Method for determining content of phenolic acid components in salvia miltiorrhiza or salvia miltiorrhiza products by using high performance liquid chromatography and application of method

Abstract

The invention relates to a method for determining the content of phenolic acid components in salvia miltiorrhiza or salvia miltiorrhiza products by using high performance liquid chromatography. The method comprises the following steps: heating a sample in a 75% ethanol solution in a water bath, extracting to obtain nine phenolic acid components: tanshinol, caffeic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, 4-hydroxyphenyllactic acid, 4-coumaroyl-4 '-hydroxyphenyllactic acid, 4-coumaroyl-3', 4 '-dihydroxyphenyllactic acid and 3, 4-coumaroyl-4'-hydroxyphenyllactic acid, redissolving the sample to be detected with methanol, and detecting the content of the salvianolic acid in the sample to be detected. High performance liquid chromatography column separation detection is adopted, qualitative treatment is carried out by retention time, and quantification is carried out by an external standard-calibration curve method. The high performance liquid chromatography established by the invention can qualitatively and quantitatively detect the content of natural phenolic acid compounds in roots and leaves of salvia miltiorrhiza, and provides a standardized method for detecting phenolic acid characteristic active components in medicinal plant salvia miltiorrhiza and products thereof in the future; effective component detection guarantee is provided for the development of new salvia miltiorrhiza products, the analysis of a synthetic route of salvianolic acid compounds and the further improvement of the quality of salvia miltiorrhiza.

Description

Technical Field

[0001] The present invention relates to the technical field of plant metabolism analysis, and in particular to a method for determining phenolic acid components in salvia miltiorrhiza or its products by high performance liquid chromatography and an application thereof. Background Art

[0002] Danshen is a traditional Chinese medicine. It is the dried root and rhizome of the plant Salvia miltiorrhiza Bge. of the Labiatae family. It tastes bitter and slightly cold, and enters the heart and liver meridians. It has the effects of removing blood stasis and relieving pain, promoting blood circulation and menstruation, clearing the heart and relieving restlessness, cooling blood and relieving pain. The chemical components of Danshen include diterpenes, phenolic acids, sesquiterpenes, alkaloids, etc. Among them, the fat-soluble diterpenoid quinone compounds tanshinone and the water-soluble salvianolic acid compounds are widely studied and are often considered to be the main pharmacological basis of Danshen. Modern research shows that salvianolic acid mainly has pharmacological effects such as inhibiting the synthesis of endogenous cholesterol in cells, preventing lipid deposition and atherosclerotic plaques, protecting the liver and protecting nerves, and has a good therapeutic effect on cardiovascular diseases.

[0003] At present, the main phenolic acids involved in the analysis of the metabolic components of Salvia miltiorrhiza stems and leaves include 3,,4-dihydroxyphenyllactic acid (danshensu), caffeic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, etc., but there is no qualitative and quantitative detection method that includes these five active ingredients at the same time; in addition, 4-hydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid and 3,4-coumaroyl-4'-hydroxyphenyllactic acid participate in the phenylalanine metabolic pathway of Lamiaceae plants, and are further catalyzed to form rosmarinic acid under the action of rosmarinic acid synthase (RAS) and cytochrome P450 family 98A subfamily oxidase. They are all key compounds involved in the analysis of the synthesis pathway of phenolic acid components in Salvia miltiorrhiza. Therefore, it is necessary to develop a method that can clarify the content of the above compounds in Salvia miltiorrhiza, which will help in the selection of sources of traditional Chinese medicine products and the quality identification and improvement of the medicinal plant Salvia miltiorrhiza. The clarification of relevant detection methods can provide strong detection guarantees for the analysis of the synthetic pathways of phenolic acid components of Salvia miltiorrhiza, the identification of enzyme functions, enzymatic characterization and rational design. Summary of the invention

[0004] In order to overcome at least one problem existing in the prior art, the present invention optimizes and designs a method for extracting phenolic acid components in Salvia miltiorrhiza or its products, which can effectively extract 9 phenolic acid components at the same time, and further develops a method for determining the contents of 9 phenolic acid components in Salvia miltiorrhiza or its products using high performance liquid chromatography. The above detection method can realize rapid screening and accurate identification of phenolic acid components, and can efficiently qualitatively and quantitatively analyze phenolic acid metabolites in plants.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The first aspect of the present invention is to provide a method for extracting phenolic acid components in salvia miltiorrhiza or its products, which comprises using 75% ethanol solution to perform water bath heating treatment on the sample at 75-90°C for 50-130min to extract phenolic acid components; preferably, the phenolic acid components include danshensu, caffeic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, 4-hydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, and 3,4-coumaroyl-4'-hydroxyphenyllactic acid.

[0007] Furthermore, the sample was heated in a water bath for 60 minutes at 85°C using a 75% ethanol solution to extract phenolic acid components. In the above steps, some active compounds are unstable and easily decomposed at high temperatures. The present invention uses a 75% ethanol solution instead of conventional methanol-water extraction, which can protect the active substances to a certain extent, optimizes the conventional heating reflux process for 120 minutes to water bath heating for 60 minutes, simplifies the extraction process and extraction time while ensuring the extraction quality, and makes the extraction steps more economical and concise.

[0008] Furthermore, the extraction method specifically includes the following steps: mixing the sample with a 75% ethanol solution at a solid-liquid ratio of 1:8 to 20 (preferably a solid-liquid ratio of 1:16), placing it at room temperature for 8 to 16 hours (preferably placing it at room temperature for 12 hours), heating it in an 85°C water bath for 60 minutes, taking it out and cooling it to room temperature, filtering it, and evaporating the filtrate for 1.5 to 3 hours (preferably using a vacuum freeze dryer to evaporate for 2 hours, and weighing to confirm that the sample has evaporated to a constant weight), and storing the treated sample at low temperature for later use (preferably stored at 4°C).

[0009] Furthermore, in the above extraction method, the sample includes Salvia miltiorrhiza rhizome, Salvia miltiorrhiza hairy root, Salvia miltiorrhiza leaf, Salvia miltiorrhiza decoction piece, and Salvia miltiorrhiza oral liquid. It is understandable that the above sample may also be other Salvia miltiorrhiza products.

[0010] Furthermore, the extraction method also includes a sample pretreatment step:

[0011] Step A, sampling of salvia miltiorrhiza or its products: fresh salvia miltiorrhiza samples and hairy roots of salvia miltiorrhiza are washed, cut into small pieces and placed in a centrifuge tube; salvia miltiorrhiza slices are cut into small pieces and placed in a centrifuge tube; salvia miltiorrhiza oral liquid is directly placed in a centrifuge tube;

[0012] Step B, quick freezing: quick freezing the fresh salvia miltiorrhiza samples and salvia miltiorrhiza hairy roots (preferably placed in an insulated bucket, added with liquid nitrogen, and covered with a lid for quick freezing); this step is omitted for salvia miltiorrhiza decoction pieces and salvia miltiorrhiza oral liquid;

[0013] Step C, grinding and crushing: the frozen fresh salvia miltiorrhiza sample and salvia miltiorrhiza hairy root are quickly transferred into a container that has been pre-cooled (preferably pre-cooled with liquid nitrogen), and quickly ground and crushed; the salvia miltiorrhiza decoction piece sample is ground and crushed; this step is omitted for the salvia miltiorrhiza oral liquid; it is understood that the above-mentioned grinding and crushing container can be a grinding container conventionally used in the art, such as a mortar and pestle;

[0014] Step D, sample evaporation: transfer the ground fresh Salvia miltiorrhiza sample and Salvia miltiorrhiza hairy root into a centrifuge tube (preferably a 2 mL centrifuge tube), evaporate for 3 hours, and weigh and record every 20 minutes for the last hour to confirm that the sample has evaporated to a constant weight; this step is omitted for Salvia miltiorrhiza decoction pieces and Salvia miltiorrhiza oral solution.

[0015] The second aspect of the present invention is to provide a method for determining the content of phenolic acid components in Salvia miltiorrhiza or its products by high performance liquid chromatography, which comprises:

[0016] Step S1, sample processing: using any extraction method described in the first aspect to process the sample to obtain phenolic acid components;

[0017] Step S2: using high performance liquid chromatography to detect the treated sample to determine the content of each phenolic acid component in the sample.

[0018] Furthermore, in the high performance liquid chromatography described in step S2, mobile phase A is 0.08% formic acid aqueous solution, mobile phase B is 100% methanol, and gradient elution is adopted; the detection wavelengths are 270nm, 286nm and 330nm. The mobile phase components of the present invention are based on the traditional commonly used 0.1% formic acid aqueous solution and methanol, and the concentration of formic acid water is reduced after ensuring the peak type standard. 0.08% formic acid aqueous solution and methanol are used as AB phases, which reduces the amount of formic acid with pungent odor and corrosiveness, and is more economical and safe.

[0019] Furthermore, the procedure of the gradient elution method is:

[0020]

[0021] In the process of constructing the method of the present invention, under the same conditions, isocratic elution and 0.08% formic acid aqueous solution and acetonitrile, 0.08% formic acid aqueous solution and methanol and acetonitrile were used to optimize the elution procedure. There was a phenomenon that the characteristic peaks of the compounds could not be completely separated, and the target compounds could not be well quantitatively detected. The above problem was effectively solved by optimizing the AB phase ratio and procedure in the above gradient elution method. The above detection method optimizes the elution procedure based on the chromatographic conditions used by the liquid chromatography-mass spectrometry method, and can efficiently detect 9 kinds of salvianolic acid components in the absence of mass spectrometry detection conditions.

[0022] Furthermore, in the high performance liquid chromatography described in step S2, the sample to be tested is re-dissolved with a methanol solution and centrifuged, and the supernatant is filtered before testing; preferably, 5 mL of a methanol solution is used to re-dissolve the sample to be tested, vortexed and mixed, and then centrifuged to filter the supernatant into a brown sample bottle for testing. The above centrifugation condition is preferably centrifuged at 10000×g for 10 min at 4°C. The above methanol solution can be a methanol solution with a concentration of 75% to 100%, such as a 75% methanol solution, a 100% methanol solution, and the like.

[0023] Furthermore, in the high performance liquid chromatography described in step S2, the liquid chromatography conditions are: the chromatographic column is C18, the particle size is 5 μm, the inner diameter is 250×4.6 mm, and the filling particles are octadecylsilane bonded silica particles; the column temperature is 20-35° C. (preferably 25° C.); the mobile phase is ultrasonicated for 5-10 min, the flow rate is 1.2 mL / min, and the injection volume is 10 μL. .

[0024] Furthermore, in step S2, each phenolic acid component in the sample is quantitatively detected using a calibration curve or a working curve. It is understandable that the calibration curve or the working curve can be drawn using conventional methods in the art.

[0025] Furthermore, the step of preparing the calibration curve includes: selecting standard application solutions of each component with appropriate concentrations, and preparing a standard series of mixed solutions with final concentrations of 1 to 100 μg / mL using 70% methanol aqueous solution; for danshensu, rosmarinic acid, salvianolic acid A and salvianolic acid B, additionally preparing 500 μg / mL and 1000 μg / mL, and drawing a calibration curve with the signal response value of the analyte as the ordinate and the concentration of the standard to be tested as the abscissa.

[0026] Furthermore, the step of formulating the working curve includes: selecting Salvia miltiorrhiza leaves, rhizomes and hairy roots with low content of each component, taking 1g of mixed powder by the 4-point method and dissolving it in 10mL of 70% methanol solution to form a blank juice, and after determining the background value, diluting the standard solution with the same volume of blank juice, thereby drawing the working curve with reference to the formulation of the calibration curve.

[0027] Furthermore, in a specific embodiment, the sample usage for detection is: 4.15g of Salvia miltiorrhiza rhizome, 2.1g of Salvia miltiorrhiza leaf, 0.1g of Salvia miltiorrhiza hairy root, 2g of Salvia miltiorrhiza decoction piece, 5mL of Salvia miltiorrhiza oral liquid.

[0028] The third aspect of the present invention is to provide an application of the extraction method described in any one of the first aspect, or the detection method described in any one of the second aspect, which is selected from at least one of the following applications: application in qualitative and quantitative analysis of phenolic acid metabolites in plants; application in analysis of the synthesis pathway of phenolic acid components of Salvia miltiorrhiza, application in screening of raw material sources for traditional Chinese medicine products, application in quality identification and improvement of the medicinal plant Salvia miltiorrhiza, and application in preparation of products containing Salvia miltiorrhiza or its phenolic acid components.

[0029] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0030] (1) The method of the present invention realizes the simultaneous extraction and detection of 9 kinds of salvianolic acid compounds including danshensu, caffeic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, 4-hydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid and 3,4-coumaroyl-4'-hydroxyphenyllactic acid in the extract of the medicinal plant Salvia miltiorrhiza, which is green and low-cost;

[0031] (2) The method of the present invention optimizes the chromatographic mobile phase and elution degree, thereby solving the problems of unseparated peaks, unreturned baseline peaks, asymmetric peaks, and tailing peaks in the spectrum when conventional high performance liquid chromatography (HPLC) is used for determination;

[0032] (3) The method of the present invention has a good linear relationship for detecting danshensu between 5 and 1000 ppm, a good linear relationship for detecting caffeic acid between 1 and 100 ppm, a good linear relationship for detecting salvianolic acid B between 5 and 1000 ppm, a good linear relationship for detecting rosmarinic acid between 1 and 1000 ppm, a good linear relationship for detecting salvianolic acid A between 5 and 1000 ppm, a good linear relationship for detecting 4-hydroxyphenyllactic acid between 10 and 100 ppm, a good linear relationship for detecting 4-coumaroyl-4'-hydroxyphenyllactic acid between 5 and 100 ppm, a good linear relationship for detecting 4-coumaroyl-3',4'-dihydroxyphenyllactic acid between 5 and 100 ppm, and a good linear relationship for detecting 3,4-coumaroyl-4'-hydroxyphenyllactic acid between 5 and 100 ppm, and has excellent quantitative detection capability;

[0033] (4) The method of the present invention takes a short time (only about 52 minutes) to detect the contents of 9 salvianolic acid compounds at one time, is simple to operate, and has high repeatability and accuracy, which provides a favorable guarantee for the analysis of the synthetic pathway of salvianolic acid components. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0035] Figure 1 This is a chromatogram of the phenolic acid extract of Salvia miltiorrhiza hairy roots in one embodiment of the present invention;

[0036] Figure 2 This is a chromatogram of the phenolic acid extract of Salvia miltiorrhiza leaves in one embodiment of the present invention;

[0037] Figure 3 This is a chromatogram of the phenolic acid extract of Salvia miltiorrhiza rhizome in one embodiment of the present invention;

[0038] Figure 4 This is a chromatogram of the phenolic acid extract of Danshen decoction pieces in one embodiment of the present invention;

[0039] Figure 5 This is a chromatogram of the phenolic acid extract of Danshen oral liquid in one embodiment of the present invention;

[0040] Figure 6 The chromatogram is a mixed solution of 9 kinds of salvianolic acid compound standards in one embodiment of the present invention; wherein 1# is a danshensu peak, 2# is a 4-hydroxyphenyllactic acid peak, 3# is a caffeic acid peak, 4# is a rosmarinic acid peak, 5# is a salvianolic acid B peak, 6# is a 4-coumaroyl-3',4'-dihydroxyphenyllactic acid peak, 7# is a 3,4-coumaroyl-4'-hydroxyphenyllactic acid peak, 8# is a salvianolic acid A peak, and 9# is a 4-coumaroyl-4'-hydroxyphenyllactic acid peak;

[0041] Figure 7 This is a standard curve fitting diagram of Danshensu in one embodiment of the present invention;

[0042] Figure 8 This is a standard curve fitting diagram of 4-hydroxyphenyllactic acid in one embodiment of the present invention;

[0043] Fig. 9 This is a standard curve fitting diagram of caffeic acid in one embodiment of the present invention;

[0044] Fig.10 This is a standard curve fitting diagram of rosmarinic acid in one embodiment of the present invention;

[0045] Fig.11 This is a standard curve fitting diagram of salvianolic acid B in one embodiment of the present invention;

[0046] Fig.12 This is a standard curve fitting diagram of 4-coumaroyl-3',4'-dihydroxyphenyllactic acid in one embodiment of the present invention;

[0047] Fig.13This is a standard curve fitting diagram of 3,4-coumaroyl-4'-hydroxyphenyllactic acid in one embodiment of the present invention;

[0048] Fig.14 This is a standard curve fitting diagram of salvianolic acid A in one embodiment of the present invention;

[0049] Fig.15 This is a standard curve fitting diagram of 4-coumaroyl-4'-hydroxyphenyllactic acid in one embodiment of the present invention;

[0050] Fig.16 This is a working curve fitting diagram of Danshensu in one embodiment of the present invention;

[0051] Fig.17 This is a working curve fitting diagram of 4-hydroxyphenyllactic acid in one embodiment of the present invention;

[0052] Fig.18 This is a working curve fitting diagram of caffeic acid in one embodiment of the present invention;

[0053] Fig.19 This is a working curve fitting diagram of rosmarinic acid in one embodiment of the present invention;

[0054] Fig. 20 This is a working curve fitting diagram of salvianolic acid B in one embodiment of the present invention;

[0055] Fig.21 This is a working curve fitting diagram of 4-coumaroyl-3',4'-dihydroxyphenyllactic acid in one embodiment of the present invention;

[0056] Fig. 22 This is a working curve fitting diagram of 3,4-coumaroyl-4'-hydroxyphenyllactic acid in one embodiment of the present invention;

[0057] Fig.23 This is a working curve fitting diagram of salvianolic acid A in one embodiment of the present invention;

[0058] Fig.24 This is a working curve fitting diagram of 4-coumaroyl-4'-hydroxyphenyllactic acid in one embodiment of the present invention. DETAILED DESCRIPTION

[0059] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the scope of protection of the present invention. The experimental methods without specific conditions in the following embodiments are usually measured according to national standards. The experimental materials without the source indicated in the following embodiments are all commercially available raw materials. The equipment used in each step in the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to the general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise specified, all parts are weight parts and all percentages are mass percentages. Unless otherwise defined or specified, all professional and scientific terms used in the present invention have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar or equal to the recorded content can be applied to the method of the present invention.

[0060] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other. The present invention is further described below in conjunction with the drawings and specific embodiments, but is not intended to be a limitation of the present invention.

[0061] In the following examples, the model of the high performance liquid chromatograph is Agilent 1260DAD VL, which has a multi-wavelength detector and can simultaneously detect up to 8 compounds with different characteristic wavelengths; the chromatographic column is Diamonsil R 5μm C18(2); Fresh rhizomes and leaves of Salvia miltiorrhiza were from Purple Flowering Salvia miltiorrhiza in Dali, Yunnan; Salvia miltiorrhiza hairy roots were cultivated in our laboratory and the provenance was from Linyi, Shandong; Salvia miltiorrhiza slices were from Beijing Tong Ren Tang Pharmaceutical Co., Ltd. and the origin was Weifang, Shandong; Salvia miltiorrhiza oral liquid was from Chongqing Fuling Pharmaceutical Factory Co., Ltd. of Taiji Group.

[0062] The following examples are merely illustrative of the present invention.

[0063] Example 1- Extraction of phenolic acid components from Salvia miltiorrhiza or its products

[0064] This embodiment is a preferred preparation process of a sample of phenolic acid extract of the Chinese medicinal material Salvia miltiorrhiza and its products, which specifically includes the following steps:

[0065] (1) Sample pretreatment;

[0066] 1) Sampling: Wash the rhizomes and leaves of 6-month-old fresh Salvia miltiorrhiza (fresh Salvia miltiorrhiza samples) and hairy roots of Salvia miltiorrhiza, dry them with absorbent paper, cut them into small pieces and put them into centrifuge tubes; cut the dried pieces into small pieces and put them into centrifuge tubes; keep the Salvia miltiorrhiza oral solution for later use.

[0067] 2) Quick freezing: Place the prepared fresh Salvia miltiorrhiza samples and Salvia miltiorrhiza hairy roots in a heat preservation bucket, add liquid nitrogen, cover with a lid, and perform quick freezing; this step is omitted for Salvia miltiorrhiza decoction pieces and Salvia miltiorrhiza oral liquid.

[0068] 3) Grinding and crushing: After freezing, the fresh Salvia miltiorrhiza samples and Salvia miltiorrhiza hairy roots are quickly transferred into a mortar that has been pre-cooled with liquid nitrogen, and quickly ground and crushed with a pestle; the dried Salvia miltiorrhiza slices are also transferred into the mortar and ground; this step is omitted for the Salvia miltiorrhiza oral solution.

[0069] 4) Sample evaporation and weighing: transfer the ground fresh Salvia miltiorrhiza samples and Salvia miltiorrhiza hairy roots into 2 mL centrifuge tubes and evaporate in a vacuum freeze-dryer for 3 h. During the last hour, weigh and record the samples every 20 min using a balance to confirm that the samples have evaporated to a constant weight. This step can be omitted for Salvia miltiorrhiza decoction pieces and oral solutions.

[0070] Sampling of solid products of Salvia miltiorrhiza: Use a 1 / 100,000 balance to accurately weigh 4.15g of Salvia miltiorrhiza rhizome, 2.1g of Salvia miltiorrhiza leaf, 0.1g of Salvia miltiorrhiza hairy root, and 2g of Salvia miltiorrhiza slices, and freeze them in a 15mL centrifuge tube for later use.

[0071] Sampling of Danshen liquid product: After mixing evenly, accurately measure 5 mL of liquid and place it in a 15 mL centrifuge tube and freeze it for later use.

[0072] (2) Extraction step of phenolic acid components of salvianolic acid;

[0073] Weigh the evaporated Salvia miltiorrhiza rhizomes and leaves, Salvia miltiorrhiza hairy roots, Salvia miltiorrhiza slices, and Salvia miltiorrhiza oral liquid samples, add 75% ethanol solution to make the solid-liquid ratio of 1:16, and place at room temperature for 12 hours, then heat and reflux in an 85°C water bath for 60 minutes, take out and cool to room temperature, filter, and evaporate the filtrate in a vacuum freeze-dryer for 2 hours. Weigh to confirm that the sample has evaporated to constant weight, and store the sample in a 4°C refrigerator for low temperature storage until use.

[0074] The compounds extracted by the above method include 9 salvianolic acid components: danshensu, caffeic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, 4-hydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid and 3,4-coumaroyl-4'-hydroxyphenyllactic acid.

[0075] Example 2-Detection of phenolic acid content in Salvia miltiorrhiza or its products

[0076] In this embodiment, the sample to be tested obtained in Example 1 is detected by high performance liquid chromatography, which specifically includes the following steps:

[0077] (1) Dissolve the sample in 5 mL of methanol solution, vortex and mix well, and then centrifuge at 10,000 × g for 10 min at 4 °C. Filter the supernatant with a 0.22 μm organic phase microporous filter membrane and transfer it into a brown sample bottle for the detection of 9 salvianolic acid compounds.

[0078] (2) Detection principle: Danshensu, 4-hydroxyphenyllactic acid, caffeic acid, rosmarinic acid, salvianolic acid B, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 3,4-coumaroyl-4'-hydroxyphenyllactic acid, salvianolic acid A, and 4-coumaroyl-4'-hydroxyphenyllactic acid in the test sample are extracted with 75% ethanol solution and pure methanol solution, filtered through a 0.22 μm organic phase microporous filter membrane, and separated by a high performance liquid chromatography column. Detection is performed using an Agilent 1260VL DAD UV-Vis multi-wavelength detector, and qualitative analysis is performed using the same retention time as the standard, and quantitative analysis is performed using the external standard-calibration curve method.

[0079] (2) Instrument testing conditions are:

[0080] Column: Diamonsil R 5μm C18(2)250×4.6mm, octadecylsilane bonded silica particles;

[0081] Column temperature: 25°C;

[0082] Mobile phase: Phase A: 0.08% formic acid aqueous solution, Phase B: methanol, mobile phase ultrasonication for 10 min;

[0083] Flow rate: 1.2 mL / min; injection volume: 10 μL;

[0084] The gradient elution conditions are shown in Table 1 below:

[0085] Table 1 - Gradient elution conditions

[0086] time 0.01min 10min 25min 52min Phase A (0.08% formic acid in water) 71 71 55 55 Phase B (Methanol) 29 29 45 45

[0087] Detection wavelength: 270nm; 286nm; 330nm.

[0088] The detection wavelength of general water-soluble components is 286nm. Except for danshensu and rosmarinic acid, the other 7 active compounds involved in this embodiment are water-soluble compounds, and the solubility in water is relatively low, and they are alcohol-soluble compounds. 4-Hydroxyphenyllactic acid was detected at a detection wavelength of 270nm; danshensu, tanshenolic acid B, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 3,4-coumaroyl-4'-hydroxyphenyllactic acid, 4-coumaroyl-4"-hydroxyphenyllactic acid and tanshenolic acid A were detected at a detection wavelength of 286nm; caffeic acid and rosmarinic acid were detected at a detection wavelength of 330nm.

[0089] When the concentration of the mixed solution of the 9 compound standards is 30 μg / mL and the injection volume is 10 μL, the response peak areas of each compound to different wavelengths are shown in Table 2. Different compounds respond differently to different ultraviolet excitation lights, and the response is highest at their respective optimal wavelengths, and the minimum detection limit is also reduced accordingly. Therefore, the present invention selects 270 nm, 286 nm, and 330 nm as the detection wavelengths of the extract.

[0090] Table 2- Response peak area of ​​each compound to different wavelengths

[0091]

[0092] (3) The chromatogram of the mixed solution of 9 salvianolic acid compound standards is as follows: Figure 6 As shown (in the mixed injection, the concentration of each standard is 100 μg / mL, the injection volume is 10 μL, among which 1# is the peak shape of danshensu, 2# is the peak shape of 4-hydroxyphenyllactic acid, 3# is the peak shape of caffeic acid, 4# is the peak shape of rosmarinic acid, 5# is the peak shape of salvianolic acid B, 6# is the peak shape of 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 7# is the peak shape of 3,4-coumaroyl-4'-hydroxyphenyllactic acid, 8# is the peak shape of salvianolic acid A, and 9# is the peak shape of 4-coumaroyl-4'-hydroxyphenyllactic acid), by Figure 6 The results show that the 9 kinds of salvianolic acid compounds have good peak separation, the peaks return to the baseline, and the peaks are symmetrical. The relevant information is shown in Table 2. There is no interference with other coexisting substances obtained in the extraction method selected by the present invention.

[0093] Table 2 Separation information of the tested salvianolic acid compounds

[0094]

[0095] (4) The spectra of the phenolic acid extracts obtained from the samples (Danshen hairy roots, Danshen leaves, Danshen rhizomes, Danshen decoction pieces, Danshen oral liquid) in the above examples are as follows: Figure 1 to Figure 5 As shown, the content of the target characteristic active ingredient in each Danshen or its product is calculated by mass fraction according to formula (1) (the calculation result can be retained to two decimal places):

[0096]

[0097] Where:

[0098] ρ—the concentration of the substance to be tested in the sample calculated from the standard curve, in milligrams per liter (mg / L);

[0099] v—the fixed volume of the sample extract, in milliliters (mL);

[0100] X—the measured concentration of the analyte, in milligrams per kilogram (mg / kg) or milligrams per liter (mg / L);

[0101] m—the amount of sample taken, in grams (g) or milliliters (mL).

[0102] Example 3-Optimization of extraction and detection process

[0103] This embodiment optimizes the extraction method involved in Example 1 and the detection method involved in Example 2, specifically including:

[0104] (1) Selection of extraction solution;

[0105] In this embodiment, an extraction solution was selected. Considering the thermal stability of salvianolic acid compounds in ethanol, ethanol heating reflux was considered for extraction. 75% ethanol solution and 100% ethanol solution were used for extraction respectively. It was found that the concentration of the target compound in the 75% ethanol solution was higher, but there were also more coexisting substances. Since the target compound has a high solubility in methanol, the 75% ethanol solution was used for extraction and then evaporated and then re-dissolved with 75% methanol solution and 100% methanol. It was found that the methanol concentration did not affect the extraction of the target compound.

[0106] The traditional process usually uses a hot reflux device to react for 120 minutes for extraction. During the process optimization process of this embodiment, it was found that heating in a water bath at 85°C for 60 minutes can achieve the same extraction effect of salvianolic acid substances in the traditional process.

[0107] (2) Selection of mobile phase and column temperature;

[0108] Mobile phase A compared pure water, 1% formic acid water and 0.08% formic acid water, and found that 0.08% formic acid water had better separation effect and peak shape; mobile phase B compared methanol and acetonitrile, and found that 0.08% formic acid water and acetonitrile could greatly shorten the elution time of target compounds, but it was difficult to separate 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 3,4-coumaroyl-4'-hydroxyphenyllactic acid and tansolic acid A. Therefore, mobile phase A was finally selected as 0.08% formic acid aqueous solution, and mobile phase B was 100% methanol.

[0109] In the selection of column oven temperature, the present invention selects 20°C, 25°C, 30°C, and 35°C to optimize the chromatographic conditions. On the basis of ensuring the separation degree and peak shape, under the column temperature condition of 25°C, all target compounds can peak as quickly as possible.

[0110] (3) Validation of the Chinese herbal medicine Danshen and its solid and liquid products;

[0111] The sample was prepared and tested using the aforementioned method. The chromatogram was as follows: Figure 1 to Figure 5The contents of each component are shown in Table 3 (the contents of each salvianolic acid substance in the table are the contents of the dry weight of fresh Salvia miltiorrhiza and its products). The results showed that the contents of danshensu, 4-hydroxyphenyllactic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, and 4-coumaroyl-3',4'-dihydroxyphenyllactic acid in Danshen oral liquid were higher than those in other Chinese medicine products, the content of caffeic acid in hairy roots of Salvia miltiorrhiza was the highest, and 3,4-coumaroyl-4'-hydroxyphenyllactic acid and 4-coumaroyl-4'-hydroxyphenyllactic acid could only be detected in hairy roots.

[0112] Table 3-Contents of phenolic acid compounds in different Chinese medicinal materials and their products (mg / kg)

[0113]

[0114]

[0115] Remarks: N / A: Below the detection limit of the method

[0116] Example 4-Verification of the applicability of the detection method for the content of phenolic acids in Salvia miltiorrhiza or its products

[0117] In this example, the content of phenolic acid components in Danshen or its products is quantitatively detected by drawing a calibration curve and a working curve, and the applicability of the quantitative detection method is further verified by precision experiments, accuracy experiments and stability experiments. The sample extraction method and the detection method refer to Examples 1 and 2, which specifically include the following steps:

[0118] (1) Quantitative detection;

[0119] 1) Calibration curve: Select the standard application solution of each component with appropriate concentration, and prepare a series of standard mixed solutions with final concentrations of 1 to 100 μg / mL with 70% methanol aqueous solution. For danshensu, rosmarinic acid, salvianolic acid A and salvianolic acid B, additional 500 μg / mL and 1000 μg / mL are prepared. Select no less than 5 concentration points according to the instrument performance and detection needs. Set the automatic injection program according to the instrument operating conditions, determine the signal response value of the analyte as the ordinate, and the concentration of the standard to be tested as the abscissa. Draw the calibration curve and calculate the regression equation. Repeat the measurement 3 times for each concentration. The results are shown in the table. Figure 7 to Figure 15 The regression equation is as follows:

[0120] The regression equation of Danshensu was: y = 5.2229x-7.3551 (R2 = 0.9991);

[0121] 4-Hydroxyphenyllactic acid regression equation: y = 3.5963x-7.0646 (R2 = 0.9992);

[0122] Caffeic acid regression equation: y = 53.715x-58.448 (R 2 =0.9993);

[0123] Rosmarinic acid regression equation: y = 24.65x-34.051 (R 2 =0.9993);

[0124] Regression equation of salvianolic acid B: y = 12.283x-35.718 (R 2 =0.9992);

[0125] 4-Coumaryl-3',4'-dihydroxyphenyllactic acid regression equation: y = 25.473x-39.578 (R 2 =0.9993);

[0126] 3,4-Coumaryl-4'-hydroxyphenyllactic acid regression equation: y = 15.111x-39.326 (R 2 =0.999);

[0127] Regression equation of salvianolic acid A: y = 28.869x-87.251 (R 2 =0.9991);

[0128] 4-Coumaryl-4'-hydroxyphenyllactic acid regression equation: y = 28.294x-45.25 (R 2 =0.9993).

[0129] The above results show that danshensu has a good linear relationship between 5-1000ppm, caffeic acid has a good linear relationship between 1-100ppm, salvianolic acid B has a good linear relationship between 5-1000ppm, rosmarinic acid has a good linear relationship between 1-1000ppm, salvianolic acid A has a good linear relationship between 5-1000ppm, 4-hydroxyphenyllactic acid has a good linear relationship between 10-100ppm, 4-coumaroyl-4'-hydroxyphenyllactic acid has a good linear relationship between 5-100ppm, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid has a good linear relationship between 5-100ppm, and 3,4-coumaroyl-4'-hydroxyphenyllactic acid has a good linear relationship between 5-100ppm.

[0130] 2) Working curve: In this embodiment, a working curve was also prepared. Salvia miltiorrhiza leaves, rhizomes and hairy roots with low content of each component were selected and 1g of mixed powder was dissolved in 10mL of 70% methanol solution by the 4-point method to form a blank concoction. After the background value was determined, the standard solution was diluted with the same volume of blank concoction to obtain the regression equation as shown below:

[0131] Danshensu regression equation: y = 4.6116x (R 2 =0.9996);

[0132] 4-Hydroxyphenyllactic acid regression equation: y = 3.7001x (R2 = 0.9998);

[0133] Caffeic acid regression equation: y = 18.368x (R 2 =0.9997);

[0134] Rosmarinic acid regression equation: y = 9.6218x (R 2 =0.9998);

[0135] Regression equation of salvianolic acid B: y = 8.3547x (R 2 =0.9999);

[0136] 4-Coumaryl-3',4'-dihydroxyphenyllactic acid regression equation: y = 22.841x (R 2 =0.9998);

[0137] 3,4-Coumaryl-4'-hydroxyphenyllactic acid regression equation: y = 12.706x (R 2 =0.9998);

[0138] Regression equation of salvianolic acid A: y = 14.776x (R 2 =0.9993);

[0139] 4-Coumaryl-4'-hydroxyphenyllactic acid regression equation: y = 2.4496x (R 2 =0.9996).

[0140] The calibration curve and working curve drawn above were used to quantify the five samples respectively, and the results were statistically analyzed. The results showed no significant difference, which indicates that the calibration curve can be used instead of the working curve, which saves the step of preparing the real blank juice in practical applications, and is more concise and efficient.

[0141] (2) Precision test;

[0142] According to literature search, preliminary experiments were conducted. Salvia miltiorrhiza leaves, rhizomes and hairy roots with relatively low concentrations of target phenolic acid compounds were selected. 1 g of mixed powder was dissolved in 10 mL of 70% methanol solution using the 4-point method to form a blank juice. After the background value was determined, 3 portions of blank juice were added with low, medium and high concentrations of standard solutions, respectively, so that the final concentrations of the added phenolic acid compounds in the juice were 10, 25 and 75 μg / mL, respectively. Each group of samples was determined 6 times at the same time. The results of the intra-batch precision test are shown in Table 4. The relative standard deviations (RSDs) were 0.68-1.35% (salvia miltiorrhiza), 0.59-0.69% (4-hydroxyphenyllactic acid), 0.6-2.79% (caffeic acid), and 0.7-1.29% ( The relative standard deviations of the measured values ​​of the following compounds were basically below 1.5%, with a small discreteness of the sample data, high precision of the instrument and good reliability of the measured values.

[0143] Table 4 - Intra-batch precision experiment

[0144]

[0145] Remarks: N / A: Below the detection limit of the method

[0146] (3) Accuracy of the method (spike recovery method);

[0147] Three standard substances of low, medium and high concentrations were added to the low concentration Salvia miltiorrhiza extract, so that the final concentrations of phenolic acid compounds in the test samples were 10, 25 and 75 μg / mL, respectively. Each concentration was measured 6 times at the same time. The test results are shown in Table 4 above. The spiked recovery rate of danshensu was between 95 and 103%, the spiked recovery rate of 4-hydroxyphenyllactic acid was between 94 and 99%, the spiked recovery rate of caffeic acid was between 97 and 104%, the spiked recovery rate of rosmarinic acid was between 93 and 106%, the spiked recovery rate of salvianolic acid B was between 94 and 98%, the spiked recovery rate of 4-coumaroyl-3',4'-dihydroxyphenyllactic acid was between 91 and 95%, the spiked recovery rate of 3,4-coumaroyl-4'-hydroxyphenyllactic acid was between 95 and 101%, the spiked recovery rate of salvianolic acid A was between 94 and 101%, and the spiked recovery rate of 4-coumaroyl-4'-hydroxyphenyllactic acid was between 94 and 100%. The national standard requires in the laboratory quality control specification that when the concentration of the measured component is between 1 and 100 mg / L, the spike recovery rate should range from 90 to 110%. The measured spike recovery rates all meet the above requirements. The above determination method has high accuracy and can be used for the detection of related components.

[0148] (4) Stability test;

[0149] The samples with a spiked concentration of 75 μg / mL in the "intra-batch precision" test (the concentration of all target compounds was 200 μg / mL) were selected and sealed in a refrigerated (4°C) environment to avoid light. The samples were tested on the same day, 3 days, 7 days, and 14 days after placement, with 6 samples tested each time. The experimental results are shown in Table 5. The decline rate of the samples within two weeks was ≤10%. The samples can be stored for at least two weeks at 4°C. The corresponding standard solutions prepared can generally be stored for one year at -20°C.

[0150] Table 5 - Sample stability test results

[0151]

[0152]

[0153] As can be seen from the above examples, it was found through testing that the content of salvianolic acid B and rosmarinic acid in the medicinal plant Salvia miltiorrhiza is the highest, followed by caffeic acid and danshensu. The main active ingredients of Salvia miltiorrhiza that work in the human body are salvianolic acid B, rosmarinic acid, danshensu, etc. Substances such as 4-coumaroyl-3',4'-dihydroxyphenyllactic acid are precursors of these active secondary metabolites. They are inactive in themselves and need to be generated through the action of various catalytic enzymes to produce end products before they can play a role in the human body. The content of different precursors varies greatly, and it is extremely important to detect active compounds and their precursors for the efficacy evaluation of salvianolic acid compounds.

[0154] The present invention finds that the extraction method of 9 kinds of phenolic acid compounds of salvianolic acid in Example 1 can directly and simultaneously extract 9 kinds of phenolic acid substances, which lays a foundation for their simultaneous detection. The extraction method is an extremely important part of the whole research, and the simultaneous extraction and detection of 9 kinds of phenolic acid compounds are also conducive to the development of Chinese medicine Salvia miltiorrhiza and its health products and medicines. The high-efficiency chromatography constructed by the present invention uses retention time for qualitative analysis and external standard-calibration curve method for quantitative analysis, and can qualitatively and quantitatively detect the content of natural phenolic acid compounds in Salvia miltiorrhiza rhizome and leaves, providing a standardized method for the detection of phenolic acid characteristic active ingredients in the medicinal plant Salvia miltiorrhiza and its products in the future, and providing effective ingredient detection guarantee for the development of new Salvia miltiorrhiza products, the analysis of the synthesis pathway of phenolic acid compounds of Salvia miltiorrhiza and the further improvement of Salvia miltiorrhiza quality.

[0155] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A method for extracting phenolic acid components from Salvia miltiorrhiza or its products, characterized in that: The method comprises: using 75% ethanol solution to heat the sample in a water bath for 50 to 130 minutes at 75 to 90° C. to extract phenolic acid components, wherein the phenolic acid components include danshensu, caffeic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, 4-hydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, and 3,4-coumaroyl-4'-hydroxyphenyllactic acid.

2. The extraction method according to claim 1, characterized in that The samples were heated in a water bath at 85°C for 60 min using 75% ethanol solution to extract phenolic acid components.

3. The extraction method according to claim 2, characterized in that The extraction method specifically comprises the following steps: mixing the sample with a 75% ethanol solution at a solid-liquid ratio of 1:8-20, placing the sample at room temperature for 8-16 hours, heating the sample in a water bath at 85°C for 60 minutes, taking the sample out, cooling it to room temperature, filtering it, evaporating the filtrate for 1.5-3 hours, and storing the processed sample at low temperature for later use.

4. The extraction method according to claim 1, characterized in that The samples include salvia miltiorrhiza rhizomes, salvia miltiorrhiza hairy roots, salvia miltiorrhiza leaves, salvia miltiorrhiza decoction pieces, and salvia miltiorrhiza oral liquid.

5. The extraction method according to claim 4, characterized in that The extraction method further comprises a sample pretreatment step: Step A, sampling of salvia miltiorrhiza or its products: washing fresh salvia miltiorrhiza samples and hairy roots of salvia miltiorrhiza, cutting them into small pieces and placing them in a centrifuge tube; cutting salvia miltiorrhiza pieces into small pieces and placing them in a centrifuge tube; directly placing salvia miltiorrhiza oral liquid in a centrifuge tube; Step B, quick freezing: quick freezing the fresh salvia miltiorrhiza samples and salvia miltiorrhiza hairy roots; this step is omitted for salvia miltiorrhiza decoction pieces and salvia miltiorrhiza oral liquid; Step C, grinding and crushing: quickly transfer the frozen fresh salvia miltiorrhiza samples and salvia miltiorrhiza hairy roots into a container that has been cooled in advance, and grind and crush quickly; grind and crush the salvia miltiorrhiza decoction piece samples; this step is omitted for salvia miltiorrhiza oral liquid; Step D, sample evaporation: transfer the ground fresh Salvia miltiorrhiza samples and Salvia miltiorrhiza hairy roots into a centrifuge tube and evaporate for 3 hours. During the last hour, weigh and record the samples every 20 minutes to confirm that the samples have evaporated to a constant weight. This step is omitted for Salvia miltiorrhiza decoction pieces and Salvia miltiorrhiza oral solution.

6. A method for determining the content of phenolic acid components in Salvia miltiorrhiza or its products by high performance liquid chromatography, characterized in that: The method comprises: Step S1, sample processing: processing the sample by using the extraction method as described in any one of claims 1 to 5 to obtain phenolic acid components; Step S2: using high performance liquid chromatography to detect the treated sample to determine the content of each phenolic acid component in the sample.

7. The method according to claim 6, characterized in that In the high performance liquid chromatography described in step S2, mobile phase A is 0.08% formic acid aqueous solution, mobile phase B is 100% methanol, gradient elution is adopted; detection wavelengths are 270nm, 286nm and 330nm.

8. The method according to claim 7, characterized in that The procedure of the gradient elution method is:

9. The method according to claim 6, characterized in that In the high performance liquid chromatography described in step S2, the sample to be tested is re-dissolved with a methanol solution and centrifuged, and the supernatant is filtered and then tested; wherein the liquid chromatography conditions are: the chromatographic column is C18, the particle size is 5 μm, the inner diameter is 250×4.6 mm, and the filling particles are octadecylsilane bonded silica particles; the column temperature is 25°C; the mobile phase is ultrasonically applied for 5-10 min, the flow rate is 1.2 mL / min, and the injection volume is 10 μL; and / or, In step S2, each phenolic acid component in the sample is quantitatively detected using a calibration curve or a working curve.

10. An application of the extraction method according to any one of claims 1 to 5, or the method according to any one of claims 6 to 9, characterized in that: The application is selected from at least one of the following applications: application in qualitative and quantitative analysis of phenolic acid metabolites in plants; application in analysis of the synthesis pathway of phenolic acid components of Salvia miltiorrhiza, application in screening of raw material sources for traditional Chinese medicine products, application in quality identification and improvement of the medicinal plant Salvia miltiorrhiza, and application in preparation of products containing Salvia miltiorrhiza or its phenolic acid components.

Images