A method for preparing carnosic acid with high purity and high transfer rate

Abstract

The invention discloses a preparation method for obtaining high-purity and high-transfer rate carnosic acid from a Chinese medicinal material rosemary. The method adopts countercurrent extraction to obtain a crude extract from the medicinal material, and then performs elution on a chromatography column to obtain high-purity carnosic acid after recrystallization. The content of carnosic acid obtained by the preparation method of the invention is as high as more than 95%, the transfer rate of the obtained product is high, and the performance is stable; and the equipment used in the preparation method is simple, which reduces the use of extraction solvents and the energy consumption required for industrial production, and is very suitable for large-scale industrial production, and has considerable economic benefits.

Description

Technical Field

[0001] The invention relates to the technical field of medicine, and in particular to a method for preparing high-purity and high-transfer rate carnosic acid from a traditional Chinese medicinal material rosemary. Background Art

[0002] Carnosic acid, chemical formula is C 20 H 28 O4 is a colorless to light yellow powder crystal, which is easily soluble in oils and insoluble in water. It has the characteristics of high efficiency, safety, and high temperature resistance. Because of the presence of both o-diphenol hydroxyl and carboxyl groups in its structure, it has a wide range of pharmacological effects, such as anti-inflammatory, antibacterial, antioxidant, and anti-tumor. It has been used in many industries such as cosmetics, medicine, and food, and has important value.

[0003] Rosemary (Rosmarinusofficinalis L.), also known as tansy, is an evergreen shrub of the genus Rosmarinus in the Lamiaceae family. It is warm in nature, spicy in taste, and has the effects of aromatic spleen strengthening, calming and tranquilizing the mind. There have been a large number of reports on its chemical components at home and abroad, including terpenes, flavonoids, organic acids, multi-branched alkanes, and amino acids. Among them, the main component with antioxidant ability is the phenolic diterpenoid compound carnosic acid.

[0004] At present, the methods for extracting carnosic acid from rosemary are mainly traditional organic solvent extraction, supercritical carbon dioxide extraction, etc., but the traditional hot reflux solvent extraction method is still the main method in industrial production. However, due to the instability of the structure of carnosic acid, it is easily degraded into carnosol during these traditional extraction methods, resulting in low purity of carnosic acid and low transfer rate of carnosic acid.

[0005] In the prior art, there are few reports on the preparation methods of high-purity carnosic acid. A Chinese patent application entitled "Preparation method of carnosic acid and its application" (CN202210514731.2) provides a method for preparing high-purity carnosic acid by dissolving rosemary extract in n-hexane, extracting with an alkaline aqueous solution, adjusting the pH with an acid, collecting the precipitate, and finally crystallizing at low temperature; another Chinese patent application entitled "A method and application of liquid-liquid extraction to separate carnosic acid and ursolic acid" (CN202210641695.6) provides a method of extracting rosemary with ethanol, concentrating the extract, and subjecting the concentrate to liquid-liquid extraction with a mixed solvent consisting of a mixture of n-hexane and dichloromethane, and finally obtaining carnosic acid with a purity greater than 80%.

[0006] The high-purity carnosic acid extraction process involved in the above-mentioned invention patent application CN202210514731.2 adopts acid to adjust the pH for precipitation, which is complicated to operate and has high equipment requirements in large-scale production. In addition, carnosic acid degradation (from carnosic acid to carnosin) is prone to occur during alkaline water treatment, resulting in a decrease in CA transfer rate, and the organic-containing wastewater after acid-base treatment puts huge environmental pressure on production enterprises; and the carnosic acid preparation method involved in the above-mentioned invention patent application CN202210641695.6 has less than ideal purity and transfer rate of carnosic acid, which is difficult to meet the requirements for high purity of carnosic acid in the medical or food fields. Summary of the invention

[0007] In order to solve the deficiencies of the above-mentioned prior art, the present invention aims to provide a method for preparing carnosic acid with high purity and high transfer rate, that is, the purity of the carnosic acid product obtained by the preparation method is not less than 95% and the total transfer rate is at least 50%; the object of the present invention is achieved by the following technical scheme:

[0008] A method for preparing high-purity and high-transfer rate carnosic acid, the method comprising the following steps:

[0009] (1) Weigh an appropriate amount of rosemary dry leaves, crush them, add an alcohol solution, perform countercurrent extraction, recover the solvent from the extract, filter or centrifuge to obtain a solid, granulate, dry, and obtain carnosic acid crude extract granules for later use;

[0010] (2) taking the carnosic acid crude extract particles obtained in step (1), loading them into a column, adding an organic solvent for elution, and recovering them to obtain a carnosic acid pretreated sample for later use;

[0011] (3) Taking the carnosic acid pretreated sample obtained in step (2), adding a low-polarity organic solvent for recrystallization, thereby obtaining the product of the present invention, wherein the carnosic acid content is greater than 95%.

[0012] Preferably, in the preparation method of the present invention, the countercurrent extraction process in step (1) is continuous countercurrent extraction, the alcohol solution is 70-90% ethanol solution, and the amount of the alcohol solution used is 4-12 times the amount of the medicinal material; in the granulation process, the raw material used is rosemary dry leaves, which are extracted, the solvent is recovered, and the precipitate is obtained by filtering or centrifugation, sieved, and granulated by a swing granulator to prepare extract granules, and the granulation mesh is 10-60 mesh;

[0013] The column loading process in step (2) is to load the carnosic acid crude extract particles into the chromatography column; the organic solvent is at least one of petroleum ether, alkanes containing 3 to 7 carbon atoms, dichloromethane, chloroform, and ethyl acetate; the amount of the organic solvent added is 5 to 12 times the amount of the carnosic acid crude extract particles, and the flow rate of the elution solvent is 0.5 to 2 BV / h;

[0014] In step (3), the low-polarity organic solvent used in the recrystallization is at least one of alkanes containing 3 to 7 carbon atoms, the recrystallization temperature is room temperature, and the crystallization time is 6 to 24 hours.

[0015] Preferably, in the above preparation method of the present invention, the alcohol solution in step (1) is a 75-85% ethanol solution, and the amount of the alcohol solution used is 6-10 times the amount of the medicinal material; the organic solvent in step (2) is n-hexane or 6# solvent;

[0016] More preferably, in the above-mentioned preparation method of the present invention, the alcohol solution in step (1) is an 80% ethanol solution, and the amount of the alcohol solution used is 8 times the amount of the medicinal material; the organic solvent in step (2) is n-hexane; the added amount is 5 to 12 times the amount of the crude extract of carnosic acid, and the flow rate of the elution solvent is 1 BV / h; the recrystallization temperature in step (3) is room temperature, and the crystallization time is 6 to 12 hours.

[0017] The best method for preparing high-purity carnosic acid according to the present invention comprises the following steps:

[0018] (1) Weigh an appropriate amount of rosemary dry leaves, crush them, use 80% ethanol solution in an amount 8 times the amount of the medicinal material, perform continuous countercurrent extraction, recover the solvent from the extract, filter or centrifuge to obtain a solid, granulate, and dry to obtain a crude extract of carnosic acid for later use;

[0019] (2) taking the carnosic acid crude extract particles obtained in step (1), loading them into a chromatography column, adding 10 times the amount of n-hexane for elution at a flow rate of 1 BV / h, and recovering the eluate to obtain a carnosic acid pretreated sample for later use;

[0020] (3) Take the carnosic acid pretreated sample obtained in step (2), add 3 times of n-hexane for recrystallization, and place it at room temperature for 12 hours to crystallize, thereby obtaining the product of the present invention. The carnosic acid content of the obtained product is greater than 95%, and the total transfer rate is greater than 50%.

[0021] The beneficial effects of the present invention are as follows:

[0022] 1. Compared with the traditional reflux extraction technology with disadvantages such as long extraction time, high solvent usage, high energy consumption and low recovery rate resulting in high cost, the present invention adopts continuous countercurrent extraction, so that the extraction solvent and the medicinal material flow in opposite directions, the two form countercurrent contact, and the concentration gradient is always maintained at a high level, ensuring continuous and sufficient contact extraction, greatly saving extraction time, reducing solvent usage and energy consumption, while minimizing the loss of carnosic acid in the extraction process, and significantly improving the recovery rate of carnosic acid; therefore, the technical solution of the present invention has the beneficial effects of simple process, cost saving, high extraction efficiency, etc.

[0023] 2. The present invention adopts the method of eluting with an organic solvent after installing a chromatography column; it is a dynamic leaching method with high solvent utilization rate, sufficient elution of carnosic acid, less elution of related impurities, and the leachate can be directly collected; at the same time, this process performs extraction at normal temperature and pressure, the equipment is simple and the capacity is large, the operation is simple and easy to use, and it is suitable for large-scale industrial production.

[0024] 3. The solvents ethanol and n-hexane used in the present invention are widely used in the food industry, such as in the production of oils and fats. They operate in a closed pipeline system at room temperature, with low organic solvent volatilization loss and high safety factor, and are suitable for large-scale production. At the same time, the extraction efficiency is high, and the degradation of carnosic acid to carnosin is avoided without heating. Therefore, the process can basically ignore the influence of different types of impurities in the medicinal materials on the final product, and is suitable for the extraction of carnosic acid from different plant sources.

[0025] Therefore, the present invention has the operability of industrial production, and the economic benefits generated will also be obvious.

[0026] The present invention is described in more detail below in conjunction with the examples. The following examples are merely illustrative, and the present invention is not limited by the examples. DETAILED DESCRIPTION Example 1

[0027] (1) Weigh 1508 kg of dried rosemary leaves (the content of carnosic acid in the medicinal material is 3.07%) and cut them into pieces of about 1 cm 2 Size, using 75% ethanol aqueous solution, continuous countercurrent extraction, feed rate of 100kg / hour, solvent flow rate of 500L / h, filtrate recovery ethanol, plate and frame filtration, the obtained solids are granulated with a swing granulator, and after drying, about 205Kg of carnosic acid extract particles are obtained for standby use;

[0028] (2) 300 g of the crude carnosic acid extract particles obtained in step (1) were loaded into a glass chromatography column, and 3 L of an organic solvent containing n-hexane was added for elution at a flow rate of 1 BV / h. The eluate was recovered to obtain a carnosic acid pretreated sample for later use;

[0029] (3) Take the carnosic acid pretreated sample obtained in step (2), add 300 ml of n-hexane, stir and heat for 30 minutes, cool, leave at room temperature for 6 hours, filter and dry the filter cake to obtain 39.12 g of carnosic acid crystals of the extract of the present invention, with a measured content of 95.66% and a transfer rate of 55.23%. Example 2

[0030] (1) Weigh 1046 kg of rosemary dry leaves (the content of carnosic acid in the medicinal material is 2.95%), cut into pieces of about 1 cm, add 80% ethanol solution, and perform continuous countercurrent extraction at a feed rate of 100 kg / hour and a solvent flow rate of 700 L / hour. Recover ethanol from the filtrate, filter on a plate and frame, and granulate using a swing granulator. After drying, 148.5 kg of carnosic acid crude extract particles are obtained and set aside;

[0031] (2) 300 g of the crude carnosic acid extract particles obtained in step (1) were loaded into a glass chromatography column, and 3.3 L of an organic solvent containing cyclohexane was added for elution at a flow rate of 1 BV / h. The eluate was recovered to obtain a carnosic acid pretreated sample for later use;

[0032] (3) Take the carnosic acid pretreated sample obtained in step (2), add 200 ml of 6# solvent, stir and heat for 30 minutes, cool, leave at room temperature for 10 hours, filter and dry the filter cake to obtain 36.64 g of the carnosic acid extract of the present invention, with a measured content of 95.16% and a transfer rate of 51.47%. Example 3

[0033] (1) 300 g of the crude extract of carnosic acid obtained in Example 1 was loaded into a glass chromatography column, and 3.6 L of an organic solvent containing petroleum ether was added for elution at a flow rate of 0.5 BV / h. The eluate was recovered to obtain a carnosic acid pretreated sample for later use;

[0034] (2) Take the carnosic acid pretreated sample obtained in the above step (1), add 500 ml of cyclohexane, heat for 30 minutes, cool, and place at room temperature for 12 hours. Filter and dry the filter cake to obtain 38.16 g of the extract of the present invention, with a measured content of 95.10% and a transfer rate of 53.56%. Example 4

[0035] (1) 300 g of the crude extract of carnosic acid obtained in Example 2 was loaded into a glass chromatography column, and 2.4 L of an organic solvent containing dichloromethane was added for elution at a flow rate of 2 BV / h. The eluate was recovered to obtain a carnosic acid pretreated sample for later use;

[0036] (2) Take the carnosic acid pretreated sample obtained in the above step (1), add 200 ml of n-heptane, heat for 10 minutes, cool, and place at room temperature for 12 hours. Filter and dry the filter cake to obtain 33.71 g of the extract of the present invention, with a measured content of 95.36% and a transfer rate of 51.57%. Example 5

[0037] (1) 300 g of the crude extract of carnosic acid obtained in Example 2 was loaded into a glass chromatography column, and 3 L of an organic solvent containing a 6# solvent was added for elution at a flow rate of 2 BV / h. The eluate was recovered to obtain a carnosic acid pretreated sample for later use;

[0038] (2) Take the carnosic acid pretreated sample obtained in step (1), add 200 ml of cyclohexane, heat for 10 minutes, cool, and place at room temperature for 12 hours. Filter and dry the filter cake to obtain 32.57 g of the carnosic acid extract of the present invention, with a measured content of 95.86% and a transfer rate of 50.08%. Example 6

[0039] (1) 300 g of the crude extract of carnosic acid obtained in Example 2 was loaded into a glass chromatography column, and 2.4 L of an organic solvent containing ethyl acetate was added for elution at a flow rate of 2 BV / h. The eluate was recovered to obtain a carnosic acid pretreated sample for later use;

[0040] (2) Take the carnosic acid pretreated sample obtained in step (1), add 200 ml of cyclohexane, heat for 10 minutes, cool, and place at room temperature for 12 hours. Filter and dry the filter cake to obtain 34.07 g of the carnosic acid extract of the present invention, with a measured content of 95.76% and a transfer rate of 52.34%. Example 7

[0041] (1) 300 g of the crude extract of carnosic acid obtained in Example 2 was loaded into a glass chromatography column, and 1.8 L of an organic solvent containing chloroform was added for elution at a flow rate of 1 BV / h. The eluate was recovered to obtain a carnosic acid pretreated sample for later use;

[0042] (2) Take the carnosic acid pretreated sample obtained in step (1), add 200 ml of cyclohexane, heat for 10 minutes, cool, and place at room temperature for 12 hours. Filter and dry the filter cake to obtain 33.61 g of the extract of the present invention, with a measured content of 95.43% and a transfer rate of 51.45%.

Claims

1. A method for preparing carnosic acid, characterized in that: The method comprises the following steps: (1) Weigh an appropriate amount of rosemary dry leaves, crush them, add an alcohol solution, perform countercurrent extraction, recover the solvent from the extract, filter or centrifuge to obtain a solid, granulate, dry, and obtain carnosic acid crude extract granules for later use; (2) taking the carnosic acid crude extract particles obtained in step (1), loading the carnosic acid crude extract particles into a chromatography column, adding an organic solvent, wherein the organic solvent is at least one of petroleum ether, an alkane containing 3 to 7 carbon atoms, dichloromethane, chloroform, and ethyl acetate, eluting, and recovering to obtain a carnosic acid pretreated sample for use; (3) taking the carnosic acid pretreated sample obtained in step (2), adding a low polarity organic solvent, wherein the low polarity organic solvent is at least one of alkanes containing 3 to 7 carbon atoms, and performing recrystallization to obtain carnosic acid, wherein the carnosic acid content is greater than 95%.

2. The method for preparing carnosic acid according to claim 1, characterized in that: In the countercurrent extraction process of step (1), the alcohol solution is a 70-90% ethanol solution, and the amount of the alcohol solution used is 4-12 times the amount of the medicinal material.

3. The method for preparing carnosic acid according to claim 1, characterized in that: In the countercurrent extraction process of step (1), the alcohol solution is a 75-85% ethanol solution, and the amount of the alcohol solution used is 6-10 times the amount of the medicinal material.

4. The method for preparing carnosic acid according to claim 1, characterized in that: In the countercurrent extraction process of step (1), the alcohol solution is an 80% ethanol solution, and the amount of the alcohol solution used is 8 times the amount of the medicinal material.

5. The method for preparing carnosic acid according to claim 1, characterized in that: In the granulation process of step (1), the raw material used is rosemary dry leaves, which are extracted, the organic solvent is recovered, and the precipitate obtained by filtering or centrifugation is sieved and granulated. The granulation size is 10 to 60 meshes.

6. The method for preparing carnosic acid according to claim 1, characterized in that: The amount of organic solvent added in step (2) is 5 to 12 times the amount of carnosic acid crude extract particles, and the flow rate of the elution solvent is 0.5 to 2 BV / h.

7. The method for preparing carnosic acid according to claim 1, characterized in that: The organic solvent in step (2) is n-hexane.

8. The method for preparing carnosic acid according to claim 1, characterized in that: In step (3), the volume of the low-polarity organic solvent added is 2 to 5 times the volume of the sample, the mixture is heated under reflux to dissolve, and crystallized at room temperature. The crystallization time is 6 to 24 hours.

9. The method for preparing high-purity carnosic acid according to claim 1, characterized in that: The low polarity organic solvent in step (3) is n-hexane.

10. The method for preparing carnosic acid according to claim 1, characterized in that: The method comprises the following steps: (1) Weigh an appropriate amount of rosemary dry leaves, crush them, use 80% ethanol solution in an amount 8 times the amount of the medicinal material, perform continuous countercurrent extraction, recover the solvent from the extract, filter or centrifuge to obtain a solid, granulate, and dry to obtain carnosic acid extract granules for later use; (2) taking the carnosic acid crude extract particles obtained in step (1), loading them into a chromatography column, adding 10 times the amount of n-hexane for elution at a flow rate of 1 BV / h, and recovering the eluate to obtain a carnosic acid pretreated sample for later use; (3) Take the carnosic acid pretreated sample obtained in step (2), add 3 times of n-hexane for recrystallization, and place it at room temperature for 12 hours to crystallize to obtain carnosic acid. The obtained product has a carnosic acid content of more than 95% and a total transfer rate of more than 50%.