Method for simultaneously detecting caffeic acid and rosmarinic acid in prunella vulgaris or its glycoside extract and application thereof

Abstract

The application discloses a method for simultaneously detecting caffeic acid and rosmarinic acid in prunella vulgaris or its glucoside extract and application thereof. The method simultaneously qualitatively identifies the caffeic acid and the rosmarinic acid in the prunella vulgaris or its glucoside extract by using a thin layer chromatography method; the caffeic acid and the rosmarinic acid are directly extracted by using a specific polar solvent extraction method, and then the content of the caffeic acid and the rosmarinic acid in the prunella vulgaris or its glucoside extract is simultaneously determined by using a high performance liquid chromatography method, with the mixed caffeic acid and rosmarinic acid reference substance as a control. The method has the advantages of high accuracy and precision, good stability and repeatability, and overcomes the complicated HPLC gradient elution method required for simultaneously detecting the caffeic acid and the rosmarinic acid in the prunella vulgaris in the past, and improves the accuracy and reproducibility of the determination. The application firstly finds that the content of the caffeic acid component with the glucoside effect in the preparation of the glucoside extract of the prunella vulgaris is significantly improved, and the corresponding prunella vulgaris conversion content is improved by more than one time.

Description

Technical Field

[0001] This invention relates to a quality analysis method for pharmaceuticals, health products, food, or cosmetics, and particularly to a method and application for the simultaneous detection of caffeic acid and rosmarinic acid in Prunella vulgaris or its sucrose extract. 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 and 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 phenylpropanoids. It possesses pharmacological effects such as anti-tumor, anti-inflammatory, antibacterial, antiviral, immunomodulatory, blood pressure-lowering, blood sugar-lowering, and blood lipid-lowering effects. Glycoside, an extract from white water lily, is an effective anti-glycation ingredient in skincare products, currently mainly imported due to its scarcity and high price. Prunella vulgaris contains abundant phenylpropanoid components, which are not only effective anti-glycation ingredients but also plentiful. The most abundant phenylpropanoid component in Prunella vulgaris is rosmarinic acid, followed by caffeic acid. Extracting rosmarinic acid and caffeic acid from Prunella vulgaris to produce glycoside extract is a viable alternative to the precious glycoside.

[0003]

[0004] Rosmarinic acid and caffeic acid both contain ortho- and tho-dihydroxyl groups, exhibiting good antioxidant, antibacterial, antiviral, and anti-inflammatory activities. They also have a good scavenging effect on glycation free radicals and are natural glycation components. There have been some reports on the detection of rosmarinic acid and caffeic acid in Prunella vulgaris, and methods for the simultaneous determination of rosmarinic acid and caffeic acid in Prunella vulgaris and its preparations have also been reported, such as "Determination of Tanshinone, Caffeic Acid and Rosmarinic Acid Content in Prunella vulgaris with Different Decoction Times by Reversed-Phase High Performance Liquid Chromatography" [China Journal of Traditional Chinese Medicine Information, 2020, 27(2):64-67]. However, all of these methods use a gradient elution method, which is relatively complicated and has relatively poor reproducibility and stability. Currently, there are no reports on TLC methods for the simultaneous detection of caffeic acid and rosmarinic acid in Prunella vulgaris and its extracts, nor on HPLC methods using constant current elution for the simultaneous detection of caffeic acid and rosmarinic acid in Prunella vulgaris and its extracts; furthermore, there are no reports of findings that the preparation of its extracts has significantly increased caffeic acid content, which has a saccharifying effect. Summary of the Invention

[0005] The primary objective of this invention is to address the aforementioned shortcomings of Prunella vulgaris and its extracts by achieving, for the first time, a TLC method for the simultaneous detection of caffeic acid and rosmarinic acid in Prunella vulgaris and its extracts. This invention also establishes, for the first time, a HPLC method for the simultaneous determination of caffeic acid and rosmarinic acid in Prunella vulgaris and its extracts using constant current elution, thereby providing a method for the simultaneous detection of caffeic acid and rosmarinic acid in Prunella vulgaris or its extracts with high accuracy and precision, good stability and repeatability.

[0006] Another object of the present invention is to provide an application of the method for simultaneously detecting caffeic acid and rosmarinic acid in the above-mentioned Prunella vulgaris or its sugar extract.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A method for simultaneously detecting caffeic acid and rosmarinic acid in Prunella vulgaris or its sucrose extract includes the following steps: simultaneously performing qualitative identification of caffeic acid and rosmarinic acid in Prunella vulgaris or its sucrose extract using thin-layer chromatography; directly extracting caffeic acid and rosmarinic acid using a specific polar solvent extraction method; then using a mixed standard of caffeic acid and rosmarinic acid as a reference; and simultaneously determining the content of caffeic acid and rosmarinic acid in Prunella vulgaris or its sucrose extract using high-performance liquid chromatography.

[0009] The sucrose extract is preferably prepared by the following steps:

[0010] (I) Extraction of phenylpropanoid components and crude polysaccharides: Using Prunella vulgaris (dried fruit spikes of Prunella vulgaris) as raw material, water extraction was performed to obtain an extract of Prunella vulgaris containing phenylpropanoid components and crude polysaccharides.

[0011] (II) Concentration of extract containing phenylpropanoids and crude polysaccharides: The extract obtained in step (I) is concentrated to obtain a concentrated solution;

[0012] (III) Separation and preparation of Prunella vulgaris extract and crude polysaccharide from Prunella vulgaris: The concentrate and ethanol were mixed evenly to obtain solution A, which contained 65-75% alcohol. Solution A was refrigerated and allowed to stand, and then filtered or vacuum filtered to obtain crude polysaccharide filtrate and ethanol filtrate of Prunella vulgaris extract.

[0013] (IV) Preparation of Prunella vulgaris extract: Take the ethanol filtrate of Prunella vulgaris extract, concentrate it, and obtain Prunella vulgaris extract.

[0014] The herbal medicine described in step (I) is obtained by cleaning and drying.

[0015] The preferred method for water extraction in step (I) is as follows: after soaking the herb Prunella vulgaris in water, decoct and reflux, and collect the decoction.

[0016] The water is preferably purified water.

[0017] The soaking time is preferably 1 to 2 hours.

[0018] The number of times the decoction is refluxed is preferably 3 to 4 times; more preferably 3 times.

[0019] The preferred steps for water extraction are as follows: add water equivalent to 18-20 times the weight of the medicinal materials and decoct for 1-1.5 hours; repeat the above steps 2-3 times; the first decoction is performed on the soaked Prunella vulgaris medicinal materials, and the repeated decoction is performed on the dregs; filter and combine the decoctions obtained from each decoction, or combine and filter; more preferably, the first decoction is performed by adding water equivalent to 20 times the weight of the medicinal materials and decoct for 1.5 hours; the repeated decoction is performed by adding water equivalent to 18 times the weight of the medicinal materials and decoct for 1 hour.

[0020] The filtration is preferably performed using a 250-400 mesh filter; more preferably using a 350 mesh filter.

[0021] The preferred method of filtration is to keep the decoction liquid warm while filtering it after each decoction.

[0022] The preferred method of concentration in step (II) is vacuum concentration.

[0023] The degree of concentration described in step (II) is preferably to concentrate to a relative density of 1.39-1.88 (measured at 70-80°C) or to concentrate to a concentration of 0.9-1.1g of Prunella vulgaris herb per 3mL of solution; more preferably, to concentrate to a relative density of 1.6-1.7 (measured at 70-75°C) or to concentrate to a concentration of 1g of Prunella vulgaris herb per 3mL of solution.

[0024] The dry solids content of the concentrate described in step (II) is 0.060–0.066 g / mL; the total dry solids content is 15–20%.

[0025] The alcohol content of solution A in step (III) is preferably 70% by volume.

[0026] The preferred temperature for refrigeration in step (III) is 2–8°C; more preferably 4–6°C.

[0027] The refrigeration time in step (III) is preferably 36 to 60 hours; more preferably 48 hours.

[0028] The filtration described in step (III) is vacuum filtration.

[0029] The filtration described in step (III) is centrifugal filtration.

[0030] The filter used for vacuum filtration or filtration in step (III) is preferably a 350-800 mesh filter; more preferably a 600 mesh filter.

[0031] The concentration method described in step (IV) is preferably vacuum concentration, which can recover ethanol.

[0032] The concentration described in step (IV) is preferably stopped when the relative density is 1.10-1.20 (measured at 60-70℃) or when the volume of the concentrated solution is equivalent to 1g of Prunella vulgaris.

[0033] The dry solids content of the Prunella vulgaris extract described in step (IV) is 0.0420-0.0460 g / ml; the total dry solids content, that is, the dry solids content in the Prunella vulgaris extract as a percentage of the total content of the Prunella vulgaris herb, is 7.6-9.6% (g / g).

[0034] The specific steps of the thin-layer chromatography detection method are as follows:

[0035] (1) Preparation of the test solution:

[0036] 1) Preparation of the Prunella vulgaris test solution:

[0037] A. Add the powdered Prunella vulgaris to a methanol-water solution for ultrasonic extraction, filter, remove the solvent from the filtrate, and obtain the residue;

[0038] B. Dissolve the residue in methanol, filter, and obtain the Prunella vulgaris test solution;

[0039] 2) Preparation of the test solution of Prunella vulgaris extract:

[0040] A. Take the extract of Prunella vulgaris and remove the solvent to obtain the residue;

[0041] B. Dissolve the residue in methanol, filter, and obtain the test solution of Prunella vulgaris extract;

[0042] (2) Preparation of reference solution: Caffeic acid and rosmarinic acid reference standards were dissolved in methanol to obtain a mixed solution of caffeic acid and rosmarinic acid, which was used as the reference solution;

[0043] (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;

[0044] (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, and you can identify whether Prunella vulgaris or its sugar extract contains caffeic acid and rosmarinic acid.

[0045] The Prunella vulgaris powder mentioned in step (1)1)A ​​is preferably obtained by the following steps: crushing Prunella vulgaris and passing it through a sieve of at least 40 mesh to obtain Prunella vulgaris powder.

[0046] The preferred sieve is a 40-50 mesh sieve.

[0047] The methanol content in the methanol aqueous solution described in step (1)1)A ​​is preferably 40-60% by volume; more preferably 50% by volume.

[0048] The amount of methanol aqueous solution mentioned in step (1)1)A ​​is preferably calculated based on 2.0-4.0g of Prunella vulgaris powder to 100-200mL of methanol aqueous solution; more preferably, it is calculated based on 2.5g of Prunella vulgaris powder to 100mL of methanol aqueous solution.

[0049] The ultrasonic extraction conditions described in step (1)1)A ​​are preferably 50-200W power and 40-60kHz frequency for 20-80 minutes; more preferably 80-150W power and 45-50kHz frequency for 30-60 minutes; and most preferably 150W power and 45kHz frequency for 45 minutes.

[0050] The filtration described in step (1)1)A ​​can be achieved through filter paper or filter membrane.

[0051] The filter membrane is preferably a 0.45 μm filter membrane.

[0052] The solvent removal method described in step (1)1)A ​​is preferably the solvent removal method by water bath heating and evaporation.

[0053] The amount of methanol used in step (1)1)B is preferably calculated as 2.0-4.0g of Prunella vulgaris powder mixed with 2-4mL of methanol; more preferably, it is calculated as 2.5g of Prunella vulgaris powder mixed with 2mL of methanol aqueous solution.

[0054] The filtration described in step (1)1)B is preferably performed using a 0.45 μm filter membrane.

[0055] The solvent removal method described in step (1)2)A is preferably the solvent removal method by water bath heating and evaporation.

[0056] The amount of methanol used in step (1)2)B is preferably calculated as 2-4 mL of methanol in the ratio of 2.0-4.0 g of Prunella vulgaris extract to 2 mL of methanol; more preferably, it is calculated as 2 mL of methanol aqueous solution in the ratio of 2.5 g of Prunella vulgaris extract to 2 mL of methanol aqueous solution.

[0057] The filtration described in step (1)2)B is preferably performed using a 0.45 μm filter membrane.

[0058] The concentration of caffeic acid in the reference solution described in step (2) is preferably 0.20 to 0.21 mg / mL; the concentration of rosmarinic acid is preferably 0.98 to 1.0 mg / mL.

[0059] The amount of the test solution used in step (3) is 4 to 10 μL; more preferably 6 μL.

[0060] The amount of the reference solution used in step (3) is 4 to 10 μL; more preferably 6 μL.

[0061] The developing agent mentioned in step (3) is preferably a toluene-ethyl acetate-isopropanol-formic acid solution; more preferably, it is a toluene-ethyl acetate-isopropanol-formic acid solution obtained by mixing at a volume ratio of 6.4-6.6:1.9-2.1:0.9-1.1:0.4-0.6; and most preferably, it is a toluene-ethyl acetate-isopropanol-formic acid solution obtained by mixing at a volume ratio of 6.5:2.0:1.0:0.5.

[0062] TLC results of Prunella vulgaris and its sucrose extract: Under UV light (254 nm), the test samples of Prunella vulgaris and its sucrose extract showed fluorescent quenched spots at the same positions as the reference standards caffeic acid and rosmarinic acid. Under UV light (365 nm), the test samples of Prunella vulgaris and its sucrose extract showed fluorescent spots of the same color at the same positions as the reference standards caffeic acid and rosmarinic acid. When comparing the TLC chromatograms of Prunella vulgaris and its sucrose extract, under the same sample amount and chromatographic conditions, there were significant differences between the chromatograms of the Prunella vulgaris sucrose extract and the Prunella vulgaris sample. The caffeic acid spots in the Prunella vulgaris sucrose extract were significantly larger than those in the Prunella vulgaris sample.

[0063] The specific polar solvent extraction method described above involves directly extracting caffeic acid and rosmarinic acid using a 45-55% (v / v) methanol aqueous solution, which can effectively extract caffeic acid and rosmarinic acid.

[0064] The methanol aqueous solution is preferably a methanol aqueous solution with a volume percentage of 50%.

[0065] The specific steps of the high-performance liquid chromatography detection method are as follows:

[0066] 1) Chromatographic conditions: The chromatographic conditions and system suitability test used octadecylsilane-bonded silica gel as the stationary phase, acetonitrile-methanol-0.2% (v / v) acetic acid aqueous solution as the mobile phase, and the detection wavelength was 330 nm; the theoretical plate number calculated based on the peaks of caffeic acid and rosmarinic acid should not be less than 10,000;

[0067] 2) Preparation of reference solution: Accurately weigh caffeic acid and rosmarinic acid reference standards, dissolve them in methanol aqueous solution and make up to volume to obtain a mixed reference solution of caffeic acid and rosmarinic acid;

[0068] 3) Preparation of the test solution:

[0069] ① Preparation of Prunella vulgaris test solution: Take Prunella vulgaris powder, weigh it accurately, place it in a stoppered conical flask, add methanol aqueous solution accurately, weigh it accurately, sonicate it, cool it, weigh it again, make up the lost weight with methanol aqueous solution, shake it well, filter it, and take the filtrate to obtain the Prunella vulgaris test solution.

[0070] ② Preparation of the test solution of Prunella vulgaris extract: Take the Prunella vulgaris extract, measure it accurately, first adjust the concentration of the obtained solution with methanol to be the same as that of the methanol aqueous solution; then add the methanol aqueous solution to the mark, shake well, filter, and take the filtrate to obtain the test solution of Prunella vulgaris extract.

[0071] 4) Determination: Accurately pipette the reference solution and the test solution separately, inject them into a liquid chromatograph, and determine the result.

[0072] The mobile phase described in step 1) is preferably obtained by mixing acetonitrile, methanol and 0.2% (v / v) aqueous acetic acid in a volume ratio of 14-16:11-13:72-74; more preferably, it is obtained by mixing acetonitrile, methanol and 0.2% (v / v) aqueous acetic acid in a volume ratio of 15:12:73.

[0073] The chromatographic column used in step 1) is preferably an Ultimate XB-C18 column.

[0074] The column temperature in the chromatography described in step 1) is 24-27°C; more preferably 25°C.

[0075] The flow rate of the mobile phase in step 1) is preferably 0.8-1.2 mL / min; more preferably 1 mL / min.

[0076] The methanol aqueous solution mentioned in step 2) is preferably a methanol aqueous solution with a concentration of 45-55% by volume; more preferably a methanol aqueous solution with a concentration of 50% by volume.

[0077] The reference solution described in step 2) contains 0.005-0.006 mg of caffeic acid and 0.05-0.06 mg of rosmarinic acid per 1 mL.

[0078] The Prunella vulgaris powder mentioned in step 3)① is preferably obtained by the following steps: crushing Prunella vulgaris and passing it through a sieve of at least 40 mesh to obtain Prunella vulgaris powder.

[0079] The methanol aqueous solution mentioned in step 3)① is preferably a methanol aqueous solution with a volume percentage of 45-55%; more preferably a methanol aqueous solution with a volume percentage of 50%.

[0080] The amount of methanol aqueous solution mentioned in step 3)① is preferably calculated as 100-300mL of methanol aqueous solution per 1.0-3.0g of Prunella vulgaris powder; more preferably, it is calculated as 100mL of methanol aqueous solution per 1.5g of Prunella vulgaris powder.

[0081] The preferred conditions for ultrasound treatment in step 3)① are 500-700W power and 30-50kHz frequency for 40-80 minutes; more preferably, 600W power and 40kHz frequency for 30-60 minutes.

[0082] The methanol aqueous solution mentioned in step 3) ② is preferably a methanol aqueous solution with a concentration of 45-55% by volume; more preferably a methanol aqueous solution with a concentration of 50% by volume.

[0083] The concentration of the test solution of the Prunella vulgaris extract described in step 3)② is preferably 1.4-1.6 g / 100 mL based on the amount of Prunella vulgaris herb; more preferably 1.5 g / 100 mL.

[0084] The volume of the reference solution and the volume of the test solution drawn in step 4) are preferably mixed in a volume ratio of 1:1.

[0085] The preferred volume of the reference solution is 10 μL.

[0086] The preferred volume of the test solution is 10 μL.

[0087] On a dried basis, Prunella vulgaris contains not less than 0.010% caffeic acid (C9H8O4) and not less than 0.010% rosmarinic acid (C... 18 H 16 08) Not less than 0.20%; the content of caffeic acid (C) in the Prunella vulgaris extract shall be calculated based on the corresponding dried weight of Prunella vulgaris. 24 H 26 O 13 The content shall not be less than 0.020%; it contains rosmarinic acid (C 18 H 16 08) Not less than 0.18%.

[0088] By comparing the HPLC chromatograms of Prunella vulgaris and its rosmarinic acid extract, based on the same amount of dried Prunella vulgaris, the absorption peak height and peak area of ​​caffeic acid in the HPLC chromatogram of the rosmarinic acid extract were significantly higher than those in the HPLC chromatogram of Prunella vulgaris. Furthermore, by using the HPLC method for simultaneous determination of caffeic acid and rosmarinic acid in this study, the content of caffeic acid, which has rosmarinic acid-derived activity, in the rosmarinic acid extract of Prunella vulgaris was significantly increased; based on the same amount of dried Prunella vulgaris, the caffeic acid content was more than doubled.

[0089] The method for simultaneous detection of caffeic acid and rosmarinic acid in Prunella vulgaris or its sucrose extract is applied in the development of Prunella vulgaris and its sucrose extract, as well as its cosmetics and related products.

[0090] Prunella vulgaris is a common medicinal herb listed in the Chinese Pharmacopoeia, with abundant wild and cultivated resources. However, due to the lack of development and utilization of its glycosaminoglycan properties, the quality control of Prunella vulgaris and its glycosaminoglycan extracts remains a blank. The present invention has the following beneficial effects:

[0091] 1. This invention is the first to realize a TLC detection method for simultaneous detection of caffeic acid and rosmarinic acid in Prunella vulgaris and its extract, providing a method and basis for the development and utilization of Prunella vulgaris and its extract;

[0092] 2. This invention establishes for the first time an HPLC method for the simultaneous determination of caffeic acid and rosmarinic acid in Prunella vulgaris and its extracts by constant current elution, providing a basis for the quality control of Prunella vulgaris and its extracts in cosmetics, health care and medicine.

[0093] 3. The detection method for simultaneous determination of caffeic acid and rosmarinic acid in Prunella vulgaris and its extracts provided by this invention has the characteristics of high accuracy and precision, good stability and repeatability. It overcomes the shortcomings of gradient elution methods, which are relatively cumbersome and have relatively poor reproducibility and stability.

[0094] 4. The detection method of the present invention found that the caffeic acid content, which has a sugar-reducing effect, was significantly increased in the preparation of Prunella vulgaris sugar extract, providing a method and basis for the development and utilization of Prunella vulgaris sugar extract in cosmetics, food, health care or medicine. Attached Figure Description

[0095] Figure 1 These are TLC images of the identification results of caffeic acid and rosmarinic acid in Prunella vulgaris under a UV lamp at 254 nm; among them, 1, 2, 3, 4, 6, and 7 are the test samples of Prunella vulgaris, and 5 is the reference standard of caffeic acid and rosmarinic acid.

[0096] Figure 2 These are TLC images of the caffeic acid and rosmarinic acid identification results in the extract of Prunella vulgaris syrup under ultraviolet light at 254 nm; among them, 1, 2, 3, 5, 6, and 7 are the test samples of Prunella vulgaris syrup extract, and 4 is the reference standard of caffeic acid and rosmarinic acid.

[0097] Figure 3 These are TLC images of the identification results of caffeic acid and rosmarinic acid in Prunella vulgaris under a 365nm UV lamp; among them, 1, 2, 3, 4, 6, and 7 are the test samples of Prunella vulgaris, and 5 is the reference standard of caffeic acid and rosmarinic acid.

[0098] Figure 4 These are TLC images of the caffeic acid and rosmarinic acid identification results in the extract of Prunella vulgaris syrup under ultraviolet light at 365 nm; among them, 1, 2, 3, 5, 6, and 7 are the test samples of Prunella vulgaris syrup extract, and 4 is the reference standard of caffeic acid and rosmarinic acid.

[0099] Figure 5 This is an HPLC chromatogram of caffeic acid and rosmarinic acid reference standards.

[0100] Figure 6 This is an HPLC chromatogram of caffeic acid and rosmarinic acid in Prunella vulgaris.

[0101] Figure 7 This is an HPLC chromatogram of caffeic acid and rosmarinic acid in the extract of Prunella vulgaris.

[0102] Figure 8 This is the HPLC chromatogram of the blank extraction solvent in Prunella vulgaris.

[0103] Figure 9 This is a linear fitting curve of a mixed standard solution of caffeic acid and rosmarinic acid. Detailed Implementation

[0104] 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.

[0105] Example 1: Simultaneous thin-layer chromatography (TLC) detection of caffeic acid and rosmarinic acid in Prunella vulgaris and its extract.

[0106] 1. Preparation of the test solution:

[0107] 1.1 Preparation of Prunella vulgaris test solutions: Take 6 portions of Prunella vulgaris sample, pulverize and break the cell wall of each sample, pass them through a 40-mesh sieve, accurately weigh 2.5g of Prunella vulgaris powder for each sample, add 100mL of aqueous solution containing 50% (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 Prunella vulgaris powder test solutions 1, 2, 3, 4, 6 and 7 respectively, for later use.

[0108] 1.2 Preparation of test solutions of Prunella vulgaris extract: Take 6 samples of Prunella vulgaris extract (samples prepared in Examples 4-6, with 2 samples prepared in each example), accurately pipette 5 mL of each sample (equivalent to 2.5 g of Prunella vulgaris herb), place them in evaporating dishes, heat in a water bath to evaporate to dryness, add 2 mL of methanol to dissolve the residue, and obtain test solutions 1, 2, 3, 5, 6, and 7 of Prunella vulgaris extract, respectively, for later use.

[0109] 2. Preparation of reference solutions: Weigh 0.41 mg of caffeic acid reference standard and 1.95 mg of rosmarinic acid reference standard into 2 mL volumetric flasks, add 2 mL of methanol to dissolve them, and prepare a solution containing approximately 0.21 mg of caffeic acid and 0.98 mg of rosmarinic acid per mL. This solution is used as the reference solution. The Prunella vulgaris test solution is numbered No. 5, and the Prunella vulgaris sucrose extract test solution is numbered No. 4 for reference.

[0110] 3. Thin-layer chromatography (TLC) detection:

[0111] According to the Thin Layer Chromatography (General Rule 0502) method in Part I of the 2015 edition of the Pharmacopoeia of the People's Republic of China, 6 μL each of the above-mentioned ① Prunella vulgaris powder test solution and reference solution were spotted separately onto the same high-performance silica gel GF plate. 254On a thin-layer plate, take 6 μL each of the above-mentioned ② Prunella vulgaris extract test solution and reference solution, and spot them separately on the same high-performance silica gel GF plate. 254 On thin-layer plates, toluene-ethyl acetate-isopropanol-formic acid (volume ratio = 6.5:2.0:1.0:0.5) was used as the developing solvent. The plates were then removed, dried, and examined under UV light at 254 nm and 365 nm, respectively.

[0112] Detection under 254 nm UV light: In the chromatogram of the Prunella vulgaris powder test sample, fluorescent quenched spots appear at the same positions as those in the chromatograms of the reference standards caffeic acid and rosmarinic acid (see...). Figure 1 In the chromatogram of the test sample of Prunella vulgaris extract, fluorescent quenched spots appeared at the same positions as those of the reference standards caffeic acid and rosmarinic acid (see...). Figure 2 ).

[0113] When examined under a 365nm UV lamp: the chromatogram of the *Prunella vulgaris* powder test sample shows fluorescent spots of the same color at the corresponding positions as those of the reference standards caffeic acid and rosmarinic acid (see...). Figure 3 In the chromatogram of the test sample of Prunella vulgaris extract, fluorescent spots of the same color appeared at the corresponding positions as those of the reference standards caffeic acid and rosmarinic acid (see...). Figure 4 ).

[0114] 4. Thin-layer chromatography (TLC) detection results and conclusions:

[0115] Under UV light at 254 nm, the chromatograms of *Prunella vulgaris* and its extract showed the same fluorescent quenched spots at the corresponding positions as the reference standards caffeic acid and rosmarinic acid. Under UV light at 365 nm, the same colored fluorescent spots were observed at the same positions as the reference standards caffeic acid and rosmarinic acid. The results demonstrate that all six *Prunella vulgaris* samples and six *Prunella vulgaris* extracts contained caffeic acid and rosmarinic acid. Furthermore, the chromatograms of caffeic acid and rosmarinic acid in the six *Prunella vulgaris* and its extracts were essentially identical.

[0116] 5. Discussion:

[0117] The chromatograms of the test samples of Prunella vulgaris and its caffeine extract also show that, under the same sample amount and chromatographic conditions, there are significant differences between the chromatograms of the Prunella vulgaris caffeine extract sample and the Prunella vulgaris sample. This indicates that some components change during the preparation of the Prunella vulgaris caffeine extract. In particular, the amount of caffeic acid in the Prunella vulgaris caffeine extract sample is significantly higher than that in the corresponding Prunella vulgaris sample, suggesting that the preparation method of the Prunella vulgaris caffeine extract is conducive to the production of caffeic acid, the effective component of caffeine.

[0118] Example 2: Detection method for simultaneous HPLC determination of caffeic acid and rosmarinic acid in Prunella vulgaris and its extract.

[0119] 1. Principles and Reagents

[0120] 1.1 Principle

[0121] Caffeic acid and rosmarinic acid contained in Prunella vulgaris and its sugar extract are polar water-soluble substances. Prunella vulgaris uses a polar solvent extraction method to extract the polar components caffeic acid and rosmarinic acid while separating them from the fat-soluble components. The sugar extract of Prunella vulgaris is directly dissolved and extracted with 50% methanol as a polar solvent, and the contents of caffeic acid and rosmarinic acid are determined by high performance liquid chromatography.

[0122] 1.2 Reagents

[0123] Acetic acid (analytical grade); methanol (analytical grade), methanol (chromatographic grade), acetonitrile (chromatographic grade); caffeic acid reference standard (batch number G22020065, content: 99.7%), TMRM Quality Testing Standard Material Center; rosmarinic acid reference standard (batch number 111871-202007, content: 98.1%), China National Institutes for Food and Drug Control; Prunella vulgaris sample (batch number 20220703), Anhui Bozhou Zhongyitang Traditional Chinese Medicine Sales Co., Ltd.

[0124] 1.3 Instruments

[0125] 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.

[0126] 2. Methods and Results

[0127] 2.1 Preparation of reference solution

[0128] Accurately weigh 0.534 mg of caffeic acid reference standard and 5.810 mg of rosmarinic acid reference standard, place them in a 100 mL volumetric flask, dissolve them in 50% (v / v) methanol and dilute to volume to obtain a mixed caffeic acid and rosmarinic acid reference standard solution.

[0129] 2.2 Preparation of test solution

[0130] 2.2.1 Preparation of Prunella vulgaris test solution: Take Prunella vulgaris, pulverize it through a 40-mesh sieve, weigh about 1.5g of Prunella vulgaris powder, accurately weigh it, place it in a stoppered conical flask, accurately add 100mL of aqueous solution containing 50% (v / v) methanol, accurately weigh it, sonicate it (power 600W, frequency 40kHz) for 60 minutes, cool it, weigh it again, replenish the lost weight with aqueous solution containing 50% (v / v) methanol, shake well, filter it, and take the filtrate to obtain the Prunella vulgaris test solution.

[0131] 2.2.2 Preparation of the test solution of Prunella vulgaris extract: Take 3 mL of Prunella vulgaris extract (equivalent to 1.5 g of Prunella vulgaris powder, prepared in Example 4), accurately measure it, place it in a 100 mL volumetric flask, add 3 mL of methanol first, then add an aqueous solution containing 50% (v / v) methanol to the mark, shake well, filter, and take the filtrate to obtain the test solution of Prunella vulgaris extract.

[0132] 2.3 Chromatographic conditions

[0133] The chromatographic column used was an Ultimate XB-C18 (4.6 mm × 250 mm, 5 μm), the column temperature was 25 ℃, the detection wavelength was 330 nm, the flow rate was 1 mL / min, and the injection volume was 10 μL for both the reference solution and the test solution. The mobile phase was acetonitrile-methanol-0.2% acetic acid aqueous solution (v / v 15:12:73). The chromatogram is shown below. Figure 5-8 .

[0134] From the chromatogram Figure 5-8 It was found that in the HPLC chromatograms of caffeic acid and rosmarinic acid reference standards, a caffeic acid absorption peak appeared at approximately 6.5 minutes of retention time, and a rosmarinic acid absorption peak appeared at approximately 20.0 minutes of retention time. In the HPLC chromatograms of Prunella vulgaris and its extract, a caffeic acid absorption peak appeared at approximately 6.5 minutes of retention time, and a rosmarinic acid absorption peak appeared at approximately 20.0 minutes of retention time. The peak shapes were consistent with those of the caffeic acid and rosmarinic acid reference standards, and the plate numbers were all above 12000, indicating good separation. However, no caffeic acid or rosmarinic acid absorption peaks were observed in the HPLC chromatogram of the blank extraction solvent. This suggests that the HPLC determination conditions for caffeic acid and rosmarinic acid in Prunella vulgaris and its extract are mature and usable. Figure 6 and Figure 7 It can also be seen that the peak height and peak area ratio of caffeic acid and rosmarinic acid in the Prunella vulgaris extract are significantly different from those in the whole Prunella vulgaris. The proportion of caffeic acid in the Prunella vulgaris extract is significantly higher, suggesting that the preparation method of the Prunella vulgaris extract is conducive to the production of caffeic acid, a component of the extract.

[0135] 2.4 Examination of Linear Relationships

[0136] Take the reference solution from section "2.1", filter it through a 0.45 μm microporous membrane, and inject 4, 8, 12, 16, and 20 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: Caffeic acid Y = 88.862X - 0.4185, R0 2 =0.9985; Rosmarinic acid Y = 29.522X - 0.1547, R 2 =0.9998.

[0137] Table 1. Experimental data on the linear relationship between caffeic acid and rosmarinic acid (n=5)

[0138]

[0139] Caffeic acid Y = 88.862X - 0.4185, R 2 =0.9985; Rosmarinic acid Y = 29.522X - 0.1547, R 2 =0.9998.

[0140] The results show that caffeic acid exhibits a good linear relationship with peak area in the range of 0.02136–0.1068 μg, and rosmarinic acid exhibits a good linear relationship with peak area in the range of 0.2324–1.1620 μg.

[0141] 2.5 Precision Test: Accurately pipette 10 μL of the mixed standard solution of caffeic acid and rosmarinic acid from section "2.1", inject continuously 6 times under the chromatographic conditions from section "2.3", and measure the peak area. The average peak area value of caffeic acid was calculated to be 4.338, with an RSD of 1.31%, and the average peak area value of rosmarinic acid was calculated to be 17.026, with an RSD of 1.10%. The RSD of the peak area values ​​of both is <2%, and the results are shown in Table 2. The results indicate that the instrument precision is good.

[0142] Table 2 Precision test data (n=6)

[0143]

[0144]

[0145] 2.6 Stability Test: Take the *Prunella vulgaris* test solution from section “2.2.2.1” and, under the chromatographic conditions from section “2.2.3”, inject 10 μl at 0 hours, 1 hour, 4 hours, 8 hours, 12 hours, and 24 hours respectively. Measure the peak area values ​​of caffeic acid and rosmarinic acid. The average peak area value of caffeic acid in the *Prunella vulgaris* test sample was calculated to be 2.219, with an RSD of 1.95%. The average peak area value of rosmarinic acid was calculated to be 21.023, with an RSD of 1.48%. The RSD of the peak areas of caffeic acid and rosmarinic acid in the sample was <2%. The results are shown in Table 3.

[0146] Table 3. Stability test data (n=6)

[0147]

[0148] The test results show that the sample solution has good stability within 24 hours.

[0149] 2.7 Reproducibility Test: Six samples of *Prunella vulgaris* from the same batch (batch number 20220703) were prepared according to the preparation method of the *Prunella vulgaris* test solution in section “2.2”. The samples were injected and analyzed under the chromatographic conditions in section “2.3”, and the contents of caffeic acid and rosmarinic acid were calculated. The average caffeic acid content in the *Prunella vulgaris* samples was 0.0197%, with an RSD of 1.26%. The average rosmarinic acid content was 0.4932%, with an RSD of 1.23%. The RSDs of both caffeic acid and rosmarinic acid in the samples were <2%. The results are shown in Table 4.

[0150] Table 4. Reproducibility test data (n=6)

[0151]

[0152] The experimental results show that the reproducibility of this determination method is good.

[0153] 2.8 Recovery Test: Six samples of Prunella vulgaris (batch number 20220703) with known content were accurately weighed, each 750 mg. An appropriate amount of reference solution equivalent to 100% of the caffeic acid and rosmarinic acid content in the sample was accurately added to each sample. The 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 "2.2.3". The contents and recoveries of caffeic acid and rosmarinic acid were calculated. The results showed that the average recovery rate of caffeic acid in the Prunella vulgaris samples was 99.89%, with an RSD of 1.80%, and the average recovery rate of rosmarinic acid was 100.27%, with an RSD of 1.14%. The results are shown in Table 5.

[0154] Table 5. Recovery rates of caffeic acid and rosmarinic acid (n=6)

[0155]

[0156] The experimental results showed that the recovery rates of caffeic acid and rosmarinic acid in the Prunella vulgaris samples were between 97.28% and 102.04%, respectively, and the recovery rates were between 98.84% and 101.71%, respectively. The samples showed good recovery upon spiking.

[0157] Example 3: Simultaneous determination of caffeic acid and rosmarinic acid content in three batches of Prunella vulgaris and its sucrose extract samples

[0158] 1. Content determination

[0159] Following the method for content determination in Example 2 above, the contents of caffeic acid and rosmarinic acid in three different batches of Prunella vulgaris samples and three different batches of Prunella vulgaris extract samples were determined, and the results are shown in Table 6.

[0160] Table 6. Results of caffeic acid and rosmarinic acid content determination in Prunella vulgaris and its extract samples (n=3)

[0161]

[0162] 2. Measurement Results

[0163] The caffeic acid content in the three batches of Prunella vulgaris samples ranged from 0.0197% to 0.0243%, and the rosmarinic acid content ranged from 0.4932% to 0.5318%. Based on the corresponding Prunella vulgaris samples, the caffeic acid content in the three batches of Prunella vulgaris extract samples ranged from 0.0497% to 0.0598%, and the rosmarinic acid content ranged from 0.3869% to 0.4314%.

[0164] 3. Discussion:

[0165] 3.1 The results of this study show that the determination of caffeic acid and rosmarinic acid in Prunella vulgaris and its extracts exhibits good linearity within the linear range, and the precision, stability, reproducibility, and recovery tests all meet the requirements. This study is the first to achieve simultaneous TLC detection of caffeic acid and rosmarinic acid in Prunella vulgaris and its extracts. It is also the first to achieve simultaneous HPLC detection of caffeic acid and rosmarinic acid in Prunella vulgaris and its extracts using constant current elution, overcoming the cumbersome HPLC method of gradient elution required for the simultaneous detection of caffeic acid and rosmarinic acid in Prunella vulgaris, thus improving the accuracy and reproducibility of the determination.

[0166] 3.2 Using the simultaneous TLC and HPLC detection methods for caffeic acid and rosmarinic acid in this study, it was discovered for the first time that the caffeic acid content, which has a saccharifying effect, was significantly increased in the process of preparing the saccharifying extract of Prunella vulgaris, with the corresponding Prunella vulgaris content increasing by more than 100%. This study also provides methods and basis for the quality control of Prunella vulgaris and its saccharifying extract.

[0167] Example 4: Preparation of Prunella vulgaris extract and crude polysaccharide from Prunella vulgaris

[0168] (1) Extraction of phenylpropanoid components and crude polysaccharides

[0169] Take 2 kg of cleaned and dried Prunella vulgaris herb, add 40000 mL of purified water, stir and mix well, soak for 1.5 hours, heat and decoct under reflux for 1.5 hours (starting the timer from boiling), filter while hot (350 mesh), and obtain 28980 mL of filtrate; add 36000 mL of purified water to the residue, decoct and reflux for 1.0 hour (starting the timer from boiling), filter while hot (350 mesh), and obtain 32620 mL of filtrate; add 36000 mL of purified water to the residue for the third time, decoct and reflux for 1.0 hour (starting the timer from boiling), filter while hot (350 mesh), and obtain 32570 mL of filtrate; combine the three filtrates to obtain approximately 93750 mL of extract of Prunella vulgaris containing phenylpropanoid components and crude polysaccharides, and set aside; discard the residue.

[0170] (2) Concentration of extract containing phenylpropanoids and crude polysaccharides

[0171] Take about 93750 mL of the extract of Prunella vulgaris containing phenylpropanoids and crude polysaccharides, concentrate it under reduced pressure to about 6000 mL (each 3 mL solution is equivalent to 1 g of Prunella vulgaris herb), stop the concentration when the relative density is 1.65 (measured at 73℃), cool it to obtain a concentrated solution, the dry solids content of the concentrated solution is 0.0599 g / mL; the total dry solids amount is 359.4 g; set aside for later use.

[0172] Calculation of total dry solids in concentrate:

[0173] Total dry solids in the concentrate (g) = Concentrate volume (mL) × Dry solids content (g / mL).

[0174] Calculation of total dry solids content in concentrate:

[0175]

[0176] (3) Preparation of Prunella vulgaris extract and crude polysaccharide from Prunella vulgaris

[0177] Take about 6000 mL of the above concentrated solution and place it in an alcohol precipitation apparatus. Slowly add 13000 mL of 95% (v / v) ethanol while stirring, so that the alcohol content in the concentrated solution reaches 65% (v / v). Stir well, refrigerate and stand for 48 hours, and filter under reduced pressure with 600 mesh filter paper to obtain about 563.7 g of Prunella vulgaris crude polysaccharide filtrate and about 16080 mL of Prunella vulgaris polysaccharide extract ethanol filtrate. Set aside for later use.

[0178] (4) Isolation and preparation of crude polysaccharides from Prunella vulgaris:

[0179] Take about 563.7g of the above-mentioned Prunella vulgaris crude polysaccharide filtrate, dry it under reduced pressure at 60℃ until the moisture content is <9%, grind it into powder, and obtain 159.97g of Prunella vulgaris crude polysaccharide extract; the yield is 8.00%.

[0180]

[0181] (5) Preparation of Prunella vulgaris extract:

[0182] Take approximately 16080 mL of the ethanol filtrate of the Prunella vulgaris extract, recover the ethanol under reduced pressure at 70℃, and concentrate the filtrate under reduced pressure to approximately 4000 mL (each 2 mL solution is equivalent to 1 g of Prunella vulgaris herb). The relative density is 1.15 (measured at 60℃). Stop concentration, cool, and obtain 4000 mL of Prunella vulgaris extract for later use. The dry solids content of the extract after cooling is 0.0445 g / mL; the total dry solids content, i.e., the dry solids in the Prunella vulgaris extract as a percentage of the Prunella vulgaris herb, is 8.90% (g / g).

[0183] Example 5: Preparation of Prunella vulgaris extract and crude polysaccharide from Prunella vulgaris (Part 2)

[0184] (1) Extraction of phenylpropanoid components and crude polysaccharides

[0185] Take 2 kg of cleaned and dried Prunella vulgaris herb, add 40000 mL of purified water, stir and mix well, soak for 1.5 hours, heat and decoct under reflux for 1.5 hours (starting the timer from boiling), filter while hot (350 mesh), and obtain 28850 mL of filtrate; add 36000 mL of purified water to the residue, decoct and reflux for 1.0 hour (starting the timer from boiling), filter while hot (350 mesh), and obtain 32550 mL of filtrate; add 36000 mL of purified water to the residue for the third time, decoct and reflux for 1.0 hour (starting the timer from boiling), filter while hot (350 mesh), and obtain 32280 mL of filtrate; combine the three filtrates to obtain approximately 93360 mL of extract of Prunella vulgaris containing phenylpropanoid components and crude polysaccharides, and set aside; discard the residue.

[0186] (2) Concentration of extract containing phenylpropanoids and crude polysaccharides

[0187] Take about 93360 mL of the extract of Prunella vulgaris containing phenylpropanoids and crude polysaccharides, concentrate it under reduced pressure to about 6000 mL (each 3 mL solution is equivalent to 1 g of Prunella vulgaris herb), stop the concentration when the relative density is 1.61 (measured at 75℃), cool it to obtain a concentrated solution, the dry solids content of the concentrated solution is 0.0598 g / mL; the total dry solids amount is 358.6 g; set aside for later use.

[0188] (3) Preparation of Prunella vulgaris extract and crude polysaccharide from Prunella vulgaris

[0189] Take about 6000 mL of the above concentrated solution and place it in an alcohol precipitation apparatus. Slowly add 16800 mL of 95% (v / v) ethanol while stirring, so that the alcohol content in the concentrated solution reaches 70% (v / v). Stir well, refrigerate and let stand for 48 hours, and filter under reduced pressure with 600 mesh filter paper to obtain about 582.2 g of Prunella vulgaris crude polysaccharide filtrate and about 19850 mL of Prunella vulgaris polysaccharide extract ethanol filtrate. Set aside for later use.

[0190] (4) Isolation and preparation of crude polysaccharides from Prunella vulgaris:

[0191] Take about 582.2g of the above-mentioned Prunella vulgaris crude polysaccharide filtrate, dry it under reduced pressure at 60℃ until the moisture content is <9%, grind it into powder, and obtain 162.98g of Prunella vulgaris crude polysaccharide extract; the yield is 8.15%.

[0192] (5) Preparation of Prunella vulgaris extract:

[0193] Take approximately 19850 mL of the ethanol filtrate of the Prunella vulgaris extract, recover the ethanol under reduced pressure at 70℃, and concentrate the filtrate under reduced pressure to approximately 4000 mL (each 2 mL solution is equivalent to 1 g of Prunella vulgaris herb). The relative density is 1.14 (measured at 60℃). Stop concentration, cool, and obtain 4000 mL of Prunella vulgaris extract for later use. The dry solids content of the extract after cooling is 0.0441 g / mL; the total dry solids content, i.e., the dry solids content in the Prunella vulgaris extract as a percentage of the Prunella vulgaris herb, is 8.81% (g / g).

[0194] Example 6: Preparation of Prunella vulgaris extract and crude polysaccharide from Prunella vulgaris (Part 3)

[0195] (1) Extraction of phenylpropanoid components and crude polysaccharides

[0196] Take 2 kg of cleaned and dried Prunella vulgaris herb, add 40000 mL of purified water, stir and mix well, soak for 1.5 hours, heat and decoct under reflux for 1.5 hours (starting from boiling), filter while hot (350 mesh), and obtain 28920 mL of filtrate; add 36000 mL of purified water to the residue, decoct under reflux for 1.0 hour (starting from boiling), filter while hot (350 mesh), and obtain 32620 mL of filtrate; add 3 kg of purified water to the residue for the third time. 6000 mL of water was decocted and refluxed for 1.0 hour (starting from boiling). The mixture was then filtered while hot (350 mesh) to obtain 32510 mL of filtrate. The residue was then added to 36000 mL of purified water for the fourth time and decocted and refluxed for 1.0 hour (starting from boiling). The residue was then filtered while hot (350 mesh) to obtain 32630 mL of filtrate. The four filtrates were combined to obtain approximately 126580 mL of extract of Prunella vulgaris containing phenylpropanoids and crude polysaccharides, which was set aside. The residue was discarded.

[0197] (2) Concentration of extract containing phenylpropanoids and crude polysaccharides

[0198] Take 126580 mL of the extract of Prunella vulgaris containing phenylpropanoids and crude polysaccharides, concentrate it under reduced pressure to about 6000 mL (each 3 mL solution is equivalent to 1 g of Prunella vulgaris herb), stop the concentration when the relative density is 1.70 (measured at 75℃), cool it to obtain a concentrated solution, the dry solids content of the concentrated solution is 0.0601 g / mL; the total dry solids amount is 360.6 g; set aside for later use.

[0199] (3) Preparation of Prunella vulgaris extract and crude polysaccharide from Prunella vulgaris

[0200] Take about 6000 mL of the above concentrated solution and place it in an alcohol precipitation apparatus. Slowly add 22500 mL of 95% (v / v) ethanol while stirring, so that the alcohol content in the concentrated solution reaches 75% (v / v). Stir well, refrigerate and stand for 48 hours, and filter under reduced pressure with 600 mesh filter paper to obtain about 678.6 g of Prunella vulgaris crude polysaccharide filtrate and about 27960 mL of Prunella vulgaris polysaccharide extract ethanol filtrate. Set aside for later use.

[0201] (4) Isolation and preparation of crude polysaccharides from Prunella vulgaris:

[0202] Approximately 678.6 g of the above-mentioned Prunella vulgaris crude polysaccharide filtrate was dried under reduced pressure at 60°C until the moisture content was <9%, and then ground into powder to obtain 176.36 g of Prunella vulgaris crude polysaccharide extract; the yield was 8.82%.

[0203] (5) Preparation of Prunella vulgaris extract:

[0204] Take approximately 27960 mL of the ethanol filtrate of the Prunella vulgaris extract, recover the ethanol under reduced pressure at 70℃, and concentrate the filtrate under reduced pressure to approximately 4000 mL (each 2 mL solution is equivalent to 1 g of Prunella vulgaris herb). The relative density is 1.16 (measured at 60℃). Stop concentration, cool, and obtain 4000 mL of Prunella vulgaris extract for later use. The dry solids content of the extract after cooling is 0.0440 g / mL; the total dry solids content, i.e., the dry solids content in the Prunella vulgaris extract as a percentage of the Prunella vulgaris herb, is 8.80% (g / g).

[0205] 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 simultaneously detecting caffeic acid and rosmarinic acid in Prunella vulgaris or its sucrose extract, characterized in that... The steps include: using thin-layer chromatography to qualitatively identify caffeic acid and rosmarinic acid in Prunella vulgaris or its extract; directly extracting caffeic acid and rosmarinic acid using a specific polar solvent extraction method; using a mixed standard of caffeic acid and rosmarinic acid as a reference; and using high-performance liquid chromatography to determine the content of caffeic acid and rosmarinic acid in Prunella vulgaris or its extract. The specific steps of the thin-layer chromatography method are as follows: (1) Preparation of the test solution: 1) Preparation of the Prunella vulgaris test solution: A. Add the powdered Prunella vulgaris to a methanol-water solution for ultrasonic extraction, filter, remove the solvent from the filtrate, and obtain the residue; B. The residue was dissolved in methanol and filtered to obtain the Prunella vulgaris test solution; 2) Preparation of the test solution of Prunella vulgaris extract: A. Take the extract of Prunella vulgaris and remove the solvent to obtain the residue; B. Dissolve the residue in methanol, filter, and obtain the test solution of Prunella vulgaris extract; (2) Preparation of reference solution: Caffeic acid and rosmarinic acid reference standards were dissolved in methanol to obtain a mixed solution of caffeic acid and rosmarinic acid, which was used as 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, and you can identify whether Prunella vulgaris or its sugar extract contains caffeic acid and rosmarinic acid. In step (1) 1) A, the methanol content in the methanol-water solution is 40-60% by volume. The amount of methanol aqueous solution mentioned in step (1) 1) A is calculated based on 2.0-4.0g of Prunella vulgaris powder mixed with 100-200mL of methanol aqueous solution; The conditions for ultrasonic extraction described in step (1) 1)A are: extraction at a power of 50-200W and a frequency of 40-60kHz for 20-80 minutes; The amount of methanol mentioned in step (1) 1) B is calculated based on 2.0-4.0g of Prunella vulgaris powder mixed with 2-4mL of methanol; The amount of methanol mentioned in steps (1) and (2)B is calculated based on 2-4 mL of methanol, which is equivalent to 2.0-4.0 g of Prunella vulgaris extract. The developing agent mentioned in step (3) is a toluene-ethyl acetate-isopropanol-formic acid solution obtained by mixing in a volume ratio of 6.4-6.6:1.9-2.1:0.9-1.1:0.4-0.6; The specific steps of the high-performance liquid chromatography 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-0.2% (v / v) acetic acid aqueous solution as the mobile phase, and the detection wavelength was 330 nm; the theoretical plate number calculated based on the peaks of caffeic acid and rosmarinic acid should not be less than 10,000; 2) Preparation of reference solution: Accurately weigh caffeic acid and rosmarinic acid reference standards, dissolve them in methanol aqueous solution and make up to volume to obtain a mixed reference solution of caffeic acid and rosmarinic acid; 3) Preparation of the test solution: ① Preparation of Prunella vulgaris test solution: Take Prunella vulgaris powder, weigh it accurately, place it in a stoppered conical flask, add methanol aqueous solution accurately, weigh it accurately, sonicate it, cool it, weigh it again, make up the lost weight with methanol aqueous solution, shake it well, filter it, and take the filtrate to obtain the Prunella vulgaris test solution. ② Preparation of the test solution of Prunella vulgaris extract: Take the Prunella vulgaris extract, measure it accurately, first adjust the concentration of the obtained solution with methanol to be the same as that of the methanol aqueous solution; then add the methanol aqueous solution to the mark, shake well, filter, and take the filtrate to obtain the test solution of Prunella vulgaris extract. 4) Determination: Accurately pipette the reference solution and the test solution separately, inject them into the liquid chromatograph, and determine the result; The mobile phase mentioned in step 1) is prepared by mixing acetonitrile, methanol and 0.2% (v / v) aqueous acetic acid in a volume ratio of 14-16:11-13:72-74. The chromatographic column used in step 1) is an Ultimate XB-C18 column; 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 45-55% by volume; The reference solution described in step 2) contains 0.005-0.006 mg of caffeic acid and 0.05-0.06 mg of rosmarinic acid per mL; Step 3)① The Prunella vulgaris powder mentioned above is obtained by the following steps: Prunella vulgaris is crushed and passed through a sieve of at least 40 mesh to obtain Prunella vulgaris powder; The methanol-water solution mentioned in step 3) ① is a methanol-water solution with a concentration of 45-55% by volume; The amount of methanol-water solution mentioned in step 3) ① is calculated based on 100-300 mL of methanol-water solution per 1.0-3.0 g of Prunella vulgaris powder; The conditions for ultrasound treatment mentioned in step 3) ① are: power 500-700W, frequency 30-50kHz, and treatment time 40-80 minutes. The methanol-water solution mentioned in step 3) ② is a methanol-water solution with a concentration of 45-55% by volume. The concentration of the test solution of the Prunella vulgaris extract described in step 3) ② is 1.4-1.6 g / 100 mL based on the amount of Prunella vulgaris herb. The volume of the reference solution and the volume of the test solution in step 4) are mixed at a volume ratio of 1:

1.

2. The method for simultaneously detecting caffeic acid and rosmarinic acid in Prunella vulgaris or its sucrose extract according to claim 1, characterized in that: The sucrose extract was prepared by the following steps: (I) Extraction of phenylpropanoid components and crude polysaccharides: Using Prunella vulgaris as raw material, water extraction was performed to obtain an extract of Prunella vulgaris containing phenylpropanoid components and crude polysaccharides. (II) Concentration of extract containing phenylpropanoids and crude polysaccharides: The extract obtained in step (I) is concentrated to obtain a concentrated solution; (III) Separation and preparation of Prunella vulgaris extract and crude polysaccharide from Prunella vulgaris: The concentrate and ethanol were mixed evenly to obtain solution A, which contained 65-75% alcohol. Solution A was refrigerated and allowed to stand, and then filtered or vacuum filtered to obtain crude polysaccharide filtrate from Prunella vulgaris and ethanol filtrate of Prunella vulgaris extract. (IV) Preparation of Prunella vulgaris extract: Take the ethanol filtrate of Prunella vulgaris extract, concentrate it, and obtain Prunella vulgaris extract.

3. The method for simultaneously detecting caffeic acid and rosmarinic acid in Prunella vulgaris or its sucrose extract according to claim 2, characterized in that: The herbal medicine described in step (I) is the herbal medicine obtained by cleaning and drying; The water extraction steps described in step (I) are as follows: after soaking the Prunella vulgaris herb in water, decoct and reflux, and take the decoction; The concentration method described in step (II) is vacuum concentration; The concentration described in step (II) is to stop when the relative density is 1.39-1.88 when measured at 70-80℃ or when each 3 mL of solution is equivalent to 0.9-1.1 g of Prunella vulgaris herb. The refrigeration temperature described in step (III) is 2–8°C; The refrigeration time mentioned in step (III) is 36 to 60 hours; The filter used for vacuum filtration or filtration in step (III) is a filter with a specification of 350-800 mesh; The concentration method described in step (IV) is vacuum concentration; The concentration described in step (IV) is stopped when the relative density is measured to be 1.10-1.20 at 60-70℃ or when the volume of the concentrated solution is equivalent to 1g of Prunella vulgaris.

4. The method for simultaneously detecting caffeic acid and rosmarinic acid in Prunella vulgaris or its sucrose extract according to claim 1, characterized in that: The Prunella vulgaris powder mentioned in step (1) 1) A is obtained by the following steps: Prunella vulgaris is crushed and passed through a sieve of at least 40 mesh to obtain Prunella vulgaris powder; The solvent removal mentioned in step (1) 1) A is achieved by removing the solvent through water bath heating and evaporation; The solvent removal mentioned in steps (1) and (2)A is achieved by removing the solvent through water bath heating and evaporation. The concentration of caffeic acid in the reference solution described in step (2) is 0.20–0.21 mg / mL; the concentration of rosmarinic acid is 0.98–1.0 mg / mL. The volume of the test solution used in step (3) is 4–10 μL; The amount of the reference solution used in step (3) is 4 to 10 μL.

5. The method for simultaneously detecting caffeic acid and rosmarinic acid in Prunella vulgaris or its sucrose extract according to claim 1, characterized in that: The specific polar solvent extraction method described above involves directly extracting caffeic acid and rosmarinic acid using a methanol aqueous solution with a volume percentage of 45-55%.

6. The method for simultaneous detection of caffeic acid and rosmarinic acid in Prunella vulgaris or its extract according to any one of claims 1 to 5, in the development of Prunella vulgaris and its extract, cosmetics and products thereof.

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