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Abstract

The present application relates to a kind of white hair silver dew's fingerprint and content detection method and its application in traditional Chinese medicine quality control, specifically including the following steps: (1) white hair silver dew is crushed after medicine and is extracted by ultrasonic, is prepared into test solution;(2) the test solution obtained in step (1) is detected by HPLC, and the HPLC diagram of test product is obtained;(3) the HPLC diagram of test product obtained in step (2) is compared with the HPLC fingerprint of white hair silver dew medicine, and the similarity of white hair silver dew medicine above 0.90 is qualified product.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of traditional Chinese medicine, and particularly relates to a fingerprint spectrum and content detection method of Dasiphora mandshurica (Maxim.) Juz. and application thereof in quality control of traditional Chinese medicine. BACKGROUND

[0002] Dasiphora mandshurica (Maxim.) Juz., also known as Yawangcha, Huaxi Yinmei, Huaxi Yinluomei, etc., was originally a plant of Rosaceae Potentilla, and is reclassified as a plant of Rosaceae Dasiphora by botanists in recent years. The resource is rich and widely distributed in arid mountain slopes, valleys, rock slopes, shrubs and mixed wood in Shaanxi, Gansu, Qinghai and other places in China, with an altitude of 1200-3400 meters. In Shaanxi, it mainly grows wild in the alpine meadow of Taibai Mountain in Qinling with an altitude of about 3000 meters. Yawangcha is recorded in Taibai Bencao and Qinling Plant Fauna as "clearing heat, benefiting brain and clearing heart". Modern clinical treatment is mainly used for treating diabetes, hypertension, hyperlipidemia, insomnia, depression and the like. It can be long-term tea drinking and can also be used for treating metabolic diseases such as diabetes. It is a medicinal and edible plant integrating treatment, health care and health preservation. Since the content of each chemical component in Dasiphora mandshurica in different regions has obvious difference, the quality effect is affected. At present, there is no unified standard for quality evaluation of Dasiphora mandshurica in China. In order to make up for the blank of quality evaluation of Dasiphora mandshurica and prevent the phenomenon of using inferior goods as good ones, the application provides a fingerprint spectrum and content detection method of Dasiphora mandshurica and application thereof in quality control of traditional Chinese medicine. SUMMARY

[0003] The application provides an HPLC fingerprint spectrum of Dasiphora mandshurica medicinal material, characterized in that the HPLC fingerprint spectrum of the Dasiphora mandshurica medicinal material is basically consistent with that of R or B when HPLC chromatographic conditions are as follows. Figure 1 R or Figure 2 B;

[0004] The HPLC chromatographic conditions are as follows.

[0005] The chromatographic column is Agilent Poroshell 120SB-C 18 , with a specification of 150mmx4.6mm, 2.7μm.

[0006] The mobile phase is acetonitrile as phase A, methanol as phase B and 0.1wt% phosphoric acid aqueous solution as phase C.

[0007] The gradient elution condition is as follows.

[0008]

[0009] The flow rate is 0.5mL·min -1Column temperature: 25℃; Detection wavelength: 254nm; Injection volume: 10μL.

[0010] Another embodiment of the present invention provides an HPLC fingerprint of the above-mentioned *Symplocos rubrum* medicinal material, characterized in that the HPLC fingerprint of the *Symplocos rubrum* medicinal material is substantially the same as... Figure 1 or Figure 2 B is consistent with the chromatographic fingerprint of no less than 16 characteristic fingerprint peaks, of which peak 7 is agrimonyin, peak 9 is isoquercitrin, peak 10 is ellagic acid, peak 11 is quercetin, and peak 12 is nepeta glycoside.

[0011] Another embodiment of the present invention provides the application of the HPLC fingerprint of the above-mentioned *Prunus armeniaca* medicinal material in the quality control and component analysis of *Prunus armeniaca* medicinal material.

[0012] Another embodiment of the present invention provides the application of the HPLC fingerprint of the above-mentioned *Silver Dewdrop* medicinal material in the quality control of *Silver Dewdrop* medicinal material, characterized in that the application includes the following steps:

[0013] (1) The white-haired silver plum medicinal material was pulverized and extracted by ultrasonication, and then prepared into a test solution;

[0014] (2) Take the test solution obtained in step (1) and perform HPLC analysis to obtain the HPLC chromatogram of the test solution. The chromatographic conditions are as follows:

[0015] The chromatographic column was an Agilent Poroshell 120SB-C. 18 Specifications: 150mm × 4.6mm, 2.7μm;

[0016] Mobile phase: Acetonitrile as phase A, methanol as phase B, and 0.1 wt% phosphoric acid aqueous solution as phase C;

[0017] Gradient elution conditions:

[0018]

[0019] Flow rate: 0.5 mL / min -1 Column temperature: 25℃; Detection wavelength: 254nm; Injection volume: 10μL;

[0020] (3) Compare the HPLC chromatogram of the test sample obtained in step (2) with the HPLC fingerprint chromatogram of the white-haired silver plum medicinal material described in this invention. The white-haired silver plum medicinal material with a similarity of 0.90 or above (preferably with a similarity of 0.95 or 0.98 or above) is a qualified product.

[0021] The preparation method of the test solution in step (1) is as follows: Pulverize the white-haired silver plum medicinal material, pass it through a 50-mesh sieve, weigh 0.2g, place it in a stoppered conical flask, accurately add 25ml of 75% methanol, seal tightly, weigh, sonicate (power 300W, frequency 50kHz) for 45 minutes, cool (restore to room temperature), weigh again, replenish the lost weight with 75% methanol, shake well, filter through a 0.45μm microporous organic filter membrane, and obtain the solution.

[0022] Another embodiment of the present invention provides a method for determining the content of agrimonin, isoquercitrin, ellagic acid, quercetin, and / or pleuronectin in the medicinal material *Prunus armeniaca*, characterized in that the method comprises the following steps:

[0023] (1) The white-haired silver plum medicinal material was pulverized and extracted by ultrasonication, and then prepared into a test solution;

[0024] (2) Take the test solution obtained in step (1) and detect it by HPLC. Substitute the peak areas of agrimonyin, isoquercitrin, ellagic acid, quercetin and / or nepeta into the corresponding linear equations. Agrimoniain: Y = 11467.78X + 704.70, isoquercitrin: Y = 51371.76X - 178.05, ellagic acid: Y = 123235.14X + 59520.84, quercetin: Y = 47720.46X + 80.43, nepeta: Y = 62818.88X - 635.79. The contents of agrimonyin, isoquercitrin, ellagic acid, quercetin and / or nepeta can be obtained.

[0025] The preparation method of the test solution in step (1) is as follows: Pulverize the white-haired silver plum medicinal material, pass it through a 50-mesh sieve, weigh 0.2g, place it in a stoppered conical flask, accurately add 25ml of 75% methanol, seal tightly, weigh, sonicate (power 300W, frequency 50kHz) for 45 minutes, cool (restore to room temperature), weigh again, replenish the lost weight with 75% methanol, shake well, filter through a 0.45μm microporous organic filter membrane, and obtain the solution.

[0026] The chromatographic conditions for HPLC detection in step (2) are as follows:

[0027] The chromatographic column was an Agilent Poroshell 120SB-C. 18 Specifications: 150mm × 4.6mm, 2.7μm;

[0028] Mobile phase: Acetonitrile as phase A, methanol as phase B, and 0.1 wt% phosphoric acid aqueous solution as phase C;

[0029] Gradient elution conditions:

[0030]

[0031] Flow rate: 0.5 mL / min -1 Column temperature: 25℃; Detection wavelength: 254nm; Injection volume: 10μL.

[0032] Compared with the prior art, the advantages of the present invention are as follows: (1) The present invention establishes the fingerprint spectrum of *Symplocos buergeriana* medicinal material for the first time by analyzing the HPLC samples of *Symplocos buergeriana* medicinal material collected at different times, providing an effective method for the quality control of *Symplocos buergeriana* medicinal material; (2) Through the Chinese medicine chromatographic fingerprint spectrum similarity evaluation system (2012 version), the common pattern of the HPLC fingerprint spectrum of *Symplocos buergeriana* medicinal material was determined, and 16 common peaks were identified. Except for sample S7 which was unqualified (similarity 0.817), the similarity of the other 9 batches of samples (S1~S6, S8~S10) was greater than 0.986, and greater than 0.900 (qualified product). Peak 7 was determined to be agrimonyin, and peak 9 was determined to be isoquercitrin. Peak 10 is ellagic acid, peak 11 is quercetin, and peak 12 is eugenol. A corresponding standard curve was established to determine the content of the components. (3) Since there are many components in white-haired silver dew, and the solubility and resolution of each component are different, multiple chromatographic conditions were investigated, and the chromatographic conditions of the present invention were finally selected. The effects of extraction solvent (30%, 50%, 75%, 100% methanol), solvent volume (10mL, 25mL, 50mL) and ultrasonic extraction time (15, 30, 45, 60min) on the extraction rate of the components to be tested were investigated respectively. The peak area and resolution of the chromatographic peaks under different extraction conditions were compared. Finally, 75% methanol was selected as the solvent, the solvent volume was 25mL, and ultrasonic extraction was performed for 45min. The mobile phases tested included methanol-water, methanol-0.1% phosphoric acid solution, acetonitrile-0.1% phosphoric acid solution, methanol-0.5% phosphoric acid solution, methanol-0.1% formic acid solution, and acetonitrile-methanol-0.1% phosphoric acid solution. The results showed that the acetonitrile-methanol-0.1% phosphoric acid three-phase system provided the best gradient elution separation effect. The Agilent TC-5C18 column (250 mm × 4.6 mm, 5 μm), Kromasil 100-3.5-C18 (150 mm × 4.6 mm, 3.5 μm), Agilent Poroshell 120SB-C18 column (150 mm × 4.6 mm, 2.7 μm), and Agilent Poroshell 120PFP column (150 × 4.6 mm, 2.7 μm) were evaluated. After comprehensive comparison of peak number, peak shape, resolution, and baseline, the Agilent Poroshell column was selected. The 120SB-C18 column employs a novel core-shell chromatography technique. Its packing material has a particle size of 2.7 μm, consisting of a solid silica core with a diameter of 1.7 μm and a porous outer layer with a thickness of 0.5 μm. This small-particle-size packing material has similar high column efficiency to sub-2 μm packing material, but the column pressure can be reduced by 40-50%, achieving separation efficiency equivalent to UPLC on a conventional HPLC chromatograph.In addition, the separation performance was examined at detection wavelengths of 230, 254, 280, 300, and 325 nm. The results showed that the chromatogram information was richest at a wavelength of 254 nm, and the peak area of ​​the main chromatographic peaks was the largest, indicating the best separation. Attached Figure Description

[0033] Figure 1 These are the HPLC fingerprints of 10 batches of *Prunus armeniaca* medicinal materials (S1-S10), with R being the reference fingerprint.

[0034] Figure 2 The HPLC chromatograms are as follows: (A) for the mixed reference standard and (B) for the test solution of the white-haired silver plum medicinal material. Peak 7 is agrimonyin, peak 9 is isoquercitrin, peak 10 is ellagic acid, peak 11 is quercetin, and peak 12 is nepeta glycoside.

[0035] Figure 3 This is a columnar stacking chart showing the content of five chemical components in 10 batches of white-haired silver nectarine. Detailed Implementation

[0036] To facilitate a further understanding of the present invention, the following embodiments are provided for more detailed description. However, these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following.

[0037] Example 1

[0038] 1. Instruments and reagents

[0039] A Shimadzu LC-2010A high-performance liquid chromatograph, equipped with a UV detector and an LC Solution workstation, and a CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., model: KQ-400DE); an SQP type 0.0001 g electronic balance and an SQP type 0.0001 g electronic balance (Beijing Sartorius Scientific Instruments Co., Ltd.). Isoquercetin (batch number: MUST-21041613) reference standard was purchased from Chengdu Mansite Biotechnology Co., Ltd.; Agrimonia pilosa extract (batch number: 230418) was purchased from Chengdu Zhibiao Chemical Pure Biotechnology Co., Ltd.; Polygonum aviculare (batch number: 21010501) was purchased from Chengdu Pufeide Biotechnology Co., Ltd.; Ellagic acid (batch number: HR1827W1) and quercetin (batch number: HR1232W12) reference standards were purchased from Baoji Chenguang Biotechnology Co., Ltd. The purity of all the above reference standards was ≥98%. Methanol was of chromatographic purity, formic acid was of mass spectrometry purity (Fisher Chemicals), water was ultrapure water, and all other reagents were of analytical grade. Ten batches of *Dasiphora mandshurica* samples were collected from June to August 2019 to 2022 in the Qinling Mountains of Baoji City, Shaanxi Province. They were identified by Associate Researcher Chen Zhiyong of the Institute of Chinese Materia Medica, Shaanxi Academy of Traditional Chinese Medicine, as flowers, leaves, and young branches of *Dasiphora mandshurica* (Maxim.) Juz., a plant belonging to the genus *Dasiphora* in the family Rosaceae.

[0040] 2. Fingerprint spectroscopy determination methods and results

[0041] 2.1.1 Chromatographic conditions

[0042] Agilent Poroshell 120SB-C 18 The chromatographic column (150 mm × 4.6 mm, 2.7 μm) was used. Acetonitrile was used as mobile phase A, methanol as mobile phase B, and 0.1% phosphoric acid aqueous solution as mobile phase C. Gradient elution was performed according to Table 1. The flow rate was 0.5 ml / min, the detection wavelength was 254 nm, the column temperature was 25 ℃, and the injection volume was 10 μL.

[0043] Table 1 Gradient elution conditions

[0044]

[0045]

[0046] 2.1.2 Preparation of the test solution

[0047] Take approximately 0.2g of white-haired silver dewberry medicinal powder that has passed through a 50-mesh sieve, weigh it accurately, place it in a 100mL conical flask, add 25mL of 75% methanol, weigh it, sonicate it for 45min, cool it, replenish the lost weight with 75% methanol, and filter it through a 0.45μm microporous organic filter membrane to obtain the final product.

[0048] 2.1.3 Precision Test

[0049] The same test solution from section "2.1.2" was used, and the chromatographic conditions from section "2.1.1" were applied. Six consecutive injections were performed, and the relative retention time and relative peak area of ​​each common peak were calculated using the retention time and peak area of ​​ellagic acid as references. The results showed that the RSD of the relative retention time of the 16 common peaks ranged from 0.02% to 0.14% (n=6), and the RSD of the relative peak area ranged from 1.21% to 2.96% (n=6), indicating good precision of the method.

[0050] 2.1.4 Stability Test

[0051] The same test solution from section "2.1.2" was analyzed at 0, 2, 4, 8, 12, and 24 hours according to the chromatographic conditions from section "2.1.1". The relative retention time and relative peak area of ​​each common peak were calculated using the retention time and peak area of ​​ellagic acid as references. The results showed that the RSD of the relative retention time of the 16 common peaks ranged from 0.01% to 0.08% (n=6), and the RSD of the relative peak area ranged from 0.43% to 3.97% (n=6), indicating that the test solution was stable within 24 hours.

[0052] 2.1.5 Repeatability Test

[0053] Take the same batch of medicinal materials (S1), prepare the test solution according to the method in section "2.1.2", prepare 6 parallel solutions, and determine them according to the chromatographic conditions in section "2.1.1". Using the retention time and peak area of ​​ellagic acid as references, calculate the relative retention time and relative peak area of ​​each common peak. The results show that the RSD of the relative retention time of the 16 common peaks is 0.02% to 0.13% (n=6), and the RSD of the relative peak area is 1.89% to 3.18% (n=6), indicating that the method has good repeatability.

[0054] 2.1.6 Establishment and Similarity Evaluation of HPLC Fingerprints

[0055] Ten batches of *Prunus armeniaca* samples were collected. Test solutions were prepared according to the method described in section "2.1.2," and chromatographic conditions were applied under the conditions described in section "2.1.1." Data files were exported in "AIA" format and then imported into the Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 version) for data analysis. Using the chromatogram of sample S1 as the reference chromatogram, a time window width of 0.2 min was set. The fingerprint chromatogram and the reference fingerprint chromatogram R(…) were generated using the multi-point correction method and the averaging method. Figure 1The similarity was calculated. By analyzing the fingerprint spectra of 10 batches of samples, 16 chromatographic peaks were identified as common peaks of the *Prunus armeniaca* medicinal material. The similarity results of the 10 batches of samples are shown in Table 2. The similarity scores of S1–S6 and S8–S10 were all greater than 0.986, indicating that the quality of these 9 batches of medicinal material was relatively stable. However, the similarity score of sample S7 was only 0.817 (less than 0.90), indicating that S7 differed significantly from the other batches of medicinal material and was therefore unqualified.

[0056] Table 2 Similarity evaluation results

[0057]

[0058] 2.1.7 Common Peak Identification

[0059] Reference standards were used to identify the chromatographic peaks, and five common peaks were identified: peak 7 (agrimonia pilosa), peak 9 (isoquercetin), peak 10 (ellagic acid), peak 11 (quercetin), and peak 12 (polygonum aviculare). Peak 10 (ellagic acid) showed good resolution, a moderate peak area, and was inexpensive and readily available; therefore, it was selected as the reference peak. The relative retention times and relative peak areas of the other common peaks were calculated.

[0060] 2.2 Determination of multiple index contents

[0061] In this study, the contents of agrimonyin (peak 7), isoquercitrin (peak 9), ellagic acid (peak 10), quercetin (peak 11), and polygonum aviculare (peak 12) in *Prunus armeniaca* were relatively high, with high separation and strong measurability. Furthermore, all five components exhibit hypoglycemic activity, consistent with the main clinical efficacy of *Prunus armeniaca* in treating diabetes. Considering the indicative nature, effectiveness, and measurability of the chemical components, agrimonyin, isoquercitrin, ellagic acid, quercetin, and polygonum aviculare were ultimately selected as the indicative components for quantitative determination. 2.2.1 Chromatographic conditions and preparation of the test solution

[0062] The chromatographic conditions are the same as in section "2.1.1", and the preparation method of the test solution is the same as in section "2.1.2". The HPLC chromatograms of the mixed reference solution and the test solution (S1) under these chromatographic conditions are shown below. Figure 2 .

[0063] 2.2.2 Preparation of mixed reference standard stock solution

[0064] Accurately weigh appropriate amounts of agrimonyin, isoquercitrin, ellagic acid, quercetin, and eugenol reference standards, dissolve them in methanol, and prepare a mixed reference stock solution containing agrimonyin (1620.0 μg / mL), isoquercitrin (79.2 μg / mL), ellagic acid (142.0 μg / mL), quercetin (90.9 μg / mL), and eugenol (157.5 μg / mL).

[0065] 2.2.3 Study on linear relationships

[0066] Accurately pipette 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 mL of the mixed reference stock solution under section "2.2.2" into 10 mL volumetric flasks, dilute to the mark with 75% methanol, and mix well. Inject the solution according to the chromatographic conditions under section "2.1.1", record the chromatogram, and determine the peak area. Plot the reference concentration on the x-axis and the peak area on the y-axis to obtain the linear regression equation, as shown in Table 3.

[0067] Table 3. Linear relationships of the five components

[0068]

[0069] 2.2.4 Precision Test

[0070] Take the mixed reference standard stock solution from section "2.2.2", dilute it 10 times with 75% methanol, and inject it under the chromatographic conditions described in section "2.1.1" for six consecutive determinations, recording the peak areas. The calculated RSDs of the peak areas of agrimonyin, isoquercitrin, ellagic acid, quercetin, and polygonum aviculare were 0.58%, 1.26%, 0.59%, 1.06%, and 0.90%, respectively, indicating that the established instrumental method has good precision.

[0071] 2.2.5 Stability Test

[0072] The same test solution was injected and measured at 0, 2, 4, 8, 12, and 24 h. The RSDs of the peak areas of agrimonyin, isoquercitrin, ellagic acid, quercetin, and eugenol were calculated to be 1.88%, 1.86%, 2.76%, 1.62%, and 2.60%, respectively, indicating that the test solution prepared by this method is stable within 24 h.

[0073] 2.2.6 Repeatability Test

[0074] Take the same batch of medicinal materials (S1), prepare the test solution according to the method in section "2.1.2", prepare 6 parallel solutions, and determine the average contents of agrimonyin, isoquercitrin, ellagic acid, quercetin, and eurythrin according to the chromatographic conditions in section "2.1.1". The calculated average contents are 60.47, 1.31, 1.67, 2.26, and 1.43 mg / g, respectively, with RSDs of 0.64%, 1.50%, 2.01%, 2.55%, and 2.10%, respectively. The results show that the method has good repeatability.

[0075] 2.2.7 Recovery Experiment

[0076] Accurately weigh 0.1 g of *Prunus armeniaca* sample (S1) with known contents of the five analytes, and weigh six portions in total. Add appropriate amounts of each reference standard to each portion and prepare the test solution according to the method described in section "2.1.2". Calculate the recovery rate of each analyte. The average recoveries of agrimonyin, isoquercitrin, ellagic acid, quercetin, and polygonum aviculare were 98.45%, 96.08%, 99.47%, 97.54%, and 95.66%, respectively, with RSDs of 1.83%, 1.77%, 2.78%, 2.63%, and 2.99%, respectively, indicating that the established method has good accuracy.

[0077] 2.2.8 Determination of Sample Content

[0078] Accurately weigh 0.2g each of the medicinal materials *Gynostemma pentaphyllum* and prepare a sample solution according to the test solution preparation method in section "2.1.2". Determine the contents of agrimonyin, isoquercitrin, ellagic acid, quercetin, and polygonum aviculare using the external standard method. The content results are shown in Table 4. Simultaneously, a stacked column chart was used to visually characterize the differences in the contents of the five chemical components, including agrimonyin. Figure 3 ).

[0079] Table 4. Results of content determination of five index components in *Prunus cerasifera* (mg / g, n=3)

[0080]

[0081] 3 Discussion

[0082] The effects of extraction solvents (30%, 50%, 75%, and 100% methanol), solvent volume (10 mL, 25 mL, and 50 mL), and ultrasonic extraction time (15, 30, 45, and 60 min) on the extraction rate of the analytes were investigated. The peak area and resolution of the chromatographic peaks under different extraction conditions were compared. Ultimately, 75% methanol was selected as the solvent, with a volume of 25 mL and ultrasonic extraction for 45 min. The mobile phases tested included methanol-water, methanol-0.1% phosphoric acid solution, acetonitrile-0.1% phosphoric acid solution, methanol-0.5% phosphoric acid solution, methanol-0.1% formic acid solution, and acetonitrile-methanol-0.1% phosphoric acid solution. The results showed that the acetonitrile-methanol-0.1% phosphoric acid three-phase system provided the best gradient elution separation effect. The following columns were evaluated: Agilent TC-5C18 (250 mm × 4.6 mm, 5 μm), Kromasil 100-3.5-C18 (150 mm × 4.6 mm, 3.5 μm), Agilent Poroshell 120SB-C18 (150 mm × 4.6 mm, 2.7 μm), and Agilent Poroshell 120PFP (150 × 4.6 mm, 2.7 μm). After comprehensive comparison of peak number, peak shape, resolution, and baseline, Agilent was selected. The Poroshell 120SB-C18 column utilizes a novel core-shell chromatography technique. Its packing material has a particle size of 2.7 μm, consisting of a 1.7 μm diameter solid silica core and a 0.5 μm thick porous outer layer. This small-particle-size packing material exhibits similar high column efficiency to sub-2 μm packing material, but with a 40-50% reduction in column pressure, achieving separation efficiency equivalent to UPLC on a conventional HPLC system. Furthermore, the separation performance was investigated at detection wavelengths of 230, 254, 280, 300, and 325 nm. The results showed that the chromatogram information was richest at 254 nm, with the largest peak area of ​​the main chromatographic peaks, indicating the best resolution.

[0083] This invention established HPLC fingerprints for 10 batches of *Gynostemma pentaphyllum*, identifying 16 common peaks. Five of these common peaks were identified using reference standards: peak 7 (agreonine), peak 9 (isoquercetin), peak 10 (ellagic acid), peak 11 (quercetin), and peak 12 (polygonum aviculare). Based on multivariate statistical analysis and the effectiveness and measurability of the indicative components, the contents of five chemical components—agreonine, isoquercetin, ellagic acid, quercetin, and polygonum aviculare—in *Gynostemma pentaphyllum* were determined. According to literature reports, the main chemical components of *Gynostemma pentaphyllum* are flavonoids, and there are currently no reports of it containing tannins. This invention discloses that the main chemical components of *Gynostemma pentaphyllum* are tannins and flavonoids, and the presence of tannins (agreonine and ellagic acid) in *Gynostemma pentaphyllum* is a first-time discovery and report. The content of indicative components is shown in the stacked plot (…). Figure 3 As can be clearly seen from the table, agrimonyin, a tannic acid compound, has the highest content among all batches of medicinal materials, and its content is absolutely dominant. The average content of agrimonyin in *Silver Root Plum* is as high as 99.91 mg / g. Tannic acids and their active metabolites, urolithin, have pharmacological effects such as anti-inflammatory, antioxidant, anti-tumor, hypoglycemic, and cardiovascular disease treatment. Agrimonyin, a representative component, is a hydrolyzable ellagic tannin dimer (molecular formula C...). 82 H 54 O 52 (Molecular weight 1870) exhibits significant anti-diabetic and anti-tumor pharmacological activities, consistent with the traditional therapeutic effects of *Symplocos buergeriana*, thus confirming the pharmacological efficacy of this medicinal material at the chemical composition level. The HPLC fingerprinting and multi-index content determination method for *Symplocos buergeriana* established in this invention can provide methods and scientific basis for the quality evaluation research of medicinal materials.

Claims

1. The application of an HPLC fingerprint of *Prunus armeniaca* medicinal material in the quality control and component analysis of *Prunus armeniaca* medicinal material, characterized in that... The HPLC fingerprint of the medicinal material *Prunus armeniaca* has 16 characteristic fingerprint chromatographic peaks, of which peak 7 is agrimonyin, peak 9 is isoquercitrin, peak 10 is ellagic acid, peak 11 is quercetin, peak 12 is eugenol, and peak 10 is a reference peak. The HPLC chromatographic conditions are as follows: The chromatographic column was an Agilent Poroshell 120 SB-C. 18 Specifications: 150 mm × 4.6 mm, 2.7 μm; Mobile phase: Acetonitrile as phase A, methanol as phase B, and 0.1 wt% phosphoric acid aqueous solution as phase C; Gradient elution conditions: Flow rate: 0.5 mL•min -1 ; Column temperature: 25℃; Detection wavelength: 254 nm; Injection volume: 10 μL; The extraction solvent for the white-haired silver dewberry medicinal material is selected from 75% methanol by volume.

2. The application according to claim 1, characterized in that... The application includes the following steps: (1) The medicinal material of white-haired silver plum was pulverized and extracted by ultrasonication, and the solution was prepared as a test sample; (2) Take the test solution obtained in step (1) and perform HPLC analysis to obtain the HPLC chromatogram of the test solution. The chromatographic conditions are as follows: The chromatographic column was an Agilent Poroshell 120 SB-C. 18 Specifications: 150 mm × 4.6 mm, 2.7 μm; Mobile phase: Acetonitrile as phase A, methanol as phase B, and 0.1 wt% phosphoric acid aqueous solution as phase C; Gradient elution conditions: Flow rate: 0.5 mL•min -1 ; Column temperature: 25℃; Detection wavelength: 254 nm; Injection volume: 10 μL; (3) Compare the HPLC chromatogram of the test sample obtained in step (2) with the HPLC fingerprint chromatogram of the white-haired silver plum medicinal material. The white-haired silver plum medicinal material with a similarity of 0.90 or higher is a qualified product. The solvent used for ultrasonic extraction in step (1) is selected from 75% methanol by volume.

3. A method for determining the content of agrimonin, isoquercitrin, ellagic acid, quercetin, and pleuronectin in the medicinal material *Prunus armeniaca*, characterized in that... The method includes the following steps: (1) The medicinal material of white-haired silver plum was pulverized and extracted by ultrasonication, and the solution was prepared as a test sample; (2) Take the test solution obtained in step (1) and detect it by HPLC. Substitute the peak areas of agrimonyin, isoquercitrin, ellagic acid, quercetin and nepeta into the corresponding linear equations. Agrimoniain: Y = 11467.78 X + 704.70, isoquercitrin: Y = 51371.76X - 178.05, ellagic acid: Y = 123235.14 X + 59520.84, quercetin: Y = 47720.46 X + 80.43, nepeta: Y = 62818.88 X - 635.

79. The contents of agrimonyin, isoquercitrin, ellagic acid, quercetin and nepeta can be obtained. The solvent used for ultrasonic extraction in step (1) is 75% methanol (v / v). The chromatographic conditions for HPLC detection in step (2) are as follows: The chromatographic column was an Agilent Poroshell 120 SB-C. 18 Specifications: 150 mm × 4.6 mm, 2.7 μm; Mobile phase: Acetonitrile as phase A, methanol as phase B, and 0.1 wt% phosphoric acid aqueous solution as phase C; Gradient elution conditions: Flow rate: 0.5 mL•min -1 ; Column temperature: 25℃; detection wavelength: 254 nm; injection volume: 10 μL.

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