The protective effects of AL-pomegranate extract on oxidatively-stressed skin cells

Abstract

The present invention discloses a regenerative potential of anthocyanin less pomegranate (AL-pomegranate or white pomegranate) extract in promoting skin health by reducing oxidative stress, inflammati

Description

[0001] The present invention relates to the field of skin hygiene, and more particularly to product and method for cosmetic composition. BACKGROUND

[0002] The following 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] Skin health is a critical aspect of overall well-being and maintaining its integrity and resilience against environmental and physiological stressors is essential. Despite advancements in dermatological treatments, challenges persist in managing skin conditions exacerbated by oxidative stress, inflammation, and microbial imbalances. These factors are commonly implicated in various skin disorders, including acne, premature aging, and inflammatory skin diseases. Oxidative stress arises from an imbalance between reactive oxygen species (ROS) and the skin's antioxidant defenses, leading to cellular damage and accelerated aging. Free radicals can induce lipid peroxidation, compromise cell membranes, and exacerbate inflammation. Current treatments often focus on alleviating symptoms but may fall short in addressing the underlying oxidative damage and inflammatory processes effectively.

[0004] Inflammatory responses are another significant factor in skin disorders directly impacting aging and acne progression. Elevated levels of pro-inflammatory cytokines, such as interleukin-1 beta (IL-1 [3), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-a), are associated with chronic inflammation and skin damage. Existing anti-inflammatory treatments sometimes offer partial relief but may not fully address the systemic inflammation contributing to skin conditions. Moreover, microbial imbalances on the skin, particularly the proliferation of acne-causing bacteria, pose challenges in effective acne management. Conventional antimicrobial treatments may address microbial overgrowth but often lack the ability to balance skin microbiota or provide long-term benefits.

[0005] The search for natural and effective solutions to enhance skin health has led to an increased interest in plant-derived compounds with antioxidant, anti-inflammatory, and antimicrobial properties. Thus, there is a pressing need for innovative approaches that integrate natural compounds to address oxidative stress, inflammation, and microbial imbalances comprehensively. Effective solutions should offer enhanced antioxidant protection, reduce inflammation, and balance skin microbiota, ultimately improving overall skin health and resilience. As a result, there exists a need for improvements over the prior art and more particularly for a more efficient way. SUMMARY OF THE INVENTION

[0006] A brief summary on one or more aspects of the present invention is provided hereinafter to enable a basic understanding for these aspects. The summary is not an exhaustive review for all the conceived aspects and is intended to neither point out all the critical or decisive factors for all the aspects, nor define the scope of any or all of the aspects. Its only aim is to provide some concepts of one or more aspects in a simplified manner as a preface of the more detailed description given later.

[0007] In one aspect, the present invention provides a dermatological composition based on AL-pomegranate extract for preventing, retarding, or arresting skin changes associated with aging and the presence of acne.

[0008] According to another aspect, this invention provides a unique AL-pomegranate extract derived from white pomegranate, rich in bioactive compounds such as polyphenols, flavonoids, and prebiotics. According to the same aspect, the extract is prepared using a controlled ethanol and water extraction process, which ensures the retention of these beneficial compounds. The primary active components include punicalagins, ellagic acid, prebiotics and a range of flavonoids known for their potent antioxidant and anti-inflammatory properties.

[0009] According to yet another aspect of the present invention, the use of AL-pomegranate extract for enhancing skin health and addressing oxidative stress-related skin conditions is validated. According to this aspect the disclosed extract is shown to significantly improve antioxidant and anti-inflammatory responses in human keratinocytes exposed to oxidative damage from hydrogen peroxide (H2O2).

[00010] In another aspect, the AL-pomegranate extract demonstrates potent antioxidant activity by enhancing the function of key enzymes. According to the same aspect, the disclosed composition is observed to increase Superoxide Dismutase (SOD) activity and restore Catalase levels in a concentration-dependent manner, reflecting its capacity to neutralize reactive oxygen species and mitigate oxidative damage.

[00011] In yet another aspect, the disclosed composition is also shown to improve Glutathione Peroxidase (GPx) activity, which is crucial for reducing hydrogen peroxide and lipid peroxides, further protecting cells from oxidative stress. According to the same aspect, the disclosed extract also effectively reduces lipid peroxidation markers, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) Oxidase, and Lipoxygenase (LOX) activities, indicating its role in decreasing lipid peroxidation and oxidative damage in keratinocytes.

[00012] According to one last aspect the AL-pomegranate extract is shown to exhibit significant anti-inflammatory properties by decreasing the activity of Cyclooxygenase-2 (COX-2) and by reducing levels of pro-inflammatory cytokines, including Interleukin-1 beta (IL-ip), Interleukin-6 (IL-6), and Tumor Necrosis Factor-alpha (TNF-a). This reduction in inflammatory markers supports the extract’s potential in alleviating inflammatory skin conditions and promoting overall skin health.

[00013] The foregoing paragraphs have been provided by way of general introduction and are not intended to limit the scope of the following claims. The described embodiments, together with further advantages, will be best understood by reference to the following detailed description taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0014] 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.

[0015] The diagrams are for illustration only, which thus is not a limitation of the present disclosure, and wherein:

[0016] FIG. 1 illustrates Cellular viabilities in AL-pomegranate post-treated cultured human keratinocytes, in accordance with an aspect of the invention.

[0017] FIG. 2 illustrates Superoxide Dismutase (SOD) activity in AL-pomegranate post-treated cultured human keratinocytes, in accordance with an aspect of the invention.

[0018] FIG. 3 illustrates Catalase activity in AL-pomegranate post-treated cultured human keratinocytes, in accordance with an aspect of the invention.

[0019] FIG. 4 illustrates GPx activity in AL-pomegranate post-treated cultured human keratinocytes, in accordance with an aspect of the invention.

[0020] FIG. 5 illustrates NADPH oxidase activity in AL-pomegranate treated human keratinocytes, in accordance with an aspect of the invention.

[0021] FIG. 6 illustrates Cyclooxygenase-2 (COX-2) activity in AL-pomegranate treated cultured Human Keratinocytes, in accordance with an aspect of the invention.

[0022] FIG. 7 illustrates LOX activity in AL-pomegranate treated cultured Human Keratinocytes, in accordance with an aspect of the invention.

[0023] FIG. 8 illustrates Interleukin-1 beta activity in AL-pomegranate treated cultured Human Keratinocytes, in accordance with an aspect of the invention.

[0024] FIG. 9 illustrates IL-6 activity in AL-pomegranate treated cultured Human Keratinocytes, in accordance with an aspect of the invention.

[0025] FIG 10 illustrates Tumor necrosis factor-alpha activity in AL-pomegranate treated cultured Human Keratinocytes, in accordance with an aspect of the invention. DETAILED DESCRIPTION OF DRAWINGS

[0026] The following is a detailed description of the various aspects of the disclosure depicted in the accompanying drawings. The different 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 invention’s aspects; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.

[0027] This invention relates to a novel AL-pomegranate extract formulated to enhance skin health by addressing key factors such as oxidative stress, inflammation, and microbial imbalances, which are known contributors to skin aging and various disorders. The extract is derived from white pomegranate, which is rich in polyphenols, flavonoids, and prebiotics— compounds that offer significant antioxidant and anti-inflammatory properties. Key bioactive compounds present in the extract include punicalagins, ellagic acid, prebiotics and a variety of flavonoids, which are recognized for their ability to neutralize reactive oxygen species (ROS) and protect the skin from oxidative damage. The extract is prepared using an ethanol and water extraction process, ensuring the retention of high levels of polyphenols, flavonoids, and prebiotics. These compounds play a crucial role in enhancing the activity of antioxidant enzymes such as catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx), which protect cells from oxidative stress. Additionally, the prebiotics in the extract help balance the skin microbiome, potentially reducing the presence of acne-causing bacteria.

[0028] Inflammation is a major factor in skin aging and the progression of acne. The polyphenols in the AL-pomegranate extract have been shown to inhibit inflammatory pathways by reducing the expression of pro-inflammatory cytokines, including TNF-a, IL-6, and IL-ip. The extract also blocks cyclooxygenase-2 (COX-2) and lipoxygenase (LOX) pathways, further mitigating inflammatory responses. Moreover, the extract has been found to modulate lipid peroxidation, a process that can lead to excessive sebum production and contribute to acne. By influencing the activity of NADPH oxidase, a key enzyme in ROS production and lipid peroxidation, the extract helps reduce oxidative stress and sebum production, thereby decreasing acne symptoms. The antioxidant properties of compounds like punicalagins and flavonoids also stabilize cell membranes and prevent oxidative damage to lipids. The AL-pomegranate extract is tested on cultured human keratinocytes subjected to oxidative injury using hydrogen peroxide. The treatment involves various concentrations of the extract, and its effects were evaluated on biomarkers including COX-2, LOX, TNF-a, IL-6, IL-ip, catalase, NADPH oxidase, SOD, and GPx. The results demonstrated that the extract significantly reduces inflammation, enhances antioxidant defenses, modulates lipid peroxidation, and mitigates acne, highlighting its potential as a natural therapeutic agent for dermatological applications and anti-aging skincare solutions.

[0029] This application covers the unique formulation and application of the AL-pomegranate extract in skincare products designed to improve skin health by minimizing oxidative stress, inflammation, and microbial imbalances.

[0030] The present methodology details the procedures employed in evaluating the effects of AL-pomegranate extract on cultured human keratinocytes (HaCaT cells) subjected to oxidative stress. Multiple tests have also been conducted to determine an optimal extract composition having the abilities to improve healthcare while having minimal negative impact. Experimental results focus on assessing the extract's potential to augment antioxidant enzyme functions, reduce lipid peroxidation, and mitigate inflammation, ultimately contributing to skin health. The steps outlined herein include the preparation of the extract, induction of oxidative injury in HaCaT cells, treatment with varying concentrations of the extract, and subsequent biochemical assays to measure relevant biomarkers.

[0031] Test Setup and Preparation

[0032] Sampling- Pomegranate (Punica granatum L.) fruit of the anthocyanin-less (AL) genotype is harvested from the field in Multan, Pakistan. The fruit is thoroughly washed with the leaves and stems removed. The cleaned fruit is freeze-dried to preserve its bioactive components. The dried fruit is then crushed, homogenized, and powdered, before storing in airtight bottles for further analysis.

[0033] Bioactive Compound Extraction - In order to extract the naturally occurring bioactives (polyphenols, flavonoids and probiotics) found within the pomegranate fruit, a 10g sample of the powdered pomegranate fruit is mixed with 100 ml of distilled water in a separatory funnel containing 50% ethanol. The mixture is filtered through Whatman No. 1 filter paper to remove sediment. The resulting filtrate is concentrated under reduced pressure using a rotary evaporator at 40°C and then lyophilized to obtain the final extract.

[0034] Injury of H2O2 to HaCaT Cells - HaCaT cells are cultured in Dulbecco's Modified Eagle's Medium High Glucose (DMEM-HG) supplemented with 100 pg / mL streptomycin, 100 U / mL penicillin, and 10% fetal bovine serum (FBS) until they reach 70-80% confluence. The medium is then replaced with fresh DMEM-HG containing 6 mM H2O2 and incubated for 4 hours to induce oxidative injury. After incubation, cells are washed with PBS, harvested for analysis, or subjected to further treatment.

[0035] Treatment of Injured HaCaT Cells with AL-Pomegranate Extract- A 10 mg / mL stock solution of AL-pomegranate extract is prepared by dissolving 10 mg of the extract in dimethyl sulfoxide (DMSO). This stock solution is diluted with DMEM to prepare various concentrations: 10 pg / mL, 20 pg / mL, 50 pg / mL, 100 pg / mL, and 150 pg / mL. The treated cells are incubated with these dilutions for 24 hours. Post-treatment, cell viability is assessed using the MTT assay, and the cells are used for additional analyses. The experimental groups are summarized in the Table 1: Table 1. Post-treated HaCaT groups with AL-pomegranate. No Groups Groups details 1 Control Untreated HaCaT cells 2 H2O2 injured HaCaT cells injured with H2O2 3 T-Pome-10 pg / ml H2O2 injured HaCaT treated with 10 pg / ml AL-pomegranate extract 4 T-Pome-20 pg / ml H2O2 injured HaCaT treated with 20 pg / ml AL-pomegranate extract 5 T-Pome-50 pg / ml H2O2 injured HaCaT treated with 50 pg / ml AL-pomegranate extract 6 T-Pome-100 pg / ml H2O2 injured HaCaT treated with 100 pg / ml AL-pomegranate extract 7 T-Pome-150 pg / ml H2O2 injured HaCaT treated with 150 pg / ml AL-pomegranate extract

[0036] MTT Assay - HaCaT cells treated with AL-pomegranate extract are subjected to the MTT assay to determine cell viability. Cells are incubated with 0.5 mg / mL MTT solution for 4 hours at 37°C. After incubation, the MTT solution is removed, and formazan crystals, which result from MTT reduction by viable cells, are dissolved in DMSO. The absorbance is measured at 540 nm using a microplate reader.

[0037] Assessment of Anti-oxidative Enzymes -

[0038] a) Superoxide Dismutase (SOD) Assay - SOD activity is measured using a commercial SOD activity assay kit. Cell lysates are prepared and incubated with the assay's substrates and detection reagents. Absorbance is measured at a microplate reader.

[0039] b) Catalase Assay - Catalase activity is assessed using a commercial catalase activity assay kit. Cell lysates are prepared and incubated with the kit's substrates and detection reagents. Absorbance is recorded using a microplate reader.

[0040] c) Glutathione Peroxidase (GPx) Assay - GPx activity is determined using a commercial GPx activity assay kit. Cell lysates are prepared and incubated with the assay's substrates and detection reagents. Absorbance is measured at a microplate reader.

[0041] Estimation of Lipid Peroxidation -

[0042] a) NADPH Oxidase Assay - The activity of NADPH oxidase is measured using a commercial NADPH oxidase activity assay kit. Cell lysates are prepared and mixed with the provided reagents. Absorbance is measured at 550 nm.

[0043] b) Cyclooxygenase-2 (COX-2) Assay - COX-2 expression is assessed using a human COX-2 ELISA kit. Cell lysates are prepared and incubated with the kit's antibodies. Absorbance is measured at 450 nm.

[0044] c) Lipoxygenase (LOX) Assay - LOX activity is measured using a commercial LOX activity assay kit. Cell lysates are prepared and incubated with the kit's substrates and detection reagents. Absorbance is measured at 234 nm.

[0045] Assessment of Anti-inflammatory Enzymes -

[0046] a) Interleukin-1 p (IL-ip) Assay: IL-ip levels are measured using a commercial IL-ip ELISA kit. Cell culture supernatants are collected, incubated with the provided antibodies, and absorbance is measured at 450 nm.

[0047] b) Interleukin-6 (IL-6) Assay: IL-6 levels are determined using a commercial IL-6 ELISA kit. Cell lysates are prepared and incubated with the capture and detection antibodies. Absorbance is measured at 450 nm.

[0048] c) Tumor Necrosis Factor-a (TNF-a) Assay: TNF-a levels are measured using a commercial TNF-a ELISA kit. Cell lysates are prepared, and the sample is incubated with the capture and detection antibodies. Absorbance is read at 450 nm.

[0049] Statistical Analysis - Data from various experimental groups are analyzed using a computer software and by using statistical tests like one-way ANOVA. Differences between groups are considered statistically significant if the P value is less than 0.05. Bonferroni's posttest is used for group comparisons.

[0050] Results and Analysis

[0051] FIG. 1 illustrates Cellular viabilities in post-AL-pomegranate treated cultured human keratinocytes, in accordance with an aspect of the present invention. It presents a bar graph showing the viability of cultured human keratinocytes after exposure to hydrogen peroxide (H2O2)-induced oxidative stress and subsequent treatment with different concentrations of AL-pomegranate extract. The data, expressed as the standard error of the mean, ± SEM, from triplicate experiments, reveals a dose-dependent protective effect of the extract, with significant increases in cell viability observed at higher concentrations of AL-pomegranate extract. This suggests that the extract effectively mitigates oxidative damage in keratinocytes, supporting its potential use in enhancing skin cell resilience against environmental stressors.

[0052] The enhanced anti-oxidative enzyme activity of the enzyme superoxide dismutase (SOD) was assessed in cultured human keratinocytes (HaCaT) that had been subjected to oxidative injury via H2O2 and then treated with varying concentrations of AL-pomegranate extract. SOD is critical in protecting cells from oxidative stress by converting superoxide radicals into less harmful molecules like oxygen and hydrogen peroxide. From the results presented in FIG. 2, the control group, which consisted of untreated keratinocytes, demonstrated an SOD activity which was measured at 0.499±0.0637, representing the baseline activity in healthy cells. However, the keratinocytes exposed to H2O2 showed a significant reduction in SOD activity, dropping to 0.216±0.0l 87. This decrease highlights the substantial oxidative stress and damage induced by H2O2. Treatment with AL-pomegranate extract led to a concentration-dependent recovery of SOD activity in the damaged cells. At the lowest concentration of 10 pg / mL, SOD activity remained similar to that of the H2O2-injured cells, indicating minimal recovery. As the concentration increased to 20 pg / mL and 50 pg / mL, there was a gradual improvement in SOD activity, with levels rising to 0.257±0.0219 and 0.294=0.0439, respectively. Further increases in the extract concentration to 100 pg / mL and 150 pg / mL resulted in more substantial recoveries, with SOD activities of 0.316±0.0144 and 0.378±0.0385, respectively. Although the highest tested concentration (150 pg / mL) did not fully restore SOD activity to the levels observed in the control group, it nonetheless demonstrated a marked improvement compared to the untreated, oxidatively stressed cells. The results indicate that AL-pomegranate extract effectively enhances SOD activity in a concentration-dependent manner, highlighting its potent antioxidative properties. The ability of the disclosed extract to restore SOD activity suggests its potential as a therapeutic agent for managing oxidative stress in skin cells. By mitigating oxidative stress, AL-pomegranate extract could be valuable in treating skin conditions associated with oxidative damage, such as acne and aging, thereby promoting overall skin health and resilience.

[0053] Analyzing the bar chart of FIG. 2, it can be observed that the SOD activity was significantly impaired in keratinocytes subjected to oxidative damage, underscoring the oxidative stress experienced by the cells. However, treatment with AL-pomegranate extract led to a dose-dependent restoration of the SOD activity. Notably, higher concentrations of the extract (100 pg / mL and 150 pg / mL) significantly improved SOD activity, approaching levels seen in untreated control cells. These results suggest that AL-pomegranate extract effectively counteracts oxidative stress in keratinocytes, restoring their antioxidant defense mechanisms.

[0054] The activity of catalase, another key antioxidant enzyme, was similarly assessed in cultured human keratinocytes that were exposed to oxidative damage induced by H2O2 and subsequently treated with various concentrations of AL-pomegranate extract, with the results presented in the bar chart of FIG. 3. Catalase is a critical antioxidant enzyme responsible for converting hydrogen peroxide into water and oxygen, thereby protecting cells from oxidative damage. The findings from FIG. 3 clearly show that catalase activity was significantly reduced in keratinocytes exposed to H2O2, indicating substantial oxidative stress and cellular injury. As observed, a catalase activity of 0.992±0.132 was recorded in untreated cells, representing baseline activity in healthy cells. Following oxidative injury from H2O2, catalase activity significantly decreased to 0.437=0.0555, indicating substantial oxidative stress and cellular damage. When the damaged keratinocytes were treated with AL-pomegranate extract, a concentration-dependent increase in catalase activity was observed. At the lowest concentration of 10 pg / mL, catalase activity remained low at 0.4±0.0529, showing minimal recovery. However, at higher concentrations of 20 pg / mL and 50 pg / mL, there was a gradual improvement in catalase activity, reaching 0.5±0.0643 and 0.52±0.07, respectively. The most significant recovery was seen at 100 pg / mL and 150 pg / mL, where catalase activity rose to 0.623=0.0845 and 0.883=0.0367, respectively. The activity at 150 pg / mL was close to the level observed in untreated control cells, indicating that the highest concentration of AL-pomegranate extract was particularly effective in restoring catalase activity. The restoration of both SOD and catalase activities in keratinocytes treated with AL-pomegranate extract underscores the extract's potent antioxidative properties. These enzymes play critical roles in protecting skin cells from oxidative damage, which is a contributing factor to various skin conditions, including acne and premature aging. The concentration-dependent enhancement of enzyme activities suggests that AL-pomegranate extract could be an effective natural therapeutic agent for mitigating oxidative stress and improving skin health. By bolstering the skin's antioxidant defenses, AL-pomegranate extract has the potential to protect against environmental insults and promote resilience in skin cells.

[0055] The activity of glutathione peroxidase (GPx), another key antioxidant enzyme, was also evaluated in cultured human keratinocytes that were subjected to oxidative injury via H2O2 and treated with varying concentrations of AL-pomegranate extract. From the results shown in FIG. 4, it is observed that GPx plays a vital role in reducing lipid hydroperoxides to their corresponding alcohols and in reducing free hydrogen peroxide to water. In untreated keratinocytes, GPx activity was measured at 0.714=0.0382. However, cells that were damaged by H2O2 exhibited a significant decrease in GPx activity, dropping to 0.36=0.0551. reflecting substantial oxidative damage. Treatment with AL-pomegranate extract resulted in a concentration-dependent restoration of GPx activity. At lower concentrations (10 pg / mL and 20 pg / mL), the recovery of GPx activity was minimal, with values of 0.327±0.00667 and 0.277±0.0406, respectively. A slight increase was observed at 50 pg / mL, where GPx activity rose to 0.333=0.0491. More substantial increases in GPx activity were observed at higher concentrations of 100 pg / mL and 150 pg / mL, with activities reaching 0.467±0.0167 and 0.523±0.0536, respectively. These findings indicate that AL-pomegranate extract effectively restores GPx activity in a dose-dependent manner, particularly at higher concentrations. The restoration of GPx activity suggests that AL-pomegranate extract can help mitigate oxidative stress by enhancing the cellular antioxidant defense system. This further supports the potential of AL-pomegranate extract as a natural therapeutic agent for protecting skin cells from oxidative damage, contributing to overall skin health and resilience.

[0056] The activity levels of NADPH oxidase, an enzyme associated with lipid peroxidation and oxidative stress, were also analyzed with the results presented in FIG. 5. The control group of untreated cells exhibited low NADPH oxidase activity, while the H2O2-damaged group showed a significant increase, indicating elevated lipid peroxidation and oxidative stress. However, treatment with AL-pomegranate extract led to a dose-dependent reduction in NADPH oxidase activity as observed from the bas chart of FIG. 5. While lower concentrations of the extract (10 pg / mL and 20 pg / mL) resulted in minimal reductions, higher concentrations (50 pg / mL, 100 pg / mL, and 150 pg / mL) showed a more pronounced decrease in NADPH oxidase activity. At the highest concentration of 150 pg / mL, NADPH oxidase activity was reduced to a level comparable to that of the control group, suggesting that AL-pomegranate extract effectively mitigates lipid peroxidation. This reduction in NADPH oxidase activity underscores the potent antioxidative effects of AL-pomegranate extract, highlighting its potential for use in protecting skin cells from oxidative stress and lipid peroxidation, which are key contributors to conditions such as acne and inflammation. These findings collectively demonstrate the ability of AL-pomegranate extract to enhance antioxidant enzyme activity and reduce oxidative damage in skin cells, supporting its potential as a natural therapeutic agent for maintaining skin health and resilience.

[0057] The effect of AL-pomegranate extract on the activity of cyclooxygenase-2 (COX-2), an enzyme associated with inflammation, was investigated in cultured human keratinocytes subjected to oxidative stress induced by hydrogen peroxide (H2O2) with the results presented in FIG. 6. COX-2 is known to play a pivotal role in the inflammatory response, particularly in conditions involving oxidative stress, such as acne. As depicted in FIG. 6, the control group, which was not exposed to oxidative stress, exhibited a baseline COX-2 activity of 0.418±0.0598. In contrast, the H2O2-damaged group showed a substantial increase in COX-2 activity to 0.783±0.0867, reflecting heightened inflammatory response due to oxidative damage. Treatment with AL-pomegranate extract resulted in a concentration-dependent reduction in COX-2 activity. At lower concentrations (10 pg / mL and 20 pg / mL), there was a modest decline in COX-2 activity to 0.67±0.0814 and 0.603±0.0664, respectively. As the concentration of the extract increased, the reduction in COX-2 activity became more pronounced. At 50 pg / mL, COX-2 activity decreased slightly to 0.61±0.0693. A significant reduction was observed at 100 pg / mL, with COX-2 activity dropping to 0.45±0.0379. The highest concentration tested, 150 pg / mL, led to the most substantial decrease in COX-2 activity to 0.37=0.0961, bringing it close to the control group level. Statistical significance was considered at p <0.05, and the values are presented as mean ± SEM based on triplicate experiments. These findings suggest that AL-pomegranate extract effectively attenuates COX-2 activity in a dose-dependent manner, thereby reducing the inflammatory response in H2O2-injured keratinocytes. The ability of AL-pomegranate extract to modulate COX-2 activity aligns with its known anti-inflammatory properties, which are largely attributed to the presence of polyphenols and flavonoids such as punicalagins. By downregulating COX-2 activity, the extract could potentially reduce inflammatory lesions and oxidative stress in the skin, promoting overall skin health and resilience. These results further bolster the potential application of AL-pomegranate extract as a natural therapeutic for managing inflammation and oxidative stress in dermatological conditions.

[0058] FIG. 7 illustrates the results of the LOX activity in cultures human keratinocytes treated with various concentrations of AL-pomegranate extract following exposure to H2O2. The data indicates a concentration-dependent reduction in LOX activity with increasing doses of the extract. Starting with an extract concentration of 10 pg / mL, LOX activity decreased to 1.58±0.179, whereas at 20 pg / mL, it further reduced to 1.38±0.156. 50 pg / mL of the extract showed LOX activity of 1.44±0.164, with a greater reduction of 1.04±0.0985 observed at extract concentration of 100 pg / mL. The highest concentration tested, 150 pg / mL, induced the most notable reduction, with LOX activity diminishing to 0.79±0.0907, under the control group level. Statistical significance is defined as p <0.05, and values are presented as mean ± SEM. The results highlight the effectiveness of AL-pomegranate extract in regulating LOX activity, which is crucial for lipid peroxidation and oxidative stress. The extract’s ability to inhibit LOX activity supports its potential use in reducing lipid peroxidation and inflammation in skin cells.

[0059] FIG. 8 depicts Interleukin-1 beta (IL-10) activity in AL-pomegranate treated cultured human keratinocytes. The bar graph illustrates the levels of IL-ip in human keratinocytes treated with different concentrations of AL-pomegranate extract following exposure to H2O2. The control group, receiving no treatment, displayed an IL-ip level of 1.07±0.071, meanwhile, the H2O2-injured group indicated a considerable surge in IL-ip levels to 1.6=0.176. signifying an amplified inflammatory reaction. Treatment with varying concentrations of AL-pomegranate extract ranging from 10 pg / mL to 150 pg / mL exhibit considerable reduction in IL-ip levels as the extract concentration is increased. The least IL-ip levels of 1.13±0.0913 were observed when treated with the highest concentration of 150 pg / mL of the AL-pomegranate extract. The results demonstrate that AL-pomegranate extract effectively reduces IL-ip levels in H2O2-injured keratinocytes in a concentration-dependent manner. By lowering IL-ip levels, AL-pomegranate extract may help diminish inflammatory responses and oxidative stress in skin cells, thereby promoting overall skin health and aiding in the management of inflammatory skin disorders like acne. Additionally, the prebiotics present in the extract may enhance these anti-inflammatory effects by supporting a beneficial microbiota, further contributing to reduced inflammation.

[0060] FIG. 9 indicates Interleukin-6 (IL-6) activity in AL-pomegranate treated cultured human keratinocytes. The bar graph depicts IL-6 levels in human keratinocytes treated with different concentrations of AL-pomegranate extract following exposure to H2O2. Significance is defined as p <0.05, with data presented as mean ± SEM. The results show a reduction in the IL-6 activity as the AL-pomegranate extract concentration gradually increased from 10 pg / mL tol50 pg / mL. The least IL-6 of 0.373±0.0882 is observed for the group treated with the highest (150 pg / mL) concentration of the Al-pomegranate extract. The results demonstrate that AL-pomegranate extract significantly reduces IL-6 levels in hydrogen peroxide-damaged keratinocytes in a concentration-dependent manner. This supports the extract's anti-inflammatory potential, particularly due to punicalagins, which are known to modulate inflammatory cytokines like IL-6. By decreasing IL-6 levels, AL-pomegranate extract can mitigate the inflammatory response and oxidative stress in skin cells, potentially improving skin health and assisting in managing inflammatory skin conditions such as acne. Additionally, the presence of flavonoids and prebiotics in the extract may enhance these antiinflammatory effects by supporting a beneficial microbial environment that can further modulate cytokine production and promote overall skin condition.

[0061] FIG. 10 illustrates Tumor necrosis factor-alpha (TNF-a) activity in AL-pomegranate treated cultured human keratinocytes. The bar graph depicts TNF-a levels in human keratinocytes treated with different concentrations (10 pg / mL to!50 pg / mL) of AL-pomegranate extract compared to the control and H2O2-injured samples. The highest extract concentration of 150 pg / mL shows the least TNF-a activity of 0.137±0.0384 comparable to the un-injured / control sample of 0.132±0.0111. These outcomes show that AL-pomegranate extract effectively reduces TNF-a levels in hydrogen peroxide-damaged keratinocytes supporting the cellular potential of pomegranate polyphenols, particularly punicalagins, known for their ability to suppress the production of pro-inflammatory cytokines like TNF-a. By decreasing TNF-a levels, AL-pomegranate extract can mitigate inflammatory responses and oxidative stress in skin cells, potentially enhancing skin health and aiding in the management of skin conditions such as blemishes. Additionally, the flavonoids and prebiotics present in the extract may further contribute to these effects by neutralizing free radicals, maintaining a beneficial microbial environment, and supporting overall skin health.

[0062] The AL-pomegranate extract disclosed herein offers several significant advantages making it a valuable ingredient for developing natural innovative skincare products aimed at enhancing skin health and treating various skin conditions. Some of the significant advantages of the present invention include;

[0063] a) The ability to prepare the extract from naturally occurring materials free from synthetic chemicals and safe for the human skin.

[0064] b) The extract is rich in polyphenols and flavonoids, which are potent antioxidants, helping protect skin cells from oxidative stress by neutralizing reactive oxygen species (ROS), thus preventing premature aging and other oxidative damage-related skin issues.

[0065] c) The presented AL-pomegranate extract has strong anti-inflammatory properties, reducing levels of pro-inflammatory cytokines and inhibiting the activity of inflammatory enzymes. This makes it effective in treating inflammatory skin conditions such as acne, eczema, and psoriasis.

[0066] d) By inhibiting NADPH oxidase, the extract decreases lipid peroxidation, which is a key factor in the development of acne and other skin conditions. This reduction leads to less sebum production, preventing clogged pores and reducing acne breakouts.

[0067] e) The prebiotics in the extract nourish beneficial bacteria on the skin, promoting a balanced microbiome which helps reduce the presence of acne-causing microbes and enhances the skin's natural defense mechanisms.

[0068] f) The extract offers broad protection against environmental stressors and endogenous oxidative agents, supporting overall skin health and resilience enabling the skin to withstand daily aggressors.

[0069] g) The extract can be incorporated into a wide range of skincare products, including creams, serums, masks, and cleansers, making it versatile for various cosmetic and therapeutic uses.

[0070] h) The effectiveness of the AL-pomegranate extract increases with concentration, allowing for customizable formulations based on specific skin needs and conditions.

[0071] i) Compounds like punicalagins and flavonoids in the extract help stabilize cell membranes, preventing oxidative damage to lipids. This action reduces sebum secretion and acne symptoms, contributing to clearer and healthier skin.

[0072] j) The use of natural ingredients like pomegranate aligns with the growing demand for eco-friendly and sustainable skincare products. This advantage appeals to environmentally conscious consumers and supports sustainable sourcing practices.

[0073] While the subject invention is described and illustrated with respect to certain preferred and alternative aspects, it should be understood that various modifications can be made to those use-cases without departing from the subject invention, the scope of which is defined in the following claims.

Claims

1. An organic dermal composition for improving skin health, comprising:an organic extract enabling antioxidant defense;bioactive compounds which reduce lipid peroxidation;bioactive compounds which mitigate inflammation;bioactive compounds which nourish the beneficial gut and skin probiotics leading to optimized epidermal probiotics balance;2. The organic dermal composition for improving skin health of claim 1, wherein the organic extract further comprises of:powdered AL-pomegranate; andan organic solvent.

3. The composition for improving skin health of claim 2, wherein the organic solvent is dimethyl sulfoxide (DMSO).

4. The composition of claim 1, wherein the bioactive compounds comprise of:polyphenols such as punicalagins;flavonoids including anthocyanins;catechins; andVitamins.

5. The composition of claim 1, wherein the agents that optimize the probiotic balance comprise of:antimicrobial peptides derived from pomegranate extract;natural acids, including ellagic acid; andtannins which can interact with microbial communities, including probiotics.

6. The composition of claim 1, wherein the components that optimize overall epidermal wellness comprises of:prebiotics that promote the growth of beneficial skin bacteria;moisturizing agents such as natural sugars and amino acids; andanti-inflammatory agents that reduce skin inflammation and oxidative stress.

7. The composition of claim 1, wherein the extract enhances antioxidative enzyme activity by significantly increasing levels of protective enzymes, including superoxide dismutase, catalase, and glutathione peroxidase, to protect skin cells from reactive oxidative species.

8. The composition of claim 1, wherein the extract inhibits NADPH oxidase enzyme activity, thereby reducing lipid peroxidation and sebaceous secretion, which are pivotal factors in acne development.

9. The composition of claim 1, wherein the extract decreases levels of pro-inflammatory cytokines and inhibits inflammatory enzyme activity, alleviating inflammation and oxidative stress within skin cells.

10. The composition of claim 1, wherein the extract contains polyphenols and flavonoids, including punicalagins, which impart robust antioxidant and anti-inflammatory properties to enhance skin defense and restoration.

11. The composition of claim 1, wherein the effectiveness of the extract increases with concentration, with higher doses notably boosting antioxidant defenses and reducing inflammatory markers.

12. The composition of claim 1, wherein the extract functions as a natural therapeutic for managing oxidative stress-linked dermal conditions, including acne and inflammation, thereby fostering overall epidermal health and resilience.

13. The composition of claim 1, wherein the prebiotics, polyphenols, and flavonoids in the extract synergistically enhance its efficacy in modulating oxidation, inflammation, and lipid peroxidation.

14. The composition of claim 1, wherein the extract contains compounds such as punicalagins and flavonoids that stabilize cell membranes and prevent oxidative damage to lipids, contributing to diminished sebum secretion and acne symptoms.

15. The composition of claim 1, wherein the extract provides comprehensive protection from environmental and endogenous oxidative invaders, supporting the maintenance of healthy, youthful skin.

16. A method of enhancing skin condition, said method comprising uniformly applying the dermal composition as claimed in claim 1.

17. The method of claim 16, comprising combating a dermal disorder.

18. The method of claim 17, wherein the dermal disorder comprises acne.

19. The method of claim 17, wherein the dermal disorder comprises of eczema.

20. The method of claim 17, wherein the dermal disorder comprises psoriasis.Amendments to the claims have been filed follows:Claims22 1 2 251. An organic dermal composition for improving the appearance and condition of skin, comprising anthocyanin-less (AL) pomegranate extract containing:bioactive compounds which reduce lipid peroxidation and improve antioxidant defense;bioactive compounds which mitigate inflammation;bioactive compounds which nourish the beneficial gut and skin probiotics leading to optimized epidermal probiotics balance;2. The organic dermal composition of claim 1, wherein the AL-pomegranate extract is obtained from powdered AL-pomegranate using an organic solvent.

3. The organic dermal composition of claim 2, wherein the organic solvent is dimethyl sulfoxide (DMSO).

4. The composition of claim 1, wherein the bioactive compounds comprise of:polyphenols such as punicalagins;flavonoids such as catechins;prebiotics and vitamins.

5. The dermal composition of claim 1, wherein the AL-pomegranate extract inherently comprises ellagic acid and tannins.

6. The non-therapeutic dermal composition of claim 1, wherein the composition further comprises naturally occurring prebiotic components that support the balance of the skin surface microbiome.

7. The non-therapeutic dermal composition of claim 1, wherein the composition providesantioxidant skin-care benefits that help protect the skin from environmental oxidative stress.

8. The non-therapeutic dermal composition of claim 1, wherein the composition contributes to reducing the appearance of excess skin oiliness by reducing lipid peroxidation and sebaceous secretion.

9. The composition of claim 1, wherein the extract decreases levels of pro-inflammatory cytokines and inhibits inflammatory enzyme activity, alleviating inflammation and oxidative stress within skin cells.

10. The composition of claim 1, wherein the extract contains polyphenols and flavonoids, including punicalagins, which impart robust antioxidant and anti-inflammatory properties to enhance skin defense and restoration.

11. The composition of claim 1, wherein the effectiveness of the extract increases with concentration, with higher doses notably boosting antioxidant defenses and reducing inflammatory markers.

12. The composition of claim 1, wherein the extract functions as a natural anti-oxidant for managing oxidative stress-linked dermal conditions, including acne and inflammation, thereby fostering overall skin health and resilience.

13. The composition of claim 1, wherein the prebiotics, polyphenols, and flavonoids in the extract synergistically enhance its efficacy in modulating oxidation, inflammation, and lipid peroxidation.

14. The dermal composition of claim 1, wherein the composition comprises punicalagins and flavonoids and contributes to maintaining the visual smoothness, surface integrity, and balanced appearance of the skin, including a reduction in the visible appearance of excess oiliness.

15. The composition of claim 1, wherein the extract provides comprehensive protection from environmental and endogenous oxidative invaders, supporting the maintenance of healthy, youthful skin.

16. A skin-care method of improving the appearance of skin, comprising uniformly applying the dermal composition of claim 1.

17. skin-care method of claim 16, wherein the method improves the appearance of oily or blemish-prone skin.

18. The skin-care method of claim 16, wherein the method improves the appearance of dry or sensitive-looking skin19. The skin-care method of claim 16, wherein the method improves the appearance of uneven or stressed-looking skin.

20. The non-therapeutic topical skin-care method of claim 16, wherein the method is performed as part of a personal skin-care routine

Images