WO2005092353A1

WO2005092353A1 - Hop extracts, production and use thereof

Info

Publication number: WO2005092353A1
Application number: PCT/EP2005/002633
Authority: WO
Inventors: Clemens Erdelmeier; Peter C. Schmidt; Roland Walker
Original assignee: Dr. Willmar Schwabe Gmbh & Co. Kg
Priority date: 2004-03-16
Filing date: 2005-03-11
Publication date: 2005-10-06
Also published as: DE102004012830A1

Abstract

The invention relates to a novel method for producing hop extracts having an increased prenylchalcone (such as xanthohumol) and prenylflavone (such as 8 or 6-prenyl-naringenin and isoxanthohumol) content, to novel extracts obtained according to said method, to medicaments or food produced using said extracts, and to the use of the inventive extracts for producing medicaments or food for the prophylaxis and treatment of pathological conditions caused by an oestrogen deficiency or by the disregulation of the sex hormone metabolism, especially the oestrogen metabolism.

Description

Hop extracts, production and use

The present invention relates to a new process for the production of hop extracts with an increased content of prenyl chalcones (such as xanthohumol) and prenyl flavones (such as 8- or 6-prenyl-naringenin and isoxanthohumol), new extracts obtainable by this process, medicaments produced with these extracts, Foodstuffs, in particular dietetic foodstuffs, food supplements and “medical food” and “dietary supplements”, and the use of the extracts for the production of such agents for the prophylaxis and therapy of disease states which are caused by a lack of estrogens or by dysregulation of the sex hormone metabolism, in particular the estrogen metabolism, caused. The aforementioned disease states can be selected from the group consisting of menopausal complaints, benign prostatic hyperplasia, osteoporosis, Alzheimer's disease, cardiovascular diseases and cancer.

The greatest importance of hops still lies in their use in beer production. Because of its bitter and aromatic substances, it is crucial for the taste of the beer. In addition, because of their antimicrobial properties, these substances have a certain importance in the preservation of beer.

The scientific evidence available on hops at the beginning of the eighties led to a positive monograph by Commission E of the then Federal Health Office. This means that the use of hops for falling asleep, restlessness and anxiety is basically permitted.

Hops have long been of medical importance as a mild sedative in folk medicine. The oxidation-sensitive α- and ß-bitter acids are probably responsible for this effect. For these ingredients, radical scavenger properties as well as the Anti-lipid peroxidation properties have been demonstrated (M. Tagashira et al., Biosci. Biotech. Biochem. 59, 740-742 (1995)). EP 0 677 289 A2 also describes pharmaceutical compositions for the treatment of osteoporosis which contain compounds from the group of α-bitter acids and α-iso-bitter acids.

In recent years, in addition to the α- and ß-bitter acids, the phenolic constituents of hops have also been increasingly investigated, and numerous other flavon-type compounds have been found in the hop plant in addition to xanthohumol 1 (JF Stevens et al., Phytochemistry 44, 1575-1585 (1997), JF Stevens et al., J. Chromat. A 832 (1-2), 97-107 (1999)). These are primarily isoprenylated flavonoids such as B. 6- or 8-prenylnaringenin 2 and 3 and isoxanthohumol 4. Stevens et al. (Phytochemistry 53, 759-775 (2000)) also examined the chemotaxonomy of hop species and hop taxa. The chemistry and the biological properties of the flavonoids in hops have been discussed in detail by Stevens et al. reports (J.F. Stevens et al., J.Am.Soc.Brew.Chem.56, 136-145 (1998)).

Hops have been repeatedly attributed to estrogenic properties. This estrogenic activity of the hops has now been confirmed. It was found that the prenylated flavones in particular are responsible for these effects (SR Milligan et al., J. Clin. Endocrinol. Metab. 84, 2249-2252 (1999)). For xanthohumol, the main chalcon of hops, pronounced cancer-preventive properties have been demonstrated (C. Gerhäuser et al. Molecular Cancer Therapeutics 1, 959-969 (2002)).

On an industrial scale, the hops are extracted either with ethanol or with liquid or supercritical carbon dioxide. The latter technique accounts for over 80% of the hop extracts used commercially. The technology of hop extraction with carbon dioxide has been extensively described by Gardner (D.S. Gardner in. Extraction of Natural Products using near-critical solvents, Ed. M.B. King and T.R. Bott, Blackie Academic & Professional, Glasgow, 1993). The supercritical carbon dioxide extracts used in the brewing industry are usually produced by extraction at pressures of 200-350 bar and 40-60 ° C.

WO 83/00701 A1 claims a process for obtaining estrogen-active substances from hops, characterized in that a carbon dioxide extract is first prepared from hops with the addition of water as an entrainer, and then the estrogen-active substances are obtained therefrom by means of ether extraction or chromatographic processes , Furthermore, the use of these substances as an additive to animal feed, for cosmetic products or as a bath additive is claimed. No information is given about the nature of these estrogenic substances.

US4507329 describes a process for the production of a supercritical hop extract, in which a selective extraction of the α-acids from hops is to be achieved by adding an organic component to the carbon dioxide.

In WO03014287 prenylchalkon- and prenylflavonhaltigen hop extracts are described, which can be obtained by a three-stage extraction process. The raw hops are first used with an apolar

Pre-extracted solvent, then extracted a second time with water and finally the desired extract is obtained by extraction with aqueous ethanol.

The present invention has for its object to provide a new simple and efficient process for the production of hop extracts with an increased content of prenyl chalcones and flavones.

The present invention is also based on the object of providing plant extracts with which pharmaceutical preparations or foods, in particular dietetic foods or

Dietary supplements as well as "medical food" and "dietary supplements" can be produced which are suitable for the prophylaxis and therapy of disease states which are caused by a lack of estrogens or by dysregulation of the sex hormone metabolism, in particular the estrogen metabolism. The aforementioned disease states can be selected from the group consisting of menopausal complaints, benign prostatic hyperplasia, osteoporosis, Alzheimer's disease, cardiovascular diseases and cancer.

Common to all the extraction processes described so far using compressed carbon dioxide is that only relatively non-polar hop ingredients such as e.g. the α- and ß-bitter acids are extracted. The more polar ingredients such as flavones, chalcones and other phenolic ingredients remain in the plant residue.

Surprisingly, it was found that hop extracts with a significantly higher prenyl chalcone and flavone content than previous extracts are obtained by a two-stage extraction with compressed C0 ₂ if the hops after a first extraction with C0 ₂ in a second, subsequent extraction with C0 ₂ with addition a cosolvent is extracted. In the present invention, lower alcohols, in particular with a chain length of C1-C4, are used as cosolvents. Particularly preferred examples are methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, 2-butanol and tertiary butyl alcohol, with methanol, ethanol, isopropanol and n-butanol being very particularly preferred. The ratio of employed in the 1st stage of extraction with CO ₂ hops is added to the 2nd stage of extraction with C0 ₂ cosolvent in the range of 1: 1 to 1:15, preferably 1: 6 to 1: 10 degrees. The extraction temperature is in the range of 40-80 ° C, preferably about 60 ° C is extracted. The extraction pressure can be set in the range from 40 to about 500 bar, preferably <500 bar, preferably it is 100-300 bar. The extraction time can be selected between 30 minutes and 5 hours, but is preferably about 3 hours. The CO ₂ throughput is in a range of 4-15 kg / h, preferably around 8 kg / h.

All hop varieties containing prenyl flavones and chalcones can be used to produce extracts according to the invention, but preferably the Hallertauer Magnum variety.

The invention is illustrated by the following examples. In the comparative example, raw hops were extracted in the usual way according to the prior art with compressed carbon dioxide. The resinous extract obtained in this way is rich in hop bitter acids (α- and ß-acids), but phenolic ingredients are not detectable. In Examples 1 and 2 of the raw hops were first pre-extracted as in the comparative example in a conventional manner with compressed C0. ₂ The drug in the extraction container was then extracted in a second step with compressed CO ₂ with the addition of a cosolvent in order to obtain the extract according to the invention. In Examples 3 to 10, a hop was used which had already been pre-extracted in analogy to the comparative example. Such a hop, ie a hop which has already been subjected to extraction with CO _{2 in} accordance with the 1st stage of the process according to the invention, is commonly referred to as hop hop.

Comparative example

240 g of chopped raw hops of the "Hallertauer Magnum" variety were extracted with 8 kg / h of CO ₂ for 3 hours at 300 bar and 60 ° C. 65.1 g (27.1%) lipophilic extract obtained. This extract was composed as follows:

Xanthohumol n.n.

8-prenyl-naringenin n.n.

6-prenyl-naringenin n.n.

Isoxanthohumol n.n. α-acids 35.4% ß-acids 18.7%

(n.n. = undetectable)

example 1

240 g of chopped raw hops of the "Hallertauer Magnum" variety were extracted with 8 kg / h of CO ₂ for 3 hours at 300 bar and 60 ° C. 65.1 g (27.1%) of lipophilic extract were obtained. The extract container remaining hop drug was then extracted a second time for 3 hours at 300 bar and 60 ° C. with 8 kg / h of CO ₂ , 2 liters of methanol being added as entrainer during the first hour, and after vacuum drying of this extract at 40 ° C. for 14 hours 9.8 g (4.1%) of extract obtained, which was composed as follows:

Xanthohumol 7.12%

8-prenyl-naringenin 0.12%

6-prenyl-naringenin 0.39%

Isoxanthohumol 0.14% α-acids 7.54% β-acids 1.42%

Example 2

600 g of raw hop pellets, produced from the "Hallertauer Magnum" type, were extracted with 8 kg / h of CO ₂ for 6 hours at 300 bar and 60 ° C. obtained 159.3 g (26.6%) of lipophilic extract. The hop pellets remaining in the extraction container were then extracted a second time at 300 bar and 60 ° C. with 8 to 10 kg / h CO ₂ for 6 hours, 4 liters of methanol being fed in as entrainer during the first 2 hours. After vacuum drying this extract at 40 ° C. for 24 hours, 23.0 g (3.3%) extract were obtained. This extract was composed as follows:

Xanthohumol 8.30%

8-prenyl-naringenin 0.15% 6-prenyl-naringenin 0.61%

Isoxanthohumol 0.22% α-acids 5.06% β-acids n.a.

Example 3

311 g of hop shoots of the "Hallertauer Magnum" variety were extracted with 8 kg / h of CO ₂ for 3 hours at 300 bar and 60 ° C., 2 liters of methanol being added as entrainer during the first hour. After vacuum drying of this extract at 40 ° C. 15.2 g (4.9%) of extract were obtained over 14 hours, which was composed as follows:

Xanthohumol 9.23%

8-prenyl-naringenin 0.17%

6-prenyl-naringenin 0.69%

Isoxanthohumol 0.23% α-acids 2.29% β-acids n.a.

Example 4

310 g of hop shoots of the "Hallertauer Magnum" variety were extracted at 300 bar and 60 ° C with 8 kg / h CO _{2 for} 3 hours, with 2 liters during the first hour Ethanol was fed as an entrainer. After vacuum drying this

Extract at 40 ° C for 14 hours gave 12.9 g (4.16%) extract. This extract was composed as follows:

Xanthohumol 12.15%

8-prenyl-naringenin 0.20%

6-prenyl-naringenin 0.75%

Isoxanthohumol 0.27% α-acids 2.00% β-acids n.a.

Example 5

312 g of hop grains of the "Hallertauer Magnum" variety were extracted with 8 kg / h of CO ₂ for 3 hours at 100 bar and 60 ° C., 2 liters of methanol being added as entrainer during the first hour. After vacuum drying of this extract at 40 ° C. 23.8 g (7.2%) of extract were obtained over 24 hours and was composed as follows:

Xanthohumol 6.82%

8-prenyl-naringenin 0.15%

6-prenyl-naringenin 0.51%

Isoxanthohumol 0.18% α-acids 0.69% β-acids n.a.

Example 6

312 g of hop grains of the "Hallertauer Magnum" variety were extracted with 8 kg / h of CO ₂ for 3 hours at 100 bar and 60 ° C., 2 liters of ethanol being fed in as entrainer during the first hour. After vacuum drying of this extract at 40 ° C. 12.5 g (4.0%) of extract were obtained over a period of 16 hours, which was composed as follows: Xanthohumol 10.16%

8-prenyl-naringenin 0.18%

6-prenyl-naringenin 0.67%

Isoxanthohumol 0.25% α-acids 1, 48% β-acids n.a.

Example 7

311 g of hop spent grains of the "Hallertauer Magnum" variety were extracted with 8 kg / h of CO ₂ for 3 hours at 300 bar and 60 ° C., 2 liters of isopropanol being added as entrainer during the first hour. After vacuum drying of this extract at 40 ° C. 7.6 g (2.4%) of extract were obtained over 24 hours and was composed as follows:

Xanthohumol 15.45%

8-prenyl-naringenin 0.28%

6-prenyl-naringenin 1.00%

Isoxanthohumol 0.48% α-acids 0.78% β-acids n.a.

Example 8

312 g of hop shoots of the "Hallertauer Magnum" variety were extracted with 8 kg / h of CO ₂ for 3 hours at 100 bar and 60 ° C., 2 liters of isopropanol being added as entrainer during the first hour. After vacuum drying of this extract at 40 ° C. 6.9 g (2.2%) of extract were obtained over a period of 24 hours, which was composed as follows:

Xanthohumol 16.97%

8-prenyl-naringenin 0.27% 6-prenyl-naringenin 1, 08%

Isoxanthohumol 0.47% α-acids 1, 5% β-acids n.

Example 9

312 g of hop grains of the "Hallertauer Magnum" variety were extracted with 8 kg / h of CO ₂ for 3 hours at 300 bar and 60 ° C., 2 liters of n-butanol being fed in as entrainer during the first hour. After vacuum drying this extract at 40 9.2 g (3.0%) of extract was obtained over a period of 24 hours and was composed as follows:

Xanthohumol 12.38%

8-prenyl-naringenin 0.22%

6-prenyl-naringenin 0.80%

Isoxanthohumol 0.38% α-acids 1.66% β-acids n.a.

Example 10

313 g of hop shoots of the "Hallertauer Magnum" variety were extracted with 8 kg / h of CO ₂ for 3 hours at 100 bar and 60 ° C., 2 liters of n-butanol being fed in as entrainer during the first hour. After vacuum drying this extract at 40 9.2 g (2.9%) of extract was obtained over a period of 24 hours and was composed as follows:

Xanthohumol 9.24% 8-prenyl-naringenin 0.16%

6-prenylnaringenin 0.59%

Isoxanthohumol 0.24% α-acids 1.36% ß-acids nn

Example 11:

The supercritical hop extracts produced were tested for estrogenic activity using a cellular estrogen receptor binding assay using the human endometrial carcinoma cell lines ECC-1 (Bergeron et al., Mol. And Cell. Endocrinol. 150, 179-187, 1999). The cells were incubated (37 ° C, 5% CO 2 in air) in DME medium (ICN, 1033126) with the addition of fetal calf serum (5%), glutamine (2 mM), antibiotic / antifungal solution (1%, Sigma, A-9909) and insulin (0.25 U / ml). Before the experiment was carried out, the adherently growing cells were detached from the bottom of the tissue culture bottles by trypsinization and adjusted to a density of 5 × 10 5 cells / ml in complete culture medium. 500 μl of cell suspension were introduced into each well of a 24-well tissue culture plate (TPP, No. 9224). After 24 h, the plates were centrifuged (350 g, 5 min, RT), the medium was discarded and the cells twice with test medium (DMEM-F12 [Sigma, D-2906], with the addition of charcoal-stripped bovine calf serum [5%], Washed glutamine [2 mM] and antibiotic / antifungal solution, and then cells were cultured in 500 µl fresh test medium for another 24 hours.

After removing 100 μl of medium, the extracts were added to the cells in a 10-fold final concentration in a volume of 50 μl. An hour later, 2,4,6,7-3H-estradiol (50μl in 0.01% ethanol, Amersham, TRK 322) was also pipetted in 10-fold final concentration (3 nM) to all substance mixtures. After an incubation for 35 min at 37 The supernatants were discarded and 500 μl of phosphate buffer (5 mM, pH 7.4 with the addition of 0.5% bovine serum albumin, 0.25 mM sucrose and 10% glycerol) were added. After 30 min at room temperature, the cells were washed twice with ice-cold phosphate buffer without BSA and then 500 μl of ice-cold 96% ethanol were added. The cells suspended in ethanol were transferred to 4 ml of Quicksafe A and the bound radioactivity in a β-counter (Packard , Tri-Carb 2300 TR). All approaches were made in duplicates. For the determination of the nonspecific binding, cells were incubated in the presence of 100 nM unlabeled estradiol. Samples without marked estradiol were used as blank values. The percentage inhibition of receptor binding was calculated in each case in comparison to a solvent control (DMSO, 0.1%) tested in parallel. The half-maximum inhibitory concentration (IC50) was determined by non-linear regression calculation.

Results: Table 1:

The extracts are many times less effective in terms of the value of pure 17ß-estradiol. However, at normal dosages, the concentrations of phytoestrogens in the organism are many times higher than those of the body's own steroids, so that the effect of the extracts in the organism corresponds approximately to that of 17ß-estradiol.

Claims

claims

1. A process for producing a dry hop extract with an increased content of prenyl chalcones and flavones, characterized by a two-stage extraction with compressed carbon dioxide, the hops first being extracted in the usual way with C0 ₂ and then the extract being separated off, in which case then in the second stage with C0 ₂ and with the addition of an organic cosolvent, a further extraction is carried out and the extract obtained is freed from the extracting agent in order to obtain the hop dry extract.

2. The method according to claim 1, wherein a lower C1-C4 alcohol is used as cosolvent.

3. The method according to claim 2, wherein the lower C1-C4 alcohol is selected from the group comprising methanol, ethanol, isopropanol and n-butanol.

4. The method according to any one of claims 1 to 3, wherein the ratio of hops used in the first stage of extraction with CO ₂ to cosolvent added in the second stage of extraction with CO _{2 is} in the range from 1: 1 to 1:15.

5. The method according to any one of claims 1 to 4, wherein the extraction temperature in both the first and in the second stage of extraction with C0 _{2 is} independently in a range from 40 to 80 ° C.

6. The method of claim 5, wherein the extraction temperature is about 60 ° C.

7. The method according to any one of claims 1 to 6, wherein the extraction pressure in both the first and in the second stage of the extraction with C0 _{2 is} independently in a range from 40 to about 500 bar.

8. The method according to claim 7, wherein the extraction pressure is in the range of 100 to 300 bar.

9. The method according to any one of claims 1 to 8, wherein the extraction time is between 30 minutes and 5 hours.

10. The method of claim 9, wherein the extraction time is about 3 hours.

11. Process according to one of claims 1 to 10, wherein the CO ₂ is located throughput information both in the first and in the second stage of extraction with C0 _{2 are} independently in a range of 4 to 15 kg / h.

12. The method according to claim 11, wherein the C0 ₂ throughput is about 8 kg / h.

13. The method according to any one of claims 1 to 12, wherein the hops are the "Hallertau Magnum" variety.

14. Extract obtainable by a process according to one of claims 1 to 13.

15. Use of the extract according to claim 14 for the production of medicaments or of food for the prophylaxis and therapy of disease states which are caused by a lack of estrogens or by dysregulation of the sex hormone metabolism, in particular the estrogen metabolism, selected from the group consisting of climacteric complaints, benign Prostatic hyperplasia, osteoporosis, Alzheimer's disease and cardiovascular diseases.

16. Use of an extract according to claim 14, for the manufacture of medicaments or of food for the prophylaxis and therapy of sex-hormone-dependent cancers.

17. Medicament or food containing an extract according to claim 14 and other customary pharmaceutically acceptable auxiliaries.