CN111135810B

CN111135810B - Preparation method of special chromatographic separation medium for cannabidiol separation

Info

Publication number: CN111135810B
Application number: CN202010072864.XA
Authority: CN
Inventors: 张维冰; 李笃信; 戈兆松
Original assignee: SOOCHOW HIGH TECH CHROMATOGRAPHY CO LTD
Current assignee: SOOCHOW HIGH TECH CHROMATOGRAPHY CO LTD
Priority date: 2020-01-22
Filing date: 2020-01-22
Publication date: 2022-08-23
Anticipated expiration: 2040-01-22
Also published as: CN111135810A

Abstract

The invention relates to a preparation method of a special chromatographic separation medium for separating cannabidiol, which is characterized by comprising the following specific steps: (1) adding elemental bromine into an organic solvent containing a certain mole number of cannabinol, and stirring; (2) adding the porous amino silicon spheres into the organic solvent, stirring, heating for reaction for 24 hours, and filtering; (3) putting the bonded silicon spheres into the organic solvent again, adding a proper amount of pentafluorophenylacetic acid, stirring, uniformly mixing, adding a catalyst, heating for reacting for 24 hours, and filtering; (4) repeatedly washing with organic solvent, and oven drying to obtain chromatographic separation medium. The chromatographic separation medium bonded with two groups has better performance than that of a bonded single group in both bonding density and separation selectivity, and particularly has better separation performance on aromatic compounds and derivatives thereof.

Description

Preparation method of special chromatographic separation medium for cannabidiol separation

Technical Field

The invention relates to the technical field of chromatographic separation, in particular to a preparation method of a chromatographic separation medium special for cannabidiol separation.

Background

Cannabidiol is a non-toxic non-addictive active substance in cannabis and has pharmacological and pharmaceutical effects of anticonvulsant spasticity, anti-epilepsy, anti-anxiety, anti-bacterial, anti-inflammatory, anti-anesthesia and nervous system protection; meanwhile, the effect of tetrahydrocannabinol on the nervous system of a human body can be effectively eliminated, and the medicine is known as an antitoxic compound. The industrial hemp has high economic value, great development potential, good development and utilization of the green resource of the industrial hemp, and wide prospect in China. Cannabis contains various phenolic substances, such as Cannabinol (CBN), Cannabidiol (CBD), Tetrahydrocannabinol (THC), Cannabigerol (CBG), etc., and due to the medicinal value of Cannabidiol (CBD), efficient extraction of high-purity Cannabidiol (CBD) is a hot point of research. The following methods are commonly used for extracting cannabidiol:

the solvent extraction method comprises the steps of filling dried hemp leaves into an extraction container, adding a solvent, carrying out reflux extraction, filtering an extracting solution, concentrating to obtain an extract, carrying out back extraction by using different solvents, gradually improving the purity of cannabidiol, wherein organic solvent (petroleum ether, n-hexane, dichloromethane and the like) residues easily exist in the traditional process, and the traditional process is wide in variety of solvents, large in solvent consumption, complex in process and low in extraction rate.

The supercritical fluid or sub-fluid extraction technology for processing the dry hemp leaves has the advantages of high efficiency, good quality and little pollution, but has high cost for industrial production, high energy consumption in the operation process, high maintenance cost, limited production capacity and low yield. The cannabidiol produced has low purity and is not suitable for the medical field. Patent CN 110314408A provides a process for extracting cannabidiol, which mainly uses supercritical carbon dioxide extraction equipment to extract and separate the obtained cannabidiol intermediate product to obtain cannabidiol, but the yield of the device is slightly low.

The chromatographic principle of column chromatography CBD purification is divided into adsorption chromatography and ion exchange chromatography, and the chromatography materials are more, including macroporous resin, polyamide, silica gel, MCI, ion exchange resin and the like. And (3) dissolving the cannabinol crude product by utilizing the adsorbability of a chromatographic material, selecting an eluent, and eluting by using the eluent to obtain a slightly pure CBD solution. However, the column chromatography purification method has the disadvantages of unreasonable process, low yield, complex reagents or materials, expensive stationary phase, difficult adsorbent regeneration, difficult solvent recycling and the like. Patent CN 110143855 a provides a method for separating and purifying cannabinol by elution using alumina as a column chromatography stationary phase. Patent CN 201810022445 discloses a preparation method of high purity cannabidiol, which adopts macroporous resin chromatography and polyamide column chromatography combined technology to carry out purification, and then solvent recrystallization.

High-speed counter current chromatography (HSCCC) is a newly developed high-efficiency liquid-liquid partition chromatography technology, which utilizes a two-phase solvent system to establish a special unidirectional hydrodynamic equilibrium in a spiral tube rotating at high speed, wherein one phase is used as a stationary phase, the other phase is used as a mobile phase, and substances are separated according to different partition coefficients in the two phases, so that sample loss, inactivation, denaturation and the like caused by non-adsorption are avoided, and the method is very suitable for separation of natural active biological components. Patent CN 110256206 a discloses a method for separating and extracting cannabidiol by high-speed counter-current chromatography, which uses chloroform and methanol as solvents, dissolves the cannabidiol extract in a mobile phase, and pumps the solution into a counter-current chromatography tube at a certain flow rate. However, the method is complicated to operate, needs professional personnel to operate, and has the highest purity of only 98 percent.

The microbiological method is to ferment the plant leaves by one or more fungi, and utilize the enzymatic reaction of the microorganism to decompose the cell walls of the plant cells, thereby improving the permeability of the hemp leaves cells and being beneficial to the permeation and dissolution of the later-stage extraction solvent. Patent CN 110041172 a discloses a process for improving cannabidiol extraction rate by treating hemp flower leaves with microorganisms, which comprises culturing fungi from hemp plant leaves, roots and stems, fermenting the hemp flower leaves, adding a mixture of various solvents, extracting, and purifying. The method is too complex in process, relates to the field of biology, and is complicated in subsequent treatment.

The chromatography is a mode with higher utilization rate in the process of separating and purifying natural products, and can be divided into adsorption chromatography, partition chromatography, ion exchange chromatography, gel chromatography and the like according to the principle, and different eluents are adopted according to different fillers. Patent CN 110304993 a discloses a method for producing high-purity cannabidiol by chromatography.

The existing technology for producing cannabidiol has many defects, such as low purity, low yield, complex steps and the like. In order to solve the problems, the invention provides a synthesis method of a chromatographic column filler special for separating cannabidiol, cannabinol and pentafluorophenethyl are bonded on silicon spheres, two groups simultaneously act on cannabidiol, thus enhancing the retention of cannabidiol, not only making up the gap that the traditional C18 single bonded filler cannot meet the existing separation requirements, but also obtaining high-purity cannabidiol, and being capable of being used for preparing chromatography and meeting the industrial production requirements.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a preparation method of a chromatographic separation medium special for cannabidiol separation.

The purpose of the invention is realized by the following technical scheme:

a method for preparing chromatographic separation medium specially used for separating cannabidiol comprises bonding cannabinol to the surface of porous amino silicon ball, and bonding with pentafluorophenylacetic acid with small volume to the middle of cannabinol, so that two groups having effect on separation are bonded to the surface of silicon ball. The method comprises the following specific steps:

(1) adding elemental bromine into an organic solvent containing cannabinol, and stirring;

the organic solvent is toluene or carbon tetrachloride and carbon disulfide, preferably carbon tetrachloride;

(2) adding the porous amino silicon spheres into the organic solvent, stirring, heating for reaction for 24 hours, and filtering;

the ratio of the simple substance bromine to the cannabinol to the porous amino silicon spheres is as follows: elemental bromine: cannabinol is 2: 5: 5-7: 9:9, the particle size of the porous amino silicon ball is 5-100 microns;

(3) placing the bonded silicon spheres in the organic solvent again, adding the pentafluorophenylacetic acid, stirring, uniformly mixing, adding the catalyst, heating for reacting for 24 hours, and filtering;

the dosage of the pentafluorophenylacetic acid is porous amino silicon spheres: pentafluorophenylacetic acid was 5: 1-5: 2;

the catalyst is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxysuccinimide, 1-hydroxybenzotriazole, preferably 1-hydroxybenzotriazole;

the dosage of the catalyst is 0.5 to 5 percent of that of the pentafluorophenylacetic acid;

(4) repeatedly washing with an organic solvent, and drying to obtain a chromatographic separation medium;

the washing times are 2-4 times, preferably 3 times;

the heating and drying temperature in the above steps is 70-80 ℃, preferably 75 ℃.

Compared with the prior art, the invention has the following positive effects:

the special medium for separating the cannabidiol is a silicon ball bonded two separation groups, has higher bond and density compared with a single bonded group, has interaction on the cannabidiol simultaneously, enhances the retention of the cannabidiol in a reversed phase chromatographic column, is easy to elute, can be used for a preparative chromatographic column, can meet the requirement of industrial continuous preparation, and has high purity of the collected cannabidiol.

Drawings

FIG. 1 is a prior art stationary phase bonding formula;

FIG. 2 is a chemical synthesis scheme of the present invention;

FIG. 3 is a crude HPLC analysis of cannabidiol;

FIG. 4 is a HPLC analysis chart of cannabidiol after separation and purification by the cannabidiol-specific medium;

FIG. 5 is a crude HPLC analysis of another cannabidiol;

FIG. 6 is a HPLC analysis chart of cannabidiol after separation and purification by the cannabidiol-specific medium;

Detailed Description

The following provides a specific embodiment of the preparation method of the chromatographic separation medium special for cannabidiol separation.

Example 1

A method for preparing chromatographic separation medium special for separating cannabidiol comprises bonding cannabinol to the surface of porous silicon spheres, and further bonding with pentafluorophenethyl group with small volume to the gaps of cannabinol molecules to make the surface of the silicon spheres have two functional groups effective for separation; the method comprises the following specific steps:

(1) adding 30g of elemental bromine and 30g of cannabinol into carbon tetrachloride in sequence, and fully and uniformly stirring;

(2) adding 20g of porous amino silicon spheres with the particle size of 5 mu m, stirring to wet the porous amino silicon spheres, reacting for 24 hours at 75 ℃, and filtering to obtain bonded silicon spheres;

(3) putting the bonded silicon spheres into carbon tetrachloride again, adding 8g of pentafluorophenylacetic acid, stirring and mixing uniformly, adding 0.3g of 1-hydroxybenzotriazole, reacting at 75 ℃ for 24 hours, and filtering to obtain bonded silicon spheres;

(4) washing bonded silicon ball with carbon tetrachloride for 3 times, drying at 75 deg.C to obtain chromatographic separation medium, and filling into chromatographic column.

Column: 15X 310mm, 5 μm,

wavelength: 210nm

Mobile phase: methanol-water

Flow rate: 10.6ml/min

The crude cannabidiol was taken and analyzed by C18 reverse phase chromatography as shown in FIG. 3.

The cannabidiol was separated and purified by means of a packed preparative chromatography column and the purified cannabidiol was analyzed by means of a C18 reverse phase chromatography column, see FIG. 4. Table 1 purity of purified cannabidiol of example 1, enriched to 99.34%;

TABLE 1

Peak(s)	Retention time (min)	Area (μ U s)	Height (mu U)	Area (%)
					1	5.955	341674	23272	0.66
2	21.803	51708496	3489116	99.34

Example 2

The method changes the mixture ratio of raw materials and a plurality of reagents to prepare the medium special for separating the cannabidiol, and comprises the following steps:

(1) sequentially adding 50g of elemental bromine and 50g of cannabinol into carbon tetrachloride, and fully and uniformly stirring;

(2) adding 38g of porous amino silicon spheres with the particle size of 10 mu m, stirring to wet the porous amino silicon spheres, reacting for 24 hours at 80 ℃, and filtering to obtain bonded silicon spheres;

(2) putting the bonded silicon spheres into carbon tetrachloride again, adding 15g of pentafluorophenylacetic acid, stirring and mixing uniformly, adding 0.5g N-hydroxysuccinimide, reacting for 24 hours at 80 ℃, and filtering to obtain bonded silicon spheres;

(3) washing bonded silicon spheres with carbon tetrachloride for 3 times, drying at 80 ℃ to obtain a chromatographic separation medium, and filling into a chromatographic column.

Column: 15X 310mm, 10 μm,

wavelength: 210nm

Mobile phase: methanol-water

Flow rate: 10.6ml/min

The crude cannabidiol was taken and analyzed by C18 reverse phase chromatography as shown in FIG. 5.

The separation and purification was performed by using the packed preparative chromatography column, and the purified cannabidiol was analyzed by using a C18 reverse phase chromatography column, as shown in FIG. 6. Table 2 shows the purity of cannabidiol after purification in case 2, which was up to 99.90% after enrichment.

TABLE 2

Peak(s)	Retention time (min)	Area (μ U s)	Height (mu U)	Area (%)
					1	6.927	21679	2890	0.10
2	23.498	22379283	1267303	99.90

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the concept of the present invention, and these modifications and decorations should also be regarded as being within the protection scope of the present invention.

Claims

1. A preparation method of a special medium for separating cannabidiol is characterized by comprising the following specific steps:

(2) adding the porous amino silicon spheres into the organic solvent, stirring, heating for reaction for 24 hours, and filtering to obtain bonded silicon spheres;

(3) placing the bonded silicon spheres in an organic solvent again, adding pentafluorophenylacetic acid, stirring, uniformly mixing, adding a catalyst, heating for reacting for 24 hours, and filtering;

the organic solvent is toluene or carbon tetrachloride or carbon disulfide;

the dosage of the porous amino silicon ball, the elemental bromine and the cannabinol is as follows: elemental bromine: cannabinol is 2: 5: 5-7: 9: 9;

in the steps (2) and (3), the heating temperature is 70-80 ℃;

in the step (3), the catalyst is N-hydroxysuccinimide or 1-hydroxybenzotriazole;

in the step (3), the amount of the catalyst is 0.5-5% of that of pentafluorophenylacetic acid;

in the step (3), the dosage of the pentafluorophenylacetic acid is porous amino silicon spheres: pentafluorophenylacetic acid was 5: 1-5: 2.

2. the method for preparing a cannabidiol separation dedicated medium as claimed in claim 1, wherein in the step (2), the particle size of the porous amino silicon spheres is 5-100 μm.

3. The method for preparing a medium dedicated for cannabidiol separation as claimed in claim 1, wherein in step (4), the number of washing is 2-4.