PREPARATIONS WITH C-METHYL FLAVONOIDS

Abstract

Suggested are preparations, comprising or consisting of (a) C-methyl flavonoids, a salt of C-methyl flavonoids and/or an extract containing C-methyl flavonoids, and (b) at least one inorganic salt or mineral substance.

Claims

1. Preparations, comprising (a) C-methyl flavonoids, a salt of C-methyl flavonoids and/or an extract containing C-methyl flavonoids, and (b) at least one inorganic salt or mineral substance. wherein the C-methyl flavonoids are selected from the group comprising Deodarin, 8-C-Methyl-Quercetin, Pinoquercetin.

2. The preparations of claim 1, characterised in that the salt of C-methyl flavonoids are selected from the group comprising the alkali metals salts, alkaline earth metals salts or ammonium salts.

3. Preparations, comprising (a) C-methyl flavonoids, a salt of C-methyl flavonoids and/or an extract containing C-methyl flavonoids, and (b) at least one inorganic salt or mineral substance. wherein the C-methyl flavonoids are selected from the group comprising Cedeodarin and Cedrin, and the inorganic salts or mineral substances are selected from the group comprising potassium chloride, potassium sulfate, chlorides, sulfates, phosphates, carbonates of magnesium and calcium, carnalite, kainite, schoenite and ammonium chloride.

4. The preparations of claim 1, characterised in that the extract containing C-methyl flavonoids are obtained from plants belonging to the plant genus Pinus by extraction with solvents selected from the group consisting of water, C.sub.1 to C.sub.4 alcohols, acetone, methyl ethyl ketone, .sup.tbutyl methyl ether, and ethyl acetate or their mixture.

5. The preparations of claim 1, characterised in that the mineral substances are selected from the group consisting of potassium, magnesium and calcium salts and their mixtures.

6. preparations of claim 3, characterised in that the mineral substance is potassium chloride.

7. The preparations of claim 1, characterised in that the preparation comprises component (a) in concentration of from about 0.01 to about 500 ppm.

8. The preparations of claim 1, characterised in that the preparation comprises component (b) in concentration of from 0.01 wt % to 5 wt %.

9. Food containing the preparations of claim 1.

10. The food of claim 9 representing bakery products or soups.

11. Pharmaceutical preparations containing the preparations of claim 1.

12. The pharmaceutical preparations of claim 11 are in liquid form.

13. The food of claim 10, characterised in that they contain C-methyl flavonoids or a salt of C-methyl flavonoids in concentrations of from about 0.01 to about 500 ppm.

14. A process for masking unpleasant, particularly bitter, astringent taste impressions of inorganic salts and food or pharmaceutical preparations containing these salts comprising or consisting of the following steps: providing food or pharmaceutical preparations containing an inorganic salt with said unpleasant, particularly bitter, astringent taste impressions, adding 0.01 to 500 ppm of C-methyl flavonoids or a salt of C-methyl flavonoids to them, wherein the C-methyl flavonoids are selected from the group comprising Cedrin, Deodarin, Cedeodarin, 8-C-Methyl-Quercetin, Pinoquercetin.

15. A method comprising using C-methyl flavonoids acid, a salt of C-methyl flavonoids and/or an extract containing C-methyl flavonoids for masking unpleasant, particularly bitter, astringent and/or liquorice-like taste impressions of inorganic salts and of food or pharmaceutical preparations containing these substances, wherein the C-methyl flavonoids are selected from the group comprising Cedrin, Deodarin, Cedeodarin, 8-C-Methyl-Quercetin, Pinoquercetin.

Description

DESCRIPTION OF THE INVENTION

[0013] A first subject-matter of the invention relates to preparations, comprising or consisting of [0014] (a) C-methyl flavonoids, a salt of C-methyl flavonoids and/or an extract containing C-methyl flavonoids, and [0015] (b) at least one inorganic salt or mineral substance.

[0016] Surprisingly, it was found that C-methyl flavonoids fully meet the complex profile of requirements described in the beginning. It improves the taste of inorganic alkali metal salts and alkaline earth metal salts having unpleasant off-flavours like metallic, bitter, acrid or chemical, especially inorganic potassium salts as well as chloride salts. It is a purely plant-based product which is fully effective also in very small concentrations of, for example, 10 μM (3 mg/l), and which thus proves to be superior to alternative prior-art substances.

[0017] Taste impressions, particularly unpleasant taste impressions that are intended to be improved within the meaning of the invention are understood as metallic, bitter, chemical and acrid taste sensations. Moreover, pleasant taste impressions, e.g. saltiness or spiciness and flavours are intended to be improved within the meaning of the invention. The overall improvement of unpleasant and pleasant tastes leads to a superior taste impression.

[0018] C-Methyl Flavonoids

[0019] C-Methyl flavonoids are flavonoids, carrying an additional methyl group which is linked to the flavonoid scaffold via carbon-carbon bond. This additional methyl group is typically adjacent to one or more phenolic hydroxyl groups. Typical representatives of methyl flavonoids are those from pine trees.

[0020] C-Methyl flavonoids are known from different plant families. They are particularly frequent in the plant families Pinaceae, Ericaceae, Myricaceae, Myrtaceae, and Annonaceae.

[0021] Typical representatives of C-methyl flavonoids are C-methylated analogs of Quercetin and Dihydroquercetin. There are compounds with one or two methyl groups.

[0022] Exemplary structural formula of C-methyl flavonoids belonging to different flavonoid classes (flavone, flavanonol, flavanon):

##STR00001##

[0023] Preferably the C-methyl flavonoids are selected from the group comprising Cedrin, Deodarin, Cedeodarin, Pinoquercetin, 8-C-Methyl-Quercetin, 6-C-Methyl-Quercetin, Pinomyrecetin.

[0024] More preferably the C-methyl flavonoids are selected from the group comprising C-methyl flavones, e.g. Pinoquercetin, 8-C-Methyl-Quercetin, Pinomyrecetin, and C-methyl-flavanonols, e.g. Cedrin.

[0025] The most preferable C-methyl flavonoids are C-methyl flavones, e.g. Pinoquercetin, 8-C-Methyl-Quercetin, Pinomyrecetin

[0026] Instead of using the actual C-methyl flavonoids itself it is also possible to use its salts, specifically its alkali metals salts, alkaline earth metals salts or ammonium salts.

[0027] According to the present application extracts containing C-methyl flavonoids can also be used, the preparation of which is known to the skilled person in the art and described by way of example, in example 1 below. Preferably, these extracts contain at least 1% (w/v), more preferably at least 5-50% (w/v) and particularly preferred about 20 to 50% (w/v) C-methyl flavonoids.

[0028] For the preparation of extracts containing C-methyl flavonoids different plant sources can be used. Preferably the extracts are obtained from the genus Pinales, more preferably from the family Pinaceae and most preferably from the genus Pinus.

[0029] The conventional different extraction methods can be used for the preparation of C-methyl flavone containing extracts. Preferably the solvents are selected from the group comprising water, C.sub.1 to C.sub.4-alcohols, acetone, methyl ethyl ketone, ethyl acetate, .sup.tbutyl methyl ether, preferably selected from the group comprising water, methanol, ethanol, and acetone. The solvents can be used alone or in a mixture.

[0030] Inorganic Salts or Mineral Substances

[0031] According to the present application the inorganic salts or mineral substances are any substances serving as sodium chloride replacers in food products, such as anorganic alkali metal salts as well as alkaline earth metal salts, for example potassium, magnesium and calcium salts and their mixtures.

[0032] Preferably the inorganic salts or mineral substances are selected from the group comprising potassium chloride, potassium sulfate, chlorides, sulfates, phosphates, carbonates of magnesium and calcium.

[0033] Carnalite, kainite, schoenite and ammonium chloride also belong to the inorganic salts or mineral substances according to the present application.

[0034] Preparations

[0035] According to the present application the preparation comprises component (a) in concentration of from about 0.01 to about 500 ppm, preferably from about 0.05 ppm to about 100 ppm, particularly preferably from about 0.1 to about 50 ppm.

[0036] According to the present application the preparation comprises component (b) in concentration of from 0.01 wt % to 5 wt %, preferably from 0.05 wt % to 2 wt %.

[0037] According to the present application the preparation comprises components (a) and (b) in the mass ratio of from 1:50 to 1:5000.

[0038] A form of preparation which is intended for commercial distribution and which is particularly suitable, for example, in form of a table salt substitute mixture, which contains 20-60% potassium chloride, 30-70% sodium chloride, about 10% other mineral salts like calcium or magnesium as well as other components like.

[0039] A further form of preparation which is intended for commercial distribution and which is particularly suitable, for example, in form of a seasoning flavour packages for food products. Such flavour packages contain mineral salts, e.g. sodium chloride; sodium nitrate; sodium sulphate; sodium citrate; sodium-Lactate; sodium gluconate; sodium glutamate; ammonium chloride; potassium chloride; potassium glutamate; potassium citrate; potassium sulphate; potassium citrates; potassium-L-lactate; potassium formate; potassium succinate; potassium fumarate; carnalite; kainite; schoenite; magnesium chloride; magnesium sulphate; magnesium carbonate; magnesium citrate; magnesium formate; magnesium gluconate; calcium chloride; calcium sulphate; calcium phosphate; calcium carbonate; calcium citrate; calcium formate; calcium gluconate; calcium glutamate; calcium glycerophosphate; essential microminerals such as iron, manganese, copper, zinc and cobalt; seasoning herbs; herbal extracts; vegetables; spices; anticaking agents like silicon dioxide, tricalcium phosphate; carbohydrates, like sucrose, glucose, lactose, fructose, glycerol; sugar alcohols, such as sorbitol, maltitol, lactitol, mannitol, xylitol; sugar substitutes; glycyrrhizin; sodium glycyrrhizinate; high intensity sweeteners, like sucralose, saccharin, aspartame, advantame, acesulfame K, cyclamate; yeast products, such as yeast extracts, autolysed yeast, hydrolysed yeast; hydrolysed animal and vegetable proteins; choline chloride; amino acids such as L-lysine, L-histidine, L-ornithine; citric acid; malic acid; tartaric acid; fumaric acid; lactic acid; acetic acid; benzoeic acid; adipic acid; gluconic acid; food polymers such as maltodextrin, dextrin, starch, polydextrose, inulin, gum arabic, guar gum, xanthan gum, Karaya gum, carrageenan, agar-agar, pectin, rice flour, sodium alginate, polyethylene glycol, sodium carboxymethyl cellulose, methyl cellulose, proteins, gluten; folic acid; ascorbic acid; glutamic acid; monopotassium L-glutamate; monosodium L-glutamate, monoammonium L-glutamate; calcium di-L-glutamate; magnesium di-L-glutamate; guanylic acid; disodium guanylate; dipotassium guanylate; calcium guanylate; inosinic acid; disodium inosinate; dipotassium inosinate; calcium inosinate; calcium 5′-ribonucleotides; disodium 5′-ribonucleotides, taurine, betaine.

[0040] Foods

[0041] A further subject-matter of the present invention relates to food which contain the preparations according to the invention. In this case it is possible to add the components individually or together, where the addition of the mixture is preferred. With respect to the taste improvement, component (a) can be used alone or as a mixture of (a) and (b).

[0042] Examples for foods, in principle, comprise baked goods, e.g. bread, dry biscuits, cakes, other baked products, confectionery (e.g. chocolates, chocolate bar products, other bar products, fruit gum, hard and soft caramels, chewing gum), alcoholic or nonalcoholic beverages (for example, coffee, tea, iced tea, wine, wine-containing beverages, beer, beer-containing beverages, liqueurs, schnapps, brandies, fruit-containing (carbonated) beverages, isotonic (carbonated) beverages, refreshing (carbonated) beverages, nectars, spritzers, fruit and vegetable juices, fruit or vegetable juice formulations), instant beverages (for example, instant cocoa beverages, instant tea beverages, instant coffee beverages, instant fruit beverages), meat products (for example, ham, fresh sausage or uncooked sausage formulations, seasoned or marinated fresh or salted meat products), eggs or egg products (dried egg, egg white, egg yolk), cereal products (for example, breakfast cereals, muesli bars, precooked ready-made rice products), dairy products (for example, milk beverages, butter milk beverages, milk ice cream, yoghurt, kefir, fresh cheese, soft cheese, hard cheese, dried milk powder, whey, whey beverages, butter, buttermilk, products containing partly or completely hydrolysed milk protein), products from soy protein or other soy bean fractions (for example, soy milk and products produced therefrom, fruit beverages with soy protein, soy lecithin-containing formulations, fermented products such as tofu or tempeh or products produced therefrom, soy sauces), products made of other plant-based protein sources, for example, oat protein beverages, fruit formulations (for example, preserves, fruit ice cream, fruit sauces, fruit fillings), vegetable formulations (for example, ketchup, sauces, dried vegetables, frozen vegetables, precooked vegetables, vegetables preserved in vinegar), snack products (for example, baked or fried potato crisps/chips or products based on potato dough, extrudates on a maize or peanut basis), products on a fat and oil basis, or emulsions of the same (for example, mayonnaise, remoulade, dressings), other ready meals and soups (for example, dried soups, instant soups, pre-cooked soups), condiments, seasoning mixtures and, particularly, seasonings, which are used, for example, in the snack industry.

[0043] Pharmaceutical Preparations

[0044] The taste problem described above does not only appear in foods, but also in pharmaceutical products in which the mineral salt needs to be masked particularly carefully.

[0045] Therefore, a further subject-matter of the invention also relates to pharmaceutical preparations, containing the preparations according to the invention. Also in this case, it is preferred to add component (a) and (b) as a mixture. Preferably, the pharmaceutical preparations are liquid products, particularly cough syrup, antipyretic agents, or antibiotics. Liquid forms of medication are particularly used in children and where the masking of the bitter taste of the active agent is technologically impossible, for example, when applying capsules, tablets or other solid forms of pharmaceuticals.

[0046] Both the foods and the pharmaceutical preparations may contain C-methyl flavonoids or a salt of C-methyl flavonoids in concentrations from about 0.01 to 500 ppm, preferably, about 0.05 ppm to 100 ppm, particular preferably 0.1 to 50 ppm

INDUSTRIAL APPLICATION

[0047] A further subject-matter of the invention relates to a process for masking unpleasant taste impressions by replacement of sodium chloride with mineral salts or enhancement of sodium chloride salty taste in foods or pharmaceutical preparations containing these substances, comprising or consisting of the following steps: [0048] (a) Providing food or pharmaceutical preparations containing an inorganic salt with said unpleasant, particularly bitter, astringent taste impressions, [0049] (b) adding 0.01 to 500 ppm of C-methyl flavonoids or a salt of C-methyl flavonoids to them.

[0050] Preferably, C-methyl flavonoids, a salt of C-methyl flavonoids and/or an extract containing C-methyl flavonoids is added in quantities such that the concentration of C-methyl flavonoids in the product is from about 0.01 to about 500 ppm, preferably, from about 0.05 ppm to about 100 ppm, particularly preferably from about 0.1 to about 50 ppm. The use of C-methyl flavonoids, a salt of C-methyl flavonoids and/or an extract of C-methyl flavonoids is also claimed for masking unpleasant taste impressions of sodium chloride replacers as well as foods or pharmaceutical preparations containing these substances, in the process of which C-methyl flavonoids, a salt of C-methyl flavonoids and/or an extract containing C-methyl flavonoids is added to these such that the concentration of C-methyl flavonoids in the product is from about 0.01 to about 500 ppm, preferably, from about 0.05 ppm to about 100 ppm, particularly preferably from about 0.1 to about 50 ppm.

EXAMPLES

Example 1

[0051] Isolation of C-methylflavonoids from Pinus sp.

[0052] Extraction

[0053] 360 g of dried bark of Pinus ayacahuite (collected in El Salvador by ProBioTec Ltd in 2002, sample receipt by AnalytiCon Nov. 15, 2002) was subsequently extracted with methanol:.sup.tbutyl methyl ether (1:1) and methanol. Both extracts were combined and dried in vacuum. Yield: 53 g.

[0054] Medium Pressure Liquid Chromatography (MPLC)

Separation ID: H-0735

[0055] Stationary phase: RP 18, 40-60μ
Mobile phase A: Water
Mobile phase B: Methanol

Gradient: 67% A to 15% A in 60 min

[0056] Flowrate: 80 ml/min
Collected fractions were analysed by HPLC-MS. Fractions containing C-methyl flavonoids were combined and the solvent was evaporated in vacuum. Yields of relevant fractions: H-0753-C: 3.53 g, H-0735-E 0.5 g.

[0057] Preparative HPLC

Separation ID: H-0735-C

[0058] Stationary phase: LichrospherSelect B, 10 μm, 250×50 mm
Mobile Phase A: 5 mMol ammoniumformate buffer, adjusted with formic acid to pH 3.0
Mobile Phase B: Methanol—acetonitril 1:1 (v/v) with 5 mMol ammoniumformate

Gradient: 24% to 39% B in 57 min

[0059] Flowrate: 80 ml/min
Detection: evaporative light scattering detector (ELSD)

Separation ID: H-0735-E

[0060] Stationary phase: LichrospherSelect B, 10 μm, 250×50 mm
Mobile Phase A: 5 mMol ammoniumformate buffer, adjusted with formic acid to pH 3.0
Mobile Phase B: Methanol—acetonitril 1:1 (v/v) with 5 mMol ammoniumformate

Gradient: 38% auf 50% B in 60 min

[0061] Flowrate: 80 ml/min

Detection: ELSD

[0062] Collected fractions were analysed by HPLC-MS. Fractions containing C-methyl flavonoids were combined and the solvent was evaporated in vacuum and analysed by H-NMR spectroscopy. The following compounds were isolated from the bark of P. ayacahuite:

TABLE-US-00001 Aus- beute Ordernummer Batch Struktur Name/CAS [mg] NP-002055 H-0735-C-06 [00002] Cedrin [75513-81-4] 131 mg NP-012227 H-0735-C-07 [00003] Deodarin [33788-39-5]  20 mg NP-012228 H-0735-C-08 [00004] Cedeodarin [31076-39-8]  45 mg NP-012356 H-0735-E-02 [00005] Pinoquercetin [491-49-6]  60 mg

[0063] All isolated compounds were characterized by the LCMS method listed in table 1:

TABLE-US-00002 TABLE 1 LCMS method for analytical characterisation of pure compounds HPLC System PE Series 200 MS System Applied Biosystems API 150 Data System Analyst 1.3 Stationäre Phase Merck Select B 250 × 4 mm, 5 μm Flussrate 1 ml/min Detektion (+/(−)-ESI, Fast-Switching-Mode ELSD (Sedex 75) UV (Merck, 254 nm) Sample 10 mg/ml in DMSO concentration Injection volume 30 μl Mobile Phase: A: 5 mM ammonium formiate and 0.1% formic acid B: Acetonitril/methanol = 1:1, 5 mM ammonium formiate und 0.1% ammonic acid (pH 3) Gradient Time [min] % A % B 00.0 85 15 30.0 0 100 35.0 0 100

Example 2

Synthesis of 6-C-Methylquercetin (Pinoquercetin) and 8-C-Methylquercetin

[0064] ##STR00006##

Step A. Synthesis of 2-methoxy-1-(2,4,6-trihydroxy-3-methylphenyl)ethanone

[0065] 2,4,6-Trihydroxytoluene (5.0 g, 35.6 mmol) was dissolved in dry ether (175 mL) under inert gas, methoxyacetyl chloride (3.59 mL, 39.3 mmol) and aluminium chloride (19.0 g, 142.7 mmol) were added at 0° C. The mixture was stirred overnight at room temperature. After complete conversion the mixture was poured into ice water, the aqueous phase was extracted with diethyl ether several times and the combined extracts were washed with water and dried with sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (Dichloromethane and methanol) to yield the title compound as an off white solid (2.9 g). MS (m/z): 213.1 [M+H.sup.+]

Step B. Synthesis of 2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-3-methoxy-6-methyl-4H-chromen-4-one and 2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-3-methoxy-8-methyl-4H-chromen-4-one

[0066] 2-methoxy-1-(2,4,6-trihydroxy-3-methylphenyl)ethanone (2.9 g, 13.6 mmol), fine powdered potassium carbonate (11.3 g, 81.7 mmol), tetrabutylammonium hydrogensulfate (6.9 g, 20.4 mmol), 3,4-dibenzyloxybenzoyl chloride (5.4 g, 27.2 mmol) were dissolved in toluene (170 mL) and were stirred at 120° C. for 6 h under nitrogen atmosphere. After cooling, the toluene was decanted and water (100 mL) was added to the remaining solid. The aqueous solution was extracted with DCM several times. The combined organic phases were washed with water and were added to the toluene solution. The organic solvents were evaporated under reduced pressure to obtain a brown residue. The residue was dissolved in a 5% potassium carbonate solution and was refluxed for 4 h. After fully conversion, the solvent was evaporated under reduced pressure and the residue was dissolved in water. The pH was adjusted to 5-7 and the aqueous solution was extracted with DCM several times. The combined extracts were washed with water and dried with sodium sulfate, and the solvent was removed under reduced pressure. The crude product was recrystallized from anhydrous ethanol to yield the title compound as an off white solid (1.65 g). MS (m/z): 359.5 [M+H.sup.+]

Step C. Synthesis of 6-C-Methylquercetin (Pinoquercetin) and 8-C-Methylquercetin

[0067] 2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-3-methoxy-6-methyl-4H-chromen-4-one and 2-(3,4-dimethoxyphenyl)-5,7-dihydroxy-3-methoxy-8-methyl-4H-chromen-4-one (1.45 g, 4.1 mmol) were dissolved in DCM (80 mL) and the solution was cooled to −10° C. A 1M solution of Boron tribromide in DCM (32.4 mL, 32.5 mmol) was added dropwise and the reaction mixture was stirred at room temperature over night. After fully conversion, water was added at 0° C. and the aqueous solution was extracted with DCM several times. The combined organic phases were dried with sodium sulfate and the solvent was removed under reduced pressure to yield the title compound as an off white solid (1.4 g). MS (m/z): 315.1 [M−H.sup.+]
The synthesized raw product LNB0247-119-A was purified by preparative HPLC:

[0068] MPLC

Separation ID: C-2433

[0069] Stationary phase: RP 18, 40-60μ
Sample amount: 2.84 g

Mobile Phase A: Water

Mobile Phase B: Methanol

Gradient: 75% A to 10% A in 60 min

[0070] Flowrate: 80 ml/min

[0071] Preparative HPLC

Separation ID: C-2433-D

[0072] Stationary phase: Kromasil C18, 10 μm, 250×50 mm
Sample amount: 1.01 g

Mobile Phase A: Water

Mobile Phase B: Acetonitril

Gradient: 31% auf 43% B in 57 min

[0073] Flowrate: 109 ml/min

Detection: ELSD

[0074] Collected fractions were analysed by HPLC-MS. Fractions containing C-methyl flavonoids were combined and the solvent was evaporated in vacuum and analysed by H-NMR spectroscopy. The following compounds were isolated:

TABLE-US-00003 yield Ordernumber Batch structure name/CAS [mg] NP-021118 C-2433-D-01 [00007] 8-C-Methyl- Quercetin [54987-82-5} 224 NP-012356 C-2433-D-02 [00008] 6-C-Methyl- Quercetin [491-49-6] 298

Example 3

[0075] Synthesis of Deodarin and Cedeodarin as Racemic Mixtures

##STR00009##

Step A. Synthesis of (E)-3-(3,4-bis(methoxymethoxy)phenyl)-1-(2,4,6-tris(methoxymethoxy)-3-methylphenyl)prop-2-en-1-one

[0076] Potassium hydroxide (2.9 g, 51.5 mmol) was dissolved in dry methanol (30 mL) under argon atmosphere. 1-(2,4,6-tris(methoxymethoxy)-3-methylphenyl)ethanone [Hossaln, M. A. et al. Bangladesh Journal of Scientific and Industrial Research 1998, 33, 94-96] (771 mg, 2.45 mmol) and 3,4-bis(methoxymethoxy)benzaldehyde [Du, G. et al. ChemMedChem 2017, 12, 183-193] (560 mg, 2.48 mmol) were dissolved in dry methanol (5 mL) and was added to the solution dropwise. The reaction mixture was stirred at room temperature overnight and after fully conversion water was added. The aqueous phase was extracted with ethyl acetate several times, the combined organic phases were washed with water, dried with sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by flash chromatography (Heptane and ethyl acetate) to yield the title compound as a white solid (980 mg). MS (m/z): 213.1 [M+H.sup.+]

Step B. Synthesis of (3-(3,4-bis(methoxymethoxy)phenyl)oxiran-2-yl)(2,4,6-tris(methoxymethoxy)-3-methylphenyl)methanone

[0077] (E)-3-(3,4-bis(methoxymethoxy)phenyl)-1-(2,4,6-tris(methoxymethoxy)-3-methylphenyl)prop-2-en-1-one (980 mg, 1.8 mmol) was dissolved in dry methanol (23 mL), a 2M sodium hydroxide solution (1.2 mL) and 30% hydrogen peroxide solution (1.19 mL, 39.6 mmol) were added and the reaction mixture was stirred at room temperature for 2 h. After fully conversion water was added and the aqueous phase was extracted with ethyl acetate several times. The combined organic phases were washed with water, dried with sodium sulfate, and the solvent was removed under reduced pressure to yield the title compound as a white solid (1000 mg). MS (m/z): 561.4 [M+Na.sup.+]

Step C. Synthesis of Deodarin and Cedeodarin

[0078] (3-(3,4-bis(methoxymethoxy)phenyl)oxiran-2-yl)(2,4,6-tris(methoxymethoxy)-3-methylphenyl)methanone (320 mg, 0.59 mmol) was dissolved in a mixture of methanol, thf and conc. HCl (8 mL, 3 mL, 0.15 mL) and the reaction mixture was stirred at 55° C. for 2 h. After full conversion water was added and the aqueous phase was extracted with ethyl acetate for several times. The combined organic phases were dried with sodium sulfate and the solvent was removed under reduced pressure to yield the title compound as an off white solid (216 mg). MS (m/z): 317.2 [M−H.sup.+]
The synthesized raw product LNB0276-152-fr30-42 was purified by preparative HPLC:

[0079] Preparative HPLC

Separation ID: C-2826-M

[0080] Stationary phase: Kromasil C18, 10 μm, 250×50 mm
Sample amount: 0.46 g

Mobile Phase A: Water

Mobile Phase B: Acetonitril

Gradient: 35% auf 45% B in 57 min

[0081] Flowrate: 80 ml/min

Detection: ELSD

[0082] Collected fractions were analysed by HPLC-MS. Fractions containing C-methyl flavonoids were combined and the solvent was evaporated in vacuum and analysed by H-NMR spectroscopy. The following compounds were isolated:

TABLE-US-00004 Order yield number Batch Structure Name/CAS [mg] NP-012227 C-2826-N-01 [00010] Deodarin [33788-39-5] (as racemic mixture)  14 NP-012228 C-2826-N-02 [00011] Cedeodarin [31076-39-8] (as racemic mixture) 127

Example 4

In-Vitro Assay

[0083] A human cell based assay system ScreenLine® based on lingual cells was established. The activation of these cells was quantified in order to detect natural substances which reduce the bitterness of potassium chloride. The change of the intracellular calcium level was measured for quantitation of the activation of the ScreenLine® (see FIGS. 1 and 2).

[0084] Establishment of the Cell-Based Measurement System

[0085] In order to detect substances with inhibitory activity, a high-throughput cell-based in-vitro measurement system was established. To this end, ScreenLine® was used that was feasible for this application based on Hochheimer, A., Krohn, M., Rudert, K., Riedel, K., Becker, S., Thirion, C. and Zinke, H. “Endogenous gustatory responses and gene expression profile of stably proliferating human taste cells isolated from fungiform papillae” Chem Senses, 39, 359-377 (2014).

Example 5

The Natural Taste of High-Purity C-Methyl Flavonoids and of Extracts Containing C-Methyl Flavonoids

[0086] The sensory evaluation of the samples was performed by a panel of five experienced assessors. The sensory tests were performed descriptively, randomized, blinded, and by means of a “sip and spit” method. To this end, the sample is moved within the mouth for a few seconds to evaluate its taste, and it is not swallowed but expectorated. The results may be taken from the following Table 2a:

TABLE-US-00005 TABLE 2a Tasting of high-purity Pinoquercetin Test Descriptive evaluation, “sip and spit” method, blinded method and randomised samples, pH neutral Test sample Purified Pinoquercetin, purity > 98% Panelists 5-7 experienced assessors Preparation Four different concentrations (1 μM, 10 μM, 25 μM, of the and 50 μM, or approximately 0.3, 3.2, 8 and 16 ppm, samples respectively) dissolved in spring water Evaluation  1 μM (0.3 ppm) clean flavor 10 μM (3.2 ppm) quite clean flavour, very slight chemical note 25 μM (8 ppm) quite clean flavour, very slight bitterness 50 μM (16 ppm) quite clean flavour, slight floral note, slight bitterness & drying

TABLE-US-00006 TABLE 2b Tasting of extracts containing Pinoquercetin Test Descriptive evaluation, “sip and spit” method, blinded method and randomised samples, pH neutral Test 1) purified Pinoquercetin, purity > 98% sample 2) commercially available C-methyl flavonoids acid, purity 85% (HPLC) 3) extract with ca. 25% Pinoquercetin Panelists 3-5 experienced assessors Preparation Three different concentrations (dissolved in 0.5% of the ethanol), comparison sample 0.5% ethanol in water, final samples concentration of the ethanol below 0.5% Sam- Sam- Sam- Evaluation Conc. ple 1 ple 2 ple 3  3 mg/l neutral neutral neutral 7.5 mg/l  neutral neutral slightly bitter 30 mg/l slightly slightly More bitter bitter bitter than samples 1 and 2, resinous

Example 6

[0087] Taste of the Combination of Pinoquercetin with Potassium Chloride

[0088] The sensory evaluation of the samples was performed twice by a team of seven experienced assessors. The results may be taken from the following Table 3:

TABLE-US-00007 TABLE 3 Tasting of high-purity Pinoquercetin + potassium chloride (KCl) Test method Descriptive and discriminative evaluation, “sip and spit” method, blinded and randomised samples, pH neutral, confidence scoring: only guessed, not sure, fairly sure, very sure recorded: sensory descriptions of assessors Test sample Purified Pinoquercetin, purity > 98% (ACD-2207) Panelists 7 experienced assessors, trained to recognise the off-flavour, in particular the bitterness, of potassium chloride Preparation Four different concentrations of Pinoquercetin (1 μM, of the 10 μM, 25 μM, and 50 μM) with 1.5% potassium samples chloride dissolved in spring water Evaluation Two assessments with 7 assessors were carried out. This resulted in 14 data points/responses.  1 μM (0.3 ppm) bitterness of KCl is reduced by the product in 64% of responses average confidence score: fairly sure to very sure dominant flavours: saltiness, enhanced saltiness 10 μM (3.2 ppm) bitterness of KCl is reduced by the product in 64% of responses average confidence score: fairly sure to very sure dominant flavour: saltiness 25 μM (8 ppm) bitterness of KCl is reduced by the product in 64% of responses average confidence score: fairly sure dominant flavour: saltiness 50 μM (16 ppm) bitterness of KCl is reduced by the product in 71% of responses average confidence score: fairly sure dominant flavour: saltiness

Example 7

Taste Modulation of Crackers

[0089] The sensory evaluation of the samples was performed by 15 naïve as well as experienced assessors. Details may be taken from the following Table 4:

TABLE-US-00008 TABLE 4 Taste evaluation of cracker salted with potassium chloride Test method Descriptive evaluation Test sample Purified Pinoquercetin, purity > 98% (ACD-2207) Panelists 15 naïve as well as experienced Preparation of Cracker thesamples 150 g flower type 405 35 g neutral plant oil 25 ml water 25 ml red wine 6 g curcuma 2 g herbs 20 g sesam 3.6 g KCl for 1.5% KCl or 2.4 g KCl for 1% KCl +/−3 mg ACD-2207 (~10-15 ppm) Evaluation Cracker with ACD-2207 was preferred, due to reduced off-flavours, enhanced saltiness as well as improved aroma profile.
The sensory profile of the cracker with potassium chloride was strongly improved by the product. The product would enable a strong reduction of sodium chloride content in crackers and related categories.

Example 8

Taste Modulation of Vegetable Bouillon

[0090] The sensory evaluation of the samples was performed by 6 naïve assessors. Details may be taken from the following Table 5:

TABLE-US-00009 TABLE 5 Taste evaluation of vegetable broth salted with potassium chloride Test method Descriptive evaluation Test sample Purified Pinoquercetin, purity > 98% (ACD-2207) Panelists 6 naïve assessors Preparation of 2.25 g vegetable broth the samples 1.2 g potassium chloride 0.45 g sodium chloride 3.8 mg ACD-2207 (~25 ppm) ad 150 ml tap water Evaluation Soup with ACD-2207 was preferred, due to reduced off-flavours, enhanced saltiness as well as improved aroma profile.
The sensory profile of the soup salted with potassium chloride was strongly improved by the product. This was also evident in soups containing a small amount of sodium chloride. Thus, the product would enable a strong reduction of sodium chloride content in soups and related categories.