Method for isolating and purifying xanthohumol

Abstract

The present invention is a method for isolating and purifying a plant-derived bioactive substance (xanthohumol) from hop plant material and by-products formed during the process of extracting hops with supercritical carbon dioxide.

Description

[Technical Field]

[0001] The present invention relates to a method for isolating and purifying a plant-derived bioactive substance (xanthohumol) from hop plant material and by-products formed during the process of extracting hops with supercritical carbon dioxide.

[0002] Xanthohumol (2',4,4'-trihydroxy-6'-methoxy-3'-(3-methylbut-2-en-1-yl)chalcone, CAS: 6754-58-1, abbreviated as Xn) is a naturally occurring substance of the prenylated chalcone family. Xn is found in the female inflorescences (hop cones) of hops (Humulus lupulus). This compound is also found in beer, where it gives it a bitter taste.

[0003] WO 2014 / 016409 discloses a pharmaceutical composition containing a therapeutic amount of xanthohumol and a roasted extract, preferably a hop extract containing xanthohumol, for the treatment and prevention of cancer. The roasted extract is selected from low-temperature or high-temperature extracts of coarsely ground or unground roasted malt, cereals, coffee, and cocoa.

[0004] Another European Patent No. 1761245 discloses a method for producing a roasted extract containing xanthohumol from a roasted product of cereals, cereal malt, or coffee and a hop extract containing Xn, and its use in the production of beverages and foods.

[0005] Chinese Patent Publication No. 101440029 discloses a method for extracting xanthohumol from powdered hops and supercritical carbon dioxide (scCO2). The recovered hop raffinate is subjected to ultrasonic extraction with alcohol, the extract is concentrated, mixed with diatomaceous earth, eluted with a mixture of alcohol and water, and filtered and concentrated at low temperature to obtain the final product, xanthohumol.

[0006] However, the processes described in the literature for extracting and isolating xanthohumol from plant material have a number of drawbacks and disadvantages, such as: i) It requires the use of large amounts of organic solvents, especially highly toxic solvents (dichloromethane, n-hexane, chloroform - for example, CN101440029, CN103524321). ii) It requires the use of large amounts of expensive materials such as silica gel (silica gel). iii) (iii) Xanthohumol irreversibly decomposes to by-products (mainly isomer isoxanthohumol, CAS: 521-48-2) and polymer oxidative degradation products (extraction using aqueous solutions of strong bases, e.g., NaOH, KOH). iv) The process has low efficiency and scalability (e.g., countercurrent chromatography techniques). v) It is not possible to obtain high-purity xanthohumol in large quantities.

[0007] Additionally, methods described for obtaining xanthohumol by chemical synthesis are inefficient and not very scalable. For example, J. Nat. Prod. 2007, 70, 1507-1509 describes a six-step chemical synthesis method for small amounts of Xn (<0.1 g) with a total yield of 10%. Furthermore, the final purification step does not allow for economically viable preparation of large quantities of xanthohumol with high pharmaceutical purity.

[0008] In view of the problems presented, it is clear that there is a need for new methods for isolating xanthohumol from plant material and by-products of hops that do not suffer from the drawbacks and disadvantages mentioned above.

[0009] The present invention aims to obtain xanthohumol of high pharmaceutical purity from plant material. According to the present invention, the developed method provides a cost-effective technology for extracting and purifying xanthohumol from widely available plant material on semi-industrial and industrial scale with low environmental impact.

[0010] The present invention is a novel method for isolating and purifying xanthohumol by extraction from plant material, comprising: - weighing an appropriate amount of plant material, then adding a weighed amount of mixture A, subjecting the contents of the extractor to a continuous extraction process at 20-90 ° C for 2-96 hours under an inert gas atmosphere, passing the soluble fraction of mixture A and plant material through a suitable ceramic flow filter system for evaporation, returning the evaporated solvent to said extractor, and leaving the solid residue X1 as waste; - Mixture A is removed from the extractor, Mixture B is added to the solid portion, the contents of the extractor are subjected to a continuous extraction process under an inert gas atmosphere at 20-90°C for 0.5-24 hours, Mixture B and the soluble fraction of the plant material are passed through a suitable ceramic flow filter system for evaporation, Mixture B is evaporated, and the solid residue X2 is used as a crude product for further processing; after the extraction is completed, the contents of the extractor are sent to a filter press, the filtrate after evaporation of Mixture B is attached to X2, and the solid residue from the filter press is used as waste X3; a weighed amount of X2 is dissolved in a mixture C to give a concentration of Xn between 0.005 and 0.5 mol / l, and the resulting solution of X2 in mixture C is subjected to a single or multiple extraction processes successively with a mixture D having a concentration between 0.005 and 1.0 mol / l, a mixture E having a concentration between 0.005 and 0.2 mol / l, a mixture F having a concentration between 0.005 and 1.0 mol / l, and a mixture G having a concentration between 0.05 and 1.0 mol / l, to obtain a solution H, said extraction processes being carried out in an inert gas atmosphere at a temperature between 10 and 50°C, preferably between 10 and 30°C, In this step of isolating and purifying -Xn, a reversible process of forming a highly water-soluble Xn salt (phenolate) is used, whereby a suspension of Xn in water is obtained after neutralization with an acidic aqueous solution from the D mixture, the H solution is set to an Xn content of 0.01-0.1 mol / l (preferentially 0.05-0.10 mol / l), and then the H solution is subjected to an alkaline-acid extraction process in stream using a liquid-liquid separator, and two solution streams, the H solution and the I mixture, are fed to a flow reactor, which is directly connected to the liquid-liquid separator, the organic stream leaving the separator is waste X4, and the aqueous stream is neutralized with the D mixture, and the extraction process is carried out in an inert gas atmosphere at a temperature of 2-35°C, preferentially 20-25°C; -Xn suspension in water is obtained, which is subjected to (a) filtration, (b) centrifugation, or (c) extraction with mixture C, and the organic phase after the extraction process is washed with 0.05-1.0 mol / L, preferably 0.1-0.2 mol / L of mixture G, preferably sodium chloride or sodium sulfate, and then dried with molecular sieves or anhydrous sodium sulfate, anhydrous magnesium sulfate, or a mixture of desiccants, and after draining the desiccants, a solution J is obtained (Xn content in the dry weight is 80-90 wt%), and after filtration (a) or centrifugation (b), the Xn precipitate is dissolved in mixture C and subjected to the same process as after process (c); A weighed amount of mixture A is added to solution J in which Xn is sparingly soluble (preferably C7-C8 saturated hydrocarbons, preferably n-heptane). The volatile fraction is evaporated until Xn begins to precipitate, the precipitated Xn is drained and washed with mixture A at 0-40°C (preferably 15-25°C), the filtrate is waste X5, the precipitate is immersed in mixture C, the insoluble precipitate is drained again, the filtrate is waste X6, the precipitate is dried under reduced pressure at 15-25°C, the dried precipitate is dissolved in mixture B, the volatile fraction is evaporated to dryness under reduced pressure at 15-35°C, preferably 25-30°C, and after vacuum drying at 15-25°C, the final product is obtained in the form of a pale yellow powder (Xn content ≥ 99.5% as determined by HPLC).

[0011] The extraction in an inert gas atmosphere is advantageously carried out at 25-50°C for 6-36 hours.

[0012] The method wherein mixture A is saturated, unsaturated, or cyclic C5-C8 hydrocarbons, toluene, xylene (ortho-xylene, meta-xylene, para-xylene, and mixtures thereof).

[0013] The method, wherein the residue X1, X2, or X3 is xanthohumol.

[0014] Mixture B is a polar organic solvent or a mixture of polar organic solvents, i.e. aliphatic alcohols of the general formula ROH (R=C1-C4 aliphatic radical), in particular methyl alcohol (MeOH), ethyl alcohol (EtOH), 2-propanol (i-PrOH), esters of the general formula R1CO2R2 (R1 / R2=saturated and unsaturated aliphatic C1-C4 hydrocarbons, R2=aromatic hydrocarbons or aromatic-aliphatic hydrocarbons (e.g. PhCH2-)), in particular ethyl acetate, liquid ethers of the general formula R1OR2 (R1 / R2=saturated and unsaturated, acyclic and cyclic aliphatic hydrocarbons C1-C4), in particular diethyl ether (Et2O), diisopropyl ether (i-Pr2O), tert-butyl methyl ether (t-BuOMe) and 2-methyltetrahydrofuran (2-MeTHF), - A method in which the solvent is a liquid polar aprotic solvent, such as N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO), and hexamethylphosphoramide (HMPA).

[0015] X in mixture C n The concentration of . 01 to 0.05 mol / l.

[0016] wherein said mixture C is a water-immiscible organic solvent or a mixture of organic solvents as defined below, preferentially a mixture of n-heptane and ethyl acetate (volume ratio v / v: 8:2 to 99:1), esters of the general formula R1CO2R2 (R1 / R2=saturated and unsaturated aliphatic C1-C4 hydrocarbons, R2=aromatic hydrocarbons or aromatic-aliphatic hydrocarbons (e.g. PhCH2-)), in particular ethyl acetate, saturated, unsaturated, cyclic C5-C8 hydrocarbons, toluene (PhMe), xylenes (ortho-xylene, meta-xylene, para-xylene, and mixtures thereof); Liquid ethers of the general formula R1OR2 (R1 / R2 = saturated and unsaturated, acyclic and cyclic aliphatic C1-C4 hydrocarbons), in particular diethyl ether (Et2O), diisopropyl ether (i-Pr2O), tert-butyl methyl ether (t-BuOMe), and 2-methyltetrahydrofuran (2-MeTHF).

[0017] The method of claim 1, wherein mixture D is an aqueous solution of an inorganic and / or organic acid containing a compound from the following list or a mixture thereof: - Citric acid, ethylenediaminetetraacetic acid (versenic acid, EDTA), trichloroacetic acid (CCl3CO2H), -Hydrochloric acid (HCl), sulfuric acid (H2SO4), phosphoric acid (H3PO4), nitric acid (HNO3), and sodium hydrogen sulfate (sodium bisulfate, NaHSO4).

[0018] The method wherein the advantageous concentration of said mixture D is 0.05 to 0.2 mol / L.

[0019] A method wherein mixture E is an aqueous solution of an inorganic salt containing transition metal cations of zinc, iron, cobalt, copper, and nickel, such as zinc chloride (ZnCl2).

[0020] The method, wherein the advantageous concentration of said mixture E is 0.05 to 0.1 mol / L.

[0021] A process wherein mixture F is an aqueous solution of a weak inorganic base and / or a weak organic base containing a compound from the following list or a mixture thereof: bicarbonates of metal cations of general formula MHCO3 (M=Li, Na, K, Cs), dihydrogen phosphates of metal cations of general formula M2HPO4 (M=Li, Na, K, Cs), monohydrogen phosphates of metal cations of general formula MH2PO4 (M=Li, Na, K, Cs), salts of monocarboxylic or polycarboxylic acids RCOOM (M=Li, Na, K, Cs, R=saturated and unsaturated aliphatic C1-C4 hydrocarbons, R2=aromatic hydrocarbons or aliphatic-aromatic hydrocarbons), Nitrogen bases of the general formula R1-N(R2)(R3) (R1 / R3=H, saturated and unsaturated aliphatic C1-C4 hydrocarbons, R2=aromatic hydrocarbons or aliphatic-aromatic hydrocarbons).

[0022] A method wherein the advantageous concentration of said mixture F is 0.05 to 0.1 mol / L.

[0023] The process wherein mixture G is an aqueous solution of inorganic salts containing cations of alkali metals (Li, Na, K, Cs) and alkaline earth metals (Be, Mg, Ca), advantageously sodium chloride, sodium sulfate, or mixtures thereof.

[0024] The method, wherein the advantageous concentration of said mixture G is 0.1 to 0.2 mol / L.

[0025] Advantages of the subject invention: The final Xn product exhibits high purity (Xn content >95 wt%). -High process efficiency enables highly efficient separation of Xn. The plant waste generated by the process according to the invention is an excellent fertilizer for use in agriculture. -Toxic solvents such as halogenated alkanes are not required. The recovery of the solvents used in the technological process according to the invention allows to reduce the amount of waste generated. -The compact production scale also leads to reduced energy consumption.

[0026] The terms used above and in the patent specification and claims have the following meanings: Xanthohumol - used interchangeably with the Polish name ksantohumol. Raw materials of plant origin - these are growths, plant materials. Growth matter - a by-product of the hop extraction process. Plant-derived bioactive substances - also called plant extracts. High purity - bioactive substance content >95% by weight. High efficiency - separation efficiency from materials >80%. Plant Material - Any part of the hops plant and / or the product formed in the process of extracting hops with supercritical carbon dioxide. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows a block diagram of a process for isolating xanthohumol from growth. [Figure 2] HPLC chromatograms of plant extracts are shown. [Figure 3] HPLC chromatogram of xanthohumol after the final purification process.

[0028] The present invention is illustrated by the following examples, which do not constitute a limitation of the present invention.

[0029] Example 1 Input materials: 1) Green-gray dust and granules (outgrowths) containing 0.1 to 50% by weight of xanthohumol. 2) Plant materials contain large amounts of non-polar and polar impurities that are difficult to remove by conventional extraction processes (aqueous extraction with water-immiscible organic solvents).

[0030] Procedure for obtaining Xn from plant material (Figure 1): a) Preliminary isolation (concentration of plant material) The plant material is suspended in n-heptane and subjected to a continuous extraction process at 20-30°C to remove non-polar impurities. The extraction temperature is then increased to 50-60°C (at which temperature some components of the plant material melt), and the process is repeated. The heptane is evaporated, and the yellow residue is filtered through a filter using a press. The dry residue is transferred to an extraction reactor, ethyl acetate is added, and the process is repeated as with the n-heptane extraction, except that xanthohumol is accumulated in a flask on a rotary evaporator. At the end of the process, the xanthohumol content in the yellowish precipitate is approximately 10-12% by weight.

[0031] The precipitate is dissolved in an appropriate amount of ethyl acetate-heptane mixture (9:1 v / v) to achieve a xanthohumol concentration of approximately 0.05 mol / L. The resulting dark solution is extracted in the presence of 0.2 M citric acid solution, 0.05 M ZnCl2, 5% NaHCO3, and brine. After drying the organic layer over anhydrous Na2SO4 and evaporating the organic solvent, a solid mixture with a xanthohumol content of 20-25% is obtained.

[0032] b) Purification The mixture is then dissolved in ethyl acetate in an amount sufficient to achieve a xanthohumol concentration of 0.05-0.1M. The solution is then filtered and subjected to continuous alkaline-acid extraction using a liquid-liquid separator. Specifically, the xanthohumol ethyl acetate solution is extracted against 0.05-0.1M NaOH solution, the organic layer is discarded, and the xanthohumol (sodium salt form) in the aqueous layer is added to 0.05M-0.1M citric acid solution, resulting in the precipitation of xanthohumol. This is then extracted against ethyl acetate and dried over anhydrous Na2SO4. Ethyl acetate and brine are added to the residue. After draining the inorganic salts, most of the ethyl acetate is evaporated, and a minimal amount of n-heptane is added to initiate the precipitation of xanthohumol from the solvent mixture. The distillation process continues until all xanthohumol has precipitated. Then, methanol is added to remove traces of ethyl acetate, and the evaporation process continues. The yellow precipitate is drained, washed with n-heptane and dried under reduced pressure to give very pure (≧99.5% as determined by HPLC) xanthohumol as a pale yellow powder.

[0033] Mixture A: 9. A water-immiscible organic solvent or mixture of organic solvents as defined below: 10. Saturated, unsaturated, and cyclic hydrocarbons C5 to C8, toluene, and xylenes (ortho-xylene, meta-xylene, para-xylene, and mixtures thereof).

[0034] Mixture B: 11. A polar organic solvent or mixture of polar organic solvents as defined below: Aliphatic alcohols of the general formula ROH (R = C1-C4 aliphatic hydrocarbons), especially MeOH, EtOH, and 2-propanol. Esters of the general formula R1CO2R2 (R1 / R2 = saturated and unsaturated aliphatic C1-C4 hydrocarbons, R2 = aromatic hydrocarbons or aromatic-aliphatic hydrocarbons (e.g. PhCH2)), especially ethyl acetate. liquid ethers of the general formula R1OR2 (R1 / R2 = saturated and unsaturated, acyclic and cyclic aliphatic C1-C4 hydrocarbons), in particular diethyl ether (Et2O), diisopropyl ether (i-Pr2O), tert-butyl methyl ether (t-BuOMe), and 2-methyltetrahydrofuran (2-MeTHF); Liquid polar aprotic solvents: N,N-dimethylformamide (DMF), N,N-dimethylacetamide, dimethyl sulfoxide (DMSO), hexamethylphosphoramide (HMPA).

[0035] Mixture C: 12. Water-immiscible organic solvents or mixtures of organic solvents as defined below, preferentially ethyl acetate / n-heptane mixtures (8:2 to 99:1 v / v). Esters of the general formula R1O2R2 (R1 / R2 = saturated and unsaturated aliphatic C1-C4 hydrocarbons, R2 = aromatic hydrocarbons or aromatic-aliphatic hydrocarbons (e.g. PhCH2)), especially ethyl acetate. Saturated, unsaturated, cyclic C5-C8 hydrocarbons, toluene, xylene (ortho-xylene, meta-xylene, para-xylene, and mixtures thereof). Liquid ethers of the general formula R1OR2 (R1 / R2 = saturated and unsaturated, acyclic and cyclic aliphatic hydrocarbons C1-C4), in particular diethyl ether (Et2O), diisopropyl ether (i-Pr2O), tert-butyl methyl ether (t-BuOMe), and 2-methyltetrahydrofuran (2-MeTHF).

[0036] Mixture D: 13. Aqueous solutions of inorganic and / or organic acids containing compounds from the following list or mixtures thereof: Citric acid, ethylenediaminetetraacetic acid (Versenoic acid, EDTA), trichloroacetic acid (CCl3CO2H), Hydrochloric acid (HCl), sulfuric acid (H2SO4), phosphoric acid (H3PO4), nitric acid (HNO3), and sodium hydrogen sulfate (sodium bisulfate, NaHSO4).

[0037] Mixture E: 14. Aqueous solutions of inorganic salts containing transition metal cations such as zinc, iron, cobalt, copper, and nickel, e.g., zinc chloride (ZnCl2).

[0038] Mixture F: 15. Aqueous solutions of weak inorganic and / or weak organic bases containing compounds from the following list or mixtures thereof: Bicarbonates of metal cations with the general formula MHCO3 (M = Li, Na, K, Cs) Dihydrogen phosphates of metal cations with the general formula M2HPO4 (M=Li, Na, K, Cs) Monohydrogen phosphates of metal cations with the general formula MH2PO4 (M=Li, Na, K, Cs) Salts of monocarboxylic or polycarboxylic acids RCOOM (M=Li, Na, K, Cs, R=saturated and unsaturated aliphatic hydrocarbons C1-C4, R2=aromatic hydrocarbons or aliphatic-aromatic hydrocarbons). Nitrogen bases of the general formula R1-N(R2)(R3) (R1 / R3=H, saturated and unsaturated aliphatic hydrocarbons C1-C4, R2=aromatic hydrocarbons or carbohydrates R).

[0039] Mixture G: 16. Aqueous solutions of inorganic salts containing cations of alkali metals (lithium, Li, Na, K, Cs) and alkaline earth metals (beryllium, Mg, Ca). Preferentially sodium chloride, sodium sulfate, or mixtures thereof.

[0040] Mixture I: 17. Aqueous solutions of strong inorganic and / or strong organic bases containing compounds from the following list or mixtures thereof: Alkali metal hydroxides of the general formula MOH (M = Li, Na, K, Cs) Orthophosphates of metal cations of general formula M3PO4 (M=Li, Na, K, Cs), Strong organic bases, such as 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU), NR1R2R3 (R-R 13 =H, C1~C6 aliphatic hydrocarbon).

[0041] Phase I 18. A weighed amount of plant-derived raw material (raw material (a) - Xn content 0.1-2.0% by weight; raw material (b) Xn content 10-40% by weight) is placed in a steel extractor equipped with a mechanical stirrer, a reflux condenser, and a dedicated filter system. A weighed amount of mixture A is then added. The contents of the extractor are continuously extracted under an inert gas atmosphere at 20-90 °C (preferentially 25-50 °C) for 2-96 hours (preferentially 12-36 hours). Mixture A and the soluble fraction of the plant material are passed through a system of suitable dedicated flow-through filters for evaporation. The evaporated solvent is returned to the extractor, and the solid residue X1 is discarded.

[0042] Phase 2 19. Mixture A is removed from the extractor and mixed B is added to a constant mass. The contents of the extractor are continuously extracted in an inert gas atmosphere at 20-90°C (preferably 20-50°C) for 0.5-24 hours (preferably 6-12 hours). Mixture B and the soluble fraction of the plant material are passed through a suitable ceramic flow-through filter system for evaporation. Mixture B is evaporated, and the solid residue X2 becomes the crude product for further processing. After the extraction is complete, the contents of the extractor are sent to a filter press. The filtrate from the evaporation of mixture B adheres to X2, and (a) the Xn content of the solid residue is 5-20% by weight (when raw material (a) is used) or 20-80% (when raw material (b) is used). The solid residue from the filter press becomes waste X3 and can be used as fertilizer.

[0043] Stage 3 20. Dissolve a weighed amount of X2 in mixture C to a concentration of a) Xn 0.005-0.5 mol / l (preferentially 0.01-0.05 mol / l), b) the same as a). The resulting solution of X2 in mixture C is subjected to successive single or multiple extraction processes with mixture D at 0.005-1.0 mol / L (preferentially 0.05-0.2 mol / L), mixture E at 0.005-0.2 mol / L (preferentially 0.05-0.1 mol / L), mixture F at 0.005-1.0 mol / L (preferentially 0.05-0.1 mol / L), and mixture G at 0.05-1.0 mol / L (preferentially 0.1-0.2 mol / L), to obtain solution H (Xn content in dry weight: 20-25 wt% when raw material (a) is used, and 30-90 wt% when raw material (b) is used). The extraction process is carried out under an inert gas atmosphere at 10-50°C (preferentially 10-30°C).

[0044] Stage 4 This stage of Xn isolation and purification involves a reversible process that forms a highly water-soluble Xn salt (phenolate), which, after neutralization with the aqueous acid from mixture D, results in a suspension of Xn in water. Xn is practically insoluble in water (1.3 mg / L at 23°C, J. Agric. Food Chem. 1999, 47, 2421-2428).

[0045] 21. The H solution is set to an Xn content of 0.01-0.1 mol / L (preferentially 0.05-0.10 mol / L), and then the H solution is subjected to an alkali-acid extraction process in stream using a liquid-liquid separator. 22. A flow reactor is fed with two solution streams: solution H (Xn in a water-immiscible organic solvent) and mixture I. 23. The flow reactor is directly connected to a liquid-liquid separator. The organic stream from the separator becomes waste X4, and the aqueous stream is subjected to a neutralization process with mixture D. The extraction process is carried out in an inert gas atmosphere at a temperature of 2 to 35 ° C (preferentially 20 to 25 ° C). 24. The result is a suspension of Xn in water, which is then (a) filtered, (b) centrifuged, or (c) extracted with a mixture of C.

[0046] After the extraction process of 25.C, the organic phase is washed with a mixture of G (preferentially sodium chloride or sodium sulfate) at a concentration of 0.05-1.0 mol / L (preferentially 0.1-0.2 mol / L), and then dried over molecular sieves or anhydrous sodium sulfate (Na2SO4), anhydrous magnesium sulfate (MgSO4), or a mixture of the listed desiccants. After draining the desiccants, a J solution (Xn content of 80-90% by weight in the dry mass) is obtained.

[0047] 26. After filtration (a) or centrifugation (b), the Xn precipitate is dissolved in the C mixture and the same procedure is followed as after process (c).

[0048] Stage 5 At this stage, final purification is performed to obtain Xn of high pharmaceutical purity (Xn content ≧99.5% by dry weight as determined by HPLC).

[0049] 27. A weighed amount of mixture A (preferentially C7-C8 saturated hydrocarbons, preferably n-heptane) in which Xn is sparingly soluble is added to solution J, and the volatile fraction is evaporated until precipitation of Xn begins. The precipitated Xn is drained and washed with mixture A at 0-40°C (preferably 15-25°C). The filtrate is waste X5. The precipitate is immersed in mixture C (preferentially a mixture of aliphatic hydrocarbons and ethers).

[0050] 28. The insoluble precipitate is redried and the filtrate is designated as waste X6. The residue is dried under reduced pressure at 15-25°C. The dried residue is dissolved in mixture B (preferentially an aliphatic alcohol) and the volatile fraction is evaporated to dryness under reduced pressure at 15-35°C (preferentially 25-30°C). After drying under vacuum at 29.15–25°C, the final product is obtained as a pale yellow powder (Xn content ≥99.5% as determined by HPLC, Figure 3 ).

Claims

1. 1. A method for isolating and purifying xanthohumol by extraction from plant material, comprising: - weighing out an appropriate amount of plant material, then adding a measured amount of mixture A, and subjecting the contents of the extractor to a continuous extraction process at 20-90°C for 2-96 hours under an inert gas atmosphere, passing the soluble fraction of mixture A and plant material through a suitable ceramic flow filter system in order to evaporate it, returning the evaporated solvent to said extractor, and leaving the solid residue X1 as waste; - Mixture A is removed from the extractor, Mixture B is added to the solid portion, the contents of the extractor are subjected to a continuous extraction process under an inert gas atmosphere at 20-90°C for 0.5-24 hours, Mixture B and the soluble fraction of the plant material are passed through a suitable ceramic flow filter system for evaporation, Mixture B is evaporated and the solid residue X2 is the crude product for further processing, after the extraction is completed the contents of the extractor are sent to a filter press, the filtrate after evaporation of Mixture B is attached to X2 and the solid residue from the filter press is waste X3; a weighed amount of X2 is dissolved in a mixture C to give a concentration of Xn between 0.005 and 0.5 mol / l, and the solution of X2 in the mixture C obtained is subjected to successive single or multiple extraction processes with a mixture D having a concentration between 0.005 and 1.0 mol / l, a mixture E having a concentration between 0.005 and 0.2 mol / l, a mixture F having a concentration between 0.005 and 1.0 mol / l and a mixture G having a concentration between 0.05 and 1.0 mol / l, to obtain a solution H, said extraction processes being carried out in an inert gas atmosphere at a temperature between 10 and 50°C, preferably between 10 and 30°C, - In this step of isolation and purification of Xn, a reversible process of forming a highly water-soluble Xn salt (phenolate) is used, whereby a suspension of Xn in water is obtained after neutralization with an acidic aqueous solution from the D mixture, the H solution is set to an Xn content of 0.01-0.1 mol / l (preferentially 0.05-0.10 mol / l), then the H solution is subjected to an alkaline-acid extraction process in stream using a liquid-liquid separator, feeding two solution streams, the H solution and the I mixture, into a flow reactor, which is directly connected to the liquid-liquid separator, the organic stream leaving the separator becomes waste X4, and the aqueous stream is neutralized with the D mixture, the extraction process being carried out in an inert gas atmosphere at a temperature of 2-35°C, preferentially 20-25°C, - a suspension of Xn in water is obtained, which is subjected to (a) filtration, (b) centrifugation or (c) extraction with mixture C, the organic phase after said extraction process is washed with 0.05-1.0 mol / l, preferably 0.1-0.2 mol / l of mixture G, preferably sodium chloride or sodium sulfate, and then dried with molecular sieves or anhydrous sodium sulfate, anhydrous magnesium sulfate or a mixture of desiccants, after discharging said desiccants a solution J is obtained (Xn content in the dry weight is 80-90 wt.%), after filtration (a) or centrifugation (b) the Xn precipitate is dissolved in mixture C and subjected to the same process as after process (c), a weighed amount of mixture A is added to solution J in which Xn is sparingly soluble (preferentially C7-C8 saturated hydrocarbons, preferably n-heptane), the volatile fraction is evaporated until precipitation of Xn begins, the precipitated Xn is discharged and washed with mixture A at 0-40°C (preferably 15-25°C), the filtrate is waste X5, the precipitate is immersed in mixture C and the insoluble precipitate is discharged again, the filtrate is waste X6, the precipitate is dried under reduced pressure at 15-25°C, the dried precipitate is dissolved in mixture B, the volatile fraction is evaporated to dryness under reduced pressure at 15-35°C, preferably 25-30°C, and after drying in vacuum at 15-25°C, the final product is obtained in the form of a pale yellow powder (Xn content by HPLC ≥ 99.5%),

2. 2. The process according to claim 1, wherein the extraction in an inert gas atmosphere is preferably carried out at 25 to 50° C. for 6 to 36 hours.

3. The mixture A is a saturated, unsaturated, or cyclic C ５ ~C ８ 10. The method of claim 1, wherein the hydrocarbons are toluene, xylene (ortho-xylene, meta-xylene, para-xylene, and mixtures thereof).

4. 2. The method of claim 1, wherein the residue X1, X2, or X3 is xanthohumol.

5. The mixture B is a polar organic solvent or a mixture of polar organic solvents, i.e., aliphatic alcohols of the general formula ROH (R=C １ ~C ４ aliphatic groups), in particular methyl alcohol (MeOH), ethyl alcohol (EtOH), 2-propanol (i-PrOH), -General formula R １ CO ２ R ２ Ester (R １ / R ２ = saturated and unsaturated aliphatic C １ ~C ４ Hydrocarbons, R ２ = aromatic hydrocarbons or aromatic-aliphatic hydrocarbons (e.g., PhCH ２ -)), especially ethyl acetate, -General formula R １ OR ２ Liquid ether (R １ / R ２ = saturated and unsaturated, acyclic and cyclic aliphatic hydrocarbons C １ ~C ４ ), especially diethyl ether (Et ２ O), diisopropyl ether (i-Pr ２ O), tert-butyl methyl ether (t-BuOMe), and 2-methyltetrahydrofuran (2-MeTHF), The method of claim 1, wherein the solvent is a liquid polar aprotic solvent, such as N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dimethylsulfoxide (DMSO), and hexamethylphosphoramide (HMPA).

6. X in mixture C ｎ The concentration of ． 2. The method of claim 1, wherein the concentration of the hydroxybenzoate is 0.01 to 0.05 mol / l.

7. 2. The method according to claim 1, wherein said mixture C is a water-immiscible organic solvent or a mixture of organic solvents as defined below, preferentially a mixture of n-heptane and ethyl acetate in a volume ratio v / v of 8:2 to 99:1: -General formula R １ CO ２ R ２ Ester (R １ / R ２ = saturated and unsaturated aliphatic C １ ~C ４ Hydrocarbons, R ２ = aromatic hydrocarbons or aromatic-aliphatic hydrocarbons (e.g., PhCH ２ -)), especially ethyl acetate, -Saturated, unsaturated, cyclic C ５ ~C ８ Hydrocarbons, toluene (PhMe), xylenes (ortho-xylene, meta-xylene, para-xylene, and mixtures thereof), -General formula R １ OR ２ Liquid ether (R １ / R ２ = saturated and unsaturated, acyclic and cyclic aliphatic C １ ~C ４ Hydrocarbons), especially diethyl ether (Et ２ O), diisopropyl ether (i-Pr ２ O), tert-butyl methyl ether (t-BuOMe), and 2-methyltetrahydrofuran (2-MeTHF).

8. 2. The method of claim 1, wherein the mixture D is an aqueous solution of an inorganic and / or organic acid containing the compounds from the following list or mixtures thereof: Citric acid, ethylenediaminetetraacetic acid (Versenoic acid, EDTA), trichloroacetic acid (CCl ３ CO2H), hydrochloric acid (HCl), sulfuric acid (H ２ SO ４ ), phosphoric acid (H ３ P.O. ４ ), nitric acid (HNO ３ ), sodium hydrogen sulfate (sodium bisulfate, NaHSO ４ .

9. 2. The method according to claim 1, wherein the concentration of said mixture D is advantageously between 0.05 and 0.2 mol / L.

10. The mixture E contains inorganic salts containing transition metal cations of zinc, iron, cobalt, copper, and nickel, such as zinc chloride (ZnCl ２ 2. The method of claim 1, wherein the aqueous solution is

11. 2. The method according to claim 1, wherein the concentration of the mixture E is advantageously between 0.05 and 0.1 mol / L.

12. 2. The method of claim 1, wherein the mixture F is an aqueous solution of a weak inorganic and / or organic base containing the compounds from the following list or mixtures thereof: -General formula MHCO ３ bicarbonates of metal cations (M=Li, Na, K, Cs), - General formula M ２ HPO ４ dihydrogen phosphates of metal cations (M=Li, Na, K, Cs), -General formula MH ２ P.O. ４ monohydrogen phosphates of metal cations (M=Li, Na, K, Cs), - salts of monocarboxylic or polycarboxylic acids RCOOM (M=Li, Na, K, Cs, R=saturated and unsaturated aliphatic C １ ~C ４ Hydrocarbons, R ２ = aromatic hydrocarbons or aliphatic-aromatic hydrocarbons), - nitrogen bases of the general formula R1-N(R2)(R3) (R1 / R3=H, saturated and unsaturated aliphatic C １ ~C ４ Hydrocarbons, R ２ = aromatic hydrocarbons or aliphatic-aromatic hydrocarbons).

13. 2. The method according to claim 1, wherein the concentration of said mixture F is advantageously between 0.05 and 0.1 mol / L.

14. 2. The method according to claim 1, wherein the mixture G is an aqueous solution of inorganic salts containing cations of alkali metals (Li, Na, K, Cs) and alkaline earth metals (Be, Mg, Ca), preferably sodium chloride, sodium sulfate, or mixtures thereof.

15. 2. The method according to claim 1, wherein the concentration of the mixture G is advantageously between 0.1 and 0.2 mol / L.

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