CN110803982A - Microwave-assisted extraction of cannabidiol and preparation method thereof - Google Patents
Microwave-assisted extraction of cannabidiol and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biomedicine, and particularly relates to a microwave-assisted cannabidiol extraction method and a preparation method thereof, wherein the cannabidiol accounts for more than or equal to 95% and less than 100% by weight, and tetrahydrocannabinol accounts for no detection; the invention provides a microwave-assisted extraction method of cannabidiol and a preparation method thereof, and through multiple creative tests, the technical problems of long extraction time, incomplete extraction and the like of the existing process are solved, and meanwhile, the problem of high-efficiency extraction equipment cost is effectively solved. The preparation method can remarkably improve the extraction rate of cannabidiol from industrial cannabis sativa and simplify the extraction steps.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a microwave-assisted extraction method of cannabidiol and a preparation method thereof.
Background
Cannabis sativa L, also known as Cannabis sativa L, is a plant of the genus Cannabis of the family Moraceae, an annual upright herb, 1-3 meters high. The Cannabidiol (CBD) component contained in the cannabis sativa is a phenolic compound which is not separated from other animals and plants at present, and the Cannabidiol (CBD) is a non-addictive component and has high medicinal value. The research shows that if cannabidiol is reasonably applied to medicine, the cannabidiol has the functions of resisting epilepsy, resisting psychosis, resisting depression, relieving pain, relieving nausea caused by cancer chemotherapy, treating asthma and the like. In recent years, scientists also find that Cannabidiol (CBD) is a powerful antioxidant, has the function of blocking the adverse effect of certain drugs on human nerves, and has a series of physiological activity functions of blocking breast cancer metastasis, resisting rheumatoid arthritis, resisting insomnia and the like. Cannabis is generally classified into two types, industrial cannabis and narcotic cannabis, according to the proportion of Tetrahydrocannabinol (THC) contained therein. The industrial cannabis belongs to low-toxicity cannabis, wherein the content of Tetrahydrocannabinol (THC) is not more than 0.3 percent at most (according to the national accepted definition method), and the industrial cannabis has no drug utilization value; the content of Tetrahydrocannabinol (THC) in the leaves and flowers of the drug marijuana is high, and the drug is high-toxicity marijuana.
Because of the medicinal value of cannabidiol, a plurality of extraction plants are established all over the world, the prior art mostly extracts the cannabidiol by using an organic solvent extraction method, which consumes a large amount of organic solvent, increases the extraction cost and pollutes the environment, and on the other hand, the organic solvent remained in the cannabidiol extract can also reduce the quality of the cannabidiol extract, so that the extraction method which improves the purity and the extraction efficiency of the cannabidiol extract and reduces the production cost is very important. In order to overcome the defects of the prior art, the invention provides the microwave-assisted extraction method of the cannabidiol and the preparation method thereof, and the method is easy to operate, high in selectivity, low in energy consumption and material consumption, short in extraction time, high in extraction purity and free of special separation steps.
Disclosure of Invention
The invention provides a microwave-assisted cannabidiol preparation method, which solves the technical problems of long extraction time, incomplete extraction and the like of the prior art through multiple creative tests and also effectively solves the problem of high-efficiency extraction equipment cost. The preparation method can remarkably improve the extraction rate of cannabidiol from industrial cannabis sativa and simplify the extraction steps.
The technical scheme of the invention is as follows:
a cannabidiol extract contains cannabidiol 95% or more and less than 100% by weight, and tetrahydrocannabinol is not detected. The preferable weight percentage content of cannabidiol in the extract is more than or equal to 97% and less than 100%, and the weight percentage content of tetrahydrocannabinol in the extract is not detected.
The preparation method of the cannabidiol extract comprises the following steps:
(1) screening and dehydrating: and (3) putting the dried industrial hemp into a grinder for grinding, and dehydrating and drying the ground and sieved industrial hemp to obtain a crude industrial hemp product (w/w) with the moisture content of less than 10%.
(2) And (3) heat treatment: and (2) putting the industrial hemp raw material obtained in the step (1) into a drying box, and drying for 2-5 hours at the drying temperature of 110-.
(3) Microwave pretreatment: and (3) putting the conversion raw material obtained in the step (2) into an extraction tank, and starting a microwave generator for pretreatment for 2-8 min.
(4) Leaching: adding an extraction solvent into the step (3), and soaking and extracting for 0.5-2 hours at normal temperature to obtain a primary extraction solution.
(5) And (3) filtering: and (4) performing pressure filtration on the primary extract obtained in the step (4).
(6) Concentration and drying: and (5) carrying out reduced pressure concentration on the filtrate obtained in the step (5), and drying to obtain the cannabidiol extract.
(7) Water precipitation: dissolving the cannabidiol crude product in a mixed organic solvent, wherein the volume to weight ratio of the mixed organic solvent to the cannabidiol crude product is 15-25:1(ml/g), and preparing a cannabidiol crude product solution; cooling the cannabidiol crude product solution to 5-10 ℃, adding the cannabidiol antisolvent, filtering and drying to obtain the cannabidiol crude product.
(8) Preparation: the cannabidiol extract is subjected to flocculation precipitation, column chromatography, crystallization and other methods to obtain cannabidiol crystals.
The screen of the pulverizer in the step (1) is 30-50 meshes; the temperature for dehydration and drying is selected from 60-80 ℃.
The extraction solvent in the step (4) is selected from alcohols of C1-C4, preferably methanol, ethanol, isopropanol or a mixed solvent thereof.
The plastid ratio of the raw material to the extraction solvent in the step (4) is selected from 3: 1-1: 3, preferably 2:1, 1:1 or 1: 2.
The filtration and separation pressure in the step (5) is 0.1-0.2MPa, and the filter screen is 400-600 meshes.
The mixed solvent in the step (7) is a mixed solvent of saturated alkane of C5-C10 and alcohol of C1-C3; the volume ratio of the saturated alkanes of C5-C10 to the alcohols of C1-C3 is selected from 1: 1-1: 3; the anti-solvent is water, and the volume ratio of the water to the cannabidiol crude product solution is 1: 1-1: 5.
Compared with the prior art, the technical scheme of the invention has the following advantages:
compared with the extraction method in the prior art, the method for extracting cannabidiol from industrial hemp through microwave pretreatment has the advantages of simple process and no need of using a large amount of organic solvent for repeated extraction; in addition, the extraction time can be greatly shortened, the extraction rate can be improved, and the method has a good application value in the pharmaceutical industry.
Detailed Description
The technical solutions of the present invention are described below with specific examples, but the scope of the present invention is not limited thereto. The embodiments described in this specification are merely illustrative of implementations of the inventive concept and the scope of the present invention should not be considered limited to the specific forms set forth in the embodiments but rather by the equivalents thereof as may occur to those skilled in the art upon consideration of the present inventive concept. While the following embodiments of the invention have been described, the invention is not limited to the specific embodiments and applications described above, which are intended to be illustrative, instructive, and not limiting. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention as defined by the appended claims.
Example 1
Microwave-assisted extraction and preparation of cannabidiol
(1) Cleaning and airing 10kg of industrial hemp, adopting an XL-60C type traditional Chinese medicine grinder, then sieving by a 40-mesh sieve, placing the flower and leaf of the industrial hemp after grinding and sieving in an oven, and dehydrating and drying at 75 ℃ to obtain a crude product of the industrial hemp.
(2) And (2) putting the industrial hemp raw material obtained in the step (1) into a drying box, flatly paving the raw material to a thickness of 1.5 cm, setting the drying temperature to 130 ℃, and drying for 4 hours to obtain the converted industrial hemp raw material.
(3) And (3) putting the conversion raw material obtained in the step (2) into a 50L extraction tank, and starting a microwave generator for pretreatment for 8 min.
(4) Adding absolute ethyl alcohol into the step (3), wherein the mass volume ratio of the absolute ethyl alcohol to the industrial hemp raw material is 1:3, and soaking for 1 hour at 60 ℃; discharging the first leaching solution, adding absolute ethyl alcohol, soaking at 60 ℃ for 0.5 hour, and mixing to obtain the primary leaching solution, wherein the mass volume ratio of the absolute ethyl alcohol to the industrial hemp raw material is 1: 2.
(5) And (4) pumping the primary extract obtained in the step (4) into a 600-mesh filter tank, boosting the pressure to 0.1MPa, and filtering.
(6) And (4) concentrating the filtrate obtained in the step (5) under reduced pressure, and drying at 90 ℃ to prepare 104g of cannabidiol extract.
(7) Dissolving 100g of the obtained cannabidiol crude product in 200ml of mixed solvent of ethanol and n-hexane (the volume ratio of the ethanol to the mixed solvent of n-hexane is 1:1) to prepare a cannabidiol crude product solution; and reducing the temperature of the prepared cannabidiol crude product solution to 5 ℃, adding pure water (the volume ratio of water to the cannabidiol crude product solution is 1:2), standing overnight at 5-10 ℃, filtering and drying to obtain 48.16 g of cannabidiol crude product (the content of cannabidiol is 34%).
(8) The obtained cannabidiol crude product is processed by flocculation precipitation, column chromatography, crystallization and other methods to obtain cannabidiol crystals 8.2g (cannabidiol content is 99.6%, tetrahydrocannabinol is not detected); the flocculant is PAC, the chromatography filler is macroporous resin, and the crystallization solvent is cyclohexane.
Example 2
Microwave-assisted extraction and preparation of cannabidiol
(1) Cleaning and airing 10kg of industrial hemp, adopting an XL-60C type traditional Chinese medicine grinder, then sieving by a 40-mesh sieve, placing the flower and leaf of the industrial hemp after grinding and sieving in an oven, and dehydrating and drying at 75 ℃ to obtain a crude product of the industrial hemp.
(2) And (2) putting the industrial hemp raw material obtained in the step (1) into a drying box, flatly paving the raw material to a thickness of 1.5 cm, setting the drying temperature to 130 ℃, and drying for 4 hours to obtain the converted industrial hemp raw material.
(3) And (3) putting the conversion raw material obtained in the step (2) into a 50L extraction tank, and starting a microwave generator for pretreatment for 5 min.
(4) Adding absolute ethyl alcohol into the step (3), wherein the mass volume ratio of the absolute ethyl alcohol to the industrial hemp raw material is 1:3, and soaking for 1 hour at 60 ℃; discharging the first leaching solution, adding absolute ethyl alcohol, soaking at 60 ℃ for 0.5 hour, and mixing to obtain the primary leaching solution, wherein the mass volume ratio of the absolute ethyl alcohol to the industrial hemp raw material is 1: 2.
(5) And (4) pumping the primary extract obtained in the step (4) into a 600-mesh filter tank, boosting the pressure to 0.1MPa, and filtering.
(6) And (4) concentrating the filtrate obtained in the step (5) under reduced pressure, and drying at 90 ℃ to prepare 106g of cannabidiol extract.
(7) Dissolving 100g of the obtained cannabidiol crude product in 200ml of mixed solvent of ethanol and n-hexane (the volume ratio of the ethanol to the mixed solvent of n-hexane is 1:1) to prepare a cannabidiol crude product solution; and reducing the temperature of the prepared cannabidiol crude product solution to 5 ℃, adding pure water (the volume ratio of water to the cannabidiol crude product solution is 1:2), standing overnight at 5-10 ℃, filtering and drying to obtain 46.3g of cannabidiol crude product (the content of cannabidiol is 36%).
(8) The obtained cannabidiol crude product is processed by flocculation precipitation, column chromatography, crystallization and other methods to obtain cannabidiol crystals 8.4g (cannabidiol content is 99.2%, tetrahydrocannabinol is not detected); the flocculant is PAC, the chromatography filler is macroporous resin, and the crystallization solvent is cyclohexane.
Example 3
Microwave-assisted extraction and preparation of cannabidiol
(1) Cleaning and airing 20kg of industrial hemp, adopting an XL-60C type traditional Chinese medicine grinder, then sieving by a 40-mesh sieve, placing the flower and leaf of the industrial hemp after grinding and sieving in an oven, and dehydrating and drying at 75 ℃ to obtain a crude product of the industrial hemp.
(2) And (2) putting the industrial hemp raw material obtained in the step (1) into a drying box, flatly paving the raw material to a thickness of 1.5 cm, setting the drying temperature to 130 ℃, and drying for 4 hours to obtain the converted industrial hemp raw material.
(3) And (3) putting the conversion raw material obtained in the step (2) into a 50L extraction tank, and starting a microwave generator for pretreatment for 6 min.
(4) Adding absolute ethyl alcohol into the step (3), wherein the mass volume ratio of the absolute ethyl alcohol to the industrial hemp raw material is 1:3, and soaking for 1 hour at 60 ℃; discharging the first leaching solution, adding absolute ethyl alcohol, soaking at 60 ℃ for 0.5 hour, and mixing to obtain the primary leaching solution, wherein the mass volume ratio of the absolute ethyl alcohol to the industrial hemp raw material is 1: 2.
(5) And (4) pumping the primary extract obtained in the step (4) into a 600-mesh filter tank, boosting the pressure to 0.1MPa, and filtering.
(6) And (4) concentrating the filtrate obtained in the step (5) under reduced pressure, and drying at 90 ℃ to prepare 216g of cannabidiol extract.
(7) Dissolving 100g of the obtained cannabidiol crude product in 200ml of mixed solvent of ethanol and n-hexane (the volume ratio of the ethanol to the mixed solvent of n-hexane is 1:1) to prepare a cannabidiol crude product solution; and reducing the temperature of the prepared cannabidiol crude product solution to 5 ℃, adding pure water (the volume of water and the cannabidiol crude product solution is 1:2), standing overnight at 5-10 ℃, filtering and drying to obtain 48.0g of cannabidiol crude product (the content of cannabidiol is 32%).
(8) The obtained cannabidiol crude product is processed by flocculation precipitation, column chromatography, crystallization and other methods to obtain 7.9g cannabidiol crystals (with the cannabidiol content of 99.8 percent, without detecting tetrahydrocannabinol); the flocculant is PAC, the chromatography filler is macroporous resin, and the crystallization solvent is cyclohexane.
Example 4
Preparation of cannabidiol
Dissolving 100g of the obtained cannabidiol crude product in 200ml of mixed solvent of ethanol and n-heptane (the volume ratio of the mixed solvent of ethanol and n-heptane is 4:1) to prepare a cannabidiol crude product solution; reducing the temperature of the prepared cannabidiol crude product solution to 5 ℃, adding pure water (the volume ratio of water to the cannabidiol crude product solution is 1:2), standing overnight at 5-10 ℃, filtering and drying to obtain 53.0g of cannabidiol crude product (the content of cannabidiol is 27%)
The obtained cannabidiol crude product is processed by flocculation precipitation, column chromatography, crystallization and other methods to obtain cannabidiol crystals 8.4g (cannabidiol content is 99.0%, tetrahydrocannabinol is not detected); the flocculant is PAC, the chromatographic filler is macroporous resin, and the crystallization solvent is n-hexane.
Detection contrast
Control 1: the extraction is carried out by the method described in example 1 of patent CN 106278828A.
Experimental example sample 1: prepared according to the method described in example 1.
Experimental example sample 2: prepared according to the method described in example 4.
The detection and analysis method comprises the following steps: an experimental instrument: hitachi L-2300 high performance liquid chromatography, Hitachi, Japan; hitachi H i ta H i L-2400 ultraviolet detector; japanese Hitachi D-2000E l ite chromatography workstation; sartorius BP210S electronic balance.
Reagent: ethanol and tetrahydrofuran are pure in chromatography and purchased from Shandong Yuwang Kogyo Co Ltd; acetic acid is chromatographically pure;
and (3) testing the sample: cannabidiol crystals, prepared by the method described in example 1.
Preparing a test solution: taking 1mg of the test solution, and preparing the test solution with the ethanol constant volume to 100 mu g/mL.
Comparison products: cannabidiol and tetrahydrocannabinol were purchased from china pharmaceutical biologicals institute.
A chromatographic column: the chromatographic column is an Agilent ZORBAX SB-C18 liquid chromatographic column 880975-902.
Preparing a mobile phase: the organic phase is a mixed solvent of ethanol and tetrahydrofuran (v/v ═ 15: 1); the aqueous phase was 0.8% aqueous sodium acetate (w/v).
Mobile phase:
a: 0.8% aqueous sodium acetate (pH adjusted to 2.5 with acetic acid) -EtOH tetrahydrofuran (90: 10);
b: 0.8% aqueous sodium acetate (pH adjusted to 2.5 with acetic acid) -EtOH tetrahydrofuran (60: 40).
Detection conditions are as follows: the flow rate is 1.0 mL/min; the detection wavelength is 220 nm; the column temperature was 35 ℃; the amount of sample was 10. mu.L.
Table 1 gradient conditions (volume ratio):
preparing reference solution and determining linear regression equation by taking cannabidiol reference solution (1mg/mL), preparing reference mother solution of 200 mug/mL cannabidiol by ethanol constant volume, and preparing cannabidiol reference solution with concentration gradient of 0.05 mug/mL, 0.5 mug/mL, 1 mug/mL, 5 mug/mL, 25 mug/mL, 50 mug/mL, 100 mug/mL and 200 mug/mL; according to the same method of cannabidiol, taking a tetrahydrocannabinol reference substance (1mg/mL) solution, preparing a reference substance mother solution of 200 mu g/mL cannabidiol by using ethanol to fix the volume, and preparing the tetrahydrocannabinol reference substance solution with the concentration gradient of 0.02 mu g/mL, 0.2 mu g/mL, 0.8 mu g/mL, 4 mu g/mL, 20 mu g/mL, 40 mu g/mL, 80 mu g/mL and 160 mu/mL.
The following linear regression equation is obtained according to the corresponding peak areas:
cannabidiol:
Y=75621X+39051,n=8(R2=0 .9995);
tetrahydrocannabinol:
Y=73973X+36298,n=8(R2=0 .9994)。
the results of the CBD content measurements of the above-mentioned control and experimental samples are shown in table 2.
Table 2 results for CBD content of each sample:
name (R) | Content of CBD | Tetrahydrocannabinol content |
Standard article | 100% | Not detected out |
Reference substance | 93 .7% | 0.27% |
Experimental example sample 1 | 99 .6% | Not detected out |
Experimental example sample 2 | 99 .0% | Not detected out |
According to the detection results, compared with the method provided by CN106278828A, the preparation method provided by the invention has the advantages that the process is simple, repeated extraction and column chromatography are not needed, and the extraction rate is up to more than 90%; in addition, the refined cannabidiol extract has the purity of over 99 percent, no tetrahydrocannabinol is detected, and the tetrahydrocannabinol meets the medicinal standard, so the preparation and refining method provided by the invention is suitable for pharmaceutical industrial production, reduces the production cost and simultaneously reduces the environmental pollution.
Claims (8)
1. A microwave-assisted extraction method of cannabidiol and a preparation method thereof are characterized by comprising the following steps:
(1) screening and dehydrating: putting the dried industrial hemp into a pulverizer to be pulverized, and dehydrating and drying the pulverized and sieved industrial hemp to prepare a crude industrial hemp product (w/w) with the moisture content of less than 10%;
(2) and (3) heat treatment: putting the industrial hemp raw material obtained in the step (1) into a drying box, and drying for 2-5 hours at the drying temperature of 110-;
(3) microwave pretreatment: putting the conversion raw material obtained in the step (2) into an extraction tank, and starting a microwave generator for pretreatment for 2-8 min;
(4) leaching: adding an extraction solvent into the step (3), and soaking and extracting for 0.5-2 hours at normal temperature to obtain a primary extraction solution;
(5) and (3) filtering: carrying out pressure filtration on the primary extract obtained in the step (4);
(6) concentration and drying: concentrating the filtrate obtained in the step (5) under reduced pressure, and drying to obtain cannabidiol extract;
(7) water precipitation: dissolving the cannabidiol crude product in a mixed organic solvent, wherein the volume to weight ratio of the mixed organic solvent to the cannabidiol crude product is 15-25:1(ml/g), and preparing a cannabidiol crude product solution; cooling the cannabidiol crude product solution to 5-10 ℃, adding the cannabidiol antisolvent, filtering and drying to obtain a cannabidiol crude product;
(8) preparation: the cannabidiol extract is subjected to flocculation precipitation, column chromatography, crystallization and other methods to obtain cannabidiol crystals.
2. The microwave-assisted extraction of cannabidiol and its preparation method as claimed in claim 1, wherein: the screen mesh of the hemp flower and leaf crushing and grinding machine in the step 1 is 30-50 meshes; the temperature for dehydration and drying is selected from 60-80 ℃.
3. The microwave-assisted extraction of cannabidiol and its preparation method as claimed in claim 1, wherein: the drying temperature of the drying box in the step 2 is 110-140 ℃, and the drying time is 2-5 hours.
4. The microwave-assisted extraction of cannabidiol and its preparation method as claimed in claim 1, wherein: and 3, starting a microwave generator for pretreatment, wherein the pretreatment time is 2-8 min.
5. The microwave-assisted extraction of cannabidiol and its preparation method as claimed in claim 1, wherein: in the step 4, the plastid ratio of the N raw material to the leaching solvent is 3: 1-1: 3, preferably 2:1, 1:1 or 1: 2.
6. The microwave-assisted extraction of cannabidiol and its preparation method as claimed in claim 1, wherein: in the step 5, the filtering and separating pressure is 0.1-0.2MPa, and the filter screen is 400-600 meshes.
7. The microwave-assisted extraction of cannabidiol and its preparation method as claimed in claim 1, wherein: the mixed solvent in the step 7 is a mixed solvent of saturated alkane of C5-C10 and alcohol of C1-C3; the volume ratio of the saturated alkanes of C5-C10 to the alcohols of C1-C3 is selected from 1: 1-1: 3; the anti-solvent is water, and the volume ratio of the water to the cannabidiol crude product solution is 1: 1-1: 5.
8. The microwave-assisted extraction of cannabidiol and its preparation method as claimed in claim 1, wherein: in the step 7, the organic solvent and the cannabidiol crude product are mixed according to the volume and weight ratio of 15-25:1(ml/g) to prepare a cannabidiol crude product solution; the cannabidiol crude product solution is cooled to 5-10 ℃.
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