[001] The present disclosure relates to the field of cosmetics in general and anti-ageing composition in particular. There is provided a composition for inhibiting the activity of elastase enzyme.
BACKGROUND OF INVENTION
[002] Skin is composed of three layers, namely, epidermis, dermis, and subcutaneous tissue. Extracellular matrix (ECM) is the outermost part of the skin and is composed of proteoglycans interwoven with matrix metallo-proteins, such as, collagen, elastin, and fibronectin. ECM primarily plays a role in providing structural framework which is essential for growth and maintaining elasticity of the skin. Collagen is the major structural protein which provides supportive framework to the cell and creates suitable environment for cell development and morphogenesis. Elastin maintains skin elasticity and resilience (Oikarinen A. International Journal of Cosmetic Science, March 25, 2004). Hyaluronic acid (HA) holds the water together and keeps body smooth, moist and lubricated. Ageing affects the elasticity and composition of various components forming the skin. There are two types of ageing which affects the skin, they are categorised into external and internal ageing. Intrinsic or internal ageing is natural ageing and is caused by changes in elasticity of the skin, internal ageing is also referred to as chronological ageing which is dependent on age. External ageing is caused by photo ageing. Photo ageing is referred to ageing of skin caused by exposure to the solar radiation.
[003] Degradation of ECM has been linked to skin ageing, and an increase in activity of certain enzymes like elastase, hyaluronidase, and collagenase has been observed with ageing (Maity N., et al. J Ethnopharmacol. 2011, 137: 1300-1305; Wary KK et al., Mol Cancer. 2003, 2: 25-36; Losso JN et al., Biotechnol Lett. 2004, 26: 901-905.). The degradation of the ECM with age results in a decrease in levels of

elastin, hyaluronidase, and collagen levels, which leads to loss of strength and flexibility in skin, thereby making the wrinkles visible on the skin. Elastase is a proteolytic enzyme which is primarily responsible for breakdown of elastin. The enzyme elastase also plays a role during wound repair process, where it is responsible for clearing foreign particles and thus facilitating tissue repair.
[004] During ageing, elasticity of the skin is decreased due to the activity of enzyme elastase which leads to sagging. At the same time, the amount of hyaluronic acid in the skin also diminishes and skin becomes dry and wrinkled.
[005] US20070166267 Al discloses a composition for treating the skin comprising an acylated short chain bioactive peptide and Lycium barbarum extract product. Also disclosed is a method for topically administering the composition in an amount therapeutically effective to reduce wrinkles by building the dermal fibroblast matrix. [006] WO2013083431 Al discloses a skin anti-ageing composition comprising myricetin or glycoside thereof, and at least one LXR alpha agonist.
SUMMARY OF INVENTION
[007] In an aspect of the present disclosure, there is provided a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2. [008] In an aspect of the present disclosure, there is provided a method for preparing the composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining at least one active; and (iii) contacting citrulline and the at least one active, to obtain the composition.
[009] 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
[0010] 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.
[0011] Figure 1 illustrates elastase inhibition displayed by individual components at 50 ug/ml and 100 ug/ml, in accordance with an embodiment of the present disclosure. [0012] Figure 2 illustrates elastase inhibition displayed by individual components at 100 ug/ml and 200 ug/ml, in accordance with an embodiment of the present disclosure. [0013] Figure 3 illustrates elastase inhibition displayed by a combination where each component is added at a concentration of 50 ug/ml, in accordance with an embodiment of the present disclosure.
[0014] Figure 4 illustrates a comparison of elastase inhibition displayed by a combination where each component is added at a concentration of 50 ug/ml against a combination where each component is added at a concentration of 100 ug/ml, in accordance with an embodiment of the present disclosure.
DETAILED DESCRIPTION OF THE INVENTION
[0015] 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.

[0016] 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.
[0017] 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.
[0018] 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".
[0019] 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.
[0020] The term "including" is used to mean "including but not limited to".
"Including" and "including but not limited to" are used interchangeably.
[0021] Carriers are substances that serve as mechanisms to improve the delivery and
the effectiveness of drugs.
[0022] A diluent (also referred to as filler, dilutant, or thinner) is a diluting agent.
[0023] An excipient is an inactive substance that serves as the vehicle or medium for
a drug or other active substance. Excipients include colouring agents, humectants,
preservatives, emollients, and combinations thereof.
[0024] For the purposes of the present document, the term "anti-ageing" refers to the
external effect of the composition of the present disclosure on the skin. The
composition of the present disclosure reduces the wrinkles on the skin thus depicts an
anti-ageing effect of the composition.

[0025] 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
i disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.
[0026] Skin ageing is a slow process which leads to gradual degradation of ECM and thus, produces wrinkle on the skin along with sagging appearance of the skin. There is an evergreen market demand for a product which claims to reduce wrinkles on skin
) and thus, gives an anti-ageing effect. There are many anti-ageing synthetic products available in the market, but such products are associated with side-effects upon extended usage. Thus, there exists a demand for products which constitute majority of natural compounds which are not only safe, but also effective upon extended usage. Also, compositions which target molecular machinery of ageing will be more
i effective in reducing the wrinkles on the skin. Thus, the present disclosure discloses a composition comprising citrulline and at least one active, wherein the at least one active is selected from the group consisting of catechine, N6-benzyladenine (N6BA), and combinations thereof, and citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2. Citrulline is a non-essential amino acid,
) catechine is a natural phenol and is a secondary metabolite produced by plant which also has antioxidant property. N6-benzyladenine is a synthetic cytokinin which is used extensively in plant tissue culture. The composition of the present disclosure synergistically inhibits the activity of elastase enzyme and thus, is an anti-ageing composition. The present composition can thus be used in various ant-wrinkle and
i anti-ageing formulations.
[0027] The present disclosure discloses a composition comprising: citrulline, and catechine, wherein citrulline and catechine w/w ratio in said composition is in the range of 1:0.5 to 1:2. The present disclosure discloses a composition comprising:

citrulline, and N6-benzyladenine, wherein w/w ratio of citrulline to N6-benzyladenine in said composition is in the range of 1:0.5 to 1:2.
[0028] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a w/w ratio range of citrulline to at least one active of 1:0.5 to 1:2 should be interpreted to include not only the explicitly recited limits of about 1:0.5 to about 1:2, but also to include sub-ranges, such as 1:0.8 to 1:1.7, 1:1 to 1:1.5, and so forth, as well as individual amounts, including fractional amounts, within the specified ranges, such as 1:0.8, 1:1, 1:1.5, for example.
[0029] 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. [0030] In an embodiment of the present disclosure, there is provided a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2. [0031] In an embodiment of the present disclosure, there is provided a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is 1:1.
[0032] In an embodiment of the present disclosure, there is provided a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, and wherein the at least one active is selected from the group consisting of catechine, N6-benzyladenine (N6BA), and combinations thereof.

[0033] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) citrulline; and (b) catechine, wherein citrulline to catechine w/w ratio
in said composition is in the range of 1:0.5 to 1:2.
[0034] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) citrulline; and (b) catechine, wherein citrulline to catechine w/w ratio
in said composition is 1:1.
[0035] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) citrulline; and (b) N6-benzyladenine, wherein citrulline to N6-
benzyladenine w/w ratio in said composition is in the range of 1:0.5 to 1:2.
[0036] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) citrulline; and (b) N6-benzyladenine, wherein citrulline to N6-
benzyladenine w/w ratio in said composition is 1:1.
[0037] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least
one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, and wherein
citrulline has a weight percentage in the range of 0.005% to 0.01% with respect to the
composition.
[0038] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least
one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, and wherein
the at least one active has a weight percentage in the range of 0.005% to 0.01% with
respect to the composition.
[0039] In an embodiment of the present disclosure, there is provided a composition
comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least
one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, and wherein
citrulline has a weight percentage in the range of 0.005% to 0.01% with respect to the
composition, and wherein the at least one active has a weight percentage in the range
of 0.005%) to 0.01%) with respect to the composition.

[0040] In an embodiment of the present disclosure, there is provided a composition comprising: (a) citrulline; and (b) catechine, wherein citrulline to catechine w/w ratio in said composition is in the range of 1:0.5 to 1:2, and wherein citrulline has a weight percentage in the range of 0.005% to 0.01% with respect to the composition, and
5 wherein catechine has a weight percentage in the range of 0.005% to 0.01% with respect to the composition.
[0041] In an embodiment of the present disclosure, there is provided a composition comprising: (a) citrulline; and (b) N6-benzyladenine, wherein citrulline to N6-benzyladenine w/w ratio in said composition is in the range of 1:0.5 to 1:2, and
0 wherein citrulline has a weight percentage in the range of 0.005%) to 0.01%> with respect to the composition, and wherein N6-benzyladenine has a weight percentage in the range of 0.005%> to 0.01%> with respect to the composition.
[0042] In an embodiment of the present disclosure, there is provided a composition comprising: (a) citrulline; (b) at least one active; (c) at least one diluent; (d) at least
5 one carrier; and (e) at least one excipient, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2. In another embodiment of the present disclosure, citrulline to the at least one active w/w ratio in said composition is 1:1. [0043] In an embodiment of the present disclosure, there is provided a composition
0 comprising: (a) citrulline; (b) at least one active; (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, and wherein the at least one active is selected from the group consisting of catechine, N6-benzyladenine (N6BA), and combinations thereof.
5 [0044] In an embodiment of the present disclosure, there is provided a composition comprising: (a) citrulline; (b) catechine; (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to catechine w/w ratio in said composition is in the range of 1:0.5 to 1:2.

[0045] In an embodiment of the present disclosure, there is provided a composition comprising: (a) citrulline; (b) catechine; (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to catechine w/w ratio in said composition is in the range of 1:0.5 to 1:2, and wherein citrulline has a weight percentage in the range of 0.005% to 0.01% with respect to the composition, and wherein catechine has a weight percentage in the range of 0.005% to 0.01% with respect to the composition.
[0046] In an embodiment of the present disclosure, there is provided a composition comprising: (a) citrulline; (b) N6-benzyladenine (N6BA); (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to N6-benzyladenine (N6BA) w/w ratio in said composition is in the range of 1:0.5 to 1:2. [0047] In an embodiment of the present disclosure, there is provided a composition comprising: (a) citrulline; (b) N6-benzyladenine (N6BA); (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to N6-benzyladenine (N6BA) w/w ratio in said composition is in the range of 1:0.5 to 1:2, and wherein citrulline has a weight percentage in the range of 0.005% to 0.01% with respect to the composition, and wherein N6-benzyladenine has a weight percentage in the range of 0.005% to 0.01% with respect to the composition. [0048] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining at least one active; and (iii) contacting citrulline and the at least one active, to obtain the composition.
[0049] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining at least

one active; and (iii) contacting citrulline and the at least one active, to obtain the composition, and wherein the at least one active is selected from the group consisting of catechine, N6-benzyladenine (N6BA), and combinations thereof. [0050] In an embodiment of the present disclosure, there is provided a method for
i preparing a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is 1:1, said method comprising: (i) obtaining citrulline; (ii) obtaining at least one active; and (iii) contacting citrulline and the at least one active, to obtain the composition. [0051] In an embodiment of the present disclosure, there is provided a method for
) preparing a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining at least one active; and (iii) contacting citrulline and the at least one active, to obtain the composition, wherein citrulline has a weight percentage in the range of 0.005% to
i 0.01% with respect to the composition, and the at least one active has a weight percentage in the range of 0.005% to 0.01% with respect to the composition. [0052] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) catechine, wherein citrulline to catechine w/w ratio in said composition is in the range of 1:0.5 to 1:2,
) said method comprising: (i) obtaining citrulline; (ii) obtaining catechine; and (iii) contacting citrulline and catechine, to obtain the composition.
[0053] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) catechine, wherein citrulline to catechine w/w ratio in said composition isl:l, said method comprising:
i (i) obtaining citrulline; (ii) obtaining catechine; and (iii) contacting citrulline and catechine, to obtain the composition.
[0054] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) catechine, wherein

citrulline to catechine w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining catechine; and (iii) contacting citrulline and catechine, to obtain the composition, wherein citrulline has a weight percentage in the range of 0.005% to 0.01% with respect to the composition, and catechine has a weight percentage in the range of 0.005% to 0.01% with respect to the composition.
[0055] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) N6 benzyladenine, wherein citrulline to N6-benzyladenine w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining N6-benzyladenine; and (iii) contacting citrulline and N6-benzyladenine, to obtain the composition.
[0056] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) N6-benzyladenine, wherein citrulline to N6-benzyladenine w/w ratio in said composition is 1:1, said method comprising: (i) obtaining citrulline; (ii) obtaining N6-benzyladenine; and (iii) contacting citrulline and N6-benzyladenine, to obtain the composition. [0057] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) N6-benzyladenine, wherein citrulline to N6-benzyladenine w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining N6-benzyladenine; and (iii) contacting citrulline and N6-benzyladenine, to obtain the composition, and wherein citrulline has a weight percentage in the range of 0.005%) to 0.01%) with respect to the composition, and N6-benzyladenine (N6BA) has a weight percentage in the range of 0.005%> to 0.01%> with respect to the composition. [0058] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; (b) at least one active; (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to

the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining at least one active; (iii) obtaining at least one diluent; (iv) obtaining at least one carrier; (v) obtaining at least one excipient; and (vi) contacting citrulline, the at least one active, the at least one diluent, the at least one carrier, and the at least one excipient, to obtain the composition. In another embodiment of the present disclosure, citrulline to the at least one active w/w ratio in said composition is 1:1.
[0059] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; (b) at least one active; (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining at least one active; (iii) obtaining at least one diluent; (iv) obtaining at least one carrier; (v) obtaining at least one excipient; and (vi) contacting citrulline, the at least one active, the at least one diluent, the at least one carrier, and the at least one excipient, to obtain the composition, and wherein the at least one active is selected from the group consisting of catechine, N6-benzyladenine (N6BA), and combinations thereof. [0060] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; (b) at least one active; (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining at least one active; (iii) obtaining at least one diluent; (iv) obtaining at least one carrier; (v) obtaining at least one excipient; and (vi) contacting citrulline, the at least one active, the at least one diluent, the at least one carrier, and the at least one excipient, to obtain the composition, and wherein citrulline has a weight percentage in the range of 0.005% to 0.01% with respect to the composition, and the at least one active has a weight percentage in the range of 0.005% to 0.01% with respect to the composition.

[0061] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; (b) catechine; (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to catechine w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method
5 comprising: (i) obtaining citrulline; (ii) obtaining catechine; (iii) obtaining at least one diluent; (iv) obtaining at least one carrier; (v) obtaining at least one excipient; and (vi) contacting citrulline, catechine, the at least one diluent, the at least one carrier, and the at least one excipient, to obtain the composition. In another embodiment of the present disclosure, citrulline to catechine w/w ratio in said composition is 1:1.
D [0062] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; (b) catechine; (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to catechine w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining catechine; (iii) obtaining at least
5 one diluent; (iv) obtaining at least one carrier; (v) obtaining at least one excipient; and (vi) contacting citrulline, catechine, the at least one diluent, the at least one carrier, and the at least one excipient, to obtain the composition, and wherein citrulline has a weight percentage in the range of 0.005% to 0.01% with respect to the composition, and catechine has a weight percentage in the range of 0.005% to 0.01% with respect
D to the composition.
[0063] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; (b) N6-benzyladenine (N6BA); (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to catechine w/w ratio in said composition is in the range of 1:0.5 to 1:2,
5 said method comprising: (i) obtaining citrulline; (ii) obtaining N6-benzyladenine (N6BA); (iii) obtaining at least one diluent; (iv) obtaining at least one carrier; (v) obtaining at least one excipient; and (vi) contacting citrulline, N6-benzyladenine (N6BA), the at least one diluent, the at least one carrier, and the at least one excipient,

to obtain the composition. In another embodiment of the present disclosure, citrulline to N6-benzyladenine (N6BA) w/w ratio in said composition is 1:1. [0064] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; (b) N6-benzyladenine (N6BA); (c) at least one diluent; (d) at least one carrier; and (e) at least one excipient, wherein citrulline to N6-benzyladenine (N6BA) w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining citrulline; (ii) obtaining N6-benzyladenine (N6BA); (iii) obtaining at least one diluent; (iv) obtaining at least one carrier; (v) obtaining at least one excipient; and (vi) contacting citrulline, N6-benzyladenine (N6BA), the at least one diluent, the at least one carrier, and the at least one excipient, to obtain the composition, and wherein citrulline has a weight percentage in the range of 0.005% to 0.01% with respect to the composition, and N6-benzyladenine(N6BA) has a weight percentage in the range of 0.005%) to 0.01%> with respect to the composition.
[0065] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining a solution A of citrulline; (ii) obtaining a solution B of the at least one active; and (iii) contacting solution A and solution B, to obtain the composition, wherein solution A is prepared in an organic solvent.
[0066] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining a solution A of citrulline; (ii) obtaining a solution B of the at least one active; and (iii) contacting solution A and solution B, to obtain the composition, wherein solution A is prepared in an inorganic solvent.

[0067] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining a solution A of citrulline; (ii) obtaining a solution B of the at least one active; and (iii) contacting solution A and solution B, to obtain the composition, wherein solution B is prepared in an organic solvent.
[0068] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) at least one active, wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining a solution A of citrulline; (ii) obtaining a solution B of the at least one active; and (iii) contacting solution A and solution B, to obtain the composition, wherein solution B is prepared in an inorganic solvent.
[0069] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) catechine, wherein citrulline to catechine w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining a solution A of citrulline; (ii) obtaining a solution B of catechine; and (iii) contacting solution A and solution B, to obtain the composition, wherein solution A is prepared in DMSO (dimethyl sulfoxide), and solution B is prepared in DMSO (dimethyl sulfoxide).
[0070] In an embodiment of the present disclosure, there is provided a method for preparing a composition comprising: (a) citrulline; and (b) N6-benzyladenine (N6BA), wherein citrulline to N6-benzyladenine (N6BA) w/w ratio in said composition is in the range of 1:0.5 to 1:2, said method comprising: (i) obtaining a solution A of citrulline; (ii) obtaining a solution B of N6-benzyladenine (N6BA); and (iii) contacting solution A and solution B, to obtain the composition, wherein solution

A is prepared in DMSO (dimethyl sulfoxide), and solution B is prepared in DMSO
(dimethyl sulfoxide).
[0071] In an embodiment of the present disclosure, there is provided a method for
inhibiting elastase enzyme activity in skin cells, said method comprising: (a)
obtaining a composition as described herein; and (b) contacting the composition of
step (a) with skin cells, wherein contacting the composition inhibits elastase enzyme
activity in skin cells.
[0072] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition exhibits elastase enzyme inhibiting
activity.
[0073] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition is used in anti-wrinkle formulations
activity.
[0074] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition inhibits skin ageing.
[0075] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition inhibits skin ageing by inhibiting the
activity of elastase enzyme.
[0076] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition can be for topical application.
[0077] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition can be in the form of a lotion.
[0078] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein the composition can be in form of an oil.
[0079] 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 serum.
[0080] 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
[0081] 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.
[0082] The examples depicted in the present section discuss the effect of the composition comprising citrulline and at least one active in inhibiting the activity of elastase enzyme. The effect of the composition of the present disclosure has been compared with compositions comprising citrulline with either oleanolic acid or carnosine. The synergistic effect of the composition of the present disclosure has been compared with the individual actives in inhibiting the activity of elastase enzyme. Materials required:
Porcine pancreatic elastase (PPE, Sigma, Type IV); 0.1M HEPES buffer (pH=7.5); 0.8mMN-Succ-(Ala) 3-nitroanilide (SANA). Oleanolic acid was procured from Sami Labs Pvt. Ltd. L-carnosine was procured from Puneeth Laboratories Pvt. Ltd. N6BA

was procured from Harison Agro Rasayan Pvt Ltd. L-citrulline was procured from BioKyowa Inc. and catechine was procured from Sigma. Protocol:
Anti-elastase activity was determined according to the method of Kraunsoe et al. (Kraunsoe et al., Biochemistry 1996, 35:9090-9096), with minor modifications. Into 96-well plates was added: 25 ul each of 0.1 M HEPES buffer (pH 7.5), test sample (50 or 100 ug/ml) and elastase (1 ug/ml). The blank wells contained HEPES buffer (75 ul), the negative control 25 ul elastase and 50 ul HEPES buffer. The solvent controls contained 25 ul each of elastase, HEPES buffer and 5% DMSO. Extract controls containing 150 ul HEPES buffer and 25 ul of the extract were colour controls of each extract tested. Plates were incubated at room temperature for 20 min after which 100 ul of the substrate N-Methoxysuccinyl- Ala-Ala-Pro-Val-p-nitroanilide (1 mM) was added and the plates were incubated for further 40-60 min at 25 °C.
Absorbance was read at 405 nm using a spectrophotometer. The percentage inhibition was calculated using the formula as below:
Inhibition {%) =■ [(^^-^ia^J/A™^] x 100
Where Acontroi is the absorbance of buffer, elastase + solvent and Atest sample is the
absorbance of buffer, elastase + test sample. All the chemicals were dissolved in 5%
DMSO, except oleanolic acid, which was dissolved in 100% DMSO.
EXAMPLE 1
Dose response of individual components in inhibiting elastase enzyme activity
[0083] Figure 1 depicts the elastase inhibition activity of citrulline (Cit), carnosine
(Car), oleanolic acid (OA), N6-benzyladenine (N6BA), and catechine (Catech) in
isolation at concentrations of 50ug/ml and lOOug/ml.
[0084] It can be appreciated from Figure 1 that citrulline, N6BA, and catechine fail to
inhibit elastase enzyme activity at either of the above-mentioned concentration.
Oleanolic acid at 50ug/ml leads to around 20% inhibition of elastase enzyme activity

and at lOOug/ml leads to more than 40% inhibition of elastase enzyme activity. In case of carnosine, a concentration of 50ug/ml does not elicit any inhibitory effect, while a concentration of lOOug/ml exhibits more than 10% inhibition of elastase enzyme activity.
[0085] The elastase inhibition activity of all the above-mentioned actives at a concentration of 200ug/ml were also tested and is depicted in Figure 2. Referring to Figure 2, N6BA, and catechine fail to inhibit elastase enzyme activity even at a concentration of 200ug/ml, whereas citrulline and carnosine are able to show a meagre inhibition of elastase inhibition activity. Amongst all, oleanolic acid exhibits significant inhibition of elastase enzyme activity.
EXAMPLE 2
Composition exhibiting elastase inhibition activity
[0086] The present example depicts the synergistic effect of the composition of the
present disclosure in inhibiting elastase enzyme activity. Following compositions
were evaluated for the ability:
Composition 1: citrulline and carnosine
Composition 2: citrulline and oleanolic acid
Composition 3: citrulline and N6B A
Composition 4: citrulline and catechine.
The compositions as referred above intend to highlight the components of the
compositions. The concentrations of the different components can differ and has been
specified in parentheses.
[0087] The compositions 3 and 4 represent the compositions disclosed in the present
disclosure. The compositions 1 and 2 comprises citrulline along with other actives.
[0088] Figure 3 depicts the result of elastase inhibition assay with the four
compositions as mentioned above, wherein the four compositions have been prepared
by combining 50ug/ml of each of the actives. It can be observed from Figure 3 that

compositions 1 and 2 exhibit around 8% and 33% inhibition of elastase enzyme activity. As the individual effects of carnosine and oleanolic acid are significant in inhibiting elastase enzyme activity. Thus, comparing the inhibitory activities of the individual components of citrulline, carnosine, and oleanolic acid, with the inhibitory activities of compositions 1 and 2 it can be concluded that the compositions 1 and 2 are not displaying significant inhibitory activities in a synergistic manner. [0089] From Figure 3, it can be appreciated that compositions 3 and 4 show a significant inhibitory effect of elastase enzyme activity. Figure 3 depicts the effect of the compositions where all the components are added at a concentration of 50ug/ml. The actives citrulline, N6BA, and catechine in isolation did not exhibit elastase enzyme inhibitory activity (Refer to Example 1) but surprisingly the composition 3 (citrulline 50ug/ml+N6BA 50ug/ml) exhibits around 30% of inhibition. Similarly, the composition 4 (citrulline 50ug/ml+catechine 50ug/ml) exhibits around 26% of elastase enzyme activity inhibition. Thus, it can be ascertained that the compositions 3 and 4 are displaying synergistic inhibition of elastase enzyme activity. [0090] Figure 4 depicts the elastase inhibition activity exhibited by the compositions in which each of the active is added at a concentration of lOOug/ml. The same has been compared with the effect of composition in which each of the active is added at a concentration of 50ug/ml. Referring to Figure 4, it can be observed that the composition 3 (citrulline 100ug/ml+N6BA lOOug/ml) exhibits around 20% inhibition, and the composition 4 (citrulline lOOug/ml+catechine lOOug/ml) exhibits around 18% inhibition of elastase activity. From Figure 2, it can be clearly observed that citrulline lOOug/ml, N6BA lOOug/ml, and catechine lOOug/ml in isolation fail to exhibit any inhibitory activity. Also, citrulline 200ug/ml, N6BA 200ug/ml, and catechine 200ug/ml in isolation does not exhibit significant inhibitory activity. Thus, the compositions 3 and 4 surprisingly show synergistic inhibitory activity in the specified combinations.

[0091] Overall, the composition comprising citrulline and N6BA exhibit synergistic effect in inhibiting the elastase enzyme activity, wherein citrulline and N6BA can be present in 50ug/ml or lOOug/ml. Similarly, the composition comprising citrulline and catechine exhibit synergistic effect in inhibiting the elastase enzyme activity, wherein citrulline and catechine can be present in 50ug/ml or lOOug/ml. Thus, the composition of the present disclosure can be used in ant-ageing formulations.
Advantages of the present disclosure:
[0092] The present disclosure discloses a composition comprising citrulline along with at least one active. The at least one active is selected from the group consisting of N6BA and catechine. The composition comprising citrulline and N6BA has been explicitly shown to exhibit synergistic inhibition of elastase activity inhibition. Similarly, the composition comprising citrulline and catechine has also been shown to exhibit synergistic inhibition of elastase activity. The composition of the present disclosure can be used in anti-ageing formulations for topical applications. Since, the composition as disclosed in the present disclosure has significant herbal content, it is expected to have lesser side effects as compared to synthetic cosmetics available in the market for anti-ageing purposes.

1. A composition comprising:
a) citrulline; and
b) at least one active,
wherein citrulline to the at least one active w/w ratio in said composition is in the range of 1:0.5 to 1:2.
2. The composition as claimed in 1, wherein citrulline to the at least one active w/w ratio in said composition is 1:1.
3. The composition as claimed in claim 1, wherein the at least one active is selected from the group consisting of catechine, N6-benzyladenine (N6BA), and combinations thereof.
4. The composition as claimed in any of the claims 1 to 3, wherein the at least one active is catechine.
5. The composition as claimed in any of the claims 1 to 3, wherein the at least one active is N6-benzyladenine (N6BA).
6. The composition as claimed in claim 1, wherein citrulline has a weight percentage in the range of 0.005% to 0.01% with respect to the composition.
7. The composition as claimed in claim 1, wherein the at least one active has a weight percentage in the range of 0.005% to 0.01% with respect to the composition.
8. The composition as claimed in claim 1, wherein the composition further comprises: (a) at least one diluent; (b) at least one carrier; and (c) at least one excipient.
9. A method for preparing the composition as claimed in any of the claims 1 to 7, said method comprising:

a) obtaining citrulline;
b) obtaining at least one active; and

c) contacting citrulline and the at least one active, to obtain the composition.
10. A method for preparing the composition as claimed in any of the claims 1 to
8, said method comprising:
a) obtaining citrulline;
b) obtaining at least one active;
c) obtaining at least one diluent;
d) obtaining at least one carrier;
e) obtaining at least one excipient; and
f) contacting citrulline, the at least one active, the at least one diluent, the at least one carrier, and the at least one excipient, to obtain the composition.

11. The composition as claimed in any of the claims 1 to 8, wherein the composition exhibits elastase enzyme inhibiting activity.
12. The composition as claimed in any of the claims 1 to 8, wherein the composition is used in anti-ageing formulations.
Date 16 February 2018
MALATHI LAKSHMIKUMARAN
IN/PA-1433
Agent for the Applicant To,
The Controller of Patents The Patent Office at Chennai