A method for extracting rosmarinic acid from rosemary

Abstract

The present application belongs to the technical field of plant active ingredient extraction, and discloses a method for extracting rosmarinic acid from rosemary. The method comprises the following steps: crushing and sieving rosemary stems and leaves, then soaking them in an ethanol solution, loading them into a glass chromatography column for column chromatography extraction after sufficient swelling, and obtaining an extract; rotary evaporating the extract to an aqueous phase, drying to obtain a rosemary water-soluble extract; dissolving the rosemary water-soluble extract in acid water, and extracting with an extraction liquid to obtain an extraction liquid; and eluting the extraction liquid through a silica gel column to obtain rosmarinic acid. The method can significantly improve the yield of rosmarinic acid and the utilization rate of raw materials, and reduce the loss of active substances in the sample by optimizing the steps and controlling the pH values of the ethanol solution and the acid water. The extraction process of the method is stable, has strong reproducibility, high consistency in multiple extraction results, is easy to scale up, and is conducive to industrial production.

Description

Technical Field

[0001] This invention belongs to the field of plant active ingredient extraction technology, specifically relating to a method for extracting rosmarinic acid from rosemary. Background Technology

[0002] In recent years, with the continuous improvement of people's living standards, the safety of artificial preservatives in food, cosmetics, and daily necessities has received increasing attention. The research and application of pure natural antioxidants has become a research hotspot in the food and chemical industries. Rosemary, a plant of the genus *Rosemary* in the Lamiaceae family, is currently recognized as a plant with high antioxidant activity and is safe and non-toxic. It is hailed as a third-generation green food antioxidant, with broad application prospects and huge market potential. Currently, commercially available rosemary antioxidants include both fat-soluble and water-soluble antioxidants. According to GB 1886.172-2016 National Food Safety Standard for Food Additives: Rosemary Extract, the main active ingredient in water-soluble antioxidants should contain no less than 5% rosmarinic acid. Rosmarinic acid has strong antioxidant capacity and can withstand temperatures above 100℃, making it widely applicable in various beverages, aquatic products, and natural water-soluble pigments. Furthermore, rosmarinic acid also possesses various activities such as antibacterial, anti-inflammatory, and antitumor activity.

[0003] Currently, the extraction methods for rosmarinic acid in industrial production are mostly solvent extraction or supercritical fluid extraction. Both methods use large amounts of solvent, resulting in low rosmarinic acid content in the extract, making subsequent separation and purification of rosmarinic acid difficult. The main methods for separating and purifying rosmarinic acid include extraction, macroporous resin adsorption, and silica gel column chromatography. Extraction methods yield rosmarinic acid with low purity; macroporous resin adsorption and silica gel column chromatography require complex elution systems with complex solvent compositions and poor reproducibility, making industrial-scale production difficult. Furthermore, current processes for extracting rosmarinic acid from rosemary generally suffer from low yields and low raw material utilization, further complicating industrial production.

[0004] Therefore, there is an urgent need to provide a method for extracting rosmarinic acid from rosemary that can improve the yield of rosmarinic acid and the utilization rate of raw materials; and the extraction process should be stable, highly reproducible, and conducive to industrial production. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a method for extracting rosmarinic acid from rosemary. The method provided by the present invention can improve the yield of rosmarinic acid and the utilization rate of raw materials; moreover, the extraction process of this method is stable, highly reproducible, and conducive to industrial production.

[0006] This invention provides a method for extracting rosmarinic acid from rosemary.

[0007] Specifically, a method for extracting rosmarinic acid from rosemary includes the following steps:

[0008] (1) Crush and sieve the rosemary stems and leaves, then soak them in an ethanol solution. After they have fully swollen, pack them into a glass chromatography column for column chromatography extraction to obtain the extract.

[0009] (2) The extract obtained in step (1) is rotary evaporated to the aqueous phase and then dried to obtain the rosemary water-soluble extract;

[0010] (3) Dissolve the rosemary water-soluble extract obtained in step (2) in acid water, and then extract it with an extractant to obtain an extractant;

[0011] (4) The extract obtained in step (3) was eluted by silica gel column chromatography to obtain rosmarinic acid.

[0012] Preferably, in step (1), the sieving process is to pass through a 10-40 mesh sieve; more preferably, in step (1), the sieving process is to pass through a 20 mesh sieve.

[0013] Preferably, in step (1), the ethanol solution is an ethanol solution with a volume fraction of 50%-80%; more preferably, the ethanol solution is an ethanol solution with a volume fraction of 50%-70%; more preferably, in step (1), the ethanol solution is an ethanol solution with a volume fraction of 50%-60%.

[0014] Preferably, in step (1), the soaking process involves soaking at room temperature (5-40℃) for 1-5 hours, such as 2 hours, 3 hours, etc.

[0015] Preferably, in step (1), the column packing height during the column chromatography extraction is 2.5-15 times the diameter of the chromatography column, the elution solvent is an aqueous ethanol solution with a volume percentage of 50%-70%, and the elution flow rate is 1-3 BV / h.

[0016] More preferably, in step (1), the column packing height during the column chromatography extraction is 5-15 times the diameter of the chromatography column, the elution solvent is an aqueous ethanol solution with a volume percentage of 50%-70%, and the elution flow rate is 1.5-2.5 BV / h.

[0017] Preferably, the column chromatography extraction process is carried out at room temperature (5-40°C).

[0018] Preferably, in step (3), the temperature of the rotary evaporation is 40-50°C; more preferably, in step (3), the temperature of the rotary evaporation is 40-45°C.

[0019] Preferably, in step (3), the process of dissolving with acidic water is to dissolve with acidic water with a pH value of 0-2.5; more preferably, in step (3), the process of dissolving with acidic water is to dissolve with acidic water with a pH value of 0-2.

[0020] Preferably, in step (3), the acidic water is a hydrochloric acid solution and / or an acetic acid solution.

[0021] Preferably, in step (3), the extract is ethyl acetate.

[0022] Preferably, in step (4), the elution process is carried out using a single solvent.

[0023] Preferably, the single solvent is ethyl acetate.

[0024] More specifically, a method for extracting rosmarinic acid from rosemary includes the following steps:

[0025] (1) Pulverize the dried rosemary stems and leaves, pass them through a 10-40 mesh sieve, and then soak them in an ethanol solution with a volume fraction of 50%-80%. After they have fully swollen, pack them into a glass chromatography column with a diameter-to-height ratio of 1:(2.5-15) and extract them by column chromatography at an elution flow rate of 1-3 BV / h to obtain the extract.

[0026] (2) The extract obtained in step (1) is rotary evaporated to the aqueous phase and then dried to obtain the rosemary water-soluble extract;

[0027] (3) Dissolve the rosemary water-soluble extract obtained in step (2) in acidic water with a pH of 0-3, and then extract with ethyl acetate to obtain the extract;

[0028] (4) Using ethyl acetate as the elution solvent, the extract obtained in step (3) was eluted by silica gel column chromatography to obtain rosmarinic acid.

[0029] This invention provides a method for extracting rosmarinic acid from rosemary. First, a water-soluble extract of rosemary is prepared using ethanol solution and column chromatography. Then, rosmarinic acid is obtained through acid-water dissolution, extraction with an extractant, and silica gel column chromatography elution. This method, through optimization of the steps and control of the pH values ​​of the ethanol solution and acid solution, can significantly improve the yield of rosmarinic acid and the utilization rate of raw materials. Furthermore, this process ensures stability, high reproducibility, and easy scale-up for large-scale production. Studies have found that using ethanol solution extraction without column chromatography, or using other methods to prepare the water-soluble extract of rosemary, results in poor reproducibility, process instability, and inconsistent product quality. Controlling the pH value of the acid solution and the dissolution process also play a crucial role in improving product stability.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) The method for extracting rosmarinic acid from rosemary provided by the present invention can significantly improve the yield of rosmarinic acid and the utilization rate of raw materials by optimizing the steps and controlling the pH value of ethanol solution and acid water, and reduce the loss of active substances in the sample; and the extraction process of this method is stable, highly reproducible, and the results of multiple extractions are highly consistent, easy to scale up, and conducive to industrial production.

[0032] (2) The method provided by the present invention can efficiently extract rosmarinic acid from rosemary, and the extraction conditions are mild, the process is simple, the reagents used are few, the cost is low, and the yield is high, making it suitable for large-scale industrial production of rosemary antioxidants. Attached Figure Description

[0033] Figure 1 The yield of the rosemary water-soluble extracts prepared in Examples 3-6 and Comparative Example 1;

[0034] Figure 2 The extraction rate is the extraction yield of the extract powders prepared in Examples 7, 8 and Comparative Examples 2-6. Detailed Implementation

[0035] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.

[0036] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0037] Example 1

[0038] A method for extracting rosmarinic acid from rosemary includes the following steps:

[0039] (1) Using dried rosemary stems and leaves as material, the raw material powder was obtained by crushing and passing through a 20-mesh sieve; then 10g of the raw material powder was added to a 60% (V / V) ethanol solution and soaked for 2.5h. After full swelling, it was packed into a 20×400mm glass chromatography column for column chromatography extraction with a diameter-to-height ratio of 1:10 and an elution flow rate of 2MV / h. 2MV of extract was collected.

[0040] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain a rosemary water-soluble extract with a yield of 1.37%.

[0041] (3) Dissolve the extract obtained in step (2) fully in hydrochloric acid aqueous solution at pH 2, and extract with an equal volume of ethyl acetate to obtain rosmarinic acid powder with a purity of over 48%.

[0042] (4) The powder in (3) was then eluted by silica gel column chromatography with ethyl acetate. The 1.5-2.0 BV eluent was collected to obtain rosmarinic acid with a purity of over 93.0%. The yield of rosmarinic acid in the whole preparation process was 70.79%.

[0043] Example 2

[0044] Using the same method as in Example 1, the same batch of rosemary stem and leaf raw materials were extracted twice more, and the purity and yield of rosmarinic acid from the two extractions were tested and calculated. The tested purities were 93.2% and 92.9%, respectively, with a relative standard deviation (RSD) of 3.18%; the tested yields of rosmarinic acid were 71.12% and 70.18%, respectively, with a relative standard deviation (RSD) of 4.48%.

[0045] As can be seen from Examples 1 and 2, the method for extracting rosmarinic acid from rosemary provided by the present invention uses fewer reagents, has a lower extraction temperature, and results in less loss of active substances in the sample; moreover, the results of multiple extractions are highly consistent and reproducible, and can be adapted to large-scale production.

[0046] Examples 3-6 and Comparative Example 1 investigated the effect of different volume fractions of ethanol solution on the extraction of rosemary water-soluble extract. The yield of the rosemary water-soluble extract was as follows: Figure 1 As shown.

[0047] Example 3

[0048] A method for extracting rosmarinic acid from rosemary includes the following steps: (1) using dried rosemary stems and leaves as material, crushing them and passing them through a 20-mesh sieve to obtain raw material powder; then taking 10g of raw material powder and adding it to a 50% (V / V) ethanol solution to soak for 2h, after fully swelling, packing it into a 20×400mm glass chromatography column for column chromatography extraction, with a diameter-to-height ratio of 1:10, an elution flow rate of 2MV / h, and collecting 2MV of extract;

[0049] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain the rosemary water-soluble extract. The yield of the extract was tested to be 1.35±0.11%.

[0050] Example 4

[0051] A method for extracting rosmarinic acid from rosemary includes the following steps: (1) using dried rosemary stems and leaves as material, crushing them and passing them through a 20-mesh sieve to obtain raw material powder; then taking 10g of raw material powder and adding it to a 60% (V / V) ethanol solution to soak for 2h, after fully swelling, packing it into a 20×400mm glass chromatography column for column chromatography extraction, with a diameter-to-height ratio of 1:10, an elution flow rate of 2MV / h, and collecting 2MV of extract;

[0052] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain the rosemary water-soluble extract. The yield of the extract was tested to be 1.34±0.07%.

[0053] Example 5

[0054] A method for extracting rosmarinic acid from rosemary includes the following steps: (1) using dried rosemary stems and leaves as material, crushing them and passing them through a 20-mesh sieve to obtain raw material powder; then taking 10g of raw material powder and adding it to a 70% (V / V) ethanol solution to soak for 2h, after fully swelling, packing it into a 20×400mm glass chromatography column for column chromatography extraction, with a diameter-to-height ratio of 1:10, an elution flow rate of 2MV / h, and collecting 2MV of extract;

[0055] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain the rosemary water-soluble extract. The yield of the extract was tested to be 1.26±0.03%.

[0056] Example 6

[0057] A method for extracting rosmarinic acid from rosemary includes the following steps: (1) using dried rosemary stems and leaves as material, crushing them and passing them through a 20-mesh sieve to obtain raw material powder; then taking 10g of raw material powder and adding it to an 80% (V / V) ethanol solution to soak for 2h, after fully swelling, packing it into a 20×400mm glass chromatography column for column chromatography extraction, with a diameter-to-height ratio of 1:10, an elution flow rate of 2MV / h, and collecting 2MV of extract;

[0058] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain the rosemary water-soluble extract. The yield of the extract was tested to be 0.96±0.03%.

[0059] Comparative Example 1

[0060] A method for extracting rosmarinic acid from rosemary includes the following steps: (1) using dried rosemary stems and leaves as material, crushing them and passing them through a 20-mesh sieve to obtain raw material powder; then taking 10g of raw material powder and adding it to a 95% (V / V) ethanol solution to soak for 2h, after fully swelling, packing it into a 20×400mm glass chromatography column for column chromatography extraction, with a diameter-to-height ratio of 1:10, an elution flow rate of 2MV / h, and collecting 2MV of extract;

[0061] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain the rosemary water-soluble extract. The yield of the extract was tested to be 0.23±0.01%.

[0062] (3) Dissolve the extract obtained in step (2) completely in hydrochloric acid aqueous solution with pH 2, and extract with an equal volume of ethyl acetate to obtain extract powder;

[0063] (4) The extract powder from (3) was then subjected to silica gel column chromatography with ethyl acetate for isocratic elution. The eluent of 1.5-2.0 BV was collected to obtain rosmarinic acid. The purity of rosmarinic acid and the yield of rosmarinic acid in the entire preparation process were tested. The purity of rosmarinic acid was 61.2%, and the yield of rosmarinic acid was 19.5%.

[0064] Examples 7-8 and Comparative Examples 2-6 investigated the effect of acid solutions with different pH values ​​on the extraction rate in step (3). The extraction rates are as follows: Figure 2 As shown.

[0065] Example 7

[0066] A method for extracting rosmarinic acid from rosemary includes the following steps:

[0067] (1) Using dried rosemary stems and leaves as material, the raw material powder was obtained by crushing and passing through a 20-mesh sieve; then 10g of the raw material powder was added to a 60% (V / V) ethanol solution and soaked for 2h. After full swelling, it was packed into a 20×400mm glass chromatography column for column chromatography extraction. The diameter-to-height ratio was 1:10, the elution flow rate was 2MV / h, and 2MV of extract was collected.

[0068] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain a rosemary water-soluble extract with a yield of 1.34%.

[0069] (3) The extract obtained in step (2) was fully dissolved in hydrochloric acid aqueous solution at pH 1, and extracted with an equal volume of ethyl acetate to obtain extract powder. The extraction rate was tested and found to be 64.7 ± 6.25%.

[0070] Example 8

[0071] A method for extracting rosmarinic acid from rosemary includes the following steps:

[0072] (1) Using dried rosemary stems and leaves as material, the raw material powder was obtained by crushing and passing through a 20-mesh sieve; then 10g of the raw material powder was added to a 60% (V / V) ethanol solution and soaked for 2h. After full swelling, it was packed into a 20×400mm glass chromatography column for column chromatography extraction. The diameter-to-height ratio was 1:10, the elution flow rate was 2MV / h, and 2MV of extract was collected.

[0073] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain a rosemary water-soluble extract with a yield of 1.34%.

[0074] (3) The extract obtained in step (2) was fully dissolved in hydrochloric acid aqueous solution at pH 2, and extracted with an equal volume of ethyl acetate to obtain extract powder. The extraction rate was tested and found to be 63.9 ± 0.30%.

[0075] Comparative Example 2

[0076] A method for extracting rosmarinic acid from rosemary includes the following steps:

[0077] (1) Using dried rosemary stems and leaves as material, the raw material powder was obtained by crushing and passing through a 20-mesh sieve; then 10g of the raw material powder was added to a 60% (V / V) ethanol solution and soaked for 2h. After full swelling, it was packed into a 20×400mm glass chromatography column for column chromatography extraction. The diameter-to-height ratio was 1:10, the elution flow rate was 2MV / h, and 2MV of extract was collected.

[0078] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain a rosemary water-soluble extract with a yield of 1.34%.

[0079] (3) The extract obtained in step (2) was fully dissolved in hydrochloric acid aqueous solution at pH 3, and extracted with an equal volume of ethyl acetate to obtain extract powder. The extraction rate was tested and found to be 28.4 ± 1.05%.

[0080] Comparative Example 3

[0081] A method for extracting rosmarinic acid from rosemary includes the following steps:

[0082] (1) Using dried rosemary stems and leaves as material, the raw material powder was obtained by crushing and passing through a 20-mesh sieve; then 10g of the raw material powder was added to a 60% (V / V) ethanol solution and soaked for 2h. After full swelling, it was packed into a 20×400mm glass chromatography column for column chromatography extraction. The diameter-to-height ratio was 1:10, the elution flow rate was 2MV / h, and 2MV of extract was collected.

[0083] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain a rosemary water-soluble extract with a yield of 1.34%.

[0084] (3) The extract obtained in step (2) was fully dissolved in hydrochloric acid aqueous solution at pH 4, and extracted with an equal volume of ethyl acetate to obtain extract powder. The extraction rate was tested and found to be 22.7 ± 0.71%.

[0085] Comparative Example 4

[0086] A method for extracting rosmarinic acid from rosemary includes the following steps:

[0087] (1) Using dried rosemary stems and leaves as material, the raw material powder was obtained by crushing and passing through a 20-mesh sieve; then 10g of the raw material powder was added to a 60% (V / V) ethanol solution and soaked for 2h. After full swelling, it was packed into a 20×400mm glass chromatography column for column chromatography extraction. The diameter-to-height ratio was 1:10, the elution flow rate was 2MV / h, and 2MV of extract was collected.

[0088] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain a rosemary water-soluble extract with a yield of 1.34%.

[0089] (3) The extract obtained in step (2) was fully dissolved in hydrochloric acid aqueous solution at pH 5, and extracted with an equal volume of ethyl acetate to obtain extract powder. The extraction rate was tested and found to be 41.3 ± 5.23%.

[0090] Comparative Example 5

[0091] A method for extracting rosmarinic acid from rosemary includes the following steps:

[0092] (1) Using dried rosemary stems and leaves as material, the raw material powder was obtained by crushing and passing through a 20-mesh sieve; then 10g of the raw material powder was added to a 60% (V / V) ethanol solution and soaked for 2h. After full swelling, it was packed into a 20×400mm glass chromatography column for column chromatography extraction. The diameter-to-height ratio was 1:10, the elution flow rate was 2MV / h, and 2MV of extract was collected.

[0093] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain a rosemary water-soluble extract with a yield of 1.34%.

[0094] (3) The extract obtained in step (2) was fully dissolved in hydrochloric acid aqueous solution at pH 6, and extracted with an equal volume of ethyl acetate to obtain extract powder. The extraction rate was tested and found to be 32.6 ± 0.15%.

[0095] Comparative Example 6

[0096] A method for extracting rosmarinic acid from rosemary includes the following steps:

[0097] (1) Using dried rosemary stems and leaves as material, the raw material powder was obtained by crushing and passing through a 20-mesh sieve; then 10g of the raw material powder was added to a 60% (V / V) ethanol solution and soaked for 2h. After full swelling, it was packed into a 20×400mm glass chromatography column for column chromatography extraction. The diameter-to-height ratio was 1:10, the elution flow rate was 2MV / h, and 2MV of extract was collected.

[0098] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain a rosemary water-soluble extract with a yield of 1.34%.

[0099] (3) The extract obtained in step (2) was fully dissolved in hydrochloric acid aqueous solution at pH 7, and extracted with an equal volume of ethyl acetate to obtain extract powder. The extraction rate was tested and found to be 15.7 ± 2.3%.

[0100] Comparative Example 7

[0101] A method for extracting rosmarinic acid from rosemary includes the following steps:

[0102] (1) Using dried rosemary stems and leaves as material, the raw material powder was obtained by crushing and passing through a 20-mesh sieve; then 10g of the raw material powder was added to a 60% (V / V) ethanol solution and soaked for 2.5h. After full swelling, it was packed into a 20×400mm glass chromatography column for column chromatography extraction with a diameter-to-height ratio of 1:10 and an elution flow rate of 2MV / h. 2MV of extract was collected.

[0103] (2) The extract collected in step (1) was rotary evaporated to the aqueous phase at 45°C and dried to obtain a rosemary water-soluble extract with a yield of 1.34%.

[0104] (3) Dissolve the extract obtained in step (2) fully in hydrochloric acid aqueous solution at pH 2, and extract with an equal volume of ethyl acetate to obtain rosmarinic acid powder with a purity of over 50%.

[0105] (4) The powder in (3) was then eluted by silica gel column chromatography with n-butanol, and the 1.5-2.0 BV eluent was collected to obtain rosmarinic acid with a purity of 75.2%. The yield of rosmarinic acid in the whole preparation process was 50.55%.

Claims

1. A method for extracting rosmarinic acid from rosemary, characterized in that, Includes the following steps: (1) Crush and sieve the rosemary stems and leaves, then soak them in a 50%-70% ethanol solution. After they have fully swollen, pack them into a glass chromatography column for column chromatography extraction to obtain the extract. (2) The extract obtained in step (1) is rotary evaporated to the aqueous phase and then dried to obtain the rosemary water-soluble extract; (3) The rosemary water-soluble extract obtained in step (2) is dissolved in acidic water with a pH of 0-2.5, and then extracted with ethyl acetate to obtain an extract; (4) The extract obtained in step (3) was eluted by silica gel column chromatography with ethyl acetate to obtain rosmarinic acid.

2. The method according to claim 1, characterized in that, In step (1), the sieving process is to pass the material through a 10-40 mesh sieve.

3. The method according to claim 1, characterized in that, In step (1), the column packing height during the column chromatography extraction is 2.5-15 times the diameter of the chromatography column, the elution solvent is an aqueous ethanol solution with a volume percentage of 50%-70%, and the elution flow rate is 1-3 BV / h.

4. The method according to claim 1, characterized in that, In step (3), the acidic water is a hydrochloric acid solution and / or an acetic acid solution.

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