A method for detecting the content of ursolic acid and total triterpenes in prunella vulgaris seed and prunella vulgaris seed oil and application thereof

Abstract

The application discloses a detection method and application of ursolic acid and total triterpenes in prunella vulgaris seed and prunella vulgaris seed oil. The detection method is characterized in that ursolic acid in prunella vulgaris seed and prunella vulgaris seed oil is qualitatively identified by using a thin layer chromatography method; oil and fat components interfering with determination of total triterpenes are separated and removed by column separation, and then the content of total triterpenes in prunella vulgaris seed and prunella vulgaris seed oil is determined by using spectrophotometry with ursolic acid or oleanolic acid as a control, so that the problems of interference and false high content in determination of the content of total triterpenes in oil components in the prior art are overcome. The method has the advantages of high accuracy and precision, good stability and repeatability, and provides a method for quality detection of triterpenes in development and utilization of prunella vulgaris seed and prunella vulgaris seed oil, and has strong specificity and good reproducibility.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of quality detection, and particularly relates to a detection method for the contents of ursolic acid and total triterpenes in Prunella vulgaris seeds and Prunella vulgaris seed oil and application thereof. BACKGROUND

[0002] Prunella vulgaris L. is a perennial herbaceous plant with medicinal and edible properties, and has been used as a medicine for thousands of years. Prunella vulgaris L. is recorded in the 2015 edition of Chinese Pharmacopoeia. Prunella vulgaris L. has a bitter and pungent taste and is cold in nature, and is attributed to the liver and gallbladder channels. Prunella vulgaris L. can clear the liver and relieve fire, improve eyesight, resolve masses and relieve swelling, and has good clinical therapeutic effects on various diseases such as red and painful eyes, photophobia and lacrimation, night pain of eyeball, dizziness and blurred 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.

[0003] Prunella vulgaris seeds are the dry mature seeds of Prunella vulgaris L. and are the reproductive seeds of Prunella vulgaris L. When Prunella vulgaris L. is harvested, the Prunella vulgaris seeds almost do not contain or rarely contain Prunella vulgaris L. due to reasons such as maturity and shedding of the Prunella vulgaris seeds and harvesting operations (such as shaking). At present, Prunella vulgaris seeds are only used for breeding Prunella vulgaris L. and have no other applications.

[0004] In the process of developing the value of Prunella vulgaris seeds, the applicant first obtained Prunella vulgaris seed oil by using Prunella vulgaris seeds as raw materials, and found that Prunella vulgaris seeds and Prunella vulgaris seed oil are rich in triterpenes such as ursolic acid. However, there is no related report on the quality control method of Prunella vulgaris seeds and Prunella vulgaris seed oil in the prior art. SUMMARY

[0005] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a detection method for the contents of ursolic acid and total triterpenes in Prunella vulgaris seeds and Prunella vulgaris seed oil.

[0006] Another purpose of the present application is to provide the application of the detection method for the contents of ursolic acid and total triterpenes in Prunella vulgaris seeds and Prunella vulgaris seed oil.

[0007] The purpose of the present application is achieved by the following technical solution: a detection method of ursolic acid and total triterpenes in Prunella vulgaris L. seeds and Prunella vulgaris L. seed oil, comprising the following steps: using thin layer chromatography detection method to qualitatively identify ursolic acid in Prunella vulgaris L. seeds and Prunella vulgaris L. seed oil; using column separation to separate and remove oil and fat components interfering with the determination of total triterpenes, and then using ursolic acid or oleanolic acid as a control, using spectrophotometry to determine the content of total triterpenes in Prunella vulgaris L. seeds and Prunella vulgaris L. seed oil.

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

[0009] (1) Preparation of test solution:

[0010] ① Preparation of test solution of Prunella vulgaris L. seeds: Prunella vulgaris L. seed powder is added to a methanol aqueous solution for ultrasonic extraction, filtered, and the obtained filtrate is subjected to solvent removal, and the residue is dissolved in methanol to obtain the test solution of Prunella vulgaris L. seed powder; wherein the concentration of methanol in the methanol aqueous solution is 70-90% by volume;

[0011] ② Preparation of test solution of Prunella vulgaris L. seed oil: Prunella vulgaris L. seed oil and petroleum ether with a boiling range of 60-90°C are uniformly mixed, and then subjected to extraction with a methanol aqueous solution to separate and obtain the lower methanol aqueous phase; the obtained methanol aqueous phase is subjected to solvent removal, and the residue is dissolved in methanol to obtain the test solution of Prunella vulgaris L. seed oil; wherein the concentration of methanol in the methanol aqueous solution is 70-90% by volume;

[0012] (2) Preparation of control solution: ursolic acid control is dissolved in methanol to serve as the control solution;

[0013] (3) Thin layer chromatography (TLC) detection: the test solution and the control solution are taken and spotted on the same high-performance silica gel G thin layer plate, developed with a developing agent, taken out, air-dried, sprayed with a color developing agent, and heated at 100-110°C until the spots are clearly colored;

[0014] (4) Observation of silica gel G thin layer plate: the silica gel G thin layer plate obtained in step (3) is placed under a daylight lamp and an ultraviolet light lamp respectively for inspection, and whether the same color spots exist in the test chromatogram at the positions corresponding to the control chromatogram is observed, so as to identify whether the Prunella vulgaris L. seeds and Prunella vulgaris L. seed oil contain ursolic acid, a triterpene component.

[0015] The Prunella vulgaris L. seed powder in step (1) is preferably obtained by the following steps: Prunella vulgaris L. seeds are crushed and broken, and passed through a sieve with a mesh size of at least 40 to obtain Prunella vulgaris L. powder.

[0016] The sieve is preferably a sieve with a mesh size of 40-50.

[0017] The conditions of the ultrasonic extraction in step (1) ① are preferably 50-200 W of power, 40-60 kHz of frequency, and 20-80 minutes of extraction; more preferably 80-150 W of power, 45-50 kHz of frequency, and 30-60 minutes of extraction; and most preferably 150 W of power, 45 kHz of frequency, and 30-60 minutes of extraction.

[0018] The concentration of the methanol in the methanol aqueous solution in step (1) ① is preferably 80% by volume.

[0019] The amount of the methanol aqueous solution in step (1) ① is preferably 30-60 mL of the methanol aqueous solution per 2.0-5.0 g of the Prunella vulgaris L. seed powder.

[0020] The removal of the solvent in step (1) ① is preferably by evaporation.

[0021] The amount of the methanol in step (1) ① is preferably 1-3 mL of the methanol aqueous solution per 2.0-5.0 g of the Prunella vulgaris L. seed powder; and more preferably 2 mL of the methanol aqueous solution per 2.0-5.0 g of the Prunella vulgaris L. seed powder.

[0022] The amount of the petroleum ether in step (1) ② is preferably 30 mL of the petroleum ether per 0.6-1.5 g of the Prunella vulgaris L. seed oil.

[0023] The concentration of the methanol in the methanol aqueous solution in step (1) ② is preferably 80% by volume.

[0024] The amount of the methanol aqueous solution in step (1) ② is preferably 30 mL of the methanol aqueous solution per 0.6-1.5 g of the Prunella vulgaris L. seed oil.

[0025] The extraction in step (1) ② is performed for 1 or more times; and preferably 2-3 times, and the methanol aqueous solutions are combined.

[0026] The removal of the solvent in step (1) ② is preferably by evaporation.

[0027] The amount of the methanol in step (1) ① is preferably 1-3 mL of the methanol aqueous solution per 0.6-1.5 g of the Prunella vulgaris L. seed powder; and more preferably 2 mL of the methanol aqueous solution per 0.6-1.5 g of the Prunella vulgaris L. seed powder.

[0028] The concentration of the ursolic acid in the control solution in step (2) is preferably 0.9-1.1 mg / mL; and more preferably 1 mg / mL.

[0029] The amount of the test sample solution in step (2) is 2-10 μL.

[0030] The amount of the control solution used in step (2) is 2-10 μL.

[0031] The developing agent used in step (3) is preferably toluene-ethyl acetate-formic acid solution; more preferably toluene-ethyl acetate-formic acid solution mixed at a volume ratio of 8.1-8.3:1.5-1.7:0.1-0.3; most preferably toluene-ethyl acetate-formic acid solution mixed at a volume ratio of 8.2:1.6:0.2.

[0032] The color developing agent used in step (3) is preferably ethanolic sulfuric acid solution; more preferably 10% (v / v) ethanolic sulfuric acid solution.

[0033] The temperature of heating used in step (3) is preferably 105°C.

[0034] The wavelength of the ultraviolet lamp used in step (4) is 365 nm.

[0035] The specific steps of the content determination are as follows:

[0036] 1) Preparation of test sample solution

[0037] ① Preparation of test sample solution of Prunella vulgaris seeds:

[0038] a. Take Prunella vulgaris seed powder 3.0-6.0 g, accurately weigh, place in a conical flask with a stopper, accurately add ethyl acetate 100 mL, accurately weigh, ultrasonic treatment, weigh again, add ethyl acetate to the original weight, place to clarify, accurately take 50 mL of supernatant, place in an evaporating dish, heat evaporation in a water bath to dryness, the residue is reserved;

[0039] b. The above residue is dissolved in 10 mL of petroleum ether with a boiling range of 60-90°C, and then transferred to a chromatographic column with a 10 g silica gel of 100-200 mesh, and the inner diameter of the column is 15-20 mm; elute with 100 mL of eluent A obtained by mixing petroleum ether with a boiling range of 60-90°C and ethyl acetate at a volume ratio of 90:10, and discard the eluate; then elute with 150 mL of eluent obtained by mixing ethyl acetate and methanol at a volume ratio of 80:20, collect the eluate, place in an evaporating dish, heat evaporation in a water bath to dryness, dissolve the residue in 80 mL of anhydrous ethanol, transfer to a 100 mL volumetric flask, make up to the mark with anhydrous ethanol, shake well, and use as the test sample solution;

[0040] Preparation of the test solution of the Prunella vulgaris seed oil: 0.30-0.80 g of the Prunella vulgaris seed oil sample was precisely weighed, dissolved in 10 mL of petroleum ether with a boiling range of 60-90°C, and then transferred to a chromatographic column with a 10 g silica gel with a particle size of 100-200 mesh. The inner diameter of the column was 15-20 mm. Elution was performed with 100 mL of an eluent A prepared by mixing petroleum ether and ethyl acetate in a volume ratio of 90:10, and the eluent was discarded. Then, 150 mL of an eluent prepared by mixing ethyl acetate and methanol in a volume ratio of 80:20 was used for elution, and the eluent was collected, evaporated to dryness in an evaporating dish on a water bath, dissolved in 80 mL of anhydrous ethanol, transferred to a 100 mL volumetric flask, diluted with anhydrous ethanol to the mark, and shaken to obtain the test solution.

[0041] 2) Preparation of the triterpene control solution: an appropriate amount of the triterpene control was precisely weighed, dissolved in anhydrous ethanol to obtain a solution containing 0.1 mg per 1 mL, and the triterpene control solution was obtained.

[0042] 3) Preparation of the standard curve: 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of the control solution were precisely measured and placed in 15 mL test tubes with stoppers, dried, cooled, and 0.2 mL of freshly prepared vanillin glacial acetic acid solution and 0.8 mL of perchloric acid were precisely added, shaken, heated in a 70°C water bath for 15 minutes, immediately placed in an ice bath for 5 minutes, taken out, 4 mL of ethyl acetate was precisely added, shaken, and the absorbance was measured at a wavelength of 545 nm with the corresponding reagents as the blank. The standard curve was plotted with the absorbance as the vertical coordinate and the concentration as the horizontal coordinate.

[0043] (4) Determination method: 0.2 mL of the test solution was precisely measured and placed in a 15 mL test tube with a stopper. The method described under the preparation of the standard curve was used from the “drying” step, and the absorbance was measured by the same method. The content of the triterpene control in the test solution was read from the standard curve, calculated, and the total triterpene content of the test sample was obtained.

[0044] The ultrasonic extraction conditions described in step 1) are preferably 50-200 W of power, 40-60 kHz of frequency, and 20-80 minutes of extraction time; more preferably 80-150 W of power, 45-50 kHz of frequency, and 30-60 minutes of extraction time; and most preferably 150 W of power, 45 kHz of frequency, and 45 minutes of extraction time.

[0045] The triterpene control described in step 2) is ursolic acid or oleanolic acid.

[0046] The vanillin glacial acetic acid solution described in step 3) is preferably obtained by the following steps: 0.5 g of vanillin was precisely weighed, dissolved in 10 mL of glacial acetic acid, and the vanillin glacial acetic acid solution was obtained.

[0047] The formula of the calculation in step 4) is as follows:

[0048] The calculation formula of the total triterpenoid content in Prunella vulgaris L. seed (powder) is shown as formula (1); and the calculation formula of the total triterpenoid content in Prunella vulgaris L. seed oil is shown as formula (2);

[0049]

[0050]

[0051] X - the content of triterpenoids in the sample, in units of grams per hundred grams (g / 100g);

[0052] m1 - the mass of the Prunella vulgaris L. seed (powder) or Prunella vulgaris L. seed oil sample, in units of grams (g);

[0053] m2 - the mass of triterpenoids in the sample solution calculated by the linear regression equation, in units of milligrams (mg);

[0054] V1 - the volume of the sample solution, in units of milliliters (mL);

[0055] V2 - the volume of the sample solution, in units of milliliters (mL).

[0056] The above detection method is applied in the development of Prunella vulgaris L. seed and Prunella vulgaris L. seed oil.

[0057] Prunella vulgaris L. is a common medicinal and edible herb recorded in Chinese Pharmacopoeia, and its wild and planted resources are very rich. However, since the Prunella vulgaris L. seed has not been developed and utilized, thousands of tons of Prunella vulgaris L. 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:

[0058] 1. The present application provides a detection method of the content of ursolic acid and total triterpenoids in Prunella vulgaris L. seed and Prunella vulgaris L. seed oil for the first time, which provides a method and basis for the development and utilization of Prunella vulgaris L. seed and Prunella vulgaris L. seed oil, and reduces the resource waste of Prunella vulgaris L. seed;

[0059] 2. The present application first discovers that Prunella vulgaris L. seed and Prunella vulgaris L. seed oil are rich in triterpenoid components represented by ursolic acid and oleanolic acid, which provides a basis for the health care and medical use of Prunella vulgaris L. seed and Prunella vulgaris L. seed oil;

[0060] 3、The application overcomes the problem of interference of oil in the determination of the content of total triterpenoid components in the oil component and the problem of false high content of total triterpenoid components (oil impurities also participate in the color reaction, leading to the problem) in the prior art; the application obtains the optimal separation condition by a large number of experiments and by exploring different column chromatography separation conditions (including but not limited to eluent) for the purpose of removing oil to the greatest extent and retaining total triterpenoid components: the eluent of petroleum ether (60-90℃): ethyl acetate = volume ratio 90:10 can remove more than 90% of oil, while the total triterpenoid components are retained on the column (determined by detecting whether the total triterpenoid components are contained in the eluted solution); the eluent of ethyl acetate:methanol = volume ratio 80:20 can completely elute the total triterpenoid on the column; thus, the detection method for the content of ursolic acid and total triterpenoid in Prunella vulgaris L. seeds and Prunella vulgaris L. seed oil provided by the application has the characteristics of high accuracy and precision, good stability and repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0061] Figure 1 is a photo of the TLC identification result of ursolic acid, a triterpenoid component in Prunella vulgaris L. seed oil, under visible light; wherein, 1, 2, 3, 5, 6 and 7 are Prunella vulgaris L. seed oil test samples, and 4 is ursolic acid reference substance.

[0062] Figure 2 is a photo of the TLC identification result of ursolic acid, a triterpenoid component in Prunella vulgaris L. seed oil, under ultraviolet light; wherein, 1, 2, 3, 5, 6 and 7 are Prunella vulgaris L. seed oil test samples, and 4 is ursolic acid reference substance.

[0063] Figure 3 is a photo of the TLC identification result of ursolic acid, a triterpenoid component in Prunella vulgaris L. seed oil, under visible light; wherein, 1, 2, 3, 4, 6 and 7 are Prunella vulgaris L. seed oil test samples, and 5 is ursolic acid reference substance.

[0064] Figure 4 is a photo of the TLC identification result of ursolic acid, a triterpenoid component in Prunella vulgaris L. seed oil, under ultraviolet light; wherein, 1, 2, 3, 4, 6 and 7 are Prunella vulgaris L. seed oil test samples, and 5 is ursolic acid reference substance.

[0065] Figure 5 is a linear fitting curve diagram of the standard solution of oleanolic acid. DETAILED DESCRIPTION

[0066] 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.

[0067] Example 1: TLC detection of ursolic acid in Prunella vulgaris L. seeds

[0068] (1) Preparation of test solution: Take 6 samples of Prunella vulgaris L. seeds, crush the hulls, pass through a 40-mesh sieve, accurately weigh 2.5 g of Prunella vulgaris L. seed powder each, add 50 mL of 80% (v / v) methanol aqueous solution, ultrasonic extraction for 45 min, filter, evaporate the filtrate to dryness in an evaporating dish in a water bath, dissolve the residue with 2 mL of methanol to obtain Prunella vulgaris L. seed powder test solutions 1, 2, 3, 5, 6 and 7, respectively, for standby.

[0069] (2) Preparation of control solution: weigh 2.01 mg of ursolic acid control, add 2 mL of methanol to dissolve, to obtain a solution containing about 1.01 mg of ursolic acid per mL, as control solution No. 4, for standby.

[0070] (3) Thin layer chromatography (TLC) detection:

[0071] According to the test of "Chinese Pharmacopoeia" Part 1 2015 edition, take 5 μL of the above two solutions respectively, point on the same high-performance silica gel thin layer plate, use toluene-ethyl acetate-formic acid (volume ratio 8.2:1.6:0.2) as developing agent, develop, take out, air dry, spray with 10% sulfuric acid ethanol solution (i.e. concentrated sulfuric acid with a concentration of 98% and anhydrous ethanol in a volume ratio of 1:9), heat at 105°C until the spots are clear, and identify under visible light and ultraviolet light respectively;

[0072] Visible light identification: in the test sample chromatogram, the same purple red spots appeared at the position corresponding to the ursolic acid chromatogram of the control; see Figure 1 .

[0073] Under ultraviolet light (365 nm), the same orange yellow fluorescent spots appeared in the test sample chromatogram at the position corresponding to the ursolic acid chromatogram of the control; see Figure 2 .

[0074] (4) TLC detection results and conclusions:

[0075] From the visible light identification: in the test sample chromatogram, the same purple red spots appeared at the position corresponding to the ursolic acid chromatogram of the control and under ultraviolet light (365 nm), the same orange yellow fluorescent spots appeared in the test sample chromatogram at the position corresponding to the ursolic acid chromatogram of the control, which proved that ursolic acid, a triterpenoid component, was contained in the 6 samples of Prunella vulgaris L. seeds. The main triterpenoid components in the chromatograms of the 6 test samples were basically the same.

[0076] Example 2 TLC detection of ursolic acid in Prunella vulgaris L. seed oil

[0077] (1) Preparation of test solution: Take 6 samples of Prunella vulgaris L. seed oil, each weighing about 0.8 g; dissolve in 30 mL of petroleum ether (60-90°C), then transfer to a separatory funnel, add 30 mL of 80% (v / v) methanol aqueous solution, shake and extract twice, each for 1 minute, separate the lower layer of methanol aqueous solution, and place it in an evaporating dish, heat in a water bath to evaporate to dryness, add 2 mL of methanol to dissolve, and obtain 1, 2, 3, 4, 6, and 7 test solutions of Prunella vulgaris L. seed oil, respectively, for use.

[0078] (2) Preparation of control solution: weigh 2.01 mg of ursolic acid control, add 2 mL of methanol to dissolve, and obtain a solution containing about 1.01 mg of ursolic acid per mL, as control solution, numbered 5, for use.

[0079] (3) Thin layer chromatography (TLC) detection:

[0080] According to the test of "Chinese Pharmacopoeia" Part 1 2015 edition, take 5 μL of the above two solutions, respectively, and point them on the same high-performance silica gel thin layer plate, use toluene-ethyl acetate-formic acid (volume ratio 8.2:1.6:0.2) as developing agent, develop, take out, dry, spray with 10% sulfuric acid ethanol solution, heat at 105°C until the spots are clear, and identify under visible light and ultraviolet light, respectively.

[0081] Visible light identification: in the test sample chromatogram, the same purple red spots appeared at the position corresponding to the ursolic acid control chromatogram; see Figure 3 .

[0082] Under ultraviolet light (365 nm), the same orange yellow fluorescent spots appeared in the test sample chromatogram at the position corresponding to the ursolic acid control chromatogram; see Figure 4 .

[0083] (4) TLC detection results and conclusions:

[0084] From the visible light identification: in the test sample chromatogram, the same purple red spots appeared at the position corresponding to the ursolic acid control chromatogram, and under ultraviolet light (365 nm), the same orange yellow fluorescent spots appeared in the test sample chromatogram at the position corresponding to the ursolic acid control chromatogram, which proved that the 6 samples of Prunella vulgaris L. seed oil contained ursolic acid, a triterpenoid component. The main triterpenoid components in the 6 test sample chromatograms were basically the same.

[0085] Example 3 Determination method of total triterpenoids in Prunella vulgaris L. seeds and Prunella vulgaris L. seed oil and methodological verification

[0086] I. Determination method of total triterpenoids in Prunella vulgaris L. seeds and Prunella vulgaris L. seed oil

[0087] 1.1 Principle

[0088] The oil and fat substances and triterpenoids in Prunella vulgaris L. seeds and Prunella vulgaris L. seed oil are separated and removed from the oil and fat substances on the chromatographic column under optimized conditions, and the obtained triterpenoids are enriched and reacted with vanillin under the action of perchloric acid to generate colored substances. At a wavelength of 545 nm, the absorbance is proportional to the content of triterpenoids. The content of triterpenoids is determined by colorimetry with oleanolic acid or ursolic acid as a control.

[0089] 1.2 Reagents

[0090] 1.2.1 Perchloric acid, anhydrous ethanol, glacial acetic acid, and vanillin are all analytical pure, and oleanolic acid or ursolic acid is a standard.

[0091] 1.2.2 Oleanolic acid standard solution (0.1 mg / mL): 10.0 mg of oleanolic acid control was dissolved in anhydrous ethanol and diluted to 100 mL.

[0092] 1.2.3 5% (v / v) vanillin-glacial acetic acid solution: 0.5 g of vanillin was accurately weighed in a 10 mL volumetric flask, dissolved in glacial acetic acid, and diluted to the calibration mark. This solution needs to be prepared immediately before use.

[0093] 1.3 Instruments

[0094] UV spectrophotometer, analytical balance, water bath, common glassware, and drying oven.

[0095] 1.4 Preparation of standard curve

[0096] 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of oleanolic acid standard solution were taken in stoppered cuvettes and placed in a drying oven to dry the solvent at 90°C. 0.2 mL of freshly prepared 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid (added perpendicularly to the liquid surface without touching the wall of the test tube to ensure thorough mixing with the reaction solution) were added, and gently shaken. Heat in a 70°C water bath (the temperature needs to be strictly controlled, and a thermometer is used to confirm the real-time temperature) for 15 min, then remove and immediately place in ice water for 5 min, and adjust to room temperature using a tap water bath. Dilute 5.0 mL of glacial acetic acid accurately with a 5 mL pipette, and shake well. Use the reagent blank as a reference to measure the absorbance value A at 545 nm within 30 min using a UV spectrophotometer. Take the absorbance A as the vertical coordinate and the mass of oleanolic acid (mg) as the horizontal coordinate to draw a standard curve, obtain the linear regression equation, and calculate the correlation coefficient.

[0097] 1.5 Sample determination

[0098] 1.5.1 Determination of Prunella vulgaris L. seed samples

[0099] (1) Summer fructus seed sample, broken wall, 40 mesh sieve, take summer fructus seed powder 5.0g, accurate weighing, placed in a conical flask with plug, accurate add ethyl acetate 100mL, accurate weighing, ultrasonic treatment (power 150W, frequency 45kHz) 45 minutes, reweigh, add ethyl acetate to the original weight, place clear, place clear, accurate suction supernatant 50mL, placed in an evaporating dish, water bath heating evaporation to dryness, the residue is dissolved in petroleum ether (60-90℃) 10mL, and then transferred to a chromatographic column with a diameter of 15-20mm which is filled with 10g (100-200 mesh) silica gel. Elute with petroleum ether (60-90℃): ethyl acetate = volume ratio 90:10 100mL, discard the eluent; then elute with ethyl acetate:methanol = volume ratio 80:20 150mL, collect the eluent, place in an evaporating dish, water bath heating evaporation to dryness, add anhydrous ethanol 80mL to dissolve, transfer to a 100mL volumetric flask, and dilute to the mark with anhydrous ethanol, shake well, as the test solution;

[0100] (2) Take 0.2mL of the test solution in a colorimetric tube with a plug, and place it in a drying oven. Dry the solvent at 90℃. Add 0.2mL of 5% vanillin-glacial acetic acid solution and 0.8mL of perchloric acid (add vertically without touching the tube wall to ensure thorough mixing with the reaction solution), shake gently, and heat in a 70℃ water bath for 15 minutes. Remove and immediately place in ice water for 5 minutes, then adjust to room temperature with a tap water bath. Accurately transfer 5.0mL of glacial acetic acid with a 5mL pipette, and dilute by shaking. Use the reagent blank as a reference to measure the absorbance A at 545nm within 30 minutes using a UV spectrophotometer. Calculate the mass of total triterpenes in the test solution by linear regression equation.

[0101] 1.5.2 Summer fructus seed oil sample determination

[0102] (1) Accurately weigh 0.50g of summer fructus seed oil sample, precisely weigh, add petroleum ether (60-90℃) 10mL to dissolve, and then transfer to a chromatographic column with a diameter of 15-20mm which is filled with 10g (100-200 mesh) silica gel. Elute with petroleum ether (60-90℃): ethyl acetate = volume ratio 90:10 100mL, discard the eluent; then elute with ethyl acetate:methanol = volume ratio 80:20 150mL, collect the eluent, place in an evaporating dish, water bath heating evaporation to dryness, add anhydrous ethanol 80mL to dissolve, transfer to a 100mL volumetric flask, and dilute to the mark with anhydrous ethanol, shake well, as the test solution;

[0103] (2) Take 0.2 mL of the test solution into a stoppered colorimetric tube and place it in a drying oven to dry the solvent at 90 °C. Add 0.2 mL of 5% vanillin-glacial acetic acid solution and 0.8 mL of perchloric acid (add perpendicularly to the liquid surface, without touching the test tube wall, to ensure thorough mixing with the reaction solution), gently shake, and heat in a water bath at 70 °C (this temperature needs to be strictly controlled; please confirm the real-time temperature with a thermometer) for 15 min. Remove and immediately cool in ice water for 5 min, then adjust to room temperature using a tap water bath. Accurately transfer 5.0 mL of glacial acetic acid into a 5 mL pipette for dilution and shake well. Using a reagent blank as a reference, measure the absorbance A at 545 nm using a UV spectrophotometer within 30 min. Calculate the mass of total triterpenes in the test solution of Prunella vulgaris seed oil using a linear regression equation.

[0104] 1.6. Calculation

[0105] 1.6.1 Calculation of total triterpenoid content in Prunella vulgaris seeds

[0106]

[0107] (1) In the formula:

[0108] X — The content of triterpenoids in the sample, in grams per 100 grams (g / 100g);

[0109] m1—mass of the Prunella vulgaris seed powder sample, in grams (g);

[0110] m2 — The mass of triterpenoids in the sample solution used for determination, calculated by a linear regression equation, in milligrams (mg);

[0111] V1—The volume of the test solution after dilution, in milliliters (mL);

[0112] V2—The volume of sample liquid used for the determination, in milliliters (mL).

[0113] 1.6.2 Calculation of total triterpenoid content in Prunella vulgaris seed oil

[0114]

[0115] (2) Where:

[0116] X — The content of triterpenoids in the sample, in grams per 100 grams (g / 100g);

[0117] m1—Mass of the Prunella vulgaris seed oil sample, in grams (g);

[0118] m2 — The mass of triterpenoids in the sample solution used for determination, calculated by a linear regression equation, in milligrams (mg);

[0119] V1 - the volume of the liquid to be measured, in milliliters (mL);

[0120] V2 - the volume of the sample solution, in milliliters (mL);

[0121] The calculation results are rounded to three significant figures.

[0122] The detection method of the triterpenoids was established according to the detection method of triterpenoids in Ganoderma lucidum in the first part of the Pharmacopoeia of the People's Republic of China (2015 edition). Both the Prunella vulgaris L. seeds and the Prunella vulgaris L. seed oil contain oil substances and triterpenoids. When the oil substances and the triterpenoids are mixed, the oil substances will interfere with the determination of the content of the triterpenoids, and the oil substances need to be separated and removed. The detection method of the triterpenoids separates and removes the oil substances on the chromatographic column with optimized conditions, and then determines the triterpenoids enriched, which is accurate and reliable.

[0123] II. Validation of the method for determining the content of total triterpenoids in Prunella vulgaris L. seeds and Prunella vulgaris L. seed oil

[0124] 1. Preparation of the standard oleanolic acid stock solution:

[0125] 0.01003 g of the oleanolic acid reference substance with a content of 98.7% (w / w) was weighed into a 100 mL volumetric flask, dissolved and diluted with anhydrous ethanol to the mark, and shaken to prepare an oleanolic acid reference substance stock solution of about 0.09900 mg / mL.

[0126] 2. Preparation of the sample solution:

[0127] The Prunella vulgaris L. seed sample was crushed and broken, passed through a 40-mesh sieve, and 5.0032 g of the broken Prunella vulgaris L. seed powder was accurately weighed into a conical flask with a stopper. 100 mL of ethyl acetate was accurately added, the weight was determined, and the sample was ultrasonically treated (power 150 W, frequency 45 kHz) for 45 minutes. The weight was determined again, ethyl acetate was added to the original weight, and the sample was left to clarify. 50 mL of the supernatant was accurately pipetted into an evaporating dish, heated on a water bath to evaporate to dryness, and the residue was dissolved in 10 mL of petroleum ether (60-90°C). The solution was transferred to a chromatographic column with a 10 g (100-200 mesh) silica gel column of 18 x 300 mm (inner diameter x length) that had been prepared. The column was eluted with 100 mL of an eluent of petroleum ether (60-90°C): ethyl acetate = volume ratio 90:10, and the eluent was discarded. Then, the column was eluted with 150 mL of an eluent of ethyl acetate:methanol = volume ratio 80:20, and the eluent was collected and evaporated to dryness on a water bath. The residue was dissolved in 80 mL of anhydrous ethanol, transferred to a 100 mL volumetric flask, and diluted with anhydrous ethanol to the mark. The mixture was shaken to prepare a test solution, which was ready for use.

[0128] 3. Linear range

[0129] The standard curve of oleanolic acid was determined by the established detection method. The absolute content of oleanolic acid was selected as 0.000 mg, 0.020, 0.040, 0.059, 0.079, 0.099, 0.119 mg on the X axis. The determination results are shown in Table 1. The mass m was taken as the abscissa, and the absorbance value A was taken as the ordinate. Linear fitting was performed, and the linear relationship between m and A was obtained. The linear regression equation and the linear correlation coefficient are shown in Table 1, and the linear fitting curve is shown in Figure 5 The method has good linear relationship and meets the requirements of quantitative analysis.

[0130] Table 1 Linear regression equation and linear correlation coefficient of oleanolic acid standard solution

[0131]

[0132]

[0133] 4. Method precision

[0134] The same batch of Prunella vulgaris seeds was determined for 6 times by the established method, and the sample amount was 5.0 g. The relative average deviation (RSD) of the 6 determination results was calculated, and the results are shown in Table 2.

[0135] Table 2 Precision test of determination method

[0136]

[0137] 5. Method repeatability test

[0138] The same batch of Prunella vulgaris seeds was determined for 6 times by the established method, and the sample amount was 5.0 g. The relative average deviation (RSD) of the 6 determination results was calculated, and the results are shown in Table 3.

[0139] Table 3 Repeatability test of determination method

[0140]

[0141] From Table 2 and Table 3, the RSD values are 1.49% and 2.25% respectively, which meets the general method requirement of RSD < 5%, and can be used for the determination of total triterpenes in Prunella vulgaris seeds and Prunella vulgaris seed oil.

[0142] 6. Method stability test

[0143] 0.8 mL of oleanolic acid standard stock solution was added to 6 colorimetric tubes, and the color development stability time was set to 15, 30, 45 min according to the method. The absorbance value was determined, and a blank comparison was made at each time point. The results are shown in Table 4.

[0144] Color stability time test

[0145]

[0146] From Table 4, it can be seen that the color stability time has better absorbance at 15 min, and the parallelism is good, and with the increase of time, the blank absorption increases; therefore, the color stability time is selected as 15 min.

[0147] 7. Sample addition recovery rate test

[0148] Take the Fructus Prunellae seed sample, crush the hull, pass through a 40 mesh sieve, accurately weigh 5.0 g (accurate to 0.0001 g) of Fructus Prunellae seed broken powder, and a total of 6 samples are weighed, 3 samples for a group, a total of two groups. After weighing the samples, operate according to the test method, and add 14.35, 14.35, 14.35, 17.61, 17.61, 17.61 mg of oleanolic acid reference substance into the samples respectively, process according to the experimental method and perform colorimetric determination, and calculate the recovery rate. The test results are shown in Table 5.

[0149] Table 5

[0150]

[0151] From Table 5, it can be seen that the average recovery rates of the two groups of different concentrations are 100.8% and 98.63% respectively, which meet the general detection method requirement of 90-110% for recovery rate.

[0152] Example 4: Content determination of total triterpenes in Fructus Prunellae seeds

[0153] (1) Preparation of test solution: Take the Fructus Prunellae seed sample, crush the hull, pass through a 40 mesh sieve, accurately weigh 5.0 g (accurate to 0.0001 g) of Fructus Prunellae seed broken powder, and a total of 3 samples are weighed, 5.0031, 5.0012, and 5.0024 g respectively, and are placed in conical flasks with stoppers. Accurately add 100 mL of ethyl acetate, accurately weigh, ultrasonic treatment (power 150 W, frequency 45 kHz) for 45 minutes, re-weigh, add ethyl acetate to the original weight, place to clarify, accurately take 50 mL of supernatant, place in an evaporating dish, and evaporate to dryness on a water bath. Dissolve the residue in 10 mL of petroleum ether (60-90°C), and then transfer to a chromatographic column with a diameter of 18 x 300 mm, which is filled with 10 g (100-200 mesh) of silica gel. Elute with 100 mL of eluent (petroleum ether (60-90°C): ethyl acetate = 90:10 by volume), and discard the eluate. Then elute with 150 mL of eluent (ethyl acetate: methanol = 80:20 by volume), collect the eluate, place in an evaporating dish, and evaporate to dryness on a water bath. Dissolve the residue in 80 mL of anhydrous ethanol, transfer to a 100 mL volumetric flask, and dilute to the mark with anhydrous ethanol. Shake well to obtain the test solution, which is ready for use.

[0154] (2) Triterpenoids control solution preparation: 0.01003 g of oleanolic acid control with a content of 98.7% was weighed into a 100 mL volumetric flask, dissolved and diluted with anhydrous ethanol to the mark, shaken well, and oleanolic acid control solution of about 0.0990 mg / mL was prepared;

[0155] (3) Preparation of standard curve: 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL of control solution was precisely measured and placed in a 15 mL test tube with a stopper, dried, cooled, and 0.2 mL of freshly prepared vanillin glacial acetic acid solution (0.5 g of vanillin was accurately weighed and dissolved in 10 mL of glacial acetic acid to obtain it) and 0.8 mL of perchloric acid were precisely added, shaken well, heated in a 70°C water bath for 15 minutes, immediately placed in an ice bath for 5 minutes, taken out, and 4 mL of ethyl acetate was precisely added, shaken well, and the corresponding reagent was used as a blank. The absorbance was measured at 545 nm wavelength according to the ultraviolet-visible spectrophotometry (general rule 0401) in the first part of the People's Republic of China Pharmacopoeia 2015 edition, and the standard curve was drawn with absorbance as the vertical coordinate and concentration as the horizontal coordinate;

[0156] (4) Content determination: 0.2 mL of test sample solution was precisely measured and placed in a 15 mL test tube with a stopper, and the method under the preparation of the standard curve was used from "drying" to operation, the absorbance was measured, and the content of triterpenoids control in the test sample solution was read from the standard curve. The results are shown in Table 6.

[0157] Table 6 Determination of the content of total triterpenoids in Prunella vulgaris L. seeds

[0158]

[0159] Example 5 Determination of the content of total triterpenoids in Prunella vulgaris L. seed oil

[0160] (1) Preparation of Prunella vulgaris L. seed oil test sample solution: three batches of Prunella vulgaris L. seed oil samples were taken, and 0.5 g (accurate to 0.0001 g) was accurately weighed, and a total of 3 sub-samples were weighed, which were 0.5011, 0.5002, and 0.5009 g, respectively. They were dissolved in 10 mL of petroleum ether (60-90°C), then transferred to a chromatographic column with a 10 g (100-200 mesh) silica gel and an inner diameter of 15-20 mm, eluted with 100 mL of eluent (petroleum ether (60-90°C): ethyl acetate = volume ratio 90:10), and the eluent was discarded. Then 150 mL of eluent (ethyl acetate:methanol = volume ratio 80:20) was used for elution, and the eluent was collected and placed in an evaporating dish. Water bath heating was used to evaporate to dryness, and the residue was dissolved in 80 mL of anhydrous ethanol, transferred to a 100 mL volumetric flask, and diluted with anhydrous ethanol to the mark. Shake well to serve as the test sample solution for future use;

[0161] (2) Preparation of triterpenoid control solution: 0.01005 g of ursolic acid control with a content of 98.2% (v / v) was weighed into a 100 mL volumetric flask, dissolved and diluted with anhydrous ethanol to the calibration mark, shaken well, and ursolic acid control solution of about 0.0987 mg / mL was prepared;

[0162] (3) Preparation of standard curve: 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL of control solution was precisely measured and placed in a 15 mL stoppered test tube, dried, cooled, 0.2 mL of freshly prepared vanillin glacial acetic acid solution (0.5 g of vanillin was precisely weighed and dissolved into 10 mL with glacial acetic acid) and 0.8 mL of perchloric acid were precisely added, shaken well, heated in a 70°C water bath for 15 minutes, immediately placed in an ice bath for 5 minutes, taken out, 4 mL of ethyl acetate was precisely added, shaken well, and the corresponding reagent was used as blank, the absorbance was determined at 545 nm wavelength according to the ultraviolet-visible spectrophotometry (general rule 0401) in the first part of the People's Republic of China Pharmacopoeia 2015 edition, and the standard curve was drawn with absorbance as the vertical coordinate and concentration as the horizontal coordinate;

[0163] (4) Content determination: 0.2 mL of test sample solution was precisely measured and placed in a 15 mL stoppered test tube, and the method under the preparation of standard curve was used from “drying” to “precisely add 4 mL of ethyl acetate, shake well”, and the same method was used to determine the absorbance, and the content of triterpenoid control in the test sample solution was read from the standard curve, and the calculation result was shown in Table 7;

[0164] Table 7 Determination of the content of total triterpenes in Prunella vulgaris seed oil

[0165]

[0166] The Prunella vulgaris seed oil used in the application is prepared by the following method:

[0167] (1) Collection of Prunella vulgaris seeds: fully mature and dry Prunella vulgaris fruit spikes were picked and placed in a clean collection device, the Prunella vulgaris seeds in the dry Prunella vulgaris fruit spikes were shaken off into the collection device, and the obtained Prunella vulgaris seeds were collected to obtain crude Prunella vulgaris seeds.

[0168] (2) Purification of Prunella vulgaris seeds

[0169] A, remove coarse impurities: the above collected crude Prunella vulgaris seeds were placed in a sieve with a mesh size of 20 meshes, shaken and sieved, so that the grass seeds with a particle size smaller than the mesh size of the sieve passed through, and the relatively coarse impurities with a particle size larger than the mesh size of the sieve were removed, to obtain coarse impurity-removed Prunella vulgaris seeds;

[0170] B. Removing fine impurities: The above-mentioned coarse Prunella vulgaris seed product from which coarse impurities are removed is placed in a sieve with a mesh size of 30, and shaken to pass the impurities with a particle size smaller than the mesh size of the sieve, so as to remove the finer impurities with a particle size smaller than the mesh size of the sieve, and collect the Prunella vulgaris seeds that cannot pass through the mesh size of the sieve, to obtain pure Prunella vulgaris seeds.

[0171] (3) Breaking the shells of Prunella vulgaris seeds:

[0172] A. The above-mentioned pure Prunella vulgaris seeds are placed in a smashing and breaking machine, and a smashing and breaking machine sieve with a mesh size of 50 is selected for smashing and breaking;

[0173] B. The smashing and breaking machine is placed in an environment with a temperature of 10±2℃;

[0174] C. The machine is started to smash, and the smashed Prunella vulgaris seed powder is placed in a sieve with a mesh size of 50, and shaken to pass the seed powder with a particle size smaller than the mesh size of the sieve, and the seed powder that cannot pass through is added to the smashing machine for further smashing and breaking until all of it passes through, to obtain broken Prunella vulgaris seed powder; the breaking rate is more than 99%.

[0175] (4) Dry compression granulation:

[0176] ① Adding and mixing: The above-mentioned broken Prunella vulgaris seed powder is mixed with dextrin (the amount of dextrin is 0.8% of the mass of the broken Prunella vulgaris seed powder), and mixed uniformly;

[0177] ② Granulation: The above-mentioned mixed powder is granulated into particles with a particle size of 0.1-0.3 cm by using a granulator with a dry compression method, and the main pressure of the granulator is selected to be 7 MPa and the side pressure is selected to be 0.7 MPa, to obtain Prunella vulgaris seed powder particles 5.07 kg.

[0178] (5) Supercritical CO2 extraction:

[0179] ① Extraction: The above-mentioned Prunella vulgaris seed powder particles 5.07 kg are placed in a supercritical CO2 extraction kettle, CO2 is used as the supercritical fluid, the extraction temperature is 45℃, the extraction pressure is 30 MPa, the extraction time is 4 hours, and the extraction flow rate is 23 kg / h;

[0180] ② Separation: Two-stage separation is adopted, and the process conditions for separation are as follows: the pressure of the first-stage separator is 12 MPa, and the separation temperature is 40℃; the pressure of the second-stage separator is 5 MPa, and the separation temperature is 30℃; yellow-green Prunella vulgaris seed oil 0.92 kg is obtained.

[0181] (6) Filtration and impurity removal: The above-mentioned Prunella vulgaris seed oil is placed in a filtration device with a mesh size of 350, and is filtered under reduced pressure, and the impurities that cannot be filtered are discarded, to obtain clear Prunella vulgaris seed oil.

[0182] (7) quality inspection and filling: the above hedyarum seed oil sample is taken for quality inspection, and after reaching the standard, it is filled with nitrogen.

[0183] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for detecting ursolic acid and total triterpenoids in Prunella vulgaris seeds and Prunella vulgaris seed oil, characterized in that... The steps include: using thin-layer chromatography to qualitatively identify ursolic acid in Prunella vulgaris seeds and Prunella vulgaris seed oil; using column separation to separate and remove oily components that interfere with the determination of total triterpenes; and then using ursolic acid or oleanolic acid as a control to determine the content of total triterpenes in Prunella vulgaris seeds and Prunella vulgaris seed oil by spectrophotometry. The specific steps of the thin-layer chromatography detection method are as follows: (1) Preparation of the test solution: ① Preparation of Prunella vulgaris seed test solution: Prunella vulgaris seed powder was added to a methanol aqueous solution for ultrasonic extraction, filtered, the solvent was removed from the filtrate, and the residue was dissolved in methanol to obtain the Prunella vulgaris seed powder test solution; wherein, the concentration of methanol in the methanol aqueous solution was 70-90% by volume. ② Preparation of the Prunella vulgaris seed oil test solution: Prunella vulgaris seed oil and petroleum ether at 60-90℃ are mixed evenly, and then extracted with a methanol-water solution. The lower methanol-water solution phase is separated. The solvent is removed from the obtained methanol-water solution phase, and the residue is dissolved in methanol to obtain the Prunella vulgaris seed oil test solution. The concentration of methanol in the methanol-water solution is 70-90% by volume. (2) Preparation of reference solution: Dissolve ursolic acid 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 G thin-layer plate, develop with the developing solvent, remove, air dry, spray with color developing agent, and heat at 100-110℃ until the spots are clearly visible; (4) Observe the silica gel G thin layer plate: Place the silica gel G thin layer plate obtained in step (3) under a fluorescent lamp and under an ultraviolet lamp respectively to examine it. Observe whether there are spots of the same color at the corresponding positions of the test sample chromatogram and the reference chromatogram. This will identify whether the seeds of Prunella vulgaris and the oil of Prunella vulgaris contain the triterpenoid component ursolic acid. The developing solvent mentioned in step (3) is a toluene-ethyl acetate-formic acid solution obtained by mixing in a volume ratio of 8.1-8.3:1.5-1.7:0.1-0.

3. The colorimetric reagent mentioned in step (3) is a sulfuric acid ethanol solution; The wavelength of the ultraviolet lamp mentioned in step (4) is 365nm.

2. The method for detecting ursolic acid and total triterpenoids in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 1, characterized in that: The Prunella vulgaris seed powder mentioned in step (1) is obtained by the following steps: crushing and breaking the cell wall of Prunella vulgaris seeds and passing them through a sieve of at least 40 mesh to obtain Prunella vulgaris seed powder; The conditions for ultrasonic extraction described in step (1) ① are: extraction at a power of 50-200W and a frequency of 40-60kHz for 20-80 minutes; The concentration of methanol in the methanol-water solution mentioned in step (1) ① is 80% by volume. The amount of methanol-water solution mentioned in step (1) ① is calculated based on 2.0-5.0g of Prunella vulgaris seed powder mixed with 30-60mL of methanol-water solution; The solvent removal mentioned in step (1) ① is achieved by evaporation to dryness; The amount of methanol used in step (1) ① is calculated based on 2.0-5.0g of Prunella vulgaris seed powder mixed with 1-3mL of methanol aqueous solution; The amount of petroleum ether used in step (1) ② is calculated based on 0.6-1.5g of Prunella vulgaris seed oil mixed with 30mL of petroleum ether; The concentration of methanol in the methanol-water solution described in step (1) ② is 80% by volume. The amount of methanol-water solution used in step (1) ② is calculated based on 0.6-1.5g of Prunella vulgaris seed oil mixed with 30mL of methanol-water solution; The extraction process described in step (1) ② is performed more than once; The solvent removal mentioned in step (1) ② is achieved by evaporation. The amount of methanol used in step (1)① is calculated based on 0.6-1.5g of Prunella vulgaris seed powder mixed with 1-3mL of methanol aqueous solution.

3. The method for detecting ursolic acid and total triterpenoids in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 2, characterized in that: The sieve in question is a 40-50 mesh sieve; The conditions for ultrasonic extraction are: extraction at a power of 80-150W and a frequency of 45-50kHz for 30-60 minutes. The amount of methanol mentioned in step (1) ① is calculated based on 2.0-5.0g of Prunella vulgaris seed powder mixed with 2mL of methanol aqueous solution; The extraction process described in step (1) ② is repeated 2 to 3 times, and the methanol-water phases are combined. The amount of methanol used in step (1) ① is calculated based on 0.6-1.5g of Prunella vulgaris seed powder mixed with 2mL of methanol aqueous solution.

4. The method for detecting ursolic acid and total triterpenoids in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 1, characterized in that: The concentration of ursolic acid in the reference solution described in step (2) is 0.9–1.1 mg / mL; The volume of the test solution used in step (2) is 2–10 μL; The amount of the reference solution used in step (2) is 2 to 10 μL.

5. The method for detecting ursolic acid and total triterpenoids in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 1, characterized in that: The heating temperature mentioned in step (3) is 105°C.

6. The method for detecting ursolic acid and total triterpenoids in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 1, characterized in that: The colorimetric agent mentioned in step (3) is a 10% (v / v) sulfuric acid ethanol solution.

7. The method for detecting ursolic acid and total triterpenoids in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 1, characterized in that: The specific steps for content determination are as follows: 1) Preparation of the test solution ① Preparation of the test solution of Prunella vulgaris seeds: a. Take 3.0-6.0g of Prunella vulgaris seed powder, weigh it accurately, place it in a stoppered conical flask, add 100mL of ethyl acetate accurately, weigh it accurately, sonicate it, weigh it again, add ethyl acetate to the original weight, let it clarify, accurately pipette 50mL of the supernatant, place it in an evaporating dish, heat it in a water bath and evaporate it to dryness, and keep the residue for later use. b. Dissolve the above residue in 10 mL of petroleum ether at 60-90℃, then transfer it to a chromatography column packed with 10 g of 100-200 mesh silica gel. The inner diameter of the chromatography column is 15-20 mm. Elute with 100 mL of eluent A, prepared by mixing petroleum ether and ethyl acetate at 60-90℃ in a volume ratio of 90:

10. Discard the eluent. Then elute with 150 mL of eluent prepared by mixing ethyl acetate and methanol in a volume ratio of 80:

20. Collect the eluent, place it in an evaporating dish, and evaporate it to dryness in a water bath. Dissolve the residue in 80 mL of anhydrous ethanol, transfer it to a 100 mL volumetric flask, and dilute to the mark with anhydrous ethanol. Shake well to obtain the test solution. ② Preparation of the test solution of Prunella vulgaris seed oil: Weigh 0.30-0.80g of Prunella vulgaris seed oil sample accurately, add 10mL of petroleum ether at 60-90℃ to dissolve, and then transfer to a chromatography column packed with 10g of 100-200 mesh silica gel with an inner diameter of 15-20mm; elute with 100mL of eluent A obtained by mixing petroleum ether and ethyl acetate at 60-90℃ in a volume ratio of 90:10, and discard the eluent; then elute with 150mL of eluent obtained by mixing ethyl acetate and methanol in a volume ratio of 80:20, collect the eluent, place it in an evaporating dish, heat it in a water bath to evaporate to dryness, dissolve the residue in 80mL of anhydrous ethanol, transfer it to a 100mL volumetric flask, dilute to the mark with anhydrous ethanol, shake well, and use as the test solution; 2) Preparation of triterpenoid reference solution: Take an appropriate amount of triterpenoid reference standard, accurately weigh it, and add anhydrous ethanol to prepare a solution containing 0.1 mg per 1 mL, which is the triterpenoid reference solution; 3) Preparation of standard curve: Accurately measure 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of the reference solution and place them in 15 mL stoppered test tubes, evaporate to dryness, cool, and accurately add 0.2 mL of freshly prepared vanillin-acetic acid solution and 0.8 mL of perchloric acid. Shake well, heat in a 70°C water bath for 15 minutes, immediately cool in an ice bath for 5 minutes, remove, accurately add 4 mL of ethyl acetate, shake well, and use the corresponding reagent as a blank. Measure the absorbance at a wavelength of 545 nm. Plot the standard curve with absorbance as the ordinate and concentration as the abscissa. (4) Determination method: Accurately measure 0.2 mL of the test solution and place it in a 15 mL stoppered test tube. Follow the method under the preparation of standard curve, starting from "evaporation", and operate in the same way to measure the absorbance. Read the content of triterpenoid reference in the test solution from the standard curve and calculate to obtain the total triterpenoid content of the test sample.

8. The method for detecting ursolic acid and total triterpenoids in Prunella vulgaris seeds and Prunella vulgaris seed oil according to claim 7, characterized in that: The conditions for ultrasonic extraction described in step 1) ① are: extraction at a power of 50-200W and a frequency of 40-60kHz for 20-80 minutes; The triterpenoid reference standard mentioned in step 2) is ursolic acid or oleanolic acid; The vanillin glacial acetic acid solution mentioned in step 3) is obtained by the following steps: accurately weigh 0.5g of vanillin, add glacial acetic acid to dissolve it into 10mL, and obtain the vanillin glacial acetic acid solution.

9. The application of the detection method according to any one of claims 1 to 8 in the development of Prunella vulgaris seeds and Prunella vulgaris seed oil.

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