FIELD OF INVENTION
[001] The present disclosure relates to the field of cosmetics, and in particular the present disclosure relates to an anti-senescent composition.
BACKGROUND OF THE INVENTION
[002] Skin aging is a multifactorial phenomenon exhibited by the appearance of fine lines and wrinkles. As the skin ages, it loses its elasticity and one of the contributors of this process is cellular senescence. Cellular senescence refers to the irreversible arrest of the cell cycle at different stages. Senescent cells secrete pro-inflammatory cytokines, chemokines, growth factors, and proteases, a phenomenon called senescence-associated secretory phenotype (SASP), which are thought to contribute to the functional decline of the skin.
[003] Both skin aging and senescence are multifactorial processes, thus determining a specific cause has been a major hurdle in skin-aging related research. A means to analyze these processes is through the use of senescence-associated biomarkers that can be found in various human tissues in vivo, and are associated with both aging and pathology. One widely used marker of replicative senescence is senescence-associated β-galactosidase (SA β-gal) activity. The enzyme β-galactosidase, a lysosomal hydrolase, is normally active at pH 4, but in senescent cells β-galactosidase is often seen to be active at pH 6 which can be detected with a simple biochemical assay. SA β-gal activity appears to be a result of increased lysosomal activity at a suboptimal pH, which becomes detectable in senescent cells due to an increase in lysosomal content. [004] Active research is being carried out to decrease senescence levels which leads to enhanced cellular activities, skin firmness, and elasticity. However, specific compounds that have a targeted effects to remedy the process of skin aging are very limited and thus there is a need to develop better formulations to reduce senescence-associated skin aging. [005] PCT/AU2014/001048 describes a method of treating or preventing DNA damage in a cell or cellular senescence of a cell or induction of the senescence associated secretory phenotype (SASP) in a cell, or for treating or preventing the effects of aging, or for preventing or treating cellular senescence and/or induction of SASP associated with high caloric intake or obesity, or for reducing the side effects of chemotherapy, radiotherapy, corticoid treatment, anti-retroviral treatment, or PPARγ (Peroxisome proliferator-activated receptor gamma) agonist treatment, comprising administering an effective amount of an NAD+ agonist.

[006] PCT/US2009/063137 describes compositions and methods used to reduce the visible signs of aging of the skin by recalibrating the expression of genes, genetic networks, and cellular pathways in the human skin, particularly using combinations of natural compounds that produce synergistic effects on the expression of genes and genetic networks.
[007] US5589483A describes a method for delaying the onset of cell senescence associated with skin aging in humans which involves administering to fibroblast cells in the human an isoquinoline PADPRP inhibitor effective to increase the maximum achievable number of cumulative population doublings of the fibroblast cells, whereby the proliferative capacity of the fibroblast cells is increased.
SUMMARY OF THE INVENTION
[008] In an aspect of the present disclosure, there is provided a composition comprising: (a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 – 1:12:2.5.
[009] In an aspect of the present disclosure, there is provided a method for ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 – 1:12:2.5; and (b) contacting the said composition with skin.
[010] In an aspect of the present disclosure, there is provided a process for preparing a composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 – 1:12:2.5, said process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; and (d) contacting xanthohumol, chrysin, and royal jelly peptide to obtain a composition.
[011] In an aspect of the present disclosure, there is provided a process for preparing a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable additives, said process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; (d) obtaining suitable additives; and (e) contacting xanthohumol, chrysin, royal jelly peptide, and suitable additives to obtain a composition, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of 1:8:0.4-1:12:2.5.

[012] 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 THE ACCOMPANYING FIGURES
[013] The following figures form 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 figures in combination with the detailed description of the specific embodiments presented
herein.
[014] Figure 1 depicts the dose dependent effect of different concentrations of xanthohumol on
human dermal fibroblasts, in accordance with an embodiment of the present disclosure.
[015] Figure 2 depicts the dose dependent effect of different concentrations of chrysin on
human dermal fibroblasts, in accordance with an embodiment of the present disclosure.
[016] Figure 3 depicts the dose dependent effect of different concentrations of royal jelly
peptide on human dermal fibroblasts, in accordance with an embodiment of the present
disclosure.
[017] Figure 4 depicts the dose dependent effect of different concentrations of GrB peptide on
human dermal fibroblasts, in accordance with an embodiment of the present disclosure.
[018] Figure 5 depicts the effect of xanthohumol (X), chrysin, (C) and royal jelly peptide (RJ)
on senescence associated beta-galactosidase (SA-β-gal) protein expression at a ratio of 1:10:2.8.
[019] Figure 6 depicts the effect of xanthohumol (X), chrysin (C) and royal jelly peptide (RJ)
on senescence associated beta-galactosidase (SA-β-gal) protein expression at a ratio of 1:10:0.7.
[020] Figure 7 depicts effect of xanthohumol (X), chrysin (C) and GrB peptide on SA-β-gal
expression.
[021] Figure 8 depicts the effect of xanthohumol (X), chrysin (C) and royal jelly peptide (RJ)
on SA-β-gal expression at a ratio of 1:10:1.4, in accordance with an embodiment of the present
disclosure.

DETAILED DESCRIPTION OF THE INVENTION
[022] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features. Definitions
[023] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected 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.
[024] 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.
[025] 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”. [026] 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.
[027] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.
[028] A composition comprising “synergistic activity” or a “synergistic composition” is a combination of compounds which exhibits increased biological or functional activity as a non-linear multiple of the biological or functional activity of the individual compounds. In other words, the combined biological or functional activity of two or more compounds being tested is significantly greater than the expected result based on independent effects of the compounds when tested separately. Synergy may be apparent only at some ranges or concentrations.

[029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference. [030] Xanthohumol (X) is the principal prenylated chalcone of the hop plant, an ingredient of beer. Xanthohumol employs a broad spectrum of chemo preventive actions towards malignant tumors in vitro and in vivo (Gerhauser et al., Mol Cancer Ther., 2002; 1(11): 959-69; Gerhäuser C, Eur J Cancer, 2005; 41(13): 1941-54). Recently, xanthohumol has been reported to activate the pro-apoptotic arm of the unfolded protein response in chronic lymphocytic leukemia (Lust et al., Anticancer Res., 2009; 29(10):3797-805).
[031] Chrysin (5, 7-dihydroxyflavone) is present at high levels in honey and propolis. It has been reported to be cytotoxic with EC50 value of 100 μM in a wide range of cell lines such as breast (MCF-7, MDA-MB-231 cells), colon (Lovo, DLD-1) and prostate cancer cells (Androutsopoulos et al., Bioorg Med Chem., 2011, 1; 19(9): 2842-9; Gan et al., Onco, 2009l; 45(10):e150-4). Moreover, it has been implicated as a Notch I signaling activator in anaplastic thyroid carcinoma (ATC) on a routine high- throughput screening (Tsao et al., Development, 2009; 136(13): 2297-307).
[032] Royal jelly is rich in amino acids (specifically 29), 10-hydroxydecenoic acid (10-HDA), lipids, sugars, vitamins, and proteins. It contains vitamins A, B complex (including folic acid and inositol), C, D and E, and also has ample levels of iron and calcium, as well as other minerals. Royal jelly also contains acetylcholine, which is needed to transmit nerve messages from cell to cell.
[033] 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.
[034] Various factors affect skin aging, which is exhibited by fine lines and wrinkles. Cellular senescence is an important contributor to the skin aging. Senescence-associated β-galactosidase activity is one of the widely used bio-markers of senescence. This enzyme can be a target to obtain a potent solution to skin aging. The present disclosure provides herbal compounds, royal

jelly peptide and their combinations that can protect against cellular senescence, and promises to
open new avenues for achieving reduced skin aging and establishing a new strategy to prevent
age associated skin problems.
[035] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 – 1:12:2.5.
[036] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said
composition is in the range of 1:10:1 – 1:10:2.
[037] In an embodiment of present disclosure, there is provided a composition comprising: (a)
xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal
jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said
composition is 1:8:1.
[038] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said is
1:8:1.4.
[039] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said
composition is in the range of 1:8:2.
[040] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said
composition is 1:10:1.
[041] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said

[042] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said
composition is 1:10:2.
[043] In an embodiment of present disclosure, there is provided a composition comprising: (a)
xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal
jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said
composition is 1:12:1.
[044] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said
composition is 1:12:1.4.
[045] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said
composition is 1:12:2.
[046] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of
1:8:0.4 - 1:12:2.5.
[047] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of
1:10:1 – 1:10:2.
[048] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (c) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:8:1.
[049] In an embodiment of present disclosure, there is provided a composition comprising: (a)
xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:8:1.4.

[050] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:8:2.
[051] In an embodiment of present disclosure, there is provided a composition comprising: (a)
xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.
[052] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.4.
[053] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:2.
[054] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:12:1.
[055] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:12:1.4.
[056] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:12:2.
[057] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 - 1:12:2.5, and said
composition is effective in ameliorating the deleterious effects of senescence associated enzymes
on skin.
[058] In an embodiment of present disclosure, there is provided a composition comprising: (a)
xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 - 1:12:2.5, and said
composition is effective in ameliorating the deleterious effects of senescence associated enzymes
on skin, and said senescence associated enzyme is selected from a group consisting of

senescence associated beta-galactosidase, mitogen activated protein kinase, superoxide
dismutase, sirtuin, and combinations thereof.
[059] In an embodiment of present disclosure, there is provided a composition comprising: (a)
xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 - 1:12:2.5, and said
composition is effective in ameliorating the deleterious effects of senescence associated enzymes
on skin, and said senescence associated enzyme is senescence associated beta-galactosidase.
[060] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 - 1:12:2.5, and said
composition is effective in ameliorating the deleterious effects of senescence associated enzymes
on skin, and said senescence-associated enzyme is mitogen activated protein kinase.
[061] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 - 1:12:2.5, and said
senescence associated enzyme is superoxide dismutase.
[062] In an embodiment of the present disclosure, there is provided a composition comprising:
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 - 1:12:2.5, and said
senescence associated enzyme is sirtuin.
[063] In an embodiment of present disclosure, there is provided a composition to ameliorate
deleterious effect of senescence associated enzymes on skin, said composition comprising (a)
xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is 1:8:1.
[064] In an embodiment of present disclosure, there is provided a composition to ameliorate
deleterious effect of senescence associated enzymes on skin said composition comprising (a)
xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is 1:8:1.4.
[065] In an embodiment of present disclosure, there is provided a composition to ameliorate
deleterious effect of senescence associated enzymes on skin, said composition comprising (a)

xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is 1:8:2.
[066] In an embodiment of present disclosure, there is provided a composition to ameliorate
deleterious effect of senescence associated enzymes on skin, said composition comprising (a)
xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is 1:10:1.
[067] In an embodiment of the present disclosure, there is provided a composition to ameliorate
the deleterious effects of senescence associated enzymes on skin, said composition comprising
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is 1:10:1.4.
[068] In an embodiment of the present disclosure, there is provided a composition to ameliorate
the deleterious effects of senescence associated enzymes on skin, said composition comprising
(a) xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is 1:10:2.
[069] In an embodiment of present disclosure, there is provided a composition to ameliorate
deleterious effect of senescence associated enzymes on skin, said composition comprising (a)
xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is 1:12:1.
[070] In an embodiment of the present disclosure, there is provided a composition to ameliorate
the effect of senescence associated enzymes on skin, said composition comprising (a)
xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is 1:12:1.4.
[071] In an embodiment of the present disclosure, there is provided a composition to ameliorate
the effect of senescence associated enzymes on skin, said composition comprising (a)
xanthohumol; (b) chrysin; and (c) royal jelly peptide, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is 1:12:2.
[072] In an embodiment of present disclosure, there is provided a composition comprising: (a)
xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives,, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of
1:8:0.4 - 1:12:2.5, and said composition is effective in ameliorating the deleterious effects of
senescence associated enzymes on skin, and said senescence associated enzyme is selected from

a group consisting of senescence associated beta-galactosidase, mitogen activated protein kinase,
superoxide dismutase, sirtuin, and combinations thereof.
[073] In an embodiment of present disclosure, there is provided a composition to ameliorate
deleterious effect of senescence associated enzymes on skin, said composition comprising: (a)
xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:8:1.
[074] In an embodiment of present disclosure, there is provided a composition to ameliorate
deleterious effect of senescence associated enzymes on skin said composition comprising: (a)
xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:8:1.4.
[075] In an embodiment of present disclosure, there is provided a composition to ameliorate
deleterious effect of senescence associated enzymes on skin, said composition comprising: (a)
xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:8:2.
[076] In an embodiment of present disclosure, there is provided a composition to ameliorate
deleterious effect of senescence associated enzymes on skin, said composition comprising: (a)
xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.
[077] In an embodiment of the present disclosure, there is provided a composition to ameliorate
the deleterious effects of senescence associated enzymes on skin, said composition comprising:
(a) xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.4.
[078] In an embodiment of the present disclosure, there is provided a composition to ameliorate
the deleterious effects of senescence associated enzymes on skin, said composition comprising:
(a) xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:2.
[079] In an embodiment of present disclosure, there is provided a composition to ameliorate
deleterious effect of senescence associated enzymes on skin, said composition comprising: (a)
xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:12:1.

[080] In an embodiment of the present disclosure, there is provided a composition to ameliorate
the effect of senescence associated enzymes on skin, said composition comprising: (a)
xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:12:1.4.
[081] In an embodiment of the present disclosure, there is provided a composition to ameliorate
the effect of senescence associated enzymes on skin, said composition comprising: (a)
xanthohumol; (b) chrysin; (c) royal jelly peptide; and (d) suitable additives, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:12:2.
[082] In an embodiment of the present the present disclosure, there is provided a composition
as described herein, wherein said composition is for topical application.
[083] In an embodiment of the present disclosure, there is provided a composition as described
herein, wherein said composition is in the form of a gel.
[084] In an embodiment of the present disclosure, there is provided a composition as described
herein, wherein said composition is in the form of a cream.
[085] In an embodiment of the present disclosure, there is provided a composition as described
herein, wherein said composition is in the form of a lotion.
[086] In an embodiment of the present disclosure, there is provided a composition as described
herein, wherein said composition is in the form of a serum.
[087] In an embodiment of the present disclosure, there is provided a composition as described
herein, wherein said composition is in aerosolized form.
[088] In an embodiment of the present disclosure, there is provided a method for ameliorating
the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a
composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of
1:8:0.4 - 1:12:2.5; and (b) contacting said composition with skin.
[089] In an embodiment of present disclosure, there is provided a method for ameliorating the
effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a
composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide; and (b)
contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide
w/w ratio in said composition is 1:8:1.

[090] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:8:1.4.
[091] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:8:2.
[092] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.
[093] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.4.
[094] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:2.
[095] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:12:1.

[096] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a
composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide; and (b)
contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide
w/w ratio in said composition is 1:12:1.4.
[097] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a
composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide; and (b)
contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide
w/w ratio in said composition is 1:12:2.
[098] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated enzyme on skin, said method comprising: (a) obtaining a
composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide; and (b)
contacting said composition with skin, wherein said composition further comprises suitable
additives.
[099] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a
composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable
additives; and (b) contacting the said composition with skin, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 – 1:12:2.5.
[100] In an embodiment of present disclosure, there is provided a method of ameliorating the
effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a
composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable
additives; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is 1:8:1.
[101] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a
composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable
additives; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is 1:8:1.4.

[102] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable additives; and (b) contacting the said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:8:2.
[103] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable additives; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.
[104] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable additives; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.4.
[105] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable additives; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:2.
[106] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable additives; and (b) contacting the said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:12:1.
[107] In an embodiment of the present disclosure, there is provided a method of ameliorating the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable additives; and (b) contacting said composition with skin, wherein and xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:12:1.4.

[108] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated enzymes on skin, said method comprising: (a) obtaining a
composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable
additives; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is 1:12:2.
[109] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated enzymes on skin as described herein, wherein said senescence
associated enzyme is selected from a group consisting of senescence associated beta
galactosidase, mitogen activated protein kinase, superoxide dismutase, sirtuin, and combinations
thereof.
[110] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated enzymes on skin as described herein, wherein said senescence
associated enzyme is senescence associated beta galactosidase.
[111] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated enzymes on skin as described herein, wherein said senescence
associated enzyme is mitogen activated protein kinase.
[112] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated enzymes on skin as described herein, wherein said senescence
associated enzyme is superoxide dismutase.
[113] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated enzymes on skin as described herein, wherein said senescence
associated enzyme is sirtuin.
[114] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated beta-galactosidase on skin, said method comprising: (a)
obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and
(b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide
w/w ratio in the said composition is in the range 1:8:0.4-1:12:2; and (b) contacting said
composition with skin.
[115] In an embodiment of present disclosure, there is provided a method of ameliorating the
effect of senescence associated beta-galactosidase on skin, said method comprising: (a) obtaining
a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (b)

contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide
w/w ratio in said composition is in 1:10:1.
[116] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated beta-galactosidase on skin, said method comprising: (a)
obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and
(b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide
w/w ratio in said composition is 1:10:1.4.
[117] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated beta-galactosidase on skin, said method comprising: (a)
obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and
(b) contacting said composition with skin, wherein xanthohumol to chrysin to royal jelly peptide
w/w ratio in said composition is 1:10:2.
[118] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated beta-galactosidase on skin, said method comprising: (a)
obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and
(iv) suitable additives; and (b) contacting said composition with skin, wherein xanthohumol to
chrysin to royal jelly peptide w/w ratio in the said composition is in the range 1:8:0.4-1:12:2; and
(b) contacting said composition with skin.
[119] In an embodiment of present disclosure, there is provided a method of ameliorating the
effect of senescence associated beta-galactosidase on skin, said method comprising: (a) obtaining
a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable
additives; and (b) contacting said composition with skin, wherein xanthohumol to chrysin to
royal jelly peptide w/w ratio in said composition is in 1:10:1.
[120] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated beta-galactosidase on skin, said method comprising: (a)
obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and
(iv) suitable additives; and (b) contacting said composition with skin, wherein xanthohumol to
chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.4.
[121] In an embodiment of the present disclosure, there is provided a method of ameliorating
the effect of senescence associated beta-galactosidase on skin, said method comprising: (a)
obtaining a composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and

(iv) suitable additives; and (b) contacting said composition with skin, wherein xanthohumol to
chrysin to royal jelly peptide w/w ratio in said composition is 1:10:2.
[122] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising: (i) xanthohumol; (ii) chrysin; and (iii) royal jelly peptide, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of
1:8:0.4 - 1:12:2.5, said process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c)
obtaining royal jelly peptide; and (d) contacting xanthohumol, chrysin, and royal jelly peptide to
obtain a composition.
[123] In an embodiment of present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin and royal jelly peptide, said process comprising:
(a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; and (d)
contacting xanthohumol, chrysin, and royal jelly peptide to obtain a composition, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition in 1:8:1.
[124] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin and royal jelly peptide, said process comprising:
(a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; and (d)
contacting xanthohumol, chrysin, and royal jelly peptide to obtain a composition, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:8:1.4.
[125] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin and royal jelly peptide, said process comprising:
(a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; and (d)
contacting xanthohumol, chrysin, and royal jelly peptide to obtain a composition, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:8:2.
[126] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin and royal jelly peptide, said process comprising:
(a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; and (d)
contacting xanthohumol, chrysin, and royal jelly peptide to obtain a composition, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.
[127] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin and royal jelly peptide, said process comprising:
(a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; and (d)

contacting xanthohumol, chrysin, and royal jelly peptide to obtain a composition, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of
1:10:1.4.
[128] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin and royal jelly peptide, said process comprising:
(a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; and (d)
contacting xanthohumol, chrysin, and royal jelly peptide to obtain a composition, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of
1:10:2.
[129] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin and royal jelly peptide, said process comprising:
(a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; and (d)
contacting xanthohumol, chrysin, and royal jelly peptide to obtain a composition, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of
1:12:1.
[130] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin and royal jelly peptide, said process comprising:
(a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; and (d)
contacting xanthohumol, chrysin, and royal jelly peptide to obtain a composition, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of
1:12:1.4.
[131] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin and royal jelly peptide, said process comprising:
(a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; and (d)
contacting xanthohumol, chrysin, and royal jelly peptide to obtain a composition, wherein
xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of
1:12:2.
[132] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising: (i) xanthohumol; (ii) chrysin; (iii) royal jelly peptide; and (iv) suitable
additives, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is
in the range of 1:8:0.4 - 1:12:2.5, said process comprising: (a) obtaining xanthohumol; (b)

obtaining chrysin; (c) obtaining royal jelly peptide; (d) obtaining suitable additives; and (e)
contacting xanthohumol, chrysin, royal jelly peptide, and suitable additives to obtain a
composition.
[133] In an embodiment of present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin, royal jelly peptide and suitable additives, said
process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly
peptide; (d) obtaining suitable additives; and (e) contacting xanthohumol, chrysin, royal jelly
peptide, and suitable additives to obtain a composition, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is 1:8:1.
[134] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin, royal jelly peptide and suitable additives, said
process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly
peptide; (d) obtaining suitable additives; and (e) contacting xanthohumol, chrysin, royal jelly
peptide, and suitable additives to obtain a composition, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is 1:8:1.4.
[135] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin, royal jelly peptide and suitable additives, said
process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly
peptide; (d) obtaining suitable additives; and (e) contacting xanthohumol, chrysin, royal jelly
peptide, and suitable additives to obtain a composition, wherein xanthohumol to chrysin to royal
jelly peptide w/w ratio in said composition is 1:8:2.
[136] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin, royal jelly peptide and suitable additives, said
process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly
peptide; (d) obtaining suitable additives; and (e) contacting xanthohumol, chrysin, royal jelly
peptide, and suitable additives to obtain a composition, wherein xanthohumol to the chrysin to
royal jelly peptide w/w ratio in said composition is 1:10:1.
[137] In an embodiment of the present disclosure, there is provided a process for preparing a
composition comprising xanthohumol, chrysin, royal jelly peptide and suitable additives, said
process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly
peptide; (d) obtaining suitable additives; and (e) contacting xanthohumol, chrysin, royal jelly

peptide, and suitable additives to obtain a composition, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of 1:10:1.4.
[138] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising xanthohumol, chrysin, royal jelly peptide and suitable additives, said process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; (d) obtaining suitable additives; and (e) contacting xanthohumol, chrysin, royal jelly peptide, and suitable additives to obtain a composition, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of 1:10:2.
[139] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising xanthohumol, chrysin, royal jelly peptide and suitable additives, said process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; (d) obtaining suitable additives; and (e) contacting xanthohumol, chrysin, royal jelly peptide, and suitable additives to obtain a composition, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of 1:12:1.
[140] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising xanthohumol, chrysin, royal jelly peptide and suitable additives, said process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; (d) obtaining suitable additives; and (e) contacting xanthohumol, chrysin, royal jelly peptide, and suitable additives to obtain a composition, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of 1:12:1.4.
[141] In an embodiment of the present disclosure, there is provided a process for preparing a composition comprising xanthohumol, chrysin, royal jelly peptide and suitable additives, said process comprising: (a) obtaining xanthohumol; (b) obtaining chrysin; (c) obtaining royal jelly peptide; (d) obtaining suitable additives; and (e) contacting xanthohumol, chrysin, royal jelly peptide, and suitable additives to obtain a composition, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of 1:12:2.
[142] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.

[143] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of the disclosure and is not intended to be taken restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
Example 1
Materials and methods
[144] Chrysin and xanthohumol were procured from Sigma Aldrich, and GrB peptide was procured from from USV limited, Mumbai. The royal jelly peptide was custom synthesized from USV limited, Mumbai and possesses the amino acid sequence of Acetylated-CGYIGSR-NH2. Human dermal fibroblast (HDF) cells were procured from LONZA, fibroblast basal media from Invitrogen and cellular senescence kit from Cell bio labs.
[145] Method of preparation of composition comprising xanthohumol, chrysin and royal jelly peptide: Xanthohumol, chrysin and royal jelly peptide at particular concentrations were dissolved in an DMSO (Dimethyl sulfoxide). Further, the composition may comprise additives, but not limited to, emollients, pH adjusters, preservatives, and any other cosmetically suitable ingredient in order to formulate various recipes for topical application and/or delivery. Such methodologies are known to a person of ordinary skill in the art.
[146] The HDF cells were exposed to 100µM H2O2 for 1 hour. Cells were then washed with phosphate buffer saline (1X PBS) and incubated with different concentrations of chrysin (0.002, 0.0015, 0.001, 0.0005 and 0.00025 %), xanthohumol (0.003, 0.002, 0.001, 0.0005, 0.00001, and 0.00005 %), royal jelly peptide (0.005, 0.0025, 0.00125, 0.000625, 0.0003125, 0.000156, 0.000078, 0.000039 %), GrB peptide (0.00125, 0.000625, 0.000313, 0.000516, 0.000078, 0.000039 %) or combinations of xanthohumol, chrysin and royal jelly peptide (as shown in Table 1 below), and 10 µM H2O2 for 24 hours.

[147] Cellular senescence assay: Senescent cells are characterized by an irreversible G1 growth arrest and altered cellular morphology which compromise the skin’s elasticity and integrity paving the way towards aging. Senescence-associated β-galactosidase (SA-β-gal) is a characteristic enzymatic marker detected at acidic pH in senescent cells, in culture, and mammalian tissues.
[148] For the senescence studies on primary human dermal fibroblast (HDF) (Lonza) cells, equal number of cells (10, 000) were seeded in 12-well plates in DMEM (Dulbecco's Modified Eagle's medium) medium containing 10% fetal calf serum (FCS) (Gibco) and penicillin (100 µg/ml) or streptomycin (100 µg/ml) and incubated at 37 °C in humidified chamber in 5% CO2. Adherent HDF cells were exposed to 250 µM hydrogen peroxide (H2O2) for one hour. After the treatment of cells with H2O2, cells were treated with the xanthohumol, chrysin, royal jelly peptide, or combinations thereof for 48 hours along with 10 µM H2O2. Subsequently, the cells were lysed and processed according to the manufacturer’s protocol (CELLBIOLABS, Catalog no. CBA-231). The principle behind this assay is that when a fluorescent dye/substrate of a certain wavelength is added to the cells/ cell lysates, due to enzymatic reaction the substrate gets converted to a product of a different wavelength, the difference in the fluorescent values are read in a fluorescence plate reader. The values are expressed in Relative fluorescence units (RFU). Briefly, cells were lysed in 1X cell lysis buffer and incubated at 4 °C for 5 minutes. The total cell lysate was then transferred to a micro centrifuge tube and centrifuged for 10 minutes at 4 °C and the cell supernatant was collected.

[149] The total protein concentration of each cell lysate sample was determined by a protein estimation method using nano-orange protein purification kit from Thermofisher. The cell lysate (approx. 50 μL) was transferred to a 96-well plate, to this 50 μL of freshly prepared 2X assay buffer was added, thoroughly mixed and incubated at 37 °C, under protection from light, for 3 hrs. To this reaction mixture, 200 μL of Stop solution was added and fluorescence was measured with a fluorescence plate reader at 360 nm (Excitation) / 465 nm (Emission).
Example 2
Results
[150] Figure 1 depicts the effect of varying concentrations of xanthohumol on the viability of
human dermal fibroblast cells. As seen in Figure 1, with decreasing concentrations of
xanthohumol ranging from 0.003 - 0.00005%, a dose dependent increase in viability of human
dermal fibroblasts from 2.1 - 135% can be seen. Based on the observed results, a concentration
of 0.00005% xanthohumol was selected for subsequent analysis which results in about 35%
increase in the viability of human dermal fibroblast cells as compared to the vehicle control,
which is 0.1% DMSO.
[151] Figure 2 depicts the effect of chrysin at varying concentrations on viability of human
dermal fibroblast cells. As seen in Figure 2, with decreasing concentrations of chrysin ranging
from 0.002 - 0.00025%, a dose dependent increase in the range of 66.1 - 115.7% in the viability
of human dermal fibroblasts was observed. Based on the results, a concentration of 0.0005%
chrysin was selected for subsequent analysis, which results in about 15% increase in the viability
of human dermal cells as compared to the vehicle control.
[152] Figure 3 depicts the effect of royal jelly peptide at varying concentrations on the viability
on human dermal fibroblast cells. As seen in Figure 3, royal jelly peptide did not affect the
viability of human dermal fibroblast cells significantly. Based on the results, concentrations
below 0.000156% were selected for subsequent analyses.
[153] From the results as shown in Figure 1, Figure 2 and Figure 3, it can be inferred that upon
exposure to a particular concentration of xanthohumol, chrysin, or royal jelly peptide, human
dermal fibroblast cells were viable and thus particular non-toxic concentrations were selected for
subsequent studies.
[154] Next, the effect of varying concentrations of GrB peptide on eliciting the viability of
human dermal fibroblast cells was investigated. As seen in Figure 4, by reducing the

concentration of GrB peptide, an increase in cell viability was observed. Based on the data, it can be inferred that concentrations of 0.000078% and below shows enhanced cell viability on human dermal fibroblast cells.
[155] For the effect of a combination of xanthohumol, chrysin, and royal jelly peptide on expression of senescence associated beta-galactosidase (SA-β-gal), various ratios were tested. As seen in Figure 5, at a combination of xanthohumol, chrysin and royal jelly peptide in w/w ratio of 1:10:2.8, no synergy was observed. The level of expression of SA-β-gal at this particular ratio was similar than the individual effects of xanthohumol and chrysin The result here thus indicates that the combination of xanthohumol at 0.00005%, chrysin at 0.0005%, and royal jelly peptide at 0.00014% does not decrease SA-β-gal protein expression significantly as compared to corresponding decrease exhibited by individual components.
[156] Figure 6, depicts the effect of another combination of xanthohumol, chrysin, and royal jelly peptide (w/w ratio of 1:10:7) on the expression of senescence associated beta-galactosidase (SA-β-gal). As seen in Figure 6, percent reduction in the expression of beta-galactosidase (SA-β-gal) when a combination of xanthohumol, chrysin and royal jelly peptide at w/w ratio of 1:10:7 was tried no synergy was observed in the inhibition of senescence associated beta galactosidase (SA-β-gal) expression in human dermal fibroblast cells. Here also, effect of a combination of xanthohumol, chrysin, and royal jelly peptide at a ratio of 1:10:7 on beta-galactosidase not significant, at 90% inhibition compared to the individual components of chrysin and royal jelly peptide, which showed an inhibition of 94 and 95 % respectively, demonstrating a clear lack of synergistic activity of the three components of xanthohumol, chrysin and royal jelly peptide at this specific ratio.
[157] Figure 7, depicts the effect of a combination of GrB peptide with xanthohumol and chrysin on expression of senescence associated beta-galactosidase (SA-β-gal) when used in the place of royal jelly peptide. This combination did not have any synergistic effect and its effect was less than the individual effect of xanthohumol and chrysin. The results indicate that the combination of xanthohumol at 0.00005%, chrysin at 0.0005% and GrB peptide at 0.00007% concentration (1:10:1.4) does not decrease SA-B-gal protein with respect to individual actives. [158] Figure 8, depicts the effect of a combination of xanthohumol, chrysin, and royal jelly peptide at a w/w ratio of 1:10:1.4 on expression of senescence associated galactosidase (SA-β-gal). Unexpectedly and surprisingly, this combination showed an enhanced inhibition of

senescence associated galactosidase (SA-β-gal) expression. There is approximately a 35% reduction in the expression of senescence associated galactosidase (SA-β-gal), which is more than the effect of xanthohumol, chrysin, or royal jelly peptide alone. The results indicate the combination of xanthohumol at 0.00005%, chrysin at 0.0005% and royal jelly peptide at 0.00007% concentrations function synergistically to inhibit senescence associated beta galactosidase (SA-β-gal). The inhibitory activity demonstrated by this combination of the constituents cannot be obtained by a mere admixture of the three and requires the presence of xanthohumol, chrysin and royal jelly peptide at very specific ratios to demonstrate distinct synergy, which cannot be obtained without undue experimentation. Further, substituting the constituents with others, such as GrB, even at the same specific ratios, as seen in Figure 7, does not produce the same result. Therefore, this synergistic and unique combination of xanthohumol, chrysin and royal jelly peptide may potentially help in decreasing cellular senescence and combating the signs of aging skin.
[159] Overall, the present disclosure provides a composition comprising xanthohumol, chrysin, and royal jelly peptide which at particular concentrations and w/w ratios exhibits a surprising and unexpected synergistic effect in inhibiting expression of senescence associated beta-galactosidase in human dermal cells. It is to be appreciated that the observed synergism in particular instances in not a result of mere aggregation of the individual properties of chrysin, xanthohumol and royal jelly peptide as chrysin or xanthohumol or royal jelly peptide alone at any concentration, or in combination at various other w/w ratios and concentrations do not show any significant enhancement of inhibition of expression of senescence associated beta-galactosidase. In fact, based on the effect of chrysin or xanthohumol or royal jelly peptide alone on inhibiting expression of senescence associated beta-galactosidase, a person skilled in the art would find motivation to assess the effect of a combination of chrysin, xanthohumol and royal jelly peptide on modulating expression of senescence associated beta-galactosidase. It is to be noted that it is contemplated that it is within the purview of a person skilled in the art to incorporate the essential disclosure of the present specification into a host of formulations, particularly for topical use.

1. A composition comprising:
(a) xanthohumol;
(b) chrysin; and
(c) royal jelly peptide,
wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of 1:8:0.4 - 1:12:2.5.
2. The composition as claimed in claim 1, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is in the range of 1:10:1 - 1:10:2.
3. The composition as claimed in claim 1, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.
4. The composition as claimed in claim 1, wherein xanthohumol to chrysin to royal jelly peptide w/w ratio in said composition is 1:10:1.4.
5. The composition as claimed in any of the claims 1-4, further comprising suitable additives.
6. The composition as claimed in any of the claims 1-5, wherein said composition is effective in ameliorating the deleterious effects of senescence associated enzymes on skin.
7. The composition as claimed in claim 6, wherein said senescence associated enzyme is selected from the group consisting of senescence associated beta-galactosidase, mitogen activated protein kinase, superoxide dismutase, sirtuin, and combinations thereof.
8. A method for ameliorating the effect of senescence associated enzymes on skin, said method comprising:

(a) obtaining the composition as claimed in any of the claims 1-5; and
(b) contacting said composition with skin.
9. A process for preparing a composition as claimed in any of the claims 1-4, said process
comprising:
(a) obtaining xanthohumol;
(b) obtaining chrysin;
(c) obtaining royal jelly peptide; and
(d) contacting xanthohumol, chrysin, and royal jelly peptide to obtain a composition.

10. A process for preparing a composition as claimed in any of the claims claim 1- 5, said process comprising:
(a) obtaining xanthohumol;
(b) obtaining chrysin;
(c) obtaining royal jelly peptide;
(d) obtaining suitable additives; and
(e) contacting xanthohumol, chrysin, royal jelly peptide, and suitable additives to obtain a composition.