Application of a fingerprint spectrum construction method of polygala or polygalae preparation in identification of polygalae preparation and its raw product

Abstract

The application relates to the technical field of traditional Chinese medicine detection and analytical chemistry, in particular to application of a fingerprint spectrum construction method of polygala or prepared polygala in identification of prepared polygala and its crude product. The fingerprint spectrum construction method comprises analysis by using ultra-high performance liquid chromatography, and the chromatographic conditions are that acetonitrile is used as mobile phase A, and 0.05%-0.2% formic acid aqueous solution is used as mobile phase B. The fingerprint spectrum has characteristics, reproducibility and operability, and can clearly show the common components and differential components of polygala and prepared polygala. The construction method can be applied to identification of prepared polygala and its crude product, and the prepared polygala and the crude product are identified according to the presence or absence of chromatographic peaks, and the method is intuitive and accurate.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of traditional Chinese medicine detection and analytical chemistry, and particularly relates to application of a construction method of a fingerprint spectrum of Polygala or prepared Polygala in identification of prepared Polygala and raw Polygala. BACKGROUND

[0002] Polygala is derived from the dried roots of Polygala tenuifolia Willd. or Polygala sibirica L. of the family Polygalaceae, has the effects of warming and bitter, pungent, and is related to the heart, kidney and lung, and has the effects of soothing the nerves, benefiting the wisdom, communicating between the heart and kidney, eliminating phlegm and reducing swelling. The 2020 edition of Chinese Pharmacopoeia records Polygala and prepared Polygala as processed products of Polygala. Polygala is prepared by removing impurities from the dried roots of Polygala, slightly washing, moistening, cutting into segments and drying. Prepared Polygala is prepared by removing impurities from the dried roots of Polygala, adding into the decoction of liquorice, and drying after the decoction is boiled to dryness. Ancient records show that raw Polygala can cause a stuffy feeling and irritate the throat, while prepared Polygala can alleviate the toxicity of irritating the throat and enhance the effect of soothing the nerves and benefiting the wisdom. Modern pharmacological studies show that traditional Chinese medicine processing can change the properties of drugs, guide the meridians of drugs, and change the tendency of the effects of drugs on the body, thereby explaining the reasons for the differences in toxicity and efficacy between Polygala and prepared Polygala.

[0003] The Pharmacopoeia records that Polygala is in the form of cylindrical segments, the outer skin is grayish yellow to gray brown, with transverse wrinkles, the cross section is brown yellow, and prepared Polygala is in the form of Polygala segments, the surface is yellow brown. It is difficult to distinguish Polygala and prepared Polygala from the appearance, and currently no other method for distinguishing Polygala and prepared Polygala has been reported. In view of the differences in pharmacological activities between Polygala and prepared Polygala, in order to ensure the safety and effectiveness of clinical medication, it is necessary to establish a method for identifying and controlling the quality of prepared Polygala and its raw product Polygala.

[0004] Traditional Chinese medicine fingerprint spectrum is a recognition of the overall effect of traditional Chinese medicine material group, can clarify the chemical composition of traditional Chinese medicine at the whole level, is one of the important indicators for quality control of traditional Chinese medicine, and can be used for judging the quality of medicinal materials, identifying the authenticity of medicinal materials, distinguishing different parts of medicinal materials, and identifying raw and processed products of medicinal materials. By establishing the fingerprint spectrum of prepared Polygala and its raw product, screening specific indicators for the differences in chemical components between the two, the two can be objectively and scientifically distinguished.

[0005] Therefore, it is necessary to provide a method for simultaneously constructing the fingerprint spectrum of Polygala and prepared Polygala, so as to realize the scientific identification of the two. SUMMARY

[0006] Based on this, one or more embodiments of the present application provide an application of a fingerprint spectrum construction method of Radix Polygalae or processed Radix Polygalae in identifying processed Radix Polygalae and its crude product, and the fingerprint spectrum obtained by the fingerprint spectrum construction method of the present application can objectively, scientifically and comprehensively reflect the inherent chemical characteristic information of Radix Polygalae and processed Radix Polygalae, and serve as a basis for quality detection and identification.

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

[0008] An application of a fingerprint spectrum construction method of Radix Polygalae or processed Radix Polygalae in identifying processed Radix Polygalae and its crude product;

[0009] The fingerprint spectrum construction method of Radix Polygalae or processed Radix Polygalae includes the following steps:

[0010] Preparation of a test sample solution, wherein the test sample solution includes an extract of Radix Polygalae or processed Radix Polygalae;

[0011] Liquid chromatography analysis of the test sample solution to establish a fingerprint spectrum of Radix Polygalae or processed Radix Polygalae;

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

[0013] Using acetonitrile as mobile phase A and using an acid aqueous solution with a volume concentration of 0.05% to 0.2% as mobile phase B for gradient elution;

[0014] The program of the gradient elution includes:

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

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

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

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

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

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

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

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

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

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

[0025] 44min~45min, mobile phase A from 30%→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 from 38%→40%;

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

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

[0031] wherein, 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 comprise one or more of the following:

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

[0034] (2) the column temperature is 38℃~42℃;

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

[0036] (4) the injection volume is 1μL~3μL;

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

[0038] In some embodiments, the preparation method of the extract of Polygalae or processed Polygalae comprises the following steps:

[0039] extracting Polygalae or processed Polygalae with an extraction solvent to obtain the extract of Polygalae or processed Polygalae;

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

[0041] optionally, the alcohol comprises methanol.

[0042] In some embodiments, the weight-to-volume ratio of the prepared polygala root and the extraction solvent is 1 g: 50-100 mL.

[0043] In some embodiments, the extraction method is ultrasonic extraction.

[0044] Optionally, the ultrasonic extraction is performed at a power of 200-300 W, a frequency of 30-50 kHz, and for a time of 20-40 min.

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

[0046] A reference solution is prepared, which comprises a control medicinal material solution and a control substance solution.

[0047] The control substance solution comprises at least one of the following control substances: 4-hydroxybenzoic acid, sebastianoside A5, sebastianoside A6, sebastianoxanthone B, ferulic acid, globuladiol A, polygalaxanthone III, micranthusoside B, 3,6'-diacylsucrose, micranthusoside A, 4-methoxycinnamic acid, polygalaxoside C, polygalasaponin B, polygalasaponin F, glycyrrhizin, apiosyl glycyrrhizin, glycyrrhetic acid, and glycyrrhizic acid.

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

[0049] Each of the control substances is mixed and dissolved in an organic solvent to prepare the control substance solution.

[0050] The organic solvent comprises methanol.

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

[0052] In some embodiments, the fingerprint of the prepared polygala root 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 is missing the peak 10, the peak 12, the peak 13, the peak 18, and the peak 36 relative to the fingerprint of the prepared polygala root.​

[0054] Wherein, taking peak 16 as a reference peak, the relative retention time of each peak is 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 product to be tested is identified as Radix Polygalae or processed Radix Polygalae by comparing the chromatogram of the product to be tested with the fingerprint of Radix Polygalae or processed Radix Polygalae, comprising:

[0061] (1) comparing the chromatogram of the product to be tested with the fingerprint of the processed Radix Polygalae, if 50 characteristic peaks consistent with the retention time of the 50 peaks of the fingerprint of the processed Radix Polygalae appear in the chromatogram of the product to be tested, the product to be tested is identified as the processed Radix Polygalae; and

[0062] (2) comparing the chromatographic spectrum of the to-be-tested product with the fingerprint spectrum of the Radix Polygalae, if the chromatographic spectrum of the to-be-tested product has 45 characteristic peaks consistent with the 45 peaks of the Radix Polygalae in the retention time, and there is no chromatographic peak at the retention time corresponding to the peak 18, the peak 10, the peak 36, the peak 12 and the peak 13, the to-be-tested product is identified as the Radix Polygalae.

[0063] The method for constructing the fingerprint spectrum of the Radix Polygalae or the processed Radix Polygalae according to the present application can comprehensively detect the components of the to-be-tested product by using appropriate chromatographic conditions, and is stable, precise and reliable. The fingerprint spectrum constructed by the method has characteristics of reproducibility and operability, can provide more rich characteristic peak information, and thus can more comprehensively reflect the quality of the sample, and provide a rapid, sensitive, objective and accurate detection method for the quality of the Radix Polygalae and the processed Radix Polygalae.

[0064] The processed Radix Polygalae is processed from the Radix Polygalae, and the traditional technology is to identify the appearance of the decoction pieces, which has certain subjectivity and ambiguity. The identification method of the Radix Polygalae and the processed Radix Polygalae according to the present application is based on the construction of the fingerprint spectrum, and the identification is realized by the presence or absence of the chromatographic peak, so that the Radix Polygalae and the processed Radix Polygalae can be identified scientifically and effectively, and the defects of the non-uniform and insufficient accuracy of the artificial identification standard are remedied. BRIEF DESCRIPTION OF DRAWINGS

[0065] In order to more clearly illustrate the technical solutions in the specific embodiments or the prior art, the drawings needed to be used in the specific embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creating any inventive labor.

[0066] Figure 1 The comparison chart of the chromatographic detection chart of the test product under different extraction solvents in section 1.2.1 of Example 1;

[0067] Figure 2 The comparison chart of the chromatographic detection chart of the test product under different extraction methods in section 1.2.2 of Example 1;

[0068] Figure 3 The comparison chart of the chromatographic detection chart of the test product under different mobile phase conditions in section 1.3.1 of Example 1;

[0069] Figure 4 The comparison chart of the chromatographic detection chart of the test product under different flow rate conditions in section 1.3.2 of Example 1;

[0070] Figure 5This is a comparison of the chromatograms of the test sample under different column temperature conditions in section 1.3.3 of Example 1;

[0071] Figure 6 This is a comparison of the chromatograms of the test sample under different column types under the conditions described in section 1.3.4 of Example 1;

[0072] Figure 7 This is a comparison of the chromatograms of the test sample under different elution procedures in section 1.3.5 of Example 1;

[0073] Figure 8 The superimposed chromatograms are of 10 batches of prepared Polygala tenuifolia test samples from Example 1;

[0074] Figure 9 The chromatogram of the prepared Polygala tenuifolia reference standard of Example 1;

[0075] Figure 10 The chromatogram of one of the reference solutions in Example 1 is shown below, with the following peaks: Peak 3: 4-hydroxybenzoic acid; Peak 4: Siberian polygalactosyl sugar A5; Peak 5: Siberian polygalactosyl sugar A6; Peak 7: Siberian polygalactosyl xanthanone B; Peak 8: ferulic acid; Peak 9: globulin A; Peak 14: Polygala tenuifolia. Ketone III, peak 15: Polygala tenuifolia glycoside B, peak 16: 3,6'-disinoyl sucrose, peak 19: Polygala tenuifolia glycoside A, peak 20: 4-methoxycinnamic acid, peak 24: Polygala tenuifolia glycoside C, peak 44: Polygala tenuifolia saponin B, peak 50: Polygala tenuifolia saponin F.

[0076] Figure 11 The chromatogram of one of the reference solutions in Example 1 is shown below, with the following peaks: Peak 10: glycyrrhizin, Peak 12: apigenin, Peak 18: glycyrrhizin, Peak 36: glycyrrhizic acid.

[0077] Figure 12 The superimposed chromatograms are of 10 batches of Polygala tenuifolia test samples from Example 1;

[0078] Figure 13 The chromatogram of the Polygala tenuifolia reference standard of Example 1;

[0079] Figure 14 This is the chromatogram of the sample tested in Example 2, determined according to the chromatographic conditions under section "1.3.6";

[0080] Figure 15 This is a cluster analysis diagram of Polygala tenuifolia and processed Polygala tenuifolia from Example 3;

[0081] Figure 16 Principal component analysis score plots of Polygala tenuifolia and processed Polygala tenuifolia from Example 3. Detailed Implementation

[0082] The application will be further described below with reference to the accompanying drawings and examples. It should be understood that these examples are intended to illustrate the application and not to limit the scope of the application. Furthermore, it should be understood that various modifications can be made to the application by those skilled in the art upon reading the teachings of the present application as set forth herein. Such modifications are intended to fall within the scope of the appended claims.

[0083] 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 herein is for describing particular embodiments only and is not intended to be limiting of the application.

[0084] The term

[0085] Unless otherwise indicated, or unless the context clearly indicates otherwise, the terms or phrases used in this application have the following meanings:

[0086] The term "and / or", as used in the application herein, is intended to cover the meaning of "and" as well as the meaning of "or", and is intended to include any and all permutations of two or more items, associated with the term, including alternate combinations like "and / or", "individually", or the like.

[0087] In this application, "preferably", "more preferably", "even more preferably", and the like are used to describe a particularly desirable implementation or example, but do not serve to limit the scope of the application in any way.

[0088] In this application, "further", "furthermore", "in addition", and the like are used to describe additional features or advantages that are not essential to the application.

[0089] In this application, the technical features described in an open-ended manner include both the closed technical solution consisting of the listed features, and the open technical solution containing the listed features.

[0090] In this application, when referring to a numerical interval (i.e. a numerical range), unless otherwise specified, the optional numerical distribution within the numerical interval is considered to be continuous, and includes both numerical end points (i.e. the minimum value and the maximum value) of the numerical range, and every numerical value between the two numerical end points. Unless otherwise specified, when the numerical interval refers only to integers within the numerical interval, it includes both end point integers of the numerical range, and every integer between the two end point integers. Furthermore, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood as including any and all sub-ranges subsumed therein.

[0091] The temperature parameters in the present application, if not particularly limited, allow for constant temperature treatment as well as for variations within a certain temperature interval. It is to be understood that the constant temperature treatment allows for fluctuations within the accuracy of the instrument control. Fluctuations within a range of, for example, ±0.5°C, ±0.4°C, ±0.3°C, ±0.2°C, ±0.1°C are allowed.

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

[0093] The present application provides a method for constructing a fingerprint spectrum of Radix Polygalae and prepared Radix Polygalae, and application of the method in identifying prepared Radix Polygalae and its raw product. The method for constructing a fingerprint spectrum, by using appropriate chromatographic conditions, can comprehensively reflect the quality of Radix Polygalae and prepared Radix Polygalae, and is stable, precise, and reproducible, thus providing a more objective basis for quality detection and sample identification.

[0094] The present application provides a method for constructing a fingerprint spectrum of Radix Polygalae and prepared Radix Polygalae, and application of the method in identifying prepared Radix Polygalae and its raw product. The method for constructing a fingerprint spectrum, by using appropriate chromatographic conditions, can comprehensively reflect the quality of Radix Polygalae and prepared Radix Polygalae, and is stable, precise, and reproducible, thus providing a more objective basis for quality detection and sample identification.

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

[0096] A test sample solution is provided, which comprises an extract of Radix Polygalae or prepared Radix Polygalae;

[0097] The test sample solution is subjected to liquid chromatography analysis to establish a fingerprint spectrum of Radix Polygalae or prepared Radix Polygalae;

[0098] The conditions for the liquid chromatography analysis comprise:

[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 is used;

[0101] The program for the gradient elution comprises:

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

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

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

[0105] 11 min to 16 min, the volume percentage of the mobile phase A is maintained at 18%;

[0106] 16min~20min, the volume percentage of the mobile phase A is increased from 18% to 20%;

[0107] 20min~21min, the volume percentage of the mobile phase A is increased from 20% to 23%;

[0108] 21min~35min, the volume percentage of the mobile phase A is maintained at 23%;

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

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

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

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

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

[0114] 64min~68min, the volume percentage of the mobile phase A is increased from 35% to 38%;

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

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

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

[0118] wherein 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 of the liquid chromatography analysis further comprise one or more of the following:

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

[0121] (2) the column temperature is 38℃~42℃;

[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 the mobile phase B includes at least one of phosphoric acid and formic acid.

[0125] In some embodiments, the method for preparing the extract of Polygalae or processed Polygalae includes the following steps:

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

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

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

[0129] In some embodiments, the use further includes the following steps:

[0130] preparing a reference solution, which includes a control medicinal material solution and a control substance solution;

[0131] the control substance solution includes at least one of the following control substances: 4-hydroxybenzoic acid, secoisolaricirecin A5, secoisolaricirecin A6, secoisolaricirecinol B, ferulic acid, globul A, Polygalae ketone III, micromeria B, 3,6'-diacylsucrose, micromeria A, 4-methoxycinnamic acid, Polygalae glycoside C, Polygalae saponin B, Polygalae saponin F, glycyrrhizin, apiosyl glycyrrhizin, glycyrrhizinic acid, and glycyrrhizinic acid.

[0132] In some embodiments, the method for preparing the control substance solution includes the following steps:

[0133] mixing the control substances, dissolving with an organic solvent to obtain the control substance solution;

[0134] the organic solvent includes methanol.

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

[0136] In some embodiments, the fingerprint of prepared Radix Polygalae 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;

[0137] The fingerprint of Radix Polygalae is missing peak 10, peak 12, peak 13, peak 18, and peak 36 relative to the fingerprint of prepared Radix Polygalae;

[0138] In which, peak 16 is the reference peak, and the relative retention time of each peak is 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 discloses a fingerprint spectrum construction method of polygala or prepared polygala, and application of the fingerprint spectrum construction method in identification of prepared polygala and raw polygala.

[0145] (1) comparing the chromatogram of the sample to be tested with the fingerprint spectrum of the prepared polygala, if 50 characteristic peaks of the sample to be tested are consistent with the 50 peaks of the fingerprint spectrum of the prepared polygala in retention time, then the sample to be tested is identified as the prepared polygala; and

[0146] (2) comparing the chromatogram of the sample to be tested with the fingerprint spectrum of the raw polygala, if 45 characteristic peaks of the sample to be tested are consistent with the 45 peaks of the fingerprint spectrum of the raw polygala in retention time, and no chromatographic peak exists at the retention time corresponding to peak 18, peak 10, peak 36, peak 12 and peak 13, then the sample to be tested is identified as the raw polygala.

[0147] In some embodiments, the method for constructing the chromatogram of the sample to be tested is as follows: the sample to be tested is extracted with an extraction solvent to obtain a sample to be tested solution;

[0148] The sample to be tested solution is subjected to liquid chromatography analysis to generate a chromatogram of the sample to be tested;

[0149] The extraction solvent and the extraction method can refer to the preparation method of the sample solution described above; the liquid chromatography analysis conditions can refer to the chromatographic detection conditions in the method for constructing the fingerprint spectrum of the raw polygala or the prepared polygala described above;

[0150] In the fingerprint spectrum, peak 16 is a characteristic peak consistent with the retention time of the 3,6'-dijesseryl sucrose reference substance solution.

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

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

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

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

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

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

[0157] If all the above conditions are met, it is identified as excellent quality.

[0158] In some embodiments, one or more of single factor variance analysis, cluster analysis and principal component analysis are used to verify the identification results in the identification method of Polygalae Radix and prepared Polygalae Radix.

[0159] In some embodiments, SPSS 20.0 software is used for single factor variance analysis.

[0160] In some embodiments, SIMCA 14.1 software is used for cluster analysis and principal component analysis.

[0161] The following are some specific examples.

[0162] In the following examples, the experimental methods are all conventional methods unless otherwise specified. In the following examples, the medicinal material raw materials, excipients, reagents, etc. are all commercially purchased 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 balance (Mettler-Toledo, Switzerland); KQ-700DE digital ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); 111B two-litre high-speed Chinese medicine grinder (Zhejiang Ruian Yongli Pharmaceutical Machinery Co., Ltd.).

[0165] The reagents are as follows:

[0166] 4-Hydroxybenzoic acid (batch number: 101149-202204, purity 100%, China National Institutes for Food and Drug Control); Siberian polygalactosyl glycoside A5 (batch number: 10096, purity 98%, Shanghai Shidander Standard Technical Service Co., Ltd.); Siberian polygalactosyl glycoside A6 (batch number: 9886, purity 95%, Shanghai Shidander Standard Technical Service Co., Ltd.); Siberian polygalactosyl xanthonone B (batch number: 15703, purity 98%, Shanghai Shidander Standard Technical Service Co., Ltd.); Polygalactosyl glycoside B (batch number: 20110201, purity 98.68%, Chengdu Pufeide Biotechnology Co., Ltd.); Ferulic acid (batch number: 110773-202316, purity 99.3%, China National Institutes for Food and Drug Control); Gamma glycoside A (batch number: 11034, purity 95%, Shanghai Shidander Standard Technical Service Co., Ltd.); Polygala tenuifolia Ketone III (Batch No.: 111850-202207, Purity 95.8%, China National Institutes for Food and Drug Control); 3,6'-Disinopyrosine Sucrose (Batch No.: 111848-202307, Purity 96.7%, China National Institutes for Food and Drug Control); Polygala tenuifolia glycoside A (Batch No.: 16614, Purity 98%, Shanghai Shidander Standard Technical Service Co., Ltd.); 4-Methoxycinnamic acid (Batch No.: 13275, Purity 98%, Shanghai Shidander Standard Technical Service Co., Ltd.); Polygala tenuifolia glycoside C (Batch No.: 14003, Purity 98%, Shanghai Shidander Standard Technical Service Co., Ltd.); Polygala tenuifolia saponin B (Batch No.: 15710, Purity 95%, Shanghai Shidander Standard Technical Service Co., Ltd.); Polygala tenuifolia saponin F (Batch No.: 15709 (95% purity, Shanghai Shidander Standard Technical Service Co., Ltd.); glycyrrhizin (batch number: 111610-202209, 95.2% purity, China National Institutes for Food and Drug Control); apigenin glycyrrhizin (batch number: DST230404, 98% purity, Lemeitian Pharmaceutical); glycyrrhizin (batch number: wkq21032002, 99.72% purity, Sichuan Weikeqi Biotechnology Co., Ltd.); glycyrrhizic acid (batch number: 15199, 98% purity, Shanghai Shidander Standard Technical Service Co., Ltd.); chromatographic grade methanol and acetonitrile (Merck, Germany); chromatographic grade formic acid (Tianjin Kemeio Chemical Reagent Co., Ltd.); water was ultrapure water (from the laboratory Milli-Q ultrapure water system), and all other reagents were analytical grade.

[0167] The materials are as follows:

[0168] Ten batches of Polygala tenuifolia medicinal materials were identified by Sun Dongmei, Chief Pharmacist of Traditional Chinese Medicine at Guangdong Yifang Pharmaceutical Co., Ltd., as dried roots of Polygala tenuifolia Willd., a plant in the Polygalaceae family. Ten batches of Polygala tenuifolia slices and ten batches of processed Polygala tenuifolia slices were prepared in-house by the laboratory of Guangdong Yifang Pharmaceutical Co., Ltd. Specific sample information is shown in Table 1.

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

[0170]

[0171] The following tests involve chromatographic detection and are performed using the following method: Accurately pipette 2 μL of the test solution (reference solution or sample solution), inject it into the ultra-high performance liquid chromatograph, and record the chromatogram.

[0172] Example 1: Constructing a fingerprint map

[0173] 1.1 Preparation of reference material

[0174] Accurately weigh 4-hydroxybenzoic acid, Siberian polygalactosyl sugar A5, Siberian polygalactosyl sugar A6, Siberian polygalactosyl oxantrone B, ferulic acid, globulin A, and polygala tenuifolia. Ketone III, Polygala tenuifolia glycoside B, 3,6'-disinyl sucrose, Polygala tenuifolia glycoside A, 4-methoxycinnamic acid, Polygala tenuifolia glycoside C, Polygala tenuifolia saponin B, and Polygala tenuifolia saponin F reference standards were accurately weighed and dissolved in methanol to prepare concentrations of 14.0000 μg / mL, 27.3420 μg / mL, 34.5325 μg / mL, 21.5600 μg / mL, 21.1175 μg / mL, 12.3629 μg / mL, 13.5850 μg / mL, 21.5550 μg / mL, and 7 μg / mL, respectively. Mixed reference solution I with concentrations of 7.0699 μg / mL, 88.1020 μg / mL, 22.4420 μg / mL, 27.0970 μg / mL, 212.6575 μg / mL, and 74.9075 μg / mL; accurately weigh appropriate amounts of glycyrrhizin, apigenin, glycyrrhizin, and glycyrrhizic acid reference standards, and add methanol to prepare mixed reference solution II with mass concentrations of 19.9920 μg / mL, 11.3680 μg / mL, 51.4555 μg / mL, and 27.3910 μg / mL, respectively.

[0175] 1.2 Investigation of the preparation method of the test sample

[0176] 1.2.1 Investigation of extraction solvent

[0177] Take approximately 0.5 g of the herbal powder (passed through a No. 2 sieve), and extract it using 25 mL of water, methanol, 50% methanol, ethanol, and 50% ethanol as extraction solvents, respectively. Sonicate for 30 min each time. Inject and detect the samples under the same preliminary experimental chromatographic conditions. The results are as follows: Figure 1 The results showed that chromatographic peaks with good shape and a large number of peaks were obtained when 50% methanol was used as the extraction solvent; therefore, 50% methanol was chosen as the extraction solvent.

[0178] 1.2.2 Examination of Extraction Methods

[0179] Take about 0.5 g of powder (pass No. 2 sieve) of the decoction pieces, and use 25 mL of 50% methanol as the extraction solvent for ultrasonic extraction and heating reflux for 30 min, respectively. The sample is injected for detection under the same pre-experimental chromatographic conditions, and the results are as shown in Table A and B in Figure 2 The results show that the ultrasonic extraction and heating reflux extraction have little difference in effect. Since the ultrasonic extraction is simple to operate, it is selected as the extraction method.

[0180] 1.2.3 Determination of test sample preparation method

[0181] Based on the above investigation results, the preparation method is determined as follows: take about 0.5 g of powder (pass No. 2 sieve) of the decoction pieces, accurately weigh, place in a conical flask with a plug, accurately add 25 mL of 50% methanol, weigh, cool, re-weigh, make up the weight loss with 50% methanol, shake well, filter, and take the filtrate, which is obtained.

[0182] 1.3 Establish chromatographic conditions

[0183] 1.3.1 Investigation of mobile phase

[0184] Prepare the mobile phase B by using different proportions of phosphoric acid and formic acid, which are 0.05% formic acid, 0.1% formic acid, 0.1% phosphoric acid, and 0.2% formic acid, respectively. Perform liquid phase detection, and the results are as shown in Table A, B, C, and D in Figure 3

[0185] 1.3.2 Investigation of flow rate

[0186] Perform detection at flow rates of 0.2 mL / min, 0.25 mL / min, and 0.3 mL / min, respectively, and the results are as shown in Table A, B, and C in Figure 4

[0187] 1.3.3 Investigation of column temperature

[0188] Perform detection at column temperatures of 20°C, 25°C, 30°C, 35°C, and 40°C, respectively, and the results are as shown in Table A, B, C, D, and E in Figure 5

[0189] 1.3.4 Investigation of chromatographic column

[0190] Use Waters ACQUITY HSS T3 (2.1 mm x 150 mm, 1.8 μm) chromatographic column, Waters ACQUITY UPLC BEH Shield RP18 (2.1 mm x 150 mm, 1.7 μm) chromatographic column, and Thermo Scientific TM Syncronis TM ​​​aQ (2.1 mm x 100 mm, 1.7 μm) chromatographic column, SHIMADZU Shim-pack GIST C18-AQ (2.1 mm x 150 mm, 1.9 μm) chromatographic column, respectively, and the results are as follows Figure 6 A, B, C and D in Table 1.

[0191] 1.3.5 Elution program investigation

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

[0193] Elution program 1:

[0194] 0-13 min, the volume percentage of mobile phase A was increased from 5% to 22%,

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

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

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

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

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

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

[0201] Elution program 2:

[0202] 0-3 min, the volume percentage of mobile phase A was increased from 5% to 8%,

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

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

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

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

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

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

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

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

[0211] Elution program 3:

[0212] 0 to 3 min, the volume percentage of mobile phase A is increased from 5% to 8%,

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

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

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

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

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

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

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

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

[0221] Elution program 4:

[0222] 0 to 3 min, the volume percentage of mobile phase A is increased from 4% to 8%,

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

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

[0225] 15 min ~ 27 min, the volume percentage of mobile phase A is raised from 19% to 21%,

[0226] 27 min ~ 33 min, the volume percentage of mobile phase A is raised from 21% to 24%,

[0227] 33 min ~ 42 min, the volume percentage of mobile phase A is raised from 24% to 29%,

[0228] 42 min ~ 43 min, the volume percentage of mobile phase A is raised from 29% to 33%,

[0229] 43 min ~ 50 min, the volume percentage of mobile phase A is raised from 33% to 35%,

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

[0231] 60 min ~ 60.1 min, the volume percentage of mobile phase A is raised from 38% to 90%,

[0232] 60.1 min ~ 62 min, the volume percentage of mobile phase A is maintained at 90%.

[0233] Elution program 5:

[0234] 0 ~ 3 min, the volume percentage of mobile phase A is raised from 4% to 8%,

[0235] 3 min ~ 7 min, the volume percentage of mobile phase A is raised from 8% to 16%,

[0236] 7 min ~ 16 min, the volume percentage of mobile phase A is raised from 16% to 18%,

[0237] 16 min ~ 17 min, the volume percentage of mobile phase A is raised from 18% to 20%,

[0238] 17 min ~ 31 min, the volume percentage of mobile phase A is raised from 20% to 21%,

[0239] 31 min ~ 33 min, the volume percentage of mobile phase A is raised from 21% to 24%,

[0240] 33 min ~ 42 min, the volume percentage of mobile phase A is raised from 24% to 29%,

[0241] 42 min ~ 43 min, the volume percentage of mobile phase A is raised from 29% to 34%,

[0242] 43 min ~ 48 min, the volume percentage of mobile phase A is maintained at 34%,

[0243] 48 min ~ 49 min, the volume percentage of mobile phase A is raised from 34% to 36%,

[0244] 49 min ~ 60 min, the volume percentage of mobile phase A is raised from 36% to 38%,

[0245] 60 min ~ 60.1 min, the volume percentage of mobile phase A is raised from 38% to 90%,

[0246] 60.1 min ~ 62 min, the volume percentage of mobile phase A is maintained at 90%.

[0247] Elution program 6:

[0248] 0 ~ 3 min, the volume percentage of mobile phase A is raised from 4% to 8%,

[0249] 3 min ~ 7 min, the volume percentage of mobile phase A is raised from 8% to 14%,

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

[0251] 13 min ~ 16 min, the volume percentage of mobile phase A is raised from 14% to 15%,

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

[0253] 17 min ~ 19 min, the volume percentage of mobile phase A is raised from 20% to 23%,

[0254] 19 min ~ 27 min, the volume percentage of mobile phase A is maintained at 23%,

[0255] 27 min ~ 28 min, the volume percentage of mobile phase A is raised from 23% to 24%,

[0256] 28 min ~ 37 min, the volume percentage of mobile phase A is raised from 24% to 29%,

[0257] 37 min ~ 38 min, the volume percentage of mobile phase A is raised from 29% to 35%,

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

[0259] 43 min ~ 56 min, the volume percentage of mobile phase A is raised from 35% to 38%,

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

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

[0262] 63.1 min ~ 65 min, the volume percentage of mobile phase A is maintained at 90%.

[0263] 1.3.6 Determination of chromatographic conditions

[0264] Based on the above investigation results, the determined chromatographic conditions are as follows: Waters CORTECS T3 (2.1 mm x 150 mm, 1.6 μm) column is used; the mobile phase is 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; 43-44 min, 28%-30% A; 44-45 min, 30%-34% A; 45-64 min, 34%-35% A; 64-68 min, 35%-38% A; 68-73 min, 38%-40% A; 73-73.1 min, 40%-90% A; 73.1-75 min, 90% A); program detection wavelength: 280 nm (0-50 min), 250 nm (50-56 min), 280 nm (56-75 min); flow rate 0.3 mL / min; column temperature 40°C; injection volume 2 μL.

[0265] 1.4 Methodology investigation

[0266] 1.4.1 Precision test

[0267] Prepare the Radix Polygalae test solution by the method under item "1.2.3", and continuously inject to determine 6 times under the chromatographic conditions under item "1.3.6", take 3,6'-di-jasminyl sucrose chromatographic peak as the reference peak (S), calculate the relative retention time RSD range of each common peak and S peak is 0.01%-0.15%, the relative peak area RSD range is 0.41%-1.79%, which indicates that the instrument precision is good.

[0268] 1.4.2 Reproducibility test

[0269] Take the prepared polygala sample, prepare the test solution according to the method in item "1.2.3", in parallel for 6 times, inject for determination under the chromatographic conditions in item "1.3.6", take the chromatographic peak of 3,6'-di-jasmine acyl sucrose No. 16 as the reference peak (S), calculate the relative retention time RSD range of each common peak to S peak, which is 0.03%-0.21%, and the relative peak area RSD range is 0.63%-4.53%, indicating that the method has good repeatability.

[0270] 1.4.3 Stability test

[0271] Take the prepared polygala test solution prepared according to the method in item "1.2.3", and inject for determination under the chromatographic conditions in item "1.3.6" at 0h, 2h, 4h, 8h, 12h, 18h and 24h after preparation respectively, take the chromatographic peak of 3,6'-di-jasmine acyl sucrose No. 16 as the reference peak (S), calculate the relative retention time RSD range of each common peak to S peak, which 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 chromatogram and determination of common peaks

[0273] 1.5.1 Construction of polygala fingerprint chromatogram

[0274] Take 10 batches of prepared polygala samples, prepare the test solution according to the method in item "1.2.3", inject for detection under the chromatographic conditions in item "1.3.6", and collect the chromatogram. Use the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012 Edition" software for data processing, establish the fingerprint chromatogram, take the No. ZYZ01 chromatogram as the reference chromatogram, take the "median" method as the control chromatogram generation method, and generate the control fingerprint chromatogram of 10 batches of prepared polygala respectively by full peak matching, and the fingerprint chromatogram is calibrated with 50 common peaks, the results are shown in Table 2, Table 3, Figure 8 、 Figure 9 .

[0275] Table 2 Common peak retention time of 10 batches of prepared polygala

[0276]

[0277]

[0278] Table 3 Common peak peak area of 10 batches of prepared polygala

[0279]

[0280]

[0281] Figure 9 and the chromatogram of the control product Figure 10-11 ​Comparison was made and 18 main components were identified, namely, peak 3 (4-hydroxybenzoic acid), peak 4 (siberian milkwort sugar A5), peak 5 (siberian milkwort sugar A6), peak 7 (siberian milkwort xanthone B), peak 8 (ferulic acid), peak 9 (sphaerioside A), peak 10 (liquiritin), peak 12 (sphaerioside A), peak 14 (milkwort ketone III), peak 15 (polygalaxanthin B), peak 16 (3,6'-di-jasminyl sucrose), peak 18 (liquiritigenin), peak 19 (polygalaxanthin A), peak 20 (4-methoxycinnamic acid), peak 24 (siberian milkwort glycoside C), peak 36 (glycyrrhizic acid), peak 44 (polygalaxanthin B), and peak 50 (polygalaxanthin F).

[0282] 1.5.2 Similarity evaluation

[0283] The 10 batches of prepared milkwort samples obtained in 1.5.1 were introduced into the traditional Chinese medicine chromatographic fingerprint similarity evaluation system, and the similarity was calculated. The results are shown in Table 4. The results show that the similarity of the 10 batches of prepared milkwort samples is greater than 0.95, indicating that the batch-to-batch difference of the different batches of prepared milkwort samples is small, and the quality is stable.

[0284] Table 4 Similarity evaluation results of samples

[0285]

[0286] 1.5.3 Construction of milkwort fingerprint

[0287] Ten batches of milkwort samples were prepared according to the method in "1.2.3", and the sample solution was detected by the chromatographic conditions in "1.3.6", and the chromatogram was collected. The data were processed by "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2012" software, and the fingerprint was established. YZ01 was used as the reference chromatogram, "median" method was used as the control chromatogram generation method, and 10 batches of milkwort control fingerprint chromatograms were generated by full peak matching. The fingerprint chromatogram has 45 common peaks, and the results are shown in Tables 5, 6, Figure 12 Figure 13

[0288] Table 5 Common peak retention time of 10 batches of milkwort

[0289]

[0290]

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

[0292] Table 6 Common peak area of 10 batches of milkwort

[0293] ​​​

[0294]

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

[0296] The Figure 13 chromatogram of the test sample was compared with that of the control sample, and 13 main components were identified, namely, peak 3 (4-hydroxybenzoic acid), peak 4 (sibiricose A5), peak 5 (sibiricose A6), peak 7 (sibiricin B), peak 8 (ferulic acid), peak 9 (bulleyanose A), peak 14 (polygalaxanthone III), peak 15 (polygalaxanthone B), peak 16 (3,6'-diacylsucrose), peak 19 (polygalaxanthone A), peak 20 (4-methoxycinnamic acid), peak 24 (polygalaxanthone C), peak 44 (polygalaxanthone B), and peak 50 (polygalaxanthone F). Figure 10-11

[0297] Example 2: A detection method for polygalae decoction pieces

[0298] The test sample (ZYZ 11) was prepared according to the method in item "1.2.3", and the test sample solution was prepared according to the method in item "1.3.6". The detection results are shown in Table 6. Figure 14

[0299] The Figure 14 chromatogram of the test sample was compared with that of the control sample, and 13 main components were identified, namely, peak 3 (4-hydroxybenzoic acid), peak 4 (sibiricose A5), peak 5 (sibiricose A6), peak 7 (sibiricin B), peak 8 (ferulic acid), peak 9 (bulleyanose A), peak 14 (polygalaxanthone III), peak 15 (polygalaxanthone B), peak 16 (3,6'-diacylsucrose), peak 19 (polygalaxanthone A), peak 20 (4-methoxycinnamic acid), peak 24 (polygalaxanthone C), peak 44 (polygalaxanthone B), and peak 50 (polygalaxanthone F). Figure 9

[0300] Example 3: Identification of polygalae decoction pieces and raw polygalae by fingerprint chromatography

[0301] 1. Construction of the fingerprint chromatogram of polygalae decoction pieces and identification of polygalae decoction pieces

[0302] The fingerprint chromatograms of polygalae decoction pieces and raw polygalae prepared according to Example 1 were compared, and it was found that the fingerprint chromatogram of raw polygalae was missing 5 peaks, namely, peak 10, peak 12, peak 13, peak 18, and peak 36, compared with the fingerprint chromatogram of polygalae decoction pieces. These 5 peaks were mainly introduced by the use of licorice in the preparation of polygalae decoction pieces. Therefore, polygalae decoction pieces and raw polygalae can be distinguished according to the presence or absence of these 5 peaks. Figure 9 Figure 13

[0303] 2. Independent T test

[0304] ​​​​​​The peak area of prepared Radix Polygalae and its raw product Radix Polygalae (part of missing chromatographic peak area is 0) was introduced into SPSS 26.0 for one-way ANOVA analysis, and the results are shown in Table 7. The peak area significance results show that the chromatographic peak changes significantly before and after processing of Radix Polygalae, and there are significant differences in the peak area of 6 peaks, namely peaks 10, 12, 13, 18, 36 and 45, among which peak 45 significantly decreases after processing, and the rest are newly added components after processing.

[0305] Table 7 Independent T-test analysis results of peak area of fingerprint (unit: mAU*min)

[0306]

[0307]

[0308] 3. Orthogonal partial least squares discriminant analysis (OPLS-DA)

[0309] In order to better investigate the main marker components of the composition difference between prepared Radix Polygalae and its raw product Radix Polygalae, orthogonal partial least squares discriminant analysis (OPLS-DA) method was used to analyze the samples. The peak area of 10 batches of prepared Radix Polygalae and Radix Polygalae (part of missing chromatographic peak area is 0) was introduced into SIMCA 14.1 after conversion for OPLS-DA analysis, and the variable projection importance (Variable Importance for the Projection, VIP value) of each index was arranged in size, and the index with VIP value > 1 was selected as the main difference component for distinguishing prepared Radix Polygalae and Radix Polygalae.

[0310] In the established OPLS-DA model of prepared Radix Polygalae and Radix Polygalae, the cumulative explanatory ability parameter R2X of independent variable was 0.995, the cumulative explanatory ability parameter R2Y of dependent variable was 0.921, and the prediction ability parameter Q2 was 0.826, all greater than 0.5, indicating that the established model had strong explanation rate and prediction rate, and the obtained OPLS-DA score plot is shown in Figure 15 , and the variable importance projection (VIP) value is shown in Figure 16 . As can be seen from Figure 15 , prepared Radix Polygalae and Radix Polygalae can be completely distinguished, in which prepared Radix Polygalae is located in the right quadrant of the score plot, and Radix Polygalae is located in the left quadrant of the score plot. As can be seen from Figure 16It can be seen that 15 VIP values are extracted with VIP value > 1 as the standard, and the importance ranking is peak 16 (3, 6'-di-glucosinolate sucrose), peak 18 (glycyrrhizin), peak 19 (tenuifolin A), peak 10 (liquiritin), peak 35, peak 44 (polygalaxoside B), peak 49, peak 4 (polygalaxin A5), peak 30, peak 29, peak 40, peak 45, peak 36 (glycyrrhizic acid), peak 50 (polygalaxin F), peak 34, and the above components are likely to be the differential markers of polygalaxin and its raw polygalaxin.

[0311] 4. Identification criteria:

[0312] In combination with the fingerprint of polygalaxin and polygalaxin decoction pieces, single factor variance analysis and OPLS-DA results, it is finally determined that the chromatographic peaks with Chinese medicine identification significance of polygalaxin and polygalaxin are 5, in turn, peak 18, peak 10, peak 36, peak 12, and peak 13.

[0313] The identification criteria of polygalaxin and polygalaxin decoction pieces can be:

[0314] If the chromatogram of the to-be-tested decoction piece appears 50 characteristic peaks consistent with the retention time of the above-mentioned fingerprint of polygalaxin decoction pieces, 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 to-be-tested decoction piece is determined to be polygalaxin.

[0315] If the chromatogram of the to-be-tested decoction piece appears 45 characteristic peaks consistent with the retention time of the above-mentioned fingerprint of polygalaxin decoction pieces, and there is no chromatographic peak at the retention time corresponding to peak 18, peak 10, peak 36, peak 12, and peak 13, then the to-be-tested decoction piece is determined to be polygalaxin.

[0316] The technical features of the above-mentioned embodiments and examples can be combined in any suitable manner. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments and examples are described, however, as long as the combinations of the technical features do not exist contradictions, they should be considered within the scope of the present disclosure.

[0317] The above embodiments only express several implementation ways of the present application, but cannot be understood as limitation to the patent scope. It should be pointed out that, for ordinary skilled in the art, several variations 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. In addition, it should be understood that, after reading the above teaching of the present application, the skilled in the art can make various changes or modifications to the present application, and the equivalent forms also fall within the protection scope of the present application. It should also be understood that, the skilled in the art can obtain the technical solutions on the basis of the technical solutions provided by the present application through logical analysis, reasoning or limited test, which all fall within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. The application of a fingerprint spectrum construction method for Polygala tenuifolia or processed Polygala tenuifolia in the identification of processed Polygala tenuifolia and its raw product; The method for constructing the fingerprint spectrum of Polygala tenuifolia or prepared Polygala tenuifolia includes the following steps: Prepare a reference solution, wherein the reference solution comprises a reference medicinal material solution and a reference standard solution; The reference solution contains the following reference substances: 4-hydroxybenzoic acid, Siberian polygalactosyl sugar A5, Siberian polygalactosyl sugar A6, Siberian polygalactosyl oxantrone B, ferulic acid, globulin A, and polygala tenuifolia. Ketone III, Polygala tenuifolia glycoside B, 3,6'-disinoyl sucrose, Polygala tenuifolia glycoside A, 4-methoxycinnamic acid, Polygala tenuifolia glycoside C, Polygala tenuifolia saponin B, Polygala tenuifolia saponin F, glycyrrhizin, apigenin glycyrrhizin, glycyrrhizin and glycyrrhizic acid. Preparation of test solution: Polygala tenuifolia or processed Polygala tenuifolia is extracted with an extraction solvent to obtain an 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%; The test solution was analyzed by liquid chromatography to establish a fingerprint spectrum of Polygala tenuifolia or processed Polygala tenuifolia. The conditions for the liquid chromatography analysis include: The chromatographic column is an octadecylsilane-bonded silica gel column; Acetonitrile is used as mobile phase A, and an aqueous solution of acid with a volume concentration of 0.05% to 0.2% is used as mobile phase B, wherein the acid in mobile phase B includes at least one of phosphoric acid and formic acid; Gradient elution was employed; The gradient elution procedure includes: The detection wavelengths are 275nm to 285nm from 0min to 50min, 245nm to 255nm from 50min to 56min, and 275nm to 285nm from 56min to 75min.

2. The application according to claim 1, characterized in that, The conditions for the liquid chromatography analysis also include one or more of the following: (1) The column temperature is 38℃~42℃; (2) The flow rate of the mobile phase is 0.28 mL / min to 0.32 mL / min; (3) The injection volume is 1 μL to 3 μL.

3. The application according to claim 1, characterized in that, The alcohol in the extraction solvent includes methanol.

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

5. The application according to claim 3 or 4, characterized in that, The extraction method is ultrasound.

6. The application according to claim 5, characterized in that, The ultrasound power is 200W to 300W, the frequency is 30kHz to 50kHz, and the duration is 20min to 40min.

7. The application according to claim 1, characterized in that, The preparation method of the reference solution includes the following steps: The reference standards are mixed and dissolved in an organic solvent to prepare the reference standard solution. The organic solvent includes methanol.

8. The application according to claim 7, characterized in that, The concentration of each of the reference standards in the reference solution is 10–100 μg / mL.

9. The application according to claim 1, characterized in that, The fingerprint spectrum of the prepared 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 spectrum of the Polygala tenuifolia is missing peaks 10, 12, 13, 18, and 36 compared to the fingerprint spectrum of the prepared Polygala tenuifolia. With peak 16 as the reference peak, the relative retention times of the 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 application according to claim 1, characterized in that, Identifying the test sample as Polygala tenuifolia or processed Polygala tenuifolia by comparing its chromatogram with the fingerprint chromatogram of Polygala tenuifolia or processed Polygala tenuifolia includes: (1) Compare the chromatogram of the test sample with the fingerprint chromatogram of the prepared Polygala tenuifolia. If the chromatogram of the test sample shows 50 characteristic peaks with retention times consistent with the 50 peaks in the fingerprint chromatogram of the prepared Polygala tenuifolia, then the test sample is identified as prepared Polygala tenuifolia; and (2) Compare the chromatogram of the test sample with the fingerprint chromatogram of Polygala tenuifolia. If the chromatogram of the test sample shows 45 characteristic peaks with the same retention time as the fingerprint chromatogram 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 sample is identified as Polygala tenuifolia.

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