Method for simultaneously extracting five phenolic acid components in salvia miltiorrhiza

Abstract

The invention provides a method for simultaneously extracting five phenolic acid components in salvia miltiorrhiza, and belongs to the technical field of traditional Chinese medicine extraction. According to the extraction method disclosed by the invention, five phenolic acid components including protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A in salvia miltiorrhiza are simultaneously extracted by utilizing an electrically driven membrane extraction technology, and the composition of a sample phase solution and a receptor phase solution and the process parameters of the extraction process are limited. According to the method, the five phenolic acid components in the radix salviae miltiorrhizae are extracted by utilizing an electrically driven membrane extraction technology, the phenolic acid components are migrated in a specific direction in a solvent, and selective permeation of the five phenolic acid components in the radix salviae miltiorrhizae is realized by matching with selectivity of a supported liquid membrane and controlling process parameters of an extraction process at the same time. The result of the embodiment shows that the extraction method provided by the invention can be used for simultaneously extracting five phenolic acid components including protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine extraction, and in particular to a method for selectively extracting five phenolic acid components in salvia miltiorrhiza. Background Art

[0002] The main water-soluble components in Danshen are phenolic acids, including a series of salvianolic acids such as salvianolic acid A and salvianolic acid B, as well as protocatechuic aldehyde, rosmarinic acid and danshensu. Among them, protocatechuic aldehyde, as a water-soluble component of Danshen discovered in the 1970s, has been confirmed by a large number of pharmacological studies to have clinical application value, including anti-ischemic stroke, anti-inflammatory, anti-angina, inhibition of platelet aggregation, and improvement of chondrocyte aging. Among the salvianolic acid components, salvianolic acid A and salvianolic acid B have the strongest antioxidant and free radical scavenging effects, among which salvianolic acid B has the highest content. Rosmarinic acid and caffeic acid have antioxidant, anti-inflammatory, and anti-tumor effects. Therefore, it is of certain research value to simultaneously extract multiple salvianolic acid components from the matrix of Danshen water extract.

[0003] At present, the commonly used methods for extracting phenolic acids from Salvia miltiorrhiza are solvent extraction, macroporous adsorption resin, column chromatography and liquid-liquid countercurrent distribution chromatography. For example, the prior art provides a method for extracting phenolic acids from Salvia miltiorrhiza using solvent extraction, which realizes the extraction of tanshinone ⅡA and salvianolic acid B from Salvia miltiorrhiza; it also provides a method for extracting and separating phenolic acids from Salvia miltiorrhiza using column chromatography, which realizes the extraction of salvianolic acid A from Salvia miltiorrhiza. It can be seen that although the extraction method provided by the prior art can realize the extraction of phenolic acids from Salvia miltiorrhiza, it can only extract one or two phenolic acids from Salvia miltiorrhiza, and still cannot realize the simultaneous extraction of five specific phenolic acids, namely protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A. Summary of the invention

[0004] The object of the present invention is to provide a method for simultaneously extracting five phenolic acid components in Salvia miltiorrhiza. The extraction method provided by the present invention can simultaneously extract five phenolic acid components, namely, protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A, in Salvia miltiorrhiza.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for simultaneously extracting five phenolic acid components from Salvia miltiorrhiza, and uses an electrically driven membrane extraction technology to simultaneously extract five phenolic acid components, namely, protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A from Salvia miltiorrhiza;

[0007] The sample phase solution of the electro-driven membrane extraction is a water extract of Salvia miltiorrhiza; the pH of the sample phase solution is 4 to 10;

[0008] The acceptor phase solution of the electro-driven membrane extraction is a NaOH solution with a molar concentration of 0.01 to 10 mmol / L;

[0009] The supported liquid membrane of the electro-driven membrane extraction comprises a fiber membrane and a separation solution adsorbed in the fiber membrane, wherein the separation solution is a mixed solution of an organic solvent and an additive;

[0010] The organic solvent is 1-ethyl-2-nitrobenzene, 5-ethylidene-2-norbornene, dodecanitrile, tributyl phosphate, tri(2-ethylhexyl) phosphate, trioctyl phosphate, n-hexanoic acid, n-heptanol, n-octanol, n-nonanol or n-decanol;

[0011] The additive is o-nitrophenyl octyl ether;

[0012] The mass ratio of the organic solvent to the additive in the solution is (85-99.9):(0.1-15);

[0013] The extraction voltage of the electrically driven membrane extraction is 100 to 200 V;

[0014] The extraction time of the electrically driven membrane extraction is 10 to 60 minutes.

[0015] Preferably, the organic solvent is 1-ethyl-2-nitrobenzene.

[0016] Preferably, the mass ratio of the organic solvent to the additive in the separation solution is (90-95):(5-10).

[0017] Preferably, the pH of the sample phase is 4.1-4.24.

[0018] Preferably, the molar concentration of the acceptor phase solution is 1 to 10 mmol / L.

[0019] Preferably, the extraction voltage of the electrically driven membrane extraction is 120-150V.

[0020] Preferably, the extraction time of the electrically driven membrane extraction is 20 to 40 minutes.

[0021] Preferably, the fiber membrane is a hollow fiber tube.

[0022] Preferably, the extraction method comprises the following steps:

[0023] (1) sealing one end of the hollow fiber tube and then immersing it in a separation solution to obtain a supported liquid membrane;

[0024] (2) injecting the acceptor phase solution into the tube of the supported liquid membrane in step (1), and placing the supported liquid membrane containing the acceptor phase solution into the sample phase solution, applying power for extraction, and obtaining five phenolic acid components: protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A.

[0025] Preferably, the extraction is carried out under stirring conditions; the stirring rate is 800-2000 rpm / min.

[0026] The invention provides a method for simultaneously extracting five phenolic acid components from Salvia miltiorrhiza, and utilizes an electric-driven membrane extraction technology to simultaneously extract five phenolic acid components, namely, protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A from Salvia miltiorrhiza; the sample phase solution of the electric-driven membrane extraction is a Salvia miltiorrhiza water extract; the pH value of the sample phase solution is 4-10; the acceptor phase solution of the electric-driven membrane extraction is a NaOH solution with a molar concentration of 0.01-10 mmol / L; the supporting liquid membrane of the electric-driven membrane extraction comprises a fiber membrane and a separation solution adsorbed in the fiber membrane, and the separation solution is The ion solution is a mixed solution of an organic solvent and an additive; the organic solvent is 1-ethyl-2-nitrobenzene, 5-ethylidene-2-norbornene, dodecanitrile, tributyl phosphate, tri(2-ethylhexyl) phosphate, trioctyl phosphate, n-hexanoic acid, n-heptanol, n-octanol, n-nonanol or n-decanol; the additive is o-nitrophenyl octyl ether; the mass ratio of the organic solvent to the additive in the solution is (85-99.9):(0.1-15); the extraction voltage of the electro-driven membrane extraction is 100-200V; and the extraction time of the electro-driven membrane extraction is 10-60min. The extraction method provided by the present invention utilizes the electro-driven membrane extraction technology to extract five phenolic acid components in Salvia miltiorrhiza, realizes the migration of phenolic acid components in a specific direction in the solvent, and realizes the adjustment of the hydrophilicity balance of the supporting membrane liquid solvent by limiting the composition of the supporting liquid membrane solvent, so that the hydrophilicity of the supporting liquid membrane solvent matches protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A, and realizes the selective permeation of the five phenolic acid components; by limiting the voltage and time of the extraction process, while ensuring the selective permeation of the five phenolic acid components, other phenolic acid substances are prevented from being extracted, and the five phenolic acid components in Salvia miltiorrhiza are extracted simultaneously. The results of the embodiment show that the extraction method provided by the present invention can realize the simultaneous extraction of five phenolic acid components of protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A in Salvia miltiorrhiza. DETAILED DESCRIPTION

[0027] The present invention provides a method for simultaneously extracting five phenolic acid components in Salvia miltiorrhiza, and utilizes an electrically driven membrane extraction technology to simultaneously extract five phenolic acid components, namely, protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A in Salvia miltiorrhiza.

[0028] In the present invention, the sample phase solution of the electro-driven membrane extraction is a water extract of Salvia miltiorrhiza; the pH of the sample phase solution is 4 to 10, preferably 4 to 5.2, and more preferably 4.1 to 4.24. In an embodiment of the present invention, the pH of the sample phase solution may specifically be 4, 4.05, 4.1, 4.2, 4.24, 5, 5.5, 8 or 10. In the present invention, by limiting the pH of the sample phase solution in the electro-driven final extraction, the stability of the overall extraction environment is ensured, the influence of the pH value on the dissociation of Salvia miltiorrhiza is avoided, the mass transfer effect is improved, and the extraction and separation of the five phenolic acid components are improved.

[0029] As an embodiment of the present invention, the pH of the Salvia miltiorrhiza aqueous extract can be adjusted using a NaOH solution with a molar concentration of 1 mmol / L.

[0030] As an embodiment of the present invention, the method for preparing the Salvia miltiorrhiza aqueous extract comprises the following steps:

[0031] Performing a first reflux extraction on the powder of the medicinal material of Danshen in ultrapure water and then filtering the extract to obtain a first filtrate and a filter residue;

[0032] The filter residue is subjected to a second reflux extraction in ultrapure water and then filtered to obtain a second filtrate;

[0033] The first filtrate and the second filtrate are combined and diluted to obtain a Salvia miltiorrhiza aqueous extract.

[0034] The invention performs a first reflux extraction on salvia miltiorrhiza powder in ultrapure water and then filters the powder to obtain a first filtrate and a filter residue.

[0035] As an embodiment of the present invention, the particle size of the Salvia miltiorrhiza medicinal material powder can be less than 1.16 mm, or can be less than 1 mm.

[0036] As an embodiment of the present invention, the mass volume ratio of the salvia miltiorrhiza powder to ultrapure water can be 1g: (6-10) mL, 1g: (6-8) mL, or 1g: 6 mL. In the present invention, by limiting the dosage ratio of the salvia miltiorrhiza powder to ultrapure water, it is ensured that the salvia miltiorrhiza powder can be fully immersed, which is conducive to the complete extraction of phenolic acid components in the salvia miltiorrhiza powder.

[0037] As an embodiment of the present invention, the temperature of the first reflux can be 98-100°C, or 98-99°C; the time of the first reflux can be 4-5h, or 4-4.5h. In the present invention, by limiting the process parameters of the first reflux operation process, the complete extraction of phenolic acid components in Salvia miltiorrhiza can be further achieved.

[0038] The present invention has no special limitation on the filtration, and the filtration operation commonly used by those skilled in the art can be adopted.

[0039] After obtaining the first filtrate and filter residue, the present invention performs a second reflux extraction on the filter residue in ultrapure water and then filters it to obtain a second filtrate.

[0040] As an embodiment of the present invention, the mass volume ratio of the Danshen medicinal material powder and ultrapure water can be 1g: (3-5) mL, 1g: (3-4) mL, or 1g: 3 mL. In the present invention, by limiting the amount of ultrapure water, sufficient extraction of the remaining phenolic acid substances in the filter residue is achieved.

[0041] As an embodiment of the present invention, the temperature of the second reflux can be 98-100°C, or 98-99°C; the time of the second reflux can be 2-3h, or 2-2.5h. In the present invention, by limiting the process parameters of the second reflux operation process, the complete extraction of phenolic acid components in the filter residue is further achieved.

[0042] The present invention has no special limitation on the filtration, and the filtration operation commonly used by those skilled in the art may be adopted.

[0043] After obtaining the second filtrate, the present invention combines the first filtrate and the second filtrate and then dilutes them to obtain the salvia miltiorrhiza water extract.

[0044] As an embodiment of the present invention, after the merging, a third filtration and centrifugation may be performed in sequence to obtain a supernatant.

[0045] As an embodiment of the present invention, the third filtration may be performed using gauze.

[0046] As an embodiment of the present invention, the centrifugal speed can be 1000-1200 rpm / min, or 1000-1100 rpm / min; the centrifugal time can be 10-15 min, or 10-12 min. In the present invention, by limiting the process parameters of the centrifugal operation, the solid impurities in the third filtrate are fully removed.

[0047] The present invention has no special limitation on the dilution operation, as long as the concentration of phenolic acid components in the obtained Salvia miltiorrhiza water extract meets the use requirements.

[0048] In the present invention, the acceptor phase solution of the electro-driven membrane extraction is a NaOH solution; the molar concentration of the NaOH solution is 0.01 to 10 mmol / L, preferably 0.1 to 10 mmol / L, and more preferably 1 to 10 mmol / L. In an embodiment of the present invention, the molar concentration of the NaOH solution may specifically be 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 mmol / L. In the present invention, by limiting the molar concentration of the acceptor phase solution, an effective electro-driven effect can be ensured, which is beneficial for the migration of the five phenolic acid substances toward the acceptor phase solution, and effectively improves the extraction and separation effect of the five phenolic acid components.

[0049] As an embodiment of the present invention, the solvent of the NaOH solution may be methanol. In the present invention, by limiting the type of solvent in the acceptor phase solution, the phenolic acid components are effectively dissolved, so that the phenolic acid components extracted into the acceptor phase solution can exist stably.

[0050] In the present invention, the supported liquid membrane of the electro-driven membrane extraction includes a fiber membrane and a separation solution adsorbed in the fiber membrane; the fiber membrane is preferably a hollow fiber tube; the separation solution is a mixed solution of an organic solvent and an additive; the organic solvent is 1-ethyl-2-nitrobenzene, 5-ethylidene-2-norbornene, dodecanitrile, tributyl phosphate, tri(2-ethylhexyl) phosphate, trioctyl phosphate, n-hexanoic acid, n-heptanol, n-octanol, n-nonanol or n-decanol, preferably 1-ethyl-2-nitrobenzene; the additive is o-nitrophenyl octyl ether. In the present invention, by limiting the composition of the supported liquid membrane, the extraction selectivity is effectively improved, the migration of five phenolic acid components, namely protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A, is ensured, and the mass transfer resistance of the above five phenolic acid components is reduced to avoid the extraction of other phenolic acid components, thereby achieving the simultaneous extraction of five phenolic acid components.

[0051] In the present invention, the mass ratio of the organic solvent to the additive in the separation solution is (85-99.9):(0.1-15), preferably (88-99):(1-12), and more preferably (90-95):(5-10). In the present invention, by limiting the mass ratio of the organic solvent to the additive in the separation solution, the affinity of the support liquid membrane to different phenolic acid components is effectively improved, and the selective separation of five phenolic acid components is achieved; while controlling the ratio of the two can improve the pore structure and chemical environment of the support liquid membrane, achieve the screening of multiple phenolic acid components, improve the extraction of five phenolic acid components, and avoid the extraction of other phenolic acid components.

[0052] In the present invention, the extraction voltage of the electro-driven membrane extraction is 100-200 V, preferably 110-180 V, and more preferably 120-150 V. In an embodiment of the present invention, the extraction voltage of the electro-driven membrane extraction may be specifically 100, 105, 110, 115, 120, 130, 150, 160, 180, 190 or 200 V. In the present invention, by limiting the extraction voltage, the driving force of solute migration is controlled to ensure that the phenolic acid components can overcome the mass transfer resistance and complete the migration of the sample phase solution to the receptor phase solution.

[0053] In the present invention, the extraction time of the electro-driven membrane extraction is 10 to 60 minutes, preferably 15 to 50 minutes, and more preferably 20 to 40 minutes. In an embodiment of the present invention, the extraction time of the electro-driven membrane extraction may be specifically 10, 12, 15, 18, 20, 30, 40, 45, 50, 55 or 60 minutes. In the present invention, by limiting the extraction time, the extraction of five phenolic acid components is guaranteed, avoiding the inability to extract five phenolic acid components simultaneously due to too short a time, and avoiding the extraction of other phenolic acid components due to too long a time.

[0054] As an embodiment of the present invention, the method for simultaneously extracting five phenolic acid components from Salvia miltiorrhiza comprises the following steps:

[0055] (1) sealing one end of the hollow fiber tube and then immersing it in a separation solution to obtain a supported liquid membrane;

[0056] (2) injecting the acceptor phase solution into the tube of the supported liquid membrane in step (1), and placing the supported liquid membrane containing the acceptor phase solution into the sample phase solution, applying power for extraction, and obtaining five phenolic acid components: protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A.

[0057] The invention seals one end of the fiber tube and then soaks it in a separation solution to obtain a supported liquid membrane.

[0058] As an embodiment of the present invention, the fiber tube can be sealed by tying one end of the fiber tube with a rope to ensure that the receptor phase solution does not leak out.

[0059] The present invention has no special limitation on the size of the hollow fiber tube, which can be set as required.

[0060] As an embodiment of the present invention, the immersion time can be 10 to 15 seconds, or 10 to 12 seconds. In the present invention, by limiting the immersion time of the fiber tube in the separation solution, the separation solution is completely infiltrated in the pore structure of the fiber tube, further improving the separation and extraction effect of the supported liquid membrane.

[0061] After the supported liquid membrane is obtained, the present invention injects the acceptor phase solution into the tube body of the supported liquid membrane, and puts the supported liquid membrane containing the acceptor phase solution into the sample phase solution, and performs extraction by powering on to obtain five phenolic acid components, namely protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A.

[0062] As an embodiment of the present invention, the extraction can stir the sample phase solution; the stirring rate can be 800-2000rpm / min, or 800-1500rpm / min. In the present invention, by limiting the stirring rate, the diffusion of phenolic acid substances to the acceptor phase solution is promoted, while avoiding excessive bubbles generated by excessive stirring. Excessive bubbles gather on the surface of the support liquid membrane and may affect the permeation of phenolic acid substances.

[0063] After the extraction is completed, the present invention rinses the receptor phase solution from the support liquid membrane and rinses the support liquid membrane with ultrapure water before combining it with the receptor phase solution to obtain five phenolic acid components: protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A.

[0064] As an embodiment of the present invention, the number of rinsing is not particularly limited, as long as the phenolic acid components obtained by extraction can be fully transferred to avoid waste of the extraction product.

[0065] The present invention provides a method for simultaneously extracting five phenolic acid components in Salvia miltiorrhiza. The phenolic acid components in Salvia miltiorrhiza are extracted by adopting an electric-driven membrane extraction technology, and the acceptor phase solution, the sample phase solution and the extraction process parameters of the electric-driven membrane extraction are limited, so as to promote the smooth migration of the five phenolic acid components in Salvia miltiorrhiza, and realize the simultaneous extraction of the five phenolic acid components of protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A in Salvia miltiorrhiza.

[0066] In order to further illustrate the present invention, the method for simultaneously extracting five phenolic acid components from Salvia miltiorrhiza provided by the present invention is described in detail below in conjunction with examples, but they should not be construed as limiting the scope of protection of the present invention.

[0067] Example 1

[0068] A method for simultaneously extracting five phenolic acid components from Salvia miltiorrhiza, using an electrically driven membrane extraction technology to extract five phenolic acid components, namely, protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A from Salvia miltiorrhiza; the method comprises the following steps:

[0069] (1) sealing one end of a fiber tube with a length of 5 cm, and then immersing the fiber tube in a separation solution composed of 1-ethyl-2-nitrobenzene and o-nitrobenzene octyl ether for 10 seconds to obtain a supported liquid membrane; wherein the mass ratio of 1-ethyl-2-nitrobenzene to o-nitrobenzene octyl ether is 95:5;

[0070] (2) injecting 15 μL of 10 mmol / L NaOH solution into the tube of the supported liquid membrane in step (1), adding 1.5 mL of Salvia miltiorrhiza aqueous extract with a pH of 4.24 into an injection vial, wherein the cap of the injection vial has two small holes, inserting two 1 cm hoses to fix a platinum wire electrode with a diameter of 0.3 mm, placing the supported liquid membrane containing the NaOH solution into the injection vial containing the Salvia miltiorrhiza aqueous extract, and placing the anode into the acceptor phase solution, placing the anode into the Salvia miltiorrhiza aqueous solution, applying power at 100 V and 800 rpm / min for extraction for 20 min, and after the extraction is completed, flushing the acceptor phase solution NaOH solution in the supported liquid membrane with a syringe, rinsing with ultrapure water for 5 times, and then combining with the NaOH solution to obtain five phenolic acid components: protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A;

[0071] The preparation method of the salvia miltiorrhiza aqueous extract with a pH of 4.24 is as follows:

[0072] 100 g of salvia miltiorrhiza powder with a particle size of 1 mm was added to 600 mL of ultrapure water, and the mixture was filtered after a first reflux at 98° C. for 4 h to obtain a first filtrate and a filter residue; wherein the mass volume ratio of the salvia miltiorrhiza powder to the ultrapure water was 1 g:6 mL;

[0073] The filter residue was added into 300 mL of ultrapure water, and the mixture was refluxed for 2 h at 98° C., and then filtered to obtain a second filtrate; wherein the mass volume ratio of the Danshen medicinal material powder to the ultrapure water was 1 g:3 mL;

[0074] After combining the first filtrate and the second filtrate, the mixture was filtered for the third time with gauze, and the filtrate was centrifuged at 1000 rpm / min for 10 min. The supernatant was diluted with ultrapure water (the mass volume ratio of Salvia miltiorrhiza powder to ultrapure water was 1 g:30 mL), and the pH of the Salvia miltiorrhiza aqueous extract was adjusted with 1 mmol / L NaOH solution to obtain a Salvia miltiorrhiza aqueous extract with a pH of 4.24.

[0075] Example 2

[0076] The difference between Example 2 and Example 1 is that the separation solution supporting the liquid membrane is a single 1-ethyl-2-nitrobenzene, and the rest is the same as Example 1.

[0077] Example 3

[0078] The difference between Example 3 and Example 1 is that the mass ratio of 1-ethyl-2-nitrobenzene to o-nitrophenyl octyl ether in the separation solution is 90:10, and the rest is the same as Example 1.

[0079] Example 4

[0080] The difference between Example 4 and Example 1 is that the pH value of the sample phase solution, the water extract of Salvia miltiorrhiza, is 4.16, and the rest is the same as Example 1.

[0081] Example 5

[0082] The difference between Example 5 and Example 1 is that the acceptor phase solution is a NaOH solution with a molar concentration of 1 mmol / L, and the rest is the same as Example 1.

[0083] Example 6

[0084] The difference between Example 6 and Example 1 is that the extraction voltage is 150V, and the rest is the same as Example 1.

[0085] Test Example 1

[0086] The recovery rates of protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A extracted by the extraction method of Example 1 were tested;

[0087] The volume of the acceptor phase solution flushed out from the support liquid membrane in Example 1 was adjusted to the same volume as the water extract of Salvia miltiorrhiza, and the NaOH solution and the water extract of Salvia miltiorrhiza after the extraction were detected by high performance liquid chromatography, and the recovery rate was calculated;

[0088] The conditions of the HPLC method are:

[0089] Chromatographic column: Agilent ZORBAX Eclipse Plus C 18 (4.6×150mm, 3.5μm);

[0090] UV detection wavelength: 280nm; column temperature: 30℃; injection volume: 20μL;

[0091] Mobile phase: A: 0.1% formic acid, B: methanol;

[0092] The gradient conditions of the mobile phase in the HPLC detection are shown in Table 1;

[0093] Table 1 Gradient conditions of mobile phase in HPLC detection

[0094]

[0095]

[0096] Weigh protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid respectively, use methanol as a solvent to prepare a standard solution with a concentration of 1 mg / mL, and dilute it to the required concentration with ultrapure water;

[0097] Establish a standard curve of protocatechuic aldehyde: select seven concentration points (0.01, 0.05, 1, 5, 10, 50, 100 μg / mL) in the range of 0.01-100 μg / mL, test each concentration point three times, perform linear fitting based on the concentration and peak area of ​​protocatechuic aldehyde, and calculate the regression line;

[0098] Establish a caffeic acid standard curve: select five concentration points (0.01, 1, 10, 50, 100 μg / mL) in the range of 0.01-100 μg / mL, test each concentration point three times, perform linear fitting based on the concentration and peak area of ​​caffeic acid, and calculate the regression line;

[0099] Establish a standard curve of salvianolic acid B: select seven concentration points (0.01, 0.05, 0.1, 1, 5, 10, 50 μg / mL) in the range of 0.01-50 μg / mL, test each concentration point three times, perform linear fitting based on the concentration and peak area of ​​salvianolic acid B, and calculate the regression line;

[0100] Establish a standard curve of rosmarinic acid: select seven concentration points (0.01, 0.05, 0.1, 1, 5, 10, 50 μg / mL) in the range of 0.01-50 μg / mL, test each concentration point three times, perform linear fitting based on the concentration and peak area of ​​rosmarinic acid, and calculate the regression line;

[0101] Establish a standard curve of salvianolic acid A: select five concentration points (0.1, 1, 5, 10, 50 μg / mL) in the range of 0.1-50 μg / mL, test each concentration point three times, perform linear fitting based on the concentration and peak area of ​​salvianolic acid A, and calculate the regression line;

[0102] Precision investigation: The intra-day stability and inter-day stability deviation values ​​at the same concentration level were evaluated, expressed as relative standard deviation (RSD%); the intra-day stability was evaluated based on the results of HPLC by performing 6 sets of electro-driven membrane extraction tests on the same day; under the same extraction conditions, two sets of electro-driven membrane extraction were performed every day for three consecutive days, and the obtained data were used to determine the inter-day stability;

[0103] The results of the methodological investigation of applying the electro-driven membrane extraction technology to the extraction of five phenolic acid components from Salvia miltiorrhiza are shown in Table 2 ;

[0104] Table 2 Methodological investigation results of applying electro-driven membrane extraction technology to the extraction of five phenolic acid components from Salvia miltiorrhiza

[0105]

[0106]

[0107] As can be seen from Table 2, the extraction method provided by the present invention has high precision and stability, can effectively improve the detection accuracy, and the extraction method has a low detection limit, and can effectively achieve the simultaneous extraction of protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A.

[0108] The recovery rate is calculated as follows:

[0109] Protocatechuic aldehyde recovery rate = (protocatechuic aldehyde concentration in NaOH solution after extraction / original protocatechuic aldehyde concentration in Salvia miltiorrhiza aqueous extract) × 100%;

[0110] The recoveries of caffeic acid, salvianolic acid B, rosmarinic acid, and salvianolic acid A were calculated in the same way as the protocatechuic aldehyde recovery;

[0111] The recoveries of protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A extracted by the method of Example 1 are shown in Table 3.

[0112] Test cases 2 to 6

[0113] The difference between Test Examples 2 to 6 and Test Example 1 is that the test objects are protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A extracted by the extraction methods of Examples 2 to 6, respectively, and the rest is the same as Test Example 1. The results are shown in Table 3;

[0114] Table 3 Recovery rates of phenolic acid components obtained by the extraction methods of Examples 1 to 6

[0115] Example Protocatechuic aldehyde Caffeic acid Salvianolic acid B Rosmarinic acid Salvianolic acid A Example 1 48.71％ 44.62％ 22.88％ 29.32％ 6.01％ Example 2 21.94％ 3.07％ 0.325％ 2.81％ 5.93％ Example 3 37.66％ 39.52％ 17.51％ 21.65％ 3.94％ Example 4 44.32％ 36.48％ 22.21％ 25.51％ 3.94％ Example 5 33.08％ 36.54％ 21.06％ 26.54％ 4.38％ Example 6 28.91％ 34.89％ 15.03％ 20.15％ 2.23％

[0116] It can be seen from Table 3 that the extraction method provided by the present invention can achieve the simultaneous extraction of five phenolic acid components, namely protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A, from Salvia miltiorrhiza.

[0117] Comparative Examples 1 to 8

[0118] The only difference between Comparative Examples 1 to 8 and Example 1 is that the organic solvents in the separation solution in the supported liquid membrane of the electro-driven membrane extraction are ethyl ether, o-nitrophenyl octyl ether, triethyl phosphate, n-dodecane, n-octanoic acid, n-decanoic acid, 4-nitroisopropylbenzene and undecyl alcohol, respectively, and the rest are the same as Example 1.

[0119] Comparative Example 9

[0120] The difference between Comparative Example 9 and Example 1 is that the pH value of the sample phase solution, the water extract of Salvia miltiorrhiza, is 10.5, and the rest is the same as Example 1.

[0121] Comparative Example 10

[0122] The difference between Comparative Example 10 and Example 1 is that the concentration of the acceptor phase solution NaOH solution is 100 mmol / L, and the rest is the same as Example 1.

[0123] Comparative Example 11

[0124] The difference between Comparative Example 11 and Example 1 is that the extraction voltage is 50 V, and the rest is the same as Example 1.

[0125] Comparative Example 12

[0126] The difference between Comparative Example 11 and Example 1 is that the extraction time is 5 minutes, and the rest is the same as Example 1.

[0127] Comparative test examples 1 to 12

[0128] The operation steps of comparative test examples 1 to 12 are the same as those of test example 1;

[0129] The extraction method provided in Comparative Example 1 can only extract protocatechuic aldehyde;

[0130] The extraction method provided in Comparative Example 2 can only extract protocatechuic aldehyde, caffeic acid, salvianolic acid B and salvianolic acid A;

[0131] The extraction method provided in Comparative Example 3 can only extract protocatechuic aldehyde, caffeic acid, salvianolic acid B and rosmarinic acid;

[0132] The extraction methods provided in Comparative Examples 4 and 6 did not extract phenolic acid components from Salvia miltiorrhiza;

[0133] The extraction methods provided in Comparative Examples 5 and 8 can only extract protocatechuic aldehyde, caffeic acid, rosmarinic acid and salvianolic acid A;

[0134] The extraction method provided in Comparative Example 7 can only extract protocatechuic aldehyde, caffeic acid and rosmarinic acid;

[0135] The extraction method provided in Comparative Example 9 can only extract protocatechuic aldehyde, caffeic acid, salvianolic acid B and rosmarinic acid;

[0136] The extraction method provided in Comparative Example 10 can only extract protocatechuic aldehyde, caffeic acid, salvianolic acid B and salvianolic acid A;

[0137] The extraction method provided in Comparative Example 11 can only extract protocatechuic aldehyde and caffeic acid;

[0138] The extraction method provided in Comparative Example 12 can only extract protocatechuic aldehyde, caffeic acid, salvianolic acid B and rosmarinic acid.

[0139] In summary, the extraction method provided by the present invention can achieve the simultaneous extraction of protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A in Salvia miltiorrhiza. Changing the process parameters of the extraction method will result in the inability to simultaneously extract the five phenolic acid components.

[0140] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously extracting five phenolic acid components from Salvia miltiorrhiza, using an electrically driven membrane extraction technique to simultaneously extract five phenolic acid components, namely, protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A from Salvia miltiorrhiza; The sample phase solution of the electro-driven membrane extraction is a water extract of Salvia miltiorrhiza; the pH of the sample phase solution is 4 to 10; The acceptor phase solution of the electro-driven membrane extraction is a NaOH solution with a molar concentration of 0.01 to 10 mmol / L; The supported liquid membrane of the electro-driven membrane extraction comprises a fiber membrane and a separation solution adsorbed in the fiber membrane, wherein the separation solution is a mixed solution of an organic solvent and an additive; The organic solvent is 1-ethyl-2-nitrobenzene, 5-ethylidene-2-norbornene, dodecanitrile, tributyl phosphate, tri(2-ethylhexyl) phosphate, trioctyl phosphate, n-hexanoic acid, n-heptanol, n-octanol, n-nonanol or n-decanol; The additive is o-nitrophenyl octyl ether; The mass ratio of the organic solvent to the additive in the separation solution is (85-99.9):(0.1-15); The extraction voltage of the electrically driven membrane extraction is 100 to 200 V; The extraction time of the electrically driven membrane extraction is 10 to 60 minutes.

2. The method according to claim 1, characterized in that: The organic solvent is 1-ethyl-2-nitrobenzene.

3. The method according to claim 1 or 2, characterized in that: The mass ratio of the organic solvent to the additive in the separation solution is (90-95):(5-10).

4. The method according to claim 1, characterized in that: The pH of the sample phase is 4.1-4.

24.

5. The method according to claim 1, characterized in that The molar concentration of the acceptor phase solution is 1 to 10 mmol / L.

6. The method according to claim 1, characterized in that The extraction voltage of the electrically driven membrane extraction is 120-150V.

7. The method according to claim 1, characterized in that The extraction time of the electrically driven membrane extraction is 20 to 40 minutes.

8. The method according to claim 1, characterized in that The fiber membrane is a hollow fiber tube.

9. The method according to claim 8, characterized in that The following steps are involved: (1) sealing one end of the hollow fiber tube and then immersing it in a separation solution to obtain a supported liquid membrane; (2) injecting the acceptor phase solution into the tube of the supported liquid membrane in step (1), and placing the supported liquid membrane containing the acceptor phase solution into the sample phase solution, applying power for extraction, and obtaining five phenolic acid components: protocatechuic aldehyde, caffeic acid, salvianolic acid B, rosmarinic acid and salvianolic acid A.

10. The method according to claim 9, characterized in that The extraction is carried out under stirring conditions; the stirring rate is 800-2000 rpm / min.

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