FIELD OF INVENTION
[001] The present disclosure relates to the field of cosmetic formulations in general and skin-aging compositions in particular. There is provided a composition comprising L-citrulline and at least one tetrapeptide to inhibit skin aging.
5 BACKGROUND OF THE INVENTION
[002] Collagen is a key structural component of skin which provides support and forms the architecture of skin. It is a protein which occurs in the form of long fibrils, and is found abundantly in fibrous tissues such as tendons, ligaments and skin. One of the key mechanisms that causes skin aging is degradation of collagen. Effective
10 strategies to stop the gradual reduction in collagen levels is either by increasing
collagen levels, or by targeting those mechanisms which degrade collagen. [003] The primary causative factor in collagen degradation is the activity of a family of proteases known as matrix metalloproteinases (MMPs). The key MMPs that cause severe damage to collagen and elastin include Matrix Metalloproteinases-1 (MMP-1),
15 MMP-3 and MMP-9. Studies have linked the activity of MMP-1 directly to the
susceptibility of accelerating the skin aging process. The degradation of collagen fibrils has been attributed to increased activity of MMP-1 in aged skin compared to young skin as observed in in vivo studies. It is well known that UV irradiation i.e. photoaging (Quan et al., J. Inv. Dermatology Symposium Proceedings,2009, 14, 20;
20 Fagot D et al., Arch Dermatol Res,2002,293:576) and, heat (J. of Inv. Dermatology,
2004, 123, 6, 1012; Brenneisen et al., Biochem J., 2002, 1; 365(Pt 1): 31) induce MMPs, specifically MMP-1. Well established anti-aging actives have been shown to prevent collagen degradation by suppression of MMP-1 levels and/ or activity (Oh Hi et al., Phytother. Res., 2009, 23(9):1299), however, there are very few reports of such
25 actives in prior art that have targeted these pathways. Hence, there is a need to

identify the actives or active combinations with certain features such as small size and demonstrating synergy in terms decreasing deleterious MMP-I levels.
SUMMARY OF THE INVENTION
[004] In an aspect of the present disclosure, there is provided a composition 5 comprising: (a) L-citrulline; and (b) at least one tetrapeptide, wherein L-citrulline to at least one tetrapeptide w/w ratio in said composition is in the range of 1:0.00013-1:0.00018.
[005] In an aspect of the present disclosure, there is provided a method of preparing a composition comprising: (a) L-citrulline; and (b) at least one tetrapeptide, said
10 method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; and (c) contacting L-citrulline, and the at least one tetrapeptide to obtain said composition, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018. [006] In an aspect of the present disclosure, there is provided a method of inhibiting
15 skin aging comprising: (a) L-citrulline; and (b) at least one tetrapeptide, said method comprising: (a) obtaining the at least one tetrapeptide; (b) obtaining L-citrulline; and (c) contacting said composition with skin, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018, and said composition inhibits skin aging.
20 [007] 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
25 subject matter.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[008] 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
5 description of the specific embodiments presented herein.
[009] Figure 1 shows the effect of the combination of L-citrulline and the tetrapeptide on the inhibition of Matrix Metalloproteinase 1 (MMP1) protein levels, in accordance with an embodiment of the present disclosure.
[0010] Figure 2 shows the effect of the combination of adenosine triphosphate (ATP)
10 and the tetrapeptide on the inhibition of Matrix Metalloproteinase 1 (MMP1) protein
levels, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0011] 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
15 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
20 [0012] 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
25 completeness, particular terms and their meanings are set forth below.

[0013] 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.
[0014] 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 5 as “consists of only”.
[0015] 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.
10 [0016] Carriers are substances that serve as mechanisms to improve the delivery and
the effectiveness of drugs.
[0017] A diluent (also referred to as filler, dilutant, or thinner) is a diluting agent.
[0018] 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, 15 preservatives, emollients, and combinations thereof.
[0019] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
[0020] 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 20 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.
[0021] Skin aging is a multi-factorial process and is visible in the form of fine lines
25 and wrinkles. One of the primary reasons for this loss in elasticity in skin is due to the
degradation of collagen. Collagen degradation occurs during the due course of aging
and one of the enzymes controlling the degradation of this protein is Matrix
Metalloproteinases 1 (MMP1). One of the methods, therefore, to attempt to delay the
process of skin aging is by targeting the activity of this enzyme. The present
5

disclosure has adopted this strategy to tackle the problem of skin aging by providing a
solution in the form of a composition comprising the amino acid- L-citrulline, and a
tetrapeptide, which at specific ratios can inhibit the activity of MMP1, thereby
subsequently reducing collagen degradation.
5 [0022] 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.
[0023] In an embodiment of the present disclosure, there is provided a composition
10 comprising: (a) L-citrulline; and (b) at least one tetrapeptide, wherein L-citrulline to
the at least one tetrapeptide w/w ratio in said composition is in the range of 1:0.00013- 1:0.00018.
[0024] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) L-citrulline; and (b) at least one tetrapeptide having the sequence Ile-
15 Glu-Pro-Asp in said composition, wherein L-citrulline to the at least one tetrapeptide
w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018.
[0025] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) L-citrulline; and (b) at least one tetrapeptide, wherein L-citrulline to
the at least one tetrapeptide w/w ratio in said composition is 1: 0.00016.
20 [0026] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) L-citrulline; and (b) at least one tetrapeptide, wherein L-citrulline
weight percentage in said composition is in the range of 0.02%- 0.5%, the at least one
tetrapeptide weight percentage in said composition is in the range of 0.00001%-
0.0001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said
25 composition is in the range of 1: 0.00013- 1:0.00018.
[0027] In an embodiment of the present disclosure, there is provided a composition comprising: (a) L-citrulline; and (b) at least one tetrapeptide having the sequence Ile-Glu-Pro-Asp in said composition, wherein L-citrulline weight percentage in said
composition is in the range of 0.02%- 0.5%, the at least one tetrapeptide weight
6

percentage in said composition is in the range of 0.00001%- 0.0001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018.
[0028] In an embodiment of the present disclosure, there is provided a composition
5 comprising: (a) L-citrulline; and (b) at least one tetrapeptide, wherein L-citrulline
weight percentage in said composition is in the range of 0.02%- 0.5%, the at least one
tetrapeptide weight percentage in said composition is in the range of 0.00001%-
0.0001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition
is 1: 0.00016.
10 [0029] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) L-citrulline; and (b) at least one tetrapeptide, wherein L-citrulline weight percentage in said composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%-0.00005%, and L-citrulline to the at least one tetrapeptide w/w ratio in said 15 composition is in the range of 1: 0.00013- 1:0.00018.
[0030] In an embodiment of the present disclosure, there is provided a composition comprising: (a) L-citrulline; and (b) at least one tetrapeptide, wherein L-citrulline weight percentage in said composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%-20 0.00005%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1: 0.00016.
[0031] In an embodiment of the present disclosure, there is provided a composition comprising: (a) L-citrulline; and (b) at least one tetrapeptide, wherein L-citrulline weight percentage in said composition is 0.25%, the at least one tetrapeptide weight 25 percentage in said composition is 0.00004%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
[0032] In an embodiment of the present disclosure, there is provided a composition comprising: (a) L-citrulline; and (b) at least one tetrapeptide, wherein L-citrulline
weight percentage in said composition is 0.125%, the at least one tetrapeptide weight
7

percentage in said composition is 0.00002%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
[0033] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) L-citrulline; and (b) at least one tetrapeptide, wherein L-citrulline
5 weight percentage in said composition is 0.625%, the at least one tetrapeptide weight
percentage in said composition is 0.00001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
[0034] In an embodiment of the present disclosure, there is provided a composition comprising: (a) L-citrulline; (b) at least one tetrapeptide; (c) at least one carrier; (d) at
10 least one diluent; and (e) at least one excipient, wherein L-citrulline to the at least one
tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018. [0035] In an embodiment of the present disclosure, there is provided a composition comprising: (a) L-citrulline; (b) at least one tetrapeptide having the sequence Ile-Glu-Pro-Asp in said composition; (c) at least one carrier; (d) at least one diluent; and (e) at
15 least one excipient, wherein L-citrulline to the at least one tetrapeptide w/w ratio in
said composition is in the range of 1: 0.00013- 1:0.00018.
[0036] In an embodiment of the present disclosure, there is provided a composition comprising: (a) L-citrulline; (b) at least one tetrapeptide; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein L-citrulline to the at least one
20 tetrapeptide w/w ratio in said composition 1: 0.00016.
[0037] In an embodiment of the present disclosure, there is provided a composition comprising: (a) L-citrulline; (b) at least one tetrapeptide; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein L-citrulline weight percentage in said composition is in the range of 0.02%- 0.5%, the at least one
25 tetrapeptide weight percentage in said composition is in the range of 0.00001%-
0.0001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018.
[0038] In an embodiment of the present disclosure, there is provided a composition comprising: (a) L-citrulline; (b) at least one tetrapeptide having the sequence of Ile-

Glu-Pro-Asp in said composition; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein L-citrulline weight percentage in said composition is in the range of 0.02%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.0001%, and L-citrulline to the at least one 5 tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018.
[0039] In an embodiment of the present disclosure, there is provided a composition comprising: (a) L-citrulline; (b) at least one tetrapeptide; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein L-citrulline w/w percentage in said composition is in the range of 0.02%- 0.5%, the at least one tetrapeptide weight
10 percentage in said composition is in the range of 0.00001%- 0.0001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition 1: 0.00016. [0040] In an embodiment of the present disclosure, there is provided a composition comprising: (a) L-citrulline; (b) at least one tetrapeptide; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein L-citrulline weight percentage
15 in said composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.00005%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018. [0041] In an embodiment of the present disclosure, there is provided a composition
20 comprising: (a) L-citrulline; (b) at least one tetrapeptide; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein L-citrulline weight percentage in said composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.00005%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition 1: 0.00016.
25 [0042] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) L-citrulline; (b) at least one tetrapeptide; (c) at least one carrier; (d) at least one diluent; and (e) at least one excipient, wherein L-citrulline weight percentage in said composition is 0.25%, the at least one tetrapeptide weight percentage in said

composition is 0.00004%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
[0043] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) L-citrulline; (b) at least one tetrapeptide; (c) at least one carrier; (d) at
5 least one diluent; and (e) at least one excipient, wherein L-citrulline weight
percentage in said composition is 0.125%, the at least one tetrapeptide weight percentage in said composition is 0.00002%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016. [0044] In an embodiment of the present disclosure, there is provided a composition
10 comprising: (a) L-citrulline; (b) at least one tetrapeptide; (c) at least one carrier; (d) at
least one diluent; and (e) at least one excipient, wherein L-citrulline weight percentage in said composition is 0.625%, the at least one tetrapeptide weight percentage in said composition is 0.00001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
15 [0045] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; and (c) contacting L-citrulline, and the at least one tetrapeptide to obtain said composition, wherein L-citrulline to the at least one tetrapeptide w/w ratio in
20 said composition is in the range of 1: 0.00013- 1:0.00018.
[0046] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide having the sequence of Ile-Glu-Pro-Asp in said composition; and (c)
25 contacting L-citrulline, and the at least one tetrapeptide to obtain said composition,
wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018.
[0047] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide,

said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one
tetrapeptide; and (c) contacting L-citrulline, and the at least one tetrapeptide to obtain
said composition, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said
composition is 1:0.00016.
5 [0048] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; and (c) contacting L-citrulline, and the at least one tetrapeptide to obtain said composition, wherein L-citrulline weight percentage in said composition is in the
10 range of 0.02%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.0001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013-1:0.00018. [0049] In an embodiment of the present disclosure, there is provided a method of
15 preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide having the sequence of Ile-Glu-Pro-Asp in said composition, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; and (c) contacting L-citrulline, and the at least one tetrapeptide to obtain said composition, wherein L-citrulline weight percentage in said composition is in the range of 0.02%- 0.5%, the at
20 least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.0001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018.
[0050] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide,
25 said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; and (c) contacting L-citrulline, and the at least one tetrapeptide to obtain said composition, wherein L-citrulline weight percentage in said composition is in the range of 0.02%- 0.5%, the at least one tetrapeptide weight percentage in said

composition is in the range of 0.00001%- 0.0001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
[0051] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; and (c) contacting L-citrulline, and the at least one tetrapeptide to obtain said composition, wherein L-citrulline weight percentage in said composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.00005%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013-1:0.00018.
[0052] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; and (c) contacting L-citrulline, and the at least one tetrapeptide to obtain said composition, wherein L-citrulline weight percentage in said composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.00005%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
[0053] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; and (c) contacting L-citrulline, and the at least one tetrapeptide to obtain said composition, wherein L-citrulline weight percentage in said composition is 0.25%, the at least one tetrapeptide weight percentage in said composition is 0.00004%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016. [0054] In an embodiment of the present disclosure, there is provided a method of
preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide,
12

said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; and (c) contacting L-citrulline, and the at least one tetrapeptide to obtain said composition, wherein L-citrulline weight percentage in said composition is 0.125%, the at least one tetrapeptide weight percentage in said composition is 5 0.00002%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
[0055] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one
10 tetrapeptide; and (c) contacting L-citrulline, and the at least one tetrapeptide to obtain said composition, wherein L-citrulline weight percentage in said composition is 0.625%, the at least one tetrapeptide weight percentage in said composition is 0.00001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
15 [0056] In an embodiment of the present disclosure there is provided a method of
preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining a solution A of L-citrulline; (b) obtaining a solution B of the at least one tetrapeptide; and (c) contacting said solution A, and said solution B to obtain said composition, wherein L-citrulline to the at least one
20 tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018.
[0057] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining a solution A of L-citrulline; (b) obtaining a solution B of the at least one tetrapeptide; and (c) contacting said solution A, and said
25 solution B, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said
composition is 1:0.00016.
[0058] In an embodiment of the present disclosure there is provided a method of
preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide,
said method comprising: (a) obtaining a solution A of L-citrulline; (b) obtaining a
13

solution B of at least one tetrapeptide; and (c) contacting said solution A, and said
solution B, wherein L-citrulline weight percentage in said composition is in the range
of 0.02%- 0.5%, the at least one tetrapeptide weight percentage in said composition is
in the range of 0.00001%- 0.0001%, and L-citrulline to the at least one tetrapeptide
5 w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018.
[0059] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining a solution A of L-citrulline; (b) obtaining a solution B of at least one tetrapeptide; and (c) contacting said solution A, and said
10 solution B, wherein L-citrulline weight percentage in said composition is in the range
of 0.02%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.0001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016. [0060] In an embodiment of the present disclosure there is provided a method of
15 preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide,
said method comprising: (a) obtaining a solution A of L-citrulline; (b) obtaining a solution B of at least one tetrapeptide; and (c) contacting said solution A, and said solution B, wherein L-citrulline weight percentage in said composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said composition is
20 in the range of 0.00001%- 0.00005%, and L-citrulline to the at least one tetrapeptide
w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018. [0061] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining a solution A of L-citrulline; (b) obtaining a
25 solution B of at least one tetrapeptide; and (c) contacting said solution A, and said
solution B, wherein L-citrulline weight percentage in said composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.00005%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.

[0062] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining a solution A of L-citrulline; (b) obtaining a solution B of the at least one tetrapeptide; and (c) contacting said solution A, and said 5 solution B, wherein said solution A is prepared in an organic solvent.
[0063] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining a solution A of L-citrulline; (b) obtaining a solution B of the at least one tetrapeptide; and (c) contacting said solution A, and said 10 solution B, wherein said solution A is prepared in an inorganic solvent.
[0064] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining a solution A of L-citrulline; (b) obtaining a solution B of the at least one tetrapeptide; and (c) contacting said solution A, and said 15 solution B, wherein said solution B is prepared in an organic solvent.
[0065] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide, said method comprising: (a) obtaining a solution A of L-citrulline; (b) obtaining a solution B of the at least one tetrapeptide; and (c) contacting said solution A, and said 20 solution B, wherein said solution B is prepared in an inorganic solvent.
[0066] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; (c) 25 obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting L-citrulline, the at least one tetrapeptide, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018.

[0067] In an embodiment of the present disclosure there is provided a method of
preparing a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide
having the sequence of Ile-Glu-Pro-Asp in said composition; (iii) at least one carrier;
(iv) at least one diluent; and (v) at least one excipient, said method comprising: (a)
5 obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; (c) obtaining the at
least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting L-citrulline, the at least one tetrapeptide, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range
10 of 1: 0.00013- 1:0.00018.
[0068] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one
15 tetrapeptide; (c) obtaining the at least one carrier, the at least one diluent, and the at
least one excipient; and (d) contacting L-citrulline, the at least one tetrapeptide, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
20 [0069] In an embodiment of the present disclosure there is provided a method of
preparing a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; (c) obtaining the at least one carrier, the at least one diluent, and the at
25 least one excipient; and (d) contacting L-citrulline, the at least one tetrapeptide, the at
least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein L-citrulline weight percentage in said composition is in the range of 0.02%- 0.5%, the at least one tetrapeptide weight percentage in said

composition is in the range of 0.00001%- 0.0001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018. [0070] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide 5 having the sequence of Ile-Glu-Pro-Asp in said composition; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; (c) obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting L-citrulline, the at least one tetrapeptide, the at least one carrier, the at least
10 one diluent, and the at least one excipient to obtain said composition, wherein L-citrulline weight percentage in said composition is in the range of 0.02%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.0001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018.
15 [0071] In an embodiment of the present disclosure there is provided a method of
preparing a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; (c) obtaining the at least one carrier, the at least one diluent, and the at least one
20 excipient; and (d) contacting L-citrulline, the at least one tetrapeptide, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein L-citrulline weight percentage in said composition is in the range of 0.02%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.0001%, and L-citrulline to the at least one
25 tetrapeptide w/w ratio in said composition is 1:0.00016.
[0072] In an embodiment of the present disclosure there is provided a method of
preparing a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide; (iii)
at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method
comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; (c)
17

obtaining the at least one carrier, the at least one diluent, and the at least one
excipient; and (d) contacting L-citrulline, the at least one tetrapeptide, the at least one
carrier, the at least one diluent, and the at least one excipient to obtain said
composition, wherein L-citrulline weight percentage in said composition is in the
5 range of 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said
composition is in the range of 0.00001%- 0.00005%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013-1:0.00018. [0073] In an embodiment of the present disclosure there is provided a method of
10 preparing a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide; (iii)
at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; (c) obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting L-citrulline, the at least one tetrapeptide, the at
15 least one carrier, the at least one diluent, and the at least one excipient to obtain said
composition, wherein L-citrulline weight percentage in said composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.00005%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
20 [0074] In an embodiment of the present disclosure there is provided a method of
preparing a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; (c) obtaining the at least one carrier, the at least one diluent, and the at
25 least one excipient; and (d) contacting L-citrulline, the at least one tetrapeptide, the at
least one carrier, the at least one diluent, and the at least one excipient to obtain said
composition, wherein L-citrulline weight percentage in said composition is 0.25%,
the at least one tetrapeptide weight percentage in said composition is 0.00004%, and
L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
18

[0075] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; (c) 5 obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting L-citrulline, the at least one tetrapeptide, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein L-citrulline weight percentage in said composition is in the range of 0.125%, the at least one tetrapeptide weight percentage in said composition is
10 in the range of 0.00002%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
[0076] In an embodiment of the present disclosure there is provided a method of preparing a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient, said method
15 comprising: (a) obtaining L-citrulline; (b) obtaining the at least one tetrapeptide; (c) obtaining the at least one carrier, the at least one diluent, and the at least one excipient; and (d) contacting L-citrulline, the at least one tetrapeptide, the at least one carrier, the at least one diluent, and the at least one excipient to obtain said composition, wherein L-citrulline weight percentage in said composition is in the
20 range of 0.625%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%, and L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
[0077] In an embodiment of the present disclosure, there is provided a composition for use in preparing formulations, said composition comprising: (a) L-citrulline; and
25 (b) at least one tetrapeptide, wherein L-citrulline to the at least one tetrapeptide w/w
ratio in said composition is in the range of 1: 0.00013- 1:0.00018.
[0078] In an embodiment of the present disclosure, there is provided a composition
for use in preparing formulations, said composition comprising: (a) L-citrulline; and
(b) at least one tetrapeptide having the sequence of Ile-Glu-Pro-Asp in said
19

composition, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018.
[0079]
[0080] In an embodiment of the present disclosure, there is provided a composition
5 for use in preparing formulations, said composition comprising: (a) L-citrulline; and
(b) at least one tetrapeptide, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
[0081] In an embodiment of the present disclosure, there is provided a composition
for use in preparing formulations, said composition comprising: (a) L-citrulline; and
10 (b) at least one tetrapeptide, wherein L-citrulline weight percentage in said
composition is in the range of 0.02%- 0.5%, the at least one tetrapeptide weight
percentage in said composition is in the range of 0.00001%- 0.0001%, and L-
citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range
of 1: 0.00013- 1:0.00018.
15 [0082] In an embodiment of the present disclosure, there is provided a composition
for use in preparing formulations, said composition comprising: (a) L-citrulline; and
(b) at least one tetrapeptide, wherein L-citrulline weight percentage in said
composition is in the range of 0.02%- 0.5%, the at least one tetrapeptide weight
percentage in said composition is in the range of 0.00001%- 0.0001%, and L-
20 citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
[0083] In an embodiment of the present disclosure, there is provided a composition
for use in preparing formulations, said composition comprising: (a) L-citrulline; and
(b) at least one tetrapeptide, wherein L-citrulline weight percentage in said
composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight
25 percentage in said composition is in the range of 0.00001%- 0.00005%, and L-
citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018. [0084] In an embodiment of the present disclosure, there is provided a composition
for use in preparing formulations, said composition comprising: (a) L-citrulline; and
20

(b) at least one tetrapeptide, wherein L-citrulline weight percentage in said
composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight
percentage in said composition is in the range of 0.00001%- 0.00005%, and L-
citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016.
5 [0085] In an embodiment of the present disclosure, there is provided a method of
inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide; and (b) contacting said composition with skin, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018, and said method inhibits skin aging.
10 [0086] In an embodiment of the present disclosure, there is provided a method of
inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide having the sequence of Ile-Glu-Pro-Asp in said composition; and (b) contacting said composition with skin, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1:
15 0.00013- 1:0.00018, and said method inhibits skin aging.
[0087] In an embodiment of the present disclosure, there is provided a method of inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide; and (b) contacting said composition with skin, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition
20 is 1:0.00016, and said method inhibits skin aging.
[0088] In an embodiment of the present disclosure, there is provided a method of inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide; and (b) contacting said composition with skin, wherein L-citrulline weight percentage in said composition is in the range of
25 0.02%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.0001%, L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018, and said method inhibits skin aging.

[0089] In an embodiment of the present disclosure, there is provided a method of
inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-
citrulline; and (ii) at least one tetrapeptide having the sequence of Ile-Glu-Pro-Asp in
said composition; and (b) contacting said composition with skin, wherein L-citrulline
5 weight percentage in said composition is in the range of 0.02%- 0.5%, the at least one
tetrapeptide weight percentage in said composition is in the range of 0.00001%-0.0001%, L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018, and said method inhibits skin aging. [0090] In an embodiment of the present disclosure, there is provided a method of
10 inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-
citrulline; and (ii) at least one tetrapeptide; and (b) contacting said composition with skin, wherein L-citrulline weight percentage in said composition is in the range of 0.02%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.0001%, L-citrulline to the at least one tetrapeptide w/w
15 ratio in said composition is 1:0.00016, and said method inhibits skin aging.
[0091] In an embodiment of the present disclosure, there is provided a method of inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide; and (b) contacting said composition with skin, wherein L-citrulline weight percentage in said composition is in the range of
20 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in
the range of 0.00001%- 0.00005%, L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013- 1:0.00018, and said method inhibits skin aging. [0092] In an embodiment of the present disclosure, there is provided a method of
25 inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-
citrulline; and (ii) at least one tetrapeptide; and (b) contacting said composition with skin, wherein L-citrulline weight percentage in said composition is in the range of 0.05%- 0.5%, the at least one tetrapeptide weight percentage in said composition is in

the range of 0.00001%- 0.00005%, L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016, and said method inhibits skin aging. [0093] In an embodiment of the present disclosure, there is provided a method of inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-5 citrulline; and (ii) at least one tetrapeptide; and (b) contacting said composition with skin, wherein L-citrulline weight percentage in said composition is 0.25%, the at least one tetrapeptide weight percentage in said composition is 0.00004%, L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016, and said method inhibits skin aging.
10 [0094] In an embodiment of the present disclosure, there is provided a method of
inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide; and (b) contacting said composition with skin, wherein L-citrulline weight percentage in said composition is 0.125%, the at least one tetrapeptide weight percentage in said composition is 0.00002%, L-citrulline
15 to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016, and said method inhibits skin aging.
[0095] In an embodiment of the present disclosure, there is provided a method of inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-citrulline; and (ii) at least one tetrapeptide; and (b) contacting said composition with
20 skin, wherein L-citrulline weight percentage in said composition is 0.625%, the at least one tetrapeptide weight percentage in said composition is 0.00001%, L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016, and said method inhibits skin aging. [0096] In an embodiment of the present disclosure, there is provided a method of
25 inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient; and (b) contacting said composition with skin, wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is in
the range of 1: 0.00013- 1:0.00018, and said method inhibits skin aging.
23

[0097] In an embodiment of the present disclosure, there is provided a method of
inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-
citrulline; (ii) at least one tetrapeptide having the sequence of Ile-Glu-Pro-Asp in said
composition; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one
5 excipient; and (b) contacting said composition with skin, wherein L-citrulline to the
at least one tetrapeptide w/w ratio in said composition is in the range of 1: 0.00013-1:0.00018, and said method inhibits skin aging.
[0098] In an embodiment of the present disclosure, there is provided a method of
inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-
10 citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one
diluent; and (v) at least one excipient; and (b) contacting said composition with skin,
wherein L-citrulline to the at least one tetrapeptide w/w ratio in said composition is
1:0.00016, and said method inhibits skin aging.
[0099] In an embodiment of the present disclosure, there is provided a method of
15 inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-
citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one
diluent; and (v) at least one excipient; and (b) contacting said composition with skin,
wherein L-citrulline weight percentage in said composition is in the range of 0.02%-
0.5%, the at least one tetrapeptide weight percentage in said composition is in the
20 range of 0.00001%- 0.0001%, L-citrulline to the at least one tetrapeptide w/w ratio in
said composition is in the range of 1: 0.00013- 1:0.00018, and said method inhibits skin aging.
[00100] In an embodiment of the present disclosure, there is provided a method
of inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-
25 citrulline; (ii) at least one tetrapeptide having the sequence of Ile-Glu-Pro-Asp in said
composition; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one
excipient; and (b) contacting said composition with skin, wherein L-citrulline weight
percentage in said composition is in the range of 0.02%- 0.5%, the at least one
tetrapeptide weight percentage in said composition is in the range of 0.00001%-24

0.0001%, L-citrulline to the at least one tetrapeptide w/w ratio in said composition is
in the range of 1: 0.00013- 1:0.00018, and said method inhibits skin aging.
[00101] In an embodiment of the present disclosure, there is provided a method
of inhibiting skin aging comprising: (a) obtaining a composition comprising(i) L-5 citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient; and (b) contacting said composition with skin, wherein L-citrulline weight percentage in said composition is in the range of 0.02%-0.5%, the at least one tetrapeptide weight percentage in said composition is in the range of 0.00001%- 0.0001%, L-citrulline to the at least one tetrapeptide w/w ratio in 10 said composition is 1:0.00016, and said method inhibits skin aging.
[00102] In an embodiment of the present disclosure, there is provided a method
of inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-
citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one
diluent; and (v) at least one excipient; and (b) contacting said composition with skin,
15 wherein L-citrulline weight percentage in said composition is in the range of 0.05%-
0.5%, the at least one tetrapeptide weight percentage in said composition is in the
range of 0.00001%- 0.00005%, L-citrulline to the at least one tetrapeptide w/w ratio in
said composition is in the range of 1: 0.00013- 1:0.00018, and said method inhibits
skin aging.
20 [00103] In an embodiment of the present disclosure, there is provided a method
of inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-
citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one
diluent; and (v) at least one excipient; and (b) contacting said composition with skin,
wherein L-citrulline weight percentage in said composition is in the range of 0.05%-
25 0.5%, the at least one tetrapeptide weight percentage in said composition is in the
range of 0.00001%- 0.00005%, L-citrulline to the at least one tetrapeptide w/w ratio in
said composition is 1:0.00016, and said method inhibits skin aging.
[00104] In an embodiment of the present disclosure, there is provided a method
of inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-25

citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one
diluent; and (v) at least one excipient; and (b) contacting said composition with skin,
wherein L-citrulline weight percentage in said composition is 0.25%, the at least one
tetrapeptide weight percentage in said composition is 0.00004%, L-citrulline to the at
5 least one tetrapeptide w/w ratio in said composition is 1:0.00016, and said method
inhibits skin aging.
[00105] In an embodiment of the present disclosure, there is provided a method
of inhibiting skin aging comprising: (a) obtaining a composition comprising: ((i) L-citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one
10 diluent; and (v) at least one excipient; and (b) contacting said composition with skin,
wherein L-citrulline weight percentage in said composition is 0.125%, the at least one tetrapeptide weight percentage in said composition is 0.00002%, L-citrulline to the at least one tetrapeptide w/w ratio in said composition is 1:0.00016, and said method inhibits skin aging.
15 [00106] In an embodiment of the present disclosure, there is provided a method
of inhibiting skin aging comprising: (a) obtaining a composition comprising: (i) L-citrulline; (ii) at least one tetrapeptide; (iii) at least one carrier; (iv) at least one diluent; and (v) at least one excipient; and (b) contacting said composition with skin, wherein L-citrulline weight percentage in said composition is 0.625%, the at least one
20 tetrapeptide weight percentage in said composition is 0.00001%, L-citrulline to the at
least one tetrapeptide w/w ratio in said composition is 1:0.00016, and said method inhibits skin aging.
[00107] In an embodiment of the present disclosure, there is provided a
composition as described herein, wherein said composition can be for topical
25 application.
[00108] In an embodiment of the present disclosure, there is provided a
composition as described herein, wherein said composition can be in the form of an aerosol.

[00109] In an embodiment of the present disclosure, there is provided a
composition as described herein, wherein said composition can be in the form of a lotion.
[00110] In an embodiment of the present disclosure, there is provided a
5 composition as described herein, wherein said composition can be in the form of a serum.
[00111] In an embodiment of the present disclosure, there is provided a
composition as described herein, wherein said composition can be in the form of an
oil.
10 [00112] 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 15 that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.
EXAMPLES
[00113] The disclosure will now be illustrated with working examples, which
is intended to illustrate the working of disclosure and not intended to take
20 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
25 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
[00114] The human dermal fibroblast, HDF-HaCaT [procured from National
Centre for Cell Science (NCSS)], Pune co-culture model was used for the study &
5 ELISA based assay method was adapted to measure Matrix Metalloproteinase-I
levels. The human MMP-1 ELISA assay kit was procured from Sigma (#RAB0361). The tetrapeptide used in this study was custom synthesized at USV Limited, Mumbai. The sequence of this peptide is Ile-Glu-Pro-Asp. The peptide was dissolved in solvent DMSO. The amino acid L-citrulline was commercially procured from Sigma-Aldrich
10 (CAS No. 372-75-8) and was dissolved in sterile H2O in this study. ATP was
commercially procured from Sigma-Aldrich (CAS No. 34369-07-8) and was dissolved in sterile H2O in this study.
[00115] HDF-HaCaT co-culture: Indian origin adult human dermal fibroblasts
(HDF) were cultured in Medium 106 containing growth factors [(LSGS Supplement,
15 Gibco #S-003-K)]. The media was replaced every alternate day till over 80%
confluency was attained. The cells were dislodged using 0.25% trypsin EDTA solution and sub-cultured by seeding 3500 cells/cm2 of the flask surface. The cultures were tested for the absence of Mycoplasma by using MycoAlert Mycoplasma Detection kit (LONZA).
20 [00116] For the cytotoxicity assay, the cells were seeded at a density of 8750
viable cells per well (100μL/well) in M106 (Gibco) medium containing growth factors [Low Serum Growth Supplement (LSGS) Kit (Gibco), fetal bovine serum, 2% v/v hydrocortisone (1 μg/ml), human epidermal growth factor, 10 ng/ml, basic fibroblast growth factor, 3 ng/ml, heparin, 10 μg/ml in 96 well cell culture plates].
25 After ~48 hr incubation, when the HDFa cells appeared to form a confluent
monolayer in the wells, the media was removed and 13750 HaCaT cells were layered
onto the HDFa matrix (100 μL/well) in M154 CF-PRF media containing 30 μM
CaCl2. The plates were further incubated overnight prior to treatment with the test
ingredients at the desired concentrations, along with appropriate vehicle and sterility
28

controls (wells with media only, without cells, respectively). The plates were
incubated in a humidified incubator at 37°(75% CO2 for 48 hrs, after which the media
from the plates was removed and replaced with 200uL of media, containing 10% (20
|iL) of Cell Titre Aqueous Solution (MTS Reagent). The plates were incubated at
5 37°C for a further 5-6 hr followed by reading at 490 nm using a Spectramax Reader.
[00117] Matrix Metalloproteinase-I (MMP-I) Assay: The ELISA based assays
were performed in triplicates and each experiment was repeated 3 times (n=3, N=3).
[00118] To culture the cells for the required experiments, cryopreserved vials
of HDFa and HaCaT were thawed in a 37°C water bath and cells were seeded into 75
10 cm2 Tissue Culture Flasks (Corning) in their respective growth media with 1X
antibiotics (Antibiotic-Antimycotic 100X from Gibco containing 10000 Units/ml Penicillin, 10000 ug/mL of Streptomycin and 25 ug/mL of Fungizone antimycotic). The flasks were incubated at 37°C in a humidified incubator with 5% CO2 environment. The media was changed every alternate day till the flasks achieved 75-
15 80% confluency. Upon attainment of confluency, cells were dislodged by
trypsinization, enumerated and cryovials were prepared at 1 million cells/vial/ml. The cryovials were preserved under the vapor phase of liquid nitrogen. Once the cell stocks were prepared, the cells were expanded for seeding in the main assay. HDF vials were cryopreserved at Passage 2 (P=2) and HaCaT cells were cryopreserved at
20 Passage 20 (P=20). HDF cells were seeded at 70,000 cells/well in 12-well cell
culture plates and incubated for 48 hr in M106 containing LSGS Growth Factors. Upon HaCaT addition (0.11 million/well), the media was changed to M154 CF-PRF containing 30 uM CaCl2 and the plates were incubated for 24 hr before the test compounds (in 1 ml media per well) were added at the concentrations mentioned. The
25 compound dilutions were prepared in M154 CF-PRF containing 30 uM CaCl2. The
treated and control cells were incubated for 48 hr at 37°C in a humidified incubator
with a 5% CO2 environment.
[00119] For MMP-1 assays, the culture supernatants were collected 48 hr post
incubation. The plates were centrifuged briefly (~ 5 min) at 300 g to remove any
29

cellular debris. 50 uL of the clarified culture supernatant was collected in 96-well U-
bottom polypropylene plates. The tubes and plates were stored at -80°C until they
were processed further. Samples were diluted 40-fold in 96-wells deep well block by
transferring 5ul of sample to 195uL of 1X assay diluent buffer supplied with the kit.
5 The contents were mixed thoroughly with a multichannel pipette to ensure uniformity
of the suspension.
[00120] Lyophilized Human MMP-1 Protein Standard vial supplied with the
kit was briefly centrifuged and 400 uL of 1X Assay Diluent was added and mixed to prepare a 0.1 ug/mL standard. 5 ul of 0.1 ug/mL MMP-1 standard was added to 995
10 uL of 1X assay diluent to achieve 500 pg/mL concentration. 2-fold serial dilution
from 500 pg/ml stock was carried out in assay diluent buffer upto 7.8 pg/ml for the standard curve. 100ul of samples (n=3) and MMP-1 standard dilutions (n=4) along with 100ul of only assay diluent (which serves as blank) was transferred to Human MMP-1 Antibody-coated ELISA Plate and incubated for 2.5 hours at room
15 temperature with gentle shaking covering the plate with ELISA plate sealer.
Meanwhile, the biotinylated detection antibody vial was briefly centrifuged and 100 uL of 1x Assay Diluent Buffer was added to prepare a detection antibody concentrate (80x). The detection antibody concentrate was then diluted by 80-fold with 1x assay diluent buffer for the final use. 20x Wash Buffer was diluted to obtain adequate
20 volume of 1x wash buffer. Samples/solution from the ELISA plates were decanted
out and washed 4 times with 1x wash buffer by filling each well with 300 ul of wash buffer using a multichannel pipette. Complete removal of liquid at each step was made sure by blot-drying the plates. 100 ul of 1x prepared biotinylated detection antibody was added to each well and the plates were incubated for 1 hour at room
25 temperature with gentle shaking. After incubation, the detection antibody solution
was discarded from the plate by decanting and the wash step was repeated with 1x wash buffer and the plates were dried by tapping against paper-towel. The HRP-Streptavidin concentrate vial was briefly spun and pipetted up and down to mix

gently before use, as precipitates may form during storage. HRP-Streptavidin
concentrate was then diluted 440-fold with 1x Assay Diluent Buffer before use.
[00121] 100μl of prepared 1x HRP-Streptavidin solution was then added to
each well and incubated for 45 minutes at room temperature with gentle shaking.
5 After incubation, the HRP-Streptavidin solution was discarded from the plate by
decanting and the wash step was repeated with 1x wash buffer and the plates were
blot-dried. 100μl of ELISA Colorimetric TMB Reagent was added to each well and
plates were incubated for 30 minutes at room temperature in the dark with gentle
shaking. 50μl of Stop Solution was added to each well and the absorbance was
10 measured at 450 nm immediately using FlexStation 3 (Molecular Devices).
Example 2
Cytotoxicity Assays for the tetrapeptide, L-citrulline and adenosine triphosphate
[00122] To determine the working range of the constituents used in the
15 composition of the present disclosure, cytotoxicity assays were conducted according
to the protocol provided above.
[00123] The tetrapeptide was tested within a concentration range of 0.00004-
0.005%. Cytotoxicity assays on the HDF-HaCaT co-culture revealed a non-toxic working concentration in the range of 0.00004- 0.00031%. Within this range, the
20 percent viability of the cells was 100% or higher. Concentrations of 0.00063% and
higher showed a drop in the viability levels. Thus, concentrations of 0.0003% or lower were used in the subsequent assays. The data is represented in Table 1 below Table 1
Percent viability Peptide conc
(%)
Average SD
0.005 1.11 1.62
0.0025 13.60 2.52

[00124] The amino acid L-citrulline was tested within a concentration range of
0.008- 1%. The cytotoxicity data reveals that L-citrulline in general demonstrates low
toxicity levels. 100% and above cell viability was observed for most of the
5 concentrations tested, only a marginal dip the percent viability was observed at a
concentration of 0.5% L-citrulline. Thus, a concentration of 0.25% and below was used the further experiments. The data is represented in Table 2 below.

[00125] Finally, the cytotoxicity levels of ATP was tested within a
concentration range of 0.008-1%. In general, the level of toxicity of ATP was high at
concentrations of 0.016% and above. The non-toxic concentration within the tested
range was at 0.008%, where the cell viability was at 101.43%. Thus concentrations of
5 0.008% and lower were tested for further experiments. The data is represented in
Table 3 below.

Table 3

ATP conc (%) Percent viability

Average SD
1 55.44 1.99
0.5 59.72 0.31
0.25 60.35 2.00
0.125 64.66 4.02
0.0625 80.75 7.09
0.031 97.13 6.45
0.016 98.98 7.40
0.008 101.43 3.92
Example 3
5 Inhibition of MMP-1 levels by the combination of L-citrulline and the
tetrapeptide
[00126] To determine whether the tetrapeptide or the L-citrulline had any
effect in the inhibition of MMP-1 levels, the actives were supplied to human dermal fibroblast cells and tested as per the protocol elucidated in Example 1.
10 [00127] The tetrapeptide was tested at three concentrations of 0.0003%,
0.0002%, and 0.00008%. At all the concentrations tested, the peptide was not able to
repress MMP-1 protein levels (Figure 1). In fact, as compared to the control, there
was an enhancement in the protein levels in the presence of the peptide.
[00128] On the other hand, L-citrulline was able to partially supress MMP-1
15 protein levels at both the concentrations tested. At 0.125% and 0.25%, L-citrulline
34

could inhibit the protein levels by 78%. Thus, L-citrulline does possess the ability to inhibit MMP-1 protein levels (Figure 1).
[00129] The combination of the tetrapeptide and L-citrulline was then tested to
determine whether the composition could show enhanced ability to inhibit the MMP-
5 1 protein. Surprisingly, the combination of L-citrulline and the tetrapeptide was able
to significantly inhibit MMP-1 protein levels (Figure 1). The maximum synergistic
effect was shown by the combination of 0.00001% peptide and 0.0625% L-citrulline
at 68%. This synergy is unexpected as the peptide by itself did not demonstrate any
ability to supress MMP-1 protein levels. Further, the observed inhibition is far more
10 than the expected inhibition values if a mere admixture of the two components is
prepared at these particular weight percentages. For instance, if the average percentage inhibition of the individual actives at 0.00008% peptide and 0.125% L-citrulline is calculated, the value that we arrive is 92.5% inhibition, which is significantly higher than the percent inhibition values of the composition at similar
15 weight percentages of the two ingredients (0.00004% peptide+ 0.125% L-citrulline,
Figure 1). Thus, the composition of L-citrulline and the tetrapeptide shows a significant synergistic effect.
[00130] Further, this synergistic activity is not apparent when the ingredients
are replaced with other actives. As seen in Figure 2, a combination of the peptide and
20 ATP did not yield the same result as the combination of L-citrulline and the peptide,
and did not show any significant reduction in MMP-1 levels as compared to ATP in isolation. Thus, using L-citrulline and the tetrapeptide is synergistic only at certain specific concentrations. Further, the composition is not -obvious to a person skilled in the art and cannot be arrived at without undue experimentation as using compositions
25 with similar constituents do not yield the same effect, demonstrating the specificity of
this particular combination to enhance MMP-1 inhibition.
Advantages of the present disclosure: Overall, the present disclosure provides a
composition comprising the amino acid L-citrulline and a custom synthesized
tetrapetptide having the sequence Ile-Glu-Pro-Asp, which is able to reduce the levels
35

of the collagen-degrading enzyme Matrix Metalloproteinases-1 (MMP-1), thereby
inhibiting the process of aging by inhibiting collagen degradation. The combination
of L-citrulline and the tetrapeptide demonstrates distinct synergism, as compared to
the actives alone, within a w/w ratio range of 1: 0.00013- 1:0.00018 of L-citrulline to
5 the tetrapeptide, and particularly at a ratio of 1: 0.00016 of L-citrulline to the
tetrapeptide. Thus, the specificity of the composition described herein to function within this specific w/w ratio range cannot be arrived at without undue experimentation and is therefore not obvious to a person skilled in the art.
36