Method for analyzing phenolic components in perilla leaves based on micellar electrokinetic chromatography technology

Abstract

The invention discloses a method for analyzing phenolic components in perilla leaves based on micellar electrokinetic chromatography. The preparation method comprises the following steps: grinding perilla leaves, and adding a methanol solution to obtain a perilla leaf extracting solution; uniformly mixing tea saponin, borax and distilled water, performing ultrasonic treatment, filtering, and adjusting the pH value to 8.0-8.4 to obtain a buffer solution; the method comprises the following steps: equivalently adding ten phenol standard substances into methanol, and mixing to obtain a standard substance solution; the perilla leaf extracting solution and the mixed standard substance solution are subjected to centrifugal treatment, supernatant is taken and subjected to electrokinetic chromatographic separation with the prepared buffer solution, and a perilla leaf electrophoresis chromatogram and a standard substance electrophoresis chromatogram are obtained; and finally, performing quantitative analysis on phenolic substances in the perilla leaves according to peak areas of the standard product electrophoresis chromatogram and the perilla leaf electrophoresis chromatogram. The natural tea saponin is applied to the micellar electrokinetic chromatography technology and used for separating the phenolic compounds in the perilla leaves, and a novel method capable of simply, rapidly, greenly and effectively analyzing the phenolic compounds in the perilla leaves is provided.

Description

Technical Field

[0001] The present invention belongs to the technical field of material analysis, and relates to a micellar electrokinetic chromatography analysis method, specifically a method for analyzing phenolic components in Perilla leaves based on micellar electrokinetic chromatography technology. Background Art

[0002] Perilla leaf refers to the dried leaves (or with buds) of the annual herb Perilla frutescens, belonging to the genus Perilla of the Lamiaceae family, and is widely distributed in China, South Korea, India, etc. As the main medicinal organ of the herb, the leaves accumulate many main medicinal components. Perilla leaf is a traditional medicinal plant with both medicinal and edible uses in China, and is widely used in barbecues, soups, stews, and is also suitable for making pickles, seasonings, side dishes, etc. The active ingredients are the key nutritional and medicinal components of Perilla leaf, including amino acids, terpenes, phenolic acids and flavonoids, which can inhibit the occurrence of cancer and improve the body's immunity. The pharmacological effects of the active ingredients, including anti-allergic, antioxidant, anti-cancer and anti-depressant effects, have also been pointed out. In recent years, due to the high medicinal and nutritional value of Perilla leaf, its active ingredients have attracted great attention. Therefore, many analytical techniques have been used to determine these active ingredients, such as micellar electrokinetic chromatography, high performance liquid chromatography, ultra-high performance liquid chromatography tandem mass spectrometry and other methods. However, these methods still have several disadvantages. They are not only costly, but also require a large amount of organic solvents, which is contradictory to the principles of green chemistry. Therefore, it is imperative to develop a more environmentally friendly and efficient detection and separation method to better meet the needs of environmental protection and separation of complex matrices.

[0003] When the concentration of surfactant in the solution exceeds the critical micelle concentration (CMC), surfactant molecules spontaneously self-assemble into micelles, forming a micellar solution. Micellar electrokinetic chromatography is a technique in which a micellar solution provides a pseudo-stationary phase (micelles), and under the action of an electric field, separation is achieved by taking advantage of the difference in the distribution of analytes between the micelles and the solution. This technique evolved from capillary zone electrophoresis and shows greater advantages in separating neutral ions. As shown in previous reports, it has been used to separate amino acids in medicinal plants, proteins of different sizes and properties, flavonoids, anthraquinones, etc. In micellar electrokinetic chromatography, different surfactants are used to separate a large number of solutes, and surfactants are mainly divided into four types: anionic, cationic, zwitterionic, and nonionic. Anionic surfactants, such as sodium dodecyl sulfate, are the most commonly used because they can form negatively charged micelles, and they interact with analytes through hydrophobic or electrostatic attraction. Cationic surfactants, such as cetyltrimethylammonium bromide, adhere to the capillary wall through the Coulomb attraction between their positively charged ions and the negatively charged silanol groups on the surface. This interaction is particularly useful when analyzing analytes with opposite charges. Zwitterionic surfactants, such as n-dimethyl-3-ammonio-1-propanesulfonate, provide the flexibility to form positively and negatively charged micelles depending on the pH value, making them suitable for a wide range of applications. Nonionic surfactants are used when charge interactions are not required, and they provide a stable micellar environment for analytes, taking Brij35 as an example. It is well known that the micellar solution, as the key component providing the pseudo-stationary phase in micellar electrokinetic chromatography, provides a longer migration window and improves the resolution and sensitivity of the separation process. Therefore, it is of great significance to explore and develop a new micellar system based on environmentally friendly surfactants and apply this new system to the separation of complex compounds.

[0004] Tea saponin is a pentacyclic triterpenoid saponin extracted from steroid and triterpene skeletons. These compounds have similar structural characteristics, including organic acids, aglycones, and sugar components. They are widely extracted from a variety of plants, including soybeans, tea seeds, and chickpeas, as well as specific marine organisms such as starfish and sea cucumbers. The hydrophilic groups are mainly concentrated at the junction between the sugar part, organic acid, and aglycone, while the hydrophobic groups are mainly located within the ligand. Recent studies have shown that due to its amphiphilic structure, tea saponin has good foaming, emulsifying, and detergency properties. Therefore, as a natural surfactant, tea saponin has been widely used in the fields of daily chemicals, food, medicine, etc. In addition, this unique structure endows tea saponin with a series of biological activities, such as anti-inflammatory, antifungal, and gastric mucosa protection. Currently, the application of tea saponin in the capillary electrophoresis separation process has not been reported. As a natural surfactant, tea saponin has important application value in micellar electrokinetic chromatography due to its unique surface activity and structural stability. However, there is currently no research invention on the application of tea saponin as a non-ionic surfactant in the field of micellar electrokinetic chromatography. Tea saponin can be used as a potential stationary phase material, providing new possibilities for the separation of a large number of compounds in complex matrices. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for analyzing phenolic components in Perilla frutescens leaves based on micellar electrokinetic chromatography technology. The present invention is based on natural tea saponin as the capillary electrophoresis stationary phase to form micellar electrokinetic chromatography technology for analyzing phenolic components in Perilla frutescens leaves.

[0006] The present invention is specifically as follows: Step (1): Use a pulverizer to crush Perilla frutescens leaves into powder. Add the Perilla frutescens leaf powder to a methanol solution with a volume concentration of 30 - 80%, add 10 - 50 g of Perilla frutescens leaf powder per liter of methanol solution, and perform ultrasonic treatment for 10 - 60 min to obtain a Perilla frutescens leaf extract.

[0007] Step (2): Prepare the buffer solution: Mix tea saponin, borax, and distilled water evenly, and perform ultrasonic treatment for 15 - 20 min to obtain a uniformly mixed solution; filter through a 0.22 - 0.45 μm organic nylon filter head, and adjust the pH value to 8.0 - 8.4 by boric acid solution or sodium hydroxide solution to obtain the buffer solution. The concentration of tea saponin in the buffer solution is 0.8% - 2.0% w / v, and the concentration of borax is 10.0 - 20.0 mM. Tea saponin is used as a surfactant with a purity of greater than or equal to 98% w / w.

[0008] The concentrations of the boric acid solution and sodium hydroxide solution are 1 M.

[0009] Step (3): Add ten kinds of phenolic standards in equal amounts to methanol. After dissolution, a mixed standard solution is obtained, and the concentration of phenolic standards in the mixed standard solution is 50 - 500 mg / L. The ten kinds of phenolic standards are: resveratrol, catechin, rutin, sinapic acid, ferulic acid, p-coumaric acid, rosmarinic acid, caffeic acid, gallic acid, and protocatechuic acid.

[0010] Step (4): Centrifuge the perilla leaf extract at a speed of 10,000 - 15,000 rpm for 2 - 5 min; take the upper layer solution and inject it into an electrophoresis vial, and perform capillary electrophoresis chromatography separation with the prepared buffer solution to obtain the perilla leaf electrophoresis chromatogram; Step (5): Centrifuge the mixed standard solution at a speed of 10,000 - 15,000 rpm for 2 - 5 min; take the upper layer solution and inject it into an electrophoresis vial; perform capillary electrophoresis chromatography separation with the prepared buffer solution to obtain the standard product electrophoresis chromatogram; Step (6): Quantitatively analyze the phenolic substances in perilla leaves according to the peak areas of the standard product electrophoresis chromatogram and the perilla leaf electrophoresis chromatogram.

[0011] The technical solutions of the present invention mainly rely on the following: 1) Tea saponin is a pentacyclic triterpenoid saponin composed of organic acids, aglycones, and sugar components. It has both hydrophilic and hydrophobic properties and can be used as a non-ionic surfactant. 2) When the concentration of the tea saponin solution reaches the critical micelle concentration, it self-assembles into micelles. The micelles contain a hydrophobic core that encapsulates compounds through hydrophobic interactions. Different compounds have different distribution abilities and migration rates in the micelle phase and the aqueous phase, thus achieving separation. 3) Different types of salts can affect the ionic strength and pH value of the buffer solution, the dissociation degree and charge state of the analyte, and cause changes in the electroosmotic velocity, thereby achieving separation.

[0012] The present invention applies natural tea saponin to micellar electrokinetic chromatography technology for separating phenolic compounds in perilla leaves. Adding natural tea saponin as a surfactant to the buffer solution is considered to be green and efficient. The present invention considers the influence of main separation factors, such as the type and concentration of the surfactant, the buffer solution system, the salt concentration in the buffer solution, and the pH value of the buffer solution, and conducts systematic optimization. The present invention provides a new method for simply, rapidly, greenly, and effectively analyzing phenolic compounds in perilla leaves. The beneficial effects of the present invention include: 1. The present invention first uses tea saponin as a surfactant in micellar electrokinetic chromatography technology. No organic solvents are added to the buffer solution and the separation process, which has the advantages of being green, pollution-free, and high separation efficiency. Through single-factor experiments, several key parameters affecting the separation of target compounds are studied and optimized.

[0013] 2. This method has a wide range of applications and can be used for the detection and separation of phenolic compounds in various medicinal materials, showing great potential in the detection and separation of phenolic compounds extracted from natural medicinal materials.

[0014] 3. The detection limit and quantification limit of the method of the present invention are 0.40 - 0.95 and 1.34 - 3.16 μg / mL respectively, with a good linear relationship and a correlation coefficient of 0.9991 - 0.9999. The method of the present invention is a potential, green and effective method for analyzing phenolic compounds in Perilla leaves. Description of the Drawings

[0015] Figure 1 Chromatograms of separation efficiency at different tea saponin concentrations; among them, 1 is resveratrol; 2 is catechin; 3 is rutin; 4 is sinapic acid; 5 is ferulic acid; 6 is p - coumaric acid; 7 is rosmarinic acid; 8 is caffeic acid, 9 is gallic acid; 10 is protocatechuic acid.

[0016] Figure 2 Chromatograms of separation efficiency of different surfactants; among them, 1 is resveratrol; 2 is catechin; 3 is rutin; 4 is sinapic acid; 5 is ferulic acid; 6 is p - coumaric acid; 7 is rosmarinic acid; 8 is caffeic acid, 9 is gallic acid; 10 is protocatechuic acid.

[0017] Figure 3 Chromatograms of separation efficiency of different buffer systems; among them, 1 is resveratrol; 2 is catechin; 3 is rutin; 4 is sinapic acid; 5 is ferulic acid; 6 is p - coumaric acid; 7 is rosmarinic acid; 8 is caffeic acid, 9 is gallic acid; 10 is protocatechuic acid.

[0018] Figure 4 Chromatograms of separation efficiency at different pH values of the buffer; among them, 1 is resveratrol; 2 is catechin; 3 is rutin; 4 is sinapic acid; 5 is ferulic acid; 6 is p - coumaric acid; 7 is rosmarinic acid; 8 is caffeic acid, 9 is gallic acid; 10 is protocatechuic acid. Detailed Embodiments

[0019] In order to make the objectives, technical solutions and advantages of the present invention more clear and 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.

[0020] Example 1. Step (1) Use a pulverizer to crush Perilla leaves into powder, pass the Perilla leaf powder through a No. 4 sieve, take 1 g and add it to 100 ml of a methanol solution with a volume concentration of 30%, and ultrasonically treat for 10 min to obtain a Perilla leaf extract.

[0021] In step (2), tea saponin, borax, and distilled water were mixed evenly, ultrasonically treated for 15 min, filtered through a 0.22-μm organic nylon filter head, and the pH value of the solution was 8.0. The pH value was adjusted to 8.4 with a 1-M sodium hydroxide solution to obtain a buffer solution. The concentration of tea saponin in the buffer solution was 0.8% w / v, and the concentration of borax was 10.0 mM. The purity of the surfactant tea saponin was 98.5% w / w.

[0022] In step (3), ten phenolic standards (resveratrol, catechin, rutin, sinapic acid, ferulic acid, p-coumaric acid, rosmarinic acid, caffeic acid, gallic acid, protocatechuic acid) were added to methanol in equal amounts, and after dissolution, a mixed standard solution was obtained. The concentration of phenolic standards in the mixed standard solution was 100 mg / L.

[0023] In step (4), the perilla leaf extract was centrifuged at 10,000 rpm for 5 min; the supernatant was taken and injected into an electrophoresis vial, and capillary electrophoresis was performed with the prepared buffer solution to obtain an electrophoresis chromatogram of perilla leaves. In step (5), the mixed standard solution was centrifuged at 12,000 rpm for 3 min; the supernatant was taken and injected into an electrophoresis vial; capillary electrophoresis was performed with the prepared buffer solution to obtain an electrophoresis chromatogram of the standards. In step (6), the phenols in perilla leaves were quantitatively analyzed based on the peak areas of the electrophoresis chromatograms of the standards and perilla leaves.

[0024] Example 2. In step (1), perilla leaves were crushed into powder using a pulverizer. The perilla leaf powder was passed through a No. 5 sieve, and 2 g was added to 100 ml of a 40% v / v methanol solution and ultrasonically treated for 20 min to obtain a perilla leaf extract.

[0025] In step (2), tea saponin, borax, and distilled water were mixed evenly, ultrasonically treated for 16 min, filtered through a 0.25-μm organic nylon filter head, and the pH value of the solution was 8.8. The pH value was adjusted to 8.0 with a 1-M boric acid solution to obtain a buffer solution. The concentration of tea saponin in the buffer solution was 1.0% w / v, and the concentration of borax was 20.0 mM. The purity of the surfactant tea saponin was 99% w / w.

[0026] In step (3), ten phenolic standards (resveratrol, catechin, rutin, sinapic acid, ferulic acid, p-coumaric acid, rosmarinic acid, caffeic acid, gallic acid, protocatechuic acid) were added to methanol in equal amounts, and after dissolution, a mixed standard solution was obtained. The concentration of phenolic standards in the mixed standard solution was 50 mg / L.

[0027] Step (4): Centrifuge the perilla leaf extract at 11,000 rpm for 4 min; take the upper layer liquid and inject it into an electrophoresis vial, and perform capillary electrophoresis chromatography separation with the prepared buffer solution to obtain the perilla leaf electrophoresis chromatogram. Step (5): Centrifuge the mixed standard solution at 10,000 rpm for 5 min; take the upper layer liquid and inject it into an electrophoresis vial; perform capillary electrophoresis chromatography separation with the prepared buffer solution to obtain the standard product electrophoresis chromatogram. Step (6): Perform quantitative analysis of phenols in perilla leaf according to the peak areas of the standard product electrophoresis chromatogram and the perilla leaf electrophoresis chromatogram.

[0028] Example 3. Step (1): Use a pulverizer to crush perilla leaves into powder, sieve the perilla leaf powder through a No. 4 sieve, take 2.5 g and add it to 100 ml of a 50% (v / v) methanol solution, and perform ultrasonic treatment for 30 min to obtain the perilla leaf extract.

[0029] Step (2): Mix tea saponin, borax, and distilled water evenly, perform ultrasonic treatment for 17 min, filter through a 0.30-μm organic nylon filter head, and adjust the pH value of the solution to 8.4 to obtain the buffer solution. The concentration of tea saponin in the buffer solution is 1.2% w / v, and the concentration of borax is 15.0 mM. The purity of the surfactant tea saponin is 98% w / w.

[0030] Step (3): Add ten kinds of phenol standards (resveratrol, catechin, rutin, sinapic acid, ferulic acid, p-coumaric acid, rosmarinic acid, caffeic acid, gallic acid, protocatechuic acid) equally to methanol, and dissolve to obtain a mixed standard solution. The concentration of the phenol standards in the mixed standard solution is 200 mg / L.

[0031] Step (4): Centrifuge the perilla leaf extract at 12,000 rpm for 3.5 min; take the upper layer liquid and inject it into an electrophoresis vial, and perform capillary electrophoresis chromatography separation with the prepared buffer solution to obtain the perilla leaf electrophoresis chromatogram. Step (5): Centrifuge the mixed standard solution at 15,000 rpm for 2 min; take the upper layer liquid and inject it into an electrophoresis vial; perform capillary electrophoresis chromatography separation with the prepared buffer solution to obtain the standard product electrophoresis chromatogram. Step (6): Perform quantitative analysis of phenols in perilla leaf according to the peak areas of the standard product electrophoresis chromatogram and the perilla leaf electrophoresis chromatogram.

[0032] Example 4. Step (1): Use a pulverizer to crush perilla leaves into powder, sieve the perilla leaf powder through a No. 5 sieve, take 3 g and add it to 100 ml of a 60% (v / v) methanol solution, and perform ultrasonic treatment for 40 min to obtain the perilla leaf extract.

[0033] Step (2): Mix saponin, borax, and distilled water evenly, perform ultrasonic treatment for 18 min, filter through a 0.35-μm organic nylon filter head, with the solution pH value being 8.3 to obtain a buffer solution. The concentration of saponin in the buffer solution is 1.4% w / v, and the concentration of borax is 12.0 mM. The purity of the surfactant saponin is 98.5% w / w.

[0034] Step (3): Equally add ten phenolic standards (resveratrol, catechin, rutin, sinapic acid, ferulic acid, p-coumaric acid, rosmarinic acid, caffeic acid, gallic acid, protocatechuic acid) to methanol, and dissolve to obtain a mixed standard solution. The concentration of phenolic standards in the mixed standard solution is 500 mg / L.

[0035] Step (4): Centrifuge the perilla leaf extract at 13,000 rpm for 3 min; take the supernatant and inject it into an electrophoresis vial, and perform electrokinetic chromatography separation with the prepared buffer solution to obtain a perilla leaf electrophoresis chromatogram. Step (5): Centrifuge the mixed standard solution at 13,000 rpm for 3 min; take the supernatant and inject it into an electrophoresis vial; perform electrokinetic chromatography separation with the prepared buffer solution to obtain a standard product electrophoresis chromatogram. Step (6): Quantitatively analyze the phenols in perilla leaves based on the peak areas of the standard product electrophoresis chromatogram and the perilla leaf electrophoresis chromatogram.

[0036] Example 5. Step (1): Use a pulverizer to crush perilla leaves into powder, pass the perilla leaf powder through a No. 4 sieve, take 4 g and add it to 100 ml of a 70% v / v methanol solution, and perform ultrasonic treatment for 50 min to obtain a perilla leaf extract.

[0037] Step (2): Mix saponin, borax, and distilled water evenly, perform ultrasonic treatment for 19 min, filter through a 0.40-μm organic nylon filter head, with the solution pH value being 8.3 to obtain a buffer solution. The concentration of saponin in the buffer solution is 0.9% w / v, and the concentration of borax is 12.0 mM. The purity of the surfactant saponin is 99% w / w.

[0038] Step (3): Equally add ten phenolic standards (resveratrol, catechin, rutin, sinapic acid, ferulic acid, p-coumaric acid, rosmarinic acid, caffeic acid, gallic acid, protocatechuic acid) to methanol, and dissolve to obtain a mixed standard solution. The concentration of phenolic standards in the mixed standard solution is 300 mg / L.

[0039] Step (4): Centrifuge the perilla leaf extract at 14,000 rpm for 2.5 min; take the supernatant and inject it into an electrophoresis vial, and perform electrokinetic chromatography separation with the prepared buffer solution to obtain a perilla leaf electrophoresis chromatogram. Step (5): Centrifuge the mixed standard solution at 14,000 rpm for 2.5 min; take the upper layer solution and inject it into the electrophoresis vial; perform capillary electrophoresis with the prepared buffer solution to obtain the standard electrophoresis chromatogram. Step (6): Quantitatively analyze the phenols in perilla leaves based on the peak areas of the standard electrophoresis chromatogram and the perilla leaf electrophoresis chromatogram.

[0040] Example 6. Step (1): Use a pulverizer to crush perilla leaves into powder, pass the perilla leaf powder through a No. 5 sieve, take 5 g and add it to 100 ml of methanol solution with a volume concentration of 80%, and perform ultrasonic treatment for 60 min to obtain the perilla leaf extract.

[0041] Step (2): Mix tea saponin, borax, and distilled water evenly, perform ultrasonic treatment for 20 min, filter through a 0.45 μm organic nylon filter head, the pH value of the solution is 8.6, and adjust the pH value to 8.1 with a 1 M boric acid solution to obtain the buffer solution. The concentration of tea saponin in the buffer solution is 1.1% w / v, and the concentration of borax is 18.0 mM. The purity of the surfactant tea saponin is 98% w / w.

[0042] Step (3): Add ten kinds of phenol standards (resveratrol, catechin, rutin, sinapic acid, ferulic acid, p-coumaric acid, rosmarinic acid, caffeic acid, gallic acid, protocatechuic acid) equally to methanol, and dissolve to obtain a mixed standard solution. The concentration of the phenol standards in the mixed standard solution is 400 mg / L.

[0043] Step (4): Centrifuge the perilla leaf extract at 15,000 rpm for 2 min; take the upper layer solution and inject it into the electrophoresis vial, and perform capillary electrophoresis with the prepared buffer solution to obtain the perilla leaf electrophoresis chromatogram. Step (5): Centrifuge the mixed standard solution at 11,000 rpm for 4.5 min; take the upper layer solution and inject it into the electrophoresis vial; perform capillary electrophoresis with the prepared buffer solution to obtain the standard electrophoresis chromatogram. Step (6): Quantitatively analyze the phenols in perilla leaves based on the peak areas of the standard electrophoresis chromatogram and the perilla leaf electrophoresis chromatogram.

[0044] Investigate the influence of different concentrations of tea saponin on the separation effect: Example 7. Step (1): Use a pulverizer to crush perilla leaves into 50-mesh powder, take 0.50 g of the powder and add it to 10 mL of methanol solution with a volume concentration of 80% to obtain the perilla leaf extract.

[0045] Step (2): Take a certain amount of saponin and borax, add them to 10 mL of distilled water and mix evenly. Then perform ultrasonic treatment for 15 min. The concentration of saponin in the solution is 0.8% w / v, the concentration of borax is 15 mM, the purity of saponin is 98% w / w. Filter through a 0.45 μm organic nylon filter head. The pH value of the solution is 8.4 to obtain a buffer solution.

[0046] Step (3): Add equal amounts of ten phenolic standards (resveratrol, catechin, rutin, sinapic acid, ferulic acid, p-coumaric acid, rosmarinic acid, caffeic acid, gallic acid, protocatechuic acid) to methanol to obtain a 200 mg / L mixed standard solution.

[0047] Step (4): Centrifuge the perilla leaf extract at 13,000 rpm for 3 min; take the supernatant and inject it into an electrophoresis vial, and perform capillary electrophoresis separation with the prepared buffer solution to obtain the perilla leaf electrophoresis chromatogram. Step (5): Centrifuge the mixed standard solution at 13,000 rpm for 3 min; take the supernatant and inject it into an electrophoresis vial; perform capillary electrophoresis separation with the prepared buffer solution to obtain the standard product electrophoresis chromatogram. Step (6): Quantitatively analyze the phenolic substances in perilla leaves according to the peak areas of the standard product electrophoresis chromatogram and the perilla leaf electrophoresis chromatogram.

[0048] The electrophoresis conditions for quantitative analysis are as follows: The separation efficiency of the separation method is indicated by measuring the resolution between compounds through Agilent 7100CE. The capillary column activation procedure is to rinse with 1 M sodium hydroxide solution for 10 min, 0.1 M sodium hydroxide solution for 10 min, ultrapure water for 5 min, and buffer solution for 5 min. The outer diameter of the capillary is 365 μm and the inner diameter is 50 μm. The separation voltage is 30 kV and the column temperature is 25 °C. The analysis results are shown in the following table: Example 8. The concentration of saponin in the prepared buffer solution is 1.4% w / v, and the others are the same as in Example 7.

[0049] Comparative Example 1. The concentration of saponin in the prepared buffer solution is 2.0% w / v, and the others are the same as in Example 7.

[0050] Comparative Example 2. The concentration of saponin in the prepared buffer solution is 0.2% w / v, and the others are the same as in Example 7.

[0051] In the micellar electrokinetic chromatography based on natural tea saponin, the concentration of tea saponin is a key factor and has a great impact on the separation efficiency. To investigate the effect of tea saponin concentration on the separation performance of micellar electrokinetic chromatography, different tea saponin concentrations from 0.2% to 2.0% w / v (Examples 7, 8 and Comparative Examples 1, 2) were studied. As Figure 1 shown, when the tea saponin concentration was 0.2% w / v (curve a in the figure), the 10 analytes could not be completely separated, and the resolution of catechin and rutin, rosmarinic acid and caffeic acid was 0.81 and 0.93 respectively, and the separation rate was very low. The possible reason for this result is that the number of nonionic micelles formed is insufficient, and a small amount of analytes can bind to the micelles, making it difficult to achieve effective separation. When the tea saponin concentration increased from 0.2 w / v to 0.8% w / v (curve b in the figure), the migration time and resolution of phenolic acid compounds and flavonoid compounds both increased. Among them, the resolution of catechin and rutin increased from 0.81 to 2.38, and the separation effect was good. When the saponin addition amount reached 0.8% w / v, the number of micelles formed allowed the analytes to be optimally distributed between the pseudo-stationary phase and the solution. However, when higher concentrations of surfactants (1.4 and 2.0% w / v) were added to the borax solution (curves c and d in the figure), the baseline was disturbed, and the resolution and the apparent mobility of some measured compounds decreased. The electroosmotic flow mobility decreased from 12.583 to 10.356×10 -5 cm 2 / V∙s. This phenomenon may be due to the increase in the viscosity of the solution with the increase in concentration, resulting in a decrease in electroosmotic flow mobility and an increase in migration time. In addition, it further affects the mobility of these measured compounds, and the longer migration time leads to the band broadening of these peaks. Therefore, 0.8% w / v tea saponin in the borax solution was selected as the appropriate concentration for the next optimization.

[0052] Investigate the effect of surfactant type on the separation efficiency: Comparative Example 3. Replace tea saponin with 3-sulfopropyl dodecyldimethyl betaine, and the others are the same as in Example 7.

[0053] Comparative Example 4. Replace tea saponin with Tween 80, and the others are the same as in Example 7.

[0054] Comparative Example 5. Replace tea saponin with Triton X-100, and the others are the same as in Example 7.

[0055] Comparative Example 6. Replace tea saponin with sodium dodecyl sulfate, and the others are the same as in Example 7.

[0056] Surfactants, as one of the key components of micellar electrokinetic chromatography, show significant differences in separation and detection for different types. Ionic and non-ionic surfactants such as saponin, 3-sulfopropyl dodecyldimethyl betaine, Tween 80, Triton X-100, and sodium dodecyl sulfate were added at a concentration of 0.8% w / v (Example 7 and Comparative Examples 3, 4, 5, 6) to evaluate their separation efficiency. As Figure 2 shown, when using 3-sulfopropyl dodecyldimethyl betaine (curve b in the figure), it can be clearly observed that only 4 of the analytes to be measured were detected, and the peak shapes of 3 of them collapsed. This may be due to the weak electroosmotic flow generated by the zwitterionic surfactant in the buffer solution, resulting in an increase in the migration time of the analytes, thereby reducing the electrophoretic mobility difference between phenolic acids and flavonoids, and the analytes to be measured could not be completely separated. When applying sodium dodecyl sulfate (curve c in the figure), the peak shapes of caffeic acid and gallic acid were irregular; at the same time, it led to the co-elution of two components, catechin and rutin. The influence of sodium dodecyl sulfate on separation may be related to the binding of the solute to the micelle surface, and strong Coulomb interactions (attraction or repulsion) also occur when the analyte is charged. Anionic analytes usually have a weak affinity for anionic sodium dodecyl sulfate micelles due to electrostatic repulsion, resulting in low separation efficiency. As common polyoxyethylene non-ionic surfactants, Tween 80 and Triton X-100 have been widely used in micellar electrokinetic chromatography. However, satisfactory baseline separation was not observed between the two groups of analytes (catechin and rutin, rosmarinic acid and caffeic acid) for Tween 80 (curve d in the figure) and Triton X-100 (curve e in the figure). At the same concentration, the current detected by the anionic surfactant (34.7 μA) was higher than that detected by the non-ionic surfactant (22 μA). The reason for this phenomenon may be that due to the hydrophobic interaction of the analyte, Tween 80 and Triton X-100 bind to the micelles, containing more hydrophilic groups - alkoxy groups, and the hydrophobicity is weaker. While saponin, as a hydrophobic compound (curve a in the figure), binds strongly to the analytes, increasing the partition coefficient between the analyte micelles and the aqueous phase and improving the resolution (for example, the resolution of catechin and rutin, rosmarinic acid and caffeic acid increased to 2.08 and 1.59 respectively). In summary, among the 5 surfactants, saponin is the best choice in terms of both peak shape and resolution, so saponin is the most suitable surfactant for further optimization.

[0057] Investigate the influence of different buffer systems on the separation efficiency: Comparative Example 7. Replace borax with sodium acetate, and the others are the same as in Example 7.

[0058] Comparative Example 8. Replace borax with ammonium acetate, and the others are the same as in Example 7.

[0059] Comparative Example 9 Sodium hydrogen phosphate was used to replace borax, and the others were the same as in Example 7.

[0060] Comparative Example 10 Tris reagent was used to replace borax, and the others were the same as in Example 7.

[0061] Different types of salts can affect the ionic strength of the buffer solution, thereby affecting the degree of dissociation and charge state of the analyte. Under the condition that other experimental conditions remain unchanged, different types of salts were selected, such as sodium acetate, ammonium acetate, sodium hydrogen phosphate, Tris reagent, and borax (Comparative Examples 7, 8, 9, 10 and Example 7), and were mixed with 0.8% w / v saponin in the buffer solution to evaluate the separation efficiency of 10 analytes. The comparative electrophoretograms of the tested compounds are as Figure 3 shown. When ammonium acetate (curve c in the figure) and sodium acetate (curve b in the figure) were used, only rosmarinic acid and caffeic acid could be detected, and the separation rate of the two analytes was 0.98. The reason for this phenomenon may be that the addition of two organic reagents, sodium acetate and ammonium acetate, affected the polarity of the buffer solution. At the same time, it affected the hydrogen bond balance between the saponin molecule and the -OH and other polar groups in the water molecule, thereby affecting the interaction between the analyte and the micelle. When Tris reagent (curve d in the figure) was added to the buffer solution, only two components were eluted and detected. Under the condition of the same amount of salt in the buffer solution, the ion mobility of the Tris reagent electroosmotic flow was lower (the current during micellar electrokinetic chromatography was 4.3 μA), resulting in a slower migration rate of the analyte, a wider peak shape, and a poorer separation effect. Seven analytes were separated and detected using sodium hydrogen phosphate (curve e in the figure), while when borax (curve a in the figure) was used as an additive in the buffer solution, all 10 analytes achieved baseline separation and a satisfactory resolution (2.93) was obtained. This is because the addition of sodium hydrogen phosphate led to higher conductivity and reduced the detection sensitivity. Considering the resolution of the compounds to be measured and the appropriate background conductivity, borax is the most effective salt that can be added to the buffer solution.

[0062] Investigate the effect of the concentration of borax in different buffer solutions on the separation efficiency: Comparative Example 11 The concentration of borax in the prepared buffer solution was 5 mM, and the others were the same as in Example 7.

[0063] Example 9 The concentration of borax in the prepared buffer solution was 10 mM, and the others were the same as in Example 7.

[0064] Example 10 The concentration of borax in the prepared buffer solution was 20 mM, and the others were the same as in Example 7.

[0065] To improve the separation selectivity and shorten the analysis time, the borax concentration in the buffer (5, 10, 15, 20 mM, Comparative Example 11, Example 9, 7, and 10) was optimized. The buffer was treated with a buffer containing 0.8% w / v saponin and borax to investigate the effect of the buffer concentration. As the buffer concentration (5 - 20 mM) increased, the ζ potential decreased, the electroosmotic flow decreased, the migration time prolonged, and the apparent mobility decreased from 12.074 to 10.831×10 -5 cm 2 / V∙s. In the 5 mM borax running buffer, the peak shapes of caffeic acid and protocatechuic acid collapsed, the peak shapes of rosmarinic acid and gallic acid became broad and asymmetric, and the resolution of gallic acid and protocatechuic acid was 0.91. When the borax concentration increased to 15 mM, good separation of 10 target analytes was achieved within a reasonable analysis time, and the resolution of gallic acid and protocatechuic acid increased to 1.90. This is because as the buffer concentration increases, the ionic strength increases, the ζ potential decreases, and the electroosmotic flow velocity decreases. The longer the migration time of the compound to be measured in the capillary, the more conducive it is to extend the time window and further improve the separation efficiency. However, when 20 mM borax was added to the buffer, the resolution did not increase significantly, and the migration time of the compounds of interest prolonged. At borax concentrations of 5, 10, 15, and 20 mM, the peak currents of flavonoids and phenolic acids were 12.3, 22.8, 32.7, and 45.1 μA, respectively, due to the enhanced ionic strength. In summary, a suitable analysis time and good resolution were obtained in the 15 mM borax buffer, so this concentration was the optimal condition.

[0066] The effect of the pH value of different buffers on the separation efficiency was investigated: Example 11. The pH value of the buffer was adjusted to 8.1, and the others were the same as in Example 7.

[0067] Comparative Example 12. The pH value of the buffer was adjusted to 8.7, and the others were the same as in Example 7.

[0068] Comparative Example 13. The pH value of the buffer was adjusted to 9.0, and the others were the same as in Example 7.

[0069] The selection of the buffer pH value is very important in micellar electrokinetic chromatography because it directly affects the overall charge of the analytes and the level of electroosmotic flow. The pH value was varied by 0.3 within the range of 8.1 - 9.0 (Example 11, Example 7, Comparative Examples 12 and 13), while keeping the buffer concentration constant at 15 mM borax and 0.8% w / v saponin. From Figure 4It can be seen from the chromatogram that at pH 8.1 (curve a in the figure), the baseline separation of the target analyte is achieved. When the pH increases to 8.4, the resolution of the 10 analytes (1.50 - 8.50) is significantly improved and the separation efficiency increases (curve b in the figure). However, at pH 8.7 (curve c in the figure), resveratrol and catechin are merged, showing a baseline with distorted peak shape. When the pH increases to 9.0 (curve d in the figure), resveratrol and catechin are eluted, and the migration times of all analytes change. The peak area of gallic acid is significantly reduced by 3.7 times compared with that under the condition of pH 8.7. The reason for this phenomenon can be explained as follows: with the increase of the buffer pH, the ionization of groups such as SiO - on the capillary wall increases, and the surface negative charge density increases. The electrostatic repulsion between phenols and the capillary wall is enhanced, making these compounds more likely to interact with micelles in the solution, thereby reducing the difference in analyte migration rates (the apparent migration rates of resveratrol and catechin are similar) and causing co-elution. Therefore, considering the separation efficiency of the 10 analytes, choosing a pH value of 8.1 - 8.4 has better effects in terms of resolution and analysis time.

Claims

1. A method for analyzing phenolic components in Perilla frutescens leaves based on micellar electrokinetic chromatography, characterized in that: Step (1): Use a pulverizer to crush Perilla frutescens leaves into powder, add the Perilla frutescens leaf powder to a methanol solution with a volume concentration of 30 - 80%, add 10 - 50 g of Perilla frutescens leaf powder to each liter of methanol solution, and perform ultrasonic treatment for 10 - 60 min to obtain a Perilla frutescens leaf extract; Step (2): Prepare a buffer solution: Mix saponin, borax, and distilled water evenly, perform ultrasonic treatment for 15 - 20 min to obtain a uniformly mixed solution; filter through a 0.22 - 0.45 μm organic nylon filter head, and adjust the pH value to 8.0 - 8.4 with boric acid solution or sodium hydroxide solution to obtain a buffer solution; the concentration of saponin in the buffer solution is 0.8% - 1.4% w / v, and the concentration of borax is 10.0 - 20.0 mM; Step (4): Centrifuge the Perilla frutescens leaf extract, take the supernatant and inject it into an electrophoresis vial, and perform micellar electrokinetic chromatography separation with the prepared buffer solution to obtain a Perilla frutescens leaf electrophoresis chromatogram; Step (5): Centrifuge the mixed standard solution, take the supernatant and inject it into an electrophoresis vial; perform micellar electrokinetic chromatography separation with the prepared buffer solution to obtain a standard product electrophoresis chromatogram; Step (6): Quantitatively analyze the phenols in Perilla frutescens leaves according to the peak areas of the standard product electrophoresis chromatogram and the Perilla frutescens leaf electrophoresis chromatogram. The saponin added in the preparation of the buffer solution is used as a surfactant, and its purity is greater than or equal to 98% w / w.

2. The method for analyzing phenolic components in Perilla leaves based on micellar electrokinetic chromatography technology according to claim 1, wherein: The concentrations of the boric acid solution and the sodium hydroxide solution are 1 M.

3. The method for analyzing phenolic components in Perilla frutescens leaves based on micellar electrokinetic chromatography technology according to claim 1, wherein: The specific centrifugation treatment in Step (4) is to centrifuge at a speed of 10000 - 15000 rpm for 2 - 5 min.

4. The method for analyzing phenolic components in Perilla leaves based on micellar electrokinetic chromatography technology according to claim 1, wherein: The specific centrifugation treatment in Step (5) is to centrifuge at a speed of 10000 - 15000 rpm for 2 - 5 min.

5. The method for analyzing phenolic components in Perilla frutescens leaves based on micellar electrokinetic chromatography technology according to claim 1, characterized in that: ​

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