CN109568389B - Preparation method of high-purity cannabinol extract - Google Patents
Preparation method of high-purity cannabinol extract Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/60—Moraceae (Mulberry family), e.g. breadfruit or fig
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/17—Preparation or pretreatment of starting material involving drying, e.g. sun-drying or wilting
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
Abstract
The invention discloses a preparation method of a cannabis phenol extract, which comprises the following steps: pulverizing the extracted part of Cannabis sativa L, and baking to obtain medicinal powder; extracting the medicinal powder with solvent to obtain extractive solution, and concentrating to obtain soft extract; extracting the thick paste with solvent, collecting the upper layer liquid, and concentrating to obtain thick paste; adjusting the pH of the soft extract to alkaline, filtering, adjusting the pH of the obtained filtrate to acidic, and filtering to obtain precipitate; dissolving the precipitate with solvent, performing column chromatography on the obtained solution, and eluting; concentrating the eluate to obtain cannabinol extract. In the method, the used raw materials, reagents and instruments are cheap and easy to obtain, and the cost is low; the high-quality extract can be obtained, a plurality of cannabinoids are reserved, resource waste is avoided, wherein the total content of the cannabinoids reaches more than 80%, particularly the THC content is less than 1 per thousand, and the product safety is high; and each preparation batch has good stability, and industrialization is easy to realize.
Description
Technical Field
The invention relates to the technical field of chemistry, in particular to a preparation method of a high-purity cannabis phenol extract.
Background
Cannabis sativa L, a plant of the Cannabis family, the Cannabis genus, Cannabis, China hemp, Cannabis sativa, Mucuna spicata, and Jute, and has important agricultural and medicinal values. Cannabis sativa contains a toxic component THC (tetrahydrocannabinol) which is hallucinogenic and can be used as a drug, and the seed is prohibited for a long time.
Because of the extremely high economic and medicinal values of the hemp, the raw material hemp specially used for industrial application is called industrial hemp for short, the Tetrahydrocannabinol (THC) content in the hemp flowers and leaves in the growth period is less than three thousandth, and the hemp has no value of extracting toxic component tetrahydrocannabinol or can be directly sucked as drugs, and can be legally planted in large scale and used for industrial development.
At present, more than 500 kinds of cannabinoids are separated from hemp plants, wherein the cannabinoids are at least 86 kinds, and the cannabinoids are terpene phenolic compounds, and researches prove that industrial hemp plants contain a plurality of cannabinoids, wherein the main phenolic compounds comprise more than eighty kinds of Cannabidiol (CBD), sub-Cannabidiol (CBDV), Cannabinol (CBN), Cannabigerol (CBG), cannabichromel (CBC), Tetrahydrocannabinol (THC) and the like. The phenolic substances are proved to have strong pharmacological activity, for example, CBD has no neurotoxicity, can block the influence of THC on the human nervous system, has obvious pharmacological activity of resisting spasm, rheumatic arthritis, anxiety and the like, and has great industrial development value; CBD and THCV can influence lipid and carbohydrate metabolism, and can be a new choice for controlling blood sugar of type 2 diabetes patients, and the like, CBDV has better anti-epileptic activity, and meanwhile, researches show that CBDV can relieve nausea symptoms and is helpful for treating gastrointestinal problems, and the combined application effect of the phenolic substances is stronger. However, THC in cannabis has the hallucinogenic effect, and the extract without THC is obtained by taking industrial cannabis as a raw material, so that a raw material for providing high-activity substances for pharmaceutical preparations is necessary.
The cannabis polyphenol extract taking cannabis phenols as main components has a similar structure, the removal difficulty of THC is high, and the research is less, and patent application CN106860492A discloses a preparation method of cannabis phenol compounds, but the method has complex steps and low yield, and THC is not removed as harmful substances, so that the method is not suitable for practical application; patent application CN106278828A discloses a method for extracting CBD from industrial cannabis sativa leaves, the purity of CBD in the finished product is high, and THC is removed, but other cannabinol compounds such as CBDV, THCV and the like are not considered in the extraction, the resources are wasted to a certain extent, and the extraction method has more complicated steps, which is not beneficial to industrial production.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a preparation process of a high-purity cannabis phenol extract, wherein the total content of cannabis phenol compounds in the extract reaches more than 80%, and the main active ingredients and the contents thereof are respectively as follows: 10% -13% of CBDV; 55% -60% of CBD; 4% -5% of CBG; 4% -5% of THCV; THC, less than one ten thousandth of the content.
In a first aspect, the present invention provides a process for the preparation of a high purity cannabinol extract.
In a second aspect, the present invention provides a highly pure cannabinol extract prepared by the above method.
The third aspect of the invention provides an application of the cannabinol extract in preparing medicines.
Specifically, the preparation method of the cannabinol extract provided by the invention comprises the following steps:
(1) pulverizing the extracted part of Cannabis sativa L, and baking to obtain medicinal powder;
(2) extracting the medicinal material powder obtained in the step (1) by using a solvent to obtain an extracting solution, and concentrating the extracting solution to obtain thick paste;
(3) extracting the thick paste obtained in the step (2) by using a solvent, taking the upper layer liquid, and concentrating the upper layer liquid to obtain thick paste;
(4) adjusting the pH of the thick paste obtained in the step (3) to be alkaline, filtering, adjusting the pH of the obtained filtrate to be acidic, and filtering to obtain a precipitate;
(5) dissolving the precipitate obtained in the step (4) with a solvent, and performing column chromatography and elution on the obtained solution;
(6) and (5) concentrating the eluent obtained in the step (5) to obtain the cannabinol extract.
Preferably, the extraction site in step (1) is selected from the group consisting of: one or a combination of more than two of hemp leaf, hemp flower, hemp root, hemp stem core and hemp seed meal in any proportion; more preferably, the extraction part is cannabis flos and/or cannabis leaves, and further preferably, the cannabis flos and/or cannabis leaves in full bloom stage are used as the cannabis flos and/or cannabis leaves.
Preferably, the cannabis in step (1) is selected from one or a combination of more than two of industrial cannabis, intermediate cannabis or medicinal cannabis; more preferably industrial hemp.
Preferably, the hemp extract part in step (1) is a hemp extract part which is primarily dried (such as drying in the sun, baking and the like) after being picked, and more preferably, the hemp extract part has a water content of 10-15% and a soil content of less than 3%.
The baking in the step (1) is beneficial to converting some other components in the hemp extract part into cannabinoids, for example, cannabinolic acid can be converted into Cannabinol (CBN) by removing carboxyl through non-enzymatic reaction, and the total content of the phenolic compounds after baking can be improved by more than 50%; preferably, the baking temperature is 100-. Preferably, the pulverization in step (1) is to pulverize the hemp extract part to 10-80 mesh (specifically, 10, 20, 30, 40, 50, 60, 70 or 80 mesh).
Preferably, the water content of the medicinal material powder in the step (1) is less than 6%.
Preferably, the solvent in step (2) is ethanol, and more preferably, the ethanol is ethanol with a concentration of 60-95% (v/v, specifically, 60%, 70%, 80%, 90% or 95%).
Preferably, the amount of the solvent used in step (2) is 5-15 times of the amount of the medicinal material (specifically, 5, 8, 10, 12 or 15 times of the amount of the medicinal material).
Preferably, the number of extraction times in step (2) is 1-5 (specifically 1, 2, 3, 4 or 5), and each extraction time is 1-3 h.
Preferably, the extraction method in step (2) is selected from: one or more of cold soaking method, ultrasonic extraction method, reflux extraction method and percolation method; more preferably, the extraction method in step (2) is a cold dipping method.
Preferably, the cold leaching extraction conditions are as follows: extracting with 5-15 times of 60-95% ethanol by cold soaking for 1-5 times, each for 1-3 hr.
The extraction in the step (3) is a key step for removing impurities, and more than 70% of the impurities can be removed.
Preferably, the extraction solvent in step (3) is selected from: one or more of ethyl acetate, n-butanol, n-hexane, petroleum ether and dichloromethane; more preferably, the extraction solvent in step (3) is n-hexane.
Preferably, the amount of the extraction solvent used in step (3) is 1-6 times of the amount of the medicinal material (specifically, 1, 2, 3, 4, 5 or 6 times of the amount of the medicinal material).
Preferably, the number of extractions in step (3) is 1-3 (specifically, 1, 2 or 3).
Preferably, the concentration temperature in step (2) and/or (3) is 50 to 70 ℃, more preferably 65 ℃.
Preferably, the relative density of the thick paste in step (2) and/or (3) is 1.05-1.35 (measured at 50 ℃) (e.g. 1.05, 1.08, 1.10, 1.20, 1.30 or 1.35).
The phenolic compound in the thick paste obtained in the step (3) can be further purified by adjusting the pH value in the step (4), and the phenolic compound can be salified by adjusting the pH value of the filtrate to be acidic after filtering, so that the salt can be converted into the corresponding phenolic compound.
Preferably, the pH adjustment to alkaline in step (4) comprises the step of adjusting the pH by adding a strong base; more preferably, the strong base is selected from: one or a combination of two or more of calcium hydroxide, barium hydroxide, potassium hydroxide, and sodium hydroxide, and in one embodiment of the invention, the strong base is sodium hydroxide.
Preferably, said basic pH in step (4) is pH10.0-12.0, more preferably pH 11.0.
Preferably, the adjusting of the pH to acidity in step (4) comprises the step of adjusting the pH by adding a weak acid; more preferably, the weak acid is selected from: one or a combination of two or more of acetic acid, sulfurous acid and phosphoric acid, and in one embodiment of the present invention, the weak acid is acetic acid.
Preferably, the acidic pH in step (4) is pH4.0-5.0, more preferably pH 4.6.
Preferably, step (4) further comprises a stirring step after the pH is adjusted to be alkaline, and more preferably, the stirring time is 0.5-2 h.
Preferably, the solvent in step (5) is selected from: one or more of ethyl acetate, n-butanol, n-hexane, petroleum ether and dichloromethane.
Preferably, the amount of the solvent used in step (5) is 0.5-2 times (specifically, 0.5, 1, 1.5 or 2 times) the amount of the medicinal material.
Preferably, the column chromatography in step (5) uses silica gel packing, and more preferably, the silica gel packing is selected from: amorphous silica gel, spherical silica gel, and silica gel powder.
Preferably, the elution mode in the step (5) is to perform gradient elution on the chromatographic column by using an elution solvent; more preferably, the elution step comprises: eluting with eluting solvent 1 to remove impurities, and eluting with eluting solvent 2 to obtain target product (i.e. cannabinol compound adsorbed in chromatographic column).
Preferably, the elution solvent is selected from: acetone-petroleum ether mixed liquor, dichloromethane-n-hexane mixed liquor, ethyl acetate-heptane mixed liquor and dichloromethane-petroleum ether mixed liquor.
Preferably, the elution solvent 1 is selected from: one of an acetone-petroleum ether mixed solution with a volume ratio of 1:1, a dichloromethane-n-hexane mixed solution with a volume ratio of 1:9 and an ethyl acetate-heptane mixed solution with a volume ratio of 1: 30.
Preferably, the amount of the elution solvent 1 is 2 to 8 times (specifically, 2, 3, 4, 5, 6, 7 or 8 times) the column volume, and more preferably 4 to 6 times the column volume.
Preferably, the elution solvent 2 is selected from: one of an acetone-petroleum ether mixed solution with a volume ratio of 7:3, a dichloromethane-n-hexane mixed solution with a volume ratio of 2:8, and an ethyl acetate-heptane mixed solution with a volume ratio of 1: 25.
Preferably, the amount of the elution solvent 2 is 2 to 8 times (specifically, 2, 3, 4, 5, 6, 7 or 8 times) the column volume, and more preferably 5 to 7 times the column volume.
Preferably, the gradient elution in the step (5) further comprises a step of eluting the THC with an elution solvent 3 after eluting to remove impurities and eluting to obtain a target product part; more preferably, the resulting eluate is destroyed, such as with concentrated acid (e.g., concentrated hydrochloric acid, concentrated nitric acid, etc.).
Preferably, the elution solvent 3 is selected from: one of an acetone-petroleum ether mixed solution with a volume ratio of 8:2, an acetone-petroleum ether mixed solution with a volume ratio of 4:6 and an ethyl acetate-heptane mixed solution with a volume ratio of 1: 10.
Preferably, the amount of the elution solvent 2 is 2 to 8 times (specifically, 2, 3, 4, 5, 6, 7 or 8 times) the column volume, and more preferably 4 to 6 times the column volume.
Preferably, the concentration in the step (6) is reduced pressure concentration; more preferably, the temperature of the concentration under reduced pressure is 55-70 deg.C, and/or the pressure is-0.10 MPa.
Specifically, in the cannabinol extract provided by the invention, the total content of cannabinol compounds is more than 80%.
Preferably, the cannabinol extract has a THC content of
The following.
Preferably, the cannabinol extract comprises one or more of CBD, CBDV, CBG and THCV; more preferably, the cannabinol extract comprises CBD, CBDV, CBG and THCV; more preferably, the cannabinol extract has a CBD content of 55-60%, a CBDV content of 10-13%, a CBG content of 4-5% and a THCV content of 4-5%.
Specifically, in the application of the cannabis phenol extract in preparation of medicines, the medicines are medicines for treating epilepsy.
The cannabinol extract can also be used as a raw material for further separating and purifying to obtain single cannabinol compounds.
The preparation method of the cannabinol extract provided by the invention has the advantages that the used raw materials, reagents and instruments are cheap and easy to obtain, the cost is low, and the adopted operation and method are simple and easy to carry out; and high quality cannabinol extract can be obtained, which retains more cannabinol compounds such as CBD, CBDV, CBG and THCV, and avoids resource waste, wherein the total content of cannabinol compoundsThe content of the psychotropic component THC is above 80 percent
The safety of the product is guaranteed; and each preparation batch has high stability, and the component content and the activity in the product are stable, so that the industrialization is easy to realize.
Drawings
FIG. 1 shows a HPLC chromatogram for detecting the extract prepared in example 1 of the present invention.
FIG. 2 shows a HPLC chromatogram for detecting the extract prepared in example 2 of the present invention.
FIG. 3 shows a HPLC chromatogram for detecting the extract prepared in example 3 of the present invention.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, as the following abbreviations and their corresponding materials appear in the present invention:
the "cannabinol extract" as used herein refers to a product extracted from a raw material of a cannabis extract part (e.g., cannabis flowers and/or leaves), which may be in a liquid or solid form and contains at least one cannabinol compound, and preferably, the "cannabinol extract" of the present invention contains at least CBD, CBDV, CBG and THCV, and the THC content is in the range of
Hereinafter, in particular, in the extract product prepared in the example of the present invention, THC was not detected.
Unless otherwise specified, the "content" referred to in the present invention is generally a mass content, such as a content of CBDV in the extract of 10% to 13%, which means: in the extract, the mass of CBDV accounts for 10-13% of the total mass of the extract.
The descriptions of the "x times of crude drug amount", "x times of medicinal material amount", and the like in the invention refer to that the volume of the adopted solvent such as n-hexane, petroleum ether, and the like is x times of the mass of the medicinal material, specifically, if the "1 time of medicinal material amount", the mass of the medicinal material is 1g, and the dosage of the adopted solvent is 1 ml.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The hemp extraction part raw materials used in the embodiment of the invention are as follows: the flower and leaf raw material is 7-9 months old mature hemp flower and leaf, dried in the shade, and is industrial hemp whose THC content is below 0.3%. Other reagents are commercially available products unless otherwise specified.
Example 1
The experimental procedure was as follows:
(1) pulverizing industrial hemp leaves into coarse powder of 60 mesh, baking at 150 deg.C for 80min to water content of 2.2%;
(2) extracting the medicinal materials obtained in the step (1) by adopting a cold soaking method, soaking for 3 hours at room temperature by using 15 times of 95% ethanol, extracting for 5 times, and mixing to obtain an extracting solution;
(3) concentrating the extractive solution obtained in step (2) at 65 deg.C to obtain soft extract (relative density 1.08, measured at 50 deg.C);
(4) extracting the thick paste obtained in the step (3) with n-hexane 6 times the amount of the medicinal materials for 3 times, stirring for 1h, taking the upper layer after layering, combining the upper layer liquid, and concentrating at 65 ℃ to obtain thick paste (the relative density is 1.08, and the relative density is measured at 50 ℃);
(5) adding sodium hydroxide into the thick paste obtained in the step (4) to adjust the pH value to 11, fully stirring for 1h, filtering, adding acetic acid into the filtrate to adjust the pH value to 4.6, and filtering to obtain insoluble precipitate;
(6) dissolving the insoluble precipitate obtained in the step (5) by using petroleum ether with the dosage of 1 time of crude drug, and performing silica gel column chromatography, wherein the acetone: eluting with petroleum ether (volume ratio: 1:1, the ratio of the components in the eluent is the volume ratio of the two respectively) by 5 times of the column volume, and recovering solvent as impurity part; acetone: eluting with petroleum ether (7: 3) for 6 times of column volume, collecting the second part of eluate, and concentrating to obtain active ingredient extract; acetone: petroleum ether (8: 2) elutes the part which is mainly tetrahydrocannabinol in 5 times of column volume, and concentrated hydrochloric acid is added for destruction after concentration;
(7) and (4) concentrating the second part of eluent obtained in the step (6) under reduced pressure at 55-70 ℃ and-0.10 MPa until the eluent is thick to obtain the cannabis phenol extract, and analyzing and detecting the content of components such as CBDV, CBD, CBG, THCV and THC in the extract.
The sample detection and analysis method comprises the following steps:
chromatographic conditions and system applicability test: octadecylsilane chemically bonded silica is used as a filling agent; acetonitrile is used as a mobile phase A, water is used as a mobile phase B, and the ratio of A (%): b (%) (80: 20) isocratic elution; the detection wavelength was 210 nm. The number of theoretical plates should not be less than 2500 calculated by CBD peak.
Preparation of control solutions: accurately weighing CBD reference substance, adding methanol to obtain reference substance solutions each containing 0.1mg per 1 ml; precisely weighing CBDV reference substance, and adding methanol to obtain reference substance solutions each containing 0.05mg per 1 ml; accurately weighing THCV reference substance, and adding methanol to obtain reference substance solutions each containing 0.01mg per 1 ml; precisely weighing CBG reference substance, and adding methanol to obtain reference substance solutions each containing 0.01mg per 1 ml; precisely weighing tetrahydrocannabinol reference substance, and adding methanol to obtain reference substance solutions each containing 0.01mg per 1 ml.
Preparation of a test solution: taking 5mg of the finished product, adding methanol to a constant volume of 50ml, filtering with a microporous filter membrane (0.45 mu m), and taking a subsequent filtrate to obtain the product.
The determination method comprises the following steps: precisely sucking 10 μ l of each of the reference solution and the sample solution, injecting into liquid chromatograph, and measuring.
The detection chromatogram of the extract is shown in FIG. 1, wherein the retention times and corresponding substances are as follows: 4.728 is CBDV, 7.466 is CBG, 7.921 is CBD, 8.772 is THCV.
As a result: in the extract prepared in this example, the total content of cannabinol compounds is 84%, and the main active ingredients and the contents thereof are respectively: CBDV, 11%; CBD, 58%; 4.3 percent of CBG; THCV, 4.1%; THC was not detected.
Example 2
The experimental procedure was as follows:
(1) pulverizing industrial hemp leaves into 60 mesh coarse powder, baking at 100 deg.C for 20min to water content of 5.1%;
(2) extracting the medicinal materials obtained in the step (1) by adopting a cold soaking method, soaking the medicinal materials for 1 hour at room temperature by using 5 times of 60% ethanol, and extracting for 1 time;
(3) concentrating the extractive solution obtained in step (2) at 65 deg.C to obtain soft extract (relative density 1.08, measured at 50 deg.C);
(4) extracting the thick paste obtained in the step (3) with n-hexane 1 time of the amount of the medicinal materials, stirring for 1h, layering, collecting the upper layer, and concentrating at 65 ℃ to obtain thick paste (the relative density is 1.08, and the concentration is measured at 50 ℃);
(5) adding sodium hydroxide into the thick paste obtained in the step (4) to adjust the pH value to 11, fully stirring for 1h, filtering, adding acetic acid into the filtrate to adjust the pH value to 4.6, and filtering to obtain insoluble precipitate;
(6) dissolving the insoluble precipitate obtained in the step (5) by using 1 time of crude drug amount of dichloromethane, and performing silica gel column chromatography, wherein the molar ratio of dichloromethane: eluting with n-hexane (1: 9) for 5 times of column volume to obtain impurity part, and recovering solvent; dichloromethane: eluting with n-hexane (2: 8) for 6 times of column volume, collecting the second part of eluate, and concentrating to obtain active ingredient extract; acetone: petroleum ether (4: 6) elutes the part which is mainly tetrahydrocannabinol in 5 times of column volume, and concentrated hydrochloric acid is added for destruction after concentration;
(7) and (4) concentrating the second part of eluent obtained in the step (6) under reduced pressure at 55-70 ℃ and-0.10 MPa until the eluent is thick to obtain the cannabis phenol extract, analyzing and detecting the content of components such as CBDV, CBD, CBG, THCV and THC in the extract, wherein the analysis and detection method refers to example 1.
The detection chromatogram of the extract is shown in FIG. 2, wherein the retention times and corresponding substances are as follows: 4.726 is CBDV, 7.455 is CBG, 7.907 is CBD, 8.769 is THCV.
As a result: in the extract prepared in this example, the total content of cannabinol compounds is 84%, and the main active ingredients and the contents thereof are respectively: CBDV, 11%; CBD, 56%; 4.2% of CBG; THCV, 4.3%; THC was not detected.
Example 3
The experimental procedure was as follows:
(1) pulverizing industrial hemp leaves into coarse powder of 60 mesh, baking at 120 deg.C for 40min to water content of 3.3%;
(2) extracting the medicinal materials obtained in the step (1) by adopting a cold soaking method, soaking for 2 hours at room temperature by using 10 times of 80% ethanol, extracting for 2 times, and mixing to obtain an extracting solution;
(3) concentrating the extractive solution obtained in step (2) at 65 deg.C to obtain soft extract (relative density 1.08, measured at 50 deg.C);
(4) extracting the thick paste obtained in the step (3) with n-hexane 2 times the amount of the medicinal materials twice, stirring for 2h, taking the upper layer after layering, combining the upper layer liquid, and concentrating at 65 ℃ to obtain thick paste (the relative density is 1.08, and the relative density is measured at 50 ℃);
(5) adding calcium hydroxide into the thick paste obtained in the step (4) to adjust the pH value to 11, fully stirring for 1h, filtering, adding acetic acid into the filtrate to adjust the pH value to 4.6, and filtering to obtain insoluble precipitate;
(6) dissolving the insoluble precipitate obtained in the step (5) by using ethyl acetate with the dosage of 1 time of crude drug, and performing silica gel column chromatography, wherein the weight ratio of ethyl acetate: eluting with heptane (1: 30) for 5 times of column volume to obtain impurity part, and recovering solvent; ethyl acetate: eluting with heptane (1: 25) for 6 times of column volume, collecting the second part of eluate, and concentrating to obtain active ingredient extract; ethyl acetate: part mainly containing tetrahydrocannabinol and eluted by heptane (1: 10) in 5 times of column volume is concentrated and destroyed by adding concentrated hydrochloric acid;
(7) and (4) concentrating the second part of eluent obtained in the step (6) under reduced pressure at 55-70 ℃ and-0.10 MPa until the eluent is thick to obtain the cannabis phenol extract, analyzing and detecting the content of components such as CBDV, CBD, CBG, THCV and THC in the extract, wherein the analysis and detection method refers to example 1.
The detection chromatogram of the extract is shown in FIG. 3, where the retention times and corresponding substances are as follows: 4.724 is CBDV, 7.452 is CBG, 7.907 is CBD, 8.757 is THCV.
As a result: in the extract prepared in this example, the total content of cannabinol compounds is 84%, and the main active ingredients and the contents thereof are respectively: CBDV, 13%; CBD, 55%; 4.6 percent of CBG; THCV, 4.4%; THC was not detected.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.
Claims (11)
1. A method for preparing cannabinol extract comprises the following steps: (1) pulverizing the extracted part of Cannabis sativa L, and baking to obtain medicinal powder; (2) extracting the medicinal material powder obtained in the step (1) by using a solvent to obtain an extracting solution, and concentrating the extracting solution to obtain thick paste; (3) extracting the thick paste obtained in the step (2) by using a solvent, taking the upper layer liquid, and concentrating the upper layer liquid to obtain thick paste; (4) adjusting the pH of the thick paste obtained in the step (3) to be alkaline, filtering, adjusting the pH of the obtained filtrate to be acidic, and filtering to obtain a precipitate; (5) dissolving the precipitate obtained in the step (4) with a solvent, and performing column chromatography and elution on the obtained solution; (6) concentrating the eluent obtained in the step (5) to obtain cannabinol extract;
the extraction part in the step (1) is cannabis flowers and/or cannabis leaves;
the cannabis flowers and/or cannabis leaves are cannabis flowers and/or cannabis leaves in full-bloom stage;
the hemp in step (1) is industrial hemp;
the baking conditions in the step (1) are as follows: the baking temperature is 100-150 ℃, and the baking time is 20-80 min; the pulverization is to pulverize the hemp extraction part to 10-80 meshes;
the extraction solvent in the step (2) is ethanol;
the dosage of the solvent in the step (2) is 5-15 times of the amount of the medicinal materials, the extraction times are 1-5 times, and the extraction time is 1-3h each time;
the extraction solvent in the step (3) is n-hexane;
the alkaline pH value in the step (4) is 10.0-12.0;
the acidic pH in the step (4) is 4.0-5.0;
the elution mode in the step (5) is to perform gradient elution on the chromatographic column by adopting an elution solvent;
the elution step comprises: eluting with an eluting solvent 1 to remove impurities, and then eluting with an eluting solvent 2 to obtain a target product part;
the elution solvent 1 is selected from: one of an acetone-petroleum ether mixed solution with a volume ratio of 1:1, a dichloromethane-n-hexane mixed solution with a volume ratio of 1:9 and an ethyl acetate-heptane mixed solution with a volume ratio of 1: 30; the elution solvent 2 is selected from: one of an acetone-petroleum ether mixed solution with a volume ratio of 7:3, a dichloromethane-n-hexane mixed solution with a volume ratio of 2:8 and an ethyl acetate-heptane mixed solution with a volume ratio of 1: 25;
the chromatographic column filler used in the step (5) is silica gel filler;
the silica gel filler is selected from: amorphous silica gel, spherical silica gel and silica gel powder;
the cannabis phenol extract prepared by the preparation method comprises CBD, CBDV, CBG and THCV;
the cannabinol extract has THC content of
The following;
the CBD content is 55-60%, the CBDV content is 10-13%, the CBG content is 4-5%, and the THCV content is 4-5%.
2. The preparation method according to claim 1, wherein the amount of the solvent used in the step (2) is 5-15 times of the amount of the medicinal material, the number of extraction times is 1-5, and each extraction time is 1-3h, and the extraction method is selected from: one or more of cold soaking method, ultrasonic extraction method, reflux extraction method and percolation method.
3. The method according to claim 1, wherein the amount of the extraction solvent used in the step (3) is 1 to 6 times the amount of the medicinal material, and the number of times of extraction is 1 to 3.
4. The method according to claim 1, wherein the concentration temperature in the step (2) and/or (3) is 50 to 70 ℃.
5. The method of claim 1, wherein the step of adjusting the pH to alkaline in step (4) comprises the step of adjusting the pH by adding a strong base.
6. The method of claim 5, wherein the strong base is selected from the group consisting of: one or more of calcium hydroxide, barium hydroxide, potassium hydroxide and sodium hydroxide; the pH adjustment to acidity in step (4) comprises a step of adjusting the pH by adding a weak acid.
7. The method of claim 6, wherein the weak acid is selected from the group consisting of: one or a combination of two or more of acetic acid, sulfurous acid and phosphoric acid.
8. The method according to claim 1, wherein the basic pH in the step (4) is pH 11.0; the acidic pH in step (4) is pH 4.6.
9. The method according to claim 1, wherein the solvent in the step (5) is selected from the group consisting of: one or more of ethyl acetate, n-butanol, n-hexane, petroleum ether and dichloromethane; the dosage of the solvent in the step (5) is 0.5 to 2 times of the amount of the medicinal materials.
10. The method according to claim 1, wherein the amount of the eluting solvent 1 is 2 to 8 column volumes; the dosage of the elution solvent 2 is 2 to 8 times of the column volume.
11. A cannabinol extract prepared by the method of any one of claims 1-10.
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