Note: Descriptions are shown in the official language in which they were submitted.
CA 02850685 2015-11-06
PROCESS FOR PRODUCING FUCOXANTHINOL EXTRACT AND METHODS OF USE
TECHNICAL FIELD
[0001] The present invention generally relates to extracts obtained from
marine invertebrates.
More specifically, the present invention relates to a method of extracting
fucoxanthinol
compositions from Echinoidea tissues/organisms, and the compositions extracted
by using the
method.
BACKGROUND ART
[0002] Carotenoid compounds have been studied intensively due to their
potential medicinal
applications. In particular, some carotenoid compounds have been shown to
display significant
anti-oxidative activity and free radical elimination characteristics, as well
as the ability to inhibit
the growth of cancer. Oxygen-free carotenoids are known as carotenes, while
those
carotenoids that contain oxygen are known as xanthophylls.
[0003] One xanthophyll that has received significant attention is fucoxanthin,
which is derived
from brown algae. Fucoxanthin is a pigmented compound in the chloroplast of
brown algae and
seaweeds that give the plants their characteristic colour. When fucoxanthin is
ingested, it is
hydrolyzed into the deacetylated form, fucoxanthinol. Studies in mice indicate
that following
ingestion of fucoxanthin, it is metabolized into fucoxanthinol in the
digestive tract, and then
further converted to amarouciaxanthin A in the liver (Hashimoto et al., [2009]
"The distribution
and accumulation of fucoxanthin and its metabolites after oral administration
in mice," British
Journal of Nutrition 102:242-248).
[0004] Research on fucoxanthin and fucoxanthinol has demonstrated a diverse
range of
activity. As with other carotenoids, fucoxanthin and its metabolites have been
shown to have
anti-oxidative effects ¨ including free radical scavenging activity and
singlet oxygen quenching.
(Sachindra et al., [2007] "Radical Scavenging and Singlet Oxygen Quenching
Activity of Marine
Carotenoid Fucoxanthin and Its Metabolites," J. Agric. Food Chem. 55:8516-
8522). In addition,
fucoxanthin has shown anti-inflammatory effects through the inhibition of
inducible nitric oxide
synthase and cyclooxygenase 2 protein expression, as well as reducing levels
of nitric oxide,
prostaglandin E2, tumor necrosis factor-a and interleukins 113 and 6 (Heo et
al. [2008]
"Cytoprotective effect of fucoxanthin isolated from brown algae Sargassum
siliquastrum against
H202-induced cell damage," Eur. Food Res. Technol. 228:145-151; and Kim et
al., [2010]
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"Evaluation of anti-inflammatory effect of fucoxanthin isolated from brown
algae in
lipopolysaccharide-stimulated RAW 264.7 macrophages," Food Chem. Toxicol.
48:2045-2051).
Reactive oxygen species generated as a result of UV radiation exposure were
also decreased
as a result of fucoxanthin (Heo and Jeon, [2009] "Protective effect of
fucoxanthin isolated from
Sargassum siliquastrum on UV-B induced cell damage," J. Photochem. Photobiol.
95:101-107)
suggesting that fucoxanthin may have a protective effect for the skin.
[0005] Fucoxanthin and fucoxanthinol also have anti-obesity effects. For
example,
fucoxanthin has been combined with pomegranate seed oil with the combination
having the
effect of reducing body and liver fat content and improving liver function, as
well as increasing
resting energy expenditure (Maeda et al., [2005] "Fucoxanthin from edible
seaweed, Undaria
pinnatifida, shows antiobesity effect through UCP1 expression in white adipose
tissues,"
Biochem. Biophys. Res. Commun. 332:392-397; Abidov et al., [2010] "The effects
of
Xanthigen TM in the weight management of obese premenopausal women with non-
alcoholic
fatty liver disease and normal liver fat," Diabetes Obes. Metab. 12:72-81).
Further research
suggests that the anti-obesity effects of fucoxanthin and its metabolites may
stem from the
regulation of expression of the uncoupling protein (UCP1) in white adipose
tissue (VVAT), which
caused a decrease of abdominal fat in both rats and mice. (Miyashita et al.,
[2011] "The allenic
carotenoid fucoxanthin, a novel marine nutraceutical from brown seaweeds," J.
Sci. Food Agric.
91:1166-1174) Maeda et al. also successfully demonstrated that fucoxanthin
decreases blood
glucose levels in diabetic mice, in addition to reducing the levels of insulin
in plasma.
[0006] Fucoxanthin and fucoxanthinol have been shown to induce apoptosis in a
number of
human cancer cell lines, including those for prostate and colon cancers, as
well as leukemia.
While the full understanding of the effects of fucoxanthinol on cancer cells
is yet to be
elucidated, it has been reported that fucoxanthinol down regulates the
peroxisome proliferator-
activated receptor y, which also contributes to its activity as an anti-
obesity compound.
(Hosokawa et al. [2004] "Fucoxanthin induces apoptosis and enhances the
antiproliferative
effect of the PPARgamma ligand, troglitazone, on colon cancer cells," Biochim.
Biophys. Acta
1675:113-119). With regard to cancer treatment, fucoxanthin has been shown to
inhibit
metastatic potential of cancer cells (Chung et al., [2013] "Marine algal
fucoxanthin inhibits the
metastatic potential of cancer cells," Biochem. Biophys. Res. Commun. 439:580-
585) in in vitro
and in vivo models. Fucoxanthinol also inhibits the cell viability in T-cell
lines infected with
human T-cell leukemia virus type 1, and adult T-cell leukemia. The
fucoxanthinol induces
apoptosis by arresting the cell cycle during the G1 phase (Ishikawa et al.,
[2008] "Antiadult T-
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cell leukemia effects of brown algae fucoxanthin and its deacetylated product,
fucoxanthinol,"
Int. J. Cancer 123:2702-2712). Fucoxanthin treatment has reduced viability of
three lines of
human prostate cancer cells, also through apoptosis (Kotake-Nara et at. [2001]
"Carotenoids
affect proliferation of human prostate cancer cells," J. Nutr. 131:3303-3306).
Fucoxanthinol has
been shown to exert a stronger effect than fucoxanthin in the
antiproliferation of prostate cancer
cells (Asai et al., [2004] "Biotransformation of fucoxanthinol into
amarouciaxanthin A in mice and
HEPG2 cells: formation and cytotoxicity of fucoxanthin metabolites," Drug
Metab. Dispos.
32:205-211) and shows almost twice the efficacy of fucoxanthin in the
inhibition of HTLV-1-
infected T-cell lines and ATL cells. (Ishikawa et al., [2008] "Antiadult T-
cell leukemia effects of
brown algae fucoxanthin and its deacetylated product, fucoxanthinol," Int. J.
Cancer 123:2702-
2712). A recent review of the anti-cancer effects of fucoxanthin and
fucoxanthinol outlines the
various mechanisms through which these compounds exert their influence (Ravi
Kumar et al.,
[2013] "Fucoxanthin: A Marine Carotenoid Exerting Anti-Cancer Effects by
Affecting Multiple
Mechanisms," Mar. Drugs 11:5130-5147).
[0007] In addition, fucoxanthin and fucoxanthinol have been shown to possess
antiangiogenic
activity, preventing the development of new blood vessels, which can be useful
in the inhibition
of tumour growth. (Sugawara et al., [2006] "Antiangiogenic Activity of Brown
Algae Fucoxanthin
and Its Deacetylated Product, Fucoxanthinol" J. Agric. Food Chem. 54:9805-
9810).
[0008] Fucoxanthin can be chemically synthesized, but the process is expensive
and the yield
is low. Accordingly, it is desirable to find a way to extract fucoxanthinol
from natural sources in
a cost effective method with an appropriate yield such that the compound can
be used in
compositions for medicinal or health purposes. Previous work has shown that
fucoxanthin can
be extracted from marine diatoms (Kim, [2012] "A Potential Commercial Source
of Fucoxanthin
Extracted from the Microalga Phaeodactylum tricomutum," Appl. Biochem.
Biotechnol.
166:1843-1855) by a solvent extraction method. Different solvent/diatom ratios
and different
temperatures resulted in different yields ranging up to 0.78% fucoxanthin.
High pressure
solvent extraction of fucoxanthin from seaweed (Eisenia bicyclis) gave maximum
extraction
results of 0.42 mg/g (Shang et al., [2011] "Pressurized liquid method for
fucoxanthin extraction
from Eisenia bicycfis (Kjellman) Setchell," J. Biosci. Bioeng. 111:237-241).
Quitain et at.
([2013], "Supercritical carbon dioxide extraction of fucoxanthin from Undaria
pinnatifida," J.
Agric. Food Chem. 61(24):5792-5797) showed that fucoxanthin can be extracted
from seaweed
(Undaria pinnatifida) using a supercritical carbon dioxide method with a range
of temperatures
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and pressures, as well as flow rates, resulting in extraction rates of 0.53 to
1.22 g extract per
100 g sample. These processes do not encompass the extraction of
fucoxanthinol.
[0009] In order to extract fucoxanthinol, which is more active than
fucoxanthin, it is necessary
to extract it from organisms that digest the seaweeds and produce the
metabolite in the
digestive tract.
[0010] Fucoxanthinol cal be extracted from sea squirt (Ascidian) or other
Echinozoa tissues
as outlined in US 8,445,028 using a method that combines tissues with a
dispersing agent and
then uses a solvent extraction process, followed by high pressure liquid-solid
extraction. Other
methods of fucoxanthinol extradion that are simpler and result in appropriate
yields are
desirable and useful for the preparation of medications using fucoxanthinol.
SUMMARY OF THE INVENTION
[0011] In the studies described herein, it is shown that fucoxanthinol
extracts may be obtained
from various organs and tissues of marine invertebrates belonging to the
Echinozoa subphylum.
[0012] In one aspect, the present invention provides an Echinozoa tissue or
organ extract
obtained by a process comprising:
= providing Echinozoa tissues or organs;
= mixing or blending the tissues or organs to achieve homogenization;
= using solvent extraction to extract the fucoxanthinol from the blended
tissues or organs;
= optionally centrifuging the solvent-tissue mixture to remove particulate
matter;
= evaporating a portion of the liquid component to remove solvent and water;
= filtering the remaining liquid; and
= evaporating the final filtered liquid to remove the majority of the water
and solvent in
order to achieve a purified fucoxanthinol extract.
[0013] In one aspect, the Echinozoa tissue and or organs are provided in a
frozen state and
thawed prior to homogenization.
[0014] In another aspect, the above-mentioned extraction process is performed
at a
temperature of about 15 C. to about 25 C. In a further aspect, the
evaporation step in the
above-mentioned extraction process is performed at a temperature of about 35
C. to about 50
C.
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[0015] In another aspect, the above-mentioned tissues or organs are (i)
tissues or organs of
the digestive tract, (ii) viscera, or (iii) a combination of (i) and (ii).
[0016] In another aspect, the present invention provides a use of (i) the
above-mentioned
extract, (ii) the above-mentioned composition, or (iii) a combination of (i)
and (ii), as a
medicament.
[0017] In another aspect, the present invention provides a use of (i) the
above-mentioned
extract, (ii) the above-mentioned composition, or (iii) a combination of (i)
and (ii), for the
preparation of a medicament.
[0018] In another aspect, the present invention provides a composition
comprising the above-
mentioned extract and a carrier. In various aspects of the present invention,
the above-
mentioned composition is an anti-oxidant composition an anti-obesity
composition, an anti-
cancer composition, or an anti-angiogenic composition. In other aspects, the
above-mentioned
composition is a composition that reduces blood glucose or increases resting
energy
expenditure.
[0019] In another aspect, the present invention provides a use of (i) the
above-mentioned
extract, (ii) the above-mentioned composition, or (iii) a combination of (i)
and (ii), for prevention
or treatment of obesity or cancer.
[0020] In another aspect, the present invention provides a use of (i) the
above-mentioned
extract, (ii) the above-mentioned composition, or (iii) a combination of (i)
and (ii), for the
preparation of a medicament for (a) decreasing or inhibiting oxidative stress
in a cell or tissue,
(b) prevention or treatment of a condition associated with oxidative stress,
(c) reducing blood
pressure, (d) increasing energy expenditure rate, (e) reducing inflammation,
(f) treating a
cancerous condition, or (f) treatment of a condition requiring a
neuroprotective effect.
[0021] In another aspect, the present invention provides a method for
preventing or treating a
condition, said method comprising administering to said subject (i) the above-
mentioned extract,
(ii) the above-mentioned composition, or (iii) a combination of (i) and (ii).
[0022] Other objects, advantages and features of the present invention will
become more
apparent upon reading of the following non-restrictive description of specific
embodiments
thereof, given by way of example only with reference to the accompanying
drawings.
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BRIEF DESCRIPTION OF THE DRAWING
[0023] Figure 1 shows a UV chromatogram for a representative sample of the
extract with
peaks for fucoxanthinol at RT 9.51 and for fucoxanthin at RT 11.50.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT OF THE PRESENT
INVENTION
[0024] The present invention relates to fucoxanthinol extracts obtained from
the tissues of
organisms in the Echinodermata phylum, specifically in the Echinozoa
subphylum. Echinozoa
are invertebrate marine animals found at all ocean depths. As a group, the
Echinozoa are
known to eat algae and seaweed species, including those algae and seaweed in
the brown
colour category, which contain fucoxanthin. When the Echinozoa eat these algae
and seaweed
species, their digestive processes metabolize the fucoxanthin to
fucoxanthinol.
[0025] The processing of various sea urchin species for food purposes is
designed to remove
the edible roe from the spiny exterior of the urchin, as well as separating
the roe from the gut
portion of the organism. The gut of the urchin, including the digestive
system, is not typically
eaten or used for other purposes. As a result, sea urchin processing
facilities typically dispose
of the sea urchin gut as a "waste product" which is sent directly to dumping
sites. The present
invention uses this former "waste product" for the extraction of
fucoxanthinol.
[0026] In some embodiments, the present invention uses the tissues of
Echinozoa from the
Diadematoidea, Arbacioida and Echinoida orders. In another embodiment, the
tissues of
Echinozoa from the Strongylocentrotidae family are used. In one embodiment,
the tissues of
the sea urchin Strongylocentrotus droebachiensis (Green Sea Urchin) are used.
S.
droebachiensis is widespread on the northern hemisphere, and has also been
harvested by
man as a food source for thousands of years.
[0027] Accordingly, in a first aspect, the present invention provides a
process for preparing an
Echinozoa tissue or organ extract comprising:
= providing Echinozoa tissues or organs;
= mixing or blending the tissues or organs to achieve homogenization;
= using solvent extraction to extract the fucoxanthinol from the blended
tissues or organs;
= optionally centrifuging the solvent-tissue mixture to remove particulate
matter;
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= evaporating a portion of the liquid component to remove solvent and
water;
= filtering the remaining liquid; and
= evaporating the final filtered liquid to remove the majority of the water
and solvent in
order to achieve a purified fucoxanthinol extract.
[0028] In some cases, it is desirable that the Echinozoa tissue and or organs
are provided in a
frozen state. The processing of a large quantity of organisms results in large
volumes of tissue
and organs that may need to be stored or transported prior to the extraction
process. In order to
prevent the decomposition of the tissues and the potential reduction of
fucoxanthinol yield, the
tissues and organs may be frozen in large quantities at the source, such as a
processing
factory. Prior to the blending or homogenizing step, the tissues must be
thawed in order to
facilitate the homogenization. As will be understood by those of skill in the
art, different
quantities of frozen tissue must be thawed for longer or shorter periods of
time depending on
the amount of tissue and the size of the frozen mass of tissue, in order that
the tissue
throughout the mass is thawed.
[0029] The starting material (i.e. Echinozoa tissue or organ) is homogenized
using
commercially-available grinders, blenders or comparable devices. For example,
complete
homogenization may be achieved at a minimum of 2500 rpm in an industrial
blender in order to
optimize the eventual fucoxanthinol yield. The homogenization of the tissue
allows for the
maximum surface area exposure, thus maximizing the yield of fucoxanthinol that
is obtained
from the extraction process. In an embodiment, the starting material is
received in a frozen
condition, and is subsequently thawed by leaving the material at room
temperature before being
ground and homogenized. The ground/homogenized material may be stored,
preferably in a
frozen state, before extraction. In an embodiment, the ground/homogenized
material is kept
frozen (e.g., at about -20 C.) before extraction. One of skill in the art
will recognize that the
ideal homogenization speed and duration will depend upon the quantity of
tissue being
homogenized.
[0030] The extraction process is performed in the presence of a solvent, such
as an alcohol or
polyol (or any combination/mixture thereof). Solvents that may be used for the
extraction
process include, for example, ethanol, methanol, propanol, n-hexane and
acetone. The
solvents may be pure (100%) solvent or may be mixed with water (for example,
80% ethanol
may be used). The homogenized material is combined with the solvent at a 1:1
v/v ratio and
thoroughly mixed for a period of time. As one of skill in the art will
realize, the length of time
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necessary for the mixing will depend upon the quantity of material. For
example, 50 kg of
homogenized tissue is allowed to mix for 8 to 12 hours per extraction.
Following the mixing
phase, the slurry is left to settle in order to separate the solvent
containing the fucoxanthinol
from the solid homogenized tissue material. In an embodiment of the present
invention, the
above-mentioned extraction process is performed at a temperature of about 15
C. to about 25
C. In a further embodiment, the evaporation step in the above-mentioned
extraction process is
performed at a temperature of about 35 C. to about 50 C.
[0031] Following the settling of the material, the liquid portion containing
the solvent and
extracted fucoxanthinol is separated from the solid material. A large portion
of the liquid portion
may be removed by pumping or decanting in a way that minimizes mixing with any
settled or
suspended solids. Fresh solvent is added to the remaining slurry for
subsequent extractions.
One of skill in the art will recognize that extractions may be performed
multiple times in order to
maximize the yield of fucoxanthinol. For example, extractions may be performed
from one to
five times in order to achieve a good yield.
[0032] The liquid portion that is removed from the slurry may be partly clear,
but a portion of it
may have a high concentration of suspended solids. This portion is likely to
be the portion that
was closest to the settled material. Therefore, a centrifugation step is
ideally used to eliminate
any such suspended solids from the liquid. Centrifugation may be performed on
a suitably sized
centrifuge depending on the amount of liquid requiring centrifugation, and
should be performed
at a speed of 2500 to 6500 rpm for 5 to 20 minutes.
[0033] The extract obtained from the top of the slurry, and the liquid from
the centrifugation
step is in the form of a liquid concentrate. The concentrate may be further
concentrated (e.g.,
evaporation of the extraction solvent and water portion of the concentrate)
using apparatus and
methods well known in the art, to obtain a solid or dry extract. In an
embodiment, the liquid
extract is dried via gaseous flow, e.g., under the flow of an inert gas, e.g.,
under (e.g.,
continuous) nitrogen flow. In another embodiment, the liquid extract is dried
via evaporation,
e.g., using a rotating evaporator or similar device. In another embodiment,
the liquid extract is
dried at ambient temperature (e.g., between about 15 C. to about 50 C., and
more particularly
between about 35 C. to about 50 C.) In addition, the evaporation may be
performed under
vacuum, such as a vacuum with a value between 72 and 35 mbar. Ideally, the
water and
solvent is evaporated at a rate between 3 and 60 L per hour.
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[0034] In order to maximize purity of the final fucoxanthinol extract, a
filtration step may be
used before the final evaporation step. The liquid from the first evaporation
may be filtered to
remove any suspended solids or precipitated proteins. Filtering may be
conducted with a filter
having a pore size of about 22 pm to about 100 pm.
[0035] Following filtration, a final evaporation step is used to produce the
oily extract
containing fucoxanthinol. The final evaporation is similar to the first
evaporation, and should be
stopped when no further water or solvent is being obtained, at which point
less than 10% of
water or solvent is left in the oil.
[0036] In another aspect, the present invention provides an Echinozoa tissue
or organ extract
containing fucoxanthinol obtained by the above-mentioned process.
[0037] In another aspect, the present invention provides a composition or
formulation (e.g., an
antioxidant composition or formulation) comprising the above-mentioned extract
and a carrier or
excipient (e.g., a pharmaceutically acceptable, cosmetically acceptable, or a
consumable
carrier/excipient).
[0038] In various embodiments, an extract of the invention may be used
therapeutically in
formulations or medicaments to prevent or treat a condition, such as a
condition associated with
oxidative stress. "Oxidative stress" as used herein generally refers to
oxidative damage in a cell,
tissue, or organ, caused by reactive oxygen species (ROS), such as free
radicals and
peroxides. The level of oxidative stress is typically determined by the
balance between the rate
at which oxidative damage is induced and the rate at which it is efficiently
repaired and
removed. Other conditions which may be prevented or treated using an extract
of the invention
include obesity and those forms of cancer which can be treated or inhibited
through the use of
fucoxanthinol. In addition, the fucoxanthinol extract of the invention may be
used therapeutically
to increase resting energy expenditure, to reduce blood glucose levels, and as
an
antiangiogenic compound.
[0039] The invention provides corresponding methods of medical treatment, in
which a
therapeutic dose of an extract of the invention is administered in a
pharmacologically acceptable
formulation, e.g. to a patient or subject in need thereof. Accordingly, the
invention also provides
therapeutic compositions comprising an extract of the invention and a
pharmacologically
acceptable excipient or carrier.
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[0040] A "therapeutically effective amount" or "effective amount" (in the
context of treatment)
refers to an amount effective, at dosages and for periods of time necessary,
to achieve the
desired therapeutic result, such as a reduction of oxidative stress and in
turn a reduction in
progression of or the amelioration of an associated condition. A
therapeutically effective amount
may vary according to factors such as the disease state, age, sex, and weight
of the individual,
and the ability of the compound to elicit a desired response in the
individual. Dosage regimens
may be adjusted to provide the optimum therapeutic response. A therapeutically
effective
amount is also one in which any toxic or detrimental effects of the compound
are outweighed by
the therapeutically beneficial effects. A "prophylactically effective amount"
or "effective amount"
(in the context of prevention) refers to an amount effective, at dosages and
for periods of time
necessary, to achieve the desired prophylactic result, such as preventing or
inhibiting the rate of
disease onset or progression of a condition associated with oxidative stress.
A prophylactically
effective amount can be determined as described above for the therapeutically
effective
amount. For any particular subject, specific dosage regimens may be adjusted
over time
according to the individual need and the professional judgement of the person
administering or
supervising the administration of the compositions.
[0041] Various embodiments of the present invention provide for the treatment
of obesity,
oxidative stress, cancer, diabetes and angiogenesis, comprising the step of
administering to a
mammal a therapeutically effective amount of the fucoxanthinol extract
obtained by the
extraction method of the invention.
[0042] The extract or composition of the present invention may be administered
in any number
of conventional dosage forms, e.g., topical, oral, parenteral, rectal,
transdermal, and the like.
Oral or rectal dosage forms include capsules, tablets, pills, powders, cachets
and suppositories.
Liquid oral dosage forms include solutions and suspensions. Parenteral
preparations include
sterile solutions and suspensions. Topical dosage forms can be creams,
ointments, lotions,
transdermal devices and the like. Except insofar as any conventional media or
agent is
incompatible with an extract of the invention, use thereof in the
pharmaceutical compositions of
the invention is contemplated. Supplementary active compounds can also be
incorporated into
the compositions.
[0043] The formulations and pharmaceutical compositions contemplated by the
above dosage
forms can be prepared with conventional pharmaceutically acceptable excipients
and additives
using conventional techniques. Such pharmaceutically acceptable excipients and
additives are
CA 02850685 2015-11-06
intended to include carriers, binders, flavorings, buffers, thickeners, color
agents, stabilizing
agents, emulsifying agents, dispersing agents, suspending agents, perfumes,
preservatives,
lubricants, etc.
[0044] Further, the composition can be prepared such that an extract of the
invention can be
administered in a controlled or time release formulation, for example in a
composition which
includes a slow release polymer for prolonged release.
[0045] Suitable pharmaceutically acceptable carriers include solvents,
coatings, dispersion
media, and the like. Solid carriers include magnesium carbonate, magnesium
stearate, talc,
sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose,
sodium
carboxymethylcellulose, low melting waxes, cocoa butter and the like. Capsules
can be made
wherein the active compound is inserted into pharmaceutically acceptable
capsules as a carrier.
The active compounds of this invention can be mixed with pharmaceutically
acceptable
excipients or can be used in finely divided powder form without excipients for
inclusion into
capsules. Similarly, cachets are included as are liposomes as known to those
skilled in the arts.
[0046] Liquid form preparations include solutions, suspensions and emulsions.
Examples
include water or water-propylene glycol solutions for parenteral injection.
Liquid preparations
can also be formulated in solution in polyethylene glycol and/or propylene
glycol, which may
contain water. Aqueous solutions suitable for oral use can be prepared by
adding the active
component in water and adding suitable colorants, flavors, stabilizing,
sweetening, solubilizing
and thickening agents as desired. Aqueous suspensions suitable for oral use
can be made by
dispersing the active component in the oil form within an emulsifier such as
TWEENTm-80 as is
known in the industry familiar with oil/water emulsions.
[0047] However, the compositions of the present invention may also be
formulated in other
convenient forms for use as a treatment, such as, an injectable solution or
suspension, a spray
solution or suspension, a lotion, a chewing gum, a lozenge, and a food or
snack item. Such
items can include any ingestible ingredient, allowing for formulation of the
extract of the present
invention into food items such as cereals, bars, gumdrops, chewable candies or
slowly
dissolving lozenges.
[0048] In various embodiments, an extract of the invention may be used
cosmetically in
formulations for such applications, e.g., to reduce or prevent effects
associated with oxidative
stress, e.g., of skin. The invention provides corresponding cosmetic methods,
in which an
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extract of the invention is applied or administered in a cosmetically
acceptable formulation, e.g.,
in a suitable topical formulation to a site of interest (e.g., an area of
skin). Accordingly, the
invention also provides cosmetic compositions comprising an extract of the
invention and a
cosmetically acceptable excipient, carrier or vehicle.
[0049] A cosmetically acceptable excipient, carrier or vehicle that may act as
a diluent,
dispersant or carrier for the extract of the invention and the other materials
that may be present
in the composition, so as to facilitate their distribution when the
composition is applied to the
skin. "Cosmetically acceptable carrier", "cosmetically acceptable excipient"
or "cosmetically
acceptable vehicle" as used herein refers to one or more compatible solid or
liquid fillers,
diluents, extenders and the like, which are cosmetically acceptable as defined
herein. The term
"compatible," as used herein, means that the components of the compositions of
this invention
are capable of being commingled with the primary active of the present
invention (e.g., the
extract described herein), and with each other, in a manner such that there is
no interaction that
would substantially reduce the efficacy of the composition under ordinary use
situations. The
type of vehicle utilized in the present invention depends on the type of
product desired. The
vehicles may also include but are not limited to one or more of organic
solvents, thickeners,
humectants, oils, silicone oils, water, emulsifiers, liquid or solid
emollients, propellants and
powders. Such mixtures may take several forms, including but not limited to
solutions,
dispersions, emulsions (0/W, W/0 or W/ONV) such as light creams, lotions,
serums, and gels.
Powders, such as chalk, talc, fullers earth, kaolin, starch, gums, colloidal
silica sodium
polyacrylate, tetra alkyl and/or trialkyl aryl ammonium smectites, chemically
modified
magnesium aluminium silicate, organically modified montmorillonite clay,
hydrated aluminium
silicate, fumed silica, carboxyvinyl polymer, sodium carboxymethyl cellulose
and ethylene glycol
monostearate can be selected.
[0050] As used herein, "lotions" are liquid cosmetics, often suspensions or
dispersions
intended for external application to the body.
[0051] As used herein, "creams" are soft cosmetic-type preparations. Creams of
the oil-in-
water (0/W) type include preparations such as foundation creams, hand creams,
shaving
creams, and the like. Creams of the water-in-oil (W/0) type include cold
creams, emollient
creams, and the like. Pharmaceutically, creams are solid emulsions containing
suspensions or
solutions of active ingredients for external application. Generally,
preparations of this type are
classified as ointments. Specifically, they belong to the emulsion-type bases.
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=
[0052] As used herein, "ointments" are semisolid preparations for external
application of such
consistency that may be readily applied to the skin. They should be of such
composition that
they soften, but not necessarily melt, when applied to the body. They serve as
vehicles for the
topical application of active ingredients and also function as protectives and
emollients for the
skin. For many years ointments were limited by definition and use to mixtures
of fatty
substances. Today, in addition to such oleaginous mixtures, there are ointment
preparations
possessing the same general consistency but entirely free of oleaginous
substances. In many
instances, they are emulsions of fatty or wax-like materials with
comparatively high proportions
of water. These emulsions may be either water-in-oil (W/O) or oil-in-water
(0/W) emulsions,
depending primarily on the selection of the emulsifying agent. Such semisolid
emulsions are
also referred to as creams. Creams and ointments containing large amounts of
insoluble
powders are referred to as pastes. Pastes are usually stiffer and more
absorptive than creams
and ointments.
[0053] For cosmetic applications, a composition of the invention comprising an
extract of the
invention may further comprise standard cosmetic ingredients, for example
those known in the
art to be used as moisturizers, stabilizers, preservatives, scents and the
like. The type of
cosmetic composition may be, for example, skin care cosmetics such as skin
lotion, emulsion,
cream, and cleansing agents; make-up cosmetics such as lipsticks and
foundation. The
cosmetics may be in any form without limitation.
[0054] The cosmetic compositions of the present invention may further comprise
one or more
cosmetic agents or dermatological active agents e.g., agents capable of
treating or preventing
any sign of aging of the skin. The active agents may be chosen, for example,
from skin
whitening agents, optical brightening agents, sunscreen agents, moisturizers,
free-radical
scavengers, keratolytic agents, vitamins, anti-elastase and anti-collagenase
agents, peptides,
fatty acid derivatives, steroids, trace elements, extracts of algae and of
planktons, enzymes and
coenzymes, flavonoids and ceramides, alpha-hydroxy acids and mixtures thereof,
and
enhancing agents.
[0055] Other cosmetically or dermatologically acceptable agents that may be
used in the
compositions of the invention include but are not limited to coloring agents
(e.g., pigments,
dyes, colorants), preservatives, perfumes and fragrances, pulverulent agents,
antiperspirants
and/or odor absorbers, natural extracts, procyannidol oligomers, urea,
caffeine, fillers,
keratolytic agents, extracts of algae, fungi, plants, yeasts or bacteria,
hydrolysed, partially
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CA 02850685 2015-11-06
hydrolysed or unhydrolysed proteins such as enzymes, antibacterial or
bactericidal agents e.g.,
2,4,4'-trichloro-2'-hydroxydiphenyl ether (triclosan) and 3,4,4'-
trichlorocarbanilide (or
triclocarban) and azelaic acid, matt-effect agents, for instance fibres,
tensioning agents, and
mixtures thereof. Amounts of such agents typically range from about 0.0001% to
about 20% by
weight of the composition.
[0056] The composition may be packaged in a suitable container. The choice of
container may
depend upon the viscosity and intended use of the composition by the consumer.
For example,
a lotion or fluid cream can be packaged in a bottle or a roll-ball applicator,
or a capsule, or a
propellant-driven aerosol device or a container fitted with a pump suitable
for finger operation.
When the composition is a cream, it can simply be stored in a non-deformable
bottle or squeeze
container, such as a tube or a lidded jar.
[0057] In another aspect, the extract and/or compositions comprising the
extract of the present
invention can be formulated for administration as foods or dietary supplements
using one or
more consumable carriers. A "consumable carrier" is herein defined as any
food, food
ingredient, or food additive, or any excipient utilized for tabletting,
encapsulation, or other
formulation of an active agent for oral administration, whether for human or
animal use. For
dietary supplements, the extract can be mixed according to methods routine in
the art. Dietary
supplements can be prepared in a variety of forms including, but not limited
to, liquid, powder, or
solid pill forms. The extract or composition of the present invention can be
administered either
alone or in combination with other compounds or extracts where combining
compounds or
extracts would lead to additive or synergistic effects. The extract and/or
composition of the
present invention can also be added directly to foods and ingested as part of
a normal meal.
Various methods are known to those skilled in the art for addition or
incorporation of such
agents into foods.
[0058] The compositions of the invention herein can comprise, consist
essentially of, or
consist of, the ingredients and components (e.g., the extract) described
herein. "Consisting
essentially of' as used herein means that the composition may include
additional ingredients,
but only if the additional ingredients do not materially alter the basic and
novel characteristics of
the claimed compositions or methods.
[0059] In an embodiment the subject is a mammal, in a further embodiment, a
human.
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. .
[0060] The present invention is illustrated in further detail by the following
non-limiting
examples.
[0061] The articles "a," "an" and "the" are used herein to refer to one or to
more than one (i.e.,
to at least one) of the grammatical object of the article. The terms
"including" and "comprising"
are used herein to mean, and are used interchangeably with, the phrases
"including but not
limited to" and "comprising but not limited to."
Example 1: Fucoxanthinol Extraction Process
[0062] A quantity of 50 kg of frozen digestive system organs and associated
tissue from S.
droebachiensis was obtained for use in the extraction process. The material
was kept at room
temperature for approximately 48 hours in order to completely defrost. The
thawed material was
homogenized in an industrial blender at 2500 RPM for approximately 30 minutes
in order to
achieve optimal homogenization.
[0063] The 50 kg of homogenized material, still at room temperature, was
combined with 50 L
of 100% ethanol to give a 1:1 v/v ratio, and allowed to mix in a large vat for
8 to 12 hours with a
mixer. The slurry was allowed to settle overnight prior to the centrifugation
step. After settling,
50 L of the liquid was pumped from the top of the extraction vat. Fresh
ethanol was added to
the slurry and the mixing and settling steps were repeated. A total of 3
extractions were
conducted.
[0064] For each extraction, the pumped liquid supernatant was subjected to
centrifugation in
1.4 L quantities at 4060 rpm for 10 minutes. Following centrifugation, the
cleared supernatant
was placed in a Heidolph Tm rotary evaporator and evaporated at 50 C under
vacuum (50
mbar). Late in the evaporation but prior to the complete evaporation of the
ethanol/water mix,
the liquid was filtered through a 45 pm filter in order to remove any
remaining solids or
precipitates.
[0065] Following filtration, the remaining liquid was subjected to evaporation
again until no
further water or solvent was able to be removed. The final liquid which
contained the extracted
fucoxanthinol had a brown colour and was of an oily consistency.
CA 02850685 2016-02-22
Example 2: Analysis of Concentration Factors
[0066] The extract containing fucoxanthinol was tested for fucoxanthin and
fucoxanthinol
content in order to determine the concentration factors comparing the raw
material to the final
extract. The concentration factors are outlined below:
Table 1: Concentration Factors for Fucoxanthin and Fucoxanthinol
Concentration
Analytes Units Raw materials Final extract
factors
Fucoxanthinol mg/100 g 8.4 250.0 29.8
Fucoxanthin mg/100g 2.8 88.4 31.6
Fat g/100 g 1.9 51.7 27.2
Analysis Method Summary
[0067] Quantitation of fucoxanthin and fucoxanthinol was carried out using
HPLC DAD
(Extraction column: C18 5 pm 15 cm x 4.6 mm; Amax=450 nm) and HPLC-MS (APCI,
Mode +,
Full Scan) with specific mass for fucoxanthinol 617=[M+H]+, 599=[(M+H) -H20]+,
581=[(M+H)-2
H20]+, 563=[(M+H)-3 H20]+ and fucoxanthin 659=[M+H]+, 641=[(M+H) -H20]+,
623=[(M+H)-2
H20]+, 605=[(M+H)-3 H20]+.
[0068] Concentration factor = Final extract/Raw materials
30
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=
Example 3: Composition of Final Extract
[0069] Samples of the final fucoxanthinol-containing extract were subjected to
standardized
tests in order to provide a composition analysis. Table 2, below, provides the
results for three
representative samples.
Table 2: Composition of Fucoxanthinol Extract
Analytes Units Sample 1 Sample 2 Sample 3
Moisture g/100g 13.9 14.9 12.4
Protein g/100g 19.5 22.3 21.3
Fat g/1 00g 53.9 48.8 52.3
Ash g/100g 6.6 8.8 6.3
Carbohydrates g/1 00g 8.9 5.2 7.8
Method Summary
= OAS-FC01: Determination of Moisture in Foods (AOAC 950.46b(a))
= OAS-FC04: Determination of Protein in Foods (AOAC 981.10)
= OAS-FC06: Determination of Fat in Foods by Acid Hydrolysis (AOAC 922.06)
= OAS-FCO2: Determination of Ash in Foods (AOAC 920.153)
= Carbohydrates: Calculation
= Calories: Calculation
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Example 4: Microbiological Analysis of Final Extract
[0070] In order to ensure that the final extract obtained was safe and
suitable for human
consumption and use in medicinal compounds or treatments, the extract was
subjected to
standardized tests to determine the microbiological parameters. The results of
three
representative samples are provided in Table 3, below.
Table 3: Microbiological Analysis of Fucoxanthinol Extract
Analytes Units Sample 1 Sample 2 Sample 3
Total Bacteria Count cfu/g <10 < 10 <10
Coliforms MPN/g <3 <3 <3
E. coil MPN/g <3 <3 <3
Staphylococcus aureus cfu/g <5 <5 <5
Salmonella 25 g nf nf nf
Yeasts/Molds g < 10 /< 10 < 10 /< 10 < 10 /< 10
Method Summary
[0071] Tests were performed according to the corresponding Compendium of
Analytical
Methods, Health Protection Branch and/or AOAC Official Methods
= Total Bacteria Count: MFHPB-18
= Coliforms: FFA05 as in MFHPB-19
= E. coil: FFA05 as in MFHPB-19
= Staphylococcus aureus : MFHPB-21
= Salmonella: MFHPB-20
= Yeasts/Molds: MFHPB-22
= nf = not found
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Example 5: Analysis for Heavy Metals Contained in Final Extract
[0072] In order to ensure that the final extract obtained was safe and
suitable for human
consumption and use in medicinal compounds or treatments, the extract was
subjected to
standardized tests to determine the amounts of any heavy metals present in the
extract. The
results of three representative samples are provided in Table 4, below.
Table 4: Heavy Metal Analysis of Fucoxanthinol Extract Samples
Analytes Units Sample 1 Sample 2 Sample 3
Arsenic mg/kg 42.3 46.1 37.1
Cadmium mg/kg <0.02 <0.02 <0.02
Lead mg/kg <0.02 <0.02 <0.02
Mercury mg/kg <0.005 <0.005 <0.005
Method Summary
[0073] Portions of the samples were prepared by Microwave Assisted Digestion
in nitric acid.
The resulting solutions were analyzed for trace elements by ICP-MS. Mercury
was analyzed by
Cold Vapour AAS.
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