U.S. patent application number 14/423995 was filed with the patent office on 2015-07-23 for extraction method.
This patent application is currently assigned to The Product Makers (Austrilia) Pty Ltd. The applicant listed for this patent is PHYTOLIN PTY LTD, The Product Makers (Australia) Pty Ltd. Invention is credited to David Kannar, Barry James Kitchen, Lance Sparrow, Gregory Yu Foo Szto.
Application Number | 20150201660 14/423995 |
Document ID | / |
Family ID | 50182261 |
Filed Date | 2015-07-23 |
United States Patent
Application |
20150201660 |
Kind Code |
A1 |
Kannar; David ; et
al. |
July 23, 2015 |
Extraction Method
Abstract
The present invention relates to a process for producing an
extract derived from sugar cane, the process comprising: i) mixing
a sugar cane derived product with ethanol to produce an extraction
mixture comprising at least about 50% v/v ethanol; ii) allowing a
precipitate to form in the extraction mixture; iii) removing the
precipitate from the extraction mixture to obtain a supernatant;
and iv) removing ethanol from the supernatant to produce the
extract derived from sugar cane. The present invention further
relates to extracts produced according to the process of the
invention. The invention also relates to the use of such extracts
in a method of lowering the available calorific value of a food or
beverage, in treating or preventing disease, and as a nutritional
supplement, dietary supplement, sports nutrition product, food
coating or pharmaceutical product.
Inventors: |
Kannar; David; (Victoria,
AU) ; Kitchen; Barry James; (Victoria, AU) ;
Sparrow; Lance; (Victoria, AU) ; Szto; Gregory Yu
Foo; (Victoria, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Product Makers (Australia) Pty Ltd
PHYTOLIN PTY LTD |
Keysborough, Victorie
Toorak, Victoria |
|
AU
AU |
|
|
Assignee: |
The Product Makers (Austrilia) Pty
Ltd
Keysborough, Victorie
AU
|
Family ID: |
50182261 |
Appl. No.: |
14/423995 |
Filed: |
August 28, 2013 |
PCT Filed: |
August 28, 2013 |
PCT NO: |
PCT/AU2013/000964 |
371 Date: |
February 25, 2015 |
Current U.S.
Class: |
424/750 ;
426/590; 426/655 |
Current CPC
Class: |
A61P 43/00 20180101;
C13B 10/06 20130101; C13B 10/14 20130101; A23L 2/60 20130101; A23L
29/30 20160801; A61K 36/899 20130101; A23L 21/00 20160801; A23L
33/125 20160801; A23L 33/10 20160801; A23L 7/00 20160801; A23L
27/33 20160801; B01D 15/08 20130101; A61P 29/00 20180101 |
International
Class: |
A23L 1/30 20060101
A23L001/30; B01D 15/08 20060101 B01D015/08; A23L 2/60 20060101
A23L002/60; A61K 36/899 20060101 A61K036/899; A23L 1/236 20060101
A23L001/236 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 28, 2012 |
AU |
2012903726 |
Claims
1. A process for producing an extract derived from sugar cane, the
process comprising: i) mixing a sugar cane derived product with
ethanol to produce an extraction mixture comprising at least about
50% v/v ethanol; ii) allowing a precipitate to form in the
extraction mixture; iii) removing the precipitate from the
extraction mixture to obtain a supernatant; and iv) removing
ethanol from the supernatant to produce the extract derived from
sugar cane.
2. The process of claim 1, wherein the process further comprises
removing water from the extract to produce an aqueous extract
having about 65.degree. Bx (Brix).
3. The process of claim 1 or claim 2, wherein the sugar cane
derived product is selected from molasses, bagasse, first expressed
juice, mill mud, clarified sugar juice, clarified syrup, treacle,
golden syrup, field trash, cane strippings and/or dunder.
4. The process of any one of the preceding claims, wherein the
process further comprises subjecting the supernatant to ion
exchange chromatography, hydrophobic interaction chromatography,
liquid LCMS and/or MALDI-TOF to produce the extract.
5. The process of any one of the preceding claims, wherein the
extraction mixture comprises about 70% to about 85% ethanol.
6. The process of any one of the preceding claims, wherein the
process further comprises a preliminary extraction.
7. The process of claim 6, wherein the preliminary extraction
comprises: i) mixing a sugar cane derived product with ethanol to
produce a preliminary extraction mixture; ii) allowing a
precipitate to form in the preliminary extraction mixture; iii)
removing the precipitate from the preliminary extraction mixture to
obtain a preliminary supernatant; and iv) subjecting the
preliminary supernatant to the process described in any one of
claims 1 to 5.
8. The process of claim 7, wherein the preliminary extraction
mixture comprises at least about 25% v/v ethanol.
9. An extract produced by the process of any one of the preceding
claims.
10. The extract of claim 9, wherein the extract comprises at least
25 mg/ml flavonoids, and/or at least 25 mg/ml polyphenols.
11. The extract of claim 9 or 10 which comprises one or more of
tricin, apigenin, luteolin, caffeic acid, hydroxycinnamic acids,
sinapic acid, and derivatives thereof.
12. The extract of any one of claims 9 to 11, wherein the extract
has .alpha.-glucosidase inhibitory activity and/or .alpha.-amylase
inhibitory activity.
13. The extract of any one of claims 9 to 12, wherein the extract
has anti-inflammatory activity.
14. A composition comprising the extract of any one of claims 9 to
13 which is a nutritional supplement, dietary supplement, sports
nutrition product, food coating or pharmaceutical composition.
15. A food or beverage comprising the extract of any one of claims
9 to 13 or the composition of claim 14.
16. A method of lowering the available calorific value of a food or
beverage, the method comprising adding the extract of any one of
claims 9 to 13, and/or the composition of claim 14, to the food or
beverage.
17. A method of treating or preventing disease in a subject, the
method comprising administering to the subject the extract of any
one of claims 9 to 13, the composition of claim 14, and/or the food
or beverage of claim 15.
18. The extract of any one of claims 9 to 13, the composition of
claim 14, and/or the food or beverage of claim 15, for use in the
treatment or prevention of disease.
19. Use of the extract of any one of claims 9 to 13, the
composition of claim 14, and/or the food or beverage of claim 15 in
the manufacture of a medicament for the treatment or prevention of
disease.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to extracts derived from sugar
cane and the subsequent processing streams (e.g., raw sugar,
molasses, bagasse, mill mud and field trash). The present invention
also relates to processes for producing the extracts. The present
invention further relates to use of the extracts to reduce the
available calorific value and/or glycaemic index of foods and
beverages, and to use of the extracts in methods of treating or
preventing diseases such as diabetes and metabolic syndrome as well
as underlying conditions including but not limited to
inflammation.
BACKGROUND
[0002] After being mechanically harvested, sugar cane is
transported to a primary mill and crushed between serrated rollers.
The crushed sugar cane is then pressed to extract the raw sugar
juice, while the bagasse (leftover fibrous material) is used for
fuel. The raw juice is then heated to its boiling point to extract
any impurities, then lime and bleaching agents are added and mill
mud is removed. The raw juice is further heated under vacuum to
concentrate and increase Brix value. The concentrated syrup is
seeded to produce bulk sugar crystals and a thick syrup known as
molasses. The two are separated by a centrifuge and the molasses
waste stream is collected for use as a low-grade animal feedstock.
The sugar refining process thus generates a large number of
products including raw juice, bagasse, mill mud, clarified juice,
molasses, dunder and sugar crystals. Dunder is produced when sugar
or molasses is fermented under controlled conditions to produce
ethanol.
[0003] The bulk sugar crystals from the above process are further
refined to produce many commercially available sugar products. For
example, the further refining may include mixing the bulk sugar
crystals with a hot concentrated syrup to soften the outer coating
on the crystals. The crystals are then recovered by centrifuge and
subsequently dissolved in hot water, a step that is sometimes
called affination. This sugar liquor is then further purified by
carbonation or phosfloatation, filtration, decolourisation and then
seeded with fine sugar crystals. Once the crystals have grown to
the requisite size, the crystals are separated from the syrup by
centrifugation, then dried, graded and packaged. There may be
several repetitions of recovering sugar crystals from the sugar
liquor. The dark sugar syrup which is left after all of the sugar
crystals have been recovered is also called refinery molasses.
[0004] Molasses and other products of the sugar refining process,
especially the thick syrups and juices, are complex mixtures of
substances. Typically they are difficult to refine further and
there are often substances in the compositions that poison standard
separating materials. Molasses and the other thick syrups and
juices typically comprise polyphenols, polysaccharides, flavonoids,
peptides and proteins, minerals, organic acids, and mono and
disaccharides. Complex polymers are also formed during processing
(e.g., melanoidins).
[0005] The key consideration with a primary sugar milling operation
is to maximise the extract and recovery of sucrose. Similarly, the
key consideration for sugar refineries processing primary mill
sugar is to improve sucrose purity. Ethanol has been used in the
recovery of sucrose from waste streams such as molasses (U.S. Pat.
No. 1,730,473) and removal of impurities (U.S. Pat. No. 4,116,712;
U.S. Pat. No. 2,000,202). Ethanol has also been used in the
isolation of individual polyphenols from dunder, including tricin,
luteolin or apigenin (WO 2004/014159). In this prior art process,
ethanol was first used in a crude clean up step to crash out
impurities and then as part of a solvent mixture to
chromatographically isolate fractions comprising specific
polyphenols.
[0006] Molasses, golden syrup and treacle have been used as a
health food since the early 20th century and there have been claims
that they are good therapies or cures for a wide range of
disorders. Recent evidence demonstrates that novel sugar cane
phytochemicals contained in, the products of the sugar cane
refining process reduce glycaemic index (GI) and therefore reduce
the risk of obesity and diabetes. However the strong taste of these
sugar cane derived products containing high molecular weight
colourants makes them unpalatable to many people and the high
viscosity of treacle and molasses makes them difficult to handle
and unstable for incorporation into other foodstuffs. The other
products of cane sugar refining such as bagasse and mill mud are
known to include potentially useful substances but their hitherto
intractable nature and instability has meant that they are usually
thrown away as waste.
[0007] Extracts derived from sugar cane containing isolated
polyphenols have been produced previously but are not as effective
as complex mixtures of polyphenols and flavonoids derived from
sugar cane products. Extracts derived from sugar cane containing
mixtures of polyphenols and flavonoids have been produced
previously but are dark coloured and bitter tasting, thus reducing
the palatability and/or appearance of the finished food or beverage
to which they are added. Accordingly, it is desirable to develop an
improved extraction method that results in a sugar cane derived
extract having an increased concentration of polyphenols, lower
colour and decreased bitterness to avoid the palatability problems
associated with known extracts. Extracts with these improved
properties would find broader use in foods, nutraceuticals and
pharmaceuticals compared to current offerings.
SUMMARY OF THE INVENTION
[0008] The present inventors have developed a process for producing
a sugar cane derived extract comprising polyphenols and/or
flavonoids in more functionally effective amounts and having
improved taste, lower colour and polysaccharide content. The
extracts of the present invention are believed to provide a
synergistic combination of polyphenols and other components that
are not contemplated in the prior art. As a consequence, the
extract can be added back to foods or beverages in smaller
quantities without significantly affecting the palatability and/or
colour of the product.
[0009] Accordingly, in one aspect the present invention provides a
process for producing an extract derived from sugar cane, the
process comprising:
[0010] i) mixing a sugar cane derived product with ethanol to
produce an extraction mixture comprising at least about 50% v/v
ethanol;
[0011] ii) allowing a precipitate to form in the extraction
mixture;
[0012] iii) removing the precipitate from the extraction mixture to
obtain a supernatant; and
[0013] iv) removing ethanol from the supernatant to produce the
extract derived from sugar cane.
[0014] The extract may be a powder, including a freeze dried powder
or dehydrated powder. Preferably, the extract is a liquid extract,
more preferably an aqueous extract.
[0015] In one embodiment, the process may further include:
[0016] i) mixing the sugar cane derived product with ethanol to
produce a preliminary extraction mixture (e.g., comprising at least
about 25% v/v ethanol);
[0017] ii) allowing a precipitate to form in the preliminary
extraction mixture; and
[0018] iii) removing the precipitate from the preliminary
extraction mixture to obtain a preliminary supernatant.
[0019] The preliminary supernatant may then be subjected to the
process of the invention described above.
[0020] As will be apparent to the skilled person, isopropanol can
be used in place of ethanol. As will also be apparent to the
skilled person, any suitable food grade polar solvents could also
be used in place of ethanol. Suitable food grade polar solvents
include butanol, acetone, ethyl acetate and/or propyl acetate.
[0021] In one embodiment, the process further comprises removing
water from the extract, preferably by evaporation under vacuum, to
produce an aqueous extract having about 65.degree. Bx (Brix).
[0022] Any suitable waste product from the sugar cane milling or
refining process may be used as the sugar cane derived product in
the process of the invention. In one embodiment, the sugar cane
derived product is selected from molasses, bagasse, first expressed
juice, mill mud, clarified sugar juice, clarified syrup, treacle,
golden syrup, field trash, cane strippings and/or dunder.
[0023] In one particular embodiment, the sugar cane derived product
is molasses or dunder, preferably molasses.
[0024] The skilled person will appreciate that in some instances
the sugar cane derived product is mixed with water in order to
enable efficient and/or uniform mixing of the sugar cane product
with ethanol to form the extraction mixture. Accordingly, in one
embodiment, the sugar cane derived product comprises a mixture of
water with a product selected from molasses, bagasse, first
expressed juice, mill mud, clarified sugar juice, clarified syrup,
treacle, golden syrup, field trash, cane strippings and/or
dunder.
[0025] In instances where the sugar cane derived product comprises
solid components, it may be desirable to first blend or homogenise
the sugar cane derived product prior to mixing it with ethanol to
form the extraction mixture. Thus, in another embodiment, the sugar
cane derived product is bagasse, field trash and/or cane strippings
blended or homogenised with water.
[0026] The skilled person can readily determine a suitable period
of time for allowing the precipitate to form in the reaction
mixture. In one embodiment, the precipitate is allowed to form for
about 10 minutes to about 24 hours prior to removing the
precipitate.
[0027] In one embodiment, the precipitate is removed from the
extraction mixture by filtration, centrifugation, and/or by
allowing the precipitate to settle.
[0028] In another embodiment, the extraction mixture is maintained
at a temperature of about 20.degree. C. to about 30.degree. C.
[0029] In yet another embodiment, the ethanol is removed from the
supernatant by evaporation under vacuum.
[0030] In one embodiment, the process is performed in batch. In an
alternative embodiment, the process is performed in continuous
flow.
[0031] In another embodiment, the process further comprises
subjecting the supernatant to membrane filtration and/or ion
exchange, hydrophobic or size exclusion chromatography and
collecting one or more flavonoid and/or polyphenol containing
fractions to produce the extract derived from sugar cane.
[0032] Preferably, the extract derived from sugar cane comprises a
mixture of flavonoids and/or polyphenols. More preferably, the
extract further includes components that exist with flavonoids
and/or polyphenols in sugar cane including minerals, vitamins,
carbohydrates, organic acids and/or fiber. More preferably, the
extract comprises a mixture of flavonoids and/or polyphenols as
glycones and aglycones.
[0033] In one embodiment, the supernatant is subject to size
exclusion membrane filtration or size exclusion chromatography.
[0034] In another embodiment, the process further comprises
subjecting the supernatant to ion exchange chromatography,
hydrophobic interaction chromatography, liquid chromatography-mass
spectrometry (LCMS) and/or matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) to produce the
extract, preferably hydrophobic interaction chromatography.
[0035] In one embodiment, the process further comprises subjecting
the supernatant to hydrophobic interaction chromatography on food
grade resin, preferably FPX66 or similar grades.
[0036] In one embodiment of the process of the invention, the
extraction mixture comprises about 70% to about 85% ethanol.
[0037] In one embodiment, the extract comprises one or more of
tricin, apigenin, luteolin, caffeic acid, hydroxycinnamic acids,
sinapic acid, and derivatives thereof.
[0038] In one embodiment, the extract produced by the process of
the invention has reduced colour per unit concentration of
polyphenols when compared to prior art compositions. The colour of
the extract may be analysed by measuring the absorbance of the
supernatant at 420 nm. Preferably, the supernatant has an
absorbance at 420 nm of about 800 to about 140 milliabsorbance
(mAU) units. The colour of the extract may also be analysed using
the ICUMSA protocol.
[0039] In another aspect, the present invention provides an extract
produced by the process of the invention.
[0040] In one embodiment, the extract produced by the process of
the invention comprises at least 25 mg/ml flavonoids and/or at
least 25 mg/ml polyphenols.
[0041] In one particular embodiment, the extract produced by the
process of the invention comprises one or more of tricin, apigenin,
luteolin, caffeic acid, hydroxycinnamic acids, sinapic acid, and
derivatives thereof.
[0042] In one embodiment, the extract produced by the process of
the invention has .alpha.-glucosidase inhibitory activity and/or
.alpha.-amylase inhibitory activity.
[0043] In one embodiment, the extract produced by the process of
the invention has anti-inflammatory activity.
[0044] In one embodiment, the extract produced by the process of
the invention has calorific value reduction properties and/or
activity which slows the flux of carbohydrates, particularly
monosaccharides, from the gut into the blood. Optionally, the
extract also has glycemix index reduction properties.
[0045] In yet another aspect, the present invention provides an
extract derived from sugar cane, wherein the extract comprises at
least 25 mg/ml flavanoids, and/or at least 25 mg/ml
polyphenols.
[0046] In one embodiment, the extract derived from sugar cane
comprises one or more of tricin, apigenin, luteolin, caffeic acid,
hydroxycinnamic acids, sinapic acid, and derivatives thereof.
[0047] In another embodiment, the extract derived from sugar cane
has .alpha.-glucosidase inhibitory activity and/or .alpha.-amylase
inhibitory activity.
[0048] In one embodiment, the extract derived from sugar cane has
anti-inflammatory activity.
[0049] In one embodiment, the extract derived from sugar cane has
calorific value reduction properties and/or activity which slows
the flux of carbohydrates, particularly monosaccharides, from the
gut into the blood. Optionally, the extract also has glycemix index
reduction properties.
[0050] In another aspect, the present invention provides a
composition comprising the extract of the invention.
[0051] In one embodiment, the composition is a nutraceutical,
dietary supplement, sports nutrition product, food coating or
pharmaceutical composition. Preferably, the composition is a
dietary supplement or a pharmaceutical composition.
[0052] In another embodiment, the composition may include one or
more additional active ingredients. Active ingredients may include,
but are not limited to, fucodian and arcabose. It is believed that
such combinations may have a synergistic effect.
[0053] In another aspect, the present invention provides a food or
beverage comprising the extract of the invention or the composition
of the invention.
[0054] In one embodiment, the food or beverage is selected from a
bakery product, crystallised sugar, confectionary, breakfast
cereal, naturally derived fiber, chemically derived fiber, dairy
product, soft drink, water, coffee, cocoa, tea, or alcoholic
beverage.
[0055] In another embodiment, the food or beverage is a soft drink
selected from a fruit juice containing beverage and a carbonated
soft drink.
[0056] In another aspect, the present invention provides a method
of lowering the glycaemic index of a food or beverage, the method
comprising adding the extract of the invention, and/or the
composition of the invention, to the food or beverage.
[0057] In another aspect, the present invention provides a method
of reducing the available calorific value of a food or beverage,
the method comprising adding the extract of the invention, and/or
the composition of the invention, to the food or beverage. In
addition, or alternatively, the method comprises administering the
composition of the invention to a subject prior to, in conjunction
with, or after consumption of the food or beverage.
[0058] In one embodiment, the food or beverage is selected from a
bakery product, crystallised sugar, confectionary, breakfast
cereal, naturally derived fiber, chemically derived fiber, diary
product, soft drink, water, coffee, cocoa, tea, or alcoholic
beverage.
[0059] In one particular embodiment, the food is crystallised
sugar.
[0060] In another aspect, the present invention provides a method
of treating or preventing disease in a subject, the method
comprising administering to the subject the extract of the
invention, the composition of the invention, and/or the food or
beverage of the invention.
[0061] In one embodiment, the disease that is treated or prevented
is diabetes and/or metabolic syndrome. In one particular
embodiment, the diabetes is type II diabetes. In another
embodiment, the diabetes is type I diabetes.
[0062] In another aspect, the present invention provides the
extract of the invention, the composition of the invention, and/or
the food or beverage of the invention, for use in the treatment or
prevention of disease.
[0063] In another aspect, the present invention provides use of the
extract of the invention, the composition of the invention, and/or
the food or beverage of the invention in the manufacture of a
medicament for the treatment or prevention of disease.
[0064] As will be apparent, preferred features and characteristics
of one aspect of the invention are applicable to many other aspects
of the invention.
[0065] Throughout this specification the word "comprise", or
variations such as "comprises" or "comprising", will be understood
to imply the inclusion of a stated element, integer or step, or
group of elements, integers or steps, but not the exclusion of any
other element, integer or step, or group of elements, integers or
steps.
[0066] The invention is hereinafter described by way of the
following non-limiting Examples and with reference to the
accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0067] FIG. 1. Analysis of polyphenols, tricin and colour (A 420
nm) in ethanolic supernatant samples. Sample 1=0% ethanol, sample
2=50% ethanol, sample 3=66% ethanol, sample 4=75% ethanol, sample
5=80% ethanol, sample 6=83% ethanol, sample 7=86% ethanol.
[0068] FIG. 2. Plot of % glucosidase inhibition vs. log 1.4
.mu.g/ml for fucoidan (control), dunder (sample 6) and molasses
(sample 3).
[0069] FIG. 3. Comparison of % PGE2 inhibition for aspirin
(control), ibuprofen (control), dunder (sample 6) and molasses
(sample 3).
[0070] FIG. 4. HPLC spectra of (a) Sample 1; (b) Sample 2; (c)
Sample 3; (d) Sample 4; (e) Sample 5; (f) Sample 6; (g) phenolics
at various retention times; and (h) flavonoids at various retention
times.
[0071] FIG. 5. Plots of % polyphenol (PP) recovery vs. % ethanol
and % colour removal vs. % ethanol of molassess feedstock samples
having 25.degree. Brix and 48.degree. Brix. x axis=% ethanol; y
axis=% polyphenol recovery and % colour removal.
[0072] FIG. 6. Flow chart of an extraction method to produce a
standardised extract according to the invention.
DETAILED DESCRIPTION
General Techniques and Definitions
[0073] Unless specifically defined otherwise, all technical and
scientific terms used herein shall be taken to have the same
meaning as commonly understood by one of ordinary skill in the art
(e.g., chemistry, biochemistry, food and nutritional science, cell
culture, molecular biology, and immunology).
[0074] [66] Unless otherwise indicated, the recombinant protein,
cell culture, and immunological techniques utilized in the present
invention are standard procedures, well known to those skilled in
the art. Such techniques are described and explained throughout the
literature in sources such as, J. Perbal, A Practical Guide to
Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 3.sup.rdedn, Cold Spring
Harbour Laboratory Press (2001), R. Scopes, Protein
Purification--Principals and Practice, 3.sup.rd edn, Springer
(1994), T. A. Brown (editor), Essential Molecular Biology: A
Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover
and B. D. Hames (editors), DNA Cloning: A Practical Approach,
Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al.
(editors), Current Protocols in Molecular Biology, Greene Pub.
Associates and Wiley-Interscience (1988, including all updates
until present), Ed Harlow and David Lane (editors) Antibodies: A
Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.
E. Coligan et al. (editors) Current Protocols in Immunology, John
Wiley & Sons (including all updates until present).
[0075] "Administering" as used herein is to be construed broadly
and includes administering an extract or composition comprising the
extract as described herein to a subject as well as providing an
extract or composition comprising the extract as described herein
to a cell.
[0076] As used herein, the terms "treating", "treat" or "treatment"
include administering a therapeutically effective amount of an
extract or composition comprising the extract as described herein
sufficient to reduce or delay the onset or progression of a
specified disease, or to reduce or eliminate at least one symptom
of the disease.
[0077] As used herein, the terms "preventing", "prevent" or
"prevention" include administering a therapeutically effective
amount of an extract or composition comprising the extract
sufficient to stop or hinder the development of at least one
symptom of the specified condition.
[0078] The term "about" as used herein refers to a range of +1-5%
of the specified value.
[0079] The term "dietary supplement" as used herein is to be
construed broadly and means a preparation or formulation which is
added to or otherwise included in a subject's normal diet, and is
present in addition to the normal diet. A dietary supplement may be
administered to a subject prior to, in conjunction with, or after
consumption of a food or beverage.
Process for Producing Extract Derived from Sugar Cane
Feedstock for the Extraction Process
[0080] After being mechanically harvested, sugar cane is
transported to a mill and crushed between serrated rollers. The
crushed sugar cane is then pressed to extract the raw sugar juice,
while the bagasse (leftover fibrous material) is typically used for
fuel. The raw juice is then heated to its boiling point to extract
any impurities, then lime and bleaching agents are added and mill
mud is removed. The raw juice is further heated under vacuum to
concentrate and increase Brix value. The concentrated syrup is
seeded to produce bulk sugar crystals and a thick syrup known as
molasses. The two are separated by a centrifuge and typically the
molasses waste stream is collected for use as a low-grade animal
feedstock.
[0081] The extract produced according to the process of the
invention can be derived from any sugar cane derived product,
including those produced during the sugar cane milling process, the
sugar cane refining process and other processes using sugar cane
products.
[0082] Accordingly, the term "sugar cane derived product" as used
herein refers to products of the sugar cane milling and refining
processes including molasses, bagasse, first expressed juice, mill
mud, clarified sugar juice, clarified syrup, treacle, golden syrup,
field trash, cane strippings, growing tips, pulp and dunder. These
sugar cane derived products comprise complex mixtures of substances
including flavonoids, flavones, and polyphenols (Duarte-Almeida et
al., 2006; Colombo et al., 2006; Payet et al., 2006; US
2012/0115941) as well as phytosterols, oligosaccharides,
polysaccharides, mono and disaccharides, organic acids (e.g. cis
and trans aconitic acid), peptides and proteins. The type and
amount of these components vary between the different sugar cane
derived products. For example, molasses includes mono and
disaccharides whereas dunder is almost free of sugar components.
This improves the palatability of extracts derived from molasses
compared with those derived from dunder, which are bitter and less
palatable. This also affects the composition of, for example,
polyphenols in extracts derived from molasses compared with dunder.
Molasses comprises polyphenols that link to the carbohydrate
backbone of mono, di and polysaccharides and cellulose and
hemicellulosic materials such as lignans etc. Accordingly, the
composition of polyphenols in molasses and dunder differ. Extracts
containing compounds such as polyphenols and flavonoids extracted
from a sugar cane derived product can be used in the production of,
for example, functional foods and beverages and nutritional
supplements. Advantageously, extracts that comprise complex
mixtures of substances display improved characteristics compared
with extracts of isolated components, such as tricin, luteolin or
apigenin, or combinations of synthetic compounds. Extracts
according to the invention therefore can comprise one or more of
tricin, apigenin, luteolin, caffeic acid, hydroxycinnamic acids,
sinapic acid, and derivatives thereof. It is believed that natural
biological mixtures can have a synergistic effect and may be better
than synthetic or individually isolated natural products.
[0083] In the process of the invention, the sugar cane derived
product is used as a feedstock and mixed with ethanol to form the
extraction mixture. Ethanol is used to extract a mixture of
flavonoids and/or polyphenols. In contrast, the prior art uses
ethanol to crash out impurities either in the extraction of sucrose
or during the isolation of individual polyphenols (WO 2004/014159).
The skilled person will understand that in order to facilitate
extraction of a mixture of flavonoids and/or polyphenols the rate
of mixing the feedstock with ethanol may need to be controlled as
well as the degree of mixing, ie homogeneity.
[0084] The skilled person will understand that in order to
facilitate mixing of the sugar cane derived product with ethanol,
the sugar cane derived product may need to be mixed with a liquid,
typically water, and/or heated in order to achieve a desired
viscosity. By way of example, molasses is viscous at room
temperature and, as a consequence, mixing of molasses and ethanol
may be difficult to achieve consistently, efficiently or uniformly
unless the viscosity of the molasses is adjusted. In embodiments of
the invention in which the sugar cane derived product is molasses,
for example, the molasses may be mixed with water at a ratio of
molasses to water of from about 75:25 to about 25:75. In one
embodiment, the ratio of molasses to water is about 50:50.
[0085] The sugar cane derived product, either mixed with water or
not, may be heated to decrease viscosity. For example, the sugar
cane derived product may be heated to about 25.degree. C. to about
60.degree. C., more preferably about 25.degree. C. to about
40.degree. C., more preferably about 26.degree. C. to about
30.degree. C.
[0086] For sugar cane derived products comprising solid material
such as bagasse, field trash and cane strippings, it is desirable
that the product is first blended or homogenised with water prior
to mixing with ethanol to form the extraction mixture. The amount
of water with which the sugar cane derived product is blended or
homogenised can be readily determined by the skilled person in
order to achieve a sugar cane derived product having a suitable
viscosity for mixing with ethanol to form an extraction
mixture.
[0087] Preferably the sugar cane derived product will have a
viscosity less than or equal to about 100 centipoise, more
preferably between about 50 to about 100 centipoise.
[0088] The high viscosity of molasses is as a result of the high
total solids (particularly soluble carbohydrates) and this is
typically measured by determination of Brix degrees. Preferably the
sugar cane derived product will have about 20.degree. to about
50.degree. Brix. Feedstocks with high Brix (>50.degree.) behave
differently to lower Brix feedstocks (<30.degree.) with respect
to the separation of polyphenols and colour by increasing levels of
ethanol. This observation is crucial in both small and large scale
separation processes.
[0089] In the process of the invention, it is also desirable that
extremes of pH be avoided in the extraction mixture. Extreme pH can
have a deleterious effect on the components of the extraction
mixture. Accordingly, in one embodiment the extraction mixture has
a pH of about pH 4 to about pH 7.5.
[0090] The extract derived from the process of the invention may be
used without further purification. Optionally, the extract may be
subjected to purification, preferably hydrophobic interaction
chromatography. The purification step removes impurities, such as
pigments that contribute to the colour of the extract. In contrast,
the prior art uses chromatography to isolate fractions of
individual polyphenols (WO 2004/014159).
Addition of Ethanol to the Sugar Cane Derived Product
[0091] To extract compounds such as polyphenols and flavonoids, the
sugar cane derived product is mixed with ethanol to form an
extraction mixture. The present inventors have found that producing
an extraction mixture comprising at least 50% v/v ethanol results
in an increase in precipitate formation in the reaction mixture.
Upon removal of the precipitate, the retained supernatant
advantageously has reduced colour and bitterness compared to a
supernatant prepared from an extraction mixture comprising less
than 50% v/v ethanol.
[0092] In addition, the present inventors found that increasing the
concentration of ethanol in the extraction mixture above 50% v/v
resulted in further reduction in the colour of the retained
supernatant once the precipitate had been removed (FIG. 1).
Accordingly, in one embodiment, the extraction mixture comprises at
least 50% v/v ethanol. In another embodiment, the extraction
mixture comprises at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, 60%, 65%, 70%, or 75% v/v ethanol, or at least 80%, 81%, 82%,
83%, 84% or 85 ethanol v/v.
[0093] The present inventors have found that the optimal
concentration of ethanol in the extraction mixture for removing
colour in the supernatant while minimising reduction in polyphenols
is about 75% to about 85% v/v. Accordingly, in one embodiment, the
extraction mixture comprises about 75% to about 85% v/v ethanol. In
another embodiment, the extraction mixture comprises about 83% v/v
ethanol.
[0094] The present inventors also found that while colour removal
was substantially achieved with a reaction mixture comprising about
70% v/v ethanol, an extraction mixture comprising about 80% to
about 83% v/v ethanol produced an extract that was stable and
produced no precipitate over a 6 month period. Thus, in one
embodiment of the process according to the invention, the
extraction mixture comprises about 80% to about 83% v/v
ethanol.
Removal of Precipitate and Ethanol
[0095] Following the formation of precipitate in the extraction
mixture, the precipitate may be removed from the mixture by any
suitable method known in the art. For example the precipitate may
be removed by centrifugation and the supernatant obtained.
Alternatively, the precipitate may be allowed to settle for a time
sufficient to allow the supernatant to be obtained while leaving
precipitate behind, such as by sedimentation under gravity for
example. The skilled person will understand that other techniques
such as filtration can be used alone or in combination with
centrifugation or sedimentation in order to produce the extract
derived from sugar cane.
[0096] Once the supernatant has been obtained the ethanol is
removed using techniques known in the art. By way of non-limiting
example, the ethanol may be removed from the supernatant by
evaporation, such as by using a rotary evaporator with a heating
bath at approximately 45.degree. C. or higher. In some instances it
may be desirable to further remove water from the supernatant to
obtain an aqueous extract having about 64-65.degree. Bx (degrees
Brix).
Multiple Extraction Process
[0097] In one embodiment of the process of the invention, the
process comprises multiple extractions.
[0098] In a preliminary extraction, a sugar cane derived product is
mixed with ethanol to produce a preliminary extraction mixture
(e.g., comprising at least about 25% v/v ethanol), a precipitate is
allowed to form in the preliminary extraction mixture and the
precipitate is removed from the preliminary extraction mixture to
obtain a preliminary supernatant.
[0099] Then, in a further extraction, the preliminary supernatant
is mixed with ethanol to produce a further extraction mixture
comprising at least about 50% v/v ethanol, a precipitate is allowed
to form in the further extraction mixture, the precipitate is
removed from the further extraction mixture to obtain a further
supernatant and ethanol is removed from the further supernatant to
produce an extract derived from sugar cane. Further ethanol can be
added to a 50% extract supernatant to an ethanol level between
75-83% ethanol. A further precipitate forms and can be recovered.
The supernatant can become the final extract.
Fractionation of the Extract
[0100] In one embodiment of the process of the invention, the
supernatant comprising ethanol, or the extract from which ethanol
has been removed, is fractionated to produce the extract derived
from sugar cane. By way of non-limiting example, the supernatant or
extract may be subject to membrane filtration, size exclusion
chromatography, ion exchange chromatography, and or hydrophobic
interaction chromatography.
[0101] There are several techniques known in the art for separating
compounds based on size. For example, it is known in the art that
components of a supernatant or extract falling within a specific
molecular weight range may be separated by size exclusion
processing methods such as gel permeation chromatography or
ultrafiltration.
[0102] Separation of components in the supernatant or extract may
also be achieved using chromatographic techniques or combinations
of techniques such as ion exchange chromatography, hydrophobic
interaction chromatography, such as for example on XAD or
preferably a food grade resin such as FPX66 resin, which may use
fractional elution by stepwise increase in pH or with suitable
solvents, liquid chromatography-mass spectrometry (LCMS) and/or
matrix-assisted laser desorption/ionization-time of flight
(MALDI-TOF) to produce the extract.
[0103] The supernatants or extracts may be further processed by
standard techniques such as but not limited to microfiltration,
reverse osmosis, gel permeation, vacuum evaporation and freeze
drying, spray drying and tunnel drying.
Testing Extracts for Biological Activity
[0104] Sugar cane derived extracts according to the invention may
be tested for biological activity using known techniques and
assays. For example, an extract according to the invention can be
tested for enzymatic activity. By way of non-limiting examples, the
extracts may be tested for .alpha.-glucosidase inhibitory activity
and/or .alpha.-amylase inhibitory activity.
[0105] As known in the art, .alpha.-glucosidase inhibitory activity
can be measured as the ability of test samples to prevent the
hydrolysis of 4-methylumbelliferyl-.alpha.-D-glucopyranosidase by
yeast (Saccharomyces cerevisiae) .alpha.-glucosidase using known
techniques. As will be understood by the skilled person, acarbose
can be included in the assay as a positive control.
[0106] In addition, .alpha.-amylase inhibitory activity may be
measured by the ability of samples to slow the rate at which
porcine pancreatic amylase hydrolyses labelled starch (E-11954,
Molecular Probes). As understood in the art, acarbose may be
included in the assay as a positive control.
[0107] An extract according to the invention can also be tested for
anti-inflammatory activity. By way of a non-limiting example, the
extracts may be tested for prostaglandin E2 (PGE2) activity. As
known in the art, PGE2 inhibitory activity can be measured by the
ability of test samples to inhibit PGE2 production in 3T3 cells
when stimulated with calcium ionophore. As understood in the art,
aspirin and ibuprofen may be included in the assay as a positive
control.
Uses of the Extracts Derived from Sugar Cane
[0108] The extracts produced according to the process of the
invention can be used in a wide variety of economically useful
applications.
Food and Beverages
[0109] In one embodiment of the invention, an extract produced by
the process of the invention is included in a food or beverage
product. Food or beverages according to the invention can be
prepared by the skilled person using known techniques. Non-limiting
examples of such food or beverage products are baked goods, dairy
type foods and drinks, snacks, etc. The amount of the extract to be
used in a food or beverage can be variable as the extracts
themselves are nutrients.
[0110] Thus, the extract may be used in any food or food product
such as but not limited to sugar, for example crystallised sugar,
confectioneries, snacks (sweet and savoury), cocoa-containing
foods, flavours, dairy products including cheeses, butter, ice
cream, yoghurt, and other dairy spreads; fat-based products
including margarines, spreads, mayonnaise, shortenings, cooking and
frying oils, and dressings; cereal-based and bakery products
including breads and pastas whether these goods are cooked, baked
or otherwise processed; confectioneries including chocolate,
candies, chewing gum, desserts, non-dairy toppings, sorbets, icings
and other fillings; and other miscellaneous food products including
eggs and egg products, processed foods such as soups and
pre-prepared pastas.
[0111] Thus, the extract may be incorporated into or added to
processed foods including but not limited to: breads, pastas,
biscuits, sauces, soups, bars, dairy products (for example, milk,
yoghurts, cheese, ice cream), cakes, ready-to-eat prepared meals,
and breakfast cereals.
[0112] In addition, the extracts may be used in beverages, whether
hot or cold including coffee, tea, cocoa, cereal, chicory and other
plant extract based beverages, alcoholic or non-alcoholic beverages
and including colas and other carbonated and non-carbonated soft
drinks, fruit juices, juice drinks, dairy-based beverages, and meal
replacement drinks.
[0113] The extracts may also be added to food grade ingedients such
as soluble fiber (e.g. oligofructosaccharide), insoluble fiber
(e.g. cocoa bean fiber, sugar cane fiber, oatbran), naturally
derived fibers (e.g., hemicelluloses, lignocelluloses), chemically
derived fibers (e.g., inulin), flour, starch, modified starch,
gelatine, or other food, to produce a unique composition or
ingredient with enhanced levels of polyphenols, flavonoids, and/or
other phytochemicals derived from sugarcane.
[0114] The extract according to the invention is particularly
useful for increasing the active phytochemical content of coffee
products without increasing bitterness. Roasting coffee beans
causes the development of the myriad desired flavours of coffee.
The roasting also causes the bitter taste of coffee which is
related to an increase in the level of the antioxidants,
chlorogenic acid lactones and phenylindanes. The more that coffee
beans are roasted, the more chlorogenic lactones and phenylindanes
are produced. The extract of the invention can be used to increase
the level of active phytochemicals (such as polyphenols) in coffee
products.
[0115] The food or beverage of the present invention can include
additional ingredients including an orally ingestible diluent or
carrier. Many orally ingestible diluents or carriers are known in
the food sciences. These include, but are not limited to,
manufactured cereals, fruit or vegetable products, beverages or
beverage concentrates, ground meat products or vegetable analogues
thereof, and any inert diluent, carrier, or excipient known in the
pharmaceutical art.
Nutritional Supplements and Sports Nutrition Products
[0116] The extract produced according to the process of the
invention may be added to or included in a nutritional supplement.
As used herein, a "nutritional supplement" is an orally ingestible
product consumed to improve overall nutrition, health, well-being,
or performance of a subject in an activity and/or an orally
ingestible product which provides additional perceived nutritional
or biological benefit to a subject. As would be understood, the
nutritional supplement may be provided in a concentrated form, thus
allowing for the addition of the nutritional supplement to a food
or drink product to allow for the consumption of a desired quantity
of an extract of the invention in a reasonable serving size.
[0117] In one embodiment the nutritional supplement is a sports
nutrition product. Thus, the extract may be added to a sports
nutrition product including, by way of non-limiting examples,
sports powders, sports drinks, energy drinks, pre-mixes, juices,
energy bars, energy gels, isotonic drinks and gelatine, starch
based or pectin jellies.
[0118] The nutritional supplements and sports nutrition products of
the current invention can include additional ingredients. In some
embodiments, more than one of the extracts of the current invention
can be included in the same nutritional supplement or sports
nutrition formulation. Other additional ingredients include any
ingestible product. Preferred additional ingredients include, but
are not limited to, other active food supplement ingredients such
as micro-nutrients such as vitamins and minerals or macro-nutrients
such as polyunsaturated fatty acids or fiber. The food additive may
also include acceptable dispersing and suspending agents, and
water. Other conventional nutritional supplements can also be
included if desired. The nutritional supplement or sports nutrition
product can take many forms including, but not limited to, powders,
liquids, tablets, capsules, solutions, concentrates, syrups,
suspensions, or dispersions.
Low GI Products
[0119] The glycaemic index or GI ranks carbohydrates according to
their effect on blood glucose levels. The lower the GI, the slower
the rise in blood glucose level will be when the food is consumed.
Some research has shown that by eating a diet with a lower GI,
people with diabetes, for example, can reduce their average blood
glucose levels. This is important in reducing the risk of
developing diabetes-related complications.
[0120] To determine a food's GI rating, measured portions of the
food containing 10-50 grams of carbohydrate are fed to at least 10
healthy people after an overnight fast. Finger-prick blood samples
are taken at 15-30 minute intervals over the next two hours. These
blood samples are used to construct a blood sugar response curve
for the two hour period. The area under the curve (AUC) is
calculated to reflect the total rise in blood glucose levels after
eating the test food. The GI rating (%) is calculated by dividing
the AUC for the test food by the AUC for the reference food
(usually glucose or white bread) and multiplying by 100. A GI value
of 55 or less is considered `low`, 56-69 is considered "medium" and
over 70 is "high".
[0121] A lower glycemic index suggests slower rates of digestion
and absorption of carbohydrates and is believed to equate to a
lower insulin demand, better long-term blood glucose control and a
reduction in blood lipids. It has been shown that individuals who
followed a low GI diet over many years were at a significantly
lower risk for developing both type 2 diabetes and associated
conditions such as cataracts as well as coronary heart disease.
High blood glucose levels or repeated glycemic "spikes" following a
meal may promote these diseases by both increasing oxidative damage
to the vasculature and via the direct increase in insulin levels.
Postprandial hyperglycemia has been considered a risk factor mainly
associated with diabetes but it is now that it also presents an
increased risk for atherosclerosis and other conditions in the
non-diabetic population.
[0122] Low-GI foods, by virtue of their slow digestion and
absorption, produce gradual rises in blood sugar and insulin levels
and have been shown to improve both glucose and lipid levels in
people with diabetes (type 1 and type 2) and have benefits for
weight control as they help control appetite and delay hunger. Low
GI diets also reduce insulin levels and insulin resistance.
[0123] Thus, the extracts produced according to the process of the
present invention may be used in food or beverages, for example, to
lower or reduce the GI of the food or beverage. By way of example,
the extracts according to the present invention in the form of a
liquid extract can be sprayed onto standard sugar (whether derived
from sugar cane or sugar beets) to produce a low GI sugar. The
extracts according to the present invention in the form of a liquid
extract can also be sprayed onto other carriers such as flour,
starch, bagasse or fiber thus increasing the levels of polyphenols
and/or flavonoids in these food ingredients.
[0124] Endogenous and "adsorbed" polyphenols and other sugarcane
phytochemicals use the fibers to protect these compounds from early
metabolic changes in the gut and increase delivery to specific
sites in the colon where these compounds reduce inflammation and
other disease processes including cyclooxygenase enzymes involved
with intestinal carcinogenesis or polyp formation (Cai et al.).
Low Available Calorific Value Products
[0125] The available calorific value of a food or beverage relates
to the amount of energy available from the food or beverage when
digested. The calories in a food or beverage, as well as the GI of
the food or beverage, which affects the rate of digestion and
absorption, contribute to the available calorific value of the food
or beverage. For example, although chocolate is a low-GI food, it
contains a high number of calories and therefore has a high
available calorific value as determined by conventional methods
including bomb calorimetry.
[0126] The extracts produced according to the process of the
present invention may be used in food or beverages, for example, to
lower or reduce the available calorific value of the food or
beverage. In addition, or alternatively, the process of the present
invention may be used to lower or reduce the available calorific
value of a food or beverage by administering the composition of the
invention, including the dietary supplement and/or pharmaceutical
composition, to a subject prior to, in conjunction with, or after
consumption of the food or beverage. In this way when the food or
beverage is digested the amount of carbohydrate absorbed is
reduced. This effectively reduces the amount of energy available
from the food or beverage when digested. By way of example, the
extracts according to the present invention in the form of a liquid
extract can be sprayed onto standard sugar (whether derived from
sugar cane or sugar beets) to produce a reduced available calorific
value sugar (sucrose). The extracts according to the present
invention in the form of a liquid extract can also be sprayed onto
other carriers such as flour, starch or fiber thus increasing the
levels of polyphenols and/or flavonoids in these food ingredients.
As will be apparent to a skilled person, a powder, such as a freeze
dried powder or dehydrated powder, can be used in place of a liquid
extract.
Therapeutic and Prophylactic Methods
[0127] In the Western diet, 50-70% of dietary calories are derived
from carbohydrate. Approximately half are from simple sugars
(glucose, fructose, sucrose, lactose, maltose and trehalose) with
the remainder coming from complex carbohydrates (hemicelluloses,
galactans, mannans and starch). Complex carbohydrates other than
starch are referred to as dietary fiber which is either partly or
totally digested in the large intestine.
[0128] The digestion of dietary carbohydrates is a physiologically
regulated process in the gastrointestinal tract. Important enzymes
in this process include salivary and pancreatic .alpha.-amylase and
intestinal .alpha.-glucosidase. Beginning in the mouth, food is
chewed and mixed with salivary .alpha.-amylase. Starch released
begins to break down immediately. This hydrolysis process slows or
stops in the stomach because of the change in pH but resumes again
in the duodenum where pancreatic .alpha.-amylase is secreted. The
result is to produce maltose and maltotriose from amylase and
maltose, maltotriose, glucose and dextrin from amylopectin.
[0129] In the 1960's and 70's, debate began about the possible
contributory role of sucrose and fructose in diabetes, obesity and
cardiovascular disease. Many research groups suggested that sugar
and fructose increased risk of diabetes and heart disease by
demonstrating that consuming large amounts elevated blood lipids,
glucose, insulin and uric acid. Accordingly the US Senate Select
Committee on Nutrition recommended that individuals reduce the
amount of sugar in their diets.
[0130] Analysis of sugar consumption from 1980 to 2003 in
Australia, the United Kingdom and United States of America reveals
that per capita consumption of refined sucrose decreased by 23%,
10% and 20% respectively. Prevalence of obesity over the same
timeframe has increased 3 fold in Australians. When all sources of
nutritive sweeteners, including high fructose corn syrups, were
considered, per capita consumption still decreased in Australia
(-16%) and the UK (-5%), but increased in the USA (+23%). This
suggests that once total energy intake has been accounted for, per
capita changes in energy from sucrose may not explain changes in
the incidence of obesity. When sucrose, glucose, or starch were
replaced with >100 g of fructose/day, a weight gain of 0.44
kg/week was observed in adults. Recent evidence demonstrates that
novel sugar cane phytochemicals reduce GI and therefore reduce the
risk of obesity and diabetes.
[0131] The principal enzymes responsible for the breakdown of
carbohydrates in the human body are .alpha.-amylase and
.alpha.-glucosidase and so inhibition of one or both of these
enzymes can result in the GI of foods being reduced. The extracts
of the invention which demonstrate inhibition of the activity of
.alpha.-glucosidase and .alpha.-amylase in vitro will delay
carbohydrate absorption in vivo, thus reducing post-prandial
increase in blood glucose. In addition, the extracts of the
invention are able to lower the GI of a food or beverage.
[0132] Thus, the extracts produced by the process of the invention
are of use in the modulation of biological pathways associated with
disease including diabetes and metabolic syndrome. Thus, in one
aspect, the present invention provides methods of treating or
preventing disease by administering to a subject an extract of the
invention, a composition of the invention, and/or a food or
beverage of the invention.
Compositions and Administration
[0133] In certain embodiments, the present invention provides
compositions comprising an extract of the invention and a suitable
carrier or excipient. In one embodiment, the composition is a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier. The extracts are incorporated into pharmaceutical
compositions suitable for administration to a mammalian subject,
e.g., a human. The extracts may be incorporated into the
pharmaceutical compositions by way of coating a dosage form. Such
compositions typically comprise the "active" compound (i.e. the
extract derived from sugar cane) and a "pharmaceutically acceptable
carrier". As used hereinafter the language "pharmaceutically
acceptable carrier" is intended to include any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. The use of such media and
agents for pharmaceutically active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active compound, such media can be used in
the compositions of the invention. Supplementary active compounds
can also be incorporated into the compositions.
[0134] Because the current extract is higher in concentration
(polyphenols (mg)/extract (ml)) of polyphenols and lower in colour
relative to other extracts from sugar, the extracts are "more
effective" in delivering a therapeutic amount of biologically
active compounds. The lower colour and organoleptic astringency of
some syrups prepared according to the present invention, broaden
their possible use and application in a range of foods and
carriers.
EXAMPLES
Example 1
Preparation of Extract
Feedstock Preparation
[0135] 100 ml of Mackay terminal molasses was measured into a glass
beaker at room temperature (RT). The weight was 140 g. Then 100 mls
of distilled water was added and stirred manually with a glass
stirring rod until most of the viscous molasses was mixed with the
water. The beaker was then placed on a magnetic stirrer and mixed
for 10-15 minutes. The temperature was held at 26-28.degree. C. The
pH of this solution was 5.4-5.6.
[0136] The final volume was 200 ml. A 1 ml sample was removed and
diluted with 1 ml water, mixed well and then a drop was placed on a
Ella refractometer. The Brix reading was 48.
Treatment with AR Ethanol (100% v/v)
[0137] The feedstock (200 ml) was placed in a glass beaker on a
magnetic stirrer and adjusted so that a clear vortex was formed,
ethanol was slowly added into the vortex to ensure rapid mixing of
the feedstock with the ethanol. Over a period of about 30 minutes,
950 ml of ethanol was added. The temperature was maintained at
26-28.degree. C. This resulted in the final ethanol level in the
mixture being 83% v/v. The mixture was stirred for another 30
minutes. A number of sub-samples were taken during the addition of
the ethanol to support the observations as the solution changed in
colour and different coloured precipitates formed as the ethanol %
increased.
Recovery of the 83% Ethanol Supernatant/Extract
[0138] The final mixture was turbid and had a coagulated black
gelatinous precipitate visibly present in the bottom of the beaker.
The supernatant was removed and centrifuged at 4000 rpm
(2500.times.g), for 5 minutes. The clear yellow supernatant was
decanted leaving the black precipitate plug in the centrifuge tube.
About 950 ml of the mixture was centrifuged and the final volume of
supernatant recovered was 880 ml.
Removal of Ethanol from the Supernatant
[0139] Ethanol was removed using a Buchi rotary evaporator under
vacuum. The bath temperature was 45.degree. C. The final
concentrate (free of any ethanol odour) volume was 62 ml and the
Brix level was 64-65. Ethanol, its azeotroph and water were all
removed during this process.
Bioactive Extract
[0140] The final bioactive extract was a dark/yellow colour, free
of any particulate material and with a fresh sweet flavour similar
to that of golden syrup or treacle.
Example 2
Preparation of Extract
Preparation of Feedstock
[0141] 200 ml of feedstock was prepared from Mackay terminal
molasses as previously described in Example 1. The Brix level was
48.
Addition of AR Ethanol (100%)
[0142] 1000 ml of ethanol was added in 200 ml increments so the
formation, colour and appearance of the precipitate could be
observed as the percentage of ethanol increased. Observations were
made at 50, 66, 75, 80 and 83% ethanol (v/v), but no samples were
collected. The final mixture volume (83% ethanol, v/v) was 1200 ml
and this was left standing at 20-25.degree. C. overnight
(approximately 18 hours). As before, the ethanol was slowly added
into a stirring vortex created by rapid magnetic stirring of the
mixture.
Recovery of the 83% Supernatant
[0143] The supernatant was recovered in the same way as described
in Example 1. The volume was 1050 ml.
Removal of Ethanol
[0144] Ethanol was removed at 45.degree. C. under vacuum as
described in Example 1. The final syrup volume was 78 ml and the
Brix level was 63-65. It had a similar, almost identical
dark/yellow colour and taste to the syrup recovered from Example
1.
Observations
[0145] At 50% v/v ethanol, an amorphous like floc/precipitate forms
and the mixture is a dark to gray colour. Its graininess is similar
to the look of curdled milk in coffee.
[0146] Further addition of ethanol to 66% v/v results in a
significant change as a sticky, gelatinous black mass comes out of
solution and starts to settle spontaneously. It appears to be
adhering to the glass beaker sides but it also rapidly sediments to
the bottom of the beaker. As this black precipitate forms the
mixture takes on a clearer yellow but still turbid appearance. The
gray amorphous precipitate that formed at 50% seems to have
disappeared or it has been "captured" by this new black
precipitate. Another possibility is that the 50% precipitate
changes (or even redissolves) between 50 and 66% ethanol. These
first 2 precipitates up to 66% ethanol are quite significant in
terms of the amount of material removed from solution.
[0147] As more ethanol is added, further cloudy caramel coloured
fine precipitates formed (small amounts) up until the final 83%
supernatant was reached. Between 66% to 75% ethanol, a further
small sticky black precipitate formed, similar to the one occurring
up to 66% ethanol.
Example 3
Preparation of Extract
Preparation of Feedstock
[0148] 100 ml of molasses from a primary sugar mill was used. The
crude molasses had a Brix of 78 using an Atago-Pal 2 digital
refractometer. The weight of the material was 145 g. As described
in the previous experiments, 100 ml of distilled water was added to
the 100 ml of molasses, mixed and stirred for 15 minutes to ensure
a homogeneous feedstock. The final feedstock had a Brix of
49-50.
Effect of Adding Increasing Amounts of Ethanol (100% v/v)
[0149] Two separate lots of 7 centrifuge tubes were used and 10 ml
of feedstock was added to each. To the first 7 tubes, distilled
water was added as follows: 0, 10, 20, 30, 40, 50 and 60 ml. To the
second 7 tubes, 100% v/v ethanol was added as follows: 0, 10, 20,
30, 40, 50 and 60 ml. All tubes were mixed and shaken 3 times
during standing at room temperature (25.degree. C.) for 90
minutes.
Removal of Precipitates
[0150] All tubes were centrifuged as described previously.
Supernatants were recovered and measured. The appearance of initial
mixtures as well as the supernatants and precipitates were recorded
by photography (data not shown) and visual descriptions.
Summary of Increasing Ethanol Levels on the Feedstock
[0151] The grey precipitate that formed at 50% ethanol was
comparably larger than all of the others and quite different in
appearance. Increasing ethanol to 66% resulted in a compact black
precipitate which was smaller in volume and any signs of the
initial grey precipitate which formed initially at 50% ethanol
disappeared. At 66% ethanol, the supernatant was clear and dark
yellow. As the ethanol level increased, a similar black precipitate
resulted, probably slightly more than that formed at 66%. As the
percentage ethanol increased, the supernatants became lighter
yellow. All supernatants from 66% up were clear (free of any
retained turbidity). A summary of these observations is provided in
Table 1.
TABLE-US-00001 TABLE 1 Summary of observations (ppt = precipitate)
Feedstock Super ppt. Vol. Tube Vol. mls Ethanol added mls % Ethanol
Vol. mls mls Comments 1 10 0 0 9.5 0.2-0.3 Small ppt 2 10 10 50
15.5 4-5 Grey ppt large 3 10 20 66 27 3-3.5 Black ppt smaller 4 10
30 75 29.5 4 Black ppt 5 10 40 80 46 4 Black ppt 6 10 50 83 55 5
Black ppt 7 10 60 86 65 6 Black ppt
Example 4
Analysis of Extract Polyphenols and Colour
[0152] Supernatants were analysed for polyphenols (colorimetric)
and colour (A 420 nm) to determine recoveries of polyphenols and
degree of removal of colour with increasing levels of ethanol. An
analysis of polyphenol levels in supernatants is provided in Table
2, an analysis of tricin in the supernatant samples is provided in
Table 3, and an analysis of supernatant sample colour is provided
in Table 4.
TABLE-US-00002 TABLE 2 Analysis of polyphenols (PP) in supernatant
samples. % Supernatant Solids PP Total PP Recovery PP (mg/mL) per
Sample EtOH (mL) % (mg/mL) (mg) % unit solids 1 0 9.5 44.8 12.71
120.70 100 28.36 2 50 15.5 33.6 5.71 88.57 73 17.01 3 66 27 13.5
2.99 80.61 67 22.12 4 75 36 8.8 1.89 67.96 56 21.45 5 80 46 6.3
1.39 64.06 53 22.11 6 83 55 4.8 0.97 53.54 44 20.28 7 86 65 4.2
0.83 54.08 45 19.81
TABLE-US-00003 TABLE 3 Analysis of tricin in supernatant samples.
Supernatant Tricin per unit Tricin Sample % EtOH Vol (mL) mg/g
solids in supematant (mg) % Recovery 1 0 9.5 0.113 0.252 1.25 100 2
50 15.5 0.062 0.186 0.92 74 3 66 27 0.039 0.288 0.92 74 4 75 36
0.018 0.206 0.45 61 5 80 46 0.016 0.246 0.60 48 6 83 55 0.010 0.200
0.43 35 7 86 65 0.006 0.149 0.33 26
TABLE-US-00004 TABLE 4 Analysis of supernatant sample colour
(colour units at A 420) Supernatant Colour Total Sample % EtOH Vol
(mL) units/ml colour % Recovery 1 0 9.5 318.8 3028.5 100 2 50 15.5
106.5 1650.1 54 3 66 27 40.0 1080.0 36 4 75 36 19.2 689.8 23 5 80
46 4.5 207.6 7 6 83 55 3.1 167.9 6 7 86 65 2.5 160.6 5
Example 5
.alpha.-Glucosidase Inhibition
[0153] .alpha.-Glucosidase inhibitory activity is measured as the
ability of test samples to prevent the hydrolysis of
4-methylumbelliferyl-.alpha.-D-glucopyranosidase by yeast
(Saccharomyces cerevisiae) .alpha.-glucosidase.
[0154] Table 5 lists the samples tested. Each sample (0.5 mL) was
weighed and freeze-dried to determine its % dry matter. The samples
were then resuspended in either dimethyl sulfoxide (DMSO) or
DMSO:water (1:1) to a concentration of 15 mg/mL, after which the
samples were serially diluted.
TABLE-US-00005 TABLE 5 Composition of samples tested Sample Sample
Description % Dry matter Solvent 1 Molasses, clarified 27.50 DMSO 2
Molasses, 50-75% ppt in H.sub.2O 10.91 DMSO--H.sub.2O 3 Molasses,
75% supernatant 43.46 DMSO 4 Dunder, clarified 22.42 DMSO 5 Dunder,
50-75% ppt in H.sub.2O 23.92 DMSO--H.sub.2O 6 Dunder, 75%
supernatant 32.07 DMSO
[0155] The substrate,
4-methylumbelliferyl-.alpha.-D-glucopyranoside, and enzyme, yeast
.alpha.-glucosidase, were prepared in the assay buffer, sodium
acetate buffer (pH 5.5). The substrate (final concentration 83
.mu.M, 45 .mu.L) was added to 96-well plates containing 45 .mu.L
enzyme (final concentration 1.7 mU/mL) and 10 .mu.L of sample. The
plate was mixed on an orbital shaker for 30 seconds and incubated
for 20 minutes at 37.degree. C. The reaction was stopped by the
addition of 100 mM sodium glycine buffer (100 .mu.L, pH 10.6), the
plate was shaken for a further 30 seconds, and the fluorescence
intensity was measured at .lamda..sub.ex 355 nm and .lamda..sub.en
460 nm. Fucoidan was used as a positive control and sodium acetate
buffer (pH 5.5) was used as a negative control.
[0156] All of the samples showed some degree of .alpha.-glucosidase
inhibitory activity. The highest activity was observed in sample 4
with IC.sub.50=64 .mu.g/mL and the lowest activity was observed in
sample 3 with IC.sub.50=1,037 .mu.g/mL. All of the samples showed
less activity than the positive control fucoidan (IC.sub.50=0.17
.mu.g/mL). Sample 6 produced a more effective reduction in enzyme
activity than sample 3. The skilled person would recognise that,
with further concentration, the extracts could contain higher
amounts of polyphenols and hence be even more effective in
inhibiting .alpha.-glucosidase activity.
Example 6
PEG2 Inhibition
[0157] The in vitro production of PEG2 from 3T3 cells was measured
using the Cayman Chemical Prostaglandin E2 monoclonal EIA (Enzyme
Immuno Assay) kit. The cells were exposed to samples 3 and 6 and
stimulated with calcium ionophore. The cell supernatants were then
assayed for PEG2 production. The cell cytotoxicity of the samples
was tested against 3T3 cells to confirm that the observed PEG2
inhibition was not due to cell cytotoxicity. Aspirin and ibuprofen
were used as positive controls.
[0158] The highest PEG2 inhibition observed for sample 3 and sample
6 was 29.90% and 42.33%, respectively, at 0.488 .mu.g/mL. As shown
in FIG. 3, the PEG2 inhibitory response of sample 6 was similar to
that of ibuprofen (42.14% at 0.488 .mu.g/mL). Inhibition of PEG2
production in the cells, relative to control cells not exposed to
the samples, indicates that the samples act as an anti-inflammatory
agent in vitro.
Example 7
Extraction of Polyphenols from Molasses Using Ion-Exchange
Resins
[0159] Ion exchange resins are sometimes used in sugar refineries
to remove waste colourants from affination liquor which is produced
after the first step towards producing a white sugar. These
colourants include melanoidins, caramels (these are Maillard
reaction products generated by the reaction between sugars and
amino acids driven by temperature and alkaline pH), and natural
colourants such as polyphenols and flavonoids. The affination
liquor is treated with a resin and sugars and a minor amount of
colourants pass through and then this material is further processed
to produce food grade molasses (quite different to primary mill
molasses). The resin is regenerated with an acid wash and the
washings containing some of the colourants and a small amount of
polyphenols are discarded. The resin is then washed with an
alkaline pH 12-13 solution which removes the bound polyphenols and
flavonoids, and this effluent is adjusted to pH 6.5 to 7.0 with
acid or preferably it is passed through an acidic resin column to
"exchange" the alkali for H.sup.+ions so the effluent pH is less
than 7. This process was reproduced in the laboratory on a small
scale.
[0160] Liquid chromatography profiles of polyphenols were monitored
initially in the affination liquor (pH 5.0-6.0) and then in the
alkaline resin extracts (pH 12-13) and then after adjusting these
extracts (pH 6.5-7.0). Polyphenols are extremely sensitive to high
pH conditions forming flavones. Such changes can modify the
bioactivity of these compounds and even reversing the pH from
alkaline to neutral does not necessarily return them to their
previous bioactive properties.
[0161] Liquid chromatography fingerprints from-alkaline pH 12-13
extracts were significantly different to fingerprints from the same
extracts but adjusted to pH 6.5-7.0. A number of major peaks in the
alkaline extracts seemed to have shifted to lower elution times on
the chromatogram, possibly reflecting the structural change to
flavones. When the pH of these alkaline extracts was adjusted to
neutral pH, the peaks shifted to longer elution times and aligned
themselves with peaks obtained from the original liquor
feedstock.
[0162] Storing the alkaline extracts for up to 14 days at room
temperature did not significantly alter the liquid chromatography
fingerprints suggesting that no further changes to flavones was
occurring with time. Further, if alkaline extracts were immediately
returned back to neutrality, polyphenol peaks also remained stable
over 14 days storage.
Example 8
Colour Reduction and Polyphenol Recovery
[0163] The present inventors have surprisingly found that colour
can be removed efficiently without significant loss of polyphenols.
When ethanol concentration was increased beyond 40% a more suitable
extract was produced.
[0164] Table 6 details the various samples tested.
TABLE-US-00006 TABLE 6 Properties of samples tested ICUMSA Total
Phenolics Sample Colour (mg CE/100 g) 1 32240 808 2 37710 1356 3
65210 1169 4 66030 1797 5 32440 943 6 10960 349
[0165] Sample 1: Townsville Terminal Molasses, which has been
clarified by centrifugation. The crude molasses (about 75 Brix) was
adjusted to 25 Brix with water and centrifuged (4,000 rpm for 10
mins). The precipitate was discarded. The initial clarified
feedstock was still 25 Brix and used to produce a 75% ethanol
supernatant, which is Sample 2. 200 ml of this clarified feedstock
was used to make the 75% super extract. Total CE/100 g=808 mg.
ICUMSA 32,240.
[0166] Sample 2: This 75% super was made by initially removing the
50% precipitate, recovering the 50% super and then adding more
ethanol to 75%. Precipitate removed, 75% super recovered and
ethanol removed. The final volume was 80 ml and it was 40 Brix.
Total CE/100mg=1356mg. ICUMSA 37,710.
[0167] Sample 3: Dunder with 22 Brix (as all the sugar had been
removed). The dunder was centrifuged as above and any precipitate
was discarded. 200 ml of this clarified dunder was used to make the
75% super extract (Sample 4). Total CE/100 mg=1169 mg. ICUMSA
65,210.
[0168] Sample 4: This 75% super was made by initially removing the
50% precipitate, recovering the 50% super and then adding more
ethanol to 75%. Precipitate removed, 75% super recovered and
ethanol removed. The final volume was 80 ml and it was 32 Brix.
[0169] Sample 5: Hydrophobic chromatography extract of Sample 4.
FPX66 material recovered from 15 ml of Sample 4. The peak removed
with 70% ethanol was bulked (aliquots were combined) and the
ethanol removed. Final volume was 25 ml.
[0170] Sample 6: Hydrophobic chromatography extract of Sample 2.
FPX66 material recovered from 15 ml of Sample 2. The peak removed
with 70% ethanol was bulked (aliquots were combined) and the
ethanol removed. Final volume was 40 ml.
[0171] The polyphenol recovery and colour reduction calculations
are detailed below.
[0172] Samples 1 & 2: (i) Polyphenol recovery--200 ml of 25
Brix Molasses=808 mg CE. After dilution, extraction and reduction,
25 Brix increased to 40 Brix (40/25=1.6) and volume reduced from
200 ml to 80 ml (200/80=2.5). Therefore, if 100% recovery of
phenols was achieved, total phenolics would have increased to 808
mg CE.times.2.5=2020 CE. Total phenolics of 1356 CE was detected
which means 1356 CE/2020 CE.times.100=67.13% of total CE was
recovered. (ii) Colour reduction--Colour should have theoretically
increased 2.5.times. from 32,240 to 80,600 ICUMSA. Colour detected
was however only 37,710 ICUMSA. Colour was therefore reduced by
(80,600/37,710) 2.14 times or 42,890 ICUMSA units.
[0173] Samples 3 & 4: (i) Polyphenol recovery--200 ml of 22
Brix Dunder=1,169 mg CE. After dilution, extraction and reduction,
22 Brix increased to 32 Brix (32/22=1.45) and volume reduced from
200 ml to 80 ml (200/80=2.5). Therefore, if 100% recovery of
phenols was achieved, total phenolics would have increased to 1,169
mg CE.times.2.5=2,922 CE. Total phenolics of 1,797 CE was detected
which means 1,797 CE/2,922 CE.times.100=61.5% of total CE was
recovered. (ii) Colour reduction--Colour should have theoretically
increased 2.5.times. from 65,210 to 163,025 ICUMSA. Colour detected
was however only 66,030 ICUMSA. Colour was therefore reduced by
(163,025/66,030) 2.5 times or 96,995 ICUMSA units.
[0174] Sample 5: (i) Polyphenol recovery--25 ml of Sample 4 made
from 15 ml starting volume. Theoretical dilution of phenolics was
therefore (15 ml/25 ml=0.6) or 1,797.times.0.6=1,078 mg CE.
Analysis of Sample 5 was 943 mg CE. This means (943 mg CE/1,078 mg
CE) 87.5% of total phenolics were recovered (12.5% were lost). (ii)
Colour reduction--Colour should have theoretically decreased 40%
from 66,030 to 39,618 ICUMSA. Colour detected was however 32,440
ICUMSA. Colour was therefore reduced by
(39,618-32,440=7,178/39,618=18.1%) or 7,178 ICUMSA units. The FPX66
chromatographic method was therefore more effective at reducing
Dunder colour without equivalent loss of phenolics.
12.5/18.1=69.1%. This means that for every ICUMSA colour unit
removed only 30.9% of the phenolics were removed.
[0175] Sample 6: (i) Polyphenol recovery--40 ml of Sample 2 made
from 15 ml starting volume. Theoretical dilution of phenolics was
therefore (15 ml/40 ml=0.375) or 1,356.times.0.375=508 mg CE.
Analysis of Sample 6 was 349 mg CE. This means (349 mg CE/508 mg
CE) 68.7% of total phenolics were recovered (31.3% were lost). (ii)
Colour reduction--Colour should have theoretically decreased 31.3%
from 37,710 to 25,907 ICUMSA. Colour detected was however 10,960
ICUMSA. Colour was therefore reduced by
(25,907-10,960=14,947/25,907=57.7%) or 14,947 ICUMSA units. The
FPX66 chromatographic method was therefore very effective at
reducing molasses colour without equivalent loss of phenolics.
57.7/31.3=54.25% This means that for every ICUMSA colour unit
removed only 54.25% of the phenolics were removed.
[0176] FIG. 4 shows the HPLC fingerprints of the samples as well as
some spectra of unidentified phenolics and flavonoids at various
retention times.
[0177] As shown in FIG. 5, the present inventors observed that
75-85% EtOH is the optimal concentration (which differs from that
reported in WO 2004/014159) as it yields maximal colour reduction
without significant loss of polyphenols.
[0178] The present inventors also observed that, when molasses is
diluted to 25 Brix and then 48 Brix and extraction is broken into
two stages, the colour of the final extract is lower and polyphenol
recovery is higher. Therefore, an extraction method to produce a
standardised extract is as set out in FIG. 6.
[0179] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
above-described embodiments, without departing from the broad
general scope of the present disclosure. The present embodiments
are, therefore, to be considered in all respects as illustrative
and not restrictive.
[0180] The steps, features, integers, compositions and/or compounds
disclosed herein or indicated in the specification of this
application individually or collectively, and any and all
combinations of two or more of said steps or features.
[0181] All publications discussed and/or referenced herein are
incorporated herein in their entirety.
[0182] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is solely for the purpose of providing a context for
the present invention. It is not to be taken as an admission that
any or all of these matters form part of the prior art base or were
common general knowledge in the field relevant to the present
invention as it existed before the priority date of each claim of
this application.
REFERENCES
[0183] Colombo et al. (2006) Phytochem Anal, 17:337-343. [0184]
Duarte-Almeida et al. (2006) Plant Foods for Human Nutrition,
61:187-192. [0185] Cai H et al. (2005) Mol Cancer Ther,
4:1287-1292. [0186] Payet et al. (2006) J Agric Food Chem,
54:7270-7276.
* * * * *