Protective effect of AL-pomegranate extract on oxidative stress skin cells

Abstract

The present invention discloses the regenerative potential of AL-pomegranate extracts in promoting skin health by addressing oxidative stress, inflammation and microbial imbalances that lead to aging and various skin conditions. The extract is prepared using ethanol and water, the extract being rich in polyphenols, flavonoids and prebiotics. The cultured human keratinocytes are exposed to oxidative damage using hydrogen peroxide and treated with different concentrations of the extract. The present invention relates to biomarkers for measuring oxidative stress (SOD, catalase, GPx), lipid peroxidation (NADPH oxidase, LOX), and inflammation (COX-2, IL-1beta, IL-6, TNF-alpha). Results show concentration-dependent recovery of antioxidant enzymes and reduction of lipid peroxidation markers. Furthermore, inflammatory biomarkers are significantly reduced, particularly at high concentrations (150 [mu] g / mL), where the improvement is close to the control group level. These findings show that the AL-pomegranate extract is effective in enhancing antioxidant defense, reducing lipid peroxidation, and reducing inflammation in keratinocytes, supporting its potential as a natural therapeutic for managing oxidative stress-related skin conditions, such as acne and inflammatory disorders. Further studies are suggested to validate these benefits in a clinical environment.

Description

Technical Field

[0001] The present invention relates to the field of skin hygiene and more particularly to products and methods for cosmetic compositions. Background Art

[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 explicitly or implicitly referenced is prior art.

[0003] Skin health is a key aspect of overall health, and maintaining its integrity and resilience to environmental and physiological stressors is critical. Despite advances in dermatological treatments, challenges remain in managing skin conditions exacerbated by oxidative stress, inflammation, and microbial imbalance. These factors are often associated with a variety of skin conditions, including acne, premature aging, and inflammatory skin diseases. Oxidative stress results from an imbalance between reactive oxygen species (ROS) and the skin's antioxidant defenses, leading to cell damage and accelerated aging. Free radicals can induce lipid peroxidation, damage cell membranes, and exacerbate inflammation. Current treatments typically focus on relieving symptoms, but may not effectively address the underlying oxidative damage and inflammatory processes.

[0004] Inflammatory response is another important factor in skin disorders that directly affects the progression of aging and acne. Elevated levels of proinflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) are associated with chronic inflammation and skin damage. Existing anti-inflammatory treatments sometimes provide partial relief, but may not completely resolve the systemic inflammation that causes skin conditions. In addition, microbial imbalances on the skin, especially the proliferation of bacteria that cause acne, pose a challenge to effective acne management. Conventional antimicrobial treatments may address the overgrowth of microorganisms, but generally lack the ability to balance the skin microbiome 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. Therefore, innovative approaches that integrate natural compounds are urgently needed to comprehensively address oxidative stress, inflammation and microbial imbalance. Effective solutions should provide enhanced antioxidant protection, reduce inflammation, and balance the skin microbiome, ultimately improving overall skin health and elasticity. Therefore, there is a need for improvements over the existing technology, and more specifically a more effective approach. Summary of the invention

[0006] The following provides a brief summary of one or more aspects of the present invention to enable a basic understanding of these aspects. This summary is not an exhaustive review of all contemplated aspects, and is neither intended to point out all key or decisive factors of all aspects, nor to define the scope of any or all aspects. Its sole purpose is to provide some concepts of one or more aspects in a simplified manner as a prelude to a more detailed description given later.

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

[0008] According to another aspect, the present invention provides a unique AL-pomegranate extract derived from white pomegranate, which is 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 main active components include punicalagin, which is known for its potent antioxidant and anti-inflammatory properties, ellagic acid, prebiotics and a series of flavonoids.

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

[0010] In another aspect, AL-pomegranate extract exhibited potent antioxidant activity by enhancing the function of key enzymes. According to the same aspect, the disclosed composition was observed to increase superoxide dismutase (SOD) activity and restore catalase levels in a concentration-dependent manner, reflecting its ability to neutralize reactive oxygen species and mitigate oxidative damage.

[0011] In yet another aspect, the disclosed composition also showed improved glutathione peroxidase (GPx) activity, which is essential for reducing hydrogen peroxide and lipid peroxides, further protecting cells from oxidative stress. According to the same aspect, the disclosed extract also effectively reduced lipid peroxidation markers, nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) oxidase and lipoxygenase (LOX) activities, indicating its role in reducing lipid peroxidation and oxidative damage in keratinocytes.

[0012] According to a final aspect, AL-pomegranate extract was shown to exhibit significant anti-inflammatory properties by reducing the activity of cyclooxygenase-2 (COX-2) and by reducing the levels of pro-inflammatory cytokines, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). This reduction in inflammatory markers supports the extract's potential in alleviating inflammatory skin conditions and promoting overall skin health.

[0013] The foregoing paragraphs are provided by way of general introduction and are not intended to limit the scope of the appended claims.The described embodiments, together with further advantages, will be better understood by reference to the following detailed description taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The accompanying drawings are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification. The drawings illustrate exemplary aspects of the disclosure and together with the description serve to explain the principles of the disclosure.

[0015] These figures are for illustration only and are not intended to limit the present disclosure, and wherein:

[0016] Figure 1 Cell viability in cultured human keratinocytes post-treated with AL-pomegranate according to aspects of the present invention is shown.

[0017] Figure 2 Shown is superoxide dismutase (SOD) activity in cultured human keratinocytes post-treated with AL-pomegranate according to aspects of the present invention.

[0018] Figure 3 Catalase activity in cultured human keratinocytes post-treated with AL-pomegranate according to aspects of the present invention is shown.

[0019] Figure 4 Shown is GPx activity in cultured human keratinocytes post-treated with AL-pomegranate according to aspects of the present invention.

[0020] Figure 5 Shown is NADPH oxidase activity in AL-pomegranate treated human keratinocytes according to aspects of the present invention.

[0021] Figure 6 Shown is cyclooxygenase-2 (COX-2) activity in cultured human keratinocytes treated with AL-pomegranate according to aspects of the present invention.

[0022] Figure 7 Shown is LOX activity in cultured human keratinocytes treated with AL-pomegranate according to aspects of the present invention.

[0023] Figure 8 Interleukin-1β activity in cultured human keratinocytes treated with AL-pomegranate according to aspects of the present invention is shown.

[0024] Fig. 9 Shown is IL-6 activity in cultured human keratinocytes treated with AL-pomegranate according to aspects of the present invention.

[0025] Fig.10 Tumor necrosis factor-α activity in cultured human keratinocytes treated with AL-pomegranate according to aspects of the present invention is shown. DETAILED DESCRIPTION

[0026] The following is a detailed description of various aspects of the invention depicted in the accompanying drawings. The various aspects are described in such detail so as to clearly convey the present disclosure. However, the amount of detail provided is not intended to limit the intended variations of the various aspects of the present invention; on the contrary, the intent is to cover all modifications, equivalents, and alternatives that fall within the spirit and scope of the present disclosure.

[0027] The present invention relates to a novel AL-pomegranate extract, which is formulated to enhance skin health by addressing key factors such as oxidative stress, inflammation and microbial imbalance, which are known contributors to skin aging and various conditions. The extract is derived from white pomegranate, which is rich in polyphenols, flavonoids and prebiotics - these compounds provide significant antioxidant and anti-inflammatory properties. The key bioactive compounds present in the extract include punicalagin, ellagic acid, prebiotics and various flavonoids, which are believed to have the 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 vital role in enhancing the activity of antioxidant enzymes such as catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPx), which protect cells from oxidative stress. In addition, the prebiotics in the extract help balance the skin microbiome, potentially reducing the presence of bacteria that cause acne.

[0028] Inflammation is a major factor in skin aging and acne progression. The polyphenols in AL-pomegranate extract have been shown to inhibit inflammatory pathways by reducing the expression of proinflammatory cytokines, including TNF-α, IL-6, and IL-1β. The extract also blocks the cyclooxygenase-2 (COX-2) and lipoxygenase (LOX) pathways, further alleviating the inflammatory response. In addition, the extract has been found to regulate lipid peroxidation, a process that can lead to excess sebum production and cause acne. By affecting the activity of NADPH oxidase, a key enzyme in ROS production and lipid peroxidation, the extract helps reduce oxidative stress and sebum production, thereby reducing acne symptoms. The antioxidant properties of compounds such as punicalagins and flavonoids also stabilize cell membranes and prevent oxidative damage to lipids. AL-pomegranate extract was tested on cultured human keratinocytes subjected to oxidative damage using hydrogen peroxide. The treatment involved various concentrations of the extract, and its effects were evaluated on biomarkers including COX-2, LOX, TNF-α, IL-6, IL-1β, catalase, NADPH oxidase, SOD, and GPx. The results showed that the extract significantly reduced inflammation, enhanced antioxidant defenses, modulated lipid peroxidation, and alleviated acne, highlighting its potential as a natural therapeutic agent for dermatological applications and anti-aging skin care solutions.

[0029] This application covers unique formulations and uses of AL-Pomegranate extract in skin care products designed to improve skin health by minimizing oxidative stress, inflammation, and microbial imbalance.

[0030] This method details the procedures used in evaluating the effects of AL-Pomegranate extract on cultured human keratinocytes subjected to oxidative stress (HaCaT cells). Multiple tests were also conducted to determine the optimal extract composition with the ability to improve health while having minimal negative effects. The experimental results focused on evaluating the extract's potential to enhance antioxidant enzyme function, reduce lipid peroxidation, and alleviate inflammation, ultimately contributing to skin health. The steps outlined herein include the preparation of the extract, the induction of oxidative damage in HaCaT cells, treatment with different concentrations of the extract, and subsequent biochemical assays to measure relevant biomarkers.

[0031] Test Setup and Preparation

[0032] sampling - Pomegranate (Punica granatum L.) fruits of the anthocyanin-free (AL) genotype were harvested from fields in Multan, Pakistan. The fruits were thoroughly washed and the leaves and stems were removed. The washed fruits were freeze-dried to preserve their bioactive components. The dried fruits were then crushed, homogenized, and powdered and then stored in sealed bottles for further analysis.

[0033] Extraction of bioactive compounds - To extract naturally occurring bioactive substances (polyphenols, flavonoids and probiotics) found in pomegranate fruit, a sample of 10 g of powdered pomegranate fruit was mixed with 100 ml of distilled water in a separatory funnel containing 50% ethanol. The mixture was filtered through Whatman No. 1 filter paper to remove the precipitate. The resulting filtrate was concentrated under reduced pressure at 40° C. using a rotary evaporator and then freeze-dried to obtain the final extract.

[0034] H 2 O 2 Damage to HaCaT cells - HaCaT cells were cultured in Dulbecco's modified Eagle's medium high glucose (DMEM-HG) supplemented with 100 μg / mL streptomycin, 100 U / mL penicillin, and 10% fetal bovine serum (FBS) until they reached 70%-80% confluence. H2O2 The medium was replaced with fresh DMEM-HG of 50 μl and incubated for 4 hours to induce oxidative damage. After incubation, the cells were washed with PBS and 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 was prepared by dissolving 10 mg of the extract in dimethyl sulfoxide (DMSO). This stock solution was diluted with DMEM to prepare various concentrations: 10 μg / mL, 20 μg / mL, 50 μg / mL, 100 μg / mL, and 150 μg / mL. The treated cells were incubated with these dilutions for 24 hours. After treatment, cell viability was assessed using the MTT assay, and the cells were used for additional analysis. The experimental groups are summarized in Table 1:

[0036] Table 1. HaCaT groups post-treated with AL-pomegranate.

[0037] serial number Group Group Details 1 Comparison Untreated HaCaT cells 2 <![CDATA[Damaged by H2O2]]> <![CDATA[HaCaT cells damaged by H2O2]]> 3 T-Pome-10μg / ml <![CDATA[HaCaT damaged by H2O2 treated with 10 μg / ml AL-pomegranate extract]]> 4 T-Pome-20μg / ml <![CDATA[HaCaT damaged by H2O2 treated with 20 μg / ml AL-pomegranate extract]]> 5 T-Pome-50μg / ml <![CDATA[HaCaT damaged by H2O2 treated with 50 μg / ml AL-pomegranate extract]]> 6 T-Pome-100μg / ml <![CDATA[HaCaT damaged by H2O2 treated with 100 μg / ml AL-pomegranate extract]]> 7 T-Pome-150μg / ml <![CDATA[HaCaT damaged by H2O2 treated with 150 μg / ml AL-pomegranate extract]]>

[0038] MTT assay - HaCaT cells treated with AL-pomegranate extract were subjected to MTT assay to determine cell viability. Cells were incubated with 0.5 mg / mL of MTT solution at 37°C for 4 hours. After incubation, the MTT solution was removed and the formazan crystals obtained by the reduction of MTT by living cells were dissolved in DMSO. The absorbance was measured at 540 nm using a microplate reader.

[0039] Assessment of antioxidant enzymes—

[0040] a) Superoxide dismutase (SOD) assay - SOD activity was measured using a commercial SOD activity assay kit. Cell lysates were prepared and incubated with the assay substrate and detection reagents. The absorbance was measured on a microplate reader.

[0041] b) Catalase Assay - Catalase activity was assessed using a commercial catalase activity assay kit. Cell lysates were prepared and incubated with the substrate and detection reagents of the kit. The absorbance was recorded using a microplate reader.

[0042] c) Glutathione Peroxidase (GPx) Assay - GPx activity was determined using a commercial GPx activity assay kit. Cell lysates were prepared and incubated with the assay substrate and detection reagents. Absorbance was measured on a microplate reader.

[0043] Assessment of lipid peroxidation—

[0044] a) NADPH Oxidase Assay - Use a commercial NADPH Oxidase Activity Assay Kit to measure NADPH oxidase activity. Prepare cell lysate and mix with the provided reagent. Measure absorbance at 550 nm.

[0045] b) Cyclooxygenase-2 (COX-2) Assay—Human COX-2 ELISA kit was used to assess COX-2 expression. Cell lysates were prepared and incubated with the antibodies of the kit. Absorbance was measured at 450 nm.

[0046] c) Lipoxygenase (LOX) Assay - LOX activity was measured using a commercial LOX activity assay kit. Cell lysates were prepared and incubated with the substrate and detection reagents of the kit. Absorbance was measured at 234 nm.

[0047] Assessment of anti-inflammatory enzymes—

[0048] a) Interleukin-1β (IL-1β) assay: IL-1β levels were measured using a commercial IL-1β ELISA kit. Cell culture supernatants were collected, incubated with the provided antibodies, and absorbance was measured at 450 nm.

[0049] b) Interleukin-6 (IL-6) assay: A commercial IL-6 ELISA kit was used to determine IL-6 levels. Cell lysates were prepared and incubated with capture and detection antibodies. The absorbance was measured at 450 nm.

[0050] c) Tumor Necrosis Factor-α (TNF-α) Assay: A commercial TNF-α ELISA kit was used to measure TNF-α levels. Cell lysates were prepared and samples were incubated with capture and detection antibodies. The absorbance was read at 450 nm.

[0051] Statistical analysis - Data from each experimental group were analyzed using computer software and by using statistical tests such as one-way ANOVA. Differences between groups were considered statistically significant if the P value was less than 0.05. Bonferroni's post hoc test was used for group comparisons.

[0052] Results and Analysis

[0053] Figure 1 Cell viability in cultured human keratinocytes after AL-pomegranate treatment according to aspects of the present invention is shown. 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. Data expressed as standard error of the mean ± SEM from three replicates revealed a dose-dependent protective effect of the extract, with a significant increase 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 the resilience of skin cells against environmental stressors.

[0054] The enhanced antioxidant enzyme activity of superoxide dismutase (SOD) was evaluated in cultured human keratinocytes (HaCaT) that had been subjected to oxidative damage via H2O2 and then treated with different concentrations of AL-pomegranate extract. SOD plays an important role in protecting cells from oxidative stress by converting superoxide radicals into less harmful molecules such as oxygen and hydrogen peroxide. Figure 2From the results presented in, the control group consisting of untreated keratinocytes showed SOD activity measured at 0.499 ± 0.0637, representing the baseline activity in healthy cells. However, keratinocytes exposed to H2O2 showed a significant decrease in SOD activity, which dropped to 0.216 ± 0.0187. This decrease highlights the extensive oxidative stress and damage induced by H2O2. Treatment with AL-pomegranate extract resulted in a concentration-dependent recovery of SOD activity in damaged cells. At the lowest concentration of 10 μg / mL, SOD activity remained similar to that of H2O2-damaged cells, indicating minimal recovery. As the concentration increased to 20 μg / mL and 50 μg / mL, there was a gradual increase in SOD activity, with levels rising to 0.257 ± 0.0219 and 0.294 ± 0.0439, respectively. Further increase in extract concentration to 100 μg / mL and 150 μg / mL resulted in greater recovery, with SOD activity of 0.316 ± 0.0144 and 0.378 ± 0.0385, respectively. Although the highest tested concentration (150 μg / mL) did not fully restore SOD activity to the level observed in the control group, it still showed significant improvement compared to untreated oxidatively stressed cells. The results showed that AL-pomegranate extract effectively enhanced SOD activity in a concentration-dependent manner, highlighting its potent antioxidant 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 alleviating oxidative stress, AL-pomegranate extract may be valuable in treating skin conditions associated with oxidative damage, such as acne and aging, thereby promoting overall skin health and elasticity.

[0055] analyze Figure 2 From the bar graph, it can be observed that SOD activity was significantly impaired in keratinocytes subjected to oxidative damage, highlighting the oxidative stress experienced by the cells. However, treatment with AL-pomegranate extract resulted in a dose-dependent restoration of SOD activity. Notably, higher concentrations of the extract (100 μg / mL and 150 μg / mL) significantly increased SOD activity, approaching the levels seen in untreated control cells. These results suggest that AL-pomegranate extract effectively counteracts oxidative stress in keratinocytes and restores their antioxidant defense mechanisms.

[0056] The activity of catalase, another key antioxidant enzyme, was similarly evaluated in cultured human keratinocytes exposed to oxidative damage induced by H2O2 and subsequently treated with various concentrations of AL-pomegranate extract, where the results are presented in Figure 3 Catalase is an important antioxidant enzyme that is responsible for converting hydrogen peroxide into water and oxygen, thereby protecting cells from oxidative damage. Figure 3The findings clearly show that catalase activity was significantly reduced in keratinocytes exposed to H2O2, indicating the presence of a large amount of oxidative stress and cell damage. As observed, a catalase activity of 0.992±0.132 was recorded in untreated cells, representing the baseline activity in healthy cells. After oxidative damage from H2O2, catalase activity was significantly reduced to 0.437±0.0555, indicating a large amount of oxidative stress and cell damage. When damaged keratinocytes were treated with AL-pomegranate extract, a concentration-dependent increase in catalase activity was observed. At the lowest concentration of 10μg / mL, catalase activity was still low at 0.4±0.0529, showing minimal recovery. However, at higher concentrations of 20μg / mL and 50μg / mL, there was a gradual increase in catalase activity, reaching 0.5±0.0643 and 0.52±0.07, respectively. The most significant recovery was seen at 100 μg / mL and 150 μg / mL, where catalase activity rose to 0.623 ± 0.0845 and 0.883 ± 0.0367, respectively. The activity at 150 μg / mL was close to the levels 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 activity and catalase activity in keratinocytes treated with AL-pomegranate extract emphasizes the potent antioxidant properties of the extract. These enzymes play a key role 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 activity suggests that AL-pomegranate extract may be an effective natural therapeutic agent for reducing oxidative stress and improving skin health. By strengthening the skin's antioxidant defenses, AL-pomegranate extract has the potential to protect against environmental insults and promote elasticity in skin cells.

[0057] The activity of glutathione peroxidase (GPx), another key antioxidant enzyme, was also evaluated in cultured human keratinocytes subjected to oxidative damage via H2O2 and treated with different concentrations of AL-pomegranate extract. Figure 4In the results shown in , it was observed that GPx plays a crucial role in reducing lipid hydroperoxides to their corresponding alcohols and in reducing free hydrogen peroxide to water. In untreated keratinocytes, GPx activity was measured to be 0.714±0.0382. However, cells damaged by H2O2 showed a significant decrease in GPx activity, down to 0.36±0.0551, reflecting the extensive oxidative damage. Treatment with AL-pomegranate extract resulted in a concentration-dependent recovery of GPx activity. At lower concentrations (10μg / mL and 20μg / 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 50μg / mL, with GPx activity rising to 0.333±0.0491. A greater increase in GPx activity was observed at higher concentrations of 100 μg / mL and 150 μg / mL, where the activity reached 0.467 ± 0.0167 and 0.523 ± 0.0536, respectively. These findings suggest that AL-pomegranate extract effectively restored GPx activity in a dose-dependent manner, especially at higher concentrations. The restoration of GPx activity suggests that AL-pomegranate extract may help alleviate 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 elasticity.

[0058] The activity levels of NADPH oxidase, an enzyme associated with lipid peroxidation and oxidative stress, were also analyzed, with the results Figure 5 The control group of untreated cells showed low NADPH oxidase activity, while the H2O2-damaged group showed a significant increase, indicating elevated lipid peroxidation and oxidative stress. Figure 5As observed in the bar graph of , treatment with AL-pomegranate extract resulted in a dose-dependent decrease in NADPH oxidase activity. While lower concentrations of the extract (10 μg / mL and 20 μg / mL) resulted in minimal decreases, higher concentrations (50 μg / mL, 100 μg / mL, and 150 μg / mL) showed more pronounced decreases in NADPH oxidase activity. At the highest concentration of 150 μg / mL, NADPH oxidase activity was reduced to levels comparable to those of the control group, indicating that AL-pomegranate extract effectively mitigated lipid peroxidation. The reduction in NADPH oxidase activity emphasizes the potent antioxidant effects of AL-pomegranate extract, highlighting its potential for protecting skin cells from oxidative stress and lipid peroxidation, which are key contributors to conditions such as acne and inflammation. Together, these findings 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 elasticity.

[0059] The effect of AL-pomegranate extract on the activity of cyclooxygenase-2 (COX-2), an enzyme involved in inflammation, was studied in cultured human keratinocytes subjected to oxidative stress induced by hydrogen peroxide (H2O2), where the results Figure 6 COX-2 is known to play a key role in inflammatory responses, particularly in conditions involving oxidative stress such as acne. Figure 6As depicted in , the control group not exposed to oxidative stress exhibited a baseline COX-2 activity of 0.418 ± 0.0598. In contrast, the H2O2-injured group showed a substantial increase in COX-2 activity to 0.783 ± 0.0867, reflecting an enhanced inflammatory response due to oxidative injury. Treatment with AL-pomegranate extract resulted in a concentration-dependent decrease in COX-2 activity. At lower concentrations (10 μg / mL and 20 μg / mL), there was a modest decrease in COX-2 activity, 0.67 ± 0.0814 and 0.603 ± 0.0664, respectively. The decrease in COX-2 activity became more pronounced as the concentration of the extract increased. At 50 μg / mL, COX-2 activity was slightly reduced to 0.61 ± 0.0693. A significant decrease was observed at 100 μg / mL, with COX-2 activity decreasing to 0.45 ± 0.0379. The highest concentration tested, 150 μg / mL, resulted in the greatest reduction 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 ​​were expressed as mean ± SEM based on three replicates. These findings suggest that AL-pomegranate extract effectively attenuates COX-2 activity in a dose-dependent manner, thereby reducing the inflammatory response in H2O2-damaged keratinocytes. The ability of AL-pomegranate extract to modulate COX-2 activity is consistent with its known anti-inflammatory properties, which is largely attributed to the presence of polyphenols and flavonoids such as punicalagin. By downregulating COX-2 activity, the extract can potentially reduce inflammatory lesions and oxidative stress in the skin, promoting overall skin health and elasticity. These results further support the potential application of AL-pomegranate extract as a natural therapeutic agent for managing inflammation and oxidative stress in dermatological conditions.

[0060] Figure 7Results for LOX activity in cultured human keratinocytes treated with various concentrations of AL-pomegranate extract after exposure to H2O2 are shown. The data demonstrate a concentration-dependent decrease in LOX activity with increasing doses of the extract. Starting from an extract concentration of 10 μg / mL, LOX activity decreased to 1.58±0.179, while at 20 μg / mL, it further decreased to 1.38±0.156. 50 μg / mL of the extract showed a LOX activity of 1.44±0.164, with a greater decrease of 1.04±0.0985 observed at an extract concentration of 100 μg / mL. The highest concentration tested, 150 μg / mL, induced the most significant decrease at the control level, with LOX activity decreasing to 0.79±0.0907. Statistical significance was defined as p<0.05, and values ​​are expressed as mean±SEM. The results highlight the effectiveness of AL-pomegranate extract in modulating LOX activity, which is crucial for lipid peroxidation and oxidative stress. The ability of the extract to inhibit LOX activity supports its potential use in reducing lipid peroxidation and inflammation in skin cells.

[0061] Figure 8 Interleukin-1β (IL-1β) activity in AL-pomegranate treated cultured human keratinocytes is depicted. The bar graph shows the levels of IL-1β in human keratinocytes treated with different concentrations of AL-pomegranate extract after exposure to H2O2. The control group that did not receive treatment showed an IL-1β level of 1.07±0.071, while the H2O2 damaged group indicated a significant increase in IL-1β levels to 1.6±0.176, indicating an enhanced inflammatory response. Treatment with different concentrations of AL-pomegranate extract ranging from 10μg / mL to 150μg / mL showed a significant decrease in IL-1β levels as the extract concentration increased. The lowest IL-1β level of 1.13±0.0913 was observed when treated with the highest concentration of 150μg / mL of AL-pomegranate extract. The results showed that AL-pomegranate extract effectively reduced IL-1β levels in H2O2 damaged keratinocytes in a concentration-dependent manner. By reducing IL-1β levels, AL-pomegranate extract can help reduce inflammatory responses and oxidative stress in skin cells, thereby promoting overall skin health and helping to manage inflammatory skin conditions such as acne. Additionally, the prebiotics present in the extract can enhance these anti-inflammatory effects by supporting a beneficial microbiota, further helping to reduce inflammation.

[0062] Fig. 9Interleukin-6 (IL-6) activity in cultured human keratinocytes treated with AL-pomegranate is indicated. The bar graph depicts IL-6 levels in human keratinocytes treated with different concentrations of AL-pomegranate extract after exposure to H2O2. Significance was defined as p<0.05, where data are presented as mean ± SEM. The results showed that IL-6 activity decreased as the concentration of AL-pomegranate extract gradually increased from 10 μg / mL to 150 μg / mL. The lowest IL-6 of 0.373 ± 0.0882 was observed for the group treated with the highest concentration (150 μg / mL) of AL-pomegranate extract. The results showed that AL-pomegranate extract significantly reduced IL-6 levels in hydrogen peroxide-damaged keratinocytes in a concentration-dependent manner. This supports the anti-inflammatory potential of the extract, especially due to punicalagins, which are known to regulate inflammatory cytokines such as IL-6. By reducing IL-6 levels, AL-pomegranate extract can reduce inflammatory responses and oxidative stress in skin cells, potentially improving skin health and helping to manage inflammatory skin conditions such as acne. In addition, the presence of flavonoids and prebiotics in the extract may enhance these anti-inflammatory effects by supporting a beneficial microbial environment, which can further modulate cytokine production and promote overall skin condition.

[0063] Fig.10 Tumor necrosis factor-α (TNF-α) activity in cultured human keratinocytes treated with AL-pomegranate is shown. The bar graph depicts TNF-α levels in human keratinocytes treated with different concentrations (10 μg / mL to 150 μg / mL) of AL-pomegranate extract compared to control samples and H2O2 damaged samples. The highest extract concentration of 150 μg / mL showed the lowest TNF-α activity of 0.137±0.0384, which is comparable to 0.132±0.0111 in the undamaged / control sample. These results indicate that AL-pomegranate extract effectively reduces TNF-α levels in hydrogen peroxide damaged keratinocytes, supporting the cellular potential of pomegranate polyphenols, particularly punicalagins, which are known for their ability to inhibit the production of proinflammatory cytokines such as TNF-α. By reducing TNF-α levels, AL-pomegranate extract can reduce inflammatory responses and oxidative stress in skin cells, potentially enhancing skin health and helping to manage skin conditions such as blemishes. Additionally, the flavonoids and prebiotics present in the extract may further promote these effects by neutralizing free radicals, maintaining a beneficial microbial environment, and supporting overall skin health.

[0064] The AL-Pomegranate extract disclosed herein provides several significant advantages, making it a valuable ingredient for developing natural, innovative skin care products designed to enhance skin health and treat various skin conditions. Some of the significant advantages of the present invention include:

[0065] a) The ability to prepare extracts from naturally occurring materials that are free of synthetic chemicals and safe for human skin.

[0066] b) The extract is rich in polyphenols and flavonoids, which are powerful antioxidants that help protect skin cells from oxidative stress by neutralizing reactive oxygen species (ROS), thereby preventing premature aging and other skin problems associated with oxidative damage.

[0067] c) The AL-pomegranate extract of the present invention has strong anti-inflammatory properties, reducing the 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.

[0068] d) By inhibiting NADPH oxidase, the extract reduces lipid peroxidation, a key factor in the development of acne and other skin conditions. This reduction results in less sebum production, preventing pore clogging and reducing acne breakouts.

[0069] e) Prebiotics in the extract nourish the beneficial bacteria on the skin, promoting a balanced microbiome, which helps reduce the presence of acne-causing microorganisms and strengthens the skin's natural defense mechanisms.

[0070] f) Extracts provide broad protection against environmental stressors and endogenous oxidants, supporting overall skin health and elasticity, enabling the skin to defend itself against daily aggressors.

[0071] g) The extract can be incorporated into a wide range of skin care products including creams, serums, masks and cleansers, making it versatile for a variety of cosmetic and therapeutic uses.

[0072] h) The effectiveness of AL-Pomegranate extract increases with concentration, allowing for customizable formulations based on specific skin needs and pathologies.

[0073] i) Compounds in the extract such as punicalagins and flavonoids help stabilize cell membranes and prevent oxidative damage to lipids. This action reduces sebum secretion and acne symptoms, contributing to clearer and healthier skin.

[0074] j) The use of natural ingredients such as pomegranate is in line with the growing demand for environmentally friendly and sustainable skin care products. This benefit appeals to environmentally conscious consumers and supports sustainable sourcing practices.

[0075] While the subject invention has been described and illustrated with respect to certain preferred and alternative aspects, it should be understood that various modifications may be made to these use-cases without departing from the subject invention, the scope of which is defined in the appended claims.

Claims

1. An organic skin composition for improving skin health, comprising: organic extracts that enable antioxidant defense; bioactive compounds that reduce lipid peroxidation; bioactive compounds that reduce inflammation; Bioactive compounds that nourish beneficial gut and skin probiotics to result in an optimized epidermal probiotic balance.

2. The organic skin composition for improving skin health according to claim 1, wherein the organic extract further comprises: Powdered AL-Pomegranate; and Organic solvents.

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

4. The composition of claim 1, wherein the biologically active compound comprises: Polyphenols, such as punicalagins; flavonoids, including anthocyanins; Catechins; and Vitamins.

5. The composition of claim 1, wherein the agent that optimizes the probiotic balance comprises: antimicrobial peptides derived from pomegranate extract; Natural acids, including ellagic acid; and Tannins, which are able to interact with microflora, including probiotics.

6. The composition of claim 1, wherein the components that optimize overall epidermal health comprise: prebiotics that promote the growth of beneficial skin bacteria; Humectants, such as natural sugars and amino acids; and Anti-inflammatory agent that reduces inflammation and oxidative stress in the skin.

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

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

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

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

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

12. The composition of claim 1, wherein the extract is used as a natural therapeutic agent for managing oxidative stress related skin conditions including acne and inflammation, thereby promoting overall epidermal health and elasticity.

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

14. The composition according to claim 1, wherein the extract comprises compounds such as punicalagins and flavonoids that stabilize cell membranes and prevent oxidative damage to lipids, helping to reduce sebum secretion and acne symptoms.

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

16. A method for improving skin condition, the method comprising uniformly applying the skin composition according to claim 1.

17. The method of claim 16 comprising combating a skin disorder.

18. The method of claim 17, wherein the skin condition comprises acne.

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

20. The method of claim 17, wherein the skin disorder comprises psoriasis.

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