Method and equipment for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction

Abstract

The present invention provides a method and apparatus for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction. The method comprises: mixing a crude product containing carnosic acid with a low-alcohol to obtain a solid-liquid mixture; placing the solid-liquid mixture in a separator; and introducing an extraction solvent into the separator from bottom to top. A liquid-solid-liquid three-phase system is formed in the separator from top to bottom, wherein the liquid phase system at the top is a carnosic acid extract; and the extraction solvent is one or more of n-hexane, n-heptane, and octane. The present invention uses a low-alcohol to dissolve the crude product containing carnosic acid, and uses a specific extraction solvent to introduce it into the separator from bottom to top, allowing it to fully contact the components to be extracted. Thus, the prepared carnosic acid product has a high content and low ursolic acid residue. This method can omit the subsequent product desolventizing process, reducing costs, and can achieve continuous operation with high separation efficiency. This provides a new industrial method for separating the active ingredients of rosemary, with a simple process and low energy consumption.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product separation, in particular to a method and equipment for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction. Background Art

[0002] Carnosic acid is a phenolic diterpenoid compound, a colorless to pale yellow powder crystal. It is readily soluble in oils and fats but insoluble in water. It is safe, non-toxic, structurally stable, and heat-resistant. Carnosic acid not only possesses antioxidant properties but also exhibits numerous pharmacological and biological activities, including anti-tumor and inflammation, brain and liver protection, and body fat reduction. Due to its excellent properties, it is widely used in pharmaceuticals, skincare products, daily necessities, health supplements, food additives, and fragrances.

[0003] Ursolic acid, also known as ursolic acid, is a pentacyclic triterpenoid compound found in plant parts such as loquat leaves and rosemary. Studies have shown that ursolic acid has multiple pharmacological effects, including antioxidant free radical protection, antioxidant stress relief, autophagy promotion, anti-tumor activity, and lipid-lowering properties.

[0004] In the existing carnosic acid and ursolic acid separation process, solvent extraction methods are mostly used, but the preparation process is relatively cumbersome, it is impossible to achieve continuous production, and the product purity is relatively low. For example, in the patent with publication number CN104447334A, rosemary extract is concentrated and then added with water to precipitate, the precipitate is extracted with petroleum ether, the extract is carnosic acid, and the remaining solid phase is ursolic acid. The ursolic acid and carnosic acid content prepared by this process is low, and the content of the two products is only about 25%. In the patent with publication number CN104447334A, ursolic acid and carnosic acid are separated by organic nanofiltration membrane. Compared with solvent extraction, this process has higher cost. In the patent with publication number CN113480592A, the ursolic acid crude product is separated by multiple solvent extraction, membrane preparation and crystallization process to carry out the separation of ursolic acid and carnosic acid products. Although the ursolic acid content prepared is high, the process is relatively complicated and it is impossible to achieve industrial continuous production. The applicant's own prior patent CN114773425A also only achieved a purity of 70-83% for carnosic acid, but a purity of only 20-25% for ursolic acid.

[0005] Therefore, it is necessary to develop a simple, efficient and continuous process for the separation of carnosic acid and ursolic acid. Summary of the Invention

[0006] The present invention provides a method and apparatus for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction, which are used to solve the defects of the prior art such as complex separation process, high energy consumption or poor separation effect, and achieve the separation effect of high carnosic acid crystal content, high ursolic acid content and low residual solvent under simple process conditions.

[0007] The present invention provides a method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction, comprising:

[0008] A crude product containing carnosic acid is mixed with a low-alcohol to obtain a solid-liquid mixture, the solid-liquid mixture is placed in a separator, and an extraction solvent is introduced into the separator from bottom to top, so that a liquid-solid-liquid three-phase system is formed from top to bottom in the separator, and the liquid phase system at the upper end is a carnosic acid extract; wherein the extraction solvent is one or more of n-hexane, n-heptane, and octane.

[0009] The present invention adopts low-alcohol to dissolve a crude product containing carnosic acid, and adopts a specific extraction solvent to be introduced into a separator from bottom to top so as to fully contact the components to be extracted, thereby achieving high carnosic acid product content and low ursolic acid residue, omitting the subsequent product desolventizing process, reducing costs, and the entire process can be carried out continuously. The process is simple, the energy consumption is low, and the method is suitable for industrial production.

[0010] According to the method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction provided by the present invention, the extraction solvent is n-hexane, or n-hexane and octane in a volume ratio of 1:0.1, or n-hexane and n-heptane in a volume ratio of 1:0.5.

[0011] According to the liquid-solid-liquid three-phase extraction method for separating carnosic acid and ursolic acid provided by the present invention, the low-alcohol solvent is an alcohol solvent with a concentration of 60% to 70%, for example, 60% ethanol, 65% ethanol, or 70% ethanol.

[0012] According to the method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction provided by the present invention, the solid-liquid ratio of the crude product containing carnosic acid to the low-alcohol is 1:(2-3) g / ml.

[0013] According to the method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction provided by the present invention, the extract obtained by the first extraction is separated, the remaining materials are mixed and then introduced into the extraction solvent again, the extract is collected, and the operation is repeated for a total of 5 to 8 extraction operations.

[0014] According to the method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction provided by the present invention, the crude product containing carnosic acid is obtained by extracting rosemary dry leaves with ethanol, concentrating them, adding water and filtering them.

[0015] In some embodiments of the present invention, the method comprises the following steps:

[0016] (1) The crude product S containing carnosic acid is dissolved in an alcohol solvent L1 having a concentration of 60% to 70%, and after sufficient stirring, a solid-liquid mixture is obtained. The mixture is added to a separator via a pressure pump. The separator is a tank device with a side paddle stirring function and can be liquid-inlet at the bottom and liquid-outlet at the top;

[0017] (2) The extraction solvent L2 is continuously introduced into the separator from the lower end, forming a liquid-solid-liquid (L2-S-L1) three-phase system from top to bottom in the separator, and the extract is continuously discharged from the upper end of the separator and collected at the outer end of the separator;

[0018] (3) After the first round of extraction, the S phase and the L1 phase in the separator are fully stirred by a side paddle (the side paddle is located between the S phase and the L1 phase);

[0019] (4) Continue to introduce the extraction solvent L2 into the separator, repeat 5 to 8 times, and collect the extract;

[0020] (5) The extracts are combined to obtain carnosic acid;

[0021] (6) After the material in the separator is released, the remaining solid material is ursolic acid.

[0022] According to the method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction provided by the present invention, the purity of carnosic acid in the extract is 70% to 80%. After collecting the extract, the solid material remaining in the separator is ursolic acid with a purity of 35% to 40%.

[0023] The method provided by the present invention can simply and efficiently separate carnosic acid and ursolic acid. The purity of the obtained carnosic acid and ursolic acid is higher than that of the prior art, and the residual soluble matter in ursolic acid is lower than that of the prior art, with the total residual soluble matter reduced to below 1000 ppm. Moreover, carnosic acid can be prepared in various forms from the extract. For example, a carnosic acid oil product can be prepared by adding auxiliary materials, and a carnosic acid powder product can be prepared by crystallization, with a powder content of 70% to 80%.

[0024] The present invention also provides a separation device, including a turnover barrel, a separator and a liquid collection barrel, wherein the discharge port of the turnover barrel is connected to the feed port at the bottom of the separator, and the discharge port at the top of the separator is connected to the feed port of the liquid collection barrel.

[0025] According to the separation device provided by the present invention, the lower side wall of the separator is provided with at least one side paddle, and the blade of the side paddle is located at the center of the cross section of the separator.

[0026] The separation equipment provided by the present invention can be used to separate carnosic acid and ursolic acid.

[0027] The present invention provides a method and device for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction. A crude product containing carnosic acid is dissolved in a low-alcohol, and a specific extraction solvent is introduced into a separator from bottom to top for full contact with the components to be extracted. Thus, a prepared carnosic acid product has a high content and low ursolic acid residue, and a subsequent product desolventizing step can be omitted, thereby reducing costs. Continuous liquid inlet and outlet can be achieved, resulting in high separation efficiency. This provides a new industrial method for separating effective components of rosemary, which has a simple process and low energy consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a structural schematic diagram of the separation equipment provided by the present invention.

[0029] Reference numerals:

[0030] 1: Turnover barrel; 2: Separator; 3: Liquid collection barrel; 4: Side slurry; 5: Pump. DETAILED DESCRIPTION

[0031] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0032] In the present invention, the content of each substance was determined using high performance liquid chromatography (HPLC) as follows:

[0033] 1. Carnosic acid content determination chromatographic conditions: chromatographic column: Waters CORTECS-C18 column (3.0 × 100 mm, 2.7 μm); mobile phase: 0.1% phosphoric acid acetonitrile: 0.1% phosphoric acid water = 23:77; detection wavelength: 280 nm; column temperature: 30°C; injection volume: 5 μL; flow rate: 0.6 mL / min.

[0034] Chromatographic conditions for ursolic acid content determination: chromatographic column: Waters CORTECS-C18 column (4.6×150 mm, 5 μm); mobile phase: 0.1% phosphoric acid in water: methanol = 12:88; detection wavelength: 210 nm; column temperature: 25°C; injection volume: 10 μL; flow rate: 1.0 mL / min.

[0035] 2. Calculation method of content (purity):

[0036] The peak area was measured by ARC liquid chromatography, and the content of the analyte (e.g., carnosic acid) was calculated based on the peak area and the formula as follows:

[0037]

[0038] Where: C 样 —the content of the substance to be tested in the sample, %; B—the intercept of the standard curve; S 样 —sample peak area; V—sample dilution multiple; C 标 —Standard sample concentration, mg / mL; M—Sample mass, g; S 样 —Sample concentration, mg / mL.

[0039] Example 1

[0040] This embodiment provides a separation device, the structural diagram of which is shown in FIG. Figure 1 As shown (the figure also includes materials), it includes a turnover barrel 1, a separator 2 and a liquid collecting barrel 3. The discharge port of the turnover barrel 1 is connected to the feed port at the bottom of the separator 2, and the discharge port at the top of the separator 2 is connected to the feed port of the liquid collecting barrel 3. The lower side wall of the separator 2 is provided with at least one side paddle 4, and the blade of the side paddle 4 is located at the center of the cross section of the separator 2. A pump 5 is provided on the pipeline between the discharge port of the turnover barrel 1 and the feed port of the separator 2. During operation, the mixture of the crude carnosic acid and the low-alcohol in the turnover barrel 1 is pumped into the separator 2 by the pump 5, and then the alkane extraction solvent is pumped into the separator 2. After extraction, the extract enters the liquid collecting barrel 3 from the discharge port of the separator 2.

[0041] Example 2

[0042] This example uses the separation equipment of Example 1 to provide a method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction, and the steps are as follows:

[0043] (1) adding 70% methanol by volume to dissolve the crude product containing carnosic acid at a solid-liquid ratio of 1:2, stirring thoroughly to obtain a solid-liquid mixture, and adding the mixture to a separator via a pressure pump;

[0044] (2) n-hexane is continuously introduced into the separator to form a liquid-solid-liquid (L2-S-L1) three-phase system from top to bottom in the separator, and the extract is continuously discharged from the upper end of the separator into the liquid collection tank;

[0045] (3) After the first round of extraction, the S phase and L1 phase in the separator were fully stirred by side paddles and then allowed to stand for 30 min;

[0046] (4) continuing to introduce n-hexane into the separator and collecting the extract;

[0047] (5) The combined extracts were concentrated and cooled to crystallize to obtain carnosic acid with a content of 79.52%;

[0048] (6) The material in the separator is released, and the remaining solid material is ursolic acid with a content of 38.66%.

[0049] Example 3

[0050] This embodiment provides a method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction, the steps of which are as follows:

[0051] (1) adding 60% ethanol by volume to the crude product containing carnosic acid at a solid-liquid ratio of 1:2 to dissolve the product, stirring the mixture to obtain a solid-liquid mixture, and adding the mixture to a separator via a pressure pump;

[0052] (2) n-hexane:octane (volume ratio 1:0.1) is continuously introduced into the separator to form a liquid-solid-liquid (L2-S-L1) three-phase system from top to bottom in the separator. The extract is continuously discharged from the upper end of the separator into the liquid collection tank;

[0053] (3) After the first round of extraction, the S phase and L1 phase in the separator were fully stirred by side paddles and then allowed to stand for 30 min;

[0054] (4) Continue to introduce n-hexane:octane (volume ratio 1:0.1) into the separator and collect the extract;

[0055] (5) The combined extracts were concentrated and cooled to crystallize to obtain carnosic acid with a content of 69.08%;

[0056] (6) The material in the separator is released, and the remaining solid material is ursolic acid with a content of 35.23%.

[0057] Example 4

[0058] This embodiment provides a method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction, the steps of which are as follows:

[0059] (1) adding 65% ethanol by volume to the crude product containing carnosic acid at a solid-liquid ratio of 1:3 to dissolve the product, stirring the mixture to obtain a solid-liquid mixture, and adding the mixture to the separator via a pressure pump;

[0060] (2) n-hexane:n-heptane (volume ratio 1:0.5) is continuously introduced into the separator to form a liquid-solid-liquid (L2-S-L1) three-phase system from top to bottom in the separator. The extract is continuously discharged from the upper end of the separator into the liquid collection tank;

[0061] (3) After the first round of extraction, the S phase and L1 phase in the separator were fully stirred by side paddles and then allowed to stand for 30 min;

[0062] (4) Continue to introduce n-hexane:n-heptane (volume ratio 1:0.5) into the separator and collect the extract;

[0063] (5) The combined extracts were concentrated and cooled to crystallize to obtain carnosic acid with a content of 71.52%;

[0064] (6) The material in the separator is discharged, and the remaining solid material is ursolic acid with a content of 36.45%.

[0065] Comparative Example 1

[0066] This comparative example provides a method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction. The specific method is the same as that of Example 2, except that in step (1), the crude product containing carnosic acid is dissolved in methanol with a volume concentration of 95%.

[0067] Finally, in step (5), the content of carnosic acid is 56.32%; in step (6), the content of ursolic acid is 15.62%.

[0068] Comparative Example 2

[0069] This comparative example provides a method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction. The specific method is the same as that of Example 3, except that the extraction solvent introduced into the separator in step (2) is petroleum ether. Finally, in step (5), the carnosic acid content is 31.24%, and in step (6), the ursolic acid content is 8.92%.

[0070] Comparative Example 3

[0071] This comparative example provides a method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction. The specific method is the same as that of Example 3, except that the extraction solvent introduced into the separator in step (2) is n-hexane:dichloromethane (volume ratio of 1:0.5). Finally, in step (5), the carnosic acid content is 67.38%, and in step (6), the ursolic acid content is 21.33%.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction, characterized in that: include: A crude product containing carnosic acid is mixed with a low-alcohol to obtain a solid-liquid mixture, the solid-liquid mixture is placed in a separator, and an extraction solvent is introduced into the separator from bottom to top, forming a liquid-solid-liquid three-phase system from top to bottom in the separator, with the liquid phase system at the top being the carnosic acid extract; the extract obtained from the first extraction is separated, the remaining materials are mixed, and the extraction solvent is introduced again, the extract is collected, and the operation is repeated for a total of 5 to 8 extraction operations; Wherein, the low-alcohol is methanol or ethanol with a volume concentration of 60% to 70%; The extraction solvent is n-hexane, or n-hexane and octane, or n-hexane and n-heptane; The crude product containing carnosic acid is obtained by extracting rosemary dry leaves with ethanol, concentrating the leaves, adding water and filtering the extracted leaves.

2. The method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction according to claim 1, characterized in that The extraction solvent is n-hexane, or n-hexane and octane in a volume ratio of 1:0.1, or n-hexane and n-heptane in a volume ratio of 1:0.

5.

3. The method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction according to claim 1, characterized in that The solid-liquid ratio of the crude product containing carnosic acid to the low-alcohol is 1:(2-3) g / ml.

4. The method for separating carnosic acid and ursolic acid by liquid-solid-liquid three-phase extraction according to any one of claims 1 to 3, characterized in that: The purity of carnosic acid in the extract is 70% to 80%; After the extraction liquid is collected, the solid material remaining in the separator is ursolic acid, and the purity of the ursolic acid is 35% to 40%.

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