% to 38%,

[0231] From 60min to 60.1min, the volume percentage of mobile phase A increased from 38% to 90%.

[0232] From 60.1min to 62min, the volume percentage of mobile phase A was maintained at 90%.

[0233] Elution procedure 5:

[0234] From 0 to 3 minutes, the volume percentage of mobile phase A increased from 4% to 8%.

[0235] From 3 to 7 minutes, the volume percentage of mobile phase A increased from 8% to 16%.

[0236] From 7min to 16min, the volume percentage of mobile phase A increased from 16% to 18%.

[0237] From 16 to 17 minutes, the volume percentage of mobile phase A increased from 18% to 20%.

[0238] From 17min to 31min, the volume percentage of mobile phase A increased from 20% to 21%.

[0239] From 31min to 33min, the volume percentage of mobile phase A increased from 21% to 24%.

[0240] From 33min to 42min, the volume percentage of mobile phase A increased from 24% to 29%.

[0241] From 42min to 43min, the volume percentage of mobile phase A increased from 29% to 34%.

[0242] From 43 to 48 minutes, the volume percentage of mobile phase A was maintained at 34%.

[0243] From 48min to 49min, the volume percentage of mobile phase A increased from 34% to 36%.

[0244] From 49min to 60min, the volume percentage of mobile phase A increased from 36% to 38%.

[0245] From 60min to 60.1min, the volume percentage of mobile phase A increased from 38% to 90%.

[0246] From 60.1min to 62min, the volume percentage of mobile phase A was maintained at 90%.

[0247] Elution procedure 6:

[0248] From 0 to 3 minutes, the volume percentage of mobile phase A increased from 4% to 8%.

[0249] From 3 to 7 minutes, the volume percentage of mobile phase A increased from 8% to 14%.

[0250] 7min~13min, the volume percentage of mobile phase A is maintained at 14%,

[0251] From 13min to 16min, the volume percentage of mobile phase A increased from 14% to 15%.

[0252] 16min~17min, the volume percentage of mobile phase A increased from 15% to 20%,

[0253] From 17min to 19min, the volume percentage of mobile phase A increased from 20% to 23%.

[0254] From 19min to 27min, the volume percentage of mobile phase A was maintained at 23%.

[0255] From 27min to 28min, the volume percentage of mobile phase A increased from 23% to 24%.

[0256] From 28min to 37min, the volume percentage of mobile phase A increased from 24% to 29%.

[0257] From 37min to 38min, the volume percentage of mobile phase A increased from 29% to 35%.

[0258] 38min~43min, the volume percentage of mobile phase A is maintained at 35%,

[0259] From 43min to 56min, the volume percentage of mobile phase A increased from 35% to 38%.

[0260] From 56min to 63min, the volume percentage of mobile phase A increased from 38% to 40%.

[0261] From 63min to 63.1min, the volume percentage of mobile phase A increased from 40% to 90%.

[0262] From 63.1min to 65min, the volume percentage of mobile phase A was maintained at 90%.

[0263] 1.3.6 Determine the chromatographic conditions

[0264] Based on the above investigation results, the chromatographic conditions determined are as follows: a Waters CORTECS T3 (2.1 mm × 150 mm, 1.6 μm) chromatographic column was used; the mobile phase was acetonitrile (A): 0.1% formic acid aqueous solution (B), gradient elution (0-3 min, 10%-13% A; 3-10 min, 13%-15% A; 10-11 min, 15%-18% A; 11-16 min, 18% A; 16-20 min, 18%-20% A; 20-21 min, 20%-23% A; 21-35 min, 23% A; 35-39 min, 23%-27% A; 39-43 min, 27%-28% A; 4 3~44min, 28%~30%A; 44~45min, 30%~34%A; 45~64min, 34%~35%A; 64~68min, 35%~38%A; 68~73min, 38%~40%A; 73~73.1min, 40%~90%A; 73.1~75min, 90%A); program detection wavelength: 280nm (0~50min), 250nm (50~56min), 280nm (56~75min); flow rate 0.3mL / min; column temperature 40℃; injection volume 2μL.

[0265] 1.4 Methodological considerations

[0266] 1.4.1 Precision test

[0267] Prepare the prepared Polygala test solution according to the method in "1.2.3", and perform continuous sampling and determination 6 times according to the chromatographic conditions in "1.3.6". Take the chromatographic peak of No. 16 3,6'-diesinapoylsucrose as the reference peak (S). The relative retention time RSD range of each common peak and the S peak is 0.01% to 0.15%, and the relative peak area RSD range is 0.41% to 1.79%, indicating that the instrument has good precision.

[0268] 1.4.2 Repeatability test

[0269] Take the prepared Polygala tenuifolia sample and prepare the test solution according to the method under "1.2.3". Prepare 6 parallel samples and inject and measure according to the chromatographic conditions under "1.3.6". Take the chromatographic peak of No. 16 3,6'-diesinapoylsucrose as the reference peak (S). The relative retention time RSD range of each common peak and the S peak is 0.03% to 0.21%, and the relative peak area RSD range is 0.63% to 4.53%, indicating that the method has good repeatability.

[0270] 1.4.3 Stability test

[0271] Take the prepared Polygala test solution prepared by the method under "1.2.3", and inject and measure it according to the chromatographic conditions under "1.3.6" at 0h, 2h, 4h, 8h, 12h, 18h and 24h after preparation, respectively. Take the chromatographic peak of No. 16 3,6'-diesinapoylsucrose as the reference peak (S), and calculate that the relative retention time RSD range of each common peak and the S peak is 0.04% ~ 0.96%, and the relative peak area RSD range is 1.75% ~ 4.35%, indicating that the test solution has good stability within 24h.

[0272] 1.5 Establishment of characteristic spectra and determination of common peaks

[0273] 1.5.1 Construction of Polygala tenuifolia fingerprint

[0274] Take 10 batches of processed Polygala samples, prepare the test solution according to the method under "1.2.3", inject and test according to the chromatographic conditions under "1.3.6", and collect chromatograms. Use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software for data processing, establish fingerprints, use ZYZ01 as the reference spectrum, use the "median" method as the reference spectrum generation method, and generate reference fingerprints of 10 batches of processed Polygala by full peak matching. A total of 50 common peaks were calibrated in the fingerprints. The results are shown in Table 2, Table 3, Figure 8 , Fig. 9 .

[0275] Table 2 Retention time of common peaks in 10 batches of Polygala tenuifolia

[0276]

[0277]

[0278] Table 3 Common peak areas of 10 batches of Polygala tenuifolia

[0279]

[0280]

[0281] Will Fig. 9 Chromatogram of the reference substance Figure 10-11By comparison, 18 main components were identified, namely peak 3 (4-hydroxybenzoic acid), peak 4 (Siberian Polygala sugar A5), peak 5 (Siberian Polygala sugar A6), peak 7 (Siberian Polygala xanthone B), peak 8 (ferulic acid), peak 9 (globuloside A), peak 10 (liquiritin), peak 12 (apigenin), peak 14 (polygala ketone III), peak 15 (polygala glycoside B), peak 16 (3,6'-dianisopylsucrose), peak 18 (liquiritigenin), peak 19 (polygala glycoside A), peak 20 (4-methoxycinnamic acid), peak 24 (polygala glycoside C), peak 36 (glycyrrhizic acid), peak 44 (polygala saponin B), and peak 50 (polygala saponin F).

[0282] 1.5.2 Similarity Evaluation

[0283] The chromatograms of the 10 batches of processed Polygala obtained in 1.5.1 were imported into the similarity evaluation system of Chinese medicine chromatographic fingerprints to calculate the similarity. The results are shown in Table 4. The results show that the similarities of the 10 batches of processed Polygala are all greater than 0.95, indicating that the differences between different batches of processed Polygala samples are small and the quality is relatively stable.

[0284] Table 4 Sample similarity evaluation results

[0285]

[0286] 1.5.3 Construction of Polygala tenuifolia fingerprint

[0287] Take 10 batches of Polygala samples, prepare the test solution according to the method under "1.2.3", inject and test according to the chromatographic conditions under "1.3.6", and collect chromatograms. Use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software for data processing, establish fingerprints, use YZ01 as the reference spectrum, use the "median" method as the reference spectrum generation method, and generate reference fingerprints of 10 batches of Polygala by full peak matching. A total of 45 common peaks were calibrated in the fingerprints. The results are shown in Tables 5, 6, Fig.12 , Fig.13 .

[0288] Table 5 Retention time of common peaks of 10 batches of Polygala tenuifolia

[0289]

[0290]

[0291] In Table 5, “ / ” means that the corresponding peak does not exist in the chromatogram.

[0292] Table 6 Common peak areas of 10 batches of Polygala

[0293]

[0294]

[0295] In Table 6, “ / ” means that the corresponding peak does not exist in the chromatogram.

[0296] Will Fig.13 Chromatogram of the reference substance Figure 10-11 By comparison, 13 main components were identified, namely peak 3 (4-hydroxybenzoic acid), peak 4 (Siberian Polygala sugar A5), peak 5 (Siberian Polygala sugar A6), peak 7 (Siberian Polygala xanthone B), peak 8 (ferulic acid), peak 9 (globuloside A), peak 14 (Polygala ketone III), peak 15 (polygala glycoside B), peak 16 (3,6'-dianisopylsucrose), peak 19 (polygala glycoside A), peak 20 (4-methoxycinnamic acid), peak 24 (polygala glycoside C), peak 44 (polygala saponin B), and peak 50 (polygala saponin F).

[0297] Embodiment 2: A kind of detection method of preparing Polygala root slices

[0298] Take the sample to be tested (ZYZ 11), prepare the solution of the sample to be tested according to the method in "1.2.3", and perform the test according to the chromatographic conditions in "1.3.6". The test results are as follows: Fig.14 shown.

[0299] Will Fig.14 and Fig. 9 The comparison revealed that the test spectrum of the sample to be tested could detect 50 characteristic peaks, and the similarity with the control characteristic spectrum was 0.995. It was determined that the quality of the sample to be tested was stable, met the quality requirements, and was a qualified product.

[0300] Embodiment 3: Identification of processed Polygala root slices and raw Polygala root fingerprints

[0301] 1. Construction of fingerprint of Polygala root slices and identification with processed Polygala root

[0302] Refer to the fingerprint of processed Polygala and Polygala prepared in Example 1 Fig. 9 and Fig.13 By comparison, it can be seen that compared with the fingerprint of processed Polygala, the fingerprint of Polygala lacks 5 peaks, namely peak 10, peak 12, peak 13, peak 18 and peak 36. These are mainly the components introduced when processed Polygala uses licorice. Therefore, processed Polygala and Polygala can be distinguished based on the presence or absence of these 5 peaks.

[0303] 2. Independence T test

[0304] The peak areas of processed Polygala pieces and raw Polygala (the peak areas of partially missing chromatographic peaks were counted as 0) were respectively imported into SPSS26.0 for one-way ANOVA, and the results are shown in Table 7. The results of peak area significance showed that the chromatographic peaks of Polygala changed significantly before and after processing, and there were significant differences in the peak areas of 6 peaks, namely peak 10, peak 12, peak 13, peak 18, peak 36 and peak 45, among which peak 45 was significantly reduced after processing, and the rest were newly added components after processing.

[0305] Table 7 Fingerprint peak area independence T test analysis results (unit: mAU*min)

[0306]

[0307]

[0308] 3. Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA)

[0309] In order to better investigate the main characteristic components of the differences between processed and raw Polygala, the orthogonal partial least squares discriminant analysis (OPLS-DA) method was used to analyze the samples. The peak areas of 10 batches of processed Polygala and Polygala (the peak areas of some missing chromatographic peaks were counted as 0) were converted and imported into SIMCA 14.1 for OPLS-DA analysis. The variable importance for the projection (VIP value) of each index was ranked, and the index with VIP value>1 was selected as the main differential component to distinguish processed Polygala and Polygala.

[0310] In the OPLS-DA model of Polygala tenuifolia and Polygala tenuifolia established, the cumulative explanatory power parameter R2X of the independent variable was 0.995, the cumulative explanatory power parameter R2Y of the dependent variable was 0.921, and the predictive power parameter Q2 was 0.826, all of which were greater than 0.5, indicating that the established model had a strong explanatory rate and predictive rate. The obtained OPLS-DA score diagram is shown in Fig.15 , variable importance projection (VIP) values ​​are shown in Fig.16 .Depend on Fig.15 It can be seen that Radix Polygalae and Radix Polygalae can be completely distinguished, with Radix Polygalae located in the right quadrant of the score graph and Radix Polygalae located in the left quadrant of the score graph. Fig.16It can be seen that a total of 15 VIP values ​​were extracted with VIP value > 1 as the standard, and the importance ranking was peak 16 (3,6'-dienasinoylsucrose), peak 18 (liquiritigenin), peak 19 (polygala tenuifolia glycoside A), peak 10 (liquiritigenin), peak 35, peak 44 (polygala saponin B), peak 49, peak 4 (Siberian polygala sugar A5), peak 30, peak 29, peak 40, peak 45, peak 36 (glycyrrhizic acid), peak 50 (polygala saponin F), and peak 34. The above components may be the differential markers of processed Polygala tenuifolia and its raw product.

[0311] 4. Identification criteria:

[0312] Combining the fingerprint spectra of processed Polygala and Polygala slices and the results of one-way ANOVA and OPLS-DA, it was finally determined that there were 5 chromatographic peaks of processed Polygala and Polygala that were significant for traditional Chinese medicine identification, namely peak 18, peak 10, peak 36, peak 12, and peak 13.

[0313] The identification criteria of processed Polygala and Polygala slices can be:

[0314] If the chromatogram of the decoction piece to be tested shows 50 characteristic peaks with the same retention time as the fingerprint spectrum of the above-mentioned processed Polygala root decoction piece, and the ratio of the peak area of ​​peak 18 to peak 16 is greater than 0.04, the ratio of the peak area of ​​peak 10 to peak 16 is greater than 0.05, the ratio of the peak area of ​​peak 36 to peak 16 is greater than 0.03, the ratio of the peak area of ​​peak 12 to peak 16 is greater than 0.03, and the ratio of the peak area of ​​peak 13 to peak 16 is greater than 0.01, then the decoction piece to be tested is determined to be processed Polygala root;

[0315] If the chromatogram of the tested medicinal piece shows 45 characteristic peaks with the same retention time as the fingerprint spectrum of the above-mentioned Polygala medicinal piece, and there are no chromatographic peaks at the retention times corresponding to peaks 18, 10, 36, 12 and 13, then the tested medicinal piece is determined to be Polygala.

[0316] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0317] The above-described embodiments only express several implementation methods of the present application, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present application, several variations and improvements can also be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided in the present application through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the attached claims, and the description and drawings may be used to explain the contents of the claims.

Claims

1. Application of a fingerprint spectrum construction method of Polygala tenuifolia or processed Polygala tenuifolia in identifying processed Polygala tenuifolia and its raw products; The fingerprint spectrum construction method of Polygala tenuifolia or processed Polygala tenuifolia comprises the following steps: preparing a test solution, wherein the test solution comprises an extract of Polygala tenuifolia or processed Polygala tenuifolia; The test solution is subjected to liquid chromatography analysis to establish a fingerprint of Polygala tenuifolia or processed Polygala tenuifolia; The conditions of the liquid chromatography analysis include: Acetonitrile is used as mobile phase A, and an aqueous solution of an acid with a volume concentration of 0.05% to 0.2% is used as mobile phase B, and gradient elution is adopted; The procedure of the gradient elution includes: 0min~3min, mobile phase A from 10%→13%; 3min~10min, mobile phase A from 13%→15%; 10min~11min, mobile phase A changes from 15% to 18%; 11min~16min, mobile phase A from 18%; 16min~20min, mobile phase A changes from 18%→20%; 20min-21min, mobile phase A from 20% to 23%; 21min~35min, mobile phase A from 23%; 35min-39min, mobile phase A from 23% to 27%; 39min~43min, mobile phase A changed from 27%→28%; 43min-44min, mobile phase A changed from 28% to 30%; 44min~45min, mobile phase A changed from 30% to 34%; 45min~64min, mobile phase A changed from 34% to 35%; 64min~68min, mobile phase A changed from 35% to 38%; 68min~73min, mobile phase A changed from 38%→40%; 73min~73.1min, mobile phase A changed from 40%→90%; 73.1min~75min, mobile phase A from 90%; Among them, the detection wavelength is 275nm-285nm at 0min-50min, 245nm-255nm at 50min-56min, and 275nm-285nm at 56min-75min.

2. The use according to claim 1, characterized in that: The conditions of the liquid chromatography analysis also include one or more of the following: (1) The chromatographic column is an octadecylsilane bonded silica gel chromatographic column; (2) Column temperature is 38°C to 42°C; (3) The flow rate of the mobile phase is 0.28 mL / min to 0.32 mL / min; (4) The injection volume is 1 μL to 3 μL; (5) The acid in the mobile phase B includes at least one of phosphoric acid and formic acid.

3. The use according to claim 1, characterized in that: The method for preparing the extract of Polygala tenuifolia or processed Polygala tenuifolia comprises the following steps: Extracting Polygala tenuifolia or processed Polygala tenuifolia with an extraction solvent to obtain an extract of the Polygala tenuifolia or processed Polygala tenuifolia; The extraction solvent is an aqueous solution of alcohol with a volume concentration of 30% to 70%; Optionally, the alcohol comprises methanol.

4. The use according to claim 3, characterized in that: The weight-to-volume ratio of the Polygala tenuifolia or processed Polygala tenuifolia to the extraction solvent is 1 g: 50-100 mL.

5. The use according to claim 3 or 4, characterized in that: The extraction method is ultrasound; Optionally, the power of the ultrasound is 200W to 300W, the frequency is 30kHz to 50kHz, and the time is 20min to 40min.

6. The use according to claim 1, characterized in that: The following steps are also included: preparing a reference solution, wherein the reference solution comprises a reference medicinal material solution and a reference substance solution; The reference substance solution includes at least one of the following reference substances: 4-hydroxybenzoic acid, Siberian Polygala sugar A5, Siberian Polygala sugar A6, Siberian Polygala xanthone B, ferulic acid, globuloside A, Polygala Ketone III, polygala glycoside B, 3,6'-dianisopylsucrose, polygala glycoside A, 4-methoxycinnamic acid, polygala glycoside C, polygala saponin B, polygala saponin F, liquiritigenin, liquiritigenin, and glycyrrhizic acid.

7. The use according to claim 6, characterized in that: The preparation method of the reference substance solution comprises the following steps: Mixing the reference substances, adding an organic solvent to dissolve the mixture, and preparing the reference substance solution; The organic solvent includes methanol.

8. The use according to claim 7, characterized in that: The concentration of each of the reference substances in the reference substance solution is 10 to 100 μg / mL.

9. The use according to claim 1, characterized in that: The fingerprint of the processed Polygala tenuifolia includes 50 peaks, namely peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, peak 9, peak 10, peak 11, peak 12, peak 13, peak 14, peak 15, peak 16, peak 17, peak 18, peak 19, peak 20, peak 21, peak 22, peak 23, peak 24, peak 25, peak 26, peak 27, peak 28, peak 29, peak 30, peak 31, peak 32, peak 33, peak 34, peak 35, peak 36, peak 37, peak 38, peak 39, peak 40, peak 41, peak 42, peak 43, peak 44, peak 45, peak 46, peak 47, peak 48, peak 49 and peak 50; The fingerprint of the Polygala root lacks the peak 10, the peak 12, the peak 13, the peak 18 and the peak 36 relative to the fingerprint of the processed Polygala root; Among them, taking peak 16 as the reference peak, the relative retention times of other peaks are as follows: Peak 1: 0.12 ± 10%; Peak 2: 0.13 ± 10%; Peak 3: 0.18 ± 10%; Peak 4: 0.20 ± 10%; Peak 5: 0.22 ± 10%; Peak 6: 0.42 ± 10%; Peak 7: 0.45 ± 10%; Peak 8: 0.46 ± 10%; Peak 9: 0.51 ± 10%; Peak 10: 0.55 ± 10%; Peak 11: 0.57 ± 10%; Peak 12: 0.59 ± 10%; Peak 13: 0.60 ± 10%; Peak 14: 0.62 ± 10%; Peak 15: 0.69 ± 10%; Peak 17: 1.04 ± 10%; Peak 18: 1.11 ± 10%; Peak 19: 1.18 ± 10%; Peak 20: 1.20 ± 10%; Peak 21: 1.32 ± 10%; Peak 22: 1.42 ± 10%; Peak 23: 1.46 ± 10%; Peak 24: 1.47 ± 10%; Peak 25: 1.57 ± 10%; Peak 26: 1.61 ± 10%; Peak 27: 1.67 ± 10%; Peak 28: 1.69 ± 10%; Peak 29: 1.87 ± 10%; Peak 30: 2.02 ± 10%; Peak 31: 2.05 ± 10%; Peak 32: 2.17 ± 10%; Peak 33: 2.25 ± 10%; Peak 34: 2.30 ± 10%; Peak 35: 2.38 ± 10%; Peak 36: 2.89 ± 10%; Peak 37: 3.17 ± 10%; Peak 38: 3.36 ± 10%; Peak 39: 3.39 ± 10%; Peak 40: 3.45 ± 10%; Peak 41: 3.51±10%; Peak 42: 3.59±10%; Peak 43: 3.61±10%; Peak 44: 3.65±10%; Peak 45: 3.72±10%; Peak 46: 3.75±10%; Peak 47: 3.77±10%; Peak 48: 3.78±10%; Peak 49: 3.80±10%; Peak 50: 3.85±10%.

10. The use according to claim 1, characterized in that: Identifying the test product as Polygala tenuifolia or processed Polygala tenuifolia by comparing the chromatogram of the test product with the fingerprint of Polygala tenuifolia or processed Polygala tenuifolia comprises: (1) comparing the chromatogram of the test product with the fingerprint of the processed Polygala tenuifolia, if the chromatogram of the test product has 50 characteristic peaks with the same retention times as the 50 peaks in the fingerprint of the processed Polygala tenuifolia, then identifying the test product as processed Polygala tenuifolia; and (2) Compare the chromatogram of the test product with the fingerprint of Polygala tenuifolia. If the chromatogram of the test product has 45 characteristic peaks with the same retention times as the 45 peaks in the fingerprint of Polygala tenuifolia, and there are no chromatographic peaks at the retention times corresponding to peaks 18, 10, 36, 12 and 13, then the test product is identified as Polygala tenuifolia.

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