Compositions comprising a cell hydrolysate and polyphenols for topical application

Abstract

A topical composition comprising a cell hydrolysate obtained from an in vitro culture and one or more polyphenols. Use of the composition in the preparation of a product for skincare, cosmetic or wound care purpose. In embodiment, the in vitro cell culture is yellow croaker swim bladder cell, and the polyphenol is resveratrol or epigallocatechin gallate (EGCG).

Description

COMPOSITIONS COMPRISING A CELL HYDROLYSATE AND POLYPHENOLS FOR TOPICAL APPLICATIONTechnical Field

[0001] This invention generally relates to novel compositions comprising a cell hydrolysate from cultivated cells and one or more polyphenols for topical application. Embodiments discussed herein also generally relate to a process of making and uses of compositions comprising a cell hydrolysate from cultivated cells and one or more polyphenols.Background

[0002] Cell hydrolysate contains abundant nutrients and protein factors that stimulate skin repair and regeneration. Products containing or derived from cell hydrolysate have been developed and formulated for topical application for the purposes of skincare, cosmetics or wound care.

[0003] Many existing skincare, wound care, and cosmetic products include either animal-sourced active ingredients or plant-derived polyphenols, or less commonly, a combination of both. Polyphenols (compounds having more than one phenolic hydroxyl group and naturally found in plants) are often added to topical products due to their strong antioxidant, anti-inflammatory, anti-cancer, and antimicrobial properties.

[0004] However, animal-sourced materials raise safety concerns as they may contain environmental contaminants (e.g. heavy metals, antibiotics, microplastics, pesticides), adventitious agents (e.g. bacteria, viruses, fungi, parasites, transmissible spongiform encephalopathy agents), and toxins (e.g. ciguatoxin, tetrodotoxin), thereby posing risks to the users. Furthermore, animal-sourced ingredients may display significant batch-to-batch variations due to changes in the environment and food / feed choices, time of harvest, disease, parasites and so on, and ultimately make it challenging to maintain sufficient level of product consistency.

[0005] Raising animals only for the purpose of obtaining some usable parts is energy and cost inefficient because a significant portion of the supplied nutrients and energy are used by animals to move around, mating, and growth of unusable parts (e.g. animal hair). In some instances, animal-sourcedingredient(s) may only contain one or several related compounds (e.g. collagen) purified from the animal materials. This limits the product’s effectiveness (due to limited availability of compounds) and adds cost due to the purification step required. Relying on ingredients obtained from wild animals also increases the difficulty of product traceability.

[0006] Last but not least, obtaining raw materials from animals may disturb the natural habitat of animals and cause animal suffering and animal sacrifice. This damages the ecosystem and raises animal welfare issues.Summary

[0007] It is an aspect of the present invention to provide a topical composition comprising a cell hydrolysate from cultivated cells and one or more polyphenols, where the topical composition generates synergistic effects and produces more cost-effective products than either the cell hydrolysate or polyphenol(s) alone.

[0008] It is another aspect of the present invention to provide a topical composition for improving skin health or wound conditions. It is another aspect of the present invention to provide a topical composition for stimulating the expression of one or more growth factors in relation to skin health, structure or functions.

[0009] It is another aspect of the present invention to provide a method of improving skin health or wound conditions using the composition described herein.

[0010] It is also an aspect of the present invention to provide a method of producing a topical composition comprising a cell hydrolysate from cultivated cells and one or more polyphenols.Brief Description of the Drawings

[0011] The disclosure may be better understood by reference to the detailed description when considered in connection with the accompanying drawings. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the disclosure.

[0012] FIG. 1 is a flowchart of a method for producing cultivated cell hydrolysate by in vitro cell culture, according to one embodiment of the present disclosure.

[0013] FIG. 2 is a flowchart of evaluation of compositions comprising cultivated cell hydrolysate and polyphenols on human skin cells, according to one embodiment of the present disclosure.

[0014] FIG. 3A-3C are charts illustrating the respective level of gene expression of (a) CTGF (FIG. 3A), (b) HBEGF (FIG. 3B) and (c) TGFA (FIG. 30) relative to the vehicle control in human epidermal keratinocyte when treated with 1 % (w / v) of cell hydrolysate and 100pM resveratrol for 24 hours, according to one embodiment of the present disclosure.

[0015] FIG. 4A-4C are charts illustrating the respective level of gene expression of (a) CTGF (FIG. 4A), (b) HBEGF (FIG. 4B) and (c) TGFA (FIG. 4C) relative to the vehicle control in human epidermal keratinocyte when treated with 1 % (w / v) of cell hydrolysate and 50pM EGCG for 24 hours, according to one embodiment of the present disclosure.Detailed Description

[0016] As mentioned in the background, cell hydrolysate and polyphenols have been used for developing products for the purposes of skincare, cosmetic or wound care.

[0017] On the one hand, many products containing cell hydrolysate are produced using animal-sourced ingredients and have various drawbacks. On the other hand, due to cost, solubility, and regulatory reasons, the concentration of polyphenols in topical products currently available for skincare, cosmetic or wound care uses is often limited. For example, resveratrol (a polyphenol from grapes) is commonly added at a level of 1 -3% (w / v) falling below the level required to achieve maximum effectiveness of the product. Higher dose may be used in the formulation to give higher efficacies or better effects, however this will increase production costs.

[0018] Considering the benefits of cell hydrolysate and polyphenols on skin, and the drawbacks of using animal-sourced ingredients, it is desirable to have a topical product containing the cell hydrolysate and polyphenols without the use of animal-sourced materials.

[0019] In view of the above, it is one aspect of the present invention to provide a topical composition comprising a cultivated cell hydrolysate from in vitro cell culture and one or more polyphenols. The topical composition of the present invention generates synergistic effects and produces more cost-effective products than either cell hydrolysate or polyphenol(s) alone. As used herein, a “synergistic effect” mean an effect when two or more substances being used together resulting in an overall effect that is greater than the sum of individual effects of any of them.

[0020] It is also an aspect of the present invention to provide an alternative method of obtaining animal-derived raw materials using in vitro culture. The derived raw materials (e.g. cell hydrolysate) according to the present invention are free of environmental contaminants, adventitious agents and allergens. It also helps to limit animal suffering and sacrifice associated with the production of animal-derived materials.

[0021] Overall, by using a formulation of a cultivated cell hydrolysate in conjunction with another beneficial ingredient polyphenol, the present invention offers the following non-exhaustive benefits:1. The levels of environmental contaminants, adventitious agents, and toxins in the final product are reduced.2. Providing a more environmentally friendly alternative to generate animal ingredients from cultivated cells for topical applications.3. Improved batch-to-batch consistency and product traceability.4. Reducing animal suffering and sacrifice associated with traditional animal- sourced materials.5. Providing cost-effective topical application products by making use of the synergistic effects between cultivated cell hydrolysate and polyphenols. These products show higher levels and a wider spectrum of benefits as compared to previous products containing just animal-sourced active ingredients or polyphenols alone.6. Reducing production costs because lower amounts of active ingredients are needed to achieve the same level of effects.

[0022] The benefits of the present invention also lead to the creation of new values that meet consumers’ needs. Topical compositions of the present invention includes active ingredients that can help with skincare and wound care with the following characteristics:(1 ) Clean label due to the high purity and non-existence of waste or harmful chemicals in the derived raw materials;(2) Sustainability due to little or low reliance on animal sources;(3) Non-chemical synthesis due to the use of biomaterials;(4) Multi-functional due to its complete molecular profile with all functional proteins naturally produced by the subject cells; and(5) Backed by scientific principles and test results.

[0023] Generating Cell Hydrolysate by In Vitro Cell Culture

[0024] Cell hydrolysate contains numerous nutrients, growth factors, extracellular matrix molecules, and may be used as an active ingredient in dietary supplements, hair care, skincare, wound care, cosmetic or food products. However, existing technologies often require extraction of cell hydrolysate directly from animals, and this approach raises safety concerns because animal-derived materials may contain environmental contaminants (e.g. heavy metal, antibiotics, microplastics, pesticides), adventitious agents (e.g. bacteria, viruses, fungi, transmissible spongiform encephalopathy agents, parasites), and toxins (e.g. ciguatoxins, tetradotoxins).

[0025] Cellular agriculture allows the production of animal-based products by cell cultivation technologies. Compared to sourcing the product directly from animals, this new approach is not only more ecologically friendly, but also provides consumers with products that are free of adventitious agents and environmental contaminants.

[0026] The present invention provides a cellular hydrolysate of cultivated cells as an ingredient for skincare, cosmetic, and wound care purposes. Cultivated cell hydrolysate contains a mixture of functional peptides, lipids, amino acids,and other nutrients from the hydrolysis of the cultivated cell that stimulate skin repair and regeneration.

[0027] In terms of the functional peptides, the cell hydrolysate of the present invention comprises all protein polypeptides and / or protein polypeptides fragments derived thereof from whole cell. In other words, the cell hydrolysate comprises multiple protein polypeptides and / or protein polypeptides fragments derived thereof instead of a single protein polypeptide. As such, the composition of the present invention is multifunctional. Hence, the cell hydrolysate generates a higher level and wider spectrum of effects as compared to only including a few active compounds purified from animal sources. This also serves as a safer alternative to cell hydrolysate extracted directly from animal.

[0028] In one aspect of the present invention, there is provided a method of producing cell hydrolysate from in vitro cell culture.

[0029] In one embodiment, the present invention provides a method of producing cell hydrolysate from in vitro cell culture, comprising the following steps:(i) providing an in vitro cell culture;(ii) harvesting the cells from the in vitro cell culture;(iii) lysing the harvested cells to release all the protein polypeptides from the harvested cells; and(iv) optionally cutting or cleaving the protein polypeptides from the lysing step, where the harvesting step further comprises the step of isolating the cells from cell culture medium through centrifugation or filtration; the lysing step further comprises at least one of the following: sonication, high pressure homogenizer, manual grinding, freeze / thaw cycles, heating, osmotic shock, cavitation, alkali and / or detergent(s), acid hydrolysis and / or enzyme(s); and the protein polypeptides from the lysing step are cut or cleaved by enzyme(s) or chemical reagent(s) comprising at least one of subtilisin, chymotrypsin, trypsin, carboxypeptidase, elastase, pepsin, proteinase K and / or cyanogen bromide.

[0030] In one embodiment, cells are grown under contaminant-free and disease-free conditions. The cultivating conditions are well controlled and do not contain environmental contaminants or adventitious agents. Specific cellclones with minimal levels of toxins (e.g. ciguatoxins and tetradotoxins) may be selected and propagated. Cells are first grown under controlled conditions using a culture medium which is either partially defined (i.e. defined basal medium supplemented with FBS / plant hydrolysates / human platelet lysate) or a chemically defined medium (i.e. medium having defined concentrations of all nutrients and growth factors) that is devoid of animal or plant components.

[0031] Once the cells are confluent, they are collected by centrifugation after being detached from the culture surface using trypsin / EDTA or any other recombinant protease (e.g. recombinant collagenase IV, TrypLe Express) (adherence cells) or aspirated from the culture container (suspension cells). Cells are lysed by sonication and digested by a desired protease of choice to generate short functional peptides. In some embodiments, short peptides are preferred because molecules having a molecular size greater than 500 Daltons (Da) may not effectively penetrate through the outermost epidermis and get absorbed by the underlying skin layers.

[0032] Finally, the soluble fraction of the cell lysate is separated from the cell debris using centrifugation, the protease is optionally inactivated by heating or dilution. A person having ordinary skill in the art will appreciate that the cultivated cell hydrolysate from a particular cell line obtained or obtainable by the present method of producing a cell hydrolysate are substantially consistent in terms of its content and quality, presenting minor or negligible batch-to-batch variation and may be used readily for the preparation of the topical applications disclosure herein. Optionally, quality control tests such as assessment of cell growth and protein content of individual batch of production may be carried out to check and ensure consistency in different batches.

[0033] As compared to animal-derived materials used in making conventional products, cell hydrolysate is a safer alternative because it is not directly obtained from the natural environment or a farm (thereby free of environmental contaminants and adventitious agents). Specific cell clones that do not produce toxins can also be selected for generating cell hydrolysate. As the cell is broken up by sonication and digested by a desired protease, an array of short peptides, lipids, amino acids, and nutrients are released from the cell. Many of these molecules have molecular sizes below 500 Da (due to hydrolysis) and may beabsorbed by the underlying skin layers. For traditional animal-sourced ingredients, only one or a few active compounds are purified. These compounds may not be readily absorbed by the skin layers if hydrolysis procedures are not performed.

[0034] Nutrients in the medium are directly supplied to cells for cell growth. Every cultivated cell is lysed to produce the cell hydrolysate. There is no waste of energy and nutrients for the growth of unused animal parts, or life processes such as animal mating and locomotion.

[0035] The cells to be cultivated or used for the production of cell hydrolysate according to the present disclosure may be of any type, and from any species. In one embodiment, fish swim bladder cells are cultivated and cell hydrolysate from the cultivated fish swim bladder cells are obtained according to the present disclosure. In one embodiment, the cultivated cells are fish cells, avian cells, other non-mammalian cells or mammalian cells.

[0036] According to some embodiments of the present invention, the cell hydrolysate comprises (i) a mixture of protein polypeptides and / or polypeptide fragments derived from collagen 1 a1 ; (ii) a mixture of protein polypeptides and / or polypeptide fragments derived from collagen 1 [31 ; (iii) a mixture of protein polypeptides and / or polypeptide fragments derived from connective tissue growth factor (CTGF); and (iv) a mixture of protein polypeptides and / or polypeptide fragments derived from Decorin. Such protein polypeptides and / or polypeptide fragments are derived from a cell line such as yellow croaker swim bladder cell line from an in vitro cell culture and enzymatically digested by protease such as Proteinase K.

[0037] In some embodiments, the cell hydrolysate composition further comprises protein polypeptides and / or polypeptide fragments derived from one or more of Lumican, Fibulin, Chondroitin, Chitosan, Glycosaminoglycan (chondroitin and heparan), Chondroadherin and Tropomyosin.

[0038] In one embodiment, the protein polypeptides and / or polypeptides fragments in the cell hydrolysate have a molecular size smaller than 500 Daltons (Da). The cell hydrolysate of the present invention can reach and take effect on the deep skin layers (dermis, hypodermis) as the hydrolysate of the present invention is small enough to pass through the stratum corneum andalso maintains the key protein domains of growth factors and cytokines to elicit their functional activities.

[0039] In summary, the method of producing cell hydrolysate (i.e. animal- derived raw materials) from in vitro cell culture of the present invention offers a lot of benefits compared to cell hydrolysate created from animals.

[0040] First, since the cells are grown under contaminant-free and disease-free conditions, the culture medium does not contain environmental contaminants or adventitious agents. As a result, the animal-derived raw materials generated will be also contaminant-free and disease-free.

[0041] Second, an animal-component-free and chemically defined medium may be used to reduce the chance of triggering an allergic reaction of the user. This is achieved by substituting animal serum and animal-derived growth factors (e.g. bovine insulin) in the medium by recombinant growth factors. Plant extract / hydro lysate is not required in a chemically defined medium.

[0042] Third, animal cells and cell hydrolysates are produced under controlled and well-defined conditions and protocols. For example, the nutritional profile (carbohydrates, amino acids, vitamins, minerals) of the basal medium is known and consistent and may be further refined using a chemically defined medium, i.e. a medium with known concentrations of all nutrients and growth factors. The exact time, place, and all methods, reagents and personnel involved in producing each batch of cultivated cell hydrolysate are known, hence the cultivated cell hydrolysate will have better batch-to-batch consistency than conventional products.

[0043] Fourth, enhance traceability. Since the supply chain for every culture medium component is known, everything could be easily traced back to the origin.

[0044] Fifth, reduce animal suffering and sacrifice. Running the production process does not require a continuous supply of animal tissues from wild animals. For example, to produce more collagen-based cream using the conventional method, one will need to harvest more animal body parts which results in more animal suffering or sacrifice. By contrast, in the present invention, the starter cells (including but not limited to stem cells, muscle cells, fibroblast cells, adipocytes) are purified from a small piece of animal tissue anddeveloped into a cell line, which may be cryopreserved and propagated indefinitely in a culture medium. The cell line may be thawed and propagated indefinitely under cell culture conditions to produce active ingredients (e.g., growth factors, ECM molecules) when needed. Therefore, the entire process is self-sustainable and causes little animal suffering / sacrifice.

[0045] Sixth, reduce waste and enhance efficiency. Nutrients in the medium are directly supplied to cells for cell growth. Every cultivated cell is lysed to produce the cell hydrolysate. Hence, the approach of the subject invention more efficient in terms of nutrition and energy input and more environmentally friendly than the existing approaches in which a significant portion of energy and nutrients supplied to animals and plants is usually spent on life processes (locomotion, mating) and the growth of unusable body parts (animal hair, toxic plant leaves / seeds).

[0046] Seventh, cultivated cell hydrolysate contains a mixture of amino acids, peptides, lipids, and other nutrients resulting from the hydrolysis of cultivated animal cells. This induces a greater response in the skin. Moreover, while conventional animal-derived ingredients require multiple purification steps due to impurities, the cell hydrolysate requires minimal purification steps. This can decrease production costs.Composition of Cultivated Cell Hydrolysate and Polyphenols

[0047] In one aspect, the present invention provides a topical composition comprising a cell hydrolysate from cultivated cells and one or more polyphenols, where the topical composition generates synergistic effects and produces more cost-effective products than either the cell hydrolysate or polyphenol(s) alone.

[0048] In various embodiments, the present invention provides a combination of a cell hydrolysate from cultivated cells and one or more polyphenols. The combination may be used as active ingredients for skin care, wound care, or cosmetic purposes, and may be formulated into a topical composition which is then applied for improving skin health or wound conditions, or for treating wounds. The combination can also induce effective responses for skin repair and regeneration, and have anti-aging effects.

[0049] Polyphenols are a class of compounds naturally found in many plant foods. More than 8,000 polyphenols have been identified in various plant species and generally divided into five classes: flavonoids, phenolic acids, lignans, stilbenes and other polyphenols. Polyphenols have been found to have antioxidant properties that may help keep human healthy and protect against various diseases. Polyphenols have been added to topical products owing to their strong antioxidant, anti-inflammatory, anti-cancer, and antimicrobial properties. However, due to solubility, cost, and regulatory issues, their levels in the final product are mostly limited to less than 5% (w / v). This sets a limit for the maximum levels and types of responses in the skin tissue following topical application.

[0050] Cultivated cell hydrolysate, which alone can already generate benefits on skin, can trigger additive or even synergistic effects when compared to polyphenols. Against the background mentioned above, the combination of cultivated cell hydrolysate and polyphenols as provided in this disclosure enables more effective skincare, cosmetic and wound care products be produced and in a more cost-effective manner than traditional products containing animal-sourced active ingredients only or polyphenols alone. Due to the synergistic interactions between cultivated cell hydrolysate and polyphenols, lower levels of the two ingredients may be used while achieving the same magnitude of responses and help reducing production costs.

[0051] To the inventors’ knowledge, the present disclosure is the first to suggest a combination of cultivated cell hydrolysate with polyphenols for topical application, and also the first to show that such combination can generate synergistic effects as compared to either one of the entities alone.

[0052] In one embodiment, the present topical composition comprises one or more polyphenols in combination with the cell hydrolysate. Any type of polyphenols, no matter natural occurring or synthetic, of any class of polyphenols (e.g. flavonoids, phenolic acids, lignans, stilbenes, and other polyphenols), may be used in conjunction with the cell hydrolysate. Examples of polyphenols include but not limited to resveratrol, epigallocatechin gallate (EGCG), tannic acid, caffeic acid, gallic acid, ferulic acid, sinapic acid, quercetin, myricetin, naringenin, taxifolin, luteolin, cyanidin, genisterin,kaempferol, catechins, anthocyanins, curcumin, secoisolariciresinol and other forms of flavonoids, phenolic acids lignans, and stilbenes.

[0053] In one embodiment, the present topical composition may further comprise other active ingredients that are beneficial to skin care, skin repair or regeneration. In one embodiment, the present topical composition may further one or more excipients such as thickeners, emulsifying agents, preservatives, antioxidants, and buffer agents. Other ingredients may also be included (but are not limited to) water, fatty acids or fatty acid esters, hyaluronic acid, emulsifiers, and antioxidants.

[0054] In various embodiments, the present topical composition may be formulated as a semi-solid preparations or a liquid emulsion or solution. For instance, the formulation may be a cream, serum, ointment, aerosol foam or spray, powder, lotion, paste or gel.

[0055] In some other embodiments, the combination of cell hydrolysate and polyphenols may be formulated or incorporated into a patch or mask that the users may apply directly on their skin.

[0056] In various embodiments, the topical composition of the present invention comprises a cultivated cell hydrolysate (0.1 -5%, w / v) and polyphenols (0.0001 - 5%, w / v) as active ingredients in skincare, cosmetic, and wound care products. In one embodiment, the topical composition of the present invention comprises a cultivated cell hydrolysate (1 -2%, w / v) and a polyphenol (0.001 - 0.005%, w / v).

[0057] In one embodiment, the amount of the cultivated cell hydrolysate in the topical composition is in the range of about 0.1 % to about 5% (w / v). In another embodiment, the amount of the cultivated cell hydrolysate in the topical composition is in the range of about 0.3% to about 3% (w / v).

[0058] In one embodiment, the amount of a polyphenol in the topical composition is in the range of about 0.0001 % to about 5% (w / v). In another embodiment, the amount of a polyphenol in the topical composition is in the range of about 1 % to about 3% (w / v).

[0059] In one embodiment, the amount of a polyphenol in the topical composition is in the range of about 1 pM to about 100 mM. In another embodiment, the amount of a polyphenol in the topical composition is in the range of about 100 pM to about 1 mM.

[0060] In various embodiments, the weight ratio of the cultivated cell hydrolysate and the polyphenol in the topical composition is in the range of 1 :1 to 1 :500. In one embodiment, the weight ratio of the cultivated cell hydrolysate and the polyphenol in the topical composition is in the range of 1 :10 to 1 :100. In another embodiment, the weight ratio of the cultivated cell hydrolysate and the polyphenol in the topical composition is in the range of 1 :50 to 1 :250.

[0061] In one embodiment, the present topical composition comprises a cell hydrolysate from cultivated fish cells and resveratrol. The amount of the cell hydrolysate from cultivated fish cells in the topical composition may be in the range of about 0.1 %-5% (w / v), about 0.5-2.5% (w / v) or about 1 %-2% (w / v), and the amount of resveratrol may be in the range of about 0.0001 % to about 5% (w / v), about 0.001 % to about 2.5% (w / v) or about 0.1 % to about 1 % (w / v). The weight ratio of the cell hydrolysate from cultivated fish cells and resveratrol may be in the range of 1 : 1 to 1 :500, 1 :5 to 1 :250, or 1 :50 to 1 : 100.

[0062] In another embodiment, the present topical composition comprises a cell hydrolysate from cultivated fish cells and EGCG. The amount of the cell hydrolysate from cultivated fish cells in the topical composition may be in the range of about 0.1 %-5% (w / v), about 0.5-2.5% (w / v) or about 1 %-2% (w / v), and the amount of EGCG may be in the range of about 0.0001 % to about 5% (w / v), about 0.001 % to about 2.5% (w / v) or about 0.1 % to about 1 % (w / v). The weight ratio of the cell hydrolysate from cultivated fish cells and EGCG may be in the range of 1 :1 to 1 :500, 1 :5 to 1 :250, or 1 :50 to 1 :100.

[0063] The efficacy of these combinations were tested in a human keratinocyte cell-based model (HaCaT cells) to evaluate their potential effects on skin repair and regeneration. As illustrated in the examples herein, while the cultivated cell hydrolysate individually functions to stimulate skin repair and regeneration, the combination of the cultivated cell hydrolysate and polyphenol generate synergistic effects producing a larger magnitude of effects or wider spectrum of effects) than just having either ingredient alone.Application of the Composition

[0064] In one aspect, the present invention provides a method of improving skin health or wound conditions using the composition described herein.

[0065] In another aspect, the present invention provides a method of enhancing the gene expression of one or more growth factors in a human skin cell (e.g. keratinocytes and basal cells) using the composition described herein. Such growth factors may affect the growth or differentiation of the skin cells and be capable of modulating skin health, structure or functions.

[0066] A person skilled in the art would appreciate that, for the demonstrated effects on skin cells brought by the cell hydrolysate and polyphenols, the composition or combination of the present disclosure may be used directly as products (or indirectly as ingredients in preparing products) for various skin care, cosmetic and wound care uses. For example, such products may elicit growth of skin cells, anti-aging effects, skin repair, skin regeneration and other positive effects on skin cells.Example 1 - Preparation of Cell Pellet and Cell Hydrolysate from In Vitro Culture

[0067] Reagent and materials

[0068] Reagents used were DMEM (Gibco, 10566016), DMEM / F12 (Gibco, 10565018), resveratrol (Sigma, R5010) and epigallocatechin gallate (EGCG) (Shengjiade). Resveratrol and EGCG were reconstituted in DMSO and were stored at -20 °C away from light until use. Materials for total RNA isolation (12183018A), first strand cDNA synthesis (18080051 ), and RNAIater (AM7020) were purchased from ThermoFisher Scientific. The real-time PCR gene array and SYBR Green master mix were purchased from Qiagen (PAHS-121Z).

[0069] The method for production of cell hydrolysate has been previously described in US Patent Number 11306342B2, the content of which is hereby incorporated by reference in its entirety.

[0070] FIG. 1 and the following steps describe a method for producing cell hydrolysate according to one embodiment of the present invention.

[0071] Preparation of the cell pellet1. Cells are seeded at 4 x 104 cells / cm2in growth medium (DMEM / F12) and kept inside a humidified incubator (37°C; 5% CO2, 95% air).2. Upon reaching 90% confluence, the growth medium is removed. Cells are washed once by PBS and detached by treatment with trypsin / EDTA inside the CO2 incubator.3. When the cells have been dislodged, stop trypsin activity by adding a growth medium. Collect cell suspension into 50 mL tubes.4. Centrifuge the cells at 400 x g for 5 min. Remove the supernatant. Resuspend the cell pellet in the medium (e.g. DMEM / F12).5. Determine the cell concentration using a Countess II Automated Cell Counter. Seed a portion of the cells for making the next batch of cell hydrolysates.6. Transfer the remaining cell suspension to a pre-weighed 50 mL tube. Centrifuge the cells 400 x g for 5 min.7. Remove the supernatant. Weigh the tube again. Record the number of cells and the mass of the cell pellet.8. Store the cell pellet at -80°C.

[0072] Preparation of Cell Hydrolysate1 . Resuspend the cell pellet in 1 mL PBS.2. Lyse the cells by sonicating the cell suspension using a Branson Digital Sonifier S450D (amplitude= 25%; 15s ON, 15s off, total= 10 cycles). Keep the tube on ice.3. Add an appropriate amount of protease to the cell suspension to break down cellular proteins into smaller peptides. Perform the digestion for 1-2 hours and keep the tube inside a 37°C water bath. Swirl the tube every 10 min.4. When the digestion is complete, clarify the cell hydrolysate by centrifugation (optional).5. If desired, inactivate the protease by heating the hydrolysate at 80°C for 15 min or by 100-fold dilution with PBS.6. Store the hydrolysate at -20 °C.Example 2 - Evaluation of Effects on Human Skin Cells

[0073] FIG. 2 and the following describe evaluation of compositions comprising cultivated cell hydrolysate and polyphenols on human skin cells, according to one embodiment of the present disclosure.

[0074] Cell culture and treatment

[0075] HaCaT human epidermal keratinocyte line is cultured in DMEM complete medium inside a humidified incubator at 37°C and 5% CO2. Cells upon confluence are split at a ratio of 1 :10 for routine maintenance. To study the effect of treatment, 1.26 x106cells / well were seeded onto 6-well plate with 2 mL DMEM complete medium. After an 8-hour incubation and cell attachment, the 2 mL DMEM complete medium was replaced with 2 mL basal DMEM medium. The plate was then returned to the incubator. After 16-hour incubation, 1 mL basal DMEM medium containing 3X concentrations of resveratrol or EGCG with or without cell hydrolysate was added to the wells. The final concentrations of treatment were 100 pM (about 22.825 mg / L) resveratrol or 50 pM (about 22.9186 mg / L) EGCG with or without 1 % (w / v) cell hydrolysate. The final concentration of DMSO in each treatment was 1 % (v / v). Cells were treated for 24 hours in the incubator.

[0076] Total RNA isolation

[0077] The treatment medium was first removed from the well. Cells on each well were then immersed with 1 mL RNAIater solution overnight at 4°C. On the next day, RNAIater solution was removed. Total RNA of the samples was isolated using a column-based kit according to the manufacturer’s instructions. Oncolumn DNase digestion was included in the isolation procedure to eliminate genomic DNA contamination. Total RNA was eluted in 40 pL nuclease-free water. The RNA quantity and purity were estimated using the NanoDrop spectrophotometer.

[0078] First-strand cDNA synthesis

[0079] Purified RNA samples were processed into single-stranded cDNA using SuperScript III reverse transcriptase according to the manufacturer’s instructions. Briefly, about 1 pg total RNA was incubated with oligo(dT)20 primer at 65°C for 5 minutes. The reaction mix was incubated on ice for 2 minutes. A cocktail containing the first-strand buffer, DTT, RNase inhibitor,dNTP and reverse transcriptase was then added to the reaction mix. The final volume of each reaction was 20 pL. The reaction was performed at 50°C for 60 minutes, followed by heating at 70°C for 15 minutes. After the reaction was completed, each cDNA sample was diluted with nuclease-free water to a final volume of 110 pL. The diluted cDNA samples were stored at -20°C until use.

[0080] Real-time PCR using gene array

[0081] SYBR Green-based real-time PCR was performed using a 96-well formatted gene array according to the manufacturer’s instructions. Each gene array contains primers of 84 genes of interest, 6 housekeeping genes and 6 reaction controls. Briefly, 100 pL diluted cDNA was mixed with 1350 pL 2X SYBR Green master mix and 1250 pL nuclease-free water to make up a 2700 pL reaction mix. 25 pL of the reaction mix was loaded to each well of the column in the reaction plate using a multichannel pipette. The reaction plate was tightly sealed with optical adhesive film and was centrifuged for 1 minute at 1000xg at room temperature before loading into LightCycler 480 from Roche. The cycling conditions as suggested by the manufacturer were followed. Cycle threshold (CT) values of the reaction were obtained by the second derivative maximum analysis method available in the machine software. Relative expression of genes was calculated by the 2-AACT method, using RPLP0 as the housekeeping gene and then normalized to the vehicle control.

[0082] Results

[0083] To examine the gene expression profile of human epidermal keratinocyte when treated with the two compositions comprising cell hydrolysate and polyphenols, real-time PCR was performed using a gene array that contains primers of 84 genes related to skin health, structure or functions.

[0084] As seen in FIG. 3A-3C and FIG. 4A-4C, the cell hydrolysate alone could upregulate the gene expression of growth factors that are essential to skin health, structure or functions such as connective tissue growth factor (CTGF), heparin-binding EGF-like growth factor (HBEGF) and transforming growth factor-alpha (TGFA) from 2.5-fold to 8.5-fold relative to the vehicle. Polyphenols tested, i.e., resveratrol and EGCG, also upregulated the expression of these genes but in a lesser extent from 1.5-fold to 2.3-fold relative to the vehicle forresveratrol (FIG. 3A-3C) and from 1.5-fold to 4-fold for EGCG (FIG. 4A-4C). Remarkably, expression of these genes was further upregulated when the cell hydrolysate was used in combination with these polyphenols. The expression of these genes was synergistically upregulated from 6.1 -fold to 10.2-fold relative to the vehicle for resveratrol (FIG. 3A-3C) and from 3.4-fold to 22.5-fold for EGCG (FIG. 4A-4C).

[0085] The results demonstrated that the cell hydrolysate when added in combination with polyphenols acted synergistically in upregulating the gene expression of growth factors related to skin health, structure or functions.

[0086] The above description is illustrative and is not restrictive. Many variations of embodiments may become apparent to those skilled in the art upon review of the disclosure. The scope embodiments should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the pending claims along with their full scope or equivalents.

[0087] One or more features from any embodiment may be combined with one or more features of any other embodiment without departing from the scope embodiments. A recitation of "a", "an" or "the" is intended to mean "one or more" unless specifically indicated to the contrary. Recitation of "and / or" is intended to represent the most inclusive sense of the term unless specifically indicated to the contrary.

[0088] While the present disclosure may be embodied in many different forms, the drawings and discussion are presented with the understanding that the present disclosure is an exemplification of the principles of one or more inventions and is not intended to limit anyone embodiment to the embodiments illustrated.

[0089] The disclosure, in its broader aspects, is therefore not limited to the specific details, representative system and methods, and illustrative examples shown and described above. Various modifications and variations may be made to the above specification without departing from the scope or spirit of the present disclosure, and it is intended that the present disclosure covers all such modifications and variations provided they come within the scope of the following claims and their equivalents.

Claims

CLAIMSWhat is claimed is:1 . A composition for topical application comprising a cell hydrolysate obtained from an in vitro culture and at least one polyphenol.

2. The composition of claim 1 , wherein the cell hydrolysate comprises substantially all protein polypeptides and / or polypeptide fragments derived substantially from all the proteins in a cell from the in vitro cell culture.

3. The composition of claim 2, wherein the polypeptides and / or polypeptide fragments have an average molecular size ranged from about 100 Daltons (Da) to about 500 Da.

4. The composition of claim 1 , wherein the in vitro cell culture is yellow croaker swim bladder cells.

5. The composition of claim 1 , wherein the polyphenol is selected from the group consisting of flavonoids, phenolic acid, lignans, and stilbenes.

6. The composition of claim 1 , wherein the polyphenol is selected from the group consisting of resveratrol, epigallocatechin gallat, tannic acid, caffeic acid, gallic acid, ferulic acid, sinapic acid, quercetin, myricetin, naringenin, taxifolin, luteolin, cyanidin, genisterin, kaempferol, catechins, anthocyanins, curcumin, and secoisolariciresinol.

7. The composition of claim 1 , wherein the composition comprises at least 0.1% (w / v) cell hydrolysate.

8. The composition of claim 1 , wherein the composition comprises at least 0.0001 % (w / v) of polyphenol.

9. The composition of claim 1 , wherein the composition comprises about 0.1 to about 5% (w / v) cell hydrolysate and about 0.0001 to about 5% (w / v) of polyphenol.

10. The composition of claim 1 , wherein the weight ratio of the cell hydrolysate to polyphenol is 1 :1 to 1 :500.

11. The composition of claim 1 , wherein the topical application is in the form of a cream, serum, ointment, aerosol foam, spray, powder, lotion, paste, gel, patch or mask.

12. The composition of claim 1 , wherein the composition is capable of enhancing the gene expression of one or more growth factors in a human skin cell, wherein the one or more growth factors are capable of affecting the growth or differentiation of said human skin cell.

13. The composition of claim 12, wherein the one or more growth factors are selected from the group consisting of connective tissue growth factor (CTGF), heparin- binding EGF-like growth factor (HBEGF) and transforming growth factor-alpha (TGFA).

14. The composition of claim 12, wherein the cell hydrolysate and the polyphenol produce synergistic effects on enhancing the gene expression of such one or more growth factors.

15. The composition of claim 14, wherein the respective quantity of the cell hydrolysate and the polyphenol in the composition is lower than the quantity of the cell hydrolysate when applied alone and the quantity of the polyphenol when applied alone for achieving the same extent of enhancement of said gene expression.

16. The composition of claim 1 , wherein the cell hydrolysate comprises(ii) a mixture of protein polypeptides and / or polypeptide fragments derived from collagen 1 a1 ;(iii) a mixture of protein polypeptides and / or polypeptide fragments derived from collagen 1 (31 ;(iv) a mixture of protein polypeptides and / or polypeptide fragments derived from connective tissue growth factor (CTGF); and(v) a mixture of protein polypeptides and / or polypeptide fragments derived from Decorin.

17. The composition of claim 16, wherein the cell hydrolysate further comprises protein polypeptides and / or polypeptide fragments derived from one or more of: lumican, fibulin, chondroitin, chitosan, chondroitin, heparan, chondroadherin and tropomyosin.

18. The composition of claim 1 , wherein the cell hydrolysate is produced by the following steps:(i) providing an in vitro cell culture;(ii) harvesting the cells from the in vitro cell culture;(iii) lysing the harvested cells to release all the protein polypeptides from the harvested cells; and(iv) optionally cutting or cleaving the protein polypeptides from the lysing step, wherein the harvesting step further comprises the step of isolating the cells from cell culture medium through centrifugation or filtration; wherein the lysing step further comprises at least one of the following: sonication, high pressure homogenizer, manual grinding, freeze / thaw cycles, heating, osmotic shock, cavitation, alkali and / or detergent(s), acid hydrolysis and / or enzyme(s); and wherein protein polypeptides from the lysing step are cut or cleaved by enzyme(s) or chemical reagent(s) comprising at least one of subtilisin, chymotrypsin, trypsin, carboxypeptidase, elastase, pepsin, proteinase K and / or cyanogen bromide.

19. Use of the composition of claim 1 in the preparation of a product for skincare, cosmetic or wound care purpose.

20. A method of enhancing the gene expression of one or more growth factors in a human skin cell using the composition of claim 1 , wherein the one or more growth factors are capable of affecting the growth or differentiation of said human skin cell.

21. The method of claim 20, wherein the one or more growth factors are selected from the group consisting of connective tissue growth factor (CTGF), heparin-binding EGF-like growth factor (HBEGF) and transforming growth factor-alpha (TGFA).

22. A method of improving skin health using the composition of claim 1.

23. A method of improving wound conditions using the composition of claim 1 .

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