Method for constructing characteristic spectrum of radix ulmi and ulmus crassifolia medicinal materials and identification method

Abstract

The present application relates to the technical field of traditional Chinese medicine identification and quality evaluation, and particularly relates to a characteristic chromatogram construction method of Sanguisorba officinalis and Sanguisorba officinalis var. longifolia medicinal materials, comprising: preparation of reference solution and test solution; detecting the reference solution and the test solution by using ultra-high liquid chromatography to obtain corresponding chromatograms, comparing the reference solution and the test solution corresponding chromatograms to obtain the characteristic chromatogram of the Sanguisorba officinalis and Sanguisorba officinalis var. longifolia medicinal materials. The present application constructs the UPLC characteristic chromatogram of Sanguisorba officinalis medicinal materials and Sanguisorba officinalis var. longifolia medicinal materials, fully and comprehensively reflects the characteristic peak information of Sanguisorba officinalis medicinal materials and Sanguisorba officinalis var. longifolia medicinal materials, the separation degree of common peaks is good, the baseline is stable, and the method is stable, precise and reproducible. By constructing the UPLC characteristic chromatogram of Sanguisorba officinalis and Sanguisorba officinalis var. longifolia medicinal materials, a rapid, comprehensive detection and identification method is provided for the quality of Sanguisorba officinalis and Sanguisorba officinalis var. longifolia medicinal materials, which can directly evaluate the quality difference of Sanguisorba officinalis medicinal materials and Sanguisorba officinalis var. longifolia medicinal materials, and effectively distinguish Sanguisorba officinalis medicinal materials of different origins.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine identification and quality evaluation technology, and in particular to a method for constructing characteristic maps and identification methods for Sanguisorba officinalis and Sanguisorba longifolia medicinal materials. Background Technology

[0002] my country boasts a vast variety of medicinal herbs and abundant resources. However, the prevalence of homonyms, variant names, and multiple sources for a single herb has led to confusion regarding the types of medicinal herbs circulating in the market, including counterfeit and substandard products. Therefore, in addition to rigorously distinguishing genuine from counterfeit herbs, it is essential to establish effective methods for species identification and quality evaluation, thereby improving the scientific rigor and precision of quality standards and ensuring the quality of medicinal herbs.

[0003] With the application of modern new technologies and methods in traditional Chinese medicine (TCM) and the widespread use of large-scale analytical instruments, TCM characteristic mapping technology has been gradually promoted. Compared with traditional TCM identification methods (original plant / animal identification, morphological identification, and microscopic identification), it can be used for both source identification (qualitative level) and quality evaluation (quantitative level), showing significant advantages. Therefore, it has gradually become the mainstream method for identifying the authenticity and source of medicinal materials. TCM characteristic mapping refers to the characteristic spectrum of common peaks of various component groups in TCM materials detected and identified after appropriate treatment using modern instrumental analysis techniques and methods. It is mainly divided into chemical component characteristic mapping and biological characteristic mapping. TCM chemical component characteristic mapping techniques include spectroscopic methods, chromatographic methods, and coupled analytical techniques. Biological characteristic mapping techniques include random amplified polymorphic DNA (RAD) technology, PCR technology, and restriction fragment length polymorphism (RFLP) analysis technology, among which DNA characteristic mapping is widely used.

[0004] Sanguisorba officinalis, or *Sanguisorba longifolia*, is the dried root of a plant in the Rosaceae family. The latter is commonly known as "Mian Di Yu". It is harvested in spring when the plant is sprouting or in autumn after the plant has withered. The fibrous roots are removed, the root is washed, and then dried, or sliced ​​while fresh and then dried. Research has shown that *Sanguisorba longifolia* is a variety of *Sanguisorba officinalis*, with very little difference between the two dominant haplotypes. Furthermore, the sites showing interspecific differences are also intraspecific sites, meaning that the DNA barcodes of *Sanguisorba officinalis* and *Sanguisorba longifolia* show little interspecific difference, making accurate differentiation between the two species impossible. The chemical components of traditional Chinese medicine (TCM) are the material basis for its clinical efficacy and a major indicator for evaluating the quality of medicinal materials. Fingerprint (characteristic) mapping technology, which reflects the integrity and dynamics of the internal chemical components of TCM, provides a new approach to TCM quality control and necessary technical support for the standardization of authentic medicinal materials, especially for the preservation and innovation of TCM.

[0005] Patent document CN113391005A discloses a high-performance liquid chromatography (HPLC) detection method and identification method for Sanguisorba officinalis, its charred slices, reference extract, and formulation granules. This invention patent establishes an HPLC characteristic spectrum of Sanguisorba officinalis, which is detected using HPLC. The detection time is 60 minutes, and the number of characteristic peaks is 7, but only 3 characteristic peaks are identified. It only distinguishes between raw and processed products and fails to identify different source varieties. Patent document CN113325095A discloses a method for constructing and detecting an UPLC fingerprint spectrum of Sanguisorba officinalis. It establishes an UPLC fingerprint spectrum of Sanguisorba officinalis, with a detection time of 60 minutes and only 4 characteristic peaks: the first peak is gallic acid, the third peak is catechin or epicatechin, and the fourth peak is ellagic acid. This method improves the accuracy and stability of Sanguisorba officinalis detection, but it fails to distinguish Sanguisorba officinalis from different source varieties. Summary of the Invention

[0006] To address the above shortcomings, this invention provides a method for constructing characteristic maps of Sanguisorba officinalis and Sanguisorba longifolia, which can be used to identify and distinguish between two different source medicinal materials. The specific technical solution is as follows:

[0007] A method for constructing characteristic maps of *Sanguisorba officinalis* and *Sanguisorba longifolia* medicinal materials includes the following steps:

[0008] (1) Preparation of reference solution: Take Sanguisorba officinalis reference material or Sanguisorba longifolia reference material, add water to extract, filter, take the filtrate, extract with ethyl acetate, combine the ethyl acetate solutions, evaporate to dryness; dissolve the residue in methanol, filter, take the filtrate as the reference solution of the reference material; separately take gallic acid, gallocatechin, catechin, ellagic acid, and 2,3,8-tri-O-methylellagic acid reference standards, add methanol to prepare a mixed solution as the reference solution of the reference standard.

[0009] (2) Preparation of test solution: Take Sanguisorba officinalis or Sanguisorba longifolia powder, add test sample extraction solvent, extract, filter, take the filtrate, extract with extraction solvent, combine the extracts and evaporate to dryness; dissolve the residue in methanol, filter, take the filtrate to obtain the test solution.

[0010] (3) Chromatographic conditions: Column: octadecylsilane-bonded silica gel as packing material; diode array detector, detection wavelength 225-235 nm; column temperature 25℃-35℃; flow rate 0.28-0.32 mL / min; gradient elution with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B;

[0011] (4) The reference solution and the test solution were detected by ultra-high performance liquid chromatography to obtain the corresponding chromatograms. The corresponding chromatograms of the reference solution and the test solution were compared to obtain the characteristic chromatograms of the medicinal materials Sanguisorba officinalis and Sanguisorba longifolia.

[0012] Preferably, in the above-mentioned method for constructing the characteristic maps of Sanguisorba officinalis and Sanguisorba longifolia medicinal materials, in step (1), the preparation of the reference solution is as follows: take Sanguisorba officinalis or Sanguisorba longifolia reference medicinal material and add water, the ratio of reference medicinal material to water is 0.2g:25-100mL, heat under reflux or ultrasonic extraction for 20-40min, filter, take the filtrate, shake with ethyl acetate 2-3 times, combine the ethyl acetate solutions, evaporate to dryness; dissolve the residue in 75% methanol, take the filtrate as the reference solution of the reference medicinal material; separately take appropriate amounts of gallic acid, gallocatechin, catechin, ellagic acid, and 2,3,8-tri-O-methylellagic acid reference standards, add 50% methanol to prepare a mixed solution containing 35-45μg of each per 1ml, as the reference solution of the reference standard.

[0013] Preferably, in the above-mentioned method for constructing the characteristic map of Sanguisorba officinalis and Sanguisorba longifolia medicinal materials, in step (2), the solvent for extracting the test sample is water or 30% to 100% methanol, and the ratio of Sanguisorba officinalis or Sanguisorba longifolia powder to the solvent for extracting the test sample is 0.2g:25 to 100mL; the extraction solvent is water-saturated n-butanol or ethyl acetate, the extraction method is reflux extraction or ultrasonic extraction, the extraction time is 15 to 60min, and the number of extractions is 1 to 3.

[0014] Preferably, in the above-mentioned method for constructing the characteristic map of Sanguisorba officinalis and Sanguisorba longifolia medicinal materials, in step (2), the solvent for extracting the test sample is water, and the ratio of Sanguisorba officinalis or Sanguisorba longifolia powder to the solvent for extracting the test sample is 0.2g:25mL; the extraction solvent is ethyl acetate, the extraction method is ultrasonic extraction, the extraction time is 30min, and the number of extractions is 2.

[0015] Preferably, in the above-mentioned method for constructing the characteristic maps of Sanguisorba officinalis and Sanguisorba longifolia medicinal materials, the gradient elution is as follows:

[0016] 0–15 min, Phase A: 2% → 10%, Phase B: 98% → 90%;

[0017] 15–30 min, Phase A: 10% → 20%, Phase B: 90% → 80%;

[0018] 30–35 min, Phase A: 20% → 35%, Phase B: 80% → 65%;

[0019] 35–45 min, Phase A: 35% → 45%, Phase B: 65% → 55%.

[0020] Preferably, in the above-mentioned method for constructing the characteristic chromatograms of Sanguisorba officinalis and Sanguisorba longifolia medicinal materials, the chromatographic column has a length of 100 mm, an inner diameter of 2.1 mm, and a particle size of 1.8 μm; the detection wavelength is 230 nm, the column temperature is 30 °C, the flow rate is 0.3 ml / min, and the theoretical plate number calculated based on the ellagic acid peak should not be less than 10,000.

[0021] Preferably, in the above-mentioned method for constructing characteristic chromatograms of Sanguisorba officinalis and Sanguisorba longifolia medicinal materials, the chromatogram of the test sample should show 7 to 8 characteristic peaks, which should correspond to the retention times of the characteristic peaks in the chromatogram of the reference medicinal materials. Among them, peaks 1, 2, 4, 6, and 8 should correspond to the retention times of the reference peaks of gallic acid, gallocatechin, catechin, ellagic acid, and 2,3,8-tri-O-methylellagic acid.

[0022] Preferably, in the above-mentioned method for constructing the characteristic spectrum of Sanguisorba officinalis and Sanguisorba longifolia medicinal materials, the peak corresponding to the ellagic acid reference peak is the S peak. The relative retention times of peaks 3, 5, and 7 with peak S are calculated, and their relative retention times should be within ±10% of the specified values, which are peak 3 - 0.48, peak 5 - 0.85, and peak 7 - 1.32.

[0023] Preferably, in the above-mentioned method for constructing the characteristic maps of Sanguisorba officinalis and Sanguisorba longifolia, the relative peak area of ​​peak 8 and peak 6 corresponding to the Sanguisorba officinalis is 0.060 to 0.230; and the relative peak area of ​​peak 8 and peak 6 corresponding to the Sanguisorba longifolia is 0 to 0.057.

[0024] A characteristic chromatographic identification method for *Sanguisorba officinalis* and *Sanguisorba longifolia* medicinal materials is proposed, using the above-mentioned method for detection and analyzing the detection results.

[0025] Compared with the prior art, the beneficial effects of the present invention are:

[0026] The present invention provides a method for constructing UPLC characteristic maps of *Sanguisorba officinalis* and *Sanguisorba longifolia* medicinal materials. These maps comprehensively reflect the characteristic peak information of both materials, exhibiting good separation of common peaks, stable baselines, and stable method with good precision and reproducibility. By constructing UPLC characteristic maps of *Sanguisorba officinalis* and *Sanguisorba longifolia*, a rapid and comprehensive detection and identification method for their quality is provided. This allows for a direct assessment of quality differences between the two materials and effectively distinguishes *Sanguisorba officinalis* from different sources. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments 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 based on these drawings without creative effort.

[0028] Figure 1 Chromatograms of the test solution under different mobile phases;

[0029] Figure 2Chromatograms of the test solution for different gradient elution regimes;

[0030] Figure 3 Chromatograms of the sample solutions for extraction with different extraction solvents were used to investigate the extraction of the sample solutions.

[0031] Figure 4 Chromatograms of the test sample solutions for different extraction methods;

[0032] Figure 5 Chromatograms of the test solution for different solvent dosages;

[0033] Figure 6 Chromatograms of the test solution under different extraction times;

[0034] Figure 7 Chromatograms of the test solution for different extraction solvents;

[0035] Figure 8 The chromatogram of the test solution for determining the number of extractions;

[0036] Figure 9 This is a chromatogram for a specific test.

[0037] Figure 10 To determine the precision of the chromatogram;

[0038] Figure 11 Chromatograms for stability testing;

[0039] Figure 12 To test the repeatability of the chromatogram;

[0040] Figure 13 Chromatograms were examined at different column temperatures;

[0041] Figure 14 Chromatograms were examined at different flow rates;

[0042] Figure 15 Chromatograms were examined for different chromatographic columns;

[0043] Figure 16 To examine chromatograms using different instruments;

[0044] Figure 17 A comparative characteristic atlas of Sanguisorba officinalis medicinal material;

[0045] Figure 18 A comparative characteristic atlas of *Sanguisorba officinalis* (long-leaved burnet) medicinal material;

[0046] Figure 19 The spectrum is an identification map of 5 common characteristic peaks. Detailed Implementation

[0047] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise defined, all technical terms used below have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of protection of the present invention. Unless otherwise specifically stated, all raw materials, reagents, instruments, and equipment used in the present invention are commercially available or can be prepared by existing methods.

[0048] instrument:

[0049] Waters H-Plus ultra-high performance liquid chromatograph (Waters Corporation, USA); electronic analytical balance (ML204 / 02, XSR204, Mettler Toledo); electronic balance (E600-2, Changshu Shuangjie Test Instrument Factory); CNC ultrasonic cleaner (KQ-500DE, Kunshan Ultrasonic Instrument Co., Ltd.).

[0050] Material:

[0051] Gallic acid reference standard (batch number 110831-201605, content calculated as 90.8%, stored at 4℃ protected from light); Sanguisorba officinalis (batch number 121286-201703) reference medicinal material was sourced from the China National Institutes for Food and Drug Control; Sanguisorba officinalis (longifolia) reference medicinal material (batch number 240100-202210) was sourced from Shanghai Hongyong Biotechnology Co., Ltd.; 23 batches of Sanguisorba officinalis medicinal material were identified by the State Key Laboratory of Quality Research in Chinese Medicine (Macau University of Science and Technology) as dried roots of Sanguisorba officinalis L., a plant of the Rosaceae family; 20 batches of longifolia Sanguisorba officinalis medicinal material were identified by the State Key Laboratory of Quality Research in Chinese Medicine (Macau University of Science and Technology) and Associate Professor Huang Haibo of Guangzhou University of Chinese Medicine as dried roots of longifolia Sanguisorba officinalis L. var. longifolia (Bert.) Yüet Li. For detailed origin information, please refer to Tables 1 and 2.

[0052] Table 1. Origin information of 23 batches of Sanguisorba officinalis medicinal materials

[0053] S1 Longxi, Gansu S13 Longxi, Gansu S2 Longxi, Gansu S14 Turpan City, Xinjiang S3 Longxi, Gansu S15 Turpan City, Xinjiang S4 Longxi, Gansu S16 Longxi, Gansu S5 Longxi, Gansu S17 Longxi, Gansu S6 Longxi, Gansu S18 Longxi, Gansu S7 Turpan City, Xinjiang S19 Longxi, Gansu S8 Turpan City, Xinjiang S20 Longxi, Gansu S9 Turpan City, Xinjiang S21 Longxi, Gansu S10 Longxi, Gansu S22 Longxi, Gansu S11 Longxi, Gansu S23 Longxi, Gansu S12 Longxi, Gansu

[0054] Table 2. Origin information of 20 batches of Sanguisorba officinalis medicinal materials

[0055] S1 Pingyi, Shandong S11 Anqing, Anhui S2 Pingyi, Shandong S12 Huai'an, Jiangsu S3 Tai'an, Shandong S13 Yancheng, Jiangsu S4 Tai'an, Shandong S14 Lianyungang, Jiangsu S5 Tai'an, Shandong S15 Lishui, Zhejiang S6 Jining, Shandong S16 Lishui, Zhejiang S7 Rizhao, Shandong S17 Quzhou, Zhejiang S8 Yantai, Shandong S18 Jiujiang, Jiangxi S9 Chuzhou, Anhui S19 Ganzhou, Jiangxi S10 Lu'an, Anhui S20 Jiujiang, Jiangxi

[0056] Reagents: Acetonitrile (chromatographic grade), water (ultrapure water), and other reagents were of analytical grade.

[0057] Example 1

[0058] 1. Proposed ultra-high performance liquid chromatography (UHPLC) detection method

[0059] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 100 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B, with gradient elution performed according to the specifications in Table 3. The detection wavelength was 230 nm, the column temperature was 30 °C, and the flow rate was 0.3 mL / min; the theoretical plate number, calculated based on the ellagic acid peak, should not be less than 10,000.

[0060] Table 3 Gradient elution regime

[0061]

[0062] Preparation of reference solution: Take 0.2g of *Sanguisorba officinalis* or *Sanguisorba longifolia* reference material, place it in an Erlenmeyer flask, add 25ml of water, heat under reflux for 30min, remove, cool, shake well, filter, and measure 15ml of the subsequent filtrate. Extract twice with ethyl acetate, 25ml each time, combine the ethyl acetate extracts, and evaporate to dryness. Dissolve the residue in 75% methanol, transfer to a 10ml volumetric flask, add 75% methanol to the mark, shake well, filter, and take the subsequent filtrate as the reference solution for the reference material. Separately, take appropriate amounts of gallic acid, gallocatechin, catechin, ellagic acid, and 2,3,8-tri-O-methylellagic acid reference standards, add 50% methanol to prepare a mixed solution containing 40μg of each per 1ml, as the reference solution for the reference standard.

[0063] Preparation of the test solution: Take 0.2 g of *Sanguisorba officinalis* or *Sanguisorba longifolia* powder (passed through a No. 4 sieve), add 25 mL of water, extract by sonication for 30 min, cool, shake well, filter, and measure 15 mL of the filtrate. Extract twice with ethyl acetate, 25 mL each time, and combine the extracts to dryness. Dissolve the residue in 75% methanol, transfer to a 10 mL volumetric flask, add 75% methanol to the mark, shake well, filter, and collect the filtrate to obtain the test solution.

[0064] Determination: Accurately pipette 1 μl of the reference solution and the test solution into the liquid chromatograph, and determine by ultra-high performance liquid chromatography. Record the chromatogram.

[0065] 2. Investigation of chromatographic conditions

[0066] 2.1 Investigation of the mobile phase

[0067] The test solution (Batch number of Sanguisorba officinalis: YC200824-04) was prepared according to the preparation method of the test solution in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method". During the test, the mobile phase A was acetonitrile. Different mobile phases B (0.1% glacial acetic acid, 0.1% phosphoric acid, 0.1% formic acid) were investigated. Elution was carried out according to the gradient elution regime in Table 4. Other chromatographic conditions were carried out according to the conditions proposed in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method".

[0068] Test results are shown Figure 1 As shown in the figure, compared with 0.1% formic acid and 0.1% glacial acetic acid, the baseline of 0.1% phosphoric acid is relatively stable and the characteristic peaks are well separated. Therefore, the acetonitrile-0.1% phosphoric acid mobile phase system was selected.

[0069] Table 4 Gradient elution regime

[0070]

[0071] 2.2 Examination of Gradient Elution System

[0072] The test solution (Batch number of Sanguisorba officinalis: YC200824-04) was prepared according to the preparation method of the test solution in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method". Different gradient elution regimes were investigated, and other conditions were performed according to the conditions proposed in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method". The gradient elution proposed in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method" (Table 1) was optimized and improved through trial and adjustment of 55 gradients, ultimately selecting the optimal gradient to ensure good separation between each characteristic peak and adjacent chromatographic peaks, thus ensuring good robustness and reproducibility of the method. Methods 18, 36, and 55 are used for illustration. The gradient elution regimes for methods 18, 36, and 55 are shown in Tables 5, 6, and 3, respectively.

[0073] Test results are shown Figure 2 The results show that the gradient elution regime (method 55) selected in this invention can achieve good separation between each characteristic peak and adjacent chromatographic peaks, and the baseline is stable.

[0074] Table 5 shows the gradient elution regime used in Method 18.

[0075]

[0076] Table 6 shows the gradient elution regime used in Method 36.

[0077]

[0078] 3. Investigation of the preparation method of the test solution

[0079] 3.1 Investigation of extraction solvent

[0080] Take 0.2g of Sanguisorba officinalis (batch number: YC200824-04) and add 25mL of the test sample extraction solvent. Examine the different polarities of the test sample extraction solvents (water, 30% methanol, 50% methanol, 75% methanol, and methanol). Extract with ultrasound for 30min, cool, shake well, filter, and take 15mL of the filtrate. Extract twice with ethyl acetate, 25mL each time. Combine the extracts and evaporate to dryness. Dissolve the residue in 75% methanol, transfer to a 10ml volumetric flask, add 75% methanol to the mark, shake well, filter, and take the filtrate to obtain the test sample solution. Detect according to the conditions specified in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method".

[0081] See results Figure 3 ,Depend on Figure 3 It can be seen that the response values ​​of peaks 2 and 3 decrease slightly as the proportion of methanol increases. Considering the peak shape, baseline, and cost savings, water is used as the extraction solvent.

[0082] 3.2 Selection of Extraction Method

[0083] Take 0.2g of Sanguisorba officinalis (batch number: YC200824-04), add 25mL of water, and investigate ultrasonic extraction and reflux extraction respectively, with an extraction time of 30min; cool, shake well, filter, and take 15mL of the filtrate, extract twice with ethyl acetate, 25mL each time, and combine the extracts and evaporate to dryness. Dissolve the residue in 75% methanol, transfer to a 10ml volumetric flask, add 75% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution. Detect according to the conditions proposed in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method".

[0084] See results Figure 4 The results showed that the chromatogram shapes and the number of chromatographic peaks of ultrasonic and reflux extraction methods were basically the same. Considering that ultrasonic extraction is convenient and simple to operate, ultrasonic extraction method was selected.

[0085] 3.3 Investigation of Solvent Usage

[0086] Take 0.2g of Sanguisorba officinalis (batch number: YC200824-04), add water, and investigate extraction with different solvent volumes (25ml, 50ml, 100ml). Extract by ultrasonication for 30min; cool, shake well, filter, and take 15mL of the filtrate. Extract twice with ethyl acetate, 25mL each time, and combine the extracts. Evaporate to dryness. Dissolve the residue in 75% methanol, transfer to a 10ml volumetric flask, add 75% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution. Detect according to the conditions specified in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method".

[0087] See results Figure 5As shown in the figure, the shape and number of chromatographic peaks of the chromatograms after extraction with different solvent volumes are basically the same. However, the response values ​​of each peak in the chromatogram extracted with 25 ml of solvent are more appropriate and the peak shape is better. Therefore, the solvent volume of 25 ml was selected.

[0088] 3.4 Examination of extraction time

[0089] Take 0.2g of Sanguisorba officinalis (batch number: YC200824-04), add 25mL of water, and extract ultrasonically. The extraction time was investigated at different times (15, 30, and 60 minutes). After cooling, shake well, filter, and take 15mL of the filtrate. Extract twice with ethyl acetate, 25mL each time. Combine the extracts and evaporate to dryness. Dissolve the residue in 75% methanol, transfer to a 10mL volumetric flask, add 75% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution. Perform the detection according to the conditions specified in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method".

[0090] The results are as follows Figure 6 As shown in the figure, the shape and number of chromatographic peaks of the chromatograms at different extraction times are basically the same, and the peak response values ​​at different extraction times are not significantly different. To ensure complete extraction and save time, an extraction time of 30 min was selected.

[0091] 3.5 Selection of Extraction Solvent

[0092] Because the polar components of Sanguisorba officinalis are complex and there are many small impurity peaks, resulting in an uneven baseline, it is considered to extract the solution and purify it appropriately before analyzing its characteristic components.

[0093] Take 0.2g of Sanguisorba officinalis (batch number: YC200824-04), add 25mL of water, and extract ultrasonically for 30min. Cool, shake well, filter, and take 15mL of the filtrate. Examine the filtrate with different polarity extraction solvents (diethyl ether, petroleum ether (60-90℃), chloroform, ethyl acetate, and water-saturated n-butanol). Extract twice with 25mL of the extraction solvent each time, and combine the extracts to evaporate to dryness. Dissolve the residue in 75% methanol, transfer to a 10mL volumetric flask, add 75% methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution. Perform the detection according to the conditions specified in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method".

[0094] The results are as follows Figure 7 As shown in the figure, the extraction capabilities of different extraction solvents for this product are as follows: petroleum ether (60-90℃) is the weakest, with almost no obvious chromatographic peaks; followed by chloroform and diethyl ether. Compared with unextracted, water-saturated n-butanol extracts a similar number of chromatographic peaks, but the baseline is poorer and there are more impurity peaks, resulting in poor separation. In contrast, the chromatogram extracted with ethyl acetate has fewer impurity peaks and better separation, with a significant improvement in baseline. Therefore, ethyl acetate was chosen as the extraction solvent.

[0095] 3.6 Examination of the number of extractions

[0096] Take 0.2g of Sanguisorba officinalis (batch number: YC200824-04), add 25mL of water, and extract ultrasonically for 30min. Cool, shake well, filter, and take 15mL of the filtrate for extraction with ethyl acetate. The extraction was tested for different extraction times (1, 2, and 3 times), with 25mL extracted each time. The extracts were combined and evaporated to dryness. The residue was dissolved in 75% methanol, transferred to a 10ml volumetric flask, and 75% methanol was added to the mark. The mixture was shaken well, filtered, and the filtrate was collected to obtain the test solution. The test was performed according to the conditions specified in "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method".

[0097] The results are as follows Figure 8 As shown in the figure, the chromatograms and the number of peaks are basically the same for extraction times 1, 2 and 3. However, the peak area response of extraction 1 is lower than that of extraction 2 and 3. The peak area responses of extraction 2 and 3 are basically the same. In order to ensure complete extraction and save solvent, extraction 2 times is selected.

[0098] 4. Methodological Validation

[0099] Ellagic acid (peak 6) has a moderate elution time, a good peak shape, and stable composition. Therefore, this study uses ellagic acid as a reference peak to calculate the relative retention time and relative peak area of ​​each common characteristic peak and the reference peak.

[0100] 4.1 Specificity test

[0101] The test solution (batch number: YC200824-04) and reference solution were prepared according to "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method"; 0.2 g of excipient (maltodextrin) was taken, and a negative sample solution was prepared according to the preparation method of the test solution under "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method". 1 μl each of the negative sample solution, test solution, and reference solution were injected into the liquid chromatograph, and analyzed under the chromatographic conditions suitable for the system. The results are as follows: Figure 9 As shown in the figure, the results indicate that the negative sample did not have a corresponding chromatographic peak at the corresponding position of the analyte in the test sample, indicating that the excipients did not interfere with the detection and that the method has good specificity.

[0102] 4.2 Precision Test

[0103] The 0-hour test solution was used in the stability study, and six consecutive injections were performed to assess the instrument's precision. The results are as follows: Figure 10 As shown in Table 7, the results show that the relative retention time (RSD) of each characteristic peak measured after six consecutive injections is less than 3.0%, indicating that the instrument has good precision.

[0104] Table 7. Precision: Relative Retention Time and Relative Peak Area

[0105]

[0106]

[0107] 4.3 Stability Test

[0108] The sample solution of *Sanguisorba officinalis* (batch number: YC200824-04) was prepared according to the method described in section "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method". The stability of the sample solution was investigated at 0, 2, 4, 6, 8, 12, 16, 20, and 24 hours. The results are shown in [Figure number missing]. Figure 11 According to Table 8, the relative retention times (RSD) of each characteristic peak were less than 3.0%, indicating that the test solution had good stability within 24 hours.

[0109] Table 8. Relative retention time and relative peak area for stability

[0110]

[0111] 4.4 Repeatability Test

[0112] Six solutions of *Sanguisorba officinalis* (batch number: YC200824-04) were prepared according to the method described in section "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method," and the repeatability of the experimental method was investigated. Results are shown below. Figure 12 According to Table 9, the relative retention time (RSD) of each characteristic peak is less than 3.0%, indicating good repeatability.

[0113] Table 9. Relative retention time and relative peak area for repeatability

[0114]

[0115]

[0116] 4.5 Durability Test

[0117] Weigh 0.2 g of the same batch of Sanguisorba officinalis powder (batch number: YC200824-04) and prepare the test solution according to the method under "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method". Based on the original chromatographic conditions, change the column temperature, flow rate, and use different brands of chromatographic columns to determine the robustness of the chromatographic conditions. Calculate the elution time of each characteristic peak and the RSD% of the relative retention time and relative peak area.

[0118] See results Figures 13-15As shown in Table 10, the results indicate that when the column temperature is 25-35℃, the flow rate is 0.28-0.32 ml / min, and different brand of columns are used, there are no significant differences in the relative retention times and relative peak areas of all characteristic peaks except for characteristic peak 1. Characteristic peak 1 has a fast elution time, and its retention time is significantly affected by column temperature and different columns. Since peak 1 is a known component peak, its retention time can be directly compared with that of the reference standard, and no specific requirement for relative retention time is needed. In conclusion, this method has good robustness.

[0119] Table 10 Durability: Relative Retention Time and Relative Peak Area

[0120]

[0121]

[0122] 4.6 Intermediate Precision Test

[0123] A batch of Sanguisorba officinalis powder (batch number: YC200824-04), approximately 0.2 g, was weighed and used to prepare a test solution according to the method described in section "1. Proposed Ultra-High Performance Liquid Chromatography Detection Method". Based on the original chromatographic conditions, the intermediate precision of the method was investigated using chromatographic columns of the same specifications and different instruments (two instruments of the same brand and model, and one instrument of a different brand). The elution times of each peak were recorded, and the relative retention times and relative peak areas (RSD values) were calculated. The results are shown below. Figure 16 See Table 11. The results show that, due to the rapid elution of peak 1, its relative retention time and relative peak area are significantly affected by different instruments, but this does not affect the identification of the original source and quality analysis of *Sanguisorba officinalis*. There are no significant differences in the relative retention time and relative peak area of ​​other characteristic peaks. The number of peaks and resolution of the chromatograms obtained from the Agilent 1290 DAD instrument are basically consistent with those obtained from the Waters H-Plus instrument, indicating that this method has good reproducibility.

[0124] Table 11 Intermediate Precision: Relative Retention Time and Relative Peak Area

[0125]

[0126]

[0127] Example 2: Construction of the Characteristic Map of Sanguisorba officinalis Medicinal Material

[0128] The characteristic spectra of 23 batches of Sanguisorba officinalis medicinal materials were determined using the proposed ultra-high performance liquid chromatography (UHPLC) method described in section "1. Proposed UHPLC Detection Method". Based on the principles of stable relative retention time and detectability in all batches with relatively high peaks, eight peaks with good repeatability and resolution were selected as characteristic peaks. The peak corresponding to the ellagic acid reference peak was designated as the S peak. The relative retention time and relative peak area of ​​each characteristic peak and the S peak were calculated, as shown in Table 12.

[0129] Table 12. Relative retention time and relative peak area of ​​23 batches of Sanguisorba officinalis medicinal materials

[0130]

[0131]

[0132] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize characteristic chromatograms of 23 batches of Sanguisorba officinalis medicinal materials, and such characteristic chromatograms were established. Figure 17 As shown.

[0133] Example 3: Construction of the Characteristic Map of Sanguisorba officinalis 'Longleaf'

[0134] The characteristic spectra of 20 batches of Sanguisorba officinalis rootstock were determined using the proposed ultra-high performance liquid chromatography method. Based on the principle that the relative retention time was stable and that the peaks could be detected in all batches of samples with relatively high peak values, seven peaks with good repeatability and resolution were selected as characteristic peaks. The peak corresponding to the ellagic acid reference peak was designated as the S peak. The relative retention time and relative peak area of ​​each characteristic peak and the S peak were calculated, as shown in Table 13.

[0135] Table 1320 shows the relative retention time and relative peak area of ​​*Sanguisorba officinalis* medicinal materials.

[0136]

[0137]

[0138] The chromatographic fingerprint similarity evaluation system for traditional Chinese medicine (2012 version) was used to synthesize chromatograms of 20 batches of Sanguisorba officinalis, and a characteristic chromatogram of Sanguisorba officinalis was established, such as... Figure 18 As shown.

[0139] Example 4: Identification of Common Characteristic Peaks

[0140] For the unknown characteristic peaks, UHPLC-Q-TOF MS high-resolution mass spectrometry identification and secondary mass spectrometry result analysis were performed, and the results were confirmed with reference standards. Ultimately, peak 1 was identified as gallic acid, peak 2 as gallocatechin, peak 4 as catechin, peak 6(S) as ellagic acid, and peak 8 as 2,3,8-tri-O-methylellagic acid. Figure 19 As shown.

[0141] Example 5: Establishment of Limits for Relative Retention Time

[0142] The methodology and validation results ultimately stipulate that the test sample chromatogram should exhibit 7–8 characteristic peaks, corresponding to the retention times of the characteristic peaks in the reference chromatogram of the medicinal material. Specifically, peaks 1, 2, 4, 6, and 8 should correspond to the retention times of the reference peaks of gallic acid, gallocatechin, catechin, ellagic acid, and 2,3,8-tri-O-methylellagic acid. The peak corresponding to the ellagic acid reference peak is designated as peak S. The relative retention times of peaks 3, 5, and 7 with peak S should be calculated and should be within ±10% of the specified values, which are 0.48 (peak 3), 0.85 (peak 5), and 1.32 (peak 7), respectively.

[0143] Example 6: Identification of Sanguisorba officinalis and Sanguisorba longifolia.

[0144] From the data in Tables 12 and 13, we can see that:

[0145] (1) The main difference between Sanguisorba officinalis and Sanguisorba longifolia is the relative peak area of ​​peak 8. Based on the actual range of values, the relative peak area limit of Sanguisorba officinalis is defined as 0.060 to 0.230, with the lower limit being the average peak area ratio of peak 8 to peak 6 (S) of 23 batches of Sanguisorba officinalis minus 1.4 times SD and the upper limit being the average peak area ratio of peak 8 to peak 12 (S). The relative peak area limit of Sanguisorba longifolia is defined as 0 to 0.057, with the lower limit being the average peak area ratio of peak 8 to peak 6 (S) of 20 batches of Sanguisorba longifolia minus 1 times SD and the upper limit being the average peak area ratio of peak 8 to peak 6 (S) of 20 batches of Sanguisorba longifolia minus 1 times SD and the upper limit being the average peak area ratio of peak 8 to peak 6 (S) of 20 batches of Sanguisorba longifolia minus 3 times SD.

[0146] (2) The actual relative peak area of ​​peak 8 in batch 20 of long-leaf Sanguisorba officinalis is 0 to 0.057 (within the specified range of 0 to 0.057), and the actual relative peak area of ​​peak 8 in batch 23 of Sanguisorba officinalis is 0.072 to 0.208 (within the specified range of 0.060 to 0.230), which can distinguish it from Sanguisorba officinalis and long-leaf Sanguisorba officinalis.

[0147] In summary, this method can effectively identify Sanguisorba officinalis and Sanguisorba longifolia, better clarify the differences between traditional Chinese medicines of different origins, and facilitate more accurate use of traditional Chinese medicine.

[0148] It should be noted that the present invention has conducted a large number of research experiments on chromatographic conditions in a long-term and extensive experimental study, but these cannot be described in detail here. The above only uses typical experiments as an illustration.

[0149] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A method for constructing characteristic maps of *Sanguisorba officinalis* and *Sanguisorba longifolia* medicinal materials, characterized in that, Includes the following steps: (1) Preparation of reference solution: Take Sanguisorba officinalis reference material or Sanguisorba longifolia reference material, add water to extract, filter, take the filtrate, extract with ethyl acetate, combine the ethyl acetate solutions, evaporate to dryness; dissolve the residue in methanol, filter, take the filtrate as reference solution of reference material; separately take gallic acid, gallocatechin, catechin, ellagic acid, 2,3,8-tri-O-methylellagic acid reference standards, add methanol to prepare a mixed solution as reference solution of reference standard; (2) Preparation of test solution: Take Sanguisorba officinalis or Sanguisorba longifolia powder, add test sample extraction solvent to extract, the test sample extraction solvent is water or 30%~100% methanol solution, filter, take the filtrate, extract with extraction solvent, the extraction solvent is water-saturated n-butanol or ethyl acetate, combine the extracts and evaporate to dryness; dissolve the residue in methanol, filter, take the filtrate to obtain the test solution; (3) Chromatographic conditions: Column: octadecylsilane bonded silica gel as packing material; diode array detector, detection wavelength 225~235nm; column temperature 25℃~35℃; flow rate 0.28~0.32mL / min; gradient elution with acetonitrile as mobile phase A and 0.1% phosphoric acid solution as mobile phase B; The gradient elution is as follows: 0~15min, Phase A: 2%→10%, Phase B: 98%→90%; 15~30min, Phase A: 10%→20%, Phase B: 90%→80%; 30~35min, Phase A: 20%→35%, Phase B: 80%→65%; 35~45min, Phase A: 35%→45%, Phase B: 65%→55%; (4) The reference solution and the test solution were detected by ultra-high performance liquid chromatography to obtain the corresponding chromatograms. The corresponding chromatograms of the reference solution and the test solution were compared to obtain the characteristic chromatograms of the medicinal materials Sanguisorba officinalis and Sanguisorba longifolia. The chromatogram of the test sample should show 7 to 8 characteristic peaks, of which peaks 1, 2, 4, 6 and 8 are gallic acid, gallocatechin, catechin, ellagic acid and 2,3,8-tri-O-methylellagic acid peaks, respectively.

2. The method for constructing characteristic maps of *Sanguisorba officinalis* and *Sanguisorba longifolia* medicinal materials according to claim 1, characterized in that, In step (1), the preparation of the reference solution is as follows: take Sanguisorba officinalis reference material or Sanguisorba longifolia reference material and add water. The ratio of reference material to water is 0.2g:25~100mL. Heat under reflux or extract with ultrasound for 20~40min. Filter and take the filtrate. Extract with ethyl acetate by shaking 2~3 times. Combine the ethyl acetate solutions and evaporate to dryness. Dissolve the residue in 75% methanol and use the filtrate as a reference solution for the reference medicinal material. Separately, take appropriate amounts of gallic acid, gallocatechin, catechin, ellagic acid, and 2,3,8-tri-O-methylellagic acid reference standards, add 50% methanol to prepare a mixed solution containing 35-45 μg of each per ml, and use this solution as a reference solution for the reference standards.

3. The method for constructing characteristic maps of *Sanguisorba officinalis* and *Sanguisorba longifolia* medicinal materials according to claim 1, characterized in that, In step (2), the ratio of Sanguisorba officinalis powder or Sanguisorba longifolia powder to the extraction solvent of the test sample is 0.2g:25~100mL; the extraction method is reflux extraction or ultrasonic extraction, the extraction time is 15~60min, and the number of extractions is 1~3 times.

4. The method for constructing characteristic maps of *Sanguisorba officinalis* and *Sanguisorba longifolia* medicinal materials according to claim 3, characterized in that, In step (2), the solvent for extracting the test sample is water, and the ratio of Sanguisorba officinalis powder or Sanguisorba longifolia powder to the solvent for extracting the test sample is 0.2g:25mL; the extraction solvent is ethyl acetate, the extraction method is ultrasonic extraction, the extraction time is 30min, and the extraction is performed twice.

5. The method for constructing characteristic maps of *Sanguisorba officinalis* and *Sanguisorba longifolia* medicinal materials according to claim 1, characterized in that, The chromatographic column is 100 mm long, 2.1 mm inner diameter, and 1.8 μm particle size; the detection wavelength is 230 nm, the column temperature is 30 °C, and the flow rate is 0.3 ml / min; the theoretical plate number calculated based on the ellagic acid peak should be no less than 10,000.

6. The method for constructing characteristic maps of *Sanguisorba officinalis* and *Sanguisorba longifolia* medicinal materials according to claim 1, characterized in that, The peak corresponding to the ellagic acid reference peak is the S peak. Calculate the relative retention times of peaks 3, 5, and 7 with peak S. The relative retention times should be within ±10% of the specified values, which are -0.48 for peak 3, -0.85 for peak 5, and -1.32 for peak 7.

7. The method for constructing characteristic maps of *Sanguisorba officinalis* and *Sanguisorba longifolia* medicinal materials according to claim 6, characterized in that, The relative peak area of ​​peak 8 and peak 6 corresponding to the *Sanguisorba officinalis* medicinal material is 0.060~0.230; the relative peak area of ​​peak 8 and peak 6 corresponding to the *Sanguisorba longifolia* medicinal material is 0~0.057.

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