Detection method of isorotundic acid glycoside in prunella vulgaris and application thereof

Abstract

The application discloses a detection method of isorotundic acid glycoside in prunella vulgaris L seeds and application thereof. The detection method comprises the following steps: performing qualitative identification on the isorotundic acid glycoside in the prunella vulgaris L seeds by using a thin layer chromatography method; directly extracting the isorotundic acid glycoside by using a specific polar solvent extraction method and separating the isorotundic acid glycoside from fat-soluble ingredients; and performing content determination on the isorotundic acid glycoside in the prunella vulgaris L seeds by using a high performance liquid chromatography method and taking the isorotundic acid glycoside reference substance as a reference. The detection method has the advantages of high accuracy and precision, good stability and repeatability, provides a method for detecting the quality of the isorotundic acid glycoside component in the development and utilization of the prunella vulgaris L seeds, and has strong specificity and good reproducibility.

Description

TECHNICAL FIELD

[0001] The application relates to a quality analysis method of a medicine, health product, food or cosmetic, in particular to a detection method of isorosmarinic acid glycoside in Prunella vulgaris seeds and application thereof. BACKGROUND

[0002] Prunella vulgaris L. of Lamiaceae, is recorded in Shennong Bencao Jing, is a perennial herb with medicinal and edible properties, and has a medicinal history of several thousand years. Prunella vulgaris L. is recorded in the 2015 edition of Chinese Pharmacopoeia. Prunella vulgaris L. tastes bitter and pungent, is cold in nature, and is attributed to the liver and gallbladder channels. Prunella vulgaris L. can clear the liver and remove fire, improve eyesight, resolve masses and eliminate swelling. Prunella vulgaris L. has good clinical therapeutic effects on various diseases such as red and painful eyes, photophobia and lacrimation, night pain of eyeball, dizziness and dim vision, scrofula, goiter, breast cancer, hypertension, lymph node tuberculosis, infiltrative pulmonary tuberculosis, simple goiter, parotitis and acute jaundice type infectious hepatitis. Modern research shows that Prunella vulgaris L. contains various chemical components, including triterpenes, sterols, flavonoids, organic acids, coumarins and other types of compounds. Prunella vulgaris L. has pharmacological effects such as anti-tumor, anti-inflammatory, antibacterial, anti-viral, immune regulation, blood pressure reduction, blood sugar reduction and blood lipid reduction. Prunella vulgaris L. is the dry mature seed of Prunella vulgaris L. and is the reproductive seed of Prunella vulgaris L. Prunella vulgaris L. seed should have the genetic active substances and effects of Prunella vulgaris L. However, the application value of Prunella vulgaris L. seed in food, health care or medicine has not been valued, and there is no related report on isorosmarinic acid glycoside, especially the quality control method of isorosmarinic acid glycoside in Prunella vulgaris L. seed.

[0003] Isorosmarinic acid glycoside (the structural formula is shown in the following formula) is also called salviaflaside, and the English name is Rosmarinic acid-3-O-glucoside. The molecular formula is C 24 H 26 O 13Molecular weight: 522.4; white crystalline powder, soluble in methanol, ethanol, DMSO and other organic solvents, pharmacological studies show that isorosmarinic acid glycoside has antibacterial, antiviral, antioxidant, anti-inflammatory activity, immunosuppressive activity, anti-thrombus effect. Isorosmarinic acid glycoside is a glycoside formed by rosmarinic acid and a molecule of glucose, which should have the same or similar pharmacological effects as rosmarinic acid. The reports of pharmacological studies of isorosmarinic acid glycoside are relatively few, while the reports of pharmacological studies of rosmarinic acid are relatively more. Modern pharmacological studies show that rosmarinic acid has antioxidant, antibacterial, anti-inflammatory, antitumor, antidepressant and anxiolytic, kidney disease improvement, liver protection and other pharmacological activities. Its effects and mechanisms are mainly as follows: (1) antioxidant effect: rosmarinic acid has strong antioxidant and free radical scavenging effects, which are related to its ortho-diphenol hydroxyl structure; (2) antibacterial and anti-inflammatory effect: rosmarinic acid has obvious inhibitory effect on Staphylococcus aureus, Micrococcus luteus, Escherichia coli, Bacillus subtilis and other bacteria, and has certain inhibitory effect on psoriasis, dermatitis, respiratory tract inflammation, acute lung injury and other inflammations; (3) antitumor effect: rosmarinic acid has obvious inhibitory effect on colorectal cancer, breast cancer, cervical cancer, leukemia, lung cancer, liver cancer and other tumors, and its antitumor mechanism may be related to improving the immune function of the body and effectively inhibiting the proliferation and invasion of tumor cells and inducing apoptosis; (4) antidepressant and anxiolytic effect: the antidepressant effect of rosmarinic acid, in vitro experiments found that rosmarinic acid can promote the proliferation of neonatal rat astrocytes, in vivo experiments found that rosmarinic acid can improve the depression-like behavior of chronic unpredictable stress depression model rats, and low-dose rosmarinic acid can play an anxiolytic effect; (5) liver protection effect: rosmarinic acid can inhibit the proliferation and differentiation of hepatic stellate cells HSCs, and resist carbon tetrachloride-induced anti-fibrosis by inhibiting the expression of TGF-β1 and CTGF; (6) kidney disease improvement effect: rosmarinic acid has the effect of improving kidney-related diseases, mainly manifested as reducing uric acid production, inhibiting glomerulonephritis and delaying chronic renal insufficiency.

[0004]

[0005] The present application first discovers that the summer grass seed is rich in isorosmarinic acid glycoside component, and first establishes a detection method for isorosmarinic acid glycoside in summer grass seed, which comprises qualitative identification of isorosmarinic acid glycoside in summer grass seed by thin layer chromatography; separation of fat-soluble components and isorosmarinic acid glycoside polar components by polar solvent extraction method, and then comparison with isorosmarinic acid glycoside reference substance, and determination of the content of isorosmarinic acid glycoside in summer grass seed by high performance liquid chromatography. The present application provides a method and basis for the development and utilization of summer grass seed and its products. SUMMARY

[0006] The primary purpose of the present application is to provide a quality control method for Prunella vulgaris L seeds, and to provide a detection method for isorotundic acid glycoside in Prunella vulgaris L seeds.

[0007] Another purpose of the present application is to provide an application of the detection method for isorotundic acid glycoside in Prunella vulgaris L seeds.

[0008] The purpose of the present application is achieved by the following technical scheme: a detection method for isorotundic acid glycoside in Prunella vulgaris L seeds, comprising the following steps: using thin layer chromatography to qualitatively identify isorotundic acid glycoside in Prunella vulgaris L seeds; using a specific polar solvent extraction method to directly extract isorotundic acid glycoside and separate it from lipid-soluble components, and then using isorotundic acid glycoside reference substance as a control, using high performance liquid chromatography to determine the content of isorotundic acid glycoside in Prunella vulgaris L seeds.

[0009] The specific steps of the thin layer chromatography detection method are as follows:

[0010] (1) Preparation of test solution:

[0011] Preparation of test solution of Prunella vulgaris L seeds:

[0012] A. Prunella vulgaris L seed powder is added to a methanol aqueous solution and ultrasonically extracted, filtered, and the obtained filtrate is subjected to solvent removal to obtain a residue;

[0013] B. The residue is dissolved in methanol and filtered to obtain a test solution of Prunella vulgaris L seed powder;

[0014] (2) Preparation of reference solution: isorotundic acid glycoside reference substance is dissolved in methanol as a reference solution;

[0015] (3) Thin layer chromatography (TLC) detection: the test solution and the reference solution are taken and spotted on the same high-performance silica gel GF254 254 thin layer plate, and developed with a developing agent, taken out and dried;

[0016] (4) Observation of silica gel GF254 254 thin layer plate: under ultraviolet light 254 nm and ultraviolet light 365 nm, respectively, identify whether the test chromatogram contains the same color spots at the positions corresponding to the reference chromatogram, so as to identify whether Prunella vulgaris L seeds contain isorotundic acid glycoside components.

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

[0018] The sieve is preferably a sieve with a mesh size of 40-50.

[0019] The content of methanol in the methanol aqueous solution in step (1) A is preferably 40-60% by volume; more preferably 50% by volume.

[0020] The amount of the methanol aqueous solution in step (1) A is preferably 50-250 mL of the methanol aqueous solution per 1.0-5.0 g of the Prunella vulgaris L. seed powder; more preferably 100 mL of the methanol aqueous solution per 2.0 g of the Prunella vulgaris L. seed powder.

[0021] The ultrasonic extraction in step (1) A is preferably performed at a power of 50-200 W and a frequency of 40-60 kHz for 20-80 minutes; more preferably at a power of 80-150 W and a frequency of 45-50 kHz for 30-60 minutes; most preferably at a power of 150 W and a frequency of 45 kHz for 45 minutes.

[0022] The filtration in step (1) A can be performed by using filter paper or filter membrane.

[0023] The filter membrane is preferably a 0.45 μm filter membrane.

[0024] The removal of the solvent in step (1) A is preferably performed by water bath heating and evaporation.

[0025] The amount of methanol in step (1) B is preferably 1-5 mL of methanol per 1.0-5.0 g of the Prunella vulgaris L. seed powder; more preferably 2 mL of methanol per 2.0 g of the Prunella vulgaris L. seed powder.

[0026] The filtration in step (1) B is preferably performed by using a 0.45 μm filter membrane.

[0027] The concentration of isorosmarinic acid in the control solution in step (2) is preferably 0.9-1.1 mg / mL; more preferably 0.95-1 mg / mL.

[0028] The amount of the test sample solution in step (3) is 2-10 μL; more preferably 4 μL.

[0029] The amount of the control solution in step (3) is 2-10 μL; more preferably 4 μL.

[0030] The developing agent in step (3) is preferably toluene-ethyl acetate-isopropyl alcohol-formic acid solution; more preferably toluene-ethyl acetate-isopropyl alcohol-formic acid solution obtained by mixing 4.4-4.6:2.4-2.6:2.4-2.6:0.4-0.6 by volume; most preferably toluene-ethyl acetate-isopropyl alcohol-formic acid solution obtained by mixing 4.5:2.5:2.5:0.5 by volume.

[0031] The specific polar solvent extraction method is direct extraction of isorosmarinic acid glycoside with 45-55% methanol aqueous solution, which can effectively extract isorosmarinic acid glycoside and separate it from fat-soluble ingredients.

[0032] The methanol aqueous solution is preferably 50% methanol aqueous solution.

[0033] The specific steps of the high performance liquid chromatography detection method are as follows:

[0034] 1) Chromatographic conditions: the chromatographic conditions and system suitability test use octadecylsilane-bonded silica gel as the filler, acetonitrile-methanol-0.2% (v / v) acetic acid aqueous solution as the mobile phase, and the detection wavelength is 319 nm; the theoretical plate number calculated according to the isorosmarinic acid glycoside peak should not be less than 8000;

[0035] 2) Preparation of reference solution: accurately weigh isorosmarinic acid glycoside reference substance, dissolve and dilute with methanol aqueous solution to obtain isorosmarinic acid glycoside reference solution;

[0036] 3) Preparation of test solution: take Prunella vulgaris seed powder, accurately weigh, put into a conical flask with a plug, accurately add methanol aqueous solution, accurately weigh, ultrasonic treatment, cool, re-weigh, make up the weight loss with methanol aqueous solution, shake well, filter, and take the filtrate, which is obtained;

[0037] 4) Determination: accurately pipette the reference solution and the test solution, inject into the liquid chromatography, and determine, which is obtained.

[0038] The mobile phase in step 1) is preferably acetonitrile, methanol and 0.2% (v / v) acetic acid aqueous solution with a volume ratio of 14-16:11-13:72-74; more preferably, acetonitrile, methanol and 0.2% (v / v) acetic acid aqueous solution with a volume ratio of 15:12:73.

[0039] The chromatographic column in the chromatography in step 1) is preferably an Ultimate XB-C18 chromatographic column.

[0040] The column temperature in the chromatography in step 1) is 24-27°C; more preferably, 25°C.

[0041] The flow rate of the mobile phase in step 1) is preferably 0.8-1.2 mL / min; more preferably, 1 mL / min.

[0042] The methanol aqueous solution in step 2) is preferably 45-55% methanol aqueous solution; more preferably, 50% methanol aqueous solution.

[0043] The concentration of the isorotundic acid glycoside control solution in step 2) is preferably 0.05-0.06 mg per 1 mL.

[0044] The Spodiopiperis Radix powder in step 3) is preferably obtained by crushing the Spodiopiperis Radix seeds to break the shells, and passing through a sieve of at least 40 meshes to obtain the Spodiopiperis Radix powder.

[0045] The methanol aqueous solution in step 3) 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.

[0046] The amount of the methanol aqueous solution in step 3) is preferably 50-150 mL per 0.5-1.5 g of the Spodiopiperis Radix seed powder; more preferably 100 mL per 1.0 g of the Spodiopiperis Radix seed powder.

[0047] The ultrasonic condition in step 3) is preferably 500-700 W of power, 30-50 kHz of frequency, for 40-80 minutes; more preferably 600 W of power, 40 kHz of frequency, for 30-60 minutes.

[0048] The volume of the control solution in step 4) and the volume of the test solution are preferably in a ratio of 1:1.

[0049] The volume of the control solution is preferably 10 μL.

[0050] The volume of the test solution is preferably 10 μL.

[0051] The Spodiopiperis Radix seed contains isorotundic acid glycoside (C 24 H 26 O 13 ) of not less than 0.35% by dry weight.

[0052] The method for detecting isorotundic acid glycoside in the Spodiopiperis Radix seed is applied to the development of the Spodiopiperis Radix seed and products thereof.

[0053] Spodiopiperis Radix is a common medicinal and edible flavor recorded in Chinese Pharmacopoeia, and its wild and planted resources are very rich. However, since the Spodiopiperis Radix seeds are not developed and utilized, thousands of tons of Spodiopiperis Radix seeds are not collected every year, and are scattered in the wild, causing resource waste. The present application has the following advantages and effects compared with the prior art:

[0054] 1. The present application provides a method for detecting isorotundic acid glycoside in Spodiopiperis Radix seeds for the first time, which provides a method and basis for the development and utilization of Spodiopiperis Radix seeds and Spodiopiperis Radix seed oil, and reduces the resource waste of Spodiopiperis Radix seeds;

[0055] 2. The application first discovers that the Prunella vulgaris seed is rich in isorotundic acid glycosides, and provides a basis for health care and medical use of the Prunella vulgaris seed;

[0056] 3. The detection method of the isorotundic acid glycoside in the Prunella vulgaris seed provided by the application has the characteristics of high accuracy and precision, good stability and repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 Figure 1 is a TLC identification result of the isorotundic acid glycoside in the Prunella vulgaris seed under the ultraviolet light 254 nm; wherein, 1, 2, 3, 4, 6, 7 are the Prunella vulgaris seed test samples, and 5 is the isorotundic acid glycoside reference substance.

[0058] Figure 2 Figure 2 is a TLC identification result of the isorotundic acid glycoside in the Prunella vulgaris seed under the ultraviolet light 365 nm; wherein, 1, 2, 3, 4, 6, 7 are the Prunella vulgaris seed test samples, and 5 is the isorotundic acid glycoside reference substance.

[0059] Figure 3 Figure 3 is an HPLC chromatogram of the isorotundic acid glycoside reference substance.

[0060] Figure 4 Figure 4 is an HPLC chromatogram of the isorotundic acid glycoside in the Prunella vulgaris seed.

[0061] Figure 5 Figure 5 is an HPLC chromatogram of the blank extraction solvent of the Prunella vulgaris seed.

[0062] Figure 6 Figure 6 is a linear fitting curve diagram of the isorotundic acid glycoside standard solution. DETAILED DESCRIPTION

[0063] The application will be further described in detail below in combination with the embodiments and the drawings, but the embodiments of the application are not limited thereto.

[0064] Example 1: TLC detection of the isorotundic acid glycoside in the Prunella vulgaris seed

[0065] (1) Preparation of the test sample solution: 6 samples of the Prunella vulgaris seed were taken, and the shells were broken and crushed through a 40-mesh sieve. 2.0 g of the Prunella vulgaris seed powder was accurately weighed, and was added into 100 mL of a 50% (v / v) methanol aqueous solution, and was ultrasonically extracted (power 150 W, frequency 45 kHz) for 45 min. The filtrate was placed in an evaporating dish, and was evaporated to dryness on a water bath. 2 mL of methanol was added to dissolve the residue, and was filtered through a 0.45 μm filter to obtain 6 test sample solutions of the Prunella vulgaris seed powder, which were used for standby.

[0066] (2) Preparation of the control solution: 1.96 mg of isorotundic acid reference substance was weighed and dissolved in 2 mL of methanol to obtain a solution containing 0.98 mg of isorotundic acid reference substance per 1 mL, which was used as the control solution, No. 5, for standby.

[0067] (3) Thin layer chromatography (TLC) detection:

[0068] According to the test of the thin layer chromatography (general rule 0502) in the first part of the Pharmacopoeia of the People's Republic of China, 2015 edition, 4 μL of each of the above two solutions was taken and spotted on the same high-performance silica gel GF plate. 254 On the thin layer plate, toluene-ethyl acetate-isopropyl alcohol-formic acid (4.5:2.5:2.5:0.5) was used as the developing agent, and after development, removal and air-drying, the plate was detected under ultraviolet light (254 nm) and ultraviolet light (365 nm), respectively.

[0069] Detection under ultraviolet light (254 nm): in the test sample chromatogram, the same fluorescence quenching spot as the isorotundic acid reference substance was observed at the position corresponding to the chromatogram of the control substance; see Figure 1 .

[0070] Detection under ultraviolet light (365 nm): in the test sample chromatogram, the same color fluorescence spot as the isorotundic acid reference substance was observed at the position corresponding to the chromatogram of the control substance; see Figure 2 .

[0071] (4) TLC detection results and conclusions:

[0072] The results of detection under ultraviolet light 254 nm and detection under ultraviolet light (365 nm) prove that the six samples of Prunella vulgaris L. seeds all contain isorotundic acid, and the isorotundic acid in the six test sample chromatograms is basically the same.

[0073] Example 2: Method for determining the content of isorotundic acid in Prunella vulgaris L. seeds and methodological verification

[0074] 1. Principle and reagents

[0075] 1.1 Principle

[0076] The isorotundic acid contained in Prunella vulgaris L. seeds is a polar water-soluble substance. The polar component isorotundic acid is extracted and separated from the fat-soluble component at the same time by using a polar solvent extraction method, and the content of isorotundic acid is determined by high-performance liquid chromatography.

[0077] 1.2 Reagents

[0078] Acetic acid, methanol analytical pure, methanol chromatographic pure, acetonitrile chromatographic pure, isorotundic acid reference substance (batch number DSTDY055001, purity: HPLC≥98%) produced by Chengdu Lumeitian Medicine Co., Ltd. / Desite Biological Products; Prunella vulgaris L. seed sample produced by Jiangxi Xinkangjian Ecological Agricultural Development Co., Ltd.

[0079] 1.3 Instruments

[0080] BY-500A high-speed universal pulverizer produced by Yongkang Sufeng Trade Co., Ltd.; U3000 series high-performance liquid chromatograph (U ultraviolet detector) produced by Thermo Fisher Scientific, Germany; BT125D type 1 / 100,000 electronic analytical balance produced by Sartorius, Germany; SB25-12TD type new zhi ultrasonic cleaner produced by Bana Cleaning Equipment Co., Ltd.; ultraviolet light; water bath; commonly used glass instruments; drying oven.

[0081] 2 Methods and results

[0082] 2.1 Preparation of reference solution

[0083] 5.51 mg of isorotundic acid reference substance was precisely weighed and placed in a 100 mL volumetric flask, dissolved and made up with 50% (v / v) methanol to obtain the isorotundic acid reference solution.

[0084] 2.2 Preparation of test solution

[0085] Preparation of Prunella vulgaris L. seed test solution: Prunella vulgaris L. seeds were crushed and broken, passed through a 40 mesh sieve, and about 1.0 g of Prunella vulgaris L. seed powder was precisely weighed and placed in a stoppered conical flask. 100 mL of 50% (v / v) methanol aqueous solution was precisely added, and the weight was precisely weighed. Ultrasonic treatment was performed (power 600 W, frequency 40 kHz) for 60 minutes, and the weight was weighed again after cooling. The lost weight was made up with 50% (v / v) methanol aqueous solution, shaken well, filtered with a 0.45 μm filter, and the filtrate was obtained to obtain the Prunella vulgaris L. seed test solution.

[0086] 2.3 Chromatographic conditions

[0087] Ultimate XB-C18 chromatographic column (4.6 mm x 250 mm, 5 μm) was used, column temperature was 25°C, detection wavelength was 319 nm, flow rate was 1 mL / min, injection volume was 10 μL for both the reference solution and the test solution, and the mobile phase was acetonitrile-methanol-0.2% acetic acid aqueous solution (15:12:73). The chromatogram is shown in Figures 3-5 .

[0088] The chromatogram is shown in Figures 3-5It can be seen that in the HPLC chromatogram of isorotundic acid control, there is an absorption peak of isorotundic acid control at the retention time of about 11.5 minutes, and in the HPLC chromatogram of Prunella vulgaris L. seed, there is an absorption peak of isorotundic acid at the retention time of about 11.5 minutes, and the peak shape is similar to that of isorotundic acid control, the separation degree is good, and the plate number of isorotundic acid is about 13000. No absorption peak of isorotundic acid is found in the HPLC chromatogram of the blank extraction solvent; it is indicated that the HPLC determination condition of isorotundic acid in Prunella vulgaris L. seed is mature and can be used.

[0089] 2.4 Linear relationship investigation

[0090] Take the control solution under item "2.1", filter it through a 0.45 μm microporous filter, and according to the chromatographic conditions under item "2.3", take 2, 4, 8, 12, 16, 20 μL respectively, take the injection amount (μg) as the abscissa and the peak area as the ordinate, draw the standard curve, and the results are shown in Table 1 and Figure 6 ; the regression equation is Y = 30.928X - 1.5175, R 2 = 0.9998.

[0091] Table 1 Linear relationship experiment data of isorotundic acid (n = 6)

[0092]

[0093] Y = 30.928X - 1.5175, R 2 = 0.9998

[0094] The above results show that isorotundic acid has a good linear relationship with the peak area in the range of 0.2204-2.2040 μg.

[0095] 2.5 Precision test: precisely take 10 μL of isorotundic acid control solution under item "2.1", and according to the chromatographic conditions under item "2.3", continuously inject 6 times, measure the peak area, calculate the average value of the peak area value of isorotundic acid as 15.596, and the RSD is 1.68%, the RSD of the peak area value is less than 2%, and the results are shown in Table 2; the results show that the instrument precision is good.

[0096] Table 2 Precision test data (n = 6)

[0097]

[0098] 2.6 Stability test: Take the Prunella vulgaris L. seed test sample solution under item 2.2, and according to the chromatographic conditions under item 2.3, respectively, at 0 hours, 1 hour, 4 hours, 8 hours, 12 hours, 24 hours, respectively, 20 μL of sample was injected, the peak area value of isorotundic acid glycoside in Prunella vulgaris L. seed test sample was measured, and the average value of the peak area value of isorotundic acid glycoside in Prunella vulgaris L. seed test sample was 14.671, and the RSD was 1.62%; the RSD of the peak area of isorotundic acid glycoside in the sample was less than 2%, and the results are shown in Table 3.

[0099] Table 3 Stability test data (n = 6)

[0100]

[0101] The test results show that the sample test solution is stable within 24 hours.

[0102] 2.7 Reproducibility test: Take the same batch of Prunella vulgaris L. seed sample (batch number 20220314), and prepare 6 Prunella vulgaris L. seed sample solutions according to the preparation method of Prunella vulgaris L. seed test sample solution under item 2.2; respectively, according to the chromatographic conditions under item 2.3, the sample was injected and determined, and the average content of isorotundic acid glycoside in Prunella vulgaris L. seed sample was 0.5234%, and the RSD was 1.88%; the RSD of the content of isorotundic acid glycoside in the sample was less than 2%. The results are shown in Table 4.

[0103] Table 4 Reproducibility test data (n = 6)

[0104]

[0105] The test results show that the reproducibility of the determination method is good.

[0106] 2.8 Recovery test: 6 portions of 0.5 g of the same batch of Prunella vulgaris L. seed sample (batch number 20220314) with known content were precisely weighed, and an appropriate amount of reference substance solution equivalent to 100% of the content of isorotundic acid glycoside in the sample was precisely added, respectively, and the test sample solution was prepared according to the method under item 2.2; then, according to the chromatographic conditions under item 2.3, the sample was injected and determined, and the content and recovery rate of isorotundic acid glycoside were calculated, and the average recovery rate of isorotundic acid glycoside in Prunella vulgaris L. seed sample was 99.81%, and the RSD was 2.21%. The results are shown in Table 5.

[0107] Table 5 Isorotundic acid glycoside addition recovery rate test (n = 6)

[0108]

[0109] The test results show that the recovery rate of isorotundic acid glycoside in Prunella vulgaris L. seed is between 97.74-102.63%, and the sample addition recovery is good.

[0110] Example 3 Determination of the content of isorotundic acid glycoside in three batches of Prunella vulgaris L. seeds

[0111] According to the method of content determination in Example 3 above, the content of isorotundic acid glycoside in three different batches of Prunella vulgaris L. seed samples was determined respectively, and the results are shown in Table 6.

[0112] Table 6 Determination results of the content of isorotundic acid glycoside in Prunella vulgaris L. seeds (n=3)

[0113]

[0114] The content of isorotundic acid glycoside in three batches of Prunella vulgaris L. seeds was measured to be between 0.4376% and 0.55234%;

[0115] Discussion: The results of this study show that the determination of the content of isorotundic acid glycoside in Prunella vulgaris L. seeds has a good linear relationship within the linear range, and the precision, stability, reproducibility, and recovery rate tests all meet the requirements. This study is the first time to find that Prunella vulgaris L. seeds contain rich isorotundic acid glycoside components, and this determination method is also the first time to establish an HPLC detection method for isorotundic acid glycoside in Prunella vulgaris L. seeds, providing a method and basis for the quality control of Prunella vulgaris L. seeds. Since isorotundic acid glycoside is a water-soluble component, it is difficult to dissolve in Prunella vulgaris L. oil (obtained by pressing or conventional supercritical CO2 extraction, see patent applications "2022102354058" and "202210235666X"), so Prunella vulgaris L. oil contains almost no isorotundic acid glycoside component.

[0116] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application are equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for detecting isorhamnetin in Prunella vulgaris seeds, characterized in that... The process includes the following steps: qualitative identification of isorhamnetin in Prunella vulgaris seeds using thin-layer chromatography; direct extraction of isorhamnetin using a specific polar solvent extraction method and separation from lipid-soluble components; comparison with isorhamnetin reference standard; and determination of isorhamnetin content in Prunella vulgaris seeds using high-performance liquid chromatography. The specific steps of the thin-layer chromatography method are as follows: (1) Preparation of the test solution: 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. Dissolve the residue in methanol, filter, and obtain the test solution of Prunella vulgaris seed powder; (2) Preparation of reference solution: Dissolve isorhamnetin reference standard in methanol to prepare 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: Place the plate under ultraviolet light at 254nm and 365nm respectively for detection; observe whether there are spots of the same color at the corresponding positions of the test sample and the reference sample in the chromatogram, and you can identify whether the seeds of Prunella vulgaris contain isorhamnetin. The developing agent mentioned in step (3) is a toluene-ethyl acetate-isopropanol-formic acid solution obtained by mixing in a volume ratio of 4.4-4.6:2.4-2.6:2.4-2.6:0.4-0.

6.

2. The method for detecting isorhamnetin in Prunella vulgaris seeds 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; The conditions for ultrasonic extraction described in step (1)A are: extraction at a power of 50-200W and a frequency of 40-60kHz for 20-80 minutes; The methanol content in the methanol-water solution described in step (1)A is 40-60% by volume. The amount of methanol-water solution used in step (1) A is calculated based on 1.0-5.0g of Prunella vulgaris seed powder mixed with 50-250mL of methanol-water solution; The amount of methanol used in step (1) B is calculated based on 1.0-5.0g of Prunella vulgaris seed powder mixed with 1-5mL of methanol.

3. The method for detecting isorhamnetin in Prunella vulgaris seeds according to claim 2, characterized in that: The conditions for ultrasonic extraction described in step (1)A are: extraction at a power of 80-150W and a frequency of 45-50kHz for 30-60 minutes; The methanol content in the methanol-water solution described in step (1)A is 50% by volume. The amount of methanol used in step (1) B is calculated based on 2.0g of Prunella vulgaris seed powder mixed with 2mL of methanol aqueous solution.

4. The method for detecting isorhamnetin in Prunella vulgaris seeds according to claim 1, characterized in that: The solvent removal mentioned in step (1)A is achieved by removing the solvent through water bath heating and evaporation; The filtration described in step (1)B is filtration using a 0.45µm filter membrane; The concentration of isorhamnetin in the reference solution described in step (2) is 0.9–1.1 mg / mL; The amount of the test solution used in step (3) is 2 to 10 μL; The amount of the reference solution used in step (3) is 2 to 10 μL.

5. The method for detecting isorhamnetin in Prunella vulgaris seeds according to claim 1, characterized in that: The specific polar solvent extraction method described above involves directly extracting isorhamnetin using a 45-55% (v / v) methanol aqueous solution.

6. The method for detecting isorhamnetin in Prunella vulgaris seeds 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-0.2% (v / v) acetic acid aqueous solution as the mobile phase, and the detection wavelength was 319 nm; the theoretical plate number calculated based on the isorhamnetin peak should not be less than 8000; 2) Preparation of reference solution: Accurately weigh isorhamnetin reference standard, dissolve and dilute with methanol aqueous solution to obtain isorhamnetin reference solution; 3) Preparation of test solution: Take the powdered Prunella vulgaris seeds, weigh accurately, place in a stoppered conical flask, add methanol aqueous solution accurately, weigh accurately, sonicate, cool, weigh again, make up the lost weight with methanol aqueous solution, shake well, filter, and take the filtrate to obtain the test solution. 4) Determination: Accurately pipette the reference solution and the test solution separately, inject them into a liquid chromatograph, and determine the result.

7. The method for detecting isorhamnetin in Prunella vulgaris seeds according to claim 6, characterized in that: 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 concentration of the isorhamnetin reference solution mentioned in step 2) is 0.05-0.06 mg of isorhamnetin per 1 mL; The Prunella vulgaris seed powder mentioned in step 3) is obtained by the following steps: crushing and breaking the cell walls 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) is a methanol-water solution with a concentration of 45-55% by volume. The amount of methanol-water solution used in step 3) is calculated based on 50-150 mL of methanol-water solution per 0.5-1.5 g of Prunella vulgaris seed powder. The ultrasound conditions described in step 3) are: power 500-700W, frequency 30-50kHz, and treatment for 40-80 minutes.

8. The method for detecting isorhamnetin in Prunella vulgaris seeds according to claim 7, characterized in that: The mobile phase mentioned in step 1) is prepared by mixing acetonitrile, methanol and 0.2% (v / v) acetic acid aqueous solution in a volume ratio of 15:12:73; The column temperature in the chromatography described in step 1) is 25°C; The flow rate of the mobile phase mentioned in step 1) is 1 mL / min; The methanol-water solution mentioned in step 2) is a methanol-water solution with a concentration of 50% by volume; The methanol-water solution mentioned in step 3) is a methanol-water solution with a concentration of 50% by volume. The ultrasound conditions described in step 3) are 600W power and 40kHz frequency for 30-60 minutes.

9. The application of the method for detecting isorhamnetin in Prunella vulgaris seeds according to any one of claims 1 to 8 in the development of Prunella vulgaris seeds and their products.

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