Method for enriching phenolic acid and flavonoid compounds in perilla leaves
The two-aqueous aqueous microextraction method of ionic liquid assisted by anionic surfactant was solved, and the problems of low extraction efficiency and environmental pollution of phenolic acids and flavonoids in traditional perilla leaves were achieved, achieving efficient and environmentally friendly extraction and enrichment effects.
Patent Information
- Application Number
- CN202510419690.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-07-08
AI Technical Summary
The extraction technology of phenolic acids and flavonoids in traditional perilla leaves has problems such as long extraction time and high organic solvent consumption, resulting in low efficiency and environmental pollution.
Anionic surfactant-enhanced ionic liquid assisted bi-aqueous microextraction (SE-IL-ATPME) method was used to form a stable bi-aqueous system by adding ionic liquid, anionic surfactant and salt. The phenolic acid and flavonoid compounds in perilla leaves were extracted after vortex and centrifugation.
It realizes efficient and environmentally friendly extraction and enrichment of phenolic acids and flavonoids, reduces the consumption of samples and reagents, and improves the extraction efficiency.
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Figure CN120275519A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of analytical chemistry, and relates to a method for enriching phenolic acids and flavonoid compounds in Perilla leaves, which is an anion surfactant-enhanced ionic liquid-assisted aqueous two-phase microextraction method. Background Art
[0002] Aqueous two-phase system (ATPS) is an emerging liquid-liquid extraction technique that has received extensive attention due to its unique characteristics, such as simple operation, environmental friendliness, and the ability to achieve rapid separation. These advantages make it highly applicable in the biological, pharmaceutical, and analytical fields. ATPS consists of two immiscible aqueous phases, which are usually formed by mixing two hydrophilic polymers, a polymer and a salt, or two salts. The separation principle of ATPS lies in the different distribution coefficients of the target analyte between the two phases, which are also affected by surface properties, molecular size, hydrophobicity, and electrostatic interactions. However, traditional ATPS usually requires a relatively large amount of samples and solvents, increasing the raw material cost and generating more waste, resulting in inevitable environmental pollution.
[0003] In recent years, ionic liquids (ILs) have been widely used in ATPS due to their unique physicochemical properties, such as low volatility, high thermal stability, and adjustable polarity. Ionic liquids can form specific interactions with target compounds, such as hydrogen bonds and electrostatic interactions, thus significantly improving the distribution efficiency and selectivity of ATPS. However, current research on ionic liquid-assisted ATPS is limited by the limited types of ionic liquids explored. Many ionic liquids either cannot form stable ATPS or exhibit insufficient phase separation behavior, which limits their practical applications. Therefore, exploring more novel ionic liquid-assisted ATPS is of great significance and value for developing more efficient extraction techniques.
[0004] Surfactants are a class of amphiphilic compounds with unique molecular structures, consisting of hydrophilic heads and hydrophobic tails. According to the nature of their hydrophilic groups, surfactants can be classified into several categories such as anionic, cationic, non-ionic, and zwitterionic surfactants. Due to their remarkable emulsifying, solubilizing, and interfacial activity properties, surfactants have been widely used in various fields such as detergents, cosmetics, and pharmaceuticals. It is worth noting that when the surfactant concentration exceeds the critical micelle concentration, a micellar solution is formed, which can encapsulate and solubilize the target analyte, thereby improving the extraction efficiency. Anionic surfactants exhibit strong emulsifying ability and interfacial activity, making them very effective in improving the partitioning behavior of other substances. Therefore, by adding anionic surfactants to ionic liquid-based ATPS, the emulsification and solubilization effects of the target compound can be significantly improved, thus optimizing its distribution between the two phases. In addition, the synergistic effect between anionic surfactants and other components of ATPS contributes to the stability of the entire system and is expected to solve the inherent limitations of traditional ATPS.
[0005] Perilla leaves, derived from the dried leaves of Perilla frutescens (L.), are widely cultivated and naturally distributed in East Asian countries, including China, Japan, and Korea. Perilla leaves have been widely used in culinary practices and traditional medicine for centuries. As an edible plant, Perilla leaves are widely used in cooking, seasoning, making tea, and as a food coloring due to their rich nutritional value and unique aroma. In addition, Perilla leaves also have medicinal values such as relieving colds, improving indigestion, antibacterial, and anti-inflammatory effects, and are a functional food with both nutritional and therapeutic dual effects. This is mainly attributed to its rich variety of bioactive components, including volatile oils, phenolic acids, and flavonoids. Traditional extraction techniques, such as maceration, ultrasound-assisted extraction, and microwave-assisted extraction, are the main extraction methods for bioactive substances in Perilla leaves. However, these methods have some significant limitations, such as longer extraction times and larger consumption of organic solvents, which reduce the extraction efficiency and cause environmental problems. Summary of the Invention
[0006] The purpose of the present invention is to provide a method for enriching phenolic acids and flavonoid compounds in Perilla leaves in view of the deficiencies of the prior art. The present invention uses anionic surfactant-enhanced ionic liquid-assisted aqueous two-phase microextraction (SE-IL-ATPME) for the extraction and enrichment of phenolic acids and flavonoid compounds in Perilla leaves. The present invention uses ionic liquid as the extraction solvent, adding traditional Chinese medicine powder, anionic surfactant, and salt, and forming ATPS after vortexing and centrifugation.
[0007] The method of the present invention adopts the following technical solutions: Step (1) Use a pulverizer to grind Perilla leaves into powder.
[0008] Step (2) Add perilla leaf powder, NaH2PO4, and sodium dodecyl sulfate into 1-hexyl-3-methylimidazolium bromide [C6MIM]Br solution in sequence to obtain a sample solution. The concentration of [C6MIM]Br solution is 100-200 mM. Add 5-10 g of perilla leaf powder, 200-400 g of NaH2PO4, and 25-45 g of sodium dodecyl sulfate into each liter of [C6MIM]Br solution.
[0009] Step (3) Adjust the pH value of the sample solution to 7.4-7.6 with ethylenediamine.
[0010] Step (4) Vortex for 120-160 s at room temperature to make the sample solution evenly mixed.
[0011] Step (5) Centrifuge the sample solution at 3000-5000 rpm for 5-15 min to form a stable aqueous two-phase system.
[0012] Step (6) Take the upper enriched phase of the aqueous two-phase system, dilute it 5-10 times with methanol, and centrifuge at 10000-15000 rpm for 2-5 min; Take the supernatant and load it into a liquid phase vial for UHPLC analysis to obtain a perilla leaf chromatogram.
[0013] Step (7) Add protocatechuic aldehyde, caffeic acid, luteoloside, rosmarinic acid, and luteolin in equal amounts into methanol, and dissolve to obtain a mixed standard solution. The concentration of each substance in the mixed standard solution is 5-50 mg / L; Centrifuge the mixed standard solution at 10000-15000 rpm for 2-5 min, take the upper layer liquid and inject it into a liquid phase vial for UHPLC analysis to obtain a standard chromatogram.
[0014] Step (8) According to the perilla leaf chromatogram and the standard chromatogram, perform quantitative analysis on phenolic acids and flavonoids in perilla leaf.
[0015] The technical solutions of the present invention mainly include: 1) Hydrogen bonds and electrostatic interactions can be formed between ionic liquids and target compounds, significantly improving the partitioning efficiency and selectivity of ATPS. 2) Anionic surfactants have strong emulsifying ability and interfacial activity, which can effectively improve the stability of ATPS and improve the partitioning of target analytes between two phases.
[0016] The present invention uses an ionic liquid as an extraction solvent, adds perilla leaf powder, an anionic surfactant, and a salt, and forms an aqueous two-phase system after vortexing and centrifuging. The present invention considers factors such as ionic liquid concentration, vortex time, salt dosage, SDS dosage, and pH value, and conducts systematic optimization. The present invention is an efficient and environmentally friendly method for extracting and enriching phenolic acids and flavonoids in perilla leaf. The beneficial effects of the present invention include: 1. The present invention demonstrates that the anionic surfactant SDS can enhance IL-ATPME, which is applicable to the one-step extraction and enrichment of phenolic acids and flavonoids in solid matrices.
[0017] 2. This method proposes a miniaturized aqueous two-phase extraction method, reducing the consumption of samples and reagents, and is an environmentally friendly and efficient method for determining hydrophobic compounds in traditional Chinese medicine.
[0018] 3. The method of the present invention is an efficient and environmentally friendly method for improving the extraction efficiency of phenolic acids and flavonoids in natural medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 It is the chromatogram of Perilla frutescens leaves (A) extracted by the method of the present invention and the chromatogram of the standard product (B); wherein 1 is protocatechuic aldehyde, 2 is caffeic acid, 3 is luteoloside, 4 is rosmarinic acid, and 5 is luteolin. Figure 2 It is the line graph of the extraction effect at different ionic liquid concentrations, wherein 1 is protocatechuic aldehyde, 2 is caffeic acid, 3 is luteoloside, 4 is rosmarinic acid, and 5 is luteolin. Figure 3 It is the line graph of the extraction effect at different vortex times, wherein 1 is protocatechuic aldehyde, 2 is caffeic acid, 3 is luteoloside, 4 is rosmarinic acid, and 5 is luteolin. Figure 4 It is the line graph of the extraction effect at different NaH2PO4 contents, wherein 1 is protocatechuic aldehyde, 2 is caffeic acid, 3 is luteoloside, 4 is rosmarinic acid, and 5 is luteolin. Figure 5 It is the line graph of the extraction effect at different sodium dodecyl sulfate contents, wherein 1 is protocatechuic aldehyde, 2 is caffeic acid, 3 is luteoloside, 4 is rosmarinic acid, and 5 is luteolin. Figure 6 It is the line graph of the extraction effect at different pH values of the sample solution, wherein 1 is protocatechuic aldehyde, 2 is caffeic acid, 3 is luteoloside, 4 is rosmarinic acid, and 5 is luteolin. DETAILED DESCRIPTION OF THE INVENTION
[0020] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0021] Example 1. Step (1) Grind Perilla frutescens leaves into powder with a pulverizer and pass through a 30-mesh sieve; Step (2): Add 2 mL of [C6MIM]Br solution with a concentration of 100 mM into a round-bottom centrifuge tube, and then successively add 10 mg of perilla leaf powder, 0.4 g of NaH2PO4, and 60 mg of sodium dodecyl sulfate to obtain a sample solution; Step (3): Adjust the pH value of the sample solution to 7.4 with ethylenediamine; Step (4): Vortex for 130 s at room temperature to mix the sample solution evenly; Step (5): Centrifuge the sample solution at 3000 rpm for 15 min to form a stable aqueous two-phase system; Step (6): Take the upper enriched phase of the aqueous two-phase system, dilute it 5 times with methanol, and centrifuge at 10000 rpm for 5 min; Take the supernatant and load it into a liquid-phase vial for UHPLC analysis to obtain the perilla leaf chromatogram; Step (7): Add equal amounts of protocatechuic aldehyde, caffeic acid, luteoloside, rosmarinic acid, and luteolin to methanol, dissolve to obtain a mixed standard solution, and the concentration of each substance in the mixed standard solution is 10 mg / L; Centrifuge the mixed standard solution at 13000 rpm for 3 min, take the upper layer solution and inject it into a liquid-phase vial for UHPLC analysis to obtain the standard chromatogram; Step (8): According to the perilla leaf chromatogram and the standard chromatogram, perform quantitative analysis on phenolic acids and flavonoids in perilla leaf.
[0022] Example 2. Step (1): Grind perilla leaf into powder with a pulverizer and pass through a 40-mesh sieve; Step (2): Add 2 mL of [C6MIM]Br solution with a concentration of 200 mM into a round-bottom centrifuge tube, and then successively add 20 mg of perilla leaf powder, 0.8 g of NaH2PO4, and 80 mg of sodium dodecyl sulfate to obtain a sample solution; Step (3): Adjust the pH value of the sample solution to 7.6 with ethylenediamine; Step (4): Vortex for 150 s at room temperature to mix the sample solution evenly; Step (5): Centrifuge the sample solution at 5000 rpm for 5 min to form a stable aqueous two-phase system; Step (6): Take the upper enriched phase of the aqueous two-phase system, dilute it 10 times with methanol, and centrifuge at 15000 rpm for 2 min; Take the supernatant and load it into a liquid-phase vial for UHPLC analysis to obtain the perilla leaf chromatogram; Step (7): Add equal amounts of protocatechuic aldehyde, caffeic acid, luteoloside, rosmarinic acid, and luteolin to methanol, dissolve to obtain a mixed standard solution, and the concentration of each substance in the mixed standard solution is 5 mg / L; Centrifuge the mixed standard solution at 10000 rpm for 5 min, take the upper layer solution and inject it into a liquid-phase vial for UHPLC analysis to obtain the standard chromatogram; Step (8) Quantitatively analyze phenolic acids and flavonoids in perilla leaves based on the perilla leaf chromatogram and the standard chromatogram.
[0023] Example 3. Step (1) Grind perilla leaves into powder with a pulverizer and pass through a 60-mesh sieve; Step (2) Add 2 mL of [C6MIM]Br solution with a concentration of 120 mM to a round-bottom centrifuge tube, and then successively add 18 mg of perilla leaf powder, 0.5 g of NaH2PO4, and 90 mg of sodium dodecyl sulfate to obtain a sample solution; Step (3) Adjust the pH value of the sample solution to 7.5 with ethylenediamine; Step (4) Vortex for 160 s at room temperature to mix the sample solution evenly; Step (5) Centrifuge the sample solution at 4500 rpm for 8 min to form a stable aqueous two-phase system; Step (6) Take the upper enriched phase of the aqueous two-phase system, dilute it 6 times with methanol, and centrifuge at 12000 rpm for 4 min; Take the supernatant and load it into a liquid-phase vial for UHPLC analysis to obtain the perilla leaf chromatogram; Step (7) Add equal amounts of protocatechuic aldehyde, caffeic acid, luteoloside, rosmarinic acid, and luteolin to methanol, and dissolve to obtain a mixed standard solution. The concentration of each substance in the mixed standard solution is 50 mg / L; Centrifuge the mixed standard solution at 15000 rpm for 2 min, take the upper layer solution and inject it into a liquid-phase vial for UHPLC analysis to obtain the standard chromatogram; Step (8) Quantitatively analyze phenolic acids and flavonoids in perilla leaves based on the perilla leaf chromatogram and the standard chromatogram.
[0024] Example 4. Step (1) Grind perilla leaves into powder with a pulverizer and pass through a 50-mesh sieve; Step (2) Add 2 mL of [C6MIM]Br solution with a concentration of 150 mM to a round-bottom centrifuge tube, and then successively add 15 mg of perilla leaf powder, 0.6 g of NaH2PO4, and 50 mg of sodium dodecyl sulfate to obtain a sample solution; Step (3) Adjust the pH value of the sample solution to 7.5 with ethylenediamine; Step (4) Vortex for 120 s at room temperature to mix the sample solution evenly; Step (5) Centrifuge the sample solution at 4000 rpm for 10 min to form a stable aqueous two-phase system; Step (6) Take the upper enriched phase of the aqueous two-phase system, dilute it 8 times with methanol, and centrifuge at 13000 rpm for 3 min; Take the supernatant and load it into a liquid-phase vial for UHPLC analysis to obtain the perilla leaf chromatogram; Step (7): Add equal amounts of protocatechuic aldehyde, caffeic acid, luteoloside, rosmarinic acid, and luteolin into methanol. After dissolution, a mixed standard solution is obtained, and the concentration of each substance in the mixed standard solution is 30 mg / L. Centrifuge the mixed standard solution at 12,000 revolutions per minute for 4 minutes, take the upper layer solution and inject it into a liquid phase vial for UHPLC analysis to obtain a standard chromatogram. Step (8): According to the perilla leaf chromatogram and the standard chromatogram, perform quantitative analysis on phenolic acids and flavonoids in perilla leaves. The perilla leaf chromatogram and the standard chromatogram are as Figure 1 shown.
[0025] The conditions for quantitative analysis by high performance liquid chromatography are as follows: The extraction effect is analyzed using an Agilent 1290 ultra-high performance liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA), which is equipped with an autosampler, a binary pump, a diode array detector, and a column oven for compound chromatographic analysis. An Agilent Poroshell 120 EC-C18 chromatographic column (4.6 mm × 100 mm, 2.7 μm) is used. Pure water (A, containing 0.1% formic acid) and acetonitrile (B) are used as the mobile phase, and the gradient program is as follows: 0 - 8 min, 17% - 18% B; 8 - 10 min, 18% - 35% B; 10 - 12 min, 35% - 60% B; 12 - 13 min, 65% - 100% B; 13 - 16 min, 100% B. The injection volume is 3 μL. The detection wavelength is 330 nm, the column temperature is 30 °C, and the flow rate is 0.6 mL / min. The analysis results are shown in the following table: Investigate the effect of ionic liquid concentration on the extraction effect: Example 5. Use a [C6MIM]Br solution with a concentration of 100 mM, and the others are the same as in Example 4.
[0026] Example 6. Use a [C6MIM]Br solution with a concentration of 200 mM, and the others are the same as in Example 4.
[0027] Comparative Example 1. Use a [C6MIM]Br solution with a concentration of 50 mM, and the others are the same as in Example 4.
[0028] Comparative Example 2. Use a [C6MIM]Br solution with a concentration of 250 mM, and the others are the same as in Example 4.
[0029] Due to the moderate alkyl chain length and good water solubility of [C6MIM]Br, it was selected as the extraction solvent in this study, which helped to promote the formation of ATPS. The effects of [C6MIM]Br solution concentrations of 50, 100, 150, 200, and 250 mM on the extraction efficiencies of protocatechuic aldehyde, caffeic acid, luteoloside, rosmarinic acid, and luteolin were systematically investigated (Examples 4, 5, 6 and Comparative Examples 1, 2). As Figure 2 shown, when the [C6MIM]Br solution concentration was increased from 50 mM to 150 mM, the peak areas of the above five target compounds increased significantly, and the effect was obvious above 100 mM. This may be attributed to the increase in the number of active sites provided by [C6MIM]Br with the increase of its concentration, thus enhancing the enrichment ability of ATPS. However, when the [C6MIM]Br concentration exceeded 150 mM, the extraction efficiencies of the five target analytes decreased significantly. It was speculated that this was due to the increase in the viscosity of the ATPS system caused by the too high [C6MIM]Br concentration, and the effect was obvious below 200 mM. Considering the enrichment efficiency and system stability comprehensively, 100 - 200 mM of [C6MIM]Br was determined as the optimal ionic liquid concentration for the subsequent experiments.
[0030] Investigate the effect of vortex time on the extraction effect: Comparative Example 3. The vortex time was set to 40 s, and the others were the same as in Example 4.
[0031] Comparative Example 4. The vortex time was set to 80 s, and the others were the same as in Example 4.
[0032] Example 7. The vortex time was set to 160 s, and the others were the same as in Example 4.
[0033] Comparative Example 5. The vortex time was set to 200 s, and the others were the same as in Example 4.
[0034] Vortexing can make the perilla leaf powder and the extraction solvent mix fully, thus promoting the formation of ATPS and significantly improving the extraction and enrichment efficiencies of the target analytes. Therefore, it is of great significance to study the effect of vortex time on the extraction rate (Examples 1, 7 and Comparative Examples 3, 4, 5). From Figure 3 it can be seen that when the vortex time increased from 40 s to 120 s, the peak area increased by nearly two times, and the extraction efficiency of the target analyte reached the maximum at 120 s. Appropriate extension of the vortex time can enhance the contact effect between the analyte and the enrichment phase, thus further improving the extraction efficiency. However, when the vortex time exceeded 120 s, the peak area tended to be stable, indicating that the target compound had been basically completely dissolved in the extraction solution at 120 s. The relative effect was better when the time was 120 - 160 s.
[0035] Investigate the effect of the amount of NaH2PO4 in the sample solution on the extraction efficiency: Comparative Example 6. The amount of NaH2PO4 is 0.2 g, and the others are the same as in Example 4.
[0036] Example 8. The amount of NaH2PO4 is 0.4 g, and the others are the same as in Example 4.
[0037] Example 9. The amount of NaH2PO4 is 0.8 g, and the others are the same as in Example 4.
[0038] Comparative Example 7. The amount of NaH2PO4 is 1.0 g, and the others are the same as in Example 4.
[0039] In the preparation process of ATPS, inorganic salts play a crucial role in the distribution of ionic liquids. An appropriate amount of inorganic salts can not only enhance the stability of ATPS but also significantly improve the efficiency of extracting target components from various aqueous matrices. In the range of 0.2 to 0.8 g of salt dosage, its effect on the extraction efficiency was investigated (Examples 4, 8, 9 and Comparative Examples 6, 7). As Figure 4 shown, with the increase of salt dosage, the extraction efficiency of the five target analytes increased significantly and reached the highest point when the salt dosage was 0.6 g. However, when the salt dosage exceeded 0.6 g, the extraction efficiency began to show a downward trend, and the effect was not good after exceeding 0.8 g. This phenomenon may be attributed to the fact that with the increase of salt amount, the salting-out effect in the solution gradually increases, promoting the phase transfer of the active ingredient and [C6MIM]Br to the upper enrichment phase and achieving efficient extraction; but excessive salt will cause water loss in the upper phase, thus destroying the stability of ATPS.
[0040] Investigate the effect of the amount of sodium dodecyl sulfate SDS on the extraction efficiency: Comparative Example 8. The dosage of sodium dodecyl sulfate is 30 mg, and the others are the same as in Example 4.
[0041] Example 10. The dosage of sodium dodecyl sulfate is 70 mg, and the others are the same as in Example 4.
[0042] Example 11. The dosage of sodium dodecyl sulfate is 90 mg, and the others are the same as in Example 4.
[0043] Comparative Example 9. The dosage of sodium dodecyl sulfate is 110 mg, and the others are the same as in Example 4.
[0044] The dosage of sodium dodecyl sulfate is a key parameter affecting the extraction efficiency. The SDS dosage within the range of 30 - 110 mg was selected to further investigate its influence on the extraction efficiency (Examples 4, 10, 11 and Comparative Examples 8, 9). The corresponding results are shown in Figure 5 . The results showed that as the SDS dosage increased from 30 mg to 110 mg, the peak area of the target compound showed a decreasing trend, which might be attributed to the decrease in extraction efficiency caused by the dilution effect, manifested as the gradually lightening color of the top phase. However, when the SDS dosage was 30 mg, the volume of the upper phase formed was small, which was not conducive to the suction process in actual operation. Considering the experimental feasibility and extraction efficiency comprehensively, 50 - 90 mg was determined as the preferred dosage.
[0045] Investigate the influence of the pH value of the sample solution on the extraction efficiency: Comparative Example 10. Adjust the pH value of the sample solution to 5.5 with acetic acid, and the others are the same as in Example 4.
[0046] Comparative Example 11. Adjust the pH value of the sample solution to 6.0 with acetic acid, and the others are the same as in Example 4.
[0047] Comparative Example 12. Adjust the pH value of the sample solution to 6.5 with acetic acid, and the others are the same as in Example 4.
[0048] Comparative Example 13. The pH value is 7.0, and the others are the same as in Example 4.
[0049] Comparative Example 14 Adjust the pH value of the sample solution to 8.0 with ethylenediamine, and the others are the same as in Example 4.
[0050] The pH value of the sample solution has a significant influence on the solubility of the target compound, which is considered to be one of the key factors determining the extraction efficiency. Adjust the pH value of the ATPS to the range of 5.5 - 8.0, and the influence of the pH value on the extraction efficiency (Example 4 and Comparative Examples 10, 11, 12, 13, 14) is as shown in Figure 6 . Except for caffeic acid and rosmarinic acid, most target compounds showed the best extraction efficiency at a pH value of 7.5. This phenomenon might be attributed to the increase in the solubility of the target analyte in the weak alkaline environment, thus enhancing its extraction performance in the ATPS. However, as the pH value further increased, the peak area showed a decreasing trend, presumably due to the degradation of some target compounds under strong alkaline conditions. Adjusting the pH value to 7.4 - 7.6 gave better extraction results.
Claims
1. A method for enriching phenolic acids and flavonoids in perilla leaves, characterized in that: Step (1): Use a pulverizer to grind perilla leaves into powder. Step (2): Sequentially add perilla leaf powder, NaH2PO4, and sodium dodecyl sulfate to a 1-hexyl-3-methylimidazolium bromide [C6MIM]Br solution to obtain a sample solution. Step (3): Adjust the pH value of the sample solution to 7.4 - 7.6 with ethylenediamine. Step (4): Vortex at room temperature for 120 - 160 s to make the sample solution evenly mixed. Step (5): Centrifuge the sample solution to form a stable aqueous two-phase system. Step (6): Take the upper enriched phase of the aqueous two-phase system, dilute it 5 - 10 times with methanol, centrifuge, take the supernatant and load it into a liquid phase vial for UHPLC analysis to obtain a perilla leaf chromatogram. Step (7): Add protocatechuic aldehyde, caffeic acid, luteoloside, rosmarinic acid, and luteolin in equal amounts to methanol, dissolve to obtain a mixed standard solution, and the concentration of each substance in the mixed standard solution is 5 - 50 mg / L; centrifuge the mixed standard solution, take the upper layer liquid and inject it into a liquid phase vial for UHPLC analysis to obtain a standard chromatogram. Step (8): According to the perilla leaf chromatogram and the standard chromatogram, perform quantitative analysis on phenolic acids and flavonoids in perilla leaves.
2. The enrichment method of phenolic acids and flavonoid compounds in perilla leaves according to claim 1, characterized in that: The concentration of the 1-hexyl-3-methylimidazolium bromide [C6MIM]Br solution is 100 - 200 mM, and 5 - 10 g of perilla leaf powder, 200 - 400 g of NaH2PO4, and 25 - 45 g of sodium dodecyl sulfate are added to each liter of [C6MIM]Br solution.
3. The method for enriching phenolic acids and flavonoids in perilla leaves according to claim 1, characterized in that: The centrifugation treatment in Step (5) is to centrifuge at 3000 - 5000 revolutions per minute for 5 - 15 minutes.
4. The enrichment method of phenolic acids and flavonoids in Perilla leaves according to claim 1, characterized in that: The centrifugation treatment in Step (6) is to centrifuge at 10000 - 15000 revolutions per minute for 2 - 5 minutes; the centrifugation treatment in Step (7) is to centrifuge at 10000 - 15000 revolutions per minute for 2 - 5 minutes.