Method for detecting caffeic acid in prunella vulgaris seed and application thereof

Abstract

The application discloses a detection method of caffeic acid in prunella vulgaris L seeds and application thereof. The detection method comprises the following steps: qualitatively identifying the caffeic acid in the prunella vulgaris L seeds by using a thin layer chromatography method; directly extracting the caffeic acid by using a specific polar solvent extraction method and separating the caffeic acid from fat-soluble components; and determining the content of the caffeic acid in the prunella vulgaris L seeds by using a high performance liquid chromatography method by comparison with a caffeic acid reference substance. The detection method has the advantages of high accuracy and precision, good stability and repeatability, and provides a quality detection method of the caffeic acid 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 or food, in particular to a detection method of caffeic acid in Prunella vulgaris seeds and application thereof. BACKGROUND

[0002] Prunella vulgaris L. is a perennial herbaceous plant with medicinal and edible properties, which has been recorded in Shennong's Herbal Classic and has a history of several thousand years of medicinal use. 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 swollen eyes, photophobia and lacrimation, night pain in the eyeball, dizziness and blurred vision, scrofula, goiter, breast cancer, hypertension, lymph node tuberculosis, infiltrative pulmonary tuberculosis, simple goiter, parotitis, acute jaundice type infectious hepatitis and the like. Modern research shows that Prunella vulgaris L. contains various chemical components, including triterpenes, sterols, flavonoids, organic acids, coumarins and the like. Prunella vulgaris L. has pharmacological effects such as anti-tumor, anti-inflammatory, antibacterial, anti-viral, immune regulation, blood pressure reduction, blood sugar reduction, blood lipid reduction and the like. Prunella vulgaris L. seeds are the dry mature seeds of Prunella vulgaris L. and are the reproductive seeds of Prunella vulgaris L. Prunella vulgaris L. seeds should have the genetic active substances and effects of Prunella vulgaris L. However, the application value of Prunella vulgaris L. seeds in food, health care or medicine has not been valued, and no relevant reports on the research of Prunella vulgaris L. seeds have been found. In particular, the quality control method of Prunella vulgaris L. seeds is blank.

[0003] Caffeic acid, also known as 3,4-dihydroxy cinnamic acid, is an organic acid with the molecular formula C9H8O4 and a molecular weight of 180.15. It is a yellow crystalline solid obtained from concentrated aqueous solutions and a hydrate obtained from dilute aqueous solutions. It has a decomposition point of 223-225° (softens at 194°) and is slightly soluble in cold water, easily soluble in hot water and cold ethanol. The main effects of caffeic acid are: 1. Increasing white blood cells and platelets: Caffeic acid is a current hemostatic agent that can increase white blood cells and platelets. It has the functions of contracting and consolidating microvessels, increasing coagulation factors, and increasing white blood cells and platelets. 2. Anti-inflammatory effect: Caffeic acid can eliminate oxygen free radicals released by neutrophils and macrophages during inflammatory reactions, significantly improving leukopenia, oxidative stress and inflammation. In addition, caffeic acid can prevent the activation of NF-κB and reduce the expression of pro-inflammatory cytokines to enhance the anti-inflammatory effect of the body. 3. Antibacterial and antiviral effects: Caffeic acid has a wide range of antibacterial effects. In vitro experiments show that it has antiviral activity, with strong inhibition of cowpox and adenovirus, followed by poliovirus type I and parainfluenza virus type III. 4. Antioxidant effect: Caffeic acid has good antioxidant properties, and its oxidative activity is closely related to its molecular structure. Caffeic acid and its analogues are effective natural antioxidants with free radical scavenging mechanisms, also known as direct antioxidant mechanisms. Caffeic acid and hydroxycinnamic acid are natural antioxidants found in plants. 5. Anti-cancer effect: Caffeic acid phenethyl ester (CAPE) is a natural anti-cancer active product. CAPE can inhibit the production of reactive oxygen species and regulate inflammatory factors at the post-transcriptional level, showing significant inhibition of cancer cell proliferation and migration. The effect of caffeic acid derivatives with caffeoyl / cinnamoyl groups on the proliferation of human breast cancer cells (MCF-7) and non-cancer cells (MCF10A) shows that caffeoyl / cinnamoyl groups have no effect on MCF10A proliferation. Selective toxicity or growth inhibition of cancer cells but not non-cancer cells is a key feature of caffeic acid compounds in anti-cancer effects. The anti-tumor activity of caffeic acid and its derivatives has attracted attention for a long time. Caffeic acid in coffee can inhibit the growth of breast cancer cells. 6. Hypoglycemic effect: ⑴ Studies have shown that diabetes is related to oxidative stress, and the antioxidant effect of caffeic acid and its derivatives has a positive effect on the treatment of diabetes. It has been found that caffeic acid can reduce the absorption of glucose by endothelial cells and promote the decomposition of liver glycogen. ⑵ Diabetic nephropathy (DN) is a common and refractory diabetic microvascular complication. Caffeic acid is a widely distributed phenolic compound in nature and has been shown to be effective in the treatment of DN. The unique and simple chemical structure of caffeic acid makes it have good antioxidant, anti-inflammatory, antiviral and other pharmacological activities, making it a good alternative drug for the treatment of diabetic nephropathy. Caffeic acid can effectively treat DN by reducing urinary protein and protecting renal function, and its pharmacological mechanism is related to hypoglycemic, autophagy, anti-inflammatory and antioxidant effects.7. Other effects: caffeic acid can improve the central excitability of rats; can increase the amount of hydrochloric acid secretion in the human stomach, and can make the pulse slow; can enhance the uterine tension, can enhance the bile secretion of rats; has the effects of increasing white blood cells, stopping bleeding and inactivating vitamin B1; can inhibit the generation of lipid peroxide in mouse brain homogenate; has the effects of shortening blood clotting and bleeding time; has the effect of resisting snake venom: 3 micrograms of caffeic acid can completely inhibit 20 micrograms of rattlesnake venom phosphodiesterase, and can be used as an anti-snake venom agent.

[0004]

[0005] The present application first discovers that the Prunella vulgaris seed contains caffeic acid components, and first establishes a detection method for caffeic acid in the Prunella vulgaris seed, which comprises the following steps: qualitatively identifying the caffeic acid in the Prunella vulgaris seed by using a thin layer chromatography method; separating the fat-soluble components and the polar components of the caffeic acid by using a polar solvent extraction method, and then comparing with a caffeic acid reference substance, and determining the content of the caffeic acid in the Prunella vulgaris seed by using a high performance liquid chromatography method. The present application provides a method and a basis for the development and utilization of the Prunella vulgaris seed and its products. SUMMARY

[0006] The primary purpose of the present application is to provide a quality control method for the Prunella vulgaris seed, which is blank, to first discover that the Prunella vulgaris seed is rich in caffeic acid components, and to first establish a detection method for the caffeic acid in the Prunella vulgaris seed, so as to provide a detection method for the caffeic acid in the Prunella vulgaris seed, which has high accuracy and precision, good stability and repeatability.

[0007] Another purpose of the present application is to provide the application of the above-mentioned detection method for the caffeic acid in the Prunella vulgaris seed.

[0008] The purpose of the present application is achieved by the following technical scheme: a detection method for the caffeic acid in the Prunella vulgaris seed, comprising the following steps: qualitatively identifying the caffeic acid in the Prunella vulgaris seed by using a thin layer chromatography method; directly extracting the caffeic acid by using a specific polar solvent extraction method and separating the fat-soluble components, and then comparing with a caffeic acid reference substance, and determining the content of the caffeic acid in the Prunella vulgaris seed by using a high performance liquid chromatography method.

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

[0010] (1) Preparation of the test solution of the sample:

[0011] Preparation of the test solution of the sample of the Prunella vulgaris seed:

[0012] A. The Prunella vulgaris seed powder is added into a methanol aqueous solution for ultrasonic extraction, filtration, removal of the solvent from the obtained filtrate, and obtaining a residue;

[0013] B. The residue is dissolved in methanol, filtration, and obtaining the test solution of the Prunella vulgaris seed powder;

[0014] (2) Preparation of the control solution: dissolve the caffeic acid control sample in methanol as the control solution;

[0015] (3) TLC detection: take the test sample solution and the control solution and spot them on the same high-performance silica gel GF plate respectively; 254 develop the plate with the developing agent, take it out, and dry it;

[0016] (4) Observe the silica gel GF plate under the UV light at 254 nm and 365 nm respectively; 254 Observe the test sample chromatogram under the UV light at 254 nm and 365 nm respectively; if there is a spot of the same color at the position corresponding to the control sample chromatogram, it can be determined that the Prunella vulgaris seed contains caffeic acid.

[0017] The Prunella vulgaris powder in step (1) is preferably obtained by the following steps: crushing the Prunella vulgaris seed to break the shell, and passing it through a sieve with a mesh size of at least 40 to obtain the Prunella vulgaris 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 calculated based on 1.0-5.0 g of the Prunella vulgaris seed powder and 50-250 mL of the methanol aqueous solution; more preferably based on 2.5 g of the Prunella vulgaris seed powder and 2 mL of the methanol aqueous solution.

[0021] The conditions of the ultrasonic extraction in step (1) A 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; most preferably 150 W of power, 45 kHz of frequency, and 45 minutes of extraction.

[0022] The filtration in step (1) A can be filtration through filter paper or filtration through a filter membrane.

[0023] The filter membrane is preferably a filter membrane with a pore size of 0.45 μm.

[0024] The removal of the solvent in step (1) A is preferably by evaporation.

[0025] The amount of methanol in step (1) B is preferably calculated based on 1.0-5.0 g of the Prunella vulgaris seed powder and 1-3 mL of methanol; more preferably based on 2.5 g of the Prunella vulgaris seed powder and 2 mL of the methanol aqueous solution.

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

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

[0028] The amount of the test solution described in step (3) is 2-10 μL; more preferably 6 μL.

[0029] The amount of the control solution described in step (3) is 2-10 μL; more preferably 6 μL.

[0030] The developing agent described in step (3) is preferably toluene-ethyl acetate-isopropanol-formic acid solution; more preferably toluene-ethyl acetate-isopropanol-formic acid solution mixed in a volume ratio of 6.4-6.6:1.9-2.1:0.9-1.1:0.4-0.6; most preferably toluene-ethyl acetate-isopropanol-formic acid solution mixed in a volume ratio of 6.5:2.0:1.0:0.5.

[0031] The specific polar solvent extraction method is direct extraction of caffeic acid with 45-55% (v / v) methanol aqueous solution, which can effectively extract caffeic acid and separate it from lipid-soluble components.

[0032] The methanol aqueous solution is preferably 50% (v / v) 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) aqueous acetic acid as the mobile phase, and the detection wavelength is 323 nm; the theoretical plate number calculated according to the caffeic acid peak should not be less than 8000;

[0035] 2) Preparation of the control solution: accurately weigh caffeic acid control, dissolve in methanol aqueous solution and dilute to volume to obtain the caffeic acid control solution;

[0036] 3) Preparation of the test solution: take the Prunella vulgaris seed powder, accurately weigh, place in a conical flask with a plug, accurately add methanol aqueous solution, accurately weigh, ultrasonically treat, cool, re-weigh, make up the weight loss with methanol aqueous solution, shake well, filter, and take the filtrate to obtain the test solution;

[0037] 4) Determination: accurately pipette the control solution and the test solution, respectively, inject into the liquid chromatography, and determine to obtain the results.

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

[0039] The column in the chromatography in step 1) is preferably an Ultimate XB-C18 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 aqueous methanol solution in step 2) is preferably an aqueous methanol solution with a concentration of 45-55% (v / v); more preferably an aqueous methanol solution with a concentration of 50% (v / v).

[0043] The concentration of the caffeic acid reference solution in step 2) is preferably 0.010-0.011 mg caffeic acid per 1 mL.

[0044] The Spica Prunellae powder in step 3) is preferably obtained by crushing the Spica Prunellae seeds to break the cell walls, and then passing through a sieve with a mesh size of at least 40 to obtain the Spica Prunellae powder.

[0045] The aqueous methanol solution in step 3) is preferably an aqueous methanol solution with a concentration of 45-55% (v / v); more preferably an aqueous methanol solution with a concentration of 50% (v / v).

[0046] The amount of the aqueous methanol solution in step 3) is preferably 50-150 mL of the aqueous methanol solution per 3-10 g of the Spica Prunellae seeds; more preferably 50 mL of the aqueous methanol solution per 5 g of the Spica Prunellae seeds.

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

[0048] The volume of the reference solution in step 4) and the volume of the test solution in step 4) are preferably mixed in a volume ratio of 1:2.

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

[0050] The volume of the test solution is preferably 20 μL.

[0051] The Prunella vulgaris seed contains not less than 40 μg / g of caffeic acid (C9H8O4) calculated as dry product.

[0052] The method for detecting caffeic acid in the Prunella vulgaris seed is applied to the development of the Prunella vulgaris seed and products thereof.

[0053] Prunella vulgaris 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 seed is not developed and utilized, thousands of tons of Prunella vulgaris seeds are not collected every year and 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 caffeic acid in the Prunella vulgaris seed for the first time, which provides a method and basis for the development and utilization of the Prunella vulgaris seed, and reduces the resource waste of the Prunella vulgaris seed.

[0055] 2. The present application first discovers that the Prunella vulgaris seed contains caffeic acid, which provides a basis for the health care and medical use of the Prunella vulgaris seed.

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

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

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

[0059] Figure 3 Fig. 3 is an HPLC chromatogram of the caffeic acid control sample.

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

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

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

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

[0064] Example 1 TLC detection of caffeic acid in Prunella vulgaris L. seeds

[0065] (1) Preparation of test solution: 6 samples of Prunella vulgaris L. seeds were taken, the hulls were broken and crushed, and then passed through a 40-mesh sieve. 2.5 g of each of the crushed Prunella vulgaris L. seeds was accurately weighed, and then added to 100 mL of a 50% (v / v) methanol aqueous solution. Ultrasonic extraction (power 150 W, frequency 45 kHz) was performed for 45 min. Filtration was performed, and the filtrate was placed in an evaporating dish and heated in a water bath to evaporate to dryness. 2 mL of methanol was added to dissolve the residue, and then 0.45 μm filtration was performed to obtain Prunella vulgaris L. seed test solutions 1, 2, 3, 4, 6, and 7, which were reserved for use.

[0066] (2) Preparation of control solution: 1.92 mg of caffeic acid was weighed, and then added to 2 mL of methanol to prepare a solution containing about 0.96 mg of caffeic acid per mL, which was used as the control solution, numbered 5, and reserved for use.

[0067] (3) TLC detection:

[0068] According to the TLC method (general test 0502) in the first part of the 2015 edition of the Pharmacopoeia of the People's Republic of China, 6 μL of each of the above two solutions was taken and spotted on the same high-performance silica gel GF plate. 254 TLC plate, and toluene-ethyl acetate-isopropanol-formic acid (6.5:2.0:1.0:0.5) was used as the developing agent. After development, removal, and air-drying, ultraviolet light (254 nm) and ultraviolet light (365 nm) were used for detection.

[0069] Ultraviolet light (254 nm) detection: in the test sample chromatogram, the same fluorescent quenching spots as the caffeic acid control were observed at the corresponding positions. Figure 1 .

[0070] Ultraviolet light (365 nm) detection: in the test sample chromatogram, the same color fluorescent spots as the caffeic acid control were observed at the corresponding positions. Figure 2 .

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

[0072] The results of ultraviolet light (254 nm) detection and ultraviolet light (365 nm) detection prove that the 6 samples of Prunella vulgaris L. seeds all contain caffeic acid components, and the caffeic acid components in the 6 test sample chromatograms are basically the same.

[0073] Example 2 Method for determining the content of caffeic acid in Prunella vulgaris L. seeds and method validation

[0074] 1 Principle and reagents

[0075] 1.1 Principle

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

[0077] 1.2 Reagents

[0078] Acetic acid (analytical pure), methanol (analytical pure), methanol (chromatographically pure), acetonitrile (chromatographically pure), caffeic acid reference substance (batch number G22020065, content: 99.7%) from the TMRM Quality Control Standard Substance Center, Prunella vulgaris L. seed sample from Jiangxi Xinkangjian Ecological Agricultural Development Co., Ltd.

[0079] 1.3 Instruments

[0080] BY-500A high-speed universal pulverizer from Yongkang Sufeng Trade Co., Ltd., U3000 series high performance liquid chromatograph (U ultraviolet detector) from Thermo Fisher Scientific, Germany, BT125D type 1 / 10 million electronic analytical balance from Sartorius, Germany, SB25-12TD type new zhi ultrasonic cleaner from Bana Cleaning Equipment Co., Ltd., ultraviolet light, water bath, commonly used glass instruments, and drying oven.

[0081] 2 Methods and Results

[0082] 2.1 Preparation of Reference Solution

[0083] 5.34 mg of caffeic acid reference substance was precisely weighed and placed in a 50 mL volumetric flask. It was dissolved and made up to volume with 50% methanol to obtain the caffeic acid reference solution.

[0084] 2.2 Preparation of Test Solution

[0085] Preparation of Prunella vulgaris L. seed test solution: Prunella vulgaris L. seeds were taken, crushed, and passed through a 40 mesh sieve. About 5.0 g of Prunella vulgaris L. seed powder was precisely weighed and placed in a stoppered conical flask. 50 mL of a 50% (v / v) methanol aqueous solution was precisely added. The weight was precisely weighed, and ultrasonic treatment was performed (power 600 W, frequency 40 kHz) for 60 minutes. After cooling, the weight was re-weighed, and the lost weight was made up with a 50% (v / v) methanol aqueous solution. After shaking well, it was filtered through a 0.45 μm filter. The filtrate was taken to obtain the Prunella vulgaris L. seed test solution.

[0086] 2.3 Chromatographic Conditions

[0087] Ultimate XB-C18 column (4.6 mm x 250 mm, 5 μm), column temperature 25 ℃, detection wavelength 323 nm, flow rate 1 mL / min, injection volume: 10 μL for reference substance solution, 20 μL for sample solution, mobile phase: acetonitrile-methanol-0.2% acetic acid (15:12:73); see Figure 1 for chromatogram Figures 3-5 .

[0088] From the chromatogram Figures 3-5 , it can be seen that in the HPLC chromatogram of the caffeic acid reference substance, an absorption peak of the caffeic acid reference substance appears at a retention time of about 6.7 minutes, and in the HPLC chromatogram of the Prunella vulgaris L. seeds, an absorption peak of caffeic acid appears at a retention time of about 6.7 minutes, and the peak shape is similar to that of the caffeic acid reference substance, with good separation and a column plate number of about 12000; and in the HPLC chromatogram of the blank extraction solvent, no absorption peak of caffeic acid is seen; indicating that the HPLC determination conditions for caffeic acid in the Prunella vulgaris L. seeds are mature and applicable.

[0089] 2.4 Investigation of linear relationship

[0090] Take the reference substance solution under item "2.1", dilute 10 times with 50% (v / v) methanol aqueous solution, filter through a 0.45 μm microporous filter, and take 4, 8, 12, 16 and 20 μL for injection under the chromatographic conditions in item "2.3", with injection amount (μg) as the abscissa and peak area as the ordinate, to draw a standard curve, the results of which are shown in Table 1 and Figure 2. Figure 6 The regression equation is Y = 59.355X - 0.0127, R 2 = 0.9996.

[0091] Table 1 Experimental data of linear relationship of caffeic acid (n = 5)

[0092]

[0093] The regression equation is Y = 59.355X - 0.0127, R 2 = 0.9996

[0094] The above results show that caffeic acid has a good linear relationship with peak area in the range of 0.0427-0.2136 μg.

[0095] 2.5 Precision test: precisely take 10 μL of the diluted 10 times caffeic acid reference substance solution under item "2.4", and continuously inject 6 times under the chromatographic conditions in item "2.3", determine the peak area, and calculate the average value of the caffeic acid peak area value as 6.380, with RSD of 1.62%, and the RSD of the peak area value is <2%, the results of which are shown in Table 2; the results show that the instrument precision is good.

[0096] Table 2 Data of precision test (n = 6)

[0097]

[0098] 2.6 Stability test: Take the Prunella vulgaris L. seed sample solution under item 2.2, and according to the chromatographic conditions under item 2.3, respectively, 20 μL of sample was injected at 0 hour, 1 hour, 4 hours, 8 hours, 12 hours and 24 hours, and the peak area value of caffeic acid was determined. The average value of the peak area value of caffeic acid in the Prunella vulgaris L. seed sample was 6.487, and the RSD was 1.13%. The RSD of the peak area of caffeic acid 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 has good stability 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 the Prunella vulgaris L. seed sample solution under item 2.2. According to the chromatographic conditions under item 2.3, the sample was injected and determined, and the average content of caffeic acid in the Prunella vulgaris L. seed sample was 54.17 μg / g, and the RSD was 1.03%. The RSD of the content of caffeic acid 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 determination method has good reproducibility.

[0106] 2.8 Recovery rate test: Precisely weigh 6 portions of the same batch of Prunella vulgaris L. seed sample (batch number 20220314) with known content, each 2.5 g, and precisely add the appropriate amount of reference substance solution equivalent to 100% of the content of caffeic acid in the sample, and prepare the sample solution 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 of caffeic acid and the recovery rate were calculated. The average recovery rate of caffeic acid in the Prunella vulgaris L. seed sample was 99.71%, and the RSD was 1.41%. The results are shown in Table 5.

[0107] Table 5 Caffeic acid spiking recovery rate test (n = 6)

[0108]

[0109] The test results show that the recovery rate of caffeic acid in the Prunella vulgaris seeds is between 98.17-101.76%, and the sample addition recovery is good.

[0110] Example 3 Determination of the content of caffeic acid in three batches of Prunella vulgaris seeds

[0111] According to the method of content determination in Example 2 above, the content of caffeic acid in three different batches of Prunella vulgaris seed samples was determined, and the results are shown in Table 6.

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

[0113]

[0114] The content of caffeic acid in three batches of Prunella vulgaris seeds was measured to be between 51.13-59.73 μg / g.

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

[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 caffeic acid in Prunella vulgaris seeds, characterized by It comprises the following steps: using thin layer chromatography to qualitatively identify caffeic acid in Prunella vulgaris L seeds; using a specific polar solvent extraction method to directly extract caffeic acid and separate it from fat-soluble ingredients, and then using caffeic acid reference substance for comparison, using high performance liquid chromatography to determine the content of caffeic acid in Prunella vulgaris L seeds; The specific steps of the thin layer chromatography are as follows: (1) Preparation of test solution: Preparation of test solution of Prunella vulgaris L seeds: A. Prunella vulgaris L seed powder is added to a methanol aqueous solution for ultrasonic extraction, filtration, removal of solvent from the obtained filtrate to obtain a residue; B. The residue is dissolved in methanol, filtered to obtain the test solution of Prunella vulgaris L seed powder; (2) Preparation of reference solution: Caffeic acid reference substance is dissolved in methanol as the reference solution; (3) Thin layer chromatography: Take the test solution and the control solution respectively on the same high-performance silica gel GF plate 254 On the thin layer plate, develop with the developing agent, take out, and dry. (4) Observation of silica gel GF 254 Thin layer plate: under the ultraviolet light 254 nm and ultraviolet light 365 nm, respectively, identify whether the test product chromatogram contains the same color spots as the control product chromatogram at the corresponding position, that is, whether the prunella vulgaris seed contains caffeic acid components. The developing agent in step (3) is a toluene-ethyl acetate-isopropyl alcohol-formic acid solution mixed in a volume ratio of 6.4-6.6:1.9-2.1:0.9-1.1:0.4-0.

6.

2. The detection method of caffeic acid in Prunella vulgaris L seeds according to claim 1, characterized in that: The Prunella vulgaris L seed powder in step (1) is obtained by the following steps: crushing the Prunella vulgaris L seeds, passing through a sieve with a mesh size of at least 40 meshes to obtain Prunella vulgaris L seed powder; The ultrasonic extraction conditions in step (1) A are 20-80 minutes of extraction at a power of 50-200 W and a frequency of 40-60 kHz; The content of methanol in the methanol aqueous solution in step (1) A is 40-60% by volume; The amount of the methanol aqueous solution in step (1) A is calculated based on 1.0-5.0 g of Prunella vulgaris L seed powder to 50-250 mL of methanol aqueous solution; The amount of methanol in step (1) B is calculated based on 1.0-5.0 g of Prunella vulgaris L seed powder to 1-3 mL of methanol.

3. The detection method of caffeic acid in Prunella vulgaris L seeds according to claim 2, characterized in that: The ultrasonic extraction conditions in step (1) A are 30-60 minutes of extraction at a power of 80-150 W and a frequency of 45-50 kHz The content of methanol in the methanol aqueous solution in step (1) A is 50% by volume; The amount of methanol in step (1) B is calculated based on 2.5 g of Prunella vulgaris L seed powder to 2 mL of methanol aqueous solution.

4. The detection method of caffeic acid in Prunella vulgaris L seeds according to claim 1, characterized in that: The removal of solvent in step (1) A is by evaporation to dryness; The filtration in step (1) B is filtration using a filter membrane with a pore size of 0.45 µm; The concentration of caffeic acid in the reference solution in step (2) is 0.9-1.1 mg / mL; The amount of the test solution in step (3) is 2-10 µL; The amount of the reference solution in step (3) is 2-10 µL.

5. The detection method of caffeic acid in Prunella vulgaris L seeds according to claim 1, characterized in that: The specific polar solvent extraction method is direct extraction of caffeic acid with a methanol aqueous solution with a volume percentage of 45-55%.

6. The detection method of caffeic acid in Prunella vulgaris L 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 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 323 nm; the theoretical plate number calculated according to the coffee acid peak should not be less than 8000; 2) Preparation of the reference solution: accurately weigh the coffee acid reference substance, dissolve and make up with the methanol aqueous solution to obtain the coffee acid reference solution; 3) Preparation of the test solution: take the Prunella vulgaris L. seed powder, accurately weigh, put into a conical flask with a plug, accurately add the methanol aqueous solution, accurately weigh, ultrasonic treatment, cool, re-weigh, make up the weight loss with the methanol aqueous solution, shake well, filter, and take the filtrate to obtain the test solution; 4) Determination: accurately take the reference solution and the test solution, inject into the liquid chromatography, and determine to obtain the test solution.

7. The detection method of coffee acid in Prunella vulgaris L. seed according to claim 6, wherein: in step 1), the mobile phase is acetonitrile, methanol and 0.2% (v / v) acetic acid aqueous solution, which are mixed in a volume ratio of 14-16:11-13:72-74; in step 1), the column temperature in the chromatography is 24-27℃; in step 1), the flow rate of the mobile phase is 0.8-1.2 mL / min; in step 2), the methanol aqueous solution is a methanol aqueous solution with a concentration of 45-55% (v / v); in step 2), the concentration of the coffee acid reference solution is 0.10-0.11 mg of coffee acid per 1 mL; in step 3), the Prunella vulgaris L. seed powder is obtained by the following steps: crushing the Prunella vulgaris L. seed, passing through a sieve with a mesh size of at least 40 to obtain the Prunella vulgaris L. seed powder; in step 3), the methanol aqueous solution is a methanol aqueous solution with a concentration of 45-55% (v / v); in step 3), the amount of the methanol aqueous solution is 50-150 mL per 3-10 g of Prunella vulgaris L. seed powder; in step 3), the ultrasonic treatment is performed at a power of 500-700 W and a frequency of 30-50 kHz for 40-80 minutes.

8. The detection method of coffee acid in Prunella vulgaris L. seed according to claim 7, wherein: in step 1), the column temperature in the chromatography is 25℃; in step 1), the flow rate of the mobile phase is 1 mL / min; in step 2), the methanol aqueous solution is a methanol aqueous solution with a concentration of 50% (v / v); in step 3), the methanol aqueous solution is a methanol aqueous solution with a concentration of 50% (v / v); in step 3), the ultrasonic treatment is performed at a power of 600 W and a frequency of 40 kHz for 30-60 minutes.

9. The detection method of coffee acid in Prunella vulgaris L. seed according to any one of claims 1-8 for use in the development of Prunella vulgaris L. seed and its products. ​ ​ ​

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