Methods for preparing compositions relating to blueguard oral for recovering from blue light photoageing, oxidative stress and inflammation

Abstract

A method for preparing composition of BlueGuard Oral for recovering from blue light, oxidative stress and inflammation. In use, the method includes the steps of, freeze drying of ingredients of BlueGuard-Oral to produce fine powder to about 80 mesh powder; and, dissolving and mixing the fine powder of ingredients of BlueGuard Oral in distilled water to make a solution of required concentrations.

Description

[0001] METHODS FOR PREPARING COMPOSITIONS RELATING TO

[0002] BLUEGUARD ORAL FOR RECOVERING FROM BLUE LIGHT

[0003] PHOTOAGEING, OXIDATIVE STRESS AND INFLAMMATION

[0004] FIELD OF THE INVENTION

[0005] Embodiments of the present invention generally relate to compositions and methods of fruit and plants powder, 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 and blue light stress on skin, UVA and UVB, and further decreasing reactive oxygen species during blue light stress.

[0006] BACKGROUND OF THE INVENTION

[0007] 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.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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, blue light 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.

[0012] 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.

[0013] 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.

[0014] High Energy Visible light (HEV), in particular, 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.

[0015] 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.

[0016] 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 blue light (HEV).

[0017] 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 blue light or UV radiation. Furthermore, prior art disclosures have described 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 blue light or UV radiation.

[0018] 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 blue light. 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.

[0019] Given these 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 powders, that can effectively protect the skin from digital ageing, including exposure to artificial lighting that contains larger quantities of HEV light. In essence, there is a pressing need for an invention that endeavors to harness novel substances extracted from plants or fruits to prevent premature skin ageing and protect the skin from the harmful effects of blue light.

[0020] SUMMARY OF THE INVENTION

[0021] 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.

[0022] In accordance with one embodiment of the present disclosure, a method is provided for preparing a composition known as BlueGuard Oral. This composition is intended for ameliorating the damaging effects of blue light exposure, oxidative stress, and inflammation. The preparation method involves a series of steps, which are detailed herein.

[0023] In the first step, the ingredients of the BlueGuard Oral composition are subjected to freeze drying. This process aims to effectively dehydrate the ingredients while preserving their structural and nutritional integrity. The freeze drying process converts the ingredients into a fine powder, which is then sieved to a granularity of approximately 80 mesh.

[0024] Following the freeze drying process, the resulting fine powder is then dissolved and mixed in distilled water. The concentration of the solution is carefully controlled to achieve the desired potency of the BlueGuard Oral composition. By employing this method, a composition is produced that is specifically designed to combat the harmful effects of blue light, oxidative stress, and inflammation.

[0025] It should be noted that while this embodiment provides one method for preparing the BlueGuard Oral composition, various modifications and variations may be implemented without departing from the scope of the present invention. Such modifications and variations are contemplated within the scope of the present disclosure. DETAILED DESCRIPTION

[0026] The present invention encompasses various embodiments primarily aimed at providing novel and inventive solutions focusing on methods for preparing a composition termed as BlueGuard Oral. This composition aids in the recovery from harmful effects of blue light, oxidative stress, and inflammation. The preparation method involves freeze-drying the ingredients of BlueGuard Oral to produce a fine powder with an approximately 80 mesh size. This powder is then dissolved and mixed in distilled water to create a solution of required concentrations. In application, the composition serves to protect cells from damage induced by blue light (BL).

[0027] In a specific embodiment of the present invention, the preparation method is extended to include passing the fine powder solution through a sterile filter to produce different dilutions in a plain medium (RPMI 1640).

[0028] In another embodiment, the fine powder solution is prepared using various dilutions such as 100ug / ml, 200ug / ml, 500ug / ml, and 1000ug / ml.

[0029] A further embodiment introduces additional steps, including obtaining a HaCaT human keratinocyte cell line and culturing the cell line with an RPMI 1640 medium complemented with 10% fetal bovine serum, 100 ll / rnl penicillin, and 100 pg / ml streptomycin solution. This culture is maintained at a temperature of 37°C in a 5% CO2 incubator.

[0030] Yet another embodiment involves culturing the HaCaT cell lines onto a 96 well plate and subsequently treating the cell lines with different powers of blue light. An additional embodiment includes the calculation of viability of the HaCaT cells using an MTT assay, a colorimetric assay for assessing cell metabolic activity. In a further embodiment, the method includes treating the HaCaT cells with an IC 50 dose of LED-BL irradiation, culturing the HaCaT cells onto a 96-well plate for irradiation to measure cell proliferation, treating the cells with increased concentrations of 100ug / ml, 200ug / ml, 500ug / ml, and 1000ug / ml of BlueGuard Oral for 24 hours, and evaluating the viability of the cells via assay. Herein, an optimum cyto-protective dose for BL stress recovery is identified as 200ug / ml. As illustrated in Fig. 1A and Fig. 1 B, 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 blue light. The bar graph in Fig. 1A illustrates the relative values of percentage viability after the MTT assay, while Fig. 1 B's line graph displays the relative percentage of viabilities on different powers of blue light.

[0031] Figures 2A and 2B show the plot of percentage (%) viabilities versus various concentrations of BlueGuard Oral 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 BlueGuard Oral.

[0032] In accordance with an additional embodiment of the present invention, the method includes calculating the Lactate Dehydrogenase (LDH) activity of the HaCaT cells via an LDH release assay, a common method for evaluating cell membrane integrity as a marker of cytotoxicity and cell viability.

[0033] In accordance with an embodiment of the present invention, ingredients of the BlueGuard Oral includes xanthones, phenolic acid, and flavonoid, Garcinia mangostana, theobroma cacao seed extract, rosemary, lutein & zeaxanthin. Those of ordinary skill in the art will appreciate that in order to assess the level of cytoplasmic enzyme lactate dehydrogenase (LDH) released from the cells LDH assay was done. LDH is released in cytosol when cells are damaged or under stress. Our results showed reduced levels of LDH released in treatment groups of 3T3 cells (Table 1 , Figure 3).

[0034] Table 1 : Relative absorbance of post treated cells

[0035] Figure 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).

[0036] 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 BlueGuard Oral, an increased rate of angiogenesis was 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 BlueGuard Oral augments the level of angiogenesis in HaCaT cells. VEGF levels were assessed via two methodologies: immunocytochemistry and Enzyme-Linked Immunosorbent Assay (ELISA). Immunocytochemistry demonstrated amplified levels of VEGF in treated HaCaT cells, whereas in cells stressed by blue light, the level of angiogenesis is generally deemed to be lower. Further quantification of angiogenic secreted protein levels was conducted by ELISA, which revealed increased VEGF levels in post-treated HaCaT cell groups when compared to blue light-stressed HaCaT cells. These results underscore the potential of BlueGuard Oral in promoting angiogenesis and mitigating the harmful effects of blue light, thus serving as a promising solution in the realm of anti-aging skincare.

[0037] Table 2: ELISA value of VEGF

[0038] Fig. 4A illustrates an immunofluorescence micrograph showing staining of VEGF, wherein green color 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 blueguard-oral, 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 post treated HaCaT cells. According to the results, treatment of cell line with blueguard lowers the collagenase and inflammation levels in HaCaT cells. Levels of collagenase and inflammation were estimated by ELISA showed reduced collagenase and inflammation levels in blueguard post treated groups as compared to blue light stressed cells. Whereas in post treated group the levels of collagenase and inflammation showed no significant difference from untreated group. Levels of inflammation were also estimated with immunostaining of TNF-a. This also strengthened our finding of lower inflammation levels in blueguard post treated cells as compared to the stressed cells.

[0039] Table 3: ELISA absorbance values of MMP-1, IL1-a and TNF-a 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).

[0040] In accordance with an embodiment of the present invention, the compositions as disclosed herein possess remarkable property of enhancing the antioxidant to combat the BL induced oxidative stress. In use, HaCaT cells’ intracellular MDA activity was measured (n = 5 replicates) before and after treatment with blueguard-oral powder. MDA activity was significantly decreased in post treated cells. The presence of blueguard-oral powder in the media significantly lowered the MDA activity.

[0041] Table 4: Absorbance values of MDA

[0042] 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).

[0043] In accordance with an embodiment of the present invention, the compositions as disclosed herein show a decreased activity of Catalase in blueguard-oral Treated HaCaT cells. In use, catalase activity was measured in HaCaT cells (n = 5 replicates) before and after treatment with blueguard-oral powder. Catalase activity was found to be lowered significantly in post treated HaCaT cells. While, in case of blue light stressed cells catalase activity was found to very high.

[0044] Table 5: Absorbance values of catalase

[0045] 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).

[0046] Those of ordinary skill in the art will appreciate that bioactives in Blueguard- Oral could potentially increase the blood flow to the skin via increased VEGF resulting in induction of anti-aging. Also, BlueGuard-Oral was proved to be a strong candidate for inflammation reduction. Furthermore, BlueGuard-Oral has remarkable property of enhancing the antioxidant 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.

[0047] Consequently, people are increasingly exposed to more and more blue light sources for longer periods due to their pervasive use and growing prominence. Blue light has a very small wavelength, and so generates a larger amount of energy.

[0048] There exist multiple reports that show that, over time, exposure to the blue end of the light spectrum may cause significant long-term harm to eyes and health problems (eyestrain, headaches, physical and mental fatigue, sleep disorders, etc.) (Austin et al., 2018; Beatty, Koh, Phil, Henson, & Boulton, 2000; Marshall, Gordon, McCauley, de Souza Filho, & Burnier, 2006; Moon et al., 2008). Blue light exposure is well known to be important for good health. Evidence has shown that visible high-energy light enhances alertness, improves memory and cognitive performance and raises the mood.

[0049] Consequently, so much blue light can interrupt the flow of circadium, which can cause nights and tiredness during the morning. However, the latest question being asked by dermatologists is: “Is this intimate interaction with technology aging our skin?”

[0050] 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 blueguard-oral 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.

[0051] 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. Both the activated VEGFR-1 and VEGFR-2 protected against UVA-induced cell death (Zhu et al., 2013) and contribute to regenerate the damaged skin by neovascularization and enhanced blood supply. These beneficial effects are significant because many times during the day we are usually exposed to blue light damage. Whereas the chronological skin aging and BL-exposed disruption indicate differences within the phenotype, the clinically aged skin phenotype can be linked with a general transition leading to the assumption that both are driven by the same cause. In addition, the disease pathways tend to include chromophores linked to mitochondria (e.g. cytochromes and flavins; Godley et al., 2005) that absorb the visible spectrum in the blue region to greatly contribute to permanent DNA damage.

[0052] In addition, most features of ageing such as genomic dysfunction (Lopez-Otin, Blasco, Partridge, Serrano, & Kroemer, 2013), accumulation of mitochondrial DNA mutations (Birch-Machin, Russell, & Latimer, 2013), depletion of telomeres, and replicative senescence (Passos et al., 2007; Velarde, Flynn, Day, Melov, & Campisi, 2012) are driven by ROS and oxidative stress.

[0053] Hence the use of antioxidants to reduce the adverse impact of scavenging ROS is an innovative method to avoid damage from blue light penetration. The health benefits of using naturally occurring botanicals for their possible protective impact against light-radiation-mediated damage referred to as "photochemopreventive effects" have been given significant significance over the last few decades (Afaq, Adhami, Ahmad & Mukhtar, 2002).

[0054] In accordance with this invention, one such natural product is blueguard-oral, one of the most potent antioxidants that originates as a second metabolite of plant in nature. It has investigated for its radical scavenger activity, supported by a catalase and MDA assays. Studies have shown that the effect on light- mediated skin loss is effectively minimized by natural antioxidant-rich botanicals (Afaq et al., 2005; Afaq, Zaid, Khan, Dreher, & Mukhtar, 2009).

[0055] As per research conducted in accordance with embodiments of the instant invention, it has been demonstrated that LED-BL exposure produces ROS formation, oxidative stress, and damage to DNA. These effects are accompanied by evidently increased formation of MMP-1 (by drastic reduction of collagen type I) and reduction of cellular viability. Possibly, DNA damages result from exposure of cell cultures to LED-BL directly through DNA absorption and indirectly via the ROS. Light energy absorption in DNA photoproducts and ROS formation is well known. Cells when treated with blueguard-oral could combat ROS by decreased lipid peroxidation. All the ingredient in blueguardoral have already been investigated for their potential antioxidant properties, here the synergistic properties of these botanicals further enhance their potential of improving the antioxidative index of the stressed cells.

[0056] The present study demonstrated that BlueGuard-Oral powder could be a potent agent which can enhance the production of factors who could potentially enhance the regenerative, antioxidative and anti-inflammatory properties of skin cells that will in turn enhances the production of collagen and reduction of stressed factors in skin cells.

[0057] 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.

[0058] 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

Claims1. A method of preparing composition of BlueGuard Oral for recovering from blue light, oxidative stress and inflammation, said method comprising the steps of: freeze drying of ingredients of BlueGuard-Oral to produce fine powder to about 80 mesh powder; dissolving and mixing said fine powder of ingredients of BlueGuard Oral in distilled water to make a solution of required concentrations, wherein said composition protects cells from blue light (BL) induced damage, and wherein an optimum cyto-protective dose for BL stress recovery is 200ug / ml.

2. The method as claimed in claim 1 , wherein said method further comprises the step of passing said fine powder solution through sterile filter solution for preparing different dilutions in plain medium (RPMI 1640)3. The method as claimed in claim 2, wherein said fine powder solution is prepared by using different dilutions of 100ug / ml, 200ug / ml, 500ug / ml and 1000ug / ml.

4. The method as claimed in claim 1 , wherein said method further comprises steps of: obtaining a HaCaT human keratinocyte cell line; and,culturing said cell line with an RPMI 1640 medium complemented with 10% fetal bovine serum, 100 ll / rnl penicillin and 100 pg / ml streptomycin solution at a temperature of 37°C in a 5% CO2 incubator.

5. The method as claimed in claim 1 , wherein said method further comprises the steps of; culturing said HaCaAT cell lines onto 96 wells plate; treating said cell lines with different powers of blue light.

6. The method as claimed in claim 5, said method further comprises the steps of calculating viability of said HaCaT cells via MTT assay.

7. The method as claimed in claim 1 , said method further comprises the steps of, treating said HaCaT cells IC 50 dose of LED-BL irradiation; 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 BlueGuard-Oral for 24 hours; and, evaluating viability of said cells via assay.

8. The method as claimed in claim 1 , wherein said method comprises the steps of calculating the LDH activity of said HaCaT cells via LDH released assay.

9. The method as claimed in claim 1 , wherein Bioactives in Blueguard-Oral are configured to increase blood flow to skin via increased VEGF resulting in induction of anti-aging.

10. The method as claimed in Claim 1 , wherein said composition of BlueGuard Oral is configured to reduce inflammation.

11. The method as claimed in Claim 1 , wherein said composition of BlueGuard Oral is configured to enhance antioxidants to combat BL induced oxidative stress.

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