U.S. patent application number 13/839260 was filed with the patent office on 2013-12-19 for long lasting absorption of flavonoids.
The applicant listed for this patent is Nestec S.A.. Invention is credited to Ivana Jankovic, Maarit Rein, Myriam Richelle, Gary . Williamson.
Application Number | 20130336941 13/839260 |
Document ID | / |
Family ID | 37709496 |
Filed Date | 2013-12-19 |
United States Patent
Application |
20130336941 |
Kind Code |
A1 |
Richelle; Myriam ; et
al. |
December 19, 2013 |
LONG LASTING ABSORPTION OF FLAVONOIDS
Abstract
The present invention relates to methods for a ong-term and
sustained release of flavonoids, in particular rhamnose-containing
flavonoids, and for prolonging the uptake of said flavonoids in the
gastro-intestinal tract. It further relates to compositions
comprising said flavonoid and .alpha.-rhamnosidase. It also
encompasses compositions comprising hesperidin and
hesperetin-7-gluoside.
Inventors: |
Richelle; Myriam; (Savigny,
CH) ; Williamson; Gary .; (Harrogate, GB) ;
Jankovic; Ivana; (Epalinges, CH) ; Rein; Maarit;
(Lutry, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nestec S.A. |
Vevey |
|
CH |
|
|
Family ID: |
37709496 |
Appl. No.: |
13/839260 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12438501 |
Oct 20, 2009 |
8475786 |
|
|
PCT/EP2007/007394 |
Aug 22, 2007 |
|
|
|
13839260 |
|
|
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Current U.S.
Class: |
424/93.42 ;
424/93.4; 424/93.44; 424/93.45; 424/93.46; 424/94.61; 514/27 |
Current CPC
Class: |
A61K 31/7048 20130101;
A61Q 19/00 20130101; C12Y 302/01006 20130101; C12N 9/2402 20130101;
C12R 1/46 20130101; A61P 1/00 20180101; A23L 33/18 20160801; A61P
17/00 20180101; C12Y 302/0104 20130101; C12R 1/25 20130101; A61K
2800/412 20130101; C12R 1/225 20130101; A61K 8/602 20130101; A61P
19/08 20180101; A61K 8/66 20130101; A61K 38/47 20130101; A61P 9/00
20180101; A61K 8/11 20130101 |
Class at
Publication: |
424/93.42 ;
424/93.4; 424/93.44; 424/93.45; 424/93.46; 424/94.61; 514/27 |
International
Class: |
A61K 38/47 20060101
A61K038/47; A23L 1/305 20060101 A23L001/305; A61K 31/7048 20060101
A61K031/7048 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 24, 2006 |
EP |
06017678.1 |
Claims
1: A product comprising: a first composition comprising a
rhamnose-containing flavonoid; and a second composition comprising
an .alpha.-rhamnosidase, and the first and second compositions do
not react with each other unless certain conditions are met.
2: The product according to claim 1, wherein the certain conditions
are selected from the group consisting of pH, temperature, water
activity and combinations thereof.
3: The product according to claim 1, wherein the certain conditions
are gastro-intestinal tract conditions.
4: The product according to claim 1, wherein the
.alpha.-rhamnosidase is only able to cleave the rhamnose-containing
flavonoid in gastro-intestinal tract conditions.
5: The product according to claim 4, wherein the
.alpha.-rhamnosidase is only able to cleave the rhamnose-containing
flavonoid at a temperature provided by the gastro-intestinal tract
conditions.
6: The product according to claim 1, wherein the product has a pH
that prevents the .alpha.-rhamnosidase from cleaving the
rhamnose-containing flavonoid.
7: The product according to claim 1, wherein the
.alpha.-rhamnosidase has been treated with an inhibitor such that
only when gastro-intestinal tract conditions are met is the
.alpha.-rhamnosidase able to cleave the rhamnose-containing
flavonoid.
8: The product according to claim 1, wherein the
.alpha.-rhamnosidase is encapsulated in a micro-organism capable of
producing .alpha.-rhamnosidase, and the product has conditions in
which the micro-organism has a metabolic rate such that the
.alpha.-rhamnosidase is not released by the micro-organism.
9: The product according to claim 8, wherein the product has a
water activity that lowers the metabolic rate of the micro-organism
to prevent the micro-organism from releasing the
.alpha.-rhamnosidase.
10: The product according to claim 9, wherein the micro-organism is
a bacterium.
11: The product according to claim 10, wherein the bacterium is
selected from Lactobacillus, Bifidobacterium, Streptococcus,
Lactococcus, Enterococcus, Bacillus, Staphylococcus, Leuconostoc,
Pediococcus, Oenococcus and combinations thereof.
12: The product according to claim 10, wherein the bacterium is
selected from the group consisting of Lactobacillus crispatus
ATCC33820, Lactobacillus crispatus CNCM I-3654, Lactobacillus
plantarum ATCC8014, Lactobacillus plantarum CNCM I-3653,
Lactobacillus gasseri CNCM I-3795 and mixtures thereof.
13: The product according to claim 8, wherein the micro-organism is
present in an amount of 10.sup.6-10.sup.10 cfu/g.
14: The product according to claim 1, wherein the second
composition is a powder.
15: The product according to claim 1, wherein the
rhamnose-containing flavonoid is hesperidin.
16: The product according to claim 1, wherein the product is a
composition selected from the group consisting of nutritional
composition, a pharmaceutical composition and a cosmetic
composition.
17: The product according to claim 1, wherein the product is
selected from the group consisting of a liquid, a solution, a
spray, a powder, a tablet, a capsule, a yoghurt, a biscuit, milk, a
beverage, chocolate, ice cream, breakfast cereal flakes, a
breakfast cereal bar, a milk powder, a soy-based product, a
non-milk fermented product, a nutritional supplement, a food
supplement, a pet food, an infant formula and combinations
thereof.
18: A product comprising: a first powder comprising a
rhamnose-containing flavonoid; and a second powder comprising an
.alpha.-rhamnosidase, and the first and second powders do not react
with each other unless certain conditions are met.
19: A method of improving aesthetics or comfort of skin, a coat, or
hair of humans or pets, the method comprising the step of orally
administering to a human or a pet in need of same a product
comprising a first composition comprising a rhamnose-containing
flavonoid and a second composition comprising an
.alpha.-rhamnosidase, and the .alpha.-rhamnosidase is only able to
cleave the rhamnose-containing flavonoid in gastro-intestinal tract
conditions.
20: A method of treating damage to the skin, coat and/or hair of
humans or pets, the method comprising the step of orally
administering to a human or a pet in need of same a product
comprising a first composition comprising a rhamnose-containing
flavonoid and a second composition comprising an
.alpha.-rhamnosidase, and the .alpha.-rhamnosidase is only able to
cleave the rhamnose-containing flavonoid in gastro-intestinal tract
conditions.
21: A method for improving skin health, the method comprising the
step of administering separately and simultaneously a first
composition comprising at least one rhamnose-containing flavonoid
and a second composition comprising .alpha.-rhamnosidase.
22: A composition comprising a mix of hesperidin and
hesperetin-7-glucoside.
23: The composition according to claim 22, wherein the hesperidin
and hesperetin-7-glucoside are present in the composition in a
ratio of between 70/30 to 50/50.
Description
PRIORITY CLAIM
[0001] This application is a continuation of U.S. application Ser.
No. 12/438,501, filed Oct. 20, 2009, which is a National Stage of
International Application No. PCT/EP2007/007394, filed on Aug. 22,
2007 which claims priority to European Patent Application No.
06017678.1, filed on Aug. 24, 2006, the entire contents of which
are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to methods for a long-term and
sustained release of flavonoids, in particular rhamnose-containing
flavonoids, and for prolonging the uptake of said flavonoids in the
gastro-intestinal tract. It further relates to compositions
comprising said flavonoid and .alpha.-rhamnosidase. It also
encompasses compositions comprising hesperidin and
hesperetin-7-glucoside.
BACKGROUND
[0003] Flavonoids, or bioflavonoids, are a ubiquitous group of
polyphenolic substances which are present in most plants,
concentrating in seeds, fruit skin or peel, bark, and flowers. A
great number of plant medicines contain flavonoids, which have been
reported by many authors as having antibacterial, antioxidant,
anti-inflammatory, antiallergic, antimutagenic, antiviral,
antineoplastic, anti-thrombotic, and vasodilatory actions.
[0004] Recent developments described in WO 2005/058255 A1 also show
the benefits of flavanone-containing compositions for improving the
skin, hair and coat health of humans or pets.
[0005] In nature, these compounds mainly occur as glycosides. This
conjugation with sugars impacts markedly their kinetics of
absorption. For instance, flavonoid glucosides have a fast and
early absorption while flavonoid rutinosides (rhamnose-glucoside)
have a slow and late absorption.
[0006] For flavonoid rutinosides such as hesperidin,
bioavailability is low and late in mammals due to the lack of the
enzyme .alpha.-rhamnosidase in the small intestine, which would
remove the rhamnose moiety of said flavonoid rutinosides. This
leads to time-consuming progression of flavonoid rutinosides in the
gastro-intestinal tract through to the colon.
[0007] Health foods and drinks containing long-acting flavonoid
glycosides are described in JP 2003-073279 whereby a mixture of
flavonoids having various degrees of glycosylation is included in
the food product such that the absorption occurs throughout the
gastro-intestinal tract.
[0008] JP 2000-078955 also describes a way to improve the
absorption of flavonoids in food or medicine by providing a mixture
of physiologically active flavonoids and derivatives thereof.
[0009] Further, Espin J. C. et al. describe in the Journal of
Agricultural and Food Chemistry, 2004, 52(20), p. 61366142, the
production of bioavailable glucoside flavonoids in fruit juices and
green tea. This is achieved by treating the fruit juices and green
tea with rhamnosidase enzymes from A.spergill us aculeatus.
[0010] There thus still remains a need to provide alternative ways
by which flavonoid compositions have a controlled effect in
mammals.
SUMMARY
[0011] Accordingly, this need is solved by the features of the
independent claims. The dependent claims further develop the
central idea of the invention.
[0012] Thus, in a first aspect of the invention, a composition
comprising at least one rhamnose-containing flavonoid and
.alpha.-rhamnosidase.
[0013] The compositions of the invention are formulated for
cosmetic, nutritional and pharmaceutical use.
[0014] In a second aspect, the invention relates to a bacterium
having .alpha.-rhamnosidase activity, which is selected from
Lactobacillus crispatus, Lactobacillus plantarum, Lactobacillus
gasseri, Lactobacillus acidophilus or Leuconostoc
mesenteroides.
[0015] The present invention relates, in a further aspect, to the
use of .alpha.-rhamnosidase in the preparation of a composition
comprising at least one rhamnose-containing flavonoid for improving
the bioefficacy and/or bioavailability of said flavonoid.
[0016] Under another aspect of the invention, the use of
.alpha.-rhamnosidase and at least one rhamnose-containing flavonoid
in the manufacture of a composition for the improvement of skin
health is provided.
[0017] Also, the cosmetic use of the compositions of the invention
represents another facet of the invention.
[0018] Also relating to the invention is a method for sustaining
and/or improving the bioavailability of rhamnose-containing
flavonoids comprising the step of providing a composition
comprising at least one rhamnose-containing flavonoid and
.alpha.-rhamnosidase, said .alpha.-rhamnosidase being in a state in
which it is essentially not able to cleave the rhamnose moiety of
said flavonoid.
[0019] A method for prolonging the plasma levels of metabolites of
rhamnose-containing flavonoids after ingestion of said flavonoid
comprising the step of orally providing a composition comprising at
least one rhamnose-containing flavonoid and .alpha.-rhamnosidase,
said .alpha.-rhamnosidase being in a state in which it is
essentially not able to cleave the rhamnose moiety of said
flavonoid, is a further aspect of the present invention.
[0020] A method for improving skin health comprising the step of
orally administering a composition comprising at least one
rhamnose-containing flavonoid and .alpha.-rhamnosidase, or orally
administering separately and simultaneously a composition
comprising at least one rhamnose-containing flavonoid and
.alpha.-rhamnosidase, said .alpha.-rhamnosidase being in a state in
which it is essentially not able to cleave the rhamnose moiety of
said flavanoid, also falls under an aspect of the present
invention.
[0021] Finally, the present invention encompasses compositions
which comprise a mix of hesperidin and hesperitin-7-glucoside,
preferably in a ratio of between 70/30 to 50/50.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The present invention is further described hereinafter with
reference to some of its embodiments shown in the accompanying
drawings in which:
[0023] FIG. 1a depicts the molecular structure of hesperidin,
[0024] FIG. 1b depicts the molecular structure of
hesperetin-7-glucoside,
[0025] FIG. 1c depicts the molecular structure of hesperetin,
and
[0026] FIG. 2 is a graph comparing the plasma hesperetin levels
depending onwhether hesperetin-7-glucoside alone is ingested,
whether hesperidin alone is ingested or whether a composition
according to the present invention is consumed.
[0027] FIG. 3 shows the disappearance of hesperidin in vitro in
conditions close to those of the small intestine (pH 6) in the
presence of pure .alpha.-rhamnosidase (CTRL+), in the presence of
entire cells of Lactobacillus gasseri CNCM I3795 (907 EC), in the
presence of broken cells of Lactobacillus gasseri CNCM I-3795 (907
CE) or in the absence of any bacteria/enzyme (CTRL-).cndot.
[0028] FIG. 4 shows the formation of hesperetin-7-glucoside at pH 6
(calculated from the starting point amount of hesperidin (100%)),
wherein CTRL+, CTRL-, 907 CE and 907 EC have the same meaning as in
FIG. 3.
[0029] FIG. 5 shows the formation of hesperetin at pH 6 when
treating hesperidin with entire or broken cells of Lactobacillus
gasseri (CNCM 1-3795)
[0030] FIG. 6 shows the disappearance of hesperidin in vitro in
conditions close to those of the stomach after digestion of a meal
(pH 4) in the presence of pure .alpha.-rhamnosidase (CTRL+), in the
presence of entire cells of Lactobacillus gasseri CNCM 1-3795 (907
EC), in the presence of broken cells of Lactobacillus gasseri CNCM
1-3795 (907 CE) or in the absence of any bacteria/enzyme
(CTRL-).
[0031] FIG. 7 shows the formation of hesperetin-7-glucoside at pH 4
(calculated from the starting point amount of hesperidin (100%)),
wherein CTRL+, CTRL-, 907 CE and 907 EC have the same meaning as in
FIG. 6.
[0032] FIG. 8 illustrates the result of TIMI experiment. Hydrolysis
of hesperidin into hesperetin-7-glucoside monitored up to 3 hours
in stomach and small intestine when using bacterial strains of
Lactobacillus gasseri (CNCM 1-3795). The figure shows that
bacterial enzymes are poorly active in the stomach, only in the
small intestine and that the amount of hydrolysed hesperidin
decreases as the compounds moves down the GI-tract.
[0033] FIG. 9 illustrates the degree of hydrolysis of hesperidin
into hesperetin-7-glucoside when using pure .alpha.-rhamnosidase
enzyme. This graph shows that pure enzyme is active in stomach but
less active in the GI-tract.
DETAILED DESCRIPTION
[0034] The present invention relates to ways in which to control
the absorption of rhamnose-containing flavonoids in mammals such
that, after ingestion of said flavonoid, the absorption occurs
equally at the upper and lower gastro-intestinal tract or
throughout the length of the gastro-intestinal tract.
[0035] Thus, the basic principle underlying the present invention,
in order to achieve a long-lasting and regulated effect, is a way
to provide, in a regulated fashion, a flavonoid which is, at least
partially, in an absorbable form throughout its passage in the
gastro-intestinal tract of a mammal. Typically, the flavonoid is at
least partially hydrolysed in the upper gastro-intestinal tract and
also in the colon.
[0036] According to the invention, the presence of
.alpha.-rhamnosidase at an early stage of digestion (stomach, small
intestine) allows, to a certain extent, partial cleavage of
rhamnose-containing flavonoids (cf. FIGS. 3-9). This results in the
flavonoids being more absorbable, earlier on in the digestion
process. Moreover, enzymes naturally present in the colon further
the cleavage process such that a sustained absorption of flavonoids
throughout their passage in the gastro-intestinal tract ensues.
[0037] Accordingly, the present invention proposes compositions
comprising at least one rhamnose-containing flavonoid and an
.alpha.-rhamnosidase, wherein the .alpha.-rhamnosidase is in a
state in which it is essentially not able to cleave the
rhamnose-containing flavonoid.
[0038] Provided the right conditions are met (environment, pH,
temperatures etc.), .alpha.-rhamnosidase enzymes normally have the
ability to cleave substrates comprising a rhamnose moiety.
[0039] In the compositions of the invention however, the enzyme is
in a state such that it is essentially prevented from carrying out
its normal function. It is only upon ingestion of the compositions,
that the environment of the enzyme is changed such that the new
conditions (pH, temperature etc.) allow the enzyme to become active
and thus to cleave the rhamnose moiety of the flavonoid.
[0040] Thus, in the compositions, uses or methods of the present
invention, the .alpha.-rhamnosidase is in a state in which it is
essentially not able to cleave the rhamnose-containing flavonoid.
Only upon ingestion of the composition is the .alpha.-rhamnosidase
able to cleave the rhamnose-containing flavonoid.
[0041] This "retarded" .alpha.-rhamnosidase activity ensures that
the cleaving of the flavonoid by the .alpha.-rhamnosidase will only
occur upon ingestion. Thus, only in gastro-intestinal tract
conditions does the .alpha.-rhamnosidase activity occur.
[0042] This can be achieved by several means, according to varying
embodiments of the invention.
[0043] For instance, this can be achieved by having the
.alpha.-rhamnosidase in the composition under conditions in which
it is "inactive", i.e. it is not able to cleave the rhamnose moiety
of the flavanoid. An inactive arhamnosidase is, for example, an
.alpha.-rhamnosidase which has been treated with an inhibitor, such
that only when the conditions of the gastro-intestinal tract are
met, the .alpha.-rhamnosidase is able to be active.
[0044] Alternatively, the conditions in the composition may be such
that the enzyme is "inactive", for example by having high pH value.
Upon ingestion, the low pH of the gastro-intestinal tract will
provide favourable conditions for the .alpha.-rhamnosidase to
become active.
[0045] According to another embodiment, this can be achieved by
separating the .alpha.-rhamnosidase in the composition from the
flavonoid. The separation is such that the .alpha.-rhamnosidase is
not in direct contact with the flavonoid.
[0046] The separation can be made, for instance, by encapsulating
.alpha.-rhamnosidase by means known in the art. Thus, the enzyme
may be encapsulated such that it is only released under
gastro-intestinal tract conditions.
[0047] Alternatively, by encapsulating the .alpha.-rhamnosidase in
a micro-organism capable of releasing .alpha.-rhamnosidase, the
.alpha.-rhamnosidase is not in direct contact with the flavonoid in
the composition. Such "bio-encapsulation" may be achieved by an
.alpha.-rhamnosidase producing strain of a micro-organism which is
kept under conditions (water activity etc.) such that the
micro-organism presents a low or zero metabolic rate. The
conditions (water activity etc.) in the composition are therefore
such that the micro-organism is not releasing the enzyme under
these conditions. The .alpha.-rhamnosidase is "bio-encapsulated"
within or on the outside of the cell walls of the
micro-organism.
[0048] Thus, in the present invention, by ".alpha.-rhamnosidase in
a state in which it is essentially not able to cleave the rhamnose
moiety of the rhamnose-containing flavonoid" is meant any form of
the enzyme as described above.
[0049] When an .alpha.-rhamnosidase-producing micro-organism is
used in the compositions or the methods of the present invention,
it is preferably a bacterium. More preferably, the micro-organism
is selected from Lactobacillus, Bifidobacterium, Streptococcus,
Lactococcus, Enterococcus, Bacillus, Staphylococcus, Leuconostoc,
Pediococcus, Oenococcus etc.
[0050] Most preferably, the micro-organism is selected from
Lactobacillus crispatus (ATCC 33820), Lactobacillus crispatus (CNCM
1-3654), Lactobacillus plantarum (ATCC 8014), Lactobacillus
plantarum (CNCM 1-3653), Lactobacillus gasseri (CNCM 1-3795) or
mixtures thereof.
[0051] Thus, a micro-organism having .alpha.-rhamnosidase activity
selected from Lactobacillus crispatus (ATCC 33820), Lactobacillus
crispatus (CNCM 1-3654), Lactobacillus plantarum (ATCC 8014),
Lactobacillus plantarum (CNCM 13653) or Lactobacillus gasseri (CNCM
1-3795) is part of the present invention.
[0052] Lactobacillus plantarum NCC 1076 and Lactobacillus crispatus
NCC 2852 were deposited on Jul. 18, 2006 under the Budapest treaty
with the Collection Nationale de Cultures de Microorganism,
Institut Pasteur, Rue du Docteur Roux, F-76724 Paris Cedex 15 and
were assigned accession numbers CNCM 1-3653 and CNCM 1-3654,
respectively.
[0053] The micro-organism is preferably present in the compositions
of the invention in an amount of 10.sup.6-10.sup.10 cfu/g. More
preferably it is present in an amount of 10.sup.9 cfu/g.
[0054] The micro-organism capable of producing .alpha.-rhamnosidase
may further be encapsulated. Encapsulation of micro-organisms is a
method well-known to the person of skill in the art.
[0055] The flavonoids used in the present invention may be selected
from any flavonoid comprising a rhamnose moiety. Any such
rhamnose-containing flavonoid may be selected from the group
consisting of hesperidin, rutin, eriotricin, naringin,
neohesperidin, diosmin, linarin, poncirin, prunin, etc. and any
possible combination from this list comprising two or more
components from the list. Preferably, the flavonoid is hesperidin
(FIG.-1a).
[0056] Hesperidin (FIG. 1a) comprises a rutinose (rhamnose-glucose)
moiety. In the presence of an active .alpha.-rhamnosidase enzyme,
the rhamnose moiety of hesperidin may be cleaved off to a certain
extent to yield hesperetin-7-glucoside (FIG. 1b). In turn,
hesperetin-7-glucoside may be further cleaved by other enzymes,
e.g. glucosidase enzymes which are present in the gastro-intestinal
tract, to give hesperetin (FIG. 1c).
[0057] Referring to FIG. 2, it can be seen that, on one hand, the
plasma level of hesperetin (FIG. 1c) upon ingestion of
hesperetin-7-glucose (FIG. 1b) shows a sharp peak shortly after
ingestion. Without wishing to be bound by theory, it is thought
that this is due to the presence of enzymes which are able to
cleave the glucose moiety off, such that hesperetin is readily
absorbable.
[0058] On the other hand, the hesperetin plasma level resulting
from the ingestion of hesperidin (FIG. 1a) appears only later and
to a smaller extent. Without wishing to be bound by theory, it is
thought that this is due to the presence of enzymes able to cleave
the rutinose moiety of hesperidin only at a later stage of passage
through the gastro-intestinal tract.
[0059] Thus, the compositions of the present invention
advantageously provide a plasma level of hesperetin which is
sustained over a longer period of time (cf. FIG. 2).
[0060] Further, according to another embodiment of the present
invention, such a sustained plasma level of hesperetin is also
provided by compositions comprising a mix of hesperidin (FIG. 1a)
and hesperetin-7-glucoside (FIG. 1b). Preferably, these are present
in the compositions in a ratio of hesperidin to
hesperetin-7-glucoside of between 70/30 to 50/50.
[0061] The amount of flavonoid in the compositions of the present
invention is such that it corresponds to an amount ranging from
0.01 mg to 1 g of the aglycone equivalent of the flavonoid
compound. Preferably, the flavonoid is present in an amount ranging
from 10 mg to 800 mg of the aglycone equivalent of the flavonoid
compound.
[0062] For instance, when hesperidin (FIG. 1a) is used in a
composition of the present invention, it is present in an amount
which will provide 0.01 mg to ig, preferably 10 mg to 800 mg of the
corresponding hesperetin (FIG. 1c). This is easily calculated by a
man of skill in the art.
[0063] In the compositions of the present invention, the
.alpha.-rhamnosidase may be present in an amount sufficient to
provide between 10-50% of the flavonoid aglycone or of a form of
glycosylated, flavanoid which is absorbable at the early stages of
digestion.
[0064] For instance, in the case where the flavanoid is hesperidin,
the amount of .alpha.-rhamnosidase used is an amount sufficient to
provide between 10-50% of hesperetin 7-glucoside (FIG. 1b) or
hesperetin (FIG. 1c) in the upper gastro-intestinal tract (small
intestine). This can be easily assessed by methods known in the
art, such as experiments with TIM-1 of TNO and in vivo confirmation
(cf. FIGS. 8 and 9)
[0065] The compositions of the present invention are preferably
formulated for use as nutritional, pharmaceutical or cosmetic
compositions.
[0066] Therefore, the compositions of the present invention may be
dry, moist, or semi-moist compositions. By "dry", is meant
compositions having a water activity below 0.6. By "semi-moist" is
meant compositions having a water activity between 0.6 and 0.9 and
by "moist" is meant composition having a water activity above
0.9.
[0067] They may be selected from liquid, dry or semi-dry
compositions such as solutions, sprays, powders, tablets, capsules,
yoghurt, biscuit, milk, beverages, chocolate, ice cream, breakfast
cereal flakes or bars, milk powders, soy-based products, non-milk
fermented products, nutritional supplements, food supplement, pet
food, infant formula etc.
[0068] For ingestion, many embodiments of oral compositions and in
particular of food supplements are possible. They are formulated by
means of the usual methods for producing sugar-coated tablets,
gelatine capsules, gels, emulsions, tablets, capsules or solutions.
In particular, the rhamnose-containing flavonoids and the
.alpha.-rhamnosidase or, in a different embodiment, the hesperidin
and hesperetin-7-glucoside may be incorporated into any other forms
of food supplements or of enriched foods, for example food bars, or
compacted or non-compacted powders. The powders can be diluted with
water, in a fizzy drink, dairy products or soya-derived products or
can be incorporated into food bars.
[0069] The compositions may comprise the usual excipients and
constituents, e.g. fatty and/or aqueous constituents, humectifying
agents, thickeners, preserving agents, texturing, flavouring and/or
coating agents, antioxidants, dyes that are usual in the food
domain.
[0070] According to a further aspect of the present invention,
.alpha.-rhamnosidase may be used in the preparation of a
composition comprising at least one rhamnose-containing flavonoid
and wherein the .alpha.-rhamnosidase is in a state in which it is
essentially not able to cleave the rhamnose moiety of said
flavonoid, for improving the bioefficacy and/or bioavailability of
said flavonoid. By "composition" is covered any composition
according to the invention as described above.
[0071] Bioefficacy is defined as the proportion of the ingested
nutrient converted to an active form of the nutrient having
significant biological effect. It is closely related to
bioavailability which is defined as the degree to which a substance
is absorbed into the systemic circulation. By improving the
bioefficacy and/or bioavailability of a flavonoid, the present
invention offers the advantage of a more effective composition with
more durable and sustained effects.
[0072] Thus, the present invention also provides a method for
sustaining and/or improving the bioavailability of
rhamnose-containing flavonoids comprising the step of providing a
composition comprising at least one rhamnose-containing flavonoid
and .alpha.-rhamnosidase, said arhamnosidase being in a state in
which it is essentially not able to cleave the rhamnose moiety of
said flavonoid.
[0073] A comparison of the plasma levels after ingestion of
hesperidin (FIG. 1a) or after ingestion of hesperetin-7-glucoside
(FIG. 1c) shows a noticeable difference to the compositions of the
present invention. Indeed, after the ingestion of the compositions
of the present invention, the hesperetin plasma levels are
maintained for a sustained period of time (FIG. 2).
[0074] Thus, the present invention further encompasses a method for
prolonging the plasma levels of metabolites of rhamnose-containing
flavonoids after ingestion of said flavonoid comprising the step of
orally providing a composition comprising at least one
rhamnose-containing flavonoid and .alpha.-rhamnosidase, said
.alpha.-rhamnosidase being in a state in which it is essentially
not able to cleave the rhamnose moiety of said flavonoid.
[0075] In the methods of the present invention, the
.alpha.-rhamnosidase may be provided separately from the
composition comprising the rhamnose-containing flavonoid. For
example, said .alpha.-rhamnosidase may be provided as a tablet,
capsule etc. to be ingested at the same time as the composition
comprising the flavonoid. Alternatively, it may be provided, for
example, as a powder to be sprinkled onto the flavonoid-containing
composition. The skilled person could readily envisage a variety of
different alternatives to the specific embodiments mentioned
herein.
[0076] Under another aspect, the compositions according to the
present invention may be used cosmetically. By "cosmetic use" is
meant a non-therapeutic use which may improve the aesthetic aspect
or comfort of the skin, coat and/or hair of humans or pets.
[0077] In this context, the cosmetic use may include preventing
damages to, and/or improving the skin, coat and/or hair of humans
or pets. Such damages include in particular actinic and ageing
damages of the skin such as dryness, irregular pigmentation
(notably freckling, lentigines, guttate hypomelanosis and
persistent hyperpigmentation), wrinkling (notably fine surface
lines and deep furrows), stellate pseudoscars, elastosis,
inelasticity, telangiectasia, venous lakes, comedones, sebaceous
hyperplasia, acrochordon and seborrhea keratosis.
[0078] The cosmetic use may also have particular benefits on hair
and coat, such as an improved hair or coat density, fibre diameter,
colour, oiliness, glossiness, sebum production and may help to
prevent hair or coat loss.
[0079] The present invention further encompasses therapeutic uses
such as dermatological uses for instance. Indeed, the use of
.alpha.-rhamnosidase and at least one rhamnose-containing
flavonoid, wherein the .alpha.-rhamnosidase is in a state in which
it is essentially not able to cleave said flavonoid, in the
manufacture of compositions for the improvement of skin health,
falls under another aspect of the invention. Said compositions may
also be used for the prevention of inflammation or for the
improvement of bone and/or cardiovascular health. By "composition"
is covered any composition according to the invention as described
above.
[0080] Under this embodiment, the compositions according to the
present invention may be utilised for treating and/or preventing
damages of the skin which are, for example, produced by a stress
situation e.g. by means of a chemical, biological or a physical
stress, e.g. by exposure to oxidants or carcinogens, exposure to
bacteria, viruses, fungi, lipids derived from surrounding cells
and/or microbes, or exposure to UV-irradiation.
[0081] These damages further comprise actinic keratoses, purpura,
cherry angiodema, basal cell carcinoma and squamous cell carcinoma,
skin burning and/or blistering, epidermal hyperplasia,
inflammation, immune suppression, and cancer, e.g. non-melanoma and
melanoma skin cancers.
[0082] The effect of the compositions according to the present
invention, on skin of humans or pets, can be measured by using
conventional methods including minimal erythemal dose (MED),
colorimetry, transepidermal water loss, DNA repair, measure of
interleukins and proteoglycans production, or collagenase activity,
barrier function or cell renewal.
[0083] Consequently, a method for improving skin health comprising
the step of orally administering a composition comprising at least
one rhamnose-containing flavonoid and .alpha.-rhamnosidase, or
orally administering separately and simultaneously a composition
comprising at least one rhamnose-containing flavonoid and
.alpha.-rhamnosidase, said .alpha.-rhamnosidase being in a state in
which it is essentially not able to cleave the rhamnose moiety of
said flavonoid, also falls under an aspect of the present
invention. This method is also useful in improving cardiovascular
and bone health.
[0084] It will be understood that the concept of the present
invention may likewise be applied as an adjuvant therapy assisting
in presently used medications. Since the compositions of the
present invention may easily be orally administered with food
material, special clinical food may be administered containing a
high amount of the objective substances.
[0085] Furthermore, the concept of the present invention may
likewise be extended to topical applications of compositions
comprising a rhamnose-containing flavonoid and an
alpha-rhamnosidase enzyme.
[0086] The present invention is further illustrated by means of the
non-limiting examples described below.
EXAMPLES
Example 1
Materials
[0087] Hesperidinase "Amano" Conc. (A.MANO PHARMACEUTICAL CO.,
LTD.)
[0088] Cleavage of Hesperidin by Bacterial Crude Extracts In
Vitro
[0089] To test if the bacterial alpha-rhamnosidases can recognise
and cleave hesperidin as a substrate, the crude extracts of the
cells grown in the presence of rhamnose are incubated with
hesperidin at pH 4 and pH 6 for 4 hours and 8 hours and the
analysis is done by HPLC. The results are presented in the table
below as a percentage of hesperidin or its derivatives based on the
total hesperidin amounts at the beginning of the reaction (0.08
mg/mL). The tests were performed with 0.08 mg hesperidin/mL, which
is an approximate concentration of hesperidin aimed for the final
product and crude extracts of 3.times.10.sup.9 bacteria/mL.
[0090] The results show that the two strains have both
arhamnosidase and .beta.-glucosidase activity and as a consequence
cleave hesperidin into hesperetin-7-glucoside and aglycone to
different extents, depending on the reaction conditions. The
results suggest that bacteria can be used for partial hesperidin
transformation into hesperetin-7-glucoside and aglycone in
situ.
TABLE-US-00001 Hesperetin- Hes- 7- Bacterial peritin glucoside
aglycone counts 4 h 8 h 4 h 8 h 4 h 8 h Control (-) pH 4 -- nd 95
nd 0 nd 0 (no enzyme) pH 6 nd 95 nd 0 nd 0 Control (+) pH 4 0 0 92
93 0 0 pH 4 (hesperedinase) pH 6 66 49 25 40 0 0 pH 6 L.
Acidophilus pH 4 3.4 .times. 10.sup.9/ml 18 5 28 18 26 42 NCC 3010
pH 6 26 10 6 5 13 27 L. Plantarum pH 4 3.4 .times. 10.sup.9/ml 12 8
5 4 62 66 NCC1313 pH 6 9 0 5 0 25 33 Nd: not cleterminecl
Example 2
[0091] 0.2 to 50 mg of hesperidin/g of product is mixed with
10.sup.7-10.sup.10 cfu/(g of product) of alpha-rhamnosidase active
bacteria. The resulting mixture is blended with a suitable carrier.
Carriers may be selected from fermented milk, yogurt, fresh cheese,
renneted milk, confectionery bar, breakfast cereal flakes or bars,
drink, milk powder, soy-based product, non-milk fermented
product.
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