A method for constructing a characteristic spectrum of the Acorus tatarinowii-Polygala tenuifolia herb pair

Abstract

This invention relates to the field of traditional Chinese medicine detection technology, specifically to a method for constructing a characteristic chromatogram of the Acorus tatarinowii-Polygala tenuifolia herb pair, comprising the following steps: preparation of a test solution; detection of the test solution by ultra-high performance liquid chromatography (UHPLC); and construction of a characteristic chromatogram of the Acorus tatarinowii-Polygala tenuifolia herb pair based on the obtained HPLC chromatogram; the conditions for UHPLC detection include: a chromatographic column packed with octadecylsilane-bonded silica gel, acetonitrile as mobile phase A, and 0.02-0.08% phosphoric acid aqueous solution as mobile phase B; elution according to a specific gradient program; the detection wavelength from 0 to t1 in the gradient program is 315-325 nm, and then the wavelength is switched to 198-208 nm; t1 is 58-62 min. Through the method for constructing the characteristic chromatogram of the Acorus tatarinowii-Polygala tenuifolia herb pair of this invention, the characteristic chromatogram of the test sample should present 23 characteristic peaks, with 15 characteristic peaks identified, thus achieving quality control of the Acorus tatarinowii-Polygala tenuifolia herb pair.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine detection, and particularly relates to a construction method of a characteristic spectrum of Acorus tatarinowii Rhizoma-Polygalae Radix. BACKGROUND

[0002] Acorus tatarinowii Schott is a dry rhizome of Acorus tatarinowii Schott in Araceae, which is warm in nature, bitter and acrid in taste, and belongs to heart and stomach channels. It has the effects of opening orifice and resolving phlegm, refreshing and invigorating the mind, and eliminating dampness and promoting appetite. Polygala tenuifolia Willd is a dry root of Polygala tenuifolia Willd or Polygala sibirica L in Polygalaceae, which is warm in nature, bitter and acrid in taste, and belongs to heart, kidney and lung channels. It has the effects of soothing the mind, communicating between heart and kidney, resolving phlegm and eliminating swelling. Acorus tatarinowii Rhizoma-Polygalae Radix (AP) is a commonly used Chinese medicine pair for invigorating the mind in clinical practice. Both of them are first recorded in Shennong's Herbal Classic. They are used together to treat "forgetfulness" and other diseases. Previous studies have found that Acorus tatarinowii Rhizoma-Polygalae Radix is the most frequently used pair of Chinese medicine in the treatment of senile dementia. The main material basis for improving memory of Acorus tatarinowii is volatile phenylpropanoid components such as asarinin, methyl eugenol, etc. The main material basis for improving memory of Polygala tenuifolia is polygalasaponins, oligosaccharide esters, ketones, etc. The combination of the two drugs usually has a pharmacological effect of "1+1>2". However, the chemical components of the medicine obtained by decocting under the acidic condition are changed in quality and quantity. The reason may be that the new chemical substances produced by the two drugs are more easily absorbed by the body than the original medicine, or the new components produced by the two drugs promote the absorption of the original chemical components in the body, thereby exerting the pharmacological effect of "1+1>2". Therefore, it is particularly important to comprehensively and efficiently control the quality of various effective components of Acorus tatarinowii Rhizoma-Polygalae Radix decocted under acidic conditions to ensure the clinical efficacy. Ketones, etc. The combination of the two drugs usually has a pharmacological effect of "1+1>2". However, the chemical components of the medicine obtained by decocting under the acidic condition are changed in quality and quantity. The reason may be that the new chemical substances produced by the two drugs are more easily absorbed by the body than the original medicine, or the new components produced by the two drugs promote the absorption of the original chemical components in the body, thereby exerting the pharmacological effect of "1+1>2". Therefore, it is particularly important to comprehensively and efficiently control the quality of various effective components of Acorus tatarinowii Rhizoma-Polygalae Radix decocted under acidic conditions to ensure the clinical efficacy.

[0003] Characteristic chromatograms of traditional Chinese medicine (TCM) are an effective means of evaluating the overall quality of TCM compound preparations. Their advantage lies in their ability to comprehensively characterize the component characteristics of all herbs in the prescription, and they are widely used in the qualitative and quantitative analysis of TCM and its compound preparations. Currently, there is no specific quality control method for the *Acorus tatarinowii*-*Polygala tenuifolia* herb pair. Only in the quality reference studies of *Kanxin San* containing the *Acorus tatarinowii*-*Polygala tenuifolia* herb pair are there quality control methods that mostly employ HPLC to establish detection methods for one or more components in combination. However, these currently published detection methods all have significant shortcomings. The main shortcoming is that the control of the active ingredients can only characterize the individual chemical components of the *Acorus tatarinowii*-*Polygala tenuifolia* herb pair before combination, and cannot accurately characterize the chemical components that change after combination. Therefore, for the *Acorus tatarinowii*-*Polygala tenuifolia* herb pair, which has complex pharmacological effects in vivo and undergoes chemical changes after combination, a specific, comprehensive, rapid, and accurate quality control method is needed, but no relevant reports have been found yet. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the deficiency of the prior art in lacking specific quality control of chemical components that undergo chemical composition changes after the combination of Acorus tatarinowii and Polygala tenuifolia, thereby providing a method for constructing a characteristic spectrum of the Acorus tatarinowii-Polygala tenuifolia drug pair to solve the above problem.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for constructing a characteristic spectrum of the Acorus tatarinowii-Polygala tenuifolia herb pair includes the following steps:

[0007] Preparation of the test solution;

[0008] The test solution was analyzed by ultra-high performance liquid chromatography, and a characteristic chromatogram of the Acorus tatarinowii-Polygala tenuifolia drug pair was constructed based on the measured liquid chromatogram.

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

[0010] Elution should be performed according to the following gradient procedure:

[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 sample solution includes an acid-water boiled extract of Acorus tatarinowii and Polygala tenuifolia.

[0014] Preferably, in the ultra-high performance liquid chromatography (UHPLC) detection conditions, the column length is 150 mm, the inner diameter 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 to 0.27 ml / min, preferably 0.25 ml / min;

[0018] And / or, the theoretical plate number calculated based on the 3,6'-disinyl sucrose peak should be no less than 5000;

[0019] And / or, using a 0.05% aqueous phosphoric acid solution as mobile phase B;

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

[0021] And / or, the gradient program further includes: changing the volume fraction of mobile phase A from 37% to 40% and the volume fraction of mobile phase B from 63% to 60% between 87 min and 108 min; and changing the volume fraction of mobile phase A from 40% to 48% and the volume fraction of mobile phase B from 60% to 52% between 108 min and 115 min. In this invention, the information after 87 min is mainly from Polygala tenuifolia, and since the peak is not identified here, the elution program for this time period can be omitted.

[0022] Preferably, the preparation process of the test solution is as follows: take the test sample, grind it finely, weigh it accurately, add solvent, weigh it, sonicate it, take it out, cool it, weigh it again, replenish the lost weight with the corresponding solvent, shake it well, filter it, and take the filtrate to obtain the test solution.

[0023] Preferably, the test sample is a pair of Polygala tenuifolia and Acorus tatarinowii herbs;

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

[0025] Preferably, the construction method further includes the step of preparing a reference solution by adding solvents to Siberian polygalactosyl sucrose A6, 3,6'-disinyl sucrose, and β-asarone, and the step of obtaining a reference chromatogram by detecting the reference solution using ultra-high performance liquid chromatography according to any one of the construction methods of claims 1-4.

[0026] Preferably, the preparation process of the reference solution is as follows: Siberian polygalactosyl sucrose A6, 3,6'-disinyl sucrose, and β-asarone are accurately weighed and added to a solvent to prepare a reference solution containing 40 μg of Siberian polygalactosyl sucrose A6, 85 μg of 3,6'-disinyl sucrose, and 280 μg of β-asarone per 1 ml; the concentration of the reference solution is not particularly required, but is only used for peak localization;

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

[0028] Preferably, the characteristic chromatogram of the test sample includes characteristic peaks 1 to 23; wherein, peaks 2, 9, and 21 are consistent with the retention times of the chromatographic peaks of Siberian polygalactosyl sugar A6, 3,6'-disinyl sucrose, and β-asarone references, respectively.

[0029] When the test sample is an acid-water boiled extract of the Acorus tatarinowii-Polygala tenuifolia drug pair, the obtained characteristic spectrum includes characteristic peaks 1-23 and peak S, which is newly added compared to the test sample solution prepared directly using the test sample solution preparation method of the Acorus tatarinowii-Polygala tenuifolia drug pair.

[0030] Preferably, peak 2 is taken as peak S1, and the relative retention time of peak 1 and peak S1 is calculated;

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

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

[0033] The specified values ​​for the relative retention times of peaks 1 to 23 and peak S are as follows:

[0034]

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

[0036] Preferably, peak 1 is Siberian polygalactosyl glycoside A5; peak 2 is Siberian polygalactosyl glycoside A6; and peak 4 is polygalactosyl glycoside. Ketone B; Peak 5 is globulin A; Peak 6 is Polygala tenuifolia. Ketone XI; Peak 7 is Polygala tenuifolia. III; Peak 8 is polygalactoside B; Peak 9 is 3,6'-disinyl sucrose; Peak 10 is polygalactoside A; Peak 11 is polygalactoside A; Peak 12 is polygalactoside C; Peak 20 is methyl eugenol; Peak 21 is β-asarone; Peak 22 is γ-asarone; Peak 23 is α-asarone.

[0037] In this invention, unless otherwise specified, all percentages in the solution are volume percentages.

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

[0039] A method for constructing a characteristic chromatogram of the Acorus tatarinowii-Polygala tenuifolia herb pair includes the following steps: preparation of a reference solution and a standard solution; ultra-high performance liquid chromatography (UHPLC) detection of the reference solution and the test solution; and construction of a characteristic chromatogram of the Acorus tatarinowii-Polygala tenuifolia herb pair based on the measured HPLC chromatograms; the HPLC detection conditions include: a chromatographic column packed with octadecylsilane-bonded silica gel, gradient elution with acetonitrile as mobile phase A and 0.02-0.08% phosphoric acid aqueous solution as mobile phase B; in the gradient program, the detection wavelength from 0 to t1 min is 315-325 nm, then the wavelength is switched to 198-208 nm; t1 is 58-62 min; the present invention uses a specific gradient program for elution (95%-77% phosphoric acid aqueous phase targets saponins, oligosaccharide esters, and...) in Polygala tenuifolia. Separation of ketone components; separation of asarone components from Acorus tatarinowii using 77%–63% phosphate aqueous phase; the test sample used in the test sample solution includes an acid-water decoction extract of the Acorus tatarinowii-Polygala tenuifolia herb pair. Based on the lack of specific quality control for the Acorus tatarinowii-Polygala tenuifolia herb pair in existing technologies, this invention uses ultra-high performance liquid chromatography (UPLC) to establish a characteristic chromatographic method that can comprehensively characterize the memory-improving material basis components in the Acorus tatarinowii-Polygala tenuifolia herb pair. The characteristic chromatogram of the test sample should show 23 characteristic peaks and one additional peak S when the test sample is the acid-water decoction extract of the Acorus tatarinowii-Polygala tenuifolia herb pair, identifying 15 characteristic peaks (namely Siberian polygala tenuifolia A5, Siberian polygala tenuifolia A6, and Polygala tenuifolia A5). Ketone B, Glucosinolate A, Polygala tenuifolia Ketone XI, Polygala tenuifolia III. Polygala glycoside B, 3,6'-disinyl sucrose, polygalain A, polygala glycoside A, polygala glycoside C, methyl eugenol, β-asarone, γ-asarone, α-asarone; Of the 23 characteristic peaks, 19 belong to the medicinal characteristics of Polygala tenuifolia, 4 belong to the medicinal characteristics of Acorus tatarinowii, and 1 belongs to the characteristic peak of the acid-water decoction extract of the Acorus tatarinowii-Polygala tenuifolia medicinal pair. From the perspective of controlling the overall quality of the Acorus tatarinowii-Polygala tenuifolia medicinal pair, multiple S peaks and accompanying controls were selected for evaluation. 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 medicinal pair. Specifically, this invention employs UPLC (Ultra-Ultra-Lipochromatographic Chromatography) with a wavelength-changing chromatographic detection method to achieve comprehensive quality control of various basic components in drug pairs. The wavelengths are combined by switching between 315-325 nm and 198-208 nm. Specifically, 315-325 nm is used before 58-62 minutes to target polygala saponins, oligosaccharide esters, and... The quality control of ketones is followed by quality control of asarone components in Acorus tatarinowii at 198–208 nm (the general detection wavelength for asarone components in Acorus tatarinowii is 257 nm; the 198–208 nm detection wavelength used in this invention is not the conventional detection wavelength for asarone; this invention uses the 198–208 nm detection wavelength to take into account the chromatographic peak of Polygala tenuifolia during the detection of the Acorus tatarinowii-Polygala tenuifolia drug pair), thereby achieving overall quality control of the Acorus tatarinowii-Polygala tenuifolia drug pair; at the same time, the method of this invention avoids the defects of non-specific drug pair quality control methods in the prior art, and can accurately characterize the chemical components and their changes after the Acorus tatarinowii-Polygala tenuifolia drug pair is combined, thereby ensuring clinical efficacy. Attached Figure Description

[0040] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0041] Figure 1 This is a characteristic spectrum of the Polygala tenuifolia-Acorus tatarinowii herb pair in Example 1 of the present invention;

[0042] Figure 2 These are the characteristic spectra of the Polygala tenuifolia-Acorus tatarinowii herb pair, the Acorus tatarinowii reference herb, and the Polygala tenuifolia reference herb in Example 1 of this invention;

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

[0044] Figure 4 These are comparative characteristic chromatograms of the Polygala tenuifolia-Acorus tatarinowii herb pairs in Example 1 and Comparative Examples 1-2 of the present invention;

[0045] Figure 5 This is a comparative characteristic spectrum of the test samples in Example 3 of the present invention when the test samples are Polygala tenuifolia, Acorus tatarinowii, or the Polygala tenuifolia-Acorus tatarinowii pair;

[0046] Figure 6 This is a comparative characteristic chromatogram of the test samples in Example 6 of the present invention when the test samples are the acid-boiled extracts of Polygala tenuifolia, Acorus tatarinowii, and the acid-boiled extracts of the Polygala tenuifolia-Acorus tatarinowii herb pair. Detailed Implementation

[0047] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0048] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0049] The characteristic chromatographic method of the Polygala tenuifolia-Acorus tatarinowii herb pair of the present invention is determined by high performance liquid chromatography (Chinese Pharmacopoeia 2020 edition, Part IV, General Chapter 0512).

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

[0051] Chromatographic column: Waters CORTECS UPLC T3 (column length 150 mm, column inner diameter 2.1 mm, particle size 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) were chromatographic grade; methanol and ethanol were analytical grade; and water was Watson's purified water.

[0057] Example 1

[0058] A method for constructing the characteristic spectrum of the Polygala tenuifolia-Acorus tatarinowii herb pair, the specific process of which is as follows:

[0059] 1. Characteristic chromatogram of the test sample

[0060] 1.1 Solution Preparation

[0061] Preparation of reference solution:

[0062] Accurately weigh appropriate amounts of Siberian polygalactoside A6, 3,6'-disinyl sucrose, and β-asarone reference standards, and add methanol to prepare a solution containing 40 μg of Siberian polygalactoside A6, 85 μg of 3,6'-disinyl sucrose, and 280 μg of β-asarone per 1 ml. This is the reference solution.

[0063] Preparation of test solution:

[0064] Take an appropriate amount of the Polygala tenuifolia-Acorus tatarinowii herb pair (Polygala tenuifolia herb: Acorus tatarinowii herb = 1:1, mass ratio), grind it into a fine powder, take about 1.0g, accurately weigh it, place it in a stoppered conical flask, accurately add 25ml of methanol, weigh it, sonicate for 30min, take it out, let it cool, add the weight, shake it well, filter it, and take the filtrate to obtain the test solution.

[0065] 1.2 Chromatographic Methods

[0066] The following chromatographic conditions were used for detection:

[0067] The column was packed with octadecylsilane and silica gel (column length 150 mm, column inner diameter 2.1 mm, particle size 1.6 μm); acetonitrile was used as mobile phase A and 0.05% phosphoric acid aqueous solution was used as mobile phase B, and gradient elution was performed according to the specifications in Table 2; the flow rate was 0.25 ml / min, the column temperature was 35 ℃, and the detection wavelength was 320 nm (320 nm was switched to 203 nm at 60 min). The theoretical plate number, calculated based on the 3,6'-disinyl sucrose peak, should not be less than 5000.

[0068] Table 2

[0069]

[0070] Accurately pipette 1 μl each of the reference solution and the test solution into the ultra-high performance liquid chromatograph and determine the results. The characteristic chromatogram of the Polygala tenuifolia-Acorus tatarinowii herb pair is shown below. Figure 1 As shown.

[0071] 1.3 Assignment of Characteristic Peaks

[0072] Collect the medicinal pair of Polygala tenuifolia and Acorus tatarinowii, as well as the reference medicinal materials for each herb, and obtain their liquid chromatography spectra according to the above method, such as... Figure 2 As shown, the common peaks were assigned medicinal properties, and characteristic peaks were determined.

[0073] Peak attribution was performed based on the negative substance benchmark of the Polygala tenuifolia-Acorus tatarinowii herbal pair, among which... Figure 1 Peaks 1 to 19 are classified as Polygala tenuifolia slices, and peaks 20 to 23 are classified as Acorus tatarinowii slices.

[0074] 1.4 Identification of Characteristic Peaks

[0075] The test solution of Polygala tenuifolia-Acorus tatarinowii herb pair and the reference solution of its main chemical components were analyzed and compared under 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 As shown.

[0076] By comparing the characteristic peaks in the reference and test solutions, peak 1 was identified as Siberian polygalactosyl sugar A5; peak 2 as Siberian polygalactosyl sugar A6; and peak 4 as polygalactosyl sugar. Ketone B; Peak 5 is globulin A; Peak 6 is Polygala tenuifolia. Ketone XI; Peak 7 is Polygala tenuifolia. Ketone III; Peak 8 is polygalactoside B (polygalactoside B); Peak 9 is 3,6'-disinyl sucrose; Peak 10 is polygalactoside A; Peak 11 is polygalactoside A; Peak 12 is polygalactoside C; Peak 20 is methyl eugenol; Peak 21 is β-asarone; Peak 22 is γ-asarone; Peak 23 is α-asarone.

[0077] 1.5 Selection of Evaluation Method

[0078] From the perspective of holistic control of the Polygala tenuifolia-Acorus tatarinowii herb pair, the multi-S peak and accompanying control were selected for evaluation, i.e., the characteristic chromatogram of the test sample ( Figure 1 The chromatogram should show 23 characteristic peaks, among which peaks 2, 9, and 21 should correspond to the retention times of the reference peaks of Siberian polygalactosyl sucrose A6, 3,6'-disinyl sucrose, and β-asarone, respectively. At a detection wavelength of 320 nm switched to 203 nm, the peak corresponding to the Siberian polygalactosyl sucrose A6 reference peak is designated as peak S1, and the relative retention time of peak 1 with peak S1 is calculated; the peak corresponding to the 3,6'-disinyl sucrose reference peak is designated as peak S2, and the relative retention times of peaks 3–19 with peak S2 are calculated; the peak corresponding to the β-asarone reference peak is designated as peak S3, and the relative retention times of peaks 20–23 with peak S3 are calculated. The relative retention times of each characteristic peak should be within ±5% of the specified values. 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 Validation

[0082] 1.6.1 Precision

[0083] Following the established characteristic spectral method, a test solution was prepared from a sample of *Acorus tatarinowii* and *Polygala tenuifolia*. The sample was injected six times consecutively to obtain characteristic spectra. At a detection wavelength of 320 nm switched to 203 nm, the peak corresponding to the Siberian polygala tenuifolia A6 reference peak was designated as peak S1, and the relative retention time of peak 1 was calculated. The peak corresponding to the 3,6'-disinoyl sucrose reference peak was designated as peak S2, and the relative retention times of peaks 3 to 19 were calculated. The peak corresponding to the β-asarone reference peak was designated as peak S3, and the relative retention times of peaks 20 to 23 were calculated. The RSD and its range relative to the specified values ​​were also calculated. The results showed that the RSD of each characteristic peak was between 0% and 0.3%, and the relative retention times of each characteristic peak were between -0.6% and 0.6% of the specified value, indicating good precision of the characteristic spectral method. See Tables 4 and 5 for details.

[0084] Table 4. Retention Schedule for Precision Examination of Feature Maps

[0085]

[0086]

[0087] Table 5. Relative Retention Time and Specified Range for Precision Examination of Feature Maps

[0088]

[0089]

[0090] 1.6.2 Repeatability

[0091] Following the established characteristic chromatogram method, six test solutions were prepared from the *Acorus tatarinowii*-*Polygala tenuifolia* herb pair and analyzed to obtain characteristic chromatograms. At a detection wavelength of 320 nm switched to 203 nm, the peak corresponding to the Siberian polygala tenuifolia A6 reference peak was designated as peak S1, and the relative retention time of peak 1 was calculated relative to peak S1. The peak corresponding to the 3,6'-disinoyl sucrose reference peak was designated as peak S2, and the relative retention times of peaks 3–19 relative to peak S2 were calculated. The peak corresponding to the β-asarone reference peak was designated as peak S3, and the relative retention times of peaks 20–23 were calculated. The RSD and its range relative to the specified values ​​were also calculated. The results showed that the RSD of each characteristic peak was between 0% and 0.5%, and the relative retention times of each characteristic peak were between -0.6% and 0.5% of the specified value, indicating good repeatability of the characteristic chromatogram. See Tables 6 and 7 for details.

[0092] Table 6. Timetable for Retention of Feature Map Repeatability Examination

[0093]

[0094]

[0095] Table 7. Relative Retention Time and Specified Value Range for Repeatability Testing of Characteristic Spectra

[0096]

[0097]

[0098] 1.6.3 Intermediate Precision

[0099] Using a Shimadzu LC-30AD with a TUV detector, and following the established characteristic spectral method, six test solutions of the *Acorus tatarinowii*-*Polygala tenuifolia* herb pair were prepared and analyzed to obtain characteristic spectra. At a detection wavelength of 320 nm switched to 203 nm, the peak corresponding to the Siberian polygala tenuifolia A6 reference peak was designated as peak S1, and the relative retention time of peak 1 was calculated. The peak corresponding to the 3,6'-disinoyl sucrose reference peak was designated as peak S2, and the relative retention times of peaks 3 to 19 were calculated. The peak corresponding to the β-asarone reference peak was designated as peak S3, and the relative retention times of peaks 20 to 23 were calculated. The RSD and its range relative to the specified values ​​were also calculated. The results showed that the relative retention time RSD 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. Compared with the relative retention time data of each characteristic peak of the six samples in the repeatability test, the relative retention time RSD ranged from 0% to 2.4%, indicating that the relative retention time of the characteristic spectrum met the analytical requirements in different instruments. See Tables 8 and 9 for details.

[0100] Table 8. Timetable for Retention of Intermediate Precision Examination of Feature Maps

[0101]

[0102]

[0103] Table 9. Relative Retention Time and Specified Range for Intermediate Precision Examination of Feature Maps

[0104]

[0105]

[0106] 1.6.4 Stability

[0107] Following the established characteristic chromatographic method, the test solution prepared from the *Acorus tatarinowii*-*Polygala tenuifolia* herb pair was analyzed at 0h, 3h, 6h, 9h, 12h, 15h, and 24h to obtain characteristic chromatograms. At a detection wavelength of 320nm switched to 203nm, the peak corresponding to the Siberian polygala tenuifolia A6 reference peak was designated as peak S1, and the relative retention time of peak 1 was calculated. The peak corresponding to the 3,6'-disinoyl sucrose reference peak was designated as peak S2, and the relative retention times of peaks 3–19 were calculated. The peak corresponding to the β-asarone reference peak was designated as peak S3, and the relative retention times of peaks 20–23 were calculated. The RSD and its range relative to the specified values ​​were also calculated. The results showed that the RSD of each characteristic peak was between 0% and 0.2%, and the relative retention times of each characteristic peak were between -0.6% and 0.6% of the specified value, indicating good stability of the chemical components in the solution over 24 hours. See Tables 10 and 11 for details.

[0108] Table 10: Timetable for Retention of Characteristic Map Stability Assessment

[0109]

[0110]

[0111] Table 11: Relative Retention Time and Specified Range for Stability Assessment of Characteristic Spectra

[0112]

[0113]

[0114] The characteristic chromatogram standard for the test sample being the *Acorus tatarinowii*-*Polygala tenuifolia* drug pair is as follows: the characteristic chromatogram of the test sample should have 23 characteristic peaks, among which peaks 2, 9, and 21 should correspond to the retention times of the reference peaks of Siberian polygala tenuifolia A6, 3,6'-disinoyl sucrose, and β-asarone, respectively. At a detection wavelength of 320-203 nm, the peak corresponding to the Siberian polygala tenuifolia A6 reference peak is designated as peak S1, and the relative retention time of peak 1 to peak S1 is calculated; the peak corresponding to the 3,6'-disinoyl sucrose reference peak is designated as peak S2, and the relative retention times of peaks 3 to 19 to peak S2 are calculated; the peak corresponding to the β-asarone reference peak is designated as peak S3, and the relative retention times of peaks 20 to 23 are calculated; and the RSD and the range relative to the specified values ​​are calculated. The relative retention times of each characteristic peak should be within ±5% of the specified values, as shown in Table 3.

[0115] Example 2

[0116] This embodiment detects the characteristic chromatograms of the Polygala tenuifolia-Acorus tatarinowii herb pair. The only difference between this embodiment and Example 1 is that a 0.02% phosphoric acid aqueous solution was used as mobile phase B; otherwise, they are identical. The relative retention times of each characteristic peak are within ±5% of the specified values, as shown in Table 12.

[0117] Example 3

[0118] This embodiment detects the characteristic chromatograms of the Polygala tenuifolia-Acorus tatarinowii herb pair. The only difference from Example 1 is the use of 0.08% phosphoric acid aqueous solution as mobile phase B; all other aspects are identical to Example 1. The characteristic chromatograms are as follows: Figure 5 As shown in Table 12, the relative retention times of each characteristic peak are within ±5% of the specified value.

[0119] Example 4

[0120] This embodiment detects the characteristic spectra of the Polygala tenuifolia-Acorus tatarinowii herb pair. The only difference from Example 1 is that the detection wavelength in the gradient program is 315 nm from 0 to 58 min, and then the wavelength is switched to 198 nm. The rest is the same as in Example 1. The relative retention times of each characteristic peak are within ±5% of the specified value, as shown in Table 12.

[0121] Example 5

[0122] This embodiment detects the characteristic spectra of the Polygala tenuifolia-Acorus tatarinowii herb pair. The only difference from Example 1 is that the detection wavelength is 325 nm for the first 62 minutes in the gradient program, and then the wavelength is switched to 208 nm. The rest is the same as in Example 1. The relative retention times of each characteristic peak are within ±5% of the specified value, as shown in Table 12.

[0123] Table 12 Relative retention times and specified values ​​under different chromatographic conditions

[0124]

[0125]

[0126] Example 6

[0127] Considering that the Polygala tenuifolia-Acorus tatarinowii herbal pair is mostly used by decoction, and that most of the herbs that can be paired with this herbal pair are acidic, an acid decoction was performed. 2g each of Polygala tenuifolia, Acorus tatarinowii, and the Polygala tenuifolia-Acorus tatarinowii herbal pair were added to 30ml of an acidic aqueous solution with the pH adjusted to 2.0 using hydrochloric acid. The mixture was heated under reflux for 1 hour, filtered, and the filtrate was concentrated to dryness. An appropriate amount of methanol was added to the residue, and the volume was adjusted to 50ml. The residue was weighed, sonicated for 30 minutes, removed, cooled, weighed again, shaken well, filtered, and the filtrate was collected to obtain the test solution.

[0128] The detection was performed according to the chromatographic conditions of Example 1, and the characteristic chromatograms after detection were compared. Figure 6 It was found that around 15 minutes, an additional characteristic peak appeared in the combination of Polygala tenuifolia and Acorus tatarinowii, and the relative peak height of some characteristic peaks changed between 20 and 25 minutes. This indicates that simultaneous acid decoction of Polygala tenuifolia and Acorus tatarinowii after combination can enhance or reduce the dissolution of some components and generate new chemical substances compared to acid decoction of individual herbs.

[0129] Specifically, through the feature maps of Examples 1 and 6 ( Figure 2 and Figure 6 The comparison shows that the extract obtained by simultaneous acid decoction of Polygala tenuifolia and Acorus tatarinowii as test samples, compared with the direct use of the Polygala tenuifolia-Acorus tatarinowii drug pair as test samples, can detect the characteristic peaks 1-23. At the same time, the detection method of the present invention can also simultaneously detect the newly added characteristic peak - peak S of the acid-decoction extract. Furthermore, by comparing the relative retention peak areas of the two test samples, it can be seen that simultaneous acid decoction of Polygala tenuifolia and Acorus tatarinowii as test samples can reduce the dissolution of corresponding components such as peaks 14, 17, 19, 20, 21, 22, and 23, and increase the dissolution of corresponding components such as peaks 1, 2, 3, 5, 8, 10, 11, and 15, compared with the Polygala tenuifolia-Acorus tatarinowii drug pair.

[0130] Comparative Example 1

[0131] The difference between this comparative example and Example 1 is that only a single wavelength (specifically 320nm) is used for detection; all other conditions are the same as in Example 1. The characteristic spectrum of the 320nm detection wavelength is as follows: Figure 4 As shown, when detected at a wavelength of 320 nm, the characteristic peaks in Acorus tatarinowii showed weak responses, 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) is used for detection, while other conditions are the same as in Example 1. The characteristic spectrum of the 203 nm detection wavelength is as follows: Figure 4 As shown, when a 203nm wavelength is used for detection, the baseline noise is relatively large, which is not conducive to the identification of characteristic peaks and the peak S cannot be detected.

[0134] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for constructing a characteristic chromatogram of a Acorus calamus-Gynostemma pentaphyllum medicinal pair, characterized in that, The method comprises the following steps: Preparation of the test sample solution and the reference solution; The test sample solution and the reference solution are detected by the ultra-high performance liquid chromatography, the characteristic chromatogram of the Acori graminei and Polygalae couplet is constructed according to the liquid chromatogram of the test sample solution, and the reference chromatogram is constructed according to the liquid chromatogram of the reference solution; The detection condition of the ultra-high performance liquid chromatography comprises: taking acetonitrile as the mobile phase A and taking 0.02-0.08% phosphoric acid aqueous solution as the mobile phase B; The elution is carried out according to the following gradient program: The detection wavelength is 315-325 nm in the gradient program from 0 to t1, and then the wavelength is switched to 198-208 nm; the t1 is 58-62 min; The test sample used in the test sample solution is the acid water boiled extract of the Acori graminei and Polygalae couplet or the Polygalae and Acori graminei couplet. In the detection condition of the ultra-high performance liquid chromatography, 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; the chromatographic column is CORTECS T3. The preparation process of the test sample solution is as follows: the test sample is finely ground, accurately weighed, and then the solvent is added and weighed, and the weight is determined again after ultrasonic treatment, cooling, and weighing again. The lost weight is made up with the corresponding solvent, shaken, filtered, and the filtered solution is obtained, which is the test sample solution; the solvent in the test sample solution is methanol. The preparation process of the reference solution is as follows: Siberian Polygalae sugar A6, 3,6'-di mustard acyl sucrose, and β-asarone are dissolved in the solvent to prepare the reference solution.

2. The construction method of claim 1, wherein, The column temperature is 33-37 ℃; And / or, the flow rate is 0.23-0.27 ml / min; And / or, the theoretical plate number calculated according to the 3,6'-di mustard acyl sucrose peak should be not less than 5000; And / or, the mobile phase B is 0.05% phosphoric acid aqueous solution; And / or, the detection wavelength is 320 nm in the gradient program from 0 to 60 min, and then the wavelength is switched to 203 nm; And / or, the gradient program further comprises: in the time from 87 min to 108 min, the volume fraction of the mobile phase A changes from 37% to 40%, and the volume fraction of the mobile phase B changes from 63% to 60%; in the time from 108 min to 115 min, the volume fraction of the mobile phase A changes from 40% to 48%, and the volume fraction of the mobile phase B changes from 60% to 52%.

3. The construction method of claim 2, wherein, The column temperature is 35 ℃; And / or, the flow rate is 0.25 ml / min.

4. The construction method of claim 1, wherein, The preparation process of the reference solution is as follows: Siberian Polygalae sugar A6, 3,6'-di mustard acyl sucrose, and β-asarone are accurately weighed, and then the solvent is added to prepare the reference solution containing Siberian Polygalae sugar A6, 3,6'-di mustard acyl sucrose, and β-asarone. And / or, the solvent in the reference solution is methanol.

5. The construction method according to any one of claims 1 to 4, characterized in that, The characteristic peaks of peaks 1-23 are included in the characteristic chromatogram of the test sample; wherein, the retention time of peaks 2, 9, and 21 is consistent with that of the chromatographic peak of the Siberian Polygalae sugar A6, 3,6'-di mustard acyl sucrose, and β-asarone reference.

6. The construction method according to claim 5, characterized in that, When the test sample is the acid water decoction extract of the Acori tatarinowii Radix et Rhizoma-Gensing Radix et Rhizoma medicinal pair, the characteristic peaks of peaks 1-23 are included in the obtained characteristic spectrum, and peak S is newly added to the test sample solution compared with the test sample solution prepared by directly using the preparation method of the Acori tatarinowii Radix et Rhizoma-Gensing Radix et Rhizoma medicinal pair.

7. The construction method of claim 6, wherein, Take peak 2 as S1 peak, and calculate the relative retention time of peak 1 and S1 peak. Take peak 9 as S2 peak, and calculate the relative retention time of peaks 3-19 and S2 peak, and calculate the relative retention time of peak S and S2 peak. Take peak 21 as S3 peak, and calculate the relative retention time of peaks 20-23 and S3 peak. The relative retention time of peaks 1-23 and peak S is as follows: The relative retention time of each characteristic peak should be within ±5% of the specified value.

8. The construction method according to claim 7, characterized in that, Peak 1 is polygalaxyl A5; peak 2 is polygalaxyl A6; peak 4 is polygalaxyl ketone B; peak 5 is globosidoside A; peak 6 is polygalaxyl ketone XI; peak 7 is polygalaxyl III; peak 8 is polygalaxyl glycoside B; peak 9 is 3,6'-diacylsucrose; peak 10 is polyganaconitine A; peak 11 is polygalaxyl glycoside A; peak 12 is polygalaxyl glycoside C; peak 20 is methyl eugenol; peak 21 is β-asarone; peak 22 is γ-asarone; and peak 23 is α-asarone.

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