A white heirloom tomato composition supporting skin lightening through modulation of melanin transfer.

Abstract

A composition for skin lightening and cell regeneration comprising white heirloom tomato extract (TOMESORAL). The composition effectively reduces melanin transfer, increases GSH levels, promotes skin

Description

[0001] Embodiments of the present invention generally relate to tomato-based compositions for skincare, and, more particularly, to methods for preparing white heirloom tomato-based compositions for anti-ageing purposes, and associated compositions thereof. BACKGROUND OF THE INVENTION

[0002] The foilowing description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0003] As it is well known, for the whitening of the skin to a lighter skin tone or for the elimination of dark spots from the skin, whitening compositions are useful. Further, these whitening or skin bleaching compositions are effective as these inhibit the formation of melanin (skin color is determined by this pigment). However, such skin bleaching or whitening compositions contain harsh chemicals, such as peroxides, glutathione, formaldehyde and the like, that has severe effects on the skin.

[0004] As disclosed in conventional prior art disclosures, the use of hydroquinone suppresses the melanocyte activity and helps in whitening of the skin. However, such less stringent composition has other disadvantages i.e., the hydroquinone is oxidized by air and light. Further, the oxidized product causes skin irritation and inflammation.

[0005] Furthermore, the prior art discloses dermatological compositions that use paucilamellar liposomal carriers that help in treating Melanoma caused by exposure to sunlight. However, such compositions are not effective in whitening the skin to a lighter skin tone. Additionally, some conventional prior art also discloses the use of naturally derived materials in dermatological compositions, such as but not limited to, lemons, oranges, cucumber, rose fruit and the like. However, such naturally derived materials limit their usefulness based on age, skin type, and color, and vary greatly in different subjects.

[0006] As disclosed in various prior art, the use of cosmetics compositions containing carotenoids with the extract of plant of the genus malus, containing polyphenols helps in skin whitening. However, these compositions containing carotenoids are typically highly colored and are sensitive to light and oxidation that considerably limits their usefulness.

[0007] Furthermore, prior art also mentions the use of polyphenolic compounds, mostly resorcinols having skin whitening properties. However, these compounds are susceptible to air and UV oxidation leading to color instability that causes loss of skin tightening efficacy. Additionally, as per related prior art, the extract obtained from fruits of acerola containing Vitamin C, are incorporated into cosmetics to provide a whitening effect. Therefore, the prior art disclosures briefly discuss the compositions that are useful for whitening of the skin. However, the element of usage to increase natural glutathione level is yet to be introduced in the market.

[0008] Carotenoids, recognized for their importance in nutrition and health, serve as precursors for retinoids, offering both health and cosmetic advantages upon accumulation in human skin (Melendez-Martinez, Mandic et al. 2022, Udensi, Loughman et al. 2022).

[0009] Notably, colorless carotenoids such as phytoene (PT) and phytofluene (PTF) have recently attracted attention due to their bioavailability and potential health benefits (Melendez-Martinez, Mandic et al. 2022, Biskanaki, Kalofiri et al. 2023, Kiki 2023).

[00010] Colorless carotenoids found in tomatoes, including phytoene, phytofluene, lutein and zeaxanthin are believed to contribute to antioxidant mechanisms, sparking discussions regarding their incorporation into nutricosmetics for improving both skin appearance and safety (Etminan, Takkouche et al. 2004, Canene-Adams, Campbell et al.2005, Giovannucci 2005, Engelmann, Clinton et al. 2011).

[0011] Tomatoes have been recognized as an important source of dietary flavonoids because of their high consumption worldwide. The total flavonoid content of different tomato types varies between 4 and 26 mg 100 (-1) g FW (Slimestad, Fossen et al. 2008). Tomatoes have considerable number of flavonoids predominantly quercetin and quercetin glycosides (isoquercitrin, rutin).

[0012] Heirlooms tomatoes, which originated from 50 years old seeds without any crossbreeding are known for their diversity of flavor, color and shape. These are white colored tomatoes, which have been investigated for various biological properties. In this study the role of heirloom tomato powder was investigated for the inhibition of melanin transfer in order to minimize cutaneous pigmentation levels. It is basically colorless carotenoids and flavonoids rich powder that can cause the decrease of melanin transfer and increase in glutathione levels. In order to prove this fact, first strategy is to define the non-cytotoxic dose of the tomato powder and for this fact determination MTT assay was used for both cell lines.

[0013] However, studies have often overlooked colorless carotenoids and flavonoids due to challenges in detection methods using typical colored carotenoid detection wavelengths (Rodriguez-Amaya 2001). Nonetheless, recent research has explored their antioxidant capacity and isomerism (Melendez-Martinez, Stinco et al. 2013, Melendez-Martinez, Paulino et al. 2014, Stinco, Escudero-Gilete et al. 2016), aligning with the growing interest in dietary compounds promoting skin appearance and safety (Madhere and Simpson 2010). Tomatoes, rich in carotenoids like lycopene, are associated with reduced risks of chronic diseases, with colorless carotenoids like phytoene and phytofluene believed to possess biological activity (Etminan, Takkouche etal. 2004, Canene-Adams, Campbell et al. 2005, Engelmann, Clinton et al. 2011). Glutathione, a potent antioxidant, has become a common skin-lightening agent, albeit with debate regarding its efficacy and safety (Exner, Wessner et al. 2000, Sonthalia, Daulatabad et al. 2016).

[0014] This study emphasizes the importance of glutathione in its reduced form (GSH) and introduces heirloom white tomatoes, enriched in carotenoids and flavonoids, as a unique source potentially offering antioxidant benefits to cells (Khachik, Carvalho et al. 2002).

[0015] As flavonoids are considered as potentially health-promoting substances, thus efforts are made to produce tomatoes with a standardized content of flavonoids and other compounds (Stewart, Bozonnet et al. 2000, Slimestad and Verheul 2005), or to elevate the concentration of especially quercetin and quercetin 3-rutinoside(rutin) (Slimestad, Fossen et al. 2008). Nutricosmetics, bridging the realms of food, nutrition, health, and cosmetics, consider colorless carotenoids and flavonoids as promising components for promoting "beauty from within," aligning with the increasing interest in dietary compounds that enhance skin appearance and safety (Madhere and Simpson 2010).

[0016] The emerging paradigm of photo protection via dietary interventions underscores the crucial role played by plant constituents, particularly carotenoids and flavonoids, in safeguarding plants against excessive light and preventing UV damage in humans (Stahl and Sies 2007, Kiefer, Weibel et al. 2010). Tomatoes, globally consumed and recognized for their flavonoid content, including quercetin and quercetin glycosides, are currently undergoing efforts to standardize and enhance flavonoid concentrations (Crozier, Lean et al. 1997, Stewart, Bozonnet et al. 2000, Slimestad and Verheul 2005, Slimestad, Fossen et al. 2008).

[0017] Tomatoes, renowned for their carotenoid content such as lycopene, have been linked to reduced risks of chronic diseases, with colorless carotenoids like phytoene and phytofluene speculated to possess bioactive properties. Glutathione, a potent antioxidant with anti-melanogenic properties, has emerged as a common skin-lightening agent, although debates persist regarding its efficacy and safety (Exner, Wessner et al. 2000, Sonthalia, Daulatabad et al. 2016).

[0018] Skin melanocytes produce melanin, which acts as a natural barrier against ultraviolet (UV) light and reduces DNA damage in skin cells (Berens, Van Den Bossche et al. 2005). But its aberrant build-up causes pigmentation disorders such as melanoma (Buitrago, Hardre et al. 2016), senile freckles, postinflammatory pigmentation, and chloasma (Ashraf, Rafiq et al. 2015). As such, controlling the pigmentation of human skin continues to be a top priority for the cosmetic and pharmaceutical industries (Kim, Hwang et al. 2015). Skin-whitening ingredients such as vitamin C, hydroquinone, arbutin, azelaic acid, retinoic acid, and kojic acid are found in many cosmeticproducts (Hakozaki, Minwalla et al. 2002, Matos, Varela et al. 2015), albeit they can have negative side effects like dermatitis, skin irritation, and death of melanocytes. As a result, the search for organic color-reducing compounds has accelerated (Wang, Chang et al. 2017). However, more research is required to determine the precise processes by which these drugs work to prevent the synthesis and transport of melanin. Melanin transport to epidermal cells is the only process that causes pigmentation, hence blocking this transfer can effectively prevent pigmentation (Ma, Zhao etal. 2010).

[0019] Given the beneficial effects associated with a significant and unique blend of colorless carotenoids and flavonoids in heirloom white tomatoes, the current study endeavors to explore the potential of carotenoids and flavonoids derived from white heirloom tomatoes in promoting skin brightening and cell regeneration by inhibiting melanin transfer to epidermis and increasing reduced glutathione (GSH).

[0020] Thus, there is a recognized need for, and it would be highly beneficial to have alternative, effective natural combination of flavonoid and carotenoid preparations having minimal adverse side effects, which are effective for skin lightening and which are suitable for the cosmetic industry. Accordingly, there remains a need in the art for innovative, novel, efficient methods for preparing white heirloom tomato-based compositions for skin lightening potential via inhibition of melanin transfer activity. SUMMARY

[0021] The summary of the invention does not necessarily disclose all the features essential for defining the invention: the invention may reside in a sub-combination of the disclosed features.

[0022] Exclusive white tomato powder derived from heirloom tomatoes, hereinafter referred to as TOMESORAL, recognizing the diverse shape, color and flavor attributes is disclosed herein. The TOMESORAL composition is rich in phytoene, phytofluene, lutein and zeaxanthin as obtained from the fruit of Organic white Heirloom tomatoes.

[0023] According to one aspect, the present disclosure provides a means of producing TOMESORAL composition by considering white heirloom tomato fruits and subjecting them to meticulous sampling and decontamination through rigorous washings before being considered suitable for testing. Subsequently, the sampled fruit was subjected to the freeze-drying method, before being powdered into the final TOMESORAL composition.

[0024] According to another aspect, the present disclosure provides a method for skin lightening and cell regeneration, comprising the administration of an effective amount of TOMESORAL. According to the same aspect, TOMESORAL contains carotenoids and flavonoids that inhibit melanin transfer activity and promote skin cell regeneration.

[0025] According to another aspect, TOMESORAL effectively reduces melanin transfer, increases GSH levels in the skin, promotes skin cell regeneration, and shows no cytotoxic effects.

[0026] According to another aspect, the present disclosure also provides optimal doses of TOMESORAL as identified to show significant recovery from skin injury.

[0027] According to yet another aspect, TOMESORAL may further reduce LDH release, indicating reduced cell damage, and lowering PAR-2 activity, indicating inhibition of melanin transfer.

[0028] According to another aspect, TOMESORAL is configured to improve skin tone and appearance.

[0029] According to a final aspect, TOMESORAL is further configured to be used for skin lightening and cell regeneration, offering a safe and effective solution for improving skin tone and appearance. Brief Description of Drawings

[0030] The accompanying drawings are included to provide a further understanding of the present disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary aspects of the present disclosure and, together with the description, serve to explain the principles of the present disclosure.

[0031] The diagrams are for illustration only, which thus is not a limitation of the present disclosure, and wherein:

[0032] Figure 1 A and C) and B and D) illustrates Bar graph and Line graph showing the relative percentage of viabilities on different concentrations of tomato powder respectively, in accordance with one of the aspects of the present disclosure.

[0033] Figure 2: A) Graph shows the levels of LDH release in A-375 and HaCaT post treated cells. Values were obtained as mean ± SEM, and * indicates a significant difference in cellular viability and LDH levels between treated groups and untreated controls (p<0.05).

[0034] Figure 3: Graph illustrates the levels of melanin in post treated cells, in accordance with an aspect of the present disclosure.

[0035] Figure 4 Graph illustrates PAR-2 activity in post treated cells, in accordance with an aspect of the present disclosure.

[0036] Figure 5: The graph displays the GSH levels in post-treated cells, in accordance with an aspect of the present disclosure. Detailed Description of the Drawings

[0037] The following is a detailed description of the different aspects of the disclosure depicted in the accompanying drawings. The described aspects are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of the defined aspects; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.

[0038] In the following description, numerous specific details are set forth in order to provide a thorough understanding of aspects of the present disclosure. It will be apparent to one skilled in the art that aspects of the present invention may be practiced without some of these specific details.

[0039] A skin care system, method and composition for improving skin tone and appearance is disclosed herein. The system constitutes of a composition derived from white heirloom tomatoes (TOMESORAL) containing colourless carotenoids and flavonoids; a cytotoxicity assessment module; an injury reduction analysis module; a melanin estimation module; a PAR-2 activity evaluation module; and a GSH level estimation module, wherein the system is configured to reduce melanin transfer, increase GSH levels, and promote skin cell regeneration without cytotoxic effects. Experiment Methodology and Setup Preparation: 1. Preparation of TOMESORAL

[0040] TOMESORAL White Tomato is an exclusive tomato powder derived from heirloom tomatoes recognized for their diverse color, shape, and flavor attributes, originating from plant seeds aged at least 50 years. These tomatoes are rich in phytoene, phytofluene, lutein and zeaxanthin obtained from the fruit of organic white heirloom tomatoes. The tomato fruits underwent meticulous sampling and decontamination through rigorous washing. Subsequently, they were subjected to the freeze-drying method as identified in the prior-art. 2. Culturing of Cell lines

[0041] The A-375 melanoma cells and HaCaT cells were seeded onto culture dishes and cultured in Dulbecco's Modified Eagle Medium-High Glucose (DMEM-HG) that is supplemented with antibiotics and 10% fetal bovine serum (FBS) until reaching the sub culturing phase, at which point they were utilized in ensuing experiments. 3. Stock and Dilution preparation

[0042] The TOMESORAL solution was prepared by dissolving the TOMESORAL powder stock in distilled water. After mixing, the solution was passed through a syringe filter, followed by preparation of its different dilutions (lOOug / ml, 200ug / ml, 500ug / ml, lOOOug / ml and 2000ug / ml) in plain medium (DMEM). 4. Treatment of Cell line with Tomato Fruit Powder

[0043] The A-375 melanoma cell line and HaCaT cell line prepared at step 2 were seeded onto a 96- well plate to assess cell proliferation. The cells were subjected to the treatment with specified concentrations of the TOMESORAL solution, as mentioned earlier, for a duration of 24 hours. Subsequently, a cell viability assay was conducted on the cells, and the collected medium was utilized for subsequent experimental analyses. 5. Cell Viability Assay

[0044] The cellular viability was assessed utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay), conducted on cells that were cultured in a 96-well plate. The cells were subjected to various concentrations of TOMESORAL solution for a duration of 24 hours. Following the treatment period, 25 pl of MTT solution was introduced, and after an additional 2-3 hours, the resultant purple-hued crystals were dissolved by treatment with a detergent like 10% SDS (sodium dodecyl sulfate) solution. Subsequently, absorbance measurements were acquired at a wavelength of 570 nm after 3 hours of incubation. 6. LDH released assay

[0045] The A-375 melanoma cell line and HaCaT cellines were treated with 6 mM H2O2 followed by exposure to varying concentrations of TOMESORAL solution for a duration of 24 hours. Subsequently, the activity of lactate dehydrogenase (LDH) in the treated A-375 and HaCaT cell lines was measured in the supernatant of all experimental groups, following the protocol provided by AMP Diagnostics, Austria. For the LDH assay, a 5 pl sample of the medium from each experimental condition was used. Specifically, 5 pl of the medium from each group was mixed with 100 pl of the assay's working reagent in a 96-well plate, followed by a 5-minute incubation period. The absorbance was then measured at a wavelength of 340 nm using a spectrophotometer. 7. Co-Culturing of Cell lines The HaCaT cells (keratinocytes) were cultured at 2.5 x 10 cells / well in six-well plates. After two days A-375 melanoma cells (melanocytes) (5 x 10^) were added to each well containing the keratinocytes and at the initial seeding ratio of keratinocytes to melanocytes of 5:1. The cells were seeded onto culture dishes and cultured in Dulbecco's Modified Eagle Medium-High Glucose (DMEM-HG) that were supplemented with antibiotics and 10% fetal bovine serum (FBS) until reaching the 70% confluency, at which point they were utilized in ensuing experiments. Keratinocytes and melanocytes at the same cell densities were cultured separately to serve as controls. The cells were subjected to the treatment with optimum concentration (130pg / ml) of tomato fruit powder for a duration of 48 hours. Different test groups were labelled according to the details presented in Table 1.

[0046] Table 1. Different test groups. Group Label Detail of group 1 UT-A375 Cultured melanocytes 2 A375-Tomes 150ug / ml Cultured melanocytes treated with TOMESORAL 130pg / ml 3 UT-HaCaT Cultured keratinocytes 4 HaCaT-Tomes 150ug / ml Cultured keratinocytes treated with TOMESORAL 150pg / ml 5 Co-Culture-A375+ HaCaT Co-cultured keratinocytes + melanocytes 6 Co-Culture-A375+ HaCaT- Tomes 150ug / ml Cultured keratinocytes + melanocytes treated with TOMESORAL 150pg / ml 8. Estimation of Melanin Content

[0047] In 6-well culture plates, different groups of cells were sown in 2 ml of media, and the cells were allowed to adhere overnight in the incubator. Optimum concentration of TOMESORAL solution (130pg / ml) was applied to the cells in MEM for a duration of 48 hours. Then the cells were detached by 0.25% trypsinethylenediamine tetraacetic acid solution (2min digestion followed by washing to separate melanocytes and then 8min digestion for keratinocyte digestion for co-culture only), centrifuged for 15 minutes at 1 200 *g at 4°C. after removing the supernatant, the treated cells were lysed with 200 pl IN NaOH and pipetted repeatedly to homogenize them. For analysis, the crude cell extracts were transferred into 96-well plates in triplicate. Relative melanin content was determined by absorbance at 405 nm using a microplate reader. 9. Estimation of Protease-Activated Receptor 2 (PAR-2) via ELISA

[0048] A solid phase sandwich enzyme-linked immunosorbent assay (ELISA) was conducted to assess the activity of Protease-Activated Receptor 2 (PAR-2) using a modified version of a previously described protocol (Wajid, Naseem et al. 2015). In brief, a 96-well plate (Coming, USA) was initially coated with rabbit polyclonal anti-PAR-2 antibody (Santa Cruz Biotechnology, USA) and incubated for 120 minutes. Following three washes with tris buffered saline (TBS), blocking was carried out using 1% bovine serum albumin (BSA). Subsequently, lOOpl of medium from both cell groups was added to each well and incubated for 60 minutes. After incubation, samples were aspirated, and the wells were rinsed thrice with TBS containing Tween-20 (TBS-T). The plate was then incubated overnight with an HRP-conjugated donkey anti-rabbit secondary antibody (Santa Cruz Biotechnology, USA) for 90 minutes. Following another round of washing, lOOpl of tetramethyl benzidine (TMB) chromogenic solution (Invitrogen Inc., USA) was added to initiate the reaction. The reaction was terminated by adding 0.18M sulfuric acid, and absorbance was measured at a wavelength of 450nm. 10. Estimation of GSH

[0049] Glutathione (GSH) levels in all the previously listed groups of Table 1 containing heirloom powder were assessed by analyzing samples from various experimental groups. GSH concentrations were quantified using GSH assay kits according to the manufacturer's protocol (Sigma Aldrich). Results and Analysis:

[0050] The data from the experimental groups were presented as the mean ± standard error of the mean (SEM) across triplicate experiments. Statistical comparisons between group means were performed using one-way analysis of variance (ANOVA), followed by Bonferroni's test to identify differences between specific groups. Statistical significance was determined by a p-value less than 0.05. 1. Cytotoxicity and Optimal Dose Indication [0051 ] The MTT assay, known for its reliability and simplicity in assessing cell cytotoxicity, was conducted. Figure 1 illustrates the relationship between percentage (%) cytotoxicity and different concentrations of the TOMESORAL solution in the A-375 melanoma cell line and HaCaT cells. It was observed that the majority of TOMESORAL concentrations did not induce significant cytotoxic effects on both cell lines. Based on these findings, the optimal dose (OC) was determined to be 130 pg / ml for the A-375 melanoma cell line and 150 pg / ml for the HaCaT cells, as depicted in Figure 1A -D.

[0052] Figure 1A and 1C Bar graph show the relative values of absorbance after MTT assay. Fig 1-B and 1-D, line graphs show the relative percentage of viabilities on different concentrations of TOMESORAL while box represents the OC value for tomato powder. Values were taken as mean ± SEM, (p<0.05). 2. Injury assessment in post-treated groups

[0053] A-375 and HaCaT Cells were further treated with 6mM H2O2 for 3 hours with the injured cells then treated with the same doses of TOMESORAL as used in the cytotoxicity experiments mentioned earlier. There was a significant decrease in cell viability with cells treated with H2O2, and a significant increase in viability after TOMESORAL treatment, showing a significant recovery from injury.

[0054] To examine the levels of injury and recovery, the cytoplasmic enzyme lactate dehydrogenase (LDH) produced by cells was measured using the LDH test. LDH is released in the cytosol when cells are injured or stressed. The experiments revealed lower levels of LDH released in A-375 and HaCaT cells’ treatment groups when compared to wounded cells even at lower doses. Based on the LDH release findings and cell viability results, an effective dose range of TOMESORAL for recovery from H2O2 caused stress was determined to be lOOug / ml- 2000ug / ml with an OC of approximately 150ug / ml, as shown in Figure 2A and 2B. 3. Melanin assessment in TOMESORAL treated cells

[0055] Melanin is primarily localized within the basal layer, also known as the Stratum basale, of the human epidermis. Within this layer, melanin is present in both melanocytes (MCs), responsible for melanin synthesis, and adjacent keratinocytes (KCs), which receive and utilize melanin. The results indicated a reduced quantity of melanin in the A-375-Tomes group, demonstrating the anti- melanogenic effects of TOMESORAL. Both groups of HaCaT did not exhibit a significant amount of melanin. However, the Co-Culture-A375+ HaCaT group showed a significant amount of melanin, despite the removal of melanocytes from the culture before melanin estimation. Furthermore, melanin synthesis occurred in melanocytes, and the transfer of melanin to keratinocytes was evident in co-cultured cells with A-375 melanocytes, explaining the expression of melanin in keratinocytes. Conversely, when these co-cultured cells were treated with TOMESORAL with melanocyte digestion was conducted post-treatment, a significantly lower amount of melanin was detected, indicating the inhibition of melanin transfer from melanocytes to keratinocytes as illustrated in Figure 3. 4. Protease-activated receptor 2 (PAR-2) activity assessment

[0056] PAR-2 participates in various physiological functions, encompassing growth, development, mitogenesis, injury responses, and cutaneous pigmentation. In keratinocytes, PAR-2 regulates growth and differentiation, with its inhibition promoting increased growth, differentiation, and regeneration of keratinocytes. Additionally, PAR-2 is implicated in the phagocytosis of melanin and can trigger Ca2+ activity within cells. Results indicated low percentage activity of PAR-2 in HaCaT cells showing the inhibition of melanin transfer that will further inhibit cutaneous pigmentation. In TOMESORAL post-treated co-cultured cells, the percentage of PAR-2 activity was significantly lowered as shown in Figure 4. 5. Increased GSH in Post Treated Groups

[0057] Co-cultured A-375 and HaCaT cells’ intracellular GSH levels were measured (n = 5 replicates) before and after TOMESORAL treatment. GSH levels were found to be significantly increased in TOMESORAL post treated cells as apparent in Figure 5.

[0058] The above provided results clearly indicate that the heirloom tomato powder, enriched in carotenoids and flavonoids, could be a potent agent for inhibition of melanin transfer and enhancing glutathione production, contributing to improved skin tone and longterm skincare benefits. The optimal dose range for effective treatment without cytotoxic effects was identified, and post-treatment with TOMESORAL concentrations showed significant recovery from injury, reduced LDH release, reduced PAR-2 activity, inhibited melanin transfer, and increased GSH levels.

[0059] In conclusion, the present disclosure discloses the usage of TOMESORAL (a white heirloom tomato extract) for skin cell regeneration, highlighting the promising prospects for skincare interventions of the colorless extract. The presented results confirm the non-cytotoxic effects of TOMESORAL and demonstrate its efficacy in mitigating melanin transfer, reducing oxidative stress, and promoting skin cell lightening.

Claims

1. A composition for skin lightening comprising essentially of white heirloom tomato extract (TOMESORAL) containing carotenoids and flavonoids inhibiting melanin transfer activity.

2. A method for preparing the composition of skin lightening comprising the steps of: washing the white heirloom tomatoes to remove impurities;freeze drying the selected tomatoes;crushing the freeze-dried tomatoes into a powder to create the extract;dissolving the tomato extract into distilled water; andfiltering the dilution and mixing with DMEM to prepare the final dilution.

3. The composition of Claim 1, wherein the optimum dilution is 150 ug / ml.

4. The composition of Claim 1, wherein the flavonoids are water soluble.

5. The composition of Claim 1, wherein the flavonoids are water insoluble.

6. A method for skin lightening, comprising the administration of an effective amount of the composition to the skin surface.

7. The method of claim 2, wherein the carotenoids in the composition are colourless.

8. The method of claim 2, wherein the composition is derived specifically from white heirloom tomatoes.

9. The method of claim 2, configured to improve skin tone and appearance.

10. A skin care system for improving skin tone and appearance, comprising essentially of: a composition derived from white heirloom tomatoes containing colourless carotenoids and flavonoids of claim 1;a cytotoxicity assessment module;an injury reduction analysis module;a melanin estimation module;a PAR-2 activity evaluation module; anda GSH level estimation module, wherein the skin care system is configured to reduce melanin transfer, increase GSH levels.

11. The skin care system of Claim 10, wherein the cytotoxicity assessment depicts the safety of the composition.

12. The skin care system of claim 10, wherein the injury reduction analysis assesses recovery from skin injury.

13. The skin care system of claim 10, wherein the PAR-2 activity evaluation measures the inhibition of melanin transfer.

14. The skin care system of claim 10, wherein the GSH level estimation measures the increase in GSH levels in the skin.

15. The skin care system of claim 10, wherein the composition reduces LDH release, indicating reduced cell damage.

16. The skin care system of claim 10, wherein the composition shows no cytotoxic effects.

17. The skin care system of claim 10, wherein it is configured to be used for skin lightening, offering a safe and effective solution for improving skin tone and appearance.LDCMApplication No: GB2416528.4Examiner:Dr Gareth MarlowClaims searched: 1-21Date of search: 23 April 2025Patents Act 1977: Search Report under Section 17Documents considered to be relevant:Category Relevant to claims Identity of document and passage or figure of particular relevance X 1 -21 WO 2022 / 238727 Al (MOMAND HOSSAY), see entire document, esp. Page 3, Claims X 1 -21 Kurdish Studies, Sept 2024, vol 12., no 5. Momand et. al., "Exploring the dual benefits of'TOMESORAL': photo-protective and skin lightening potential of carotenoids and flavonoids in a preliminary controlled trial", pg 949-965. see entire document, esp. Sections 5.3.2, 5.5, Conclusion, avalable from; View of Exploring the Dual Benefits of'TOMESORAL': Photo-Protective and Skin Lightening Potential of Carotenoids and Flavonoids in a Preliminary Controlled Trial <https: / / kurdishstudies.net / menu- X 1 -21 script / index, php / ks / article / view / 3 3 80 / 2294> CN 110192994 A (KUNLUN TECH RESEARCH GUANGZHOU CO LTD), see esp. ExamplesCategories:X Document indicating lack of novelty or inventive step A Document indicating technological background and / or state of the art. Y Document indicating lack of inventive step if P Document published on or after the declared priority date but combined with one or more other documents of before the filing date of this invention. same category. & Member of the same patent family E Patent document published on or after, but with priority date earlier than, the filing date of this application.Field of Search:International Classification:Subclass Subgroup Valid From A61K 0008 / 9789 01 / 01 / 2017 A61Q 0019 / 02 01 / 01 / 2006Application No: GB2416528.4Claims searched: 1-17Examiner: Dr Gareth MarlowDate of search: 29 October 2025Patents Act 1977Further Search Report under Section 17Documents considered to be relevant:Category Relevant to claims Identity of document and passage or figure of particular relevance v A 1 - 17 WO 2022 / 238727 Al (MOMAND HOSSAY), see entire document, esp. page 4 and Claims X 1 - 17 CN 110192994 A (KUNLUN TECH RESEARCH GUANGZHOU CO LTD), see esp. examples X 1 - 17 Kurdish Studies, Sept 2024, vol 12, no. 5, Momand et. al. "Exploring the dual benefits of'TOMESORAL': photoprotective and skin lightening potential of carotenoids and flavonoids in a preliminary controlled trial", pg 949 - 965 see esp. Sections 5.3.2, 5.5 and Conclusion A - Drying Technology, 2010, 28(5), Gaware et. al., "Drying of tomato using different methods: comparison of dehydration and rehydration kinetics", pg 651-658 see esp. Freeze DryingCategories:X Document indicating lack of novelty or inventive step A Document indicating technological background and / or state of the art. Y Document indicating lack of inventive step if P Document published on or after the declared priority date but combined with one or more other documents of same category. before the filing date of this invention. & Member of the same patent family E Patent document published on or after, but with priority date earlier than, the filing date of this application.Field of Search:International Classification:Subclass Subgroup Valid From A61K 0008 / 9789 01 / 01 / 2017Subclass Subgroup Valid From A61Q 0019 / 02 01 / 01 / 2006

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