阅读说明：本技术 使用液：液色谱法纯化***素的方法 (Use solution: method for purifying cannabinoids by liquid chromatography ) 是由 X·纳达尔鲁拉 于 2019-01-29 设计创作，主要内容包括：本说明书公开了使用独特的两相溶剂体系和液-液色谱法如离心分配色谱法(CPC)或逆流色谱法(CCC)从植物材料纯化一种或多种大麻素的方法。本说明书还提供了纯化的大麻素比如CBG、CBGA、CBGV、CBD、CBDA、CBDV、THC、THCA和THCV,包括通过公开的方法产生的这些大麻素中的一种或多种的组合物,和采用这种纯化的大麻素和组合物治疗疾病或病症的方法。(The specification discloses methods for purifying one or more cannabinoids from plant material using a unique biphasic solvent system and liquid-liquid chromatography, such as Centrifugal Partition Chromatography (CPC) or Counter Current Chromatography (CCC). The specification also provides purified cannabinoids such as CBG, CBGA, CBGV, CBD, CBDA, CBDV, THC, THCA and THCV, compositions comprising one or more of these cannabinoids produced by the disclosed methods, and methods of treating diseases or conditions using such purified cannabinoids and compositions.)

1. A method of purifying one or more cannabinoids from plant material comprising a plant, plant resin or plant extract, the method consisting essentially of:

(a) mixing the plant material with a solvent selected from pentane, hexane, heptane, petroleum ether, cyclohexane, dichloromethane, chloroform, tetrahydrofuran, diethyl ether, toluene, benzene, ethanol, methanol, isopropanol, acetone, acetonitrile, ethyl acetate, butane, propane, 1,1,1, 2-tetrafluoroethane (R134a) or liquid, subcritical or supercritical CO₂Or a mixture thereof, to form a solvent mixture that extracts the one or more cannabinoids from the plant material, wherein the solvent mixture has an original volume;

(b) for THC-type extracts, one or more solvents selected from hexane: ethanol: water, pentane: acetonitrile and hexane: acetonitrile, wherein the ratio of pentane: acetonitrile system and the hexane: the acetonitrile system optionally includes ethyl acetate and/or water as modifiers; for CBD-type extracts, the ratio of hexane: ethanol: adding a biphasic solvent system of water to the extract; and for CBG-type extracts, the ratio of hexane: ethanol: adding a biphasic solvent system of water to the extract; and

(c) carrying out the liquefaction using the biphasic solvent system of step b): liquid chromatography, thereby purifying the one or more cannabinoids.

2. The method of claim 1, wherein for THC-type extracts, the biphasic solvent system is hexane: ethanol: and (3) water.

3. The method of claim 1, wherein for THC-type extracts, the biphasic solvent system is a mixture of pentane: ethyl acetate: acetonitrile: and (3) water.

4. The method of claim 1, wherein for THC-type extracts, the biphasic solvent system is hexane: ethyl acetate: acetonitrile: and (3) water.

5. The method of claim 1, wherein for CBD-type extracts, the biphasic solvent system is hexane: ethanol: and (3) water.

6. The method of claim 1, wherein for CBG-type extracts, the biphasic solvent system is hexane: ethanol: and (3) water.

7. The method as claimed in claim 1, wherein an extract of cannabis of chemical form I or II is used to purify THC, THCA, THCV, THCVA, CBN or CBV and fractionate the CBD-and CBG-type cannabinoids.

8. The method as claimed in claim 1, wherein an extract of cannabis of chemical type II or III is used to purify CBD, CBDA, CBDVA or CBDV and fractionate the THC-and CBG-type cannabinoids.

9. The method as claimed in claim 1, wherein an extract of cannabis chemotype IV is used to purify CBG, CBGA, CBGVA or CBGV and fractionate the CBD-and THC-type cannabinoids.

10. The method of claim 1, wherein the liquid: the liquid chromatography is Centrifugal Partition Chromatography (CPC) or countercurrent chromatography (CCC).

11. The method of claim 1, wherein after step a), the solvent mixture is reduced to dryness or to about 50% or less of the original volume of the solvent mixture in step (a) to a concentration of at least one of: concentrating the one or more cannabinoids prior to liquid chromatography.

12. The process according to claim 1, wherein the solvent mixture of step (a) is purified prior to step (b).

13. The method of claim 1, wherein prior to step (a), one or more cannabinoids present in the plant material are decarboxylated by heating the plant material.

14. The method of claim 11, wherein after the solvent mixture is reduced to dryness, the dry extract product of the solvent mixture is dissolved in ethanol, frozen at a temperature of-20 ℃ to 4 ℃, filtered to remove precipitated material and reduced to dryness prior to purification by liquid-liquid chromatography.

15. The method of claim 10, using a rotor design quantum CPC or CPC PRO.

16. The method of claim 10, using a rotor design quantum CPC or CPC PRO, wherein the total test time is 12-20 minutes, independent of rotor volume.

17. The method of claim 8, wherein the CBD, CBDA, CBDVA, or CBDV is in liquid: the step of liquid chromatography is followed by crystallization.

18. The method of claim 9, wherein the CBG, CBGA, CBGVA or CBGV is in liquid: the step of liquid chromatography is followed by crystallization.

19. The process of claim 1, wherein the plant material is first incubated with a non-polar solvent selected from the group consisting of petroleum ether, pentane, hexane, and heptane to form a solvent mixture, the non-polar solvent extracting the one or more cannabinoids from the plant material to form the solvent mixture.

20. The method of claim 1, wherein the plant material is first contacted with a solvent selected from the group consisting of pentane, hexane, heptane, petroleum ether, cyclohexane, dichloromethane, trichloro-methaneMethane, tetrahydrofuran, diethyl ether, toluene, benzene, ethanol, methanol, isopropanol, acetone, acetonitrile, ethyl acetate, butane, propane, 1,1,1, 2-tetrafluoroethane (R134a) or liquid, subcritical or supercritical CO₂Or a mixture thereof; filtration, decantation or centrifugation; reducing to dryness; and then incubated with a non-polar solvent selected from the group consisting of petroleum ether, pentane, hexane, and heptane to form a solvent mixture, the non-polar solvent extracting the one or more cannabinoids from the plant material to form the solvent mixture.

21. The process according to claim 1, wherein after step (a) the one or more cannabinoids present in the plant material and extract are decarboxylated by heating the solvent mixture, wherein the solvent mixture is the original volume, the concentrated volume or a dry extract obtained from evaporation of the original volume of the solvent mixture to dryness.

22. The method of claim 15, wherein the rotor design is CPC 1000 PRO.

23. The method of claim 15, wherein the rotor has a rotor volume of 1 liter, the injection is 50mL, the flow rate of the mobile phase (pentane or hexane phase) of the biphasic solvent system during the assay is 200mL/min, and the flow rate of the stationary phase (ethanol or acetonitrile phase) of the biphasic solvent system during the extrusion phase of the assay is 350 mL/min.

24. The process of claim 1, wherein acetonitrile is used: a gradient of aqueous mobile liquid phase, re-purifying the fraction of THC contaminated with CBC or the fraction of THCV contaminated with CBN on a C-8 or C-18 coated silica solid stationary phase using solid-liquid chromatography selected from gravity, flash or preparative HPLC.

25. The method of claim 1, wherein the cannabinoid is selected from the group consisting of CBD, CBDA, and CBDV.

26. The method of claim 1, wherein the cannabinoid is selected from the group consisting of CBG, CBGA, and CBGV.

27. The method of claim 1, wherein the cannabinoid is selected from THC, THCA, and THCV.

Technical Field

The present invention relates to the separation of cannabinoid compounds using a unique biphasic solvent system and liquid-liquid chromatography, such as Centrifugal Partition Chromatography (CPC) or Counter Current Chromatography (CCC).

Background

Cannabis is a flowering plant genus, whose species are distinguished by plant phenotype and secondary metabolite characteristics. Cannabis is a genus that includes: cannabis, including cultivated Cannabis (Cannabis sativa L) and all subspecies, putatively Indian Cannabis (Cannabis indica Lam.) species, wild Cannabis (Cannabis ruderalis Janisch), and hybrids and varieties thereof, as discussed further below. The cannabis plant has been cultivated for a variety of uses, including the manufacture of fiber (cannabis), medical uses, and recreational pharmaceutical uses. Cannabis is also commonly referred to as cannabis leaf.

Cannabis is now widely recognized as having substantial benefits for a variety of medical uses. For example, cannabis is widely used by various levels of society to treat a variety of diseases, disorders and conditions, including but not limited to nausea, pain relief (e.g., chronic pain, cancer-related pain or neuropathic pain), glaucoma, loss of appetite, mucosal inflammation, inflammatory diseases (e.g., crohn's disease), neurodegenerative diseases, epilepsy (affecting children and adults), seizures, diabetes, leprosy, fever, obesity, asthma, urinary tract infections, cough, anorexia associated with weight loss in aids patients, graft-versus-host disease, glioma, perinatal asphyxia and post-traumatic stress disorder (PTSD), and autoimmune diseases (e.g., multiple sclerosis).

In many countries, one of the most common ways of using cannabis for medical use is by smoking. Medical marijuana, while proven beneficial in certain indications, has its drawbacks. For example, the amount of active ingredient may vary depending on the differences that exist in the plant species and the varying growth conditions that lead to variation within the species. As a result, it may be difficult to maintain control of the correct dosage of medicinal cannabis due to fluctuations in the active ingredient. Another disadvantage of smoking medical cannabis is the negative impact of some components of cannabis tobacco. In addition to the required cannabinoids, smoke from plant matter includes carcinogens. In addition, the use of cannabis in large quantities by ingestion is associated with an accelerated decline in lung function (pulmonary decline).

Cannabinoids are compounds which are active at human cannabinoid receptors and are responsible for causing many of the pharmacological effects of cannabis. Cannabinoids of plant origin (also known as phytocannabinoids) are abundant in cannabis. Two known cannabinoids that are present in relatively high concentrations in cultivated cannabis are tetrahydrocannabinolic acid (THCA) or its decarboxylation products Tetrahydrocannabinol (THC) and cannabidiolic acid (CBDA) or its decarboxylation products Cannabidiol (CBD). THC induces psychoactive (calming) effects, analgesic effects, antioxidant effects and increases appetite. However, THC is also associated with a number of negative or adverse side effects, including, but not limited to, reduced short-term memory, dry mouth, impaired visual perception and motor skills, red eyes (i.e., blood streaks), increased anxiety, sporadic infarctions, stroke, paranoia, acute psychosis, mental retardation (reduced mental retardation), hallucinations, bizarre behavior, irrational panic attacks, unreasonable thoughts, and a variety of other cognitive and social problems. On the other hand, CBD is becoming a popular cannabinoid for medical purposes due to its lack of psychoactive properties at typical doses, unlike THC. In addition, CBD was found to have neuroprotective and ameliorative effects in patients with epilepsy, schizophrenia and parkinson's disease. Thus, patients and healthcare providers exhibit a preference for CBD because patients need to work, drive, and move clearly while receiving treatment.

Cannabinoid compounds have been purified from plant cultivated cannabis using a variety of chromatographic techniques. For example, flash chromatography on silica gel, C8 or C18; preparative HPLC on silica gel column C8 or C18; and supercritical CO on silica gel₂Chromatography. However, these chromatographic processes are cumbersome and expensive.

Therefore, there is a need for a simple and inexpensive method for selectively purifying and concentrating medically beneficial cannabinoids. In addition, it would be desirable to develop pharmaceutical formulations that include higher levels of beneficial cannabinoids. However, THC and THCA can also be purified by this method from cultivated cannabis plants and extracts rich in or low in THC-THCA.

Centrifugal Partition Chromatography (CPC) and Counter Current Chromatography (CCC) can be used, for example, to extract and enrich compounds from plant extracts on analytical, semi-preparative and preparative scales. CPC and CCC are liquid-liquid chromatography methods that use mainly biphasic solvents. It enables complex mixtures of substances to be separated from crude extracts with little loss. CPC and CCC are easier and cheaper than liquid chromatography (HPLC) because no matrix effects and irreversible adsorption on solid phase occur. Cannabinoids have been purified using CPC, but not using the solvent system described in this patent application (see, e.g., Hazekamp et al, "advanced isolation of Cannabinoids from Cannabis sativa by centrifugal functional isolation Chromatography", Journal of Liquid Chromatography & related technologies, Vol.27, No. 15, No. 2004, 1/11, p.2421, p.2439, XP 055081, ISSN: 1082-6076, DOI: 10.1081/JLC-200028170; see also WO 2016/135346). These systems have longer test times, less sample loading and only moderate yields.

The present disclosure addresses these and other problems by providing methods for the separation and purification of cannabinoid compounds using solvent systems and Centrifugal Partition Chromatography (CPC) or Counter Current Chromatography (CCC). In the case of CPC, the use of a quantum CPC rotor (arman) or CPC 1000pro (gilson) has significantly reduced time on the centrifuge, and large sample loads. By this method, cannabinoid compounds can be obtained in high yields with a purity of 95% or higher.

Disclosure of Invention

In one aspect, the invention provides a method of purifying one or more cannabinoids from plant material including plants, plant resins or plant extracts, the method consisting essentially of:

(a) mixing plant material with at least one solvent selected from pentane, hexane, heptane, petroleum ether, cyclohexane, dichloromethane, chloroform, tetrahydrofuran, diethyl ether, toluene, benzene, ethanol, methanol, isopropanol, acetone, acetonitrile, ethyl acetate, butane, propane, 1,1,1, 2-tetrafluoroethane (R134a) or liquid, subcritical or supercritical CO₂Or a mixture thereof, to form a solvent mixture, the solvent mixture extracting one or more cannabinoids from the plant material, wherein the solvent mixture has an original volume;

(b) for THC-type extracts, one or more solvents selected from hexane: ethanol: water, pentane: acetonitrile and hexane: acetonitrile, two-phase solvent system solvent mixture was added, wherein pentane: acetonitrile system and hexane: the acetonitrile system optionally includes ethyl acetate and/or water as modifiers; for CBD-type extracts, the ratio of hexane: ethanol: adding a two-phase solvent system of water to the extract; and for CBG-type extracts, the ratio of hexane: ethanol: adding a two-phase solvent system of water to the extract; and

(c) carrying out the liquefaction using the biphasic solvent system of step b): liquid chromatography to purify the one or more cannabinoids.

In one embodiment, for THC-type extracts, the biphasic solvent system is hexane: ethanol: and (3) water. In another embodiment, for THC-type extracts, the biphasic solvent system is pentane: ethyl acetate: acetonitrile: and (3) water. In another embodiment, for THC-type extracts, the biphasic solvent system is hexane: ethyl acetate: acetonitrile: and (3) water. In another embodiment, for CBD-type extracts, the biphasic solvent system is hexane: ethanol: and (3) water. In another embodiment, for CBG-type extracts, the biphasic solvent system is hexane: ethanol: and (3) water.

In one embodiment, an extract of cannabis of chemical form I or II is used to purify THC, THCA, THCV, THCVA, CBN or CBV and fractionate CBD-and CBG-type cannabinoids. In another embodiment, an extract of cannabis of chemical type II or III is used to purify CBD, CBDA, CBDVA or CBDV and fractionate THC-type and CBG-type cannabinoids. In another embodiment, an extract of cannabis chemotype IV is used to purify CBG, CBGA, CBGVA or CBGV and fractionate CBD-and THC-type cannabinoids.

In one embodiment, acetonitrile is used: a gradient of aqueous mobile liquid phase, re-purifying the fraction of THC contaminated with CBC or the fraction of THCV contaminated with CBN on a C-8 or C-18 coated silica solid stationary phase using solid-liquid chromatography selected from gravity, flash or preparative HPLC.

In one embodiment, the ratio of liquid: the liquid chromatography is Centrifugal Partition Chromatography (CPC) or countercurrent chromatography (CCC).

In one embodiment, after step a), the solvent mixture is reduced to dryness or to about 50% or less of the original volume of the solvent mixture in step (a) such that the ratio of water: concentrating the one or more cannabinoids prior to liquid chromatography.

In one embodiment, the solvent mixture of step (a) is purified prior to step (b). In another embodiment, the one or more cannabinoids present in the plant material are decarboxylated by heating the plant material prior to step (a). In another embodiment, after reducing the solvent mixture to dryness, the dried extract product of the solvent mixture is dissolved in ethanol, frozen at a temperature of-20 ℃ to 4 ℃, filtered to remove precipitated material and reduced to dryness prior to purification by liquid-liquid chromatography.

In one embodiment, the method of CPC uses a rotor design quantum CPC or CPC PRO. In another embodiment, the method of CPC uses a rotor design quantum CPC or CPCPPRO, where the total test time is 12-20 minutes, independent of the rotor volume. In another embodiment, the CPC rotor has a rotor volume of 1 liter, the injection is 50mL, the flow rate of the mobile phase (pentane or hexane phase) of the two-phase solvent system during the experiment is 200mL/min, and the flow rate of the stationary phase (ethanol or acetonitrile phase) of the two-phase solvent system during the extrusion phase of the experiment is 350 mL/min.

In one embodiment, the CBD, CBDA, CBDVA or CBDV is in liquid: the step of liquid chromatography is followed by crystallization. In another embodiment, the CBG, CBGA, CBGVA or CBGV is in liquid: the step of liquid chromatography is followed by crystallization.

In one embodiment, the plant material is first incubated with a non-polar solvent selected from the group consisting of petroleum ether, pentane, hexane, and heptane to form a solvent mixture, which non-polar solvent extracts one or more cannabinoids from the plant material to form the solvent mixture.

In one embodiment, the plant material is first contacted with a solvent selected from pentane, hexane, heptane, petroleum ether, cyclohexane, dichloromethane, chloroform, tetrahydrofuran, diethyl ether, toluene, benzene, ethanol, methanol, isopropanol, acetone, acetonitrile, ethyl acetate, butane, propane, 1,1,1, 2-tetrafluoroethane (R134a) or liquid, subcritical or supercritical CO₂Or a mixture thereof; filtration, decantation or centrifugation; reducing to dryness; and then incubated with a non-polar solvent selected from the group consisting of petroleum ether, pentane, hexane, and heptane to form a solvent mixture, the non-polar solvent extracting one or more cannabinoids from the plant material to form the solvent mixture.

In one embodiment, after step (a), the one or more cannabinoids present in the plant material and the extract are decarboxylated by heating a solvent mixture, wherein the solvent mixture is the original volume, the concentrated volume or a dry extract obtained by evaporating the original volume of the solvent mixture to dryness.

In one embodiment, acetonitrile is used: a gradient of aqueous mobile liquid phase, re-purifying the fraction of THC contaminated with CBC or the fraction of THCV contaminated with CBN on a C-8 or C-18 coated silica solid stationary phase using solid-liquid chromatography selected from gravity, flash or preparative HPLC.

In one embodiment, the cannabinoid is selected from CBD, CBDA, and CBDV. In another embodiment, the cannabinoid is selected from the group consisting of CBG, CBGA, and CBGV. In another embodiment, the cannabinoid is selected from THC, THCA and THCV.

Other aspects of the specification disclose methods of treating diseases or conditions using purified cannabinoids and pharmaceutical compositions comprising one or more cannabinoids produced by the disclosed methods. Non-limiting examples of diseases or conditions include pain, schizophrenia, convulsions, inflammation, anxiety, depression, neurodegenerative diseases, stroke, traumatic brain injury, cancer, migraine, arthritis, chronic pain, nausea and vomiting, anorexia, glaucoma, glioma, epilepsy, asthma, perinatal asphyxia, graft versus host disease, addiction, dependency and withdrawal symptoms, multiple sclerosis, spinal cord injury, tourette's syndrome, dystonia or tardive dyskinesia.

Drawings

Not applicable.

Detailed Description

The invention provides a use solution: a method for separating and purifying one or more cannabinoids from a plant extract by liquid chromatography. Non-limiting examples of cannabinoids include Tetrahydrocannabinol (THC), Tetrahydrocannabivarin (THCV), tetrahydrocannabinolic acid (THCA), Cannabidiol (CBD), Cannabivarin (CBDV), cannabidiolic acid (CBDA), Cannabigerol (CBGV), Cannabigerol (CBG) and cannabigerolic acid (CBGA) from plants belonging to the cannabis genus.

Liquid: liquid chromatography

The disclosed method provides a use solution: liquid chromatography process for the purification of cannabinoids from plant extracts. The optional crystallization step(s) may be carried out in liquid: the step of liquid chromatography is performed before or after. Alternatively, without a crystallization step, only liquid: and (4) performing liquid chromatography. In one embodiment, the ratio of liquid: the liquid chromatography step comprises counter current chromatography or centrifugal partition chromatography. In aspects of chromatographic embodiments, a chromatographic step is applied after each crystallization step described below (e.g., after steps (c), (e), (h), or (i)). In one embodiment, a CPC/CCC chromatography step is applied prior to (e.g., after) the crystallization step described below.

Both CCC and CPC are liquid-liquid based chromatographic methods in which both the stationary phase and the mobile phase are liquid. Permanent adsorption of the analyte onto the column is avoided by eliminating the solid support and high analyte recovery is achieved. The instrument can be easily switched between normal and reverse phase modes of operation by merely changing the mobile and stationary phases. For liquid chromatography, the operation is limited by the composition of the column and the commercially available instruments. Almost any pair of immiscible solutions can be used for liquid-liquid chromatography as long as the stationary phase can be successfully retained. In one embodiment, the mobile phase is an organic and/or non-polar agent and the stationary phase is water and/or a polar agent.

Liquid: the solvent cost of liquid chromatography is also typically lower than that of High Performance Liquid Chromatography (HPLC) and eliminates the cost of purchasing and disposing of solid adsorbents. Another advantage is that experiments performed in the laboratory can be scaled to industrial volumes. When performing GC or HPLC in large volumes, resolution is lost due to problems with surface/volume ratio and flow dynamics; this can be avoided when both phases are liquid.

In one embodiment, the mobile organic phase may comprise pentane, petroleum ether, hexane, cyclohexane or heptane. In one embodiment, the stationary phase may comprise ethanol, methanol, isopropanol, acetone, acetonitrile, and/or water. In one embodiment, the mobile phase is pentane, hexane, cyclohexane or heptane and the stationary phase is water and ethanol, methanol or isopropanol. In one embodiment, the mobile phase is pentane or heptane and the stationary phase is acetone and/or acetonitrile, possibly using water as modifier.

In Counter Current Chromatography (CCC) and Centrifugal Partition Chromatography (CPC), a two-phase system is used. In one embodiment of the presently described process, the two-phase system comprises hexane: ethanol: and (3) water. One embodiment uses a ratio of (20:13:7) or 20:12:8 to isolate CBG-type cannabinoids (CBG, CBGA, CBGVA and CBGV). One embodiment uses a ratio of (20:14:6) to isolate CBD-type cannabinoids (CBD, CBDA, CBDVA and CBDV). One embodiment uses a ratio of (20:17:3) to separate THC-type cannabinoids (THC, THCA, THCVA and THCV). One embodiment uses a gradient reversed phase assay with a mixture of ethanol and water as the mobile phase, gradually increasing the concentration of ethanol from a ratio (20:12:8) to (20:18:2), replacing hexane with pentane, heptane and/or cyclohexane and ethanol with methanol or isopropanol, and using an organic phase of pentane or hexane as the mobile phase or two-phase system.

In one embodiment, the two-phase system is pentane with or without ethyl acetate or water as modifiers: acetonitrile or hexane: acetonitrile for the isolation of THC-type cannabinoids. In one embodiment, for THC-type extracts, the ratio of pentane: acetonitrile in a ratio of 10:10 to 7:3 by volume, e.g., pentane: ethyl acetate: acetonitrile: water (10:0:10:0) to pentane: ethyl acetate: acetonitrile: water (7:3:7: 3). In another embodiment, the ratio of hexane: the ratio of acetonitrile is 10:10 to 7:3 by volume, for example, hexane: ethyl acetate: acetonitrile: water (10:0:10:0) to hexane: ethyl acetate: acetonitrile: water (7:3:7: 3). Preferred solvent ratios for THC-type cannabinoids are pentane: ethyl acetate: acetonitrile: the water is (19:1:19:1) by volume or (9:1:9:1) by volume. These two systems are also useful for CBD and CBG-type extracts. For CBD-type extracts, pentane: ethyl acetate: acetonitrile: the ratio of water is preferably (8:2:8:2) by volume and for CBG-type extracts, pentane: ethyl acetate: acetonitrile: the ratio of water was (7:3:7:3) by volume.

Another embodiment of the process comprises a mixture having hexane in the ratio ranging from (20:20:1) to (20:1:20) and from (20:1:5) to (20:1:10) and from (1:20:10) to (30:20: 1): ethanol: a two-phase system of water. For example, the ratio of hexane to ethanol may be in the range of about 1:20 to about 20:1, e.g., about 1:20, about 1:10, about 3:20, about 4:20, 5:20, about 6:20, about 7:20, about 8:20, about 9:20, about 10:20, about 11:20, about 12:20, about 13:20, about 14:20, about 15:20, about 16:20, about 17:20, about 18:20, about 19:20, about 20:19, about 20:18, about 20:17, about 20:16, about 20:15, about 20:14, about 20:13, about 20: 12. about 20:11, about 20:10, about 20:9, about 20:8, about 20:7, about 20:6, about 20:5, about 20:4, about 20:3, about 20:2, or about 20: 1. Similarly, the ratio of ethanol to water may be in the range of about 20:1 to about 1:20, e.g., about 1:20, about 1:10, about 3:20, about 4:20, 5:20, about 6:20, about 7:20, about 8:20, about 9:20, about 10:20, about 11:20, about 12:20, about 13:20, about 14:20, about 15:20, about 16:20, about 17:20, about 18:20, about 19:20, about 20:19, about 20:18, about 20:17, about 20:16, about 20:15, about 20:14, about 20:13, about 20: 12. about 20:11, about 20:10, about 20:9, about 20:8, about 20:7, about 20:6, about 20:5, about 20:4, about 20:3, about 20:2, or about 20: 1.

In one aspect, the ratio of hexane: ethanol: the ratio of water is from (20:19:1) to (20:8:12) and hexane and methanol are replaced by pentane, heptane and/or cyclohexane and/or isopropanol is replaced by ethanol, the organic phase of pentane or hexane being used as mobile phase or two-phase system. In particular, a two-phase system hexane in a ratio (20:13: 7): ethanol: water was used to isolate CBG-type cannabinoids, in a ratio of (20:14:6) two phase system hexane: ethanol: water for separation of CBD-type cannabinoids and a two phase system hexane in a ratio (20:17: 3): ethanol: water was used to isolate THC-type cannabinoids or a gradient reverse phase experiment using a mixture of ethanol and water as the mobile phase, gradually increasing the concentration of ethanol from a ratio (20:12:8) to (20:18: 2).

Another embodiment is the process of the invention wherein a two phase system of hexane: ethanol: water and hexane with pentane, heptane and/or cyclohexane and ethanol with methanol and/or isopropanol, in the chromatography techniques of CPC and CCC, the organic phase of pentane or hexane is used as mobile phase for the separation and/or purification of any species and chemical type present in the plant from cannabisThe extract of (a) is treated with pentane, hexane, heptane, petroleum ether, cyclohexane, dichloromethane, chloroform, tetrahydrofuran, diethyl ether, toluene, benzene, ethanol, methanol, isopropanol, acetone, acetonitrile, ethyl acetate, butane, propane, refrigeration gas (e.g., 1,1,1, 2-tetrafluoroethane (R134a)) or liquid, subcritical or supercritical CO₂Or a mixture of these solvents.

Accordingly, embodiments of the methods of the invention include performing Counter Current Chromatography (CCC) or Centrifugal Partition Chromatography (CPC) before or after each crystallization step (e.g., after steps (c), (e), (h), or (i) shown below) to isolate and purify cannabinoids: tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCV), tetrahydrocannabinolic acid (THCA), tetrahydrocannabinolic acid (THCVA), Cannabidiol (CBD), Cannabidiol (CBDV), cannabidiolic acid (CBDA), cannabidiolic acid (CBDVA), Cannabinol (CBN), Cannabinol (CBV), Cannabigerol (CBGV), Cannabigerol (CBG), cannabigerolic acid (CBGVA) and cannabigerolic acid (CBGA).

Crystal of cannabinoid

In one embodiment, a method of purifying one or more cannabinoids from a plant material comprises a) incubating the plant material with a first non-polar solvent to form a first solvent mixture, the first non-polar solvent extracting the one or more cannabinoids from the plant material; b) reducing the volume of the first solvent mixture to about 50% or less of the original volume of the first solvent mixture in step (a) by concentrating the one or more cannabinoids; c) incubating the reduced first solvent mixture in a manner that crystallizes the one or more cannabinoids; d) incubating one or more crystallized cannabinoids with a second non-polar solvent to form a second solvent mixture; and e) incubating the second solvent mixture in a manner that crystallizes the one or more cannabinoids, thereby resulting in purification of the one or more cannabinoids. The disclosed process further provides that the crystallized cannabinoid or cannabinoids of step (c) may be purified prior to step (d) using, for example, filtration resulting in collection of a mother liquor. The mother liquor can be collected and incubated in a manner to crystallize the one or more cannabinoids. Step (a) may be repeated one or more times. Steps (d) and (e) may be repeated one or more times until the purity of the cannabinoid or cannabinoids is 95% or higher.

In one embodiment, a method of purifying one or more cannabinoids from a plant material comprises a) incubating the plant material with a first non-polar solvent to form a first solvent mixture, the first non-polar solvent extracting the one or more cannabinoids from the plant material; b) filtering the first solvent mixture; c) reducing the volume of the first solvent mixture to about 50% or less of the original volume of the first solvent mixture in step (a) by concentrating the one or more cannabinoids; d) incubating the reduced first solvent mixture in a manner that crystallizes the one or more cannabinoids; e) purifying the one or more crystallized cannabinoids in step (d) using filtration, resulting in a mother liquor being collected; f) incubating the one or more crystallized cannabinoids with a second non-polar solvent to form a second solvent mixture, wherein the second solvent mixture dissolves at least 50% of the one or more crystallized cannabinoids; g) incubating the second solvent mixture in a manner that crystallizes the one or more cannabinoids; and h) purifying the one or more crystallized cannabinoids in step (g) using filtration, thereby resulting in purification of the one or more cannabinoids, resulting in collection of a mother liquor. The disclosed method may further comprise: i) purifying the one or more crystallized cannabinoids using filtration, resulting in a mother liquor being collected; and j) incubating the mother liquor in a manner that crystallizes the one or more cannabinoids. Step (a) may be repeated one or more times. Steps (i) and (j), steps (f) and (g), and steps (f), (g) and (h) may be repeated one or more times until the purity of the cannabinoid or cannabinoids is 95% or higher.

In one embodiment, a method of purifying one or more cannabinoids from a plant material comprises a) incubating the plant material with a first non-polar solvent to form a first solvent mixture, the first non-polar solvent extracting the one or more cannabinoids from the plant material; b) filtering the first solvent mixture; c) reducing the volume of the first non-polar solvent in the filtrate obtained in step (b) by evaporation; d) incubating the reduced first solvent mixture in a manner that crystallizes the one or more cannabinoids; e) removing the first non-polar solvent by vacuum filtration; f) further reducing the amount of the first non-polar solvent from the filtrate of (e) by evaporation; g) incubating the one or more crystallized cannabinoids with a second non-polar solvent to form a second solvent mixture, wherein the second solvent mixture dissolves at least 50% of the one or more crystallized cannabinoids; h) incubating the second solvent mixture in a manner that crystallizes the one or more cannabinoids; i) removing the second non-polar solvent by vacuum filtration and preserving the obtained crystals; and j) adding sufficient non-polar solvent per gram of cannabinoid to dissolve and recrystallize the crystals obtained in step (i).