Cosmetic composition stem cell-derived callus extract of punica granatum

Abstract

The invention relates to cosmetic and dermocosmetic compositions comprising stem cell-derived callus extracts from Punica granatum obtained via standardized in vitro plant stem cell cultures. The extracts are rich in polyphenols, flavonoids, ellagic acid, anthocyanins, tannins, vitamins C, E, and K, and amino acids, providing potent antioxidant, anti-inflammatory, regenerative, moisturizing, and brightening effects. These compositions stimulate collagen synthesis, enhance skin elasticity and firmness, protect against oxidative stress, reduce redness and irritation, and promote overall skin renewal. Formulations include aqueous-based ampoules, essences, or serums containing 0.5-5% by weight of the extract, optionally combined with humectants, skin-soothing agents, antioxidants, and stabilizers. The invention further discloses optimized methods for callus induction, extraction, and formulation, ensuring reproducibility, high bioactive yield, stability, scalability, and sustainable production. This provides a scientifically validated, eco-friendly cosmetic solution delivering comprehensive skin benefits, addressing anti-aging, regeneration, hydration, and brightening needs.

Description

[0001] Description

[0002] Title of Invention: Cosmetic composition stem cell-derived callus extract of Punica granatum

[0003] TECHNICAL FIELD

[0004] The present invention relates to cosmetic and dermocosmetic compositions containing callus extract derived from in vitro stem cell cultures of Punica granatum. The invention focuses on aqueous-based formulations such as ampoules, essences, and serums comprising 0.5-5% by weight of the extract in combination with cosmetically acceptable excipients including humectants, skinsoothing agents, and stabilizers. A distinctive feature of the invention is the stem cell-derived callus extract of Punica granatum, which provides enhanced anti-aging, skin-regeneration, hydration, elasticity, and brightening effects. By employing advanced in vitro plant tissue culture techniques, the invention provides a reproducible and sustainable source of bioactive compounds suitable for large-scale industrial application in the cosmetic and dermocosmetic markets.

[0005] BACKGROUND ART

[0006] Plant-derived extracts have long been used in cosmetic and therapeutic applications due to their content of polyphenols, flavonoids, terpenes, and other bioactive metabolites with antioxidant, anti-inflammatory, and protective activities. Conventional extracts from mature plant tissues, however, face challenges including seasonal variability, environmental dependence, and inconsistent bioactive content, which limit their reproducibility and scalability in industrial formulations.

[0007] Like animals, multicellular plants consist of differentiated tissues alongside unique stem cells responsible for growth and regeneration. Plant stem cells are undifferentiated cells capable of selfrenewal and differentiation into multiple specialized cell types. Found in meristems, stem cell- derived callus tissue, injury sites, and developing embryos, they can be isolated from various plant parts including leaves, fruits, stems, flowers, roots, root caps, and seeds. Stem cell-derived callus formation involves dedifferentiation during somatic embryogenesis, generating stem cells that can produce new plant tissue. Meristematic cells at shoot and root tips continuously support plant growth throughout the plant’s life cycle. The term "plant stem cells" also encompasses extracts derived from these cells through processes such as cell lysis or partial digestion. This broad spectrum of stem cell sources provides a renewable and versatile foundation for plant regeneration and numerous biotechnological applications.

[0008] Differentiated plant cells produce a wide range of secondary metabolites and protective compounds that have evolved to defend the plant against environmental stressors, pests, and diseases. Organs such as roots, flowers, fruits, and seeds synthesize antioxidants, pigments, and signaling molecules essential for plant survival. In contrast, stem cells primarily focus on growth and regeneration, producing a distinct and often more concentrated profile of bioactive substances with potent biological activities.

[0009] Consumer demand for safe, effective, and sustainable skincare solutions has increasingly emphasized natural plant extracts in cosmetic formulations. While conventional extracts from mature plant tissues are celebrated for their antioxidant, moisturizing, and skin-soothing effects, their production is vulnerable to environmental variability, seasonal changes, and supply limitations. These factors can cause inconsistencies in quality, potency, and availability, posing significant challenges for large-scale cosmetic manufacturing.

[0010] Plant stem cell biotechnology offers a promising alternative. Callus cultures -aggregates of undifferentiated plant cells grown under controlled in vitro conditions-represent a renewable and standardized source of bioactive compounds. These cultures can yield metabolites with unique protective and regenerative properties, overcoming the limitations of conventional plant extracts.

[0011] Among promising botanical sources, Punica granatum is rich in polyphenols, flavonoids, and other bioactive compounds that contribute to antioxidant, anti-inflammatory, and skin-regenerating activities. Stem cell-derived callus extracts of Punica granatum provide enhanced protection against oxidative stress, support skin repair, improve hydration, and promote elasticity and brightening effects.

[0012] Punica granatum callus stem cells are a rich source of bioactive compounds, including polyphenols, flavonoids, tannins, anthocyanins, ellagic acid, vitamins C and E, amino acids, and minerals, which collectively provide powerful antioxidant, anti-inflammatory, and skinregenerating effects. These compounds help neutralize free radicals, protect skin cells from oxidative stress, and prevent premature aging, while growth factors and signaling molecules produced by the stem cells stimulate collagen synthesis, cell renewal, and overall skin regeneration, enhancing elasticity and firmness. The extract also exhibits strong anti-inflammatory activity, reducing redness, irritation, and inflammation, and its anti-aging properties help inhibit collagen breakdown and wrinkle formation. Additionally, the vitamins and minerals support hydration, brighten skin, and even out tone by modulating melanin production. Using Punica granatum callus stem cells offers a sustainable, cruelty-free alternative to animal-derived stem cells, providing a reproducible and eco-friendly source of potent regenerative ingredients suitable Accordingly, there remains a need for stable, sustainable, and scientifically validated cosmetic formulations capable of delivering multiple skin benefits, anti-aging, regeneration, hydration, elasticity, and brightening, through standardized stem cell-derived callus extracts produced from Punica granatum stem cell culture technology.

[0013] SUMMARY OF INVENTION

[0014] The present invention relates to novel cosmetic and dermocosmetic compositions comprising stem cell-derived callus extracts obtained from in vitro plant stem cell cultures of Punica granatum. These extracts are produced through rigorously standardized callus induction and extraction protocols, ensuring reproducible yields, high concentrations of bioactive compounds, and scalable production suitable for industrial application. Punica granatum callus extracts are rich in polyphenols, flavonoids, ellagic acid, anthocyanins, tannins, vitamins C, E, and K, and amino acids, providing potent antioxidant, anti-inflammatory, regenerative, moisturizing, and brightening effects. The extracts stimulate collagen synthesis, enhance skin elasticity and firmness, protect against oxidative stress, reduce redness and irritation, and promote overall skin renewal.

[0015] The cosmetic compositions are provided as aqueous-based preparations, such as ampoules, essences, or serums, containing 0.5% to 5% by weight of the callus extract. Optionally, these formulations include humectants, skin-soothing agents, antioxidants, and stabilizers to maintain stability and optimize dermal delivery. The formulations demonstrate long-term stability under accelerated storage conditions and are readily compatible with large-scale industrial manufacturing. The invention further discloses optimized methods for the induction and cultivation of stem cell- derived callus to maximize metabolite accumulation, extraction techniques that preserve bioactivity while providing reproducible yields, and formulation strategies that maintain activity within cosmetically acceptable carriers. By utilizing plant stem cell technology, the invention addresses significant limitations of conventional mature plant extracts, including variability in bioactive content, dependence on seasonal and environmental factors, and sustainability concerns.

[0016] Accordingly, the invention provides a scientifically validated, reproducible, and eco-friendly cosmetic solution delivering comprehensive skin benefits, including anti-aging, regeneration, moisturization, skin brightening, elasticity improvement, and protection against oxidative damage, thereby fulfilling unmet needs in the cosmetic and dermocosmetic sectors.

[0017] TECHNICAL PROBLEM

[0018] Existing cosmetic formulations utilizing plant extracts face substantial limitations that compromise both their efficacy and commercial viability. Many conventional products rely on extracts obtained from mature plant tissues, which exhibit significant variability in active compound content due to environmental factors, seasonal fluctuations, and inconsistent raw material supply. This variability adversely affects the stability, reproducibility, and biological activity of the final cosmetic compositions, limiting their claimed benefits such as anti-aging, skin regeneration, hydration, and brightening.

[0019] In addition, traditional extraction methods from differentiated plant tissues often produce heterogeneous mixtures of bioactive compounds with unstable chemical profiles, making consistent product performance across manufacturing batches difficult to achieve. These challenges hinder large-scale production and the reliable commercialization of natural cosmetic products.

[0020] Accordingly, there remains a pressing need for a standardized, renewable, and sustainable source of highly potent bioactive compounds that can be produced under controlled conditions. Such a source would enable cosmetic compositions with reproducible quality, enhanced efficacy, and multifunctional skin benefits, addressing the limitations observed in prior art.

[0021] SOLUTION TO PROBLEM that rely on mature plant tissue extracts by employing stem cell-derived callus extracts obtained from in vitro cultures of Punica granatum. This extract is both novel and optimized to utilize the concentrated bioactive compounds characteristic of plant stem cells, including polyphenols, flavonoids, vitamins, amino acids, and fatty acids, which are often present at lower or inconsistent levels in conventional plant extracts.

[0022] Production of these Punica granatum stem cell-derived callus extracts under rigorously controlled in vitro conditions ensures a standardized, renewable, and scalable supply of highly effective bioactive ingredients. This process significantly enhances reproducibility, stability, and efficacy in cosmetic applications. Controlled cultivation and extraction minimize batch-to-batch variability caused by environmental, seasonal, and harvesting factors, thereby overcoming a major drawback of prior art formulations.

[0023] Furthermore, the invention incorporates Punica granatum stem cell-derived callus extracts into advanced cosmetic delivery systems, such as oil-in-water (O / W) emulgel formulations with tailored rheological and sensory properties. These systems maintain the biological integrity and stability of the extract while providing superior skin compatibility, improved absorption, and a lightweight, non-greasy texture. Such compositions ensure long-term formulation stability even under stress conditions such as elevated temperatures or repeated freeze-thaw cycles.

[0024] Plant stem cells possess unique biological characteristics that distinguish them from differentiated plant tissues and make them particularly suitable for advanced cosmetic applications. These undifferentiated cells exhibit self-renewal and totipotency, allowing continuous biosynthesis of essential metabolites that support plant defense and regeneration. When cultured in vitro, Punica granatum stem cells can produce highly concentrated levels of secondary metabolites, such as polyphenols, flavonoids, antioxidants, vitamins, and amino acids, in a controlled and reproducible manner, independent of environmental fluctuations. Consequently, extracts derived from such callus cultures display enhanced bioactivity, purity, and stability. Moreover, these metabolites have been demonstrated to promote skin regeneration, collagen synthesis, antioxidant protection, and anti-inflammatory effects in human skin models. Therefore, the incorporation of Punica granatum stem cell-derived callus extracts provides a sustainable and scientifically advanced solution that fulfills both the technical and commercial demands of next-generation dermocosmetic products. ADVANTAGES EFFECTS OF INVENTION

[0025] This invention provides a significant improvement over existing skincare products by offering a reproducible, multifunctional cosmetic solution. Punica granatum stem cell-derived callus extracts deliver comprehensive benefits, including anti-aging, skin regeneration, moisturizing, and brightening effects. The metabolites produced by these stem cells exhibit enhanced antioxidant, anti-inflammatory, and regenerative properties that are not achievable with conventional mature plant extracts.

[0026] Standardized in vitro callus culture production ensures consistent bioactive compound profiles and overcomes supply chain and quality inconsistencies inherent to natural sources, reliably fulfilling commercial manufacturing requirements. The incorporation of these extracts into innovative emulgel or aqueous-based formulations further optimizes delivery of bioactive compounds to the skin and enhances consumer acceptability, thereby increasing product market competitiveness.

[0027] Overall, the combination of Punica granatum stem cell-derived callus extracts, controlled biotechnological production, and advanced formulation techniques provides a novel, effective, and sustainable cosmetic innovation aligned with consumer demand for natural and scientifically validated products.

[0028] DESCRIPTION OF EMBODIMENTS

[0029] We have demonstrated that stem cell-derived callus extracts from Punica granatum contain bioactive compounds with potent antioxidant, anti-inflammatory, and skin-regenerating properties. The extract is rich in polyphenols, flavonoids, ellagic acid, vitamins, and amino acids, which collectively support skin regeneration, improve elasticity, enhance hydration, and reduce inflammation. Incorporating Punica granatum callus-derived extracts into cosmetic formulations effectively promotes skin repair, provides strong antioxidant protection, and brightens the overall complexion.

[0030] In certain embodiments, the plant stem cell product comprises exosomes isolated from Punica granatum stem cells, derived from callus cultures initiated from leaves, fruits, or embryos. This composition promotes skin regeneration, enhances skin elasticity, and reduces wrinkles by stimulating collagen production while exhibiting low cytotoxicity. The invention encompasses various cosmetic forms, including creams, lotions, masks, serums, and aqueous ampoules suitable for human skin application.

[0031] Further embodiments include a variety of Punica granatum stem cell-derived callus products designed to protect the skin, enhance regeneration, and provide anti-aging effects through bioactive compounds produced by cultured plant cells. These extracts can be formulated alone or in combination with humectants, skin-soothing agents, antioxidants, and stabilizers to optimize skin delivery and maintain stability under storage conditions.

[0032] Additionally, the invention utilizes supernatant fluid derived from Punica granatum callus cultures containing a mixture of growth factors and metabolites. This ingredient is employed in cosmetic formulations to moisturize, repair, and strengthen the skin barrier, providing a sustainable and scientifically validated approach for next-generation dermocosmetic products.

[0033] DETAILED DESCRIPTION OF INVENTION

[0034] The present invention provides cosmetic and dermocosmetic compositions comprising callus extracts derived from stem cell cultures of Punica granatum, offering superior skin care benefits by leveraging the unique regenerative and bioactive properties of plant stem cells. These stem cell-derived callus extracts are produced using advanced in vitro plant tissue culture techniques, ensuring a sustainable, standardized, and renewable source of potent bioactive compounds such as polyphenols, flavonoids, ellagic acid, vitamins, amino acids, and other secondary metabolites. The extracts exhibit enhanced antioxidant, anti-inflammatory, moisturizing, and skin regeneration effects, collectively contributing to anti-aging, wrinkle prevention, skin brightening, pore refinement, barrier reinforcement, and improvement of skin elasticity and texture.

[0035] The compositions may take various forms, including aqueous ampoules, essences, serums, creams, and lotions, with Punica granatum callus-derived extracts incorporated at effective concentrations ranging from 0.5% to 5%, optionally combined with humectants, skin-soothing agents, antioxidants, and bioactive enhancers to maximize efficacy and skin compatibility. Callus cultures are initiated from meristematic or injury-induced plant tissues and propagated under controlled laboratory conditions to ensure purity, uniformity, and high yield of desired phytochemicals. Extraction involves gentle cell lysis, followed by concentration and stabilization using suitable excipients, liposomes, or encapsulation technologies to preserve bioactivity and facilitate skin penetration.

[0036] This invention addresses the limitations of conventional plant extracts by eliminating variability due to environmental, seasonal, and harvesting factors, providing highly reproducible cosmetic formulations with scientifically validated claims and consumer-appealing natural ingredients.

[0037] Basic Formulation (Light O / W Emulgel) - Weight Percent

[0038] Phase A - Aqueous Phase

[0039] • Deionized water: qs to 100%

[0040] • Glycerin: 3.0%

[0041] • Propanediol: 3.0% (enhances solubilization and dispersion of extract powders)

[0042] • Sepinov EMT 10 (HEA / SAc TA copolymer): 0.30% (electrolyte- and extract-resistant gelling agent)

[0043] • Disodium EDTA (chelating agent): 0.10%

[0044] • Allantoin: 0.20%

[0045] • Sodium hyaluronate (blend of high and low molecular weight): 0.15%

[0046] • pH Buffer: Citric acid / sodium hydroxide solution adjusted to final pH 5.0-5.5 (target pH 5.3)

[0047] Phase B - Oil Phase (Light, Non-Greasy)

[0048] • Caprylic / Capric Triglyceride: 3.0%

[0049] • C12-15 Alkyl Benzoate: 2.0%

[0050] • Squalane: 1.0%

[0051] • Dimethi cone (100-200 cSt): 1.0%

[0052] • Glyceryl Stearate Citrate: 1.2%

[0053] Cetearyl Alcohol: 0.8% Cetearyl Glucoside: 0.4%

[0054] • Tocopherol (Vitamin E): 0.5%

[0055] Phase C - Cool Down (<40°C)

[0056] • Punica granatum stem cell-derived callus extract (water-soluble powder): 3.0%

[0057] • Niacinamide: 4.0% (brightening and skin barrier enhancement)

[0058] • 3-O-Ethyl Ascorbic Acid: 1.5% (stable at target pH; enhances brightening and antioxidant effects)

[0059] • Panthenol (Vitamin B5): 1.0%

[0060] • Bisabolol: 0.20%

[0061] • Preservative Germaben IE 1.0% (suitable for O / W emulsions with pH 3-7)

[0062] Total non-water phase: approx. 23.25%

[0063] Water: approx. 76.75% to 100%

[0064] Rationale for Formulation Design:

[0065] • Compatibility with water-soluble extract powders is achieved using glycerin / propanediol blend in the aqueous phase, facilitating wetting and gradual powder solubilization, minimizing skin irritation.

[0066] • Anti-inflammatory synergy is achieved through the combination of bisabolol, panthenol, allantoin, and polyphenols from Punica granatum extracts.

[0067] • Brightening and anti-aging system includes 4% niacinamide + 1.5% 3-O-ethyl ascorbic acid, optimized for pH 5-5.5, without hydroquinone-related risks.

[0068] • Lightweight, non-greasy sensory profile achieved via light esters, caprylic / capric triglyceride, and a small dose of dimethicone; tocopherol protects the oil phase from oxidation. • Stability up to 45°C maintained through acrylate-thioether polymer, EDTA, and tocopherol. Recommended testing includes accelerated aging at 45°C, thermal cycling, and freeze-thaw cycles.

[0069] Variants for Skin Types:

[0070] • Dry Skin: Increase capry lie / capric triglyceride to 5.0%, squalane to 2.0%, dimethicone to 1.2%, glycerin to 4.0%, panthenol to 1.5%. Adjust Sepinov EMT 10 to 0.35% or cetearyl alcohol to 1.0% to maintain viscosity; adjust water accordingly.

[0071] • Oily Skin (Ultra-light / Matt): Capry lie / capric triglyceride 2.0%, alkyl benzoate 1.5%, squalane 0.5%, dimethicone 0.7%, propanediol 3.5% for enhanced cooling. Optionally, 0.3-0.5% spheronized silica for mattifying; maintain Sepinov EMT 10 at 0.30% to preserve texture.

[0072] Compatibility Notes:

[0073] • Avoid polycationic surfactants (e.g., polyquaterniums) with high polyphenol extracts to prevent haze / precipitation.

[0074] • Niacinamide stable at pH 5-5.5; avoid below pH 5.

[0075] • 3-O-ethyl ascorbic acid compatible and more stable than phosphate derivatives.

[0076] • Germaben II suitable for O / W emulsions at pH 3-7; avoid high loads of mild nonionic surfactants. PET (preservative efficacy testing) mandatory.

[0077] • Ethanol only for temporary solubilization (<2%), preferably omitted in final formulation.

[0078] Recommended Stability Testing:

[0079] • 6 months accelerated aging at 45 °C with controls at 40°C and 25 °C

[0080] • Thermal cycling 4°C 40°C, 6-8 cycles

[0081] • Freeze / thaw -5°C 25°C, 3 cycles

[0082] Evaluation: phase separation, rheology / viscosity, pH, color / odor, centrifugation, microbiological challenge tests (Gram ± bacteria, fungi) EXAMPLES

[0083] Example 1 : Callus culture initiation

[0084] Stem cell-derived callus cultures are initiated from sterilized explants (leaves, stems, or fruit segments) of Punica granatum on Murashige and Skoog (MS) medium supplemented with auxins and cytokinins. Cultures are maintained at 25°C in the dark. Friable callus forms within 2-4 weeks and is subcultured every 3 weeks to maintain high vitality and uniform phytochemical content.

[0085] Example 2: Extraction of callus-derived bioactive compounds

[0086] Harvested callus is suspended in phosphate-buffered saline or water and subjected to gentle cell disruption via high-pressure homogenization. The suspension is centrifuged to remove debris, and the supernatant is collected. Extracts can be concentrated by lyophilization or vacuum concentration and stabilized using preservatives and encapsulation agents such as liposomes to enhance bioactivity and skin penetration.

[0087] Example 3 : Anti-aging ampoule formulation (Aqueous base)

[0088] • Punica granatum callus extract 2%

[0089] • Glycerin 5%

[0090] • Butylene glycol — 3%

[0091] • Panthenol 1%

[0092] • Sodium hyaluronate —> 0.1%

[0093] • Phenoxyethanol —>■ 0.5%

[0094] • Purified water — balance to 100%

[0095] The solution is homogenized under sterile conditions and packaged in single-use ampoules. The formulation is stable for at least 12 months at 25°C.

[0096] Example 4: Hydrating emulgel

[0097] • Punica granatum callus extract 3%

[0098] • Carbomer 0.3%

[0099] Dimethicone — > 1 % Glycerin 4%

[0100] • Tocopherol acetate 0.5%

[0101] • Neutralizing agent — q.s.

[0102] • Preservative — 0.5%

[0103] • Purified water — balance to 100%

[0104] The emulgel is lightweight, non-greasy, and retains >90% antioxidant activity after accelerated stability testing at 40°C for 3 months.

[0105] Example 5 : Brightening serum

[0106] • Punica granatum callus extract 1.5%

[0107] • Niacinamide 2%

[0108] • Sodium hyaluronate 0.2%

[0109] • Allantoin 0.3%

[0110] • Propylene glycol 4%

[0111] • Preservative 0.5%

[0112] • Purified water balance to 100%

[0113] Pilot testing (n=15) demonstrated measurable improvements in skin brightness after 4 weeks.

[0114] Example 6: In vitro bioactivity testing

[0115] • Antioxidant activity: DPPH and ABTS assays show >80% radical scavenging at 50 pg / mL.

[0116] • Anti-inflammatory activity: Human keratinocytes treated with 1% callus extract for 24 h exhibit reduced TNF-a and IL-6 expression.

[0117] Example 7: Clinical evaluation

[0118] A randomized controlled trial with topical application of the formulated essence twice daily for 8 weeks in adult volunteers showed: Significant improvement in skin hydration and elasticity (cutometry)

[0119] • Reduction in wrinkle depth (profilometry)

[0120] • Enhanced overall skin brightness

[0121] These results confirm the multifunctional skin benefits of Punica granatum callus-derived formulations.

Claims

CLAIMSWhat is claimed is:

1. A cosmetic composition for skin care comprising:- stem cell-derived callus extract of Punica granatum wherein the extracts are obtained from in vitro stem cell-derived callus cultures under controlled conditions, and wherein the combination provides enhanced anti-aging, skin-regeneration, moisturizing, and brightening effects compared to individual extracts.

2. The cosmetic composition according to claim 1 , wherein the total concentration of the stem cell- derived callus extracts ranges from 0.5% to 5% by weight of the final formulation.

3. The cosmetic composition according to claim 1 or 2, wherein the extracts are formulated into aqueous-based ampoules, essences, serums, or emulsions.

4. The cosmetic composition according to any preceding claim, wherein the synergistic effect is demonstrated by at least one of the following:(a) enhanced antioxidant activity in DPPH or ABTS assay compared to the sum of the individual extracts,(b) greater reduction in pro-inflammatory cytokines (TNF-a, IL-6) in human keratinocyte assays,(c) clinically significant improvement in skin hydration, elasticity, or wrinkle depth compared to compositions containing only one extract.

5. The cosmetic composition according to any preceding claim, further comprising one or more cosmetic excipients selected from humectants, skin-soothing agents, or antioxidants, wherein the formulation is stable under accelerated storage conditions.

6. A method for improving skin condition comprising topically applying the cosmetic composition of any one of claims 1-5 to human skin, thereby reducing wrinkles, improving hydration, and enhancing skin brightness.