Note: Descriptions are shown in the official language in which they were submitted.
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PCT Patent Application
Methods of Purifying Cannabinoids Using Liquid:Liquid Chromatography
[1] This application claims priority to and is a continuation of U.S. Patent
Application 15/882,516, filed on
January 29, 2018. The present application is also related to U.S. Patent
Application 15/707,524, filed
September 18, 2017, now U.S. Patent 10,155,708, which claims priority to and
is a continuation-in-part of
U.S. Patent Application 15/004,848, filed on January 22, 2016, now U.S. Patent
9,765,000, which claims
the benefit of U.S. Provisional Patent Application 62/106,644, filed on
January 22, 2015, the contents of
which are each hereby incorporated by reference in their entirety.
TECHNICAL FIELD
[2] The present invention relates to the isolation of cannabinoid compounds
using unique biphasic solvent
systems and liquid-liquid chromatography as centrifugation partitioning
chromatography (CPC) or counter
current chromatography (CCC).
BACKGROUND OF THE INVENTION
[3] Cannabis is a genus of flowering plants whose species are distinguished by
plant phenotypes and
secondary metabolite profiles. Cannabis is a genus include: Cannabis sp.
including Cannabis sativa L. and
all subspecies, the putative species Cannabis indica Lam., Cannabis ruderalis
Janisch, and hybrids and
varieties thereof, as discussed further below. Cannabis plants have been
cultivated for a variety of uses
including making fibers (hemp), medicinal use and recreational drug use.
Cannabis is also commonly
known as marijuana.
[4] Cannabis has now been generally acknowledged as having substantial
benefits for various medical
uses. For example, cannabis is regularly used by a wide cross-section of
society to treat a variety of
maladies, ailments and symptoms including, but not limited to, nausea, pain
relief (such as chronic pain,
cancer related pain, or neuropathic pain), glaucoma, lack of appetite, mucous
membrane inflammation,
inflammatory diseases (such as Crohn's disease), neurodegenerative disease,
epilepsy (that affects
children and adults), seizures, diabetes, leprosy, fever, obesity, asthma,
urinary tract infections, coughing,
anorexia associated with weight loss in AIDS patients, graft-versus-host
disease, glioma, perinatal asphyxia
and post-traumatic stress disorder (PTSD) and autoimmune disease (such as
multiple sclerosis).
[5] One of the most common ways that cannabis is used for medicinal use in
many countries is through
smoking. Smoking medical cannabis, although proven to be beneficial in certain
indications, has
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disadvantages. For example, the amounts of active ingredients may differ
depending on the differences
present in plant varietals as well as changing growing conditions which result
in intravarietal variations. As
a result, it can be difficult to maintain control over the proper dosing of
medicinal cannabis due to active
ingredients fluctuations. Another disadvantage of smoking medical cannabis is
the negative impact of some
of the constituents of cannabis smoke. The smoke from the plant matter
comprise carcinogens in addition
to the desired cannabinoids. In addition, heavy cannabis use through smoking
has been associated with
accelerated pulmonary decline.
[6] Cannabinoids are compounds active on cannabinoid receptors in humans and
are responsible for
eliciting many of the pharmacological effects of cannabis. Cannabinoids of
plant origin, also known as
phytocannabinoids, are abundant in Cannabis. Two known cannabinoids which are
present in relatively
high concentrations in Cannabis sativa L. are tetrahydracannabinolacid (THCA)
or its decarboxylated
product tetrahydracannabinol (THC) and cannabidiolic acid (CBDA) or its
decarboxylated product
cannabidiol (CBD). THC elicits psychoactive (calming) effects, analgesic
effects, antioxidant effects and to
increase appetite. However, THC is also associated with many negative or
undesirable side effects
including, but are not limited to, decreased short-term memory, dry mouth,
impaired visual perception and
motor skillsõ red (i.e., blood shot) eyes, increased anxiety, occasional
infarction, stroke, paranoia, acute
psychosis, lowered mental aptitude, hallucinations, bizarre behavior,
irrational panic attacks, irrational
thoughts and various other cognitive and social problems. On the other hand,
CBD is increasingly
becoming a popular cannabinoid for medicinal purposes because unlike THC, CBD
is non-psychoactive at
typical doses. In addition, CBD was found to have neuroprotective effects and
to have ameliorative effects
in patients with epilepsy, schizophrenia and Parkinson's disease. Accordingly,
patients and healthcare
providers are exhibiting a preference for CBD because patients need to work,
drive and function with clarity
while undergoing treatment.
[7] Diverse chromatographic techniques have been used purify cannabinoid
compounds from the plant
Cannabis sativa. For example, Flash chromatography on silica gel, C8 or C18;
preparative HPLC on silica
gel columns, C8 or C18; and supercritical CO2 chromatography on silica gel.
However, these
chromatographic processes are tedious and expensive.
[8] Thus, what is needed is a simple and less expensive process that
selectively purifies and concentrates
medically beneficial cannabinoids. In addition, it is also desirous to develop
medicinal formulations
comprising higher levels of beneficial cannabinoids. However, THC and THCA can
also be purified by this
method from THC-THCA rich or THC-THCA low Cannabis sativa L. plant and
extracts.
[9] Centrifugation partitioning chromatography (CPC) and counter current
chromatography (CCC) can be
used, e.g., in the extraction and enrichment of compounds from plant extracts
in analytical, semi-
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preparative and preparative scale. CPC and CCC are a liquid-liquid
chromatography methods using a
mostly two-phase solvent. It enables an almost loss-free separation of complex
mixtures of substances
from crude extracts. CPC and CCC as compared to liquid chromatography (HPLC)
are easier and also
cheaper, because matrix effects and irreversible adsorption on solid phases do
not occur. Cannabinoids
have been purified using CPC, but not using the solvent systems described in
this patent application (see,
e.g., Hazekamp, et al., "Preparative Isolation of Cannabinoids from Cannabis
sativa by Centrifugal Partition
Chromatography", Journal of Liquid Chromatography & Related Technologies, vol.
27, no. 15, 11 January
2004 (2004-01-11), pages 2421 -2439, XP055202081, ISSN: 1082-6076, DOI:
10.1081/JLC-200028170;
see also W02016/135346). These systems have long run times, less sample load
and only moderate
yields.
[10] The present disclosure solves these and other problems by providing a
method for isolating and
purifying cannabinoid compounds using a solvent system and centrifugation
partition chromatography
(CPC) or counter current chromatography (CCC). In the case of CPC, there is
significantly less time on the
centrifuge and a large sample load, using the Quantum CPC rotor (ARMEN) or the
CPC 1000 PRO
(GILSON). By means of this procedure it is possible to obtain high yields of
cannabinoid compounds having
a purity of 95% or more.
SUMMARY OF THE INVENTION
[11] In one aspect, the present invention provides a method of purifying one
or more cannabinoids from a
plant material including a plant, a plant resin or a plant extract, the method
consisting essentially of the
following steps:
(a) incubating the plant material with a solvent selected from the group
consisting of pentane, hexane,
heptane, petroleum ethers, cyclohexane, dichloromethane, trichloromethane,
tetrahydrofurane,
diethyl ether, toluene, benzene, ethanol, methanol, isopropanol, acetone,
acetonitrile, ethyl acetate,
butane, propane, 1,1,1,2-Tetrafluoroethane (R134a) or, liquid, subcritical or
supercritical CO2 or
mixes thereof to form a solvent mixture which extracts the one or more
cannabinoids from the plant
material, wherein the solvent mixture has an original volume;
(b) for THC-type extracts, adding to the solvent mixture a biphasic solvent
system selected from the
group consisting of hexane:ethanol:water, pentane:acetonitrile and
hexane:acetonitrile, wherein
the pentane:acetonitrile system and the hexane:acetonitrile system optionally
include ethyl acetate
and/or water as a modifier; for CBD-type extracts, adding to the extract a
biphasic solvent system
of hexane:ethanol:water; and for CBG-type extracts, adding to the extract a
biphasic solvent system
of hexane:ethanol:water; and
(c) performing liquid:liquid chromatography using a biphasic solvent system of
step b), thereby purifying
the one or more cannabinoids.
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[12] In one embodiment, for the THC-type extracts the biphasic solvent system
is hexane:ethanol:water is
at a ratio of (20:17:3) by volume. In another embodiment, for the THC-type
extracts the biphasic solvent
system is pentane:ethyl acetate:acetonitrile:water at a ratio from (10:0:10:0)
to (7:3:7:3) by volume. In
another embodiment, for the THC-type extracts the biphasic solvent system is
hexane:ethyl
acetate:acetonitrile:water at a ratio from (10:0:10:0) to (7:3:7:3) by volume.
In another embodiment, for the
CBD-type extracts the biphasic solvent system is hexane:ethanol:water at a
ratio of (20:14:6) by volume.
In another embodiment, for the CBG-type extracts the biphasic solvent system
is hexane:ethanol:water at
a ratio of at a ratio of (20:12:8) or (20:13:7) by volume.
[13] In one embodiment, an extract of chemotype I or II Cannabis sativa L. is
used to purify THC, THCA,
THCV, THCVA, CBN or CBV and fractionate the CBD-type and CBG-type
cannabinoids. In another
embodiment, an extract of chemotype ll or III Cannabis sativa L. is used to
purify CBD, CBDA, CBDVA or
CBDV and fractionate the THC-type and CBG-type cannabinoids. In another
embodiment, an extract of
chemotype IV Cannabis sativa L. is used to purify CBG, CBGA, CBGVA or CBGV and
fractionate the CBD-
type and THC-type cannabinoids.
[14] In one embodiment, fractions of THC contaminated by CBC or the fractions
of the THCV
contaminated with CBN are re-purified using solid-liquid chromatography
selected from the group
consisting of gravity, Flash or preparative HPLC over 0-8 or 0-18 coated
silica solid stationary phase,
using a gradient of acetonitrile:water mobile liquid phase.
[15] In one embodiment, the liquid:liquid chromatography is centrifugation
partitioning chromatography
(CPC) or is counter current chromatography (CCC).
[16] 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) thereby concentrating
the one or more cannabinoids
before the liquid:liquid chromatography.
[17] In one embodiment, the solvent mixture of step (a) is purified prior to
step (b). In another embodiment,
prior to step (a), the one or more cannabinoids present in the plant material
are decarboxylated by heating
the plant material. In another embodiment, after the solvent mixture is
reduced to dryness, a dry extract
product of the solvent mixture is dissolved in ethanol, chilled at a
temperature from -20 C to 4 C, filtered
to remove precipitated material and reduced to dryness before purification by
liquid-liquid chromatography.
[18] 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 CPC PRO,
wherein the total run
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time is 12-20 minutes, independent of rotor volume. In another embodiment, the
CPC rotor has a rotor
volume of 1 liter, a sample injection of 50 mL, a flow rate of a mobile phase
(pentane or hexane phase) of
the biphasic solvent system of 200 mL/min during the run, and a flow rate of a
stationary phase (the
ethanolic or acetonitrile phase) of the biphasic solvent system of 350 mL/min
during the extrusion phase of
the run.
[19] In one embodiment, the CBD, CBDA, CBDVA or CBDV is crystalized after the
step of liquid:liquid
chromatography. In another embodiment, the CBG, CBGA, CBGVA or CBGV is
crystalized after the step
of liquid:liquid chromatography.
[20] 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 extracts
the one or more cannabinoids from the plant material to form the solvent
mixture.
[21] In one embodiment, the plant material is first incubated with a solvent
selected from the group
consisting of pentane, hexane, heptane, petroleum ethers, cyclohexane,
dichloromethane,
trichloromethane, tetrahydrofurane, diethyl ether, toluene, benzene, ethanol,
methanol, isopropanol,
acetone, acetonitrile, ethyl acetate, butane, propane, 1,1,1,2-
Tetrafluoroethane (R134a) or, liquid,
subcritical or supercritical CO2 or mixes thereof; filtered, decanted or
centrifuged; reduced 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 which extracts the one or more
cannabinoids from the plant
material to form the solvent mixture.
[22] In one embodiment, after step (a) the one or more cannabinoids present in
the plant material and
extracts are decarboxylated by heating the solvent mixture, wherein the
solvent mixture is the original
volume, a concentrated volume or a dry extract obtained from evaporation to
dryness of the original volume
of the solvent mixture.
[23] In one embodiment, fractions of THC contaminated by CBC or the fractions
of the THCV contaminated
with CBN are re-purified using solid-liquid chromatography selected from the
group consisting of gravity,
Flash or preparative HPLC over 0-8 or 0-18 coated silica solid stationary
phase, using a gradient of
acetonitrile:water mobile liquid phase.
[24] In one embodiment, the cannabinoid is selected from the group consisting
of 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 the group consisting
of THC, THCA and THCV.
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[25] Other aspects of the present specification disclose methods of treating a
disease or condition using
purified cannabinoids and pharmaceutical compositions comprising one or more
cannabinoid produced by
the disclosed methods. Non-limiting examples of a disease or condition include
pain, schizophrenia,
convulsion, inflammation, anxiety, depression, neurodegenerative disease,
stroke, traumatic brain injury,
cancer, migraines, arthritis, chronic pain, nausea and vomiting, anorexia,
glaucoma, glioma, epilepsy,
asthma, perinatal asphyxia, graft-versus-host disease, addiction, symptoms of
dependency and withdrawal,
multiple sclerosis, spinal cord injury, burette's syndrome, dystonia, or
tardive dyskinesia.
BREIF DESCRIPTION OF THE DRAWINGS
[26] Not applicable.
DETAILED DESCRIPTION OF THE INVENTION
[27] The present invention provides a method for isolating and purifying one
or more cannabinoids from a
plant extract using liquid:liquid chromatography. Non-limiting examples of a
cannabinoid include
tetrahydrocannabinol (THC), tetrahydrocannabidivarin (THCV),
tetrahydrocannabinolic acid (THCA),
cannabidiol (CBD), cannabidivarin (CBDV), cannabidiolic acid (CBDA),
cannabigerovarin (CBGV),
cannabigerol (CBG), and cannabigerol acid (CBGA)from a plant belonging to the
genus Cannabis.
Liquid:Liquid Chromatography
[28]The disclosed method provides for purification of cannabinoids from a
plant extract using a liquid:liquid
chromatographic step. An optional crystallization step(s) may be performed
before or after the step of
liquid:liquid chromatography. Alternatively, no crystallization step is
used, only the liquid:liquid
chromatographic step. In one embodiment, the liquid:liquid chromatography step
includes countercurrent
chromatography or centrifugal partition chromatography. In an aspect of the
chromatographic embodiment,
the chromatographic step is applied after each crystallization step described
below (e.g. after step (c), (e),
(h) or (i)). In one embodiment the CPC/C00 chromatographic step is applied
prior to the crystallization step
described below (e.g. after step (b)).
[29] Both CCC and CPC are liquid-liquid based chromatographic methods, where
both the stationary phase
and the mobile phase are liquids. By eliminating solid supports, permanent
adsorption of the analyte onto
the column is avoided, and a high recovery of the analyte can be achieved. The
instrument is also easily
switched between normal-phase and reversed-phase modes of operation simply by
changing the mobile
and stationary phases. With liquid chromatography, operation is limited by the
composition of the columns
and media commercially available for the instrument. Nearly any pair of
immiscible solutions can be used
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in liquid-liquid chromatography provided that the stationary phase can be
successfully retained. In one
embodiment, the mobile phase is organic and/or non-polar, and the stationary
phase is the aqueous and/or
polar reagent.
[30] Solvent costs for liquid liquid chromatography are also generally lower
than for high-performance liquid
chromatography (HPLC), and the cost of purchasing and disposing of solid
adsorbents is eliminated.
Another advantage is that experiments conducted in the laboratory can be
scaled to industrial volumes.
When GC or HPLC is carried out with large volumes, resolution is lost due to
issues with surface-to-volume
ratios and flow dynamics; this is avoided when both phases are liquid.
[31] In one embodiment, the mobile organic phase may include pentane,
petroleum ether, hexane,
cyclohexane, or heptane. In one embodiment, the stationary phase may include
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 with the possible use of water as a modifier.
[32] In countercurrent chromatography (CCC) and centrifugal partition
chromatography (CPC), a two-phase
system is used. In one embodiment of the presently recited methods, the two-
phase system includes
hexane:ethanol:water used at ratios of (20:19:1) to (20:8:12). One embodiment
uses ratios of (20:13:7) or
20:12:8 for isolation of CBG-type cannabinoids (CBG, CBGA, CBGVA and CBGV).
One embodiment uses
ratios of (20:14:6) for isolation of CBD-type cannabinoids (CBD, CBDA, CBDVA
and CBDV). One
embodiment uses ratios of (20:17:3) for isolation of THC-type cannabinoids
(THC, THCA, THCVA and
THCV). One embodiment uses a gradient reverse phase run with ethanol and water
mix as mobile phase
increasing the concentration of ethanol gradually from the ratio (20:12:8) to
(20:18:2), with substitutions of
pentane, heptane and/or cyclohexane with hexane and methanol or isopropanol
instead of ethanol, with
the organic phase of pentane or hexane as mobile phase or the two-phase
system.
[33] In one embodiment the two phase system is pentane:acetonitrile or
hexane:acetonitrile with or without
ethyl acetate or water as a modifier for the isolation of THC-type
cannabinoids. In one embodiment for
THC-type extracts, the ratio of pentane:acetonitrile is from 10:10 to 7:3,
e.g., pentane:ethyl
acetate:acetonitrile:water (10:0:10:0) to pentane:ethyl
acetate:acetonitrile:water (7:3:7:3) by volume. In
another embodiment the ratio of hexane:acetonitrile is from 10:10 to 7:3,
e.g., hexane:ethyl
acetate:acetonitrile:water (10:0:10:0) to hexane:ethyl
acetate:acetonitrile:water (7:3:7:3) by volume.
Preferred solvent ratios for THC-type cannabinoids are pentane:ethyl acetate:
acetonitrile:water at
(19:1:19:1) by volume or (9:1:9:1) by volume. These two systems can also be
used for CBD and CBG-type
extracts. For CBD-type extracts the ratio of pentane:ethyl
acetate:acetonitrile:water is preferably (8:2:8:2)
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by volume and for CBG-type extracts the ratio of pentane:ethyl
acetate:acetonitrile: water is (7:3:7:3) by
volume.
[34] Another embodiment of the present methods includes a two-phase system
having
hexane:ethanol:water at ratios 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). For example the ratio of hexane to ethanol may be
range from 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 20:20, 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 range from 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 20:20, 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.
[35] In one aspect the ratio of hexane:ethanol:water is (20:19:1) to
(20:8:12), and with substitutions of
pentane, heptane and/or cyclohexane with hexane and methanol and/or
isopropanol instead of ethanol,
with the organic phase of pentane or hexane as mobile phase or the two-phases
system. In particular, the
ratios of the two-phase system hexane:ethanol:water are (20:13:7) for
isolation of CBG-type cannabinoids,
(20:14:6) for isolation of CBD-type cannabinoids and (20:17:3) to isolate THC-
type cannabinoids or using
a gradient reverse phase run with ethanol and water mix as mobile phase
increasing the concentration of
ethanol gradually from the ratio (20:12:8) to (20:18:2).
[36] Another embodiment is the method of the invention, wherein the two-phase
system,
hexane:ethanol:water is used, and substitutions of pentane, heptane and/or
cyclohexane with hexane and
methanol and/or isopropanol instead of ethanol, with the organic phase of
pentane or hexane as mobile
phase in the chromatographic techniques of CPC and CCC for isolating and/or
purifying the cannabinoids
that are present in extracts made with pentane, hexane, heptane, petroleum
ethers, cyclohexane,
dichloromethane, trichloromethane, tetrahydrofurane, diethyl ether, toluene,
benzene, ethanol, methanol,
isopropanol, acetone, acetonitrile, ethyl acetate, butane, propane,
refrigerant gases (e.g., 1,1,1,2-
Tetrafluoroethane (R134a)) or, liquid, subcritical or supercritical CO2 or
mixes of these solvents from any
variety and chemotype of the Cannabis sativa L. plant.
[37] Therefore, an embodiment of the method of the invention includes before
or after each crystallization
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step (e.g., after step (c), (e), (h) or (i) as shown below) a countercurrent
chromatography (CCC) or a
centrifugal partition chromatography (CPC) are carried out to isolate and
purify the cannabinoids:
tetrahydrocannabinol (THC), tetrahydrocannabidivarin (THCV),
tetrahydrocannabinolic acid (THCA),
tetrahydrocannabidivarinic acid (THCVA), cannabidiol (CBD), cannabidivarin
(CBDV), cannabidiolic acid
(CBDA), cannabidivarinic acid (CBDVA), cannabinol (CBN), cannabivarin (CBV),
cannabigerovarin
(CBGV), cannabigerol (CBG), cannabigerovarinic acid (CBGVA) and cannabigerol
acid (CBGA).
Crystallization of Cannabinoids
[38] 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 which extracts
the one or more cannabinoids from a 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) in a manner that concentrates
the one or more cannabinoids; c) incubating the reduced first solvent mixture
in a manner that crystalizes
the one or more cannabinoids; d) incubating the one or more crystalized
cannabinoids with a second non-
polar solvent to form a second solvent mixture; and e) incubating the second
solvent mixture in a manner
that crystalizes the one or more cannabinoids, thereby resulting in the
purification of one or more
cannabinoids. The disclosed methods further provide that the one or more
crystalized cannabinoids of step
(c) may be purified prior to step (d), using, e.g., filtration that results in
a collection of a mother liquor. The
mother liquor may be collected and incubated in a manner that crystalizes 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 one or more cannabinoids is 95% or more.
[39] 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 which extracts
the one or more cannabinoids from a 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) in a manner that concentrates the one or more cannabinoids; d)
incubating the reduced first solvent
mixture in a manner that crystalizes the one or more cannabinoids; e)
purifying the one or more crystalized
cannabinoids in step (d) using filtration that results in a collection of a
mother liquor; 0 incubating the one
or more crystalized 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
crystalized cannabinoids; g)
incubating the second solvent mixture in a manner that crystalizes the one or
more cannabinoids; and h)
purifying the one or more crystalized cannabinoids of step (g) using
filtration that results in a collection of a
mother liquor, thereby resulting in the purification of one or more
cannabinoids. The disclosed methods
may further comprise: i) purifying the one or more crystalized cannabinoids
using filtration that results in a
collection of a mother liquor; and j) incubating the mother liquor in a manner
that crystalizes the one or more
cannabinoids. Step (a) may be repeated one or more times. Steps (i) and (j),
steps (f) and (g) and steps (0,
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(g) and (h) may be repeated one or more times until the purity of the one or
more cannabinoids is 95% or
more.
[40] 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 which extracts
the one or more cannabinoids from a plant material; b) filtering the first
solvent mixture; c) reducing by
evaporation, the volume of the first non-polar solvent in the filtrate
obtained in step (b); d) incubating the
reduced first solvent mixture in a manner that crystalizes the one or more
cannabinoids; e) removing the
first non-polar solvent by vacuum filtering; f) further reducing the amount of
first non-polar solvent from the
filtrate of (e) by evaporation; g) incubating the one or more crystalized
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 crystalized cannabinoids; h) incubating the second solvent
mixture in a manner that
crystalizes the one or more cannabinoids; i) removing the second non-polar
solvent by vacuum filtering and
saving the crystals obtained; and j) adding sufficient non-polar solvent per
gram of cannabinoid to dissolve
the crystals obtained in step (i) and recrystallizing.
[41] Aspects of the present specification disclose, in part, incubating the
plant material with a first non-polar
solvent to form a first solvent mixture which extracts the one or more
cannabinoids from a plant material.
The extract obtained from a plant can be obtained by maceration in a non-polar
solvent. A "non-polar
solvent" as used herein includes a liquid non-polar solvent comprising lower
05-012, or 05-08 straight chain,
or branched chain alkanes. Non-limiting examples of the non-polar solvent
include pentane, hexane,
petroleum ether (60-80 C bp), cyclohexane, heptane, chloroform, benzene,
toluene, or diethyl ether. In one
embodiment, the non-polar solvent used in any one of or all of the present
extraction steps is hexane. In
one aspect of this embodiment, at least one of the extraction and/or
purification steps for extraction of CBG
and/or CBGA is performed with hexane. In another embodiment, the non-polar
solvent used in any one of
or all of the present extraction steps is pentane or petroleum ether (40-60 C
bp). In one aspect of this
embodiment, one or more of the extraction and/or purification steps for
extraction/purification of CBD is
performed with pentane or petroleum ether (40-60 C bp). In another embodiment,
the non-polar solvent
used in any one of or all of the present extraction steps is heptane. In one
aspect of this embodiment, one
or more of the extraction and/or purification steps for
extraction/purification of THCA is performed with
heptane.
[42] Besides the particular non-polar solvent, extraction of the one or more
cannabinoids from a plant
material is a function of temperature, time and number of extraction steps. In
aspects of this embodiment,
incubating the plant material with a non-polar solvent occurs for a time
period of, e.g., at least 5 minutes, at
least 10 minutes, at least 15 minutes, for at least 30 minutes, for at least
45 minutes, for at least 1 hour, for
at least 1.25 hours, for at least 1.5 hours, for at least 1.75 hours, for at
least 2 hours, for at least 2.25 hours,
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for at least 2.5 hours, for at least 2.75 hours, for at least 3.0 hours, for
at least 3.25 hours, for at least 4.5
hours, for at least 4.75 hours, or for at least 5.0 hours. In other aspects of
this embodiment, incubating the
plant material with a non-polar solvent occurs for a time period of, e.g., at
most 5 hours, for at most 4.75
hours, for at most 4.5 hours, for at most 4.25 hours, for at most 4.0 hours,
for at most 3.75 hours, for at
most 3.5 hours, for at most 3.25 hours, for at most 3.0 hours, for at most
2.75 hours, for at most 2.5 hours,
for at most 2.25 hours, for at most 2.0 hours, for at most 1.75 hours, for at
most 1.5 hours, for at most 1.25
hours, for at most 1.25 hours, for at most 1.0 hours, for at most 45 minutes,
for at most 30 minutes, or for
at most 15 minutes. In yet other aspects of this embodiment, incubating the
plant material with a non-polar
solvent occurs for a time period of, e.g., about 15 minutes to about 5 hours,
about 30 minutes to about 5
hours, about 45 minutes to about 5 hours, about 1 hour to about 5 hours, about
1 hour to about 4 hours,
about 1 hour to about 3.5 hours, about 1 hour to about 3.0 hours, about 1 hour
to about 2.25 hours, about
1 hour to about 2 hours, about 1 hour to about 1.75 hours, about 1 hour to
about 1.5 hours, about 30
minutes to about 1.5 hours, about 30 minutes to about 1.25 hours, about 30
minutes to about 1 hour, about
45 minutes to about 1.75 hours, about 45 minutes to about 1.5 hours, about 45
minutes to about 1.25 hours,
or about 45 minutes to about 1 hour.
[43] In aspects of this embodiment, incubating the plant material with a non-
polar solvent occurs at a
temperature of, e.g., 0 C or higher, 4 C or higher, 8 C or higher, 12 C or
higher, 16 C or higher, 20 C or
higher or 24 C or higher, 28 C or higher, 32 C or higher, 36 C or higher, 40 C
or higher, 44 C or higher,
48 C or higher, 52 C or higher, 56 C or higher or 60 C or higher. In other
aspects of this embodiment,
incubating the plant material with a non-polar solvent occurs at a temperature
of, e.g., 0 C or lower, 4 C or
lower, 8 C or lower, 12 C or lower, 16 C or lower, 20 C or lower, 24 C or
lower, 28 C or lower, 32 C or
lower, 36 C or lower, 40 C or lower, 44 C or lower, 48 C or lower, 52 C or
lower, 56 C or lower or 60 C or
lower. In other aspects of this embodiment, incubating the plant material with
a non-polar solvent occurs
at a temperature of, e.g., about 0 C to about 4 C, about 0 C to about 8 C,
about 0 C to about 12 C, about
0 C to about 16 C, about 0 C to about 20 C, about 0 C to about 24 C, about 0 C
to about 28 C, about 0 C
to about 32 C, about 0 C to about 36 C, about 0 C to about 40 C, about 0 C to
about 44 C, about 0 C to
about 48 C, about 0 C to about 52 C, about 0 C to about 56 C, about 0 C to
about 60 C, about 4 C to
about 8 C, about 4 C to about 12 C about 4 C to about 16 C, about 4 C to about
20 C, about 4 C to about
24 C, about 4 C to about 28 C, about 4 C to about 32 C, about 4 C to about 36
C, about 4 C to about
40 C, about 4 C to about 44 C, about 4 C to about 48 C, about 4 C to about 52
C, about 4 C to about
56 C, about 4 C to about 60 C, about 8 C to about 12 C, about 8 C to about 16
C, about 8 C to about
20 C, about 8 C to about 24 C, about 8 C to about 28 C, about 8 C to about 32
C, about 8 C to about
36 C, about 8 C to about 40 C, about 8 C to about 44 C, about 8 C to about 48
C, about 8 C to about
52 C, about 8 C to about 56 C, about 8 C to about 60 C,about 12 C to about 16
C, about 12 C to about
20 C, about 12 C to about 24 C, about 12 C to about 28 C, about 12 C to about
32 C, about 12 C to about
36 C, about 12 C to about 40 C, about 12 C to about 44 C, about 12 C to about
48 C, about 12 C to about
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52 C, about 12 C to about 56 C, about 12 C to about 60 C, about 16 C to about
20 C, about 16 C to about
24 C, about 16 C to about 28 C, about 16 C to about 32 C, about 16 C to about
36 C, about 16 C to about
40 C, about 16 C to about 44 C, about 16 C to about 48 C, about 16 C to about
52 C, about 16 C to about
56 C, about 16 C to about 60 C, about 20 C to about 24 C, about 20 C to about
28 C, about 20 C to about
32 C, about 20 C to about 36 C, about 20 C to about 40 C, about 20 C to about
44 C, about 20 C to about
48 C, about 20 C to about 52 C, about 20 C to about 56 C, about 20 C to about
60 C, about 24 C to about
28 C, about 24 C to about 32 C, about 24 C to about 36 C, about 24 C to about
40 C, about 24 C to about
44 C, about 24 C to about 48 C, about 24 C to about 52 C, about 24 C to about
56 C, about 24 C to about
60 C, about 28 C to about 32 C, about 28 C to about 36 C, about 28 C to about
40 C, about 28 C to about
44 C, about 28 C to about 48 C, about 28 C to about 52 C, about 28 C to about
56 C, about 28 C to about
60 C, about 32 C to about 36 C, about 32 C to about 40 C, about 32 C to about
44 C, about 32 C to about
48 C, about 32 C to about 52 C, about 32 C to about 56 C, about 32 C to about
60 C, about 36 C to about
40 C, about 36 C to about 44 C, about 36 C to about 48 C, about 36 C to about
52 C, about 36 C to about
56 C, about 36 C to about 60 C, about 40 C to about 44 C, about 40 C to about
48 C, about 40 C to about
52 C, about 40 C to about 56 C, about 40 C to about 60 C, about 44 C to about
48 C, about 44 C to about
52 C, about 44 C to about 56 C, about 44 C to about 60 C , about 48 C to about
52 C, about 48 C to about
56 C, about 48 C to about 60 C, about 52 C to about 56 C, about 52 C to about
60 C or about 52 C to
about 60 C.
[44] Aspects of the present specification disclose, in part, purifying the
solvent mixture. In an aspect of this
embodiment, the solvent mixture is purified by filtration.
[45] Aspects of the present specification disclose, in part, reducing the
volume of the solvent mixture in a
manner that concentrates the one or more cannabinoids, by at least 50% of the
original volume to dryness.
In aspects of this embodiment, the volume of the first solvent mixture is
reduced by evaporation. In aspects
of this embodiment, the volume of the first solvent mixture is reduced by,
e.g., 60% or less, 50% or less,
45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less,
15% or less, 10% or less,
5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the original
volume of the first solvent
mixture used to extract the one or more cannabinoids from a plant material. In
aspects of this embodiment,
the volume of the first solvent mixture is reduced by, e.g., about 0.1% to
about 5%, about 0.1% to about
10%, about 0.1% to about 15%, about 0.1% to about 20%, about 0.1% to about
25%, about 0.1% to about
30%, about 0.1% to about 35%, about 0.1% to about 40%, about 0.1% to about
45%, about 0.1% to about
50%, about 0.5% to about 5%, about 0.5% to about 10%, about 0.5% to about 15%,
about 0.5% to about
20%, about 0.5% to about 25%, about 0.5% to about 30%, about 0.5% to about
35%, about 0.5% to about
40%, about 0.5% to about 45%, about 0.5% to about 50%, about 1% to about 15%,
about 1% to about
20%, about 1% to about 25%, about 1% to about 30%, about 1% to about 35%,
about 1% to about 40%,
about 1% to about 45%, about 1% to about 50%, about 1% to about 55%, about 1%
to about 60%, 5% to
about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about
25%, about 5% to about
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30%, about 5% to about 35%, about 5% to about 40%, about 5% to about 45%,
about 5% to about 50%,
about 5% to about 55%, about 5% to about 60%, about 10% to about 15%, about
10% to about 20%, about
10% to about 25%, about 10% to about 30%, about 10% to about 35%, about 10% to
about 40%, about
10% to about 45%, about 10% to about 50%, about 10% to about 55%, about 10% to
about 60%, about
15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to
about 35%, about
15% to about 40%, about 15% to about 45%, about 15% to about 50%, about 15% to
about 55%, about
15% to about 60%, about 20% to about 25%, about 20% to about 30%, about 20% to
about 35%, about
20% to about 40%, about 20% to about 45%, about 20% to about 50%, about 20% to
about 55%, about
20% to about 60%, about 25% to about 30%, about 25% to about 35%, about 25% to
about 40%, about
25% to about 45%, about 25% to about 50%, about 25% to about 55%, about 25% to
about 60%, about
30% to about 35%, about 30% to about 40%, about 30% to about 45%, about 30% to
about 50%, about
30% to about 55%, about 30% to about 60%, about 35% to about 40%, about 35% to
about 45%, about
35% to about 50%, about 35% to about 55%, about 35% to about 60%, about 40% to
about 45%, about
40% to about 50%, about 40% to about 55%, about 40% to about 60%, about 45% to
about 50%, about
45% to about 55%, about 45% to about 60%, about 50% to about 55%, about 50% to
about 60% or about
55% to about 60%.
[46] Aspects of the present specification disclose, in part, incubating the
reduced solvent mixture in a
manner that crystalizes one or more cannabinoids. Generally, crystallization
of the one or more
cannabinoids in the reduced first solvent mixture is a function of temperature
and time. In aspects of this
embodiment, the reduced first solvent mixture is incubated at a temperature
of, e.g., -70 C or higher, -60 C
or higher, -50 C or higher, -40 C or higher, -30 C or higher, -20 C or higher
or 0 C or higher, 4 C or higher,
8 C or higher, 12 C or higher, 16 C or higher, 20 C or higher, 24 C or higher
or 28 C or higher. In other
aspects of this embodiment, the reduced first solvent mixture is incubated at
a temperature of, e.g., -70 C
or lower, -60 C or lower, -50 C or lower, -40 C or lower, -30 C or lower, -20
C or lower or 0 C or higher,
4 C or lower, 8 C or lower, 12 C or lower, 16 C or lower, 20 C or lower, 24 C
or lower or 28 C or lower. In
yet other aspects of this embodiment, the reduced first solvent mixture is
incubated at a temperature of,
e.g., about -70 C to about 40 C, -70 C to about 30 C, -70 C to about 20 C, -70
C to about 10 C, -70 C to
about 0 C, -20 C to about 40 C, -20 C to about 30 C, -20 C to about 20 C, -20
C to about 10 C, -20 C to
about 0 C, about 0 C to about 5 C, about 0 C to about 10 C, about 0 C to about
15 C, about 0 C to about
20 C, about 0 C to about 25 C, about 0 C to about 4 C, about 0 C to about 8 C,
about 0 C to about 12 C,
about 0 C to about 16 C, about 0 C to about 20 C, about 0 C to about 24 C,
about 0 C to about 28 C,
about 4 C to about 8 C, about 4 C to about 12 C about 4 C to about 16 C, about
4 C to about 20 C, about
4 C to about 24 C, about 4 C to about 28 C, about 8 C to about 12 C, about 8 C
to about 16 C, about 8 C
to about 20 C, about 8 C to about 24 C, about 8 C to about 28 C, about 12 C to
about 16 C, about 12 C
to about 20 C, about 12 C to about 24 C, about 12 C to about 28 C, about 16 C
to about 20 C, about 16 C
to about 24 C, about 16 C to about 28 C, about 20 C to about 24 C, about 20 C
to about 28 C or about
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24 C to about 28 C.
[47] In aspects of this embodiment, the reduced first solvent mixture is
incubated for a time period of, e.g.,
1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or
more, 6 hours or more, 7
hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 12 hours or
more, 14 hours or more,
16 hours or more, 18 hours or more, 20 hours or more, 22 hours or more, 24
hours or more, 28 hours or
more, 32 hours or more, 36 hours or more, 40 hours or more, 44 hours or more,
48 hours or more, 52 hours
or more, 56 hours or more, 60 hours or more, 64 hours or more, 68 hours or
more, 72 hours or more, 76
hours or more, 80 hours or more, 84 hours or more, 88 hours or more, 92 hours
or more or 96 hours or
more. In other aspects of this embodiment, the reduced first solvent mixture
is incubated for a time period
of, e.g., 1 hour or less, 2 hours or less, 3 hours or less, 4 hours or less, 5
hours or less, 6 hours or less, 7
hours or less, 8 hours or less, 9 hours or less, 10 hours or less, 12 hours or
less, 14 hours or less, 16 hours
or less, 18 hours or less, 20 hours or less, 22 hours or less, 24 hours or
less, 28 hours or less, 32 hours or
less, 36 hours or less, 40 hours or less, 44 hours or less, 48 hours or less,
52 hours or less, 56 hours or
less, 60 hours or less, 64 hours or less, 68 hours or less, 72 hours or less,
76 hours or less, 80 hours or
less, 84 hours or less, 88 hours or less, 92 hours or less or 96 hours or
less. In yet other aspects of this
embodiment, the reduced first solvent mixture is incubated for a time period
of, e.g., about 1 hour to about
12 hours, about 1 hour to about 24 hours, about 1 hour to about 36 hours,
about 1 hour to about 48 hours,
about 1 hour to about 60 hours, about 1 hour to about 72 hours, about 1 hour
to about 84 hours, about 1
hour to about 96 hours, about 2 hours to about 12 hours, about 2 hours to
about 24 hours, about 2 hours
to about 36 hours, about 2 hours to about 48 hours, about 2 hours to about 60
hours, about 2 hours to
about 72 hours, about 2 hours to about 84 hours, about 2 hours to about 96
hours, about 4 hours to about
12 hours, about 4 hours to about 24 hours, about 4 hours to about 36 hours,
about 4 hours to about 48
hours, about 4 hours to about 60 hours, about 4 hours to about 72 hours, about
4 hours to about 84 hours,
about 4 hours to about 96 hours, about 6 hours to about 12 hours, about 6
hours to about 24 hours, about
6 hours to about 36 hours, about 6 hours to about 48 hours, about 6 hours to
about 60 hours, about 6 hours
to about 72 hours, about 6 hours to about 84 hours, about 6 hours to about 96
hours, about 8 hours to
about 12 hours, about 8 hours to about 24 hours, about 8 hours to about 36
hours, about 8 hours to about
48 hours, about 8 hours to about 60 hours, about 8 hours to about 72 hours,
about 8 hours to about 84
hours, about 8 hours to about 96 hours, about 12 hours to about 24 hours,
about 12 hours to about 36
hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours,
about 12 hours to about 72
hours, about 12 hours to about 84 hours, about 12 hours to about 96 hours,
about 16 hours to about 24
hours, about 16 hours to about 36 hours, about 16 hours to about 48 hours,
about 16 hours to about 60
hours, about 16 hours to about 72 hours, about 16 hours to about 84 hours,
about 16 hours to about 96
hours, about 24 hours to about 36 hours, about 24 hours to about 48 hours,
about 24 hours to about 60
hours, about 24 hours to about 72 hours, about 24 hours to about 84 hours,
about 24 hours to about 96
hours, about 36 hours to about 48 hours, about 36 hours to about 60 hours,
about 36 hours to about 72
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hours, about 36 hours to about 84 hours, about 36 hours to about 96 hours,
about 48 hours to about 60
hours, about 48 hours to about 72 hours, about 48 hours to about 84 hours,
about 48 hours to about 96
hours or about 72 hours to about 96 hours.
[48] Aspects of the present specification disclose, in part, purifying the one
or more cannabinoids which
are crystalized after incubation in the reduced solvent mixture. In an aspect
of this embodiment, purification
of the one or more crystalized cannabinoids is performed using filtration that
results in a collection of a
mother liquor.
[49] Aspects of the present specification disclose, in part, incubating the
one or more crystalized
cannabinoids with a second non-polar solvent to form a second solvent mixture.
Incubation of the one or
more crystalized cannabinoids with a second non-polar solvent to form a second
solvent mixture at least
partially dissolves the one or more crystalized cannabinoids. In aspects of
this embodiment, incubation of
the one or more crystalized cannabinoids with a second non-polar solvent to
form a second solvent mixture
dissolves, e.g., at least 50%, at least 55%, at least 60%, at least 65%, at
least 70%, at least 75%, at least
80%, at least 85%, at least 90% or at least 95% of the one or more crystalized
cannabinoids. In other
aspects of this embodiment, incubation of the one or more crystalized
cannabinoids with a second non-
polar solvent to form a second solvent mixture dissolves, e.g., at most 50%,
at most 55%, at most 60%, at
most 65%, at most 70%, at most 75%, at most 80%, at most 85%, at most 90% or
at most 95% of the one
or more crystalized cannabinoids. In yet other aspects of this embodiment,
incubation of the one or more
crystalized cannabinoids with a second non-polar solvent to form a second
solvent mixture dissolves, e.g.,
about 50% to about 95%, about 55% to about 95%, about 60% to about 95%, about
65% to about 95%,
about 70% to about 95%, about 75% to about 95%, about 80% to about 95%, about
85% to about 95%,
about 90% to about 95%, about 50% to 100%, about 55% to 100%, about 60% to
100%, about 65% to
100%, about 70% to 100%, about 75% to 100%, about 80% to 100%, about 85% to
100%, about 90% to
100% or about 95% to 100%.
[50] Aspects of the present specification disclose, in part, purifying the one
or more crystalized
cannabinoids obtained from a second solvent mixture, as described above for
the solvent mixture. In an
aspect of this embodiment, the one or more crystalized cannabinoids is
purified using filtration that results
in a collection of a mother liquor.
[51] The disclosed methods may further comprise, incubating the mother liquor
in a manner that crystalizes
the one or more cannabinoids. The one or more cannabinoids can be crystalized
using the same
temperature and time conditions used to crystalizes the one or more
cannabinoids from the reduced solvent
mixture described above.
[52] The result of the disclosed methods is a substantially pure preparation
of one or more cannabinoids.
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A "substantially pure" preparation of a cannabinoid or a cannabinoid acid is
defined as a preparation having
a chromatographic purity (of the desired cannabinoid or cannabinoid acid) of
90% or greater, 91% or
greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96%
or greater, 97% or greater,
98% or greater or 99% or greater as determined by area normalisation of an
HPLC profile or by a
quantification percent of purity respect a certified commercial standard.
[53] In an aspect of this embodiment, the disclosed methods result in the
purification of CBGA having a
purity that is 90% or greater, 91% or greater, 92% or greater, 93% or greater,
94% or greater, 95% or
greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater as
determined by area
normalisation of an HPLC profile or by a quantification percent of purity
respect a certified commercial
standard. In an aspect of this embodiment, the disclosed methods result in the
purification of CBG having
a purity that is 90% or greater, 91% or greater, 92% or greater, 93% or
greater, 94% or greater, 95% or
greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater as
determined by area
normalisation of an HPLC profile or by a quantification percent of purity
respect a certified commercial
standard. In an aspect of this embodiment, the disclosed methods result in the
purification of CBD having
a purity that is 90% or greater, 91% or greater, 92% or greater, 93% or
greater, 94% or greater, 95% or
greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater as
determined by area
normalisation of an HPLC profile or by a quantification percent of purity
respect a certified commercial
standard.
[54] The term "crude cannabinoid", "raw cannabinoid" or "product enriched in a
given cannabinoid"
encompasses preparations having at least 60%, at least 65%, at least 70%, at
least 75%, at least 80%, at
least 85%, or at least 90% chromatographic purity for the desired cannabinoid.
Such a product will generally
contain a greater proportion of impurities, non-target materials and other
cannabinoids than a "substantially
pure" preparation.
Cannabinoids
[55] The cannabinoids purified by the disclosed methods are not particularly
limited and include
cannibigerol-type (CBG-type) cannabinoids; cannaibichromene-type cannabinoids
(CBC-type);
cannabidiol-type cannabinoids (CBD-type); tetrahydracannabinol-type
cannabinoids (THC-type);
cannabinol-type cannabinoids (CBN-type); and derivatives thereof. The
cannabinoid derivatives may not
themselves be cannabinoids. However, their chemistry is recognized as being
derived from cannabigerol,
cannabinol, or cannabidiol. For instance, cannabinoids of interest include the
following and their
corresponding acids: CBG (Cannabigerol), CBC (Cannabichromene), CBL
(Cannabicyclol), CBV
(Cannabivarin), THCV (Tetrahydrocannabivarin), CBDV (Cannabidivarin), CBCV
(Cannabichromevarin),
CBGV (Cannabigerovarin), CBGM (Cannabigerol Monomethyl Ether), THC
(tetrahydrocannabinol), CBT
(Cannabicitran-type), Iso-THC (Iso-Tetrahydrocannabinol-type) and CBE
(Cannabielsoin-type). In fresh
plant material of Cannabis, most cannabinoids are present in the form
carboxylic acid known as acidic
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cannabinoids or "cannabinoid acids". The free phenolic forms of the
cannabinoids are also known as neutral
cannabinoids.
[56] The disclosed methods may be used to extract/purify cannabinoids or
cannabinoid acids from any
plant material known to contain such cannabinoids or cannabinoid acids. The
source for the cannabinoids
is not limited, but can include plant material. The term "plant material"
encompasses a plant or plant part
(e.g. bark, wood, leaves, stems, roots, flowers, fruits, seeds, berries or
parts thereof) as well as exudates,
resins, and plant extracts, and includes material falling within the
definition of "botanical raw material" in the
Guidance for Industry Botanical Drug Products Draft Guidance, August 2000, US
Department of Health and
Human Services, Food and Drug Administration Centre for Drug Evaluation and
Research.
[57] The disclosed methods may be used to extract/purify cannabinoids or
cannabinoid acids from any
plant material known to contain such cannabinoids or cannabinoid acids. Most
typically, but not necessarily,
the "plant material" will be derived from one or more cannabis plants. Plants
from which cannabinoids may
be isolated include: Cannabis sp. including Cannabis sativa L. and all
subspecies, the putative species
Cannabis indica Lam., Cannabis ruderalis Janisch, and hybrids and varieties
thereof, as discussed further
below. The Cannabis sativa L. plant can be of the variety Carma, Aida,
Octavia, Juani or any other variety
of the chemotype IV, whose main cannabinoid is CBG or CBGA (Meijer EP, Hammond
KM. The inheritance
of chemical phenotype in Cannabis sativa L. (II): Cannabigerol predominant
plants. Euphytica. 2005. 145:
189-198.) or from any variety belonging to the chemotype ll or III, whose main
cannabinoid is CBD or
CBDA or even from a variety from the chemotype I, whose main cannabinoid is
THC or THCA (de Meijer
EP, Bagatta M, Carboni A, Crucitti P, Moliterni VM, Ranalli P, Mandolino G.
The inheritance of chemical
phenotype in Cannabis sativa L. Genetics. 2003. Jan; 163(1):335-46.)
[58] In one embodiment, the disclosed methods use material from the plant
Cannabis sativa L. variety
belonging to chemotype IV, having CBGA/CBG as main cannabinoids. In another
embodiment, the
disclosed methods use material from the plant Cannabis sativa L. variety
belonging to chemotype III, having
CBDA/CBD as main cannabinoids. In another embodiment, the disclosed methods
use material from the
plant Cannabis sativa L. variety belonging to chemotype II, having THCA-
CBDA/THC-CBD as main
cannabinoids. In yet another embodiment, the disclosed methods use material
from the plant Cannabis
sativa L. variety belonging to chemotype I, having THCA/THC as the main
cannabinoids.
[59] The term "cannabis plant(s)" encompasses wild type Cannabis sativa and
also variants thereof,
including cannabis chemovars (varieties characterised by virtue of chemical
composition) which naturally
contain different amounts of the individual cannabinoids, also Cannabis sativa
L. subspecies indica
including the variants var. indica and var. kafiristanica, Cannabis indica and
also plants which are the result
of genetic crosses, self-crosses or hybrids thereof. The term "cannabis plant
material" is to be interpreted
accordingly as encompassing plant material derived from one or more cannabis
plants. For the avoidance
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of doubt, it is hereby stated that "cannabis plant material" includes herbal
cannabis and dried cannabis
biomass.
[60] "Decarboxylated cannabis plant material" refers to cannabis plant
material which has been subject to
a decarboxylation step in order to convert cannabinoid acids to the
corresponding free cannabinoids.
Resins and Extracts
[61] "Resin" as used herein includes resins produced from any of the plant
types discussed above, and in
one embodiment, includes products of the stalked resin glands of Cannabis sp.,
including the putative
species Cannabis indica, the species Cannabis sativa and Cannabis ruderalis,
and hybrids or varietals
thereof. These stalked resin glands may be from female, unfertilized or
fertilized plants or from dioecious
or monoecious varieties of Cannabis.
[62] The method of the invention makes it possible to isolate the cannabinoids
of interest (e.g., CBG, CBGA,
CBGVA, CBD, CBDA, CBDVA, THC, THCA or THCVA) by crystallization with a non-
polar solvent (e.g.,
hexane, pentane, heptane or petroleum ethers), from the plant, resin or the
extracts obtained from the plant,
wherein the crystallization is before or after a liquid liquid chromatography
step. In some cases, the extract
of the resin or plant is first obtained by extracting with pentane, hexane,
heptane, petroleum ethers,
cyclohexane, dichloromethane, trichloromethane, tetrahydrofurane, diethyl
ether, toluene, benzene,
ethanol, methanol, isopropanol, acetone, acetonitrile, ethyl acetate, butane,
propane, refrigerant gases
(e.g.: 1,1,1,2-Tetrafluoroethane (R134a)) or, liquid, subcritical or
supercritical CO2 or mixes of these
solvents. In this embodiment, the disclosed method obtains the cannabinoids of
interest (e.g., CBG, CBGA,
CBGVA, CBD, CBDA, CBDVA, THC, THCA or THCVA) with a purity of 60% to 96%,
which will be called
"raw" with a high yield and further with a purity of at least 60%, at least
61%, at least 62%, at least 63%, at
least 64% at least 65%, at least 66%, at least 67%, at least 68%, at least
69%, least 70%, at least 71%, at
least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least
77%, at least 78%, at least 79%,
at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least
85%, at least 86%, at least
87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at
least 94% or at least 95% (the yield ranges between 50%-90% depending on the
type of plant raw material
or the type of extract). With subsequent recrystallizations of this "raw"
composition in a non-polar solvent
(e.g., hexane, pentane, heptane or petroleum ethers), it is possible to obtain
a purity greater than 90%,
greater than 91%, greater than 92%, greater than 93%, greater than 94%,
greater than 95%, greater than
96%, greater than 97%, greater than 98%, greater than 99%, of e.g., CBG, CBGA,
CBGVA, CBD, CBDA,
CBDVA, THC, THCA or THCVA.
[63] The non-polar solvent used to obtain an extract is not particularly
limited, the method of the invention
offers good results with extracts obtained with any of pentane, hexane,
heptane, cyclohexane, petroleum
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ethers, dichloromethane, trichloromethane, tethrahydrofurane, toluene,
benzene, diethyl ether, ethanol,
methanol, isopropanol, acetone, acetonitrile, ethyl acetate, butane, propane,
refrigerant gases (e.g.:
1,1,1,2-Tetrafluoroethane (RI 34a)) and liquid, subcritical or supercritical
CO2 or mixes of these solvents.
Isolation of Cannabinoid Acids
[64] In embodiments where the method is to be used for the isolation of
cannabinoid acids, an acidified
extraction solvent to prepare the initial extract may optionally be used to
ensure the extraction of high levels
of cannabinoid acids. The primary purpose of this acidification is to
prevent/minimise ionisation of the
cannabinoid acid, which could otherwise adversely affect the purification
process. In one embodiment, the
method uses acidified non-polar solvents, of the types described above.
Acidification may be achieved by
the addition of a small volume of acid to the solvent. Generally, it is
sufficient to add a relatively weak acid,
such as acetic acid. For any given purification process the optimal amount and
type of acid used may be
determined empirically. An example of an acidified solvent is 0.1% acetic acid
in hexane. Other solvents
include pentane, hexane, heptane, cyclohexane, petroleum ethers,
dichloromethane, trichloromethane,
tethrahydrofurane, diethyl ether, ethanol, methanol, isopropanol, acetone,
acetonitrile, ethyl acetate,
butane, propane, refrigerant gas 1,1,1,2-Tetrafluoroethane (RI 34a), liquid
002, subcritical CO2 or
supercritical CO2 or mixes of these solvents This is the extraction solvent of
choice for preparing an initial
extract from the starting plant material in the preparation of cannabinoid
acids.
Isolation of Cannabiderol, Cannabidiol or Tetrahvdrocannabinol-Prior
Decarboxvlation
[65] In embodiments of the method where it is desired to purify free
cannabinoids, rather than the
cannabinoid acids, the plant material may be subjected to a decarboxylation
step. The purpose of the
decarboxylation step is to convert cannabinoid acids present in the plant
material to the corresponding free
cannabinoids. Decarboxylation may be carried out by heating the plant material
to a defined temperature
for a suitable length of time. Decarboxylation of cannabinoid acids is a
function of time and temperature,
thus at higher temperatures a shorter period of time will be taken for
complete decarboxylation of a given
amount of cannabinoid acid. In selecting appropriate conditions for
decarboxylation consideration must,
however, be given to minimizing thermal degradation of the desirable,
pharmacological cannabinoids into
undesirable degradation products, particularly thermal degradation of ,6,9 THC
to cannabinol (CBN).
[66] Thus, in another embodiment of the present methods, cannabinoids, e.g.,
CBG, CBGA, CBGV, CBD,
CBDA, CBDV, THC, THCA or THCV are isolated and purified, and in which prior to
performing step (a), the
plant material, resin or extracts from the plant are decarboxylated for at
least about 1 hour, 1.1 hours, 1.2
hour, 1.3 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9
hours, 2 hours, 2.1 hours, 2.2
hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7 hours, 2.8 hours, 2.9
hours, 3 hours, 3.1 hours, 3.2
hours, 3.3 hours, 3.4 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5
hours, 6 hours, 6.5 hours, 7 hours,
7.5 hours, 8 hours at around 60 C, 65 C, 70 C, 75 C, 80 C, 85 C, 90 C, 95 C,
100 C, 105 C, 110 C, 111 C,
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112 C, 113 C, 114 C, 115 C, 116 C, 117 C, 118 C, 119 C, 120 C, 121 C, 122 C,
123 C, 124 C, 125 C,
126 C, 127 C, 128 C, 129 C or 130 C, 135 C, 140 C, 145 C, 150 C, 155 C, 160 C,
165 C, 170 C, 175 C,
or 180 C. In one embodiment, the decarboxylation is performed for at least 2
hours at a temperature of
120 C. In one embodiment, the decarboxylation is performed for at least 1
hours at a temperature of 150 C.
[67] In one embodiment, the decarboxylation is performed at a temperature of
at least 60 C, 65 C, 70 C,
75 C, 80 C, 85 C, 90 C, 95 C, 100 C, 105 C, 110 C, 115 C, 120 C, 125 C, 130 C,
135 C, 140 C, 145 C,
150 C, 155 C, 160 C, 165 C, 170 C, 175 C, or 180 C. In one embodiment, the
decarboxylation is
performed at a temperature of at most 175 C, 170 C, 165 C, 160 C, 155 C, 150
C, 145 C, 140 C, 135 C,
130 C, 125 C, 120 C, 115 C, 110 C, 100 C, 95 C, 90 C, 85 C, 80 C, 75 C, 70 C,
65 C, or 60 C. In one
embodiment, the decarboxylation is performed at a temperature ranging from 60
C to 180 C, ranging from
70 C to 175 C, 75 C to 170 C, 80 C to 165 C, 85 C to 160 C, 90 C to 155 C, 95
C to 150 C, 100 C to
145 C, 105 C to 140 C, 110 C to 135 C, 115 C to 130 C, or 120 C to 130 C.
[68] Another embodiment is the method, wherein cannabigerol (CBG), cannabidiol
(CBD), cannabidivarin
(CBDV), tetrahydrocannabinol (THC), tetrahydrocannabidivarin (THCV),
cannabinol (CBN), cannabivarin
(CBV) and/or cannabigerovarin (CBGV) are isolated and purified, and in which
prior to performing step (a),
the plant material or resin of said plant are decarboxylated at least at 120
C for 2 hours.
[69] Another embodiment is the method, wherein step (a) is repeated at least
once. In one embodiment,
step (a) is repeated 2 times or 3 times. Another embodiment is the method,
wherein time in step (a) is at
least about 60 minutes.
[70] Another embodiment is the method, wherein step (i) is repeated at least
once. In one embodiment,
step (i) is repeated 2 times or 3 times.
Characterization of Resultant Product
[71] In one embodiment, the present methods obtain a substantially pure
cannabinoid product. A
"substantially pure" preparation of a cannabinoid or a cannabinoid acid is
defined as a preparation having
a chromatographic purity (of the desired cannabinoid or cannabinoid acid) of
greater than 90%, greater
than 91%, greater than 92%, greater than 93%, greater than 94%, greater than
95%, greater than 96%,
greater than 97%, greater than 98%, greater than 99% and greater than 99.5%,
as determined by area
normalisation of an HPLC profile or by quantification by HPLC with a certified
commercial standard.
[72] Purity of CBG, CBGA, THC, THCA, THCV, CBDA, CBDV and CBD are expressed as
HPLC
quantification with certified commercial standard from Sigma-Aldrich. To
evaluate the purity of CBGV we
used the relative area by GCMS instrument, using the EIC (extractor ion
chromatography) chromatogram
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to guarantee the correct measurement.
[73] The HPLC conditions used to test the cannabinoid purity where the
following: Column: Mediterranean
Sea, C18, 3pm size particle, 250mm x 4.6mm; Mobil phase: Water and Methanol
with formiate ammonium;
Det.: DAD, 210nm (CBG and CBD) and 270nm (CBGA); Inj.: 10pL; Oven: 34 C.
Products obtained by Methods
[74] The present methods obtain a composition which includes a substantially
pure cannabinoid or
cannabinoid acid in liquid or solid form. For instance, the final product may
be applied while in its crystalline
form or may be further dissolved or formulated into a liquid, powder or
compressed tablet. In one
embodiment, the present methods obtain a crystalline cannabinoid in powder
form. In another embodiment,
the present methods obtain a cannabinoid solution.
[75] The product obtained herein may be incorporated or formulated into
products suitable for
pharmaceutical purposes, recreational ingestion (e.g., food supplements,
nutriceuticals), or as recreational
inhalants (e.g., cigarettes and/or oils or liquids for electronic
cigarettes/vape/hookah products, or incense).
[76] Of course, working with cannabis plants and cannabinoids may require a
government license or
approval in some territories, but may often be obtained for medicinal
purposes. That said, the present
methods do not exclude the use of the product as a non-medicinal product, with
the appropriate government
approvals.
Pharmaceutical product
[77] The present methods in one embodiment produce a product which may be
included in a
pharmaceutical product, medicinal preparation, or medicament (hereinafter
"pharmaceuticals"). Such
pharmaceutical products may be formulated as liquids, tablets, capsules,
microcapsules, nanocapsules,
trans-dermal patches, gels, foams, oils, aerosols, nanoparticulates, powders,
creams, emulsions, micellar
systems, films, sprays, ovules, infusions, teas, decoctions, suppositories,
etc.
[78] Products obtained by the present methods may be included in a
pharmaceutical composition including
a compound of the present product or a pharmaceutically acceptable salt or
solvate thereof, together with
a pharmaceutically acceptable excipient. In an aspect of this embodiment, a
pharmaceutical composition
comprises CBGA, CBG, CBGV, CBDA, CBD, CBDV, THCA, THC, THCV or any combination
thereof.
[79] The term "excipient" is used herein to describe any ingredient other than
the compound of the
invention. The choice of excipient will to a large extent depend on factors
such as the particular mode of
administration, the effect of the excipient on solubility and Pharmaceutical
compositions suitable for the
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delivery of compounds of the present invention and methods for their
preparation will be readily apparent
to those skilled in the art. Such compositions and methods for their
preparation may be found, for example,
in "Remington's Pharmaceutical Sciences", 19th Edition (Mack Publishing
Company, 1995).
[80] The compounds of the invention may be administered orally. Oral
administration may involve
swallowing, so that the compound enters the gastrointestinal tract, or buccal
or sublingual administration
may be employed by which the compound enters the blood stream directly from
the mouth. Formulations
suitable for oral administration include both solid and liquid formulations.
[81] Solid formulations include tablets, capsules (containing particulates,
liquids, microcapsules, or
powders), lozenges (including liquid-filled lozenges), chews, multi- and nano-
particulates, gels, solid
solutions, liposomal preparations, microencapsulated preparations, creams,
films, ovules, suppositories
and sprays.
[82] Liquid formulations include suspensions, solutions, syrups and elixirs.
Such formulations may be
employed as fillers in soft or hard capsules and typically comprise a carrier,
for example, water, ethanol,
polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and
one or more emulsifying agents
and/or suspending agents. Liquid formulations may also be prepared by the
reconstitution of a solid, for
example, from a sachet.
[83] The compounds of the invention may also be used in fast-dissolving, fast-
disintegrating dosage forms
such as those described in Expert Opinion in Therapeutic patents, 11(6), 981-
986, by Liang and Chen
(2001).
[84] For tablet dosage forms, depending on dose, the drug may make up from 1
weight '3/0 to 80 weight '3/0
of the dosage form, more typically from 5 weight '3/0 to 60 weight '3/0 of the
dosage form.
[85] In addition to the drug, tablets generally contain a disintegrant.
Examples of disintegrants include
sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl
cellulose, croscarmellose
sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline
cellulose, lower alkyl-
substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium
alginate. Generally, the
disintegrant will comprise from 1 weight '3/0 to 25 weight '3/0 or from 5
weight '3/0 to 20 weight '3/0 of the dosage
form.
[86] Binders are generally used to impart cohesive qualities to a tablet
formulation. Suitable binders include
microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and
synthetic gums,
polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and
hydroxypropyl methylcellulose.
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[87] Tablets may also contain diluents, such as lactose (monohydrate, spray-
dried monohydrate,
anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol,
microcrystalline cellulose, starch and
dibasic calcium phosphate dihydrate.
[88] Tablets may also optionally comprise surface active agents, such as
sodium lauryl sulfate and
polysorbate 80, and glidants such as silicon dioxide and talc. When present,
surface active agents may
comprise from 0.2 weight % to 5 weight % of the tablet, and glidants may
comprise from 0.2 weight % to 1
weight % of the tablet.
[89] Tablets also generally contain lubricants such as magnesium stearate,
calcium stearate, zinc stearate,
sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl
sulphate. Lubricants
generally comprise from 0.25 weight % to 10 weight /0, from 0.5 weight % to 3
weight % of the tablet.
[90] Other possible ingredients include anti-oxidants, colourants, flavouring
agents, preservatives and
taste-masking agents.
[91] Exemplary tablets contain up to about 80% drug, from about 10 weight % to
about 90 weight % binder,
from about 0 weight % to about 85 weight % diluent, from about 2 weight % to
about 10 weight %
disintegrant, and from about 0.25 weight % to about 10 weight % lubricant.
[92] Tablet blends may be compressed directly or by roller to form tablets.
Tablet blends or portions of
blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or
extruded before tabletting.
The final formulation may comprise one or more layers and may be coated or
uncoated; it may even be
encapsulated.
[93] The formulation of tablets is discussed in "Pharmaceutical Dosage Forms:
Tablets", Vol. 1, by H.
Lieberman and L. Lachman (Marcel Dekker, New York, 1980).
[94] Consumable oral films are typically pliable water-soluble or water-
swellable thin film dosage forms
which may be rapidly dissolving or mucoadhesive and typically comprise a
compound of formula (I), a film-
forming polymer, a binder, a solvent, a humectant, a plasticiser, a stabiliser
or emulsifier, a viscosity-
modifying agent and a solvent. Some components of the formulation may perform
more than one function.
The film-forming polymer may be selected from natural polysaccharides,
proteins, or synthetic hydrocolloids
and is typically present in the range 0.01 to 99 weight /0, more typically in
the range 30 to 80 weight /0.
Other possible ingredients include anti-oxidants, colorants, flavourings and
flavour enhancers,
preservatives, salivary stimulating agents, cooling agents, co-solvents
(including oils), emollients, bulking
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agents, anti-foaming agents, surfactants and taste-masking agents. Films in
accordance with the invention
are typically prepared by evaporative drying of thin aqueous films coated onto
a peelable backing support
or paper. This may be done in a drying oven or tunnel, typically a combined
coater dryer, or by freeze-
drying or vacuuming.
[95] Solid formulations for oral administration may be formulated to be
immediate and/or modified release.
Modified release formulations include delayed-, sustained-, pulsed-,
controlled-, targeted and programmed
release.
[96] Suitable modified release formulations for the purposes of the invention
are described in U.S. Pat. No.
6,106,864. Details of other suitable release technologies such as high energy
dispersions and osmotic and
coated particles are to be found in "Pharmaceutical Technology On-line",
25(2), 1-14, by Verma et al (2001).
The use of chewing gum to achieve controlled release is described in WO
00/35298.
[97] The compounds of the invention may also be administered directly into the
blood stream, into muscle,
or into an internal organ. The products obtained by the present methods can
also be administered
parenterally (for example, by subcutaneous, intravenous, intraarterial,
intrathecal, intraventricular,
intracranial, intramuscular, or intraperitoneal injection). Parenteral
formulations are typically aqueous
solutions which may contain excipients such as salts, carbohydrates and
buffering agents (in one
embodiment, to a pH of from 3 to 9), but, for some applications, they may be
more suitably formulated as
a sterile non-aqueous solution or as a dried form to be used in conjunction
with a suitable vehicle such as
sterile, pyrogen-free water.
[98]Formulations for parenteral administration may be formulated to be
immediate and/or modified release.
Modified release formulations include delayed-, sustained-, pulsed-,
controlled-, targeted and programmed
release. Thus the compound of the invention may be formulated as a solid, semi-
solid, or thixotropic liquid
for administration as an implanted depot providing modified release of the
active compound. Examples of
such formulations include drug-coated stents and poly(dl-lactic-
coglycolic)acid (PGLA) microspheres.
[99]The compounds obtained by the present methods may also be administered
topically to the skin or
mucosa, that is, dermally or transdermally. Typical formulations for this
purpose include gels, hydrogels,
lotions, solutions, creams, ointments, dusting powders, cosmetics, oils, eye
drops, dressings, foams, films,
skin patches, wafers, implants, sponges, fibres, bandages and microemulsions.
Liposomes may also be
used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum,
white petrolatum, glycerin,
polyethylene glycol and propylene glycol. Penetration enhancers may be
incorporated--see, for example, J
Pharm Sci, 88 (10), 955-958, by Finnin and Morgan (October 1999).
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[100] Other means of topical administration include delivery by
electroporation, iontophoresis,
phonophoresis, sonophoresis and microneedle or needle-free (e.g., Powderject.
TM., Bioject.TM., etc.)
injection.
[101] Formulations for topical administration may be formulated to be
immediate and/or modified release.
Modified release formulations include delayed-, sustained-, pulsed-,
controlled-, targeted and programmed
release.
[102] The compounds of the invention may be administered rectally or
vaginally, for example, in the form
of a suppository, pessary, or enema. Cocoa butter is a traditional suppository
base, but various alternatives
may be used as appropriate.
[103] Formulations for rectal/vaginal administration may be formulated to be
immediate and/or modified
release. Modified release formulations include delayed-, sustained-, pulsed-,
controlled-, targeted and
programmed release.
[104] The compounds of the invention may be combined with soluble
macromolecular entities, such as
cyclodextrin and suitable derivatives thereof or polyethylene glycol-
containing polymers, in order to improve
their solubility, dissolution rate, taste-masking, bioavailability and/or
stability for use in any of the
aforementioned modes of administration.
[105] Drug-cyclodextrin complexes, for example, are found to be generally
useful for most dosage forms
and administration routes. Both inclusion and non-inclusion complexes may be
used. As an alternative to
direct complexation with the drug, the cyclodextrin may be used as an
auxiliary additive, i.e. as a carrier,
diluent, or solubiliser. Most commonly used for these purposes are alpha-,
beta- and gamma-cyclodextrins,
examples of which may be found in International Patent Applications Nos. WO
91/11172, WO 94/02518
and WO 98/55148.
[106] Pharmaceutical compositions for inhalation or insufflation include
solutions and suspensions in
pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof,
and powders. The liquid or
solid pharmaceutical compositions can contain suitable pharmaceutically
acceptable excipients. In some
embodiments, the pharmaceutical compositions are administered by the oral or
nasal respiratory route for
local or systemic effect. Pharmaceutical compositions in pharmaceutically
acceptable solvents can be
nebulized by use of inert gases. Nebulized solutions can be inhaled directly
from the nebulizing device or
the nebulizing device can be attached to a face mask tent, or intermittent
positive pressure breathing
machine. Solution, suspension, or powder pharmaceutical compositions can be
administered, e.g., orally
or nasally, from devices that deliver the formulation in an appropriate
manner.
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[107] The pharmaceutical composition described herein may be combined with the
administration of
another drug or active ingredient. Thus, the present products may be used to
alleviate, minimize or prevent
not only a disease or condition, but a side effect of another treatment
regime.
Recreational Products
[108] In one embodiment, the purified cannabinoids obtained by the present
methods may be included
in compositions such as oils (both for topical administration as massage oil,
or to be burned or aeresolized),
incense, cosmetics, bath oils, perfumes, makeup, food seasonings, toothpastes,
ingestible solids (e.g., as
a powder included in or on foods) or liquids (e.g., teas), etc.
[109] For instance, a product produced by the present methods may be included
in a "vape" product
containing propylene glycol, glycerine, vegetable glycerine, aqueous
glycerine, and optionally flavorings. In
one aspect, the "vape" product may also include other drugs, such as nicotine.
Methods of Treating a Condition
[110] The pharmaceutical products described herein may be administered to
treat or reduce the
symptoms of a disease or condition. In one embodiment, the present products
may be administered to
treat pain, Schizophrenia, convulsion, inflammation, anxiety or panic,
depression (including unipolar or
bipolar mood disorder and syndromel depression etc.), as a neuroprotective
(i.e., for treatment of
neurodegenerative disease, stroke, traumatic brain injury), cancer, graft-
versus-host disease, migraines,
arthritis, chronic pain (including neuropathic pain), nausea and vomiting,
anorexia, glaucoma, glioma,
epilepsy (that affects children and adults), asthma, perinatal asphyxia,
addiction (and symptoms of
dependency and withdrawal), movement disorders evidencing spasticity (in
multiple sclerosis and spinal
cord injury), Tourette's syndrome, dystonia, and tardive dyskinesia.
[111] In particular methods embodiments, treatment methods reduce, decrease,
suppress, limit, control
or inhibit the presence of one or more symptoms associated with a condition;
reduce, decrease, suppress,
limit, control or inhibit side-effects of another pharmaceutical treatment;
reduce, decrease, suppress, limit,
control or inhibit the symptoms of addiction. In additional particular methods
embodiments, treatment
methods include administration of an amount of the present product sufficient
to increase, induce, enhance,
augment, promote or stimulate an immune response against the condition; or
decrease, reduce, inhibit,
suppress, prevent, control, or limit the spread of the condition within a
subject or patient, or between
subjects or patients. In further particular methods embodiments, treatment
methods include administration
of an amount of the present products sufficient to protect an individual from
a pathology related to the
condition, or reduce, decrease, limit, control or inhibit susceptibility to a
pathology related to the condition.
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Reagents for the Performance of the Present Method
[112] In yet another embodiment the present invention includes reagents for
the purification of
cannabinoids. Such reagents include hexane (for CBG and CBGA), pentane and
petroleum ether 40-60 C
bp (for CBD), heptane and petroleum ether 60-80 C bp (for THCA) for the
crystallization of the cannabinoid,
and optionally reagents for the liquid chromatography such as ethanol,
methanol, or isopropyl, or heptane,
acetone, and acetonitrile.
Aspects
Aspects of the present specification can also be described as follows:
1. A method of purifying one or more cannabinoids from a plant material, the
method comprising a)
incubating the plant material with a first non-polar solvent to form a first
solvent mixture which extracts
the one or more cannabinoids from a 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) in a manner that
concentrates the one or more cannabinoids; c) incubating the reduced first
solvent mixture at a
temperature range of between about -70 C to about 40 C in a manner that
crystalizes the one or more
cannabinoids; d) incubating the one or more crystalized 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 crystalized cannabinoids; and e) incubating the second solvent mixture
at a temperature range
of between about -70 C to about 40 C in a manner that crystalizes the one or
more cannabinoids,
thereby resulting in the purification of one or more cannabinoids.
2. The method according to embodiment 1, wherein the plant material is a plant
extract or a plant resin.
3. The method according to embodiment 1 or embodiment 2, wherein the plaint
material is derived from
the genera Cannabis.
4. The method according to any one of embodiments 1-3, wherein the plaint
material is derived from a
Cannabis sativa L, Cannabis indica Cannabis ruderalis, hybrids thereof or
varietals thereof.
5. The method according to embodiment 4, wherein the Cannabis sativa L.
varietal comprises a
Chemotype I varietal, Chemotype II varietal, a Chemotype III varietal or a
Chemotype IV varietal.
6. The method according to embodiment 4, wherein the Cannabis sativa L.
varietal comprises a Carma
varietal, a AIDA varietal, a SARA varietal, a PILAR varietal, a Futura 75
varietal, MONIEK varietal, or a
60.2/1/9 experimental varietal.
7. The method according to any one of embodiments 1-6, wherein prior to step
(a), the plant material is
treated to decarboxylate one or more cannabinoids present in the plant
material.
8. The method according to any one of embodiments 1-7, wherein the first non-
polar solvent of step (a)
comprises pentane, hexane, heptane, cyclohexane, petroleum ether,
dicloromethane, tricloromethane,
tethrahydrofurane, diethyl ether, ethanol, methanol, isopropanol, acetone,
acetonitrile, ethyl acetate,
butane, propane, refrigeration gas 1,1,1 ,2-Tetrafluoroethane (R134a), liquid
CO2, subcritical CO2 and
supercritical CO2.
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9. The method according to any one of embodiments 1-8, wherein the one or
more cannabinoids comprise
tetrahydrocannabinol (THC), tetrahydrocannabidivarin (THCV),
tetrahydrocannabinolic acid (THCA),
tetrahydrocannabidivarinic acid (THCVA), cannabinol (CBN), cannabivarin (CBV),
cannabidiol (CBD),
cannabidivarin (CBDV), cannabidiolic acid (CBDA), cannabidivarinic acid
(CBDVA), cannabigerol
(CBG), canabigerovarin (CBGV), canabigerovarinic acid (CBGV) or cannabigerolic
acid (CBGA).
10. The method according to any one of embodiments 1-9, wherein in step (a)
the first solvent mixture is
incubated at least 5 minutes.
11. The method according to embodiment 10, wherein in step (a) the first
solvent mixture is incubated at
about 10 minutes to about 1500 minutes.
12. The method according to embodiment 11, wherein in step (a) the first
solvent mixture is incubated at
about 30 minutes to about 120 minutes.
13. The method according to any one of embodiments 1-12, wherein step (a) is
repeated at least once.
14. The method according to embodiment 13, wherein step (a) is repeated three
times.
15. The method according to any one of embodiments 1-14, wherein in step (b),
the volume of the first
solvent mixture is reduced to about 1% to about 50% of the original volume of
the first solvent mixture
in step (a).
16. The method according to embodiment 15, wherein in step (b), the volume of
the first solvent mixture is
reduced to about 0.1% to about 15% of the original volume of the first solvent
mixture in step (a).
17. The method according to embodiment 15, wherein in step (b), the volume of
the first solvent mixture is
reduced to about 16% to about 50% of the original volume of the first solvent
mixture in step (a).
18. The method according to any one of embodiments 1-17, wherein in step (b),
the volume of the first
solvent mixture is reduced by evaporation.
19. The method according to any one of embodiments 1-18, wherein in step (c),
the reduced first solvent
mixture is incubated at a temperature range of between about -20 C to about 30
C.
20. The method according to embodiment 19, wherein in step (c), the reduced
first solvent mixture is
incubated at a temperature range of between about 0 C to about 25 C.
21. The method according to embodiment 20, wherein in step (c), the reduced
first solvent mixture is
incubated at a temperature range of between about 4 C to about 8 C
22. The method according to any one of embodiments 1-21, wherein in step (c),
the reduced first solvent
mixture is incubated for a time period of at least 30 minutes, at least 1 hour
or at least 2 hours.
23. The method according to embodiment 22, wherein in step (c), the reduced
first solvent mixture is
incubated for a time period of between 1 hour and 96 hours.
24. The method according to embodiment 23, wherein in step (c), the reduced
first solvent mixture is
incubated for a time period of between 2 hour and 72 hours.
25. The method according to embodiment 24, wherein in step (c), the reduced
first solvent mixture is
incubated for a time period of between 4 hour and 48 hours.
26. The method according to embodiment 25, wherein in step (c), the reduced
first solvent mixture is
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incubated for a time period of between 6 hour and 24 hours.
27. The method according to embodiment 26, wherein in step (c), the reduced
first solvent mixture is
incubated for a time period of between 12 hour and 24 hours.
28. The method according to any one of embodiments 1-27, wherein step (c)
further comprises seeding
the reduced solvent mixture with a cannabinoid.
29. The method according to embodiment 28, wherein the cannabinoid used to
seed the reduced solvent
mixture comprises a purified cannabinoid, a partially purified cannabinoid or
crude extract comprising
a cannabinoid.
30. The method according to any one of embodiments 1-29, wherein the second
non-polar solvent of step
(d) comprises pentane, hexane, heptane, petroleum ethers, cyclohexane,
dichloromethane,
trichloromethane, tetrahydrofurane, diethyl ether, toluene, benzene, ethanol,
methanol, isopropanol,
acetone, acetonitrile, ethyl acetate, butane, propane, refrigerant gases
(e.g.: 1,1,1,2-Tetrafluoroethane
(RI 34a)) or, liquid, subcritical or supercritical CO2 or mixes of these
solvents.
31. The method according to any one of embodiments 1-30, wherein in step (d),
the second solvent mixture
dissolves at least 75% of the one or more crystalized cannabinoids.
32. The method according to embodiment 31, wherein in step (d), the second
solvent mixture dissolves at
least 85% of the one or more crystalized cannabinoids.
33. The method according to embodiment 32, wherein in step (d), the second
solvent mixture dissolves at
least 95% of the one or more crystalized cannabinoids.
34. The method according to any one of embodiments 1-33, wherein in step (d),
the second solvent mixture
is incubated at a temperature range of between about 30 C to about 60 C.
35. The method according to embodiment 34, wherein in step (d), the second
solvent mixture is incubated
at a temperature range of between about 40 C to about 50 C.
37. The method according to any one of embodiments 1-35, wherein in step (d),
the second solvent mixture
is incubated for a time period of at least 6 minutes.
38. The method according to embodiment 37, wherein in step (d), the second
solvent mixture is incubated
for a time period of between 0.25 hour and 4 hours.
39. The method according to any one of embodiments 1-38, wherein in step (e),
the second solvent mixture
is incubated at a temperature range of between about -20 C to about 30 C.
40. The method according to embodiment 39, wherein in step (e), the second
solvent mixture is incubated
at a temperature range of between about 0 C to about 25 C.
41. The method according to embodiment 40, wherein in step (e), the second
solvent mixture is incubated
at a temperature range of between about 4 C to about 8 C
42. The method according to any one of embodiments 1-41, wherein in step (e),
the second solvent mixture
is incubated for a time period of at least 6 minutes, at least 1 hour, at
least 2 hours, at least 3 hours or
at least 4 hours.
43. The method according to embodiment 42, wherein in step (e), the second
solvent mixture is incubated
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for a time period of between 0.1 hour and 96 hours.
44. The method according to embodiment 43, wherein in step (e), the second
solvent mixture is incubated
for a time period of between 2 hour and 72 hours.
45. The method according to embodiment 44, wherein in step (e), the second
solvent mixture is incubated
for a time period of between 4 hour and 48 hours.
46. The method according to embodiment 45, wherein in step (e), the second
solvent mixture is incubated
for a time period of between 6 hour and 24 hours.
47. The method according to embodiment 46, wherein in step (e), the second
solvent mixture is incubated
for a time period of between 12 hour and 24 hours.
48. The method according to any one of embodiments 1-47, wherein the one or
more crystalized
cannabinoids of step (c) is purified prior to step (d).
49. The method according to embodiment 48, wherein the purification is
performed using filtration that
results in a collection of a mother liquor.
50. The method according to embodiment 49, further comprising incubating the
mother liquor at a
temperature range of between about -70 C to about 40 C in a manner that
crystalizes the one or more
cannabinoids.
51. The method according to embodiment 50, further comprising f) purifying the
one or more crystalized
cannabinoids using filtration that results in a collection of a mother liquor;
and g) incubating the mother
liquor at a temperature range of between about -70 C to about 40 C in a manner
that crystalizes the
one or more cannabinoids.
52. The method according to any one of embodiments 1-52, wherein steps (f) and
(g) are repeated at least
once.
53. The method according to embodiment 52, wherein steps (f) and (g) are
repeated 2 times.
54. The method according to embodiment 53, wherein steps (f) and (g) are
repeated 3 times.
55. The method according to any one of embodiments 1-54, wherein steps (d) and
(e) are repeated at least
once.
56. The method according to embodiment 50, wherein steps (d) and (e) are
repeated 2 times.
57. The method according to embodiment 51, wherein steps (d) and (e) are
repeated 3 times.
58. The method according to any one of embodiments 1-57, wherein the first
solvent mixture of step (a) is
purified prior to step (b).
59. The method according to embodiment 58, wherein the purification is
performed using filtration.
60. The method according to any one of embodiments 1-59, wherein the one or
more crystalized
cannabinoids of step (e) is filtered.
61. The method according to any one of embodiments 1-60, further comprising
performing liquid:liquid
chromatography after one or more of steps (b) or (d).
62. The method according to embodiment 61, wherein the liquid:liquid
chromatography is counter current
chromatography (CCC) or centrifugal partition chromatography (CPC).
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63. The method according to embodiment 62, wherein the mobile organic phase
includes pentane, hexane,
cyclohexane, or heptane.
64. The method according to embodiment 62, wherein the stationary phase
includes ethanol, methanol,
isopropanol, acetone, acetonitrile and/or water.
65. The method according to embodiment 62, wherein the mobile phase is
pentane, hexane, cyclohexane,
or heptane and the stationary phase is water and ethanol, methanol, or
isopropanol. In one
embodiment, the two-phase system is hexane:ethanol:water at ratios of
(20:19:1) to (20:8:12) and
wherein hexane may be substituted by pentane, heptane and/or cyclohexane and
wherein ethanol may
be substituted by methanol and/or isopropanol instead of ethanol, with the
organic phase of pentane
or hexane as mobile phase or the two-phase system. In one embodiment the
ratios of the two-phase
system, hexane:ethanol: water are (20:13:7) for CBG-type cannabinoids
(20:14:6) for CBD-type
cannabinoids and (20:17:3) for THC-type cannabinoids or using a gradient
reverse phase run with
ethanol and water mix as mobile phase increasing the concentration of ethanol
gradually from the ratio
(20:12:8) to (20:18:2).
66. The method according to embodiment 62, wherein the mobile phase is
pentane, hexane or heptane
with or without ethyl acetate as a modifier, and the stationary phase is
acetone and/or acetonitrile with
or without water as a modifier. In one embodiment the two phase system is
pentane:acetonitrile or
hexane:acetonitrile with or without ethyl acetate or water as a modifier, at
ratios of (10:0:10:0) to
(7:3:7:3). In one embodiment for THC-type cannabinoids the ratio of
pentane:acetonitrile is from 10:10
(e.g., pentane:ethyl acetate:acetonitrile:water
(10:0:10:0)) to 7:3:7:3 pentane:ethyl
acetate:acetonitrile:water by volume. In another embodiment the ratio of
hexane:acetonitrile is from
10:10 (e.g., hexane:ethyl acetate:acetonitrile:water (10:0:10:0)) to 7:3:7:3
hexane:ethyl
acetate:acetonitrile:water by volume. Preferred solvent ratios for THC-type
cannabinoids are
pentane:ethyl acetate: acetonitrile:water at 19:1:19:1 by volume or 9:1:9:1 by
volume.
67. A purified cannabinoid produced by the method according to any one of
embodiments 1-66.
68. A pharmaceutical composition comprising a purified cannabinoid produced by
the method according
to any one of embodiments 1-66.
69. The pharmaceutical composition of embodiment 68, further comprising a
pharmaceutically acceptable
excipient or carrier.
70. A method of treating a disease or condition comprising administering the
cannabinoid produced by the
method according to any one of embodiments 1-66 to a subject in need thereof.
71. The method of treating a disease or condition of embodiment 70, wherein
the disease or condition is
pain, schizophrenia, convulsion, inflammation, anxiety, depression,
neurodegenerative disease, stroke,
traumatic brain injury, cancer, migraines, arthritis, chronic pain, nausea and
vomiting, anorexia,
glaucoma, epilepsy, asthma, addiction, symptoms of dependency and withdrawal,
multiple sclerosis,
spinal cord injury, Tourette's syndrome, dystonia, or tardive dyskinesia.
72. A method of purifying a cannabinoid from a plant material, the method
comprising: a) incubating the
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plant material with a first non-polar solvent to form a first solvent mixture
which extracts the one or more
cannabinoids from a 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) in
a manner that concentrates the one or more cannabinoids; d) incubating the
reduced first solvent
mixture at a temperature range of between about -70 C to about 40 C in a
manner that crystalizes the
one or more cannabinoids; e) purifying the one or more crystalized
cannabinoids in step (d) using
filtration that results in a collection of a mother liquor; f) incubating the
one or more crystalized
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 crystalized
cannabinoids; g) incubating the
second solvent mixture at a temperature range of between about -70 C to about
40 C in a manner that
crystalizes the one or more cannabinoids; and h) purifying the one or more
crystalized cannabinoids of
step (g) using filtration that results in a collection of a mother liquor,
thereby resulting in the purification
of one or more cannabinoids
73. The method according to embodiment 72, wherein the mother liquor of step
(e) and/or step (h) is
incubated at a temperature range of between about -70 C to about 40 C in a
manner that crystalizes
the one or more cannabinoids.
74. The method according to embodiment 73, further comprising i) purifying the
one or more crystalized
cannabinoids using filtration that results in a collection of a mother liquor;
and j) incubating the mother
liquor at a temperature range of between about -70 C to about 40 C in a manner
that crystalizes the
one or more cannabinoids.
75. The method according to embodiment 74, wherein steps (i) and (j) are
repeated at least once.
76. The method according to embodiment 75, wherein steps (i) and (j) are
repeated 2 times.
77. The method according to embodiment 76, wherein steps (i) and (j) are
repeated 3 times.
78. The method according to any one of embodiments 72-77, wherein steps (f)
and (g) are repeated at
least once.
79. The method according to embodiment 78, wherein steps (f) and (g) are
repeated 2 times.
80. The method according to embodiment 79, wherein steps (f) and (g) are
repeated 3 times.
81. The method according to any one of embodiments 72-80, wherein steps (f),
(g) and (h) are repeated at
least once.
82. The method according to embodiment 81, wherein steps (f), (g) and (h) are
repeated 2 times.
83. The method according to embodiment 82, wherein steps (f), (g) and (h) are
repeated 3 times.
84. The method according to any one of embodiments 72-83, wherein the plant
material is a plant extract
or a plant resin.
85. The method according to any one of embodiments 72-84, wherein the plaint
material is derived from
the genera Cannabis.
86. The method according to any one of embodiments 72-85, wherein the plaint
material is derived from
Cannabis sativa L., Cannabis indica Cannabis ruderalis, hybrids thereof or
varietals thereof.
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87. The method according to embodiment 86, wherein the Cannabis sativa L.
varietal comprises a
Chemotype II varietal, a Chemotype III varietal or a Chemotype IV varietal.
88. The method according to embodiment 86, wherein the Cannabis sativa L.
varietal comprises a Carma
varietal, a AIDA varietal, a SARA varietal, a PILAR varietal, a Futura 75,
MONIEK varietal or a 60.2/1/9
experimental varietal.
89. The method according to any one of embodiments 72-88, wherein prior to
step (a), the plant material
is treated to decarboxylate one or more cannabinoids present in the plant
material.
90. The method according to any one of embodiments 72-89, wherein the first
non-polar solvent of step (a)
comprises pentane, hexane, heptane, cyclohexane, petroleum ether,
dicloromethane, tricloromethane,
tethrahydrofurane, diethyl ether, benzene, toluene, ethanol, methanol,
isopropanol, acetone,
acetonitrile, ethyl acetate, butane, propane, refrigeration gas 1,1,1,2-
Tetrafluoroethane (RI 34a), liquid
002, subcritical CO2 and supercritical 002.
91. The method according to any one of embodiments 72-90, wherein the one or
more cannabinoids
comprise tetrahydrocannabinol (THC), tetrahydrocannabidivarin (THCV),
tetrahydrocannabinolic acid
(THCA), cannabidiol (CBD), cannabidivarin (CBDV), cannabidiolic acid (CBDA),
cannabigerol (CBG),
cannabigerovarin (CBGV) or cannabigerolic acid (CBGA).
92. The method according to any one of embodiments 72-91, wherein in step (a)
the first solvent mixture
is incubated at least 5 minutes.
93. The method according to embodiment 92, wherein in step (a) the first
solvent mixture is incubated at
about 10 minutes to about 1500 minutes.
94. The method according to embodiment 93, wherein in step (a) the first
solvent mixture is incubated at
about 30 minutes to about 120 minutes.
95. The method according to any one of embodiments 72-94, wherein step (a) is
repeated at least once.
96. The method according to embodiment 95, wherein step (a) is repeated twice.
97. The method according to embodiment 96, wherein step (a) is repeated 3
times.
98. The method according to any one of embodiments 72-97, wherein in step (c),
the volume of the first
solvent mixture is reduced to about 5% to about 50% of the original volume of
the first solvent mixture
in step (a).
99. The method according to embodiment 98, wherein in step (c), the volume of
the first solvent mixture is
reduced to about 1% to about 15% of the original volume of the first solvent
mixture in step (a).
100. The method according to embodiment 98, wherein in step (c), the volume of
the first solvent mixture
is reduced to about 15% to about 50% of the original volume of the first
solvent mixture in step (a).
101. The method according to any one of embodiments 72-100, wherein in step
(c), the volume of the first
solvent mixture is reduced by evaporation.
102. The method according to any one of embodiments 72-101, wherein in step
(d), the reduced first solvent
mixture is incubated at a temperature range of between about -20 C to about 30
C.
103. The method according to embodiment 102, wherein in step (d), the reduced
first solvent mixture is
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incubated at a temperature range of between about 0 C to about 25 C.
104. The method according to embodiment 103, wherein in step (d), the reduced
first solvent mixture is
incubated at a temperature range of between about 4 C to about 8 C
105. The method according to any one of embodiments 72-104, wherein in step
(d), the reduced first solvent
mixture is incubated for a time period of at least 30 minutes, at least 1 hour
or at least 2 hours.
106. The method according to embodiment 105, wherein in step (d), the reduced
first solvent mixture is
incubated for a time period of between 1 hour and 96 hours.
107. The method according to embodiment 106, wherein in step (d), the reduced
first solvent mixture is
incubated for a time period of between 2 hour and 72 hours.
108. The method according to embodiment 107, wherein in step (d), the reduced
first solvent mixture is
incubated for a time period of between 4 hour and 48 hours.
109. The method according to embodiment 108, wherein in step (d), the reduced
first solvent mixture is
incubated for a time period of between 6 hour and 24 hours.
110. The method according to embodiment 109, wherein in step (d), the reduced
first solvent mixture is
incubated for a time period of between 12 hour and 24 hours.
111. The method according to any one of embodiments 72-110, wherein step (d)
further comprises seeding
the reduced solvent mixture with a cannabinoid.
112. The method according to embodiment 111, wherein the cannabinoid used to
seed the reduced solvent
mixture comprises a purified cannabinoid, a partially purified cannabinoid or
crude extract comprising
a cannabinoid.
113. The method according to any one of embodiments 72-112, wherein the second
non-polar solvent of
step (f) comprises pentane, hexane, heptane, cyclohexane, petroleum ether,
dichloromethane,
trichloromethane, tethrahydrofurane, diethyl ether, benzene, toluene, ethanol,
methanol, isopropanol,
acetone, acetonitrile, ethyl acetate, butane, propane, refrigeration gas
1,1,1,2-Tetrafluoroethane
(R134a), liquid CO2, subcritical CO2 and supercritical CO2.
114. The method according to any one of embodiments 72-113, wherein in step
(f), the second solvent
mixture dissolves at least 75% of the one or more crystalized cannabinoids.
115. The method according to embodiment 114, wherein in step (f), the second
solvent mixture dissolves
at least 85% of the one or more crystalized cannabinoids.
116. The method according to embodiment 115, wherein in step (f), the second
solvent mixture dissolves
at least 95% of the one or more crystalized cannabinoids.
117. The method according to any one of embodiments 72-116, wherein in step
(f), the second solvent
mixture is incubated at a temperature range of between about 30 C to about 60
C.
118. The method according to embodiment 117, wherein in step (f), the second
solvent mixture is incubated
at a temperature range of between about 40 C to about 50 C.
119. The method according to any one of embodiments 72-118, wherein in step
(f), the second solvent
mixture is incubated for a time period of at least 6 minutes.
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120. The method according to embodiment 119, wherein in step (0, the second
solvent mixture is incubated
for a time period of between 0.1 hour and 4 hours.
121. The method according to any one of embodiments 72-120, wherein in step
(g), the second solvent
mixture is incubated at a temperature range of between about -20 C to about 30
C.
122. The method according to embodiment 121, wherein in step (g), the second
solvent mixture is incubated
at a temperature range of between about 0 C to about 25 C.
123. The method according to embodiment 122, wherein in step (g), the second
solvent mixture is incubated
at a temperature range of between about 4 C to about 8 C
124. The method according to any one of embodiments 72-123, wherein in step
(g), the second solvent
mixture is incubated for a time period of at least 6 minutes, at least 1 hour,
at least 2 hours, at least 3
hours or at least 4 hours.
125. The method according to embodiment 124, wherein in step (g), the second
solvent mixture is incubated
for a time period of between 0.1 hour and 96 hours.
126. The method according to embodiment 125, wherein in step (g), the second
solvent mixture is incubated
for a time period of between 2 hour and 72 hours.
127. The method according to embodiment 126, wherein in step (g), the second
solvent mixture is incubated
for a time period of between 4 hour and 48 hours.
128. The method according to embodiment 127, wherein in step (g), the second
solvent mixture is incubated
for a time period of between 6 hour and 24 hours.
129. The method according to embodiment 128, wherein in step (g), the second
solvent mixture is incubated
for a time period of between 12 hour and 24 hours.
130. The method according to any one of embodiments 72-129, wherein the
temperature in steps (d) and
(g) is at most about 4 C for CBGA/CBG purification and step (d) is at most -20
C for CBD purification.
131. The method according to any one of embodiments 72-130, further comprising
performing liquid liquid
chromatography after one or more of steps (c), (e) or (h).
132. The method according to embodiment 131, wherein the liquid:liquid
chromatography is counter current
chromatography (CCC) or centrifugal partition chromatography (CPC).
133. The method according to embodiment 131 or embodiment 132, wherein the
mobile organic phase
includes pentane, hexane, cyclohexane, or heptane.
134. The method according to any one of embodiments 131-132, wherein the
stationary phase includes
ethanol, methanol, isopropanol, acetone, acetonitrile and/or water.
135. The method according to embodiment 131 or embodiment 132, wherein the
mobile phase is pentane,
hexane, cyclohexane, or heptane and the stationary phase is water and ethanol,
methanol, or
isopropanol.
136. The method according to embodiment 131 or embodiment 132, wherein the
mobile phase is pentane,
hexane or heptane with or without ethyl acetate as a modifier, and the
stationary phase is acetone
and/or acetonitrile with or without water as a modifier.
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137. The method according to any one of embodiments 72-136, further comprising
performing counter
current chromatography (CCC) or centrifugal partition chromatography (CPC)
after the steps (e) or (h)
to isolate, purify or repurify the cannabinoids tetrahydrocannabinol (THC),
tetrahydrocannabidivarin
(THCV), tetrahydrocannabinolic acid (THCA), tetrahydrocannabidivarinic acid
(THCVA), cannabinol
(CBN), cannabivarin (CBV) cannabidiol (CBD), cannabidivarin (CBDV),
cannabidiolic acid (CBDA),
cannabidivarinic acid (CBDVA), cannabigerol (CBG), cannabigerovarin (CBGV),
cannabigerovarinic
acid (CBGVA) and cannabigerolic acid (CBGA).
138. The method according to any one of embodiments 131-137, wherein the
chromatography uses a two-
phase system, hexane:ethanol:water at ratios of (20:19:1) to (20:8:12) and
wherein hexane may be
substituted by pentane, heptane and/or cyclohexane and wherein ethanol may be
substituted by
methanol and/or isopropanol instead of ethanol, with the organic phase of
pentane or hexane as mobile
phase or the two-phase system. The chromatography also uses a two phase system
pentane:acetonitrile or hexane:acetonitrile with or without ethyl acetate or
water as a modifier, at ratios
of (10:0:10:0) to (7:3:7:3).
139. The method according to any one of embodiments 131-138, wherein the
ratios of the two-phase
system, hexane:ethanol: water are (20:13:7) for CBG-type cannabinoids
(20:14:6) for CBD-type
cannabinoids and (20:17:3) for THC-type cannabinoids or using a gradient
reverse phase run with
ethanol and water mix as mobile phase increasing the concentration of ethanol
gradually from the ratio
(20:12:8) to (20:18:2). For THC-type extracts the ratio of
pentane:acetonitrile is from 10:10 (e.g.,
pentane:ethyl acetate:acetonitrile:water (10:0:10:0)) to 7:3:7:3 pentane:ethyl
acetate:acetonitrile:water
by volume. In another embodiment the ratio of hexane:acetonitrile is from
10:10 (e.g., hexane:ethyl
acetate:acetonitrile:water (10:0:10:0)) to 7:3:7:3 hexane:ethyl
acetate:acetonitrile:water by volume.
Preferred solvent ratios for THC-type cannabinoids are pentane:ethyl acetate:
acetonitrile:water at
19:1:19:1 by volume or 9:1:9:1 by volume.
140. The method according to any one of embodiments 72-139, wherein
cannabigerol (CBG),
cannabigerovarin (CBGV), cannabidiol (CBD), cannabidivarin (CBDV),
cannabivarin (CBV),
cannabinol (CBN), tetrahydrocannabidivarin (THCV) or tetrahydrocannabinol
(THC) are isolated and
purified and prior to step (a), the plant material, resin or extracts of said
plant are decarboxylated at
about at least 120 C for at least 1 hour.
141. The method according to any one of embodiments 72-139, wherein
cannabigerol (CBG),
cannabigerovarin (CBGV), cannabidiol (CBD), cannabidivarin (CBDV),
cannabivarin (CBV),
cannabinol (CBN), tetrahydrocannabidivarin (THCV) or tetrahydrocannabinol
(THC) is isolated and
purified, and prior to step (a), the plant, plant material, plant extract, or
resin are decarboxylated by
hydrodistillation (steam distillation) at least at 90 C for 2 hours.
142. A purified cannabinoid produced by the method according to any one of
embodiments 72-141.
143. A pharmaceutical composition comprising a purified cannabinoid produced
by the method according
to any one of embodiments 72-141.
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144. The pharmaceutical composition of embodiment 143, further comprising a
pharmaceutically
acceptable excipient or carrier.
145. A method of treating a disease or condition comprising administering the
cannabinoid produced by the
method according to any one of embodiments 72-141 to a subject in need
thereof.
146. The method of treating a disease or condition of embodiment 145, wherein
the disease or condition is
pain, schizophrenia, convulsion, inflammation, anxiety, depression,
neurodegenerative disease,
stroke, traumatic brain injury, cancer, migraines, arthritis, chronic pain,
nausea and vomiting, anorexia,
glaucoma, epilepsy, asthma, addiction, symptoms of dependency and withdrawal,
multiple sclerosis,
spinal cord injury, Tourette's syndrome, dystonia, or tardive dyskinesia.
147. The method according to any one of embodiments 7 or 89, wherein the plant
material is heated
between 100 C to 160 C in order to decarboxylate one or more cannabinoids
present in the plant
material.
148. The method according to embodiment 147, wherein the plant material is
heated between 120 C to
150 C in order to decarboxylate one or more cannabinoids present in the plant
material.
149. The method according to embodiments 147 or 148, wherein the plant
material is heated for a time
period of at least 30 minutes.
150. The method according to embodiment 149, wherein the plant material is
heated for a time period of
about 1 hour to about 3 hours.
151. The method according to any one of embodiments 1-150, wherein the one or
more cannabinoids
purified is CBGA, CBG, CBGV, CBDA, CBD, CBDV, THCA, THC, THCV or any
combination thereof.
152. The method according to embodiments 151, wherein the CBGA has a purity of
90% or greater, 91%
or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater,
96% or greater, 97% or
greater, 98% or greater or 99% or greater as determined by area normalisation
of an HPLC profile or
by a quantification percent of purity respect a certified commercial standard.
153. The method according to embodiments 151, wherein the CBG, CBGA or CBGV
has a purity of 90%
or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater,
95% or greater, 96% or
greater, 97% or greater, 98% or greater or 99% or greater as determined by
area normalisation of an
HPLC profile or by a quantification percent of purity respect a certified
commercial standard.
154. The method according to embodiments 151, wherein the CBD, CBDA or CBDV
has a purity of 90% or
greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95%
or greater, 96% or
greater, 97% or greater, 98% or greater or 99% or greater as determined by
area normalisation of an
HPLC profile or by a quantification percent of purity respect a certified
commercial standard.
155. The method according to embodiments 151, wherein the THC, THCA, or THCV
has a purity of 90% or
greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95%
or greater, 96% or
greater, 97% or greater, 98% or greater or 99% or greater as determined by
area normalisation of an
HPLC profile or by a quantification percent of purity respect a certified
commercial standard.
156. The pharmaceutical composition of any one of embodiments 68, 69, 143 or
144, wherein the purified
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cannabinoid is CBG, CBGA, CBGV, CBD, CBDA, CBDV, THC, THCA, or THCV or any
combination
thereof.
157. The pharmaceutical composition of embodiment 156, wherein the purified
cannabinoid is CBGA or
CBGV having a purity of 90% or greater, 91% or greater, 92% or greater, 93% or
greater, 94% or
greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99%
or greater as
determined by area normalisation of an HPLC profile or by a quantification
percent of purity respect a
certified commercial standard.
158. The pharmaceutical composition of embodiment 156, wherein the purified
cannabinoid is CBG having
a purity of 90% or greater, 91% or greater, 92% or greater, 93% or greater,
94% or greater, 95% or
greater, 96% or greater, 97% or greater, 98% or greater or 99% or greater as
determined by area
normalisation of an HPLC profile or by a quantification percent of purity
respect a certified commercial
standard.
159. The pharmaceutical composition of embodiment 156, wherein the purified
cannabinoid is THC, THCA
or THCV having a purity of 90% or greater, 91% or greater, 92% or greater, 93%
or greater, 94% or
greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99%
or greater as
determined by area normalisation of an HPLC profile or by a quantification
percent of purity respect a
certified commercial standard.
160. The pharmaceutical composition of embodiment 156, wherein the purified
cannabinoid is CBD, CBDA,
or CBDV having a purity of 90% or greater, 91% or greater, 92% or greater, 93%
or greater, 94% or
greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater or 99%
or greater as
determined by area normalisation of an HPLC profile or by a quantification
percent of purity respect a
certified commercial standard.
161. The method according to any one of embodiments 1-155, wherein a
substantially pure preparation of
one or more cannabinoids is achieved without the use a chromatographic
technique.
162. The method according to embodiment 161, wherein a substantially pure
preparation of CBGA or CBGV
is achieved without the use a chromatographic technique.
163. The method according to embodiment 161, wherein a substantially pure
preparation of CBG is
achieved without the use a chromatographic technique.
164. The method according to embodiment 161, wherein a substantially pure
preparation of CBD, CBDA,
or CBDV is achieved without the use a chromatographic technique.
165. The method according to embodiment 161, wherein a substantially pure
preparation of THC, THCA or
THCV is achieved without the use a chromatographic technique.
EXAMPLES
[113] The following non-limiting examples are provided for illustrative
purposes only in order to facilitate
a more complete understanding of representative embodiments now contemplated.
These examples
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should not be construed to limit any of the embodiments described in the
present specification, including
those pertaining to the compounds, pharmaceutical compositions, or methods and
uses disclosed herein.
Example 1:
Purification of Cannabinoids CBD and CBDV using Centrifugation Partitioning
Chromatography
[114] Maceration of 100 g of plant material of the variety Theresa with
CBD/CBDV was carried out in 1 L
of petroleum ether (40-60 C bp) for one hour. This procedure was repeated two
times with 0.75 L of
petroleum ether (40-60 C bp). The plant material was filtered, and the
petroleum ether evaporated down
completely to achieve 9.1 g of dry extract. This 9.1 g of extract was
decarboxylated at 150 C for 2 hours
obtaining 8.7 g of decarboxylated extract. 8 g of the extract was dissolved
with hexane at a volume of 50
mL and then used as a sample for injection in the CPC 1000 PRO (Gilson) before
called CPC-Quantum
(Armen) of 1 L rotor volume. We use the biphasic solvent system
hexane:ethanol:water at proportions of
20:14:6, the flow rate of the mobile phase (hexane phase) is 200 mL/min during
the run, and changed to
350 mL/min in the extrusion and change of the stationary phase (ethanolic
phase). The complete run last
14 minutes, with 2 phases: run with pumping mobile phase that is from 10
minutes and an extrusion phase
of 4 minutes. The use of solvent per run is from 2 L of hexane and 1.4 L of
Ethanolic phase. The total
solvent used is from 3.4 L per run. We obtain 2.9 g of CBD in the fraction
from minute 4:55 to 7:20 and 0.9
g of CBDV in the fraction from the minute 7:20 to 10. The purity of the
evaporated fractions was > 95% after
recrystallization in petroleum ether (40-60 C bp) the CBD or after wash or
recrystallization in petroleum
ether (40-60 C bp) or hexane from the CBDV.
Example 2:
Purification of Cannabinoids CBD and CBDA using Centrifugation Partitioning
Chromatography
[115] Maceration of 100 g of plant material of the variety Sara with CBDA was
carried out in 1 L of
petroleum ether (40-60 C bp) for one hour. This procedure was repeated two
times with 0.75 L of petroleum
ether (40-60 C bp). The plant material was filtered, and the petroleum ether
evaporated down completely
to achieve 15 g of dry extract. 8 g of the extract was dissolved with hexane
at a volume of 50 mL and then
used as a sample for injection in the CPC 1000 PRO (Gilson) before called CPC-
Quantum (Armen) of 1 L
rotor volume. We use the biphasic solvent system hexane:ethanol:water at
proportions of 20:14:6, the flow
rate of the mobile phase (hexane phase) is 200 mL/min during the run, and
changed to 350 mL/min in the
extrusion and change of the stationary phase (ethanolic phase). The complete
run last 15 minutes, with 2
phases: run with pumping mobile phase that is from 11 minutes and an extrusion
phase of 4 minutes. The
use of solvent per run is from 2.2 L of hexane and 1.4 L of Ethanolic phase.
The total solvent used is from
3.6 L per run. We obtain 0.7 g of CBD in the fraction from minute 5:31 to 7.20
and 3.7 g of CBDV in the
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fraction from the minute 7.20 to 11. The purity of the evaporated fractions
was > 95% after dryness of the
CBDA and after recrystallization in petroleum ether (40-60 C bp) the CBD.
Example 3:
Purification of Cannabinoids CBG and CBGV using Counter Current Chromatography
[116] Maceration of 100 g of plant material of the variety Juani with CBG/CBGV
was carried out in 1 L of
hexane for one hour. This procedure was repeated two times with 0.75 L of
hexane. The plant material
was filtered, and the hexane evaporated down completely to achieve 6 g of dry
extract. This 6 g of extract
was decarboxylated at 150 C for 2 hours obtaining 5.6 g of decarboxylated
extract. 3,5 g of the extract was
dissolved with hexane at a volume of 20 mL and then used as a sample for
injection in the LabPrep CCC
(AECS) in the coil of 750 mL and 2.8 mm I.D. We use the biphasic solvent
system hexane:ethanol:water at
proportions of 20:12:8, the flow rate of the mobile phase (hexane phase) is 25
mL/min during the run, and
changed to 35 mL/min in the extrusion and change of the stationary phase
(ethanolic phase). The complete
run last 75 minutes, with 2 phases: run with pumping mobile phase that last 60
minutes and an extrusion
phase of 25 minutes. The use of solvent per run is from 1.5 L of hexane and
0.875 L of Ethanolic phase.
The total solvent used is from 2.375 L per run. We obtain 0.7 g of CBG in the
fraction from minute 20 to 31
and 3.7 g of CBGV in the fraction from the minute 45 to 57. The fraction from
minute 32 to 45 contains 0.1g
of a mixture of CBG/CBGV. The purity of the evaporated fractions was > 95%
after dryness of the CBGV
and after recrystallization in hexane for the CBG. CBGV was easily
recrystallized in hexane in order to
obtain higher purity.
Example 4:
Purification of Cannabinoids THCA and THC using Counter Current Chromatography
[117] Maceration of 100 g of plant material of the variety Moniek with
THC/THCA was carried out in 1 L
of hexane for one hour. This procedure was repeated two times with 0.75 L of
hexane. The plant material
was filtered, and the hexane evaporated down completely to achieve 26 g of dry
extract. This 26 g of extract
was decarboxylated at 120 C for 2 hours obtaining 24 g of decarboxylated
extract. 2 g of the extract was
dissolved with hexane at a volume of 20 mL and then used as a sample for
injection in the LabPrep CCC
(AECS) in the coil of 750 mL and 2.8 mm I.D. We use the biphasic solvent
system hexane:ethanol:water at
proportions of 20:17:3, the flow rate of the mobile phase (hexane phase) is 25
mL/min during the run, and
changed to 35 mL/min in the extrusion and change of the stationary phase
(ethanolic phase). The complete
run last 70 minutes, with 2 phases: run with pumping mobile phase that last 45
minutes and an extrusion
phase of 25 minutes. The use of solvent per run is from 1.125 L of hexane and
0.875 L of Ethanolic phase.
The total solvent used is from 2 L per run. We obtain 0.8 g of THCA in the
fraction from minute 30 to 35
and 0.25 g of THC in the fraction from the minute 38 to 43. The fraction from
minute 35 to 38 contains 0.2g
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of a mixture of THCA/THC. The purity of the evaporated fractions was > 95%
after dryness. THCA can be
recrystallized in heptane in order to increase the purity at higher
percentages.
Example 5:
Purification of Cannabinoids THC, THCV and CBV (CBNIO using Counter Current
Chromatography
[118] Maceration of 100 g of plant material of the experimental breeding cross
pollinated 60.1 /4/4/8 x
51.2/8/2 with THC/THCV was carried out in 1 L of hexane for one hour. This
procedure was repeated two
times with 0.75 L of hexane. The plant material was filtered, and the hexane
evaporated down completely
to achieve 9 g of dry extract. This 9 g of extract was decarboxylated at 120 C
for 2 hours obtaining 7.9 g of
decarboxylated extract. 1.5 g of the extract was dissolved with hexane at a
volume of 20 mL and then used
as a sample for injection in the LabPrep CCC (AECS) in the coil of 750 mL and
2.8 mm I.D. We use the
biphasic solvent system hexane:ethanol:water at proportions of 20:17:3, the
flow rate of the mobile phase
(hexane phase) is 25 mL/min during the run, and changed to 35 mL/min in the
extrusion and change of the
stationary phase (ethanolic phase). The complete run last 75 minutes, with 2
phases: run with pumping
mobile phase that last 50 minutes and an extrusion phase of 25 minutes. The
use of solvent per run is from
1.250 L of hexane and 0.875 L of Ethanolic phase. The total solvent used is
from 2.125 L per run. We obtain
0.6 g of THC in the fraction from minute 22 to 27 and 0.15 g of THCV in the
fraction from the minute 32 to
38. The fraction from minute 27 to 32 contains 0.1 g of a mixture of THC/THCV.
The fraction from minute
40 to 48 contains 0.035 g of CBV. The purity of the evaporated fractions was >
95% after dryness.
Example 6:
Purification of Cannabinoids THC + THCV using Counter Current Chromatography
[119] Maceration of 100 g of plant material of the experimental breeding cross
pollinated 60.1 /4/4/8 x
51.2/8/2 with THC/THCV was carried out in 1 L of hexane for one hour. This
procedure was repeated two
times with 0.75 L of hexane. The plant material was filtered, and the hexane
evaporated down completely
to achieve 9 g of dry extract. This 9 g of extract was decarboxylated at 120 C
for 2 hours obtaining 7.9 g of
decarboxylated extract. 0.5 g of the extract was dissolved with hexane at a
volume of 5 mL and then used
as a sample for injection in the LabPrep CCC (AECS) in the coil of 155 mL and
0.8 mm I.D. We use the
biphasic solvent system hexane:acetonitrile at proportions of 10:10, the flow
rate of the mobile phase
(hexane phase) is 8 mL/min during the run, and changed to 15 mL/min in the
extrusion and change of the
stationary phase (acetonitrile phase). The complete run last 102 minutes, with
2 phases: run with pumping
mobile phase that last 90 minutes and an extrusion phase of 12 minutes. The
use of solvent per run is from
0.720 L of hexane and 0.180 L of acetonitrile phase. The total solvent used is
from 0.9 L per run. We obtain
0.2 g of THC in the fraction from minute 45 to 58 and 0.057 g of THCV in the
fraction from the minute 65 to
83. The fraction from minute 59 to 64 contains 0.01 g of a mixture of THC/THCV
+ CBCV. The fraction from
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minute 39 to 45 contains 0.04 g of THC + CBC. The purity of the evaporated
fractions that contain one
cannabinoid was > 95% after dryness.
Example 7:
Purification of Cannabinoids THC + THCV using Counter Current Chromatography
[120] Maceration of 100 g of plant material of the experimental breeding cross
pollinated 60.1 /4/4/8 x
51.2/8/2 with THC/THCV was carried out in 1 L of hexane for one hour. This
procedure was repeated two
times with 0.75 L of hexane. The plant material was filtered, and the hexane
evaporated down completely
to achieve 9 g of dry extract. This 9 g of extract was decarboxylated at 120 C
for 2 hours obtaining 7.9 g of
decarboxylated extract. 0.5 g of the extract was dissolved with hexane at a
volume of 5 mL and then used
as a sample for injection in the LabPrep CCC (AECS) in the coil of 155 mL and
0.8 mm I.D. We use the
biphasic solvent system hexane:ethyl Acetate:acetonitrile:water at proportions
of 9 :1 : 9 :1, the flow rate of
the mobile phase (hexane phase) is 8 mL/min during the run, and changed to 15
mL/min in the extrusion
and change of the stationary phase (acetonitrile phase). The complete run last
72 minutes, with 2 phases:
run with pumping mobile phase that last 60 minutes and an extrusion phase of
12 minutes. The use of
solvent per run is from 0.480 L of hexane:ethyl acetate and 0.180 L of
acetonitrile : water phase. The total
solvent used is from 0.660 L per run. We obtain 0.21 g of THC in the fraction
from minute 22 to 37 and
0.051 g of THCV in the fraction from the minute 47 to 58. The fraction from
minute 38 to 46 contains 0.01
g of a mixture of THC/THCV. The purity of the evaporated fractions that
contain one cannabinoid was >
95% after dryness.
Example 8:
Purification of Cannabinoids THC and THCA using Centrifugation Partitioning
Chromatography
[121] Maceration of 100 g of plant material of the variety Moniek with
THC/THCA was carried out in 1 L
of hexane for one hour. This procedure was repeated two times with 0.75 L of
hexane. The plant material
was filtered, and the hexane evaporated down completely to achieve 26 g of dry
extract. This 26 g of extract
was decarboxylated at 120 C for 2 hours obtaining 24 g of decarboxylated
extract. 5 g of the extract was
dissolved with hexane at a volume of 50 mL and then used as a sample for
injection in the CPC 1000 PRO
(Gilson) before called CPC-Quantum (Armen) of 1 L rotor volume. We use the
biphasic solvent system
hexane:ethanol:water at proportions of 20:14:6, the flow rate of the mobile
phase (hexane phase) is 200
mL/min during the run, and changed to 350 mL/min in the extrusion and change
of the stationary phase
(ethanolic phase). The complete run last 13 minutes, with 2 phases: run with
pumping mobile phase that is
from 9 minutes and an extrusion phase of 4 minutes. The use of solvent per run
is from 1.8 L of hexane
and 1.4 L of Ethanolic phase. The total solvent used is from 3.2 L per run. We
obtain 2.9 g of THCA in the
fraction from minute 4.45 to 5.40 and 0.7 g of a mixture of THCA/THC in the
fraction from the minute 5.40
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to 7.20. The purity of the evaporated fractions was > 95% after dryness of the
THCA.
Example 9:
Purification of Cannabinoids THC and THCV using Centrifugation Partitioning
Chromatography
[122] Maceration of 100 g of plant material of the variety Raquel with
THC/THCV was carried out in 1 L
of hexane for one hour. This procedure was repeated two times with 0.75 L of
hexane. The plant material
was filtered, and the hexane evaporated down completely to achieve 16 g of dry
extract. This 16 g of extract
was decarboxylated at 120 C for 2 hours obtaining 14 g of decarboxylated
extract. 4 g of the extract was
dissolved with hexane at a volume of 50 mL and then used as a sample for
injection in the CPC 1000 PRO
(Gilson) before called CPC-Quantum (Armen) of 1 L rotor volume. We use the
biphasic solvent system
hexane:ethanol:water at proportions of 20:14:6, the flow rate of the mobile
phase (hexane phase) is 200
mL/min during the run, and changed to 350 mL/min in the extrusion and change
of the stationary phase
(ethanolic phase). The complete run last 15 minutes, with 2 phases: run with
pumping mobile phase that is
from 11 minutes and an extrusion phase of 4 minutes. The use of solvent per
run is from 2.2 L of hexane
and 1.4 L of Ethanolic phase. The total solvent used is from 3.6 L per run. We
obtain 2.4 g of THC in the
fraction from minute 6.25 to 7.25 and 0.42 g of THCV in the fraction from the
minute 8.10 to 9.00. The purity
of the evaporated fractions was > 95% after dryness of the THC and THCV. The
14 g of extract were
purified with 3 run of CPC obtaining 7.2g of THC >95% purity and 1.25g of THCV
>95% purity.
[123] 1 g of the fraction pool from minute 7.23 to 8.09 of the 3 anterior run,
a mix of THC, THCV and
CBN, was purified by flash chromatography over a 120g column of C18 bounded
silica with
water:acetronitrile as a mobile phase in gradient mode at a 40 mL/min flow.
TIME (min) % Water (10% acetonitrile) % Acetonitrile (10%
water)
0 35 65
11 15 85
20 0 100
28 0 100
28.1 35 65
[124] The total run was 28 minutes and the total solvent use was 1.12 L. We
obtain 0.3 g of THCV in the
fraction from minute 20.00 to 22.00, 0.2 g of CBN in the fraction from the
minute 22.00 to 24.00 and 0.32 g
of THC in the fraction from the minute 24.00 to 28.00. The purity of the
evaporated fractions was > 95%
after dryness of the THCV and THC.
[125] To further purify the THC fraction, 1.2 g of the THC with purity >90%
but contaminated with CBC
was purified with flash chromatography on a column of 120 g of C18 bounded
silica using a
acetonitrile:water in gradient mode and a mobile phase at flow of 40m1/min.
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TIME (min) % Water (10% acetonitrile) % Acetonitrile (10%
water)
0 35 65
11 15 85
20 0 100
30 0 100
30.1 35 65
[126] The total run was 30.1 minutes and the total solvent use was 1.2 L. We
obtain 1.1 g of THC in the
fraction from minute 24.00 to 28.00 and 0.05 g of CBC in the fraction from the
minute 22.00 to 24.10. The
purity of the evaporated fraction was > 97.5% after dryness of the THC. 5
injections of 1.2 g were made in
the same 120 g column of 018 bounded silica obtaining 5.4 g of THC >97.5%
purity.
Example 10:
Purification of Cannabinoids THC and THCV using Centrifugation Partitioning
Chromatography
[127] Maceration of 100 g of plant material of the variety Raquel with
THC/THCV was carried out in 1 L
of hexane for one hour. This procedure was repeated two times with 0.75 L of
hexane. The plant material
was filtered, and the hexane evaporated down completely to achieve 16 g of dry
extract. This 16 g of extract
was decarboxylated at 120 C for 2 hours obtaining 14 g of decarboxylated
extract. 5 g of the extract was
dissolved with hexane at a volume of 50 mL and then used as a sample for
injection in the CPC 1000 PRO
(Gilson) before called CPC-Quantum (Armen) of 1 L rotor volume. We use the
biphasic solvent system
hexane:acetonitrile at proportions of 1:1, the flow rate of the mobile phase
(hexane phase) is 200 mL/min
during the run, and changed to 350 mL/min in the extrusion and change of the
stationary phase (acetonitrile
phase). The complete run last 31 minutes, with 2 phases: run with pumping
mobile phase that is from 25
minutes and an extrusion phase of 4 minutes. The use of solvent per run is
from 5.0 L of hexane and 1.4 L
of acetonitrile phase. The total solvent used is from 6.4 L per run. We obtain
2.3 g of THC in the fraction
from minute 13.23 to 17.30 and 0.51 g of THCV in the fraction from the minute
21.55 to 24.10. The purity
of the evaporated fractions was > 95% after dryness of the THC and THCV.
Example 11:
Purification of Cannabinoids THC and THCV using Centrifugation Partitioning
Chromatography
[128] Maceration of 100 g of plant material of the variety Raquel with
THC/THCV was carried out in 1 L
of hexane for one hour. This procedure was repeated two times with 0.75 L of
hexane. The plant material
was filtered, and the hexane evaporated down completely to achieve 16 g of dry
extract. This 16 g of extract
was decarboxylated at 120 C for 2 hours obtaining 14 g of decarboxylated
extract. 5 g of the extract was
dissolved with hexane at a volume of 50 mL and then used as a sample for
injection in the CPC 1000 PRO
(Gilson) before called CPC-Quantum (Armen) of 1 L rotor volume. We use the
biphasic solvent system
hexane:ethyl acetate:acetonitrile:water at proportions of 9:1:9:1, the flow
rate of the mobile phase (hexane:
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ethyl acetate phase) is 200 mL/min during the run, and changed to 350 mL/min
in the extrusion and change
of the stationary phase (acetonitrile:water phase). The complete run last 21
minutes, with 2 phases: run
with pumping mobile phase that is from 17 minutes and an extrusion phase of 4
minutes. The use of solvent
per run is from 3.4 L of hexane:ethyl acetate phase and 1.4 L of
acetonitrile:water phase. The total solvent
used is from 4.8 L per run. We obtain 2.0 g of THC in the fraction from minute
13.23 to 17.30 and 0.40 g of
THCV in the fraction from the minute 21.55 to 24.10. The purity of the
evaporated fractions was > 95% after
dryness of the THC and > 90% of the THCV. The 14g of extract was purified by 3
runs of CPC obtaining
1.2g of THCV fraction.
[129] To further purify the THCV fraction, 1.2 g of the THCV with purity >90%
but contaminated with CBN
was purified with flash chromatography on a column of 120 g of 018 silica
using a acetonitrile:water in
gradient mode and a mobile phase at flow of 40m1/min.
TIME (min) % Water (10% acetonitrile) % Acetonitrile (10%
water)
0 35 65
11 15 85
20 0 100
24 0 100
24.1 35 65
[130] The total run was 24 minutes and the total solvent use was 0.96 L. We
obtain 0.84 g of THCV in
the fraction from minute 20.00 to 22.00 and 0.40 g of CBN in the fraction from
the minute 22.00 to 24.10.
The purity of the evaporated fraction was > 95% after dryness of the THCV.
[131] The starting material is cannabis extract (whatever solvent is used in
the production of the extract)
and if it is decarboxylated or not, even if it is "winterized" (solved in
ethanol, chilled at 4 C or -20 C and
filtered from the precipitated material) or not.
[132] We use different proportions of the solvents in the biphasic solvent
system depending of which
cannabinoid we want to purify: To purify THC, THCA, THCV, THCVA, CBN or CBV we
use the biphasic
system hexane:ethanol:water at proportions of 20:17:3 in volume or
pentane:acetonitrile or
hexane:acetonitrile with or without the use of ethyl acetate and or water as
modifiers. To purify CBD, CBDA,
CBDVA and/or CBDV we use the biphasic system hexane:ethanol:water at
proportions of 20:14:6 in
volume. To purify CBG, CBGA, CBGVA and/or CBGV we use the biphasic system
hexane:ethanol:water
at proportions of 20:12:8 in volume.
[133] We use a CPC-Quantum (ARMEN) or CPC 1000 PRO (GILSON) of 1 L rotor
volume, the sample
injection is 50 mL (the g of extract depends on extract type and solvent
system used), the flow rate of the
mobile phase (hexane or pentane phase) is 200 mL/min during the run, and
changed to 350 mL/min in the
extrusion and change of the stationary phase (ethanolic or acetonitrile
phase). The complete run varies
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from 12 to 31 minutes, with 2 phases: run with pumping mobile phase that is
from 8 to 27 minutes and an
extrusion phase of 4 minutes. The use of solvent per run is from 1.6 to 5.4 L
of hexane phase and 1.4 L of
ethanolic or acetonitrile phase. The total solvent used is from 3 to 6.8 per
run.
[134] Using extracts with the main cannabinoid at 40% to 60% the maximum load
of a THC-Type extract
is 5 g/L of rotor. The maximum load for a CBD-Type extract is 12g/L of rotor.
The maximum load for CBG-
Type extract is 15g/L of rotor.
[135] Using extracts with the main cannabinoid at 40% to 60% the maximum
recovery or yield of a THC-
Type extract in pure cannabinoid is 2.8 g/run. The maximum recovery or yield
of a CBD-Type extract is 6g/
run. The maximum recovery or yield of a CBG-Type extract is 7g/L of rotor.
[136] In closing, it is to be understood that although aspects of the present
specification are highlighted
by referring to specific embodiments, one skilled in the art will readily
appreciate that these disclosed
embodiments are only illustrative of the principles of the subject matter
disclosed herein. Therefore, it
should be understood that the disclosed subject matter is in no way limited to
a particular compound,
composition, article, apparatus, methodology, protocol, and/or reagent, etc.,
described herein, unless
expressly stated as such. In addition, those of ordinary skill in the art will
recognize that certain changes,
modifications, permutations, alterations, additions, subtractions and sub-
combinations thereof can be made
in accordance with the teachings herein without departing from the spirit of
the present specification. It is
therefore intended that the following appended claims and claims hereafter
introduced are interpreted to
include all such changes, modifications, permutations, alterations, additions,
subtractions and sub-
combinations as are within their true spirit and scope.
[137] Certain embodiments of the present invention are described herein,
including the best mode known
to the inventors for carrying out the invention. Of course, variations on
these described embodiments will
become apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventor
expects skilled artisans to employ such variations as appropriate, and the
inventors intend for the present
invention to be practiced otherwise than specifically described herein.
Accordingly, this invention includes
all modifications and equivalents of the subject matter recited in the claims
appended hereto as permitted
by applicable law. Moreover, any combination of the above-described
embodiments in all possible
variations thereof is encompassed by the invention unless otherwise indicated
herein or otherwise clearly
contradicted by context.
[138] Groupings of alternative embodiments, elements, or steps of the present
invention are not to be
construed as limitations. Each group member may be referred to and claimed
individually or in any
combination with other group members disclosed herein. It is anticipated that
one or more members of a
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group may be included in, or deleted from, a group for reasons of convenience
and/or patentability. When
any such inclusion or deletion occurs, the specification is deemed to contain
the group as modified thus
fulfilling the written description of all Markush groups used in the appended
claims.
[139] Unless otherwise indicated, all numbers expressing a characteristic,
item, quantity, parameter,
property, term, and so forth used in the present specification and claims are
to be understood as being
modified in all instances by the term "about." As used herein, the term
"about" means that the characteristic,
item, quantity, parameter, property, or term so qualified encompasses a range
of plus or minus ten percent
above and below the value of the stated characteristic, item, quantity,
parameter, property, or term.
Accordingly, unless indicated to the contrary, the numerical parameters set
forth in the specification and
attached claims are approximations that may vary. For instance, as mass
spectrometry instruments can
vary slightly in determining the mass of a given analyte, the term "about" in
the context of the mass of an
ion or the mass/charge ratio of an ion refers to +/-0.50 atomic mass unit. At
the very least, and not as an
attempt to limit the application of the doctrine of equivalents to the scope
of the claims, each numerical
indication should at least be construed in light of the number of reported
significant digits and by applying
ordinary rounding techniques.
[140] Use of the terms "may" or "can" in reference to an embodiment or aspect
of an embodiment also
carries with it the alternative meaning of "may not" or "cannot." As such, if
the present specification
discloses that an embodiment or an aspect of an embodiment may be or can be
included as part of the
inventive subject matter, then the negative limitation or exclusionary proviso
is also explicitly meant,
meaning that an embodiment or an aspect of an embodiment may not be or cannot
be included as part of
the inventive subject matter. In a similar manner, use of the term
"optionally" in reference to an embodiment
or aspect of an embodiment means that such embodiment or aspect of the
embodiment may be included
as part of the inventive subject matter or may not be included as part of the
inventive subject matter.
Whether such a negative limitation or exclusionary proviso applies will be
based on whether the negative
limitation or exclusionary proviso is recited in the claimed subject matter.
[141] Notwithstanding that the numerical ranges and values setting forth the
broad scope of the invention
are approximations, the numerical ranges and values set forth in the specific
examples are reported as
precisely as possible. Any numerical range or value, however, inherently
contains certain errors necessarily
resulting from the standard deviation found in their respective testing
measurements. Recitation of
numerical ranges of values herein is merely intended to serve as a shorthand
method of referring
individually to each separate numerical value falling within the range. Unless
otherwise indicated herein,
each individual value of a numerical range is incorporated into the present
specification as if it were
individually recited herein.
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[142] The terms "a," "an," "the" and similar references used in the context of
describing the present
invention (especially in the context of the following claims) are to be
construed to cover both the singular
and the plural, unless otherwise indicated herein or clearly contradicted by
context. Further, ordinal
indicators ¨ such as "first," "second," "third," etc. ¨ for identified
elements are used to distinguish between
the elements, and do not indicate or imply a required or limited number of
such elements, and do not
indicate a particular position or order of such elements unless otherwise
specifically stated. All methods
described herein can be performed in any suitable order unless otherwise
indicated herein or otherwise
clearly contradicted by context. The use of any and all examples, or exemplary
language (e.g., "such as")
provided herein is intended merely to better illuminate the present invention
and does not pose a limitation
on the scope of the invention otherwise claimed. No language in the present
specification should be
construed as indicating any non-claimed element essential to the practice of
the invention.
[143] When used in the claims, whether as filed or added per amendment, the
open-ended transitional
term "comprising" (and equivalent open-ended transitional phrases thereof like
including, containing and
having) encompasses all the expressly recited elements, limitations, steps
and/or features alone or in
combination with unrecited subject matter; the named elements, limitations
and/or features are essential,
but other unnamed elements, limitations and/or features may be added and still
form a construct within the
scope of the claim. Specific embodiments disclosed herein may be further
limited in the claims using the
closed-ended transitional phrases "consisting of" or "consisting essentially
of" in lieu of or as an amended
for "comprising." When used in the claims, whether as filed or added per
amendment, the closed-ended
transitional phrase "consisting of" excludes any element, limitation, step, or
feature not expressly recited in
the claims. The closed-ended transitional phrase "consisting essentially of"
limits the scope of a claim to
the expressly recited elements, limitations, steps and/or features and any
other elements, limitations, steps
and/or features that do not materially affect the basic and novel
characteristic(s) of the claimed subject
matter. Thus, the meaning of the open-ended transitional phrase "comprising"
is being defined as
encompassing all the specifically recited elements, limitations, steps and/or
features as well as any optional,
additional unspecified ones. The meaning of the closed-ended transitional
phrase "consisting of" is being
defined as only including those elements, limitations, steps and/or features
specifically recited in the claim
whereas the meaning of the closed-ended transitional phrase "consisting
essentially of" is being defined as
only including those elements, limitations, steps and/or features specifically
recited in the claim and those
elements, limitations, steps and/or features that do not materially affect the
basic and novel characteristic(s)
of the claimed subject matter. Therefore, the open-ended transitional phrase
"comprising" (and equivalent
open-ended transitional phrases thereof) includes within its meaning, as a
limiting case, claimed subject
matter specified by the closed-ended transitional phrases "consisting of" or
"consisting essentially of." As
such embodiments described herein or so claimed with the phrase "comprising"
are expressly or inherently
unambiguously described, enabled and supported herein for the phrases
"consisting essentially of" and
"consisting of."
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[144] All patents, patent publications, and other publications referenced and
identified in the present
specification are individually and expressly incorporated herein by reference
in their entirety for the purpose
of describing and disclosing, for example, the compositions and methodologies
described in such
publications that might be used in connection with the present invention.
These publications are provided
solely for their disclosure prior to the filing date of the present
application. Nothing in this regard should be
construed as an admission that the inventors are not entitled to antedate such
disclosure by virtue of prior
invention or for any other reason. All statements as to the date or
representation as to the contents of these
documents is based on the information available to the applicants and does not
constitute any admission
as to the correctness of the dates or contents of these documents.
[145] Lastly, the terminology used herein is for the purpose of describing
particular embodiments only,
and is not intended to limit the scope of the present invention, which is
defined solely by the claims.
Accordingly, the present invention is not limited to that precisely as shown
and described.
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