FIELD OF INVENTION
[001] The present disclosure relates to the field of cosmetic formulations in general and skin-lightening compositions in particular. There is provided a composition comprising dihydrooxyresveratrol and Coenzyme Q10 for reducing skin pigmentation.
BACKGROUND OF INVENTION
[002] Skin pigmentation is a manifestation of presence of melanin pigment within keratinocytes. Melanin is synthesised in melanocytes under tight regulation from enzyme tyrosinase (membrane-bound copper-containing glycoprotein). The pigment is synthesised and stored in specialised organelle known as melanosomes. The melanosomes are further transported to keratinocytes which are distributed in skin and hair to render the visible colour. Melanin production can be regulated by targeting tyrosinase enzyme by developing an inhibitor of tyrosinase. The steps leading to the distribution of melanosomes can also be considered for regulating skin pigmentation. There are a number of studies directed on developing tyrosinase inhibitors (Mishima, Yutaka, et al. Pigment Cell & Melanoma Research 1.6, 1988, 367-374; Solano, Francisco, et al. Pigment Cell Research 19.6, 2006, 550-571) but additional studies covering novel approaches on regulation of skin pigmentation are warranted.
[003] Filopodia play a principal role in transfer of melanosomes to keratinocytes. Filopodia are essentially actin-based structures arising from neuronal growth cones and function in neuronal path finding (Davenport, Roger W., et al. Nature, 1993, 361.6414, 721-724.). A protein belonging to Rho GTPase family of intracellular protein known as CDC42 enhances filopodia formation, thereby increasing melanosome transport (Scott, Glynis, et al., Journal of Cell Science, 2002, 115 (7), 1441-1451).

[004] Thus, targeting the different players involved in the melanogenesis pathway presents a means to control skin pigmentation. There are different approaches to tackle this problem.
[005] US20080305059A1 discloses skin lightening/even toning compositions that
are provided for reducing skin pigmentation of normal skin and for lightening hyper-
pigmented skin with the said compositions comprising (i) highly purified
hexylresorcinol which is substantially free or resorcinol, (ii) optionally, at least one
other skin lightening agent, and (iii) a dermatologically acceptable carrier.
[006] US3856934A discloses synergistic compositions for depigmentation by
topical application which comprise a mixture of hydroquinone, retinoic acid and a
corticosteroid formulated in a pharmaceutically-cosmetically acceptable vehicle.
[007] US6123959A discloses aqueous compositions comprising liposomes of
phospholipids, and at least one competitive inhibitor of an enzyme for the synthesis
of melanin, in combination with at least one non-competitive inhibitor of an enzyme
for the synthesis of melanin.
[008] However, despite the plethora of compositions and treatments a need still remains to find effective solutions for hyperpigmentation of skin.
SUMMARY OF INVENTION
[009] In an aspect of the present disclosure, there is provided a composition comprising: (a) dihydrooxyresveratrol; and (b) Coenzyme Q10, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1.
[0010] In an aspect of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; and (c) contacting dihydrooxyresveratrol, and Coenzyme Q10 to obtain said

composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1.
[0011] In an aspect of the present disclosure, there is provided a method of regulating skin pigmentation comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; and (c) contacting said composition with skin, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1 and said composition inhibits skin pigmentation.
[0012] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0013] The following drawings form a part of the present specification and are
included to further illustrate aspects of the present disclosure. The disclosure may be
better understood by reference to the drawings in combination with the detailed
description of the specific embodiments presented herein.
[0014] Figure 1 shows the cell viability data for dihydrooxyresveratrol, in accordance
with an embodiment of the present disclosure.
[0015] Figure 2 shows the cell viability data for Coenzyme Q10 (Co Q10), in
accordance with an embodiment of the present disclosure.
[0016] Figure 3 shows the cell viability data for oxyresveratrol, in accordance with an
embodiment of the present disclosure.
[0017] Figure 4 shows the cell viability data for rapamycin, in accordance with an
embodiment of the present disclosure.

[0018] Figure 5 shows the cell viability data for epigallocatechin gallate, in
accordance with an embodiment of the present disclosure.
[0019] Figure 6 shows the synergistic effect of the combination of
dihydrooxyresveratrol and Coenzyme Q10 (Co Q10) on the inhibition of CDC42
protein expression, in accordance with an embodiment of the present disclosure.
[0020] Figure 7 shows the effect of dihydrooxyresveratrol and epigallocatechin
gallate (EGCG) on CDC42 protein expression, in accordance with an embodiment of
the present disclosure.
[0021] Figure 8 shows the base peak chromatogram of keratinocyte cells treated with
vehicle control, in accordance with an embodiment of the present disclosure.
[0022] Figure 9 shows the base peak chromatogram of keratinocyte cells treated with
α-MSH hormone, in accordance with an embodiment of the present disclosure.
[0023] Figure 10 shows the base peak chromatogram of keratinocyte cells treated
with dihydrooxyresveratrol, in accordance with an embodiment of the present
disclosure.
[0024] Figure 11 shows the base peak chromatogram of keratinocyte cells treated
with α-MSH hormone and dihydrooxyresveratrol, in accordance with an embodiment
of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions
[0026] For convenience, before further description of the present disclosure, certain
terms employed in the specification, and examples are delineated here. These
definitions should be read in the light of the remainder of the disclosure and
understood as by a person of skill in the art. The terms used herein have the meanings
recognized and known to those of skill in the art, however, for convenience and
completeness, particular terms and their meanings are set forth below.
[0027] The articles “a”, “an” and “the” are used to refer to one or to more than one
(i.e., to at least one) of the grammatical object of the article.
[0028] The terms “comprise” and “comprising” are used in the inclusive, open sense,
meaning that additional elements may be included. It is not intended to be construed
as “consists of only”.
[0029] Throughout this specification, unless the context requires otherwise the word
“comprise”, and variations such as “comprises” and “comprising”, will be understood
to imply the inclusion of a stated element or step or group of element or steps but not
the exclusion of any other element or step or group of element or steps.
[0030] Carriers are substances that serve as mechanisms to improve the delivery and
the effectiveness of drugs.
[0031] A diluent (also referred to as filler, dilutant, or thinner) is a diluting agent.
[0032] An excipient is an inactive substance that serve as the vehicle or medium for a
drug or other active substance. Excipients include colouring agents, humectants,
preservatives, emollients, and combinations thereof.
[0033] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.
[0034] Dihydrooxyresveratrol is an analog of oxyresveratrol, and having a tyrosinase
inhibition activity higher than oxyresveratrol. Higher tyrosinase inhibitory activity of
dihydrooxyresveratrol is predicted to be because of its bibenzyl structure.

[0035] Coenzyme Q10 is also known as ubiquinone. It contains a polyisoprene chain having 10 isoprene units. It is key component of the mitochondrial respiratory chain for ATP synthesis. It is mainly studied for its potent antioxidant activities. [0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
[0037] Skin pigmentation can be localised or widespread. The broad categories of the disorders are: depigmentation, hyperpigmentation and hypopigmentation. There are more than 125 genes that are involved either directly or indirectly in regulation of skin pigmentation. One of the key factors responsible for pigmentation is the transport and distribution of melanosomes to keratinocytes present in hair and skin. CDC42 have been reported to play a crucial role in transportation of melanosomes to keratinocytes by accelerating the movement of filopodia. The present disclosure is about developing a synergistic compound which inhibits the expression of CDC42 protein thereby inhibiting skin pigmentation.
[0038] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.
[0039] In an embodiment of the present disclosure, there is provided a composition comprising: (a) dihydrooxyresveratrol; and (b) Coenzyme Q10, wherein dihydrooxyresveratrol to the Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1.
[0040] In an embodiment of the present disclosure, there is provided a composition comprising: (a) dihydrooxyresveratrol; and (b) Coenzyme Q10, wherein

dihydrooxyresveratrol to the Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:0.5.
[0041] In an embodiment of the present disclosure, there is provided a composition comprising: (a) dihydrooxyresveratrol; and (b) Coenzyme Q10, wherein dihydrooxyresveratrol to the Coenzyme Q10 w/w ratio in said composition is 1:0.4. [0042] In an embodiment of the present disclosure, there is provided a composition comprising: (a) dihydrooxyresveratrol; and (b) Coenzyme Q10, wherein dihydrooxyresveratrol weight percentage in said composition is in the range of 0.0001% - 0.003%, Coenzyme Q10 weight percentage in said composition is in the range of 0.0002%-0.003%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1.
[0043] In an embodiment of the present disclosure, there is provided a composition comprising: (a) dihydrooxyresveratrol; and (b) Coenzyme Q10, wherein dihydrooxyresveratrol weight percentage in said composition is in the range of 0.0001% - 0.003%, Coenzyme Q10 weight percentage in said composition is in the range of 0.0002%-0.003%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is 1:0.4.
[0044] In an embodiment of the present disclosure, there is provided a composition comprising: (a) dihydrooxyresveratrol; and (b) Coenzyme Q10, wherein dihydrooxyresveratrol weight percentage in said composition is in the range of 0.001% - 0.003%, Coenzyme Q10 weight percentage in said composition is in the range of 0.0008%- 0.002%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio ranges from 1:0.2 – 1:1.
[0045] In an embodiment of the present disclosure, there is provided a composition comprising: (a) dihydrooxyresveratrol; and (b) Coenzyme Q10, wherein dihydrooxyresveratrol weight percentage in said composition is in the range of 0.001% - 0.003%, Coenzyme Q10 weight percentage in said composition is in the range of 0.0002% -0.002%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is 1:0.4.

[0046] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) dihydrooxyresveratrol; and (b) Coenzyme Q10, wherein
dihydrooxyresveratrol weight percentage in said composition is 0.0024%, Coenzyme
Q10 weight percentage in said composition is 0.001%, and dihydrooxyresveratrol to
Coenzyme Q10 w/w ratio is 1:0.4.
[0047] In an embodiment of the present disclosure, there is provided a composition
reducing skin pigmentation comprising: (a) dihydrooxyresveratrol; (b) Coenzyme
Q10; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient,
wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is in the range of 1:0.2 –
1:1.
[0048] In an embodiment of the present disclosure, there is provided a composition
reducing skin pigmentation comprising: (a) dihydrooxyresveratrol; (b) Coenzyme
Q10; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient,
wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is in the range of 1:0.2 –
1:0.5.
[0049] In an embodiment of the present disclosure, there is provided a composition
reducing skin pigmentation comprising: (a) dihydrooxyresveratrol; (b) Coenzyme
Q10; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient,
wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is 1:0.4.
[0050] In an embodiment of the present disclosure, there is provided a composition
reducing skin pigmentation comprising: (a) dihydrooxyresveratrol; (b) Coenzyme
Q10; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient,
wherein dihydrooxyresveratrol weight percentage in said composition is in the range
of 0.0001% - 0.003%, Coenzyme Q10 weight percentage in said composition is in the
range of 0.0002% - 0.003%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio
in said composition is in the range of 1:0.2 – 1:1.
[0051] In an embodiment of the present disclosure, there is provided a composition
reducing skin pigmentation comprising: (a) dihydrooxyresveratrol; (b) Coenzyme
Q10; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient,

wherein dihydrooxyresveratrol weight percentage in said composition is in the range of 0.0001% - 0.003%, Coenzyme Q10 weight percentage in said composition is in the range of 0.0002% - 0.003%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4.
[0052] In an embodiment of the present disclosure, there is provided a composition reducing skin pigmentation comprising: (a) dihydrooxyresveratrol; (b) Coenzyme Q10; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein dihydrooxyresveratrol weight percentage in said composition is in the range of 0.001% - 0.003%, co-enzyme Q 10 weight percentage in said composition is in the range of 0.0008% - 0.002%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1.
[0053] In an embodiment of the present disclosure, there is provided a composition reducing skin pigmentation comprising: (a) dihydrooxyresveratrol; (b) Coenzyme Q10; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein dihydrooxyresveratrol weight percentage in said composition is in the range of 0.001% - 0.003%, co-enzyme Q 10 weight percentage in said composition is in the range of 0.0008% - 0.002%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is 1:0.4.
[0054] In an embodiment of the present disclosure, there is provided a composition reducing skin pigmentation comprising: (a) dihydrooxyresveratrol; (b) Coenzyme Q10; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein dihydrooxyresveratrol weight percentage is 0.0024%, Coenzyme Q10 weight percentage is 0.001%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is 1:0.4.
[0055] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; and (c) contacting dihydrooxyresveratrol and Coenzyme Q10 to

obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is in the range of 1:0.2 – 1:1.
[0056] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; and (c) contacting dihydrooxyresveratrol and Coenzyme Q10 to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is in the range of 1:0.2 – 1:0.5.
[0057] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; and (c) contacting dihydrooxyresveratrol and Coenzyme Q10 to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is 1:0.4.
[0058] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; and (c) contacting dihydrooxyresveratrol and Coenzyme Q10 to obtain said composition, wherein dihydrooxyresveratrol weight percentage in said composition is in the range of 0.0001% - 0.003%, Coenzyme Q10 weight percentage in said composition is in the range of 0.0002% - 0.003%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1. [0059] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; and (c) contacting dihydrooxyresveratrol and Coenzyme Q10 to obtain said composition, wherein dihydrooxyresveratrol weight percentage in said composition is in the range of 0.0001% - 0.003%, Coenzyme Q10 weight percentage

in said composition is in the range of 0.0002% - 0.003%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4.
[0060] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; and (c) contacting dihydrooxyresveratrol and Coenzyme Q10 to obtain said composition, wherein dihydrooxyresveratrol weight percentage in said composition is in the range of 0.001% - 0.003%, Coenzyme Q10 weight percentage in said composition is in the range of 0.0008% - 0.002%; and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1. [0061] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; and (c) contacting dihydrooxyresveratrol and Coenzyme Q10 to obtain said composition, wherein dihydrooxyresveratrol weight percentage in said composition is in the range of 0.001% - 0.003%, Coenzyme Q10 weight percentage in said composition is in the range of 0.0008% - 0.002%; and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4.
[0062] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; and (c) contacting dihydrooxyresveratrol and Coenzyme Q10 to obtain said composition, wherein dihydrooxyresveratrol weight percentage in said composition is 0.0024%, Coenzyme Q10 weight percentage in said composition is 0.001%, and dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4.
[0063] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; (ii) Coenzyme Q10; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said

method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting dihydrooxyresveratrol, Coenzyme Q10, the at least one carrier, the at least one diluent and the at least one excipient, to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1.
[0064] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; (ii) Coenzyme Q10; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting dihydrooxyresveratrol, Coenzyme Q10, the at least one carrier, the at least one diluent and the at least one excipient, to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:0.5.
[0065] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; (ii) Coenzyme Q10; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting dihydrooxyresveratrol, Coenzyme Q10, the at least one carrier, the at least one diluent and the at least one excipient, to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is 1:0.4.
[0066] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol having a weight percentage in said composition in the range of 0.0001% - 0.003%; (ii) Coenzyme Q10 having a weight percentage in said composition in the range of 0.0002% -0.003%; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one

excipient, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting dihydrooxyresveratrol, Coenzyme Q10, the at least one carrier, the at least one diluent and the at least one excipient, to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1.
[0067] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol having a weight percentage in said composition in the range of 0.0001% - 0.003%; (ii) Coenzyme Q10 having a weight percentage in said composition in the range of 0.0002% -0.003%; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting dihydrooxyresveratrol, Coenzyme Q10, the at least one carrier, the at least one diluent and the at least one excipient, to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4. [0068] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol having a weight percentage in said composition in the range of 0.001% - 0.003%; (ii) Coenzyme Q10 having a weight percentage in said composition in the range of 0.0008% - 0.002%; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting dihydrooxyresveratrol, Coenzyme Q10, the at least one carrier, the at least one diluent and the at least one excipient, to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1.

[0069] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol having a weight percentage in said composition in the range of 0.001% - 0.003%; (ii) Coenzyme Q10 having a weight percentage in said composition in the range of 0.0008% - 0.002%; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting dihydrooxyresveratrol, Coenzyme Q10, the at least one carrier, the at least one diluent and the at least one excipient, to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4.
[0070] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol having a weight percentage in said composition of 0.0024%; (ii) Coenzyme Q10 having a weight percentage in said composition of 0.001%; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining dihydrooxyresveratrol; (b) obtaining Coenzyme Q10; (c) obtaining the at least one carrier; (d) obtaining the at least one diluent; (e) obtaining the at least one excipient; and (f) contacting dihydrooxyresveratrol, Coenzyme Q10, the at least one carrier, the at least one diluent and the at least one excipient, to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4. [0071] In an embodiment of the present disclosure, there is provided a method comprising: (a) obtaining a composition comprising: (i) dihydrooxyresveratrol, and (ii) Coenzyme Q10; and (b) contacting said composition with skin, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1 and said composition inhibits skin pigmentation.
[0072] In an embodiment of the present disclosure, there is provided a method comprising: (a) obtaining a composition comprising: (i) dihydrooxyresveratrol, and (ii) Coenzyme Q10; and (b) contacting said composition with skin, wherein

dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4 and said composition inhibits skin pigmentation.
[0073] In an embodiment of the present disclosure, there is provided a method comprising: (a) obtaining a composition comprising: (i) dihydrooxyresveratrol; (ii) Coenzyme Q10; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient; and (b) contacting said composition with skin wherein said composition inhibits skin pigmentation and wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is 1:0.2 – 1:1.
[0074] In an embodiment of the present disclosure, there is provided a method
comprising: (a) obtaining a composition comprising: (i) dihydrooxyresveratrol; (ii)
Coenzyme Q10; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one
excipient; and (b) contacting said composition with skin, wherein
dihydrooxyresveratrol to Coenzyme Q10 w/w ratio is 1:0.4 and said composition inhibits skin pigmentation.
[0075] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol; (b) obtaining a solution B of Coenzyme Q10; and (c) contacting solution A and solution B to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1.
[0076] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol; (b) obtaining a solution B of Coenzyme Q10; and (c) contacting solution A and solution B to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:0.5.
[0077] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol;

(b) obtaining a solution B of Coenzyme Q10; and (c) contacting solution A and solution B to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4.
[0078] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (i) dihydrooxyresveratrol having a weight
percentage in said composition in the range of 0.0001% - 0.003%; and (ii) Coenzyme
Q10 having a weight percentage in said composition in the range of 0.0002% -
0.003%, said method comprising: (a) obtaining a solution A of
dihydrooxyresveratrol; (b) obtaining a solution B of Coenzyme Q10; and (c) contacting solution A and solution B to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1.
[0079] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (i) dihydrooxyresveratrol having a weight
percentage in said composition in the range of 0.0001% - 0.003%; and (ii) Coenzyme
Q10 having a weight percentage in said composition in the range of 0.0002% -
0.003%, said method comprising: (a) obtaining a solution A of
dihydrooxyresveratrol; (b) obtaining a solution B of Coenzyme Q10; and (c) contacting solution A and solution B to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4. [0080] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol; (b) obtaining a solution B of Coenzyme Q10; and (c) contacting solution A and solution B to obtain said composition, wherein said solution A is prepared in organic solvent.
[0081] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme Q10, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol;

(b) obtaining a solution B of Coenzyme Q10; and (c) contacting solution A and
solution B to obtain said composition, wherein said solution A is prepared in
inorganic solvent.
[0082] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme
Q10, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol;
(b) obtaining a solution B of Coenzyme Q10; and (c) contacting solution A and
solution B to obtain said composition, wherein said solution B is prepared in organic
solvent.
[0083] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme
Q10, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol;
(b) obtaining a solution B of Coenzyme Q10; and (c) contacting solution A and
solution B to obtain said composition, wherein said solution B is prepared in
inorganic solvent.
[0084] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme
Q10, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol;
(b) obtaining a solution B of Coenzyme Q10; and (c) contacting solution A and
solution B to obtain said composition, wherein said solution A is prepared in DMSO
(dimethyl sulfoxide).
[0085] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (i) dihydrooxyresveratrol; and (ii) Coenzyme
Q10, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol;
(b) obtaining a solution B of Coenzyme Q10; and (c) contacting solution A and
solution B to obtain said composition, wherein said solution B is prepared in DMSO
(dimethyl sulfoxide).
[0086] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (i) dihydrooxyresveratrol; (ii) Coenzyme Q10;

(iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol; (b) obtaining a solution B of Coenzyme Q10; (c) obtaining a solution C of carriers, diluents and excipients; and (d) contacting solution A, solution B, solution C to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1.
[0087] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; (ii) Coenzyme Q10; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol; (b) obtaining a solution B of Coenzyme Q10; (c) obtaining a solution C of carriers, diluents and excipients; and (d) contacting solution A, solution B, solution C to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2- 1:0.5.
[0088] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol; (ii) Coenzyme Q10; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol; (b) obtaining a solution B of Coenzyme Q10; (c) obtaining a solution C of carriers, diluents and excipients; and (d) contacting solution A, solution B, solution C to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4.
[0089] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol having a weight percentage in said composition in the range of 0.0001% - 0.003%; (ii) Coenzyme Q10 having a weight percentage in said composition in the range of 0.0002% -0.003%; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol; (b) obtaining a solution B of Coenzyme Q10; (c) obtaining a

solution C of carriers, diluents and excipients; and (d) contacting solution A, solution B, solution C to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is in the range of 1:0.2 – 1:1. [0090] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol having a weight percentage in said composition in the range of 0.0001% - 0.003%; (b) Coenzyme Q10 having a weight percentage in said composition in the range of 0.0002% - 0.003%; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol; (b) obtaining a solution B of Coenzyme Q10; (c) obtaining a solution C of carriers, diluents and excipients; and (d) contacting solution A, solution B, solution C to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4.
[0091] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) dihydrooxyresveratrol having a weight percentage in said composition of 0.0024%, (ii) Coenzyme Q10 having a weight percentage in said composition of 0.001%; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining a solution A of dihydrooxyresveratrol; (b) obtaining a solution B of Coenzyme Q10; (c) obtaining a solution C of carriers, diluents and excipients; and (d) contacting solution A, solution B, solution C to obtain said composition, wherein dihydrooxyresveratrol to Coenzyme Q10 w/w ratio in said composition is 1:0.4.
[0092] In an embodiment of the present disclosure, there is provided a composition described herein, wherein said composition reduces skin pigmentation.
[0093] In an embodiment of the present disclosure, there is provided a composition described herein, wherein said composition reduces skin pigmentation by inhibiting expression of CDC42 protein.
[0094] In an embodiment of the present disclosure, there is provided a composition described herein, wherein said composition can be used for topical application.

[0095] In an embodiment of the present disclosure, there is provided a composition
described herein, wherein said composition can be in the form of aerosol.
[0096] In an embodiment of the present disclosure, there is provided a composition
described herein, wherein said composition can be in the form of a lotion.
[0097] In an embodiment of the present disclosure, there is provided a composition
described herein, wherein said composition can be in the form of an oil.
[0098] In an embodiment of the present disclosure, there is provided a composition
described herein, wherein said composition can be in the form of a serum.
[0099] Although the subject matter has been described with reference to specific
embodiments, this description is not meant to be construed in a limiting sense.
Various modifications of the disclosed embodiments, as well as alternate
embodiments of the subject matter, will become apparent to persons skilled in the art
upon reference to the description of the subject matter. It is therefore contemplated
that such modifications can be made without departing from the spirit or scope of the
present subject matter as defined.
EXAMPLES
[00100] The disclosure will now be illustrated with working examples, which
is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1:
Material and Methods
[00101] The dihydrooxyresveratrol used for the experiments was procured
from Sami Labs, India and the subsequent stocks were prepared in DMSO. The
Coenzyme Q10 and epigallocatechin gallate were purchased from Sigma.
Oxyresveratrol was purchased from Sami Labs, India and its stock was prepared in
DMSO. Human keratinocyte cells (HaCaT cells) were procured from ATCC, India.
The DMEM media used for the maintenance of cell lines was purchased from Gibco,
USA. The Cellular Viability kit was purchased from Invitrogen, India.
[00102] Cell culture conditions: Cell culture experiments involved protocols
for 5 days. On day 1, HaCaT cells were seeded in T-25 flask and incubated with 5%
CO2 at 37°C. On day2, HaCaT cells were exposed to 5µM αMSH for 72 hours, the
actives were treated simultaneously with αMSH induction. αMSH is a melanin
stimulating hormone, it is responsible for pigmentation in the skin. On Day 5, the
cells were lysed and processed for proteomics analysis as per the protocol.
[00103] Cell viability assay: HaCaT cells were cultured in DMEM medium.
For conducting the experiments, 90% confluent cells were trypsinised and suspended in DMEM at 2X105 cells/ml. Cell viability assay were carried out in 96-well plates (2X104 cells/well). After 24 hours, the cells were treated with four different concentrations of dihydrooxyresveratrol, Coenzyme Q10, oxyresveratrol, rapamycin and epigallocatechin gallate respectively and incubated for 72 hours. The media was then removed and 20µl of 5mg/ml solution of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was added to each well. The formazan crystals were solubilized following addition of 100µl of DMSO and absorbance was read at 540nm.
[00104] Proteomic analysis: The HaCaT cells were treated with
dihydrooxyresveratrol (0.002%), oxyresveratrol (0.002%), rapamycin (0.002%) and αMSH (5µM). For this analysis, 30µg of total protein of each sample (untreated and treated HaCaT cells) was considered for in-solution digestion method. The peptide

mixtures were separated using a reverse-phase column nano-high performance liquid
chromatography (HPLC) using pico-fit columns (Thermo) (0.075mmX120mm, C18).
The peptides were eluted using a gradient 5 to 90% acetonitrile (95%) at a flow rate
of 350nl/min for 60 min. A linear trap quadrapole (LTQ) orbitrap velos mass
spectrometer equipped with nano-electron source ionization (ESI) source was used to
acquire spectra. A 75µm metal emitter spray tip was used and the spray voltage was
set at 2 kV. The instrument was operated in data-dependent mode with dynamic
exclusion enabled. The tandem mass spectrometry (MS/MS) spectra on the top 20
most abundant peptide ions in full MS scan were obtained. The normalized collision-
induced dissociation (CID) was set at 35% for MS/MS. All MS/MS spectra were
searched against the human protein database from Uniprot using SEQUEST
algorithm incorporated in proteome discoverer 1.4(using trypsin as cleavage enzyme).
[00105] In-Solution digestion method: This protocol pertains to the digestion
of proteins in the solution. For digestion, 30µg of cell protein extract was resuspended in 47µl of 6M urea, 50mM Ammonium bicarbonate solution, 1µl of 250mM TCEP (tris [2 carboxyethyl phosphine]) was added in solution and the sample was incubated at 37°C for 30 min. Further, 2µl of 625mM Iodoacetamide was added and the sample was incubated in dark at room temperature for 45 min. Finally, in-solution digestion was carried by addition of 450µl trypsin in 50mM Ammonium bicarbonate with protein digest ratio of 50:1. The digested peptides were purified with OASIS 1 ml cartridge system from Waters Scientific (WAT094225).
Example 2:
Cytotoxicity assays for dihydrooxyresveratrol, Coenzyme Q10, oxyresveratrol, rapamycin and epigallocatechin gallate
[00106] Cytotoxicity assays were performed with HaCaT cells and above
mentioned actives for assessing the viability of the cells and hence deciding the working concentrations of the compounds.

[00107] The HaCaT cells did not show any significant reduction of viability
over the concentration of dihydrooxyresveratrol ranging from 0.000615% to 0.0048%
(Figure 1).
[00108] On performing viability tests with Coenzyme Q10, the HaCaT cells
did not show any significant reduction in viability. The concentrations tested for
Coenzyme Q10 ranged from 0.001% to 0.008% (Figure 2).
[00109] Various concentrations of oxyresveratrol ranging from 0.0006% to
0.0048% were tested on HaCaT cell line. The cells did not show any significant cell
death even at the highest concentration of 0.0048% (Figure 3).
[00110] HaCaT cells were treated with rapamycin with the concentrations
ranging from 0.002% to 0.016%. No significant reduction in the cell viability was
observed with rapamycin (Figure 4).
[00111] On treating the HaCaT cells with different concentrations of
epigallocatechin gallate (0.0006% - 0.0048%), no significant reduction in the viability
of HaCaT cells was observed (Figure 5).
[00112] This experiment shows that none of the working concentrations
belonging to this range of the said actives affects the cell viability of HaCaT cells.
Therefore, the difference in the protein expression can only be because of the effects
of different actives involved and not because of reduction in number of viable cells.
Example 3:
Synergistic effects of dihydrooxyresveratrol and Coenzyme Q10 on the expression of
CDC42 protein
[00113] Figure 6 demonstrates the effect of different actives on inhibiting
proteins involved in modulating melanosome transport functions. The protein levels
presented are quantified using a proteomic score derived from the proteome software
specified above. The ability of the said proteins to suppress or increase melanogenesis
is related to its activity on filopodia formation. Thus, if the actives are able to
significantly reduce the levels of alpha-msh induced CDC42 protein, they are

potential candidates use for the reduction of skin pigmentation through suppression of
CDC42 protein expression.
[00114] Among the actives tested, only dihydrooxyresveratrol (at a
concentration of 0.0024%) and Co Q10 (at a concentration of 0.001%), were able to
moderately reduce the levels of alpha-msh induced CDC42 protein expression.
Dihydrooxyresveratrol had a proteomics score of close to 12 which represents a 40%
inhibition compared to cells treated with alpha-msh alone.
[00115] Dihydrooxyresveratrol and Co Q10 were then tried in combination.
Remarkably, the combination of dihydrooxyresveratrol and Co Q10 treatment on
cells showed a dramatic reduction in CDC42 protein levels at approximately 75%
reduction as compared to cells treated only with alpha-msh. This combination
therefore shows a surprising synergistic effect as the levels are approximately 2-fold
lower than the actives used alone.
[00116] This effect cannot be obtained by the mere substitution of one of the
components with another active. As seen in Figure 7, the combination of
dihydrooxyresveratrol and epigallocatechin gallate - a polyphenol with known
antioxidant and UV -protectant properties- did not show any synergism in the
inhibition of melanocyte production, even though the compounds were used at the
same concentration as the synergistic composition (0.0024% dihydrooxyresveratrol
and 0.001% epigallocatechin gallate).
[00117] Epigallocatechin gallate (0.001%) is capable of inhibiting CDC42
protein expression. However, in combination with dihydrooxyresveratrol, no
synergistic effect can be observed in inhibition of CDC42 expression.
Example 4:
Representative base peaks resulting from various HaCaT cell treatments
[00118] The HaCaT cells were treated with various actives followed by in-
solution digestion method. They were further subjected to proteomic analysis according to the said protocol.

[00119] Figure 8 – 11 shows the base peak chromatogram pattern for various
samples. The abundance in number of peaks represents the quality of sample fractionation occurring during the LC-MS/MS (Liquid Chromatography coupled Mass Spectrometry) run. Higher the number of peaks, higher the number of proteins identified using proteome discoverer software.
[00120] Advantages of the present disclosure: Overall, the present subject
matter relates to a composition comprising dihydrooxyresveratrol and Coenzyme Q10, which at particular ratios and concentrations is able to repress key proteins in the melanosome transport pathway. At a 1:0.4 ratio of dihydrooxyresveratrol to Coenzyme Q10, this composition shows a surprisingly enhanced inhibition of alpha-msh induced CDC42 protein expression, which is approximately 14% more than when the actives are used in isolation and is a 75% reduction compared to a treatment with alpha-msh alone. Thus, this composition demonstrates distinct synergism in the inhibition of melanosome transport proteins and further, cannot be obtained without undue experimentation as a substitution of the ingredients with other melanogenesis suppressors does not yield the same result. The present invention is thus not obvious to a person skilled in the art and has potential applications in formulations to inhibit skin pigmentation.