CN116768725A - Method for extracting rosmarinic acid from rosemary - Google Patents
Method for extracting rosmarinic acid from rosemary Download PDFInfo
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- CN116768725A CN116768725A CN202310732407.2A CN202310732407A CN116768725A CN 116768725 A CN116768725 A CN 116768725A CN 202310732407 A CN202310732407 A CN 202310732407A CN 116768725 A CN116768725 A CN 116768725A
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- DOUMFZQKYFQNTF-WUTVXBCWSA-N (R)-rosmarinic acid Chemical compound C([C@H](C(=O)O)OC(=O)\C=C\C=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-WUTVXBCWSA-N 0.000 title claims abstract description 122
- 241001529742 Rosmarinus Species 0.000 title claims abstract description 68
- ZZAFFYPNLYCDEP-HNNXBMFYSA-N Rosmarinsaeure Natural products OC(=O)[C@H](Cc1cccc(O)c1O)OC(=O)C=Cc2ccc(O)c(O)c2 ZZAFFYPNLYCDEP-HNNXBMFYSA-N 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 61
- DOUMFZQKYFQNTF-MRXNPFEDSA-N rosemarinic acid Natural products C([C@H](C(=O)O)OC(=O)C=CC=1C=C(O)C(O)=CC=1)C1=CC=C(O)C(O)=C1 DOUMFZQKYFQNTF-MRXNPFEDSA-N 0.000 title claims abstract description 61
- TVHVQJFBWRLYOD-UHFFFAOYSA-N rosmarinic acid Natural products OC(=O)C(Cc1ccc(O)c(O)c1)OC(=Cc2ccc(O)c(O)c2)C=O TVHVQJFBWRLYOD-UHFFFAOYSA-N 0.000 title claims abstract description 61
- 239000000284 extract Substances 0.000 claims abstract description 95
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 68
- 238000000605 extraction Methods 0.000 claims abstract description 55
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000004440 column chromatography Methods 0.000 claims abstract description 24
- 238000007873 sieving Methods 0.000 claims abstract description 21
- 239000002253 acid Substances 0.000 claims abstract description 20
- 238000002791 soaking Methods 0.000 claims abstract description 19
- 238000001035 drying Methods 0.000 claims abstract description 18
- 238000001704 evaporation Methods 0.000 claims abstract description 18
- 239000011521 glass Substances 0.000 claims abstract description 18
- 238000010898 silica gel chromatography Methods 0.000 claims abstract description 10
- 241001530490 Salvia rosmarinus Species 0.000 claims abstract description 4
- 238000011068 loading method Methods 0.000 claims abstract description 4
- 238000010298 pulverizing process Methods 0.000 claims abstract description 4
- 235000015639 rosmarinus officinalis Nutrition 0.000 claims abstract description 4
- 230000008961 swelling Effects 0.000 claims abstract description 4
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical group CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 54
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical group Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 24
- 239000002904 solvent Substances 0.000 claims description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 4
- 238000004090 dissolution Methods 0.000 claims description 3
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 3
- 239000002994 raw material Substances 0.000 abstract description 34
- 238000009776 industrial production Methods 0.000 abstract description 8
- 241000196324 Embryophyta Species 0.000 abstract description 4
- 239000013543 active substance Substances 0.000 abstract description 3
- 239000004480 active ingredient Substances 0.000 abstract description 2
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 239000000843 powder Substances 0.000 description 42
- 239000000243 solution Substances 0.000 description 40
- 238000010828 elution Methods 0.000 description 17
- 239000000463 material Substances 0.000 description 15
- 239000000203 mixture Substances 0.000 description 15
- 238000012360 testing method Methods 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 11
- 239000012071 phase Substances 0.000 description 11
- 238000000638 solvent extraction Methods 0.000 description 11
- 239000007864 aqueous solution Substances 0.000 description 10
- 238000000622 liquid--liquid extraction Methods 0.000 description 10
- 239000003963 antioxidant agent Substances 0.000 description 7
- 239000008346 aqueous phase Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 230000003078 antioxidant effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 238000010829 isocratic elution Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000002714 Extracts of rosemary Substances 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 235000019306 extracts of rosemary Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 229940092258 rosemary extract Drugs 0.000 description 2
- 235000020748 rosemary extract Nutrition 0.000 description 2
- 239000001233 rosmarinus officinalis l. extract Substances 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 241000207923 Lamiaceae Species 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 239000012156 elution solvent Substances 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 235000019261 food antioxidant Nutrition 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000000194 supercritical-fluid extraction Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/56—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C67/00—Preparation of carboxylic acid esters
- C07C67/48—Separation; Purification; Stabilisation; Use of additives
- C07C67/58—Separation; Purification; Stabilisation; Use of additives by liquid-liquid treatment
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Extraction Or Liquid Replacement (AREA)
Abstract
The invention belongs to the technical field of extraction of plant active ingredients, and discloses a method for extracting rosmarinic acid from rosemary. The method comprises the following steps: pulverizing stem and leaf of Rosmarinus officinalis, sieving, soaking in ethanol solution, swelling, loading into glass chromatographic column, and performing column chromatography to obtain extractive solution; rotary evaporating the extractive solution to water phase, and drying to obtain herba Rosmarini officinalis water-soluble extract; dissolving herba Rosmarini officinalis water-soluble extract with acid water, and extracting with extractive solution to obtain extractive solution; and eluting the extract by silica gel column chromatography to obtain rosmarinic acid. According to the method, the yield and the raw material utilization rate of rosmarinic acid can be obviously improved and the loss of active substances in a sample can be reduced by optimizing the steps and controlling the pH values of the ethanol solution and the acid water; the method has the advantages of stable extraction process, strong repeatability, high consistency of repeated extraction results, easy amplification and contribution to industrial production.
Description
Technical Field
The invention belongs to the technical field of extraction of plant active ingredients, and particularly relates to a method for extracting rosmarinic acid from rosemary.
Background
In recent years, with the continuous improvement of the living standard of people, the problem of using safety of artificial preservatives in foods, cosmetics and daily necessities is increasingly receiving attention, and the research and development and application of pure natural antioxidants are becoming research hotspots in the fields of foods and chemical industry. Rosemary is a plant of the genus Labiatae, is a plant which has higher antioxidation effect, is safe and nontoxic, is praised as a third-generation green food antioxidant, and has wide application prospect and great market potential. Currently, commercially available rosemary antioxidants include both lipid-soluble antioxidants and water-soluble antioxidants. According to GB 1886.172-2016 food safety national standard food additive rosemary extract, the content of the main active component rosmarinic acid in the water-soluble antioxidant is not less than 5%, and the rosmarinic acid has strong oxidation resistance and can resist high temperature of above 100 ℃, so that the water-soluble antioxidant can be widely applied to various beverages, aquatic products and natural water-soluble pigments. Meanwhile, rosmarinic acid also has various activities such as antibacterial, anti-inflammatory and anti-tumor activities.
At present, most of the extraction methods of rosmarinic acid in industrial production are solvent extraction methods or supercritical fluid extraction methods, a large amount of solvents are used in both methods, the content of rosmarinic acid in the obtained extract is low, and the subsequent separation and purification of rosmarinic acid are difficult. The separation and purification method of rosmarinic acid mainly comprises an extraction method, a macroporous resin adsorption method and a silica gel column chromatography method. The rosmarinic acid prepared by the extraction method has lower purity; the macroporous resin adsorption method and the silica gel column chromatography are used for purifying rosmarinic acid, a complex elution system is needed, the composition of an elution solvent is complex, the repeatability is poor, and the industrial production is difficult to realize. In addition, the prior art has the problems of low yield of the rosmarinic acid extracted from the rosemary, low raw material utilization rate and more difficulty in industrial production.
Therefore, there is a need to provide a method for extracting rosmarinic acid from rosemary, which can improve the yield and the raw material utilization rate of rosmarinic acid; and the extraction process is stable, the reproducibility is strong, and the industrial production is facilitated.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. To this end, the present invention proposes a method for extracting rosmarinic acid from rosemary. The method provided by the invention can improve the yield and the raw material utilization rate of rosmarinic acid; the method has stable extraction process and strong repeatability, and is beneficial to industrial production.
The invention provides a method for extracting rosmarinic acid from rosemary.
Specifically, the method for extracting rosmarinic acid from rosemary comprises the following steps:
(1) Pulverizing stem and leaf of Rosmarinus officinalis, sieving, soaking in ethanol solution, swelling, loading into glass chromatographic column, and performing column chromatography to obtain extractive solution;
(2) Rotary evaporating the extract obtained in the step (1) to a water phase, and then drying to obtain a rosemary water-soluble extract;
(3) Dissolving the rosemary water-soluble extract obtained in the step (2) by adopting acid water, and extracting by adopting an extract liquid to obtain an extract liquid;
(4) Eluting the extract obtained in the step (3) by silica gel column chromatography to obtain rosmarinic acid.
Preferably, in step (1), the sieving is performed by passing through a 10-40 mesh sieve; further preferably, in step (1), the sieving is performed by passing through a 20 mesh sieve.
Preferably, in the step (1), the ethanol solution is an ethanol solution with a volume fraction of 50% -80%; further preferably, the ethanol solution is an ethanol solution with a volume fraction of 50% -70%; more preferably, in step (1), the ethanol solution is a 50% -60% ethanol solution by volume fraction.
Preferably, in step (1), the soaking is performed at room temperature (5-40 ℃) for 1-5 hours. Such as 2 hours, 3 hours, etc.
Preferably, in the step (1), the column height is 2.5-15 times of the diameter of the chromatographic column in the column chromatography extraction process, the eluting solvent is 50-70% ethanol water solution by volume percent, and the eluting flow rate is 1-3BV/h.
Further preferably, in the step (1), the column height is 5-15 times of the column diameter during the column chromatography extraction, the eluting solvent is 50-70% ethanol water solution by volume percent, and the eluting flow rate is 1.5-2.5BV/h.
Preferably, the column chromatography extraction process is performed at room temperature (5-40 ℃).
Preferably, in step (3), the temperature of the rotary evaporation is 40-50 ℃; further preferably, in step (3), the temperature of the rotary evaporation is 40-45 ℃.
Preferably, in the step (3), the process of dissolving by acid water is dissolving by acid water with a pH value of 0-2.5; further preferably, in the step (3), the process of dissolving with acid water is dissolving with acid water having a pH value of 0 to 2.
Preferably, in step (3), the acid water is a hydrochloric acid solution and/or an acetic acid solution.
Preferably, in step (3), the extract is ethyl acetate.
Preferably, in step (4), the elution is performed with a single solvent.
Preferably, the single solvent is ethyl acetate.
More specifically, a method for extracting rosmarinic acid from rosemary, comprising the steps of:
(1) Pulverizing dried stem and leaf of Rosmarinus officinalis, sieving with 10-40 mesh sieve, soaking in 50-80% ethanol solution, swelling, loading into glass chromatographic column with diameter-to-height ratio of 1 (2.5-15), and extracting by column chromatography at eluting flow rate of 1-3BV/h to obtain extractive solution;
(2) Rotary evaporating the extract obtained in the step (1) to a water phase, and then drying to obtain a rosemary water-soluble extract;
(3) Dissolving the rosemary water-soluble extract obtained in the step (2) by adopting acid water with the pH value of 0-3, and then extracting by adopting ethyl acetate to obtain an extract;
(4) And (3) eluting the extract obtained in the step (3) by silica gel column chromatography by taking ethyl acetate as an eluting solvent to obtain rosmarinic acid.
The method for extracting rosmarinic acid from rosemary provided by the invention comprises the steps of preparing a water-soluble extract of rosemary by using an ethanol solution and column chromatography extraction method, and then preparing rosmarinic acid by acid water dissolution, extractant extraction and silica gel column chromatography elution. The method can remarkably improve the yield and the raw material utilization rate of rosmarinic acid by optimizing the steps and controlling the pH values of the ethanol solution and the acid water; the process step can stabilize the extraction process, has strong repeatability, is easy to amplify, and can be used for large-scale production. It is found that the extraction method is poor in reproducibility, unstable in process and nonuniform in quality of the prepared product due to the fact that the column chromatography extraction is not carried out after the ethanol solution is adopted for extraction and the water-soluble rosemary extract is prepared in other modes. The pH value of the acid water is controlled, and the dissolution process plays an important role in improving the stability of the product.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the method for extracting rosmarinic acid from rosemary, provided by the invention, the yield and the raw material utilization rate of rosmarinic acid can be obviously improved by optimizing the steps and controlling the pH values of the ethanol solution and the acid water, and the loss of active substances in a sample can be reduced; the method has the advantages of stable extraction process, strong repeatability, high consistency of repeated extraction results, easy amplification and contribution to industrial production.
(2) The method provided by the invention can be used for efficiently extracting rosmarinic acid from rosemary, has the advantages of mild extraction condition, simple process, few reagents, low cost and high yield, and is suitable for large-scale industrial production of rosemary antioxidant.
Drawings
FIG. 1 is the yields of water-soluble extracts of rosemary prepared in examples 3-6 and comparative example 1;
FIG. 2 shows the extraction rates of the extract powders prepared in examples 7, 8 and comparative examples 2 to 6.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
Example 1
A method for extracting rosmarinic acid from rosemary, comprising the steps of:
(1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 60% (V/V) ethanol solution is added for soaking for 2.5 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) Rotary evaporating the extract collected in the step (1) to water phase at 45 ℃, and drying to obtain rosemary water-soluble extract, wherein the yield of the extract is 1.37%;
(3) Fully dissolving the extract obtained in the step (2) by using hydrochloric acid aqueous solution with pH of 2, and performing liquid-liquid extraction by using equal volume of ethyl acetate to obtain rosmarinic acid powder with the purity of more than 48%;
(4) And (3) performing silica gel column chromatography isocratic elution on the powder in the step (3) by using ethyl acetate, and collecting the eluent of 1.5-2.0BV to obtain rosmarinic acid with the purity of more than 93.0%, wherein the yield of the rosmarinic acid in the whole preparation process is 70.79%.
Example 2
The same procedure as in example 1 was used to extract the same batch of rosemary stem and leaf materials twice, and the purity and yield of the twice extracted rosmarinic acid were tested and calculated. The purity was tested to be 93.2% and 92.9% respectively, and the relative standard deviation RSD was 3.18%; the yields of rosmarinic acid were tested to be 71.12%, 70.18% and the relative standard deviation RSD to be 4.48%.
As can be seen from examples 1 and 2, the method for extracting rosmarinic acid from rosemary provided by the invention uses less reagent, has low extraction temperature, and has small loss of active substances in the sample; and the method has the advantages of high result consistency and good reproducibility, and is suitable for large-scale mass production.
Examples 3-6 and comparative example 1 studied the effect of different volume fractions of ethanol solutions on the extraction of water-soluble extracts of rosemary, the yields of which are shown in figure 1.
Example 3
A method for extracting rosmarinic acid from rosemary, comprising the steps of: (1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 50% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) And (3) rotationally evaporating the extracting solution collected in the step (1) to an aqueous phase at 45 ℃, and drying to obtain the rosemary water-soluble extract, wherein the yield of the test extract is 1.35+/-0.11%.
Example 4
A method for extracting rosmarinic acid from rosemary, comprising the steps of: (1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 60% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) And (3) rotationally evaporating the extracting solution collected in the step (1) to an aqueous phase at 45 ℃, and drying to obtain the rosemary water-soluble extract, wherein the yield of the test extract is 1.34+/-0.07%.
Example 5
A method for extracting rosmarinic acid from rosemary, comprising the steps of: (1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 70% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) And (3) rotationally evaporating the extracting solution collected in the step (1) to an aqueous phase at 45 ℃, and drying to obtain the rosemary water-soluble extract, wherein the yield of the test extract is 1.26+/-0.03%.
Example 6
A method for extracting rosmarinic acid from rosemary, comprising the steps of: (1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 80% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) And (3) rotationally evaporating the extracting solution collected in the step (1) to an aqueous phase at 45 ℃, and drying to obtain the rosemary water-soluble extract, wherein the yield of the test extract is 0.96+/-0.03%.
Comparative example 1
A method for extracting rosmarinic acid from rosemary, comprising the steps of: (1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 95% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) And (3) rotationally evaporating the extracting solution collected in the step (1) to an aqueous phase at 45 ℃, and drying to obtain the rosemary water-soluble extract, wherein the yield of the test extract is 0.23+/-0.01%.
(3) Fully dissolving the extract obtained in the step (2) by using a hydrochloric acid aqueous solution with the pH of 2, and performing liquid-liquid extraction by using an equal volume of ethyl acetate to obtain extract powder;
(4) And (3) performing isocratic elution on the extract powder in the step (3) by using ethyl acetate through silica gel column chromatography, and collecting the eluent of 1.5-2.0BV to obtain rosmarinic acid. The purity of rosmarinic acid and the yield of rosmarinic acid in the whole preparation process were tested. The purity of rosmarinic acid was 61.2%, and the yield of rosmarinic acid was 19.5%.
Examples 7-8 and comparative examples 2-6 the effect of acid solutions of different pH values on the extraction rate in step (3) was investigated, the extraction rate being shown in FIG. 2.
Example 7
A method for extracting rosmarinic acid from rosemary, comprising the steps of:
(1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 60% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) Rotary evaporating the extract collected in the step (1) to water phase at 45 ℃, and drying to obtain rosemary water-soluble extract, wherein the yield of the extract is 1.34%;
(3) And (3) fully dissolving the extract obtained in the step (2) by using a hydrochloric acid aqueous solution with the pH of 1, performing liquid-liquid extraction by using an equal volume of ethyl acetate to obtain extract powder, and testing the extraction rate, wherein the extraction rate is 64.7+/-6.25%.
Example 8
A method for extracting rosmarinic acid from rosemary, comprising the steps of:
(1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 60% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) Rotary evaporating the extract collected in the step (1) to water phase at 45 ℃, and drying to obtain rosemary water-soluble extract, wherein the yield of the extract is 1.34%;
(3) And (3) fully dissolving the extract obtained in the step (2) by using a hydrochloric acid aqueous solution with the pH of 2, performing liquid-liquid extraction by using an equal volume of ethyl acetate to obtain extract powder, and testing the extraction rate, wherein the extraction rate is 63.9+/-0.30%.
Comparative example 2
A method for extracting rosmarinic acid from rosemary, comprising the steps of:
(1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 60% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) Rotary evaporating the extract collected in the step (1) to water phase at 45 ℃, and drying to obtain rosemary water-soluble extract, wherein the yield of the extract is 1.34%;
(3) And (3) fully dissolving the extract obtained in the step (2) by using a hydrochloric acid aqueous solution with the pH of 3, performing liquid-liquid extraction by using an equal volume of ethyl acetate to obtain extract powder, and testing the extraction rate, wherein the extraction rate is 28.4+/-1.05%.
Comparative example 3
A method for extracting rosmarinic acid from rosemary, comprising the steps of:
(1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 60% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) Rotary evaporating the extract collected in the step (1) to water phase at 45 ℃, and drying to obtain rosemary water-soluble extract, wherein the yield of the extract is 1.34%;
(3) And (3) fully dissolving the extract obtained in the step (2) by using a hydrochloric acid aqueous solution with the pH of 4, performing liquid-liquid extraction by using an equal volume of ethyl acetate to obtain extract powder, and testing the extraction rate, wherein the extraction rate is 22.7+/-0.71%.
Comparative example 4
A method for extracting rosmarinic acid from rosemary, comprising the steps of:
(1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 60% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) Rotary evaporating the extract collected in the step (1) to water phase at 45 ℃, and drying to obtain rosemary water-soluble extract, wherein the yield of the extract is 1.34%;
(3) And (3) fully dissolving the extract obtained in the step (2) by using a hydrochloric acid aqueous solution with the pH of 5, performing liquid-liquid extraction by using an equal volume of ethyl acetate to obtain extract powder, and testing the extraction rate, wherein the extraction rate is 41.3+/-5.23%.
Comparative example 5
A method for extracting rosmarinic acid from rosemary, comprising the steps of:
(1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 60% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) Rotary evaporating the extract collected in the step (1) to water phase at 45 ℃, and drying to obtain rosemary water-soluble extract, wherein the yield of the extract is 1.34%;
(3) And (3) fully dissolving the extract obtained in the step (2) by using a hydrochloric acid aqueous solution with the pH of 6, performing liquid-liquid extraction by using an equal volume of ethyl acetate to obtain extract powder, and testing the extraction rate, wherein the extraction rate is 32.6+/-0.15%.
Comparative example 6
A method for extracting rosmarinic acid from rosemary, comprising the steps of:
(1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 60% (V/V) ethanol solution is added for soaking for 2 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) Rotary evaporating the extract collected in the step (1) to water phase at 45 ℃, and drying to obtain rosemary water-soluble extract, wherein the yield of the extract is 1.34%;
(3) And (3) fully dissolving the extract obtained in the step (2) by using a hydrochloric acid aqueous solution with the pH of 7, performing liquid-liquid extraction by using an equal volume of ethyl acetate to obtain extract powder, and testing the extraction rate, wherein the extraction rate is 15.7+/-2.3%.
Comparative example 7
A method for extracting rosmarinic acid from rosemary, comprising the steps of:
(1) Taking dried rosemary stems and leaves as materials, crushing, and sieving with a 20-mesh sieve to obtain raw material powder; then 10g of raw material powder is taken, 60% (V/V) ethanol solution is added for soaking for 2.5 hours, the mixture is fully swelled and then is filled into a 20X 400mm glass chromatographic column for column chromatography extraction, the diameter-to-height ratio is 1:10, the elution flow rate is 2MV/h, and 2MV extract is collected;
(2) Rotary evaporating the extract collected in the step (1) to water phase at 45 ℃, and drying to obtain rosemary water-soluble extract, wherein the yield of the extract is 1.34%;
(3) Fully dissolving the extract obtained in the step (2) by using hydrochloric acid aqueous solution with pH of 2, and performing liquid-liquid extraction by using equal volume of ethyl acetate to obtain rosmarinic acid powder with the purity of more than 50%;
(4) And (3) performing silica gel column chromatography isocratic elution on the powder in the step (3) by using n-butanol, and collecting the eluent of 1.5-2.0BV to obtain rosmarinic acid with the purity of 75.2%, wherein the yield of rosmarinic acid in the whole preparation process is 50.55%.
Claims (10)
1. A method for extracting rosmarinic acid from rosemary, comprising the steps of:
(1) Pulverizing stem and leaf of Rosmarinus officinalis, sieving, soaking in ethanol solution, swelling, loading into glass chromatographic column, and performing column chromatography to obtain extractive solution;
(2) Rotary evaporating the extract obtained in the step (1) to a water phase, and then drying to obtain a rosemary water-soluble extract;
(3) Dissolving the rosemary water-soluble extract obtained in the step (2) by adopting acid water, and extracting by adopting an extract liquid to obtain an extract liquid;
(4) Eluting the extract obtained in the step (3) by silica gel column chromatography to obtain rosmarinic acid.
2. The method of claim 1, wherein in step (1), the sieving is performed by passing through a 10-40 mesh sieve.
3. The method according to claim 1, wherein in step (1), the ethanol solution is 50% -80% by volume of ethanol solution.
4. A method according to claim 3, wherein the ethanol solution is a 50% -70% volume fraction ethanol solution.
5. The method according to claim 1, wherein in the step (1), the column height is 2.5-15 times the column diameter during the column chromatography extraction, the eluting solvent is 50-70% ethanol water solution by volume, and the eluting flow rate is 1-3BV/h.
6. The method according to claim 1, wherein in the step (3), the acid water dissolution is carried out by using acid water having a pH of 0 to 2.5; preferably, the process of dissolving by acid water adopts acid water with pH value of 0-2 for dissolving.
7. The method according to claim 6, wherein in step (3), the acid water is a hydrochloric acid solution and/or an acetic acid solution.
8. The process according to claim 1, wherein in step (3) the extract is ethyl acetate.
9. The method of claim 1, wherein in step (4), the eluting is performed with a single solvent.
10. The method of claim 9, wherein the single solvent is ethyl acetate.
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