A method for preparing a novel composition having potential to recover photoageing, oxidative stress and inflammation induced by blue light

Abstract

A composition for improving skin health comprises Momordica cochinchinensis (Gac) yellow fruit extract; Theobroma cacao (cacao) seeds extract; Citrus paradise Macfad. (Grapefruit) fruit extract; Garci

Description

Embodiments of the present invention generally relate to compositions and methods of fruit and plants extract and using them to protect the skin against digital ageing caused by exposure to electronic devices, and, more particularly, to methods for preparing compositions aimed at increasing resistance against blue light (BL) and BL induced stress on skin, UVA and UVB, and further decreasing reactive oxygen species during BL stress. BACKGROUND OF THE INVENTION The present disclosure is generally directed towards comprehending and elucidating the primary causes of physiological ageing and the multitude of effects that it encompasses, particularly in relation to the human body. It is understood that ageing is fundamentally an accumulation of diverse physiological changes over time that are inevitable and intrinsic to the living process. Two primary theories have been identified that provide substantial insight into this complicated phenomenon, namely the Oxygen-Free Radical Theory and the Telomere Theory. The Telomere Theory posits a comprehensive explanation for the ageing process of telomeres, which are critical components of the linear chromosomes in eukaryotic cells. Telomeres function to safeguard the integrity of the chromosome, and as such, are integral to cellular health and vitality. It is observed that with each successive cell division, there occurs a gradual shortening of the replicated DNA strand and correspondingly, a reduction in the length of the telomere in the chromosome. This process of telomeric attrition is an integral part of the ageing process according to the Telomere Theory. Moreover, it is further elucidated that the length of a telomere is subject to fluctuations, particularly under conditions of oxidative stress. Notably, the telomeric DNA is observed to be less resilient to oxidative stress compared to other genomic regions, and hence, recovery from oxidative damage in telomeric DNA is generally less effective and efficient. The Oxygen-Free Radical Theory, on the other hand, provides an alternative perspective on the ageing process, emphasizing the detrimental impacts of oxidative stress. This stress, resulting from environmental factors such as air pollution and excessive sunlight exposure, enhances the presence of radicals within a living organism. These radicals can wreak havoc on key skin components such as hyaluronic acid, elastin, collagen, and the connective tissue of the corium, thereby contributing to the formation of skin wrinkles. Moreover, an increased generation of radicals can stimulate an overproduction of melanin, which subsequently leads to adverse skin conditions such as discoloration, freckles, and wrinkles. The ageing process, it should be noted, does not occur uniformly across individuals. It has been observed to manifest at varying rates, and to varying degrees due to numerous external factors that induce premature skin ageing. Such factors encompass exposure to sunlight, BL exposure, and exposure to harmful UVB radiation. These factors trigger a range of undesirable biochemical changes in the skin including inflammation and damage to DNA and cellular organelles, a condition commonly referred to as photo-damage. This, in turn, augments the risk of numerous skin conditions, including but not limited to wrinkles, age spots, sunburn, skin redness and skin sagging, fine lines, loss of elasticity, increased sagging, loss of firmness, uneven skin tone, coarse surface texture, and mottled pigmentation. Accordingly, the present disclosure elucidates on the intricate mechanisms underlying the physiological ageing process, with a specific focus on its impact on skin health and appearance. The disclosure further provides valuable insights into the external factors that influence this process and potentially exacerbate premature skin ageing. The present disclosure further illuminates the deleterious effects of prolonged exposure to compact fluorescent light (CFL) bulbs, light-emitting diodes (LEDs), and electronic device screens and displays that utilize LED backlights. These sources commonly emit light in the ranges of 380-500 nm and 400-500 nm respectively, contributing significantly to a phenomenon now referred to as 'digital ageing'. This phenomenon, characterized by skin damage, increased risk of skin cancer, and premature skin ageing, is an emerging concern in our increasingly digital society. High Energy Visible light (HEV) has been found to expedite skin ageing by promoting the overexpression of damaging free radicals and compromising the skin barrier function. Existing conventional disclosures have noted the existence of various cosmetic compositions, often derived from fruit or plant powders, designed to shield the skin from UVB and UVA light exposure. However, these formulations appear to have overlooked the need to protect the skin from the effects of visible light and HEV wavelengths. Furthermore, conventional sunscreen compositions are predominantly designed to absorb light at wavelengths below 380 nm. To date, there has not been identified a single sunscreen agent capable of providing comprehensive protection from all harmful wavelengths that affect the skin. Even combinations of various sunscreen agents in cosmetic compositions have failed to provide complete protection from all harmful wavelengths, especially longer wavelength solar radiation (>380 nm), including the damaging effects of CFL- and LED-light exposure above 380 nm. In addition, as disclosed in prior art, conventional methods of anti-ageing utilizing cosmetic products comprising retinol, retinoic acid, retinyl palmitate, and the like are well known. However, the effects of such products are not always satisfactory, and many of these products have been associated with skin irritation and fail to provide protection from indoor BL (HEV). Moreover, certain prior art discloses topical cosmetic compositions that incorporate dapsone, yet these compositions were principally utilized for the treatment of acne and did not offer protection against harmful BL or UV radiation. Furthermore, some prior art does disclose the use of cosmetic formulations comprising oak extract, grape seed, and green tea to protect the skin against air pollutants. However, these compositions similarly do not offer protection against the harmful effects of BL or UV radiation. It should also be noted that numerous cosmetic compositions that include ascorbic acid, alpha tocopherol, or Superoxide Dismutase (SOD) as free radical eliminators are commercially available. However, these cosmetic formulations have not been shown to offer protection against the harmful effects of BL. Moreover, such products have often been criticized for their economic inefficiency and lack of satisfactory effects, largely due to the instability of the chemical mixtures. Given the shortcomings in the current state of the art, there is an urgent need for the development of herbal cosmetic compositions, potentially based on fruit or plant extracts, that can effectively protect the skin from digital ageing, including exposure to artificial lighting comprising largely of HEV light. In essence, there is a pressing need for a composition that endeavours to harness novel substances extracted from plants or fruits to prevent premature skin ageing, and protect the skin from the harmful effects of BL. SUMMARY OF THE INVENTION The embodiments of the present disclosure have several features, no single one of which is solely responsible for their desirable attributes. Without limiting the scope of the present embodiments as expressed by the claims that follow, their more prominent features will now be discussed briefly. After considering this discussion, and particularly after reading the section entitled “Detailed Description”, one will understand how the features of the present embodiments provide advantages. In accordance with one embodiment of the present disclosure, an organic fruit I plant based composition for ameliorating the damaging effects of BL exposure, oxidative stress, and inflammation. According to the same embodiment, the composition may comprise of extracts from Momordica cochinchinensis (Gac) yellow fruit, Theobroma Cacao (cacao) seeds, Citrus paradise Macfad. (Grapefruit) fruit, Garcinia mangostana (mangosteen) fruit, and Rosmarinus officinalis (Rosemary) leaves. According to another embodiment of the present disclosure, a method for preparing the said composition is provided. In the first step, the ingredients of the disclosed composition, namely Momordica cochinchinensis (Gac) yellow fruit, Theobroma cacao (cacao) seeds, Citrus paradise Macfad. (Grapefruit) fruit, Garcinia mangostana (mangosteen) fruit, and Rosmarinus officinalis (Rosemary) leaves may be subjected to freeze-drying to effectively dehydrate the ingredients while preserving their structural and nutritional integrity. The freeze-drying process converts the ingredients into a fine powder, which may then be sieved to a granularity of approximately 80 mesh. Following the freeze drying process, the resulting fine powder may then be dissolved and mixed in phosphate buffered saline (PBS) to achieve the desired potency of the disclosed composition. It should be noted that while these embodiments provide a method for preparing the disclosed composition, various modifications and alterations may be implemented without departing from the scope of the present disclosure. Such modifications and variations are contemplated within the scope of the present disclosure. DETAILED DESCRIPTION The present invention encompasses various embodiments primarily aimed at providing novel and inventive solutions focusing on methods for preparing a composition aimed at fighting the adverse effects of exposure to BL. This composition includes phytochemical resources including Momordica cochinchinensis (Gac) yellow fruit, Theobroma cacao (cacao) seeds, Citrus paradise Macfad. (Grapefruit) fruit, Garcinia mangostana (mangosteen) fruit, and Rosmarinus officinalis (Rosemary) leaves. The preparation method involves freeze-drying the aforementioned ingredients to produce a fine powder with an approximately 80 mesh size. This powder is then dissolved and mixed in PBS to create a stock solution of required concentration. In accordance with a specific embodiment of the present invention, the preparation method may be extended to include passing the fine powder solution through a sterile filter to produce different dilutions in a plain medium (DMEM). According to one of the embodiments, the fine powder solution is prepared using various dilutions such as 100ug / ml, 200ug / ml, 500ug / ml, and 1000ug / ml. Testing is then performed by obtaining a HaCaT (human keratinocytes) cell line and culturing the cell line with an DMEM medium complemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 pg / ml streptomycin solution. This culture is maintained at a temperature of37°C in a 5% CO2 incubator. The experimentation may further involve culturing the HaCaT cell lines onto a 96 well plate and subsequently treating the cell lines with different powers of BL. Cellular metabolic activity may then be observed by calculating the viability of HaCaT cells using a colorimetric MTT assay. Table 1 provides a summary of this test setup with the exposure times and corresponding irradiation power used for different test setups. Table 1: Blue light stress intensity and time of exposure Irradiation power Exposure time 5 J / cm2 15 mins 10 J / cm2 30 mins 15 J / cm2 30 mins 25 J / cm2 1.5 hrs 45 J / cm2 1.5 hrs 50 J / cm2 3 hrs 85 J / cm2 3 hrs In order to monitor cell proliferation, the HaCaT cells are treated with an IC 50 dose of LED-BL irradiation, followed by treating the cells with varying concentrations of the disclosed composition (100ug / ml, 200ug / ml, 500ug / ml, and 1000ug / ml) for 24 hours to evaluate the cell via MTT assay. An optimum composition strength of 200ug / ml is selected for further testing. As illustrated in Fig. 1A and Fig. 1B, the MTT assay was performed, which is a reliable and simple method for the measurement of cell cytotoxicity. The figures show the plot of percentage (%) viabilities versus various powers of BL. The bar graph in Fig. 1A illustrates the relative values of percentage viability after the MTT assay, while Fig. 1B's line graph displays the relative percentage of viabilities on different powers of BL. Figures 2A and 2B show the plot of percentage (%) viabilities versus various concentrations of the disclosed composition powder. The bar graph in Fig. 2A illustrates the relative values of percentage viabilities after the MTT assay, while Fig. 2B's line graph displays the relative percentage of viabilities on different concentrations of the disclosed composition. The Lactate Dehydrogenase (LDH) activity of the HaCaT cells treated with an optimum concentration (200pg / ml) of the disclosed composition via an LDH assay is measured for evaluating cell membrane integrity as a marker of cytotoxicity and cell viability. LDH is released in cytosol when cells are damaged or under stress, the results presented in Table 2 and Fig. 3 indicate a reduction in excreted LDH levels in the treated group of HaCaT cells, showing the efficacy of the disclosed composition. Table 2: Relative absorbance of post treated cells Groups Control BL stress 200ug / ml treated Absorban ce (SEM) 0.0643±0. 0004 0.149±0.0 101 0.0579±0. 0011 Fig. 3 depicts the expression analysis of Lactate Dehydrogenase (LDH) release, where the graph signifies the LDH release in post-treated cells. The recorded values represent the mean ± SEM, with the asterisk (*) indicating a significant difference between the LDH levels of treated groups compared to the untreated controls (p<0.05). As per a particular embodiment of the present invention, the disclosed compositions include bioactives, potent elements capable of increasing angiogenesis, thereby inducing anti-aging effects. In application, upon treating HaCaT cells with the disclosed composition, an increased rate of angiogenesis is observed. This was estimated by gauging the level of the angiogenetic protein, Vascular Endothelial Growth Factor (VEGF), in post-treated HaCaT cells. The ensuing results suggest that treatment of the cell line with the optimum composition of the disclosed composition augments the level of angiogenesis in HaCaT cells. The VEGF levels were assessed via two methodologies: immunocytochemistry and Enzyme-Linked Immunosorbent Assay (ELISA). Immunocytochemistry demonstrated amplified levels of VEGF in HaCaT cells treated with the optimum concentration of the disclosed composition, whereas in cells stressed by BL, the level of angiogenesis is generally deemed to be lower. Further quantification of angiogenic secreted protein levels was conducted by ELISA, which revealed (as shown in Table 3) increased VEGF levels in post-treated HaCaT cell group (treated with optimum composition of the disclosed composition) when compared to BL-stressed HaCaT cells. These results underscore the potential of the disclosed composition in promoting angiogenesis and mitigating the harmful effects of BL, thus serving as a promising solution in the realm of anti-aging skincare. Table 3: ELISA value of VEGF Groups Control Blue light stress 200ug / ml Absorbanc e (SEM) 0.531±0.01 85 0.178±0.0 182 0.512±0.03 90 Fig. 4A illustrates an immunofluorescence micrograph showing staining of VEGF, wherein green colour intensity shows the level of protein expression, and Fig. 4B’s graph shows the angiogenesis levels in post treated cells. Values were taken as mean ± SEM and * shows the significant difference between VEGF levels of treated groups and untreated controls (p<0.05). In accordance with an embodiment of the present invention, the compositions as disclosed herein have proven to be a strong candidate for inflammation reduction. In use, after treating HaCaT cells with the disclosed composition, reduced collagenase and inflammation was observed in stressed cells via estimating the level of protein MMP-1 and inflammatory markers IL1-a and TNF- a in HaCaT cells treated with the optimum concentration (200pg / ml)of the disclosed composition. According to the results, treatment of cell line lowers the collagenase and inflammation levels in HaCaT cells. Levels of collagenase and inflammation as estimated by ELISA showed reduced collagenase and inflammation levels in post treated groups as compared to BL stressed cells. Additionally, the post treated group showed similar levels of collagenase and inflammation as that of untreated group (control). The results of this study are presented in Table 4. Table 4: ELISA absorbance values of MMP-1, IL1-a and TNF-a Groups Control Blue light stress 200ug / ml MMP-1 0.345±0.02 17 0.753±0.02 76 0.354±0.024 3 IL1-a 0.240±0.01 90 0.783±0.04 09 0.290±0.019 4 TNF-a 0.344±0.02 99 0.633±0.01 84 0.346±0.023 3 Fig. 5A’s graph shows the MMP-1 levels in post treated cells, Fig. 5B’s graph shows the levels of IL-1 a in post treated cells, Fig. 5C’s graph shows the TNF-a level in post treated cells, and Fig.5D’s Immunofluorescence micrograph showing staining of TNF-a. Green color intensity shows the level of protein expression. Values were taken as mean ± SEM and * shows the significant difference between TNF-a levels of treated groups and untreated controls (p<0.05). In order to observe the antioxidant properties of the disclosed composition, HaCaT cells’ intracellular MDA activity was measured (n = 5 replicates) before and after treatment with the disclosed composition powder. MDA activity was significantly decreased in post treated cells. Table 5 shows the absorbance values measured in this study. Table 5: Absorbance values of MDA Groups Control Blue light stress 200ug / ml Absorbanc e (SEM) 0.341±0.01 63 0.818±0.0 353 0.432±0.02 20 As illustrated in Figure 6, an analysis of MDA and the corresponding bar graph shows the MDA levels in post treated HaCaT cells. Values were taken as mean ± SEM and * shows the significant difference between MDA levels of treated groups and controls (p<0.05). In accordance with an embodiment of the present invention, the compositions as disclosed herein show a decreased activity of Catalase in the disclosed composition treated HaCaT cells. In use, catalase activity was measured in HaCaT cells (n = 5 replicates) before and after treatment with the optimal concentration of the disclosed composition powder. Catalase activity was found to be lowered significantly in post treated HaCaT cells. While, in case of BL stressed cells catalase activity was found to be very high. Table 6 shows the results of catalase absorbance. Table 6: Absorbance values of catalase Groups Control Blue light stress 200ug / ml Absorbanc e (SEM) 0.835±0.07 34 1.4810.09 88 0.865±0.05 46 Figure 7 illustrates an activity of catalase, wherein the graph represents the levels of catalase in post treated HaCaT cells. Values were expressed as mean ± SEM and * shows the significant difference between MDA levels of treated groups and controls (p<0.05). Those of ordinary skill in the art will appreciate that bioactives in the disclosed composition could potentially increase the blood flow to the skin via increased VEGF resulting in induction of anti-aging. Also, the disclosed composition was proven to be a strong candidate for inflammation reduction as well as an active enhancer of the antioxidant activity to combat the BL-induced oxidative stress. Generally, it is well known that throughout the years, the advancement of optical screen technologies has advanced dramatically, and many electrical devices now use LED backlight technologies to enhance light and visibility of the screen. These LEDs emit very intense blue waves of light. The products using this technology include mobile phones, laptops, iPads and flat screen televisions. Consequently, people are increasingly exposed to more and more BL sources for longer periods due to their pervasive use and growing prominence. BL has a very small wavelength, and so generates a larger amount of energy. Accordingly, as per analysis related to the present invention, it is reported that LED-BL in keratinocytes at 45 J / cm2 has adverse effects on the skin, including premature aging, by releasing free radicals that trigger cell-damage. At the same time, the results demonstrated that the disclosed composition protects skin keratinocytes by damage induced by LED-BL avoiding ROS formation and decreasing MMP-1, preserving collagen type I production, and maintaining the proliferation expression without influencing cell viability. In use, vascular endothelial growth factor (VEGF) promotes angiogenesis and plays important roles both in physiological and pathological conditions. VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and are specific to endothelial cells but constitutively expressed in normal human keratinocytes. The disclosed invention, through multiple tests, has demonstrated to be a potent agent to enhance the production of factors who could potentially enhance the regenerative, antioxidative and anti-inflammatory properties of skin cells that will in turn enhance the production of collagen and reduction of stressed factors in skin cells. The conditional language used herein, such as, among others, “can,” “could,” “might,” “may,” “e.g.,” and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and / or steps. Thus, such conditional language is not generally intended to imply that features, elements and / or steps are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and / or steps are included or are to be performed in any particular embodiment. The terms "comprising," "including," 'having," and the like are synonymous and are used inclusively, in an open-ended fashion, and do not exclude additional elements, features, acts, operations, and so forth. Also, the term "or" is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term "or" means one, some, or all of the elements in the list. While there has been shown and described the preferred embodiment of the instant invention it is to be appreciated that the invention may be embodied otherwise than is herein specifically shown and described and that, within said embodiment, certain changes may be made in the form and arrangement of the parts without departing from the underlying ideas or principles of this invention as outlined in the Claims appended herewith. Therefore, the appended claims are to be construed to cover all equivalents falling within the true scope and spirit of the invention.

Claims

1. A composition to improve skin health comprising of:Momordica cochinchinensis (Gac) yellow fruit extract;Theobroma cacao (cacao) seeds extract;Citrus paradise Macfad. (Grapefruit) fruit extract;Garcinia mangostana (mangosteen) fruit extract; andRosmarinus officinalis (Rosemary) leaves extracts.

2. The composition of Claim 1, wherein an optimal in-vitro cyto-protective concentration is 200ug / ml.

3. A method of preparing the said composition to improve skin health, comprising the steps of:freeze drying the different components to produce fine powder to about 80 mesh powder; anddissolving and mixing the said fine powder of ingredients in saline to make a stock solution of the required concentration;4. The method of Claim 3, wherein the saline is the phosphate buffered saline.

5. The method as claimed in Claim 3, wherein the said method further comprises thestep of passing the said fine powder solution through sterile filter solution for preparing different dilutions from stock solution in plain medium (DMEM without FBS).

6. The method as claimed in Claim 5, wherein the said fine powder solution is prepared by using different dilutions of 100ug / ml, 200ug / ml, 500ug / ml and 1000ug / ml.

7. The method as claimed in Claim 3, wherein the said method further comprises steps of:obtaining a HaCaT (human keratinocyte) cell line; and,culturing said cell line with DMEM medium complemented with 10% fetal bovine serum (FBS), 100 U / ml penicillin and 100 pg / ml streptomycin solution at a temperature of 37°C in a 5% CO2 incubator.

8. The method as claimed in Claim 3, wherein the said method further comprises the steps of;culturing said HaCaAT cell lines onto 96 wells plate; andtreating the said cell lines with different powers (5 J / cm2, 10 J / cm2,15 J / cm2, 25 J / cm2, 45 J / cm2, 50 J / cm2 and 85 J / cm2) of blue light.

9. The method as claimed in claim 8, where the said method further comprises the steps of calculating viability of said HaCaT cells via MTT assay after blue light irradiation.

10. The method as claimed in Claim 3, wherein the said method further comprises the steps of:treating LED-BL irradiated HaCaT cells (irradiated with IC 50 dose of LED-BL);culturing said HaCaT cells onto a 96-well plate for irradiation, for measurement of cell proliferation;treating said cells with increased concentrations of 100ug / ml, 200ug / ml, 500ug / ml and 1000ug / ml of the said composition for 24 hours; andevaluating viability of said cells via MTT assay.

11. The method as claimed in Claim 3, wherein the said method comprises the steps of calculating the LDH activity of the said composition treated with LED-BL irradiated HaCaT cells via LDH released assay.

12. The method as claimed in Claim 3, wherein the bioactives in the said composition are configured to increase blood flow to skin via increased VEGF resulting in induction of anti-aging in BL irradiated HaCaT cells.

13. The method as claimed in Claim 3, wherein the said composition is configured to reduce inflammation in BL irradiated HaCaT cells.

14. The method as claimed in Claim 3, wherein the said composition is configured to enhance antioxidants to combat BL induced oxidative stress in BL irradiated HaCaT cells.

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