Enzymatic demethylation of flavonoids

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. The method of producing 8-prenylnaringenin in vitro involves: a) providing a first composition containing a culture of Eubacterium limosum or Peptostreptococcus productus bacterial cells, having 5-alkoxyflavonoid-dealkylating activity, and b) bringing a second composition containing 5-alkoxyflavonoids into contact with said first composition to facilitate dealkylation of said 5-alkoxyflavonoids with said bacterial cell culture. Disclosed is a combination for obtaining 8-prenylnaringenin in vitro and use of the culture of Eubacterium limosum or Peptostreptococcus productus bacterial cells, having 5-alkoxyflavonoid-dealkylating activity to obtain 8-prenylnaringenin in vitro. The method of enhancing 5-alkoxyflavonoid-dealkylating activity of the culture of Eubacterium limosum or Peptostreptococcus productus bacterial cells involves a step for inoculating said bacterial cell culture on a medium containing 5-alkoxyflavonoids, and a step for selecting a colony with the highest 5-alkoxyflavonoid-dealkylating activity. The Eubacterium limosum LMG P-23546 strain has 5-alkoxyflavonoid-dealkylating activity and is capable for converting isoxanthohumol to 8-prenylnaringenin.

EFFECT: invention increases efficiency of producing a phytoextractant such as 8-prenylnaringenin.

14 cl, 9 dwg, 4 tbl, 6 ex

 

The technical field

The present invention relates to phytoestrogens and their receipt, and also to pharmaceutical compositions and dietary supplements that include phytoestrogens.

The level of technology

Hops (Humulus Lupulus L.) has been used for centuries as the main raw materials for brewing, which gave the beer a bitter taste and a special smell. In the last few years this plant has attracted increased attention as a source of prenylflavonoids, a subclass of flavonoids, containing non-polar prenolol a side chain that is associated with one of the phenol rings. They are present in lupulina glands found at the base bracteoles hop cones of the female plant. Of these prenylflavonoids the most attention now draw two chalcone (xanthohumol (X) and desmethyltramadol (DMX)) and three flavanone (azaxanthone (IX), 8 prenylnaringenin (8-PN) and 6-prenylnaringenin (6-PN)) (Figure 2) due to their potential health effects. X identified as a strong anti-cancer agent, while for 8-PN has been shown that it is one of the most potent phytoestrogens, identified to date, with a much higher activity than the well-known soy phytoestrogens. It was demonstrated that 8-PN in vivo exhibits estrogenic activity, etc is depreda bone loss in rats the inhibition of angiogenesis and metastasis, and it has been shown that 8-PN shows antiandrogenna activity.

X is present as the dominant pranidhana in female hop cones in concentrations up to 1% (wt./weight), while DMX is present in lower concentrations (De Keukeleire et al. (2003) J. of Agric. and Food Chem. 51, 4436-4441). The ratio X/DMX different varieties of hops. By isomerization X is transformed into IX, and DMX turns into 8-PN 6-PN.

Estrogenic effects of hops were discovered in the last decade. Hop baths used for the treatment of gynecological diseases, and women who spent hops were often observed menstrual disorders. In 1999, Milligan et al. [J. Clin. Endocrinol. Metab. 84, 2249-2252] identified a new phytoestrogen hop, 8-prenylnaringenin. Although it is much weaker than 17β-estradiol (<1%), it is one of the most potent phytoestrogens, identified to date, with much more activity than other phytoestrogens derived from soy, such as compounds genistein and daidzein.

It was discussed whether the effects of estrogens from food and/or environment is a risk factor for health, such as endocrine disruption. In the case of hop prenylflavonoids beer is their main food source. In the United the States in 2001, the average beer consumption per person has grown by calculations about 225 ml of beer per day (USDA, 2003). When suggested that if this amount was consumed in the form most common in USA brand light pilzenskoe beer (500-1000 mcg prenylflavonoids/l beer), daily consumption of prenylflavonoids would be about 0.14 mg However, found in beer concentrations (and, thus, the average consumption) strongly depend on the brewing process, so strong Beers contain up to 4 mg prenylflavonoids/l While X represents the dominant prenylflavonoids present in hops (0,1-1% of dry weight), most of it is transformed into IX by thermal isomerization in the brewing process. Thus, IX is the most common prenylflavonoids found in beer, and is present in concentrations of 500 ág/l (light pilzenskoe beer) up to 4 mg/l (strong beer). Similarly, DMX turns into 8-PN, reaching final concentrations in beer to 100 μg of 8-PN/L. But, despite the high activity of 8-PN, the total estrogenic activity in the beer remains in 500-1000 times less than the concentration of dangerous from the point of view of the in vivo activity (~100 mg/l) (Milligan et al. (2002) Reproduction 123, 235-242). In addition, many Beers prepare now with the use of extracts of hops instead of whole hops that provides reduced concentrations of 8-PN or its complete absence. So, basically this is consistent with the that, based on current knowledge, there are no harmful health effects may not be caused by phytoestrogens with an average consumption of beer.

On the other hand, now many data is correlated intentional consumption phytoestrogen with possible health benefits (Magee & Rowland (2004) Br. J. Nutr. 91, 513-531). Besides beer, sold dietary supplements based on hops with a statement effects such as breast enlargement or reduction of the tides. All of the effects of phytoestrogens on health are potentially the result of the combination of multiple individual with a variety of phytochemicals and possibly additive or opposing activities. Until now only the isoflavones and lignans considered important phytoestrogens in the human diet, especially because of the concentration in beer 8-PN was considered too low for positive or negative effects on health.

Several patent publications describe the beneficial health effects of dietary flavonoids, for example, the use IX to prevent a decrease in bone density (WO04089359), the use of extracts of hops for medicines that have estrogenic properties (WO02085393), and the use of IX or X in food products with the application of anti-inflammatory or anti-aging properties (patent WO03090555). In addition to the, it was also suggested the use of 8-PN in cosmetic products for skin care (SA).

For in vivo to make actions identified in vitro, food flavonoids must be absorbed from the intestine and reach their targets unchanged. Mainly Monomeric flavonoids are without modifications through the stomach into the small intestine where it can cause absorption from the intestine to the mesenteric circulatory system. In vitro studies revealed when the suction significant biotransformation X in the liver (Yilmazer et al. (2001a) FEBS Lett. 491, 252-256) and 8-PN (Nicolic et al. (2004) Drug Metabolism and Disposition 32, 272-279). However, the degree of dietary intake of polyphenols in the small intestine rather limited (10-20%), thus, it is assumed that a large portion of flavonoids reaches the colon. Naringenin, not predominandy analogue of 8-PN, showed intense microbial biotransformation in the intestine, including the splitting of the ring and degidroksilirovanie (Rechner et al. (2004) Free It. Biol. Med. 36, 212-225) with subsequent absorption and excretion. Little is known about intestinal transformation of prenylflavonoids. Nookandeh et al. (2004) Phytochemistry 65, 561-570, introduced X to rats at 1000 mg/kg body weight and allocated 22 metabolite of faeces. However, most (89%) received flavonoids was not modified Agricultural Remaining fraction consisted of small amounts of various metal is of Bolotov, including a number IX. Avula et al. (2004) [J. Chromatogr. Sci. 42:378-382] carried out a similar experiment with rats and found mainly not modified X, and also a number of unidentified metabolites.

The possibility that IX can act as proestrogen suggested Coldham et al. (2002) Food Addit. Contam. 19:1138-1147. The assumption was based on the ability of the liver to the various biotransformation, which involves demethylation. However, the impact on IX liver microsomes did not lead to increased estrogenic activity, which concluded that 8-PN was not made. On the contrary, Nicolic et al. describe what liver microsomes can demetilirovanie IX, but not X (Nicolic et al. (2005) J. of Mass Spectrom. 40, 289-299). However, it was shown that in addition to the demethylation microsome assay also modify the side prenolol chain with obtaining, in the end, the great variety of minor degradation products. Schaefer et al. (2003) (J. Steroid Biochem. Mol. Biol. 84, 359-360) identified low levels of 8-PN in the urine of two test subjects after oral consumption IX, and linked this fact with demethylation in the liver.

In addition to the liver, the large intestine is also an important place of transformation of substances in the body. The large intestine contains ~1012microorganisms/cm3(about 400 different species) with a huge catalytic and hydrolytic is Kim potential. The importance of this microbial community in the metabolism of phytoestrogens tend to be well-studied. Wang et al. (2000) Chem. Pharm. Bull. 48, 1606-1610, identified two types of bacteria responsible for the transformation of Lignano, and Decroos et al. (2005) Arch. Environ. 183, 45-55, has recently provided a microbial consortium capable of transformation soy phytoestrogen of daidzein in equal. In addition, it was shown that the bioavailability of phytoestrogens enhance some intestinal bacteria because they contain β-glucosidase, which are necessary for the hydrolysis of glycosides of phytoestrogens (Rowland et al. (2003) Br. J. Nutr 89, s45-S58). Thus, it is assumed that the intestinal microbiota is an important factor for the bioavailability of phytoestrogens (Turner et al. (2003) Nutr. Rev. 61, 204-213).

As the economic interest are only present in female hop cones essential oil and alpha-acid how important ingredients of brewing, various techniques have been developed for the extraction of hops with the specific purpose to extract only these compounds. On the one hand, CO2at the moment, is the most common solvent in the production of extracts of hops (Palmer & Ting (1995) Food Chem. 52, 345-352). Compared with the ways in which the use of traditional organic solvents (ethanol, hexane, methanol or methylene chloride), CO2extraction PR is more selective extracts, which in the production of beer can be used as a good alternative to whole hops or hop pellets. CO2extracts form the basis for a large number of additionally allocated and refined products, such as ISO-alpha-acids and restored derivatives. Another way for additional purification of CO2-extracts by removing unwanted prenylflavonoids described in the patent US3794744.

On the other hand, various methods have been developed for specific receipt and cleaning of prenylflavonoids (mainly X). Examples of these methods of extraction are described in the patent US 4121040 and in the German patent DE19939350. Using these processes, it is easy to get xanthohumol, therefore, manifested little interest in the development of the method of chemical synthesis X. However, 8-PN is much more difficult to obtain from natural extracts because of its low concentrations present in the hop shot. Thus, strategies have been developed for the synthesis, to obtain 8-PN by prenisolone commercially available naringenin. First 8-PN received from naringenin or fluoroacetophenone by unproductive non-selective With direct-prenisolone. Effective small-scale synthesis was performed using a rearrangement of Clausena catalyzed by europium(III) (Gester et al. (2001) Tetrahedron 57, 1015-1018)Nedavno in the European patent EP1524269 was described products in industrial scale, based on this method.

Despite a comprehensive industrial use of hops and extracts of hops, there is no efficient way of getting from a natural source of bioactive Premierbank phytoestrogens, such as 8-PN.

The invention

The aim of the present invention is to propose an efficient method for the production of bioactive Premierbank phytoestrogens, such as 8-PN, from 5-alkoxylated, which can be obtained from natural source, as well as pharmaceutical compositions and dietary supplements that use such bioactive prenisolone phytoestrogens.

In the first aspect of the present invention provides a composition having 5-alkoxyphenyl-transfersno (5-AO-FT) and/or 6'-alkoxyalkane-transfersno (6'-AO-CT) activity. More specifically, the invention provides a composition having 5-methoxyflavone-transfersno (5-MO-FMT) and/or 6'-Methoxychlor-transfersno (6'-MO-CMT) activity. The following embodiment of the present invention relates to compositions capable of deaccelerate prenisolone 5-alkoxylated and/or prenisolone 6'-alkoxyalkane. In a particular embodiment of the present invention provides a composition capable of deaccelerate 6'-alkoxyalkane xanthohumol (X) and/or 5-alkoxylated isaxanli umol (IX). Thus, the compositions of the present invention is capable of production of bioactive phytoestrogens, more specifically Premierbank phytoestrogens, more specifically 8-PN.

According to a specific embodiment of the composition, with 5-alkoxysilane - and/or 6'-alkoxyalkane dealkylase activity is a composition comprising or derived from material of animal origin, more specifically prokaryotic origin. More specifically, the compositions of the present invention include bacterial cells or extracts of supernatant or other purified or partially purified material such bacterial cells. A specific embodiment of the present invention relates to compositions comprising homoacetogenic bacteria such as Eubacterium sp. or Peptostreptococcus sp., more specifically, Eubacterium limosum, or Peptostreptococcus productus or their extracts, supernatant or other purified or partially purified material based on them.

Another specific embodiment of the invention includes bacterial strains and/or compositions, including cells, their extracts, supernatant or other purified or partially purified material based on them, which was enriched products 5-alkoxyphenyl-transferases (5-AO-FT) and/or 6'-alkoxyalkane-transferases (6'-AO-CT) activity, more specifically using popcorn the x incubations with 5-alkoxylated, such as 5-methoxyflavone.

Another specific embodiment of the invention includes compositions comprising 5-methoxy(prenyl)the flavonoid methyltransferase and/or 6'-methoxy(prenyl)Halcon the methyltransferase from homoacetogenic bacterial strain, more specifically from Eubacterium sp., and even more specifically from Eubacterium limosum.

A specific embodiment of the compositions according to the present invention relates to compositions comprising enriched activity 5-alkoxylated transferase (5-AO-FT) and/or 6'-alkoxyalkane transferase (6'-AO-CT), obtained from a bacterial strain of Eubacterium limosum, deposited at the Belgian Coordinated collections of Microorganisms (BCCM) in the BCCM/LMG collection under Deposit number LMG P-23546.

In the following aspect the present invention provides methods of production of phytoestrogens, including dealkylation 5-alkoxylated position 5 or dealkylation 6'-alkoxyalkane at the corresponding position 6', wherein they carry out in vitro using eukaryotic or prokaryotic material of animal origin. In a specific embodiment of the methods used to obtain 8-PN. According to a specific embodiment of dealkylation in the methods according to the invention is a demethylation and is carried out using eukaryotic or prokaryotic the ical material of animal origin. More specifically, not the animal material is a bacterial strain, more specifically homoacetogenic bacteria or purified, or partially purified fractions or components based on it, such as partially treated or highlighted enzymes. A specific embodiment relates to dialkylamino using material from Eubacterium sp. or Peptostreptococcus sp., such as Eubacterium limosum. The following specific embodiment of the method according to the invention include methods dealkylation Premierbank 5-alkoxylated and/or Premierbank 6'-alkoxyalkane.

According to the following specific embodiment suggests ways dealkylation 5-alkoxylated and/or 6'-alkoxyalkane vegetable origin, more specifically with the origin of the hops. According to specific embodiments of the ways dealkylation 6'-alkoxyalkane xanthohumol and/or 5-alkoxylated of azaxanthone.

Another aspect of the invention is the application of bacterial cell lines for in vitro dealkylation 5-alkoxylated and/or 6'-alkoxyalkane, more specifically for demethylation of 5-methoxyflavone and/or 6'-metaxalone. More specifically, the bacterial cells are cells of homoacetogenic bacterial strain, such as Eubacterium limosum. The following specific embodiment is predstavljaet the application of bacterial cells, in which increased production of 5-alkoxylated transferases (5-AO-FT) and/or 6'-alkoxyalkane-transferases (6'-AO-CT) activity, for example, using repeated incubations with 5-alkoxysilanes, such as 5-methoxyflavone.

In another aspect of the invention provides methods of production of phytoestrogens in vitro, which include stage a) providing a bacterial strain of bacteria, more specifically homoacetogenic bacteria or extract based on it and b) introducing into contact with the composition comprising 5-alkoxylated, more specifically 5-methoxyflavone and/or 6'-alkoxyalkane, more specifically 6'-metaxalone bacterial strain or extract thereof so as to enable dealkylation 5-alkoxylated and/or 6'-alkoxyalkane using bacterial strain or extract. Optionally, the methods further include the identification and/or purification of the obtained dialkylamino flavonoid.

Specific embodiments of these methods are methods that include the preparation of an extract of the bacterial strain, which additionally includes a step of enrichment and optional purification of bacterial extract to contain enriched or purified 5-alkoxyphenyl-transfersno (5-AO-FT) activity and/or enriched or the eyes of the military 6'-alkoxyalkane-transfersno (6'-AO-CT) activity.

Additional or alternative methods of the present invention include stage enrichment products bacterial strain 5-AO-FT and/or 6'-AO-CT activity with repeated incubations with 5-alkoxysilanes, such as 5-methoxyflavone.

In another aspect of the invention features a 5-methoxyphenylalanine methyltransferase or 6'-methoxypsoralen methyltransferase from Eubacterium limosum.

In another aspect of the invention offers the pharmaceutical compositions and dietary supplements, including bioactive phytoestrogens obtained using the methods of the present invention.

In another aspect of the invention offers the pharmaceutical compositions and dietary supplements that includes two components for simultaneous or sequential introduction, where the first component includes homoacetogenic bacterium, or an extract or component based on it, with 5-alkoxyphenyl-transfersno (5-AO-FT) and/or 6'-alkoxyalkane-transfersno (6'-AO-CT) activity, and the second component comprising 5-alkoxyphenyl or 6'-alkoxyalkane or their source, such as an extract of hops. According to specific embodiments, the flavonoid is a 6'-alkoxyalkane xanthohumol or 5-alkoxylated azaxanthone. The following specific embodiments relate to pharmaceutical compositions and dietary add the am according to the invention, where homoacetogenic the bacterium is a Eubacterium limosum. Optional bacteria in the pharmaceutical compositions according to the invention is represented in the form of a composition for specific delivery in the rectum.

In the present invention are described that IX can be demetrious to 8-PN using living organisms of animal origin, such as bacteria of the intestines of man or animals, especially vertebrates or mammals, and that IX can, thus, act as proestrogen. The present invention additionally identified microorganisms capable of transforming IX into 8-PN, for example, in vitro production of 8-PN using cultures of such microorganisms. Additionally, the present invention proposes a method of selection of other strains that are able to quantitatively obtain 8-PN from IX.

The present invention additionally demonstrated that the process of transformation of methylated precursors flavonoid phytoestrogens in vivo using microbial flora is very variable and depends on the composition of the microbial flora of the individual (between individuals or the same individual at different times). Probably this has important consequences for the effects of phytoestrogens on individuals. Indeed, extracts of hops in beer and food or supplements less variety of the IX gene is present in higher concentrations, than 8-PN.

Using the methods of production of activated phytoestrogens (in vitro or in vivo) in the present invention further provides an interesting alternative or addition to modern food extracts of hops. Unpredictable output when converting methylated precursors flavonoid phytoestrogens (e.g., IX) in their active demetilirovanie connection can be monitored using in vitro prior conversion or in vivo/in situ dealkylation. This allows you to monitor the effect of the active component on each individual, despite individual differences in intestinal microflora, or specifically to take these differences into account.

DETAILED description of the INVENTION

Understood that the drawings illustrate the present invention but should not be considered that you have in mind any limitations of the invention presented here incarnations.

Figure 1: Basic structure of flavonoids.

Figure 2: Structure of prenylflavonoids hops and their transformation.

Figure 3: Response to estrogen (mean + STD.) faecal culture (C), incubated with IX (0 and 8 days of incubation) (n=3).

Figure 4: Transformation IX (25 mg/l) human fecal cultures (E-L) 8-PN in 3 days [mean + STD. (n=3)].

Figure 5: Transformation of azaxanthone (IX) 8-ol is solarenemy (8-PN) with intestinal bacteria from 51 human individual. Individuals were assigned in ascending order products 8-PN, and the results are presented as a value showing the percent conversion of IX into 8-PN (n=3) (+/- STD. off).

6: Conversion of IX (25 mg/l) 8-PN with P. productus (three cultures: Inc 1 Inc 2 Inc 3). The disappearance IX (filled symbols) and the production of 8-PN

(empty symbols) were detected for 13 days.

7: Conversion of IX into 8-PN after adding culture of E. Limosum to fecal culture (E. Limosum as a percentage from 0% (fecal sample) to 100% (not infected culture of E. Limosum)) (n=3).

Fig: Conversion of IX into 8-PN in the microbial ecosystem modeling human intestine, under conditions enabling the activation of methylated methylflavone. PF+ TWIN branch SHIME.

Figure 9: Conversion of IX into 8-PN in the microbial ecosystem modeling human intestine, under conditions which prevent the activation of methylated methylflavone.

Definition

For this application we used the following abbreviations:

X: xanthohumol;

DMX: desmethyltramadol;

IX: azaxanthone;

8-PN: 8 prenylnaringenin;

6-PN: 6-prenylnaringenin;

5-AO-FT: 5-alkoxylated alkyltransferase;

5-MO-FMT: 5-methoxyflavone methyltransferase;

6'-AO-CT: 6'-alkoxyalkane alkyltransferase;

6'-MO-CMT: 6'-Methoxychlor methyltransferase.

The term "flavonoids which refers to a group of organic molecules, based on the skeleton of 15 carbon atoms containing chromanone ring and having a second aromatic ring b In position 2, 3 or 4 (Figa). Figa demonstrates the traditional numbering for deputies, which is also used in the present invention. Subgroups of flavonoids are balcony, flavanones, flavones, flavanols, and isoflavones. Balcony (Pigv) are isomers of flavanones (Figs). Figv and 2 demonstrate the traditional numbering balkanov. Flavanones are different from flavones (Fige) the fact that they lack double bonds at positions 2, 3. Flavones (Fige) are flavonoids, deprived 3-one group of flavanols (Fige). Isoflavones are flavonoids, where the phenyl ring In localized at position 3 (Fig.1F). All these subgroups have the ketone function in position 4.

The term "prenylflavonoids"used in the present invention, refers to the flavonoids containing nonpolar prenolol side chain attached to one of the phenolic rings. Prisilna chain often is in position 8, but may also be in the position 6, or in both positions 6 and 8 [hakanah prisilna chain localized in position 3' and/or 5']. In the hops prenylflavonoids mainly located in lupulina glands located at the base bracteoles in hop cones of the female plant. Other natural East is czniki of prenylflavonoids represent for example, Dendrolobium lanceolatum, Sophora flavescens, Sophora tomentosa, Artocarpus communus and Marshallia grandiflora. Examples of prenylflavonoids are balcony (such as xanthohumol (X) and desmethyltramadol (DMX), dehydrocyclization) and flavanones (such as azaxanthone (IX), 8 prenylnaringenin (8-PN) and 6-prenylnaringenin (6-PN)).

The term "geranyllinalool" refers to the flavonoid containing nonpolar geranyloxy side chain attached to one of the phenolic rings. Examples are tetrahydroxyphenylchlorin, 6-geranylgeranyl, 3'-geranylgeranylation and 8-geranylgeranyl. All these geranylgeranyl compounds were isolated from hop cones and 8-geranylgeraniol was attributed to estrogenic activity (Milligan et al. (2000) J. Clin. Endocrinol. Metab. 85, 4912-4915).

The term "enzymatic dealkylation or demethylation", used here, refers to the removal of compounds with the use of enzyme alkyl or a methyl group, respectively.

The term "5-alkoxy dealkylation or 5-methoxy demethylation", used here, refers to the removal of the alkyl group of the alkoxy group or the methyl of the methoxy (-OCH3) groups, respectively, localized in position 5 of the flavonoid (the numbering of the ring you can see in Figa). In this context, "the 5-methoxy and 5-O-methyl" have the same value.

The term "5-alkoxy(prenyl)flavo aid transferase (5-AO-(P)FT)" refers to an enzyme able to provide a 5-alkoxy dealkylation 5-alkoxy(prenyl)of flavonoids.

A particular group of flavonoids are balcony, where the numbering of the ring is different. Thus, with regard to balkanov, the present invention relates to the removal of alkyl groups from balkanologie compounds, more particularly to a 6'-alkoxy demethylation, i.e. to remove the methyl group of the alkoxy groups such as methoxy (-OCH3)localized in the 6' position of chalcone (the numbering of the ring you can see in Figv). Here "6'-methoxy and 6'-O-methyl-" have the same value. Enzymes, providing 6'-alkoxy dealkylation and, more specifically, 6'-methoxy demethylation is also designated here as "6'-alkoxy(prenyl)Halcon transferase (6'-AO-(P)CT and 6'-methoxy(prenyl)Halcon methyltransferase (6'-MO-(P)CMT)", respectively.

The term "microorganism"is used here, includes bacteria, and fungi. It refers to the individual strains of microorganisms, microbial consortia or microbial communities, such as the microbial community of the gut of animals or any other part of the body of animals (including humans), or from any source in the environment.

The term "in vitro method"used in the context of the present invention relates to a method performed outside of multicellular organisms, and includes both methods, the implementation is slow in the absence of living cells (using for example, lysed cells, protein extracts or recombinant proteins), and the processes carried out with the use of living cells, more specifically the cultures of the selected cells. If you refer to in vitro methods, it is thus assumed that exclude such processes as occurring in intact cones of hops or in the intestines of living zivotic.

When referring to "in situ" dealkylase, demetrious activity in vivo, it is assumed that it occurs in one or more specific organ of the body.

When referring here to the "bacteria"refers to both aerobic and anaerobic bacteria.

"Homoacetogenic", in relation to the bacteria are anaerobic bacteria, which reduce CO2to acetate or oxidized to acetate through the path of acetyl-CoA. Typical homoacetogenic bacteria are, for example, Acetoanaerobium noterae, Acetobacterium woodii, Acetobacterium wieringae, Acetogenum kivui, Acetitomaculum ruminis, Clostridium aceticum, Clostridium thermoaceticum, Clostridium formicoaceticum, Desulfotomaculum orientis, Sporomusa paucivorans, Peptostreptococcus sp. and Eubacteum sp.

The terms first, second, third and the like in the description and in the claims is used to denote differences between similar elements and are not necessary for describing a sequential or chronological order. It should be understood that thus the term t is Auda interchangeable under appropriate circumstances, and the here described embodiment according to the invention is able to act in a different order than described or illustrated here.

According to the present invention the enzymatic dealkylation and, more specifically, 5-alkoxy dealkylation of flavonoids can be done with the help of enzymes, generally designated as 5-alkoxy-transferase, more specifically using a 5-methoxyflavone methyltransferase (5-MO-FMT) and/or 6'-Methoxychlor methyltransferase (6'-MOE-CMT), as well as with intact live or inactivated cells or cell material), which produces a 5-alkoxylated alkyltransferase or enzyme 6'-alkoxyalkane alkyltransferase using lysate of such cells, using fraction of the lysate of such cells (e.g., membrane or cytoplasm), using enriched or purified protein fraction that includes the specified 5-alkoxylated alkyltransferase and/or 6'-alkoxyalkane alkyltransferase, or with expressed recombinant 5-alkoxylated alkyltransferase and/or 6'-alkoxyalkane alkyltransferase. If recombinant dealkilirovanny enzyme is secreted by the cells, can be used in a suitable environment. If the recombinant enzyme localized in the cytoplasm, the secretion signals can be added to recombinant DNA to which the protein returns, which can be isolated from the growth medium.

The invention offers alkoxy-dealkylase, more specifically enzymes capable of removing alkyl groups from alkoxysilanes. The alkyl group may be a linear or branched alkyl. More specifically, the alkyl group is a C1-C6alkyl. In a particular embodiment alkyl is stands.

According to the present invention offers a 5-alkoxy-alkyltransferase, more specifically 5-alkoxylated alkyltransferase that have prokaryotic or eukaryotic animal origin, including 5-alkoxy-alkyltransferase, originating from a plant cell or a microorganism.

The first aspect of the present invention relates to cells, extracts and enriched with partially purified or purified proteins (as well as to compositions that include one of these components or more)capable of dialkylamino 5-alkoxylated, more specifically, capable of demethylation of 5-methoxyflavone and/or 6'-metaxalone. According to a specific embodiment of the present invention cells, extracts and proteins, including the activity of 5-AO-FMT (and/or 6'-AO-CMT), are of bacterial origin. More specifically, the bacteria are homoacetogenic bacteria. The following embodiment of the image is etenia refers to homoacetogenic bacteria, selected from the Eubacterium species, and Peptostreptococcus. Homoacetogenic bacteria can be grown under anaerobic conditions with sugars, one-compounds such as formate, methanol, co and CO2plus H2as with alkoxycarbonyl aromatic compounds as carbon source. Bacterial strains with increased or enriched prenylflavonoids dealkylase or demetrious activity can be obtained by selection, based on the repeated incubation with an appropriate substrate, as herein described in the examples section. Enrichment activity, as here indicated, refers to activity, from about 1.5 to 10 times higher than the activity in the original strain, more specifically up to ~3 times higher than the activity in the original strain. Additionally or alternatively, the enrichment method of the present invention provides enzymatic activity, which reaches 90-100% conversion of the substrate (using, for example, 25 mg/l IX). Thus, the present invention also relates to a method of enrichment of 5-alkoxylated transferases (5-AO-FT) and/or 6'-alkoxyalkane transferases (6'-AO-CT) activity of bacterial strains, including the stage of incubation of strain on medium containing 5-alkoxysilanes, such as 5-methoxyphenylalanine (or 6'-Methoxychlor), more specifically including article is Dios dispersion of bacteria in the environment, including IX (or X), with subsequent breeding colonies with higher production. More specifically, strain several times sown on the environment, including the substrate, as, for example, 2-10 times, more specifically 3-4 times, with subsequent breeding colony with the highest 5-alkoxyphenyl-transferases (5-AO-FT) and/or 6'-alkoxyalkane-transferases (6'-AO-CT) activity. This activity can be measured, for example, on the basis of the product of the final reaction product. According to a specific embodiment of this method of enrichment is carried out on homoacetogenic bacterial strain, more specifically on the strain of Eubacterium or Peptostreptococcus, more specifically E. limosum or P. productus. A specific example of enriched bacterial strain of Eubacterium limosum deposited Willy Verstraete 15 March 2006 in the Belgian Coordinated collections of Microorganisms (BCCM) in the BCCM/LMG collection under Deposit number LMG P-23546, Laboratorium voor Microbiologie, Universiteit Gent (UGent), K.L. Ledeganckstraat 35, B-9000 Gent, Belgium.

Thus, the present invention proposes a method of producing enriched partially purified and/or purified 5-alkoxylated transferase, more specifically 5-methoxyphenylalanine methyltransferase, which includes the stage of getting a bacterial strain, more specifically homoacetogenic strain, such as Eubacterium limosum with enhanced/enriched 5AO-FT activity, more specifically took the military 5MO-FMT activity and enrichment, partial purification or purification of the enzyme was performed using classical methods of purification, including precipitation with ammonium sulfate, ion-exchange chromatography and gel filtration chromatography.

In the present invention are available bacterial strains as a source, where the alkyl or methyltransferase is present in high concentrations and/or where there is a natural mutant with high activity. In both cases, refer to the "enriched" bacterial strain.

According to another embodiment of the invention the cell, including 5-alkoxy dealkylase activity, more specifically 5-methoxyflavone demetrious activity (and/or 6'-Methoxychlor demetrious activity), is a transgenic cell obtained by introducing into a microorganism or plant cell a DNA sequence that encodes a 5-alkoxy-alkyltransferase, more specifically 5-MO-FMT (and/or 6'-MO-CMT). Genetically modified plant cells enriched with 5-MO-FMT (and/or 6'-MO-CMT) activity, which can be combined with high natural or artificially induced level of flavonoids (e.g., methylflavone), resulting “in planta” production of phytoestrogens. Thus, in the present invention are available, but are not limited to, genetically modificar the bathrooms plants with increased content of phytoestrogens, such as hop plants.

According to another embodiment of the cells, extracts and proteins, including 5-alkoxy dealkylase activity, more specifically 5-methoxy demetrious activity, are plant cells Humulus lupulus or other plants, where it is synthesized 8-PN, such as Marshallia grandiflora and Sophora tomentosa. For large-scale transformations, you can host a screening plants, where high natural transformation X or IX into 8-PN in order to detect natural mutants in 5-MO-FMT (and/or 6'-MO-CMT) or the super-expression of 5-MO-FMT (and/or 6'-MO-CMT).

Cells or compositions can be tested for the presence of activity of 5-AO-FT (and/or 6'-AO-CT), more specifically 5-MO-FMT (and/or 6'-MO-CMT), using a test where to explore the transformation of the test substrate 5-O-methylated flavonoid (6'-O-methylated chalcone) in his demetriadou form. The test substrate is preferably predominandy 5-methoxyflavone. According to a particular embodiment of the compound IX is used as a substrate, and the transformation of 8-PN can be tested using mass spectrometry, HPLC or other analytical method. The enzymatic specificity of the cell extract or the composition comprising 5-methoxy demetrious activity, you can test using flavonoid in the form of a substrate with methoxypropane others is Azaniah. For example, a suitable substrate for the test is tangeretin, which contains metoxygroup in positions 4', 5, 6, 7 and 8. This test allows to distinguish between dealkylase activity 5-AO-FT (and/or 6'-AO-CT) or demetrious activity 5-MO-FMT (and/or 6'-MO-CMT) of bacterial origin and demetrious activity of microsomes mammalian or plant cells. Extracts of bacterial or plant cells containing 5-methoxy demetrious activity, receive standard methods of protein extraction. Purified proteins with 5-MO-FMT (and/or 6'-MO-CMT) activity, get methods protein purification together with the screening on the activity of purified fractions.

The second aspect of the invention relates to the use of bacteria, producing 5-alkoxylated a transferase (5-AO-FT) and/or 6'-alkoxyalkane a transferase (6'-AO-CT), or, more specifically 5-methoxyflavone the methyltransferase (5-MO-FMT) and/or 6'-methoxy alkalotolerant (6'-MO-CMT), or extract, or purified proteins based on them, including this activity for dealkylation or demethylation of natural or synthetic 5-methoxyflavone, including prenisolone or geranylgeranyl 5-methoxyflavanone.

In a particular embodiment of the invention for cost-effective in vitro production of 8-PN and related compounds used cells, more to ncrete microorganisms, able to convert 5-methylated flavonoids, such as IX, 8-PN. Developed various embodiments, such as incubation, for example, bacterial demetrious strain with extracts of hops or (partially) purified derived from hops compounds; application of, for example, bacterial demetrious strain, cell extract or, ultimately, a suitable environment on top of extracts of hops or (partially) purified derived from hops compounds; or immersing the latter in a medium containing the strain, for example, demetrious bacteria or cell extract or, ultimately, in a suitable environment, possibly with subsequent inactivation of strain over time. Thus, the present invention also provides methods of cost-effective large-scale in vitro production of phytoestrogens.

According to the present invention, the bacteria containing 5-AO-FT (and/or 6'-MO-CT) activity, extracts and/or proteins, can be used for the production of active estrogen, more specifically phytoestrogens from 5-alkoxysilanes. One specific embodiment of the present invention relates to the use of 5-MO-FMT and/or 6'-MO-CMT activity for demethylation of plant flavonoids, more specifically derived from hops (Humulus lupulus). The following specific embodiment of the present invention relates to transformed the Yu IX into 8-PN, or demethylation derivatives IX to derivatives of 8-PN, having essentially the same biological activity. According to a specific embodiment of certain derivatives demetilirovanny of prenylflavonoids, such as 8-PN, can be obtained using the initial modification patterns readily available methylated precursors with subsequent demethylation according to the present invention, whereby receive demetilirovanie derivatives prenylflavonoids. Possible modifications are such as the inclusion of side chains or saturated, or unsaturated bonds.

In addition, cells and extracts of the present invention containing 5-AO-FT and/or 6'-AO-CT, more specifically 5-MO-FMT and/or 6'-MO-CMT activity, can be used to dealkylation 5-alkoxysilanes. More specifically, according to the present invention were developed as well as other substrates 5-methoxyflavanone (or 6'-metaxalone)other than IX (or X), such as 5-methoxyflavanone or 6'-metaxalone containing substituents in positions 4, 6, 7, 8, 2', 3', 4', 5' and 6' (according to the numbering of flavonoids), each of which was independently selected from the group consisting of H, C1-C6alkyl group, a C1-C6alkoxygroup and C1-C6acyl group, halogen, a longer carbon chains,aromatic derivatives, one or more residue(s) of sugar or sugar alcohols, ethers, esters, phosphates, sulfates, amines, etc.

According to a specific embodiment of the invention 5-alkoxylated, more specifically 5-methoxyflavone characterized by a hydroxyl group in position 7 and/or a double bond between positions 2 and 3 and/or hydroxyl group in position 4'. More specifically the present invention relates to the use of 5-MO-FMT activity for demethylation of 5-methoxyflavanone compounds, including prenolol or geranyloxy group at position 6 and/or 8. This prenolol or geranyloxy group optionally can be further modified by modifications such as the introduction of a double bond, transformation into isoprenoids and substitution, but not limited to these modifications. In certain embodiments, these 5-methoxyflavanone are 6'-metaxalone or 5-methoxyflavanone, including their prenisolone and geranylgeranyl version. In specific embodiments they are selected from the group xanthohumol (X) (2',4,4'-trihydroxy-3'-prenyl-6'-Methoxychlor) (numbering balkanov depicted on Figv and 2) and azaxanthone (IX) (5-O-methyl-8-prenylnaringenin), or derivatives thereof, with such essentially biological activity (or by demethylation of these compounds are a result of the connection is such essentially biological activity).

Another embodiment of the present invention relates to the use of bacteria or extracts from them, including 5-alkoxylated transfersno (5-AO-FT) and/or 6'-alkoxyalkane transfersno (6 AO-ST) activity for dealkylation of compounds selected from the group of the following molecules contains in addition to the 5-metoxygroup: 4'-acetyl-7-prenoxdiazine, (+/-)-(E)-8-(4"-hydroxyisopropyl)naringenin (8-PN-OH), (+/-)-((E)-8-(4"-oxoazetidin)naringenin (8-PN=O) and 6.8-diphenylalanine.

The present invention offers improved ways of obtaining demetilirovanny of prenylflavonoids in vitro using non-animal eukaryotic or prokaryotic material. It is of particular interest for those demetilirovanny of prenylflavonoids that have commercial value, such as 8-PN. As mentioned above, 8-PN demonstrates in vivo estrogenic activity, prevents loss of bone mass, inhibits angiogenesis and metastasis and manifests antiandrogenna activity. Therefore, the compounds obtained by the methods of the present invention, can be used to treat or prevent diseases such as osteoporosis and cancer. Demetilirovanie prenylflavonoids and geranyllinalool with estrogenic properties, obtained using the methods of the present invention may be incorporated is in intended for human consumption or animal food or food additives, such as drinks, including beer, as well as in cosmetic products intended for use on skin care person or animal. Thus, the present invention still offers improved methods for the production of such food products or food additives and medicines.

Another aspect of the present invention relates to in situ activation of methylated flavonoids in the intestine or any other part of the body of man or animal by injecting the cells, cell extracts or purified enzymes from 5-MO-FMT or 6'-MO-CMT activity. According to a specific embodiment, the introduction of 5-MO-FMT and/or 6'-MO-CMT activity combined with the introduction of methylated flavonoids or their source separately or in a single composition, simultaneously or sequentially. As a composition, including enzymatic activity, and composition, comprising a substrate, can be represented in the form of pharmaceutical compositions or food supplements. Sources methylated flavonoids include, but are not limited to, parts of the plant (e.g., hops), or extracts, or purified methylated flavonoid compounds. Using products demetilirovanny of prenylflavonoids in situ, more specifically, with products of 8-PN offers an alternative method of treatment and/or prevention of diseases and is of ostani, but not limited to, which can be treated with estrogen, diseases such as bone loss, pathological angiogenesis, metastasis, and suggested use as a antiandrogenna therapy.

Optional can be developed different strategies introduction for specific delivery to the colon alkyl flavonoids. This can be achieved, for example, by encapsulating the composition, which leads to the release in the colon, or by conjugation with obtaining a conjugate selected from the group consisting of glucuronide, sulfate, acetate, propionate, glucoside, acetylpyrazine, malonylglucosides and mixtures thereof.

Another aspect of the present invention relates to pharmaceutical compositions and/or food additive comprising or consisting of a cell extract or purified protein on its basis, with a 5-MO-FMT and/or 6'-MO-CMT activity. According to a particular embodiment of this aspect of the invention the cell is a cell or a microorganism, more specifically bacterial cell homoacetogenic, such as homoacetogenic Eubacterium and Peptostreptococcus. Optional pharmaceutical compositions or dietary supplements additionally include a power methylated prenylflavonoids or geranylgeraniol, which demetilirovanny compounds exhibit strong is th estrogenic activity. This source may be a plant extract (especially hops) or enriched fraction. It can also be synthetic methylated prenylflavonoids. As microorganisms and methylated flavonoids can be represented as/with separate pharmaceutical carriers for simultaneous or sequential injection, or can be combined in the same pharmaceutical carrier, distributed homogeneous or asymmetrically. Accordingly, the invention provides a combination pharmaceutical compositions, combinations, pharmaceutical compositions and dietary supplements, and combinations of food additives. In addition, the present invention provides methods of treatment, including stage sequential or simultaneous administration of the pharmaceutical compositions of the present invention to the needy in this patient. Similarly, in the present invention proposes the use of compositions comprising 5-alkoxyalkyl-transfersno activity and/or compositions described here includes 5-alkoxyphenyl, for the industrial manufacture of a medicinal product. Typically, the compositions of the present invention is administered in the form of estrogen supplementation and/or used in estrogenzawisimaya therapy need in this patient.

The number of methylated IX or X (which will be de atilirovanie to 8-PN), which must be entered is in the range between 10 and 20000 micrograms/day/75 kg, between 50 and 10,000 micrograms/day/75 kg, or between 50 and 7000 micrograms/day/75 kg, for example about 5, 10 or 20 milligrams/day/75 kg Methylated forms less powerful flavonoids can be respectively introduced in large doses after comparing estrogenic activity of this demetilirovanny form 8-PN.

In another aspect of the present invention features a pharmaceutical composition including bacteria or bacterial extract with 5-alkoxy-alkyltransferase activity of the present invention and a pharmaceutical carrier. In order to achieve optimal efficiency, the pharmaceutical carrier is preferably releases the microorganisms in the colon. Specific drug delivery in the colon are well known and are described, for example, Chourasia & Jain (2004) Drug. Deliv. 11(2), 129-148. Now available various strategies for specific release of drugs in the colon, including the formation of prodrugs, coated with pH-sensitive polymers, application-specific colon biodegradable polymer systems with controlled time-release osmotic systems and delivery systems drugs to control blood pressure. Among the variety of the ways of achieving specific release of the drug in the colon - application-specific colon biodegradable polymers shows great promise. Polysacharides are bacterial enzymes that are available in sufficient quantities to be used for specific drug delivery to the colon. Based on this method, explored a variety of polysaccharides for a specific release of drugs in the colon. These polysaccharides include pectin, guar-gum, amylose, inulin, dextran, chitosan and chondroitin sulfate. This family of natural polymers is attractive for drug delivery, since it includes polymers with a large number of the substituted groups, a wide limit on the molecular weight, varying chemical compositions and, for the most part, low toxicity and, in addition, the high stability of biodegradation. The most preferred feature of these substances is permission for their use as pharmaceutical excipients.

To prevent degradation from the inside dialkylamino bacteria of the present invention, the bacteria can be placed first in not degradiruem bacteria pharmaceutical carrier and then coated with polymer, which can collapse under the action of the microbial flora of the colon. Then BA is the criteria can be released, for example, through mechanisms controlled pH controlled time release of the drug or taking advantage of the increase luminale pressure in the colon, due to the strong peristalsis, as described Leopold (1999) Med Klin. 94 Suppl 1, 6-11.

Also known as system-specific delivery to the colon, which do not depend on the enzymatic activity of intestinal microorganisms. For example, European patent EP0673645 describes the system-specific drug delivery to the colon, which includes three parts: (1) intersolubility coating to prevent penetration of the gastric fluid in the delivery system, thus preventing any release of the drug in the stomach; (2) degradiruem polymer layer, which is exposed to and partially degraded during passage through the upper gastrointestinal tract, and (3) the inner part, which is containing the active ingredient(s) in the usual tablet or granule, which is easily desintegrated and sequentially releases the drug in the specific place of delivery - the colon after the erosion of the erodible polymer layer.

European patent application OR describes pharmaceutical compositions with a property specific controlled visvobodi the Oia active elements, which have a pharmacological effect in the intestine, and more specifically in the colon and terminal part of the ileum. The active element is prepared in multicomponent multi-dose form and cover at least two diaphragms, one with pH-dependent solubility and other insoluble but permeable to intestinal fluids. While the coated active element remains in the stomach and in the initial part of the intestine, that is, until the pH is less than 5.5, it is not released. Only when he reaches the place where the pH is higher (small intestine or colon), is pH-dependent dissolution of the membrane in order to begin the release of the active element. From this point the second membrane, which is pH-independent, but permeable to liquids intestine, acts by slowing and controlling the dissolution of the medicinal product within the tract of the small intestine-colon.

ER describes the composition with one or more probiotic microorganisms, such as Eubacterium, and a carrier to deliver the microorganisms in the colon. The carrier is modified or not modified resistant starch, more particularly starch with a high amylose content, which acts as a growth or maintenance medium for microorganisms in tools the Oh intestine. Patent application US 2004175389 describes a pharmaceutical composition for the preservation of probiotic bacteria during passage through the stomach with the possibility of release only in the intestine, more specifically in the colon, and which is practically not active in water and, accordingly, has a long shelf life. Medicinal composition consists essentially anhydrous mixture of probiotic bacteria with monovalent alginate salts, where the mixture has been formed and maintained, essentially anhydrous environment. Alginate salts include sodium alginate and potassium alginate, but does not include divalent salts, such as magnesium alginate or calcium alginate. Mainly for medicinal composition serves intersolubility coating (for example, gelatin or cellulose encapsulation).

It should be understood that although specific embodiments, specific constructions and configurations, as well as the materials discussed in the next section examples, they are only illustrative, and can be made various changes or modifications in form and detail, without going beyond borders and essence of this invention.

EXAMPLES

Example 1: Demethylation of azaxanthone (IX) using faecal cultures of human

Fecal samples were obtained from 12 healthy men aged 20 to 35 years and were bonacini from a to L. Not one of these patients had no history of gastrointestinal disease, and patients had not received antibiotics within 3 months prior to delivery of the sample. Were prepared fecal suspension from 20%(wt./about.) fresh fecal samples by homogenization of faeces in a phosphate buffer salt (0,1M, rn)containing 1G/l of thioglycolate sodium as a reducing agent. Material in the form of particles were removed by centrifugation (1 min, 500xg). The supernatant was then defined as "culture".

The ability of the cultures obtained from fecal samples A, B, C and D (hereinafter referred to as culture A-D)to destroy or transform prenylflavonoids IX hop tested by incubation of faecal cultures (10% (vol./about.)) in a nutrient medium with cardio-cerebral extract (0.5 g/l cysteine-HCl) 25 mg/l of azaxanthone for 8 days under anaerobic conditions. Incubation of extracts at 0 and day 8 tested for the biologically active transformation products IX using yeast test systems for the evaluation of estrogenic activity, according to De Boever et al. (2001) Env. Health Perspectives 109, 691-697, based on the work Routledge & Sumpter (1996) Environ. Toxicol. Chem. 15, 241-248. Briefly, Saccharomyces cerevisiae transformed human genome estrogen receptor (ERα), together with expressing plasmids containing responsive elements and LacZ reporter gene (to deruosi the enzyme β-galactosidase). The activity of β-galactosidase activity was quantitatively assessed at 540 nm using the conversion of chromogenic substances chlorophenol red-β-D-galactopyranoside in hlorfenilovy red. Response Biotest is expressed as the absorbance at 540 nm divided by the optical density at 630 nm [(A/A630)net]. Estrogenic activity of the samples was expressed in a percentage equivalent to 10 nm estradiol (E2), which caused 100% response Biotest on estrogenic activity. Biotests were performed in 96-well cards, in which 10 μl of the test compounds were tested, and incubated with 240 μl of genetically modified yeast (an optical density of 0.25 at 610 nm). Serial dilution of test compounds were made in dimethyl sulfoxide, which enables to obtain a dose-responsive curves for dose (y-axis) depending on the activity (x-axis). The data were processed using the 4-parameter logistic model using a Marquardt-Levenberg algorithm (Sigmaplot 4.0, SPSS Inc., Chicago, Illinois, USA) (De Boever et al. (2001) Env. Health Perspectives 109, 691-697).

The results are presented in figure 3. None of the incubations does not demonstrate estrogenic response at day 0. After 8 days in a strong increase in estrogenic properties observed in culture (Figure 3), but not in cultures A, B and D (data not shown). These results reveal the ability of fecal cult of the market With turning IX in connection with the increased estrogenic properties. To further test this transformation, culture A-D (10% (vol./about.)) were incubated for 8 days in a nutrient medium with cardio-cerebral extract (0.5 g/l cysteine-HCl) with X or IX at a concentration of 25 mg/l under anaerobic conditions, and the transformation products were detected using HPLC (table 1). It has been proven that IX is resistant to transformation in cultures A, B and D, which corresponds to the results obtained using the yeast test systems for the evaluation of estrogenic activity. However, in a culture With received almost 40% of 8-PN, which explains the increase in estrogenic properties of culture With. X slightly turned in IX in all samples, but, as has already been detected, during the incubation X with autoclaved cultures, this transformation was not the enzymatic isomerization. In culture has also detected a small amount of 8-PN, which resulted from the conversion of IX human faecal bacteria.

These results demonstrate the ability of intestinal bacteria to transform X or IX into 8-PN through a process of enzymatic O-demethylation metoxygroup in position 5 components. But not all cultures had the opportunity to carry out this reaction. Thus, the remaining culture E-L (10% (vol./about.)) were incubated for 3 days in PI is athelney environment with cardio-cerebral extract (0.5 g/l cysteine-HCl) with IX with a concentration of 25 mg/l under anaerobic conditions (Figure 4). Microbial O-demethylation IX were detected only in samples E, J and K.

This example demonstrates that methylated prenylflavonoids are not metabolically inert after ingestion, but may be activated in biologically (more) active demetilirovanie derivatives. However, this ability to transform highly dependent on the composition and activity of the microbial community of the gut, because activation IX is found only in one third of the tested samples.

To further explore these internal individual differences, all incubated 51 fecal sample within 3 days in culture medium with cardio-cerebral extract (0.5 g/l cysteine-HCl)containing IX at a concentration of 25 mg/l, under anaerobic conditions (Figure 5). The results are presented as % of production 8-PN in relation to the concentration of incubated IX, and the samples had to increase capacity for the production of 8-PN. Data were analyzed using two-stage cluster analysis and found three groups (labeled a, b and c) with significantly different values (P<0,01, Well-Wallis).

These data demonstrate that activation of methylated prenylflavonoids depends on the microbial community of the gut, separating individuals on strong (group C, 16%), medium (group b, 22%) and weak (group a, 63%) producers of 8-PN. In the main final impact on the active components will depend largely on the combination of the concentration of the precursor and the potential transformation of the microbial community of the gut.

Example 2. The use of microorganisms to produce compounds with estrogenic properties type 8-prenylnaringenin

This example describes the ability of two well-characterized intestinal anaerobic bacteria for conversion of IX into 8-PN.

Eubacterium limosum ATCC 8486 and Peptostreptococcus productus ATCC 27340 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). E. limosum were incubated for 13 days in a nutrient medium with cardio-cerebral extract (0.5 g/l cysteine-HCl), with IX and 8-PN in a concentration of 25 mg/l, under anaerobic conditions (table 2). This strain was able to turn IX into 8-PN. The strain was not subjected to additional degradation of 8-PN, since all 8-PN, provided as a substrate could be obtained after 13 days of incubation. This path is completely different from the metabolic pathway, which is observed in liver microsomes, where 8-PN in the future significantly metabolized [Nikolic et al. (2005) J. of Mass Spectrom. 40, 289-299; Nikolic et al. (2004) Drug Metabolism and Disposition 32, 272-279].

Table 2
Transformation IX and 8-PN using E. limosum
Eubacterium limosum
Substrate% Get *
XIX8-PN6-PN
IXn51,4(4,6)36,4(11,6)n
8-PNnn98,3(1,0)0,4(0,1)
*the results are presented as average (AVG off.) the molar obtain X, IX, 8-PN or 6-PN, expressed as a percentage
n: below detection

Due to the ability of E. limosum. to transform IX into 8-PN, the selection process was carried out to obtain a strain that is able to quantitatively produce 8-PN. The selection process consisted of 6 parallel incubations E. Limosum. [culture obtained from single colonies] with 25 mg/l IX, and incubation for 8 days. Then the culture that produced the greatest number of 8-PN, selectively and used for inoculation for the next round of 6 parallel incubations (table 3). While in the first round of selection of the lowest level of production was only 2%, then through the three stages of the breeding discovered it increased to 82%, and most effektivnaya culture transformed all entered IX into 8-PN. The average production of all six incubations in each round increased from 22.5% to 90.5 per cent, and the standard deviation was reduced from 20% to 7% after the selection process. This means that only after three rounds selectively strain that was transformed almost all IX (high average), and was stable (low standard deviation).

Table 3
Selection of E. Limosum, producing 8-PN, by 3 repeated incubations
Molar % conversion of IX into 8-PN
Round of selectionIIIIII
The lowest value2,124,382,1
The highest value46,579,4102,5
The average value (AVG off.)22,5(19,3)57,9(19,6)90,5(6,9)

To test the ability of P. productus to carry out the enzymatic preframe the s IX into 8-PN, the strain was incubated three times for 13 days in a nutrient medium with cardio-cerebral extract (0.5 g/l cysteine-HCl)25 mg/l IX under anaerobic conditions. The samples were analyzed every 2 days and determined the concentration of IX and 8-PN (6). Depending on the incubation of P. productus transformed from 10% to 50% incubated IX into 8-PN. This fact demonstrates that this strain is also suitable for the production of 8-PN. P. productus strains could further discriminates on increased demetrious activity that logically, as described for E. limosum.

A specific example of the bacterial strain of Eubacterium limosum, which was enriched, was deposited Willy Verstraete in the Belgian Coordinated collections of Microorganisms (BCCM) in the BCCM/LMG collection under Deposit number LMG P-23546 March 15.

Example 3. The use of microorganisms for the conversion of methylated prenylflavonoids in a fermentation installation

Experiment with fermentation periodic action with makeup developed to apply selectively strain of Eubacterium limosum, as obtained above, for the conversion of methylated prenylflavonoids in a fermentation installation. Fermentation was carried out in a fermenter Braun patch BIOSTAT®M (2 l Chan)filled with 1.5 liters of a nutrient medium with cardio-cerebral extract (0.5 g/l cysteine-HCl). Subsequently, the fermenter, sterilize the Ali by autoclaving for 30 min at 121°C. Before inoculation in the fermenter create anaerobic conditions by purging system for 1H with gaseous nitrogen. After that, the fermenter was inoculable 2-day culture of E. Limosum and added to the fermentation liquid 25 mg/l IX. Fermentation was carried out at 37°C for 2 weeks without pH control. From day 1, three times a day was added to the reactor at a rate of 10 ml/min 200 ml anaerobic nutrient medium with cardio-cerebral extract (0.5 g/l cysteine-HCl)containing 25 mg/l IX, and simultaneously removed from the system the contents of the fermenter with a speed of 200 ml/min. From the outflow took 10 ml of sample for chemical analysis. This was done on days 0, 1, 2, and then every 2 days. Data were represented as % conversion (8-PN/(IX + 8-PN)). There was obtained a conversion of IX into 8-PN 0% (day 0), 43% (day 1) and 100% (day 2 and subsequent days).

This example demonstrates that selectively strain capable of turning IX vysokopetrovsky 8-PN using strategies based on fermentation, leading to applications such as obtaining products with estrogenic properties of the predecessors, to purify the desired compound for use as an ingredient for other applications or to activate predecessor in extracts of hops or other plant extracts, including methylated prenylflavonoids.

Example 4: Add the pressure strain initiates ex vivo conversion of IX into 8-PN

The most effective strain of E. limosum, obtained in the result of the experiment on selection of example 2, were added to are not able to transform into normal culture conditions In example 1 to evaluate the ability of this strain to initiate the production of 8-PN in a complex environment fecal suspension. The strain was added to the culture in proportions in the range of 0% to 100% (vol./vol.). The mixture is incubated with 10% (vol./about.) 25 mg/l IX for seven days in a nutrient medium with cardio-cerebral extract (0.5 g/l cysteine-HCl) in anaerobic conditions. The concentration of 8-PN were detected every two days (Fig.7). The results show that with increasing addition of E. limosum products 8-PN was increased. Equal amount of culture E. limosum and fecal sample (100% figure 7) was each other competition in the conversion of IX into 8-PN, but even adding 1% half added IX transformed into 8-PN in one day. Interestingly, the maximum concentration of 8-PN was achieved in all incubations on the first day, which reveals that the entire available IX immediately transformed. No further conversion of 8-PN was not detected, because the concentration of 8-PN on the first day and the seventh did not differ significantly (Student T-test, p>0,05).

This example demonstrates that selectively strain was able to turn IX vysokopetrovsky 8-PN in complex environments the fecal culture, leading to possible applications such as in situ transformation of precursors into products with estrogenic properties with other good materials such as extracts of hops or other plant extracts, including methylated prenylflavonoids.

Example 5. The transformation of methylated prenylflavonoids in a dynamic in vitro simulation model of the intestinal tract

The next stage was to apply a dynamic in vitro simulation model of the intestinal tract, in order to demonstrate the in situ transformation of precursors such as IX products with estrogenic properties, such as 8-PN (simulator of the human intestinal microbial ecosystem (SHIME)), (Molly et al. (1993) Appl. Environ. Biotechnol. 39, 254-258). SHIME consists of a sequence of five reactors that represent different parts of the gastrointestinal tract of man. The first two reactors (stomach [reactor 1] and small intestine [reactor 2]) are elements of the periodic filling to simulate different stages of eating and digestion, with peristaltic pumps, feed a certain number of SHIME food (3 times/day), and pancreatic and bile fluid in the stomach and in the office of the duodenum, and consuming appropriate reactors at regular intervals. The last three sections (ACC. rising abadon what I [reactor 3], transverse colon [reactor 4] and the descending colon [reactor 5]) are a flow-through reactor with stirrer with constant volume and pH control. The duration of sedimentation and pH in different vats were chosen in order to reproduce in vivo conditions in different parts of the gastrointestinal tract. The passage of food in the small intestine imitated in the reactor 2 by adding 60 ml of artificial pancreatic and biliary fluids, Pancreatin and NaHCO3. The temperature was maintained at 37°C With a thermostat, and the system was maintained under anaerobic conditions by purging with N2for 15 minutes every day. Seed material was prepared from fecal matter, as described by De Boever et al. (2000) J. Nutrition 130, 2599-2606. The reactors 3, 4 and 5 were filled with nutrient medium and the pH was adjusted to the appropriate pH range. At the end was added 50 ml of inoculum in the last three reactor.

For this experiment, two of these systems were combined as two completely separate reactor, which are managed by the same pumps (pumps with two head, which allows you to add exactly the same amount of fluid in both systems), have identical pH and temperature control, and which receive the same liquid diet. Thus, all parameters are fully controlled and would is and identical with the exception of the intestinal microbial community in the system 2, which can be entered separately. In this case, we have introduced a community that is able to activate methylated prenylflavonoids (PF+) and which cannot (PF-). After two weeks of stabilization period, during which time added to normal SHIME food, 25 mg/l IX was introduced to SHIME food for 4 weeks (day 15 and 44). In the last two weeks selectively strain of Eubacterium limosum of example 2 was also introduced in the first part of the colon to simulate the application of strain as a probiotic (day 30-44).

Fig.7 and 8 show the concentration of IX and 8-PN in parts of the ascending colon, transverse colon and descending colon for PF+ and PF - communities. In PF+ Department found activation methoxylamine of prenylflavonoids in remote parts of the colon, when he introduced only IX (day 15-30), whereas no transformation is not met in the PF - branch. After adding the bacterial strain (starting from day 30) activating potential increased PF+ branch and also in the PF - branch, and products estrogen-active 8-PN were detected in a remote part of the colon.

The example demonstrates that selectively strain of example 2 had the ability to activate methylated prenylflavonoids in conditions that mimic human intestine.

Example 6: Dem who nstrate in vivo 5-alkoxyphenyl demetrious ability selektirovanii strain of example 2

Experiment with arsenicism and infected human flora (HFA) rats was carried out to test the ability selektirovanii strain of example 2 to activate methylated prenylflavonoids in vivo. Only 12 axenically rats used for the study. When rats were 5 weeks of age, 3 HFA rats were infected by oral administration through a gastric tube freshly isolated, homogenized fecal culture, which has prenylflavonoids demetrious activity. These HFA rats were designated as PF+ rats.

At the same time 3 HFA rats were infected by oral administration through a gastric tube freshly isolated, homogenized fecal culture without prenylflavonoids demetrious activity. These HFA rats were designated as PF - rats. All rats were kept in a separate, closed total cells prior to the experiment.

After 3 weeks of stabilization of microbial cultures in the intestine of rats was started the first experiment. After placing rats in individual chambers for studies of the metabolism of each rat daily was administered 2 mg IX/kg body weight for 5 days and each day was collected total urine for 24 hours After 3 days quantitatively assessed in the urine IX and 8-PN. The transformation of [8-PN/(IX+8-PN)] are presented in Table 4. Then the rats were placed back in the General cledith two weeks before the second part of the experiment.

Here 6 axenically rats were infected with a strain of E. limosum example 2 in 7 days by daily oral administration through a gastric tube Iog9 bacterial suspension. On the second day, rats were placed in individual chambers for studies of metabolism for total collection of urine for 24 hours Starting from the 2nd day on day 7 rats were injected 7.2 mg IX/kg body weight by oral administration via a stomach tube. On the 7th day quantitatively assessed in the urine IX and 8-PN. The transformation of [8-PN/(IX+8-PN)] are presented in Table 4.

Table 4
Average and Wed off. % excretion in urine 8-PN/(IX+8-PN) in the total urine within 24 hours
IXIX + E. limosum
The average value (AVG off.)The average value (AVG off.)
PF+55,3 (9,1)
PF-23,6 (10,4)
Axenically0,0 (0,0)41,1 (16,8)

This example demonstrates that activateimmediately of prenylflavonoids is exclusively microbial phenomenon, because exericise rats did not produce 8-PN. In addition, differences in the ability of intestinal transformation lead to different allocation of 8-PN, because in the urine PF+ rats degree 8-PN was higher compared to PF - rats. Finally, this example reveals that selectively strain of E. limosum can activate methylated prenylflavonoids in vivo, because exericise rats have started to produce 8-PN after infection by bacteria.

1. The way to obtain 8-prenylnaringenin in vitro, including:
a) providing a first composition comprising a culture of bacterial cells of the species Eubacterium limosum or species Peptostreptococcus productus, with 5-alkoxyphenyl-dealkylase activity, and
b) bringing into contact the second composition comprising 5-alkoxyphenyl, with the above first composition so as to make it possible dealkylation of these 5-alkoxylated using the specified culture of bacterial cells.

2. The method according to claim 1, where the specified 5-alkoxyphenyl are 5-methoxyflavanone.

3. The method according to claim 1 which further includes enrichment dealkylase activity of this culture of bacterial cells by repeated incubations with 5-methoxyphenylalanine.

4. The method according to claim 1, where the specified 5-alkoxylation is azaxanthone.

5. The method according to claim 1, where the specified 5-alkoxylated them is no vegetable origin.

6. The method according to claim 5, where this plant is hops.

7. The use of the culture of bacterial cells of the species Eubacterium limosum or species Peptostreptococcus productus, with 5-alkoxyphenyl-dealkylase activity, to obtain 8-prenylnaringenin in vitro.

8. Combination to obtain 8-prenylnaringenin in vitro, comprising a first pharmaceutical composition comprising a culture of bacterial cells of the species Eubacterium limosum or species Peptostreptococcus productus, with 5-alkoxyphenyl-dealkylase activity, and a second pharmaceutical composition comprising a source of 5-alkoxysilanes.

9. The combination of claim 8, where the specified source 5-alkoxysilanes is an extract of hops.

10. The combination of claim 8 or 9, where the specified 5-alkoxysilane is azaxanthone.

11. The combination of claim 8, where this culture of bacterial cells presents in the form of a composition for specific delivery to the colon.

12. The enrichment method 5-alkoxyphenyl-dealkylase activity culture of bacterial cells of the species Eubacterium limosum or species Peptostreptococcus productus, including the state of sowing the specified culture of bacterial cells on the environment, including 5-alkoxysilane, and the stage of the breeding colony, which has the highest 5-alkoxyphenyl-dealkylase activity.

13. The method according to item 12, where the specified stage again, at least on the times.

14. Selectively bacterial strain of Eubacterium limosum LMG P-23546, with 5-alkoxyphenyl-dealkylase activity and capable of turning azaxanthone 8 prenylnaringenin.



 

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FIELD: biotechnology, microbiology, medicine.

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FIELD: biotechnology, microbiology, agriculture.

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3 tbl, 2 ex

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2 tbl, 10 ex

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2 tbl, 10 ex

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3 tbl, 2 dwg, 5 ex

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9 cl, 8 tbl, 2 ex

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EFFECT: improved preparing method, valuable veterinary properties of preparation.

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