CN111440184A

CN111440184A - Method for preparing high-purity carnosol

Info

Publication number: CN111440184A
Application number: CN202010482363.9A
Authority: CN
Inventors: 尚小雅; 李金杰; 王欣
Original assignee: Beijing Union University
Current assignee: Beijing Union University
Priority date: 2018-01-30
Filing date: 2018-01-30
Publication date: 2020-07-24
Anticipated expiration: 2038-01-30
Also published as: CN108358945B; CN111440184B; CN108358945A

Abstract

A method for preparing high-purity carnosol comprises the steps of dissolving rosemary extract with water-saturated ethyl acetate, mixing dissolved solution normal-phase silica gel samples, loading the samples onto a column by a dry method, performing gradient elution with chloroform, methanol and acetic acid (100-95) to (0-5) to (0-1 ‰), combining the same components, performing reduced pressure concentration to obtain carnosol with the purity of about 85%, performing Sephadex L H-20 gel column chromatography purification, eluting a mobile phase with chloroform-methanol or petroleum ether-chloroform-methanol, combining the same components, performing reduced pressure evaporation to obtain the carnosol with the purity of more than 98%, and obtaining the high-purity carnosol with the content of more than or equal to 98% from the rosemary extract.

Description

Method for preparing high-purity carnosol

The present application is a divisional application of the patent application "a method for simultaneously preparing high purity carnosol and carnosic acid from rosemary extract", original application No.: 201810092815.5, filing date originally: year 2018, month 1 and day 30.

Technical Field

The invention relates to a method for preparing high-purity natural antioxidant active ingredient carnosol from rosemary extract, in particular to a separation method of Sephadex L H-20 gel column chromatography with a remarkable effect on separating a large amount of carnosol.

Background

Rosemary is a natural antioxidant, and carnosol and carnosic acid are main antioxidant components of the rosemary, so that the demand of edible antioxidant products is increased along with the rapid development of the functional food industry; with the successful introduction of rosemary into China and large-area cultivation, the method for preparing the high-purity antioxidant active monomer from the rosemary or the extract thereof, which is simple, easy and cheap, is found, and has good use value and practical significance.

There are several patents reporting the extraction of antioxidant actives from rosemary: CN 1698587a reports a method for extracting rosemary total phenols, wherein the sum of the contents of the main components carnosol and carnosic acid is more than 13%; CN 101402864A, wherein the content of the main antioxidant active ingredients of carnosol and carnosic acid is more than 50 wt%; the content of carnosol and carnosic acid in the rosemary extract obtained from CN 105669432A is 40-45%. However, up to now, there are many patents reporting a method of obtaining high purity carnosic acid by separation and purification from rosemary only: CN 102115442A utilizes preparative phosphoric acid-silica gel column chromatography to obtain carnosic acid with the purity of 92%; CN 104058955A can be used for obtaining carnosic acid with purity of more than 98% by silica gel column chromatography; separating and purifying CN 101851158A by medium pressure chromatography, and recrystallizing with organic solvent to obtain carnosic acid with purity of 98%; CN 101113133A herba Rosmarini officinalis is extracted with weak base aqueous solution of disodium EDTA, the pH of the filtrate is adjusted with acid, the polarity of the filtrate is adjusted with organic solvent to obtain precipitate, and the precipitate is dried to obtain carnosic acid with purity of 99.42%. There are also methods for obtaining carnosol only by isolation and purification from rosemary: purifying CN 103319495A with macroporous adsorbent resin to obtain crude carnosol, and crystallizing with hydrophilic organic solvent to obtain carnosol with purity of 95%; CN 102171217A is prepared by crystallizing herba Rosmarini officinalis extractive solution with acetic acid at a certain ratio to obtain carnosol with purity of 85.6%; patent CN 101835782A and patent CN 101835783 a report that carnosol with purity of more than 90% is obtained by using a method for catalyzing the production of carnosol from carnosic acid.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for preparing large quantities of high-purity carnosol, which is simple, easy to operate, high in yield and suitable for large-scale production.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method of preparing high purity carnosol, the method comprising the steps of:

I. dissolving herba Rosmarini officinalis extract with water saturated ethyl acetate, filtering, and removing insoluble substances;

mixing the filtrate with normal phase silica gel, loading the mixture into a column by a dry method, loading the mixture by a dry method, performing gradient elution by chloroform, methanol and acetic acid (100-95): (0-5): (0-1 per thousand) (volume ratio), and combining the same components to obtain 5 components, wherein the components are respectively as follows: a small polar impurity part, a carnosol crude product, a carnosic acid crude product, a component with the main component of ursolic acid and a large polar impurity part;

and III, purifying the crude carnosol obtained in the step I by using hydroxypropyl sephadex, eluting a mobile phase by using chloroform to methanol (2-2.5) to 1 (volume ratio) or petroleum ether to chloroform to methanol (2-5) to 1 (volume ratio) at equal degrees, combining the same components, and evaporating to dryness under reduced pressure to obtain the carnosol with the content of more than or equal to 98%.

The method as described above, preferably, the rosemary extract is an ethanol aqueous extract of rosemary flowers or leaves.

The method preferably comprises the steps of crushing rosemary flowers or leaves, carrying out ultrasonic extraction for 60-90 minutes by using ethanol water solution with the volume percentage concentration of (50-70)% according to the liquid-material ratio of (10-15) to 1 (L/Kg), continuously extracting for 2-4 times, combining extracting solutions, concentrating under reduced pressure and evaporating to dryness to obtain the rosemary extract.

The method as described above, preferably, the gradient elution in step I is carried out five times, and the mobile phase is sequentially: chloroform; chloroform, methanol and acetic acid, the volume ratio is 100: 0: 1 per mill; chloroform, methanol and acetic acid in the volume ratio of 100 to 1; chloroform, methanol and acetic acid, the volume ratio is 100: 2: 1 per mill; chloroform, methanol and acetic acid, the volume ratio is 95: 5: 1 per mill.

In the method, the length of the hydroxypropyl glucan gel column in the step II is preferably 120-150 cm; loading columns by a wet method, and loading samples by the wet method; the flow rate of the mobile phase in the hydroxypropyl dextran gel is 2-6 ml/min.

Preferably, the ratio of chloroform to methanol in step II is 2.5: 1 (by volume), or the ratio of petroleum ether to chloroform to methanol is 5: 1 (by volume).

The method has the beneficial effects that only one-time normal phase silica gel adsorption column is used, so that adsorptive pigment impurities in the sample are removed, and the loss of the sample caused by dead adsorption due to repeated sample loading of the normal phase silica gel is avoided.

Drawings

FIG. 1 is a detection scheme of high purity carnosol HP L C;

FIG. 2 shows carnosol¹H-NMR chart;

FIG. 3 is carnosol¹³C-NMR chart;

Detailed Description

EXAMPLE 1 preparation of Rosmarinus officinalis extract

10Kg of dried rosemary leaves are crushed and sieved by a 20-mesh sieve, the solvent is ultrasonically extracted by using 60 percent ethanol water solution by volume percentage concentration, the liquid-material ratio is 12: 1 (L/Kg), the ultrasonic time is 75min, three times of continuous extraction are carried out, the extracting solutions are combined, and the rosemary extract is obtained after decompression, concentration and evaporation to dryness, wherein the carnosol content is 10.28 wt%.

Example 2 preparation of high purity carnosol from Rosmarinus officinalis extract

Normal phase silica gel column separation and purification

500g of the rosemary extract prepared in example 1 was added to 5000ml of ethyl acetate saturated with water, dissolved by sonication for 0.5 hour, and the insoluble matter was removed by filtration. The filtrate was mixed with 500g of normal phase silica gel (160-. Gradient elution was performed, with the following mobile phases and extracts, in order of elution:

(1) the mobile phase is chloroform, the elution volume is 5L, and the elution components are mainly small polar impurity parts;

(2) the mobile phase is chloroform, methanol and acetic acid, the volume ratio is 100: 0: 1 per mill, the elution volume is 10L, and the main component is carnosol with the content of about 85 percent;

(3) the mobile phase is chloroform, methanol and acetic acid, the volume ratio is 100: 1: 1 ‰, the elution volume is 15L, and the main component is carnosic acid with content of 90%;

(4) the mobile phase is chloroform, methanol and acetic acid, the volume ratio is 100: 2: 1 per mill, the elution volume is 10L, and the main component is ursolic acid;

(5) the mobile phase is chloroform, methanol and acetic acid, the volume ratio is 95: 5: 1 per mill, the elution volume is 5L, and the main component is a large polar impurity part.

Mixing the same components, recovering solvent under reduced pressure to obtain 1.2g of carnosol with content of 98% or more, and separating the carnosol with content of 85% with gel column in the next step.

(II) gel column separation and purification

And (2) repeatedly purifying the carnosol component containing about 85 wt% obtained in the step (I) by using hydroxypropyl Sephadex L H-20 gel, loading the gel column with 150cm in length by using a wet method, loading the gel column by using the wet method, eluting a mobile phase by using chloroform and methanol at a volume ratio of 2.5: 1, wherein the flow rate of the mobile phase is 5ml/min, the chromatography time is 490min, combining the same components, and evaporating to dryness under reduced pressure to obtain 41.5g of the carnosol with high content of more than or equal to 98%.

(III) HP L C assay

HP L C detection is carried out on the carnosol obtained in the step (II), and the result is shown in figure 1.

Liquid chromatograph: waters 2545 model 2998 detector

A chromatographic column: waters Xbridge TM Shield RP 18 Column (4.6 x 250mm, 5 μm)

Flow rate 1m L/min

Detection wavelength: 284nm

Sample introduction amount: 10 μ l

Preparing solution of a test sample and a reference sample: chromatographic grade methanol dissolution

Detecting the mobile phase of carnosol: 70: 30: 3 thousandths of methanol, water and glacial acetic acid

The sethoxydim peak emergence time: 13.76min

(IV) measurement of one-dimensional nuclear magnetic resonance Spectroscopy

Detecting the carnosol sample with the content of more than or equal to 98% obtained in the step (II) by using an INOVA-500 nuclear magnetic resonance instrument (Varian corporation, USA) to obtain one-dimensional nuclear magnetic hydrogen spectrum and carbon spectrum data, and the result is shown in attached figure 2 and figure 3.

1. Spectral data of carnosol

¹H-NMR(DMSO，500MHz)：0.78(3H，s，H-18)，0.81(3H，s，H-19)，1.12(3H，d，J＝7.0Hz，H-16)，1.23(3H，d，J＝7.0Hz，H-17)，3.22(1H，m，H-15)，5.46(1H，s，H-7)，6.70(1H，s，H-14)；

¹³C-NMR(DMSO，100MHz)：28.81(C-1)，18.57(C-2)，40.55(C-3)，34.18(C-4)，47.87(C-5)，29.26(C-6)，76.95(C-7)，134.25(C-8)，131.57(C-9)，44.95(C-10)，143.30(C-11)，143.07(C-12)，121.87(C-13)，111.28(C-14)，26.20(C-15)，22.82(C-16)，22.69(C-17)，31.38(C-18)，19.44(C-19)，175.54(C-20)。

Comparing with the known literature, the structure of the carnosol is finally confirmed.

Example 3 preparation of high purity carnosol from Rosmarinus officinalis extract

Normal phase silica gel column separation and purification

The carnosol component with the content of about 85 percent is obtained by adopting the same method as the step (one) of the example 2.

(II) gel column separation and purification

And (2) repeatedly purifying the carnosol component containing about 85 wt% obtained in the step (I) by using hydroxypropyl Sephadex L H-20 gel, loading the gel column with a wet method, loading the gel column with the wet method, eluting the mobile phase by using chloroform and methanol at a volume ratio of 2: 1, wherein the flow rate of the mobile phase is 3ml/min, the chromatography time is 500min, combining the same components, and evaporating to dryness under reduced pressure to obtain 40.8g of the carnosol with high content of more than or equal to 98%.

(III) HP L C assay

And (3) respectively carrying out HP L C detection on the carnosol obtained in the step (II), wherein the detection conditions are the same as those of the step (III) in the example 2, and the peak emergence time of the carnosol is similar to that of the example 2.

Example 4 preparation of high purity carnosol from Rosmarinus officinalis extract

Normal phase silica gel column separation and purification

(II) gel column separation and purification

And (2) repeatedly purifying the carnosol component containing about 85 wt% obtained in the step (one) by using hydroxypropyl Sephadex L H-20 gel, wherein the length of the gel column is 150cm, the gel column is filled by a wet method, the sample is loaded by the wet method, the mobile phase is eluted by petroleum ether, chloroform and methanol in a volume ratio of 5: 1, the flow rate of the mobile phase is 6ml/min, the chromatography time is 490min, the same components are combined, and the mixture is evaporated to dryness under reduced pressure to obtain 41.3g of the carnosol with high content of more than or equal to 98%.

(III) HP L C assay

HP L C detection is carried out on the carnosol obtained in the step (II), the detection conditions are the same as those of the step (III) in the example 2, and the result is that the peak time of the carnosol is similar to that of the example 2.

Example 5 preparation of high purity carnosol from Rosmarinus officinalis extract

Normal phase silica gel column separation and purification

(II) gel column separation and purification

And (2) repeatedly purifying the carnosol component containing about 85 wt% obtained in the step (one) by using hydroxypropyl Sephadex L H-20 gel, wherein the length of a gel column is 150cm, the gel column is filled by a wet method, the sample is loaded by the wet method, the mobile phase is eluted by petroleum ether, chloroform and methanol in a volume ratio of 2: 1, the flow rate of the mobile phase is 5ml/min, the chromatography time is 500min, the same components are combined, and the mixture is evaporated to dryness under reduced pressure to obtain 41.2g of the carnosol with high content of more than or equal to 98%.

(III) HP L C assay

Comparative example 1 dissolution test of rosemary extract

Six portions of the rosemary extract prepared in example 1 were weighed out, 10g portions were divided into six groups, each group was dosed with 100 ml: ultrasonically dissolving petroleum ether, chloroform, ethyl acetate, acetone, 80% ethanol and water saturated ethyl acetate for 0.5 h, filtering to remove insoluble substances, concentrating the filtrate under reduced pressure to obtain extract, weighing, detecting and calculating the content of carnosol.

The specific grouping operation is as follows:

a first group: dissolving 10g herba Rosmarini officinalis extract in 100ml petroleum ether under ultrasonic for 0.5 hr, filtering to remove insoluble substances, and concentrating the filtrate under reduced pressure to obtain extract 3.9g, carnosol content is 14.15%, and calculating carnosol absolute content is 0.5519 g. The petroleum ether cannot completely dissolve the carnosol in the extract.

Second group: dissolving 10g herba Rosmarini officinalis extract with 100m1 chloroform under ultrasonic for 0.5 hr, filtering to remove insoluble substances, and concentrating the filtrate under reduced pressure to obtain extract 5.0g, carnosol content is 15.10%, and calculating absolute content of carnosol to be 0.7550 g. Chloroform dissolution does not completely dissolve the carnosol in the extract.

Third group: dissolving 10g herba Rosmarini officinalis extract with 100m ethyl acetate 1 for 0.5 hr, filtering to remove insoluble substances, and concentrating the filtrate under reduced pressure to obtain extract 5.6g, which contains carnosol 16.01% and calculated as absolute carnosol 0.8966 g. The carnosol in the extract cannot be completely dissolved out even if the ethyl acetate is dissolved.

And a fourth group: dissolving 10g herba Rosmarini officinalis extract in 100ml acetone under ultrasonic for 0.5 hr, filtering to remove insoluble substances, and concentrating the filtrate under reduced pressure to obtain extract 6.0g, carnosol content is 15.87%, and absolute carnosol content is calculated to be 0.9522 g. Acetone is dissolved, and the carnosol in the extract cannot be completely dissolved out.

And a fifth group: dissolving 10g herba Rosmarini officinalis extract in 100ml 80% ethanol under ultrasonic for 0.5 hr, filtering to remove insoluble substances, and concentrating the filtrate under reduced pressure to obtain extract 6.5g, carnosol content is 15.42%, and calculating carnosol absolute content to be 1.0023 g. Dissolving with 80% ethanol to completely dissolve carnosol in the extract.

A sixth group: dissolving 10g herba Rosmarini officinalis extract in 100ml water saturated ethyl acetate under ultrasonic condition for 0.5 hr, and concentrating the filtrate under reduced pressure to obtain extract 6.3g, wherein the carnosol content is 16.27%, and the calculated absolute content of carnosol is 1.0250 g. Dissolving with water saturated ethyl acetate, can completely dissolve carnosol in the extract, remove large polar impurities to the utmost extent, and recover solvent easily.

Claims

1. A method for preparing high purity carnosol, comprising the steps of:

mixing the filtrate with normal phase silica gel, loading the filtrate on a column by a dry method, loading the filtrate on a dry method, and mixing the filtrate with chloroform: methanol: carrying out gradient elution on acetic acid (100-95): (0-5): (0-1 ‰) (volume ratio), and combining the same components to obtain 5 components, wherein each component is as follows: a small polar impurity part, a carnosol crude product, a carnosic acid crude product, a component with the main component of ursolic acid and a large polar impurity part;

and III, purifying the crude carnosol obtained in the step II by using a hydroxypropyl sephadex column, and using chloroform as a mobile phase: methanol (2-2.5) 1 (volume ratio) or petroleum ether: chloroform: and (2-5) methanol are subjected to isocratic elution, the same components are combined, and the components are evaporated to dryness under reduced pressure to obtain the carnosol with the content being not less than 98%.

2. The method of claim 1, wherein the rosemary extract is an aqueous ethanol extract of rosemary flowers or leaves.

3. The method of claim 1, wherein: in the step I, the rosemary extract is dissolved by using water saturated ethyl acetate.

4. The process according to claim 1, wherein the gradient elution in step I is carried out in five times, with the mobile phases being, in order: chloroform; chloroform, methanol and acetic acid, wherein the volume ratio is 100: 0: 1 per mill; chloroform, methanol and acetic acid, wherein the volume ratio is 100: 1: 1 per mill; chloroform, methanol and acetic acid, wherein the volume ratio is 100: 2: 1 per mill; chloroform, methanol and acetic acid, wherein the volume ratio is 95: 5: 1 per mill.

5. The method according to claim 1, wherein the length of the hydroxypropyl sephadex column in the step II is 120-150 cm; loading columns by a wet method, and loading samples by the wet method; the flow rate of the mobile phase in the hydroxypropyl dextran gel is 2-6 ml/min.

6. The method according to any one of claims 1 to 5, wherein the ratio of chloroform: methanol 2.5: 1 (volume ratio), or petroleum ether: chloroform: methanol 5: 5: 1 (volume ratio).