U.S. patent application number 16/637584 was filed with the patent office on 2020-07-09 for use of polyphenol containing sugar cane extracts for preventing, improving or treating a skin condition.
The applicant listed for this patent is The Product Makers (Australia) Pty Ltd. Invention is credited to Matthew FLAVEL, Barry KITCHEN, Gregor MACNAB, Shane MITCHELL, Julian NEOH.
Application Number | 20200215145 16/637584 |
Document ID | / |
Family ID | 65273025 |
Filed Date | 2020-07-09 |
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United States Patent
Application |
20200215145 |
Kind Code |
A1 |
MITCHELL; Shane ; et
al. |
July 9, 2020 |
USE OF POLYPHENOL CONTAINING SUGAR CANE EXTRACTS FOR PREVENTING,
IMPROVING OR TREATING A SKIN CONDITION
Abstract
The present disclosure is in the field of the prevention,
improvement or treatment of skin conditions, for example skin
conditions associated with skin aging and/or skin pigmentation
and/or wound healing and/or psoriasis and/or acne. The disclosure
provides extracts derived from sugar cane comprising polyphenols
for the prevention, improvement or treatment of skin conditions,
including skin conditions associated with skin aging and/or skin
pigmentation and/or wound healing and/or psoriasis and/or acne.
Inventors: |
MITCHELL; Shane;
(Keysborough, Victoria, AU) ; KITCHEN; Barry;
(Keysborough, Victoria, AU) ; MACNAB; Gregor;
(Keysborough, Victoria, AU) ; NEOH; Julian;
(Keysborough, Victoria, AU) ; FLAVEL; Matthew;
(Keysborough, Victoria, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Product Makers (Australia) Pty Ltd |
Keysborough, Victoria |
|
AU |
|
|
Family ID: |
65273025 |
Appl. No.: |
16/637584 |
Filed: |
August 8, 2018 |
PCT Filed: |
August 8, 2018 |
PCT NO: |
PCT/AU2018/050826 |
371 Date: |
February 7, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 17/10 20180101;
A61K 36/899 20130101; A61K 9/0014 20130101; A61Q 19/08 20130101;
A61Q 19/10 20130101; A61P 17/04 20180101; A61Q 7/00 20130101; A61K
9/0019 20130101; A61P 17/02 20180101; A61K 9/06 20130101; A61Q
19/02 20130101; A61K 31/191 20130101; A61P 17/06 20180101; A61K
8/9794 20170801; A61P 17/08 20180101; A61P 17/00 20180101; A61P
17/14 20180101 |
International
Class: |
A61K 36/899 20060101
A61K036/899; A61K 31/191 20060101 A61K031/191; A61P 17/02 20060101
A61P017/02; A61P 17/04 20060101 A61P017/04; A61P 17/06 20060101
A61P017/06; A61P 17/08 20060101 A61P017/08; A61P 17/10 20060101
A61P017/10; A61P 17/14 20060101 A61P017/14; A61Q 19/02 20060101
A61Q019/02; A61Q 19/08 20060101 A61Q019/08; A61Q 7/00 20060101
A61Q007/00; A61K 8/9794 20060101 A61K008/9794; A61K 9/00 20060101
A61K009/00; A61K 9/06 20060101 A61K009/06 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 9, 2017 |
AU |
2017903170 |
Claims
1. A method for preventing, improving or treating a skin condition
in a subject, the method comprising topical or injection
administration of a composition comprising from about 0.05 wt % to
about 50 wt % of an extract derived from sugar cane to the subject,
the extract comprising from about 10 catechin equivalent (CE) g/L
to about 50 CE g/L of polyphenols or from about 100 CE mg/g to
about 500 CE mg/g of polyphenols.
2. The method of claim 1, wherein the composition comprises from
about 0.05 wt % to about 10 wt % of the extract.
3. The method of claim 2, wherein the composition comprises from
about 0.05 wt % to about 5 wt % of the extract.
4. The method of claim 1, wherein the skin condition is selected
from the group consisting of atopic dermatitis, eczema, psoriasis,
dry skin, oily skin, and pruritic skin.
5. (canceled)
6. The method of claim 1, wherein the skin condition is selected
from the group consisting of wrinkles, fine lines, dark spots, age
spots, mottled pigmentation, skin pigmentation, melasma, darkened
skin, skin elasticity, dark circles under the eyes and changes
associated with skin aging.
7. (canceled)
8. (canceled)
9. The method of claim 1, wherein the skin condition is acne.
10. The method of claim 1, wherein the skin condition is wound
healing.
11. The method of claim 1, wherein the skin condition is hair
loss.
12. The method of claim 1, wherein the prevention, improvement or
treatment of the skin condition provides skin moisturisation, skin
exfoliation, skin lightening or colour reduction, skin pigmentation
reduction, skin redness reduction, skin flushing reduction,
inflammation reduction, fine line reduction, wrinkle reduction,
wrinkle depth reduction, skin dryness reduction, skin roughness
reduction, enhanced skin radiance, enhanced skin tone, enhanced
skin clarity, enhanced skin firmness, enhanced skin tightness,
enhanced skin elasticity, and/or enhanced overall skin
appearance.
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. The method of claim 10, wherein the improvement or treatment of
wound healing provides decreased formation of scar tissue, improved
healing time, improved appearance of the healed skin, protection of
the healing skin tissue from oxidative damage, and/or prevention or
treatment of the wound from infection.
18. The method of claim 1, wherein administration is: i) topical;
or ii) by injection.
19. (canceled)
20. The method of claim 1, wherein the composition is administered:
i) once daily; or ii) twice daily.
21. (canceled)
22. (canceled)
23. (canceled)
24. The method of claim 18, wherein the composition is in the form
of a cream, serum or gel.
25. The method of claim 1, wherein the extract comprises from about
15 CE g/L to about 40 CE g/L of polyphenols or about 150 CE mg/g to
about 400 CE mg/g of polyphenols.
26. The method of claim 25, wherein the extract comprises from
about 20 CE g/L to about 30 CE g/L of polyphenols or from about 200
CE mg/g to about 300 CE mg/g of polyphenols.
27. The method of claim 1, wherein the extract is derived from a
sugar cane derived product selected from the group consisting of
molasses, massecuite, bagasse, first expressed juice, mill mud,
clarified sugar juice, clarified syrup, treacle, golden syrup,
field trash, cane strippings, dunder and combinations thereof.
28. (canceled)
29. The method of claim 1, wherein the composition comprises lactic
acid and/or glycolic acid.
30. An extract derived from sugar cane for preventing, improving or
treating a skin condition, the extract comprising from about 10
catechin equivalent (CE) g/L to about 50 CE g/L of polyphenols or
from about 100 CE mg/g to about 500 CE mg/g of polyphenols, and
wherein the medicament is formulated to be administered topically
or by injection and comprises from about 0.05 wt % to about 50 wt %
of the extract derived from sugar cane.
31. A composition comprising from about 0.05 wt % to about 50 wt %
of an extract derived from sugar cane for use in preventing,
improving or treating a skin condition in a subject, the extract
comprising from about 10 catechin equivalent (CE) g/L to about 50
CE g/L of polyphenols or from about 100 CE mg/g to about 500 CE
mg/g of polyphenols, and wherein the use is topical or by
injection.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority from Australian
Provisional Patent Application No 2017903170 filed on 9 Aug. 2017,
the content of which is incorporated herein by reference.
TECHNICAL FIELD
[0002] The present disclosure is in the field of the prevention,
improvement or treatment of skin conditions, for example skin
conditions associated with skin aging and/or skin pigmentation
and/or wound healing and/or psoriasis and/or acne. The disclosure
provides extracts derived from sugar cane comprising polyphenols
for the prevention, improvement or treatment of skin conditions,
including skin conditions associated with skin aging and/or skin
pigmentation and/or wound healing and/or psoriasis and/or acne.
BACKGROUND
[0003] The dermis layer of skin includes three crucial components:
collagen, elastin, and glycosaminoglycans (GAGs). These components
form the majority of the Extracellular Matrix (ECM). Proteoglycans
are also present in the ECM.
[0004] Collagen is a structural protein found in connective
tissues, such as skin, tendons, ligaments, cartilage, bones, teeth,
heart valves, and the cornea. Collagen is composed of long fibres
of protein and provides strength to the skin.
[0005] Elastin is a fibrous protein that is highly stretchy and
resilient, providing elasticity to the skin. Elastin allows skin to
resume its shape after stretching, contracting or pinching.
[0006] GAGs are polysaccharides. GAGs are water-binding substances
and in combination with water, GAGs create a fluid that fills the
space between the collagen and elastin fibers in the dermis, giving
it turgidity (bounce). There are various GAGs in the dermis, the
most common being: hyaluronic acid, chondroitin sulfate, keratin
sulfate, dermatan sulfate, heparin sulfate, and heparin.
[0007] Deterioration of the physical appearance of skin (the skin's
condition) typically appears as a result of aging processes. As
skin ages, it becomes thinner and more easily damaged and its
ability to heal itself is decreased. Among other things, skin aging
is noted by a decrease in volume and elasticity.
[0008] As skin ages, quantitative and qualitative changes in
collagen, elastin and GAGs in the skin occurs, leading to
roughness, wrinkles and sagging of the skin. Melanin in the skin
becomes unevenly distributed causing uneven tone, freckles and age
spots. Sweat- and oil-secreting glands in the skin also decrease,
leaving the skin dry and thin.
[0009] The effects of aging on the skin may also be promoted by
habitual facial expressions, sun damage, smoking, poor hydration or
nutrition, high stress levels, environmental pollution, alcohol or
drug abuse, and various other factors.
[0010] Prevention, improvement and/or treatment methods for the
deterioration of the physical appearance of skin may include sun
protection, daily skin care routines, use of antioxidants,
cessation of smoking and/or improved diet and the like. There are
also cosmetic and/or therapeutic products and procedures for the
physical appearance of skin available on the market. Examples of
cosmetic products include products containing retinoids, vitamin C
or alpha hydroxy acids. Examples of cosmetic procedures include
Botox, chemical peels, dermabrasion, laser treatments and dermal
fillers.
[0011] The cosmetic products and procedures available on the market
have both some dangers and inadequacies for treating and/or
ameliorating the aging and effects of aging of human skins.
Additionally, the cosmetic procedures available are not suitable
for self-administration and can add to increased costs associated
with receiving treatment.
[0012] Other areas of cosmetic importance in relation to skin
conditions include wound healing, psoriasis, acne and hair loss.
Sufferers of psoriasis, acne and hair loss often suffer from
embarrassment. Treatment of wounds can improve healing time,
prevent infection and reduce scarring. Moreover, inflammation,
which is associated with each of wound healing, psoriasis and acne
causes skin redness, swelling and discomfort. Hence, reducing
inflammation can therefore lead to improved wound healing,
treatment of psoriasis and treatment of acne.
[0013] Accordingly, there is a need for improved formulations
related to the treatment or management of skin aging and/or
pigmentation and/or the treatment of wound healing and/or psoriasis
and/or acne and/or hair loss.
[0014] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification 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 disclosure
as it existed before the priority date of each claim of this
application.
SUMMARY
[0015] In one aspect of the disclosure there is provided a method
for preventing, improving or treating a skin condition in a
subject, the method comprising topical or injection administration
of a composition comprising from about 0.05 wt % to about 50 wt %
of an extract derived from sugar cane to the subject, the extract
comprising from about 10 catechin equivalent (CE) g/L to about 50
CE g/L of polyphenols or from about 100 CE mg/g to about 500 CE
mg/g of polyphenols.
[0016] In another aspect of the disclosure there is provided use of
an extract derived from sugar cane in the manufacture of a
medicament for preventing, improving or treating a skin condition,
the extract comprising from about 10 catechin equivalent (CE) g/L
to about 50 CE g/L of polyphenols or from about 100 CE mg/g to
about 500 CE mg/g of polyphenols, and wherein the medicament is
formulated to be administered topically or by injection and
comprises from about 0.05 wt % to about 50 wt % of the extract
derived from sugar cane.
[0017] In another aspect of the disclosure there is provided a
composition comprising from about 0.05 wt % to about 50 wt % of an
extract derived from sugar cane for use in preventing, improving or
treating a skin condition in a subject, the extract comprising from
about 10 catechin equivalent (CE) g/L to about 50 CE g/L of
polyphenols or from about 100 CE mg/g to about 500 CE mg/g of
polyphenols, and wherein the use is topical or by injection.
[0018] In one embodiment, the composition comprises from about 0.05
wt % to about 10 wt % of the extract.
[0019] In one embodiment, the composition comprises from about 0.05
wt % to about 5 wt % of the extract.
[0020] In one embodiment, the skin condition is selected from the
group consisting of atopic dermatitis, eczema, psoriasis, dry skin,
oily skin, and pruritic skin.
[0021] In one embodiment, the skin condition is psoriasis.
[0022] In one embodiment, the skin condition is selected from the
group consisting of wrinkles, fine lines, dark spots, age spots,
mottled pigmentation, skin pigmentation, melasma, darkened skin,
skin elasticity, dark circles under the eyes and changes associated
with skin aging.
[0023] In one embodiment, the skin condition is selected from the
group consisting of wrinkles, fine lines, skin elasticity, and
changes associated with skin aging.
[0024] In one embodiment, the skin condition is selected from the
group consisting of dark spots, age spots, mottled pigmentation,
skin pigmentation, melasma, darkened skin, dark circles under the
eyes and changes associated with skin aging.
[0025] In one embodiment, the skin condition is acne.
[0026] In one embodiment, the skin condition is wound healing.
[0027] In one embodiment, the skin condition is hair loss.
[0028] In one embodiment, the prevention, improvement or treatment
of the skin condition provides skin moisturisation, skin
exfoliation, skin lightening or colour reduction, skin pigmentation
reduction, skin redness reduction, skin flushing reduction,
inflammation reduction, fine line reduction, wrinkle reduction,
wrinkle depth reduction, skin dryness reduction, skin roughness
reduction, enhanced skin radiance, enhanced skin tone, enhanced
skin clarity, enhanced skin firmness, enhanced skin tightness,
enhanced skin elasticity, and/or enhanced overall skin
appearance.
[0029] In one embodiment, the prevention, improvement or treatment
of the skin condition provides skin moisturisation, skin
exfoliation, skin redness reduction, skin flushing reduction, skin
dryness reduction, enhanced skin radiance, enhanced skin tone,
enhanced skin clarity, enhanced skin firmness, enhanced skin
tightness, enhanced skin elasticity, and/or enhanced overall skin
appearance.
[0030] In one embodiment, the prevention, improvement or treatment
of the skin condition provides inflammation reduction.
[0031] In one embodiment, the prevention, improvement or treatment
of the skin condition provides fine line reduction, wrinkle
reduction, wrinkle depth reduction, enhanced skin elasticity,
and/or skin roughness reduction.
[0032] In one embodiment, the prevention, improvement or treatment
of the skin condition provides skin lightening or colour reduction
and/or skin pigmentation reduction.
[0033] In one embodiment, the improvement or treatment of wound
healing provides decreased formation of scar tissue, improved
healing time, improved appearance of the healed skin, protection of
the healing skin tissue from oxidative damage, and/or prevention or
treatment of the wound from infection.
[0034] In another embodiment, the administration is topical.
[0035] In another embodiment, the administration is by
injection.
[0036] In another embodiment, the composition is administered twice
daily.
[0037] In another embodiment, the composition is administered once
daily.
[0038] In one embodiment, the skin is on the face, neck, hands
and/or back.
[0039] In one embodiment, the skin is on the face.
[0040] In one embodiment, the composition is in the form of a
cream, serum or gel.
[0041] In one embodiment, the composition comprises the extract
comprises from about 15 CE g/L to about 40 CE g/L of polyphenols or
about 150 CE mg/g to about 400 CE mg/g of polyphenols.
[0042] In one embodiment, the extract comprises from about 20 CE
g/L to about 30 CE g/L of polyphenols or from about 200 CE mg/g to
about 300 CE mg/g of polyphenols.
[0043] In one embodiment, the extract is derived from a sugar cane
derived product selected from the group consisting of molasses,
massecuite, bagasse, first expressed juice, mill mud, clarified
sugar juice, clarified syrup, treacle, golden syrup, field trash,
cane strippings, dunder and combinations thereof.
[0044] In one embodiment, the sugar cane derived product is
molasses.
[0045] In one embodiment, the composition comprises lactic acid
and/or glycolic acid.
[0046] Any embodiment herein shall be taken to apply mutatis
mutandis to any other embodiment unless specifically stated
otherwise.
[0047] The present disclosure is not to be limited in scope by the
specific embodiments described herein, which are intended for the
purpose of exemplification only. Functionally-equivalent products,
compositions and methods are clearly within the scope of the
disclosure, as described herein.
[0048] Throughout this specification, unless specifically stated
otherwise or the context requires otherwise, reference to a single
step, composition of matter, group of steps or group of
compositions of matter shall be taken to encompass one and a
plurality (i.e. one or more) of those steps, compositions of
matter, groups of steps or group of compositions of matter.
BRIEF DESCRIPTION OF DRAWINGS
[0049] Whilst it will be appreciated that a variety of embodiments
of the disclosure may be utilised, in the following, we describe a
number of examples of the disclosure with reference to the
following drawings:
[0050] FIG. 1 exhibits an exemplary process for the preparation of
extracts derived from molasses.
[0051] FIG. 2 exhibits another exemplary process for the
preparation of extracts derived from molasses.
[0052] FIG. 3 exhibits base peak chromatograms (FTMS negative) of
three extracts from molasses obtained by the process of FIG. 1 and
analysed by LCMS. A) resin bound sample, B) resin unbound sample,
and C) 74 Brix sample.
[0053] FIG. 4 exhibits .sup.1H NMR spectrum of three extracts from
molasses obtained by the process of FIG. 1 in D.sub.2O with TSP (at
0.00 ppm) as reference. A) resin bound sample, B) resin unbound
sample, and C) 74 Brix sample. Arrows indicate associated peak
signals to specific sugars: nine arrows pointing up--sucrose; two
arrows pointing down and two arrows pointing diagonally
down--glucose; two arrows pointing down in the
middle--fructose.
[0054] FIG. 5 exhibits expanded 0.6-3.2 ppm region of the .sup.1H
NMR spectrum of the resin unbound (B) and resin bound (A) extracts
obtained by the process of FIG. 1 in D.sub.2O with TSP as
reference.
[0055] FIG. 6 exhibits expanded 5.0-10.0 ppm region of the .sup.1H
NMR spectrum of the resin unbound (B) and resin bound (A) extracts
obtained by the process of FIG. 1 in D.sub.2O with TSP as
reference.
[0056] FIG. 7 exhibits the spectra of three extracts from molasses
analysed by GC-MS. A) resin unbound sample (Extract B) prepared
according to the process in FIG. 1, B) resin bound sample (Extract
A) prepared according to the process in FIG. 1, and C) resin bound
sample (Extract D) prepared according to the process in FIG. 2.
[0057] FIG. 8 exhibits a LCMS spectrum of a representative extract
derived from sugar cane molasses prepared according to Example
3.
[0058] FIG. 9 exhibits a process for the preparation of extracts
derived from dunder.
[0059] FIG. 10 exhibits LCMS spectra for sugar cane dunder starting
material (A) and an extract of sugar cane dunder prepared according
to Example 4 (B).
[0060] FIG. 11 exhibits a process for the preparation of extracts
derived from dunder and molasses.
[0061] FIG. 12 exhibits a 58 year old subject over the 12 week
trial. (A) It exhibits the subject's face before the 12 week trial.
(B) After the 12 week trial the subject measured a 49% wrinkle
reduction and a 65% reduction in skin roughness.
[0062] FIG. 13 exhibits a 58 year old subject over the 12 week
trial. (A) It exhibits the subject's face before the 12 week trial.
(B) After the 12 week trial the subject measured a 37% wrinkle
reduction.
[0063] FIG. 14 exhibits a subject suffering from psoriasis over the
6 week trial. (A) It exhibits the subject's knee before the 6 week
trial. (B) It exhibits the subject's knee after 2 weeks of
continuous use of a composition comprising an extract derived from
sugar cane of the present disclosure. (C) It exhibits the subject's
knee after 4 weeks of continuous use of a composition comprising an
extract derived from sugar cane of the present disclosure. (D) It
exhibits the subject's knee after 6 of weeks of continuous use of a
composition comprising an extract derived from sugar cane of the
present disclosure.
[0064] FIG. 15 exhibits a subject suffering from psoriasis over the
3 month trial. (A) It exhibits the subject's knee before the 3
month trial. (B) It exhibits the subject's knee after 1 month of
continuous use of a composition comprising an extract derived from
sugar cane of the present disclosure. (C) It exhibits the subject's
knee after 3 months of continuous use of a composition comprising
an extract derived from sugar cane of the present disclosure.
[0065] FIG. 16 (A) exhibits the elastase inhibition of an extract
derived from sugar cane of the present disclosure; and (B) exhibits
the elastase inhibition of the control compound
N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone.
[0066] FIG. 17 (A) exhibits the collagenase inhibition of an
extract derived from sugar cane of the present disclosure; and (B)
exhibits the collagenase inhibition of control compound
1,10-phenanthroline.
[0067] FIG. 18 (A) exhibits the tyrosinase inhibition of an extract
derived from sugar cane of the present disclosure; (B) exhibits the
tyrosinase inhibition of control compound kojic acid.
[0068] FIG. 19 (A) exhibits the inhibition of melanin production by
an extract derived from sugar cane of the present disclosure; and
(B) exhibits the inhibition of melanin by the control compound
chloroquine.
[0069] FIG. 20 (A) exhibits the anti-proliferative effects of an
extract derived from sugar cane of the present disclosure on mouse
melanoma cells, B16; (B) exhibits 24 hour cell survival of mouse
melanoma cells in the presence of an extract derived from sugar
cane of the present disclosure; (C) exhibits 48 hour cell survival
of mouse melanoma cells in the presence of an extract derived from
sugar cane of the present disclosure; (D) exhibits 72 hour cell
survival of mouse melanoma cells in the presence of an extract
derived from sugar cane of the present disclosure.
[0070] FIG. 21 (A) exhibits the activation of telomerase by an
extract derived from sugar cane of the present disclosure; and (B)
exhibits the activation of telomerase by the control compound
cycloastragenol.
[0071] FIG. 22 (A) exhibits the inhibition of MMP-1 by an extract
derived from sugar cane of the present disclosure; and (B) exhibits
the inhibition of MMP-1 by control compound avobenzone.
[0072] FIG. 23 exhibits the activation of Nrf2 by an extract
derived from sugar cane of the present disclosure.
[0073] FIG. 24 exhibits a representative binding curve for an
extract derived from sugar cane of the disclosure against nuclear
factor .kappa.B (NF-.kappa.B).
[0074] FIG. 25 exhibits a representative binding curve for an
extract derived from sugar cane of the disclosure against tumor
necrosis factor .alpha. (TNF-.alpha.).
[0075] FIG. 26 exhibits a representative inhibition curve for an
extract derived from sugar cane of the disclosure against
prostaglandin E2 (PGE.sub.2).
[0076] FIG. 27 (A) Exhibits representative inhibition curves for an
extract derived from sugar cane of the disclosure against
cyclooxygenase-1 (COX-1), (B) Exhibits representative inhibition
curves for an extract derived from sugar cane of the disclosure
against cyclooxygenase-2 (COX-2).
[0077] FIG. 28 exhibits the anti-bacterial efficacy testing of the
sugar cane extracts of the present disclosure at different
concentrations (0.1-10 mg/ml) (black bars) against the growth of
(A) Escherichia coli, (B) Staphylococcus epidermidis, (C)
Staphylococcus aureus, (D) Staphylococcus mutans and (E)
Propionibacterium acnes, in blood agar following 24-72 h incubation
at 37.degree. C. Positive control penicillin-streptomycin is shown
as white bars. Error bars represent standard deviation of the
means, using well and disc methods.
[0078] FIG. 29 exhibits representative examples of bacterial growth
zones of inhibition caused by different concentrations of the sugar
cane extracts of the present disclosure (0.1-10 mg/ml) on tryptone
soya blood agar plates; disc or well methods are shown. Inhibition
by sugar cane extracts of the present disclosure for (A)
Escherichia coli, (B) Staphylococcus epidermidis, (C)
Staphylococcus aureus, (D) Staphylococcus mutans, (E)
Propionibacterium acnes and (F) Bacterial growth inhibition in the
presence of penicillin-streptomycin.
DETAILED DESCRIPTION
General Techniques and Definitions
[0079] 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, cell culture, molecular biology,
pharmacy, cosmetology, and dermatology). Further, unless otherwise
required by context, singular terms shall include pluralities and
plural terms shall include the singular. Thus, as used in this
specification and the appended claims, the singular forms "a", "an"
and "the" include plural referents unless the context clearly
indicates otherwise. Thus, the term "an subject" means "one or more
subjects" unless the context clearly indicates otherwise.
[0080] "Administering" as used herein is to be construed broadly
and includes administering an extract or a 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.
[0081] The phrase "an effective amount" as used herein, refers to
an amount which is sufficient to elicit the biological or medical
response of a tissue, system, animal or human that is being sought
by the researcher, veterinarian, medical doctor or other clinician.
Undesirable effects, e.g. side effects, are sometimes manifested
along with the desired effect; hence, a practitioner balances the
potential benefits against the potential risks in determining what
an appropriate "effective amount" is. The exact amount required
varies from subject to subject, depending on the species, age and
general condition of the subject, mode of administration and the
like. Thus, it may not be possible to specify an exact "effective
amount". However, an appropriate "effective amount" in any
individual case may be determined by one of ordinary skill in the
art using routine experimentation. The effective amount in this
context includes an amount required to treat or prevent or improve
a skin condition. By "ameliorate" is included relieving of adverse
symptoms, inducing a state of comfort or wellbeing or removing or
reducing biochemical, physiological or clinical markers of the
disease or the condition.
[0082] The terms "treating", "treat", "treatment", "improving",
"improve" or "improvement", as used herein, include administering
an effective amount of an extract of the present disclosure or a
composition comprising the extract sufficient to reduce or delay
the onset or progression of a specified condition, or to reduce or
eliminate at least one symptom of the condition. As would be
understood by those skilled in the art of treating or improving a
skin condition, the term "treatment" includes that the skin
condition is cured, however, it does not necessarily mean that the
skin condition is completely cured.
[0083] The terms "preventing" or "prevent" as used herein, include
administering an effective amount of an extract of the present
disclosure or a composition comprising the extract sufficient to
avoid the onset of a specified condition, or to avoid at least one
symptom of the condition. As would be understood by those skilled
in the art of preventing a skin condition, the term "preventing"
includes that the skin condition is completely prevented, however,
it does not necessarily mean that the skin condition is completely
prevented.
[0084] "Subject" as used herein refers to an animal, such as mammal
including a human who can benefit from the extracts derived from
sugar cane, compositions containing the extracts and methods and
uses described herein. There is no limitation on the type of animal
that could benefit from the presently described extracts derived
from sugar cane, compositions containing the extracts and methods
and uses. A subject regardless of whether a human or non-human
animal may be referred to as an individual, subject, animal, host
or recipient as well as patient. of the present disclosure have
applications in human medicine, human cosmetics, and veterinary
medicine.
[0085] The term "about" as used herein refers to a range of +/-5%
of the specified value.
[0086] The term "CE", or "catechin equivalent" as used herein is a
measure of total polyphenolic content, expressed as mg catechin
equivalents/g crude material or g catechin equivalents/L crude
material.
[0087] The term "GAE", or "gallic acid equivalent" as used herein
is a measure of total polyphenolic content, expressed as mg gallic
acid equivalents/g extract derived from sugar cane or g gallic acid
equivalents/L extract derived from sugar cane.
[0088] The term "free amino acids" as used herein refers to amino
acids which are singular molecules and structurally not attached to
peptide bonds which are attached to other amino acids.
[0089] The term "sugar cane derived product" as used herein refers
to products of the sugar cane milling and refining processes
including, but not limited to, sugar, molasses, massecuite,
bagasse, first expressed juice, mill mud, clarified sugar cane
juice, clarified syrup, treacle, golden syrup, field trash, cane
strippings, leaves, growing tips, pulp and dunder and combinations
thereof. Dunder is the residue produced when a product such as
sugar or molasses is fermented to give, for example, ethanol. Sugar
cane dunder is also referred to as biodunder, stillage or vinasse.
As used herein, the terms "dunder", "bio-dunder", "stillage" and
"vinasse" are equivalent and used interchangeably.
[0090] Throughout this specification, various aspects and
components of the invention can be presented in a range format. The
range format is included for convenience and should not be
interpreted as an inflexible limitation on the scope of the
invention. Accordingly, the description of a range should be
considered to have specifically disclosed all the possible
sub-ranges as well as individual numerical values within that
range, unless specifically indicated. For example, description of a
range such as from 1 to 5 should be considered to have specifically
disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5,
from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual
and partial numbers within the recited range, for example, 1, 2, 3,
4, 5, 5.5 and 6, unless where integers are required or implicit
from context. This applies regardless of the breadth of the
disclosed range. Where specific values are required, these will be
indicated in the specification.
[0091] 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.
Extracts Derived from Sugar Cane to Prevent, Improve or Treat a
Skin Condition
[0092] It has been previously demonstrated that sugar cane waste
and sugar cane extracts provide various benefits to human beings
and animals. For example, sugar cane waste has been used for feed
for animals and for a source to the bio-fuel industry. It has also
been reported that some sugar cane extracts containing
phytochemicals may be used as a nutritional supplements to provide
a boost of energy and that some sugar cane extracts containing
phytochemicals have the ability to lower the glycaemic index (GI)
of foods and beverages. Lowering the GI of foods and beverages has
potential applications, such as in reducing the risk of, and
regulating and/or managing, conditions such as obesity and
diabetes.
[0093] Certain documents provide processes for producing sugar cane
extracts and the use of such extracts in methods of lowering the
available calorific value of foods and/or beverages, in treating or
preventing diseases, and as a nutritional supplements, dietary
supplements, food ingredients, food modifiers, sports nutrition
products, food coatings and/or pharmaceutical products (e.g.
WO/2014/032100, WO/2012/106761).
[0094] However, the use of extracts derived from sugar cane
comprising a specific range of polyphenol content has not
previously been described in the application of preventing,
improving or treating a skin condition. The present inventors have
surprisingly found that the polyphenol containing extracts derived
from sugar cane of the present disclosure can be used to prevent,
improve or treat various types of skin conditions.
[0095] The extracts derived from sugar cane of the present
disclosure have been demonstrated to treat or to improve various
types of skin conditions. The skin conditions include, for example,
atopic dermatitis, acne, eczema, psoriasis, dry skin, oily skin,
pruritic skin, wrinkles, fine lines, dark spots, age spots, mottled
pigmentation, skin pigmentation, melasma, darkened skin, redness,
flushing, inflammation, skin elasticity, dark circles under the
eyes and changes associated with skin aging, hair loss and wound
healing.
[0096] The effects of the treatment or improvement include skin
lightening, fine line reduction, wrinkle reduction, wrinkle depth
reduction, improvement of skin radiance, tone and clarity, skin
inflammation reduction, improvement of skin firmness, tightness,
and elasticity, skin moisturisation, improvement of skin's overall
appearance, skin colour reduction, skin redness reduction, skin
flushing reduction, skin dryness reduction and skin roughness
reduction. The effects of the treatment or improvement also include
reduced itchiness and flakiness. When the skin condition is wound
healing, the effects of the treatment or improvement include
reduced inflammation and therefore decreased formation of scar
tissue, improved healing time and appearance of the healed skin,
protection of the healing skin from oxidative damage and
antibacterial properties which aid in the prevention of the wound
from infection or treatment of the infection, which can delay the
healing process.
Exemplary Processes for Producing Extracts Derived from Sugar
Cane
[0097] A suitable process for producing the extract derived from
sugar cane may be determined by one of ordinary skill in the art.
Exemplary processes are provided below.
Feedstock for the Extraction Process
[0098] 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 raw sugar juice and
leaves fibrous material known as bagasse (typically used as 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 the 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.
[0099] The extracts produced according to the processes of the
disclosure 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.
[0100] Accordingly, the term "sugar cane derived product" as used
herein refers to products of the sugar cane milling and refining
processes including, but not limited to, molasses, massecuite,
bagasse, first expressed juice, mill mud, clarified sugar juice,
clarified syrup, treacle, golden syrup, field trash, cane
strippings, growing tips, pulp, dunder and combinations thereof. In
one embodiment, the sugar can derived product is molasses or
dunder. In another embodiment, the sugar can derived product is
molasses. In another embodiment, the sugar can derived product is
dunder. In another embodiment, the sugar cane derived product is a
combination of molasses and dunder. In another embodiment, the
sugar cane derived product is massecuite. In another embodiment,
the sugar cane derived product is bagasse. In another embodiment,
the sugar cane derived product is first expressed juice. In another
embodiment, the sugar cane derived product is mill mud. In another
embodiment, the sugar cane derived product is clarified sugar cane
juice. In another embodiment, the sugar cane derived product is
clarified syrup. In another embodiment, the sugar cane derived
product is treacle. In another embodiment, the sugar cane derived
product is golden syrup. In another embodiment, the sugar cane
derived product is field trash. In another embodiment, the sugar
cane derived product is cane strippings. In another embodiment, the
sugar cane derived product is leaves. In another embodiment, the
sugar cane derived product is growing tips. In another embodiment,
the sugar cane derived product is pulp.
[0101] Sugar cane derived products generally comprise complex
mixtures of substances including, but not limited to, polyphenols,
phytosterols, monosaccharides, disaccharides, oligosaccharides,
polysaccharides, organic acids, amino acids, peptides, proteins,
vitamins, and minerals.
[0102] As would be understood by one of ordinary skill in the art,
polyphenols are compounds characterized by the presence of multiple
phenol structural units. Polyphenols may be classified into
sub-groups by their chemical structure. Examples of sub-groups of
polyphenols include, but are not limited to, flavonoids (including
flavones, flavanols, flavonols), hydroxybenzoic acids,
hydroxycinamic acids, catechins, proanthocyanidins, anthocyanidins,
stilbenes, lignans, and phenolic acids. The polyphenols of sugar
cane derived products also include conjugates such as, for example,
glycosides, glucosides, galactosides, galacturonides, ethers,
esters, arabinosides, sulphates, phosphates, aldopentoses (xylose,
arabinose) and aldohexoses.
Exemplary Processes Involving an Extraction Step
[0103] One exemplary process with molasses as the sugar cane
derived product is depicted in FIG. 1.
[0104] In one process for producing extracts of the disclosure, the
sugar cane derived product is used as a feedstock and mixed with a
suitable solvent such as ethanol to form an extraction mixture.
[0105] One of ordinary skill in the art will understand that in
order to facilitate mixing of the sugar cane derived product with a
suitable solvent such as ethanol, the sugar cane derived product
may need to be mixed with a liquid, for example but not limited to
water, and/or heated in order to achieve a desired viscosity. In
one embodiment of the disclosure in which the sugar cane derived
product is molasses, for example, the molasses may be mixed with a
liquid, for example, water to achieve a desired viscosity. The
sugar cane derived product, either mixed with a liquid or not, may
be heated to decrease viscosity.
[0106] For sugar cane derived products comprising solid material
such as bagasse, field trash and cane shippings, it is desirable
that the product is first blended or homogenised with a liquid, for
example but not limited to water, prior to mixing with ethanol to
form the extraction mixture. The amount of a liquid with which the
sugar cane derived product is blended or homogenised can be readily
determined by one of ordinary skill in the art in order to achieve
a sugar cane derived product having a suitable viscosity for mixing
with ethanol to form an extraction mixture.
[0107] In one embodiment, the sugar cane derived product will have
a viscosity less than or equal to about 100 centipoise. In another
embodiment, the sugar cane derived product will have a viscosity of
between about 50 to about 100 centipoise. In another embodiment,
the sugar cane derived product will have a viscosity of between
about 50 to about 80 centipoise.
[0108] 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. In one
embodiment, the sugar cane derived product may have about
10.degree. to about 80.degree. Brix. In another embodiment, the
sugar cane derived product may have about 20.degree. to about
70.degree. Brix. In another embodiment, the sugar cane derived
product may have about 200 to about 50.degree. Brix. In another
embodiment, the sugar cane derived product may have about
30.degree. to about 60.degree. Brix. In another embodiment, the
sugar cane derived product may have about 40.degree. to about
50.degree. Brix.
Addition of Ethanol to the Sugar Cane Derived Product
[0109] To extract compounds such as polyphenols, the sugar cane
derived product may be mixed with ethanol to form an extraction
mixture. In one embodiment, the extraction mixture comprises at
least about 50% v/v ethanol. In another embodiment, the extraction
mixture comprises at least about 55%, 60%, 65%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% or 85% v/v
ethanol.
[0110] The optimal concentration of ethanol in the extraction
mixture for removing colour in the supernatant while minimising
reduction in polyphenols is about 70% to about 85% v/v. In one
embodiment, the extraction mixture comprises about 65% to about 75%
v/v ethanol. In one embodiment, the extraction mixture comprises
about 70% to about 80% v/v ethanol. In one embodiment, the
extraction mixture comprises about 70% to about 75% v/v ethanol. In
one embodiment, the extraction mixture comprises about 75% to about
80% v/v ethanol. In one embodiment, the extraction mixture
comprises about 80% to about 85% v/v ethanol. In one embodiment,
the extraction mixture comprises about 80% to about 83% v/v
ethanol. In one embodiment, the extraction mixture comprises about
65% v/v ethanol. In another embodiment, the extraction mixture
comprises about 70% v/v ethanol. In another embodiment, the
extraction mixture comprises about 75% v/v ethanol. In another
embodiment, the extraction mixture comprises about 80% v/v ethanol.
In another embodiment, the extraction mixture comprises about 83%
v/v ethanol. In another embodiment, the extraction mixture
comprises about 85% v/v ethanol.
[0111] In the process of the disclosure, it may be 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. In another embodiment, the
extraction mixture has a pH of about pH 4 to about pH 6. In another
embodiment, the extraction mixture has a pH of about pH 4 to about
pH 5.
Removal of Precipitate and Ethanol
[0112] 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 may be 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, for example, by sedimentation under
gravity. One of ordinary skill in the art 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.
[0113] 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
increase the Brix value of the supernatant. In one embodiment the
process provides an extract having at least about 60.degree. Bx
(degrees Brix).
[0114] In some instances the Bx value of the extract derived from
sugar cane is at least about 65.degree. Bx. In some instances the
Bx value of the extract derived from sugar cane is at least about
70.degree. Bx. In some instances the Bx value of the extract
derived from sugar cane is about 60-65.degree. Bx. In some
instances the Bx value of the extract derived from sugar cane is
about 65-70.degree. Bx. In some instances the Bx value of the
extract derived from sugar cane is about 64-65.degree. Bx. In some
instances the Bx value of the extract derived from sugar cane is
about 70-75.degree. Bx.
Fractionation of the Extract Derived from Sugar Cane
[0115] In one embodiment of the process of the disclosure, the
supernatant comprising ethanol, or the extract derived from sugar
cane from which ethanol has been removed may be used without
further processing. Optionally the supernatant comprising ethanol,
or the extract derived from sugar cane from which ethanol has been
removed may be subjected to purification or fractionation.
[0116] A purification step may remove impurities, such as pigments
that contribute to the colour of the extract derived from sugar
cane. By way of non-limiting example, the supernatant or the
extract derived from sugar cane may be subject to a purification
step which includes, one or more or of, membrane filtration, size
exclusion chromatography, ion exchange chromatography, and/or
hydrophobic interaction chromatography. In one embodiment, the
supernatant or extract may be subjected to hydrophobic interaction
chromatography.
[0117] 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.
[0118] Separation of components in the supernatant and/or the
extract derived from sugar cane may also be achieved using
chromatographic techniques or combinations of techniques. In one
embodiment, chromatographic techniques include, but are not limited
to, ion exchange chromatography, hydrophobic interaction
chromatography, liquid chromatography-mass spectrometry (LCMS)
and/or HPLC. Appropriate stationary and mobile phases of any
chromatographic technique used will be readily determined by one of
ordinary skill in the art. Appropriate elution techniques will also
be readily determined by one of ordinary skill in the art.
Chromatographic techniques may utilise fractional elution by
stepwise increase in pH or with suitable solvents.
[0119] In one embodiment, the supernatant and/or the extract
derived from sugar cane is subjected to one or more chromatographic
techniques. In one embodiment, the supernatant and/or the extract
derived from sugar cane is subjected to hydrophobic interaction
chromatography. In one embodiment, the supernatant and/or the
extract derived from sugar cane is subjected to hydrophobic
interaction chromatography with an XAD, sephadex LH-20 or FPX66
resin. In one embodiment, the supernatant and/or the extract
derived from sugar cane is subjected to sephadex LH-20 resin. In
one embodiment, the supernatant and/or the extract derived from
sugar cane is subjected to XAD resin. In one embodiment, the
supernatant and/or the extract derived from sugar cane is subjected
to FPX66 resin.
[0120] The supernatant and/or the extract derived from sugar cane
may also be processed by standard techniques such as, but not
limited to, microfiltration, reverse osmosis, gel permeation,
vacuum evaporation and freeze drying, spray drying and/or tunnel
drying.
Exemplary Processes without an Extraction Step
[0121] Another exemplary process with molasses as the sugar cane
derived product is depicted in FIG. 2. In this process for
producing extracts of the disclosure, the molasses and is not mixed
with ethanol in a preliminary step. The extract derived from sugar
cane may be obtained from a process without the addition of ethanol
in the first step (FIG. 2).
[0122] To obtain the extract derived from sugar cane, molasses may
first diluted in a liquid, for example but not limited to water, to
a desired Brix value. In one embodiment, the molasses is diluted to
about 20.degree. Bx with water. The components of the diluted
solution may be subjected to one or more chromatographic techniques
known in the art, for example by passing over a FPX66 ion exchange
resin. A range of components from the molasses bind to the ion
exchange resin beads and these components are collected later in
the process as the bound fraction. The unbound fraction is eluted
and may or may not be processed further. Once the unbound fraction
has been removed from the system, ethanol may be used to elute the
bound fraction. In one embodiment, 75% ethanol is used to elute the
bound fraction. Following elution, the ethanol may be evaporated
from the solution. Any method for removing the ethanol may be
employed, including for example, heat exchange and evaporation. In
one embodiment, ethanol is removed by evaporation.
Exemplary Processes with Multiple Filtration Steps
[0123] Another exemplary process for producing an extract according
to the disclosure is described below. This exemplary process
involves multiple filtration steps. This exemplary process with
dunder as the sugar cane derived product is depicted in FIG. 9.
[0124] Sugar cane dunder is allowed to settled overnight (typically
eight hours) in a V-bottom tank. The supernatant is then subjected
to a number of filtration steps. The skilled person will understand
that a variety of filtration steps (such as, for example,
microfiltration or ultrafiltration) may be performed and the
appropriate filtration steps will be readily determined by the
skilled person.
[0125] In one embodiment, the supernatant is subjected to
sequential microfiltration. In one embodiment the supernatant is
sequentially filtered through: (i) a 5 micron filter; (ii) a 1
micron filter; (iii) a 0.5 micron filter; and (iv) a 0.1 micron
filter. The skilled person would understand that a variety of
filters could be used in the process to remove the desired sediment
and undissolved matter. Exemplary filters are stainless steel
filters, ceramic filters and cellulose filters.
[0126] The filtered supernatant is subsequently concentrated to
remove water providing the extract. Any method for removing the
water may be employed, including for example, heat exchange and
evaporation. In one embodiment, the filtered supernatant is
concentrated in a heat exchanger to remove water until the desired
Brix level of the extract is achieved. In one embodiment, the
process provides an extract having at least about 40.degree. Bx. In
one embodiment, the Bx value of the extract is at least about
50.degree. Bx. In one embodiment, the Bx value of the extract is at
least about 55.degree. Bx. In one embodiment, the Bx value of the
extract is at least about 60.degree. Bx. In one embodiment, the Bx
value of the extract is at least about 70.degree. Bx. In one
embodiment, the Bx value of the extract is about 45-55.degree. Bx.
In one embodiment, the Bx value of the extract is about 50.degree.
Bx. In one embodiment, the Bx value of the extract is about
50-55.degree. Bx. In one embodiment, the Bx value of the extract is
about 55-60.degree. Bx. In one embodiment, the Bx value of the
extract is about 50-70.degree. Bx.
Exemplary Processes with Mixtures of Sugar Cane Derived
Products
[0127] Another exemplary process for producing an extract according
to the disclosure is described below. This exemplary process with a
combination of dunder and molasses as the sugar cane derived
product is depicted in FIG. 11.
[0128] Sugar cane mill molasses is mixed with settled sugar cane
dunder (as described above) and stirred well to provide a mixture
with the desired Brix level. The skilled person will understand
that in order to facilitate mixing of the molasses and dunder, a
liquid, for example but not limited to water, may be added. The
liquid may be added to the molasses and/or the dunder prior to
combining the two or the liquid may be added to the combined
molasses and dunder. Additionally, heat may be applied to achieve a
desired viscosity. In one embodiment, the combined mixture of
molasses and dunder is about 50-55.degree. Bx. In one embodiment,
the combined mixture of molasses and dunder is about 50.degree. Bx.
In one embodiment, the combined mixture of molasses and dunder is
about 55.degree. Bx. In one embodiment, the combined mixture of
molasses and dunder is at least about 50.degree. Bx. In one
embodiment, the combined mixture of molasses and dunder is at least
about 60.degree. Bx. In one embodiment, the combined mixture of
molasses and dunder is at least about 70.degree. Bx.
[0129] The combined mixture of molasses and dunder is maintained at
a constant temperature (for example between 20-25.degree. C.) and
ethanol (for example 95% food grade ethanol) is added and stirred
to ensure that the ethanol is evenly and quickly dispersed. Ethanol
is added until the desired ethanol level is reached. The desired
ethanol content can be from about 50% v/v to about 90% v/v. The
desired ethanol content can be about 50, 51, 52, 53, 54, 55, 56,
57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90%
v/v. In one embodiment, the desired ethanol level is at least about
60% v/v. In one embodiment, the desired ethanol level is at least
about 70% v/v. In one embodiment, the desired ethanol level is at
least about 80% v/v. In one embodiment, the desired ethanol level
is about 60-70% v/v. In one embodiment, the desired ethanol level
is about 70-80% v/v. In one embodiment, the desired ethanol level
is about 75% v/v. In one embodiment, the desired ethanol level is
about 76% v/v.
[0130] The addition and mixing of ethanol may lead to the formation
of a gelatinous precipitate. The precipitate in the mixture is
allowed to settle and the supernatant is removed, by, for example
decantation and/or filtration. In one embodiment, the supernatant
is decanted. In one embodiment, the supernatant is filtered. In one
embodiment, the supernatant is decanted and filtered.
[0131] The ethanol is removed from the supernatant to provide the
extract. Any method for removing the ethanol may be employed,
including for example, heat exchange and evaporation. In one
embodiment, the ethanol is removed by evaporation until the desired
Brix level of the extract is achieved. In one embodiment, the
process provides an extract having at least about 50.degree. Bx. In
one embodiment, the Bx value of the extract is at least about
60.degree. Bx. In one embodiment, the Bx value of the extract is at
least about 70.degree. Bx. In one embodiment, the Bx value of the
extract is at least about 80.degree. Bx. In one embodiment, the Bx
value of the extract is about 50-60.degree. Bx. In one embodiment,
the Bx value of the extract is about 60-70.degree. Bx. In one
embodiment, the Bx value of the extract is about 70-80.degree. Bx.
In one embodiment, the Bx value of the extract is about
65-75.degree. Bx. In one embodiment, the Bx value of the extract is
about 75.degree. Bx. In one embodiment, the Bx value of the extract
is about 70.degree. Bx.
Extracts Derived from Sugar Cane
[0132] As described above, extracts derived from sugar cane
generally comprise complex mixtures of substances including, but
not limited to, polyphenols, phytosterols, oligosaccharides,
polysaccharides, monosaccharide, disaccharides, organic acids,
amino acids, peptides, proteins, vitamins, and minerals.
[0133] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises at least about 10 CE g/L of
polyphenols or at least about 150 mg CE/g of polyphenols. As
explained above, the term "CE", or "catechin equivalent" is a
measure of total polyphenolic content, expressed as mg catechin
equivalents/g extract derived from sugar cane or g catechin
equivalents/L extract derived from sugar cane.
[0134] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises at least about 10, 15, 20, 25, 30,
35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 CE g/L
of polyphenols.
[0135] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises at least about 100, 110, 120, 130,
140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 250,
275, 300, 325, 350, 375, 400, 425, 450, 500, 525, 550, 575, 600,
625, 650, 675, 700, 725, 750, 775 or 800 mg CE/g of
polyphenols.
[0136] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises from about 10 CE g/L to about 70
CE g/L of polyphenols or from about 100 CE mg/g to about 700 CE
mg/g of polyphenols.
[0137] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises from about 10 CE g/L to about 60
CE g/L of polyphenols or from about 100 CE mg/g to about 600 CE
mg/g of polyphenols.
[0138] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises from about 10 catechin equivalent
(CE) g/L to about 50 CE g/L of polyphenols or from about 100 CE
mg/g to about 500 CE mg/g of polyphenols.
[0139] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises from about 15 catechin equivalent
(CE) g/L to about 40 CE g/L of polyphenols or from about 150 CE
mg/g to about 400 CE mg/g of polyphenols.
[0140] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises from about 20 catechin equivalent
(CE) g/L to about 30 CE g/L of polyphenols or from about 200 CE
mg/g to about 300 CE mg/g of polyphenols.
[0141] In one embodiment, the extract derived from sugar cane of
the disclosure comprises from about 20 CE g/L to about 27 g CE/L of
polyphenols or from about 200 CE mg/g to about 270 CE mg/g of
polyphenols.
[0142] In one embodiment, the extract derived from sugar cane of
the disclosure comprises from about 27 CE g/L to about 35 g CE/L of
polyphenols or about 270 CE mg/g to about 350 CE mg/g of
polyphenols.
[0143] In one embodiment, the extract derived from sugar cane of
the disclosure comprises from about 35 CE g/L to about 40 g CE/L of
polyphenols or from about 350 CE mg/g to about 400 CE mg/g of
polyphenols.
[0144] In one embodiment, the extract derived from sugar cane of
the disclosure comprises from about 40 CE g/L to about 50 g CE/L of
polyphenols or from about 400 CE mg/g to about 500 CE mg/g of
polyphenols.
[0145] In one embodiment, the extract derived from sugar cane of
the disclosure comprises from about 45 CE g/L to about 50 g CE/L of
polyphenols or about 450 CE mg/g to about 500 CE mg/g of
polyphenols.
[0146] The extract derived from sugar cane of the present
disclosure may contain the flavonoid class of polyphenols. The
extract derived from sugar cane may contain flavonoids in any
amount. In one embodiment, the extract derived from sugar cane of
the disclosure comprises at least about 1 CE g/L of flavonoids or
at least about 10 CE mg/g of flavonoids.
[0147] In one embodiment, the extract derived from sugar cane of
the disclosure comprises from about 1 CE g/L to about 15 CE g/L of
flavonoids or from about 10 CE mg/g to about 150 CE mg/g of
flavonoids. In one embodiment, the extract derived from sugar cane
of the disclosure comprises from about 3 CE g/L to about 10 CE g/L
of flavonoids or about 30 CE mg/g to about 100 CE mg/g of
flavonoids. In one embodiment, the extract derived from sugar cane
of the disclosure comprises about 5 CE g/L to about 8 CE g/L of
flavonoids or about 50 CE mg/g to about 80 CE mg/g of flavonoids.
In one embodiment, the extract derived from sugar cane of the
disclosure comprises about 6 CE g/L to about 8 CE g/L of flavonoids
or about 60 CE mg/g to about 80 CE mg/g of flavonoids. In one
embodiment, the extract derived from sugar cane of the disclosure
comprises about 6.5 CE g/L to about 7.5 CE g/L of flavonoids or
about 65 CE mg/g to about 75 CE mg/g of flavonoids.
[0148] The extract derived from sugar cane of the present
disclosure may contain the proanthocyanidin class of polyphenols.
The extract derived from sugar cane may contain proanthocyandins in
any amount. In one embodiment, the extract derived from sugar cane
of the present disclosure comprises at least about 1.5 CE g/L of
proanthocyanidins or at least about 15 CE mg/g of
proanthocyanidins. In one embodiment, the extract derived from
sugar cane of the disclosure comprises at least about 1.8 CE g/L of
proanthocyanidins or at least about 18 CE mg/g of
proanthocyanidins. In one embodiment, the extract derived from
sugar cane of the disclosure comprises about 1.5 CE g/L to about
2.5 CE g/L of proanthocyanidins or about 15 CE mg/g to about 25 CE
mg/g of proanthocyanidins. In one embodiment, the extract derived
from sugar cane of the disclosure comprises about 1.8 CE g/L to
about 2.2 CE g/L of proanthocyanidins or about 18 CE mg/g to about
22 CE mg/g of proanthocyanidins.
[0149] The extract derived sugar cane of the present disclosure may
be a liquid extract. In one embodiment, the liquid extract is a
syrup.
[0150] The extract derived from sugar cane of the present
disclosure may be in a powder form. In one embodiment, the powder
form is a freeze dried powder form, or a dehydrated powder form or
a spray dried powder form.
[0151] The polyphenols of the extract derived from sugar cane of
the disclosure include, but are not limited to, one or more of
syringic acid, chlorogenic acid, caffeic acid, vanillin, sinapic
acid, vitexin, p-coumaric acid, ferulic acid, gallic acid, vanillic
acid, diosmin, diosmetin, apigenin, vitexin, orientin,
homoorientin, swertisin, tricin, (+)catechin, (-)catechin gallate,
(-)epicatechin, quercetin, kaempherol, myricetin, rutin,
schaftoside, isoschaftoside, luteolin, scoparin and/or derivatives
thereof. The polyphenols of the extract derived from sugar cane of
the present disclosure may also include, but are not limited to,
one or more of hydroxycinnamic acid, isoorientin, swertiajaponin,
neocarlinoside, isovitexin, vicenin, and/or derivatives
thereof.
[0152] The polyphenols of the extract derived from sugar cane also
include conjugates, such as, for example, glycosides, glucosides,
galactosides, galacturonides, ethers, esters, arabinosides,
sulphates, phosphates, aldopentoses (xylose, arabinose) and
aldohexoses.
[0153] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises syringic acid, chlorogenic acid,
caffeic acid, vanillin, sinapic acid, diosmin, diosmetin, apigenin,
vitexin, orientin, homoorientin, swertisin, and tricin and/or
derivatives thereof.
[0154] In one embodiment, the extract derived from sugar cane of
the disclosure comprises syringic acid, chlorogenic acid and
diosmin and/or derivatives thereof.
[0155] In one embodiment, the extract derived from sugar cane of
the disclosure comprises syringic acid. In one embodiment, the
extract derived from sugar cane of the disclosure comprises
chlorogenic acid. In one embodiment, the extract derived from sugar
cane of the disclosure comprises diosmin. In one embodiment, the
extract derived from sugar cane of the present disclosure comprises
caffeic acid. In one embodiment, the extract derived from sugar
cane of the present disclosure comprises vanillin. In one
embodiment, the extract derived from sugar cane of the present
disclosure comprises sinapic acid. In one embodiment, the extract
derived from sugar cane of the present disclosure comprises
vitexin. In one embodiment, the extract derived from sugar cane of
the present disclosure comprises p-coumaric acid. In one
embodiment, the extract derived from sugar cane of the present
disclosure comprises ferulic acid. In one embodiment, the extract
derived from sugar cane of the present disclosure comprises gallic
acid. In one embodiment, the extract derived from sugar cane of the
present disclosure comprises vanillic acid. In one embodiment, the
extract derived from sugar cane of the present disclosure comprises
diosmetin. In one embodiment, the extract derived from sugar cane
of the present disclosure comprises apigenin. In one embodiment,
the extract derived from sugar cane of the present disclosure
comprises orientin. In one embodiment, the extract derived from
sugar cane of the present disclosure comprises homoorientin. In one
embodiment, the extract derived from sugar cane of the present
disclosure comprises swertisin. In one embodiment, the extract
derived from sugar cane of the present disclosure comprises tricin.
In one embodiment, the extract derived from sugar cane of the
present disclosure comprises (+)-catechin. In one embodiment, the
extract derived from sugar cane of the present disclosure comprises
(-)-catechin gallate. In one embodiment, the extract derived from
sugar cane of the present disclosure comprises (-)-epicatechin. In
one embodiment, the extract derived from sugar cane of the present
disclosure comprises quercetin. In one embodiment, the extract
derived from sugar cane of the present disclosure comprises
kaempherol. In one embodiment, the extract derived from sugar cane
of the present disclosure comprises myricetin. In one embodiment,
the extract derived from sugar cane of the present disclosure
comprises rutin. In one embodiment, the extract derived from sugar
cane of the present disclosure comprises schaftoside. In one
embodiment, the extract derived from sugar cane of the present
disclosure comprises isoschaftoside. In one embodiment, the extract
derived from sugar cane of the present disclosure comprises
luteolin. In one embodiment, the extract derived from sugar cane of
the present disclosure comprises scoparin. In one embodiment, the
extract derived from sugar cane of the present disclosure comprises
hydroxycinnamic acid. In one embodiment, the extract derived from
sugar cane of the present disclosure comprises isoorientin. In one
embodiment, the extract derived from sugar cane of the present
disclosure comprises swertiajaponin. In one embodiment, the extract
derived from sugar cane of the present disclosure comprises
neocarlinoside. In one embodiment, the extract derived from sugar
cane of the present disclosure comprises isovitexin. In one
embodiment, the extract derived from sugar cane of the present
disclosure comprises vicenin.
[0156] In one embodiment, syringic acid, chlorogenic acid and
diosmin are the three most abundant polyphenols of the extract
derived from sugar cane of the disclosure.
[0157] In one embodiment, the extract derived from sugar cane of
the disclosure comprises about 5-20 .mu.g/g dry weight of syringic
acid. In one embodiment, the extract derived from sugar cane of the
disclosure comprises about 7-15 .mu.g/g dry weight of syringic
acid. In one embodiment, the extract derived from sugar cane of the
disclosure comprises about 10-12 .mu.g/g dry weight of syringic
acid. In one embodiment, the extract derived from sugar cane of the
disclosure, when present, comprises about 10.9 .mu.g/g dry weight
of syringic acid. The extract derived from sugar cane may be in a
syrup form.
[0158] In one embodiment, the extract derived from sugar cane of
the disclosure comprises about 50-200 .mu.g/g dry weight of
syringic acid. In one embodiment, the extract derived from sugar
cane of the disclosure comprises about 90-130 .mu.g/g dry weight of
syringic acid. In one embodiment, the extract derived from sugar
cane of the disclosure comprises about 100-120 .mu.g/g dry weight
of syringic acid. In one embodiment, the extract derived from sugar
cane of the disclosure comprises about 107 .mu.g/g dry weight of
syringic acid. The extract derived from sugar cane may be in a
powder form.
[0159] In one embodiment, the extract derived from sugar cane of
the disclosure comprises about 1-15 .mu.g/g dry weight of
chlorogenic acid. In one embodiment, the extract derived from sugar
cane of the disclosure comprises about 3-10 .mu.g/g dry weight of
chlorogenic acid. In one embodiment, the extract derived from sugar
cane of the disclosure comprises about 5-8 .mu.g/g dry weight of
chlorogenic acid. In one embodiment, the extract derived from sugar
cane of the disclosure comprises about 6.53 .mu.g/g dry weight of
chlorogenic acid. The extract derived from sugar cane may be in a
syrup form.
[0160] In one embodiment, the extract derived from sugar cane of
the disclosure comprises about 30-150 .mu.g/g dry weight of
chlorogenic acid. In one embodiment, the extract derived from sugar
cane of the disclosure comprises about 60-90 .mu.g/g dry weight of
chlorogenic acid. In one embodiment, the extract derived from sugar
cane of the disclosure comprises about 70-80 .mu.g/g dry weight of
chlorogenic acid. In one embodiment, the extract derived from sugar
cane of the disclosure comprises about 74 .mu.g/g dry weight of
chlorogenic acid. The extract derived from sugar cane may be in a
powder form.
[0161] In one embodiment, the extract derived from sugar cane of
the disclosure comprises about 10-30 .mu.g/g dry weight of diosmin.
In one embodiment, the extract derived from sugar cane of the
disclosure comprises about 15-25 .mu.g/g dry weight of diosmin. In
one embodiment, the extract derived from sugar cane of the
disclosure comprises about 18-21 .mu.g/g dry weight of diosmin. In
one embodiment, the extract derived from sugar cane of the
disclosure comprises about 19-45 .mu.g/g dry weight of diosmin. The
extract derived from sugar cane may be in a syrup form.
[0162] In one embodiment, the extract derived from sugar cane of
the disclosure comprises about 100-300 .mu.g/g dry weight of
diosmin. In one embodiment, the extract derived from sugar cane of
the disclosure comprises about 190-260 .mu.g/g dry weight of
diosmin. In one embodiment, the extract derived from sugar cane of
the disclosure comprises about 210-240 .mu.g/g dry weight of
diosmin. In one embodiment, the extract derived from sugar cane of
the disclosure comprises about 227 .mu.g/g dry weight of diosmin.
The extract derived from sugar cane may be in a powder form.
[0163] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 7-15 .mu.g/g dry weight of
syringic acid, and/or about 4-9 .mu.g/g dry weight of chlorogenic
acid, and/or about 0.1-0.5 .mu.g/g dry weight of caffeic acid,
about 0.05-0.3 .mu.g/g dry weight of vanillin, and/or about 0.1-0.3
.mu.g/g dry weight of sinapic acid, and/or about 15-25 .mu.g/g dry
weight of diosmin, and/or about 0.1-0.4 .mu.g/g dry weight of
orientin, and/or about 0.4-0.9 .mu.g/g dry weight of swertisin,
and/or about 0.05-0.3 .mu.g/g dry weight of disomentin. The extract
derived from sugar cane may be in a syrup form.
[0164] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 10-12 .mu.g/g dry weight of
syringic acid, and/or about 5-8 .mu.g/g dry weight of chlorogenic
acid, and/or about 0.2-0.4 .mu.g/g dry weight of caffeic acid,
and/or about 0.1-0.2 .mu.g/g dry weight of vanillin, and/or about
0.1-0.25 .mu.g/g dry weight of sinapic acid, and/or about 18-21
.mu.g/g dry weight of diosmin, and/or about 0.2-0.3 .mu.g/g dry
weight of orientin, and/or about 0.5-0.8 .mu.g/g dry weight of
swertisin, and/or about 0.1-0.2 .mu.g/g dry weight of disomentin.
The extract derived from sugar cane may be in a syrup form.
[0165] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 10.9 .mu.g/g dry weight of
syringic acid, and/or about 6.53 .mu.g/g dry weight of chlorogenic
acid, and/or about 0.29 .mu.g/g dry weight of caffeic acid, and/or
about 0.153 .mu.g/g dry weight of vanillin, and/or about 0.18
.mu.g/g dry weight of sinapic acid, and/or about 19.45 .mu.g/g dry
weight of diosmin, and/or about 0.245 .mu.g/g dry weight of
orientin, and/or about 0.69 .mu.g/g dry weight of swertisin, and/or
about 0.15 .mu.g/g dry weight of disomentin. The extract derived
from sugar cane may be in a syrup form.
[0166] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 90-130 .mu.g/g dry weight of
syringic acid, and/or about 60-90 .mu.g/g dry weight of chlorogenic
acid, and/or about 4-10 .mu.g/g dry weight of caffeic acid, and/or
about 1-4 .mu.g/g dry weight of vanillin, about 1-3 .mu.g/g dry
weight of sinapic acid, and/or about 190-260 .mu.g/g dry weight of
diosmin, and/or about 3-7 .mu.g/g dry weight of orientin, and/or
3-8 .mu.g/g dry weight of swertisin, and/or about 0.05-0.3 g/g dry
weight of disomentin. The extract derived from sugar cane may be in
a powder form.
[0167] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 100-120 .mu.g/g dry weight
of syringic acid, and/or about 70-80 .mu.g/g dry weight of
chlorogenic acid, and/or about 6-8 .mu.g/g dry weight of caffeic
acid, about 2-3 .mu.g/g dry weight of vanillin, and/or about
1.5-2.5 .mu.g/g dry weight of sinapic acid, and/or about 210-240
.mu.g/g dry weight of diosmin, about 4-5 .mu.g/g dry weight of
orientin, 4-6 .mu.g/g dry weight of swertisin, and/or about 0.1-0.2
.mu.g/g dry weight of disomentin. The extract derived from sugar
cane may be in a powder form.
[0168] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 107 .mu.g/g dry weight of
syringic acid, and/or about 74 .mu.g/g dry weight of chlorogenic
acid, and/or about 7.5 .mu.g/g dry weight of caffeic acid, and/or
about 2 .mu.g/g dry weight of vanillin, and/or about 1.7 .mu.g/g
dry weight of sinapic acid, and/or about 227 .mu.g/g dry weight of
diosmin, and/or about 4.5 .mu.g/g dry weight of orientin, 5.2
.mu.g/g dry weight of swertisin, and/or about 0.16 .mu.g/g dry
weight of disomentin. The extract derived from sugar cane may be in
a powder form.
[0169] The extract derived from sugar cane of the present
disclosure may contain a range of organic acids that are found
naturally in sugar cane. These organic acids may include, but are
not limited to, aconitic (cis- and trans-), oxalic, citric,
tartaric, lactic, glycolic, succinic, malic, fumaric and shikimic
acids. In one embodiment, the extract derived from sugar cane
contains higher levels of citric and malic acids than other organic
acids. In one embodiment, the extract derived from sugar cane
contains lactic acid. In one embodiment, the extract derived from
sugar cane contains glycolic acid. In one embodiment, the extract
derived from sugar cane contains oxalic, citric, tartaric, lactic,
glycolic, succinic and malic acids. In another embodiment, the
extract derived from sugar cane contains low to trace amounts of
oxalic, citric, tartaric, lactic, glycolic, succinic and malic
acids. In another embodiment, the two most abundant organic acids
in the extract derived from sugar cane are trans- and cis-aconitic
acids.
[0170] The extract derived from sugar cane of the present
disclosure may contain trans- and/or cis-aconitic acids. In one
embodiment, the extract derived from sugar cane of the present
disclosure comprises trans-aconitic in amount of about
10,000-40,000 mg per kg and/or cis-aconitic in amount of about
3,000-7,000 mg/kg. In one embodiment, the extract derived from
sugar cane of the present disclosure may contain trans-aconitic in
an amount of about 17,000-30,000 mg per kg and/or cis-aconitic in
amount of about 4,000-6,500 mg/kg. In one embodiment, the extract
derived from sugar cane of the present disclosure may contain
trans-aconitic in amount of about 20,000-25,000 mg per kg and/or
cis-aconitic in amount of about 5,000-5,500 mg/kg.
[0171] The extract derived from sugar cane of the present
disclosure may contain lactic acid and/or glycolic acid. In one
embodiment, the extract derived from sugar cane of the present
disclosure comprises lactic acid in amount of about 100-5,000 mg
per kg. In one embodiment, the extract derived from sugar cane of
the present disclosure comprises lactic acid in amount of about
100-3,000 mg per kg. In one embodiment, the extract derived from
sugar cane of the present disclosure comprises lactic acid in
amount of about 100-1,000 mg per kg. In one embodiment, the extract
derived from sugar cane of the present disclosure comprises lactic
acid in amount of about 100-500 mg per kg. In one embodiment, the
extract derived from sugar cane of the present disclosure comprises
glycolic acid in amount of about 10-5,000 mg per kg. In one
embodiment, the extract derived from sugar cane of the present
disclosure comprises glycolic acid in amount of about 100-5,000 mg
per kg. In one embodiment, the extract derived from sugar cane of
the present disclosure comprises glycolic acid in amount of about
100-3,000 mg per kg. In one embodiment, the extract derived from
sugar cane of the present disclosure comprises glycolic acid in
amount of about 100-1,000 mg per kg. In one embodiment, the extract
derived from sugar cane of the present disclosure comprises
glycolic acid in amount of about 100-500 mg per kg.
[0172] The extract derived from sugar cane of the present
disclosure may contain amino acids. In one embodiment, the total
amino acids levels of the extract derived from sugar cane of the
present disclosure is about 50,000-80,000 .mu.g per gram, or about
60,000-70,000 .mu.g per gram, or about 65,000 .mu.g per gram. In
one embodiment, about 10-40% of these total amino acids are
essential amino acids. In one embodiment, about 15-30% of these
total amino acids are essential amino acids. In one embodiment,
about 20-25% of these total amino acids are essential amino
acids.
[0173] The extract derived from sugar cane of the present
disclosure may contain free amino acids. In one embodiment, the
extract derived from sugar cane of the present disclosure comprises
about 10,000-50,000 .mu.g of free amino acids per gram. In one
embodiment, the extract derived from sugar cane of the present
disclosure may contain about 20,000-35,000 .mu.g of free amino
acids per gram. The extract derived from sugar cane of the present
disclosure may contain about 25,000-30,000 .mu.g of free amino
acids per gram.
[0174] As defined above, the term "free amino acids" as used herein
refers to amino acids which are singular molecules and structurally
not attached to peptide bonds which are attached to other amino
acids.
[0175] The extract derived from sugar cane of the present
disclosure may contain leucine, a branched chain essential amino
acid. In one embodiment, the concentration of leucine in the
extract derived from sugar cane, is about 1-5 mM, or about 1.5-4
mM, or about 2-3 mM. In one embodiment, the amount of leucine in
the extract derived from sugar cane is about 1,000-20,000 .mu.g per
gram, or about 1,000-10,000 .mu.g per gram, or about 1,000-5,000
.mu.g per gram, or about 1,000-2,000 .mu.g per gram, or about
5,000-10,000 .mu.g per gram, or about 10,000-20,000 .mu.g per
gram.
[0176] The extract derived from sugar cane of the present
disclosure may contain minerals. In one embodiment, the extract
derived from sugar cane derived from sugar cane contains minerals
that are found naturally in sugar cane. In one embodiment, the
extract derived from sugar cane derived from sugar contains one or
more minerals including, but not limited to, potassium, sodium,
calcium, magnesium, iron, zinc, selenium and chromium.
[0177] In one embodiment, the extract derived from sugar cane
contains minerals bound to the polyphenols. In one embodiment, the
extract derived from sugar cane contains divalent ions bound to the
polyphenols. In one embodiment, the extract derived from sugar cane
contains calcium, magnesium and/or iron bound to the polyphenols.
In one embodiment, the extract derived from sugar cane contains
iron bound to the polyphenols.
[0178] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 20,000-32,000 mg of
potassium per kilogram, and/or about 300-600 mg of sodium per
kilogram, and/or about 800-1,300 mg of calcium per kilogram, and/or
about 3,000-6,000 mg of magnesium per kilogram, and/or about 40-90
mg of iron per kilogram, and/or about 3-10 mg of zinc per kilogram,
and/or about 500-900 g of selenium per kilogram and/or about
1,000-1,600 .mu.g of chromium per kilogram. The extract derived
from sugar cane may be in a syrup form.
[0179] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 25,000-27,000 mg of
potassium per kilogram, and/or about 400-500 mg of sodium per
kilogram, and/or about 1,000-1,200 mg of calcium per kilogram,
and/or about 4,000-5,500 mg of magnesium per kilogram, and/or about
55-75 mg of iron per kilogram, and/or about 5.5-7.5 mg of zinc per
kilogram, and/or about 700-850 .mu.g of selenium per kilogram,
and/or about 1,200-1,400 .mu.g of chromium per kilogram. The
extract derived from sugar cane may be in a syrup form.
[0180] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 26,000 mg of potassium per
kilogram, and/or about 450 mg of sodium per kilogram, and/or about
1,090 mg of calcium per kilogram, and/or about 4,700 mg of
magnesium per kilogram, and/or about 65 mg of iron per kilogram,
about 6.6 mg of zinc per kilogram, and/or about 786 .mu.g of
selenium per kilogram and/or about 1,300 .mu.g of chromium per
kilogram. The extract derived from sugar cane may be in a syrup
form.
[0181] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 50-350 mg of potassium per
kilogram, and/or about 5-70 mg of sodium per kilogram, and/or about
7,000-10,000 mg of calcium per kilogram, and/or about 1,000-3,000
mg of magnesium per kilogram, and/or about 500-1,300 mg of iron per
kilogram. The extract derived from sugar cane may be in a powder
form.
[0182] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 100-250 mg of potassium per
kilogram, and/or about 10-50 mg of sodium per kilogram, and/or
about 8,000-9,000 mg of calcium per kilogram, and/or about
1,500-2,500 mg of magnesium per kilogram, and/or about 800-1,000 mg
of iron per kilogram. The extract derived from sugar cane may be in
a powder form.
[0183] In one embodiment, the extract derived from sugar cane of
the present disclosure comprises about 190 mg of potassium per
kilogram, and/or about 30 mg of sodium per kilogram, and/or about
8,800 mg of calcium per kilogram, and/or about 2,000 mg of
magnesium per kilogram, and/or about 890 mg of iron per kilogram.
The extract derived from sugar cane may be in a powder form.
[0184] The extract derived from sugar cane of the present
disclosure may contain monosaccharides, disaccharides,
oligosaccharides and/or polysaccharides. Examples of these include,
but are not limited to, sucrose, glucose, galactose, xylose,
ribose, mannose, rhamnose, fructose, maltose, lactose, maltotriose,
xylopyarnose, raffinose, 1-kestose, theanderose, 6-kestose, panose,
neo-kestose and nystose, glucans and xylans.
[0185] The extract derived from sugar cane of the present
disclosure may contain fiber. The fiber may be present in the
extract as obtained by the process or fiber may be added to the
extract. The term "fiber" as used herein refers to indigestible
portion of food derived from plants. The fiber may be soluble or
insoluble fiber. Non-limiting examples of fiber include, sugar cane
fiber, oat bran, flour (including, for example soy, rice, wheat,
bran, rye, corn, sorghum, potato), modified starch, gelatin,
non-starch polysaccharides such as arabinoxylans, cellulose, chia
fiber, psyillium fiber, fenugreek fiber and many other plant
components such as resistant starch, resistant dextrins, inulin,
lignin, chitins, pectins, beta-glucans, and oligosaccharides. In
one embodiment, the extract derived from sugar cane of the present
disclosure contains sugar cane fiber. In one embodiment, the
extract derived from sugar cane of the present disclosure contains
flour. In one embodiment, the extract derived from sugar cane of
the present disclosure contains modified starch. In one embodiment,
the extract derived from sugar cane of the present disclosure
contains cellulose. In one embodiment, the extract derived from
sugar cane of the present disclosure contains chia fiber. In one
embodiment, the extract derived from sugar cane of the present
disclosure contains pysillium fiber. In one embodiment, the extract
derived from sugar cane of the present disclosure contains
fenugreek fiber.
[0186] In one embodiment, the fiber is present in the extract of
the present disclosure. In one embodiment, the fiber is added to
the extract of the present disclosure.
[0187] It may be desirable that extremes of pH of the extract
derived from sugar cane or the supernatant of the present
disclosure be avoided. In one embodiment, the pH of the extract or
the supernatant derived from sugar cane of the present disclosure
is in the range of about 3 to about 7, or about 3 to about 6, or
about 4 to about 5.5, or about 4.5 to about 5, or about 4.6 to
about 4.8.
[0188] The Brix value of the extract derived from sugar cane of the
present disclosure may vary. In some instances the Bx value of the
extract is at least about 40.degree. Bx (degrees Brix). In some
instances the Bx value of the extract is at least about 50.degree.
Bx. In some instances the extract of the present disclosure has at
least about 60.degree. Bx (degrees Brix). In some instances the Bx
value of the extract is at least about 65.degree. Bx. In some
instances the Bx value of the extract is at least about 70.degree.
Bx. In some instances the Bx value of the extract is about
50-75.degree. Bx. In some instances the Bx value of the extract is
about 50-70.degree. Bx. In some instances the Bx value of the
extract is about 60-65.degree. Bx. In some instances the Bx value
of the extract is about 50-60.degree. Bx. In some instances the Bx
value of the extract is about 55.degree. Bx. In some instances the
Bx value of the extract is about 60-65.degree. Bx. In some
instances the Bx value of the extract is about 64-65.degree. Bx. In
some instances the Bx value of the extract is about 65-70.degree.
Bx. In some instances the Bx value of the extract is about
70-75.degree. Bx. In some instances the Bx value of the extract is
about 75-80.degree. Bx.
Compositions, Methods and Uses of the Extracts Derived from Sugar
Cane
[0189] The extracts derived from sugar cane of the present
disclosure may be added to compositions and may have application in
various uses and methods.
[0190] In one aspect of the disclosure there is provided a
composition comprising an extract derived from sugar cane
comprising polyphenols of the present disclosure for preventing,
improving or treating a skin condition.
[0191] In one embodiment, there is provided a composition
comprising an extract derived from sugar cane for use in
preventing, improving or treating a skin condition, the extract
comprising from about 10 catechin equivalent (CE) g/L to about 50
CE g/L of polyphenols or from about 100 CE mg/g to about 500 CE
mg/g of polyphenols.
[0192] The extracts derived from sugar cane of the present
disclosure, together with a conventional adjuvant, carrier or
diluent, may be placed into the form of pharmaceutical or
cosmeceutical compositions and unit dosages thereof, and in such
form may be employed as solids, such as tablets, powders or filled
capsules, liquids as solutions, suspensions, emulsions (including
microemulsions), syrups, elixirs or capsules filled with the same,
lotions, creams, serums, gels, ointments and oils.
[0193] Compositions include those for oral, topical and injection
administration. In one embodiment, the composition is for oral
administration. In another embodiment, the composition is for
topical administration. In another embodiment, the composition is
for injection administration.
[0194] In one embodiment, the composition is in the form of a
tablet or capsule. In one embodiment, the composition is in the
form of a tablet. In one embodiment, the composition is in the form
of a capsule. In one embodiment, the composition is in the form of
a liquid. In one embodiment, the composition is in the form of a
syrup.
[0195] In one embodiment, the composition is in the form of a
lotion, cream, serum or gel. In one embodiment, the composition is
in the form of a cream, serum or gel. In one embodiment, the
composition is in the form of a lotion. In one embodiment, the
composition is in the form of a cream. In one embodiment, the
composition is in the form of a serum. In one embodiment, the
composition is in the form of a gel.
[0196] The compositions of the present disclosure may take the form
of any type of product that is desired. For example, but not
limited to, the compositions may be face and/or body creams or
gels, hand and/or foot creams or gels, shower and/or bath products
(including for example, soaps, hand and/or body washes, shower
gels, face washes, cleansers, toners, exfoliators, shampoos,
conditioners) and sun care products (including for example sun
creams, sun blocks and sun screens as lotions, sprays, creams or
gels and after-sun products).
[0197] In one embodiment, the composition is a face and/or body
cream or gel, hand and/or foot cream or gel, shower and/or bath
product or sun care product. In one embodiment, the composition is
a face and/or body cream or gel. In one embodiment, the composition
is a face cream or gel. In one embodiment, the composition is a
body cream or gel. In one embodiment, the composition is a hand
and/or foot cream or gel. In one embodiment, the composition is a
hand cream or gel. In one embodiment, the composition is a shower
and/or bath product. In one embodiment, the composition is a sun
care product.
[0198] The composition comprising an extract derived from sugar
cane of the present disclosure, contains one or more
pharmaceutically and/or cosmeceutically acceptable carriers,
diluents and/or excipients. By "pharmaceutically and/or
cosmeceutically acceptable" it is meant the carrier, diluent or
excipient must be compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof. A
composition may also be described inter alia as a medicament or
formulation.
[0199] The compositions may also include excipients and other
additives, including but not limited to, gelling agents,
thickeners, plasticizers, stabilisers, moisturisers, emollients,
penetration enhancers, detergents, colouring agents, wetting or
emulsifying agents, humectants, surfactants, transport enhancers,
pH adjusting agents, preservatives, fragrances and the like.
[0200] The compositions may include any and all solvents,
dispersion media, coatings, anti-bacterial and anti-fungal agents,
isotonic and absorption delaying agents and the like. The use of
such media and agents for pharmaceutical and cosmeceutical
compositions is well known in the art and except insofar as any
conventional media or agent is incompatible with the active
ingredient; their use in the compositions as described herein is
contemplated.
[0201] Penetration enhancers can be selected from the group, but
are not limited to, propylene glycol, calcium chelators such as
EDTA, methylated P-cyclodextrin, and polycarboxylic acids;
surfactants such as sodium lauryl sulfate, sodium dodecyl sulfate,
carnitine, carnitine esters, and tween; bile salts such as sodium
taurocholate; fatty acids such as oleic and linoleic acid; and
non-surfactants such as dialkyl sulfoxides; E-flux inhibitors such
as D-a-tocopheryl polyethylene glycol 1000 succinate (TPGS), and
peppermint oil; chitosan and chitosan derivatives such as N-methyl
chitosan, N-trimethyl chitosan, mono-N-carboxymethyl chitosan,
quaternized chitosan derivatives; SNAC (N-(8-(2-hydroxybenzoyl)
amino) caprylate) and SNAD
(N-(10-(2-hydroxybenzoyl)amino)-decanoate); N-acylated non-alpha
amino acids; Gelucire 44/14 or Vitamin E TPGS; CARBOPOL.RTM. 934P;
others known to those of ordinary skill in the art; and
combinations thereof.
[0202] Gelling agents can be selected from the group, but are not
limited to, acacia, alginic acid, bentonite, carbopols, carbomer
940, carbomer 941, gelatin, carbomer copolymer, aluminum
monostearat, dextrin, magnesium aluminum silicate, silicon dioxide,
sodium alginate, triethanolamine, polyvinyl alcohol, pectin,
methylcellulose, hydroxypropyl cellulose, aqueous thickening agents
such as neutral, anionic-cationic polymers and other materials
known to one of ordinary skill in the art and mixtures thereof. pH
adjusting agents can be selected from the group, but are not
limited to, triethanolamine, triethylamine, diethylmethylamine,
ethyldimethylamine, isopropyldimethylamine.one or more adipic
acids, glycines, citric acids, calcium hydroxides, magnesium
aluminometasilicates, buffers, typically Bronsted-Lowry and/or
Lewis acids and/or bases, or any combinations thereof and other
materials known to one of ordinary skill in the art.
[0203] Moisturisers can be selected from the group, but are not
limited to, polyethylene glycol, propylene glycol, dipropylene
glycol, 1,3-butylene glycol, glycerin, diglycerin, xylitol,
maltitol, maltose, D-mannitol, glucose, fructose, sodium
chondroitin sulfate, sodium hyaluronate, sodium lactate,
glucosamine, cyclodextrin, cococaprylate/caprate and other
materials known to one of ordinary skill in the art and mixtures
thereof.
[0204] Emulsifiers can be selected from the group, but are not
limited to, sodium lauryl sulfate, ceteth-20, laureth-3, glyceryl
stearate, polyethylene glycol, macrogol cetostearyl ether, stearic
acid, stearyl alcohol, polysorbate 60, Irish moss, Tween 80,
sorbitol monostearate.glycol esters, fatty acids, fatty alcohols,
fatty acid glycol esters, fatty esters, fatty ethers, esters of
glycerin, esters of propylene glycol, fatty acid esters of
polyethylene glycol, fatty acid esters of polypropylene glycol,
esters of sorbitol, esters of sorbitan anhydrides, carboxylic acid
copolymers, esters and ethers of glucose, ethoxylated ethers,
ethoxylated alcohols, alkyl phosphates, polyoxyethylene fatty ether
phosphates, fatty acid amides, acyl lactylates, soaps, polyethylene
glycol 20 sorbitan monolaurate (polysorbate 20), polyethylene
glycol 5 soya sterol, steareth-2, steareth-20, steareth-21,
ceteareth-20, PPG-2 methyl glucose ether distearate, ceteth-10,
polysorbate 80, cetyl phosphate, potassium cetyl phosphate,
diethanol amine cetyl phosphate, polysorbate 60, glyceryl stearate,
PEG-100 stearate, tragacanth gum, 10-30 alkyl acrylate
crosspolymers and other materials known to one of ordinary skill in
the art and mixtures thereof.
[0205] Emollients can be selected from the group, but are not
limited to, liquid vaseline. paraffinum liquidum, petrolatum,
proplylene glycol, fatty acid esters, mineral oil including
dimethicone, waxes including white wax, spermacetic wax, squalene,
cetearyl alcohol, cetostearyl alcohol, stearyl alcohol,
2-Octyldodecanol, mineral oil USP, light mineral oil NF, liquid
paraffin BP, light liquid paraffin BP, candelilla wax, sweet almond
oil, apricot oil, emu oil, argan oil, glycerin, coconut oil, grape
seed oil, honey, lanolin and other materials known to one of
ordinary skill in the art and mixtures thereof.
[0206] Surfactants can be selected from the group, but are not
limited to, a polysorbate, polyoxyethylene (20) sorbitan
monostearate, polyoxyethylene (20) sorbitan monooleate, a
polyoxyethylene fatty acid ester, Myrj 45, Myrj 49, Myrj 52 and
Myrj 59; a polyoxyethylene alkylyl ether, polyoxyethylene cetyl
ether, polyoxyethylene palmityl ether, polyethylene oxide hexadecyl
ether, polyethylene glycol cetyl ether, a sucrose ester, a partial
ester of sorbitol, sorbitan monolaurate, sorbitan monolaurate a
monoglyceride, a diglyceride, isoceteth-20, a sucrose ester, or
selected from the group consisting of steareth 2, glyceryl
monostearate/PEG 100 stearate, Glyceryl Stearate, Steareth-21, peg
40 stearate, polysorbate 60, polysorbate 80, sorbitan stearate,
laureth 4, Sorbitan monooleate, ceteareth 20, steareth 20, ceteth
20, Macrogol Cetostearyl Ether, ceteth 2, PEG-30
Dipolyhydroxystearate, sucrose distearate, polyoxyethylene (100)
stearate, PEG 100 stearate, laureth 4, cetomacrogol ether, cetearyl
alcohol, cetearyl glucoside, oleyl alcohol, steareth-2, diisopropyl
adipate, capric/caprilic triglicerides, polysorbate 20, montanov 68
(cetearyl alcohol (and) cetearyl glucoside), sharonmix 824 (a
liquid blend of methyl paraben, ethyl paraben and propyl
paraben--in phenoxyethanol), Simusol 165 (glyceryl stearate and
PEG-100 stearate), methyl glucose sequistearate, Peg 30
dipolyhydroxystearate, sucrose stearic acid esters, sorbitan
laureth, sorbitan stearate, sodium lauryl sulfate, and mixtures
thereof.
[0207] Preservatives can be selected from the group, but not
limited to, methylparaben and propylparaben and the salts thereof
(e.g. sodium or potassium salts), sodium benzoate, diazolidinyl
urea, phenoxyethanol, DMDM hydantoin, sorbic acid, benzyl alcohol,
formaldehyde, triclosan.methylisothiazolinone,
methylchloroisothiazolinone, caffeine, citric acid, benzoic acid,
butylated hydroxytoluene, propylene glycol, organic acids, esters
of parahydroxybenzoic acid(methyl, ethyl, propyl and butyl esters
of parahydroxy benzoic acid, and their sodiumsalts etc), chloform,
chlorocresol, quaternary ammonium compounds and butylated
hydroxyanisole, and the mixtures thereof.
[0208] Solvents can be selected from the group, but not limited to,
ethyl alcohol, polyethylene glycol, propylene glycol, isopropyl
alcohol, purified water and other materials known to one of
ordinary skill in the art and mixtures thereof.
[0209] Thickeners can be selected from the group, but not limited
to, beeswax, cocoa butter, shea butter, wool wax, cetyl alcohol,
gum acacia, gum tragacanth, locust bean gum, guar gum,
hydroxypropyl guar, xanthan gum, cellulose gum, sclerotium gum,
carrageenan gum, karaya gum, cellulose gum, rosin, anionic polymers
such as polyacrylic acid, carboxymethylcellulose, methylcellulose,
hydroxyethylcellulose, hydroxypropylcellulose,
hydroxymethylcellulose, polyethylene glycol, acrylic acid polymers,
PEG-150 distearate, decyl alcohol, SMDI copolymer, faponite XLG,
ethyl cellulose, natrosol and other materials known to one of
ordinary skill in the art and mixtures thereof.
[0210] Plasticizers can be selected from the group, but not limited
to, glycerol, propylene glycol or another glycol, peppermint oil,
eucalyptol oil, geranyl acetate or geraniol, phthalate, sebacate
and citrate esters, triacetin, sorbitol, sucrose, triethyl citrate,
dibutyl phthalate and other materials known to one of ordinary
skill in the art and mixtures thereof.
[0211] Compositions of the present disclosure may be formulated,
for example, by employing conventional solid or liquid vehicles or
diluents, as well as additives of a type appropriate to the mode of
desired administration (for example, excipients, binders,
preservatives, stabilizers, flavours, lubricants, disintegrating
agents, etc.) according to techniques such as those well known in
the art of formulation. The composition comprising an extract
derived from sugar cane may be contained within matrixes,
nanoparticles, liposomes, vesicles, microcapsules, microspheres and
the like, or within a solid particulate material.
[0212] For example, but not limited to, a binder such as gum,
acacia, corn starch or gelatin; excipients such as dicalcium
phosphate; a disintegrating agent such as corn starch, potato
starch, alginic acid and the like; a lubricant such as magnesium
stearate; and a sweetening agent such as sucrose, lactose or
saccharin may be included in the composition of the present
disclosure.
[0213] The topical compositions of the present disclosure, e.g. in
the form of lotions, creams or gels, may contain acceptable
diluents, carriers and other excipients to impart the desired
texture, consistency, viscosity and appearance. Acceptable
diluents, carriers and other excipients are familiar to those
skilled in the art and include, but are not limited to, skin
penetration enhancers, ethoxylated and nonethoxylated surfactants,
fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil,
coconut oil, and mineral oil), cocoa butter waxes, silicon oils,
buffering agents, cellulose derivatives, emulsifying agents such as
non-ionic organic and inorganic bases, preserving agents, wax
esters, steroid alcohols, triglyceride esters, phospholipids such
as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol
esters, hydrophilic lanolin derivatives, and hydrophilic beeswax
derivatives.
[0214] In one embodiment, the composition comprises a skin
penetration enhancer.
[0215] Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills or capsules may be coated with shellac,
sugar or both. A syrup or elixir may contain sucrose as a
sweetening agent, and/or methyl and propylparabens as
preservatives, and/or a dye and/or a flavouring. Any material used
in preparing any dosage unit form should be pharmaceutically pure
and substantially non-toxic in the amounts employed.
[0216] Generally, injection compositions of the present disclosure
are low-viscosity, sterile formulations. The injection compositions
may contain, in addition to the extract derived from sugar cane,
other ingredients typically used in such products, such as
antimicrobials, hydration agents, tissue bulking agents or tissue
fillers, preservatives, emulsifiers, natural or synthetic oils,
solvents, surfactants, detergents, buffers, gelling agents,
antioxidants, fillers, thickeners, powders, viscosity-controlling
agents and water, and optionally including anaesthetics, anti-itch
actives, conditioning agents, minerals, silicones or derivatives
thereof, amino acids and vitamins.
[0217] Injectable compositions of the present disclosure may be in
the form of controlled-release or sustained-release compositions
which comprise the extract derived from sugar cane and a material
such that they are released within the tissue in a controlled
manner over time. The composition comprising the extract derived
from sugar cane may be contained within matrixes, nanoparticles,
liposomes, vesicles, microcapsules, microspheres and the like, or
within a solid particulate material, all of which is selected
and/or constructed to provide release of the extract derived from
sugar cane over time.
[0218] The compositions of the present disclosure may conveniently
be presented in dosage unit form and may be prepared by any of the
methods well known in the art of formulation. Exemplary techniques
for formulation of the compositions of the present disclosure may
be found in "Remington's Pharmaceutical Sciences", Mack Publishing
Co., Easton Pa., 22.sup.nd edition, 2012. Some methods include the
step of bringing the extract derived from sugar cane of the present
disclosure, into association with the carrier which constitutes one
or more accessory ingredients. In general, the compositions are
prepared by uniformly and intimately bringing the extract derived
from sugar cane of the present disclosure, into association with a
liquid carrier or a finely divided solid carrier or both, and then,
if necessary, shaping the product into the desired formulation. In
the composition the extract derived from sugar cane is included in
an amount sufficient to produce the desired effect upon the skin
condition. As used herein, the term "composition" is intended to
encompass a product comprising the specified ingredients in the
specified amounts, as well as any product which results, directly
or indirectly, from combination of the specified ingredients in the
specified amounts.
[0219] The compositions of the present disclosure may also comprise
other therapeutically active compounds which can be applied in the
prevention, improvement and treatment of skin conditions. Selection
of the appropriate active compounds for use in combination therapy
may be made by one of ordinary skill in the art, according to
conventional pharmaceutical principles. The combination of active
compounds may act synergistically to effect the prevention,
improvement or treatment of the various skin conditions. Using this
approach, one may be able to achieve therapeutic efficacy with
lower dosages of each active compound, thus reducing the potential
for adverse side effects.
[0220] The compositions of the present disclosure may also
comprise, for example, alpha hydroxyl acids (e.g. glycolic, lactic,
tartaric, and citric acids), beta hydroxyl acids (e.g. salicylic
acid), hydroquinone, kojic acid, benzaldehyde-O-alkyloximes,
retinol, tretinoin (and other vitamin A derivatives), L-ascorbic
acid, hyaluronic acid, vitamin C and derivatives thereof, vitamin E
and derivatives thereof, copper peptide, alpha-lipoic acid,
dimethylaminoethanol, coenzyme Q-10, salicylic acid and benzoyl
peroxide.
[0221] In one embodiment the compositions of the present disclosure
include lactic acid and/or glycolic acid. Lactic acid and glycolic
acid have previously been used in skin products for their ability
to hydrate (moisturise) and exfoliate. The dual function of
exfoliation and moisturisation removes damaged, dry and dead skin
cells and promotes growth of new cells. Lactic acid and glycolic
acid have also previously been used in skin products for their
ability to lighten and brighten the skin. Extracts derived from
sugar cane comprising lactic acid and/or glycolic acid of the
present disclosure are therefore useful in preventing, improving or
treating skin conditions.
[0222] In one embodiment, the compositions of the present
disclosure comprise lactic acid and/or glycolic acid in amount of
about 0.01-30 wt % based upon the total weight of the composition.
In one embodiment, lactic acid and/or glycolic acid is present in
an amount from about 0.05 wt % to about 25 wt %. In one embodiment,
lactic acid and/or glycolic acid is present in an amount from about
5 wt % to about 20 wt %. In one embodiment, lactic acid and/or
glycolic acid is present in an amount from about 1 wt % to about 20
wt %. In one embodiment, lactic acid and/or glycolic acid is
present in an amount from about 0.1 wt % to about 10 wt %. In one
embodiment, lactic acid and/or glycolic acid is present in an
amount from about 1 wt % to about 10 wt %. In one embodiment,
lactic acid and/or glycolic acid is present in an amount from about
1 wt % to about 5 wt %. In one embodiment, lactic acid and/or
glycolic acid is present in an amount from about 0.05 wt % to about
5 wt %. In one embodiment, lactic acid and/or glycolic acid is
present in an amount from about 1 wt % to about 3 wt %. In one
embodiment, lactic acid and/or glycolic acid is present in an
amount from about 3 wt % to about 8 wt %. In one embodiment, lactic
acid and/or glycolic acid is present in an amount from about 5 wt %
to about 10 wt %. In one embodiment, lactic acid and/or glycolic
acid is present in an amount from about 5 wt % to about 20 wt
%.
[0223] When other therapeutically active compounds are employed in
combination with the extracts derived from sugar cane of the
present disclosure, they may be used, for example, in amounts as
noted in the Physicians' Desk Reference (PDR) or as otherwise
determined by one of ordinary skill in the art.
[0224] The compositions of the present disclosure comprise the
extracts derived from sugar cane of the present disclosure in an
amount of at least 0.05 wt % based upon the total weight of the
composition. The percentage of the extract derived from sugar cane
in the compositions may, of course, be varied and may be between
about 0.05 wt % to about 50 wt % based upon the total weight of the
composition. The amount of the extracts derived from sugar cane of
the present disclosure in compositions is such that a suitable
dosage will be obtained.
[0225] In one embodiment, the extract derived from sugar cane is
present in an amount from about 0.05 wt % to about 50 wt % based
upon the total weight of the composition. In one embodiment, the
extract derived from sugar cane is present in an amount from about
1 wt % to about 30 wt % based upon the total weight of the
composition. In one embodiment, the extract derived from sugar cane
is present in an amount from about 5 wt % to about 20 wt % based
upon the total weight of the composition. In one embodiment, the
extract derived from sugar cane is present in an amount from about
1 wt % to about 20 wt % based upon the total weight of the
composition. In one embodiment, the extract derived from sugar cane
is present in an amount from about 0.1 wt % to about 10 wt % based
upon the total weight of the composition. In one embodiment, the
extract derived from sugar cane is present in an amount from about
1 wt % to about 10 wt % based upon the total weight of the
composition. In one embodiment, the extract derived from sugar cane
is present in an amount from about 1 wt % to about 5 wt % based
upon the total weight of the composition. In one embodiment, the
extract derived from sugar cane is present in an amount from about
0.05 wt % to about 10 wt % based upon the total weight of the
composition. In one embodiment, the extract derived from sugar cane
is present in an amount from about 0.5 wt % to about 5 wt % based
upon the total weight of the composition. In one embodiment, the
extract derived from sugar cane is present in an amount from about
0.5 wt % to about 2.5 wt % based upon the total weight of the
composition. In one embodiment, the extract derived from sugar cane
is present in an amount from about 1 wt % to about 3 wt % based
upon the total weight of the composition. In one embodiment, the
extract derived from sugar cane is present in an amount from about
2.5 wt % to about 5 wt % based upon the total weight of the
composition. In one embodiment, the extract derived from sugar cane
is present in an amount from about 2 wt % to about 6 wt % based
upon the total weight of the composition. In one embodiment, the
extract derived from sugar cane is present in an amount from about
3 wt % to about 8 wt % based upon the total weight of the
composition. In one embodiment, the extract derived from sugar cane
is present in an amount from about 5 wt % to about 10 wt % based
upon the total weight of the composition. In one embodiment, the
extract derived from sugar cane is present in an amount from about
5 wt % to about 20 wt % based upon the total weight of the
composition.
[0226] In the prevention, improvement or treatment of skin
conditions, an appropriate dosage level will generally be about 10
to 5000 mg of the extract derived from sugar cane of the present
disclosure per day which can be administered in single or multiple
doses. In one embodiment, the dosage level will be about 10 to
about 5,000 mg per day. In one embodiment, the dosage level will be
about 10 to about 1,000 mg per day. In one embodiment, the dosage
level will be about 10 to about 500 mg per day. In one embodiment,
the dosage level will be about 100 to about 1,000 mg per day. In
one embodiment, the dosage level will be about 100 to about 2,000
mg per day. In one embodiment, the dosage level will be about 100
to about 3,000 mg per day. In one embodiment, the dosage level will
be about 100 to about 4,000 mg per day. In one embodiment, the
dosage level will be about 100 to about 5,000 mg per day. In one
embodiment, the dosage level will be about 250 to about 4,000 mg
per day. In one embodiment, the dosage level will be about 500 to
about 3,000 mg per day. In one embodiment, the dosage level will be
about 1,000 to 2,000 mg per day. In one embodiment, the dosage
level will be about 2,000 to 5,000 mg per day. In one embodiment,
the dosage level will be about 2,000 to 4,000 mg per day. In one
embodiment, the dosage level will be about 2,000 to 3,000 mg per
day.
[0227] It will be understood, however, that the specific dosage
level and frequency of dosage for any particular patient may be
varied and will depend upon a variety of factors including the
activity of the specific extract derived from sugar cane employed,
the metabolic stability and length of action of that extract
derived from sugar cane, the age, body weight, general health, sex,
diet, mode and time of administration, rate of excretion, drug
combination, the severity of the particular condition, and the
subject undergoing therapy.
Preventing, Improving and/or Treating Skin Conditions with the
Extracts Derived from Sugar Cane
[0228] The extracts derived from sugar cane and compositions
comprising the extracts of the present disclosure can be used for
preventing, improving or treating skin conditions. The skin
conditions include, for example, atopic dermatitis, acne, eczema,
psoriasis, dry skin, oily skin, pruritic skin, wrinkles, fine
lines, dark spots, age spots, mottled pigmentation, skin
pigmentation, melasma, darkened skin, redness, flushing,
inflammation, skin elasticity, dark circles under the eyes, changes
associated with skin aging, hair loss and wound healing.
[0229] For preventing, improving or treating many of these skin
conditions, the extracts derived from sugar cane of the present
disclosure provide their beneficial effects by: inhibiting or
activating key enzymes in the skin; possessing a high antioxidant
and anti-inflammatory activity; and inhibiting particular
pathogenic bacteria.
[0230] For improving wound healing, the extracts derived from sugar
cane of the present invention provide their beneficial effects by
influencing one or more phases of the healing process. For example:
reducing inflammation and thereby preventing scar tissue; by
improving the healing time and appearance of the healed skin; by
protecting the healing skin from oxidative damage; and by
preventing or treating infection.
[0231] The present inventors have surprisingly found that the
extracts derived from sugar cane of the present disclosure inhibit
the collagen degrading enzymes, including for example collagenase
and matrix metalloproteinase-1 (MMP-1), and the elastin degrading
enzymes, including for example elastase. See Examples 11 and 16.
Wrinkle formation in the skin is accompanied by a decrease in skin
elasticity and the curling of elastic fibers (such as elastin) in
the dermis. Elastase inhibitors suppress elastase activity
(degradation of elastin) and prevent the damage of dermal elastin,
thus helping mitigate wrinkle formation.
[0232] The collagen content of skin is the net balance between
collagen synthesis and collagen breakdown. It is known that with
age collagen synthesis in the skin is reduced. Additionally,
environmental stress such as smoking, UV exposure, pollution and
inflammation stimulate the production of collagen-degrading enzymes
that causes collagen breakdown. Collagenase and MMP-1, two of the
key collagen-degrading enzymes, are considered to be central to the
causes of skin aging. Direct inhibition of these enzymes can be an
effective approach to mitigate collagen breakdown in the skin
thereby improving skin health, general condition and tone and
reduction of wrinkles.
[0233] The colour of mammalian skin is determined by many factors,
one of which is the production and distribution of melanin. Melanin
is essential in protecting skin against UV radiation, but over
production of melanin is also a major consequence of UV damage and
the aging process that induces pigmentation disorders such as
freckles and senile lentigo (i.e., age spots). Excessive melanin
has also been viewed as a melanoma precursor. Melanin inhibition is
a desirable effect sought in various fronts of cosmetic industry to
achieve skin whitening, lessen aging appearance, and preventing
melanoma.
[0234] The present inventors have also surprisingly found that the
extracts derived from sugar cane of the present disclosure inhibit
the tyrosinase enzyme. See Example 12. In the melanin biosynthesis
process, tyrosinase is the key enzyme that catalyses the first step
of melanogenesis. Previous studies have shown that melanin
reduction and skin whitening and/or lightening can be achieved, at
least partially, by deactivation of tyrosinase. Therefore,
tyrosinase inhibitors have become increasingly important in
cosmetic and medicinal products used in the prevention or reduction
of hyperpigmentation, in skin whitening and/or lightening, to
lessen the aging appearance and to prevent melanoma.
[0235] The extracts derived from sugar cane of the present
disclosure also activate telomerase. See Example 15. Telomerase is
an enzyme which builds telomeres, the regions of repetitive
sequences at each end of chromosomes in most eukaryotes. Telomeres
protect the end of the chromosome from DNA damage and become
shortened through recursive cell division thereby causing cells to
age. Telomerase can slow, stop or perhaps even reverse the telomere
shortening that happens with age, however, the amount and activity
of telomerase also declines with age. Activation of telomerase can
reverse skin cell aging and revert the skin to a more youthful
physical and genetic state.
[0236] The extracts derived from sugar cane of the present
disclosure possess a high antioxidant capacity and may also provide
their beneficial effects through this antioxidant activity. The
extracts derived from sugar cane of the present disclosure
demonstrated antioxidant activity against all six common free
radicles (reactive oxygen species (ROS)), i.e., peroxyl radical,
hydroxyl radical, superoxide anion, singlet oxygen, peroxynitrite,
and hypochlorite, that are generated in cells and the body as a
result of oxidative stress. Antioxidant activity against all the
six common free radicals of an extract derived from sugar cane of
the present disclosure is well demonstrated in Tables 32 and
33.
[0237] Furthermore, the present inventors have surprisingly found
that extracts derived from sugar cane of the present disclosure
stimulate the production of Nuclear factor (erythroid derived
2)-like 2 (Nrf2) in human cells. See Example 19. Nrf2 is a
redox-sensitive transcription factor that binds to antioxidant
response elements (ARE) to regulate the expression of antioxidant
enzymes that protect against oxidative damage triggered by injury
and inflammation. Activation of the Nrf2 pathway (both topical and
internal/systemic) has been found to have a wide range of
beneficial effects on skin, including reduced rates of skin
cancers, protection from ultraviolet radiation, reduced
inflammation, irritation and redness, reduction of wrinkles and
improvement in skin tone, enhanced barrier function, and improved
wound healing.
[0238] The extracts derived from sugar cane of the present
disclosure also inhibit Nuclear Factor .kappa.B (NF-.kappa.B), a
protein complex that is involved in cellular responses to stimuli
such as stress and free radicals, ultraviolet irradiation, oxidized
LDL, and bacterial or viral antigens. See Example 20. It has been
associated with inflammation and plays a major role in the aging
process of the skin. Reducing or inhibiting inflammation improves
skin tone, appearance, reduces swelling, improves healing time and
can decrease the formation of scar tissue in wound healing.
[0239] The present inventors have also surprisingly found that
extracts derived from sugar cane of the present disclosure inhibit
Tumor Necrosis Factor (TNF)-.alpha.. See Example 21. TNF-.alpha. is
a pro-inflammatory cytokine that triggers downstream cellular
feedback loops governing inflammation. TNF-.alpha. has been
identified as an inflammation trigger and precursor. Reducing or
inhibiting inflammation improves skin tone, appearance, reduces
swelling, improves healing time and can decrease the formation of
scar tissue in wound healing.
[0240] The extracts derived from sugar cane of the present
disclosure also inhibit prostaglandin E.sub.2 synthesis. See
Example 22. PGE.sub.2 is the major prostaglandin produced by
cyclooxygenase enzymes in the skin and is a potentiator of acute
inflammation. Thus, inhibiting PGE.sub.2 reduces or inhibits
inflammation, leading to improved skin tone, appearance, reduced
swelling, improved healing time and decreased formation of scar
tissue.
[0241] The extracts derived from sugar cane of the present
disclosure inhibit Cyclooxygenases-2 (COX-2). See Example 23. COX-2
catalyzes the conversion of arachidonic acid to prostaglandin (PG)
H.sub.2, the precursor of PGs and thromboxane and plays an
important role in inflammation and pain. Hence, COX-2 inhibition
can reduce symptoms of inflammation. The reduction or inhibition of
inflammation improves skin tone, appearance, reduces swelling,
improves healing time and can decrease the formation of scar tissue
in wound healing.
[0242] The extracts derived from sugar cane of the present
disclosure also inhibit the growth of pathogenic bacteria which can
cause topical skin infections and acne.
[0243] See Example 24. Examples of pathogenic bacteria include, but
are not limited to, Stapholococcus aureus, Staphylococcus
epidermidis, Propionibacterium acnes and Escherichia coli. The
antibacterial properties of the extracts of the present disclosure
also aids in the treatment of wounds by preventing or treating
infection, which can delay the healing process.
[0244] In one embodiment, the extracts derived from sugar cane of
the present disclosure can be used in methods for preventing skin
conditions. In one embodiment, the extracts derived from sugar cane
of the present disclosure can be used in methods for improving skin
conditions. In one embodiment, the extracts derived from sugar cane
of the present disclosure can be used in methods for treating skin
conditions.
[0245] In one aspect of the disclosure there is provided a method
for preventing, improving or treating a skin condition in a
subject, the method comprising administering an effective amount of
an extract derived from sugar cane comprising polyphenols of the
present disclosure. In one embodiment, there is provided a method
for preventing, improving or treating a skin condition in a subject
in need thereof, the method comprising administering an effective
amount of an extract derived from sugar cane to the subject, the
extract comprising from about 10 catechin equivalent (CE) g/L to
about 50 CE g/L of polyphenols or from about 100 CE mg/g to about
500 CE mg/g of polyphenols.
[0246] In another aspect of the disclosure there is provided an
extract derived from sugar cane comprising polyphenols of the
present disclosure for use in preventing, improving or treating a
skin condition in a subject. In one embodiment, there is provided
an extract derived from sugar cane for use in preventing, improving
or treating a skin condition in a subject, the extract comprising
from about 10 catechin equivalent (CE) g/L to about 50 CE g/L of
polyphenols or from about 100 CE mg/g to about 500 CE mg/g of
polyphenols.
[0247] In another aspect of the disclosure there is provided the
use of an extract derived from sugar cane comprising polyphenols of
the present disclosure in the manufacture of a medicament for
preventing, improving or treating a skin condition. In one
embodiment, there is provided the use of an extract derived from
sugar cane in the manufacture of a medicament for preventing,
improving or treating a skin condition, the extract comprising from
about 10 catechin equivalent (CE) g/L to about 50 CE g/L of
polyphenols or from about 100 CE mg/g to about 500 CE mg/g of
polyphenols.
[0248] As would be understood by those skilled in the art, the
methods and uses described herein may be for therapeutic or
cosmetic benefits. In one embodiment, the methods and uses are
therapeutic. Therapeutic methods and uses relate to the prevention,
improvement or treatment of a skin disease or disorder as well as
the alleviation of the symptoms of pain and suffering of the skin
disease or disorder.
[0249] In one embodiment, there is provided a therapeutic method
for preventing, improving or treating a skin condition in a
subject, the method comprising administering a therapeutically
effective amount of an extract derived from sugar cane comprising
polyphenols of the present disclosure.
[0250] In another embodiment, the methods and uses are cosmetic.
Cosmetic methods and uses are designed to beautify the skin,
improve the appearance of the skin and generally improve the
aesthetics of the skin. In one embodiment, there is provided a
cosmetic method for preventing, improving or treating a skin
condition in a subject, the method comprising administering an
effective amount of an extract derived from sugar cane comprising
polyphenols of the present disclosure.
[0251] The extracts derived from sugar cane comprising polyphenols
of the present disclosure may be used in the prevention,
improvement or treatment of any relevant skin condition. The skin
condition may be selected from the group including, for example,
atopic dermatitis, acne, eczema, psoriasis, dry skin, oily skin,
pruritic skin, wrinkles, fine lines, dark spots, age spots, mottled
pigmentation, skin pigmentation, melasma, darkened skin, redness,
flushing, inflammation, skin elasticity, dark circles under the
eyes, changes associated with skin aging, hair loss and wound
healing.
[0252] In one embodiment, the skin condition is atopic dermatitis.
In one embodiment, the skin condition is acne. In one embodiment,
the skin condition is eczema. In one embodiment, the skin condition
is psoriasis. Psoriasis is a chronic autoimmune disease
characterized by patches of red, itchy and scaly skin, which can be
painful.
[0253] In one embodiment, the skin condition is dry skin. In one
embodiment, the skin condition is oily skin. In one embodiment, the
skin condition is pruritic skin. In one embodiment, the skin
condition is wrinkles. In one embodiment, the skin condition is
fine lines. In one embodiment, the skin condition is dark spots. In
one embodiment, the skin condition is age spots. In one embodiment,
the skin condition is mottled pigmentation. In one embodiment, the
skin condition is skin pigmentation. In one embodiment, the skin
condition is melasma. In one embodiment, the skin condition is
darkened skin. In one embodiment, the skin condition is redness. In
one embodiment, the skin condition is flushing. In one embodiment,
the skin condition is inflammation. In one embodiment, the skin
condition is skin elasticity. In one embodiment, the skin condition
is dark circles under the eyes. In one embodiment, the skin
condition is changes associated with skin aging.
[0254] In one embodiment, the skin condition is selected from dry
skin, oily skin, pruritic skin, wrinkles, fine lines, dark spots,
age spots, mottled pigmentation, skin pigmentation, melasma,
darkened skin, redness, flushing, inflammation, skin elasticity,
dark circles under the eyes and changes associated with skin aging.
In another embodiment the skin condition is wrinkles, fine lines,
dark spots, age spots, mottled pigmentation, skin pigmentation,
melasma, darkened skin, skin elasticity, dark circles under the
eyes and changes associated with skin aging.
[0255] The prevention, improvement or treatment of the skin
condition may provide one or multiple benefits. The prevention,
improvement or treatment of the skin condition may provide
benefits, including for example, skin moisturisation, skin
exfoliation, skin lightening or colour reduction, skin pigmentation
reduction, skin redness reduction, skin flushing reduction,
inflammation reduction, fine line reduction, wrinkle reduction,
wrinkle depth reduction, flakiness reduction, itchiness reduction,
skin dryness reduction, skin roughness reduction, enhanced skin
radiance, enhanced skin tone, enhanced skin clarity, enhanced skin
firmness, enhanced skin tightness, enhanced skin elasticity,
enhanced aesthetic appearance of the skin and/or enhanced overall
skin appearance. In one embodiment, the prevention, improvement or
treatment of the skin condition provides skin moisturisation. In
one embodiment, the prevention, improvement or treatment of the
skin condition provides skin exfoliation. In one embodiment, the
prevention, improvement or treatment of the skin condition provides
skin lightening or colour reduction. In one embodiment, the
prevention, improvement or treatment of the skin condition provides
skin pigmentation reduction. In one embodiment, the prevention,
improvement or treatment of the skin condition provides skin
redness reduction. In one embodiment, the prevention, improvement
or treatment of the skin condition provides skin flushing
reduction. In one embodiment, the prevention, improvement or
treatment of the skin condition provides inflammation reduction. In
one embodiment, the prevention, improvement or treatment of the
skin condition provides fine line reduction. In one embodiment, the
prevention, improvement or treatment of the skin condition provides
wrinkle reduction. In one embodiment, the prevention, improvement
or treatment of the skin condition provides wrinkle depth
reduction. In one embodiment, the prevention, improvement or
treatment of the skin condition provides flakiness reduction. In
one embodiment, the prevention, improvement or treatment of the
skin condition provides itchiness reduction. In one embodiment, the
prevention, improvement or treatment of the skin condition provides
skin dryness reduction. In one embodiment, the prevention,
improvement or treatment of the skin condition provides skin
roughness reduction. In one embodiment, the prevention, improvement
or treatment of the skin condition provides enhanced skin radiance.
In one embodiment, the prevention, improvement or treatment of the
skin condition provides enhanced skin tone. In one embodiment, the
prevention, improvement or treatment of the skin condition provides
enhanced skin clarity. In one embodiment, the prevention,
improvement or treatment of the skin condition provides enhanced
skin firmness. In one embodiment, the prevention, improvement or
treatment of the skin condition provides enhanced skin tightness.
In one embodiment, the prevention, improvement or treatment of the
skin condition provides enhanced skin elasticity. In one
embodiment, the prevention, improvement or treatment of the skin
condition provides enhanced aesthetic appearance of the skin. In
one embodiment, the prevention, improvement or treatment of the
skin condition provides enhanced overall skin appearance.
[0256] In one embodiment, the skin condition is a hair loss
condition and the extracts derived from sugar cane comprising
polyphenols of the present disclosure may be used in the
prevention, improvement or treatment of a hair loss condition. The
hair loss condition may be selected from the group including, for
example, male-pattern hair loss, female-pattern hair loss, alopecia
areata, and a thinning of hair known as telogen effluvium. A hair
loss condition includes that commonly referred to as `balding`,
`baldness` and `hair thinning`.
[0257] Hair follicle growth occurs in cycles. Each cycle consists
of a long growing phase (anagen), a short transitional phase
(catagen) and a short resting phase (telogen). At the end of the
resting phase, the hair falls out (exogen) and a new hair starts
growing in the follicle beginning the cycle again. Hair is
naturally lost during the `resting` telogen phase. Problems occur
and hair loss becomes visible when there are too many resting hair
follicles. The longer they remain resting, the greater the chance
of permanent hair loss. Without wishing to be bound by theory, the
extracts derived from sugar cane comprising polyphenols of the
present disclosure activate resting hair follicles (`follicular
activation`) and can therefore be used to prevent, improve or treat
hair-loss conditions.
[0258] In one embodiment, the hair loss condition is balding. In
one embodiment, the hair loss condition is baldness. In one
embodiment, the hair loss condition is hair thinning. In one
embodiment, the hair loss condition is male-pattern hair loss. In
one embodiment, the hair loss condition is female-pattern hair
loss. In one embodiment, the hair loss condition is alopecia
areata. In one embodiment, the hair loss condition is telogen
effluvium.
[0259] In one embodiment, the skin condition is wound healing and
the extracts derived from sugar cane of the present disclosure may
be used in the improvement or treatment of wound healing. Skin
wounds can include trauma, burns, abrasions, lacerations,
ulcerations, skin cancers, infection or underlying medical
conditions such as diabetes and wounds from surgical
procedures.
[0260] Wound healing is a natural restorative response to tissue
injury which generates resurfacing, reconstitution, and restoration
of the tensile strength of injured skin. The wound healing stages
are made up of three basic phases--the inflammatory phase which
consists of the establishment of homeostasis, and inflammation; the
proliferative phase, which consists of granulation, contraction and
epithelialisation and the remodelling phase, which eventually
determines the strength and appearance of the healed skin. The
symptoms of inflammation include heat, redness, swelling and pain.
Whilst inflammation is involved in the first stage of the wound
healing process, if it persists for too long, inflammation can
delay recovery and may result in increased scar tissues.
[0261] Without wishing to be bound by theory, the extracts derived
from sugar cane of the present disclosure provide their beneficial
effects by influencing one or more phases of the healing process
such as by reducing inflammation and thereby preventing scar
tissue. In addition, the high antioxidant activity of the extracts
of the present disclosure play a significant role in the process of
wound healing by improving the healing time and appearance of the
healed skin and by protecting the healing skin from oxidative
damage. Further, the extracts of the present disclosure have
antibacterial properties which aid in the prevention or treatment
of the wound from infection, which can delay the healing
process.
[0262] In one embodiment, the improvement or treatment of wound
healing provides reduced inflammation. In one embodiment, the
improvement or treatment of wound healing provides decreased
formation of scar tissue. In one embodiment, the improvement or
treatment of wound healing provides improved healing time. In one
embodiment, the improvement or treatment of wound healing provides
improved appearance of the healed skin. In one embodiment, the
improvement or treatment of wound healing provides protection of
the healing skin from oxidative damage. In one embodiment, the
improvement or treatment of wound healing provides prevention or
treatment of the wound from infection.
[0263] In the methods and uses of the present disclosure, the
extracts derived from sugar cane may be administered orally,
topically or by injection. In the methods or uses of the present
disclosure, the extracts may be administered in the form of a
composition as described herein and/or as known by one of ordinary
skill in the art.
[0264] The frequency of administration of the extract derived from
sugar cane or a composition comprising the extract derived from
sugar cane, may be as required to provide the desired prevention,
improvement or treatment of the skin condition. As would be
understood by one of ordinary skill in the art, the frequency of
administration of the extract derived from sugar cane or a
composition comprising the extract derived from sugar cane, may
depend on the amount or dosage of the extract. A higher amount or
dosage of the extract derived from sugar cane may result in less
frequent administration being required. A lower amount or dosage of
the extract derived from sugar cane may result in more frequent
administration being required. The administration of the extract
derived from sugar cane or a composition comprising the extract
derived from sugar cane, may be for a short period or for an
extended or continuous period, depending on the skin condition.
[0265] The frequency of administration may be daily, twice daily,
thrice daily, every 1-3 days, every 1-5 days, weekly, fortnightly,
monthly, bi-monthly, every 1-3 months, every 1-6 months, every 6
months, or yearly. In one embodiment, the frequency of
administration is daily. In one embodiment, the frequency of
administration is twice daily. In one embodiment, the frequency of
administration is weekly. In one embodiment, the frequency of
administration is fortnightly. In one embodiment, the frequency of
administration is monthly. In one embodiment, the frequency of
administration is bi-monthly. In one embodiment, the frequency of
administration is every 1-3 months. In one embodiment, the
frequency of administration is every 1-6 months. In one embodiment,
the frequency of administration is every 6 months. In one
embodiment, the frequency of administration is yearly.
[0266] The methods and uses may be useful for any mammal with a
skin condition. The mammal may, for example, be primates, such as
humans, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats
or other bovine, ovine, equine, canine, feline, rodent or murine
species. In one embodiment, the mammal is human.
[0267] As would be understood by one of ordinary skill in the art,
the skin may be any skin or part thereof of a subject. For example,
but not limited to, the skin may be the skin on the face, neck,
hands, chest, arms, legs, shoulders, back and feet. In one
embodiment, the skin is on the face, neck, hands and/or. In one
embodiment, the skin is on the face. In one embodiment, the skin is
on the neck. In one embodiment, the skin is on the hands. In one
embodiment, the skin is on the back.
EXAMPLES
[0268] Example 1 provides illustrative and non-limiting examples of
characterisation of the extracts derived from sugar cane of the
present disclosure.
Example 1. Characterisation of Extracts Derived from Sugar Cane
[0269] In order to characterise the types and quantity of
polyphenols in extracts derived from sugar cane, some extracts were
analysed by Liquid Chromatography-Mass Spectrometry (LCMS) and by
NMR spectroscopy.
[0270] The three samples A, B, and C were fractions from molasses
(FIG. 1). All the samples were stored at -20.degree. C.
TABLE-US-00001 TABLE 1 Extract fractions from molasses Code Sample
Name Description A FPX66 bound fraction Brown syrup B FPX66 unbound
fraction Light yellow syrup C 74 Brix Dark brown syrup
[0271] One mL of each of the samples were transferred into
pre-weighed vials in duplicate and then freeze-dried for 3 days to
obtain dry mass (Table 2). One replicate of each of the samples was
analysed by NMR spectroscopy and the other replicate of each of the
samples was used for quantitative analysis of polyphenols by
LCMS.
TABLE-US-00002 TABLE 2 Moisture content of samples Wt. of Wt. of
Repli- 1.0 Loss in dried % Mois- Anal- Sample cate mL, g wt., g
extract, g ture ysis A: a 1.0568 0.8471 0.2097 80.16 NMR Bound b
1.0683 0.8559 0.2124 80.12 LCMS Fraction B: a 1.1324 0.7761 0.3563
68.54 NMR Unbound b 1.1288 0.7730 0.3558 68.48 LCMS Fraction C: a
1.0300 0.2491 0.7809 24.18 NMR 74 Brix b 1.1690 0.2751 0.8939 23.53
LCMS Fraction
[0272] The 74 Brix sample was fractionated by C18 solid phase
extraction (SPE) to remove the sugars and obtain more concentrated
phenolic components. One mL was diluted in Milli-Q water and eluted
through a Waters 3 mL SPE Vac C18 cartridge that was initially
activated with MeOH and then conditioned with Milli-Q water. The
polar components were eluted with 6 mL Milli-Q water which was
discarded. The remaining metabolites on the SPE cartridge were then
eluted with 2.times.3 mL MeOH into a pre-weighed vial and the
solvent was evaporated to dryness under nitrogen gas. The 74 Brix
SPE-MeOH fraction was further dried overnight in the freeze dryer
and then weighed to obtain the dry weight of fraction (55.6 mg).
The extract was reconstituted in 200 .mu.L 80:20 MeOH--H.sub.2O
(concentration=278 mg/mL) and analysed on the LCMS.
Reference Standards
[0273] Table 3 lists the reference standards used for the
qualitative analysis of phenolic compounds by LCMS. Standard
solutions were prepared either in MeOH or 1:1 MeOH--H.sub.2O.
Fourteen of the standards were used for quantitative analysis of
phenolic compounds by LCMS and a range concentrations was prepared
from stock solutions indicated in Table 3 using 80:20
MeOH--H.sub.2O as diluent.
TABLE-US-00003 TABLE 3 List of reference standards used for LCMS
analysis Stock Molecular. Molecular Concentration, Code Compound
Formula Wt., g/mol .mu.g/mL S01 Syringic acid
C.sub.9H.sub.10O.sub.5 198.17 6,000 S02 Caffeic acid
C.sub.9H.sub.8O.sub.4 180.16 600 S03 Vanillin C.sub.8H.sub.8O.sub.3
152.15 60 S4 Sinapic acid C.sub.11H.sub.12O.sub.5 224.21 115 S5
Tricin C.sub.17H.sub.14O.sub.7 330.29 100 S6 Chlorogenic acid
C.sub.16H.sub.18O.sub.9 354.31 1,900 S7 Diosmin
C.sub.28H.sub.32O.sub.15 608.54 1,000 S8 Diosmetin
C.sub.16H.sub.12O.sub.6 300.26 100 S9 Apigenin
C.sub.15H.sub.10O.sub.5 270.24 10 S10 Vitexin
C.sub.21H.sub.20O.sub.10 432.38 100 S11 Orientin
C.sub.21H.sub.20O.sub.11 448.38 90 S12 Homoorientin
C.sub.21H.sub.20O.sub.11 448.38 40 S13 Swertisin
C.sub.22H.sub.22O.sub.10 446.40 21 S14 Myricetin
C.sub.15H.sub.10O.sub.8 318.24 400
Nuclear Magnetic Resonance (NMR) Spectroscopy
[0274] Approximately 1 g of the samples were freeze dried and the
dried residue was taken up in at least 1 mL of D.sub.2O (Cambridge
Isotopes) with 2 mM of 3-(trimethylsilyl)propionic-2,2,3,3-d.sub.4
acid sodium salt (TSP, Sigma Aldrich 269913) and 0.5% sodium azide
(NAN.sub.3). Six hundred L of each sample was transferred into 5 mm
NMR tubes and analysed. .sup.1H (700.13 MHz) and .sup.13C NMR
(176.07 MHz) spectra were acquired using a Bruker Avance III NMR
spectrometer with cryoprobe and TopSpin v3.2 software.
Qualitative Analysis by Liquid Chromatography-Mass Spectroscopy
(LCMS)
[0275] The samples were analysed by LCMS. The negative MS data was
analysed using Genedata software and after pre-processing (RT
restriction to exclude sugars, noise removal, cluster
identification, etc.). 4,250 features were identified across all
samples. There were 4,196 features identified in sample A (FPX66
bound fraction), 1,127 in sample B (FPX66 unbound fraction), and
178 in C (74 Brix sample) (FIG. 3).
[0276] A number of phenolic compounds were identified in the
extracts by comparison to the 42 standards analysed: vanillin,
apigenin, orientin, vitexin, caffeic acid, chlorogenic acid,
syringic acid, diosmin, swertisin, homoorientin, diosmetin, sinapic
acid (trace amount), myricetin (trace amount), tricin (trace
amount).
[0277] Table 4 exhibits polyphenol amounts in extracts derived from
sugar cane from LCMS analysis in .mu.g/gram dry weight basis.
TABLE-US-00004 TABLE 4 Polyphenol amounts in exemplary extracts
derived from sugar cane of the present disclosure 74 Brix sample
(C) FPX66 bound sample (A) Polyphenol in .mu.g/g in .mu.g/g
Syringic Acid 10.9 107.57 Caffeic Acid 0.29 7.54 Vanillin 0.153
2.13 Sinapic Acid 0.18 1.73 Tricin 0.03 0.4 Chlorogenic Acid 6.53
74.29 Diosmin 19.45 227 Diosmetin 0.15 0.16 Apigenin 0.001 0.01
Vitexin 0.084 1.62 Orientin 0.245 4.5 Homoorientin 0.041 0.58
Swertisin 0.69 5.25
Qualitative Analysis by Nuclear Magnetic Resonance (NMR)
Spectroscopy
[0278] All the samples showed the dominant presence of sucrose and
glucose with fructose present in lower amounts (FIG. 4). The
samples A, B and C showed well resolved peaks in the 3-5 ppm region
where the sugar signals are expected.
[0279] Metabolites such as organic acids and amino acids were
identified through database comparison in Chenomx.TM. and the Human
Metabolome Database (www.hmdb.ca). These metabolites were in either
or both the bound and unbound fractions (FIGS. 5 and 6).
[0280] Organic acids identified were acetate, pyruvate, and
formate; and amimo acids identified were isoleucine, valine, methyl
succinate, hydroxybutyrate, alanine, proline, methionine,
sarcosine, asparagine. Trigonelline, which is an alkaloid typically
present in coffee was also identified (FIG. 6).
[0281] Amino acids identified were isoleucine, valine, methyl
succinate, hydroxybutyrate, alanine, proline, methionine,
sarcosine, asparagine.
[0282] Total amino acids, free amino acids, essential amino acids
and leucine, minerals of the extract were measured by using
standard technique.
[0283] Table 5 exhibits mineral concentration of an extract derived
from sugar cane of the present disclosure in mg/Kg dry weight
basis. The concentration of selenium and chromium is shown in
.mu.g/kg dry weight basis.
TABLE-US-00005 TABLE 5 Mineral composition of extracts derived from
sugar cane of the present disclosure 74 Brix FPX66 bound FPX66
bound Anions Sample (C) sample (A) sample (A) Potassium 26,000
mg/kg 100-250 mg/kg 190 mg/kg Sodium 450 mg/kg 10-50 mg/kg 30 mg/kg
Calcium 1,090 mg/kg 8,000-9,000 mg/kg 8,800 mg/kg Magnesium 4,700
mg/kg 1,500-2,500 mg/kg 2,000 mg/kg Iron 65 mg/kg 800-1000 mg/kg
890 mg/kg Zinc 6.6 mg/kg Not detected Not detected Selenium 786
.mu.g/kg Not detected Not detected (.mu.g/kg) Chromium 1,300
.mu.g/kg Not detected Not detected (.mu.g/kg)
Analysis by Gas Chromatography-Mass Spectroscopy (GC-MS)
[0284] In order to characterise the types of compounds in extracts
derived from sugar cane molasses, extracts A, B and D were
additionally analysed by Gas Chromatography-Mass Spectrometry
(GC-MS). The three extracts A, B, and D were fractions from
molasses (FIGS. 1 and 2).
Polar Metabolite Derivatization
[0285] All samples were dissolved in 10 .mu.L of 30 mg/mL
methoxyamine hydrochloride in pyridine and derivatized at
37.degree. C. for 120 minutes with mixing at 500 rpm. The samples
were incubated for 30 minutes with mixing at 500 rpm after addition
of both 20 .mu.L N,O-bis-(trimethylsilyl)trifluoroacetamide (BSTFA)
and 1 .mu.L retention time standard mixture [0.029% (v/v)
n-dodecane, n-pentadecane, n-nonadecane, n-docosane, n-octacosane,
n-dotriacontane, n-hexatriacontane dissolved in pyridine]. Each
derivatized sample was allowed to rest for 60 min prior to
injection.
GC-MS Instrument Conditions
[0286] Samples (1 .mu.L) were then injected into a GC-MS system in
split (1:20 split ratio) or splitless mode, comprised of a Gerstel
PAL3 Autosampler, a 7890B Agilent gas chromatograph and a 5977B
Agilent quadrupole MS (Agilent, Santa Clara, USA). The Mass
Spectrometer was adjusted according to the manufacturer's
recommendations using tris-(perfluorobutyl)-amine (CF43). A J&W
Scientific VF-5MS column (30 m long with 10 m guard column, 0.25 mm
inner diameter, 0.25 .mu.m film thickness) was used. The injection
temperature was set at 250.degree. C.; the Mass Spectrometer
transfer line at 290.degree. C., the ion source adjusted to
250.degree. C. and the quadrupole at 150.degree. C. Helium (UHP
5.0) was used as the carrier gas at a flow rate of 1.0 mL/minute.
The following temperature program was used; injection at 70.degree.
C., hold for 1 minute, followed by a 7.degree. C./minute oven
temperature, ramp to 325.degree. C. and a final 6 minute heating at
325.degree. C. Mass spectra were recorded at 2 scans/s with an
50-600 m/z scanning range.
Data Processing and Statistical Analysis
[0287] Both chromatograms and mass spectra were processed using the
Agilent MassHunter Workstation Software, Quantitative Analysis,
Version B.07.01/Build 7.1.524.0. Mass spectra of eluting compounds
were identified using the commercial mass spectra library NIST 08
(http://www.nist.gov), the public domain mass spectra library of
Max-Planck-Institute for Plant Physiology, Golm, Germany
(http://csbdb.mpimp-golm.mpg.de/csbdb/dbma/msri.html) and the
in-house mass spectral library. All matching mass spectra were
additionally verified by determination of the retention time by
analysis of authentic standard substances. If a specific metabolite
had multiple TMS derivatives, the metabolite with the greater
detector response and better peak shape within the dynamic range of
the instrument was selected.
[0288] The results of the GC-MS analysis (Tables 6-9) confirmed the
LCMS study and many additional compounds were detected. Each sample
contained more than 100 different identified compounds. These
mainly included a diverse range of acids, polyphenols, sugars and
phytosterols, many of which have well characterised beneficial
effects on health. For example, potential active ingredients for
anti-acne and psoriasis were identified including azelaic acid,
salicylic acid, gycolic acid. Arbutin, an antioxidant, which
inhibits tyrosinase, and is known to be skin-whitening agent was
also identified. Of the compounds identified, 19 compounds were
found to be specific to Extract B. The GC-MS traces of the extracts
are shown in FIG. 7.
TABLE-US-00006 TABLE 6 Identified peaks and retention times of
Extract A Compound Retention Time CONT: 1038.1 6.252 Pyruvic Acid
(1TMS) 7.00900 Lactic Acid (2TMS) 7.12300 Glycolic Acid 7.38700
Oxalic Acid (2TMS) 7.56100 CONT 1228 8.17000 3-Hydroxypropanoic
acid 8.49900 3-hydroxy-pyridine (1TMS) 8.52000 CONT: 1228 8.76250
n-Heptanoic acid (1TMS) 9.38600 Malonic Acid (2TMS) 9.54500 Benzoic
acid (1TMS) 10.4667 DL-Serine (2TMS) 10.523 Phosphoric acid (3TMS)
10.686 Succinic Acid (2TMS) 11.553 DL-Glyceric Acid (3TMS) 11.772
Uracil (2TMS) 11.997 Fumaric Acid (2TMS) 12.222 n-Nonanoic acid
(1TMS) 12.432 Thymine (2TMS) 13.12 DL-Aspartic acid (2TMS) 13.562
DL-Malic Acid (3TMS) 14.622 DL-Lactic Acid Dimer (2TMS) 14.861
2,4-dihydroxy Butanoic Acid 13.256 (3TMS) Salicylic acid (2TMS)
15.071 Erythronic acid (4TMS) 15.509 DL-Phenylalanine (1TMS) 15.807
trans-Cinnamic acid (TMS) 15.845 3-Hydroxy-Benzoic Acid 16.046
4-Hydroxyphenylethanol 16.128 3-Hydroxy-3-methylglutaric 16.5649
4-hydroxy-Benzoic Acid 17.099 1,4-lactone Pentonic acid 17.12
(3TMS) 1,4-lactone, 2-methoximine 17.286 Gluconic acid Arabinose
17.419 1-O-methyl alpha D 18.703 2,Deoxy-pentos-3-ylose 18.8857
3-deoxy-Glucosone 18.961 Vanillic acid (2TMS) 19.271
2-Keto-L-gluconic acid (5TMS) 19.308 4-hydroxy-cis-cinnamic acid
19.642 Azelaic acid (2TMS) 19.715 Shikimic acid (4TMS) 19.818
D(-)-Quinic acid (5TMS) 20.477 Fructose MX1 20.641 Fructose MX2
20.785 1,5-lactone Gluconic acid 20.958 Glucose MX1 21.019
Unknown21.85 21.285 Glucose MX2 21.285 1-Ethylglucopyranoside
21.649 Sorbitol 21.841 4-hydroxy trans-cinnamic 21.924 Gluconic
acid (6TMS) 22.463 Unknown 22.614 22.614 Unknown 22.650 22.65
1-o-methyl beta-D- 22.926 Galactopyranoside (4TMS)
3,4-dimethoxy-trans- 23.13 n-Hexadecanoic acid 23.274 Inositol
23.718 n-Octadecan-1-ol (Steryl 23.793 trans-Ferulic acid (2TMS)
23.944 3,4-dimethoxy-trans 24.471 2-O-Glcyerol-beta-D- 24.896
galactopyranoside (6TMS) trans-Sinapic acid (2TMS) 25.852
1-Benzylglucopyranoside 27.474 Thymidine (3TMS) 27.745 Uridine
(3TMS) 28.267 Unknown_28.673 28.384 Unknown_28.722 28.722
Unknown_29.097 29.097 Salicylic acid 29.515 (Arbutin) Hydroquinone-
29.614 beta-D-glucopyranoside (5TMS) Unknown 29.700 29.7 Adenosine
(3TMS) 29.861 Unknown_29.995 29.9987 Sucrose 30.073 Cellobiose MX1
30.74 Trehalose 31.14 Turanose MX1 31.273
TABLE-US-00007 TABLE 7 Identified peaks and retention times of
Extract B Compound Retention time Pyruvic Acid (1TMS) 6.99800 DL-
Lactic Acid (2TMS) 7.128 Glycolic Acid 7.38260 Oxalic Acid (2TMS)
7.55710 CONT 1140 8.44700 3-Hydroxypropanoic Acid 8.49810 (2TMS)
CONT 1228 8.76800 DL-Isoleucine (1TMS) 9.15240 Malonic Acid (2TMS)
9.54010 Methylmalonic Acid 9.63460 (2TMS) Cont. 1228.8 9.77600
4-Hydroxy Butyric Acid 10.11450 (2TMS) Cont: 1249.3 10.2423 Glycine
(3TMS) 10.3667 Cont: 1256.7 10.4578 DL-Serine (2TMS) 10.52 Glycerol
10.598 Thymidine 10.85 DL-Threonine (2TMS) 11.1777 Cont 1308
11.2255 Succinic acid (2TMS) 11.5521 DL-Glyceric Acid (3TMS)
11.7654 Glutaric acid (2TMS) 11.93 Fumaric Acid (2TMS) 12.2187
n-nonanoic acid 12.4298 2-Methyl-1,3-butanediol 12.5264
2-methyl-1,2-propanediol 12.9108 (2TMS) Unknown_503_459_13.1919
13.1919 2,4-dihydroxy-Butanoic acid 13.2575 (3TMS) DL-2-methyl
Malic 13.8718 Acid)3TMS) n-Decanoic acid (1TMS) 14.168 DL-Malic
Acid (3TMS) 14.6195 DL-Lactic acid dimer 14.8606 (2TMS) Glutaric
Acid (2TMS) 15.0406 Salicylic acid (2TMS) 15.067
Unknown_408_393_15.1528 15.1528 DL-Pyroglutamic Acid 15.2894 (2TMS)
2-Hydroxyglutaric Acid 15.353 (3TMS) hydrocarbon contamination
15.4061 Erythronic acid (4TMS) 15.5038 Threonic Acid (4TMS) 15.796
Unknown_16.0060 16.066 2,3-Dihydroxybutanedioic 16.3982 acid (4TMS)
Unknown_16.441 16.439 3-Hydroxy-3-methylglutaric 16.5604 acid
(3TMS) Unknown 16.796 4-hydroxy-Benzoic Acid 17.077 (2TMS)
2-Hydroxyhexanedioic acid 17.175 (3TMS) 1,4-lactone, 2methoximine
17.262 Gluconic acid Unknown 17.426 2,4,5-Trihydroxypentanoic acid
17.5502 (4TMS) 3- Hydroxysebacic acid 18.581 (3TMS) Ribonic acid
(5TMS) 19.2434 2-Keto-L-gluconic acid (5TMS) 19.3067 3
-deoxy-Glucosone 19.53 Shikimic acid (4TMS) 19.829 unknown_20.027
20.0266 D-Psicose (5TMS) (also known 20.163 as Allulose)
Unknown_20.419 20.419 Fructose MX1 20.652 Fructose MX2 20.796
Glucose MX1 21.018 Glucose MX2 21.296 Mannitol 21.463
Ethylglucopyranoside (4TMS) 21.7186 4-hydroxy-trans cinnamic acid
21.954 unknown_22.015 22.015 Gulonic acid (6TMS) 22.076
Unknown_22.276 22.276 Gluconic acid (6TMS) 22.507 Unknown_22.6352
22.6352 Galactonic acid (6TMS) 22.692 1-O-methyl-beta-D- 22.946
Galactopyranoside (4TMS) 1-Ethylglucopyranoside 22.9484 (4TMS)
n-hexadecanoic acid (1TMS) 23.305 Inositol 23.696 Unknown_23.963
23.9661 Unknown_24.227 24.2272 Unknown_24.307 24.3072 trans-Caffeic
acid (3TMS) 24.4705 Unknown_24.740 24.7404 Unknown_24.904 24.9038
Unknown_24.9560 24.956 n-Octadecanoic acid (1TMS) 25.82659
Unknown_26.411 Unknown_27.229 (Helicin) Salicylaldehyde-beta-
29.095 D-glucoside (Arbutin) Hydroquinone-beta- 29.612
D-glucopyranoside (5TMS) Sucrose 30.084 Cellobiose MX1 30.762
Maltose 31.128 Trehalose 31.195 3-caffeoyl-trans-Quinic acid
34.8481 5-caffeoyl-trans-Quinic Acid 35.617 Campesterol (1TMS)
36.361 Raffinose 36.95 1-Kestose 37.083
TABLE-US-00008 TABLE 8 Identified peaks and retention times of
Extract D Compound RT CONT: 1038.1 6.252 Pyruvic Acid (1TMS)
7.05400 Lactic Acid (2TMS) 7.16200 Glycolic Acid 7.42600 Oxalic
Acid (2TMS) 7.58700 CONT 1228 8.17000 3-Hydroxypropanoic acid
(2TMS) 8.52500 3-hydroxy-pyridine (1TMS) 8.55100 CONT: 1228 8.76800
DL-Isoleucine (1TMS) 9.18700 n-Heptanoic acid (1TMS) 9.40500
Malonic Acid (2TMS) 9.56600 Benzoic acid (1TMS) 10.4667 DL-Serine
(2TMS) 10.537 Phosphoric acid (3TMS) 10.75 Thymidine (BP) 10.857
Maleic Acid (2TMS) 11.375 Succinic Acid (2TMS) 11.565 DL-Glyceric
Acid (3TMS) 11.78 Itaconic acid (2TMS) 12.072 Fumaric Acid (2TMS)
12.23 n-Nonanoic acid (1TMS) 12.438 Nicotinic acid (1TMS) 12.749
Cytosine (2TMS) 12.822 2,4-dihydroxy Butanoic Acid 13.262 (3TMS)
2-methyl-DL-Malic Acid (3TMS) 14.319 Salicylic acid (2TMS) 14.432
3,4,5,6-D4-Salicylic acid (2TMS) 14.555 DL-Malic acid (3TMS) 14.626
Nicotinimide (1TMS) 14.691 DL-Lactic acid dimer (2TMS) 14.863
Glutaric acid (2TMS) 15.049 DL-Pyroglutamic acid (2TMS) 15.296
2-Hydroxyglutatic acid (3TMS) 15.365 Erythronic acid (4TMS) 15.508
Trihydroxybenzene (3TMS) 15.53 DL-Phenylalanine (1TMS) 15.82
trans-Cinnamic acid (TMS) 15.845 3-Hydroxy-Benzoic Acid (2TMS)
16.045 4-Hydroxyphenylethanol (2TMS) 16.128 2-oxo-Glutaric acid
(1MEOX) 16.16 (2TMS) 3-Hydroxy-3-methylglutaric acid 16.564 (3TMS)
4-hydroxy-Benzoic Acid (2TMS) 17.087 1,4-lactone Pentonic acid
(3TMS) 177.116 1,4-lactone, 2-methoximine 17.286 Gluconic acid
Arabinose 17.419 1-O-methyl beta-D- 18.701 Galactopyranoside
2,Deoxy-pentos-3-ylose 18.883 dimethoxyamine cis-Aconitic acid
(3TMS) 18.951 4-Hydroxyphenylpropionic acid 19.192 (2TMS) Vanillic
acid (2TMS) 19.261 2-Keto-L-gluconic acid (5TMS) 19.309
3-deoxy-Glucosone 19.531 4-hydroxy-cis-cinnamic acid 19.641 Azelaic
acid (2TMS) 19.709 Shikimic acid (4TMS) 19.818 D(-)-Quinic acid
(5TMS) 20.481 Fructose MX1 20.63 Fructose MX2 20.785 1,5-lactone
Gluconic acid 20.954 Glucose MX1 20.996 Glucose MX2 21.285 Sorbitol
21.841 4-hydroxy trans-cinnamic acid 21.913 Gluconic acid (6TMS)
22.462 1-o-methyl beta-D- 22.535 Galactopyranoside (4TMS)
3,4-dimethoxy-trans-Cinnamic acid 23.127 n-Hexadecanoic acid (1TMS)
23.277 Inositol 23.718 n-Octadecan-1-ol (Steryl alcohol) 23.791
trans-Ferulic acid (2TMS) 23.937 trans-Caffeic acid (3TMS) 24.468
2-O-Glcerol-beta-D- 25.514 galactopyranoside (6TMS)
1-Methyl-beta-D-galactopyranoside 25.517 (4TMS) trans-Sinapic acid
(2TMS) 25.85 1-Benzylglucopyranoside 27.471 Thymidine (3TMS) 27.74
Uridine (3TMS) 28.272 Salicylaldehyde-beta-D-glucoside 29.521 (TMS)
Salicylic acid glucopyranoside 29.522 (5TMS) (Arbutin)
Hydroquinone-beta-D- 29.924 glucopyranoside (5TMS) Unknown 29.704
29.704 Adenosine (3TMS) 29.866 Sucrose 30.073 Cellobiose MX1 30.751
Maltose 31.129 Trehalose 31.151 Turanose MX1 31.273 Guanosine
31.638 Melibiose MX1 32.406 beta-D-Glucopyranuronic acid 33.164
(5TMS) Galactinol 33.573 3-p-coumaroyl-trans-Quinic acid 33.658
Unknown_33.736 33.736 3-caffeoyl-trans-Quinic acid 34.859
Unknown_34.708 35.32 4-Caffeoyl-trans-Quinic Acid 35.418
Unknown_35.549 35.55 5-Caffeoyl-trans-Quinic Acid 35.619 1-Kestose
37.083
TABLE-US-00009 TABLE 9 Library of individual compounds detected
across each sample Extract Extract Extract Compounds A B D Notes
Hydroquinone-beta-D- x x x antioxidant, glucopyranoside inhibits
tyrosinase, (Arbutin) (5TMS) whitening agent.
Salicylaldehyde-beta-D- x x O-glucoside glucoside (Helicin)
1,4-lactone Pentonic x x sugar acid acid (3TMS) 1,4-lactone, 2- x x
x sugar acid methoximine Gluconic acid 1,5-lactone Gluconic acid x
x sugar acid 1-Benzylglucopyranoside x x glucoside
1-Ethylglucopyranoside x x glucoside (4TMS) 1-Kestose x x inulin
1-O-methyl alpha D x found in cereals Mannopyranoside and alfalfa
(4TMS) 1-o-methyl beta-D- x x x glucoside Galactopyranoside (4TMS)
2,Deoxy-pentos-3-xylose x x amine dimethoxyamine (2TMS) 2,3- x
antioxidant, Dihydroxybutanedioic one of the main acid (4TMS)
Tartatic acid acids found in wine; acidifier 2,4,5- x fatty acid
Trihydroxypentanoic acid (4TMS) 2,4-dihydroxy-Butanoic x x x
carboxylic acid acid (3TMS) 2-Hydroxyglutaric Acid x x fatty acid
(3TMS) 2-Keto-L-gluconic acid x x x sugar acid (5TMS)
2-O-Glycerol-beta-D- x x glucoside galactopyranoside (6TMS)
2-oxo-Glutaric acid x sugar acid (1MEOX) (2TMS) 3-Hydroxysebacic
acid x fatty acid (3TMS) 3,4-dimethoxy-trans x x phenolic acid
Cinnamic Acid 3,4,5,6-D4-Salicylic acid x carboxylic acid (2TMS)
3-caffeoyl-trans-Quinic x x x cyclic polyol acid 3-deoxy-Glucosone
x x x malliard reaction product 3-Hydroxy-Benzoic Acid x x
carboxylic acid (2TMS) 3-Hydroxy-3- x x x fatty acid methylglutaric
acid (3TMS) 3-Hydroxypropanoic x x x carboxylic acid Acid (2TMS)
3-hydroxy-pyridine x x pyridine (1TMS) 3-p-coumaroyl-trans- x x
cyclic polyol Quinic acid 4-Caffeoyl-trans-Quinic x x cyclic polyol
Acid 4-Hydroxy Butyric Acid x relevant for cancer, (2TMS) diabetes,
inflammation 4-hydroxy trans-cinnamic x x x phenolic acid acid
4-hydroxy-Benzoic Acid x x carboxylic acid (2TMS)
4-hydroxy-cis-cinnamic x x phenolic acid acid
4-Hydroxyphenylethanol x x antioxidant phenol (2TMS) (Tyrosol) 4- x
carboxylic acid Hydroxyphenylpropionic acid (2TMS)
5-caffeoyl-trans-Quinic x x x cyclic polyol Acid Adenosine (3TMS) x
x nucleoside Arabinose x x aldopentose Azelaic acid (2TMS) x x
anti-acne and psoriasis Benzoic acid (1TMS) x x carboxylic acid
beta-D-Glucopyranuronic x o-glucuronides acid (5TMS) Campesterol
(1TMS) x phytosterol, antioxidant and hypocholesterolemic
Cellobiose MX1 x x x disaccharide cis-Aconitic acid (3TMS) x
organic acid Cytosine (2TMS) x nucleobase D(-)-Quinic acid x x
cyclic polyol (5TMS) DL-2-methyl Malic Acid x x organic acid (3TMS)
DL-Isoleucine (1TMS) x x organic acid DL-Lactic Acid (2TMS) x x x
organic acid DL-Malic Acid (3TMS) x x x organic acid DL-Serine
(2TMS) x x x organic acid DL-Threonine (2TMS) x anti-oxidant
DL-Aspartic acid (2TMS) x organic acid DL-Glyceric Acid x x x sugar
acid (3TMS) DL-Lactic acid dimer x x x organic acid (2TMS)
DL-Phenylalanine x x organic acid (1TMS) DL-Pyroglutamic Acid x x
organic acid (2TMS) D-Psicose (5TMS) (also x monosaccharide known
as Allulose) Erythronic acid (4TMS) x x x sugar acid
Ethylglucopyranoside x glucoside (4TMS) Fructose MX1 x x x
monosaccharide Fructose MX2 x x x monosaccharide Fumaric Acid
(2TMS) x x x carboxylic acid Galactinol x x galactose metabolism
intermediate Galactonic acid (6TMS) x organic acid Gluconic acid
(6TMS) x x x sugar acid Glucose MX1 x x x monosaccharide Glucose
MX2 x x x monosaccharide Glutaric acid (2TMS) x x fatty acid
Glycerol x polyol compound Glycine (3TMS) x amino acid Glycolic
Acid x x x organic acid Guanosine x nucleoside Gulonic acid (6TMS)
x organic acid Inositol x x x carbocyclic sugar Itaconic acid
(2TMS) x organic acid Maleic Acid (2TMS) x organic acid Malonic
Acid (2TMS) x x x organic acid Maltose x x disaccharide Mannitol x
sugar alcohol Melibiose MX1 x x reducing disaccharide Methylmalonic
Acid x carboxylic acid (2TMS) n-Decanoic acid (1TMS) x
anti-bacteria, anti- fungal, nematocide n-Heptanoic acid (1TMS) x x
fatty acid n-hexadecanoic acid x x x fatty acid (1TMS) Nicotinic
acid (Niacin) x Vitamin B3 form (1TMS) Nicotinimide (1TMS) x
Vitamin B3 form n-Nonanoic acid (1TMS) x x x fatty acid
n-Octadecan-1-ol (Steryl x x fatty alcohol alcohol) n-Octadecanoic
acid x hypocholestelomic (1TMS) Oxalic Acid (2TMS) x x x organic
acid Phosphoric acid (3TMS) x x inorganic acid Pyruvic Acid (1TMS)
x x x organic acid Raffinose x trisaccharide Ribonic acid (5TMS) x
sugar acid Salicylic acid (2TMS) x x x anti- acne/antipsoriasis
Salicylic acid x x glucoside glucopyranoside (5TMS) Shikimic acid
(4TMS) x x x cyclic polyol Sorbitol x x sugar alcohol Stigmasterol
x phytosterol Succinic acid (2TMS) x x x organic acid Sucrose x x x
disaccharide Threonic Acid (4TMS) x sugar acid Thymidine x x x
nucleotide Thymine (2TMS) x nucleobase trans-Caffeic acid x x
phenolic acid (3TMS) trans-Cinnamic acid x x phenolic acid (TMS)
trans-Ferulic acid x x phenolic acid (2TMS) trans-Sinapic acid x
phenolic acid (2TMS) Trehalose x x x disaccharide Turanose MX1 x x
reducing disaccharide Uracil (2TMS) x nucleobase Uridine (3TMS) x x
nucleoside Vanillic acid (2TMS) x x phenolic acid
[0289] Example 2 to Example 6 provide illustrative and non-limiting
examples of the preparation and characterisation of extracts
derived from sugar cane of the present disclosure.
Example 2. Sugar Cane Extracts Derived from Molasses
[0290] Example sugar cane extracts of the present disclosure were
prepared from molasses as follows.
[0291] Sugar cane molasses was diluted with de-ionised water, mixed
well to give a final Brix of 50'. This mixture was held between
20-25.degree. C. and 95% food grade ethanol added with overhead
stirring to ensure that the ethanolic mixture was evenly and
quickly dispersed. This step was continued until the final ethanol
content reached 76% v/v. During this time, a gelatinous precipitate
formed. The precipitate was allowed to settle and the supernatant
was decanted and filtered under vacuum in a Buchner Funnel through
a Whatman GFA filter paper grade 1. The ethanol was subsequently
removed under reduced pressure in a Buchi Rotary Evaporator at
45.degree. C. Evaporation was continued under reduced pressure at
50-55.degree. C. to give a syrup with a final Brix of 70.degree.
with a bitter sweet aroma. Characterisation of exemplary syrups
obtained by this method is shown in Table 10.
TABLE-US-00010 TABLE 10 Properties of sugar cane extracts prepared
from molasses Property Extract 1 Extract 2 Brix (.degree. Bx) 65-70
70.degree. (+/-2) @ 20.degree. C. pH 4-5 4.6 (+/-0.2) @ 20.degree.
C. Density (g/mL) 1.25-1.35 1.35 (+/-0.05 @ 20.degree. C.) Colour
Absorbance 69.1 -- 420 Absorbance 270 708 -- Ratio A270/A420 10 --
Total Polyphenol 16,500 Minimum 20,000 (mg/L as gallic acid
equivalents) Total Flavonoids 2800 Minimum 7,000 (mg/L as catechin
equivalents) ORAC 5.0 -- Minimum 2.5 mol/kg CAA -- Minimum 2.5
mol/kg as trolox equivalents Conductivity 138,800 -- (.mu.S/cm)
Calcium 5100 mg/kg 400-1,300 mg/L Iron 110 mg/kg 10-100 mg/L
Magnesium 1800 mg/kg 2,400-5,500 mg/L Potassium 26,000 mg/kg
20,000-40,0000 mg/L Sodium 23 mg/100 g 60-80 mg/100 mL Zinc --
0.3-0.8 mg/100 mL Selenium -- 0.03-0.09 mg/100 mL Chromium --
0.03-0.140 mg/100 mL
Example 3. Fractionated Sugar Cane Extracts Derived from
Molasses
[0292] In general, the title fractionated sugar cane extracts may
be prepared using hydrophobic chromatography procedures. Extracts
prepared using the processes described in Example 2 and any sugar
cane derived product may be used as feedstocks for chromatography.
The hydrophobic resin used for chromatography may be a food grade
resin.
[0293] In a representative preparation, FPX66 resin (Dow, Amberlite
FPX66, food grade)) was pre-treated by washing with de-ionised
water, ethanol and then finally with de-ionised water following the
manufacturer's instructions. The washed resin was filtered under
vacuum through a Buchner Funnel using Whatman filter paper grade 1
(1 m pore size). The resin granules were then used as is.
[0294] De-ionised water was added to sugar cane molasses with
constant stirring until the Brix reached 20.degree.. To a beaker
containing 1 litre of the 20.degree. Brix feedstock (maintained at
20-25.degree. C.) and mounted on a magnetic stirrer, 500 g of wet
weight pre-treated resin was added with gentle stirring to ensure
effective mixing of the resin granules with the feedstock. The
mixing was continued for 10 min at which point the mixture was
filtered under vacuum and the resin was collected.
[0295] The collected resin was washed by resuspension in de-ionised
water (1 litre). This step was repeated.
[0296] The washed resin was then suspended in 1 litre 70% ethanol
solution in de-ionised water, stirred for 10 mins and the filtrate
was collected by vacuum filtration. This was repeated twice more
with 1 litre batches of the 70% ethanolic solution with each
filtrate being collected. Finally, the three 70% ethanolic
filtrates were combined and the ethanol removed by evaporation
under reduced pressure. The aqueous fraction was lyophilised or
spray-dried into a free flowing brown powder with a moisture
content of 0.3-2.0% w/w. The properties of the ethanolic fraction
are shown below in Table 11.
TABLE-US-00011 TABLE 11 Properties of an extract derived from sugar
cane molasses Properties Ethanol fraction Colour Absorbance at 420
nm 10 (1% in solution @ 20.degree. C.) Absorbance at 270 nm 180 (1%
in solution @ 20.degree. C.) Ratio A270 nm/A420 nm 19 (1% in
solution @ 20.degree. C.) Total Polyphenol Minimum 200 (mg/g gallic
acid equivalent) Total Flavonoid Minimum 50 (mg/g catechin
equivalent) Calcium (mg/kg) 840 Iron (mg/kg) 77 Magnesium (mg/kg)
2300 Potassium (mg/kg) 1100 Sodium (mg/g) 1700 Zinc (mg/kg) 48
Selenium (mg/kg) 0.18 Chromium (mg/kg) 1.8
[0297] FIG. 8 exhibits a LCMS spectrum of a representative extract
derived from sugar cane molasses using this process.
Example 4. Sugar Cane Extracts Derived from Dunder
[0298] A scheme for the preparation of the title sugar cane
extracts is shown in FIG. 9.
[0299] Sugar cane dunder was allowed to settled overnight for eight
hours in a V-bottom tank. The supernatant was then subjected to
sequential microfiltration through: (i) a 5 micron filter; (ii) a 1
micron filter; (iii) a 0.5 micron filter; and (iv) a 0.1 micron
filter.
[0300] The filtered supernatant was subsequently concentrated in a
heat exchanger to remove water to provide the liquid extract with
55.sup.0Bx.
[0301] The properties of an extract derived from dunder is shown
below in Table 12.
TABLE-US-00012 TABLE 12 Properties of an extract derived from
dunder Properties Sugar cane extract Brix 55.degree. (+/-2) @
20.degree. C. pH .sup. 4.6 (+/-0.2) @ 20.degree. C. Density 1.28
g/mL (+/-0.05) @ 20.degree. C. Colour Absorbance 420 190-280
Absorbance 270 2300-3000 Ratio A270/A420 10-15 Total Polyphenol
Minimum 45,000 (mg/L as gallic acid equivalent) Total Flavonoid
Minimum 10,000 (mg/L as catechin equivalent) Conductivity (uS/m)
250,000-350,000 Calcium (mg/kg) 3,000-4,000 Iron (mg/kg) 100-150
Magnesium (mg/kg) 3,000-5,000 Potassium (mg/kg) 30,000-40,000
Sodium (mg/kg) 2,000-3,000 Zinc (mg/100 g) 0.5-1.5 Selenium (mg/100
g) 0.02-0.05 Chromium (mg/100 g) 0.20-0.5
[0302] FIG. 10 exhibits example LCMS spectra for sugar cane dunder
starting material (A) and an extract of sugar cane derived dunder
(B) in accordance with the above process.
Example 5. Hybrid Sugar Cane Extracts Derived from a Combination of
Sugar Cane Molasses and Dunder
[0303] A scheme for the preparation of the title sugar cane
extracts is shown in FIG. 11.
[0304] Sugar cane mill molasses was diluted with water and mixed
with settled sugar cane dunder (as described above) and stirred
well to provide a mixture with 50.degree. Bx. The combined mixture
of molasses and dunder was maintained at a constant temperature of
between 20-25.degree. C. and 95% food grade ethanol added and
stirred to ensure that the ethanol was evenly and quickly
dispersed. Ethanol was added until the ethanol level was 76%
v/v.
[0305] The addition and mixing of ethanol led to the formation of a
gelatinous precipitate. The precipitate in the mixture was allowed
to settle and the supernatant was removed by decantation and vacuum
filtration in a Buchner funnel through a Whatman GFA filter paper
1.
[0306] The ethanol was removed from the supernatant under vacuum in
a Buchi rotary evaporator at 45.degree. C. Evaporation of water
from the supernatant was performed under vacuum at 50-55.degree. C.
until the final syrup reaches 70.degree. Bx.
[0307] Table 13 shows the properties of the hybrid sugar cane
extract obtained.
TABLE-US-00013 TABLE 13 Properties of a hybrid sugar cane extract
Property Sugar cane extract Brix .sup. 70.degree. (+/-2) @
20.degree. C. pH 4.6 (+/-0.2) @ 20.degree. C. Density 1.35 (+/-0.05
@ 20.degree. C.) Colour Absorbance 420 90-120 Absorbance 270
1900-2300 Ratio A270/A420 15-30 Total Polyphenol Min 30,000 (mg/L
as gallic acid milligrams per litre equivalent) (as gallic acid
equivalents) Total Flavonoid Minimum 10,000 (mg/L as catechin
equivalent) Conductivity (.mu.S/m) 180,000-200,000 Calcium (mg/kg)
80-160 Iron (mg/kg) 2-8 Magnesium (mg/kg) 300-600 Potassium (mg/kg)
2000-4000 Sodium (mg/kg) 60-180 Zinc (mg/100 g) 1.5-3.0 Selenium
(mg/100 g) 0.04-0.09 Chromium (mg/100 g) 0.015-0.50
Example 6. Characteristics of Extracts Derived from Sugar Cane
TABLE-US-00014 [0308] TABLE 14 Two example extracts derived from
sugar cane of the present disclosure Extract 3 prepared Extract 4
prepared according to the according to the process of Example 2
process of Example 3 Brix 65-70 Brix 68-70 Brix pH 5.3-5.9 4.5-4.7
Density 1.25-1.35 1.35 Colour Absorbance 420 69.1 65 Absorbance 270
708 1506 Ratio A270/A420 10 23 Total Polyphenol 16,500
24,000-28,000 (mg/L gallic acid equivalent) Total Flavonoid 2800
5900 (mg/L catechin equivalent) Conductivity (us) 138,800 57,200
Calcium (mg/kg) 5100 1614 Iron (mg/kg) 110 52 Magnesium (mg/kg)
1800 2250 Potassium (mg/kg) 26,000 21,000 Sodium (mg/100 g) 23
47
[0309] Table 15 exhibits a component comparison between molasses
and extracts derived from sugar cane of the present disclosure.
TABLE-US-00015 TABLE 15 A comparison between molasses and an
extract derived from sugar cane of the present disclosure. Extract
derived from Components Molasses sugar cane Total solids (g/L) 80.5
70 Fructose (g/L) 131.9 74.5 Glucose (g/L) 107.1 52.8 Sucrose (g/L)
473.8 343 Total sugars (g/L) 722.8 470 Ratio (Fructose +
Glucose/Sucrose) 0.50 0.41 Total polyphenol (mg GAE/L) 20,000
25,000-28,000 Antioxidants (ABTS mg GAE/L) 7,000-8,500
10,500-11,500 Calcium (mg/L) 5746 2145 Magnesium (mg/L) 2374 3003
Sodium (mg/L) 303 605 Potassium (mg/L) 20,794 27170
[0310] Example 7 to Example 24 provide illustrative and
non-limiting examples of activities of the extracts derived from
sugar cane of the present disclosure.
Example 7
[0311] Compositions comprising an extract derived from sugar cane
of the present disclosure were tested on human subjects to evaluate
the use in preventing, improving or treating skin conditions.
[0312] A panel of human subjects was convened to evaluate the use
of a composition comprising an extract derived from sugar cane of
the present disclosure in preventing, improving or treating skin
conditions.
[0313] Application of the compositions was topically to the face of
the human subjects. The biophysical effects of the topically
applied treatment composition was evaluated pre- and
post-application when used by the same participants. 11 subjects
were recruited for the study, with results tabulated for all 10
subjects who completed to 12 weeks.
[0314] Wrinkle reduction was determined using silicone impressions
and profilometry and skin blemishes using a Minolta
Spectrophotometer. In addition, a photographic record was taken of
each of the test participants.
Test Material Handling
[0315] Each of the test samples of extracts derived from sugar cane
of the present disclosure was assigned a unique laboratory code
number and entered into a daily log identifying the lot number,
sample description, date received and tests requested.
Standards for Inclusion in the Study
[0316] i. Individuals nominally between the ages 40 to 65 years.
[0317] ii. Individuals not taking medication or under the care of a
physician for a period of one month prior to commencement and
throughout the entire test period. [0318] iii. Individuals who have
completed a preliminary medical history. [0319] iv. Individuals who
have read, understood and signed an informed consent document.
[0320] v. Individuals who understand instructions for use and are
willing to cooperate with the program as stated. [0321] vi.
Individuals free of any dermatological or systemic disorder that
would interfere with the results, at the discretion of the
Investigator. [0322] vii. Individuals able to cooperate with the
investigator and the research staff, be willing to complete the
full course of the study. [0323] viii. Individuals with
self-described dry skin. Standards for Exclusion from the Study
[0324] i. Individuals who are under doctor's care. [0325] ii.
Individuals who are currently taking medication which in the
opinion of the investigator would mask or interfere with the
results. [0326] iii. Individuals with any history of sensitivity to
cosmetics in general and moisturisers in particular. [0327] iv.
Individuals with any form of skin cancer, or any disease that would
interfere with the test results. [0328] v. Individuals diagnosed
with chronic skin allergies. [0329] vi. Female volunteers who
indicate that they are pregnant or nursing an infant. [0330] vii.
Individuals with excessive hair on the test sites. [0331] viii.
Individuals with known hypersensitivity to cosmetic products.
Preparation
[0332] In order to precondition the test sites and keep topical
treatments constant for all test subjects, the participants were
required to abstain from use of moisturizers and skin treatments on
the test area for a period of 10 days prior to study commencement.
At the completion of the 10 days `washout` period, the participants
were required to return to the test facility at the time specified
by the technician for the study commencement.
Product Application Instructions
[0333] On the first day of the study, the participants were trained
on how to apply. The treatment composition was then evenly applied
to the faces. The participants were required to continue to use the
materials as instructed, that is, twice per day, morning and
evening.
Instrumental Description Profilometry (Wrinkles and Roughness)
[0334] At each visit, a single silicone replica was made of the
target area and a photographic record was kept of this target for
subsequent relocation. The samples were stored in controlled
conditions for comparative measurement. Comparative analysis of
skin profilometry was conducted, using surface roughness and
wrinkle depth analysis. Concurrent use of other moisturiser or skin
care products did not occur in the skin area under study. The
height of the replicated wrinkles were measured using Miyomoto
Surftest profilometer. Ry (depth) and Ra (mean roughness) were
recorded at each time of measuring operation. The area scanned from
each sample was clearly mapped so as to determine the same area in
respective two month sampling.
Instrumental Description L*a*b* Colour Measurement
[0335] A Minolta Chromometer hand held spectrophotometer was
utilized. This tri-stimulus instrument was utilised to determine
colour values and changes vis background colour in an unpigmented
adjoining area. Specular Component Included (SCI) values were
documented.
Digital Photography
[0336] At each time point, a series of high resolution digital
photographs were collected. The subject was presented with a clean
face, with hair pulled off the face, with no jewelry (unless
permanent) and with a black drape used to standardize clothing.
Subject positioning was reproduced upon return visit. A light booth
was used so as to provide controlled reproducible light conditions.
The booth consists of an array of 8 equally spaced fluorescent
tubes in a semicircular configuration. The software driven system
allows the position and expression of the test subjects to be
aligned to a high degree. Lux values were calibrated and
documented.
Results
[0337] Table 16 exhibits the results of the skin roughness test. On
average, skin roughness was reduced by 17% at 8 weeks and 20% at 12
weeks of the use of the treatment preparation.
TABLE-US-00016 TABLE 16 Results of the skin roughness test Start
Date: End Date: All measurements in um Skin Rougness Ra T = 0 T = 1
T = 2 week week Week 0 8 12 Subject ID Age Race Ra Ra Ra M068 58 A
8.7 4.51 3.08 M570 44 C 7.51 8.62 6.43 M638 57 C 2.59 2.69 2 M549
58 C 11.94 9.55 9.15 M041 59 A 6.53 4.43 6 M664 52 C 6.12 7.89 5.5
M661 49 C 3.67 3.27 3.85 M632 51 A 5.57 3.75 5.68 M612 60 A 3.68
4.41 4.48 M618 56 A 6.14 4.05 3.71 M394 54 C 8.67 5.87 6.97 n = 11
Average 6.465 5.367 5.168 Difference 17% 20.1% (% improvement)
[0338] Table 17 exhibits the results of the wrinkle depth test. On
average, wrinkle depth was reduced by 9% at 8 weeks and 20% at 12
weeks of the use of the treatment preparation.
TABLE-US-00017 TABLE 17 Results of the wrinkle depth test Wrinkle
Depth Ry T = 0 T = 1 T = 2 Subject week 0 week 8 week 12 ID Age
Race Ry Ry Ry M068 58 A 46 35.5 23.6 M570 44 C 35 37 29.2 M638 57 C
35 32.7 27.8 M549 58 C 56.5 41 35.6 M041 59 A 37.9 35.9 36.5 M664
52 C 47.9 47.1 35.6 M661 49 C 30 37.5 32.1 M632 51 A 27.5 22.2 23
M612 60 A 45 46.5 43.2 M618 56 A 39.4 31.9 34.4 M394 54 C 37.5 31.4
28.7 n = 11 Average 39.791 36.245 31.791 Difference (% 8.9% 20.1%
improvement)
[0339] FIG. 12 exhibits a 58 year old subject before (FIG. 12A) and
after the 12 week trial (FIG. 12B). After the 12 week trial the
subject measured a 49% wrinkle reduction and a 65% reduction in
skin roughness.
[0340] FIG. 13 exhibits another 58 year old subject before (FIG.
13A) and after the 12 week trial (FIG. 13B). After the 12 week
trial the subject measured a 37% wrinkle reduction.
[0341] Table 18 exhibits the results of the colour reduction and
pigmented spot test. On average, the pigmented area was reduced in
colour by 2% at 8 weeks of the use of the treatment
preparation.
TABLE-US-00018 TABLE 18 Colour reduction and pigmented spot test L*
Value SCI Pigmented Spot Subject ID Age T = 0 T = 1 T = 2 M041 60
56.41 57.28 57.30 M394 54 56.94 58.41 58.46 M632 50 56.67 57.03
57.26 M661 49 57.53 58.63 59.16 M664 52 57.01 57.65 57.35 M570 44
56.46 60.70 58.98 M612 60 56.73 58.24 58.41 M549 58 55.82 56.39
56.79 M638 57 55.09 56.23 56.39 M618 56 53.14 53.30 53.38 M068 58
54.69 56.13 55.22 56.0 57.3 57.2 Average % vis Initial 102.2%
102.0% L*a*b* L* Whitening Black = 0 Scale White = 100
[0342] Table 19 exhibits the results of the colour
reduction--surrounding area test. A Minolta Colour Computer was
used. On average, the surrounding area was reduced in colour by
less than 1% at 8 weeks of the use of the treatment
preparation.
TABLE-US-00019 TABLE 19 Colour reduction - surrounding area test L*
Value SCI Surrounding area Subject ID Age T = 0 T = 1 T = 2 T = 3
M041 60 61.25 62.54 61.73 0.00 M394 54 62.37 63.47 62.52 0.00 M632
50 64.48 65.28 65.77 0.00 M661 49 61.22 61.42 60.65 0.00 M664 52
63.58 63.87 63.85 0.00 M570 44 61.35 63.00 62.49 0.00 M612 60 60.75
61.79 62.47 0.00 M549 58 60.87 61.21 61.58 0.00 M638 57 61.76 62.12
61.51 0.00 M618 56 62.07 61.62 61.76 0.00 M068 58 63.08 62.41 62.24
0.00 62.1 62.6 62.4 0.0 Average % vis Initial 100.9% 100.6% 0.0%
L*a*b* L* Whitening Black = 0 Scale White = 100
Adverse Events
[0343] No adverse effects were observed.
Example 8. Psoriasis Study 1
[0344] A composition comprising an extract derived from sugar cane
of the present disclosure was tested on a human subject to evaluate
the use in preventing, improving or treating psoriasis.
[0345] Application of the composition was topically to the affected
skin area twice a day. The biophysical effects of the topically
applied treatment composition was evaluated pre-application, during
the application process and post-application.
[0346] A photographic record shown in FIG. 14 was taken of the test
participant. (A) exhibits the subject's knee before the 6 week
trial, (B) exhibits the subject's knee after 2 weeks of continuous
use of a composition comprising an extract derived from sugar cane
of the present disclosure, (C) exhibits the subject's knee after 4
weeks of continuous use of a composition comprising an extract
derived from sugar cane of the present disclosure, (D) exhibits the
subject's knee after 6 of weeks of continuous use of a composition
comprising an extract derived from sugar cane of the present
disclosure.
[0347] The subject had suffered from psoriasis for 35 years and had
tried many different types of diets, creams, tablets and natural
remedies. Some of these were at great expense with minimal short
term effects and no relief from the itching and unsightly plaques
caused by the psoriasis. The subject reported that the composition
reduced the size of the affected areas and relieved the itching and
that the affected areas begun to return to normal skin colour and
tone after using the composition. The subject also reported that
the composition was gentle on the skin, "user" friendly and
moreover did not leave any oily residue. The subject had tried
other treatments which only improved the affected areas for short
periods and did not have any long term effects, causing frustration
and disappointment. Furthermore, some of the ingredients of the
other treatments caused damage to the subject's clothing.
Example 9. Psoriasis Study 2
[0348] A composition comprising an extract derived from sugar cane
of the present disclosure was tested on a human subject to evaluate
the use in preventing, improving or treating psoriasis.
[0349] Application of the composition was topically to the affected
skin area twice a day. The biophysical effects of the topically
applied treatment composition was evaluated pre-application, during
the application process and post-application.
[0350] FIG. 15 is a photographic record taken of the test
participant. (A) exhibits the subject's knee before the 3 month
trial, (B) exhibits the subject's knee after 1 month of continuous
use of a composition comprising an extract derived from sugar cane
of the present disclosure, (C) exhibits the subject's knee after 3
months of continuous use of a composition comprising an extract
derived from sugar cane of the present disclosure.
Example 10. Elastase Inhibitory Assay
[0351] As described above, wrinkle formation in the skin is
accompanied by a decrease in skin elasticity and the curling of
elastic fibres such as elastin in the dermis. Elastase inhibitors
suppress elastase activity and prevent the damage of dermal
elastin, thus helping mitigate wrinkle formation.
[0352] An elastase inhibition assay was performed to determine the
elastase inhibition potential of an extract derived from sugar cane
of the present disclosure. Two batches (replicate 1 and replicate
2) of an extract derived from sugar cane of the present disclosure
in a concentration of from 0.12 mg/mL to 250 mg/mL were tested for
the ability to inhibit elastase.
[0353] To measure elastase inhibitory potential of an extract
derived from sugar cane of the present disclosure, a test sample of
an extract derived from sugar cane was mixed and incubated with
N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone in tris-HCl
buffer for 15 min. Then elastase was added to the mixture and
incubated for 15 min. The inhibition rate was measured at 410 nm
and calculated as follows:
Elastase inhibition ( % ) = 1 - ( Abs sample + elastase - Abs blank
- elastase Abs blank + elastase - Abs sample - elastase ) .times.
100 ##EQU00001##
[0354] Wherein, [0355] Abs.sub.sample+elastase=absorbance of test
sample after incubation with elastase, [0356]
Abs.sub.sample-elastase=absorbance of test sample before incubation
with elastase, [0357] Abs.sub.blank+elastase=absorbance of blank
after incubation with elastase, and [0358]
Abs.sub.blank-elastase=absorbance of blank before incubation with
elastase.
[0359] N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone, a
human leukocyte elastase (HLE) inhibitor, was used as an assay
control. Two batches (replicate 1 and replicate 2) of
N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone in a
concentration of from 0.12 .mu.M to 250 .mu.M were used in the
control assay.
[0360] Table 20(a) exhibits the relationship between concentrations
of the extract derived from sugar cane of the present disclosure
and elastase inhibition. Table 20(b) exhibits the relationship
between the concentrations of
N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone and elastase
inhibition.
TABLE-US-00020 TABLE 20 (a) Elastase inhibition assay results
Sample Inhibition (%) Conc. (mg/ml) Replicate 1 Replicate 2 Average
CV(%) 250.00 94.65% 94.68% 94.67% 0.02 125.00 77.55% 77.89% 77.72%
0.31 62.50 68.33% 66.50% 67.41% 1.92 31.25 61.05% 63.56% 62.31%
2.84 15.63 48.05% 55.52% 51.78% 10.19 7.81 39.72% 44.51% 42.12%
8.04 3.91 25.08% 35.29% 30.18% 23.91 1.95 12.91% 21.21% 17.06%
34.39 0.98 5.62% 13.00% 9.31% 56.11 0.49 2.06% 4.42% 3.24% 51.50
0.24 -1.76% 3.62% 0.93% 410.18 0.12 -1.48% -2.62% -2.05% -39.26
TABLE-US-00021 TABLE 20 (b) Elastase inhibition assay results
N-methoxysuccinyl- Ala-Ala-Pro-Val- chloromethyl ketone Inhibition
(%) Conc. (uM) Relicate 1 Replicate 2 Average CV(%) 250.00 87.63%
87.79% 87.71% 0.13 125.00 87.08% 87.26% 87.17% 0.14 62.50 86.04%
86.22% 86.13% 0.15 31.25 84.26% 84.46% 84.36% 0.17 15.63 81.14%
80.78% 80.96% 0.31 7.81 76.17% 76.35% 76.26% 0.16 3.91 68.45%
69.03% 68.74% 0.60 1.95 58.83% 59.60% 59.21% 0.92 0.98 45.26%
46.08% 45.67% 1.27 0.49 29.92% 30.65% 30.29% 1.70 0.24 18.31%
18.17% 18.24% 0.55 0.12 10.14% 10.54% 10.34% 2.71
[0361] Table 21 exhibits maximum degree of inhibition and
concentration used in the assay and EC.sub.50 (effective
concentration at 50% of maximal inhibition) for an extract derived
from sugar cane of the present disclosure in comparison to the
assay control, N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl
ketone.
TABLE-US-00022 TABLE 21 Elastase inhibition assay results Maximum
Concentration Elastase inhibition inducing the Inhibition Assay
achieved maximum inhibition EC.sub.50 An extract derived 94.67%
250.00 mg/mL 14.38 mg/mL from sugar cane N-methoxysuccinyl- 87.71%
250 .mu.M 0.83 .mu.M Ala-Ala-Pro-Val- (0.42 .mu.g/mL) chloromethyl
ketone
[0362] FIG. 16(A) exhibits the elastase inhibition of an extract
derived from sugar cane of the present disclosure. The EC.sub.50
observed was 14.38 mg/mL.
[0363] FIG. 16(B) exhibits the elastase inhibition of the control
compound N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethyl ketone. The
EC.sub.50 observed was 0.83 .mu.M.
[0364] An extract derived from sugar cane of the present disclosure
demonstrated elastase inhibition activity. At a concentration of
250 mg/mL, an extract derived from sugar cane of the present
disclosure has achieved maximum elastase inhibition of 94.67%. The
EC.sub.50 of the extract was 14.38 mg/mL.
Example 11. Collagenase Inhibition Assay
[0365] As described above, the collagen content of skin is the net
balance between collagen synthesis and collagen breakdown. It is
known that with age collagen synthesis in the skin is reduced.
Additionally, environmental stress such as smoking, UV exposure,
pollution and inflammation stimulate the production of
collagen-degrading enzymes that causes collagen breakdown.
Collagenase and MMP-1, two of the key collagen-degrading enzymes,
are considered to be central to the causes of skin aging. Direct
inhibition of these enzymes can be an effective approach to
mitigate collagen breakdown in the skin thereby improving skin
health, general condition and tone and reduction of wrinkles.
[0366] A collagenase inhibition assay was performed to determine
the collagenase inhibition of an extract derived from sugar cane of
the present disclosure. Two batches (replicate 1 and replicate 2)
of an extract derived from sugar cane of the present disclosure in
a concentration of from 0.17 mg/mL to 350 mg/mL were tested for the
ability to inhibit collagenase.
[0367] To measure collagenase inhibition potential of an extract
derived from sugar cane of the present disclosure, a test sample of
an extract derived from sugar cane was mixed sequentially with
type-I collagen substrate and a diluted collagenase enzyme
solution. After incubation for 30 minutes at 37.degree. C.,
fluorescence was measured at excitation maxima of 495 nm and
emission maxima of 515 nm. The inhibition percentage was calculated
as follows.
Collagenase inhibition activity ( % ) = ( 1 - Abs sample - Abs
blank Abs positve control - Abs negative control ) .times. 100
##EQU00002##
[0368] Wherein, [0369] Abs.sub.sample=absorbance of the sample
test, [0370] Abs.sub.blank=absorbance of the sample blank, [0371]
Abs.sub.positive control=absorbance of the positive control, and
[0372] Abs.sub.negative control=absorbance of the negative
control.
[0373] 1,10-phenanthroline was used as a positive assay control.
Two batches (replicate 1 and replicate 2) of 1,10-phenanthroline in
a concentration of from 1.7 .mu.M to 3500 .mu.M were used in the
positive control assay. A negative control was performed with a
buffer and a collagen substrate but without collagenase enzyme.
[0374] Table 22(a) exhibits the relationship between concentrations
of the extract derived from sugar cane of the present disclosure
and collagenase inhibition. Table 22(b) exhibits the relationship
between concentrations of 1,10-phenanthroline and collagenase
inhibition.
TABLE-US-00023 TABLE 22(a) Collagenase inhibition assay results
Sample Inhibition (%) Conc. (mg/ml) Replicate 1 Replicate 2 Average
CV(%) 350.00 89.73% 90.23% 89.98% 0.39 175.00 77.75% 81.61% 79.68%
3.42 87.50 65.46% 62.90% 64.18% 2.82 43.75 46.80% 53.88% 50.34%
9.95 21.88 38.86% 42.29% 40.58% 5.97 10.94 24.99% 31.28% 28.13%
15.80 5.47 9.08% 14.16% 11.62% 30.89 2.73 -5.54% -2.96% -4.25%
-42.96 1.37 -19.31% -14.76% -17.03% -18.89 0.68 -28.14% -22.59%
-25.37% -15.48 0.34 -33.41% -29.42% -31.41% -9.00 0.17 -39.84%
-32.26% -36.05% -14.88
TABLE-US-00024 TABLE 22(b) Collagenase inhibition assay results
1,10-phenanthroline Inhibition (%) Conc. (uM) Relicate 1 Replicate
2 Average CV(%) 3500.00 79.75% 78.24% 78.99% 1.35 1750.00 76.83%
77.71% 77.27% 0.81 875.00 77.01% 77.72% 77.37% 0.64 437.50 76.63%
77.39% 77.01% 0.70 218.75 75.14% 75.78% 75.46% 0.61 109.38 71.77%
71.54% 71.66% 0.23 54.69 57.26% 59.74% 58.50% 3.00 27.34 25.64%
24.25% 24.95% 3.95 13.67 -7.40% -6.00% -6.70% -14.72 6.84 -22.85%
-20.46% -21.65% -7.83 3.42 -27.08% -24.45% -25.76% -7.24 1.71
-29.63% -28.47% -29.05% -2.82
[0375] Table 23 exhibits maximum degree of inhibition and
concentration used in the assay and EC.sub.50 (effective
concentration at 50% of maximal inhibition) for an extract derived
from sugar cane of the present disclosure in comparison to the
assay control, 1,10-phenanthroline.
TABLE-US-00025 TABLE 23 Collagenase inhibition assay results
Maximum Concentration Collagenase inhibition inducing the
Inhibition Assay achieved maximum inhibition EC.sub.50 An extract
derived 89.98% 350.00 mg/mL 29.65 mg/mL from sugar cane
1,10-phenanthroline 78.99% 3.50 mM 26.85 .mu.M (4.84 .mu.g/mL)
[0376] FIG. 17(A) exhibits the collagenase inhibition of an extract
derived from sugar cane of the present disclosure. The EC.sub.50
observed was 29.65 mg/mL.
[0377] FIG. 17(B) exhibits the collagenase inhibition of the
control compound 1,10-phenanthroline. The EC.sub.50 observed was
26.85 .mu.M.
[0378] An extract derived from sugar cane of the present disclosure
demonstrated collagenase inhibition activity. At a concentration of
350 mg/mL, an extract derived from sugar cane of the present
disclosure achieved maximum elastase inhibition of 89.98%. The
EC.sub.50 of the extract was 29.65 mg/mL.
Example 12. Tyrosinase Inhibition Assay
[0379] As described above, colour of mammalian skin is determined
by many factors, one of which is the production and distribution of
melanin pigmentation. In melanin biosynthesis process, tyrosinase
is the key enzyme that catalyses the first step of melanogenesis.
Substantial studies have shown that melanin reduction and
skin-whitening can be achieved, at least partially, by deactivating
of tyrosinase. Therefore, tyrosinase inhibitors have become
increasingly important in cosmetic and medicinal products used in
the prevention of hyperpigmentation.
[0380] A tyrosinase inhibition assay was performed to determine the
tyrosinase inhibition potential of an extract derived from sugar
cane of the present disclosure. Two batches (replicate 1 and
replicate 2) of an extract derived from sugar cane of the present
disclosure in a concentration of from 0.31 mg/mL to 10 mg/mL were
tested for the ability to inhibit tyrosinase.
[0381] To measure tyrosinase inhibition potential of an extract
derived from sugar cane of the present disclosure, tyrosinase was
added to a mixture of a test sample solution containing an extract
derived from sugar cane, L-DOPA, and phosphate buffer (pH 6.8),
which was then incubated at 37.degree. C. for 5 hours. Tyrosinase
activity was determined through the amount of dopachrome production
in the mixture, which was measured by optical density at 450 nm.
Dopachrome is a cyclization product of L-DOPA, an intermediate in
the biosynthesis of melanin.
[0382] Kojic acid, a known tyrosinase inhibitor, was used as a
positive assay control. One sample of kojic acid in a concentration
from 3.91 .mu.M to 1000 .mu.M was used in the positive control
assay.
[0383] Table 24(a) exhibits the relationship between concentrations
of the extract derived from sugar cane of the present disclosure
and tyrosinase inhibition. Table 24(b) exhibits the relationship
between the concentrations of kojic acid and tyrosinase
inhibition.
TABLE-US-00026 TABLE 24(a) Tyrosinase inhibition assay results
Sample Inhibition (%) Conc. (mg/ml) Replicate 1 Replicate 2 Average
10.00 87.65% 88.72% 88.19% 5.00 61.18% 65.10% 63.14% 2.50 23.59%
29.13% 26.36% 1.25 -14.95% -11.11% -13.03% 0.63 -23.18% -18.88%
-21.03% 0.31 -17.83% -32.45% -25.14%
TABLE-US-00027 TABLE 24(b) Tyrosinase inhibition assay results
Kojic Acid Inhibition (%) Conc. (uM) Result 1000.00 90.12% 500.00
75.45% 250.00 52.40% 125.00 30.45% 62.50 12.35% 31.25 1.23% 15.63
-4.66% 7.81 -9.33% 3.91 -11.66%
[0384] Table 25 exhibits maximum degree of inhibition and
concentration used in the assay and EC.sub.50 (effective
concentration at 50% of maximal inhibition) for an extract derived
from sugar cane of the present disclosure in comparison to the
assay control, kojic acid.
TABLE-US-00028 TABLE 25 Tyrosinase inhibition assay results Maximum
Concentration Tyrosinase inhibition inducing the inhibition Assay
achieved maximum inhibition EC.sub.50 An extract derived 88.19%
10.00 mg/mL 3.62 mg/mL from sugar cane kojic acid 90.12% 1000 .mu.M
226.50 .mu.M (32.2 .mu.g/mL)
[0385] FIG. 18(A) exhibits the tyrosinase inhibition of an extract
derived from sugar cane of the present disclosure. The EC.sub.50
observed was 3.62 mg/mL.
[0386] FIG. 18(B) exhibits the tyrosinase inhibition of the control
kojic acid. The EC.sub.50 observed was 226.50 .mu.M.
[0387] An extract derived from sugar cane of the present disclosure
demonstrated tyrosinase inhibition activity. At a concentration of
10 mg/mL, an extract derived from sugar cane of the present
disclosure has achieved maximum elastase inhibition of 88.19%. The
EC.sub.50 of the extract was 3.62 mg/mL. The EC.sub.50 observed was
226.50 .mu.M.
Example 13. Cellular Melanin Pigmentation Inhibition Assay
[0388] As described above, melanin is essential in protecting human
skin against UV radiation, but over production of melanin is also a
major consequence of UV damage and aging process that induces
pigmentation disorders such as freckles and senile lentigo (i.e.,
age spots). Excessive melanin has also been viewed as a melanoma
precursor. Melanin inhibition is a desirable effect sought in
various fronts of cosmetic industry to achieve skin whitening,
lessen aging appearance, and preventing melanoma.
[0389] A melanin production inhibition assay was performed to
determine the potential of an extract derived from sugar cane of
the present disclosure to inhibit melanin production. Two batches
(replicate 1 and replicate 2) of an extract derived from sugar cane
of the present disclosure in a concentration of from 1.56 mg/mL to
50 mg/mL were tested for the ability to inhibit melanin production
in human skin cells.
[0390] To measure melanin pigmentation inhibition potential of an
extract derived from sugar cane of the present disclosure, a test
sample of an extract derived from sugar cane, in combination with
theophylline, was added to a pre-incubated human skin cell culture.
After incubating for 72 hours, the cells were washed with
phosphate-buffered saline (PBS), lysed in 1 M NaOH, and boiled for
5 minutes to solubilize the melanin. The inhibition rates of the
lysates were measured at 490 nm and expressed as a percentage
relative to the value of the untreated control.
[0391] Chloroquine, a known melanin production inhibitor, was used
as an assay control. Two batches (replicate 1 and replicate 2) of
chloroquine in a concentration from 0.31 .mu.M to 10 .mu.M were
used in the control assay.
[0392] Table 26(a) exhibits the relationship between concentrations
of the samples of an extract derived from sugar cane of the present
disclosure and melanin production inhibition.
[0393] Table 26(b) exhibits the relationship between concentrations
of chloroquine and their melanin production inhibition.
TABLE-US-00029 TABLE 26(a) Melanin production inhibition assay
results Sample Melanin inhibition (%) Conc (mg/ml) Replicate 1
Replicate 2 Average CV 50.00 78.57% 80.28% 79.43% 2% 25.00 51.91%
56.31% 54.11% 6% 12.50 -0.43% -2.63% -1.53% -102% 6.25 1.04% -5.07%
-2.02% -214% 3.13 -1.16% 0.55% -0.31% -396% 1.56 -1.90% -0.67%
-1.28% -67%
TABLE-US-00030 TABLE 26(b) Melanin production inhibition assay
results Chloroquine Melanin inhibition (%) Conc (.mu.M) Replicate 1
Replicate 2 Average CV 10.00 85.66% 90.55% 88.11% 4% 5.00 79.79%
86.40% 83.09% 6% 2.50 76.61% 75.63% 76.12% 1% 1.25 36.50% 32.10%
34.30% 9% 0.63 15.47% 16.69% 16.08% 5% 0.31 1.77% 0.06% 0.92%
132%
[0394] Table 27 exhibits maximum degree of inhibition and
concentration used in the assay and EC.sub.50 (effective
concentration at 50% of maximal inhibition) for an extract derived
from sugar cane of the present disclosure in comparison to the
assay control, chloroquine.
TABLE-US-00031 TABLE 27 Melanin production inhibition assay results
Maximum Concentration Melanin production inhibition inducing the
inhibition Assay achieved maximum inhibition EC.sub.50 An extract
derived 79.43% 50.00 mg/mL 23.98 mg/mL from sugar cane Chloroquine
88.11% 10 .mu.M 1.43 .mu.M (0.74 .mu.g/mL)
[0395] FIG. 19(A) exhibits the melanin production inhibition of an
extract derived from sugar cane of the present disclosure. The
EC.sub.50 of the extract was 23.98 mg/mL.
[0396] FIG. 19(B) exhibits the melanin production inhibition of the
control chloroquine. The EC.sub.50 of the extract was 1.43
.mu.M.
[0397] An extract derived from sugar cane of the present disclosure
demonstrated melanin production inhibition activity. At a
concentration of 50 mg/mL, an extract derived from sugar cane of
the present disclosure has achieved maximum melanin production
inhibition of 79.43%. The EC.sub.50 of the extract was 23.98
mg/mL.
Example 14. Melanoma Anti-Proliferative Assay
[0398] The anti-proliferative effects of an extract derived from
sugar cane of the present disclosure were assessed in the mouse
melanoma cancer cell line, B 16.
[0399] The rapid colourimetric assay for cellular growth and
survival, Thiazolyl Blue Tetrazolium Bromide (MTT) assay was used
to asses cell proliferation. The MTT assay is a robust and reliable
method which acquires the signal of mitochondrial performances and
hence assesses cellular viability.
[0400] NAD(P)H-dependent cellular oxidoreductase enzymes of the
mitochondria are capable of reducing the tetrazolium dye MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its
insoluble formazan, which has a purple colour. The reduction
reaction takes place only when mitochondrial enzymes are active and
therefore conversion can be directly related to the number of
viable cells. The absorbance of the purple solution can be
quantified by measuring a wavelength by a spectrophotometer.
[0401] Mouse melanoma cancer cells were seeded in 96 well plates
and an extract derived from sugar cane of the present disclosure
added at varying concentrations (0-500 .mu.g/ml) over 6 days, with
media change containing an extract derived from sugar cane of the
present disclosure on day 3. Cultures were grown in a humidified
incubator at 5% CO.sub.2 and 37.degree. C. Cellular proliferation
was assessed via spectrophotometry (Biorad microplate reader, 6.0)
using wavelength 570 nm on days 3-6. The Experiment was conducted
in triplicate.
[0402] Dose dependent anti-proliferative effects of an extract
derived from sugar cane of the present disclosure were observed in
the mouse melanoma cancer cell line B16. A partial effect was
observed at 200 .mu.g/ml (p<0.05) with complete
antiproliferative effect being observed at 400 .mu.g/ml
(p<0.05).
[0403] FIG. 20(A) exhibits the anti-proliferative effects of an
extract derived from sugar cane of the present disclosure on mouse
melanoma cells, B16.
[0404] To ensure that the extract derived from sugar cane of the
present disclosure was inhibiting proliferation in the melanoma
cell line and not causing toxicity to cells, melanoma cells were
cultured in the presence (400 .mu.g/ml) or absence of an extract
derived from sugar cane of the present disclosure for 24-72 hours
and imaged.
[0405] FIG. 20(B) exhibits 24 hour cell survival of mouse melanoma
cells in the presence of an extract derived from sugar cane of the
present disclosure.
[0406] FIG. 20(C) exhibits 48 hour cell survival of mouse melanoma
cells in the presence of an extract derived from sugar cane of the
present disclosure.
[0407] FIG. 20(D) exhibits 72 hour cell survival of mouse melanoma
cells in the presence of an extract derived from sugar cane of the
present disclosure.
Example 15. Cellular Telomerase Activity Assay
[0408] As described above, telomerase is an enzyme which builds
telomeres, the regions of repetitive sequences at each end of
chromosomes in most eukaryotes. Telomerase can slow, stop or
perhaps even reverse the telomere shortening that happens with age,
however, the amount and activity of telomerase also declines with
age. Activation of telomerase can reverse skin cell aging and
revert the skin to a more youthful physical and genetic state.
[0409] A cellular telomerase activity assay determines the ability
of a test material to stimulate telomerase activity in human cells,
which translates to the material's anti-aging potential.
[0410] A cellular telomerase activity assay was performed to
determine telomerase activity potential of an extract derived from
sugar cane of the present disclosure. Two batches (replicate 1 and
replicate 2) of an extract derived from sugar cane of the present
disclosure in a concentration of from 0.16 mg/mL to 5 mg/mL were
tested for the telomerase activity potential.
[0411] In the cellular telomerase activity assay, telomerase
activity level of human cells treated with (and without) a test
sample of an extract derived from sugar cane of the present
disclosure was monitored. Specifically, a telomeric repeat
amplification protocol (TRAP assay) was used for the assay. The
TRAP assay was divided into three main steps: extension,
amplification, and detection of telomerase products. In the
extension phase, telomeric sequences were added to the telomerase
substrate, a non-telomeric oligonucleotide, through the action of
telomerase present in the cell extract. The products of the
extension step were amplified by PCR using specific primers and
finally were analyzed via electrophoresis.
[0412] Cycloastragenol, a known telomerase activator, was used as
an assay control. Two batches (replicate 1 and replicate 2) of
cycloastragenol in a concentration from 0.06 .mu.M to 3 .mu.M were
used in the control assay.
[0413] Table 28(a) exhibits the relationship between concentrations
of an extract derived from sugar cane of the present disclosure and
telomerase activation. Table 28(b) exhibits the relationship
between the concentrations of ycloastragenol and telomerase
activation.
TABLE-US-00032 TABLE 28(a) Cellular telomerase activity assay
results Sample Telomerase activity Conc (mg/ml) Replicate 1
Replicate 2 Average CV 5.00 5097.83 5891.83 5494.83 10% 2.50
2885.83 3722.83 3304.33 18% 1.25 881.83 2576.83 1729.33 69% 0.63
107.83 615.83 361.83 99% 0.31 662.83 384.83 523.83 38% 0.16 -93.17
480.83 193.83 209%
TABLE-US-00033 TABLE 28(b) Cellular telomerase activity assay
results Cycloastragenol Telomerase activity Conc (.mu.M) Replicate
1 Replicate 2 Average CV 3.00 20995.83 22822.83 21909.33 6% 1.00
15590.83 15521.83 15556.33 0% 0.50 7834.83 7167.83 7501.33 6% 0.25
5415.83 5631.83 5523.83 3% 0.13 2080.83 1752.83 1916.83 12% 0.06
925.83 1848.83 1387.33 47%
[0414] Table 29 exhibits a concentration of half-maximal telomerase
activation of an extract derived from sugar cane of the present
disclosure in comparison to the assay control, cycloastragenol.
TABLE-US-00034 TABLE 29 Cellular telomerase activity assay results
Cellular Telomerase Activity Assay EC.sub.50 An extract derived
from sugar cane 2.94 mg/mL Cycloastragenol 0.79 .mu.M (0.39
.mu.g/mL)
[0415] FIG. 21(A) exhibits the telomerase activation of an extract
derived from sugar cane of the present disclosure. The EC.sub.50
observed was 2.94 mg/mL.
[0416] FIG. 21(B) exhibits the telomerase activation of the control
cycloastragenol. The EC.sub.50 observed was 0.79 .mu.M.
[0417] An extract derived from sugar cane of the present disclosure
demonstrated telomerase enhancing activities. The EC.sub.50 of the
extract was 2.94 mg/mL.
Example 16. MMP-1 Inhibition Assay
[0418] As described above, when human skin experiences
environmental stress such as UV irradiation, the skin cells undergo
a number of changes including increased level of free radicals,
sunburn inflammation (erythema), tanning, etc. At the molecular
level, environmental stress causes DNA damage, increases
pro-inflammatory cytokine levels (e.g. interleukins), and increases
matrix metalloproteinases (MMPs) levels that cause excessive
breakdown of connective tissue components and increased wrinkle
appearance.
[0419] The UV protection function of an extract derived from sugar
cane of the present disclosure on skin using human skin cells was
investigated. MMP-1, one of the key matrix metalloproteinases
(MMPs) that is viewed as an indicator for tissue integrity
breakdown and wrinkle formation, was used as a biomarker for skin
damage. MMP-1 inhibitors have been related to skin aging prevention
and therapy.
[0420] An MMP-1 inhibition assay was performed to determine the
MMP-1 inhibition potential of an extract derived from sugar cane of
the present disclosure. Two batches (replicate 1 and replicate 2)
of an extract derived from sugar cane of the present disclosure in
a concentration of from 0.31 mg/mL to 5 mg/mL were tested for the
ability to inhibit MMP-1 in human skin cells.
[0421] In order to determine the UV protection ability of a test
sample of an extract derived from sugar cane of the present
disclosure, human skin cells were treated with (and without) the
test sample followed by UV irradiation. The treated cells were then
disrupted by subjection to several freeze-thaw cycles, and the
proteins were obtained by centrifugation. Using the collected
lysate, zymography was used to determine the enzymatic activity of
secreted MMP-1.
[0422] In the zymography, the proteins were separated by
electrophoresis under denaturing (sodium dodecyl sulfate, SDS),
nonreducing conditions. The separation occurred in a polyacrylamide
gel containing a collagen substrate that was co-polymerized with
the acrylamide. During electrophoresis, the SDS caused the MMPs to
denature and become inactive. After electrophoresis, the gel was
washed and subsequently incubated in an appropriate activation
buffer. During this incubation, the concentrated, renatured MMPs in
the gel digested the collagen substrate. After incubation, the gel
was stained with Coomassie.RTM. Blue, and the MMPs were detected as
clear bands against a blue background of undegraded substrate. The
clear bands in the gel was quantified by densitometry.
[0423] Avobenzone, a known MMP-1 inhibitor, was used as an assay
control. Two batches (replicate 1 and replicate 2) of avobenzone in
a concentration from 0.31 .mu.M to 10 .mu.M were used in the
control assay.
[0424] Table 30(a) exhibits the relationship between concentrations
of an extract derived from sugar cane of the present disclosure and
MMP-1 inhibition. Table 30(b) exhibits the relationship between the
concentrations of avobenzone and MMP-1 inhibition.
TABLE-US-00035 TABLE 30(a) MMP-1 inhibition assay results Sample
MMP-1 Inhibition (%) Conc (mg/mL) Replicate 1 Replicate 2 Average
CV 5.00 101.62% 104.43% 103.0% 1.9% 2.50 95.83% 96.74% 96.3% 0.7%
1.25 70.66% 67.03% 68.8% 3.7% 0.63 23.00% 24.84% 23.9% 5.5% 0.31
10.96% 11.0%
TABLE-US-00036 TABLE 30(b) MMP-1 inhibition assay results
Avobenzone MMP-1 inhibition (%) Conc (%) Replicate 1 Replicate 2
Average CV 10.00 95.44% 99.52% 97.5% 3.0% 5.00 85.34% 96.03% 90.7%
8.3% 2.50 77.44% 82.22% 79.8% 4.2% 1.25 56.96% 39.99% 48.5% 24.8%
0.63 22.51% 65.72% 44.1% 69.3% 0.31 5.0% 5.0%
[0425] Table 31 exhibits maximum degree of inhibition and
concentration used in the assay and EC.sub.50 (effective
concentration at 50% of maximal inhibition) for an extract derived
from sugar cane of the present disclosure in comparison to the
assay control, avobenzone.
TABLE-US-00037 TABLE 31 MMP-1 inhibition assay results Maximum
Concentration MMP-1 inhibition inhibition inducing the Assay
achieved maximum inhibition EC.sub.50 An extract derived .sup. 103%
5 mg/mL 1.05 mg/mL from sugar cane Avobenzone 97.50% 10% 1.22%
[0426] FIG. 22(A) exhibits the MMP-1 inhibition of an extract
derived from sugar cane of the present disclosure. The EC.sub.50
observed was 1.05 mg/mL.
[0427] FIG. 22(B) exhibits the MMP-1 inhibition of the control
compound avobenzone.
[0428] An extract derived from sugar cane of the present disclosure
demonstrated MMP-1 inhibition activity. At a concentration of 5
mg/mL, an extract derived from sugar cane of the present disclosure
has achieved maximum MMP-1 inhibition of 103%. The EC.sub.50 of the
extract was 1.05 mg/mL.
Example 17. Oxygen Radical Absorbance Capacity (ORAC)
[0429] Oxygen Radical Absorbance Capacity (ORAC) tests are among
the most acknowledged methods that measure antioxidant scavenging
activity against oxygen radicals that are known to be involved in
the pathogenesis of aging and many common diseases. ORAC 6.0
consists of six types of ORAC assays that evaluate the antioxidant
capacity of a material against six primary reactive oxygen species
(ROSs, commonly called "oxygen radicals") found in humans: peroxyl
radical, hydroxyl radical, superoxide anion, singlet oxygen,
peroxynitrite, and hypochlorite. This is a comprehensive panel that
evaluates the antioxidant capacity of a material against oxygen
radicals.
[0430] The ORAC 6.0 tests are based on evaluating the capacity of
an interested material to protect a probe (a fluorescent probe or
chromagen) from its damage by ROSs. In all ORAC assays, an ROS
inducer is introduced to the assay system. The ROS inducer triggers
the release of a specific ROS, which would degrade the probe and
cause its emission wavelength or intensity change. When an
antioxidant material presents in the environment, the antioxidant
absorbs the ROS and preserves the probe from degradation. The
degree of probe preservation indicates the antioxidant capacity of
the material.
[0431] An ORAC 6.0 test was performed on human skin cells to
determine oxygen radical absorbance capacity of an extract derived
from sugar cane of the present disclosure. An ROS inducer was
introduced to an ORAC assay system. The ROS inducer triggered
release of a specific ROS, which degraded the probe and caused its
emission wavelength or intensity change. When an antioxidant
material presents in a test sample, the antioxidant absorbs the ROS
and preserved the probe from degradation. The degree of probe
preservation indicated the antioxidant capacity of the material.
Trolox was used as the reference standard. The test results were
expressed as mole Trolox equivalency per millilitre of a test
sample.
[0432] Table 32 exhibits the results of an ORAC 6.0 test of an
extract derived from sugar cane of the present disclosure. The
results were expressed as mole Trolox equivalency (.mu.mole TE) per
gram of a tested material.
TABLE-US-00038 TABLE 32 ORAC results for an extract derived from
sugar cane of the present disclosure Analysis Result Units ORAC
against peroxyl radicals 226.15 .mu.mole TE/milliliter ORAC against
hydroxyl radicals 1015.09 .mu.mole TE/milliliter ORAC against
peroxynitrite 11.45 .mu.mole TE/milliliter ORAC against super oxide
anion 64.88 .mu.mole TE/milliliter ORAC against singlet oxygen
163.78 .mu.mole TE/milliliter ORAC against hypochlorite 186.92
.mu.mole TE/milliliter ORAC 6.0 (sum of above) 1668.27 .mu.mole
TE/milliliter
TABLE-US-00039 TABLE 33 ORAC results for 4 extracts derived from
sugar cane of the present disclosure Results Analysis Extract A
Extract B Extract C Extract D ORAC against peroxyl 303 258 265
2,336 radicals ORAC against hydroxyl 1,902 1,179 1,220 13,785
radicals ORAC against 25 32 34 255 peroxynitrite ORAC against super
121 82 74 450 oxide anion ORAC against singlet 348 279 263 2,011
oxygen ORAC 5.0 (sum of 2,699 1,830 1,856 18,837 above) ORAC
against -- 94 107 620 hypochlorite ORAC 6.0 (sum of -- 1,924 1,963
19,457 above)
[0433] The data in Table 33 demonstrates that the extracts of the
present disclosure are efficient at scavenging 6 well-characterised
and biologically relevant oxidants. The individual ORAC values
against each oxidant and the combined total ORAC 6 value
demonstrates that the extracts of the present disclosure are
powerful antioxidants against a range of oxidant species of
biological significance.
Example 18. Cellular Antioxidant Assay (CAA)
[0434] CAA analyses the capacity of a material to protect a
fluorescent probe (as a marker) from damage by reactive oxygen
species (ROS) in intracellular environment.
[0435] A CAA was performed to determine cellular antioxidant
activity of an extract derived from sugar cane of the present
disclosure. In the CAA, peroxyl radical was used as the ROS, and
human skin cells was used as the cellular model. Quercetin, a known
antioxidant, was used as the standard.
[0436] In this CAA, pre-treated human skin cells were incubated
with a test sample of an extract derived from sugar cane of the
present disclosure and a probe, such as 2',7'-dichlorofluorescin
diacetate (DCFH-DA). After incubation for 1 hour, an exogenous
source of peroxyl radicals, such as
2,2'-azobis(2-methylpropionamididine) dihydrochloride (ABAP) was
added. In the presence of the peroxyl radical, the probe oxidised
into a fluorescent product. Fluorescence of the product was
monitored with emission at 538 nm and excitation at 485 nm at
regular time points to determine the extent of oxidation, and CAA
was calculated as follows:
CAA unit=100-(.intg.A.sub.sample/.intg.A.sub.control).times.100
Wherein,
[0437] .intg.A.sub.sample=integrated area under the fluorescence
versus time curve for the test sample, and [0438]
.intg.A.sub.blank=integrated area under the fluorescence versus
time curve for the blank.
[0439] Cells treated with samples that have antioxidant activity
had lower fluorescence compared with cells that were not treated
with antioxidants. Quercetin, a known antioxidant, was used as a
reference standard, and the results were expressed as .mu.mole
quercetin equivalency per gram of the test sample.
[0440] Table 34 exhibits the results of CAA of an extract derived
from sugar cane of the present disclosure. The results are
expressed as .mu.mole quercetin equivalency per gram of a tested
material.
TABLE-US-00040 TABLE 34 CAA results for an extract derived from
sugar cane of the present disclosure Analysis Result Units CAA
13.84 .mu.mole QE/gram
TABLE-US-00041 TABLE 35 CAA results for 5 extracts derived from
sugar cane of the present disclosure Sugar cane extract Results
(.mu.mol QE/gram) Extract I 48.16 Extract II 56.21 Extract III
61.37 Extract IV 67.35 Extract V 229.12
[0441] CAA is used to observe the antioxidant capabilities of a
substance in a living cellular context, rather than as an abstract
chemical reaction. This technique is designed to give a detailed
understanding of the mechanisms, bioavailability, uptake, and
metabolism of the antioxidant compounds in a cell culture
environment that reflects the complexity of a biological system. A
high CAA value indicates that an antioxidant compound has been able
to enter the cell which indicates bioavailability, without
negatively affecting the cell which would indicate toxicity. As a
reference, the Kakadu Plum (Terminalia ferdinandiana) has been
suggested to have the highest Vitamin C concentration of any fruit
in the world (Brand et al. 1982). Consequently, it is acknowledged
to be an extremely efficient antioxidant. Kakadu Plum has been
reported to return a CAA value of 71.5.+-.11.3 QE/gram (Tan et al.
2011). The sugar cane extracts disclosed returned CAA values
slightly lower, within or significantly higher than this range.
This demonstrates that the sugar cane extracts of the present
disclosure provide powerful antioxidant protection in both in vitro
and in vivo contexts.
Example 19. Cellular Nrf2 Activation Assay
[0442] Cellular Nrf2 activation assay determines the potential of a
test material stimulating the production of Nrf2 in human cells.
Nrf2 serves as a biomarker for anti-oxidation and anti-inflammatory
capacity. Nrf2 is a redox-sensitive transcription factor that binds
to antioxidant response elements (ARE) to regulate the expression
of antioxidant enzymes that protect against oxidative damage
triggered by injury and inflammation. Activation of the Nrf2
pathway has been found to have a wide range of beneficial effects
on skin, including reduced rates of skin cancers, protection from
ultraviolet radiation, reduced inflammation, irritation and
redness, reduction of wrinkles and improvement in skin tone,
enhanced barrier function, and improved wound healing.
[0443] A cellular Nrf2 activation assay was performed to determine
Nrf2 activation potential of an extract derived from sugar cane of
the present disclosure. Two batches (replicate 1 and replicate 2)
of an extract derived from sugar cane of the present disclosure in
a concentration of from 15.625 .mu.g/mL to 500 .mu.g/mL were tested
for the ability to activate Nrf2 in human cells.
[0444] In order to measure the effect of a test sample of an
extract derived from sugar cane of the present disclosure on Nrf2
activity, a reporter gene assay was used. Human cells were
transfected with an Nrf2/antioxidant response element (ARE)
reporter gene. An ARE reporter comprises of tandem repeats of the
ARE transcriptional response element (upstream of firefly
luciferase) and a renilla luciferase plasmid under the control of
the cytomegalovirus promoter, as an internal control.
Transcriptional activity of Nrf2 was determined by measurement of
luciferase activities in the transfected human cells as assessed
using an appropriate reporter assay kit and a plate reader.
[0445] Table 36 exhibits the results of the cellular Nrf2
activation assay of an extract derived from sugar cane of the
present disclosure.
TABLE-US-00042 TABLE 36 Cellular Nrf2 activation assay results Nrf2
pathway activity Sample Standard Conc. (.mu.g/mL) Replicate 1
Replicate 2 Average deviation 500 73715.08 53623.08 63669.03
14207.189 250 20837.08 31168.08 26002.58 7305.120 125 12246.08
6352.08 9299.08 4167.687 62.5 2279.08 3046.08 2662.58 542.351 31.25
1466.08 1247.08 1356.58 154.856 15.625 1220.08 1072.08 1146.08
104.652
[0446] An extract derived from sugar cane of the present disclosure
demonstrated Nrf2 activation activity as shown in FIG. 23. At a
concentration of 500 .mu.g/mL, an extract derived from sugar cane
of the present disclosure has achieved Nrf2 activation of 63669.
The EC.sub.50 of the extract was 631.2 .mu.g/mL. This data
demonstrates that the extract derived from sugar cane of the
present disclosure activates Nrf2 and therefore has anti-oxidation
and anti-inflammatory capacity.
Example 20. Nuclear Factor .kappa.B Study
Description
[0447] Nuclear Factor .kappa.B (NF-.kappa.B) is a protein complex
that is involved in cellular responses to stimuli such as stress
and free radicals, ultraviolet irradiation, oxidized LDL, and
bacterial or viral antigens. It plays a key role in regulating the
immune response to infection and has been associated with
inflammation and plays a major role in the skin aging process.
Further, genomic studies have linked psoriasis with mediators in
the NF-.kappa.B pathway.
[0448] Suppression of NF-.kappa.B limits the production of
pro-inflammatory gene expression and reduces the level of
inflammation. Therefore inhibition of NF-.kappa.B is used as an
indicator of anti-inflammatory activity.
Methodology
[0449] The assay of NF-.kappa.B inhibition follows a procedure
where a test material is absorbed into human cells. A
proinflammatory cytokine is then introduced to the human cells to
mimic cellular stress, which would normally induce NF-.kappa.B
activation leading to inflammation. If an NF-.kappa.B inhibiting
material is present in the cellular environment, the material
inhibits NF-.kappa.B activation and the degree of inhibition can be
monitored via NF-.kappa.B expression. NF-.kappa.B expression level
of the human cells, treated with and without the test material,
under the stressed condition are therefore monitored and compared
to assess the NF-.kappa.B inhibition effect of a material.
[0450] Human cells were first treated with or without a
representative powdered extract derived from sugar cane of the
present disclosure (extract of Example 3) to allow for natural
absorption of the extract into the cells. Maximum percentage of
NF-.kappa.B expression inhibition induced by the powdered extract
was reported as with the concentration used that induced the
maximum inhibition of NF-.kappa.B expression. The half maximal
inhibitory concentration (EC.sub.50) was calculated. Assay results
are shown in Table 37.
TABLE-US-00043 TABLE 37 Nuclear factor .kappa.B activation assay
results Sugar cane extract of Example 3 Inhibition (%) Conc.
(.mu.g/mL) Replicate 1 Replicate 2 Average 178.13 92.04 104.19
98.12 89.06 66.45 76.33 71.39 44.53 53.73 58.42 56.08 22.27 12.37
15.49 13.93 11.13 7.96 14.00 10.98 5.57 4.83 13.86 9.35
[0451] The response curve for this data is shown in FIG. 24. A
maximum inhibition of 98.12% was observed. The concentration that
induced the maximum inhibition was 178 .mu.g/mL. The calculated
half-maximal response (EC.sub.50) was calculated to be 632.1
.mu.g/mL. This data demonstrates that the extract derived from
sugar cane of the present disclosure inhibits NF-.kappa.B
indicating anti-inflammatory activity.
Example 21. TNF-.alpha. Study
Description
[0452] Tumor necrosis factor (TNF)-.alpha. is a pro-inflammatory
cytokine (small proteins that impact cell signalling) that triggers
downstream cellular feedback loops governing inflammation.
TNF-.alpha. has been identified as an inflammation trigger and
precursor and is released quickly at wound tissues, initiating
inflammation. Further, high levels of proinflammatory cytokines,
including TNF-.alpha. have been detected in psoriatic skin lesions.
Thus, TNF-.alpha. inhibitors have potential as anti-inflammatory
agents for the skin.
Methodology Human cells are first treated with or without powdered
sugar cane extract (extract of Example 3) to allow for natural
absorption of the material into the cells. Then, the cells are
stressed with an inflammation inducer, which would normally
stimulate TNF-.alpha. production then further develop into
inflammation through series of cellular signalling. If a
TNF-.alpha. inhibitor presents in the cellular environment, the
material inhibits TNF-.alpha. production and the degree of
inhibition is assessed by level of decreased TNF-.alpha.
production. TNF-.alpha. production level of the human cells,
treated with and without a test material, under the stressed
condition is monitored and compared to assess the TNF-.alpha.
inhibition effect of the test material. The maximum percentage of
TNF-.alpha. expression inhibition induced by the tested sugar cane
extracts was reported as was the concentration used that induced
the maximum inhibition of TNF-.alpha. expression. Assay results are
shown in Table 38.
TABLE-US-00044 TABLE 38 Cellular TNF.alpha. inhibition assay
results Sugar cane extract of Example 3 Inhibition (%) Conc.
(.mu.g/mL) Replicate 1 Replicate 2 Average 178.13 99.20 94.80 97.00
89.06 92.36 92.66 92.51 44.53 59.36 59.94 59.65 22.27 20.00 30.35
25.18 11.13 13.65 10.63 12.14 5.57 -3.73 5.06 0.66
[0453] The response curve for this data is shown in FIG. 25. A
maximum inhibition of 97% was observed. The concentration that
induced the maximum inhibition was 178 .mu.g/mL. The calculated
half-maximal response (IC.sub.50) was calculated to be 36.31
.mu.g/mL. This data demonstrates that the extract derived from
sugar cane of the present disclosure inhibits TNF-.alpha.
indicating anti-inflammatory activity.
Example 22. Prostaglandin E.sub.2 (PGE.sub.2) Study
Description
[0454] PGE.sub.2 is a primary product of arachidonic acid
metabolism controlled by cyclooxygenase enzymes. It is a potent
mediator of inflammation and plays a critical role in increasing
vascular permeability, fever generation, tumor growth and wound
healing. In particular, PGE.sub.2 affects keratinocyte
proliferation, differentiation and angiogenesis. Drugs used to
inhibit PGE.sub.2 synthesis have shown to control inflammation,
pain and fever.
[0455] Assaying the inhibition of PGE.sub.2 expression follows a
procedure whereby a test material is absorbed into mammalian cells.
Cells are stressed with an inflammation inducer, which would
normally stimulate PGE.sub.2 production that would further develop
into inflammation through series of cellular signalling. However,
if a PGE.sub.2 inhibitor is presents in the cellular environment,
the material inhibits PGE.sub.2 production and the degree of
inhibition is assessed by level of decreased PGE.sub.2 production.
PGE.sub.2 production level in cells, treated with and without a
test material, under the stressed condition is monitored and
compared to assess the PGE.sub.2 inhibition effect of the test
material.
Methodology
[0456] Mammalian cells were first treated with or without powdered
sugar cane extract (extract of Example 3) to allow for natural
absorption of the material into the cells. The maximum percentage
of PGE.sub.2 expression inhibition induced by the powdered extract
and the concentration used that induced the maximum inhibition of
PGE.sub.2 expression were reported. The half maximal effective
concentration (EC.sub.50) was calculated. The assay results are
shown in Table 39.
TABLE-US-00045 TABLE 39 Cellular PGE.sub.2 inhibition assay results
Conc. of powdered sugar cane extract of Example 3 (.mu.g/mL)
Inhibition of PGE2 (%) 183.13 58.29 91.56 44.08 45.78 45.72 22.89
30.97 11.45 24.41 5.72 -8.38
[0457] The response curve for this data is shown in FIG. 26. A
maximum inhibition of 58.29% was observed. The concentration that
induced the maximum inhibition was 183.13 .mu.g/mL. The calculated
half-maximal response (IC.sub.50) was calculated to be 91.62
.mu.g/mL. This data demonstrates that the extract derived from
sugar cane of the present disclosure inhibits PGE.sub.2 indicating
anti-inflammatory activity.
Example 23. Cyclooxygenases-1 (COX-1) and Cyclooxygenases-2 (COX-2)
Inhibition Assays
Description
[0458] Cyclooxygenases-1 (COX-1) inhibitors are among the important
targets for treatment of inflammation related diseases. COX has two
well-known isoforms, COX-1 and COX-2, which are similar in their
amino-acid sequences and identity. COX-2 predominates at sites of
inflammation and is involved in chronic inflammation observed in
chronic lesions.
Methodology
[0459] COX-1 and COX-2 inhibition assays were used to assess the
inhibition capability of representative powdered extracts derived
from sugar cane of the present disclosure (extract of Example 3) by
monitoring the extracts' impact on the activity of a specific COX
enzyme. The assays compare the enzymatic activity of the target COX
in the presence with and without the material of interest to
determine the inhibition potential of the material. The results
were expressed as the concentration of the tested material used to
achieve 50% of COX inhibition (IC.sub.50), if 50% of inhibition has
been achieved. If the IC.sub.50 value could not be calculated, the
maximum percentage of COX inhibition achieved, and the
concentration of the material used that induced the maximum
inhibition were reported. COX-1 and COX-2 results are shown in
Table 40 and Table 41 respectively.
TABLE-US-00046 TABLE 40 COX-1 assay results COX-1 inhibition (%)
Standard Conc. (.mu.g/mL) Replicate 1 Replicate 2 Average deviation
500.00 16.73 6.62 11.68 7.15 250.00 -3.67 4.83 0.58 6.01 125.00
5.73 -5.65 0.04 8.05 62.50 1.14 10.11 5.63 6.35 31.25 4.83 6.62
5.72 1.27 15.63 8.38 3.92 6.15 3.16
[0460] The inhibition curve for the COX-1 data is shown in FIG. 27
panel A. The maximum inhibition is 11.68% and the calculated
effective concentration at maximum inhibition is 500.00
.mu.g/mL.
TABLE-US-00047 TABLE 41 COX-2 assay results COX-2 inhibition (%)
Standard Conc. (.mu.g/mL) Replicate 1 Replicate 2 Average deviation
500.00 46.52 44.98 45.70 1.02 250.00 25.78 30.20 27.99 3.13 125.00
18.97 26.43 22.70 5.27 62.50 11.56 9.99 10.78 1.10 31.25 13.09 9.99
11.54 2.19 15.63 9.20 6.79 8.00 1.71
[0461] The inhibition curve for the COX-2 data is shown in FIG. 27
panel B. The maximum inhibition was observed to be 47.70%. The
calculated effective concentration at maximum inhibition was 500.00
.mu.g/mL.
[0462] This demonstrates that the extract of sugar cane of the
present disclosure was selective for COX-2 over COX-1. Further,
this data demonstrates that the extract derived from sugar cane of
the present disclosure inhibits COX-2 indicating anti-inflammatory
activity.
Example 24. Inhibitory Effect of the Extracts on Microbial
Growth
[0463] As described above, a variety of pathogenic microorganisms
thrive in the skin. To measure the inhibitory potential of an
extract derived from sugar cane of the present disclosure on
microorganisms, a sample of an extract derived from sugar cane of
the present disclosure was tested for its effectiveness as an
anti-bacterial agent.
Bacterial Strains and Materials
[0464] Bacterial strains, Staphylococcus aureus ATC.RTM. 25923,
Staphylococcus epidermidis ATC.RTM. 14990, Streptococcus mutans
ATC.RTM. 25175 and Escherichia coli ATC.RTM. 25922 were purchased
from In Vitro Technologies (Noble Park, VIC Australia).
Propionibacterium acnes ATCC.RTM. 6919 was ordered from Thermo
Scientific.TM. Culti-Loops.TM. (Waltham, Mass. USA). Tryptone soya
agar with 5% horse blood plates, and, tryptone soya broth were
purchased from Thermo Scientific.TM.. Petri dishes (90.times.15
mm), loop inoculating PS blue, anaerobic jars (2.5 Litre) and
anaerobic Gen Sachets were purchased from Thermo Scientific.TM..
Penicillin-streptomycin was obtained from Sigma Aldrich (Castle
Hill, NSW Australia).
Culturing and Growth Conditions
[0465] All strains used were in freeze dried form and transferred
into enrichment sterilized tryptone soya broth (TSB) media. The
cultures were propagated 3 times to optimize growth at 37.degree.
C. and at different incubation times, depending on the strain type;
Escherichia coli was incubated for 24 hours (h), Staphylococcus
aureus, Staphylococcus epidermidis and Streptococcus mutans were
incubated for 48 h, and Propionibacterium acnes for 72 h.
Propionibacterium acnes (loop form) was ready to culture directly
into blood agar. There were no differences in the preparation of
agar plates for disc and well diffusion assays. Briefly, 100 .mu.L
of a bacterial suspension was added into 6 mm wells/discs of
tryptone soya agar plates with 5% horse blood (TSAB).
Extract Derived from Sugar Cane and Control Solutions
[0466] The extract derived from sugar cane was prepared according
to the process of FIG. 1.
[0467] Stock solutions of the extract were prepared as previously
described (Taguri et al., 2004) with small modifications. Briefly,
the extract powder was dissolved in sterilized water and filtered
through Advantec cellulose acetate filters (0.2 mm pore size; 25 mm
diameter). The range of concentrations of extract solution was
serially diluted in 14 stages (0.1, 0.5, 0.7, 0.8, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10 mg/ml). The negative control used was sterilized
water and the positive control was penicillin-streptomycin
(consisting of 10,000 units/ml penicillin G sodium salt and 10
mg/ml streptomycin sulfate).
Antimicrobial Activity of the Extract
[0468] Antibacterial tests were carried out using the Oxford cup
assay with minor modifications (Shang et al., 2014). After
autoclaving, TSB media was cooled to 50.degree. C. Susceptibility
of the test organism to the extract was determined by employing the
standard disk or well diffusion technique. Wells 6 mm in diameter
were made in TSAB plates. The bacterial suspension in TSB was
spread and plated on blood agar media. For the disk diffusion test,
sterile paper discs (6 mm) were added of the test sample (100
.mu.l) and placed onto the inoculated blood agar surface. After
cultivation at 37.degree. C. for (24, 48 or 72 h) under anaerobic
conditions in an anaerobic jar containing AnaeroGen sachets to
maintain anaerobic conditions, the resulting inhibition zone
diameters were measured. Using the well (6 mm) diffusion technique,
100 l/well of the sample was added and the size of the inhibition
zone of growth was measured after 24 h or 48 h at 37.degree. C. All
experiments were repeated for each bacterial strain at least 3
times.
Statistical Analysis
[0469] Analysis of data (one-way ANOVA) was used to determine any
significant differences (p<0.05) in the diameter of the
inhibition zones, using Minitab 17 software. All data are expressed
as the mean of triplicate .+-.standard deviation.
The Extract Inhibits Growth of Escherichia coli
[0470] Escherichia coli is a gram-negative anaerobic bacteria
mostly found in the intestine. Most strains of Escherichia coli are
harmless and constitute part of the normal gut flora and are
involved in vitamin K synthesis. However, some Escherichia coli
serotypes are often responsible for food poisoning and disease
(i.e. gastroenteritis, hemorrhagic colitis, Crohn's disease,
travellers' diarrhea, urinary tract infections, sepsis, pneumonia)
and wound infections. Antibiotics are used to treat Escherichia
coli infections and to decrease the course of illness, though, the
rate of bacterial resistance to commonly used antibiotics is
increasing and antibiotics are more commonly not recommended.
[0471] Growth of Escherichia coli was evaluated in TSAB plates
containing a series of wells/discs loaded with a range of
concentrations of the extract at 37.degree. C. following 24 h
incubation period. The diameters of the inhibition zone around the
wells/discs increased gradually with increasing concentrations of
the extract, starting at 0.7 mg/ml and up to 10 mg/ml (p<0.05,
compared to negative control). The lowest growth inhibition zone
was 1.5 mm with at 0.7 mg/ml concentration of the extract whilst
the highest inhibition zone was 10.6 mm at concentrations 9 mg/ml
and 10 mg/ml (FIGS. 28A and 29A). The inhibition zone of the
positive control penicillin-streptomycin reached 35 mm and the
negative control showed no inhibition zone.
The Extract Inhibits Growth of Staphylococcus epidermidis
[0472] Staphylococcus epidermidis is a gram-positive anaerobic
bacterium part of the normal skin flora which is non-pathogenic
although those with compromised immune systems are at risk of
developing infection. In addition, Staphylococcus epidermidis is
commonly hospital acquired infection, in addition to drug users and
those patients needing catheters and prosthetic heat valves; hand
washing has been used in hospitals to reduce Staphylococcus
epidermidis contamination and spread. Due to it being part of the
normal skin flora, Staphylococcus epidermidis has developed
resistance to commonly used antibiotics. Of interest,
Staphylococcus epidermidis is often present in affected acne
vulgaris pores together with Propionibacterium acnes.
[0473] Staphylococcus epidermidis was grown on TSAB plates
containing a series of wells loaded with a fixed amount of the
extract (100 .mu.l/well or disc) with increasing concentrations of
the extract from 0.1-10 mg/ml. After 48 h of incubation at
37.degree. C., the diameters of the growth inhibition zone were
measured. In general, the inhibition zone diameters gradually
increased from 5 mm to 10.4 mm between 2-5 mg/ml (p<0.05) after
which there was a plateau from 5-10 mg/ml concentration of the
extract (FIG. 28B, 29B). The inhibition zone of the positive
control penicillin-streptomycin reached 30 mm and the negative
control and doses of the extract less than 2 mg/ml showed no
inhibition zones.
The Extract Inhibits Growth of Staphylococcus aureus
[0474] Staphylococcus aureus (gram-positive bacterium) part of the
normal flora of the skin, nose and respiratory tract, is not
commonly pathogenic but can cause minor to severe life threatening
infections. Staphylococcus aureus infects wounds and causes
impetigo and septicaemia. With the emergence of antibiotic
resistant Staphylococcus aureus strains this has become a problem
worldwide and accounts up to 50,000 deaths each year in the USA
alone.
[0475] Staphylococcus aureus was cultured for 48 h on TSAB plates
at 37.degree. C. with a series of wells/discs containing different
concentrations of the extract. Antibacterial activity at varying
concentrations are shown in FIG. 28C. The diameters of bacterial
growth inhibition zones around the wells/discs were clearly
inhibited between 2-10 mg/ml of the extract, with weak and unclear
inhibition between 0.5-1 mg/ml concentrations of the extract. There
were no significant differences in zone inhibition between
concentration 2-8 mg/ml but were significant compared to 0.5-1
mg/ml concentrations (p<0.05). The highest diameters of
Staphylococcus aureus growth inhibition zones were at concentration
9-10 mg/ml of approximately 10.8 mm which was significantly higher
than that at <8 mg/ml (p<0.05) (FIGS. 28C and 29C). The
inhibition zone of the positive control penicillin-streptomycin
reached 35 mm and the negative control and doses of the extract
less than 0.5 mg/ml showed no inhibition zones.
The Extract Inhibits Growth of Streptococcus mutans
[0476] Streptococcus mutans (gram positive anaerobic bacterium)
mostly found in the oral cavity plays a crucial role in tooth
decay, oral diseases and certain cardiovascular diseases.
Streptococcus mutans are also part of the normal oral flora in
humans and in canines and contribute to infections of wounds caused
by bites. Increased use of fluoride based toothpastes and oral
rinses in recent years in order to decrease Streptococcus mutans
growth has resulted in the emergence of Streptococcus mutans
resistant strains.
[0477] Streptococcus mutans was added to wells in TSAB plates at
37.degree. C. for 48 h. When inhibition zones were examined in
relation to a range of concentrations of the extract, no
Streptococcus mutans bacterial inhibition zones were observed at
concentrations less than 6 mg/ml. However, at concentrations
between 7-10 mg/ml of the extract, significant inhibition zones
were noted (p<0.05) with concentrations 9-10 mg/ml being most
significant of about 15 mm (FIGS. 28D and 29D). In comparison to
positive control penicillin-streptomycin the bacterial inhibition
zone was 30 mm and the negative control and doses of the extract
less than 7 mg/ml showed no inhibition zones.
The Extract Inhibits Growth of Propionibacterium acnes
[0478] Propionibacterium acnes, also a gram-positive anaerobic
bacterium is closely linked to acne, blepharitis and atopic
dermatitis, and is susceptible to a vast number of antibiotics,
natural anti-microbials (i.e. tea tree oil, citrus oil, honey) and
over the counter anti-bacterial chemicals. However, in recent years
there has been an emergence of antibiotic resistant
Propionibacterium acnes strains which has resulted in a problem
worldwide.
[0479] The anti-bacterial growth of Propionibacterium acnes in the
presence of different concentrations of the extract was examined at
37.degree. C. and 72 h of incubation. Zones of inhibition showed
that concentrations of the extract had good growth inhibition
activity (17-18 mm) only at concentrations 9-10 mg/ml (FIGS. 28E
and 29E). The inhibition zone of the positive control
penicillin-streptomycin (FIG. 29F) was in the order of 30 mm and
the negative control and doses of the extract less than 9 mg/ml
showed no inhibition zones.
Example 25
[0480] Example 25 provides illustrative and non-limiting examples
of compositions comprising extracts derived from sugar cane of the
present disclosure.
TABLE-US-00048 Hand cream Ingredient Ingredient Quantity Vegesorb
Cream Base* (ex. Vegesorb) 948.250 grams Extract derived from sugar
cane of the 50.000 grams present disclosure White Peony Fragrance
(ex. ABP 1.750 grams (Perfumis)) *Vegesorb Cream Base contains
purified water, Glycerin (plant derived), glyceryl monosterate
(plant derived), almond oil (plant derived), apricot oil (plant
derived), PEG-20 sterate (plant derived), cetostearyl alcohol
(plant derived), stearic acid (plant derived), cetostearyl
octanoate (plant derived), Vitamin E (plant derived),
phenoxyethanol (purpose designed water soluble preservative),
diazolidinylurea (purpose designed water soluble preservative),
methyl hydroxybenzoate (purpose designed water soluble
preservative).
[0481] The hand cream contains 5% of an extract derived from sugar
cane of the present disclosure.
[0482] The hand cream was prepared by the following method:
1. Add the Vegesorb base to main vessel; 2. Add the extract derived
from sugar cane and mix well; 3. Add the White Peony fragrance and
mix well; and 4. Pack in appropriate packaging.
TABLE-US-00049 Face cream Ingredient Ingredient Quantity Vegesorb
Cream Base* (ex. Vegesorb) 948.500 grams Extract derived from sugar
cane of the 50.000 grams present disclosure Lily of the Valley
Fragrance (ex. ABP 1.500 grams Perfumis) *Vegesorb Cream Base
contains purified water, Glycerin (plant derived), glyceryl
monosterate (plant derived), almond oil (plant derived), apricot
oil (plant derived), PEG-20 sterate (plant derived), cetostearyl
alcohol (plant derived), stearic acid (plant derived), cetostearyl
octanoate (plant derived), Vitamin E (plant derived),
phenoxyethanol (purpose designed water soluble preservative),
diazolidinylurea (purpose designed water soluble preservative),
methyl hydroxybenzoate (purpose designed water soluble
preservative).
[0483] The face cream contains 5% of an extract derived from sugar
cane of the present disclosure.
[0484] The face cream was prepared by the following method:
1. Add the Vegesorb base to main vessel; 2. Add the extract derived
from sugar cane and mix well; 3. Add the Lilly of the Valley
fragrance and mix well; and 4. Pack in appropriate packaging.
TABLE-US-00050 Frangipani face cream Ingredient Ingredient Quantity
(%) Frangipani Fragrance 0.15 Extract derived from sugar cane of
the 5.0 present disclosure Vegesorb Cream Base* 94.85 *Vegesorb
Cream Base contains purified water, Glycerin (plant derived),
glyceryl monosterate (plant derived), almond oil (plant derived),
apricot oil (plant derived), PEG-20 sterate (plant derived),
cetostearyl alcohol (plant derived), stearic acid (plant derived),
cetostearyl octanoate (plant derived), Vitamin E (plant derived),
phenoxyethanol (purpose designed water soluble preservative),
diazolidinylurea (purpose designed water soluble preservative),
methyl hydroxybenzoate (purpose designed water soluble
preservative).
[0485] The face cream was prepared using a similar method to that
described above.
TABLE-US-00051 BB face cream Ingredient Ingredient Quantity (%)
Extract derived from sugar cane of the 2.0 present disclosure
Medium skin tone BB cream* 94.85 *BB Cream contains water,
dimethicone, glycerin, prunus armeniaca kernel oil/apricot kernel
oil, oryza sativa bran oil/ricebran oil, propanediol, ethylhexyl
methoxycinnamate, stearic acid, butyrospermum parkii butter/shea
butter, palmitic acid, peg-100 stearate, glyceryl stearate, peg-20
stearate, stearyl alcohol, cera alba/beeswax,
acrylamide/sodiumacryloyldimethyltaurate copolymer, phenoxyethanol,
parfum/fragrance, tocopheryl acetate, isohexadecane,
hydroxypropyltetrahydropyrantriol, caprylyl glycol, propylene
glycol, dimethiconol, vigna aconitifolia/vigna aconitifolia seed
extract, polysorbate 80, pentaerythrityl tetra-di-t-butyl
hydroxyhydrocinnamate, sodium cocoyl glutamate, disodium edta,
hydrolyzed soyprotein, linalool, benzyl salicylate, limonene,
cinnamic acid, phloroglucinol trimethyl ether, benzyl alcohol,
geraniol, citral, titanium dioxide and iron oxides.
[0486] The face cream was prepared using a similar method to that
described above.
TABLE-US-00052 Face mask Ingredient Ingredient Quantity (%) Facial
Serum Base* 69.75 Chamomile Floral Water 25 Extract derived from
sugar cane of the 5 present disclosure Pink Grapefruit Fragrance
0.25 *Facial Serum Base contains purified water, glycerine, aloe
vera leaf juice, xanthan gum, phenoxyethanol, butylene glycol,
caprylyl glycol, polysorbate 20, hydrolyzed marine collagen,
carbomer, triethanolamine, rosehip oil, evening primrose oil,
jojoba oil, apple fruit extract, melon fruit extract, sorbitol,
ethylhexylglycerin, beta-glucan, potassium sorbate, sodium
benzoate, portulaca extract, seaweed extract, green tea leaf
extract, citric acid, 1,2-hexanediol, grapefruit seed extract,
benzoic acid, Vitamin C.
[0487] The face mask was prepared using a similar method to that
described above.
TABLE-US-00053 Aloe Vera soap Ingredient Ingredient Quantity Aloe
Vera Natural Melt & Pour Soap 965.00 grams Base* (ex. Aussie
Soap Supplies) Extract derived from sugar cane of the 25.000 grams
present disclosure Wildflower Honey Fragrance (ex. Aussie 10.00
grams Soap Supplies) *Aloe Vera Natural Melt & Pour Soap Base
contains coconut oil, palm oil, safflower oil, glycerine (Kosher,
of vegetable origin), aloe vera gel, purified water, sodium
hydroxide, sorbitol, propylene glycol (made from vegetable
glycerine), sorbitan oleate, oat protein.
[0488] The soap contains 2.5% of an extract derived from sugar cane
of the present disclosure.
[0489] The soap was prepared by the following method:
1. Weigh the required amount of soap and gently melt; 2. Once Soap
is melted, add the extract derived from sugar cane and Fragrance,
slowly mix, but do not let boil; 3. Pour into selected moulds, and
set at ambient temp. Do not move while base is resetting; and 4.
Remove from mould. Cut to selected size and wrap.
TABLE-US-00054 Wildflower and honey soap Ingredient Ingredient
Quantity (%) Frangipani Fragrance 1.0 Extract derived from sugar
cane of the 2.5 present disclosure Aloe vera soap base* 96.5 *Aloe
Vera Soap Base contains coconut oil, palm oil, safflower oil,
glycerine (Kosher, of vegetable origin), aloe vera gel, purified
water, sodium hydroxide, sorbitol, propylene glycol (made from
vegetable glycerine), sorbitan oleate, oat protein.
[0490] The soap was prepared using a similar method to that
described above.
TABLE-US-00055 After sun care lotion Ingredient Ingredient Quantity
Aloe Vera Gel Base* (ex. New 873.950 grams Directions) Extract
derived from sugar cane of the 25.000 grams present disclosure
50:50 Xanthan:Acacia Blend 0.0500 grams Glycerine 100.000 grams
Aloe and Cucumber Fragrance (ex. ABP 1.000 grams Perfumis) *Aloe
Vera Gel Base contains water, phenoxyethanol, Carbomer,
ethylhexylglycerin, sodium hydroxide, glycerin, aloe barbadensis
leaf juice powder, disodium EDTA.
[0491] The after sun care lotion contains 2.5% of an extract
derived from sugar cane of the present disclosure.
[0492] The after sun care lotion was prepared by the following
method:
1. Weigh the Aloe Vera Gel Base;
[0493] 2. Add Glycerine to the base and mix well; 3. Add the
extract derived from sugar cane to the mix and mix well; 4. Add
xanthan and acacia blend to the blend and mix well until the
product thickens; 5. Add fragrance to the base mix well; and 6.
Pack into appropriate packaging.
TABLE-US-00056 Shower gel Ingredient Ingredient Quantity Hand and
Body Wash Base* (ex. Aussie 941.300 grams Soap Supplies) Extract
derived from sugar cane of the 30.000 grams present disclosure
Energy Fragrance (ex. Aussie Soap 28.700 grams Supplies) *Hand and
Body Wash Base contains aqua, organic potassium cocoate, organic
potassium oleate, lauryl betaine, organic glycerine from organic
sunflower oil, benzyl alcohol, decyl glucoside, potassium citrate.
The shower gel contains 3% of an extract derived from sugar cane of
the present disclosure.
[0494] The shower gel was prepared by the following method:
1. Add the unscented Hand and Body Wash Base to main vessel; 2. Add
the extract derived from sugar cane and mix well. 3.3 Add the
fragrance and mix well. 3.4 Pack in to appropriate packaging.
TABLE-US-00057 Lemon Verbena Shower gel Ingredient Ingredient
Quantity (%) Lemon Verbena Fragrance 2.87 Extract derived from
sugar cane of the 3.0 present disclosure Shower Gel Base* 94.13
*Shower Gel Base contains aqua, organic potassium cocoate, organic
potassium oleate, lauryl betaine, organic glycerine from organic
sunflower oil, benzyl alcohol, decyl glucoside, potassium
citrate.
[0495] The shower gel was prepared using a similar method to that
described above.
TABLE-US-00058 Energy Shower gel Ingredient Ingredient Quantity (%)
Energy Fragrance 2.87 Extract derived from sugar cane of the 3.0
present disclosure Shower Gel Base* 94.13 *Shower Gel Base contains
aqua, organic potassium cocoate, organic potassium oleate, lauryl
betaine, organic glycerine from organic sunflower oil, benzyl
alcohol, decyl glucoside, potassium citrate.
[0496] The shower gel was prepared using a similar method to that
described above.
TABLE-US-00059 Shampoo - conditioning Ingredient Ingredient
Quantity Shampoo - Conditioning Base* (ex. 973.00 grams Aussie Soap
Supplies) Extract derived from sugar cane of the 25.000 grams
present disclosure Bamboo and Lily Fragrance (ex. ABP 2.000 grams
Perfumis) *Shampoo - Conditioning Base contains aqua, sodium
lauroyl methyl isethionate, cocamidopropyl betaine, lauryl
glucoside, sodium chloride, sodium methyl isethionate,
phenoxyethanol, sodium lauroyl isethionate, sodium benzoate,
trisodium sulfosuccinate, polyquaternium 10, lauric acid, zinc
dilaurate, trisodium ethylenediamine disuccinate, sodium
laurate.
[0497] The shampoo-conditioning contains 2.5% of an extract derived
from sugar cane of the present disclosure.
[0498] The shampoo-conditioning was prepared by the following
method:
1. Add the Unscented Shampoo-Conditioning base to main vessel; 2.
Add the extract derived from sugar cane and mix well; 3. Add the
fragrance and mix well; and 4. Pack in to appropriate
packaging.
TABLE-US-00060 Dual action extra strength heat gel Ingredient
Ingredient Quantity (%) Dencorub base 95 Extract derived from sugar
cane of the 5.0 present disclosure
[0499] The heat gel was prepared using a similar method to that
described above.
TABLE-US-00061 Face cream Ingredient Extract derived from sugar
cane of the present disclosure aqua cetearyl alcohol cetyl alcohol
glycerin .alpha.-arbutin ceteareth-20 isononyl isononanoate phenoxy
ethanol niacinamide tocopheryl acetate disodium EDTA BHT
[0500] It will be appreciated by one of ordinary skill 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.
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