A method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil and its application

Abstract

This invention discloses a method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil, and its application. The method includes qualitative identification of rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil using thin-layer chromatography; direct extraction of rosmarinic acid using a specific polar solvent extraction method and separation from fat-soluble components; and determination of rosmarinic acid content in Prunella vulgaris seeds and Prunella vulgaris seed oil using high-performance liquid chromatography (HPLC) after comparison with a rosmarinic acid reference standard. This method has advantages such as high accuracy and precision, good stability and repeatability, and provides a method for quality detection of rosmarinic acid components in the development and utilization of Prunella vulgaris seeds and Prunella vulgaris seed oil, exhibiting strong specificity and good reproducibility.

Description

Technical Field

[0001] This invention relates to a quality analysis method for pharmaceuticals, health products, or food, and particularly to a method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil, and its application. Background Technology

[0002] Prunella vulgaris L., a plant belonging to the Lamiaceae family, is a perennial herb with dried fruit spikes. First recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), it gets its name from the fact that it withers after the summer solstice. It is a medicinal and edible plant with a medicinal history spanning thousands of years and is now included in the 2015 edition of the *Chinese Pharmacopoeia*. Prunella vulgaris has a bitter and pungent taste, is cold in nature, and enters the liver and gallbladder meridians. It can clear liver heat, improve eyesight, and reduce swelling. It has good clinical therapeutic effects on various diseases, including red and swollen eyes, photophobia, tearing, night pain in the eyes, dizziness, scrofula, goiter, breast cancer, hypertension, lymph node tuberculosis, infiltrative pulmonary tuberculosis, simple goiter, mumps, and acute icteric infectious hepatitis. Modern research shows that Prunella vulgaris contains various chemical components, including triterpenes, sterols, flavonoids, organic acids, and coumarins. It possesses pharmacological effects such as anti-tumor, anti-inflammatory, antibacterial, antiviral, immunomodulatory, antihypertensive, hypoglycemic, and hypolipidemic properties. Prunella vulgaris seeds are the dried, mature seeds of the plant Prunella vulgaris L. (Lamiaceae family). As the reproductive seeds of Prunella vulgaris, they should possess the genetically active substances and effects of the herb. However, their application value in food, health care, or medicine has not yet received sufficient attention, and no related reports have been found. In particular, quality control methods for Prunella vulgaris seeds and Prunella vulgaris seed oil are lacking.

[0003] Rosmarinic acid (RA) is a polyphenolic acid with the chemical name R(+)2-[3-(3,4-dihydroxyphenyl)-1-oxo-2-propenyl]oxy-3,4-dihydroxyphenylpropionic acid. It is widely distributed in plants of the Lamiaceae, Boraginaceae, and Cucurbitaceae families and is one of the main active ingredients of Prunella vulgaris. Modern pharmacological studies have shown that rosmarinic acid has various pharmacological activities, including antioxidant, antibacterial, anti-inflammatory, antitumor, antidepressant, anti-anxiety, kidney disease improvement, and liver protection. Its main effects and mechanisms of action are: (1) Antioxidant effect: Rosmarinic acid has a strong antioxidant effect and can scavenge free radicals in the body. This effect is related to the structure of its ortho-dihydroxyl group; (2) Antibacterial and anti-inflammatory effect: Rosmarinic acid has a significant inhibitory effect on bacteria such as Staphylococcus aureus, Micrococcus luteus, Escherichia coli, and Bacillus subtilis. It also has a certain inhibitory effect on various inflammations such as psoriasis, dermatitis, respiratory tract inflammation, and acute lung injury; (3) Antitumor effect: Rosmarinic acid has a significant inhibitory effect on various tumors such as colorectal cancer, breast cancer, cervical cancer, leukemia, lung cancer, and liver cancer. Its antitumor mechanism may be related to improving the body's immune function and effectively inhibiting the proliferation and invasion of tumor cells. (3) Involved in apoptosis; (4) Antidepressant and anti-anxiety effects: Rosmarinic acid has antidepressant effects. In vitro experiments have shown that rosmarinic acid can promote the proliferation of astrocytes in newborn rats. In vivo experiments have shown that rosmarinic acid can improve depressive-like behavior in rats with chronic unpredictable stress depression. Low doses of rosmarinic acid can exert an anti-anxiety effect; (5) Hepatoprotective effect: Rosmarinic acid can inhibit the proliferation and differentiation of hepatic stellate cells (HSCs) and counteract carbon tetrachloride-induced antifibrosis by inhibiting the expression of TGF-β1 and CTGF; (6) Effects on improving kidney diseases: Rosmarinic acid has the effect of improving kidney-related diseases, mainly manifested in reducing uric acid production, inhibiting glomerulonephritis, and delaying chronic renal insufficiency.

[0004] During the research process of developing the value of Prunella vulgaris seeds, the applicant of this application discovered for the first time that Prunella vulgaris seeds and Prunella vulgaris seed oil are rich in rosmarinic acid components. However, there are no reports in the prior art regarding the detection of rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of existing technologies and provide a method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil. This application discloses for the first time a method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil. This method utilizes a specific polar solvent extraction method to directly extract rosmarinic acid and separate it from fat-soluble components. High-performance liquid chromatography (HPLC) is then used to determine the content of rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil. This method has advantages such as high accuracy and precision, good stability and repeatability.

[0006] Another object of the present invention is to provide an application of the above-mentioned method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil.

[0007] The objective of this invention is achieved through the following technical solution: a method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil, comprising the following steps: qualitative identification of rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil using thin-layer chromatography; direct extraction of rosmarinic acid using a specific polar solvent extraction method and separation from fat-soluble components; comparison with rosmarinic acid reference standard; and determination of rosmarinic acid content in Prunella vulgaris seeds and Prunella vulgaris seed oil using high-performance liquid chromatography.

[0008] The specific steps of the thin-layer chromatography detection method are as follows:

[0009] (1) Preparation of the test solution:

[0010] ① Preparation of the test solution of Prunella vulgaris seeds:

[0011] A. Add the powdered Prunella vulgaris seeds to a methanol aqueous solution for ultrasonic extraction, filter, remove the solvent from the filtrate, and obtain the residue;

[0012] B. The residue was dissolved in methanol to obtain a solution of Prunella vulgaris seed powder for testing; wherein the concentration of methanol in the methanol-water solution was 70-90% by volume.

[0013] ② Preparation of Prunella vulgaris seed oil test solution: Prunella vulgaris seed oil and petroleum ether with a boiling range of 60-90℃ are mixed evenly, and then extracted by shaking with methanol aqueous solution. The lower methanol aqueous solution phase is separated and obtained. The solvent is removed from the obtained methanol aqueous solution phase, and the residue is dissolved in methanol to obtain the Prunella vulgaris seed oil test solution. The concentration of methanol in the methanol aqueous solution is 70-90% by volume.

[0014] (2) Preparation of reference solution: Rosmarinic acid reference standard was dissolved in methanol to prepare the reference solution;

[0015] (3) Thin-layer chromatography (TLC) detection: Apply the test solution and the reference solution separately to the same high-performance silica gel GF plate. 254 Develop the thin-layer plate with a developing solvent, remove it, and let it air dry;

[0016] (4) Observe the silica gel GF 254 Thin-layer plate: Examine under ultraviolet light at 254nm and 365nm respectively; observe whether there are spots of the same color at the corresponding positions of the test sample chromatogram and the reference sample chromatogram to identify whether Prunella vulgaris seeds and Prunella vulgaris seed oil contain rosmarinic acid.

[0017] The Prunella vulgaris powder mentioned in step (1) is preferably obtained by the following steps: crushing and breaking the cell wall of Prunella vulgaris seeds and passing them through a sieve of at least 40 mesh to obtain Prunella vulgaris powder.

[0018] The preferred sieve is a 40-50 mesh sieve.

[0019] The preferred conditions for ultrasonic extraction in step (1) ①A are extraction at a power of 50-200W and a frequency of 40-60kHz for 20-80 minutes; more preferably, extraction at a power of 80-150W and a frequency of 45-50kHz for 30-60 minutes; and most preferably, extraction at a power of 150W and a frequency of 45kHz for 30-60 minutes.

[0020] The methanol concentration in the methanol aqueous solution described in step (1) ①A is preferably 80% by volume.

[0021] The amount of methanol aqueous solution mentioned in step (1) ①A is preferably calculated based on 2.0-5.0g of Prunella vulgaris seed powder mixed with 30-60mL of methanol aqueous solution.

[0022] In step (1)①A, the solvent removal is preferably carried out by evaporation.

[0023] The amount of methanol used in step (1) ①B is preferably calculated based on 2.0-5.0g of Prunella vulgaris seed powder mixed with 1-3mL of methanol aqueous solution; more preferably, it is calculated based on 2.0-5.0g of Prunella vulgaris seed powder mixed with 2mL of methanol aqueous solution.

[0024] The amount of petroleum ether used in step (1) ② is preferably calculated based on 0.6-1.5g of Prunella vulgaris seed oil to 30mL of petroleum ether.

[0025] The methanol concentration in the methanol aqueous solution described in step (1) ② is preferably 80% by volume.

[0026] The amount of methanol aqueous solution mentioned in step (1) ② is preferably calculated based on 0.6-1.5g of Prunella vulgaris seed oil to 30mL of methanol aqueous solution.

[0027] The extraction in step (1) ② is performed more than once; preferably twice, and the methanol-water phases are combined.

[0028] The preferred shaking time in step (1)② is 1 min per shaking.

[0029] The solvent removal method described in step (1) ② is preferably the solvent removal by evaporation.

[0030] The amount of methanol used in step (1)② is preferably calculated based on 0.6-1.5g of Prunella vulgaris seed powder mixed with 1-3mL of methanol aqueous solution; more preferably, it is calculated based on 0.6-1.5g of Prunella vulgaris seed powder mixed with 2mL of methanol aqueous solution.

[0031] The concentration of rosmarinic acid in the reference solution described in step (2) is preferably 0.9 to 1.1 mg / mL; more preferably 1 mg / mL.

[0032] The amount of the test solution used in step (2) is 2 to 10 μL.

[0033] The amount of the reference solution used in step (2) is 2 to 10 μL.

[0034] The developing agent mentioned in step (3) is preferably a cyclohexane-ethyl acetate-isopropanol-formic acid solution; more preferably a cyclohexane-ethyl acetate-isopropanol-formic acid solution obtained by mixing in a volume ratio of 6.6-6.8:1.3-1.5:1.5-1.7:0.2-0.4; and most preferably a cyclohexane-ethyl acetate-isopropanol-formic acid solution obtained by mixing in a volume ratio of 6.7:1.4:1.6:0.3.

[0035] The specific steps of the high-performance liquid chromatography detection method are as follows:

[0036] 1) Chromatographic conditions: The chromatographic conditions and system suitability test used octadecylsilane-bonded silica gel as the stationary phase, acetonitrile-methanol-0.1% (v / v) phosphoric acid aqueous solution as the mobile phase, and the detection wavelength was 330 nm; the theoretical plate number calculated based on the rosmarinic acid peak should not be less than 8000;

[0037] 2) Preparation of reference solution: Take an appropriate amount of rosmarinic acid reference standard, accurately weigh it, dissolve it in methanol aqueous solution and dilute to volume to obtain rosmarinic acid reference solution; the methanol aqueous solution is a methanol aqueous solution with a volume percentage of 45-55%;

[0038] 3) Preparation of the test solution

[0039] ① Preparation of Prunella vulgaris seed test solution: Take Prunella vulgaris seed powder, accurately weigh it, place it in a stoppered conical flask, accurately add 45-55% (v / v) methanol aqueous solution, accurately weigh it, sonicate it, cool it, weigh it again, replenish the lost weight with 45-55% (v / v) methanol aqueous solution, shake well, filter it, and take the filtrate to obtain the test solution;

[0040] ② Preparation of Prunella vulgaris seed oil test solution: Take Prunella vulgaris seed oil, accurately weigh it, place it in a stoppered conical flask, accurately add 45-55% (v / v) methanol aqueous solution, sonicate, cool, weigh it again, replenish the lost weight with 45-55% (v / v) methanol aqueous solution, shake well, let stand to allow the emulsion to separate into clear layers, take the 45-55% (v / v) methanol aqueous solution portion, filter it, and take the filtrate to obtain the test solution;

[0041] 4) Determination method: Accurately pipette equal volumes of the reference solution and the test solution, inject them into a liquid chromatograph, and determine the result.

[0042] The mobile phase described in step 1) is preferably obtained by mixing acetonitrile, methanol and 0.1% (v / v) phosphoric acid aqueous solution in a volume ratio of 17-19:15-17:65-67; ​​more preferably, it is obtained by mixing acetonitrile, methanol and 0.1% (v / v) phosphoric acid aqueous solution in a volume ratio of 18:16:66.

[0043] The chromatographic column used in step 1) is preferably an Ultimate XB-C18 column.

[0044] The injection volume in the chromatogram described in step 1) is 5-10 μL; more preferably 10 μL.

[0045] The column temperature in the chromatography described in step 1) is 24-27°C; more preferably 25°C.

[0046] The flow rate of the mobile phase in step 1) is preferably 0.8-1.2 mL / min; more preferably 1 mL / min.

[0047] The methanol aqueous solution mentioned in step 2) is preferably a methanol aqueous solution with a concentration of 50% by volume.

[0048] The concentration of the rosmarinic acid reference solution mentioned in step 2) is preferably 0.04-0.05 mg of rosmarinic acid per 1 mL.

[0049] The Prunella vulgaris powder mentioned in step 3)① is preferably obtained by the following steps: crushing and breaking the cell wall of Prunella vulgaris seeds and passing them through a sieve of at least 40 mesh to obtain Prunella vulgaris powder.

[0050] The methanol aqueous solution mentioned in step 3)① is preferably a methanol aqueous solution with a concentration of 50% by volume.

[0051] The amount of methanol-water solution mentioned in step 3)① is preferably calculated based on 50-150 mL of methanol-water solution per 0.5-1.5 g of Prunella vulgaris seed powder.

[0052] The preferred conditions for ultrasound in step 3)① are 500-700W power and 30-50kHz frequency for 40-80 minutes; more preferably 600W power and 40kHz frequency for 60 minutes.

[0053] The methanol aqueous solution mentioned in step 3)② is preferably a methanol aqueous solution with a concentration of 50% by volume.

[0054] The amount of methanol aqueous solution mentioned in step 3) ② is preferably calculated as 50-150 mL of methanol aqueous solution per 0.4-1.0 g of Prunella vulgaris seed oil; more preferably, it is calculated as 100 mL of methanol aqueous solution per 0.8 g of Prunella vulgaris seed oil.

[0055] The preferred conditions for ultrasound treatment in step 3) ② are 500-700W power and 30-50kHz frequency for 40-80 minutes; more preferably, 600W power and 40kHz frequency for 60 minutes.

[0056] The volume of aspirated in step 4) is preferably 10 μL.

[0057] This product, calculated on a dried basis, contains rosmarinic acid (C...). 18 H 16 08) Not less than 0.45%; Prunella vulgaris seed oil contains rosmarinic acid (C 18 H 16 08) Not less than 0.60%.

[0058] The specific polar solvent extraction method described above involves directly extracting rosmarinic acid using a 45-55% (v / v) methanol aqueous solution, which effectively extracts rosmarinic acid and separates it from fat-soluble components.

[0059] The methanol aqueous solution is preferably a methanol aqueous solution with a volume percentage of 45-55%.

[0060] The Prunella vulgaris seed oil mentioned above is prepared by supercritical CO2 extraction using a polar entrainer; preferably, it is prepared through the following steps:

[0061] A. Mix the cell-wall broken Prunella vulgaris seed powder and polar entrainer evenly; then, evenly fill the mixed powder containing polar entrainer into a supercritical CO2 extraction vessel, heat the supercritical CO2 extraction vessel to a temperature of 30-60℃, pressurize it to 10-25MPa by introducing supercritical CO2 fluid, stop the pump, and statically soak and extract for 20-60 minutes; after static soaking and extraction, start dynamic extraction and start timing when the temperature and pressure of the separator reach the set values, and the extraction time is 100-180 minutes;

[0062] B. A two-stage separation process is adopted, with the following conditions: first-stage desorber pressure of 9–13 MPa and separation temperature of 37–43℃; second-stage desorber pressure of 4–6 MPa and separation temperature of 27–33℃; resulting in yellow-green Prunella vulgaris seed oil.

[0063] C. Place the obtained Prunella vulgaris seed oil in a filter device with a pore size equal to or smaller than 350 mesh for vacuum filtration, discarding impurities that cannot be filtered out, to obtain clear Prunella vulgaris seed oil.

[0064] The application of the method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil in the development of Prunella vulgaris seeds and Prunella vulgaris seed oil.

[0065] Prunella vulgaris is a common medicinal herb listed in the Chinese Pharmacopoeia, possessing both wild and cultivated resources. However, due to the lack of development and utilization of its seeds, tens of thousands of tons of seeds are left uncollected and scattered in wastelands every year, resulting in resource waste. This invention offers the following advantages and effects compared to existing technologies:

[0066] 1. This invention provides a method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil for the first time, providing a method and basis for the development and utilization of Prunella vulgaris seeds and Prunella vulgaris seed oil, and reducing the waste of Prunella vulgaris seeds.

[0067] 2. This invention is the first to discover that Prunella vulgaris seeds and Prunella vulgaris seed oil are rich in rosmarinic acid components, providing a basis for the health care and medicinal use of Prunella vulgaris seeds and Prunella vulgaris seed oil;

[0068] 3. The method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil provided by the present invention has the characteristics of high accuracy and precision, good stability and repeatability. Attached Figure Description

[0069] Figure 1 The images show the TLC identification results of rosmarinic acid in Prunella vulgaris seeds under a 254 nm UV lamp; where 1, 2, 3, 5, 6, and 7 are the test samples of Prunella vulgaris seeds, and 4 is the reference standard of rosmarinic acid.

[0070] Figure 2 The images show the TLC identification results of rosmarinic acid in Prunella vulgaris seeds under ultraviolet light at 365 nm; among them, 1, 2, 3, 5, 6, and 7 are the test samples of Prunella vulgaris seeds, and 4 is the reference standard of rosmarinic acid.

[0071] Figure 3 The images show the TLC identification results of rosmarinic acid, a triterpenoid component in Prunella vulgaris seed oil, under a UV lamp at 254 nm. Among them, 1, 2, 3, 5, 6, and 7 are the test samples of Prunella vulgaris seed oil, and 4 is the reference standard of rosmarinic acid.

[0072] Figure 4The images show the TLC identification results of ursolic acid, a triterpenoid component in Prunella vulgaris seed oil, under ultraviolet light at 365 nm. Among them, 1, 2, 3, 5, 6, and 7 are the test samples of Prunella vulgaris seed oil, and 4 is the reference standard of rosmarinic acid.

[0073] Figure 5 This is the HPLC chromatogram of rosmarinic acid reference standard.

[0074] Figure 6 This is an HPLC chromatogram of rosmarinic acid in Prunella vulgaris seeds.

[0075] Figure 7 This is the HPLC chromatogram of rosmarinic acid in Prunella vulgaris seed oil.

[0076] Figure 8 This is an HPLC chromatogram of the blank extraction solvent in Prunella vulgaris seed (oil).

[0077] Figure 9 This is a linear fitting curve of the rosmarinic acid standard solution. Detailed Implementation

[0078] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0079] Example 1: Thin-layer chromatography (TLC) detection of rosmarinic acid in Prunella vulgaris seeds

[0080] (1) Preparation of test solutions: Take 6 samples of Prunella vulgaris seeds, crush and break the cell wall of each sample, pass them through a 40-mesh sieve, accurately weigh 2.5g of each sample of Prunella vulgaris seed powder, add 50mL of aqueous solution containing 80% (v / v) methanol, extract by ultrasonication (150W power, 45kHz frequency) for 45min, filter, place the filtrate in an evaporating dish, heat in a water bath to evaporate to dryness, add 2mL of methanol to dissolve the residue, and obtain test solutions of Prunella vulgaris seed powder 1, 2, 3, 5, 6 and 7 respectively, for later use.

[0081] (2) Preparation of reference solution: Weigh 1.81 mg of rosmarinic acid reference standard, add 2 mL of methanol to dissolve it, and prepare a solution containing about 0.91 mg of rosmarinic acid per 1 mL. This solution is designated as reference solution No. 4 and is kept for later use.

[0082] (3) Thin-layer chromatography (TLC) detection:

[0083] According to the thin-layer chromatography method (General Rule 0502) in Part I of the 2015 edition of the Pharmacopoeia of the People's Republic of China, 5 μL of each of the above two solutions were applied separately to the same high-performance silica gel GF plate. 254On the thin-layer plate, cyclohexane-ethyl acetate-isopropanol-formic acid (volume ratio 6.7:1.4:1.6:0.3) was used as the developing solvent. After development, the plate was removed and dried. It was then examined under UV light at 254 nm and UV light at 365 nm, respectively.

[0084] Detection under 254 nm UV light: In the chromatogram of the test sample, fluorescent quenching spots appear at the corresponding positions as in the chromatogram of the reference standard rosmarinic acid; see... Figure 1 .

[0085] Examine under ultraviolet light (365nm): In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard rosmarinic acid; see... Figure 2 .

[0086] (4) Thin-layer chromatography (TLC) detection results and conclusions:

[0087] The results of detection under 254nm ultraviolet light and 365nm ultraviolet light showed that all six samples of Prunella vulgaris seeds contained rosmarinic acid, and the chromatograms of rosmarinic acid in the six samples were basically the same.

[0088] Example 2: Thin-layer chromatography (TLC) detection of rosmarinic acid in Prunella vulgaris seed oil

[0089] (1) Preparation of test solutions: Take 6 portions of Prunella vulgaris seed oil samples, weigh about 0.8g of each; add 30mL of petroleum ether (60-90℃) to dissolve each sample, then transfer each sample to a separatory funnel, add 30mL of aqueous solution containing 80% (v / v) methanol to each sample, shake and extract twice, shaking for 1 minute each time, let stand to separate the layers, separate the lower layer of methanol aqueous solution, place each sample in an evaporating dish, heat in a water bath to evaporate to dryness, add 2mL of methanol to dissolve each residue, and obtain test solutions No. 1, 2, 3, 5, 6 and 7 of Prunella vulgaris seed oil for later use.

[0090] (2) Preparation of reference solution: Weigh 1.81 mg of rosmarinic acid reference standard, add 2 mL of methanol to dissolve it, and prepare a solution containing about 0.91 mg of rosmarinic acid per 1 mL. This solution is designated as reference solution No. 4 and is kept for later use.

[0091] (3) Thin-layer chromatography (TLC) detection:

[0092] According to the thin-layer chromatography method (General Rule 0502) in Part I of the 2015 edition of the Pharmacopoeia of the People's Republic of China, 4 μL of each of the above two solutions were applied separately to the same high-performance silica gel GF plate. 254On the thin-layer plate, cyclohexane-ethyl acetate-isopropanol-formic acid (volume ratio 6.7:1.4:1.6:0.3) was used as the developing solvent. After development, the plate was removed and dried. It was then examined under UV light at 254 nm and UV light at 365 nm, respectively.

[0093] Detection under 254 nm UV light: In the chromatogram of the test sample, fluorescent quenching spots appear at the corresponding positions as in the chromatogram of the reference standard rosmarinic acid. Figure 3 .

[0094] Examine under ultraviolet light (365nm): In the chromatogram of the test sample, fluorescent spots of the same color appear at the corresponding positions as in the chromatogram of the reference standard rosmarinic acid. Figure 4 .

[0095] (4) Thin-layer chromatography (TLC) detection results and conclusions:

[0096] The results of detection under 254nm ultraviolet light and 365nm ultraviolet light showed that all six samples of Prunella vulgaris seed oil contained rosmarinic acid, and the chromatograms of rosmarinic acid in the six samples were basically the same.

[0097] Example 3: Determination of Rosmarinic Acid Content in Prunella Vulgaris Seeds and Prunella Vulgaris Seed Oil and Methodological Validation

[0098] 1. Principles and Reagents

[0099] 1.1 Principle

[0100] Rosmarinic acid, contained in Prunella vulgaris seeds and Prunella vulgaris seed oil, is a polar water-soluble substance. The polar component rosmarinic acid was extracted using a polar solvent extraction method and separated from the fat-soluble components. The content of rosmarinic acid was determined by high performance liquid chromatography.

[0101] 1.2 Reagents

[0102] Phosphoric acid (analytical grade), methanol (analytical grade), methanol (chromatographic grade), acetonitrile (chromatographic grade), rosmarinic acid reference standard (batch number 111871-202007, content: 98.1%), China National Institutes for Food and Drug Control. Prunella vulgaris seed sample, Jiangxi Xinkangjian Ecological Agriculture Development Co., Ltd.

[0103] 1.3 Instruments

[0104] BY-500A high-speed universal pulverizer, Yongkang Sufeng Industry and Trade Co., Ltd.; Thermo Fisher U3000 series high-performance liquid chromatograph (U UV detector), Thermo Fisher Scientific, Germany; BT125D type 1 / 100,000 electronic analytical balance, Sartorius, Germany; SB25-12TD type Xinzhi ultrasonic cleaner, Bona Cleaning Equipment Co., Ltd.; UV lamp; water bath; common glassware; drying oven.

[0105] 2. Methods and Results

[0106] 2.1 Preparation of reference solution

[0107] Accurately weigh 4.09 mg of rosmarinic acid reference standard, place it in a 100 mL volumetric flask, dissolve it in 50% (v / v) methanol aqueous solution and dilute to volume to obtain rosmarinic acid reference standard solution.

[0108] 2.2 Preparation of test solution

[0109] 2.2.1 Preparation of Prunella vulgaris seed test solution: Take Prunella vulgaris seeds, crush and break the cell wall, pass through a 40-mesh sieve, weigh about 1.0 g of Prunella vulgaris seed powder, accurately weigh it, place it in a stoppered conical flask, accurately add 100 mL of aqueous solution containing 50% (v / v) methanol, accurately weigh it, sonicate (power 600W, frequency 40kHz) for 60 minutes, cool, weigh it again, replenish the lost weight with aqueous solution containing 50% methanol, shake well, filter, and take the filtrate to obtain the Prunella vulgaris seed test solution.

[0110] 2.2.2 Preparation of Prunella vulgaris seed oil test solution: Weigh approximately 0.8 g of Prunella vulgaris seed oil accurately and place it in a stoppered conical flask. Accurately add 100 mL of an aqueous solution containing 50% (v / v) methanol. Sonicate the solution (600 W power, 40 kHz frequency) for 60 minutes, cool, and weigh again. Make up the lost weight with an aqueous solution containing 50% (v / v) methanol, shake well, and allow the emulsion to separate into clear layers. Take the portion of the aqueous solution containing 50% (v / v) methanol, filter it, and take the filtrate to obtain the Prunella vulgaris seed oil test solution.

[0111] 2.3 Chromatographic conditions

[0112] The chromatographic column used was an Ultimate XB-C18 (4.6 mm × 250 mm, 5 μm), column temperature 25 ℃, detection wavelength 330 nm, flow rate 1 mL / min, injection volume 10 μL, and mobile phase was acetonitrile-methanol-0.1% (v / v) phosphoric acid aqueous solution (volume ratio 18:16:66); the chromatogram is shown below. Figure 5-8 .

[0113] From the chromatogram Figure 5-8 It can be seen that in the HPLC chromatogram of rosmarinic acid reference standard, an absorption peak of rosmarinic acid reference standard appears at a retention time of about 10.3 minutes. In the HPLC chromatograms of Prunella vulgaris seeds and Prunella vulgaris seed oil, an absorption peak of rosmarinic acid appears at a retention time of about 10.3 minutes, and the peak shape is consistent with that of rosmarinic acid reference standard, indicating good separation. However, no absorption peak of rosmarinic acid is seen in the HPLC chromatogram of the blank extraction solvent. This suggests that the HPLC determination conditions for rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil are mature and usable.

[0114] 2.4 Examination of Linear Relationships

[0115] Take the reference solution from section "2.1", filter it through a 0.45 μm microporous membrane, and inject 5, 10, 15, 20, 25, and 30 μL of the solution under the chromatographic conditions described in section "2.3". Plot a standard curve with the injection volume (μg) on ​​the x-axis and the peak area on the y-axis. The results are shown in Table 1 and [Table data would be inserted here]. Figure 9 The regression equation is: Y = 39.886X - 0.0115, R0 2 =0.9993.

[0116] Table 1. Experimental data on the linear relationship of rosmarinic acid (n=6)

[0117]

[0118] Y = 39.886X - 0.0115, R 2 =0.9993

[0119] The above results indicate that rosmarinic acid exhibits a good linear relationship with peak area in the range of 0.2045–1.2270 μg.

[0120] 2.5 Precision test: Accurately pipette 10 μL of the rosmarinic acid reference solution under section “2.1”, and inject it 6 times consecutively under the chromatographic conditions under section “2.3”. Measure the peak area and calculate the average peak area value of rosmarinic acid as 16.261, with an RSD of 0.69%. The RSD of the peak area value is <2%, and the results are shown in Table 2. The results indicate that the instrument has good precision.

[0121] Table 2 Precision test data (n=6)

[0122]

[0123] 2.6 Stability Test: The *Prunella vulgaris* seed sample solution (section 2.2.1) and the *Prunella vulgaris* seed oil sample solution (section 2.2.2) were injected at 0, 1, 4, 8, 12, and 24 hours respectively, according to the chromatographic conditions (section 2.3). The peak area of ​​rosmarinic acid was measured. The average peak area of ​​rosmarinic acid in the *Prunella vulgaris* seed sample was calculated to be 23.557, with an RSD of 0.99%. The average peak area of ​​rosmarinic acid in the *Prunella vulgaris* seed oil sample was calculated to be RSD = 0.99%, with an RSD of 0.93%. The RSDs of the peak areas of rosmarinic acid in both samples were <2%, as shown in Table 3.

[0124] Table 3. Stability test data (n=6)

[0125]

[0126] The test results show that the sample solution has good stability within 24 hours.

[0127] 2.7 Reproducibility Test: Six sample solutions of *Prunella vulgaris* seeds (batch number 20220314) were prepared according to the preparation method of the *Prunella vulgaris* seed test solution in section “2.2.1”. Six sample solutions of *Prunella vulgaris* seed oil (batch number 20220318) were prepared according to the preparation method of the *Prunella vulgaris* seed oil test solution in section “2.2.2”. The rosmarinic acid content was determined under the chromatographic conditions in section “2.3”. The average rosmarinic acid content in the *Prunella vulgaris* seed samples was 0.5894%, with an RSD of 0.98%. The average rosmarinic acid content in the *Prunella vulgaris* seed oil samples was 0.7969%, with an RSD of 0.94%. The RSDs of the rosmarinic acid content in both samples were <2%. The results are shown in Table 4.

[0128] Table 4. Reproducibility test data (n=6)

[0129]

[0130] The experimental results show that the reproducibility of this determination method is good.

[0131] 2.8 Recovery Test: Six samples of *Prunella vulgaris* seed (batch number 20220314) with known content and 0.5 g each were accurately weighed. An appropriate amount of reference solution equivalent to 100% of the rosmarinic acid content in each sample was accurately added, and test solutions were prepared according to the method described in section "2.2.1". Six samples of *Prunella vulgaris* seed oil (batch number 20220318) with known content and 0.4 g each were accurately weighed. An appropriate amount of reference solution equivalent to 100% of the rosmarinic acid content in each sample was accurately added, and test solutions were prepared according to the method described in section "2.2.2". The samples were then injected and analyzed under the chromatographic conditions described in section "2.3". The rosmarinic acid content and recovery rate were calculated. The average recovery rate of rosmarinic acid in the *Prunella vulgaris* seed samples was 99.22%, with an RSD of 1.70%. The average recovery rate of rosmarinic acid in the *Prunella vulgaris* seed oil samples was 100.30%, with an RSD of 1.89%. The results are shown in Table 5.

[0132] Table 5. Recovery rate of rosmarinic acid (n=6)

[0133]

[0134] The experimental results showed that the recovery rate of rosmarinic acid in Prunella vulgaris seeds was between 97.11% and 101.26%, and the recovery rate of rosmarinic acid in Prunella vulgaris seed oil was between 97.58% and 102.67%. The recovery of the two batches of samples was good.

[0135] Example 4: Determination of rosmarinic acid content in three batches of Prunella vulgaris seeds and Prunella vulgaris seed oil

[0136] Following the method for content determination in Example 3 above, the content of rosmarinic acid in three different batches of Prunella vulgaris seeds and three different batches of Prunella vulgaris seed oil samples was determined, and the results are shown in Table 6.

[0137] Table 6. Results of rosmarinic acid content determination in Prunella vulgaris seeds and Prunella vulgaris seed oil samples (n=3)

[0138]

[0139] The content of rosmarinic acid in the three batches of Prunella vulgaris seeds was measured to be between 0.5431% and 0.7073%; the content of rosmarinic acid in the three batches of Prunella vulgaris seed oil was measured to be between 0.6886% and 0.9563%.

[0140] Discussion: The results of this study indicate that the determination of rosmarinic acid content in Prunella vulgaris seeds and Prunella vulgaris seed oil showed good linearity within the linear range, and the precision, stability, reproducibility, and recovery tests all met the requirements.

[0141] The Prunella vulgaris seed oil used in the experiment was prepared by supercritical CO2 extraction using a polar entrainer. Prunella vulgaris seed oil prepared by pressing (see patent application "2022102354058") or conventional supercritical CO2 extraction (see patent application "202210235666X") contains little or no rosmarinic acid.

[0142] The Prunella vulgaris seed oil used in this invention is prepared by the following method:

[0143] (1) Collection of Prunella vulgaris seeds: Harvest fully mature and dried Prunella vulgaris fruit spikes, place them in a clean collection device, shake the Prunella vulgaris seeds from the dried Prunella vulgaris fruit spikes into the collection device, collect the obtained Prunella vulgaris seeds, and obtain 5.94 kg of crude Prunella vulgaris seeds.

[0144] (2) Cleaning of Prunella vulgaris seeds

[0145] A. Removing coarse impurities: Using a 20-mesh sieve, place 5.94 kg of the collected Prunella vulgaris seed crude product in the sieve, shake and sieve to allow the seeds with a particle size smaller than the sieve mesh to pass through, and remove the coarser impurities with a particle size larger than the sieve mesh to obtain 5.63 kg of Prunella vulgaris seed crude product with coarse impurities removed.

[0146] B. Removing fine impurities: Using a 30-mesh sieve, place 5.63 kg of the crude Prunella vulgaris seeds (after removing coarse impurities) in the sieve, shake and sieve to allow impurities smaller than the sieve mesh to pass through, removing the finer impurities smaller than the sieve mesh. Collect the Prunella vulgaris seeds that cannot pass through the sieve mesh to obtain 5.09 kg of pure Prunella vulgaris seeds.

[0147] (3) Cell wall disruption of Prunella vulgaris seeds:

[0148] A. Place the above-mentioned purified Prunella vulgaris seeds into a pulverizer and use a 40-mesh sieve for pulverizing and breaking down the cells.

[0149] B. Place the blender in an environment of 10±2℃; or install a cooling circulating water device on the outside of the heating part of the blender and keep the blending temperature below 15℃.

[0150] C. Start the crushing machine. Place the crushed Prunella vulgaris seed powder in a 40-mesh sieve and shake it through the sieve. Allow the seed powder with a particle size smaller than the sieve mesh to pass through. Add the seed powder that cannot pass through to the crusher for further crushing and cell wall breaking until all of them pass through. Obtain 4.88 kg of crushed Prunella vulgaris seed powder with a cell wall breaking rate of over 99%.

[0151] (4) Mix with the entrainer:

[0152] ① Add entrainer and mix: Add the polar entrainer methanol to the above-mentioned broken cell wall Prunella vulgaris seed powder raw material. The ratio of Prunella vulgaris seed powder to methanol entrainer is 800 ml of methanol entrainer for every 1 kg of Prunella vulgaris seed powder, and mix evenly.

[0153] ② Filling: The powder, which is a uniform mixture of the above-mentioned Prunella vulgaris seed powder and methanol entrainer, is uniformly filled into the supercritical CO2 extraction vessel.

[0154] (5) Supercritical extraction:

[0155] ① Static soaking extraction: The supercritical CO2 extraction vessel filled with the above-mentioned Prunella vulgaris seed mixture powder is heated to 45°C, and supercritical CO2 fluid is introduced to pressurize to 20MPa. The pump is then stopped, and static soaking extraction is performed for 40 minutes.

[0156] ② Dynamic extraction: After static soaking and extraction for 40 minutes, dynamic extraction begins and timing begins when the temperature and pressure of the separator reach the set values ​​(i.e., the separation conditions in ③ below). The extraction time is 120 minutes.

[0157] ③ Separation: Two-stage separation was adopted. The separation process conditions were: pressure of 10 MPa and separation temperature of 37℃ in the first stage separator; pressure of 4 MPa and separation temperature of 27℃ in the second stage separator; 0.95 kg of yellow-green Prunella vulgaris seed oil was obtained, and the oil yield was 18.6% based on the weight of the cleaned Prunella vulgaris seeds.

[0158] (6) Filtration and impurity removal: Take the above-mentioned Prunella vulgaris seed oil and place it in a filter device with a pore size of 350 mesh for vacuum filtration. Discard the impurities that cannot be filtered out to obtain 0.93 kg of clear Prunella vulgaris seed oil.

[0159] (7) Quality inspection and filling: The above-mentioned Prunella vulgaris seed oil samples were taken for quality testing, and after meeting the standards, they were filled with nitrogen gas.

[0160] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil, characterized in that: The process includes the following steps: using thin-layer chromatography to qualitatively identify rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil; directly extracting rosmarinic acid using a specific polar solvent extraction method and separating it from fat-soluble components; then using rosmarinic acid reference standard as a reference and determining the content of rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil using high-performance liquid chromatography. The specific steps of the thin-layer chromatography method are as follows: (1) Preparation of the test solution: ① Preparation of the test solution of Prunella vulgaris seeds: A. Add the powdered Prunella vulgaris seeds to a methanol aqueous solution for ultrasonic extraction, filter, remove the solvent from the filtrate, and obtain the residue; B. The residue was dissolved in methanol to obtain a solution of Prunella vulgaris seed powder for testing; wherein the concentration of methanol in the methanol-water solution was 70-90% by volume. ② Preparation of Prunella vulgaris seed oil test solution: Prunella vulgaris seed oil and petroleum ether with a boiling range of 60-90℃ are mixed evenly, and then extracted with methanol aqueous solution by shaking. The lower methanol aqueous solution phase is separated. The solvent is removed from the obtained methanol aqueous solution phase, and the residue is dissolved in methanol to obtain the Prunella vulgaris seed oil test solution. The concentration of methanol in the methanol aqueous solution is 70-90% by volume. (2) Preparation of reference solution: Rosmarinic acid reference standard was dissolved in methanol to prepare the reference solution; (3) Thin-layer chromatography detection: Apply the test solution and the reference solution separately to the same high-performance silica gel GF plate. 254 Develop the thin-layer plate with a developing solvent, remove it, and let it air dry; (4) Observe the silica gel GF 254 Thin-layer plate: Examine under ultraviolet light at 254nm and 365nm respectively; observe whether there are spots of the same color at the corresponding positions of the test sample chromatogram and the reference sample chromatogram to identify whether Prunella vulgaris seeds and Prunella vulgaris seed oil contain rosmarinic acid. The developing agent mentioned in step (3) is a cyclohexane-ethyl acetate-isopropanol-formic acid solution obtained by mixing in a volume ratio of 6.6-6.8:1.3-1.5:1.5-1.7:0.2-0.

4.

2. The method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 1, characterized in that: The Prunella vulgaris seed powder mentioned in step (1) is obtained by the following steps: crushing the Prunella vulgaris seeds to break the cell wall and passing them through a sieve of at least 40 mesh to obtain Prunella vulgaris seed powder; Step (1) ① The concentration of methanol in the methanol-water solution mentioned in A is 80% by volume; The concentration of methanol in the methanol-water solution described in step (1) ② is 80% by volume. Step (1) ① The amount of methanol aqueous solution mentioned in A is calculated based on 2.0-5.0g of Prunella vulgaris seed powder mixed with 30-60mL of methanol aqueous solution; The amount of methanol mentioned in step (1) ①B is calculated based on 2.0-5.0g of Prunella vulgaris seed powder mixed with 1-3mL of methanol aqueous solution; The amount of petroleum ether used in step (1) ② is calculated based on 0.6-1.5g of Prunella vulgaris seed oil mixed with 30mL of petroleum ether; The amount of methanol-water solution used in step (1) ② is calculated based on 0.6-1.5g of Prunella vulgaris seed oil mixed with 30mL of methanol-water solution; The amount of methanol used in step (1) ② is calculated based on 0.6-1.5g of Prunella vulgaris seed powder mixed with 1-3mL of methanol aqueous solution; The concentration of rosmarinic acid in the reference solution described in step (2) is 0.9–1.1 mg / mL; The volume of the test solution used in step (2) is 2–10 μL; The amount of the reference solution used in step (2) is 2 to 10 μL.

3. The method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 1, characterized in that: The conditions for ultrasonic extraction described in step (1) ①A are 50-200W power and 40-60kHz frequency for 20-80 minutes; Step (1) ① The solvent removal method described in A is to remove the solvent by evaporation; The extraction process described in step (1) ② is performed more than once; The shaking time mentioned in step (1) ② is 1 minute per shaking; The solvent removal mentioned in step (1) ② is achieved by evaporation.

4. The method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 1, characterized in that: The specific steps of the high-performance liquid chromatography detection method are as follows: 1) Chromatographic conditions: The chromatographic conditions and system suitability test used octadecylsilane-bonded silica gel as the stationary phase, acetonitrile-methanol-0.1% (v / v) phosphoric acid aqueous solution as the mobile phase, and the detection wavelength was 330 nm; the theoretical plate number calculated based on the rosmarinic acid peak should not be less than 8000; 2) Preparation of reference solution: Take an appropriate amount of rosmarinic acid reference standard, accurately weigh it, dissolve it in methanol aqueous solution and dilute to volume to obtain rosmarinic acid reference solution; the methanol aqueous solution is 45-55% by volume. 3) Preparation of the test solution ① Preparation of Prunella vulgaris seed test solution: Take Prunella vulgaris seed powder, weigh accurately, place in a stoppered conical flask, add 45-55% (v / v) methanol aqueous solution accurately, weigh accurately, sonicate, cool, weigh again, replenish the lost weight with 45-55% (v / v) methanol aqueous solution, shake well, filter, and take the filtrate to obtain the test solution; ② Preparation of Prunella vulgaris seed oil test solution: Take Prunella vulgaris seed oil, accurately weigh it, place it in a stoppered conical flask, accurately add 45-55% (v / v) methanol aqueous solution, sonicate, cool, weigh it again, replenish the lost weight with 45-55% (v / v) methanol aqueous solution, shake well, let stand to allow the emulsion to separate into clear layers, take the 45-55% (v / v) methanol aqueous solution portion, filter it, and take the filtrate to obtain the test solution; 4) Determination method: Accurately pipette equal volumes of the reference solution and the test solution, inject them into a liquid chromatograph, and determine the result.

5. The method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 4, characterized in that: The mobile phase mentioned in step 1) is prepared by mixing acetonitrile, methanol and 0.1% (v / v) phosphoric acid aqueous solution in a volume ratio of 17-19:15-17:65-67; The chromatographic column used in step 1) is an Ultimate XB-C18 column; The injection volume in the chromatogram described in step 1) is 5-10 μL; The column temperature in the chromatography described in step 1) is 24-27℃; The flow rate of the mobile phase mentioned in step 1) is 0.8-1.2 mL / min; The methanol-water solution mentioned in step 2) is a methanol-water solution with a concentration of 50% by volume; The concentration of the rosmarinic acid reference solution mentioned in step 2) is 0.04-0.05 mg of rosmarinic acid per 1 mL; The Prunella vulgaris seed powder mentioned in step 3) ① is obtained by the following steps: crushing and breaking the cell wall of Prunella vulgaris seeds, and passing them through a sieve of at least 40 mesh to obtain Prunella vulgaris seed powder; The methanol-water solution mentioned in step 3) ① is a methanol-water solution with a concentration of 50% by volume; The amount of methanol-water solution mentioned in step 3) ① is calculated based on 50-150 mL of methanol-water solution per 0.5-1.5 g of Prunella vulgaris seed powder; The methanol-water solution mentioned in step 3) ② is a methanol-water solution with a concentration of 50% by volume. The amount of methanol-water solution mentioned in step 3) ② is calculated based on 50-150 mL of methanol-water solution per 0.4-1.0 g of Prunella vulgaris seed oil.

6. The method for detecting rosmarinic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 4, characterized in that: The conditions for ultrasound treatment mentioned in step 3) ① are: power 500-700W, frequency 30-50kHz, and treatment time 40-80 minutes. The ultrasound conditions described in step 3) ② are 500-700W power, 30-50kHz frequency, and 40-80 minutes of treatment.

7. The application of the detection method according to any one of claims 1 to 6 in the development of Prunella vulgaris seeds and Prunella vulgaris seed oil.

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