U.S. patent application number 16/248921 was filed with the patent office on 2019-07-18 for pharmaceutical preparations.
The applicant listed for this patent is Symrise AG. Invention is credited to Elke Kock, Jakob Ley, Kathrin Liszt, Veronika Somoza, Sabine Widder.
Application Number | 20190216770 16/248921 |
Document ID | / |
Family ID | 47628003 |
Filed Date | 2019-07-18 |
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United States Patent
Application |
20190216770 |
Kind Code |
A1 |
Kock; Elke ; et al. |
July 18, 2019 |
PHARMACEUTICAL PREPARATIONS
Abstract
Pharmaceutical preparations are proposed, comprising
bitter-masking acting substances for reducing and/or inhibiting
gastric acid secretion induced by food constituents.
Inventors: |
Kock; Elke; (Neumarkt,
AT) ; Ley; Jakob; (Holzminden, DE) ; Somoza;
Veronika; (Weidling, AT) ; Liszt; Kathrin;
(Rechnitz, AT) ; Widder; Sabine; (Holzminden,
DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Symrise AG |
Holzminden |
|
DE |
|
|
Family ID: |
47628003 |
Appl. No.: |
16/248921 |
Filed: |
January 16, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14761474 |
Nov 4, 2015 |
10226445 |
|
|
PCT/EP2014/050742 |
Jan 15, 2014 |
|
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16248921 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23F 3/405 20130101;
A61K 9/4875 20130101; G01N 33/5044 20130101; A23L 2/52 20130101;
A61K 9/0056 20130101; A61K 9/0058 20130101; A23F 5/465 20130101;
A61K 31/353 20130101; A23V 2002/00 20130101; A61K 31/365 20130101;
A61K 9/19 20130101; A23L 33/10 20160801; A61K 47/22 20130101; A61K
9/0095 20130101; A61K 31/341 20130101; A23L 27/86 20160801 |
International
Class: |
A61K 31/365 20060101
A61K031/365; A61K 31/353 20060101 A61K031/353; A23F 3/40 20060101
A23F003/40; A61K 31/341 20060101 A61K031/341; A23F 5/46 20060101
A23F005/46; G01N 33/50 20060101 G01N033/50; A23L 2/52 20060101
A23L002/52; A61K 47/22 20060101 A61K047/22; A23L 27/00 20060101
A23L027/00; A23L 33/10 20060101 A23L033/10 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 17, 2013 |
EP |
13151578.5 |
Claims
1-15. (canceled)
16. A pharmaceutical preparation, comprising (a) a food constituent
inducing gastric acid secretion, and (b) a bitter-masking acting
substance for reducing and/or inhibiting gastric acid secretion
induced by said food constituent, wherein the bitter-masking acting
substance (component b) is homoeriodictyol and/or eriodictyol, the
food constituent inducing gastric acid secretion (component a) is
selected from the group consisting of caffeine, theobromine,
theophylline, tartaric acid, racemic acid, malic acid, succinic
acid, salicin, arbutin, neohesperidin, eriocitrin, neoeriocitrin,
narirutin, naringin, phloridzin, trilobatin, gallic and ellagic
esters of carbohydrates, galloylated catechins and epicatechins and
oligomers thereof, proanthocyanidins, procyanidins, thearubigin,
quercetin, quercitrin, rutin, taxifolin, myricetin, myricitrin,
limonin, nomilin, lupolone, humolone, stevioside, rubodid,
rebaudioside A, rebaudioside C, glycyrrhizin, glycyrrhetic acid and
oleuropein and mixtures thereof, the food constituent is present in
an amount of from about 3 to about 300 ppm, calculated on the
preparation, and said bitter-masking acting substance is present in
an amount effective to reduce or inhibit proton secretion in HGT-1
cells upon ingestion.
17. The preparation as claimed in claim 16, wherein said
preparation comprises the bitter-masking acting substance and the
food constituent which form components (a) and (b) in the weight
ratio of 1:100 to 1:1.
18. A composition for oral administration comprising the
preparation of claim 16.
19. The composition as claimed in claim 18, wherein said
composition is in the form of bakery products, confectionery,
alcoholic or non-alcoholic drinks, meat products, eggs or egg
products, cereal products, milk products, products made from soy
protein or other soybean fractions, fruit preparations, vegetable
preparations, snack foods, products based on fat and oil or
emulsions of the same, other ready meals and soups, spices, spice
mixtures and seasonings.
20. The composition as claimed in claim 18, wherein said
composition is in the form of a capsule.
21. The preparation as claimed in claim 16, wherein (b) the
bitter-masking acting substance is present in an amount of from
about 3 to about 300 ppm.
22. The preparation as claimed in claim 21, wherein (b) the
bitter-masking acting substance is present in an amount of from
about 10 to about 100 ppm.
23. A composition for oral administration comprising a
pharmaceutical preparation, comprising (a) a food constituent
inducing gastric acid secretion and selected from the group
consisting of caffeine, theobromine, theophylline, tartaric acid,
racemic acid, malic acid, succinic acid, salicin, arbutin,
neohesperidin, eriocitrin, neoeriocitrin, narirutin, naringin,
phloridzin, trilobatin, gallic and ellagic esters of carbohydrates,
galloylated catechins and epicatechins and oligomers thereof,
proanthocyanidins, procyanidins, thearubigin, quercetin,
quercitrin, rutin, taxifolin, myricetin, myricitrin, limonin,
nomilin, lupolone, humolone, stevioside, rubodid, rebaudioside A,
rebaudioside C, glycyrrhizin, glycyrrhetic acid and oleuropein and
mixtures thereof, (b) a bitter-masking acting substance for
reducing and/or inhibiting gastric acid secretion induced by said
food constituent and which is homoeriodictyol and/or eriodictyol,
and (c) one of bakery products, confectionery, alcoholic drinks,
meat products, eggs or egg products, cereal products, milk
products, products made from soy protein or other soybean
fractions, fruit preparations, vegetable preparations, snack foods,
products based on fat and oil or emulsions of the same, other ready
meals and soups, spices, spice mixtures and seasonings, wherein the
food constituent is present in an amount of from about 3 to about
300 ppm, calculated on the preparation, and said bitter-masking
acting substance is present in an amount effective to reduce or
inhibit proton secretion in HGT-1 cells upon ingestion.
24. The preparation as claimed in claim 23, wherein the food
constituent is caffeine.
25. The preparation as claimed in claim 23, wherein (b) the
bitter-masking acting substance is present in an amount of from
about 10 to about 100 ppm.
26. The preparation as claimed in claim 24, wherein said
preparation comprises the bitter-masking acting substance and the
food constituent which form components (a) and (b) in the weight
ratio of 1:100 to 1:1.
Description
FIELD OF THE INVENTION
[0001] The invention is in the fields of pharmaceuticals and
foodstuffs and relates to preparations comprising active
ingredients against secretion of gastric acid induced by food
constituents.
PRIOR ART
[0002] The stimulation of gastric acid secretion is an important
mechanism to initiate the digestion of food, especially
protein-rich food. It may be quite positive in terms of digestion
to activate short-term moderate additional secretion. However, if
gastric acid is excessively secreted due to external stimuli and
the pH in the stomach is thus greatly reduced, this generally leads
to acute discomfort or acid reflux. If this condition persists for
a relatively long time or gastric acid secretion is chronically
highly activated, inflammatory conditions of the gastric mucosa and
the esophagus may be induced, which in turn, as a long-term
consequence, trigger ulcers and, in the worst case, even malignant
tissue alterations up to stomach cancer and esophageal cancer.
[0003] Gastric acid secretion is also induced, in addition to
general stimuli such as food intake or hunger states etc, by
diverse low molecular weight food constituents. For example, Liszt
et al. in J. Agric Food Chem. 60, 7022-7030 (2012) describe that
the fruit acids malic acid and succinic acid increasingly present
in white wine make a substantial contribution to excess acid
secretion which can however be mitigated again by alcohol in the
case of tartaric acid. Hop constituents, e.g. via beer consumption,
can also induce release of protons into the stomach contents (cf.
Walker et al. in J. Agric Food Chem. 60, 1405-1412 (2012), in which
a trend of the relative bitterness and the activity on proton
secretion can be observed. Also coffee, and in particular the
caffeine present therein, is able to significantly stimulate
gastric acid secretion, as has been shown by Rubach et al., in Mol.
Nutr. Food Res. 56, 325-335 (2012).
[0004] Classically, two major groups of active ingredients are used
for reducing the gastric acid secretion described above: firstly,
neutralization of the gastric acid to increase the pH with the aid
of basic materials such as sodium hydrogen carbonate, calcium
carbonate, basic aluminum hydroxide or magnesium hydroxide etc.;
secondly, decreasing the gastric acid secretion by direct blocking
of the acetyl choline receptors (M.sub.3 type) to be found on the
secretory cells (parietal cells) by active ingredients such as
pirenzepin or, more frequently, histamine H.sub.2-receptors, e.g.
by active ingredients such as cimetidine, ranitidine or famotidine;
as a third mode of action, the ATP-driven proton pump of the
parietal cells is directly modulated (omeprazole).
[0005] The mechanism of the stimulatory activity of the
aforementioned food constituents is generally unclear. It is
therefore also not possible to predict which low molecular weight
substances used for or in food (except basic substances due to
their buffer effect) may even possibly reverse the effect and thus
may reduce the excess secretion.
[0006] From the article of M. T. Montforte et al. entitled
"Protective Effect of Calamintha officinalis (monk's leaves)
against alcohol-induced gastric mucosa injury in rats", it is
apparent that extracts of Calamintha officinalis, known in Italy as
"culinary herb" under the name Mentuccia, have a gastroprotective
effect in animal experiments. The extracts here comprise
polyphenols, catechins, tannins and terpenes such as eriocitrin,
eriodyctiol, benzoic acid, chlorogenic acid and others. The
gastroprotective effect was confirmed in this case by the reduced
formation of stomach tumors (cf. Phytother. Res. 26(6), pp. 839-844
(2012).
[0007] The publication of M. Trautmann et al. entitled
"Aspirin-like drugs, ethanol-induced rat gastric injury and mucosal
eicosanoid release" relates to the influence of aspirin and other
active ingredients similar to salicylic acids on the release of
leukotrienes and prostaglandins. In this case, salicylamide is
known as one of the substances which inhibit the secretion of
leukotrienes and thus has a gastroprotective effect (cf. Europ. J.
Pharmocol. 201(1), pp. 53-58 (1991).
[0008] The article of R. Magous et al. entitled "Leukotrienes
stimulate acid secretion from isolated gastric parietal cells" is
concerned with leukotrienes, which stimulate a mild contraction of
the muscles in the stomach region. It should be noted that specific
leuokotrienes stimulate acid secretion in isolated gastric cells
from hares (cf. Biochem. Biophys. Res. Comm. 114(3), pp. 897-900
(1983).
[0009] The publication of M. B. Eswaran et al. entitled
"Gastroprotective activity of Cinnamonium tamala leaves on
experimental gastric ulcers in rats" relates to the
gastroprotective properties of, for example, eugenol and other
active ingredients from the leaves of Cinnamomum tamala (cf. J.
Ethnopharm. 128(2), pp. 537-540 (2010).
[0010] The document of G. Kaithwas et al. entitled "Evaluation of
antiulcer and antisecretory potential of Linum usitatissimum fixed
oil." relates to the use of linseed oil for gastroprotective
purposes (cf. Inflammopharm. 18(3), pp. 137-145 (2010).
[0011] This publication of I. Gurbuz et al. entitled
"Anti-ulcerogenic lignans from Taxus boccata" concerns the use of
lignans, spefically resinols from Taxus in cancer therapy (cf. Z.
f. Naturforsch. 59c, pp. 233-236 (2004).
[0012] U.S. Pat. No. 4,865,847 (GOSSWEIN) relates to a method for
preventing stimulation of gastric juices, in which chlorogenic acid
is absorbed together with the food.
[0013] It is known from the publication of J. Ramage entitled
"Inhibition of food stimulated acid secretion by misoprostol" that
the formation of gastric acid by means of food may be avoided if a
synthetic prostaglandin E analog is used (cf. Br. J. Clin. Pharmac.
19, pp. 9-12 (1985).
[0014] The object of the invention, therefore, was to find low
molecular weight substances, preferably natural substances, which
can be used for or in food, which are characterized by better
compatibility and higher performance compared to the known active
ingredients and where the acid secretion induced by the
aforementioned food constituents can be reduced or even completely
prevented.
DESCRIPTION OF THE INVENTION
[0015] The invention relates to a pharmaceutical preparation
comprising bitter-masking acting substances for reducing and/or
inhibiting gastric acid secretion induced by food constituents.
[0016] It has been found, surprisingly, that substances which are
capable of masking the bitterness of many foods, also at the same
time reduce or even inhibit the secretion of gastric acid induced
by these substances. In particular, it was found that substances of
the group (a), such as homoeriodictyol, eriodictyol, matairesinol,
lariciresinol or enterolactone, can in combination again reduce or
completely block the gastric acid-stimulating effect of the
substances of the group (b), such as caffeine or theobromine.
[0017] This is all the more astonishing, since up to now it has
been assumed that the masking substances only dock onto certain
taste receptors and do not even have a pharmacological effect. The
use of the masking substances for the stated purpose has the
further advantage here that they have already proven to be
palatable.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The present invention will be described in greater detail
with reference to the accompanying drawings, in which
[0019] FIG. 1 illustrates a graph of proton secretion on exposure
to caffeine or caffeine/enterolactone, and
[0020] FIG. 2 illustrates a graph of proton secretion on exposure
to enterolactone.
BITTER-MASKING SUBSTANCES (COMPONENT A)
[0021] The bitter-masking substances which can form component (a)
are selected from the group formed by: [0022] hydroxyflavanones,
particularly eriodictyol, eriodictyol-7-methyl ether,
homoeriodictyol, and sodium, potassium, calcium, magnesium or zinc
salts thereof, especially those which are described in the European
patent application EP 1258200 A2; [0023] hydroxybenzamides,
particularly those which are described in the international patent
application WO 2006 024587 A2; [0024] hydroxyphenylalkanediones,
particularly gingerdione-[2] and gingerdione-[3], and especially
those which are described in the international patent application
WO 2007 003527 A2; [0025] gamma-aminobutyric acid, particularly
those which are described in the international patent application
WO 2005 096841 A1; [0026] 4-hydroxydihydrochalcones, particularly
phloretin and those which are disclosed in the international patent
application WO 2007 107596 A1; [0027] vanillyl lignans,
particularly matairesinol, lariciresinol, hydroxymatairesinol which
are disclosed, for example, in the international patent application
WO 2012 146,584, and particularly also [0028] enterolactone.
[0029] Bitter-Masking Food Constituents Inducing Secretion of
Gastric Acid (Component b)
[0030] These substances comprising the component (b) are selected
from the group formed by: [0031] xanthines, particularly caffeine,
theobromine and theophylline; [0032] fruit acids, particularly
tartaric acid, racemic acid, malic acid and succinic acid; [0033]
phenolic glycosides, particularly salicin and arbutin; [0034]
flavanone glycosides, particularly neohesperedin, eriocitrin,
neoeriocitrin, narirutin and naringin; [0035] dihydrochalcone
glycosides, particularly phloridzin, trilobatin; [0036]
hydrolyzable tannins, particularly gallic or ellagic esters or
carbohydrates, e.g. pentagalloyl glucose; [0037] non-hydrolyzable
tannins, particularly galloylated catechins or epicatechins and
oligomers thereof, e.g. proanthyocyanidins or procyanidins,
thearubigins, flavones and glycosides thereof, particularly
quercetin, quercitrin, rutin, taxifolin, myricetin, myrictrin;
[0038] terpenoid bitter substances, particualrly limonin, nomilin,
lupolone and humolone; [0039] triterpene glycosides, particualrly
steviosides, rubusoside, stevioside, rebaudioside A, rebaudioside
C, glycyrrhizin (glycyrrhizic acid) and glycyrrhetic acid; and
[0040] iridoid bitter substances such as oleuropein and mixtures
thereof.
Preferred Embodiments
[0041] The highest reduction or complete inhibition of the induced
gastric acid secretion was observed in preparations which comprised
caffeine and/or theobromine as component (b) and to which was added
one of the following substances of component (a), namely
homoeriodictyol (1), eriodictyol (2), matairesinol (3),
lariciresinol (4) and/or enterolactone (5). Corresponding
preparations are therefore particularly preferred.
[0042] The bitter-masking substances which can form component (a)
are selected from the group formed by:
[0043] The preferred compounds are shown again in the following
Figure:
##STR00001##
[0044] In principle, the therapeutic preparations may comprise both
the substances which form component (a) and (b), in each case in
amounts of approximately 0.1 mg/kg (corresponding to 0.1 ppm) to
approximately 1% by weight. However, the substances are preferably
present in each case in amounts of approximately 0.5 to
approximately 1000 mg/kg (corresponding to approximately 0.5 to
approximately 1000 ppm), preferably of approximately 1 to
approximately 500 mg/kg, more preferably of approximately 3 to
approximately 300 mg/kg, particularly preferably of approximately 5
to approximately 200 mg/kg and most preferably of approximately 10
to 100 mg/kg.
[0045] The preparations according to the invention may comprise the
substances which form components (a) and (b) in the weight ratio of
approximately 1:100 to approximately 1:1, preferably 1:90 to
approximately 1:2 and particularly of approximately 1:5 to
approximately 1:10.
[0046] Oral Preparations
[0047] The present invention further relates to a preparation for
oral administration, specifically providing nutrition or enjoyment,
comprising as already elucidated in detail above
(a) bitter-masking acting substances and (b) substances which
induce the secretion of gastric acid.
[0048] The preparations providing nutrition or enjoyment (i.e.
suitable for consumption) are, in the context of this invention,
e.g. bakery products (e.g. bread, dry biscuits, cakes, other baked
goods), confectionery (e.g. chocolates, chocolate bar products,
other bar products, fruit gums, hard and soft caramels, chewing
gum), alcoholic or non-alcoholic drinks (e.g. coffee, tea, wine,
wine-based drinks, beer, beverages containing beer, liqueurs,
spirits, brandies, fruit-containing carbonated beverages, isotonic
drinks, soft drinks, nectars, fruit and vegetable juices, fruit or
vegetable juice preparations), instant drinks (e.g. instant cocoa
drinks, instant tea drinks, instant coffee drinks), meat products
(e.g. ham, fresh or cured sausage, spiced or marinated fresh or
cured meat), eggs or egg products (dried egg, egg white, egg yolk),
cereal products (e.g. breakfast cereals, muesli bars, precooked
instant rice products), dairy products (e.g. milk drinks, ice milk,
yogurt, kefir, cream cheese, soft cheese, hard cheese, dried milk
powder, whey, butter, buttermilk, partially or fully hydrolyzed
milk protein-containing products), products made from soy protein
or other soybean fractions (e.g. soy milk and products manufactured
therefrom, preparations containing soy lecithin, fermented products
such as tofu or tempeh or products manufactured therefrom, soy
sauce), fruit preparations (e.g. jams, fruit ice cream, fruit
sauces, fruit fillings), vegetable preparations (e.g. ketchup,
sauces, dried vegetables, frozen vegetables, precooked vegetables,
pickled vegetables, cooked vegetables), snack foods (e.g. baked or
fried potato chips or reconstituted potato products, bread dough
products, extrudates based on maize or peanut), products based on
fat and oil or emulsions of the same (e.g. mayonnaise, remoulade,
dressings, seasoning preparations), other ready meals and soups
(e.g. dried soups, instant soups, precooked soups), spices, spice
mixtures and especially seasonings which are used in the snack food
sector.
[0049] The oral preparations in the context of the invention may
also serve as semi-finished goods for the preparation of further
orally consumable preparations. The preparations in the context of
the invention may also be in the form of capsules, tablets (oral
and/or gastric disintegrating tablets), sugar-coated pills,
granules, pellets, mixtures of solids, dispersions in liquid
phases, as emulsions, as powders, as solutions, as pastes or as
other swallowable or chewable preparations as food supplements.
[0050] Preference is given here to incorporating one or more of the
substances of group (a) in an orally consumable preparation,
particularly in the form of preparations which can be swallowed
(e.g. drinks, dispersions, emulsions, pastes, capsules which can
dissolve in the mouth or stomach, tablets, compressed products,
soft caramels, sugar-coated pills, granules, pellets, fruit gums)
or in preparations not intended to be swallowed (e.g. chewing gum,
hard caramels) releasing sufficient concentrations of substances of
group (a) in an effective concentration.
[0051] The substances of group (a) to be used in accordance with
the invention in preparations suitable for consumption in
accordance with the invention, particularly in the preferred
concentrations to be used in accordance with the invention, have no
appreciable taste of their own and in particular they have no
unpleasant or objectionable flavors at the (preferred or
particularly preferred) concentrations employed. The substances of
group (a) are each capable alone and/or in mixtures to reduce or to
completely block the bitterness of one or more of the substances of
group (b).
[0052] The substance(s) of group (a) and also preparations
according to the invention comprising the substance(s) of group
(a), can be fed orally in time sequence before, during or after
oral administration of the substance(s) of group (b) or
preparations comprising the substance(s) of group (b).
[0053] Particularly advantageous here is the known fact that the
substances of group (a) are regularly capable of partially or
completely reducing the bitter (unpleasant) taste of the substances
of group (b) and therefore can significantly increase the
acceptability of the preparations. This is particularly the case if
the substances of group (b) are applied at the same time as the
substances of group (a).
[0054] In the case of subsequent oral administration of the
substance(s) of group (a), preparation forms are preferred which
comprise the substance(s) of group (a) in a form which causes
delayed release.
[0055] In an alternative to the simultaneous oral administration,
preparations may also be suitable which have one or more of the
unpleasant tasting and gastric acid-stimulating substances of group
(b) in an effective amount with respect to gastric acid stimulation
and simultaneously comprise one or more of the substances of group
(a) in an effective amount with respective to gastric acid
stimulation.
[0056] In principle, the oral preparations may comprise--be they
finished or semi-finished products--both the substances which form
component (a) and (b), in each case in amounts of approximately 0.1
mg/kg (corresponding to 0.1 ppm) to approximately 1% by weight.
[0057] However, the substances are preferably present in each case
in amounts of approximately 0.5 to approximately 1000 mg/kg
(corresponding to approximately 0.5 to approximately 1000 ppm),
preferably of approximately 1 to approximately 500 mg/kg, more
preferably of approximately 3 to approximately 300 mg/kg,
particularly preferably of approximately 5 to approximately 200
mg/kg and most preferably of approximately 10 to 100 mg/kg.
[0058] The preparations according to the invention may comprise the
substances which form components (a) and (b) in the weight ratio of
approximately 1:100 to approximately 1:1, preferably 1:90 to
approximately 1:2 and particularly of approximately 1:5 to
approximately 1:10.
INDUSTRIAL APPLICABILITY
[0059] A preferred method for determining the desired effect of
substances of group (a) on decreasing the effect of substances of
group (b) consists of measuring the intracellular pH of HGT-1 cell
cultures (human gastric tumor cell line) after treatment with the
test substances as described in Liszt, K. I.; Walker, J.; Somoza,
V. J. Agric Food Chem. 2012, 60, (28), 7022-7030. To date, only the
somatostatin receptor (SSTR2) on the cell surface of gastric
acid-secreting cells has been described as a target for reducing
the secretion; this receptor has also been described in HGT-1 To
date, these cells have only ever been brought into contact with
potential SSTR2 regulators; the co-administration of bitter
agonists and potential antagonists has not been described to
date.
[0060] Two further aspects of the present invention relate firstly
to a non-therapeutic method for reducing and/or inhibiting
secretion of gastric acid induced by food constituents, in which an
effective amount of a bitter-masking acting substance is orally
administered to a human or animal, and secondly the use of
bitter-masking acting substances for reducing and/or inhibiting the
secretion of gastric acid induced by food constituents. With regard
to the nature of the substances and the amounts used thereof,
reference is made to the embodiments above which are fully
incorporated here.
EXAMPLES
[0061] Cell Model
[0062] Human parietal cells, also known as human gastric tumor cell
line (HGT-1), are used as cell model. These were provided by Dr. C.
Laboisse (Laboratory of Pathological Anatomy, Nantes, France). The
cells were cultured under standard conditions at 37.degree. C., 5%
CO.sub.2 in DMEM (Dulbecco's Modified Eagle's Medium) with 4 g/l
glucose, 10% FBS, 2% L-glutamine and 1% penicillin/streptomycin.
For the measurement of the intracellular proton concentration, the
cells are treated with trypsin/EDTA, the viability determined by
means of trypan blue staining and 100 000 cells/well seeded in
black 96-well plates.
Example 1
[0063] Determination of the Intracellular pH of HGT-1 Cell Cultures
in the Presence of the Substances of Group (a) and Substances of
Group (b)
[0064] To measure the intracellular pH, the dye
1,5-carboxyseminaphthorhodafluoro acetoxymethyl ester (SNARF-1-AM)
is used. The cells in the 96-well plates are washed once with
Krebs-Hepes buffer (KRHP) and incubated at 37.degree. C. and 5%
CO.sub.2 for 30 min with the dye dissolved in KRHP at a
concentration of 3 .mu.M. The cells are then washed twice with KRHP
and substances of group (b), for example 3 mM caffeine or 0.3 mM
theobromine, alone or in combination with substances of group (a),
for example, homoeriodictyol (HED) or eriodictyol or matairesinol,
lariciresinol or enterolactone, are applied in volumes of 100 .mu.l
at various concentrations in phenol red-free DMEM; as additional
control experiments, the abovementioned substances of group (a) are
tested alone. Homoeriodictyol is dissolved in double-distilled
water while eriodictyol, matairesinol, lariciresinol or
enterolactone are dissolved in ethanol (EtOH). The final
concentration of solvent which is added to the cells is at most 1%.
The fluorescent dye is excited at a wavelength of 488 nm and the
emission measured at 580 nm and 640 nm. The ratio of the
fluorescence values at 580 nm to 640 nm is compared to a
calibration curve from which the pH can be determined. For the
calibration curve, the cells are treated with a potassium phosphate
buffer of different pH values from pH 7.2-8.2 and 2 .mu.M
nigericin. Nigericin equilibrates the intracellular and
extracellular pH such that the intracellular pH may be defined. The
intracellular W concentration is determined from the intracellular
pH. The intracellular proton index (IPX) is calculated by log 2
transformation of the ratio of treated cells and untreated cells
(control). The results given here are stated as percentage changes
compared to untreated control cells (cf. Malte Rubach, R. L.,
Elisabeth Seebach, Mark M. Somoza, Thomas Hofmann; Somoza, a. V.,
Mol Nutr Food Res. in press, 2011; Rubach, M.; Lang, R.; Hofmann,
T.; Somoza, V., Ann N Y Acad Sci 2008, 1126, 310-4; Rubach, M.;
Lang, R.; Skupin, C.; Hofmann, T.; Somoza, V., J Agric Food Chem
2010, 58, 4153-61; Weiss, C.; Rubach, M.; Lang, R.; Seebach, E.;
Blumberg, S.; Frank, O.; Hofmann, T.; Somoza, V., J Agric Food Chem
2010, 58, 1976-85; Liszt, K. I.; Walker, J.; Somoza, V., J Agric
Food Chem 2012; Walker, J.; Hell, J.; Liszt, K. I.; Dresel, M.;
Pignitter, M.; Hofmann, T.; Somoza, V., J Agric Food Chem 2012, 60,
1405-12). The number of replicates specified refers to the
technical replicates (tr) or the number or total replicates (n),
which results from the number of technical replicates multiplied by
the number of biological replicates.
[0065] Given in Table 1A below is the percentage increase of the
proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by DMEM or DMEM with 1% ETOH or 1 mM
histamine or 3 mM caffeine with and without 1% EtOH. The data are
presented as mean values and mean standard deviations, n=21-37,
tr=6. Statisitics: one-way Anova with post-hoc test according to
Dunn. Significant differences (p<0.05) are indicated by
letters.
TABLE-US-00001 TABLE 1A Comparative Example, increase in secretion
due to substances of group (b). Test substance T/C [%] SEM Control
(DMEM) 0.150 1.00 a EtOH 1% 14.44 2.61 b 1 mM Histamine 27.42 3.17
b 3 mM Caffeine 48.87 3.64 d 3 mM Caffeine + 1% EtOH 44.92 3.51
d
[0066] Given in Table 1B below is the percentage increase of the
proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by DMEM or DMEM with 1% ETOH or 1 mM
histamine or 0.3 mM theobromine with and without 1% EtOH. The data
are presented as mean values and mean standard deviations, n=21-37,
tr=6. Statisitics: one-way Anova with post-hoc test according to
Dunn. Significant differences (p<0.05) are indicated by
letters.
TABLE-US-00002 TABLE 1B Comparative Example, increase in secretion
due to substances of group (b). Test substance T/C [%] SEM DMEM
(control) 0.15 1.00 a 1% EtOH 14.44 2.61 b 1 mM Histamine 27.42
3.17 b, c 0.3 mM Theobromine 17.51 2.65 b 0.3 mM Theobromine + 1%
EtOH 32.11 2.49 c, d
[0067] Given in Table 2A below is the percentage increase of the
proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by DMEM (control) or homoeriodictyol
(HED) at various concentrations. The data are presented as mean
values and mean standard deviations, n=4, tr=6. Statisitics:
one-way Anova with post-hoc test according to Dunn. Significant
differences (p<0.05) are indicated by letters.
TABLE-US-00003 TABLE 2A Influence on the secretion by substances of
group (a). Test substance T/C [%] SEM DMEM (control) -1.43 2.01 a
0.003 mM HED -8.04 3.88 a 0.03 mM HED 12.70 4.02 a 0.3 mM HED -3.66
5.24 a
[0068] Given in Table 2B below is the percentage increase of the
proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by DMEM (control) or eriodictyol at
various concentrations. The data are presented as mean values and
mean standard deviations, n=3-7, tr=6. Statisitics: one-way Anova
with post-hoc test according to Dunn. Significant differences
(p<0.05) are indicated by letters.
TABLE-US-00004 TABLE 2B Influence on the secretion by substances of
group (a). Test substance T/C [%] SEM Control (DMEM) 0.98 2.30 a
0.03 mM Eriodictyol -10.77 11.16 a 0.3 mM Eriodictyol -11.67 7.25
a
[0069] Given in Table 2C below is the percentage increase of the
proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by 3 mM caffeine or 3 mM caffeine in
combination with matairesinol at various concentrations. The data
are presented as mean values and mean standard deviations, n=8,
tr=6. Statisitics: one-way Anova with post-hoc test according to
Dunn. Significant differences (p<0.05) are indicated by
letters.
TABLE-US-00005 TABLE 2C Influence on the secretion by substances of
group (a). Test substance T/C [%] SEM DMEM (control) -0.63 1.70 a
0.03 mM Matairesinol -9.40 3.73 a 0.3 mM Matairesinol -31.69 2.95
b
[0070] Given in Table 2D below is the percentage increase of the
proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by DMEM (control) or lariciresinol at
various concentrations. The data are presented as mean values and
mean standard deviations, n=8, tr=6. Statisitics: one-way Anova
with post-hoc test according to Dunn. Significant differences
(p<0.05) are indicated by letters.
TABLE-US-00006 TABLE 2D Influence on the secretion by substances of
group (a). Test substance T/C [%] SEM Control (DMEM) 0.15 1.93 a
0.03 mM Lariciresinol 6.40 4.46 a 0.3 mM Lariciresinol -2.04 2.78
a
[0071] Given in Table 2E below and also FIG. 1 is the percentage
increase of the proton secretion in HGT-1 cells compared to
untreated controls after 10 minutes stimulation by DMEM (control)
or enterolactone at various concentrations. The data are presented
as mean values and mean standard deviations, n=8, tr=6.
Statisitics: one-way Anova with post-hoc test according to Dunn.
Significant differences (p<0.05) are indicated by letters.
TABLE-US-00007 TABLE 2E Influence on the secretion by substances of
group (a). Test substance T/C [%] SEM Control (DMEM) -2.28 1.80 a
0.03 mM Enterolactone -2.76 3.75 a 0.3 mM Enterolactone -27.31 4.43
a
[0072] Given in Table 3A below is the percentage increase of the
proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by 3 mM caffeine or 3 mM caffeine in
combination with homoeriodictyol (HED) at various concentrations.
The data are presented as mean values and mean standard deviations,
n=4, tr=6. Statisitics: one-way Anova with post-hoc test according
to Dunn. Significant differences (p<0.05) are indicated by
letters.
TABLE-US-00008 TABLE 3A Decrease in the secretion, triggered by
substances of group (b), by substances of group (a) Test substance
T/C [%] SEM 3 mM Caffeine 54.79 5.61 a 3 mM Caffeine + 0.03 mM HED
37.99 6.70 a 3 mM Caffeine + 0.3 mM HED 20.18 5.99 b
[0073] Given in Table 3B below is the percentage increase of the
proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by 3 mM caffeine or 3 mM caffeine in
combination with eriodictyol at various concentrations. The data
are presented as mean values and mean standard deviations, n=4,
tr=6. Statisitics: one-way Anova with post-hoc test according to
Dunn. Significant differences (p<0.05) are indicated by
letters.
TABLE-US-00009 TABLE 3B Decrease in the secretion, triggered by
substances of group (b), by substances of group (a) Test substance
T/C [%] SEM 3 mM Caffeine 54.05 4.37 a 3 mM Caffeine + 0.03 mM
Eriodictyol 29.69 8.82 a 3 mM Caffeine + 0.3 mM Eriodictyol -33.96
7.55 b
[0074] Given in Table 3C below is the percentage increase of the
proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by 3 mM caffeine or 3 mM caffeine in
combination with matairesinol at various concentrations. The data
are presented as mean values and mean standard deviations, n=4,
tr=6. Statisitics: one-way Anova with post-hoc test according to
Dunn. Significant differences (p<0.05) are indicated by
letters.
TABLE-US-00010 TABLE 3C Decrease in the secretion, triggered by
substances of group (b), by substances of group (a) Test substance
T/C [%] SEM 3 mM Caffeine 38.75 2.63 a 3 mM Caffeine + 0.03 mM
Matairesinol 14.39 3.55 b 3 mM Caffeine + 0.3 mM Matairesinol
-20.43 4.82 c
[0075] Reproduced in Table 3D below is the percentage increase of
the proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by 3 mM caffeine or 3 mM caffeine in
combination with lariciresinol at various concentrations. The data
are presented as mean values and mean standard deviations, n=5,
tr=6. Statisitics: one-way Anova with post-hoc test according to
Dunn. Significant differences (p<0.05) are indicated by
letters.
TABLE-US-00011 TABLE 3D Decrease in the secretion, triggered by
substances of group (b), by substances of group (a) Test substance
T/C [%] SEM 3 mM Caffeine 52.41 4.16 a 3 mM Caffeine + 0.03 mM
Lariciresinol 46.40 3.98 a 3 mM Caffeine + 0.3 mM Lariciresinol
21.57 3.78 b
[0076] Reproduced in Table 3E below and also FIG. 2 is the
percentage increase of the proton secretion in HGT-1 cells compared
to untreated controls after 10 minutes stimulation by 3 mM caffeine
or 3 mM caffeine in combination with enterolactone at various
concentrations. The data are presented as mean values and mean
standard deviations, n=5, tr=6. Statisitics: one-way Anova with
post-hoc test according to Dunn. Significant differences
(p<0.05) are indicated by letters.
TABLE-US-00012 TABLE 3E Decrease in the secretion, triggered by
substances of group (b), by substances of group (a) Test substance
T/C [%] SEM 3 mM Caffeine 34.12 2.86 a 3 mM Caffeine + 0.03 mM
Enterolactone 23.41 2.56 a 3 mM Caffeine + 0.3 mM Enterolactone
-18.25 3.39 b
[0077] Reproduced in Table 3F below is the percentage increase of
the proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by 0.3 mM theobromine or 0.3 mM
theobromine in combination with homoeriodictyol (HED) at various
concentrations. The data are presented as mean values and mean
standard deviations, n=3, tr=6. Statisitics: one-way Anova with
post-hoc test according to Dunn. Significant differences
(p<0.05) are indicated by letters.
TABLE-US-00013 TABLE 3F Decrease in the secretion, triggered by
substances of group (b), by substances of group (a) Test substance
T/C [%] SEM 0.3 mM Theobromine 20.92 3.37 a 0.3 mM Theobromine +
0.003 mM HED 21.56 2.33 a 0.3 mM Theobromine + 0.03 mM HED 13.79
3.50 a 0.3 mM Theobromine + 0.3 mM HED -16.92 3.27 b
[0078] Reproduced in Table 3G below is the proton secretion in
HGT-1 cells after 10 minutes stimulation by 0.3 mM theobromine or
0.3 mM theobromine in combination with eriodictyol at various
concentrations. The data are presented as mean values and mean
standard deviations, n=3, tr=6. Statisitics: one-way Anova with
post-hoc test according to Dunn. Significant differences
(p<0.05) are indicated by letters.
TABLE-US-00014 TABLE 3G Decrease in the secretion, triggered by
substances of group (b), by substances of group (a) Test substance
T/C [%] SEM 0.3 mM Theobromine 47.64 5.28 a 0.3 mM Theobromine +
0.03 mM Eriodictyol 23.95 4.17 b 0.3 mM Theobromine + 0.3 mM
Eriodictyol -42.86 9.73 c
[0079] Reproduced in Table 3H below is the proton secretion in
HGT-1 cells after 10 minutes stimulation by 0.3 mM theobromine or
0.3 mM theobromine in combination with matairesinol at various
concentrations. The data are presented as mean values and mean
standard deviations, n=3, tr=6. Statisitics: one-way Anova with
post-hoc test according to Dunn. Significant differences
(p<0.05) are indicated by letters.
TABLE-US-00015 TABLE 3H Decrease in the secretion, triggered by
substances of group (b), by substances of group (a) Test substance
T/C [%] SEM 0.3 mM Theobromine 34.12 2.86 a 0.3 mM Theobromine +
0.03 mM Matairesinol 23.41 2.56 b 0.3 mM Theobromine + 0.3 mM
Matairesinol -18.25 3.39 c
[0080] Reproduced in Table 3I below is the percentage increase of
the proton secretion in HGT-1 cells compared to untreated controls
after 10 minutes stimulation by 0.3 mM theobromine or 0.3 mM
theobromine in combination with lariciresinol at various
concentrations. The data are presented as mean values and mean
standard deviations, n=3, tr=6. Statisitics: one-way Anova with
post-hoc test according to Dunn. Significant differences
(p<0.05) are indicated by letters.
TABLE-US-00016 TABLE 3I Decrease in the secretion, triggered by
substances of group (b), by substances of group (a) Test substance
T/C [%] SEM 0.3 mM Theobromine 30.81 3.44 a 0.3 mM Theobromine +
0.03 mM Lariciresinol 31.73 5.80 a 0.3 mM Theobromine + 0.3 mM
Lariciresinol 17.55 3.67 a (t-test p < 0.05)
[0081] As follows from Tables 1A and 1B, the proton secretion in
the HGT-1 cells is stimulated by substances of group (b), in this
case caffeine or theobromine, which effectively triggers acid
secretion into the extracellular space. The substances of group (a)
alone lead either to no significant effect (Tables 2A, 2B and 2D)
or even to a significant decrease of the constitutive acid
secretion (Table 2C). The stimulation of proton secretion by 3 mM
caffeine (Tables 3A-3E) or 0.3 mM theobromine (Tables 3F-3I) could
be reduced or completely inhibited by additional administration of
the substances of group (a), particularly homoeriodictyol,
eriodictyol, matairesinol, lariciresinol or enterolactone at a
concentration of 0.3 mM.
Application Example 1
[0082] Spray-Dried Preparation as Semi-Finished Goods for Preparing
Finished Goods.
[0083] Drinking water is placed in a container and maltodextrin and
gum arabic dissolved therein. The substances of group (a) are then
emulsified in the carrier solution using a Turrax. The temperature
of the spray solution should not exceed 30'C. The mixture is then
spray-dried (nominal temperature at the start: 185-195.degree. C.,
nominal temperature at the end: 70-75.degree. C.). The spray-dried
semi-finished goods comprise ca. 18-22% of the compounds of group
(a).
TABLE-US-00017 TABLE 4 Semi-finished goods composition - amounts in
% by weight. Preparation A B C D E Drinking water 60.8 60.8 60.8
60.8 60.8 Maltodextrin from wheat 24.3 24.3 24.3 24.3 24.3 Gum
arabic 6.1 6.1 6.1 6.1 6.1 Matairesinol 8.8 -- 4.4 -- --
Homoeriodictyol -- 8.8 4.4 -- -- Eriodictyol -- -- -- 8.8 --
Enterolactone -- -- -- -- 8.8
Application Example 2
[0084] Drinkable Iced Tea Preparation
[0085] The compounds of group (a) were each pre-dissolved at 10% in
ethanol. Black tea extract was dissolved in water and stirred in a
glass beaker together with sugar, a flavoring preparation (peach
flavor) and the ethanolic solutions of the compounds of group
(a).
TABLE-US-00018 TABLE 5 Iced tea composition - amounts in % by
weight. Preparation A B Black tea extract 1.4 1.4 Water 89.5 89.5
Flavoring preparation (peach type) 0.65 0.65 Sugar 7.0 7.0 Citric
acid (crystalline) 1.2 1.2 Ascorbic acid 0.2 0.2 Matairesinol in
ethanol (10%) 0.05 -- Homoeriodictyol in ethanol (10%) -- 0.05
Application Example 3
[0086] Gelatine Capsules without Substances of Group (b) for Direct
Consumption after Application of Substances of Group (b).
[0087] The gelatine capsules suitable for direct consumption were
prepared according to WO 2004/050069 and had a diameter of 5 mm and
the ratio by weight of core material to shell material was 90:10.
The capsules opened in the mouth within less than 10 seconds and
dissolved completely in less than 50 seconds.
TABLE-US-00019 TABLE 6 Gelatine capsule composition - amounts in %
by weight. Ingredients A B C Gelatine shell: Glycerol 2.014 2.014
2.014 Gelatine 240 Bloom 7.91 7.91 7.91 Sucralose 0.065 0.065 0.065
Allura red 0.006 -- 0.011 Brilliant blue 0.005 0.011 -- Core
composition Vegetable oil triglyceride (coconut oil 79.49 68.55
58.55 fraction) Orange flavoring comprising 1% by weight 10.0 -- --
homoeriodictyol based on the total weight of the flavoring.
Peppermint flavoring comprising 1% by -- 20.0 -- weight
matairesinol based on the total weight of the flavoring. Cherry
flavoring comprising 1% by weight -- -- 28.65 enterolactone based
on the total weight of the flavoring. Rebaudioside A 98% 0.05 0.05
-- 2-Hydroxypropylmenthylcarbonate 0.33 0.20 --
2-Hydroxyethylmenthylcarbonate -- 0.20 1.00 (1R,3R,4S)
Menthyl-3-carboxylic acid -- 0.55 -- N-ethylamide (WS-3)
(-)-Menthyl lactate (Frescolat ML) -- 0.30 -- Vanillin 0.07 --
0.10
Application Example 4
[0088] Chewing Gum without Substances of Group (b)
[0089] Parts A to D are mixed and kneaded intensively. The crude
mass may be processed, for example in the form of thin strips, to
form ready-to-consume chewing gum.
TABLE-US-00020 TABLE 7 Chewing gum composition - amounts in % by
weight. Part Preparation A B A Chewing gum base, "Jagum T" company
30.00 30.00 B Sorbitol, powdered 39.00 39.00 Isomalt .RTM.
(Palatinit GmbH) 9.50 9.50 Xylitol 2.00 2.00 Mannitol 3.00 3.00
Aspartame .RTM. 0.10 0.10 Acesulfame .RTM. K 0.10 0.10 Emulgum
.RTM. (Colloides Naturels, Inc.) 0.30 0.30 C Sorbitol, 70% 14.00
14.00 Glycerol 1.00 1.00 D Mint flavoring comprising 1%
matairesinol based 1.00 -- on the total weight of the flavoring.
Tutti frutti flavoring comprising 1% -- 2.00 homoeriodictyol based
on the total weight of the flavoring.
Application Example 5
[0090] Use in a Soluble Cappuccino Drink
[0091] The specified raw materials are mixed. In each case 12.5 g
of the prepared instant cappuccino powder are dissolved in 150 ml
of hot water.
TABLE-US-00021 TABLE 8 Cappuccino drink composition - amounts in %
by weight. Preparation A B Coffee extract, spray-dried 14.0 16.0
Sugar 28.3 25.3 Fat powder 18.2 18.2 Coffee whitener, foaming 30.0
28.0 Hydrocolloids/emulsifiers 1.8 1.8 Lactose 4.7 4.7
Semi-finished product A from application example 1 3.0 --
Semi-finished product B from application example 1 -- 6.0
Application Example 6
[0092] Tea Drink
[0093] The tea and the semi-finished goods are mixed and packed in
tea bags made of filter paper. To use, one tea bag is infused in
100-250 ml of boiling water and left to draw for 2-5 min.
TABLE-US-00022 TABLE 9 Tea drink composition - amounts in % by
weight. Preparation A B C Black tea, Ceylon, leaf product 94.00 --
-- Green tea, China, leaf product -- 91.90 -- Mate tea, Peru, leaf
product -- -- 95.00 Semi-finished product A from application
example 2 6.0 -- -- Semi-finished product B from application
example 2 -- 8.0 -- Semi-finished product C from application
example 2 -- -- 5.0 Flavor (lemon type) -- 0.1 --
Application Example 7
[0094] Use in a Bitter Chocolate
[0095] A bitter chocolate was prepared from the following raw
materials and subsequently poured into rectangular forms.
TABLE-US-00023 TABLE 10 Chocolate composition - amounts in % by
weight. Preparation A B Cocoa mass 55.55 55.55 Cocoa butter 11.70
11.70 Sugar 29.50 29.50 Skimmed milk 3.00 3.00 Lecithin 0.2 0.2
Vanillin 0.035 0.035 10% Matairesinol in ethanol -- 0.05 10%
Homoeriodictyol in ethanol 0.05 --
Application Example 8
[0096] Sugar-Free Hard Caramel
[0097] Palatinit was mixed with water and the mixture melted at
165.degree. C. and subsequently cooled to 115.degree. C. The
remaining constituents were added and, after mixing, poured into
molds, removed from the molds after solidification and then
individually packaged.
TABLE-US-00024 TABLE 11 Caramel composition - amounts in % by
weight. Preparation A B C D Palatinat, type M 75.00 74.00 75.50
75.00 Citric acid -- 1.0 0.5 -- Water 24.88 24.842 23.88 24.844
Yellow coloring -- 0.01 -- -- Red coloring -- -- 0.01 -- Blue
coloring 0.01 -- -- 0.01 Peppermint flavoring 0.1 -- -- 0.1 Lemon
flavoring -- 0.1 -- -- Red fruit flavoring -- -- 0.1 --
Rebaudioside A 98% -- 0.040 -- 0.040 Hesperetin -- 0.001 -- 0.001
Phloretin -- 0.002 -- -- Homoeriodictyol 0.010 0.005 -- 0.005
Matairesinol -- 0.005 -- -- Eriodictyol -- -- 0.010 --
Enterolactone -- -- -- 0.005
* * * * *