A method for effectively separating cannabidiol, cannabidivarin, tetrahydrocannabinol

Abstract

The application discloses a method for effectively separating cannabidiol, cannabidivarin and tetrahydrocannabinol. The application uses a chromatographic separation and purification method, uses a coordination resin as a filler, and uses a mixed solution of polar and non-polar solvents as an eluent, so as to separate a cannabidiol pure product with a purity of greater than or equal to 99% from a cannabidiol crude product. The cannabidiol and the analogs tetrahydrocannabinol and cannabidivarin are completely separated by one-step purification. The ligand in the coordination resin is used to form a coordination complex with CBD, so that the retention time difference between CBD, THC and CBDV is increased, and thus a good separation effect is achieved. The CBD, CBDV and THC can be completely separated by selecting specific coordination resins and specific elution conditions, and the separation selectivity is good. The purity of the cannabidiol obtained by the separation method can be higher than 99%, the yield can be higher than 80%, and the separation method is suitable for the separation and purification of high-purity CBD.

Description

TECHNICAL FIELD

[0001] The application relates to a method for effectively separating cannabidiol, cannabidivarin and tetrahydrocannabinol, in particular to a method for separating cannabidiol, cannabidivarin and tetrahydrocannabinol by using coordination chromatography, and belongs to the technical field of biological medicines. BACKGROUND

[0002] Cannabidiol (CBD) is a natural product extracted from a cannabis plant, has good medicinal value, has the effects of anti-tumor, neuroprotection, metabolism and immune regulation, anti-inflammatory, anti-oxidation, cardiovascular protection, antibacterial and the like. The main chemical components contained in cannabis include cannabidiol (CBD), tetrahydrocannabinol (THC) and cannabidivarin (CBDV) in addition to CBD. THC is a hallucinogenic component in cannabis and has neuroactivity and is widely considered to be the most important active substance in cannabis, but due to the hallucinogenic effect and addiction of THC, it can be used to refine drugs, which seriously endangers social health. CBDV is a phytocannabinoid naturally existing in a cannabis plant, which is similar in structure to CBD. The two analogs are difficult to remove by traditional chromatography and crystallization methods, resulting in great difficulty in separation and purification of CBD and low yield.

[0003] Cannabidiol is not only a natural active ingredient with great development prospect in the fields of medicine, food, health care products, daily chemical products and the like, and can antagonize the hallucinogenic effect of THC in the human body, so it is extremely concerned by the public.

[0004] The molecular formula of cannabidiol is C 21 H 30 O2, which is generally white to light yellow crystals, has a melting point of 66-67 DEG C, is almost insoluble in water, and is soluble in organic solvents such as ethanol, methanol, benzene, chloroform and the like. The structure of CBD is as follows:

[0005]

[0006] At present, the purification process of CBD still has defects such as small batch yield, low yield and low product purity, and most of the current technical means are complex in process and tedious in extraction and purification procedures, which are not suitable for industrial production. The purification process of CBD mainly includes resin adsorption and chromatographic separation. Among them, chromatographic separation includes silica gel chromatography, alumina chromatography and polyamide column chromatography process.

[0007] CN111747826A, CN110386861A, CN110156568A and the like adopt macroporous resin adsorption and silica gel column chromatography process to purify cannabidiol, but the process has small load, low yield and insufficient industrial production.

[0008] CN111960930A adopts a three-stage purification process, AB-8 macroporous resin gradient elution, then elution by silica gel column, and finally preparation liquid chromatography by C18 filler to obtain a high-purity cannabidiol solution. Although the product purity is high, the process is complicated and the production cost is high, which is not suitable for industrial production.

[0009] CN110655453A discloses a method for extracting and separating secondary cannabidiol, using organic solvent extraction to enrich secondary cannabidiol, purifying and enriching by polyamide resin column, neutral alumina and bonded silica gel column, and obtaining high-purity secondary cannabidiol by crystallization. This process requires multiple purification processes, is complicated to operate, requires high equipment, has high production cost, and is not suitable for industrial production.

[0010] The above commonly used separation processes have certain limitations: the macroporous adsorption resin used in most current separation processes is difficult to completely separate CBD, CBDV and THC, and often requires multiple purifications, which also leads to low final yield, complicated process, and cannabidiol purity that does not meet the requirements; polyamide resin will adsorb and resolve both CBD and THC, making it difficult to separate the two, and high-purity CBD solution can only be obtained by combining other processes. The silica gel is disposable, which has high production cost. At the same time, the silica gel column has small loading capacity, which ultimately leads to low extraction efficiency. The C18, alumina and silica gel fillers used in the above separation techniques have high production cost, and the column bed pressure of these materials is high, which requires high equipment and is difficult to industrialize. For separation processes that require multiple purifications, the consumption of eluent is large and more complex, causing greater environmental pollution. SUMMARY

[0011] The technical problem to be solved by the present application is that the main components in the crude cannabis obtained from cannabis plants include cannabidiol, secondary cannabidiol and tetrahydrocannabinol, which are extremely similar in structure, so the existing separation and purification technology of cannabidiol cannot completely separate the three, and multiple purification processes are required, which is complicated to operate, requires elution under medium-high pressure, has high production cost, and is not suitable for industrial production.

[0012] To solve the above technical problems, the present application provides a method for effectively separating cannabidiol, secondary cannabidiol and tetrahydrocannabinol, using coordination resin as filler and adopting chromatographic separation and purification method to separate cannabidiol from cannabidiol crude product, wherein the coordination resin is one of HZ-6560, HZ-6563, HZ-6566 and HZ-6567.

[0013] Preferably, in the method for separating and purifying, the mixture of the polar organic solvent A and the non-polar organic solvent B is used as the eluent.

[0014] Preferably, the polar organic solvent A is a mixture of one or more of methanol, ethanol, acetonitrile, acetone, n-butanol, ethyl acetate, dichloromethane and chloroform; and the non-polar organic solvent B is a mixture of one or more of petroleum ether, methylcyclohexane, toluene, xylene, pentane, n-hexane and 1,2-dichloroethane.

[0015] Preferably, the volume ratio of the polar organic solvent A to the non-polar organic solvent B in the eluent is 20:80 to 80:20.

[0016] More preferably, the polar solvent is ethyl acetate and / or chloroform; the non-polar solvent is n-hexane; and the volume ratio of the polar solvent to the non-polar solvent is 20 to 40:60 to 80.

[0017] Preferably, the method for separating and purifying comprises the steps of pretreatment, sample loading and elution of the chromatographic column filled with the coordination resin.

[0018] Preferably, the step of pretreating the chromatographic column comprises sequentially activating with anhydrous ethanol solution and equilibrating with the eluent.

[0019] Preferably, the sample loading amount in the sample loading step is 5 to 15 mg / mL of column volume.

[0020] Preferably, the elution is isocratic elution, the isocratic elution process is to flush the chromatographic column with the eluent, the flow rate of the elution is 0.2 to 5 column volumes per hour, the volume of the eluent is 9 to 12 column volumes, and then the elution is ended, the component rich in cannabidiol is collected, and the cannabidiol pure product is obtained after concentration.

[0021] Compared with the prior art, the present application has the following beneficial effects:

[0022] (1) The present application can realize complete separation of CBD, CBDV and THC by using the coordination chromatography separation method, the ligand in the coordination resin forms a coordination complex with CBD, thereby increasing the retention time difference between CBD, THC and CBDV, so that a good separation effect is achieved; the present application obtains a method for separating and purifying cannabidiol by selecting a specific coordination resin under specific elution conditions, the separation selectivity is good, the purity of the cannabidiol pure product purified by the method reaches more than 99%, the yield reaches more than 80%, and the method is suitable for separating and purifying high-purity CBD.

[0023] (2) In view of the shortcomings of the prior art, the present invention provides a novel process for effectively separating cannabidiol, hypocannabidiol and tetrahydrocannabinol. The purification method of cannabidiol provided by the present invention has high purity, and can completely separate cannabidiol, hypocannabidiol and tetrahydrocannabinol in only one purification process. It is simple and convenient to operate, has low equipment requirements, low solution consumption, is environmentally friendly, and reduces production costs. It is suitable for large-scale production.

[0024] (3) The present invention can completely separate CBD from its analogues CBDV and THC through one-step purification, and the purity of the separated cannabidiol can reach higher than 99%. The separation method of the present invention has a simple process flow, can achieve the separation effect at room temperature and pressure, has low equipment requirements, is simple and controllable to operate, and has a stable implementation process. The method of the present invention only requires one-step purification process, without the need for multiple purification steps, thereby achieving higher yield and reducing production costs. At the same time, the obtained product has high purity and high yield, which is suitable for industrial production. Attached Figure Description

[0025] Figure 1 This is the HPLC chromatogram of crude cannabidiol from Example 1;

[0026] Figure 2 This is an HPLC peak area diagram of cannabidiol, hypocannabidiol, and tetrahydrocannabinol eluted by coordination chromatography in Example 1;

[0027] Figure 3 This is the HPLC chromatogram of pure cannabidiol obtained after chromatography in Example 1. Detailed Implementation

[0028] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0029] In the following examples, unless otherwise specified, the reagents, packing materials and experimental equipment used are all commercially available products; the chromatography column used has a size of 21.6×126mm, and coordination chromatography packing material is used as the chromatography column packing material, with a column volume of 36mL.

[0030] Example 1

[0031] This embodiment provides a method for purifying cannabidiol, the steps of which are as follows:

[0032] Crude cannabidiol with a purity of 56.74% was analyzed by high-performance liquid chromatography (HPLC) as follows: Figure 1The sample was dissolved in n-hexane solution and then loaded, with a loading amount / colum volume of 6.5 mg / mL. The column filler was HZ-6560 type coordination resin, which was pre-processed by activating with anhydrous ethanol solution and then equilibrated with a mixed solution of ethyl acetate and n-hexane in a volume ratio of 30:70. After loading, isocratic elution was performed at 25°C under normal pressure with a flow phase of ethyl acetate and n-hexane in a volume ratio of 30:70, at a flow rate of 1.0 BV / h. The fractions were collected in sections, and the peak area results of cannabidiol, cannabidivarin and tetrahydrocannabinol in each fraction are shown in Table 1. Figure 2 The fractions rich in CBD were combined and concentrated to obtain a cannabidiol product. HPLC analysis showed that the purity of the cannabidiol product was 99.2%, and CBDV and THC were not detected. The HPLC results are shown in Table 2. Figure 3 The yield of CBD was calculated to be 83.3%.

[0033] Example 2

[0034] This example provides a method for purifying cannabidiol, with the following steps:

[0035] A crude cannabidiol product with a purity of 56.74% was taken, and its high performance liquid chromatography (HPLC) results are shown in Table 3. Figure 1 The sample was dissolved in n-hexane solution and then loaded, with a loading amount / colum volume of 6.5 mg / mL. The column filler was HZ-6560 type coordination resin, which was pre-processed by activating with anhydrous ethanol solution and then equilibrated with a mixed solution of ethyl acetate and n-hexane in a volume ratio of 30:70. After loading, isocratic elution was performed at 25°C under normal pressure with a flow phase of ethyl acetate and n-hexane in a volume ratio of 30:70, at a flow rate of 1.0 BV / h. The fractions were collected in sections, and the peak area results of cannabidiol, cannabidivarin and tetrahydrocannabinol in each fraction are shown in Table 1.

[0036] Example 3

[0037] A crude cannabidiol product with a purity of 56.74% was taken, and its high performance liquid chromatography (HPLC) results are shown in Table 3. Figure 1The crude cannabidiol product with a purity of 56.74% was dissolved in n-hexane solution and then loaded onto the column with a loading amount of 6.5 mg / mL per column volume. The column filler was HZ-6560 type coordination resin, and the column was pretreated before use. First, the column was activated with anhydrous ethanol solution, and then equilibrated with a mixed solution of ethyl acetate and n-hexane with a volume ratio of 50:50. After loading, isocratic elution was performed at 25°C under normal pressure using a mobile phase of ethyl acetate and n-hexane with a volume ratio of 50:50 at a flow rate of 1.0 BV / h. The fractions enriched in CBD were collected and combined, and then concentrated to obtain the cannabidiol product. HPLC analysis showed that the purity of the cannabidiol product was 98.1%, and the CBDV content was 0.23%.

[0038] Example 4

[0039] The crude cannabidiol product with a purity of 56.74% was dissolved in n-hexane solution and then loaded onto the column with a loading amount of 6.5 mg / mL per column volume. The column filler was HZ-6560 type coordination resin, and the column was pretreated before use. First, the column was activated with anhydrous ethanol solution, and then equilibrated with a mixed solution of ethyl acetate and n-hexane with a volume ratio of 50:50. After loading, isocratic elution was performed at 25°C under normal pressure using a mobile phase of ethyl acetate and n-hexane with a volume ratio of 50:50 at a flow rate of 1.0 BV / h. The fractions enriched in CBD were collected and combined, and then concentrated to obtain the cannabidiol product. HPLC analysis showed that the purity of the cannabidiol product was 98.1%, and the CBDV content was 0.23%. Figure 1 The crude cannabidiol product with a purity of 56.74% was dissolved in n-hexane solution and then loaded onto the column with a loading amount of 6.5 mg / mL per column volume. The column filler was HZ-6560 type coordination resin, and the column was pretreated before use. First, the column was activated with anhydrous ethanol solution, and then equilibrated with a mixed solution of ethyl acetate and n-hexane with a volume ratio of 50:50. After loading, isocratic elution was performed at 25°C under normal pressure using a mobile phase of ethyl acetate and n-hexane with a volume ratio of 50:50 at a flow rate of 1.0 BV / h. The fractions enriched in CBD were collected and combined, and then concentrated to obtain the cannabidiol product. HPLC analysis showed that the purity of the cannabidiol product was 98.1%, and the CBDV content was 0.23%.

[0040] Example 5

[0041] The crude cannabidiol product with a purity of 56.74% was dissolved in n-hexane solution and then loaded onto the column with a loading amount of 6.5 mg / mL per column volume. The column filler was HZ-6560 type coordination resin, and the column was pretreated before use. First, the column was activated with anhydrous ethanol solution, and then equilibrated with a mixed solution of ethyl acetate and n-hexane with a volume ratio of 50:50. After loading, isocratic elution was performed at 25°C under normal pressure using a mobile phase of ethyl acetate and n-hexane with a volume ratio of 50:50 at a flow rate of 1.0 BV / h. The fractions enriched in CBD were collected and combined, and then concentrated to obtain the cannabidiol product. HPLC analysis showed that the purity of the cannabidiol product was 98.1%, and the CBDV content was 0.23%. Figure 1 The crude cannabidiol product with a purity of 56.74% was dissolved in n-hexane solution and then loaded onto the column with a loading amount of 6.5 mg / mL per column volume. The column filler was HZ-6560 type coordination resin, and the column was pretreated before use. First, the column was activated with anhydrous ethanol solution, and then equilibrated with a mixed solution of ethyl acetate and n-hexane with a volume ratio of 50:50. After loading, isocratic elution was performed at 25°C under normal pressure using a mobile phase of ethyl acetate and n-hexane with a volume ratio of 50:50 at a flow rate of 1.0 BV / h. The fractions enriched in CBD were collected and combined, and then concentrated to obtain the cannabidiol product. HPLC analysis showed that the purity of the cannabidiol product was 98.1%, and the CBDV content was 0.23%.

[0042] As can be seen from Examples 1-5 above, the purification method for cannabidiol provided by the present invention can effectively separate cannabidiol, hypocannabidiol, and tetrahydrocannabinol. Under the elution conditions of Example 1, the purity of the purified cannabidiol can reach over 99%, and CBDV and THC were not detected. The elution conditions of Example 1 are optimal. Under the elution conditions of Example 2, CBDV and THC were also not detected, and the purity was 98.6%. Under the elution conditions of Examples 3-5, only trace amounts of CBDV were detected (CBDV content < 0.05%).

[0043] The product contained 0.25% (CBD), with no detected THC, and a purity exceeding 98%. Therefore, in the eluent of this invention, the volume ratio of polar organic solvent A to non-polar organic solvent B is 20:80 to 80:20, and elution is performed at a flow rate of 0.2-5 column volumes / hour. Elution is terminated after 9-12 column volumes, and the CBD-rich component is collected to obtain a high-purity cannabidiol solution. The coordination chromatography separation method of this invention can effectively separate cannabidiol from its analogues, hypocannabidiol and tetrahydrocannabinol, thereby obtaining high-purity cannabidiol.

[0044] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A method for effectively separating cannabidiol, hypocannabidiol, and tetrahydrocannabinol, characterized in that, Pure cannabidiol was obtained from crude cannabidiol by using a coordination resin as a packing material and chromatographic separation and purification method. The coordination resin is one of HZ-6560, HZ-6563, HZ-6566 and HZ-6567. The chromatographic separation and purification method includes: pretreatment, sample loading, and elution steps for a chromatography column packed with coordination resin; the pretreatment step for the chromatography column includes: activation with anhydrous ethanol solution and equilibration with eluent in sequence; the elution is isocratic elution, and the isocratic elution process is to wash the chromatography column with eluent, the elution flow rate is 0.2-5 column volumes / hour, the volume of eluent is 9-12 column volumes, then the elution is stopped, the fraction rich in cannabidiol is collected, and after concentration, pure cannabidiol is obtained; In the chromatographic separation and purification method, a mixture of polar and non-polar solvents is used as the eluent; the polar solvent is at least one selected from methanol, ethanol, acetonitrile, acetone, n-butanol, ethyl acetate, dichloromethane, and chloroform; the non-polar solvent is at least one selected from petroleum ether, methylcyclohexane, toluene, xylene, pentane, n-hexane, and 1,2-dichloroethane.

2. The method for effectively separating cannabidiol, hypocannabidiol, and tetrahydrocannabinol according to claim 1, characterized in that, The volume ratio of polar solvent to non-polar solvent in the eluent is 20:80 to 80:

20.

3. The method for effectively separating cannabidiol, hypocannabidiol, and tetrahydrocannabinol according to claim 2, characterized in that, The polar solvent is ethyl acetate and / or chloroform; the non-polar solvent is n-hexane; the volume ratio of the polar solvent to the non-polar solvent is 20~40:60~80.

4. The method for effectively separating cannabidiol, hypocannabidiol, and tetrahydrocannabinol according to claim 1, characterized in that, The sample loading amount in the sample loading step is 5-15 mg / mL column volume.

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