HPLC (High Performance Liquid Chromatography) fingerprint spectrum detection method of Angelica Koreana
Through the HPLC fingerprint detection method, the problem of insufficient research on the quality standards of Korean angelica was solved, a characteristic and systematic quality control method was established, the identification and quality control of Korean angelica was realized, and the standardized production of medicinal materials and clinical drug safety were supported.
Patent Information
- Application Number
- CN202510942641.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-09
- Publication Date
- 2025-09-05
AI Technical Summary
Existing technologies have little research on the quality standards of Korean Angelica, and lack scientific quality control methods, which affects the standardization of medicinal materials and the safety of clinical use.
High performance liquid chromatography (HPLC) fingerprint detection method was used to establish the fingerprint of Angelica sinensis by preparing test and reference solutions. Gradient elution and UV detection were used, and 20 common peaks were identified. In particular, 7-demethylxanthin was used as the reference peak for similarity analysis.
It has achieved the characteristic and systematic quality control of Korean angelica, broken through the limitations of traditional single ingredient testing, provided a reliable identification and quality control method, ensured the precision and repeatability of the test results, and supported the quality grading and origin traceability of medicinal materials.
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Figure CN120594714A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of medicinal material quality control, and in particular to an HPLC fingerprint detection method for Korean angelica. Background Art
[0002] Korean angelica is the source plant of angelica in Chinese Korean medicine (abbreviated as Korean medicine). It is the root of the Apiaceae plant Korean angelica (Angelica gigas Nakai), also known as wild angelica. It is distributed in various provinces in Northeast China, the Korean Peninsula and Japan. Korean angelica often grows on high mountain slopes above 1,000 meters above sea level and prefers sandy and rocky soil. It is distributed wild in various counties and cities in the Changbai Mountain area and is also cultivated.
[0003] The root of Korean angelica has the effects of dispelling wind and unblocking meridians, promoting blood circulation and relieving pain, and moisturizing and lubricating the intestines. It is primarily used to treat dysmenorrhea, traumatic injuries, blood deficiency, and constipation. Literature reports indicate that Korean angelica contains a variety of chemical components, including coumarins, volatile oils, organic acids, and sugars. The Korean Pharmacopoeia stipulates that the total content of the coumarin compounds purpurogenol, purpurogenol, and purpurogenol angelate in authentic Korean angelica must not be less than 6.0%. These chemical components have various pharmacological activities, including anti-cancer and anti-inflammatory properties.
[0004] While extensive research has been conducted both domestically and internationally on the chemical composition and pharmacological effects of Korean angelica, little research has been conducted on the quality standards for this ethnic medicine. This study, using high-performance liquid chromatography (HPLC) fingerprint analysis, aims to establish comprehensive, systematic, and scientific quality standards for Korean angelica. This study is of vital importance for standardizing and regulating the quality of Korean angelica and ensuring the safety and effectiveness of clinical medication. This study fills a gap in the quality standards for Korean angelica and provides a scientific basis for establishing these standards. Summary of the Invention
[0005] The main purpose of the present invention is to provide an HPLC fingerprint detection method for Korean angelica, and the obtained Korean angelica high-performance liquid fingerprint can provide a reliable basis for the identification and quality control of Korean angelica.
[0006] To achieve the above object, the HPLC fingerprint detection method of Angelica sinensis proposed in the present invention comprises the following steps: Step 1: Preparation of test solution: Grind the Korean angelica root to be tested, extract it with 70% ethanol by ultrasonic, and filter to obtain the test solution.
[0007] Step 2, preparation of reference solution: taking chlorogenic acid, ferulic acid, 7-demethyl cyperifoliin, peucedanum danshol and peucedanum danshol angelic acid ester reference substances, dissolving them in 70% ethanol, and diluting to volume to prepare the reference solution; Step 3, high performance liquid chromatography detection: accurately aspirate the test solution and the reference solution, inject them into a high performance liquid chromatograph for detection, the high performance liquid chromatograph contains a UV detector, the detection wavelength is set to 325 nm, the chromatographic column is Promosil C18 (4.6 mm × 250 mm, 5 μm), the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution is used, the flow rate is 1.0 mL / min, and the column temperature is 30 ° C; Step 4. Fingerprint construction and analysis: Record the chromatogram of the test solution from 0 to 70 minutes, and use the Chinese Pharmacopoeia Committee's Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System to import the chromatogram of the test solution, perform multi-point correction and data matching, and obtain the fingerprint and perform similarity analysis.
[0008] Preferably, the specific steps of preparing the test solution are: taking 0.5 g of Korean angelica medicinal material powder (passed through a 40-mesh sieve), accurately weighing it, placing it in a stoppered conical flask, accurately adding 10 mL of 70% ethanol, tightly plugging the stopper, weighing its mass, ultrasonically extracting for 40 minutes, cooling to room temperature, making up the lost weight with 70% ethanol, shaking well, and filtering with a 0.45 μm microporous filter membrane to obtain the test solution.
[0009] Preferably, the specific steps for preparing the reference solution are: accurately weighing 2 mg each of chlorogenic acid, ferulic acid, 7-demethylcorticosterol, purpurogenol and purpurogenol angelic acid ester reference substances, placing them in a 10 mL volumetric flask, adding 70% ethanol and shaking, and adjusting the volume to the scale to prepare a solution with a concentration of 200 μg / mL. After taking appropriate amounts and mixing them, a reference solution is obtained, and the solution is filtered with a 0.45 μm microporous filter membrane to obtain a reference solution.
[0010] Preferably, the procedure of the gradient elution is as follows: At 0 min, mobile phase A was 12% acetonitrile solution, and mobile phase B was 88% 0.1% phosphoric acid in water; At 10 min, mobile phase A was 18% acetonitrile solution, and mobile phase B was 82% 0.1% phosphoric acid in water; At 25 min, mobile phase A was 20% acetonitrile solution, and mobile phase B was 80% 0.1% phosphoric acid in water; At 45 min, mobile phase A was 50% acetonitrile solution, and mobile phase B was 50% 0.1% phosphoric acid in water; At 60 min, mobile phase A was 50% acetonitrile solution, and mobile phase B was 50% 0.1% phosphoric acid in water; At 70 minutes, mobile phase A was 60% acetonitrile solution, and mobile phase B was 40% 0.1% phosphoric acid aqueous solution.
[0011] Preferably, the fingerprint has 20 common peaks, of which peak 17 is the reference peak, i.e. 7-demethylcorticosterol, and the relative retention times of the peaks are: Peak 1: 0.160; Peak 2: 0.172; Peak 3: 0.265; Peak 4: 0.378; Peak 5: 0.398; Peak 6: 0.406; Peak 7: 0.477; Peak 8: 0.508; Peak 9: 0.577; Peak 10: 0.601; Peak 11: 0.617; Peak 12: 0.630; Peak 13: 0.681; Peak 14: 0.713; Peak 15: 0.734; Peak 16: 0.956; Peak 17 is the reference peak; Peak 18: 1.260; Peak 19: 1.278; Peak 20: 1.307.
[0012] Preferably, the relative retained peak areas of the common peaks in the fingerprint are: Peak 1: 2.924; Peak 2: 0.065; Peak 3: 0.423; Peak 4: 0.163; Peak 5: 0.287; Peak 6: 0.679; Peak 7: 0.053; Peak 8: 0.887; Peak 9: 0.366; Peak 10: 1.676; Peak 11: 0.200; Peak 12: 0.255; Peak 13: 0.210; Peak 14: 0.087; Peak 15: 0.085; Peak 16: 0.043; Peak 17 is a reference peak; Peak 18: 24.332; Peak 19: 22.403; Peak 20: 0.286.
[0013] Preferably, the injection volume of the HPLC detection is 10 μL, and the detection time is 70 min.
[0014] Preferably, the Korean angelica is the dried root of the Umbelliferae plant Korean angelica, collected from various places in South Korea and Northeast China.
[0015] An HPLC fingerprint of Korean angelica obtained by the method according to any one of claims 1 to 8, wherein the fingerprint comprises 20 common peaks, with 7-demethylcorticosterol as the reference peak, and the relative retention time and relative peak area are as described in claims 5-6.
[0016] Preferably, the HPLC fingerprint is used in the authenticity identification, quality control and origin tracing of Korean Angelica sinensis.
[0017] The present invention provides an HPLC fingerprint detection method for Korean Angelica sinensis, which has the following beneficial effects: (1) The HPLC fingerprint detection method of Korean Angelica was based on the test results of 10 batches of Korean Angelica samples collected from different origins. 20 common peaks with stable retention time and peak area were selected to mark the fingerprint of the chemical components of Korean Angelica. The common peak area was greater than 90% of the total peak area, and the non-common peak area was less than 10% of the total peak area, indicating good repeatability, characteristic and systematicity. After similarity evaluation, the similarity of the fingerprints of Korean Angelica collected from different origins was greater than 0.90, with good correlation, and the establishment of the fingerprint of Korean Angelica was completed. This method is simple and reliable and can be used as an effective method for the identification and quality control of Korean Angelica.
[0018] (2) The HPLC fingerprint detection method of Korean angelica uses HPLC fingerprint technology to simultaneously analyze the retention time and peak shape characteristics of 20 common peaks in Korean angelica, covering multiple active ingredients such as chlorogenic acid, ferulic acid, and coumarins, breaking through the limitations of traditional single ingredient detection.
[0019] (3) The fingerprint method established by the HPLC fingerprint detection method of Korean angelica optimizes the separation conditions through gradient elution, so that the common peaks are well separated, and the precision, repeatability and stability are verified to ensure the reliability of the detection results. It can quantify the composition differences of Korean angelica from different origins, and combine cluster analysis to achieve origin traceability, providing data support for the quality grading and standardized production of medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.
[0021] Figure 1 is the HPLC chromatogram of the reference substance; Figure 2 is the HPLC chromatogram of the test sample; Figure 3 This is the HPLC chromatogram of the precision experiment of Korean Angelica sinensis; Figure 4 This is the HPLC chromatogram of the reproducible experiment of Korean Angelica sinensis; Figure 5 This is the HPLC chromatogram of the stability experiment of Korean Angelica sinensis; Figure 6 This is the common peak fingerprint of Korean Angelica sinensis; Figure 7 The HPLC fingerprints of 10 batches of Korean Angelica sinensis; R: total map; Step 1: Sandaogou, Hunchun; Step 2: Liangshui, Tumen; Step 3: West slope of Changbai Mountain; Step 4: West of Liangshui River; S5: Chunhua, Hunchun; S6: East of Liangshui River; S7: Shengli, Hunchun; S8: Daejeon, South Korea; S9: Iksan, South Korea; S10: Tianqiaoling, Wangqing; Figure 8 This is the cluster analysis result of 10 batches of Korean Angelica.
[0022] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION
[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0024] See also Figure 1-8 The present invention provides an HPLC fingerprint detection method for Angelica sinensis, which specifically comprises the following steps: Step 1: Preparation of test solution: Grind the Korean angelica root to be tested, extract it with 70% ethanol by ultrasonic, and filter to obtain the test solution.
[0025] Step 2. Preparation of reference solution: Accurately weigh 2 mg each of chlorogenic acid, ferulic acid, 7-demethylcorticosterol, purpurogenol and purpurogenol angelic acid ester reference substances, place them in a 10 mL volumetric flask, add 70% ethanol and shake well, dilute to the scale to make a solution with a concentration of 200 μg / mL, take appropriate amounts of each and mix them to obtain a reference solution, filter with a 0.45 μm microporous membrane to obtain the reference solution.
[0026] Step 3, high performance liquid chromatography detection: Accurately aspirate the test solution and the reference solution, inject them into a high performance liquid chromatograph for detection, the high performance liquid chromatograph contains a UV detector, the detection wavelength is set to 325 nm, the chromatographic column is Promosil C18 (4.6 mm × 250 mm, 5 μm), the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution is used, the flow rate is 1.0 mL / min, and the column temperature is 30 ° C.
[0027] Step 4. Fingerprint construction and analysis: Record the chromatogram of the test solution from 0 to 70 minutes, and use the Chinese Pharmacopoeia Committee's Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System to import the chromatogram of the test solution, perform multi-point correction and data matching, and obtain the fingerprint and perform similarity analysis.
[0028] The gradient elution procedure is as follows: At 0 min, mobile phase A was 12% acetonitrile solution, and mobile phase B was 88% 0.1% phosphoric acid in water; At 10 min, mobile phase A was 18% acetonitrile solution, and mobile phase B was 82% 0.1% phosphoric acid in water; At 25 min, mobile phase A was 20% acetonitrile solution, and mobile phase B was 80% 0.1% phosphoric acid in water; At 45 min, mobile phase A was 50% acetonitrile solution, and mobile phase B was 50% 0.1% phosphoric acid in water; At 60 min, mobile phase A was 50% acetonitrile solution, and mobile phase B was 50% 0.1% phosphoric acid in water; At 70 minutes, mobile phase A was 60% acetonitrile solution, and mobile phase B was 40% 0.1% phosphoric acid aqueous solution.
[0029] There are 20 common peaks in the fingerprint, of which peak 17 is the reference peak, namely 7-demethylcorticoidin. The relative retention times of the peaks are: Peak 1: 0.160; Peak 2: 0.172; Peak 3: 0.265; Peak 4: 0.378; Peak 5: 0.398; Peak 6: 0.406; Peak 7: 0.477; Peak 8: 0.508; Peak 9: 0.577; Peak 10: 0.601; Peak 11: 0.617; Peak 12: 0.630; Peak 13: 0.681; Peak 14: 0.713; Peak 15: 0.734; Peak 16: 0.956; Peak 17 is the reference peak; Peak 18: 1.260; Peak 19: 1.278; Peak 20: 1.307.
[0030] In an embodiment of the present invention 1. Material selection: Test medicinal materials: S1: Sandagou, Hunchun; S2: Liangshui, Tumen; S3: West slope of Changbai Mountain; S4: West of Liangshui River; S5: Chunhua, Hunchun; S6: East of Liangshui River; S7: Shengli, Hunchun; S8: Daejeon, South Korea; S9: Iksan, South Korea; S10: Tianqiaoling, Wangqing.
[0031] Reference substances: chlorogenic acid reference substance (Shanghai Yuanye Biotechnology Co., Ltd.); ferulic acid reference substance (Shanghai Yuanye Biotechnology Co., Ltd.); 7-demethylcorticosterol reference substance (Shanghai Yuanye Biotechnology Co., Ltd.); purpurogenol reference substance (Baoji Chenguang Biotechnology Co., Ltd.); purpurogenol angelic acid ester reference substance (Baoji Chenguang Biotechnology Co., Ltd.).
[0032] Reagents: phosphoric acid (analytical grade, Beijing Hongjing Chemical Factory); formic acid (analytical grade, Tianjin Komeiou Chemical Reagent Co., Ltd.); acetic acid (glacial acetic acid, analytical grade, Gongzhuling Chemical Reagent Factory); trifluoroacetic acid (chromatographic grade, Shanghai Aladdin Biochemical Technology Co., Ltd.); ethanol (analytical grade, Tianjin Komeiou Chemical Reagent Co., Ltd.); methanol (chromatographic grade, Shenyang Huadong Reagent Factory); acetonitrile (chromatographic grade, Tianjin Huadong Reagent Factory); ultrapure water (homemade).
[0033] Instruments: FA2004 electronic balance (Shanghai Liangping Instrument Co., Ltd.); filtration vacuum device (Zhengzhou Great Wall Science and Technology Industry and Trade Co., Ltd.); RRH-100 universal high-speed crusher (Okailife (Hong Kong) Industrial Co., Ltd.); pipette (100 L, Henan Brothers Instrument Equipment Co., Ltd.); pipette (200 L, France); Primaide-5430 secondary array tube (DAD) detector (Hitachi, Japan); 2400 high-performance liquid chromatograph (Hitachi, Japan); ultrasonic cleaning degasser (Kunshan Ultrasonic Instrument Co., Ltd.); Primaide-1410 UV detector (Hitachi, Japan); Primaide chromatography workstation (Hitachi, Japan).
[0034] 2. Methods and Results 2.1.1 Selection of detection wavelength In order to obtain the most abundant information on the chemical components of Angelica sinensis in the fingerprint, a diode array detector (DAD) was used to scan the entire band from 200 to 400 nm. After a comprehensive investigation of the peak area, peak shape and number of chromatographic peaks, it was found that the peak shape and separation were good when the detection wavelength was 325 nm. Therefore, 325 nm was used as the detection wavelength to establish the HPLC fingerprint.
[0035] 2.1.2 Mobile phase selection Gradient elution was performed using methanol-water and acetonitrile-water systems as the mobile phases, respectively. The results showed that the chromatogram of Korean Angelica obtained by elution with acetonitrile-water system was better, but the separation was not ideal. In order to improve the separation, formic acid, acetic acid, phosphoric acid, and trifluoroacetic acid were added to the aqueous phase to improve the peak shape and separation. The results showed that the peak shape was better when 0.1% phosphoric acid aqueous solution was used as the mobile phase. Therefore, acetonitrile-0.1% phosphoric acid aqueous solution was selected as the mobile phase, and the elution time program is shown in Table 1.
[0036] 2.1.3 Determination of experimental conditions The chromatographic column was Promosil C18 (4.6 mm × 250 mm, 5 m); the flow rate was 1.0 mL / min; the column temperature was 30°C; the detection wavelength was 325 nm; the injection volume was 10 L; the mobile phase was acetonitrile-0.1% phosphoric acid water; and the detection time was 70 min.
[0037] 2.2 Preparation of sample solution 2.2.1 Preparation of reference solution Accurately weigh 2 mg of each of the reference substances: chlorogenic acid, ferulic acid, 7-demethylxanthin, purpurogenol, and purpurogenol angelate. Place the solution in a 10 mL volumetric flask. Add 70% ethanol, shake well, and dilute to the mark to a concentration of 200 g / mL. Take appropriate amounts of each and mix to obtain a reference solution. Filter through a 0.45 µm microporous membrane and set aside.
[0038] 2.2.2 Preparation of test solution Accurately weigh 0.5 g of Korean Angelica sinensis powder (passed through a 40-mesh sieve) and place it in a stoppered conical flask. Accurately add 10 mL of 70% ethanol, tightly plug the stopper, weigh its mass, ultrasonicate for 40 minutes, cool to room temperature, make up the lost weight with 70% ethanol, shake well, let it stand, take the supernatant and filter it through a microporous filter membrane (0.45 μm), and take the filtrate to obtain the product.
[0039] 2.3 Establishment of Korean Angelica fingerprint like Figure 1 、 Figure 2 As shown, according to the above chromatographic conditions, batches 1 to 10 of the test samples were sampled and analyzed, and 20 common peaks with a separation degree greater than 1.0 were determined; by comparing the UV absorption spectrum, retention time and the addition of mixed reference solution to the test samples, it was confirmed that peaks 1, 4, 17, 18, and 19 were the chromatographic peaks of chlorogenic acid, ferulic acid, 7-demethyledulosin, purpurogenol, and purpurogenol angelic acid ester, respectively; among them, peak 17 (7-demethyledulosin) had a stable retention time and no interference from other peaks, so it was used as the reference peak to calculate the relative retention times and relative peak areas of the 20 common peaks, as shown in Tables 2 and 3; wherein relative retention time = retention time of each component peak / retention time of the reference peak, relative peak area = peak area of each component peak / peak area of the reference peak.
[0040] Table 2 Relative retention times of common peaks in the fingerprints of 10 batches of Korean Angelica Table 3 Relative peak areas of common peaks in the fingerprints of 10 batches of Korean Angelica As shown in Tables 2 and 3, the RSD of the relative retention time of the common peaks in the HPLC fingerprints of 10 batches of Korean Angelica from different origins was 0.054%-0.300%, which was less than 3%, indicating good reproducibility; however, the RSD value of the relative peak area was 27%-92%, which showed significant differences. This indicates that the chemical component contents of the 10 batches of Korean Angelica from different origins were different due to their different growth environments.
[0041] 2.4 Similarity Calculation and Comparison Prepare the test sample solution according to the method under "2.2" and inject the sample into the system using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A Edition" software provided by the Chinese Pharmacopoeia Committee. Import the chromatogram data of 1 to 10 batches of test samples into the system, set S1 as the reference spectrum, and use the "median" method to generate the reference spectrum. After multi-point correction and automatic matching, a common pattern was established. Based on the matched data, 20 common peaks (such as Figure 6 ), and calculated the similarity between Korean Angelicae Sinensis from different origins and the common pattern. The results are shown in Table 4.
[0042] 2.5 Methodological Validation 2.5.1 Precision experiment Accurately pipette 10 L of the same sample solution and inject it 5 times in a row according to the chromatographic conditions under 2.1. Use the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A Version" software to examine and calculate the retention time and peak area of each major chromatographic peak with Peak 17 (7-demethylcorticoidin) as the reference peak. The results show that the standard deviation of the relative retention time and relative peak area of each major chromatographic peak is less than 3%, indicating that the instrument has good precision. Figure 3 , Table 5.
[0043] Table 5-1 Precision test results of Korean Angelica fingerprint (relative retention time) Table 5-2 Precision test results of Korean Angelica fingerprint (relative peak area) 2.5.2 Repeatability Experiment Five aliquots of the same test solution were accurately drawn and analyzed using the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A Version" software, using Peak 17 (7-demethylcorticoidin) as the reference peak. The retention time and peak area of each major chromatographic peak were calculated. The results showed that the standard deviation of the relative retention time and relative peak area of each major chromatographic peak was less than 3%, indicating good reproducibility of the method. Figure 4 , Table 6) Table 6-1 Results of the reproducibility test of the fingerprint of Korean Angelica sinensis (relative retention time) Table 6-2 Results of the reproducibility test of the fingerprint spectrum of Korean Angelica sinensis (relative peak area) 2.5.3 Stability test Accurately pipette 10 L of the same test solution and inject it at 0 h, 4 h, 8 h, 12 h, and 24 h according to the chromatographic conditions under 2.1. Use the "Chinese Herbal Medicine Chromatographic Fingerprint Similarity Evaluation System 2004A Version" software to examine and calculate the retention time and peak area of each major chromatographic peak with Peak 17 (7-demethylcorticoidin) as the reference peak. The results show that the standard deviation of the relative retention time and relative peak area of each major chromatographic peak is less than 3%, indicating that the test solution is stable within 24 h. Figure 5 , Table 7) Table 7-1 Results of stability test of Korean Angelica fingerprint (relative retention time) Table 7-2 Results of stability test of Korean Angelica fingerprint (relative peak area) 2.6 Cluster Analysis The test solution was prepared according to the method under "2.2" and sampled for analysis. The peak area values of the 10 batches of samples were used as variables. SPSS 19.0 statistical analysis software was used to perform a systematic cluster analysis on the samples. Euclidean distance was used as the sample measurement, and the vertical axis was the sample number. The cluster analysis results of 10 batches of Korean Angelica medicinal materials were obtained, see Figure 8 According to the cluster analysis results, Korean angelica medicinal materials from different origins can be clustered into six categories. S2 and 3 are clustered into one category, S6 and 7 are clustered into one category, S4 and 5 are clustered into one category, S8 and 9 are clustered into one category, S10 is clustered into one category, and S1 is clustered into one category, which can be used as a reference for quality evaluation.
[0044] 3. Summary This study established a high-performance liquid chromatography (HPLC) fingerprint detection method for Korean angelica. By systematically optimizing chromatographic conditions (detection wavelength 325 nm, Promosil C18 column, acetonitrile-0.1% phosphoric acid gradient elution), 20 common peaks were identified from 10 batches of Korean angelica from different origins, with Peak 17 (7-demethylcorticoidin) serving as the reference peak. The relative retention time RSDs of all peaks were less than 3%, demonstrating good reproducibility. The common peaks accounted for over 90% of the fingerprint area, while the non-common peaks accounted for less than 10%. Similarity evaluation showed that the similarity of samples from different origins was greater than 0.90, demonstrating the method's significant specificity and systematicity.
[0045] Methodological validation results demonstrated that the method demonstrated excellent precision, reproducibility, and stability, with the sample solution remaining stable for 24 hours, making it reliable for component analysis of Korean angelica. Combined with cluster analysis, 10 sample batches were clustered into six categories based on origin, providing data support for quality grading and origin traceability of the medicinal material.
[0046] This fingerprint detection method breaks through the limitations of traditional single ingredient detection and realizes the simultaneous analysis of multiple active ingredients such as chlorogenic acid, ferulic acid, and purpurogenol in Korean angelica. It provides a scientific basis for the authenticity identification, quality control and standardized production of Korean angelica, and is of great significance to promoting the standardized development of Korean ethnic medicinal materials.
[0047] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. All equivalent structural transformations made by using the contents of the present invention description and drawings under the inventive concept of the present invention, or direct / indirect application in other related technical fields are included in the patent protection scope of the present invention.
Claims
1. A HPLC fingerprint detection method for Angelica sinensis, characterized in that: The following steps are involved: Step 1: Preparation of test solution: Grind the Korean angelica root to be tested, extract with 70% ethanol by ultrasonic, and filter to obtain the test solution; Step 2, preparation of reference solution: taking chlorogenic acid, ferulic acid, 7-demethyl cyperifoliin, peucedanum danshol and peucedanum danshol angelic acid ester reference substances, dissolving them in 70% ethanol, and diluting to volume to prepare the reference solution; Step 3, high performance liquid chromatography detection: accurately aspirate the test solution and the reference solution, inject them into a high performance liquid chromatograph for detection, the high performance liquid chromatograph contains a UV detector, the detection wavelength is set to 325 nm, the chromatographic column is Promosil C18 (4.6 mm × 250 mm, 5 μm), the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution is used, the flow rate is 1.0 mL / min, and the column temperature is 30 ° C; Step 4. Fingerprint construction and analysis: Record the chromatogram of the test solution from 0 to 70 minutes, and use the Chinese Pharmacopoeia Committee's Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System to import the chromatogram of the test solution, perform multi-point correction and data matching, and obtain the fingerprint and perform similarity analysis.
2. The HPLC fingerprint detection method for Angelica sinensis according to claim 1, wherein: The test solution is prepared as follows: 0.5 g of Angelica sinensis powder (passed through a 40-mesh sieve) is accurately weighed, 10 mL of 70% ethanol is added, and ultrasonic extraction is performed for 40 minutes. The weight loss is compensated and then filtered to obtain the test solution.
3. The HPLC fingerprint detection method for Angelica sinensis according to claim 1, wherein: The reference solution is prepared as follows: 2 mg each of chlorogenic acid, ferulic acid, 7-demethyl cyperus rotundus, peucedanum arvense and peucedanum angelic acid ester reference substances are taken, the volume is diluted to 10 mL with 70% ethanol, and the mixture is filtered to obtain the reference solution.
4. The HPLC fingerprint detection method for Angelica sinensis according to claim 1, wherein: The procedure of the gradient elution is as follows: At 0 min, mobile phase A was 12% acetonitrile solution, and mobile phase B was 88% 0.1% phosphoric acid in water; At 10 min, mobile phase A was 18% acetonitrile solution, and mobile phase B was 82% 0.1% phosphoric acid in water; At 25 min, mobile phase A was 20% acetonitrile solution, and mobile phase B was 80% 0.1% phosphoric acid in water; At 45 min, mobile phase A was 50% acetonitrile solution, and mobile phase B was 50% 0.1% phosphoric acid in water; At 60 min, mobile phase A was 50% acetonitrile solution, and mobile phase B was 50% 0.1% phosphoric acid in water; At 70 minutes, mobile phase A was 60% acetonitrile solution, and mobile phase B was 40% 0.1% phosphoric acid aqueous solution.
5. The HPLC fingerprint detection method for Angelica sinensis according to claim 1, wherein: The fingerprint has 20 common peaks, of which peak 17 is the reference peak, i.e. 7-demethylcorticoidin. The relative retention times of the peaks are: Peak 1: 0.160; Peak 2: 0.172; Peak 3: 0.265; Peak 4: 0.378; Peak 5: 0.398; Peak 6: 0.406; Peak 7: 0.477; Peak 8: 0.508; Peak 9: 0.577; Peak 10: 0.601; Peak 11: 0.617; Peak 12: 0.630; Peak 13: 0.681; Peak 14: 0.713; Peak 15: 0.734; Peak 16: 0.956; Peak 17 is the reference peak; Peak 18: 1.260; Peak 19: 1.278; Peak 20: 1.
307.
6. The HPLC fingerprint detection method for Angelica sinensis according to claim 1, wherein: The relative retention peak areas of the common peaks in the fingerprint are: Peak 1: 2.924; Peak 2: 0.065; Peak 3: 0.423; Peak 4: 0.163; Peak 5: 0.287; Peak 6: 0.679; Peak 7: 0.053; Peak 8: 0.887; Peak 9: 0.366; Peak 10: 1.676; Peak 11: 0.200; Peak 12: 0.255; Peak 13: 0.210; Peak 14: 0.087; Peak 15: 0.085; Peak 16: 0.043; Peak 17 is a reference peak; Peak 18: 24.332; Peak 19: 22.403; Peak 20: 0.
286.
7. The HPLC fingerprint detection method for Angelica sinensis according to claim 1, wherein: The injection volume of the HPLC detection was 10 μL, and the detection time was 70 min.
8. The detection method according to any one of claims 1 to 7, characterized in that The Korean angelica is the dried root of the Umbelliferae plant Korean angelica, which is collected from various places in South Korea and Northeast China.
9. An HPLC fingerprint of Angelica sinensis obtained by the method according to any one of claims 1 to 8, characterized in that: The fingerprint spectrum contains 20 common peaks, with 7-demethylated cork pepper as the reference peak, and the relative retention time and relative peak area are as described in claims 5-6.
10. Application of the HPLC fingerprint of claim 9 in authenticity identification, quality control and origin tracing of Korean Angelica sinensis.