A method for determining the content of phenolic acid components in Salvia miltiorrhiza or its products using high performance liquid chromatography and its application.

Abstract

This invention relates to a method for determining the content of phenolic acids in *Salvia miltiorrhiza* or its products using high-performance liquid chromatography (HPLC). The sample is extracted with 75% ethanol solution in a water bath to obtain nine phenolic acid components: tanshinone, caffeic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, 4-hydroxyphenyllactic acid, 4-coumaryl-4'-hydroxyphenyllactic acid, 4-coumaryl-3',4'-dihydroxyphenyllactic acid, and 3,4-coumaryl-4'-hydroxyphenyllactic acid. The sample is redissolved in methanol, and separation and detection are performed using HPLC column chromatography. Qualitative analysis is based on retention time, and quantification is performed using the external standard-calibration curve method. The HPLC method established in this invention can qualitatively and quantitatively detect the content of natural phenolic acid compounds in the rhizomes and leaves of *Salvia miltiorrhiza*, providing a standardized method for the detection of characteristic active components of phenolic acids in the medicinal plant *Salvia miltiorrhiza* and its products. It also provides effective component detection assurance for the development of new *Salvia miltiorrhiza* products, the elucidation of the synthetic pathways of phenolic acid compounds in *Salvia miltiorrhiza*, and further quality improvement of *Salvia miltiorrhiza*.

Description

Technical Field

[0001] This invention relates to the field of plant metabolism analysis technology, and in particular to a method for determining phenolic acid components in Salvia miltiorrhiza or its products using high performance liquid chromatography and its application. Background Technology

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

[0003] Currently, the main phenolic acids involved in the analysis of the metabolic components of Salvia miltiorrhiza stems and leaves include 3,4-dihydroxyphenyllactic acid (tanshinone), caffeic acid, salvianolic acid B, rosmarinic acid, and salvianolic acid A. However, there is still no qualitative and quantitative detection method that simultaneously includes all five active ingredients. In addition, 4-hydroxyphenyllactic acid, 4-coumaryl-4'-hydroxyphenyllactic acid, 4-coumaryl-3',4'-dihydroxyphenyllactic acid, and 3,4-coumaryl-4'-hydroxyphenyllactic acid participate in the phenylalanine metabolic pathway of Lamiaceae plants. Under the action of rosmarinic acid synthase (RAS) and cytochrome P450 family 98A subfamily oxidase, they are further catalyzed to form rosmarinic acid. These are all key compounds involved in the study of the synthesis pathway of phenolic acid components in Salvia miltiorrhiza. Therefore, it is essential to develop a method that can clearly define the content of the above compounds in Danshen. This will help in the selection of sources for traditional Chinese medicine products and the quality identification and improvement of the medicinal plant Danshen. Furthermore, the clarification of relevant detection methods can provide strong detection support for the analysis of the synthetic pathways of tanshinone phenolic acid components, the identification of enzyme functions, enzymatic characterization, and rational design. Summary of the Invention

[0004] To overcome at least one problem in the existing technology, the present invention optimizes the extraction method of phenolic acid components in Salvia miltiorrhiza or its products, which can simultaneously and effectively extract nine kinds of phenolic acid components. Furthermore, a method for determining the content of nine kinds of phenolic acid components in Salvia miltiorrhiza or its products using high performance liquid chromatography has been developed. The above detection method can achieve rapid screening and accurate identification of phenolic acid components, and can efficiently perform qualitative and quantitative analysis of phenolic acid metabolites in plants.

[0005] To achieve the above objectives, 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 from Salvia miltiorrhiza or its products, wherein the sample is subjected to a water bath heating treatment at 75-90°C for 50-130 minutes using a 75% ethanol solution to extract the phenolic acid components; preferably, the phenolic acid components include tanshinone, caffeic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, 4-hydroxyphenyllactic acid, 4-coumaryl-4'-hydroxyphenyllactic acid, 4-coumaryl-3',4'-dihydroxyphenyllactic acid, and 3,4-coumaryl-4'-hydroxyphenyllactic acid.

[0007] Furthermore, the sample was subjected to a 60-minute water bath heating treatment at 85°C using a 75% ethanol solution to extract phenolic acid components. In the above steps, some active compounds are unstable and easily decompose at high temperatures. This invention uses a 75% ethanol solution instead of the conventional methanol-water extraction, which can provide a certain degree of protection for the active substances. The conventional 120-minute reflux heating process is optimized to a 60-minute water bath heating process, simplifying the extraction process and time while ensuring extraction quality, making the extraction steps more economical and concise.

[0008] Further, the extraction method specifically includes the following steps: mixing the sample with 75% ethanol solution at a material-to-liquid ratio of 1:8 to 20 (preferably 1:16), placing it at room temperature for 8 to 16 hours (preferably 12 hours), heating it in an 85°C water bath for 60 minutes, removing it and cooling it to room temperature, filtering it, and evaporating the filtrate for 1.5 to 3 hours (preferably evaporating it in a vacuum freeze dryer for 2 hours, and weighing it to confirm that the sample has been evaporated to constant weight), and storing the treated sample at low temperature for later use (preferably 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 slices, and Salvia miltiorrhiza oral liquid. It is understood 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 Salvia miltiorrhiza hairy roots are washed, cut into small pieces, and placed in centrifuge tubes; Salvia miltiorrhiza slices are cut into small pieces and placed in centrifuge tubes; Salvia miltiorrhiza oral liquid is placed directly into centrifuge tubes;

[0012] Step B, quick-freezing: Quick-freeze the fresh Salvia miltiorrhiza sample and Salvia miltiorrhiza hairy roots (preferably placed in an insulated container, add liquid nitrogen, and cover for quick-freezing); this step is omitted for Salvia miltiorrhiza slices and Salvia miltiorrhiza oral liquid.

[0013] Step C, Grinding and pulverizing: Quickly transfer the frozen fresh Salvia miltiorrhiza sample and Salvia miltiorrhiza hairy roots into a container that has been pre-cooled (preferably pre-cooled with liquid nitrogen) and grind and pulverize rapidly; grind and pulverize the Salvia miltiorrhiza slices sample; this step is omitted for Salvia miltiorrhiza oral liquid; it is understood that the above grinding and pulverizing container can be a grinding container commonly used in the art, such as a mortar and pestle;

[0014] Step D, Sample Drying: Transfer the ground and pulverized fresh Salvia miltiorrhiza sample and Salvia miltiorrhiza hairy roots into centrifuge tubes (preferably 2mL centrifuge tubes) and dry for 3 hours. Weigh and record the sample every 20 minutes in the last hour to confirm that the sample has been dried to constant weight. This step is omitted for Salvia miltiorrhiza slices and Salvia miltiorrhiza oral liquid.

[0015] A 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 using high performance liquid chromatography, comprising:

[0016] Step S1, Sample processing: The sample is processed using any of the extraction methods described in the first aspect to obtain phenolic acid components;

[0017] Step S2: The processed sample is analyzed by high performance liquid chromatography to determine the content of each phenolic acid component in the sample.

[0018] Furthermore, in the high-performance liquid chromatography (HPLC) method described in step S2, mobile phase A is a 0.08% formic acid aqueous solution, and mobile phase B is 100% methanol, using a gradient elution method; the detection wavelengths are 270 nm, 286 nm, and 330 nm. Based on the commonly used 0.1% formic acid aqueous solution and methanol, this invention reduces the concentration of formic acid aqueous solution after ensuring peak standardization, using 0.08% formic acid aqueous solution and methanol as phases A and B. This reduces the amount of formic acid used, which has an irritating odor and is corrosive, making it more economical and safer.

[0019] Furthermore, the procedure for the gradient elution method is as follows:

[0020]

[0021] In the construction of the method of this invention, under the same conditions, optimization of elution procedures using isocratic elution and elution programs with 0.08% formic acid aqueous solution and acetonitrile, 0.08% formic acid aqueous solution and methanol, and acetonitrile, all resulted in the incomplete separation of compound characteristic peaks, hindering the quantitative detection of target compounds. Optimization of the AB phase ratio and procedure in the aforementioned gradient elution methods effectively solved these problems. The above detection method optimizes the elution procedure based on the chromatographic conditions used in liquid chromatography-mass spectrometry (LC-MS), enabling efficient detection of nine tanshinone acid compounds even in the absence of mass spectrometry detection conditions.

[0022] Further, in the high-performance liquid chromatography (HPLC) method described in step S2, the sample to be tested is reconstituted with methanol solution and centrifuged. The supernatant is then filtered before detection. Preferably, the sample to be tested is reconstituted with 5 mL of methanol solution, vortexed to mix, centrifuged, and the supernatant is filtered and transferred to a brown sample vial before detection. The centrifugation conditions are preferably 4°C at 10000×g for 10 min. The methanol solution can be a 75%–100% methanol solution, such as a 75% methanol solution or a 100% methanol solution.

[0023] Further, in the high-performance liquid chromatography (HPLC) method described in step S2, the HPLC conditions are as follows: a C18 column with a particle size of 5 μm and an inner diameter of 250 × 4.6 mm, and packed with octadecylsilane-bonded silica gel particles; a column temperature of 20–35℃ (preferably 25℃); ultrasonication of the mobile phase for 5–10 min; a flow rate of 1.2 mL / min; and an injection volume of 10 μL.

[0024] Furthermore, in step S2, a calibration curve or working curve is used to quantitatively detect each phenolic acid component in the sample. It is understood that the calibration curve or working curve can be plotted using methods conventional in the art.

[0025] Furthermore, the steps for developing the calibration curve include: selecting standard solutions of each component at appropriate concentrations and preparing a series of mixed standard solutions with final concentrations of 1–100 μg / mL using 70% methanol aqueous solution; for tanshinone, rosmarinic acid, salvianolic acid A, and salvianolic acid B, additional solutions of 500 μg / mL and 1000 μg / mL are prepared respectively. The calibration curve is plotted with the signal response value of the analyte as the ordinate and the concentration of the standard as the abscissa.

[0026] Furthermore, the steps for developing the working curve include: selecting leaves, rhizomes, and hairy roots of Salvia miltiorrhiza with low content of each component, taking 1g of mixed powder using the quartering method, dissolving it in 10mL of 70% methanol solution to form a blank drug solution, measuring the background value, and then diluting the standard solution with the same volume of blank drug solution, thereby drawing the working curve with reference to the development of the calibration curve.

[0027] Furthermore, in one specific embodiment, the sample usage for testing is as follows: 4.15g of Salvia miltiorrhiza rhizome, 2.1g of Salvia miltiorrhiza leaf, 0.1g of Salvia miltiorrhiza hairy root, 2g of Salvia miltiorrhiza slices, and 5mL of Salvia miltiorrhiza oral liquid.

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

[0029] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects:

[0030] (1) The method of the present invention enables the simultaneous extraction and detection of nine tanshinone compounds, namely tanshinone, caffeic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, 4-hydroxyphenyllactic acid, 4-coumaryl-4'-hydroxyphenyllactic acid, 4-coumaryl-3',4'-dihydroxyphenyllactic acid and 3,4-coumaryl-4'-hydroxyphenyllactic acid, from the extract of the medicinal plant Salvia miltiorrhiza. It is green and low-cost.

[0031] (2) The method of the present invention optimizes the chromatographic mobile phase and elution degree, and solves the problems of peak shape not being separated, peak not returning to baseline, peak shape asymmetry, and peak shape tailing in the spectrum when using conventional high performance liquid chromatography (HPLC) for determination;

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

[0033] (4) The method of the present invention can detect the content of nine tanshinone compounds in a short time (only about 52 minutes), and is simple to operate, highly repeatable and accurate, providing a favorable guarantee for the analysis of the synthesis pathway of tanshinone components. Attached Figure Description

[0034] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings:

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

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

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

[0038] Figure 4 This is a chromatogram of the phenolic acid extract of tanshinone slices 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 This is a chromatogram of a mixed solution of nine salvianolic acid compounds as standard solutions in one embodiment of the present invention; wherein 1# is the peak shape of tanshinone, 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-coumaryl-3',4'-dihydroxyphenyllactic acid, 7# is the peak shape of 3,4-coumaryl-4'-hydroxyphenyllactic acid, 8# is the peak shape of salvianolic acid A, and 9# is the peak shape of 4-coumaryl-4'-hydroxyphenyllactic acid.

[0041] Figure 7 This is a standard curve fitting diagram of tanshinone 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] Figure 9 This is a standard curve fitting diagram of caffeic acid in one embodiment of the present invention;

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

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

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

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

[0048] Figure 14 This is a standard curve fitting diagram of tanshinone A in one embodiment of the present invention;

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

[0050] Figure 16 This is a curve fitting diagram of tanshinone in one embodiment of the present invention;

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

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

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

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

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

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

[0057] Figure 23 This is a curve fitting diagram of tanshinone A in one embodiment of the present invention;

[0058] Figure 24 This is a curve fitting diagram of 4-coumaryl-4'-hydroxyphenyllactic acid in one embodiment of the present invention. Detailed Implementation

[0059] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments without specific conditions are generally determined according to national standards. Experimental materials in the following embodiments without specified sources are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0060] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0061] In the following examples, the high-performance liquid chromatograph (HPLC) was an Agilent 1260DAD VL, which features a multi-wavelength detector capable of simultaneously detecting up to eight compounds with different characteristic wavelengths; the chromatographic column was a Diamonsil column. R 5μm C18(2); fresh Salvia miltiorrhiza rhizomes and leaves were from Salvia miltiorrhiza var. purpurea in Dali, Yunnan; Salvia miltiorrhiza hairy roots were from Salvia miltiorrhiza var. purpurea cultivated in our laboratory, with the seed source being Linyi, Shandong; Salvia miltiorrhiza slices were from Beijing Tongrentang Pharmaceutical Co., Ltd., with the place of origin being Weifang, Shandong; Salvia miltiorrhiza oral liquid was from Chongqing Fuling Pharmaceutical Factory Co., Ltd. of Taiji Group.

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

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

[0064] This embodiment describes a preferred preparation procedure for a phenolic acid extract sample of the traditional Chinese medicine Danshen and its products, specifically including the following steps:

[0065] (1) Sample pretreatment;

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

[0067] 2) Quick-freezing: Place the prepared fresh Salvia miltiorrhiza samples and Salvia miltiorrhiza hairy roots in an insulated container, add liquid nitrogen, cover and quick-freeze; this step is omitted for Salvia miltiorrhiza slices and Salvia miltiorrhiza oral liquid.

[0068] 3) Grinding and pulverizing: After freezing, quickly transfer the fresh Salvia miltiorrhiza sample and Salvia miltiorrhiza hairy roots into a mortar that has been pre-cooled with liquid nitrogen, and grind and pulverize them quickly with a pestle; similarly, transfer the dried Salvia miltiorrhiza slices sample into a mortar and grind and pulverize them; this step is omitted for Salvia miltiorrhiza oral liquid.

[0069] 4) Drying and weighing the sample: Transfer the ground and pulverized fresh Salvia miltiorrhiza sample and Salvia miltiorrhiza hairy roots into a 2mL centrifuge tube and dry them in a vacuum freeze dryer for 3 hours. In the last hour, weigh the sample every 20 minutes and record the result to confirm that the sample has been dried to constant weight. This step is omitted for Salvia miltiorrhiza slices and oral liquid.

[0070] Sampling of solid products of Salvia miltiorrhiza: Using a balance of 1 / 100,000, 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 into 15mL centrifuge tubes and freeze for later use.

[0071] Sampling of Danshen liquid products: After mixing evenly, accurately measure 5 mL of liquid and place it in a 15 mL centrifuge tube for freezing and storage.

[0072] (2) Extraction steps of tanshinone phenolic acid components;

[0073] Weigh the dried Salvia miltiorrhiza rhizomes, leaves, hairy roots, processed slices, and oral liquid samples. Add 75% ethanol solution to make the material-to-liquid ratio 1:16. After standing at room temperature for 12 hours, heat and reflux in an 85℃ water bath for 60 minutes. Remove and cool to room temperature, filter, and dry the filtrate in a vacuum freeze dryer for 2 hours. Weigh to confirm that the sample has been dried to constant weight, and store the sample in a 4℃ refrigerator for later use.

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

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

[0076] This embodiment uses high-performance liquid chromatography (HPLC) to detect the sample obtained in Example 1, specifically including the following steps:

[0077] (1) The sample to be tested was reconstituted with 5 mL of methanol solution, vortexed and mixed, and then centrifuged at 10000×g for 10 min at 4℃. The supernatant after centrifugation was filtered through a 0.22 μm organic phase microporous membrane and transferred into a brown sample bottle for the detection of 9 kinds of tanshinone acid compounds.

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

[0079] (2) The instrument testing conditions are as follows:

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

[0081] Column temperature: 25℃;

[0082] Mobile phase: Phase A: 0.08% formic acid aqueous solution, Phase B: methanol; mobile phase was sonicated 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 solution) 71 71 55 55 Phase B (methanol) 29 29 45 45

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

[0088] The detection wavelength for water-soluble components is generally 286 nm. Of the nine active compounds involved in this example, only tanshinone and rosmarinic acid are water-soluble; the other seven compounds have low solubility in water and are classified as alcohol-soluble. 4-Hydroxyphenyllactic acid was detected at a wavelength of 270 nm; tanshinone, salvianolic acid B, 4-coumaryl-3',4'-dihydroxyphenyllactic acid, 3,4-coumaryl-4'-hydroxyphenyllactic acid, 4-coumaryl-4'-hydroxyphenyllactic acid, and salvianolic acid A were detected at a wavelength of 286 nm; and caffeic acid and rosmarinic acid were detected at a wavelength of 330 nm.

[0089] When the concentration of the mixed solution of the nine compound standards was 30 μg / mL and the injection volume was 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 light, with the highest response at their respective optimal wavelengths and the corresponding lowest detection limits also decreasing. Therefore, this invention selects 270 nm, 286 nm, and 330 nm as the detection wavelengths of the extract.

[0090] Table 2 - Peak areas of each compound at different wavelengths

[0091]

[0092] (3) The chromatogram of the mixed solution of nine salvianolic acid compounds as shown in the figure. Figure 6 As shown (in the mixed injection, the concentration of each standard is 100 μg / mL, and the injection volume is 10 μL, where 1# is the peak shape of tanshinone, 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-coumaryl-3',4'-dihydroxyphenyllactic acid, 7# is the peak shape of 3,4-coumaryl-4'-hydroxyphenyllactic acid, 8# is the peak shape of salvianolic acid A, and 9# is the peak shape of 4-coumaryl-4'-hydroxyphenyllactic acid), from Figure 6 The results show that the peaks of the nine salvianolic acid compounds are well separated, the peak averages return to the baseline, and the peak shapes are symmetrical. Relevant information is shown in Table 2. They do not interfere with other coexisting compounds obtained by the extraction method used in this invention.

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

[0094]

[0095] (4) The spectra of phenolic acid extracts obtained from the samples (Salvia miltiorrhiza hairy root, Salvia miltiorrhiza leaf, Salvia miltiorrhiza rhizome, Salvia miltiorrhiza slices, Salvia miltiorrhiza oral liquid) in the above embodiments are as follows: Figures 1-5 As shown, the content of the target characteristic active ingredient in each Danshen or its product is expressed as a mass fraction and calculated according to formula (1) (the calculation result can be retained to two decimal places):

[0096]

[0097] In the formula:

[0098] ρ—The concentration of the analyte in the sample is calculated from the standard curve, in milligrams per liter (mg / L);

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

[0100] X—The measured concentration of the analyte, expressed 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 in Embodiment 1 and the detection method in Embodiment 2, specifically including:

[0104] (1) Selection of extraction solution;

[0105] In this embodiment, the extraction solution was selected. Considering the thermal stability of tanshinone compounds in ethanol, the extraction was carried out by heating and reflux of ethanol. Extraction was performed using 75% ethanol solution and 100% ethanol solution, respectively. It was found that the concentration of the target compound was higher in the 75% ethanol solution, but there were also more coexisting substances. Since the target compound has high solubility in methanol, after extraction in 75% ethanol solution, the solution was evaporated and then redissolved in 75% methanol solution and 100% methanol solution. It was found that the methanol concentration did not affect the extraction of the target compound.

[0106] Traditional processes typically use a hot reflux apparatus to react for 120 minutes for extraction. In this embodiment, during process optimization, it was found that heating in an 85°C water bath for 60 minutes can achieve the same extraction effect on tanshinone-containing phenolic acids as the traditional process.

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

[0108] Mobile phase A was compared with pure water, 1% formic acid solution and 0.08% formic acid solution. It was found that 0.08% formic acid solution was the best mobile phase for separation effect and peak shape. Mobile phase B was compared with methanol and acetonitrile. It was found that 0.08% formic acid solution and acetonitrile could significantly shorten the peak time of the target compounds. However, it was difficult to separate 4-coumaryl-3',4'-dihydroxyphenyllactic acid, 3,4-coumaryl-4'-hydroxyphenyllactic acid and salvianolic acid A. Therefore, mobile phase A was finally selected as 0.08% formic acid solution and mobile phase B was 100% methanol.

[0109] In terms of column oven temperature selection, this invention optimizes chromatographic conditions by choosing 20℃, 25℃, 30℃, and 35℃. Under the condition of 25℃ column temperature, all target compounds can elute as quickly as possible while ensuring resolution and peak shape.

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

[0111] Samples were prepared and tested using the aforementioned method, and the chromatogram is shown below. Figures 1-5As shown in Table 3, the content of each component is as follows (the content of each tanshinone acid in the table is the content of fresh tanshinone and its products by dry weight). The results show that the contents of tanshinone, 4-hydroxyphenyllactic acid, rosmarinic acid, salvianolic acid A, salvianolic acid B, and 4-coumaryl-3',4'-dihydroxyphenyllactic acid in tanshinone oral liquid are higher than those in other traditional Chinese medicine products. The content of caffeic acid is the highest in the hairy roots of tanshinone. 3,4-coumaryl-4'-hydroxyphenyllactic acid and 4-coumaryl-4'-hydroxyphenyllactic acid can only be detected in the hairy roots.

[0112] Table 3 - Content of phenolic acid compounds (mg / kg) in different Chinese herbal medicines, including Danshen and its products.

[0113]

[0114]

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

[0116] Example 4 - Applicability verification of the method for detecting the content of phenolic acid components in Danshen or its products

[0117] This embodiment uses calibration curves and working curves to quantitatively detect the content of phenolic acid components in Salvia miltiorrhiza or its products. Precision, accuracy, and stability experiments further validate the applicability of the quantitative detection method. The sample extraction and detection methods are described in Examples 1 and 2, and specifically include the following steps:

[0118] (1) Quantitative detection;

[0119] 1) Calibration Curve: Select standard solutions of appropriate concentrations for each component and prepare a series of mixed standard solutions with final concentrations of 1–100 μg / mL using 70% methanol aqueous solution. For tanshinone, rosmarinic acid, salvianolic acid A, and salvianolic acid B, additional solutions of 500 μg / mL and 1000 μg / mL are prepared. Select no fewer than 5 concentration points according to instrument performance and detection needs. Refer to the instrument operating conditions and set up an automatic sample injection program. Plot the calibration curve with the signal response value of the analyte as the ordinate and the concentration of the standard as the abscissa, and calculate the regression equation. Each concentration is measured three times. The results are shown in [Figure number missing]. Figures 7-15 The regression equation is shown below:

[0120] The regression equation for tanshinone is: y = 5.2229x - 7.3551 (R² = 0.9991);

[0121] The regression equation for 4-hydroxyphenyllactic acid is: y = 3.5963x - 7.0646 (R² = 0.9992).

[0122] The regression equation for caffeic acid is: y = 53.715x - 58.448 (R²) 2 =0.9993);

[0123] The regression equation for rosmarinic acid is: y = 24.65x - 34.051 (R²) 2 =0.9993);

[0124] The regression equation for salvianolic acid B is: y = 12.283x - 35.718 (R²) 2 =0.9992);

[0125] Regression equation for 4-coumaryl-3',4'-dihydroxyphenyllactic acid: y = 25.473x - 39.578 (R²) 2 =0.9993);

[0126] The regression equation for 3,4-coumaryl-4'-hydroxyphenyllactic acid is: y = 15.111x - 39.326 (R²) 2 =0.999);

[0127] The regression equation for salvianolic acid A is: y = 28.869x - 87.251 (R²) 2 =0.9991);

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

[0129] The above results indicate that tanshinone exhibits good linearity in the range of 5-1000 ppm, as do caffeic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, 4-hydroxyphenyllactic acid, 4-coumaryl-4'-hydroxyphenyllactic acid, 4-coumaryl-3',4'-dihydroxyphenyllactic acid, and 3,4-coumaryl-4'-hydroxyphenyllactic acid.

[0130] 2) Working Curve: In this embodiment, a working curve was also prepared. 1g of the mixed powder from *Salvia miltiorrhiza* leaves, rhizomes, and hairy roots with relatively low content of each component was taken using the quartering method and dissolved in 10mL of 70% methanol solution to form a blank solution. After measuring the background value, the standard solution was diluted with the same volume of blank solution to obtain the regression equation shown below:

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

[0132] The regression equation for 4-hydroxyphenyllactic acid is: y = 3.7001x (R² = 0.9998).

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

[0134] The regression equation for rosmarinic acid is: y = 9.6218x(R) 2 =0.9998);

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

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

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

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

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

[0140] The five samples were quantified using the calibration curve and working curve plotted above, and the results were statistically analyzed. The results showed no significant difference, indicating that the calibration curve can be used instead of the working curve, which saves the step of preparing a real blank drug solution in practical applications, making it simpler and more efficient.

[0141] (2) Precision test;

[0142] Based on literature review and preliminary experiments, 1g of mixed powder from *Salvia miltiorrhiza* leaves, rhizomes, and hairy roots with relatively low concentrations of target phenolic acids was dissolved in 10mL of 70% methanol solution using the quartering method to form blank extracts. After determining the background value, three blank extracts were taken and three standard solutions of low, medium, and high concentrations were added respectively, so that the final concentrations of each phenolic acid compound in the extracts were 10, 25, and 75 μg / mL, respectively. Each sample was measured six times simultaneously. The results of the intra-batch precision test are shown in Table 4. The relative standard deviations (RSDs) were 0.68–1.35% (tanshinone), 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 compounds were 0.66–1.58% (rosmarinic acid), 1.07–1.49% (4-coumaryl-3',4'-dihydroxyphenyllactic acid), 1.03–1.17% (3,4-coumaryl-4'-hydroxyphenyllactic acid), 0.79–1.89% (coumaryl-A), and 0.5–1.2% (4-coumaryl-4'-hydroxyphenyllactic acid), except for caffeic acid which had an RSD of 2%. The relative standard deviations of the measured values ​​of the other compounds were generally below 1.5%, indicating small sample data dispersion, high instrument precision, and good reliability of the measured values.

[0143] Table 4 - Intra-batch precision test

[0144]

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

[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 tanshinone extract to make the final concentrations of each phenolic acid compound in the sample 10, 25 and 75 μg / mL, respectively. Each concentration was measured 6 times. The experimental results are shown in Table 4 above. The recoveries of tanshinone, 4-hydroxyphenyllactic acid, caffeic acid, rosmarinic acid, salvianolic acid B, 4-coumaryl-3',4'-dihydroxyphenyllactic acid, 3,4-coumaryl-4'-hydroxyphenyllactic acid, salvianolic acid A, and 4-coumaryl-4'-hydroxyphenyllactic acid were 94-100% respectively. The national standard requires that when the concentration of the analyte is between 1 and 100 mg / L, the spiked recovery rate should be between 90 and 110%. The spiked recovery rates measured all meet the above requirements. The above determination method has high accuracy and can be used for the detection of relevant components.

[0148] (4) Stability test;

[0149] The samples spiked at a concentration of 75 μg / mL (all target compounds were 200 μg / mL) were selected for the "intra-batch precision" test. They were stored in a cool (4°C) environment, protected from light and sealed. Measurements were taken on the first day, 3 days, 7 days, and 14 days after storage, with 6 samples measured each time. The results are shown in Table 5. The sample degradation rate was ≤10% within two weeks. The samples could be stored at 4°C for at least two weeks, and the corresponding standard solutions prepared could generally be stored at -20°C for one year.

[0150] Table 5 - Results of Sample Stability Experiment

[0151]

[0152]

[0153] As shown in the above examples, testing revealed that the highest contents of salvianolic acid B and rosmarinic acid were found in the medicinal plant *Salvia miltiorrhiza*, followed by caffeic acid and tanshinone. The main active components of *Salvia miltiorrhiza* that function in the human body include salvianolic acid B, rosmarinic acid, and tanshinone. Substances such as 4-coumaryl-3',4'-dihydroxyphenyllactic acid, as precursors to these active secondary metabolites, are inactive on their own and require the action of various catalytic enzymes to generate final products before they can exert their effects in the human body. The contents of different precursor substances vary greatly, making the simultaneous detection of active compounds and their precursors crucial for the efficacy evaluation of salvianolic acid compounds.

[0154] This invention discovers that the extraction method for the nine tanshinone compounds described in Example 1 can directly and simultaneously extract these nine tanshinone compounds, laying the foundation for their simultaneous detection. This extraction method is a crucial part of the entire study, and the simultaneous extraction and detection of the nine tanshinone compounds is beneficial for the development of the traditional Chinese medicine Danshen, as well as its health products and pharmaceuticals. The high-efficiency chromatographic method constructed in this invention uses retention time for qualitative analysis and external standard-calibration curve method for quantitative analysis. It can qualitatively and quantitatively detect the content of natural phenolic acid compounds in the rhizomes and leaves of Danshen, providing a standardized method for the detection of characteristic active ingredients of phenolic acids in the medicinal plant Danshen and its products. It also provides effective component detection assurance for the development of new Danshen products, the elucidation of the synthetic pathways of tanshinone compounds, and further quality improvement of Danshen.

[0155] The specific embodiments of the present invention have been 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 to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A method for determining the content of phenolic acids in *Salvia miltiorrhiza* or its products using high-performance liquid chromatography, characterized in that, The method includes: Step S1, Sample processing: The sample is processed using the extraction method for phenolic acid components from Salvia miltiorrhiza or its products to obtain phenolic acid components; Step S2: The processed sample is analyzed by high performance liquid chromatography to determine the content of each phenolic acid component in the sample. The extraction method of phenolic acid components in Danshen or its products includes: heating the sample in a water bath at 75-90°C for 50-130 min using 75% ethanol solution to extract phenolic acid components, wherein the phenolic acid components include tanshinone, caffeic acid, salvianolic acid B, rosmarinic acid, salvianolic acid A, 4-hydroxyphenyllactic acid, 4-coumaryl-4'-hydroxyphenyllactic acid, 4-coumaryl-3',4'-dihydroxyphenyllactic acid, and 3,4-coumaryl-4'-hydroxyphenyllactic acid; The method for extracting phenolic acid components from Salvia miltiorrhiza or its products further includes a sample pretreatment step: Step A: Sampling of Salvia miltiorrhiza or its products: Fresh Salvia miltiorrhiza samples and Salvia miltiorrhiza hairy roots are washed, cut into small pieces, and placed in centrifuge tubes; Salvia miltiorrhiza slices are cut into small pieces and placed in centrifuge tubes; Salvia miltiorrhiza oral liquid is placed directly into centrifuge tubes; Step B, quick-freezing: Quick-freeze the fresh Salvia miltiorrhiza samples and Salvia miltiorrhiza hairy roots; this step is omitted for Salvia miltiorrhiza slices and Salvia miltiorrhiza oral liquid. Step C, Grinding and Powdering: Quickly transfer the frozen fresh Salvia miltiorrhiza sample and Salvia miltiorrhiza hairy roots into a pre-cooled container and grind and powder them rapidly; grind and powder the Salvia miltiorrhiza slices sample; omit this step for Salvia miltiorrhiza oral liquid. Step D, Sample Drying: Transfer the ground and pulverized fresh Salvia miltiorrhiza sample and Salvia miltiorrhiza hairy roots into centrifuge tubes and dry for 3 hours. Weigh and record the sample every 20 minutes in the last hour to confirm that the sample has been dried to constant weight. This step is omitted for Salvia miltiorrhiza slices and Salvia miltiorrhiza oral liquid. In the high performance liquid chromatography method described in step S2, mobile phase A is 0.08% formic acid aqueous solution, mobile phase B is 100% methanol, and gradient elution is used; the detection wavelengths are 270 nm, 286 nm and 330 nm. The procedure for the gradient elution method is as follows: In the high-performance liquid chromatography (HPLC) method described in step S2, the sample to be tested is reconstituted with methanol solution and centrifuged. The supernatant is then filtered before detection. The HPLC conditions are as follows: C18 column with a particle size of 5 µm and an inner diameter of 250 × 4.6 mm; octadecylsilane-bonded silica gel particles are used as packing material; column temperature is 25℃; mobile phase is sonicated for 5-10 min at a flow rate of 1.2 mL / min; and injection volume is 10 µL. In step S2, calibration curves or working curves are used to quantitatively detect each phenolic acid component in the sample.

2. The method according to claim 1, characterized in that, In the extraction method of phenolic acid components from Danshen or its products, the sample is subjected to a water bath heating treatment at 85°C for 60 min using a 75% ethanol solution to extract the phenolic acid components.

3. The method according to claim 2, characterized in that, The extraction method of phenolic acid components in Danshen or its products specifically includes the following steps: the sample is mixed with 75% ethanol solution at a material-to-liquid ratio of 1:8~20, placed at room temperature for 8~16 h, heated in an 85℃ water bath for 60 min, removed and cooled to room temperature, filtered, the filtrate is evaporated for 1.5~3 h, and the processed sample is stored at low temperature for later use.

4. The method according to claim 1, characterized in that, The samples included Salvia miltiorrhiza rhizome, Salvia miltiorrhiza hairy root, Salvia miltiorrhiza leaf, Salvia miltiorrhiza slices, and Salvia miltiorrhiza oral liquid.

5. An application of the method as described in any one of claims 1 to 4, characterized in that, The application is selected from at least one of the following: application in qualitative and quantitative analysis of phenolic acid metabolites in plants; application in the analysis of the synthetic pathway of phenolic acid components in tanshinone; application in the screening of raw material sources for traditional Chinese medicine products; application in the quality identification and improvement of the medicinal plant tanshinone; and application in the preparation of products containing tanshinone or its phenolic acid components.

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