Method for separating natural substance mixtures by means of SCPC

Abstract

The invention relates to a method for separating natural substance mixtures, in particular those consisting of plant extracts, and for isolating and purifying and obtaining same, by means of sequential centrifugal partition chromatography (sCPC).

Claims

1. A method for separating and/or purifying one or more cannabinoids from a Cannabis plant extract, comprising a step of performing at least one liquid-liquid partition chromatography on the Cannabis plant extract to separate and/or purify the one or more cannabinoids, wherein in performing the at least one liquid-liquid partition chromatography a continuous change of the stationary phase to the mobile phase and vice versa takes place, and wherein one or more fractions are removed to separate and/or purify the one or more cannabinoids.

2. The method according to claim 1, wherein at least one rotor is used.

3. The method according to claim 2, wherein a rotation of more than 2,000 rpm takes place.

4. The method according to claim 1, wherein the Cannabis plant extract contains less than 40 wt % fat/lipids.

5. The method according to claim 1, wherein the Cannabis plant extract is obtained from a first solvent selected from the group consisting of alcohols, water, hydrocarbons, and mixtures thereof, and the soluble parts are used.

6. The method according to claim 1, wherein the Cannabis plant extract is a Cannabis sativa extract.

7. The method according to claim 1, wherein the stationary phase and mobile phase are independently selected from the group consisting of hydrocarbons having 5-8 carbon atoms, acetonitrile, ethyl acetate, t-butyl methyl ether, alcohols, and water, so that a two-phase system can be obtained.

8. The method according to claim 7, wherein the solvents at least contain: a.) n-heptane and/or acetonitrile, n-heptane/acetonitrile; b.) n-heptane/ethyl acetate/acetonitrile; c.) n-heptane/ethyl acetate/t-butyl methyl ether/acetonitrile; d.) n-heptane/ethyl acetate/methanol/water; e.) n-heptane/ethanol/water.

9. The method according to claim 7, wherein the stationary phase and mobile phase are independently selected from n-heptane, acetonitrile, ethyl acetate, t-butyl methyl ether, water, ethanol, methanol, n-heptane and/or acetonitrile, so that a two-phase system can be obtained.

10. A method for separating and/or purifying one or more cannabinoids from a Cannabis plant extract, comprising at least one liquid-liquid partition chromatography step, wherein a continuous change of the stationary phase to the mobile phase and vice versa takes place, wherein at least one cannabinoid is removed.

11. The method according to claim 10, wherein at least one rotor is used.

12. The method according to claim 11, wherein a rotation of more than 2,000 rpm takes place.

13. The method according to claim 10, wherein the Cannabis plant extract contains less than 40 wt % fat/lipids.

14. The method according to claim 10, wherein the Cannabis plant extract is obtained from a first solvent selected from the group consisting of alcohols, water, hydrocarbons, and mixtures thereof, and the soluble parts are used.

15. The method according to claim 10, wherein the one or more cannabinoids is cannabidiol or tetrahydrocannabinol.

16. The method according to claim 7, wherein the Cannabis plant extract is a Cannabis sativa extract.

17. The method according to claim 10, wherein the stationary phase and mobile phase are independently selected from the group consisting of hydrocarbons having 5-8 carbon atoms, acetonitrile, ethyl acetate, t-butyl methyl ether, alcohols, and water, so that a two-phase system can be obtained.

18. The method according to claim 10, wherein the solvents at least contain: a.) n-heptane and/or acetonitrile, n-heptane/acetonitrile; b.) n-heptane/ethyl acetate/acetonitrile; c.) n-heptane/ethyl acetate/t-butyl methyl ether/acetonitrile; d.) n-heptane/ethyl acetate/methanol/water; e.) n-heptane/ethanol/water.

19. The method according to claim 17, wherein the stationary phase and mobile phase are selected from n-heptane, acetonitrile, ethyl acetate, t-butyl methyl ether, water, ethanol, methanol, n-heptane and/or acetonitrile, so that a two-phase system can be obtained.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a principal cycle for an sCPC.

(2) FIG. 2 shows an HPLC chromatogram for the starting material as described in Example 2.

(3) FIG. 3 shows the Batch partition chromatography (CPC) as described in Example 2.

(4) FIG. 4 shows the Continuous Partition Chromatography (sCPC) as described in Example 2.

(5) FIG. 5 shows the HPLC chromatogram of the ascending side as described in Example 2.

(6) FIG. 6 shows the HPLC chromatogram of the descending side as described in Example 2.

(7) FIG. 7 shows an HPLC chromatogram for the starting product Essi Agni casti as described in Example 3.

(8) FIG. 8 shows the Batch partition chromatography (CPC) as described in Example 3.

(9) FIG. 9 shows a chromatogram for True Moving Bed (TMB) Purification Step 1 for Pre-enrichment as described in Example 3.

(10) FIG. 10 shows a DAD spectrum of purified casticin after TMB as described in Example 3.

(11) FIG. 11 shows HPLC chromatogram of purified casticin after TMB in Example 3.

(12) FIG. 12 shows an HPLC chromatogram of the starting product Essi Agni casti as described in Example 3.

(13) FIG. 13 shows a GC chromatogram of the starting product oleum chamomillae as described in Example 4.

(14) FIG. 14 shows the Batch partition chromatography (CPC) as described in Example 4.

(15) FIG. 15 shows a chromatogram for TMB Purification Step 1 Continuous Partition Chromatography (sCPC) as described in Example 4.

(16) FIG. 16 shows a chromatogram for TMB Purification Step 2 Continuous Partition Chromatography (sCPC) as described in Example 4.

(17) FIG. 17 shows a DAD spectrum of purified chamazulene after TMB Purification Step 2 as described in Example 4.

(18) FIG. 18 shows a GC chromatogram of purified chamazulene after TMB Purification Step 2 as described in Example 4.

(19) FIG. 19 shows the Batch partition chromatography (CPC) as described in Example 5.

(20) FIG. 20 shows a chromatogram for TMB Purification Step 1 Continuous Partition Chromatography (sCPC) as described in Example 5.

(21) FIG. 21 shows a chromatogram for TMB Purification Step 2 Continuous Partition Chromatography (sCPC) as described in Example 5.

(22) FIG. 22 shows an HPLC chromatogram of purified primulasaponin 1 after TMB Purification Step 2 as described in Example 5.

(23) The following examples and figures serve only to explain the invention without limiting the invention to said examples.

EXAMPLE 1

Description of Equipment

(24) Equipment:

(25) Continuous liquid-liquid chromatography True Moving Bed (TMB) 500 system, with a 2×250 mL rotor, 100-3,000 rpm speed, up to 100 mL/min flow rate, 100 bar maximum pressure of Armen Instrument (Saint-Ave, France) or 12 L production system with a 2×6 L rotor.

(26) Additional Equipment: 2× eluent pump, up to 100 mL/min flow rate; 2× sample delivery pump, up to 50 mL/min flow rate; 2× diode array detector Ecom, 4 wavelengths, 200-600 nm detection range, 6 kg; 2× fraction collector LS-5600, incl. rack set made of stainless steel, 20 kg

(27) Operating Principle:

(28) The sample or substance mixture is injected continuously onto both rotors in phases, and the sample components are separated according to their polarity and their liquid-liquid distribution coefficients K using a suitable two-phase solvent system. After the system is started, a stationary, steady state is established. Product side A can then be removed at one rotor by means of a fraction collector. Alternately, product side B can be removed at the second rotor by mean of a further fraction collector. The detectors are used to monitor the separation.

EXAMPLE 2

Industrial Hemp Extract with Depletion of THC

(29) Extraction of Industrial hemp extract THC-free: Extraction of Cannabis herb using CO.sub.2, decarboxylation at 120° C. in a vacuum, dissolution in n-heptane/ethanol/water,

(30) Method:

(31) Parameters:

(32) TABLE-US-00001 Rotation: 2,600 rpm Ascending mode: Flow rate elution: 30 mL/min Flow rate injection: 10 mL/min/5 mL/min Pump duration: 90 sec Descending mode: Flow rate elution: 30 mL/min Flow rate injection: 10 mL/min/5 mL/min Pump duration: 99 sec Flow rate = flow rate

(33) HPLC Chromatogram Starting Material (see FIG. 2):

(34) Content analysis: CBD: 51.54%, D9-THC: 1.21%, CBN: 1.38%

(35) Batch Partition Chromatography (CPC) (see FIG. 3):

(36) Separation of 3.05 g starting product (50 g/L) in 30 min in batch operation with n-heptane/ethanol/water (5:4:1.4)—the vertical line at 17:00 describes the separation line in the later continuous partition chromatography.

(37) Continuous Partition Chromatography (sCPC)(see FIG. 4):

(38) Separation in continuous operation (50 g/L) with the sCPC with n-heptane/ethanol/water (5:4:1.4)

(39) Grey: UV signal descending side, black: UV signal ascending side

(40) HPLC chromatogram of the ascending side (see FIG. 5):

(41) Content analysis: CBD: 0.00%, D9-THC: 2.63%, CBN: 2.84% HPLC chromatogram of the descending side (see FIG. 6):

(42) Content analysis: CBD: 96.22%, D9-THC: 0.00%, CBN: 0.00%

(43) Comparison of the productivities for industrial hemp extract separation:

(44) TABLE-US-00002 Consumption Mass Solvent per g flow consumption Product System [g/h] [L/h] [L/g] Continuous True 34.81 8.1 0.23 Moving Bed 2 × 250 mL rotor Batch CPC 500 mL 6.10 1.8 0.30 rotor

EXAMPLE 3

(45) Purification of Casticin

(46) Parameters:

(47) TABLE-US-00003 Rotation: 2,600 rpm Ascending mode: Flow rate elution: 15 mL/min Flow rate injection: 5 mL/min Pump duration: 30 sec Descending mode: Flow rate elution: 15 mL/min Flow rate injection: 5 mL/min Pump duration: 60 sec

(48) HPLC Chromatogram Starting Product Essi Agni casti (see FIG. 7):

(49) Solvent system: n-heptane/ethanol/water (5:4:1) (v/v/v)

(50) Content of casticin in the dry extract: 0.145% (m/m)

(51) Batch partition chromatography (CPC) (see FIG. 8):

(52) Separation of 1 g Essi Agni casti in batch operation with n-heptane/ethyl acetate/ethanol/water (7:10:7:10)—the vertical lines at 22:00 and 28:00 describe the separations in the later continuous partition chromatography.

(53) True Moving Bed Purification Step 1 for Pre-enrichment (see FIG. 9):

(54) Content of casticin: 60.3% (m/m)

(55) True Moving Bed Purification Step 2 for Pre-enrichment: DAD spectrum (see FIG. 10) and HPLC chromatogram (see FIG. 11), purified casticin after TMB

(56) Content of casticin: 98.3% (m/m)

(57) HPLC chromatogram of the starting product Essi Agni casti (see FIG. 12):

(58) Content of casticin in the dry extract: 0.145% (m/m)

(59) Solvent system: n-heptane/ethyl acetate/ethanol/water (7:10:7:10) (v/v/v/v)

EXAMPLE 4

(60) Purification of Chamazulene

(61) GC chromatogram of the starting product oleum chamomillae (see FIG. 13):

(62) Content of chamazulene in essential oil: 15.8% (m/m)

(63) Batch partition chromatography (CPC) (see FIG. 14):

(64) Separation of 0.5 g oleum chamomillae in batch operation with n-heptane/TBME/acetonitrile (4:2:4)—the blue lines describe the separations in the later continuous partition chromatography.

(65) Chamazulene 1. TMB step

(66) Parameters:

(67) TABLE-US-00004 Rotation: 2,600 rpm Ascending mode: Flow rate elution: 15 mL/min Flow rate injection: 5 mL/min Pump duration: 90 sec. Descending mode: Flow rate elution: 15 mL/min Flow rate injection: 5 mL/min Pump duration: 75 sec.

(68) Solvent system: n-heptane/TBME/acetonitrile (4:2:4) (v/v/v)

(69) 1. TMB Step Continuous Partition Chromatography (sCPC) (see FIG. 15):

(70) Separation of oleum chamomillae (25 g/L) in continuous operation with the sCPC with n-heptane/ethyl acetate/ethanol/water (7:10:7:10) as Purification Step 1

(71) Content of chamazulene after pre-enrichment TMB1: 70.3% (m/m)

(72) 2. TMB step

(73) Parameters:

(74) TABLE-US-00005 Rotation: 2,600 rpm Ascending mode: Flow rate elution: 15 mL/min Flow rate injection: 5 mL/min Pump duration: 30 sec. Descending mode: Flow rate elution: 15 mL/min Flow rate injection: 5 mL/min Pump duration: 60 sec.

(75) Solvent system: n-heptane/TBME/acetonitrile (4:2:4) (v/v/v)

(76) 2. TMB Step Continuous Partition Chromatography (sCPC) (see FIG. 16):

(77) Separation of the material from TMB Step 1 (25 g/L) in continuous operation with the sCPC with n-heptane/TBME/acetonitrile (4:2:4) as Purification Step 2.

(78) DAD spectrum (see FIG. 17) and GC chromatogram (see FIG. 18) purified chamazulene after TMB Purification Step 2:

(79) Content of chamazulene: 99.0% (m/m)

EXAMPLE 5

(80) Purification of Primulasaponins

(81) Essi Primula veris

(82) Content of primulasaponin 1 in the extract: ˜20% (m/m)

(83) Batch partition chromatography (CPC) (see FIG. 19):

(84) Separation of 0.1 g Essi Primula veris in batch operation with n-heptane/ethyl acetate/water (6:5:4)—the blue lines describe the separations in the later continuous partition chromatography 1. TMB step

(85) Parameters:

(86) TABLE-US-00006 Rotation: 2,600 rpm Ascending mode: Flow rate elution: 15 mL/min Flow rate injection: 5 mL/min Pump duration: 90 sec. Descending mode: Flow rate elution: 15 mL/min Flow rate injection: 5 mL/min Pump duration: 75 sec.

(87) Solvent system: n-heptane/ethyl acetate/water (6:5:4) (v/v/v) 1. TMB Step Continuous Partition Chromatography (sCPC) (see FIG. 20):

(88) Separation of Essi Primula veris (5 g/L) in continuous operation with the sCPC with n-heptane/ethyl acetate/water (6:5:4) as Purification Step 1

(89) Content of primulasaponin 1 after pre-enrichment TMB1: 60.1% (m/m)

(90) 2. TMB step

(91) Parameters:

(92) TABLE-US-00007 Rotation: 2,600 rpm Ascending mode: Flow rate elution: 15 mL/min Flow rate injection: 5 mL/min Pump duration: 30 sec. Descending mode: Flow rate elution: 15 mL/min Flow rate injection: 5 mL/min Pump duration: 60 sec.

(93) Solvent system: n-heptane/ethyl acetate/water (6:5:4) (v/v/v)

(94) 2. TMB Step Continuous Partition Chromatography (sCPC) (see FIG. 21):

(95) Separation of the material from TMB step 1 (5 g/L) in continuous operation with the sCPC with n-heptane/ethyl acetate/water (6:5:4) as Purification Step 2

(96) HPLC chromatogram purified primulasaponin 1 after TMB Purification Step 2 (see FIG. 22):

(97) Content of primulasaponin 1: 84.0% (m/m)