INCLUSION COMPOUND CONTAINING NON-PSYCHOACTIVE CANNABINOID AND METHOD FOR PREPARTION THEREOF

Abstract

Disclosed are an inclusion compound containing a non-psychoactive cannabinoid, and a method for preparation thereof. The inclusion compound is prepared by means of the following method: a cannabis extract is subjected to, in sequence, dissolving in organic solvent, incorporation of inclusion agent, analysis and removal of impurities in water-organic solvent, and vacuum drying and precipitation, and thus obtained. In the inclusion compound, psychoactive ingredients such as tetrahydrocannabidiol (THC) and Δ9-tetrahydrocannabinol (THCV) may be completely removed, effectively enriching the non-psychoactive ingredients such as cannabidiol (CBD), cannabigerol (CBG), cannabichromene (CBC), and so on with a total content of greater than 50%. The processing technique of the preparation method is simple to perform, and is conducive to commercialization.

Claims

1. An inclusion compound containing non-psychoactive cannabinoid, wherein raw materials of the inclusion compound comprise a cannabis extract and an inclusion agent, and the weight ratio of the inclusion agent to a crude drug raw material of the cannabis extract is 0.01-10:1.

2. The inclusion compound according to claim 1, wherein the cannabis extract comprises non-psychoactive cannabinoid and psychoactive cannabinoid, and the inclusion compound contains substantially no psychoactive cannabinoid.

3. The inclusion compound according to claim 2, wherein the inclusion compound is obtained by adding a cannabis extract solution dropwise to a inclusion agent solution, stirring, separating and precipitating, and dissolving a resulting precipitate in a water-organic solvent, then separating and precipitating a resulting mixture, and drying the resulting precipitate in a vacuum.

4. The inclusion compound according to claim 3, wherein the temperature of the adding and stirring step is 25° C.−80° C.; wherein a volume ratio of water to organic solvent is 1:1-5 in water-organic solvent, wherein the organic solvent is selected from one or a combination of two or more of ethanol, methanol, isopropanol, ethyl acetate and acetone in any proportion; wherein the inclusion agent solution is an aqueous solution of the inclusion agent, wherein a weight ratio of water to the crude drug raw material of the cannabis extract is 10-40:1.

5. The inclusion compound according to claim 1, wherein the inclusion agent is a cyclodextrin or a derivative thereof, preferably, the cyclodextrin is selected from one or a combination of two or more of α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin in any proportion; the cyclodextrin derivative is selected from one or a combination of two or more of 2-hydroxypropyl cyclodextrin, 3-hydroxypropyl cyclodextrin, branched cyclodextrin, methylated cyclodextrin, and low molecular weight β-cyclodextrin polymers, carboxymethyl cyclodextrin, cyclodextrin sulfate, cyclodextrin phosphate, cyclodextrin nitrate, cyclodextrin-epichlorohydrin cross-linked polymer and large cyclodextrin series in any proportion; the crude drug material of the cannabis extract is selected from one or a combination of two or more of hemp leaves, hemp flowers, hemp roots, hemp straws, and hemp seeds in any proportion, preferably hemp flowers and/or hemp leaves.

6. A method for preparing the inclusion compound according to claim 1, wherein the method comprises the following steps: (1) dissolving the cannabis extract with the organic solvent to obtain the cannabis extract solution; (2) weighing the inclusion agent and dissolving in water to obtain the inclusion agent solution; (3) adding the solution obtained in step (1) dropwise to the inclusion agent solution obtained in step (2), stirring at 25° C.−80° C., and then refrigerating after cooling to room temperature, filtering to obtain a filter cake 1; (4) adding the water-organic solvent into the filter cake 1 obtained in step (3), stirring, dissolving, and filtering to obtain a filter cake 2; (5) vacuum drying the filter cake 2 obtained in step 4 to obtain the inclusion compound containing non-psychoactive cannabinoid.

7. The preparation method according to claim 6, wherein the cannabis extract in step (1) is a thick paste obtained by extracting the crude drug raw material, concentrating the extract, purifying, and concentrating; the crude drug raw material is selected from one or a combination of two or more of hemp leaves, hemp flowers, hemp roots, hemp straws and hemp seeds in any proportion, preferably hemp flowers and/or hemp leaves.

8. The preparation method according to claim 6, wherein the inclusion agent in step (3) is cyclodextrin or a derivative thereof, preferably, the cyclodextrin is selected from one or a combination of two or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin in any proportion; the cyclodextrin derivative is selected from one or a combination of two or more of 2-hydroxypropyl cyclodextrin, 3-hydroxypropyl cyclodextrin, branched cyclodextrin, methylated cyclodextrin, and low molecular weight β-cyclodextrin polymers, carboxymethyl cyclodextrin, cyclodextrin sulfate, cyclodextrin phosphate, cyclodextrin nitrate, cyclodextrin-epichlorohydrin cross-linked polymer and large cyclodextrin series in any proportion; the organic solvent in steps (1) and (4) is independently selected from one or a combination of two or more of ethanol, methanol, isopropanol, ethyl acetate, and acetone in any proportion.

9. The preparation method according to claim 6, wherein the weight ratio of the inclusion agent in step (2) to the crude drug raw material of the cannabis extract in step (1) is 0.01-10:1; the weight ratio of the water in step (2) to the crude drug raw material of the cannabis extract in step (1) is 10-40:1; the stirring temperature in step (3) is 50-70° C.; the stirring time in step (3) is 0.5-4 hours; the refrigeration time in step (3) is 12-24 hours; in the water-organic solvent in step (4), the volume ratio of water to organic solvent is 1:1-5; the stirring temperature in step (4) is 20-30° C.; the stirring time in step (4) is 0.5-3 hours.

10. The application of the inclusion compound according to claim 1 in the preparation of medicines, medical beauty products, and cosmetics.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] FIG. 1 is a liquid chromatogram of an ethanol solution in step (2) in Example 1 of the invention.

[0066] FIG. 2 is a liquid chromatogram of filtrate 1 in step (5) in Example 1 of the invention.

[0067] FIG. 3 is a liquid chromatogram of filtrate 2 in step (5) in Example 1 of the invention.

[0068] FIG. 4 is a liquid chromatogram of the vacuum-dried product of the filter cake 2 in step (5) in Example 1 of the invention.

DETAILED DESCRIPTION

[0069] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the field of the invention, such as the following abbreviations and their corresponding substances appearing in the invention are:

[0070] CBDV cannabidivarin

[0071] CBD cannabidiol

[0072] CBG cannabigerol

[0073] THCV Δ.sup.9-tetrahydrocannabinol

[0074] THC tetrahydrocannabidiol

[0075] CBC cannabichromene

[0076] In the invention, the term “cannabinoid” refers to secondary metabolites unique to the cannabis plant containing the molecular structure of alkyl and monoterpenoid groups, for example, CBD, THC, CBDV, CBG, THCV, CBN, CBC, etc. (As described in “Chen X, Yang M, Guo HY (2011). Research advances in cannabinoids of Cannabis sativa. Chin Bull Bot 46, 197-205”). Psychoactive substances refer to chemical substances affecting human emotions and behaviors, changing the state of consciousness, and causing dependence. In the invention, the term “psychoactive cannabinoid” refers to cannabinoid with the above-mentioned psychoactive, such as THC, CBN, THCV, etc., while “non-psychoactive cannabinoid” refers to cannabinoid that do not have the above-mentioned psychoactive, such as CBD, CBDV, CBG, CBC, etc.

[0077] The term “crude drug” refers to pure natural traditional Chinese medicine materials without processing or simple processing of plant, animal, and mineral. In the invention, the crude drug of the cannabis extract is cannabis medicinal material, and the medicinal parts of cannabis medicinal material include leaves, flowers, roots, stalks, seeds, etc.

[0078] In order to be able to understand the technical content of the invention more clearly, the following examples are specifically described in detail, the purpose of the following examples is only to better understand the content of the invention and not to limit the protection scope of the invention.

Example 1

[0079] The preparation steps of the inclusion compound containing non-psychoactive cannabinoid are as follows:

[0080] (1) 10 kg medicinal material of hemp flower and leaf was added with 100 kg of 75% ethanol, and the resulting mixture was stirred and extracted at room temperature for 1 hour, extracted for 2 times, and then the resulting extracts were combined and concentrated under reduced pressure to a density of 1.014 (50° C.), and purified by a styrene-type macroporous resin column, the sample after loaded was left standing for 30 minutes, and then eluted with 5 times column volume of purified water, 3 times column volume of 55% ethanol, 6 times volume of 85% ethanol in turn, then the 85% ethanol eluate was collected and concentrated under reduced pressure to obtain a thick paste;

[0081] (2) the thick paste obtained in step (1) was added with 95% (v/v, the same below) ethanol, stirred for dissolution to obtain an ethanol solution;

[0082] (3) 50 kg β-cyclodextrin was weighed and added into 300 kg of purified water, and then the resulting mixture was heated and stirred for dissolution at 60° C. to obtain an inclusion agent solution;

[0083] (4) the ethanol solution obtained in step (2) was slowly added into the inclusion agent solution obtained in step (3), and the resulting mixture was stirred at 60° C. for 4 hours until the addition was completed, after cooling to room temperature, the resulting mixture was refrigerated for 18 hours;

[0084] (5) the refrigerated liquid obtained in step (4) was filtered to collect a filtrate 1 and a filter cake 1, the filter cake 1 was added into 65% ethanol, and the resulting mixture was stirred for dissolution at 30° C. for 1 hour, and then filtered to collect a filtrate 2 and a filter cake 2, the collected filter cake 2 was dried in a vacuum environment to obtain the inclusion compound of non-psychoactive cannabinoids;

[0085] (6) the content of CBDV, CBD, CBG, THCV and THC in the vacuum dried product of the ethanol solution obtained in step (2), filtrate 1, filtrate 2 and filter cake 2 obtained in step (5) were detected respectively.

[0086] The sample detection and analysis methods are as follows:

[0087] Chromatographic conditions and system applicability test: octadecyl silane bonded silica gel was used as a filler, 0.1% formic acid aqueous solution as mobile phase A, 0.1% formic acid in acetonitrile as mobile phase B, the elution procedure is as follows, the detection wavelength was 220 nm, and the number of theoretical plates calculated by CBD was not less than 5000.

[0088] When the elution time was 0-6 minutes, the volume concentration of the mobile phase: mobile phase A: 30%, and mobile phase B: balance; when the elution time was 6-12 minutes, the volume concentration of the mobile phase: mobile phase A: 30% to 23% in a gradient, and mobile phase B: balance; when the elution time was 12-22 minutes, the volume concentration of the mobile phase: mobile phase A: 23%, and mobile phase B: balance; when the elution time was 22-22.2 minutes, the volume concentration of the mobile phase: mobile phase A: 23% to 30% in a gradient, and mobile phase B: balance; and when the elution time was 22.2-26 minutes, the volume concentration of the mobile phase: mobile phase A: 30%, and mobile phase B: balance.

[0089] Preparation of reference solution: CBD reference substance was precisely weighed and added with methanol to obtain a reference substance solution containing 0.15 mg per 1 ml; CBDV reference substance was precisely weighed and added with methanol to obtain a reference substance solution containing 0.01 mg per 1 ml; THCV reference substance was precisely weighed and added with methanol to obtain a reference substance solution containing 0.01 mg per 1 ml; CBG reference substance was precisely weighed and added with methanol to obtain a reference substance solution containing 0.01 mg per 1 ml; THC reference substance was precisely weighed and added with methanol to obtain a reference substance solution containing 0.01 mg per 1 ml; CBC reference substance was precisely weighed and added with methanol to obtain a reference substance solution containing 0.01 mg per 1 ml.

[0090] Preparation of test solution:{circle around (1)}1.5 mL the obtained ethanol solution or filtrate was measured and added with methanol to make the volume to 5 mL, then filtered with a microporous membrane (0.45 μm) to obtain an additional filtrate; {circle around (2)}2.5 g the resulting filter cake was measured and added with methanol to make the volume to 50 mL, then filtered with a microporous membrane (0.45 μm) to obtain an additional filtrate.

[0091] Determination method: the reference solution and the test solution were precisely absorbed 10 μL each and injected into the liquid chromatography for determination.

[0092] The detection chromatogram of the ethanol solution is shown in FIG. 1, where the retention time and corresponding substances are as follows: 5.105 is CBDV, 8.099 is CBG, 8.517 is CBD, 8.384 is THCV, 15.354 is THC, and 19.106 is CBC.

[0093] The detection chromatogram of filtrate 1 is shown in FIG. 2, where the retention time and corresponding substances are as follows: 5.111 is CBDV, 8.253 is CBG, 8.536 is CBD, 9.402 is THCV, and 15.367 is THC.

[0094] The detection chromatogram of filtrate 2 is shown in FIG. 3, where the retention time and corresponding substances are as follows: 5.101 is CBDV, 8.518 is CBD, 9.3976 is THCV, 15.363 is THC, and 19.099 is CBC.

[0095] The detection chromatogram of the dried substance of filter cake 2 is shown in FIG. 4, in which the retention time and corresponding substances are as follows: 5.073 is CBDV, 8.049 is CBG, 8.467 is CBD, and 19.011 is CBC.

[0096] Results: the inclusion compound containing non-psychoactive cannabinoids prepared in this example, the total content of cannabinoids was 55.5%, and THCV and THC were not detected.

Example 2

[0097] The preparation steps of the inclusion compound containing non-psychoactive cannabinoid are as follows:

[0098] (1) 10 kg medicinal material of hemp flower and leaf was added with 100 kg of 95% ethanol, and the resulting mixture was stirred and extracted at room temperature for 0.5 hour, extracted for 4 times, and then the resulting extracts were combined and concentrated under reduced pressure to a density of 1.014 (50° C.), and purified by a styrene-type macroporous resin column, the sample after loaded was left standing for 30 minutes, and then eluted with 5 times column volume of purified water, 3 times column volume of 55% ethanol, 6 times volume of 85% ethanol in turn, then the 85% ethanol eluate was collected and concentrated under reduced pressure to obtain a thick paste;

[0099] (2) the thick paste obtained in step (1) was added with 95% (v/v, the same below) isopropanol, stirred for dissolution to obtain an isopropanol solution;

[0100] (3) 10 kg 2-hydroxypropyl-β-cyclodextrin was weighed and added into 100 kg of purified water, and then the resulting mixture was heated and stirred for dissolution at 70° C. to obtain an inclusion agent solution;

[0101] (4) the isopropanol solution obtained in step (2) was slowly added into the inclusion agent solution obtained in step (3), and the resulting mixture was stirred at 70° C. for 0.5 hours until the addition was completed, after cooling to room temperature, the resulting mixture was refrigerated for 12 hours;

[0102] (5) the refrigerated liquid obtained in step (4) was filtered to collect a filtrate 1 and a filter cake 1, the filter cake 1 was added into 80% isopropanol, and the resulting mixture was stirred for dissolution at 20° C. for 3 hour, and then filtered to collect a filtrate 2 and a filter cake 2, the collected filter cake 2 was dried in a vacuum environment to obtain the inclusion compound of non-psychoactive cannabinoids;

[0103] (6) the content of CBDV, CBD, CBG, THCV and THC in the vacuum dried product of the filter cake 2 obtained in step (5) was detected, and the detection methods and steps refer to the relevant methods and steps in Example 1.

[0104] Results: the inclusion compound containing non-psychoactive cannabinoids prepared in this example, the total content of cannabinoids was 50.5%, and THCV and THC were not detected.

Example 3

[0105] The preparation steps of the inclusion compound containing non-psychoactive cannabinoid are as follows:

[0106] (1) 10 kg medicinal material of hemp flower and leaf was added with 150 kg of 75% ethanol, and the resulting mixture was stirred and extracted at room temperature for 1 hour, extracted for 3 times, and then the resulting extracts were combined and concentrated under reduced pressure to a density of 1.014 (50° C.), and purified by a styrene-type macroporous resin column, the sample after loaded was left standing for 30 minutes, and then eluted with 5 times column volume of purified water, 3 times column volume of 55% ethanol, 6 times volume of 85% ethanol in turn, then the 85% ethanol eluate was collected and concentrated under reduced pressure to obtain a thick paste;

[0107] (2) the thick paste obtained in step (1) was added with an appropriate amount of acetone, stirred for dissolution to obtain an acetone solution;

[0108] (3) 100 kg 3-hydroxypropyl-β-cyclodextrin was weighed and added into 400 kg of purified water, and then the resulting mixture was heated and stirred for dissolution at 50° C. to obtain an inclusion agent solution;

[0109] (4) the acetone solution obtained in step (2) was slowly added into the inclusion agent solution obtained in step (3), and the resulting mixture was stirred at 50° C. for 2 hours until the addition was completed, after cooling to room temperature, the resulting mixture was refrigerated for 24 hours;

[0110] (5) the refrigerated liquid obtained in step (4) was filtered to collect a filtrate 1 and a filter cake 1, the filter cake 1 was added into 50% acetone, and the resulting mixture was stirred for dissolution at 25° C. for 0.5 hour, and then filtered to collect a filtrate 2 and a filter cake 2, the collected filter cake 2 was dried in a vacuum environment to obtain the inclusion compound of non-psychoactive cannabinoids;

[0111] (6) the content of CBDV, CBD, CBG, THCV and THC in the vacuum dried product of the filter cake 2 obtained in step (5) was detected, and the detection methods and steps refer to the relevant methods and steps in Example 1.

[0112] Results: the inclusion compound containing non-psychoactive cannabinoids prepared in this example, the total content of cannabinoids was 52.4%, and THCV and THC were not detected.

Example 4

[0113] The preparation steps of the inclusion compound containing non-psychoactive cannabinoid are as follows:

[0114] (1) 10 kg raw medicinal material of hemp flower and leaf was taken, and the thick paste of cannabis extract was prepared by referring to the experimental steps (1)-(7) of Example 1 of the Chinese application CN109568389A.

[0115] (2) the thick paste obtained in step (1) was added with an appropriate amount of ethanol, stirred for dissolution to obtain an ethanol solution;

[0116] (3) 30 kg β-cyclodextrin was weighed and added into 200 kg of purified water, and then the resulting mixture was heated and stirred for dissolution at 65° C. to obtain an inclusion agent solution;

[0117] (4) the acetone solution obtained in step (2) was slowly added into the inclusion agent solution obtained in step (3), and the resulting mixture was stirred at 65° C. for 4 hours until the addition was completed, after cooling to room temperature, the resulting mixture was refrigerated for 20 hours;

[0118] (5) the refrigerated liquid obtained in step (4) was filtered to collect a filtrate 1 and a filter cake 1, the filter cake 1 was added into 80% ethanol, and the resulting mixture was stirred for dissolution at 25° C. for 1.5 hour, and then filtered to collect a filtrate 2 and a filter cake 2, the collected filter cake 2 was dried in a vacuum environment to obtain the inclusion compound of non-psychoactive cannabinoids;

[0119] (6) the content of CBDV, CBD, CBG, THCV and THC in the vacuum dried product of the filter cake 2 obtained in step (5) was detected, and the detection methods and steps refer to the relevant methods and steps in Example 1.

[0120] Results: the inclusion compound containing non-psychoactive cannabinoids prepared in this example, the total content of cannabinoids was 65.8%, and THCV and THC were not detected. According to the Chinese application CN109568389A, the cannabis extract obtained in Example 1 contains 4.1% THCV. It can be seen that the hydrate prepared by the method of the invention can retain non-psychotropic cannabinoids with high selectivity and eliminate psychoactive cannabinoids.

[0121] The above are only preferred examples of the invention and are not intended to limit the invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the invention shall be included in the protection scope of the invention.