CN114685416A - Method for separating cannabinoid - Google Patents
Method for separating cannabinoid Download PDFInfo
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- CN114685416A CN114685416A CN202011613795.5A CN202011613795A CN114685416A CN 114685416 A CN114685416 A CN 114685416A CN 202011613795 A CN202011613795 A CN 202011613795A CN 114685416 A CN114685416 A CN 114685416A
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- cannabinol
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- phase extraction
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- VBGLYOIFKLUMQG-UHFFFAOYSA-N Cannabinol Chemical compound C1=C(C)C=C2C3=C(O)C=C(CCCCC)C=C3OC(C)(C)C2=C1 VBGLYOIFKLUMQG-UHFFFAOYSA-N 0.000 claims abstract description 103
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/78—Ring systems having three or more relevant rings
- C07D311/80—Dibenzopyrans; Hydrogenated dibenzopyrans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/58—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring other than with oxygen or sulphur atoms in position 2 or 4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/94—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems condensed with rings other than six-membered or with ring systems containing such rings
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
- Extraction Or Liquid Replacement (AREA)
Abstract
The invention provides a separation method of cannabinoids. The method for isolating cannabinoids comprises the following steps: (1) performing solid phase extraction, purification and concentration on a raw material sample to obtain a section I containing tetrahydrocannabinol and cannabinol and a section II containing cannabichromene and cannabinol; (2) and respectively carrying out column chromatography separation and purification on the I section containing the tetrahydrocannabinol and the cannabinol and the II section containing the cannabichromene and the cannabinol to obtain a tetrahydrocannabinol primary product, a cannabinol primary product, a cannabichromene primary product and a cannabinol primary product. According to the invention, wax, grease and pigment are effectively removed by adopting a solid-phase extraction and purification mode, the sectional purification effect is superior to other technical means, and the conventional column chromatography separation and purification preparation is combined, so that the purposes of recovering and separating cannabidiol, cannabinol, cannabichromene and cannabinol with high purity and high yield are realized, the destruction cost of the tail material containing tetrahydrocannabinol is reduced, and the production amplification can be realized.
Description
Technical Field
The invention relates to the technical field of extraction and separation of chemical components of natural plants, in particular to a separation method of magnetic cannabinoid.
Background
Industrial hemp (Cannabis sativa L.) is a plant of Cannabis of Cannabaceae, the content of Tetrahydrocannabinol (THC) in the flower and leaf in the growth period is less than three per thousand, and the industrial hemp can be legally planted in large scale and industrially developed and utilized. Industrial cannabis flowers and leaves contain cannabinoid and non-cannabinoid families and are widely used in the medical field. The cannabinoid family is mainly divided into Tetrahydrocannabinol (THC), Cannabinol (CBN), Cannabidiol (CBD), Cannabigerol (CBG), Cannabidivarin (CBDV), cannabichromene (CBC), cannabigerol (THCV), tetrahydrocannabinol, etc.; the main classes of non-cannabinoids are: cannabinoids, other terpenoids, beta-sitosterols; these extracts are widely used in the medical industry.
With the continued development of Cannabidiol (CBD), the medicinal value of other related cannabinoids is also increasingly recognized. For example, US8470874B2 teaches that natural or synthetic CBC and its derivatives prepared by them have a specific therapeutic effect in treating mood disorders such as depression and show a strong anti-inflammatory and antibacterial effect. More than one Cannabinol (CBL) is reported as one of the active ingredients, and no single-component pharmacological and efficacy data exist. Patent US20030158191a1 teaches the use of Cannabinol (CBN) and its derivatives in immune system disorders such as hiv disease and neoplastic diseases.
However, due to the hemp extract tails fraction, it usually contains a large amount of fatty waxes and pigments and is complex in composition. The most advanced treatment currently is molecular distillation, from which the cannabinoids are separated as a light phase, but provided that the distillation must be carried out with preferential wax removal and that only a mixture in one boiling range is obtained. At present, no industrial preparation method for materials with the characteristics exists.
CN112010738A discloses a chromatographic separation method of cannabinoid compounds, which comprises the step of carrying out chromatographic separation on a cannabis extract by using an aqueous ethanol solution, and comprises the following steps: (1) washing impurities, namely washing impurities by using an ethanol aqueous solution; (2) gradient elution, adopting ethanol water solution as gradient elution solvent, and respectively collecting eluent of 1-2BV, eluent of 2.5-4BV and eluent of 4-6 BV; (3) and (3) concentrating, namely concentrating the eluates collected in the step (2) respectively. The method can directly separate high-purity hypocannabidiol, cannabidiol and tetrahydrocannabinol; the method adopts a reverse separation mode in the whole process for improving the purity, but the reverse separation is undoubtedly not suitable for the factory tailings containing a large amount of wax, pigment and grease, and the reverse separation mode in the whole process also brings great difficulty to the concentration of post-treatment and is not suitable for industrial production.
CN111315459A discloses a process for the purification and isolation of cannabinoids from dried cannabis and cannabis leaves using simulated moving bed chromatography for the continuous purification of cannabinoids extracted from dried cannabis and cannabis leaves by purification steps of filtration, decolourisation, activation or decarboxylation, dewaxing, polishing and crystallisation and a continuous simulated moving bed process recovery step to purify cannabinoids, in particular cannabidiol and tetrahydrocannabinol, which is complicated with respect to waxiness, pigment and grease removal processes and fails to recover pure cannabinol, cannabichromene and cannabicyclol.
Therefore, the development of a process suitable for industrial processing of extraction plant tailings and efficient recovery and separation of various cannabinoids is a major research focus in the field.
Disclosure of Invention
In view of the deficiencies of the prior art, it is an object of the present invention to provide a method for isolating cannabinoids. The separation method provided by the invention realizes the purpose of recovering and separating cannabidiol, cannabinol, cannabichromene and cannabinol with high purity and high yield, and reduces the destruction component of the tetrahydrocannabinol-containing tailings.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for isolating a cannabinoid, the method comprising in particular the steps of:
(1) performing solid phase extraction, purification and concentration on a raw material sample to obtain a section I containing tetrahydrocannabinol and cannabinol and a section II containing cannabichromene and cannabinol;
(2) and respectively carrying out column chromatography separation and purification on the I section containing the tetrahydrocannabinol and the cannabinol and the II section containing the cannabichromene and the cannabinol to obtain a tetrahydrocannabinol primary product, a cannabinol primary product, a cannabichromene primary product and a cannabinol primary product.
In the invention, wax, grease and pigment are effectively removed by adopting a solid phase extraction and purification mode, the sectional purification effect is superior to other technical means (such as molecular distillation), and the conventional column chromatography separation and purification preparation is combined, so that the aims of recovering and separating cannabidiol, tetrahydrocannabinol, cannabinol, cannabichromene and cannabinol with high purity and high yield are realized, the destruction component of the tail material containing the tetrahydrocannabinol is reduced, and the method can realize production amplification.
Preferably, in the step (1), the content of tetrahydrocannabinol in the raw material sample is 0.3% or less by mass, and may be, for example, 0.3%, 0.25%, 0.2%, 0.15%, 0.1%, 0.05%, or the like.
Preferably, in the step (1), the cannabidiol content of the raw material sample is 2-20% by mass, the cannabinol content of the raw material sample is 1-10% by mass, the cannabichromene content of the raw material sample is 0.5-5% by mass, and the cannabinol content of the raw material sample is 2-20% by mass.
The cannabidiol may be present in an amount of 2-20% by mass, for example, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20% by mass, based on 100% by mass of the raw material sample.
The cannabinol content may be 1-10% by mass, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or the like, based on 100% by mass of the raw material sample.
The content of the cannabichromene may be 0.5 to 5% by mass, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or the like, based on 100% by mass of the raw material sample.
The content of cannabicyclol may be 2 to 20% by mass, for example, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20% or the like, based on 100% by mass of the raw material sample.
Preferably, in step (1), the raw material sample is cannabidiol extraction plant tailings or industrial cannabis extract raw material.
Wherein, the cannabidiol extraction plant tailing refers to that the rest part is collectively called extraction plant tailing after CBD extraction is complete; the industrial hemp extract material is alcohol extract of CBD or other organic solvent extract. Namely, the raw material sample can be industrial hemp with THC content below 0.3%, and the residual tailings are obtained after CBD is extracted by a Han Union pharmaceutical extraction factory; or the flower and leaf raw material is mature hemp flower and leaf of 7-9 months, and after the initial drying by farmers, the industrial hemp alcohol extract with THC content below 0.3 percent is also suitable for the preparation of the method.
Preferably, in step (1), the raw material sample is selected from any one of industrial cannabis, intermediate cannabis or medicinal cannabis or a combination of at least two of industrial cannabis;
preferably, in the step (1), the part extracted from the industrial hemp extract raw material is any one or a combination of at least two of hemp flowers, hemp leaves, hemp roots, hemp stalk cores or hemp seed meal.
Preferably, in step (1), the solid phase extraction is performed at a low temperature of-10 to 10 ℃, for example, -10 ℃, -5 ℃, 0 ℃, 5 ℃, 10 ℃ and the like.
Wherein, the low temperature condition is that a cold circulating liquid with the temperature of minus 10 ℃ to 10 ℃ is introduced into the chromatography column jacket, so that wax, pigment and most of grease are deposited on the top end of the column packing, and the subsequent treatment is easy.
Preferably, in step (1), the filler used for solid-phase extraction purification comprises any one of activated clay, diatomite, calcium phosphate, calcium carbonate, polyamide powder or silica gel or a combination of at least two of the above.
The filler adsorbent used in the invention can be regenerated properly and dried for repeated use (for example, clay and calcium phosphate can draw out the polluted parts such as wax at the top end, and can be repeatedly used after being treated at the temperature of 300 ℃ and 500 ℃ for 1-5 h), theoretically more than 100 times, and basically no production waste is generated.
Preferably, the particle size of the polyamide powder is 90 to 120 mesh, and may be, for example, 90 mesh, 95 mesh, 100 mesh, 105 mesh, 110 mesh, 115 mesh, 120 mesh, or the like.
Preferably, the particle size of the silica gel is 100-500 meshes, for example, 100 meshes, 200 meshes, 300 meshes, 400 meshes, 500 meshes and the like can be realized.
Preferably, the particle diameters of the above-mentioned other fillers, other than the polyamide powder and the colloidal silica, are each independently 30 to 300 mesh, and may be, for example, 30 mesh, 40 mesh, 50 mesh, 60 mesh, 80 mesh, 100 mesh, 150 mesh, 200 mesh, 250 mesh, 300 mesh, or the like.
Preferably, in step (1), the solid phase extraction purification is performed by using a solid phase extraction column.
Preferably, in step (1), the solid phase extraction column has a diameter-height ratio of 1 (1-3), and may be, for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, or the like.
In the invention, the diameter-height ratio of the solid phase extraction column is limited to 1 (1-3), so that the requirement of primary separation can be met, if the extraction column is shorter and has smaller diameter, the separation degree is reduced, and the cross superposition boundary of each separation section is unclear; if the extraction column is shorter in diameter and larger in height, although the separation effect is improved, the separation is slow, the risk of column blockage is more likely to occur, larger positive air pressure is needed for auxiliary flushing, and the waste of the filler adsorbent is caused.
Preferably, in the step (1), the mass ratio of the total amount of the filler in the solid phase extraction column to the raw material sample is 1 (0.25-5), and may be, for example, 1:0.25, 1:0.3, 1:0.4, 1:0.5, 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.5, 1:3, 1:4, 1:5, etc., preferably 1 (0.5-2).
Preferably, in step (1), the solid phase extraction is dry loading or wet loading.
Preferably, the dry loading is specifically: mixing part of the filler with a raw material sample to form a solid dispersion system, and transferring the solid dispersion system into a solid phase extraction column filled with the filler; the mass ratio of the partial filler to the raw material sample is 1 (0.25-2), and may be, for example, 1:0.25, 1:0.3, 1:0.4, 1:0.5, 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, etc., preferably 1 (0.5-1).
Preferably, the wet loading is specifically: mixing an organic solvent with a raw material sample to form a liquid dispersion system, and transferring the liquid dispersion system into a solid phase extraction column filled with a filler; the volume-to-mass ratio of the organic solvent to the raw material sample is 1mL (0.25-5) g, and may be, for example, 1mL:0.25g, 1mL:0.5g, 1mL:1g, 1mL:1.5g, 1mL:2g, 1mL:3g, 1mL:4g, 1mL:5g, or the like, preferably 1mL (0.5-2) g.
Preferably, the organic solvent for wet loading is an elution solvent, preferably any one or a combination of at least two of hexane, heptane or ethanol aqueous solution with a concentration of 40-65 v% (e.g. 40 v%, 45 v%, 50 v%, 55 v%, 60 v%, 65 v%, etc.).
Preferably, the wet loading is assisted by heating during dissolution or size mixing at 50-75 deg.C, such as 50 deg.C, 55 deg.C, 60 deg.C, 65 deg.C, 70 deg.C, 75 deg.C, etc.
Preferably, in step (1), the elution solvent used in the solid-phase extraction purification comprises any one of alkane, halogenated alkane, ester, alcohol, ketone, organic acid or water or a combination of at least two of them.
Preferably, the alkane is selected from any one of petroleum ether, diethyl ether, n-hexane or n-heptane or a combination of at least two of the same.
Preferably, the halogenated alkane is selected from dichloromethane and/or chloroform.
Preferably, the ester is selected from ethyl acetate and/or butyl acetate.
Preferably, the alcohol is selected from any one of methanol, ethanol or isopropanol or a combination of at least two thereof.
Preferably, the ketone is selected from acetone and/or butanone.
Preferably, the organic acid is selected from any one of formic acid, acetic acid or trifluoroacetic acid or a combination of at least two thereof.
In the present invention, the elution solvent used may be a binary solvent system such as a mixed solution of n-hexane and ethyl acetate, a mixed solution of ethyl acetate and acetone, or an aqueous ethanol solution of 45 to 100 v% (for example, 45 v%, 50 v%, 60 v%, 70 v%, 80 v%, 90 v%, 100 v%, etc.), an aqueous isopropanol solution of 85 v% or more (for example, 85 v%, 90 v%, 95 v%, etc.); the elution solvent used may also be a ternary solvent system, such as n-hexane, a mixture of ethyl acetate and formic acid, ethanol, a mixture of formic acid and water, and the like.
Preferably, the elution is carried out under the medium-low pressure condition of 0.05-15MPa, such as 0.05MPa, 0.06MPa, 0.08MPa, 0.1MPa, 0.2MPa, 0.4MPa, 0.6MPa, 0.8MPa, 1MPa, 2MPa, 4MPa, 6MPa, 8MPa, 10MPa, 12MPa, 14MPa, 15MPa, etc.
Preferably, in the step (1), during the solid phase extraction and purification process, the cannabidiol remaining in the raw material sample is obtained by eluting with the first solvent, then the cannabidiol is obtained by eluting with the second solvent to obtain the segment I containing tetrahydrocannabinol and cannabinol, and finally the segment II containing cannabichromene and cannabinol is obtained by eluting with the third solvent.
Preferably, in step (1), the first solvent is a mixture of alcohol or ketone, organic acid and water. That is, the first solvent may be a mixture of alcohol, organic acid and water, or a mixture of ketone, organic acid and water.
Preferably, in the step (1), the first solvent comprises the following components in percentage by volume, based on the total volume of the first solvent being 100%: 45-90% (for example, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, etc.) of an alcohol or ketone, 0.05-5% (for example, 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, 5%, etc.) of an organic acid, and the balance of water.
Preferably, the organic acid is formic acid, which may be present in an amount of 0.5 to 3% by volume, for example 0.5%, 1%, 2%, 3%, etc. The organic acid can promote the elution segmentation to be more definite, and the target component to be more concentrated.
Preferably, in the step (1), the second solvent is a mixed solution of alcohol and ester, a mixed solution of ketone and ester, an aqueous solution of alcohol or an aqueous solution of ketone.
Preferably, the alcohol to ester volume ratio is (30-60: 1, e.g., 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, etc., and the ketone to ester volume ratio is (30-60: 1, e.g., 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, etc.
Preferably, the alcohol is present in the aqueous alcohol solution in an amount of 90-100% by volume, e.g., 90%, 92%, 94%, 96%, 98%, 100%, etc.
Preferably, the ketone is present in the aqueous solution in an amount of 70-90% by volume, e.g., 70%, 75%, 80%, 85%, 90%, etc.
Preferably, in step (1), the solvent III is any one of alkane, halogenated alkane or ester or a combination of at least two of alkane, halogenated alkane or ester.
Preferably, in step (1), the concentration temperature is 50-65 ℃, for example, 50 ℃, 52 ℃, 55 ℃, 58 ℃, 60 ℃, 63 ℃, 65 ℃ and the like, and the concentration pressure is-0.10 to-0.065 MPa, for example, -0.10MPa, -0.09MPa, -0.08MPa, -0.07MPa, -0.065MPa and the like.
Preferably, in step (1), the concentration to a dry extract with a relative density of 1.05-1.15, for example, may be 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.12, 1.14, 1.15, etc.
The dry extract refers to the state without distillate after 5 minutes of vacuum concentration.
Preferably, in the step (2), the sections I and II are separated and purified independently by adopting forward column chromatography or reverse column chromatography.
Preferably, the reverse column chromatography uses any one of reverse ODS silica gel (e.g. C4, C8, C18, amino, glycosyl, phenyl, etc.), polyamide powder or MCI resin powder containing a special bond.
Preferably, the eluent for reverse phase column chromatography is selected from any one of alcohol, ketone, ester or water or the combination of at least two of the above.
Preferably, step (2) is followed by step (3): cracking and destroying the primary tetrahydrocannabinol product, adjusting neutrality or inactivating and then discarding; concentrating and drying the primary cannabinol product, the primary cannabichromene product and the primary cannabicyclol product respectively.
Preferably, in the step (3), the destruction is performed by oxidation, reduction, an enzyme method, acid-base cleavage or thermal cleavage.
Preferably, in step (3), the concentration temperature is 50-65 ℃, for example, 50 ℃, 52 ℃, 55 ℃, 58 ℃, 60 ℃, 63 ℃, 65 ℃ and the like, and the concentration pressure is-0.10 to-0.065 MPa, for example, -0.10MPa, -0.09MPa, -0.08MPa, -0.07MPa, -0.065MPa and the like.
Preferably, in the step (3), the drying includes any one of vacuum drying, freeze drying, near infrared drying, forced air drying or microwave drying or a combination of at least two thereof.
Preferably, in the step (3), the drying temperature is 30 to 65 ℃, and may be, for example, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, 65 ℃ or the like.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a separation and purification process of high-purity CBC, CBN and CBL, which has high transfer rate and purity of more than 95 percent, and simultaneously carries out purification and enrichment on the THC part to be destroyed again and reduces the destruction cost of the Tailings (THC) by more than 80 percent.
(2) The invention adopts temperature-controllable solid phase extraction, can effectively remove wax, grease and pigment, has better sectional purification effect than other technical means (such as molecular distillation), is combined with conventional chromatographic preparation, can realize production amplification and has excellent industrialized prospect.
(3) The adsorbent used in the invention can be regenerated properly and used repeatedly after being dried (for example, the clay and the calcium phosphate can draw out the polluted parts such as wax at the top end, and can be used repeatedly after being treated at the temperature of 300 ℃ and 500 ℃ for 1-5 h), theoretically more than 100 times, and basically no production waste is generated.
Drawings
FIG. 1 is a high performance liquid chromatogram of the CBD extraction plant tailings provided in example 1.
Fig. 2 is a high performance liquid chromatogram of the THC and CBN containing segment I provided in example 1.
FIG. 3 is a high performance liquid chromatogram of stage II containing CBC and CBL provided in example 1.
FIG. 4 is a high performance liquid chromatogram of the CBC product provided in example 1.
FIG. 5 is a high performance liquid chromatogram of the CBL product provided in example 1.
Fig. 6 is a high performance liquid chromatogram of the CBN final product provided in example 1.
Detailed Description
The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings. It should be understood by those skilled in the art that the specific embodiments are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
Example 1
The present embodiment provides a method for separating a cannabinoid, which specifically includes the following steps:
(1) taking 100g of CBD extraction plant tailings (the CBD extraction plant tailings contain 2.75 wt% of CBD, 8.85 wt% of THC, 10.57 wt% of CBN, 2.92 wt% of CBC and 21.43 wt% of CBL, as shown in figure 1), adding 50mL of normal hexane, mixing the slurry at the temperature of 60 ℃ with the assistance of the slurry, then adding 100g of activated clay while hot, mixing the mixture uniformly, and cooling the mixture to room temperature to form a solid dispersion system; transferring the solid dispersion into a short column (diameter-height ratio of 4:5.5, 240mL) containing 75g of activated clay and 25g of diatomite, flushing 300mL of the column with 65 v% ethanol aqueous solution at 10 ℃ and recovering 4.2g of CBD section (CBD content of 65.53%) for later use; then flushing 500mL of column with 80 v% ethanol water solution to obtain a section I containing THC and CBN; then compressed air is introduced for pressure drying, and 350mL of normal hexane is used for flushing the column to obtain a section II containing CBC and CBL; finally, the target fraction I eluate 40.36g (as shown in FIG. 2) and fraction II eluate 26.88g (as shown in FIG. 3) were obtained by sequentially concentrating at 60 ℃ and-0.08 mpa.
(2) Eluting the I-section eluate with 200-mesh 300-mesh silica gel column (diameter-height ratio of 3:45, 320mL) sequentially with mixed solution (450mL) of n-hexane, ethyl acetate and formic acid at volume ratio of 99.45:0.5:0.05 to obtain THC primary product; obtaining a CBN primary product solution by using a mixed solution (550mL) of normal hexane, ethyl acetate and formic acid with the volume ratio of 98.45:1.5: 0.05;
passing the eluate of II stage through silica gel column (diameter-height ratio of 2:35, 105mL), and sequentially eluting with n-hexane (200mL) to obtain CBL primary product solution; eluting with a mixed solution (150mL) of n-hexane and ethyl acetate at a volume ratio of 98.5:1.5 to obtain a CBC primary product solution.
(3) Mixing THC-containing components, adding 11.20g in total, cracking and destroying with nitric acid, adjusting pH to 7.0, and discarding; concentrating the other three primary solutions at 65 deg.C and-0.09 MPa to obtain paste, vacuum drying the three primary solutions at 50 deg.C under-0.10 MPa for 48 hr to obtain 9.66g CBN, 20.01g CBL, and 2.65g CBC. Samples were taken and tested by HPLC for CBC purity of 98.25% (as shown in FIG. 4), CBL purity of 99.26% (as shown in FIG. 5) and CBN purity of 98.87% (as shown in FIG. 6).
Example 2
The present embodiment provides a method for separating a cannabinoid, which specifically includes the following steps:
(1) taking 1000g of industrial hemp flower and leaf alcohol extract (the CBD content is 14.55 wt%, the THC content is 0.25 wt%, the CBN content is 7.55 wt%, the CBC content is 1.51 wt%, and the CBL content is 1.41 wt%) to add into 1000mL of 60 v% ethanol water solution, homogenizing at 45 ℃ to form a liquid dispersion system; transferring the liquid dispersion system to a solid phase extraction short column (diameter-height ratio is 40:55, 6L) filled with 2000g of polyamide powder (90-120 meshes), flushing the column 12L with 45 v% ethanol water solution at the column temperature of 0 ℃, and recovering 278g (CBD content is 52.18%) of components rich in CBD and CBDV for later use; washing 10L of the column with 90 v% ethanol water solution to obtain a section I containing THC and CBN; finally, compressed air is introduced to be pressed and dried, and ethyl acetate is used for washing the column 9L to obtain a II section containing CBC and CBL; respectively and sequentially concentrating at 50 ℃ and-0.09 MPa to obtain 121.76g of target I section eluate and 75.83g of target II section eluate.
(2) Loading the eluate of the section I onto MCI column (75-120 μm, diameter/height ratio of 6:45, 1.28L), isocratically eluting with 85 v% isopropanol water solution for 2.8L to obtain THC primary product solution and CBN primary product solution in sequence; subjecting the eluate of II stage to MCI column (75-120 μm, diameter/height ratio of 6:45, 1.28L), isocratically eluting with 85 v% isopropanol water solution for 1.8L to obtain CBC primary solution, and washing column with isopropanol for 1.6L to obtain CBL primary solution.
(3) All the components containing THC are combined, and 57.52g in total are destroyed by thermal cracking at 350 ℃ and then discarded; concentrating the other three primary solutions at 65 deg.C under-0.09 MPa to obtain paste, vacuum drying at 50 deg.C under-0.10 MPa for 48 hr to obtain final product of CBN 60.72g, CBL 13.18g and CBC 12.75 g. Sampling, and determining the purity of CBN to be 99.05%, CBL to be 98.97% and CBC to be 98.86% by HPLC.
Example 3
The present embodiment provides a method for separating a cannabinoid, which specifically includes the following steps:
(1) taking 1000g of CBD extraction plant tailings (the CBD content of the CBD extraction plant tailings is 2.82 wt%, the THC content of the CBC is 9.75 wt%, the CBN content of the CBN is 11.57 wt%, the CBC content of the CBC is 2.96 wt%, and the CBL content of the CBD extraction plant tailings is 21.43 wt%), adding 500mL of n-heptane, and homogenizing under the assistance of 50 ℃ to form a liquid dispersion system; transferring the liquid dispersion to a solid phase extraction short column (length-diameter ratio of 10:30, 1.2L) filled with 1.62kg of calcium phosphate and 0.18kg of diatomite (120-200 mesh); after the column temperature is reduced to-10 ℃, washing 1.8L of column with ethanol, and recovering 48.06g (with the CBD content of 57.98%) of components rich in CBD and the like for later use; introducing compressed air to press dry the chromatographic column, and flushing the chromatographic column by using a mixed solution (2.5L) of n-heptane, ethyl acetate and formic acid with a volume ratio of 99:0.5:0.5 to obtain a section I containing CBC and CBL; flushing the column with a mixed solution (2L) of n-heptane and ethyl acetate at a volume ratio of 90:10 to obtain a section II containing THC and CBN; sequentially concentrating at 50 deg.C and-0.09 MPa to obtain target I section eluate 376.58g and II section eluate 240.27 g.
(2) Loading the eluate of the above section I onto MCI column (75-120 μm, length-diameter ratio of 6:45, 1.28L), isocratically eluting with 90 v% ethanol water solution for 2.4L to obtain CBC primary product solution, and washing column with ethyl acetate for 1.8L to obtain CBL primary product solution. Subjecting the eluate of II stage to YMC 4 silica gel chromatography column (60-210 μm, length-diameter ratio of 6:45, 1.2L), isocratic eluting with mixed solution of n-heptane, ethyl acetate and formic acid at volume ratio of 98:1.5:0.5 for 3.2L to obtain THC primary product solution and CBN primary product solution;
(3) all the components containing THC are combined, the total amount is 146.25g, and the THC is cracked and destroyed by acid potassium permanganate at 70 ℃, neutralized and discarded; concentrating the other three primary solutions at 65 deg.C under-0.09 Mpa to obtain paste, vacuum drying at 50 deg.C under-0.10 MPa for 48 hr to obtain 112.30g CBN, 202.11g CBL and 26.85g CBC. The sample was taken and the purity of CBC was 98.61%, CBL 99.66% and CBN 99.19% by HPLC.
Example 4
This example provides a method for separating cannabinoids, which differs from example 1 in that the ratio of the diameter to the height of the short column of the solid phase extraction in step (1) is 1:0.8, and the other steps are the same as in example 1.
Example 5
This example provides a method for separating cannabinoids, which differs from example 1 in that the ratio of the diameter to the height of the short column of the solid phase extraction in step (1) is 1:5, and the other steps are the same as in example 1.
Example 6
This example provides a method for separating cannabinoids, which differs from example 1 in that 100g of CBD extraction plant tailings are taken in step (1), 50mL of n-hexane is added, the mixture is slurried with the aid of 60 ℃, then 25g of activated clay is added while hot, and the other steps are the same as in example 1.
Example 7
This example provides a method for separating cannabinoids, which is different from example 1 in that 100g of CBD extraction plant tailings are taken in step (1), 50mL of n-hexane is added, the mixture is slurried with the aid of 60 ℃, then 200g of activated clay is added while it is hot, and the other steps are the same as example 1.
Example 8
This example provides a separation method of cannabinoids, which is different from example 1 in that in step (2), the above-mentioned I-stage eluate is loaded on 200-300 mesh silica gel column (diameter/height ratio is 3:45, 320mL), and then sequentially eluted by a mixed solution (450mL) of n-hexane and ethyl acetate in a volume ratio of 99.5:0.5 to obtain a THC crude product, and other steps are the same as example 1.
Example 9
This example provides a method for separating a cannabinoid, which is different from example 1 in that, in step (2), a CBN primary solution is obtained as a mixed solution (550mL) of n-hexane and ethyl acetate in a volume ratio of 98.5: 1.5.
Comparative example 1
The present comparative example provides a method for the isolation of cannabinoids, the method specifically comprising the steps of:
(1) 100g of CBD extraction plant tailings are taken, wherein the CBD content is 2.75%, the THC content is 8.85%, the CBN content is 10.57%, the CBC content is 2.92% and the CBL content is 21.43%. Adding 50mL of n-hexane, mixing the slurry at the temperature of 60 ℃, adding 100g of activated clay while the slurry is hot, uniformly mixing the mixture, and cooling the mixture to room temperature to form a solid dispersion system; transferring the solid dispersion into a short column (diameter-height ratio of 4:5.5, 240mL) containing 75g of activated clay and 25g of diatomite, flushing 300mL of the column with 65 v% ethanol aqueous solution at 10 ℃ and recovering 4.2g of CBD section (CBD content of 65.53%) for later use; then compressed air is introduced for drying, and the weight ratio of normal hexane 98: 780mL of column was washed with ethyl acetate 2, and the solvent was recovered by concentration under reduced pressure to obtain 68.62g of a mixed eluate containing THC, CBN, CBC and CBL.
(2) Passing the mixed eluate through 200-300 mesh silica gel column (diameter-height ratio of 3:45, 320mL), sequentially; eluting with n-hexane (200mL) to obtain CBL primary solution; eluting with mixed solution (450mL) of n-hexane, ethyl acetate and formic acid at a volume ratio of 99.45:0.5:0.05 to obtain THC primary product; eluting with a mixed solution (250mL) of n-hexane, ethyl acetate and formic acid at a volume ratio of 98.45:1.5:0.05 to obtain a CBC primary solution; a CBN primary solution was obtained as a mixed solution (500mL) of n-hexane, ethyl acetate and formic acid at a volume ratio of 98.45:1.5: 0.05.
(3) Mixing the THC-containing components, adding 20.17g in total, cracking and destroying with nitric acid, adjusting pH to 7.0, and discarding; in addition, the other three primary solution were concentrated to paste at 65 ℃ and-0.09 MPa, and the three primary pastes were dried under vacuum at 50 ℃ and-0.10 MPa for 48 hours to obtain 13.78g of CBN, 25.79g of CBL, and 4.29g of CBC. The sample was taken and the purity of CBC was 58.97%, CBL 79.19% and CBN 63.92% by HPLC.
Comparative example 2
The present comparative example provides a method for the isolation of cannabinoids, the method specifically comprising the steps of:
(1) 100g of CBD extraction plant tailings are taken, wherein the CBD content is 2.75%, the THC content is 8.85%, the CBN content is 10.57%, the CBC content is 2.92% and the CBL content is 21.43%. Adding 150mL of normal hexane, mixing the slurry at the temperature of 60 ℃, then adding a 200-mesh 300-mesh silica gel column (the diameter-height ratio is 3:45 and 320mL) while the slurry is hot, cooling to room temperature (25.5 ℃) after the sample loading is finished, and eluting by the normal hexane (450mL) in sequence to obtain a CBL primary product solution; eluting with mixed solution (450mL) of n-hexane, ethyl acetate and formic acid at a volume ratio of 99.45:0.5:0.05 to obtain THC primary product; eluting with a mixed solution (320mL) of n-hexane, ethyl acetate and formic acid at a volume ratio of 98.45:1.5:0.05 to obtain a CBC primary solution; a CBN primary solution was obtained as a mixed solution (500mL) of n-hexane, ethyl acetate and formic acid at a volume ratio of 98.45:1.5: 0.05.
(2) Mixing THC-containing components, adding 25.33g in total, cracking with nitric acid, destroying, adjusting pH to 7.0, and discarding; in addition, the other three primary solution were concentrated to paste at 65 ℃ and-0.09 MPa, and then the three primary pastes were dried under vacuum at 50 ℃ and-0.10 MPa for 48 hours to obtain 17.66g of CBN, 38.15g of CBL and 8.05g of CBC. Samples were taken and the CBC purity was 31.04%, CBL purity 53.85%, CBN purity 48.79% by HPLC.
Performance testing
The purity detection and the recovery calculation were performed on the finished products provided in examples 1 to 9 and comparative examples 1 to 2, respectively, wherein the purity detection was performed by HPLC, and the specific test methods are as follows:
chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; using acetonitrile as mobile phase A and 0.1% formic acid-water as mobile phase B, and carrying out gradient elution according to the following A (%) and B (%) time table: 72:28 elution for 0 to 11.5 min; 11.5min to 24min to 90: 10; 24min to 26min down to 72: 28; the detection wavelength was 210 nm. The number of theoretical plates is not less than 2500 calculated according to CBN peak;
preparation of control solutions: precisely weighing CBN, CBC, CBL, THC and CBD reference substances, and adding methanol-water (1:1) to prepare a mixed reference substance solution containing 0.03mg per l mL to obtain the final product.
Preparation of a test solution: taking raw materials (tailing sample/extract sample) or about 8-12mg of three dried cannabinoid finished products, precisely weighing, placing in a 250mL measuring flask, adding 20mL of acetonitrile, performing ultrasonic treatment for 10 minutes, adding acetonitrile to dilute to a scale, shaking up, filtering with a microporous filter membrane (0.45pm), and taking a subsequent filtrate to obtain the cannabinoid.
The determination method comprises the following steps: precisely sucking 10 μ L of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The purity of some cannabinoid XX of the raw material is (the XX peak area of the sample is multiplied by 0.03 multiplied by 250)/(the XX peak area of the reference sample is multiplied by the actual weighing value of the sample) multiplied by 100 percent
The recovery of a certain cannabinoid XX is calculated as follows:
some cannabinoid XX recovery ═ (weight of some cannabinoid XX finished product x purity)/(weight of raw material x purity of some cannabinoid XX) × 100%
Wherein cannabinoid XX is selected from CBN, CBC, CBL, THC and CBD.
The specific test results are shown in table 1:
TABLE 1
The test data in table 1 show that the purity of CBC, CBL and CBN obtained by the separation method of cannabinoids of the present invention can reach more than 98%, and the recovery rate can reach more than 89%, thus demonstrating that the temperature-controlled solid phase extraction technology of the present invention, in combination with a specific chromatography method, can realize the segmentation and fine separation of complex components, and finally can obtain a plurality of cannabinoid finished products with purity of more than 95%, and the recovery rates are all more than 70%, and the process is relatively simple.
The applicant states that the present invention is illustrated by the above examples to describe the isolation of cannabinoids, but the present invention is not limited to the above examples, i.e. it is not meant to imply that the present invention must rely on the above examples to be practiced. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitution of each raw material sample and addition of auxiliary components, selection of specific modes, etc., of the product of the present invention, falls within the scope and disclosure of the present invention.
Claims (10)
1. A method for isolating cannabinoids, the method comprising the steps of:
(1) performing solid phase extraction, purification and concentration on a raw material sample to obtain a section I containing tetrahydrocannabinol and cannabinol and a section II containing cannabichromene and cannabinol;
(2) and respectively carrying out column chromatography separation and purification on the I section containing the tetrahydrocannabinol and the cannabinol and the II section containing the cannabichromene and the cannabinol to obtain a tetrahydrocannabinol primary product, a cannabinol primary product, a cannabichromene primary product and a cannabinol primary product.
2. The method for separating cannabinoids as claimed in claim 1, wherein in step (1), the mass content of tetrahydrocannabinol in the raw material sample is 0.3% or less;
preferably, in the step (1), the cannabidiol content of the raw material sample is 2-20% by mass, the cannabinol content of the raw material sample is 1-10% by mass, the cannabichromene content of the raw material sample is 0.5-5% by mass, and the cannabinol content of the raw material sample is 2-20% by mass.
3. The method of isolating cannabinoids as claimed in claim 1 or 2, wherein in step (1), the raw material sample is cannabidiol extraction plant tailings or industrial cannabis extract raw material;
preferably, in the step (1), the raw material sample is selected from any one or a combination of at least two of industrial cannabis, intermediate cannabis or medicinal cannabis;
preferably, in the step (1), the part extracted from the industrial hemp extract raw material is any one or a combination of at least two of hemp flowers, hemp leaves, hemp roots, hemp stem cores or hemp seed meals.
4. The method of isolating cannabinoids as claimed in any of claims 1-3, wherein in step (1), the solid phase extraction is performed at a low temperature, wherein the low temperature is-10 to 10 ℃;
preferably, in the step (1), the filler used for the solid-phase extraction purification comprises any one of activated clay, diatomite, calcium phosphate, calcium carbonate, polyamide powder or silica gel or a combination of at least two of the activated clay, the diatomite, the calcium phosphate, the calcium carbonate, the polyamide powder or the silica gel;
preferably, the particle size of the polyamide powder is 90-120 meshes;
preferably, the particle size of the silica gel is 100-500 meshes.
5. The method of isolating cannabinoids as claimed in any of claims 1-4, wherein in step (1), the solid phase extraction purification is performed using a solid phase extraction column;
preferably, in the step (1), the diameter-height ratio of the solid phase extraction column is 1 (1-3);
preferably, in the step (1), the mass ratio of the total amount of the filler in the solid phase extraction column to the raw material sample is 1 (0.25-5), preferably 1 (0.5-2);
preferably, in step (1), the solid phase extraction adopts dry loading or wet loading;
preferably, the dry loading is specifically: mixing part of the filler with a raw material sample to form a solid dispersion system, and transferring the solid dispersion system into a solid phase extraction column filled with the filler; the mass ratio of the partial filler to the raw material sample is 1 (0.25-2), preferably 1 (0.5-1);
preferably, the wet loading is specifically: mixing an organic solvent with a raw material sample to form a liquid dispersion system, and transferring the liquid dispersion system into a solid phase extraction column filled with a filler; the volume mass ratio of the organic solvent to the raw material sample is 1mL (0.25-5) g, preferably 1mL (0.5-2) g;
preferably, the organic solvent for wet loading is an elution solvent, preferably any one of hexane, heptane or ethanol water solution with concentration of 40-65 v%, or a combination of at least two thereof.
6. The process for the isolation of cannabinoids as claimed in any of claims 1-5, wherein in step (1), the elution solvent used for the solid phase extraction purification comprises any one or a combination of at least two of alkanes, halogenated alkanes, esters, alcohols, ketones, organic acids or water;
preferably, the alkane is selected from any one or a combination of at least two of petroleum ether, diethyl ether, n-hexane or n-heptane;
preferably, the halogenated alkane is selected from dichloromethane and/or chloroform;
preferably, the ester is selected from ethyl acetate and/or butyl acetate;
preferably, the alcohol is selected from any one of methanol, ethanol or isopropanol or a combination of at least two thereof;
preferably, the ketone is selected from acetone and/or butanone;
preferably, the organic acid is selected from any one of formic acid, acetic acid or trifluoroacetic acid or a combination of at least two thereof.
7. The method of separating cannabinoids as claimed in any of claims 1-6, wherein in step (1), during the solid phase extraction and purification process, the cannabidiol remaining in the raw material sample is eluted with solvent one, then eluted with solvent two to obtain a first fraction containing tetrahydrocannabinol and cannabinol, and finally eluted with solvent three to obtain a second fraction containing cannabichromene and cannabinol;
preferably, in the step (1), the first solvent is a mixed solution of alcohol or ketone, organic acid and water;
preferably, in the step (1), the first solvent comprises the following components in percentage by volume, based on the total volume of the first solvent being 100%: 45-90% of alcohol or ketone, 0.05-5% of organic acid and the balance of water;
preferably, the organic acid is formic acid, and the volume content of the formic acid is 0.5-3%;
preferably, in the step (1), the second solvent is a mixed solution of alcohol and ester, a mixed solution of ketone and ester, an aqueous solution of alcohol or an aqueous solution of ketone;
preferably, the volume ratio of alcohol to ester is (30-60):1, the volume ratio of ketone to ester is (30-60): 1;
preferably, in the alcohol aqueous solution, the volume of the alcohol is 90-100%;
preferably, in the aqueous solution of the ketone, the volume of the ketone accounts for 70-90%;
preferably, in step (1), the solvent III is any one of alkane, halogenated alkane or ester or a combination of at least two of alkane, halogenated alkane or ester.
8. The process for the isolation of cannabinoids as claimed in any of claims 1-7, wherein in step (1), the temperature of the concentration is 50-65 ℃, and the pressure of the concentration is-0.10 to-0.065 MPa;
preferably, in the step (1), the concentration is carried out until a dry extract with the relative density of 1.05-1.15 is obtained.
9. The method of isolating cannabinoids as claimed in any of claims 1-8, wherein in step (2), said stages I and II are independently isolated and purified by forward column chromatography or reverse column chromatography;
preferably, the reverse column chromatography adopts any one of reverse ODS silica gel, polyamide powder or MCI resin powder containing special bonding;
preferably, the eluent for reverse phase column chromatography is selected from any one of alcohol, ketone, ester or water or the combination of at least two of the above.
10. The method of isolating cannabinoids as claimed in any of claims 1-9, further comprising step (3) after step (2): cracking and destroying the primary tetrahydrocannabinol product, adjusting neutrality or inactivating and then discarding; concentrating and drying the primary cannabinol product, the primary cannabichromene product and the primary cannabicyclol product respectively;
preferably, in the step (3), the destruction is performed by using a chemical reaction method of oxidation, reduction, an enzyme method, acid-base cracking or thermal cracking;
preferably, in the step (3), the concentration temperature is 50-65 ℃, and the concentration pressure is-0.10 to-0.065 MPa;
preferably, in step (3), the drying includes any one of vacuum drying, freeze drying, near infrared drying, forced air drying or microwave drying or a combination of at least two of them;
preferably, in step (3), the temperature of the drying is 30-65 ℃.
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