METHOD FOR PREPARATION OF A CANNABINOID EXTRACT FROM HEMP

Abstract

The invention relates to a method for extraction and separation of cannabinoids from industrial hemp, designed for medicinal purposes, and also the preparation of an extract, not containing tetrahydrocannabinol, and the preparation of maximum refined individual cannabinoids.

The advantage of the method according to the invention consists in the preparation of an extract from hemp, which contains at a high percentage medically useful cannabinoids and doesn't contain undesirable admixtures and tetrahydrocannabinol, so that it can be used without any restrictions as a pharmaceutical. Moreover, the method allows the possibility of separation, if required, into individual useful cannabinoids as pure compounds, in ecological terms, without environmental pollution, as it is according to the most synthetic methods. The possibility of producing pure compounds represents a great contribution to the research of substances, related to a concrete medical application and the preparation of various combinations thereof, with the objective of expansion the field of application. The method is also cost-effective.

The method consists in that the extract, obtained in accordance with various methods, undergoes a centrifugal countercurrent liquid-liquid chromatography, as the operation includes a centrifugation of solvents and the extract, obtained during the previous operations; the solvents form two phases, the phase, which the extract is dissolved in, is mobile, and the other one is stationary, whereby the mobile phase passes through the stationary phase, wherein several amounts of the components of the extract content are captured; this passing of the mobile phase through the stationary phase is repeated many times, until separation of the desired substances, which are analyzed in a familiar way, whereby as stationary phase solvents are used, which are selected from the group of straight-chain and branched-chain hydrocarbons, produced from crude oil, straight-chain and/or branched-chain alcohols, straight-chain and/or branched-chain ketones, straight-chain and/or branched chain carboxylic acids, straight-chain and/or branched-chain nitriles, gases in supercritical and subcritical condition, like carbon dioxide, nitrogen, nitrogen oxides, water with modified acidity with or without salts of organic and non-organic substances dissolved therein, as for example NaSO.sub.3, carbonate compounds or mixtures of the above-mentioned solvents, and as mobile phase solvents are used, which are selected from the group of straight-chain and branched-chain hydrocarbons, produced from crude oil, straight-chain and/or branched-chain alcohols, straight-chain and/or branched-chain ketones, straight-chain and/or branched chain carboxylic acids, straight-chain and/or branched-chain nitriles, gases in supercritical and subcritical condition, like carbon dioxide, nitrogen, nitrogen oxides, water with modified acidity by organic and/or inorganic acids and bases, as well as Lewis acids and bases in the interval from 0 to 14 pH, with or without salts of organic and non-organic substances dissolved therein, as for example NaSO.sub.3, carbonate compounds, or mixtures of the above-mentioned solvents, while the choice of the solvents between the two phases is conditional on that, they shall be different and immiscible with each other; the centrifugation revolutions and the flow speed of the mobile phase are designed (calculated or determined experimentally) depending on the total phase volume; thus at the finish of the process the tetrahydrocannabinols and/or the tetrahydrocannabinol acids are separated from the remaining cannabinoids and/or cannabinoid acids in the solution, the other cannabinoids in the process can also be separated as a pure substance, separate in a solution and the solvents are evaporated respectively, in order to obtain a pure substance.

Claims

1. Method for preparation of a cannabinoid extract from hemp, in particular the preparation of an extract, not containing tetrathydrocannabinol, and the preparation of maximum refined individual cannabinoids by means of extraction and chromatography, characterized in that the extract, obtained according to different methods, undergoes a centrifugal countercurrent liquid-liquid chromatography, and the operation comprises a centrifugation of solvents and the extract obtained during the previous operations, the solvents form two phases, the phase, in which the extract is solved, is mobile, and the other one is stationary, whereby the mobile phase pass throughout the stationary phase, wherein some quantifies of the components, contained in the extract, are captured and/or delayed, this passing of the mobile phase through the stationary phase is repeated many-times until separation of the desired substances, whereby as a stationary phase solvents are used, which are selected from the group of straight-chain and branched-chain hydrocarbons, produced from crude oil, straight-chain and/or branched-chain alcohols, straight-chain and/or branched-chain ketones, straight-chain and/or branched chain carboxylic acids, straight-chain and/or branched-chain nitriles, solvents in supercritical or subcritical condition, like methane, propane, butane, carbon dioxide, nitrogen, nitrogen oxides, pure water, water with modified acidity with or without salts of organic and non-organic substances dissolved therein, as for example sodium stearate, NaSO.sub.3, carbonate compounds or mixtures of the above-mentioned solvents, and as a mobile phase solvents ate used, selected horn the group of straight-Chain and branched-chain hydrocarbons, produced from crude oil, straight-chain and/or branched-chain alcohols, straight-chain and/or branched-chain ketones, straight-chain and/or branched chain carboxylic acids, straight-chain and/or branched-chain nitriles, pure water, as well as water with modified acidity with or without salts of organic and non-organic substances dissolved therein, as for example sodium stearate, NaSO.sub.3, carbonate compounds, solvents in supercritical or subcritical condition, like methane, propane, butane, carbon dioxide, nitrogen, nitrogen oxides, pure water, as well as water with modified acidity, with or without salts of organic and non-organic substances dissolved therein, as for example sodium stearate, NaSO.sub.3, carbonate compounds or mixtures of the above-mentioned solvents, while the choice of the solvents between the two phases is conditional on that, they shall be different and immiscible with each other, the centrifugation revolutions and the flow speed of the mobile phase are designed (calculated or determined experimentally) depending on the total phase volume, at the finish of the process the tetrahydrocannabinols and/or the tetrahydrocannabinol acids are separated from the remaining cannabinoids and/or cannabinoid acids in the solution, and the other cannabinoids in the process can also be separated as a pure substance separate in a solution. The solvents are evaporated respectively, in order to obtain a pure substance. The process can be repeated for refining.

2. Method according, to claim 1, characterized in that extraction takes place while dry and/or fresh plant matter, and/or mechanical extract, and/or chemical extract from industrial hemp Cannabis stiva L. is extracted non-selectively or selectively using solvents under supercritical or subcritical conditions from the group of carbon dioxide, pure water, water with modified acidity, with or without salts of organic and non-organic substances dissolved therein like, for example, sodium stearate, NaSO.sub.3, carbonate compounds and others, nitric oxides, nitrogen, hydrocarbons, separated from crude oil, for example pentane, hexane, toluol, 3-methylpentane, higher and lower alcohols, ketones, nitrile compounds, halogenated hydrocarbons, separated from crude oil, and/or mixture of these solvents, with or without cosolvents from the group of hydrocarbons, separated from crude oil, for example pentane, hexane, toluol, 3-methylpentane, higher and lower alcohols, ketones, nitrile compounds, halogenated, hydrocarbons, separated from crude oil and/or the relative mixture thereof, at temperatures from 20 to 380 C. and at a pressure from 10 to 1000 bar, while the process can take place through soaking from 1 min to 24 hours, or through continuous processing with fresh and/or recirculating solvent, in some cases the pressure of the solution is decreased in a separate vessel and if required, a winterization is carried out, in order to separate the waxes; at the next step, the pressure of the extract solution, obtained at the previous stage, is reduced until separation of the cannabinoids and/or the cannabinoid acids, and the related substances of the solvent in the form of resin, including in some cases volatile substances, in that case the volatile substances are separated at the next stage by heating from 5 to 300 C. or freezing from 100 to 5 C. by means of evacuating or creating a subatmospheric pressure, while the volatile substances in the flow are either condensed, or transported throughout water with modified acidity, with or without salts of organic and non-organic substances dissolved therein like, for example, sodium stearate, NaSO.sub.3, carbonate compounds and others, and/or an organic solvent horn the group of hydrocarbons, separated from crude oil, for example pentane, hexane, toluol, 3-methylpentane, higher and lower alcohols, ketones, nitrile compounds, halogenated hydrocarbons and/or mixture of these solvents.

3. Method in accordance with claim 1, characterized in that the extraction takes place with solvents from the group, consisting of hydrocarbons, separated from crude oil, for example pentane, hexane, toluol, 3-methylpentane; lower and higher alcohols, for example ethanol, isopropyl; ketones, for example acetone; nitrile compounds, for example acetonitrile; halogenated hydrocarbons, for example chloroform and dichloromethane, under application of soaking at intervals from 1 min to 24 hours; flushing, whereby the solvent and the plant matter are in contact for a few seconds reflux or ultrasound assisted extraction for a time period from 1 min. up to 24 hours.

4. Method according to claim 1, characterized in that the mechanical extract is produced through collection from the plant surface of resin and trichomes, which contain useful cannabinoids in quantity.

5. Method in accordance with claim 1, characterized in that the mechanical extract is obtained by means of freezing or desiccation of trichomes and chopping, and/or screening, and/or blowing.

6. Method according to claim 1, characterized in that depending of the method of extraction and due to the fact, that an extract with different content of undesirable and/or targeted components can be obtained, alternative intermediate operations before the next operation, namely chromatography, are applied for example: decarboxylation with neutral cannabinoid as a targeted component, winterization in the presence of undesirable wages; liquid-liquid extraction in the presence of water-soluble undesirable substances in the extract; flushing through an adsorbent and/or molecular sieve, and/or glass material, and/or sand, in the presence of polar and/or non-polar undesirable, and/or targeted substances; filtration or combination thereof.

Description

BRIEF EXPLANATION OF THE FIGURES

[0019] FIG. 1: HPLC of plant matter.

[0020] FIG. 2: Fraction sequence.

[0021] FIG. 3: HPLC of integrated pure fractions.

[0022] Table 1: Molar solubility of THC at different temperatures and pressure in supercritical carbon dioxide.

[0023] Table 2: Molar solubility of CBD at different temperatures and pressure in supercritical carbon dioxide.

[0024] Table 3: Molar solubility of CBG at different temperatures and pressure in supercritical carbon dioxide

[0025] Table 4: Molar solubility of CBN at different temperatures and pressure in supercritical carbon dioxide

EXEMPLARY EMBODIMENT 1: ISOLATION OF THC FROM A TOTAL HEMP EXTRACT

[0026] In accordance with this embodiment the industrial hemp is harvested and the seed is separated from the straw. The straw is collected and it can be desiccated or fresh extracted. According to the example described here, the straw is desiccated, separated from the seeds, chopped and pulverised. This embodiment requires 1000 g leaf and fruit tips, as well as flower heads of industrial hemp containing tetrahydrocannabinol (THC) 0.06%, tetrahydrocannabinol acid (THCA) 0.01%, cannabidiol acid (CBDA) 1,16%, cannabigerol acid (CBGA) 0.1%, cannabinol (CBN) 0.1% (FIG. 1), and is aimed at the isolation of the psychoactive THC and the respective acid form from the remaining components of the hemp extract.

[0027] Also, the given plant matter contains a low amount of THC, which can be extracted with a determined quantity of SCCD, so that only CBD, CBG and CBN, which will be extracted in a second step for preparation of a hemp extract without THC, remain in the plant matter.

TABLE-US-00001 TABLE 1 Molar solubility of THC at different temperatures and pressure in supercritical carbon dioxide t = 40 C. t = 50 C. t = 60 C. P (bar) 10.sub.4y P (bar) 10.sub.4y P (bar) 10.sub.4y 130 0.20 0.02 130 0.26 0.03 130 0.31 0.03 190 0.66 0.07 150 0.44 0.04 150 0.70 0.07 200 0.66 0.07 170 0.71 0.07 170 1.56 0.16 230 0.71 0.07 190 0.76 0.08 200 1.66 0.17 250 0.81 0.08 200 1.32 0.13 220 2.12 0.21 220 1.41 0.14 230 2.85 0.29 230 2.00 0.20

TABLE-US-00002 TABLE 2 Molar solubility of CBD at different temperatures and pressure in supercritical carbon dioxide t = 40 C. t = 50 C. t = 60 C. P (bar) 10.sub.4y P (bar) 10.sub.4y P (bar) 10.sub.4y 110 1.00 0.10 120 0.95 0.09 115 0.90 0.09 120 1.20 0.12 125 1.60 0.16 120 1.20 0.12 125 1.30 0.13 130 1.89 0.19 125 1.61 0.16 130 1.30 0.13 135 1.88 0.19 130 1.85 0.18 140 1.42 0.14 140 2.22 0.22 145 1.75 0.17 145 1.61 0.16 155 2.50 0.25 160 2.00 0.20 155 1.70 0.17 165 2.69 0.27 165 1.82 0.18 160 1.63 0.16 195 2.71 0.27 170 1.75 0.17 170 1.89 0.19 175 1.89 0.19

TABLE-US-00003 TABLE 3 Molar solubility of CBG at different temperatures and pressure in supercritical carbon dioxide t = 40 C. t = 50 C. t = 60 C. P (bar) 10.sub.4y P (bar) 10.sub.4y P (bar) 10.sub.4y 130 1.15 0.12 130 1.20 0.12 130 1.31 0.13 155 1.20 0.12 150 1.21 0.12 150 1.39 0.14 175 1.19 0.12 160 1.21 0.12 170 1.60 0.16 185 1.23 0.12 165 1.33 0.13 180 1.79 0.18 195 1.30 0.13 170 1.36 0.14 185 1.80 0.18 200 1.30 0.13 180 1.34 0.13 190 1.92 0.19 205 1.50 0.15 190 1.45 0.14 200 2.01 0.20 210 1.60 0.16 200 1.53 0.15 210 2.08 0.21 210 1.69 0.17

TABLE-US-00004 TABLE 4 Molar solubility of CBN at different temperatures and pressure in supercritical carbon dioxide t = 40 C. t = 50 C. t = 60 C. P (bar) 10.sub.4y P (bar) 10.sub.4y P (bar) 10.sub.4y 130 1.23 0.12 130 2.43 0.24 130 2.41 0.24 140 1.28 0.13 135 3.00 0.30 135 2.30 0.13 145 1.30 0.13 140 3.69 0.37 150 3.01 0.30 150 1.46 0.15 145 3.69 0.37 155 3.31 0.33 155 1.61 0.16 150 3.80 0.38 160 3.29 0.33 160 1.80 0.18 155 3.90 0.39 170 3.50 0.35 170 1.92 0.19 160 3.90 0.39 180 3.20 0.32 180 2.01 0.20 170 4.50 0.45 200 3.20 0.32 190 2.16 0.22 180 4.25 0.43 200 2.34 0.23

[0028] The chopped straw is heated to 105 C. for 50 min and is extracted. In the first step 1 kg SCCD at 170 bar and 60 C. is used, and in the second step 10 kg SCCD at 130 bar and 40 C. are used. During the next step the solvent pressure is reduced to 50 bar in a separate vessel. Subsequently, the carbon dioxide is separated from the obtained extract by means of depressurization. The extract is heated to 40 C. under vacuum 10 torr and the volatile substances are isolated. The obtained extract is dissolved in the organic solvent ethanol in a ratio of 1 kg:10 l and it is freezed at 20 C. The coagulated waxes are separated by filtration and the liquid extract is concentrated by evaporation. 510 mg extract from the first step is dissolved to 5 ml in hexane. The dissolved extract undergoes a centrifugal partition chromatography (CPC), which is a type of countercurrent liquid-liquid chromatography. In this particular case a filler for a solvent system with capacity 100 ml is used. The selected system is hexane/acetone/acetonitrile in volume ratios 5:2:3. The process takes place in an ascending mode, while acetone/acetonitrile is used as stationary phase, and hexane as mobile phase. The flow speed is 5 ml/min and the rotation speed is 600 rpm. The dissolved extract is injected into the system. Fractions of 5 ml each are collected and the fractions, containing THC, are separated, while the remaining fractions are integrated. The fractions, containing other cannabinoids and THC, undergo a CPC in the same way, until pure THC is isolated. All fractions except THC are integrated. They are concentrated and the yield represents some percentage; subsequently the obtained substance is desiccated and/or dissolved in a new solvent and thus a total extract from hemp without THC is obtained as a result.

Exemplary Embodiment 2: Preparation of Pure Substances

[0029] According to this embodiment the industrial hemp is harvested and the seed is separated from the straw. The straw is collected and it can be desiccated or fresh extracted. In accordance with the example described here, the straw is desiccated, separated from the seeds, chopped and pulverised. The chopped straw is extracted. This embodiment requires 100 g leaf and fruit tips, as well as flower heads of industrial hemp according to Example 1. In that case the isolation of cannabinoid acids as pure substances is targeted.

[0030] Pentane is used as a solvent at room temperature. 100 g chopped straw is soaked in the solvent in ratio of 1 kg:15 l and is let to stay for 24 hours, while it is mixed every hour. The solvent is percolated from the straw and it is evaporated under reduced pressure until the preparation of 5 ml solution. It is put on a filter bed of glass filter, filed with sand. The extract is flushed through a water solution of 0.2 M NaOH until the full washing out of the saturated color and the streaming of a clear spurt of the solution, whereby the cannabinoid acids in the water solution are isolated from the neutral cannabinoids. A hydrochloric acid is added to the solution up to pH=1.9 in order to precipitate the acid cannabinoids, which are filtrated. 2.5 g cannabinoid acids, obtained from the last process, are dissolved in 5 ml methanol. This dissolved extract undergoes a centrifugal partition chromatography (CPC), which is a type of countercurrent liquid-liquid chromatography. In this particular case a filler for a solvent system with capacity 100 ml is used. The partition system is hexane/methanol/water in volume ratios 5:3:2. The water is acidified by 0.25 mM formic acid. The process takes place in a descending mode, while methanol/water is used as mobile phase, and hexane as stationary phase. The flow speed is 4 ml/min and the rotation speed is 500 rpm. The dissolved extract is injected into the system. Fractions of 10 ml each with pure substances are collected in a solution (FIG. 2). Fractions with more than one component undergo the process once again. The fractions are analysed by means of thin-layer chromatography, uniform fractions are integrated and concentrated, and desiccated and/or dissolved in a new solvent, and thus pure cannabinoid acids are obtained as a result (FIG. 3).