Method for simultaneously detecting various phenolic acids and flavonoid active substances in perilla frutescens

Abstract

The invention provides a method for simultaneously detecting various phenolic acids and flavonoid active substances in perilla frutescens, which is characterized in that perilla leaves, stems and seeds are taken as detection objects, an ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) technology is adopted, a mixed reference substance solution and a to-be-detected sample solution are prepared, chromatography-mass spectrometry detection parameters are optimized, and the content of various phenolic acids and flavonoid active substances in the perilla frutescens is determined. 15 active substances can be accurately quantified at the same time; the method has the advantages that the pretreatment is simple and convenient, the analysis time is only 10 minutes, the detection sensitivity is high, the detection limit is as low as 0.002 g / L, the linear relation is good, R2 is greater than or equal to 0.9987, the precision, the accuracy and the stability all meet the detection standard, and the detection range covers 1-5000 g / L. The method provides reliable technical support for quality evaluation, product control and standardized production of perilla frutescens, is suitable for detection of perilla frutescens related products, and promotes industrial development of medicinal and edible substances.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine component detection technology, specifically to a method for simultaneously detecting multiple phenolic acids and flavonoid active substances in perilla. Background Technology

[0002] Perilla ( Perilla frutescens (L.) Britt. is an annual herb belonging to the genus Perilla in the family Lamiaceae. It was among the first plants in my country recognized as having both edible and medicinal properties, with its leaves, stems, and seeds all possessing significant edible and medicinal value. It is included in the Pharmacopoeia of the People's Republic of China. In terms of consumption, perilla leaves can be eaten directly or used as a condiment and raw material for health foods. Perilla oil, pressed from perilla seeds, is rich in α-linolenic acid and has excellent health benefits. In terms of medicinal use, perilla leaves relieve exterior syndromes and dispel cold, regulate qi and harmonize the stomach; perilla stems regulate qi, relieve chest tightness, alleviate pain, and calm the fetus; perilla seeds lower qi, resolve phlegm, relieve cough and asthma, and moisten the intestines to promote bowel movements. The entire plant has significant medicinal value.

[0003] The "food and medicine homology" characteristic of perilla is closely related to its various active ingredients, among which phenolic acids and flavonoids are the core active components, possessing multiple biological activities such as antioxidation, anti-inflammation, antibacterial, anti-allergy, antiviral, antitumor, neuroprotection, and cardiovascular protection. With increasing public emphasis on healthy eating and high-quality raw materials, the need for quality control of perilla and its products is becoming increasingly urgent, and the accurate quantification of active ingredients is a key aspect of perilla quality evaluation.

[0004] Existing methods for detecting active ingredients in perilla mainly employ high-performance liquid chromatography-ultraviolet spectrometry (HPLC-UV). However, this method has significant drawbacks: First, it detects only a single component, typically only 3-5 components, making it difficult to comprehensively reflect the quality of perilla. Second, the detection time is long, requiring 30-60 minutes for a single analysis, resulting in low efficiency and failing to meet the needs of batch sample testing. Third, the sensitivity is low, with detection limits mostly above 5 μg / L, making it difficult to detect low-content active ingredients. Fourth, the detection range is narrow, unable to adapt to the significant differences in content of active ingredients in different parts of perilla (leaves, stems, and seeds), and the various active ingredients in perilla also vary considerably due to different origins, varieties, processing methods, and other factors.

[0005] Therefore, there is a need for a detection method with higher sensitivity, selectivity, and accuracy, a wider detection range, and the ability to handle large differences in content. Summary of the Invention

[0006] The present invention aims to at least partially solve one of the technical problems in the above-mentioned technologies, namely, to provide a method for simultaneously detecting multiple phenolic acids and flavonoids in perilla, thereby solving the problems of limited component coverage, low efficiency, insufficient sensitivity, and narrow detection range of existing detection methods, and providing reliable technical support for the quality evaluation, product control, standardized production, and efficient utilization of resources of perilla.

[0007] Therefore, this invention proposes a method for simultaneously detecting multiple phenolic acids and flavonoids in perilla, which includes the following steps: Preparation of mixed reference solutions: Weigh out 6 kinds of phenolic acid standards and 9 kinds of flavonoid standards respectively, prepare single substance standard stock solutions with methanol as solvent, mix the single substance standard stock solutions, and dilute to volume with methanol to obtain mixed reference solutions; then, use 75% methanol as solvent to prepare a series of mixed reference solutions with different known concentrations. Preparation of the test sample solution: After drying the perilla sample, crush it and sieve it. Take the perilla sample powder, add 75% methanol, vortex oscillate, sonicate, cool and vortex oscillate again. Take the supernatant and filter it through a membrane to obtain the test sample solution. Sample determination and content calculation: The mixed reference solution and the test sample solution were injected into an ultra-high performance liquid chromatography-tandem mass spectrometer for determination. A standard curve was plotted with the content of each active substance in the mixed reference solution as the x-axis and the corresponding quantitative ion peak area as the y-axis. Based on the quantitative ion peak area of ​​each active substance in the test sample solution and the standard curve, the content of 15 active substances in the test sample was calculated.

[0008] According to the method of the present invention, this method uses ultra-high performance liquid chromatography-tandem mass spectrometry to determine the content of 6 phenolic acids and 9 flavonoids in perilla. The method features simple pretreatment (requiring only three steps: pulverization, extraction, and filtration, without complex purification steps), rapid analysis, high sensitivity, good accuracy and precision, and a wide detection range. It can simultaneously analyze 15 active ingredients and is suitable for both high-content (e.g., baicalin) and low-content (e.g., quercetin) components. It is applicable to the detection of all components in perilla leaves, stems, and seeds, providing a more comprehensive reflection of perilla quality. The method has a limit of detection (LOD) as low as 0.002 μg / L and a limit of quantitation (LOQ) of 0.007–3.980 μg / L, far superior to traditional methods. The linear correlation coefficient R² ≥ 0.9987, intra-day precision RSD 1.99–3.88%, and inter-day precision RSD The concentration ranges from 2.86% to 9.96%, with spiked recoveries ranging from 89.75% to 109.51%, meeting the requirements for trace component detection and quantitative accuracy. This provides a technical basis for the quality evaluation and product control of perilla, and promotes the healthy development of food-medicine homologous substances in China.

[0009] Optionally, the perilla sample includes perilla leaves, perilla stems, and perilla seeds.

[0010] Optionally, phenolic acids include rosmarinic acid, caffeic acid, ferulic acid, gallic acid, chlorogenic acid, and protocatechuic acid; flavonoids include luteolin, luteolinin, baicalin, apigenin, catechin, rutin, quercetin, kaempferol, and isorhamnetin.

[0011] Optionally, the chromatographic parameters of the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) are as follows: C18 column, column temperature 35–45 °C, injection volume 0.8–1.2 µL, flow rate 0.25–0.35 mL / min; mobile phase includes phase A and phase B, phase A being 0.08–0.12% formic acid in water and phase B being 0.08–0.12% formic acid in acetonitrile; the injection solution is a mixture of methanol-acetonitrile-isopropanol-water with a volume ratio of 1:1:1:1; the gradient elution program is as follows: 0–0.5 min, 5% B; 0.5–6 min, 5%–45% B; 6–6.01 min, 45%–70% B; 6.01–8 min, hold at 70% B; 8–8.01 min, 70%–5% B; 8.01–10 min, hold at 5% B until stop. These chromatographic conditions enable the effective separation of 15 active substances within 10 minutes, reducing the analysis time by 3 to 6 times compared to the traditional HPLC-UV method, thus meeting the needs of batch sample detection.

[0012] Optionally, the mass spectrometry parameters of the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) are as follows: ion source: heated electrospray ionization; scanning polarity: positive and negative ion switching mode; spray voltage: 4000 V for positive ions, 3500 V for negative ions; sheath gas flow rate: 4.58 L / min; auxiliary gas flow rate: 12.11 L / min; ion transport capillary temperature: 320℃; vapor temperature: 100℃; scanning mode: selective reaction monitoring; collision gas: high-purity argon, pressure: 0.2 Pa; scan cycle time: 1 s.

[0013] Furthermore, the characteristic ions and corresponding mass spectrometry parameters of the 15 active ingredients are shown in Table 1 below: Table 1 Mass spectrometry parameters of various active ingredients

[0014] Note: These are quantitative ions.

[0015] Optionally, in the sample solution to be tested, the ratio of perilla sample powder to 75% methanol is 1:80~1:120 g:mL; the parameters for vortex oscillation are: 2~4 min for the first vortex oscillation, and 20~40 s for vortex oscillation after ultrasonic treatment; the conditions for ultrasonic treatment are: temperature 35~45℃, power 200~300 W, frequency 45~55 kHz, and treatment time 25~35 min; the filter membrane is a 0.20~0.24 µm PALL GHP filter membrane.

[0016] Furthermore, the preparation parameters for single-substance standard stock solutions are as follows: the stock solution concentrations for rosmarinic acid, caffeic acid, gallic acid, chlorogenic acid, protocatechuic acid, ferulic acid, luteolin, catechin, rutin, and kaempferol are 1000 µg / mL; the stock solution concentrations for luteolinoside, baicalin, apigenin, quercetin, and isorhamnetin are 100 µg / mL; the mass concentration of the mixed reference solution is 10 μg / mL; the concentration range of the series of mixed reference solutions is: rosmarinic acid 10~5000 µg / L, caffeic acid 1~5000 µg / L, ferulic acid 10~5000 µg / L, gallic acid 10~5000 µg / L, chlorogenic acid 10~5000 µg / L, protocatechuic acid 10~5000 µg / L, luteolinoside 10~5000 µg / L. µg / L, luteolin 10~5000 µg / L, baicalin 5~5000 µg / L, apigenin 1~2000 µg / L, catechin 10~5000 µg / L, rutin 5~5000 µg / L, quercetin 1~5000 µg / L, kaempferol 1~5000 µg / L, isorhamnetin 1~5000 µg / L.

[0017] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0018] Figure 1 The total ion chromatogram of 15 active substances in the mixed control standard according to an embodiment of the present invention is shown. Figure 2 The extraction ion chromatogram of 15 active substances in the mixed reference standard according to an embodiment of the present invention; Figure 3 The total ion chromatogram of 15 active substances in the test sample perilla leaves according to an embodiment of the present invention; Figure 4 The above is an extraction ion chromatogram of 15 active substances in the test sample perilla leaves according to an embodiment of the present invention; Figure 5 The total ion chromatogram of 15 active substances in perilla seeds, the sample to be tested according to an embodiment of the present invention; Figure 6 The above is an extraction ion chromatogram of 15 active substances in perilla seeds, a sample to be tested according to an embodiment of the present invention. Figure 7 The total ion chromatogram of 15 active substances in the perilla stem sample according to an embodiment of the present invention is shown. Figure 8 The above is an extraction ion chromatogram of 15 active substances in the perilla stem sample according to an embodiment of the present invention. In the diagram, the numbers represent: 1. Gallic acid; 2. Protocatechuic acid; 3. Catechin; 4. Chlorogenic acid; 5. Caffeic acid; 6. Rutin; 7. Baicalin; 8. Luteolin; 9. Ferulic acid; 10. Rosmarinic acid; 11. Luteolin; 12. Quercetin; 13. Apigenin; 14. Kaempferol; 15. Isorhamnetin. Detailed Implementation

[0019] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0020] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0021] The test materials used in this invention are all common commercial products, which can be purchased on the market or prepared by known methods.

[0022] Instruments: Thermo U3000 ultra-high performance liquid chromatograph (Thermo Scientific, USA), TSQ Endura triple quadrupole mass spectrometer (Thermo Scientific, USA); Vortex Genius 3 circular oscillator (IKA GmbH, Germany); multi-functional pulverizer (Ningbo Zhao Ji Electric Appliance Co., Ltd.); BP 211D 0.0001 g balance (Sartorius Corporation); TP-202 electronic balance (Beijing Sartorius Scientific Instruments Co., Ltd.); KQ-500DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd.).

[0023] Materials: The raw materials for the experiment, perilla leaves, seeds and stems, were produced in Sanming City, Fujian Province, and collected from the planting base of Mingbawei Industrial Research Institute in Sanming City.

[0024] Reagents: Rosmarinic acid (CAS No.: 20283-92-5, batch number 2421348, purity 99.9%), caffeic acid (CAS No.: 331-39-5, batch number 2425349, purity 99.0%), ferulic acid (CAS No.: 1135-24-6, batch number 2560280, purity 99.8%), gallic acid (CAS No.: 149-91-7, batch number 2430867, purity 99.8%). The following compounds are present: chlorogenic acid (CAS No.: 327-97-9, batch number: 2590287, purity 98.6%), protocatechuic acid (CAS No.: 99-50-3, batch number: 2429543, purity 97.6%), luteolin (CAS No.: 5373-11-5, batch number: 2563357, purity 98.0%), and luteolin (CAS No.: 491-70-3, batch number: 2433292, purity 99.7%). The following compounds are listed: 97.8% purity, baicalin (CAS No.: 27740-01-8, batch number 2576010, purity 94.9%), apigenin (CAS No.: 520-36-5, batch number 2567295, purity 95.9%), catechin (CAS No.: 154-23-4, batch number 2564122, purity 98.0%), and quercetin (CAS No.: 117-39-5, batch number 2420393). The following substances were used: Kaempferol (CAS No.: 480-19-3, batch number: 2566069, purity 98.9%) (Shanghai Anpuyun Experimental Supplies Co., Ltd.); Kaempferol (CAS No.: 520-18-3, batch number: KB372599, purity 98.2%) and Rutin (CAS No.: 153-18-4, batch number: KB384983, purity 98.9%) (Shanghai Yuanye Biotechnology Co., Ltd.). PALLGHP Acrodisc needle filter (13 mm × 0.22 μm, Waters, USA); Acetonitrile and methanol (chromatographic grade, Tedia, USA); Formic acid and acetic acid (analytical grade, Shanghai Guoyao Group Chemical Reagent Co., Ltd.).

[0025] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0026] Example Preparation of mixed reference solution Accurately weigh 10 mg each of the six phenolic acid standards and the nine flavonoid standards (accurate to 0.01 mg), and prepare single-substance standard stock solutions using methanol as solvent. The mass concentrations are as follows: 1000 μg / mL for rosmarinic acid, caffeic acid, gallic acid, chlorogenic acid, protocatechuic acid, ferulic acid, luteolin, catechin, rutin, and kaempferol; and 100.0 μg / mL for luteolinoside, baicalin, apigenin, quercetin, and isorhamnetin. Store at 4°C protected from light.

[0027] Take 100 μL each of the standard stock solutions of rosmarinic acid, caffeic acid, gallic acid, chlorogenic acid, protocatechuic acid, ferulic acid, luteolin, catechin, rutin, and kaempferol, and 1 mL each of the standard stock solutions of luteolinoside, baicalin, apigenin, quercetin, and isorhamnetin, and place them in a 10 mL volumetric flask. Dilute to volume with methanol to prepare a mixed reference solution with a mass concentration of 10 μg / mL.

[0028] Using 75% methanol as solvent, prepare 8-12 series of mixed reference solutions containing different known concentrations of the following: rosmarinic acid 10-5000 µg / L, caffeic acid 1-5000 µg / L, ferulic acid 10-5000 µg / L, gallic acid 10-5000 µg / L, chlorogenic acid 10-5000 µg / L, protocatechuic acid 10-5000 µg / L, luteolin 10-5000 µg / L, baicalin 5-5000 µg / L, apigenin 1-2000 µg / L, catechin 10-5000 µg / L, rutin 5-5000 µg / L, quercetin 1-5000 µg / L, and kaempferol 1-5000 µg / L. µg / L, isorhamnetin 1~5000 µg / L.

[0029] Preparation of the sample solution to be tested After the perilla sample was naturally air-dried, it was pulverized using a pulverizer, and the powder was passed through a 20-mesh sieve for later use. Take 0.2 g of perilla powder (accurate to 0.0001 g), add 20 mL of 75% methanol (accurate to 0.01 mL), seal tightly, vortex for 3 min, sonicate (power 250 W; frequency 50 kHz) for 30 min, cool to room temperature, vortex for 30 s to mix, take 1 mL of the supernatant, and filter through a 0.22 µm PALL GHP membrane to obtain the sample to be tested. The perilla sample included perilla leaf samples, perilla stem samples, and perilla seed samples. The PALL GHP membrane has broad chemical compatibility with solutions of water and organic solvents, and is suitable for the 75% methanol extraction solvent used in this example.

[0030] Instrument testing: 1 µL of the mixed reference standard and the test sample solution were injected into the ultra-high performance liquid chromatography-tandem mass spectrometry for determination. A standard curve was plotted with the content of each active substance in the mixed reference standard solution as the abscissa (x) and the peak area of ​​the corresponding quantitative ion as the ordinate (y). Based on the peak area of ​​the quantitative ion of each active substance in the test sample solution and the established standard curves of each active ingredient, the content of 15 active substances in the test sample was calculated.

[0031] The chromatographic conditions were as follows: ACCQ-TAG ULTRA C18 column (2.1 mm × 100 mm, 1.7 μm); column temperature: 40℃; injection volume: 1 µL; flow rate: 0.3 mL / min; mobile phase: phase A was 0.1% formic acid in water, phase B was 0.1% formic acid in acetonitrile; syringe wash buffer: methanol-acetonitrile-isopropanol-water (1:1:1:1); gradient elution conditions: 0–0.5 min, 5% B; 0.5–6 min, 5%–45% B; 6–6.01 min, 45%–70% B; 6.01–8 min, hold at 70% B; 8–8.01 min, 70%–5% B; 8.01–10 min, hold at 5% B until stop. These chromatographic parameters enabled the effective separation of 15 active substances within 10 min. The syringe wash buffer effectively cleaned the injection needle without leaving any residue.

[0032] Mass spectrometry conditions: Ion source: heated electrospray ionization (H-ESI); Scan polarity: positive and negative ion switching mode; Spray voltage: 4000 V for positive ions, 3500 V for negative ions; Sheath gas: 4.58 L / min; Auxiliary gas: 12.11 L / min; Ion transport capillary temperature: 320℃; Vapor temperature: 100℃; Scan mode: selective reaction monitoring; Collision gas: high-purity argon (purity >99.995%), pressure: 0.2 Pa. Scan cycle time: 1 s. The characteristic ions and corresponding mass spectrometry parameters of the 15 active ingredients are shown in Table 1.

[0033] Table 1 Mass spectrometry parameters of various active ingredients

[0034] Note: These are quantitative ions.

[0035] Data processing The total ion chromatogram and extracted ion chromatogram of 15 active substances in a 1000 µg / L mixed reference solution are as follows: Figure 1 and Figure 2A standard curve was plotted with the content of each active substance as the abscissa (x) and the peak area of ​​the corresponding quantitative ion as the ordinate (y). The linear equation and correlation coefficient were calculated. The limit of quantitation (LOD) and limit of quantitation (LOQ) were determined based on signal-to-noise ratios S / N=3 and S / N=10. The results are shown in Table 2. All 15 active substances showed good linearity within their respective linear ranges. 2 ≥0.9987.

[0036] Table 2. Determination of linear relationships for various active substances

[0037] The accuracy, precision, repeatability, stability, and spike recovery of the method were investigated. Precision and accuracy assessment: A mixed reference solution with a mass concentration of 1000 µg / L was prepared and injected six times consecutively within one day for three consecutive days. The relative standard deviation (RSD) was calculated using the results of the six mixed reference solutions on the same day to assess intra-day precision; the RSD was calculated using the results of the mixed reference solutions from three consecutive days to assess inter-day precision. The intra-day and inter-day precision results are shown in Table 3. The intra-day precision RSD for the 15 active substances was 1.99–3.88%, and the inter-day precision RSD was 2.86–9.96%, indicating that the method of this application has good intra-day and inter-day precision.

[0038] Repeatability and stability studies: Six samples of perilla leaves from the same batch were used to prepare six parallel test solutions. The RSD of the results from these six parallel samples was calculated to assess the repeatability of the samples. One sample solution was then analyzed at 0, 2, 6, 10, 18, and 24 hours, and the RSD of the same sample at each time point was calculated to assess the sample stability of the method. The repeatability and stability results are shown in Table 3. The RSD range of the parallel determination of 15 active substances in the samples was all within 0.83%–8.86%, indicating that the method has good repeatability. The RSD range of the contents of the 15 active substances in the test samples within 24 hours was all within 0.89%–9.95%, indicating that the test solution was stable within 24 hours.

[0039] Spike recovery study: Six samples of perilla leaf with known content were accurately weighed and added in equal amounts to a mixed reference solution. Kaempferol and isorhamnetin, which were not detected, were added at a concentration of 50 µg / L to prepare the test solution. The peak area was measured, and the average recovery rate and RSD were calculated to evaluate the sample spike recovery rate of the method. The spike recovery results are shown in Table 3. The average recovery rates were all between 89.75% and 109.51%, and the RSDs were between 1.95% and 10.87%, indicating that the method of this application has good accuracy.

[0040] Table 3. Analysis of the accuracy, precision, repeatability, stability, and spiked recovery rate of the method.

[0041] Note: - indicates not detected, and no repeatability and stability studies were conducted.

[0042] Determination of actual samples: After the perilla leaves, stems, and seeds were naturally air-dried, they were placed in a cool, ventilated place for later use. Before the experiment, they were pulverized using a grinder, and the powder was passed through a 20-mesh sieve. Three portions of perilla leaf, stem, and seed powder (0.2 g each, accurate to 0.0001 g) were weighed out and analyzed according to the preparation method of the test solution. The total ion chromatogram and extracted ion chromatogram of 15 active substances in the perilla leaf were as follows. Figure 3 and Figure 4 The total ion chromatogram and extracted ion chromatogram of 15 active substances in the perilla seeds to be tested were as follows: Figure 5 and Figure 6 The total ion chromatogram and extracted ion chromatogram of 15 active substances in the perilla stem sample were as follows: Figure 7 and Figure 8 Based on the peak area of ​​the quantitative ions of each active substance in the test solution and the established standard curve, the content of the sample was calculated. The results are shown in Table 4. It can be seen that the content of different active ingredients varies greatly in the same part of Perilla frutescens, and the distribution of the same ingredient in different parts has a certain degree of diversity.

[0043] Table 4. Content of 15 active substances in different parts of perilla.

[0044] In summary, according to embodiments of the present invention, an analytical method for simultaneously detecting six phenolic acids and nine flavonoid active substances in perilla using ultra-high performance liquid chromatography-tandem mass spectrometry is provided. This method features simple pretreatment, rapid analysis, high sensitivity, good accuracy and precision, and a wide detection range, enabling the simultaneous analysis of 15 active substances with significant differences in content. It provides a technical basis for the quality evaluation and product control of perilla, and offers guidance for the cultivation and standardized production of perilla. It can be applied in multiple fields such as extract quality control, production process improvement, and finished product quality testing, providing a new reference for the efficient utilization of perilla resources and the development of functional products, and promoting the healthy development of food-medicine homologous substances in China.

[0045] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0046] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for simultaneously detecting multiple phenolic acids and flavonoids in perilla, characterized in that, Includes the following steps: Preparation of mixed reference solutions: Weigh out 6 kinds of phenolic acid standards and 9 kinds of flavonoid standards respectively, prepare single substance standard stock solutions with methanol as solvent, mix the single substance standard stock solutions, and dilute to volume with methanol to obtain mixed reference solutions; then, use 75% methanol as solvent to prepare a series of mixed reference solutions with different known concentrations. Preparation of the test sample solution: After drying the perilla sample, crush it and sieve it. Take the perilla sample powder, add 75% methanol, vortex oscillate, sonicate, cool and vortex oscillate again. Take the supernatant and filter it through a membrane to obtain the test sample solution. Sample determination and content calculation: The mixed reference solution and the test sample solution were injected into an ultra-high performance liquid chromatography-tandem mass spectrometer for determination. A standard curve was plotted with the content of each active substance in the mixed reference solution as the x-axis and the corresponding quantitative ion peak area as the y-axis. Based on the quantitative ion peak area of ​​each active substance in the test sample solution and the standard curve, the content of 15 active substances in the test sample was calculated.

2. The method as described in claim 1, characterized in that, The perilla sample includes perilla leaves, perilla stems, and perilla seeds.

3. The method as described in claim 1, characterized in that, Phenolic acids include rosmarinic acid, caffeic acid, ferulic acid, gallic acid, chlorogenic acid, and protocatechuic acid; flavonoids include luteolin, luteolinin, baicalin, apigenin, catechin, rutin, quercetin, kaempferol, and isorhamnetin.

4. The method as described in claim 1, characterized in that, The chromatographic parameters of the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) instrument were as follows: C18 column, column temperature 35–45 °C, injection volume 0.8–1.2 µL, flow rate 0.25–0.35 mL / min; mobile phase consisted of phase A (0.08–0.12% formic acid in water) and phase B (0.08–0.12% formic acid in acetonitrile); the eluent was a mixture of methanol, acetonitrile, isopropanol, and water at a volume ratio of 1:1:1:1; the gradient elution program was as follows: 0–0.5 min, 5% B; 0.5–6 min, 5%–45% B; 6–6.01 min, 45%–70% B; 6.01–8 min, hold at 70% B; 8–8.01 min, 70%–5% B; 8.01–10 min, hold at 5% B until stop.

5. The method as described in claim 1, characterized in that, The mass spectrometry parameters of the ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS) system were as follows: ion source: heated electrospray ionization; scanning polarity: positive and negative ion switching mode; spray voltage: 4000 V for positive ions, 3500 V for negative ions; sheath gas flow rate: 4.58 L / min; auxiliary gas flow rate: 12.11 L / min; ion transport capillary temperature: 320℃; vapor temperature: 100℃; scanning mode: selective reaction monitoring; collision gas: high-purity argon, pressure: 0.2 Pa; scan cycle time: 1 s.

6. The method as described in claim 5, characterized in that, The characteristic ions and corresponding mass spectrometry parameters of the 15 active ingredients are shown in Table 1 below: Table 1 Mass spectrometry parameters of various active ingredients Note: These are quantitative ions.

7. The method as described in claim 1, characterized in that, In the sample solution to be tested, the ratio of perilla sample powder to 75% methanol is 1:80~1:120 g:mL; the parameters for vortex oscillation are: 2~4 min for the first vortex oscillation, and 20~40 s for vortex oscillation after ultrasonic treatment; the conditions for ultrasonic treatment are: temperature 35~45℃, power 200~300 W, frequency 45~55 kHz, and treatment time 25~35 min; the filter membrane is a 0.20~0.24 µm PALL GHP filter membrane.

8. The method as described in claim 2, characterized in that, The preparation parameters for single-substance standard stock solutions are as follows: the stock solution concentrations for rosmarinic acid, caffeic acid, gallic acid, chlorogenic acid, protocatechuic acid, ferulic acid, luteolin, catechin, rutin, and kaempferol are 1000 µg / mL; the stock solution concentrations for luteolinoside, baicalin, apigenin, quercetin, and isorhamnetin are 100 µg / mL; the mass concentration of the mixed reference solution is 10 μg / mL; the concentration ranges of the series of mixed reference solutions are: rosmarinic acid 10~5000 µg / L, caffeic acid 1~5000 µg / L, ferulic acid 10~5000 µg / L, gallic acid 10~5000 µg / L, chlorogenic acid 10~5000 µg / L, protocatechuic acid 10~5000 µg / L, and luteolinoside 10~5000 µg / L. µg / L, luteolin 10~5000 µg / L, baicalin 5~5000 µg / L, apigenin 1~2000 µg / L, catechin 10~5000 µg / L, rutin 5~5000 µg / L, quercetin 1~5000 µg / L, kaempferol 1~5000 µg / L, isorhamnetin 1~5000 µg / L.

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