DECARBOXYLATED CANNABIS RESINS, USES THEREOF AND METHODS OF MAKING SAME
20190038663 ยท 2019-02-07
Inventors
- Lakshmi Premakanth KOTRA (Toronto, CA)
- Melissa Maureen Lewis (Brampton, CA)
- Ewa Wasilewski (Etobicoke, CA)
- Har Grover (Toronto, CA)
Cpc classification
A61K2236/00
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
B01D11/0211
PERFORMING OPERATIONS; TRANSPORTING
A61K31/352
HUMAN NECESSITIES
International classification
A61K31/352
HUMAN NECESSITIES
Abstract
The disclosure relates to decarboxylated cannabis resins and methods of making the decarboxylated cannabis resins by extraction and decarboxylation of cannabinoids from Cannabis species using microwaves and solvents. The disclosure also relates to use of the decarboxylated cannabis resins for making pharmaceutical products comprising same.
Claims
1. A decarboxylated cannabis resin, wherein the resin comprises decarboxylated cannabinoids from a Cannabis spp. plant, such that 9-tetrahydrocannabidiolic acid (9-THCA) and cannabidiolic acid (CBDA) cannabinoids in the plant are each independently 90% to 100% decarboxylated to yield 9-tetrahydrocannabinol (THC) and cannabidiol (CBD) respectively in the resin.
2. The decarboxylated cannabis resin of claim 1, wherein the resin further comprises cannabinol (CBN).
3. The decarboxylated cannabis resin of claim 1, where the resin is substantially free of solvent.
4. A pharmaceutical product comprising (i) the decarboxylated cannabis resin of claim 1 and (ii) a suitable pharmaceutically acceptable carrier or excipient.
5. A natural health product comprising the decarboxylated cannabis resin of claim 1.
6. A method of extracting and decarboxylating cannabinoids from cannabis, the method comprising: (i) extracting cannabinoids by contacting the cannabis with a solvent thereby forming a cannabis extract; and (ii) decarboxylating the cannabinoids in the cannabis extract by subjecting the cannabis extract to microwaves at temperature of about 100-200 C. in a sealed container for a time period sufficient to form the corresponding decarboxylated cannabinoids in the cannabis extract.
7. The method of claim 6, wherein before the step (i) of extracting, the cannabis is broken down to produce cannabis of a size and form suitable for extraction.
8. The method of claim 6, wherein the extracting and decarboxylating steps occur concurrently.
9. The method of claim 6, wherein the solvent is selected from the group consisting of: 80-100% ethanol, ethylene glycol, isopropanol, and a combination of any of the above.
10. The method of claim 6, wherein the cannabis is completely submerged in the solvent in the sealed container.
11. The method of claim 6, wherein the time period is about 15-75 minutes and wherein the temperature is between about 130 C. to about 180 C.
12. The method of claim 6, wherein the microwaves have a frequency of about 2.45 GHz.
13. The method of claim 6, wherein the decarboxylating step (ii) occurs under a pressure of about 2-22 bar.
14. The method of claim 6, comprising the step of (iii) removing the solvent from the cannabis extract, thereby producing a cannabis resin.
15. The method of claim 6, wherein the extracted and decarboxylated cannabinoids are recovered in the form of isolated compounds.
16. The method of claim 14, wherein the cannabis resin is subjected to winterization for the removal of waxes.
17. The method of claim 1, wherein the cannabis is dried cannabis.
18. (canceled)
19. The method of claim 6, wherein the cannabis is a plant part selected from the group consisting of a trichome, a cannabis female inflorescence, a flower bract, a cannabis stalk, a cannabis leaf, and combinations thereof.
20. (canceled)
21. The method of claim 6, wherein the cannabis remains in contact with the solvent during the decarboxylating step.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0044] In order that the subject matter may be readily understood, embodiments are illustrated by way of examples in the accompanying drawings, in which:
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DETAILED DESCRIPTION
[0078] As previously described, tetrahydrocannabinol (THC), or more precisely its main isomer .sup.9-THC, is the principal psychoactive constituent (or cannabinoid) of cannabis. Unlike THC, its corresponding acid THC-A (or .sup.9-THC-A for its main isomer) is a non-psychoactive cannabinoid found in raw and live cannabis.
[0079] .sup.9-THC is only one of the family of compounds known as cannabinoids. For example, .sup.8-THC is a double bond isomer of .sup.9-THC and is a minor constituent of most varieties of cannabis. The major chemical difference between the two compounds is that .sup.9-THC is easily oxidized to cannabinol (CBN) whereas .sup.8-THC does not oxidize to cannabinol as it is very stable. .sup.8-THC, for the most part, produces similar psychometric effects as does .sup.9-THC, but is generally considered to be 50% less potent than .sup.9-THC. On the other hand, CBD has no or limited psychometric activity on its own when administered to humans, however as discussed below CBD comprises other therapeutic qualities. In the cannabis plant, CBGA is a precursor and cannabinol (CBN), a metabolite of THC. As cannabis matures, THC gradually breaks down to CBN which also has psychoactive properties.
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[0082] Turning to
[0083] The extracted and decarboxylated cannabinoids are optionally recovered. Recovery can include filtering the solvent from the extract of cannabis plant material to isolate the decarboxylated cannabinoids.
[0084] In some embodiments, the process comprises an extraction step before, during or after decarboxylation. In some embodiments, the process of the disclosure comprises more than one extraction step.
[0085] In some embodiments, the decarboxylation step can occur before, during or after extraction.
[0086] In an embodiment of the disclosure, a one-step method for extraction and decarboxylation is used, wherein the cannabis plant material (that is suitably prepared, e.g. optionally dried and broken down as described herein) is placed in a suitable extracting solvent (e.g. a pharmaceutically acceptable solvent such as ethanol, glycerol, and isopropanol, and other solvents as is known to those skilled in the art, kept in suspension or solution (e.g. by stirring, agitation, shaking or other means known to those skilled in the art) and subjected to microwave radiation while stirring at a temperature, pressure and time to obtain suitably extracted and decarboxylated cannabinoids that can be recovered for use as either a mixture or individual chemical components (e.g. for use as a therapeutic or pharmaceutical product). Further in some embodiments, the solvent can be food grade oil and/or a medium chain triglyceride, for example, coconut oil.
[0087] Such a method is more efficient in converting the cannabinoid acid into its decarboxylated form than other known methods of extraction and decarboxylation (such as simple heating).
[0088] Particularly,
[0089]
Drying 102
[0090] At step 102, cannabis plant material can be dried to reduce water/moisture content. Herein, cannabis plant material encompasses any cannabis plant, including but not limited to Cannabis sativa, Cannabis indica and Cannabis ruderalis, and all subspecies thereof (for example, Cannabis sativa subspecies indica including the variants var. indica and var. kafiristanica), including wild or domesticated type Cannabis plants and also variants thereof, including cannabis chemovars (varieties characterized by virtue of chemical composition) which naturally contain different amounts of the individual cannabinoids 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. Cannabis plant material includes live or fresh cannabis and dried cannabis. In addition, any part of the cannabis plant may be used, including but not limited to trichomes, flower buds, flower bracts, leaves, stalk and any other plant part that may contain cannabinoids. Also, although the female plants may produce a higher concentration of cannabinoids than male plants, both female (including feminized plants) and male plants can be used.
[0091] Optional drying step 102 can be used to remove excess moisture from the cannabis plant material prior to the cannabis plant material undergoing extraction and decarboxylation. Removing water content from the cannabis plant material can help to provide even heating at later stages in the extraction and/or decarboxylation process. Alternatively, fresh cannabis plant material (e.g. from the plant directly) can be used for the subsequent break down, extraction and decarboxylation steps. Herein, drying of the cannabis plant material at step 102 can occur by any means, for example in an oven at temperatures in the range of 60-75 C, or similar conditions, or using a vacuum oven or similar conditions over several hours, for example 4 hours, or 6 hours or 8 hours, depending on the amount of moisture. During the drying process, when heating is employed using heating elements in the ovens or infrared heating among other processes, some of the cannabinoid carboxyl acids forms could be converted into their decarboxylated cannabinoid forms.
Break Down 104
[0092] At break down step 104, cannabis plant material can be broken down to produce a cannabis plant material of a size and form suitable for extraction and decarboxylation by subjecting to microwave heating.
[0093] Trichomes (i.e. resin glands) of the cannabis plant material are nearly microscopic, mushroom-like protrusions from the surface of the buds, fan leaves, and the stalk. While relatively complex, trichomes are comprised primarily of a stalk and a head. The production of cannabinoids such as THC occurs predominantly in the head of the trichome. Cannabinoids are concentrated in the trichomes of the plant. The trichome is built to easily shed from the cannabis plant material surface.
[0094] The term a size and form suitable for extraction and decarboxylation refers to a reduction in the particle size of the cannabis plant material fragments.
[0095] Herein, breakdown of the cannabis plant material at step 104 can occur by any mechanical means including by crushing, smashing, grinding, pulverizing, macerating, disintegration or equivalent processes as are known to those skilled in the art that reduce the cannabis plant material into small pieces suitable in size and form for extraction and/or decarboxylation.
[0096] In one example embodiment, sonication can also be used to loosen the cannabis plant material in contact with an appropriate solvent such as ethanol, and/or by breaking down cellular membranes making it suitable for extraction and/or decarboxylation. In another example embodiment, maceration can be performed with a mortar and pestle to produce a cannabis plant material of a size and form suitable for extraction and/or decarboxylation.
[0097] In certain embodiments, the cannabis plant material is reduced in size such that its particle size is within a range of 1 mm to 10 mm.
Extraction 106
[0098] Upon completion of break down step 104, extraction step 106 may be performed. It should be noted that extraction step 106 may occur as a separate step to decarboxylation step 108 either before or after decarboxylation step 108, or as will be described below, extraction step 106 and decarboxylation step 108 may occur concurrently. For the avoidance of doubt it should be understood that any extraction, including but not limited to sonication in the presence of a solvent, reflux (Soxhlet) extraction and supercritical fluid extraction (SFE) may occur before or after decarboxylation step 108. In addition, it should also be understood that break down (104), extraction (106) and decarboxylation (108) may also occur in one step.
[0099] In one embodiment, extraction step 106 can comprise contacting cannabinoids from the broken down cannabis material that is the product of break down step 104 with a solvent.
[0100] In some embodiments, the solvent treatment in extraction step 106 removes non-cannabinoid impurities to leave a substantially pure preparation of cannabinoids. It has been shown that non-polar, liquid solvents may be useful for this function. Suitable non-polar solvents therefore include essentially any non-polar solvents which are substantially less polar than the cannabinoids, such that impurities which are more polar than the cannabinoids are removed by treatment with the solvent. Filtration and other methods as is known to those skilled in the art can also be used to remove impurities.
[0101] Useful non-polar solvents include, but are not limited to, C5-C12 straight chain or branched chain alkanes, or carbonate esters of C1-C12 alcohols. The more volatile C5-C12 alkanes may be particularly useful, as they are more easily removed from the extract. Further, solvents that have been approved for use in pharmaceutical compositions, such as ethanol (e.g. 95% ethanol) may be particularly useful.
[0102] Particularly useful solvents include pentane, hexane, heptane, iso-octane and ethanol, and/or mixtures thereof or the like as is known to those skilled in the art.
[0103] In one embodiment of extraction step 106, broken down cannabis plant material can be added to a solvent and concurrently sonicated.
[0104] Herein, sonication refers to the application of ultrasonic vibration (e.g. >20 kHz) to fragment cells, macromolecules and membranes of the dried or undried cannabis plant material. Ultrasonic vibration can be provided by any means known in the art.
[0105] In one exemplary embodiment, sonication of a mixture of cannabis plant material and solvent can occur for 5-25 minutes at 25 C., where the ratio of cannabis plant material and solvent is such that all cannabis plant material is submerged in the solvent completely in the reaction vessel.
[0106] Upon the completion of sonication of the mixture of cannabis plant material and solvent, the solvent is removed from the mixture. Removal of the solvent can occur by any means known in the art, including but not limited to filtration and/or evaporation. One embodiment for filtering after sonication is vacuum filtering over a glass sintered funnel to separate the resultant extract in the filtrate and the plant material. The latter can then be subjected to further extractions such as Soxhlet or other solvent extractions as is known to those skilled in the art, for example, SFE.
[0107] In yet another embodiment of extraction step 106, cannabinoids can be extracted from cannabis plant material that is broken down in step 104 by reflux (Soxhlet) extraction.
[0108] During reflux (Soxhlet) extraction, cannabis plant material that is broken down in step 104 is generally suspended above a heated solvent in a receptacle. The solvent is heated to reflux in a distillation flask such that solvent vapor travels up a distillation arm and floods into the receptacle housing raw cannabis material. A condenser suspended above the raw cannabis material ensures that any solvent vapor rising above the raw cannabis material cools and subsequently drips back down into the receptacle housing the raw cannabis material. The receptacle slowly fills with warm solvent such that cannabinoids begin to dissolve into the warm solvent. When the receptacle fills, it is emptied by a siphon such that the solvent is returned to the distillation flask. This cycle may be allowed to repeat many times, over hours or days.
[0109] Preferably, reflux (Soxhlet) extraction occurs at a solvent temperature higher than the boiling point of the corresponding solvent used for extraction and is conducted over a period of approximately 3 to 5 hours.
[0110] Once extraction is complete, removal of the solvent can occur by any means known in the art, including but not limited to filtering and/or evaporation as previously described.
[0111] In place of either sonication or reflux (Soxhlet) extraction as described above, another embodiment of extraction step 106 encompassed by the subject application is the extraction of cannabinoids from cannabis plant material by SFE.
[0112] SFE refers to a process of separating one or more components (extractant) from another (matrix) using supercritical fluids as the extracting solvent. Extraction is usually from a solid matrix (e.g. cannabis plant material), but can also be from liquids or resinous material (for example, hash oil).
[0113] Although numerous supercritical fluids can be used, carbon dioxide (CO.sub.2) is the most commonly used supercritical fluid for SFE. In other exemplary embodiments, CO.sub.2 can be modified by co-solvents such as ethanol or methanol as is known to those skilled in the art.
[0114] Extraction conditions for supercritical fluids are above the critical temperature (for example, 31 C. for CO.sub.2) and critical pressure (for example, 74 bar for CO.sub.2). Addition of modifiers such as but not limited to ethanol can require altering these extraction conditions.
[0115] An exemplary SFE system contains a pump for CO.sub.2 (as well as any other solvents), a pressure cell to contain the cannabis material, a means of maintaining pressure in the system and a collecting vessel. The liquid is pumped to a heating zone, where it is heated to supercritical conditions. It then passes into the extraction vessel, where it rapidly diffuses into the solid matrix and dissolves the cannabis material to be extracted. The dissolved material (for example, cannabinoids) is swept from the extraction cell into a separator at lower pressure, and the extracted material settles out. The CO.sub.2 can then be cooled, re-compressed and recycled, or discharged to atmosphere.
[0116] Herein, the temperature of the SFE extraction performed at extraction step 106 can, in some embodiments, be in the range of 35-55 C.
[0117] Further the pressure the SFE extraction performed at extraction step 106 can in some embodiments be in the range of 65-85 bar.
[0118] SFE in the present disclosure occurs at about 40 C. with a back pressure regulator pressure of 12 MPa and the extracted compounds are monitored using a photodiode array of 200-600 nm (monitoring at 254 nm). The acquisition time and method times of the system can each vary by a few minutes up to 60 minutes, ideally between 15 and 30 minutes, depending on the ratio of supercritical fluid and the co-solvent is altered for the extraction.
[0119] In specific embodiments, SFE can be carried out multiple times in succession. In such embodiments, the SFE is a fractional SFE.
[0120] As previously described for sonication with a solvent and reflux (Soxhlet) extraction, once SFE is complete, removal of the solvent can occur by any means known in the art, including but not limited to filtering and/or evaporation.
Microwave-Assisted Extraction and Decarboxylation 108
[0121] Decarboxylation of phytocannabinoid acids such as .sup.9-THC-A is a function of the time and temperature of the reaction. For instance, the decarboxylation of concentrated .sup.9-THC-A in solution into .sup.9-THC and the degradation of .sup.9-THC vary with temperature. Temperature controls are therefore important for controlling desired ratios of decarboxylation products. The use of conventional household microwaves in the processing of cannabis has been discussed in the literature, however, with mixed, inconsistent results and not necessarily specifically for extraction in a solvent and decarboxylation. Further, in order to obtain 100% decarboxylation, the temperature must be sustained over a period of time without burning of the cannabis material or boiling/evaporation of the solvent. If the temperature is higher than the boiling point of the solvent employed, the solvent will boil over and/or evaporate. In order to sustain the temperature over the required period of time to fully decarboxylate the cannabinoids but not burn the cannabis plant material or boil/evaporate the solvent with the cannabis, the microwave vessel (i.e. the sealed container) must be under pressure. Sealing the vessel or container ensures pressure in the vessel or container.
[0122] As shown in
[0123] Microwave assisted extraction and decarboxylation 108 can comprise suspending cannabis plant material in a solvent and subjecting the mixture to microwaves in a closed container at a temperature, pressure and time sufficient to form decarboxylated cannabinoids.
[0124] Herein, the term microwaves refer to a form of electromagnetic radiation with wavelengths ranging from one meter to one millimeter; with frequencies between 300 MHz (100 cm) and 300 GHz (0.1 cm).
[0125] Further, solvent treatment in microwave assisted extraction and decarboxylation step 108 is again to remove non-cannabinoid impurities to leave a substantially pure preparation of cannabinoids. As such, non-polar, liquid solvents are useful for this function. In one embodiment, ethanol is used as the liquid solvent in microwave assisted extraction and decarboxylation step 108. In another embodiment, 95% ethanol is used as the liquid solvent in microwave assisted extraction and decarboxylation step 108.
[0126] As ethanol has a boiling point of 78 C. and the decarboxylation process of cannabis is temperature dependent (as described above), temperature control is important in microwave-assisted extraction step 108.
[0127] In one exemplary embodiment, suitable conditions to promote decarboxylation of CBDA and THCA to CBD and THC, respectively, are to suspend the cannabis plant material in a solvent (such as ethanol) and then subject this mixture to electromagnetic radiation (for example, microwaves) of a wavelength in the range of 10.sup.6-10.sup.9 nm and a frequency of 300 MHz-300 GHz. In one embodiment, the conditions further include, for example, the following: temperature range of 40-250 C., temperature increase of 2-5 C./sec, pressure range of 0-20 bar (2 MPa, 290 psi), microwave power range of 0-400 W at 2.45 GHz or minor variations and adjustments to suit a particular solvent and/or reaction conditions to reach the required temperature and accomplish decarboxylation. If stirring is required, a variable magnetic stirrer (300-900 RPM) may be used.
[0128] In another exemplary embodiment, microwave assisted extraction and decarboxylation can be performed at a temperature in the range of 100-200 C. (including all possible integers and fractions of integers in this range, for example, 163.5 C.), 130-170 C., 150-170 C. or 130-150 C.
[0129] In another exemplary embodiment, microwave assisted extraction and decarboxylation can be performed at a pressure in the range of 2-22 bar (including all possible integers and fractions of integers in this range), for example, 10.7 bar or 17 bar or 18 bar or 19 bar or 20 bar or 21 bar.
[0130] The cannabis plant material can be suspended in a solvent and subjected to microwaves at frequency and wavelength of 2.45 GHz and 1.2210.sup.8 nm, respectively, and a temperature in the range of 130-190 C.
[0131] The raw cannabis material can be suspended in a solvent and subjected to microwaves at frequency and wavelength of 2.45 GHz and 1.2210.sup.8 nm, respectively, and a temperature in the range of 150-190 C. and the solvent is ethanol.
[0132] In another embodiment, cannabis plant material can be suspended in a solvent and the mixture stirred for a defined period of time (e.g. 0-30 sec or a reasonable length of time so as to suspend the material) before being subjected to microwaves. In one embodiment, the defined period is 30 seconds.
[0133] In another embodiment, cannabis material can be suspended in a solvent and the mixture can be stirred while being subjected to microwaves. In one embodiment, the defined period is 10 minutes and in another embodiment the defined period is 20 minutes. A table of working microwave variables is provided below for reference.
TABLE-US-00001 TABLE 1 Total Pre- Power Holding Reaction Temperature time stirring Stir rate supplied Power Pressure ( C.) (mins) (sec) (rpm) (W) (W) (Bar) 150 20 30 900 400 60 12 150 20 30 900 400 60 11 150 20 30 900 400 60 10 150 20 30 900 400 55 13 150 20 30 900 400 60 13 150 20 30 900 400 60 12 170 15 30 900 400 70 18 150 10 30 600 295 38 8.5 150 10 30 600 270 40 8.5 150 10 30 600 360 40 9 150 10 0 600 400 75 8 150 10 0 600 400 75 8.5 150 10 0 600 400 75 8.5 100 30 0 600 150 38 1 100 30 0 600 113 38 2 100 30 0 600 145 30 1 150 10 30 600 265 60 8 150 10 30 600 255 38 9 150 10 30 600 260 40 8.5 170 10 30 600 310 40 17 150 10 30 600 280 38 10 130 10 30 600 250 30 5 100 5 30 600 400 30 2 100 5 30 600 390 40 2
[0134] The parameters below can be set by the user, depending on the type of microwave equipment employed and the options available for user settings: [0135] Power (OFF)=Constant power or the maximum power applied when heating the reaction mixture. [0136] Initial Power (OFF)=Power applied initially when heating the reaction mixture. [0137] Fixed Hold Time (ON)=If ON, the time countdown starts when the target temperature or target pressure is reached, i.e. the initial time taken to reach the set temperature or pressure is not included in the heating time. [0138] Pressure (OFF)=Target pressure for reaction. [0139] Cooling (OFF)=If OFF, cooling is not applied during the heating process. [0140] Absorption (NORMAL)=If NORMAL, power applied is initially between 200 and 400 W, depending on the target temperature. [0141] Temperature=Target temperature. (Note: The temperature is monitored by an external infrared sensor that measures the surface temperature of the glass vial in real time.) [0142] Total time=Total time for all steps. [0143] Pre-stirring=Stirring time before heating process. [0144] Stir-rate=Rotational speed of magnetic stir bar.
[0145] The parameters below were observed and extrapolated.
[0146] Power supplied=Power used to achieve the target temperature.
[0147] Holding power=Power used to maintain the target temperature.
[0148] Reaction pressure=Maximum pressure during the reaction.
[0149] In some embodiments, the decarboxylated cannabinoid product can be used directly or further processed, purified or recovered prior to use.
Recovery 110
[0150] Optionally, after being subjected to microwaves, a preparation of decarboxylated cannabinoids can be recovered from the resulting suspension at recovery step 110.
[0151] In one embodiment, extracted and decarboxylated cannabinoids are recovered by filtering the solvent from the extract of cannabis plant material to isolate the decarboxylated cannabinoids or decarboxylated cannabinoid comprising fraction.
[0152] In another embodiment, extracted and decarboxylated cannabinoids are recovered by filtering through an appropriate Celite pad and/or activated carbon (e.g. charcoal) to obtain clarified solution for subsequent processing or use. In this embodiment, Celite can be placed in a glass sintered funnel and then layered with activated carbon. Filtering agents can be washed with ethanol via vacuum filtration and extract can be dissolved in appropriate volume of suitable solvent such as ethanol and transferred to a funnel. Vacuum can then be applied and the filtering agent can be washed with the solvent until cannabinoids are completely eluted. The resulting filtrate can then be concentrated to dryness (e.g. at 25 C.). Someone with skill in the art can also conceive employing functionalized membranes, cellulose filters or the like to accomplish the above recovery task, instead of Celite and activated carbon pad.
[0153] Alternatively, the resulting preparation of decarboxylated cannabinoids from step 108 can be collected and subsequently processed according to any of the extraction methods described in step 106, including but not limited to sonication, reflux (Soxhlet) extraction and/or SFE.
Uses and Products
[0154] The decarboxylated cannabis resins can be used (i) directly as a medicine, natural health product, or for recreational use, or (ii) as a raw material in the preparation of products, such as pharmaceutical, natural health products, or recreational use products for known uses of decarboxylated cannabinoid(s), such as known therapeutic or psychoactive uses.
[0155] In one embodiment, the decarboxylated resin of the disclosure can be used directly, for example as a medicine or as a natural health product.
[0156] In one embodiment, a pharmaceutical composition can be formed comprising the resulting decarboxylated cannabinoid resin produced by the methods disclosed, which can further comprise a suitable pharmaceutical acceptable carrier or excipient.
[0157] The pharmaceutical compositions of the present disclosure can additionally comprise one or more further active pharmaceutical ingredients in addition to decarboxylated cannabinoids.
[0158] Pharmaceutical compositions can be prepared in various dosage forms depending on the desired use and mode of administration, whether it is oral (e.g., tablet or liquid forms), a mist or other forms (aerosol, inhaler or intravenous suitable formulations), as desired, using techniques well known in the art.
[0159] Pharmaceutically acceptable carriers or excipients for various different dosage forms are well-known in the art and include carriers, diluents, fillers, binders, lubricants, disintegrants, glidants, colorants, pigments, taste masking agents, sweeteners, flavorants, plasticizers, and any acceptable auxiliary substances such as absorption enhancers, penetration enhancers, surfactants, co-surfactants, and specialized oils. The proper excipient(s) is (are) selected based in part on the dosage form, the intended mode of administration, the intended release rate, and manufacturing reliability. Examples of common types of excipients include various polymers, waxes, calcium phosphates, and sugars.
[0160] A person of skill in the art would reference known methods of pharmaceutical preparations, such as described in Remington: The Science and Practice of Pharmacy, 22.sup.nd Edition, Edited by Allen, Loyd V., Jr, 2012.
[0161] The present disclosure is described in the following Examples, which are set forth to aid in the understanding of the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims which follow thereafter.
Examples
[0162] Experiment 1: Decarboxylated Cannabis Resin
[0163] Medicinal cannabis was subjected to extraction and chemical analysis by UPLC-MS.
[0164] Extraction Methodologies
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[0166] UPLC-MS Methodology. Cannabinoid standards, cannabis extracts and cannabinoids in the donor samples were analyzed using Waters ACQUITY UPLC H-Class System equipped with Quaternary Solvent Manager and Sample Manager FTN. The detector used to monitor the samples was Waters MS 3100 mass spectrometer. Benzophenone, caffeine or .sup.9-THC-d3 was used as an internal standard. Conditions are listed in Table 2.
TABLE-US-00002 TABLE 2 UPLC-MS Chromatographic conditions UPLC-MS Conditions Column: BEH (2.1 50 mm, C.sub.18, 1.7 m) Mass scan range (ESI +ve and ve): 150-500 m/z Flow rate: 0.6 mL/min Solvent: H.sub.2O (0.1% formic acid; solvent A) MeOH (0.1% formic acid; solvent B) Gradient conditions: 0-4.5 min: 30% A/70% B - 100% B 4.5-5.0 min: 100% B 5.0-5.2 min: 100% B - 30% A/70% B 5.2-6.0 min: 30% A/70% B
[0167] Extraction Results.
TABLE-US-00003 TABLE 3 Quantities of extracts and major cannabinoids obtained from extraction Methods A-D of Strain I. Cannabinoid in resin (%) Plant Extract Total .sup.9-THC Total CBD Method used (g) isolated (g) (THCA + THC) (CBDA + CBD) A 1.51 0.453 31.2 0.5 66.1 1.4 B 2.00 0.616 16.7 0.2 33.3 0.6 C 1.00 0.277 30.4 0.9 59.3 1.9 D 1.01 0.211 29.4 7.6 33.8 8.6
TABLE-US-00004 TABLE 4 Quantities of extracts and major cannabinoids obtained after Method D (closed system microwave extraction). Extract Cannabinoid in resin (%) Strain Plant used (g) isolated (g) .sup.9-THC CBD I 1.01 0.0003 0.211 0.01 29.4 7.6 33.8 8.6 II 1.00 0.0004 0.154 0.004 1.9 0.2 37.6 4.1 III 1.00 0.002 0.210 0.009 49.8 0.9 0
[0168] Chemical Analyses by UPLC-MS.
[0169] Mass spectra of Strain 1 before (
[0170] Closed system microwave extraction provides simultaneously extraction and decarboxylation of the cannabinoids, as observed during chemical analysis by UPLC-MS.
[0171] Experiment 2: Further Comparison of Various Extraction Methods, Decarboxylation of Cannabinoids and Production of Standard Curves
[0172] Method 1A: Ultrasonic Extraction (Sonication).
[0173] General Procedure: [0174] 1. Dried plant material was weighed and macerated using a mortar and pestle. [0175] 2. Solvent was added (25-130 mL) and mixture sonicated for 5 mins at 25 C. [0176] 3. Solvent was decanted and filtered over a glass sintered funnel using a vacuum filtration. [0177] 4. Steps 2 and 3 were repeated twice with the remaining fibre material. [0178] 5. Filtrate was concentrated to dryness (at 25 C.) then weighed (green resin).
[0179] Table 5 below shows the results of sonication of three common strains of cannabis.
TABLE-US-00005 TABLE 5 Results of sonication extraction with various solvents on three strains of cannabis. Solvent Isopropanol/ Strain Hexanes hexanes (1:1) Ethanol Cannabis strain 1 Plant 0.256 1.0013 0.246 (THC: 7.18/CBD: Used (g) 1.5084 8.6) Resin 0.0617 0.4748 0.1802 produced (g) 0.4531 Cannabis strain 2 Plant N/A N/A 2.0018 (THC: 0/CBD: 9) Used (g) Resin N/A N/A 0.4795 produced (g) Cannabis strain 3 Plant N/A 2.9475 (THC: 18.6/CBD: 0) Used (g) Resin N/A N/A 1.1611 produced (g)
[0180] Method 1B: Filtration over Celite/Activated Carbon.
[0181] Following Method 1A (described above), extracts were subjected to filtration over Celite and activated carbon in order to eliminate the green colour of extracts. The results are shown in Table 6.
[0182] General Procedure: [0183] 1. Celite was placed in a glass sintered funnel, then layered with activated carbon. [0184] 2. Extract was dissolved in 1 mL ethanol and transferred to funnel. [0185] 3. Vial that contained extract was washed twice with 1.5 mL ethanol and transferred to funnel. [0186] 4. Vacuum was then applied and filtering agent washed with ethanol until filtrate was no longer UV active (60-70 mL). [0187] 5. Filtrate was concentrated to dryness (at 25 C.), then weighed (orange resin).
TABLE-US-00006 TABLE 6 Results of extract filtration with Celite/Activated Carbon for three strains of cannabis Strain Extract used (g) Extract isolated (g) Cannabis strain 1 0.2265 0.2095 (THC: 7.18/CBD: 8.6) Cannabis strain 2 0.2026 0.1717 (THC: 0/CBD: 9) Cannabis strain 3 0.4984 0.4561 (THC: 18.6/CBD: 0)
[0188] Method 2: Soxhlet Extraction
[0189] General Procedure: [0190] 1. Dried plant material was weighed and macerated using a mortar and pestle [0191] 2. Crushed material was then transferred to a cellulose extraction thimble (43123 mm; 2 mm thickness) [0192] 3. Thimble was then inserted into a large extractor (size: 55/50) [0193] 4. Solvent (400 mL) and stir bar were added to a round bottom flask, which was then placed in the suitable DrySyn heating block and connected to the extractor [0194] 5. Extractor was then connected to a large condenser (size: 55/50) and refluxing was done at 120 C. for 3.5 hrs [0195] 6. Once refluxing was complete, the solvent was concentrated to dryness (at 25 C.) then weighed (green resin)
[0196] Table 7 below provides the results of Soxhlet extraction of cannabis according to the forgoing procedure.
TABLE-US-00007 TABLE 7 Results of Soxhlet extraction of cannabis. Strain Amount of plant used (g) Extract isolated (g) Cannabis 2.0021 0.6163 (THC: 7.18/CBD: 8.6)
[0197] Method 3: Supercritical Fluid Extraction (SFE)
[0198] General Procedure: [0199] 1. Dried plant material was weighed and macerated using a mortar and pestle [0200] 2. Crushed plant material was transferred to a 10 mL extraction vessel and subjected to either of the following conditions below [0201] 3. All fractions were combined and concentrated to dryness (at 25 C.) then weighed (green resin)
[0202] Method 3A: SFE Conditions
TABLE-US-00008 Solvent A = CO.sub.2 Solvent B = ethanol Temperature = 40 C. BPR = 12 MPa PDA = 200-600 nm (monitoring at 254 nm) Acquisition time = 30 mins Method time = 30.2 mins [0203] i. 5 mins static with 1:1 A/B [0204] ii. 25 mins dynamic with 1:1 A/B (Flow rate=10 mL/min; make up pump=0.2 mL/min) [0205] iii. Fractions were collected every 5 mins
[0206] Method 3B: SFE Conditions
TABLE-US-00009 Solvent A = CO.sub.2 Solvent B = ethanol Temperature = 40 C. BPR = 12 MPa PDA = 200-600 nm (monitoring at 254 nm) [0207] i. 15 mins dynamic with 100% A (Flow rate=10 mL/min) [0208] Fractions were collected every 7.5 mins [0209] Acquisition time=15 mins Method time=15.2 mins [0210] ii. 30 mins dynamic with 80:20 A/B (Flow rate=10 mL/min; make up pump=1 mL/min) [0211] Fractions were collected every 5 mins [0212] Acquisition time=30 mins Method time=30.2 mins
[0213] Table 8 below shows the results of SFE according to the conditions outlined in Methods 3A and 3B for cannabis.
TABLE-US-00010 TABLE 8 Results of SFE extraction of cannabis according to two sets of conditions. Amount of Extract Strain Conditions plant used (g) isolated (g) Cannabis Method 3A 1.0047 0.2164 (THC: 7.18/CBD: 8.6) Method 3B 1.0043 0.2561
[0214] Method 4: Microwave-Assisted Extractions with Ethanol (MAE) Followed by SFE.
[0215] Suitable conditions to promote decarboxylation of CBDA and THCA to CBD and THC, respectively, were determined with MAE.
[0216] The solvent used for this extraction was ethanol. However, since ethanol has a boiling point of 78 C., the highest temperature that could be achieved when heating only ethanol in a sealed vessel under microwave conditions had to be determined.
[0217] General Procedure: [0218] 1. Ethanol (11 mL) and a stir bar were placed in a 20 mL microwave vial which was then sealed. [0219] 2. General microwave conditions: [0220] a. Pre-stirring=30 secs [0221] b. Run time=15 mins [0222] c. Absorption=Normal
[0223] The results are shown in Table 9 below.
TABLE-US-00011 TABLE 9 Determination of highest temperature that could be achieved when heating only ethanol in a sealed vessel under microwave conditions. Temperature Attempt # ( C.) Results 1 200 Maximum system pressure attained; Run aborted due to high pressure 2 150 Run completed 3 190 Maximum system pressure attained; Run aborted due to high pressure 4 180 Maximum system pressure attained; Run aborted due to high pressure 5 170 Run completed
[0224] Once the maximum temperature that ethanol could be heated was determined, the following conditions were performed with cannabis:
[0225] General Procedure: [0226] 1. Dried plant material was weighed and macerated using a mortar and pestle [0227] 2. Crushed plant material was transferred to a 20 mL or 5 mL microwave vial along with a stir bar [0228] 3. Ethanol (11 mL or 3 mL) was added to the vial such that the plant material was completely submerged. The vial was then sealed and subjected to the microwave conditions below: [0229] a. Pre-stirring=30 secs [0230] b. Run time=10 mins [0231] c. Absorption=Normal [0232] 4. The suspension was filtered and the filtrate and plant fibre collected separately [0233] 5. Filtrate was concentrated and plant fibre subjected to the SFE conditions below:
TABLE-US-00012 Solvent A = CO.sub.2 Solvent B = ethanol Temperature = 25 C. BPR = 12 MPa PDA = 200-600 nm (monitoring at 254 nm) Acquisition time = 20 mins Method time = 20.2 mins [0234] 6. Gradient from 100% A to 50%; 0.1 mins-15 mins (Flow rate=10 mL/min; make up pump=1 mL/min) [0235] 7. Fractions were collected every 7.5 mins
[0236] The results are shown in Table 10 below.
TABLE-US-00013 TABLE 10 Extraction of cannabis by Microwave and SFE at various microwave temperatures Extract Extract Temper- Amount isolated isolated ature of plant after after Strain Method ( C.) used (g) microwave (g) SFE (g) Cannabis spp. A 100 1.0012 0.3246 N/A (THC: 7.18/ B 130 0.2554 0.0592 0.0008 CBD: 8.6) C 150 0.2543 0.063 0.0005 D 170 0.2554 0.0644 0.0025
[0237] Table 14 (provided below) shows the analyses and quantification of the cannabinoids.
[0238] Method 5. SFE/Soxhlet/Sonication Extraction Followed by Microwave of the Resin (for Decarboxylation).
[0239] General Procedure: [0240] 1. Resin isolated from Methods 1A (ethanolic extract), 2 and 3A were dissolved in 3-3.5 mL ethanol and transferred to a 5 mL microwave vial. [0241] 2. A stir bar was added and the vial sealed and subjected to microwave conditions below: [0242] a. Temperature=150 C. [0243] b. Pre-stirring=30 secs [0244] c. Run time=10 mins [0245] d. Absorption=Normal [0246] 3. The reaction mixture was then concentrated.
[0247] The results are shown in Table 11 below.
TABLE-US-00014 TABLE 11 Weights of resins after subjecting to microwave heating. Resin isolated Amount of after Strain Method resin (g) microwave (g) Cannabis spp. 1A (Sonication) 0.2223 0.1692 (THC: 7.18/CBD: 8.6) 2 (Soxhlet) 0.2651 0.2211 3A (SFE; 50:50) 0.2164 0.1884
[0248] Chromatography Analyses (HPLC/MS/PDA)
[0249] The chromatographic profiles of the cannabis extracts were determined by LC-PDA-MS equipped with a Waters 2545 binary gradient module LC, Waters PDA2998 photodiode array detector (190-800 nm) and a Waters 3100 mass spectrometer (60-2000 Da).
[0250] LC was performed on an X-Bridge analytical C18 column (4.6 mm150 mm, 5 um I.D.) with 1.5 mL/min flow rate. Mass spectra were recorded using ESI (+ve) mode. The injection samples were filtered using Millex-GV Syringe Filters (0.22 m, EMD Millopore).
[0251] Chromatographic conditions were as follows: [0252] Mobile phase: [0253] A: Water/0.1% formic acid [0254] B: Methanol/0.1% formic acid [0255] Gradient: [0256] 0 to 25 min: 30% A/70% B.fwdarw.100% B [0257] 25 to 28 min: 100% B [0258] 28 to 30 min: 100% B.fwdarw.30% A/70% B [0259] 30 to 35 min: 30% A/70% B [0260] Injection volume: 10 L [0261] Flow rate: 1.5 mL/min [0262] Total run time: 35 min
[0263] UPLC/MS.
[0264] The cannabis extracts and cannabinoid standards were analyzed using Waters ACQUITY UPLC H-Class System equipped with Quaternary Solvent Manager, Sample Manager FTN, Acquity UPLC BEH column (2.150 mm, C18, 1.7 m). The sample injection plate and the column were maintained at 15 C. and 40 C., respectively. The detector used to monitor the samples was Waters MS 3100 mass spectrometer.
[0265] Chromatographic conditions were as follows:
[0266] Mobile phase: [0267] A: Water/0.1% formic acid [0268] B: Methanol/0.1% A formic acid [0269] Gradient: [0270] 0 to 4.5 min: 30% A/70% B.fwdarw.100% B [0271] 4.5 to 5.0 min: 100% B [0272] 5.0 to 5.2 min: 100% B.fwdarw.30% A/70% B [0273] 5.2 to 6.0 min: 30% A/70% B [0274] Injection volume: 2 L [0275] Flow rate: 0.6 mL/min [0276] Total run time: 6 min
[0277] Standard Curves for Cannabinoids:
[0278] Standard cannabinoids samples were purchased from Cerilliant-Certified Reference Standards in the form of 1.0 mg/mL solution in methanol.
[0279] 1. .sup.8-Tetrahydrocannabinol (.sup.8-THC, Cat #. T-032, Lot FE10011501)
[0280] 2. .sup.9-Tetrahydrocannabinol (.sup.9-THC, Cat #. T-005, Lot FE05271502)
[0281] 3. .sup.9-Tetrahydrocannabinolic acid A (THCA-A, Cat #. T-093, Lot ER02101506)
[0282] 4. .sup.2-Cannabidoil (CBD, Cat #. C-045, Lot FE012881502)
[0283] 5. Cannabidiolic acid (CBDA, Cat #. C-144. Lot FE0181602)
[0284] 6. Cannabinol (CBN, Cat #. C-046, Lot FE06081502)
[0285] The chemical structures of cannabinoids 1-6 are provided in
[0286] Working stock solution of each standard sample was prepared using water/0.1% formic acid and methanol/0.1% formic acid. The final concentration of each stock sample was 50 g/mL in 30% water/0.1% formic acid and 70% methanol/0.1% formic acid.
[0287] The stock samples (50 g/mL) were diluted with mobile phase (30% water/0.1% formic acid and 70% methanol/0.1% formic acid) to obtain the following concentrations:
[0288] 0, 0.1, 0.5, 1.0, 2.5, 5.0, 7.5, and 10.0 g/mL
[0289] Not all the concentrations were included in the construction of the standard curve. Some of the cannabinoids (e.g. 0.1 or 10.0 g/mL) were excluded due to very low signal of saturation level.
[0290] Each concentration was run in triplicate. 2 L injections were made and the signal was recorded for up to 6 minutes. SIR+ve (311, 315, and 359 m/z) or SIR ve (313 and 357) and mass scan (150-500 m/z) in positive mode were monitored and recorded. SIR chromatograms were integrated and the AUC was plotted vs. concentration (g/mL).
TABLE-US-00015 TABLE 12 Cannabinoids standard curve - summary. Reference compound Retention time (min) Range (g/mL) .sup.2-CBD 1.70 0.1-10.0 CBD-A 1.90 0.01-1.0 CBN 2.33 0.1-7.5 .sup.9-THC 2.58 0.1-7.5 .sup.8-THC 2.70 0.1-10.0 THCA-A 3.42 0.01-1.0
[0291] Standard curves for each of cannabinoids 1-6 as described above are provided in
[0292]
[0293]
[0294]
[0295]
[0296]
[0297]
[0298] Table 13 provides the concentration (g/mL) of cannabinoids in extracts obtained using microwave extraction method at different temperatures. The solid material (plant fiber) leftover after the microwave reaction was exposed to SFE extraction (method 3A). The total volume of each microwave reaction was 3 mL.
TABLE-US-00016 TABLE 13 Concentration (g/mL) of cannabinoids in extracts obtained using microwave extraction method at different temperatures Extraction conditions .sup.8-THC .sup.9-THC THCA CBD CBN Microwave, 100 C., 10 min; 0 554 358 500 252 No SFE done for this sample N/A N/A N/A N/A N/A Microwave, 130 C., 10 min 0 7046 225 7565 443 SFE 0 243 0 302 32 Microwave, 150 C., 10 min 0 7201 0 9408 508 SFE 0 86 0 135 82 Microwave, 170 C., 10 min 0 6502 0 8566 495 SFE 0 936 0 1321 120
[0299]
[0300] Table 14 shows the amount of cannabinoids in the cannabis extracts, after subjecting to Method 5. See also Table 11. Note: CBN appears to be formed during the microwave based decarboxylation of THCA. CBDA was not quantified.
TABLE-US-00017 TABLE 14 Amount of cannabinoids in the cannabis extracts, after subjecting to Method 5 .sup.8-THC .sup.9-THC .sup.9-THCA-A .sup.2-CBD CBN mg/g mg/g mg/g mg/g mg/g Sonication 0 0 69.47 4.93 0 (Method 1A) Soxhlet alone 0 44.32 40.71 25.4 0 (Method 2) SFE alone 0 1.91 36.91 3.68 0 (Method 3A) Sonication + w 0 107.18 0 145.1 9.53 (Method 1A and 5) Soxhlet + w 0 113.98 0 161.75 10.08 (Method 2 and 5) SFE + w 0 96.48 0 122.66 8.96 (Method 3A and 5) W = microwave ND = not determined
[0301] A plot of the above data is shown in
[0302] Table 15 shows the yield (mg/g of plant material) of cannabinoids in the extracts obtained using microwave extraction method at different temperatures. The solid material (plant fiber) left over after the microwave reaction was exposed to SFE extraction (method 3A). The total volume of each microwave reaction was 3 mL. CBDA was not quantified.
TABLE-US-00018 TABLE 15 Yield (mg/g of plant material) of cannabinoids in extracts obtained using microwave extraction method at different temperatures .sup.8- .sup.9- THC THC THCA-A CBD CBN Conditions mg/g mg/g mg/g mg/g mg/g Microwave 100 C., 10 min 0 45 29 41 3 No SFE N/A N/A N/A N/A N/A Microwave, 130 C., 10 min 0 83 3 89 5 Follow-up SFE 0 3 0 4 0 Microwave, 150 C., 10 min 0 85 0 111 6 Follow-up SFE 0 1 0 2 1 Microwave, 170 C., 10 min 0 76 0 101 6 Follow-up SFE 0 11 0 16 1
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[0304]
[0305]
[0306]
[0307]
[0308]
[0309]
[0310]
[0311]
[0312]
[0313]
[0314]
[0315]
[0316]
[0317]
[0318] Summary of Results for Experiment 2
[0319]
[0320] Table 5 shows the use of various solvents during ultrasonic extraction. There were also minimal amounts of cannabis plant material lost during the recovery filtration process (see Table 6). This also appears to be similar recovery across different extraction methodssolvent, Soxhlet, SFE extraction (See Tables 7 and 8).
[0321] When conducting cannabinoid extraction/decarboxylation in ethanol using a microwave, due to the boiling point of ethanol, it was shown that extraction/decarboxylation of cannabinoids using a microwave is best conducted at temperatures below 180 C., for example at 160 C.10 C. (e.g. +/the acceptable standard of error). It was also shown under the conditions used that conversion of THCA to THC (the desired decarboxylated product) was better at 130 C. than at 100 C., and that if conducted using microwave alone versus microwave and SFE, temperatures above 130 C., for example from 150 C. to 170 C. showed more efficient conversion. (See Tables 13 and 15 as well as
[0322]
[0323]
[0324]
[0325]
[0326] From the above results, it appears that the significant decarboxylated product THC results from microwave extraction and that the addition of a second extraction step, such as SFE, does not appear to change the cannabinoid profile. It further shows that CBD as well as THC components are present in extract after microwave extraction alone or with microwave and SFE extractions combined.
[0327] In summary, the present disclosure shows that extraction and decarboxylation of cannabis plant material can be done concurrently using a microwave, set at a temperature below the boiling point of the extraction solvent, such as ethanol, without the need for a separate extraction step. This optimizes decarboxylated cannabinoid formation and recovery and can produce a more consistent and reproducible product with consistent and reproducible efficacy and therapeutic results.
[0328] An extraction step can also be included before use of a microwave. Although an extraction step after the microwave step is possible, it is not necessary.
[0329] Experiment 3: Supercritical Fluid Extraction (SFE)
[0330] General Procedure: [0331] 1. Dried plant material was weighed and macerated using a mortar and pestle [0332] 2. Crushed plant material was transferred to a 10 mL extraction vessel and subjected to either of the following conditions below [0333] 3. Fractions from each run were collected every 5 mins and combined and concentrated to dryness (at 25 C.) then weighed (green resin) [0334] 4. Extraction was done three times with same plant fibre
[0335] A) SFE conditions
TABLE-US-00019 Solvent A = CO.sub.2 Solvent B = ethanol Temperature = 25 C. BPR = 12 MPa PDA = 200-600 nm (monitoring at 254 nm)
[0336] Flow rate=10 mL/min; make up pump=1 mL/min
[0337] Acquisition time=30 mins Method time=30.2 mins
[0338] i. 0.1 min-25 mins; gradient of 0-50% B in A
[0339] 26 mins; 100% A
[0340] 30 mins; 100% A
[0341] Table 16 below shows the corresponding results.
TABLE-US-00020 TABLE 16 Weights of extract after SFE. Strain Run Extract isolated (g) Variety 1 = 1.0023 g 1 0.2634 (THC: 7.18/CBD: 8.6) 2 0.0038 3 0.010
[0342] Experiment 4: Microwave-Assisted Decarboxylation of Extract with Ethanol (MAE)
[0343] General Procedure: [0344] 1. Extract isolated from the SFE method described above was dissolved in 5-10 mL ethanol and an appropriate volume transferred to 5 mL microwave vials [0345] 2. Added additional volume of ethanol and a stir bar to the 5 mL microwave vials [0346] 3. The vials were sealed and subjected to one of two microwave conditions below: [0347] (a) Temperature=150 C.; run time=10 mins; stir rate=600 rpm; absorption=Normal [0348] (b) Temperature=100 C.; run time=30 mins; stir rate=600 rpm; absorption=Normal [0349] 4. The solution was then concentrated at 35 C. after transferring to 20 mL vials
[0350] The results are shown in Table 17 below.
TABLE-US-00021 TABLE 17 Weights of the resins after subjecting to microwave heating. Amount of Extract resin used isolated after Strain Method (g) microwave (g) Variety 1 (a) 0.0832 0.0212 (THC: 7.18/CBD: 8.6) (b) 0.0832 0.0612 (a) 0.2095 0.1589 Variety 2 (a) 0.1693 0.1114 (THC: 0/CBD: 9) Variety 3 (a) 0.2722 0.1759 (THC: 18.6/CBD: 0)
[0351] Experiment 5: Additional Microwave-Assisted Extractions with Ethanol (MAE), Optimization of Conditions for Decarboxylation
[0352] General Procedure: [0353] 1. Dried plant material was weighed and macerated using a laboratory blender at 22,000 rpm for 60 secs [0354] 2. Crushed plant material was re-weighed and transferred to a 20 mL microwave vial along with a stir bar [0355] 3. Ethanol (10 mL) was added to the vial which was then sealed and subjected to the microwave conditions below: [0356] (a) Temperature=170 C.; run time=15 mins; pre-stirring=30 sec; stir rate=900 rpm; absorption=Normal [0357] (b) Temperature=150 C.; run time=20 mins; pre-stirring=30 sec; stir rate=900 rpm; absorption=Normal [0358] 4. The suspension was filtered and the filtrate and plant fibre collected separately [0359] 5. Filtrate was then concentrated at 35 C., then transferred to a 20 mL vial using ethanol and again concentrated at 35 C., then stored in the refrigerator
[0360] The results are shown in Table 18 below.
TABLE-US-00022 TABLE 18 Amount of Amount of plant plant Extract before after isolated mac- mac- after eration eration microwave Strain Method (g) (g) (g) Variety 1 (a) 1.0062 0.8256 0.2265 (THC: 7.18/CBD: 8.6) Variety 2 (a) 1.0042 0.8382 .sup.a (THC: 0/CBD: 9) (b) 1.0050 0.8293 0.2155 Variety 3 (b) 1.0063 0.7883 0.1166 (THC: 18.6/CBD: 0) .sup.aDesired temperature could not be achieved due to pressure build-up; cap of vial popped off causing solvent and plant fibre to escape from vial.
[0361] Since condition (b) in step 3 above proved successful at that scale, subsequent decarboxylations were performed using that microwave condition and were done in triplicate. However, the following modifications were made:
[0362] Step 1: Blend at 18,000 rpm for 4 secs
[0363] Step 4: Filtration done over celite/activated carbon (as previously described in an earlier report)
[0364] Step 5: Filtrate was then concentrated at 35 C., weighed, transferred to a 20 mL vial using ethanol, then stored in the refrigerator
[0365] The results are shown in Table 19 below.
TABLE-US-00023 TABLE 19 Amount Amount of plant of plant Average before after Extract mac- mac- Extract isolated eration eration isolated (g) Strain Runs (g) (g) (g) SD Variety 1 1 1.0079 0.9260 0.2044 0.2105 (THC: 7.18/CBD: 8.6) 2 1.0084 0.9594 0.2184 0.007 3 1.0080 0.9254 0.2088 Variety 2 1 1.0032 0.8287 0.1624 0.1654 (THC: 0/CBD: 9) 2 1.0038 0.8467 0.1721 0.006 3 1.0040 0.8585 0.1617 Variety 3 1 1.0017 0.9169 0.2225 0.2212 (THC: 18.6/CBD: 0) 2 1.0054 0.8952 0.2068 0.014 3 1.0033 0.9021 0.2343
[0366] This experiments shows the consistency of the extraction method.
[0367] Experiment 6: Cannabis Extractions (1 Gram Scale)
[0368] General Procedure (1.0 g Batch; Before Maceration): [0369] 1. Dried plant material was weighed and macerated using a laboratory blender at 18,000 rpm for 4 secs [0370] 2. Crushed plant material was re-weighed (0.875 g) and transferred to a 20 mL microwave vial along with a stir bar [0371] 3. Ethanol (10 mL) was added to the vial which was then sealed and subjected to the microwave conditions below: [0372] Temperature=150 C.; run time=20 mins; pre-stirring=30 sec; stir rate=900 rpm; absorption=Normal [0373] 4. The suspension was filtered over celite/activated carbon and the filtrate and plant fibre collected separately [0374] 5. Filtrate was then concentrated at 35 C., then transferred to a 20 mL vial using ethanol and again concentrated at 35 C., then stored in the refrigerator [0375] 6. Decarboxylations were performed in triplicate
[0376] Winterization Procedure: [0377] 1. Resin was dissolved in ethanol (10 mL/g) and heated at 40 C. for 5 mins in a water bath [0378] 2. Vial containing extract solution was cooled to 75 C. using a dry ice/acetone bath for 3-4 hrs [0379] 3. Solution was filtered with a pre-weighed syringe filter in 20 mL vials Filter specifications: MillexGV (sterile), Low Protein Binding Durapore (PVDF) Membrane; 0.22 m pore size; 33 mm diameter [0380] 4. Filter was washed with ethanol that had been cooled for 5 mins at 75 C. using a dry ice/acetone bath [0381] 5. Filtrate was concentrated at 35 C. and extract was weighed [0382] 6. Syringe filter was weighed after 2-3 days drying in the fumehood
TABLE-US-00024 TABLE 20 The quantities of cannabis plants used and the amounts of extract obtained before and after winterization. Extract Extract Plant Plant isolated isolated before after before after maceration maceration winter- winter- Strain Runs (g) (g) ization (g) ization (g) Variety 1 1 1.008 0.9369 0.2105 0.1962 (THC: 7.18/ 2 0.0003 0.019 0.007 0.007 CBD: 8.6) 3 Variety 2 1 1.004 0.8446 0.1654 0.1541 (THC: 0/ 2 0.0004 0.015 0.006 0.004 CBD: 9) 3 Variety 3 1 1.003 0.9047 0.2212 0.2095 (THC: 18.6/ 2 0.0019 0.011 0.014 0.009 CBD: 0) 3
TABLE-US-00025 TABLE 21 The quantities of cannabinoids (as % of resin and mg/g of plant) in the extracts isolated, before and after winterization. Cannabinoid in resin before Cannabinoid in resin after winterization winterization .sup.9- .sup.9- .sup.9- .sup.9- CBD CBD THC THC CBD CBD THC THC Strains Runs (%) (mg/g) (%) (mg/g) (%) (mg/g) (%) (mg/g) Variety 1 1 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. (THC: 2 7.18/CBD: 3 8.6) Variety 2.sup.b 1 n.d. n.d. 37.6 4.1 67.1 7.1 1.9 0.1 3.5 0.2 (THC: 2.sup.a 0/CBD: 9) 3.sup.a Variety 3.sup.b 1 n.d. n.d. 49.8 0.8 119.0 1.4 (THC: 2.sup.a 18.6/CBD: 0) 3.sup.a n.d. = not determined; samples were used up before re-analysis with internal standard was performed. .sup.aAverage calculations based on runs 2 and 3 only.
Summary of results: Extracting and decarboxylating 1 gram scale batch of cannabis was successful.
[0383] Experiment 7: Larger Scale MAE (3.75 g Batch; Before Maceration) Using the Modified Conditions [0384] 1. Dried plant material was weighed (3.75 g per batch) and macerated using a laboratory blender at 18,000 rpm for 4 secs. [0385] 2. Crushed plant material was re-weighed and transferred to 320 mL microwave vials along with stir bars (1.2 g of plant fibre per vial) [0386] 3. 95%-100% Ethanol (12 mL) was added to the vial which was then sealed and subjected to the microwave conditions below: [0387] Temperature=150 C.; run time=30 mins; pre-stirring=30 sec; stir rate=900 rpm; absorption=Normal [0388] Note: Time for decarboxylation was increased to 30 mins since analysis revealed that decarboxylation was incomplete at that scale [0389] 4. The 320 mL vials were combined after decarboxylation and the suspension was filtered and the filtrate and plant fibre collected separately [0390] 5. Filtrate was then concentrated at 35 C., then transferred to a 20 mL vial using ethanol and again concentrated at 35 C., weighed and stored in the refrigerator [0391] 6. Decarboxylations were performed in duplicate
[0392] The results are shown in Table 22 below.
TABLE-US-00026 TABLE 22 Amount Amount of plant of plant Average before after Extract mac- mac- Extract isolated eration eration isolated (g) Strain Runs (g) (g) (g) SD Variety 1 1 3.7922 3.6268 0.9967 0.9437 (THC: 7.18/CBD: 8.6) 2 3.7228 3.5800 0.8906 0.075 Variety 2 1 .sup.b 1.2427 .sup.c .sup.c (THC: 0/CBD: 9) Variety 3 1 3.7100 3.5978 1.0658 1.1572 (THC: 18.6/CBD: 0) 2 3.8012 3.6035 1.2486 0.1293 .sup.bPlant fibre is supplied as pulverized buds. Size was appropriate, therefore no further maceration was done. .sup.cDesired temperature of 150 C. could not be achieved due to pressure build-up. [0393] Summary of results: From the results above, it can be concluded that larger scale microwave assisted extraction was successful, at 3-4 grams scale. This method can be scaled up into multi-gram and larger scales with appropriate adjustments to conditions.
[0394] Experiment 8: Large Scale Microwave-Assisted Extractions with Ethanol (MAE)
(A) General Procedure (3.75 g Batch; Before Maceration):
[0395] 1. Dried plant material was weighed and macerated using a laboratory blender at 18,000 rpm for 4 secs [0396] 2. Crushed plant material was re-weighed and transferred to 320 mL microwave vials along with stir bars (1.2 g of plant fibre per vial) [0397] 3. 95%-100% Ethanol (12 mL) was added to the vial which was then sealed and subjected to the microwave conditions below: [0398] Temperature=150 C.; run time=30 mins; pre-stirring=30 sec; stir rate=900 rpm; absorption=Normal [0399] Note: Time for decarboxylation was increased to 30 mins since analysis revealed that decarboxylation was incomplete at that scale [0400] 4. All 20 mL vials were combined after decarboxylation and the suspension was filtered and the filtrate and plant fibre collected separately (first batch) [0401] 5. Filtrate was then concentrated at 35 C., then transferred to a 20 mL vial using ethanol and again concentrated at 35 C., weighed and stored in the refrigerator [0402] 6. Decarboxylations were performed in duplicate
[0403] Winterization Procedure:
[0404] 1. Same as previously described
TABLE-US-00027 TABLE 23 The quantities of cannabis plants used and the amounts of extract obtained before and after winterization. Plant Extract isolated Extract before Plant after before isolated after maceration maceration winterization winterization Strain Runs (g) (g) (g) (g) Variety 1 1.sup.a 3.7575 0.049 3.6034 0.033 0.9967 0.9437 0.075 0.7794 (THC: 2 0.8906 7.18/CBD: 8.6) Variety 2 1 1.2427 .sup.b n.d. n.d n.d n.d (THC: 0/CBD: 9) Variety 3 1 3.7556 0.064 3.6007 0.004 1.0618 1.1543 0.131 0.7984 0.8973 0.140 (THC: 2 1.2468 0.9961 18.6/CBD: 0) n.d. = not determined. Desired temperature of 150 C. could not be achieved due to pressure build-up; nothing further was done with buds. .sup.aNo winterization was performed on run 1; winterization done on run 2 only. .sup.b Plant fibre is supplied as pulverized buds. Size was appropriate, therefore no further maceration was done.
TABLE-US-00028 TABLE 24 The quantities of cannabinoids (as % of resin and mg/g of plant) in the extracts isolated, before and after winterization. Cannabinoid in resin before Cannabinoid in resin after winterization winterization .sup.9- .sup.9- .sup.9- .sup.9- CBD CBD THC THC CBD CBD THC THC Strains Runs (%) (mg/g) (%) (mg/g) (%) (mg/g) (%) (mg/g) Variety 1 1 n.d. n.d. n.d. n.d. 39.0 2.2 84.9 4.7 30.3 1.4 66.1 3.0 (THC: 2.sup.a 7.18/CBD: 8.6) Variety 3 1 68.6 8.4 219.0 2.7 61.1 4.6 153.2 35.0 (THC: 2 18.6/CBD: 0) n.d. = not determined; samples were used up before re-analysis with internal standard was performed. .sup.aCalculations based on run 2 only.
(B) General Procedure (7.5 q Batch; Before Maceration):
[0405] 1. Dried plant material was weighed and macerated using a laboratory blender at 18,000 rpm for 10 secs [0406] 2. Crushed plant material was re-weighed and transferred to 620 mL microwave vials along with stir bars (1.2 g of plant fibre per vial) [0407] 3. See steps 3-6 in (A) above
[0408] Winterization Procedure:
[0409] 1. Same as previously described
TABLE-US-00029 TABLE 25 The quantities of Variety 1 (THC: 7.18/CBD: 8.6) used and the amounts of extract obtained before and after winterization. Amount of Amount of Extract Extract plant before plant after isolated before isolated after maceration maceration winterization winterization Runs (g) (g) (g) (g) 1 7.5361 3.6156 1.0154 0.8115 2 0.002 0.064 0.030 3 7.5063 3.6640 0.9327 0.8247 4 0.026 0.014 0.022
TABLE-US-00030 TABLE 26 The quantities of cannabinoids (as % of resin and mg/g of plant) in the Variety 1 extract isolated, before and after winterization. Cannabinoid in resin after Cannabinoid in resin before winterization winterization .sup.9- CBD CBD .sup.9-THC .sup.9-THC CBD CBD THC .sup.9-THC Runs (%) (mg/g) (%) (mg/g) (%) (mg/g) (%) (mg/g) 1 40.6 3.9 113.7 3.7 31.1 2.0 87.3 0.1 31.6 5.5 70.8 9.7 23.1 4.1 51.6 7.3 2 3 29.2 0.1 74.3 0.9 20.9 1.0 53.3 3.0 44.0 2.9 99.0 4.7 31.3 0.7 70.3 0.2 4
(C) General Procedure (4.0 and 7.0 g Batches; Before Maceration):
[0410] 1. Dried plant material was weighed and macerated using a laboratory blender at 18,000 rpm for 10 secs [0411] 2. Crushed plant material was re-weighed and transferred to 20 mL microwave vials along with stir bars (1.2 g of plant fibre per vial) 13. For 4.0 g batch, see steps 3-5 in (A) above [0412] 4. For 7.0 g batch, see steps 3-5 in (A) above
[0413] Winterization Procedure:
[0414] 1. Same as previously described
TABLE-US-00031 TABLE 27 The quantity of Variety 1 plant (THC: 7.18/CBD: 8.6) used and the amount of extract obtained before and after winterization. Amount of Amount of Extract Extract plant before plant after isolated before isolated after maceration maceration winterization winterization Runs (g) (g) (g) (g) 1 4.0325 3.8199 0.8889 0.7358 2 7.1293 6.8717 1.5244 1.4101
TABLE-US-00032 TABLE 28 The quantities of cannabinoids (as % of resin and mg/g of plant) in the Variety 1 extract isolated, before and after winterization. Cannabinoid in resin after Cannabinoid in resin before winterization winterization .sup.9- CBD CBD .sup.9-THC .sup.9-THC CBD CBD THC .sup.9-THC Runs (%) (mg/g) (%) (mg/g) (%) (mg/g) (%) (mg/g) 1 30.2 0.0 70.3 0.1 27.1 0.4 63.0 0.0 38.4 6.1 73.9 11.8 32.1 7.5 61.8 14.4 2 33.6 2.9 74.5 6.5 28.9 3.2 64.2 7.1 42.5 0.5 87.2 1.1 37.0 2.9 75.9 5.9 [0415] Summary of results: Extracting and decarboxylating 3.75 gram scale batch of cannabis was successful.
[0416] Experiment 9: Larger Scale Microwave-Assisted Extractions with Ethanol (MAE) and Winterization [0417] 1. Dried plant material was weighed (7.5 g per batch) and macerated using a laboratory blender at 18,000 rpm for 10 secs [0418] 2. Crushed plant material was re-weighed and transferred to 620 mL microwave vials along with stir bars (1.2 g of plant fibre per vial) [0419] 3. 95%-100% Ethanol (12 mL) was added to the vial which was then sealed and subjected to the microwave conditions below: [0420] Temperature=150 C.; run time=30 mins; pre-stirring=30 sec; stir rate=900 rpm; absorption=Normal [0421] 4. The 320 mL vials were combined after decarboxylation and the suspension was filtered and the filtrate and plant fibre collected separately [0422] 5. Filtrate was then concentrated at 35 C., then transferred to a 20 mL vial using ethanol and again concentrated at 35 C., weighed and stored in the refrigerator [0423] 6. Resin was dissolved in ethanol (10 mL/g) [0424] 7. Vials containing extract solution were cooled to 75 C. using a dry ice/acetone bath for 4 hrs [0425] 8. Solution was filtered with a pre-weighed syringe filter in 20 mL vials [0426] Filter specifications: MillexGV (sterile), Low Protein Binding Durapore (PVDF) Membrane, 0.22 m pore size, 33 mm diameter [0427] 9. Filter was washed with ethanol that had been cooled for 5 mins at 75 C. using a dry ice/acetone bath [0428] 10. Filtrate was concentrated at 35 C. and vacuum dried for 2 days at 40 C. (via water bath), then extract was weighed [0429] 11. Syringe filter was weighed after 2-3 days drying in the fumehood
[0430] The results are shown in Table 29 below.
TABLE-US-00033 TABLE 29 Amount of mg of plant Amount of cannabinoid/ before plant after Extract g of maceration maceration isolated plant (%) Strain Runs (g) (g) (g) THC CBD Variety 1 1 7.5063 3.682 0.8403 7.0 10.7 (THC: 2 3.6459 0.8091 7.7 12.2 7.18/CBD: 8.6) [0431] Summary of results: 7.5 g large scale batch of cannabis extraction and decarboxylation was successful. These experiments demonstrate that the methods of the disclosure consistently extract and decarboxylate cannabinoids and can be used on a commercial scale.
[0432] Experiment 10: Winterization
[0433] Winterization is a procedure typically used to remove waxes and other partially soluble materials at 010 C. temperature range. This process may not be applicable, if there are no waxes present in the extract, or such hydrophobic molecules are broken down, and would be solidify at the ice-bath or below-zero temperatures.
[0434] Experiment 10A
To remove waxes, the solutions of extract which had been stored in the refrigerator (5 C.) for 1-3 weeks were manipulated as follows: [0435] 1. Solution was filtered using a syringe filter in 20 mL vials [0436] Filter specifications: MillexGV (sterile) [0437] Low Protein Binding Durapore (PVDF) Membrane [0438] 0.22 m pore size [0439] 13 m diameter [0440] 2. Filtrate was concentrated at 35 C. and vacuum dried for 3 days at 40 C. (via water bath) [0441] 3. Extracts were then re-weighed
[0442] Experiment 10E3 [0443] 1. Resin was dissolved in 95%-100% ethanol (10 mL/g) and heated at 40 C. for 5 mins in a water bath [0444] 2. Vial containing extract solution was cooled to 75 C. using a dry ice/acetone bath for 3-3.75 hrs [0445] 3. Solution was filtered with a pre-weighed syringe filter in 20 mL vials (see above for filter specifications); filter was washed with ethanol that had been cooled for 5 mins at 75 C. using a dry ice/acetone bath [0446] 4. Filtrate was concentrated at 35 C. and extract was weighed [0447] 5. Syringe filter was weighed after 2-3 days drying in the fumehood
[0448] The results are shown in Tables 30a and b below.
TABLE-US-00034 TABLE 30a Amount of extract isolated before and after winterization methods After 1.sup.st After 2.sup.nd Before 1.sup.st winterization winterization winterization and drying and drying Strain Runs (g) (g) (g) Variety 1 1 0.2044 0.2111 0.2031 (THC: 7.18/CBD: 8.6) 2 0.2184 0.2345 0.1964 3 0.2088 0.1981 0.1891 Variety 2 1 0.1624 0.1677 0.1580 (THC: 0/CBD: 9) 2 0.1721 0.1622 0.1508 3 0.1617 0.1677 0.1536 Variety 3 1 0.2225 0.2088 0.1996 (THC: 18.6/CBD: 0) 2 0.2068 0.2221 0.2145 3 0.2343 0.2243 0.2145
TABLE-US-00035 TABLE 30b Average extract isolated before and after winterization methods After 1st After 2nd Before 1st winterization winterization winterization and drying and drying Strain Runs (g) (g) (g) Variety 1 1 0.2105 0.2146 0.1962 (THC: 7.18/CBD: 8.6) 2 0.007 0.018 0.007 3 Variety 2 1 0.1654 0.1659 0.1541 (THC: 0/CBD: 9) 2 0.006 0.003 0.004 3 Variety 3 1 0.2212 0.2184 0.2095 (THC: 18.6/CBD: 0) 2 0.014 0.008 0.009 3 [0449] Tables 31a and b below show the amount of cannabinoid (in milligrams) in the extract per gram pf plant material isolated before and after winterization methods
TABLE-US-00036 TABLE 31a Amount of cannabinoid (in milligrams) in the extract per gram pf plant material Before 1.sup.st 1.sup.st 2.sup.nd winterization winterization winterization (mg/g) (mg/g) (mg/g) Strains Runs CBD .sup.9-THC CBD .sup.9-THC CBD .sup.9-THC Variety 1 1 92.9 65.1 98.3 76.2 157.4 109.0 (THC: 2 114.9 82.3 114.3 91.5 124.7 93.7 7.18/CBD: 3 115.5 85.7 113.7 93.8 184.5 138.1 8.6) Variety 2 1 200.5 113.7 149.7 (THC: 0/CBD: 2 211.6 109.5 147.3 9) 3 146.9 131.2 161.9 Variety 3 1 406.1 207.7 235.4 (THC: 2 185.6 274.8 236.2 18.6/CBD: 0) 3 232.9 301.0 283.4
TABLE-US-00037 TABLE 31b Average cannabinoid (in milligrams) in the extract per gram pf plant material Before 1.sup.st winterization 1.sup.st winterization 2.sup.nd winterization (mg/g) (mg/g) (mg/g) Strains Runs CBD .sup.9-THC CBD .sup.9-THC CBD .sup.9-THC Variety 1 1 107.8 12.9 77.7 11.0 108.8 9.1 87.2 9.6 155.5 29.9 113.6 22.6 (THC: 2 7.18/CBD: 3 8.6) Variety 2 1 186.3 34.6 118.1 11.5 153.0 7.8 (THC: 0/CBD: 2 9) 3 Variety 3 1 274.9 116.1 261.2 48.1 251.7 27.5 (THC: 2 18.6/CBD: 0) 3
[0450] Summary of Results:
[0451] Winterization of extracts was successful in removing waxes from the extract.
[0452] Experiment 11: Chemistry of Medicinal Cannabis Before and after Decarboxylation
[0453] Methodology
[0454] Extraction:
[0455] Dried plant material (1 g) was weighed and transferred to a mortar and was macerated using a pestle. The crused plant material was then transferred into a 10 mL vessel and was subjected to supercritical fluid extraction (SFE), with supercritical CO.sub.2 as solvent A and ethanol as solvent B. The photodiode array detector was set to monitor wavelengths in the range of 200-600 nm and the back pressure regulator was set to 12 MPa. The SFE conditions used were: flow rate=10 mL/min (CO.sub.2 and slave pumps) and 1 mL/min (make-up pump); temperature=25 C.; gradient: 100% A50% A (0.1-25 mins), 100% B (25-26 mins) and 100% A (26-30 mins). Once the method was completed, all fractions were combined and concentrated to dryness under reduced pressure (at 25 C.) to afford 0.28 g of a green sticky resin. This was used for further work-up and analyses.
[0456] Activation:
[0457] Activation of phytocannabinoids was conducted by subjecting cannabis extract to heat using microwaves. A 5 mL-size microwave vial was charged with cannabis extract (27.72 mg) dissolved in ethanol (2 mL). The vial was sealed and was subjected to heat for 10 min at 150 C. in a pressure vessel to afford a green sticky extract. This was concentrated to dryness at 35 C. to obtain the activated cannabis extract as a resin (21.2 mg).
[0458]
[0459] Table 32.
[0460] Potential changes in chemical composition after decarboxylation of strain I cannabis extract. Left column indicates the potential compounds that were present in cannabis extract obtained through a supercritical fluid extraction (SFE), but not in the decarboxylated resin. This extract was then subjected to heating conditions using microwave technology, and the right column shows the new chemicals that were identified, which were not present in the cannabis extract prior to employing microwave technology described in this disclosure.
TABLE-US-00038 TABLE 32 New Compounds Found Upon Compounds Lost Upon Activation Activation Roughanic acid (Fatty acid) Kaempferol (Flavonol) -Linolenic acid (Fatty acid) Luteolin (Flavonol) Quercetin (Flavonoid) 4,7-Dimethoxy-1,2,5-trihydroxyphenanthrene (Non-cannabinoid) 4,5-Dihydroxy-2,3,6-trimethoxy-9,10- 5-Methyl-4-pentyl-2,6,2-trihydroxybiphenyl dihydrophenanthrene (Non-cannabinoid) (Non-cannabinoid) .sup.9-Tetrahydrocannabiorcolic acid (C.sub.1- 5-Methyl-4-pentylbiphenyl-2,2,6-triol (Non- Cannabinoid Acid) cannabinoid) Cannabigerol (C.sub.5-Neutral Cannabinoid) Cannabichromevarin (C.sub.3-Neutral Cannabinoid) Cannabichromanon (Neutral Cannabinoid) Cannabicyclovarin (C.sub.3-Neutral Cannabinoid) Cannabigerovarinic acid (C.sub.5-Cannabinoid Acid) Cannabidivarin (C.sub.3-Neutral Cannabinoid) 6,7-cis/trans-Epoxycannabigerol (Cannabinoid .sup.7-cis-iso-Tetrahydrocannabivarin (C.sub.3-Neutral Derivative) Cannabinoid) 7-Hydroxycannabichromane (Cannabinoid .sup.9-tetrahydrocannabivarin (C.sub.3-Neutral Derivative) Cannabinoid) C.sub.4-Tetrahydrocannabinolic acid (C.sub.4-Cannabinoid Chrysoeriol (Flavone) Acid) Cannabitriol (Neutral Cannabinoid) C.sub.4-Cannabidiol (C.sub.4-Neutral Cannabinoid) Cannabiripsol (Neutral Cannabinoid) C.sub.4-Tetrahydrocannabinol (C.sub.4-Neutral Cannabinoid) Cannabigerolic Acid (C.sub.5-Cannabinoid Acid) Cannabifuran (Neutral Cannabinoid) 7R-Cannabicoumaronic Acid (Cannabinoid Acid) Cannabinol (C.sub.5-Neutral Cannabinoid) Tetrahydrocannabinolic acid-8-one (Cannabinoid Cannabinodiol (C.sub.5-Neutral Cannabinoid) Acid Derivative) 5-Acetoxy-6-geranyl-3-n-pentyl-1,4- 10-oxo-.sup.6a-tetrahydrocannabinol (Cannabinoid benzoquinone (Non-cannabinoid) Derivative) 4-Acetoxycannabichromene (Cannabinoid 7R-Cannabicourmarone (Neutral Cannabinoid) Derivative) Cannabielsoic Acid-/ (Cannabinoid Acid) Cannabichromanone-D (Neutral Cannabinoid) 6,7-trans/cis-Epoxycannabigerolic acid Cannabidiol Monoethylether (Cannabinoid (Cannabinoid Acid Derivative) Derivative) .sup.9-Tetrahydrocannabinolic acid + C.sub.2H.sub.2O Cannabielsoic Acid-A/B (Cannabinoid Acid) (Cannabinoid Acid Derivative) Orientin (Flavone) Sesquicannabigerol (Cannabinoid Derivative) 4-Terpenyl-.sup.9-Tetrahydrocannabinolate (Cannabinoid Acid Ester) -Terpenyl-.sup.9-Tetrahydrocannabinolate (Cannabinoid Acid Ester) Bornyl/epi-bornyl-.sup.9-Tetrahydrocannabinolate (Cannabinoid Acid Ester) /-Fenchyl .sup.9-Tetrahydrocannabinolate (Cannabinoid Acid Ester)
[0461] Closed system, microwave extraction provided the simultaneous extraction and decarboxylation of the cannabinoids. In the native cannabis extract, 63 compounds could be observed (
[0462] Experiment 12: Solvent-Free Decarboxylated Cannabis Resin
[0463] General Procedure
[0464] To remove the solvent from the decarboxylated resin, the following procedures are used:
distillers or rotary evaporators are used to evaporate solvents employed in extraction and decarboxylation process, concentrate and obtain solvent-free decarboxylated resin. During the evaporation of solvent, a higher temperature than ambient temperature is used to facilitate faster evaporation of the solvent. In addition, a vacuum may be used to facilitate removal of solvent at lower pressure than the atmospheric pressure. In general, such processes are well established and known to those skilled in the art.
[0465] The resulting decarboxylated cannabis resin may comprise less than 5% solvent, or the resin can be solvent-free.
[0466] Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, but also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.
[0467] Numerical data may be presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5, but also to include individual numerals such as 2, 3, 4, and sub-ranges such as 1 to 3, 2 to 4 etc. The same principle applies to ranges reciting only one numerical value, such as less than about 4.5, which should be interpreted to include all of the above-recited values and ranges. Further, such an interpretation should apply regardless of the breadth of the range or the characteristic being described.
[0468] While the foregoing disclosure has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art, from a reading of the disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure in the appended claims.
[0469] All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.