Note: Descriptions are shown in the official language in which they were submitted.
WO 2022/238727
PCT/1B2021/053926
METHODS FOR PREPARING WHITE HEIRLOOM TOMATO-BASED
COMPOSITIONS FOR SKIN LIGHTENING VIA ENHANCED
GLUTATHIONE, AND ASSOCIATED COMPOSITIONS THEREOF
FIELD OF THE INVENTION
Embodiments of the present invention generally relate to compositions for
tomato-based compositions, and, more particularly, to methods for preparing
white heirloom tomato-based compositions for anti-ageing purposes, and
associated compositions thereof.
BACKGROUND OF THE INVENTION
As it is well known that for the whitening of the skin to a lighter skin tone
or for
the elimination of dark spots from the skin, whitening compositions are
useful.
Further, these whitening or skin bleaching compositions are effective as these
inhibits the formation of melanin (Skin colour is determined by this pigment).
However, such skin bleaching or whitening compositions contain harsh
chemical, such as peroxides, glutathione, formaldehyde and the like, that has
severe effects on the skin.
As disclosed in conventional prior art disclosure, the use of hydroquinone
suppresses the melanocyte activity and helps in whitening of the skin.
However,
such less stringent composition has other disadvantage i.e., the hydroquinone
is oxidised by air and light. Further, the oxidised product causes skin
irritation
and inflammation.
Furthermore, there has been disclosed in the prior art disclosure about the
dermatological compositions that use paucilamellar liposomal carriers that
help
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in treating Melanoma caused by exposure to sunlight. However, such
compositions are not effective in whitening the skin to a lighter skin tone.
Additionally, some conventional prior art disclosure discloses the use of
naturally derived materials in dermatological compositions, such as but not
limited to, lemons, oranges, cucumber, rose fruit and the like. However, such
naturally derived materials limit their usefulness based on age, skin type,
colour
and vary greatly in different subjects.
As disclosed in various prior art disclosures, the use of cosmetics
composition
containing carotenoids with the extract of plant of the genus malus,
containing
polyphenols helps in skin whitening. However, these compositions containing
carotenoids are typically highly coloured and are sensitive to light and
oxidation
that considerably limits their usefulness.
Furthermore, prior art disclosures also disclose the use of polyphenolic
compounds, mostly resorcinols having skin whitening properties. However,
these compounds being susceptible to air and UV oxidation leads to colour
instability that causes loss of skin tightening efficacy. Additionally, as per
related
prior art disclosure, the extract obtained from fruits of acerola containing
Vitamin C, are incorporated into cosmetics to provide a whitening effect.
Therefore, the prior art disclosures briefly discuss about the compositions
that
are useful for whitening of the skin. However, the element of usage to
increase
natural glutathione level is yet to be introduced in the market.
Thus, there is a recognized need for, and it would be highly beneficial to
have
alternative, effective natural cocktail of flavonoid and carotenoid
preparations
having minimal adverse side effects, which are effective for whitening skin
and
which are suitable for the cosmetic industry. Accordingly, there remains a
need
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in the art for innovative, novel, efficient methods for preparing white
heirloom
tomato-based compositions for anti-ageing purposes, and associated
compositions thereof.
SUMMARY OF THE INVENTION
The embodiments of the present disclosure have several features, no single
one of which is solely responsible for their desirable attributes. Without
limiting
the scope of the present embodiments as expressed by the claims that follow,
their more prominent features will now be discussed briefly. After considering
this discussion, and particularly after reading the section entitled "Detailed
Description", one will understand how the features of the present embodiments
provide advantages.
In one embodiment, a method for preparing white heirloom tomato-based
composition for anti-ageing purposes, the method including the steps of,
sampling and decontaminating a tomato fruit sample via vigorous washing;
freeze-drying and homogenising the tomato fruit sample to produce tomato fruit
powder; preparing tomato fruit powder stock solution using the tomato fruit
powder; and, dissolving and mixing the tomato fruit powder stock solution
(100mg/m1) in distilled water.
DETAILED DESCRIPTION
Various embodiments of the present invention are aimed at providing novel and
inventive solutions focused on methods for preparing white heirloom tomato-
based compositions for anti-ageing purposes, and associated compositions
thereof.
In accordance with an embodiment of the present invention, a method for
preparing white heirloom tomato-based composition for anti-ageing purposes,
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the method including the steps of, sampling and decontaminating a tomato fruit
sample via vigorous washing; freeze-drying and homogenising the tomato fruit
sample to produce tomato fruit powder; preparing tomato fruit powder stock
solution using the tomato fruit powder; and, dissolving and mixing the tomato
fruit powder stock solution (100mg/m1) in distilled water.
In accordance with an embodiment of the present invention, the method further
includes the step of passing the tomato fruit powder stock solution through
syringe filter for preparing different dilutions in plain medium (DMEM). In
use,
the tomato fruit powder stock solution is prepared by using dilutions of
10Oug/ml, 200ug/ml, 500ug/ml, 1000ug/m1 and 2000ug/ml.
In accordance with an embodiment of the present invention, the method further
includes steps of, obtaining a NIH 3T3 (mouse fibroblast) cell line from a
cell
tissue culture; and, culturing the cell line in Dulbecco's Modified Eagle's
Medium
High Glucose (DMEM-HG) with 10% foetal bovine serum (FBS). In use, the
method further includes the steps of, treating the NIH 3T3 (mouse fibroblast)
cell lines with tomato fruit powder; culturing the cell lines onto 96 well
plates;
treating the cell lines with 6mm H202 for approx. 4 hours to create ROS
mediated cellular damage; and, treating injured cells with the concentrations
(10Oug/ml, 200ug/ml, 500ug/ml, 1000ug/m1 and 2000ug/m1) of the tomato fruit
powder stock solution for a period of 24 hours.
In accordance with an embodiment of the present invention, the method further
includes the step of, calculating viability of the NIH 3T3 cells via MTT
assay,
evaluating the Glutathione reductase and reduced Gluthathione (GSH) levels
in the NIH 3T3 cells, and, calculating the LDH activity of the NIH 3T3 cells
via
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LDH released assay. In use, the heirloom white tomato fruit includes phytoene
and Phytofluene.
In accordance with an embodiment of the present invention, Figure 1
illustrates
3T3 cell line, as the plot of cytotoxicity percentage (%) versus various
concentrations of Tomesol heirloom white tomato (Tomesoral), wherein lower
concentrations of tomato powder did not show significant cytotoxicity on the
cell
lines, whereas, the higher concentration like 1mg or 2mg showed some
cytotoxicity. Subsequently, on the basis of cytotoxicities, IC50 value was
calculated as 1.986mg/ml.
Also, the embodiment of the invention in Figure 1 (a) illustrates the Bar
graph
which further shows the relative values of absorbance after treating the 3T3
cells with MTT (3-(4, 5-dimethylthiazol-2-y1) - 2, 5-diphenyltetrazolium
bromide)
assay.
Furthermore, the embodiment of the invention as illustrated in Figure 1 (b)
shows line graph that depicts the relative percentage of viabilities on
different
concentrations of tomato powder while box represents the IC50 value for
tomato powder. Values were taken as mean SEM and * shows the significant
difference between viabilities of treated groups and untreated controls
(p<0.05).
In accordance with an embodiment of the present invention, as illustrated in
Figure 2(a), the relative absorbance of post treated cells, measuring the
reduced levels of cytoplasmic enzyme LDH (Lactate dehydrogenase) released
(during cell injury or stress), in treatment groups of 3T3 cells, after
performing
LDH assay. Also, the embodiment of the invention as illustrated in Figure 2
(b)
shows the Expression analysis of LDH release in post treated cells. Values
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were taken as mean SEM and *shows the significant difference between LDH
levels of treated groups and untreated controls (p<0.05).
In accordance with an embodiment of the present invention, Figure 3 (a) shows
relative absorbance of post treated cells that further shows an increased
Glutathione Reductase in Post Treated Groups; 3T3 cells 'intracellular
glutathione reductase (GR) activity was measured (n = 5 replicates) before and
after treatment with Tomesol heirloom white tomato (Tomesoral).
Subsequently, Glutathione reductase (GR) activity was significantly increased
in post treated cells, wherein, the presence of white tomato powder in the
media
significantly boosted GR activity from 2.24 0.152 mU/mL prior to treatment to
5.07 0.323 mU/mL in the presence of Tomesol heirloom white tomato
(Tomesoral) at a concentration of 500ug/ml. However, the addition of more
tomato powder to a concentration of 1mg/m1 and 2mg/m1 will not boost the
levels further. The peak value is observed in 500ug/m1 concentration.
In accordance with an embodiment of the present invention, Figure 3(b) shows
the expression analysis of reduced glutathione (GSH). Reduced glutathione
(GSH) of H202 injured 3T3 cells was estimated (n = 5 replicates) pre and post-
treatment with heirloom tomato powder. GSH was significantly increased in
tomato powder treated cells. The presence of white tomato powder in the media
significantly enhances GSH as untreated cells has value of 0.347 0.0152 while
treated groups have GSH levels as: T.TP-10Oug/m1 (0.417 0.0426), T.TP-
200ug/m1 (0.524 0.0426), T.TP-500ug/m1 (0.630 0.0323), T.TP-1mg/m1
(0.630 0.0104) and T.TP-2mg/m1 (0.629 0.0199) compared to stressed cells
(0.201 0.0139). The optimum dose of tomato powder is 500ug/ml. The addition
of more tomato powder to a concentration of 1mg/m1 and 2mg/m1 will not boost
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the levels further. Further, Graph shows the reduced glutathione (GSH) levels
in post treated cells. Values were taken as mean SEM and * shows the
significant difference between GR levels of treated groups and untreated
controls (p<0.05). Therefore, the present invention shows the use of Tomesol
heirloom white tomato (Tomesoral) that is rich in carotenoids (phytoene and
phytofluene) and flavonoids (chalconaringenin and rutin) unique natural
cocktail
which were specifically targeted to increase and forces glutathione level to
increase excessively. In use, the heirloom white tomato fruit includes
phytoene
and Phytofluene.
The conditional language used herein, such as, among others, "can," "could,"
"might," "may," "e.g.," and the like, unless specifically stated otherwise, or
otherwise understood within the context as used, is generally intended to
convey that certain embodiments include, while other embodiments do not
include, certain features, elements and/or steps. Thus, such conditional
language is not generally intended to imply that features, elements and/or
steps
are in any way required for one or more embodiments or that one or more
embodiments necessarily include logic for deciding, with or without author
input
or prompting, whether these features, elements and/or steps are included or
are to be performed in any particular embodiment. The terms "comprising,"
"including," 'having," and the like are synonymous and are used inclusively,
in
an open-ended fashion, and do not exclude additional elements, features, acts,
operations, and so forth. Also, the term "or" is used in its inclusive sense
(and
not in its exclusive sense) so that when used, for example, to connect a list
of
elements, the term "or" means one, some, or all of the elements in the list.
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While there has been shown and described the preferred embodiment of the
instant invention it is to be appreciated that the invention may be embodied
otherwise than is herein specifically shown and described and that, within
said
embodiment, certain changes may be made in the form and arrangement of the
parts without departing from the underlying ideas or principles of this
invention
as outlined in the Claims appended herewith. Therefore, the appended claims
are to be construed to cover all equivalents falling within the true scope and
spirit of the invention.
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