FIELD OF INVENTION
[001] The present disclosure relates to the field of skin-aging in general, and the role of reduced collagen synthesis in skin aging, in particular. There is provided herein a composition comprising apple fruit cellular extract and adenosine triphosphate and uses thereof.
BACKGROUND OF THE INVENTION
[002] There is a variety of factors that governs the process of skin aging. Among other things, the process is primarily characterized by a decrease in synthesis of extracellular matrix (ECM) proteins like collagen and elastin, increase in protease activity leading to degradation of ECM, and decrease in epidermal turnover. Skin aging manifests in the form of fine lines and wrinkles on facial skin and other parts of the body. As the skin aging process progresses, variations in the desquamation pattern are seen. Desquamation refers to the scraping off of an outer layer of the skin, which disrupts the epidermal barrier resulting in the loss of natural moisturizing factors. Uncontrolled dehydration severely affects ECM maintenance and continuity as a result of which skin becomes dry and thin. Increase in the levels of collagen by boosting the collagen synthesis, can decreases facial fine lines (MinYaar et al., J. Invest. Dermatol. Symp. Proc.; 7:51 -58; Ganceviciene et al., Dermatoendocrinol.; 4(3):308-319; 2012).
[003] There are plenty of prior art documents disclosing various
compounds/compositions aimed at ameliorating the problem of skin aging, some of which include retinol, epidermal growth factors, ascorbic acid and argireline, among others. However, the problem of skin aging has still not been adequately addressed and therefore there is a need to identify new formulations.
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SUMMARY OF INVENTION
[004] The subject matter described herein is directed towards a synergistic composition comprising apple fruit cellular extract and adenosine triphosphate. [005] In an aspect of the present disclosure, there is provided a composition comprising: (a) apple fruit cellular extract; and (b) adenosine triphosphate, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:10-1:20.
[006] In an aspect of the present disclosure, there is provided a method of inhibiting the appearance of skin-aging, said method comprising:
(a) obtaining a composition comprising of (i) apple fruit cellular extract; and (ii) adenosine triphosphate; and
(b) contacting said composition with skin,
wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:10-1:20.
[007] In an aspect of the present disclosure, there is provided a method of preparing a composition comprising: (a) apple fruit cellular extract; and (b) adenosine triphosphate, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:10-1:20, said process comprising the steps of
(a) obtaining apple fruit cellular extract;
(b) obtaining adenosine triphosphate; and
(c) contacting apple fruit cellular extract and adenosine triphosphate to obtain said
composition.
[008] 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.
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BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[009] 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.
[0010] Figure 1 depicts the graphical representation of the effect of apple fruit
cellular extract (ACE) at different concentrations on cell viability, in accordance with
an embodiment of the present disclosure.
[0011] Figure 2 depicts the graphical representation of the effect of adenosine
triphosphate (ATP) at various percent concentrations on cell viability, in accordance
with an embodiment of the present disclosure.
[0012] Figure 3 depicts the graphical representation of the percentage increase of
collagen synthesis at 0.0006% of the combination of ACE and ATP (AA), in
accordance with an embodiment of the present disclosure.
[0013] Figure 4 depicts the graphical representation of the effect of 0.0002% of ACE
and ATP (AA) on collagen synthesis, in accordance with an embodiment of the
present disclosure.
[0014] Figure 5 depicts the graphical representation of the effect of 0.002% of ACE
and ATP (AA) on collagen synthesis, in accordance with an embodiment of the
present disclosure.
[0015] Figure 6 depicts the retention time for chlorogenic acid standard.
[0016] Figure 7 depicts the retention time of chlorogenic acid in the ACE.
DETAILED DESCRIPTION OF THE INVENTION
[0017] 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.
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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.
Definition s
[0018] 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.
[0019] 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.
[0020] 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”.
[0021] 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.
[0022] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.
[0023] 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.
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[0024] The present disclosure relates to the problem of skin aging which is a by-product of a decrease in the synthesis of collagen and elastin proteins. The present invention provides a synergistic formulation of apple fruit extract and adenosine triphosphate which enhance collagen protein synthesis, thus providing a solution to slow down the skin aging process by prolonging collagen production.
[0025] 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.
[0026] 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.
[0027] In an embodiment of the present disclosure there is provided a composition comprising: (a) apple fruit cellular extract; and (b) adenosine triphosphate, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:10-1:20
[0028] In an embodiment of the present disclosure, there is provided a composition for inhibiting skin-aging, said composition comprising (a) apple fruit cellular extract; (b) adenosine triphosphate; and (c) suitable carriers, diluents, and excipients.
[0029] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said cosmetically suitable carriers, diluents, and excipients are selected from the group consisting of sunscreen agents, antioxidants, stabilizers, emollients, solvents, preservatives and combinations thereof.
6

[0030] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein apple fruit cellular extract to adenosine triphosphate w/w
ratio in said composition is 1:10-1:20, and wherein said composition synergistically
enhances collagen protein synthesis.
[0031] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein apple fruit cellular extract to adenosine triphosphate w/w
ratio in said composition is 1:14.7, and wherein said composition synergistically
enhances collagen protein synthesis.
[0032] In an embodiment of the present disclosure, there is provided a composition
comprising:
(a) apple fruit cellular extract having a weight percentage in said composition in the
range of 0.000025-0.00011%; and
(b) adenosine triphosphate having a weight percentage in said composition in the
range of 0.00037- 0.0017%, wherein apple fruit cellular extract to adenosine
triphosphate w/w ratio in said composition is 1:10-1:20
[0033] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein adenosine triphosphate weight percentage in said
composition is 0.00056%.
[0034] In an embodiment of the present disclosure, there is provided a composition
as described herein, wherein apple fruit cellular extract weight percentage in said
composition is 0.000038%.
[0035] In an embodiment of the present disclosure, there is provided a composition
comprising:
(a) apple fruit cellular extract having weight percentage of 0.000038% in said
composition is; and
(b) adenosine triphosphate having weight percentage of 0.00056% in said
composition,
wherein said composition synergistically enhances collagen protein synthesis.
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[0036] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:14.7, wherein:
(a) apple fruit cellular extract weight percentage in said composition is in the range of
0.000025-0.00011%; and
(b) adenosine triphosphate weight percentage in said composition is in the range of
0.00037- 0.0017%.
[0037] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:14.7, wherein,
(a) apple fruit cellular extract weight percentage in said composition is 0.000038%;
and
(b) adenosine triphosphate weight percentage in said composition is 0.00056%,
wherein said composition synergistically enhances collagen protein synthesis.
[0038] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said composition can be for topical application.
[0039] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said composition can be in the form of an aerosol.
[0040] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said composition can be in the form of a lotion.
[0041] In an embodiment of the present disclosure, there is provided a composition as described herein, wherein said composition can be in form of an oil.
[0042] 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.
[0043] In an embodiment of the present disclosure, there is provided a method of preparing a composition comprising: (a) apple fruit cellular extract; (b) adenosine triphosphate; and (c) cosmetically suitable carriers, diluents, and excipients.
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[0044] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein apple fruit cellular extract to
adenosine triphosphate w/w ratio in said composition is in the range of 1:10-1:20.
[0045] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein apple fruit cellular extract to
adenosine triphosphate w/w ratio in said composition is 1:14.7.
[0046] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein apple fruit cellular extract
weight percentage in said composition is in the range of 0.000025-0.00011%.
[0047] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein adenosine triphosphate weight
percentage in said composition is in the range of 0.00037- 0.0017%.
[0048] In an embodiment of the present disclosure, there is provided a method of
preparing a composition as described herein, wherein:
(a) apple fruit cellular extract weight percentage in said composition is in the range
of 0.000025-0.00011%; and
(b) adenosine triphosphate weight percentage in said composition is in the range of
0.00037- 0.0017%.
[0049] In an embodiment of the present disclosure, there is provided a method of preparing a composition as described herein, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:10-1:20, wherein:
(a) apple fruit cellular extract weight percentage in said composition is in the range of
0.000025-0.00011%; and
(b) adenosine triphosphate weight percentage in said composition is in the range of
0.00037- 0.0017%.
[0050] In an embodiment of the present disclosure, there is provided a method of preparing a composition as described herein, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:14.7, wherein:
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(a) apple fruit cellular extract weight percentage in said composition is in the range of
0.000025-0.00011%; and
(b) adenosine triphosphate weight percentage in said composition is in the range of
0.00037- 0.0017%.
[0051] In an embodiment of the present disclosure, there is provided a method of preparing a composition as described herein, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:14.7, wherein:
(a) apple fruit cellular extract weight percentage in said composition is 0.000038%; and
(b) adenosine triphosphate weight percentage in said composition is 0.00056%. [0052] In an embodiment of the present disclosure, there is provided a method of preparation of apple fruit cellular extract, said method comprising:
(a) obtaining callus mass developed from apple fruit explant;
(b) culturing tissue obtained from callus mass under constant shaking in the absence
of light for a time period in the range of 20-30 days to obtain apple stem cells;
(c) processing said apple stem cells into a paste;
(d) adding at least one preservative having a weight percentage of 0.2-0.4%, and at least one antioxidant having a weight percentage in the range of 0.1-0.3% to said paste; and
(e) lyophilizing said paste from step (d) for a period of 15-20 hours to obtain apple fruit cellular extract.
[0053] In an embodiment of the present disclosure, there is provided a method of preparation of apple fruit cellular extract, said method comprising:
(a) obtaining callus mass developed from apple fruit explant;
(b) culturing tissue obtained from callus mass under constant shaking in the absence
of light for a time period in the range of 28 days to obtain apple stem cells; and
(c) processing said apple stem cells into a paste;
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(d) adding at least one preservative at a weight percentage of 0.2-0.4%, and at least one antioxidant at a weight percentage in the range of 0.1-0.3% to said paste; and
(e) lyophilizing said paste for a period of 15-20 hours to obtain apple fruit cellular extract.
[0054] In an embodiment of the present disclosure, there is provided a method of preparation of apple fruit cellular extract, said method comprising:
(a) obtaining callus mass developed from apple fruit explant;
(b) culturing tissue obtained from callus mass under constant shaking in the absence
of light for a time period of 28 days to obtain apple stem cells; and
(c) processing said apple stem cells into a paste;
(d) adding at least one preservative having weight percentage of 0.3 %, and at least one antioxidant having weight percentage in the range of 0.2 % to said paste; and
(e) lyophilizing said paste for a period of 15-20 hours to obtain apple fruit cellular extract.
[0055] In an embodiment of the present disclosure, there is provided a method of preparation of apple fruit cellular extract, said method comprising:
(a) obtaining callus mass developed from apple fruit explant;
(b) culturing tissue obtained from callus mass under constant shaking in the absence
of light for a time period of 28 days to obtain apple stem cells; and
(c) processing said apple stem cells into a paste;
(d) adding at least one preservative having weight percentage of 0.3 %, and at least one antioxidant having weight percentage in the range of 0.2 % to said paste; and
(e) lyophilizing said paste for a period of 18 hours to obtain apple fruit cellular extract.
[0056] In an embodiment of the present disclosure, there is provided a method of preparation of apple fruit cellular extract, said method comprising: (a) obtaining callus mass developed from apple fruit explant;
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(b) culturing tissue obtained from callus mass under constant shaking in the absence
of light for a time period of 28 days to obtain apple stem cells; and
(c) processing said apple stem cells into a paste;
(d) adding at least one preservative having weight percentage of 0.2-0.4%, and at least one antioxidant having weight percentage in the range of 0.1-0.3 % to said paste; and
(e) lyophilizing said paste for a period of 18 hours to obtain apple fruit cellular extract.
[0057] In an embodiment of the present disclosure, there is provided a method of
preparation of apple fruit cellular extract as described herein, wherein at least one
preservative is selected from the group consisting of sodium benzoate, acetic acid,
nisin, propionic acid, nitrates, and nitrites.
[0058] In an embodiment of the present disclosure, there is provided a method of
preparation of apple fruit cellular extract as described herein, wherein the
preservative is sodium benzoate.
[0059] In an embodiment of the present disclosure, there is provided a method of
preparation of apple fruit cellular extract as described herein, wherein at least one
antioxidant is selected from the group consisting of L-ascorbic acid, citric acid,
butylated hydroxy anisole, butylated hydroxy toluene.
[0060] In an embodiment of the present disclosure, there is provided a method of
preparation of apple fruit cellular extract as described herein, wherein the antioxidant
is L-ascorbic acid.
[0061] In an embodiment of the present disclosure, there is provided a method of
preparation of apple fruit cellular extract, said method comprising:
(a) obtaining callus mass developed from apple fruit explant;
(b) culturing tissue obtained from callus mass under constant shaking in the absence
of light for a time period in the range of 20-30 days to obtain apple stem cells; and
(c) processing said apple stem cells into a paste;
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(d) adding sodium benzoate having weight percentage of 0.2-0.4%, and L-ascorbic acid having weight percentage in the range of 0.1-0.3%, to said paste; and
(e) lyophilizing said paste for a period of 15-20 hours to obtain apple fruit cellular extract.
[0062] In an embodiment of the present disclosure, there is provided a method of preparation of apple fruit cellular extract, said method comprising:
(a) obtaining callus mass developed from apple fruit explant;
(b) culturing tissue obtained from callus mass under constant shaking in the absence
of light for a time period of 28 days to obtain apple stem cells; and
(c) processing said apple stem cells into a paste;
(d) adding sodium benzoate having weight percentage of 0.2-0.4%, and L-ascorbic acid having weight percentage in the range of 0.1-0.3 %, to said paste; and
(e) lyophilizing said paste for a period of 18 hours to obtain apple fruit cellular extract.
[0063] In an embodiment of the present disclosure, there is provided a method of preparation of apple fruit cellular extract, said method comprising:
(a) obtaining callus mass developed from apple fruit explant;
(b) culturing tissue obtained from callus mass under constant shaking in the absence
of light for a time period of 28 days to obtain apple stem cells; and
(c) processing said apple stem cells into a paste;
(d) adding sodium benzoate having weight percentage of 0.3 %, and L-ascorbic acid having weight percentage in the range of 0.2 % to said paste; and
(e) lyophilizing said paste for a period of 18 hours to obtain apple fruit cellular extract.
[0064] In an embodiment of the present disclosure, there is provided a method of inhibiting skin-aging, said method comprising:
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(a) obtaining a composition comprising of (i) apple fruit cellular extract; (ii) adenosine triphosphate; and (iii) suitable carriers, diluents, and excipients; and
[0065] (b) contacting said composition with skin, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:10-1:20.
[0066] In an embodiment of the present disclosure, there is provided a method of inhibiting skin-aging as described herein, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:14.7, and wherein said method synergistically enhances collagen protein synthesis.
[0067] In an embodiment of the present disclosure, there is provided a method of inhibiting skin-aging as described herein, wherein apple fruit cellular extract weight percentage in said composition is in the range of 0.000025-0.00011% and adenosine triphosphate weight percentage in said composition is in the range of 0.00037-0.0017%, and wherein said method synergistically enhances collagen protein synthesis.
[0068] In an embodiment of the present disclosure, there is provided a method of inhibiting skin-aging as described herein, wherein apple fruit cellular extract weight percentage in said composition is 0.000038%, and wherein said method synergistically enhances collagen synthesis.
[0069] In an embodiment of the present disclosure, there is provided a method of inhibiting skin-aging as described herein, wherein adenosine triphosphate weight percentage in said composition is 0.00056%, and wherein said method synergistically enhances collagen synthesis.
[0070] In an embodiment of the present disclosure, there is provided a method of inhibiting skin-aging as described herein, wherein apple fruit cellular extract weight percentage in said composition is 0.000038% and adenosine triphosphate weight percentage in said composition is 0.00056%, and wherein said method synergistically enhances collagen protein synthesis.
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[0071] In an embodiment of the present disclosure, there is provided a method of cosmetically inhibiting skin-aging as described herein, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:10-1:20, wherein:
(a) apple fruit cellular extract weight percentage in said composition is in the range of
0.000025-0.00011%; and
(b) adenosine triphosphate weight percentage in said composition is in the range of
0.00037- 0.0017%;
wherein said method synergistically enhances collagen synthesis.
[0072] In an embodiment of the present disclosure, there is provided a method of cosmetically inhibiting skin-aging as described herein, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:14.7, wherein:
(a) apple fruit cellular extract weight percentage in said composition is in the range of
0.000025-0.00011%; and
(b) adenosine triphosphate weight percentage in said composition is in the range of
0.00037-0.0017%;
wherein said method synergistically enhances collagen synthesis.
[0073] In an embodiment of the present disclosure, there is provided a method of cosmetically inhibiting skin-aging as described herein, wherein apple fruit cellular extract to adenosine triphosphate w/w ratio in said composition is 1:14.7, wherein:
(a) apple fruit cellular extract weight percentage in said composition is 0.000038%; and
(b) adenosine triphosphate weight percentage in said composition is 0.00056%; wherein said method synergistically enhances collagen synthesis.
[0074] 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
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that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.
EXAMPLES
[0075] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and 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
Material and Methods
[0076] Preparation of Apple fruit callus from solid culture: Healthy royal gala apple fruit were selected and surface sterilized with 1% sodium hypochlorite for 10 minutes and 1% fungicide (Bavistin) for 10 minutes, followed by 0.05% mercuric chloride for 4 minutes. Apple fruit were washed with sterile water after each treatment under sterile conditions. Fruit tissues were collected and treated with antioxidant solution containing 200 mg PVP, 100 mg myo-inositol and 100 mg ascorbic acid in 1L sterile water for 5-10 minutes.
[0077] The tissue was excised into pieces and inoculated on callus induction media: MS- Salts with 1mg/ L NAA, 3% sucrose as carbon source, 100 mg/L inositol, 200
16

mg/L PVP, 5 mg/L citric acid, gelling agar 0.7%, pH 5.8) and incubated at temperature 24±2 oC in the dark. Callus initiation was observed after 21 days.
[0078] The callus induced from the apple fruit was excised and multiplied on callus multiplication media with the same ingredients except using MS- Salts with 2,4-D in the place of NAA.
[0079] Preparation of apple fruit cell suspension culture: Apple fruit callus at 1-2 gm/100 ml of liquid media was weighed. This was then inoculated into liquid callus multiplication media comprising MS- Salts with 1mg/ L 2,4-D, 3% sucrose as carbon source, 100mg/L inositol, 200mg/L PVP, 5mg/L citric acid, pH 5.8. The cells were cultured at 120-140 RPM at a temperature of 24±2 oC without light and harvested after 28 days.
[0080] Apple fruit cellular extract preparation: Apple fruit stem cells were initially ground in a blender to make a fine paste. To this 0.3% (w/w) of sodium benzoate (preservative) and 0.2% (w/w) of L-ascorbic acid (anti-oxidant) were added and mixed thoroughly. The apple fruit stem cell paste was taken in to a freeze drying flask and was frozen using a 1:4 mixture of liquid nitrogen and acetone. The flask was connected to a lyophilizer and the content was dried for 18 hours. The freeze dried fluffy off-whitish powder was obtained which was stored at -20 OC in an air-tight container. The stock solution of apple fruit cellular extract and adenosine triphosphate (ATP) at a concentration of 4.7% was prepared, comprising 4.4% ATP and 0.3% apple fruit cellular extract.
[0081] Testing cellular iron uptake: Human Adult dermal fibroblasts (LONZA, India) were seeded in 24- well plates (Costar) in Fibroblast basal media (LONZA). The culture was incubated in 5% CO2 in the incubator and was allowed to reach confluency to form a fibroblast bed. HaCaT keratinocytes (NCCS, India) at a seeding density of 0.05 million cells per well were seeded on the fibroblast bed. Medium 154CF⁄PRF (GIBCO) containing 0.03mM calcium chloride was used while seeding HaCaT keratinocytes. After 24 hrs, the medium was replenished with actives and
17

incubated in 5% CO2 incubator. Cells were harvested after 72 hrs for total collagen estimation. Sircol assay carried out based on manufacturer’s protocol (BIOCOLOR).
Example 2
Biomarker analysis apple fruit cellular extract
[0082] The apple fruit cellular extract so obtained was analysed for specific
biomarker standards. For this purpose, the amount of chlorogenic acid was analysed
using High Performance Liquid Chromatography (HPLC) (Figure 6 and Figure 7).
The chlorogenic acid content in the lyophilised apple cellular extract was determined
as 0.001%.
Example 3
Cellular cytotoxicity measurements of apple fruit cellular extract and adenosine
triphosphate
[0083] Cellular cytotoxicity to human adult dermal fibroblasts at various weight
percentages of apple fruit extract was evaluated in order to determine optimum
dosage levels for activity against inhibiting aging by enhancing levels of collagen
synthesis. As seen in Figure 1, apple fruit cellular extract at various weight
percentages (0.00002-0.017%) does not significantly cause cellular toxicity. In
particular, the cell extract at 0.006%-0.0002% does not show any adverse effect on
cell viability.
[001] Similar to Figure 1, the effect of adenosine triphosphate at various weight percentages on cellular toxicity was also evaluated. As seen in Figure 2, adenosine triphosphate in weight percentages of (0.00001%-0.001%) does not cause cellular toxicity compared to vehicle alone control. In particular, the ATP concentration range from 0.0001%-0.001% does not show any adverse effect on cell viability. [002] Based on the effect of apple fruit cellular extract and adenosine triphosphate on cellular toxicity as shown in Figure 1 and Figure 2 respectively, the concentrations
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of 0.0002%, 0.0006% and 0.002% were chosen to test the effect of the composition on enhancing collagen synthesis.
Example 4
Effect of the combination of apple fruit cellular extract and adenosine
triphosphate on collagen synthesis
[0084] The effect of the composition of apple fruit cellular extract and ATP was tested to determine its effect on collagen protein expression. This composition consisted of apple fruit cellular extract at a weight percentage of 0.3% and ATP at a weight percentage of 4.4%. Thus the composition tested represents a 1:14.7 ratio of apple fruit cellular extract to ATP.
[0085] For the purposes of testing, the stock was diluted to a working concentration of 0.0006% and the effect of the composition at this percentage concentration was tested. As demonstrated in Figure 3, both ATP and apple fruit cellular extract do not affect collagen protein expression in isolation. Cells incubated with ATP and apple fruit cellular extract showed collagen protein expression at 99% and 95%, respectively, similar to the vehicle control. Thus, it can be inferred that in isolation, ATP and apple fruit cellular extract, have negligible effects in affecting collagen synthesis.
[0086] Surprisingly, at a concentration of 0.0006%, the combination of apple fruit cellular extract and ATP remarkably increased collagen protein expression levels at 130% (Figure 3). Thus, at the percentage composition of 0.0006%, a 1:14.7 w/w ratio of apple fruit cellular extract: adenosine triphosphate functions synergistically to enhance collagen synthesis at levels exceeding which the components by themselves could achieve.
[0087] Further, as is seen in Figures 4 and 5, the synergistic effect observed at a 1:14.7 w/w ratio of apple fruit cellular extract: adenosine triphosphate, is specific to this ratio alone. In Figure 4, a lower concentration of the combination of the two
19

constituents at 0.0002% does not show any significant enhancement of collagen levels. Similarly, a higher concentration of 0.002% apple fruit cellular extract: adenosine triphosphate, does not show any synergism to enhance collagen synthesis.
[0088] Overall, the present disclosure provides a composition comprising of apple fruit cellular extract and adenosine triphosphate that at particular w/w ratios synergistically enhances collagen synthesis. Further, at the particular w/w ratio of 1:14.7, there is observed a synergistic increase in collagen protein synthesis. As collagen synthesis is associated with inhibiting skin aging symptoms such as appearance of wrinkles, and fine lines, enhanced collagen synthesis, using a composition as described herein is likely to be useful in cosmetics which yield good results against appearance of fine lines and wrinkles associated with aging.
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