Method for separating and purifying xanthohumol

Abstract

The present invention is a method for separating and purifying a plant-derived bioactive substance (xanthohumol) from plant material of humulus lupulus and by-products formed during extraction of humulus lupulus with supercritical carbon dioxide.

Description

Technical Field

[0001] The present invention relates to a method for separating and purifying plant-derived bioactive substances (xanthohumol) from hop plant materials and by-products formed during the extraction of hops using supercritical carbon dioxide. Background Art

[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 in the prenylated chalcone family. Xn is found in the female inflorescence of hops (hop flower heads). This compound is also found in beer, giving it a bitter taste.

[0003] The specification of WO2014 / 016409 discloses a pharmaceutical composition for treating and preventing cancer, comprising a therapeutic amount of xanthohumol and a roasted extract, and advantageously, the pharmaceutical composition comprises a hop extract containing xanthohumol. The roasted extract is selected from a cold extract or a hot extract of coarsely ground or unground roasted malt, grains, coffee and cocoa.

[0004] From the specification of another application EP1761245B, it is known how to produce a roasted extract containing xanthohumol from a product containing roasted cereals, cereal malt or coffee and hop extract containing Xn, and the use of the roasted extract in the production of beverages and foods.

[0005] The specification of CN101440029 discloses a method for extracting xanthohumol from powdered hops and supercritical carbon dioxide (scCO2). The collected 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, filtered and concentrated at low temperature to obtain the final product xanthohumol.

[0006] However, the methods described in the literature for extracting and isolating xanthohumol from plant materials have many drawbacks and disadvantages, such as:

[0007] i) It is necessary to use a large amount of organic solvents, especially highly toxic solvents (dichloromethane, n-hexane, chloroform - such as CN101440029, CN103524321);

[0008] ii) It requires the use of large amounts of expensive materials, such as silica gel;

[0009] iii) (iii) irreversible decomposition of xanthohumol into by-products (mainly the isomer isoxanthohumol, CAS: 521-48-2) and polymeric oxidative degradation products (extraction using aqueous solutions of strong bases such as NaOH, KOH);

[0010] iv) low process efficiency and scalability (e.g., countercurrent chromatography technology);

[0011] v) It is impossible to obtain a large amount of high-purity xanthohumol.

[0012] Furthermore, the methods described for obtaining xanthohumol by chemical synthesis are inefficient and not scalable. For example, the paper J. Nat. Prod. 2007, 70, 1507-1509 describes how to synthesize a small amount of Xn (<0.1 g) by a six-step chemical synthesis with an overall yield of 10%. Moreover, the final purification step does not allow the economically feasible preparation of large quantities of xanthohumol with high pharmaceutical purity.

[0013] In view of the problems presented, there is clearly a need for a new method for isolating xanthohumol from hop plant material and by-products which does not suffer from the above-mentioned disadvantages and inconveniences. Summary of the invention

[0014] The object of the present invention is to obtain xanthohumol of high pharmaceutical purity from plant materials. According to the present invention, the developed process provides a technology for the extraction and purification of xanthohumol from widely available plant materials on a semi-technical and industrial scale, which is cost-effective and has a low environmental impact.

[0015] The present invention is a novel method for separating and purifying xanthohumol by extracting from plant materials, the method comprising the following contents:

[0016] Weigh an appropriate amount of plant material and then add a measured amount of mixture A, wherein the contents in the extractor are subjected to a continuous extraction process at 20°C-90°C under an inert gas atmosphere for 2h-96h, the mixture A and the soluble part of the plant material are evaporated through a suitable ceramic flow filtration system, the evaporated solvent is returned to the extractor, and the solid residue X1 is waste;

[0017] Removing mixture A from the extractor, adding mixture B to the solid matter, wherein the extractor contents are subjected to a continuous extraction process for 0.5h-24h at 20°C-90°C under an inert gas atmosphere; evaporating mixture B and the soluble portion of the plant raw material through a suitable ceramic flow filtration system, wherein mixture B is evaporated, and the solid residue X2 is a primary product for further processing; after the extraction is completed, the extractor contents are swept into a filter press, and the filtrate after evaporation of mixture B adheres to X2, and the solid residue from the filter press is waste material X3;

[0018] A weighed amount of X2 is dissolved in a mixture C to obtain a concentration of Xn of 0.005 mol / l-0.5 mol / l, and the obtained solution of X2 in the mixture C is subjected to a continuous single or multiple extraction process with a mixture D having a concentration of 0.005 mol / l-1.0 mol / l, a mixture E having a concentration of 0.005 mol / l-0.2 mol / l, a mixture F having a concentration of 0.005 mol / l-1.0 mol / l, and a mixture G having a concentration of 0.05 mol / l-1.0 mol / l to obtain a solution H, wherein the extraction process is carried out at a temperature of 10° C.-50° C., preferably 10° C.-30° C., in an inert gas atmosphere;

[0019] In the step of separation and purification of Xn, a reversible process of forming a readily water-soluble Xn salt (phenolate) is used, wherein the Xn salt, after neutralization with an acidic aqueous solution from the D-mixture, obtains a suspension of Xn in water, the H-solution is set to an Xn content of 0.01 mol / l-0.1 mol / l (preferably 0.05 mol / l-0.10 mol / l), after which the H-solution is subjected to an alkali-acid extraction process in flow using a liquid-liquid separator, two solution streams are injected into a flow reactor - the H solution and the I mixture, the flow reactor is directly connected to the liquid-liquid separator, the organic stream from the separator is the waste X4, the water stream is neutralized with the mixture D, and the extraction process is carried out at a temperature of 2°C-35°C, preferably 20°C-25°C, in an inert gas atmosphere;

[0020] A suspension of Xn in water is obtained, which is (a) filtered, (b) centrifuged or (c) extracted with a mixture C; the organic phase after the extraction process is washed with a mixture G, which preferably has a concentration of 0.05 mol / l-1.0 mol / l, preferably 0.1 mol / l-0.2 mol / l of sodium chloride or sodium sulfate, and then dried with a molecular sieve or anhydrous sodium sulfate, magnesium sulfate or a mixture of the above desiccants; after removing the desiccant, a solution J (with a content of Xn of 80 wt%-90 wt% on a dry weight basis) is obtained; after (a) filtering or (b) centrifuging, the Xn precipitate is dissolved in the mixture C and treated similarly to that after treatment (c);

[0021] A measured amount of mixture A is added to solution J, wherein Xn is slightly soluble in mixture A (preferably C7-C8 saturated hydrocarbons, preferably n-heptane), the volatile parts are volatilized until precipitation of Xn begins, the precipitated Xn is discharged and washed with mixture A at 0°C-40°C (preferably 15°C-25°C), the filtrate constituting waste X5, the precipitate is impregnated with mixture C, the insoluble precipitate is discharged again, the filtrate constituting waste X6, the precipitate is dried at 15°C-25°C under reduced pressure, the dried precipitate is dissolved in mixture B, the volatile parts are evaporated to dryness at 15°C-35°C, preferably 25°C-30°C, under reduced pressure, and the final product is obtained after vacuum drying at 15°C-25°C in the form of a pale yellow powder (Xn content ≥99.5% according to HPLC determination).

[0022] A method wherein the extraction in an inert gas atmosphere is advantageously carried out at 25°C-50°C for 6h-36h.

[0023] A method, wherein the mixture A is saturated, unsaturated or cyclic C5-C8 hydrocarbons, toluene, xylene (o-xylene, m-xylene, p-xylene and mixtures thereof).

[0024] A method wherein the residue X1, residue X2 or residue X3 is xanthohumol.

[0025] A method, wherein the mixture B is a polar organic solvent or a mixture of polar organic solvents, namely:

[0026] Fatty alcohols of the general formula ROH, wherein R = C1-C4 aliphatic group, in particular methanol (MeOH), ethanol (EtOH), 2-propanol (i-PrOH);

[0027] Esters of the general formula R1CO2R2, wherein R1 / R2 = saturated or unsaturated aliphatic C1-C4 hydrocarbons, R2 = aromatic hydrocarbons or aromatic-aliphatic hydrocarbons (e.g. PhCH2-), in particular ethyl acetate;

[0028] Liquid ethers of the general formula R1OR2, where 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);

[0029] Liquid polar aprotic solvents such as N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO) and hexamethylphosphoramide (HMPA).

[0030] A method wherein the concentration of Xn in the mixture C is 0.01 mol / l - 0.05 mol / l.

[0031] A process, wherein the mixture C is a water-immiscible organic solvent or a mixture of organic solvents as defined below, preferably a mixture of n-heptane and ethyl acetate (volume ratio v / v is 8:2 to 99:1):

[0032] Esters of the general formula R1CO2R2, wherein R1 / R2 = saturated or unsaturated aliphatic C1-C4 hydrocarbons, R2 = aromatic hydrocarbons or aromatic-aliphatic hydrocarbons (e.g. PhCH2-), in particular ethyl acetate;

[0033] Saturated, unsaturated, cyclic C5-C8 hydrocarbons, toluene (PhMe), xylene (o-xylene, m-xylene, p-xylene and mixtures thereof);

[0034] Liquid ethers of the general formula R1OR2, where 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).

[0035] A method, wherein the mixture D is an aqueous solution of an inorganic acid and / or an organic acid containing a compound selected from the following list or a mixture thereof: citric acid, ethylenediaminetetraacetic acid (edetic acid, EDTA), trichloroacetic acid (CCl3CO2H), hydrochloric acid (HCl), sulfuric acid (H2SO4), phosphoric acid (H3PO4), and nitric acid (HNO3), sodium bisulfate (sodium acid sulfate, NaHSO4).

[0036] A process wherein the advantageous concentration of the mixture D is 0.05 mol / l - 0.2 mol / l.

[0037] A process in which mixture E is an aqueous solution of an inorganic salt of a transition metal cation containing zinc, iron, cobalt, copper and nickel, such as zinc chloride (ZnCl2).

[0038] A process wherein the advantageous concentration of the mixture E is 0.05 mol / l - 0.1 mol / l.

[0039] A method, wherein the mixture F is an aqueous solution of a weak inorganic base and / or a weak organic base containing a compound selected from the following list or a mixture thereof:

[0040] Bicarbonates of metal cations, with the general formula MHCO3, where M = Li, Na, K, Cs;

[0041] Dihydrogen phosphates of metal cations, with the general formula M2HPO4, where M = Li, Na, K, Cs;

[0042] Monohydrogen phosphates of metal cations, with the general formula MH2PO4, where M = Li, Na, K, Cs;

[0043] Salts of mono- or polycarboxylic acids RCOOM, where M = Li, Na, K, Cs, R = saturated or unsaturated aliphatic C1-C4 hydrocarbons, R2 = aromatic hydrocarbons or aliphatic-aromatic hydrocarbons;

[0044] Nitrogen bases, with the general formula R1-N(R2)(R3), where R1 / R3 = H, saturated and unsaturated aliphatic C1-C4 hydrocarbons, and R2 = aromatic hydrocarbons or aliphatic-aromatic hydrocarbons.

[0045] A process wherein the advantageous concentration of the mixture F is 0.05 mol / l - 0.1 mol / l.

[0046] A process in which the mixture G is an aqueous solution of an inorganic salt containing cations of alkali metals (Li, Na, K, Cs) and alkaline earth metals (Be, Mg, Ca), advantageously sodium chloride, sodium sulfate or mixtures thereof.

[0047] A process wherein the advantageous concentration of the mixture G is 0.1 mol / l - 0.2 mol / l.

[0048] Advantages of the subject invention:

[0049] The final Xn product showed high purity (Xn content>95% by weight);

[0050] High process efficiency, allowing efficient Xn separation;

[0051] The plant waste produced by the method according to the invention is an excellent fertilizer for use in agriculture;

[0052] No toxic solvents such as halogenated alkanes are required;

[0053] Recycling the solvents used in the technological process according to the invention allows reducing the amount of waste generated;

[0054] Compact production scale also means less energy consumption.

[0055] The terms used above and in the patent specification and claims have the following meanings:

[0056] Xanthohumol: Used interchangeably with the Polish name ksantohumol.

[0057] Raw materials of plant origin: These are by-products, plant materials.

[0058] By-products: By-products of the hop extraction process.

[0059] Phytogenic bioactive substances: Also called plant extracts.

[0060] High purity: >95% biologically active substance by weight.

[0061] High efficiency: Material separation efficiency >80%.

[0062] Plant material: All parts of the hop plant and / or products formed during the extraction of hops with supercritical carbon dioxide. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 A block diagram of a method for separating xanthohumol from by-products is shown.

[0064] Figure 2 HPLC chromatograms of plant extracts are shown.

[0065] Figure 3 Shown is the HPLC chromatogram of xanthohumol after the final purification process. DETAILED DESCRIPTION

[0066] The present invention is illustrated by the following high performance examples, which are not intended to limit the present invention.

[0067] Example 1

[0068] Input Materials:

[0069] 1) Greenish grey dust and particles (by-products) with a xanthohumol content of 0.1% to 50% by weight.

[0070] 2) Plant materials contain a large amount of non-polar and polar impurities that are difficult to remove by traditional extraction methods (water extraction with water-immiscible organic solvents).

[0071] Steps for obtaining Xn from plant materials ( Figure 1 ):

[0072] a) Pre-separation (enrichment of plant raw materials)

[0073] The plant raw material is suspended in n-heptane and continuous extraction is carried out at 20°C-30°C to remove non-polar impurities. Then, the extraction temperature is increased to 50°C-60°C (some components of the plant raw material melt at this temperature) and the process is repeated. The heptane is evaporated and the yellow residue is filtered through a filter using pressure. The dried residue is transferred to an extraction reactor, ethyl acetate is added, and a process similar to the n-heptane extraction is repeated, except that now xanthohumol will accumulate in a flask on a rotary evaporator. At the end of the process, the content of xanthohumol in the light yellow precipitate is about 10%-12% by weight.

[0074] The precipitate was dissolved in an appropriate amount of ethyl acetate-heptane mixture (9:1, v / v) to obtain a xanthohumol concentration of ~0.05 mol / L. The resulting dark solution was 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, the organic solvent was evaporated to obtain a solid mixture with a xanthohumol content of 20%-25%.

[0075] b) Purification

[0076] The mixture is then dissolved in an amount of ethyl acetate to obtain a xanthohumol concentration of 0.05M-0.1M; the solution is then filtered and subjected to alkaline-acid flow extraction using a liquid-liquid separator. Specifically, the solution of xanthohumol in ethyl acetate is extracted with a 0.05M-0.1M NaOH solution, the organic layer is discarded, and the xanthohumol (in the form of a sodium salt) in the aqueous layer is added to a 0.05M-0.1M citric acid solution that causes the precipitation of xanthohumol, which is then extracted with ethyl acetate and dried over anhydrous Na2SO4. Ethyl acetate and brine are added to the residue. After the inorganic salts are removed, most of the ethyl acetate is evaporated, and a small amount of n-heptane is added to induce the precipitation of xanthohumol from the solvent mixture. The evaporation process is continued until all of the xanthohumol is precipitated, after which methanol is added to remove traces of ethyl acetate, and the evaporation process is continued. The yellow precipitate is removed, washed with n-heptane, and dried under reduced pressure to obtain xanthohumol as a light yellow powder with very high purity (≥99.5% as determined by HPLC).

[0077] Mixture A:

[0078] 9. Water-immiscible organic solvents or mixtures of organic solvents as defined below:

[0079] 10. Saturated, unsaturated or cyclic C5-C8 hydrocarbons, toluene, xylene (o-xylene, m-xylene, p-xylene and mixtures thereof).

[0080] Mixture B:

[0081] 11. A polar organic solvent or a mixture of polar organic solvents as defined below:

[0082] Fatty alcohols of the general formula ROH, where R = C1-C4 aliphatic hydrocarbon, in particular MeOH, EtOH and 2-propanol;

[0083] Esters of the general formula R1CO2R2, where R1 / R2 = saturated or unsaturated aliphatic C1-C4 hydrocarbons, R2 = aromatic hydrocarbons or aromatic-aliphatic hydrocarbons (e.g. PhCH2), especially ethyl acetate;

[0084] Liquid ethers of the general formula R1OR2, where 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);

[0085] Liquid polar aprotic solvents, N,N-dimethylformamide (DMF), N,N-dimethylacetamide, dimethyl sulfoxide (DMSO), hexamethylphosphoramide (HMPA).

[0086] Mixture C:

[0087] 12. A water-immiscible organic solvent or a mixture of organic solvents as defined below, preferably a mixture of ethyl acetate / n-heptane (8:2-99:1, v / v):

[0088] Esters of the general formula R1CO2R2, wherein R1 / R2 = saturated or unsaturated aliphatic C1-C4 hydrocarbons, R2 = aromatic hydrocarbons or aromatic-aliphatic hydrocarbons (e.g. PhCH2-), in particular ethyl acetate;

[0089] Saturated, unsaturated, cyclic C5-C8 hydrocarbons, toluene, xylene (o-xylene, m-xylene, p-xylene and mixtures thereof);

[0090] Liquid ethers of the general formula R1OR2, where 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).

[0091] Mixture D:

[0092] 13. An aqueous solution of an inorganic acid and / or an organic acid comprising a compound selected from the following list or a mixture thereof:

[0093] Citric acid, ethylenediaminetetraacetic acid (edetic acid, EDTA), trichloroacetic acid (CCl3CO2H), hydrochloric acid (HCl), sulfuric acid (H2SO4), phosphoric acid (H3PO4), nitric acid (HNO3) and sodium bisulfate (sodium acid sulfate, NaHSO4).

[0094] Mixture E:

[0095] 14. Aqueous solutions of inorganic salts of transition metal cations containing zinc, iron, cobalt, copper and nickel, for example zinc chloride (ZnCl2).

[0096] Mixture F:

[0097] 15. An aqueous solution of a weak inorganic base and / or a weak organic base comprising a compound selected from the following list or a mixture thereof:

[0098] Bicarbonates of metal cations, with the general formula MHCO3, where M = Li, Na, K, Cs;

[0099] Dihydrogen phosphates of metal cations, general formula M2HPO4, where M = Li, Na, K, Cs;

[0100] Monohydrogen phosphates of metal cations, with the general formula MH2PO4, where M = Li, Na, K, Cs;

[0101] Salts of mono- or polycarboxylic acids RCOOM, where M = Li, Na, K, Cs, R = saturated or unsaturated aliphatic C1-C4 hydrocarbons, R2 = aromatic hydrocarbons or aliphatic-aromatic hydrocarbons;

[0102] Nitrogen base, general formula R1-N(R2)(R3), where R1 / R3 = H, saturated and unsaturated aliphatic C1-C4 hydrocarbons, R2 = aromatic hydrocarbons or carbohydrates R.

[0103] Mixture G:

[0104] 16. An aqueous solution of an inorganic salt containing cations of alkali metals (Li, Na, K, Cs) and alkaline earth metals (Be, Mg, Ca), preferably sodium chloride, sodium sulfate or a mixture thereof.

[0105] Mixture I:

[0106] 17. An aqueous solution of a strong inorganic base and / or a strong organic base comprising a compound selected from the following list or a mixture thereof:

[0107] Alkali metal hydroxides, with the general formula MOH, where M = Li, Na, K, Cs;

[0108] Orthophosphates of metal cations, with the general formula M3PO4, where M = Li, Na, K, Cs;

[0109] Strong organic bases, for example, 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU), NR1R2R3, where RR 13 =H, C1-C6 aliphatic hydrocarbon.

[0110] Phase 1

[0111] 18. A weighed amount of raw material of plant origin (raw material (a): Xn content of 0.1%-2.0% by weight; raw material (b): Xn content of 10%-40% by weight) is placed in a steel extractor equipped with a mechanical stirrer, a reflux condenser and a dedicated filter system. Then a measured amount of mixture A is added. The extractor contents are continuously extracted at 20°C-90°C (preferably 25°C-50°C) under an inert gas atmosphere for 2h-96h (preferably 12h-36h). Mixture A and the soluble part of the raw material of the plant are evaporated through a suitable system of dedicated flow-through filters. The evaporated solvent is returned to the extractor, and the solid residue X1 is waste.

[0112] Phase 2

[0113] 19. Remove mixture A from the extractor and add mixture B to constant weight. The extractor contents are continuously extracted at 20°C-90°C (preferably 20°C-50°C) in an inert gas atmosphere for 0.5h-24h (preferably 6h-12h). Mixture B and the soluble part of the plant material are evaporated through a suitable ceramic flow-through filtration system. Mixture B is evaporated and the solid residue X2 is the primary product for further processing. After the extraction is completed, the extractor contents are diverted to a filter press. The filtrate after evaporation of mixture B adheres to X2 (a) and the solid residue Xn contains 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 is waste X3, which can be used as fertilizer.

[0114] Phase 3

[0115] 20. Dissolve a weighed amount of X2 in mixture C to obtain a) a concentration of Xn of 0.005 mol / l-0.5 mol / l (preferably 0.01 mol / l-0.05 mol / l), b) the same as a). The obtained solution of X2 in mixture C is subjected to a continuous single or multiple extraction process with mixture D of 0.005mol / l-1.0mol / l (preferably 0.05mol / l-0.2mol / l), mixture E of 0.005mol / l-0.2mol / l (preferably 0.05mol / l-0.1mol / l), mixture F of 0.005mol / l-1.0mol / l (preferably 0.05mol / l-0.1mol / l) and mixture G of 0.05mol / l-1.0mol / l (preferably 0.1mol / l-0.2mol / l) to obtain solution H (when raw material (a) is used, the Xn content is 20wt%-25wt% based on dry weight, and when raw material (b) is used, the Xn content is 30wt%-90wt%). The extraction process is carried out at 10°C-50°C (preferably 10°C-30°C) in an inert gas atmosphere.

[0116] Phase 4

[0117] At the stage of Xn isolation and purification, a reversible process is utilized to form a readily water-soluble Xn salt (phenolate), which, after neutralization with an acidic aqueous solution from mixture D, gives 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).

[0118] 21. The H solution is set to have an Xn content of 0.01 mol / l-0.1 mol / L (preferably 0.05 mol / l-0.10 mol / L), and then the H solution is subjected to an alkali-acid extraction process in flow using a liquid-liquid separator.

[0119] 22. Two solution streams are injected into the flow reactor - solution H (Xn in a water-immiscible organic solvent) and mixture I.

[0120] 23. The flow reactor is directly connected to the liquid-liquid separator. The organic stream from the separator is waste X4, the water stream is neutralized with mixture D, and the extraction process is carried out at a temperature of 2°C-35°C (preferably 20°C-25°C) in an inert gas atmosphere.

[0121] 24. The result is a suspension of Xn in water which is (a) filtered, (b) centrifuged or (c) extracted with a mixture of C.

[0122] 25. After the extraction process of C, the organic phase is washed with a mixture of G (preferably sodium chloride or sodium sulfate) with a concentration of 0.05 mol / l-1.0 mol / l (preferably 0.1 mol / l-0.2 mol / l), and then dried with molecular sieves or anhydrous sodium sulfate (Na2SO4), magnesium sulfate (MgSO4) or a mixture of the listed desiccants. After the desiccants are discharged, a J solution (Xn content of 80 wt%-90 wt% on a dry weight basis) is obtained.

[0123] 26. After (a) filtration or (b) centrifugation, dissolve the Xn precipitate in the C mixture and perform operations similar to those after treatment (c).

[0124] Phase 5

[0125] At this stage, final purification is performed to obtain Xn of high pharmaceutical purity (Xn content ≥ 99.5% on a dry weight basis as determined by HPLC).

[0126] 27. A measured amount of mixture A in which Xn is sparingly soluble (preferably C7-C8 saturated hydrocarbons, preferably n-heptane) is added to solution J, and the volatile parts are allowed to evaporate until precipitation of Xn begins. The precipitated Xn is drained off and washed with mixture A at 0°C-40°C (preferably 15°C-25°C). The filtrate constitutes waste X5. The precipitate is impregnated with mixture C (preferably a mixture of aliphatic hydrocarbons and aliphatic ethers).

[0127] 28. The insoluble precipitate is dried again and the filtrate is waste X6. The residue is dried at 15°C-25°C under reduced pressure. The dried residue is dissolved in mixture B (preferably in fatty alcohol) and the volatile part is evaporated to dryness at 15°C-35°C (preferably 25°C-30°C) under reduced pressure.

[0128] 29. After vacuum drying at 15°C-25°C, the final product is obtained as a light yellow powder (according to HPLC determination, Xn content ≥ 99.5%, Figure 3 ).

Claims

1. A method for separating and purifying xanthohumol by extraction from plant materials, characterized in that: Weigh an appropriate amount of plant material and then add a measured amount of mixture A, wherein the contents in the extractor are subjected to a continuous extraction process at 20°C-90°C under an inert gas atmosphere for 2h-96h, the mixture A and the soluble part of the plant material are evaporated through a suitable ceramic flow filtration system, the evaporated solvent is returned to the extractor, and the solid residue X1 is waste; Removing mixture A from the extractor, adding mixture B to the solid matter, wherein the extractor contents are subjected to a continuous extraction process for 0.5h-24h at 20°C-90°C under an inert gas atmosphere; evaporating mixture B and the soluble portion of the plant raw material through a suitable ceramic flow filtration system, wherein mixture B is evaporated, and the solid residue X2 is a primary product for further processing; after the extraction is completed, the extractor contents are swept into a filter press, and the filtrate after evaporation of mixture B adheres to X2, and the solid residue from the filter press is waste material X3; A weighed amount of X2 is dissolved in a mixture C to obtain a concentration of Xn of 0.005 mol / l-0.5 mol / l, and the obtained solution of X2 in the mixture C is subjected to a continuous single or multiple extraction process with a mixture D having a concentration of 0.005 mol / l-1.0 mol / l, a mixture E having a concentration of 0.005 mol / l-0.2 mol / l, a mixture F having a concentration of 0.005 mol / l-1.0 mol / l, and a mixture G having a concentration of 0.05 mol / l-1.0 mol / l to obtain a solution H, wherein the extraction process is carried out at a temperature of 10° C.-50° C., preferably 10° C.-30° C., in an inert gas atmosphere; In the step of separation and purification of Xn, a reversible process of forming a readily water-soluble Xn salt (phenolate) is used, wherein the Xn salt, after neutralization with an acidic aqueous solution from the D-mixture, obtains a suspension of Xn in water, the H-solution is set to an Xn content of 0.01 mol / l-0.1 mol / l (preferably 0.05 mol / l-0.10 mol / l), after which the H-solution is subjected to an alkali-acid extraction process in flow using a liquid-liquid separator, two solution streams are injected into a flow reactor - the H solution and the I mixture, the flow reactor is directly connected to the liquid-liquid separator, the organic stream from the separator is the waste X4, the water stream is neutralized with the mixture D, and the extraction process is carried out at a temperature of 2°C-35°C, preferably 20°C-25°C, in an inert gas atmosphere; A suspension of Xn in water is obtained, and the suspension is subjected to (a) filtration, (b) centrifugation or (c) extraction with a mixture C; the organic phase after the extraction process is washed with a mixture G, which preferably has a concentration of 0.05 mol / l-1.0 mol / l, preferably 0.1 mol / l-0.2 mol / l of sodium chloride or sodium sulfate, and then dried with a molecular sieve or anhydrous sodium sulfate, magnesium sulfate or a mixture of the above desiccants; after removing the desiccant, a solution J (with a content of Xn of 80 wt%-90 wt% on a dry weight basis) is obtained; after (a) filtration or (b) centrifugation, the Xn precipitate is dissolved in the mixture C and subjected to a treatment similar to that after treatment (c); A measured amount of mixture A is added to solution J, wherein Xn is sparingly soluble in mixture A (preferably C7-C8 saturated hydrocarbons, preferably n-heptane), the volatile parts are volatilized until precipitation of Xn begins, the precipitated Xn is discharged and washed with mixture A at 0°C-40°C (preferably 15°C-25°C), the filtrate constituting waste X5, the precipitate is impregnated with mixture C, the insoluble precipitate is discharged again, the filtrate constituting waste X6, the precipitate is dried at 15°C-25°C under reduced pressure, the dried precipitate is dissolved in mixture B, the volatile parts are evaporated to dryness at 15°C-35°C, preferably 25°C-30°C, under reduced pressure, and the final product is obtained after vacuum drying at 15°C-25°C in the form of a pale yellow powder (Xn content ≥99.5% according to HPLC).

2. The method according to claim 1, characterized in that The extraction in an inert gas atmosphere is advantageously carried out at 25° C. to 50° C. for 6 h to 36 h.

3. The method according to claim 1, characterized in that The mixture A is saturated, unsaturated or cyclic C5-C8 hydrocarbons, toluene, xylene (o-xylene, m-xylene, p-xylene and mixtures thereof).

4. The method according to claim 1, characterized in that: The residue X1, the residue X2 or the residue X3 is xanthohumol.

5. The method according to claim 1, characterized in that The mixture B is a polar organic solvent or a mixture of polar organic solvents, namely: Fatty alcohols of the general formula ROH, wherein R = C1-C4 aliphatic group, in particular methanol (MeOH), ethanol (EtOH), 2-propanol (i-PrOH); Esters of the general formula R1CO2R2, wherein R1 / R2 = saturated or 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, where 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 such as N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMA), dimethyl sulfoxide (DMSO) and hexamethylphosphoramide (HMPA).

6. The method according to claim 1, characterized in that X in the mixture C n The concentration is 0.01mol / l-0.05mol / l.

7. The method according to claim 1, characterized in that The mixture C is a water-immiscible organic solvent or a mixture of organic solvents as defined below, preferably a mixture of n-heptane and ethyl acetate (volume ratio v / v is 8:2 to 99:1): Esters of the general formula R1CO2R2, wherein R1 / R2 = saturated or 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), xylene (o-xylene, m-xylene, p-xylene and mixtures thereof); Liquid ethers of the general formula R1OR2, where 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).

8. The method according to claim 1, characterized in that: The mixture D is an aqueous solution of an inorganic acid and / or an organic acid containing a compound selected from the following list or a mixture thereof: citric acid, ethylenediaminetetraacetic acid (edetic acid, EDTA), trichloroacetic acid (CCl3CO2H), hydrochloric acid (HCl), sulfuric acid (H2SO4), phosphoric acid (H3PO4), nitric acid (HNO3), sodium bisulfate (sodium acid sulfate, NaHSO4).

9. The method according to claim 1, characterized in that: Advantageously, the concentration of the mixture D is 0.05 mol / l-0.2 mol / l.

10. The method according to claim 1, characterized in that The mixture E is an aqueous solution of an inorganic salt of transition metal cations containing zinc, iron, cobalt, copper and nickel, such as zinc chloride (ZnCl2).

11. The method according to claim 1, characterized in that Advantageously, the concentration of the mixture E is 0.05 mol / l-0.1 mol / l.

12. The method according to claim 1, characterized in that The mixture F is an aqueous solution of a weak inorganic base and / or a weak organic base containing a compound selected from the following list or a mixture thereof: Bicarbonates of metal cations, with the general formula MHCO3, where M = Li, Na, K, Cs; Dihydrogen phosphates of metal cations, with the general formula M2HPO4, where M = Li, Na, K, Cs; Monohydrogen phosphates of metal cations, with the general formula MH2PO4, where M = Li, Na, K, Cs; Salts of mono- or polycarboxylic acids RCOOM, where M = Li, Na, K, Cs, R = saturated or unsaturated aliphatic C1-C4 hydrocarbons, R2 = aromatic hydrocarbons or aliphatic-aromatic hydrocarbons; Nitrogen bases, with the general formula R1-N(R2)(R3), where R1 / R3 = H, saturated and unsaturated aliphatic C1-C4 hydrocarbons, and R2 = aromatic hydrocarbons or aliphatic-aromatic hydrocarbons.

13. The method according to claim 1, characterized in that Advantageously, the concentration of the mixture F is 0.05 mol / l - 0.1 mol / l.

14. The method according to claim 1, characterized in that The mixture G is an aqueous solution of an inorganic salt containing cations of alkali metals (Li, Na, K, Cs) and alkaline earth metals (Be, Mg, Ca), advantageously sodium chloride, sodium sulfate or a mixture thereof.

15. The method according to claim 1, characterized in that Advantageously, the concentration of the mixture G is 0.1 mol / l-0.2 mol / l.

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