Industrial rosmarinic acid extraction method based on ragwort and medical use of rosmarinic acid

Abstract

The invention discloses a method for extracting rosmarinic acid from ragwort fruit, which is characterized in that the method comprises the following steps: extraction: ragwort fruit is crushed and sieved, and acid water is added for ultrasonic extraction; extraction: the extract is dispersed in warm water and then extracted with an organic solvent; enrichment: the extract is analyzed in a macroporous resin chromatography column with ethanol of appropriate concentration, and the analyzed solution is collected. Crystallization: the analyzed solution is concentrated to one tenth of the volume under reduced pressure, cooled and allowed to stand, and the precipitate is collected. Purification: the precipitate is dissolved and filtered with acid water again, and the filtrate is repeatedly extracted and crystallized to obtain rosmarinic acid crystals; drying: the crystals are dried to obtain pure rosmarinic acid. The method has a simple process flow, low production cost, and the organic solvent can be recycled, and the obtained product has high purity and good quality.

Description

Technical Field

[0001] The invention relates to the field of traditional Chinese medicine extraction, and in particular to an industrial extraction method of rosmarinic acid. Background Art

[0002] Rosmarinic acid (RosA) is a natural phenolic acid active substance with a structure as shown in Formula 1 below. It is named after it was first isolated from rosemary and has been approved by the U.S. Food and Drug Administration (FDA) as a "public safety food". It has significant pharmacological effects in anti-inflammation, free radical scavenging, anti-thrombotic, anti-platelet aggregation, immunomodulatory and antibacterial, anti-depressant, and inhibiting the proliferation of glomerular mesangial cells.

[0003]

[0004] Providing a large amount of high-purity rosmarinic acid is of great significance for its drug development. However, the currently known industrial extraction methods of high-purity rosmarinic acid are cumbersome, with high production cost, low purity, and the limitations of being difficult to realize industrialization, which are not satisfactory. For example, the method disclosed in Chinese patent CN107382729A, the process is based on Labiatae plants such as rosemary as raw materials, and due to the influence of other impurity components, an ultrafiltration membrane needs to be used, which greatly limits the flux of the method and is not suitable for large-scale industrial production; the method disclosed in Chinese patent CN107032994 needs to use an expensive Sephadex LH-20 chromatographic column, so its method is also not suitable for large-scale industrial production; the extraction method disclosed in Chinese patent CN10656478, based on Perilla as raw material, and needs to use biological enzymes, and this method cannot provide high-purity rosmarinic acid.

[0005] The fruit of Cordia dichotoma Forst.f is the dried mature fruit of the Boraginaceae plant. This medicinal material mainly relies on imports and has a long history of application in Uyghur medicine. Uyghur medicine believes that the fruit of Cordia dichotoma Forst.f has the effect of generating dampness and heat, ripening abnormal black bile, moistening the lungs and throat, relieving cough and reducing phlegm, clearing voice and quenching thirst, and promoting bowel movements and urination. It is used for dry cold or black bile diseases, dry throat and itchy throat, uncomfortable cough, loss of voice and thirst, urinary difficulties, and constipation. Chemical composition studies have shown that the plant is rich in phenolic acids, flavonoids, steroids and alkaloids, among which phenolic acid compounds are the basis of the pharmacological activity of the plant, and its main component is the homologue of rosmarinic acid, which may become a resource plant for rosmarinic acid. However, there is no method for separating and preparing rosmarinic acid monomers from Cordia dichotoma Forst.f and reports on the application of rosmarinic acid in anti-diabetic drugs. Summary of the invention

[0006] During the development of resource plants in Xinjiang, the inventors accidentally discovered that the fruit of the ragwort tree can serve as a good source of rosmarinic acid. At the same time, due to its special ingredients, a large amount of high-purity rosmarinic acid can be obtained in a relatively simple way.

[0007] The present application provides a method for preparing rosmarinic acid suitable for industrial production. The preparation method has a simple process flow, low production cost, and recyclable organic solvents. The prepared product has high purity and good quality, and is particularly suitable for industrial production.

[0008] Specifically, the present invention provides an industrial extraction method of rosmarinic acid, characterized in that: the fruit of Cordia dichotoma Forst.f is used as a raw material, and the method comprises the following steps:

[0009] Extraction process: crush the dried fruit raw material of the ragwort, place it in an acidic aqueous solution, the ratio of the raw material to the acidic aqueous solution is 1:20-30 by weight, and filter to obtain an extract;

[0010] Organic solvent extraction step: concentrating the extract, adding an equal volume of organic solvent for extraction, and concentrating under reduced pressure to obtain an extract;

[0011] Macroporous adsorption resin enrichment process: the extract is loaded with macroporous adsorption resin, impurities are washed with pure water, and then desorbed with 40-60% (volume) ethanol aqueous solution, and the eluent is collected. The macroporous adsorption resin is a styrene or divinylbenzene type non-polar macroporous adsorption resin with a particle size of 155-165 μm and a surface area of ​​500-600 m 2 / g;

[0012] Supersaturation process: combine the eluates, concentrate under reduced pressure to 10% to 13% volume, cool to 4°C to 10°C, and filter to obtain crude rosmarinic acid;

[0013] Purification process: add water to dissolve the crude rosmarinic acid and filter it. The weight ratio of crude rosmarinic acid to water is 1:10-20. Adjust the pH value of the solution to 2-3 and let it stand for 5-10 minutes. Extract it with an equal volume of ethyl acetate. Concentrate the extract under reduced pressure, cool it, and crystallize it to obtain pure rosmarinic acid.

[0014] In a preferred embodiment of the present invention, the pH value of the acidic aqueous solution in the extraction step should be 3-4.

[0015] In a preferred embodiment of the present invention, the extraction process temperature is controlled below 60° C., ultrasonic-assisted extraction is performed, the power is 100-140 W, and the extraction time is 60 to 100 min.

[0016] In a preferred embodiment of the present invention, the organic solvent in the organic solvent extraction step is n-butanol, and the extraction time is 30 minutes.

[0017] In a preferred embodiment of the present invention, the macroporous adsorption resin is styrene or divinylbenzene type non-polar macroporous adsorption resin is HP20.

[0018] In a preferred embodiment of the present invention, in the supersaturation process, the temperature is 50-55° C. and the vacuum degree is -0.07 MPa to -0.09 MPa.

[0019] Compared with the prior art, the present application is described in detail as follows:

[0020] 1. Compared with the traditional preparation method, this method is simple to operate, easy to recycle, safe and non-toxic, high in purity and quality, and effectively reduces the separation cost.

[0021] 2. The fruit of the brand-new rosemary source plant, Rauwolfia rapa, has been developed. The fruit is rich in resources in Xinjiang. Although it has been reported that rosmarinic acid exists in Rauwolfia rapa, it is rare that the components in the plant that are difficult to separate from rosmarinic acid (rosmarinic acid methyl ester / ethyl ester exists in the plant, and other polyphenol components are less) can be efficiently removed by the method of the present invention. It is particularly suitable that the components similar to rosmarinic acid in the contained ingredients can be removed by the method of the present invention, or the components that are difficult to separate are less, making it possible to obtain a large amount of high-purity rosmarinic acid at low cost.

[0022] 3. Experimental verification shows that the purity of the rosmarinic acid obtained by the preparation method provided by the present invention can reach HPLC>96.0% or more. Furthermore, the preparation method of the present invention also includes: nuclear magnetic resonance spectroscopy and high-performance liquid chromatography to detect the obtained rosmarinic acid product, effectively ensuring the stability and controllability of the product quality.

[0023] 4. Since high-purity rosmarinic acid raw materials are guaranteed, the inventors tested the pharmacological activity of rosmarinic acid in anti-diabetes. Cell experiments showed that rosmarinic acid has the pharmacological activity of promoting cellular uptake of glucose. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the HPLC analysis results of rosmarinic acid obtained in a comparative example of the present invention;

[0025] Figure 2 This is a graph showing the results of HPLC analysis of rosmarinic acid prepared by the method of the present invention;

[0026] Figure 3 Rosmarinic acid 1 H-NMR spectrum;

[0027] Figure 4 Rosmarinic acid13 C-NMR spectrum

[0028] Figure 5 This is a graph showing the results of rosmarinic acid glucose uptake activity. DETAILED DESCRIPTION

[0029] The various elements of the invention are discussed in more detail below.

[0030] The present invention provides an industrial extraction method of rosmarinic acid, which is characterized in that: the fruit of Cordia dichotoma Forst.f is used as a raw material, and the method comprises the following steps:

[0031] Extraction process: crush the dried fruit raw material of the ragwort, place it in an acidic aqueous solution, the ratio of the raw material to the acidic aqueous solution is 1:20-30 by weight, and filter to obtain an extract;

[0032] Organic solvent extraction step: concentrating the extract, adding an equal volume of organic solvent for extraction, and concentrating under reduced pressure to obtain an extract;

[0033] Macroporous adsorption resin enrichment process: the extract is loaded with macroporous adsorption resin, impurities are washed with pure water, and then desorbed with 40-60% (volume) ethanol aqueous solution, and the eluent is collected. The macroporous adsorption resin is a styrene or divinylbenzene type non-polar macroporous adsorption resin with a particle size of 155-165 μm and a surface area of ​​500-600 m 2 / g;

[0034] Supersaturation process: combine the eluates, concentrate under reduced pressure to 10% to 13% volume, cool to 4°C to 10°C, and filter to obtain crude rosmarinic acid;

[0035] Purification process: add water to dissolve the crude rosmarinic acid and filter it. The weight ratio of crude rosmarinic acid to water is 1:10-20. Adjust the pH value of the solution to 2-3 and let it stand for 5-10 minutes. Extract it with an equal volume of ethyl acetate. Concentrate the extract under reduced pressure, cool it, and crystallize it. It can be cooled at 4°C to 10°C to achieve crystallization, so as to obtain pure rosmarinic acid.

[0036] The HPLC purity of rosmarinic acid obtained by this method can reach more than 95%, and after optimizing the conditions, it can reach more than 97%.

[0037] As the resource source of rosmarinic acid of the present invention, the inventors have discovered the fruit of the fruit of the rosmarinic acid tree, which has extensive resources in Xinjiang. In the preliminary research and analysis of the impurity components, the researchers believe that the polyphenol components similar to rosmarinic acid are mainly concentrated in the methyl ester and ethyl ester of rosmarinic acid. In the early stage of the scheme exploration, ethanol reflux treatment was used for extraction, and then an organic solvent extraction process similar to the above and a macroporous adsorption resin process were used. The results of HPLC chromatography of the obtained crude extract are shown in the attached Figure 1 .

[0038] Therefore, the feature of using acidic water extraction in the present invention is important. If acidic water extraction is not performed but the commonly used ethanol method is used, it is difficult to quickly obtain high-purity rosmarinic acid.

[0039] The present invention uses styrene or divinylbenzene type non-polar particles with a particle size of 155 to 165 μm and a surface area of ​​500 to 600 m 2 / g macroporous adsorption resin is important. The inventors have found that this type of macroporous adsorption resin is particularly suitable for adsorbing rosmarinic acid compounds and separating them from other components in the fruit of the ragwort. Other types of macroporous adsorption resins, such as multifunctional acrylate macroporous adsorption resins and acrylamide macroporous adsorption resins, are not suitable for the method of the present invention and cannot obtain high-purity rosmarinic acid.

[0040] In the purification process of the present invention, it is also very important to dissolve the crude rosmarinic acid in water and adjust the pH value of the solution to 2-3. Through such steps, a high-purity pure product can be precipitated after extraction with ethyl acetate. If the acidity adjustment is not performed or the pH is not appropriate, the high-purity pure rosmarinic acid of the present invention cannot be obtained.

[0041] The preparation method of the present invention as a whole does not use a large amount of organic solvents, and even the amount of ethanol used is very small. The column chromatography with poor industrial production efficiency is not used. The purification and refinement of rosmarinic acid is achieved by using specific raw material plants, accurately adjusting the pH value of the extraction, and a specific type of macroporous adsorption resin, and accurately adjusting the pH value of the extraction process. The method is particularly suitable for large-scale industrial production.

[0042] The inventors analyzed the reasons why the present invention can efficiently obtain rosmarinic acid. First, it is related to the raw material plant. The components contained therein can only enrich rosmarinic acid in the preparation method of the present invention; a specific type of macroporous adsorption resin can efficiently enrich rosmarinic acid, and the precise control of the pH value twice in the whole process is conducive to the specific selection of rosmarinic acid from rosmarinic acid homologues. In addition, the inventors of the present invention speculate that it is possible that the appropriate control of the pH value can promote the conversion of the rosmarinic acid esters rich in the plant into rosmarinic acid online during the extraction process, thereby not only reducing the difficulty of impurity separation, but also increasing the content of the target substance. The technical solution of the present invention has a strong integrity, and the combination of various elements is very important. Even when the inventors tried to use the various steps in the method of the present invention for extracting rosemary from Lamiaceae plants, they did not obtain an ideal pure product.

[0043] In a preferred embodiment of the present invention, the pH value of the acidic aqueous solution in the extraction step should be 3-4.

[0044] In a preferred embodiment of the present invention, the extraction process temperature is controlled below 60°C during the extraction step, and ultrasonic-assisted extraction can be performed to shorten the extraction time. For example, using a power of 100-140W can shorten the extraction time to 60-100min.

[0045] In a preferred embodiment of the present invention, the organic solvent in the organic solvent extraction step is preferably n-butanol. From the perspective of polarity matching, the solvent can improve the extraction efficiency, and a good recovery efficiency can be obtained with an extraction time of less than 30 minutes.

[0046] The method for separating and purifying rosmarinic acid from the fruit of the present invention is specifically described below using examples.

[0047] Example

[0048] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0049] The fruits and leaves of the Rhizoma Corydalis used in the present invention are purchased from Hotan Chinese medicinal materials market in Xinjiang Uygur Autonomous Region.

[0050] The purity test in the following experiments was carried out by HPLC, and the specific conditions were as follows: the HPLC model was: a Waters 2695 semi-preparative HPLC instrument (Waters Company, USA, with a detector system of Waters 2996).

[0051] The chromatographic conditions of the high performance liquid chromatography are: acetonitrile as mobile phase A, 0.1% by volume formic acid aqueous solution as mobile phase B, gradient elution; flow rate 1.0 ml / min; column temperature 25° C.; injection volume 20 μL; detection wavelength 254 nm; wherein the gradient elution program is: elution time 20 min, volume ratio A:B=90:10→0:100.

[0052] Example 1

[0053] (1) Extraction: 5 kg of dried fruits of the Rhizoma Corydalis were crushed and sieved, placed in an aqueous solution with a solid-liquid ratio of 1:25 (w / v), and extracted at 55°C for 60 min with an ultrasonic power of 100 W to obtain an extract;

[0054] (2) Extraction: The extract was concentrated under reduced pressure to one-tenth of its volume, and n-butanol solvent was added for extraction for 30 min. The extract was concentrated under reduced pressure to one-tenth of its volume at a temperature of 50° C. and a vacuum degree of -0.07 MPa to -0.08 MPa to obtain an extract;

[0055] (3) Enrichment: The extract was diluted and then precipitated on non-polar macroporous resin HP20 (particle size 155-165 μm, surface area 500-550 m 2 / g, Mitsubishi Corporation, Japan) were separated, impurities were washed with pure water, and then desorbed with 40% (volume fraction) ethanol aqueous solution, and the analytical solution was collected;

[0056] (4) Crystallization: Combine the analytical solutions, concentrate under reduced pressure to one-tenth of the volume at 50°C and a vacuum degree of -0.07 MPa to -0.08 MPa, cool and allow to stand, and collect the crude rosmarinic acid;

[0057] (5) Purification: The precipitate was dissolved in water, the pH value was adjusted to 2.0, and the precipitate was extracted with ethyl acetate for 3 times. The extracts were combined, and concentrated to an appropriate volume under reduced pressure at 50° C. and a vacuum degree of -0.07 MPa to -0.08 MPa. The precipitate was cooled in a freezing room and crystallized to obtain pure rosmarinic acid.

[0058] (6) Drying: After drying the crystals obtained in the above three times, 3.4 g of rosmarinic acid with a purity of 96.5% can be obtained.

[0059] Its purity test results refer to Figure 2 .

[0060] Embodiment 2:

[0061] (1) Extraction: 5 kg of dried fruits of the Rhizoma Corydalis were crushed and sieved, placed in an aqueous solution with a solid-liquid ratio of 1:25 (w / v), and extracted at 55°C for 70 min with an ultrasonic power of 110 W to obtain an extract;

[0062] (2) Extraction: The extract was concentrated under reduced pressure to one-tenth of its volume, and n-butanol solvent was added for extraction for 30 min. The extract was concentrated under reduced pressure to one-tenth of its volume at a temperature of 50° C. and a vacuum degree of -0.07 MPa to -0.08 MPa to obtain an extract;

[0063] (3) Adsorption: The diluted extract was subjected to non-polar macroporous resin HP20 (particle size 155-165 μm, surface area 500-550 m 2 / g, Mitsubishi Corporation, Japan) for separation, impurities were washed with pure water, and then desorbed with 50% (volume) ethanol aqueous solution, and the analytical solution was collected;

[0064] (4) Crystallization: Combine the analytical solutions, concentrate under reduced pressure to one-tenth of the volume at 50°C and a vacuum degree of -0.07 MPa to -0.08 MPa, cool and allow to stand, and collect the crude rosmarinic acid;

[0065] (5) Purification: Dissolve the precipitate in water, adjust the pH to 2.0, extract with ethyl acetate three times, combine the extracts, and concentrate to an appropriate volume under reduced pressure at 50° C. and a vacuum degree of -0.07 MPa to -0.08 MPa, cool in a freezing room, and crystallize to obtain pure rosmarinic acid;

[0066] (6) Drying: After drying the crystals obtained in the above three times, 3.5 g of rosmarinic acid with a purity of 97.4% can be obtained.

[0067] Embodiment 3:

[0068] (1) Extraction: 5 kg of dried fruits of the Rhizoma Corydalis were crushed and sieved, placed in an aqueous solution of pH = 4.0 with a solid-liquid ratio of 1:25 (w / v), and extracted at 60°C for 80 min with an ultrasonic power of 120 W to obtain an extract;

[0069] (2) Extraction: The extract was concentrated to one-tenth of its volume under reduced pressure, and n-butanol solvent was added for extraction for 30 min. The extract was concentrated to one-tenth of its volume under reduced pressure at a temperature of 55° C. and a vacuum degree of -0.07 MPa to -0.08 MPa to obtain an extract;

[0070] (3) Adsorption: The diluted extract was added to a non-polar macroporous resin D4020 (particle size 155-165 μm, surface area 540-580 m 2 / g, Nankai University Chemical Plant) for separation, washing with pure water to remove impurities, then desorbing with 60% (volume) ethanol aqueous solution, collecting the analytical solution;

[0071] (4) Crystallization: Combine the analytical solutions, concentrate under reduced pressure to one-tenth of the volume at 55°C and a vacuum degree of -0.07 MPa to -0.08 MPa, cool and allow to stand, and collect the crude rosmarinic acid;

[0072] (5) Purification: Dissolve the precipitate in water, adjust the pH to 3.0, extract with ethyl acetate three times, combine the extracts, and concentrate to an appropriate volume under reduced pressure at 55° C. and a vacuum degree of -0.07 MPa to -0.08 MPa, cool in a freezing room, and crystallize to obtain pure rosmarinic acid;

[0073] (6) Drying: The crystals obtained in the above three times were dried to obtain 3.4 g of rosmarinic acid with a purity of 96.0%.

[0074] Embodiment 4:

[0075] (1) Extraction: 5 kg of dried fruits of the Rhizoma Corydalis were crushed and sieved, placed in an aqueous solution of pH = 4.0 with a solid-liquid ratio of 1:25 (w / v), and extracted at 60° C. for 90 min with an ultrasonic power of 130 W to obtain an extract;

[0076] (2) Extraction: The extract was concentrated to one-tenth of its volume under reduced pressure, and n-butanol was added for extraction for 30 min. The extract was concentrated to one-tenth of its volume under reduced pressure to obtain an extract;

[0077] (3) Adsorption: The diluted extract was subjected to non-polar macroporous resin HP20 (particle size 155-165 μm, surface area 500-550 m 2 / g, Mitsubishi Corporation, Japan) for separation, impurities were washed with pure water, and then desorbed with 60% (volume) ethanol aqueous solution, and the analytical solution was collected;

[0078] (4) Crystallization: Combine the analytical solutions, concentrate under reduced pressure to one-tenth of the volume, cool and allow to stand, and collect the crude rosmarinic acid;

[0079] (5) Purification: Dissolve the precipitate in water, adjust the pH to 3.0, extract with ethyl acetate three times, combine the extracts, and concentrate to an appropriate volume under reduced pressure at 55° C. and a vacuum degree of -0.07 MPa to -0.08 MPa, cool in a freezing room, and crystallize to obtain pure rosmarinic acid;

[0080] (6) Drying: After drying the crystals obtained in the above three times, 3.4 g of rosmarinic acid with a purity of 96.7% can be obtained.

[0081] Comparative Example 1

[0082] In this comparative example, except that ethanol reflux was used in the extraction process, other parameters were the same as those in Example 1.

[0083] This comparative example can obtain 1.8 g of rosmarinic acid with a purity of 74.7%.

[0084] Comparative Example 2

[0085] In this comparative example, except that the pH value of the acid water in the extraction process is 2, other parameters are the same as those in Example 1.

[0086] This comparative example can obtain 2.6 g of rosmarinic acid with a purity of 88.4%.

[0087] Comparative Example 3

[0088] In this comparative example, except that the pH value of the acid water in the extraction process was 5, other parameters were the same as those in Example 1.

[0089] This comparative example can obtain 2.0 g of rosmarinic acid with a purity of 78.7%.

[0090] Comparative Example 4

[0091] In this comparative example, except that the macroporous resin in step 3 is replaced by a medium polar resin AB-8 (particle size 130-140 μm, surface area 480-520 m 2 / g, Nankai University Chemical Plant), other parameters are the same as in Example 1.

[0092] This comparative example can obtain 2.5 g of rosmarinic acid with a purity of 87.3%.

[0093] Comparative Example 5

[0094] In this comparative example, except that the macroporous resin in step 3 is a high polarity resin DA-201 (particle size 155-165 μm, surface area ≥ 200 m 2 / g, Nankai University Chemical Plant), other parameters are the same as in Example 1.

[0095] This comparative example can obtain 2.1 g of rosmarinic acid with a purity of 82.4%.

[0096] Comparative Example 6

[0097] In this comparative example, except that the pH value was adjusted to 1 when the precipitate was dissolved in water during the purification process, other parameters were the same as those in Example 1.

[0098] This comparative example can obtain 2.0 g of rosmarinic acid with a purity of 75.1%.

[0099] Comparative Example 7

[0100] In this comparative example, except that the pH value was adjusted to 4 when the precipitate was dissolved in water during the purification process, other parameters were the same as those in Example 1.

[0101] This comparative example can obtain 2.5 g of rosmarinic acid with a purity of 86.2%.

[0102] Comparative Example 8

[0103] In this comparative example, except that ethanol reflux was used in the extraction process, other parameters were the same as those in Example 3.

[0104] This comparative example can obtain 1.7 g of rosmarinic acid with a purity of 72.8%.

[0105] Comparative Example 9

[0106] In this comparative example, except that the pH value of the acid water in the extraction process is 2, other parameters are the same as those in Example 3.

[0107] This comparative example can obtain 2.1 g of rosmarinic acid with a purity of 77.5%.

[0108] Comparative Example 10

[0109] In this comparative example, except that the pH value of the acid water in the extraction process is 5, other parameters are the same as those in Example 3.

[0110] This comparative example can obtain 2.4 g of rosmarinic acid with a purity of 84.1%.

[0111] Comparative Example 11

[0112] In this comparative example, except that the macroporous resin in step 3 is replaced by a medium polar resin AB-8 (particle size 130-140 μm, surface area 480-520 m 2 / g, Nankai University Chemical Plant), other parameters are the same as in Example 3.

[0113] This comparative example can obtain 2.3 g of rosmarinic acid with a purity of 87.6%.

[0114] Comparative Example 12

[0115] In this comparative example, except that the macroporous resin in step 3 is a high polarity resin DA-201 (particle size 155-165 μm, surface area ≥ 200 m 2 / g, Nankai University Chemical Plant), other parameters are the same as in Example 3.

[0116] This comparative example can obtain 2.1 g of rosmarinic acid with a purity of 81.2%.

[0117] Comparative Example 13

[0118] In this comparative example, except that the pH value was adjusted to 1 when the precipitate was dissolved in water during the purification process, other parameters were the same as those in Example 3.

[0119] This comparative example can obtain 1.8 g of rosmarinic acid with a purity of 70.0%.

[0120] Comparative Example 14

[0121] In this comparative example, except that the pH value was adjusted to 3 when the precipitate was dissolved in water during the purification process, other parameters were the same as those in Example 3.

[0122] This comparative example can obtain 2.5 g of rosmarinic acid with a purity of 85.6%.

[0123] Comparative Example 15

[0124] The dried rosemary leaves purchased from the Hotan Chinese Medicine Market in Xinjiang Uygur Autonomous Region were used as raw materials. The experiment was carried out in the same manner as in Example 1 except that the Rhizoma Cyperi fruits were replaced.

[0125] This comparative example can obtain 2.4 g of rosmarinic acid with a purity of 62.4%.

[0126] Pharmacological experiment part:

[0127] Glucose uptake activity screening:

[0128] Differentiated L6 cells were cultured at 1×10 4 ~5×10 4 The density of cells / well was seeded in a black 96-well plate, and 100 μL of α-MEM medium was added to each well to incubate the cells for 12 hours until the cells were full. A blank control group, a positive control group and a test sample group were set up, and three sub-wells were set up in each group. Subsequently, 100 μL of α-MEM medium containing 2-NBDG and the test sample (30 μg / mL) was added to the stable L6 cells and incubated in a constant temperature incubator for 30 minutes. After 30 minutes, the 96-well plate was fully centrifuged at 400g in a centrifuge for 5 minutes, and the supernatant was discarded. After that, 200 μL of the kit buffer was added to all cell wells, mixed with a pipette, and centrifuged at 400g for 5 minutes at room temperature using a centrifuge. After discarding the kit buffer solution, 100 μL of the kit analysis reagent was added to each well.

[0129] The fluorescence microplate reader was set to analyze, detect and record the fluorescence intensity of the fluorescent-labeled glucose in the cells of each well at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0130] The NC group was the blank control group, and the PC group was the positive control group for insulin. The glucose uptake in the NC group was taken as the unit uptake. The results showed that rosmarinic acid could promote the uptake of glucose by L6 cells, and the uptake was 1.75 times that of the blank group.

[0131] Its activity test results refer to Figure 5 .

Claims

1. A method for industrial extraction of rosmarinic acid, characterized in that: Ragwood Cordia dichotoma The fruit of Forst.f is used as raw material, and the method comprises the following steps: Extraction process: crush the dried fruit raw material of the ragwort tree, place it in an acidic aqueous solution, the pH value of the acidic aqueous solution is 3-4, the ratio of the raw material to the acidic aqueous solution is 1:20-30 by weight, and filter to obtain an extract; Organic solvent extraction step: concentrating the extract, adding an equal volume of organic solvent for extraction, and concentrating under reduced pressure to obtain an extract; Macroporous adsorption resin enrichment step: the extract is loaded with macroporous adsorption resin, impurities are washed with pure water, and then desorbed with 40-60% ethanol aqueous solution by volume, and the eluent is collected. The macroporous adsorption resin is a styrene or divinylbenzene type non-polar macroporous adsorption resin with a particle size of 155-165 µm and a surface area of ​​500-600 m 2 / g; Supersaturation process: combine the eluates, concentrate under reduced pressure to 10%~13% volume, cool to 4℃~10℃, and filter to obtain crude rosmarinic acid; Purification process: add water to dissolve the crude rosmarinic acid and filter it, the weight ratio of the crude rosmarinic acid to water is 1:10-20, adjust the pH value of the solution to 2-3 and let it stand for 5-10 minutes, extract it with an equal volume of ethyl acetate, concentrate the extract under reduced pressure, cool it, and crystallize it to obtain pure rosmarinic acid; The macroporous adsorption resin is styrene or divinylbenzene type non-polar macroporous adsorption resin HP20.

2. The industrial extraction method of rosmarinic acid according to claim 1, characterized in that: During the extraction process, the extraction temperature was controlled below 60 °C, and ultrasonic-assisted extraction was performed with a power of 100-140 W and an extraction time of 60-100 min.

3. The industrial extraction method of rosmarinic acid according to claim 1, characterized in that: The organic solvent in the organic solvent extraction step is n-butanol, and the extraction time is 30 min.

4. The industrial method for extracting rosmarinic acid according to claim 1, wherein: In the supersaturation process, the temperature is 50-55° C. and the vacuum degree is -0.07 MPa to -0.09 MPa.

Images