NZ719709B2

NZ719709B2 - Process for the preparation of xanthohumol

Info

Publication number: NZ719709B2
Application number: NZ719709A
Authority: NZ
Inventors: Mariusz Gago
Original assignee: Axen Bio Group Sp Z Oo
Priority date: 2013-10-04
Filing date: 2014-09-16
Publication date: 2021-01-06

Abstract

The invention provides a process for the preparation of xanthohumol, wherein a xanthohumol-containing extract is mixed with water, a transition metal salt solution is added to the obtained mixture, and then the obtained xanthohumol precipitate is collected and dried to obtain xanthohumol of the purity higher than 90%. ty higher than 90%.

Description

Process for the preparation of xanthohumol The invention provides a process for the preparation of high purity xanthohumol from post-extraction spent hops, gran ulated hop plant or hops. Due to the high purity of the ob tained xanthohumol it could be successfully used in dietary supplements, beverages and as a therapeutic product ingredi ent .

Xanthohumol (Xn), or ( (E)[2,4-dihydroxymethoxy(3- methylbutenyl)-phenyl](4-hydroxyphenyl)-propen-l-one) is a naturally-occurring compound of the group of prenylated chalcones, the main source of it being female inflorescence of Humulus Lupus (hop). It is present only in traces in the beer.

The compound is composed of two benzene rings linked by the a, (3 - unsaturated carbonyl moiety: HO. OCH3 OH O Xanthohumol (Xn).

The presence of a free hydroxy group in the molecule makes it easily isomerizable to the proper flavanone - an intermedi- ate at the flavonoid biosynthesis pathway. As opposed to other ingredients of hops, xanthohumol is more lipophilic, the fea ture being connected with the presence of the prenylated sub- stituent in its structure. It should be noted that in living biological systems, the prenylated (farnesyl or geranylgera- nyl) moieties facilitate attachment of numerous intracellurar molecules to a cell membrane, determining their proper activi ties. Likewise, it is suggested that in the case of xanthohu mol, such moieties can significantly modulate biological ac tivity of the compound, and what is more, influence its physi cal-chemical properties and sub-cellular distribution.

In vitro and in vivo studies suggest also that xanthohumol reduces inflammatory and oxidative processes by means of vari ous mechanisms. The leading ones are: elimination of oxygen free radicals and their reactive forms (RTF), cyclooxygenase (COX I and COX II) activity inhibition, reduction in prosta glandin, NO, TNFa, NFKB production and neutralization of mem brane phospholipide peroxidation.

Antioxidative and antiinflammatory activity of xanthohumol is connected with the presence of the hydroxy groups and the prenylated substituent in its molecule. Of importance is also the a, p unsaturated carbonyl moiety. The moiety partici pates in the Michel reaction with biologically important nu- cleophiles eg. : L-cysteine in proteins and other sulfhydryl group-containing molecules. In that way, covalent adducts which cause the loss of biological activity of various mole cules bound by xanthohumol are obtained.

The broad spectrum of xanthohumol activity stems probably from the compound's ability to bind important biological mole cules. One of the best known examples is the influence of xan thohumol on polymerase a synthetase, topoisomerase I, alkaline phosphatase, aromatase, diacylglycerol acyltransferase, MAPK signalling pathway proteins, FAK kinase, Akt and STAT or NF-KB transcription factors. Xn-induced reduction of activities of said proteins induces pleiotropic cell effects as a conse quence of proliferation, cell cycle, adhesion and migration disturbances in tumorous cells.

Noteworthy is also information concerning its neuroprotec tive properties. In the rat model of MCAO-induced focal ische mia, which reflects focal brain ischemia in vivo, xanthohumol significantly reduces the brain ischemic necrosis area. Xan thohumol is thus suggested to reduce also a neurotoxic effect induced by the p-amyloid peptide (AfJ) . A|3 is known to mediate the free-radical mechanism and is one of the main causes of Alzheimer's disease, by increasing production of free radicals and peroxidation of lipids in nerve cells, which consequently lead to their apoptosis.

Further, apart from its effect on receptors, the mechanism of xanthohumol's activity could be connected to its selective activity towards lipid membranes. Studies on mechanisms of in teraction of xanthohumol with model lipid membranes indicate major modification of dynamic and structural properties of a membrane. Even very small concentrations of xanthohumol strongly interact with hydrophilic fragments of lipid membrane to influence bilayer thickness changes. It is also known that neonicotinoids, due to their high hydrophobicity, penetrate lipid membranes very easily. Interaction of xanthohumol with lipid membrane can significantly reduce its penetration by ne onicotinoids, which could explain its strong activity, apart from blocking nicotine receptors.

Concerning its chemical structure, xanthohumol is a poly phenol, however, as opposed to polyphenols, it is scarcely soluble in hot water, but dissolves readily in alcohols or wa ter/alcohol mixtures.

A good source of xanthohumol is the post-extraction spent hops, which comprises wastes from the production of hop ex tract used by brewing industry for the manufacture of beer.

The post-extraction spent hops are estimated to contain up to 1% of xanthohumol, depending on the species of a hop plant.

Numerous technologies for the preparation of xanthohumol are known in the art. The application DE 199 39 350 A1 de scribes a process for the preparation of a xanthohumol- enriched hop extract, wherein mixtures of water and ethanol were used for extraction. The obtained extract contained 5 to % of xanthohumol.

The patent EP 1 424 385 B1 discloses a method of extract ing xanthohumol from the raw xanthohumol-containing material by use of compressed CO2 as a solvent under a pressure above 500 bar and at temperatures above 60oC.

The application CN 101440029 discloses a method of ex tracting xanthohumol from the hop plant and xanthohumol- containing products, wherein the extraction with an organic solvent assisted by ultrasounds is conducted, followed by mix ing with diatomite, filtering, washing out with water/methanol mixture, concentrating, precipitating and drying to obtain xanthohumol of the 86% purity.

The patent EP 2 187 899 B1 discloses a process for the preparation of a composition of the high xanthohumol content from a hop plant, wherein a salt is added to a xanthohumol- containing solution to the concentration in the range of from 0.05 to 5.0M, followed by increasing pH to from 10.5 to 12.0, filtering the obtained precipitate, acidifying the filtrate to pH 7-8 to obtain the xanthohumol precipitate of the 40-95% pu rity.

The patent application US 11/790,365 discloses a process for the preparation of the powder of the high (60-90%) xantho humol content, comprising suspending a xanthohumol-enriched extract in an alkaline solution, separating insoluble prod ucts, neutralizing and precipitating the dissolved xanthohumol with an acid and isolating the product.

Patent CN 101433592 discloses use of various adsorption resins to prepare high purity xanthohumol.

In conclusion, analysis of the prior art discloses une quivocally that there is a need for developing a simple and inexpensive process for the preparation of high purity xantho humol .

The problem is solved by a newly developed process for the preparation of xanthohumol from post-extraction spent hops, granulated hop plant or hops.

Thus, the invention relates to a process for the prepara tion of xanthohumol, wherein: a) a xanthohumol-containing extract is mixed with wa ter; b) a transition metal salt solution is added to the ob tained mixture; c) the obtained xanthohumol precipitate is collected and dried to obtain xanthohumol of the purity higher than 90%.

Preferably, in step b) the concentration of the salt in the mixture is adjusted within the range of from 0.001M to 10M, more preferably in step b) the concentration of the salt in the mixture is adjusted within the range of from 0.001M do 0.05M or above 5M.

Preferably, additionally after the salt is added in step bl) the solution is alkalized to the pH level above 7; b2) the first precipitate is filtered off, and the fil trate is acidified to the pH level below 7; b3) the acidified filtrate is concentrated to obtain the xanthohumol precipitate.

Preferably, in step bl) the solution is alkalized to the pH level in the range of 7.5 do 10.5 or above 12.

Preferably, the xanthohumol-containing extract is obtained by extraction of the post-extraction spent hops, hops, granu- lated hop plant or a mixture thereof with an organic water- miscible solvent, the solvent being used in an amount of from 0.1 to 10 liters per 1 kg of the raw material.

Preferably, as an organic water-miscible solvent, ketones and alcohols or a mixture thereof is used, more preferably, as an organic water-miscible solvent acetone, methanol, ethanol, propanol or a mixture thereof is used.

Preferably, the extraction is conducted at a temperature in the range of from 5 to 650C.

Preferably, the xanthohumol-containing extract is mixed with water at a ratio in the range of from 0.1 to 5 liters of water per 1 liter of the extract, more preferably the xantho humol-containing extract is mixed with water at a ratio in the range of from 1 to 3 liters of water per 1 liter of the ex tract.

Preferably, in step b) a concentration of a transition metal salt is provided within the range of from 0.01M to 0.05M.

Preferably, as a transition metal salt a copper(II) salt or a zinc(II) salt is used.

Preferably, as a transition metal salt a chloride salt, a nitrate salt, or a sulfate salt is used.

Preferably, as a transition metal salt copper (II) chlo ride, copper (II) sulfate or zinc (II) chloride or zinc(II) sul fate is used.

The process according to the invention allows to obtain xanthohumol of the purity of at least 90% (usually above 95%) in a good yield. The process according to the invention do not require use of complex chromatography systems or supported resins.

Purity of the obtained xanthohumol was determined by the HPLC technique on the SUPELCOSIL LC-PAH column of 15 cm x 4,6 mm, 5 jam grain size, column temperature of 200C, Phase A - 95% acetonitrile 0,3% HCOOH; Phase B 2% acetonitrile 0,3% HCOOH, 2 ml/min flow rate, 20 jal injection, detection at the wavelength of 370 and 290 nm.

The invention was illustrated by the following working ex amples .

Example 1 250 g of post-extraction spent hops were weighed out and poured over with acetone (0.75 L) . After mechanical stirring for 1 h the extract formed was filtered through a filter pa per. The obtained filtrate (0.65 L) was flushed with water (1.3 L) . Then the solution was added with an aqueous solution of CuCla (the concentration of 10 g/1) at the volume of 300 ml per 1 liter of the filtrate (the final concentration of 0,022M). After adding copper chloride, the pH of the solution was stabilized at ~4,5. Practically immediately after copper chloride pouring, precipitation in the extract occurred of ag glomerates of a green substance of residual humulons and chlo rophyll separating from a yellow solution where xanthohumol was found. After an hour, NaOH was added to precipitate an ex cess of copper by raising the pH to about 10. After 1 h, pre cipitation was observed of a muddy mass and copper hydroxide precipitate at the bottom of the vessel. The precipitates were filtered off on a filter paper. The obtained filtrate was acidified with HC1 to pH 6 and concentrated on a rotary evapo rator to yield a crystalline xanthohumol precipitate which was filtered off and dried in an oven (temperature of 50-60oC) to give 206 mg of xanthohumol of 97.8% HPLC purity. The yield of the xanthohumol extraction process was 82% based on the total content of xanthohumol in the spent hops.

Example 2 Xanthohumol was obtained as in Example 1, except that af ter adding copper chloride, the precipitated green sludge was filtered off and the filtrate was concentrated on a rotary evaporator to yield the crystalline xanthohumol precipitate of the 90.8% HPLC purity.

Example 3 Xanthohumol was obtained as in Example 1, except that cop per chloride was substituted by a zinc sulfate (ZnSCM) solution at the concentration of 10 g/1. After addition of the zinc salt solution, the pH of the solution was stabilized at ~6, and precipitation was observed. After about an hour NaOH was added with the pH increase to about 10. After 1 h further pre cipitate was observed at the bottom of the vessel. The precip itate was filtered off on a filter paper. The obtained fil trate was acidified with HC1 do pH 6 and concentrated on a ro tary evaporator to yield the crystalline xanthohumol precipi tate, which was filtered off and dried in an oven (the temper ature of 50-60oC) to give xanthohumol of the 97.8% HPLC purity with the 7 9% yield based on the total content of xanthohumol in the spent hops.

Example 4 250 g of post-extraction spent hops were weighed out and poured over with 0,75 liters of pure methanol. After mechani cal stirring for 1 h the formed extract was filtered on a fil ter paper. The obtained filtrate was poured over with water in a volume ratio of 1:2 (one part of the filtrate to two parts of water) . Then the solution was added with the aqueous solu tion of CuCl2 (at the concentration of 10 g/1) in a volume of 300 ml per 1 liter of the filtrate (the final concentration of the salt of 0,022M). After 1 h, precipitation in the extract was observed of agglomerates of a green substance separating from a yellow solution. NaOH was then added to raise the pH to . After 1 h, a muddy green precipitate was observed at the bottom of the vessel. The formed extract was filtered on a filter paper. The obtained extract was acidified with HC1 to pH 6. The remaining amounts of the solvent were removed on an evaporator and a suspension of xanthohumol crystals was ob tained. The suspension was filtered through a filter paper to yield xanthohumol, which was dried in an oven at 550C to give 196 mg of xanthohumol of the 95.8% purity with the yield of about 78% based on the total content of xanthohumol in the spent hops.

Example 5 Xanthohumol was obtained as in Example 4, except that in stead copper (II) chloride, copper (II) sulfate (CuSO-j) was used at the concentration of 10 g/1 and xanthohumol was obtained with the 94.8% purity.

Example 6 Xanthohumol was obtained as in Example 1, except that higher concentrations of copper(II) chloride were used in the range of 0.022M to 0.044M. Xanthohumol was obtained with the purity ranging from 96.3 - 97.8%.

Example 7 Xanthohumol was obtained as in Example 1, except that the ratio of the extract from the first step to the amount of ware added of 1:1 and the CuCla concentration of 0.022M were used, to yield xanthohumol of the 96.8% purity.

Example 8 Xanthohumol was obtained as in Example 1, except that 2- propanol in place of acetone and the CuClz concentration of 0,022M were used to yield xanthohumol with the 93.8% purity.

Comparative Example 1 250 g of post-extraction spent hops were weighed out and poured over with acetone (0.75 L) . After mechanical stirring for 1 h the extract formed was filtered through a filter pa per. The obtained filtrate was concentrated to yield xanthohu mol of the 32.8% purity.

Comparative Example 2 250 g of post-extraction spent hops were weighed out and poured over with acetone (0.75 L) . After mechanical stirring for 1 h the extract formed was filtered through a filter pa per. The obtained filtrate (0.65 1) was poured over with water (1.3 1). Then the solution was added with the aqueous solution of NaCl (at the concentration of 30 g/1) at the volume of 300 ml per 1 liter of the filtrate. Then, NaOH was added to raise the pH to about 12. The precipitate formed was filtered off through a filter paper. The obtained filtrate was acidified with HC1 to pH 5 and concentrated on a rotary evaporator to obtain a xanthohumol precipitate, which was filtered off and dried in an oven (the temperature of 50-60oC) to yield xantho humol of the 84.8% HPLC purity.

Comparative Example 3 Xanthohumol was obtained as in Comparative Example 2, ex cept that NaCl was not used. Xanthohumol was obtained of the 58% HPLC purity - as a result of alkalization to pH 12 only.

Based on the above-described working examples, it could be ascertained that the process according to the invention allows to obtain xanthohumol of a high purity by an exceptionally simple and inexpensive process. It is obvious for a person skilled in the art that xanthohumol obtained by the inventive process could be further purified according to known methods.

Claims

1. Claims: 1. A process for the preparation of xanthohumol, character ized in that: a) a xanthohumol-containing extract is mixed with wa ter; b) a transition metal salt solution is added to the ob tained mixture; c) the obtained xanthohumol precipitate is collected and dried to obtain xanthohumol of the purity higher than 90%. characterized in that in step b) The process of claim 1, the concentration of the salt in the mixture is adjusted with in the range of from 0,001M to 10M. characterized in that in step b) 3. The process of claim 2, the concentration of the salt in the mixture is adjusted with in the range of from 0,001M to 0,05M. characterized in that 4. The process of any of claims 1-3, further after adding the salt in step b: bl) the solution is alkalized to the pH level above 7; b2) the first precipitate is filtered off, and the fil trate is acidified to the pH level below 7; b3) the acidified filtrate is concentrated to obtain the xanthohumol precipitate. The process of claim 4, characterized in that in step bl) the solution is alkalized to the pH level in the range of from 7, 5 to 10,5 . 6 • The process of any of claims 1-5, characterized in that the xanthohumol-containing extract is obtained by extraction of post-extraction spent hops, hops, granulated hop plant or a mixture thereof with an organic water-miscible solvent, said solvent being used in an amount of from 0,1 to 10 liters per 1 kg of the raw material. 7. The process of claim 6, characterized in that as an organic water-miscible solvent, ketones and alcohols or a mixture thereof are used. 0 The process of claim 7, characterized in that as an or ganic water-miscible solvent, acetone, methanol, ethanol, pro- panol or a mixture thereof is used. 9. The process of any of claims 1-8, characterized in that the extraction is conducted at a temperature in the range of from 5 to 650C. 10. The process of any of claims 1-9, characterized in that the xanthohumol-containing extract is mixed with water at a ratio in the range of from 0,1 to 5 liters of water per 1 li ter of the extract. 11. The process of claim 10, characterized in that the xantho humol-containing extract is mixed with water at a ratio in the range of from 1 to 3 liters of water per 1 liter of the ex tract . 12. The process of any of claims 1-11, characterized in that in step b) the concentration of a transition metal salt is provided within the range of from 0,011X1 to 0,05M. 13. The process of any of claims 1-12, characterized in that as a transition metal salt, a copper (II) salt or a zinc(II) salt is used. 14. The process of any of claims 1-12, characterized in that as a transition metal salt, a chloride salt, a nitrate salt, or a sulfate salt is used. 15. The process of any of claims 1-14, characterized in that as a transition metal salt, copper (II) chloride, copper(II) sulfate or zinc(II) chloride or sulfate zinc (II) is used.