CN111470953A - Method for extracting and separating high-purity cannabidiol from low-content industrial cannabis sativa leaves - Google Patents

Method for extracting and separating high-purity cannabidiol from low-content industrial cannabis sativa leaves Download PDF

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CN111470953A
CN111470953A CN202010335132.5A CN202010335132A CN111470953A CN 111470953 A CN111470953 A CN 111470953A CN 202010335132 A CN202010335132 A CN 202010335132A CN 111470953 A CN111470953 A CN 111470953A
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alcohol
cannabidiol
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CN111470953B (en
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张赪
高月静
任超鑫
智旭亮
贾晓妮
刘琼
智彩辉
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Sunresin New Materials Co ltd Xi'an
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    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
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    • C07—ORGANIC CHEMISTRY
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    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
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    • C07C37/70—Purification; separation; Use of additives, e.g. for stabilisation by physical treatment
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Abstract

The invention discloses a method for extracting and separating high-purity Cannabidiol (CBD) from low-content industrial cannabis sativa leaves, which adopts the modes of leaching, macroporous resin adsorption and desorption, reverse chromatography and crystallization to finally obtain a cannabidiol pure product with the purity of more than 99%. For separating other cannabinoids, extraction and decolorization processes can be added between leaching and macroporous resin adsorption and desorption processes, and reverse phase chromatography is performed by using polymer reverse phase packing, so that separation of cannabidiol suboxydol (CBDV) Cannabinol (CBN) Tetrahydrocannabinol (THC) in the flower leaves can be completed. The extraction method has the advantages of high extraction, extraction and decoloration process yield and high impurity removal rate; the macroporous resin and the reversed phase column have large chromatographic capacity and good separation effect. The method meets the requirements of development of a plurality of industrial hemp products, has great application advantages in the field of pharmacy, and is suitable for industrial popularization.

Description

Method for extracting and separating high-purity cannabidiol from low-content industrial cannabis sativa leaves
Technical Field
The invention relates to a method for extracting and separating high-purity cannabidiol from low-content industrial cannabis sativa leaves, belonging to the technical field of plant extraction and purification.
Technical Field
Cannabidiol (CBD) is a natural component extracted from the cannabis plant. Cannabidiol has the molecular formula C21H30O2The character of the crystal is white to light yellow sticky or crystal, the melting point is 66-67 ℃, the crystal is almost insoluble in water, and the crystal is dissolved in organic solvents such as ethanol, methanol, ether, benzene, chloroform and the like. Cannabidiol has the function of blocking the adverse effect of certain polyphenols on the nervous system of a human body, has a series of physiological activity functions of blocking breast cancer metastasis, treating epilepsy, resisting rheumatoid arthritis, resisting insomnia and the like, has a good effect on treating multiple sclerosis, and has a very wide future application prospect of CBD. The prior extraction and purification process and technical means have many defects, low yield, small yield and low purity of the final product. In addition, most of the existing technical means have single process production mode, the extraction and purification procedures are not uniform, and the industrial production level is difficult to achieve.
At present, the hemp flowers and leaves are extracted by solvent extraction, microwave extraction, supercritical extraction and the like. The enrichment and purification separation of cannabidiol mainly comprises two modes of macroporous adsorption and resolution and chromatographic separation.
In the related patents, the solvent extraction of CN110066216A adopts a hot reflux extraction method, which is not economical and has a large content of polar impurities in the extract. The microwave extraction and the supercritical extraction related in the inventions of CN110156568A and CN110386861A are both completed by special equipment, the equipment has high cost and small yield, the related extraction method has single target object, and no collection means is provided for other effective components.
In the target substance enrichment and purification mode, CN110066216A adopts the extraction modes of solvent extraction, macroporous resin enrichment and silica gel column chromatography, and the solvent extraction of the invention is a single-concentration solvent for extracting cannabidiol, so that the enrichment of polar impurities in the extract is more, the loading capacity of the silica gel column chromatography is small, and finally, the extraction efficiency is low and the process energy consumption is high; CN110156568A adopts microwave extraction, macroporous resin adsorption and column chromatography separation to extract and purify cannabidiol, and the defects of the macroporous adsorption mode and the silica gel column chromatography mode adopted by chromatography are small in load and insufficient in industrial yield; CN110386861A adopts a large supercritical and macroporous resin purification mode, the process has high equipment cost, single extraction target and low extraction efficiency for low-content hemp flower leaves; CN110283049A the invention adopts ultrahigh pressure extraction and macroporous resin adsorption mode, wherein the macroporous adsorption mode adopts homogeneous phase selective adsorption, the cannabidiol adsorption amount is small, and the final investment cost is large.
The chromatographic separation filler adopted in the purification and separation of the invention CN110066216A, CN110156568A, CN110386861A, CN110066217A, CN110283049A, CN109970518A and CN109851480A is silica or silica-based compound filler. The filler has the disadvantages of small loading capacity, low industrialized yield, poor separation effect and serious filler attenuation.
Disclosure of Invention
In order to solve the problems of low extraction rate, poor complete production, large equipment investment and poor separation effect of cannabinoids except cannabidiol in the production of the existing industrial hemp leaves, the invention provides a method for extracting and purifying high-purity cannabidiol and other cannabinoids from low-content industrial hemp leaves.
The method adopts gradient extraction, namely alcohol extraction of cannabinoids in different concentration systems, so that the content of cannabinoids in the extract can be improved to the maximum extent, and the enrichment of polar impurities is reduced; the macroporous resin adsorption enrichment process can greatly improve the loading capacity by loading the sample in a suspension state, and is favorable for industrial popularization; high-purity CBDV, CBN and more than 99% of CBD crystals can be obtained by adopting a reverse chromatography and crystallization process mode of the polymer microspheres consisting of polyacrylate-styrene-divinylbenzene, and compared with a chromatographic separation mode of a single purification process, the method has the characteristics of good separation effect, large carrying capacity, high repeated utilization rate and the like, and also provides a saving-type process for systematically extracting Cannabidiol (CBDV) and Cannabidiol (CBD) in the cannabis sativa leaves.
In order to realize the purpose of separating and extracting high-purity Cannabidiol (CBD), the method provided by the invention comprises the steps of leaching, adsorption and resolution, reversed-phase column chromatography and crystallization, and specifically comprises the following steps:
step one, leaching:
(1) pulverizing industrial hemp flower and leaf with CBD content of 0.1-2.5%, wherein the average particle size of the pulverized flower and leaf is 0.2-2 mm;
(2) leaching with 6-15 times of water at 10-80 deg.C for 0.5-4 hr, performing solid-liquid separation, repeatedly leaching for 1-3 times, and allowing the solid flower leaf to go to the next step;
(3) leaching with 10-40% (V/V) alcohol solution 6-15 times the weight of the flower and leaf at 10-80 deg.C for 0.5-4 hr, performing solid-liquid separation, repeating leaching for 1-3 times, and allowing the solid flower and leaf to go to the next step;
(4) leaching with 70-100% (V/V) alcohol 6-15 times of the weight of the flower and leaf at 10-80 deg.C for 0.5-4 hr, performing solid-liquid separation, repeatedly leaching for 1-3 times, mixing, collecting leaching solution, and allowing the solid flower and leaf to enter the next step;
(5) washing the leaves with pure water 1-3 times the weight of the leaves, performing solid-liquid separation after washing, repeatedly washing for 1-3 times, wherein the washing liquid can be applied in the step (3) in the next period, and the leaves solid is dried to be used as an organic fertilizer;
(6) concentrating the leaching liquor by 3-20 times to form a suspension to obtain a concentrated leaching liquor, and entering the next step;
step two, adsorption and desorption of macroporous resin:
(1) loading the macroporous resin into a column by using 1-3BV alcohol aqueous solution with the concentration of 30-60% (V/V) by a wet method, wherein the filling height is not less than 50 cm;
(2) 2-6BV of 30-60% (V/V) alcohol water-soluble equilibrium resin column is used, and the linear flow velocity in the equilibrium process is not higher than 5 cm/min;
(3) adding alcohol into the concentrated leaching liquor obtained in the previous step, adjusting the alcohol concentration to 30-60% (V/V), forward loading to a macroporous resin column, loading the macroporous resin column with 30-100 g/L (cannabidiol pure product/liter macroporous), wherein the linear speed in the loading process is not higher than 5cm/min, and enabling effluent liquid to enter an alcohol recovery system;
(4) washing impurities with 1-5BV of 30-60% (V/V) alcohol water solution at linear velocity not higher than 5cm/min, and recycling the effluent liquid into an alcohol recycling system;
(5) resolving with 3-15BV 60-90% (V/V) alcohol water solution at linear velocity not higher than 5cm/min, collecting resolved solution, and concentrating to obtain extract;
(6) regenerating the macroporous resin by using a regenerated solvent aqueous solution with the concentration of 2-6BV and 90-100% (V/V), wherein the linear velocity is not higher than 5cm/min in the regeneration process, the effluent liquid enters a regenerated solvent recovery system, and the resin column enters the next cycle of column balancing step;
step three, reversed phase chromatography:
(1) using reversed phase filler as stationary phase of chromatographic column, homogenizing reversed phase filler with 1-3CV concentration of 0-100% (V/V) alcohol water solution, and wet loading column with height not lower than 25cm and loading pressure not higher than 10 MPa;
(2) using 2-CV alcohol water-soluble equilibrium chromatographic column with concentration of 30-60% (V/V), and the linear flow velocity in the equilibrium process is not higher than 10 cm/min;
(3) dissolving the extract obtained in the adsorption and desorption process in 50-80% (V/V) alcohol water solution to obtain 10-60 g/L cannabidiol solution, loading the cannabidiol solution to a chromatographic column in a forward direction, loading the cannabidiol solution with 10-40 g/L (cannabidiol pure product/liter of filler), wherein the linear speed is not higher than 10cm/min in the loading process, and enabling effluent liquid to enter an alcohol recovery system;
(4) washing impurities with 4-15CV of 50-80% (V/V) alcohol water solution at linear velocity not higher than 10cm/min, and recovering alcohol from the effluent;
(5) resolving with 4-15CV 60-90% (V/V) alcohol water solution at linear velocity not higher than 10cm/min, collecting Cannabidiol (CBD) component solution, and concentrating to obtain extract;
(6) regenerating the chromatographic column by using a regenerated solvent aqueous solution with the 2-6CV concentration of 90-100% (V/V), wherein the linear velocity is not higher than 10cm/min in the regeneration process, enabling effluent liquid to enter a regenerated solvent recovery system, and enabling the chromatographic column to enter the next cycle of column balancing step.
Step four, crystallization:
(1) dissolving cannabidiol component extract with 1-3 times of organic solvent at 10-60 deg.C;
(2) cooling to-20-50 deg.C, maintaining the temperature for 16-72h, and separating out crystal;
(3) solid-liquid separation, and drying the solid to obtain cannabidiol crystals;
(4) the liquid solvent enters an organic solvent recovery system.
Furthermore, the alcohol in the process step refers to monohydric alcohol with the number of carbon atoms not more than 4, which can be mutually dissolved with water in any proportion, and includes but is not limited to one or more of methanol, ethanol, n-propanol, isopropanol and butanol;
the organic solvent is slightly water-soluble or water-insoluble organic solvent, and includes but is not limited to one or a mixture of more of ethyl acetate, propyl acetate, butyl acetate, isobutyl acetate, ether, anisole, methyl isobutyl ketone, diisobutyl ketone, 1, 2-dichloroethane, 1,1, 1-trichloroethane, cyclohexane, 1, 2-dichloroethylene, dichloromethane, pentane, n-hexane, cyclohexane, heptane, methylcyclohexane, toluene, xylene, cumene and petroleum ether;
the macroporous resin is polymer small balls formed by copolymerization of polyacrylate-styrene-divinylbenzene, the particle size range of the small balls is 0.3-1.5mm, and the pore diameter range is
Figure BDA0002466290030000041
The specific surface area is not less than 500m2The cross-linking degree is 50-80%, and the polyacrylate accounts for 5-20% of the weight of the monomer.
The reversed phase filler is a filler which contains hydrophobic groups and separates substances with different polarities by using intermolecular force difference. Including but not limited to C18-bonded silica gel, C12-bonded silica gel, C8-bonded silica gel, phenyl-bonded silica gel, butyl-bonded silica gel, and polymeric reverse phase fillers containing ester groups, butyl groups, phenyl groups, octyl groups, dodecyl groups, hexadecyl groups, vinyl groups, allyl groups, vinyl benzenes, or divinyl benzenes;
the concentration refers to a process for increasing the content of solute in the solution, and includes but is not limited to distillation, reduced pressure distillation, thin film evaporation, nanofiltration, freeze-drying and the like.
For extracting purified Cannabidiol (CBDV) and cannabidiol from low-content industrial cannabis sativa leaves
The demand of cannabinoids such as (CBD) and Cannabinol (CBN) and the like needs to use a polymer reversed phase filler instead of a conventional reversed phase filler on the basis of the scheme, and an extraction and decoloration step needs to be added before a macroporous resin adsorption and desorption step to ensure the separation effect and the service life of the polymer chromatographic filler. The specific process steps are as follows:
step one, leaching:
(1) pulverizing low content industrial hemp leaves to average particle size range of 0.2-2 mm;
(2) leaching with 6-15 times of water at 10-80 deg.C for 0.5-4 hr, performing solid-liquid separation, repeatedly leaching for 1-3 times, and allowing the solid flower leaf to go to the next step;
(3) leaching with 10-40% (V/V) alcohol solution 6-15 times the weight of the flower and leaf at 10-80 deg.C for 0.5-4 hr, performing solid-liquid separation, repeating leaching for 1-3 times, and allowing the solid flower and leaf to go to the next step;
(4) leaching with 70-100% (V/V) alcohol 6-15 times of the weight of the flower and leaf at 10-80 deg.C for 0.5-4 hr, performing solid-liquid separation, repeatedly leaching for 1-3 times, mixing, collecting leaching solution, and allowing the solid flower and leaf to enter the next step;
(5) washing the leaves with pure water 1-3 times the weight of the leaves, performing solid-liquid separation after washing, repeatedly washing for 1-3 times, wherein the washing liquid can be applied in the step (3) in the next period, and the leaves solid is dried to be used as an organic fertilizer;
(6) concentrating the leaching solution by 3-20 times to form suspension to obtain concentrated leaching solution, and performing the next step.
Step two, extraction:
(1) extracting the concentrated extract with organic solvent at 0.3-3 times of the volume of the concentrated extract, at 0-40 deg.C for 10-60 min, standing for separating liquid, mixing the organic solvent extract in step (2), and recovering alcohol from the concentrated extract;
(2) repeating the step (1) for 0-2 times, and mixing the extracts for temporary storage;
(3) performing impurity washing treatment by using 0-30% (V/V) alcohol-water solution and the temporarily stored extraction liquid in the step (2), wherein the dosage of the alcohol-water solution is 1-3 times of the volume of the extraction liquid in the step (2), the extraction temperature is 0-40 ℃, the extraction time is 10-60 minutes, standing and separating liquid are performed after extraction is completed, the organic solvent extraction liquid enters the next step, and the alcohol solution enters an alcohol recovery system;
(4) repeating the step (3) for 0-5 times until the 0-30% (V/V) alcohol solution is colorless or yellowish after layering, and temporarily storing the organic solvent extract;
(5) extracting with 60-85% (V/V) alcoholic solution and the organic solution in step (4), wherein the dosage of the alcoholic solution is 3-5 times the volume of the organic solution in step (4), the extraction temperature is 0-40 ℃, the extraction time is 10-60 minutes, standing and separating are carried out after the extraction is finished, and the 60-85% (V/V) alcoholic solution enters the next step;
(6) repeating the step (5) for 2-6 times, combining and collecting 60-85% (V/V) alcohol solution, entering the next step, and entering the organic solution into an organic solvent recovery system.
Step three, decoloring:
(1) adding the collected 60-85% (V/V) alcohol-water extraction solution into a certain amount of activated carbon, and stirring for 0.5-4 h;
(2) performing solid-liquid separation, removing the activated carbon, leaching the filter cake for 0-4 times by using 60-85% (V/V) alcohol aqueous solution with the volume 1-3 times that of the filter cake, and collecting the liquid, namely the target object decolorization liquid;
(3) washing the filter cake with pure water to obtain a washing liquid, and feeding the washing liquid into an alcohol recovery system;
(4) concentrating the decolorized solution by 5-20 times to obtain suspension with concentration of 1-20 g/L (cannabidiol pure product/liter suspension), keeping at 80-110 deg.C for 2-6 hr under nitrogen protection, storing, and performing adsorption and desorption;
step four, adsorption and desorption of macroporous resin:
(1) loading the macroporous resin into a column by using 1-3BV alcohol aqueous solution with the concentration of 30-60% (V/V) by a wet method, wherein the filling height is not less than 50 cm;
(2) using 2-6BV alcohol water-soluble equilibrium resin column with the concentration of 30-60% (V/V), wherein the linear flow velocity in the equilibrium process is not higher than 5 cm/min;
(3) adding alcohol into the concentrated leaching solution obtained in the previous step, adjusting the alcohol concentration to 30-60% (V/V), loading the sample to a macroporous resin column in a forward direction, stopping loading the sample when the CBD concentration detected at the outlet of the resin column is less than 0.01mg/ml, wherein the linear speed of the loading process is not higher than 5cm/min, and enabling the effluent to enter an alcohol recovery system;
(4) washing impurities with 1-5BV of 30-60% (V/V) alcohol water solution at linear velocity not higher than 5cm/min, and recycling the effluent liquid into an alcohol recycling system;
(5) resolving with 3-30BV 60-90% (V/V) alcohol water solution at linear velocity not higher than 5cm/min, collecting resolved solution, and concentrating to obtain extract;
(6) regenerating the macroporous resin by using a regenerated solvent aqueous solution with the concentration of 2-6BV and 90-100% (V/V), wherein the linear velocity is not higher than 5cm/min in the regeneration process, the effluent liquid enters a regenerated solvent recovery system, and the resin column enters the next cycle of column balancing step.
Step five, reversed phase chromatography:
(1) using polymer reverse phase filler as the stationary phase of chromatographic column, homogenizing with 1-3CV concentration 0-100% (V/V) alcohol water solution, and wet loading to obtain column with height not lower than 25cm and pressure not higher than 10 MPa;
(2) using 2-6CV concentration 30-60% (V/V) alcohol water-soluble equilibrium chromatographic column, the linear flow velocity in the equilibrium process is not higher than 10 cm/min;
(3) dissolving the extract obtained in the adsorption and desorption process in 50-80% (V/V) alcohol water solution to obtain cannabidiol solution with the concentration of 10-60 g/L, loading the cannabidiol solution to a chromatographic column in a forward direction, controlling the loading capacity to be 10-40 g/L (CBD pure product/liter filler) and the linear speed of the loading process to be not higher than 10cm/min, and enabling effluent to enter an alcohol recovery system;
(4) performing gradient analysis by using 4-15CV (concentration of 50-95% (V/V) alcohol water solution with linear velocity not higher than 10cm/min in the analysis process, respectively collecting each gradient analysis solution to obtain sub-Cannabidiol (CBDV), Cannabidiol (CBD), Tetrahydrocannabinol (THC) and Cannabinol (CBN) analysis solutions, and respectively concentrating to obtain extract;
(5) regenerating the chromatographic column by using a regenerated solvent aqueous solution with the 2-6CV concentration of 90-100% (V/V), wherein the linear velocity is not higher than 10cm/min in the regeneration process, enabling effluent liquid to enter a regenerated solvent recovery system, and enabling the chromatographic column to enter the next cycle of column balancing step.
Step six, crystallization:
(1) dissolving cannabidiol component extract with 1-3 times of organic solvent at 10-60 deg.C;
(2) cooling to-50- -20 deg.C, maintaining the temperature for 16-72h, and separating out crystal;
(3) solid-liquid separation, and drying the solid to obtain cannabidiol crystals;
(4) the liquid solvent enters an organic solvent recovery system.
More specifically, the alcohol refers to monohydric alcohol with the number of carbon atoms not more than 4, which can be mutually dissolved with water in any proportion, and includes but is not limited to one or a mixture of methanol, ethanol, n-propanol, isopropanol and butanol;
the organic solvent is slightly water-soluble or water-insoluble organic solvent, and includes but is not limited to one or a mixture of more of ethyl acetate, propyl acetate, butyl acetate, isobutyl acetate, ether, anisole, methyl isobutyl ketone, diisobutyl ketone, 1, 2-dichloroethane, 1,1, 1-trichloroethane, cyclohexane, 1, 2-dichloroethylene, dichloromethane, pentane, n-hexane, cyclohexane, heptane, methylcyclohexane, toluene, xylene, cumene and petroleum ether;
the macroporous resin is polymer small balls formed by copolymerization of polyacrylate-styrene-divinylbenzene, the particle size range of the small balls is 0.3-1.5mm, and the pore diameter range is
Figure BDA0002466290030000071
Specific surface area ofBelow 500m2The cross-linking degree is 50-80%, and the polyacrylate accounts for 5-20% of the weight of the monomer.
The reverse filler is polymer microsphere composed of polyacrylate-styrene-divinylbenzene, the particle diameter range of the microsphere is 30-150 μm, and the pore diameter range is
Figure BDA0002466290030000081
Specific surface area range 400-600m2The cross-linking degree is 40-80%, and the weight of acrylic ester is 5-20% of the total weight of the monomers.
The concentration refers to a process for increasing the content of solute in the solution, and includes but is not limited to distillation, reduced pressure distillation, thin film evaporation, nanofiltration, freeze-drying and the like;
in the invention, the low-content industrial hemp flower and leaf is the industrial hemp flower and leaf with the CBD content of 0.1-2.5%.
The above embodiments do not limit the technical solutions of the present invention in any way, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.
The content of cannabidiol product crystals purified by the process is more than 99%, other cannabinoid components, Cannabidiol (CBDV) and Cannabinol (CBN) are effectively enriched and purified, and the cannabidiol product crystals can be used as raw materials for medicine production. The invention has the following advantages:
1. by adopting gradient extraction, namely extracting the cannabinoids by alcohol with different concentration systems, the content of the cannabinoids in the extract can be improved to the maximum extent, and the enrichment of polar impurities is reduced;
2. the extraction and the decoloration process are combined for use, so that the nonpolar impurities can be effectively removed, the service life of the resin filler can be prolonged, and the production cost is reduced;
3. the macroporous resin adsorption enrichment process greatly improves the loading capacity by loading the sample in a suspension state, and has obvious industrial advantages;
4. the high-purity CBD can be obtained by adopting the process modes of reverse phase chromatography and crystallization, and the whole process is combined, so that the resin loading capacity is improved, and the production cost is reduced;
5. the combined process of polymer reverse phase chromatography and crystallization can effectively and systematically purify and separate Cannabidiol (CBDV), Cannabidiol (CBD) and Cannabinol (CBN);
6. the combination of decolorization and decarboxylation by heating is adopted to effectively convert cannabidiolic acid (CBDA) into Cannabidiol (CBD), thereby improving the process yield.
The process has the advantages of complete operation, less equipment investment, environmental protection, economy, large chromatographic separation capacity, flexible adjustment of yield according to production requirements and high industrialization degree.
Drawings
FIG. 1, example 1 extraction of step phase detection profiles.
FIG. 2 shows a liquid phase detection pattern in the macroporous resin adsorption and analysis step of example 1.
FIG. 3 is a liquid phase detection pattern of the reverse phase chromatographic separation step of example 1.
FIG. 4 is a liquid phase detection pattern of the crystallization step of example 1.
FIG. 5, example 8 extraction of step phase detection profiles.
FIG. 6, liquid phase detection profile of the extraction step of example 8.
FIG. 7, liquid phase detection profile of the decolorization step of example 8.
FIG. 8 shows a liquid phase detection pattern in the macroporous resin adsorption and desorption step of example 8.
FIG. 9, example 8 liquid phase detection profile of reversed phase chromatographic separation procedure.
FIG. 10, liquid phase detection profile of the crystallization step of example 8.
Detailed Description
Example one
Leaching, namely 500g of industrial hemp flowers and leaves with the CBD content of 0.6% (w/w), crushing to the average particle size of 1mm, sieving to obtain 400g of powder, adding 2.4L of pure water, stirring, heating to 70 ℃, preserving heat for 4h, filtering, adding 2.4L 30% (V/V) ethanol into filter residue, stirring, heating to 70 ℃, preserving heat for 4h, filtering, adding 2L 90 (V/V) ethanol into the filter residue, stirring, heating to 70 ℃, preserving heat for 4h, filtering, collecting filtrate, adding 2L 90% (V/V) ethanol into the filter residue, stirring, heating to 70 ℃, preserving heat for 4h, filtering, combining the filtrate to obtain 4L leaching liquor, 45 ℃, 100mbar, rotary evaporation and concentration to 0.26L, detecting the CBD concentration in concentrated suspension liquid phase to be 8.8 g/L, the extraction rate to be 95.3%, and detecting the liquid phase as shown in figure 1.
Adsorption and desorption of macroporous resin, namely, 45ml of L X-T83 (produced by Xian lan Xiao science and technology New Material Co., Ltd.) macroporous resin is filled into a column by a wet method with 30% (V/V) ethanol water solution, the diameter of the chromatographic column is 1cm, the filling height is 57cm, 30% (V/V) ethanol water solution is used for balancing the macroporous resin column, the flow rate is 1.5ml/min, and the column is balanced for 1h, 90% ethanol is supplemented to the concentrated suspension obtained in the previous step, 520ml of the total is loaded onto the macroporous resin column, the forward loading is carried out, the effluent with the flow rate of 1.5ml/min enters an ethanol recovery system, 30% (V/V) ethanol water solution is used for washing impurities, the flow rate is 1.5ml/min, the effluent is washed for 30min, the effluent enters an alcohol recovery system, 70% (V/V) ethanol water solution is used for desorption, the column volume is 8 times of the column volume, the flow rate is 1.5ml/min in the desorption process, the desorption solution is collected and concentrated to obtain an extract, the CBD content is 33%, the yield is 95.2%, the liquid phase detection atlas is shown in the attached figure, the drawing, the regeneration process of the macroporous resin regeneration.
Reverse phase chromatography separation, wherein 47ml of L X-T83SS chromatographic resin (produced by Xian lan Xiao science and technology New Material Co., Ltd.) is added with 80ml of 50% ethanol, wet column packing is carried out, the diameter of the resin column is 10mm, the height is 600mm, the column packing pressure is 10Mpa, 30% ethanol is used for balance after column packing is completed, the flow rate is 4.7ml/min and balance is 40min, 9.6g of the extract is added with 120ml of 50% ethanol for stirring and dissolution, 30ml of the extract is taken out and loaded, the flow rate is kept at 4.7ml/min by 16.8 g/L (CBD pure product/reverse phase filler), after sample loading is finished, 60% ethanol is used for washing for 3h, 70% ethanol is used for elution for 1h, 60% eluent in 3h and all 70% ethanol eluent at about 0.6L ℃, 100mbar, rotary evaporation concentration is carried out until the extract is 0.834g, the content of liquid phase detection is 88% of high and medium CBD, 93%, and.
And (3) crystallization: dissolving the extract with 5ml n-hexane at 25 deg.C. Cooling to-40 deg.C after dissolution, maintaining the temperature for 48h, centrifuging, and drying to obtain cannabidiol crystal with purity of 99.4% 0.72g, and liquid phase detection chromatogram shown in figure 4.
Example two
Leaching, namely 500g of flowers and leaves of industrial hemp with the CBD content of 0.5% (w/w), crushing to the average particle size of 0.5mm, adding 3.2L of purified water, stirring, heating to 20 ℃, preserving heat for 3.5h, filtering, adding 3.2L 10% (V/V) methanol into filter residue, stirring, heating to 20 ℃, preserving heat for 3.5h, filtering, adding 3.2L 90% (V/V) methanol into the filter residue, stirring, heating to 40 ℃, preserving heat for 2h, filtering, collecting filtrate, adding 3.2L 90% (V/V) methanol into the filter residue, stirring, heating to 40 ℃, preserving heat for 2h, filtering, combining the filtrate to obtain 6.4L leaching liquor, 45 ℃, 100mbar, concentrating by rotary evaporation to 0.52L, detecting the concentration of concentrated suspension liquid phase, wherein the CBD concentration is 4.6 g/L, and the extraction rate is 96.2%.
Adsorption and desorption of macroporous resin, namely, filling 45ml of L X-T83 (produced by Seisan blue Xiao science and technology New Material Co., Ltd.) macroporous resin into a column by a wet method with 40% (V/V) methanol water solution, wherein the diameter of the column is 1cm, the filling height is 57cm, balancing the macroporous resin column by 40% (V/V) methanol water solution, the flow rate is 1.5ml/min, balancing for 1h, loading 520ml of the concentrated suspension obtained in the previous step into the macroporous resin column, loading the suspension in the forward direction, feeding the effluent at the flow rate of 1.5ml/min into a methanol recovery system, washing impurities by 40% (V/V) methanol water solution, the flow rate is 1.5ml/min, washing impurities for 30min, feeding the effluent into an alcohol recovery system, analyzing by 85% (V/V) methanol water solution, the analysis volume is 7 times the column volume, the flow rate in the analysis process is 1.5ml/min, collecting the analysis solution, concentrating the analysis solution into an extract, obtaining 6.4g of extract, the content of CBD, the effluent at 93.1%, regenerating the yield of the resin by 95% acetone water solution, regenerating the process, and feeding the.
Reverse phase chromatography separation, wherein 47ml of L X-2000 chromatographic resin (produced by Xian lan Xiao science and technology New materials Co., Ltd.) is added with 80ml of 50% methanol, wet column packing is carried out, the diameter and height of the resin column are 10mm, the column packing pressure is 10Mpa, after column packing is finished, 50% methanol is used for balancing, the flow rate is 4.7ml/min and balancing is 40min, 80ml of 70% methanol is added into the extract obtained in the previous step, stirring and dissolving are carried out, 30ml of the extract is sampled, the flow rate is kept at 4.7ml/min by loading 17.9 g/L (CBD pure product/reverse phase filler), after sample loading is finished, 75% methanol is used for washing for 3h, 85% methanol is used for eluting for 1h, 0.6L is collected, the temperature is controlled at 100mbar, rotary evaporation is carried out for concentration, the extract is carried out until the content of CBD.
And (3) crystallization: dissolving the extract with 5ml n-hexane at 25 deg.C. After the dissolution is finished, the temperature is reduced to minus 30 ℃, the temperature is kept for 48 hours, and the cannabidiol crystal with the purity of 99.4 percent is obtained by centrifugation and drying.
EXAMPLE III
Leaching, namely 500g of industrial hemp flowers and leaves with the CBD content of 1% (w/w), crushing to the average particle size of 1mm, adding pure water 4L, stirring, heating to 30 ℃, preserving heat for 3 hours, filtering, adding 4L 10% (V/V) n-propanol into filter residues, stirring, heating to 30 ℃, preserving heat for 3 hours, filtering, adding 4L 95% (V/V) n-propanol into the filter residues, stirring, heating to 50 ℃, preserving heat for 2 hours, filtering, collecting filtrate, adding 4L 95% (V/V) n-propanol into the filter residues, stirring, heating to 50 ℃, preserving heat for 2 hours, filtering, combining the filtrate to obtain 8L leaching liquor, 45 ℃, 100mbar, rotary evaporation and concentration to 0.56L, detecting the CBD concentration in the concentrated suspension liquid phase to be 8.5 g/L and the extraction rate to be 95.2%.
Adsorption and desorption of macroporous resin, namely, 45ml of L X-22 (produced by Xian lan blue Xiao science and technology New Material Co., Ltd.) macroporous resin is filled into a column by a 40% (V/V) n-propanol aqueous solution wet method, the diameter of the chromatographic column is 1cm, the filling height is 57cm, the macroporous resin column is balanced by 40% (V/V) n-propanol aqueous solution, the flow rate is 1.5ml/min, and the balance is 1h, 560ml of the concentrated suspension obtained in the previous step is loaded onto the macroporous resin column and the effluent is loaded in the forward direction, the flow rate of the effluent is 1.5ml/min and enters a n-propanol recovery system, the effluent is washed by 40% (V/V) n-propanol aqueous solution, the flow rate is 1.5ml/min and the effluent is 30min and enters an alcohol recovery system, the effluent is analyzed by 70% (V/V) n-propanol aqueous solution, the volume is 8 times of the column volume, the flow rate of the analysis process is 1.5ml/min, the analysis solution is collected and concentrated to the extract, the CBD content is 34%, the yield is 95.3%, the effluent is regenerated by 90% acetone aqueous solution, the regeneration process, the flow;
reversed-phase chromatographic separation, namely adding 47ml of reversed-phase chromatographic packing (DAISOGE L SP-120-40-ODS-RPS, purchased from Japan Kao Co., Ltd.) into 80ml of 50% isopropanol, filling the mixture into a column by a wet method, wherein the diameter of the resin column is 10mm, the height of the resin column is 600mm, the pressure of the filled column is 10Mpa, the balance is carried out by using 50% (V/V) isopropanol after the column filling is finished, the flow rate is 4.7ml/min, the balance is 40min, 80m of 60% isopropanol is added into the extract obtained in the previous step, stirring and dissolving are carried out, 30ml of the extract is taken, the loading amount of the extract is 16.8 g/L (CBD pure product/reversed-phase packing) and the flow rate is 4.7ml/min, after the loading is finished, the eluent is washed for 3h by 60% (V/V) isopropanol, eluted for 1h, 0.6L, 100mbar is collected, rotary evaporation and concentration is carried out until the content.
And (3) crystallization: dissolving the extract with 5ml n-hexane at 25 deg.C. After the dissolution is finished, the temperature is reduced to minus 30 ℃, the temperature is kept for 48 hours, and the cannabidiol crystal with the purity of 99.4 percent is obtained by centrifugation and drying.
Example four
Leaching, namely 500g of industrial hemp flowers and leaves with the CBD content of 5% (w/w), crushing to the average particle size of 2mm, adding pure water 6L into a sieve mesh, stirring, heating to 80 ℃, preserving heat for 0.5h, filtering, adding 6L 40% (V/V) isopropanol into filter residues, stirring, heating to 80 ℃, preserving heat for 0.5h, filtering, adding 6L 100% (V/V) isopropanol into the filter residues, stirring, heating to 80 ℃, preserving heat for 0.5h, filtering, collecting filtrate, adding 6L 100% (V/V) isopropanol into the filter residues, stirring, heating to 80 ℃, preserving heat for 0.5h, filtering, combining the filtrate to obtain 12L leaching liquor, 45 ℃, 100mbar, concentrating by rotary evaporation to 2L, detecting the CBD concentration in the concentrated suspension to be 12.1 g/L and the extraction rate to be 96.7%.
Adsorption and desorption of macroporous resin, namely, 45ml of L X-AB-8 (produced by Seisan blue, Xiao science and technology New materials Co., Ltd.) macroporous resin is filled into a column by a wet method with 60% (V/V) isopropanol water solution, the diameter of the chromatographic column is 1cm, the filling height is 57cm, the 60% (V/V) isopropanol water solution is used for balancing the macroporous resin column, the flow rate is 1.5ml/min, and the column is balanced for 1h, 2000ml of the suspension concentrated in the previous step is loaded onto the macroporous resin column, the suspension is loaded forwards, the suspension with the flow rate of 1.5ml/min enters an isopropanol recovery system, 40% (V/V) isopropanol water solution is used for impurity washing, the flow rate is 1.5ml/min, the suspension is washed for 30min, and the suspension enters an alcohol recovery system, 70% (V/V) isopropanol water solution is used for desorption, the desorption volume is 8 times of the column volume, the flow rate in the desorption process is 1.5ml/min, the desorption solution is collected and concentrated to obtain 68.87g of extract, the content of CBD is 33.8%, the yield is 96.3%, the regeneration of the resin by using 90% acetone water solution, the regeneration process, the;
reverse phase chromatography separation, namely adding 80ml of 50% (V/V) isopropanol into 47ml of Kromasil 100-16-C18 (produced by kromasil corporation, Sweden), packing the mixture into a column by a wet method, wherein the diameter of the resin column is 10mm, the height of the resin column is 600mm, the pressure of the packed column is 10Mpa, the balance is carried out by using 60% (V/V) isopropanol after the column packing is finished, the flow rate is 4.7ml/min, the balance is 40min, the extract obtained in the previous step is added with 60% (V/V) isopropanol 80m, stirred and dissolved, 30ml of the solution is sampled, the flow rate is kept at 4.7ml/min by loading 16.8 g/L (CBD pure product/reverse phase filler), the eluent is washed for 3h after the sampling is finished, the eluent is eluted for 1h by 70% isopropanol, 0.6L, 45 ℃, 100mbar is collected by rotary evaporation and concentrated until the content of the extract is 0.834g, the content of CBD in the extract is.
And (3) crystallization: dissolving the extract with 5ml n-hexane at 25 deg.C. After the dissolution is finished, the temperature is reduced to-10 ℃, the temperature is kept for 16h, and the cannabidiol crystal with the purity of 99.4 percent is obtained by centrifugation and drying.
EXAMPLE five
Leaching, namely 500g of industrial hemp flower and leaf with the CBD content of 1% (w/w), crushing to the average particle size of 0.2mm, sieving, adding 2.4L of pure water, stirring, heating to 10 ℃, preserving heat for 4h, filtering, adding 2.4L 10% (V/V) n-butyl alcohol into filter residue, stirring, heating to 10 ℃, preserving heat for 4h, filtering, adding 2.4L 70% (V/V) n-butyl alcohol into filter residue, stirring, heating to 80 ℃, preserving heat for 0.5h, filtering, collecting filtrate, adding 2.4L 70% (V/V) n-butyl alcohol into filter residue, stirring, heating to 80 ℃, preserving heat for 0.5h, filtering, combining filtrate to obtain 4.8L leaching liquor, 45 ℃, 100mbar, rotary evaporation and concentration to 0.4L, detecting the CBD concentration in the concentrated suspension liquid phase to be 12.1 g/L, and the extraction rate to be 96.5%.
And (3) extraction: adding 120ml of n-hexane into the concentrated solution of the extract, extracting at 10 ℃ for 60 minutes, standing for separating liquid after extraction is finished, and combining n-hexane extract liquid. Extracting with 60ml of water solution at 40 ℃ for 60 minutes, and standing for liquid separation after extraction. Extracting with 700ml 60% (V/V) isopropanol solution and the n-hexane solution at 10 deg.C for 60 min, standing, separating, mixing and collecting 80% (V/V) ethanol solution with CBD concentration of 1.4 mg/ml.
Decolorizing, namely adding 0.2 time of activated carbon into a 60% (V/V) isopropanol water extraction solution phase, stirring for 4 hours, removing the activated carbon by suction filtration, leaching a filter cake for 4 times by using the 60% (V/V) isopropanol water solution, collecting liquid to obtain a target object decolorized solution, washing the filter cake by using purified water to obtain a washing liquid, introducing the washing liquid into an alcohol recovery system, concentrating the decolorized solution by 5 times to form a suspension with the concentration of 20 g/L (cannabidiol pure product/liter suspension), protecting the suspension by using nitrogen after concentration, and converting for 6 hours at 80 ℃.
Adsorption and desorption of macroporous resin, namely, 45ml of L X-T83 (produced by the New science and technology Material Co., Ltd.) macroporous resin is filled into a column by a 60% (V/V) isopropanol aqueous solution wet method, the diameter of the chromatographic column is 1cm, the filling height is 57cm, the macroporous resin column is balanced by the 60% (V/V) isopropanol aqueous solution, the flow rate is 1.5ml/min, and the column is balanced for 1h, 2000ml of the suspension concentrated in the previous step is loaded onto the macroporous resin column, the suspension is loaded forwards, the effluent with the flow rate of 1.5ml/min enters an isopropanol recovery system, the effluent with the flow rate of 40% (V/V) isopropanol aqueous solution is used for washing impurities, the flow rate is 1.5ml/min, the impurities are washed for 30min, and the effluent enters an alcohol recovery system, the macroporous resin is desorbed by the 70% (V/V) isopropanol aqueous solution, the desorption volume is 8 times of the column volume, the flow rate of the desorption process is 1.5ml/min, the desorption solution is collected and concentrated to the extract, the extract is obtained, the CBD content is 34.2%, the yield is 97.3%, the regeneration of the macroporous resin is regenerated by the acetone aqueous solution, the regeneration process;
reverse phase chromatography separation, wherein 47ml of L X-2000 chromatography resin (produced by Xian lan Dai science and technology New materials Co., Ltd.) is added with 80ml of 50% (V/V) isopropanol, the column is packed by a wet method, the diameter of the resin column is 10mm, the height of the resin column is 600mm, the column packing pressure is 10Mpa, after the column packing is completed, 50% isopropanol is used for balancing, the flow rate is 4.7ml/min and the balance is 40min, 70% (V/V) isopropanol is added into the extract obtained in the previous step, the mixture is stirred and dissolved, 30ml of the mixture is sampled, the flow rate is kept at 16.8 g/L (CBD pure product/reverse phase filler) for 4.7ml/min, after the sampling is completed, 60% (V/V) isopropanol is used for washing for 3h, 70% (V/V) isopropanol is used for eluting for 1h, 2h before 60% isopropanol is collected, 0.6L is concentrated, CBDV component is obtained, 0.47g of the rest eluent is collected, 0.6L, 45 ℃, 100 ℃ is concentrated by rotary evaporation until the content of the eluent is detected to be 93%.
And (3) crystallization: dissolving the extract with 5ml n-hexane at 25 deg.C. After the dissolution is finished, the temperature is reduced to-40 ℃, the temperature is kept for 48 hours, and the cannabidiol crystal with the purity of 99.4 percent is obtained by centrifugation and drying.
EXAMPLE six
Leaching, namely 500g of industrial hemp flower and leaf with the CBD content of 0.5% (w/w), crushing to the average particle size of 0.5mm, sieving, adding 3.2L of pure water, stirring, heating to 20 ℃, preserving heat for 3.5 hours, filtering, adding 3.2L 10% (V/V) isopropanol into filter residue, stirring, heating to 20 ℃, preserving heat for 3.5 hours, filtering, adding 3.2L 90% (V/V) isopropanol into the filter residue, stirring, heating to 40 ℃, preserving heat for 2 hours, filtering, collecting filtrate, adding 3.2L 90% (V/V) isopropanol into the filter residue, stirring, heating to 40 ℃, preserving heat for 2 hours, filtering, combining the filtrates to obtain 6.4L leaching liquor, 45 ℃, 100mbar, performing rotary evaporation and concentration to 0.4L, detecting the CBD concentration in the suspension liquid phase, wherein the CBD concentration is 6.09 g/L and the extraction rate is 97.5%.
And (3) extraction: adding 400ml of n-hexane into the concentrated solution of the extract, extracting at 10 ℃ for 50 minutes, standing for separating liquid after extraction is finished, and combining n-hexane extract liquid. Extracting with 800ml 10% (V/V) isopropanol solution at 20 deg.C for 40min, and standing for separating. Extracting with 1600% (V/V) 70% isopropanol solution and the n-hexane solution at 10 deg.C for 50 min, standing, separating, mixing and collecting 80% (V/V) ethanol solution with CBD concentration of 1.4 mg/ml.
Decoloring, namely adding 10 times of activated carbon into 80% (V/V) isopropanol water extraction solution phase, stirring for 2 hours, removing the activated carbon by suction filtration, leaching filter cakes for 2 times by using 70% (V/V) isopropanol water solution, collecting liquid to obtain target substance decoloring liquid, washing the filter cakes by purified water to obtain washing liquid, feeding the washing liquid into an alcohol recovery system, concentrating the decoloring liquid by 5 times to obtain suspension with the concentration of 20 g/L (cannabidiol pure product/liter suspension), protecting the suspension by nitrogen after concentration, and converting for 5 hours at 90 ℃.
Adsorption and desorption of macroporous resin, namely, 45ml of L X-T83 (produced by Seisan blue, Xiao science and technology New Material Co., Ltd.) macroporous resin is filled into a column by a wet method with 30% (V/V) isopropanol water solution, the diameter of the chromatographic column is 1cm, the filling height is 57cm, 30% (V/V) isopropanol water solution is used for balancing the macroporous resin column, the flow rate is 1.5ml/min, and the column is balanced for 1h, 400ml of the suspension concentrated in the previous step is loaded into the macroporous resin column, the suspension is loaded forwards, the effluent with the flow rate of 1.5ml/min enters an isopropanol recovery system, 30% (V/V) isopropanol water solution is used for impurity washing, the flow rate is 1.5ml/min, the impurity is washed for 30min, and the effluent enters an alcohol recovery system, 60% (V/V) isopropanol water solution is used for desorption, the column volume is 8 times of the desorption volume, the flow rate in the desorption process is 1.5ml/min, the desorption solution is collected and concentrated to an extract, the extract is obtained, the CBD content is 34.1%, the yield is 96.1%, and the regeneration process is carried out by using 90% acetone water solution, the regeneration process of the;
reverse phase chromatographic separation, wherein 47ml of L X-2000 chromatographic resin (produced by Xian lan Xiao science and technology New Material Co., Ltd.) is added with 80ml of 50% (V/V) isopropanol, the column is packed by a wet method, the diameter of the resin column is 10mm, the height of the resin column is 600mm, the column packing pressure is 10Mpa, after the column packing is finished, the 50% isopropanol is used for balancing, the flow rate is 4.7ml/min and the balance is 40min, the extract in the last step is added with 70% (V/V) isopropanol 80m and stirred for dissolving, 30ml of the extract is sampled, the flow rate is kept at 4.7ml/min by loading 16.8 g/L (CBD pure product/reverse phase filler), after the sampling is finished, 60% (V/V) isopropanol is washed for 3h, 70% (V/V) isopropanol is eluted for 1h, the eluent is collected at 0.6L, 100mbar, the extract is evaporated and concentrated to 0.84g by rotation, the high and medium CBD content is.
And (3) crystallization: dissolving the extract with 5ml n-hexane at 25 deg.C. After the dissolution is finished, the temperature is reduced to minus 30 ℃, the temperature is kept for 48 hours, and the cannabidiol crystal with the purity of 99.4 percent is obtained by centrifugation and drying.
EXAMPLE seven
Leaching, namely 500g of industrial hemp flower and leaf with the CBD content of 1% (w/w), crushing to the average particle size of 0.8mm, sieving, adding pure water 4L, stirring, heating to 30 ℃, preserving heat for 3 hours, filtering, adding 4L 10% (V/V) isopropanol into filter residue, stirring, heating to 30 ℃, preserving heat for 2 hours, filtering, adding 4L 90% (V/V) isopropanol into filter residue, stirring, heating to 50 ℃, preserving heat for 2 hours, filtering, collecting filtrate, adding 4L 90% (V/V) isopropanol into filter residue, stirring, heating to 50 ℃, preserving heat for 2 hours, filtering, combining filtrate to obtain 8L leaching liquor, 45 ℃, 100mbar, rotary evaporation and concentration to 0.5L, detecting the CBD concentration in the concentrated suspension liquid to be 9.63 g/L and the extraction rate to be 96.3%.
And (2) extracting, namely adding 1L volume of n-hexane into the concentrated solution of the extract, extracting at the temperature of 30 ℃ for 30 minutes, standing and separating after extraction is finished, combining n-hexane extract, extracting by using 1L (V/V) 30% isopropanol solution, extracting at the temperature of 30 ℃ for 20 minutes, extracting for 20 minutes, standing and separating after extraction is finished, extracting by using 3L (V/V) 80% isopropanol solution and the n-hexane solution in the previous step, extracting at the temperature of 30 ℃ for 30 minutes, standing and separating after extraction is finished, combining and collecting 80% (V/V) alcohol solution, wherein the CBD concentration is 1.5 mg/ml.
Decoloring, namely adding 15 times of activated carbon into 80% (V/V) isopropanol water extraction solution phase, stirring for 1h, removing the activated carbon by suction filtration, leaching a filter cake for 2 times by using 80% (V/V) isopropanol water solution, collecting liquid to obtain a target object decoloring solution, washing the filter cake by purified water to obtain a washing liquid, feeding the washing liquid into an alcohol recovery system, concentrating the decoloring solution by 5 times to obtain suspension with the concentration of 20 g/L (cannabidiol pure product/liter suspension), protecting the suspension by nitrogen after concentration, and converting for 3h at 100 ℃.
Adsorption and desorption of macroporous resin, namely, 45ml of L X-T83 (produced by Seisan blue, Xiao science and technology New Material Co., Ltd.) macroporous resin is filled into a column by a wet method with 30% (V/V) isopropanol water solution, the diameter of the chromatographic column is 1cm, the filling height is 57cm, the 30% (V/V) isopropanol water solution is used for balancing the macroporous resin column, the flow rate is 1.5ml/min, the column is balanced for 1h, 300ml of the suspension concentrated in the previous step is loaded into the macroporous resin column, the suspension is loaded forwards, the effluent with the flow rate of 1.5ml/min enters an isopropanol recovery system, 30% (V/V) isopropanol water solution is used for impurity washing, the flow rate is 1.5ml/min, the impurity is washed for 30min, the effluent enters an alcohol recovery system, the 60% (V/V) isopropanol water solution is used for desorption, the column volume is 8 times of the column volume, the flow rate in the desorption process is 1.5ml/min, the desorption solution is collected and concentrated to obtain 13.64g of extract, the CBD content is 33.9%, the yield is 96.1.1%, the regeneration process is carried out by using 90% acetone water solution, the regeneration process;
reverse phase chromatographic separation, wherein 47ml of L X-T83SS chromatographic resin (produced by Xian lan Xiao science and technology New Material Co., Ltd.) is added with 80ml of 50% (V/V) isopropanol, the column is filled by a wet method, the diameter of the resin column is 10mm, the height of the resin column is 600mm, the column filling pressure is 10MPa, the 50% (V/V) isopropanol is used for balancing after the column filling is finished, the flow rate is 4.7ml/min and the column filling is 40min, 70% (V/V) isopropanol 80m is added into the extract in the previous step, stirring and dissolving are carried out, 30ml of the extract is sampled, the loading capacity is 16.8 g/L (CBD pure product/reverse phase filler) is kept at the flow rate of 4.7ml/min, the eluent is washed for 3h by 60% (V/V) isopropanol 834 after the sampling is finished, the eluent is eluted for 1h by 70% (V/V) and collected for 0.6L ℃, the temperature is 100mbar, the eluent is concentrated by rotary evaporation until the content of.
And (3) crystallization: dissolving the extract with 5ml n-hexane at 25 deg.C. After the dissolution is finished, the temperature is reduced to minus 30 ℃, the temperature is kept for 48 hours, and the cannabidiol crystal with the purity of 99.4 percent is obtained by centrifugation and drying.
Example eight
Leaching, namely 1kg of industrial hemp flower and leaf with the CBD content of 0.7% (w/w), crushing to the average particle size of 2mm, sieving by using a sieve, adding 8L of pure water, stirring, heating to 80 ℃, preserving heat for 2 hours, filtering, adding 6L 30% (V/V) ethanol into filter residue, stirring, heating to 70 ℃, preserving heat for 2 hours, filtering, adding 6L 90% (V/V) ethanol into the filter residue, stirring, heating to 70 ℃, preserving heat for 2 hours, filtering, collecting filtrate, adding 6L 90% (V/V) ethanol into the filter residue, stirring, heating to 70 ℃, preserving heat for 2 hours, filtering, combining the filtrate to obtain a 12L leaching solution, 45 ℃, 100mbar, rotary evaporation and concentration to 0.66L, detecting the CBD concentration in the concentrated suspension by using a liquid phase detection method, wherein the extraction rate is 10.3 g/L, the liquid phase detection spectrum is shown in figure 5.
And (2) extracting, namely adding 0.66L volume of n-hexane into concentrated solution of the extract, extracting at the temperature of 20 ℃, extracting for 10 minutes, standing and separating after extraction is finished, extracting twice, combining n-hexane extract liquor 1.33L, extracting by using a 30% (V/V) ethanol solution of 1.33L, extracting at the temperature of 20 ℃, extracting for 10 minutes, standing and separating after extraction is finished, retaining n-hexane components, extracting by using a 70% (V/V) ethanol solution of 4L and the n-hexane solution in the previous step, extracting at the temperature of 20 ℃, extracting for 10 minutes, standing, separating, extracting for 3 times after extraction is finished, combining and collecting a 70% (V/V) ethanol solution 12L with the concentration of 0.55mg/ml, the recovery rate in the extraction process is 97.2%, and a liquid phase detection map is shown in figure 6.
Decolorizing, adding 33g of activated carbon into 70% (V/V) ethanol water extraction solution, stirring for 2h, removing the activated carbon by suction filtration, rinsing the filter cake for 1 time by using 70% (V/V) ethanol water solution, collecting the liquid, namely the target object decolorized solution, washing the filter cake by using purified water, obtaining a washing liquid, feeding the washing liquid into an alcohol recovery system, carrying out rotary evaporation at the temperature of 45 ℃ and 100mbar, concentrating the decolorized solution to 650ml, carrying out suspension with the concentration of 10.1 g/L (cannabidiol pure product/liter suspension), carrying out nitrogen protection on a system after concentration, carrying out conversion at the temperature of 110 ℃ for 2h, and obtaining a liquid phase detection map shown in figure 7.
Adsorption and desorption of macroporous resin 130ml L X-T83 (produced by Xian lan Xiao science and technology New Material Co., Ltd.) macroporous resin is filled with 50% (V/V) ethanol water solution by wet method, the diameter of the chromatographic column is 1.6cm, the filling height is 65cm, 50% (V/V) ethanol water solution is used for balancing the macroporous resin column, the flow rate is 1.5ml/min, the column is balanced for 2h, 650ml of 90% ethanol is added into 650ml of the concentrated suspension obtained in the previous step, the mixture is uniformly mixed and loaded onto the macroporous resin column, the mixture is loaded forwards, the effluent with the flow rate of 1.5ml/min enters an ethanol recovery system, 50% (V/V) ethanol water solution is used for washing impurities with the flow rate of 1.5ml/min, the effluent enters an ethanol recovery system, 70% (V/V) ethanol water solution is used for desorption, the column volume is 9 times of the column volume, the flow rate of the desorption process is 1.5ml/min, the desorption solution is collected and concentrated to an extract, the extract is obtained, the CBD content is 44.6%, the yield is 96.1%, the liquid phase chromatography detection is shown in the attached drawing, the liquid phase detection, the regeneration process is carried out, the regeneration of the.
Reverse phase chromatography separation, wherein 47ml of L X-T83SS chromatographic resin (produced by Xian lan Xiao science and technology New Material Co., Ltd.) is added with 80ml of 60% (V/V) ethanol, the column is packed by a wet method, the diameter of the resin column is 10mm, the height of the resin column is 600mm, the column packing pressure is 10Mpa, after the column packing is completed, the 60% (V/V) ethanol is used for balancing, the flow rate is 4.7ml/min, the balance is 40min, 70% (V/V) ethanol is added into the extract obtained in the previous step, the mixture is stirred and dissolved, 15ml of the mixture is loaded, the flow rate is kept at 4.7ml/min by 25 g/L (CBD pure product/reverse phase filler), after the sample loading is completed, the 60% (V/V) isopropanol is eluted for 2h, the 70% (V/V) isopropanol is eluted for 1h, 60% of 0.6L, 70% of eluent is collected respectively, the liquid phase eluent is collected at 0.3L ℃, the temperature is 100mbar, the rotary evaporation concentration is carried out to the extract, the CBDV component is respectively obtained at 0.77g, the CBD.
And (3) crystallization: dissolving the extract with 5ml n-hexane at 25 deg.C. Cooling to-50 deg.C after dissolution, maintaining the temperature for 46h, centrifuging, and drying to obtain cannabidiol crystal 0.738g with purity of 99.8%, and liquid phase detection chromatogram shown in figure 10.

Claims (20)

1. A method for extracting and separating high-purity cannabidiol from low-content industrial cannabis sativa leaves is characterized by comprising the following steps:
step one, leaching: leaching the crushed low-content industrial hemp flowers and leaves by adopting alcohol water solutions with different concentrations, combining high-concentration alcohol leaching liquor, and reducing the alcohol content in the leaching liquor by concentration and evaporation;
step two, adsorption and desorption of macroporous resin:
adsorbing the target components in the concentrated leaching liquor by using macroporous resin, washing impurities by using a low-concentration alcohol solution, resolving the high-concentration alcohol solution, collecting the resolved solution, and concentrating the resolved solution into an extract;
step three, reversed phase chromatography:
purifying by using reversed phase filler as stationary phase and alcohol-water solution as mobile phase to obtain extract, separating cannabidiol and tetrahydrocannabinol, collecting cannabidiol component, and concentrating to obtain extract;
step four, crystallization: dissolving the cannabidiol extract by using an organic solvent, crystallizing at low temperature to obtain crystals, and performing solid-liquid separation and drying to obtain the high-purity cannabidiol crystals.
2. The method as claimed in claim 1, wherein an extraction and decolorization process is added between the first leaching step and the second macroporous resin adsorption and desorption process, and a polymer reversed phase packing is used for reversed phase chromatography, wherein the extraction and decolorization process comprises the following steps:
(1) and (3) extraction: extracting the concentrated extract by using an organic solvent, standing, separating, extracting and separating organic solvent components by using pure water, a low-concentration alcohol solution and a high-concentration alcohol solution respectively, combining and collecting high-concentration alcohol extract, and entering the next step;
(2) and (3) decoloring: decoloring the extract liquor in the last step by using active carbon, collecting filtrate after solid-liquid separation, and reducing the alcohol content in the decolored liquid by concentration and evaporation;
step three, reversed phase chromatography:
and (3) purifying the extract in the last step by using a polymer reversed phase filler as a stationary phase, separating the hypocannabinol, the cannabidiol, the cannabinol and the tetrahydrocannabinol, respectively collecting each component, and concentrating to obtain the extract.
3. The method as claimed in claim 1, wherein the step one leaching process comprises the following steps:
(1) pulverizing industrial hemp leaves to an average particle size range of 0.2-2 mm;
(2) leaching with 6-15 times of water at 10-80 deg.C for 0.5-4 hr, performing solid-liquid separation, repeatedly leaching for 1-3 times, and allowing the solid flower leaf to go to the next step;
(3) leaching with 10-40% (V/V) alcohol solution 6-15 times the weight of the flower and leaf at 10-80 deg.C for 0.5-4 hr, performing solid-liquid separation, repeating leaching for 1-3 times, and allowing the solid flower and leaf to go to the next step;
(4) leaching with 70-100% (V/V) alcohol 6-15 times of the weight of the flower and leaf at 10-80 deg.C for 0.5-4 hr, performing solid-liquid separation, repeatedly leaching for 1-3 times, mixing, collecting leaching solution, and allowing the solid flower and leaf to enter the next step;
(5) concentrating the leaching solution by 3-20 times to form suspension to obtain concentrated leaching solution, and performing the next step.
4. The method for extracting and separating high-purity cannabidiol from low-content industrial cannabis sativa leaves as claimed in claim 1, wherein the adsorption and desorption process of the macroporous resin in step two comprises the following steps:
(1) loading the macroporous resin into a column by using 1-3BV alcohol aqueous solution with the concentration of 30-60% (V/V) by a wet method, wherein the filling height is not less than 50 cm;
(2) using 2-6BV alcohol water-soluble equilibrium resin column with the concentration of 30-60% (V/V), wherein the linear flow velocity in the equilibrium process is not higher than 5 cm/min;
(3) adding alcohol into the concentrated leaching liquor obtained in the last step, adjusting the alcohol concentration to 30-60% (V/V), loading the sample to a macroporous resin column in a positive direction, stopping loading the sample when the CBD concentration detected at the outlet of the resin column is more than 0.01mg/ml, and keeping the linear speed of the loading process not higher than 5 cm/min;
(4) washing impurities with 1-5BV alcohol water solution with concentration of 30-60% (V/V), wherein the linear velocity of the impurity washing process is not higher than 5 cm/min;
(5) resolving with 3-15BV 60-90% (V/V) alcohol water solution at linear velocity not higher than 5cm/min, collecting resolved solution, and concentrating to obtain extract;
(6) regenerating the macroporous resin by using 2-6BV of regenerated solvent aqueous solution with the concentration of 90-100% (V/V), wherein the linear speed in the regeneration process is not higher than 5cm/min, and the resin column is ready to enter the column balancing step of the next period.
5. The method of claim 1, wherein the three-step reverse phase chromatography comprises the steps of:
(1) using reversed phase filler as stationary phase of chromatographic column, homogenizing reversed phase filler with 1-3CV concentration of 0-100% (V/V) alcohol water solution, and wet loading column with height not lower than 25cm and loading pressure not higher than 10 MPa;
(2) using 2-6CV concentration 30-60% (V/V) alcohol water-soluble equilibrium chromatographic column, the linear flow velocity in the equilibrium process is not higher than 10 cm/min;
(3) dissolving the extract obtained in the adsorption and desorption process in 50-80% (V/V) alcohol water solution to obtain cannabidiol solution with the concentration of 10-60 g/L, loading the cannabidiol solution to a chromatographic column in the forward direction, controlling the loading capacity to be 10-40 g/L (CBD pure product/liter of filler), and keeping the linear speed of the loading process to be not higher than 10 cm/min;
(4) washing impurities with 4-15CV (concentration of 50-80% (V/V) alcohol water solution at linear velocity not higher than 10 cm/min;
(5) resolving with 4-15CV 60-90% (V/V) alcohol water solution at linear velocity not higher than 10cm/min, collecting Cannabidiol (CBD) component solution, and concentrating to obtain extract;
(6) regenerating the chromatographic column by using a regeneration solvent aqueous solution with the 2-6CV concentration of 90-100% (V/V), wherein the linear velocity is not higher than 10cm/min in the regeneration process, and the chromatographic column is ready to enter the column balancing step of the next period.
6. The method of claim 1, wherein the four-step crystallization process comprises the following steps:
(1) dissolving cannabidiol component extract obtained by reversed phase chromatography with 1-3 times of organic solvent at 10-60 deg.C;
(2) cooling to-50-20 deg.C, and maintaining for 16-72h to allow crystal to be fully precipitated;
(3) solid-liquid separation, and solid drying to obtain cannabidiol crystal.
7. The method as claimed in claim 1, wherein the cannabidiol content of the low-content industrial cannabis sativa leaves is 0.1-2.5%.
8. The method as claimed in claim 1, wherein the alcohol is monohydric alcohol with carbon number not greater than 4, which is miscible with water in any ratio, and includes but not limited to methanol, ethanol, and one or more of n-propanol, isopropanol and butanol.
9. The method as claimed in claim 1, wherein the organic solvent is a slightly water-soluble or water-insoluble organic solvent, and includes but is not limited to one or more of ethyl acetate, propyl acetate, butyl acetate, isobutyl acetate, ether, anisole, methyl isobutyl ketone, diisobutyl ketone, 1, 2-dichloroethane, 1,1, 1-trichloroethane, cyclohexane, 1, 2-dichloroethylene, dichloromethane, pentane, n-hexane, cyclohexane, heptane, methylcyclohexane, toluene, xylene, cumene, petroleum ether.
10. The method as claimed in claim 1, wherein the macroporous resin is polymer beads copolymerized from polyacrylate-styrene-divinylbenzene, the particle size of the beads is 0.3-1.5mm, and the pore size of the beads is within the range of 0.3-1.5mm
Figure FDA0002466290020000041
The specific surface area is not less than 500m2The cross-linking degree is 50-80%, and the polyacrylate accounts for 5-20% of the weight of the monomer.
11. The method as claimed in claim 1, wherein the reverse phase filler is a filler containing hydrophobic groups for separating polar substances with different intermolecular force difference, including but not limited to C18 bonded silica gel, C12 bonded silica gel, C8 bonded silica gel, phenyl bonded silica gel, butyl bonded silica gel, and polymer reverse phase filler containing ester group, butyl group, phenyl group, octyl group, dodecyl group, hexadecyl group, vinyl group, allyl group, vinylbenzene or divinylbenzene.
12. The method as claimed in claim 1, wherein the concentration includes but is not limited to distillation, distillation under reduced pressure, thin film evaporation, nanofiltration, lyophilization, etc.
13. The method for extracting and separating high-purity cannabidiol from low-content industrial cannabis sativa leaves as claimed in claim 2, wherein the extraction process comprises the following steps:
(1) extracting the concentrated extract by using an organic solvent, wherein the dosage of the organic solvent is 0.3-3 times of the volume of the concentrated extract, the extraction temperature is 0-40 ℃, the extraction time is 10-60 minutes, standing and separating the extract after the extraction is finished, and temporarily storing and combining the organic solvent extract to the step (2);
(2) repeating the step (1) for 0-2 times, and mixing the extracts for temporary storage;
(3) performing impurity washing treatment by using 0-30% (V/V) alcohol-water solution and the temporarily stored extraction liquid in the step (2), wherein the dosage of the alcohol-water solution is 1-3 times of the volume of the extraction liquid in the step (2), the extraction temperature is 0-40 ℃, the extraction time is 10-60 minutes, standing and separating liquid are performed after extraction is completed, and the organic solvent extraction liquid enters the next step;
(4) repeating the step (3) for 0-5 times until the 0-30% (V/V) alcohol solution is colorless or yellowish after layering, and temporarily storing the organic solvent extract;
(5) extracting with 60-85% (V/V) alcoholic solution and the organic solution in step (4), wherein the dosage of the alcoholic solution is 3-5 times the volume of the organic solution in step (4), the extraction temperature is 0-40 ℃, the extraction time is 10-60 minutes, standing and separating are carried out after the extraction is finished, and the 60-85% (V/V) alcoholic solution enters the next step;
(6) repeating the step (5) for 2-6 times, combining and collecting 60-85% (V/V) alcohol solution, and entering the next step.
14. The method for extracting and separating high-purity cannabidiol from low-content industrial cannabis sativa leaves as claimed in claim 2, wherein the decolorization process comprises the following steps:
(1) adding the collected 60-85% (V/V) alcohol-water extraction solution into a certain amount of activated carbon, and stirring for 0.5-4 h;
(2) performing solid-liquid separation, removing the activated carbon, leaching the filter cake for 0-4 times by using 60-85% (V/V) alcohol aqueous solution with the volume 1-3 times that of the filter cake, and collecting the liquid, namely the target object decolorization liquid;
(3) washing the filter cake with pure water;
(4) concentrating the decolorized solution by 5-20 times to obtain suspension with concentration of 1-20 g/L (cannabidiol pure product/liter suspension), treating at 80-110 deg.C under nitrogen protection for 2-6 hr, storing, and performing adsorption and desorption.
15. The method of claim 2, wherein the three-step reverse phase chromatography comprises the steps of:
(1) using polymer reverse phase filler as the stationary phase of chromatographic column, homogenizing with 1-3CV concentration 0-100% (V/V) alcohol water solution, and wet loading to obtain column with height not lower than 25cm and pressure not higher than 10 MPa;
(2) using 2-6CV concentration 30-60% (V/V) alcohol water-soluble equilibrium chromatographic column, the linear flow velocity in the equilibrium process is not higher than 10 cm/min;
(3) dissolving the extract obtained in the adsorption and desorption process in 50-80% (V/V) alcohol water solution to obtain cannabidiol solution with the concentration of 10-60 g/L, loading the cannabidiol solution to a chromatographic column in a forward direction, and loading the cannabidiol solution with the capacity of 10-40 g/L (CBD pure product/liter of filler), wherein the linear speed in the loading process is not higher than 10 cm/min;
(4) performing gradient analysis by using 4-15CV (concentration of 50-95% (V/V) alcohol water solution with linear velocity not higher than 10cm/min in the analysis process, respectively collecting each gradient analysis solution to obtain sub-Cannabidiol (CBDV), Cannabidiol (CBD), Tetrahydrocannabinol (THC) and Cannabinol (CBN) analysis solutions, and respectively concentrating to obtain extract;
(5) regenerating the chromatographic column by using a regeneration solvent aqueous solution with the 2-6CV concentration of 90-100% (V/V), wherein the linear velocity is not higher than 10cm/min in the regeneration process, and the chromatographic column is ready to enter the column balancing step of the next period.
16. The method as claimed in claim 14, wherein the amount of activated carbon added is 0.2-20 times the weight of cannabidiol in the extract.
17. The method as claimed in claim 2, wherein the organic solvent is a slightly water-soluble or water-insoluble organic solvent, and includes but is not limited to one or more of ethyl acetate, propyl acetate, butyl acetate, isobutyl acetate, ether, anisole, methyl isobutyl ketone, diisobutyl ketone, 1, 2-dichloroethane, 1,1, 1-trichloroethane, cyclohexane, 1, 2-dichloroethylene, dichloromethane, pentane, n-hexane, cyclohexane, heptane, methylcyclohexane, toluene, xylene, cumene, petroleum ether.
18. The method as claimed in claim 2, wherein the reverse phase filler is polymer microspheres of polyacrylate-styrene-divinylbenzene, the particle size of the microspheres is 30-150 μm, and the pore size of the microspheres is in the range of 30-150 μm
Figure FDA0002466290020000061
Specific surface area range 400-600m2The cross-linking degree is 40-80%, and the weight of acrylic ester is 5-20% of the total weight of the monomers.
19. The method for extracting and separating high purity cannabidiol from low content industrial cannabis sativa leaves as claimed in any one of claims 4, 5 and 15, wherein the regeneration solvent is water soluble or slightly water soluble alcohol, ester, ether, ketone, amide and sulfone solvents, including but not limited to methanol, ethanol, isopropanol, butanol, ethyl formate, ethyl acetate, propyl acetate, butyl acetate, isobutyl acetate, dioxane, diethyl ether, anisole, acetone, methyl ethyl ketone, butanone, methyl isobutyl ketone, diisobutyl ketone, dimethylformamide, dimethylacetamide and dimethylsulfoxide.
20. The method of claim 1 for extracting and separating high purity cannabidiol from low content industrial cannabis sativa leaves, comprising the steps of:
(1) leaching, namely 1kg of industrial hemp flower and leaf with the CBD content of 0.7% (w/w), crushing to the average particle size of 2mm, sieving by using a sieve, adding 8L of pure water, stirring, heating to 80 ℃, preserving heat for 2 hours, filtering, adding 6L 30% (V/V) ethanol into filter residue, stirring, heating to 70 ℃, preserving heat for 2 hours, filtering, adding 6L 90% (V/V) ethanol into the filter residue, stirring, heating to 70 ℃, preserving heat for 2 hours, filtering, collecting filtrate, adding 6L 90% (V/V) ethanol into the filter residue, stirring, heating to 70 ℃, preserving heat for 2 hours, filtering, combining the filtrate to obtain 12L leaching liquor, concentrating to 0.66L by rotary evaporation at 45 ℃, and detecting the CBD concentration in the concentrated suspension liquid phase to be 10.3 g/L;
(2) extracting, namely adding 0.66L volume of n-hexane into concentrated solution of the extract, extracting at 20 ℃ for 10 minutes, standing and separating after extraction is finished, extracting twice, combining n-hexane extract liquor 1.33L, extracting by using 30% (V/V) ethanol solution of 1.33L, extracting at 20 ℃ for 10 minutes, standing and separating after extraction is finished, retaining n-hexane component, extracting by using 70% (V/V) ethanol solution of 4L and the n-hexane solution in the previous step, extracting at 20 ℃ for 10 minutes, standing and separating after extraction is finished, extracting for 3 times, combining and collecting 70% (V/V) ethanol solution 12, wherein the concentration of 12L is 0.55 mg/ml;
(3) decolorizing, namely adding 33g of activated carbon into a 70% (V/V) ethanol water extraction solution phase, stirring for 2 hours, removing the activated carbon by suction filtration, leaching a filter cake for 1 time by using 70% (V/V) ethanol water solution, collecting liquid, namely target object decolorized liquid, washing the filter cake by purified water, obtaining washing liquid, feeding the washing liquid into an alcohol recovery system, carrying out rotary evaporation at the temperature of 45 ℃ and 100mbar to concentrate a suspension of the decolorized liquid to 650ml and the concentration of 10.1 g/L (cannabidiol pure product/liter suspension), carrying out nitrogen protection on a system after concentration, and converting for 2 hours at the temperature of 110 ℃;
(4) adsorption and desorption of macroporous resin, namely, loading 130ml L X-T83 macroporous resin produced by Xian lan Xiao science and technology New materials Co., Ltd into a column by a 50% (V/V) ethanol water solution wet method, wherein the diameter of the chromatographic column is 1.6cm, the filling height is 65cm, balancing the macroporous resin column by 50% (V/V) ethanol water solution, the flow rate is 1.5ml/min, balancing for 2h, adding 650ml 90% ethanol into 650ml concentrated suspension obtained in the previous step, uniformly mixing, loading the mixture into the macroporous resin column, loading the mixture in the forward direction, feeding the effluent with the flow rate of 1.5ml/min into an ethanol recovery system, washing the effluent with 50% (V/V) ethanol water solution at the flow rate of 1.5ml/min, washing the effluent with the ethanol water solution of 70% (V/V), resolving the column volume which is 9 times of the column volume, feeding the flow rate of 1.5ml/min in the resolution process, collecting the resolution solution, concentrating the extract to obtain 14.15g of extract, the CBD content of 44.6%, the yield of 96.1.1%, regenerating the acetone aqueous solution, regenerating the macroporous resin;
(5) reverse phase chromatography, namely adding 80ml of 60% (V/V) ethanol into 47ml of L X-T83SS chromatographic resin produced by Xian lan Heng Xiao science and technology New Material Co., Ltd, filling a column by a wet method, wherein the diameter of the resin column is 10mm, the height of the resin column is 600mm, the column filling pressure is 15MPa, after the column filling is finished, the 60% (V/V) ethanol is used for balancing, the flow rate is 4.7ml/min and 40min are used, 70% (V/V) ethanol is added into the extract obtained in the previous step, stirring and dissolving are carried out, 15ml of the extract is obtained, the flow rate is kept at 25 g/L (CBD pure product/reverse phase filler) for 4.7ml/min, after the sample filling is finished, the 60% (V/V) isopropanol is used for eluting for 2h, 70% (V/V) isopropanol is used for eluting for 1h, 60% of eluent is respectively collected, 0.6L, 70% of eluent is collected, 0.3L, 45 ℃, 100mbar, rotary evaporation is concentrated to the extract, and;
(6) and (3) crystallization: dissolving the above extract with 2.4ml n-hexane at 25 deg.C, cooling to-50 deg.C, maintaining for 46h, centrifuging, and drying to obtain cannabidiol crystal.
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