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CN-121994957-A - Method for simultaneously detecting various phenolic acids and flavonoid active substances in perilla

CN121994957ACN 121994957 ACN121994957 ACN 121994957ACN-121994957-A

Abstract

The invention provides a method for simultaneously detecting various phenolic acids and flavonoid active substances in perilla, which takes perilla leaves, stems and seeds as detection objects, adopts an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) technology, optimizes chromatographic-mass spectrometry detection parameters by preparing a mixed reference substance solution and a sample solution to be detected, realizes simultaneous accurate quantification of 15 active substances, has simple pretreatment, has high detection sensitivity, low detection limit of 0.002 mug/L and good linear relation, ensures that R 2 is more than or equal to 0.9987, ensures that the precision, the accuracy and the stability meet detection standards, and covers 1-5000 mug/L. The method provides reliable technical support for quality evaluation, product control and standardized production of the purple perilla, is suitable for detection of purple perilla related products, and promotes industrialized development of medicinal and edible homologous substances.

Inventors

  • LIU HONG
  • LIU QINGMEI
  • LIU GUANGMING
  • LIU WENMEI
  • ZOU ZEHUA

Assignees

  • 集美大学

Dates

Publication Date
20260508
Application Date
20260206

Claims (8)

  1. 1. A method for simultaneously detecting various phenolic acids and flavonoid active substances in perilla, which is characterized by comprising the following steps: preparing mixed reference substance solutions by respectively weighing 6 phenolic acid standard substances and 9 flavone standard substances, preparing single substance standard stock solutions by taking methanol as a solvent, mixing the single substance standard stock solutions, and fixing the volume by using methanol to prepare mixed reference substance solutions; The preparation of the sample solution to be tested comprises the steps of drying a perilla sample, crushing and sieving, taking perilla sample powder, adding 75% methanol, carrying out vortex oscillation, ultrasonic treatment and cooling, carrying out vortex oscillation again, and taking a supernatant fluid filter membrane to obtain the sample solution to be tested; And (3) sample measurement and content calculation, namely respectively injecting the mixed reference substance solution and the sample solution to be measured into an ultra-high performance liquid chromatography tandem mass spectrometer for measurement, drawing a standard curve by taking the content of each active substance in the mixed reference substance solution as an abscissa x and the corresponding quantitative ion peak area as an ordinate y, and calculating the content of 15 active substances in the sample to be measured according to the quantitative ion peak areas and the standard curve of each active substance in the sample solution to be measured.
  2. 2. The method of claim 1, wherein the perilla sample comprises perilla leaf, perilla stem, and perilla seed.
  3. 3. The method of claim 1, wherein the phenolic acid comprises rosmarinic acid, caffeic acid, ferulic acid, gallic acid, chlorogenic acid, protocatechuic acid, and the flavonoid comprises luteolin, scutellarin, apigenin, catechin, rutin, quercetin, kaempferol, isorhamnetin.
  4. 4. The method of claim 1, wherein the chromatographic parameters of the ultra-high performance liquid chromatography tandem mass spectrometer are C18 chromatographic column, the column temperature is 35-45 ℃, the sample injection amount is 0.8-1.2 mu L, the flow rate is 0.25-0.35 mL/min, the mobile phase comprises A phase and B phase, the A phase is 0.08-0.12% formic acid water, the B phase is 0.08-0.12% acetonitrile formate, the needle washing liquid is a mixed liquid of methanol-acetonitrile-isopropanol-water, the volume ratio is 1:1:1:1, the gradient elution procedure is 0~0.5 min,5%B;0.5~6 min,5% -45%B;6~6.01 min,45% -70% B, 6.01-8 min, 70%B;8~8.01 min,70% -5% B is 8.01-10 min, and 5% B is kept until stopping.
  5. 5. The method of claim 1, wherein the mass spectrometry parameters of the ultra-high performance liquid chromatography tandem mass spectrometer are ion source heating electrospray ionization, scan polarity positive and negative ion switching mode, spray voltage of positive ions 4000V, negative ions 3500V, sheath gas flow of 4.58L/min, auxiliary gas flow of 12.11L/min, ion transfer capillary temperature of 320 ℃, vapor temperature of 100 ℃, scan mode of selective reaction monitoring, collision gas of high purity argon, and pressure of 0.2 Pa. The scanning cycle time is 1 s.
  6. 6. The method of claim 5, wherein characteristic ions and corresponding mass spectral parameters of the 15 active ingredients are as follows in table 1: Table 1 mass spectral parameters of the various active ingredients Note that: To quantify ions.
  7. 7. The method of claim 1, wherein the ratio of the purple perilla sample powder to 75% methanol in the sample solution to be tested is 1:80-1:120 g/mL, the parameters of vortex oscillation are that the first vortex oscillation is 2-4 min, the vortex oscillation is 20-40 s after ultrasonic treatment, the ultrasonic treatment condition is that the temperature is 35-45 ℃, the power is 200-300W, the frequency is 45-55 kHz, the treatment time is 25-35 min, and the filter membrane is a 0.20-0.24 mu m PALL GHP filter membrane.
  8. 8. The method of claim 2, wherein the preparation parameters of the single-substance standard stock solution are rosmarinic acid, caffeic acid, gallic acid, chlorogenic acid, protocatechuic acid, ferulic acid, luteolin, catechin, rutin and kaempferol, the stock solution concentration of luteolin, scutellarin, apigenin and isorhamnetin is 1000 mug/mL, the mass concentration of the mixed reference solution is 10 mug/mL, the concentration of the series mixed reference solution is in the range of 10-5000 mug/L of rosmarinic acid, 1-5000 mug/L of caffeic acid, 10-5000 mug/L of ferulic acid, 10-5000 mug/L of gallic acid, 10-5000 mug/L of chlorogenic acid, 10-5000 mug/L of protocatechuic acid, 10-5000 mug/L of luteolin, 5-5000 mug/L of luteolin, 1-2000 mug/L of apigenin, 10-5000 mug/L of quercetin, 1-5000 mug/L of isorhamnospermin and 1-5000 mug/L of kaempferol.

Description

Method for simultaneously detecting various phenolic acids and flavonoid active substances in perilla Technical Field The invention relates to the technical field of detection of traditional Chinese medicine components, in particular to a method for simultaneously detecting various phenolic acids and flavonoid active substances in perilla. Background Perilla frutescens (Perilla frutescens (L.) Britt.) is annual herb plant of Perilla genus of Labiatae family, and is a first-approved "medicinal and edible homologous" plant in China, and its leaves, stems and seeds have important edible and medicinal values, and are recorded in pharmacopoeia of the people's republic of China. In the aspect of eating, the perilla leaf can be directly eaten or used as a seasoning and a health-care food raw material, the perilla oil squeezed by the perilla seeds is rich in alpha-linolenic acid, has good health-care effect, and in the aspect of medicine, the perilla She Jiebiao has the effects of dispelling cold, promoting qi and harmonizing stomach, regulating qi and relieving middle-jiao, relieving pain and preventing miscarriage, and the perilla seed has obvious whole plant medicinal value. The characteristic of 'medicine and food homology' of the purple perilla is closely related to various active ingredients contained in the purple perilla, wherein phenolic acids and flavonoid substances are core functional ingredients, and the purple perilla has multiple biological activities such as antioxidation, anti-inflammatory, antibacterial, antiallergic, antiviral, antitumor, neuroprotection, cardiovascular and cerebrovascular protection and the like. Along with public importance on healthy diet and high-quality raw materials, the quality control requirements of perilla and products thereof are increasingly urgent, and the accurate quantification of active ingredients is a key link of the quality evaluation of the perilla. The existing method for detecting the active ingredients of the perilla mainly adopts a high performance liquid chromatography ultraviolet spectroscopy (HPLC-UV), but has the obvious defects that firstly, the detection ingredients are single, only 3-5 ingredients can be usually detected, the quality condition of the perilla is difficult to comprehensively reflect, secondly, the detection time is long, the single analysis needs 30-60 minutes, the efficiency is low, the detection requirements of batch samples cannot be met, thirdly, the sensitivity is low, the detection limit is more than 5 mug/L, the low-content active ingredients are difficult to detect, fourthly, the detection range is narrow, the active ingredients with larger content difference in different parts (leaves, stems and seeds) of the perilla cannot be adapted, and the difference of various active ingredients in the perilla is larger due to factors such as different production places, varieties and processing methods. Therefore, a detection method with higher sensitivity, selectivity and accuracy, wider detection range and larger content difference is needed for analysis. Disclosure of Invention The invention aims to solve at least one of the technical problems in the technology to a certain extent, namely, provides a method for simultaneously detecting various phenolic acids and flavonoid active substances in perilla, solves the problems of less component coverage, low efficiency, insufficient sensitivity and narrow detection range of the existing detection method, and provides reliable technical support for quality evaluation, product control, standardized production and efficient resource utilization of the perilla. Therefore, the invention provides a method for simultaneously detecting various phenolic acids and flavonoid active substances in perilla, which comprises the following steps: preparing mixed reference substance solutions by respectively weighing 6 phenolic acid standard substances and 9 flavone standard substances, preparing single substance standard stock solutions by taking methanol as a solvent, mixing the single substance standard stock solutions, and fixing the volume by using methanol to prepare mixed reference substance solutions; The preparation of the sample solution to be tested comprises the steps of drying a perilla sample, crushing and sieving, taking perilla sample powder, adding 75% methanol, carrying out vortex oscillation, ultrasonic treatment and cooling, carrying out vortex oscillation again, and taking a supernatant fluid filter membrane to obtain the sample solution to be tested; And (3) sample measurement and content calculation, namely respectively injecting the mixed reference substance solution and the sample solution to be measured into an ultra-high performance liquid chromatography tandem mass spectrometer for measurement, drawing a standard curve by taking the content of each active substance in the mixed reference substance solution as an abscissa x and the corresponding quantitative ion peak area as an or