Method for constructing specific chromatogram of rhizoma acori graminei-polygala tenuifolia drug pair

Abstract

The invention relates to the technical field of traditional Chinese medicine detection, in particular to a construction method of a specific chromatogram of a rhizoma acori graminei-polygala tenuifolia drug pair, which comprises the following steps: preparing a test solution; carrying out ultra-high performance liquid chromatography detection on the test solution, and constructing a specific chromatogram of the rhizoma acori graminei-polygala tenuifolia drug pair according to a detected liquid chromatogram; the detection conditions of the ultra-high performance liquid chromatography are as follows: octadecylsilane chemically bonded silica is taken as a chromatographic column of a filler, acetonitrile is taken as a mobile phase A, and a 0.02-0.08% phosphoric acid aqueous solution is taken as a mobile phase B; eluting according to a specific gradient program; the detection wavelength of 0 to t1 in the gradient program is 315 to 325 nm, and then the wavelength is switched to 198 to 208 nm; the t1 ranges from 58 min to 62 min. According to the construction method of the specific chromatogram of the rhizoma acori graminei-polygala tenuifolia drug pair, 23 characteristic peaks should be shown in the specific chromatogram of a test sample, 15 characteristic peaks are identified, and quality control over the rhizoma acori graminei-polygala tenuifolia drug pair is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine detection, and particularly relates to a method for constructing a characteristic fingerprint of the medicine pair of Acorus tatarinowii Rhizoma - Polygalae Radix. Background Art

[0002] Acorus tatarinowii Rhizoma is the dried rhizome of the plant Acorus tatarinowii Schott of the Araceae family. It is warm in nature, pungent and bitter in taste, and belongs to the heart and stomach meridians; it has the effects of resuscitating the mind by inducing resuscitation and resolving phlegm, improving intelligence and awakening the mind, and promoting digestion by removing dampness. Polygalae Radix is the dried root of the plant Polygala tenuifolia Willd or Polygala sibirica L of the Polygalaceae family. It is warm in nature, bitter and pungent in taste, and belongs to the heart, kidney and lung meridians; it has the effects of calming the mind and improving intelligence, connecting the heart and kidney, reducing phlegm, and detumescence. The medicine pair of Acori Tatarinowii Rhizoma - Polygalae Radix (AP) is a commonly used medicine pair for improving intelligence in clinical practice. Both of these two herbs were first recorded in "Shennong Ben Cao Jing". Because both of them can calm the mind and improve intelligence, and resolve phlegm and induce resuscitation, and their combination has a stronger effect, they have been commonly used for the treatment of diseases such as "amnesia" since ancient times. Previous studies have found that "Acorus tatarinowii Rhizoma - Polygalae Radix" is the medicine pair with the highest frequency of occurrence in traditional Chinese medicine prescriptions for treating senile dementia. The main material basis for Acorus tatarinowii Rhizoma to improve memory is volatile phenylpropanoid components, such as asarone, methyleugenol, etc.; the main material basis for Polygalae Radix to improve memory is polygala saponins, oligosaccharide esters, ketones, etc. After the two herbs are combined, they usually play a pharmacological role of "1 + 1 > 2". And the herbs usually combined with this medicine pair are mostly acidic herbs. Under this acidic condition, the chemical components of the decocted medicine have changed in terms of quality and quantity. The reason may be that the new chemical substances produced by the two are more easily absorbed by the body than the original drugs, or the new components produced by the two herbs promote the absorption of the original chemical components in the body, thus playing a pharmacological role of "1 + 1 > 2". Therefore, it is particularly important to comprehensively and efficiently control the quality of various active ingredients in the decoction of the medicine pair of Acorus tatarinowii Rhizoma - Polygalae Radix under acidic conditions to ensure clinical efficacy.

[0003] The characteristic chromatogram of traditional Chinese medicine is an effective means to evaluate the overall quality of compound traditional Chinese medicine preparations. Its advantage lies in being able to comprehensively characterize the component characteristics of all medicinal flavors in the preparation prescription, and it is widely used in the qualitative and quantitative analysis of traditional Chinese medicine and its compounds. Currently, there is no specific quality control method for the Shichangpu-Yuanzhi herb pair. It can only be seen in the quality research of the reference substance of KaiXinSan containing the Shichangpu-Yuanzhi herb pair. Most of its quality control methods use HPLC to establish detection methods for one type of component or a combination of multiple types of components. However, there are obvious defects in these currently disclosed detection methods. The main defect is that the control of active ingredients can only characterize the respective chemical components of the Shichangpu-Yuanzhi herb pair before compatibility, and cannot accurately characterize the chemical components that change after the compatibility of the Shichangpu-Yuanzhi herb pair. Therefore, for the Shichangpu-Yuanzhi herb pair with complex pharmacological effects in the body and chemical component changes after compatibility, a specific quality control method that can comprehensively, quickly, and accurately characterize the quality is needed, but there is no relevant report yet. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect of the prior art lacking specific quality control for the chemical components that change after the compatibility of the Shichangpu-Yuanzhi herb pair, so as to provide a method for constructing the characteristic chromatogram of the Shichangpu-Yuanzhi herb pair to solve the above problems.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for constructing the characteristic chromatogram of the Shichangpu-Yuanzhi herb pair, comprising the following steps:

[0007] Preparation of the test solution;

[0008] Perform ultra-high performance liquid chromatography detection on the test solution, and construct the characteristic chromatogram of the Shichangpu-Yuanzhi herb pair according to the measured liquid chromatogram;

[0009] The conditions for the ultra-high performance liquid chromatography detection include: a chromatographic column filled with octadecylsilane-bonded silica gel, with acetonitrile as mobile phase A and 0.02 - 0.08% phosphoric acid aqueous solution as mobile phase B;

[0010] Perform elution according to the following gradient program:

[0011]

[0012] In the gradient program, the detection wavelength from 0 to t1 is 315 - 325 nm, and then the wavelength is switched to 198 - 208 nm; t1 is 58 - 62 min;

[0013] The test sample used in the test solution includes the acid-water decoction extract containing the Shichangpu-Yuanzhi herb pair.

[0014] Preferably, in the conditions for detection by ultra - performance liquid chromatography, the column length of the chromatographic column is 150 mm, the inner diameter of the column is 2.1 mm, and the particle size is 1.6 μm;

[0015] and / or, the chromatographic column includes but is not limited to CORTECS T3;

[0016] and / or, the column temperature is 33 - 37 °C, preferably 35 °C;

[0017] and / or, the flow rate is 0.23 - 0.27 ml / min, preferably 0.25 ml / min;

[0018] and / or, the number of theoretical plates calculated by the 3,6'-di - sinapoyl - sucrose peak should be not less than 5000;

[0019] and / or, an aqueous solution of 0.05% phosphoric acid is used as mobile phase B;

[0020] and / or, the detection wavelength from 0 to 60 min in the gradient program is 320 nm, and then the wavelength is switched to 203 nm;

[0021] and / or, the gradient program further includes: in the time from 87 min to 108 min, the volume fraction of mobile phase A changes from 37% to 40%, and the volume fraction of mobile phase B changes from 63% to 60%; in the time from 108 min to 115 min, the volume fraction of mobile phase A changes from 40% to 48%, and the volume fraction of mobile phase B changes from 60% to 52%. In the present invention, after 87 min, it is mainly the information of Polygala tenuifolia Willd., and since the peaks here are not identified, the elution program for this time period may not be retained.

[0022] Preferably, the preparation process of the test solution is as follows: Take the test sample, grind it finely, weigh it accurately, add the solvent, weigh again, perform ultrasonic treatment, take it out, let it cool, weigh again, make up the lost weight with the corresponding solvent, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0023] Preferably, the test sample is the medicinal pair of Polygala tenuifolia Willd. and Acorus tatarinowii Schott;

[0024] and / or, the solvent in the test solution is methanol.

[0025] Preferably, the construction method further includes the steps of preparing a reference solution by using polygalacic acid A6, 3,6'-di - sinapoyl - sucrose, β - asarone and a solvent, and detecting the reference solution by the ultra - performance liquid chromatography method in the construction method according to any one of claims 1 - 4 to obtain a reference chromatogram.

[0026] Preferably, the preparation process of the reference substance solution is as follows: Take sibiricose A6, 3,6'-di-O-sinapoyl-sucrose, and β-asarone, weigh accurately, add a solvent to prepare a reference substance solution containing 40 μg of sibiricose A6, 85 μg of 3,6'-di-O-sinapoyl-sucrose, and 280 μg of β-asarone per 1 ml; there is no special requirement for the concentration of the reference substance solution, which is only used for peak positioning;

[0027] And / or, the solvent in the reference substance solution is methanol.

[0028] Preferably, the characteristic chromatogram of the test sample includes characteristic peaks of peak 1 to peak 23; among them, the retention times of peak 2, peak 9, and peak 21 are consistent with the chromatographic peaks of the reference substances of sibiricose A6, 3,6'-di-O-sinapoyl-sucrose, and β-asarone, respectively.

[0029] When the test sample is the acid-water decoction extract of Acori Tatarinowii-Radix Polygalae drug pair, the obtained characteristic chromatogram includes characteristic peaks of peak 1 - peak 23 and a new peak S added to the test sample solution prepared by directly using the preparation method of the test sample solution for the Acori Tatarinowii-Radix Polygalae drug pair.

[0030] Preferably, taking peak 2 as S1 peak, calculate the relative retention time of peak 1 and S1 peak;

[0031] And / or, taking peak 9 as S2 peak, calculate the relative retention times of peak 3 - peak 19 and S2 peak, and calculate the relative retention time of peak S and S2 peak;

[0032] And / or, taking peak 21 as S3 peak, calculate the relative retention times of peak 20 - peak 23 and S3 peak;

[0033] Among them, the specified values of the relative retention times of peak 1 - peak 23 and peak S are as follows:

[0034]

[0035] The relative retention time of each characteristic peak should be within the range of ±5% of the specified value.

[0036] Preferably, peak 1 is sibiricose A5; peak 2 is sibiricose A6; peak 4 is tenuifolin ketone B; peak 5 is globularic acid A; peak 6 is tenuifolin ketone XI; peak 7 is tenuifolin Ⅲ; peak 8 is polygalacin B; peak 9 is 3,6'-di-O-sinapoyl-sucrose; peak 10 is xanthoangelol A; peak 11 is polygalacin A; peak 12 is polygalacin C; peak 20 is methyl eugenol; peak 21 is β-asarone; peak 22 is γ-asarone; peak 23 is α-asarone.

[0037] In the present invention, unless otherwise specified, the percentages of the solutions are all percentages by volume.

[0038] The technical solution of the present invention has the following advantages:

[0039] A method for constructing a characteristic spectrum of a herb pair of Acorus tatarinowii and Polygala tenuifolia comprises the following steps: preparing a reference solution and a standard solution; performing ultra-high performance liquid chromatography detection on the reference solution and the test solution, and constructing a characteristic spectrum of the herb pair of Acorus tatarinowii and Polygala tenuifolia according to the measured liquid chromatogram; the conditions for the liquid chromatography detection include: using a chromatographic column with octadecylsilane bonded silica gel as a filler, acetonitrile as a mobile phase A, and 0.02-0.08% phosphoric acid aqueous solution as a mobile phase B for gradient elution; in the gradient program, the detection wavelength from 0 to t1min is 315-325nm, and then the wavelength is switched to 198-208nm; t1 is 58-62min; the invention performs elution according to a specific gradient program (95%-77% phosphoric acid aqueous phase is used for saponins, oligosaccharide esters and Separation of ketone components; 77% to 63% phosphoric acid aqueous phase for separation of asarone components in Acorus tatarinowii); the test sample used in the test solution includes an acid-boiled extract of the Acorus tatarinowii-Polygala tenuifolia drug pair. Based on the problem that the prior art lacks specific quality control of the Acorus tatarinowii-Polygala tenuifolia drug pair, the present invention uses ultra-performance liquid chromatography (UPLC) to establish a characteristic spectrum method that can comprehensively characterize the material basis components of the Acorus tatarinowii-Polygala tenuifolia drug pair that improve memory. The characteristic spectrum of the test sample should present 23 characteristic peaks and 1 newly added peak S when the test sample is the acid-boiled extract of the Acorus tatarinowii-Polygala tenuifolia drug pair, and 15 characteristic peaks (Siberian Polygala tenuifolia A5, Siberian Polygala tenuifolia A6, Polygala tenuifolia A7, and Polygala tenuifolia) are identified. Ketone B, globuloside A, Polygala tenuifolia Ketone Ⅺ, Polygala tenuifolia Ⅲ, Polygala glycoside B, 3,6'-dienasinoylsucrose, Polygala flavescens A, Polygala glycoside A, Polygala glycoside C, methyl eugenol, β-asarone, γ-asarone, α-asarone); among the 23 characteristic peaks, 19 were attributed to the characteristic peaks of Polygala tenuifolia, 4 were attributed to the characteristic peaks of Acorus tatarinowii, and 1 was attributed to the acid-boiled extract of the Acorus tatarinowii-Polygala tenuifolia drug pair. From the perspective of overall control of the Acorus tatarinowii-Polygala tenuifolia drug pair, multiple S peaks and accompanying controls were selected for evaluation, and the specified values and ranges of each characteristic peak (peak 1-peak 23 and peak S) relative to the S peak were determined, thereby achieving overall quality control of the Acorus tatarinowii-Polygala tenuifolia drug pair. Specifically, the present invention uses UPLC to achieve a more comprehensive quality control of various basic components in the drug pair by a method of switching the chromatographic detection wavelength; wherein the wavelength is switched from 315 to 325 nm to 198 to 208 nm for combination, specifically: 58 to 62 minutes before 315 to 325 nm for polygala saponins, oligosaccharide esters and Quality control of ketones, and then quality control of asarone components in Acorus tatarinowii at 198 - 208 nm (the general detection wavelength of asarone components in Acorus tatarinowii is 257 nm, and the detection wavelength of 198 - 208 nm adopted in the present invention is not the conventional detection wavelength of asarone. In the process of detecting the Acorus tatarinowii - Polygala tenuifolia drug pair by the detection wavelength of 198 - 208 nm, the chromatographic peaks of Polygala tenuifolia can be taken into account), thereby realizing the overall quality control of the Acorus tatarinowii - Polygala tenuifolia drug pair; meanwhile, the method of the present invention avoids the defects of the non - specific drug pair quality control method in the prior art, can accurately characterize the chemical components and their changes after the compatibility of the Acorus tatarinowii - Polygala tenuifolia drug pair, and thus ensures the clinical efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0041] Figure 1 is the characteristic chromatogram of the Polygala tenuifolia - Acorus tatarinowii drug pair in Example 1 of the present invention;

[0042] Figure 2 is the characteristic chromatogram of the Polygala tenuifolia - Acorus tatarinowii drug pair, the control medicinal material of Acorus tatarinowii, and the control medicinal material of Polygala tenuifolia in Example 1 of the present invention;

[0043] Figure 3 is the peak identification chromatogram of the Polygala tenuifolia - Acorus tatarinowii drug pair and its main chemical component reference substance solution in Example 1 of the present invention;

[0044] Figure 4 is the control characteristic chromatogram of the Polygala tenuifolia - Acorus tatarinowii drug pair in Example 1 and Comparative Examples 1 - 2 of the present invention;

[0045] Figure 5 is the control characteristic chromatogram when the test samples are Polygala tenuifolia medicinal material, Acorus tatarinowii medicinal material, and the Polygala tenuifolia - Acorus tatarinowii drug pair in Example 3 of the present invention;

[0046] Figure 6 is the control characteristic chromatogram when the test samples are the acid - water boiled extract of polygalacic acid, the acid - water boiled extract of Acorus tatarinowii, and the acid - water boiled extract of the Polygala tenuifolia - Acorus tatarinowii drug pair in Example 6 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0047] The following embodiments are provided to better further understand the present invention. It is not limited to the described optimal implementation manner, and does not limit the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other prior arts falls within the protection scope of the present invention.

[0048] For those embodiments where specific experimental steps or conditions are not indicated, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments whose manufacturers are not indicated, they are all conventional reagent products that can be obtained through commercial purchase.

[0049] The characteristic chromatogram method of the polygala tenuifolia - acorus tatarinowii drug pair of the present invention is determined by high performance liquid chromatography (General Principles 0512, Part IV of the Chinese Pharmacopoeia 2020 Edition).

[0050] Instruments: Waters ACQUITY UPLC H-Class ultra high performance liquid chromatograph, TUV Detector ultraviolet detector, Empower 3 chromatographic workstation; ME104E electronic balance (Mettler Toledo), JY2002 electronic balance (Mettler Toledo), KQ-500DB ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.); electronic constant temperature water bath DZKW-4 (Beijing Zhongxing Weiye Instruments Co., Ltd.).

[0051] Chromatographic column: Waters CORTECS UPLC T3 (column length is 150 mm, column inner diameter is 2.1 mm, particle size is 1.6 μm).

[0052] The test drugs are shown in Table 1 below:

[0053] Table 1

[0054]

[0055]

[0056] Reagents: Acetonitrile and phosphoric acid (Fisher Chemical) are chromatographically pure, methanol and ethanol are analytically pure, and water is purified water from Watson.

[0057] Example 1

[0058] A method for constructing the characteristic chromatogram of the polygala tenuifolia - acorus tatarinowii drug pair is as follows:

[0059] 1. Characteristic chromatogram of the test sample

[0060] 1.1. Solution preparation

[0061] Preparation of the reference substance solution:

[0062] Appropriately weigh a proper amount of polygalacin A6, 3,6'-di-O-sinapoyl-sucrose, and β-asarone reference substances, and accurately weigh them. Dissolve them in methanol to prepare a solution containing 40 μg of polygalacin A6, 85 μg of 3,6'-di-O-sinapoyl-sucrose, and 280 μg of β-asarone per 1 ml, and you will obtain the reference substance solution.

[0063] Preparation of the test solution:

[0064] Take an appropriate amount of the pair of Polygala tenuifolia and Acorus tatarinowii (the mass ratio of Polygala tenuifolia medicinal material to Acorus tatarinowii medicinal material is 1:1), grind it finely, take about 1.0 g, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of methanol, weigh it, ultrasonicate for 30 min, take it out, let it cool, make up the weight, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0065] 1.2 Chromatographic method

[0066] Detection is carried out under the following chromatographic conditions:

[0067] Use octadecylsilane chemically bonded silica as the filler (column length is 150 mm, column inner diameter is 2.1 mm, particle size is 1.6 μm); use acetonitrile as mobile phase A and 0.05% phosphoric acid aqueous solution as mobile phase B, and perform gradient elution according to the regulations in Table 2 below; the flow rate is 0.25 ml / min, the column temperature is 35 °C, the detection wavelengths are 320 nm (the wavelength is switched to 203 nm at 60 min for 320 nm), and the theoretical plate number calculated based on the peak of 3,6'-di-O-sinapoyl-sucrose should be not less than 5000.

[0068] Table 2

[0069]

[0070] Precisely pipette 1 μl each of the reference substance solution and the test solution, inject them into an ultra-high performance liquid chromatograph, and perform the determination. The characteristic chromatogram of the pair of Polygala tenuifolia and Acorus tatarinowii is shown in Figure 1 as follows.

[0071] 1.3 Attribution of characteristic peaks

[0072] Collect the pair of Polygala tenuifolia and Acorus tatarinowii and the control medicinal materials of each medicinal flavor, and obtain their liquid chromatograms according to the above method, as shown in Figure 2 as follows, and conduct the attribution of the common peaks to the medicinal flavors to determine the characteristic peaks.

[0073] According to the negative substance reference of the pair of Polygala tenuifolia and Acorus tatarinowii, perform peak attribution, among which Figure 1 peaks 1 to 19 are attributed to the cut pieces of Polygala tenuifolia, and peaks 20 to 23 are attributed to the cut pieces of Acorus tatarinowii.

[0074] 1.4 Identification of characteristic peaks

[0075] Take the test solution of the Polygala tenuifolia-Waistcoat Turmeric Rhizome medicinal pair and the reference solutions of the main chemical components, and conduct detection and comparison according to the above chromatographic conditions to determine the characteristic peaks in the characteristic chromatogram of the test solution. The identification results are shown in Figure 3 the following figure.

[0076] By comparing the characteristic peaks in the reference solution and the test solution, it was determined that peak 1 was polygala saponin A5; peak 2 was polygala saponin A6; peak 4 was tenuigenin B; peak 5 was globular adenoside A; peak 6 was tenuigenin XI; peak 7 was tenuigenin III; peak 8 was polygala glycoside B (tenuifoliside B); peak 9 was 3,6'-di-sinapoyl sucrose; peak 10 was xanthopuccine A; peak 11 was polygala glycoside A; peak 12 was polygala glycoside C; peak 20 was methyl eugenol; peak 21 was β-asarone; peak 22 was γ-asarone; peak 23 was α-asarone.

[0077] 1.5. Selection of evaluation method

[0078] From the perspective of overall control of the Polygala tenuifolia-Waistcoat Turmeric Rhizome medicinal pair, it was determined to select multiple S peaks and accompanying controls for evaluation, that is, the characteristic chromatogram of the test solution ( Figure 1 ) should show 23 characteristic peaks. Among them, peak 2, peak 9, and peak 21 should be consistent with the retention times of the reference chromatographic peaks of polygala saponin A6, 3,6'-di-sinapoyl sucrose, and β-asarone reference substances, respectively. At the detection wavelength switched from 320 nm to 203 nm, the peak corresponding to the polygala saponin A6 reference peak was the S1 peak, and the relative retention time of peak 1 and the S1 peak was calculated; the peak corresponding to the 3,6'-di-sinapoyl sucrose reference peak was the S2 peak, and the relative retention times of peaks 3-19 and the S2 peak were calculated; the peak corresponding to the β-asarone reference peak was the S3 peak, and the relative retention times of peaks 20-23 and the S3 peak were calculated; the relative retention times of each characteristic peak should be within the range of ±5% of the specified value. The specified values are shown in Table 3 below:

[0079] Table 3

[0080] Peak number 1 2 3 4 5 6 7 8 Relative retention time specified value 0.90 1.00 0.53 0.60 0.63 0.68 0.70 0.76 Peak number 9 10 11 12 13 14 15 16 Relative retention time specified value 1.00 1.02 1.09 1.29 1.34 1.41 1.53 1.59 Peak number 17 18 19 20 21 22 23 Relative retention time specified value 1.82 1.90 1.99 0.96 1.00 1.03 1.12 .

[0081] 1.6. Methodological verification

[0082] 1.6.1. Precision

[0083] According to the characteristic spectrum method established above, a sample of the Acori Tatarinowii-Radix Polygalae drug pair was taken to prepare a test solution, and it was injected continuously for 6 times to obtain a characteristic spectrum. At a detection wavelength of 203 nm switched at 320 nm, the peak corresponding to the reference peak of polygala saponin A6 was the S1 peak, and the relative retention time of peak 1 and the S1 peak was calculated; the peak corresponding to the reference peak of 3,6'-di-O-sinapoyl-sucrose was the S2 peak, and the relative retention time of peaks 3 to 19 and the S2 peak was calculated; the peak corresponding to the reference peak of β-asarone was the S3 peak, and the relative retention time of peaks 20 to 23 was calculated; and the RSD and the range from the specified value were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was between 0 and 0.3%, and the relative retention time of each characteristic peak was between -0.6% and 0.6% of the specified value, indicating that the precision of this characteristic spectrum was good. See Tables 4 and 5 for details.

[0084] Table 4 Retention Time Table for Precision Investigation of Characteristic Spectrum

[0085]

[0086]

[0087] Table 5 Table of Relative Retention Time and Specified Value Range for Precision Investigation of Characteristic Spectrum

[0088]

[0089]

[0090] 1.6.2 Repeatability

[0091] According to the characteristic spectrum method established above, 6 test solutions were prepared from samples of the Acori Tatarinowii-Radix Polygalae drug pair for injection analysis to obtain a characteristic spectrum. At a detection wavelength of 203 nm switched at 320 nm, the peak corresponding to the reference peak of polygala saponin A6 was the S1 peak, and the relative retention time of peak 1 and the S1 peak was calculated; the peak corresponding to the reference peak of 3,6'-di-O-sinapoyl-sucrose was the S2 peak, and the relative retention time of peaks 3 to 19 and the S2 peak was calculated; the peak corresponding to the reference peak of β-asarone was the S3 peak, and the relative retention time of peaks 20 to 23 was calculated; and the RSD and the range from the specified value were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was between 0 and 0.5%, and the relative retention time of each characteristic peak was between -0.6% and 0.5% of the specified value, indicating that the repeatability of this characteristic spectrum was good. See Tables 6 and 7 for details.

[0092] Table 6 Retention Time Table for Repeatability Investigation of Characteristic Spectrum

[0093]

[0094]

[0095] Table 7 Relative retention time and specified value range table for the reproducibility investigation of characteristic chromatograms

[0096]

[0097]

[0098] 1.6.3, Intermediate precision

[0099] Using Shimadzu LC-30AD and TUV detector, according to the above-established characteristic chromatogram method, six test solution samples were prepared from the Acorus tatarinowii-Schizandrae tenuifolia drug pair sample for injection analysis to obtain characteristic chromatograms. At the detection wavelength of 203 nm switched at 320 nm, the peak corresponding to the reference peak of sibiricose A6 was the S1 peak, and the relative retention time of peak 1 and the S1 peak was calculated; the peak corresponding to the reference peak of 3,6'-di-O-sinapoyl-sucrose was the S2 peak, and the relative retention time of peaks 3 to 19 and the S2 peak was calculated; the peak corresponding to the reference peak of β-asarone was the S3 peak, and the relative retention time of peaks 20 to 23 was calculated; and the RSD and the range from the specified value were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was between 0 and 0.3%, and the relative retention time of each characteristic peak was between -1.8 and 3.5% of the specified value. Comparing with the relative retention time data of each characteristic peak of the six samples in the reproducibility investigation, the RSD range of the relative retention time was 0% to 2.4%, indicating that the relative retention time of this characteristic chromatogram between different instruments met the analysis requirements. See Tables 8 and 9 for details.

[0100] Table 8 Retention time table for the intermediate precision investigation of characteristic chromatograms

[0101]

[0102]

[0103] Table 9 Relative retention time and specified value range table for the intermediate precision investigation of characteristic chromatograms

[0104]

[0105]

[0106] 1.6.4, Stability

[0107] According to the characteristic spectrum method established above, the test solution prepared from the Acori Tatarinowii-Radix Polygalae medicine pair sample was injected for analysis at 0h, 3h, 6h, 9h, 12h, 15h, and 24h respectively to obtain the characteristic spectrum. At a detection wavelength switched from 320nm to 203nm, the peak corresponding to the reference peak of polygala saponin A6 was the S1 peak, and the relative retention time of peak 1 and the S1 peak was calculated; the peak corresponding to the reference peak of 3,6'-di-O-sinapoyl-sucrose was the S2 peak, and the relative retention time of peaks 3 to 19 and the S2 peak was calculated; the peak corresponding to the reference peak of β-asarone was the S3 peak, and the relative retention time of peaks 20 to 23 was calculated; and the RSD and the range from the specified value were calculated. The results showed that the RSD of the relative retention time of each characteristic peak was between 0 and 0.2%, and the relative retention time of each characteristic peak was between -0.6% and 0.6% of the specified value, indicating that the chemical components in the solution had good stability within 24 hours. See Tables 10 and 11 for details.

[0108] Table 10 Retention Time Table for the Stability Investigation of the Characteristic Spectrum

[0109]

[0110]

[0111] Table 11 Table of the Range between the Relative Retention Time and the Specified Value for the Stability Investigation of the Characteristic Spectrum

[0112]

[0113]

[0114] That is, the standard of the characteristic spectrum for the test product of the Acori Tatarinowii-Radix Polygalae medicine pair is as follows: there should be 23 characteristic peaks in the characteristic spectrum of the test product. Among them, the retention times of peak 2, peak 9, and peak 21 should be consistent with those of the reference chromatographic peaks of polygala saponin A6, 3,6'-di-O-sinapoyl-sucrose, and β-asarone respectively. At a detection wavelength of 320 - 203nm, the peak corresponding to the reference peak of polygala saponin A6 was the S1 peak, and the relative retention time of peak 1 and the S1 peak was calculated; the peak corresponding to the reference peak of 3,6'-di-O-sinapoyl-sucrose was the S2 peak, and the relative retention time of peaks 3 to 19 and the S2 peak was calculated; the peak corresponding to the reference peak of β-asarone was the S3 peak, and the relative retention time of peaks 20 to 23 was calculated; and the RSD and the range from the specified value were calculated. The relative retention time of each characteristic peak should be within the range of ±5% of the specified value, and the specified value is shown in Table 3.

[0115] Example 2

[0116] In this example, the characteristic chromatogram of the polygala-tatarinowii rhizome drug pair was detected. The difference from Example 1 was only that 0.02% phosphoric acid aqueous solution was used as mobile phase B, and the rest was the same as in Example 1. The relative retention time of each characteristic peak was within the range of ±5% of the specified value, as shown in Table 12.

[0117] Example 3

[0118] In this example, the characteristic chromatogram of the polygala-tatarinowii rhizome drug pair was detected. The difference from Example 1 was only that 0.08% phosphoric acid aqueous solution was used as mobile phase B, and the rest was the same as in Example 1. The characteristic chromatogram was as Figure 5 shown, and the relative retention time of each characteristic peak was within the range of ±5% of the specified value, as shown in Table 12.

[0119] Example 4

[0120] In this example, the characteristic chromatogram of the polygala-tatarinowii rhizome drug pair was detected. The difference from Example 1 was only that the detection wavelength from 0 to 58 min in the gradient program was 315 nm, and then the wavelength was switched to 198 nm, and the rest was the same as in Example 1. The relative retention time of each characteristic peak was within the range of ±5% of the specified value, as shown in Table 12.

[0121] Example 5

[0122] In this example, the characteristic chromatogram of the polygala-tatarinowii rhizome drug pair was detected. The difference from Example 1 was only that the detection wavelength from 0 to 62 min in the gradient program was 325 nm, and then the wavelength was switched to 208 nm, and the rest was the same as in Example 1. The relative retention time of each characteristic peak was within the range of ±5% of the specified value, as shown in Table 12.

[0123] Table 12 Relative retention time and specified value under different chromatographic conditions

[0124]

[0125]

[0126] Example 6

[0127] Considering that the polygala-tatarinowii rhizome drug pair is mostly used by decocting with water, and at the same time, through retrieval, the medicinal flavors compatible with its drug pair are mostly acidic drugs, so it was decocted with acid water. 2 g of polygala root, 2 g of acorus tatarinowii rhizome, and 2 g of the polygala-tatarinowii rhizome drug pair were taken respectively, 30 ml of acid aqueous solution with pH adjusted to 2.0 with hydrochloric acid was added, heated under reflux for 1 hour, filtered, the filtrate was concentrated to dryness, the residue was added with an appropriate amount of methanol and fixed volume to 50 ml, weighed, sonicated for 30 min, taken out, cooled, weighed again, shaken well, filtered, and the subsequent filtrate was taken to obtain the test solution.

[0128] Detection was carried out according to the chromatographic conditions of Example 1, and the characteristic chromatograms after detection were compared. See Figure 6 , and it was found that there was one more characteristic peak in the compatibility pair of Polygala tenuifolia-WA Shi Changpu around 15 min, and the relative peak heights of some characteristic peaks in the range of 20 min - 25 min changed. This indicates that compared with decocting with acid separately for each medicinal material, decocting with acid synchronously after the compatibility of Polygala tenuifolia-WA Shi Changpu can enhance or reduce the dissolution of some components and produce new chemical substances.

[0129] Specifically, through the comparison of the characteristic chromatograms of Example 1 and Example 6 ( Figure 2 and Figure 6 ), it can be known that: when the extract obtained by decocting with acid synchronously after the compatibility of Polygala tenuifolia-WA Shi Changpu is used as the test sample, compared with directly using the medicinal pair of Polygala tenuifolia-WA Shi Changpu as the test sample, the characteristic peaks of Peak 1 - Peak 23 can be detected. At the same time, the detection method of the present invention can also synchronously detect the newly added characteristic peak - Peak S of the extract decocted with acid. At the same time, through the comparison of the relative retention peak areas of the two test samples, it can be seen that decocting with acid synchronously after the compatibility of Polygala tenuifolia-WA Shi Changpu can reduce the dissolution of the corresponding components such as Peak 14, Peak 17, Peak 19, Peak 20, Peak 21, Peak 22, Peak 23, etc. compared with the medicinal pair of Polygala tenuifolia-WA Shi Changpu, and can increase the dissolution of the corresponding components such as Peak 1, Peak 2, Peak 3, Peak 5, Peak 8, Peak 10, Peak 11, Peak 15, etc.

[0130] Comparative Example 1

[0131] The difference between this comparative example and Example 1 is that only a single wavelength (specifically 320 nm) was used for detection, and other conditions were the same as those in Example 1. The characteristic chromatogram at the detection wavelength of 320 nm is as shown in Figure 4 . When detected at a wavelength of 320 nm, the responses of the characteristic peaks in the Acorus tatarinowii Schott medicinal material were weak, and the methyl eugenol peak and γ-asarone peak could not be detected.

[0132] Comparative Example 2

[0133] The difference between this comparative example and Example 1 is that only a single wavelength (specifically 203 nm) was used for detection, and other conditions were the same as those in Example 1. The characteristic chromatogram at the detection wavelength of 203 nm is as shown in Figure 4 . When detected at a wavelength of 203 nm, the baseline noise was large, which was not conducive to the identification of characteristic peaks, and Peak S could not be detected.

[0134] Obviously, the above examples are only for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A method for constructing a characteristic fingerprint of the Acori Tatarinowii-Radix Polygalae drug pair, characterized in that, It includes the following steps: Preparation of the test solution; Performing ultra-high performance liquid chromatography (UHPLC) detection on the test solution, and constructing a characteristic chromatogram of the Acori Tatarinowii-Radix Polygalae herb pair based on the measured liquid chromatogram; The conditions for the UHPLC detection include: a chromatographic column filled with octadecylsilane-bonded silica gel, with acetonitrile as mobile phase A and an aqueous phosphoric acid solution with a concentration of 0.02 - 0.08% as mobile phase B; Elution is carried out according to the following gradient program: In the gradient program, the detection wavelength is 315 - 325 nm from 0 to t1, and then the wavelength is switched to 198 - 208 nm; t1 is 58 - 62 min; The test sample used in the test solution includes an acid-boiled extract containing the Acori Tatarinowii-Radix Polygalae herb pair.

2. The construction method according to claim 1, wherein Among the conditions for the UHPLC detection, the column length of the chromatographic column is 150 mm, the column inner diameter is 2.1 mm, and the particle size is 1.6 μm; And / or, the chromatographic column includes CORTECS T3; And / or, the column temperature is 33 - 37°C, preferably 35°C; And / or, the flow rate is 0.23 - 0.27 ml / min, preferably 0.25 ml / min; And / or, the theoretical plate number calculated based on the 3,6'-di-O-sinapoyl-sucrose peak should be not less than 5000; And / or, an aqueous phosphoric acid solution with a concentration of 0.05% is used as mobile phase B; And / or, the detection wavelength is 320 nm from 0 to 60 min in the gradient program, and then the wavelength is switched to 203 nm; And / or, the gradient program further includes: from 87 min to 108 min, the volume fraction of mobile phase A changes from 37% to 40%, and the volume fraction of mobile phase B changes from 63% to 60%; from 108 min to 115 min, the volume fraction of mobile phase A changes from 40% to 48%, and the volume fraction of mobile phase B changes from 60% to 52%.

3. The construction method according to claim 1 or 2, characterized in that, The process for preparing the test solution is as follows: Take the test sample, grind it finely, accurately weigh it, add a solvent, weigh it again, perform ultrasonic treatment, take it out, let it cool, weigh it again, make up the lost weight with the corresponding solvent, shake well, filter, and take the subsequent filtrate to obtain the test solution.

4. The construction method according to claim 3, characterized in that, The test sample is the Radix Polygalae-Acori Tatarinowii herb pair; And / or, the solvent in the test solution is methanol.

5. The construction method according to any one of claims 1-4, characterized in that, The construction method further includes the steps of preparing a reference solution by using sibiricose A6, 3,6'-di-O-sinapoyl-sucrose, and β-asarone plus a solvent, and performing UHPLC detection on the reference solution according to the UHPLC method in any one of claims 1 - 4 to obtain a reference chromatogram.

6. The construction method according to claim 5, wherein The process for preparing the reference solution is as follows: Take sibiricose A6, 3,6'-di-O-sinapoyl-sucrose, and β-asarone, accurately weigh them, and add a solvent to prepare a reference solution containing sibiricose A6, 3,6'-di-O-sinapoyl-sucrose, and β-asarone per unit volume; And / or, the solvent in the reference solution is methanol.

7. The construction method according to any one of claims 1-6, characterized in that, The characteristic spectrum of the test sample includes characteristic peaks from peak 1 to peak 23; wherein, peak 2, peak 9, and peak 21 are consistent with the retention times of the chromatographic peaks of Siberian polygala sugar A6, 3,6'-dicorinoyl sucrose, and β-asarone reference substances, respectively.

8. The construction method according to claim 7, characterized in that: When the test sample is an acid-boiled extract having the Acorus calamus-Polygala tenuifolia drug pair, the obtained characteristic spectrum includes characteristic peaks of peaks 1 to 23 and a peak S newly added to the test sample solution prepared by the test sample solution preparation method directly using the Acorus calamus-Polygala tenuifolia drug pair.

9. The construction method according to claim 8, characterized in that, Taking peak 2 as the S1 peak, calculate the relative retention time between peak 1 and S1 peak; Taking peak 9 as peak S2, calculate the relative retention time of peaks 3 to 19 and peak S2, and calculate the relative retention time of peak S and peak S2; Taking peak 21 as the S3 peak, calculate the relative retention time of peaks 20 to 23 and the S3 peak; The specified values of the relative retention times of peaks 1 to 23 and peak S are as follows: The relative retention time of each characteristic peak should be within ±5% of the specified value.

10. The construction method according to claim 9, characterized in that Peak 1 is polygalacic acid A5; Peak 2 is polygalacic acid A6; Peak 4 is tenuifolin Ketone B; Peak 5 is globularicoside A; Peak 6 is tenuifolin Ketone XI; Peak 7 is tenuifolin III; Peak 8 is polygalacin B; Peak 9 is 3,6'-di-O-sinapoyl-sucrose; Peak 10 is polygalaflavone A; Peak 11 is polygalacin A; Peak 12 is polygalacin C; Peak 20 is methyl eugenol; Peak 21 is β-asarone; Peak 22 is γ-asarone; Peak 23 is α-asarone.

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