Isolation of pure cannabinoids from <i>Cannabis</i>

Abstract

Δ.sup.9-Tetrahydrocannabinol (Δ.sup.9-THC or THC) and cannabidiol (CBD) are major constituents of the Cannabis plant that have pharmacological properties with potential therapeutic value. This invention is directed to processes for large scale isolation of these two and other cannabinoids from the Cannabis sativa plant. This is accomplished through the discovery that protected amino acid esters of the cannabinoids are easier to separate using normal phase silica column chromatography. Mild base hydrolysis of the esters regenerates the free cannabinoids in a purified form. The invention is also applicable to the isolation of other cannabinoids from Cannabis extracts.

Claims

1. A process for the isolation and purification of cannabinoids from Cannabis plant material of different varieties comprising the following steps: a) extracting the plant material using an organic extraction solvent of supercritical fluid with or without modifier followed by evaporation of the extraction solvent to yield crude extract; b) optionally winterizing the crude extract prior to derivatization to remove hydrocarbons and waxes and derivatizing the crude extract, or optionally thin film distilled extract with a t-boc-amino acid to convert cannabinoids to their t-boc-amino acid esters; c) purifying the derivatized extract using normal phase column chromatography to isolate individual esters of different cannabinoids; d) base hydrolyzing the isolated individual cannabinoid ester to regenerate the free cannabinoid with high purity greater than 90% pure; and e) optionally re-chromatographing the isolated cannabinoids to increase purity to a desired level.

2. The process of claim 1, where the extraction solvent is a hydrocarbon.

3. The process of claim 1, where extraction is carried out using supercritical fluid using CO2 with or without a modifier.

4. The process of claim 1, where the crude extract is subjected to a thin film distillation step under reduced pressure to increase the cannabinoids content of the extract.

5. The process of claim 1, where the crude extract is winterized to remove hydrocarbons and waxes.

6. The process of claim 4, where the thin film distilled extract is winterized to remove hydrocarbons and waxes.

7. The process of claim 1, where the t-boc-amino acid is selected from tryptophan, glutamine, alanine, or phenylalanine.

8. The process of claim 1, where a cannabinoid of interest is Δ.sup.9-tetrahydrocannabinol.

9. The process of claim 1, where a cannabinoid of interest is cannabidiol.

10. The process of claim 1, where a cannabinoid of interest is Δ.sup.9-tetrahydrocannabivarin.

11. The process of claim 1, where a cannabinoid of interest is cannabigerol.

12. The process of claim 2, wherein the hydrocarbon is an n-pentane, hexane or heptane.

13. The process of claim 3, wherein the modifier is acetone, ethanol or methanol.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The features, aspects and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts, wherein:

(2) FIG. 1 is a schematic representation to produce CBD;

(3) FIG. 2 is a schematic representation to produce THC, CBD and CBG;

(4) FIG. 3 is a schematic representation to produce THC; and

(5) FIG. 4 is a schematic representation to produce THCV.

DETAILED DESCRIPTION OF THE INVENTION

(6) The present invention relates to a process for the isolation and purification of cannabinoids from Cannabis plant material of different varieties. The present process comprises the following steps:

(7) a) extracting the plant material using optionally an organic solvent of supercritical fluid with or without modifier followed by evaporation of the extraction solvent to yield a crude extract;

(8) b) optionally winterizing the crude extract prior to derivatization to remove hydrocarbons and waxes and derivatizing the crude extract, or optionally thin film distilled extract with a t-boc-amino acid to convert cannabinoids to their t-boc-amino acid esters;
c) purifying the derivatized extract using normal phase column chromatography to isolate individual esters of different cannabinoids;
d) base hydrolyzing the isolated individual cannabinoid ester to regenerate the free cannabinoid with high purity (>90% pure-100% pure); and
e) optionally re-chromatographing the isolated cannabinoids to increase purity to a desired level.

(9) The process extraction solvent in the present process is a hydrocarbon such as, for example, n-pentane, hexanes or heptane. The extraction is carried out using supercritical fluid using CO.sub.2 with or without a modifier such as acetone, ethanol or methanol.

(10) The crude extract is subjected to a thin film distillation step under reduced pressure to increase the cannabinoid content of the extract.

(11) The t-boc-amino acid is selected from but not limited to tryptophan, glutamine, alanine and phenylalanine.

(12) The cannabinoid of interest in the present process can be Δ.sup.9-tetrahydrocannabinol (Δ.sup.9-THC or THC), cannabidiol (CBD), Δ.sup.9-tetrahydrocannabivarin (THCV) or cannabigerol (CBG).

Example No. 1

(13) Production of CBD from the Decarboxylated Extract of a High CBD Variety of Cannabis

(14) Extraction and Decarboxylation:

(15) Air-dried and powdered buds (274 g) of a high CBD variety of Cannabis (3.0% CBD) were extracted by maceration at room temperature with hexanes (Fisher, H-302-4) for 24 hours (1.0 L hexanes×3). The hexanes extracts were combined and evaporated under vacuum to give 19.5 g of extract which was decarboxylated by heating at 130° C. for 30 minutes to give 16.6 g of decarboxylated extract (40% CBD by GC/FID analysis).

(16) Chemical Derivatization:

(17) A portion (7.8 g) of the decarboxylated extract was dissolved in 100 mL methylene chloride (DCM, Fisher, D37-4) to which dimethylaminopyridine (DMAP) (65 mg) was added, and the reaction mixture stirred at room temperature for 10 minutes, (mixture A). In another 1 L round bottom flask, Boc-Trp-OH (15.8 g, 2.1 eq, AnaSpec. Inc, 510-791-9560) was mixed with DCC (10.7 g, 2.1 eq., Alfa Aesar, A13016) and DCM (100 mL), the reaction mixture was stirred at room temperature for 10 minutes, (mixture B). Mixture B was then added to mixture A, followed by stirring at room temperature for 15 minutes for the reaction completion, which was confirmed by Si gel Thin Layer Chromatography (Si-TLC) using ethyl acetate (EtOAc, Fisher, E145-4): DCM (5:95) as the mobile phase. After the reaction was complete, 400 mL of hexanes was added and the mixture was cooled in the freezer for 4 hours followed by filtration through a filter funnel. The filtrate was evaporated to dryness under reduced pressure to give 29.0 g of the dried reaction mixture.

(18) Column Chromatography:

(19) The reaction mixture (29 g), dissolved in 20 mL of DCM, was applied to the top of a silica gel (750 g, Silicycle 60 A, R 100303) column (dimensions: 5×80 cm). Elution was carried out using 2% EtOAc/DCM, and four fractions (A-D) were collected. Fraction B (9.3 g), which was rich in the CBD adduct was evaporated and the residue was used in the next step.

(20) Hydrolysis of CBD Adduct Fraction (Fraction B):

(21) The CBD adduct fraction (9.3 g) was dissolved in 3 mL methanol (MeOH, Fisher, A452-4), then 10 mL KOH (5N) was added and the mixture stirred at room temperature for a few minutes to allow complete hydrolysis which was checked by TLC. After completion of hydrolysis, HCl (5N) was added to neutralize the excess base and release the free CBD, followed by extraction with DCM. The organic layer was separated and dried in vacuo and then 100 mL hexanes was added to precipitate any excess reagents which are removed by filtration. The filtrate was evaporated to dryness under reduced pressure to yield 3.0 g of crude CBD (89.9% by GC/FID analysis).

(22) Column Chromatography of the Impure CBD:

(23) The impure CBD fraction (3.0 g) was dissolved in 5 mL DCM then applied to a Si gel column eluted with an isocratic mixture of EtOAc:hexanes (2.5:97.5) to give four fractions. The results are summarized in Table 1.

(24) TABLE-US-00001 TABLE 1 CBD Fractions by Process of Example 1 Fraction Weight (g) Analysis for CBD 1-2 0.075 — 3 1.875 100% 4-9 0.634 98.8%  10-11 0.552  1.8%

(25) Fractions 3 and 4-9 were combined and crystallized from hexanes to give 2.2 g of CBD as pale yellow cubic crystals with 100% purity. The purity of CBD was confirmed by GC/FID, GC/MS and NMR spectroscopic analysis. The overall yield of this process starting from decarboxylated extract to the pure CBD was 70.5%. FIG. 1 shows the schematic representation of this process.

Example No. 2

(26) Production of CBD from the Decarboxylated and Winterized Distillate of an Extract of CBD Rich Variety of Cannabis

(27) Plant Material Extraction:

(28) Dried and powdered Cannabis plant material (3.00 Kg) of CBD rich variety of Cannabis (4.03% CBD) was extracted by maceration in hexanes (20 L×2) followed by evaporation under reduced pressure to give 182.3 g dried extract (52.2% CBD).

(29) Thin Film Distillation of the Crude Extract:

(30) The hexane extract (182.3 g) was decarboxylated by heating in an oil bath for 30 minutes at 130° C., to give 162 g of decarboxylated extract. The process was monitored by TLC analysis and GC/FID of the TMS derivative or by HPLC to confirm complete decarboxylation. The decarboxylation step is essential to prevent frothing of the extract during the distillation process. The decarboxylated extract (153.0 g) was subjected to distillation using thin film distillation with the following conditions:

(31) Vacuum: 44 m Torr

(32) Temperature: 199° C.

(33) Rotation: 300 rpm

(34) Flow rate: 2 mL/min.

(35) The produced distillate (98.0 g) has golden yellow color and its CBD content is 67.7% (Recovery of CBD in this step is 70.0%). Volatile substances (7.88 g, 11.2% CBD) and residue (37.10 g, 25.5% CBD) were also collected after the distillation was completed.

(36) Winterization of the Distillate:

(37) Crude Cannabis extract as well as the thin film distillate are known to contain high concentration of waxes and hydrocarbons. These could be substantially removed or reduced by the following process, referred to as “Winterization Process”.

(38) The distillate (98.0 g) was dissolved in 200-proof ethanol at a ratio of 1 part extract to 12 parts ethanol (1.17 L). The ethanol solution of the distillate was then placed in the freezer (−20° C.) for 4 hours. While cold, the precipitated material was filtered out using a filter funnel and the filter was rinsed with 100 mL of ice cold ethanol. The filtered ethanolic solution of the distillate was evaporated to dryness to give 84.0 g residue (78% CBD content). The CBD recovery in this step is 99.0%.

(39) Chemical Derivatization:

(40) A portion of the above residue (14.0 g, 78% CBD) was dissolved in 50 mL DCM and to which 120 mg of DMAP was added, and the reaction mixture was stirred for 10 minutes (mixture A). In another 1 L round bottom flask, Boc-Trp-OH (28.4 g, 2.1 eq.) was mixed with DCC (19.2 g, 2.1 eq.) in 200 mL DCM and the reaction mixture was stirred at room temperature for 10 minutes (mixture B). Mixture B was added to mixture A then the reaction mixture was stirred till the reaction was complete (approximately 15 minutes) which was confirmed by Si gel Thin Layer Chromatography (Si-TLC) using Ethyl acetate (EtOAc, Fisher, E145-4):DCM (5:95) as the mobile phase. After completion of the reaction, 700 mL hexanes was added to the reaction product and kept in the freezer for 4 hours then filtered. The filtrate was evaporated to dryness under reduced pressure to give 46.5 g dried distillate adduct.

(41) Column Chromatography:

(42) A portion of the crude distillate adduct (22.4 g) was dissolved in 50 mL DCM and applied on the top of a Si gel column (750 g) and the material eluted with 2% EtOAc/DCM. Seventeen fractions were collected and similar fractions are combined based on TLC analysis. The results are shown in Table 2.

(43) TABLE-US-00002 TABLE 2 CBD Fractions by Process of Example 2 Fraction Weight (g) Analysis for cannabinoids 1-7  2.67 — 8 1.90 CBD adduct of 23% CBD contents 9-17 14.4 CBD adduct rich fraction of 27% CBD content

(44) The CBD adduct Fraction (14.4 g) was subjected to alkaline hydrolysis as above to give 4.7 g of CBD (82% CBD contents).

(45) Column Chromatography of the Crude CBD:

(46) The crude CBD fraction (4.7 g) was dissolved in 5 mL DCM then applied to a Si gel column eluted with 2.5% EtOAc/hexanes to yield three fractions. The results are summarized in Table 3.

(47) TABLE-US-00003 TABLE 3 Crude CBD Fractions by Process in Example 2 Fraction Weight (g) Analysis for CBD 1-6 2.85 99.98%  7-11 0.358 98.2% 12-16 1.266 —

(48) Fractions 1-6 and 7-11 were combined and crystallized from hexanes to give 3.18 g of CBD as pale yellow cubic crystals with 100% purity. The overall yield of CBD in this process starting from the decarboxylated extract to the pure CBD was 61%.

(49) The schematic representation of this process is shown in FIG. 1.

Example No. 3

(50) Production of CBD from an Extract of an Intermediate Variety of Cannabis Containing High CBD and High THC

(51) Plant Material Extraction:

(52) Air dried powdered Cannabis plant (0.81 Kg) of Intermediate variety CBD variety (6.3% CBD and 2.96% THC) was extracted by maceration in hexanes (5.0 L×2) followed by evaporation under reduced pressure to yield 126.0 g dry extract (42.8%. CBD, 20.4% THC). Portion of the extract (27.14 g) was decarboxylated by heating in an oil bath at 130° C. for 30.0 min. to give 23.6 g of the decarboxylated extract (45.7% CBD, 19.4% THC).

(53) Chemical Derivatization and Purification of the CBD and THC Adducts:

(54) The decarboxylated extract (23.6 g) was chemically derivatized as before, using t-Boc-Trp-OH, to produce adduct. 1.2 L hexanes was added to the reaction product and kept in the freezer for 4 hours then filtered. The filtrate was evaporated to dryness under reduced pressure to give 79.75 g dried distillate adduct. Portion of the adduct (26.43 g) was purified by Si gel column chromatography (800 g Si) eluted with 2% EtOAc/DCM. Five fractions were collected, and details are shown in the next Table.

(55) TABLE-US-00004 TABLE 4 CBD Fractions by Process of Example 3 Fraction Weight (g) Analysis for Cannabinoids 1-6 1.2 —  7-15 3.73 43% THC contents 16-17 0.84 — 18-19 9.3 32% CBD content 20-22 10.0
Purification of CBD:

(56) The CBD rich column fraction (9.3 g) was hydrolyzed by dissolving in 3 mL MeOH, then 10 mL KOH (5N) were added and stirred at room temperature for 5 min. to allow complete hydrolysis which was checked by TLC. HCl 6N was added till neutralization followed by extraction with DCM. The organic layer was separated and dried in vacuo and then 100 mL hexanes were added to precipitate any excess reagents followed by filtration. The filtrate was evaporated till dryness under reduced pressure to yield CBD (90%), 3.0 g of that was purified by Si gel column chromatography using EtOAc/hexanes (2.5%) as an eluent, 50 mL fractions were collected. The result was shown in Table 5.

(57) TABLE-US-00005 TABLE 5 CBD Fractions by Process of Example 3 Fraction Weight (g) Analysis for Cannabinoids 1-3 0.046 — 4-6 1.89 CBD 100%  7-10 0.146 CBD 97% 11-15 0.108 CBD 84%, CBG 8.3% 16-17 0.11 CBD 0.3%, CBG 98.9% 18-19 0.09 —

(58) The CBD from fractions 4-6 was crystallized from hexanes to yield 1.89 g CBD as pale yellow crystals (100% pure), with over all yield of 54% starting from the decarboxylated extract.

(59) Purification of THC:

(60) The THC rich column fraction 7-15 (3.73 g) was chromatographed over Si gel column eluted with 2% EtOAc/DCM (50 mL/fraction) to give 2.2 g pure THC adduct which was hydrolyzed using KOH (5N) to yield 0.96 g THC (97.49% purity) with an overall yield of 60% starting from the decarboxylated extract. The impurities in the THC are basically due to CBN (2.51%), which is a known oxidation product of THC normally found in pharmaceutical grade THC. The schematic representation of the production of THC and CBD was shown in FIG. 2.

Example No. 4

(61) Production of THC from the Distillate of an Extract of High Potency Variety of Cannabis (High THC)

(62) Plant Material Extraction:

(63) Air-dried and powdered high potency Cannabis plant material (17.20 Kg) of high THC content (>8%) was extracted at room temperature by overnight maceration and percolation with hexanes (80 L×2), followed by evaporation under reduced pressure at 60° C. to give 2.32 kg dry extract (66.4% THC).

(64) Thin Film Distillation of the Crude Extract:

(65) High potency extract (630 g) was heated at 130° C. for 30 min then subjected to distillation using thin film distillation still under the following conditions:

(66) Vacuum: 120 m Torr

(67) Temperature: 199° C.

(68) Rotation: 300 rpm

(69) Flow rate: 2 mL/min.

(70) After completion of the distillation process, the distillate (465 g; 73.8% yield) was obtained along with volatile oil fraction (57 g) and undistilled residue (70 g). The THC content of the distillate was 71.5%.

(71) Chemical Derivatization of the Distilled Extract:

(72) A portion (103.9 g) of the distillate was winterized by ethanol to remove waxes and hydrocarbons to produce 85.0 g (77% THC) winterized distillate, of which 39.5 g was chemically derivatized as following:

(73) The distillate (39.5 g) was dissolved in 100 mL methylene chloride (DCM, Fisher, D37-4) to which was added DMAP (120 mg) and the reaction mixture was stirred at room temperature for 10 minutes (mixture A). In another 2 L round bottom flask, Boc-Trp-OH (45.0 g, 1.2 eq, AnaSpec. Inc, 510-791-9560) was mixed with DCC (31.1 g, 1.2 eq., Alfa Aesar, A13016) then 100 mL DCM were added and the reaction mixture was stirred at room temperature for 10 minutes (mixture B). Mixture B was added to mixture A and the reaction mixture was stirred at room temperature until the reaction was completed (approx. 15 mins.), which was confirmed by Si gel Thin Layer Chromatography (Si-TLC) using EtOAc:DCM (5:95) as mobile phase. After the reaction completion, 2 L hexanes was added and the precipitated reagents were filtered. The filtrate was evaporated to dryness under reduced pressure to give 116.0 g dried distillate adduct.

(74) Column Chromatography:

(75) The reaction mixture (116.0 g) was dissolved in 100 mL of DCM and loaded on the top of a silica gel column (2.3 Kg, Silicycle 60 A, R 100303). Isocratic elution was performed using 2% EtOAc/DCM. Three fractions (A-C) were collected as summarized in Table 6.

(76) TABLE-US-00006 TABLE 6 THC and CBD fractions by the process described in Example 4 Fraction Weight (g) Analysis for Cannabinoids A 25.0 Complex mixture and reagents B 20.0 THC adduct and CBN adduct C 50.0 THC adduct
Hydrolysis of THC Adduct:

(77) Fraction C (50.0 g) was subjected to hydrolysis using 5N KOH to give 23 g THC with 98.90% purity. The purity of THC was determined by GC/FID and GC/MS analysis. The overall yield of THC from this process starting from the distilled extract is 75%. The schematic representation of this process was shown in FIG. 3.

Example No. 5

(78) Production of THC from the Distillate of an Extract of a High THC Variety of Cannabis

(79) Extraction and Decarboxylation:

(80) Dried plant material (31.126 kg) of high THC content Cannabis (9.96%) was extracted by hexanes (140 L) in 200 L percolator, for 12 hours, then the extract was drained and evaporated under reduced pressure at 60° C. till dryness. The dried extract was heated at 100° C. for 5 hours till complete decarboxylation which was detected by TLC to give 4.16 Kg extract (THC content 52.9%).

(81) Distillation of the Decarboxylated Extract:

(82) A portion of the above extract (1.38 kg) was distilled using thin film distillation as above to give 870 g distillate. The distillate was dissolved in 10 L EtOH and kept in the freezer for 4 hours, then filtered to remove waxes and hydrocarbons. The filtrate was concentrated under reduced pressure to yield 667 g of winterized extract.

(83) Chemical Derivatization:

(84) A portion of the distillate (50.0 g) was chemically derivatized by reaction with t-Boc-try-OH and DCC as above to produce distillate adduct. 2.5 L hexanes was added and the precipitated reagents were filtered. The filtrate was evaporated to dryness under reduced pressure to give 110 g dried distillate adduct.

(85) Purification of the Distillate Adduct to Produce THC Adduct:

(86) The adduct was dissolved in DCM and applied on a silica gel column (2.0 Kg), eluted with 2% EtOAc/DCM, to produce 4 fractions A-D in Table 7.

(87) TABLE-US-00007 TABLE 7 THC and CBD fractions by the process described in Example 5 Fraction Weight (g) Analysis for Cannabinoids A 7.9 Complex mixture and reagents B 13.0 THC/CBN adduct, C 37.0 THC adduct D 30.1 —

(88) Hydrolysis of THC adduct (37.0 g) was performed using 5N KOH to produce 15.9 g THC of 98.2% purity. The overall yield of THC in the process starting from the decarboxylated extract is 60.2%. FIG. 3 shows the schematic representation of this process.

Example No. 6

(89) Purification of Δ.sup.9-THCV from a High THCV Cannabis Fraction

(90) Preparation of Reaction Material:

(91) A fraction rich in Δ.sup.9-THCV (69.0% THCV, 1.70 g) was dissolved in 50 mL methylene chloride (DCM, Fisher, D37-4) to which 4-Dimethyl aminopyridine (10.0 mg) was added, and reaction mixture stirred at room temperature for 10 minutes, (mixture A). In another 500 mL round bottom flask, Boc-Gln-OH (1.62 g, 1.2 eq, Aldrich, 408441) was mixed with DCC (1.5 g, 1.2 eq., Alfa Aesar, A13016) and DCM (50 mL), and the reaction mixture was stirred at room temperature for 10 minutes, (mixture B). Mixture B was then added to mixture A, followed by stirring at room temperature for 15 minutes for the reaction completion, which was confirmed by Si gel Thin Layer Chromatography (Si-TLC) using ethyl acetate (EtOAc, Fisher, E145-4):DCM (5:95) as the mobile phase. After the reaction was complete, 100 mL hexanes was added and the mixture was cooled in the freezer for 4 hours, followed by filtration through a filter funnel. The filtrate was evaporated to dryness under reduced pressure to give 4.3 g of the dried reaction mixture.

(92) Column Chromatography:

(93) The reaction mixture (4.3 g) dissolved in 5 mL of DCM was applied to the top of a silica gel column (65.0 g, Silicycle 60 A, R 100303). Elution was carried out using 5% EtOAc/hexanes, and four fractions (A-D) were collected. Fraction C (917 mg), which contains pure THCV adduct, was hydrolyzed using 5N KOH to give 419 mg Δ.sup.9-THCV with 95.0% purity. The purity was determined using GC/FID and GC/MS. The overall yield of Δ.sup.9-THCV is 35%. FIG. 4 shows the schematic representation of this process.

Example No. 7

(94) Purification of CBG:

(95) The column fraction 16-17 (0.11 g, 98.9% purity) from experiment No. 3 was subjected to crystallization from hexanes to give 0.10 g CBG as white needles with 100.0% purity as determined by GC/FID and GC/MS. The schematic representation of this process was shown in FIG. 2.

(96) Preparation of CBD from a High CBD Cannabis Extract

(97) In the case of a Cannabis extract that has a high CBD content and a low THC content, purification of CBD can be carried out much more efficiently without prior derivatization.

(98) The crude extract (hexanes extract or supercritical fluid extract) could be winterized and the winterized extract subjected to a process of thin film distillation and the distillate chromatographed directly without derivatization.

(99) Below are examples of the processes.

Example No. 8

(100) Production of CBD from a High CBD Distillate

(101) Three batches, 5.0 g each of High CBD Winterized distillate (78% CBD and 10% THC) were subjected to Si gel CC (100 g silica each) eluted with 2.5% EtOAc/hexanes. Four fractions 200 mL each were collected and analyzed by TLC using DCM as an eluent. Fractions 2 and 3 were combined and concentrated under vacuum, then dissolved in 20 mL hexanes, and then kept in the freezer for 15 hrs. The supernatant was decanted and the crystals were washed with cold hexanes (5 mL). The yield and the GC analysis are shown in Table 8.

(102) TABLE-US-00008 TABLE 8 CBD Produced by Process in Example 8 CBD CBD Other Batch # Wt. (g) Recovery purity THC % cannabinoids % B# 1-CBD 3.2 g 81.4% 99.2% 0.22 0.15% THCV 0.36% other peak B# 2-CBD 2.4  61.53% 99.3% 0.28 0.08% THCV 0.30% other peak B# 3-CBD 2.86 72.77% 99.2% 0.35 0.08% THCV 0.29% other peak Average 2.83 g 71.9% 99.23% 0.28% 0.3% other CBD and peak THC in the 0.1% THCV crystals

Example No. 9

(103) Production of CBD Crystals from a High CBD Distillate

(104) Two high CBD extracts (300 g) were distilled by thin film distillation, then winterized with ethanol to give two winterized distillates (215 g and 205 g). Each winterized distillate was subjected to Si gel CC followed by crystallization to yield three batches of pure CBD (>99.9% purity) as follow:

(105) a—Winterized distillate Batch #1 (215 g, 75.05% CBD and 2.2% THC) was subjected to silica gel cc chromatography (3.0 kg silica) isocratically eluted with 2.5% EtOAc/hexanes. Eleven fractions (2 L each) were collected and examined by TLC (DCM as eluent), then similar fractions were combined to give three main fractions (A-C). Fraction B that showed single spot was crystallized from hexanes at room temperature to give 106.05 g of pure CBD (99.93% purity).

(106) b—Winterized distillate Batch #2 (205 g, 75.7% CBD and 2.2% THC) was also subjected to silica gel cc chromatography (3.0 kg silica) then crystallization as described for batch #1 to produce 108.2 g of crystalline CBD with purity=99.95%.

(107) Table 9 shows the yield and the purity (by GC analysis) of two batches of CBD.

(108) TABLE-US-00009 TABLE 9 CBD Produced by Process in Examole 9 CBD CBD Other Batch # Wt. (g) Recovery purity THC % cannabinoids % B# 1-CBD 215 g 65.75% 99.93% <0.01 0.07% B# 2-CBD 205 g 68.5% 99.95% <0.01 0.05% Average 2.10 g 67.1% 99.94% <0.01 0.06% CBD and THC in the crystals

REFERENCES

(109) 1. ElSohly, M. A.; Harland, E.; and Waller, C. W.; Cannabinoids in glaucoma II: The effect of different cannabinoids on the intraocular pressure of the rabbit; Curr. Eye Res., 3(6): 841-850, 1984. 2. El-Mallakh, R. S.; Marihuana and migraine, Headache, 27(3):442-443, 1987. 3. Volfe, Z.; Dvilansky, I. A.; and Nathan, I.; cannabinoids in block release of serotonin from platelets induced by plasma from migraine patients; Int. J. Clin. Pharmacol. Res., 5(4):243-246, 1985. 4. Maurer, M.; Henn, V.; Dirtrich, A.; and Hofmann, A.; Delta-9-tetrahydrocannabinol shows antispastic and analgesic effects in single case double-blind trial; Eur. Arch. Psychiatry Clin. Neurosci., 240(1): 1-4, 1990. 5. McLendon, D. M.; Harris, R. T.; and Maule, W. F.; Suppression of the cardiac conditioned response by delta-9-tetrahydrocannabinol: A comparison with other drugs; Psychopharmacology, 50(2): 159-163, 1976. 6. ElSohly, Mahmoud A.; Stanford, Donald F.; Harland, Ernest C.; Hikal, Ahmed H.; Walker, Larry A.; Little, Thomas L., Jr.; Rider, James N.; and Jones, Alan B.; Rectal bioavailability of delta-9-tetrahydrocannabinol from the hemisuccinate ester in monkeys; J. Pharm. Sci., 80(10): 942-945, 1991. 7. ElSohly, Mahmoud A.; Little, Thomas L., Jr. Hikal, Ahmed; Harland, Ernest; Stanford, Donald F. and Walker, Larry; Rectal bioavailability of delta-9-tetrahydrocannabinol from various esters; Pharmacol., Biochem., Behav., 40(3): 497-502, 1991. 8. Mattes, Richard D.; Shaw, Leslie M.; Edling-Owens, Judy; Engelman, Karl; and ElSohly, Mahmoud A.; Bypassing the first-pass effect for the therapeutic use of cannabinoids: Pharmacol., Biochem., Behav., 44(3): 745-77, 1993. 9. Mather L.; Cannabinoid pharmacotherapy: past, present and future: Minerva Anestesiologica, 71(7-8):405-12, 2005. 10. Brenneisen, R.; ElSohly, M. A.; Henn, V.; and Spiess, Y.; The effect of orally and rectally administered delta-9-tetrahydrocannabioi on spasticity: A pilot study with 2 patients; Inter. J. Clin. Pharmacol. and Therapeutics, 34(10): 446-452, 1996. 11. Cunha, Jomar M., E. A. Carlini, Aparecido E. Pereira, Oswaldo L. Ramos, Camila Pimentel, Rubens Gagliardi, W. L. Sanvito, N. Lander, and R. Mechoulam. “Chronic administration of cannabidiol to healthy volunteers and epileptic patients.” Pharmacology 21: 175-185, 1980. 12. Mechoulam, Raphael, Linda A. Parker, and Ruth Gallily. “Cannabidiol: an overview of some pharmacological aspects.” The Journal of Clinical Pharmacology 42, no. S1: 11S-19S, 2002. 13. Schier, Alexandre Rafael de Mello, Natalia Pinho de Oliveira Ribeiro, Jaime Eduardo Cecilio Hallak, Jose Alexandre S. Crippa, Antonio E. Nardi, and Antonio Waldo Zuardi. “Cannabidiol, a Cannabis sativa constituent, as an anxiolytic drug.” Revista Brasileira de Psiquiatria 34 (: 104-110, 2012. 14. Brenda E. Porter, Catherine Jacobson Report of a parent survey of cannabidiol-enriched cannabis use in pediatric treatment-resistant epilepsy). Epilepsy & Behavior, 29, 5745-577, 2013. 15. Lola Weissd, Michael Zeira, Raphael Mechoulam, Shimon Salvin, Ruth Gallity. Treating or preventing diabetes with Cannabidiol. U.S. Pat. No. 8,071,641 B2, issued Dec. 6, 2011. 16. Lannotti, Fabio Arturo; Hill, Charlotte L.; Leo, Antonio; Alhusaini, Ahlam; Soubrane, Camille; Mazzarella, Enrico; Russo, Emilio; Whalley, Benjamin J.; Di Marzo, Vincenzo; Stephens, Gary J. Nonpsychotropic Plant Cannabinoids, Cannabidivarin (CBDV) and Cannabidiol (CBD), Activate and Desensitize Transient Receptor Potential Vanilloid 1(TRPV1) Channels in Vitro: Potential for the Treatment of Neuronal Hyperexcitability ACS Chemical Neuroscience, 5(11): 1131-1141, 2014. 17. Prakash S. Nagarkatti, Mitzi Nagarkatti, Use of cannbaindiol in the treatment of autoimmune hepatitis US patent Aug. 14, 2012. 18. Geoffrey Guy, Bettina Platt, (2014). Cannabinoid-containing plant extracts as neuroprotective agents, U.S. Pat. No. 8,673,368. 19. Gaoni Y, Mechoulam R. Isolation, structure, and partial synthesis of an active constituent of hashish. J. Am. Chem. Soc. 86(8): 1646-1647, 1964. 20. ElSohly, Mahmoud A.; and Ross, Samir A.; Method of preparing delta-9-tetrahydrocannabinol; U.S. Pat. No. 6,365,416, B1: Apr. 2, 2002. 21. Duchek, John Robert; Cannabinoid crystalline derivatives and process of cannabinoid purification; U.S. Pat. No. 7,402,686 B2: Jul. 22, 2008. 22. Adams, R.; Hunt, M.; Clark, J. H.; Structure of cannabidiol, a product isolated from the marihuana extract of Minnesota wild hemp; J. Amer. Chem. Soc. (62): 196-200, 1940. 23. Adams, R.; Pease, D. C.; Clark, J. H.; Isolation of cannabinol, cannabidiol from red oil of Minnesota wild hemp; J. Amer. Chem. Soc. (62): 2194-2196, 1940. 24. Hazekamp, A.; Simon, R.; Looman, A. P.; Sengers, M.; Zweden, R. V.; and Verpoorte, R.; Preparative isolation of cannabinoids from Cannabis sativa by centrifugal Partition Chromatography; J. Liquid Chroma. and Related Tech., 27: 2421-2439, 2004. 25. Appendio, G.; Gibbons, S.; Giana, A.; Pagani, A.; Starvi, M.; Smith, E.; and Mukhlesur, R., Antibacterial cannabinoids from Cannabis sativa: A structure-activity study; J. Nat. Prod., 71: 1427-1430, 2008. 26. Flockhart Ian, Ehjeatley Gray, Dring Su, Lesley Archer. Method of preparing Cannabidiol from plant material; U.S. Pat. No. 0,167,283: Jul. 27, 2006.